editor’s note dear theriogenology friends, clinical theriogenology journal, managed by the society for theriogenolgy, is entering its 13th year. i am very thankful to many colleagues including the founding editor (bob youngquist) and the copy editor (john kastelic) for helping me to grow as an editor during the last 3 years. furthermore, members of the editorial board have been a tremendous help. i am extremely thankful to our management office (charles franz and tara beasley) and omnipress to generate every issue in a timely and orderly manner. i appreciate the encouragement and support from the leadership of society for theriogenology and american college of theriogenologists. as i was organizing the manuscripts for this issue, i went back and reread the mission of our journal. yes, our journal continues to provide a venue for our students, residents, practitioners, and clinical investigators in our profession and in allied professions to publish their scholarly work. in addition, conference and symposium papers and conference abstracts also add value to our journal. i appreciate the outstanding job of our conference leaders and session chairs and i am very thankful for colleagues who give their time and effort to submit quality manuscripts and abstracts, and work with me and the reviewers to refine and improve their publication. the scientific and abstract committee of the american college of theriogenologists enables our journal to publish quality abstracts. chairs and members of this committee have worked untiringly to fulfil their remits and responsibilities. i do recall my days of serving on this committee and it was a wonderful learning experience. this issue has 2 manuscripts that describe laparoscopy ovariectomy technique in 2 species addressing 2 clinical situations. i sincerely encourage colleagues to create case and technique reports to make our journal more beneficial. in addition to excellent reviews that are published in the conference issue, we welcome review papers similar to the 1 published in this issue. we have many talented and experienced scholars in our field and allied fields who can create high-quality reviews in many applied aspects of theriogenology. as i have been in the last 3 years, i will personally continue to reach out to colleagues to create reviews and look forward to publishing 1 or 2 this year. please do help me! in this regard, igor canisso suggested to have series of reviews in comparative topics across species or very species-specific topics that would be of general interest. creating reviews (both mini and series) by theriogenology residents will enable them to develop their writing skills under the tutelage of their mentors. for example, richard hopper and maria ferrer are working with their residents to write reviews on clinical topics. i was very much encouraged by the number of research reports submitted for consideration last year and i am confident that we can do much more in 2021. i implore colleagues working in various theriogenology arenas to submit research reports, an area that our journal has to definitely improve. i earnestly desire to receive more manuscripts in all 4 areas (research, clinical case(s), technique, and review) of our journal in 2021. please help our journal to continue to grow! please feel free to write to me (petera@purdue.edu) if you have questions or suggestions. regards, augustine clinical theriogenology • volume 13 number 1 • march 202123 1 contact elizabeth whitt eliawhit@vols.utk.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9410, http://dx.doi.org/10.58292/ct.v15.9410 case report fetal mummification in a goat elizabeth whitt,a hope patrick,b pablo jarrin,a tulio pradoa acollege of veterinary medicine, bdepartment of animal science, university of tennessee, knoxville, tn, usa abstract a 5-year pet doe (unknown breed) was presented (~ 1 month after her expected day of kidding) with a history of vaginal discharge and fetal membranes hanging from the vulva. she was bright, alert, and responsive with an elevated heart rate and respiratory rate. blood chemistry panel was unremarkable. vaginal speculum examination was attempted; however, her vulva was 1 inch in length (dorsal to ventral commissure) making it very hard. transabdominal uterine ultrasonography revealed no fetus and uterus had an irregular echogenicity. a small fetus was identified via transrectal uterine ultrasonography. decision was to perform cesarean surgery (left flank approach). a mummified fetus was extracted from the left uterine horn. keywords: goat, fetus, mummification, cesarean surgery background in utero death of a fetus after bone development1 (without undergoing autolysis and expulsion) becomes a mummy. mummification is possibly due to dry and hypoxic uterine environment that alters normal autolytic process. apparently, dehydration is a major factor that prevents bacterial survival. mummification has been reported in monotocous and polytocous species,2 mostly in the latter.1 not much is reported in goats. diagnosis, treatment, and causes are presented. case presentation a 5-year pet doe (unknown breed) was presented (~ 1 month after her expected day of kidding) with a history of vaginal discharge and fetal membranes hanging from the vulva. doe was recently adopted with an unknown history regarding herd status, disease prevalence, and management. physical examination revealed elevated heart rate (130/beats per minute [bpm]) and respiratory rate (30/bpm) with decreased rumen contractions. doe had a hypoplastic vulva. vaginal palpation revealed an enlarged and firm urethra, reddish brown mucoid discharge, and fetal membranes. no vaginal lacerations or other abnormalities were noticed. cervix felt closed on digital examination. vaginal speculum examination was attempted; however, her vulva was 1 inch in length (dorsal to ventral commissure) making it very hard and furthermore proper size speculum was not available. transabdominal uterine ultrasonography revealed no fetus and uterus with irregular echogenicity. a small fetus was identified via transrectal uterine ultrasonography. there were bony structures (appearance of thorax), but no heartbeat was detected. differential diagnoses were fetal mummification or maceration. prostaglandin treatment was not considered (may not expel all fetal and bacterial content). owners elected surgical option. an abortion panel (to determine fetal cause of death and mummification) was offered, but owners declined. prior to cesarean surgery, doe was given subcutaneous ceftiofur (6.6 mg/kg) and thiamine (20 mg/kg daily), intravenous flunixin meglumine (1.1 mg/kg) and butorphanol (0.1 mg/kg). thiamine was given because doe was anorexic, and as part of regular supportive care. during surgery, patient was given intravenous lactate ringer’s (140 ml/h). a  mummified fetus with small partial placental membranes (figures 1 and 2) was removed. uterus was lavaged and abdomen was infused with 1.5 ml of penicillin g procaine (10,000 iu/kg), and intravenous morphine (0.1 mg/kg) was given after surgery. the patient was quiet, alert, and responsive the morning after the procedure. the patient had no interest in eating throughout the night, and there was no rumination. outcome the patient was sent home the next day because of positive recovery after surgery. the patient was prescribed oral meloxicam (15 mg once a day for 7 days). owners were advised to observe her closely (placing her in a dry place separate from herd) and ensure that her appetite remained stable and that there was no sign of bloody feces (due to nonsteroidal mailto:eliawhit@vols.utk.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9410 2 citation line: clinical theriogenology 2023, 15, 9410, http://dx.doi.org/10.58292/ct.v15.9410 antiinflammatory drug treatment); instructed to observe for muscle fasciculations, ruminations, diarrhea, inappetence, abdominal distention, and lethargy; and to watch for redness, heat, swelling, pain, and discharge. discussion fetal mummification occurs in animals when a series of factors are met in a certain time and order. it is most often observed in animals that are polytocous compared to monotocous species.1 in pigs there is a tendency for genetic predisposition.3 in small ruminants, infectious agents have been the prominent cause. majority of diseases that cause mummification affect fetal membranes and future fertility. antiinflammatory agents are used.4 not much is known about the specific viability issues since does are commonly culled. infectious diseases responsible for mummification are toxoplasmosis and chlamydophila infection, border disease, and coxiella.5 additionally, uterine torsions can  result in mummification.6 viral infections in small ruminants do not damage the uterus whereas bacterial infections with resulting fetal maceration7 have a poor prognosis for future breeding. although prostaglandin f2a treatment is recommended for goats similar to cattle,8 it was not attempted in this case. learning points • fetal mummification in goats is a rare and uncommon occurrence with unknown etiology • treatment option is surgical approaches to excise the fetus • prostaglandin f2a treatment is not always recommended references 1. lefebvre rc: fetal mummification in the major domestic species: current perspectives on causes and management. vet med (auckl) 2015;6:233–244. doi: 10.2147/vmrr.s59520 2. yadav sk: fetal mummification in a cat. acta sci vet 2021;3:2582–3183. 3. wang s, wu p, wang, k, et al: transcriptome analysis reveals key genes and pathways associated with piglet fetal mummification. genome 2021;64:1029–1040. doi: 10.1139/gen-2021-0026 4. kumar a, gautam d, verma, nk, et al: successful management of fetal maceration in a doe: a case report. j pharm innov 2017;7:148–149. 5. baird an, bychawski s, edmondson, ma, et al: theriogenology of sheep and goats. in: pugh dg, baird, an: editors. sheep and goat medicine. 2nd edition, elsevier; st. louis: mo, 2012. p. 150–230. 6. kumar p, saini p, thakur a, et al: a rare case of fetal mummification in a gaddi doe due to uterine torsion. explor anim med res 2017;7:223–224. 7. katiyar r, rautela r: fetal maceration in goat: a case report. int j sci environ 2016;5:2323–2326. 8. lefebvre rc, saint-hilaire e, morin i, et al: retrospective case study of fetal mummification in cows that did not respond to prostaglandin f2alpha treatment. can vet j 2009;50:71–76. figure 1. mummified fetus in uterine horn figure 2. mummified fetus http://dx.doi.org/10.58292/ct.v15.9410 https://doi.org/10.2147/vmrr.s59520 https://doi.org/10.1139/gen-2021-0026 physiology and reproductive techniques for the whitetail deer physiology and reproductive techniques for whitetail deer jason coe, micheal bringans advanced deer genetics, hudson oaks, tx abstract purpose is to briefly explain the physiology of whitetail deer and to cover reproductive management and artificial insemination techniques. also, to cover some more advanced reproductive technology such as embryo transfer and in vitro fertilization. keywords: cervid, laparoscopic, transcervical, embryo introduction the commercial farming of whitetail deer is a relatively recent agricultural enterprise that came about to supply trophy animals to the hunting preserve industry. whitetail deer range over most of north and central america and are the most hunted big game species in north america. as many as 30 subspecies have been identified, with 16 residing in the us. the 2 that are economically important to deer farming are odocoileus virginianus borealis and texanus, the northern whitetail and the texas whitetail respectively. these subspecies typically grow the largest antlers and in the case of northern whitetail deer, there is also an increase in body size. to help advance the propagation of better genetics, artificial insemination has been employed to use different genetics in areas where the movement of live deer is not allowed. this has given rise to a very profitable industry. physiology whitetail deer are seasonal breeders with rut brought on by decreasing day lengths. as the days begin to shorten from midsummer into fall, there is an increase in hormones in both males and females of the species. in males, this triggers the hardening of the antlers with rubbing to remove the velvet, an increase in size of the secondary sex glands and testes, and beginning of spermatogenesis. more aggressive rutting behaviors start appearing and the bachelor groups start sparring for dominance and eventually break up before the main rut occurs. female will phase from the anestrous period and after a number of silent ovulations, will come into the first behavioral estrous cycle. any female not bred in the first cycle will continue to cycle at 21 to 29 day intervals until pregnancy occurs or increasing day lengths bring anestrous phase.1 estrous cycle of the doe is very similar to that of other ruminant species, in that luteolysis of corpus luteum from the previous ovulation decreases in blood progesterone concentrations and increases i estradiol concentrations produced by maturing follicles. a surge in luteinizing hormone from the pituitary gland in response to estradiol ultimately induces ovulation,2 and the cycle starts over. timing of rut varies from region to region, sometimes up to 8 weeks, but deer from a particular region will maintain the timing of rut even if moved to another location. this is important to remember when planning the timing of breeding, as the northern deer tend to rut in late october to mid-november, whereas texas deer will rut in late november through december. this can be manipulated somewhat; however, timing too far outside their normal time frame will adversely affect conception rates. whitetail does are polyovulatory, with most mature does producing 1.6 1.8 offspring on average. yearling does are more likely to produce single fawns. reproductive management and artificial insemination whitetail deer as a group are exceptionally fertile and respond well to manipulation of the cycle during natural rut. many of the protocols used in other small ruminant species translate well to whitetail does. the most commonly estrus synchronization currently used in the industry is a 14 day cidr protocol. a 0.3 g cidr (eazi-breed cidr sheep insertstm, zoetis animal health, parsippany, nj) is inserted into the vagina on day 0 and removed on day 14, at which time pmsg or ecg is given to synchronize ovulation. breeding should occur 54 62 hours after cidr removal. alternately, cidr can clinical theriogenology • volume 12 number 3 • september 2020268 be left for 14 days, then removed without pmsg treatment; however, breeding should be delayed a few hours (60 64 hours). in our experience, this protocol produces substantially lower conception rates compared to pmsg use. in both protocols, gnrh is given at artificial insemination (ai) to induce ovulation. artificial insemination can be accomplished in 1 of 2 ways (transcervical or laparoscopy assisted). transcervical ai is less technical of the 2 approaches and equipment involved is less expensive than that used with laparoscopic ai, thus making it a popular choice for some producers. the does are most often bred without sedation after being secured in a handling chute. a vaginal speculum is inserted and the external os cervix is located. any mucus should be removed. a goat or cattle insemination pipette is then introduced through the os and manipulated through the rings of cervix to deposit semen just cranial to cervix in the body of uterus. the downfall of the procedure is that the rings of the cervix are difficult to manipulate, and depositing semen in the cervix will result in decreased conception rates overall. conception rates vary but usually will average around 65%. for optimal conception after transcervical ai, 12 15 million progressively motile sperm are recommended. technician experience and patience will greatly influence the outcome. laparoscopic ai is more technical and the equipment used is much more expensive than transcervical ai. the minimum equipment needed is a quality rigid laparoscope of sufficient length to allow the technician to comfortably observe the uterus, a surgical or ai cart capable of holding the animal in dorsal recumbency with the head down at approximately a 45 degree angle, a good light source, 1 trochar and 2 cannulas, a source of filtered air or carbon dioxide for insufflation, aspics, and other disposable items. the procedure starts with a surgical prep and scrub of the caudal abdomen adjacent to the mammary glands. two stab incisions are then made with a number 15 blade through the linea, approximately 10 cm below the junction of the udder. another incision is made 6 8 cm off the midline on either side. filtered air or carbon dioxide are then introduced to sufficiently insufflate the abdomen to allow the trochar and cannula to be introduced without damaging internal organs in this area. once the cannulas are in place, the laparoscope is introduced, as well as the aspic containing the semen. semen is deposited by placing the needle of the aspic through the wall of the uterine horn into the lumen. ideally semen should be deposited in distal third of uterine horn ipsilateral to the dominant follicle, or split between both uterine horns if the ovaries are not observed. by depositing semen closer to oviducts, less semen can be used (~6 8 million progressively motile sperm) and conception rates are generally 10 15% better than transcervical ai. once the procedure is mastered it only takes a few minutes to perform. complications include rupture of the bladder, rumen, or uterus with introduction of the trochar. these ruptures should be repaired immediately to prevent peritonitis. advance reproductive technology more advanced reproductive procedures are also being used in whitetail deer. multiple ovulation embryo transfer (moet) has become increasingly popular in recent years as protocols have successfully been adapted to whitetail does. embryo recovery rates average around 5.5 embryos per doe. a multi injection program utilizing porcine derived fsh has been successfully adapted. the dosage varies based on a number of criteria, but fsh is administered every 12 hours for 4 days, beginning 3 days prior to cidr removal. there is also a new recombinant fsh that recently became available through compounding that is showing promise. the procedure for recovery of the embryos is the same as for ewes and other small ruminant species. animal is anesthetized and placed in dorsal recumbency with the head down at approximately 30 degrees. uterus is exteriorized and an appropriately sized foley is placed in the base of the uterine horn near uterine body. then a tom cat catheter or 18 gauge, iv catheter is placed in the lumen near the tip of the horn and flushed retrograde with 50 60 ml of flush media into an embryo concentration filter or petri dish. incisions into the uterus should be stitched in an inverting pattern to avoid adhesion formation, and care should be taken with handling the uterus as whitetail are extremely prone to scar formation. the body wall and skin are then closed and appropriate antibiotics are given prior to reversal of the anesthetic. once recovered, embryos can be transferred within a few hours fresh, or frozen for storage. for implanting embryos, a laparoscope is used to identify the corpus luteum. the clinical theriogenology • volume 12 number 3 • september 2020 269 embryo is then placed into a tom cat catheter and the catheter is used to deposit the embryo into the lumen of the uterine horn ipsilateral to the cl. as deer are polyovulatory, 2 embryos are usually placed per recipient. implantation rates of fresh embryos in whitetail is exceptionally high, often ~90%. implantation rate for frozen embryos decreases to ~65%. in vitro fertilization is a technology that has made tremendous advancements in recent years in other ruminant species. there is currently some work being done with whitetails; however, its use is not commonplace. conflict of interest there are no conflicts of interest to declare. references 1. jacobson h: reproductive management of white-tailed deer. in: youngquist rs, threlfall wr: editors. current therapy in large animal theriogenology. 2nd edition, philadelphia; saunders: 2007. p. 965-969. 2. asher gw: reproductive cycles in female cervids. in: youngquist rs, threlfall wr: editors. current therapy in large animal theriogenology. 2nd edition, philadelphia; saunders: 2007. p. 921-931. clinical theriogenology • volume 12 number 3 • september 2020270 018_ms-018 coe clinical theriogenology 2022; 14: 131 the 2022 bartlett address in light of the current events in eastern europe that has produced millions of people seeking a new life in a new land, i thought i would share some of my ancestry and how it has influenced my career decisions and approach to life. when my paternal grandfather, joseph sertich was 3 years old, his mother died giving birth to his baby sister. four years later while joseph and his father were cutting timber in a forest near nova kapala, yugoslavia (just east of zagreb, croatia) joseph’s father was crushed by a falling tree, leaving his 2 children orphans. local farmers were willing to look after joseph as he had experience caring for sheep and goats, but my grandfather always said that his sister died of a broken heart. it is easy to understand that what likely was an unthrifty 4-year-old girl did not thrive as an orphan. my grandfather worked on farms in croatia and germany and eventually made his way to the united states in 1911. he married my grandmother, effie polyan and they managed the boarding house at the nemacolin coal mine in southwestern pennsylvania. when cars became more readily available and men began to commute to work from home, the boarding house closed. in 1938 he built and operated sertich general store in carmichaels, pennsylvania with his wife and 4 children, my father being the youngest. there was a slaughterhouse behind the store where they finished livestock and provided custom dressing of stock, that produced beef, lamb, pork, chickens and a variety of smoked meats/sausages for sale in the store. this is where i grew up (lived above the store) as my parents took over management of the operation in 1955. in spite of 2 milk venders selling dairy products in our store, we always kept our own milk cow so we could have real milk for our family consumption. at 12 years of age my maternal grandfather, josef klimek immigrated to chicago, illinois in 1909 from mszana gorna, poland with his oldest sister, anna and her husband. when their father died, anna, being the eldest of the family returned to poland to deal with her father’s funeral and settle family affairs. world war i broke out and anna never returned to the united states. josef made his way to southwestern pennsylvania’s coal region and worked at the richeyville coal mine. he met mr. kois who suggested he might want to marry 1 of his 5 daughters. my grandfather thought it was a good idea and married rose, the youngest and biggest of the kois girls. eventually they saved enough money working at the mine to buy a dairy farm in carmichaels. josef got work at the nemacolin coal mine. my grandmother rose managed the dairy, milking up to 35 cows twice a day and had 8 children of which 6 survived. my parents met after at the end of world war ii and soon married, had 3 sons (twins and a singleton) and then me. i was able to spend much of my youth with both sets of my grandparpatricia sertich department of clinical studies-new bolton center school of veterinary medicine university of pennsylvania, kennett square, pa ents and all the animals at the store and dairy farm. to make my animal experience complete, i had the good fortune that my father had a keen interest in horses. he had a horse as a teenager that unfortunately broke its leg while he was in the navy during the war. when his children came along, he was keen to provide us all with ponies to ride and drive. none of my brothers maintained an interest in horses once they were able to legally drive. my dad fanned the flames of my horse passion and we enjoyed many horse activities together. looking back at things i can recall 3 distinct instances that sparked my interest in animal reproduction. the first was when i was 5 or 6 and my brother john’s paint mare foaled. i had known that the mare was expecting a foal but was baffled when one morning a rambunctious filly was running around the mare. i saw that the mare had a bright streak of blood on her white hip and was puzzled that that the filly could have come out through that bloody spot. the second instance was in 5th grade when i asked my science class teacher, mr. burchani how chickens reproduced when they did not appear to have any of the special body parts that were needed to copulate. i was disappointed that he never was able to give me an answer to this question. and the third instance when i wanted to know more about animal reproduction was when i became a senior in our greene county 4-h horse and pony club. i struggled to complete my senior project workbook which at the time required us to draw a picture of a mare’s reproductive tract and label all its parts. there were no books in our local library about horse breeding and i certainly was not going to ask the 2 elderly horsemen who led our 4-h club about female reproduction. i finally met another senior at the district horse show who had completed her workbook. she allowed me to scrutinize her genital tract drawing and the labels which i carefully copied with tracing paper, not really understanding any of the drawing and ashamed to admit it. so, i headed off to college with some burning questions in my head. during the summers when i was in undergraduate school i volunteered on weekdays at the waynesburg animal hospital for a general practitioner, dr. elmer marx. i would meet dr. marx at the hospital at 7 am and we would head out driving around southwestern pa doing large animal farm calls which included a lot of equine work throughout the day. we would return to the animal hospital on most days by 6 pm to start the small and large animal appointments at the hospital. although the appointments for the day were usually not finished, i’d leave for home around dark so i could get back early the next morning. i started at west virginia university in the school of pharmacy. after 1 or 2 days shadowing a pharmacist, i quickly realized this clinical theriogenology 2022; 14: 132 was not the field for me. i completed most of the pharmacy foundation courses while floating along in general studies. i discovered the school of agriculture when i met a pre-vet major, sarah beamer in a physical education swim class. i quickly changed to animal science and through a work study job soon found myself being the teaching assistant for the introductory survey course in animal science. my work study boss, dr. h. e. ‘doc‘ kidder and dr. e. keith inskeep were course organizers for physiology of reproduction. it was in that course that i discovered my interest in reproduction and the answers to so many questions. dr. kidder was west virginia university’s prevet advisor and i soon found myself applying to pennsylvania’s veterinary school, the university of pennsylvania. but after 2 veterinary school rejections, it was dr. inskeep that mentored me and facilitated my applications to graduate programs in reproductive physiology. keith inskeep was a pioneer in pgf2α research in sheep and he was an excellent teacher and role model. graduate school landed me in laramie, wyoming resulting in a master of science in animal science at the university of wyoming investigating the role of placental lactogen on fetal growth and development in undernourished ewes under the guidance of dr. colon kaltenbach and dr. tom dunn. we used a surgically instrumented ovine fetus model to collect fetal blood samples in late pregnancy. we had a great graduate group that included david forest, michael moseley, gary sides, david walters and we all helped each other with our projects. the team did a lot of hard work and had a lot of fun collecting research data at the university of wyoming’s agriculture experiment station which had been the wyoming territorial prison and working cattle on a few ranches throughout the state. graduate school provided some great training and life experiences, but i wanted to do work in horse reproduction and realized that i still needed to be a veterinarian. so, after my third application to the university of pennsylvania i made the move to philadelphia for veterinary school. my first contact with the reproduction team was during a lecture by their resident, gordan woods. he invited me to palpate teaching mares at the georgia and philip hofmann research center for animal reproduction located on new bolton center campus that next saturday morning. i had the good fortune to still qualify for work study and was promptly able to land a student work study job at the hofmann center that continued for 3 years. during that time, i was exposed to the work of terry blanchard, john hurtgen, wendel cooper, marolo garcia and sue mcdonnell. i had developed aspirations to be a breeding farm manager but the year i was graduating from veterinary school the section of reproduction had not identified a resident. a faculty member encouraged me to apply for the residency position since i knew the routine for handling cases at hofmann center. that was in 1983 and i never left new bolton center. we did a fair amount of cattle work my first few years at new bolton center along with a steady equine case load. learned to do stallion work with bob kenney and dickson varner, mare evaluations and their breeding management with bob kenney and j. stanley brown and obstetrical work with terry blanchard. katrin hinrichs and i were residents together. we were busy with clinic case work and teaching but also had considerable freedom to pursue projects of our own interest. at a client’s request and encouragement, i started a clinical equine embryo transfer program that also provided opportunities for research projects investigating cooled transported embryos and ovariectomized embryo recipients. dr. kenney seemed to have a generous supply of research monies for small projects and encouraged everyone to always ask questions and pursue answers. the best thing about working at a university is getting to know and work with so many great people from around the world: and the worst things about working at a university is that most of those people (students and colleagues) leave and go on to other places. and change is inevitable. as faculty and staff members came and went our section slowly lost much of its cattle work. the beef cattle work greatly decreased when the pennsylvania division of king ranch was sold and our dairy herd work was transferred to our ambulatory field service section. but the equine reproduction work has thrived and evolved. the racehorse reproduction work has been replaced by many horse breeds with more diverse disciplines whose registries and owners embrace the advanced reproductive services and quality medical care we offer. so how has all of this influenced my approach to life. here are some of the things that i think are important and have given me much satisfaction. • always shake the milk jug vigorously before pouring it on your corn flakes. • cultivate young people’s curiosity and provide them stimulating activities as they may be pivotal moments in their lives. • be perceptive to the needs of students, facilitate their academic growth and celebrate their accomplishments. • respect the past and embrace change. • encourage others to question and pursue answers. provide opportunities for investigation. • expect people to do their best. people who respect you will rise to meet your expectations. thank you for this recognition and support patricia l. sertich ms, vmd, diplomate act dr. patricia l. sertich is an associate professor-clinician educator in reproduction and behavior at the university of pennsylvania, school of veterinary medicine. patricia obtained a bachelor of science in animal and veterinary science from west virginia university in 1977 and a master of science in animal science from the university of wyoming in 1979. after earning a veterinary degree from the university of pennsylvania in clinical theriogenology 2022; 14: 133 1983 she completed a residency in large animal reproduction in 1985 and became a diplomate in the american college of theriogenologists in 1988. based at the georgia and philip hofmann research center for animal reproduction at new bolton center since 1983, dr. sertich evaluates large animals for breeding soundness and develops treatment plans to optimize their fertility. dr. sertich provides consultation on reproductive issues of patients in the george widner large animal hospital and is a critical member of the high-risk pregnancy management team. as a diplomate in the american college of theriogenologists (act), she has served on the act certifying examination committee which determines board certification for veterinarians specializing in animal reproduction. dr. sertich has mentored many act diplomates and received numerous teaching awards including 3 carl j. norden distinguished teaching awards and the christian r. and mary f. lindback foundation teaching award. as faculty liaison for the student chapter of the society for theriogenology she can facilitate training for those students to develop excellent clinical skills, provide opportunities to gain clinical experience and connect them with practices that will direct them to a satisfying and productive career in veterinary medicine. editor™s note dear theriogenology friends, there is a tremendous therapeutic potential for extracellular vesicles in assisted reproductive technology. dr. jennifer nagashima, from the smithsonian conservation biology institute, wrote a review for us (first manuscript in this issue). i am truly thankful for jen’s time and effort and for 2 reviewers for their due diligence. jen is also working to create an endnote style for our journal. in addition, 6 colleagues have promised to create reviews for our journal and i am looking forward to receiving them. dr. stuart burns, a practitioner with > 40 years of experience and a long-standing member of the sft, took the initiative to study mare reproductive loss syndrome. his efforts have resulted in 2 papers (second and third manuscripts in this issue). in my opinion, this sends a clear message that practitioners can skillfully apply their experience and available wealth of information to create research reports. i extend my appreciation to the 4 persons who reviewed these papers and provided critical insights. e.f. benson uses the phrase ‘providential privilege of helping others’ in one of his fiction books. as for me, helping our journal 2 hours each day is a ‘providential privilege’. i do exercise kindness and firmness when handling each manuscript, with an ultimate goal to assist authors to feel proud about their work and concurrently, to maintain quality. on a sad note, 4 of 48 (8%) manuscripts were rejected based on many factors; sending ‘regret’ notes for me are not best moments of this remit. i am very thankful for theriogenology mentors for supporting our journal and look forward to receiving more case/research/review/technique reports. i do thank my mentors (clinical and research) for pushing me to write well and i am still improving, with dr. john kastelic’s help. i told this story to a colleague recently during our organizations’ leadership meeting and is well worth repeating. my first case report that i created during my residency days was not received well by my mentor; his request to ‘please remove my name from this case report’ helped me to realize my inadequacy. it took me many more hours of hard work and half a dozen more drafts to earn my mentor’s approval. i am also thankful to dr. bill bosu for his time and effort to improve my scientific wordsmith skills. finally, our journal can gain further reputation if color figures are used. i am grateful to authors who are willing to pay $200/page for publishing in color (otherwise, our journal does not have any page charges). i strongly encourage theriogenology training programs to set money aside in their budgets to support this endeavor. my prayers and hope that everyone stay healthy and well in these troubled times. sincerely, augustine . clinical theriogenology • volume 12 number 2 • june 202088 1 contact william whitler william.whitler@oregonstate.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9320, http://dx.doi.org/10.58292/ct.v15.9320 technique report canine transcervical insemination: history and technique william whitler department of clinical sciences, carlson college of veterinary medicine, oregon state university, corvallis, or abstract artificial insemination in the dog was first described nearly 250 years ago. until the 1970s, the process remained predominantly in depositing semen in the cranial vagina. initially, surgery was required to successfully deposit frozen–thawed semen in uterus. however, societal demands to eliminate the need for general anesthesia and surgery for breeding have led to development of reliable and successful transcervical insemination procedures. common barriers to perform successful transcervical inseminations include equipment expense and developing skills. in addition to practice, adopting suggestions provided by the author will improve practitioner’s success. keywords: canine, dog, artificial insemination, transcervical, endoscope, history, technique history and refinements advances in veterinary medicine often lagged behind human medicine; however, the field of artificial insemination (ai) is an exception. anecdotal reports of the first human ai date backs to henry iv (nicknamed ‘henry the impotent’) of england (1425–1474), whose wife produced a daughter joanna.1 however, veterinary literature alleges that the first ai was performed by an arab chief in 1322, who stole semen from the stallion of a rival chief to place in his mares.2 human and dog sperm were first described by leeuenhoek and ham in the netherlands in 1678, but it was not until 1784 that spallanzani described the birth of three pups after ai of a dog.1 one of the earliest reports of successful canine pregnancy with frozen semen was in 1969.3 however, it was not until 1981 that the american kennel club would allow a litter of pups conceived using frozen semen to be registered.4 references to first registry dates in other countries are not available. transcervical insemination (tci) was first reported in the mid to late 1960s, but there was skepticism about the actual ability to do so blindly.3,5 by the mid-1980s, surgical ai became the most commonly recommended procedure for inseminating dogs using frozen semen because of its short lifespan in the reproductive tract. in the early 1990s, reports of laparoscopic ai appeared.6 tci in dogs using a norwegian catheter designed for foxes was introduced in 1975 but did not gain much popularity in the united states due to its steep learning curve and level of difficulty, especially in larger and obese dogs.7 endoscope-assisted tci (using a human cystoscope) was described in 1973.7 refinements in this procedure have evolved, and today, there are instruments available specifically designed for tci in dogs. some tci equipment manufacturers offer a variety of diameters and lengths of scopes for various size dogs and breeds. surgical ai has certain advantages over endoscope-assisted tci. first, with surgical ai, it is possible to confirm ovulation at insemination and visually/digitally evaluate reproductive tract for pathology. second, with surgical ai, semen can be distributed into both uterine horns, whereas in tci, presumably, whole semen is deposited into a uterine horn or the uterine body. this theoretical advantage toward an increased chance of pregnancy or litter size does not withstand scrutiny as conception rates are reported to be higher using endoscopic tci compared to surgical ai.8 a possible explanation of lower conception rate with surgical ai is due to the stress of surgery and/or anesthesia.8 apparently, this explanation was rejected since there was no difference in conception rate or litter size between dogs bred naturally and via laparoscopic ai.6 this implies that the natural act of canine coitus is as stressful as general anesthesia and surgical insemination. although laparoscopic ai and surgical ai were not compared, comparisons of laparoscopic ovariectomy and ovariohysterectomy via laparotomy indicated that laparoscopic surgery may *presented at the 2022 society for theriogenology conference (canine symposium), published after peer-review. mailto:william.whitler@oregonstate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9320 2 citation line: clinical theriogenology 2023, 15, 9320, http://dx.doi.org/10.58292/ct.v15.9320 or may not take longer, and postoperatively, the patient appeared to be slightly less painful.9,10 since the incision length and amount of tissue handling in a surgical ai are substantially less than an ovariohysterectomy, the last remaining variable is the insemination process itself. studies directly comparing fertility rates using laparoscopic ai versus conventional surgical ai are unlikely to ever be performed for ethical and financial reasons. although the cause of lower fertility rates of surgical ai compared to tci may never be elucidated, some clients still insist on surgical ai. an advantage of endoscope-assisted tci over surgical ai is the ability to perform intrauterine insemination without the need for general anesthesia and surgery. another advantage of endoscope-assisted tci is that it allows multiple inseminations during the fertilization window of estrus. this is not typically performed in surgical ai, as it would require additional general anesthesia and surgery within a 96-hour window. although there was no significant difference in conception rates between single and double inseminations, pregnancy rate in the group inseminated twice was numerically lower (62%) than a single insemination (69%).8 the mechanism by which multiple endoscope-assisted tci procedures performed in an estrous period reduce pregnancy rate remains unclear. the decision to breed twice was based on client preference and/or low numbers of live morphologically normal sperm and availability of semen.8 these authors also determined serum progesterone concentrations as often as daily in some cases to decide on insemination timing. although ideal, that level of accuracy may not always be obtainable for every patient. frozen semen quality can vary even within samples collected from the same male, making the decision to perform endoscope-assisted ai more than once in estrus, which is still a ‘judgement call’. in addition to the advantages of safety, cost, and ability to perform more than one insemination, tci allows visualization of vaginal wall crenulation and any other vaginal abnormalities that might affect normal vaginal delivery (e.g. septum and adhesions). timing of ai is determined by a few methods. if a practitioner is routinely performing vaginal cytologies as a method, large and giant breeds pose a problem (not  able to reach the cranial vagina) with the standard 6-inch swab (sterile polyester tipped applicators, puritan medical products, guilford, me). to solve this problem, the length of the swab can be increased by inserting 1 cm of the swab handle into the lumen of a 0.5 ml semen straw, held in place with a drop of cyano-acrylic glue and allowed a few minutes to dry. technique improvements sedation sedation for an endoscope-assisted tci is practitioner’s discretion. in author’s practice, sedation has rarely been used except for an occasional uncooperative dog. other practitioners report sedating almost every dog for endoscope-assisted tci. to some degree, the decision to sedate may depend on the availability of assistance to restrain the dog. author prefers to use a minimal amount of dexmetdetomidine (dexdomitor®, orion pharma, espoo, finland; zoetis inc, kalamazoo, mi) for most fidgety dogs to have them remain standing. an occasional aggressive dog that resents manipulation of the vulva may require higher doses of dexmetdetomidine and a muzzle. although most estrous dogs tolerate the endoscope-assisted tci procedure with little perceived discomfort, occasionally, a dog will violently and/or vocally object to the procedure. topical vaginal application of lidocaine is an alternate method instead of sedation. author instills 1–2 ml of lidocaine (lidocaine 2%, vet one, mwi, boise, id) via a closed end 3.5 french tom cat catheter (argyle™ [closed end catheter with 5.5 inch adapter] coviden, llc, mansfield, ma) that is inserted into the caudal vagina, and 5 minutes allowed for topical desensitization. difficulty navigating cranial vagina transcervical insemination scope is occasionally difficult to navigate beyond cranial vagina (approximately the level of the pubis). author has not arrived at a consistently reliable way to advance the scope except by: (1) increasing insufflation of the vagina; (2) increasing pressure on the endoscope; and (3) rotating the scope 180 degrees to alter the field of view (or some combination of all 3). improving external cervical os visualization in author’s practice, a ‘shunt’ (tci shunt system, minitube, verona, wi) with an inflatable cuff and sealing o-ring is utilized for majority of moderate-size dogs. digital pressure on the vulvar lips around endoscope shaft is applied on smaller dogs and those that resist shunt insertion. if shunt is difficult to insert, careful evaluation for a vestibulo-vaginal septal remnant is indicated. occasionally, these septal remnants can be overlooked if the endoscope is small enough to reach the cervix because caudal vagina and vestibule escape from examination during initial insertion. if identifying external os is difficult, stimulating defecation typically improves visualization. defecation can be stimulated using the ‘match in the anus’ technique. whether the phosphorus on the match head makes a difference is debated. most endoscope-assisted tcis are performed without applying pressure to the ventral abdomen, but occasionally, manipulation of the cervix transabdominally is attempted when visual identification of the external cervical os is difficult. majority of endoscope-assisted tcis performed in author’s practice are with the patient standing on an adjustable height table that comfortably accommodates various size dogs. although most endoscope-assisted tci procedures are performed with operator standing, if tci is difficult, performing the procedure sitting on an adjustable stool may facilitate success. apparently, subtle difference in orientation helps in some cases. occasionally, the cranial vagina is filled with estrual fluids, making external os visualization difficult. if the procedure is performed toward end of estrus, sheets of epithelial cells occasionally occlude the camera and even the external cervical os. aspiration of the fluid with the catheter can improve visualization. if the dog is large enough, passing a 16-inch rayon swab (scopettes®, birchwood laboratories, eden prairie, mn) into the cranial vagina to absorb fluids and dislodge shed epithelium can be helpful. swabs can either be purchased individually packaged sterile or can be steam-autoclaved (but the plastic shaft does bow slightly during autoclaving). http://dx.doi.org/10.58292/ct.v15.9320 citation line: clinical theriogenology 2023, 15, 9320, http://dx.doi.org/10.58292/ct.v15.9320 3 catheterizing cervix cervix of the dog typically points in a ventral direction with some variation from straight downward to slightly to either side. extending the catheter a minimum, typically ≤ 1 cm, and gently lifting the external cervical os with the scope and catheter while simultaneously advancing both sometimes allow cervical entry. excessive downward or lateral orientation of cervix can be problematic to inseminate and, in some cases, necessitates putting a slight curve or ‘crook’ in the tip of the catheter with the stylet in place. extent of curve varies among clinicians and even among dogs. if catheter tip can be started into cervix but becomes difficult to advance, withdrawing the stylet 1–2 cm allows it to ‘find its own way’ by becoming more flexible. if the stylet is removed partially and reinserted, care must be taken not to allow stylet tip to exit the ‘eye’ on the side of the catheter. this can be accomplished by visualizing the opening and ensuring that the stylet wire goes beyond the ‘eye’ into the tip either through the scope or after removing the scope or catheter from vagina. seemingly counter intuitive, larger dog cervices appear harder to traverse than smaller dogs. author speculates that this is due to higher rigidity of a larger cervix (makes lifting of the external cervical os to straighten it out more difficult). if a smaller (4 french) catheter becomes difficult, use of a 5  or 6 french catheter might improve success. although 8 french catheters are available, they are used almost exclusively for diagnostic purposes in author’s practice. insemination author typically does not thaw frozen semen until the catheter has successfully been advanced into uterus. this necessitates additional labor but avoids semen remaining in thawed condition for an excessive interval if catheter insertion becomes difficult. chilled semen is kept in the shipper, whereas fresh semen is kept at room temperature away from light until inseminated. if fresh or chilled semen volume is excessive, semen is centrifuged to an amount adequate for dog size. in some instances, it is a ‘judgement call’ with regards to whether centrifugation or retrograde leakage will result in the greatest loss of sperm. insemination proceeds slowly with constant visualization of the external cervical os for retrograde leakage. following insemination, catheter retrieval involves removing the entire scope with catheter still extended rather than retracting the catheter into the scope first. in the author’s practice, one catheter broke at withdrawal to scope before removal, necessitating surgery to retrieve the retained piece from uterus. final thoughts from an animal pain and stress perspective, endoscope-assisted tci is superior to surgical ai when frozen semen used. in parts of europe (norway, sweden, uk), canine surgical ai has been banned because it is considered as an invasive technique interfering with animal welfare.11 international breeding rules of the federation cynologique internationale (fci) states that ‘dogs should be able to reproduce naturally. ai should not be used on animals which have not reproduced naturally before. exceptions can be made by the national canine organizations to improve the health of the breed, for the welfare of the bitch or to preserve or increase the genetic pool of the breed’.12 although most of europe, asia, south america, and oceania are fci members, united kingdom, united states, and canada are not. it will be interesting to monitor if these philosophies become universal in the future. for the ultimate question to our profession regarding endoscope-assisted tci is, just because we can, should we? conflict of interest authors disclose no conflicts of interest. acknowledgment author expresses gratitude to charles estill, eleas wu, vanessa peixoto de souza, and olivia stricklin for cohorts in the adventure of trial and error to find some of the procedures described. references 1. ombelet w, van robays j: artificial insemination history: hurdles and milestones. facts views vis obgyn 2015;7:137–143. 2. bowen jm: artificial insemination in the horse. equine vet j 1969;1:98–110. doi: 10.1111/j.2042-3306.1969.tb03355.x 3. fielden ed: artificial insemination in the dog. n z vet j 1971;19:178–184. doi: 10.1080/00480169.1971.33962 4. given w: the 411 on frozen semen. canine chronicle 2017; 42(8):134–138. 5. leonard ep: dogs. in: perry e, ed. the artificial insemination of farm animals. 4th ed. new brunswick, nj: rutgers university press; 1968, p. 310. 6. silva ld, onclin k, snaps f, et al: laparoscopic intrauterine insemination in the bitch. theriogenology 43;1995:615–623. doi: 10.1016/0093-691x(94)00067-5 7. mason sj: current review of artificial insemination in dogs. vet clin n am small anim pract 2018;48:567–580. doi: 10.1016/j. cvsm.2018.02.005 8. mason sj, rous nr: comparison of endoscopic-assisted transcervical and laparotomy insemination with frozen-thawed dog semen: a retrospective clinical study. theriogenology 2014; 82:844–850. doi: 10.1016/j.theriogenology.2014.06.014 9. shariati e, bakhtiari j, khalaj a, et al: comparison between two portal laparoscopy and open surgery for ovariectomy in dogs. vet res forum 2014;5:219–223. 10. davidson e, moll h, payton m: comparison of laparoscopic ovariectomy and ovariohysterectomy in dogs. vet surg 2004;33:62–69. doi: 10.1111/j.1532-950x.2004.04003.x 11. quartuccio m, biondi v, liotto l, et al: legislative and ethical aspects of canine artificial insemination in the 21st century. italian  j anim sci 2020;19:630–643. doi: 10.1080/1828 051x.2020.1775503 12. international breeding rules of the fci (federation cynologique internationale). [cited 2022 jan 12]. available from: http://www. fci.be/medias/ele-reg-en-10990.pdf http://dx.doi.org/10.58292/ct.v15.9320 http://dx.doi.org/10.1111/j.2042-3306.1969.tb03355.x http://dx.doi.org/10.1080/00480169.1971.33962 http://dx.doi.org/10.1016/0093-691x(94)00067-5 http://dx.doi.org/10.1016/j.cvsm.2018.02.005 http://dx.doi.org/10.1016/j.cvsm.2018.02.005 http://dx.doi.org/10.1016/j.theriogenology.2014.06.014 http://dx.doi.org/10.1111/j.1532-950x.2004.04003.x http://dx.doi.org/10.1080/1828​051x.2020.1775503 http://dx.doi.org/10.1080/1828​051x.2020.1775503 http://www.fci.be/medias/ele-reg-en-10990.pdf http://www.fci.be/medias/ele-reg-en-10990.pdf clinical theriogenology 2022; 14: 378 intromission failure in a stallion dale kelley, justin roscoe, alex wittorff, adam bassett, fabio pinaffi, reed holyoak oklahoma state university college of veterinary medicine, stillwater, ok abstract determining the cause of poor libido in stallions can be challenging. many factors can cause disturbance in libido (e.g., lameness or physical pain, hormonal, age, experience, and handling). identifying the cause of poor libido is important to determine the best treatment approach. a 6-year thoroughbred stallion was presented for failure of intromission when attempting to breed mares. stallion was recently retired from racing. stallion was placed on hormonal therapy that did not change stallion’s aberrant reproductive behavior. on presentation no obvious physical causes were identified and the stallion displayed masturbation behavior. through the review of video recordings of the breeding sessions, modifications were made that eventually resulted in the stallion successfully breeding mares without hormonal intervention. this case illustrated how video recordings of breeding activity can help in determining cause of reproductive dysfunction. furthermore, interpretation of peripheral steroid hormone concentrations needs to be carried out with caution. keywords: stallion, reproductive behavior, hormone therapy, video recording background a common problem for stallions during first introduction to breeding is inadequate sexual interest or response.1,2 they may be slow to achieve and/or maintain erection, act confused, conflicted, and are easily distracted by ordinary environmental sounds, activities or reactions of the mare.3 some stallions may initially appear interested but quickly lose interest whereas others achieve erection but then not proceed normally to mount or may mount and appear confused about how to proceed.1 lack of libido can contribute to these aberrant reproductive behaviors. primary lack of libido, defined as a lack of sexual drive after maturity, is rare in stallions.4 many factors can cause disturbance in libido (e.g., musculoskeletal problems, physical trauma, hormonal causes, age, experience, and handling).4,5 after ruling out physical causes of poor libido, typically hormonal causes are examined via measuring serum testosterone concentrations. in men, low libido was considered to be caused by lower testosterone concentrations;6,7 however, an epidemiological study8 in men concluded that poor libido should not be interpreted as evidence of lower testosterone concentrations. one must interpret hormonal data cautiously as testosterone therapy in normal stallions does not necessarily enhance libido, but does alter sperm production.8 interestingly, stallions with normal libido had testosterone concentrations < 1,000 pg/ml (personal communication, molgorzata pozor, may 6, 2022) and bachelor stallions had lower testosterone concentrations than harem stallions despite having similar masturbation patterns.9 it is important to rule out physical and hormonal causes to determine if there is a behavioral component associated with failed intromission. we determined the cause of failed intromission in a stallion and had the problem corrected. case presentation a 6-year thoroughbred stallion was presented on may 5, 2022, for failure of intromission when attempting to breed mares. stallion was purchased for a stud farm off the racetrack in late january of 2022. stallion had neither bred a mare nor had semen collected before. stallion failed to obtain erection when exposed to tease mares; however, he mounted several mares. in february, stallion’s testosterone concentrations were tested and was given intravenously 80 mg of testosterone every other day. in march 2022, the stallion was sent to a referral veterinary hospital. during this time, he was exposed to ovariectomized tease mares, treated with diazepam (0.1 mg/kg; hospira, lake forest, il) once, 5 to 10 minutes prior to the second breeding session, and gnrh (cystorelin®, merial, duluth, ga) 50 µg intravenously 1 hour prior to and a second dose (50 µg) immediately before fourth breeding session, but failed to mount any mares. testosterone concentrations on days 4, 6, 8, and 9 after admission were 1,080, 780, 240, and 440 pg/ml, respectively. in addition, 80 mg of testosterone cypionate was given intramuscularly thrice (on days 5, 7, and 9) during this period. clinical theriogenology 2022; 14: 379 on presentation in our facility, stallion appeared normal during physical examination. during first 3 days, stallion was taken out of the stall, hand walked, introduced to the breeding shed, and allowed to be penned with tease mares. he displayed little interest in mares and never obtained erection. it was noted on the third day, while being groomed, stallion extended his penis and began masturbating. this behavior was repeated several times while being groomed in subsequent grooming sessions. treatment based on testosterone concentrations (341.6 pg/ml) estimation (sample collected on may 9), stallion was given intravenously 5,000 iu of hcg (choulon, intervet, summit, nj). all sessions in the breeding shed were video recorded and analyzed later. on entering the shed, stallion had erection and was allowed to mount the mare; however, intromission was not achieved. an attempt was made to collect the stallion using an artificial vagina but was unsuccessful. after the sessions the video recordings were analyzed, and 3 features were noted. first, after mounting, stallion failed to remain centered behind the mare, his hips moving laterally to both sides of the mare’s hips. stallion primarily placed his hips to mare’s right side. second, stallion turned his head counterclockwise on the longitudinal plane of his neck and gripped mare’s mane for stability. lastly, mare’s excess movement appeared to disrupt the stallion. on the next day, stallion was brought into the breeding shed and exposed to the same estrous mare from the previous day. based on the video input from the previous session, several adjustments were made. to minimize mare’s movement, mare was hobbled and sedated with 2 mg of intravenous detomidine hydrochloride (dormosedan®, orion pharmaceuticals, finland, distributed by zoetis animal health, kalamazoo, mi). a breeding shroud was placed on mare’s withers with the idea that this would allow him to grip the shroud without turning his head and enable him to stay more centered behind the mare. an artificial vagina was prepared, and stallion was brought into the breeding shed. stallion obtained erection, and an attempt was made to wash the penis with warm water and rolled cotton.10 stallion allowed his penis to be washed and maintained erection. next, stallion was allowed to mount the mare and semen collection was attempted that was successful at second attempt. semen was then analyzed. similar procedure was repeated on may 11, 12, 13, and 15 and each time an ejaculate was obtained to reinforce the behavior prior to transitioning to live covering a mare.11 no drugs or medications were given to the stallion during this time. on may 16, peripheral testosterone concentrations were 988.1 pg/ml. acupuncture examination revealed no abnormalities. later, stallion was brought into the breeding shed and exposed to a mare (sedated, restrained in breeding hobbles with a breeding shroud on and tail wrapped) in estrus. stallion obtained erection, and penis was washed with warm water and rolled cotton. after several unsuccessful attempts to live cover the mare, 10 iu of oxytocin (oxytocin, bimeda, mtc animal health, cambridge, ontario, canada) was given intravenously. intromission was not achieved after 2 more attempts and 5 mg of dinoprost (lutalyse®, zoetis animal health), was given intramuscularly. our group has observed pelvic thrust movements in stallions, after prostaglandin f2α treatment, presumably in response to smooth muscle contractions.12 since the stallion would mount the mare but failed to produce pelvic thrusts, prostaglandin f2α was given to induce these movements in the stallion. after several more attempts the stallion had intromission and ejaculation. a sample was aspirated from the mare’s vagina using a sterile pipette and viewed under a microscope for confirmation of ejaculation. after covering the mare, the stallion was sedated with 3 mg of intravenous detomidine hydrochloride. internal reproductive organs were palpated and examined via transrectal ultrasonography that revealed no abnormalities. testes, epididymides, and spermatic cords were then palpated and examined via ultrasonography. this examination was not performed earlier to minimize stress to stallion that could potentially affect behavior. no testicular, epididymal or spermatic cord abnormalities were detected. on may 17, stallion was brought into the breeding shed and exposed to an estrous mare that was sedated, restrained in breeding hobbles with a breeding shroud and tail wrap. stallion obtained erection, and the penis was washed with warm water and rolled cotton. several mounting attempts were made to allow the stallion to achieve intromission without pharmacological intervention; however, these were unsuccessful. dinoprost (5 mg) was then given intramuscularly and after several more mounting attempts, intromission and ejaculation were achieved. a sample was aspirated from the mare’s vagina using a sterile pipette and viewed under a microscope for confirmation of ejaculation. from may 18 to june 2, the stallion was brought into the breeding shed and exposed to a sedated and restrained estrous mare 5 times. during each exposure, the stallion obtained erection, and penis was washed with warm water and rolled cotton. stallion had intromission and ejaculation between second to fourth jump. each time, a sample was aspirated from the mare’s vagina using a sterile pipette and viewed under a microscope for ejaculation confirmation. it was observed that mares displaying a standing strong estrus were bred much faster (typically 2 3 mounts to achieve intromission and ejaculation) compared to mares that displayed a weak standing estrus (typically 3 5 mounts to achieve intromission and ejaculation). outcome after 9 sessions in the breeding shed, stallion successfully bred mares (erection, intromission, and ejaculation) without pharmacological intervention. during this time, stallion was regularly used to tease mares moved into a large pen, once daily. teasing was performed due to owner’s request to determine if stallion would learn to tease mares. only once he had erection with a mare while teasing mares. interestingly, we used this mare as a mount mare, to collect semen from this stallion. discussion there are many potential causes (e.g., physical, hormonal, and behavioral) for a stallion not able to breed.1,4 physical clinical theriogenology 2022; 14: 380 examination on presentation did not reveal any potential physical causes. it must be noted that physical and lameness examinations can fail to detect physical causes of failure to breed mares and thus observing the mounting behavior can potentially yield more information than traditional physical examination. video review of mounting behavior suggested no physical issues, also an acupuncture scan did not reveal any sensitivities of trigger points indicative of musculoskeletal pain or anxiety. behavior cause was diagnosed, and further diagnostics determined a physical cause was deemed unlikely for stallion’s intromission failure. stallion’s positive clinical response enabled intromission and ejaculation possible and eventually leading to semen collection using a mount mare. prior to presentation to our facility, stallion was receiving testosterone supplementation, perhaps due to low serum testosterone concentrations or to increase libido. testosterone concentrations of 1,000 pg/ml are considered normal (personal communication, sue mcdonnell, may 9, 2022) in stallions.13 this stallion historically had testosterone concentrations higher than these concentrations and failed to mount mares. additionally, since the stallion began to display masturbation behavior low libido seemed unlikely, and a behavioral component appeared most probable.14 libido is defined as sexual arousal and response.15 it has been suggested that the occurrence of masturbation in stallions with poor or absence of libido in the presence of mares is due to psychological causes as opposed to physical causes.14 during first week, 1 dose of hcg was given. this was for 2 reasons. first, the owner failed to bring testosterone medication and it was unavailable at the clinic at the time. second, since stallion had been receiving testosterone prior to arrival, we were concerned that abrupt discontinuation of testosterone treatment could have a negative effect on libido.16 video recordings of stallion behavior allows for repeated views of a situation and has been utilized in research settings16 with tie stalls and 3.05 x 3.05 m pens. a high intraand inter-observer reliability existed between live and recorded behavior.16 thus, video recording in a clinical setting is a valuable source of information to determine the cause of stallions’ reproductive dysfunction. recorded video allows individuals to focus on safety in the breeding shed without missing observing potentially valuable physical or behavioral signs. for example, a mare initially was used with only breeding hobbles (for stallion safety) to try to have as natural circumstance as possible to facilitate stallion interest. on video review, it was noticed how stallion gripped mare’s mane with his mouth contributed to his lateral hip movement and thus a breeding shroud was added in future sessions. this allowed stallion to grip the shroud more ergonomically and improved alignment of the stallion’s hips with the mare’s hips. this example highlights the use of video review to identify the cause of a problem and make effective changes. in our experience the ideal angle of a video would be lateral to the stallion with the entire animal in the video frame. if room allows in the breeding shed, a video from the opposite side of the handler can be advantageous as to avoid the handler obscuring the horse. it must be noted that there are 2 potential disadvantages of video recording from the opposite side of the handler. first, the handler may not see the person recording the video and could result in stallion injuring the person recording. second, the video does not record what may be occurring with the person using the av or manipulating penis into a mare. for these reasons we prefer to record from the same side of stallion the handler is located. lastly, allowing stallion several days to adjust to the facility was important in this case. this not only allowed the stallion time to adjust to the surroundings but also allowed us time to observe his behavior. stallion was shy and cautious. stallion introduction to the breeding shed combined with positive experiences (e.g., grooming) allowed him to become more comfortable with his new surroundings and with personnel working with him. this is most evident by eventual masturbation behavior he displayed when being groomed. with shy stallions, the handling environment can greatly influence the outcome.2 if the environment is not relaxed and positive, stallion may never become comfortable, and progress toward becoming a breeding stallion may not be possible. learning points • video sessions in the breeding shed can be helpful in determining a physical or lameness cause of reproductive dysfunction and provide information to adjust the breeding process thereby increasing the chance of a successful outcome. • determining the cause of poor libido can be challenging. a patient and methodical approach is required to determine the cause and thus the best actions to treat the stallion. • interpretation of peripheral steroid hormone concentrations needs to be carried out with caution as libido and behavior do not necessarily result in cause and effect. conflict of interest none to declare. references 1. mcdonnell sm: advances in diagnostics and therapeutic techniques in breeding behavior disorders in stallions. vet clin n am equine pract 2016;32:513-519. 2. mcdonnell sm: abnormal sexual behavior. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; john wiley & sons: 2011. 3. martin jr bb, mcdonnell sm, love cc: musculoskeletal and neurologic disease as a common factor in sexual behavior dysfunction and subfertility in breeding stallion. compend contin educ pract vet 1998;20:1159-1168. 4. klug e, bartman c, gehlen h: diagnosis and therapy of copulatory disorders of the stallion. pferdeheilkunde 1999;15:494-502. 5. hart bl: gonadal androgen and sociosexual behavior of male mammals: a comparative analysis. psychol bull 1974;81:383-400. 6. morley je: testosterone and behavior. clin geriatr med 2003;19:605-616. 7. matsumoto am: andropause: clinical implications of the decline in serum testosterone levels with aging in men. j gerontol: series a 2002;57:m76-m99. clinical theriogenology 2022; 14: 381 8. travison tg, morley je, araujo ab, et al: the relationship between libido and estosterone levels in aging men. j clin endocrinol metab 2006;91:2509-2513. 9. berndtson we, hoyer jh, squires el, et al: influence of exogenous testosterone on sperm production, seminal quality and libido of stallions. j reprod fertil suppl 1979;19-23. 10. mcdonnell sm, murray sc: bachelor and harem stallion behavior and endocrinology1. biol reprod 2018;52:577-590. 11. mcdonnell sm: starting a novice breeding stallion. clin tech equine pract 2007;6:232-238. 12. mills ds: applying learning theory to the management of the horse: the difference between getting it right and getting it wrong. equine vet j 1998;30:44-48. 13. andersson ke, forman a: effects of prostaglandins on the smooth muscle of the urinary tract. acta pharmacologica et toxicologica 1978;43:90-95. 14. mcdonnell sm: stallion behavior and endocrinology: what do we really know? proc am assoc equine pract 1995;41:18-19. 15. wilcox s, dusza k, houpt k: the relationship between recumbent rest and masturbation in stallions. j equine vet sci 1991;11:23-26. 16. dascanio jj: evaluation of sexual behavior in the stallion. equine reproductive procedures. 2014. p. 354-356. 1 contact lauren newsom lauren.newsom@oregonstate.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9221 research report accuracy of radiographic fetal count in ewes camille ogdon, charles estill, kelly van scoyk and lauren newsom carlson college of veterinary medicine, oregon state university, corvallis, or, usa abstract accurate fetal count is important for farm animal species as the number of fetuses can affect management decisions in both research and production settings. documented methods of pregnancy diagnosis include radiography, progesterone assays, transrectal palpation, and transrectal and transabdominal ultrasonography; however, there is variability in fetal count accuracy with each of these methods. abdominal radiography evaluation in 13 pregnant ewes among observers of various skill levels was compared retrospectively with the known number of fetuses determined using computed tomography. overall accuracy using abdominal radiography across skill levels for determining fetal counts correctly was 79%. accuracy decreased as the number of fetuses increased, with accuracies for singleton, twin, and triplet pregnancies being 92, 72, and 50%, respectively. additionally, observer experience was inversely related to radiographic fetal count accuracy. keywords: sheep, pregnancy, radiograph, ultrasound, abdominal computed tomography, fetal count, litter size, pregnant ewe, fetal skeleton introduction use of radiographs to determine fetal counts is a common practice in veterinary medicine because of their wide availability and affordability, particularly in patients who are small enough for abdominal radiography. many aspects of pregnancy can be evaluated using radiographs, including fetal size, position, number, and fetal viability.1 there are several practical methods of pregnancy diagnosis in ewes, including radiography, hormonal assays, pregnancy-associated protein assays, and ultrasonography.2 although only radiography, progesterone assays, and ultrasonography have the capability to discern between single and multiple fetuses; varying degrees of accuracy for absolute number of fetuses have been reported for these modalities. having an accurate radiographic fetal count can be important to sheep producers with valuable breeding animals, as larger litter sizes may result in increased production, although litters that are too large may put the ewe’s health at risk. at our institution, inaccurate small ruminant radiographic fetal counts are not uncommon regardless of observer experience or specialty training. litter size is important for research purposes where a specific number of fetuses may be desired for subjects to be included or excluded in a study. difficulty performing abdominal radiography in sheep is mostly because of overall patient size necessitating increased radiographic exposure in order to produce radiographs with clearly identifiable fetal structures. superimposition with the abdominal organs, particularly the rumen and colon, creates a challenging situation when attempting to identify and accurately count fetal skeletal structures. radiographs are a practical, affordable, and widely available method to determine pregnancy status and to attempt fetal counts in small ruminants whose average pregnancy length is between 144 and 151 days. mineralization of the fetal skeleton can be detected radiographically as early as 41 days with components of the skull, ribs, and hindlimbs visible.3 radiographic pregnancy diagnosis can be as high as 100%, and radiographs are reported to be 90% accurate in determining fetal counts after day 70–90 of pregnancy.2,4 full fetal skeletons have been radiographically identified as early as day 58 of pregnancy in goats.4 if there is no observable mineral skeletal formation of a fetus by day 75 of pregnancy, the doe is considered not pregnant.4 though fetal count accuracy increases later in pregnancy due to increased fetal size and mineralization, there are occasions when an accurate count may be desired earlier in pregnancy. serum progesterone concentrations were higher in ewes with multiple litter sizes compared to singleton pregnancies. however, there is considerable overlap between serum concentrations for different litter sizes and reliability for this test is low. in addition, there is no evidence that this test can determine an exact count of fetuses beyond the differentiation between none, one, and multiple fetuses.5 two types of transabdominal ultrasonographic imaging are available to assist with pregnancy diagnosis and prediction of fetal number, including b-mode ultrasonography alone or in combination with color doppler. b-mode ultrasonography with mailto:lauren.newsom@oregonstate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9221 2 citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9221 color doppler is the most accurate method to diagnose pregnancy, with up to 100% accuracy when performed >60 days postbreeding.6 accuracy was 100% in diagnosing pregnancy as early as day 39.7 when used by an experienced sonographer, color doppler ultrasonography had 93% accuracy for distinguishing between single or multiple fetuses when performed between day 80 and 95 of pregnancy8; however, overall accuracy decreased as litter size increased. accuracy was 31% for detecting triplet pregnancies and an overall accuracy of 78% for all types of pregnancies.7 real-time b-mode transabdominal ultrasonography can be used to determine fetal counts after day 50 of pregnancy, but with an unknown degree of accuracy.9 transrectal abdominal sonography has also been used successfully to determine pregnancy status, though this method was not reliable in determining an accurate fetal count when performed in the first 25 days of pregnancy and when multiple fetuses were present.2 apparently, computed tomography (ct) use for diagnosis of pregnancy and fetal counts in small ruminants has not been reported. ct is often cost prohibitive and geographically unavailable for most producers; however, this imaging modality can be used to determine fetal counts quickly and accurately. ct also results in increased radiation exposure to the developing fetus and mother. there is evidence that increased radiation exposure in early pregnancy (≤ day 30) can inhibit development of limbs in sheep10; however, there is limited information regarding the safe and acceptable dose of radiation in fetal sheep. the first objective was to determine the accuracy of radiographic fetal counts in pregnant sheep among observers of various skill levels. the second objective was to determine if there is a correlation in accuracy between novice and experienced observers. we hypothesized that increased experience increases radiographic fetal count accuracy and with increased fetal numbers, radiographic fetal count accuracy decreases. materials and methods digital standing lateral abdominal radiographs of 13 healthy, pregnant ewes between day 76 and 90 after breeding were evaluated retrospectively for determination of fetal number. only ewes that had abdominal radiographs in our institutions picture archiving and communication system (pacs) system (novarad, version 8.7.11, novarad corporation, american fork, ut) and a definitively known number of fetuses determined either via ct, cesarean surgery, or necropsy, were considered. ewes were identified by  searching both our pacs and hospital information system (his) systems. radiographs were anonymized before evaluation via our pacs system. participants were blinded to the definite number of fetuses for each ewe and previous imaging reports. radiographs were evaluated by a board-certified radiologist, a 1st-year radiology resident, a 4th-year veterinary student, and a board-certified theriogenologist. interpretation of radiographs relied on existing reviewer experience, with no guidelines in place for how to interpret radiographs. fetal counts were recorded in separate microsoft excel sheets for each observer and compared to known accurate fetal counts for each ewe. percent accuracy was calculated by dividing the number of radiographically observed fetuses by the definite number of fetuses present, multiplied by 100. percent accuracy was calculated for each individual observer and each number of fetuses (singleton, twins, and triplet). overall percent accuracy for all observers and for each number of fetuses was also calculated. results thirteen ewes that had abdominal radiographs and definite fetal number determined via ct were included. radiographic interpretation results of each observer and for each ewe, in addition to the definite fetal count confirmed via ct are provided (table 1). overall accuracy was 79%, ranging between 67 and 92% depending on the observer (table 2). overall accuracies for singleton, twin, and triplet pregnancies were 92, 72, and 50%, respectively (table 2). seven out of 13 ewes had at least 1 observer report an incorrect fetal number, and 6/13 cases reported an inaccurately low fetal count. ewe 13 had 2 confirmed fetuses, although only 50% of the observers accurately reported this number (figure 2). only 1 case (ewe 12) had 3 fetuses that were correctly reported by all reviewers (figure 1). in 1 case (ewe  8), a single observer reported a radiographic fetal count higher than the definite table 1. radiographic interpretation by each observer ewe number of fetuses identified on radiograph confirmed count radiologist radiology resident veterinary student theriogenologist ewe 1 2 1 2 2 2 ewe 2 1 1 1 1 2 ewe 3 2 2 2 1 2 ewe 4 2 2 2 2 2 ewe 5 1 1 1 1 3 ewe 6 2 2 2 2 2 ewe 7 1 2 2 2 2 ewe 8 1 1 1 2 1 ewe 9 1 1 1 1 1 ewe 10 1 1 1 1 1 ewe 11 2 2 2 2 2 ewe 12 3 3 3 3 3 ewe 13 1 2 2 1 2 http://dx.doi.org/10.58292/ct.v15.9221 citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9221 3 number. ewe 5 was reported by all reviewers to only have 1 fetus (figure 3); however, she had a definite fetal count of 3. surprisingly, accuracy decreased with increased observer experience with the 4th-year veterinary student having the highest overall accuracy at 92% and the observer with the most years of clinical experience had the lowest overall accuracy at 67%. discussion based on our findings, radiographic fetal count accuracy in sheep is only 79% across observers regardless of the number of fetuses, although increased litter size was associated with reduced accuracy for all observers. seven out of 13 cases (53.8%) had at least observer report an inaccurate fetal count, most of which (6/7) were inaccurately low. this is likely in part due to summation with intra-abdominal organs and overall increased size of the ewe resulting in reduced radiographic detail. only 2/13 ewes had triplet pregnancies. radiographic fetal count in ewe 12 (with triplets) was correctly evaluated by all observers, regardless of skill level. ewe 5 was reported by all reviewers to only have 1 fetus; however, ct examination identified 3 mineralized fetuses. on reevaluation of the radiographs from this ewe, a single, ill-defined spine was observed to be summating with the colon that was full of formed feces. a third fetus was not radiographically detectable in this ewe. without the knowledge of three fetuses in this case (confirmed later via ct), experienced observers were unconvinced that the mineral summating with the colon was consistent with a fetus. hence our hypothesis that increased fetal number reduces radiographic fetal count accuracy is accepted; however, further evaluation with higher sample size is necessary. sonography and color doppler are commonly used by experienced practitioners to confirm pregnancy in small ruminants, and color doppler ultrasonography is up to 93% accurate table 2. calculated accuracy (%) of interpretations radiologist radiology resident veterinary student theriogenologist overall accuracy accuracy 75 83 92 67 79% singleton accuracy 100 100 100 67 92% twin accuracy 63 75 88 63 72% triplet accuracy 50 50 50 50 50% figure 1. radiograph of a triplet pregnancy of ewe 12 (table 1) with 3 fetuses correctly identified by all reviewers figure 2. radiograph of a twin pregnancy of ewe 13 (table 1) correctly identified by 50% of the reviewers figure 3. radiograph of a confirmed triplet pregnancy of ewe 5 (table 1) with only 1 fetus identified by all reviewers http://dx.doi.org/10.58292/ct.v15.9221 4 citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9221 for  determining whether 1 or multiple fetuses are present.8 however, similar to radiography, sonographic accuracy decreased as litter size increased and sonography was only 31% accurate for detecting triplet pregnancies.7 ultrasonographic evaluation does require skill and experience and as a result is subject to human error. modern radiography is predominantly a digital modality, and images can be shared and evaluated by observers of various skill levels in various geographic locations as needed for consultation. at our institution, radiography is used primarily for research due to concern for unacceptable sonographic count accuracy if ≥ 2 are present, particularly when differentiation between twins and triplet pregnancies is absolutely necessary for inclusion or exclusion in a study. ct findings confirmed that radiography is unreliable to differentiate between twins and triplet pregnancies. interestingly, radiographic fetal count accuracy was inversely related to observer experience and training, with the 4th-year veterinary student having the highest overall accuracy at 92%. therefore, our hypothesis that increased experience leads to improved observer accuracy of radiographic fetal counts is rejected. there are several possible reasons, including the low overall number of ewes, random chance or a fundamental difference in how the observers evaluated the studies. for example, on evaluation of pregnant ewe radiographs, 2 observers may convincingly notice 1 fetus and question a second fetus. one observer may report only 1 fetus as this is the number they are certain of whereas the second observer reports 2 fetuses due to the concern of missing a fetus. the authors believe that this fundamental difference in observer interpretation reflects the realities of clinical practice, clinician preference, and overall confidence in personal skills. in  this study design, an option for documenting questionability of a fetus was not considered and it was required that each observer make a final decision about fetal number. it is possible that creating an additional option during radiographic evaluation for documenting a questionable fetus may improve overall accuracy numbers and agreement among observers. the small number of ewes studied is a limitation; repeating with a larger number of ewes may yield different results. performing repeat radiographs at various stages of pregnancy, including late pregnancy, may help to determine time points at which radiographs are most useful and most accurate. ionizing radiation is a veterinary occupational safety hazard created by radiography and ct, but not with sonography. in all situations, the benefit of obtaining diagnostic quality images should outweigh the risk of ionizing radiation exposure to people and animals involved in the imaging study. increased patient size requires an increased radiation dose to obtain diagnostic quality radiographs. this in turn results in increased patient and human exposure due to scatter radiation. majority of small ruminant radiographs at our institution are performed standing with a sedated patient and people present in the room to hold the patient and the cassette. regular training and rules are in place to reduce radiation exposure to personnel involved in each study. furthermore, personnel wear leaded devices (apron, thyroid shield, gloves, and eyeglasses) to protect from scatter radiation. personnel are also instructed to increase their distance from the primary beam and the patient by holding their arms straight and to avoid having any of their own anatomy within the primary beam. ct imaging of sedated small ruminants at our institution is a  common practice with sick and injured animals. this  modality is priced competitively and provides exponentially more  information compared to radiography. ct is a superior  modality in reducing human exposure to ionizing radiation. in either radiography or ct, there is ionizing radiation exposure to ewe and fetus. according to the national council on radiation protection and measurements and the environmental protection agency, the average radiation exposure from an abdominal radiography study and abdominal ct study in a person is 0.7 and 10 millisieverts, respectively.11 this equates to a 14–15 fold difference in radiation dose to the ewe and fetus when a ct scan is performed rather than radiography. risk to a fetus when exposed to ionizing radiation depends both on the dose of radiation and the fetal age at imaging.12 human fetal radiation dose from an abdominal ct study ranged from 1.3 to 35 milligray.12 in  people, a fetal radiation dose exposure between 10 and 20 milligray could increase the risk of childhood leukemia 1.5–2 fold above the existing rate of 1 in every 3,000  children.12 radiation exposure risk to sheep fetus is currently unknown, but could have similar risks to what are reported for people. conclusion it is important to inform producers or researchers requesting accurate fetal counts that radiographs may yield inaccurately low numbers. if accurate fetal numbers are critical for production or research decisions, other noninvasive imaging techniques (e.g., ct), can be considered to obtain a definite number as long as the benefits outweigh the risks. acknowledgement the authors thank the imaging technicians of oregon state university’s diagnostic imaging department for obtaining diagnostic studies. conflict of interest and funding none to declare. research project did not receive any specific grant from funding agencies (public, commercial, or not-forprofit sectors). references 1. thrall de: the uterus, ovaries, and testes. in: textbook of veterinary diagnostic radiology. 6th ed. st louis, mo: elsevier; 2013, pp. 758–761. 2. karen a, kovács p, beckers jf, et al: pregnancy diagnosis in sheep: review of the most practical methods. acta vet brno 2001;70: 115–126. doi: 10.2754/avb200170020115 3. wenham g: a radiographic study of early skeletal development in  foetal sheep. j agric sci (tor) 1981;96:39–44. doi: 10.1017/ s0021859600031853 4. barker cav, cawley aj: radiographic detection of fetal numbers in goats. can vet j 1967;8:59–61. 5. chauhan fs, waziri ma: evaluation of rectal-abdominal palpation technique and hormonal diagnosis of pregnancy in small ruminants. indian j anim reprod 1991;12:63–67. 6. ishwar ak: pregnancy diagnosis in sheep and goats: a review. small rumin res 1995;17:37–44. doi: 10.1016/0921-4488(95)00644-z. 7. jones ak, gately re, mcfadden kk, et al: transabdominal ultrasound for detection of pregnancy, fetal and placental http://dx.doi.org/10.58292/ct.v15.9221 http://dx.doi.org/10.2754/avb200170020115 http://dx.doi.org/10.1017/s0021859600031853 http://dx.doi.org/10.1017/s0021859600031853 https://doi.org/10.1016/0921-4488(95)00644-z citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9221 5 landmarks, and fetal age before day 45 of gestation in the sheep. theriogenology 2016;85:939–945. doi: 10.1016/j.theriogenology. 2015.11.002 8. fukui y, kobayashi m, tsubaki m, et al: comparison of two ultrasonic methods for multiple pregnancy diagnosis in sheep  and indicators of multiple pregnant ewes in the blood. anim reprod sci 1986;11:25–33. doi: 10.1016/0378-4320(86) 90099-0 9. lone sa, gupta sk, kumar n, et al: recent technologies for pregnancy diagnosis in sheep and goat: an overview. int j sci environ technol 2016;5:1208–1216. 10. rajtova v, horak j: the effect of acute irradiation on the development of limbs in sheep. biologia (bratislava) 1976;31: 171–178. 11. kase kr, strom dj, thomadsen br, et al: ionizing radiation exposure of the population of the united states. report no. 160. ncrp; 2009. [cited 2022 december 1]. available from: www.epa.gov. 12. copel j, el-sayed y, heine rp et al: the american college of obstetricians and gynecologists committee opinion: guidelines for diagnostic imaging during pregnancy and lactation. obstet gynecol 2017;130:e210–e216. doi: 10.1097/aog.000000000 0002355 http://dx.doi.org/10.58292/ct.v15.9221 http://dx.doi.org/10.1016/j.theriogenology.​2015.11.002 http://dx.doi.org/10.1016/j.theriogenology.​2015.11.002 http://dx.doi.org/10.1016/0378-4320(86)​90099-0 http://www.epa.gov http://dx.doi.org/10.1097/aog.0000000000002355 http://dx.doi.org/10.1097/aog.0000000000002355 2019 bartlett address: comparative theriogenology: international perspectives the 2019 bartlett address comparative theriogenology: international perspectives ahmed tibary comparative theriogenology section, department of veterinary clinical sciences college of veterinary medicine, washington state university, pullman, wa introduction imagine my great surprise when, instead of telling me about his latest cases or adventures in reindeer theriogenology, dr. isaac bott unexpectedly informed me that i was selected for the bartlett award. i was speechless. it is beyond my wildest dreams to be selected for such an honor. i am grateful and humbled knowing there are a great number of deserving people within our society and college. as is customary, i am asked to stand before my peers and friends and deliver the bartlett address. i would like to share with you my experiences over the years and the impact this specialty has had on my professional and personal life. beginnings my family has a long agricultural tradition. our farms included not only crops and tomatoes but also a little bit of every traditional production animal species (cattle, sheep, and goat). my main interest was horses. my father was a member of our hometown racing commission (one of only 3 race tracks in the country at that time). therefore, i grew up involved with thoroughbreds and anglo-arab breeding and racing. i spend all my free time sailing, riding and going to the track weekly during the racing season. i have to admit that i was – and still am very fond of cats. i owned a breeding colony of siamese. it was no surprise to anyone that i ended up selecting veterinary studies when i graduated from high school. i obtained my “doctorate en medecine vétérinaire” in 1980, from the only veterinary school in morocco, the “institut agronomique et vétérinaire hassan ii". the 6 year education program paralleled that of european schools and in fact many of my professors were from french, belgian or german veterinary schools. the veterinary curriculum was developed with novel ideas and included amongst other subjects a requirement in rural sociology, economics, and statistics, for which i remain very grateful because of how much this background helped me later. i developed an interest in reproductive medicine in my 4th year of veterinary medicine, guided by my first mentors, german veterinarians dr. gero hanschke and professor dr. peter glatzel with whom i had my first publications. dr. peter glatzel was my main advisor and mentor for my dvm thesis which allowed me to introduce a structured breeding soundness examination for stallions at the national stud farms and particularly for the barb and arab-barb horses for the first time. i also became involved in donkey semen cryopreservation from two endangered species the catalan and poitou which were heavily used for mule production. this experience was the final impetus to become a theriogenologist. thanks to the support of my advisor, i was hired as an instructor at the veterinary college, where i practiced as a clinician in the area of reproduction which included all aspects of reproductive medicine and surgery, neonatology, and bovine mastitis. during these years and under the leadership of professor lahlou-kassi, i had the opportunity to meet many reproductive specialists from around the world who were interested in moroccan prolific sheep and horses. my professional development did not become a reality until i obtained a scholarship to go to the university of minnesota for advanced training through a us-aid program. minnesota experience what a wonderful experience it was! i arrived in minnesota in july 1982, and after a short intensive english program i started my graduate studies in the theriogenology section. there i found the 175 clinical theriogenology • volume 11, number 3 • september 2019 perfect union between a great theriogenology faculty (drs. ray zemjanis, brad seguin, shirley johnston, norm williamson, and mel fahning) and a great reproductive physiology faculty in animal science (drs. ed graham, bo crabo, and al hunter). all these people have greatly and positively impacted my learning, professional and personal growth. i was most fortunate to get involved with faculty from other units notably dr. ashley robinson and dr. don johnson who mentored me in epidemiology and international work, and dr. al weber who introduced me to the fascinating world of cytogenetics. i was surrounded by a wonderful group of residents and graduate students many of whom have become successful theriogenologists and leaders in the field (drs. harry momont, farhat vahdat, hisham fahmi, fran smith, doug freeman, carlos gradil, miguel fortin, rebecca davies, juan samper, peggy root, stefano romagnoli, juan romano, jane barber, gary nie, atushi tajima, and sue swanson). it is within this group that i learned the science of gamete physiology and cryopreservation, fundamentals of clinical theriogenology and was initiated in advanced techniques in theriogenology which i put into practice as soon as i got back to morocco upon finishing my ms degree and completing my prelim to continue my phd research in morocco. back to morocco education that i received in minnesota opened many doors to me and within a few months i was in charge of the cryopreservation lab and equine reproduction at my school. i was very fortunate to have the confidence of several large equine (royal stud farms, national stud farms, equestrian federation) and bovine (santa gertrudis ranches and dairies) operations. i met other mentors who helped me along the way, in particular dr. abdelhafid yacoubi who included me in the reproductive program of a large herd of santa gertrudis (ranch adarouch). from 1985 1989, i participated in training of veterinarians and providing clinical services to large and small animal clients. however, i believe that my major contribution was to serve as a mentor for 2 women; drs. samira manar and nadia lotfi who wanted to work with large animals and who are now leaders in their respective fields (small ruminant reproduction and bovine reproduction). i was fortunate that the field was wide open to engage in many activities in research and development. i got involved in dairy cattle management, helping to introduce dairychamp® (which i translated to french), extension activities (ram breeding soundness examination) and even served as the director of the first veterinary program in peace corps. i was fortunate to be requested to help with reproductive issues on wildlife at the national zoological park working on large cats (particularly the barbary lions), gazelles, buffaloes, and other creatures, thanks to my friend and colleague dr. brahim haddane. i left morocco in the winter of 1989, with the promise to then general director of the iavhassan ii, dr. m’hamed sedrati who was a great mentor and supporter, to continue serving the institution whenever needed. did i say that in addition to wanting to discover new horizons in my discipline, i have also left something very dear in minnesota? my soon to be wife brigitte. back to minnesota i was welcomed again into the theriogenology team at minnesota where i worked as a clinical assistant professor and helped with dr. graham’s research and teaching as he was in fading health. the support i received in those uncertain years will remain forever engraved in my memory. it is during this second “tour” that i had the great chance to meet and learn from dr. patty olson and dr. jerry olson who mentored me along with all the other faculty for the theriogenology boards. i kept my contacts with my alma mater and continued to provide lectures and even initiated a series of books on equine reproduction in french. 176clinical theriogenology • volume 11, number 3 • september 2019 abu dhabi years in the fall of 1991, i was asked to travel to abu dhabi for a consultation on a project on camel embryo transfer that my colleague, dr. abdelhaq anouassi, had initiated with the veterinary technician i trained (mr. mustapha adnani). i had never worked on camels before, except to help colleagues with ultrasonography for their research. dr. anouassi had done tremendous work at inra-france on camel reproductive endocrinology and i was bringing a clinical approach to this lab. together, we spent long days working on development of reproductive evaluation techniques in camels which allowed us to publish a book on camelid theriogenology after 7 years. we expanded the labs to include equine with the collaboration of friends (drs. ahmed abdelhamid, kamal pasha, andrew daglish, and peter davis). my only regret during those years is not to have published more on our clinical observations on camels and equine, notably outbreaks of habronemiasis in stallions and the effect of high heat and humidity on reproductive function in horses. i was lucky that my contract was only for the breeding season which left me 4 months to travel back to morocco and the usa to work on my publications and to teach. throughout that time, i wanted to return to my passion: veterinary education and research, and was looking for a position in theriogenology. a break came finally in 1998 where i interviewed both at the university of massachusetts equine reproduction (dr. doug freeman, chair) and at wsu for a dairy cattle position (dr. shirley johnston, chair). i was offered both but opted to go to wsu because it is was tenure-track and was a veterinary program which would allow me to continue my involvement with clinical training and research. wsu: a new beginning i arrived pullman in the summer of 1998, already 20 years ago! dr. mushtaq memon, was mainly doing small animal theriogenology and dr. duane michelson was in charge of bovine reproduction. i thank them both for their encouragement and for introducing me to their clients. my position was in field disease investigation unit (fdiu) as a reproduction specialist. having had most of my work experience outside of the usa university system, i accepted (not without a challenge to my pride, i have to admit) to start from the beginning and go through all the painful processes of pretenure, tenure, promotion etc. i was very lucky to have started with fdiu under the leadership of dr. clive gay. it was a wonderful opportunity for professional growth and i am very grateful for his guidance as well as that of all the members of the team (drs. larry fox, john gay, and dale hancock). i was able to participate in his research on lupine toxicosis. the work in the fdiu was exciting, but my heart was in developing a clinical theriogenology program. i was allowed under the leadership of dr. warwick bayly to move into the clinic and develop the comparative theriogenology service. obviously, no service can be developed without the support of other critical clinical specialties and i am indebted to my colleagues in equine and agricultural animal medicine and surgery for their support. in particular, dr. claude ragle was very supportive and became a family friend, kidnaping me once to go on one of his mule rides in the wilderness. during my tenure at wsu, i was able to make progress toward tenure due to the support from colleagues from the college and from animal science, in particular drs. jerry reeves and phil senger. it is the support of these fine colleagues and that of the students who honored me with so many teaching awards including the norden distinguished teacher awards in 2001 and 2005 that allowed me to “survive’ the pretenure part of my career. the comparative theriogenology program began to strengthen in 2007 when ms. shirley sandoval joined me as the lead veterinary instruction technologist. her practical experience and her passion for her work allowed us to provide several services. we initially started capitalizing on the lack of services for camelids. i had never touched an alpaca prior to my arrival to wsu and soon we were receiving referrals from several states. from there we went on to develop an equine theriogenology program, then a small ruminants service. the program continued to strengthen thanks to wonderful 177 clinical theriogenology • volume 11, number 3 • september 2019 residents and graduate students (drs. cheryl fite, jacobo rodriguez, lisa pearson, yessinia picha, aymen elzawam, hamid alkar, alexis campbell, salman waqas, michella ciccarelli, agustin ruiz, and cristian patino). all had an impact on the program that went beyond my expectation. i pause here to thank dr. harmon rogers who involved them all in small animal theriogenology. in research, i continued working with my colleagues in abu dhabi on camel projects, but on campus i was fortunate to be included in research programs of dr. subramaniam srikumaran on big horn sheep and dr. jon oatley on spermatogonia stem cells transfer. my return to the us allowed me to finally be able to actively participate in the society for theriogenology and the american college of theriogenologists, and later the theriogenology foundation. it was a great honor for me to serve in the leadership of these organizations. this allowed me to get to know other clinicians and researchers from across north america and beyond, with similar interest. i felt and still feel welcomed by all. on international work being able to participate in research and continuing education abroad is certainly one of the highlights of my life and professional career. as a young veterinarian in morocco, i was on the receiving end of teaching from visiting professors and scientists. it is only natural that i would want to return the favor. i was given this opportunity by several people as i continued to learn. first and foremost, i never lost contact with my alma mater which continues to nourish my professional and personal life and where i continue to collaborate in teaching, research and continuing education with colleagues and former students. it is in the spirit of international work and thanks to my continuous involvement with veterinary education in north africa that i was able to associate some colleagues from wsu and other theriogenologists such as dr. carla carleton to my work there. one of the activities i am most proud of is that i was able to use my experience and all i have learned on camels in the middle east to train wonderful veterinarians and technicians from the 91st and 96th us civil affair battalions, who in turn could help many people who depend on these animals for their livelihood. i thank professors jorg and christina aurich for involving me in their equine course and resident seminars in vienna. professors luis losino and marcelo miragaya have been wonderful friends and made me discover many aspects of equine and camelid science in argentina and chile. professors julio sumar and edwin mellisho provided opportunities for involvement in peru. dr. marco alvarenga gave me the opportunity to discover brazilian equine industry. these international events allowed me to share experiences with several great theriogenologists. i have often wondered why i got all these invitations. i always came to the same conclusion, there are aspects of these international interactions that go beyond technical or scientific expertise. people like to share their experiences in a respectful and fun atmosphere. this is why i always try to involve my previous residents in international work. i am very grateful to dr. jacobo rodriguez and dr. alexis campbell who help me with camel projects in qatar. international involvement includes also the ability to receive overseas students. unfortunately, such experiences are slowly disappearing particularly for students from developing countries. at wsu, and under the leadership of dr. erik stauber, i was fortunate to work with international veterinary students who later become theriogenologists, drs. natali krekler, maria ferrer, gislaine dujovne. i know that this an effort of the theriogenology foundation through the zemajin funds which has benefited one of my graduate students (dr. y. picha). concluding remarks and pet peeves i cannot end without sharing some pet-peeves that i have developed over the years. the list is long but i limited to top 4. 1) do not use the verb “utilize” when “use” is sufficient, 2) we do not do 178clinical theriogenology • volume 11, number 3 • september 2019 “preg check” nor do we “sleeve’ or “arm”, we do pregnancy diagnosis and reproductive evaluations, 3) we do not do rectal palpation but “transrectal palpation”, 4) last, it may not be true for all but when you are so invested in your specialty, everything that touches it is personal. i have to conclude with some sort of words of wisdom. for me it is simple: what goes around comes around, so i tried to offer young aspiring theriogenologists what others had offered to me throughout my career. i have been asked frequently in recent months about when i will be retiring. my answer is, why retire from such an exciting profession and specialty? many of the concepts of what theriogenology is, laid out by my predecessors, still apply. i urge all theriogenologists to think big, think international and may you all continue to enjoy our specialty in health and happiness! 179 clinical theriogenology • volume 11, number 3 • september 2019 180clinical theriogenology • 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/marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice   luteinizing hormone receptor expression in lymphoma is not affected by body weight, sex, immunophenotype, or tumor stage in gonadectomized dogs khawla zwida, alyssa vedus, michelle kutzler department of animal and rangeland sciences, oregon state university, corvallis, or abstract purpose of this investigation was to determine if luteinizing hormone receptor (lhr) expression in canine lymphoma was affected by body weight, sex, immunophenotype (b cell, t cell) or tumor stage. formalin-fixed, paraffin-embedded lymphoma tissue samples from spayed and neutered dogs (n = 40), representing multiple breeds and mixed breeds, were subjected to routine immunohistochemical techniques using a polyclonal lhr antibody. percentage of cells positive for lhr and the staining intensity (scored 0 3) were determined at 400 x magnification. data were expressed as mean ± standard deviation and significance was defined as p < 0.05. differences between sex and tumor phenotype, body weight and tumor stage were compared. all tumor samples had cells positive for lhr. however, percentage of cells expressing lhr and its immunostaining intensity varied among individuals. there were no significant differences in percentage of lhr positive cells or staining intensity within sex or immunophenotype. additionally, there was no significant association between percentage of lhr positive cells or staining intensity within body weight or tumor stage. we concluded that increased risk of lymphoma in spayed and neutered dogs was not related to body weight, sex, immunophenotype or tumor stage. however, it is possible that risk of lymphoma may be related to increased lhr activation following gonadectomy. keywords: b cell, t cell, gonadectomized dogs, lymphoma, immunohistochemistry introduction canine lymphoma is the most common hematopoietic neoplasm in the dog, accounting for up to 24% of all canine cancers.1,2 lymphoma can be derived from either b cell or t cell lymphocytes. immunophenotyping is possible because b cell lymphomas typically express cell surface proteins cd79a, cd20, cd21, and pax5, whereas t cell lymphomas typically express the unique cell surface protein cd3.3 symptoms and treatment options vary depending on the lymphoma’s predominant cell type (i.e. b or t cells).4 although canine lymphoma is typically treated by chemotherapy, b cell lymphomas become resistant to chemotherapy after subsequent treatments and t cell lymphomas often do not respond well to chemotherapy from the onset of treatment.5 prognosis following diagnosis with canine lymphoma is also dependent on the immunophenotype of lymphoma, with average survival times shorter for t cell than b cell lymphomas (183 versus 365 days).6 in general, t cell lymphomas also have shorter remissions than b cell lymphomas.7-9 basset hound, beagle, bernese mountain dog, boxer, bulldog, bull mastiff, cocker spaniel, doberman, german shepherd, golden retriever, labrador retriever, rottweiler, shih tzu, st. bernard, and any terrier breed are at higher risk for developing lymphoma, whereas dachshunds and pomeranians are relatively lower risk breeds.2,8,10,11 additionally, breed appears to influence the lymphoma immunophenotype. boxers are more likely to develop t cell lymphomas, whereas basset hounds and cocker spaniels are more likely to develop b cell lymphomas.12 body weight may or may not be a risk factor for lymphoma. although body weight in mixed breed dogs had no influence in lymphoma occurrence,13 it had an influence in purebred dogs; 9 lymphomas were more common in medium and large breed dogs compared to toy and small breed dogs.9 additionally, smaller body weight dogs (< 17 kg) were reported to have longer median survival times14,15 or no difference in median survival times1,16 when compared to dogs with greater body weight. however, body condition was not reported in these studies. it is noteworthy that dogs with an underweight body condition had significantly shorter survival times than dogs in ideal or overweight body condition.17 survival from lymphoma may be influenced by sex. in humans, men generally had shorter survival times than women for most cancers, including lymphoma.18-20 this is similar to what was clinical theriogenology • volume 13 number 1 • march 202137   observed in dogs with advanced multicentric lymphoma; intact male dogs had significantly shorter remission and survival times than intact female dogs.10 luteinizing hormone receptors (lhr) are expressed in canine lymphoma tissue and isolated canine lymphoma cells.21 however, the influence of body weight or sex on lhr expression was not evaluated. additionally, the influence of immunophenotype or tumor stage on lhr expression was also not evaluated. based on canine lymphoma findings,6-10,14,15 it was hypothesized that greater body weight male dogs in advanced tumor stages with t cell lymphomas have higher lhr expression than lesser body weight female dogs in earlier tumor stages with b cell lymphomas. materials and methods archived tissue samples (canine comparative oncology and genomics consortium [bethesda, md]) from 40 purebred and mixed breed dogs (spayed females n = 24; neutered males n = 16) weighing 7 56 kg were used. weight categories were similar to an earlier study;13 < 10 kg (n = 4), 10 19 kg (n = 8), 20 29 kg (n =14), 30 39 kg (n = 10), and > 40 kg (n = 4). immunophenotype was available for 26 of 40 tumors (table). tumors were staged using the world health organization system: stage i, single node or lymphoid tissue in single organ; stage ii, regional multiple lymph nodes involvement; stage iii, generalized lymph node involvement; stage iv, stages i iii with liver and/or spleen involvement; and stage v, stages i iv with blood or bone marrow involvement.21 additionally, samples from each primary tumor were formalin-fixed, paraffin-embedded, and sectioned onto charged slides for lhr immunohistochemistry. all slides were deparaffinized, rehydrated, subjected to heat-induced epitope retrieval (#s1700, dako, carpinteria, ca). endogenous peroxidase activity was inactivated with 3% hydrogen peroxide and nonspecific binding was blocked (protein block serum-free [#x0909, dako]). either rabbit polyclonal antihuman lhr antibody (#nls1436, novus biologics, centennial, co) was applied at 1:100 dilution or rabbit negative control (#nc495h, biocare medical, pacheco, ca) was applied to slides. this antigen retrieval method and antibody concentration were optimal for normal and neoplastic canine lymphatic tissue.21 slides were treated with one step horse radish peroxidaseconjugated polymer antirabbit igg (#ih-8064-custom-orsu, immunobioscience, mukilteo, wa), followed by nova red peroxidase substrate (#sk4800, vector laboratories, burlingame, ca). slides were counter-stained with hematoxylin, dehydrated, and mounted. percentage of cells positive for lhr was recorded and staining intensity (scored 0 3; figure 1) was determined at 400 x magnification from 5 randomly selected fields by 1 person (av) blinded to the identity of the samples. additionally, a histology score (h-score) was assigned to each tumor sample.23,24 staining intensity (0 3) for a fixed field was multiplied by the percentage of cell positive for lhr. differences between sex and tumor phenotype were compared (welch two sample t-test in the free statistical package r [version 1.2.1355, boston, ma]). differences in bodyweight and tumor stage were compared (simple linear regression [microsoft excel, version 14.5.2, redmond, wa]). additionally, differences in body weight group were compared (one-way anova). data were expressed as mean ± standard deviation and significance was defined as p < 0.05. results although there were many purebred dogs included in the data set, mixed breed dogs were overrepresented (table). most breeds were only represented by 1 individual, except labrador retrievers (n = 3), german shorthaired pointers (n = 2), poodles (n = 2), miniature schnauzers (n = 2), and boston terriers (n = 2). irrespective of breed, all lymphoma tissue samples contained cells positive for lhr. however, percentage of cellular expression and staining intensity varied among individuals (figure 1). body weight had no significant influence and there was no significant association between the percentage of lhr positive cells (r2 = 0.021) or staining intensity (r2 = 0.077). there were also no significant differences in the percentage of lhr positive cells, staining intensity or h-score when compared by sex (figure 2) or lymphoma phenotype (figure 3). there was no significant association between the percentage of lhr positive cells (r2 = 0.0017) or staining intensity (r2 = 0.063) with regard to tumor stage. clinical theriogenology • volume 13 number 1 • march 2021 38   table. gonadectomized dogs with lymphoma studied: body weight, sex, tumor phenotype, tumor stage on the percentage of lhr positive cells, staining intensity, and histology score (h-score) female (f); male (m); not available (na) id breed sex weight (kg) tumor phenotype tumor stage percent positive staining intensity h-score 5678 miniature schnauzer f 7 na 3 10 2.5 25 5682 shih tzu f 7.2 na 5 10.5 1 10.5 5656 boston terrier f 8 na 2 10.5 3 31.5 5664 pembroke welsh corgi f 9.5 na 3 6.8 3 20.4 5676 miniature schnauzer f 11.4 na 3 3 2.5 7.5 5698 shetland sheepdog f 12.6 b cell 3 23.5 3 70.5 5707 cocker spaniel f 14 t cell 3 10.3 3 30.7 5657 poodle m 15.2 b cell 5 5.75 2.5 14.4 5702 boston terrier m 16 b cell 3 20 2 40 5675 mixed breed f 17.3 na 5 16.6 2 33.2 5666 vizsla f 19 b cell 3 20.8 1.5 31.2 5713 mixed breed f 19.7 na 3 7.5 3 22.5 5689 labrador retriever f 21 t cell 5 7.5 2 15 5715 mixed breed m 21.4 b cell 3 3 2 6 5709 poodle f 22 b cell 4 3.6 2 7.2 5708 mixed breed f 22.6 b cell 3 9 2.5 22.5 5662 german shorthair pointer f 24.5 na 4 10.8 3 32.4 5672 german shorthair pointer f 26 na 4 9.5 2 19 5681 mixed breed m 26 b cell 4 21.8 2 43.6 5655 samoyed f 26.7 na 2 17.65 3 52.9 5691 boxer m 28.6 t cell 3 15 2 30 5659 labrador retriever f 28.6 na 2 19 2 38 5710 mixed breed f 28.8 b cell 3 6.5 2 13 5684 dalmatian m 29 b cell 3 9 2 18 5714 mixed breed m 29.3 b cell 4 2 1.5 3 5720 mixed breed m 29.5 b cell 4 10 1.5 15 5694 bernese mountain dog m 30 t cell 3 4.3 1.5 6.4 5660 labrador retriever f 30 t cell 5 5.9 2 11.8 5668 mixed breed m 31.6 b cell 4 13.4 2 26.8 5690 mixed breed m 32 b cell 3 7.8 2 15.6 5667 basset hound f 34.6 na 3 8.2 3 24.6 5658 australian shepherd m 35.2 t cell 5 10.3 2 20.6 5688 mixed breed m 35.2 b cell 3 10 1 10 5671 mixed breed f 37.2 b cell 4 5 3 15 5706 black/tan coonhound f 37.5 na 3 3.6 2 7.2 5704 mixed breed f 38 b cell 4 26.5 0.5 13.2 5685 irish setter m 46.5 b cell 4 7 3 21 5703 mixed breed m 50 b cell 3 8 2 16 5680 bullmastiff f 52 b cell 5 7.3 0.5 3.6 5705 saint bernard m 56 na 5 6.3 2.5 15.6 clinical theriogenology • volume 13 number 1 • march 202139 figure 1. representative images for luteinizing hormone receptors staining intensity in canine lymphoma (a: staining intensity = 1; b: staining intensity = 2; c: staining intensity = 3). nova red; bar = 10 μm. negative control in upper right inset. arrows illustrate examples of cells stained positive. 40 clinical theriogenology • volume 13 number 1 • march 2021   figure 2. effect of sex (neutered male versus spayed female) on luteinizing hormone receptor (lhr) staining intensity (a), percentage of positive cells (b), and h-score (c). there was no significant effect of sex on lhr expression (p > 0.05). figure 3. effect of lymphoma phenotype (b cell versus t cell) on luteinizing hormone receptor (lhr) staining intensity (a) and percentage of positive cells (b), and h-score (c). there was no significant effect of phenotype on lhr expression (p > 0.05). clinical theriogenology • volume 13 number 1 • march 202141   discussion certain breeds are predisposed to developing lymphoma.2,8-12,25 since there were not enough dogs represented by any single breed in the current study to compare the expression of lhr, the effect of body weight was examined. in agreement with previous research on lhr expression in the bladder,26 body weight did not influence lhr expression in canine lymphoma. it is important to note that body condition score was not available in the current study, which could have been a confounding factor, since underweight dogs with lymphoma were reported to have significantly shorter survival times.17 sex had a role in remission and survival times following lymphoma diagnosis.1,9,16,27 therefore, we sought to determine if lhr expression in canine lymphoma differed by sex. although there was no significant influence of sex on lhr expression, it is noteworthy that the case material available for this study was unfortunately limited to only spayed and neutered dogs; these dogs may have an increased risk of developing lymphoma, depending on their breed.25 compared to intact females and males, lymphoma is 4 times more common in spayed and neutered viszlas.28 occurrence of lymphoma was higher in spayed than intact females. 10 absence of gonadal hormones in the dogs sampled for the current study may have masked effects of sex on lhr expression. further research is needed on susceptible breeds to determine if lhr expression within lymphoma tissue is increased in gonadectomized compared to intact dogs. lymphoma phenotype often influences receptor expression. for example, most human b cell lymphomas depended on the expression of a b cell receptor for continued growth.29 in dogs, the retinoid receptor is expressed higher in t cell lymphomas compared to b cell lymphomas.30 therefore, 1 objective of the current study was to determine if lhr expression changed with lymphoma phenotype. although more b cell lymphomas were present in the data set (n = 20) than t cell lymphomas (n = 6), there was no significant difference between phenotypes in the relative percentage of cells expressing lhr, nor in the relative intensity of lhr staining. receptor expression also can vary with advancing tumor stage. for example, estrogen receptor beta was expressed higher in human b cell lymphoma with tumor stage of iii or iv.31 alternatively, lhr expression in human ovarian cancer was higher in stages i and ii compared to stages iii and iv.32 in the current study, lhr expression varied widely within tumor stage, similar to reported expression of steroid hormone receptors in canine and human mammary tumors.33-35 lack of association between receptor expression and tumor stage could result from the nonstatic expression of lhr in tumors, as reported with lipoprotein receptors in canine lymphoma.36 activation of lhr in luteal,37,38 trophoblast,39 or canine t cell lymphoma cells40 resulted in dosedependent cell proliferation. because circulating lh concentrations were significantly higher in spayed and neutered dogs,41 it is possible that lhr activation has a role in the etiopathogenesis of canine lymphoma. additional research is needed to determine if reducing lh concentrations in spayed and neutered dogs with a gonadotropin-releasing hormone agonist prolongs survival time in dogs with canine lymphoma. acknowledgement pfizer canine comparative oncology and genomics consortium biospecimen repository for access to study specimens and christina mazcko for assistance with patient data. conflict of interest authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding oregon state university small grants incentive program and undergraduate research scholarship and arts program. clinical theriogenology • volume 13 number 1 • march 2021 42   references 1. macewen eg, hayes aa, matus re, et al: evaluation of some prognostic factors for advanced multicentric lymphosarcoma in the dog: 147 cases (1978-1981). j am vet med assoc 1987;190:564-568. 2. vail dm, macewen, eg, young km: canine lymphoma and lymphoid leukemia. in: withrow sj, macewen eg: editors. small animal clinical oncology, 3nd edition, philadelphia; wb saunders: 2001. p. 558-590. 3. caniatti m, roccabianca p, scanziani e, et al: canine lymphoma: immunocytochemical analysis of fine-needle aspiration biopsy. vet pathol 1996;33:204-212. 4. burke, a: lymphoma in dogs: symptoms, diagnosis, and treatment. american kennel club. november 3, 2016. accessed april 22, 2018. available from: https://www.akc.org/expert-advice/health/common-conditions/lymphoma-indogs-symptoms-diagnosis-and-treatment/. 5. marconato l, gelain me, comazzi s: the dog as a possible animal model for human non-hodgkin lymphoma: a review. hematol oncol 2013;31:1-9. 6. fan tm: overview of canine lymphoma circulatory system. mvm. 2018. accessed april 21, 2018. available from: https://www.merckvetmanual.com/circulatory-system/canine-lymphoma/overview-of-canine-lymphoma. 7. greenlee pg, filippa da, quimby fw, et al: lymphomas in dogs. a morphologic, immunologic, and clinical study. cancer 1990;66:480-490. 8. teske e. canine malignant lymphoma: a review and comparison with human non‐hodgkins lymphoma. vet q 1994;16:209-219. 9. valli ve, kass ph, san myint m, et al: canine lymphomas. vet path 2013; 50;738-748. 10. villamil ja, henry cj, hahn aw, et al: hormonal and sex impact on the epidemiology of canine lymphoma. j cancer epidemiol 2009;2009:591753. 11. comazzi s, marelli s, cozzi m, et al: breed-associated risks for developing canine lymphoma differ among countries: an european canine lymphoma network study. bmc vet res 2018;14:232. 12. dobson, jm: breed-predispositions to cancer in pedigree dogs. isrn vet sci 2013;2013:941275. 13. hart bl, hart la, thigpen ap, et al: assisting decision-making on age of neutering for mixed breed dogs of five weight categories: associated joint disorders and cancers. front vet sci 2020;7:472. 14. garrett ld, thamm dh, chun r, et al: evaluation of a 6-month chemotherapy protocol with no maintenance therapy for dogs with lymphoma. j vet intern med 2002;16:704-709. 15. valerius kd, ogilvie gk, mallinckrodt ch, et al: doxorubicin alone or in combination with asparaginase, followed by cyclophosphamide, vincristine, and prednisone for treatment of multicentric lymphoma in dogs: 121 cases (1987-1995). j am vet med assoc 1997;210:512-516. 16. deravi n, berke o, woods jp, et al: specific immunotypes of canine t cell lymphoma are associated with different outcomes. vet immunol immunopathol 2017;191:5-13. 17. romano fr, heinze cr, barber lg, et al: association between body condition score and cancer prognosis in dogs with lymphoma and osteosarcoma. j vet intern med 2016;30:1179-1186. 18. afshar n, english dr, thursfield v, et al: differences in cancer survival by sex: a population-based study using cancer registry data. cancer causes control 2018;29:1059-1069. 19. radkiewicz c, johansson alv, dickman pw, et al: sex differences in cancer risk and survival: a swedish cohort study. eur j cancer 2017;84:130-140. 20. cook mb, katherine am, susan sd, et al: "sex disparities in cancer mortality and survival." cancer epidemiol biomarkers prev 2011;20:1629-1637. 21. ettinger am, gust sk, kutzler ma: luteinizing hormone receptor expression by nonneoplastic and neoplastic canine lymphocytes. am j vet res 2019;80:572-577. 22. owen ln: tnm classification of tumours in domestic animals. 1st edition, geneva; world health organization: 1980. 23. hirsch fr, varella-garcia m, bunn pa jr, et al: epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. j clin oncol 2003;21: 3798-3807. 24. john t, liu g, tsao ms: overview of molecular testing in non-small-cell lung cancer: mutational analysis, gene copy number, protein expression and other biomarkers of egfr for the prediction of response to tyrosine kinase inhibitors. oncogene 2009;28:s14-s23. 25. hart bl, hart la, thigpen ap, et al: assisting decision-making on age of neutering for 35 breeds of dogs: associated joint disorders, cancers, and urinary incontinence. front vet sci 2020;7:388. 26. reichler im, welle m, sattler u, et al: comparative quantitative assessment of gnrhand lh-receptor mrna expression in the urinary tract of sexually intact and spayed female dogs. theriogenology 2007;67:1134-1142. 27. keller et, macewen eg, rosenthal rc, et al: evaluation of prognostic factors and sequential combination chemotherapy with doxorubicin for canine lymphoma. j vet intern med 1993;7:289-295. 28. zink mc, farhoody p, elser se, et al: evaluation of the risk and age of onset of cancer and behavioral disorders in gonadectomized vizslas. j am vet med assoc 2014;244:309-319. 29. küppers r: mechanisms of b-cell lymphoma pathogenesis. nat rev cancer 2005;5:251-262. 30. de mello sch, valli ve, kitchell be: detection of retinoid receptors in non-neoplastic canine lymph nodes and in lymphoma. can vet j 2014;55:1219-24. erratum in: can vet j 2014;55:106. clinical theriogenology • volume 13 number 1 • march 202143   31. faknuam s, assanasen t, ruangvejvorachai p, et al: estrogen receptor beta expression and prognosis of diffuse large b cell lymphoma. hematology 2018;23:235-241. 32. cui j, guo y, you z: [lh-cg receptor protein expression in epithelial ovarian cancer]. zhonghua fu chan ke za zhi 1997;32:742-745. 33. millanta f, calandrella m, bari g, et al: comparison of steroid receptor expression in normal, dysplastic, and neoplastic canine and feline mammary tissues. res vet sci 2005;79:225-232. 34. bardou vj, arpino g, elledge rm, et al: progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases. j clin oncol 2003;21:1973-1979. 35. pichon mf, broet p, magdelenat h, et al: prognostic value of steroid receptors after long-term follow-up of 2257 operable breast cancers. br j cancer 1996;73:1545-1551. 36. ceres k, fitzgerald h, quiznon ks, et al: immunohistochemical labeling of low-density lipoprotein receptor and scavenger receptor class b type 1 are increased in canine lymphoma. front vet sci 2019;11:340. 37. menon k: regulation of luteinizing hormone receptor expression by an rna binding protein: role of erk signaling. indian j med res 2014;140, suppl s1:112-119. 38. davis js, may jv, keel ba: mechanisms of hormone and growth factor action in the bovine corpus luteum. theriogenology 1996;45:1351-1380. 39. reshef e, lei zm, rao cv, et al: the presence of gonadotropin receptors in nonpregnant human uterus, human placenta, fetal membranes, and decidua. j clin endocrinol metab 1990;70:421-430. 40. flint c, gust s, kutzler ma: luteinizing hormone receptor mediated proliferation of isolated canine t lymphoma cells. clinical theriogenology 2019;11:477. 41. beijerink nj, buijtels jj, okkens ac, et al: basal and gnrh-induced secretion of fsh and lh in anestrous versus ovariectomized bitches. theriogenology 2007;67:1039-1045. clinical theriogenology • volume 13 number 1 • march 2021 44 introduction trichomonads were first reported as a cause of bovine infertility in france in 1888.1 in the us, it was first noted in 1932 in pennsylvania dairy cows2 and in 1958 in western us beef herds.3 currently, t. foetus has been practically eliminated from intensively managed cattle populations around the world where the management includes limited commingling of cattle and artificial insemination is commonly practiced. however, it remains endemic in some herds in the western us managed under range conditions with natural service.1 trichomoniasis exerts its most prominent impacts in a cattle operation through its negative effects on herd reproductive performance, resulting in fewer pregnant cows and consequently fewer calves for sale. additional factors related to trichomoniasis that exert a negative influence on the profitability of a beef cattle operation include feed and other maintenance costs for nonproductive cows, replacement costs of t. foetus-infected bulls and nonproductive females, testing costs to control t. foetus, and reduced weaning weights due to late-born calves.4,5 it is difficult to estimate the prevalence of bovine trichomoniasis with any degree of confidence. estimates in california6 and florida7 had a herd-level prevalence of 15.8 and 28.8%, respectively, whereas bull-level prevalence was 4.1 and 6.0%, respectively. in colorado and nebraska abattoirs, only 0.172% of bulls’ samples were culture positive for t. foetus.8 recent prevalence estimates of infected bulls in alabama9 and tennessee10 were 0.27 and 0.18%, respectively, with a prevalence estimate of 2.17% for wyoming herds.11 although the prevalence of bovine trichomoniasis is typically review of bovine trichomoniasis jeff ondrak 6u ranch, steele city, ne abstract bovine trichomoniasis, caused by the protozoa tritrichomonas foetus, results in reproductive failure and substantial financial losses. it was first reported in the us in 1932 and in the western beef herds in the us in 1958. western herds, along with herds in certain areas of florida, are considered the traditional range for this disease in the us. since characteristics of the causative organism are well known, its control and even eradication are possible. background information of t. foetus as it relates to etiology, pathogenesis, diagnosis, and control is provided. keywords: tritrichomonas foetus, cattle, trichomoniasis lower in nonendemic areas, its potential for substantial economic loss from this disease makes it a concern for the cattle industry. the following narrative summarizes the basics in bovine trichomoniasis regarding its etiology, pathogenesis, diagnosis, and control. etiology tritrichomonas foetus is a spindleto pear-shaped single-cell protozoa with 3 anterior flagella. it has an undulating membrane along the length of its body containing an accessory filament at its margin and a single posterior flagellum. the organism ranges from 9 25 μm in length and 3 -15 μm in width, and replicates through asexual longitudinal binary fission.12 although the life cycle of this protozoa does not include a cyst form, it can develop into a pseudocyst.13 the organism has an affinity for the bovine reproductive tract due to its limited oxygen concentrations, ample food supply in host’s red and white blood cells, and bacteria. hence it is considered an obligate parasite of the bovine reproductive tract.12 in addition, tritrichomonas foetus is capable of surviving temperatures used to store frozen semen for artificial insemination.14 pathogenesis transmission natural transmission of t. foetus is considered strictly venereal and occurs during coitus.15 the rate of natural transmission is high, with many naïve susceptible females becoming infected after a single exposure through coitus to an infected bull with clinical theriogenology 2021; 13: 226 as few as 200 t. foetus organisms.16 bulls became chronically infected after 104 t. foetus organisms were experimentally inoculated into prepuce and some bulls became infected from inoculates containing as few as 102 organisms.17 impact in the female tritrichomonas foetus can be isolated from the female bovine reproductive tract as early as 4 days after introduction.18 however, it does not interfere with conception or maternal recognition of pregnancy19 nor induce any macroor microscopic lesions in the reproductive tract until 50 days of pregnancy, which are characterized by mild inflammatory changes and eventual fetal loss in a majority of the infected females up to 95 days after exposure.20 early fetal death and abortion are followed by early return to estrus in females, the most common clinical sign. fetal loss is followed by 2 6 months of infertility, as the immune system cleared the parasite from the reproductive tract.19 this immune response led to a limited amnestic response to repeated t. foetus infection as demonstrated in multiple studies, with the longest estimated duration of partially protective immunity being < 15 months.21 with some notable exceptions (namely pyometra and chronic infection) complete clearance of t. foetus from the female reproductive tract typically occurs in 5 20 weeks.18 pyometra may be an earliest clinical sign of t. foetus infection in a cow herd;22 in affected cows, purulent debris in the uterine lumen frequently had large numbers of t. foetus.2 cows were infective for as long as 300 days23 or 22 months postbreeding.24 carrier cows had infection through normal pregnancy with t. foetus isolated up to 9 weeks25 or 63 97 days26 after delivery of an apparently normal calf. impact in the male tritrichomonas foetus is primarily an inhabitant of the superficial layers of the penile and preputial epithelium, but not routinely present in other locations of the male reproductive tract.27,28 the absence of tissue invasion may be the reason for a limited immune response in t. foetus-infected bulls.29 lack of pathological changes and failure of the immune response to eliminate t. foetus from the preputial cavity leads to chronic infection in older bulls. this relationship between age and t. foetus carrier bull status may be due to development of crypts in penile and preputial epithelium.12 however, a more recent study disputes this longstanding dogma.30 other unknown individual specific factors may have a role in development of a t. foetus infection and carrier bull status. in summary, the absence of macro and microscopic pathological changes and a limited immunological response to t. foetus infection in bulls result in a lack of clinical signs in infected bulls and development of inapparent, chronically infected bulls. herd-level impact clinical signs in a herd are the culmination of individual responses. consequently, there are increases in: number of nonpregnant cows, incidence of pyometra and abortion, and delayed expected calving dates. percent nonpregnant cows increased substantially (57%31 and 45.3%32) with subsequent decreases in annual calf crop. a computer model predicted a 20 40% prevalence of t. foetus infection in bulls would lead to a 14 50% reduction in annual calf crop.4 additionally, a second common herd-based clinical sign is a prolonged calving interval. cows exposed to t. foetus infected bulls had calving intervals of 96.5 and 98.9 days longer than nonexposed cows during the first and second years of herd infection, respectively.21 herd records would specifically identify this herd-based clinical sign, or it may be recognized due to an increased number of cows calving late in the calving season or lower weaning weights due to younger calves at weaning. diagnosis one of the main aspects of controlling bovine trichomoniasis is identifying infected bulls. the following discussion on the diagnosis of bovine trichomoniasis will focus exclusively on the diagnostic process in a bull. sample collection a modified version of a glass vaginal pipette and rubber bulb technique used in earlier studies to collect samples from female cattle was equally effective for aspirating samples from bull preputial cavities.33 this device has been adapted to utilize a plastic infusion pipette, typically 45 53 cm in length and 0.497 0.571 cm in diameter, with a 12 or 20 ml disposable syringe. general collection technique includes the following: long hairs at the preputial orifice are clipped short to reduce contamination of the sampling device. a syringe is attached to an infusion pipette and the free end of the pipette is passed into the prepuce. when the free end of the pipette reaches the fornix of the prepuce, suction is applied with the syringe. while maintaining suction with the syringe, the pipette is moved forward and backward over the surface of the penis and prepuce several times to scrape smegma into the pipette. prior to removing the pipette from prepuce, the negative pressure in the syringe is gently released to avoid pulling sample into the syringe. after this is accomplished, the pipette is removed, and sample examined. a faint pink color in the sample is acceptable as the mucous membrane of the prepuce is mildly abraded during the collection process. however, overly aggressive scraping can lead to bleeding which may reduce the quality of the sample. if the sample is of adequate quality and volume, it is transferred to the appropriate clinical theriogenology 2021; 13: 227 transport container. if a larger sample is needed, the pipette is reintroduced into the prepuce following the guidelines listed in the previous steps to collect additional smegma. after an adequate sample is recovered, it should be immediately placed in an appropriate medium for transport and testing. maintenance of the sample maintenance of t. foetus preputial specimens has been an important preanalytical concern for trichomoniasis diagnostic testing since the early days of investigation. specimens not subjected to direct examination were frequently collected and maintained in a solution whose purpose was to preserve t. foetus viability until they could be inoculated into laboratory media for incubation or placed directly into the laboratory incubator. however, the use of various solutions, broths and media strictly for specimen transport has been eliminated in the us following availability of direct inoculation into a growth medium at the collection location. this commercial transport and enrichment medium has a plastic cultivation envelope containing a proprietary medium (inpouch™tf, biomed diagnostics, inc., white city, or). inoculated samples should be protected from extreme temperatures, as cold or heat may influence ability to detect t. foetus.34,35 culture the inpouch™tf is examined in a wet mount fashion,36 by fixing the lower portion of the pouch in a plastic clip (size of a microscope slide and provided by the manufacturer) and placing the clip on a compound microscope. systematic scanning of the pouch for motile organisms morphologically consistent with t. foetus should be done daily for several minutes and repeated for 6 consecutive days before declaring the specimen negative.37 using bright field microscopy (100 400 x) to examine samples collected with inpouch™tf from virgin bulls, trichomonads were observed with morphological and motility characteristics consistent with t. foetus. further testing of the trichomonads through staining, scanning electron microscopy, and pcr revealed that the organisms had 4 anterior flagellae, similar to lower-bowel commensal trichomonads (tetratrichomonas pavlovi or tetratrichomonas buttreyi). perhaps these organisms were transferred to the prepuce in feces during sodomy.38 therefore, culture specificity is no longer regarded as 100%. polymerase chain reaction to overcome concerns with t. foetus culture sensitivity and specificity, investigators examined the value of pcr as a trichomoniasis diagnostic assay, based on the assumption that amplification of dna segments specific to t. foetus would reduce or eliminate false positives. furthermore, testing specificity is increased by reducing false negatives, as the test is not influenced by number of organisms.39 control the specific steps to a trichomoniasis control program should be guided by the t. foetus infection status of the herd, the local prevalence of trichomoniasis, the herd owner’s aversion to the risk of a disease incursion and balancing the cost and benefits of the various aspects of a trichomoniasis control program. infected herds based on the information provided above, recommendations for t. foetus elimination from infected herds include the following. sample and test all herd bulls 3 times, regardless of the test used, and cull all test positive bulls or all herd bulls in positive herd. this limits the time and money spent on testing while eliminates any risk of misidentifying a bull as t. foetus-negative that would allow the organism to remain in the herd. however, this option carries a financial burden that may be unacceptable to the herd owner. cull all nonproductive females (i.e. those nonpregnant at the end of the breeding season or those that fail to deliver a live calf prior to the next breeding season) or establish 2 distinct female management groups based on their potential for t. foetus infection, with virgin heifers and cows delivering live calves in 1 group and nonproductive cows in the other. this option involves the risk of maintaining t. foetus in the herd through nonproductive cows and requires fastidious management, including absolute isolation of nonproductive cows from all other cattle and utilization of artificial insemination or bulls exclusive to this group. consider vaccinating all females with an approved trichomoniasis vaccine (trichguard®, boehringer ingelheim vetmedica, inc., ingelheim, germany). this will not prevent infection; however, it may reduce fetal losses associated with infection and the period of infection.40,41 finally, implement prevention strategies for high-risk herds (refer below). high-risk uninfected herds herds are considered high risk for the introduction of t. foetus based on a relatively high local prevalence of the disease and utilization of management practices that increase the risk of introduction. suggested strategies for prevention of t. foetus in high-risk herds include the following. plan a pasture utilization program to minimize contact with neighboring cattle. utilize proper artificial insemination protocols with semen from a reputable source in specific management groups or eliminate the entire herd to greatly reduce the risk of t. foetus transmission. maintain a young bull battery to reduce the rate of transmission and potential development of chronic carrier bulls. isolate and/or test cattle if unplanned commingling with clinical theriogenology 2021; 13: 228 neighboring herds has occurred. females should be isolated from the rest of the herd until after the breeding season and their pregnancy status can be confirmed. bulls should be isolated and tested (3 tests at weekly intervals) to ensure that they are t. foetus negative. restrict the length of the breeding season to < 120 days to reduce the opportunity for transmission of the disease within the herd and to more easily monitor reproductive performance. institute a surveillance testing program. surveillance testing should occur between the end of a breeding season and the beginning of the next and include a single test of all bulls in the herd. the advantage of surveillance testing performed closely after the breeding season is early detection of infection that allows time to develop and implement a complete trichomoniasis control program before the next breeding season. bulls tested under this program must not be exposed to cows prior to the next breeding season. alternatively, test all bulls immediately before the breeding season. this the advantage of timing the test to coincide with an annual breeding soundness examination, thereby reducing the number of times bulls are handled. however, this may not allow sufficient time for appropriate management of the disease before the start of the breeding season. finally, implement prevention strategies for low-risk herds, as described below. low-risk uninfected herds herds are considered low risk for the introduction of t. foetus based on a low local prevalence of the disease and the utilization of management practices that reduce the risk of the introduction of trichomoniasis into the herd. suggested strategies for prevention of t. foetus in low-risk herds include the following. develop communication networks with neighbors to ensure rapid notification if trichomoniasis is diagnosed in a neighboring herd. monitor fences and cattle to rapidly identify when unplanned commingling with another herd has occurred so immediate steps can be taken to address the problem and reduce the risk of introducing trichomoniasis. maintain herd records to monitor herd reproductive performance and identify animals within management groups for early detection of a potential trichomoniasis incursion and efficient management of the outbreak. observe interand intra-state animal health regulations regarding trichomoniasis and other diseases. regulations to protect the livestock industries of each state should be considered as a barrier to trichomoniasis introduction and not as a complete prevention program at the herd level. purchase replacement animals, preferably virgin bulls and heifers, from a reputable source. purchase of nonvirgin bulls and cows, especially, from herds with unknown reproductive performance, increases the risk of trichomoniasis introduction. accepted risks in purchasing nonvirgin replacements may be greatly reduced by having a source that has excellent herd reproductive performance with bulls tested negative or by purchasing pregnant cows. conclusion bovine trichomoniasis has plagued the us cattle population since the 1930’s and continues to inflict reproductive loss and subsequently financial loss in the industry. understanding the characteristics of this disease associated with its etiology, pathogenesis, diagnosis provides a logical and practical approach to control of the causative organism, tritrichomonas foetus. conflict of interest none to report. references 1. skirrow sz, bondurant rh: bovine trichomoniasis. vet bull 1988;58:591-603. 2. emmerson ma: trichomoniasis in cattle. j am vet med assoc 1932;81:636-640. 3. fitzgerald pr, johnson ae, thorne j, et al: trichomoniasis in range cattle. vet med 1958;53:249-252. 4. rae do: impact of trichomoniasis on the cow-calf producer’s profitability. j am vet med assoc 1989;194:771-775. 5. villarroel a, carpenter te, bondurant rh: development of a simulation model to evaluate the effect of vaccination against tritrichomonas foetus on reproductive efficiency in beef herds. j am vet med assoc 2004;65:770-775. 6. bondurant rh, anderson ml, blanchard p, et al: prevalence of trichomoniasis among california beef herds. j am vet med assoc 1990;196:1590-1593. 7. rae do, crews je, greiner ec, et al: epidemiology of tritrichomonas foetus in beef bull populations in florida. theriogenology 2004;61:605-618. 8. grotelueschen dm, cheney j, hudson db, et al: bovine trichomoniasis: results of a slaughter survey in colorado and nebraska. theriogenology 1994;42:165-171. 9. rodning sp, wolfe df, carson rl, et al: prevalence of tritrichomonas foetus in several subpopulations of alabama beef bulls. theriogenology 2008;69:212-217. 10. jones bm, whitlock bk, strickland lg, et al: prevalence of tritrichomonas foetus in tennessee beef bulls. clinical theriogenology 2015;7:333. 11. yao c, bardsley kd, litzman ea, et al: tritrichomonas foetus infection in beef bull populations in wyoming. j bacteriol parasitol 2011;2:117. 12. bondurant rh, honiberg bm: trichomonads of veterinary importance. in: kreier jp: editor. parasitic protozoa. 9th edition, san diego; academia press: 1994. p. 112-188. 13. granger bl, warwood sj, benchimol m, et al: transient invagination of flagella by tritrichomonas foetus. parasitol res 2000;86:699-709. 14. clark bl, white mb, banfield jc: diagnosis of trichomonas foetus infection in bulls. aust vet j 1971;47:181-183. 15. bartlett de: trichomonas foetus infection and bovine reproduction. clinical theriogenology 2021; 13: 229 j am vet med assoc 1947;8:343-352. 16. clark bl, dufty jh, parsonson im. studies on the transmission of tritrichomonas foetus. aust vet j 1977;53:170-172. 17. clark bl, parsonson im, dufty jh: experimental infection of bulls with tritrichomonas foetus. aust vet j 1974;50:189-191. 18. murname d: field and laboratory observations on trichomoniasis of dairy cattle in victoria. aust vet j 1959;35:80-83. 19. bondurant rh: diagnosis, treatment and control of bovine trichomoniasis. compend contin educ pract vet 1985;7:179-186. 20. parsonson im, clark bl, dufty jh: early pathogenesis and pathology of tritrichomonas foetus infection in virgin heifers. j comp path 1976;86:59-66. 21. clark bl, dufty jh, parsonson im: the effect of tritrichomonas foetus infection on calving rates in beef cattle. aust vet j 1983;60:71-74. 22. rae do, crew je. tritrichomonas foetus. vet clin north am food anim pract 2006;22:595-611. 23. mancebo oa, russo am, carabajal ll, et al: persistence of tritrichomonas foetus in naturally infected cows and heifers in argentina. vet parasitol 1995;59:7-11. 24. alexander gi: an outbreak of bovine trichomoniasis in queensland and its control. aust vet j 1953;29:61-66. 25. skirrow sz: identification of trichomonad-carrier cows. j am vet med assoc 1987;191:553-554. 26. goodger wj, skirrow sz: epidemiologic and economic analysis of an unusually long epizootic of trichomoniasis in a large california dairy herd. j am vet med assoc 1986;189:772-776. 27. parsonson im, clark bl, dufty j: the pathogenesis of tritrichomonas foetus infection in the bull. aust vet j 1974;50:421-423. 28. rhyan jc, wilson kl, wagner b, et al: demonstration of tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally infected bulls. vet pathol 1999;36:406-411. 29. soto p, parma ae: the immune response in cattle infected with tritrichomonas foetus. vet parasitol 1989;33:343-348. 30. strickland l, edmondson m, maxwell h, et al: surface architectural anatomy of the penile and preputial epithelium of bulls. clinical theriogenology 2014;6:445-451. 31. barling ks, field rw, snowden kf, et al: acute trichomoniasis and sub-optimal fertility in a cow/calf herd: an investigation and case management. bov pract 2005;39:1-5. 32. alstad ad, krogh d, fischer k et al: trichomoniasis in a beef herd. vet med 1984;79:708-709. 33. hammond dm, bartlett de: establishment of infection with trichomonas foetus in bulls by experimental exposure. am j vet res 1943;4:61-65. 34. clavijo a, erol e, sneed l, et al: the influence of temperature and simulated transport conditions of diagnostic samples on real-time polymerase chain reaction for detection of tritrichomonas foetus dna. j vet diagn invest 2011;23:982-985. 35. bryan la, campbell jr, gajadhar aa: effects of temperature on the survival of tritrichomonas foetus in transport, diamond’s and inpouch tf media. vet rec 1999;144:227-232. 36. cobo er, favetto ph, lane vm, et al: sensitivity and specificity of culture and pcr of smegma samples of bulls experimentally infect with tritrichomonas foetus. theriogenology 2007;68:853-860. 37. parker s, campbell j, gajadhar a: comparison of the diagnostic sensitivity of a commercially available culture kit and a diagnostic culture test using diamond’s media for diagnosing tritrichomonas foetus in bulls. j vet diagn invest 2003;15:460-465. 38. bondurant rh, gajadhar a, campero cm, et al: preliminary characterization of a tritrichomonas foetus-like protozoan isolated from preputial smegma of virgin bulls. bov pract 1999;33:124-127. 39. morgan um, thompson rca, smith hv, et al: molecular detection of parasitic protozoa. parasitology 1998;117:73-85. 40. kvasnicka wg, taylor rel, huang jc, et al: investigations of the incidence of bovine trichomonasis in nevada and of the efficacy of immunizing cattle with vaccines containing tritrichomonas foetus. therio 1989;31:963-971. 41. hall mr, kvasnicka wg, hanks d, et al: improved control of trichomoniasis with trichomonas foetus vaccine. agri pract 1993;14:29-34. clinical theriogenology 2021; 13: 230 review of bovine trichomoniasis clinical theriogenology 2022; 14: 55 perinatal mortality in horses and camelids dalen agnew department of pathobiology and diagnostic investigation college of veterinary medicine, michigan state university, east lansing, mi abstract the neonatal health of foals and crias is affected by the quality of the uterine and placental environment before birth, threats they face during birth, and vulnerabilities of the first week of life. when identifying the cause of neonatal death, careful examination of the dam, fetal membranes, and the fetus is critical. the most common threats for horses and camelids are failure of passive transfer, bacterial sepsis, diarrhea, and congenital defects. identification of compatible lesions and confirmation with laboratory testing will allow better management of the herd. keywords: perinatal mortality, neonatal mortality, horses, south american camelids introduction the transition period between birth and early days of life (neonatal or perinatal life is usually defined as the first week of life) is likely the most challenging and high-risk period for any animal. half of foal mortality occurs in first 30 days (5.8% of live births), mostly (3.3%) in the first 2 days.1 in south american camelids, ~ 20 80% of crias do not survive the first year, whereas in north america, 5% loss is more typical.2 causes of neonatal death overlap considerably with those of abortion and stillbirth, (including infectious, traumatic, developmental, genetic, toxic, and nutritional causes). only causes of neonatal death will be discussed in this review. general approach to investigate perinatal mortality understanding the normal progression from intrauterine to terrestrial life and identifying deviations from that path are important. critical points of concern are: placental sufficiency in late pregnancy, any disruptions during delivery leading to delay, dystocia or hypoxia, trauma at birth or early life, environmental stressors such as cold, dampness or heat, and ingestion of inadequate amounts of good quality colostrum. a thorough breeding and medical history of the dam is critical to identify whether the fetus is premature and whether the dam and fetus might be susceptible to potential infectious diseases. unlike production animals, most horses and camelids are observed closely during parturition and the conditions and circumstance of the neonate’s birth can be well documented. for instance, mares give birth during night and camelids during daylight hours; variations from these norms are possible, but should trigger greater scrutiny and may predict more insidious problems.2 in addition, a complete postmortem examination of the neonate and a complete physical and reproductive examination of the mare or hembra is required. it is important that fetal membranes are carefully examined. placental abnormalities may be predictive of future problems despite apparent vitality of foal or cria. evidence of placental insufficiency such as villous hypoplasia or infection such as chronic vasculitis (thickened tortuous vessels) or chorionic infarction (thinned translucent sharply demarcated regions of chorioallantois) may indicate that the fetus may develop weakness and poor postnatal development or sepsis within first few days. in large breeding facilities, barn managers can be trained to evaluate fetal membranes and save them for more detailed inspection by the veterinarian during regular business hours. observation of postnatal nursing and confirmation of passive transfer of antibodies via colostrum are also crucial to predicting neonatal survival. failure of passive transfer (fpt) is the cause of at least 13% of neonatal equine deaths, and likely is associated with many more.1 complete fpt in foals is typically defined as a serum igg concentrations of < 400 mg/ dl on day 1, and partial fpt as serum igg concentration of 400 800 mg/dl. in crias, an estimated 9% had fpt.3 in crias, > 1,000 mg/dl of igg is expected on days 1 or 2. serum total protein can be used, but is a less accurate estimate and should be used as a last resort, with < 4.9 g/dl being compatible with fpt in 2-day old crias.4 some foals and crias survive and thrive despite fpt, but it can often lead to life-threatening sepsis, various infections, and hypoglycemia, and complicate other comorbidities. prematurity is considered when a foal is born earlier than ~ 320 335 days of pregnancy and when crias are born < 335 days; however, there is considerable variation in pregnancy lengths, seasonal variations, and some neonates will appear clinical theriogenology 2022; 14: 56 premature even if born within the expected range (these are often called ‘dysmature’).2 low birth weight, lethargy, tendon laxity, and floppy ears may suggest dysmaturity or prematurity. in crias, ears may be bent backwards and unerupted incisors are often observed. in foals, domed or prominent forehead has been frequently reported. a poor suckle reflex can also lead to fpt. these animals are especially prone to hypothermia and metabolic derangements including hyperor hypoglycemia. a lack of surfactant in these animals can lead to hypoventilation and hypoxia. causes of mortality in foals neonatal sepsis is the leading cause of death. at least 24% of bacteremic foals have been associated with maternal factors such as placentitis or dystocia.5 general cleanliness, fpt, and husbandry factors are associated with sepsis. postnatal umbilical, gastrointestinal, and respiratory portals of infection are also associated with the development of life-threatening bacteremia. blood cultures are definitive and can help guide therapy if taken antemortem. gross lesions include tacky and red mucous membranes, interstitial pneumonia, adrenal hemorrhage, hepatitis, splenitis, meningitis, uveitis, arthritis, osteomyelitis, enteritis, serosal hemorrhages, and often thrombosis of major arteries. ileus can also be associated with sepsis leading to a whole new list of complications.6 gram-negative bacteria, (especially, escherichia coli [e. coli]), are considered the most common cause; however, e. coli can often be an innocent bystander or opportunist, so identification of virulence factors by pcr is necessary. other bacteria, including gram-positive bacteria, should not be discounted, as they can also be substantial causes of neonatal sepsis. fungi, particularly candida albicans, can also have a role, particularly in immunocompromised foals.7 gastrointestinal conditions include diarrhea, enterocolitis, gastric and duodenal ulcers, meconium impaction, and congenital defects such as atresia ani. bacterial infectious causes include primarily clostridium perfringens (type a and c primarily in foals < 10 days old) and clostridium difficile, and salmonella sp. all leading to hemorrhagic and necrotizing enterocolitis. rotavirus and to a much lesser extent, coronavirus, are viral causes of neonatal diarrhea. microvilli are blunted, leading to a malabsorptive, voluminous, watery diarrhea.8,9 gastric ulceration is another very common condition in foals, most often observed in neonates in the squamous mucosa adjacent to the margo plicatus.9,10 duodenal ulceration is more common in older foals. both conditions can develop quickly and perforation may occur before clinical signs are evident. causation is still hotly debated, but it is likely multifactorial and complicated, involving environmental and physiologic stress, nonsteroidal anti-inflammatory medications, diet, or other comorbidities. meconium impaction is the most common cause of colic in neonatal foals, but it should be differentiated from more serious and likely fatal intestinal or anal atresia and intestinal aganglionosis. meconium is a yellow-green collection of sloughed cells and intestinal mucus mixed with amniotic fluid that represent the foal’s first defecation. impaction of this material is common, usually easily treated, and rarely is a cause of mortality. in contrast, intestinal atresia is uniformly fatal without surgical intervention. cause of intestinal atresia has been speculated to be ischemia secondary to vascular supply disruption to a section of intestine, possibly associated with intestinal displacements. in calves, there was an association with transrectal palpation and atresia, but no similar association was reported in other species. a grading system has been proposed:11 type i membrane atresia (a diaphragm occludes the lumen) type ii cord atresia (a fibrous cord-like remnant of intestine) type iiia blind end atresia (a segment is missing with a mesenteric defect) type iiib blind end atresia (the distal segment is coiled) type iv multiple sites of atresia prognosis for these cases is guarded, even with surgical intervention. intestinal aganglionosis is a hereditary condition, associated with the lethal white syndrome, an autosomal recessive mutation. submucosal and myenteric ganglia are absent from the distal small intestine to the colon leading to ileus. due to the extent of the lesion, these horses are usually euthanized. genetic testing (presence of endothelin b receptor genotypes) is now available for this disease for diagnosis and genetic management of possible carriers.12 ‘dummy foal’ or neonatal maladjustment syndrome (formerly known as hypoxic-ischemic encephalopathy) is a condition observed in foals that experience hypoxia for a variety of reasons during the perinatal period.13-16 placental disease and dystocia17 are the most common causes, but any prolonged or repeated hypoxia can result in a foal with a variety of neurologic signs including stupor, abnormal posture, unwillingness or inability to nurse, abnormal facial movement, head-pressing, and seizures. neonatal isoerythrolysis, resulting in hemolytic anemia in foals, is a relatively uncommon but serious disease observed in ~ 1 2% of horses and up to 10% of mules.18,19 this condition occurs when a mare produces alloantibodies against a red blood cell (rbc) antigen present on a foal’s rbcs, and the foal ingests those alloantibodies in colostrum. foals are initially normal, but develop icterus, pigmenturia, weakness, lethargy, and ultimately cardiovascular collapse. a hemolytic cross-match assay is the most reliable test. kernicterus has been reported in affected foals with prolonged hemolysis that demonstrate neurologic disease and death. histopathology demonstrates the deposition of unconjugated bilirubin and neuronal necrosis in the gray matter of the cerebellum, basal ganglia, subthalamic nuclei, and the hippocampus.19 as in any neonate, a variety of congenital conditions can also be observed that may or may not have a genetic basis, including those already mentioned and cardiac conditions like ventricular septal defect, heart valvular defects, cerebelclinical theriogenology 2022; 14: 57 lar hypoplasia (or abiotrophy), tendon laxities and contractions, microphthalmia, cleft palate, and umbilical defects.20 schistosoma reflexus, polydactyly, and other lesions are rarely observed. causes of mortality in south american crias bacterial sepsis, often associated with fpt, is a common cause of neonatal death. gram-negative bacteria, especially e. coli, are most common, but others include beta-hemolytic streptococcus and listeria monocytogenes. culture of lung, liver, and spleen can be useful, but lesions can be quite variable depending on the organs most affected. histologically, the presence of megakaryocytes in the pulmonary vasculature is strong evidence of a systemic and exhaustive immune response. multifocal to diffuse interstitial pneumonia, hepatitis, splenitis, nephritis, hypopion, and meningoencephalitis can be observed in varying degrees, and in peracute cases, there may be few if any lesions. notably, septic joints are less common in crias than ruminant and equine neonates. brain and vertebral abscesses are another possible sequela to sepsis. infection with mycoplasma haemolamae is a condition observed commonly in north and south america, and is similar to hemotropic mycoplasmas observed in cattle, pigs, and cats.2 they are small, 0.5 µm diameter round, ring, or linear organisms identified at the periphery of red blood cells on blood films. they are primarily transmitted by insect vectors or iatrogenically, but vertical transmission has been documented with crias infected at 1 day of age before colostrum ingestion. these organisms are associated with weight loss, anemia, weakness, and lethargy. these are often easily observed on blood films, but low-level infections may require pcr. similar to foals, neonatal diarrhea is a major threat to young crias. differentials should include bacterial endotoxemia or enterotoxemia, coccidiosis, cryptosporidiosis, coronavirus, rotavirus, or bovine viral diarrhea virus and coinfections should also be considered. bacterial culture should identify most bacterial causes; however, if e. coli is identified, pcr for virulence factors is recommended to differentiate commensal e. coli and pathogenic strains. salmonellosis has very rarely been identified in septicemic crias, but has not been associated with diarrhea.21 iatrogenically induced clostridium difficile and infections with clostridium perfringens have been reported but are not common. multiple coccidians can be an issue, but particularly in young camelids, eimeria macusaniensis is the most significant and can lead to lethargy, weight loss, anorexia, diarrhea, and unfortunately, sudden death.22 with coccidiosis, necrosis and villous loss are the primary lesions noticed throughout the jejunum and ileum, though fibrosis may be prominent in longer standing cases. the duodenum and colon are typically spared. organisms are often present in the deep crypts, but in heavy infections, the entire villus is affected. even in massive infections, however, fecal examinations for eimeria macusaniensis are often negative. cryptosporidia, likely c. parvum, has been identified frequently in cases of neonatal cria diarrhea and has a similar pathogenesis to observed in cattle. antemortem detection can be challenging and the use of acid-fast stains on fecal smears is likely the most effective test (simple, fast, and sensitive), whereas pcr can also be useful. postmortem gross lesions are few, other than liquid feces, but the organisms can be identified microscopically on the villus tips as 5 10 µm basophilic spheres. several studies have also identified giardia sp. leading to villus atrophy and malabsorptive diarrhea. rotavirus is a common viral cause of diarrhea in young crias, particularly in south america, and is highly transmissible, but usually it must be accompanied by other pathogens or conditions to lead to death. gross lesions are limited to liquid diarrhea. rotaviral diarrhea has 3 mechanisms: destruction of enterocytes leading to malabsorption and villus atrophy, release of vasoactive agents leading to ischemia and enteric nervous stimulation, and production of a nonstructural protein that acts as a secretory enterotoxin.8 coronaviruses in camelids have also been well-documented and can be severe; however, the severity is likely related to the involvement of other pathogens and stress.21 bovine viral diarrhea virus (bvdv) is a known entity to bovine practitioners, but bvdv can also infect camelids leading to clinical disease. crias, in particular, can be persistent carriers, transmitting infections to new herds. reported clinical signs are similar to cattle: diarrhea, unthrifty condition, abortion, and birth defects depending on the time of infection of either the cria or dam. pcr is considered the test of choice, with spleen, thymus, ileum (with peyer’s patches), lung, or whole blood being best specimens. serum can be used for paired serological testing, but currently, is not definitive in camelids. persistent infections are best confirmed with positive pcr tests conducted 3 4 weeks apart.23 respiratory disease in crias has some unique features due to their adaptations to high altitudes. their oxyhemoglobin dissociation curves distinctly differ from other domestic mammals, with a shift to left allowing for an increased affinity for oxygen in the lungs.24 thus they are less susceptible to negative effects of hypoxia after pneumonia, heart defects, atelectasis, prolonged recumbency, or other pulmonary conditions. ‘dummy crias’ that have had perinatal hypoxia can often survive without serious effects, if they are given supportive care, and cerebral necrosis or other lesions may be less severe than expected in crias that have died after severe hypoxic events. in crias, particularly in north american populations, congenital defects are more common than other domestic species.24 common defects include choanal atresia, cleft palate, cerebellar hypoplasia, prognathism and brachygnathism, hydrocephalus, cerebellar hypoplasia, deafness, atresia ani, vulvar hypoplasia, cardiac defects, and skeletal abnormalities including angular limb deformities, arthrogryposis (sometimes associated with brain defects), polydactyly, kyphosis, and scoliosis. not all these defects are hereditary, but many are and reporting of this information is important for breeders to make informed decisions on mating choices. conclusion the most critical period in any mammal’s life is the time of parturition and adjustment to life outside the uterus. establishment of a strong immune response via passive transfer of immunoglobins in colostrum and development of an independent immunocompetence that will last the rest of animal’s life is a common feature of normal camelid and equine clinical theriogenology 2022; 14: 58 neonates. when death occurs during the neonatal period, evaluation of prepartum environment (dam’s health and placental quality) and examination of the fetus is critical. foals and crias share many of the same threats during this period, but these potential problems can be addressed by careful husbandry, including accurate diagnosis of neonatal deaths for better herd management. conflict of interest author has no conflict of interest. no funding was received for this publication. references 1. veterinary services center for epidemiology and animal health: equine mortality in the united states, 2015. aphis info sheet 2017. 2. whitehead ce, cebra c: neonatology and neonatal disorders. in: cebra c, anderson de, tibary a, van saun rj, johnson lw: editors, llama and alpaca care 1st edition, st. louis; elseveier: 2014. p. 552575. 3.garmendia ae, palmer gh, demartini jc, et al: failure of passive immunoglobulin transfer: a major determinant of mortality in newborn alpacas (lama pacos). am j vet res 1987;481472-1476. 4. weaver dm, tyler jw, marion rs, et al: evaluation of assays for determination of passive transfer status in neonatal llamas and alpacas. j am vet med assoc 2000;216:559-563. 5. stewart aj, hinchcliff kw, saville wj, et al: actinobacillus sp. bacteremia in foals: clinical signs and prognosis. j vet intern med 2002;16:464-471. 6. bauer aj, schwarz nt, moore ba, et al: ileus in critical illness: mechanisms and management. curr opin crit care 2002;8:152-157. 7. sanchez lc: equine neonatal sepsis. vet clin north am equine pract 2005:273-293. 8. magdesian kg: neonatal foal diarrhea. vet clin north am equine pract 2005:295-312. 9. uzal fa, plattner bl, hostetter jm: alimentary system. in: maxie mg: editor, pathology of domestic animals vol 2, 6th edition, philadelphia; elsevier: 2014. p.1-257. 10. ryan ca, sanchez lc: nondiarrheal disorders of the gastrointestinal tract in neonatal foals. vet clin north am equine pract 2005:313-332. 11. benamou ae, blikslager at, sellon dc: intestinal atresia in foals. comp cont ed pract vet 1995;17:1510-1517. 12. magdesian kg, williams dc, aleman m, et al: evaluation of deafness in american paint horses by phenotype, brainstem auditoryevoked responses, and endothelin receptor b genotype. j am vet med assoc 2009;235:1204-1211. 13. palmer ac, rossdale pd: neuropathological changes associated with the neonatal maladjustment syndrome in the thoroughbred foal. res vet sci 1976;20:267-275. 14. madigan je, haggett ef, pickles kj, et al: allopregnanolone infusion induced neurobehavioral alterations in a neonatal foal: is this a clue to the pathogenesis of neonatal maladjustment syndrome? equine vet j 2012;44(suppl):109-112. 15. aleman m, weich km, madigan je: survey of veterinarians using a novel physical compression squeeze procedure in the management of neonatal maladjustment syndrome in foals. animals 2017;7:69-80. 16. aleman m, mccue pm, chigerwe m, et al: plasma concentrations of steroid precursors, steroids, neuroactive steroids, and neurosteroids in healthy neonatal foals from birth to 7 days of age. j vet intern med 2019;33:2286-2293. 17. giles rc, donahue jm, hong cb, et al: causes of abortion, stillbirth, and perinatal death in horses: 3,527 cases (1986-1991). j am vet med assoc 1993;203:1170-1175. 18. alexandra j, burton l, mittel d, et al: emergency treatment of mules and donkeys. in: orsini ja, divers tj: editor, equine emergencies, 4th edition, philadelphia; wb saunders: 2014.p. 646-651. 19. giguere s, polkes ac: immunological disorders in neonatal foals. 2005;241-272. 20. crowe mw, swerczek tw: equine congenital defects. am j vet res 1985;46:353-358. 21. whitehead ce: neonatal diseases in llamas and alpacas. vet clin north am food anim pract 2009:367-384. 22. cebra ck, valentine ba, schlipf jw, et al: eimeria macusaniensis infection in 15 llamas and 34 alpacas. j am vet med assoc 2007;230:94-100. 23. nelson dd, duprau jl, wolff pl, et al: persistent bovine viral diarrhea virus infection in domestic and wild small ruminants and camelids including the mountain goat (oreamnos americanus). front microbiol 2016;6:1415. 24. reynafarje c, faura j, villavicencio d, et al: oxygen transport of hemoglobin in high-altitude animals (camelidae). j appl physiol 1975;38:806-810. 25. fowler me, bravo pw: congenital/hereditary conditions. in: fowler me, bravo pw: editors, medicine and surgery of camelids, 3rd edition, ames, blackwell: 2010, p.525-528. pregnancy analytics app adds value to pregnancy diagnosis in beef herds pregnancy analytics app adds value to pregnancy diagnosis in beef herds robert larson department of clinical sciences, college of veterinary medicine kansas state university manhattan, ks abstract determining pregnancy status of beef cattle is an important service both as a major source of income for veterinary practices and a valuable information resource for cow-calf producers. however, value of pregnancy status information should be enhanced to give veterinarians a competitive advantage against alternate methods for identifying nonpregnant cows. obtaining fetal age estimates is vital to create charts to evaluate and display pregnancy percentages by 21 day periods. evaluating breeding seasons by 21 day periods and by animal age and/or other management groups is useful to practitioners investigating herds with reproduction shortfalls by identifying specific periods within breeding seasons when cows did not becoming pregnant. in addition, easy-to-read charts can enhance communication between veterinarian and beef producer by illustrating results of fetal aging. once fetal age data are collected and organized for analyses, conclusions or further questions will present themselves. nutrition, genetics, animal husbandry, male and female reproductive soundness and health all affect distribution of pregnancy within a herd. by combining transrectal uterine examination to determine pregnancy status with chute-side analysis and graphing of information, veterinarians can readily identify strengths and weaknesses within herd management. keywords: beef cattle, pregnancy diagnosis, histogram introduction determining pregnancy status of beef cattle is an important veterinary service. for veterinary practices, it is a major source of income. for cow/calf clients it is a cost-effective source of valuable information. however, to increase value of this service and for veterinarians to have competitive advantage compared to other persons and alternate methods, veterinarians should gain additional information from pregnancy status determination and use key metrics to optimize ranch productivity. beef cow reproduction is limited by 2 key factors: first, a relatively long infertile interval after calving and second, only 60 70% of successful matings of fertile cattle results in a viable pregnancy. approximately 30 40% of fertile matings result in either failure of fertilization or death of the early embryo, but in most situations, the mated, but non-pregnant cow will express estrus and ovulate a fertile oocyte about 21 days after her last ovulation and will have another 60 70% probability of conceiving and maintaining a pregnancy. cows with 3 opportunities to be bred (each with a 60 70% probability of a successful pregnancy) have ~ 95% probability of being pregnant at mid-pregnancy examination. if nearly all cows in a herd calved early enough so that they resume fertile cycles by the 21st day of the next breeding season and bulls are fertile and able to successfully breed, the ideal pregnancy pattern will be ~ 60 65% pregnant in first 21 days of breeding season, 85 90% pregnant by 42nd day of breeding and ~ 95% pregnant after 63 days of breeding (figure 1). bci pregnancy analytics app: gathering pregnancy data chute-side beef cattle institute developed a pregnancy analytics app to assist veterinarians collect data, process it rapidly and create valuable graphs and charts to allow diagnostic analytics of the pregnancy distribution. this app is used by veterinarians to enhance monitoring and evaluating cowherd breeding season success. ability to visualize the percentage of cows becoming pregnant each 21 days of the breeding season can provide important information to identify contributing causes for situations when a lower than desired percentage of herd becomes pregnant, or to identify areas for improved reproductive efficiency. until now, collecting and evaluating information chute-side was difficult. clinical theriogenology • volume 12 number 3 • september 2020232 figure 1. histogram of the percentage of cows that conceive and maintain a viable pregnancy if nearly all cows ovulate a fertile oocyte during the first 21st days of the breeding season and are bred by fertile bulls and 60 70% of available (nonpregnant) cows conceive and maintain a viable pregnancy in each subsequent 21 day breeding period. data required by this app are starting and ending dates for the breeding season and an estimate of fetal age for each pregnancy. additional information such as cow id, cow age, body condition score, and breed (or other descriptor) can be added to enhance value of pregnancy status information. after data are entered, estimated conception dates are generated and histograms created. these pregnancy patterns can help identify most likely contributing factors when investigating herds with a lower than desired percent pregnant.1,2 veterinarians can be fairly precise estimating fetal age early in pregnancy; however, ability to estimate fetal age accurately decreases as pregnancy progresses.3 therefore, to confidently place cows within fetal age groups, pregnancy diagnosis should occur < 120 days after breeding season initiation. ability to place animals within fairly tight 21 day periods is a great advantage for veterinarians when evaluating the herds’ recent past and optimizing future herd management. the ideal distribution for a 63 day breeding season should resemble figure 1. producers should strive for nutritional and management systems that allow > 60% of exposed animals to become pregnant in the first 21 breeding season days,4 with the majority of remaining animals becoming pregnant in the second 21 day period and no more than 5% of the herd being classified as nonpregnant. another way to evaluate pregnancy distribution data is to determine percent of the available (nonpregnant) cattle that become pregnant each 21 day period. as the breeding season advances and pregnant cattle are no longer available to be bred, the herd percentage that becomes pregnant each 21 days is not same as the percentage of available (nonpregnant) cattle that becomes pregnant each 21 days. this important measure is displayed as a table by clicking “% pregnancy success”. evaluating the percent of the herd that became pregnant each 21 days does not directly provide accurate visualization of how fertility is changing over the breeding season. the pregnancy distribution displayed in figure 2 provides evidence that 25% of nonpregnant cows became pregnant in the first 21 day period. however, it is not as clear that in the second 21 day period, 40% of nonpregnant cows became pregnant (30% of herd), and in the third period, 65% of nonpregnant cows became pregnant (30% of herd), and finally, that in the fourth period, 65% of nonpregnant cows became pregnant. clinical theriogenology • volume 12 number 3 • september 2020 233 figure 2. reporting % pregnancy success as a percent of available (nonpregnant) cows at the start of each 21 day period that became pregnant within each 21 day period provides a more direct indication of changing fertility over the breeding season. based on expected pregnancy success when cow and bull fertility is optimal, the “% pregnancy success” goal should be between 60 and 70% for every 21 day period of the breeding season. using the herd represented in figure 2, by the third 21 days, there is no problem with fertility in cows or bulls. the % pregnancy success values clearly indicate that reproductive problems in this herd occurred during the first 2 cycles, whereas fertility was optimal in the last 2 breeding season cycles. bci pregnancy analytics app: interpreting charts and tables at pregnancy diagnosis, veterinarians can estimate fetal age and evaluate palpable or ultrasonographic characteristics of nonpregnant reproductive tracts (figure 3). if low pregnancy percentage is due to failure to conceive due to cows not resuming fertile cycles postpartum or bulls failing to deliver fertile semen, reproductive tract examination should reveal characteristics of a nonpregnant uterus with no indication of previous pregnancy or uterine pathology. typical timing of pregnancy diagnosis relative to reasons for early gestation loss due to noninfectious (e.g. heat or transportation stress) or infectious (e.g. trichomoniasis) causes may or may not be associated with still-detectable uterine involution or pathology. because infectious agents or toxins causing pregnancy losses often occur in late pregnancy just prior to or following examination for pregnancy status, examination of nonpregnant reproductive tracts due to recent abortion should reveal some reproductive tracts with characteristics of involution or uterine pathology. once all data collected at pregnancy diagnosis are organized, in-depth and efficient evaluation of herd reproductive success can be conducted. reasons for low pregnancy percentage during any 21 day period can be placed into one of 3 categories: 1) inadequate percentage of females having fertile estrous cycles; 2) bulls not able to deliver adequate amounts of fertile semen; or 3) infectious or noninfectious agents prevented or ended pregnancy. pregnancy analytics app charts and graphs, along with reproductive tract and cow body condition physical examination findings at pregnancy diagnosis, can guide history questions, further physical examination, herd record evaluation, and diagnostic laboratory testing to assist evaluation of possible rule-outs as likely or unlikely causes of undesired pregnancy distributions. clinical theriogenology • volume 12 number 3 • september 2020234 inadequate percentage of animals were cycling by the 21st day of breeding although figure 1 depicts an ideal herd, many times evaluation of herd pregnancy status data reveals a distinctly different pregnancy distribution. figure 4 illustrates a very common distribution. in this situation, the percentage of nonpregnant cows would not necessarily indicate a problem if the breeding season lasts long enough; therefore, further evaluation is needed to begin a diagnostic workup. figure 4. typical pregnancy distribution for herd with 50% of cows cycling by end of first 21 days of breeding season. a common reason for a pregnancy distribution similar to figure 4 is that a similar pregnancy distribution the previous year resulted in many cows calving in third or later 21 day period of the calving season. the typical amount of time from calving to resumption of fertile cycles (postpartum anestrus period) for 90% of a herd’s mature cows is 60 80 days5 and for primiparous cows, it is closer to 100 120 days.6 if the breeding season begins on the same date as the previous year (and the breeding season lasts 63 days), the breeding season will commence 62 82 days postpartum and end 125 145 days postpartum for cows calving in first 21 day period of previous calving season. therefore, all early-calving cows (including primiparous cows) are expected to express estrus and be bred several times during the breeding season. cows calving in the second 21 day period will be figure 3. timing of pregnancy diagnosis relative to reasons for low pregnancy percentage indicates that failure to conceive due to cows not resuming fertile cycles postpartum or bulls failing to deliver fertile semen to cow’s reproductive tract occurred many weeks prior to examination, early gestational loss due to noninfectious (e.g. stress) or infectious causes (e.g. trichomoniasis) occurred a few weeks to a few months prior to examination, and late pregnancy loss due to infectious agents or toxins occurred just prior to or following examination. clinical theriogenology • volume 12 number 3 • september 2020 235 41 61 days postpartum at the start of breeding season and 104 124 days past calving 63 days later. once again, this timing should allow mature cows to resume cycles and have multiple opportunities to be bred during the breeding season. primiparous cows calving in the second 21 day period should also resume cycling early enough in breeding season to have 1 or 2 opportunities to be bred. in contrast, for cows that calve in the fourth 21 day period, calving has just finished as breeding begins and for those in the fifth 21 day period, the breeding season begins prior to the time they calve. limited interval from calving to breeding season initiation essentially eliminates the potential for nursing cows, and in particular, primiparous cows, to rebreed early in the breeding season. without implementing culling, nutrition, and heifer development changes in herds with flat pregnancy distributions similar to the herd depicted by figure 4, it is very difficult to positively influence the percentage becoming pregnant in first 21 days of subsequent breeding seasons. reasons that herds with a previously ideal calving distribution can deteriorate to a less-than-ideal situation includes animals too thin at calving, poor postpartum cowherd nutrition, subfertile bulls, or infectious or non-infectious pregnancy loss.5,7,8,9 bulls failed to deliver adequate amounts of fertile semen if reproductive performance is initially adequate indicating that conception occurred and pregnancy was maintained early in the breeding season, veterinarians can assume that fertile bulls were mating fertile, cycling cows, the herd was free of pregnancy wasting disease, and the postpartum anestrus period and energy reserves (as indicated by body condition score) were not problematic. a sharp decline in percent pregnancy success during the breeding season should initiate an investigation to identify potential testicular, reproductive tract, or musculoskeletal problems that prevented production or delivery of fertile semen or whether herd replacements brought in after start of breeding season could have introduced venereal disease. figure 5 illustrates a problem that is quite common in herds with 1 bull for each breeding pasture. although multiple-bull breeding pastures are more resilient to breeding failure due to bulls being unable to successfully breed cows compared to single-bull pastures, because of potential problems arising from injuries due to bull-on-bull fighting, social dominance by subfertile bulls, and isolation of groups of cows in extensive breeding pasture without 1 or more bulls present, multiple-bull pastures can also have poor reproductive efficiency due to bull problems and can have a pregnancy distributions similar to figure 5. figure 5. pregnancy distribution in a herd where a high percentage of cows are cycling at breeding season initiation and bulls are successfully breeding cows, but acute onset of bull infertility occurring late in the first 21 day period or early in the second 21 day period of the breeding season (e.g. injury, disease, etc.) resulted in reduced herd fertility followed by partial recovery. clinical theriogenology • volume 12 number 3 • september 2020236 the breeding season in this example (figure 5) starts with a high percentage of fertile cows cycling and good bull fertility. because 55% of the herd becomes pregnant in the first 21 day period, veterinarians can be confident that the prebreeding feeding/supplementation program offered adequate nutrients for a fairly high level of reproductive performance. it is also evident that bulls were able to cover the breeding pasture, find cows displaying estrus, and successfully breed cycling cows. the dramatic decrease in pregnancy percentage during the second 21 day period is strong evidence for bull infertility due to testicular or musculoskeletal insult.10 incremental increase in the percentage of available (nonpregnant) cows bred in each subsequent 21 day period in figure 5 indicates bull fertility is gradually returning. in a situation where veterinarians evaluated overall pregnancy percent but not pregnancy distribution for the herd depicted by figure 5, the percentage of nonpregnant cows would indicate herd fertility problems, although the cause of high nonpregnancy would not be evident. with limited information, nutrition or cow fertility could be suspected. and, as with this example, a bull may pass a breeding soundness examination both before and after being placed in the breeding pasture without revealing that a fertility problem existed during breeding season. by categorizing and displaying information gained at pregnancy diagnosis, the problem cause becomes more obvious. a bull breeding soundness examination (including a through physical examination) at the time the breeding season problem is discovered, may supply information about penile, testicular, foot and leg or other musculoskeletal problems that commenced during the breeding season. however, lack of identifiable pathology following the breeding season does not rule-out a physical (locomotion, mounting, intromission) or semen quality problem several weeks to months earlier. infectious or noninfectious agents prevented or ended pregnancy in situations when early pregnancy loss leads to negative pregnancy distribution effects, the problem occurred after breeding season initiation and before pregnancy diagnosis. in addition to the pregnancy distribution effect of pregnancy loss, in some situations, nonpregnant cows may exhibit palpable evidence of an involuting uterus at pregnancy diagnosis. noninfectious pregnancy loss very early in pregnancy due to environmental stress should not result in uterine pathology and would not be expected to have negative carry-over effects in the next 21 day breeding season period. in contrast, noninfectious pregnancy loss after maternal recognition of pregnancy (~ 13 days after estrus) will delay returns to normal fertility until after the embryo is resorbed or expelled and hypothalamic-pituitary axis has resumed normal estrous cycle activity – which may be later than the 21 day period following initial conception. early, noninfectious pregnancy loss that occurs before pregnancy is detectable by transrectal palpation or ultrasonography is unlikely to be differentiated from failure to conceive. infectious pregnancy loss may result from fertilization failure or very early embryonic death; therefore, transrectal palpation or ultrasonographic examination is indistinguishable from failure to conceive or early noninfectious pregnancy loss. however, because many common causes of infectious pregnancy loss in north america have peak incidence after mid-gestation when pregnancy is typically diagnosed, it is expected that palpable evidence of previous pregnancy will be detected. the length of time that palpable evidence would be evident is influenced by the stage of pregnancy at the time of pregnancy loss and whether or not the loss was accompanied by uterine pathology. infection with trichomonas foetus (trich), a protozoa transmitted during breeding, is an important cause of pregnancy loss in north america because it is diagnosed in many cattle-dense areas and can cause a high percentage of exposed cows to resorb or abort their pregnancy. the pregnancy distribution of a herd infected with trich will vary depending on what the distribution would have been without infection and the timing of trich introduction. if trich entered herd prior to breeding season initiation so that a high percentage of bulls are already infected, cows will become pregnant at a time similar to last year’s breeding season, but infected cows are likely to lose their pregnancies ~ 15 80 days into pregnancy. a period of female infertility is expected to last for another 2 6 months as a result of infection. the magnitude of loss is expected to clinical theriogenology • volume 12 number 3 • september 2020 237 approach 30 50% of exposed cows. however, if trich entered the herd during breeding season or fewer bulls were infected at the start of breeding season but the number of infected bulls increased as the breeding season progressed, then the pregnancy distribution is greatly influenced by what the distribution would have been without trich exposure, and the speed at which additional bulls became infected. other causes of early pregnancy loss (e.g. campylobacter fetus ss venerialis, bluetongue virus, leptospira borgpetersenii serovar hardjo type hardjobovis, bovine viral diarrhea virus) will have a similar effect on the pregnancy distribution, although magnitude of pregnancy loss is not expected to be as high as with trich.11,12 infectious and toxic causes of pregnancy loss commonly expressed in mid to late pregnancy include: bovine herpes virus 1 (infectious bovine rhinotracheitis), bovine viral diarrhea virus, neospora caninum, leptospira sp., pine-needle toxicosis, and others.13,14 pregnancy losses in mid to late pregnancy are likely to occur after pregnancy diagnosis time and the effect is not limited to a single period of the pregnancy distribution. if pregnancy losses occurred prior to pregnancy diagnosis, evidence of that loss is likely to be apparent during transrectal examination of the nonpregnant uterus of some of the affected cows. second-level analysis of pregnancy data to capture more information from fetal aging, the distribution of breeding dates can be analyzed not only by 21 day intervals, but also by category within 21 day intervals. for example, the herd depicted in figure 6 has a pregnancy percentage of 94.5%, which meets the goal for a 63 day breeding season. in addition, 61.8% of the herd became pregnant during the first 21 days of the breeding season – which exceeds the 60% cutoff associated with good cow and bull fertility at breeding season initiation. from these observations, one could classify this herd as having normal fertility, with no nutritional or reproductive management problems. looking at percent pregnancy success, during the first 21 days of the breeding season, 62% of available cows became pregnant. in the second 21 days, 55.6% of nonpregnant cows became pregnant; which is 21% of the herd. in the third 21 days, 68% of available cows became pregnant; which is 11.5% of the herd. these measures of reproductive success are not alarming, but there is an indication that fertility may be suboptimal during the second 21 days and closer examination of data is warranted. if data collected at pregnancy diagnosis for the herd depicted in figure 6 are further analyzed by age categories for each 21-day period (figure 7), the pregnancy distribution for primiparous cows indicates a clear problem. diagnostic information provided by percent pregnancy success indicates that whereas mature cows perform very well throughout the breeding season and primiparous cows performed well during in first 21 days, pregnancy success dramatically decreases during the second 21-day period for primiparous cows before returning to 70% for the final 21 days of breeding. that primiparous cows performed well the first 21 days of the breeding season is important to recognize because my bias when primiparous cows perform worse than mature cows is that the deficit is because it took them longer to begin fertile cycles after calving and they performed poorly early in the breeding season. however, information provided by the pregnancy analytics app for this herd indicates that primiparous cows did not have delayed return to estrus; in fact, the problem was confined to the second 21 day period. without second-level analysis, i would probably assume that heifers were too thin when they calved or that the producer should move the heifers’ breeding season so that they could calve earlier than mature cows. because of information provided by second-level analysis of data collected at pregnancy status determination, i am able to identify “which” cattle were not pregnant and “when” during the breeding season fertility was reduced; for this example, i am most interested in investigating bull issues confined to the second 21 day period of the breeding season. having this type of analytics available immediately after pulling the palpation sleeve off is not diagnostic per se. however, as i am talking to a producer, i can confine my history questions to primiparous cows during the second 21 days of the breeding season (which for this herd would have been the last 2 weeks of june through the first week of july). if i pursue diagnostic testing, i will focus my testing on bulls in the primiparous cow breeding pasture. clinical theriogenology • volume 12 number 3 • september 2020238 figure 6. herd with a good pregnancy distribution that has a hidden problem figure7. second-level analysis of pregnancy data from a herd with suboptimal fertility not apparent when evaluating overall herd pregnancy distribution. conclusion information gathered at pregnancy diagnosis is very valuable to both veterinarians and beef producers, particularly if fetal age is estimated within 21 day periods. despite the importance of reproductive performance to cowherd profitability and sustainability, without an efficient and convenient method to collect and analyze pregnancy status data, that value is difficult to capture. nutrition, genetics, animal husbandry, reproductive soundness and health influence distribution of pregnancy within a herd. by combining transrectal palpation or ultrasonographic imaging of the reproductive tract to determine pregnancy status with analysis and graphing of information, veterinarians can identify when during the breeding season which specific categories of cows did not become pregnant. the pregnancy analytics app allows a veterinarian’s knowledge and skill to be augmented by efficient digital data entry and rapid generation of commonly used herd reproductive assessments to enhance communication between veterinarian and producer. more information about the bci pregnancy analytics app is available at https://ksubci.org/pregnancy-analytics-mobile-app/ clinical theriogenology • volume 12 number 3 • september 2020 239 conflict of interest there are no conflicts of interest to declare. references 1. spire mf: breeding season evaluation of beef herds. in: howard jf: editor, current veterinary therapy, 2nd edition, philadelphia; wb saunders: 1986, p. 808. 2. randle rf: production medicine considerations for enhanced reproductive performance in beef herds. vet clin n am 1993:9:404-415. 3. bretzlaff k: a pictorial guide to bovine pregnancy diagnosis. vet med 1987;82:295-304. 4. kasari t, gleason d: herd management practices that influence total beef calf production – part i. comp cont ed pract vet 1996;18:823-832. 5. richards mw, spitzer jc, warner mb: effect of varying levels of postpartum nutrition and body condition at calving on subsequent reproductive performance in beef cattle. j anim sci 1986;62:300-306. 6. short re, bellows ra, staigmiller rb, et al: physiological mechanisms controlling anestrus and infertility in postpartum beef cattle. j anim sci 1990;68:799-816. 7. selk ge, wetteman rp, lusby ks, et al: relationships among weight change, body condition and reproductive performance of range beef cows. j anim sci 1988;66:3153-3159. 8. spitzer jc, morrison dg, wettemann rp, et al: reproductive response and calf birth and weaning weights as affected by body condition at parturition and postpartum weight gain in primiparous beef cows. j anim sci 1995;73:1251-1257. 9. utter sd, houghton pl, corah lr, et al: factors influencing first-service conception and overall pregnancy rates in commercial beef heifers. ks agri exp sta contribution no. 94-373-s; 1994, p. 107-110. 10. barth ad, oko rj: abnormal morphology of bovine spermatozoa. ames; iowa state university press: 1989. p.130-192. 11. larson rl: evaluating information obtained from pregnancy examination in beef herds. vet med 1999;94:566-576. 12. bondurant rh: selected diseases and conditions associated with bovine conceptus loss in the first trimester. theriogenology 2007;68:461-473. 13. larson r: diagnosing the cause of bovine abortion and other perinatal deaths. vet med 1996;91:478-486. 14. anderson ml: infectious causes of bovine abortion during midto late-gestation. theriogenology 2007;68:474-486. clinical theriogenology • volume 12 number 3 • september 2020240 014_ms-014 r.larson 1 color figures 3,6,7 contact margaret v. root kustritz rootk001@umn.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9596, http://dx.doi.org/10.58292/ct.v15.9596 52 research report comparison of accuracy of machine-generated or human generated captions of zoom live lectures in a comparative theriogenology course margaret root kustritz,a ryan rupprecht,b perle zhitnitskiyc adepartment of veterinary clinical sciences, university of minnesota college of veterinary medicine, st. paul, mn, usa boffice of academic and student affairs, university of minnesota college of veterinary medicine, st. paul, mn, usa cdepartment of veterinary population medicine, university of minnesota college of veterinary medicine, st. paul, mn, usa abstract captions are available with captured lectures for student review. in this study, automatically generated captions from zoom, kaltura, and youtube were compared for accuracy with captions generated by a human being. also investigated was the effect of speaker on accuracy of captioning – does the speed with which someone speaks or their accent alter accuracy of captioning? youtube was by far the most accurate of the automatic captioning systems. there were numerous mistakes made by zoom and kaltura and some significantly altered meaning. mistakes were due to the transcribing systems, not to things specific to the presenters. instructors should get in the habit of reviewing tran scripts of their lectures to ensure students are not misled.  keywords: accommodations, accessibility, public speaking introduction lecture capture is a widely accepted tool in health sciences education. lecture capture systems vary from simple cameras on a tripod with a microphone to pick up the speaker’s voice; to a webcam and computer microphone to capture lectures in a digital conference system; to extensive systems that capture audio, a video following the speaker as they move or demonstrate things, and the visual displayed through the lecture hall projector. students use captured lectures to permit them to listen to lectures that they were unable to attend in-person or at presentation, to ensure their own notes are accurate, to review complex or confusing subjects as they study, and to prepare for examinations.1 23 –4 availability of captions permits students to ‘watch’ the video in spaces where they cannot play the audio aloud (e.g. library or other quiet study space).5 students appreciate the flexibility this tool affords and feel that it supports their wellbeing.6,7 many of these lecture capture systems also create captions, either in real-time or after the video is saved. captions generally are created as transcribed text of spoken information, not inclusive of audio tracks from videos or other audible content not provided by the presenter or participants and may be time-stamped. captions are beneficial for student learning in many ways. historically, captions were provided for students with specific learning needs (e.g. hearing loss) but it has been demonstrated that provision of captions can enhance learning in students with a variety of other disabilities including autism spectrum disorder, attention deficit hyperactivity disorder, or dyslexia.5 a written transcript is also helpful for those students for whom english is not their first language.3 this may be increasingly necessary as we strive to create a more diverse student body, which may be associated with increasing responsiveness to accessibility concerns.4 students without a diagnosed disability also benefit from captioning. acoustic and visual stimuli are processed in different areas of the brain and taking in information through both channels can strengthen learning as higher level neurologic processes must be employed to integrate and encode these different inputs into a coherent piece of knowledge.1,8,9 joint presentation of auditory and visual data increases comprehension compared to presentation of auditory data alone.9 captioning is accomplished by a human or through an automated system. use of any system may help to overcome certain limitations of the speakers; some literature suggests that mailto:rootk001@umn.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9596 citation line: clinical theriogenology 2023, 15, 9596, http://dx.doi.org/10.58292/ct.v15.9596 53 students may benefit from having a written transcript if the speaker has an accent or speaks very quickly or if audio quality is poor.5,10–12 human captioners are very accurate, with reported accuracy of over 95% for those with some level of familiarity with the content they are captioning and only a slightly lower accuracy (93%) for those who are less familiar with the content.13,14 a human being can readily adjust to a speaker’s accent, speed of presentation, and volume, and can work around background noise and other distractions.12 a human captioner could also ask for things known to be more difficult to catch in a lecture and to be provided with these ahead of time, for example, technical terms, proper nouns, and acronyms or other terms that contain letters or numbers.15 human captioners can be very accurate if they caption after production, where they are watching a saved video and thus can slow it down or speed it up as needed and repeat sections where the speech is unclear. human captioners also ensure that the spelling, grammar, and punctuation are accurately transcribed so as to create an easily readable document.11 human captioning is expensive and may require scheduling such that there is substantial lapse of time between presentation of the lecture and availability of captions, limiting timely review of lectures by students. speech recognition systems are used for automatic captioning and vary greatly in accuracy depending on the system used and the level of specialized terminology in the discipline of the presentation. automatic captioners are not generally as accurate as humans and do not produce documentation as an easily readable document, with some using no punctuation at all or otherwise providing a ‘stream of consciousness’ of information that may be difficult for novices to understand out of context.16,17 speech recognition systems may or may not include fillers (‘um’ or ‘er’) and transcribe words that sound the closest to what was said whether or not their transcription makes any sense. discipline-specific terminology is not well transcribed and may be presented in a manner that changes the intended meaning.13 automatic captioning is less expensive than human captioning and can be done in real-time or after production without need for scheduling so the lecture and associated captions can be made available soon after the presentation. at our college, lecture capture has been used for over 10 years. over that time, faculty have become more accepting of the value of this tool and students have come to expect that all lectures will be captured. technology used also has varied over time. evaluation of new technology options was underway at the onset of the covid-19 pandemic when our college had to make an abrupt switch from in-person to virtual lectures. the digital conference system that was supported by the university was zooma and our college has continued to use zoom as the pandemic had its course. currently, lectures are offered either in-person and on zoom concurrently or completely on zoom. zoom lectures are available to students and faculty with captions generated by kalturab; lectures are uploaded within 24 hours but captions may take longer to load dependent on usage of the system, which is shared by colleges across the university. kaltura captions are automatically appended to these videos using automatic speech recognition, which is described by kaltura as up to 90% accurate, and are posted in canvas,c the learning management system of our college. this is the a.https://zoom.us/ b.https://corp.kaltura.com/ c.https://www.instructure.com/canvas default captioning method at our college. faculty are recommended by kaltura to review and edit their captions; at present, our college does not require this, and it is not known how many faculty review their captions for accuracy. zoom also creates captions if requested by the presenter, and these are visible in real-time and are sent to the presenter or host of the meeting as a text file after the session is ended. videos also can be uploaded to youtubed and captions generated through that website; however, this is not commonly done at our college. captions created using kaltura, zoom, and youtube are generated by machine learning algorithms and details of these systems are proprietary. machine learning algorithms are subject to limitations in quality due to how their algorithm is built, vocabulary available within the captioning system, effect of background noise, the clarity and volume of the presenter’s voice, presence of multiple overlapping voices, mispronunciations by the speakers, and accents including dialects. comparative theriogenology is a 3-credit course offered in third year spring. a variety of instructors participate in the course. lectures and review sessions are offered live via zoom and those sessions are recorded. the lectures and associated captions generated by kaltura are posted in the course canvas site for student access. the goal of this study was to evaluate what factors may make automatic captioning more or less accurate for a cohort of instructors in a course. materials and methods data were collected from 6 instructors in the course. for each, a 25-minute segment from the beginning of their presentation (after initial greetings and assurances that the technology was working) was evaluated for number of homonym errors, number of other errors, and number of medically significant errors, and speaking rate (words per minute [wpm]) with and without repeaters (e.g. ‘um’ and ‘er’). an accurate transcript was created for each presentation by the first author after listening to each presentation at 0.5 speed and at full speed. speaking rate was calculated from the corrected transcript. machine-generated transcripts were generated in kaltura, zoom, and youtube. a trained captioner who was not a medical expert created the human-generated transcripts.e youtube and zoom generated transcripts with time stamps and no punctuation. kaltura and the human captioner generated documents that looked more like course notes, with punctuation. the first author compared the transcripts from kaltura, zoom, youtube, and the human captioner to the accurate transcripts. homonyms were not counted as errors in any system. errors included missed words, additional words, and incorrect words. a given error was counted either as an overall error or a medically significant error; the latter changed the meaning in ways that would have taught the reader something that was medically incorrect. comparison of characteristics of speakers by captioning system was analyzed by either the paired or unpaired student’s t-test. correlation between speaking rate and number of errors was evaluated by calculation of the pearson correlation coefficient. overall errors and medically significant errors by system for all speakers were analyzed using anova and the paired t-test. significance was set at p < 0.05. d.https://www.youtube.com/ e.themaritimer at fiverr http://dx.doi.org/10.58292/ct.v15.9596 https://zoom.us/ https://corp.kaltura.com/ https://www.instructure.com/canvas https://www.youtube.com/ http://themaritimer at fiverr 54 citation line: clinical theriogenology 2023, 15, 9596, http://dx.doi.org/10.58292/ct.v15.9596 results speaker characteristics are summarized (table). there were no differences in number of overall errors in any system for men versus women, or for those for whom english was their first language versus those with a different first language. for those with accents versus those without accents, those with accents had fewer (p = 0.03) overall errors than those without only in kaltura. there was no demonstrated correlation between words per minute and number of overall errors or number of medically significant errors.  number of overall errors was much larger than the number of medically significant errors. the human captioner and youtube were not different and both were superior to zoom and kaltura, both for number of overall errors (p < 0.0001) and number of medically significant errors (p = 0.0003) (figures 1 and 2). an example of a significant error was the following – response when students were asked the 3 most common postpartum disorders in dogs – ‘hypercalcemia, meteorities, and mastodons’ or ‘hypoglycemia, detritus, and mastitis’ – should be ‘hypocalcemia, metritis, and mastitis’. medical and scientific terms were commonly poorly transcribed but common words also were poorly transcribed including sow (south, cell, stylist) and milk (nuke, pill, melt). favorites were ‘evil looting’ for involution and ‘arctic eye shadow’ for dr. caixeta. discussion the default captioning system used by our college is much less accurate than one other commercial system or the human captioner. knowing the extent of inaccuracy of the current system is valuable in helping the college plan for what technologies are best supported for lecture capture and captioning. this study was limited in that only 6 individuals were evaluated and only 1 lecture was evaluated for each. more accurate data may have been generated by a wider data collection. creating the gold-standard transcript and comparing all others to that gold-standard was very time-consuming; specific effort would have to be granted for a greater breadth of data collection. another limitation is a practical one regarding the need for these data and future data collection. we do not have an easy way to determine how many students access captions or transcripts or how frequently a given student does so. if few students are using the captions because of their inaccuracy, we are missing a great opportunity to help support student learning but if many are accessing the captions and are managing despite the inaccuracies, we may be creating a solution for a problem that does not exist. finally, our college is part of a larger unit and has somewhat limited capability to say what technologies it will and will not support, so any conversations about changing from the default system currently, will require extensive documentation and discussion. among the different transcript-generating software evaluated in this study, youtube was the most accurate. this is in alignment with previously published literature that reported a 98% total accuracy when youtube was used for the transcript of a college-level recorded lecture.18 when comparing the accuracy of transcription, there were no significant differences based on the presenter’s gender; this result was unexpected and at odds with a previous report of significant decrease of accuracy when the speaker was female.19 this difference may be due to the limited sample size in this study. figure 1. mean number of overall errors per captioned lecture by captioning system figure 2. mean number of medically significant errors per captioned lecture by captioning system table. instructor characteristics characteristic speaker 1 speaker 2 speaker 3 speaker 4 speaker 5 speaker 6 male/female m f m f m f english as first language n y y y y n accent? y n y n n y words per minute 146 165 179 199 158 124 fillers/100 words 4.7 4.9 2.6 0.2 1.3 10.3 http://dx.doi.org/10.58292/ct.v15.9596 citation line: clinical theriogenology 2023, 15, 9596, http://dx.doi.org/10.58292/ct.v15.9596 55 this study did not evaluate whether speaking very quickly or having a strong accent alters student learning. a study evaluating speed of speech demonstrated that students were more engaged with presenters spoke moderately quickly (172 wpm) or very quickly (213 wpm) compared to when they spoke slowly (116 wpm).20 understanding the spoken word requires one to map acoustic sensory input to stored representations of sounds, grammar, and syntax, and listening to someone with a strong accent can require more cognitive effort and slow one’s ability to understand.21 speakers who use more than 1.3 filler words per 100 content words are deemed by listeners as less credible, which may impair engagement and learning.22 only 1 of the 6 speakers in this study was below that limit, with most speakers well above that limit. in this study, characteristics of the speakers were not associated with changes in captioning. speakers who know they speak slowly or extremely quickly, have a strong accent, or use many filler words may wish to strongly encourage students to use captions to ensure they are fully understanding what was presented. multiple studies have identified that the availability of captions is associated with increased focus, increased understanding, better retention, and overall higher academic achievement.4,23–25 caption transcripts also are searchable that makes finding specific parts of lectures to be rewatched more efficient for learners.1,23 concerns have been expressed about cognitive load on students required to read captions while watching and listening to a video but students surveyed in a study did not report feeling undue pressure from this activity.24 caption transcripts may be generated without punctuation or other attention to what makes for a readable document. in one study, transcripts were shown to be most valuable for learners if they were read while the students were hearing the video.1 some authors suggest that poor captions are worse than no written transcript at all as they may confuse the student regarding what is correct and trying to read the poorly transcribed wording may distract the student from hearing important information.4,11 all transcripts should be evaluated for accuracy and edited as needed; faculty must be given time to complete this as part of their teaching effort. some literature describes students, either individually or collectively, doing this editing as a part of the course in which they are enrolled.17,26 in this study, human captioning was not statistically different from youtube but the mean number of overall errors was lower for youtube (168) than for the human captioner (191). technology is improving and at least one study has already demonstrated automatic captioning that is as accurate as human captioning.27 organizations should pay attention to advances in automatic captioning systems to take advantage of improved technology. conclusion projections in education are that more virtual training and more use of technology in all aspects of education will be the norm as we emerge from the covid-19 pandemic.28,29 this means that technologies such as captioning will need to be better understood and resources put toward making them as efficient and useful as possible. in this study, mistakes were due to the transcribing systems, not to specific characteristics of the presenters. however, speakers can improve student engagement and decrease cognitive load by watching how quickly they speak, by practicing their speech to minimize the number of filler words used, and to encourage students to use caption transcripts when reviewing captured lectures. all transcribing systems made some mistakes and instructors should get in the habit of reviewing transcripts of their lectures to ensure students are not misled. students can best use these transcripts to search for specific areas of lectures to be rewatched, and by reading the transcript while hearing the presentation. conflict of interest none. authors’ contribution dr. root kustritz conceptualized the study, identified the resource for human evaluation of captions, gathered data from transcripts and analyzed, drafted the paper, and completed revisions requested by the reviewer. mr. rupprecht uploaded videos to youtube and downloaded captions, investigated mechanisms used by mechanical captioning systems, and reviewed the paper before submission. dr. zhitnitskiy provided input on the study and provided an 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from: https://canadianaudiologist.ca/improving-accessibility -with captioning-an-overview-of-the-current-state-of-technology [cited 15 july 2022]. 12. lasecki ws, bigham jp: real-time captioning with the crowd. acm digital library: interactions 2014;21:50–55. doi: 10.1145/ 2594459 13. lasecki ws, miller cd, sadilek a, et al: real-time captioning by groups of non-experts. proceedings of the 25th annual acm symposium on user interface software and technology, october 2012; 23–34. doi: 10.1145/2380116.2380122 14. wald m: creating accessible educational multimedia through editing automatic speech recognition captioning in real-time. interact tech smart educ 2006;3:131–141. doi: 10.1108/17415650 680000058 15. takeuchi y, kojima d, sano s, et al: detection of input-difficult words by automatic speech recognition for pc captioning. in: miesenberger, k., kouroupetroglou, g. (eds) computers helping people with special needs. icchp 2018. lecture notes in computer science, vol 10896. springer, cham. doi: 10.1007/ 978-3-319-94277-3_32 16. kent m, ellis k, latter n, et al: the case for captioned lectures in australian higher education. tech trends 2018;62:158–165. doi: 10.1007/s11528-017-0225-x 17. borgaonkar r: captioning for classroom lecture video. thesis: university of houston: 2013. 18. millett p: accuracy of speech-to-text captioning for students who are deaf or hard of hearing. j educ ped rehab audiol 2021;25: 1–13. 19. tatman r: gender and dialect bias in youtube’s automatic captions. proc acl workshop on ethics in natural language processing, valencia; association for computational linguistics: 2017;53–59. 20. simonds bk, meyer kr. quinlan mm, et al: effects of instructor speech rate on student affective learning, recall, and perceptions of nonverbal immediacy, credibility, and clarity. comm res report 2006;23;187–197. 21. van engen kj, peelle je: listening effort and accented speech. front hum neurosci 2014;8:577. doi: 10.3389/fnhum.2014.00577 22. duvall e, robbins a, graham t, et al: exploring filler words and their impact. schwa lang linguistics 2014;11:35–49. 23. ellis k, kent m, peaty g: captioned recorded lectures as a mainstream learning tool. m/c j 2017;20. doi: 10.5204/mcj.1262 24. whitney m, dallas b: captioning online course videos: an investigation into knowledge retention and student perception. minneapolis, mn: proc acm technical symposium on computer science education; 2019. 25. ranchal r, taber-doughty t, guo y, et al: using speech-recognition for real-time captioning and lecture transcription in the classroom. ieee transactions learn technol 2013;6:299–311. doi: 10.1109/tlt.2013.21 26. wald m: using speech recognition transcription to enhance learning from lecture recordings. international conference on education and new developments, budapest, hungary. 23–25 jun 2018;111–115. 27. xiong w, droppo j, huang x, et al: achieving human parity in conversational speech recognition. microsoft technical report. msr-tr-2016-71, 2017; available from: https://www.microsoft. com/en-us/research/wp-content/uploads/2016/11/ms_parity.pdf [cited 10 may 2023]. 28. guppy n, verpoorten d, boud d, et al: the post-covid-19 future of digital learning in higher education: views from educators, students, and other professionals in six countries. br j educ technol 2022;53:1750–1765. doi: 10.1111/bjet.13212 29. pew research center. experts say the ‘new normal’ in 2025 will be far more tech-driven, presenting more big challenges, 2021. pewresearch.org/internet/2021/02/18/experts-say-the-new normal-in-2025-will-be-far-more-tech-driven-presenting-morebig-challenges/. http://dx.doi.org/10.58292/ct.v15.9596 https://doi.org/10.1080/0020739x.2016.1238518 https://canadianaudiologist.ca/improving-accessibility​-with-captioning-an-overview-of-the-current-state-of-technology https://canadianaudiologist.ca/improving-accessibility​-with-captioning-an-overview-of-the-current-state-of-technology https://doi.org/10.1145/2594459 https://doi.org/10.1145/2594459 https://doi.org/10.1145/2380116.2380122 https://doi.org/10.1108/17415650680000058 https://doi.org/10.1108/17415650680000058 https://doi.org/10.1007/978-3-319-94277-3_32 https://doi.org/10.1007/978-3-319-94277-3_32 https://doi.org/10.1007/s11528-017-0225-x https://doi.org/10.3389/fnhum.2014.00577 https://doi.org/10.5204/mcj.1262 https://doi.org/10.1109/tlt.2013.21 https://www.microsoft.com/en-us/research/wp-content/uploads/2016/11/ms_parity.pdf https://www.microsoft.com/en-us/research/wp-content/uploads/2016/11/ms_parity.pdf https://doi.org/10.1111/bjet.13212 http://pewresearch.org/internet/2021/02/18/experts-say-the-new-normal-in-2025-will-be-far-more-tech-driven-presenting-more-big-challenges/ http://pewresearch.org/internet/2021/02/18/experts-say-the-new-normal-in-2025-will-be-far-more-tech-driven-presenting-more-big-challenges/ http://pewresearch.org/internet/2021/02/18/experts-say-the-new-normal-in-2025-will-be-far-more-tech-driven-presenting-more-big-challenges/ use of frozen semen in bitch use of frozen semen in bitch stuart mason monash veterinary clinic, oakleigh east, victoria, australia abstract frozen semen was first used successfully in the bitch in 1969. initially semen was inseminated by vaginal insemination and subsequently, intrauterine techniques were used, achieving higher pregnancy rates than vaginal insemination. for optimal results with freezing and using frozen semen, careful collection, assessment, freezing and handling techniques must be employed. it is important when inseminating frozen semen to consider extender constituents and to make appropriate dilutions with thaw media to reduce glycerol concentrations in the extender to safe levels. however, a difficulty with using frozen semen in dogs is that many extender recipes are proprietary and constituents are unknown. poor handling of frozen semen straws (through relabelling and holding straws out of liquid nitrogen for > 15 20 seconds) detrimentally affect both motility of thawed sperm and membrane integrity and are likely to reduce pregnancy rates keywords: frozen semen, bitch, canine, artificial insemination introduction frozen semen was first used successfully in the bitch in 1969.1 initially semen was inseminated by vaginal insemination,2 but subsequently, intrauterine techniques were utilised, resulting in higher pregnancy rates than vaginal insemination.3-5 initially insemination by laparotomy under general anaesthesia was utilised; however, in 1972, kell andersen developed a non-surgical intrauterine insemination technique using the norwegian catheter.6 as fresh and fresh chilled semen have higher pregnancy rates than frozen semen, the latter should be reserved for situations wherein use of fresh or chilled semen is impossible or not viable. since the first successful frozen semen insemination, its use has increased and pregnancy rates have improved. in most parts of the world, frozen semen is used to introduce new genetics from deceased dogs or dogs in remote countries. in some countries, such as australia and new zealand, there are strict quarantine laws precluding and inhibiting importation of dogs or semen. testing of animals to enter such countries is a protracted process, making it uneconomical and not in the best welfare interests of the bitch or dog to move for a short interval for breeding. chilled semen is ruled out due to testing requirements and so frozen semen is the only viable option. there are several contributing factors to a successful pregnancy with the use of frozen semen, including fertility of the dog, collection method, freezing method, transport and handling of the semen, fertility of the bitch, timing of the bitch’s cycle, and insemination method and the operator responsible for any of these procedures.7-9 many of these factors are impossible to control; however, some factors are within the control of the people involved and how these operations are performed can dramatically affect the end result of the insemination. semen collection ideally only proven fertile males should have their semen frozen. peak fertility of male dogs is believed to be between 2 and 5 years of age (observed, unpublished data by author), so it is recommended to collect dogs for freezing between these ages. the male should be prevented from urinating in the immediate interval preceding collection, as urination during this time will increase the chance of urospermia during semen collection. semen should be collected from the male in the presence of an estrous bitch for best results. a comfortable and relaxed environment should be provided for semen collection. adequate time for the male and female to become acquainted is important for both the dogs to relax and also for the male to become adequately stimulated. allowing the male to mount the bitch may be beneficial and the male should be minimally restrained with minimal distractions. manual stimulation of the bulbus glandis through the prepuce and gentle stimulation of the tip of the penis will help stimulate erection and ejaculation. the prepuce should be reflected back over the bulbus glandis by the operator, as obtaining an erection with the bulbus glandis within the prepuce can lead to significant pain which would be expected to reduce semen quality. the dog ejacuates in 3 fractions and the second, semen-rich fraction, should be separated 197 clinical theriogenology • volume 11, number 3 • september 2019 from the other 2 prostatic fractions. poorly fractionated samples may be centrifuged and washed before freezing, although it is believed that excessive manipulation of semen may have negative impacts on fertility. semen assessment a difficulty with freezing semen is adequately assessing a sample as suitable to freeze before knowing true parameters. it is not recommended to utilise semen with raw semen values of less than 70% motility, 60% normal morphology, or samples contaminated with blood, pus or urine. in most cases, motility of dog sperm appears to decrease by 20 30% from prefreezing values, so a rough calculation should be performed based on total number of sperm, initial motility (to predict motility of thawed sperm) and approximate morphology values to determine if there will be any usable samples after freezing. sperm concentration and motility the sperm-rich fraction is accurately measured to ascertain the volume collected (in general 0.5 -1.5 ml). an aliquot of semen is then taken and diluted to enable accurate assessment of total number of sperm and motility. the author will take 10 µl of the semen rich fraction and add it to 180 µl of thaw media extender (andropro ai thaw, minitube, verona, wi) to obtain a 1:9 dilution. the aim is to end up with a concentration of <50 million sperm/ml to allow for the sperm to be ‘free moving’ and obtain a more accurate motility value and total count. dilution rate may be altered depending on the concentration of the original sample. to aid in dilution, approximate sperm concentrations based on colour/density of the gross sample are shown in table 1. table 1. approximate sperm concentration rates of canine semen samples by gross appearance. creamy, grainy semen 750 1500 x 106 sperm/ml full cream milk like semen 400 750 x 106 sperm/ml skim milk like semen 250 400 x 106 sperm/ml translucent semen < 250 x 106 sperm/ml the author uses a computer assisted semen analysis (casa) system (spermvision, minitube, verona, wi) to determine concentration and motility. other options include a hemocytometer, makler chamber, spermacue, isperm, other casa systems and the nucleocounter sp200. for the hemocytometer, makler chamber and spermacue, sperm motility assessment is made via visual estimation which may counteract any sperm concentration accuracy. all sperm counting systems have their limitations and must be used appropriately for most accurate results.10, 11 total number of sperm in the sample are recorded, as is progressive motility (sperm that swim their body length in 1 second) not total motility (sum of progressive and non-progressively motile sperm). sperm morphology sperm morphology may be obtained using a wet mount prep or stained slide. a minimum of 100 sperm should be assessed and each sperm categorised into normal and their specific abnormal form. sperm with multiple abnormalities are classically identified by the abnormality closest to the sperm head. the minimum acceptable standard for canine sperm morphology is 60% normal sperm with no more than 20% of any 1 type of abnormality. motility of thawed sperm values are proportional to percentage of normal sperm and also initial raw motility values. sperm viability sperm viability (membrane integrity) is becoming easier to assess in practice using the hyperosmotic swelling test (host), live/dead eosin nigrosin stains or fluorescent microscopy with viability stains (propridium iodide and sybr green). membrane integrity is believed to be related to semen fertility.12 semen samples with higher percentage viability should freeze better. semen freezing canine semen is frozen in either straws or pellets. extenders used to freeze semen are usually a 1 or 2 step process, with the latter being more common. sperm frozen at 200 x 106/ml is likely to 198clinical theriogenology • volume 11, number 3 • september 2019 yield better results for thawed samples than other concentrations.13 semen extenders contain a sugar source (usually fructose), a buffer to control ph, antibiotics (to protect the medium from growth, not prevent infection in the bitch), a membrane stabiliser (egg yolk or skim milk) and a cryoprotectant (glycerol). glycerol is one of the most important constituents to consider with using frozen semen effectively. to protect semen from freeze-thaw damage, extender needs to contain a minimum of 4 % glycerol.14 however, once thawed for insemination, if the glycerol concentration is > 2.5%, longevity of thawed sperm is reduced.13 there are several commercial extenders with differing freeze protocols that need to be followed. unfortunately, most are proprietary recipes and concentration of glycerol is not disclosed. some of the more common canine semen freezing extenders and glycerol concentrations of those that are known are shown in table 2. table 2. semen extenders and glycerol concentration. extender glycerol (%, w/v) clone unknown canirep (uppsala) 7.5 canipro (minitube, verona, wi) 4 synbiotics (zoetis) unknown synthetic (imv) 8 triladyl 4 camelot unknown icsb unknown semen freezing extenders which are frozen containing egg yolk before distribution appear to result in more sperm agglutination than those which utilise fresh egg yolk addition at the time of freezing (observed unpublished data by author). each semen freeze extender has a recipe that must be followed to create extended semen ready for freezing. whilst semen and extenders are equilibrating, straws or vials need to be prepared and permanently labelled for later identification. it is recommended to label straws or vials with the dog’s name, breed, microchip number and date in long format (e.g. 15th january 2019). semen freezing in pellets dry ice is utilised to freeze semen. small holes are made in the surface of the dry ice. the extended semen sample is distributed 1 drop at a time into each well using a glass pasteur pipette, whilst continually mixing the extended semen to ensure sperm don’t settle. each drop will snap freeze to form a pellet, which are then transferred off the dry ice into liquid nitrogen. one pellet is thawed in thaw media and motility assessed. once this is done, the number of pellets required for an insemination can be calculated: [total number of sperm x % morphologically normal sperm x motility after thawing (%)]/number pellets. pellets are then distributed into separate vials, with each vial being intended for 1 insemination. semen freezing in straws semen and extender mix are refrigerated to 4°c. labelled straws are also equilibrated to 4°c. each straw is filled with 0.5 ml of extended semen and the fluid is brought up to the wick of the straw to proceed to the cement. the other end of the straw is sealed and then the straw is given a short flick to move the air bubble from the sealed end to the centre of the straw (this prevents straws from exploding on thawing). thawing semen for insemination pellets pellets are emptied into a whirl pack bag containing either 1 ml of saline or thaw media in a 37°c water bath. pellets are massaged through the bag to encourage thawing. the author uses a step thaw technique, wherein pellets are added to 1 ml of warmed thaw media (ai thaw, minitube, verona, wi) and then an additional 2 ml of thaw media is slowly added. this technique slowly changes osmolarity of the semen extender and improves motility of thawed sperm.13 199 clinical theriogenology • volume 11, number 3 • september 2019 straws straws may be thawed at 37°c for 1 minute or 70°c for 8 seconds, with the latter resulting in greater longevity of thawed samples.15 thawed semen is then emptied into a whirl pack bag containing thaw media at 37°c. the amount of thaw media to use is recommended by the extender manufacturer and should come as instructions with semen. however, as semen often arrives with no paperwork, the amount of thaw media used for various extenders by the author is shown in table 3. table 3. volume of thaw media (andropro ai, minitube, verona, wi) used by author when inseminating frozen semen. extender volume andropro ai (ml) per straw clone 1.5 canirep (uppsala) 1.5 canipro (minitube, verona, wi) 0.5 synbiotics (zoetis) 1-1.5 synthetic (imv) 1.5 triladyl 0.5 camelot 3 (1 then 2 ml step thaw) icsb 3 (1 then 2 ml step thaw) insemination of frozen semen it is universally accepted that for best results, frozen semen should be inseminated intrauterine in bitches.3,8,9,16,17 whilst tci is more successful than surgical intrauterine insemination if performed appropriately,8 the method of insemination should be 1 with which the operator is most confident. it is accepted that the lifespan of frozen semen is indefinite while frozen. however, as sperm are not dead, there will still be consumption of energy sources from the frozen semen extender and accumulation of metabolic waste products over time. therefore, for older frozen semen to be successfully used, more semen than originally planned may be needed. it is well published that for best pregnancy results a minimum of 150 million live, morphologically normal sperm should be inseminated on 1 occasion if the estrous cycle is timed well.3,18-21 however, if the estrous cycle is timed poorly, then 2 inseminations 24 48 hours apart of the aforementioned number of motile sperm should be inseminated.20,21 if estrous cycle timing is well done, then the only benefit to an additional insemination is an average of 1 additional puppy per litter.20,21 the universally accepted value is to inseminate 100 million live, morphologically normal sperm; however, there are limited published data to support this.8 although some operators advocate the use of differing numbers of sperm for different-sized bitches, to the author’s knowledge, there are no good published data to support this. to ensure enough sperm are inseminated, the recommended number of straws (or vials) should be thawed when the insemination is ready to occur. the author uses casa (spermvision, minitube, verona, wi) to assess semen, due to its reliability and speed. for this, 3 µl of thawed semen is placed in a leja chamber and analysed for concentration and motility. if the number of motile sperm is <100 million, then more semen is thawed (if available) until >100 million motile sperm are attained. the final volume is then inseminated via tci. if surgical insemination is used, the sample would have to be centrifuged to a more usable volume and then inseminated. an alternative to casa is to quickly count number of sperm in the sample using a makler chamber. for this, 20 µl of thawed semen is added to 5 µl of 5% formalin saline and 10 µl of the immobilised solution placed in the makler chamber for analysis. motility is guesstimated from the original sample and then number of motile sperm in the thawed sample calculated. timing the estrous cycle for insemination of frozen semen timing of the estrous cycle to determine the best time for insemination of frozen semen is best undertaken using a combination of vaginal cytology, progesterone assay and vaginoscopy. althogh lh assays may be beneficial, compared to the other 3 techniques, their use requires more frequent visits and blood sampling to not miss the lh surge.22,23 for best results, frozen semen is inseminated after ovulation, when maximal crenulation of the vagina is evident on vaginoscopy, before the bitch enters diestrus.9 200clinical theriogenology • volume 11, number 3 • september 2019 handling effects of frozen semen on motility and likely fertility frozen semen straws or vials are recommended to be stored in liquid nitrogen or liquid nitrogen vapour for minimal disturbance to sperm quality. some technicians advocate relabelling straws that have been incorrectly identified or inappropriately labelled, which will restrict the import/export of frozen semen. special freezerbonz ii® (brady labxpert, mexico) labels are used as they will stick to frozen straws. to determine if relabelling of semen straws affects sperm motility and quality, 9 straws of frozen semen were individually cut in half within a bath of liquid nitrogen. one of the halves of each straw was relabelled by quickly removing it from the liquid nitrogen, wiping the surface with a tissue to remove ice and then applying the freezerbondz ii® label and then this half straw was immediately returned to the liquid nitrogen bath. the process of relabelling took <20 seconds and contents remained visually frozen. each half straw was then individually thawed in 0.5 ml andropro ai (minitube, verona, wi) in a water bath at 37°c for 1 minute. motility of each sample was assessed using casa (spermvision, minitube, verona, wi). viability (membrane integrity) was assess using cybr14/pi staining using fluorescent microscopy. results are shown in table 4. based on paired sample student’s t test, there were differences (p < 0.05) between relabelled and non-relabelled straws for both motility and viability. this would indicate that this procedure should not be performed, as it is likely to reduce fertility. furthermore, removal of straws/vials from liquid nitrogen should be for very short intervals to avoid damaging semen. table 4. motility (%) and viability (%) results of each relabelled (label) and non-relabelled (no label) half straw. motility 1 minute motility 2 minutes motility 10 minutes viability straw number no label label no label label no label label no label label 1 47 33 43 34 48 42 2 36 31 43 42 45 37 3 36 10 22 11 33 9 25.7 4 4 39 26 40 21 31 20 31.4 10.8 5 49 29 45 27 45 29 44.1 20.7 6 48 46 47 46 53 43 41.9 25 7 50 40 50 38 48 41 54 14.9 8 52 33 57 34 52 36 50 22.1 conclusion whilst a thorough understanding of the techniques of freezing and thawing dog semen is important, it is equally important to have an understanding of extender constituents to ensure semen is being handled adequately to maximise pregnancy rates. frozen semen handling is equally important, as relabelling or holding frozen straws or pellets out of liquid nitrogen for even short intervals will decrease sperm quality. conflict of interest none to declare. references 1. seager s: successful pregnancies utilizing frozen dog semen. ai dig 1969;17:26. 2. anderson k: fertility of frozen dog semen. acta vet scand 1972;13:128-130. 3. linde-forsberg c, strom holst b, govette g: comparison of fertility data from vaginal vs intrauterine insemination of frozen-thawed dog semen: a retrospective study. theriogenology 1999;52:11-23. 4. fontbonne a, badinand f: canine artificial insemination with frozen semen: comparison of intravaginal and intrauterine deposition of semen. j reprod fertil suppl 1993;47:325-327. 5. silva ld, onclin k, snaps f, et al: laparoscopic intrauterine insemination in the bitch. theriogenology 1995;43:615-623. 6. andersen k: insemination with frozen dog semen based on a new insemination technique. zuchthygiene 1975;10:1-4. 7. hollinshead fk, hanlon dw: factors affecting the reproductive performance of bitches: a prospective cohort study involving 1203 inseminations with fresh and frozen semen. theriogenology 2017;101:62-72. 8. mason sj, rous nr: comparison of endoscopic-assisted transcervical and laparotomy insemination with frozenthawed dog semen: a retrospective clinical study. theriogenology 2014;82:844-850. 201 clinical theriogenology • volume 11, number 3 • september 2019 9. mason sj: a retrospective clinical study of endoscopic-assisted transcervical insemination in the bitch with frozenthawed dog semen. reprod domest anim 2017;52 suppl 2:275-280. 10. hansen c, vermeiden t, vermeiden jp, et al: comparison of facscount af system, improved neubauer hemocytometer, corning 254 photometer, spermvision, ultimate and nucleocounter sp-100 for determination of sperm concentration of boar semen. theriogenology 2006;66:2188-2194. 11. douglas-hamilton dh, smith ng, kuster ce, et al: capillary-loaded particle fluid dynamics: effect on estimation of sperm concentration. j androl 2005;26:115-122. 12. kasimanickam r, nebel rl, peeler id, et al: breed differences in competitive indices of holstein and jersey bulls and their association with sperm dna fragmentation index and plasma membrane integrity. theriogenology 2006;66:1307-1315. 13. pena a, linde-forsberg cb: effects of spermatozoal concentration and post-thaw dilution rate on survival after thawing of dog spermatozoa. theriogenology 2000;54:703-718. 14. pena ai, barrio f, quintela la, et al: effect of different glycerol treatments on frozen-thawed dog sperm longevity and acrosomal integrity. theriogenology 1998;50:163-174. 15. rota a, linde‐forsberg c, vannozzi j, et al: cryosurvival of dog spermatozoa at different glycerol concentrations and freezing/thawing rates. reprod domest anim 1998;33:355-361. 16. burgess dm, mitchell ke, thomas pg: coeliotomy-assisted intrauterine insemination in dogs: a study of 238 inseminations. aust vet j 2012;90:283-290. 17. pretzer sd, lillich rk, althouse gc: single, transcervical insemination using frozen-thawed semen in the greyhound: a case series study. theriogenology 2006;65:1029-1036. 18. linde-forsberg c, forsberg m: results of 527 controlled artificial inseminations in dogs. j reprod fertil suppl 1993;47:313-323. 19. linde-forsberg c, forsberg m: fertility in dogs in relation to semen quality and the time and site of insemination with fresh and frozen semen. j reprod fertil suppl 1989;39:299-310. 20. thomassen r, farstad w, krogenaes a, et al: artificial insemination with frozen semen in dogs: a retrospective study. j reprod fertil suppl 2001;57:341-346. 21. thomassen r, sanson g, krogenaes a, et al.: artificial insemination with frozen semen in dogs: a retrospective study of 10 years using a non-surgical approach. theriogenology 2006;66:1645-1650. 22. concannon pw: endocrinologic control of normal canine ovarian function. reprod domest anim 2009;44 suppl 2:3-15. 23. concannon pw, hansel w, visek wj: the ovarian cycle of the bitch: plasma estrogen, lh and progesterone. biol reprod 1975;13:112-121. 202clinical theriogenology • volume 11, number 3 • september 2019 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left 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/metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice fithis stud™s a dud!flcanine semen evaluation protocols and pitfalls “this stud’s a dud!”canine semen evaluation protocols and pitfalls joann randall animal hospital of woodstock woodstock, il abstract properly performed canine semen evaluation is a major component of breeding soundness examination. various types of equipment and protocols are available to practitioners and laboratory technicians to accurately assess sample concentration, motility and morphology. however, it is vital that precise laboratory standardization and quality control procedures be followed to optimize accuracy and usefulness of a semen evaluation. keywords: semen evaluation, canine, dog, breeding soundness examination applications of a semen evaluation importance of an accurately performed semen evaluation as part of a breeding soundness examination cannot be over emphasized. results are used to monitor dogs in a breeding program and those undergoing therapies for subfertility or infertility. complete semen evaluation should always be done as part of pre-purchase examination or evaluation of chilled or frozen semen for artificial insemination. no single parameter from semen evaluation that practitioner can use to accurately assess fertilizing capability of a dog or predict outcome of a successful litter.1,2 however, breeding recommendations to maximize likelihood for successful pregnancies are possible using dependable and precise semen evaluation results. breeding soundness examination and paperwork obtaining a detailed history and performing a thorough physical examination prior to collecting a dog is vital, as there are many outside factors that may affect quality and accuracy of semen that might otherwise be overlooked.3 society for theriogenology (sft; www.therio.org) has a comprehensive breeding soundness examination form (refer next page) to record reproductive history, pedigree, physical examination and semen evaluation, to promote consistency and accuracy. laboratory setting prior to collecting dog, microscope stage, slides and coverslips should all be pre-warmed to 37 cº to avoid any temperature shock when transferring samples for evaluation.4 correct labeling of all slides, test tubes and containers used in evaluation with a permanent marker is essential for record accuracy. semen analysis reliability world health organization laboratory manual recommends standardized, evidence-based procedures for human semen analysis to improve reliability, accuracy and comparability of results from laboratories.5 unfortunately, these protocols are deficient in most canine commercial laboratories and private practices. this is a dilemma not only for researchers trying to analyze data, but also for practitioners depending on semen analysis accuracy for breeding recommendations or to monitor success of canine reproductive therapies. semen volume volume of ejaculate collected is not correlated to semen quality, but is required to calculate semen concentration,2 so must be accurately recorded prior to starting any laboratory testing. volume of first fraction varies from 1 5 ml and second fraction from 1 3 ml. clinical theriogenology • volume 12 number 3 • september 2020204 semen color subjectively, second fraction is normally opalescent. blood causes a pink or red discoloration possibly from either prostatic disease or penile trauma. a yellow color could indicate urine contamination or riboflavin (often present from dogs on raw diets) whereas no color often is an indicator of few to no sperm in the sample. a green hue could indicate an inflammatory exudate or excess smegma.6 canine breeding soundness evaluation guidelines established by the society for theriogenology 761 tiger oak drive, pike road, al 36064-3063  (334) 395-4666 copyright 2016 society for theriogenology – for use of members only akc reg #/other id # ______________________________________ exam date: __________________________________ call name: ________________________________ registered name: _______________________________________________________ client name: _______________________________ address: ______________________________________________________________ breed: ____________________________________ color: _____________________________________ date of birth: ________________ history reason for evaluation: ____________________________________________________ date of last litter: _________________brucellosis test: positive/negative date of test: __________________test used_________________ infertile relatives: ___________________________________________________________________________________________________ physical examination body condition: ____________________________________________ weight: _________________ pounds/kilograms pertinent health problems: ___________________________________________________________________________________________ penis/prepuce: ____________________________________________ scrotum: ________________________________________________ prostate:_________________________________________________________________________________________________________ epididymides: (r) _________________________________________ (l) ______________________________________________________ testes width: (r) ___________ (l) ___________ spermatic cords:________________________________________________________ testes consistency: (r) __________________ (l) __________________ masses/fluid/pain/other: __________________________________ semen collection & evaluation libido/ease of collection: poor / fair / good / excellent teaser bitch present: yes/no stage of cycle: ______________________ color volume (ml) concentration (sperm/ml) total sperm/ejaculate fraction 1 __________ ________ fraction 2 __________ ________ ___________________ _________________ fraction 3 __________ total motile spermatozoa: ________% progressively motile spermatozoa: ________% speed/velocity of motility: 0-5________ morphology: stain(s) utilized: _______________________________________________________ % normal: ________________________ number cells counted: 100/200 head defects (%): __________________________________________________________________________________________________ midpiece defects (%): _______________________________________________________________________________________________ tail defects (%): ___________________________________________________________________________________________________ other defects (%)___________________________________________________________________________________________________ longevity: extender(s) used: _________________________________________________ diluent rate: _______________________ motility: 24 hours_________ 48 hours_________ cytology: fraction(s) evaluated: __________________________ stain(s) utilized: ___________________________________ presence of rbc, wbc, epithelial cells, bacteria, germ cells (0 – 4+): _________________________________________________________________________________________________________________ conclusions / comments / interpretation signed: ________________________________________ clinic name: _______________________________________________________ (member – society for theriogenology) address: __________________________________________________________ clinical theriogenology • volume 12 number 3 • september 2020 205 semen ph validity of this parameter is controversial and it is not on the sft breeding soundness evaluation form. unfractionated canine semen ph ranges from 6.4 6.8. inaccuracy of ph paper strips along with newer antibiotics that ionize at multiple ph values decreases ph testing validity in dogs.7 semen motility evaluation of sperm motion cannot be used to accurately predict fertilizing potential of sperm in a given sample when used for artificial insemination.8 motility evaluation should be done as soon as possible after semen collection.9 there are 2 main methods for evaluating progressive motility, which normally should be > 70% for dog sperm. first method is a subjective evaluation of semen motility and involves assessing a drop of semen flattened between a microscope slide and a coverslip. normal sperm should traverse microscopic field in ~ 2 3 seconds in a forward progressive movement.2 motility is best assessed using a properly aligned phase contrast microscope at 250x magnification.10 layer of sperm should be only 1 layer thick so that motility can be accurately assessed in 1 plane and there should be enough room between sperm to assess individual sperm movement. if viewer detects several layers of sperm, sample should be diluted with an isotonic diluent and a less concentrated sample is viewed to improve subjective assessment. one drop of semen from a pasteur pipette (pipette capacity, ~ 7 ml) is the correct volume to allow for even distribution and movement of sperm under a 22 x 22 mm coverslip placed on a slide.11 use of a smaller coverslip (18 x 18 mm) may put less pressure on sperm and allow more free movement. sperm motility is generally highest at center of coverslip and decreases towards edges. therefore, viewer should assess those regions across coverslip equator and average motility observed in several viewing fields to increase subjective score accuracy. it is recommended that 2 trained viewers assess slide and average motility value of 2 independent results is recorded. velocity of movement is also recorded, usually on a scale of 0 5 (with 0 being no movement, and 5 being very fast progression). it is important to differentiate total motility from progressive motility; to do this, individual sperm movement is assessed. if sample is too concentrated, nonmotile sperm may be pushed around by motile ones, leading to an inflated motility score. second method for evaluating semen motility is computer-assisted sperm analysis system (casa), an expensive, automated system that visualizes and digitizes successive sperm images, processes them, and then analyzes information giving precise data regarding motion of individual sperm. when properly calibrated and used with appropriate software parameter settings, a casa system provides both accurate and precise information on detailed kinematic parameters such as curvilinear velocity, straight line velocity, and amplitude of lateral head displacement.12,13 however, machine parameters such as imaging hardware and software, recording quality and camera frame rate can alter motility results.14 concentration of sperm, extenders, amount of debris, crystals and immotile dead sperm heads present in sample also alter motility results when using casa systems.13 these issues, compounded by intensive staff training and continuous standardization and machine maintenance, make casa use challenging in private practice. sperm concentration and number sperm reservoir depletion decreases sperm concentration,15 so frequency and interval of prior collections before performing an evaluation should be known. sperm concentration can be determined manually or using automated methods. once the concentration is known, total sperm number is calculated by multiplying concentration (# sperm/ml) by total volume (ml) of ejaculate collected. in dog, normal total number of sperm in ejaculate is 300 million 2 billion.2 sperm production is dependent on grams of testicular tissue; total sperm number is ~ 10 million sperm per pound of body weight. for example, a typical value for a 10-pound dog would be 100 million sperm. clinical theriogenology • volume 12 number 3 • september 2020206 manual hemocytometer has long been considered the gold standard for precisely counting canine sperm. however, the nucleocounter® sp-100™ (chemo-metec a/s, allerod, denmark) discussed below has replaced it in recent years.16,17 this manual method is very inexpensive and accurate when performed by trained staff. it is important to use specified cover slip corresponding to hemocytometer and to count both chamber sides. if there is more than 10% discrepancy between sides, chambers should be refilled, equilibrated and recounted. to maintain errors < 5%, it is recommended to count a minimum of 400 sperm per sample.18 care must be taken to properly fill chambers and count all sperm heads in designated counting grid only once to improve accuracy. differences in sperm concentration between casa systems and hemocytometer are largely due to the segre-silberberg effect, which does not occur when using a hemocytometer due to a deeper sample chamber compared to most disposable slides used in automated systems.17 problems with sperm count are similar to assessing motility, sperm count can be inaccurate when low or high concentration semen samples are analyzed. casa reliability is very dependent on users technical competence.18-20 another automated method is the nucleocounter ® sp-100™. sperm treated with a detergent are aspirated into a cassette lined with propidium iodide, which crosses cell membrane and binds specifically to dna. fluorescently labeled sperm are then quantified. because sperm identification is so specific to dna, there is no interference from debris, concentration, or extenders.21pipetting must be done correctly for this machine to provide accurate counts. photometric devices such as the spermacue® or densimeter ® that depend on quantitative light transmission measurement through a diluted semen sample are easy to use, and are relatively inexpensive. samples that contain a lot of debris (e.g. leukocytes, bacteria, cytoplasmic droplets, etc.), are extremely concentrated, or very dilute should not be run using photometric devices, as results will be inaccurate.20 only raw semen can be used in photometric devices; extended semen can only be used if t extender has no optical density (i.e. is clear). flow cytometry using detection of light scatter and fluorescence of individual sperm is a very precise and accurate method to determine sperm concentration. however, equipment cost, need for skilled technicians, and involved sample preparation techniques limit this technique to research. quality control is critical, irrespective of whatever form of counter is used. when semen is evaluated grossly and microscopically, an estimate of sperm concentration should be performed. motility samples should be assessed for extraneous cellular debris (epithelial cells, wbc, rbc) that may interfere with accurate counting. if semen is centrifuged, the size of the sperm pellet can be used to estimate total sperm in the ejaculate. if the count calculated by whatever method is used does not match with estimated count based on the above factors, count should be repeated or calculated using another (ideally more accurate) method. sperm morphology assessment of sperm morphology is a subjective evaluation susceptible to large discrepancies between evaluators. a 79.4% inter-laboratory variability is reported in assessing canine sperm morphology.22 phase contrast or differential interference contrast microscopy is one technique to view sperm fixed with formol buffered saline as a wet mount, thereby limiting damage done to sperm using staining techniques necessary for light microscopy.2,23 specific structural defects are known to be associated with male infertility. staining techniques help visualize sperm defects when viewed under oil immersion, but compared to phase microscopy, the staining preparation technique may contribute to morphologic artifacts.24 four common staining techniques (conventional, dipping and blotting, direct mixing and ignition) were compared and it was concluded that as long as slides were made carefully according to protocols, percentage of abnormal sperm was constant across all 4 techniques.25 eosin-nigrosin (hancock’s) stain (available from sft) is a common stain for sperm morphology. typically, 1 drop of semen is mixed with 1 drop of stain, spread like a blood smear and allowed to dry. dark slide background from nigrosin facilitates visualization of sperm head, midpiece and tail. this stain is a “vital” stain, meaning that eosin is able to permeate damaged plasma membranes of dead sperm, clinical theriogenology • volume 12 number 3 • september 2020 207 staining them pink, compared to white or clear live sperm.2,20 sometimes there is only partial staining of sperm and staining may not be consistent if there are fat globules in seminal fluid2 making it more challenging to clearly designate sperm as alive or dead. round cells (germ cells, erythrocytes and leukocytes) and acrosomes cannot be easily differentiated. giemsa-wright stain (diff-quick ™ baxter healthcare, miami fl; romanowski stain) is a quick, inexpensive, 3-step stain that most clinics use for blood smears. slide is immersed 5 minutes sequentially in fixative, safranin, and crystal violet, lightly rinsed with water to remove stain and then dried. artifacts may be minimized by drying the stained smear on a slide warmer set at 37 degrees c or by blowing.2 this stain is good for round cells but cannot be used to assess acrosome and cytoplasmic droplets are difficult to observe. spermac stain is more labor intensive and costly, however, an excellent stain for evaluating acrosome, equatorial, midpiece and tail regions of sperm.20 normal percentage of morphologically normal sperm (mns) for canines should be > 80%6,9 and fertility appears to be affected if there is < 60% mns.2 a minimum of 100 sperm are evaluated and the abnormal cells classified as: 1) primary (occurring during spermatogenesis) or secondary (occurring during maturation or sample preparation); 2) major (negatively affecting fertility) or minor (not associated with fertility); or 3) compensable (improved fertility by increasing sperm numbers) or non-compensable (fertility not increased by increasing sperm numbers). other cell types in semen samples in addition to sperm, semen samples may contain erythrocytes, leukocytes, epithelial cells and immature germ cells. these are usually noted on the breeding soundness evaluation sheet as numbers of cell type/100 sperm. advanced tests many specialized stains, assays, and tests are available to evaluate sperm morphology, function, chromatin, membrane integrity, etc. in dogs displaying suboptimal fertility with normal semen evaluation values. one example is the hypoosmotic swelling test, used to evaluate membrane integrity of sperm by assessing tail curling after incubation in hypoosmotic solution. conclusion routine semen evaluation is an integral part of a canine breeding soundness exam, easy to perform and a valuable tool to evaluate sperm. results will only be reliable and accurate if equipment is routinely maintained and calibrated. to minimize error, laboratory personnel should undergo training protocols and run standardized procedures with quality control. perhaps someday we will be able to perform a semen evaluation and, using a new parameter, reliably predict fertilizing potential. conflict of interest there are no conflicts of interest to declare. references 1. hesser a, darr c, gonzales k, et al: semen evaluation and fertility assessment in a purebred dog breeding facility. theriogenology 2017;87:115-123. 2. root kustritz mv: the value of canine semen evaluation for practitioners. theriogenology 2007;68:329-337. 3. purswell bj, althouse gc, root kustritz mv: guidelines for using the canine breeding soundness evaluation form. proc society for theriogenology 1994; p.46-60. 4. mordecai j: semen collection, evaluation, and cooled shipment in the canine. clinical theriogenology 2010;2:222-229. 5. world health organization (who). who laboratory manual for the examination and processing of human semen. switzerland:who press;2010 6. johnston sd: performing a complete canine semen evaluation in a small animal hospital. in: vet clin north amer small anim pract 1991.p. 545-551. 7. root kustritz mv: applied small animal andrology. in: chenoweth pj, lorton sp, editors. animal andrology theories and applications. wallingford (uk); cab international: 2014. p.183. clinical theriogenology • volume 12 number 3 • september 2020208 8. amann rp, waberski d: computer-assisted sperm analysis (casa): capabilities and potential developments. theriogenology 2014;81:5-17. 9. robert ma, jayaprakash g, pawshe m, et al: collection and evaluation of canine semen-a review. international journal of science, environment and technology 2016;5:1586-1595. 10. amann rp: a critical review of methods for evaluation of spermatogenesis from seminal characteristics. j androl 1981;2:37-58. 11. nothling jo, dos santos ip: which fields under a coverslip should one assess to estimate sperm motility? theriogenology 2012;77:1686-1697. 12. amann rp, katz df: reflections on casa after 25 years. j androl 2004;25:317-325. 13. vasan ss: semen analysis and sperm function tests: how much to test? indian j urol 2011;27:41-48. 14. boryshpolets s, kowalski rk, dietrich gj, et al: different computer-assisted sperm analysis (casa) systems highly influence sperm motility parameters. theriogenology 2013;80:758-765. 15. boucher jh, foote rh, kirk rw: the evaluation of semen quality in the dog and the effects of frequency of ejaculation upon semen quality, libido, and depletion of sperm reserves. cornell vet1958;1:67-86. 16. burns g: clinical methods for counting canine sperm: automated and manual techniques. clinical theriogenology 2015;7:153-156. 17. kuster c: sperm concentration determination between hemacytometer and casa systems: why they can be different. theriogenology 2005;64:614-617. 18. brito lfc, althouse gc, aurich c, et al: andrology laboratory review: evaluation of sperm concentration. theriogenology 2016;85:1507-1527. 19. davis ro, katz df: operational standards for casa instruments. j andrology 1993;14:385-394. 20. fraser ns: equipment for your andrology laboratory. clinical theriogenology 2018;10:197-199. 21. lorton sp: evaluation of semen in the andrology laboratory. in: chenoweth pj, lorton sp, editors. animal andrology theories and applications. wallingford (uk): cab international; 2014. p.105-109. 22. punjabi u, wyns c, mahmoud a, et al: fifteen years of belgian experience with external quality assessment of semen analysis. andrology 2017;4:1084-1093. 23. descanio jj: wet mount evaluation of sperm. in: descanio j, mccue p, editors. equine reproductive procedures. hoboken (nj):wiley-blackwell;2014:389-391. 24. root kustritz mv, olson pn, johnston so, et al: the effects of stains and investigators on assessment of morphology of canine spermatozoa. j am an hosp assoc 1998;34:348-352. 25. rao ck: effect of the technique of staining on sperm morphology. br vet j 1957;113:13-16. clinical theriogenology • volume 12 number 3 • september 2020 209 010_ms-10 randall 57 contact reed holyoak reed.holyoak@okstate.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9597, http://dx.doi.org/10.58292/ct.v15.9597 research report impact of transrectal palpation on teaching mares: a retrospective study* sheila megehee,a candace lyman,b reed holyoaka adepartment of veterinary clinical sciences, oklahoma state university college of veterinary medicine, stillwater, ok, usa, bdepartment of clinical sciences, auburn university college of veterinary medicine, auburn, al, usa abstract transrectal palpation (trp) in mares is considered a noxious and potentially life-threatening procedure, particularly if performed by inexperienced individuals. a retrospective study was conducted using trp records maintained at the oklahoma state university college of veterinary medicine ranch. a total of 5,801 trp were performed from 2019 to 2021 by veterinary students. we hypothesized that in mares the incidence of rectal wall injury and use of intravenous sedation for behavior modification would be low. additionally, we hypothesized that averse behaviors during trp would not be a reason commonly cited for mare retirement from the teaching herd. injury incidence rate was 0.76% (44/5,801) and no injury required surgical correction or resulted in a mare’s death. sedation was used to minimize averse behaviors 0.34% of the time (20/5,801) and 11 out of 48 mares received sedation. individual mare use ranged from 1 to 22 years and 10 of 48 mares retired from 2019 to 2021. no retirement was related to averse behaviors or injury experienced during trp. findings indicated that student-generated rectal wall injury to teaching mares was at an extremely low rate of occurrence. we also suggest that in live-animal trp laboratories, animal welfare is not compromised, and it should not be cited as a reason to minimize live animal use within the veterinary curriculum. keywords: transrectal palpation, behavior, injury, attrition rate introduction historically, transrectal palpation (trp) of mares by students was commonplace; however, recently, the practice has been minimized at many veterinary schools across the united states due to the potential rectal injury and animal welfare concerns. such a risk of injury to the rectal wall has been a reason for teaching institutions to reconsider the use of institution-owned teaching animals and replacing live animal laboratories with model simulators. there is an increasing use of model simulators and replacement of live animal laboratories to teach veterinary students in response to these concerns. however, removal of live animals from the veterinary curriculum would substantially minimize the amount of real-life experience to veterinary students. 1 ‘hands-on training involving animals is an important component of agricultural research and teaching; additionally, there is no substitute of another animal or simulation for production research’.1 the clinical skill of trp is critical for * abstract (clinical theriogenology 2022:14;283) presented at the 2022 society for theriogenology conference large animal veterinarians, not only in breeding management, but also in pregnancy diagnosis and workup of cases of colic. a few reports documented the level of risk or safety of trp when included in the veterinary curriculum. however, a recent study concluded that there were no rectal injuries due to trp and ultrasonographic examination in recipient mares by students who had received 6 weeks of training. none of the mares experienced any pathological condition, including any degree of rectal injury, and specific therapy was never deemed necessary after students’ palpations.2 animal welfare is an important aspect to consider when live animals are used for teaching and anything that can be done to improve this for the teaching animals should be considered. however, recent studies have begun to determine how stressful trp is in mares. transrectal palpation examinations induced a moderate stress response indicated by increased heart rate and cortisol release. however, frequency of the trp was the main stressor and trp every 6 hours substantially increased the mares’ heart rates.3 results from 2 other studies also demonstrated that the cortisol response from trp was lower than horses that were transported and was lower in mailto:reed.holyoak@okstate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9597 58 citation line: clinical theriogenology 2023, 15, 9597, http://dx.doi.org/10.58292/ct.v15.9597 mares that experienced student trp compared to mares standing undisturbed in palpation stocks.3,4,5 our institution prides itself on the ability to prepare students for day 1 of veterinary practice. through hands-on laboratories, students are allowed to implement the skills and knowledge that they have acquired in the classroom setting and apply it directly to live animals. this study had 3 objectives: 1. identify the incidence of mare rectal injury associated with trp in teaching mares, 2. identify the number of intravenous sedation utilized for averse behaviors (i.e. behaviors interpreted as ‘dislike’), and 3. identify the length of individual mares’ use/cited reasons for retirement. materials and methods transrectal palpation records (2019–2021) from the oklahoma state university college of veterinary medicine ranch. during this period, the ranch had 48 teaching mares and their ages ranged from 4 to 26 years. the herd had several breeds (quarter horses, paints, thoroughbreds, and 1 friesian sport horse). data were collected from 2nd and 3rd year laboratory courses, senior student clinical rotations, and other student teaching opportunities. laboratories and clinical rotations were limited to a maximum of 6 students per session under the supervision of a clinician, resident, and intern. second and 3rd year laboratory is an elective class open to all students with varying degrees of horse experience, and transrectal palpation and ultrasonography experience. this elective allowed for an introduction and ability to learn the basic skills of trp, ultrasonography, and equine theriogenology. clinical rotations are offered throughout the breeding season wherein senior veterinary students gained experience with trp and were allowed to track individual mares throughout their estrous cycle while performing a wide range of reproductive procedures including breeding soundness examinations and mock embryo flushes. veterinary students transrectally palpated and performed ultrasonography of the reproductive tracts of mares through all stages of pregnancy. the teaching mares were used throughout the year for miscellaneous laboratories and various teaching purposes. documented mare injuries from transrectal palpation included any amount of blood on rectal sleeves and any degree of rectal wall tearing/compromise. individual mare medical records documenting date of entry into the teaching herd, date of retirement, and the reason for retirement were analyzed. although not the first method selected to quiet an agitated mare, our approved institutional animal care and use committee teaching protocol allowed for the use of sedation on a case-by-case basis in mares displaying averse behaviors to ensure mare and student safety. listed averse behaviors were excessive pawing or reluctance to stand still in the stocks in such a way that safety was compromised. mares were sedated intravenously with either 100 mg xylazine (anased, patterson veterinary, loveland, co), 200 mg acepromazine maleate (acepromazine, mwi animal health, boise, id), or 1.5 – 5 mg detomidine hydrochloride (dormosedan, zoetis, parsipanny, nj) depending on the mare and her behavior. the standing iacuc protocol allowed a maximum of 5 transrectal examinations in a mare per laboratory and a maximum of 3 laboratories per week. therefore, it was possible to transrectally palpate each mare a maximum of 15 times in a 7-day period. if at any point a mare had mucosal irritation (blood on palpation sleeve), she was removed from the palpation herd for a minimum of 2 weeks. if there is any indication of a rectal tear or injury, that mare was thoroughly evaluated by a veterinarian and appropriate steps were taken to treat the mare as needed. results total number of palpations by veterinary students from 2019 to 2021 were 5,801. forty-four mare rectal injuries (table 1) occurred, and the injury rate was 0.76% (44/5,801). none of the injury required surgical correction or resulted in death of a mare. the injuries consisted of mucosal irritation/abrasions, as evidenced either by blood on the examination sleeves, visual or digital identification of a tear, or both; defects to the rectal wall were not higher than minor grade 1 rectal tear < 2 cm in length. injuries occurred in 28/48 mares with 12 of these mares had multiple injuries throughout the years. sedation was used in 0.34% (20/5,801) times and 11 of 48 mares received sedation. sedation was given on multiple occasions to 5 mares, and 1 mare needed sedation 6 times from 2019 to 2021 (table 2). individual mare use ranged from 1 to 22 years and 10 of 48 mares retired from 2019 to 2021. no retirement was related to averse behaviors or injury experienced during trp (table 3). retirement or reasons teaching mares left the herd included humane euthanasia, vascular injury, aging out of program, or enrolled in a research project. discussion based on these data there was a low rate of injury to teaching mares from trp by veterinary students. none of the injuries that occurred in the teaching mares required surgical correction or resulted in full-thickness rectal wall tears. depending on the extent of mare’s injury, mares were removed from the teaching herd to recover and treated appropriately. there was no injury that resulted in a mare’s death or retirement from the teaching herd. this low rate was observed with inexperienced students and with inexperienced mares that entered the herd during this period that were new to trp. there has been concern, particularly in cattle, that inexperienced students transrectally palpating the uterus may cause pregnancy loss; however, there was not an increased risk of pregnancy loss when students performed trp on cattle that were early in pregnancy.6 students came with a wide range of experience, some might have palpated a mare for the first time during these laboratories whereas more experienced students were able to follow the whole reproductive tract on transrectal ultrasonographic examination. although injuries occurred during trp, it was observed that under our conditions they were infrequent and evidence of common injuries observed (e.g., blood on the sleeve) were manageable with full recovery of the mare. authors observed that students were readily willing to self-report evidence of injury to a mare, knowing that there was also no cause for concern of reprimand, to ensure that the mare received an appropriate examination by the veterinarian present. intravenous sedation was used infrequently for the modification of averse behaviors in mares. not all mares needed sedation for trp and it was observed that mares that received sedation did not need it consistently. one could postulate that we selected among donated mares only those with good personalities or trained mares; however, we did not choose mares. all mares were donated. mares came with a variety of training http://dx.doi.org/10.58292/ct.v15.9597 citation line: clinical theriogenology 2023, 15, 9597, http://dx.doi.org/10.58292/ct.v15.9597 59 table 1. total times teaching mares were transrectally palpated by veterinary students from 2019 to 2021 and the injuries that occurred from transrectal palpation mare/year 2019 2020 2021 total injuries 610 16 6 145 167 187 0 0 103 103 165 26 47 69 142 158 18 39 62 119 mucosal irritation 6/15/2020, rectal bleeding 8/24/2020 181 0 45 127 172 173 32 2 85 119 136 31 0 0 31 blood on sleeve 8/28/2019 59 8 70 88 166 190 0 0 91 91 178 11 79 113 203 small rectal tear 10/19/2021, rectal abrasion 10/25/2021 953 32 42 98 172 blood on sleeve 2/11/2020 130 24 0 0 24 168 21 67 0 88 blood on sleeve 8/27/2019, 9/3/2019, 3/2/2020 344 7 35 49 91 188 0 0 119 119 blood on sleeve 5/25/2021 184 0 46 123 169 blood on sleeve 2/22/2021 177 4 21 0 25 blood on sleeve 3/2/2020 163 28 72 80 180 182 5 27 5 37 180 11 17 55 83 blood on sleeve 6/4/2021 191 0 0 3 3 121 7 15 53 75 123 12 39 87 138 small rectal tear 0.5 cm 9/10/2019, blood on glove 11/1/2021 157 19 68 89 176 rectal scratch 1/2 inch 3/20/2019, blood on sleeve 2/6/2020 125 39 55 54 148 blood on glove 5/27/2021 156 1 69 88 158 172 15 83 120 218 171 28 66 151 245 186 0 0 119 119 blood on sleeve 3/1/2021 143 2 19 9 30 blood on sleeve 2/24/2020 28 14 71 107 192 small rectal tear 8/31/2021 18 7 63 103 173 mucosal roughening 4/22/2019, blood on sleeve 3/2/2020, blood on sleeve 5/20/2020, rectal irritated 8/21/2020 146 22 60 43 125 blood on sleeve 6/3/2021, small rectal tear 9/13/2021 678 11 0 0 11 blood on sleeve 8/27/2019 142 19 63 114 196 blood on sleeve 4/29/2021 179 18 46 85 149 small tear 1 cm long 8/23/2019, blood on sleeve 2/27/2020, blood on sleeve 3/23/2021 919 32 44 12 88 blood on sleeve 8/23/2019, 8/27/2019 167 19 50 79 148 183 20 41 107 168 blood on sleeve 2/22/2021 135 13 63 80 156 898 47 7 0 54 small superficial tear 4/5/2019, blood on sleeve 2/12/2020 122 10 53 12 75 0 0 0 18 18 small rectal abrasion 10/11/2021 155 14 61 108 183 blood on glove 10/25/2021, 11/2/2021 166 18 55 84 157 small rectal abrasion 8/31/2021 185 2 16 137 155 blood on sleeve 6/21/2021 835 20 48 61 129 169 13 0 0 13 1 cm rectal tear partial thickness 5/16/2019, blood on sleeve 6/6/2019       total 5,801 total incidents = 44 http://dx.doi.org/10.58292/ct.v15.9597 60 citation line: clinical theriogenology 2023, 15, 9597, http://dx.doi.org/10.58292/ct.v15.9597 table 2. number of times teaching mares were transrectally palpated by veterinary students from 2019 to 2021 and the amount of sedation and times sedation utilized for averse behaviors mare/year 2019 2020 2021 total sedation needed 610 16 6 145 167 187 0 0 103 103 1 ml xylazine 3/16/2021 165 26 47 69 142 158 18 39 62 119 181 0 45 127 172 173 32 2 85 119 dorm 0.3 ml 10/5/2021 for lab, dorm 0.3 ml 14 d preg check 6/14/2019 136 31 0 0 31 dorm 0.3 ml 10/8/2019 for bse 59 8 70 88 166 190 0 0 91 91 178 11 79 113 203 953 32 42 98 172 130 24 0 0 24 168 21 67 0 88 dorm 0.3 ml for biopsy 6/5/2020 344 7 35 49 91 dorm 0.3 ml for palp 8/20/2021, dorm 0.3 ml for palp 9/21/2020, 9/14/2020, 8/31/2020, ace 2 ml for palp 8/31/2020, dorm 0.4 ml 10/7/2019 for bse 188 0 0 119 119 dorm 0.3 ml for palp 10/5/2021, xylazine 1 ml 3/16/2021 184 0 46 123 169 177 4 21 0 25 163 28 72 80 180 182 5 27 5 37 180 11 17 55 83 191 0 0 3 3 121 7 15 53 75 123 12 39 87 138 157 19 68 89 176 125 39 55 54 148 156 1 69 88 158 172 15 83 120 218 171 28 66 151 245 186 0 0 119 119 xylazine 1 ml 3/16/2021, dorm 0.5 ml 2/19/2021 for lavage 143 2 19 9 30 28 14 71 107 192 18 7 63 103 173 146 22 60 43 125 678 11 0 0 11 142 19 63 114 196 dorm 0.5 ml for lavage post embryo aspiration 3/24/2021 179 18 46 85 149 919 32 44 12 88 167 19 50 79 148 183 20 41 107 168 135 13 63 80 156 898 47 7 0 54 dorm 0.4 ml for bse 10/7/2019, dorm 0.15 ml for bse 5/24/2019 122 10 53 12 75 0 0 0 18 18 dorm 0.3 ml 10/5/2021 155 14 61 108 183 166 18 55 84 157 185 2 16 137 155 835 20 48 61 129 169 13 0 0 13 dorm 0.3 ml for 6/7/2019       total 5,801 total times sedation was utilized: 20 http://dx.doi.org/10.58292/ct.v15.9597 citation line: clinical theriogenology 2023, 15, 9597, http://dx.doi.org/10.58292/ct.v15.9597 61 table 3. teaching mares summary (age, breed, joining date, and reasons for leaving) mare birth date breed joined herd euthanized/left program 610 1/1/2008 – 14 years qh 2016 187 4/15/2017 – 4.8 years paint 2020 165 2/6/2002 – 19.9 years paint 2017 158 4/18/1999 – 22.8 years aqha 2016 181 8/6/2004 – 17.5 years aqha 2019 173 3/7/2007 – 14.9 years aqha 2017 136 1/1/2003 – 19 years aqha 2010 euthanized for rr lameness 1/23/2020 59 1/1/2003 – 19 years apha 2015 190 1/1/2009 – 13 years paint 2020 178 5/7/2003 – 18.7 years aqha 2018 953 3/3/2000 – 21.9 years aqha 2007 130 3/25/2005 – 16.8 years aqha 2010 euthanized 7/25/2019 for lameness 168 5/20/1996 – 25.7 years aqha 2017 euthanized 11/9/2020 for lacerations 344 4/30/2005 – 16.7 years aqha 2019 euthanized 8/20/2021, jumped through window 188 4/3/2017 – 4.8 years paint 2020 184 5/22/2008 – 13.7 years friesian sport horse 2019 177 2/27/2004 – 17.9 years tb 2018 retired due to age/hard keeper 4/13/2020 163 4/4/2008 – 13.8 years tb 2017 182 4/12/2008 – 13.8 years aqha 2019 180 3/15/2008 – 13.8 years aqha 2019 191 1/1/2018 – 4 years aqha 2021 121 5/10/2000 – 21.7 years aqha 2009 123 3/26/2005 – 16.8 years aqha 2009 157 2/28/2002 – 19.9 years aqha 2016 9/28/2021 ruptured cranial mesentery artery 125 4/14/2003 – 18.8 years aqha 2009 156 5/12/2003 – 18.7 years qh 2016 172 3/23/2011 – 10.8 years apha 2017 171 4/10/2010 – 11.8 years apha 2017 186 1/1/2017 – 5 years paint 2020 143 3/31/2010 – 11.8 years aqha 2012 28 3/25/2011 – 10.8 years aqha 2018 18 4/11/2012 – 9.8 years aqha 2018 146 5/6/1999 – 22.7 years tb 2013 678 1/1/2000 – 22 years aqha 2009 euthanized for lameness 10/23/2019 142 1/1/2004 – 18 years qh-appy x 2012 179 4/12/2000 – 21.8 years aqha 2019 919 5/21/2000 – 21.7 years aqha 2005 euthanized 7/6/2021 found down in pasture 167 1/1/1998 – 24 years aqha 2017 183 1/1/2002 – 20 years aqha 2019 135 4/19/2005 – 16.7 years aqha 2010 898 3/5/2001 – 20.9 years tb 2004 retired due to age 4/13/2020 122 2/26/2005 – 16.9 years aqha 2009 0 9/10/2013 – 8.3 years qh 2020 155 5/4/2004 – 17.7 years aqha 2015 166 1/6/2007 – 15 years aqha 2017 185 2/22/2007 – 14.9 years aqha 2019 835 4/1/1995 – 26.8 years aqha 2002 169 1/1/2012 – 10 years tb 2017 left for research project 9/30/2019 http://dx.doi.org/10.58292/ct.v15.9597 62 citation line: clinical theriogenology 2023, 15, 9597, http://dx.doi.org/10.58292/ct.v15.9597 and handling. some were older and used as riding companions and some were young 4-year maidens that were only halter-broken. to help acclimate new mares to the palpation herd, we routinely brought them into the palpation barn and into the stocks where we initially allowed them to just get used to standing in the stocks. students may groom the mares while they stand and pet them for positive reinforcement. slowly they were introduced to trp and each mare was allowed to progress at her own rate. all mares were eventually placed in the palpation herd without issue. it could be perceived that observed averse behaviors were due to dislike of the trp procedures; however, if trp was a noxious procedure, one would expect teaching mares to progressively develop aversion to behaviors and require increased use of sedation. this was not demonstrated in our herd. a plausible cause for such behaviors aside from the trp procedures themselves is the period it took for students to palpate. the period mares stood in the stocks could influence their behavior, especially if they had sore feet or arthritis that could be causing them discomfort. to try to relieve this, we installed rubber mats for the mares to stand on in the stocks that provided a limited amount of comfort from the concrete floors. potential causes for averse behaviors were mares being separated momentarily from their ‘buddies,’ boredom, or mittelschmerz (ovulatory pain, merriam-webster). this could explain why some mares needed sedation multiple times depending on the day and since they did not need sedation every time they were palpated. research is underway to identify the stress response from trp in mares. teaching mares had the lowest cortisol concentrations and there was no major disturbance in mare’s welfare.7 teaching horses may be accustomed to these procedures since there was low heart rates and no increase in stress responses.8 as trp began, mean pony mare heart rate was 32.46 ± 4.83 beats per minute (bpm) and after 4 minutes of trp, the mean heart rate was 34.6 ± 11.80 bpm; heart rates increased after trp while waiting in stocks for a longer period than usual after palpation procedure.5 with these results, one could consider that the act of trp may not be a primary stressor and perhaps stressors (e.g., duration a mare is actively palpated, duration a mare is standing in confinement stocks, or simply a deviation from normal routine had a larger role). another study by the same author determined salivary and fecal glucocorticoid metabolites concentrations. author concluded that mares accustomed to palpation did not activate their hypothalamic-pituitary-adrenal axis during student palpations.9 further research is needed to identify exactly how stressful trp is to teaching mares and to identify various stress factors. we examined individual mare records to determine if there were mares retired from the teaching herd due to averse behaviors or trp-associated injury. no mares retired from the teaching herd because of averse behaviors or injury. we recognize that trp can certainly result in an injury (e.g., rectal tear or serious rectal injury) leading to retirement or death of a mare. a study10 compared student proficiencies from live-animal based learning versus simulator learning in which students who trained repeatedly on horses performed better than simulator trained students in both trp and ultrasonographic examination. another study focusing on trp for pregnancy diagnosis in cattle observed that student simulation training resulted in lower sensitivity in pregnancy diagnosis in cows that were < 6 months pregnant.11 we believe that although a simulator is a great supplement to learning, the simulator cannot provide veterinary students horsemanship, reproductive tract pathology, and real-life experiences. theoretical knowledge can be gained from reading a textbook but until it is supplemented with ‘experiential knowledge,’ information alone has no benefit. in laboratory sessions, students first caught mares from the herd and haltered them. this involved entering an area that had multiple horses and evaluating each horse’s personality. some mares were easier to catch than others, a valuable lesson in itself, and many of our students haltered a mare for the first time during these laboratories. throughout this whole process from start to finish, students were working alongside the horses and learned valuable ‘horse sense’ obtained only through experience. learning how to safely work with horses is a necessary skill in clinical practice for the safety of the horse and the veterinarian. although students can learn how normal reproductive tract looks and feels like via stimulators, they will not learn normal variations and pathology of reproductive tract, a limitation of stimulators. teaching mares’ variation in age allowed students to appreciate the differences in ovaries (i.e., ovarian senescence), uterine position (i.e., pendulous uterus), and cervical function (i.e., fibrotic cervix). throughout the season mares might have had various conditions (e.g., endometritis) wherein students themselves were able to identify the condition via ultrasonography, obtained culture samples from the mare themselves, and treated accordingly. witnessing and participating in this diagnostic work-up and treatment process matched what students will experience in clinical practice. with a teaching herd on site, not only will students have the opportunity to practice reproductive procedures, but also had opportunities to use their knowledge and practice their clinical skills in other areas of equine veterinary medicine. for example, if a teaching mare had ocular discharge or walked up to feed bin with a sudden lameness, under clinician supervision students performed diagnostics and provided treatments. these experiences cannot be accomplished with a simulator, and these are critical skills that will allow veterinary students to be properly prepared when they are in practice. this study supported the concept that students improve their trp skills via teaching mares. these data indicated that while injuries can occur, they occur at a very low rate at which most are not as serious as previously believed. transrectal palpation did not appear to be a noxious procedure where increased use of sedation is needed in response to averse behaviors. instead, most mares became acclimated to the procedures and did not needed sedation over time. none of the mares in the teaching herd retired or were removed due to injury or averse behaviors indicating that mares became acclimated to the procedures. this does not mean life-threatening injuries are impossible, just that they are infrequent. although simulators in the veterinary curriculum serve as an adjunct to developing skills and may reduce both student anxiety and the number of live animals or repeat procedures needed in order to assure student competency, the risk of injury to the teaching mares does not preclude their use in providing real-life experiential education that is essential for development of competent and confident veterinary students. conflict of interest none. http://dx.doi.org/10.58292/ct.v15.9597 citation line: clinical theriogenology 2023, 15, 9597, http://dx.doi.org/10.58292/ct.v15.9597 63 authors’ contribution all authors were involved in record-keeping and instruction; sheila megehee wrote the first draft and candace lyman and reed holyoak reviewed and edited the manuscript. acknowledgement dennis wilbourn’s years of dedication and care of teaching mares are acknowledged. references 1. guide a: adsa-asas-psa guide for the care and use of agricultural animals in research and teaching. champaign, il, usa; adsa, asas, psa: 2020. 2. camillo f, fanelli d, rota a, et al: teaching palpation of the mare’s uterus and ovaries to veterinary medicine students using a herd of embryo recipients. j equine vet sci 2020;89:103087. doi: 10.1016/j.jevs.2020.103087 3. speights ks, tipton ba, bubb rr, clark-price sc, et al: quantitative analysis of stress following student palpation per rectum in the mare. national veterinary scholars symposium. 2022:285. 4. ille n, aurich c, aurich j: physiological stress responses of mares to gynecologic examination in veterinary medicine. j equine vet sci 2016;43:6–11. doi: 10.1016/j.jevs.2016.04.010 5. van vollenhoven e, fletcher l, page pc, et al: heart rate variability in healthy, adult pony mares during transrectal palpation of the reproductive tract by veterinary students. j equine vet sci 2017;58:68–77. doi: 10.1016/j.jevs.2017.08.013 6. bond rl, midla lt, gordon ed, et al: effect of student transrectal palpation on early pregnancy loss in dairy cattle. j dairy sci 2019;102:9236–9240. doi: 10.3168/jds.2019-16515 7. berghold p, möstl e, aurich c: effects of reproductive status and management on cortisol secretion and fertility of oestrous horse mares. anim repro sci 2007;102:276–285. doi: 10.1016/j. anireprosci.2006.11.009 8. guinnefollau l, bolwell cf, gee ek, et al: horses’ physiological and behavioural responses during undergraduate veterinary practical teaching classes. appl anim behav sci 2021;241:105371. doi: 10.1016/j.applanim.2021.105371 9. van vollenhoven e, grant cc, fletcher l, et al: salivary glucocorticoid and fecal glucocorticoid metabolite concentrations in pony mares during transrectal palpation of the reproductive tract by veterinary students. j equine vet sci 2018;70:7–12. doi: 10.1016/j. jevs.2018.07.010 10. nagel c, ille n, aurich j, et al: teaching of diagnostic skills in equine gynecology: simulator-based training versus schooling on live horses. theriogenology 2015;84:1088–1095. doi: 10.1016/j. theriogenology.2015.06.007 11. annandale a, annandale ch, fosgate gt, et al: training method and other factors affecting student accuracy in bovine pregnancy diagnosis. j vet med educ 2018;45:224–231. doi: 10.3138/ jvme.1016-166r1 http://dx.doi.org/10.58292/ct.v15.9597 https://doi.org/10.1016/j.jevs.2020.103087 https://doi.org/10.1016/j.jevs.2016.04.010 https://doi.org/10.1016/j.jevs.2017.08.013 https://doi.org/10.3168/jds.2019-16515 https://doi.org/10.1016/j.anireprosci.2006.11.009 https://doi.org/10.1016/j.anireprosci.2006.11.009 https://doi.org/10.1016/j.applanim.2021.105371 https://doi.org/10.1016/j.jevs.2018.07.010 https://doi.org/10.1016/j.jevs.2018.07.010 https://doi.org/10.1016/j.theriogenology.2015.06.007 https://doi.org/10.1016/j.theriogenology.2015.06.007 https://doi.org/10.3138/jvme.1016-166r1 https://doi.org/10.3138/jvme.1016-166r1 the future is coming! new technologies being applied to reproduction in animals and humans the future is coming! new technologies being applied to reproduction in animals and humans katrin hinrichs department of clinical studies, new bolton center university of pennsylvania school of veterinary medicine, kennett square, pa abstract advances in genome sequencing, gene editing and computational analyses have brought the genetic future rushing toward us. gene editing is currently being used extensively in research, to explore function of genes via overand under-expression, and to produce animal disease models and animals useful for production of medically important components. through computational analysis of nucleotide sequence versus performance, disease or other morphological characteristics, tens of thousands of genetic variants associated with a large number of traits have been identified in many species, and selection based on genetic merit, even in preimplantation embryos, is ongoing. advances are also being made in understanding and manipulation of primordial germ cell and oocyte development, such that functional oocytes have been produced from somatic cells; and in extended in vitro culture of embryos past the implantation stage. combined with work on development of an artificial womb, the potential to achieve extra corporeal pregnancy seems plausible. many of these advances appear to warrant veterinary, breed registration or ethical oversight, so it is important that veterinarians be aware of new achievements in these areas. keywords: oocyte, embryo, stem cells, gene editing, uterus gene editing claudia klein at the 2019 society for theriogenology annual conference presented an in-depth review of methods for gene editing and provided useful references regarding these techniques.1 gene editing methods have come to the forefront; most useful are talens (transcription activator-like effector nucleases) and crispr (clustered regularly interspaced short palindromic repeats). talens are proteins. they recognize specific sections of dna via similar mechanisms to those that transcription factors use: the protein structure of the talen binds only to a specific nucleotide sequence. talens are linked to effector proteins. crispers are rna nucleotide sequences. one section of the crisper is designed to be complementary to the target dna nucleotide sequence and so recognizes and binds to that specific section of dna; the second section of the crispr molecule is associated with an effector protein. both talens and crisprs work via the effector proteins associated with them. for gene editing, the linked effector protein is an endonuclease (an enzyme that cuts dna); the specific endonuclease cas9 (crispr-associated protein 9) is typically linked to crispr. when a talen or crispr is bound to the recognized nucleotide sequence, the endonuclease will cut the dna at a specific nucleotide, adjacent to the location of the talen or crispr. since the first use of guided endonucleases to induce site-specific dna breaks,2-4 many other kinds of effector proteins have been linked to these targeting molecules: proteins that cause a gene to “turn on” and start transcription; proteins that inhibit transcription and even proteins that deliver epigenetic markers (reviewed5). linkage of these proteins to targeting molecules that recognize specific nucleotide sequences allows the investigator to modify the dna sequence, gene activity, or epigenetic profile of targeted areas of genome. the most straightforward use of gene editing tools is to create a mutation that causes a gene to be inactive. for this, editing tools are designed to make a double-stranded cut in dna, which the cell finds difficult to repair while maintaining fidelity. double-stranded repair thus is typically associated with changes to the nucleotide sequence resulting in reading frame shifts in dna, and thus in a mutation that can completely inactivate gene. it is also possible to change the sequence of a gene in a directed way, by making a break and providing the cell with multiple copies of a template, i.e. pieces of dna with the desired nucleotide sequence. the cell uses the template to direct repair of the cut section, resulting in the desired genome clinical theriogenology • volume 12 number 3 • september 2020 173 sequence in some cells. remarkably, it is not necessary in all cases to provide a template; if the homologous chromosome has a different sequence for the targeted gene (which thus is not affected by the editing tool), mammalian cells can use the homologous chromosome as a template to fix a double strand break.6 in this way, cells that are heterozygous for an undesired mutation can “fix” the gene sequence in mutated gene, without introduction of foreign dna. gene editing tools can be used to produce animals with a desired genetic modification in 2 main ways: 1) by editing somatic cells, selecting a cell with the desired modification, and performing somatic cell nuclear transfer (scnt); or 2) by injecting the editing tool into a zygote or early embryo, thereby directly editing the genome of the zygote/embryo and thus all resulting embryonic cells. a third area of intense interest is using these tools to correct disease-related genetic mutations in living animals (gene therapy). gene editing has been used in almost all domestic species, including horses (embryos with knockout for myostatin, produced by scnt)7 and dogs (gene therapy to correct the muscular dystrophy gene;8 production of myostatin-knockout dogs via zygote injection).9 it has also been applied to a wide variety of nondomestic animals, including macaques, ferrets, koi, zebrafish, squid, shrimp beetles, butterflies, and bees, to study genetic factors related to their behavior and health.10,11 the ethics of gene editing to “fix” mutated genes in human embryos is currently being debated. problems with polled gene-edited calves stories involving gene editing have recently been in the news. an american company, recombinetics, published a report in 2016, announcing production of 2 crossbred dairy bulls produced via scnt with somatic cells edited with talens, that were consequently homozygous for the polled allele.12 the company termed this method “precision breeding” (http://recombinetics.com/naturallyhornless-cattle/). they proposed that this would be a good model for introduction of gene-edited livestock into food production, as progeny of these animals would not require debudding to remove horns, a procedure with animal welfare concerns. however, genome sequencing of the gene-edited bulls’ progeny(university of california, davis) revealed a bacterial antibiotic-resistance gene in some calves’ dna (apparently, transferred to cells during the editing process) that was not detected in the original testing of the bulls.13 these findings represent a dramatic setback to possibility of acceptance of geneedited livestock for enhancement of production, both because of presence of “off-target” changes in the animals’ dna and because the problem was not revealed during initial genetic testing. birth of gene-edited human babies in a second event regarding gene editing, a scientist in china, he jiankui, announced via a video released on youtube in november, 2018, the birth of human twins which he claimed had undergone gene editing as zygotes. the editing was to induce a genetic modification, δ32 mutation in the ccr5 gene, which when naturally occurring confers resistance to entry of a common variant of hiv virus. this human experimentation violated chinese regulations banning genome editing on human embryos, as well as basic scientific and ethical guidelines (https://www.nytimes.com/2018/12/05/health/crispr-geneediting-embryos.html) and was condemned by scientists in china and abroad.14 the scientific ethics committee of academic divisions of the chinese academy of sciences stated that “the theory is not reliable, the technology is deficient, the risks are uncontrollable, and ethics and regulations prohibit the action.”14 the gene-edited embryos were apparently biopsied to confirm presence of mutation, and then transferred, resulting in birth of twin girls. however, further evaluation of the process used revealed that the editing process was not precisely directed, and that both children have a mosaic of different mutations of the targeted gene, which may or may not confer resistance to hiv. he jiankui was fired by his university, and in december 2019 was sentenced to 3 years in prison; the court found that this researcher and his collaborators forged ethical approvals and did not inform the physicians transferring the embryos of their origin. clinical theriogenology • volume 12 number 3 • september 2020174 use of gene-edited mice and time-lapse photography to study embryo development the study of embryo development has progressed markedly with the use of time-lapse, withinincubator photography, currently utilized extensively in human assisted reproductive technology (art; reviewed15) and recently applied to clinical equine ivp embryos.16-20 work in mouse embryos has gone a step further: using the precise and efficient new gene editing tools, mice have been produced in which key cytoplasmic components were labeled with fluorescent markers. combining these markers with fluorescent molecular labels and using time-lapse fluorescent microscopy, it is possible to visualize changes in cytoskeletal, nuclear and cell-lineage specific components during embryo development.21 using information gained in this manner, scientists are beginning to outline morphogenetic mechanisms involved in early embryo and blastocyst development.22 embryo biopsy, testing and selection cattle the availability of immense amounts of production data on bulls, cows, and their progeny has allowed identification of hundreds of thousands of genetic markers (single polymorphic nucleotides, snps); tens of thousands of which have been associated with desirable traits, both in beef and dairy cattle. microarrays for rapid detection of production-related snps are marketed commercially and have been used since the mid 2000s23 to select calves for genetic merit at birth, resulting in rapid and meaningful gains in productivity.24 these genetic selection methods are now being applied to embryos, via cells derived from embryo biopsy, to allow selection of embryos before transfer.25 the ability to select embryos with high genetic merit, combined with the ability to recover competent oocytes from gonadotropin-treated heifer calves as young as 2 months of age for in vitro embryo production,26 are expected to result in greatly accelerated genetic gain through decreasing generation time. horses methods for effective biopsy and genetic analysis of both in vitro-produced and in vivo-derived equine embryos have been developed.27,28 currently, this procedure is used clinically, mainly for testing embryos for presence of disease-related mutations29 or for fetal sex.30 because horses lack the immense database found in cattle, information on genetic markers for performance in horses is more difficult to generate; however, an increasing amount of information is available in this area. in a 2010 report, variants of myostatin gene, apparent as snps, were found to be strongly associated with success of horses at different racing distances.31 subsequently, equine snps were identified that are associated with “gaitedness”, e.g. found in horses that deviate from standard two-beat trot and instead perform running walk, rack, singlefoot, etc.;32 with size, e.g. miniature, pony, horse, draft horse;33 breed type;34 and with selection for speed in australian thoroughbreds, via snps in a chromosome location associated with neuromuscular junction signaling.35 because effective methods for embryo biopsy are being developed in horses, the potential for preimplantation genetic selection in horses, as done in cattle, is growing.. humans preimplantation genetic testing (pgt) has been performed on in vitro-produced embryos since the 1990’s in couples known to carry disease-related mutations.36 the term pgt refers to evaluating genetics of cells recovered on biopsy of embryo (embryonic blastomere of an early-cleavage embryo -no longer used because of deleterious effects on embryo -or trophoblast of a blastocyst). pgt can be divided into several types of assessments. testing for a specific allele, typically 1 carrying a diseaserelated mutation (recently re-termed pgt-m; m for monogenic) was developed first. subsequently, methods to evaluate the entire genome of cells obtained by biopsy were introduced, to determine aneuploidy (duplication or deletion of chromosome segments or entire chromosomes). such genome-wide screening is currently termed pgt-a (a for aneuploidies). this screening was initially done by comparative genomic hybridization and is now done via genome sequencing. the jury is still out as to whether employing pgt-a to screen embryos before transfer actually increases the chances of live birth, clinical theriogenology • volume 12 number 3 • september 2020 175 as there can be aneuploidies in the trophoblast that are not reflected in the embryo proper and the time needed for results to be available precludes fresh transfer of embryos.37 the ability to evaluate the entire genome of embryos, however, has led to the possibility of selecting embryos for desired traits. gender selection of human embryos (without a medical reason such as to avoid an x-linked disease) is illegal in some countries, but is legal, and offered, in other countries, including the us. human art labs are already offering selection for eye color and possibly other characteristics, and methods to select other traits, including intelligence (selection against genes known to be associated with a low iq), have been developed and companies plan to market them (https://genomicprediction.com/). human art centers may soon be calculating a polygenic “risk score” for each embryo, based on known genetic markers of disease susceptibility, to allow selection of embryos likely to produce the healthiest individuals. recent advances with application to species conservation major barriers for scnt use in conservation of endangered species are: 1) availability of oocytes from appropriate species, i.e. oocytes having species-specific mitochondria and cytoplasm compatibility with species’ genome, to use as host oocytes for scnt; and 2) availability of a suitable recipient female – that is, a female that is fertile, cyclically synchronous with the embryo, capable of gestating the transferred embryo and in which embryo transfer can be performed (for example, there is no established method to transfer embryos to the rhinoceros uterus). however, several recent advances have potential for application in this area: production of functional oocytes from somatic cells this possibility in mice was reported in 2012.38 somatic cells were first induced to be pluripotent by inserting genes coding for pluripotency factors, then these induced pluripotent stem cells (ipscs) were caused to differentiate to primordial germ cell-like cells (pgcs). these were aggregated with embryonic ovarian cells and transplanted to the ovarian bursa of recipient females. the somatic-origin cells differentiated to oocytes and were recovered from the recipient ovary at the germinal vesicle stage and matured in vitro, fertilized in vitro and yielded live young. in 2019, production of oocytes from somatic cells (granulosa cells) without transgenesis (using only chemical stimulation), using a similar series of procedures, was reported.39 this is a huge leap forward toward eventual use of somatic cells for derivation of species-specific oocytes, even from totally extinct species. many zoological parks have tissue or fibroblast samples from numerous species stored in liquid nitrogen; these somatic cells could be used for production of species-specific oocytes. lack of embryonic ovarian cells from desired species to use for aggregating with somatic-origin pgcs should not be a barrier, since aggregation of oocytes, recovered from primordial follicles, with ovarian cells of other species is compatible with production of functional oocytes.40 the prospect of deriving oocytes from somatic cells in this manner has great application in assisted reproduction to aid conservation of endangered species. mitochondrial-identical oocytes could be used as host oocytes for somatic cell nuclear transfer, to produce clones using stored tissue samples. even more exciting is prospect of such oocytes could be fertilized using stored sperm from males selected to produce offspring with greatest genetic diversity possible. in this manner, the population genetic diversity could be maximized and the genetics of animals for which only sperm is available could be reintroduced into the population. production of embryo-like structures directly from stem cells the term “totipotent” is used to refer either to a cell capable of developing into a complete organism (e.g. a fertilized oocyte), or to a cell that can differentiate into any cell type of an organism. however, it was felt that these are 2 different issues.41 an oocyte can develop into entire organism after fertilization or nuclear transfer, whereas an embryonic stem cell (capable of contributing to any tissue of organism) cannot generate and organize entire organism. the term “plenipotent” has been proposed for this second, less capable cell.41 clinical theriogenology • volume 12 number 3 • september 2020176 however, recent work has begun to blur these lines. production of embryo-like structures from aggregated human stem cells is possible.42 through a gel-based 3-d environment created by a microfluidic device, stem cells were induced to recapitulate early aspects of epiblast and amnion development, including cell line differentiation, lumen formation, polarity in the embryonic sac, specification of primordial germ cells and, notably, cells with primitive streak markers. derivation of induced pluripotent stem cells from somatic cells has been reported in several endangered species, including mandrill and northern white rhinoceros.43 thus, in the future, embryos might be derived directly from pluripotent stem cells. this opens the door to use of stored somatic cells to produce mitochondrial-identical clones of endangered and even extinct species without need for oocytes, or for going through the laborious steps of deriving primordial germ cells and then oogonia from stem cells. culturing embryos in vitro past blastocyst stage monkey embryos have been cultured in vitro for up to 20 days.44 embryos underwent differentiation to developmentally-appropriate cell lines and stages, including embryonic disc formation, amniotic and yolk sac cavitation, and differentiation of primordial germ cell-like cells. human embryos have also been cultured in vitro past 7 days, with cell lineage differentiation, but culture was terminated at 14 days for ethical reasons.45,46 the ability to culture embryos in vitro to the point of differentiation and cell lineage specification is both unsettling, in terms of producing fetuses “in a test tube” and promising, in terms of being able to propagate embryos from endangered species without the need for a suitable recipient female. it appears that research will continue to push the ability to support embryonic development in vitro further and further into pregnancy. artificial womb an in vitro method to support development of lambs during last 4 weeks of pregnancy has been developed.47 lambs were removed from the uterus and placed in a sterile polyethylene bag that was cannulated to allow continuous fluid exchange in an artificial “amnion,” and an oxygenating circuit was connected to the fetal umbilical cord. extensive work was performed to optimize pressures, circuit flows, oxygenation and other mechanical aspects to reproduce the environment of the gravid uterus and support normal development of the lamb. with the optimized system, lambs at a stage equivalent to extreme prematurity in human infants survived for 4 weeks, had stable hemodynamics and normal growth, including lung development and brain maturation. lambs were removed, ventilated and then euthanized to assess tissue development. the potential application of this technique in art for endangered species is immense. as in vitro early embryo culture methods continue to progress further toward supporting embryo development through the placental and fetal stages, perhaps these methods for supporting the fetus after organogenesis and umbilical cord formation will be applicable earlier and earlier in pregnancy, with the eventual possibility of supporting the entire pregnancy of an embryo/fetus without a species-compatible recipient female. conclusion in many ways, science is continuing to explore techniques proposed for decades by sciencefiction writers in an imagined future. embryo production in vitro is commonplace; genetic selection is being used widely in cattle and is also utilized in human embryos, currently largely to avoid transferring embryos with devastating genetic diseases. gene editing is being performed for research but has much interest from commercial sector, including human art. it is difficult to predict future applications of these approaches; the ethical lines that society draws in these areas appear to move, as past innovative technology becomes commonplace and accepted. conflict of interest: none to report. clinical theriogenology • volume 12 number 3 • september 2020 177 references 1. klein c: a primer to genome editing. clinical theriogenology 2019;11:181-185. 2. gasiunas g, barrangou r, horvath p, et al: cas9-crrna ribonucleoprotein complex mediates specific dna cleavage for adaptive immunity in bacteria. proc natl acad sci u s a. 2012 sep 25;109:e2579-e2586. 3. cong l, ran fa, cox d, et al: multiplex genome engineering using crispr/cas systems. science 2013;339:819-823. 4. christian m, cermak t, doyle el, et al: targeting dna double-strand breaks with tal effector nucleases. genetics 2010;186:757-761. 5. broeders m, herrero-hernandez p, ernst mpt, et al: sharpening the molecular scissors: advances in gene-editing technology. iscience 2020;23:100789. 6. wu y, liang d, wang y, et al: correction of a genetic disease in mouse via use of crispr-cas9. cell stem cell 2013;13:659-662. 7. vichera g, viale d, olivera r, et al: generation of myostatin knockout horse embryos using clustered regularly interspaced short palindromic repeats/crispr-associated gene 9 and somatic cell nuclear transfer. reprod fertil dev 2019;31:136. 8. amoasii l, hildyard jcw, li h, et al: gene editing restores dystrophin expression in a canine model of duchenne muscular dystrophy. science 2018;362:86-91. 9. zou q, wang x, liu y, et al: generation of gene-target dogs using crispr/cas9 system. j mol cell biol 2015;7:580-583. 10. reardon s: welcome to crispr's gene-modified zoo. nature 2016;mar 10 2016:https://www.scientificamerican.com/article/welcome-to-crispr-s-gene-modified-zoo/. 11. prabhune m: crispr'd animals: uncommon model organisms in genetic research. the bench 2019;september 18, 2019:https://www.synthego.com/blog/crispr-animals. 12. carlson df, lancto ca, zang b, et al: production of hornless dairy cattle from genome-edited cell lines. nat biotechnol 2016;34:479-481. 13. young ae, mansour ta, mcnabb br, et al: genomic and phenotypic analyses of six offspring of a genome-edited hornless bull. nat biotechnol 2020;38:225-232. 14. li j-r, walker s, nie j-b, et al: experiments that led to the first gene-edited babies: the ethical failings and the urgent need for better governance. j zhejiang univ sci b 2019;20:32-38. 15. milewski r, ajduk a: time-lapse imaging of cleavage divisions in embryo quality assessment. reproduction 2017;154:r37-r53. 16. marzano g, mastrorocco a, zianni r, et al: altered morphokinetics in equine embryos from oocytes exposed to dehp during ivm. mol reprod dev 2019;86:1388-1404. 17. lewis n, schnauffer k, hinrichs k, et al: morphokinetics of early equine embryo development invitro using time-lapse imaging, and use in selecting blastocysts for transfer. reprod fertil dev 2019;12;1851-1861. 18. brooks ke, daughtry bl, metcalf e, et al: assessing equine embryo developmental competency by time-lapse image analysis. reprod fertil dev 2019;12:1840-1850. 19. martino na, marzano g, mastrorocco a, et al: use of time-lapse imaging to evaluate morphokinetics of invitro equine blastocyst development after oocyte holding for two days at 15 degrees c versus room temperature before intracytoplasmic sperm injection. reprod fertil dev 2019;12:1862-1873. 20. meyers s, burruel v, kato m, et al: equine non-invasive time-lapse imaging and blastocyst development. reprod fertil dev 2019;12:1874-1884. 21. zenker j, white md, gasnier m, et al: expanding actin rings zipper the mouse embryo for blastocyst formation. cell 2018;173:776-791. 22. white md, zenker j, bissiere s, et al: instructions for assembling the early mammalian embryo. dev cell 2018;45:667-679. 23. schaeffer lr: strategy for applying genome-wide selection in dairy cattle. journal of animal breeding and genetics = zeitschrift fur tierzuchtung und zuchtungsbiologie. 2006;123:218-223. 24. garcía-ruiz a, cole jb, vanraden pm, et al: changes in genetic selection differentials and generation intervals in us holstein dairy cattle as a result of genomic selection. proc natl acad sci u s a 2016;113:e3995-e4004. 25. shojaei saadi ha, vigneault c, sargolzaei m, et al: impact of whole-genome amplification on the reliability of pretransfer cattle embryo breeding value estimates. bmc genomics. 2014 oct 12;15:889. 26. bernoco d, bailey e: frequency of the scid gene among arabian horses in the usa. anim genet 1998;29:41-42. 27. choi yh, gustafson-seabury a, velez ic, et al: viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis. reproduction 2010;140:893-902. 28. choi yh, penedo mct, daftari p, et al: accuracy of preimplantation genetic diagnosis in equine in vivo-recovered and in vitro-produced blastocysts. reprod fertil dev 2016;28:1382-1389. 29. choi yh, park rh, penedo mc, et al: progress report on clinical application of preimplantation genetic diagnosis in 36 equine embryos. j equine vet sci 2018;66:191. 30. riera fl, roldan je, espinosa jm, et al: application of embryo biopsy and sex determination via polymerase chain reaction in a commercial equine embryo transfer program in argentina. reprod fertil dev 2019;12:1917-1925. 31. hill ew, gu j, eivers ss, et al: a sequence polymorphism in mstn predicts sprinting ability and racing stamina in thoroughbred horses. plos one. 2010 jan 20;5:e8645. clinical theriogenology • volume 12 number 3 • september 2020178 32. andersson ls, larhammar m, memic f, et al: mutations in dmrt3 affect locomotion in horses and spinal circuit function in mice. nature 2012;488:642-646. 33. makvandi-nejad s, hoffman ge, allen jj, et al: four loci explain 83% of size variation in the horse. plos one. 2012;7:e39929. 34. gurgul a, jasielczuk i, semik-gurgul e, et al: a genome-wide scan for diversifying selection signatures in selected horse breeds. plos one. 2019;14:e0210751. 35. han h, mcgivney ba, farries g, et al: selection in australian thoroughbred horses acts on a locus associated with early two-year old speed. plos one. 2020;15:e0227212. 36. handyside ah, kontogianni eh, hardy k, et al: pregnancies from biopsied human preimplantation embryos sexed by y-specific dna amplification. nature 1990;344:768-770. 37. toft clf, ingerslev hj, kesmodel us, et al: a systematic review on concurrent aneuploidy screening and preimplantation genetic testing for hereditary disorders: what is the prevalence of aneuploidy and is there a clinical effect from aneuploidy screening? acta obstet gynecol scand 2020;feb10. 38. hayashi k, saitou m: generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. nat protocols 2013;8:1513-1524. 39. tian c, liu l, ye x, et al: functional oocytes derived from granulosa cells. cell rep 2019 2019/12/24/;29:4256-67.e9. 40. eppig jj, wigglesworth k: development of mouse and rat oocytes in chimeric reaggregated ovaries after interspecific exchange of somatic and germ cell components. biol reprod 2000;63:1014-1023. 41. condic ml: totipotency: what it is and what it is not. stem cells and development. 2014;23:796-812. 42. zheng y, xue x, shao y, et al: controlled modelling of human epiblast and amnion development using stem cells. nature 2019;573:421-425. 43. ben-nun if, montague sc, houck ml, et al: induced pluripotent stem cells from highly endangered species. nat methods 2011;8:829-831. 44. niu y, sun n, li c, et al: dissecting primate early post-implantation development using long-term in vitro embryo culture. science 2019;nov15;366. 45. deglincerti a, croft gf, pietila ln, et al: self-organization of the in vitro attached human embryo. nature 2016;533:251-254. 46. shahbazi mn, jedrusik a, vuoristo s, et al: self-organization of the human embryo in the absence of maternal tissues. nat cell biol 2016;18:700-708. 47. partridge ea, davey mg, hornick ma, et al: an extra-uterine system to physiologically support the extreme premature lamb. nat commun 2017 apr 25;8:15112. clinical theriogenology • volume 12 number 3 • september 2020 179 006_ms-006 hinriches transcervical insemination in bitch transcervical insemination in bitch stuart mason, nicole rous, aimee lougher monash veterinary clinic, oakleigh east, victoria, australia abstract to perform a successful transcervical insemination, the operator must be able to accurately stage the estrous cycle, have a thorough understanding of the anatomy of the bitch and confidence to perform the procedure diligently. estrous cycle timing is best undertaken using a combination of lh assay, vaginal cytology, progesterone assay and vaginoscopy. transcervical insemination results in higher pregnancy rates with both fresh semen when compared to vaginal insemination and frozen semen when compared to surgical (laparotomy) insemination. using this technique, a pregnancy rate of 86% was attained from 1850 inseminations using fresh semen with an average of 5.4 puppies per litter. in addition, utilising the same insemination technique, a pregnancy rate of 77% was attained from 940 inseminations when > 150 million frozen-thawed dog sperm were inseminated. in regards to frozen semen, there was a trend for greater pregnancy rate with increasing sperm numbers; however, in this study, there was no benefit to using > 150 200 million sperm. keywords: frozen semen, bitch, canine, artificial insemination, transcervical insemination introduction transcervical insemination (tci) in the bitch is becoming more common in practice, with increased availability of endoscopic equipment and increased awareness of the welfare of breeding bitches. in order to perform a successful tci, the operator must be able to accurately stage the estrous cycle, have a thorough understanding of the anatomy of the bitch and confidence to perform the procedure diligently. results are presented here related to 1850 fresh semen tci’s, 111 chilled semen tci’s and 940 frozen semen tci’s over a 14 year interval. timing of insemination the most common cause of pregnancy failure in the bitch is poor timing of insemination or breeding.1 bitches spontaneously ovulate a primary oocyte arrested in prophase i of the second stage of meiosis.2, 3 the luteinizing hormone (lh) surge occurs ~ 2 days before ovulation.4,5 the lh surge stimulates granulosa cells to luteinise and produce progesterone, which may aid in resumption of meiosis of the oocytes.3 after ovulation, a corpus luteum forms due to luteinization of thecal and granulosa cells. the corpus luteum secretes progesterone, the hormone solely responsible for pregnancy maintenance in the bitch. six days after ovulation, the bitch enters diestrus.6 laboratory tests and procedures for staging bitch’s cycle lh assay lh concentrations are undetectable until the point of the lh surge. daily serum analysis of lh is required due to the short, varied, duration of the lh surge in the bitch (24 60 hours). there is a rising phase of up to 12 24 hours and then a decline over 12 36 hours.7 the only readily available lh tests are qualitative lh tests, which give a positive result at concentrations > 1 ng/ml. even with daily lh testing, it is possible to not detect the lh surge, since lh concentrations vary at the peak of the lh surge (4 14 ng/ml7,8), and there is often a slow rise and fall. therefore, lh assays on their own are not recommended, since the lh surge occurs 3 8 days before oocyte fertilisation,8 coupled with the possibility of anovulatory cycles. progesterone assay progesterone concentrations rise from the lh surge and can be used as an indirect indicator of ovulation. progesterone continues to rise through estrus and early diestrus, with the cervix closing under the influence of increasing progesterone. progesterone is most commonly measured today by chemoluminescence, with serum being recommended rather than plasma.9 collection of blood for progesterone is best done on a fasted sample. refrigeration of samples within the first 2 hours after collection will reduce measured progesterone concentrations.9 203 clinical theriogenology • volume 11, number 3 • september 2019 the lh surge occurs at ~6 nmol/l (2 ng/ml) and ovulation at 15 25 nmol/l (4 8 ng/ml), with ovulation being deemed complete with values > 30 nmol/l (10 ng/ml).10 in the authors’ opinion, there is no ‘magical’ value of progesterone at which fertility is maximal; changes in values are more important to help identify ovulation. consistency of progesterone analysis is important, as different analytical machines will give different results. vaginal cytology the cytology of the vagina changes as a result of changing estrogen concentrations from developing ovarian follicles causing hyperplasia of the vagina, represented by increasing cornification of vaginal cytology samples. as the vagina undergoes hypertrophy, surface cells suffer effects of reduced oxygenation which presents as increasing enlargement of the cytoplasm, nuclear pyknosis and cornification of surface cells.11 percentage of anuclear cells increases to 50% of the cornified cells by the start of estrus. six days after ovulation, diestrus ensues, characterised by an influx of neutrophils and parabasal cells, reducing the percentage of cornified vaginal epithelial cells.6 vaginal cytology alone is a poor indicator of the ideal time to breed;6 however, in conjunction with progesterone assays and vaginoscopy, it aids in determination of the phase of the cycle (proestrus, estrus, diestrus or anestrus) and the appropriateness of insemination.12,13 vaginoscopy vaginoscopy is performed using a 25 cm long, 11 mm diameter welch allyn sigmoidoscope. under the influence of rising progesterone and increasing estrogen concentrations after the lh surge, the deep oedematous folds of proestrus, begin to flatten out and reduce. as estrogen continues to decline and progesterone continues to rise through estrus, the hypertrophy and hyperplasia of the vagina reduces, resulting in a cobblestone appearance of the vagina (crenulation), indicating ovulation is complete and the bitch is in the fertile period. by the start of diestrus, the vagina returns to a flat state with no significant folds in the mucosa.14 to determine the appropriate time for breeding/insemination a combination of progesterone assay, lh assay, vaginal cytology, and vaginoscopy are used.12 transcervical insemination procedure anatomy of the bitch understanding the anatomy of the bitch is important to ensure a successful tci. the external genitalia of the female begins at the vulva, the entrance to the vestibule. the vestibule contains the clitoris and the urethral opening. the vestibule is quite short, with the lumen heading at approximately a 45° angle from horizontal towards the lumbar vertebrae of the bitch. the vestibule terminates at the cingulum which is important for ensuring a tied mating. from the cingulum, the vagina extends forwards approximately parallel to the lumbar spine. the vagina of the bitch is quite long and extends to the level of the cervix. caudal to the cervix are 3 dorsomedian folds which consist of 3 tubercles (caudal, middle and cranial). the cranial vagina ends at the cervix, which protrudes caudally into the vagina, creating a cavity between the cervical os and the cranial vagina (fornix). the cervical lumen extends cranially from the cervical os, and in most cases, the cervical os faces down towards the floor of the vagina. the uterus of the bitch is bicornuate, extending cranially from the short body attached to the cervix in 2 distinct horns to the uterine tubes and ovaries. transcervical insemination procedures there are 2 described methods of transcervical insemination: the norwegian catheter and endoscopic assisted transcervical insemination. results of tci presented in this paper were all via the endoscopic assisted method. norwegian catheter norwegian catheter (gartnerservice, haslum, norway, jan@basbergdata.no) was the first developed nonsurgical intrauterine device in 1975.15 it consists of an outer nylon sheath with an inner metal stylet with a blunted, rounded distal tip. the procedure is performed in any size of bitch without the need of any anesthesia. with the bitch standing, restrained on a table, the operator will 204clinical theriogenology • volume 11, number 3 • september 2019 transabdominally palpate the cervix of the bitch while passing the catheter into the vagina of the bitch with the opposing hand. while holding the cervix steady with the thumb and index finger, the stylet is passed into the cervical os and through the cervix of the bitch. the semen is then inseminated through the stylet into the uterine lumen.16,17 the procedure is quick, simple and cheap to perform; however, the learning curve is steep and the procedure difficult on large breed bitches. unlike surgical insemination, the procedure may be performed more than once if required, as there is no anesthesia or surgery.17 stimulation of the vestibule during this procedure is likely to stimulate uterine contractions resulting in an insemination process more like a natural mating than that obtained by surgical insemination.18 the norwegian catheter may be used for fresh, fresh chilled or frozen semen. the majority of published reports on the use of frozen semen in the bitch pertain to the use of the norwegian catheter.17,19-23 endoscopic assisted transcervical insemination a procedure commonly known as tci, was first described in 1993 with the use of a cystoscope.24 endoscopic assisted transcervical insemination (eiu) is simple to learn with persistence and care; however, it is a technique wherein all steps must be monitored closely to maximize results.25, 26 nowadays, most operators use a uterorenoscope, as this endoscope is longer and thinner (in diameter) than the cystoscope, allowing it to be used more adequately in all bitches from toys to giant breeds.25,26 the cystoscope was often too large in diameter for toy bitches and too short to reach the cervix of large bitches. the procedure is performed with the bitch standing, restrained on a table. using a uterorenoscope, cold xenon, halogen, or lcd light source, and camera with images projected onto a monitor, the operator is able to pass the endoscope to the level of the cervical os. the endoscope is initially passed into the vagina and at this point, air is instilled into the vagina via a rectal insufflation pump attached to the endoscope (30200 rectal insufflation bulb welch allyn, skaneatele, ny). the use of a vaginal shunt (minitube, verona, wi) may aid in sealing the vagina to maintain vaginal distension. the endoscope is advanced cranially past the dorsomedian folds to the cervix. the cervical os is visualised on the ventral aspect of the cervix in most cases. a ch 5 (or ch 4) tci catheter (minitube) is advanced through the endoscope and through the cervical os. once the catheter is within the cervix, the stylet is removed and the catheter only advanced into the body of the uterus. the tci catheter has marks at increments of 1 cm to aid in positioning the opening to the catheter within the body of the uterus. the semen is then inseminated through the catheter, slowly, watching for leakage at the cervical os,24-27 and massaging the vulva to promote uterine contractions. the semen is inseminated first, followed by prostate fluid (fresh semen; if prostate fluid is not diseased) or additional extender (fresh chilled, frozen semen) until the uterus is deemed full (appearance of fluid at the cervical os).25,26 slow insemination will allow for insemination of large volumes.26 eiu is suitable for fresh, fresh chilled and frozen semen. eiu is more successful than surgical insemination when performed appropriately,25 and eiu is more successful than vaginal insemination with fresh semen.28,29 unlike with the norwegian catheter, with eiu, the cervix is visualised so it is guaranteed the catheter is intrauterine 100% of the time.24-27 why perform transcervical insemination? comparative studies of vaginal insemination of fresh semen with eiu have ~ 33 35% higher pregnancy rate with eiu.28,29 this is likely a result of semen not actually being put at the cervical os in many vaginal inseminations, and additionally intravaginal insemination requires sperm to traverse the cervix into the uterus, in contrast to tci wherein the sperm are placed directly intrauterine. it is most probable that with intravaginal insemination, a reduced number of sperm traverse the cervix into the uterus than the number placed in the vagina. this loss of sperm entering the uterus is counteracted by the recommendation of > 1 vaginal insemination; however, pregnancy results are still lower than tci.28,29 although transcervical insemination by eiu or norwegian catheter can be repeated, there is no benefit to > 1 insemination, unless timing of insemination is poor.19,20 with frozen semen use in bitches, many advocates of surgical insemination have discounted tci as wasting more semen by doing the procedure twice (compared to 1 surgical insemination). however, pregnancy rate of 1 tci is widely published,23-27,30 and more recently, pregnancy rate of tci compared to surgical 205 clinical theriogenology • volume 11, number 3 • september 2019 insemination.25 the authors recommend performing 1 intrauterine insemination with optimal timing of insemination.26 publications describing 2 inseminations by norwegian catheter or eiu were only recommended and performed in situations when timing was poor (to cover a larger possible window of fertilization)19,20 or when quality of semen was poor.25 if timing of insemination is poor, and intrauterine insemination occurs in diestrus, there is both a reduced expected pregnancy rate22,31,32 and an increased probability of inducing ceh and/or pyometra whether it be by laparotomy, eiu or norwegian catheter.33-36 surgical insemination continues to be advocated by many, as sperm are placed closer to the oviduct than with tci. however, studies on uterine motility concluded site of insemination within the uterus is irrelevant.37,38 the higher success rate of tci with frozen semen than surgical insemination is most likely due to lack of stress of surgery and anaesthesia, the presence of uterine contractions during the procedure and lack of effects of wound healing and inflammation.18,25,26 aside from the reduced success rate, ethical issues of surgical insemination (anesthesia, surgery, healing, risk of death from anesthesia, production of anti-sperm antibodies via blood sperm contact) lead many to question why the procedure continues to be advocated and hence why tci should be promoted. results of 1850 fresh semen, 111 chilled semen and 940 frozen semen tci’s (2005 2019) using the aforementioned technique of timing and insemination, pregnancy rates were attained as detailed in table 1 (fresh and chilled semen) and table 2 (frozen semen). table 1. results of tci inseminations using fresh and chilled semen in the bitch. these results are presented and analysed in relationship to the number and quality of sperm inseminated. however, it must be remembered that many other factors affect the outcome of any artificial insemination such as, but not limited to, genetic history, uterine health, health status of the male when frozen, age of frozen semen when used and health status of the bitch inseminated through estrus and early pregnancy. most of this information was unavailable in data collection for this study and hence is not discussed. fresh semen results to the authors’ knowledge, there is no other study with such a large number of results of fresh semen inseminations using the endoscope assisted technique. until now, most large published studies pertaining to the use of fresh semen in the bitch are related to vaginal and transcervical insemination using the norwegian catheter.16,21,22 two papers comparing pregnancy rate of fresh semen via vaginal insemination to eui reported a higher pregnancy rate with eui, with pregnancy fresh semen chilled semen total number performed 1850 111 average motility (%) 80.4 (39 95) 70.5 pregnancy rate (%) 86 67 pregnancy rate < 150 x 106 sperm (%) 82 pregnancy rate > 150 x 106 sperm (%) 88 67 pregnancy rate < 70% motility (%) 68 75 pregnancy rate > 70% motility (%) 87 64 resorption numbers (%) 0.8 0 average number sperm x 106 730 (25 3318) 513 (155 1290) average number of pups 5.42 (1 19) 6.67 (4 11) 206clinical theriogenology • volume 11, number 3 • september 2019 table 2. results of tci inseminations using frozen semen in the bitch. rates similar to those attained in this study.28,29 when analysing pregnancy, rate, there was no significant difference between the under and over 150 million progressively motile sperm (p = 0.5189, graphpad quickcalc), however, there was a significant difference when samples were used with > 70% progressively motile sperm than < 70% progressively motile sperm (p < 0.05, graphpad quickcalc). this concurs with current recommendations for sperm motility for optimal results. there were very few samples with < 150 million progressively motile sperm in this study, and this is likely to have impacted the analysis of inseminating over or under 150 million progressively motile sperm with fresh semen. frozen semen results pregnancy rates for frozen semen are consistent with those reported in other studies.17,19-23,25,26,29,40 as per previous studies, there is a trend with greater pregnancy rate with more progressively motile sperm inseminated.25,26 however in contrast to other studies,39,40 there was no significant difference in pregnancy rate with > 200 million progressively motile sperm (p = 1, graphpad quickcalc). this may be due to the small numbers of inseminations in the >200 million group, however as comparable results (pregnancy) were attained to other studies,17,19-23,25,26 and higher results than those advocating the use of > 200 million sperm,39,40 use of > 200 million sperm is possibly unwarranted. it is the authors’ belief that adequate sperm should be used. however, conservation of sperm for future pregnancy attempts is equally important. additionally, use of semen with < 40% progressive motility or < 30% progressive motility did not significantly reduce pregnancy rates (p = 0.0564 and p = 0.2566 respectively, graphpad quickcalc); however, there was a trend to higher pregnancy rate with higher motility in thawed samples. conclusion with good estrous cycle timing, semen handling and insemination technique, pregnancy rates in bitches using fresh, chilled or frozen semen can be maximised. conflict of interest none to declare. references 1. johnson sd, root-kustritz mv, olson pns: editors. canine and feline theriogenology. philadelphia: saunders; 2001. 2. tsutsui t: gamete physiology and timing of ovulation and fertilization in dogs. j reprod fertil suppl 1989;39:269-275. frozen semen total number performed 940 average motility (%) 50.87 pregnancy rate (%) 69 pregnancy rate < 100 million sperm (%) 62 pregnancy rate 100 150 million sperm (%) 70 pregnancy rate > 150 million sperm (%) 77 pregnancy rate 150 200 million sperm (%) 80 pregnancy rate > 200 million sperm (%) 70 pregnancy rate < 40% pms (%) 57 pregnancy rate > 40% pms (%) 76.60 pregnancy rate < 30% pms (%) 55 resorption (%) 18 average number sperm x 106 127 (10 781) average number of pups 5.04 (1 16) 207 clinical theriogenology • volume 11, number 3 • september 2019 3. reynaud k, fontbonne a, marseloo n, et al.: in vivo meiotic resumption, fertilization and early embryonic development in the bitch. reproduction 2005;130:193-201. 4. phemister rd, holst pa, spano js, et al.: time of ovulation in the beagle bitch. biol reprod 1973;8:74-82. 5. concannon pw, mccann jp, temple m: biology and endocrinology of ovulation, pregnancy and parturition in the dog. j reprod fertil suppl 1989;39:3-25. 6. root kustritz mv: managing the reproductive cycle in the bitch. vet clin north am small anim pract 2012;42:423-437. 7. concannon pw: endocrinologic control of normal canine ovarian function. reprod domest anim 2009;44 suppl 2:3-15. 8. concannon pw, hansel w, visek wj: the ovarian cycle of the bitch: plasma estrogen, lh and progesterone. biol reprod 1975;13:112-121. 9. volkmann dh: the effects of storage time and temperature and anticoagulant on laboratory measurements of canine blood progesterone concentrations. theriogenology 2006;66:1583-1586. 10. jeffcoate ia, lindsay fe: ovulation detection and timing of insemination based on hormone concentrations, vaginal cytology and the endoscopic appearance of the vagina in domestic bitches. j reprod fertil suppl 1989;39:277-287. 11. olson pn tm, wykes pm, nett tm: vaginal cytology. part i. a useful tool for staging the canine oestrous cycle. compend contin educ pract vet 1984;6:288-298. 12. goodman mf: canine ovulation timing. probl vet med 1992;4:433-444. 13. silva ld, onclin k, verstegen jp: cervical opening in relation to progesterone and oestradiol during heat in beagle bitches. j reprod fertil 1995;104:85-90. 14. lindsay fef: the normal endoscopic appearance of the caudal reproductive-tract of the cyclic and non-cyclic bitch post-uterine endoscopy. journal of small animal practice 1983;24:1-15. 15. andersen k: insemination with frozen dog semen based on a new insemination technique. zuchthygiene 1975;10:1-4. 16. linde-forsberg c: editor artificial insemination. esavs-evssar-envn reproduction in companion, exotic and laboratory animal; nantes:2005. 17. linde-forsberg c: achieving canine pregnancy by using frozen or chilled extended semen. vet clin north am small anim pract 1991;21:467-485. 18. england gc, moxon r, freeman sl: stimulation of mating-induced uterine contractions in the bitch and their modification and enhancement of fertility by prostatic fluid. reprod domest anim 2012;47 suppl 6:1-5. 19. thomassen r, farstad w, krogenaes a, et al.: artificial insemination with frozen semen in dogs: a retrospective study. j reprod fertil suppl 2001;57:341-346. 20. thomassen r, sanson g, krogenaes a, et al.: artificial insemination with frozen semen in dogs: a retrospective study of 10 years using a non-surgical approach. theriogenology 2006;66:1645-1650. 21. linde-forsberg c, forsberg m: results of 527 controlled artificial inseminations in dogs. j reprod fertil suppl 1993;47:313-323. 22. linde-forsberg c, forsberg m: fertility in dogs in relation to semen quality and the time and site of insemination with fresh and frozen semen. j reprod fertil suppl 1989;39:299-310. 23. linde-forsberg c, strom holst b, govette g: comparison of fertility data from vaginal vs intrauterine insemination of frozen-thawed dog semen: a retrospective study. theriogenology 1999;52:11-23. 24. wilson ms: non-surgical intrauterine artificial insemination in bitches using frozen semen. j reprod fertil suppl 1993;47:307-311. 25. mason sj, rous nr: comparison of endoscopic-assisted transcervical and laparotomy insemination with frozenthawed dog semen: a retrospective clinical study. theriogenology 2014;82:844-850. 26. mason sj: a retrospective clinical study of endoscopic-assisted transcervical insemination in the bitch with frozen-thawed dog semen. reprod domest anim 2017;52 suppl 2:275-280. 27. wilson ms: transcervical insemination techniques in the bitch. vet clin north am small anim pract 2001;31:291-304. 28. eilts b, editor fertility in a canine breeding colony using vaginal artificial insemination or transcervical insemination of fresh semen. anzcvs science week; 2013; gold coast, queensland, australia. 29. daskin a, tekin n, akcay e: the effect of transcervical intrauterine and intravaginal insemination methods on fertility in dogs. turkish journal of veterinary & animal sciences 2003;27:235-239. 30. pretzer sd, lillich rk, althouse gc: single, transcervical insemination using frozen-thawed semen in the greyhound: a case series study. theriogenology 2006;65:1029-1036. 31. tsutsui t, takahashi f, hori t, et al.: prolonged duration of fertility of dog ova. reprod domest anim 2009;44 suppl 2:230-233. 32. verstegen jp, silva ld, onclin k: determination of the role of cervical closure in fertility regulation after mating or artificial insemination in beagle bitches. j reprod fertil suppl 2001;57:31-34. 33. nomura k: histological evaluation of canine deciduoma induced by silk suture. j vet med sci 1995;57:9-16. 34. nomura k: induction of a deciduoma in the dog. j vet med sci 1994;56:365-369. 35. nomura k, funahashi h: histological characteristics of canine deciduoma induced by intrauterine inoculation of e. coli suspension. j vet med sci 1999;61:433-438. 36. nomura k, nishida a: histological variations of canine deciduoma induced in non pregnant horn at different stages of unilateral pregnancy. j vet med sci 1998;60:623-626. 37. fukushima fb, malm c, henry m, et al.: site of intrauterine artificial insemination in the bitch does not affect sperm distribution within the uterus. reprod domest anim 2010;45:1059-1064. 208clinical theriogenology • volume 11, number 3 • september 2019 38. tsutsui t, shimizu t, ohara n, et al.: relationship between the number of sperms and the rate of implantation in bitches inseminated into unilateral uterine horn. japanese journal of veterinary science 1989;51:257-263. 39. burgess dm, mitchell ke, thomas pg: coeliotomy-assisted intrauterine insemination in dogs: a study of 238 inseminations. aust vet j 2012;90:283-290. 40. hollinshead fk, hanlon dw: factors affecting the reproductive performance of bitches: a prospective cohort study involving 1203 inseminations with fresh and frozen semen. theriogenology 2017;101:62-72. 209 clinical theriogenology • volume 11, number 3 • september 2019 210clinical theriogenology • volume 11, number 3 • september 2019 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 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[ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice mare reproductive loss syndrome: septic setae or toxin? mare reproductive loss syndrome: septic setae or toxin? stuart burns,a lenn harrisonb aburns equine veterinary service, paris, ky buniversity of kentucky veterinary diagnostic laboratory, lexington, ky abstract abortion is characteristic of mare reproductive loss syndrome and equine amnionitis and fetal loss syndrome. exposure to lepidopteran caterpillars has been associated with these syndromes and 2 hypotheses were proposed. penetrating septic setae hypothesis asserted that caterpillar setae served as a mechanical means of introducing pathogenic bacteria into fetoplacental unit causing abortion. a second hypothesis suggested that an unidentified toxin was responsible for inciting inflammatory tissue changes, resulting in abortion. paper will summarize historical perspectives, to evaluate whether oral bacteria can induce an epithelial or endothelial response, to discuss inflammatory cascade process (implicated in abortion) and provide information for lack of evidence for setae or bacteria as causative agents (not able to induce inflammation) for these syndromes. keywords: caterpillars, abortion, septic setae, toxin, bacteria introduction mare reproductive loss syndrome (mrls) was described during 2001 and 2002 breeding seasons in ohio river valley involving ohio, kentucky, tennessee and west virginia. in decreasing order of frequency, mrls had 5 clinical presentations: early pregnancy loss, late pregnancy loss, fibrinous pericarditis, panophthalmitis and actinobacillus meningoencephalitis.1 resulting progeny loss altered a 50 year economic model of thoroughbred marketing when largest sales company in lexington, ky terminated its summer sale of most elite yearlings due to a high rate of fetal loss in mares with early conception dates (february and march of 2001) that would have provided more mature yearlings for july sales. further expenses incurred in search for mrls etiology totaled, from all sources, $1,903,553 ($694,615 from the kentucky thoroughbred association/kentucky thoroughbred owners and breeders and agriculture development board; $295,938 from grayson-jockey club research foundation; $80,000 from university of kentucky; and $423,000 in 2002 plus $410,000 in 2003 from the usda agriculture research service).2 affected tissues usually had some degree of inflammation. no lesions were identified as pathognomonic. fifteen to 20% of mrls late pregnancy losses had no identifiable lesions (harrison 2005 review of mrls case records of university of kentucky veterinary diagnostic laboratory ([ukvdl] in 2001 and 2002, unpublished data). septic setae and toxin hypotheses remain unproven1 and it has not been determined if causative factor is mechanical transport of bacteria into gravid uterus, noxious effect of a toxin, or combination of both.3 septic setae hypothesis asserts that horses consume live eastern tent caterpillars (etc; malacosoma americanum) that are covered by fine hairs (setae). setae separate from caterpillars in mare’s digestive tract and penetrate intestinal mucosa, mechanically carrying with them pathogenic bacteria. setae have small barbs that interact with motile tissue, enabling them to “ratchet” their way through epithelium and carry pathogenic bacteria into blood stream. septic material is then spread throughout body. small setae fragments also enter circulation and these contaminated fragments penetrate capillary endothelium to introduce bacteria into the placenta, pericardium or ocular globe. bacteria proliferate in invaded sites and cause early and late pregnancy losses, pericarditis and panophthalmitis, collectively called mrls.4 as oral commensal bacteria (alpha streptococcus and actinobacillus) were isolated in mrls late abortions, perhaps these bacteria were transported hemotogeneously from mare’s oral cavity to fetoplacental unit. hence, an experiment was designed to determine if intravenous administration of oral bacterium can cross protective capillary endothelial barrier in pregnant mares. clinical theriogenology • volume 12 number 2 • june 2020102 bacteria isolation of bacteria from late pregnancy losses was a most consistent finding; it occurred in 90% of mrls cases examined.5 alpha streptococci were isolated from 223 cases (51%), actinobacillus from 74 (17%) and a combination of these 2 organisms from 8 (1.8%) cases. in total, these organisms were associated with 70.4% of all mrls abortions in 2001.6 records on 3527 abortions submitted to ukvdl revealed that 628 abortions were caused by bacterial infections. presence of alpha streptococci in 26, and actinobacillus in 14 cases,7 a frequency of only 1.6%. thus, presence of alpha streptococci and actinobacillus represents a unique component of mrls rarely observed in other cases of bacteria-induced equine abortion. alpha streptococci and actinobacillus spp. are common oral commensal microflora that act as biological barriers to infection by competing with pathogens for food and space on digestive tract mucosa.8 these organisms are adapted to live on tissue surfaces, including oral mucosa, where they easily outcompete pathogens.9 cell walls of these 2 species of bacteria do not contain pathogen associated molecular patterns (pamps) that are associated with pathogenic microbes. absence of these patterns allows these bacteria to live on oral mucosa without stimulating an inflammatory response from oral mucosal epithelium. materials and methods an experiment in compliance with usda terrestrial animal health standards commission and chapters 7 and 8 under the use of animals in research and education (p. 1 12), was designed to evaluate effects of an alpha streptococcal septicemia on vascular endothelium and fetal survival. a dental wedge was used to expose oral cavity of a pregnant mare to obtain swab samples. samples were collected with a covered swab (kalayjian industries inc., signal hill, ca) passed over her soft palate mucosa and streaked onto blood agar (becton, dickinson and company, sparks, md). this mare had a previous history of mrls and possibly harbored a unique strain of alpha streptococcus not present in normal population. alpha streptococcus colonies were selected from resulting mixed growth based on morphology, chemical analysis and incomplete hemolysis of red blood cells on culture plates. these colonies were recultured in pure form on blood agar. individual colonies of alpha streptococccus were then removed from the surfaces of 4 culture plates using a sterile swab and mixed in 250 ml of sterile saline. three pregnant mares were given 10, 20, and 40 ml of this stock bacterial suspension intravenously, daily for 3 consecutive days, whereas 1 control mare received 40 ml sterile saline intravenously. mares at 40, 47, 55, and 60 days of pregnancy were selected for this experiment, as ultrasonically observable fetal fluid echogenicity from days 30 85 is a reliable indicator for agent’s presence causing mrls.1 immediately after first injection, stock suspension was streaked onto blood agar using a 1 µl calibrated loop. concentration of colony forming units in suspension was calculated to be 4 6 x 109/ml. an intradermal injection of 0.2 ml of this bacterial suspension was followed by a biopsy 96 hours after injection. each mare’s rectal temperature was recorded at 12 hour intervals and complete blood counts and fibrinogen concentrations were done every 24 hours for first 5 days. transrectal ultrasonography (5 mhz transducer) was performed every 48 hours over 20 days to evaluate fetal heartbeat and fetal fluid echogenicity. results biopsy revealed that dermis contained multifocal perivascular cuffs of low numbers of neutrophils and minimal to low numbers of macrophages, lymphocytes, and eosinophils. inflammatory cells infrequently moved into adjacent dermis. endothelial cells were occasionally hypertrophied and vascular lumen contained marginated neutrophils. infectious agents were not overtly evident. fetal fluids remained anechoic in both untreated control mares and all 3 mares injected with alpha streptococcus. mares had no febrile responses nor hematological changes. fibrinogen, a marker for inflammation, did not increase in any mare and none had clinical signs of septicemia nor response to intravenous infusion of bacterial suspension. all remained pregnant 30 days after last administration of alpha streptococcus suspension and delivered normal foals. clinical theriogenology • volume 12 number 2 • june 2020 103 discussion unaffected endothelial barrier prevented transfer of bacteria to fetoplacental unit. alpha streptococcus from oral cavity must somehow breach oral mucosal epithelium to gain entry into bloodstream for transport to distant sites in the body where mrls is expressed. neutrophils in oral cavity possess iga fe receptors at higher levels than neutrophils in blood. azurophilic granules contain myeloperoxidase, defensins, neutral serine proteases, lysozyme and bactericidal/permeability increasing protein. these azurophilic granules are primarily designed for killing microorganisms, although some specific granules also have ability to solubilize collagen.10 increased oral mucosal permeability is caused by this neutrophilic activity, but alpha streptococccus isolated from mrls abortions did not appear to have ability to initiate such a neutrophilic response. an increased mucosal permeability or physical penetration is necessary for alpha streptococcus on mucosal surface to cross through mucosal epithelium. this altered permeability was not induced. pure cultures of alpha streptococcus at this dosage and duration were unable to affect vascular endothelium or increase capillary permeability. unaffected endothelial barrier prevented transfer of bacteria to fetoplacental unit. conclusion alpha streptococcus was unable to penetrate vascular endothelium and enter the fetoplacental unit to cause mrls unless transported by some other means. setae urticating agents associated with lepidopteran setae may reduce ability of gastrointestinal mucosa and epithelial barriers of placenta to prevent bacteraemia.11 septic setae hypothesis suggests that etc setae serve as mechanism that breaches oral mucosal epithelium and vascular endothelium to allow bacteria to move from mare’s oral cavity to fetus. setae of etc are a visual and tactile cue that caterpillar is both toxic and unpalatable.12 a second function of these setae is to disrupt airflow over cuticle, thereby reducing heat loss by convection (fitzgerald 2004 personal communication). etc setae are soft and are not shed. they are covered by microscopic projections oriented toward tip of setae. thus, setae would have to migrate through tissue blunt end first for these projections to act as “barbs”, unlike processionary caterpillar (pc; ochrogaster lunifer) where projections face base of setae, enabling tip to penetrate moving tissue while barbs prevent retrograde movement.13 these setae are hollow, filled with an unidentified toxin and easily shed. setae of etcs are composed of inert substances that, by themselves, induce minimal inflammatory tissue changes. “urticating hairs” on caterpillars are so because of presence of some irritant causing molecules produced by the caterpillar, not by setae alone.14 setae alone are unable to stimulate an acute inflammatory response (diaz 2006, personal communication). etc setae were ground to a powder to allow passage through a 25 gauge needle and resuspended in sterile saline. this mixture was then sterilized by 5kgy ionizing radiation. a series of intradermal injections of this material containing 2 µg etc setae (0.2 µg setae protein) and 20 µg etc setae (2 µg seatae protein) were administered. subsequent welt size was evaluated to demonstrate an inflammatory response at 0, 6, 24, 30, 48, and 54 hours post injection and biopsies were obtained 24 hours post injection. none of injections containing setae alone induced skin welts, thereby indicating that no acute inflammatory response was produced by exposed tissue cells. setae fragments caused some necrosis and elicited a foreign body response.15 tissue migration of etc setae in experimental situations appeared to be limited to no deeper than mucosa or submucosa of intestinal tract, as observed in horse, pigs,16 goats, and rats.1 presence of setae in mucosa and submucosa induced formation of microgranulomas which are defined as “an inflammatory lesion characterized by chronic inflammation with mononuclear cell infiltration and extensive fibrosis”.17 there was no evidence of deeper migration of etc setae into the tissue. presence of setae-induced microgranulomas in intestinal mucosa and submucosa has only been demonstrated in experimental settings when etcs were fed or gavaged. no microgranulomas containing setae were reported in any adult equine gastrointestinal tracts examined at ukvdl in 2001 and 2002. caterpillar setae are inert and cannot induce inflammation beyond a foreign body response. circulating setae fragments would have to clinical theriogenology • volume 12 number 2 • june 2020104 escape from the microgranulomas and enter circulation to then breach the vascular endothelium and allow entry of bacteria into placenta, pericardial fluid and anterior and posterior chambers of the globe. setae alone have no ability to induce active inflammation. setae must gain entry into the oral cavity before they can serve as mechanical means to breach oral epithelium and vascular endothelial barriers. it is suggested that horses ingest caterpillars unknowingly. however, equids are highly selective feeders, able to sort out individual components of mixed feeds that an individual may not prefer. this is accomplished by using highly sensitive olfactory receptors and a vomeronasal organ (in nasal passages) that is very responsive to pheromones and other airborne molecules. horses have sensitive vibrissae on their muzzles that enable them to identify small plants for ingestion and avoid consumption of foreign materials. their sense of taste has not been as fully investigated, but palatability preferences are known. sense of taste, texture and smell would suggest ability to sense and avoid consuming foreign substances (gustafson 2016, personal communication) such as 1 g caterpillars, unless they were a preferred item. there are no reports of horses in their natural environment willingly consuming etcs, in spite of widespread exposure to them in 2001 and 2002. furthermore, horses chose to avoid consuming water contaminated by etcs if a source of fresh water was also available. horses will consume macerated or whole etcs that are mixed with sweet feed in an experimental setting,16 but this does not represent natural exposure or confirm ingestion in their normal environment. it was unlikely horses would readily consume etcs in a field setting.18 conclusion setae of etc are soft, inert and not shed. they appear unable to migrate through tissue any deeper than the submucosa. there is no evidence to support consumption, unless mixed with feed. therefore, we inferred that setae do not contribute to the etiology of mrls. some other explanation must be sought to identify method of passage of bacteria through epithelial/endothelial barrier, possibly inflammation. inflammation because some degree of inflammation in affected tissues was most frequently observed, it was widely held that etiological agent(s) must be capable of inducing an inflammatory response. pathogenesis of this syndrome may be found among inflammatory cascade of molecular and cellular responses that lead to inflammation. evaluation through this matrix may clarify relative role of setae, bacteria or toxin(s). a brief review of events that occur during an inflammatory cascade will aid in evaluation of both hypotheses. increased capillary permeability can be caused by inflammation. any inflammatory process must start with an insult to epithelial or endothelial cells. this insult may be traumatic, hypothermic, hyperthermic, and toxic or infectious agents.8 damaged cells produce multiple molecular signals that initiate inflammatory response. these include free heparin sulphate, normally restricted to intact cell membrane and mast cell granules. this plus fibrinogen and cellular proteins are released which activate macrophages to produce proinflammatory cytokines. these substances are recognized by toll-like receptors (tlrs) located on cell membranes of multiple cell types such as leucocytes, epithelial cells and endothelial cells. thrombin is released by damaged cells, initiating procoagulation activity and acting on fibrinogen to produce insoluble strands of fibrin that are deposited within capillaries. histamine is released by mast cell granules that stimulate production of glycoprotein p-selectin receptors from endothelial cells. this protein binds with l-selectin on neutrophils’ surface, causing them to slow down within capillaries and eventually stop and become bound to vascular endothelium by platelet activating factor (paf). dead or damaged cells release adenosine that activates mast cells to degranulate and release heparin and proinflammatory cytokines. damaged cell walls release phospholipids that react with phospholipase to produce arachidonic acid. this interacts with 5-lipooxygenase to create 4 leukotrienes, all potent proinflammatory molecules. three of these are vasoactive and increase vascular permeability. arachidonic acid also reacts with cyclooxygenase to produce 4 proinflammatory prostaglandins. molecules released by damaged tissue serve as signals to immune system’s “sentinel” cells (macrophages, clinical theriogenology • volume 12 number 2 • june 2020 105 mast cells and dendritic cells), vast majority of which are in connective tissue and/or just under any epithelial or endothelial barrier. these sentinel cells are the primary source of inflammatory cytokines and chemokines.9 tumor necrosis factor alpha (tnfα) is key mediator of acute inflammation. this family of inflammatory cytokines contains at least 18 related proteins that regulate cellular activation. tnfα family has a broad systemic effect, as tnfα receptors are on almost all nucleated cells. this family of cytokines is main stimulant to macrophages and mast cells to produce interleukin 1 (il1) and interleukin 6 (il6), both of which are potent inflammatory cytokines. tnfα in combination with il1 triggers critical changes in endothelium of small blood vessels to induce microvascular thrombosis and capillary leakage. chemokines are proinflammatory cytokines, with 50 chemokines identified. their primary roles are as chemotactic factors and leukocyte activators. production of inflammatory cytokines, leukotrienes, and prostaglandins serve as overlapping and redundant signals to immune system that tissue insult and damage has occurred. this inflammatory cascade is similar in response to any tissue damage, either infectious or noninfectious. all tissue damage must be managed by innate branch of immune system. introduction of infectious agents extends inflammatory response to involve acquired immunity branch of immune system. main difference between these 2 branches of immunity lies in their use of receptors to recognize invaders. innate system uses preexisting receptors on macrophages, neutrophils, dendrite cells and natural killer cells that are able to bind to molecular patterns common to many microbes. cell walls of gram positive organisms are characterized by complex peptidoglycans, whereas cell walls of gram-negative organisms contain peptidoglycans covered by a layer of lipopolysaccharides. innate system receptors bind to these molecular patterns that are not present in mammalian tissues. these arrangements are called pamps, recognized by receptors as foreign and “dangerous”. important receptors are toll-like receptors (tlrs) on macrophages, mast cells, dendritic cells, eosinophils and epithelial cells in respiratory and intestinal mucosa. there are at least 10 pre-existing tlr types that have specific binding sites for specific pamps. known tlrs, alone or in combination, can recognize almost all known pathogens. binding of tlrs and pamps causes cell genes to form and release cytokines in various quantities, qualities and combinations in response to specific antigens.17 it is this genetic control that makes an organism pathogenic in 1 species, but not in another. this sequence of events is summarized in the flow sheet that describes the inflammatory cascade (figure). notice how many paths lead to increased capillary permeability and leakage; this paradigm can explain all unusual components observed in mrls. conclusion some toxic irritant(s) that could induce the signs of inflammation, resulting in increased capillary permeability, would allow bacteria to enter the fetoplacental unit. unidentified irritants associated with lepidopteran caterpillars may provide this toxin. other observations twenty eight mares aborted in australia in 2004 following exposure to pc. these abortions were named equine amnionitis and fetal loss syndrome (eafl). mares involved were mid late pregnancy and clinical presentation and necropsy findings were identical to mrls cases observed 2 years previously in us.19 most mares aborted with no signs of impending abortion, but some had signs of mild colic prior to onset of delivery. inflammatory changes in amnion and amniotic portions of umbilical cord, and inner surface of allantoic lining of allantochorion were observed. there were variable pathological changes in fetus and frequent recovery of multiple species of environmental bacteria, usually nonpathogenic, from fetus and/or placenta.19 nonpathogenic isolates were also characteristic of mrls. clinical theriogenology • volume 12 number 2 • june 2020106 thrombin insoluble leucocytes strands of tlrs fibrin epithelial cells fibrinogen endothelial cells cellular activate proinflammatory cytokines protein macrophage heparin sulphate histamine p-selectin + paf neutrophils bind to endothelium l selectin cxcl 8 neutrophils insult to epithelial/ endothelial tnf +il-1 capillary microthrombosis and leakage cells heparin and proinflammatory cytokines adenosine tlrs on mast cells tryptase + tnf vasodilation and leakage dead or damaged cells 4 leukotrienes phospholipids of which 3 are vasoactive + phospholipase + 5-lipooxygenase stimulates macrophages, mast cells and inflammatory dendritic cells cytokines, arachidonic acid tnf, il-1, il-6 etc. cyclooxydase 4 proinflammatory prostaglandins figure. inflammatory cascade (adapted9 with permission) clinical theriogenology • volume 12 number 2 • june 2020 107 processionary caterpillar was confirmed as causative factor of eafl experimentally by gavaging 100 g of whole pcs into 4 mares between 194 and 205 days of pregnancy daily for 5 days;3 3 mares aborted within 11 days after first administration. subsequently, 4 groups (3 mares per group) were gavaged with 0, 1, 2, or 5 g of shed pc exoskeleton daily for 5 days. one mare of each group given 1 or 2 g of pc exoskeleton aborted and 2 of 3 mares given 5 g exoskeleton aborted.3 furthermore, early pregnancy loss, a feature of mrls but not of field cases of eafl, was produced in 2 of 6 mares between 45 and 55 days of pregnancy, on days 6 and 7 following gavages of 5 g pc exoskeleton daily for 5 days.3 tissues were examined from 4 mares, 1 untreated control and 3 gavaged with 50 g of whole pcs daily for 5 days.11 setae and/or fragments were present in all regions of gastrointestinal tract of treated mares, but none in control. endometritis was present in treated mares and setae fragments were observed in 1 mare’s uterus. tissue response to presence of setae ranged from none to microabcesses. horses are not only animals sensitive to etiological agent(s) of mrls. pregnant pigs could also be affected by exposure to etcs.16 five gilts in mid-pregnancy were each fed 40 g/day of macerated etcs for 10 days; 2 of 5 treated gilts aborted their entire litters, whereas none of 5 untreated control gilts aborted. treated gilts were necropsied 1 3 days after abortion. streptococci were isolated from fetuses of 2 litters that aborted and also from those in 3 litters that did not abort. caterpillar setae were observed in alimentary tract of all 3 gilts fed etcs. a syndrome similar to mrls and eafl was reported in dromedary camels in western sahara.20 rainfall is scarce in this desert region but when it occurs, it is accompanied by rapid growth of acacia trees. even more infrequently, this plant growth supports an outbreak of caterpillars of lasiocampipae family (etcs and pcs belong to this family) responsible for mrls and eafl. camels ingest these caterpillars while browsing on acacia leaves, following which, pregnant camels have acute abortions or deliver weak calves. some of these newborn calves exhibit bilateral hyphemia, also observed in some foals affected by mrls. many of these compromised newborn calves suffer a failure of passive antibody transfer, followed by sepsis and death. this syndrome, called duda by native pastoralists, has been recognized for many years, though apparently no formal investigations have been undertaken. mice were evaluated as a potential animal model suitable for mrls research.21 mice do not abort their conceptuses but resorb them in response to a stressor. mice were 5 days pregnant and each test group was treated for 14 days. group 1 (n = 4) received 0.5 g saline by gavage and group 2 (n = 4) received 0.4 saline by intraperitoneal injection. group 3 (n = 7) received 19 mg fresh caterpillar frass by gavage. group 4 (n = 8) received 200 mg frozen etcs by gavage and group 5 (n = 7) received 20 setae plucked from frozen etcs and injected intraperitoneally. gilmer type gland located at base of each setae may serve as a storage site for a chemical toxin. mice were euthanized on day 19 of pregnancy and necropsied. mice in groups 1, 2, 3, and 4 had no abnormalities of uterus or fetuses, whereas 3 of group 5 mice that received intraperitoneal injections of setae had resorbed all their fetuses and had acute suppurative inflammation at implantation sites; bacterial isolates from fetuses were seratia marcescens and lantoea agglumerans. this experimental design could not determine if resorptions were in response to setae alone or to a toxin associated with setae. conclusion “septic setae hypothesis” specifies that bacteria that become septic are pathogens. furthermore, 70% of isolates associated with mrls were nonpathogenic oral commensals that do not stimulate an inflammatory response from a normal immune system. if mrls is an infectious disease, it should comply with koch’s postulates. more than 20 microorganisms were isolated from various tissues in mrls cases of 2001and 2002. no bacteria could be isolated from 16% of mrls cases.6 proposed infectious etiology failed to comply with koch’s postulates in that syndrome can apparently occur with no bacteria present. it is further unlikely that 20 bacteria could all induce an identical clinical and tissue response to qualify as multiple etiological agents. evidence therefore suggests that bacteria are not etiological agents of mrls as they, alone, appear unable to cross an intact epithelial or endothelial barrier. hence, they would require clinical theriogenology • volume 12 number 2 • june 2020108 mechanical assistance from penetrated setae. septic setae hypothesis was questionable, as it appeared that setae, bacteria, or a combination thereof induced inflammation associated with mrls. increased capillary permeability caused by inflammation would allow septic bacteria to cross both epithelial and endothelial barriers. setae, bacteria, and/or unidentified toxin associated with etc appear to induce inflammation when consumed by horses, pigs, and camels and when injected intraperitoneally in mice. absence of an identified toxin, observations that camels will ingest whole caterpillars, that both mrls and eafl can be reproduced by ingestion or gavage of lepidopteran caterpillars, that clinical and tissue responses to this exposure are identical and that setae were present in 1 mare’s uterus suffering eafl, all lend support to septic setae hypothesis currently favored for mrls/eafl. however, evidence suggests that neither bacteria or setae, or a combination thereof, is the etiological agent(s) of these syndromes. tissues affected by mrls have an extensive capillary supply adjacent to anatomical structures that provide an enclosed compartment containing fluid i.e. placenta with allantoic and amniotic fluids, pericardium with pericardial fluid and globe containing vitreous. this, coupled with increased capillary permeability and microthrombosis caused by inflammatory cascade, are the foundation of mrls. acknowledgement laboratory support was donated by robert henthorn’s bluegrass veterinary laboratory. funding of animals, their care and miscellaneous expenses were provided by burns family trust. conflict of interest authors have no conflicts of interest. references 1. sebastian mm, bernard wv, riddle tw, et al: environmental pathology review paper: mare reproductive loss syndrome. vet pathol 2008;45:710-722. 2. brown k: mrls research funding exceeds $2 million: more needed. http//www.bloodhorse.com/horseracing/articles/179878/mrls-research-funding-exceeds-2-million-more-needed 2003. 3. cawdell-smith aj, todhunter kh, anderson st, et al: caterpillars and equine fetal loss: an australian perspective. tenth international symposium on equine reproduction 2010; p. 349-350. 4. tobin t, harkins jp, roberts jf, et al: the mare reproductive loss syndrome and the eastern tent caterpillar ii: a toxicokinetic/clinical evaluation and a proposed pathogenesis: septic penetrations setae. int j appl res vet med 2004;2:142-158. 5. williams nm, bolin dc, donahue jm, et al: gross and histopathological correlates of mare reproductive loss syndrome. first workshop on mare reproductive loss syndrome 2003; p. 24-25. 6. donahue j, sells s, giles r, et al: bacteria associated with mare reproductive loss syndrome: late fetal losses. first workshop on mare reproductive loss syndrome 2003; p. 27-28. 7. giles rc, donahue jm, hong cb, et al: causes of abortions, stillbirths and perinatal deaths: 3527 cases (1986-1991). j am vet med assoc 1993;203:1170-1175. 8. adams a: an overview on the immune system. role of immunology in equine health symposium 2015; p. 1-10. 9. tizard ir: how inflammation is triggered. in: tizard ir: editor. veterinary immunology: an introduction. 7th edition, philadelphia; elsevier: 2004. p. 10-23. 10. selders ss, fetz ae, radic mz, bowlin gl: an overview of the role of neutrophils in innate immunity, inflammation and host-biomaterial integration, regen biomater 2017;4:55-68. 11. todhunter kh, cawdell-smith aj, begg ap, et al: caterpillars and equine fetal loss: aspects of histology. tenth international symposium on equine reproduction 2010; p. 350-357. 12. fitzgerald td: predation and antipredation. in: eickwort gc: editor. the tent caterpillars, ithaca; comstock pub assoc: 1995. p. 167-203. 13. todhunter kh, cawdell-smith aj, bryden wl, et al: processionary caterpillar setae and equine fetal loss: histopathology of experimentally exposed mares. vet pathol 2014;51:1117-1130. 14. diaz jh: the evolving global epidemiology, syndromic classification, management, and prevention of caterpillar envenoming. am j trop med hyg 2005;72:347-357. 15. mcdowell k, williams n: immune system alteration associated with mrls, ktob foundation mrls 2005 update. 2005; p. 1-2. 16. mcdowell kj, webb ba, williams nm: invited review: the role of caterpillars in mare reproductive loss syndrome: a model for environmental causes of abortion. j anim sci 2010;88:1379-1387. clinical theriogenology • volume 12 number 2 • june 2020 109 17. tizard ir: macrophages and later stages of inflammation. in: tizard ir: editor. veterinary immunology: an introduction. 7th edition, philadelphia; elsevier: 2004. p. 34-48. 18. fitzgerald td, jeffers pm, mantella d: depletion of host-derived cyanide in the gut of the eastern tent caterpillar, malacosoma americanum. j chem ecol 2002;28:257-268. 19. perkins n: equine amnion and foetal loss. in: final report to the hunter valley equine research center, australian veterinary animal health service 2005; p. 16, 46-47. 20. volpato g, dinaro a, rossi d, saleh, et al: everybody knows; but the rest of the world: the case of a caterpillar-borne reproductive loss syndrome in dromedary camels observed by sahrawi pastoralists of western sahara. j ethnobiol ethnomed 2013;9:5. 21. sebastian m, harkins d, jackson c, et al: a laboratory animal model of mare reproductive loss syndrome: preliminary evaluation of a mouse model. first workshop on mare reproductive loss syndrome 2003. p. 51-53. clinical theriogenology • volume 12 number 2 • june 2020110 2019 reproductive microbiome alterations: canine pyometra   reproductive microbiome alterations: canine pyometra michelle kutzler oregon state university, corvallis, or abstract pyometra is a life-threatening uterine infection that affects 20 25% of reproductively intact bitches. canine pyometra is characterized by accumulation of leukocytes and exudate within the uterine lumen. although exact temporal and progressive mechanisms underlying its pathogenesis are not fully understood, a combination of endocrine, structural, inflammatory and bacterial factors are likely involved in most cases. pyometra develops during luteal phase; progesterone has a key role in establishment of infection with opportunistic bacteria. cystic endometrial hyperplasia results from proliferation of endometrial glands and is a predisposing factor for development of pyometra. endometrial proliferation factors exacerbate uterine innate immune responses. whereas escherichia coli is the most common organism isolated in up to 90% of cases with a positive culture, genes encoding for adhesins, toxins, and other factors increase virulence of certain strains. a presumptive diagnosis of pyometra is based on clinical signs and laboratory tests, with a confirmatory diagnosis based on b-mode ultrasonography, with or without doppler ultrasonography, detecting an enlarged, fluid-filled uterus with increased blood flow. broad-spectrum antibiotics with minimal nephrotoxicity are needed to prevent septicemia, but antibiotics alone will not resolve pyometra. safest and most effective treatment is surgical removal of infected uterus and ovaries. medical management of pyometra may be indicated to improve general status of the bitch prior to surgery or treatment in valuable breeding bitches. protocols using prostaglandin f2α or aglepristone are discussed in detail. keywords: aglepristone, cystic endometrial hyperplasia, escherichia coli, prostaglandin f2α, virulence factors introduction pyometra is a life-threatening bacterial infection of the uterus that affects 20 25% of reproductively intact bitches.1-5 however, incidence of pyometra may be > 50% in certain high-risk breeds, indicating that there may be genetic factors predisposing to development of pyometra.2 mean age for pyometra diagnosis in bitches is 7 years (range: 4 months to 18 years).2 etiopathogenesis canine pyometra is characterized by an accumulation of leukocytes and exudate within uterine lumen. although exact temporal and progressive mechanisms underlying pathogenesis are not fully understood, a combination of endocrine, structural, inflammatory and bacterial factors are likely involved in most cases. endocrine factors pyometra develops during luteal phase; progesterone has a key role for establishment of infection with opportunistic bacteria. during diestrus, progesterone induces endometrial proliferation, endometrial gland secretion, myometrial quiescence and cervical closure.6-7 progesterone not only inhibits endometrial bactericidal activity, it also specifically increase the affinity of endometrium to escherichia coli (e. coli).8 in addition, repeated exposure to endogenous and/or exogenous progestogens has an important role in pathogenesis of cystic endometrial hyperplasia, which also has a key role in development of pyometra.9-12 structural factors cystic endometrial hyperplasia (ceh) results from proliferation of endometrial glands and subsequent endometrial cyst formation. ceh is accompanied by severe changes in endometrial cell proliferation and apoptosis, coinciding with dysregulation in expression of apoptosis regulatory proteins (e.g. bcl2, bax).13 these alterations cause excessive proliferation, combined with deregulation of 639 clinical theriogenology • volume 11, number 4 • december 2019   apoptosis, which then causes insufficient endometrial regeneration.13 role of progestogens in pathogenesis of ceh is exacerbated by estradiol 17β.6-7 ceh is the most common uterine disease in dogs.14 incidence of ceh increases with age from < 4% in bitches under 3 years of age to > 50% in bitches by 7 years.15 it is important to note that not all cases of pyometra involve ceh. normal uterine defence mechanisms against pyometra include: (1) a potent local immune response with leukocytes and antibodies, (2) well-regulated endometrial secretions and (3) myometrial contractions.7 however, functional patency of the cervix also has an important structural role. with a functionally patent cervix (open pyometra), mucopurulent to hemorrhagic exudate can exit the uterus. when cervix is not patent (closed pyometra), bacterial laden exudate accumulates within uterine lumen, resulting in endometrial atrophy.16 closed pyometras are associated with more severe illness compared to open pyometras, but not poorer outcomes as measured by postoperative hospitalization.3 with respect to incidence, open pyometra occurs more commonly (65 75%) than closed pyometra (25 35%).3,5 inflammatory factors lipopolysaccharide (lps) from bacterial cell walls elicits a strong inflammatory response. uterine inflammation in response to lps is associated with upregulation of endometrial genes related to the innate immune response. in response to this inflammation, endometrial proliferation factors exacerbate the inflammatory reaction.17 these factors are: (1) insulin-like growth factor (igf1), (2) endometrial remodelling (via matrix metalloproteinases) and (3) inflammatory response genes (e.g. chemokine ligand 2 (cxcl2), secretory leukocyte peptidase inhibitor (spli)). based on global endometrial transcriptomic profiling by microarray, many genes are upregulated ~ 10 77 times in uteruses of bitches with pyometra.18 pyometra can be histologically classified as either hyperplastic or atrophic.19 endometrial thickness and ratio of endometrial to myometrial thickness are increased 3-fold in hyperplastic pyometra compared to a normal uterus.19 histologically, a uterus associated with atrophic pyometra has severe thinning of endometrial mucosa (with thin cuboidal epithelial cells) with concurrent hypertrophy of myometrium.19 these 2 forms have differential regulation of inflammatory cytokines and enzymes in the prostaglandin synthetic pathway in the endometrium.19 for example, a relative fold increase in interleukin 8 (il8) of 1.82 times occurs in atrophic pyometra compared to hyperplastic pyometra.19 in addition, expression of cyclooxygenase type-2 (cox2) and prostaglandin f synthase (pgfs) are significantly upregulated (3.75 and 3.15 times in atrophic pyometra compared to hyperplastic pyometra, respectively).19 this results in greater expression of proliferative ki-67 marker in endometrial stromal cells in response to the bacterial agent.13 during inflammation, cox2 is an important source of prostaglandins and thromboxane a2. cox-2 interacts directly with chemokines that are overexpressed in pyometra (e.g. cxcl8/il8, cxcl14).17 overexpression of cxcl14 contributes to inflammatory cell infiltration into uterine lumen, due to its chemoattractant action in monocytes and natural killer cells.20 however, cxcl10 has antimicrobial activity against e. coli.21 in stromal cells, cxcl10 may be involved in recruitment and potentiation of t helper 1 response.22 because il8 and cxcl14 are induced by cox2, selective cox2 inhibitors may mitigate inflammatory response during pyometra.17 cox2 is also downstream of toll-like receptor (tlr) signalling after activation by endogenous s100 proteins and other stimuli (e.g. lps). conserved pathogen-associated molecular patterns synthesized by microorganisms (lps) are recognized by tlr to initiate an innate immune response. normal canine endometrial epithelial and stromal cells express tlr4, but both tlr2 and tlr4 are upregulated in pyometra. tlr mediated immune surveillance is an important component in uterine defence mechanisms.23 continuous activation of tlr by both endogenous and exogenous stimulus can lead to an exacerbated inflammatory response in pyometra. lps interacts with lactoferrin (ltf) on the surface of bacteria to activate tlr4 on surfaces of phagocytes and epithelial cells.17 both ltf gene and protein are overexpressed in pyometra.24 calcium-binding proteins of the s100 family (e.g. s100a8, s100a9, s100a12) have been identified as endogenous danger-associated molecular patterns (damps).17 damps are intracellular 640clinical theriogenology • volume 11, number 4 • december 2019   molecules released following cell death and are recognized by the innate immune system. s100a8 and s100a9 stimulate production of several pro-inflammatory cytokines (e.g. tumor necrosis factor (tnf), il6, il1b, il8).17 another op upregulated gene in pyometra is secretory leukocyte peptidase inhibitor (slpi) gene.17 slpi encodes for an antimicrobial peptide secreted by epithelial tissues. spli modulates infection and inflammation by neutralizing lps and reducing activation of tlrs.17 bacteria positive uterine cultures have been obtained during various stages of the reproductive cycle in bitches without reproductive problems.25 common bacteria in uteruses of healthy bitches reflect bacterial flora of vagina and cervix.25 presence of mixed bacteria in uterus during proestrus and estrus is not uncommon in dogs, presumably due to cervical dilation.25 presence of intrauterine bacteria cultured from uterine lumen following elective ovariohysterectomies during diestrus is counterintuitive. in 1 study, > 10% of “normal” diestrous uteruses yielded positive bacterial cultures.25 organisms isolated included enterococcus sp., bacillus sp., and undefined gram positive species.25 furthermore, ~ 16% of bitches believed to have pyometra have negative bacteriologic findings following ovariohysterectomy.25 however, there were no attempts to detect anaerobic organisms or isolate other bacteria such as mycoplasma sp., leptospira sp., or chlamydia sp.25 with respect to pyometra, e. coli is the most common organism isolated in 90% of cases with positive culture.26-27 however, other bacteria are also cultured (table 1), with enterococcus avium isolated from the uterus of a dog with an emphysematous pyometra. in > 90% of cases of canine pyometra, only a single bacterial species was cultured.25 it is important to mention that when > 1 bacterial species is isolated from canine pyometra, e. coli is always isolated.25 escherichia coli strains of these that are isolated from pyometra are those that normally inhabit canine intestine and those present in urinary tract infections.28 escherichia coli isolated from canine pyometra is mainly assigned to phylogenetic group b2 and characterized by a high number of table 1. bacteria other than escherichia coli cultured from cases of canine pyometra.25,72 bacteria frequency (%) β-hemolytic streptococcus 15 citrobacter spp. 10 unidentified enterobacteriaceae 5.9 33.3 streptococcus spp. 5.1 klebsiella spp. 4.2 enterococcus spp. 3.4 actinomycetaceae spp. 2.5 staphylococcus spp. 2.5 16.7 pseudomonas spp. 1.7 2.0 undefined gram negatives 0.8 undefined gram positives 0.8 proteus 0.8 16.7 uropathogenic e. coli (upec) virulence factor (vf) genes and pathogenicity-associated island markers.28 escherichia coli have vf genes encoding for adhesins (including fimh, papc and papgiii), toxins (including hlya/f, usp and asta), and other factors (e.g. those involved in escape from host defences and iron uptake).28 frequency of vf genes in e. coli isolates from canine pyometra are summarized in table 2. escherichia coli isolates with > 2 adhesin genes imply that a combination of genes confers an 641 clinical theriogenology • volume 11, number 4 • december 2019   advantage.28 strongly virulent strains of e. coli (e.g. nau-b) contain 7 virulence genes. when strongly virulent strains of e. coli are inoculated into female dogs, they produce symptoms of pyometra with an earlier onset and greater severity than weakly virulent strains of e. coli (e.g. nau-i) with fewer virulence genes.28 alpha-hemolysin (hlya) vf gene was detected in 35 52% of e. coli pyometra cases.27,29-30 hlya is an rtx pore-forming exotoxin that contributes to virulence of e. coli by inducing tissue damage and a compromised early uterine immune response.27 table 2. virulence genes frequency in escherichia coli isolates from canine pyometra.28,73 gene encodes for frequency expressed (%) fimh adhesin 91.3 irp-2 yersiniabactin 91.3 fim type 1 fimbriae 90.9 fyua yersiniabactin 82.6 hlya hemolysin 30.4 63.6 sfa s fimbriae 63.6 pap p fimbriae 63.6 papc p fimbriae 30.4 63.6 papgiii adhesin 26.1 63.6 iron salmochelin 56.5 usp uropathgenic-specific protein 39.1 54.5 trat escape from host defenses 47.8 sfad/e adhesin 34.8 tsh adhesin 34.8 cnf-1 toxin 26.1 pape/f adhesin 21.7 iss escape from host defenses 21.7 iuta aerobactin system 17.4 ompt escape from host defenses 17.4 cvac escape from host defense 17.4 hlyf toxin 17.4 iucd aerobactin system 17.4 iucc aerobactin system 13.0 asta toxin 13.0 papgii adhesin 4.3 at high concentrations, hlya is able to lyse erythrocytes and nucleated host cells. at low (sublytic) concentrations, hlya can disrupt the immune signalling and cytoskeletal components.31 endometrial epithelial cells had high sensitivity to cytotoxic effect of hlya, suggesting that β-hemolytic e. coli induces earlier damage to the epithelial glandular cells than nonhemolytic e. coli strains.32 diagnosis a presumptive diagnosis of pyometra is based on clinical signs and laboratory test results, with a confirmatory diagnosis made with b-mode ultrasonography, with or without doppler ultrasonography, detecting an enlarged, fluid-filled uterus with increased blood flow. clinical signs and laboratory test results clinical signs common in pyometra include depression, anorexia, polydipsia/polyuria, vomiting and vaginal discharge.5,33-34 anorexia, polydipsia/polyuria, vomiting, and moderate to severe dehydration did not differ significantly between cases of open and closed pyometra.3 leukocytosis, with neutrophilia and left shift, lymphopenia and monocytosis are characteristic findings in pyometra, accompanied by normocytic, normochromic regenerative anemia.5 if present, leukopenia was the most important predictive factor for prolonged postoperative hospitalization, as it was associated with an 18-fold increased risk of peritonitis.5 642clinical theriogenology • volume 11, number 4 • december 2019   concomitant cystitis, proteinuria, and hypoalbuminemia usually resolve after treatment of the pyometra, but severe proteinuria that remains may predispose to renal failure.35 renal dysfunction is common. contributing factors to renal dysfunction are: endotoxemia, glomerular dysfunction, renal tubular damage and decreased response to antidiuretic hormone.35-36 blood urea nitrogen > 30 mg/dl and creatinine concentrations > 1.5 mg/dl have been associated with death.33 hypercholesterolemia, increased serum alkaline phosphatase (alp) and coagulation impairment have also been reported.36-38 systemic inflammatory response syndrome in ~ 50% of cases, pyometra can progress into systemic inflammatory response syndrome (sirs), a form of sepsis and endotoxemia that causes multi-organ dysfunctions.4,39-41 sirs is more common in dogs with closed pyometra than an open pyometra.3 clinical diagnostic criteria for sirs are nonspecific but consist of tachycardia and tachypnea, with a similar hemogram as previously described for pyometra. in pyometra cases diagnosed with sirs, there are increases in blood concentrations of: c-reactive protein (crp), prostaglandin 15-ketodihydro-pgf2α metabolite, serum amyloid a and haptoglobin. in addition, there were high serum il8 concentrations in dogs sirs, suggesting il8 may contribute to development of systemic disease in dogs with pyometra. despite being potentially life-threatening, mortality due to pyometra is relatively low (3 10%).5,42 ultrasonography in cases of hyperplastic pyometra, proliferative response can be detected with b-mode ultrasonography as a thickened uterine wall.15 in addition, increased expression of vegf-a and its receptors during pyometra cause increased blood flow (both peak systolic flow and end diastolic flow) from reduced vascular resistance.15 females diagnosed with ceh and mucometra should be monitored by doppler ultrasound during diestrus for early pyometra identification, to prevent development of systemic disease.15 treatment pyometra is an emergency condition that requires rapid medical and/or surgical intervention to minimize long-term health complications and prevent death. early intervention increases chances of survival.43 surgical management safest and most effective treatment is surgical removal of infected uterus and ovaries, (ovariohysterectomy). anesthetic risks associated with this surgery are heightened due to a compromised status. uterus may be large, friable, and prone to rupture, so it is important to handle it carefully. abdominal cavity should be protected from accidental leakage of purulent material from uterine laceration or uterine tubes/ovarian bursa opening. this is accomplished by packing off uterus with moistened laparotomy pads.43 prior to surgery, patient is stabilized with adequate intravenous fluid therapy to correct hypotension (60 ml/kg + % dehydration x body weight/100), hypoperfusion, shock, dehydration, acid-base balance and electrolyte abnormalities, coagulation disturbances, and organ dysfunctions.44 intensive postoperative monitoring is essential and in uncomplicated cases, 1 to 2 days of postoperative hospitalization is usually adequate.43 broad-spectrum antibiotics with minimal nephrotoxicity are needed to prevent septicemia (e.g. amoxicillin-clavulanic acid 12.5 mg/kg twice a day). antibiotics alone for treatment of pyometra will improve general status and may prevent progression but will not resolve the condition. antibiotics should be continued for > 2 weeks.45 antibiotic drug selection and route of administration should be based on bacterial culture, sensitivity tests and pharmacokinetics for achieving optimal effect.43 the initial choice of antibiotics should be effective against the most likely pathogen (e. coli) and adjusted after culture and sensitivity results to a narrow-spectrum alternative.43,46 in 1 study, 90% of e. coli pyometra isolates were sensitive to ampicillin.47 intravenous administration may be superior to prevent systemic effects of sepsis.46 643 clinical theriogenology • volume 11, number 4 • december 2019   medical management medical management of pyometra may be indicated to improve the general status of the bitch prior to surgery or in the bitch that cannot undergo general anesthesia for other reasons. in addition, medical management may be indicated for treatment of pyometra in valuable breeding bitches. however, the efficacy of medical management depends on clinical presentation (e.g. stability of patient, patency of cervix) and presence of underlying problems (e.g. cystic endometrial hyperplasia, ovarian cyst or tumor).48 goal of medical management is to evacuate uterine contents by dilating cervix and stimulating myometrial contractility. protocols using prostaglandin f2α (pgf2α) and progesterone antagonists (aglepristone) are described in table 3. aglepristone can be used to successfully treat either open or closed pyometras, with cervical opening occurring within 25.8 ± 12.3 hours after initial treatment.16,48-50 however, pgf2α treatment is contraindicated in closed pyometra due to its potential for forcing purulent material retrograding to uterine tubes through ovarian bursae and into peritoneal cavity or through rupture of uterine wall.51 administration of prostaglandin e2 (either intravaginally or orally) may result in cervical relaxation sufficient to allow pgf2α but this remains to be studied.45,52 intrauterine drainage and lavage via transcervical catheterization followed by instillation of intrauterine antibiotics may facilitate recovery in refractory cases.53-56 with respect to complications of medical management, ~ 20% of pyometra patients experienced complications, most commonly peritonitis (10 12% of cases) followed by chronic pyelonephritis, urinary tract infection, myocarditis, and disseminated intravascular coagulation.3,5,40,57-59 persistent proteinuria and urinary protein-creatinine indicate renal disease that requires special attention.35 other reported complications include uveitis, intracranial thromboemboli, bacterial osteomyelitis, pericarditis, septic arthritis, incisional swelling, dehiscence, urethral trauma, recurrent estrus, uterine stump pyometra, fistulous tracts, urinary incontinence, septic shock and death.35,58-59 severe complications associated with pyometra include sepsis, septic shock, peritonitis, disseminated bacterial infection organ dysfunctions and death.58,60-61 proportion of dogs with complications did not differ significantly between open and closed pyometras.3 despite this, it is prudent to obtain signed owner consent prior to treatment, regarding potential risks necessary to obtain prior to extra label drug usage.3 in addition, aglepristone is not currently approved for use in north america. prognosis following medical management of pyometra with pgf2α or aglepristone, prognosis for survival and fertility is considered guarded to good. average fertility rate following medical management is 70% (range, 14 100%).43,51,62-64 fertility rates are even higher in younger (< 5 years) bitches and those that have no other uterine or ovarian pathology.50 average reported long-term success (resolution of clinical illness) following medical management of canine pyometra with pgf2α is 86% (range, 46 100%).4849,54,62-70 combining pgf2α with cabergoline (a dopamine agonist) results in resolution rates from 83 90.5%.66-67 resolution rate with aglepristone alone is lower (60%) than with a combination of aglepristone and cloprostenol (84%).16 breeding on subsequent estrus is frequently recommended after medical management of pyometra in attempt to avoid recurrence.43 mean recurrence rate following medical management with either treatment is ~ 20% (range, 0 85%).43,49-50,63,71 it is important to mention that medical management of bitches with recurrence has been successful. at least in some breeds, pregnancy is considered to slightly reduce the risk for pyometra recurrence, which points to genetic differences in protective factors.10,39 however, pregnancy is not completely protective, and developing pyometra during pregnancy has been reported in several cases.15 644clinical theriogenology • volume 11, number 4 • december 2019   table 3. medical management of canine pyometra using prostaglandin f2α (pgf2α) or aglepristone. mechanism of action protocol efficacy adverse effects pgf2α pgf2α is both luteolytic and uterotonic. efficacy of treatment is correlated to repeated administration rather than overall total dose administered. comparison of uterotonic effect in diestrus bitches of pgf2α at a dosage of 250 µg/kg compared to 50 µg/kg resulted in uterine contractility for 32 and 23 minutes, respectively.51,75 pgf2α is most effective when administered in repeated low doses for 8 10 days. administer natural pgf2α at a dosage of 10 50 µg/kg sc every 4 6 hours.7,45,68-70 administer cloprostenol at a dosage of 1 3 µg/kg sc every 12 24 hours.62,74 following first treatment, copious discharge will be observed coming from vulva for first 48 hours and then will start to decrease. hemorrhagic discharge may be observed 4 5 days into treatment. evacuation of uterine contents greatly improves bitch’s physical condition. treatment efficacy is confirmed by ultrasonographic clearance of uterine contents 10 14 days after first treatment. adverse effects are dose-dependent and develop within minutes and last for ~ 1 hour.62 adverse effects include hypothermia and shivering, increased frequency of defecation (± diarrhea), hypersalivation and vomiting. administer atropine (25 µg/kg sc) 15 minutes before pgf2α and walk bitch for 15 20 minutes after administration to lessen symptoms. brachycephalic breeds may also be predisposed to bronchospasm.45,52 dosage calculations must be done correctly to prevent serious adverse effects, including hypovolemic shock, ventricular tachycardia and death.43 aglepristone aglepristone is a progesterone receptor antagonist that competitively binds progesterone receptors and decreases intrauterine progesterone concentration. aglepristone does not have direct uterotonic activity. administer aglepristone at a dosage of 10 mg/kg sc on days 1, 3, 6 and 9 of treatment.63 if uterine contents are still visible on day 15, administer another injection on day 15 and on day 30. results can be improved by giving cloprostenol (1 µg/kg sc) from days 3 7.16 when a combination of aglepristone and cloprostenol is administered, there was no significant difference in success rates between bitches with open versus closed pyometra.16 adverse effects have not been reported when using aglepristone alone. vomiting has been reported when used in combination with cloprostenol.16 645 clinical theriogenology • volume 11, number 4 • december 2019   references 1. wallace gb, casal ml: a review of pyometra in small animal medicine: incidence, pathophysiology, clinical diagnosis, and medical management. clinical theriogenology 2018;10:435-452 2. jitpean s, hagman r, strom holst b, et al: breed variations in the incidence of pyometra and mammary tumours in swedish dogs. reprod domestic anim 2012;47 suppl 6:347-350. 3. jitpean s, ambrosen a, emanuelson u, et al: closed cervix is associated with more severe illness in dogs with pyometra. bmc vet res 2017;13:11. doi: 10.1186/s12917-016-0924-0. 4. jitpean s, pettersson a, höglund ov, et al: increased concentrations of serum amyloid a in dogs with sepsis caused by pyometra. bmc vet res 2014;10:273. 5. jitpean s, strom-holst b, emanuelson u, et al: outcome of pyometra in female dogs and predictors of peritonitis and prolonged postoperative hospitalization in surgically treated cases. bmc vet res 2014;10:6. 6. pretzer sd: clinical presentation of canine pyometra and mucometra: a review. theriogenology 2008;70:359-363. 7. verstegen j, dhaliwal g, verstegen-onclin k: mucometra, cystic endometrial hyperplasia, and pyometra in the bitch: advances in treatment and assessment of future reproductive success. theriogenology 2008;70:364-374. 8. krekeler n, marenda ms, browning gf, et al: uropathogenic virulence factor fimh facilitates binding of uteropathogenic escherichia coli to canine endometrium. comp immunol microbiol infect dis 2012;35:461-467. 9. dow c: the cystic hyperplasia-pyometra complex in the bitch. j comp pathol 1959;69:237-251. 10. niskanen m, thrusfield mv: associations between age, parity, hormonal therapy and breed, and pyometra in finnish dogs. vet rec 1998;143:493-498. 11. chen ymm, lee cs, wright pj: the roles of progestogen and uterine irritant in the maintenance of cystic endometrial hyperplasia in the canine uterus. theriogenology 2006;66:1537-1544. 12. teunissen ghb: the development of endometritis in the dog and the effect of oestradiol and progesterone on the uterus. acta endocrinol (copenhagen) 1952;9:407-420. 13. reusche n, beineke a, urhausen c, et al: proliferative and apoptotic changes in the healthy canine endometrium and in cystic endometrial hyperplasia. theriogenology 2018;114:14-24. 14. de bosschere h, ducatelle r, vermeirsch h, et al: cystic endometrial hyperplasia-pyometra complex in the bitch: should the two entities be disconnected? theriogenology 2001;55:1509-1519. 15. moxon r, whiteside h, england gcw: prevalence of ultrasound-determined cystic endometrial hyperplasia and the relationship with age in dogs. theriogenology 2016;86:976–980. 16. fieni f, topie e, cogny a: medical treatment of pyometra in dogs. reprod domestic anim 2014;49 suppl 2:28-32. 17. voorwald fa, marchi fa, villacis ra, et al: molecular expression profile reveals potential biomarkers and therapeutic targets in canine endometrial lesions. plos one 2015;10:e0133894. doi: 10.1371/journal.pone.0133894. 18. bukowska d, kempisty b, zawierucha p, et al: microarray analysis of inflammatory response-related gene expression in the uteri of dogs with pyometra. j biol regul homeost agents 2014;28:637-648. 19. singh lk, patra mk, mishra gk, et al: endometrial transcripts of proinflammatory cytokine and enzymes in prostaglandin synthesis are upregulated in the bitches with atrophic pyometra. vet immunol immunopathol 2018;205:65-71. 20. hara t, tanegashima k: pleiotropic functions of the cxc-type chemokine cxcl14 in mammals. j biochem 2012;151:469-476. 21. cole am, ganz t, liese am, et al: cutting edge: ifn-inducible elr-cxc chemokines display defensin-like antimicrobial activity. j immunol 2001;167:623-627. 22. dufour jh, michelle d, liu mt, et al: ifn-gamma inducible protein 10 (ip-10; cxcl10)-deficient mice reveal a role for ip-10 in effector t cell generation and trafficking. j immunol 2002;168:3195-3204. 23. silva e, leitão s, henriques s, et al: gene transcription of tlr2, tlr4, lps ligands and prostaglandin synthesis enzymes are up-regulated in canine uteri with cystic endometrial hyperplasia-pyometra complex. j reprod immunol 2010;84:66-74. 24. kida k, baba e, torii r, et al: lactoferrin expression in the canine uterus during the estrous cycle and with pyometra. theriogenology 2006;66:1325-1333. 25. rubio a, boyen f, tas o, et al: bacterial colonization of the ovarian bursa in dogs with clinically suspected pyometra and in controls. theriogenology 2014;82:966-971. 26. dhaliwal gk, wray c, noakes de: uterine bacterial flora and uterine lesions in bitches with cystic endometrial hyperplasia (pyometra). vet rec 1998;143:659-661. 27. mateus l, henrique s, merino c, et al: virulence genotypes of escherichia coli canine isolates from pyometra, cystitis and fecal origin. vet microbiol 2013:166:590-594. 28. maluta rp, borges ca, berldo lg, et al: frequencies of virulence genes and pulse field gel electrophoresis fingerprints in escherichia coli isolates from canine pyometra. vet j 2014;202:393-395. 29. chen ymm, wright pj, lee c-s, et al: uropathogenic virulence factors in isolates of escherichia coli from clinical cases of canine pyometra and feces of healthy bitches. vet microbiol 2003;94:57-69. 30. siqueira a, ribeiro m, leite d, et al: virulence factors in escherichia coli strains isolated from urinary tract infection and pyometra cases and from feces of healthy dogs. res vet sci 2009;86:206-210. 646clinical theriogenology • volume 11, number 4 • december 2019   31. wiles tj, mulvey ma: the rtx pore-forming toxin alpha-hemolysin of uropathogenic escherichia coli: progress and perspectives. future microbiol 2013;8:73-84. 32. henriques s, silva e, silva mf, et al: immunomodulation in the canine endometrium by uteropathogenic escherichia coli. vet res 2016;47:114. doi 10.1186/s13567-016-0396-z. 33. kuplulu s, vural mr, demirel a, et al: the comparative evaluation of serum biochemical, haematological, bacteriological and clinical findings of dead and recovered bitches with pyometra in the postoperative process. acta vet-beograd 2009;59:193-204. 34. borresen b: pyometra in the doga pathophysiological investigation. ii. anamnestic, clinical and reproductive aspects. nord vet med 1979;31:251-257. 35. maddens b, heiene r, smets p, et al: evaluation of kidney injury in dogs with pyometra based on proteinuria, renal histomorphology, and urinary biomarkers. j vet intern med 2011;25:1075-1083. 36. asheim a: renal function in dogs with pyometra. 8. uterine infection and the pathogenesis of the renal dysfunction. acta pathol microbiol immunol scand 1964;60:99-107. 37. borresen b, skrede s: pyometra in the doga pathophysiological investigation v. the presence of intrahepatic cholestasis and an “acute phase reaction.”. nord vet med 1980;32:378-386. 38. plavec t, celinsek b, dolinar k, et al: haemostasis impairment in bitches with pyometra. acta vet beograd 2006;56:529-540. 39. hagman r, kindahl h, lagerstadt as: pyometra in bitches induces elevated plasma endotoxin and prostaglandin f2alpha metabolite levels. acta vet scand 2006;47:55-68. 40. fransson ba, lagerstadt a-s, bergstrom a, et al: c reactive protin, tumor necrosis fact alpha, and interleukin-6 in dogs with pyometra and sirs. j vet emerg crit care 2007;17:373-381. 41. karlsson i, hagman r, johannisson a, et al: cytokines as immunological markers for systemic inflammation in dogs with pyometra. reprod domest anim 2012;47 suppl 6:337-341. 42. egenvall a, hagman r, bonnett bn, et al: breed risk of pyometra in insured dogs in sweden. j vet intern med 2001;15:530-538. 43. hagman r: pyometra in small animals. vet clin north am small anim pract 2018;48:639-661. 44. fantoni d, shih ac: perioperative fluid therapy. vet clin north am small anim pract 2017;47:423-434. 45. lopate c: pyometra, cystic endometrial hyperplasia (hydrometra, mucometra, hematometra). in: greco ds, davidson ap, editors. blackwell’s five-minute veterinary consult clinical companion, small animal endocrinology and reproduction. hoboken, nj; wiley-blackwell: 2017. p. 53-62. 46. declue a: sepsis and the systemic inflammatory response syndrome. in: ettinger sj, feldman ec, cote e, editors. textbook of veterinary internal medicine: diseases of the dogs and cat. 8th edition, st louis; elsevier: 2016. p. 554-560. 47. hagman r, greko c: antimicrobial resistance in escherichia coli isolated from bitches with pyometra and from urine samples from other dogs. vet rec 2005;157:193-196. 48. fieni f: clinical evaluation of the use of aglepristone, with or without cloprostenol, to treat cystic endometrial hyperplasia-pyometra complex in bitches. theriogenology 2006;66:1550-1556. 49. trasch k wehrend a, bostedt h: follow-up examinations of bitches after conservative treatment of pyometra with the antigestagen aglepristone. j vet med anat physiol pathol clin med 2003;50:375-379. 50. jurka p, max a, hawrynska k: age related pregnancy results and further examination of bitches after aglepristone treatment of pyometra. reprod dom anim 2010;45:525-529. 51. nelson rw, feldman ec, stabenseld gh: treatment of canine pyometra and endometritis by pgf2alpha. j am vet med assoc 1982;181:899-903. 52. greer m: canine reproduction and neonatology a practical guide for veterinarians, veterinary staff and breeders. jackson, wy; teton newmedia:2015. 53. lagerstedt as, obel n, stavenborn m: uterine drainage in the bitch for treatment of pyometra refractory to prostaglandin f2a. j small anim pract 1987;28:215-222. 54. gurbulak k, pancarci m, ekici h, et al: use of aglepristone and aglepristone 1 intrauterine antibiotic for the treatment of pyometra in bitches. acta vet hung 2005;53:249-255. 55. gabor g, siver l, szenci o: intravaginal prostaglandin f2 alpha for the treatment of metritis and pyometra in the bitch. acta vet hung 1999;47:103-108. 56. de cramer kg: surgical uterine drainage and lavage as treatment for canine pyometra. j s afr vet assoc 2010;81:172-177. 57. kenney kj, matthiesen dt, brown no, et al: pyometra in cats: 183 cases (1979-1984). j am vet med assoc 1987;191:1130-1132. 58. marretta sm, matthiesen dt, nichols r: pyometra and its complications. probl vet med 1989;1:50-62. 59. wheaton lg, johnson al, parker aj, et al: results and complications of surgical treatment of pyometra: a review of 80 cases. j am anim hosp assoc 1987;25:563-568. 60. conti-patara a, de araújo caldeira j, de mattos-junior e, et al: changes in tissue perfusion parameters in dogs with severe sepsis/septic shock in response to goal-directed hemodynamic optimization at admission to icu and the relation to outcome. j vet emerg crit care 2012;22:409-418. 647 clinical theriogenology • volume 11, number 4 • december 2019   61. heiene r, kristiansen v, teige j, et al: renal histomorphology in dogs with pyometra and control dogs, and long-term clinical outcome with respect to signs of kidney disease. acta veterinaria scandinavica 2007;49:13. doi:10.1186/17510147-49-13 62. jena b, rao ks, reddy kcs, et al: comparative efficacy or various therapeutic protocols in the treatment of pyometra in bitches. vet med 2013;58:271-276. 63. contri a, gloria a, carluccio a, et al: effectiveness of a modified administration protocol for the medical treatment of canine pyometra. vet res commun 2015;39:1-5. 64. gogny a, fieni f: aglepristone: a review on its clinical use in animals. theriogenology 2016;85:555-66. 65. ros l, holst bs, hagman r: a retrospective study of bitches with pyometra, medically treated with aglepristone. theriogenology 2014;82:1281-1286. 66. england gc, freeman sl, russo m: treatment of spontaneous pyometra in 22 bitches with a combination of cabergoline and cloprostenol. vet rec 2007;160:293-296. 67. corrada y, arias d, rodriguez r, et al: combination dopamine agonist and prostaglandin agonist treatment of cystic endometrial hyperplasia-pyometra complex in the bitch. theriogenology 2006;66:1557-1559. 68. feldman ec, nelson rw: cystic endometrial hyperplasia/pyometra complex. in: feldman ec, nelson rw, editors. endocrinology and reproduction. 3rd edition, st louis, mo; saunders: 2004. p. 852-867. 69. arnold s, hubler m, casal m, et al: use of low dose prostaglandin for the treatment of canine pyometra. j small anim pract 1988;29:303-308. 70. sridevi p, balasubramanian s, devanathan t, et al: low dose prostaglandin f2 alpha therapy in treatment of canine pyometra. indian vet j 2000;77:889-890. 71. gobello c, castex g, klima l, et al: a study of two protocols combing aglepristone and cloprostenol to treat open cervix pyometra in the bitch. theriogenology 2003;60:901-908. 72. karlsson i, hagman r, johannisson a, et al: multiplex cytokine analyses in dogs with pyometra suggest involvement of kc-like chemokine in canine bacterial sepsis. vet immunol immunopathol 2016;170:41-46. 73. qian c, hou j: escherichia coli virulence influences the roles of sex hormone receptors in female dogs with simulated pyometra. exp therapeut med 2017;14: 3013-3021. 74. davidson a: female and male infertility and subfertility. in: nelson rw, couto cg, editors. small animal internal medicine. 5th edition, st louis, mo;elsevier: 2014. p. 951-965. 75. burke tj: prostaglandin f2a in treatment of pyometra-metritis. vet clin north am 1982;12:107-109. 648clinical theriogenology • volume 11, number 4 • december 2019 untitled 2020 cryopreservation of epididymal and electroejaculated bull semen using liposomeand egg yolkbased extenders cryopreservation of epididymal and electroejaculated bull semen using liposomeand egg yolkbased extenders zachary turner,a igor canisso,c giorgia podico,c edgar garrett,c jamie stewart,c parker henley,d daniel shike,d robyn ellerbrock,a david hurley,b roberto palomares,b maria ferrera adepartment of large animal medicine and bdepartment of population health college of veterinary medicine, university of georgia, athens, ga cdepartment of veterinary clinical medicine, college of veterinary medicine and ddepartment of animal sciences, university of illinois, urbana, il abstract objective of this study was to compare cryosurvival of epididymal (ep) and electroejaculated (ee) sperm cryopreserved in liposomeor egg yolkbased extenders. aliquots of ee sperm (n = 10 bulls) were cryopreserved with a liposome(optixcell; eeo) or egg yolk-based extender (botubov; eeb). bulls were castrated and sperm from each cauda epididymis were cryopreserved with optixcell (epo) or botubov (epb). postthaw total motility was higher in semen frozen in botubov than optixcell (p = 0.029). in addition, sperm distance and velocity parameters, and lateral head displacement were higher in semen frozen in optixcell (p = 0.01). plasma and acrosomal membrane integrity were higher with botubov (eeb 60 ± 3%, epb 59.7 ± 4%; mean ± sem) than optixcell (eeo 37.8 ± 6%, epo 25.8 ± 6%) (p < 0.0001). capacitated cell percentage was lower with botubov (eeb 2 ± 0.4%, epb 0.9 ± 0.3%) than optixcell (eeo 6.9 ± 2.4%, epo 4.2 ± 1.4%) (p < 0.0001). fewer ep sperm had high mitochondrial potential (epb 6.6 ± 4.4%, epo 14.6 ± 6.2%) than ee sperm (eeb 46.7 ± 9.8%, eeo 41 ± 14.6%) (p = 0.0003), with no difference between extenders. keywords: bull, freezing, semen, diluents, cryotolerance introduction egg yolk-based semen extenders have been the industry standard for cryopreservation of bovine semen for many years.1 however, there are growing concerns about the use of egg yolk, due to potential for microbial contamination, bacterial endotoxin release, presence of steroid hormones and lack of quality standards since egg-yolk composition and viral and bacterial contamination, are often not evaluated.2 liposome-based semen extenders are becoming acceptable alternatives to animalbased products.1 the cryoprotective mechanism of egg yolk is through sequestration of seminal plasma proteins by low-density lipoproteins, reducing cholesterol and phospholipid effluxes and preventing increases in membrane fluidity.3,4 since seminal plasma proteins secreted by accessory sex glands are not present in epididymal samples, it would be relevant to determine the response of epididymal sperm to cryopreservation in egg-yolk based extenders. although the cryoprotective mechanism of liposomes has not been determined, it has been suggested to be mediated via modification of plasma membrane composition by incorporating molecules of interest into the plasma membrane, or exchanging lipids and cholesterol.5 many studies were conducted to determine an ideal semen extender for bull semen cryopreservation, but only a few studies compared egg yolk-based and liposome-based extenders to cryopreserve bovine semen.6,7 in those studies, liposome-based extenders supported higher or similar postthaw motility of ejaculated sperm than egg yolk-based extenders but lower antioxidant protection.6,7 however, those studies were carried out with dairy bulls collected with an artificial vagina under controlled laboratory conditions.6,7 on farm semen collection for cryopreservation from beef bulls typically requires electroejaculation, which yields samples with variable concentrations of seminal plasma. apparently, no studies have compared egg yolk-based and liposome-based extenders to freeze semen from bos taurus beef bulls collected via electroejaculation versus epididymal harvest. assessment of epididymal sperm provides opportunity to study the effects of semen extenders on cryosurvival, as this type of sample contains only seminal plasma proteins from epididymides. epididymal sperm also have a larger proportion of cytoplasmic droplets when compared to ejaculated samples.8 these differences may influence sperm response to cryopreservation and their interaction with semen extenders. bovine epididymal sperm has been routinely cryopreserved using egg yolkbased extenders,9-17 but studies testing liposome-based extenders for cryopreservation of epididymal sperm are lacking. in addition, most protocols for epididymal sperm freezing were developed for clinical theriogenology • volume 12 number 1 • march 202029 bos indicus or holstein bulls.9-14 cryopreservation of epididymal sperm from beef bulls of bos taurus breeds has faced disappointingly low success, with postthaw total motility ranging from 10 to 16%.15,16 thus, there is a critical need to improve cryopreservation protocols for these breeds to improve postthaw semen quality, and use of a different type of semen extender may improve postthaw semen quality. collection and cryopreservation of epididymal sperm is often performed at postmortem to preserve genetics of valuable bulls that face terminal disease or sudden death. obtaining acceptable postthaw motility is critical to provide acceptable pregnancy rates with epididymal sperm. electroejaculation may provide an alternative for collection and preservation of semen from genetically valuable bulls with a terminal disease or sudden death, or for bulls in stud centres refusing to mount a teaser. ejaculated sperm were reported to have similar postthaw motility and acrosomal integrity, less intact membranes, and higher in vitro fertilization rates than epididymal sperm.10,11,14 objective of this study was to evaluate functional sperm parameters associated with cryotolerance in epididymal and electroejaculated sperm frozen with 2 semen extenders. based on studies conducted in dairy breeds and bos indicus bulls, and on authors’ experience, it was hypothesized that there are no differences in response to cryopreservation between electroejaculated and epididymal sperm in beef bulls under the conditions of our study. it was also hypothesized that the liposome-based extender is superior to an egg yolk-based extender for ejaculated and epididymal sperm, due to differences in cryoprotective mechanisms. materials and methods bulls bulls were cared for in accordance with guidelines from the guide for the care and use of agricultural animals in research and teaching. all experimental procedures were reviewed and approved by the institutional animal care and use committee at the university of illinois in urbana-champaign (protocol # 17240). ten simmangus beef bulls (bos taurus) were available for the study at the university of illinois. they were housed in pens at the university of illinois beef unit in urbana, il. bulls were kept in groups of 3 per pen and fed a total mixed ratio (63.4% dry matter, 90.7% organic matter, 7.9% crude protein, 55.8% neutral detergent fiber, 32.7% acid detergent fiber, 1.7% ether extract). bulls were 15.1 ± 0.05 months old (range 14 16), weighed 585 ± 17 kg (range 522.7 679.6), and had a body condition score of 5.8 ± 0.2 in a scale of 1 9 (range 5 9) (mean ± sd). scrotal circumference was measured with a scrotal tape at the widest point of the scrotum. the average scrotal circumference was 40.5 ± 0.6 cm (range 36 42). experimental design one ejaculate was collected from all bulls using electroejaculation. after semen evaluation, each ejaculate was divided into 2 aliquots, which were then cryopreserved with 2 semen extenders. one aliquot was cryopreserved using the liposome-based extender optixcell (imv technologies, maple grove, ms; treatment eeo), whereas the other was cryopreserved using the egg-yolk based extender botubov (botupharma, scottsdale, az; treatment eeb). botubov is a tris-based medium with 20% egg yolk, aminoacids, sugars, buffers, and surfactant orvus es paste.12 optixcell is free of animal-derived components and contains liposomes with phospholipids for cryoprotection. however, the exact composition of both extenders is proprietary and has not been disclosed. bulls were castrated 3 days later and sperm were collected from epididymis using a swim-up technique. sperm from 1 epididymal tail from each bull were collected and cryopreserved with optixcell (treatment epo), whereas sperm from contralateral epididymis were collected and cryopreserved with botubov (treatment epb). sperm concentration, motility, and morphology were evaluated prior to cryopreservation. semen was cryopreserved and stored in liquid nitrogen at -196°c for 6 months until evaluation. after thawing, sperm motility, longevity of sperm motility, and hypoosmotic tests were performed at the university of illinois. two straws from each treatment were shipped to the university of georgia in a dry shipper for evaluation of membrane integrity, membrane fluidity, acrosomal integrity, and mitochondrial potential using flow cytometry. samples were thawed and processed for evaluation in the andrology laboratory of the department of large animal medicine. clinical theriogenology • volume 12 number 1 • march 2020 30 flow cytometry was performed in the flow cytometry laboratory, a facility shared between the departments of large animal medicine and population health. sperm kinematics and functional parameters were compared among treatments. semen collection with electroejaculation electroejaculation was performed with a transrectal 60 mm upright bull probe with electroejaculator set on the programmed cycle (pulsator iv, lane manufacturing inc., denver, co). when an inadequate sample was collected, as determined by low volume or absence of sperm motility, a manual cycle of the electroejaculator was used by an experienced clinician. spermrich fractions were collected in a 15 ml conical tube. each ejaculate was divided into 2 aliquots. one aliquot was extended in botubov at a 1:1 (v:v) ratio and the other aliquot was extended in optixcell at similar ratio. extenders were prewarmed to 37°c prior to mixing with semen. extended semen was stored in 15 ml conical tubes and placed in an insulated styrofoam box at room temperature until the arrival at the illinois veterinary teaching hospital within 30 minutes after collection. sperm collection from epididymis three days after semen collection with electroejaculation, bulls were castrated using a newberry knife and an emasculator. once restrained in chute, bull’s rectum was emptied. perineal area was cleaned and scrubbed for a pudendal block. lidocaine (10 ml/side) was infused at the level of anus, 5 10 cm lateral to the edge of the anus. thereafter, a spinal needle was inserted deeply parallel through the rectum guided by transrectal palpation. then, 40 ml of lidocaine were infused. needle was withdrawn, and rectum was massaged for diffusion of lidocaine near pudendal nerve. procedure was repeated in similar manner in the opposite side. scrotum was cleaned and aseptically scrubbed with betadine and 70% isopropyl alcohol. spermatic cords were blocked with 10 ml of lidocaine. newberry knife was used to cut the bottom of the scrotum; testes were then exposed via incision to spermatic fascia. emasculator was applied to spermatic cords for 3 minutes. then, spermatic cords were transfixed and ligated twice before being transected with the emasculator. spermatic cords were ligated to avoid leakage of sperm. testes and epididymides were placed in a plastic bag, covered with sterile 0.9% sodium chloride solution at room temperature and placed in a styrofoam box at room temperature until arrival at the illinois veterinary teaching hospital within 30 minutes. upon arrival, cauda epididymides were dissected away from the testis and rinsed with 0.9% sodium chloride solution. cauda epididymides, including at least 3 cm of the proximal ductus deferens, were separated with a #10 scalpel blade and transferred to a petri dish. connective tissue containing superficial blood vessels around the cauda epididymis was removed by dissection with scissors. tissue was minced with a scalpel blade and covered with 10 ml of warm semen extender (botubov or optixcell). after incubation at room temperature for 10 minutes, the medium was aspirated and used for cryopreservation. one epididymis from each bull was processed with optixcell, and the other with botubov. cryopreservation after initial dilution, sperm concentration in each aliquot of electroejaculated and epididymal samples was determined using a nucleocounter (chemometec inc., bohemia, ny). each aliquot was further diluted with the corresponding extender to achieve a final concentration of 40 x 106 sperm/ml. semen was loaded and sealed into 10 french straws (0.5 ml), with a fully automated machine (mpp uno, minitube, germany) in a cold-room at 5oc. straws were then equilibrated at 5°c for 3 5 hours. after equilibration, straws were placed horizontally on a rack, 5 cm above liquid nitrogen for 15 minutes. then, straws were plunged in liquid nitrogen, loaded into 10 mm goblets, and transferred to storage in liquid nitrogen tank. six months after freezing, straws from each aliquot were thawed in a water bath at 38°c for 30 seconds for evaluation of postthaw semen quality. clinical theriogenology • volume 12 number 1 • march 202031 semen analysis at the university of illinois, semen volume was determined using a graduated 15 ml centrifuge tube. sperm motility was evaluated with a computer assisted sperm analysis (spermvision, minitube of america, verona, wi) prior to freezing. semen was placed in a 20 µl sperm analysis chamber over the heated specimen stage at 38°c. motion parameters were assessed from all cells present in 7 fields with an x 20 phase-contrast objective. parameters analyzed included total motility (tm, %), progressive motility (pm, %), average path velocity (vap, µm/second), straight line velocity (vsl, µm/second), curvilinear velocity (vcl, µm/second), average path distance (dap, μm), curvilinear path distance (dcl, μm), straight line distance (dsl, μm), straightness of the average path (str), linearity of the curvilinear path (lin), and amplitude of lateral head displacement (alh, µm). default manufacturer’s setting for the assessment of bovine semen was used: frame capture speed rate 60 hz; cell size (min/max) 18/60 µm2; threshold straightness 50%; vap cutoff 56 µm/second; and vsl cutoff 28 µm/second. after freezing, 1 straw was thawed from each treatment at 38°c for 30 seconds. semen was placed in 2 ml tubes in a water bath at 38°c for 4 hours and sperm motility was evaluated every 30 minutes for 4 hours. phase contrast microscopy was used for assessment of sperm morphology. a single operator performed all evaluations. sperm were diluted 1:10 (v:v) with formalin buffered solution and a 5 μl drop was placed on a microscope slide. evaluation was done at 100 x magnification under oil immersion and 100 sperm were classified based on morphological characteristics. following the new guidelines from the society for theriogenology regarding the classification of sperm morphology, sperm with distal droplets were considered normal.18 at the university of georgia, 1 or 2 straws were thawed from each treatment. sperm were washed in warm dulbecco’s phosphate buffered solution by centrifugation at 600 x g for 5 minutes to remove semen extender. one aliquot was used for evaluation of mitochondrial potential using the fluorescent probe 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolyl-carbocyanine iodide (jc-1, cat. no. t3168, molecular probes, eugene, or). one microgram of jc-1 was added to 1 ml of sperm suspension containing 5 x 106 sperm, together with 8 μl of a 1.2 mm solution of propidium iodide (pi). samples were incubated at 38°c for 15 minutes and evaluated under fluorescence microscopy. one hundred membrane-intact sperm (pi negative) were classified as having high (orange) or low (green) mitochondrial potential. another aliquot was used for evaluation of membrane fluidity using the fluorescent dye merocyanine 540 (m540, cat. no. m25471, molecular probes). the vital stain yo-pro 1 (cat. no. y3603, molecular probes) was added to identify viable cells. merocyanine 540, 1.5 µl of a 1mm solution in dmso and 1 µl of a 25 µm solution of yo-pro 1 in dmso were added to a 1 ml suspension containing 5 x 106 sperm. cells were incubated for 10 minutes at 38°c. using flow cytometry, cells were classified as membrane-intact capacitated sperm (mic, yo-pro 1 negative and m540 positive), membrane-damaged capacitated sperm (mdc, yo-pro 1 positive and m540 positive), membrane-intact noncapacitated sperm (minc, yo-pro 1 negative and m540 negative), and membrane-damaged noncapacitated sperm (mdnc, yo-pro 1 positive and m540 negative). a final aliquot was used for evaluation of acrosome integrity using peanut agglutinin labelled with fluorescein isothiocyanate (fitc-pna; cat. no. f-2301-1, ey laboratories, san mateo, ca). for this, 1 µl of a 1 µg/ml solution of fitc-pna and 1 µl of a 2.4 mm solution of pi were added to a 1 ml suspension containing 5 x 106 sperm. semen was incubated at 38 °c for 10 minutes. using flow cytometry, cells were classified as membrane-intact acrosome-intact sperm (miai, pi negative and fitc-pna negative), membrane-intact acrosome-damaged sperm (miad, pi negative and fitc-pna positive), membrane-damaged acrosome-intact sperm (mdai, pi positive and fitc-pna negative), or membrane-damaged acrosome-damaged sperm (mdad, pi positive fitc-pna positive). fluorescence activated flow cytometry was performed at the university of georgia (accuri c6 plus, bd biosciences, san jose, ca). from each sample, 10,000 events in the forward and 90º light scatter population representing whole sperm were analysed using 0.2 μm filtered 18 mega-ohm ultrapure water as sheath fluid. a gate containing sperm was selected based on dot plot distribution of forward (size) versus side scatter (complexity parameter) to eliminate debris and somatic cells from the analysis. green and red signals were detected using a 5 m watt blue argon laser (488 nm) and emission filters (535 ± 30 nm for green and 585 ± 30 nm for red). baseline background fluorescence clinical theriogenology • volume 12 number 1 • march 2020 32 signal was initially evaluated in unstained samples. the control area was marked on unstained samples with < 1% of cells registering as positive for both signals. compensation for fitc emission into the pi detector or vice versa was done by establishing quadrants on sperm labelled only with pi or fitc, followed by electronic subtraction of the fitc emission into the pi detector and pi emission into the fitc detector. similar compensation was performed for m540 and yo-pro 1. after colour compensation, fluorescence emission data were collected with logarithmic amplification for green fluorescence (fitc and yo-pro 1 using fl1 detector) and orange-red fluorescence (pi and m540 using fl3 detector). the control quadrant (lower left, ll) was marked on unstained samples to include < 1% of cells as positive in the upper left (ul), upper right (ur) and lower right (lr) quadrants. statistical analysis statistical analysis was performed using sas package (sas institute, cary, nc). distribution of data was tested for normality using a shapiro wilk test. data not normally distributed underwent logarithmic transformation. results from normally distributed or transformed data were expressed as mean ± sem. because data were balanced, arithmetic and ls means coincided. means of morphology parameters were compared between ejaculated and epididymal sperm using a paired student’s t-test. motility and flow cytometry data were analyzed using a mixed model, with bull as random effect, and source, time (when applicable) and extender as fixed effects. where an effect or interaction of source and extender was identified, pairwise comparisons were performed using least square means. comparisons among treatments within time points were performed using proc glm and tukey’s test when an interaction of source, extender and time was identified in the mixed model. significance was considered when p < 0.05. results when new guidelines from the society for theriogenology were followed, the percentage of normal sperm did not differ between ejaculated (69.1 ± 4.3%) and epididymal samples (67.8 ± 5.5%). however, when distal droplets and distal midpiece reflexes were counted separately, the percentage of morphologically normal sperm was higher in ejaculated (57.5 ± 5%) than epididymal samples (26.4 ± 4.5%; p = 0.0005), due to a lower percentage of ejaculated sperm with distal droplets (ee 11.6 ± 3.6% versus ep 41.4 ± 6.7; p = 0.002; table). table: morphological characteristics of ejaculated and epididymal bovine sperm. morphology parameter (%) ejaculated sperm (n = 10) epididymal sperm (n = 10) normal 57.5 ± 5a 26.4 ± 4.5b proximal droplets 10.9 ± 3 12.9 ± 4.7 distal droplets 11.6 ± 3.6a 41.4 ± 6.7b abnormal heads 2.5 ± 1.2 2.7 ± 0.8 abnormal midpieces 1 ±0.4 1.6 ± 0.7 abnormal acrosomes 0.2 ± 0.1 0.1 ± 0.1 coiled tails 3.2 ± 0.8 1.5 ± 0.8 kinked tails 6.9 ± 2.2 7.3 ± 2.6 detached heads 6.2 ± 2.9 5.7 ± 3.1 a,bwithin a row, means without a common superscript differed (p < 0.05) total motility was affected by time (p < 0.0001), extender (p < 0.0001) and their interaction (p = 0.029), but not source of sperm. all postthaw samples had lower motility than pre-freeze samples. however, semen frozen in botubov had higher total motility than in optixcell immediately postthaw and after 3 hours of incubation (p < 0.05; figure 1a). progressive motility was affected by time (p < 0.0001), but neither by source nor extender (figure 1a). all postthaw samples had lower progressive motility than prefreeze samples. there was an interaction among extender, time and source in all distance and velocity parameters (p = 0.01). in general, ejaculated sperm frozen in optixcell had higher dap, dsl, dcl, vap, vsl, and vcl than other treatments (figure 1 c h; p < 0.05). sperm frozen in botubov had higher str and lin, but lower alh than sperm frozen in optixcell (figure1 i k; p < 0.05). clinical theriogenology • volume 12 number 1 • march 202033 figure. 1 a k. sperm motion parameters before freezing (prefr), and 0 4 hours postthaw (pt0 pt4) in electroejaculated and epididymal bovine sperm frozen in an egg-yolk (botubov) or liposome-based (optixcell) semen extender. eeb = electroejaculated, botubov; eeo = electroejaculated, optixcell; epb = epididymal, botubov; epo = epididymal, optixcell; dcl = curvilinear distance, dap = distance of average path, dsl = straight line distance, vcl = curvilinear velocity, vap = average path velocity, vsl= straight line velocity, lin = linearity, str = straightness, alh = amplitude of lateral head displacement. a-c within a time point, means without a common superscript differed (p < 0.05). percentage of membrane-intact and membrane-damaged cells with capacitation-like associated changes in membrane fluidity differed with extender (imc p < 0.0001; dmc p = 0.001), but not source (figure 2 a b). there was a lower percentage of capacitated sperm in semen frozen in botubov than optixcell. percentage of sperm with intact membranes and intact acrosomes (imia) clinical theriogenology • volume 12 number 1 • march 2020 34 was higher in sperm frozen in botubov than optixcell (p < 0.0001), with no effect of source (figure 2 c). there was an interaction of extender with source for percentage of sperm with damaged membranes and damaged acrosomes, with electroejaculated semen frozen in botubov having the lowest value. the other 2 acrosomal parameters (dmia, imda) did not differ with extender or source. percentage of cells with low mitochondrial potential was higher in epididymal than ejaculated sperm, with no difference between extenders (p = 0.0003; figure 2d), whereas the opposite was true for percentage of sperm with high mitochondrial potential (p = 0.03). figure 2 a d. percentage of sperm with intact membranes and capacitated (imc), damaged membranes, capacitated (dmc) (panel a); intact membranes, noncapacitated (imnc), damaged membranes, noncapacitated (dmnc) (panel b); damaged membrane, intact acrosome (dmia), damaged membrane, damaged acrosome (dmda), intact membrane, intact acrosome (imia), intact membrane, damaged acrosome (imda) (panel c); and with high and low mitochondrial potential (panel d) in electroejaculated and epididymal bovine sperm frozen in an egg-yolk (botubov) or liposome-based (optixcell) semen extender. eeb = electroejaculated, botubov; eeo = electroejaculated, optixcell; epb = epididymal, botubov; epo=epididymal, optixcell. a,bwithin an end point, means without a common superscript differed (p < 0.05). discussion the ability of 2 extenders to support cryosurvival of epididymal and ejaculated beef bull sperm was assessed in this study. based on in vitro sperm parameters, we inferred that egg-yolk based extender (botubov) provided superior cryoprotection compared to the liposome-based extender (optixcell) in electroejaculated semen from beef bulls. postthaw motility was higher in semen frozen in an egg-yolk based extender than sperm frozen in the liposome-based extender, regardless of sperm source. although markers for oxidative stress were not assessed herein during incubation at 38oc, we inferred that egg-yolk based extender was superior to control oxidative stress during the thermal longevity test. findings of a previous study that compared liposomeand egg yolk-based extenders to cryopreserve bull semen appeared to support this suggestion, as sperm frozen in egg-yolk based extender had lower production of intracellular superoxide compared to sperm frozen in liposomebased extender.7 this was apparently the first study comparing the liposome-based extender optixcell and the egg yolk-based extender botubov to cryopreserve electroejaculated semen or epididymal sperm from beef bulls. previous studies with dairy bulls ejaculated into an artificial vagina concluded that whereas a liposome-based extender yielded higher or similar postthaw motility than an egg yolk-based extender, antioxidant protection was lower with the liposome-based extender.6 other authors reported higher postthaw motility and fertility of buffalo semen frozen in optixcell than tris-egg yolk extender.19-21 although acrosomal and membrane integrity were higher with optixcell in 1 study,19 there was no difference between extenders in 2 other studies.20,21 apparent differences in findings may be explained by breed or species differences in seminal plasma composition, as well as clinical theriogenology • volume 12 number 1 • march 202035 differences in seminal plasma contribution or composition associated with semen collection methods. furthermore, the egg yolk-based extender botubov is supplemented with amino acids and sugars beneficial for cryopreservation of bovine semen. although the formulation is proprietary, presumably composition of the extender used also accounted for the differences in findings between this study and previous ones. furthermore, botubov was superior to traditional tris-egg yolk extenders for freezing sex-sorted semen,22 perhaps due to botbov’s apparently more complex composition (e.g. amino acids). sperm frozen in liposome-based extender had higher values of distance and velocity parameters and lateral head displacement and lower straightness and linearity than sperm frozen in the egg yolk-based extender. this motility pattern has been associated with sperm capacitation.23 capacitated sperm have a change in their motion pattern called hyperactivation; distinguishing characteristics are increased vcl and alh, and decreased lin.23 indeed, sperm frozen in liposomebased extender had a higher percentage of cells with capacitation-associated membrane fluidity changes. cryopreservation induces a significant loss and alteration of phospholipids and cholesterol in the sperm membrane.23 loss of cholesterol is a key event in capacitation, leading to premature capacitation in cryopreserved sperm.24,25 moreover, changes in cholesterol/phospholipid ratio, or loss of decapacitating proteins, increase sensitivity of cryopreserved sperm to capacitating agents.24,25 additional studies should assess if in vitro findings affect in vivo or in vitro fertility. presence of capacitated sperm in frozen thawed bovine semen is correlated to poor field fertility, based on 56-day nonreturn rates after artificial insemination.26,27 therefore, ability of semen extenders to prevent cryocapacitation is important in providing appropriate cryosurvival. as ejaculated sperm are mixed with secretions from accessory sex glands, the sperm plasma membrane is subjected to interactions with various molecules, particularly proteins, present in secretions from these glands.28 interestingly, percentage of sperm with intact membranes and intact acrosomes was higher in both epididymal and ejaculated sperm frozen in botubov than optixcell, suggesting that the eggyolk based extender was a superior option regardless of the presence of seminal plasma. sperm frozen in the liposome-based extender also had numerically higher percentage of damaged acrosomes, although the difference was only significant in ejaculated semen, likely due to the low number of animals used. the lower percentage of sperm with intact acrosomes could represent poorer preservation of acrosomal integrity during freezing and thawing, or a higher prevalence of spontaneous acrosome reactions associated with cryocapacitation when a liposome-based extender was used. seminal plasma contains a family of closely related proteins, collectively called binder of seminal plasma proteins (bsp). these proteins bind to the sperm membrane choline phospholipids at ejaculation and potentiate sperm capacitation by stimulating cholesterol and phospholipid efflux from the sperm membrane.28 egg yolk lipoproteins specifically bind to and scavenge bsp proteins, preventing their interaction with the sperm plasma membrane.28 thus, egg yolk lipoproteins reduce cholesterol and phospholipid efflux and reduce sperm susceptibility to cryocapacitation.3 in addition, sperm membrane also incorporates cholesterol and phospholipids from egg yolk lipoproteins, increasing their content during incubation.2 although epididymal sperm lack exposure to seminal plasma, egg yolk might have increased sperm cryotolerance through direct modification of the sperm plasma membrane composition. however, this warrants further studies. although liposomes also sequester bsp proteins with a similar efficiency to egg yolk, incorporation of lipids into the plasma membrane of sperm cells is lower and less efficient when liposomes are used.29 this may in part explain the better cryoprotective results obtained with the egg yolk-based extender when compared to liposome-based extender, even in epididymal sperm (that lacks seminal plasma exposure). reports comparing freezing ability of bovine epididymal and ejaculated sperm are scarce and sometimes contradictory. although sperm motility did not differ, epididymal sperm had more intact membranes after freezing/thawing than ejaculated sperm.10,11 whereas acrosome integrity was similar in 1 study, more acrosomal damage was reported in epididymal sperm in another study.10,11 although 1 study reported similar percentage of cleaved embryos on day 3 and blastocyst development on day 7 after in vitro fertilization8, lower cleavage rate (cleaved embryos on day 3/mature oocytes) was reported with frozen epididymal sperm than ejaculated sperm in another study.14 however, in the current study, there were no differences between ejaculated and epididymal sperm in postthaw motility, plasma and acrosomal membrane integrity, or membrane fluidity. high percentage of clinical theriogenology • volume 12 number 1 • march 2020 36 proximal and distal droplets observed in epididymal sperm in this study were expected, as bulls enrolled in this study were young and epididymal sperm have a high percentage of cytoplasmic droplets, although many are shed during ejaculation. regardless of the semen extender used, frozen/thawed epididymal sperm had a remarkably lower mitochondrial potential than ejaculated sperm in this study. cytoplasmic droplets are enriched for enzymes involved in energy production and phosphorylation.30 perhaps the higher percentage of cytoplasmic droplets in epididymal sperm resulted in higher energy and ros production, thus leading to mitochondrial damage. conversely, mitochondrial potential increases during transit through the epididymis.29 mitochondrial potential was not evaluated herein before freezing, but it is possible that differences exist between epididymal and ejaculated sperm mitochondrial function. moreover, the association between mitochondrial function in epididymal and ejaculated sperm and fertility is not known. conclusion contrary to expectations, the egg yolk-based semen extender studied herein provided better cryoprotection than liposome-based extender, with a higher protective action on sperm motility, membrane and acrosome integrity, and membrane fluidity. epididymal sperm had higher or similar postthaw motility, acrosome integrity or membrane fluidity than ejaculated sperm. however, mitochondrial potential was lower. it remains to be determined if superior in vitro results obtained with an egg yolk-based extender translates into better in vivo and in vitro fertility when compared to a liposome-based extender. conflict of interest authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. references 1. sathe s, shipley c: cryopreservation of semen. in: hoper r: editor. bovine reproduction. 1st edition, ames; wiley-blackwell: 2013, p. 662-670. 2. bousseau s, brillard j, marquant-le guienne b, et al: comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. theriogenology 1998;50:699-706. 3. bergeron a, crête mh, brindle y, et al: low-density lipoprotein fraction from hen’s egg yolk decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane. biol reprod 2004;70:708-717. 4. manjunath p, nauc v, bergeron a, et al: major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen’s egg yolk. biol reprod 2002;67:1250-1258. 5. sullivan r, saez f: epididymosomes, prostasomes, and liposomes: their roles in mammalian male reproductive physiology. reproduction 2013;146:r21-35. 6. fleish a, malama e, witschi u, et al: effects of an extension of the equilibration period up to 96 hours on the characteristics of cryopreserved bull semen. theriogenology 2017;89:255-262. 7. miguel-jimenez s, mogas t, peña ai, et al: post-thaw changes in sperm membrane and ros following cryopreservation of dairy bull semen using four different commercial extenders. proc 30th annual meeting of the brazilian embryo technology society, foz do iguazu, pr, brazil, aug 25-27th. anim reprod 2016;13:573 (abs). 8. rao ra, bane a, gustafsson bk: changes in the morphology of spermatozoa during their passage through the genital tract in dairy bulls with normal and impaired spermatogenesis. theriogenology 1980;14:1-12. 9. almeida fc, silva sv, souza hm, et al: effects of glycerol, equilibration time and antioxidants on post-thaw functional integrity of bovine spermatozoa directly obtained from epididymis. andrologia 2017;49, e12623. https://doi.org/10.1111/and.12623 10. bertol maf, weiss rr, kozicki le, et al: in vitro and in vivo fertilization potential of cryopreserved spermatozoa from bull epididymides stored for up to 30 hours at ambient temperature (18 °c-20 °c). theriogenology 2016;86: 1014-1021. 11. cunha atm, carvalho jo, kussano nr, et al: bovine epididymal spermatozoa: resistance to cryopreservation and binding ability to oviductal cells. cryobiology 2016;73:348-355. 12. papa pm, papa fo, oliveira la, et al: different extenders in the cryopreservation of bovine epididymal spermatozoa. anim reprod sci 2015;161:58-63. 13. lopes g, soares l, ferreira p, et al: tris-egg yolk-glycerol (tey) extender developed for freezing dog semen is a good option to cryopreserve bovine epididymal sperm cells. reprod dom anim 2015;50:97-103. 14. chaveiro a, cerqueira c, silva j, et al: evaluation of frozen thawed cauda epididymal sperms and in vitro fertilizing potential of bovine sperm collected from the cauda epididymal. iranian j vet res 2015;16:188-193. clinical theriogenology • volume 12 number 1 • march 202037 15. losano jda, angrimani dsr, rui br, et al: the addition of docosahexaenoic acid (dha) and antioxidants (glutathione peroxidase and superoxide dismutase) in extenders to epididymal sperm cryopreservation in bulls. zygote 2018;26:199-206. 16. nichi m, rijsselaere t, losano jda, et al: evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential, membrane integrity, sperm motility and in vitro fertilization in bovine epididymal sperm. reprod dom anim 2017;52:257-263. 17. martins cf, rumpf r, pereira dc, et al: cryopreservation of epididymal bovine spermatozoa from dead animals and its uses in vitro embryo production. anim reprod sci 2007;101:326-331. 18. koziol jh, armstrong cl. manual for breeding soundness examination of bulls. 2nd edition, society for theriogenology: 2018. 19. ansari ms, rakha ba, akhter s, et al: optixcell improves the postthaw quality and fertility of buffalo bull sperm. theriogenology 2016;85:528-532. 20. singh ak, kumar a, honparkhe m, et al: comparison of in vitro and in vivo fertilizing potential of buffalo bull semen frozen in egg yolk-,soya bean lecithin-and liposome-based extenders. reprod dom anim 2018;53:195-202. 21. kumar p, saini m, kumar d, et al: liposome-based semen extender is suitable alternative to egg yolk-based extender for cryopreservation of buffalo (bubalus bubalis) semen. anim reprod sci 2015;159:38-45. 22. freitas cp, dell’aqua-junior ja, papa fo, et al: the effect of tris and botu-bov for bovine sexed sperm cryopreservation. reprod fertil dev 2008;20:211 (abs). 23. kahiravan p, kalatharan j, karthikeya g, et al: objective sperm motion analysis to assess dairy bull fertility using computer-aided system – a review. reprod dom anim 2011;46:165-172. 24. pini t, leahy t, de graaf sp: sublethal sperm freezing damage: manifestations and solutions. theriogenology 2018;118:172-181. 25. comier n, sirard ma, bailey jl: premature capacitation of bovine spermatozoa is initiated by cryopreservation. j androl 1997;18:461-468. 26. kuroda k, fukushima m, harayama h: premature capacitation of frozen thawed spermatozoa from subfertile japanese black cattle. j reprod dev 2007;53:1079e86. 27. thundathil g, j larsson, s mapletoft, et al: relationship between the proportion of capacitated spermatozoa present in frozen thawed bull semen and fertility with artificial insemination. int j androl 1999;22:366e73. 28. manjunath p, therien i.: role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation. j reprod immunol 2002; 53:109-119. 29. le guillou j, ropers mh, gaillard c, et al: sequestration of bovine seminal plasma proteins by different assemblies of phosphatidylcholine: a new technical approach. colloids and surfaces b: biointerfaces 2016;140:523-530. 30. yuan s, zheng h, zheng z, et al: proteomic analyses reveal a role of cytoplasmic droplets as an energy source during epididymal sperm maturation. plos one 8, e77466. doi:10.1371/journal.pone.0077466 clinical theriogenology • volume 12 number 1 • march 2020 38 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (adobe rgb \0501998\051) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages 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/usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice 2020 management of hematometra in a boer doe management of hematometra in a boer doe nicole poirier,a joe smith,b ryan breuer,a alanna farrell,a cassandra klostermann,a chia tseng,c amanda kreuder,b caitlin wileyb alloyd veterinary medical center bveterinary diagnostic and production animal medicine cveterinary clinical sciences iowa state university, ames, iowa abstract a 3 year old, boer doe in late gestation was presented for an elective cesarean section due to a previously diagnosed abdominal wall hernia. she was estimated to be 147 days pregnant, based on date of artificial insemination. physical examination, abdominal ultrasonography, blood chemistry analyses and complete blood count were done; there was a mild anemia, but no other significant findings. twelve hours after cesarean section, the patient was dull and on further examination was tachycardic, tachypneic and anemic with a packed cell volume of 10%. two liters of whole blood were transfused. differential diagnoses included hemoabdomen or hematometra. next day an exploratory laparotomy was performed and revealed a distended, blood-filled uterus, confirmed to be a hematometra. medical therapy included intravenous fluids, aminocaproic acid, pantoprazole, antibiotics, and antiinflammatories. subsequent follow up revealed that the patient had resumed normal behavior. keywords: cesarean section, transfusion, alfaxalone, aminocaproic acid, hematometra background spontaneous intrauterine hemorrhage (hematometra) after caesarean section has not been reported in goats. case presentation a 77.0 kg, 3 year old boer doe in late gestation was presented to the iowa state university food animal and camelid hospital in early winter for an elective cesarean section, due to a previously diagnosed abdominal body wall hernia, identified in the previous autumn after embryo recovery procedure. doe reported to be systemically healthy with normal appetite for a diet consisting of a quart of grain twice daily and free choice hay. there was no other prior medical history. on presentation (day 0), the doe appeared bright, alert and responsive, and had a pendulous abdomen (figure 1). rectal temperature (100.2ºf, normal: 100 103.5ºf), pulse rate (80 beats per minute, normal 70 90), and respiratory rate (40 breaths per minute, normal 15 40) were within limits. her mucous membranes were pale pink, with a normal capillary refill time (< 2 seconds). cardiopulmonary auscultation (normal sinus rhythm), ruminal contractions (2 per minute) and fecal production were within normal limits. abdominal ultrasonography revealed a severe thinning of abdominal wall, ~ 24 x 13 cm, that extended from xiphoid process to inguinal region. uterus contained 2 viable fetuses, of which 1 was positioned within hernia sac. fetal heart rates were approximately 150 and 140 beats per minute (consistent with a reported value of multiple fetuses at 145 days gestation, 140 ± 6.4 beats per minute).1 an abdominal dressing was placed to support hernia. preoperative complete blood count and chemistry panel revealed mild anemia [22% packed cell volume (pcv)]. dexamethasone (10 mg, 2 mg/ml, agrilabs, peachtree city, ga) was administered intramuscularly (im) for fetal maturation and an elective cesarean section was scheduled for the next morning. next day (day 1), a jugular catheter was placed. flunixin meglumine (1.1 mg/kg, prevail®, vetone, meridian, id) was given intravenously (iv) and ceftiofur sodium (2.2 mg/kg, naxcel®, zoetis, parsippany, nj) was given im. moderate ketonemia (1.5 mmol/l), stable mild anemia (pcv 22%) and normal total protein (6.3 g/dl) were noted. general anesthesia was induced with alfaxalone (2.5 mg/kg iv; alfaxan multidose, jurox inc., kansas city, mo). general anesthesia was maintained with sevoflurane (etsevo 1.5 2.3%; sandoz inc., clinical theriogenology • volume 12 number 1 • march 202039 princeton, nj) in 100% oxygen. a lumbosacral epidural was performed with preservative-free bupivacaine (0.06%; auromedics pharma llc., e. winsor, nj) and buprenorphine (0.01 mg/kg; buprenex®, reckitt benckiser healthcare (uk) ltd., hull, england) for reduction of anesthetic requirement and analgesia. lactated ringer’s solution was administered throughout the procedure. routine monitoring equipment included electrocardiography, invasive blood pressure monitor, pulse oximetry, capnography, and temperature, and expired inhalant concentration were recorded. spontaneous ventilation was maintained during anesthesia. phenylephrine (0.1 0.3 µg/kg/min, iv; hikma pharmaceuticals, columbus, oh) was titrated to maintain mean arterial pressure above 70 mm hg. arterial blood gas was performed and revealed a mild hypoglycemia and mild hypocalcemia, which were corrected with 2.5% dextrose supplementation in fluids and 23% calcium borogluconate (38 mg/kg, iv, agrilabs) respectively. cesarean section was performed via a left-flank approach in right lateral recumbency following standard aseptic and surgical techniques. an approximately 15.0 cm dorsal-to-ventral incision was created in left flank region utilizing a #10 scalpel blade through skin, subcutaneous tissue, external abdominal oblique, internal abdominal oblique, and transversus abdominis muscle layers, and into peritoneal cavity. uterus was exteriorized and packed off with 2 lap sponges and ~ a 12 cm incision was created in the lateral wall of left uterine horn. two viable kids were extracted from the uterus, without complications. immediately after delivery, kids were scored 9/10 and 10/10, respectively, utilizing a modified apgar scoring system (table). uterine incision was closed utilizing 0 polydioxanone suture (pds ii®, ethicon, basking ridge, nj) in utrecht pattern. a second layer was inverted with the same 0 pds in utrecht pattern over initial uterine closure. peritoneum and transversus abdominis were closed in a simple continuous pattern using 0 pds. internal abdominal oblique and external abdominal oblique were then closed in a simple continuous pattern using 0 pds. subcutaneous layer was closed using 0 pds in a simple continuous pattern and skin was closed using 0 braunamid (tuttlingen, germany) in a fordinterlocking pattern. a liquid aluminum bandage was applied to the incision. figure 1. at presentation, doe had an extensive ventral abdominal wall hernia and was in late term gestation. ventral body wall was supported with bandaging material (a). after cesarean section, bandaging material was reapplied for continued support and to facilitate tissue contraction and development of a fibrous ring for future hernia repair (b). a b clinical theriogenology • volume 12 number 1 • march 2020 40 table. modified apgar scoring system (based on veronesi et al: 2) used for evaluation of kids after alfaxalone administration. scores of 7 10 indicate no distress; 4 6 moderate distress and 0 3 severe distress. patient recovered uneventfully from general anesthesia. due to inadequate colostrum supply and severity of the hernia, it was elected to maintain her 2 kids on bottle feedings. patient received flunixin meglumine (1.1 mg/kg, iv; vetone) and ceftiofur sodium (2.2 mg/kg, im; zoetis) every 12 hours after surgery. kids remained bright and alert and consumed colostrum replacer readily. approximately 12 hours after cesarean section, patient exhibited frequent vocalization, rapid decline in mentation, tachypnea and pale mucous membranes. severe anemia was documented (pcv 10%) and she continued to decompensate over the next several hours, becoming anorexic, hypothermic, tachycardic, tachypneic, hyperglycemic, hypoproteinemic and hyperlactatemic. mild abdominal effusion was noted on transabdominal ultrasound. patient received supplemental oxygen via face mask (5 liters/minute), resuscitative fluid boluses, and ɛ-aminocaproic acid (40 mg/kg, iv, american regent, shirley, ny). additionally, 2 liters of whole blood was transfused following standard transfusion protocol, with no adverse reactions, as described below. a pretransfusion dose of flunixin meglumine (1.1 mg/kg, iv) was given. during transfusion, patient’s temperature, heart rate, and respiratory rate were monitored initially every 5 minutes, with a rate of 385 ml/hour (5 ml/kg/hour). after no initial signs of reaction, the rate was increased to 770 ml/hour (10 ml/kg/hour) and vitals were recorded every 10 minutes. to finish the transfusion, the patients’ rate was increased to 1540 ml/hour (20 ml/kg/hour), with vital parameters recorded every 15 minutes. patient was closely monitored for transfusion reactions including facial swelling, urticaria, fever, respiratory distress, anaphylactic reactions, and piloerection. although no adverse reactions were recorded, respiratory rate remained elevated throughout transfusion and the patient began to cough 20 minutes into transfusion, which ceased 10 minutes later. following first liter of transfusion, her pcv increased to 13%. abdominal ultrasonography at this point revealed significant effusion (suspected to be hemorrhage) within the abdominal cavity in the vicinity of uterus. there was clinical uncertainty whether abdominal ultrasonography reflected a hemoabdomen due to uterine rupture, or a hematometra. an abdominocentesis was inconclusive. exploratory laparotomy was elected to determine cause of anemia and a second liter of whole blood transfusion was initiated. general anesthesia was induced with ketamine (2 mg/kg, iv; zetaminetm, vetone, boise, id) and propofol (2 mg/kg iv; propoflotm, zoetis) and maintained on sevoflurane (etsevo 0.9 -1.3%) in 100% oxygen. a ketamine (20 µg/kg/min) and fentanyl (0.05 µg/kg/min; hospira inc., lake forest, il) were given for intraoperative analgesia at constant rate infusion. routine monitoring equipment was used as previously described. to maintain normotension, a dopamine (2 µg/kg/min; hospira inc., lake forest, il) was given during the procedure at constant rate infusion. arterial blood gas revealed mild hypocalcemia post-blood transfusion; therefore, 23% calcium borogluconate (56 mg/kg, iv) was given. patient was placed in lateral recumbency and the previous surgical incision was reopened to expose abdominal contents. there was no evidence of excessive free fluid or blood in the abdominal cavity. hematometra was confirmed via fine needle aspiration. remainder of the abdominal exploration was unrewarding. abdominal incision was closed in a routine fashion. clinical theriogenology • volume 12 number 1 • march 202041 patient recovered well from general anesthesia with normalization of her vital parameters, mentation, and activity. a balanced polyionic fluid with 20 mg/kg of aminocaproic acid was continued. pain was managed with morphine (0.1 mg/kg, iv) and flunixin meglumine (1.1 mg/kg, iv). nasal oxygen supplementation was discontinued. on day 3, doe was quiet but alert and resting comfortably. her appetite, mentation, and vital physical parameters remained normal. her last dose of aminocaproic acid was given (4 doses total) and intravenous fluids discontinued. tarry fecal matter was noted and a strong positive fecal occult blood test confirmed gastrointestinal bleeding. doe was started on pantoprazole (1 mg/kg iv, protonix®, pfizer, new york, ny). at this time the patient had normal lactate, glucose, and ketones. her pcv was 22% with a total protein of 6.2 g/dl; however, a newly developed hyperfibrinogenemia was noted (600 mg/dl). this elevation was attributed to surgical inflammatory process occurring within the uterus after cesarean section. on the third day after initial surgery, a small amount of blood-tinged vaginal discharge was observed. her pcv remained stable at 25% with a total protein of 6.8 g/dl. her abdominal hernia support bandage was changed and her incision was evaluated to be healing appropriately. on day 6, flunixin meglumine was discontinued and pantoprazole was discontinued after multiple negative fecal occult blood tests. patient continued to have bloody vaginal discharge that waxed and waned from mild to prominent over next several days. patient’s intravenous catheter was removed on day 8 of hospitalization. at this time, pcv remained stable at 26%. patient continued to have normal vital parameters and her appetite and energy were appropriate. on the evening of day 9, patient became febrile and tachycardic. flunixin meglumine (1.1 mg/kg, iv) and procaine penicillin g (22,000 iu/kg, im, agrilabs) were administered every 12 hours. patient did not have any other abnormal signs at this time. her udder was not swollen, painful, or hot on palpation. patient continued to have mild bloody vaginal discharge but her appetite and attitude remained adequate. despite continued mild bloody vaginal discharge, patient showed marked clinical improvement. normal uterine involution was observed on transabdominal ultrasonography at this stage with small amount of fluid. no free abdominal fluid was observed and surgical site was healing appropriately. on day 9, a complete blood count revealed persistent hyperfibrinogenemia, mild mature neutrophilia with toxic changes, and anemia. on day 10, transabdominal ultrasonography findings of uterus remained unchanged and her pcv improved to 22%. uterine lavage was performed and a minimal amount of brown/red tinged mucus-like fluid was recovered. it was suspected that fetal membranes were autolyzed and passing appropriately within the vaginal discharge observed. on day 12, ceftiofur sodium was discontinued. patient continued to have stringy, bloody vaginal discharge but her clinical condition remained stable in the subsequent days. there appeared to be no further development of metritis, mastitis or concurrent infection. treatment treatment included cesarean section, hypovolemic shock management, blood transfusion, pain management, antibiotics, and aminocaproic acid therapy. twelve hours after cesarean section, patient received supplemental oxygen via mask (5 liters/minute), intravenous fluids, and aminocaproic acid. two liters of whole blood was transfused; her pcv prior to first transfusion was 10% and then increased to 13%. after second transfusion, pcv increased to 19%. outcome patient experienced intra-uterine hemorrhaging following cesarean section and a transfusion of 2 liters of whole blood was necessary for stabilization. an exploratory laparotomy was performed revealing a hematometra. patient was maintained on systemic broad-spectrum antibiotics, a nonsteroidal antiinflammatory and a proton pump inhibitor throughout the course of hospitalization. uterine involution was monitored periodically using transabdominal ultrasound. patient was noted to resume normal behavior and was later determined to be a good surgical candidate for an abdominal hernia repair. clinical theriogenology • volume 12 number 1 • march 2020 42 discussion hematometra following cesarean sections has apparently not been reported in goats. there are reports of spontaneous uterine hemorrhage in other veterinary species, often related to the presence of cystic endometrial hyperplasia. severe hematometra was reported in a german shepherd dog with cystic endometrial hyperplasia. an abdominal ultrasonography demonstrated that uterine body and cranial vaginal lumen were fluid-filled and containing hyperechoic, polyploid masses suspected to be blood clots.3 in the current case, abdominal ultrasonography led to suspicion of an acute hemorrhage due to visualization of fluid within abdomen in the location of uterus. diagnostic tests supported an acute hemorrhage, but the source was unknown. with evidence of acute hemorrhage, an exploratory laparotomy was necessary to confirm the source of bleeding. in this case, the procedure revealed a distended, fluidfilled uterus. this same presentation was observed in the case of a german shepherd dog where the procedure revealed a mildly enlarged uterus. however, an ovariohysterectomy was performed on the dog and histopathology confirmed a severe, cystic, endometrial hyperplasia and pyometra complex.3 cases of canine hematometra have also been associated with uterine torsion or other uterine disease such as a pyometra, in addition to toxicities such as rodenticide exposure.4 perhaps many cases of canine hematometra are asymptomatic and uncomplicated until they lead to uterine torsion or other uterine pathology.5 spontaneous hemorrhage after cesarean section in goats has not been reported. it is not known at this time what was the exact mechanism for development of hematometra in this doe. fetal membranes were not forcibly removed during surgery, and uterus was closed in routine fashion. no significant hemorrhage was noted during surgery. perhaps the nature of induction contributed to this finding. this case involved hematometra in a goat with a concurrent severe abdominal wall hernia. goats often have forms of hernias, both acquired and congenital. abdominal, inguinal and scrotal hernias are usually acquired whereas umbilical hernias are congenital in this species.6 patient in this case had an acquired, severe abdominal hernia. although a very rare complication, a hernia may interfere with normal parturition. due to the extent of the hernia in this doe, an elective cesarean section was recommended, due to the risk of complications with vaginal delivery as a gravid uterus can become entrapped in an abdominal hernia, making vaginal delivery difficult.6 presence of an abdominal hernia can pose the risk of uterine incarceration or strangulation.7 other risks that have been reported in humans with a gravid uterus and abdominal hernia include abortion, preterm or dysfunctional labor, and intrauterine growth retardation leading to fetal death.7 risk of uterine strangulation is an indication for early hospitalization and elective cesarean section, with subsequent hernia repair.8 by performing an elective cesarean section, these complications may be avoided. selection of an appropriate anesthesia protocol for cesarean sections is important to minimize risks to doe and kid(s). alfaxalone is a neurosteroidal anesthetic agent characterized by a smooth and rapid induction, short duration of action, and quiet recovery.9 alfaxalone does not accumulate in tissues at clinical doses, making this useful in food animal species.9 alfaxalone’s mechanism is a synthetic neuroactive steroid that acts on gabaa receptors in the central nervous system producing general anesthesia.9 it is a reliable and smooth induction agent in sheep, goats, and calves.10 in the present case, use of alfaxalone was demonstrated to be clinically useful in inducing anesthesia. alfaxalone has not caused major adverse effects in previous studies, and this agent can provide excellent induction or shortterm anesthesia as a single bolus.10 alfaxalone is associated with dose-dependent and administration ratedependent respiratory depression and arterial vasodilaton, although these effects are transient and mild at clinically useful doses. however, these cardiovascular and respiratory depressant effects can become significant in critically ill patients. in goat kids, alfaxalone appeared to be a suitable induction agent.11 alfaxalone does not appear to have detrimental effects in neonates when used as an induction agent prior to caesarean section. alfaxalone was associated with higher apgar scores and overall neonatal vitality in puppies during the first 60 minutes after delivery compared to propofol; however, 3 month survival rate did not differ between groups.12 two kids in this case had apgar scores of 9 and 10 respectively, indicating that alfaxalone may be safe for kids during a caprine cesarean section. drugs that should be clinical theriogenology • volume 12 number 1 • march 202043 avoided include xylazine, ketamine, and methoxyflurane due to the association of increased risk for neonatal and maternal mortality.13 aminocaproic acid is an antifibrinolytic agent that has been used in veterinary medicine in cases of acute hemorrhage.14 hyperfibrinolysis has been shown to occur in dogs with severe, acute blood loss and antifibrinolytic agent therapy may be indicated in these cases.14 greyhounds in particular are predisposed to excessive fibrinolysis after surgery. aminocaproic acid has been beneficial as a perioperative measure in this patient population.14 generally, any animal at risk for hemorrhage after surgery may benefit from antifibrinolytic agents as treatment with aminocaproic acid has been shown to enhance clot strength in patients with hyperfibrinolysis.14 our patient was given injectable aminocaproic acid after a 50% decrease in her pcv 12 hours after surgery. a loading dose of 40 mg/kg was administered iv, followed by 3 additional doses of 20 mg/kg. each dose was diluted in 1 liter of balanced polyionic solution and given as a constant rate infusion. aminocaproic acid has been well tolerated by dogs, but its use has not been reported in goats. no adverse effects were observed in dogs for any dose of aminocaproic acid.14 aminocaproic acid as a procoagulant therapy, as described for other species, appears to be a potentially useful adjunct therapy for anemic goats due to acute hemorrhage. currently no prospective studies regarding the use of pantoprazole in goats exist. efficacy of pantoprazole in alpacas15 has been reported and has been shown to increase ph of third compartment. case reports involving other ruminant species demonstrate the use of pantoprazole in a goat,16 a sheep17 and yak bulls18 with no perceived complications. in humans, long-term use of pantoprazole has been associated with neutropenia and thrombocytopenia,19 which were not observed in our doe, although the history of transfusion could complicate these observations. based on this case and other ruminant cases, pantoprazole may be a safe short-term therapy for suspected abomasal ulcers. in this case, patient received 2 liters of whole blood, with a resulting increase in pcv from 10 to 19%. no transfusion reactions were noted (reported transfusion reaction rate was 16% in small ruminants in 1 study).20 this case highlights other issues of management of the doe with respect to cesarean sections. while not common, the need for a transfusion may require access to donors or blood products. while not commonly used, multiple drugs utilized for the management of this case were not labelled for goats and were thus used in an extra-label fashion. withdrawal times for alfaxalone and aminocaproic acid for goats are required by the food animal residue avoidance databank. no tissue residue data has been recorded to base a withdrawal time for alfaxalone in goats. however, the drug is rapidly cleared from plasma and plasma half-lives in cats, dogs, and horses range from 30 minutes to 1.5 hours for alfaxalone. furthermore, no tissue or residue data has been recorded for aminocaproic acid in any food producing species. in general, there is limited literature on aminocaproic acid. there has been only one pharmacokinetic study in horses demonstrating plasma half-life of 2.3 hours following a 20 minute cri at 3.5 mg/kg/minute. aminocaproic acid is mainly used in humans and the drug concentrations were equal or higher in milk than plasma. a minimum of a 6 week withdrawal interval has been recommended for this drug in the past, but further research is required regarding testing for aminocaproic acid. learning points • while not common, spontaneous hemorrhage following caesarean section should be considered as a differential in weak or anemic goats after uterine surgery. • alfaxalone did not appear to have detrimental effects to neonatal kids when used as an induction agent prior to caesarean section. • aminocaproic acid procoagulant therapy, as described for other species, appeared to be a potential useful therapy for goats with acute and ongoing blood loss from hematometra. clinical theriogenology • volume 12 number 1 • march 2020 44 references 1. walsh p, carvallo chaigneau fr, anderson m, et al: adverse effects of a 10-day course of ibuprofen in holstein calves. j vet pharmacol ther 2016;39:518-521. 2. veronesi mc, panzani s, faustini m, et al: an apgar scoring system for routine assessment of newborn puppy viability and short-term survival prognosis. theriogenology 2009;72:401-407. 3. troxel mt, cornetta am, pastor kf, et al: severe hematometra in a dog with cystic endometrial hyperplasia/pyometra complex. j am anim hosp assoc 2002;38:85-89. 4. padgett s, stokes j, tucker r, et al: hematometra secondary to anticoagulant rodenticide toxicity. journal of the american animal hospital association 1998;34:437-439. 5. barrand k: unilateral uterine torsion associated with haematometra and cystic endometrial hyperplasia in a bitch. veterinary record 2009;164:19-20. 6. al-sobayil fa, ahmed af: surgical treatment for different forms of hernias in sheep and goats. j vet sci 2007; 8:185-191. 7. hcini n, chelli d, boudaya f, et al: herniated gravid uterus growing in an incisional hernia: study of three cases. hernia 2016;20:633-636. 8. emegoakor c, dike e, emegoakor f: unusual complications of incisional hernia. ann med health sci res. 4. india 2014. p. 971-974. 9. maney jk, shepard mk, braun c, et al: a comparison of cardiopulmonary and anesthetic effects of an induction dose of alfaxalone or propofol in dogs. vet anaesth analg 2013;40:237-244. 10. camburn ma: use of alphaxalone-alphadolone in ruminants. vet rec 1982;111:166-167. 11. gibbons rw: anaesthesia of kids with alphaxalone/alphadolone. vet rec 1986;119:339c. 12. doebeli a, michel e, bettschart r, et al: apgar score after induction of anesthesia for canine cesarean section with alfaxalone versus propofol. theriogenology 2013;80:850-854. 13. ryan sd, wagner ae: cesarean section in dogs: anesthetic management. compendium on continuing education for the practicing veterinarian 2006;28:44-56. 14. brown jc, brainard bm, fletcher dj, et al: effect of aminocaproic acid on clot strength and clot lysis of canine blood determined by use of an in vitro model of hyperfibrinolysis. am j vet res 2016;77:1258-1265. 15. smith gw, davis jl, smith sm, et al: efficacy and pharmacokinetics of pantoprazole in alpacas. j vet intern med 2010;24:949-955. 16. smith j, klostermann c, harm t, et al: abomasal hamartoma in a la mancha wether. veterinary record case reports 2017;5:e000515. 17. viall ak, larios mora a, brewer mt, et al: what is your diagnosis? nasal discharge from a sheep. vet clin pathol 2018;47:503-504. 18. smith js, sheley m, chigerwe m: aspiration pneumonia in two tibetan yak bulls (bos grunnies) as a complications of ketamine-xylazine-butorphanaol anesthesia for recument castration. j zoo wildl med 2018;49:242-246. 19. yu z, hu j, hu y: neutropenia and thrombocytopenia induced by proton pump inhibitors: a case report. drug saf case rep 2018;5:28. doi: 10.1007/s40800-018-0093-0. 20. luethy d, stefanovski d, salber r, et al: prediction of packed cell volume after whole blood transfusion in small ruminants and south american camelids: 80 cases (2006-2016). j vet intern med 2017;31:1900-1904. (editor’s note: online edition of the manuscript has color photographs) clinical theriogenology • volume 12 number 1 • march 202045 clinical theriogenology • volume 12 number 1 • march 2020 46 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (adobe rgb \0501998\051) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype true /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false 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false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /converttocmyk /destinationprofilename (u.s. web coated \(swop\) v2) /destinationprofileselector /usename /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 0.864000 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice clinical theriogenology 2022; 14: 70 perinatal mortality in dogs and cats christopher premanandan,a dalen agnewb acollege of veterinary medicine, the ohio state university, columbus, oh bcollege of veterinary medicine, michigan state university, east lansing, mi abstract canine and feline perinatal loss is a frequent and frustrating event for practitioners and breeders. working on these cases can also be enigmatic and difficult for diagnostic pathologists to determine the cause of death. typical conditions associated with canine perinatal loss include infectious disease, sepsis, pneumonia, congenital abnormalities, and trauma. fading puppy syndrome is also a consideration. currently, feline perinatal loss is not well characterized. maternal health, a critical factor in perinatal losses, should also be assessed. collecting an endometrial biopsy could constitute a critical part of this evaluation; however, more investigation is needed to correlate endometrial lesions to fertility. keywords: abortion, fetal loss, dogs, cats, diagnostic pathology, perinatal mortality, necropsy introduction neonatal period in dogs is not well defined but is often referred to as a period from birth to 30 days. additionally, the early neonatal period is defined as the first 7 days of life and the late neonatal period is considered 1 4 weeks. perinatal mortality can be defined as death that take place before first week of life in addition to stillbirth. defined percentages of litter loss in dogs are most frequently recorded in breeding colonies. of all mortalities 55.6% occurred before the first week of life (including stillbirth) with a perinatal death percentage of 11.9%.1 perinatal loss in 10,810 litters in a norwegian kennel club study was 8%.2 only limited information is available regarding feline perinatal loss, and studies performed were less discriminatory and lacked clear distinction between early and late neonatal loss. however, the percent of stillbirth has been characterized. in 14 litters, 4.7% of the kittens were stillbirths.3 analysis of a database comprised of french breeder litters described a rate of 8.5% over a 3-year period.4 causes of perinatal death it is important to specify the types of infectious diseases that are related to stillbirth and the death of puppies and kittens within the first week of life. since infectious diseases commonly affect litters in the late neonatal period, confirmation of similar infectious agents as a cause of stillbirth or early neonatal loss is more difficult.5 in addition, there is conflicting information regarding the incidence of perinatal loss due to infectious disease.2,6 causes of perinatal loss in dogs comments fading puppy syndrome pathogenesis unknown; refer to subheading below bacterial pathogens beta-hemolytic streptococcus spp., escherichia coli, brucella canis7,8 viral pathogens canine herpesvirus-1, canine adenovirus-1, canine parvovirus-2 congenital defects losses from 1.0 to 2.2%1,9 parasitic pathogens toxoplasma gondii10 maternal infections refer to bacterial and viral pathogens maternal trauma n/a dystocia n/a many of the etiologies listed above are not limited to the perinatal period, specifically, infectious agents that overlap with canine abortion and late neonatal loss. causes of perinatal loss due to infectious disease are fairly well established despite the difficulty in detecting pathogens in many cases. however, many noninfectious causes are poorly characterized. specifically, noninfectious placental disease is not well characterized in dogs and cats. guidelines for the clinical theriogenology 2022; 14: 71 pathologic evaluation of human placenta are provided by the college of american pathologists.11 these guidelines classify the causes into 3 major categories; maternal conditions, fetal conditions, and placental abnormalities. maternal conditions include conditions such as placental abruption, systemic disorders at term, and severe oligoamnios. stillbirth and perinatal death are among the criteria of the fetal category. placental conditions include thrombosis, umbilical cord torsions, and retroplacental hemorrhage. placental abruption, hematoma and infarction are well-established causes of growth restriction and fetal death in humans.12 placental abruption represents hemorrhage in the retroplacental region at the location of the basal plate. abruption is also most commonly associated with this location; however, hemorrhage at the margin of the placental disc is possible.13 ischemic lesions can be categorized as fetal vascular malformation or maternal vascular malformation. fetal vascular malformation is typically associated with macroscopic abnormalities such as umbilical cord torsion, true umbilical cord knots, or excessive umbilical cord lengths.14 these parallels exist in horses and cattle; however, these are not well characterized or reported in dogs and cats. maternal vascular malformation is related to the remodeling process that takes place in the maternal vasculature. spiral arteries are a unique vascular arrangement in the primate placenta in which trophoblastic invasion results in modified blood flow to the fetal membrane. ultimately, this results in less vascular resistance and increased blood flow to the placental disc. abnormalities in this process can result in a myriad of lesions in the chorioallantois including infarction, hypoplasia, and retroplacental hematoma formation.14 infarction can result from both maternal and fetal vascular malformation; however, maternal vascular malformation appears to be the most commonly associated predisposing cause. infarct affecting > 5% of the nonperipheral placental structure is worthy of consideration.14 vascular abnormalities, particularly ischemia, have not been well characterized in canine and feline labyrinths. authors have commonly observed coagulation necrosis adjacent to the marginal hematomas in normal neonates. however, areas of coagulation necrosis and hemorrhage have also been observed in the middle region of the labyrinth in cases where the cause of fetal loss could not be determined from the analysis of fetal tissue. the potential for acute or chronic placental disease induced by vascular events should be considered and investigated in dogs and cats. fading puppy syndrome respiratory disease is a common feature of what many breeders call ‘fading puppy syndrome,’ a frequent cause of death in neonates. recent and ongoing research at the michigan state university suggests that developmental lung disease is likely a major cause of these cases, similar to a condition in human pediatric medicine.15 histologically, pulmonary dysplasia includes underdeveloped alveoli, pulmonary artery medial hypertrophy, increased numbers of thin-walled venous profiles, and increased capillary profiles. the 3-d reconstructions of the affected lungs have vascular abnormalities suggestive of arteriovenous shunts. continued research, including genetic analyses, is required to better understand this condition in dogs and humans and will require rigorous examination of cases of respiratory disease in puppies. canine endometrial disease similar to cattle and horses, a means to assess the health and quality of the endometrium while maintaining the animal’s fertility, is important. endometrial biopsies have been the mainstay of endometrial diagnostics in horses for some time, and to a lesser extent in ruminants. however, this technique has been difficult to apply to the bitch for several reasons: 1. zonary placentation and the regional nature of the placental sites make it difficult to extrapolate the findings from 1 endometrial sample to the entire surface of the uterus. 2. the size of the patient requires small instrumentation, blind sampling, and advanced operator technical skill. 3. the sensitivity of the endometrium to trauma during diestrus (sampling during this period may induce cystic endometrial hyperplasia). however, given these limitations, there is still value to identify endometrial lesions in real-time. general procedure is as follows: sampling should ideally occur at the end of diestrus (progesterone is < 1 ng/ml), but it may be possible during anestrus or proestrus. dogs are restrained manually (and sedated if necessary). an endoscope with a 5-french rigid instrument port is inserted transvaginally and upon visualization of the cervix, a 5-french biopsy instrument is passed transcervically into the uterus. collection of at least of 3 separate samples is suggested. sanitizing the endoscope is not required between samples. samples can be fixed in 10% neutral buffered formalin, 4% paraformaldehyde, or bouin’s solution, according to pathologist’s preference. careful handling of very small and delicate samples is critical as they can easily be crushed or lost. a 26-gauge needle may help in removing tissue from the biopsy instrument. some caution is necessary during anestrus, as the endometrium is so thin-walled that perforation of the uterus is very easy and the sample may be only adipose tissue. however, no negative sequela was noted from these events. full-thickness endometrial samples can also be taken during a laparotomy. it is tempting to take these samples during a caesarian section; however, these samples can be difficult to interpret as inflammation, necrosis, and cystic change are a normal part of mid to late involution, placental delivery, and involution and other changes are likely masked by these features. lesions described as most prevalent in cases of canine subfertility (lack of pregnancy or fetal resorption) include chronic endometritis, cystic endometrial hyperplasia, and endometrial fibrosis.16-18 endometritis and fibrosis were not described during the immediate postpartum period in a study of 98 bitches at various stages of involution.19 the relative value of biopsies taken during diestrus versus other stages of the estrous cycle has not been determined. however, since canine endometrium morphology undergoes a variety of histological changes during the estrous cycle, evaluation by a pathologist familiar with canine endometrium is advisable. clinical theriogenology 2022; 14: 72 pyometra was not detected in transcervical endometrial biopsies; however, fibrosis, cystic endometrial hyperplasia, and inflammation (equal to the full thickness sections) were observed in endometrial samples obtained after ovariohysterectomy.20 all samples with pyometra were from diestrous animals. therefore, it is more likely that the sampling procedure itself induced pyometra. however, authors have identified multiple cases of pyometra and endometritis using transcervical endometrial biopsies. whereas additional studies are needed, transcervical endometrial biopsies offer additional diagnostic information for bitch infertility, including repeated abortions. feline endometrial disease although there are many similarities between canine and feline placentation grossly, substantial variations can be observed microscopically, primarily in the chorionic labyrinth. feline placental site involution studies are very limited and consequently it is difficult to elucidate endometrial association in subfertility. although it is tempting to apply the knowledge that we have gleaned from bitch endometrium to queen, the endometrial lesions associated with feline infertility has to be explored. a systematic study of the lesions that affect feline endometrium and subsequent correlation of these lesions to subfertile queens would be an ideal starting point. conclusion investigation of canine and feline perinatal loss requires collaboration and cooperation among the owner, clinician, and pathologist as the determining specific causes can be difficult. perhaps managing expectations is a critical aspect of the process. majority of investigations often result in negative histological and ancillary diagnostic tests. however, the value of excluding infectious, particularly transmissible, causes of the perinatal death is often overlooked in this process. diagnostic testing for infectious agents is well established and readily available in most commercial and state diagnostic laboratories. critical investigations remain for noninfectious causes of death that may involve placental insufficiency and endometrial disease. additionally, further investigation of potential causes of ‘fading puppy syndrome’ is necessary as the pathogenesis of this condition is poorly understood. conflict of interest authors have no conflicts of interest. no funding was received for this publication. references 1. potkay s, bacher jd: morbidity and mortality in a closed foxhound breeding colony. lab anim sci 1977;27:78-84. 2. tønnessen r, borge ks, nødtvedt a, et al: canine perinatal mortality: a cohort study of 22 breeds. theriogenology 2012;77:17881801. 3. root mv, johnston sd, olson pn: estrous length, pregnancy rate, gestation and parturition lengths, litter size, and juvenile mortality in the domestic cat. j am anim hosp assoc 1995;31:429-433. 4. fournier a, masson m, corbière f, et al: epidemiological analysis of reproductive performances and kitten mortality rates in 5,303 purebred queens of 45 different breeds and 28,065 kittens in france. reprod domest anim 2017;52(suppl 2):153-157. 5. sturgess k: infectious diseases of young puppies and kittens. in: simpson gm, england gcw, harvey m: editors. manual of small animal reproduction and neonatology. british small animal veterinary association: 1998. p. 159-166. 6. lawler df: care and diseases of neonatal puppies and kittens. in: kirk’s current veterinary therapy x, philadelphia; wb saunders:1989. p.1325-1333. 7. jones rl: special considerations for appropriate antimicrobial therapy in neonates. vet clin north am small anim pract 1987:17:577-620. 8. vela ai, falsen e, simarro i, et al: neonatal mortality in puppies due to bacteremia by streptococcus dysgalactiae subsp. dysgalactiae. j clin microbiol 2006;44:666-668. 9. andersen ac puppy production to the weaning age. j am vet med assoc 1957;130:151-158. 10. bresciani kd, costa aj, toniollo gh, et al: transplacental transmission of toxoplasma gondii in reinfected pregnant female canines. parasitol res 2009;104:1213-1217. 11. langston c, kaplan c, macpherson t, et al: practice guideline for examination of the placenta: developed by the placental pathology practice guideline development task force of the college of american pathologists. arch pathol lab med 1997;121:449-476. 12. aurioles-garibay a, hernandez-andrade e, romero r, et al: prenatal diagnosis of a placental infarction hematoma associated with fetal growth restriction, preeclampsia and fetal death: clinicopathological correlation. fetal diagn ther 2014;36:154-161. 13. mittal n, byard rw, dahlstrom je: a practical guide to placental examination for forensic pathologists [published correction appears in forensic sci med pathol 2020 jan 8]. forensic sci med pathol 2020;16:295-312. 14. heider a: fetal vascular malperfusion. arch pathol lab med 2017;14:1484-1489. 15. tsoi, m, frantz, b, galambos, c, et al: the use of three dimensional images reconstruction to characterize lung vascular abnormalities in canine developmental lung disease. american college of veterinary pathology annual conference. 2019. 16. fontaine e, levy x, grellet a, et al: diagnosis of endometritis in the bitch: a new approach. reprod domest anim 2009;44(suppl 2):196-199. 17. mir f, fontaine e, albaric o, et al: findings in uterine biopsies obtained by laparotomy from bitches with unexplained infertility or pregnancy loss: an observational study. theriogenology 2013;79:312322. 18. gifford at, scarlett jm, schlafer dh: histopathologic findings in uterine biopsy sample from subfertile bitches: 399 cases (1990-2005). j am vet med assoc 2014;244:180-186. 19. al-bassam ma, thomson rg, o’donnell l: normal postpartum involution of the uterus in the dog. can j comp med 1981;45:217232. 20. christensen bw, schlafer dh, agnew dw, et al: diagnostic value of transcervical endometrial biopsies in domestic dogs compared with full-thickness uterine sections. reprod domest anim 2012;47(suppl 6):342-346. artifactual disruption of sperm midpiece in extended stallion semen artifactual disruption of sperm midpiece in extended stallion semen anne beckham,a camilo hernandez-avilés,a charles love,a larry johnson,b sheila teague,a mariah pearson,a kat lacaze,a dickson varnera adepartment of large animal clinical sciences, bdepartment of integrative biosciences college of veterinary medicine and biomedical sciences texas a&m university, college station, tx abstract microscopic examination of stallion sperm morphology provides a useful criterion for assessing semen quality and reproductive capabilities of a stallion. in previous investigations, personnel in our laboratory noted an apparent artifactual defect of the sperm midpiece when stallion semen was mixed with various commercial extenders prior to formalin fixation for sperm morphologic analysis. we coined this defect “stippled midpiece.” objective was to determine the etiology of this previously unreported defect by investigating the induced morphologic change using light and electron microscopic techniques. sixteen diluents/extenders in 2 sperm exposure times were compared prior to fixation for their effect on the incidence of stippled midpieces. raw semen was used as a control. the abnormality was most prevalent (p < 0.05) when semen was mixed with diluents containing casein, or milk and egg yolk. in contrast, diluents containing albumin or no protein were similar (p > 0.05) and exhibited the lowest (p < 0.05) incidence of stippled midpieces among diluent groups. diluents containing protein exhibited a higher (p < 0.05) incidence of stippled midpieces compared to raw semen. intrinsic sperm quality was not correlated with the incidence of stippled midpieces when semen was formalin-preserved in raw form. however, sperm quality was correlated with the incidence of stippled midpieces when semen was mixed with certain diluents prior to formalin fixation. this relationship was most pronounced when diluent contained milk or casein. transmission electron microscopic findings corroborated that of light microscopy for the location and structural features of the stippled midpieces. keywords: stallion, sperm, midpiece, morphology, fixation introduction comprehensive semen evaluation is imperative to understand the reproductive capability of a stallion and for any projected diagnoses of subfertility or infertility.1,2 microscopic examination of sperm morphology provides a useful criterion for assessing the quality of semen.3-5 semen samples may be sent to a reference laboratory for evaluation of semen characteristics and to provide insight into the reproductive capabilities of a stallion. to protect sperm function for cooled or frozen transport prior to analysis or artificial insemination, semen is diluted with a semen extender.6-8 semen can also be subjected to standard centrifugation techniques, or to centrifugal fractionation techniques, to select an enriched population of viable and morphologically normal sperm in an ejaculate.9-16 through morphologic evaluation of several semen samples sent to our laboratory, an apparent artifactual disruption of the sperm midpiece was observed on an inconsistent basis. the observed defect was readily detectable when examining wet mounts of sperm using differential interference contrast (dic) or phase contrast microscopy (figure 1), or by bright field microscopic evaluation of stained (eosin-nigrosin) smears of sperm (figure 2). the severity of the change was variable (figure 3). this disruption was initially attributed to improper semen handling or semen processing techniques while considering that it could also be a reflection of a stallion’s intrinsic sperm quality. this same midpiece disruption was observed in our laboratory following centrifugal fractionation of stallion semen through a silanated silica-particle solution (equipure™, nidacon, mölndal, sweden) and resuspension of sperm in a milk-based extender, followed by fixation in buffered formol saline. objective was to further investigate this novel disruption in sperm morphologic structure, an abnormality we have termed “stippled midpiece.” clinical theriogenology • volume 12 number 4 • december 2020493 figure 1. normal sperm and sperm with stippled midpiece (phase contrast or differential interference contrast microscopy) figure 2: normal sperm compared to sperm exhibiting stippled midpieces (bright-field microscopy eosin-nigrosin staining) a. normal sperm b. sperm with stippled midpiece c. two sperm with stippled midpieces a. normal sperm, as viewed with differential interference contrast microscopy b. with phase contrast microscopy c. sperm with stippled midpiece, as viewed with differential interference contrast microscopy d. with phase contrast microscopy a b d b a b c a b c figure 3. severity of stippled midpieces as viewed with differential interference contrast microscopy a. mild b. moderate c. marked c clinical theriogenology • volume 12 number 4 • december 2020 494 materials and methods stallions and semen collection all animal procedures were conducted following institutional animal ethics committee guidelines. stallions were of light breed, healthy, sexually active, and mature (aged 7 24 years). ejaculates were collected using an artificial vagina (missouri-model; nasco, ft. atkinson, wi) equipped with an in-line nylon micromesh filter (animal reproduction systems, chino, ca) to permit collection of gel-free semen. immediately prior to semen collection, the artificial vagina was lubricated with approximately 5 ml of nonspermicidal lubricant (clarity® a.i. lubricating jelly; aurora pharmaceutical, llc, northfield, mn). an insulated thermal jacket was used to protect semen in the semen receptacle (animal reproduction systems) from light and temperature fluctuations during transport to the adjacent laboratory. semen was collected using a breeding phantom in the presence of an ovariectomized mare. prior to each semen collection attempt, stallion’s penis was rinsed with warm water and dried thoroughly with paper towels. semen processing and analysis following semen collection, gel-free semen volume was measured by weight and immediately placed in an incubator (37ºc) to protect sperm from light and changes in temperature. sperm concentration and viability were measured17,18 using a fluorescence-based cell counter (nucleocountersp100™, chemometec, a/s, allerød, denmark). sperm motion characteristics were analyzed using a computer-assisted sperm motion analysis system (casma, ivos ii version 1.9.1, hamilton thorne biosciences, beverly, ma,) and measured.18 motility endpoints were percentage of total sperm motility, progressive sperm motility, curvilinear and straightness, and velocity (μm/s). sperm morphology was evaluated9 in wet mounts using differential interference contrast microscopy (1563 x magnification, olympus bx60, olympus america, inc., melville, ny). for analysis, aliquots (1.25 μl) of each sample were applied to a glass microscope slide (vwr® micro slides, vwr international, llc, west chester, pa) and overlain with a 22 x 22 mm no. 1.5 cover glass. a total of 100 sperm per sample were evaluated for morphology and evidence of midpiece stippling. each sperm was scored for morphologic characteristics and for presence or absence of a stippled midpiece. sperm morphology was classified19 and sperm were assigned to the following categories: normal, abnormal head, abnormal acrosome, proximal droplet, proximal droplet only (with no other defects), distal droplet, distal droplet only (with no other defects), abnormal midpiece, bent midpiece, bent tail, coiled tail, and broken tail. percentages of normal sperm and each morphologic defect were recorded, along with percentage of stippled midpieces. transmission electron microscopy transmission electron microscopy (tem) was used to provide further information about alterations in ultrastructural morphology of sperm induced by sperm manipulation prior to fixation. raw semen (negative control), semen diluted 1:1 (semen:extender) with extender (inra-96® [inra96]; imv technologies, l’aigle, france), and semen diluted 1:1 with phosphate buffered saline (pbs; corning cellgro, manassas, va) were processed for tem imaging. two ejaculates from a stallion that exhibited a high incidence of stippled midpieces when exposed to semen extender prior to fixation were used to identify the ultrastructural details of sperm with stippled midpieces. for tem processing, 0.5 ml of either raw or diluted semen was added to 15 ml tubes containing 6 ml of 2% glutaraldehyde in 0.1 m cacodylate and exposed for 1 hour. tubes were centrifuged at 500 x g for 10 minutes, and the supernatant was removed. pelleted sperm were resuspended and layered over a 0.1 m cacodylate buffer. sperm were washed by centrifugation at 500 x g for 10 minutes. pelleted sperm were transported to the image analysis laboratory, college of veterinary medicine and biomedical sciences, texas a&m university for further processing and analysis. at the image analysis laboratory, samples were incubated with 3% (v/v) glutaraldehyde in clinical theriogenology • volume 12 number 4 • december 2020495 0.1 m cacodylate buffer at room temperature for 1 hour. following incubation, samples were washed 3 times for 10 minutes each time, in water at room temperature. samples were then fixed and stained for 30 minutes in 1% osmium tetroxide in 0.1 m cacodylate buffer at room temperature for 30 minutes, washed 3 times for 10 minutes each time, in water on a shaker at room temperature, and stained for 2 hours in saturated aqueous uranyl acetate. following fixation and staining, a final wash was performed for 10 minutes, 3 times, in water on a shaker at room temperature. samples were dehydrated using increasing concentrations of ethanol (30, 50, 80, 95, and 100%) and then incubated for 15 minutes with 100% propylene oxide at room temperature. using propylene oxide:resin (1:1; epon 812), samples were incubated for 30 minutes at room temperature and then were set for 16 hours in 100% resin at room temperature. finally, the samples were transferred to beem® (better equipment for electron microscopy, bronx, ny) specimen embedding capsules on a glass slide and baked at 65ºc for 48 hours. ultrathin sections (100 nm thick) were cut using a ultramicrotome (leica em uc6, leica microsystems inc., buffalo grove, il) equipped with a diatome diamond knife. transmission electron microscope used was a fei morgagni™ 268 (fei company hillsboro, or) equipped with a megaview iii ccd (olympus, tokyo, japan) camera. each sample was scanned to capture sagittal views of sperm midpieces at appropriate magnification to view the ultrastructural detail (~ 7,000 40,000 x magnification). the tem imaging was used to augment our findings using dic microscopy, which were viewed as an artifactual disruption of the sperm midpiece. sperm were examined to obtain sagittal views in order to evaluate the integrity of the sperm plasma membrane and alignment of mitochondria. sperm were compared to previous reports describing the accepted ultrastructural detail of stallion sperm midpieces in order to distinguish normal and abnormal midpiece structure.22-27 sperm were classified as normal or abnormal in regard to midpiece structure. experimental design three ejaculates from 5 stallions (n = 15 ejaculates) were collected. semen aliquots were mixed with 16 diluents at a 1:1 (v/v) dilution ratio: equipure™ 80% silica particle solution (equipure; protein-free); puresperm™ 80% silica particle solution (puresperm; protein-free); redigrad™ 80% silica particle solution (redigrad; ge healthcare bio-sciences ab, uppsala, sweden; contained albumin); inra96 extender (contains a fractionated milk component, native phosphocaseinate); botusemen™ extender (botu-s; botupharma usa, phoenix, az [contains milk]); botusemen™ special extender (botu-sp; botupharma usa [contains milk with added cholesterol]); botusemen™ turbo extender (botu-t; botupharma usa [contains milk and added pentoxifyline]); botusemen™ gold extender (botu-g; botupharma usa [contains casein with added cholesterol]); tamu extender (tamu; prepared in laboratory [contains milk]), tyrode’s albumin lactate pyruvate media (talp; prepared in laboratory [contains albumin]); e-z-freezin® mfr5 (mfr5; animal reproduction systems [contains both milk and egg yolk, and glycerol as a permeable cryoprotectant]), e-z freezin® lactoseedta (le; animal reproduction systems [contains egg yolk and glycerol as a permeable cryoprotectant without milk]), e-z freezin® cryomax™ le (c-le; animal reproduction systems [contains egg yolk and glycerol and methylformamide as permeable cryoprotectants without milk]); e-z freezin® cryomax™ mfr5 (c-mfr5; animal reproduction systems [contains milk, egg yolk, glycerol, and methylformamide]); and hepes-glucose-lactose media (hgll; prepared in laboratory [protein-free]);28 commercial pbs (pbs; corning cellgro, manassas, va [protein-free]). raw semen (raw) was used as a negative control. to determine if there was an influence of semen exposure time to extender prior to fixation, subsets of all diluent treatments were exposed to semen for 1 hour prior to fixation (t1h), or immediately fixed following dilution in diluent (t0h). all samples were fixed at a 1:9 dilution ratio (v:v; semen:fixative) with 4.75% buffered formalin saline (hancock formula).20 morphologic analysis using dic microscopy was conducted following a 24-hour fixation period. in total, 34 samples per ejaculate were evaluated (17 experimental groups, including 16 diluents and raw semen as a control group, and 2 exposure times per group prior to fixation). in total, 510 fixed semen samples were evaluated using dic microscopy. sperm morphologic analysis was conducted by an evaluator who was blinded to treatments, but not stallion identification. clinical theriogenology • volume 12 number 4 • december 2020 496 data analyses data were analyzed using proprietary software (sas 9.4, sas institute inc., cary, nc). distribution of data was determined using the shapiro-wilk test. most of the data were not normally distributed; therefore, a rank-transformation of data was conducted (proc rank) prior to analysis using a general linear model (proc glm). a ls-means tukey adjustment test was used for mean separation when treatment f-ratios were significant (p < 0.05). linear relationships between stippled midpieces and various features of sperm quality were analyzed using a spearman correlation method (proc corr). untransformed data are provided in text and tables for ease of interpretation. results variability among individual stallions for certain endpoints was detected (tables 1 and 2). percent morphologically normal sperm and progressive sperm motility were lower (p < 0.05) for 1 stallion (stallion e) compared to 4 remaining stallions. one stallion (stallion a) exhibited a higher (p < 0.05) percentage of morphologically normal sperm compared to other stallions and 2 stallions (stallions a and b) had a higher (p < 0.05) percentage of progressively motile sperm. stallion a had higher (p < 0.05) percentages of total sperm motility and viability compared to stallion e. main effects (p < 0.05) of diluent and time were detected for percent stippled midpieces. however, diluent-by-time interaction (p > 0.05) was not detected. effects of diluent type on the percentage of stippled midpieces are presented (table 3). inra96 yielded a higher (p < 0.05) incidence of stippled midpieces compared to other diluents except mfr5 and c-mfr5. percent stippled midpieces was lowest (p < 0.05) for diluents pbs, hgll, redigrad, equipure, puresperm, and raw. type of permeable cryoprotectant did not impact percentage of stippled midpieces, as values were similar (p > 0.05) between diluents mfr5 and c-mfr5, and between diluents le and c-le. exposure of semen to various diluents for 1 hour prior to fixation yielded a slightly higher (p > 0.05) incidence of stippled midpieces (5.5 ± 6.15) compared to immediate fixation (4.2 ± 4.9). when combining diluents that contained common protein sources, diluents containing no protein (equipure, puresperm, hgll, pbs) or albumin only (redigrad, talp) yielded a lower (p < 0.05) percent of stippled midpieces compared with diluents that had milk or egg-yolk (table 4). diluents containing egg yolk only as a protein source (le and c-le) yielded lower (p < 0.05) percent stippled midpieces than diluents containing casein (inra96, botu-g), milk only (tamu, botu-s, botu-sp, botu-t), or a combination of milk and egg yolk (mfr5, c-mfr5). raw semen (control) yielded the lowest (p < 0.05) percentage of stippled midpieces except for semen diluted in media containing no protein (equipure, puresperm, hgll, pbs) prior to fixation a stallion-by-diluent interaction (p < 0.05) was detected. effect of 6 representative diluents on the percentage of stippled midpieces by stallion is illustrated (table 5). percentage of stippled midpieces was low and similar (p > 0.05) among all stallions for groups raw and c-le. for diluents containing casein (inra96), milk (tamu), or milk and egg yolk (c-mfr5) the percentage of stippled midpieces was higher (p < 0.05) for stallion e (lowest sperm quality) compared to stallion a (highest sperm quality). measures of semen quality summarized (tables 1 and 2). within group inra96, stallions a and d had a lower (p < 0.05) percentage of stippled midpieces than stallion e stallions b, c, and e exhibited a similar (p > 0.05) incidence of stippled midpieces. in group equipure, percentages of stippled midpieces were low for all stallions (range 0 2%) but were slightly lower (p < 0.05) in stallion d than stallion e. spearman correlation coefficients, as depicted in table 6, revealed that no correlations (p > 0.05) were detected between percentage of stippled midpieces and various measures of sperm quality in raw semen. negative correlations (p < 0.05) between percent stippled midpieces and percent morphologically normal sperm, total motility, and progressive motility were detected in all other treatment groups). the correlations between percent stippled midpieces and percentages of normal sperm, clinical theriogenology • volume 12 number 4 • december 2020497 table 1. percentage of specific sperm morphologic characteristics for 3 ejaculates from 5 stallions (mean ± sd, range in parentheses) a-d within columns, means without a common superscript differed (p < 0.05) nrm = morphologically normal sperm; ah = abnormal heads (%); aa = abnormal acrosomes (%); dh = detached heads (%); pd = proximal droplets (%); pd only = proximal droplets only (with no other sperm defect) (%); dd = distal droplets only (with no other sperm defect) (%); amp = abnormal midpieces (%); bmp = bent midpieces (%); bt = bent tails (%); ct = coiled tails (%); bk = broken tails (%); pgc = premature germ cells (%)   total motility, and progressive motility were stronger in diluents containing milk proteins (groups casein, milk and egg yolk, and milk) than in group no protein. positive correlations (p < 0.05) were observed between stippled midpieces and percent bent tails and broken tails in all treatment groups except the raw semen control. positive correlations (p < 0.05) were observed between percent coiled tails and percentage stippled midpieces in all treatment groups, including raw semen. of all sperm morphologic defects examined, only percent proximal droplets were negatively correlated (p < 0.05) to percent stippled midpieces. this was observed consistently in groups casein, milk and egg yolk, and milk. transmission electron microscopy similar to dic microscopy studies, ultrastructural morphologic alterations of the sperm midpiece was observed following induction by exposure to a milk-based extender (inra96) prior to fixation. only 1 stallion was used in this trial, as we selected the stallion with the highest incidence of stippled midpieces based on dic microscopy in prior experiments. in this tem experiment, we confirmed presence of stippled midpieces by dic microscopy in semen exposed to extender prior to formalin fixation. normal sperm midpieces were observed within all samples (figure 4), consistent with what was observed using dic microscopy. however, sperm exhibiting disruption within the midpiece were only evident in samples diluted with inra96 extender prior to formalin fixation (figure 5). disrupted midpieces (figure 5), exhibited a loss of the plasma membrane, disorder or misalignment of mitochondria, and a serrated appearance. a clear distinction was observed when comparing normal sperm (figure 4) to those exhibiting an apparent disruption of the sperm midpiece (figure 5). stallion nrm ah aa dh pd pd only dd dd only amp bmp bt ct bk a 79 ± 8 (70-84)a 1 ± 1 (0-2)b 3 ± 1 (2-4)a 1 ± 1 (0-1)c 7 ± 5 (2-11)b 6 ± 4 (2-9)a 7 ± 4 (4-12)b 2 ± 1 (1-3)b 3 ± 2 (1-5)d 3 ± 2 (1-4)a 5 ± 3 (2-8)b 3 ± 1 (2-4)b 1 ± 2 (0-3)a b 56 ± 8 (51-65)b 6 ± 3 (3-8)ab 3 ± 1 (2-4)a 3 ± 2 (2-5)abc 15 ± 2 (1316)ab 11 ± 1 (11-12)a 5 ± 5 (1-10)b 3 ± 4 (0-7)b 19 ± 4 (16-24)a 9 ± 4 (6-13)a 3 ± 3 (1-6)b 4 ± 1 (4-5)b 1 ± 1 (0-1)a c 58 ± 8 (50-66)b 7 ± 1 (7-8)ab 5 ± 6 (1-11)a 4 ± 1 (3-5)ab 10 ± 4 (7-15)b 8 ± 6 (2-14)a 17 ± 6 (1224)ab 8 ± 4 (6-13)ab 5 ± 3 (3-8)cd 3 ± 2 (1-5)a 8 ± 2 (6-10) ab 6 ± 2 (4-8)ab 1 ± 2 (0-3)a d 57 ± 6 (52-64)b 14 ± 2 (12-15)a 2 ± 2 (1-4)a 3 ± 2 (1-4)bc 18 ± 2 (16-20)a 13 ± 4 (8-16)a 9 ± 7 (2-15)b 7 ± 6 (1-12)ab 10 ± 4 (7-15)bc 6 ± 2 (4-8)a 3 ± 1 (2-3)b 3 ± 1 (2-4)b 1 ± 1 (0-1)a e 17 ± 2 (16-19)c 11 ± 8 (5-20)ab 11 ± 2 (10-13)a 8 ± 3 (5-10)a 18 ± 6 (12-24)a 6 ± 4 (3-10)a 38 ± 3 (34-40)a 14 ± 1 (13-15)a 12 ± 2 (11-14)b 8 ± 3 (6-11)a 27 ± 3 (24-30)a 12 ± 4 (8-16)a 1 ± 1 (0-2)a clinical theriogenology • volume 12 number 4 • december 2020 498 table 2. sperm output, motion characteristics, and viability for 3 ejaculates from 5 stallions (mean ± sd, range in parenthesis) a-cwithin columns, means without a common superscript differed (p < 0.05) tsn = total sperm number (x 109); conc = concentration (x 106/ml); tmot = total motility (%); pmot = progressive motility (%); vcl = curvilinear velocity (µm/s); str = straightness ([vap/vcl]100; %); viab = sperm viability (%); tpmsn = total progressively motile sperm in the ejaculate (total sperm number (x 109) x progressive motility (%)). table 3. effect of diluent type on the percentage of stippled midpieces in stallion sperm following fixation in buffered formol saline (3 ejaculates from 5 stallions; mean ± sd) a-f means without a common superscript differed (p < 0.05) *inra96 = inra-96® extender; mfr5 = e-z freezin® mfr5 equine semen extender; c-mfr5 = e-z freezin® cryomax™ mfr5 equine semen extender; botu-g = botusemen™ gold extender; tamu = milk-glucose-sucrose-based extender; botu-t = botusemen™ turbo extender; botu-sp = botusemen™ special extender; botu-s = botusemen™ extender; le = e-z freezin® le equine semen extender; cle = e-z freezin® cryomax™ le equine semen extender; talp = tyrode’s albumin lactate pyruvate; pbs = corning® phosphate-buffered saline; hgll = hepes-glucose-lactose media; redigrad = redigrad™ 80% silica particle solution; equipure = equipure™ 80% silica particle solution; puresperm = puresperm™ 80% silica particle solution; raw = raw semen. stallion tsn conc tmot pmot vcl str viab tpmsn a 4 ± 1 (4-5)b 140 ± 46 (100-191)bc 85 ± 2 (84-87)a 63 ± 5 (57-66)a 220 ± 31 (184-243)a 68 ± 6 (62-72)ab 85 ± 3 (82-87)a 3 ± 1 (2-3)ab b 10 ± 1 (9-12)a 194 ± 17 (179-213)abc 77 ± 3 (75-81)ab 55 ± 2 (53-57)a 241 ± 10 (233-253)a 67 ± 2 (65-69)ab 78 ± 8 (72-87)ab 5 ± 1 (5-6)a c 10 ± 4 (7-14)a 326 ± 114 (252-458)a 77 ± 8 (69-85)ab 38 ± 6 (32-42)b 199 ± 79 (107-245)a 58 ± 13 (49-73)b 79 ± 5 (74-84)ab 4 ± 2 (2-6)a d 6 ± 1 (5-7)ab 108 ± 40 (63-140)c 78 ± 2 (76-80)ab 45 ± 5 (40-49) b 223 ± 3 (220-225)a 58 ± 2 (56-60)b 79 ± 1 (78-80)ab 3 ± 1 (2-3)ab e 5 ± 1 (4-6)b 246 ± 73 (182-326) ab 25 ± 9 (15-32)b 18 ± 9 (9-26)c 149 ± 3 (146-152)a 77 ± 2 (76-80)a 58 ± 12 (45-69)b 1 ± 0 (0-1)b treatment* stippled midpieces (%) inra96 12 ± 1a mfr5 12 ± 1ab c-mfr5 11 ± 1ab botu-g 8 ± 1bc tamu 6 ± 1cd botu-t 5 ± 1d botu-sp 5 ± 1d botu-s 5 ± 1d le 3 ± 1e c-le 3 ± 0.4e talp 2 ± 0.3e pbs 2 ± 0.4f hgll 1 ± 0.2f redigrad 1 ± 0.3f equipure 1 ± 0.2f puresperm 1 ± 0.2f raw 1 ± 0.2f clinical theriogenology • volume 12 number 4 • december 2020499 table 4. effect of diluent composition on the percentage of stippled midpieces in stallion sperm following fixation in bufferedformol saline (3 ejaculates from each of 5 stallions; mean ± sd) diluent type* stippled midpieces (%) casein only 10 ± 7a milk and egg yolk 11 ± 8a milk only 6 ± 4b egg yolk only 3 ± 3c albumin only 2 ± 2d no protein 1 ± 2de raw semen 1 ± 1e a-e means without a common superscript differed (p < 0.05) *casein only = inra-96® extender and botusemen™ gold extender; milk and egg yolk = e-z-freezin® mfr5 and e-z freezin® cryomax™ mfr5; milk only = botusemen™ extender, botusemen™ special extender, botusemen™ turbo extender, and tamu extender; egg yolk only = e-z-freezin® lactose-edta and e-z freezin® cryomax™ le; albumin only = redigrad™ 80% silica particle solution and talp (tyrode’s albumin lactate pyruvate) media; no protein = equipure™ 80% silica particle solution, puresperm™ 80% silica particle solution, hepes-glucose-lactose media (hgll), and corning® (commercial) pbs. table 5. percentage of stippled midpieces by stallion following dilution in selected commercially available semen diluents. (3 ejaculates from each of 5 stallions; mean ± sd) stallion raw inra96 c-mfr5 c-le tamu equipure a 1 ± 1a 6 ± 3c 5 ± 3c 2 ± 2a 3 ± 3b 1 ± 1 ab b 1 ± 1a 11 ± 3ab 8 ± 4bc 3 ± 1a 5 ± 2a 2 ± 1ab c 1 ± 1a 11 ± 2ab 11 ± 6ab 2 ± 1a 8 ± 5a 1 ± 1ab d 0 ± 0a 8 ± 3bc 7 ± 3bc 2 ± 2a 6 ± 1a 0 ± 0b e 2 ± 2a 23 ± 6a 24 ± 6a 5 ± 3a 11 ± 4a 2 ± 1a a-cwithin columns, means without a common superscript differed (p < 0.05) raw = raw semen; inra96 = inra-96® extender; c-mfr5 = e-z freezin® cryomax™ mfr5; c-le = e-z freezin® cryomax™ le; tamu = tamu extender; equipure = equipure™ 80% silica particle solution figure 1. three images of normal sperm midpieces, as viewed with transmission electron microscopy a. raw semen sample; b. raw semen sample; c. semen sample in inra96 extender b  c a  clinical theriogenology • volume 12 number 4 • december 2020 500 table 6. spearman correlation coefficients (r value and p value) between semen diluent type and various sperm morphologic or motion characteristics for the percentage of stippled midpieces (3 ejaculates from 5 stallions) ns = not significant nrm = normal; ah = abnormal heads (%); aa = abnormal acrosomes (%); dh = detached heads (%); pd = proximal droplets (%); pd only = proximal droplets only (with no other sperm defect) (%); dd = distal droplets only (with no other sperm defect) (%); amp = abnormal midpieces (%); bmp = bent midpieces (%); bt = bent tails (%); ct = coiled tails (%); bk = broken tails (%); pgc = premature germ cells (%); tmot = total motility (%); pmot = progressive motility (%); vcl = curvilinear velocity (µm/s); str = straightness ([vap/vcl]100; %); casein = inra-96® extender and botusemen™ gold extender; milk and egg yolk = e-z-freezin® mfr5 and e-z freezin® cryomax™ mfr5; milk = botusemen™ extender, botusemen™ special extender, botusemen™ turbo extender, and tamu extender; egg yolk = e-z-freezin® lactose-edta and e-z freezin® cryomax™ le; albumin = redigrad™ 80% silica particle solution and talp (tyrode’s albumin lactate pyruvate) media; no protein = equipure™ 80% silica particle solution, puresperm™ 80% silica particle solution, hepesglucose-lactose media (hgll), and corning® (commercial) raw semen casein milk and egg yolk milk egg yolk albumin no protein nrm ns -0.6344 0.0027 -0.8530 <.0001 -0.5722 0.0001 -0.6846 0.0009 -0.6584 0.0016 -0.4705 0.0022 ah ns ns 0.54409 0.0131 0.3681 0.0194 ns ns ns ka ns 0.59603 0.0055 ns 0.3651 0.0205 ns ns ns dh ns 0.54581 0.0128 0.64378 0.0022 0.5920 <.0001 0.5032 0.0237 ns ns pd ns -0.4785 0.0328 -0.5587 0.0104 -0.4161 0.0076 ns ns ns pd only ns -0.5770 0.0077 -0.5730 0.0083 -0.5111 0.0008 ns -0.4994 0.0250 -0.3681 0.0194 dd ns 0.6165 0.0038 0.5068 0.0226 0.5772 <.0001 ns 0.4832 0.0309 0.3251 0.0406 dd only ns 0.6149 0.0039 0.5646 0.0095 0.6399 <.0001 ns ns 0.3789 0.0159 amp ns ns 0.4537 0.0445 ns ns ns ns bmp ns ns 0.4689 0.0370 ns ns ns ns bt ns 0.5461 0.0127 0.5092 0.0218 0.48660 0.0015 0.5722 0.0084 0.6482 0.0020 0.4824 0.0016 ct 0.7865 0.0070 0.6466 0.0021 0.7943 <.0001 0.5277 0.0005 0.6259 0.0032 0.7770 <.0001 0.6287 <.0001 bk ns 0.6222 0.0034 0.6877 0.0008 0.7218 <.0001 0.5513 0.0118 0.7308 0.0003 0.5299 0.0004 pgc ns ns ns ns ns tmot ns -0.7977 <.0001 -0.9109 <.0001 -0.7496 <.0001 -0.6531 0.0018 -0.6536 0.0018 -0.5955 <.0001 pmot ns -0.7350 0.0002 -0.8066 <.0001 -0.7879 <.0001 -0.4590 0.0418 -0.4810 0.0318 -0.3854 0.0140 vcl ns -0.4718 0.0357 -0.4876 0.0292 -0.4979 0.0011 ns ns ns str ns ns ns ns ns 0.5889 0.0063 0.3877 0.0134 clinical theriogenology • volume 12 number 4 • december 2020501 figure 2. four images of disrupted sperm midpieces observed in sperm extended in inra96 extender sample prior to formalin fixation, as viewed with transmission electron microscopy discussion evaluation of sperm morphology is one of the standard procedures used to assess the breeding potential of a stallion and evaluation of formalin-fixed wet mount specimens is commonplace for this purpose. evaluation of wet mount samples allows for a more detailed analysis of sperm with enhanced ability to better detect acrosome defects, nuclear vacuoles, and midpiece abnormalities when compared to stained semen smears. furthermore, the use of wet mount samples for evaluation of sperm morphology reduces the likelihood of artifactual abnormalities, such as detached heads, bent tails, broken tails, and coiled tails.29,30 to our knowledge, artifactual disruption of the sperm midpiece following previous exposure of sperm to semen diluents has not been reported. microscopically, this iatrogenic structural abnormality is characterized by a distinct disruption in the mitochondrial region that is revealed by a stippling appearance at the level of midpiece. stippled-midpiece abnormality was first observed in our laboratory following morphologic analysis of semen samples processed using density-gradient centrifugation and resuspension of sperm pellets in a milk-based extender. the apparent disruption of the sperm midpiece was initially attributed to improper semen handling or semen processing techniques. however, we also considered it to be a possible reflection of a stallion’s intrinsic sperm quality. a preliminary experiment was conducted to provide a more detailed description of the midpiece abnormality. a higher incidence of stippled midpieces was detected following density-gradient centrifugation protocol and resuspension of sperm in milk-based media as compared to raw semen, following formalin fixation and examination by differential interference contrast microscopy. based on these results, it appeared that the defect was a result of semen processing. however, it remained unclear if the disruption was due to improper semen handling, centrifugation, prefixation diluent type, time of fixation, or fixation method. further preliminary experiments were conducted to evaluate the various effects of semen-diluent type, fixative type and concentration, time of exposure to diluent prior to fixation, and time of fixation on the incidence of stippled midpieces. results suggested an increased incidence of stippled sperm midpieces following prior exposure of semen to diluents containing protein. a higher percentage of stippled midpieces was particularly evident in diluents containing milk. the stippled midpiece abnormality was detected when semen was exposed to diluents prior to fixation without a centrifugation step. therefore, centrifugation was determined not to be a contributing factor of the disruption. clinical theriogenology • volume 12 number 4 • december 2020 502 the present study allowed for a more thorough analysis of the effects of diluent type and time of exposure to diluent prior to fixation on the incidence of stippled midpieces and incorporated an expanded sample size. five stallions with varying semen quality were used in this experiment to determine if stallion influenced the incidence of stippled midpieces. a clear distinction of sperm quality between 2 stallions (stallion e lowest semen quality and stallion a highest semen quality) was observed, based on sperm morphologic and motion characteristics. initial analysis of 16 semen diluents revealed that semen exposure to diluents containing milk or milk products, or, to a lesser extent egg yolk, exhibited a higher incidence of stippled midpieces, as compared to diluents containing no protein, or to raw semen. diluents were then grouped according to those containing milk only, casein only, milk and egg yolk, egg yolk only, or albumin only as exogenous sources of protein in order to compare with diluents containing no protein or raw semen (control). this categorization of semen diluents revealed an effect of diluent protein type on incidence of stippled midpieces. this distinction was most pronounced in semen diluents containing casein or a combination of milk and egg yolk, where the highest incidence of stippled midpieces was detected following formalin fixation. however, presence of any exogenous protein in diluents resulted in a higher incidence of stippled midpieces compared to the raw semen control. diluents containing no protein yielded similar results to the raw semen control. in addition to diluent composition, intrinsic semen quality had an impact in the incidence of stippled midpieces. a stallion effect was not detected in fixed raw semen but was detected in semen diluted in extender containing casein, milk, or a combination of milk and egg yolk, prior to formalin fixation. under these conditions, an increased incidence of stippled midpieces was noted in the stallion with lower semen quality as compared to the stallion with higher semen quality. to further support this finding, significant correlations were observed between the percentage of stippled midpieces and percent morphologically normal sperm (negative correlation), or percent total or progressive sperm motility (negative correlations) for all diluent groups except the raw semen control. therefore, we inferred that intrinsic sperm quality did not impact the incidence of stippled midpieces when raw semen was formalin-preserved. however, sperm quality did impact the incidence of stippled midpieces when semen was mixed with certain diluents prior to formalin-fixation. the effect appeared to be most pronounced when the diluent contained milk, casein, or a combination of milk and egg yolk. an extender-fixative interaction was responsible for the stippled midpiece abnormality and there were correlations between stallion semen quality and incidence of stippled midpieces. the stallion with lowest semen quality had a higher incidence of the stippled midpieces when sperm were exposed to fixative following dilution in extender. inherently, such stallions have reduced fertility, so an extenderfixative assay could conceivably be used as a laboratory test of stallion fertility. we consider it questionable, however, that such an ancillary test in a bse would be of practical value. an effect of diluent type on the incidence of stippled midpieces was observed throughout this study. semen extenders are used to protect sperm during in vitro storage; however, the specific mechanism(s) of the protective effects of milk and egg yolk are largely unknown.31 these animal-based biological products consist of a variety of substances that cannot be accurately standardized, allowing for variation in consistency among batches. this variation may be responsible for the discrepancies observed within diluent types containing milk or egg yolk and the incidence of stippled midpieces observed. in order to reduce inconsistencies within milk, fractionation of milk through microfiltration, ultrafiltration, diafiltration, and freeze-drying has been performed to prepare purified extracts.35 among the fractions tested, french workers determined that native phosphocaseinate, composed of total micellar caseins, to be the milk component responsible for most of the protective benefits on cool-stored equine sperm.36 in the present study, a relatively high incidence of stippled midpieces was observed in diluents containing casein. therefore, simple standardization of extender composition did not resolve the issue of stippled midpieces when semen was mixed with extender prior to formalin fixation. use of an extender (beltsville f3) containing casein disabled evaluation of sperm morphology, possibly due to casein precipitation.37 this observation provides possible explanation as to why a higher incidence of stippled midpieces was observed in casein-based extenders in the current study. possibly, clinical theriogenology • volume 12 number 4 • december 2020503 extensive postfixation protein precipitation occurred within all extenders containing protein, thereby leading to the stippling pattern in the sperm mitochondrial region. sperm midpiece stippling was rarely detected in raw semen following formalin fixation. stippled midpiece appearance that was quite evident in this study was also detected with phase contrast or dic microscopy and bright-field microscopy of eosin-nigrosin stained sperm. distinction between normal sperm midpieces and stippled sperm midpieces was pronounced (figures 1 3). although sperm morphologic evaluation was conducted using dic microscopy in the present study, images were also captured using phase contrast optics and eosin-nigrosin staining to illustrate the abnormality using various methods commonly used for evaluating sperm morphology. transmission electron microscopy allowed for ultrastructural examination of extended semen prior to fixation and corroborated the abnormality viewed using dic microscopy, revealing loss of the plasma membrane, disorder or misalignment of mitochondria, and a serrated appearance of the midpiece region of some affected sperm. semen from only 1 stallion was used for the tem experiment. as such, it is possible that this stallion may not be representative of a population of stallions. further studies are needed to address this point. we selected this stallion simply because its sperm had the highest incidence of stippled midpieces when exposed to extender prior to fixation. we were interested in examining this defect at the ultrastructural level. several sperm midpiece defects have been previously described, including midpiece reflex (simple bent or folded midpiece), segmental aplasia of the mitochondrial sheath, fractured or swollen midpiece (thick, pseudodroplet), roughened midpiece (corkscrew), dag-like defect, disrupted sheath (filamentous), duplicated midpiece, and stump tail.3,4,22,29 abnormalities of sperm midpiece have been well documented and are considered to have a deleterious effect on stallion fertility.3,4 midpiece abnormalities are most commonly associated with impaired spermatogenesis as well as iatrogenic or artifactual defects due to temperature shock during semen processing.38,39 these abnormalities are viewed as intrinsic in nature, whereas the stippled midpiece abnormality described in the present study is iatrogenically induced. stippled midpieces can have some structural similarities to other abnormalities, such as segmental aplasia of the mitochondrial sheath, disrupted sheath, or swollen or roughened midpiece, some of which characterize sperm damage after cooling or a freeze-thaw cycle.22,24,29,32,40 reports comparing extender types that include morphologic analysis have been published, although these reports differ from the present study due to the method of evaluation or method of classification. one study did not subject sperm to fixation prior to morphologic analysis nor did these investigators evaluate or enumerate abnormal sperm.41 sperm morphology of semen extended in either milk or egg yolk-based extender prior to fixation was evaluated.42 however, the stippled midpiece abnormality was not reported and abnormal midpieces were not among the most prevalent abnormalities observed.42 one stallion in that study exhibited 25% midpiece defects. however, this stallion was reported to have low fertility, and no images of midpiece defects were provided. in a study evaluating morphology of human sperm, wet preparations yielded the highest percentage of midpiece morphologic defects (48.6 ± 11. l).43 although not explicitly indicated, it is presumed that raw semen samples were evaluated and no images of the midpiece defects were provided. it is then difficult to discern if the same artifactual midpiece disruptions were observed. the results of the present study suggest that the defect is a result of a reaction involving semen diluents and fixative, and possibly seminal plasma, and can be considered to be an artifactual change. due to the significance of sperm morphology for evaluation of the stallion for breeding soundness, it is important to the industry to determine an optimal method for preserving extended semen prior to sperm morphologic evaluation to avoid such artifactual changes. to address this, future studies could be directed at optimizing evaluation of flash frozen extended semen samples to avoid the addition of buffered formal saline for analysis of sperm morphology. however, caution is required because acrosome disruption can be caused by freezing and thawing of raw semen (unpublished results). the incidence of stippled midpieces observed was highest among diluents containing animal-sourced protein, as compared to diluents containing no protein or to raw semen. it would be beneficial to further examine protein types and concentrations in such diluents to deduce any potential relationships between protein clinical theriogenology • volume 12 number 4 • december 2020 504 type/concentration and incidence of stippled midpieces. evaluation of plant-based products as an additive to semen extenders could also be conducted. future investigations could be directed at evaluating susceptibility of individual sperm to midpiece stippling to determine why certain sperm appear more susceptible to acquisition of this artifactual defect. this notion could be further assessed by dual fluorescent/dic imaging of individual sperm to compare mitochondrial function and presence or absence of midpiece stippling. effect of seminal plasma on the incidence of stippled midpieces has also not been studied. as such, it might be prudent to examine sperm morphology of extended-fixed epididymal sperm. it is also perplexing that the stippling appearance was only observed in the midpiece region of sperm and not in the head or principal-piece regions. isolation of membranes from these various segments of sperm prior to dilution in extender and fixation may allow for closer scrutiny of the relative susceptibilities of regionalized sperm membranes to artifactual stippling. conclusion mixing of ejaculated semen with certain extender types produced an artifactual midpiece defect in sperm. we have coined this defect “stippled midpiece.” this defect can confound interpretation of sperm morphology but can be largely avoided by fixation of raw semen for sperm morphologic analysis. conflict of interest authors declare no conflicts of interest acknowledgement authors thank dr. joseph szule, image analysis laboratory, college of veterinary medicine and biomedical sciences, texas a&m university for his help with processing and analysis by transmission electron microscopy used for this study. funding legends premier stallion season auction fund, texas a&m university references 1. griffin pg: the breeding soundness examination in the stallion. j equine vet sci 2000;20:168-171. 2. love cc: sperm quality assays: how good are they? the horse perspective. anim reprod sci 2018;194:63-70. 3. love cc: relationship between sperm motility, morphology and the fertility of stallions. theriogenology 2011;76: 547-557. 4. love cc, varner dd, thompson ja: intra-and inter-stallion variation in sperm morphology and their relationship with fertility. j reprod fertil (suppl) 2000;56:93-100. 5. aitken rj: sperm function tests and fertility. int j androl 2006;29:69-75. 6. aurich c: semen extenders for cooled semen (europe). in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition,ames; wiley-blackwell: 2011. p. 1336-1340. 7. brinsko sp: semen extenders for cooled semen (north america). in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition,ames; wiley-blackwell: 2011. p. 1341-1343. 8. sieme h: semen extenders for frozen semen. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition,ames; wiley-blackwell: 2011. p. 2964-2971. 9. edmond aj, brinsko sp, love cc, et al: effect of centrifugal fractionation protocols on quality and recovery rate of equine sperm. theriogenology 2012;77:959-966. 10. macpherson ml, blanchard tl, love cc, et al: use of a silane-coated silica particle solution to enhance the quality of ejaculated semen in stallions. theriogenology 2002;58:317-320. 11. varner dd, love cc, blanchard tl, et al: breeding-management strategies and semen-handling techniques for stallions–case scenarios. proc am assoc equine pract 2010; p. 215-226. 12. varner dd, love cc, brinsko sp, et al: semen processing for the subfertile stallion. j equine vet sci 2008. 13. johannisson a, morrell jm, thorén j, et al: colloidal centrifugation with androcoll-e™ prolongs stallion sperm motility, viability and chromatin integrity. anim reprod sci 2009;116:119-128. 14. morrell jm, dalin am, rodriguez‐martinez h: comparison of density gradient and single layer centrifugation of stallion spermatozoa: yield, motility and survival. equine vet j 2009;41:53-58. 15. morrell jm, mari g, kútvölgyi g, et al: pregnancies following artificial insemination with spermatozoa from problem stallion ejaculates processed by single layer centrifugation with androcoll‐e. reprod domest anim 2011;46:642-645. clinical theriogenology • volume 12 number 4 • december 2020505 16. morrell jm, rodriguez‐martinez h, johannisson a: single layer centrifugation of stallion spermatozoa consistently selects the most robust spermatozoa from the rest of the ejaculate in a large sample size. equine vet j 2010;42:579585. 17. foster ml, varner dd, hinrichs k, et al: agreement between measures of total motility and membrane integrity in stallion sperm. theriogenology 2011;75:1499-1505. 18. waite ja, love cc, brinsko sp, et al: factors impacting equine sperm recovery rate and quality following cushioned centrifugation. theriogenology 2008;70:704-714. 19. kenney rm, hurtgen j, pierson r, et al: clinical fertility evaluation of the stallion. j soc theriogenol 1983;ix, part ii:3-100 . 20. hancock jl: the morphology of boar spermatozoa. j r microsc soc 1956;76:84-97. 21. foster ml, love cc, varner dd, et al: comparison of methods for assessing integrity of equine sperm membranes. theriogenology 2011;76:334-341. 22. varner dd, johnson l: from a sperm's eye view-revisiting our perception of this intriguing cell. proc am assoc equine pract 2007; p. 104-177. 23. fawcett dw: the mammalian spermatozoon. dev biol 1975;44:394-436. 24. eddy em: the spermatozoon. in: knobil e, neill jd: editors. the physiology of reproduction, san diego; elsevier academic press: 2006. p. 3–54. 25. amann rp, graham jk: spermatozoal function. in: mckinnon ao, voss jl: editors. equine reproduction. philadelphia; lea & febiger: 1993. p. 715-745. 26. pesch s, bostedt h, failing k, et al: advanced fertility diagnosis in stallion semen using transmission electron microscopy. anim reprod sci 2006;91:285-298. 27. veeramachaneni dn, moeller cl, sawyer hr: sperm morphology in stallions: ultrastructure as a functional and diagnostic tool. vet clin north am equine pract 2006;22:683-692. 28. magistrini m, couty i, palmer e: interactions between sperm packaging, gas environment, temperature and diluent on fresh stallion sperm survival. acta vet scand (suppl) 1992;88:97-110. 29. brito lf: evaluation of stallion sperm morphology. clin tech equine pract 2007;6:249-264. 30. brito lf, greene lm, kelleman a, et al: effect of method and clinician on stallion sperm morphology evaluation. theriogenology 2011;76:745-750. 31. aurich c: factors affecting the plasma membrane function of cooled-stored stallion spermatozoa. anim reprod sci 2005;89:65-75. 32. aitken rj, baker ma: oxidative stress and male reproductive biology. reprod fertil dev 2004;16:581-588. 33. eddy em, o´brien da: the spermatozoon. in: knobil e, neill jd: editors. the physiology of reproduction, new york; raven press: 1994. p. 29-77. 34. kankofer m, kolm g, aurich j, et al: activity of glutathione peroxidase, superoxide dismutase and catalase and lipid peroxidation intensity in stallion semen during storage at 5 °c. theriogenology 2005;63:1354-1365. 35. pierre a, fauquant j, le graet y, et al: préparation de phosphocaséinate natif par microfiltration sur membrane. le lait 1992;72:461-474. 36. batellier f, magistrini m, fauquant j, et al: effect of milk fractions on survival of equine spermatozoa. theriogenology 1997;48:391-410. 37. pursel vg, johnson la: glutaraldehyde fixation of boar spermatozoa for acrosome evaluation. theriogenology 1974;1:63-68. 38. hurtgen jp: evaluation of the stallion for breeding soundness. vet clin north am equine pract 1992;8:149-165. 39. baumber-skaife j: evaluation of semen. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition,ames; wiley-blackwell: 2011. p. 1278-1291. 40. barth ad, oko rj: abnormal morphology of bovine spermatozoa, ames; iowa state university press: 1989. p. 214228. 41. stanic p, tandara m, sonicki z, et al: comparison of protective media and freezing techniques for cryopreservation of human semen. eur j obstet gynecol reprod biol 2000;91:65-70. 42. morrell jm, johannisson a, dalin a-m, et al: sperm morphology and chromatin integrity in swedish warmblood stallions and their relationship to pregnancy rates. acta veterinaria scandinavica 2008;50:2. 43. meschede d, keck c, zander m, et al: influence of three different preparation techniques on the results of human sperm morphology analysis. int j androl 1993;16:362-369.   clinical theriogenology • volume 12 number 4 • december 2020 506 2019 art of measuring progesterone: understanding immunoassays art of measuring progesterone: understanding immunoassays elizabeth schooley, graham bilbrough, tiffany glavan idexx, westbrook, me abstract measurement of progesterone has become an essential component of canine reproductive medicine. reliable measurement of steroid hormones is challenging. in veterinary medicine, we depend on immunoassays to determine concentrations of circulating progesterone. immunoassay is the general term for a diagnostic technique that relies on antibodies to detect a compound within a biologic sample. there are several types of immunoassays available, which have innate differences. therefore, to correctly apply the results to a clinical scenario, it is essential for the practicing veterinarian to understand the method they are utilizing. data from development of a new in-house progesterone test are reviewed and a variety of immunoassay methods, assay validation, and future trends within diagnostic industry are discussed. understanding these principles will help the clinician choose an assay which will best suit their diagnostic needs. keywords: diagnostic testing, progesterone, canine, immunoassay, liquid chromatography-mass spectrometry introduction and background progesterone is a female reproductive hormone. measurement of progesterone in blood (plasma or serum) from bitch is an important element to predict and confirm ovulation to determine optimal breeding time and maximize fertility,1 to predict parturition2 and to investigate reproductive abnormalities.3 catalyst® progesterone is a new immunoassay from idexx that is designed to provide prompt and reliable in-clinic measurement of progesterone in canine blood samples. it works with both idexx catalyst one® and idexx catalyst dx® chemistry analyzers. it has a reportable range of 0.6 63.6 nmol/l. in clinical practice, various methods have been used to monitor progesterone in bitch. for many decades, radioimmunoassay (ria) (coat-a-count® radioimmunoassay, siemens health care diagnostics inc., los angeles, ca) was regarded as a gold standard; however, the originally validated assay was discontinued in 20144 and liquid chromatography-mass spectrometry (lc-ms) has been proposed as the gold standard.5 because lc-ms is not typically available in veterinary reference laboratories, chemiluminescent immunoassay (clia) (immulite®, siemens medical solutions diagnostics, los angeles, ca) analyzers is more widely used. however, despite strong correlation between clia and reference methods,6 clinically significant bias between methods has also been demonstrated.4 it is further complicated by differing performance between iterations of the clia methodology.7 for progesterone assays used with canine samples, it is important to have accuracy and precision in the range associated with ovulation 9.5 31.2 nmol/l or lower.4 for this study, range of clinical interest was defined as 0 32.0 nmol/l. objectives of the study were to evaluate the performance of catalyst progesterone by a method comparison to lc-ms (reference method), evaluate the performance of 2 iterations of the clia methodology by a method comparison to lc-ms, and to evaluate precision of catalyst progesterone using control fluids (precision study). materials and methods data were collated in microsoft office excel 2016 before being exported to jmp® 14.0.0 for statistical analysis, where appropriate, using the method comparison add-in from sas institute. 627 clinical theriogenology • volume 11, number 4 • december 2019 method comparison study blood samples were collected from 60 bitches visiting 2 veterinary hospitals for breeding management during september and october of 2018. all samples were collected in the periovulatory period. some patients were sampled on multiple days (range 1 7 venipunctures), allowing 101 comparisons to be made. samples used for this study were residual blood samples after clinical diagnostic testing was performed and animals were not subjected to additional venipuncture to collect study samples. we complied with the idexx reference laboratory sample retention policy. refer table for details. table. sample types and handling for the progesterone assays hospital samples clia catalyst® progesterone lc-ms a 32 bitches; 52 comparisons • clia1 (immulite 1, siemens medical solutions diagnostics, los angeles, ca; immulite®/ immulite 1000 progesterone [catalog number: lkpw1]) • serum • gel barrier tubes were not used as the package insert details a time-dependent decrease in progesterone concentrations • analyzed within 24 hours after collection at idexx reference laboratories by laboratory technicians • catalyst dx® chemistry analyzer • lithium heparin plasma • gel barrier tubes were not used as the operator’s guide indicates they are not suitable • analyzed within 4 hours after collection at the hospital by veterinary technicians • lc-ms at idexx r&d • lithium heparin plasma • samples stored at 4oc and analyzed in batches within 1 week after collection b 28 bitches: 49 comparisons • clia2000 (immulite 2000, siemens medical solutions diagnostics, los angeles, ca; immulite® 2000 progesterone [catalog number: l2kpw6]) • serum • gel barrier tubes were not used as the package insert details a time-dependent decrease in progesterone concentrations • analyzed within 24 hours after collection at idexx reference laboratories by laboratory technicians • catalyst dx® chemistry analyzer • lithium heparin plasma • gel barrier tubes were not used, as the operator’s guide indicates they are not suitable • samples stored at 4oc and analyzed at idexx r & d by laboratory technicians • lc-ms at idexx r & d • lithium heparin plasma • samples stored at 4oc and analyzed in batches within 1 week after collection for each venipuncture, within 30 minutes: 1. serum was harvested from blood collected in tubes without anticoagulant. 2. lithium heparin plasma was separated from the red blood cells and divided into 2 aliquots. no samples were excluded. all results were below the upper limit of the respective dynamic ranges of the assays. any results below the lower limit of the dynamic range (0.6 nmol/l for all assays) were assigned to 0.6 nmol/l. passing and bablok linear regression analysis was completed for various pairs of methodology. correlation coefficients were interpreted as follows: r = 0.90 1.0 defined very high correlation; 0.70 0.89, high correlation; 0.50 0.69, moderate correlation; 0.30 0.49, low correlation; and 0 0.29, little, if any, correlation.8 regression analysis was used for statistical evidence of systematic error (constant and/or proportional bias). confidence intervals of 95% for the y-intercept 628clinical theriogenology • volume 11, number 4 • december 2019 that did not include the value zero were considered evidence of constant bias. confidence intervals of 95% for the slope that did not include the value 1.0 were considered evidence of proportional bias. precision was assessed by repeated analysis of 2 control fluids in the range of clinical interest. each fluid was analyzed 8 times per day (4 in the morning, 4 in the evening) for 10 days to give a total of 80 replicates. total percentage coefficient of variation (cv) was calculated as the ratio of the standard deviation to the mean of the concentration. the greater the cv, the greater the dispersion of results around the mean. results method comparison study results are summarized in figure 1a d. clia1, clia2000, and catalyst® progesterone all demonstrated very high correlation to the reference method. for catalyst® progesterone, this was shown in both groups of samples. there was no evidence of constant or proportional bias for clia1 and catalyst® progesterone. for clia2000, there was a constant bias (intercept = 0.17 ng/ml; with confidence limits of 0.38 0.86 nmol/l) and proportional bias (slope = 0.75; with confidence limits of 0.70 0.80). the new method of catalyst® progesterone had a total cv of < 10% at both concentration levels. figure 1a. passing and bablok plots for the agreement of progesterone evaluated by two methods in samples collected during the periovulatory period. dashed line represented the identity line (x = y), solid line represents regression line, and shaded area represents the confidence interval for regression line. hospital a: comparison of catalyst progesterone and the reference method (lc-ms); n = 52, r = 0.99. 629 clinical theriogenology • volume 11, number 4 • december 2019 figure 1b. passing and bablok plots for the agreement of progesterone evaluated by two methods in samples collected during the periovulatory period. dashed line represented the identity line (x = y), solid line represents regression line, and shaded area represents the confidence interval for regression line. hospital b: comparison of catalyst progesterone and the reference method (lc-ms); n = 49, r = 0.98. figure 1c. passing and bablok plots for the agreement of progesterone evaluated by two methods in samples collected during the periovulatory period. dashed line represented the identity line (x = y), solid line represents regression line, and shaded area represents the confidence interval for regression line. hospital a: comparison of clia1 and the reference method (lc-ms); n = 52, r = 0.98. 630clinical theriogenology • volume 11, number 4 • december 2019 figure 1d. passing and bablok plots for the agreement of progesterone evaluated by two methods in samples collected during the periovulatory period. dashed line represented the identity line (x = y), solid line represents regression line, and shaded area represents the confidence interval for regression line. hospital b: comparison of clia2000 and the reference method (lc-ms); n = 49, r = 0.99. conclusion catalyst progesterone demonstrated very good correlation (r = 0.98; r = 0.99) to the study reference method of lc-ms and good precision in the range of clinical interest. both iterations of the clia demonstrated very good correlation to the reference method (clia1: r = 0.98; clia2000: r = 0.99). however, for clia2000, there was a marked proportional bias (slope = 0.75) to the reference method. this does not imply that clia2000 is unsuitable for clinical use; rather, it emphasizes the need for consistent analytical methodology and sample type when trending progesterone concentrations. catalyst progesterone produces accurate and precise results when used to quantify progesterone in plasma samples from bitches. this new immunoassay provides a reliable and convenient option to measure canine progesterone in-house. conflict of interest authors are employed by idexx laboratories inc., westbrook, me. references 1. concannon p, hansel w, mcentee k: changes in lh, progesterone and sexual behavior associated with preovulatory luteinization in the bitch. biol reprod 1977;17:604-613. 2. de cramer kgm, nöthling jo: the precision of peri-oestrous predictors of the date of onset of parturition in the bitch. theriogenology 2017;96:153-157. 3. meyers-wallen vn: unusual and abnormal canine estrous cycles. theriogenology 2007;68:1205-1210. 4. nöthling jo, de cramer kgm: comparing the values of progesterone in the blood of bitches as measured with a chemiluminescence immunoassay and a radioimmunoassay. reprod dom anim 2018;53:1136-1141. 5. stanczyk fz, lee js, santen rj: standardization of steroid hormone assays: why, how, and when? cancer epidemiol biomarkers prev 2007;16:1713-1719. 6. kutzler ma, mohammed ho, lamb sv, et al: accuracy of canine parturition date prediction from the initial rise in preovulatory progesterone concentration. theriogenology 2003;60:1187-1196. 7. schmicke m, urhausen c, wolf k, et al: evaluation of the blood progesterone concentration in the bitch measured by chemiluminescence immunoassay at the day of ovulation. tierärztl prax ausg k kleintiere heimtiere 2016; 44:317-322. 8. zady mf: z-12: correlation and simple least squares regression. westgard qc website. www.westgard.com/lesson42.htm. accessed january 15, 2019. 631 clinical theriogenology • volume 11, number 4 • december 2019 http://www.westgard.com/lesson42.htm 632clinical theriogenology • volume 11, number 4 • december 2019 untitled omniblank: andrology laboratory review: evaluation of sperm motility augustine peter,a♦ leonardo brito,b♦ gary althouse,c christine aurich,d peter chenoweth,e natalie fraser,f cheryl lopate,g charles love,h gaia luvoni,i dagmar waberskij adepartment of veterinary clinical sciences, college of veterinary medicine purdue university, west lafayette, in bstgenetics, navasota, tx cdepartment of clinical studies, new bolton center, school of veterinary medicine university of pennsylvania, kennett square, pa dcentre for artificial insemination and embryo transfer university of veterinary sciences, vienna, austria eschool of veterinary sciences, college of public health medical & veterinary sciences james cook university, townsville, queensland, australia fnatalie fraser, school of veterinary medicine, university of queensland, galton, australia greproductive revolutions, case road ne, aurora, or hdepartment of large animal clinical sciences college of veterinary medicine and biomedical sciences, texas a&m university college station, tx idepartment of health, animal science and food safety università degli studi di milano, milan, italy junit for reproductive medicine of clinics/clinic for pigs and small ruminants university of veterinary medicine, hannover, germany ♦cofirst authors, other coauthors in alphabetical order abstract this review is an effort by the andrology taskforce of the association of applied animal andrology, american college of theriogenologists, european college of animal reproduction, and society for theriogenology. it is intended to serve as a reference on methods to evaluate sperm motility in domestic animals and to contribute to adoption of best practices in veterinary andrology laboratories and semen processing centers. sperm motility evaluation topics covered include sample preparation, subjective evaluation, computer-assisted semen analysis, and use of sperm quality analyzers. emphasis is given to principles of the methods, equipment, performing evaluation, and common mistakes and/or pitfalls. in addition, precision and accuracy of the various methods are discussed. introduction semen analysis constitutes the most important clinical laboratory test currently available to evaluate male fertility. in domestic animals, this analysis is commonly used to: attest breeding soundness, characterize semen samples for trade, diagnose suberfertility/infertility, and guide clinical and management decisions. there are 2 major quantifiable traits involved in semen analysis, i.e. sperm number and semen quality. the first of these was addressed by this task force in a previous publication1 which represented the first of a series. semen quality includes sperm vitality, motility, morphology, and seminal fluid composition, aspects of which will be addressed in subsequent publications, with the current work focusing on sperm motility assesment. although sperm motility evaluation is generally considered an essential routine component of semen analysis, it is sometimes viewed dissmissively, with results taken for granted without proper validation.2 nonetheless, studies have reported interlaboratory coefficients of variation (refer precision and accurancy of methods) > 20% for sperm motility.2,3,4 this illustrates the difficulties in comparing results among laboratories and in generalizing the findings of scientific studies. recognition of relative subjectivity, and variability, in sperm motility assessment, particularly as traditionally perfomed, has led to a number of attempts to develop more objective and consistent methods, some of which are described herein. this review is intended to address the lack of formal training materials for semen analyis laboratory personnel and to serve as a reference on methods to evaluate sperm motility. clinical theriogenology • volume 13 number 1 • march 2021 24 sample preparation species-specific considerations differences in reproductive biology, including testicular size, sperm production capacity, epididymal sperm storage capacity, and ejaculate volume contribute to physiological differences in sperm motility among species. methods of semen collection, sexual stimulation, and the environment can also affect ejaculate parameters. in addition, ejaculates of some species consist of distinct fractions that differ in number of sperm, thereby affecting sperm motility evaluation. a critical objective is to obtain a representative sample that can be used for evaluation. sperm motility reflects an essential functional sperm trait that allows sperm to reach the site of fertilization and fertilize oocytes. sperm motility is a complex phenomenon that is finely regulated by its environment. ions, and metabolic and enzymatic factors influence sperm kinematics to various extents, depending on the responsiveness of sperm subpopulations in the semen sample.5,6 additionally, the type of extender and dilution may affect sperm motility traits, at least in the first hours after dilution before sperm can adapt to a changed micromilieu.7 as suggested for dogs,8 sperm motility, in general, should be performed as soon as possible after collection in all species or at least within 30 minutes. if possible, sperm motility should be first assessed in raw semen, because addition of extenders may change the natural environment and compromise the assessment of male intrinsic sperm motility values. since temperature dependence of sperm motility is broadly acknowledged, assessment of motility in a heated environment at 38°c, with a possible range of 37 39°c, is standard in andrology laboratories. differences in ejaculate composition and sperm metabolism mandate differing techniques of sample preparation and evaluation. for example, the possibility to estimate mass motility is restricted to raw semen of species with high sperm density ejaculates as in ruminants.9 boar sperm are unique in that preserved semen is incubated at 38°c prior to motility assessment to have optimal results. dog semen is usually collected in fractions and sperm motility is assessed without prostatic fluid, as that may induce sperm hypermotility.10 in addition, sperm motility estimation in liquid or frozen semen might require different sample preparations, depending on the preservation method. resuspension and incubation in specific postthaw extender might be required, according to semen processing center instructions. in most species, 30 minutes incubation is generally recommended to display stable sperm motility from extended and cooled samples. some extender manufacturers stipulate extender-specific incubation times, ranging from 2 to 30 minutes. subjective assessment of sperm motility principles sperm, unlike other cells, are designed to function principally outside the body which produces them. the functional environment of sperm is essentially a foreign object adrift in an external aqueous fluid or engulfed within a new biological niche. although capable of intense activity, sperm generally remain quiescent until ejaculation.11 assessment of motility of ejaculated sperm is considered an important measure of function. hence, in veterinary practice, subjective evaluation of sperm motility has been a fundamental component of assessing the potential fertility of males. besides, determination of sperm motility score, to some extent, enables practitioners to decide the number of sperm per dose.12 the concept of gross or wave motion and individual sperm motility was adopted by the society for theriogenology for evaluation of bull semen13 and later adopted to other ruminant species. traditionally, gross and individual sperm motility has been evaluated subjectively using light microscopy at magnifications ranging from 100 400 x, a method that is rapid, simple, and inexpensive.14 gross motility is estimated under lower (100 x) magnification without a coverslip and is scored in a scale (e.g. 0 4; 0 5; or 'poor,' 'fair,' 'good,' and 'very good'). gross motility results from the interaction of individual sperm motility with sperm concentration and refers to the swirl pattern observed. gross motility ranges from no swirl (score 0 or poor) to a fast distinct swirls referred as 'waves' and 'eddies' (score 4 5 or very good).9,15 it shoulde be noted that gross motility is not a clinical theriogenology • volume 13 number 1 • march 202125 measure of individual sperm motility patterns since immoitle sperm are also carried along with motile sperm. determination of individual progressive motility involves the use of higher (200 400 x) magnification and mostly in a diluted sample with a coverslip. the scoring system is based on percent progressive motile sperm. when every sperm in a field has rapid, progressive, forward motion, then the score is ~ close to 100%.15 progressively motile sperm are those in which the sperm exhibit forward motion in a 'relatively' straight line. for most species, the concept of individual progressive motility is intended to describe the population of sperm that are moving in a forward manner. in stallions, sperm motility is often represented as both total or progressive. gross sperm motility is not analyzed because the sperm concentration of stallion ejaculates, in general, is lower than ruminant ejaculates. total sperm motility is the percent of sperm that display any type of movement, including those that lack forward motility but exhibit tail movement and includes those that are actively moving forward (i.e. progressively motile). definition of progressive sperm motility in stallions varies considerably amongst examiners and laboratories. stallion sperm do not typically have straight trajectories, and this may be due to the abaxial attachment of midpiece to the head of sperm in a relatively high percentage of the sperm population. this is considered to be a normal occurrence. for example, the stallion manual published in 1983 by the society for theriogenology described progressive motility as follows: 'the most important and critical aspect of motility is progressive motility, i.e. sperm are actively moving forward. large, circular motion of some normal sperm is mostly due to high incidence of normal, abaxial connections between sperm head and neck'. later, it was suggested16 that 'the percent of sperm that appear to be morphologically normal and moving relatively straight across the microscopic field. since stallion sperm have abaxial midpieces, they sometimes move in a circle. movement of sperm in small, very tight circles are considered undesirable' and it was further described17as follows: 'for a spermatozoon to be called progressively motile, it must move across the microscopic field reasonable rapidly and with each back and forth lash of the tail, the head must rotate 360°'. suggested standards prescribed for progressive motility are not consistent across species. furthermore, depending on the qualifier (threshold) adopted, an animal can be classified as a 'satisfactory breeder' or an 'exceptional breeder.' for example, in small ruminants, an animal with 30% progressive sperm motility in raw semen is classified as a satisfactory breeder and one with 90% progressive motility is classified as an exceptional breeder, provided they meet the requirements of other parameters of a breeding soundness evaluation. it is also important to recognize that the acceptable percent progressively motile sperm is not similar across organizations and countries and its description is beyond the scope of this review. for example, society for theriogenology uses a score of 30% for bulls,18 whereas the western canadian association of bovine practitioners uses a score of 60%.15 in addition, for dogs, it is 70% (sperm must be moving in a straight line)19 and for stallions it is 60%.20 for boar semen, estimation of gross or total motility (instead of progressive motility) is recommended, as boar sperm tend to display circular movement, which in some cases was explained by the abaxial tail attachment to the head.21 although the evaluation of gross motility is subjective, determination of individual progressive motility is even more so. the degree of inaccuracy in the evaluation of gross and especially individual progressive motility has profound clinical consequences (i.e. 'pass' or 'fail'), especially when sperm motility is performed as the only sperm quality test. since the introduction of individual progressive motility as a method of determining sperm quality, knowledge of sperm kinetics and factors that alter sperm motility has expanded. we understand that some changes in sperm trajectory may not influence sperm viability or fertility. for example, immotile sperm can be viable; temperature reversibly (an artifact) alters the type of sperm motility (progressive to nonprogressive and vice versa) as well as the velocity; and removal of most of seminal plasma will improve the longevity of motility. equipment microscope type and magnification can affect the estimation of sperm motility. brightfield type microscopes provide a flat image with relatively poor detail discrimination, resulting in poor identification of immotile sperm. properly aligned phase contrast or differential interference contrast microscopes are recommended and considered essential for accurate motility estimation.22 the clinical theriogenology • volume 13 number 1 • march 2021 26 contrast in the image field provides a more detailed and clearer image of both motile and immotile sperm in the field of view. microscopes must be equipped with appropriate objectives and a heated stage at 37 39°c. a second associated heated stage and or warming cabinet should also be available to warm everything (e.g. pipette tips, slides, and cover slips) that contacts semen. for preserved boar semen evaluation, a water bath or dry block heater is required. all material must be intact, clean and stored protected from dust. slide and coverslip type (plastic versus glass) can affect the quality of sperm motion. slides should be glass and clean. depending on the manufacturer, glass slides can vary considerably in their clarity and cleanliness. if a slide is cloudy or dirty, it should be cleaned with alcohol, rinsed thoroughly with distilled water, and dried to assure no toxic residue is present. coverslips should also be glass, as that assures a more even distribution of semen compared to a plastic coverslip. avoid leaving finger marks while handling slides and cover slips. evaluation any circumstances that may have compromised sperm motility (e.g. failures or fluctuations in storage temperature, storage duration, and semen packaging) must be recorded. semen samples should be evaluated immediately after collection or at the latest within 30 minutes after collection for all species. if it has to be transported to a laboratory, the evaluation should be performed immediately on arrival. variation can be expected, if there is a delay in evaluation. if samples are not evaluated immediately following collection and poor sperm motility is recorded, the evaluator must take this into account and reexamine the male, ensuring optimal handling conditions. if evaluation of the sample is delayed, raw sperm should be diluted in a suitable extender immediately following collection. at extended intervals following semen collection, deleterious effects of dehydration, ph and temperature changes, and metabolic waste products might become apparent. semen is a nonhomogeneous suspension and sperm tend to sediment. therefore, immediately before taking an aliquot, careful mixing of the semen container without creating air bubbles or sample foaming is required. type of wet preparation as determined by droplet size and dimensions of the glass slide cover slip systems or the measurement chamber has a major influence on motility estimates.23 it is considered that currently applied subjective motility measurement is limited to analysis of 2 dimensional movements of sperm. hence, in vitro conditions need to be thoroughly defined and standardized. based on these aspects, it is important to recognize the relative value of subjective motility estimates and resultant limits for inter-laboratory comparisons. placement of semen on the microscope slide depends on both the coverslip dimensions (i.e. 22 x 22 mm) and 'drop size' used. a prewarned pipette tip is used to deposit 5 10 µl semen on a warm microscope slide and overlaid with a prewarmed coverslip. volume of semen and dimensions of coverslip must be standardized, so that ideally the analyses are done on a preparation of fixed depth of ~ 20 µm. lower sample depth may impair rotational motion, whereas higher sample depth hinder assessment by creating a multilayer of sperm. for example, a 6.5 µl sample covered with an 18 x 18 mm coverslip (area 324 square mm) provides a depth of 20.1 µm, whereas 10 µl of semen delivered covered with a 22 x 22 mm coverslip (area 484 square mm) provides a depth of 20.7 µm.23 clarity can be improved with the use of # 11/2 cover slips compared to # 1 cover slips. sample volume and size of cover slips must be standardized for each species, and for raw and diluted semen. assessment of raw semen may require dilution to minimize sperm overlay. dilution however, may alter sperm kinematics as outlined above. alternatively, the droplet size can be reduced, but a sample depth should not be < 10 µm. sperm from some samples are more susceptible to adhere to a coverslip and thereby give the impression that the sperm are immotile. upon closer inspection, it will become apparent that sperm heads are immotile, although midpieces and tails are vigorously moving. this is usually an artefactual change that is alleviated once semen is extended. dilution of raw semen to a specific sperm concentration (e.g. 20 40 x 106 sperm/ml) allows the evaluation of individual sperm trajectories and provides fewer sperm for the examiner to evaluate in a field. this is critical in evaluating total and progressive sperm motility. for example, for bull semen samples, an acceptable dilution is to have 10 µl of semen diluted in 790 µl of physiological saline.24 raw semen should be diluted in a medium/extender that does not reduce sperm motility. if motility estimates are lower in extended semen than raw semen, then alternative extender batches or clinical theriogenology • volume 13 number 1 • march 202127 formulations should be used. reduced motility in the extended sample can only be diagnosed if the motility of the raw sample has been evaluated. if sample concentration is standardized, then the number of sperm visualized per field will be uniform across samples. sperm concentration of the semen sample evaluated will also create artifacts. more concentrated samples tend to look better than less concentrated samples of similar motility; this tends to be a perception rather than real difference, for several reasons. in more concentrated samples, more motile sperm occupy the microscope image, and thus collide with immotile sperm, giving the perception that immotile sperm are also motile. human eyes tend to follow motile rather than immotile objects, and thus immotile sperm may be ignored. the wet preparation is examined under lower magnification to verify a clear layer of sperm that is in sample homogeneity without air bubbles. the limitation of poor image quality is not only the inability to clearly visualize relevant features, but in the case of evaluating sperm motility, may result in an artificially high motility values, since the only sperm evaluated may be those that are motile. the quality of the microscope can influence the degree of subjectivity and variability, resulting in erroneous scores. freshly prepared wet preparations are assessed as soon as sperm are no longer drifting. total motility and progressive motility are estimated at 200 400 x magnification by assessing the amount of sperm activity to the nearest 5%. multiple fields (≥ 5) should be evaluated, avoiding the periphery of the coverslip where motility is often lower. fields near sample edges and those with lower motility should be excluded from assessment, due to potential artifacts. 25 if motility estimations are below threshold values, a second sample should be prepared to confirm the findings. sperm are only considered motile if a flagellar beating can be observed. progressively moving sperm have either a linear movement or move in large circles. nonprogressive sperm move in small circles with a diameter < sperm’s length. sperm are classified as immotile if they are static and do not have flagellar beating. percent total motile sperm is estimated or calculated from the total percent sum of progressive and nonprogressive sperm. sperm velocity of movement (scale of 0 -5) determination26 provides supplemental information. mistakes and pitfalls regardless of the method of semen collection (e.g. artificial vagina, gloved hand, electroejaculation), all components that come in contact with the semen and environmental temperature have the potential to negatively impact sperm motility. therefore, materials that contact semen should be scrutinized for potential spermicidal influence and should be maintained at 37 39°c to prevent cold shock. these materials include the artificial vagina, collection cone, disposable gloves, collection receptacle, glass microscope slides, coverslips, and pipettes. as previously mentioned, the microscope stage should also be temperature controlled to assure that sperm motility is maintained. sperm motility can be easily affected by heating, chilling, and contamination with urine or other fluids, including soap.15 the size of the semen drop should be consistent; therefore, use of a pipette to measure droplet size is essential. variation in droplet size can create artefactual changes in sperm motility. a small drop that does not cover the whole coverslip can result in low motility due to the thinness of the drop causing resistance and impeding the ability of the sperm to move freely. conversely, large drops result in significant drifting of sperm, making discernment between the drift of an immotile sperm and a motile sperm challenging. the semen drop used should represent the sample of sperm, as an error may be caused by assessment of a nonrepresentative sample of sperm. computer-assisted semen analysis principles computer-assisted semen analysis (casa) refers to the automated assessment of motility of individual sperm. casa involves hardware (video camera, video-frame grabber, and computer) and software designed to acquire and digitize successive images of sperm, process and analyze the sequence of images, and finally provide information on the kinematics of individual sperm.27,28 results of sperm motility analysis by casa are considered more accurate, objective, and precise than other analyses.29 various casa systems for analysis of sperm motility are available. clinical theriogenology • volume 13 number 1 • march 2021 28 sperm motility is completely dependent on functions of the sperm midpiece and tail, whereas the sperm head does not influence the movement, but moves in response to the flagellum. nevertheless, casa will assess movement of the sperm head because technically this is easier to follow as a focal point for analysis27 since it can be more readily captured owing to its size and lumniosity. furthermore, as a direct relationship between head movement and flagellar beats can be assumed, analysis of sperm head movements provides significant information on differences in sperm kinematics under varying conditions. with most systems, casa depends on sperm images via dark field, negative or positive phase-contrast microscopy. the image of the sperm head is digitized and the number of pixels covered by the sperm head is determined. the computer recognizes any object that falls within an designated size range of a sperm head. this range has to be defined and depends on the species. after identification of the sperm head, its coordinate position on the microscope field is calculated based either on the center of the sperm head (also termed centroid) or on the brightest spot of the sperm head.30,31 after all sperm heads in a single field or frame have been identified and located, the next frame is analyzed. the head of an individual sperm is recognized on the consecutive frame if it appears within a zone of probability (i.e. a circle of a particular radius around the sperm head). this radius depends on the maximum distance a sperm would be expected to move within the time period. it is usually set by the user and is influenced by the medium used to dilute the semen. finally, the trajectory of motion is reconstructed for each sperm based on its centroid trajectory.27 dynamic values (sperm motion parameters) are then calculated (table 1). these usually include sperm motion velocity (vcl: curvilinear velocity; vsl: straight-line velocity; and vap: average path velocity), velocity ratios (lin: linearity; str: straightness; and wob: wobble) and sperm wobble characteristics (alh: amplitude of lateral head displacement; and bcf: beat-cross frequency). some sperm motion parameters may not be comparable among casa systems, due to differences in the algorithms used to compute them (e.g. vap, alh).31 further analysis of datasets by cluster analysis allows for grouping of sperm into subpopulations; however, the biological meaning of these subgroups still has to be established. 27,28 future developments of casa technique aim for capturing clearer images, such that captured sperm motion trajectory would more accurately reflect the actual sperm motion characteristics.32 one key feature of casa is the frame rate (number of images per second) of the video camera. frame rates were low (~ 4 5 hz) in early systems, but today 30 60 hz are routinely used and casa systems with 80 100 hz are available. for reliable analysis of mammalian sperm motion characteristics, > 50 hz is recommended27 and is commonly used. frame rate influences the shape of the trajectory, with low frame rates providing tracks that appear much simpler than in reality.30,32 minimal sampling time is 0.5 seconds to aquire reliable kinematic values of sperm trajectory in human sperm.33 if disposable specimen chambers are used, chamber depth must allow for unconstrained sperm movement and will therefore depend on the species-specific movement pattern of sperm. furthermore, chamber depth must match optics to achieve an appropriate depth of focus.33 for a 10 x objective, the chamber depth cannot exceed 20 µm to provide reliable tracking of sperm.34 a depth of 10 or 20 µm is recommended, but will not always provide optimal space for physiologic swimming of sperm in certain species.30 a 30 µm chamber (eflow) is currently used. it is clear that casa has already evolved substantially and is currently superior to other techniques in the research and clinical setting.35 future casa systems will probably be able to integrate several sperm quality parameters with motility36 and also use 3 dimensional tracking of sperm movement and flagellar analysis through flagellar and sperm tracking.37,38 equipment the casa system consists of a phase contrast microscope, video camera and a computer (hardware) with specific software. a list of commercial manufacturers of casa is provided elsewhere.39 evaluation common steps in evaluation include instumentation settings, sample preparation, and assesement of motility. protocols may differ, depending on the device developed and species of interest. clinical theriogenology • volume 13 number 1 • march 202129 table 1. explanations for some common casa kinematic parameters, their abbreviations and dimensions 27 abbreviation meaning explanation dimension vcl curvilinear velocity total distance that the sperm head covers in the sampling period µm/s vsl straight-line velocity straight-line distance between the first and last points of the trajectory µm/s vap average-path velocity distance the spermatozoon has travelled in the average direction of the movement in the observation period µm/s lin linearity (vsl/vcl) x 100 linearity of the curvilinear path % str straightness (vsl/vap) x 100 linearity of the average path % wob wobble (vap/vcl) x 100 oscillation of the actual path about the average path % alh amplitude of lateral head displacement width of the lateral movement of the sperm head, it is calculated as the total width of the head trajectory µm/s bcf beat-cross frequency the number of times the sperm head crosses the direction movement, calculated by counting the number of times the curvilinear path crosses the average path per second hz mistakes and pitfalls despite the application of many automation principles to casa, manual intervention is still necessary for correction.31 technical errors are mainly based on failure to discriminate between sperm and nonsperm objects, and between immotile and motile objects.34 a major problem of casa is related to the standardization and optimization of the equipment and procedures. internal image settings such as minimum contrast, frame rate or analysis time, which are important for identification and reconstruction of the trajectory of sperm, clearly influence the results.40 it is unwise to assume that outcome values from 2 different types of casa system, analyzing the same sample, would be almost identical even if end points of study were the same. hardware configurations and software algorithms could differ. second, conditions to study sperm motion are a compromise. sperm are suspended in a medium unlike any they will encounter in vivo. the suspension is viewed in a chamber (or droplet) in which sperm accumulate at interfaces between the suspension and air, or chamber wall, where they swim differently from the interface.30 a summary of factors that may affect casa measurements of sperm motility and recommedations to avoid variations and misestimations are reported.39 they can be summarized under 4 major categories (sample and slide, microscope, hardware and software, and user). other factors that can influence the outcome are sperm concentration, settings, medium (seminal plasma and extender used for anlysis alter sperm kinematics), sampling time, chamber size (alter velocity and motion pattern of sperm since sperm tend to associate with surfaces because of surface tension). analysis should be postponed until specimen drift is gone, i.e. all visible flow of medium has ceased. perhaps the greatest misunderstanding surrounding casa is the implicit acceptance that it provides a 'gold standard' for sperm motion. casa cannot and should not serve as a 'gold standard' in respect to type or nature of motion, because we do not know how sperm, regardless of species, should swim at any stage of their sojourn through the female reproductive tract.41 direct comparisons among laboratories might not be possible, because the estimation of end points is affected by the settings, type of slide/chamber used during testing, preparation of sperm for analysis, and operator regulated selection of microscopic fields for examination. role of training staff is very crucial in quality control and periodical evaluation is essential. there are no perfect systems, but a crucial point is that standarization and validation of the system are performed and optimization of protocols are assured.39 sperm quality analyzer principles the term sperm quality analyzer (sqa), represents a proprietary technology in which fluctuations in optical density (od) are recorded using a modified light beam as it passes through a clinical theriogenology • volume 13 number 1 • march 2021 30 sperm population contained in a thin glass capillary. a photometric cell registers od variations as electrical pulses that are digitally transformed to provide a sperm motility index (smi).42 in turn, the smi is claimed to provide an indication of overall sperm sample quality, including sperm concentration, progressive motility and normal morphology. 42 today, several hardware models are available with specific software for various species. studies using sqa/smi methodologies have been conducted in various species43 including humans, cattle, pigs, dogs, horses44 (sqa-ve [vequine]) and birds. claimed advantages over traditional systems include relative speed of operation, reduced cost, and less human error, whilst providing automated data recording,45 all of which represent potential advantages for industry application. good statistical relationships have been reported for motile sperm concentrations46 within prescribed sperm dilution windows, although similar relationships with sperm morphology or fertility are not as well established.47 utility of the sqa (i.e. an sqa-vb) as an industry screening tool was demonstrated in an israeli study, in which progressively motile sperm numbers were strongly associated with pregnancy rates following 8000 bovine inseminations.48 some of the units provide information on percent progressive motility and total motility, as well as values for velocity, concentration of raw sample, and percentage of morphologically normal sperm.49 equipment equipment has to be selected based on the species. for example, sqa 11-c was used for bulls,47 dogs,50 and boars,46 sqa-vp was used for boars51 and sqa-ve was used for stallions.49 evaluation each version of sqa is species specific and it is currently not possible to provide detailed information on protocol for each unit. the reader is encouraged to follow manufacturers' insructions carefully to avoid errors. certain versions are meant for raw semen, whereas some are intended for extended semen and others for postthaw motility evaluation. to provide an example, a study used block heaters that were preheated for at least 15 minutes at 40ºc for the sample and empty capillaries (to be loaded in the system) to attain a temperature of 37ºc. the raw samples were incubated in the block heater for 5 minutes prior to testing. mistakes and pitfalls depending on the comparisons to other methods (e.g. light microscopy and casa) and species, results may or may not validate all aspects of a sqa device that is evaluated.51 version and methodolgy differences have to be recognized when an equipment is chosen. some units may be good for parameters other than motility; however, may be insufficiently accurate to determine motility for both raw and extended semen. although auomated motility analyzers could lead to an increased standardization in and between laboratories, automated devices need to be validated for repeatability. precision and accuracy of methods used to determine sperm motility precision (or repeatability) of sperm motility results is usually reported as the coefficient of variation (cv) for results obtained across technicians or laboratories (inter-assay cv) or from multiple evaluations from the same sample (intra-assay cv). several factors affect the cv observed in a study, such as the number of samples, the range of motility of the samples, the number of persons performing the evaluations, and the number of replicates per semen sample. studies investigating inter-technician or inter-laboratory cv of subjective sperm motility evaluation may use fresh semen samples when technicians work in the same laboratory (or during training workshops), whereas videos or frozen semen samples are used in studies where samples are distributed across multiple laboratories. reported inter-laboratory or inter-technician cvs for sperm motility subjective estimates are summarized in table 2. considerable variation is observed among studies, but most report cvs > 15%. the only multilaboratory study involving animal samples (bovine frozen semen) reported cvs ranging from 20 to 55% in 3 separate experiments. reported intratechnician cvs in human studies are mostly < 15% (table 3), whereas intra-laboratory cvs ranged widely in veterinary laboratories evaluating frozen bovine samples (table 4). several studies have demonstrated that variation is much greater when different motile sperm populations (e.g. rapid progressive, progressive, and nonprogressive) are categorized subjectively when compared to simply determining total sperm motility,4,52 suggesting that laboratories should clinical theriogenology • volume 13 number 1 • march 202131 refrain from adopting the former evaluation approach. experience does not necessarily improve subjective sperm motility evaluation precision, as demonstrated by the relatively unchanged variation observed over time in studies with laboratories participating in external quality control programs.4,53 however, targeted training can significantly reduce intraand inter-technician variation of sperm motility evaluations.52,54 reported intra-technician cvs for casa sperm motility are mostly < 10% (table 5). however, in a multilaboratory study with frozen-thawed bovine semen, casa evaluation of sperm motility from frozen and thawed bovine samples resulted in very good precision in some laboratories (cvs < 12%) but not in others (table 4), demonstrating that the adoption of casa does not necessarily result in improved precision.1 proper training and use of standard protocols are essential to achieve adequate precision when using casa for semen evaluation.55 table 2. inter-technician or inter-laboratory coefficients of variation for sperm motility evaluated subjectively. sample species n technicians or laboratories parameter cv (%) reference fresh human 20 4 total motility 8.2 dunphy et al56 frozen human 5 10 total motility 21.0 neuwinger et al3 progressive motility 22.0 fresh human 17 12 total motility 21.8 auger et al54 video human 128 7 total motility 5.5 brazil et al a52 progressive motility 25.1 video human 26 40* total motility 13.9 -19.2* alvarez et al4 progressive motility 17.3 27.0* video human 59 121 total motility 13.8 punjabi et al53 progressive motility 15.1 frozen bovine 10 5 motility 54.7 brito2** 2 motility 45.0 4 motility 20.2 *range for 9 separate trials (test periods).**data reanalyzed according to method of evaluation. table 3. intra-technician coefficients of variation for total sperm motility evaluated subjectively species n technicians cv (%) reference human 20 4 15.6 dunphy et al56 human 5 8.4 cooper et al57 human 5 12 26.2 auger et al54 human 509 4 3.5 10.1 brazil et alb58 studies in veterinary andrology laboratories using the sqa have reported conflicting results related to the method’s precision (table 6). a crucial detail of experimental design must be considered and is likely behind various reports, and that is whether replicate results are obtained simply by repeating the reading on the same preparation or replicates involve the preparation of a completely different subsample. obviously, the variation with the former design is expected to be much lower than the variation with the latter. low cv’s have been reported in sqa studies with canine, porcine, and human semen, but studies in other species have had much greater variation. with extended bovine semen samples, cvs for smi between 2 separate capillaries ranged from 2.1 to 58.8%, although cvs could be reduced to acceptable levels (< 10%) when sample concentration ranged from 35 to 705 x 106 sperm/ml.47 the cvs for sqa sperm motility from the analysis of 5 straws from 10 frozen-thawed bovine semen samples ranged from 13 to 78% (mean 47.3%), a variation considerably greater than that observed when using subjective evaluation or casa.1 repeatability for sqa total and progressive motility was considered only just acceptable for extended equine semen and cvs were at least twice the cvs of casa.49 clinical theriogenology • volume 13 number 1 • march 2021 32 table 4. intra-laboratory coefficients of variation (%) for sperm motility according to evaluation method from 10 frozenthawed bovine semen batches evaluated in five replicates. laboratories included semen processing centers and veterinary university laboratories in the united states and brazil (adapted from brito2). table 5. intra-technician coefficients of variation for sperm motility evaluated using casa species n parameter cv (%) reference bovine 4 motility 6.0 budworth et al59 canine 8 total motility 0.06 iguer-ouada and verstegen60 progressive motility 0.15 porcine 30 total motility 3.1 vyt et al46 progressive motility 6.5 bovine 10 total motility 3.1 5.8* ehlers et al55 *range before and after training table 6. intra-technician coefficients of variation for sperm motility evaluated using the semen quality analyzer (sqa) species n parameter cv (%) reference canine 14 sperm motility index 0.025 0.46* rijsselaere et al61 porcine 30 sperm motility index 6.6 vyt et al46 bovine 5 sperm motility index 2.1 58.8* hoflack et al47 porcine 50 motility 3.2 rodriguez et al51 bovine 10 motility 47.3 brito2 *range for different samples determining the accuracy of sperm motility estimates is more complex, as it requires a method to be defined as the 'gold standard' against which estimates obtained using other methods are compared. defining a 'gold standard' for sperm motility is a controversial subject, as no 'gold standard' exists. however, any method should probably be compared to subjective evaluation from trained technicians. accuracy is determined using a variety of statistical methods, including simple correlation, regression, comparison of means, estimation of percentage differences, and bland-altman plots. the difference between subjective and casa total sperm motility was 2.0% (95% ci = 0.8 3.2%) for extended canine semen and -8.1% (95% ci = -13.3 -2.9%) for extended equine semen.49 inter-laboratory cvs ranging from 18 to 45% were observed when casa was used for evaluation of frozen bovine semen.2 these observations highlighted the several differences among various casa systems in hardware and in algorithms used for sperm detection and tracking. moreover, classification of sperm as motile is based on user-defined thresholds of velocity and mean range subjective laboratory 2 12.7 5 25 laboratory 3 13.0 0 25 laboratory 4 14.6 0 25 laboratory 5 15.1 7 35 laboratory 6 16.3 9 22 laboratory 7 17.7 7 31 laboratory 8 18.9 9 29 laboratory 9 22.7 7 56 laboratory 10 26.5 0 55 laboratory 11 32.4 17 56 laboratory 12 54.0 16 111 casa laboratory 13 8.5 5 18 laboratory 14 8.6 4 12 laboratory 15 11.9 7 17 laboratory 16 15.2 5 44 laboratory 17 16.9 9 36 laboratory 18 19.6 7 42 clinical theriogenology • volume 13 number 1 • march 202133 straightness; therefore, caution must be exercised with the notion that sperm motility estimates from various laboratories are comparable simply because casa systems were used for analysis. the smi with sqa system ranged from 275 to 305 and casa sperm motility ranged from 82 to 84% in extended porcine semen stored for 3 days.46 extended bovine semen samples with percent of total and progressive sperm motility determined subjectively ranged from 60 to 85% and from 10 to 85%, respectively and smi ranged from 9 to 527.47 the correlation of smi with motility and progressive motility evaluated subjectively was low (-0.17 0.04) for extended dog semen and low to moderate (0.49 to 0.71) for extended porcine semen.46 the obvious issue with smi, as is the case for any type of index, is the inability to properly discern deficiencies in sperm concentration, motility, and morphology, as 1 variable may compensate for the other. conversely, semen samples with identical smi values may have very different individual semen characteristics. this greatly limits the ability to offer a proper interpretation of the analysis. motility ranged from 9 to 57% in frozen-thawed bovine samples evaluated using the sqa and from 11 to 48% in samples evaluated subjectively or using casa. the difference between sqa and all other methods was ~ ± 20%.2 the difference between sperm motility assessed with sqa (66%) and casa (72%) was -6.4% (95% ci = -32.8 20%), whereas the difference from with subjective evaluation (64%) was 1.7% (95% ci = -27.5 30.9%) in extended porcine semen samples.40 the agreement of sqa sperm motility with subjective evaluation was nonexistent for extended equine samples (95% ci = -34.3 35.9%).49 review of the available literature indicates that caution must be used when comparing sperm motility results from various laboratories or even from the same laboratory when strict standardization and internal quality control procedures are not followed. precision and accuracy favor the use of wellvalidated casa systems for evaluation of sperm motility over subjective evaluation. additional studies are required before the sqa can be recommended for evaluation of animal semen. conclusion semen analysis constitutes the most important clinical laboratory test currently available to evaluate male fertility. sperm motility estimations should not be regarded as absolute measurements but, rather, they have a relative value that needs to be interpreted in the context of specific systems and settings. as suggested,62 it is important to optimize quality of semen evalution in veterinary practice by creating standardized protocols for evaluation of all semen parameters and updating those protocols as needed, thereby creating some form of quality control for the clinic laboratory. conflict of interest none to declare. references 1. brito lfc, althouse ga, aurich c, et al: andrology laboratory review: evaluation of sperm concentration. theriogenology 2016;85:1507-1527 2. brito lf: a multilaboratory study on the variability of bovine semen analysis. theriogenology 2016;85:254-266. 3. neuwinger j, behre hm, nieschlag e: external quality control in the andrology laboratory: an experimental multicenter trial. fertil steril 1990;54:308-314. 4. alvarez c, castilla ja, ramírez jp, et al: external quality control program for semen analysis: spanish experience. j assist reprod genet 2005;22:379-387. 5. gagnon c, de lamirande e: controls of sperm motility. in: de jong c, barrat c: editors, the sperm cell, 1st edition, cambridge; university press: 2006. p. 108-133. 6. mishra ak, kumar a, swain dk, et al: insights into ph regulatory mechanisms in mediating spermatozoa functions. vet world 2018;11:852-858. 7. johnson la, weitze kf, fiser p, et al: storage of boar semen. anim reprod sci 2000;62:143-172. 8. robert ma, jeyaprakash g, pawshe m, et al: collection and evaluation of canine semen. a review. int j environ sci te 2016;5:1586-1595. 9. david i, kohnke p, lagriffoul g, et al: mass sperm motility is associated with fertility in sheep. anim reprod sci 2015;161:75-81. 10. rota a, milani c, romagnoli s: effect of post-thaw dilution with autologous prostatic fluid on dog semen motility and sperm acrosome status. theriogenology 2007;67:520-525. 11. bishop dw: sperm motility. physiol rev1962;42;1-59. 12. dejarnette jm: the effect of semen quality on reproductive efficiency. vet clin north am food anim pract 2005;21:409-418. 13. blom e: a comparing-chamber for microscopic examination of undiluted bull semen. br vet j 1946;102:252-259. 14. alexander jh: bull breeding soundness evaluation: a practitioner’s perspective. theriogenology 2008;70:469-472. clinical theriogenology • volume 13 number 1 • march 2021 34 15. palmer cw: management and breeding soundness of mature bulls. vet clin north am food anim pract 2016;32:479-495. 16. pickett bw and back dg. procedures for preparation, collection, evaluation, and insemination of stallion semen. the colorado state university animal reproduction lab, 1987. 17. pickett bw and back dg. procedures for preparation, collection, evaluation, and insemination of stallion semen. 3rd edition, published by animal reproduction systems. 2002. p. 27. 18. ball l, ott rs, mortimer rg, et al: manual for breeding soundness examination in bulls. proc annu conf society for theriogenology, hastings, ne 1983. 19. threlfall wr: semen collection and evaluation. in: root-kustritz mv: editor, small animal theriogenology, 1st edition, philadelphia; elsevier: 2002, p. 98. 20. kenney rm: clinical fertility evaluation of the stallion. proc annu conf society for theriogenology, hastings, ne 1983. 21. müller e, brandl g: investigations on the motility and morphology of boar spermatozoa. dtsch tierarztl wochenschr 1975;82:153-155. 22. lorton sp: evaluation of semen in the andrology laboratory. in: chenoweth p, lorton sp: editors, animal andrology, boston; cabi: 2014. p.100-135. 23. laboratory manual for the examination and processing of human semen: cooper tg: editor, 5th edition, geneva; world health organization: switzerland 2010. 24. hoflack g, opsomer g, van soom a, et al: comparison of sperm quality of belgian blue and holstein friesian bulls. theriogenology 2006;66:1834-184. 25. nöthling jo, dos santos ip: which fields under a coverslip should one assess to estimate sperm motility? theriogenology 2012;77:1686-1697. 26. randall j: “this stud’s a dud!”. canine semen evaluation protocols and pitfalls. clinical theriogenology 2000;12:204-209. 27. hoflack g, opsomer g, van soom a, et al: comparison of sperm quality of belgian blue and holstein friesian bulls. theriogenology 2006;66:1834-184. 28. mortimer st, 2000. casa – practical aspects. j androl 2020;21:515-524. 29. martínez-pastor f, tizado je, garde jj, et al: statistical series: opportunies and challenges of sperm motility subpopulation analysis. theriogenology 2011;75:783-795. 30. krause w, viethen g: quality assessment of computer-assisted semen analysis (casa) in the andrology laboratory. andrologia 1999;31:125-129. 31. amann rp, waberski d: computer-assisted sperm analysis (casa): capabilities and potential developments. theriogenology 2014;81:5-17. 32. lu jc, huan yf, lü nq: computer-aided sperm analysis: past, present and future. andrologia 2014;46:329-338. 33. valverde a, madrigal m, caldeira c, et al. effect of frame rate capture frequency on sperm kinematic parameters and subpopulation structure definition in boars, analysed with a casa-mot system. reprod domest anim 2019;54:167-175. 34. eshre (andrology special interest group). guidelines on the application of casa technology in the analysis of spermatozoa. hum reprod 1998;13:142-145. 35. mortimer st, van der horst g, mortimer d, et al: the future of computer-aided sperm analysis. asian j androl 2015;17:545-553. 36. van der horst g, maree l, du plessis ss: current perspectives of casa applications in diverse mammalian spermatozoa. reprod fertil dev 2018;30:875-888. 37. van der horst g: computer aided sperm analysis (casa) in domestic animals: current status, three d tracking and flagellar analysis. anim reprod sci 2020 sep;220:106350. doi: 10.1016/j.anireprosci.2020.106350. epub 2020 apr 4. pmid: 32305213. 38. yániz jl, silvestre ma, santolaria p et al: casa-mot in mammals: an update. reprod fertil dev. 2018;30:799809. 39. soler c, picazo-bueno já, micó v, et al: effect of counting chamber depth on the accuracy of lensless microscopy for the assessment of boar sperm motility. reprod fertil dev 2018;30:924-934. 40. yeste m, bonet s, rodríguez-gil je: evaluation of sperm motility with casa-mot: which factors may influence our measurements? reprod fertil dev 2018;6:789-798. 41. rijsselaere t, van soom a, tanghe s, et al: new techniques for the assessment of canine semen quality: a review. theriogenology 2005;64:706-719. 42. amann rp, katz df. reflections on casa after 25 years. j androl 2004;25:317-325. 43. bartoov b, ben-barak j, mayevsky a, et al: sperm motility index: a new parameter for human sperm evaluation. fertil steril 1991;56:108-112. 44. bartoov b, kalay d, mayevsky a: sperm motility analyzer (sma), a practical tool of motility and cell concentration determinations in artificial insemination centers. theriogenology 1981;15:173-182. 45. hoogewijs mk, de vliegher sp, govaere jl, et al: influence of counting chamber type on casa outcomes of equine semen analysis. equine vet j 2012;44:542-549. 46. lammers j, splingart c, barrière p, et al: double-blind prospective study comparing two automated sperm analyzers versus manual semen assessment. j assist repro genet 2014;31:35-43. 47. vyt p, maes d, rijsselaere t, et al: motility assessment of porcine spermatozoa: a comparison of methods reprod domest anim 2004;39:447-453. 48. hoflack g, rijsselaere t, maes d, et al: validation and usefulness of the sperm quality analyzer (sqa ii-c) for bull semen analysis. reprod domest anim 2005;40:237-244. 49. chenoweth pj, zeron y, shalit u, et al: pregnancy rates in dairy cattle inseminated with different numbers of progressively motile sperm. clinical theriogenology 2010;2:389. clinical theriogenology • volume 13 number 1 • march 202135 50. hoogewijs m, de vliegher s, de schauwer c, et al: validation and usefulness of the sperm quality analyzer v equine for equine semen analysis. theriogenology 2011;75:189-194. 51. rijsselaere t, van soom a, maes d, et al: use of the sperm quality analyzer (sqa ii-c) for the assessment of dog sperm quality. reprod domest anim 2002;37:158-163. 52. rodríguez al, rijsselaere t, bijttebier j, et al: effectiveness of the sperm quality analyzer (sqa-vp) for porcine semen analysis. theriogenology 2011;75:972-977. 53. brazil c, swan sh, tollner cr, et al: quality control of laboratory methods for semen evaluation in a multicenter research study. j androl 2004a;25:645-656. 54. punjabi u, wyns c, mahmoud a, et al: fifteen years of belgian experience with external quality assessment of semen analysis. andrology 2016;4:1084-1093. 55. auger j, eustache f, ducot b, et al: intraand inter-individual variability in human sperm concentration, motility and vitality assessment during a workshop involving ten laboratories. hum reprod 2000;15:2360-2368. 56. ehlers j, behr m, bollwein h, et al: standardization of computer-assisted semen analysis using an e-learning application. theriogenology 2011;76:448-454. 57. dunphy bc, kay r, barratt cl, et al: quality control during the conventional analysis of semen, an essential exercise. j androl 1989:10:378-385. 58. cooper tg, neuwinger j, bahrs s, et al: internal quality control of semen analysis. fertil steril 1992l;58:172-178. 59. brazil c, swan sh, drobnis ez, et al: standardized methods for semen evaluation in a multicenter research study. j androl 2004b;25:635-644. 60. budworth pr, amann rp, chapman pl: relationships between computerized measurements of motion of frozenthawed bull spermatozoa and fertility. j androl 1988;9:41-54. 61. iguer-ouada m, verstegen jp: evaluation of the "hamilton thorn computer-based automated system" for dog semen analysis. theriogenology 2001;55:733-749. 62. rijsselaere t, van soom a, maes d, et al: effect of technical settings on canine semen motility parameters measured by the hamilton-thorne analyzer. theriogenology 2003;60:1553-1568. 63. root kustritz mv: the value of canine semen evaluation for practitioners. theriogenology 2007;68:329-337. clinical theriogenology • volume 13 number 1 • march 2021 36 clinical theriogenology 2022; 14: 59 failure of pregnancy in ruminants dalen agnew department of pathobiology and diagnostic investigation college of veterinary medicine, michigan state university, east lansing, mi abstract failure of pregnancy in ruminants is a substantial source of economic loss around the world. a small number of abortions should be expected even in a healthy herd or flock. identification of the cause can be challenging and often requires extensive laboratory diagnostic support. success requires an accurate history, and a complete tissue sample collection, including placenta and serum from the dam. testing on more than a single case in an outbreak may be necessary. multiple bacterial, viral, and protozoal agents have a role and there are also noninfectious causes such as vitamin and mineral deficiencies, and intoxications. keywords: abortion, failure of pregnancy, cattle, sheep, goats introduction and general approach to investigating failure of pregnancy approaching ruminant abortion requires having as complete a sample set as possible. whereas there are a few pathognomonic lesions, abortions are notoriously unrevealing grossly and even microscopically. autolysis is usually advanced and lesions are often subtle. we frequently have to resort to laboratory testing for a diagnosis and even so, a disappointing number of cases will result in no diagnosis. certainly, in the face of an outbreak, multiple aborted fetuses from multiple dams may provide a better chance at finding a diagnosis. a complete history, including nutrition, parity, and health history of the dam, is critical. a minimum sample set would include: 1. fetal membrane (chorioallantois, not amnion), cotyledonary and intercotyledonary, fixed and fresh 2. lung, fixed and fresh 3. liver, fixed and fresh 4. kidney, fixed and fresh 5. brain, fixed and fresh 6. thyroid, fixed and fresh 7. tongue, fixed 8. skin, fixed (eyelid) 9. spleen, fresh 10. thymus, fresh 11. stomach contents, fresh 12. dam serum, acute (at abortion) and convalescent (3 4 weeks later) alternatively, fetal thoracic fluid can be used for antibody detection, though hemolysis in the sample can substantially impair many assays. note that even though samples appear autolyzed, they will still have value, especially for molecular testing. brain is often especially liquefied due to autolysis and incomplete myelination, but will congeal when poured into formalin and lesions can be identified histologically. small ruminants and cattle have similar reproductive anatomy and physiology; however, do not necessarily have similar lesions nor respond equally to similar pathogens.1-18 whereas, every abortion should be noted by the herd or flock manager, and the value of a full diagnostic necropsy assessed, a certain rate of abortions is expected. in a dairy herd, for instance, a rate of 3 5% fetal loss is expected.19 deviations from a herd’s expected rate is often the impetus for an investigation. infectious causes of abortion lesions associated with infectious pathogens generally lead to either inflammatory lesions or teratogenic effects (viruses). history, gross findings, and histopathology will often provide sufficient evidence for the experienced diagnostician to make a presumptive diagnosis, but laboratory confirmation is usually necessary, including culture, immunohistochemistry, and/or polymerase chain reaction (pcr). viruses among the viruses, herpesviruses are the most ubiquitous and include infectious bovine rhinotracheitis (bovine herpesvirus-1) and caprine herpesvirus-1. gross and histologic lesions are multifocal necrosis of the liver, lung, adrenals, and kidneys, but with no placental lesions.17,24 confirmation is generally by pcr or rising serum titers in the dam. bovine clinical theriogenology 2022; 14: 60 herpesvirus-4 is associated with a higher risk of abortions, but a clear connection is elusive.5 more commonly, however, viral agents disrupt development and teratogenic effects are the presenting complaint. pestiviruses, including bovine viral diarrhea virus, border disease, and classical swine fever, lead to embryonic death and resorption early in pregnancy, but abortion in late pregnancy, abortion, stillbirth, or neonatal death are the result, with fetal developmental abnormalities, mummification, and weakness.20 arthrogryposis, a condition where the limbs are contorted and stiff, is usually associated with brain lesions such as hydrancephaly, porencephaly, and hydrocephalus. border disease can affect the hair as well, causing hair-like deformities in wool breeds of sheep. cache valley virus and some strains of bluetongue virus can cause similar lesions in cattle and small ruminants. foreign animal disease viruses such as schmallenberg, wesselsbron, and akabane viruses should also be considered if appropriate. differentials for limb and musculoskeletal developmental anomalies such as these would include toxic plants such as veratrum californicum (sheep), lathrhyrus spp., lupines, and locoweed.2,15 vitamin a deficiency has also been identified as a cause of developmental lesions in ruminants. bacteria bacteria are likely the more common cause of abortion. importantly, several are zoonotic and appropriate precautions are warranted when conducting postmortem investigations. ideally, all ruminant abortions should be conducted in a biosafety hood, but this is rarely practical, so gloves, boots and apron, goggles, and a properly fitted respirator are reasonable precautions. brucellosis in cattle is caused by brucella abortus; in sheep brucella ovis and in sheep and goats, brucella melitensis are the most important agents in this genus.10 however, there are likely other closely related species that are sporadic causes. these are reportable diseases. severe placentitis is the most common lesion, characterized by granular necrotic debris on the cotyledons and an opaque thickened and leathery intercotyledonary chorion. thick adherent pasty brown to tan material fills the intercotyledonary regions. the fetus is usually fresh. microscopically, neutrophils and macrophages fill the edematous connective tissue and the walls of chorionic vessels. trophoblast cytoplasm is frequently stuffed with small short gram-negative rods. culture, pcr, and immunohistochemistry can specifically identify the organism. serological tests are also used to identify infections, particularly on a herd or flock basis; however, caution should be exercised when interpreting these tests in regions where vaccination is used as false positives can provoke unwarranted regulatory concerns. mastitis can be a sequela to infection and can lead to chronic shedding and human exposure in the milk. coxiella burnetii is quite common in small ruminants, but is also a possible cause of abortion in cattle, though much rarer.14,26 this organism is zoonotic and considered a ‘select agent’ by the cdc due to its infectivity and potential for use as a bioterrorist weapon. in small ruminants, late-term abortion, stillbirths, and neonatal death are the common presentations. usually there are no fetal lesions, but thick suppurative cotyledonary and especially intercotyledonary plaques are characteristic. this is a condition where examination of fetal membranes is critical to the diagnosis. histologically, trophoblasts are also packed with small rods that can be noted with he stains, but gimenez stains can highlight them. routine serology is not widely used to investigate this disease in the us. chlamydia abortus (previously known as chlamydophila abortus or chlamydia psittaci immunophenotype 1 is also a common cause of infectious abortion in small ruminants and rarely in cattle.4 similar late-term abortions, stillbirths, and neonatal deaths are typical, and the gross and histologic lesions are very similar, including the presence of small rods within trophoblasts. unlike coxiellosis cases, however, fetal lesions such as hepatic, splenic, and lymph node necrosis are sometimes observed. immunohistochemistry and pcr are the best methods to confirm the diagnosis – serology can be misleading due to the ubiquity of the organism in the environment. in cattle, the lesions are typically much more subtle and may consist only of vasculitis and edema in the chroioallantoic stroma. campylobacteriosis in small ruminants is an entirely different disease than in cattle.23 in sheep and goats it is caused by campylobacter fetus subsp. fetus, campylobacter jejuni subsp. jejuni, and campylobacter lari, and is spread by the fecal oral route leading to bacteremia in the dam and subsequent placentitis and fetal sepsis. the lesions are primarily cotyledonary, with necrosis and suppurative exudate; the intercotyledonary space is often edematous, but there are not dense mats of necrotic and suppurative debris as in chlamydiosis, coxiellosis, or brucellosis. similar to these diseases, trophoblasts may contain dense mats of organisms within trophoblasts. fetal targetoid regions of liver necrosis are typically noted, along with peritoneal and pleural fluid accumulation and fibrin. these organisms can be cultured with special media (modified cary-blair, for instance) and there are specific immunohistochemical stains to make a diagnosis. wet mounts of abomasal contents or cotyledonary imprints may show these organisms darting motility with darkfield or phase contrast microscopy. in contrast, campylobacter fetus subsp venerealis is a venereally transmitted disease which is primarily associated with infertility, early embryonic death, and only rarely, abortion in cattle, though cattle can rarely be infected by other species of campylobacter.16 fibrinous intercotyledonary placentitits and fibrin in the thoracic, pericardial, and peritoneal cavities, and splenomegaly are usually observed. culture is again possible, but pcr and ihc are often more commonly used in laboratory diagnosis. listeria monocytogenes and listeria ivanovii are considered pathogens in silage-fed animals and usually in the winter, though cases are reported in pasture raised ruminants.6 infections can result in neurological disease in adults or abortion, but rarely both. regardless, there is septicemia in the dam and they are usually ill. retained fetal membranes postabortion and metritis can also be a problem. edema in the intercotyledonary placenta and necrosis in the cotyledons are present, and fetal lesions include necrotizing hepatitis, nephritis, pneumonia, myocarditis, with pleuritic, peritonitis, and pericarditis. cultures (especially if the lab can use cold enhancement) are usually diagnostic, and gram staining will demonstrate gram-positive organisms on cytology or histology. clinical theriogenology 2022; 14: 61 leptospirosis is caused by a variety of serovars, often with regional and host-specific variations in susceptibility and vector. in cattle, leptospira borgpetersenii is specifically adapted to cattle; in sheep, abortions attributed to leptospirosis are much rarer and most are due to l. hardjo, l. pommona, l. catellonis, and l. icterohaemorrhagiae.13 maternal ingestion and bacteremia are the usual routes for the bacterium to get to the placenta and fetus, though placental lesions are usually subtle. grossly, fetuses are usually markedly autolyzed, making diagnosis challenging. fetal lesions are often restricted to kidney or liver, and may be minimal. pcr on dam urine, fetal membranes, or fetal kidney is the confirmatory test of choice, though some labs can still perform the challenging fluorescent antibody assays on kidney impression smears.27 serology is challenging, in that each serovar must be individually assayed, and vaccination and past infections may result in deceivingly high titers. note that aborting dams may shed large numbers of organisms in the urine and uterine discharge and these organisms are zoonotic. salmonellosis is a fairly common cause of abortion in cattle, especially in dairy, but is very rare in small ruminants. salmonella enterica serovar dublin is most commonly associated, but other serotypes have been reported. infection is usually via the digestive tract, with maternal bacteremia resulting in placental and fetal infection. retained fetal membranes and severe fetal autolysis are common with these infections. what lesions might rarely be present are likely obscured by the autolysis. microscopically, mild inflammation of fetal membranes and necrotizing hepatitis are observed, with other organs less frequently affected. bovine foothills abortion (epizootic bovine abortion) is a disease of local, but significant, importance, causing abortion and neonatal deaths in cattle grazing pastures in the foothills of california, nevada, and oregon. pajaroellobacter abortibovis has been implicated in the most recent research, and are carried to the cows by the pajaroello tick (ornithodoros coriaceus).1 the dam is not affected, but fetal mortality can be as high as 90%. fetuses have markedly enlarged and firm livers, spleens, and lymph nodes, and petechiae around the eyes, mucous membranes, and in the thymus. ascites is also commonly reported. mycoplasma sp. and ureaplasma sp. (especially, ureaplasma diversum) are commensal organisms in the female reproductive tract and are attributed to some abortions.7 however, the difficulty in identifying these organisms and the likelihood of contamination during sampling make it difficult to confirm their role in many of these cases. identifying the organism in fetal lung or some other protected site is important to establish it as a cause in a particular case. lesions ascribed to mycoplasma spp. and ureaplasma spp. are primarily associated with vasculitis, including edema, necrosis, hemorrhage, and fibrin exudation. conjunctivitis has been reported as well. interestingly, the amnion is often the most affected part of the placenta, which is unique among the abortifacient agents described here. arguably, the most frequent bacteria isolated from abortions are opportunists. maternal bacteremia can result in spread to the uterus and infection of the placenta and fetus. suppurative placentitis and fetal septicemia are often observed. rumen acidosis in the dam is a frequently associated condition, with subsequent spread of the bacteria via ruminal ulcers into the bloodstream and then to the placenta. to confirm a bacterial cause of an abortion, the bacterium of interest should be isolated in nearly pure culture and the bacteria from compatible lesions via gram stain or immunohistochemistry. fungi in cattle and to a lesser extent, sheep and goats, mycotic infections are generally hematogenous in origin and do not ascend through cervix as is common in horses.29 portals of entry into the dam are likely in the digestive tract, respiratory tract, or skin wounds. aspergillus sp. are most common, but others include mucor spp. rhizopus spp. and candida spp. these are typically sporadic, but can be epidemic when there is a common insult to all the pregnant females in a herd (such as an outbreak of rumen acidosis). placentitis is usually very severe with thick plaques of necrotic debris, suppurative and fibrinous exudate, vasculitis, and fibrosis. often there are no fetal lesions, but if present, bronchopneumonia or dermatitis are the most common. identifying the mycotic organisms in the lesions is usually not difficult microscopically, but gömöri methenamine silver stain or periodic acid–schiff can be used to characterize the fungus. speciation is possible by culture of fetal membranes or abomasal contents. protozoa protozoan are economically important causes of ruminant abortion. in cattle, neospora caninum is the most important with a world-wide distribution and up to 60% seroprevalence rate in us herds.8 it is especially important in that it can be maintained in the cow chronically, repeatedly transmitted to the fetus during repeated pregnancies, and carried congenitally by asymptomatic calves into the next generation. infection can also occur horizontally via ingestion of oocytes from dogs or coyotes. abortions generally occur during the second trimester (an important comparative point to other causes) and the fetus is markedly autolyzed with abundant serosanguinous fluid in the peritoneal, pleural, and pericardial cavities. subtle white streaks or foci are sometimes present in the heart or tongue. histologically, foci of central necrosis surrounded by mononuclear cells are characteristic, though ihc or pcr is usually required to identify organisms in the lesions. thoracic fluid can also be used to document an antibody response in the fetus. care should be taken, however, since the incidence of neosporosis is so high in the cattle population, simply identifying organisms (or a serologic response) in an aborted fetus does not ascribe causation. in contrast to neosporosis, toxoplasmosis is much more common in small ruminants and is associated with feed contaminated with cat feces containing toxoplasma gondii.9 depending on the stage of pregnancy, the outcome may be embryonic death and resorption early in pregnancy or mummification, stillbirth, or neonatal death later in gestation. gross lesions are typically restricted to the placenta, with multifocal region of cotyledonary necrosis and mineralization. histologically, lesions are in the brain and the placenta: mononuclear inflammation in the cotyledons surrounding regions of minclinical theriogenology 2022; 14: 62 eralization and necrosis and regions of necrosis and gliosis in the brain. keep in mind that even autolyzed and liquefied brain tissue can be diagnostic histologically after formalin fixation. identification of fetal antibodies in fetal thoracic fluid can also be helpful. tritrichomonas foetus is another important protozoan cause of abortion and infertility in cattle with similarities in transmission and pathogenesis to campylobacter fetus subsp. venerealis. infertility and early embryonic loss are typical, whereas abortion and pyometra are lesser common manifestations.22 the disease is largely maintained via inapparent bull carriers, so it is observed most frequently in range situations where natural service is used. placental and fetal lung lesions are noted when abortion does occur. histologically, intralesional trichomonads are within the edematous placental stroma and bronchopneumonia with abundant intra-alveolar trichomonads in the lungs. multinucleated giant cells are often also present. immunohistochemistry can aid in finding organisms in tissue samples, but culture using specific media (e.g. inpouchtm) or pcr of fetal membranes, fetal abomasal contents, uterine or vaginal fluid, or preputial scrapings from an infected bull is the most definitive. ideally, purification of the sample by culture followed by pcr is the best approach. noninfectious causes of abortion veterinarians tend to focus on infectious causes of disease in animals, yet often noninfectious causes can be more prevalent and may indeed act as a predisposing or exacerbating factor for infectious pathogens.11 nutrition has an important role in development and growth of the fetus and deficiencies can have a serious impact on fertility. specific deficiencies that can lead to failure of pregnancy include copper, selenium, vitamin e, vitamin a, and iodine.15 copper deficiency can lead to implantation failure, early embryonic death or later in pregnancy can lead to demyelinization, depigmentation, and abnormal haircoat or wool. iodine deficiency is associated with goiter, and usually results in stillbirths and weak neonates.3,15 iodine concentrations are best measured in the thyroid gland itself, so a laboratory should have developed reference values in this organ. true iodine deficiencies are possible, but often are associated with goitrogenic plants such as brassica spp., kale, or beets (and beet by-products). vitamin e and selenium deficiency have long been known for causing white muscle disease, though this is observed much more rarely today. more likely, are subclinical deficiencies that result in decreased fertility, decreased immunocompetence of the dam and fetus, and other more subtle physiologic issues (yamini b, personal communication).28 vitamin a deficiency has been associated with multiple developmental anomalies of the bones, eyes, brain, immunocompromise, and infertility.18 in sheep, this can be associated with the use of beet pulp as an energy supplement for ewes. vitamin a levels are normally lower in fetuses, so appropriate reference values should be used. toxic plants can also be a factor in abortion and developmental anomalies in ruminants. many defects are associated with plant toxicities at a specific point in pregnancy. for instance, veratrum californicum must be ingested by the ewe on day 14 or 15 of pregnancy to produce the characteristic cyclopia that is described with this toxicity.15 ponderosa pine causes both systemic illness and abortion with retained fetal membranes in cattle.25 locoweed (astragalus and oxytropis sp.) can lead to abortions and skeletal and neurologic abnormalities. lists of possible toxic plants are long, regionally specific, and often speculative, so it is important to examine the pasture and stomach contents carefully when investigating possible plant intoxications.12,21 conclusion failure of pregnancy in ruminants can be a substantial cause of financial loss to producers. diagnosis can be frustrating as gross lesions are often inapparent and laboratory testing can take time and additional resources. additionally, a diagnosis is not always possible. however, a thorough knowledge of the possible causes, a good history, and a complete sample set, including fetal membranes and dam serum, from multiple cases will usually provide an answer that can guide management changes to alleviate the abortion problem. conflict of interest author has no conflict of interest. no funding was received for this publication. references 1. anderson ml, kennedy pc, blanchard mt, et al: histochemical and immunohistochemical evidence of a bacterium associated with lesions of epizootic bovine abortion. j vet diagn invest 2006;18:7680. 2. anderson ml: disorders of cattle. in: njaa, b: editor. kirkbride’s diagnosis of abortion and neonatal loss in animals. 4th edition, ames; wiley-blackwell: 2012. p.13-48. 3. bíres j, bartko p, weissová t: jódopénia kôz prícinou kongenitálnej strumy u kozliat [iodine deficiency in goats as a cause of congenital goiter in kids]. vet med (praha) 1996;41:133-138. 4. borel n, thoma r, spaeni p: chlamydia-related abortions in cattle from graubunden, switzerland. vet pathol 2006;43:702-708. 5. chastant-maillard s: impact of bovine herpesvirus 4 (bohv-4) on reproduction. transbound emerg dis 2015;62:245-251. 6. dhama k, karthik k, tiwari r, et al: listeriosis in animals, its public health significance (food-borne zoonosis) and advances in diagnosis and control: a comprehensive review. vet q 2015;35:211-235. 7. díaz jm, prieto a, lópez g, et al: association of ureaplasma diversum with reproductive disease in cattle. n z vet j 2019;67:249-256. 8. dubey jp, buxton d, wouda w: pathogenesis of bovine neosporosis. j comp path 2006; 134:267-289. 9. dubey jp: toxoplasmosis in sheep – the last 20 years. vet parasitol 2009;163:1-14. 10. głowacka p, żakowska d, naylor k, et al: brucella virulence factors, pathogenesis and treatment. pol j microbiol 2018;67:151-161. 11. hidiroglou m: trace element deficiencies and fertility in ruminants: a review. j dairy sci 1979;62:1195-1206. 12. james lf, panter ke, nielsen db, et al: the effect of natural toxins on reproduction in livestock. j anim sci 1992;70:1573-1579. clinical theriogenology 2022; 14: 63 13. loureiro ap, lilenbaum w: genital bovine leptospirosis: a new look for an old disease. theriogenology 2020;141:41-47. 14. macías-rioseco m, riet-correa f, miller mm, et al: bovine abortion caused by coxiella burnetii: report of a cluster of cases in uruguay and review of the literature. j vet diagn invest 2019;31:634-639. 15. moeller rb: disorders of sheep and goats. in: njaa b: editor: kirkbride’s diagnosis of abortion and neonatal loss in animals. 4th edition, ames, wiley-blackwell, 2012. p.49-88. 16. morrell el, barbeito cg, odeón ca, et al: histopathological, immunohistochemical, lectinhistochemical and molecular findings in spontaneous bovine abortions by campylobacter fetus. reprod domest anim 2011;46:309-315. 17. nandi s, kumar m, manohar m, et al: bovine herpes virus infections in cattle. anim health res rev 2009;10:85-98. 18. nicholson jw, cunningham hm: retained placenta, abortions and abnormal calves from beef cows fed all-barley rations. can vet j 1965;6:275-281. 19. norman hd, miller rh, wright jr, et al: factors associated with frequency of abortions recorded through dairy herd improvement test plans. j dairy sci 2012;95:4074-4084. 20. osburn bi, castrucci g: diaplacental infections with ruminant pestiviruses. arch virol suppl 1991;3:71-78. 21. riet-correa f, medeiros rm, schild al: a review of poisonous plants that cause reproductive failure and malformations in the ruminants of brazil. j appl toxicol 2012; 32:245-254. 22. rhyan jc, stackhouse ll, quinn wj: fetal and placental lesions in bovine abortion due to tritrichomonas foetus. vet pathol 1988;25:350-355. 23. sahin o, yaeger m, wu z, et al: campylobacter-associated diseases in animals. annu rev anim biosci 2017;5:21-42. 24. smith kc: herpesviral abortion in domestic animals. vet j 1997;153:253-268. 25. stegelmeier bl, gardner dr, james lf, et al: the toxic and abortifacient effects of ponderosa pine. vet pathol 1996;33:22-28. 26. van den brom r, van engelen e, roest hi, et al: coxiella burnetii infections in sheep or goats: an opinionated review. vet microbiol 2015;181:119-129. 27. vidal s, kegler k, greub g, et al: neglected zoonotic agents in cattle abortion: tackling the difficult to grow bacteria. bmc vet res 2017;13:373. 28. waldner cl, blakley b: evaluating micronutrient concentrations in liver samples from abortions, stillbirths, and neonatal and postnatal losses in beef calves. j vet diagn invest 2014;26:376-389. 29. walker rl: mycotic bovine abortion. in: youngquist rs and threlfall wr: editors. current veterinary therapy in large animal theriogenology. 2nd edition, st. louis; saunders elsevier: 2007. p. 417. introduction the nonsurgical control of reproduction in the bitch is a huge challenge and has a potentially large market in a near future. in europe, many owners fear surgical approach and consider ovariectomy as mutilation. progestins are the only officially approved drugs in europe for this purpose, but they have many undesirable effects. development of medical contraception is however extremely complex because it should, in theory, be used in companion animals as well as in stray dogs. however, the expectations are very different in each case. this article presents the main prospects for medical contraception of bitches. progestins there are several types of progestins. synthetic analogues of progesterone are the most commonly used compounds for prevention or suppression of estrus in dogs.1 they act by blocking the production and/or the release of gnrh from the hypothalamus, most likely by negative feedback. estrus in bitches is prevented more frequently by depot formulations of progestins (medroxyprogesterone acetate, delmadinone acetate, and proligestone). to suppress signs of estrus without any side update on medical control of reproduction in bitch with a focus on deslorelin implant alain fontbonne, cindy maenhoudt ecole nationale vétérinaire d’alfort, centre d’etude en reproduction des carnivores maisons-alfort (paris), france abstract medical control of reproduction in the bitch is a huge challenge. currently, only progestins are officially marketed for this purpose but they have undesirable effects. subcutaneous implants containg gnrh agonist deslorelin have been released in the european market. they may be used ‘off-label’ to prevent cyclicity in adult bitches. a 4.7 mg deslorelin implant (after an initial ‘flare-up’ in increases in pitutary release of gonadotropins [fsh and lh] and subsequent downregulation) prevents estrus for ~ 10 months on an average, with large individual variations. the major disadvantage of using deslorelin implants in adult bitches is the initial induction of estrus soon after implantation. currently, there are no 100% satisfactory protocols aviable to prevent this effect. additionally, deslorelin treatment may sometimes cause side effects (e.g. prolonged estrus, ovarian cysts, prolonged lactation or even and rarely uterine disease). ultrasonographic examination of ovaries and uterus is mandatory to ensure that there are no pathologies (e.g. ovarian cysts or cystic endometrial hyperplasia) present prior to treatment. if the bitch does not develop any side effects in the first weeks after first implantation, she can be reimplanted regularly several times and even life-long without any subsequent problem. a single treatment with a deslorelin implant does not compromise fertility of the bitch in subsequent estruses. treatment of prepubertal bitches before 6 months of age is useful to prevent induced estrus. however, it delays puberty and therefore prevents external genital development during treatment, but it does not seem to have any subsequent effect on growth or health. future approaches of contraception in bitches are in use of aromatase inhibitors, immunocontraception, especially antignrh immunization, drugs counteracting the effects of kisspeptins, and genetically mediated contraception. keywords: bitch, contraception, progestins, deslorelin, kisspeptins, immuno-contraception effects, it is recommended to treat bitches after the third day after the beginning of proestrus (when parabasals cells are still present in vaginal smear). oral megesterol acetate (0.05 or 0.01 mg/kg) was effecive for a year with less side effects when treated during anestrus.2 later, a higher dose (0.55 mg/kg/day for 32 days) was recommended based on sucessful treatment in anestrus in 98% of anestrus animals (n = 200).3 in contrast to past protocols, it is no longer recommended to repeat treatment throughout life.1 this is due to the concern about potential side effects. although still controversial, the main concern is the proliferation of mammary parenchyma, potentially leading to the development of benign or malignant mammary gland tumours after repeated treatment. among others, skin discoloration at the site of injection, anabolic effects, increased appetite with subsequent weight gain, insulin resistance and suppression of the adrenocortical axis are commonly reported side effects. pyometra may occasionally develop, especially when progestins are used in diestrus under endogenous progesterone secretion. proligestone is a most recent type of progestin released to the market in europe, and clinical theriogenology 2021; 13: 146 it has less progestational activity.1 its side effects on the uterus may therefore be weaker than older drugs. deslorelin subcutaneous implants gnrh agonists mimic the action of endogenous gnrh. deslorelin is a super-agonist in which the stability of the molecule and its affinity to the gnrh receptor are increased. it interferes with the pituitary gland via gnrh receptors and thus fsh/lh secretion. in europe, it is marketed under the form of a slow-release subcutaneous implant (suprelorin®) that contains either 4.7 or 9.4 mg of deslorelin. the action of deslorelin occurs in 2 phases. first, in the days after implantation, a stimulation phase occurs leading to increased lh and fsh release that may induce signs of estrus. this ‘flare-up’ effect has a short duration (2 4 weeks). it is followed by pituitary inhibition due to a postreceptor mechanism leading to inhibition of the messenger rna (mrna) encoding the β-subunits of the gonadotropins.4 consequently, plasma concentrations of fsh and lh are at basal concentations and thereby estrus is prevented. however, this effect is temporary and therefore limits the use of deslorelin for control of dog overpopulation (stray dogs). conversely, deslorelin is an option to be considered as an alternative to surgical spay in pets, because a new implant can be administered at the time of annual vaccination. use in adult bitches deslorelin has the major disadvantage of inducing estrus in nearly 100% of adult bitches before the desired inhibitory phase.5 this initial induction of estrus just after implantation limits its use in practice. several attempts have been made to avoid the consequences of this ‘flare-up’ effect. to date, none of the protocols is 100% effective.6 implantation of deslorelin in bitches during diestrus when progesterone concentrations are high (> 5 ng/ml) was 1 approach. however, estrus was observed in some implanted bitches, even when plasma progesterone concentrations were > 60 ng/ml.6 further attempts were made with the concomitant or preimplantation treatment of progestagens, acylin (gnrh antagonist), anastrazole (aromatase inhibitor), clomiphene acetate (antiestrogen) or osaterone acetate (antiandrogen).6 additionaly, deslorelin implants had adverse effects in adult bitches.7 signs such as persistent estrus, ovarian cysts, lactation, and behavioural changes were reported.8 however, the occurrence of these side effects seems rather unpredictable, and only a few individuals were affected. all implanted bitches should be closely monitored, especially during the ‘flare-up’ period, because the majority of side effects, although uncommon, are observed during that time. prolonged estrus, ovarian cysts, and pyometra were observed in a bitch implanted with deslorelin after hcg (human chorionic gonadotropin) treatment to induce ovulation.9 in most cases, removal of the implant solved the problem within 15 days.8 however, in some cases, ovariohysterectomy had to be performed. in a retrospective study7, uterine pathologies were observed in bitches with or without clinical signs of postimplantation estrus. in these cases, the treatment of choice would also be surgical removal. implantation is recommended during anestrus10 since treatment during diestrus (high progesterone concentrations) increased the risk of uterine diseases (e.g. pyometra). once the animal is no longer under the effect of the gnrh agonist, cyclicity returned and future fertility was not affected.11 the duration of estrus prevention was 10.2 ± 5.1 months on average with 4.7 mg implants, but it varied considerably from 2 27 months.6,8 often, the first natural estrus that follows implantation is norma, and the bitch ovulates normally. for bitches reimplanted several times, suppression of estrous signs was observed up to 4 years.12 this individual variation of estrus suppression is unpredictable and should be considered a further limitation. therefore, prior to using this implant in breeding bitches, this possible side effect should be discussed with the owner, especially because it is not possible to predict when the bitch will come back into estrus. suppression of reproductive cyclicity was successfully achieved in 6 of 10 bitches for 1 4 years.12 no behavioral and local or general side effects were observed in any of the treated bitches. according to these authors, the 4.7 mg deslorelin implant may work well for suppression of cyclicity if implanted in diestrus and reimplanted at intervals of 4.5 months. deolrelin 9.4 mg implant may be more suitable for this use, although its efficacy may also be shorter than 12 months. according to these authors, owner compliance is an important limiting factor. our clinical experience is that if the bitch does not develop any side effects in the first weeks after the first implantation, she can be reimplanted regularly several times and even life-long without any problems. we assessed fertility (ovulation rate, pregnancy rate, and litter size) in induced estrues after implantation.11 animals were grouped according to treatment responses: group 1 – females exhibiting signs of estrus (n = 19); group 2 – reimplanted (4.7 mg deslorelin acetate) females exhibiting subsequent spontaneous estrus (n = 7); and group 3 – females (4.7 mg deslorelin acetate) evaluated at subsequent spontaneous estrus (n = 13). there were no differences between induced estrus and posttreatment spontaneous estrus in groups 1 and 2 (short-term treatment) nor between spontaneous estrus in group 3 (long-term treatment). treatment with 4.7 mg deslorelin implant did not compromise the bitches’ fertility in subsequent estruses in any group. deslorelin implant in older bitches (> 5 years) should be used with caution, as potentially the return to cyclicity may be prolonged (> 27 months) and fertility may be affected due to age. it is recommended that owners sign an informed consent, as this is ‘off-label’ use.10 in all cases, it is mandatory before implanting a bitch to examine the ovaries and the uterus by ultrasound (to ensure that no ovarian cysts nor cystic endometrial hyperplasia are present).10 based on our own experience, sedation is usually not necessary to insert the implant under the skin. however, it is recommended to clip and disinfect the skin in order to avoid any local complications. due to potential side effects, we recommend to implant the bitch subcutaneously posterior to the umbilical area.5 in case of any further problems, the implant can be easily removed with a small skin incision under slight sedation. other authors prefer implanting under the skin of the medial part of the rear leg.13 the matrix of the implant is biocompatible and dissolves very slowly. clinical theriogenology 2021; 13: 147 it is not necessary to remove the implant and furthermore it is difficult to remove the implant several months after implantation. use in prepubertal bitches the major interest of implanting a prepubertal bitch is to prevent the onset of estrus without inducing estrus. the age at which an animal is implanted is very important, as several studies indicated that all bitches implanted > 6 months of age had signs of induced estrus. deslorelin implant in 4-month-old females postponed estrus until at least 13 months of age; however, estrus was induced in bitches that received the implant at 7 months of age.14 nevertheless, deslorelin implants can be safely used in 4-month-old prepubertal bitches without causing a ‘flare-up effect’ that commonly occurs in adult anestrous females during the first month after deslorelin implantation.15 similar observations were made in another study.16 deslorelin implants appeared to be a safe and reliable approach to reversibly postpone puberty in female dogs < 6 months. no estrous signs were observed for 13 24 months and 8 15 months (observation period) after treatment with a 4.7 or a 9.4 mg deslorelin implant, respectively.16 a 4.7 or 9.4 mg deslorelin implant was used in crossbreed prepubertal female dogs (n = 13, 4 5.1 months old) as a long-term and reversible contraceptive.17 puberty was postponed until 82.7 ± 8.9 weeks in all dogs treated with deslorelin implants (versus 61.9 ± 9.7 weeks in nonimplanted bitches) and epiphyseal closure was delayed, without apparent side effects. juvenile vaginitis was observed in 75% of the treated females between weeks 10 and 72, but resolved spontaneously.17 later, the authors assessed the effects of prepubertal use of deslorelin implants on luteal function after first spontaneous estrus. the long-term delay of puberty by a deslorelin implant in prepubertal female dogs aged 4.2 months appeared to have no negative carry-over effects on subsequent reproductive activity and ovarian functionality.18 a 4.7 or a 9.4 mg deslorelin implant delayed puberty in 4 month old female dogs and did not cause uterine disturbances. 19 another study20 evaluated effects of repeated treatment of a 4.7 mg deslorelin implant in 4.5 month old female dogs. no estrous signs were recorded after 3 consecutive treatments at 4.5 month intervals, demonstrating that puberty was efficiently postponed. regular closure of bone physes was observed, but hip dysplasia was recorded in 2 deslorelin-treated dogs. juvenile vaginitis was also reported in 75% of the cases; nonetheless, they recovered within 2 weeks after implantation. although deslorelin inhibits ovarian activity, a potential consequence is postponement of external genital development and maintenance at an infantile stage. we investigated the use of deslorelin 4.7 mg (group 1) and 9.4 mg (group 2) subcutaneous implants for the postponement of puberty in bitches < 6 months of age.21 no bitch in group 1 had any sign of induced estrus soon after implant, and owners recorded no clinical side effects. bitches had their first estrus between 13 24 months after implantation and had an elevated plasma progesterone concentrations (> 15 ng/ml), indicating that ovulation had occured. none of the bitches that underwent puberty had any abnormality of fur, growth or development of external genitalia. no bitch in group 2 had estrous signs until the end of the observation period (8 15 months). other potential uses of deslorelin subcutaneous implants gnrh receptors have been identified in several tissues (mammary gland, genital tract, bladder, and hair follicles) and deslorelin subcutaneous implants have been tried in several ‘off-label’ indications.22,23 medical control of reproduction in the bitch: areas of research hormonal manipulation aromatase inhibitors have been used for estrus prevention in foxes.24 there are some unpublished data in the bitch using a drug called finrozole. in an experimental trial conducted at the university of kuopio in finland for patent reasons (ep 1399158 a1), using oral finrozole at a dose of 3 mg/dog/day from the first day of estrous signs for 21 days, it was observed that in 2 of 6 dogs, estrogen secretion decreased to the extent that the signs of estrus disappeared. however, in 4 dogs, although estrogen concentrations decreased, estrous signs persisted. the weak response was obviously due to the very low dose used.24 however, as the fox has reproductive physiology closely resembling dog, aromatase inhibitors may be an option in the near future.25 antiprogesterone drug aglepristone (alizin®, virbac) treatment in the preovulatory period of the bitch in our studies did not block ovulation.26 however, it had a contraceptive effect by delaying oocyte maturation and preventing progression of sperm in the oviduct. contraceptive vaccines three types of contraceptive vaccines are possible:27 ‘classic’ subunit vaccines, containing antigenic fragments of the target; dna vaccines allowing cells to produce antigens directly to elicit an immune response (the vaccine is thus introducing into certain cells of the organism the gene coding for the vaccine antigen); or recombinant vaccines produced by genetic engineering that use a microbial vector that has been rendered harmless (the genes coding for the vaccine target are introduced into the vector microorganism). antignrh immunization appears ideal, because this hormone is the ‘heart’ of reproductive function. however, gnrh molecule, a simple decapeptide (10 amino acids) is not very immunogenic. to create a vaccine, it must therefore be conjugated with more antigenic substances. another approach is to combine the action of a toxin to destroy the target. an antagonist or an antignrh antibody conjugated with a toxin, once introduced into the target cell, can destroy it. however, gnrh neurons are difficult to reach because of the blood-brain barrier. in addition to gnrh, immunization trials with fsh or its receptor can lead to sterility by action on the sertoli cells in males and on the granulosa cells in females. immunization trials against lh or its receptor have clinical theriogenology 2021; 13: 148 allowed the bitch to achieve reversible infertility for about a year.28 vaccines against local targets27 (e.g. proteins of the zona pellucida) have several side effects (ovarian cysts, ovarian inflammation, prolonged proestrus or estrus, and abnormal hormone profiles). currently, contraceptive vaccines are not available in carnivores.29 there are local reactions related to adjuvants, the duration of efficacy is extremely variable among individuals and their development is made very difficult by ethical concerns and the cost of studies. kisspeptins canids are particularly sensitive to kisspeptins. injection of kp-10, a kisspeptin agonist, causes a faster and greater increase in gonadotropins compared to other mammals.30 therefore, kisspeptins are interesting therapeutic targets for the suppression of fertility in dogs and cats. among several kisspeptin analog molecules developed in recent years, only the kisspeptin agonist canine kp-10 and kisspeptin antagonist p271 and p234 peptides (tested for antagonistic properties on the kisspeptin receptor) were tested in dogs. although they exhibited antagonistic activity in many mammalian species, these molecules had no substantial effect in the bitch, irrespective of the phase of the estrous cycle,30,31 suggesting that the canine kisspeptinergic network is species specific, and remains to be studied. a kisspeptin vaccine, kissi, injected intramuscularly into lambs 3 times at an interval of 3 weeks, induced a strong antibody response and resulted in the suppression of gonadal function and sexual behaviour,32 demonstrating that it can be used as a novel target for developing a dna immunocastration vaccine. it would be effective in domestic carnivores because of adequate conservation of the kisspeptin structure between species. inhibition of kisspeptin synthesis by silencing rna (sirna) is also an interesting method that is being researched. kisspeptins are promising therapeutic targets for nonsurgical sterilization of domestic carnivores. advances in research in experimental rodents, ruminants, and even humans, suggest new uses of these molecules in dogs. however, switching from 1 species to another is not easy and makes studies in target species necessary in order to develop the optimal solution. gene silencing genetically mediated contraception (‘gene silencing’) is probably one of the promising approaches for development of new techniques for contraception.29 the principle is to inhibit the genes that activate certain functions of reproduction. gene silencing is basically blocking the expression of a particular gene, usually through the use of an interfering rna (rnai). the disruption is carried out at the level of the transcription of the gene, or of its protein translation, causing inhibition of gene expression. there is no doubt that this is an extremely vast field of research and currently very promising. conflict of interest none to report. references 1. romagnoli s, sontas h: prevention of breeding in the female. in: england g, von heimendahl a: editors. bsava manual of canine and feline reproduction and neonatalogy, 2nd edition, gloucester, united kingdom, bsava publisher: 2010. p. 23-33. 2. harris, t.w, wolchuk, n. the suppression of estrus in the dog and cat with long-term administration of synthetic progestational steroids. am j vet res 1963;24:1003-1006. 3. burke tj, reynolds ha jr: megestrol acetate for estrus postponement in the bitch. j am vet med assoc 1975;167:285-288. 4. navarro c, schober pa. pharmacodynamics and pharmacokinetics of a sustained-release implant of deslorelin in companion animals. proceedings 7th international symposium on canine and feline reproduction 2012;177-178. 5. fontaine e, mir f, vannier f, et al: induction of fertile oestrus in the bitch using deslorelin, a gnrh agonist. theriogenology 2011;76:1561-1566. 6. maenhoudt c, santos nr, fontaine e, et al: results of gnrh agonist implants in oestrous induction and oestrous suppression in bitches and queens. reprod domest anim 2012;47:393-397. 7. palm j, reichler im: the use of deslorelin acetate (suprelorin®) in companion animal medicine. schweizer archiv für tierheilkunde 2012;154:7-12. 8. fontaine e: maîtrise de la folliculogénèse chez la chienne à l’aide d’agonistes de la gnrh. phd thesis, paris 2012 9. arlt sp, spankowsky s, heuwieser w: follicular cysts and prolonged oestrus in a female dog after administration of a deslorelin implant. n z vet j 201;59:87-91. 10. maenhoudt c, santos nr, fontbonne a: manipulation of the oestrous cycle of the bitch-what works… for now. reprod domest anim 2018;53:44-52. 11. borges p, fontaine e, maenhoudt c, et al: fertility in adult bitches previously treated with a 4.7 mg subcutaneous deslorelin implant. reprod domest anim 2015;50:965-971. 12. romagnoli s, stelletta c, milani c, et al: clinical use of deslorelin for the control of reproduction in the bitch. reprod domest anim 2009;44:36-39. 13. walter b, otzdorff c, brugger n, et al: estrus induction in beagle bitches with the gnrh-agonist implant containing 4.7 mg deslorelin. theriogenology 2011;75:1125-1129. 14. trigg te, doyle ag, walsh jd, et al: a review of advances in the use of the gnrh agonist deslorelin in control of reproduction. theriogenology 2006;66:1507-1512. 15. kaya d, aslan s, kaya s, et al: clinical and endocrine short-term effects of gnrh analogue deslorelin in prepubertal bitches: does a “flare-up” occur? kafkas universitesi veteriner fakültesi dergisi 2013;19:1-6. 16. fontaine e, maenhoudt c, mir f, et al: postponement of puberty using gnrh agonist implants in bitches of different breeds. proceedings of the international symposium on canine and feline reproduction. 2012:74. 17. kaya d, schäfer-somi s, kurt b, et al: clinical use of deslorelin implants for the long-term contraception in prepubertal bitches: clinical theriogenology 2021; 13: 149 effects on epiphyseal closure, body development, and time to puberty. theriogenology 2015;83:1147-1153. 18. kaya d, gram a, kowalewski mp, et al: expression of gnrh receptor in the canine corpus luteum, and luteal function following deslorelin acetate-induced puberty delay. reprod domest anim 2017;52:1104-1112. 19. schäfer-somi s, kaya d, sözmen m, et al: pre-pubertal treatment with a gnrh agonist in bitches – effect on the uterus and hormone receptor expression. reprod domest anim 2018;53:1-9. 20. marino g, rizzo s, quartuccio m, et al: deslorelin implants in pre-pubertal female dogs: short-and long-term effects on the genital tract. reprod domest anim 2014;49:297-301. 21. fontbonne a: control of reproduction in bitches with the use of sub-cutaneous implants containing the gnrh agonist deslorelin. proceedings european society for domestic animal reproduction congress 2012:12. 22. reichler im, hubler m, jöchle w, et al: the effect of gnrh analogs on urinary incontinence after ablation of the ovaries in dogs. theriogenology 2003;60:1207-1216. 23. reichler im, welle m, eckrich c, et al: spaying-induced coat changes: the role of gonadotropins, gnrh and gnrh treatment on the hair cycle of female dogs. vet dermatol 2008;19:77-87. 24. banting a, beasley s, lindeberg h, et al: the effect of an aromatase inhibitor, finrozole, on reproductive functions of arctic foxes. j vet pharmacol ther 2012;35:163-164. 25. lindh l, lindeberg h, banting a, et al: administration of aromatase inhibitor mpv-2213ad to blue fox vixens (vulpes lagopus) as a model for contraception in female dogs. theriogenology 2020;152:53-63. 26. reynaud k, saint-dizier m, tahir mz et al: progesterone plays a critical role in canine oocyte maturation and fertilization. biol reprod 2015;93:87:1-9. 27. munks mw: progress in development of immunocontraceptive vaccines for permanent non-surgical sterilization of cats and dogs. reprod domest anim 2012;47:223-277. 28. saxena bb, clavio a, singh m, et al: modulation of ovarian function in female dogs immunized with bovine luteinizing hormone receptor. reprod domest anim. 2002;37:9-17. 29. rhodes l: new approaches to non-surgical sterilization for dogs and cats: opportunities and challenges. reprod domest anim 2017;52:327-331. 30. albers-wolthers ch, de gier j, rutten vp, et al: the effects of kisspeptin agonist canine kp-10 and kisspeptin antagonist p271 on plasma lh concentrations during different stages of the estrous cycle and anestrus in the bitch. theriogenology 2016;86:589-595. 31. albers-wolthers chj, de gier j, walen m, et al: in vitro and in vivo effects of kisspeptin antagonists p234, p271, p354, and p356 on gpr54 activation. plos one 2017;12:e0179156 32. han y, liu g, jiang x, et al: kiss1 can be used as a novel target for developing a dna immunocastration vaccine in ram lambs. vaccine 2015;33:777-782. clinical theriogenology 2021; 13: 150 update on medical control of reproduction inbitch with a focus on deslorelin implant 64 contact andre nault naulta@umn.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9598, http://dx.doi.org/10.58292/ct.v15.9598 review report overview of educational research in theriogenology margaret root kustritz,a andre naultb adepartment of veterinary clinical sciences, university of minnesota college of veterinary medicine, st. paul, mn, usa, buniversity of minnesota libraries, university of minnesota college of veterinary medicine, st. paul, mn, usa abstract a review of the literature identified 59 studies specific to educational research in theriogenology. provision of this comprehensive list of educational research in theriogenology will provide a resource for educators and encourage collaboration within the american college of theriogenologists and society for theriogenology. keywords: educational research, theriogenology, search string, cabicode educational research, also called scholarship of teaching and learning (sotl), is a growing aspect of research in veterinary medi cine. a recent review of all veterinary educational research from north american colleges of veterinary medicine established before 1985 identified 544 articles, with most published in the journal of veterinary medical education or the journal of the american veterinary medical association.1 thirty-eight of the articles in that study were published in discipline-specific journals, including clinical theriogenology; only 1 article was published in the journal theriogenology, in 1995.2 citations for publications of educational research in theriogenology were directly solicited from practicing theriogenologists using various list-serves. a literature search was conducted on march 21, 2022 of both pubmed/medline and cab abstracts in order to cover well-established journals and the more obscure gray and foreign language literature. in pubmed, the best results were obtained by performing 2 keyword searches: 1. theriogenology and education produced 341 results, and 2. ‘animal reproduction’ and education produced 240 results. there were 54 duplicates when these searches were combined, leaving 527 records. in cab abstracts, the best results were obtained by using this search string: (animal breeding or castration or spay or theriogenology or animal reproduction).mp. and (veterinary schools or veterinary education or student*).mp. where mp includes the abstract, title, original title, broad terms, heading words, identifiers, and cabicode record fields. this search produced 474 records and 46 were duplicates with the pubmed search and removed, producing a total of 955 records. endnote software was used for managing and deduplication of records. additionally, the literature cited section of included studies were examined to identify any additional research worthy of inclusion. the few manuscripts directly submitted by the authors were verified as having been captured by the literature review. authors sorted through all records, applying the following inclusion criteria: 1. does the paper discuss veterinary student education? 2. does the paper discuss theriogenology or animal reproduction? after this screening, a total of 59 papers remained (appendix). there were limitations in this study. it is possible that not all educational research papers in theriogenology were identified by direct solicitation or literature review, especially for publications in journals with limited availability. other investigators may have chosen to include some different manuscripts, as there is some subjectivity in what is deemed to be veterinary student education, especially when considering programs varying in length that may or may not incorporate undergraduate training, and what is deemed theriogenology or animal reproduction, as there is overlap with basic sciences and with some clinical sciences, including surgery and medical imaging. barriers to educational research often are barriers of institutional culture, including lack of recognition of educational research as valid research, lack of faculty development and support for educational research, lack of funding, and lack mailto:naulta@umn.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9598 citation line: clinical theriogenology 2023, 15, 9598, http://dx.doi.org/10.58292/ct.v15.9598 65 of a community of practice to provide mentoring and collaborations.1,3,4 it is our hope that providing this comprehensive list of educational research in theriogenology will provide a resource for educators and encourage collaboration within the american college of theriogenologists and society for theriogenology for performance of further research. conflict of interest none. authors’ contribution dr. root kustritz conceptualized the study, solicited papers directly from colleagues, reviewed papers identified by literature review, drafted the paper, and completed revisions requested by the reviewer. mr. nault performed the literature review and helped prepare the electronic supplement containing all the citations and uploaded it to the university’s institutional repository for readers of clinical theriogenology. both authors have read and approved the final version of the manuscript and have agreed to the submission. references 1. root kustritz mv, nault a: measuring productivity and impact of veterinary education-related research at the institutional and individual levels using the h-index. j vet med educ 2020;47:414–420. doi: 10.3138/jvme.0618-072r1 2. mier y teran c, galina cs, russell: who is publishing in theriogenology and on what subjects? theriogenology 1994; 42:727–737. doi: 10.1016/0093-691x(94)90441-k 3. baillie s, hunt ja, ruohoniemi m, et al: academics’ experiences in veterinary educational research: results of an international survey. j vet med educ 2021; 49:650–661. doi: 10.3138/jvme-2021-0079 4. fogelberg k, hunt j, baillie s: young and evolving: a narrative of veterinary educational research from early leaders. educ health prof 2022;4:124–133. doi: 10.4103/ehp.ehp_22_21 http://dx.doi.org/10.58292/ct.v15.9598 https://doi.org/10.3138/jvme.0618-072r1 https://doi.org/10.1016/0093-691x(94)90441-k https://doi.org/10.3138/jvme-2021-0079 https://doi.org/10.4103/ehp.ehp_22_21 66 citation line: clinical theriogenology 2023, 15, 9598, http://dx.doi.org/10.58292/ct.v15.9598 anderson sl, miller l, gibbons p, et al: development and validation of a bovine castration model and rubric. j vet med educ 2021;48(1):96–104. doi: 10.3138/jvme.2018-0016 annandale a, may ce, van der leek ml, et al: effect of a high-intensity one-week training programme and student-level variables on the bovine transrectal palpation and pregnancy diagnosis skills of final-year veterinary students. vet rec 2020;187(11):e99. doi: 10.1136/vr.105909 annandale a, annandale h, may k, holm d: the bovine pregnancy diagnosis challenge: evidence-based evolution of a teaching 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college of veterinary medicine and a non-profit animal shelter. j vet med educ 2008;35(4):637–640 doi: 10.3138/jvme.35.4.637 sprecher dj, farmer ja, nebel rl, mather ec: the educational implications of reproductive problems identified during investigations at michigan dairy farms. theriogenology 1995;43(2):373–380. doi: 10.1016/0093-691x(94)00030-x tivers ms, travis trd, windsor rv, moore ah: questionnaire study  of  canine neutering techniques taught in uk veterinary schools and those used in practice. j small anim pract 2005;46(9): 430–435. doi: 10.1111/j.1748-5827.2005.tb00341.x wade cm, fenwick dc: attitudes of veterinarians to undergraduate education in genetics and the use of genetics in veterinary occupations. aust vet j 1996;73(1):1–5. doi: 10.1111/j.1751-0813.1996.tb09944.x williamson ja, johnson jt, anderson s, spangler d, stonerook m, dascanio jj: a randomized trial comparing freely moving and zonal instruction of veterinary surgical skills using ovariohysterectomy models. j vet med educ 2019;46(2):195–204. doi: 10.3138/ jvme.0817-009r wilson jh, martinson kl, turner ta, et al: community service experience, veterinary student training, and owner education via castration clinics for horses of economically challenged owners in the usa. the 6th international colloquium on working equids: learning from others. proceedings of an international colloquium, new delhi, india, 29 november – 2 december 2010; 2010:357–359. [cited 1 april 2023] available from: https://www. thebrooke.org/sites/default/files/research/sixth%20colloquium/ education-to-engage-the-next-generation.pdf. http://dx.doi.org/10.58292/ct.v15.9598 https://doi.org/10.3389/fvets.2018.00095 https://doi.org/10.3389/fvets.2018.00095 https://doi.org/10.3138/jvme.35.4.637 https://doi.org/10.1016/0093-691x(94)00030-x https://doi.org/10.1111/j.1748-5827.2005.tb00341.x https://doi.org/10.1111/j.1751-0813.1996.tb09944.x https://doi.org/10.3138/jvme.0817-009r https://doi.org/10.3138/jvme.0817-009r https://www.thebrooke.org/sites/default/files/research/sixth%20colloquium/education-to-engage-the-next-generation.pdf https://www.thebrooke.org/sites/default/files/research/sixth%20colloquium/education-to-engage-the-next-generation.pdf https://www.thebrooke.org/sites/default/files/research/sixth%20colloquium/education-to-engage-the-next-generation.pdf 1 contact kenton morgan kenton.morgan@zoetis.com © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9269, http://dx.doi.org/10.58292/ct.v15.9269 research report evaluation of an equine multi-antigen rabies combination vaccine in pregnant mares glenn blodgett,a chris morrow,b scott bailey,c jaye mccracken,d natanya nieman,e deborah amodie,f kenton morganf a6666 ranch, guthrie, tx; bmobile veterinary practice, amarillo, tx; cclaiborne farm, paris, ky; dhagyard equine medical institute, lexington, ky; ewinstar farm, versailles, ky; fzoetis, parsippany, new jersey abstract mares are commonly vaccinated during pregnancy, especially in the late third trimester, with multiple vaccine products containing a variety of antigens. currently, a new core antigens vaccine containing rabies virus, tetanus toxoid, eastern equine encephalomyelitis virus, western equine encephalomyelitis virus, and west nile virus with and without the inclusion of venezuelan equine encephalomyelitis virus is used in later term mares. use of this vaccine has not been evaluated for safety in pregnant broodmares. we determined the safety of this vaccine in pregnant mares as it pertains to live foaling rate and per cycle pregnancy rate. findings indicated that the use of a new multi-antigen rabies combination vaccine had no impact on live foaling rate nor per cycle pregnancy rates. keywords: broodmares, pregnancy, vaccination, rabies, live foaling rates, per cycle pregnancy rates introduction it is a common management practice to immunize mares in late pregnancy in an effort to have adequate antibody concentrations in colostrum for passive transfer to the newborn foal and to provide disease protection for the mare. the american association of equine practitioners (aaep) recommends this practice in their vaccination guidelines.1 many vaccine products used for this purpose do not carry a usda ‘safe for use in pregnant mare’ label claim. one common vaccine antigen used in pregnant mares for this purpose is the rabies virus. when this study was initiated, none of the rabies vaccines approved for use in horses carried a pregnant mare safety claim.2–7 the most recently approved rabies vaccines for use in nonpregnant horses include 2 products: core eq innovator™ (zoetis, parsippany, nj) that includes killed rabies virus, tetanus toxoid, eastern equine encephalomyelitis (eee) virus, western equine encephalomyelitis (wee) virus, and west nile (wn) virus; and core eq innovator + vee™, zoetis (parsippany, nj) that is similar to core eq innovator™ and also includes venezuelan equine encephalomyelitis (vee) virus. it is common in southern us states, especially those bordering mexico, to include vee in their vaccination programs. these products differ from other combination equine vaccines in that they contain all of the core equine disease antigens; rabies virus, tetanus toxoid, eee virus, wee virus, and wn virus as prescribed in aaep vaccination guidelines.1 although rabies vaccines are frequently used in pregnant mares, there has been no objective safety evaluation for this purpose. due to the core antigens combination in these vaccines, their relatively recent entry into the equine market and their common use in pregnant mares, a retrospective evaluation was initiated to determine the safety of these products in pregnant mares as it pertains to live foaling rate. a similar retrospective evaluation was used previously to assess the safety of an equine vaccine in pregnant mares.8 objectives were to determine the effects of a multi-antigen rabies combination vaccination, with and without the inclusion of vve virus antigen, on live foal ing rate in broodmares, and per cycle pregnancy rate. mailto:kenton.morgan@zoetis.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9269 2 citation line: clinical theriogenology 2023, 15, 9269, http://dx.doi.org/10.58292/ct.v15.9269 materials and methods for this study the definition of ‘live foal’ was a foal that is delivered and is able to rise and nurse on its own. any foal not meeting this definition was counted as a ‘dead foal’. also, any pregnancy loss for any reason after administering the core antigens vaccines was also counted as a dead foal. live foaling rate was defined as the number of mares delivering live foals/ number of mares diagnosed in foal prior to receiving their normal regimen of late pregnancy vaccinations. live foaling rate was calculated for the year prior to incorporating the core antigens vaccines (multi-antigen rabies virus, tetanus toxoid, eee virus, wee virus, wn virus ± vee virus combination vaccine) in their pregnant mare populations and for the year after the use of the core antigens vaccines. breeding, foaling, and vaccination records of pregnant mares from 4 farms and a veterinary practice (referred to as ‘farm 4’) in various areas of the country were reviewed. two farms (farm 1 and 3) provided data sets to determine per cycle pregnancy rate (a parameter used to evaluate reproductive efficiency9,10) before and after incorporating core antigens vaccines. in farm 3, core antigens vaccination was given on a fixed date (july 21) to pregnant mares. consequently, these mares would have been in their first or second trimester, depending upon their respective successful breeding dates. in all other data sets, the average time of vaccination was 29 days +/14 days of mares’ calculated foaling date. these mares received additional vaccine boosters in their third trimester; however, these vaccines did not contain a rabies virus antigen. in all, records from 975 thoroughbred and quarter horse broodmares of varying age and parity were evaluated. of which, 458 mares had data for the year before incorporating core antigens vaccines with or without vee virus and 517 mares had data for the year after the farms began using these vaccines. within these data, there was a subset of 370 mares that had data for 2 consecutive years for this evaluation. it was ascertained from participating farms and attending veterinarians that no substantial changes were instituted at these farms regarding feeding, husbandry, and breeding or other management practices for these 2 years. data analyses live foaling rate was determined for each farm individually and for all farms collectively. live foaling rate was defined as a binary variable (1 = yes, 0 = no) and was analyzed by a generalized linear mixed model approach for repeated measures. using sas proc glimmix procedure (sas 9.4, cary, nc) combined live foaling rate of farms was analyzed with a model that considered time (before or after) as a fixed effect and farm and residual error as random effects. animal id-within-farm was the subject and time was the repeated factor. covariance structure in the repeated measures analysis was investigated using compound symmetry, first-order auto-regression and unstructured model. the assumption that gave the minimum value of the akaike’s information criterion was selected in the final analysis. analyses utilized a binomial error and logit link. treatment least squares means were compared by twosided t-test. the 5% level of significance was used to assess differences and for comparison of least square means. analyses by farm were performed in a similar manner with only residual error as random effect. results there were no differences (p > 0.05) in live foaling rate before and after incorporation of core antigens vaccines with or without vee virus among farms individually or collectively (tables 1–3). live foaling rate was not different (p > 0.05) in the subset of 370 mares for which there were data for 2 consecutive years (table 4). there were no differences (p > 0.05) in per cycle pregnancy rate before and after the incorporation of core antigen vaccines with or without vee virus in 2 farms (table 5). discussion breeding operations routinely use a number of vaccines containing a wide variety of antigens for pregnant mares. vaccine antigens used in broodmares studied varied from operation to operation. at a minimum, these antigens included rabies virus, tetanus toxoid, eee virus, wee virus, wn virus, equine influenza virus, and equine herpes virus type i and 4. some farms also utilized vee virus, rotavirus, botulism toxoid, potomac horse fever, autogenous salmonella, and autogenous table 2. live foaling rate in mares before and after receiving core antigens vaccine containing vee virus (note: farm 4 data did not converge, ls means for year after was 100% with small sample size therefore, no statistical analysis could be completed) pregnant mares/live foals live foaling % (least square means) p farm 1 year before 56/53 94.71 0.3853 year after 83/81 97.61 farm 4 year before 33/30 90.90 year after 10/10 100 table 1. live foaling rate for mares before and after receiving core antigens vaccine without vee virus pregnant mares/live foals live foaling % (least square means) p farm 2a year before 36/33 91.85 0.5413 year after 60/57 95.06 farm 2b year before 37/34 92.08 0.6838 year after 52/49 94.35 farm 3 year before 101/94 93.20 0.7830 year after 101/95 94.18 farm 5 year before 195/177 91.72 0.5100 year after 211/196 93.41 http://dx.doi.org/10.58292/ct.v15.9269 citation line: clinical theriogenology 2023, 15, 9269, http://dx.doi.org/10.58292/ct.v15.9269 3 clostridium difficile antigens. these vaccine antigens represented varied products and manufacturers. the only variable in the farms studied was the incorporation of the multi antigen rabies combination vaccine that replaced the same antigens previously used but were now in a single-dose vaccine formulation. results indicated that the new core antigen vaccines with and without vee virus had no impact on live foaling rates. broodmare populations on any given farm may vary from year to year. this can be due to a number of factors that include: retiring older broodmares from the herd, retiring mares due to disease or injury, death of individual mares, sale of mares, purchase of new mares and bringing young or maiden mares into broodmare populations. breeding and foaling records evaluated included broodmares that were and were not present on farms for 2 consecutive years of this study. this was done to attain as complete an evaluation of the vaccine as possible, while emulating the normal turnover of mares within a breeding operation. it is noteworthy that there was a numerical increase (although not significant) in live foaling rate after incorporating the new vaccines. per cycle pregnancy rate is a parameter that can be used to evaluate breeding efficiency. although there were no differences in per cycle pregnancy rate, during the 2 years that were evaluated, it is acknowledged that stallion fertility might have had an impact. findings indicated that incorporation of new core antigens vaccines had no impact on live foaling rate or per cycle pregnancy rate; however, these results do not constitute a safety claim for use in pregnant mares. conclusion there were no differences in live foaling rate nor per cycle pregnancy rate after incorporating the core antigens combination vaccines with and without vee virus. note: since study completion, these multi-antigen rabies combination vaccines with and without vee virus have received the following label claim: ‘use of 1 dose is safe during the third trimester of pregnancy in mares’ from the usda. conflict of interest and funding senior author (kenton morgan) and deborah amodie are employees of zoetis. references 1. american association of equine practitioners. vaccination guidelines. available from: https://aaep.org/guidelines/vaccination-guidelines/vaccination-and-passive-transfer [cited 4 june 2021]. 2. elanco labels. available from: https://www.elancolabels.com/us/ rabvac-3-50-dose-1-ml [cited 30 september 2022]. 3. compendium of veterinary products. available from: https://bayerall.cvpservice.com/product/view/1047551 [cited 30 september 2022]. 4. compendium of veterinary products. available from: https://bayerall.cvpservice.com/product/view/1028340 [cited 30 september 2022]. 5. zoetis us. available from: https://www.zoetisus.com/contact/ pages/product_information/msds_pi/pi/core_eq_innovator.pdf [cited 4 june 2021]. 6. zoetis us. available from: https://www.zoetisus.com/contact/ pages/product_information/msds_pi/pi/core_eq_innovator_v. pdf [cited 4 june 2021]. table 3. live foaling rate in mares before and after receiving core antigens vaccines with or without vee virus on all farms pregnant mares/live foals live foaling % (least square means) p year before 458/421 91.17 0.1396 year after 517/488 94.53 table 4. live foaling rate in 370 mares before and after receiving core antigens vaccines with or without vee virus for 2 consecutive years pregnant mares/live foals live foaling % (least square means) p year before 370/341 92.81 0.5705 year after 370/345 93.82 table 5. per cycle pregnancy rate from farms 1 and 3 before and after receiving core antigens vaccine with or without vee virus number of mares per cycle pregnancy rate (least square means) p year before 141 1.23 0.6645 year after 159 1.21 http://dx.doi.org/10.58292/ct.v15.9269 https://aaep.org/guidelines/vaccination-guidelines/vaccination-and-passive-transfer https://aaep.org/guidelines/vaccination-guidelines/vaccination-and-passive-transfer https://www.elancolabels.com/us/rabvac-3-50-dose-1-ml https://www.elancolabels.com/us/rabvac-3-50-dose-1-ml https://bayerall.cvpservice.com/product/view/1047551 https://bayerall.cvpservice.com/product/view/1047551 https://bayerall.cvpservice.com/product/view/1028340 https://bayerall.cvpservice.com/product/view/1028340 https://www.zoetisus.com/contact/pages/product_information/msds_pi/pi/core_eq_innovator.pdf https://www.zoetisus.com/contact/pages/product_information/msds_pi/pi/core_eq_innovator.pdf https://www.zoetisus.com/contact/pages/product_information/msds_pi/pi/core_eq_innovator_v.pdf https://www.zoetisus.com/contact/pages/product_information/msds_pi/pi/core_eq_innovator_v.pdf https://www.zoetisus.com/contact/pages/product_information/msds_pi/pi/core_eq_innovator_v.pdf 4 citation line: clinical theriogenology 2023, 15, 9269, http://dx.doi.org/10.58292/ct.v15.9269 7. merck usa. available from: https://merckusa.cvpservice.com/ product/basic/view/1047551 [cited 4 june 2021]. 8. vest dj, cohen nd, berezowski cj, et al: evaluation of administration of west nile virus vaccine to pregnant broodmares. j am vet  med assoc 2004;225:1894–1897. doi: 10.2460/javma. 2004.225.1894 9. blanchard tl, varner dd, schumacher j, et al: evaluation of breeding records. in: elizabeth mf, ed. manual of equine reproduction. 2nd ed. st. louis, mo: mosby; 2003, pp. 229–237. 10. love cc: evaluation of reproductive efficiency. in: samper jc, ed. equine breeding management and artificial insemination. 2nd ed. philadelphia, pa: wb saunders; 2009, pp. 289–294. http://dx.doi.org/10.58292/ct.v15.9269 https://merckusa.cvpservice.com/product/basic/view/1047551 https://merckusa.cvpservice.com/product/basic/view/1047551 http://dx.doi.org/10.2460/javma.2004.225.1894 http://dx.doi.org/10.2460/javma.2004.225.1894 introduction bovine trichomoniasis was first reported in the us in 19321 and continues to cause reproductive failure in us herds. because this disease transmission under natural conditions is strictly venereal, it should be amenable to control and even eradication. although eradication has occurred in some cattle populations, the causative organism, tritrichomonas foetus, continues to circulate through cattle herds across the us. the most substantial challenge to successfully eliminate this organism from a cattle population is the ability to consistently and accurately identify infected animals for their removal from the breeding herd. the diagnostic process consists of 3 basic phases: preanalytical (sample collection and handling), analytical (performance of the diagnostic test), and postanalytical (test result reporting and interpretation). examples of preanalytical errors include inappropriate test request, misidentification of the sample, sample collection and handling, and storage and transportation of the sample. examples of analytical errors include equipment malfunction, sample mix-ups, and undetected quality control failure. excessive turnaround period, improper data entry, incorrect interpretation, and inappropriate follow up plan are examples of postanalytical errors. most errors that occur during the entire diagnostic process occur in the preand postanalytical phases.2 the purpose of this discussion is to examine the 3 phases of diagnostic testing as they relate specifically to bovine trichomoniasis, with a focus on preand postanalytical phases. preanalytical considerations sampling location distribution of t. foetus in the preputial cavity of bulls was diagnosis of bovine trichomoniasis jeff ondrak 6u ranch, steele city, ne abstract bovine trichomoniasis was first identified in the us in 1930s as a cause of reproductive failure in cattle and its impact continues. because bovine trichomoniasis transmission is strictly venereal under natural breeding conditions, its control and eradication are feasible. however, failure to consistently and accurately identify infected animals and culling them have been major challenges. generally, errors in the 3 diagnostic phases (preanalytical, analytical, and postanalytical) prevent correct classification of tritrichomonas foetus status of tested animals. the purpose of this review is to define these phases of diagnostic testing and discuss potential errors in each phase.. keywords: tritrichomonas foetus, cattle, trichomoniasis described in detail in 1943.3 highest number of organisms were at the midshaft and caudal portion of the free penis, followed by the prepuce surrounding penis and then the galea glandis, and the prepuce near the preputial opening.3 this information has been used as the basis for recommending the collection of preputial samples from the fornix of the prepuce for trichomoniasis testing. although this recommendation is still supported4 further work is necessary to unequivocally confirm that fornix is the optimal site for collecting samples. sampling devices a variety of sampling devices and sampling techniques have been used to collect preputial samples for t. foetus testing including preputial lavage, cotton swabs, washing of artificial vaginas, specialized collection devices, and uterine infusion pipettes.5 however, without strong evidence supporting 1 method over another, the technique of utilizing a uterine infusion pipette and syringe has become the most widely accepted method for t. foetus sample collection in the us. the pizzle stick (lane manufacturing, inc., denver co) is a recently marketed product that is based on a t. foetus sampling device developed in the soviet union and described in 1969.6 the current device is a long plastic hollow rod with circular grooves around the diameter of the rod at the end that appears to provide an atraumatic sample collection option. although the original device was superior to pipette aspiration for sample collection6,7 the diagnostic sensitivity between these 2 methods was not different.8 a recently developed t. foetus sampling device (trichit, morris clinical theriogenology 2021; 13: 231 livestock products, delavan, wi) has a uterine infusion pipette with a small plastic collection cup affixed to 1 end. the cup is designed to increase the scraping circumference of the pipette while also allowing the sample to be aspirated into the pipette. the purpose of the collection cup is to increase the volume of sample collected while doing so in a less traumatic manner. efficacy of trichit over other devices remains unknown. another more recent technique described as a direct swabbing of the extended penis and prepuce with a gauze sponge has been proposed as a means for collecting a sample from a wider area of the penis and prepuce while allowing the collector the opportunity to avoid areas of gross contamination.9 because there is limited evidence to support any technique or device over another, the best approach is to determine a procedure that works best for the operator and is approved by the laboratory that tests collected samples. sample quality determining what constitutes a quality preputial sample for t. foetus testing is a challenge considering the variety of potential contaminates present in the prepuce including soil, feces, urine, semen, blood, and other organisms. recent reports10-13 emphasized the need to better understand this preanalytical aspect of trichomoniasis testing. an investigation into vaginitis in a group of cows in switzerland involved testing for t. foetus.10 although polymerase chain reaction (pcr) testing of vaginal samples indicated a substantial number of t. foetus positive cows, further investigation revealed these to be false-positive tests due to a cross reaction with simplicimonas-like organisms. an earlier report also identified false-positive pcr results due to this trichomonad in samples from bulls.11 bacteria in samples may also provide diagnostic challenges to correctly identify t. foetus infected bulls. bacterial contamination, if not inhibited in medium, had a negative impact on diagnostic sensitivity.12 among biological materials (blood, semen, and urine) urine had a negative effect on the correct classification of the t. foetus status of the sample.13 these reports not only highlighted the need to understand the limitations of the tests used to analyze t. foetus samples but also recognized the importance of reducing and eliminating contamination. sample handling once an adequate sample has been collected, the conditions under which it is transported to a diagnostic laboratory for testing can have a substantial impact on the results of the test. despite several studies examining the influence of cold temperatures on both culture and pcr testing, not much is known regarding the effects of higher temperatures on t. foetus survival and its relationship to test accuracy. although temperatures above 98.6°f affected the ability to culture t. foetus positive samples, they had no negative influence on the ability of pcr to identify the organism, even though the growth rate of the organism was affected.14 exposure of inoculated pouches (with fewer organisms) to higher temperatures (39, 68, 98.6, and 107.6°f for 24, 48, and 72 hours, respectively) were positive to culture and pcr tests except for 107.6°f.15 authors attributed this discrepancy to a difference in the number of organisms inoculated and the cutoff values used for the pcr. however, both studies supported the recommendation to protect all t. foetus samples from temperatures above 98.6°f. analytical considerations testing samples for pcr was described in 1997 that provided a substantial improvement over traditional culture-based testing protocols.16 despite advances made in pcr techniques (improved workload management for laboratories, decreased turnaround times for reporting results, and potentially improved analytical sensitivity and specificity versus the traditional culture techniques), it has not necessarily improved the overall diagnostic process for bovine trichomoniasis.17-19 the apparent lack of diagnostic improvement for trichomoniasis may have more to do with our expectations of a diagnostic test than the assay itself. an excellent review20 of diagnostic test terminology summarized the following. analytical sensitivity is an assay’s ability to detect a very low concentration of a given substance or organism in a biological sample, whereas diagnostic sensitivity is the assay’s ability to detect an individual with the condition in the population. for trichomoniasis, the pcr technique has been recognized to have extremely high analytical sensitivity with the theoretical ability to detect as few as 2 t. foetus organisms per ml sample.18 however, the ability of a test to detect a t. foetus-infected animal in a population is our priority and that is an example of diagnostic sensitivity. the diagnostic sensitivity of pcr is ~ 98%.21 analytical specificity is an assay’s ability to exclusively identify a target substance or organism in a sample, whereas diagnostic specificity is the ability of the assay to correctly identify an individual in a population who does not have the disease in question. the diagnostic specificity of pcr for trichomoniasis testing is ~ 98%.17,18 postanalytical considerations while a variety of errors may occur in the postanalytical phase, the focus of the discussion for this phase of the diagnostic process will be on interpretation of results. diagnostic test sensitivity and specificity receive much attention, but they are characteristics of the test itself. positive and negative predictive values are clinically relevant information related to the clinical theriogenology 2021; 13: 232 diagnostic process. positive predictive value is the probability that an individual that tests positive has the disease of interest. negative predictive value is the probability that an individual that tests negative does not have the disease of interest.20 in other words, predictive values give some indication as to how strongly the results of the test can be trusted to represent the true infection status of an animal. predictive values are greatly influenced by the prevalence of the disease in a population tested, as described in the following examples using these assumptions: the prevalence of trichomoniasis in the test population is 1%, the diagnostic sensitivity of the test is 98%, and the diagnostic specificity of the test is 98%. if 10,000 bulls fit the description of a test population, then we would expect to have 100 (10,000 x 0.01) infected bulls in the 10,000 bulls tested. with a diagnostic sensitivity of 98%, 98 (100 x 0.98) of the 100 infected bulls would result in a positive test and 2 (100 minus 98) of the 100 infected bulls would result in a negative test. because 100 of the 10,000 bulls are infected, then 9,900 (10,000 minus100) of the bulls would be uninfected. with a diagnostic sensitivity of 98%, 9,702 (9,900 x 0.98) of 9,900 uninfected bulls would result in a negative test and 198 (9,900 minus 9702) of the 9,900 uninfected bulls would result in a positive test. the positive predictive value is calculated by dividing the number of infected bulls which tested positive by the total number of test positive bulls. in this example, the positive predictive value is 0.331 (98/296) or the probability of a test positive bull being infected with t. foetus is 33%. the negative predictive value is calculated by dividing the number of uninfected bulls that tested negative by the total number of test negative bulls. for this example, the negative predictive value is rounded to 1.00 (9702/9704), which means that the probability of a test negative bull not being infected with t. foetus is very close to 100%. if the same assumptions are made regarding diagnostic sensitivity and diagnostic specificity, but the prevalence is changed to 20% as might be expected in trichomoniasis infected herds, then the positive predictive value is 0.925 and the negative predictive value is 0.995. in other words, the level of confidence in a positive test is lower in low prevalence situations, but much higher in high prevalence situations. in both scenarios the confidence in a negative test was high. these examples highlight the need for carefully interpreting trichomoniasis test results, regardless of the purported diagnostic sensitivity and diagnostic specificity. conclusion eradication of t. foetus from the us cattle population has proven elusive for a variety of reasons with consistent, accurate identification of infected animals as 1 of these contributing factors. although the process of diagnosing trichomoniasis may be imperfect, paying close attention to details in the steps of diagnostic process reduces preanalytical, analytical, and postanalytical errors and ultimately will enhance the prospects for control and perhaps eradication of this disease in the future. conflict of interest none to report. references 1. emmerson ma: trichomoniasis in cattle. j am vet med assoc 1932;81:636-640. 2. plebani m: the detection and prevention of errors in laboratory medicine. ann clin biochem 2010;47:101-110. 3. hammond dm, bartlett de: the distribution of trichomonas foetus in the preputial cavity of infected bulls. am j vet res 1943;4:143-149. 4. rhyan jc, willson kl, wagner b, et al: demonstration of tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally infected bulls. vet pathol 1999;36:406-411. 5. ondrak jd: tritrichomonas foetus prevention and control in cattle. vet clin north am food anim pract 2016;32:411-423. 6. sutka p, katai pl: rapid demonstration of bull trichomonadosis in unstained smear preparations from preputial scrapings. acta vet hung 1969;19:385-389. 7. tedesco lf, errico f, del baglivi lp: diagnosis of tritrichomonas foetus infection in bulls using two sampling methods and a transport medium. aust vet j 1979;55:322-324. 8. parker s, campbell j, ribble c, et al: comparison of two sampling tools for diagnosis of tritrichomonas foetus in bulls and clinical interpretation of culture results. j am vet med assoc 1999;215:231-235. 9. dewell ga, phillips pe, dohlman tm, et al: validation of a gauze sponge sampling methodology to detect tritrichomonas foetus by realtime pcr. j vet diagn invest 2016;28:595-598. 10. frey cf, müller n, stäuber n, et al: simplicimonas-like dna in vaginal swabs of cows and heifers cross-reacting in the real-time pcr for t. foetus. vet parasitol 2017;237:30-36. 11. schommer s, younger s, fales wh: cross-reaction of simplicimonas spp. trichomonads in tritrichomonas foetus assays. proc annu conf am assoc vet lab diagn 2011; p. 34. 12. clothier ka, villanueva m, torain a, et al: effects of bacterial contamination of media on the diagnosis of tritrichomonas foetus by culture and real-time pcr. vet parasitol 2015;208:143-149. 13. clothier ka, mcnabb b, torain a, et al: effects of biological materials and collection media on pcr detection of tritrichomonas foetus. open j anim sci 2019;9:121-128. 14. davidson jm, ondrak jd, anderson aa, et al: evaluation of effects of high incubation temperature on results of protozoal culture and realtime pcr testing for tritrichomonas foetus inoculated in a commercially available self-contained culture media system. j am vet med assoc 2011;239:1589-1593. 15. clavijo a, erol e, sneed l, et al: the influence of temperature and simulated transport conditions of diagnostic samples on real-time polymerase chain reaction for the detection of tritrichomonas foetus dna. j vet diagn invest 2011;23:982-985. 16. felleisen rsj: comparative sequence analysis of 5.8s rrna genes and internal transcribed spacer (its) regions of trichomonadid protozoa. parasitology 1997;115:111-119. clinical theriogenology 2021; 13: 233 17. cobo er, favetto ph, lane vm, et al: sensitivity and specificity of culture and pcr of smegma samples of bulls experimentally infected with tritrichomonas foetus. theriogenology 2007;68:853-860. 18. mukjufhi n, irons pc, michel a, et al: evaluation of a pcr test for the diagnosis of tritrichomonas foetus infection in bulls: effects of sample collection method, storage and transport medium on the test, theriogenology 2003;60:1269-1278. 19. ondrak, jd, keen je, rupp gp, et al: repeated sampling and testing by culture and pcr to detect tritrichomonas foetus carrier bulls in an infected nebraska herd. j am vet med assoc 2010;237:1068-1073. 20. saah aj, hoover dr: “sensitivity” and “specificity” reconsidered: the meaning of these terms in analytical and diagnostic settings. ann intern med 1997;126:91-94. 21. mutto aa, giambiaggi s, angel so: pcr detection of tritrichomonas foetus in preputial bull fluid without prior dna isolation. vet parasitol 2006;136:357-361. clinical theriogenology 2021; 13: 234 diagnosis of bovine trichomoniasis clinical theriogenology 2022; 14: 102 uroperitoneum in a preparturient mare giuliana gianino,a jamie kopper,b jarrod troy,b kevin kersh,b alanna farrell,b eleas wu,a michael yaeger,c paul merkatoris,a* katheryn johnson,b theresa beachlera aveterinary diagnostic and production animal medicine bveterinary clinical sciences, cveterinary pathology college of veterinary medicine, iowa state university, ames, ia *current address: department of surgical sciences, school of veterinary medicine university of wisconsin-madison, madison, wi abstract a 12-year pregnant thoroughbred mare at ~ 340 days of pregnancy was presented for evaluation of lethargy, anorexia, and colic signs. reproductive examination was unremarkable; however, uroperitoneum was diagnosed via transabdominal ultrasonography, serum biochemistry, and peritoneal fluid analysis. mare was medically stabilized for 24 hours. to promote fetal maturation, 80 mg dexamethasone was given prior to caesarean section and exploratory celiotomy. a ventral urinary bladder wall rupture and multiple descending colon mesenteric rents were identified and repaired. foal was delivered alive, treated for neonatal maladjustment syndrome, and discharged after 14 days. seven days after surgery, severe complications of the mare led to its abrupt decline in health and sudden death. keywords: mare, uroperitoneum, bladder rupture, caesarean section, dexamethasone background uroperitoneum is an insidious and potentially fatal disease process in horses.1,2 although most commonly diagnosed in foals,1,3 uroperitoneum secondary to bladder rupture is uncommon in adult horses and reportedly associated with parturition,2,4-7 urolithiasis,8,9 or trauma.10 clinical signs typically present from 48 to 72 hours after rupture and consist of lethargy, anorexia, variable urination (anuria, pollakiuria, or stranguria), tachycardia, tachypnea, abdominal distension, or cardiac arrest.3 hematologic findings often include azotemia, hyperkalemia, hyponatremia, hypochloremia, and metabolic acidosis.3 this report describes uroperitoneum secondary to urinary bladder rupture in a preparturient mare, that to the authors’ knowledge, has not previously been reported. this case underscores the importance of including uroperitoneum after urinary bladder rupture as a differential for late-term pregnant mares and highlights the possibility for safe use of maternal dexamethasone treatment after 335 days of pregnancy to hasten fetal maturity prior to caesarean section. case presentation a multiparous 12-year thoroughbred mare ~ at 340 days of pregnancy was referred to iowa state university’s lloyd veterinary medical center for evaluation of a 24-hours history of lethargy, decreased appetite, and persistent colic. at presentation, mare exhibited muscle fasciculations and weight shifting. she was tachycardic (72 beats/minute), tachypneic (48 breaths/minute), and afe brile with a normal rectal temperature (37.9°c). there was marked mammary gland development, with a slightly sticky, opaque mammary secretion. remainder of the physical examination was unremarkable. a complete blood count identified an elevated hematocrit (51.7%; reference range [rr]: 34 45%), and a mild leukocytosis (12.41 × 103/µl; rr: 5.0 11.0 × 103/µl) characterized by moderate mature neutrophilia (10.9 × 103/µl; rr: 2.1 6.7 × 103/µl). serum biochemistry included marked azotemia (bun 47 mg/dl and creatinine 9.8 mg/dl; rr: 14 21 mg/dl and 1-2.1 mg/dl, respectively), elevated muscle enzymes (ck 1198 iu/l and ast 680 iu/l; rr: 74 426 iu/l and 100 465 iu/l, respectively), hyponatremia (135 meq/l; rr: 137 145 meq/l), hypochloremia (94.0 meq/l; rr: 102 114 meq/l), hyperglycemia (148 mg/dl; rr: 80 113 mg/dl), and hyperbilirubinemia (7.48 mg/dl; rr: 0.7 2.4 mg/dl). venous blood gas analysis had alkalemia (ph 7.458), decreased ionized calcium (1.36 mmol/l; rr: 1.4 1.8 mmol/l), mild hyponatremia (133.3 mmol/l; rr: 137 145 mmol/l), and hypochloremia (94 mmol/l; rr: 102 114 mmol/l). transcutaneous transabdominal ultrasonography of caudoventral abdomen revealed a single live fetus in anterior presentation, with a normal fetal heartrate (73 beats/minutes) and anechoic fetal fluids of subjectively normal volume. mare’s cranioventral abdomen contained ~ 10 15 cm of hypoechoic free peritoneal fluid. remaining ultrasonographic clinical theriogenology 2022; 14: 103 evaluation was unremarkable. neither placental separation nor edema was evident on transrectal ultrasonography. combined thickness of the uterus and placenta (0.99 cm) was within normal limits. mammary secretions contained 200 ppm calcium (stall-side calcium carbonate test [foalwatch™ titrets®, chemetrics inc., midland, va]). peritoneal abdominocentesis yielded ~ 10 ml of large volume of mildly serosanguinous fluid with increased bun (70 mg/dl), creatinine (30 mg/dl), and potassium (8.7 meq/l), and decreased sodium (128.0 meq/l) and chloride (92.0 meq/l). diagnosis was uroperitoneum secondary to suspected urinary bladder rupture. treatment mare mare was hospitalized (day 1) for stabilization prior to exploratory laparotomy and elective caesarean section. a foley urinary catheter (jorgensen labs, loveland, co) and 32-french right cranioventral abdominal drain (teleflex, morrisville, nc) were placed to facilitate urine drainage. intravenous fluid therapy (ringer’s lactate solution [lactated ringer’s injection usp, hospira inc., lake forest, il]) was initiated at a rate of 1.5 ml/ kg/hour. a constant rate infusion ([cri]; 0.04 mg/kg/minute) of lidocaine (vetone® lidocaine 2%, sparhawk laboratories inc., lenexa, ks) was also initiated. mare was given intravenously flunixin meglumine ([1.1 mg/kg every 12 hours]; vetone® prevail, mwi animal health, boise, id). to promote fetal maturation, 80 mg of dexamethasone (vetone® dexamethasone injection 2 mg/ml, bimeda-mtc animal health inc., cambridge, ontario, canada) was given intramuscularly to the mare. at 24 hours, transcutaneous transabdominal ultrasonography revealed the fetus to be tachycardic (130 beats/minute), consistent with fetal stress.11 general anesthesia was induced with intravenous ketamine ([3.8 mg/kg]; ketaset® ketamine hcl injection, zoetis inc., parsippany, nj) and intravenous propofol ([0.5 mg/kg]; propoflo™ propofol injectable emulsion, zoetis inc.) and was maintained on isoflurane inhalant (isosol isoflurane usp, vedco inc., st. joseph, mo). a live filly was delivered via caesarean section.12 a 5 cm ventral urinary bladder defect was identified and cystorrhaphy performed.13 two 8 10 cm and 2 (3 5 cm) descending colon mesenteric rents were identified and repaired. a 32-french abdominal drain was placed cranial to the ventral midline incision followed by routine ventral midline incisional closure. mild ataxia was noted during anesthetic recovery, and upon standing, mare became pyrexic (39.2°c), tachycardic, and tachypneic. repeat serum biochemistry revealed markedly elevated muscle enzymes (ck 64,894 iu/l and ast 1728 iu/l). urine was collected via urinary catheter, and pigmenturia observed. mare was treated with external cooling techniques and a single 0.1 mg/kg dose of intravenous morphine (morphine sulfate injection usp, hospira inc., lake forest, il). intravenous fluid therapy (ringer’s lactate [3 ml/kg/hour]) was initiated and lidocaine cri was reinitiated (0.04 mg/kg/minute). penicillin g procaine ([2.2 × 104 iu/kg every 12 hours]; vetone® penone pro™ injectable suspension usp, vedco inc.) was given intramuscularly and gentamicin ([6.6 mg/kg every 24 hours]; vetone® gentamicin sulfate solution, mwi animal health) was given intravenously. mare was also given intravenously 5 iu of oxytocin im (vetone® oxytocin injection, bimeda-mtc animal health inc.) every 2 hours to promote fetal membrane expulsion. overnight, the mare remained bright and alert, demonstrating good appetite and interest in her foal. daily abdominal lavage was performed through the indwelling abdominal drain. uterine lavage (~ 30 40 liters [5 10 liters per instillation]) of nonsterile 0.9% saline was performed, releasing a portion of the chorioallantois from the caudal uterus, and was repeated twice daily until resolution of the retained fetal membranes. intramuscular oxytocin treatment was increased to 10 iu (0.014 iu/kg every 2 hours). a small weight was affixed to the chorioallantois to provide passive traction. intravenous fluid therapy was decreased to 1.5 ml/kg/hour and cri of lidocaine was discontinued. at 30 hours postoperatively, mare displayed tachycardia (60 beats/minute) with diffuse muscle fasciculations. a nasogastric tube was passed with no net reflux and a cri of lidocaine (0.04 mg/kg/minute) was reinitiated; after treatment mare appeared more comfortable and passed adequate feces. on day 4 (48 hours postoperation), fetal membranes were still retained, and prophylactic ice boot therapy was initiated (cordura ice boots, jacks’ manufacturing, washington c.h., oh). by day 5, the mare appeared comfortable with normal vital parameters. intravenous fluid therapy and the cri of lidocaine were again discontinued. on day 6, fetal membranes were completely removed. approximately a 20 cm in diameter, roughly circular, soft, fluctuant mass acutely developed cranioventral and craniolateral to the left flank with an ultrasonographic appearance was consistent with acute subcutaneous hematoma and no evidence of abdominal herniation or body wall muscle rupture. abdominal drain was removed, and a hernia belt (cm equine heal belt, cm equine products, norco, ca) was placed over the caudal abdomen encompassing the mass. eighteen hours after initial observation, it was noted that the mass had increased in size cranially. intravenous fluid therapy with ringer’s lactate solution (3 ml/kg/ hour after 10 liters bolus) and a cri of lidocaine (0.04 mg/kg/ minute) were reinitiated. all other treatments continued as described. on day 8, the hematoma in the left flank had increased in size to encompass left stifle, and mare was markedly painful and reluctant to ambulate. that evening, mare became acutely painful, tachycardic, and displayed colic signs. butorphanol tartrate ([0.02 mg/kg]; torbugesic®, zoetis inc.) was given intravenously with no resolution of clinical signs. abdominocentesis yielded opaque, yellow peritoneal fluid with an elevated lactate (5.6 mmol/l). transcutaneous abdominal ultrasonography and transrectal palpation findings were unremarkable and nasogastric intubation yielded 4 liters of spontaneous reflux. colic signs continued and morphine (0.13 mg/kg) was given intramuscularly, to provide comfort throughout the night. foal filly was initially fed via indwelling nasogastric tube due to signs of mild neonatal maladjustment syndrome and was proclinical theriogenology 2022; 14: 104 phylactically treated intravenously with ceftiofur sodium ([5 mg/kg every 12 hours] naxcel® ceftiofur sodium sterile powder, zoetis inc.). foal was given 3 liters of equine plasma (immunoglo™ 1700 normal equine plasma, mg biologics inc., ames, ia). due to mare’s declining health, filly was separated and successfully grafted to a nurse mare. outcome mare on the morning of day 9 (7 days after surgery), mare became acutely painful, collapsed, and died following suspected cardiovascular collapse. necropsy revealed ~ 35 cm of small intestinal loops protruding from ventral midline incision with dark serous to hemorrhagic fluid drainage. serosal surfaces of all abdominal viscera were hyperemic and coated in fibrinous mats. subcutaneous tissue in the region of the left flank was markedly expanded by clear, glistening fluid. approximately 40 cm in diameter in the region of left lateral body wall contained muscle fibers that were extensively frayed and separated by hemorrhage and edema. caesarean section closure was intact, with minimal fetid fluid and fibrinous material within uterine lumen. cystorrhaphy site was intact. on histopathology, the left thigh subcutis contained severe hemorrhage and vasculitis, with occasional fibrin thrombi and degenerate neutrophilic/lymphocytic infiltrates with fewer macrophages in the surrounding tissue. foal filly remained systemically healthy throughout hospitalization, with no clinical evidence of pulmonary prematurity, and was discharged at 14 days of age. at the most recent communication with the filly’s owner, the filly was ~ 8 months old and reported healthy. discussion this report described a case of uroperitoneum secondary to bladder rupture in a late-term pregnant mare not associated with parturition. urinary bladder rupture in adult horses has been reported following trauma,10 obstructive urolithiasis,8,9 and parturition in mares.2,4-7 it is generally believed that the mechanism by which the urinary bladder in foaling mares ruptures during parturition is either acutely by impingement of a distended bladder between the rim of mare’s pelvis and foal during forceful abdominal contractions or several days after parturition by necrosis of the urinary bladder wall following an ischemic, traumatic injury sustained during foaling.4 to the authors’ knowledge, this is a first case report that described a ruptured urinary bladder in a late-term pregnant mare not associated with parturition and without history or evidence of trauma or lower urinary tract obstruction. uroperitoneum secondary to a bladder wall rupture was suspected after hematologic and peritoneal fluid analysis with confirmation at surgery. without history of external trauma, observation of external trauma clinical signs, or ante or postmortem evidence to support a urinary obstruction that may have led to chronic overdistension of the bladder in this patient, an explanation for the bladder rupture could be that impingement of the bladder wall between bony structures of the mare and foal combined with possible fetal movement caused it to tear acutely. location of the defect in this mare may further support this theory, as it was on the ventral aspect of the bladder that is commonly the location of bladder wall tears suspected to occur during foaling.2,5,6 it is possible that while the mare was displaying colic signs prior to presentation, some combination of abdominal straining and rolling produced an intraabdominal pressure comparable to that generated during parturition in foaling mares and that the bladder became compromised in a similar way. impingement leading to an acute tear of the bladder wall seems more likely than one resulting from ischemic necrosis in this case, as there was no evidence of devitalized bladder tissue noted during surgery or on necropsy 7 days later. this case also demonstrated the use of a single dose of dexamethasone at ~ 340 days of pregnancy to promote fetal maturity and survivability of a near-term foal. duration of pregnancy in thoroughbred mares ranged from 306 to 390 days with a mean length of 347 days.14 approximately 5 days prior to parturition in horses, maturation of the fetal hypothalamic-pituitary-adrenal axis and a marked rise in fetal cortisol concentrations are responsible for a final wave of maturation of multiple organ systems that prepare the foal for extrauterine life.15,16 maternal dexamethasone treatment has been used to mimic this rise in cortisol and hasten fetal maturity prior to induced parturition or caesarean section, to improve foal survivability in the postnatal period; however, reduced postnatal growth rate or adrenal suppression have sometimes been reported after corticosteroid use after 320 days of pregnancy.17,18 a typical protocol is to give 100 mg of dexamethasone intramuscularly to the mare once daily for 3 4 days after 320 days of pregnancy; however this is not always possible to achieve in cases where the mare’s or fetus’s clinical condition acutely worsens or warrants immediate delivery.17,18 because 340 days is within the normal range of pregnancy in thoroughbred mares, and without prior foaling history, it cannot be proven that the dexamethasone received by the mare in this case report contributed substantially to her filly’s physiologic maturity or postdelivery survival; however, no adverse effects on the foal’s short-term health were observed. apparently, maladaptive neonatal maladjustment syndrome diagnosed in this filly was not associated with maternal dexamethasone treatment, but rather due to the immaturity of the hypothalamic-pituitary-adrenal axis and hypoxemic stress reported in premature foals and those delivered via caesarean section, respectively.19-21 thus, a single intramuscular injection of dexamethasone at a dose of 100 mg between 335 to 340 days of pregnancy may hasten fetal maturity and improve postnatal survivability; however, further study is needed to validate this protocol. in summary, this case demonstrated the importance of including bladder rupture as a differential diagnosis for late-term, preparturient pregnant mares presenting with a history of colic signs or abdominal straining and that a single intramuscular injection of dexamethasone may be considered to safely hasten fetal maturity prior to caesarean section. clinical theriogenology 2022; 14: 105 learning points • bladder ruptures occur in late-term pregnant mares, even prior to parturition. • behavioral colic in late-term pregnant mares may predispose to acute bladder injury via increased abdominal pressure and bony impingement of the bladder wall. • dexamethasone (80 mg) given intramuscularly12 hours prior to caesarean section between 335 340 days of pregnancy had no adverse effects on the foal. conflict of interest authors have no conflicts of interest to declare. acknowledgement authors thank the technical and support staff of the iowa state university lloyd veterinary medical center’s equine intensive care unit for their dedication and assistance during the management of this case. references 1. richardson dw, kohn cw: uroperitoneum in the foal. j am vet med assoc 1983;182:267-271. 2. rodgerson dh, spirito ma, thorpe pe, et al: standing surgical repair of cystorrhexis in two mares. vet surg 1999;28:113-116. 3. vaala we: manifestations and management of disease in foals: distended and/or painful abdomen, specific conditions. in: smith bp: editor. large animal internal medicine. 5th edition, st. louis; elsevier-mosby: 2015. p. 275-276. 4. nyrop ka, debowes rm, cox jh, et al: rupture of the urinary bladder in two postparturient mares. comp cont educ pract vet 1984;6:510-513. 5. jones pa, sertich ps, johnston jk: uroperitoneum associated with ruptured urinarybladder in a postpartum mare. aust vet j 1996;74:354-358. 6. higuchi t, nanao y, hiroyuki s: repair of urinary bladder rupture through a urethrotomy and urethral sphincterotomy in four postpartum mares. vet surg 2002;31:344-348. 7. stephen jo, harty ms, hollis ar, et al: a non-invasive technique of standing surgical repair of urinary bladder rupture in a post-partum mare: a case report. ir vet j 2009;62:734-736. 8. mccue pm, brooks pa, wilson wd: urinary bladder rupture as a sequela to obstructive urethral calculi. vet med 1989;84:912-914. 9. gibson kt, trotter gw, gustafson sb: conservative management of uroperitoneum in a gelding. j am vet med assoc 1992;200:16921694. 10. beck c, dart aj, mcclintock sa, et al: traumatic rupture of the urinary bladder in a horse. aust vet j 1996;73:154-155. 11. reef vb, vaala we, worth lt, et al: transabdominal ultrasonographic evaluation of the fetus and intrauterine environment in healthy mares during late gestation. vet radiol ultrasound 1995;36:533-541. 12. woodie jb: uterus and ovaries. in: auer ja, stick ja, kummerle jm, et al: editors. equine surgery. 5th edition, st. louis; elsevier: 2019. p. 1084-1086. 13. woodie jb: bladder – surgical procedures. in: auer ja, stick ja, kummerle jm, et al:editors. equine surgery. 5th edition, st. louis; elsevier: 2019. p. 1136-1138. 14. ewert m, lüders i, böröcz j, et al: determinants of gestation length in thoroughbred mares on german stud farms. anim reprod sci 2018;191:22-23. 15. rossdale pd, silver m, comline rs, et al: plasma cortisol in the foal during the late fetal and early neonatal period. res vet sci 1973;15:395-397. 16. fowden al, forhead aj, ousey jc: endocrine adaptations in the foal over the perinatal period. equine vet j 2012;44:130-139. 17. ousey jc, kölling m, kindahl h, et al: maternal dexamethasone treatment in lat gestation induces precocious fetal maturation and delivery in healthy thoroughbred mares. equine vet j 2011;43:424429. 18. rossdale pd, jeffcott lb: problems encountered during induced foaling in pony mares. vet rec 1975;97:371-372. 19. hess-dudan f, rossdale pd: neonatal maladjustment syndrome and other neurological signs in the newborn foal: part 1. equine vet educ 1996;8:24‐32. 20, hess-dudan f, rossdale pd: neonatal maladjustment syndrome and other neurologicalsigns in the newborn foal: part 2. equine vet educ 1996;8:279-283. 21. diesch tj, mellor dj: birth transitions: pathophysiology, the onset of consciousness and possible implications for neonatal maladjustment syndrome in the foal. equine vet j 2013;45:656-660. over 200 genetic mutations (variations, deletions, and insertions) have now been associated with development of a clinical disease in dogs and cats and an additional 100 or so companion animal diseases have been identified without a mutation yet identified. as veterinarians have become better at understanding and preventing nutritional and infectious diseases, disease prevalence has decreased; however, we are faced with several familial disease, particularly, but not limited to, the purebred dog or cat. several diagnostic laboratories now offer genetic tests for several canine and feline diseases. samples may be submitted in a variety of forms including buccal swabs, blood samples in edta and semen straws, and samples can be submitted with or without the involvement of a veterinarian. however, the role of a veterinarian is critical in interpretation of genetic results. although submission and running of genetic tests is not complicated, interpretation of the results and how it may impact an individual animal or a breeding program can be quite complicated and requires the medical knowledge of a veterinarian to help an owner understand the many complicated aspects of genetics, including variations in penetrance and expression. without careful counseling and discussion with a trained veterinary professional, a pet owner or breeder could lose enthusiasm and trust in genetic testing and we could lose this important tool. once genetic testing becomes available for a disease, counseling a pet owner or breeder on how to apply test results is very important. a breed association may be quick to immediately remove animals with a mutation from the breeding pool. although this approach may appear as a good idea, it can have substantial negative impact on the breed. if the mutant gene exists in 40% of a cat or dog population, a sudden reduction role of genetic testing for diseases of companion animals and genetic counseling in modern veterinary medicine kathryn meurs department of clinical sciences, college of veterinary medicine north carolina state university raleigh, nc abstract several hundred genetic mutations have now been associated with development of clinical diseases in dogs and cats. many diagnostic testing laboratories now offer genetic tests and samples may be easily submitted. however, interpretation of the results and how it may impact an individual animal or a breeding program can be quite complicated. regardless, with their veterinary medical knowledge, the veterinarian has a critical role in interpreting genetic results. keywords: genetics, mutation, inherited, breeding in 40% of the gene pool could dramatically alter the breed. additionally, due to complicated genetic phenomenons noted in many genetic diseases, not all animals that have the mutation will develop a clinical form of the disease. these concepts need to be carefully weighed against the issues of continuing to produce animals that may carry a mutation and could potentially suffer from the disease. for the breeding animal, recommendations to continue breeding should be based on many factors including size of the breed association, type of disease, risk that the animals produced will develop disease from the mutation, and severity of the disease. positive attributes of the animal should be considered. besides the development of healthier animals through breeding practices, genetics are a key way for veterinarians to become more proactively involved in patient care. ability to develop a proactive management plan for animals that test positive for a disease mutation is the true benefit of clinical genetics. although identification of mutations can be used for testing and developing a breeding program, it is unlikely that we can ever completely remove many of these variants from the population. larger gains will be in the value of testing young animals for these mutations and developing clinical management plans that can be applied for the lifespan of that pet to provide the best possible care. veterinary medicine needs to move away from being ‘reactive-responding’ once a disease has been diagnosed, to become as proactive as possible, identify animals at risk of a disease, and implement management plans to prevent or alter outcomes. role 1 © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9599, http://dx.doi.org/10.58292/ct.v15.9599 foreword welcome to this special issue of clinical theriogenology centered around the topic of educational research. great thanks to dr. augustine peter and to this journal for supporting this work and to those colleagues who provided manuscripts. in this special issue, you will see a spectrum of types of educational research publications including a review and archiving of educational research specific to theriogenology, prospective studies, retrospective studies, and descriptions of best practices. a recent review of educational research in all veterinary disciplines demonstrated great variability between schools in how much educational research was generated. we know from the literature and from anecdotes that this is due to variability in how educational research is accepted as valid research and how much time, training, and other support are provided to permit faculty, residents, and staff to do this work. it is our hope that by providing this body of information, we can encourage each other to look at our teaching with an eye for how we can monitor changes and better share that information with each other. annett annandale henry annandale margaret root kustritz guest editors http://dx.doi.org/10.58292/ct.v15.9599 17 contact annett annandale, annett.annandale@murdoch.edu.au © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9591, http://dx.doi.org/10.58292/ct.v15.9591 review article a variety of teaching interventions to improve bovine transrectal palpation training annett annandale,a,b dietmar holma afaculty of veterinary science, university of pretoria, pretoria, south africa bschool of veterinary medicine, murdoch university, perth, australia abstract pregnancy diagnosis by transrectal palpation (trp) is a frequently performed procedure in bovine practice and an important competency for veterinary graduates. student training for these skills has been identified as challenging, and reported training outcomes highlighted that students’ pregnancy diagnosis accuracy was an area that needed improvement. this led to a series of investigations evaluating specific teaching approaches aimed at optimizing trp training and improving learning outcomes while decreasing the number of palpations needed to become competent, or in other words to ‘fast-track’ the transrectal palpation skill learning process. gathered data were used to develop and implement research-based innovative teaching ideas, including optimized simulator training, in-training assessment methods, additional training opportunities linked to or not directly linked to the trp skill itself, and the use of technology to improve skills training. this paper is a summary of these teaching interventions and elaborates on the background of those and why implementation is expected to improve student competence. furthermore, even though the investigations described here are specific to bovine trp and pregnancy diagnosis skills, they highlight an approach that can be applied to other clinical hands-on skills. keywords: transrectal palpation, bovine pregnancy diagnosis, competency, clinical skills training introduction bovine pregnancy diagnosis (pd) via transrectal palpation (trp) is a frequently performed procedure in bovine practice,1 of economic importance,2–11 and an important competency for veterinary graduates.12 training of bovine trp and pd is challenging, however, as it requires extensive exposure to trp in live cows to achieve competency.13–16 veterinarians often refer to the anecdotal ‘magic 10,000,’ that is the number of rectal palpations assumed to be necessary to ensure palpation competence. the origin of this number is unknown but indicates the perceived amount of practice necessary to confidently perform bovine pds. it is possible that it is a link to ericsson’s concept of deliberate practice where a practice time of at least 10,000 hours is stipulated to lead to expert status in a specific skill.17 the australian veterinary association also previously recommended a minimum of 2,000 live cow palpations before attempting the competency examination.18,19 some publications evaluated students’ trp and pd performance after training and how many palpations are required to ensure competence,13–15,20 and it was suggested that 200 live cow palpations were insufficient to establish trp competence for all 10 students in the evaluated group.13 compared to many other skills, trp and pd training is further complicated by the nature of the procedure; demonstration of the technique by an instructor and observation of a student palpating a live cow are not possible as execution of the procedure cannot be seen.13,16,19,21 in addition to these challenges, veterinary schools are expected to graduate entry-level veterinarians with advanced skills,22,23 while welfare and ethical concerns about the use of live animals for training purposes, large student cohorts, budget constraints, and difficulty sourcing cadaver material limit hands-on experience and training opportunities in general,22,24–35 thus influence pd training for veterinary students at teaching institutions due to a shortage of palpation opportunities.13–16,21 furthermore, it seems difficult for students to get additional bovine pd and trp exposure outside the veterinary course.15,36 described training strategies to overcome some of the challenges listed above are exposure to abattoir-derived organs followed by live cow palpations,19,36 the use of cows at abattoirs prior to slaughter,15 student exposure to bovine trp and pd during extramural studies,16,21 or ambulatory clinic rotations,15,21 and the use of a computer-assisted learning tool.19 furthermore, a variety of rectal examination simulators such as the veterinary simulator industries’ bovine mailto:annett.annandale@murdoch.edu.au http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9591 18 citation line: clinical theriogenology 2023, 15, 9591, http://dx.doi.org/10.58292/ct.v15.9591 theriogenology model,a minitube’s ’henryetta’ model,b the breed’n betsy® rectal examination simulators,c and the haptic cowd are commercially available, and several studies evaluated the use and implementation of rectal examination simulators in veterinary training programs.13,16,36–40 while training of bovine trp and pd has been identified as challenging,13–16,19 only limited data were available on student performance in bovine trp and pd.13,14 recent investigations into trp and pd training outcomes highlighted that students’ pd accuracy was an area that needed improvement.36,41,42 this led to a series of projects evaluating specific teaching approaches aimed at optimizing trp training and improving learning outcomes while decreasing the number of palpations needed to become competent, or in other words to ‘fast-track’ the trp skill learning process.43 gathered data were used to develop and implement research-based innovative teaching ideas, including optimized simulator training, in-training assessment methods, additional training opportunities linked to or not directly linked to the trp skill itself, and the use of technology to improve skills training. recommended evidence-based teaching approaches the teaching approaches are listed below. this paper elaborates on the background of those and why implementation is expected to improve student competence for bovine trp and pd. for the context of this paper, sensitivity of pd is defined as students’ ability to correctly identify pregnant cows, whereas specificity is defined as students’ ability to correctly identify non-pregnant cows. in summary, we recommend: 1. repeated rectal examination simulator-based training in conjunction with live cow exposure early in the curriculum. 2. teaching efforts focusing on specificity of pd. 3. teaching efforts focusing on stage corrected sensitivity and early pregnancy stages. 4. encouraging students to use an exercise program like the ‘bovine pregnancy diagnosis improvement exercise program’ (http://icarus.up.ac.za/vetmlp/) to improve grip strength and indirectly palpation abilities. 5. use of the ‘mini-cow palpation box’ to develop coordination and fine motor skills to estimate object sizes without visualization. 6. introduction of a trp objective structured clinical examination (osce) (on simulator and/or non-pregnant live cows, depending on availability) as a compulsory formative in-training assessment (‘rite of passage’ examination) before advancing to pd training on live cows. 7. use of the validated trp osce on non-pregnant live cows as well as a validated pd assessment on live cows to ensure training outcomes and track student progress during advanced trp and pd training sessions. a. veterinary simulator industries, calgary, canada (https://vetsimulators. com/). b. minitube tiefenbach, germany (https://www.minitube.com/). c. breed’n betsy® victoria, australia (http://www.breednbetsy.com.au/). d. virtalis ltd, cheshire, united kingdom (https://www.virtalis.com/ haptic-cow/). 8. implementing focused access to live cow palpations for students with an interest in production animal practice during later stages of the curriculum. 9. ensuring supervision and quality feedback during palpation sessions. repeated rectal examination simulator-based training in conjunction with live cow exposure early in the curriculum the first recommendation is based on a validation study comparing rectal examination simulator versus live animal pd training.36 it revealed that students’ ability to detect pregnancy in cows > 6 months pregnant was similar for training on simulators and live cows. however, pd training on simulators, compared to training on live cows, was associated with lower student pd sensitivity for cows < 6 months pregnant. this was an interesting outcome since the breed’n betsy® simulators are specifically designed for early pregnancy detection and also because the live cows used for training purposes in that study were all > 6 months pregnant.36 a potential reason for this paradox was thought to be that overcoming the unusual experience of trp may be more difficult than the actual ability to palpate.36 absence of rectal peristalsis, anal sphincter tone, and other internal organs (e.g., urinary bladder and rumen) make reproductive tract palpation easier on a simulator than in the live animal, and the simulator experience may not impart the feeling of palpating a live cow. also, simulator palpations were suspected to require various arm movements and muscle activations than the same procedure on live animals. while a follow-up investigation to elaborate on this hypothesis concluded that there is no difference in muscle activation for simulator and live cow palpations, it revealed that muscle relaxation between individual trp steps for simulator palpations was more distinct for simulator compared to live cow palpations. this was thought to be due to the absence of rectal peristalsis in simulators, and a potential reason for a difference in simulator and live cow training outcome. while it was demonstrated that rectal examination simulators cannot replace live animal training, implementation of simulators into veterinary teaching programs offers remarkable additional training opportunities beyond live cow training that can help reduce the requirements of live animal training. these findings were in accordance with other studies evaluating the use and implementation of rectal examination simulators in veterinary training programs.13,16,37–40 in the case of trp and pd, simulator training was superior to theoretical instruction only,39 and additional simulator training substantially improved students’ performance after live cow palpations.16 therefore, repeated simulator-based training in conjunction with live cow exposure is recommended to optimize learning outcomes.13,16,36,39 another finding related to the use of simulators is that timing of simulator training in relation to student experience within the veterinary course is important.20,36 the proposal of simulator training early in the curriculum stems from student feedback where 4th year students (of a 6 year course) without any prior live cow pd experience were exclusively positive about their simulator experience, whereas students with previous live cow pd experience made negative remarks about the simulator exposure.36 this finding was endorsed by final-year student questionnaire feedback showing that refresher rectal examination simulator training at these later stages in a student’s career was not perceived as beneficial by the majority of students.20 it has previously been described that satisfaction data should be considered, as student perceptions of a model will affect their motivation to use it.44,45 since students felt the model was useful at a specific http://dx.doi.org/10.58292/ct.v15.9591 http://icarus.up.ac.za/vetmlp/ https://vetsimulators.com/ https://vetsimulators.com/ https://www.minitube.com/ http://www.breednbetsy.com.au/ https://www.virtalis.com/haptic-cow/ https://www.virtalis.com/haptic-cow/ citation line: clinical theriogenology 2023, 15, 9591, http://dx.doi.org/10.58292/ct.v15.9591 19 point in their veterinary education, they will likely be motivated to practice on it.44 enjoying a procedure enhances positive emotions that have been reported to facilitate successful learning.46 therefore, rectal examination simulator training should be implemented early in the curriculum before but in conjunction with live cow exposure. teaching efforts focusing on specificity of pd pd specificity is defined as the students’ ability to correctly identify non-pregnant cows. correctly and reliably identifying non-pregnant cows is especially important for farmers due to the economic implications of a false positive pd. while students are not expected to be at the same competence level as experienced practitioners, a specificity of 41 and 42% reported for 4th and 5th year students, respectively, had been identified as a problem area,36,41,42 and teaching efforts focusing on specificity of pd are recommended. strategies to accommodate this part of the training are repeated simulator training exposure before live cow access, assessment of basic palpation skills before advancing to live cows, and demonstrating competence in non-pregnant cow palpations before advancing to pds. this approach increased student specificity to 67%.20 teaching efforts focusing on stage corrected sensitivity and early pregnancy stages the stage of pregnancy is one of the factors associated with students’ bovine pd accuracy. while overall pd sensitivity can be defined as correctly identifying pregnancy, stage corrected sensitivity takes the pregnancy stage into consideration. this has been defined as correctly identifying the stage of pregnancy within 1 month of the actual stage of pregnancy for all pregnancy stages up to 4 months, and within 2 months of the actual stage of pregnancy for pregnancy stages beyond 4 months. while the mean overall reported student pd sensitivity was 61 and 82%, stage corrected sensitivity was only 31 and 43%, respectively (in the same cohort of students).36,42 furthermore, students’ sensitivity of pregnancy detection was repeatedly higher for cows more than 6 months pregnant.36,42 this confirms the need to concentrate training efforts on palpations during early pregnancy stages. encouraging students to use an exercise program like the ‘bovine pregnancy diagnosis improvement exercise program’ to improve grip strength and indirectly palpation abilities the fairly unusual and tiring physical activity of bovine trp sparked an idea for a novel approach to improve students’ trp and pd skills. the effect of a physiotherapy exercise program on students’ trp and pd skills was evaluated.42 the 2 main findings were that students who participated in the exercise program and students who had a grip strength of more than 30 kg performed better in pds. results from this investigation initiated a subsequent electromyography (emg) study taking these findings a step further and led to the development of the ‘bovine pd improvement exercise program.’47 the emg study suggested that hand muscles, extensors and flexors of the forearms, shoulder stabilizers, back muscles, and core muscles are strongly activated during trps, confirming the role that forearm muscles, and therefore grip strength, play during trp.42,47 it was also apparent that muscle endurance is more important than total muscle strength. based on these findings, a 3-month exercise program targeting exactly those muscle groups was developed with the help of an experienced biokineticist.47 the program is divided into 3 levels starting with easier entry level exercises and building up to more advanced exercises. it is available to students via the following link: http://icarus. up.ac.za/vetmlp/. the program uses readily available exercise equipment and is suitable to any exercise level, and instructions are given through narrated video clips (figure 1). this online program enables students to choose when and where they would like to exercise and only requires the participant to exercise for 30 minutes, 3 times a week. it can be done in conjunction with any other fitness or exercise activity. the program not only increases grip strength and therefore indirectly improves trp accuracy but also increases stamina and wellbeing, shapes arms nicely, and adds some fun to busy study schedules. part of implementing this intervention should be student awareness of the critical threshold for grip strength,42 and what their own grip strength is at the onset of trp and pd training. if this is done early enough, it might increase student motivation and drive to execute an exercise program as described here to improve grip strength. grip strength measurement is easily done using inexpensive commercially available dynamometers.42 use of the ‘mini-cow palpation box’ to develop coordination and fine motor skills to estimate object sizes without visualization results of a recent study41 suggested that bovine trp osce scores can predict students’ future pd accuracy, and students’ ability to estimate ovarian size was positively correlated to pd sensitivity. based on these findings, a ‘mini cow palpation box’, similar to a palpation box described,14 was developed to supplement trp training opportunities prior to students’ first live cow trps.41 it aims to develop coordination and fine motor skills in order to estimate object sizes without visualization. three-dimensional objects of varying sizes from 2 to 8 cm are labeled with the correct length, width, and height measurements. the objects are placed in a large plastic box with hand entrance holes to ensure palpation and size estimation of objects without visualization (figure 2). a ruler to measure finger width and instructions on how to use the palpation box are attached to the box. this additional teaching tool helps improve students’ fine motor, trp, and pd skills before their first live cow palpations and assists students to prepare for formative in training assessments as described earlier. introduction of a trp osce (on simulator and/or non-pregnant live cows, depending on availability) as a compulsory formative in-training assessment (‘rite of passage’ examination) before advancing to pd training on live cows it became apparent that students’ ability to estimate ovarian size and to confirm the presence or absence of a corpus luteum was correlated with higher pd sensitivity,41 whereas students’ ability to confirm the presence or absence of a follicle was positively correlated with higher pd specificity.20 based on these results and the recommended approach to introduce a trp osce as a formative assessment for ongoing feedback throughout the trp training,41 a compulsory in-training http://dx.doi.org/10.58292/ct.v15.9591 http://icarus.up.ac.za/vetmlp/ http://icarus.up.ac.za/vetmlp/ 20 citation line: clinical theriogenology 2023, 15, 9591, http://dx.doi.org/10.58292/ct.v15.9591 simulator osce assessment was implemented within the veterinary reproduction module at the faculty of veterinary science, university of pretoria, south africa. during this assessment, students must palpate 3 breed’n betsy® simulator cows and fill in an osce sheet to indicate size of ovaries and the presence or absence of ovarian structures. students can choose when they want to do the assessment and repeat the assessment as many times as necessary to pass. passing this ‘rite of passage’ assessment is a requirement before students are allowed to advance to live cow palpations. the aim is to verify that students have demonstrated basic palpation skills before live cow access is granted to ensure more efficient live cow use. if rectal examination simulators are not available, a modified assessment can be implemented where students still must describe object sizes and identify structures resembling follicles or corpora lutea in a simulated setting. use of the validated trp osce on non-pregnant live cows as well as a validated pd assessment on live cows to ensure training outcomes and track student progress during advanced trp and pd training sessions in order to evaluate training success, training outcomes have  to be reliably measured. previous studies describing a b c d e f figure 1*. screenshots of the ‘bovine pregnancy diagnosis improvement exercise program app’ as seen on smartphone accessed through: http://icarus.up.ac.za/vetmlp/. (a) homepage with the welcome video and all links of the exercise program. (b) link with the introduction video to the exercise equipment. (c) link to download the exercise program overview. (d) link to exercise level 1: videos demonstrating the exercises. (e) links to the different exercise levels give additional information and remind users to track their progress via grip strength measurements. (f) examples of 2 level 3 exercises. *source47 http://dx.doi.org/10.58292/ct.v15.9591 http://icarus.up.ac.za/vetmlp/ citation line: clinical theriogenology 2023, 15, 9591, http://dx.doi.org/10.58292/ct.v15.9591 21 posttraining student performance in bovine trp and pd involved small student cohorts, and trp skills evaluation was based on various assessment criteria for each study.13–15 to make outcome measurements more objective and repeatable, 2 trp assessment methods were developed. the first described assessment measures of students’ pd accuracy on live cows.36 the set-up and execution was subsequently refined and optimized.20,41,42 the second assessment is a trp osce that had the ability to predict students’ future pd accuracy.41 it was also tested for validity and reliability.41 results suggested that students categorized to have competent palpation skills based on the overall score achieved in the osce were more likely to correctly identify non-pregnant cows (i.e., have better pd specificity), whereas students’ ability to estimate ovarian size was positively correlated to pd sensitivity. other individual osce components that were predictive of higher pd accuracy were students’ ability to identify uterine position and diagnose absence of intrauterine fluid correctly. these assessments can be implemented to ensure training outcomes and track student progress during advanced trp and pd training sessions. furthermore, using a combination of these 2 methods to evaluate students’ trp and pd skills (a trp osce on non-pregnant cows followed by a structured assessment of pd accuracy in a real-life farm situation) can bridge the gap between practical competence and clinical performance of the same skill (trp skills in this case).48 this is important as osce assessments are conducted under simulated examination conditions, and they do not necessarily provide valid information on the candidate’s ability to perform the skill in real-life situations.48,49 implementation of focused access to live cow palpations for students with an interest in production animal practice during later stages of the curriculum usually only a small proportion of veterinary students are interested in food animal career.36,41,50 upbringing location, figure 2*. ‘mini cow palpation box’. (a) modified plastic box with hand entrance holes. (b) three-dimensional objects of varying sizes inside the box. all objects are labeled with the correct length, width, and height measurements in centimeters. (c) students using the palpation box. (d) student instruction/information sheet on the palpation box. *source41 http://dx.doi.org/10.58292/ct.v15.9591 22 citation line: clinical theriogenology 2023, 15, 9591, http://dx.doi.org/10.58292/ct.v15.9591 gender, and previous pet ownership influence the career choice of veterinary students,50–55 and 2 of the key demographics were positively correlated with a career in mixed or food animal practice: an upbringing on a small town or rural area and being male.51,52,54,55 work experience on a farm54 and other agricultural exposure were positively associated with a preference for food animal employment.50 an approach to improve pd accuracy for students truly interested in large animals is to create additional palpation opportunities and find practice placements for those students, which are ways to overcome the difficulty associated with large student cohorts. focusing on smaller groups of students who will be more likely to use the skills was investigated in a study looking at the effect of a 1-week high-intensity training program on final-year veterinary students’ pd accuracy.20 this approach was successful as students’ pd sensitivity and pd specificity were 88 and 67%, respectively. this was a notable improvement to 2 previous studies that had involved entire 4th and 5th year cohorts (table).36,42 particular important is the improvement in specificity that had previously been identified as a problem area. whereas students were previously only able to correctly identify 1 out of 3 non-pregnant cows,36,42 it increased to 2 out of 3.20 the fact that the intense 1-week training program was at a later stage in the students’ career when they would have been exposed to the initial trp and pd trainings during their 4th year, followed by additional trp exposure during clinic rotations and electives in their final year had an effective strategy on 2 distinct levels. first, more advanced veterinary students are expected to have more competent basic palpation skills, which makes intense training more effective. second, targeting students interested in food animal practice decreases the cohort size and helps overcome the difficulty to find sufficient training and assessment opportunities. ensuring supervision and quality feedback during palpation sessions private practice placements were part of a study evaluating an intense 1-week trp and pd training program.20 the number of live cow palpations by students during the practice placements varied from 20 to 600. fifty percent of students palpated <100 cows, and the other half palpated >100 cows. interestingly, there was no correlation between the number of cows palpated during the private practice placements within the training program and posttraining trp osce scores, or student pd sensitivity or pd specificity.20 this is in contrast to previous findings where there was a positive correlation between number of cows palpated and students’ trp skills.13 a possible explanation for this could be based on the concept of ‘deliberate practice,’17 which refers to ‘intense, repetitive performance, in a controlled setting, of intended cognitive or psychomotor skills within a focused domain; rigorous skills assessment to identify deficiencies and errors; specific, informative feedback on how to correct them; and ongoing practice, with progressive increases in level of difficulty, yielding gradual, continuous improvement in skills performance’.17,26,56 the effect of deliberate practice on expert status in many professional disciplines, including chess, athletics, music, and medicine, was made known,17,57 and its importance for veterinary education has been highlighted.26,58 therefore, it is likely that the quality of supervision and feedback provided by the veterinarian might be more important than the number of cows palpated to improve trp and pd skills. students who palpated >300 cows during private practice placements probably received less feedback and supervision from the veterinarian due to time pressure compared to students who palpated fewer cows.20 in support of this statement, student feedback highlighted the time and effort veterinarians took to teach them that was particularly evident among practice placements where fewer cows were palpated. this reasoning could be further substantiated by the fact that only previous trp experience with a veterinarian was identified as a student level variable associated with a higher student pd specificity compared to previous student trp experience without veterinary supervision,36 underlining the effect of expert supervision. this highlights the importance of feedback and supervision and is a strategy to decrease the number of live cow palpations necessary to achieve competency.43 this aspect should also be communicated when establishing relationships with private practitioners for student training. arrangements for sufficient supervision and feedback should be a vital part of private practice placements. conclusion this teaching tip demonstrates how training innovations can be combined with traditional training to optimally prepare students for limited live animal exposure. the aim was not to replace live animal training but to fast-track student skills as an effective approach to compensate for a shortage of live animal training opportunities. it demonstrates how researchbased innovative teaching ideas, assessment methods, and additional training opportunities linked to or not directly linked to the skill itself, and the use of technology can be implemented to improve skills training. it also highlights that collaborations with experts in other fields, like physiotherapists, biokineticists, and information technology staff in our case, can help advance new and alternative ideas. while the investigations described here are specific to bovine trp and pd skills, it is an approach that can be applied to many other clinical hands-on skills. conflict of interest authors have no conflicts of interest to report. table. overall student pd accuracy, sensitivity, and specificity for 4th, 5th, and final-year student cohorts year 201436 201641 201820 4th yearstudent cohort 5th yearstudent cohort final yearstudent cohort overall student pd accuracy (%) 71 61 82 sensitivity (%) 87 79 88 specificity (%) 41 42 67 http://dx.doi.org/10.58292/ct.v15.9591 citation line: clinical theriogenology 2023, 15, 9591, http://dx.doi.org/10.58292/ct.v15.9591 23 authors’ contribution annett annandale: conceptualization, writing, review, and editing. dietmar holm: conceptualization, review, and editing. references 1. morin de, constable pd, troutt hf, et al: individual animal medicine and animal production skills expected of entry-level veterinarians in bovine practice. j amer vet med assoc 2002;221:959–968. doi: 10.2460/javma.2002.221.959 2. romano je, fahning ml: effects of early pregnancy diagnosis by per rectal palpation of the amniotic sac on pregnancy loss in dairy cattle. j amer vet med assoc 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malone e, rendahl a: identification of student lifestyle characteristics associated with training choices to drive targeted admissions in veterinary medicine. educ health prof 2020;3:70–76. doi: 10.4103/ehp.ehp_2_20 56. george jh, doto fx: a simple five-step method for teaching clinical skills. fam med 2001;33:577–578. 57. issenberg sb, mcgaghie wc, petrusa er, et al: features and uses of high-fidelity medical simulations that lead to effective learning: a beme systematic review. med teach 2005;27:10–28. doi: 10.1080/01421590500046924 58. annandale a, scheepers e, fosgate gt: the effect of ovariohysterectomy model practice on surgical times for final-year veterinary students’ first live-animal ovariohysterectomies. j vet med educ 2020;47:44–55. doi: 10.3138/jvme.1217-181r1 http://dx.doi.org/10.58292/ct.v15.9591 https://doi.org/10.3138/jvme.32.4.468 https://doi.org/10.3138/jvme.32.4.468 https://doi.org/10.1501/vetfak_0000002804 https://doi.org/10.3138/jvme.35.4.595 https://doi.org/10.3138/jvme.1016-166r1 https://doi.org/10.3138/jvme.1016-166r1 https://doi.org/10.3138/jvme.32.1.79 https://doi.org/10.1097/sih.0b013e3181e369bf https://doi.org/10.1097/sih.0b013e3181e369bf https://doi.org/10.1136/vr.105022 https://doi.org/10.3138/jvme.2019-0043 https://dspace.library.uu.nl/handle/1874/397523 https://dspace.library.uu.nl/handle/1874/397523 https://doi.org/10.7205/milmed-d-13-00255 https://doi.org/10.3138/jvme.0815-138r1 https://doi.org/10.3138/jvme.0815-138r1 https://doi.org/10.3138/jvme.0611.068r1 https://doi.org/10.3138/jvme-2020-0039 https://doi.org/10.3138/jvme.33.4.578 https://doi.org/10.3138/jvme.36.1.7 https://doi.org/10.3138/jvme.32.4.491 https://doi.org/10.4103/ehp.ehp_2_20 https://doi.org/10.1080/01421590500046924 https://doi.org/10.3138/jvme.1217-181r1 introduction embryonic mortality and abortion are major causes of severe economic losses to the livestock industry.1,2 pregnancy loss in ruminants varies with geographic area and period. in only ~ 30% of midand late-term pregnancy losses in cattle and 57% in sheep, was a specific cause identified.3-5 despite the development of new diagnostic tools and the discovery of new infectious agents that cause abortion in ruminants diagnostic efficiency has not improved.6 infectious agents are reported as major causes of pregnancy loss. some infectious diseases that cause abortion in ruminants have zoonotic potential and are a public health concern.7 noninfectious causes of pregnancy loss, particularly during the embryonic period, may be as important as infectious causes; however, specific causes are rarely determined. although not strictly a cause of pregnancy loss, neonatal mortality during the first few days after birth may be an indicator of intrauterine disease, and thus, they should be considered among the causes of pregnancy loss.6,8 diagnosis of pregnancy loss in ruminants involves an investigative effort among owner, veterinary practitioner, and veterinary diagnostic laboratory. veterinary practitioners have an important role in educating farmers about the risks of zoonotic causes of pregnancy loss in ruminants andrés de la concha-bermejillo,a juan romanob atexas a&m veterinary medical diagnostic laboratory blarge animal clinical sciences, college of veterinary medicine & biomedical sciences texas a&m university, college station, tx abstract pregnancy loss in ruminants is a major economic loss to producers and highlights the importance of control measures to prevent its cause. although pregnancy loss may occur at any stage of pregnancy, early loss is usually unnoticed and samples are not submitted for diagnosis. pregnancy loss is detected by observations for return to estrus, transrectal palpation or ultrasonography, and blood tests for specific antigens. submissions to a laboratory often consist of 4to 5-month-old, or older, aborted fetuses and/or maternal serum. consequently, reports of pregnancy loss in ruminants are biased towards second and third trimester causes of pregnancy loss, because the chance of collecting a fetus or fetal membranes and submitting them to a laboratory improves as pregnancy advances. causes of pregnancy loss in ruminants are noninfectious and infectious. among the former are genetic defects, toxins, nutritional deficiencies, iatrogenic, drugs, and environmental causes. infectious causes include bacteria, fungi, viruses, and protozoa. a major concern of many infectious causes of pregnancy loss in ruminants is their zoonotic potential. a specific cause is identified in only 30 70% of late-term abortion samples submitted to a diagnostic laboratory. adequate specimen collection and proper handling are essential prerequisites for accurate test results. ideally, multiple concurrent tests are necessary for accurate diagnosis. main causes of pregnancy loss in ruminants are discussed. keywords: ruminants, abortion, diseases, pregnancy loss ruminant abortion, and on how to safely submit a fetus, fetal membranes, and maternal serum to the diagnostic laboratory.5 furthermore, veterinary diagnostic laboratories have an important role to identify the causes of pregnancy loss and in surveillance of infectious diseases that cause reproductive failure.9 adequate specimen collection and specimen handling are essential prerequisites for accurate test results. for any laboratory test procedure, the value of the test may be compromised by using specimens that have not been properly collected, labelled, handled or stored prior to testing.6,9 currently, because many of the infections causes of abortion in small ruminants are zoonotic, examination of small ruminant aborted fetuses and fetal membranes in some diagnostic laboratories are performed in a biosafety cabinet, and the use of n95 or a powered air purifying respirator is mandatory. in some us laboratories, the standard procedure when pursuing the cause of abortion in small ruminants requires testing for coxiella burnetii by reverse transcription polymerase chain reaction (rt-pcr) before proceeding with other testing. when rt-pcr results are positive for c. burnetti, no additional tests are done and all tissues are discarded following biosecurity guidelines. clinical theriogenology 2021; 13: 181 main causes of pregnancy loss in ruminants with an emphasis on infectious causes are discussed. transmission, pathology, and diagnostic methods of infectious causes of pregnancy loss in ruminants are highlighted. more extensive causes of pregnancy loss in cattle and small ruminants are reviewed in other papers included in these proceedings. sample submission diagnostic success of pregnancy loss in ruminants can be improved by promptly submitting suitable samples by overnight delivery service. when possible, the entire fetus and fetal membranes should be submitted chilled on ice using primary, secondary, and tertiary leak proof containers. the chorioallantoic membrane is most useful for establishing the cause of abortion because it often has microscopic lesions and in some cases the etiologic agent can be demonstrated by microscopic examination or by ancillary tests.11 if submission of the entire fetus is not possible, a field necropsy should be performed at a site that guarantees convenience, safety, and biosecurity.12 fetal brain, heart, lung, thymus, liver, spleen, kidney, adrenal gland, and skeletal muscle should be submitted to laboratory in 10% buffered formalin. a second set of similar tissues should be submitted chilled on ice in individual, labelled whirl-pak or ziploc bags for bacterial culture and molecular analyses.13 fetal blood from heart, fetal thoracic or abdominal fluids, and maternal serum should be submitted for identifying antibodies against important causes of infectious abortion.12,14 common causes of pregnancy loss in ruminants embryonic mortality and early pregnancy loss are usually unnoticed and often samples are not submitted. submissions usually consist of aborted fetuses of at least 4to 5-months of gestational age and/or maternal serum. consequently, reports of pregnancy loss in ruminants are biased towards second and third trimesters because the chances of collecting a fetus or fetal membranes and submitting them to diagnostic laboratory improves as pregnancy advances.15 although infectious agents are most frequently diagnosed, efforts to diagnose noninfectious causes of pregnancy loss are equally important.16,17 embryonic mortality embryonic mortality is a major cause of economic loss in ruminants; it reduces conception rates, thereby impacting production and profitability. causes of embryonic mortality in cattle are varied and include genetic, inbreeding, low progesterone concentrations, severe postpartum negative energy balance, and infectious diseases.18 methods for detection of early pregnancy loss include: observations for return to estrus, transrectal palpation, transrectal ultrasonography, and blood tests for specific antigens.19 noninfectious causes of pregnancy loss noninfectious causes of pregnancy loss include genetic, toxic, nutritional, medications, and environmental. fetal malformation and subsequent abortion, or the birth of abnormal offspring can be due to genetic abnormalities (e.g. arthrogryposis multiplex congenita20 and bovine arachnomelia syndrome21). a nonsense mutation in the apaf1 gene responsible for a lethal effect (holstein haplotype1), caused an estimated 525,000 spontaneous abortions worldwide over the past 35 years, accounting for ~ $420 million in losses. holstein haplotype1 was traced to the ancestor holstein sire pawnee farm arlinda chief born in 1962, a bull considered the second most influential sire in the holstein breed history.22 a description of all genetic abnormalities that cause pregnancy loss in ruminants is beyond the scope of this paper; an extensive review is available.23 toxic plants associated with pregnancy loss in ruminants include juniper (juniperus communis), locoweed (oxytropis and astragalus), pinus ponderosa, and perennial broomweed (gutierrezia microcephala).24 ingestion of veratrum californicum, a plant that grows primarily in the high mountain ranges of the western us, at about the 12 14th day of pregnancy can cause congenital cyclopia and other defects of the cranium and central nervous system in lambs, in addition to prolonged pregnancy.25 nitrates and mycotoxins are among the toxic causes of abortion in cattle.26-29 malnutrition and negative energy balance cause pregnancy loss.18 congenital nutritional muscular dystrophy caused by vitamin e and selenium deficiency is uncommon, but was reported as a cause of pregnancy loss in sheep and cattle.30 iodine deficiency and its excess was associated with abortion, stillbirths, and weak newborn calves and goats. goitrogenic compounds present in several species of brassica spp. and certain pharmacological agents, such as sulfonamides and thiouracil, induced congenital hyperplastic goiter in fetuses when dams were exposed.31,32 vitamin a deficiency in pregnant cows was suspected in cases of perinatal calf mortalities.33 dams fed a vitamin a deficient ration delivered dead, weak, uncoordinated, and blind calves.34 medications such as prostaglandin f2α can induce luteolysis and pregnancy loss in cattle and goats.35,36 ewes treated with netobimin, a benzimidazole compound, on day 17 of pregnancy delivered lambs with fetal skeletal and congenital renal malformations.37 heat stress has major effects on fertility and embryonic survival in lactating dairy cows.38,39 other causes and more in-depth explanations of noninfectious pregnancy loss and laboratory detection are described.3,4,10 infectious causes of pregnancy loss brucellosis brucellosis is a zoonotic disease caused by several brucella species and transmitted from animals to humans by ingestion clinical theriogenology 2021; 13: 182 of contaminated food products, direct contact with infected animals or inhalation of aerosols.40 brucellosis in cattle is caused more often by brucella abortus and less often by brucella melitensis. abortion in the mid-and late-term is the main clinical outcome in cattle, followed by birth of weak calves, perinatal mortality, reduced milk yield, and failure to conceive. orchitis is the most prominent lesion in infected bulls.41 brucellosis in humans is characterized by undulant fever, general malaise, miscarriage, and arthritis.42 brucella melitensis is the most virulent species of the brucella genus and the main cause of abortion in goats and sheep in many parts of the world. it is also the main agent responsible for human brucellosis, predominantly an occupational disease.43 clinical, pathological, and epidemiological features of sheep and goat brucellosis due to b. melitensis are similar to b. abortus infection in cattle.44 epididymitis and infertility in rams are most common clinical manifestation of sheep infected with b. ovis. placentitis and abortion occur occasionally in pregnant ewes.45 in contrast to other brucella species, b. ovis lacks zoonotic potential.46 brucella can be transmitted via horizontal or vertical routes. the main way all brucella species are disseminated amongst animals is through contact with fetal membranes, fetal fluids, and vaginal discharges expelled by infected animals. organisms shed in the milk of infected animals may transmit infection to the newborn. bulls may spread infection through semen.43,47 pregnant cattle infected with b. abortus may develop placentitis and abort during the last trimester of pregnancy, but generally do not abort in subsequent pregnancies. intercotyledonary areas of fetal membranes are thickened and have a yellow, leathery appearance. cotyledons appear swollen and covered with yellow to brown exudate. some aborted fetuses had bronchopneumonia.47 infected females that give birth to normal offspring shed the organism in fetal membranes, fetal fluids, and vaginal discharges. in nonpregnant animals, the infection is usually asymptomatic. diagnosis of brucellosis in infected fetuses is established by isolation of the organism from fetal membranes, fetal stomach content or lung. several pcr protocols for identification of brucella dna are used primarily in cultures. brucella antibodies in maternal serum can be detected by serological tests including the rose bengal test, serum agglutination tests, complement fixations test, coombs test, complement fixation tests, and more recently the immunocapture-agglutination technique.40 b. suis and b. melitensis infections in cattle may interfere with serological diagnosis of b. abortus infection in cattle.48 campylobacteriosis campylobacter spp. are important animal pathogens and opportunistic human pathogens. several species and subspecies of campylobacter cause pregnancy loss and infertility in ruminants.49 mammal-associated campylobacter fetus comprises 2 subspecies: c. fetus subsp venerealis and c. fetus subsp fetus, both of which are well-known causes of reproductive failures in ruminants.50 c. fetus subsp venerealis causes bovine genital campylobacteriosis characterized by infertility and abortion. c. fetus subsp fetus was the campylobacter species most frequently isolated from abortions in sheep. since the 1990s, c. jejuni has become an important campylobacter species associated with ovine abortion in the us. most isolates of c. jejuni from sheep abortions are resistant to tetracyclines, the only approved drug for treating infection in sheep in the us.51 transmission of campylobacter spp. is by ingestion of water contaminated with feces of infected animals. infection of pregnant ewes with c. fetus fetus or c. jejuni causes late term abortion, stillbirth, and/or the birth of weak lambs. average abortion rate generally is ~ 25%, but may be as high as 70%. intercotyledonary fetal membranes of aborted fetuses are edematous and cotyledons are yellow with necrotizing and suppurative inflammation and vasculitis. some aborted fetuses exhibit fibrinous peritonitis, multifocal areas of hepatic necrosis ranging from 1 mm to 5 cm in diameter, and suppurative bronchopneumonia. although uncommon, some ewes die due to endometritis and bacteremia.52 diagnosis is by bacterial isolation and identification from fetal membranes and tissues of aborted fetuses. pcr and dna sequencing are valuable tools for confirming phenotypic tests.53 chlamydiosis chlamydial abortion is usually due to infection with chlamydia abortus that also causes stillbirth, and birth of weak offspring in sheep and goats. infection in sheep is known as ovine enzootic abortion and is characterized by vaginitis, endometritis, mastitis, late term abortion, stillbirth, and birth of weak neonates that often die within 48 hours of birth. abortion usually occurs in the last 2 3 weeks of pregnancy, and up to 30% of ewes may be affected in naïve flocks. infection in goats is similar to infection in sheep. cattle, pigs, horses, wild ruminants, and humans are infrequently affected.54 susceptible animals acquire infection through exposure of oral, palpebral and genital mucosa to fluids, and tissues of infected fetuses and vaginal discharges. fetal membranes of aborted fetuses have diffuse purulent inflammation characterized by thickened and dark-red to brown cotyledons. histologically, there was loss of the cotyledonary and intercotyledonary epithelium and accumulation of necrotic debris, fibrin, neutrophils, and mononuclear leukocytes and thrombotic vasculitis in the stroma. focal hepatic necrosis, necrosis of the lung, spleen, and infrequently brain was observed in some aborted fetuses.52 chlamydia rt-pcr of fetal membranes and fetal kidney is currently the preferred method to diagnose chlamydiosis in aborted fetuses.55 furthermore, the organism can be identified clinical theriogenology 2021; 13: 183 in fetal membranes and fetal tissues by immunohistochemistry. several formats of enzyme-linked immunosorbent assay (elisa) and complement fixation tests are used to detect c. abortus in maternal serum.56 coxiellosis coxiellosis, often referred to as q (query) fever, is a highly infectious zoonotic disease caused by the intracellular bacterium coxiella burnetii that primarily affects goats, sheep, and less often cattle.7 coxiella burnetii infection of nonpregnant animals is usually asymptomatic, but can cause abortion, stillbirths, endometritis, mastitis, and infertility in pregnant small ruminants and occasionally in cattle. between 5 50% and in some cases up to 90% of pregnant ewes or goats may abort. the organism is shed in urine, feces, milk, vaginal fluids, semen, and placental and fetal fluids.57 self-limited acute febrile disease is the most common manifestation in humans infected with c. burnetii. abortions and stillbirth may occur in pregnant women. in a small percentage of infected humans, q fever progresses to a chronic form characterized by valvular endocarditis, osteoarthritis, encephalitis, and/or chronic inflammation in other organs.58 the main lesion in cases of abortion in ruminants consists of fibrinonecrotic inflammation of fetal membranes. grossly, intercotyledonary areas are thickened with a leathery appearance and are covered with white-yellow or brownish-red exudate. cotyledons are swollen and exhibit a yellow or brownish-red discoloration. microscopically, chorionic epithelium exhibits necrosis and infiltration of leukocytes on the surface. stroma of intercotyledonary areas have severe infiltration of leukocytes. large numbers of intracytoplasmic coccobacilli that are visible in hematoxylin and eosin-stained sections can be observed in the cytoplasm of chorionic trophoblast cells. organisms are gram-negative and have a magenta color when stained with gimenez stain, but they still must be differentiated from other gram-negative intercellular bacteria, such as brucella or campylobacter, by immunohistochemistry.7,57 currently, the preferred diagnostic method of q fever in aborted fetuses is amplification of c. burnetii-specific genomic dna by rt-pcr from fetal membranes and tissues. several serologic tests including indirect immunofluorescence, elisa and complement fixation test are used for the detection of c. burnetii antibodies in maternal serum. after experimental infection, c. burnetii phase ii specific antibodies, both igm and igg, can be detected 2 weeks postinfection and remain increased for up to 13 weeks. antibodies directed against c. burnetii phase i also increase, but 4 weeks later compared to phase ii antibodies. serum antibodies in infected animals can be detected for months to years.57 isolation of the microorganism in cell culture, embryonated chicken eggs and laboratory animals is considered dangerous and is rarely used.59 foothill abortion epizootic bovine abortion, also known as ‘foothill abortion’, is a vector borne disease of cattle that graze in the mountainous regions of california, southern oregon, and western nevada. it is caused by pajaroellobacter abortibovis bacteria that is transmitted by argasid tick ornithodoros coriaceus (pajaroello tick).60,61 abortion usually occurs in the last trimester of pregnancy exclusively in naïve heifers or cows when introduced in endemic areas between ~138 -183 days of pregnancy. abortion generally occurs three months or longer after exposure of pregnant dams to infected ticks. affected fetuses may induce their own delivery and may be born weak dying shortly after birth.62 some of the aborted fetuses have severe abdominal distention caused by ascites, severe generalized lymphadenomegaly and splenomegaly, and petechial hemorrhages in the mucous membranes. the thymus has areas of hemorrhage and edema and the liver is swollen and nodular. multifocal, discrete areas of pale discoloration are observed in many organs, but especially in the heart and kidney. histologically, the most characteristic lesion is inflammation of the thymus. the lymph nodes and spleen have lymphoid hyperplasia. severe periportal infiltration of mononuclear leukocytes and multifocal areas of histiocytic infiltration are observed in the pulmonary septa. vasculitis can be observed in the lung, brain, and meninges.63 recently, a live vaccine under conditional license was approved for cattle 6 months of age and older, and for nonpregnant females 60 days prior to breeding. foothill abortion vaccine was safe with 100% seroconversion and > 95% protection. histophilosis histophilosis, caused by the gram-negative bacterium histophilus somni, was associated with numerous clinical syndromes in ruminants including thrombotic meningoencephalitis, bronchopneumonia, polysynovitis, septicemia, mastitis, and sporadic abortion. aborted fetuses had histologic lesions including cerebral and myocardial vasculitis and thrombosis. isolation of the organism and detection of h. somni dna by rt-pcr are the routine techniques used in the diagnosis of h. somni induced abortion.64 leptospirosis leptospirosis is an important zoonotic disease caused by over 260 antigenically distinct serovars belonging to 25 serogroups grouped in 9 pathogenic species, 5 intermediate, and 6 saprophytic species of leptospira a gram-negative bacterium belonging to the spirochetales order.65,66 transmission is by contact with contaminated water or soil or by direct contact with urine from animals or fetal membranes and tissues and fluids of aborted animals.67 clinical theriogenology 2021; 13: 184 in the us, chronic infection of cattle with leptospira serovars hardjo and pomona produce reproductive problems manifested as early repeat breeding, early embryonic death, subfertility, abortions, fetal mummification, stillbirth, retained fetal membranes, premature births, and the birth of weak and/or low-weight calves. relative to cattle, sheep and goats can be resistant to leptospira infection.68 depending on the serovar, the abortion rate in susceptible cattle may be as high as 50% with l. pomona and between 3 to 10% with l. hardjo. aborted fetuses are often autolyzed. histologically, multifocal renal tubular necrosis or nonsuppurative interstitial nephritis and meningitis can be observed in some aborted fetuses.52 most cases of leptospirosis are currently detected by rt-pcr amplification of bacterial dna from the fetal kidney. serologic diagnosis of leptospirosis can be challenging particularly in vaccinated animals and is often used for the determination of the herd immune status, rather than to establish the cause of pregnancy loss. some cows infected with l. hardjo that eventually abort may have high microagglutination test (mat) antibody titers at abortion, but up to 40% had no detectable antibodies or nonsignificant titers.69 listeriosis listeria monocytogenes, serovars 4b and 5 (l. ivanovii), are the etiological agent of listeriosis in small ruminants. listeria have a wide distribution in the environment and may be recovered from dust, vegetation, decaying materials, soil, water, sewage, animal feeds, silage, and natural habitats.70 the main form of spread among animals is by ingestion of water or food contaminated by infected fetal membranes, feces or vaginal discharges. in small ruminants, infection with listeria monocytogenes may cause abortion, encephalitis, meningitis, septicemia, and gastroenteritis. more often, these syndromes occur separately. although serovar 5 (l. ivanovii) is less common, it is highly pathogenic in sheep and caused abortion in cattle.52 abortion in small ruminants and cattle generally occurs during the last trimester of pregnancy. aborted fetuses are often autolyzed. inflammation of fetal membranes and pinpoint areas of necrosis can be observed in the liver, heart, lung, kidney, and brain of aborted fetuses. bacterial isolation from fetal membranes, fetal stomach content or fetal tissues is the diagnostic method of choice. better results were obtained with a cold enrichment procedure.71 severe disease characterized by abortion and encephalitis is more often observed in pregnant woman and immunocompromised individuals.72 salmonellosis salmonella spp. are frequently associated with enteritis and diarrhea in ruminants, but are also a cause of abortion in cattle, sheep and goats, and are zoonotic agents. salmonella abortus-ovis and salmonella montevideo are bacterial pathogens that can cause abortions and stillbirths in pregnant ewes and goats, and mortality in neonates.73,74 in naive flocks, as many as 60% of all susceptible ewes and does may abort as a result of salmonella infection. if this disease agent becomes endemic in a flock, abortions are usually sporadic; only young animals and new sheep introduced into the flock tend to be affected. ewes and does may become carriers after aborting. ewes and does that abort may be asymptomatic prior to aborting, or have fever, depression, and diarrhea. metritis and retained fetal membranes, bacteremia and death can occur after abortion. fetal membranes are thickened, gray to red or yellow due to necrotizing suppurative inflammation and vasculitis with presence of coccobacilli in the cytoplasm of trophoblast cells. autolyzed fetuses had signs of fetal stress characterized by diffuse yellow-green staining of the skin with meconium.52 salmonella enterica subsp. enterica serovar dublin (s. dublin) may, in the course of a systemic infection, colonize the placenta and fetus and cause placentitis, abortion, and stillbirth in cattle.75 organisms can be isolated from fetal membranes and fetal abomasal contents. ureaplasma diversum infection ureaplasma diversum is a common inhabitant of vagina and prepuce of cattle. this organism was isolated from field cases of calf pneumonia, keratoconjunctivitis, mastitis, seminal vesiculitis, granular vulvitis, endometritis, salpingitis, and abortions.76 abortions, stillbirth, and birth of weak calves occurred sporadically; however, outbreaks involved multiple animals.77 yersiniosis yersinosis is a zoonotic disease caused by yersinia pseudotuberculosis that infects sheep, goats, cattle, humans, and other animal species. transmission occurs by ingestion of contaminated food or water.78 abortions in small ruminants generally occurred in the last 2 weeks of pregnancy. inflammation of fetal membranes and multifocal hepatic necrosis are the most frequent macroscopic lesions in aborted fetuses. fetal membranes are thickened, edematous and yellow. multifocal areas of hepatic necrosis are observed in some aborted fetuses. some aborted fetuses had microscopic evidence of necrotizing inflammation of fetal membranes with vasculitis and bronchopneumonia.79-81 this organism can be isolated from stomach contents, fetal membranes, lung and other fetal tissues, and from the uterus of aborting sheep and goats.81 mycotic abortion mycotic abortions in cattle are usually sporadic. aspergillus fumigatus is the most common cause of mycotic abortion in cattle with other fungi of the genera absidia spp., mucor spp., clinical theriogenology 2021; 13: 185 rhizopus spp., mortierella wolfii, candida spp., and torulopsis being less common.82 severe inflammation of fetal membranes is a frequent lesion characterized by necrosis, thickening and cupping of the cotyledons and leathery thickening and yellow discoloration of the intercotyledonary area.83 fetal dermatitis, characterized by raised circular epidermal plaques occurs in ~ 25% of the cases.84 diagnosis is established by culture and isolation. microscopically, fungal hyphae can be observed in the lesions in hematoxylin and eosin-stained sections or sections stained with gomori’s methenamine silver and periodic acid-schiff stains. fungi can also be identified in fresh tissues, or in paraffin-embedded sections by panfungal pcr.85 bluetongue virus bluetongue virus (btv) is an orbivirus of the family reoviridae family composed of 28 serotypes. btv is transmitted to ruminants by several species of biting midges (culicoides spp.) and caused thrombo-hemorrhagic fevers mainly in sheep. in the us, the main vector of btv endemic serotypes is culicoides sonorensis (c sonorensis; previously known as c varipennis). c. insignis was identified in the southeastern us. apparently, some new btv serotypes (btv-25, btv-26, btv-27) were transmitted horizontally without the involvements of the vector.86 clinical disease is more common in sheep and is characterized by fever, depression, salivation, facial swelling, panting, nasal discharge, hyperemia of the muzzle, lips, ears, oral ulceration, and coronitis. morbidity can be as high as 100% and mortality can range from 0 30%.87 in the us, btv-induced brain malformations and abortion in sheep and cattle occur infrequently and are the result of infection with live-attenuated btv strains present in vaccines licensed only for sheep.88 during the 2006 european btv outbreak, the ability of the field strain btv-8 cross the placenta and infect the fetus was a major concern because of transplacental transmission as high as 33% and an increase in the numbers of abortions, stillbirths and fetal deformities in cattle, including hydranencephaly.89-91 in these cases, btv can be detected by rt-pcr in fetal splenic tissue. isolation of infectious virus or the presence of btv antibodies in fetuses was reported in a few cases. immunotolerance was observed in a few animals.92-93 serological assays for the detection of btv antibodies in maternal serum include complement fixation, virus neutralization, agar gel immunodiffusion test, and elisa. virus isolation is by inoculation of susceptible sheep or embryonated chicken eggs with heparinized blood or homogenized lymph nodes, spleen, or lung. subsequent adaptation to cell culture and serotyping of the virus may be necessary. blood and tissue samples should be kept at 4˚c. the most common diagnostic test is rt-pcr which has a reported high sensitivity and specificity; however, virus was not present in some positive rt-pcr cases.87 infectious bovine rhinotracheitis primary infection with bovine herpes virus -1 (bhv-1) may result in several clinical manifestations including infectious bovine rhinotracheitis (ibr), abortion, infectious pustular vulvovaginitis, and systemic infection in neonates.94 after initial infection with bhv-1, the virus establishes a lifelong latent infection in the nervous sensory ganglia and pharyngeal tonsils. reactivation of bhv-1 from latency can occur after stressful situations including transportation, calving and treatment with corticosteroids.95,96 the primary immune response developed after bhv-1 natural exposure or vaccination is able to successfully control viral recrudescence in a latent carrier. abortion and fatal systemic disease in neonates are the most severe consequences of respiratory infections of seronegative cows with virulent strains of bhv-1.94 after infection in pregnant cows, bhv-1 may stay latent in the placenta and invade the fetus after several weeks. once the fetus is infected, it dies quickly and remains in utero for several days before expulsion. fetal autolysis is usually present because of rapid death of the fetus. the subcutis is edematous and red-tinged. large amounts of red-tinged fluid were present in the thoracic and abdominal cavity and pericardium.52 small, discrete white nodules are observed in the liver. histologically, discrete areas of necrosis were identified in the liver, kidneys, spleen, lungs, and adrenal glands. intranuclear inclusions characteristic of herpesvirus infections are difficult to find in hematoxylin and eosin-stained tissue sections including sections from the adrenal glands. fetal membrane lesions consist of necrosis and vasculitis. abortion generally occurs between 5 to 8 months of pregnancy. use of live modified bhv-1 vaccines in pregnant animals that did not have antibodies from a previous vaccination were at increased risk of pregnancy loss.97 in the us, a number of bovine abortions occurred following the use of bhv-1 vaccines that in part caused confusion about safety of modified-live virus products. use of inactivated vaccines is safer for pregnant animals and animals with an unknown pregnancy status. immunization of females with inactivated bhv-1 vaccine prior to breeding built protection against pregnancy loss.97 prevalence of abortion has diminished in regions that have fewer bhv-1 naïve populations. the most important practice for the control of bhv-1 transmission is detection and elimination of bhv-1 semen samples. virus isolation and bhv-1 dna amplification by rt-pcr from fetal lung or liver or from edta whole-blood or semen of adult animals are the preferred methods of diagnosis. detection of bhv-1 maternal antibodies is by virus neutralization or elisa.98 bovine viral diarrhea bovine viral diarrhea is an important infectious production disease in most cattle-producing countries worldwide clinical theriogenology 2021; 13: 186 caused by bovine viral diarrhea virus (bvdv), a member of the genus pestivirus in the flaviviridae family. currently, phylogenetic analysis has identified 21 pestivirus subtypes (bvdv1a-u) and 4 pestivirus subtypes 2 (bvdv2a-2d). four pestivirus h subtypes (hobi a d) have been identified in europe. bovine viral diarrhea viruses also are of cytopathic (cp) and noncytopathic (ncp) biotypes.99-101 in adult immunocompetent cattle, bvdv infection often causes subclinical disease with manifestations ranging from a mild transient infection to more severe respiratory disease lasting 2 3 weeks characterized by fever, nasal discharge, pneumonia and even death. animals that recover from this form of bvdv infection clear the virus and develop lifelong immunity. the infection also has been associated with diarrhea and hemorrhagic syndrome.102 countries that have implemented control and/or eradication programs have, on average, 1.5 times lower pooled bvdv prevalence at animal and herd levels compared to countries without intervention measures.103 the outcome of bvdv fetal infection is complex and varies depending on the stage of pregnancy, the ability of fetus to mount an immune response and the bvdv biotype. apparently, embryos are resistant to infection until they hatch from the zona pellucida ~ day 10 of pregnancy.104 during embryonic development and up to fetal differentiation, bvdv infection causes embryonic death. in general, when infection with ncpbvdv occurs after fetal differentiation and up to 6 months of pregnancy, embryonic death, mummification, fetal malformation, abortion, immunotolerance, the birth of persistently infected calves, stillbirth or the birth of weak or undersized or apparently normal calves, and fetal malformation may occur. later stages of pregnancy have limited susceptibility to infection and calves are born with precolostral antibodies to bvdv.105, 106 fetal infection with ncpbvdv before the development of immunocompetence (between ~ days 45 and 145 of pregnancy) can result in fetal immunotolerance and persistently infected (pi) calves that are born alive.107 these in utero-infected calves have no antibodies against bvdv, continuously shed large amounts of virus and are the main source of the virus for other herd mates. pi animals generally die by 2 years of age, often of mucosal disease that occurs when ncpbvdv mutates into cpbvdv causing super infection. bvd superinfection also result when pi animals are superinfected with field strains cpbvdv or are vaccinated with cytopathic modified live virus vaccine. after the fetus develops immunocompetence (~ 150 days of pregnancy), it is possible to have the birth of clinically normal calves with bvdv precolostral serum antibodies.108 in aborted fetuses, a battery of diagnostic tests including virus isolation, rt-pcr and demonstration of bvdv antibodies in serum or fluid from the thorax or abdomen should be done in parallel. immunohistochemistry, rt-pcr and antigen capture elisa in ear notches are used to detect pis. virus neutralization tests are used for the detection of bvdv antibodies in serum.109 detection of pis, implementation of biosecurity measures and vaccination are important practices for the control of bvd in herds. bvdv vaccines available in the us include killed virus and modified live virus.110 cache valley virus cache valley virus (cvv) is an arbovirus of the family bunyaviridae that is endemic in north america and infects a wide range of domestic and wild animals, and humans. the virus is transmitted by the bite of competent vectors of several species of aedes, anopheles, coquillettidia and culiseta genuses including ae. japonicus, ae. scapularis, ae. sollicitans, ae. taeniorhynchus, ae. vexans, an. punctipennis, an. quadrimaculatus, co. perturbans and cu. inornata.111 cvv infection is usually asymptomatic in adult sheep, goats and cattle. cvv infection during pregnancy may result in embryonic mortality, fetal mummification, fetal malformation, abortion, stillbirth and pregnancy loss in sheep and goats and less often cattle.112,113 a limited number of human case reports have described severe illness including meningitis.111 congenital malformation in sheep and goats may include one or more of the following: arthrogryposis, kyphosis, torticollis, pelvic limb hemimelia, maxillary prognathism, hydranencephaly, and cerebellar hypoplasia.114 in utero experimental infection of pregnant ewes at day 35 of pregnancy resulted in necrosis of the central nervous system and skeletal muscles at 7 14 days post-infection, and hydrocephalus, micromyelia and muscular loss at 21 28 postinfection.115 cvv infections in aborted fetuses and stillbirths can be diagnosed using serum neutralization tests because fetuses are able to mount an antibody response and clear the virus. experimental infection of pregnant ewes with cvv ~ 35 days of pregnancy had low virus antigen and rna signal in tissues by day 56 of pregnancy and cleared the virus by day 75.116 virus isolation in full-term malformed fetuses is unsuccessful. experimental infection of pregnant sheep with two california serogroup bunyaviruses (lacrosse virus and san angelo virus) and a bunyamwera serogroup member (main drain virus) induced a range of lesions including arthrogryposis, hydrocephalus, fetal death, axial skeletal deviations, anasarca, and oligohydramnios.117 fetal teratogenesis in sheep, goats, and cattle have been described in natural and experimental infections with other related exotic bunyaviruses including akabane virus, schmallenberg virus, rift valley fever virus and wesselsbron disease virus.118,119 caprine herpesvirus-1 caprine herpesvirus -1 (cphv-1) belongs to the subfamily of clinical theriogenology 2021; 13: 187 alphaherpesviruses that contains 7 genetically-related viruses. cphv-1 is closely related to bohv-1, the cause of infectious bovine rhinotracheitis. late term abortions and gastroenteric and respiratory disease in 12-week-old kids have been associated with caphv-1 infection.120 up to 50% of pregnant does in a herd may experience late term abortions or the birth of stillborn kids. numerous white, pinpoint foci ranging from 1 to 2 mm may be observed in the liver and lungs of aborted fetuses. microscopically, discrete areas of coagulative necrosis can be observed in liver, lung, thymus and less frequently other organs. intranuclear inclusions bodies in the periphery of the areas of necrosis. in adult goats, cphv-1 infections are general subclinical, but vulvovaginitis and balanoposthitis occurs infrequently.121amplification of cphv-1 by rt-pcr and virus isolation are used for diagnosis. the virus neutralization test is used for the demonstration of cphv-1antibodies in maternal serum.122 neosporosis neospora caninum, the cause of neosporosis, is an apicomplexan parasite and one of the most important causes of pregnancy loss in cattle. n. caninum also causes abortion in sheep and goats. the life cycle of n. caninum is similar to that of toxoplasma gondii in small ruminants, but cattle are the intermediate host and dogs and coyotes the definitive host of n. caninum.123 there are 2 modes of transmission: vertical from dam to offspring; and horizontal from its definitive host, the dog, to cow, sheep or goat, with vertical transmission being more common. dogs generally acquire infection by ingesting fetal membranes or tissues from infected aborted fetuses.124 abortion in cattle due to n. caninum is more common between 5 and 7 months of pregnancy. in naïve herds, 30% or more of pregnant heifers or cows abort over a period of several months. once, the infection becomes endemic in a herd, the abortion rate is about 5% per year and persists for several years. transplacental infection may also result in, fetal mummification, birth of weak compromised calves, or the birth of clinically normal infected neonates. these normal infected neonates remain persistently infected and eventually transmit infection transplacentally to their progeny and preserve neosporosis in the herd. cows that abort due to neospora infection do not show other clinical signs. apparently, risk of abortion in subsequent pregnancies is lower.123 aborted fetuses are often autolyzed or may be partially or fully mummified. focal areas of inflammation can be observed microscopically in the brain, brain stem, skeletal muscle, and heart. rarely, the organism can be recognized in the brain by standard hematoxylin and eosin staining, but can be demonstrated by immunohistochemistry. fetal membranes have necrosis of the trophoblast in cotyledons, with normal intercotyledonary areas. congenitally infected full-term calves may be born weak, show ataxia or are born clinically asymptomatic but have high serum titers of precolostral serum antibodies.52 numerous diagnostic tests including rt-pcr, histopathology and fetal serology should be done concurrently in abortion cases for the accurate diagnosis of neosporosis. serum antibodies in maternal serum detected by elisa are used to determine the infectious status of individual animals, but the presence of maternal antibodies alone is not a proof of the cause of pregnancy loss.125 the most important measure for the control of neosporosis is to identify and eliminate congenitally infected heifers and to reduce postnatal transmission from definitive hosts.126 sarcocystosis sarcocystois is a genus of cyst-forming coccidian and the cause of sarcocystosis. sarcocystois spp. are obligatory 2-host life cycle apicomplexa parasites that includes herbivores as intermediate hosts and carnivores as definitive hosts. infection is common but the majority of infected cattle, sheep, and goats are asymptomatic. acute fatal disease develops occasionally and is characterized by fever, edema, and jaundice. animals exhibit inappetence, weight loss, decreased milk production, endometritis, and neurological signs. pregnant animals may abort during the acute phase. abortion may be indirect by premature induction of parturition due to the release of prostaglandin f2α during acute disease or by direct infection of the fetus. aborted fetuses exhibit extensive microscopic lesions consisting of multifocal areas of necrosis and mononuclear cell infiltration in the brain and meninges. similar lesions can be observed in the heart, kidney, liver, lung, and fetal membranes. organism can be identified by immunofluorescence in frozen sections of tissues.52,127 toxoplasmosis toxoplasmosis, caused by the apicomplexan parasite toxoplasma gondii, is a major cause of abortion in sheep and goats.128 simultaneous infection with chlamydia abortus and coxiella burnetii occurs in some cases. most sheep and goats become infected with t. gondii by ingesting food or water contaminated with sporulated oocysts shed by cats. less than 4% of persistently infected sheep transmit the parasite in utero to the fetus.129,130 infection during the first part of pregnancy is likely to result in early embryonic death and resorption. later in pregnancy, infected ewes and does may exhibit fetal mummification, abortion, stillbirth, and birth of weak offspring. infection of pregnant ewes and does with t. gondii in naive herds may result in abortion storms. sheep and goat develop humoral and cellular immune responses after infection that provide effective protection against pregnancy loss in subsequent pregnancies.129 on gross examination, the cotyledons of aborted fetuses have numerous small white nodules ranging from 1 to 3 mm. clinical theriogenology 2021; 13: 188 in few affected fetuses, there is evidence of focal myocarditis, pneumonitis or encephalitis. the organisms can be demonstrated in formalin fixed, paraffin embedded tissues by immunohistochemistry. the preferred technique for the diagnosis of t. gondii in aborted fetuses is rt-pcr.52 presence of maternal serum antibodies is an indication of exposure, but does not prove t. gondii as the cause of abortion. trichomoniasis trichomoniasis is an economically important infectious venereal disease of cattle caused by tritrichomonas foetus, a protozoan flagellated parasite. infected bulls are asymptomatic but carry the protozoa in their preputial sheath and penis and are the source of infection for cows and heifers.132 in some bulls, the protozoa are harbored in the distal urethra. infection occurs at coitus and continues for some time after infection. most infected animals clear the infection spontaneously within a few weeks to a few months. after clearing the infection, cows can become pregnant and carry a fetus to term. the infection may cause embryonic mortality, pyometra, and infrequently mid-pregnancy abortion.133,134 lesions include, mild inflammation of fetal membranes characterized by edema, and small amounts of white to yellow exudate and mild necrosis of cotyledons. generally, fetal lesions are not present, but large numbers of organism can be detected in the fetal fluids and stomach.52,135 some aborted fetuses had suppurative bronchopneumonia.16 in aborted 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calves infected with bluetongue virus. vet rec 2008;162:490-491. https://doi.org/10.1136/ vr.162.15.490-b. 89. desmecht d, vanden bergh r, sartelet a, et al: evidence for transplacental transmission of the current wild-type strain of bluetongue virus serotype 8 in cattle. vet rec 2008;163:50-52. https://doi. org/10.1136/vr.163.2.50. 90. vercauteren g, miry c, vandenbussche f, et al: bluetongue virus serotype 8-associated congenital hydranencephaly in calves. transbound emerg dis 2008;55:293–298. https://doi.org/10.1111/j.1865-1682.2008.01034.x. 91. williamson sm, scholes sfe, welchman dde b, et al: bluetongue virus serotype 8-associated hydranencephaly in two calves in south-eastern england. vet rec 2010;167:216-218. https://doi.org/10.1136/vr.c3302. 92. de clercq k, de leeuw i, verheyden b, et al: transplacental infection and apparently immunotolerance induced by a wild-type bluetongue virus serotype 8 natural infection. transbound emerg dis 2008;55:352359. https://doi.org/10.1111/j.1865-1682.2008.01044.x. 93. zanella g, durand b, sellal e, et al: bluetongue virus serotype 8: abortion and transplacental transmission in cattle in the burgundy region, france, 2008–2009. theriogenology 2012;77:65-72. https:// doi.org/10.1016/j.theriogenology.2011.07.015. 94. muylkens b, thiry j, kirten p, et al: bovine herpesvirus 1 infection and infectious bovine rhinotracheitis. vet res 2007;38:181-209. https:// doi.org/10.1051/vetres:2006059. 95. sheffy be, davies dh: reactivation of a bovine herpesvirus after corticosteroid treatment. exp biol med 1972;140:974-976. https://doi. org/10.3181/00379727-140-36592. 96. thiry e, saliki j, schwers a, et al: parturition as a stimulus of ibr virus reactivation. vet rec 1985;116:599-600. https://doi.org/10.1136/ vr.116.22.599. 97. zimmerman a, buterbaugh r, herbert j, et al: efficacy of bovine herpesvirus-1 inactivated vaccine against abortion and stillbirth in pregnant heifers. j am vet med assoc 2007;231:1386-1389. 98. mahajan v, banga hs, deka d, et al: comparison of diagnostic tests for diagnosis of infectious bovine rhinotracheitis in natural cases of bovine abortion. j comp pathol 2013;149:391-401. https://doi. clinical theriogenology 2021; 13: 191 org/10.1016/j.jcpa.2013.05.002. 99. schweizer m, peterhans e: pestiviruses. annu rev anim biosci 2014;2:141-163. https://doi.org/10.1146/annurev-animal-022513-114209. 100. ridpath jf: bvdv genotypes and biotypes: practical implications for diagnosis and control. biologicals 2003;31:127-131. https://doi. org/10.1016/s1045-1056(03)00028-9. 101. smith db, meyers g, bukh j, et al: proposed revision to the taxonomy of the genus pestivirus, family flaviviridae. j gen virol 2017;98:2106-2112. https://doi.org/10.1099/jgv.0.000873. 102. uzal f, plattner b, hostetter j: alimentary system. in: maxie mg: editor. jubb, kennedy and palmer’s pathology of domestic animals, vol. 3. 6th edition, st. louis, mo; elsevier: 2016. p. 122-130. 103. scharnböck b, roch f-f, richter v, et al: a meta-analysis of bovine viral diarrhoea virus (bvdv) prevalences in the global cattle population. sci rep 2018;8:14420. https://doi.org/10.1038/ s41598-018-32831-2. 104. vanroose g, nauwynck h, soom av, et al: replication of cytopathic and noncytopathic bovine viral diarrhea virus in zona-free and zona-intact in vitro-produced bovine embryos and the effect on embryo quality 1. biol reprod 1998;58:857-866. https://doi. org/10.1095/biolreprod58.3.857. 105. moennig v, liess b: pathogenesis of intrauterine infections with bovine viral diarrhea virus. vet clin north amer: food anim pract 1995;11:477-487. https://doi.org/10.1016/s0749-0720(15)30462-x. 106. agerholm js, hewicker-trautwein m, peperkamp k, et al: virus-induced congenital malformations in cattle. acta vet scand 2015;57:54. https://doi.org/10.1186/s13028-015-0145-8. 107. kelling cl, topliff cl: bovine maternal, fetal and neonatal responses to bovine viral diarrhea virus infections. biologicals 2013;41:20-25. https://doi.org/10.1016/j.biologicals.2012.09.006. 108. givens md, marley msd: infectious causes of embryonic and fetal mortality. theriogenology 2008;70:270-285. https://doi. org/10.1016/j.theriogenology.2008.04.018. 109. hou p, xu y, wang h, et al: detection of bovine viral diarrhea virus genotype 1 in aerosol by a real time rt-pcr assay. bmc vet res 2020;16:114. https://doi.org/10.1186/s12917-020-02330-6. 110. fulton rw, cook bj, payton me, et al: immune response to bovine viral diarrhea virus (bvdv) vaccines detecting antibodies to bvdv subtypes 1a, 1b, 2a, and 2c. vaccine 2020;38:4032-4037. https://doi.org/10.1016/j.vaccine.2020.03.058. 111. waddell l, pachal n, mascarenhas m, et al: cache valley virus: a scoping review of the global evidence. zoonoses pub health 2019;66:739-758. https://doi.org/10.1111/zph.12621. 112. de la concha-bermejillo a: cache valley virus is a cause of fetal malformation and pregnancy loss in sheep. small rum res 2003;49:1-9. https://doi.org/10.1016/s0921-4488(03)00050-6. 113. de la concha-bermejillo a: cache valley virus. in: chase c, lutz k, mckenzie e: editors. blackwell’s five-minute veterinary consult ruminant. 2nd edition, hoboken, nj; john wiley & sons: 2017. p. 140-141. 114. harvey j, smith j, jackson n, et al: cache valley virus as a cause of fetal abnormalities in a litter of three boer kids. vet rec case rep 2019;7:e000725. https://doi.org/10.1136/vetreccr-2018-000725. 115. rodrigues hoffmann a, welsh cj, varner pw, et al: identification of the target cells and sequence of infection during experimental infection of ovine fetuses with cache valley virus. j virol 2012;86:4793-4800. https://doi.org/10.1128/jvi.06858-11. 116. rodrigues hoffmann a, dorniak p, filant j, et al: ovine fetal immune esponse to cache valley virus infection. j virol 2013;87:55865592. https://doi.org/10.1128/jvi.01821-12. 117. collisson ew, edwards jf, de la concha bermejillo a, et al: ovine fetal malformations induced by in utero inoculation with main drain, san angelo, and lacrosse viruses. am j trp med hygiene 1997;56:171-176. https://doi.org/10.4269/ajtmh.1997.56.171. 118. de regge n, van den berg t, georges l, et al: diagnosis of schmallenberg virus infection in malformed lambs and calves and first indications for virus clearance in the fetus. vet microbiol 2013;162:595-600. https://doi.org/10.1016/j.vetmic.2012.11.029. 119. oberst rd: viruses as teratogens. vet clin north amer: food anim pract 1993;9:23-31. https://doi.org/10.1016/ s0749-0720(15)30668-x. 120. williams n, vickers m, tramontin r, et al: multiple abortions associated with caprine herpesvirus infection in a goat herd. j am vet med assoc 1997;211:89-91. 121. chénier s, montpetit c, hélie p: caprine herpesvirus-1 abortion storm in a goat herd in quebec. can vet j 2004;45:241-243. 122. roperto f, pratelli a, guarino g, et al: natural caprine herpesvirus 1 (cphv-1) infection in kids. j comp pathol 2000;122:298-302. https://doi.org/10.1053/jcpa.1999.0375. 123. dubey jp, schares g, ortega-mora lm: epidemiology and control of neosporosis and neospora caninum. clin microbiol rev 2007;20:323-367. https://doi.org/10.1128/cmr.00031-06. 124. reichel mp, wahl lc, hill fi: review of diagnostic procedures and approaches to infectious causes of reproductive failures of cattle in australia and new zealand. front vet sci 2018;5:222. https://doi. org/10.3389/fvets.2018.00222. 125. dubey jp, schares g, ortega-mora lm: epidemiology and control of neosporosis and neospora caninum. clin microbiol rev 2007;20:323-367. https://doi.org/10.1128/cmr.00031-06. 126. anderson ml, andrianarivo ag, conrad pa: neosporosis in cattle. anim reprod sci 2000;60-61:417-431. https://doi.org/10.1016/ s0378-4320(00)00117-2. 127. hong c, giles jr r, newman l, et al: sarcocystosis in an aborted bovine fetus. j am vet med assoc 1982;181:585-588. 128. edwards jf, dubey jp: toxoplasma gondii abortion storm in sheep on a texas farm and isolation of mouse virulent atypical genotype t. gondii from an aborted lamb from a chronically infected ewe. vet parasitol 2013;192:129-136. https://doi.org/10.1016/j. vetpar.2012.09.037. 129. dubey jp: toxoplasmosis in sheep—the last 20 years. vet parasitol 2009;163:1-14. https://doi.org/10.1016/j.vetpar.2009.02.026. 130. hill d, dubey jp: toxoplasma gondii: transmission, diagnosis and prevention. clin microbiol infect 2002;8:634-640. https://doi. org/10.1046/j.1469-0691.2002.00485.x. 131. sánchez-sánchez r, vázquez p, ferre i, et al: treatment of toxoplasmosis and neosporosis in farm ruminants: state of knowledge and future trends. ctmc 2018;18:1304-1323. https://doi.org/10.21 74/1568026618666181002113617. 132. fitzgerald pr: bovine trichomoniasis. vet clin north amer: food anim pract 1986;2:277-282. https://doi.org/10.1016/ s0749-0720(15)31237-8. 133. bondurant rh: pathogenesis, diagnosis, and management clinical theriogenology 2021; 13: 192 of trichomoniasis in cattle. vet clin north amer: food anim pract 1997;13:345-361. https://doi.org/10.1016/s0749-0720(15)30346-7. 134. parsonson im, clark bl, dufty jh: early pathogenesis and pathology of tritrichomonas foetus infection in virgin heifers. j comp pathol 1976;86:59-66. https://doi.org/10.1016/0021-9975(76)90028-1. 135. rhyan jc, stackhouse ll, quinn wj: fetal and placental lesions in bovine abortion due to tritrichomonas foetus. vet pathol 1988;25:350355. https://doi.org/10.1177/030098588802500503. 136. michi an, favetto ph, kastelic j, et al: a review of sexually transmitted bovine trichomoniasis and campylobacteriosis affecting cattle reproductive health. theriogenology 2016;85:781-791. https://doi. org/10.1016/j.theriogenology.2015.10.037. clinical theriogenology 2021; 13: 193 pregnancy loss in ruminants 2018: the equine endometrial microbiome the equine endometrial microbiome g. reed holyoak,a candace c. lyman,a xuwen wieneke,b udaya desilvab adepartment of veterinary clinical sciences, center for veterinary health sciences, oklahoma state university, stillwater, ok; bdepartment of animal science, oklahoma state university, stillwater, ok context our purpose is to examine an alternative idea to describing the reproductive tract microbiome in the mare. defining the reproductive tract microflora in any species including the mare lies in the need to be able to determine the physiologic state of the microbiome in healthy patients in order to develop a deeper understanding of the impact dysbiosis can have on overall health. with the background information we have, and the advancement of sequencing techniques at our disposal it may be time to challenge the veterinary dogma of a “sterile uterus” and adjust our clinical practices accordingly. keywords: mare, paradigm shift, endometrial microbiota, 16s rdna introduction it has been standard dogma that although the vagina is colonized with commensal bacteria, the uterus is maintained as a sterile environment1 and a failure to eliminate bacteria, sperm and inflammatory products from the uterus after breeding will result in endometritis and reduced fertility.2 while relatively more is known about the microbiome of the human reproductive tract, much less is known about the microbial communities residing in the reproductive tract of animals. as with the mare, in cattle it has long been held that bacterial contamination during the periparturient period lead to endometritis or metritis in the cow.3 in all of our farm animal species these dogmata were based on the results from media based culture systems. although culture-based studies have laid out the foundation of our understanding of the uterine microbiota, almost three decades ago there was an indication that culture based methodologies in identifying microbial populations may underestimate diversity and overestimate the role of culturable bacteria.4 focusing on culturable bacteria, often the rarer members of microbial communities, enhances the risk of missing those microbes that are more abundant.5,6 recently we completed a study in the canine reproductive tract documenting with 16s rdna based metagenomics the presence and diversity of both vaginal and uterine microbiomes at the various stages of the estrous cycle, concluding that both the endometrium and the vagina have rich microbial ecosystems.7 additionally, a study utilizing cultureindependent 16s ribosomal rna (rrna) sequencing of the bovine and ovine vaginal microbiota from ectocervicovaginal lavages revealed that cow and ewe vaginal microbiota are unique from previously described vaginal microbial ecosystems.8 it is clear from these studies that culture-based diagnostic systems miss the great diversity present in both diseased and healthy reproductive tracts and express a need for metagenomic analytics. we began our metagenomics investigations in the mare to document the underestimation of microbial diversity with common culture methods relative to the presence of microbial dna using the approach six years ago.9 this study grew from a study utilizing molecular identification performed by sequencing of pcr products of 16s rdna using 63f and 1389r primers which suggested separate populations of microbiota between the equine uterus and vagina.10 differences were shown between vaginal and uterine microbial populations. that while there was a demonstrated transfer of a vaginal subpopulation into the uterus post-breeding there remained distinct differences between the vaginal and endometrial microbiomes.10 however, neither of these studies clearly defined the endometrial microbiota. the purpose of describing the reproductive tract microflora in any species including the mare lies in the clinical need to be able to determine the physiologic state of the microbiome in healthy patients in order to develop a deeper understanding of the impact dysbiosis can have on overall health. understanding the ramifications that broad-spectrum antibiotic use can have on the uterine normal flora should dictate to veterinarians and breeders that treating a breeding mare with antibiotics, in the absence of an understanding of the endometrial microbiome may be counterproductive. with the background clinical theriogenology • volume 10, number 3 • september 2018273 information we have and the advancement of sequencing techniques at our disposal it is time to more deeply investigate the dogma of a “sterile uterus” and adjust our clinical practices accordingly if needed. the purpose of this report is to discuss what we have learned in our efforts to characterize the equine microbiome in the non-pregnant mare. materials and methods animals twenty nine mares between the age of 8 and 18 years of age, with no history of reproductive problems were used in the study. the mares were given a complete pre-study breeding soundness examination to include only those without clinical signs of endometritis. small volume lavage (svl) all mares had uterine luminal endometrial microbiota samples collected via 150 ml of saline infused via a double guarded system, wherein the first guard is inserted into the cervix and an internal tube containing a catheter is advanced into the uterus, saline is infused, massaged within the lumen and aspirated back and the tubing then retracted before removing back through the vaginal vault. this sampling technique has been shown to have the highest return rate for microbial collection.11 microbiota analysis small volume lavage samples were spun down to collect bacterial cells and total dna from the sediment extracted using a commercial dna isolation kit (qiamp dna mini kit, qiagen, germantown, md) and following manufacturer’s instructions. a ~250 bp fragment from ribosomal v4 region was pcramplified from resulting dna. amplicon sequencing was performed by molecular research llc (mr dna), shallowater, tx using their established protocols.12 briefly, a library was built using truseq® dna pcr-free sample preparation kit (illumina inc., san diego, ca) following instructions and index codes were added. the library was quality tested on a qubit 3.0 fluorometer (thermoscientific, waltham, ma) and an agilent bioanalyzer 2100 system (agilent technologies, santa clara, ca). the library was sequenced on an illumina hiseq2500 platform (illumina) and 250bp paired-end raw reads were generated. sequence analysis mothur 1.37 was used for data mining following the miseq standard operating procedure (sop).13 briefly, paired-end reads were assembled and assigned to each sample based on their unique barcode and then truncated by removing barcodes and primer sequences. reads that were over 273 bp in length, those that contained homoploymeric tracts of longer than 8 bp in length, those that had more than one mismatch against primer sequences, contained undetermined bases, ambiguities, or did not align to v4 hypervariable region were removed from the analysis. alignment of v4 region against silva rrna ref nr12314 was performed using mothur (needleman-wunsch algorithm). uchime was used to remove chimeric sequences.15 the rest of the analyses were performed following mothur miseq standard operating procedures. sequences were assigned to operational taxonomic units (otus) based on 97% similarity. statistical analyses neighbor-joining trees were generated using megan community edition.16 the shannon diversity index was calculated using mothur and principal coordinate analyses (pcoa) was calculated based on the distance matrix. results 1.1 million sequence reads of v4 region of 16s rrna gene were generated in this study with an average of ~40,000 reads per mare. upon analysis, we identified 160 unique genera of bacteria belonging clinical theriogenology • volume 10, number 3 • september 2018 274 to 18 different phyla. there were significant differences among animals with some clustering based on the source of animals. we defined a core microbiome for equine uterus at phylum level as phyla that were present at least at 1% level in all samples where they were identified and present in at least 90% of samples. the core microbiome at phylum level across all samples were; proteobacteria (100%), firmicutes (100%), bacteroidetes (96.2%) and actinobacteria (100%). we defined the core microbiome at genus level as present in at least 60% of the samples and are present at least at 0.5% in samples where the organism is detected. the core microbiome at genus level across all samples were; pseudomonas (100%), porphyromonas (73.1%) and streptococcus (61.4%). there were animal source specific core microbiomes. discussion the mammalian vagina is known to harbor a rich microbial ecosystem, while the human vaginal microbiome is well-described,17-23 only a few non-human vaginal microbiomes have been described to date.8,24-26 the endometrium on the other hand, was considered a sterile environment1,27 until a few years ago when that paradigm was challenged by the observation that the human placenta harbors a small but diverse microbiome.27,28 this new view is, however, not without debate.29 in addition to the human work, a few studies have been performed exploring the microbiome of the bovine and ovine endometrium and placenta.30-33 while the microbiota of the cow and ewe were similar, the bovine vaginal microbiome showed greater diversity compared to the ovine and both were considerably different from those reported in the human and non-human primates. last year schnobrich, et al. described “next generation sequencing” (microgendx, formally pathogenius laboratory, lubbock, tx) of samples from 10 clinically normal mares and compared those results to traditional methods of diagnosing infectious endometritis.34 the main problem with the analyses is that the dna sequences were compared against a bank of known human and equine pathogens, therefore they did not consider all possible normal non-culturable bacteria that have heretofore been undocumented in the normal microbiota of the mare. also last year a second abstract was presented very similar to one we published in 20119 wherein uterine fluid samples obtained from mares were subjected to metagenomic dna sequencing of the l6s rrna gene. these mares were followed around the time of ovulation/artificial insemination (n=10) and during early pregnancy (n=10). the metagenomic sequencing identified over 200 bacterial species in both culture negative and culture positive samples demonstrating as we have that the uterus is not a sterile site at any point during and after estrus. proteobacteria and bacteroidetes phyla were statistically associated with culture positive samples according to the bonferroni correction. their pilot study strongly correlates with ours in evidencing of the presence of a complex bacterial microbiome of organisms that fails to grow using routine uterine culture methods.35 finally, from the same proceedings a third abstract was published describing the metagenomic analysis of the equine placental microbiome. fecal, oral, and vaginal samples were taken from pregnant mares within 30 days of foaling, as well as the gravid and non-gravid regions of the chorioallantois at the time of foaling (n=4). genomic dna was isolated from all samples, and the bacterial 16s ribosomal rna gene was amplified by pcr. similarly they reported a relative abundance of bacterial species within the chorioallantois. the three phyla represented in the gravid horn were firmicutes, proteobacteria, bacteroidetes, with the same three phyla plus actinobacteria in the non-gravid horn. the most abundant phyla within the oral, fecal, and vaginal samples firmicutes and proteobacteria were also detected in the chorioallantois. the authors noted that the most abundant bacterial phyla in gravid and nongravid chorioallantois share significant overlap, suggesting similar, but not identical, environments within different compartments of the chorioallantois. they also reported that phyla of relatively high abundance in oral and vaginal samples corresponded to those found in the chorioallantois, indicating possible associations between placental and extra-placental microbiota. however they found significant differences between the gravid horn and the fecal samples.36 clearly, this is a fast moving and clinically relevant area of investigation within the field of equine reproductive medicine and health. further controlled studies are underway in determining the clinical theriogenology • volume 10, number 3 • september 2018275 viability of this diagnostic tool in identifying clinical and subclinical cases of infectious endometritis and the role of specific components of the microbiome in the promotion of fertility and maintenance of pregnancy. in this study we have provided the largest and most comprehensive analysis of equine uterine microbiomes to date and have established the core microbiome of the healthy uterine endometrium. we have also demonstrated that the uterine microbiome of mares raised at deferent geographical locations differ from each other. conclusion we conclude from our results as well as past studies that culture-based systems miss the great diversity present in both diseased and healthy reproductive tracts. given this diversity, there is much to be studied relative to the intra-microbiota interactions and species-intrinsic factors that may be more relevant to the maintaining a state of balance and health and that an imbalance of these factors maybe more important in the development of uterine disease than the abundance of any given bacterial species. funding the author(s) received financial support for the research in-part from the bullock endowed professorship, oklahoma state university (to grh) and by the oklahoma agricultural experiment station (to ud). references 1. ansbacher r, boyson wa, morris ja: sterility of the uterine cavity. am j obstet gynecol 1967;99:394-396. 2. troedsson mh: breeding-induced endometritis in mares. vet clin north am equine pract 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zhou x, brown cj, abdo z, et al: differences in the composition of vaginal microbial communities found in healthy caucasian and black women. isme j 2007;1:121-133. 21. srinivasan s, liu cz, mitchell cm, et al: temporal variability of human vaginal bacteria and relationship with bacterial vaginosis. plos one;2010;5. 22. srinivasan s, hoffman ng, morgan mt, et al: bacterial communities in women with bacterial vaginosis: high resolution phylogenetic analyses reveal relationships of microbiota to clinical criteria. plos one. 2012;7. 23. marrazzo jm: vaginal biofilms and bacterial vaginosis: of mice and women. j inf dis 2013;207:1481-1483. 24. barfod kk, roggenbuck m, hansen lh, et al: the murine lung microbiome in relation to the intestinal and vaginal bacterial communities. bmc microbiol 2013;13:303. 25. yildirim s, yeoman cj, janga sc, et al: primate vaginal microbiomes exhibit species specificity without universal lactobacillus dominance. isme j 2014;8:2431-2444. 26. yang x, yang j, wang hn, et al: normal vaginal bacterial flora of giant pandas (ailuropoda melanoleuca) and the antimicrobial susceptibility patterns of the isolates. j zoo wildl med 2016;47:671-675. 27. giudice lc: challenging dogma: the endometrium has a microbiome with functional consequences! am j obstet gynecol 2016;215:682-683. 28. aagaard k, ma j, antony km, et al: the placenta harbors a unique microbiome. sci transl med 2014;6:237. 29. lauder ap, roche am, sherrill-mix s, et al: comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota. microbiome 2016;4:29. 30. santos tma, gilbert ro, bicalho rc: metagenomic analysis of the uterine bacterial microbiota in healthy and metritic postpartum dairy cows. j dairy sci 2011;94:291-302. 31. santos tma, bicalho rc: diversity and succession of bacterial communities in the uterine fluid of postpartum metritic, endometritic and healthy dairy cows. plos one. 2012;7(12). 32. machado v, oikonomou g, bicalho m, et al: investigation of postpartum dairy cows' uterine microbial diversity using metagenomic pyrosequencing of the 16s rrna gene. vet microbiol 2012;159:460-469. 33. peng y, wang yh, hang sq, et al: microbial diversity in uterus of healthy and metritic postpartum holstein dairy cows. folia microbiol 2013;58:593-600. 34. schnobrich mr, atwood k, barr b, et al: sequencing, culture and cytology results in 10 clinically normal mares. clin therio 2017;9:443. 35. sathe s, leiken a, plummer a: metagenomic sequencing of the uterine microbial environment during estrus and early pregnancy in mares. clin therio 2017;9:453. 36. xia yw, cornelius aj, donnelly cg, et al: metagenomic analysis of the equine placental microbiome. clin therio 2017;9:452. clinical theriogenology • volume 10, number 3 • september 2018277 figure. microbes isolated from non-culturable methods significantly differ from culture-based methods. a uterine swab obtained from a single mare were either analyzed using 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice editorial editorial dear friends of theriogenology, welcome to the conference issue (september) of our journal. but for time and effort freely provided by so many, this conference issue would not have made it. session chairs were very gracious in gathering manuscripts from presenters. scientific abstract committee has done a fine job in organizing abstracts (i do remember the days when i served on this committee). special thanks to ms. tara beasley of our management office for collecting documents and being a go-to-person for this important endeavor. as always, it is so good to have a scholar and friend in dr. john kastelic who amidst all his tight schedule helped us in copy-editing 25 manuscripts and 79 abstracts, thank you john! time spent in corresponding with colleagues (students scholars) and time spent in completing (orderly and decently) this task, all well worth their effort. many responded with alacrity and sincerity to my early morning e-mails. thanks to them all for their patience with me and to my wife of 43 years who kept me going each morning with an indian tea (thank you damayanthy!). believe me, i learned quite a bit in the last 3 months after spending ~ 200 hours. i am truly thankful for this providential assignment. we are truly thankful to omnipress for accommodating us so well. in our effort to bring a consistent and uniform pattern of formatting, editing, and copy-editing to our journal, we might have made a few unintentional mistakes and errors. please forgive us and we will do a better job for the next conference issue. please feel free to catch me at the conference to share your thoughts and suggestions. looking forward to work with incoming session chairs, members of scientific abstract committee, leaders of our organizations, our management office (dr. charles franz, executive director and ms. tara beasley, director of meetings and events), and dr. john kastelic for the 2020 conference issue. sincerely, augustine 173 clinical theriogenology • volume 11, number 3 • september 2019 174clinical theriogenology • volume 11, number 3 • september 2019 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false 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/convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice 82 contact muhammad-salman waqas salman.waqas@wsu.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9639, http://dx.doi.org/10.58292/ct.v15.9639 review report cesarean section in camelids: indications, technique, survival, and postoperative fertility muhammad-salman waqasa, alexis campbella, eduardo arroyoa, isaac bottb, abdelhak anouassic and ahmed tibarya acomparative theriogenology, department of veterinary clinical sciences, college of veterinary medicine, and center for reproductive biology, washington state university pullman, wa, usa bmountain west animal hospital, springville, ut, usa cveterinary research center, abu dhabi, uae abstract cesarean section is the most common surgery of the reproductive tract in camelids. however, limited information is available on indication, surgical approach, dam survival, postpartum complications, and neonatal survival following cesarean section in camelids. the objective of this study was to retrospectively evaluate medical records on 84 camels, 29 alpacas, and 3 llamas to evaluate the above factors. incomplete cervical dilatation (56%; n = 65/84) and uterine torsion (59.4%, n = 19/32) were the most common indications for cesarean section in camels and south american camelids (sacs), respectively. the neonatal survival (sac: 46.9%, n = 15/32; camels: 76.2%, n = 64/84) was acceptable, and postoperative fertility was excellent (sacs = 75.86%, camels = 72.6%). keywords: camelid; dystocia; surgery; cesarean; postpartum introduction camelids present several gestation and parturition characteristics that distinguish them from other domestic animals. they demonstrate almost exclusively (~98%) left horn pregnancies,1 epitheliochorial microcotyledonary placentation, and a short but explosive second stage (fetal expulsion) of labor. following parturition there is rapid uterine involution, resumption of ovarian activity, and restoration of fertility within 2 weeks of parturition. incidence of dystocia in camelids is reported to range from 2 to 8%.2–4 most cases of dystocia are resolved with non-surgical obstetrical manipulation.3 causes of dystocia reported in camelids include fetal maldispositions (head deviations, shoulder or carpal flexure, bilateral hip flexure), failure of cervical dilation, and uterine torsion.2,4,5 due to the diffuse placentation, premature detachment during dystocia can lead to fetal hypoxia followed by death or postnatal compromise. because of this risk, dystocia in camelids should be handled with the same degree of urgency as for mares.3 in addition, camelids have a narrow pelvic inlet and pelvic canal, and the cervix and vagina are more prone to laceration. thus, per vaginum obstetrical manipulations should be limited in time and force because of the high risk for complications such as vaginal adhesions and cervical lacerations.6 fetotomy is often contraindicated, particularly in alpacas, leaving cesarean section as the only option to resolve dystocia when vaginal manipulation fails to deliver the neonate.3 cesarean section is the most common reproductive emergency surgical procedure in large animal practice, including camelids.7 in ruminants, cesarean section is common and carries a favorable prognosis for both the dam and the offspring. the most common indications for cesarean section in ruminants is feto-maternal disproportion, uterine torsion, and severe fetal maldisposition.8–10 similar indications have been proposed for camelids.3,11,12 the surgical approaches for cesarean section in camelids include left flank (most common), low flank/ventrolateral and ventral midline (for llamas and alpacas only). in alpacas and llamas, a left flank or ventral midline approach can be used for cesarean section; however, a flank approach is recommended for field conditions and uncomplicated cases. a ventral midline approach under general anesthesia is recommended for cases of uterine torsion or cases of compromised uterine health. for camels, a higher left flank approach is recommended as compared to a ventrolateral (low flank) approach (described by elias13) which predisposes to herniation.7 postsurgical information on survival of neonates and dams and fertility post-cesarean section in camelids is limited.14 mailto:salman.waqas@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9639 citation line: clinical theriogenology 2023, 15, 9639, http://dx.doi.org/10.58292/ct.v15.9639 83 the objectives of this retrospective study were to evaluate indications for cesarean section, surgical techniques employed, dam and neonatal survival rates, and postoperative fertility. throughout the paper unless a specific species is mentioned, the term south american camelids (sac) will be used to refer to alpacas (vicugna pacos) and llamas (lama glama). the term camel will refer primarily to dromedary camels (camelus dromedarius). the term camelids will be used to include all three species. materials and methods medical records from alpacas and llamas that were admitted to the washington state university veterinary teaching hospital (wsu-vth) comparative theriogenology service for dystocia, medical records for alpacas from mountain west animal hospital (mwah), springville, utah, and medical records from camels from the veterinary research center (vrc), abu dhabi (uae) were reviewed. only those alpacas, llamas, and camels having undergone a cesarean section (21 alpacas and 3 llamas at wsu, 8 alpacas at mwah, and 84 camels at vrc) were included in this retrospective study. information retrieved from the medical records included the age of the female at presentation (information on age of camels was not available), time to initial presentation, indication for cesarean section, anesthetic protocol, surgical technique employed, survival of the dam and neonate, postoperative complications, and postoperative fertility of the dam. follow-up information was obtained by email or telephone interview with owners. owners were asked whether any complications developed after discharge from the hospital, the breeding history following discharge, whether dystocia occurred during subsequent pregnancies, if additional cesarean sections were performed in subsequent pregnancies, and the number of live neonates delivered in subsequent pregnancies. follow-up information for camels was obtained through review of medical records of embryo transfer recipients 1-year after surgery. the only information available for alpacas from mwah was age and parity of dam, cause of dystocia, postoperative complication, dam survival and fertility, and neonatal survival. all other relevant information on sacs was from wsu patients only. descriptive statistics were performed to determine the relative incidence of specific causes of dystocia, which led to cesarean section. results eighty-four camels, 29 alpacas (21 from wsu and 8 from mwah) and 3 llamas met the inclusion criteria for the study. one alpaca underwent 2 cesarean sections (2 years apart) and both were included in this study. sac females were either referred (45.8%; n = 11) or seen as a primary patient by the wsu-vth comparative theriogenology service (54.2%; n = 13). mean (± sem) age of sac females was 5.1 ± 0.47 years (range 2–10). information on age of camels was not available. the proportions of maiden (46.8%, n = 15) and multiparous sac females (53.2%, n = 17) were similar (p > 0.05). majority of female camels (92.9%, n = 78) were multiparous and either bred naturally (n = 11) or received an embryo at 8 days post-ovulation (n = 73). the mean (± sem) gestational age at the time of presentation for sac was 353.8 ± 2.49 days (range 329–376). the mean (± sem) gestational age at the time of presentation for camels was 376.1 ± 6.2 days (range 278–393) for naturally mated females and 374.1 ± 1.4 days (range 352–398) for embryo transfer recipients. the time to referral ranged from 0 to 72 h for sac females. in camels, the time from observation of the second stage of labor to surgery was less than 6 h for embryo transfer recipients and between 8 and 48 h in naturally mated females. failure of cervical dilation was identified as the most common indication necessitating a cesarean section (56%; n = 65) followed by fetal maldisposition (22.41%, n = 26) and uterine torsion (19.82, n = 23). failure of cervical dilation was the most common cause of dystocia (73.8%, n = 62) in camels. all the affected camels were part of a study on embryo recipients where pregnancy was maintained by daily progesterone injection until 2 weeks prior to the due date. the 2 remaining cases were alpacas, 1 of which demonstrated failure of cervical dilation due to fetal bilateral carpal flexure and ventral head/neck deviation. uterine torsion was the predominant cause of dystocia in alpacas and llamas (overall 59.4%, n = 19; alpacas 58.60%, 17/29; llamas 66.7%, 2/3). uterine torsion was significantly less common in camels (4.8%; n = 4). most sac uterine torsions were clockwise (80%; 12/15) and the degree of uterine torsion ranged from 90 to 360 degrees. in cases of sac uterine torsion, cesarean section was performed as a primary modality of treatment (73.68%, n = 14) or following failed rolling attempts (26.32%; n = 5). in 1 alpaca, the uterine torsion was resolved successfully with rolling technique; however, bilateral hip flexure of the neonate was detected resulting in a cesarean section. another alpaca developed uterine torsion as a maiden and in a subsequent pregnancy, both resulting in cesarean section. she was not rebred following the second cesarean section. other indications for a cesarean section in sacs included fetal maldisposition (25%, n = 8) and twins (6.3%, n = 2). fetal maldisposition was the second most common indication for cesarean section in sacs and the maldispositions included fetal bilateral hip flexure, fetal lateral head/neck deviation and fetal bilateral carpal flexion. the 2 dystocias associated with a twin pregnancy were in 1 llama and 1 alpaca. in camels, 21.4% (n = 18) of the cesarean sections were performed due to dystocia by fetal maldisposition. twin pregnancy was not an indication for any camel cesarean sections. fetal maldisposition was also the second most common indication (21.4%, n = 18) for cesarean section in camels. pre-surgical hematology was performed in 87.5% (n = 21) of sac cases at wsu only. complete blood count (cbc) and serum biochemistry were performed in 71.4% (n = 15) of sac cases, with only a packed cell volume (pcv) and total protein (tp) being performed in 28.6% (n = 6) of cases. the most common abnormalities identified were leukocytosis due to neutrophilia (80%; 12/15), elevated creatinine kinase (40%; 6/15), hypocalcemia (<9.0 mg/dl) (20%; 3/15), and elevated liver enzymes (13.3%; 2/15). both cases demonstrating elevated liver enzymes pre-surgically demonstrated prolonged systemic clinical illness prior to surgery due to hepatic lipidosis. the first case was an alpaca that presented on referral for decreased appetite and lethargy of unknown duration. hematology demonstrated leukocytosis due to neutrophilia, elevated fibrinogen, elevated gamma-glutamyl transferase (ggt), elevated aspartate aminotransferase (ast), elevated alkaline phosphatase (ap), and hyperglycemia. following hospitalization parturition was induced with http://dx.doi.org/10.58292/ct.v15.9639 84 citation line: clinical theriogenology 2023, 15, 9639, http://dx.doi.org/10.58292/ct.v15.9639 250 mcg cloprostenol, im (estrumate®, merck, madison, nj) cesarean section was performed because of lack of progression and evidence of fetal death and placental detachment. the female was non-responsive following anesthesia and was euthanized shortly after completion of surgery. the second case was an alpaca that was hospitalized for monitoring following diagnosis of a < 180-degree counterclockwise uterine torsion. during hospitalization, the female demonstrated progressive anorexia and development of hypoglycemia, elevated ast, and elevated ggt. supportive care was initiated, and parturition was induced using 250 mcg cloprostenol, im. a cesarean section was elected due to failure to progress to the second stage of labor 24 h after induction. the female and neonate were discharged from the wsu-vth without further complications. cesarean section was performed via left paralumbar fossa approach (80.17%; n = 93: 84 camels, 7 alpacas, 2 llamas) or ventral midline approach (19.83%; n = 23; 22 alpacas, 1 llama). all camel cesarean sections used a left paralumbar fossa approach. in contrast, a ventral midline approach was used for 71.86% (n = 23/32) of sac cases. detailed data on anesthetic management was available for 87.5% (n = 21) of sac cases at wsu. sedation prior to anesthesia was used in 57.1% (12/21) of cases. sedation consisted of 0.05–0.1 mg/ kg butorphanol tartrate im (torbugesic®, zoetis, kalamazoo, mi) (29.2%; 7/21) and/or 0.11–0.2 mg/kg diazepam iv or im (diazepam, dash, upper saddle river, nj) (20.8%; 5/21). anesthesia was induced using propofol (0.6–2 mg/kg, iv; propoflo®, zoetis, kalamazoo, mi) (57.1%; 12/21), guaifenesin (1–1.5 mg/kg 10% solution, iv; guaifenesin®, wedgewood, swedesboro, nj) (4.8%; 1/21), ketamine (2–6 mg/kg, iv or im; ketaset®, zoetis, kalamazoo,mi) (4.8%; 1/21), guaifenesin (guaifenesin®, wedgewood) + ketamine (ketaset®, zoetis) (14.3%; 3/21), or masked inhalation of an anesthetic agent in oxygen (19%; 4/21). all sac females were intubated with an endotracheal tube and maintained on isoflurane (isoflurane, piramel, talangana, india) (85.7%; 18/21) or sevoflurane (sevoflurane, akron, lak forst, il) (14.3%; 3/21) in oxygen. in all camel cases (100%; n = 76) surgery was performed with the female restrained in sternal recumbency. sedation was obtained with xylazine (0.25 mg/kg, iv; anased®, akorn, lake forest, il) alone or in combination with butorphanol (0.05 mg/kg, iv; torbugesic®, zoetis). local analgesia was provided with an inverted l block using lidocaine (4–5 mg/kg; lidocaine, vetone®, mwi, boise, id). caudal epidural analgesia with lidocaine (0.2 mg/kg; lidocaine, vetone®, mwi) was performed on only 7.9% (6/76) of camels. the flank procedure is performed in sedated and sternally restrained (cushed) animal using an inverted ‘l’ block local anesthesia. an oblique incision parallel to the direction of the quadriceps in the ‘cushed’ animal is made, on an imaginary line drawn from the tuber coxae to the base of the last rib, in the paralumbar fossa. a single-layer inverting pattern for uterine closure is suggested in cases where healthy uterine tissue is present and double-layer closure is recommended in cases where the uterus is compromised. retained fetal membranes, incisional infection, herniation, peritonitis, intestinal adhesions, and infertility are the most common postoperative complications. herniation and incisional infection due to myasis are especially common in camel cesarean sections performed in the field conditions.7 overall dam survival rate was 91.38% (n = 106) and was similar for sac (90.6%; n = 29) and camels (91.7%; n = 77). causes of death included peritonitis (n = 3), septic metritis (n = 2), and 1 case each of anesthetic complication, vaginal evisceration, and systemic compromise. the most frequent non-lethal postoperative complications were retained placenta (failure to deliver the placenta within 6 h of surgery) (15.5%, n = 18; sac 12.5%, n = 4; camels 16.7%, n = 14), surgical site infection or dehiscence (12.07%, n = 14; sac 3.1%, n = 1; camels 15.5%, n = 13; mostly due to myiasis) and metritis (6.03%, n = 7; sac 3.1%, n = 1; camels 7.1%, n = 6). in camels, complications from incisional site dehiscence due to maggot infestation were reduced with administration of local and systemic ivermectin treatment. other complications included one case each of radial nerve paralysis, sepsis, anemia, and anorexia. radial nerve paralysis and sepsis occurred in the same case. anorexia was observed following accidental laceration of the small intestine during cesarean section in 1 alpaca. the laceration was surgically repaired, and subsequent peritonitis was not observed. all postoperative complications in sac resolved prior to discharge. one alpaca developed clinical endometritis after discharge from the hospital and was successfully treated on the farm. neonatal survival was 68.1% (n = 79) with 46.9% (n = 15/32) for sacs and 76.2% (n = 64) for camels. in sac, 86.7% of the neonates were delivered alive but 1 was euthanized (demonstrated signs of prematurity) and 1 died (demonstrated signs of dysmaturity and respiratory distress) within 24 h following delivery. camel calf viability at delivery, after 1-week of life, and at weaning was 81.6%, 75% and 73.7% respectively. complete placental separation was present in all camel cases where the calf was delivered dead or died within 15 min after surgery. causes of death in the first week included sepsis (n = 2), encephalopathy (n = 2) and trauma (n = 1). overall postsurgical fertility was 73.28% (n = 85). postsurgical fertility was 75.86% in sacs with at least 1 offspring being born following cesarean section. of the surviving sac females (n = 29) reproductive records were available for 21 females following cesarean section. the owners reported no complications following patient discharge from the wsu-vth. breeding information was not available on five sac females because they were sold (n = 2), retired from breeding following cesarean section (n = 2), or had not been rebred at the time of follow-up (n = 1). postsurgical fertility was 72.6% for camels. of the surviving camels (n = 77), 48 were used as recipients in an embryo transfer program 1-year after surgery. of those, 70.8% (n = 34) became pregnant following 1 to 3 embryo transfers. other camel pregnancies were by natural mating. overall, postsurgical lifetime pregnancy establishment was not different between sac (75.86%) and camels (70.8%). discussion dystocia is relatively rare in camelids but constitutes the majority of reproductive emergencies in these species.2,3,5,7 in cases of dystocia the health and welfare of both the dam and the neonate must be considered.2 there are limited studies evaluating factors associated with cesarean section and outcome in camelids. dystocia of fetal origin seems to be more frequent in camels than in sac. most of the dystocias of fetal origin are due to postural abnormalities. although twinning is rare in camelids,2 2 cases of twins resulting in a cesarean section were observed each in an alpaca and llama in this study. these findings are consistent with other retrospective study in sacs;14 however, in that study a higher percentage of failure of cervical dilation in sacs was observed compared to our study. failure of cervical dilation has been associated with progesterone supplementation during pregnancy in camelids.15 http://dx.doi.org/10.58292/ct.v15.9639 citation line: clinical theriogenology 2023, 15, 9639, http://dx.doi.org/10.58292/ct.v15.9639 85 the survival rate of sac females in our study was slightly higher (90.9%) than in the retrospective study by miller et al (88%).14 overall survival rate for camels and sac was similar to those reported for ruminants (81 to 97.8%) in uncomplicated cases.16,17 survival rates in referral centers can be higher even in complicated cases because of the availability of more advanced care. fetal viability of sac in this study (46.9%) is slightly lower that that reported by miller et al. (59%).14 this could be due to differences in management of dystocia and time to presentation. in our conditions, most dystocia cases present after owners and referring veterinarians have attempted manipulation and the fetus is already dead at presentation to hospital. transportation time for patients traveling to the wsu-vth can also be extensive in some cases, resulting in fetal death prior to the cesarean section being performed. it is important to note that in cases where the neonate was deemed alive via transabdominal ultrasonography directly prior to the cesarean section survival rate was good (86%). neonatal survival rates in other species following cesarean section are variable. ruminants have good fetal survival rate because the second stage of parturition is normally longer.17 in contrast, the percentage of foals delivered alive following cesarean section due to dystocia is very low (11–25%), with survival rates to discharge ranging from 5 to 25%. the foal survival in elective cesarean was 58% compared to 25% for cesareans following a dystocia.18 foal survival is affected by the time from dystocia to cesarean. when duration of stage ii increases beyond 40  min, the incidence of stillbirth and foal mortality post delivery significantly increases.19 this low survival rate in equine fetuses in cases of dystocia is due primarily to fetal hypoxia due to placentation separation.18 a similar trend exists in camelids which also have a microcotyledonary diffuse placentation. miller et al. reported no significant relationship between the presence of uterine torsion and fetal death.14 similarly, in the present study 57% of the neonates delivered by cesarean section following a uterine torsion were discharged alive from the death. retained fetal membranes is described as the most common post-surgical complication in all large domestic animals.8,9,16–18 in the present study the incidence of retained fetal membranes was 15.5% which is considerably lower than that reported (88%) in the only other study on cesarean section outcome in sac.14 there may be a difference in the duration of time following surgery to define the presence of retained fetal membranes between these studies. our criterion was to classify retained fetal membranes if fetal membranes were still present more than 24 h following surgery. in most cases, we observed expulsion of the fetal membranes within 24 h without intensive management. possibly miller et al.14 used a more traditional definition of retained fetal membranes where the placenta is considered retained after 3 h. alternatively, this difference may reflect the fact that a significant number of our cases were in an advanced stage of placental detachment upon presentation. additionally, the higher incidence of retained fetal membranes observed in camelids in the miller et al14 study may be due to the higher percentage of failure of cervical dilation observed in their dataset. it is important to note that retention of the fetal membranes was not detrimental to dam survival rate or postsurgical fertility. there is limited information regarding the postoperative fertility in camelids following cesarean section. in ruminants, the prognosis for post-surgical fertility is generally good.17,20,21 in mares, postoperative foaling rates are considerably decreased (<40%) in the first year after cesarean section. however, foaling rate tend to improve in subsequent years.18 in our study, 75.86% of the sac females that were rebred, conceived, and delivered a live neonate compared to 90.5% in another retrospective study on llamas and alpacas.14 the reason for this discrepancy is not clear. in camels, there are no other reports on post-cesarean section fertility. overall, in absence of complications, the prognosis for fertility after cesarean section in camelids seems similar to that of ruminants. conclusion this retrospective study demonstrates that survival of the dam and neonate is excellent following cesarean section if intervention is early. cesarean section in camelids can be considered as a field procedure as in ruminants. dystocia due to failure of cervical dilation and uterine torsion were the most common indications for cesarean section in camels and sacs, respectively. the high rate of failure of cervical dilation in this case series is likely due to the maintenance of pregnancy with exogenous progesterone administration. postoperative fertility following cesarean section is generally good, but more observations are needed to determine risk factors for loss of fertility and neonatal survival. conflict of interest authors disclose no conflicts of interest. references 1. bianchi cp, gallelli mf, herrera jm, et al: current knowledge about the processes of luteolysis and maternal recognition of pregnancy in camelids. reprod domest anim 2023;58:3–9. doi: 10.1111/rda.14269 2. tibary a, rodriguez j, sandoval s: reproductive emergencies in  camelids. theriogenology 2008;70:515–534. doi: 10.1016/j. theriogenology.2008.04.024 3. tibary a, rodriguez js, anouassi a, et al: management of dystocia in camelids. american association of bovine practitioners conference proceedings; 2008 september 25–27; charlotte, north carolina; 2008. p. 166–176. 4. ali a, derar d, tharwat m, et al: dystocia in dromedary camels: prevalence, forms, risks and hematobiochemical changes. anim  reprod sci 2016;170:149–156. doi: 10.1016/j.anireprosci. 2016.05.004 5. pearson lk, rodriguez js, tibary a: uterine torsion in late gestation alpacas and llamas: 60 cases (2000–2009). small rumin res 2012;105:268–272. doi: 10.1016/j.smallrumres. 2011.12.008 6. rodriguez js, pearson lk, tibary a: parturition and obstetrics. in: cebra c, van-saun rj, anderson de, et al: editors. llama and alpaca care. 1st edition, st. louis, mo; elsevier: 2014. p. 274–286. 7. tibary a, campbell a, rodriguez j, et al: urogenital surgery in camelids. clin theriogenol 2020;12:271–291. 8. newman kd: bovine cesarean section in the field. vet clin north am food anim pract 2008;24:273–293. doi: 10.1016/j.cvfa. doi: 2008.02.009 http://dx.doi.org/10.58292/ct.v15.9639 https://doi.org/10.1111/rda.14269 https://doi.org/​10.1016/j.theriogenology.2008.04.024 https://doi.org/​10.1016/j.theriogenology.2008.04.024 https://doi.org/10.1016/j.anireprosci.​2016.05.004 https://doi.org/10.1016/j.anireprosci.​2016.05.004 https://doi.org/10.1016/j.smallrumres.​2011.12.008 https://doi.org/10.1016/j.cvfa.2008.02.009 https://doi.org/10.1016/j.cvfa.2008.02.009 86 citation line: clinical theriogenology 2023, 15, 9639, http://dx.doi.org/10.58292/ct.v15.9639 9. alexander d: bovine caesarean section 1. on-farm operations. in pract 2013;35:574–588. doi: 10.1136/inp.f6679 10. alexander d: bovine caesarean section 2. difficult caesareans (potential pitfalls and how to overcome them). in pract 2014;36:15–26. doi: 10.1136/inp.f7197 11. tibary a, anouassi a: surgery of the reproductive tract in camels. recent adv camelid reprod a 2000;1008–1025. 12. tibary a, anouassi a: obstetrics in camels. recent adv camelid reprod a 2001;1005:0501. 13. elias e: left ventrolateral cesarean section in three dromedary camels (camelus dromedarius). vet surg 1991;20:323–325. doi: 10.1111/j.1532-950x.1991.tb01277.x 14. miller ba, brounts sh, anderson de, et al: cesarean section in  alpacas and llamas: 34 cases (1997–2010). j am vet med assoc 2013;242:670–674. doi: 10.2460/javma. 242.5.670 15. pearson lk, tibary a: clinical management of postpartum hemorrhage following failure of cervical dilation in an alpaca. clin theriogenol 2014;6:111–117. 16. scott pr: ovine caesarean operations: a study of 137 field cases. br vet j 1989;145:558–564. doi: 10.1016/0007-1935(89) 90118-8 17. brounts sh, hawkins jf, baird an, et al: outcome and subsequent fertility of sheep and goats undergoing cesarean section because of dystocia: 110 cases (1981–2001). j am vet med assoc 2004;224:275–281. doi: 10.2460/javma.2004.224.275 18. abernathy-young kk, leblanc mm, embertson rm, et al: survival rates of mares and foals and postoperative complications and fertility of mares after cesarean section: 95 cases (1986–2000). j  am vet med assoc 2012;241:927–934. doi: 10.2460/ javma.241.7.927 19. mccue pm, ferris ra: parturition, dystocia and foal survival: a retrospective study of 1047 births. equine vet j 2012;44:22–25. doi: 10.1111/j.2042-3306.2011.00476.x 20. lyons na, karvountzis s, knight-jones tjd: aspects of bovine caesarean section associated with calf mortality, dam survival and subsequent fertility. vet j 2013;197:342–350. doi: 10.1016/j. tvjl.2013.01.010 21. hiew mw, baird an, constable pd: clinical signs and outcomes  of beef cattle undergoing cesarean section because of dystocia. j am vet med assoc 2018;252:864–872. doi: 10.2460/ javma.252.7.864 http://dx.doi.org/10.58292/ct.v15.9639 https://doi.org/10.1136/inp.f6679 https://doi.org/10.1136/inp.f7197 https://doi.org/10.1111/j.1532-950x.1991.tb01277.x https://doi.org/10.2460/javma.​242.5.670 https://doi.org/10.1016/0007-1935(89)90118-8 https://doi.org/10.2460/javma.2004.224.275 https://doi.org/10.2460/javma.241.7.927 https://doi.org/10.2460/javma.241.7.927 https://doi.org/10.1111/j.2042-3306.2011.00476.x https://doi.org/10.1016/j.tvjl.2013.01.010 https://doi.org/10.1016/j.tvjl.2013.01.010 https://doi.org/10.2460/javma.252.7.864 https://doi.org/10.2460/javma.252.7.864 introduction in 2021, the american college of theriogenologists celebrates its 50th anniversary. such an historical occasion warrants a very succinct, but necessary, reminder of the original purpose that led to the creation of an organization dedicated to the study of animal reproduction, society for theriogenology (sft), and the veterinary specialty that today we celebrate, theriogenology. during its early days, the sft established a series of guidelines for determining the reproductive potential of bulls. such guidelines eventually became what is today known as the society for theriogenology manual for breeding soundness examination (bse) of bulls, initially published in 1983, reviewed in 1992, and revised in 2018.1 given the increasing interest of veterinarians, reproductive physiologists, and owners in the development of a standardized method for selecting and predicting the potential fertility of males in food and companion animals, similar guidelines were also established for boars (1984),2 small ruminants (1980),3 dogs (1992),4 and stallions (1983).5 despite some differences in the breeding management and expectations amongst various breeding industries, most of the guidelines for the male bse follow a similar approach. these include the analysis of general health status, evaluation of the reproductive tract, libido and mating ability assessment, semen analysis, evaluation for venereally transmitted diseases (including those that are reportable), and analysis of breeding records (when available). the stallion bse was a product of a committee led by prominent theriogenologists and equine practitioners, including robert prebreeding season/prepurchase examination of the stallion: testicular ultrasonography, semen evaluation, and considerations camilo hernández-avilés, charles love department of large animal clinical sciences, college of veterinary medicine and biomedical sciences texas a&m university, college station, tx abstract the stallion breeding soundness examination (bse), as initially contemplated by the society for theriogenology, was intended to serve as a tool for selecting stallions that under good breeding management and mare fertility, were expected to efficiently render at least 75% of > 40 mares pregnant when bred naturally or 120 mares when bred artificially in 1 breeding season. since its initial introduction 38 years ago, major changes in the equine breeding industry have occurred. yet, the original purpose of the bse has remained somehow unchanged and a bse still provides to both practitioners and owners valuable information regarding the reproductive potential of the stallion and reproductive management options that could be implemented for optimizing fertility of a particular stallion. this manuscript discusses some aspects of bse that warrant special considerations, due to their implementation under field conditions and their interpretation regarding the potential fertility of stallions. keywords: stallion, breeding soundness, testes, semen quality, fertility kenney and john hurtgen (university of pennsylvania), robert pierson (colorado state university), don witherspoon (lexington, kentucky), and john simons (lexington, kentucky). this manual also incorporated comments from worldwide leaders in the study of equine reproduction at the time, including john hughes (university of california at davis), atwood asbury (university of florida), wendell cooper (university of pennsylvania), hans merkt (university of veterinary medicine at hannover, germany), marcel vandeplassche (ghent university, belgium), wladyslaw bielanski and marian tischner (krakow agriculture university, poland), among others. the list of individuals who in major or minor extents contributed to what is nowadays the stallion bse leads us to appreciate how this manual attempted to be internationally inclusive, scientifically based, and applicable for field situations by theriogenologists and general equine practitioners. from a comparative standpoint, it is worthwhile to remind practitioners and trainees that bulls, rams, bucks, and boars are mostly selected based on reproductive performance. other aspects related to genetic merit or physical conformation are only taken into consideration after a male has been proven to be potentially fertile. likewise, the results of a bse conducted in a food animal will assist in identifying males that do not have certain physical, behavioral, and reproductive characteristics above a previously established cut-off value. such individuals will be eliminated from the breeding population. in contrast, the genetic merit or pedigree, sports performance, or conformation clinical theriogenology 2021; 13: 289 will be always a priority when selecting a stallion for breeding purposes. the main goal of the stallion bse will be, thus, to identify the cause(s) of potential reduced fertility, and pursue, when possible, an alternative for the breeding management. exceptions to this will be cases of stallions with hereditary conditions (i.e. herda, hypp), or physical abnormalities that will largely hamper their breeding performance (i.e. wobbler syndrome, neurologic disease). as such, it will be very uncommon to cull stallions from a breeding farm due to results of a bse. salient points of stallion bse although the main objective of the present document is not to review thoroughly what is or what is not included in the original manual for the stallion bse,5 it is important to ponder certain concepts mentioned in that document that might warrant special attention by the audience. some of these include: 1. ‘the examination (bse) will assist in identifying the cause(s) of reduced fertility and the findings used to develop guidelines for the management of the stallion to enable it to achieve its maximum fertility’. a stallion that does not ‘pass’ a bse can still be fertile if adequate alternatives for breeding management are done. some stallions perhaps will never ‘pass’ a bse, and yet, can be utilized as studs with relative success. 2. ‘it is realized that this examination may not invariably and reliably predict the level of fertility that any particular stallion will achieve under a multitude of management conditions’. assuming that the bse will confidently predict the reproductive potential of a stallion is erroneous. this is particularly true when the breeding management and intrinsic fertility of the mares bred to a stallion are not taken into consideration. 3. ‘fertility predictions are made on the basis of the findings at the time of examination. we realize intrinsic fertility may vary over a period of time’. the bse will only determine the reproductive potential of a stallion for a specific time: the day of examination. it cannot be expected that the results of a bse will predict the performance of a stallion in the years to come, not even in the same breeding season in which the stallion is evaluated. 4. ‘there is no single physical or seminal parameter which is satisfactorily correlated with the fertility of the stallion and the best combination of measures remains to be determined’. practitioners, scientists, and breeders have always attempted to find a single test that can reliably predict male fertility. the only test that might fulfill this purpose is breeding fertile females under good management conditions. obviously, in vivo fertility trials are not an option when determining fertility potential of stallions. as such, the bse should always take a holistic approach. this implies the inclusion of multiple sperm quality assays that are logical and have scientific merit. also, interpretation of these assays should not be only based on high or low values compared to an arbitrary cut-off. rather, their interpretation should take into consideration the potential causes of a ‘low’ or unusual value and their implications to the breeding method and management used for that particular stallion. 5. ‘fertility evaluation of stallions is not a precise science… the major variables affecting the results are the innate fertility of the band of mares the stallion is bred to, the overall management of both the stallion and the mares, the veterinary management, and last, but not least, the quality of the performance of the tests as well as the quality of the interpretation of all findings’. this last sentence summarizes the 4 aspects that were discussed above. of interest is the phrase regarding the quality of tests and the interpretation of all findings during bse. practitioners must be aware that any sperm quality test is prone to errors, due to the nature of the test, errors when performing such evaluations, or the limitations and competence of the evaluator. the concept ‘garbage in – garbage out’ is very relevant in this situation. impact of new techniques for equine breeding management on stallion bse at this point, it is also worthwhile to recall the suggestions in the stallion bse manual regarding the expected levels of fertility that should be achieved by potentially satisfactory breeders. a stallion would be classified as ‘satisfactory’ if his physical, behavioral, and reproductive characteristics would allow him to render pregnant at least 75% of 40 mares (30/40 mares) bred by natural breeding, or 120 mares (90/120 mares) bred by artificial insemination. in modern equine breeding industry, it is not uncommon to find very popular stallions to be booked to 100 200 mares in the thoroughbred or > 200 mares where artificial insemination is allowed (i.e. quarter horses and warmbloods). additionally, advances in semen preservation technologies and artificial insemination, including cooled storage, freezing/thawing, low-dose insemination, estrus, and ovulation synchronization, and more recently intracytoplasmic sperm injection (icsi), have allowed high fertility to be achieved in selected individuals that otherwise would not be considered as potentially fertile, based on the guidelines established initially by the manual. the readers are referred to some previous works that have illustrated specific circumstances of breeding management for the subfertile stallion to achieve optimal fertility.6-9 that many stallions have benefited from the use of modern methods for semen processing, storage, and insemination does not imply that the manual for the stallion bse is outdated nor useless. this manual presents a very effective and thorough framework to investigate obvious causes of potential subfertility in stallions, and can be also used as a starting point to monitor the reproductive performance of stallions that just began their reproductive career. hence, theriogenologists, trainees, and general practitioners are encouraged to adopt the bse as the method of choice for the estimation of reproductive potential in stallions, either when presented the first time for stud, before every breeding season, after the occurrence of any clinical theriogenology 2021; 13: 290 health problem that might impact his reproductive potential, or as part of a prepurchase examination. testicular evaluation in the context of bse evaluation of scrotal contents in the stallion has received special attention by practitioners and owners, particularly in the case of the thoroughbred. in this industry, when a stallion is retired to stud, it is common by the 2 parties involved (buyer and seller), to sign a policy for first-season subfertility insurance. if complaints regarding the breeding performance of the stallion appear while in his first season as a stud, a veterinarian is required to conduct an exam on behalf of the policy underwriter. most of these examinations are based on assessment of the stallion’s general health status and evaluation of scrotal contents, particularly testicular size. unfortunately, veterinarians are asked to render an opinion about the potential fertility of the stallion in question, without the option of examining other aspects of reproductive performance, including libido, mating ability, ejaculatory function, or semen quality. the results of the firstseason subfertility insurance are used to judge if the stallion is likely to achieve a minimum seasonal pregnancy rate of 60% when booked to a defined number of mares. in addition, it is also expected that the veterinarian recommends the maximum number of covers per day (or per week) that should be allowed for that stallion.10 stallion testicular size can be determined by measuring either total scrotal width (tsw, cm) or total testicular volume (ttv, cm3). total scrotal width, is the largest measurement taken directly across both testes and the scrotum, commonly done with calipers. light breed stallions had a mean (± sd) of 10 ± 0.9 cm tsw when measured during the physiological breeding season (april september).11 a difference in tsw was noticed for stallions ≥ 7 years old when compared to stallions ≤ 6 years old (10.9 cm versus 9.6 -10 cm, respectively; p < 0.05). similar values (10.1 10.8 cm tsw) were reported for standardbred stallions, located either at the racetrack or the breeding farm.12 in that study, younger stallions that also were actively trained or were exposed to medications while at the racetrack had smaller tsw than nonmedicated or breeding stallions. values for tsw for thoroughbred or standardbred stallions (n = 106, 3 6 years) recently retired from racing were 9.2 10.3 cm.13 these workers indicated that 20% of stallions that did not fulfill the criteria to be considered as a ‘satisfactory prospective breeder’ also had a tsw < 8 cm. it is commonly assumed a tsw value > 8 cm as ‘normal’ for breeding stallions.14 yet, to date, studies in which a direct relationship between tsw and stallion fertility are lacking, and probably it would not be possible to establish such a relationship, given the complex nature of stallion fertility and breeding management. determination of total testicular volume (ttv, cm3) is performed either by estimating testicular measurements using a caliper or ultrasonography (figure 1). for both techniques, the stallion should be placed in a palpation stock and sedated with the aid of α2-agonists given intravenously (e.g. xylazine hydrochloride: 1.1 mg/kg or detomidine hydrochloride, 20 40 µg/kg). this helps in the descent of both testes into the scrotal sac, facilitating reliable measurement. regardless of the method used, the practitioner must estimate 3 parameters (length – l, width – w, and height – h) for each testis. given that the shape of the stallion testis resembles an ellipsoid, testicular volume can be confidently calculated by using a formula to estimate the volume of an ellipsoid: 4/3 π x l/2 x w/2 x h/2 or 0.5236 x l x w x h, inputting values in cm from each figure 1. estimation of total testicular volume (ttv, cm3) by measuring 3 dimensions of each testis: a. length; b. width; c. height. images taken using an anatomical specimen are also shown for proper orientation of the ultrasound beam. clinical theriogenology 2021; 13: 291 testicular measurement.15 results obtained from each testis are added, and the combined testicular volume is also expressed as the combined testicular weight in grams (g). similar results of ttv can be obtained when using either caliper or ultrasound measures.15 yet, the use of ultrasonography for determining ttv might be advantageous, given that more precise measurements of the testicular parenchyma are obtained, without the interference of scrotal skin or other abnormalities such as hydroceles that can artifactually increase ttv. substantial inter-operator variability can be expected when using either technique; therefore, readers are referred to a method used by the authors to properly determine ttv with ultrasonography.16 the relationship between tsw or ttv and daily sperm output (dso) has been established.15,17,18 this is of great help for estimating the number of mares that can be bred daily, or the number of seminal doses that can be produced for artificial insemination using fresh, cooled, or frozen/thawed semen. tsw, as determined by caliper measurements, accounted for up 60% of the variation in daily sperm output of stallions whose semen was collected for 10 consecutive days.17 ttv, as determined either by caliper or ultrasound measures, accounted for up to 85% of the variation in daily sperm output of stallions whose semen was collected for 7 consecutive days.15 in that study, the formula that best established a relationship between ttv and dso was (0.024 x ttv)-1.26 (figure 2). an additional calculation is replacing 1.26 by 0.76.18 this is to establish a range of dso for the estimated ttv in that particular stallion. based on previous studies,15,18 mature stallions (4 6 years), including thoroughbreds, standardbreds, warmblood, and pony stallions, have an average ttv of 250 cm3 using the regression formulae mentioned above, it can be expected that such stallions produce 4.74 5.24 x 109 sperm if collected when at dso. under ideal conditions, the relationship between ttv and dso should be performed in stallions that have been collected for several days, to deplete extragonadal sperm reserves and have a better appreciation of the amount of sperm that the testes are capable to produce on a daily basis.17,19 however, attempting to collect a stallion daily for 7 10 days is impractical, due to monetary and time limitations. even when the stallion dso is affected by the testicular size, the earliest in which dso is expected to be stabilized is after 3 5 consecutive days of semen collections in stallions with small testes (148 245 cc3).20 hence, it is often easier to collect 2 ejaculates, 1 hour apart – as described in the stallion bse manual – to have a crude approximation of the potential dso for that particular stallion. the application of ttv and predicting dso is limited unless the efficiency of the testes to produce sperm is not considered. some stallions are presented with small testes, yet high testicular efficiency, whereas other stallions can have larger but inefficient testes. rather than comparing ttv and dso against certain reported values, it is more convenient to determine if the amount of sperm ejaculated by that stallion corresponds to his predicted dso. this concept, known as spermatogenic efficiency, is the product of actual dso/predicted dso, where actual dso is the total sperm number obtained after collecting semen from the stallion at dso. it becomes obvious that normal spermatogenic efficiency should be as close as possible to 100%. a dramatic decrease in spermatogenic efficiency can be a consequence of transient testicular dysfunction, or permanent testicular degeneration, depending on the chronicity of the insult to the testes. a marked reduction in spermatogenic efficiency is most common in aged stallions with declining testicular function; often this is the earliest indicator of testicular dysfunction despite no change in testicular size.21 as a side note, figure 2. the linear relationship between total testicular volume (ttv, cm3) and daily sperm output (dso, 1 x 109 sperm), as determined by the formula (0.024 x ttv)-1.26. adapted from love et al.15 clinical theriogenology 2021; 13: 292 the spermatogenic efficiency per gram of testicular parenchyma has been reported in the stallion. for mature, sexually active stallions, it is estimated that 1 gram of functional testicular parenchyma can produce 14 18 x 106 sperm/day.22,23 in aged stallions, values approaching 6 8 x 106/g/day are concurrent with a decrease in total sperm number in each ejaculate at dso (< 2 x 109 sperm), and reduced pregnancy rates.21 this suggests that spermatogenic efficiency and spermatogenic efficiency per gram of testis are useful indicators of general testicular function in the stallion, and their estimation should always be included when evaluating testicular size during the bse. semen analysis and its potential relationship to stallion fertility perhaps, semen analysis and its interpretation has been considered as the main component of the stallion bse. although this assumption is not completely erroneous, judging the potential fertility of stallions based solely on the results of a spermiogram can lead to clinical errors. the document presented in this issue by authors discusses to a greater extent the methods that are commonly used for evaluation of semen quality in the stallion. yet, it is worthwhile to briefly discuss some of these techniques in the present document. historically, semen analysis of the stallion bse included estimation of total sperm numbers in the ejaculate, being the product of the gel-free seminal volume (ml) multiplied by the sperm concentration per ml (1 x 106), the estimation of both total and progressive sperm motility, and the evaluation of sperm morphology characteristics. these semen assays were considered to allow practitioners to conduct a bse under field conditions, using only limited and inexpensive equipment. also, the inclusion of these types of tests has its origins in what was previously done in other species, such as the bull, and what other equine researchers at the time had also observed.24-26 such analyses were mainly conducted using light, or phase-contrast microscopy for estimation of sperm motility and morphology, and direct enumeration methods for quantification of sperm numbers (e.g. hemocytometer). although the stallion bse manual recommended phase-contrast microscopy for assessing sperm motility, and particularly for sperm morphology, these authors also realized that using techniques compatible with a light microscope, such as stained smears, was more convenient for some field situations. thus, most practitioners in the field prefer to use light microscopy-based techniques for semen analysis. however, under certain circumstances, such techniques might represent a potential limitation for objective analysis of stallion sperm quality, leading to misinterpretation and misjudgment of a stallion’s potential fertility. based on the stallion bse manual, a potentially fertile stallion should ejaculate at least 1 x 109 morphologically normal, progressively motile sperm in the second ejaculate each month of the year, after 1 week of sexual rest. workers in the netherlands, using a similar evaluation system to the stallion bse manual, reported a lower threshold for the number of morphologically normal, progressively motile sperm in the ejaculate (500 600 x 106) can be considered as acceptable for breeding stallions.27 however, using such ‘threshold values’ is problematic, particularly in stallions with excellent sperm quality that require considerably fewer sperm to render fertile mares pregnant under adequate breeding management, or stallions that have acceptable semen quality and yet require to be bred to few mares for optimal fertility. this is particularly challenging when evaluating stallions for artificial insemination programs, given that certain minimum values for sperm numbers must be accommodated when breeding mares (a.k.a. ‘breeding dose’). even when some standards have been set for what is known as a seminal dose (e.g. fresh semen on the farm = 500 x 106 progressively motile sperm; cooled semen = 1,000 x 106 progressively motile sperm; frozen semen = 250 x 106 progressively motile sperm), these standards are merely based on industry expectations rather than reliable scientific data. for some stallions, these standards might represent an inefficient use of semen, due to their high intrinsic fertility, whereas for other stallions, greater numbers of sperm should be included in a ‘dose’ to achieve optimal fertility. also, it is important to acknowledge that many breeding scenarios and management systems can confound the expectations of semen quality and its relationship with stallion fertility. a clear example of this is thoroughbred stallions that have ‘low’ sperm quality but can still manage a relatively large book of mares with appropriate reproductive management of both the stallion and mares.28 a common question asked by practitioners and owners when conducting a stallion bse is to what extent do these semen quality tests reliably predict fertility? if the relationship between the seminal traits of a particular stallion(s) and his (their) fertility is high (often estimated in research scenarios by product-moment correlation analyses), then it is assumed that the prediction capacity of the semen assays is good. an example of this would be a stallion with excellent semen quality that deposits sufficient numbers of ‘good’ sperm in the uterus of a fertile mare. a similar scenario will be that of a stallion with poor semen quality, who is bred to fertile mares and does not render them all pregnant. in both cases, the relationship between sperm quality and fertility will be linear (either positive or negative), leading to the assumption of a high predictive value of semen analysis over fertility. nonetheless, many of the studies that had attempted to establish a relationship between stallion semen quality and fertility have not taken into consideration either intrinsic differences amongst stallions in sperm quality or potential differences in mare intrinsic fertility and breeding management for that particular stallion.29,30 hence, for some practitioners and scientists, the relation between semen quality and fertility appears nonexistent. to the authors’ knowledge, few published studies have attempted to demonstrate the relationship between sperm quality traits and stallion fertility, when mare intrinsic fertility and breeding management are somehow controlled.31-36 of these, it is worth mentioning that stallions with high values of sperm motility (total, not necessarily clinical theriogenology 2021; 13: 293 progressive), viability (i.e. plasma membrane intactness), normal morphology, and dna intactness (as determined by the sperm chromatin structure assay), tend to have higher fertility. yet, in these studies, a combination of these assays, rather than the use of just 1, has allowed establishing a direct relationship between semen quality and fertility. this latter aspect emphasizes the concept of using a holistic approach whenever evaluating semen quality in a stallion as part of the bse. another common question regarding semen analysis whilst conducting a bse is the use of threshold values to describe the quality of an ejaculate, and thus, the potential fertility of a stallion. in other domestic species, threshold values have been established for percent progressive motility (e.g. > 30% for ruminants,1 > 70% for boars,2 > 70% for dogs4), and percent morphologically normal sperm (~ 70 75% for the species aforementioned). although the stallion bse manual does not contemplate a minimum percent of morphologically normal nor progressively motile sperm, it is common to observe in many academic and nonacademic publications that such values are assumed to range from 60 70% for fertile stallions.14, 24-27 we agree that the higher the percentage of morphologically normal sperm and motile (not necessarily progressively motile) sperm in the ejaculate, the higher will be the potential fertility of the stallion. it is common to observe stallions with relatively ‘low’ percentages of both progressively motile sperm, or morphologically normal sperm that also have adequate fertility. such stallions might ejaculate enough sperm to compensate for the ‘lack’ of progressively motile or morphologically normal sperm. this is most common in thoroughbred stallions, although similar results can occur in stallions used for artificial insemination. data from 2 stallions with distinctinctly different fertility rates, despite 1 of them having somehow ‘better’ sperm quality, are shown in table 1. the sperm motility and viability in both stallions are similar and can be considered high. sperm dna integrity for both stallions were also similar to those in fertile stallions.33 two noticeable differences in terms of sperm quality can be observed: 1) whereas the aqha stallion had a lower testicular size, his sperm quality parameter stallion a (6-year aqha) stallion b (10-year tb) total testicular volume (cm3) 214 413 total sperm numbers (at dso; x 109) 3.9 4.4 3.6 5.7 spermatogenic efficiency (%) 89 66 sperm total motility (%) 80 90 73 78 sperm progressive motility (%) 68 85 47 59 morphologically normal sperm (%) 56 61 47 59 abnormal heads (%) 6 18 10 13 detached heads (%) 0 1 7 22 proximal droplets (%) 12 26 58 67 distal droplets (%) 3 7 4 11 bent midpieces (%) 0 2 1 5 bent tails (%) 0 2 3 compα-t (%) 8 10 14 25 mare book 41 47 54 166 seasonal pregnancy rate (%) 47 49 77 91 per cycle pregnancy rate (%) 27 28 59 69 table 1. semen quality of a 6 year old aqha stallion with excellent sperm quality and low reproductive efficiency (2 breeding seasons), and a 10 year old thoroughbred stallion with acceptable sperm quality and high reproductive efficiency (5 breeding seasons) dso = daily sperm output compα-t = cells outside the main population (dna-damaged sperm, scsa) clinical theriogenology 2021; 13: 294 testes were more efficient in producing sperm than those of the tb stallion. 2) the tb stallion had a lower percentage of morphologically normal sperm due to the presence of a considerable proportion of detached heads and proximal droplets. why then did the aqha stallion had such lower reproductive efficiency? perhaps, the tb stallion ejaculated enough morphologically normal sperm into the mare’s uterus to compensate for the presence of abnormal sperm. one might assume that only morphologically normal sperm can be both motile and viable, but based on research in our laboratory, sperm with either proximal or distal droplets are more likely to be viable than morphologically normal sperm (or: 4.95; p < 0.0001).37a different scenario would have occurred if this tb stallion was used for a program of artificial insemination with cooled or frozen/thawed semen, since morphologic abnormalities observed can negatively impact the longevity of stored sperm. also, even though the percent of morphologic abnormalities in the tb was considerably ‘high,’ this type of sperm abnormality perhaps did not interfere with his fertility, as neither proximal nor distal droplets negatively impact the likelihood of stallion sperm to establish a pregnancy when natural cover is used.38 for the aqha stallion, the picture is more complicated because this stallion has adequate-to-excellent sperm quality; thus, an explanation for his reduced fertility can not be obtained based on the bse. one might argue that his testicular size could be a limitation for obtaining adequate fertility, but his testes were more efficient in producing sperm than in the tb stallion, and the aqha stallion bred fewer mares than the tb. at this point, we speculate that the cause of reduced fertility in the aqha stallion was due to breeding management, rather than to intrinsic sperm quality. in stallions with acceptable sperm quality, mare intrinsic fertility and breeding management are the main limiting factors for adequate fertility when artificial insemination with cooled semen is used.39. evaluation of breeding records: the good, the bad, and the ugly if evaluation of scrotal contents or semen quality in the stallion might present a certain degree of difficulty for some practitioners, the analysis of the reproductive history or breeding records can become the most challenging part of the bse. one of the reasons is the lack of a consensus when defining reproductive efficiency. many definitions of fertility can be equally true, depending on to whom the question is asked. for instance, the owner of the stallion will be interested in the number of foals that are born during the season after breeding his stallion to a group of mares. this definition of fertility is known as foaling rate (fr), and in many equine breeding industries, is the most important measure of breeding efficiency, given its economic implications. foaling rate is not a fair measure of stallion fertility, just because mares can become pregnant at an efficient rate due to the high intrinsic fertility of both the mare and the stallion, and foaling rates might be reduced due to unexpected events, such as abortion or perinatal losses. another definition of fertility is the seasonal pregnancy rate (spr), being the number of mares that became pregnant after being bred to a stallion during the breeding season divided by the total number of mares bred to that stallion.40 this parameter was historically considered the definitive measure of fertility and has been used in various studies that attempted to estimate the relationship between sperm quality and stallion fertility. although it seems to be a logical parameter for the quantification of fertility, some stallions can have a high spr despite being subfertile. these stallions can have similar spr to fertile stallions, but be less efficient and require more breedings to achieve a high spr. it is considered that under well-managed breeding systems, spr should be at least 75%. a better definition of intrinsic stallion fertility is the per cycle pregnancy rate (pc-pr), which is the total number of mares pregnant at the end of the season divided by the total number of mare cycles bred.40 the assumption is that all mares that are bred are of normal fertility and cyclicity and that breeding occurred when the mare was in standing heat and close to ovulation. in general terms, determining pc-pr allows eliminating certain confounding factors that can affect stallion fertility and might facilitate identification of highly fertile stallions that can render pregnant the mares of their book in fewer cycles, from those subfertile stallions that require multiple cycles. in general terms, pc-pr for commercial stallions approaches at least 50 ¬60%; therefore, on average, a stallion will require 2 cycles or less to render mares pregnant. still, this parameter can be problematic when addressing causes of stallion subfertility, particularly in mares bred via artificial insemination, as they are often bred multiple times in the same cycle, or in mares bred either by live cover or artificial insemination due to the intrinsic fertility of the mares bred to that stallion. hence, it would be advantageous to determine the first-cycle pregnancy rate (fc-pr), that is the number of mares pregnant to the first breeding of the season, divided by the total number of mares pregnant that season.40 by using this parameter, negative effects of inherently subfertile mares on stallion fertility can be minimized. however, this parameter is oftentimes hard to measure, particularly without adequate breeding records. of this last parameter, caution must be exercised for mares that foal later in the breeding season, as they have a lower chance of getting bred and conceive on the first cycle, which in turn also would artifactually decrease fc-pr for that stallion. a final consideration regarding the evaluation of breeding records is to determine the types of mares booked to a particular stallion, and expectations of the stallion’s owner regarding his fertility. breeding a fertile stallion to maiden or foaling mares is very common when the stallion is at his first(s) season(s) at stud. if the resulting foals are of high merit, then it is very likely that this stallion will be booked for subsequent seasons to mares of high fertility. conversely, it is also common to observe highly fertile stallions to be bred to barren mares when the stallion’s popularity decreases, which might reduce his fertility due to ‘nonstallion factors,’ as illustrated in table 3. the mare class (maiden, barren, ‘slipped’, in-foal, not bred) that is bred to a clinical theriogenology 2021; 13: 295 stallion can reveal very important information regarding the intrinsic fertility of that sire. as such, practitioners should become familiarized with various definitions for mare class in the breeding context, and its impact on the potential fertility of a stallion. a more detailed description of this is provided.40,41 regarding the second aspect, theriogenologists and general practitioners often ignore what are the expectations of the owner regarding the potential fertility of a stallion. it is assumed that if a stallion can render pregnant at least 40 mares by natural cover or 120 mares by artificial insemination, then he can potentially have more mares booked to him. yet, some owners (particularly in the artificial insemination industry) do not desire to breed so many mares. for some of them, having their mares bred to that stallion in question, or perhaps some mares locally are enough reward to their economical and time investment in the stallion. we have had instances in which owners present their stallions for a bse, and their desire is not to breed more than 10 20 mares, mostly via ‘live cover’. in those cases, such individuals will be exceptionally fertile if their breeding management is adequate. eventually, some of these stallions are presented in subsequent breeding seasons for ‘reevaluation’ and either the owners decide to increase or decrease their books. adjustments regarding the breeding management of these stallions are done accordingly. should we still judge stallion fertility assuming that the bse is a ‘pass’ or ‘fail’ exam? last, but not least, a consideration regarding the classification system for breeding stallions warrants some discussion. in food animals, any male that does not fulfill the criteria set for being considered as a potentially ‘satisfactory breeder’ is classified either as ‘questionable’ or ‘unsatisfactory’. the ‘questionable’ category is used for males that might ‘pass’ a bse after a certain interval if some parameters regarding physical health or semen quality can be improved. the ‘unsatisfactory’ category is used for males that definitively do not possess the physical, behavioral, and reproductive characteristics required to ‘pass’ a bse, and that will not improve with time. for the stallion bse manual, the criteria that define whether or not this individual will pass are: • demonstrates good libido, as indicated by short reaction time, to move freely, to find and mount the mare (or breeding dummy), to make intromission, and to promptly ejaculate semen free of urine and/or blood (either in the mare’s vagina or an artificial vagina). • the penis must be of normal size and shape and be free of lesions of an inflammatory nature. • the bacteria recovered from the semen and 2 urethral swabs should be inconsistent in type and reduced in number after ejaculation. there should not be colonies of the organisms of contagious equine metritis (taylorella equigenitalis). in addition, multiple pure cultures, or an unexplained increase in colony count on the second ejaculate are considered reproductive tract infections and necessitate further investigation and clarification. • there should not be an indication of equine infectious anemia as indicated by a negative coggins test. • there should be 2 scrotal testes and epididymides of palpably normal size, shape, and texture. • the stallion should have the potential ability to ejaculate at least 1 x 109 morphologically normal, progressively motile sperm in the second ejaculate after 1 week of sexual rest, each month of the year. if a stallion meets all these criteria, he is classified as a ‘satisfactory prospective breeder’. if he is borderline in 2 or more criteria, he is considered ‘questionable’, and if he is very low in 2 or more criteria or has severe permanent shortcomings, he is considered ‘unsatisfactory’. some of these criteria can be seen as an ‘all or none’ (e.g. those regarding the presence of venereal organisms in the reproductive tract and semen), the occurrence of cryptorchidism or aplasia of the internal reproductive organs (i.e. epididymis), or those mentioned above regarding the general health status of the stallion. yet, rather than considering that a stallion passed his bse because he fulfilled all the criteria previously mentioned, theriogenologists and general practitioners should see the bse as a clinical tool instead of a ‘checklist’ to complete. if a stallion does not display adequate libido, has the presence of urine or blood in the ejaculate, presents some lesions in the penile integument, or his sperm number and quality is marginal, how can i approach those ‘issues’, and offer potential management to maximize this stallion’s fertility? certainly, many conditions that are noticed in breeding stallions can be class of mare spr pc-pr barren 2/3 (67%) 1/7 (29%) foaling 12/22 (86%) 19/32 (59%) slipped 2/2 (100%) 2/4 (50% all classes 23/27 (85%) 23/43 (53%) table 3. fertility results from a 22-year-old thoroughbred stallion with a book of 27 mares clinical theriogenology 2021; 13: 296 easily diagnosed and managed, if not treated nowadays. hence, we desist from classifying stallions based on these 3 categories when conducting a bse, and we prefer to have a clear discussion with the owner/agent of that stallion, hoping to find the best clinical, ethical, and scientific approach to enhance stallion fertility. we hope the present document will serve as the basis for academic discussions among theriogenologists, general practitioners, academicians, owners, and agents, to encourage the use of the stallion bse manual, taking into consideration the new evidence that is daily published regarding male reproductive physiology and fertility. the practitioner should also be concerned about the concentration of the sperm sample they have received (either cooled or frozen). evaluation of sperm motility alone is an insufficient measure of sperm quality. excellent sperm motility associated with low sperm numbers can result in low fertility. acknowledgments authors recognize the immense efforts of the committee that initially established the guidelines for the stallion bse manual, as well as the dedication of many colleagues that dedicated their academic life to preserve and take to the next level what was envisioned by the pioneers in the discipline of stallion reproduction. although many names came to our minds when thinking about stallion reproduction, the authors express their gratitude to their mentors and friends bob kenney, terry blanchard, and dickson varner. other individuals who are not mentioned here were not ignored on purpose. conflict of interest none to declare. funding legends premier stallion season auction, texas a&m university references 1. koziol jh, armstrong cl: manual for breeding soundness examination of bulls, 2nd edition. mathews, al. society for theriogenology 2018. 2. hurtgen jp: 1984. manual for the reproductive examination of the boar. hastings, ne. society for theriogenology 1984. 3. ott rs, memon ma: breeding soundness examinations of rams and bucks, a review. theriogenology 1980;13: 155-164. 4. purswell bj, althouse gc, root kustritz mv: guidelines for using the canine breeding soundness evaluation form. theriogenology handbook, sa-c1 (8/92). 1992. 5. kenney rm, hurtgen j, pierson r, et al: manual for clinical fertility evaluation of the stallion. hastings, ne. society for theriogenology 1983. 6. blanchard tl, love cc, thompson ja, et al: role of reinforcement breeding in a natural service mating program. proc am assoc equine pract 2006;52:384-386. 7. blanchard tl, morehead jp, ramsey j, et al: how to use planned multiple matings per estrus to improve pregnancy rates achieved by some aged subfertile thoroughbred stallions. proc am assoc equine pract 2008;54:364-366. 8. varner dd, love cc, brinsko sp, et al: semen processing for the subfertile stallion. j equine vet sci 2008;28: 677-685. 9. blanchard tl, brinsko sp, love cc, et al: case study of processing and insemination techniques: attempts to improve fertility of an aged stallion with dilute semen of poor quality. j equine vet sci 2012;32:5-11. 10. blanchard tl, brinsko sp, love cc, et al: how to use testicular measurements for first-season subfertility insurance considerations in thoroughbred stallions. proc am assoc equine pract 2008;54:374-379. 11. thompson dl, pickett bw, squires el, et al: testicular measurements and reproductive characteristics in stallions. j reprod fertil suppl. 1979;27:13-17. 12. woods gl, garcia mc, kenney rm: variations in testicular size of standardbred stallions. proc am assoc equine pract 1980;26:117-121. 13. blanchard tl, varner dd: evaluating breeding soundness in stallions – 5: predicting potential fertility. vet med 1997;815-818. 14. pickett bw, voss jl, bowen ra, et al: seminal characteristics and total scrotal width (t.s.w.) of normal and abnormal stallions. proc am assoc equine pract 1987;33:487-518. 15. love cc, garcía mc, riera fr, et al: evaluation of measures taken by ultrasonography and caliper to estimate testicular volume and predict daily sperm output in the stallion. j reprod fertil suppl 1991;44:99-105. 16. love cc: how to measure testes size and evaluate scrotal contents in the stallion. proc am assoc equine pract 2014; 60:302-308. 17. gebauer mr, pickett bw, voss jl, et al: reproductive physiology of the stallion: daily sperm output and testicular measurements. j am vet med ass 1974;165:711-713. 18. love cc, garcía mc, riera fr, et al: use of testicular volume to predict daily sperm output in the stallion. proc am assoc equine pract 1990;36:15-21. 19. varner dd, schumacher j, blanchard tl, et al: diseases and management of breeding stallions. goleta, ca. american veterinary publications 1991. p. 104-105. 20. thompson ja, love cc, stich kl, et al: a bayesian approach to prediction of stallion daily sperm output. theriogenology 2004;62:1607-1617. 21. blanchard tl, brinsko sp, varner dd, et al: progression of reproductive changes accompanying testicular dysfunction in aging thoroughbred stallions: case studies. proc am assoc equine pract 2013;59:532-536. 22. johnson l, varner dd, thompson dl jr: effect of age and season on the establishment of spermatogenesis in the horse. j reprod fertil suppl 1991;44:87-97. 23. johnson l, thompson dl jr: age-related and seasonal variation in the sertoli cell population, daily sperm production, and serum concentrations of follicle-stimulant hormone, luteinizing hormone, and testosterone in stallions. biol reprod 1983;29:777-789. 24. kenney rm, kingston rs, rajamannon ah, et al: stallion semen characteristics for predicting fertility. proc am assoc equine pract 1971;17:53-67. clinical theriogenology 2021; 13: 297 25. bielanski w: the evaluation of stallion semen in aspects of fertility control and its use for artificial insemination. j reprod fertil suppl. 1975;23:19-24. 26. dott hm: morphology of stallion spermatozoa. j reprod fertil suppl 1975;23:19-24. 27. stout tae, colenbrander b: reproductive parameters of draft horse, friesian, and warmblood stallions. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition. ames, ia: wiley blackwell: 2011. p. 1362-1366. 28. blanchard tl, varner dd, love cc, et al: management options for the aged breeding stallion with declining testicular function. j equine vet sci 2012;32:430-435. 29. voss jl, pickett bw, squires el: stallion spermatozoal morphology and motility and their relationship to fertility. j am vet med assoc 1981;178:287-289. 30. dowsett kf, pattie wa: characteristics and fertility of stallion semen. j reprod fert suppl. 1982;32:1-8. 31. jasko dj, lein hd, foote rh: determination of the relationship between sperm morphologic classifications and fertility in stallions: 66 cases (1987 – 1988). j am vet med assoc 1990;197:389-394. 32. jasko dj, little tv, lein dh, et al: comparison of spermatozoal movement and semen characteristics with fertility in stallions: 64 cases (1987 – 1988). j am vet med assoc 1992;200:979-985. 33. love cc, kenney rm: the relationship of increased susceptibility of sperm dna to denaturation and fertility in the stallion. theriogenology 1998;50:955-972. 34. love cc: relationship between sperm motility, morphology and the fertility of stallions. theriogenology 2011;76:547-557. 35. love cc, noble jk, standridge sa, et al: the relationship between sperm quality in cool-shipped semen and embryo recovery rate in horses. theriogenology 2015;84:1587-1593. 36. barrier-battut i, kempfer a, becker j, et al: development of a new fertility prediction model for stallion semen, including flow cytometry. theriogenology 2016;86:1111-1131. 37. brinsko sp, spooner ja, blanchard tl, et al: relationships among morphologic characteristics and membrane integrity of stallion sperm. clinical theriogenology 2011;3:55-60. 38. love cc, varner dd, thompson ja: intraand inter-stallion variation in sperm morphology and their relationship with fertility. j reprod fertil suppl 2000;56:93-100. 39. brinkerhoff jm, love cc, thompson ja, et al: influence of mare age, pre-breeding mare status, breeding method, and stallion on first cycle pregnancy rates on a large commercial breeding farm. anim reprod sci 2010; 121s:s159. 40. love cc: the role of breeding record evaluation in the evaluation of the stallion for breeding soundness. proc soc therio 2003;68-77. 41. blanchard tl. the subfertile stallion. in: samper jc, pycock jf, mckinnon ao, editors: current therapy in equine reproduction. 1st edition. st. louis, mo: saunders elsevier. p. 237-243. clinical theriogenology 2021; 13: 298 prebreeding season/prepurchase examination of thestallion: testicular ultrasonography, semen evaluation,and considerations how outdated perceptions have reshaped dog marketplace how outdated perceptions have reshaped dog marketplace patti strand,a frances smith,b john new,c barbara reichmana anational animal interest alliance, portland, or, borthopedic foundation for animals, columbia, mo cbiomedical and diagnostic services, university of tennessee, knoxville, tn abstract rates of household dog ownership in the united states have never been higher, according to the american veterinary medical association, which has tracked this statistic since 1982.1 likewise, market research shows significant growth in the number of u.s. dogs, with 1 firm estimating the number in 2017 at 89.7 million, an increase of 24% since 2000.2 this widely documented upturn in dog ownership is especially noteworthy in view of parallel decreases in number of dogs being produced in the united states by long-standing sources. this reduction affects both mixes and purebreds and is confirmed by multiple sources, including purebred registries, shelter statistics and breeder databases. objectives of this study were to examine consumer perceptions and preferences regarding dog acquisition and identify how these trends and perceptions impact availability, sustainability, type and quality of pet dogs. the study confirmed high levels of dog ownership reported by other studies, assessed public’s intention to acquire dogs in the future and identified preferences for several dog characteristics. the perception of dog overpopulation strongly influenced the type of dog preferred and where to acquire it. a majority of respondents was unaware of the large number of rescue dogs routinely imported into their area for adoption, a practice that keeps local supply and demand for pet dogs in equilibrium and obscures decreases in locally produced dogs. keywords: breeds, overpopulation, consumers, shelters, rescues, importation, purebreds introduction given the popularity of dogs, the economic activity surrounding their acquisition and the size of the pet industry, which is estimated to surpass $72 billion in 2018,3 little attention has been paid to the changing sources of dogs, nor the decline in many traditional sources of dogs available to consumers.4 these trends raise questions about sustainability of us dog ownership, at least from traditional sources. number, source and quality of pet dogs available in the us impacts a wide variety of stakeholders—pet businesses, animal professionals, hobbyists and pet owners alike. many have livelihood stakes in the information. some are deeply committed to animal health and welfare or breed preservation, whereas others simply have a personal interest in the source of their future dog. evolution of sources, preferences and qualitative differences affect all stakeholders. in the us today, there are 4 major dog sources:  casual breeders producing puppies from family dogs, both mixes and purebreds;  hobbyists and sportsmen producing dogs with specific characteristics for work, canine sports and as pets, mostly purebreds;  commercial breeders providing pet dogs, mostly purebreds, for american consumers; and  animal rescues and shelters, placing strays and dogs that have been relinquished by their original owners, as well as dogs acquired from other shelters and rescues from within and outside of us. of the 4 sources listed above, the only source showing an increase in supply and in placements is the nonprofit shelter and rescue category.5 it should be acknowledged that in addition to importation, shelters have also increased placements by expanding the definition of adoptable to include dogs that were formerly excluded from adoption due to health or temperament issues. high rates of neutering, restrictive dog breeding laws, economic downturns, the ease of international travel and development of the internet have all affected composition of the dog marketplace and the overall decline of us dog breeding. however, these factors alone do not explain related shifts in 225 clinical theriogenology • volume 11, number 3 • september 2019 preferences for pet dog types and sources, or why the decades-long desire for purebreds has waned at a time when overall dog ownership has increased. with all categories of dog breeding in the us on the decline and dog overpopulation solved in many but not all regions of the country, rescues and shelters have expanded their operations to fill the void. thirty years ago, animal shelters in most regions of the country were filled with local dogs but shelters in many states today would not have enough dogs to remain viable without importing rescue dogs from regions where surplus dogs still exist.6,7 today, rescue dog importation or humane relocation as its advocates call it, has become the dominant source of dogs in many regions of the country. significantly, the public remains largely unaware of where local shelter and rescue dogs originate; they still perceive that dog overpopulation is a serious problem, a belief that influences where they choose to acquire a family dog. there are no central databases or government oversight bodies that license or otherwise require shelters and rescues to be registered; however, estimates place the number of brick-and-mortar shelters at approximately 3,500 and rescues at 10,000 and growing. with the demand for dogs estimated at 8 million annually and anticipated to reach 9.2 million by 2036, some commentators assert that there simply are not enough shelter dogs in the us to meet demands.8 a constant stream of rescue dogs keeps supply and demand in equilibrium in many parts of the us, transforming humane societies into de facto pet stores. many northern shelters now import the majority of their adoptable dogs—thousands of dogs each year—a practice that displaces local dog breeding.9 the unlimited supply of dogs available through rescue transport programs is sufficient to fill market demand, impacting not only casual breeding but careful breeding done to maintain and improve long-standing breeds. genetic diversity has contracted, along with the steep decline in breed populations since the mid 1990s. significant gene pool shrinkage has already occurred, threatening many purposebred dogs that have been created over many generations, even hundreds of years. once gone, they cannot be replaced. foreign rescue dogs, especially from developing countries and territories that have street dog populations estimated in the millions, can easily meet the number of dogs required by the american public. the lack of veterinary care available in these countries, however, assures that imported dogs will harbor many diseases and parasites that are rarely found in the us.10 moreover, many of the dogs being imported today are feral street dogs with little, if any, past veterinary care or socialization. purebred dogs represent a shrinking percentage of the total dog population, so it warrants asking whether consumers now prefer mixed breed dogs to purebreds. likewise, increasing acquisition of older shelter and rescue dogs raises questions about the public’s attitude toward acquiring dogs as puppies. do today’s families prefer to obtain older dogs that were previously owned rather than acquiring puppies and raising them as they have in the past? notably, when respondents were asked what characteristics were important to them when considering acquiring a dog, predictability and purebred status were at the top of the list and acquiring puppies was important to a majority of survey respondents. in other words, the stated preferences of survey takers were often at odds with their actions as consumers. there are innumerable reasons why preferences expressed by consumers could be at odds with their actual purchasing practices, but 1 factor that has not been adequately discussed in the research literature is the role of humane marketing campaigns produced by national animal welfare organizations and local humane societies and rescue groups. for more than 30 years, local and national animal rights and welfare organizations have promoted legislative and marketing campaigns to end pet overpopulation, inhumane breeding practices and several other animal welfare problems. the campaigns have achieved great success, but their sensational approach has also triggered severe declines in the number of healthy, well-socialized dogs being produced by acceptable sources. the campaign messages stereotyped all dog breeding as harmful and reinforced an unreasonable concern over dog overpopulation, even as these problems were being resolved. today, rescue and shelter dog adoptions are often promoted using a type of cause marketing campaign that ties a product or goal to a worthwhile cause, while linking alternative choices to negative consequences. in the case of dog acquisition, the adoption of a rescue or shelter dog is marketed as the 226clinical theriogenology • volume 11, number 3 • september 2019 most humane choice, as a choice that will prevent shelter dogs from being euthanized. such campaigns assert that buying a puppy will harm a shelter dog’s chance of finding a home, that adding a new nonrescue dog to the population will lead to a shelter dog’s death. these campaign messages have been accepted by popular culture and are widely distributed in print and broadcast media and presented as news. some have even appeared on billboards. secondary messages convey that dog breeding is inhumane and that mixed breed dogs are healthier than purebreds.11 markets continually evolve in response to changing consumer demand. if the public no longer chooses dogs of specific breeds, prefers acquiring older dogs rather than puppies, or perceives that rescued shelter dogs are equally healthy and temperamentally as sound as those available from breeders or retail outlets, the market changes to reflect these choices. likewise, if the satisfaction of acquiring a dog that they believe might otherwise face euthanasia is greater to an individual than getting one with characteristics they prefer, but faces no such peril, types of dogs available will shift in response. the survey suggests that the public prefers puppies with predictable traits and purebreds over mixed-breed dogs, but often selects dogs that lack these qualities to prevent shelter dog euthanasia. even if this perception is false, if consumers believe it to be true, the supply of purebreds and puppies from traditional sources will continue to decline, being replaced by dogs from national and international rescue channels. the unanswered question is whether prospective dog owners whose choices are influenced by their perceptions of us dog overpopulation and other humane issues would continue to choose rescued shelter dogs over traditional american bred sources if they were aware that:  many rescues and shelters are importing dogs to meet local demand, not only from the states that still have surpluses, but from foreign breeders and the streets of foreign countries;  the welfare of most imported dogs is not protected at the source or during transport;  they often harbor infectious and zoonotic diseases and parasites, including lethal ones; and  avoiding us-bred purebreds and even mixed breed puppies threatens the future availability of a healthy source of american bred dogs, including purebreds. recognizing these supply chain changes is important so that long-standing as well as new sources of dogs can be evaluated for quality and sustainability. significantly, the public remains largely unaware of these changes, believing in and making personal and policy decisions based on outdated facts. materials and methods this study examined elements contributing to changes in the composition of the dog marketplace and dog ownership in the us. the objective was to examine consumer perceptions and preferences regarding dog acquisition and to identify how these trends and perceptions impact pets available to americans now and in the future. consumers were surveyed about their perceptions, preferences, and future plans for adding a dog to their family. the aim of this research was to examine these trends and assess their impact on preservation of breeds and sustainability of a healthy source of american-bred dogs. an online survey was completed by 1,666 individuals, from which a sample of 1,000 respondents between the ages of 28 and 70 was selected who in aggregate are representative of the u.s. population by gender (46.3% men and 53.7% women), income and geographic region. the survey asked questions about current dog ownership, plans for future pet dog acquisition, consumer preferences for various characteristics (e.g. size, breed, sex), and consumer beliefs regarding dog overpopulation. additional materials included data from government and institutional sources, purebred registries, media and open records searches. this research was conducted between january 2013 february 2019. 227 clinical theriogenology • volume 11, number 3 • september 2019 results findings background and general attitudes of respondents out of 1,000 respondents, 51% (507) currently owned at least 1 dog, whereas 49% (493) did not. although a slight majority of all respondents currently owned a dog, a large majority of 64% (640) viewed dog ownership itself as important or very important. of the dog owners, 58% (295) owned at least 1 purebred dog and 53% (269) at least 1 mixed-breed dog 11% (57) owned at least 1 purebred and 1 mixed-breed dog). purebred dogs among current purebred dog owners (295), 41% have registered their most recently acquired purebred dog with a breed registry [e.g., american kennel club (akc), united kennel club, continental kennel club], 49% have not, and 10% of respondents were unsure. excluding the 10% of dogs whose registry was unknown, 70% of those not registered were reportedly eligible. the remaining 30% were reportedly not eligible. sources of currently owned dogs current owners were asked the source of their most recently acquired purebred and mixed-breed dog, separately. there were 16 options to choose from, including “other,” if a respondent’s source was not provided in the survey. the top 4 responses for purebred dog owners (295) were serious in-home breeder (56), casual in-home breeder (48), friend/relative/neighbor (44), or gift (42). out of all responses, 116 purebred dogs (39%) came directly from breeders. the top 4 responses for mixed breed dog owners (269) were animal shelters (59), friend/relative/neighbor (52), stray (24), or “other” (23). overall, 35% (93) mixed breed dog owners obtained their dogs directly from rescues or shelters. anticipated acquisition of a new dog in the next 5 years overall, 38% (376) of all respondents anticipated acquiring a dog within the next 5 years. however, respondents who currently owned a dog were nearly 3 times as likely to anticipate acquiring another dog: 44% (342/778) of current dog owners versus 15% (34/222) of non-owners. important characteristics of the (376/1,000) respondents who anticipated acquiring a dog within the next 5 years, 68.35% (257) said they would actively pursue a purebred dog or puppy. health at time of acquisition an overwhelming majority of all respondents, 863, considered health of the animal (health being at the time of acquisition and throughout his/her life) a high priority in their decision to acquire a new dog. of these 863, the top response when asked where they would look for a new dog, was animal shelter (297), more than twice as many as the second highest response, “serious, in-home breeder” (143). respondents who disagreed with the statement “it is okay to purchase dogs instead of adopting them from a rescue or shelter.” were even more likely to view an animal shelter as the best place to acquire a healthy dog over a serious, in-home breeder: 155 versus 24. predictable temperament (trainability, stability, affection toward the family, etc.) was considered a high priority when acquiring a new dog by 86% of respondents (861). of these respondents, 33% (284) would look first at an animal shelter to acquire a new dog, whereas 17% (148) would go to a serious, inhome breeder, and 14% (121) would seek a new dog from a friend, relative, or neighbor. for 63% (630) of all respondents, finding a dog to meet a specific family need (e.g. being nonallergenic, getting along with current dog(s), living comfortably in an apartment) was a high priority in their decision making. 228clinical theriogenology • volume 11, number 3 • september 2019 of these 630 respondents, 31% (197) would look first in an animal shelter to find a dog to meet their specific need, whereas 18% (115) would seek a breeder, and 12% (75) a friend, relative, or neighbor. if potential performance ability was a high priority (187), respondents believed that dogs from a breeder (40), friend, relative, or neighbor (29), or an animal shelter (27) were the top 3 sources that were most likely to meet these criteria. of the other 16 possible responses (e.g. pet store, gift), none were selected by more than 13 respondents. when all respondents were asked how important it was for them to acquire their next dog as a puppy, slightly more than half (502) said it was important or very important, whereas 28% (294) said it was not important or of no importance to acquire their next dog as a puppy. of the 502 respondents who considered it important or very important to acquire their next dog as a puppy, 23% (114) would look first at a serious, in-home breeder, 20% (102) at an animal shelter, and 15% (77) from a friend, relative or neighbor. for respondents who believe that there is a surplus of dogs in the us, 23% (72) would look first at an animal shelter, 21% (66) at a serious, in-home breeder, and 15% (48) from a friend, relative or neighbor. beliefs dog surplus in the united states all respondents were asked if they believed that there is a surplus of dogs in the us: 64% (637) answered yes, 10% (97) answered no and the remaining 27% were not sure. respondents whose households who own at least one dog were more likely to believe there is a surplus of dogs in the us at 70%. of the 637 respondents who believed that there is a surplus of dogs in the us, 56% (356) think the surplus of dogs is getting worse, 26% (165) think it is about the same, while 9% don’t know or think it is improving (59 and 57 respectively). beliefs towards purchase of dogs with the choices of strongly agree, agree, no opinion, disagree or strongly disagree, respondents were asked if it was “the okay to purchase dogs instead of adopting them from a rescue or shelter.” among all respondents, 34.6% agreed or strongly agreed with statement, 34.4% disagreed or strongly disagreed, and 31.0% had no opinion. respondents who believed that there is a surplus of dogs in the us were more likely to disagree or strongly disagree with the statement (41%, 259), whereas respondents who didn’t believe that there is a surplus of dogs in the us were more likely to agree (46%, 45). demographics also played a role in respondents’ views: female respondents were 28% likely to agree or strongly agree with the statement, and 41% likely to disagree or strongly disagree. attitudes towards importation and adoption of dogs from various sources most respondents (66.6%) were not aware that some animal shelters import dogs from other states or other countries. 44% of respondents (437) were supportive of importation of dogs from other states, 26% (260) were not. when asked about importing dogs from other countries, only 16% of respondents (156) were supportive, while 61% (606) were not. even of the 156 respondents who were supportive of importing dogs from foreign countries, only 41% (64) would adopt a dog from a foreign country rather than a local dog. discussion the practice of keeping dogs as pets developed following the industrial revolution and became an ever-expanding and permanent part of american life. today, us household dog ownership rates are among the highest in the world.12 the primary sources of pet dogs throughout the 20th century were home-based breeders (both casual and purebred fanciers), and animal shelters. between and following the world wars, a commercial sector sprang up to meet demand, which included pet dog breeders, wholesalers, transporters and retailers. in the 1980s, rescue networks organized primarily by akc breed 229 clinical theriogenology • volume 11, number 3 • september 2019 clubs joined the dog marketplace, conscientiously rehoming dogs of their breed when they landed in animal shelters or other trouble. toward the end of the 20th century and at the beginning of the 21st century, internet sellers, online adoption groups like petfinder.com, and rescue relocation transport programs became active players in the dog marketplace. registration records dating from 1884 from the akc, the largest purebred registry in the us, provide a well-documented history of purebred dog ownership and current trends. its records show nearly continuous growth from just under 5,000 purebred registrations at the beginning of the 20th century until 1992, when registrations peaked at 1.5 million and thereafter commenced a steep decline, although there has been a measured rebound in recent years. purebred dogs have long been a major component and indicator of the total pet dog population in the us, so the steep decline nearly 70% since 1993 reflected in akc registration data should be noted by dog owners and pet industry professionals alike. it signifies a little noticed, but major change in the overall us dog marketplace. many small and large scale breeders alike have quit producing purebred dogs and as this happens, gene pools contract and survival of many breeds is challenged. akc registration data as well as state licensing data for commercial breeders in pennsylvania and missouri also confirm this remarkable shift. another important factor influencing dog population reductions is the increasing percentage of household dogs that are neutered in the unites states. alongside the uniquely high rates of dog ownership in 20th century america, was a huge surplus of unwanted dogs that were euthanized each year. midcentury it was still commonplace for unaltered dogs to roam the streets and for families to allow their pets to reproduce. this changed with the growing availability and acceptance of neutering in the last half of the 20th century, as americans accepted the idea that preventing unwanted litters was part of being a responsible pet owner. slowly but steadily over more than 4 decades, numbers of dogs entering shelters began to decrease. many articles refer to the 1970s as a turning point for shelter dog euthanasia. shelter data covering the period confirm the trend.13 campaigns to end euthanasia of shelter dogs have been enormously successful. today, 85% of household dogs in the us have been neutered.14 because of widespread neutering, other sources of dogs readily available in the past such as mixed-breed dogs acquired from family, friends, neighbors, and local animal shelters, are also declining animal welfare organizations report that euthanasia of shelter dogs has dropped by at least 80%, despite huge increases in the population of both human and household dogs over the same interval.15 animal shelters in many parts of the country claim a live release rate of 90% or more; a rate, which in no kill parlance, means that no adoptable animals were euthanized. despite steady and conspicuous progress in reducing shelter dog populations and euthanasia, new and aggressive legislative pet overpopulation campaigns targeting breeders were launched in the late 1980s. instead of traditional campaign messages designed to change pet owner behavior through education, the new campaigns used disparagement and shock (public pet executions). the campaigns introduced a new paradigm in which breeding itself, no matter how responsible or humane, was labeled as the problem and graphically linked to shelter dog deaths.16 these campaigns opposed all breeding until euthanasia of shelter dogs stopped and promoted the idea that breeding or buying a dog is tantamount to killing a shelter dog. “don’t breed or buy while shelter dogs die” became a campaign mantra, and is still prominent in popular culture today.17 cause marketing campaigns to end dog overpopulation coupled with a lack of awareness about the status of shelter dog euthanasia and dog importation programs provide context for why so many prospective dog owners view shelters and rescues as the most ethical locations to get their next dog. thirty-four percent of survey respondents thought purchasing a dog, rather than obtaining a dog from a rescue or shelter, is wrong. news accounts suggest that the importation of foreign dogs is increasing. data from the few states that collect such information as well as advertisements on importing rescue and shelter websites show that the number of puppies being imported is growing. colorado statistics show that number of dogs that are 120 days old or less hovers right around 50%. this should concern animal welfare advocates; perhaps us shelters, rescues and foreign breeders are now producing rescue dogs for the us market. the us still has a very high rate of overall dog ownership, but the study showed that prospective dog owners 230clinical theriogenology • volume 11, number 3 • september 2019 are confused about the differences among available sources of dogs. a significant number of respondents who indicated a desire for a specific breed and predictable traits, ideally a puppy, also indicated that they planned to get their next dog from a rescue or shelter. their preferences and perceptions do not match the current dog marketplace. the vast majority of dogs in shelters are not purebred dogs or puppies.18 in addition to saving lives, dog relocation programs aim to provide relief to overburdened shelters so that they can use their resources to develop improvements such as public education and low-cost spay/neuter programs. if the number of surplus dogs produced at the source does not decrease as a result of the transport program, the net result will be little more than an unregulated pipeline of dogs, supplying rescue dogs to destination cities but without having to meet the regulatory requirements of retail and wholesale suppliers. the public is not yet aware that the issue of homeless pet dogs has been solved in many regions of the us. a majority of our survey respondents still believe that there is a shelter dog surplus problem with the majority believing that the problem has not improved or is getting worse. a strong majority was also unaware that many of the dogs available at their local shelter or rescue group are not local animals, but from shelters and rescues from other states or even foreign countries. if current dog marketplace trends continue, demand may exceed supply from all but foreign sources. conclusion americans love dogs, but outdated perceptions about dog overpopulation and a lack of awareness about dog importation programs are quietly reshaping the dog marketplace and threatening the future availability of healthy puppies and well socialized dogs. shelter and rescue data from colorado department of agriculture (the best source of statewide rescue and shelter data), sheds light on effects dog relocation programs have on the marketplace. the number of dogs taken in by colorado’s shelters and rescues steadily increases. an uninformed observer might conclude that the number of surplus dogs in the state is increasing.19 in fact, out-of-state dogs are flowing into colorado rescues and shelters at rates high enough to meet current demand and greatly increase earlier shelter population levels, which were in steep decline prior to initiation of importation. in 2017 alone, the number of imported dogs was 31,707.20 that same year they adopted out 60,892 dogs, which was 30,232 more than they placed in 2000. the number of imported dogs and adoptions increase annually. colorado’s shelter data are uniquely robust. most states provide little if any such information and many shelters do not publish data regarding animals they take in and place. even though a given animal shelter may not share its data publicly, it is still collected for internal use. having information about animal shelter intakes, adoptions and sources is essential for planning. it is especially helpful in estimating how many dogs can be imported and adopted out in a given region while keeping local supply and demand in equilibrium. colorado provides the best example of how the current import-shelter model operates and shows how importation masks the fact that there are no longer enough local dogs to meet demand and that shelters are becoming the leading source of pet dogs in their communities. as rhode island state veterinarian dr. scott marshall put it, “…there’s some evidence that the rescue groups are a new model for the pet shop industry.” in many instances he says we’ve supplanted pet shops, “…a well-regulated industry with a lot of state and federal regulations and shifted those animals into other channels with less oversight and a lot less transparency.”21 the main reason this subject has not been thoroughly covered in the past is because of the lack of accessible, verifiable data. us shelters and rescues operate with very little oversight despite some being multimillion-dollar operations. there is no central database collecting and publishing shelter/rescue statistics on a nationwide basis. to make sense of the data, researchers have to extrapolate from key data sources, review numerous pieces of information and evaluate trends. based on materials we have received through open records requests that show state-to-state shipments of dogs, import records maintained by states like massachusetts and connecticut and statistics published by a few key shelters, it is reasonable to estimate that no more than about 15 states still have surplus problems. colorado’s data are an excellent model for extrapolation of trends. data from other states and from big city humane societies that publish their statistics show the same trends. even though it is difficult it is to report these data with precision, trends are unmistakable. 231 clinical theriogenology • volume 11, number 3 • september 2019 if these trends continue americans may someday find that carefully produced and socialized puppies are rarely available from us sources and that many of the breeds they grew up with and enjoyed in the past are no longer available. instead they may discover that pet dog breeding has been outsourced to nonprofit institutions in this country and to foreign countries where veterinary care, concern for animal welfare, husbandry and breed preservation is decades behind the us or absent. acknowledgement authors acknowledge assistance of michelle kutzler and angie niles. research supported by the national animal interest alliance. conflict of interest authors have no conflicts of interest to declare. references 1. american veterinary medical association. move over rover; poultry as pets on the rise. 2018. available from: https://www.avma.org/news/pressroom/pages/avma-releases-latest-stats-on-pet-ownership-and-veterinary-care.aspx 2. number of dogs in the united states from 2000 to 2017 (in millions). 2018. available from: https://www.statista.com/statistics/198100/dogs-in-the-united-states-since-2000/ 3. appa national pet owners survey: american pet products association. 2018. available from: https://americanpetproducts.org/uploads/memservices/gpe2017_npos_seminar.pdf 4. american kennel club: dog registration statistics from 1884–2017. available from: akc. new york, ny 5. naia shelter project. national animal interest alliance. graph showing increase in colorado dog intakes 2000–2017. available from: http://shelterproject.naiaonline.org/shelter_data/shelter/354/2/received 6. shelter overpopulation: replacing myth with math. 2010. available from: http://www.shelteroverpopulation.org/books/replacing_myth_with_math.pdf 7. naia shelter project. national animal interest alliance. graph based on statistics gathered by the colorado department of agriculture statistics showing out-of-state rescue dogs transferred into colorado shelters and rescue available from: http://shelterproject.naiaonline.org/shelter_data/shelter/354/2/transfered_infromoutside area 8. kavin k: does america have enough dogs for all the people who want one? the washington post. 2017 february 8. available from: https://washingtonpost.com/news/animalia/wp/2017/02/08/does-america-have-enough-dogs-for-allthe people-who-want-one?utm_term=.e2c1ba72c07d 9. naia shelter project. national animal interest alliance. graph based on statistics published by oregon humane society of dogs imported into their shelter. 2005–2017. available from: shelterproject.naiaonline.org/shelter_data/shelter/198/2/transfered_in_from_outside_area 10. world health organization. neglected tropical diseases. 2017. available from: https://www.who.int/neglected_diseases/diseases/en/ 11. colwill r: research at brown university. don’t breed or buy while shelter dogs die: 8 reasons to adopt a shelter dog. 2019. available from:http://www.brown.edu/research/colwill_lab/cbp/8reasonstoadopt.htm 12. pet insure i e. the rise of pet population. 2014 august 22. available from: https://www.petinsure.ie/rise-petpopulation/ 13. mach a: christian science monitor. behind the big drop in euthanasia for america’s dogs and cats. 2012 february 10. available from: www.csmonitor.com/usa/society/2012/0210/behind-the-big-drop-in euthanasia-for-america-s-dogs-and-cats 14. appa national pet owners survey. american pet products association, 2017–2018. available from: appa 2017– 2018 survey. page 19 https://americanpetproducts.org/uploads/memservices/gpe2017_npos_seminar.pdf 15. serrie j: pet euthanasia rates decline at us shelters. fox news. 2014 april 21. available from: http://foxnews.com/us/2014/04/21/pet-euthanasia-rates-decline-at-us-shelters.html 16. corwin m: los angeles times. pet sterilization becomes law in san mateo county animals: beginning in 1992, dog and cat owners must have them neutered or get a breeding license. 1990 december 19. available from: http://articles.latimes.com/1990-12-19/news/mn-6411_1_san-mateo-county 17. humane society of the united states. adopt don’t shop marketing campaign, 2014 july 30. available from: slideshare.net/gabydcl/adopt-dont-shop-marketing-campaign-for-the-humane-society 18. national animal interest alliance. survey of shelter dog composition: mutts vs. purebreds. 2015 july. available f from: http://shelterproject.naiaonline.org/purebred/ 19. naia shelter project. national animal interest alliance. graph based on statistics gathered by the colorado department of agriculture showing total dogs received by colorado rescues and shelters from all sources. 2000–2017. available from: http://shelterproject.naiaonline.org/shelter_data/shelter/354/2/received 232clinical theriogenology • volume 11, number 3 • september 2019 20. naia shelter project. national animal interest alliance. graph based on statistics gathered by the colorado department of agriculture showing dogs received by colorado rescues and shelters from out-of-state rescues and shelters. 2000–2017. available from: http://shelterproject.naiaonline.org/shelter_data/shelter/354/2/transfered_in_from_outside_area 21. liberman ea: look at rhode island’s animal welfare system. rhode island monthly.2016 february 11. available from: http://www.rimonthly.com/rhode-islandmonthly/february-2016/a-look-at-rhode-islands-animal-welfare 233 clinical theriogenology • volume 11, number 3 • september 2019 234clinical theriogenology • volume 11, number 3 • september 2019 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/useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice 1 contact heriberto rodriguez-martinez heriberto.rodriguez-martinez@liu.se © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9406, http://dx.doi.org/10.58292/ct.v15.9406 review report (invited) clinical applications of flow cytometric sperm analyses fernando peña,a heriberto rodriguez-martinezb alaboratory of equine reproduction and equine spermatology, veterinary teaching hospital, university of extremadura, cáceres, spain bdepartment of biomedical and clinical sciences, division of obstetrics and gynaecology, linköping university, linköping, sweden abstract animal andrology has benefitted by flow cytometry use (to analyze various sperm components). examples include its use to explore dna intactness, to sort sperm for chromosomal sex, or to explore via suitable fluorophores the functional intactness of essential attributes (e.g. plasma membrane). these explorations can provide the clinician with evidence of the extent of cell death, a major variable in using semen for breeding. such gains have evolved over the past decade(s) through the production of versatile markers, including biomarkers, the design of simpler protocols, and the availability of more user-friendly and less expensive benchflow cytometers. the present review summarizes the current state of flow cytometry use within animal andrology. it attempts to be critical of research gains, separating them for clinical relevance and focusing on its use to examine the early events that occur in the labile sperm plasma membrane (leading to cell death) – a most relevant marker for clinical decisions during diagnosis and prognosis of fertility when using ejaculated sperm (either unprocessed or processed) for cooling, freezing, or sperm sorting for in vitro fertilization or artificial insemination. keywords: semen analysis, stallion, flow cytometry, sperm membrane, livestock introduction clinical veterinary andrology attempts to assess the health of a breeding male. it certainly includes examination of genital organs, the ability to mate, and a thorough examination of collected semen, pursuing a species-specific general breeding soundness evaluation.1 decades of animal selection and dedicated clinical work have yielded substantial knowledge for early identification of potentially infertile sires, preventing their use for the widespread and most successful reproductive biotechnology, artificial insemination (ai). furthermore, laboratory examination of semen has provided steady support for accurate diagnoses, particularly when novel methods and equipment have been presented.2 despite methods for assessing semen normality and, particularly, sperm structure and function have been a target for andrological and spermatological research, the conventional evaluation of the ejaculate made by most clinicians has been restricted to determinations of sperm number, motility, and very seldom, morphology.3–5 this slim battery of semen parameters has historically been considered sufficient to potentially separate fertile from infertile.6 yet, the proportion of subfertile sires is still sufficiently high to cause losses on the level of billions of euros or dollars, yearly. these losses are often recognized too late, either when conception is clinically assessed, when nonreturn figures are indirectly measured, or when offspring is absent or lower than expected. although sire fertility is evidently a limiting factor, most fertility research seldom focuses on the male or the quality of the used sperm.6 one of the reasons behind is that although sperm biomarkers related to fertility exist, their use by the clinician is still burdened by requirements of high technical know-how, expensive instrumentation, and the inability of the researchers to communicate their diagnostic value, mostly due to insufficient standardization of valuable protocols since the use flow cytometry is becoming common in established companies.6 another factor to consider is the complex nature of fertility depending on numerous factors and their interactions.2,6,7 innumerable methods are now available for semen/sperm evaluation, including those examining the capability of sperm mailto:heriberto.rodriguez-martinez@liu.se http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9406 2 citation line: clinical theriogenology 2023, 15, 9406, http://dx.doi.org/10.58292/ct.v15.9406 to interact with the surrounding fluids (seminal plasma, intralumenal female genital fluids and extension artificial media we use for preservation, in vitro culture, and sorting), the lining epithelia of the internal female tract, or the coatings of the oocyte.8–11 currently, we can explore not only sperm plasma membrane status, various sperm organelles, sperm genome, and its transcriptome but also the capacity of sperm to fertilize and to initiate early embryo development in vitro.12–17 in addition, we are helped by several computer-assisted motility analyzers, of morphology screeners, and by flow cytometers of various complexity (and cost), including cutting-edge imagebased fluorescence correlation spectroscopy for high-throughput cell image acquisition.6,18–20 authors advise the readers to be acquainted with some of the excellent reviews available to grasp this development.20,21 for the clinician, perhaps the most relevant take-home message is to remember that sperm are highly differentiated cells, programmed to interact and react to the environment and prompt to suffer changes that inexorably lead to sperm death, and hence inability to participate in fertilization, and perhaps, to cause deleterious changes in the accompanying sperm.22 our intention with this review is to remind the clinician of the basic and essential diagnostic values of determining the percentages of live sperm in the sample for its fate. this concept that calls for methods that can identify sperm changes before death is established, considering sperm death is irreversible. although the integrity of the plasma membrane is rapidly lost when necrotic cell death occurs, subtle changes characterize other forms of sperm demise. numerous flow cytometry (fc) protocols have been developed to identify necrotic sperm and also various degrees of plasma membrane alteration occurring prior to sperm death.23–27 in this paper, we provide an update on the various forms of death in sperm, and how these forms are reflected on various fc protocols, establishing a critical review of the current methods for the determination of the integrity and functionality of sperm plasma membrane. a particular call is made for the value of using multiparametric fc that, aided by computational analysis (cytobank), can simultaneously identify multiple forms of sperm damage. although these multiple commercial bench-top fcs are available, the authors recommend the use of modern equipment with upgrade possibility (e.g. activation of new lasers), and having a friendly and open software. necrosis/apoptosis/ferroptosis/spermptosis/ premature capacitation although the existence of classical necrosis in sperm is widely accepted, the presence of various forms of programmed cell death has been under debate. the transcriptionally silent nature of sperm has been claimed to make programmed forms of cell death unlikely in these cells; however, currently, it is considered that sperm are programmed for cell death as a measure to promote the silent removal of redundant sperm for the female reproductive tract.28 moreover, although protein synthesis in sperm is negligible, the incorporation of new proteins through microvesicle trafficking and, especially, posttranslational modifications of existing proteins modulate sperm function. despite all sperm are activated through cell death, this may occur at various velocities and factors, i.e. oxidative stress, triggering and accelerating the process. although the terms programmed cell death and apoptosis have been used indistinctly, these terms are not synonymous. various forms of programmed cell death may share similar molecular mechanisms; however, all these forms are not identical. necrosis is characterized by a rapid loss of membrane integrity. however, in various forms of programmed cell death, more subtle changes occur, especially in a cell as highly specialized as sperm. capacitation is the maturational process that sensitizes sperm to recognize and fertilize the oocyte; this process causes substantial plasma membrane modifications. capacitation involves the removal of cholesterol from the plasma membrane, removal of coating materials from the membrane, a rise in intracellular ca2+, an increase in intracellular camp, and a dramatic increase in tyrosine phosphorylation. removal of cholesterol from the membrane is preceded by its oxidation, stimulated by bicarbonate, and the formation of oxysterols,29–31 which are depleted from the sperm membrane by albumin. the process prepares the sperm to fertilize an oocyte; however, most of the sperm in the ejaculate will never reach an egg and will die within the female reproductive tract and only a small subpopulation of sperm fully capacitate,32,33 thus mechanisms for immunologically silent elimination of redundant sperm are likely. capacitation shares molecular mechanisms with what begins to be considered special forms of programmed cell death in sperm: redox-regulated, intracellular ca2+ increase and membrane destabilization occur. however, capacitation implies a dramatic increase in tyrosine phosphorylation and changes in the pattern of motility to hyperactivated motility, whereas programmed cell death ends with cessation of any kind of motility in sperm. finally, apoptotic sperm release ‘find me/eat me’ signals to achieve a silent removal of redundant sperm. the inhibition of glutathione  peroxidase  4 (gpx4) leads to a specific form of cellular demise termed ferroptosis.34 this is a form of programmed cell death that is characterized by the failure of glutathione (gsh)-dependent antioxidant defenses that can be triggered by the inhibition of gpx4 or slcta11/xct, the antiporter that exchanges extracellular cystine for intracellular glutamate. cystine is used for gsh synthesis, a mechanism recently described in stallion sperm.27,35 it is possible that this form of cell death is present in sperm.26 induction of apoptosis or necrosis in sperm if the presence of apoptosis in ejaculated sperm is a remnant of spermatogenesis (as stated by the abortive apoptosis theory), most sperm are already programmed to experience programmed cell death; thus, the concept is that more than induced, in sperm, the programmed cell death process can be accelerated. however, classical inductors of apoptosis have been successfully used in stallion sperm, although betulinic acid seems more effective to induce apoptosis than staurosporine or thapsigargin,36 which is in agreement with the predominance on the mitochondrial pathway of apoptosis in sperm.37 in sperm, cryopreservation has been constantly associated with the production of apoptotic changes related to the oxidative stress caused by freezing and thawing. changes in the integrity of the plasma membrane although the integrity of the plasma membrane is rapidly lost when necrotic cell death occurs, subtle changes occur under other forms of sperm demise. the traditional approach for the determination of viability in sperm biology has been using dye exclusion assays; the combination of the permeable dna binding probe sybr-14 and the impermeable dna binding probe propidium iodide is widely used and is marketed as a kit for sperm viability assay. this assay became very popular, especially in the veterinary world, since its introduction in the early 90s of the past century. this combination was appropriate for the single laser (blue) of the cytometers commonly http://dx.doi.org/10.58292/ct.v15.9406 citation line: clinical theriogenology 2023, 15, 9406, http://dx.doi.org/10.58292/ct.v15.9406 3 used in the andrology labs at that time. during the first decade of the present century, more sophisticated assays started to be introduced in spermatology; these techniques allowed to disclose distinct physiological states of the membranes in addition to necrotic cells, and these assays identified the permeability of the membranes, the order of the lipid packaging, the transposition of specific phospholipids, the polarity of the membrane, and assays able to identify the peroxidation of membrane lipids. assays identifying changes in membrane permeability the probe that has gained popularity in the spermatology field is yopro-1. initially, this probe was used as an alternative to red emission viability dyes, when merocyanine-540 was used.38–40 however, yopro-1 not only is able to stain necrotic cells but can also use specific channels to cross intact membranes.41–43 moreover, live sperm may pump out yopro-1, whereas depletion of atp may result in accumulation of yopro-1 in the sperm.44 this property allows the identification of sperm, with intact membranes, but in a compromised physiological situation; therefore, this dye is used to identify apoptotic cells and to identify increased membrane permeability due to lipid peroxidation. an interesting report uses the yopro-1 assay to sort sperm of better quality (low dna fragmentation) in human sperm since dna fragmentation of the dna in sperm is due to oxidative attack, sperm experiencing oxidative stress also have increased membrane permeability,45 and these sperm can be easily identified and removed.46 lipid peroxidation of the membrane increases membrane permeability: assays identifying the transposition of phospholipids among the changes that oxidative stress induces is the increased permeability of the plasmalemma.45 lipid peroxidation is thus the consequence of oxidative stress and is a chain reaction, with peroxidation products created close to each other. the identification of the transposition of specific phospholipids from the inner to the outer membrane constitutes a classical assay for the detection of apoptotic cells. the annexin-v assay is a popular technique to identify the presence of phosphatidylserine (ps) in the outer leaflet of the plasmalemma. under normal conditions, ps and phosphatidylethanolamine are present in the interleaflet of the plasmalemma. when the cell enters apoptosis, ps translocated to the outer membrane constituting an ‘eat me’ signaling (presence of ps signals the cells that will be phagocytosed in a silent, noninflammatory manner). this is especially important for the elimination of redundant sperm from the female genitalia, and failure in this mechanism may lead to an uncontrolled inflammatory reaction to semen and posterior endometritis. polarity of the plasma membrane an anionic bis oxonol dye (disbac2)47 enters depolarized cells where they bind to the intracellular proteins of membranes and exhibit enhanced fluorescence and red-orange spectral shifts. increased depolarization results in more influx of the anionic dye, and thus an increase in fluorescence. conversely, hyperpolarization is indicated by a decrease in fluorescence. in contrast to cationic carbocyanines, anionic bis oxonols are largely excluded from mitochondria and are primarily sensitive to plasma membrane potential.48 the use of flow cytometry allows determining, at the single cell level, many distinct characteristics of sperm. recently, changes in the potential of the membrane have been studied in relation to capacitation and cryopreservation.33,49 these changes relate to modifications in intracellular na+ that can also be monitored using flow cytometry. the potential of the sperm membrane can be determined with the probe disbac2 combined with a vitality dye to exclude dead sperm from the analysis. depolarization of the membrane has been reported in relation to apoptotic changes,49 whereas capacitation is linked to hyperpolarization.33 interestingly, this occurs only in a subpopulation of live, intact acrosome sperm and not in the whole ejaculate. depolarization of the membrane has been related to apoptotic changes in somatic cells and in sperm. thus, combined staining has been employed to determine if depolarized sperm correspond also to sperm having apoptotic features. these experiments demonstrate that depolarized sperm are also caspase 3 positive, express ps translocation, and have increased intracellular na+ and collapsed mitochondria.49 active sperm caspases: denouncing sperm senescence senescent sperm express active caspase 3.37,50,51 depending of the presence of prosurvival factors, caspase 3 remains inactive due to the phosphorylation of protein kinase b (pkb or akt).43,51,52 if prosurvival factors are lost or oxidative stress reaches a threshold, caspase 3 is activated and sperm senescence and death are triggered.37,53 cryopreservation triggers this phenomenon, and surviving sperm experience accelerated senescence.52,54–57 active caspase 3 can be detected using cellevent caspase-3 green detection reagent, which consists of a four-amino-acid peptide (devd) conjugated to a nucleic acid-binding dye. this cell-permeant substrate is intrinsically nonfluorescent because the devd peptide inhibits the ability of the dye to bind to dna. after the activation of caspase 3 in apoptotic cells, the devd peptide is cleaved, enabling the dye to bind to dna and produce a bright, fluorogenic response with an absorption/emission maxima of 502/530 nm. flow cytometry data output and clinical interpretation an example (figure 1) of a multiparametric panel as an output of stallion sperm studied with a modern flow cytometer (cytoflex lx), providing data relevant for the clinician to evaluate the current and potential status of a semen sample. description of this four-color experiment implies that the information provided has to be depicted in several dot plots since the number of 2d plots increases exponentially with the number of colors in the analysis. additionally, the establishment of gates is performed manually. these are confounding data for a nonspecialist and ought to be simplified without losing relevant information, for instance, using self-learning artificial intelligence techniques. an example of such is presented (figure 2), using the flo self-organizing maps for visualization and organization of flow cytometry data. with this technique, all the characteristics of the sperm in the sample can be visualized at a glance, facilitating comparisons among samples and identification of subtle changes. in the example, aliquots of the same stallion ejaculate were extended and incubated at 37ºc for 3 hours in 2 distinct media. sperm were stained with a 4 colors protocol, to identify live, apoptotic (caspase 3 and annexin v) and necrotic sperm (live/dead viakrom 808®). a quick view of this panel, comparing the 2 incubation sets (1 versus 2), allows the observer to see that there are more viable sperma in 1a than in mapping 2. in the http://dx.doi.org/10.58292/ct.v15.9406 4 citation line: clinical theriogenology 2023, 15, 9406, http://dx.doi.org/10.58292/ct.v15.9406 figure 1. example of a multiparametric panel for the study of stallion sperm. (a) gate for the sperm, where sperm events are identified based on forward (fsc) and side scatter; events having higher area in the fsc-a are removed from the analysis since they represent doublets and clumps. (b) to remove debris, the sample is stained with a permeable dna binding probe (hoechst 33342), and only dna bearing particles, mostly sperm, are stained. unstained particles (debris) are removed from the analysis. (c) combination of h33342 and cellevent®; this combination of probes identifies 3 sperm populations corresponding to live, apoptotic, and necrotic sperm. (d) double staining with 3 probes to identify apoptotic changes, cellevent®, to identify caspase 3 positive sperm and annexin-v to identify sperm having transposition of phosphatidylserine to the outer leaflet of the membrane. three populations are easily identified. (e) combination of cellevent®, and the fixable live/dead probe viakrom 808®. this combination easily identifies 3 populations: live, apoptotic, and necrotic sperm. (f) combination of h33342 and the fixable live/dead probe viakrom 808®; this combination of probes identifies 3 types of populations: live sperm, dead sperm, and damaged sperm (2 & 3 positive for viakrom 808®). a b c d e f debris c as p 3 l/d viakrom 808 sperm doublets and clumps sperm live apoptotic necrotic annexin-v neg caspase 3 neg caspase 3 + annexin-v + caspase 3+ n ec ro tic live dead s s c -h f s c -a fsc-a h33342 c as p 3 a nn ex in -v h33342 casp 3 l/d viakrom 808 h 33 34 2 http://dx.doi.org/10.58292/ct.v15.9406 citation line: clinical theriogenology 2023, 15, 9406, http://dx.doi.org/10.58292/ct.v15.9406 5 figure 2. use of artificial intelligence techniques to simplify the output of flow cytometry analysis. figure presents a flo self-organizing mapping for visualization and organization of flow cytometry data where all the characteristics of stallion sperm in aliquots of the same ejaculate, extended, and incubated at 37ºc for 3 hours in 2 media (1 versus 2) are presented and visualized at a glance. here, sperm were stained with a 4 colors protocol to identify live, apoptotic (caspase 3 and annexin v), and necrotic sperm (live/ dead viakrom 808®) in particular clusters. the gains in time are relevant, for instance, after live sperm (red circles) comparing the 2 incubation panels (1 versus 2). in 1a, there are more viable sperm than in panel 2, where there is a larger abundance of sperm having several apoptotic markers. interestingly as well, panel 2 depicts the presence of many live sperm having phosphatidylserine transposition to the outer membrane, representing unstable membranes. anexin+ apoptotic necrotic spermatozoa viable 1 metacluster 1 2 3 4 5 6 7 8 9 10 a b anexin+ apoptotic necrotic spermatozoa viable c d 2 metacluster 1 2 3 4 5 6 7 8 9 10 http://dx.doi.org/10.58292/ct.v15.9406 6 citation line: clinical theriogenology 2023, 15, 9406, http://dx.doi.org/10.58292/ct.v15.9406 latter, sperm having various apoptotic markers are more abundant (see 2d). moreover, many live sperm exhibit phosphatidylserine transposition to the outer membrane, representing unstable membranes. such sperm are at risk, and the clinician benefits from knowing all this battery of data and its quantification before deciding the fate of the ejaculate or the use of a processed sample for further use. hopefully, there shall be gains in diagnostics and in resulting fertility. concluding remark cytomics, combining novel designed motility analyzers, multiparametric flow cytometers, and enhanced digital imaging together with the incorporation of artificial intelligence in the analysis, shall dominate the landscape of andrological laboratories and enable quick determinations on huge sperm numbers for markers highly relevant to sperm function and, hence, for fertility. such advancements shall, however, hardly replace essential clinical andrological practice. ethical permission housing, handling, and collection of semen that have been used during the analyses described in this review have followed the european community directive 2010/63/eu, 22/09/2010, and current spanish (law 6/2913 june 11th and european directive 2010/63/eu) and swedish legislation (sjvfs 2017:40). studies involved had previously been scrutinized and approved by the ethical committee of the university of extremadura (pid2021-122351ob-i00 and ib20008) and the swedish regional committee for ethical approval of animal experiments (linköpings djurförsöksetiska nämnd, linköping, sweden, permit numbers 75-12 (10/02/2012), id1400 (02/02/2018), and dnr 03416-2020 (26/03/2020)). conflict of interest authors declare no (financial, commercial, or personal) conflict of interest. author contribution authors contributed equally to the design and writing. acknowledgments authors thank former and present students, post-docs, and collaborators engaged in many of the studies reviewed. funding these studies were made possible by grants from the ministry of science-european fund for regional 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real-time ultrasonography and development of the means by which follicular wave emergence can be controlled have provided new practical approaches to superstimulation of donors for in vivo and in vitro embryo production. although some embryo transfer practitioners still initiate superstimulatory treatments during midcycle, elective control of follicular wave emergence and ovulation have greatly impacted on-farm embryo transfer, especially when large groups of donors need to be superstimulated concurrently. a combination of estradiol and progestins has been the most common treatment for synchronization of follicular wave emergence for many years. however, in countries where estradiol cannot be used, practitioners have turned to alternative treatments, e.g. mechanical follicle ablation or gonadotropin releasing hormone administration, for synchronization of follicle wave emergence for superstimulation. in vitro embryo production also depends on synchronization of follicle wave emergence. as bos indicus cattle have high antral follicle populations, large numbers of oocyes can be obtained by ovum pick up without superstimulation. however, in bos taurus donors, synchronization of follicular wave emergence and superstimulation with follicle stimulating hormone is necessary to obtain high numbers of oocytes by ovum pick up and blastocysts following in vitro fertilization. treatments for synchronization of follicular wave emergence and superstimulation are practical, easy to administer by farm personnel and facilitate application of both in vivo and in vitro embryo technologies in cattle. introduction objective of ovarian superstimulatory treatments in cattle is to obtain tmaximum number of viable embryos by stimulating growth of antral follicles and ovulation of competent oocytes.1 usual regimen for in vivo embryo production has been twice-daily intramuscular (im) treatments with fsh for 4 or 5 days. forty eight or 72 hours after initiation of treatment, prostaglandin f2α (pgf2α) is administered to induce luteolysis. estrus occurs in 36 48 hours, with ovulations beginning 24 36 hours later.2,3 many practitioners prefer decreasing follicle stimulating hormone (fsh) dose schedules, whereas others use constant-dose schedules.1 others treat with pgf2α twice on the third day of the treatment protocol, whereas others prefer to treat with pgf2α twice on the fourth day. furthermore, many do not treat with fsh on the day after administration of pgf2α. 1although there would appear to be no experimental data supporting 1 approach over others, recent experiments have indicated that follicle maturation and ovulation rate can be improved in at least some donors if fsh treatments are administered over 6 or 7 days.3,4 objective of this manuscript is to briefly summarize existing protocols for superstimulating donors for in vivo and in vitro embryo production and to present information on evolution of this technology in south america. manipulation of ovarian function for superstimulation two very important factors influencing variability in superstimulatory response are intrinsic number of antral follicles in donors and stage of follicular development at initiation of fsh treatments. response can be predicted by antral follicle counts done with ultrasonography,5,6 or measurement of circulating concentrations of anti müllerian hormone (amh; bos taurus,7,8 bos indicus9). high antral follicle counts have resulted in more ovulations and more transferable embryos than low antral follicle counts.10 similarly, top quartile of circulating amh values was associated with a greater superovulatory 337 clinical theriogenology • volume 11, number 3 • september 2019 response than the lowest quartile.11 therefore, selection of donors based on antral follicle counts or amh concentrations may be important for predictable embryo production. conventional protocol of initiating ovarian superstimulation during midcycle was based on anecdotal and experimental evidence suggesting a greater superovulatory response when gonadotropin treatments were initiated between days 8 and 12 of the cycle.1 it is now known that midcycle is the approximate time of emergence of the second follicular wave.12 however, day of second wave emergence varies between wave types (1 or 2 days later in 2 versus 3 wave cycles). in this regard, nasser et al.13 reported that superovulatory response was greater when gonadotropin treatments were initiated at the time of follicle wave emergence; 1 day asynchrony resulted in a reduced ovarian response. the necessity of waiting until midcycle to initiate fsh treatment implies monitoring estrus, the obligatory delay and an inability to group donors. an alternative is to superstimulate donors following synchronization of follicular wave emergence.14 follicle ablation transvaginal ultrasound guided follicle ablation followed by fsh treatments 1 or 2 days later is very efficacious,15,16 but requires specialized skill and equipment and is difficult to apply in the field. however, follicle aspiration for opu/ivf will synchronize wave emergence and thus, embryos could be produced both in vitro and in vivo in succession, from the same donor.17 estradiol and progesterone the preferred approach for synchronization of follicular wave emergence prior to superstimulation is administration of 5 mg estradiol 17 plus 50 or 100 mg progesterone and insertion of a progestin device 4 days before initiating fsh treatments. experimental18 and commercial14 results have shown that embryo production following this treatment at unknown stages of the estrous cycle is comparable to that initiated 8 12 days after observed estrus. although estradiol 17 and its esters are available in most countries in south america, this is not the case in many other countries around the world, necessitating the use of alternatives to synchronize follicle wave emergence prior to superstimulation. gonadotropin releasing hormone attempts to synchronize follicular wave emergence for superstimulation with gonadotropin releasing hormone (gnrh) were initially unsuccessful; however, subsequent field data were more promising. in these cases, gnrh was administered 1.5 3.0 days after insertion of a progestin device which may have increased probability of a luteinizing hormone (lh)-responsive follicle. indeed, bó et al.19 reported on the strategic use of pgf2α, a progestin device and gnrh to induce ovulation prior to initiating fsh treatments. a persistent follicle was induced by giving pgf2α progestin device insertion; following administration of gnrh 7 days later, ovulation occurred in > 95% of cattle. superstimulation initiated 36 hours after gnrh (with the progestin device remaining in place) resulted in a superovulatory response that did not differ from controls. more recently, hinshaw et al.20 reported no difference in superovulatory response whether gnrh was administered 2 or 7 days after insertion of a progestin device. extended superstimulatory treatment protocols an early study provided rationale for a hypothesis that superstimulatory treatment may recruit follicles into the wave and allow small follicles to attain medium or large diameters.21 based on this notion, attempts have been made to increase the superovulatory response by adding equine chorionic gonadotropin (ecg) treatment prior to initiating fsh treatments. pretreatment with ecg 2 days before the conventional fsh treatment protocol resulted in a numerically greater number of transferable embryos (6.7 ± 1.2 versus 4.9 ± 0.9) in an unselected group of donors22 and a significantly greater number of transferable embryos in donors defined as poor responders (3.6 ± 0.6 versus 1.0 ± 0.2).3 338clinical theriogenology • volume 11, number 3 • september 2019 more recently, we evaluated the superovulatory response and embryo recovery in donors treated with either 4 or 7 day fsh superstimulatory treatment protocols.4 twenty four beef cows were blocked by numbers of follicles ≤ 5 mm at wave emergence and placed into either a 4 or 7 day fsh treatment protocol utilizing the same total dose of 400 mg fsh (folltropin-v; vetoquinol inc., canada) administered twice-daily at a constant daily dosage. mean number of ovulations detected by ultrasonography was greater in the 7 day treatment group (30.9 ± 3.9 versus 18.3 ± 2.9, p = 0.01), consistent with a numerically greater number of follicles ≥ 10 mm just prior to ovulation (27.5 ± 4.1 versus 19.5 ± 2.6; p = 0.11). moreover, ovulations occurred more synchronously in the 7 day group (93% of ovulations occurred 12 36 hours post lh, compared to 66% in the 4 day group) suggesting that superstimulated follicles were more mature and capable of responding to an lh stimulus. although total number of ova and embryos, fertilized ova and transferable embryos did not differ statistically, all end points favored the 7-day group. in addition, when data from cows with fertilization failure were removed, number of transferable embryos tended to be higher in the 7 day group (7.6 ± 1.7 versus 4.2 ± 1.5; p = 0.07). in another study,23 a 7 day superstimulation protocol was used to investigate influence of progesterone on follicle growth, ovulation and oocyte competence. beef cows were superstimulated with 25 mg of fsh twice daily for 4 or 7 days. again, the superstimultory response (number of large follicles just prior to insemination) was greater (p < 0.05) in the 7 day group and numbers of ovulations (15.4 versus 11.6) and embryos (6.7 versus 5.9) were numerically higher in the 7 day group. duration of treatment appears to be responsible for increases in superstimulatory response, rather than fsh dose. in the 2 studies noted above, number of ovulatory-sized follicles just prior to ovulation was greater following 7 days of superstimulation than 4 days, whether total dose of fsh was greater23 or the same.4 in addition, there was no evidence that more follicles were recruited, as total numbers of follicles at the end of fsh treatment was the same as that at the beginning of fsh treatment. microarray analysis of large antral follicles as overcoming follicle selection is a key factor for ovarian superstimulation, exogenous fsh is used to prevent regression of subordinate follicles so that they assume qualities of a dominant follicle. in a recent study of follicles undergoing a 4 day superstimulation protocol, gene expression in granulosa cells was altered compared to a single, naturally occurring dominant follicle.24,25 expression of growth-related genes similar to the prelh stage of follicle growth (even though lh had been administered) and those involved in oxidative stress response were upregulated in granulosa cells of follicles undergoing a 4 day fsh superstimulation protocol compared to a preovulatory follicle of an unstimulated follicular wave. genes related to a disturbance in angiogenesis were also upregulated in superstimulated follicles. we speculate that gene expression during a 7 day superstimulation protocol may be more similar to the naturally occurring single preovulatory follicle. acquisition of lh receptors is commonly used as a marker for dominance and a prerequisite for establishment of ovulatory capacity.26,27 recently, evaluation of the expression of lhr mrna in granulosa cells of superstimulated and unstimulated follicles 12 hours after progesterone device removal revealed that expression of lhr was decreased following superstimulation.28 consequently, follicles following the conventional 4 day fsh superstimulation protocol seemed to have been delayed in maturational development and response to lh compared to a naturally occurring single preovulatory follicle.24,25 these findings further support the concept that following a 4 day superstimulation treatment protocol, some follicles do not have sufficient time to mature, upregulate expression of appropriate genes, and acquire capacity to ovulate. use of equine chorionic gonadotropin to replace the last 4 follicle stimulating hormone applications in search of possible improvements to the superstimulatory treatment protocol, price et al.29 reported that during superstimulatory treatment, pulses of lh diminish shortly after the first application of fsh and are accentuated during the last applications and the preovulatory period. this occurs due to an increase in estradiol concentrations as a consequence of high steroidogenic activity of superstimulated 339 clinical theriogenology • volume 11, number 3 • september 2019 cows.29 however, lh is essential for final growth of superstimulated follicles and for complete maturation of oocytes.30 equine chorionic gonadotropin (ecg) is a complex glycoprotein with fsh and lh activity.31 in cattle, this gonadotropin has prolonged duration of action, due its sialic acid content (10 15%).32 a remarkable feature of ecg that has been exploited in multiple experimental and commercial contexts is its ability to express fsh activity in nonequid species.33 in the early days of bovine embryo transfer, ecg was used to induce superovulation in donor cows.1 however, its long half-life, which was a feature for induction of superovulation with a single administration, resulted in multiple anovulatory follicles at the time of embryo collection and poor embryo quality (reviewed1,33). more recently, the last 2 doses of fsh in a superstimulation protocol have been replaced by different doses of ecg, with the intention of providing more lh support to the growing follicles. some studies reported beneficial effects of the association of fsh and ecg,34,35,36,37whereas other studies did not detect benefits.38,39 although administration of ecg near the end of the fsh treatment protocol did not always improve the superovulatory response, it was certainly not detrimental and raised some interest in using ecg to simplify the superstimulation protocol and decrease costs, as ecg is usually less expensive than pituitary extracts containing fsh. therefore, we designed a study to evaluate the superovulatory response and embryo production in beef donors using 8 twice-daily injections of fsh or an alternative protocol in which the last 4 fsh treatments were replaced with a single injection of ecg.40 twelve (experiment 1) and 18 (experiment 2) mature bonsmara donor cows were superstimulated twice at a 46 day interval in a crossover design. follicular wave emergence was synchronized by administration of estradiol17β at insertion of a progestin device, with superstimulation initiated 4 d later. donors in the control group received 8 applications of folltropin-v im (total dose: 300 mg) in a twice-daily decreasing dosage schedule over 4 days, whereas donors in the fsh + ecg group received only the first 4 applications of folltropin-v (total dose: 220 mg) and 48 h after initiating treatment, 800 iu of ecg im in a single dose. all donors received pgf2α im in the am and pm of the same day and intravaginal devices were removed in the am of the next day. all cows also received gnrh 24 hours later and were inseminated with frozen and thawed semen from 2 bulls, 12 and 24 hours later. ova and embryos were collected and evaluated according to the iets standards 7 days after administration of gnrh. in experiment 2, donors were treated only with fsh + ecg. the total dosage of folltropin-v was 200 mg and ecg dosage was either 800 or 600 iu. results of both experiments are presented in table 1. in experiment 1, the fsh (control) group produced more (p < 0.01) fertilized oocytes, but there were no differences in number of transferable embryos. in experiment 2, there were no differences between the fsh + 800 ecg and fsh + 600 ecg groups in any parameter evaluated. in conclusion, replacement of the last 4 injections of fsh by a single administration of either 600 iu or 800 iu of ecg decreased the number of treatments required in a superovulation program without negatively affecting production of transferable embryos. table 1. mean ± sem embryo production in bonsmara donors treated with fsh or fsh + ecg.* n total ova/embryos fertilized ova transferable embryos experiment 1 fsh 12 11.7 ± 2.5 10.5 ± 2.3ª 5.7 ± 1.4 fsh + 800 iu ecg 12 9.6 ± 1.5 6.8 ± 1.0b 5.3 ± 1.0 experiment 2 fsh + 800 iu ecg 18 6.7 ± 0.7 5.4 ± 0.8 3.6 ± 0.7 fsh + 600 iu ecg 18 6.1 ± 1.1 4.3 ± 1.0 3.7 ± 0.8 abmeans without a common superscript are different (p < 0.05). *donors were given 8 intramuscular injections of fsh at 12-hour intervals (fsh group) or the last 4 fsh treatments were replaced by a single intramuscular injection of ecg (fsh + ecg group). 340clinical theriogenology • volume 11, number 3 • september 2019 manipulation of follicular development for in vitro embryo production in vitro production of embryos, together with obtaining oocytes by ultrasound guided follicular aspiration, known worldwide as ovum pick up (opu), are reproductive biotechnologies that have advanced greatly in the last 10 years. this technology is highly developed in brazil, where 57% of in vitro embryos that are transferred in the world are produced.41 as indicated earlier, bos indicus cattle have more follicles recruited per wave as compared to bos taurus breeds, resulted in recovery of more oocytes after opu.42,43,44 likewise, in field data from our laboratory, brahman-influenced synthetic breeds produce significantly more viable oocytes and transferable blastocysts than bos taurus breeds (table 2).45 table 2. mean ± sem effects of donor breed on number of viable oocytes aspirated by opu and embryos produced in vitro without superstimulation. breed n viable oocytes blastocysts dairy bos taurus (holstein) 620 8.0 ± 0.2a 1.6 ± 0.1a beef bos taurus (angus, hereford, bonsmara) 229 11.6 ± 0.5b 3.0 ± 0.2b bos indicus (brahman) 52 15.8 ± 1.4c 6.8 ± 0.9c bos indicus x bos taurus (brangus, braford) 1045 19.3 ± 0.4d 5.3 ± 0.2c abwithin a column, means without a common superscript are different (p < 0.01). studies were conducted to evaluate effects of synchronizing follicular wave emergence and superstimulation on number and quality of cumulus-oocyte complexes (cocs) recovered by opu and in vitro embryo production.46,47 the most important conclusions of these studies were: 1) synchronizing follicle wave emergence prior to opu increased numbers of cocs obtained and blastocysts produced in bos taurus, but not bos indicus cattle; 2) treatment with estradiol and progesterone and dominant follicle removal (dfr) were equally effective in synchronization of follicular wave emergence for opu; and 3) superstimulatory treatment with fsh increased number and quality of oocytes obtained by opu in bos taurus breeds, but not in bos indicus donors. in an experiment conducted in brazil with holstein donors,48 all cows received a progestin device and 2 mg of estradiol benzoate (day 0). cows in the control group received no additional treatments, whereas cows in the fsh group received a total dose of 200 mg of folltropin-v administered in twice-daily treatments on days 4 and 5. the progestin device was removed and opu was performed on day 7 (40 hours after last injection of fsh in the treatment group). there were no differences between groups (p = 0.92) in numbers of follicles aspirated per opu session (17.2 ± 1.3 versus 17.1 ± 1.1 in the control and fsh treated cows, respectively); however, fsh-treated cows had a higher blastocyst rate (34.5%, 89/258 versus 19.8%, 55/278, p < 0.001) and more transferable embryos per opu session than those of the control group (3.0 ± 0.5 versus 1.8 ± 0.4, p = 0.02). it was concluded that superstimulation of holstein donors with fsh before opu increased efficiency of in vitro embryo production by increasing oocyte and embryo quality. in addition, nonlactating donors had a higher percentage of in vitro blastocyst development and produced more embryos per opu session than lactating cows. in a later study, similar results were obtained when the 4 doses of fsh were replaced by a single injection of 200 mg of folltropin-v diluted in a 0.5% hyaluronic acid solution (map-5, vetoquinol).49 two other studies were performed in angus donors. in the first study,50 administration of fsh resulted in more cocs obtained by opu. in the second study (ongarato et al. unpublished), multiparous, nonlactating angus cows, were randomly allocated to 2 treatment groups and treated twice in a crossover design. follicular wave emergence was synchronized with estradiol 17β and progesterone, plus a progestin device. four days later (day 4) donor cows received either 160 mg folltropin-v diluted in 4 ml of 0.5% hyaluronan by a single im injection or received no fsh (control group). cocs were obtained by 341 clinical theriogenology • volume 11, number 3 • september 2019 opu 72 hours later (day 7). results are summarized in table 3. number of viable cocs was significantly higher in fsh-treated donors than in controls. although administration of fsh prior to opu has been a common practice for increasing the available follicular population for opu, most studies have adopted conventional twice daily treatments with fsh, with either positive results in number of oocytes collected and embryos produced58,49,50,51 or no significant differences in oocyte collection and embryo production in holstein cows.52 we understand that in north america, most practitioners superstimulate donors with fsh prior to opu. table 3. mean + sem numbers of total and viable cumulus oocyte complexes (cocs) recovered and number of blastocysts produced following superstimulation of angus donors. group cocs blastocysts total viable total single fsh (n = 9) 21.4 ± 2.4a 14.1 ± 1.6a 4.2 ± 0.8 control (n = 9) 15.9 ± 2.7b 10.6 ± 2.0b 2.7 ± 0.7 p value 0.02 0.02 0.13 abwithin a column, means without a common superscript are different (p < 0.05) other studies compared effectiveness of multiple vs a single administration of fsh prior to opu. chaubal et al.53 compared administration of 200 mg of fsh diluted in saline, in multiple injections, a single im injection or 2 subcutaneous injections (120 mg and 80 mg respectively) in angus cows and reported lower oocyte numbers when fsh was administrated in a single im injection. ooe et al.54 reported that 20 mg fsh in 30% polyvinylpyrrolidone as a single im treatment prior to opu in cyclic and pregnant cows resulted in satisfactory follicular responses and oocyte recovery rates. in another study, 100 mg of fsh diluted in saline and administered as a single im injection in holstein cows, resulted in a significant increase in number of follicles aspirated, but number of oocytes recovered per animal per session and blastocyst production were unaffected compared to nonfsh treated donors.55 finally, sakaguchi et al.56 described a different and innovative approach, using a single epidural administration of fsh via caudal vertebrae in japanese black cows, reporting a higher superstimulatory response and embryo production compared to cows in the control group that received multiple im injections of fsh for 3 days. obviously, these results need further investigation. application of in vivo and in vitro embryo production in south america the commercial embryo transfer industry began in north america in the early 1970s and the technology soon spread to south america.1 brazil and argentina have consistently ranked in the top 5 countries outside north america and europe in production of in vivo-derived (ivd) embryos. viana41 reported recently that more than 992,289 ivd and 495,054 in vitro produced (ivp) bovine embryos were generated worldwide in 2017. north america accounted for > 59% (292,755) of ivd embryos, whereas south america only counted for 10% (49,230). conversely, distribution of ivp embryos were similar in north (475,696; 48%) and south (453,685; 46%) america. this is the first report in which north america produced more ivp than ivd embryos, whereas in south america, number of ivp embryos has exceeded the number of ivd embryos for > 10 years. the use of ivp in brazil has increased rapidly since 2000, driven primarily by bos indicus breeds which have large numbers of antral follicles from which large numbers of high-quality oocytes can be recovered without superstimulation. viana et al.57 reported that embryo transfer accounted for 19.7% of all “zebu” calves registered in brazil between 2005 and 2015. 342clinical theriogenology • volume 11, number 3 • september 2019 in vitro embryo production in brazil increased approximately 3.5 times between 2005 and 2016, while numbers of ivd embryos decreased approximately 3 times over the same interval (viana, personal communication). commercial ivp in brazil has been reported to have gone through 3 phases.58 the initial phase involved use of proven donors of high genetic merit in both beef and dairy cattle and numbers of ivd and ivp embryos increased similarly. the second phase of growth occurred between 2003 and 2010, driven largely by the need to produce replacement bulls. in 2005, at the peak of this phase, 90.0% of ivp was in beef breeds, with nelore accounting for 82.7% of all embryos. the third phase has involved use of sex selected sperm and is associated with a shift in ivp from beef breeds to bos taurus dairy breeds. in 2014, ivp in dairy breeds increased by 46.5% (69.0% of total), exceeding that of beef breeds for the first time. conclusion use of protocols that control follicular development and ovulation facilitate widespread application of assisted reproductive technologies. current protocols are practical and easy to perform by field staff. in superovulation schemes, estradiol is very effective in synchronizing emergence of a follicular wave, but is not available in many countries. protocols using dominant follicle removal or gnrh are effective alternatives. lengthened superstimulation protocols are also an interesting alternative, as they allow the time necessary for all growing follicles to acquire the ability to ovulate. conversely, 4 day treatment protocols appear to not allow sufficient time for all follicles to acquire this capacity. regarding in vitro production of embryos, cattle with bos indicus influence adapt very well to this technology as they have a high antral follicle population; consequently, more oocytes are obtained by opu than in bos taurus breeds. in bos taurus donors, synchronization of follicular wave emergence and use of fsh has resulted in collection of more cocs by opu and in vitro production of a higher percentage of blastocysts. conflict of interest there are no conflicts of interest to declare references 1. bó ga, mapletoft rj: historical perspectives and recent research on superovulation in cattle. theriogenology 2014;81:38-48. 2. bó ga, baruselli ps, chesta pm, et al: the timing of ovulation and insemination schedules in superstimulated cattle. theriogenology 2006;65:89-101. 3. bó ga, carballo guerrero d, adams gp: alternative approaches to setting up 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embryo production. theriogenology 1999;51:37-46. 30. oliveira acs, mattos mcc, bastos mr, et al: efficiency of superstimulatory protocol p-36 associated with the administration of ech and lh in nelore cows. theriogenology 2014;82:715-719 31. murphy bd, martinuk sd: equine chorionic gonadotrophin. endocrine reviews 1991;12:27-44. 32. schams s, menzer ch, schallenberger e, et al: some studies on pregnant mare serum gonadotrophin (pmsg) and on endocrine responses after application for superovulation in cattle, in: control of reproduction the cow. commission of the european communities, luxembourg, 1978; p. 25-80. 33. murphy bd: equine chorionic gonadotropin: an enigmatic but essential tool. anim reprod 2012: 9:223-230. 34. barros cm, barcelos ac, gouvea lm, et al: improvement of a superovulatory protocol in nelore cows: replacing the last two doses of pfsh by ecg. reprod fertil dev 2008:20:152 (abstract). 35. mattos mc, bastos mr, guardieiro mm, et al: improvement of embryo production by the replacement of the last two doses of porcine follicle-stimulating hormone with equine chorionic gonadotropin in sindhi donors. anim reprod sci 2011;125:119-123. 36. reano i, carballo d, tribulo a, et al: effect of the addition of ecg to superovulatory treatments with folltropin-v in the embryo production of embryo donors. proc viii international symposium on animal reproduction, irac, cordoba, argentina, 2009; p. 7 (abstract). 37. cifuentes e, quevedo l, hoyos a, et al: effect of the application of ecg in donor cows of superovulated embryos with folltropin-v. proc viii international symposium on animal reproduction, irac, cordoba, argentina, 2009; p. 9 (abstract). 38. sartori r, guardieiro mm, barros cm, et al: lack of improvement on embryo production by the replacement of the last two doses of pfsh by ecg in superovulated nelore heifers. reprod fertil dev 2009;21:245 (abstract). 39. davis rl, arteaga a, hasler jf: addition of equine chorionic gonadotropin to a traditional follicle stimulating hormone protocol for superovulation of bos taurus beef cows. reprod fertil dev 2012;24:224-225 (abstract). 40. barajas jl, cedeño a, andrada s, et al: embryo production using follicle stimulating hormone (fsh) or fsh + equine chorionic gonadotropin in beef donors. reprod fertil dev 2019;31:223 (abstract). 41. viana j. 2107 statistics of embryo production and transfer in domestic farm animals. embryo transfer newsletter 2018;36:8-25. 42. watanabe yf, souza ha, mingoti rd, et al: number of oocytes retrieved per donor during opu and its relationship with in vitro embryo production and field fertility following embryo transfer. anim reprod 2017;14:635-644. 344clinical theriogenology • volume 11, number 3 • september 2019 43. pontes jhf, nonato-junior i, sanches bv, et al: comparison of embryo yield and pregnancy rate between in vivo and in vitro methods in the same nelore (bos indicus) donor cows. theriogenology 2009;71:690-697. 44. baruselli ps, sa filho mf, ferreira rm, et al: manipulation of follicle development to ensure optimal oocyte quality and conception rates in cattle. reprod dom anim 2012; 47 (suppl. 4):134-141. 45. bernal b, revol j, oviedo jm, et al: influence of breed and season on in-vitro embryo production. reprod fertil dev 2016;28:255 (abstract). 46. ongaratto fl, rodriguez-villamil p, tribulo a, et al: effect of follicle wave synchronization and gonadotropin treatments on the number and quality of cumulus-oocyte complex obtained by ultrasound-guided ovum pick-up in beef cattle. anim reprod 2015;12:876-883. 47. baruselli ps, batista eos, vieira lm, et al: factors that interfere with oocyte quality for in vitro production of cattle embryos: effects of different developmental & reproductive stages. anim reprod 2016;13:264-272. 48. vieira lm, rodrigues ca, castro netto a, et al: superstimulation prior to the ovum pick-up to improve in vitro embryo production in lactating and non-lactating holstein cows. theriogenology 2014;82:318-324. 49. vieira lm, rodrigues ca, castro netto a, et al: efficacy of a single intramuscular injection of porcine fsh in hyaluronan prior to ovum pick-up in holstein cattle. theriogenology 2015;84:1-10. 50. ongaratto f, tribulo a, ramos m, et al: oocyte recovery rates and in-vitro blastocyst production in cattle treated with a single injection of folltropin-v diluted in a slow release formulation. reprod fertil dev 2011;23:202-203 (abstract). 51. blondin p, vigneault c, nivet al, et al: improving oocyte quality in cows and heifers what have we learned so far? anim reprod 2012;9:281-289. 52. oliveira lh, sanches cp, seddon as, et al: short communication: follicle superstimulation before ovum pick-up for in vitro embryo production in holstein cows. j dairy sci 2016;99:9307-9312. 53. chaubal sa, ferre lb, molina ja, et al: hormonal treatments for increasing the oocyte and embryo production in an opu-ivp system. theriogenology 2007;67:719-728. 54. ooe m, rajamahendran r, boediono a, et al: ultrasound guided follicle aspiration and ivf in dairy cows treated with fsh after removal of the estrous cycle j vet med sci 1997;59:371-376. 55. bungartz l, lucas-hahn a, rath d, et al: collection of oocytes from cattle via follicular aspiration aided by ultrasound with or without gonadotropin pretreatment and in different reproductive stages. theriogenology 1995;43:667-675. 56. sakaguchi k, ideta a, yanagawa y et al: effect of a single epidural administration of follicle-stimulating hormone via caudal vertebrae on superstimulation for in vivo and in vitro embryo production in japanese black cows. j reprod dev 2018;64:451-455. 57. viana jhm, figueiredo acs, siqueira l: brazilian embryo industry in context: pitfalls, lessons, and expectations for the future. anim reprod 2017;14:476-481. 58. sartori r, prata ab, figueiredo acs, et al: update and overview on assisted reproductive technologies (arts) in brazil. anim reprod 2016;13:300-312. 345 clinical theriogenology • volume 11, number 3 • september 2019 346clinical theriogenology • volume 11, number 3 • september 2019 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 524288 /lockdistillerparams false /maxsubsetpct 100 /optimize false /opm 0 /parsedsccomments true /parsedsccommentsfordocinfo false 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>> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice mini mammal matters: reproduction and common reproductive issues in small mammals tara harrison department of clinical sciences, college of veterinary medicine north carolina state university, raleigh, nc abstract exotic small mammals (or zoological companion animals) have become common pets. having a basic understanding of their reproduction and reproductive anatomy is helpful. common conditions that they develop should be understood. prevalence of conditions and most common conditions vary by species. awareness of these common conditions enables the veterinary practitioner to provide adequate medical therapy and treatments to these animals and to continue to improve their quality of life. keywords: guinea pig, rabbit, hedgehog, reproduction, neoplasia introduction small mammals are increasingly becoming common pets and are presented in veterinary practice. common animals kept as pets include rabbits, guinea pigs, chinchillas, ferrets, mice, rats, and sugar gliders. there are other species (e.g., groundhogs, prairie dogs, and degus) that also have reproductive issues; however, these species are not as common. it is not unusual for an owner to have reproductive questions regarding their animal or for the animal to experience abnormalities throughout the course of their lives. among small mammal pets, only rabbits and ferrets are commonly ovariohysterectomized or castrated, consequently, other species have more reproductive issues. a brief overview of reproductive anatomy and common reproductive concerns of a few pet species is provided. rabbits female rabbit anatomy is unique in that rabbit does not have a uterine body, instead rabbits have 2 separate uterine horns that open into vagina. rabbits are induced ovulators and do not have a reproductive cycle; however, they appear to have receptiveness every 4 6 days. rabbits ovulate ~ 10 hours after receptivity. duration of pregnancy in rabbits is 30 32 days. litter sizes can vary from 4 12 depending on the parity and size of the rabbit with larger and multiparous rabbits producing larger litters. it is recommended to perform ovariohysterectomy or castration on rabbits prior to 6 months of age. surgery for females is similar to other animals; however, tissues are more friable hence typically a spay hook is not used. other difference is excess fat-tissue in the female that poses a challenge to identify vessels and ovaries.1 castration in males is commonly via the scrotal approach and should be performed as closed or ‘open to closed castration’ to prevent herniation through the inguinal canal.1 rabbits develop numerous lesions of their reproductive tract. most common neoplasia is uterine adenocarcinoma, known to metastasize to lungs, liver, brain, or bone marrow. in rabbits presented with vaginal discharge, common uterine lesions were endometrial hyperplasia (44% [24/54]) and uterine adenocarcinoma (33% [18/54]);2 4 of these rabbits that presented with mammary adenocarcinoma had concurrent uterine disorders. rabbits with endometrial hyperplasia had a mean age of 4.5 years whereas rabbits with adenocarcinoma had a mean age of 6.1 years. upon presentation with adenocarcinoma, 8 rabbits were euthanized due to declined health or metastases. rabbits that underwent ovariohysterectomy as treatment for adenocarcinoma, 8/29 (28%) survived for 22 months or longer after surgery. rabbits (5 6 years) had 79.1% prevalence of adenocarcinoma.2 mammary gland abnormalities in rabbits were associated with endometrial hyperplasia or adenocarcinoma in rabbits.3,4 ovarian neoplasia has been reported; however, the prevalence is low.4 other lesions of rabbit ovaries reported were follicular cysts, cystic rete ovarii, widespread ovarian necrosis with dystrophic calcification, and ovarian adenoma.5 guinea pigs females have bicornuate uterus with short uterine body and paired uterine horns. there is 1 cervix that opens into vagina. guinea pigs have a vaginal closure membrane that opens at estrus and at parturition ~ day 26 of pregnancy.6 sexual maturity is ~ 2 months in females and 3 months in males. females can breed year-round; however, their peak reproductive age is 3 20 months, nevertheless, they reproduce until 5 years.6 guinea pigs have spontaneous ovulation. duration of pregnancy is typically 65 71 days. impending parturition can be determined by a gap of 15 mm in pubic symphysis, ~ 2 days prior to parturition.6 this width can expand to 25 mm or more at parturition. pubic separation may not occur adequately in guinea pigs that are bred after 7 8 months of age that can result in dystocia.6 it is not uncommon for guinea pigs to develop cystic ovaries. additional presenting clinical signs include bilateral nonpruritic alopecia of the flanks in female guinea pigs. cysts originating from rete ovarii are common in guinea pigs, with 58% prevalence.7 ovarian cysts were observed in 94% of guinea pigs 18 months old.8 clinical signs include abdominal distention, clitoral hypertrophy, depression, hyperkeratosis and/ or hyperpigmentation of the nipples, pain, rounded, fluctuant mass, tachypnea, vaginal bleeding, and nonpruritic alopecia of trunk or inguinal area.9 diagnosis is typically via clinical signs and ultrasonography. ovariohysterectomy is the treatment of choice and success with hormone injections (e.g., gnrh agonists) was variable.9,10 uterine hyperplasia and neoplasia tends to occur in animals that are > 6 years of age whereas those that are 7 12 months of age had a higher prevalence of uterine inflammation.5 routine castration of guinea pigs can be performed via scrotal approach; however, closure of the inguinal ring is important to prevent herniation of intestines or urinary bladder into scrotum.10 postoperative abscesses are not uncommon in guinea pigs’ castration surgeries. chinchillas chinchilla females have 2 uterine horns. presence of 2 cervices was discussed; however, the following is currently known.11 chinchillas have 2 uteri and 2 cervical canals housed in 1 anatomical cervix; officially termed as ‘uterus duplex bicollis and vaginal simplex’ (2 uteri, 2 cervices, and 1 vagina). although reproductive complications of chinchillas are not commonly reported in female chinchillas, they do occur. penile encirclement of hair in some male chinchillas may result in ischemic damage to penis and should be observed for and included in a routine examination. ferrets in us, most ferrets are ovariohysterectomized or castrated at 6 8 weeks of age. in other countries, ferrets are left intact or ‘chemically sterilized’, typically with a deslorelin (gnrh agonist) implant. females are sexually mature at 8 12 months of age. duration of pregnancy is 39 42 days. pseudopregnancy lasts 40 42 days.6 ferrets are seasonally polyestrous. in male ferrets, testes enlarge during december july when spermatogenic activity occurs. females are in persistent estrus from end of march to early august. prolonged estrus or persistent estrus cause severe anemia, thrombocytopenia, and bone marrow suppression. fifty-five percent of thrombocytopenic females were in persistent estrus with a mortality rate of 40%.6 ferrets can have a hematocrit of < 25%.12 treatment involves stabilizing the ferret with blood transfusions, supportive care, and ovariohysterectomy. gonadectomy is discouraged as it has been linked with development of hyperadrenocorticism.13 urethral obstruction can occur in ferrets due to hyperadrenocorticism from an enlarged prostate.12 cryptorchidism is reported in < 1% of ferrets.14 rodents – mice and rats benign fibroadenomas of the mammary gland are common in rats. these growths tend to be locally invasive and can develop anywhere along the mammary chain. surgical treatment is the treatment of choice. secondary or additional masses can develop shortly after surgery, or other complications (e.g., seromas or dehiscence) can occur. although treatment with deslorein implants was attempted, it was not successful in decreasing occurrence.15 performing ovariohysterectomy prior to development of mammary fibroadenomas, or by 5 7 months of age, reduced the development of these spontaneous tumors by 95%.16 mice, however, tend to develop malignant mammary adenocarcinomas and have been associated with mouse mammary tumor virus, and are commonly used in human breast cancer research.17 hedgehogs hedgehogs have 2 uterine horns. uterine neoplasia is not uncommon. neoplasia include endometrial stromal nodules (13.3%), nonneoplastic endometrial polyps (6.67%), and mammary gland adenocarcinoma (7.6%).18 primary neoplasia of uterus and ovary was adenosarcoma followed by stromal sarcoma, and leiomyosarcoma.19 mammary gland adenocarcinoma was observed in 9% of cases, 8.49% reproductive disorders and in 1 case of pyometra.20 adenomas have also been reported. reproductive neoplasia were diagnosed in females from 2 to 5 years of age.19 ovariohysterectomy of affected animals prolonged their lives and should be performed where possible.10 sugar gliders these animals are marsupials and have 2 uterine horns, and 2 lateral vaginas that open into 1 cul-de-sac divided by a septum.6 males have large prostate, 2 pairs of cowper’s glands (bulbourethral glands), have pendulous scrotum, and bifid penis.6 external urethral opening is located at the base of penis so if there is penile trauma the distal penis can be amputated.10 sugar gliders are easily castrated via electrocautery and the pendulous stalk of testes is incised.10 sugar gliders are known to self-mutilate postoperatively and should be monitored. conclusion species-specific differences exist in reproductive anatomy and reproductive disorders. thorough understanding of each species and better knowledge of their conditions will enable appropriate diagnosis and therapy for each animal. conflict of interest none to declare funding none to declare references 1. szabo z, bradley k, cahalane ak: rabbit soft tissue surgery. vet clin north am exot anim pract 2016;19:159-188. 2. walter b, poth t, böhmer e, et al: uterine disorders in 59 rabbits. vet rec 2010;166:230-233. 3. greene hs, strauss js: multiple primary tumors in the rabbit. cancer 1949;2:673-691. 4. saito k, nakanishi m, hasegawa a: uterine disorders diagnosed by ventrotomy in 47 rabbits. j vet med sci 2002;64:495-497. 5. bertram ca, klopfleisch r, müller k: ovarian lesions in 44 rabbits (oryctolagus cuniculus). j vet med sci 2017;79:1994-1997. 6. katherine q, christoph m, connie o, et al: ferrets, rabbits and rodents e-book : clinical medicine and surgery. philadelphia; saunders: 2020. 7. nielsen td, holt s, ruelokke ml, et al: ovarian cysts in guinea pigs: influence of age and reproductive status on prevalence and size. j small anim pract 2003;44:257-260. 8. hong cc: spontaneous papnlary cystadenocarcinoma of the ovary in dunkin-hartley guinea pigs. lab anim 1980;14:39-40. 9. bean ad: ovarian cysts in the guinea pig (cavia porcellus). vet clin north am exot anim pract 2013;16:757-776. 10. miwa y, sladky kk: small mammals: common surgical procedures of rodents, ferrets, hedgehogs, and sugar gliders. vet clin north am exot anim pract 2016;19:205-244. 11. jarrett cl, jarrett tr, harvey sb, et al: the uterus duplex bicollis, vagina simplex of female chinchillas. j am assoc lab anim sci 2016;55:155-160. 12. di girolamo n: disorders of the urinary and reproductive systems in ferrets. ferrets, rabbits, and rodents. 4th edition. amsterdam; elsevier. 2020;39-54. 13. shoemaker nj, schuurmans m, moorman h, et al: correlation between age at neutering and age at onset of hyperadrenocorticism in ferrets. j am vet med assoc 2000;216:195-197. 14. gobikrushanth m, zachar ek, singh n, et al: theriogenology question of the month. j am vet med assoc 2022;260:170-173. 15. vergneau-grosset c, peña l, cluzel c, et al: evaluation of deslorelin implant on subsequent mammary tumors of rats (rattus norvegicus). j exot pet med 2019;31:108-116. 16. planas-silva md, rutherford tm, stone mc: prevention of age-related spontaneous mammary tumors in outbred rats by late ovariectomy. cancer detect prev 2008;32:65. 17. cardiff rd, kenney n: mouse mammary tumor biology: a short history. adv cancer res 2007;98:53-116. 18. okada k, kondo h, sumi a, et al: a retrospective study of disease incidence in african pygmy hedgehogs (atelerix albiventris). j vet med sci 2018;80:1504-1510. 19. heatley jj, mauldin ge, cho dy: a review of neoplasia in the captive african hedgehog (atelerix albiventris). seminars in avian and exotic pet medicine. elsevier: 2005 p. 182-192. 20. gardhouse s, eshar d: retrospective study of disease occurrence in captive african pygmy hedgehogs (atelerix albiventris). isr j vet med 2015;70:32-36. m 2 contact augustine t. peter petera@purdue.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9601, http://dx.doi.org/10.58292/ct.v15.9601 editorial clinical theriogenology is breaking new ground! dear theriogenology friends, earlier in the year, our journal became open access and now the journal is ‘delivering’ its first special issue on educational research in theriogenology. i am profoundly thankful to guest editors drs. margret root kustritz, annett annandale and henry annandale, and authors of this special issue’s manuscripts for their contributions to our journal. please thank these colleagues; they have invested much time and effort for a very good cause! in preparation (formatting, editing, and copyediting along with dr. john kastelic) toward publication, i had to read the manuscripts quite a few times. i enjoyed reading and learning something new each time! we are positive that readers (including our global colleagues) will find this special issue very informative and useful. the manuscripts are arranged in the following order: application of theriogenology in year-one practice; teaching interventions; enhancing learning vis abstract reflections; use of animals, models, micro skills, and captions in theriogenology teaching; impact on animals; and overview on educational research in theriogenology. it is my fervent desire and earnest expectation to have the remaining special issues published as early as possible. therefore, i urge and implore my colleagues (guest editors) and authors to follow up on their commitments! i am eagerly waiting to hear from our guest editors. please feel free to write to me (petera@purdue.edu) if you have any questions or concerns about this or future special issues. thank you all! regards, augustine mailto:petera@purdue.edu http://dx.doi.org/10.58292/ct.v15.9601 mailto:petera@purdue.edu _goback _hlk114167287 thank you for this honor. who am i? i spent most of my career as a practicing veterinary anatomic pathologist on faculty at the veterinary college at cornell university. during this time, i developed a keen interest in diseases that involve reproductive organs of domestic animals. a substantial amount of my time was spent as the pathologist-in-charge of the surgical pathology or necropsy pathology services of the ny state diagnostic laboratory. what a fantastic position to be in; what more could one possibly hope for? for over 30 years, colleagues from around the country and occasionally from around the world would send us tissues and carcasses from cases they had worked up. they were seeking help. the needed answers. that was our job as diagnosticians, to give them answers. but the selfish reality is that we had all this material, all these cases given to us for study and use in our teaching, research and training programs. such a gift, such an opportunity. to paraphrase: if you build a... if you provide a service, the cases will come (my field of dreams). the more cases, the greater our experience became and the better we got at providing answers. the formula was simple: offer a service (providing answers), express enthusiasm, do good work, be timely, and relish the opportunities you are presented with. a great place to find and to established a niche. you are known by your work. you are known by the niche you occupy. my background i was trained at cornell during the 1960’s and 1970’s. i worked for 2 years in a mixed animal practice on the western edge of albany, ny. in 1977, i left practice and began an anatomic pathology residency and graduate training program at the university of georgia. i then moved into an academic appointment in anatomic pathology at cornell in 1982 were i worked until my retirement in 2014. additional study and board certifications (acvp, act, and acvm) followed during the first few years of as a faculty member. once tenured, there arose opportunities for sabbatical leave. the 2021 bartlett address find your niche donald schlafer emeritus professor, college of veterinary medicine cornell university, ithaca, ny my first sabbatical involved a fellowship at the university of oxford, then a few years later i had 2 stints at the san diego zoo as a visiting scientist. these times included the chance to work with the comparative placentologist/pathologist dr. kurt benireschke. part of my last sabbatical leave was as a visiting professorship at the university of padova in italy teaching some classes in theriogenology and pathology courses. i had many memorable experiences during those times. i will briefly mention 3 that i recall fondly. i had learned early on that one needs to seek out opportunities. that turned out to be good advice. i won a scholarship during my undergraduate pre-veterinary years to study in sweden for a year. shortly after arriving there, i began to plan the year. i somewhat boldly wrote a letter to the nobel committee asking if i might attend the ceremonies (1969) in stockholm that december. a couple of weeks later, a ticket for the ceremony arrived. that december, i took a train down to stockholm from uppsala and i checked into a student hostel. getting dressed in a rented tuxedo in the student hostel generated some attention and interesting comments as did the ride in the subway across the city. i made my way through the crowd watching outside stockholm’s opera house (it seemed that nearly everyone else was arriving in large cars). after presenting my invitation, as i was waiting in the main foyer, i was approached by an older, very distinguished gentleman with a very large greying beard and with his chest covered with medals. as he came up to me, he handed me his invitation, and, in swedish, asked me to show him where his seat was. after a moment’s reflection, to give myself time to mentally compose my response in swedish, rising slightly taller and with all the propriety i could muster, i told him that i was afraid i could not, as i was also a guest. indeed, a fond memory that i cherish still. as for the ceremony itself, king carl gustaf xvi of sweden presided. my seat was in the very last row, way up in the top balcony next to the guy running the lights (he was the only one there not wearing a tux). i was relishing my good luck. a few years later, i had the opportunity to meet royalty in the form of a sheep. during a trip to the rosland institute in scotland, i gave a talk on comparative placentation to the group of scientists who were working on animal cloning. this was after the world learned that this group of researchers had succeeded clinical theriogenology 2021; 13: 115 in producing the world’s first cloned mammal. after my talk, i was graciously taken to meet dolly (figure 1). (my wife has suggested i might be aging myself, so, if you do not know who dolly the sheep is, i suggest you look her up.) more part of the fetus than the mother) for research. this particular hospital clinic was limited to care for, and delivery of women considered to have high-risk pregnancies. a considerably mutually beneficial relationship existed between patients and the pregnancy/placental researcher working a floor above. the wait was never very long. a system had been worked out over the years in which by one of the researchers, or a technician who needed access to fresh placental tissues at a particular time on any day (literally), would carry a beeper. when a patient was entering the later stages of delivery, one of the delivery nurses would send a message to the beeper to let us know we should quickly come down to collect the placenta that would come shortly after. that particular day was a special one for me as arrangements had been made for me to be in the delivery room for a delivery. the delivered baby was a girl. she and her placenta were normal in every way. beautiful baby, beautiful placenta. that delivery was nearly 21 years ago now. i wonder what her life has been like? as a veterinarian, i have been admonished for using the word ‘comparative,’ as in comparative veterinary pathology, or comparative veterinary medicine. the point being, that by nature and definition, most of our work as veterinarians involves multiple species. perhaps one might get away with using the term comparative placental pathology if it includes aspects of human reproduction? finding a niche my work in surgical and necropsy pathology gave me exposure to a large amount of case material. over time there was a steady increase in reproductive cases. there were more referrals and invitations to speak at meetings. a niche in reproductive pathology was developing. training became an important and very rewarding part of my job. with faculty from cornell’s theriogenology section, we started holding joint rounds on a regular basis. these become the nidus that brought us together and became an important training activity for both pathology and theriogenology residents. dr. rick miller at the university of guelph and i had shared cases and had been on programs together. we enjoyed working with each other. with his enthusiastic cooperation, we started to hold regular traveling ‘repro rounds’ across the border. these became a twice a year exchange. guelph residents and graduate students would come to ithaca once every 6 months, stay with our residents and join us for a couple of days of case presentations and talks. about 6 months later we would go with our trainees to guelph, staying with their students and trainees and again spend a lively time, sharing cases and presentations. this went on for many years with considerable success. reproductive pathology was alive and well. figure 1. a visit to the roslin institute in scotland provided the opportunity to meet dolly a third story i will tell occurred on a day in 1990 while i was working in the placental lab at the john radcliff hospital at oxford university. i was the ‘token’ veterinarian in the unit. i was being exposed to the inner working of a university obstetric unit. i was standing in the corner of a delivery room trying to be as inconspicuous as possible, holding a medium sized stainless bowl under one arm as i awaited the delivery. i had come for a placenta. after the baby was born (my gracious) the placenta was delivered. i carried ‘my’ placenta up one floor of steps that led from the delivery suite to the back of the placental trophoblastic research lab for examination, sampling and further processing. i had been in the lab about 6 months into my fellowship, working in a research group investigating the pathogenesis of preeclampsia in young women. there were usually between 5 and 10 deliveries each day in the delivery unit. i had gone down those back steps that join the lab and delivery suite to retrieve placentas many times before. nearly all patients delivering in the hospital would sign consent forms donating their placentas, (technically clinical theriogenology 2021; 13: 116 the work was fulfilling, and the opportunities seemed endless. i was finding a niche. that is an interesting term; as it comes from the old french ‘nichier’ which means ‘to nest’ (the free dictionary). a niche is a place ‘well suited’ to the person who occupies it. it is being unique for a given person, not unlike a pair of well-worn slippers. it is also an architectural feature. a niche is a recess in wall in which an object, or stature is placed (figure 2). figure 2. a structural niche in a wall. «niche» is derived from a word that means ‘a nest’. (image is from https:// www.shellwoodcraft.com/images/products/custom-niche805-x-452.jpg) given sufficient time, personal interests, abilities and circumstances, we develop areas of interest followed by expertise. our niche can be identified by the work we are confident in, and happy to be doing. this is also a good way to look at one’s professional development. it is a helpful concept in assessing and guiding others in their careers. it allows for one to define their life’s work. with time, my niche continued to evolve with a greater focus on placentation, placental pathology, and placental pathophysiology. do you have a niche? can you describe it in 2 sentences? if colleagues were to read them, would they recognize the niche as yours? graduate dissertation research i spent 3 years on plum island doing my dissertation research in virology and reproductive pathology of african swine fever (asf). outbreaks of asf had spread to 3 countries in the western hemisphere during the 1970’s and the us government was concerned. abortions had been reported as the most consistent clinical manifestations during some outbreaks in haiti and the dominican republic. the western hemisphere viral isolates circulating in those countries caused less lethal infections than african isolates, so recognition of less dramatic forms of the disease was important. my research confirmed that in controlled laboratory settings, asf infection caused pregnant swine to abort and we studied the pathogenesis of those abortions. in another series of studies, we looked for evidence of immune protection against asf. no vaccine that is effective in preventing asf had or has been developed to date. there were scattered field reports that some pigs that had recovered from infection were somewhat protected when rechallenged with the same isolates. we designed studies to determine if passive protection through ingestion of colostrum collected from recovered sows (yes, i was milking sows) and given to normal piglets could be demonstrated by challenging those piglets with viral inoculations. the results showed that the clinical course was indeed lessened, confirming that some degree of immunity could be transferred between animals. after a time, asf infections were stopped in south america and in the caribbean islands, lessoning the immediate threat, but massive outbreaks have spread throughout domestic swine populations in eastern europe, russia, china (2018; oie reports) and far east over the last few years. mortality rates of 65% percent occurred. reports in the popular press suggest that over one-quarter of the pigs in world have died from asf! while i was doing asf research, neither i nor anyone else could have predicted that asf outbreaks would become such a crisis to the swine industries of the world. africa swine fever has become a great swine pandemic! knowledge gained through diagnostic cases the large number of cased that came through our diagnostic services provided a rich source of material for study. a few examples of insights gained through our regular service work: • enhanced interpretation of endometrial lesions in endometrial biopsies from mares and bitches. • gross and histologic diagnostic findings in abortion cases; many species. • conduction of thorough placenta examinations. • recognition and characterization of both macroscopic and microscopic lesions of diagnostic significance in clinical theriogenology 2021; 13: 117 placental tissues. • histopathology assessment of canine uterine biopsy material collected by trans-cervical procedures. • tissue changes of diagnostic importance in canine testicular biopsy. • recognition and characterization of immune-mediated orchitis in dogs. • recognition of lesions of diagnostic importance during gonadal histopathology. • characterization of abnormalities of sexual differentiation placental research i was very fortunate to have started at the veterinary college about the same time that dr. peter nathanielsz joined the faculty. peter is an md fetal physiologist with extensive experience using various research approaches to study fetal life in utero. he quickly established a large research program at the veterinary college. he was very generous in helping me gain experience in the use of surgical instrumentation in various fetal sheep models. our research included studying the fetal effects of endotoxin exposure on sheep pregnancy, placentomal eversion, and development of placental embolization techniques to model placental infarction leading to placental insufficiency leading to fetal growth retardation. after some years, my research interests turned to using chronic fetal instrumentation to investigation of placental function (figure 3) in other species. i established research projects studying placental function in the fetal calf and foal based on implanted probes. these studies later involved use of surgically implanted flow probes to quantitatively study umbilical and uterine blood flow dynamics under varying conditions (figure 4). some aspects of our placental research will be described in more detail during my address. acknowledgements as i was preparing this paper, i made a list of my mentors and colleagues who each deserve sincere thanks and acknowledgment. but the list grew to considerable lengths, so much so it become obvious i could not include them. i also became very fearful that i might leave someone out. let me give a heartfelt ‘thank you’ to each and all and ask you to know that you share this honor with me. any career successes i have had are due in most part to generous good friends and colleagues and mentors, wonderful parents, figure 3. a normal equine fetus in utero encased in an amnion and surrounded by its chorioallantoic placenta. a major challenge to study of fetal life in utero is to gain access to various fetal and placental tissues in as unperturbed a state as possible. figure 4. investigating life in-utero placental function research using chronically instrumented animals. this drawing shows placement of doppler flow probes (small square boxes) around fetal and maternal vessels, enabling detailed study of blood flow to the fetus’s placenta and simultaneously maternal blood flow to the uterus. and to many unique and fortunate circumstances. good fortune has brought many opportunities and wonderful experiences. a broadened niche in the world of theriogenology and pathology. we acknowledge the efforts extended by my fellow veterinary pathologists, drs. chris premanandan and robert foster for presenting this year’s reproductive pathology symposium. thank you. i would especially like to mention dr. kurt benirschke. we shared strong interests related to placentation and placental pathology. he was very good to me. kurt passed on in september 2018. he represented the strongest of links between the disciplines of human and animal reproductive clinical theriogenology 2021; 13: 118 biology. i fondly remember kurt’s friday afternoon’s placental ‘trim’ sessions. placentas contained in storage ‘pots’ that had been collected from all deliveries that week would be removed from the pathology refrigerator and with great care weighed, measured, photographed and studied before being sampled for histopathology, genetic testing, culture, etc. dr benirschke figure 5. dr. kurt benirschke examining a normal giraffe placenta with me at the san diego zoo. he was an authority on placentation and placental pathology. was a fountain of knowledge, knew everyone it seemed, and had a great following in the human reproduction and animal conservation fields. he was equally at home examining placenta from women, or from any of an amazing number of different animals (figure 5). among his many achievement was the establishment of ‘the frozen zoo’ in 1972 at the san diego zoo. this unique repository of cells, gametes, and genetic material is and will have unimaginable benefits in the future. dr. benirschke also created a web-based resource on placentation, filled with information about the reproductive cycle and tens of species with detailed description of placentation and placental disease of each. if you have not visited his website (placentation.ucsd.edu) on comparative placentation developed based on his work at the san diego zoo, please do. it is superb. the niche kurt occupied was very broad and very deep. again, thank you for this honor. clinical theriogenology 2021; 13: 119 clinical theriogenology 2021; 13: 120 the 2021 bartlett address clinical theriogenology 2022; 14: 130 editor’s note dear theriogenology friends, greetings and welcome to the 2022 conference issue of clinical theriogenology journal. many colleagues invested their time and effort toward making this conference issue a reality! we are especially grateful to the leadership of our organizations, conference chair, session chairs, symposium chair, abstract committee and chair, and student abstracts chair. in addition to sharing their expertise at the conference, speakers (all but 2) have created manuscripts to accompany their presentations. we do thank them. session chairs’ help in processing the papers is highly commendable. a few years ago, it was dr. robyn wilborn, past president of sft, along with dr. charles franz and sft leaders, streamlined the process of gathering abstracts and conference papers, and compiling them for the editor. once again, ms. tara beasley of our sft office did a fine job. tara created an excel spreadsheet, was in constant touch with session chairs (and authors), abstract committee, and finally enabled me to download the manuscripts and abstracts for further processing (editing, formatting, and copyediting). in all, for 2022, we are able to publish 26 manuscripts and 114 abstracts. despite our (me and copy editor dr. john kastelic) 8 decades of collective scientific manuscript writing and editing experience, and burning ‘a few midnight candles’ this time around, we might have missed capturing a few errors and also might have altered unintentionally the intended meanings of authors. our sincere apologies! wish i had time to share with authors of abstracts and 2 production animal papers for their approval. deadline to meet omnipress placed us in a very tight spot. i want to acknowledge my trusted colleague john’s passion, patience, and perseverance for copy editing. how true it is, if you give an additional remit to a busy person, the person will not only say ’yes’ but will also do a fine job! on the brighter side, it is a delight to observe the increase in both quantity and quality of abstracts. student abstracts reflect the enthusiasm students have for theriogenology and the dedication of our colleagues to our discipline. omnipress leadership and staff deserve our appreciation. finally, my heartfelt thanks to my dear wife, damayanthy peter, for all the support! please feel free to write to me (petera@purdue.edu), if you have questions or concerns regarding our conference issue, since i will not be able to attend this year’s conference. enjoy the conference and benefit from the proceedings. regards, augustine editor™s note dear theriogenology friends, hopefully this conference issue will be very useful to follow along for those who will be participating in the ‘virtual platform’ of the conference. to others who are not attending virtually, there is wealth of information on these pages. conference leaders invited scholars working in various aspects of theriogenology to share their expertise and truly we are the beneficiaries of their willingness to contribute amidst their busy schedule for the benefit of many. we are also thankful for the session chairs for their time and efforts to plan and deliver this meeting. theriogenology will continue to make progress in all aspects of its mission; quality abstracts submitted to the committee is only 1 proof of it. i thank the abstract committee for their dedication and perseverance. research and case abstracts from undergraduate students are a ‘breath of fresh air’ and surely theriogenology will flourish because of strong mentorship, critical thinking, and intellectual creativity that exist in our discipline. it was so encouraging to receive a following note (in quotes) from a student in response to my request to approve an edited abstract ‘i approve of the edits. thank you for your hard work on this, as i’m sure reformatting all the abstracts for consistency is no easy feat. it is greatly appreciated.’ all students who participated and dr. james alexander, organizer of student abstracts, deserve our appreciation. normally, the journal does not publish presentation of a ‘theriogenologists of the year’ awardee. we did it this year, since i believe it will motivate many as they read the ‘theriogenology journey’ of our colleague, professor claire card. theriogenology, will continue to shine in academia and in practice for it has the best and the brightest talent and it is the ‘back-bone of veterinary medicine’. please be sure to read professor ahmed tibary’s surgery paper; our discipline will have a tremendous impact in academia, if we continue to foster and grow reproductive surgery in our programs in collaboration with surgeons. i will be remiss, if i do not recognize the efforts of our ‘point person’ for conference papers and abstracts in our management office, ms. tara beasley. tara, thank you for your patience and persistence. i also sincerely appreciate professor john kastelic’s relentless effort to bring consistency to copyediting. regards, augustine clinical theriogenology • volume 12 number 3 • september 2020 167 003_ms-003 editor's note 1 contact jamie stewart jlstewart13@vt.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9382, http://dx.doi.org/10.58292/ct.v15.9382 research report use of deslorelin acetate to advance ovulation in goats for timed artificial insemination jamie stewart,a alyssa helms,a sherrie clark,a george perry,b elizabeth frieden,a elizabeth lee,a sarah legg,a mishta tak,a grant waldrop,a kevin pelzera avirginia-maryland college of veterinary medicine, virginia polytechnic institute and state university, blacksburg, va, usa btexas a&m agrilife research and extension center, overton, tx, usa abstract the objective of this study was to determine the efficacy of deslorelin in advancing ovulation for timed artificial insemination (ai) protocols in goats. we hypothesized that deslorelin treatment advances the onset of ovulation and improves ai pregnancy rates. does were synchronized using a 5-day cidr (controlled internal drug releasing insert) protocol with prostaglandin treatment at cidr insertion. for experiment 1, does received 0.2 mg of intramuscular deslorelin (n = 9) or saline (control, n = 10) at cidr removal. serial blood collections and transrectal ultrasonography were performed to assess ovarian dynamics and identify ovulation. for experiment 2, does received 0.2 mg of intramuscular deslorelin (n = 42), 5 ml of pmsg (pregnant mare serum gonadotropin)/hcg (human chorionic gonadotropin) (n = 42), or were left untreated (control, n = 42) at cidr removal, and were subsequently bred by transcervical ai with fresh semen after 48 to 56 hours. pregnancy diagnosis was performed at 50 and 90 days after ai. in experiment 1, compared to control does, deslorelin-treated does had an increased (p < 0.01) number of ovulations and increased (p < 0.01) serum estradiol concentrations from 48 to 72 hours after cidr removal. serum progesterone concentrations did not differ between treatments. in experiment 2, there was a main effect (p = 0.02) of treatment on pregnancy rates, with control does tending (p = 0.06) to have greater pregnancy rates than those treated with deslorelin or pmsg/hcg. deslorelin treatment also resulted in decreased (p = 0.04) breeding season pregnancy rates and increased (p = 0.05) number of cycles to achieve pregnancy compared to control does. these results demonstrated that deslorelin not only has a super-ovulatory effect but also, at the dose given, can negatively impact does’ subsequent ovarian function and ability to achieve pregnancy. keywords: does, estrus synchronization, deslorelin, sucromate, breeding introduction meat goat production has increased in popularity in the united states (us) due to the growing population of ethnic and faith-based groups who consume goat meat.1 as a result of this trend, the us has shifted from being a net exporter to a net importer of goat meat,1 suggesting a nationwide need to increase goat productivity. one of the biggest barriers to productivity in goat herds is the increasing prevalence of antiparasitic resistance, leading to increased morbidity and mortality among herds.2 the best strategy to combat growing antiparasitic resistance is to focus on the selection of parasite-resistant animals for breeding and dissemination of these valuable genetics.3 the quickest and most effective way to introduce disease-resistant genetics in a goat herd is by using artificial insemination (ai) with frozen semen. the use of fixed time ai for goats in the us is limited due to the lack of availability of commercially approved drugs.4 protocols using a combination of controlled internal drug releasing inserts (cidrs or sponges) containing progesterone combined with pregnant mare serum gonadotropin (pmsg) and/or prostaglandins have been used extra-label with much success.5 in small ruminants, pmsg is preferred for ovulation advancement due to its long half-life6 and follicle stimulating hormone (fsh) activity that causes superovulation and increased kidding numbers.7,8 in the us, pg600 (merck animal health, madison, nj, usa) is the only commercially available product that contains purified pmsg, which is combined with human chorionic gonadotropin (hcg). although the use of pg600 produces acceptable pregnancy rates with natural mating,9 the inclusion of hcg, which has luteinizing hormone activity, may result in unpredictable ovulation mailto:jlstewart13@vt.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9382 2 citation line: clinical theriogenology 2023, 15, 9382, http://dx.doi.org/10.58292/ct.v15.9382 timing and premature luteinization in small ruminants and is not ideal for use with timed insemination. additionally, repeated use of pmsg for estrus synchronization causes antipmsg antibodies to form, reducing its effectiveness over time.8,10 there is, therefore, a need to investigate alternative ovulation-stimulating products for use in goats. gonadotropin releasing hormone (gnrh) agonists are commonly used in cattle synchronization protocols but have a much shorter half-life than pmsg, which can stimulate estradiol secretion for several days in goats.6 however, long-acting gnrh agonists were developed that may produce similar superovulation effects as pmsg without the risk of decreasing efficacy with repeated use. the incorporation of a deslorelin implant in a timed insemination protocol of lactating dairy cows with low body condition scores improved corpus luteum (cl) function and enhanced pregnancy rates.11 the use of deslorelin implants also induced ovulation in nonlactating dairy cows and heifers12 and tended to decrease pregnancy losses in lactating dairy cows.13 more recently, sucromate (thorn bioscience, louisville, kentucky, usa), a controlled release deslorelin acetate product, was developed and approved for ovulation induction in mares. when given to mares with a follicle size ≥ 35 mm, ~ 90% ovulated within 48 hours.14 interestingly, ovulation rates also increased from 4% to 79% when sucromate was given to mares during the seasonal transition period.15 goats, like horses, are seasonal breeders; therefore, the use of deslorelin for inducing ovulation in does outside the breeding season or during seasonal transition warrants investigation. despite its proven effectiveness for advancing ovulation onset in both transitional and cycling mares, there is no information regarding its use in small ruminants. the objective of this study was to determine whether deslorelin treatment is effective in advancing ovulation in does to synchronize estrus for timed ai. we hypothesized that deslorelin treatment advances onset of ovulation and improves ai pregnancy rates in goats. materials and methods animals our institutional animal care and use committee approved (protocol #19-078) all experimental procedures. adult boer or boer-crossbred does from the virginia department of corrections goat herd in halifax, virginia, were enrolled in this study. experiment 1 (deslorelin effect on ovulation) twenty does (10 mature does and 10 doelings) were enrolled in experiment 1. does were blocked by age and then randomly and equally distributed between 2 replicates for september and october 2019 (n = 5 mature does and 5 doelings per replicate) experiments. within each replicate, does were similarly blocked by age and distributed randomly and equally between 2 treatments. all does were in good physical health prior to enrollment (famacha score < 3, minimum body condition score of 2/5, and no evidence of lameness or illness). estrus was synchronized using a 5-day protocol as described.5 at day -5, a cidr insert containing 0.3 g of progesterone (eazi-breedtm cidr® sheep insert, zoetis, parsippany-troy hills, nj, usa) was placed vaginally, and 10 mg of intramuscular dinoprost tromethamine (lutalyse, zoetis, usa) was administered. at cidr removal, does were given 1 of 2 treatments: 1. 0.2 mg of intramuscular deslorelin acetate (0.1 ml; n = 10; sucromatetm, thorn bioscience, louisville, kentucky, usa) or 2. 0.1 ml of intramuscular saline (n = 10). time 0 was designated as the time of treatment and cidr removal. the dosage of deslorelin was extrapolated from the dosage used in mares.14 one doe in the deslorelin-treated group was later excluded from analyses after a cystic cl was diagnosed via ultrasonography (confirmed by serum progesterone assay) that persisted throughout the duration of the sampling period. jugular catheters were placed in does on the day before treatment and cidr removal (day -1) to facilitate blood sampling. blood was collected at 0 and 12 hours and then every 4 hours from 24 to 72 hours after treatment. serum estradiol concentrations were measured using radioimmunoassay, as described.16,17 interassay coefficient of variation (cv) was 9.1%, and intraassay cv was 4.5% for the estradiol assay. serum progesterone concentrations were measured by radioimmunoassay at 0 and 72 hours and at 8 or 9 days after treatment.16,18 interassay cv was 8.4%, and intraassay cv was 5.5% for the progesterone assay. does were examined for signs of estrus (immobility reflex, tail flagging, and vaginal mucus) during each sampling period after being exposed to a buck-scented item. transrectal ultrasonography (exapad mini, universal imaging, bedford hills, new york, usa) was performed to record ovarian structures, determine interval to ovulation, and identify total number of ovulations in does every 12 hours from 0 to 72 hours after treatment. interval to ovulation was recorded at the first time point when a previously visualized dominant follicle (≥ 5 mm) was no longer visible. the number of ovulations was determined by the total number of dominant follicles that ovulated from either ovary within the 72-hour experimental period. experiment 2 (deslorelin effect on pregnancy per ai outcomes) does were synchronized as described in experiment 1 using a 5-day cidr protocol with 10 mg dinoprost tromethamine treatment at cidr insertion in october 2019. at cidr removal, does were stratified by body condition score and randomly allocated to 1 of 3 treatment groups: 1. 0.1 ml of intramuscular deslorelin (n = 42); 2. 5 ml of intramuscular pmsg/hcg (pg600, merck animal health, madison, nj, usa [n = 42]); or 3. untreated (control, n = 42). between 48 and 56 hours after cidr removal, transcervical ai was performed using fresh semen collected from 3 different bucks on the same day. does were stratified by treatment group and body condition score, and randomly allocated to receive semen from 1 of 3 bucks. does bred to buck 1 underwent ai first (~ 48 to 50 hours after cidr removal). the next group of does were bred to buck 2 (~ 48 and 52 hours after cidr removal). the last group of does were bred to buck 3 (~ 50 to 56 hours after cidr removal). semen was collected from respective bucks immediately before each ai group and diluted with optixcell extender (osseo, mn, usa) to achieve an appropriate volume to inseminate all does in each group. total sperm number per dose was not determined, but a clinician examined each sample under light microscopy on farm to ensure that extended sperm concentration and motility were subjectively adequate (concentration > 100 × 106/ml; motility > 70%) before performing ai. http://dx.doi.org/10.58292/ct.v15.9382 citation line: clinical theriogenology 2023, 15, 9382, http://dx.doi.org/10.58292/ct.v15.9382 3 all does were inseminated transcervically by a single ai technician. each goat was manually restrained on a show stand, and the external os of the cervix was visualized using a vaginal speculum and pen light. upon visualization of the cervix, an ai gun, preloaded with 0.5 ml of warm, fresh, extended semen, was passed through the speculum into the external os of the cervix. the gun was passed through a minimum of 2 cervical rings before semen was deposited. at ~ 18 days after ai, each group of does was turned out with their assigned buck for natural cover over a 40-day breeding season. transabdominal ultrasonography was performed at days 50 and 90 after ai to assess pregnancy status by ai and subsequent natural cover. approximate gestational age, as determined by fetal size, was used to determine how many cycles the does took to achieve pregnancy by natural cover. data analyses data were analyzed using r (https://www.r-project.org/). doe was used as the experimental unit. interval to ovulation and number of ovulations were found to be not normally distributed by a shapiro wilk test and analyzed using a kruskalwallis test. interval to estrus and duration of estrus were normally distributed and analyzed using a welch two sample t-test. anova with repeated measures was used to assess the effects of treatment, time, and treatment by time interactions on serum hormone concentrations using doe id as a random variable. coefficient covariance was computed by heteroscedasticity-correct covariance matrices. post-hoc tests were analyzed using a pairwise comparison with a bonferroni adjustment. the binomial outcomes of ai and breeding season pregnancy rates were analyzed using a generalized linear mixed-effects model (glmer procedure) in r with treatment and buck included as fixed variables and doe id included as a random variable. significance was declared at p ≤ 0.05 with tendencies discussed between p = 0.06 and 0.09. results experiment 1 the effects of deslorelin on ovulation dynamics are summarized (table 1). there were no differences in interval to estrus (p = 0.82) after treatment or estrus duration (p = 0.3) between deslorelin-treated and control does. similarly, interval to ovulation after treatment did not differ (p = 0.13) between control and deslorelin-treated does. interestingly, there was an increase (p = 0.007) in the number of follicles ovulated in deslorelin-treated does compared to control does (figure 1). table 1. ovulatory dynamics, as determined by transrectal ultrasonography, in does following treatment with saline (control) or intramuscular 0.1 ml deslorelin (0.2 mg) at cidr removal for experiment 1 treatment interval to estrus duration of estrus interval to ovulation # follicles ovulated control 39 ± 3.0 20 ± 4.0 61 ± 3.3 1.5 ± 0.2 deslorelin 40 ± 3.9 15 ± 2.8 53 ± 3.3 2.9 ± 0.3 p = 0.82 p = 0.3 p = 0.13 p = 0.007 treatment with deslorelin did alter neither interval to estrus or interval to ovulation after cidr removal nor estrus duration. however, does treated with deslorelin had an increase in the number of follicles ovulated. cidr: controlled internal drug releasing insert. figure 1. representative images from serial transrectal ultrasonography of does treated with intramuscular saline (control, left) versus deslorelin (right) for experiment 1. at 48 hours after controlled internal drug releasing insert (cidr) removal, an average of 1.5 preovulatory follicles (ranging from 1 to 3) were observed in control does, whereas an average of 3 preovulatory follicles (ranging from 1 to 4) were observed in deslorelin-treated does. follicles are denoted by dashed lines. http://dx.doi.org/10.58292/ct.v15.9382 https://www.r-project.org/ 4 citation line: clinical theriogenology 2023, 15, 9382, http://dx.doi.org/10.58292/ct.v15.9382 deslorelin-treated does had increased (p ≤ 0.05) serum estradiol concentrations from 48 to 72 hours after cidr removal (figure 2a) compared to control does. serum progesterone concentrations did not differ (p = 0.66) between treatments (figure 2b). experiment 2 there was a main effect (p = 0.02) of treatment on ai pregnancy rates, whereas buck tended to affect (p = 0.06) ai pregnancy rates (table 2). on post-hoc analyses, control does tended to have greater (p = 0.06) ai pregnancy rates (26%, 11/42) compared to does treated with deslorelin (7%, 3/42) or pmsg/hcg (7%, 3/42). does bred by buck 1 also tended to have greater (p = 0.09) ai pregnancy rates (24%, 10/42) than those bred by buck 2 (7%, 3/42), but did not differ (p = 0.17) from buck 3 (9.5%, 4/42). there was a main effect (p = 0.05) of treatment on breeding season pregnancy rates, but no buck effect (p = 0.6). does treated with deslorelin had lower (p = 0.04) breeding season pregnancy rates (74%, 29/39) compared to does treated with pmsg/hcg (97.5%, 39/40) or untreated controls (100%, 36/36). of the does that became pregnant by natural cover, deslorelin-treated does required an increased number of cycles to achieve pregnancy (1.6 ± 0.1 cycles) compared to untreated control does (1.3 ± 0.1 cycles; p = 0.05), but did not differ (p = 0.53) from pmsg/hcg-treated does (1.4 ± 0.1 cycles). there was also no difference (p = 0.29) in number of cycles between pmsg/hcg-treated does and untreated control does. discussion the current study was the first to investigate the use of deslorelin acetate as an ovulation advancement drug in an estrus figure 2. serum hormone concentrations of does following treatment with 0.1 ml intramuscular saline (control) or deslorelin (0.2 mg) at controlled internal drug releasing insert (cidr) removal for experiment 1. a. serum estradiol concentrations increased (p ≤ 0.05) in does from 48 to 72 hours after treatment and cidr removal (denoted by asterisks); b. serum progesterone concentrations did not differ (p = 0.66) between treatments. http://dx.doi.org/10.58292/ct.v15.9382 citation line: clinical theriogenology 2023, 15, 9382, http://dx.doi.org/10.58292/ct.v15.9382 5 synchronization protocol for timed ai in goats. in the us, there are currently no approved drugs for ovulation advancement in small ruminants. historically, the use of pmsg or a combination pmsg/hcg product (pg600) that is labeled for the induction of estrus in peri-pubertal gilts has been used extra-label in goats with varying success rates. these drugs are purified from mammalian tissue, rather than being generated in a recombinant form. therefore, issues with antibody formation following repeated use of pmsg have been reported, which may diminish its effectiveness in superovulation and ovulation advancement in small ruminants.8,10 additionally, the inconsistent availability of these drugs in the us justifies the need to evaluate alternative options for ovulation advancement in small ruminants. in cattle, gnrh agonists have been widely used for decades within estrus synchronization protocols. these products are small, recombinant proteins that do not appear to generate an immune response after administration. however, their half-life is much less than that of pmsg due to the ease that they can be degraded by endopeptidases and nonspecific exopeptidases present in the pituitary gland, liver, and kidney.19 in goats, the long half-life of pmsg aids in allowing for selection of multiple dominant follicles, resulting in a superovulation effect that increases kidding rates. the use of nano-delivered drugs has been proposed as an alternative way of delivering synchronization drugs, with the use of nano-gnrh having improved luteal function in pregnant goats.19,20 unfortunately, nano-drugs are not commercially available, so their current usefulness to producers is minimal. we utilized a commercially available gnrh agonist (deslorelin) that is suspended in sucrose acetate isobutyrate (saib) and is labeled for estrus advancement in horses (sucromate). the saib vehicle is a highly viscous and hydrophobic polymer that allows for a sustained and prolonged release of a drug. authors in a recent study reported that supplementing sucromate with sulpiride and estradiol resulted in their longterm delivery after a single treatment in horses.21 to the authors’ knowledge, the current study is the first to evaluate the use of saib for sustained-delivery of a synchronization drug in goats. neither the interval to estrus, duration of estrus, or interval to ovulation differed between deslorelin and saline-treated control does in experiment 1. the interval to estrus reported herein (39 to 40 hours after cidr removal) is consistent with that reported in boer goats synchronized with progesterone sponges and pmsg (500 iu) outside the natural breeding season.22 on the contrary, we noted the estrus duration to be 15 to 20 hours, which is significantly shorter than that reported in an earlier study (38 to 40 hours).22 however, that study utilized vasectomized bucks to visualize behavioral estrus, whereas we did not have access to a buck and relied on a buck-scented item that may have diminished the observed estrual behavior over time. in contrast to these findings, interval to estrus was decreased in dairy goats treated with either pmsg (33 hours; single dose of 750 iu at norgestomet implant removal) or fsh administration (32 hours; twice daily injections of descending doses of 4 to 1 mg over 4 days, starting 48 hours before implant removal) compared to untreated controls (48 hours).23 the discrepancy in findings highlights the vast differences that can be observed among different breeds of goats, especially among those intended for meat versus dairy. further studies assessing the onset and duration of estrus after deslorelin treatment in several breeds of goats and with alternative dosages are, therefore, warranted to best determine its potential usage. this study is the first to report a superovulation effect of a gnrh agonist in goats. using ultrasonography, we directly observed an ovulation rate of 3 in deslorelin-treated does versus an ovulation rate of 1.5 in saline-treated control does in experiment 1. ovulation rates can vary among goat breeds24 but have been reported similarly in boer does at 1.7.25 similar superovulation effects were reported in ewes treated with pmsg/hcg within the breeding season. in that study, the ovulation rate increased to 3.25 in ewes treated with 5 ml pmsg/hcg compared to 2.17 in those treated with 5 ml saline and 2.29 in those treated with 1.5 ml pmsg/hcg.26 consistent with ultrasonography findings, we observed an overall increase in serum estradiol concentrations in deslorelin-treated does compared to control does. serum estradiol concentrations peaked at ~ 40 hours after cidr removal in both deslorelin-treated and control does, which is consistent with other synchronization studies in goats.27 in goats treated with a single superovulation dose of pmsg (1,000 iu), the duration of elevated estradiol was 3.6 days, compared to 0.5 day in those treated with twice daily fsh injections (8 mg on day 12, 4 mg on day 13, 2 mg on day 14, and 1 mg on day 15).27 we observed a similar trend, where the estradiol concentrations in control does began to decline after peaking at 40 hours, reaching baseline values at ~ 52 hours after cidr removal (a total duration of 12 hours or 0.5 day). on the contrary, the deslorelin-treated does experienced a prolonged elevation of estradiol throughout the remaining experimental period of 72 hours after cidr removal (32+ hours duration). unfortunately, samples were not collected beyond this time point, so we were unable to determine the duration of estradiol elevation in these animals. further studies that extend sample collection beyond 72 hours after cidr removal would be needed to determine the exact duration of elevated estradiol after deslorelin treatment. it would also be worthwhile to explore the timing of deslorelin treatment within this synchronization protocol, its use within other synchronization protocols, or its use in conjunction with an alternative prostaglandin source (e.g. cloprostenol) to best assess its effects on estradiol concentrations and estrus duration. despite the observed superovulation effect in the current study, postovulatory progesterone concentrations did not differ between deslorelin and saline-treated control does in experiment 1. biodegradable implants that release deslorelin in vitro over a 4-day period have been previously investigated in cattle28 and can both suppress ovarian activity at a high table 2. ai pregnancy rates in does untreated (control) or treated with deslorelin or pmsg/hcg and subjected to transcervical ai for experiment 2 control (%) deslorelin (%) pmsg/hcg (%) buck 1 50 (7/14) 7 (1/14) 14 (2/14) buck 2 7 (1/14) 7 (1/14) 7 (1/14) buck 3 21 (3/14) 7 (1/14) 0 (0/14) total 26 (11/42) 7 (3/42) 7 (3/42) there was a main effect (p = 0.02) of treatment on ai pregnancy rates, whereas buck tended (p = 0.06) to affect ai pregnancy rates. on post-hoc analyses, control does tended (p = 0.06) to have greater ai pregnancy rates compared to does treated with  deslorelin or pmsg/hcg. does bred by buck 1 also tended (p = 0.09) to have greater ai pregnancy rates than those bred by buck 2; however, they did not differ (p = 0.17) from buck 3. pmsg/hcg: pregnant mare serum gonadotropin/ human chorionic gonadotropin; ai: artificial insemination. http://dx.doi.org/10.58292/ct.v15.9382 6 citation line: clinical theriogenology 2023, 15, 9382, http://dx.doi.org/10.58292/ct.v15.9382 dosage (2,100 μg) or increase plasma progesterone and luteinizing hormone concentrations at a lower dosage (700 μg).29 the use of a deslorelin implant (700 μg) in a timed insemination protocol for nonlactating holstein cows had a greater rate of increase of plasma progesterone up to 15 days after ovulation due to accessory cl formation compared to untreated or buserelin (8 μg)-treated cows.11,29 on the contrary, another study in nonlactating dairy cows and heifers identified minimal to no difference in plasma progesterone concentrations in cows given either 750 or 1,000 μg deslorelin implants during estrus synchronization.12 they did, however, report a significant increase near day 10 in cows, given a 450 μg deslorelin implant compared to gonadorelin-treated controls.12 these studies highlighted important doseand time-dependent effects of deslorelin treatment on cl formation and function. though no differences in progesterone were detected in the current study, we were limited by only acquiring 1 sample after ovulation to measure progesterone. follow-up studies with serial ultrasonography and blood collections during the diestrus period would be needed to better measure the potential luteotrophic effect of deslorelin treatment in does after ovulation. although experiment 1 discovered a superovulation effect with deslorelin treatment, the results of experiment 2 identified its deleterious effect on fertility in does. one potential issue with fertility rates may have been the timing of ai. although not statistically significant, interval to ovulation in deslorelin-treated does in experiment 1 was numerically decreased (53 hours) compared to untreated controls (61 hours). the high variability in interval to ovulation may account for the lack of significance, and a difference may have been detected if we had increased the frequency of ultrasonography examinations. overall, the greatest ai pregnancy rates were achieved in does bred to buck 1, followed by buck 3 and then buck 2. due to logistical limitations, we had to breed each of the stratified groups to 1 male at a time. therefore, does bred to buck 1 underwent ai first at ~ 48–50 hours after cidr removal, which may have contributed to improved overall ai rates. within this group bred by buck 1, 50% of control does became pregnant versus 7% of deslorelin-treated does and 14% of pmsg/hcg-treated does. there is also a likelihood that sperm quality or concentration was better for certain bucks, since this was not extensively evaluated on site and may explain why ai pregnancy rates were intermediate for buck 3 (ai interval from 50 to 56 hours after cidr removal) and worst for buck 2 (ai interval from 48 to 52 hours). within the group bred by buck 3, 21% of control does became pregnant versus 7% of deslorelin-treated does and 0% of pmsg/ hcg-treated does. there were no differences in ai pregnancy rates among the does bred to buck 2 (7% for all treatment groups). based on these findings, it is possible that a hastened onset to ovulation could have caused decreased ai pregnancy rates in both of the treatment groups compared to the control group. further studies are warranted to better determine an appropriate interval to ai when using deslorelin within a doe estrus synchronization protocol. a more likely explanation for decreased fertility in the current study is an overstimulation of the ovaries with both deslorelin and pmsg/hcg treatment. several outcomes observed in the deslorelin-treated does of the study support this theory: the prolonged estradiol secretion in experiment 1; the increased number of cycles to breed back with bucks in experiment 2; and the decreased overall pregnancy rates in experiment 2. consistent with our findings, pregnancy rates decreased to 37.5% in ewes treated with 5 ml pmsg/hcg compared to those treated with 1.5 ml pmsg/hcg (87.5%) or 5 ml saline (75%) within the breeding season.26 also similar to our findings with deslorelin-treated does, serum estradiol concentrations increased in ewes treated with 5 ml pmsg/hcg compared to those treated with 1.5 ml pmsg/hcg or control.26 in lactating dairy cows, a deslorelin dose of 750 μg resulted in decreased ai pregnancy rates (28%) compared to 450 μg deslorelin dose (41%) or gonadorelin-treated control cows (39%), suggesting a dose-dependent effect.13 after resynchronization of nonpregnant cows in that study, only 9.6% of cows treated with the 750 μg deslorelin dose and 17% of cows treated with the 450 μg deslorelin dose became pregnant at the second ai compared to 25% of gonadorelin-treated control cows.13 not surprisingly, the decrease in second ai pregnancy rates was due to a result of suppressed ovarian activity in both deslorelin-treated groups, regardless of dose.13 delayed follicular growth, the development of follicular cysts, and failed ovulations in subsequent estrous cycles were also reported in lactating and nonlactating cows treated with deslorelin implant dosages ranging from 450 μg to 2,100 μg.11–13,28,29 these findings are in line with the results of the current study that suggest the deslorelin-treated does experienced a disruption in their cycles, likely due to suppressed ovarian activity. future studies are needed to determine an ideal dosage of deslorelin for ovulation advancement in goats without altering subsequent ovarian activity. in mares, a single intramuscular injection of 1.8 mg of deslorelin acetate, suspended in saib (sucromate), was sufficient for inducing ovulation when a dominant follicle was present.30 on average, this would equate to a dosage of ~ 2 μg/kg. we extrapolated this dosage to a ~ 68-kg goat, which would be 136 μg. sucromate has a concentration of 1.8 mg (1,800 μg) per ml, so the extrapolated volume for a goat is 0.076 ml. due to the low volume and high viscosity of the solution, we elected to round up the volume to 0.1 ml (200 μg) to facilitate its administration. compared to other gnrh agonists, this dosage is high. in general, 50 μg of the gnrh agonist gonadorelin diacetate tetrahydrate is commonly given to goats undergoing estrus synchronization in the us.31 in other countries, 25 μg lecirelin is used for synchronizing ovulation in does.32 the reported dosage for buserelin is even lower at 4 μg per doe.33,34 in lactating dairy cows, both 450 μg (~ 0.7 μg/kg) and 750 μg (~ 1.2 μg/kg) deslorelin dosages have been investigated, with the larger dose causing a decrease in pregnancy rates, but both doses causing subsequent ovarian suppression.13 these previous studies, combined with the results presented herein, provide a starting point for determining adequate dosing and timing for the use of deslorelin in saib for estrus synchronization in goats and require further investigation. if investigated, the feasibility of diluting sucromate with another carrier (e.g. saline) also needs assessment since dosing below the 200 μg used in the current study would not be feasible without being able to adjust the volume and concentration. in conclusion, the results did not support the use of the commercially available deslorelin product, sucromate, for synchronization of estrus in goats at the dose of 200 μg per doe. the findings presented herein suggest that, at the dose given (200 μg), deslorelin exerted a superovulation effect on ovaries of treated does. this effect overstimulated the ovaries and caused subsequent ovarian suppression, altering the doe’s ability to be rebred. further research is needed to investigate alternative dosing regimens on ovulation and subsequent cl http://dx.doi.org/10.58292/ct.v15.9382 citation line: clinical theriogenology 2023, 15, 9382, http://dx.doi.org/10.58292/ct.v15.9382 7 function in small ruminants. additionally, there is a need to thoroughly evaluate its use in synchronization protocols to ensure that the best time for ai is determined. acknowledgments the authors thank the farm managers and employees at the virginia department of correction for their support and allowing us to use their goat herd throughout this 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30. squires el, simon bw: evaluation of a new sustained-release deslorelin acetate for induction of ovulation in mares. proc am asoc equine pract 2011;57:53–54. 31. bowdridge ec, knox wb, whisnant cs, et al: ncsynch: a novel, progestagen-free protocol for ovulation synchronization and timed artificial insemination in goats. small rumin res 2013;110:42–45. doi: 10.1016/j.smallrumres.2012.07.025 32. cosentino io, balaro mfa, leal fsc, et al: ovarian activity in dairy saanen goats subjected to a short-term ovulation induction protocol and a single injection of lecirelin (gnrh analog) given 28 h or 34 h after progestin pre-treatment. small rumin res 2020;191:106–214. doi: 10.1016/j.smallrumres.2020.106214 33. al yacoub an, gauly m, sohnrey b, et al: fixed-time deep uterine insemination in pgf2α-synchronized goats. theriogenology 2011;76:1730–1735. doi: 10.1016/j.theriogenology.2011.07.005 34. holtz w, sohnrey b, gerland m, et al: ovsynch synchronization and fixed-time insemination in 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ifce, prc, nouzilly, france bfaculty of sciences and techniques, university of tours, tours, france ccentre d'etude en reproduction des carnivores (cerca) alfort national veterinary school maisons-alfort, france dneocare, umr inra/envt 1225 ihap reproduction université de toulouse, france abstract although the dog and cat both belong to the order carnivorous, their reproductive physiology is quite different. the dog is a nonseasonal, monoestrus species with spontaneous ovulation only twice a year and with an atypical, postovulatory oocyte maturation. furthermore, the application of reproductive in vitro biotechnologies such as in vitro maturation (ivm), in vitro fertilization (ivf), intracytoplasmic sperm injection (icsi) or cloning remains quite a challenge in dogs. by contrast, the cat is a polyestrus seasonal species, with ovulation induced several times a year and typical preovulatory oocyte maturation. as a result, manipulation of ovarian physiology is feasible and in vitro reproductive biotechnologies are as efficacious as in cattle. the first part of this review describes the main facts associated with folliculogenesis in the dog and cat (initiation of growth of primordial follicles, appearance of the zona pellucida, formation of antrum, nuclear and cytoplasmic maturation of oocyte, expression of receptors to gonadotrophins, and expression of steroidogenic enzymes). second part focuses on oocyte maturation, fertilization, and in vivo early embryo development in both species. last part discusses in vitro reproductive biotechnologies (ivm, ivf, and ivd), embryo transfer, and more recent biotechnologies (cloning, in vitro folliculogenesis, and vitrification of oocytes and follicles). innovations in the dog are still limited by various characteristics (long ovarian cycle, difficult in vitro oocyte maturation) whereas in the cat, many in vitro techniques are applicable that may also be extended to wild feline species. keywords: folliculogenesis, biotechnologies, embryo transfer, dog, cat introduction although the dog and cat belong to same order (carnivorous), their reproductive physiology is quite different. dog is nonseasonal monoestrus, with spontaneous ovulation only twice a year. thus, manipulation of ovarian physiology (e.g. to reduce interval between ovulations, to induce ovulation or superovulation, or to produce embryos) is generally difficult. furthermore, application of reproductive in vitro biotechnologies (in vitro maturation [ivm], in vitro fertilization [ivf], intracytoplasmic sperm injection [icsi], and cloning) remains quite a challenge in the dog and only a few research teams have mastered them. by contrast, the cat is seasonally polyestrus with ovulation induced several times a year. as a result, manipulation of ovarian physiology (e.g. stimulation or induction of ovulation) is feasible and in vitro reproductive biotechnologies are as efficient as in cattle. our purpose is to review available information on: 1) main events occurring during in vitro follicle and oocyte growth, in vivo fertilization, and early embryo development; 2) in vitro biotechnologies and embryo transfer; and 3) other biotechnologies. in vivo follicle and oocyte growth, fertilization, and early embryo development in dog in vivo follicle and oocyte growth contrary to what occurs in many mammals (e.g. mouse, cow, woman, and others), in dog, ovarian follicles appear after birth. only oogonia are present in the fetal ovary. first primordial follicles (oocyte surrounded by granulosa cells) appear ~ 1 month after birth.1 these follicles make up the definitive stock of oocytes. some of them will start growing only years later. when growth starts, oocyte diameter clinical theriogenology • volume 12 number 3 • september 2020 189 increases and granulosa cells appear modified (rounded/cuboidal). as they increase in number, first follicles with an antral cavity appear at 4 months of age. as an antrum is formed, granulosa cells differentiate, distinguishing external cells, close to basal membrane from cells in the cumulus, close to the oocyte. as in other mammalian species, a number of changes take place as the oocyte and follicle increase in size. changes (figure 1) include formation of zona pellucida around oocyte, multiplication of granulosa cells (200 400 cells when antrum appears), 8 and first cells in theca, expression of fsh and lh receptors and ability to produce steroids (steroidogenic enzymes). figure 1. main events in dog folliculogenesis.1-7 during anestrus, some follicles reach 1 mm in diameter.1 oocyte is surrounded by granulosa cells of cumulus, with many contacts between them and oocyte, leading to continuous transfer of many compounds (pyruvate, growth factors, rnas, and organelles) from somatic cells to oocytes (figure 2). figure 2. scanning electronic microscopy image of a dog cumulus-oocyte complex showing numerous "pseudopod-like" connections between the cumulus cells (cc) and the oocyte (oo) (a) and between the cumulus cells (b). inside a follicle, the oocyte gradually acquires its ability to resume meiosis (nuclear maturation) and later acquires ability for fertilization and early embryo development (cytoplasmic maturation). a unique feature is the number of follicles with multiple oocytes (polyoocytic follicles) in the ovaries of dogs and clinical theriogenology • volume 12 number 3 • september 2020190 cats.1,9-11 in dog, as many as 14% of follicles have several oocytes (with a maximum of 17 oocytes in 1 follicle) while it represents < 1 2% in other mammals.12 they may reach ovulation but represent only 4 7% of preovulatory oocytes. among them, a single oocyte in the follicle appears to be of good quality.13 after puberty, at each cycle, larger follicles with antrum are being recruited to initiate terminal growth. growth of this follicle may be visible by ultrasonography towards the middle and end of anestrus.14 once estrus begins, follicle growth accelerates and under stimulation by lh at the preovulatory stage, follicle wall becomes thicker. immediately prior to ovulation, a protuberance may be visible at the very site where the oocyte is released (figure 3). right after ovulation, follicles are transformed into corpora lutea. figure 3. dog ovaries during periovulatory stage, site (arrow) of future oocyte expulsion is visible immediately before ovulation (a), whereas small haemorrhagic corpora lutea (arrows) are visible right after ovulation. monitoring ovulation in dogs is possible by various means15 such as lh or endocrine assays, possibly coupled with ultrasonography, depending on the veterinary clinic. indeed, as a result of preovulatory luteinization of ovarian follicles, blood progesterone concentrations gradually rise from basal concentrations to 2 ng/ml at lh peak and 3 10 ng/ml at ovulation.16-17 thus, it is possible to predict ovulation and have the followup in real time by ultrasonography.18,19 with 2 ultrasonographies a day, disappearance of follicles can be confirmed. however, not all follicles will transform into corpora lutea; some become filled with blood right after ovulation and may thus appear anechogenic. follicle size at ovulation is related to dog breed and may vary from 4.5 mm in small breeds to 8 10 mm in larger breeds.20,21 at ovulation, oocytes are surrounded by mucified granulosa cells and are drawn into the infundibulum to enter the oviduct, but are still unfertilizable. then, the oocyte loses the external layers of cumulus and migrates rapidly to middle section of oviduct (total length, 510 cm). several important events take place in oviduct: resumption of oocyte meiosis, selection of sperm,22,23 and initiation of embryo development.24 resumption of meiosis leads to first polar body expulsion (2 3 days after ovulation) from oocyte leading to metaphase ii stage that is ready for fertilization.25,26 at that stage, it becomes possible to collect in vivo-matured oocytes. two research teams have been able to routinely collect oocytes in vivo and have achieved in vitro fertilization and generated genetically modified animals or clones.27-30 ability to reach the fertilizable metaphase ii stage after extraction from the follicular environment increases from 17 to 80% for oocytes from small follicles (< 500 µm) versus those from larger follicles (~ 2 mm). 7 maturation of oocyte cytoplasm is more difficult to investigate, as it would require examination of oocytes from various follicle sizes (e.g. 24, 4 6, and 6 8 mm). after fertilization, these oocytes should be examined through fertilization and embryo development until blastocyst stage. several papers reported results obtained with oocytes from large follicles.27,31 unfortunately, in vitro culture conditions are still suboptimal, so that the exact time of cytoplasmic maturation acquisition remains uncertain. as mentioned earlier, blood concentrations in progesterone in dog at ovulation and shortly thereafter are extremely high (> 10 times those in cattle or women). presumably, these high concentrations of progesterone have significant biological effects. thus, progesterone receptors (e.g. clinical theriogenology • volume 12 number 3 • september 2020 191 aglepristone) treatment several days before ovulation induced a delay in resumption of meiosis and increased oocyte survival. it also inhibited the upward movement of sperm in oviduct.32 since oocytes are not readily fertilizable at ovulation, artificial insemination (ai) of thawed sperm, in order to obtain embryos or a pregnancy, should be done up to 2 days after ovulation. by contrast, in natural breeding, sperm survive much longer (up to 11 days),25,33 making much earlier breeding before ovulation possible. following fertilization, embryo development not only relies on oocyte reserves, but also requires exchanges with the environment in oviduct to obtain metabolites needed for further development. hormone assays, coupled with ultrasonography, made it possible for us to describe precise timing of embryo development after ai in the dog26,34 (figure 4 and table). following morula stage, 8.5 9 days after ovulation, embryos crosses utero-oviductal junction and start uterine entry.35 prior to implantation, embryos migrate frequently to uterine horn on opposite side36. implantation takes place ~ 17 days after ovulation.37 figure 4. dog embryos collected in vivo at 2 pronuclei (a), 2 or 4 cell (b), 8 cell (c), morula (d), early blastocyst (e), and expanded blastocysts (f) stages. table. timing of embryo development in vivo in the dog (note: fertilization takes place 2-3 days after ovulation) challenges to in vitro maturation, fertilization, and embryo development in vitro oocyte maturation and fertilization in dog have been investigated for several decades38 and many obstacles were encountered. however, major progress has been achieved in the last 20 years. reproductive in vitro biotechnologies represent an important research tool in that they may elucidate the biological mechanisms of oocyte maturation, fertilization, and embryo development, in particular the respective role of environmental factors (e. g. endocrine control, metabolism, growth factors, proteins, and intercellular communications). these biotechnologies also make it possible to preserve genetic resources (e.g. freezing of oocytes, sperm, embryos, ovarian cortex, and testicular pulp) for valuable dog clinical theriogenology • volume 12 number 3 • september 2020192 models in biomedical area (refer online mendelian inheritance in animals website [omia.org] that lists animal models for human diseases, 450 canine models) or to preserve threatened wild canid species. in vitro maturation of dog oocytes purpose of in vitro oocyte maturation (ivm) is to obtain a large number of fertilizable oocytes. after ovariectomy and careful dissection of ovary, oocytes are selected and cultured in vitro in a maturation medium, using bovine models. in other mammals, when oocytes are collected from ovaries/follicles, there is an immediate resumption of meiosis in all competent oocytes (oocytes that reached a sufficient degree of nuclear maturation). for example, in cattle and mice, > 80% of oocytes have resumed their meiosis and become fertilizable after 24 hours. in dog, by contrast, oocyte reaches metaphase ii stage and thus becomes fertilizable after 3 4 days in culture and with < 20% yield.39 as mentioned above, folliculogenesis and thus oocyte maturation within the follicle is a process lasting for months. final folliculogenesis, at ovulation, makes it possible to collect good quality oocytes but takes place only twice a year in dog. collecting oocytes from an ovary during anestrus results in oocytes that are still quite immature. furthermore, ovaries collected by ovariectomy may still be at prepubertal stage, that is 2 3 months only after appearance of first follicles with antrum. these oocytes are even more immature than anestrous oocytes, with poor synthesis of proteins and limited connections with granulosa cells.40 many research teams have attempted to produce in vitro-matured fertilizable canine oocytes and to identify oocytes that should be used (according to age, breed, stage of cycle), to determine which media, oxygen supply, growth factors, proteins, hormones should be introduced and which cells to use in coculture (reviewed 39,41,42). overall, the results have been disappointing. major problem appears to be oocyte size compared to follicle size.43 thus, using oocytes from large follicles makes it possible to increase the proportion of fertilizable oocytes after in vitro maturation.44 yet, rates of success remain low, which may be related to cytoplasm quality (e.g. number and organization of organelles such as mitochondria and cortical granule) in those in vitro-matured oocytes reaching the metaphase ii stage, which remain largely below those obtained with oocytes ovulated in vivo.45 current investigations attempt to mimic in vivo intraovarian environment with maturation in pure follicular fluid46or in diluted follicular fluid,47 or within an oviduct. research on oviductal environment, which started 20 years ago,48 is now designed to determine the composition of oviductal fluid and physiology of cells in oviductal epithelium31,49,50 since exposure of oocytes to oviductal environment is essential for normal fertilization.51 variety of culture conditions have been tested such as synthetic oviductal fluid medium52, use of microvesicles produced by oviductal cells,50,53 cell cultures on plastic dishes or porous inserts,49 oviductal explants54 (fragments of oviductal epithelium), or ligated oviduct.55 however, maturation rates hardly reach 20% and canine oocytes collected during anestrus, even transferred in vivo into an oviduct of dogs in estrus, are not able to adapt, despite having a potentially optimal environment.34 some investigations were conducted in cattle, to increase the rate of metaphase ii oocytes and primarily the rate of fertilization and embryos at the blastocyst stage (currently reaching 30 60%). these investigations were designed to temporarily block meiosis resumption, right after rupture within the follicle, in order to give the oocyte a chance to complete its cytoplasmic maturation. several compounds such as roscovitine and dbcamp were tested in anestrus canine oocytes without significant success.57,57 thus, the problem of immaturity of anestrus oocytes is far from being solved. as a result, up to this day, a single early pregnancy was reported after imv-ivf-ivd58 but no puppy was born. birth of pups following embryo transfer (from oocytes, matured in vivo, and fertilized in vitro) was a major progress.27 in vitro fertilization and embryo development in vitro fertilization is now routinely carried out in a number of species. progress in reproductive biotechnologies in dog is in part from applying results obtained in wild canids (e.g. silver fox). large numbers of ovaries collected on farms rearing foxes for fur production made it possible to describe maturation of oocytes (ovulated at an immature stage like in dog), in vitro fertilization and transfer.59-62 clinical theriogenology • volume 12 number 3 • september 2020 193 altogether, rates of in vitro maturation and fertilization in the silver fox are close to those obtained in dog. surgical embryo transfer has led to the birth of progeny.63 however, in vitro fertilization and embryo development remain problematic in the dog, due to a number of causes. cytoplasm quality of canine oocytes is generally poor, zona pellucida undergoes changes during culture and sperm capacitation has not been perfected.64-65 microinjection of sperm into oocyte (or icsi), allowing a normal monospermic fertilization, has been reported in dog, however, is not routinely used in laboratories.66,67 several teams have described in vitro fertilization and occasionally obtained embryos and even puppies.27 however, bringing together dog sperm and oocytes in vitro does not routinely lead to successful fertilization, because sperm can enter immature oocytes,64,68 typically ending in a high rate of polyspermia44,69 and sperm pronucleus abnormalities.37,65 however, oocytes collected at follicular phase stage have been used successfully to obtain embryos with as many as 30% undergoing cleavage after fertilization using oocytes from follicles > 2 mm.70 following fertilization, embryo development is initiated; however, it is blocked at the 8 cell stage when the embryo becomes autonomous and develops its own gene expression (stage of maternal to zygotic transition).71 blocking at 8 cell stage suggests that conditions of embryo culture are still not optimal. however, it appears that co-culture with embryonic fibroblasts may improve development of canine embryos to 16 cell/morula stages72 and that some recently described culture media might enable development of embryos up to the blastocyst stage.73,74 in vivo collection, freezing, and transfer of canine embryos as stated above, in vitro production of canine embryos is quite problematic. another possibility is to produce in vivo embryos after ovulation, followed by natural breeding or artificial insemination (reviewed69,75). collecting embryos in vivo may be achieved by flushing the oviduct to collect embryos at an early stage (2 cell morula) or rinsing uterus to obtain embryos at morula and blastocyst stages. a major difficulty remains in getting access to oviduct without ovariectomy, since oviduct is hidden in a lipid-rich ovarian bursa and infundibulum is difficult to handle. by contrast, collecting embryos in uterus is simple and requires only a laparotomy, followed by uterine rinsing.76,77 it may also be achieved by a nonsurgical endoscopic technique, with the introduction of a suitable catheter into cervix, which may allow to keep the female donor as a reproducer.78,79 embryos obtained this way may then be transferred to a recipient female or can be frozen. canine embryos from 1 donor female are difficult to obtain in large numbers, as well in vitro (as described above) as in vivo because female dogs do not respond to superovulation treatments and because the number of embryos obtained is limited by ovulation rate. pioneering research in canine embryo transfer was started in the ‘80s.25,76,80,81 transfer of fresh embryos required optimal synchronization between donor and recipient cycles. at that time, method to synchronize cycles was not available, hence many experimental kennels were needed to routinely obtain females in estrus that could be used as embryo recipients. ten years ago, only 45 puppies were born in the world after transfer to 57 recipient females (reviewed69). since then, a number of puppies were born after oocyte micromanipulation (cloning), and after ivf and transfer.51,82 currently, estrus control, suppression or stimulation, can be obtained using gnrh agonists. these treatments are efficient and free from negative impacts on subsequent fertility.83-85 thus it becomes possible, towards end of anestrus period, to induce estrus in a dog within a few days.86 generally, embryo is transferred into uterus because oviductal access is difficult. even at oviductal stages (2 16 cells), embryos can be successfully implanted in uterus. in the absence of efficient synchronization treatment, cryopreservation of embryos may allow postponement of embryo transfer until a recipient female is available. conditions for optimal cryoconservation of dog embryos remain an area of investigation, including: duration of exposure to cryoprotectants and concentration of cryoprotecting agents, evaluation of cryotolerance beyond morphological evaluation and conditions of embryo transfer after freezing and thawing to obtain pregnancies remain to be well defined. two freezing processes are used, namely, slow freezing and vitrification. regarding slow freezing, glycerol and ethylene glycol were evaluated and gave contradictory results. in a study involving 20 embryos, it appeared that glycerol alone was able to preserve zona pellucida structure.87 however, clinical theriogenology • volume 12 number 3 • september 2020194 similar post-thaw viability was established in glycerol and ethylene glycol when 50 blastocysts were used.88 in vitrification, high concentrations of cryoprotectant agents are being used, embryos are placed in a 1 5 µl microdrop and lowering of temperature is very rapid, with direct immersion in liquid nitrogen. this ultrarapid freezing is designed to avoid crystal development within cells and appeared proper for dog embryos. thus, after vitrification in 1 µl microdrops in a cryotop system, early canine embryos (zygote to 16 cells) survived freezing better (90 100% of survival after thawing) than embryos at morula (50%) or blastocyst (40%) stages.78 these investigators also transferred 77 embryos to 9 recipient females and obtained 5 pregnancies, 4 to term, and 7 puppies were born. a lag time of 1 or 2 days between donor and recipient cycle stage of the donor was not a deterrent to pregnancy.78 furthermore, a recent study89 compared slow freezing and cryotop method vitrification in 89 in vivo collected embryos at various stages (8 cell to blastocysts). after cryopreservation (30 embryos by slow freezing and 35 by vitrification) and surgical transfer in recipient females (1 6 embryos per recipient), they succeeded in obtaining 2 pregnancies (1 puppy per recipient), but only with vitrified embryos. reproductive biotechnologies under development for preservation of dog genetics somatic cloning somatic cloning, also termed somatic cell nuclear transfer, which consists of injecting somatic cell into a mature oocyte, was first described in the dog in 2005.28 indeed, the first dog obtained by cloning, snuppy, a male afghan hound, was recloned recently.90 since then, the korean research team has specialized in the collection of mature oocytes followed by micromanipulation. right after transfer of nucleus, potentially cloned embryos are transferred into an oviduct using an appropriate catheter (tom cat 3.5fr) in order to avoid blocking of canine embryos in culture at 8 16 cell stage.91 the technique has been considerably improved and its yield in number of puppies born related to the number of injected oocytes has made progress. south korean investigators have applied cloning to a number of situations: pet dogs, working dogs (e.g. scent detection dogs), preservation of breeds (e.g. sapsaree and the gyeongju donggyeong dogs), preservation of wild species (e.g. grey wolf and coyote), not to mention the creation of transgenic and canine biomedical models of human disease (reviewed82). currently, it is possible to obtain cloning of a dog or a cat in south korea, in china or in us for approximately 30,000€ per cat and 45,000 50,000€ per dog. thus, the sinogene company in beijing reported that it had cloned as many as 40 dogs and cloned its first cat in 2019. similarly, the south korean sooam company reported it had cloned 800 pet animals. in parallel, the us company viagen pets also offers cloning. cryopreservation of ovarian cortex/oocytes and in vitro folliculogenesis some other reproductive biotechnologies could be used for purposes of fundamental research or for preservation of biodiversity. in vitro folliculogenesis (dissection of ovarian follicles followed by culture for several days or weeks) may answer a number of questions regarding the growth of follicles and oocytes.92-95 for instance, in mice, one may, over an interval of 3 weeks, culture primary follicles until the preovulatory stage and obtain newborns.96 however, in dog, as in large mammals, folliculogenesis is a very lengthy process and culturing follicles for months remains quite a challenge and no progeny has been obtained so far anywhere in the world. however, in women, cryoconservation of the whole ovary or of fragments of ovarian cortex followed by autografting has been successfully used to preserve fertility prior to some gonadotoxic anticancer therapy. following the termination of cancer therapy, the ovary or cortex fragments can be reimplanted and can restore ovarian function, leading to growth of the uterus and eventually pregnancy and the birth of a baby. this procedure for fertility preservation (freezing + grafting) has successfully generated dozens of babies.97 in dogs, these techniques are still in an experimental stage. however, canine follicles survived in frozen ovaries and autografting was possible with resumption of follicle growth.99 another way to preserve female genetic material is cryoconservation of oocytes. in humans, this technique is widely used since the development of clinical theriogenology • volume 12 number 3 • september 2020 195 vitrification. two attempts of vitrification have been reported in dogs. freezing immature oocytes has already been tested and 65% of the cumulus-oocyte-complexes were reported to have adequate morphology after vitrification by the cryotop method.100 similarly, wolf oocytes survived vitrification.101 further studies are needed to examine oocyte survival and developmental potential after vitrification, according to the presence of cumulus cells and maturation stage at cryopreservation. research in this area may benefit from the results of vitrification in pigs,102 whose oocyte is also rich in lipids, like that in the dog. another technique commonly used in cattle, in vivo follicle puncture, also called ovum pick up (opu), may be quite useful in dogs. it is feasible in dog; however, it requires having a dog at similar preovulatory stage, good ultrasound equipment to clearly visualize follicles, and a laboratory close by to manipulate the oocytes (observation, culture or micromanipulation). following opu, oocytes or cumulus-oocyte-complexes might be cryopreserved or kept for maturation. in vivo follicle and oocyte growth, fertilization, and embryo development in cat similar to dogs, a cat ovary starts developing during fetal life (oogonia migration/oogenesis); however, first primordial follicles appear approximately 1 month after birth.103 histology of follicle and oocyte growth have been reported.103-106 primordial follicles, primary, secondary, and preantral follicles measure 50, 80, 130, and 150 µm respectively and contain an oocyte whose size is 40, 60, 90, and 100 µm respectively.106 zona pellucida appears in secondary follicle and antral cavity appears when follicle reaches 220 µm.106-107 at that stage, oocyte reaches 110 µm (together with zona pellucida) and nuclear maturation is completed. at the time of ovulation, oocyte size is ~ 125 130 µm.108 receptors for fsh in granulosa cells and for lh in theca cells can be detected in 200-µm small antral follicles.109 in granulosa cells, lh receptors appear when antral follicles reach at least 800 µm. similar to dog, cat ovary contains a large number of polyoocytic follicles, ~ 4%, with a recorded maximum of 10 oocytes in a single follicle.9,103,110 preovulatory diameter is 3 4 mm and may be measured via laparotomy or by laparoscopy.111,112 ultrasonography may be used113,114 without anaesthesia, particularly, in docile animals. regular assessment of behavior, vaginal smears and/or ultrasonography is needed, however, as these parameters are poorly correlated.114 ovulation can be induced by vaginal stimulation, inducing a peak in lh concentration, or may be obtained by hcg injection. ovulation may also be spontaneous, without coitus.115 meiosis resumption resumes within ovary and fertilization may take place as soon as oocyte reaches oviduct. after in vivo fertilization, embryos at 1 4, 5 8, 9 16 cell, and morula stages were present in oviduct ~ 28 34, 4046, 64 -70, and 88 94 hours respectively after ovulation.116 beyond 112 118 hours postovulation, compact morulas and blastocysts were present in uterus.116 prior to implantation, embryos migrate frequently to uterine horn on the opposite side.116 in vitro oocyte maturation, fertilization, and embryo development in cat contrary to dog, cat is a species in which oocyte maturation, fertilization and in vitro embryo development can be achieved with a rate of success similar to those obtained in cattle. thus, already more than 30 years ago, the first cat embryos were obtained after in vitro fertilization (but with in vivo matured oocytes).108,117 using oocytes collected from 55 female cats after ovarian stimulation (pmsg + hcg) made it possible to obtain as much as 80% of matured oocytes and 35 45% fertilization rate, with < 5% polyspermia.108 after transfer of embryos into oviduct following laparotomy (54 embryos in 5 recipient cats), 4 litters and a total of 10 kittens were born. pregnancy rates were satisfactory after transfer of embryos into uterus. however, embryo survival and thus the number of kittens obtained were higher after transfer into an oviduct.118 furthermore, using in vivo matured oocytes, the rate of embryo development was high: out of 100 oocytes, 75% divided after in vitro fertilization, 66% developed to the morula stage, and 18% to blastocyst stage.119 even with oocytes collected with no hormonal stimulation after ovariectomy, results can be favourable. thus, > 45% of oocytes reach metaphase ii stage after 32 38 hours of maturation,120 and after in vitro fertilization (46 56% of fertilization rate), 30 40% of embryos develop.121 furthermore, in vitro development is similar to in vivo.122 however, since cat is a seasonal species, further testing was carried out to evaluate impact of period of the year on developmental competence of oocytes used for in clinical theriogenology • volume 12 number 3 • september 2020196 vitro fertilization. in a study123 on nearly 7000 cumulus cell oocyte complexes, a seasonal effect was observed, with 45 55% embryos cleaved after fertilization and 50 70% of these embryos developing up to the blastocyst stage, but with lower rates of success in fall and winter seasons (october march). in icsi, even with epididymal or testicular sperm, 37% of embryo developed to morula stage and some births of kittens were reported.125 after cryoconservation of testicular tissue followed by microinjection of sperm into oocytes matured in vitro, embryos could be obtained, which were then frozen and transferred to several females, leading to birth of several kittens.126 ease of assisted reproductive technologies achievement in cat has led to research on wild felids (reviewed127-130). for example, 30 years ago, embryos of a leopard cat (felis bengalensis) were obtained after ovarian stimulation by pmsg + hcg treatment followed by follicle puncture via laparoscopy, in vivo fertilization and early embryo development.131 very recently, the first cheetah pups were born as the result of in vitro fertilization and embryo transfer achieved by scientists of the smithsonian conservation biology institute (https://www.si.edu). some other teams, such as that of bill swanson (cincinnati zoo and botanical garden), have become real experts in followup/induction of ovulation in domestic cats and also wild felids. this research was applied to the production of animals in zoos and in the wild, to building genetic stocks of sperm and embryos and to maintenance of animals of biomedical interest. indeed, as in dog, a number of feline models can be used in biomedical research (omia.org, 218 feline models of human disease), and the international conference on canine and feline genetics and genomics is devoted to these specific models. other reproductive biotechnologies for the preservation of cat genetics somatic cloning the first cat obtained by somatic cloning, "copycat", was born in 2001.132 genetics saving and clone company started its business and produced a first cat, "little nicky" sold for $50,000 in 2004. this company kept producing cloned cats until 2006. in the world, cat cloning can be done in south korea, in china or in us (refer above, section "dog cloning "). a cloned cat can, as the dog, be re-cloned.133 furthermore, numerous attempts at interspecific cloning were carried out. somatic cells of wild felids were injected into oocytes of domestic cats and some small wild felids (e.g. african wild cat134) were obtained this way (reviewed135,136). collection and preservation of the female genetic potential in vitro folliculogenesis in the cat was also explored. it is a powerful tool to study biological mechanisms and one way to stock genetic potential in a biobank.137,138 indeed, preantral follicles are quite numerous in ovaries and can survive cryopreservation.139 cryoconservation of immature cat oocytes by vitrification with the cryotop system has been described.140 high rates of oocyte survival were reported with resumption of maturation after thawing in ~ 25 40% of oocytes. after icsi (freezing may alter zona pellucida, and thus prevent fertilization), as many as 20% of embryos may reach the morula stage. some kittens could be obtained using vitrification technique.141 conclusion altogether, despite the complexity of canine model (2 ovulations per year, limited number of mature oocytes available and requirement of an experimental kennel), research efforts in reproductive biotechnologies are in full development, with the efforts of a few research teams in the world. on the contrary, in cat, numerous in vitro techniques are applicable that may also be extended to wild feline species. development of worldwide communications gave the scientific community a chance to collaborate. thus international embryo technology society created a committee on companion animals, nondomestic, and endangered species with the objective of sharing research progress in this area (https://www.iets.org/comm_candes.asp). clinical theriogenology • volume 12 number 3 • september 2020 197 acknowledgments we thank animal keepers and cerca (centre d'etude en reproduction des carnivores) for their help with 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production of cat embryos in modified synthetic oviduct fluid medium: effects of season and ovarian status. biol reprod 2001;65:9-13. https://doi.org/10.1095/biolreprod65.1.9. 124. bogliolo l, leoni g, ledda s, et al: intracytoplasmic sperm injection of in vitro matured oocytes of domestic cats with frozen-thawed epididymal spermatozoa. theriogenology 2001;56:955-967. https://doi.org/10.1016/s0093691x(01)00621-5. 125. gómez mc, pope ce, harris r, et al: births of kittens produced by intracytoplasmic sperm injection of domestic cat oocytes matured in vitro. reprod fertil dev 2000;12:423-433. https://doi.org/10.1071/rd00126. 126. tharasanit t, buarpung s, manee-in s, et al: birth of kittens after the transfer of frozen-thawed embryos produced by intracytoplasmic sperm injection with spermatozoa collected from cryopreserved testicular tissue. reprod domest anim 2012;47 suppl 6:305-308. https://doi.org/10.1111/rda.12072. 127. pelican km, wildt de, pukazhenthi b, et al: ovarian control for assisted reproduction in the domestic cat and wild felids. theriogenology 2006;66:37-48. https://doi.org/10.1016/j.theriogenology.2006.03.013. 128. amstislavsky s, lindeberg h, luvoni g: reproductive technologies relevant to the genome resource bank in carnivora. reprod domest anim 2012;47:164-175. https://doi.org/10.1111/j.1439-0531.2011.01886.x. 129. jewgenow k, braun bc, dehnhard m, et al: research on reproduction is essential for captive breeding of endangered carnivore species. reprod domest anim 2017;52 suppl 2:18-23. https://doi.org/10.1111/rda.12836. 130. thongphakdee a, sukparangsi w, comizzoli p, et al: reproductive biology and biotechnologies in wild felids. theriogenology 2020. https://doi.org/10.1016/j.theriogenology.2020.02.004. 131. goodrowe kl, miller am, wildt de. in vitro fertilization of gonadotropin-stimulated leopard cat (felis bengalensis) follicular oocytes. j exp zool 1989;252:89-95. https://doi.org/10.1002/jez.1402520112. 132. shin t, kraemer d, pryor j, et al: a cat cloned by nuclear transplantation. nature 2002;415:859. https://doi.org/10.1038/nature723. 133. yin xj, lee hs, yu xf, et al: production of second-generation cloned cats by somatic cell nuclear transfer. theriogenology 2008;69:1001-1006. https://doi.org/10.1016/j.theriogenology.2008.01.017. 134. gómez mc, pope ce, giraldo a, et al: birth of african wildcat cloned kittens born from domestic cats. cloning stem cells 2004;6:247-258. https://doi.org/10.1089/clo.2004.6.247. 135. gómez mc, pope ce, dresser bl: nuclear transfer in cats and its application. theriogenology 2006;66:72-81. https://doi.org/10.1016/j.theriogenology.2006.03.017. 136. gómez mc, pope ce: cloning endangered felids by interspecies somatic cell nuclear transfer. methods mol biol 2015;1330:133-152. https://doi.org/10.1007/978-1-4939-2848-4_13 137. jewgenow k, paris mcj: preservation of female germ cells from ovaries of cat species. theriogenology 2006;66:93100. https://doi.org/10.1016/j.theriogenology.2006.03.010. 138. campos lb, praxedes écg, saraiva mva, et al: advances and challenges of using ovarian preantral follicles to develop biobanks of wild mammals. biopreserv biobank 2019;17:334-441. https://doi.org/10.1089/bio.2018.0130. 139. martins jla, lopes md, de souza ff, et al: cat preantral follicle survival after prolonged cooled storage followed by vitrification. cryobiology 2018;81:94-100. https://doi.org/10.1016/j.cryobiol.2018.02.004. 140. fernandez-gonzalez l, jewgenow k: cryopreservation of feline oocytes by vitrification using commercial kits and slush nitrogen technique. reprod domest anim 2017;52 suppl 2:230-234. https://doi.org/10.1111/rda.12837. clinical theriogenology • volume 12 number 3 • september 2020202 141. pope ce, gómez mc, kagawa n, et al: in vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer. theriogenology 2012;77:531-538. https://doi.org/10.1016/j.theriogenology.2011.08.028. clinical theriogenology • volume 12 number 3 • september 2020 203 009_ms-009 reynaud introduction all gorilla species and subspecies (gorilla spp) are either endangered or critically endangered (international union for conservation of nature [iucn] primate specialist group). of the 2 species with 2 subspecies each (total of 4 subspecies), the only subspecies managed in north american zoos are western lowland gorillas (g. gorilla gorilla). as of 2019 there were ~ 354 individuals in 48 facilities accredited by the association of zoos and aquariums (aza).1 these animals are managed collaboratively with oversight by the gorilla species survival plan (ssp), led by a group of individuals with expertise in gorillas that works to minimize inbreeding and maximize genetic diversity. the gorilla ssp makes recommendations for breeding specific animals, taking into account their relatedness to the rest of the population, location, individual health, personality, genetic value, and available space in zoos.1 in the wild, western lowland gorillas live in tight-knit family groups averaging 10 individuals and consisting of an adult breeding male (silverback), a group of females, and their offspring.2 occasionally, the groups have multiple males. reproduction is relatively slow, with inter-birth intervals of 4 6 years, low birth rates (< 0.2 offspring per female per year) and high juvenile mortality rates (up to 65% by 3 years of age).2 reproduction in zoos is usually better than in the wild, in part due to better nutrition and markedly lower infant mortality rates; however, zoo clinical challenge gorilla infertility anneke moresco,a,b,c elizabeth cutchin,c scott larsen,a michael stern,a,e linda penfold,f sushan han,c betsy stringer,a michelle vallois,a jennifer barfieldc adenver zoo, denver, co; breproductive health surveillance program, morrison, co ccollege of veterinary medicine and biomedical sciences, colorado state university, fort collins, co danimal sciences, colorado state university, fort collins co; ephiladelphia zoo, philadelphia, pa fsouth-east zoo alliance for reproduction and conservation, yulee, fl abstract reproductive monitoring and intervention are challenging in nondomestic species where manual restraint is often not possible or potentially dangerous for animals and the clinician. as a result, performing even simple procedures (e.g. acquiring blood or vaginal smears or using ultrasonography) on such species may not be possible without full anesthesia, as is the case for gorilla. therefore, whenever possible, noninvasive techniques are preferred as tools for reproductive monitoring. yet, for a large proportion of nondomestic species, many basic parameters (e.g. cycle length, duration of follicular and luteal phases, or ovulatory mechanism) are not known, so diagnosis and treatment of reproductive abnormalities are even more challenging. similarly in males, many needed diagnostic procedures require either noninvasive techniques and/or anesthesia. an example of the clinical approach and challenges to breeding problems in gorillas in zoos is presented. keywords: endometrial biopsy, gorilla, great apes, infertility, semen, vaginoscopy, zoo inter-birth intervals are similar, ~ 4 years.3 despite reproduction rates in zoos being slightly higher than in the wild, zoo clinicians are confronted with unique challenges when working on reproductive issues with this species. some of these challenges are technical in nature, whereas others are secondary to a lack of baseline data. this report describes the clinical evaluation of a pair recommended to breed and the findings that may have contributed to the female’s subfertility/infertility. case history this case describes an adult female western lowland gorilla (27 years) at the denver zoo recommended to breed with a proven adult male (26 years) also housed at the denver zoo. timeline of events is provided (figure 1). the case female was considered to be overweight (body condition score 7/9), during both anesthetic events detailed here. the female in question had given birth to 1 live offspring (at a previous institution), and was on oral contraception [ethynodiol diacetate (1 mg) and ethinyl estradiol (50 µg) zovia 1/50e (nurk™), mayne pharma us, raleigh, nc]. treatment was discontinued when breeding between her and the male was attempted. clinical theriogenology 2021; 13: 402 behavioral management first attempts to encourage reproduction focused on husbandry and behavioral management as these techniques are the most natural, noninvasive and usually very low cost. mating was occasionally observed and regularly suspected by animal care staff during the initial period of introductions, but did not result in pregnancy. over the following several years, the male became increasingly aggressive to this female; although this was thought largely due to redirected aggression as the dominant female did not like the male to breed with other females, interfered with breeding attempts and would start fights over food that caused the male to aggress on this female. these led to occasional (1 or 2 per year) mild to moderate injuries to the case female and ultimately led to the social group being restructured (figure 1). sometimes when pairs are kept together for extended intervals sexual activity decreases; therefore, a technique that can be employed to increase interest is to separate the male from the female and reintroduce them at estrus. however, accurately knowing when the female is receptive so that introductions can be appropriately timed is a key factor in the success of this approach. some primates advertise the period around ovulation by developing large perineal (sexual) swellings.4 gorillas, however, do not exhibit obvious sexual skin swelling associated with fertile periods in their estrous cycle and visually determining the period of estrus is more challenging than in primates with perineal swellings.5 with the goal of separating this female from the male and reintroducing them at the time of ovulation, steps were taken to pinpoint the day of ovulation in this female. monitoring for ovulation in humans, over-the-counter urine dipstick tests for ovulation may be used to determine the time of ovulation. these tests qualitatively measure human luteinizing hormone (lh) concentrations in urine; human lh and lh in great apes is similar enough that these tests are effective,6 and have been used to detect ovulation in gorillas.7 however, this testing was considered cost-prohibitive as it would require testing the female at least every other day for a minimum of 2 cycles in order to obtain a baseline cycle and then a comparison cycle when introductions would be attempted based on predicted time of ovulation. ovulation generally occurs midway between menses; therefore, a second option to predict ovulation was to monitor menses. in gorillas, menses is subtle but can be confirmed with urine reagent strips (multistix®, siemens, munich, germany) to detect blood in the morning urine. this method is easy and economical. urine was sampled every other day over the course of 16 months. with a few exceptions, menses occurred at relatively regular intervals from march 2013 until september 2013 (cycles 1-5), and menstrual cycles became more irregular after that (september 2013 october 2014; from cycle 6 onwards) (figure 2). a third option for detecting ovulation was to monitor ovarian hormone metabolites in feces over the course of several ovarian cycles. this requires fecal sample collection, storage, shipping, and laboratory analysis, so there is a lag between endocrine events and when results are obtained. although there were substantial costs associated with this monitoring method, fecal hormone analysis was performed in parallel to the urine multistix® reagent strips (to monitor blood in the urine as an indicator of menses) so that the accuracy of reagent strips as a proxy for fecal hormones could be assessed. fecal samples were collected 3 times weekly and stored frozen (– 80°c) until analysis. fecal samples were lyophilized and pulverized; ~ 0.5 gram was first extracted with 5 ml of double deionized water, vortexed and centrifuged and 3 ml of supernatant was removed and frozen (– 20°c). furthermore, 500 µl of supernatant were vortexed with 5 ml of petroleum ether, and snap frozen in a figure 1. timeline of the social changes in the gorilla group. ocp – oral contraceptive pill clinical theriogenology 2021; 13: 403 methanol dry ice bath. the organic phase was poured into a 16 x 150 mm tube containing 500 µl deionized h2o. the second tube was then vortexed for 2 minutes, snap frozen in a dry ice methanol bath and the washed organic phase was poured into a 12 x 75 mm glass tube, which was placed into a heating block and the petroleum ether evaporated under a stream of nitrogen. a second extraction of the original 500 µl aliquot was performed using the same protocol to yield the final washed extract. the dried extract was reconstituted in 0.5 ml phosphate-buffered saline containing 0.1% gelatin and progesterone quantified by radioimmunoassay. although initially menses appeared very regular (figure 2), menstrual intervals became irregular during the observation period. fecal progesterone metabolites indicated the female was undergoing repeated luteinization and luteolysis (figure 3). dates with positive results from urine reagent strips frequently followed progesterone peaks as would be expected, and menses occurred at the end of the luteal phase. however, over the period monitored, progesterone did not increase to peak concentrations during several cycles and menses were not detected (e.g. february, april, and june 2014). differential diagnoses for the irregular bleeding included breakthrough bleeding, occasional anovulatory cycles (obesity or premenopausal transition), inflammatory/infectious processes, and malignancy. although the level of blood in urine did not occur in a predictable time figure 2. female gorilla was monitored for menses between march 2013 and october 2014. duration of menstrual cycles (from first day of menses). with 1 exception, menstrual cycles from march 2013 through september 2013 (cycles # 1-5) were regular. cycles became irregular september 2013 through october 2014 (cycles # 6 17). the vertical line indicates the shift from regular to irregular cycles. figure 3. fecal progesterone (p4) metabolites (open circles/solid line) and hematuria score on urine reagent strips (solid diamonds/ dashed line) in a female gorilla with failure to conceive clinical theriogenology 2021; 13: 404 relative to the progesterone peaks, lack of blood in the urine (extended menstrual cycles) did correspond to extended interluteal phases as measured via fecal progesterone metabolites. male reproductive examination because further evaluation of the female would be more invasive, it was chosen to perform a full reproductive evaluation of the male. the male was examined under full anesthesia (induction: 220 mg tiletamine/zolazepam via intramuscular hand-injection (telazol®, zoetis, kalamazoo, mi) and 7 mg medetomidine (wildlife pharmaceuticals, windsor, co); maintenance: isoflurane (1 2%) via endotracheal tube (vetone, mwi, boise, id). during the physical examination, there were no notable abnormal findings, including in the external genitalia. ultrasonography of the external genitalia was not performed due to time constraints of anesthesia. testes were normal when palpated, testicular length and width were determined using calipers, and testicular volume was calculated (total combined volume = 27.5 cc). the prostate felt soft and enlarged (70 x 40 mm) compared to a previous report (15 x 7 mm).8,9 after the urinary bladder was manually expressed to the extent possible, semen was collected via electroejaculation using a rectal probe (31 mm diameter) and giving stimuli at low voltage (2 4 v, 40 -100 ma). progressive sperm motility was assessed by examining semen at 37°c. semen ph (7.4 8.3) was determined using ph strips (em science, gibbstown, nj) and volume determined using an adjustable eppendorf pipettor (brinkman instruments, westbury, ny). sperm concentration was determined using a hemocytometer and an aliquot of raw semen fixed in 0.3% glutaraldehyde was used to determine the percentage of morphologically normal sperm, assessed under a phase contrast microscope at x1000. despite efforts to evacuate the bladder, the ejaculate was contaminated with urine and no motile sperm were observed. total sperm (10 x 106 sperm) were within the range considered fertile.9 observed morphological abnormalities were consistent with urine contamination and precluded assessment of percent normal morphology. however, most sperm heads were morphologically normal and the ejaculate was considered fertile. however, serum testosterone concentration (959 pg/ml) was below published concentrations for fertile male gorillas (4,137 pg/ml ± 2,191),9 and may have reduced libido. female reproductive examination because noninvasive diagnostics and the examination of the male did not yield an explanation for the failure to conceive, a comprehensive reproductive examination on the female was performed under full anesthesia. the examination included external examination, vaginoscopy, vaginal swab culture, ultrasonography, and transcervical uterine biopsy. additionally, a blood sample was collected to determine serum concentrations of the following hormones: estradiol, progesterone, antimüllerian hormone [amh], follicle stimulating hormone [fsh], and luteinizing hormone [lh]). anesthesia was induced via intramuscular hand injection with 115 mg tiletamine/intramuscular zolazepam (telazol®, zoetis) and 3.34 mg medetomidine (wildlife pharmaceuticals). anesthesia was maintained with isoflurane (1.5 2%) via endotracheal tube. general physical examination findings were normal except that the animal continued to be over-conditioned. vaginoscopy revealed a moderate amount of foreign material (straw, sticks/woody material, and hair). after removing the organic material, superficial ulcerations on the cervical mucosa were visible, although the remaining mucosa was normal (pink and without abrasions). no sample for culture was taken due to the contamination. vaginal cytology sample was of moderate cellularity, with epithelial cells as the predominant cell type. epithelial cells were characterized by distinct, round, basophilic nuclei, large cytoplasm, angular margins, and were fairly uniform in appearance. there were few to moderate numbers of red blood cells, rare leukocytes, and large amount of mixed population bacteria. the vaginal vault was cleaned and rinsed with a dilute betadine solution. ultrasonography was performed with a transvaginal transducer (4 9 mhz; fazone cb, fujifilm, valhalla, ny). the uterus was in axial position, had a normal appearing myometrium, but irregular endometrial lining with a possible soft tissue mass arising from the dorsal wall of the uterus. the right ovary appeared normal with a structure consistent with an ovarian follicle (nearing ovulation). however, the left ovary had a complex cyst with solid and cystic components (figure 4), the solid component protruded into the cyst (possibly pedunculated). based on the female gorilla’s last date of menses, she was expected to be in the late follicular phase at the time of the examination, consistent with the large follicle on the right ovary. uterus and ovary sizes were similar to published dimensions for gorillas.10 structures on the left ovary were considered potentially malignant. after the external cervical os was cleaned with betadine solution, a pederson’s speculum was placed to facilitate introduction of an endometrial suction curette (pipelle® coopersurgical, trumbull, ct) through the cervix. this device was used to obtain multiple endometrial biopsies; histopathology revealed a marked, subacute to chronic lymphoplasmacytic and neutrophilic erosive endometritis. based on culture and sensitivity results, a course of azithromycin (500 mg for 1 day and 250 mg for 5 days, tagi pharma, inc., south beloit, il) was prescribed. a follow-up examination was scheduled to perform surgery to remove the left ovary. at the follow-up examination (~ 17 months after the initial examination), vaginal evaluation revealed minimal foreign material and cytology was consistent with vaginitis (mixed bacterial population with rare yeast and suppurative inflammation). transvaginal ultrasonography revealed a normal right clinical theriogenology 2021; 13: 405 ovary with multiple small follicles and no visible luteal tissue, and a normal left ovary with no visible follicles. the abnormal structure on the left ovary could no longer be visualized and the surgery was cancelled, as the high risk of abdominal adhesions in gorillas outweighed the benefits of exploratory laparotomy. additional transcervical suction endometrial biopsies were obtained and revealed minimal inflammation and no evidence of neoplasia. serum hormone concentrations ruled out perimenopausal transition and included: estradiol (45.7 pg/ml), progesterone (0.2 ng/ml), amh (1.25 ng/ml), fsh (0.075 mlu/ml), and lh (< 0.1 mlu/ml). estradiol and progesterone concentrations were compatible with a follicular phase. serum amh concentration in this female was similar to median concentrations in women 40 years old,12 and higher than for female gorillas 2135 years old (1.07 ng/ml, n = 8),13 and was interpreted as not compatible with ovarian senescence. during menopause in humans, concentrations of fsh rise substantially during transition into menopause; however, this change has not been studied in gorillas.14 similarly, fsh concentrations were not considered consistent with ovarian senescence in women. although the female remained without a specific diagnosis, she was transferred to a different facility to place her in a better social environment since that would be the first hurdle in solving breeding problems. she was well integrated in her new group, and matings were observed; however, no pregnancy has resulted from those matings. discussion although a final diagnosis was not reached, there were multiple factors potentially hindering reproduction. in women, obesity is associated with increased risk of anovulation (reviewed15). obesity may have played a role initially; however, later the female was in better body condition. although she was still relatively young, under human care, female gorillas tend to experience many more infertile cycles than in the wild and ‘ovarian senescence’ may occur at an earlier age. endometrial biopsies revealed mild uterine pathology that may have negatively affected fertility, although at the follow-up this appeared to have resolved. in the end, the female was transferred to a different group with the goal of addressing mate incompatibility. however, that she has still not become pregnant in spite of favorable social dynamics indicates there may be additional factors contributing to infertility. this case is an example of the challenges faced by zoo clinicians working with nondomestic species. general challenges for work with zoo animals include the inability to obtain blood samples without anesthesia, the lack of documented normal ranges for every species held in zoos, the need for nonstandard sizes of instruments and equipment, the need to plan involved procedures and then reassess due to new information. in gorillas in particular it also includes the fact that they are prone to abdominal adhesions and abscesses, changing the risk-to-benefit considerations of exploratory laparotomy.11 this case also illustrates solutions to some of these challenges such as animal care staff working to habituate animals to new situations and voluntarily administration of medication, the development of noninvasive monitoring methods, including low-tech methods as an alternative to more expensive methods. lastly, it illustrates the collaboration needed to work up 1 animal: keepers, supervisors, veterinary staff, and national groups such as those advising on matches of genetic value (ssp). figure 4. transvaginal ultrasonogram from a gorilla with failure to conceive. u = uterus, * = ovarian follicle on the right ovary, arrowheads indicate a complex cyst in the left ovary clinical theriogenology 2021; 13: 406 conflict of interest there are no conflicts of interest to declare. acknowledgments authors thank the animal care and veterinary staff at the denver zoo, in particular michelle valois for the collection of multistix® data and fecal samples. authors also thank drs. terry nett and matt allen for their support in the endocrine laboratory and dr. a. pimentel for reviewing the manuscript. references 1. lukas k, elsner r, moyse j, et al: population analysis & breeding and transfer plan, western lowland gorilla (gorilla gorilla gorilla). 2019. 2. robbins mm, bermejo m, cipolletta c, et al: social structure and life‐history patterns in western gorillas (gorilla gorilla gorilla). am j primatol 2004;64:145-159. 3. sievert j, karesh wb, sunde v: reproductive intervals in captive female western lowland gorillas with a comparison to wild mountain gorillas. am j primatol 1991;24:227-234. 4. deschner t, heistermann m, hodges k, et al: timing and probability of ovulation in relation to sex skin swelling in wild west african chimpanzees, pan troglodytes verus. anim behav 2003;66:551-560. 5. nadler rd: cyclicity in tumescence of the perineal labia of female lowland gorillas. anat rec 1975;181:791-798. 6. gould kg, faulkner jr: development, validation, and application of a rapid method for detection of ovulation in great apes and in women. fertil steril 1981;35:676-682. 7. abello mt, flamme g: monitoring ovulatory cycles in western lowland gorillas (gorilla gorilla gorilla) at barcelona zoo. int zoo yearb 2005;39:185-191. 8. oerlich tm: pelvic and perineal anatomy of the male gorilla: selected observations. anat rec 1978;191:433-446. 9. gould k, kling o: fertility in the male gorilla (gorilla gorilla): relationship to semen parameters and serum hormones. am j primatol 1982;2:311-316. 10. morais jb. ultrasonographic assessment of reproductive diseases in gorillas and other captive great apes. thesis, universidade tecnoca de lisboa. 2013. 11. mylniczenko nd: a preliminary report in intra-abdominal abscesses in captive western lowland gorillas (gorilla gorilla gorilla). proc am assoc zoo vet 2003;62-66. 12. shebl o, ebner t, sir a, et al: age-related distribution of basal serum amh level in women of reproductive age and a presumably healthy cohort. fertil steril 2011;95:832-834. 13. moresco a, stern m, erikson d, et al: anti-müllerian hormone as an accurate indicator of ovarian reserve in gorillas. in: international gorilla workshop; 2016. 14. tepper pg, randolph jr jf, mcconnell ds, et al: trajectory clustering of estradiol and follicle-stimulating hormone during the menopausal transition among women in the study of women’s health across the nation (swan). j clin endocr metab 2012;97:2872-2880. 15. metwally m, li t, ledger w: the impact of obesity on female reproductive function. obes rev 2007;8:515-523. clinical theriogenology 2021; 13: 407 contact annett annandale annett.annandale@murdoch.edu.au © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9595, http://dx.doi.org/10.58292/ct.v15.9595 research report effect of foosball practice on veterinary students’ bovine artificial insemination skills mia norton,a annett annandalea,b aschool of veterinary medicine, murdoch university, perth, australia; bfaculty of veterinary science, university of pretoria, pretoria, south africa abstract live cow artificial insemination (ai) opportunities are limited at training institutions, and this study explored a novel approach to better prepare students for the task. since the arm and wrist movement while playing foosball (fb) is like picking up the cervix during ais, the effect of playing fb on veterinary students’ ai performance was investigated. objective was to determine if playing fb would improve students bovine ai ability through muscle memory development. fourth year (of a 5 year course) veterinary students (n = 28) either took part in the fb cohort (n = 17) or the nonfb cohort (n = 11). the fb cohort played 30 minutes of fb 3 times/week for 8 weeks. both cohorts then participated in ai training using bovine cadaver uteruses before they were assessed on their ability to place an ai pipette into a live cow’s uterus. although there was no difference between cohorts (p = 0.58) there was a higher proportion of successful students in the fb (0.35) compared to the nonfb cohort (0.18). small cohort size, the fact that only 11 students completed the allocated fb play time, and participants’ transrectal palpation (trp) inexperience may have influenced the result. this should, however, not discourage further investigations into alternative teaching approaches. furthermore, this study highlighted the importance of assessing competence for clinical skills that are required to learn new skills first (trp in this case) before attempting to teach a new, more advanced skill (ai in this case). this is likely to be applicable for many other clinical skills. keywords: artificial insemination, cattle, foosball, clinical skills training introduction bovine artificial insemination (ai) is a valuable large animal clinical skill that has an impact on cattle herd profitability, especially on dairy farms,1 improves the genetic quality of herds,2,3 controls disease and is cost effective compared to natural mating.4 inefficiency in ai can dramatically affect fertility,4 produce variable or low calving rates and will ultimately have a substantial economic impact on farmers.5 although large dairy operations mostly utilize technicians for ais, veterinarians are expected to have experience and knowledge with ai process in their role as a dairy consultant, or to efficiently implement ai programs for small scale or hobby farmers.1 it is therefore an important skill for veterinary students to learn. passing a pipette through the cervix for ai does not differ from passing a pipette or other instruments for diagnostic or treatment reasons and further highlights the importance of learning this skill. however, live cow training opportunities at university level are scarce.1 modern veterinary programs have a strong focus on the development of clinical skills,6 with an expectation to produce graduates with advanced technical skills.7 developing competence in the required skills depends on opportunities for sustained, deliberate hands-on practice while learning.7–9 providing sufficient opportunities for skills acquisition is influenced by various factors including increased student numbers,7,8 budget restrictions, and the welfare and ethical concerns surrounding the use of live animals for student training.7,10 the ‘five step method for teaching clinical skills’11 outlines a method for teaching psychomotor skills in an organized and successful way that aids students. this method has its benefits in training clinical skills in medical and veterinary programs where the entirety of the skill can be observed. however, there are skills (e.g., transrectal palpation (trp) and ai in large animal veterinary practice) that cannot be observed. bovine ai simulators (e.g., 1breed’n betsy® simulator, 2minitube ‘henryetta’ insemination model and 3bovine breeder™ ai simulator) were developed as 1. breed’n betsy, https://www.breednbetsy.com.au/ 2. minitube, https://www.minitube.com/catalog/en/henryetta-artificial-cow-ai training-model-p2353/ 3. realityworx, https://www.realityworks.com/product/bovine-breeder-artificial insemination-simulator/ mailto:annett.annandale@murdoch.edu.au http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9595 https://www.breednbetsy.com.au/ https://www.minitube.com/catalog/en/henryetta-artificial-cow-ai-training-model-p2353/ https://www.minitube.com/catalog/en/henryetta-artificial-cow-ai-training-model-p2353/ https://www.realityworks.com/product/bovine-breeder-artificial-insemination-simulator/ https://www.realityworks.com/product/bovine-breeder-artificial-insemination-simulator/ citation line: clinical theriogenology 2023, 15, 9595, http://dx.doi.org/10.58292/ct.v15.9595 47 training alternatives. these simulators provide excellent additional training opportunities and help to overcome some of the challenges faced with live animal training.12 however, limitations around implementing simulator training at teaching institutions include the expense to purchase simulators, labor, time, and intense small group teaching that is required for effective training.12 the need to overcome restraints around teaching certain skills has led to the development of alternate approaches to skills training.13,14 a few recent studies in veterinary education have tested a concept where learning or training outside of the main task has been used to improve performance required for the main task. one study considered how training in fine arts could improve veterinary students’ observational skills when learning cytology, assuming that deconstructing art helps develop an understanding for why things look how they do and apply these skills to other images.13 another study, focused on physical training, identified that veterinary students who took part in a 6 week exercise program were able to more accurately perform pregnancy diagnoses (pd) via trp in cows compared to students who did not participate in the exercise program.14 as demonstrated,15 alternate approaches to teaching have not only been utilized in veterinary sciences. a recently developed concept that could be applied to improve clinical skills acquisition in veterinary education is called the ‘predictor micro skills concept’.12 this concept aims to identify factors that influence a student’s ability to perform a certain skill. these factors are then used to identify micro skills involved in the performance of the overall skill. micro skills are defined as part tasks or components of the overall skill that can be learned and developed separately to the main task. once trained in micro skills, it is expected that the training and learning of the main skill will be more efficient. this concept was based on investigations aimed at improving bovine pd via trp.12 if this concept is applied to bovine ai as the skill, passing an ai pipette through the cervix would be a micro skill. since correct placement of the ai pipette into the uterus during ai is considered as one of the most challenging components of the procedure, it would be advantageous to better prepare students for this part of the ai process. passing an ai pipette through the cervix and into the uterus is facilitated by grasping the cervix and moving it cranially,16 actions like the wrist and hand actions during playing foosball (fb) (figure  1). the ‘predictor’ in this case that is expected to enhance acquisition of the micro skill, is muscle memory produced by repeated table fb exposure. muscle memory is the common term used to describe motor learning and motor memory.17 motor memory is the formation of multiple long-term memories within the nervous system,18 produced through repeated practice of a specific motion.19,20 motor memory is why humans apparently never forget how to swim, ride a bike, or drive a car;18 it allows motor skills to be performed spontaneously or in a more automatic fashion.19 there are currently no publications investigating the effect of muscle memory development on skills acquisition in veterinary education. activities (e.g., fb) are easy to implement at veterinary schools and could be used to improve student preparation for limited live cow ai training opportunities. the aim was to determine the effect of 8 weeks fb bootcamp on veterinary students’ ability to pass an ai pipette through the cervix and into a cow’s uterus. it was hypothesized that students with exposure to fb practice are more likely to successfully pass an ai pipette through the cervix and into a cow’s uterus compared to students without fb exposure. materials and methods study population and recruitment this prospective cohort study consisted of 2 student cohorts in the 4th year of the veterinary medicine course at murdoch university, perth, western australia. all students participating had previous basic bovine trp training within the 4th year veterinary reproduction module with no prior live cow ai training or experience nor additional trp experience outside the veterinary course. the 2 study cohorts were the fb exposure group (gfb) and the nonfb group (g0). participants in gfb took part in an 8-week fb bootcamp, attended an ai training session and an ai assessment where the task was to place an ai pipette into the uterus of a live cow. participants in g0 only attended the ai training and the assessment. participants in gfb were required to be available to take part in fb practice, and must not have had any previous hand, wrist, or arm injuries. participants in both, gfb and g0, needed to be available for ai training and assessment. students were recruited to each cohort by voluntary sign up after receiving information on the project in the form of a flyer posted to the 4th year veterinary students’ facebook group. students were able to choose whether they wanted to be part of gfb or g0. students interested in either group were given further information in the form of an information letter. both cohorts then attended a brief information session on the project and to sign a consent form to confirm participation in the study. withdrawal from the study was possible for participants at any stage. figure 1. hand and wrist movements during bovine artificial insemination* versus foosball source: *https://www.journalofdairyscience.org/article/s0022-0302(89)79346-2/pdf http://dx.doi.org/10.58292/ct.v15.9595 https://www.journalofdairyscience.org/article/s0022-0302(89)79346-2/pdf 48 citation line: clinical theriogenology 2023, 15, 9595, http://dx.doi.org/10.58292/ct.v15.9595 foosball exposure participants in gfb completed 8 weeks of fb practice. two fb tables were placed in an easily accessible venue at murdoch university. during the 8 weeks, gfb participants were encouraged to play fb at least 3 times a week for 30 minutes. students were able to use any free time to play fb (e.g., lunch breaks or between classes), and fb tables were accessible during weekends. fb tables were cleaned at the end of each week by an investigator as a covid-19 precaution; however, students could clean the tables and handles whenever they felt necessary using the disinfectant available at each of the fb tables. a facebook group was created for students in gfb to communicate with each other about playing times, and for communication purposes between investigators and participants. artificial insemination training during the last week of fb practice for gfb, both cohorts (gfb and g0) participated in a 2-hour ai training session. the training session took place in a teaching laboratory at the veterinary school. complete, fresh bovine reproductive tracts including cervix and vagina were sourced from an abattoir for use at the training session. the first half hour of the session was a lecture and a demonstration of the ai procedure by an experienced veterinarian using a fresh cadaver uterus. as this project was focused on the grasping of cervix and passing a pipette through the cervix and into the uterine body, this was the focus of ai training rather than the complete ai process. eight abattoir-derived uteruses were hung in frames in a position resembling true anatomic positioning of the uterus in a live cow. the frames were ~ 1.5 meter high and contained a bony bovine pelvis in which the uteruses were positioned. this allowed participants to practice the ai process without being able to see the uterus, relying on feel to correctly place the pipette. the remaining fresh uteruses were laid out on a table to allow students to familiarize themselves with the structures of the cervix and uterus. participants also had access to a video detailing the process of bovine ai (https://www. youtube.com/watch?v=h_svuit3gtk). after the initial explanation of the procedure, participants moved between activities and practiced the ai procedure themselves. an experienced veterinarian was available to help and provided feedback. cattle study population and synchronization nonpregnant (n = 25) cows from the murdoch university veterinary farm were used for the ai assessment component of the project. an estrus synchronization program was implemented to ensure that cows were in estrus or proestrus for the ai assessment. cows were kept at the murdoch university veterinary farm in their usual surroundings for the duration of this study. whenever cows were required by investigators, they were moved from their paddock into purpose-built cattle yards by the farm manager. cows were moved into cattle examination crushes for any procedures. the crush facilities had 12 examination crushes in 1 large open sided shed. lowstress stock handling principles were always adhered to when moving and handling cows. prior to synchronization, trp was performed by an experienced veterinarian to assess reproductive organs and to exclude any reproductive abnormalities. a modified controlled internal drug releasing device (cidr)-ob synchronization protocol was used. each cow had an eazibreed™ cidr® (zoetis) containing 1.38 grams of progesterone placed intravaginally (day 1). after 7 days, cidrs were removed and 24 hours later (day 8) 1 ml of intramuscular bomeroltm (estradiol benzoate, 1 mg/ml, elanco) was given. the ai assessment took place 3 days later (day 11). after ai assessment, cows received 1 ml intramuscular juramate® (prostaglandin, 250 µg/ml, jurox). artificial insemination assessment seven days after ai training, both gfb and g0 cohorts attended a live cow ai assessment where the participants’ ability to pass an ai pipette through the cervix and into the uterus of a nonpregnant cow was assessed. on the ai assessment day, 7 cows were brought into the crush facilities at a time. prior to any student palpations, cows were palpated by an experienced veterinarian to determine estrous cycle stage allowing investigators to choose cows. any cow not in estrus or proestrus was released and another cow was brought for assessment. this palpation also allowed emptying of feces prior to students’ palpation. students worked in pairs of 1 gfb and 1 g0 participant. there was 1 cow allocated to each pair of students. each student had 10 minutes to attempt passing the ai pipette through the cervix. while 1 student was passing the ai pipette, their partner timed them and held the cow’s tail out of the way. roles were switched after pipette placement was checked by an experienced veterinarian and the second student attempted to pass the ai pipette. pipette placement was evaluated transrectally by an experienced veterinarian and recorded as correct (tip of ai pipette felt in the uterine body) or incorrect (tip of ai pipette not felt in the uterine body). if there was any sign of mucosal bleeding during rectal palpations, students had to stop performing the procedure immediately, and the cow was removed from the project. the cow was examined by an experienced veterinarian and not used for any further palpations. the student was given a new cow to complete the assessment. data collection data collection was carried out using a hard copy paper sheet for the ai assessment and included: the name of the student, their student number, the ear tag number of the cow they used, which hand was used to rectally palpate, and whether they were successful in passing the ai pipette into the uterine body of the cow based on assessor feedback. ‘yes’ and ‘no’ indicated if the student was successful or not in passing the ai pipette. data collected on the cows included ear tag number, whether they were in estrus or proestrus, and whether they had mucosal bleeding or any other health concerns after student palpations. data were then transferred to an electronic spreadsheet after ai assessment. gfb participant questionnaire participants in gfb filled in a short anonymous hard copy questionnaire after completion of the ai assessment. the questions were aimed at getting feedback on the 8 weeks of fb practice and to better understand limitations of the study. questionnaire data was then transferred into an electronic data sheet. data analysis chi square test for independence was used to assess the relationship between pipette placement outcome and exposure (gfb versus g0) (https://epitools.ausvet.com.au/). ethical considerations animal ethics for this project was approved by the animal ethics committee of murdoch university (protocol http://dx.doi.org/10.58292/ct.v15.9595 https://www.youtube.com/watch?v=h_svuit3gtk https://www.youtube.com/watch?v=h_svuit3gtk https://epitools.ausvet.com.au/ citation line: clinical theriogenology 2023, 15, 9595, http://dx.doi.org/10.58292/ct.v15.9595 49 r3353/21). human ethics for this project was approved by the human ethics committee of murdoch university (protocol 2021/146). results study population a total of 28 students enrolled in this study. seventeen students took part in gfb and 11 in g0. there were 21 female students and 7 male students across both groups that equals 75 and 25% of the study population, respectively. of the 21  female students, 12 took part in gfb and 9 in g0. five of the 7 male students were in gfb and 2 were in g0. the study cohort is representative of the class (n = 98 students) that has a gender distribution of 83 and 17% for female and male students, respectively. passing ai pipette on ai assessment day, 6 participants from gfb (6/17, 35%) and 2 participants from g0 (2/11, 18%) were successful in passing the ai pipette into the body of the cow’s uterus (figure 2). the proportions of each group to be successful in passing the ai pipette were 0.35 and 0.18 for gfb and g0, respectively. participants in gfb demonstrated a higher proportion of successful pipette placements compared to g0. overall, there was no difference (p = 0.58) between students with ts practice compared to students without fb practice in their ability to pass an ai pipette into cow’s uterus. questionnaire feedback of the 16 gfb participants who completed the questionnaire, all said they had enjoyed playing fb for the 8 weeks. two students indicated that they were able to play fb for the required time each week and 14 students indicated they were unable to complete the required playing time each week. five of the 14 students were unable to complete the required playing time said that they did not make up the playing time during following weeks and 9 students indicated that they were able to make up playing time. main reasons for students missing fb playing time were not being on campus enough and absence during the semester break. one participant mentioned that the number of fb tables was insufficient. fifteen out of 16 respondents said they would keep playing fb for fun after completion of the project. estrus synchronization of the 25 synchronized cows, 16 were used for ai assessment. of the 16 cows used on the assessment day, 15 were in estrus and 1 in proestrus. three cows used for ai assessment had some mucosal bleeding noted after the second student palpation. these cows were examined by an experienced veterinarian before removal from examination crushes. there were 2 cows with mucosal bleeding after the first student palpation. these cows were removed from the project immediately after examination by the veterinarian and another cow was brought in for the second student assessment in each case. discussion we aimed at identifying an alternative training approach to the skills required for bovine ai. it follows a recent trend in veterinary and nonveterinary education where alternative training methods have been utilized to improve acquisition of important clinical and nonclinical skills.13,14,15 the main finding of this study was that the relationship between ts practice and veterinary students’ ability to pass an ai pipette into the uterus of a cow was not different (p > 0.05). although not significant, the proportion of successful students in gfb (0.35) was more than that in g0 (0.18). this suggested that ts practice may have had some effect on the student’s ability to pass an ai pipette into the uterus of a cow. this study relied heavily on gfb participant commitment and ability to play ts for the required amount of time each week to develop muscle memory, as this requires repeated practice of a specific motion.19,20 fourteen of the 16 gfb participants who completed the questionnaire indicated that they were unable to play fb for the required time each week, which is expected to have negatively impacted muscle memory development. nine of the 14 students were able to make up playing time when they missed a fb session; however, this inconsistency in fb playing could have also influenced muscle memory development. lecturefree weeks were the main reason to miss fb sessions amongst gfb participants. some students indicated they missed fb practice for an entire 2 weeks. the 2-week break was difficult to avoid when planning the timeline for the project. however, if all students in gfb had managed to consistently play fb for the required time each week it may have had an influence on the results. the building of motor memory or the learning of a new motor skill generally has 2 phases, the first being acquisition of the skill and the second being consolidation through practice and repetition of the skill.21 long term recall of the skill depends on proper consolidation.21 although humans have the capacity to learn and retain many motor memories, forming motor memory for a skill can be interrupted by the learning of different motor skills.22 the motor memory for a new skill is fragile immediately after practice, and if another skill is learnt shortly afterwards interference of the first skill may occur.22 it is for this reason that motor memory development may not have been optimal during this project. students are learning many different new motor skills throughout the veterinary course. students may have gone from playing fb to a surgery skills class, which may have interrupted formation of the motor memory from fb practice and may have ultimately affected results. furthermore, missing fb sessions is expected to have had an influence on the consolidation of the motor memory. transrectal palpation experience is another factor to consider. although all participants were from the same year level and 2 6 9 11 g0 gfb nu m be r of s tu de nt s passing ai pipette passed ai pipette didn’t pass ai pipette figure 2. number of successful and unsuccessful participants at passing ai pipette into cow’s uterus for gfb (6/17, 35% successful) and g0 (2/11, 18% successful) http://dx.doi.org/10.58292/ct.v15.9595 50 citation line: clinical theriogenology 2023, 15, 9595, http://dx.doi.org/10.58292/ct.v15.9595 had a similar baseline level of trp experience and training, it is not known if they had indeed basic palpation competence. since trp competence requires extensive training and live cow palpation practice,23,24 it is possible that most students taking part in this study would not have been competent due to limited live cow training opportunities.25 although the ai process does not require trp examination of the entire reproductive tract, it is essential to locate and be able to grasp the cervix. one student from gfb commented on the questionnaire that lack of trp training and competence made the live cow ai task difficult due to inability to locate the cervix and pick it up. furthermore, live cow trp can cause arm muscle fatigue in those not used to it as it requires a set of unique physical efforts.14 this may have made the ai task more difficult for students. therefore, a future approach to ai skills acquisition should ensure that participants do have the necessary trp competence as could be evaluated by an objective trp assessment.26 there may also be an emotional component to successfully perform bovine trp and ai that may have influenced the results of this study. veterinary students’ empathy levels towards animals and views on animal welfare27,28 could have caused concern among students around the amount of pressure or force that can safely be applied during trp and ai without harming cows. since this is something learned by experience and exposure to live cow training, participants’ inexperience in trp could have influenced their ability to pick up the cervix and carry out the ai task. this study was designed so that the ai assessment set-up resembled real-life as closely as possible. the cattle used for ai assessment were synchronized and in estrus as would be expected in a real-life ai setting. cattle were examined by an experienced veterinarian to ensure correct cycle stage before used for the ai assessment. by ensuring that cattle were in estrus, any extra difficulty in performing the procedure was removed. the cycle stage of the cows can therefore be excluded as a reason for students’ inability to pass the ai pipette into the uterus. one promising finding of this study came from the questionnaire in which students indicated that they had fun during the 8 weeks of fb practice. fifteen out of the 16 respondents to the questionnaire also indicated that they would continue to play fb after completion of the study. this may have implications in helping to reduce stress amongst veterinary students. it is well known that veterinary education programs in australia and overseas are challenging and demanding programs.29,30 the work load is extensive and can cause students to feel chronically overwhelmed by constant pressure.30 the stress experienced by students while studying veterinary medicine has been recognized to stem from factors like the large number of contact hours, and the pressure to pass highstakes examinations, as well as financial pressures and less time for social activities.31 literature on coping with stress indicates that employing a range of stress management strategies is best.32 foosball is something that is easy to make available at veterinary schools and may help students break up busy study schedules and reduce stress by providing a source of fun. a major limitation of this study was the small sample size and the uneven distribution of participants among the study cohorts. an ideal sample size was initially calculated based on a population size of 100 at a 95% confidence level and a confidence interval of 0.95, and would have been 80 participants in total, 40 per cohort. however, due to budget restrictions limiting the number of fb table purchases, cattle availability, and covid-19 pandemic restrictions and constraints due to student availability, student numbers per cohort were adjusted to 24. due to difficulty in recruiting students to the project, the cohorts were much smaller than initially planned for with 17 and 11 participants in gfb and g0, respectively. the difficulty recruiting students to the project may have been due to the time commitment required to take part in fb practice as well as ai training and assessment. the 4th year of veterinary education at murdoch university has a demanding class timetable and students may not have wanted to add other commitments to their schedule. timing of the project also meant that ai training and assessment occurred during the final weeks of the trimester making it quite close to their final examinations, which may have also contributed to lack of student interest in the project. students may have also lacked an interest in large animal veterinary practice, and therefore in a bovine ai project. another shortfall of the study is that tracking of playing time for gfb participants was not done. it would have added valuable data as consistency of playing fb could have been evaluated as a direct variable affecting participants’ ability to pass a pipette through the cervix or not. it could have potentially also been used to assess if participants who took longer to pass the pipette were those who played less. this kind of evidence would have strengthened the findings and should be included in any similar future investigations. conclusion although results did not support our hypothesis, there were various factors, including, small cohort sizes and incomplete gfb participant commitment to the required fb playing time that may have influenced the results unfavorably. therefore, this result should not discourage veterinary educators from further investigating alternative teaching approaches to help improve the teaching and learning of important clinical skills at veterinary schools. furthermore, this study highlights the importance of assessing competence for clinical skills that are required to learn new skills first (trp in this case) before attempting to teach the new, more advanced skill (ai in this case). this is likely to be applicable for many other clinical skills. acknowledgement authors thank the staff at the murdoch university veterinary farm for providing the cattle and facilities required to make this study possible. that gratitude also extends to the staff at the production animal hospital at murdoch university for their assistance in acquiring equipment and consumables, and the murdoch veterinary students association for providing the space for students to play fb. conflict of interest authors have no conflict of interest to report. authors’ contributions mia norton: conceptualization, project execution and data collection, writing, review and editing. http://dx.doi.org/10.58292/ct.v15.9595 citation line: clinical theriogenology 2023, 15, 9595, http://dx.doi.org/10.58292/ct.v15.9595 51 annett annandale: conceptualization, project execution and data collection, review and editing. references 1. dalton jc, robinson jq, price wj, et al: artificial insemination of cattle: description and assessment of a training program for veterinary students. j dairy sci 2021;104:6295–6303. doi: 10.3168/ jds.2020-19655 2. vishwanath r: artificial insemination: the state of the art. theriogenology 2003;59:571–584. doi: 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https://doi.org/10.1016/j.tins.2005.10.003 https://doi.org/10.1016/j.amjsurg.2016.10.018 https://doi.org/10.1016/j.amjsurg.2016.10.018 https://doi.org/10.1038/nn0907-1085 https://doi.org/10.1111/sms.12791 https://doi.org/10.1371/journal.pone.0017451 https://doi.org/10.3138/jvme.36.4.451 https://doi.org/10.3138/jvme.1116-172r https://doi.org/10.1111/j.1439-0531.2006.00705.x https://doi.org/10.1136/vr.105022 https://doi.org/10.1136/vr.146.10.269 https://doi.org/10.3390/vetsci6010019 https://doi.org/10.3138/jvme.32.2.213 https://doi.org/10.3138/jvme.32.2.170 https://doi.org/10.1111/avj.13070 https://doi.org/10.3138/jvme.32.2.201 evolution of fertility programs for lactating dairy cows evolution of fertility programs for lactating dairy cows paul fricke department of dairy science, university of wisconsin, madison, wi abstract this paper reviews the key concepts that lead to the development of fertility programs for highproducing lactating dairy cows using gonadotropin releasing hormone (gnrh) and prostaglandin f2α (pgf2α). the key factor affecting fertility to fixed time artificial insemination (ftai) is the response to each hormonal treatment of the ovsynch protocol. although not required for fertility, cows ovulating to the first gnrh treatment of the ovsynch protocol (g1) have greater pregnancies per artificial insemination (p/ai) than cows failing to ovulate. the use of presynchronization treatments that increase the percentage of cows initiating the ovsynch protocol during early diestrus increases the percentage of cows with medium progesterone (p4) concentrations at g1 and with high p4 concentration at pgf2α. however, some cows fail to completely regress the corpus luteum (cl) after a single pgf2α treatment, particularly cows that initiate the ovsynch protocol in a low p4 environment and cows with a young cl at the time of treatment with pgf2α. addition of a second pgf2α treatment increased the percentage of cows with complete luteal regression and p/ai. the use of fertility programs that include the concepts described in this review have resulted in more p/ai than inseminating cows after an induced estrus at first insemination. in addition, extending these concepts to resynch inseminations and the implementation of an aggressive reproductive management program for first and subsequent inseminations results in reproductive performance that is unprecedented for high-producing holstein dairy cows. keywords: fertility programs, reproduction, dairy cow introduction synchronization protocols have been incorporated widely into reproductive management programs by most dairy farms in us.1,2 at first glance, it may seem that the newly released reproductive management strategies for dairy cows protocol published by the dairy cattle reproduction council (dcrc) offers many options. in reality, reproductive management strategies have generally consolidated into a few management options depending on the extent to which farms want to use artificial insemination (ai) to a detected estrus versus fixed time artificial insemination (ftai). it is important to clarify that there is not one “right way” to approach reproductive management on all dairy farms. many strategies can be implemented to achieve excellent 21 day pregnancy rates by increasing the ai service rate alone.3 newer fertility programs increase both service rate as well as pregnancies per artificial insemination (p/ai).4 each individual farm must implement a plan to submit cows for first ai and to identify nonpregnant cows and return them to ai service to maximize their 21 day pregnancy rate. dairy farmers, dairy veterinarians, and dairy consultants are continually challenged to stay current on the latest recommendations for synchronization protocols. an excellent and up-to-date source of information on synchronization protocols can be found at dcrc web site: http://www.dcrcouncil.org/. protocols recommended by the dcrc are reviewed and updated by researchers who develop and test these protocols, and are based on the latest peer-reviewed research published in scientific literature. purpose of this paper is to overview the key research underlying development of fertility programs for lactating dairy cows. detection of estrus followed by fixed time artificial insemination artificial insemination to a detected estrus continues to play an important role in the overall reproductive management program on almost all dairy farms.1 use of detection of estrus alone for submitting lactating dairy cows for first ai, however, generally results in poor reproductive performance because of 2 broad limitations associated with detection of estrus. first limitation is with the human element (i.e. visual observation of estrus) in which dairy personnel must visually observe estrous behavior. many technologies were developed and introduced throughout the years to help overcome 317 clinical theriogenology • volume 11, number 3 • september 2019 problems with the human element of detection of estrus. these technologies include pressure-activated heat mount devices and androgenized females,5 tail chalking, pedometry,6 and radiotelemetry.7,8 dogs have even been trained to detect estrus related odors in dairy cows.9 more recently, activity monitoring systems that use accelerometer technology to detect increased physical activity associated with behavioral estrus have been widely adopted by dairy farms. a second limitation of detection of estrus pertains to the biology of the high producing dairy cow. cow related biological factors that limit detection of estrus include the effect of high milk production on the duration of estrus,10 ovulation failure after expression of estrus and ovulation without accompanying estrous behavior,11,12 and anovular conditions in dairy cows.13 taken together, these human and cow related issues substantially limit ai service rates and 21 day pregnancy rates in dairy herds that rely on detection of estrus alone for submitting cows for ai. a long-standing goal of reproductive biologists was to develop a hormonal synchronization protocol that would allow for ftai, thereby increasing the ai service rate. this goal was realized in 1995 with publication of the ovsynch protocol, a synchronization protocol in which 3 sequential hormonal treatments are used to control ovarian function.14 in the first field trial that evaluated the ovsynch protocol for reproductive management,15 lactating dairy cows managed using only ftai without detection of estrus had fewer median days to first ai (54 versus 83) and fewer days open (99 versus 118) than cows inseminated to estrus, whereas p/ai to first ai was similar (37 versus 39% for ftai versus estrus, respectively) even though cows managed using ftai were inseminated earlier postpartum. to deal with cows failing to be detected in estrus, some farms submit cows for first ai from the end of the voluntary waiting period to 80 days in milk based on a detected estrus, followed by submission of cows failing to be detected in estrus to an ovsynch protocol and ftai. because of human and cow related limitations for detection of estrus, all farms can increase reproductive performance by combining detection of estrus with use of ovsynch and ftai for cows failing to be detected in estrus. presynchronization methods used before fixed time artificial insemination presynchronization strategies were initially developed when it was reported that initiation of an ovsynch protocol between days 5 12 of the estrous cycle resulted in more p/ai than initiation of the protocol earlier or later during the estrous cycle.16-18 there are 2 broad categories of presynchronization strategies: 1) presynchronization using prostaglandin f2α (pgf2α) and 2) presynchronization that combines gonadotropin releasing hormone (gnrh) and pgf2α. presynchronization using prostaglandin f2α first presynchronization strategy tested used 2 pgf2α treatments administered 14 days apart, with the second pgf2α treatment preceding the first gnrh treatment of an ovsynch protocol by 12 days (i.e. presynch-ovsynch).19 when only cycling cows were included in the statistical analysis, p/ai to ftai increased from 29% for cows submitted to an ovsynch protocol to 43% for cows submitted to a presynch-ovsynch protocol. two things need to be clarified regarding this presynchronization strategy. first, the authors never intended that cows be inseminated to estrus during the protocol, as is now commonly practiced. in fact, a recent meta-analysis of 3 randomized controlled studies including 1,689 cows concluded that inseminating cows that show estrus after the second pgf2α treatment of a presynchovsynch protocol decreased p/ai compared to when all cows were allowed to complete the protocol and receive ftai.20 this decrease in p/ai occurs because cycling cows that are presynchronized so that the ovsynch protocol is initiated at an optimal stage of the estrous cycle are removed from the ftai protocol, thereby negating the presynchronization effect. second, the 2 pgf2α treatments preceding the ovsynch protocol were never intended to “clean the uterus”, although this effect could certainly be beneficial. an updated meta-analysis on the effect of pgf2α therapy on bovine endometritis that included 9 experiments in 8 eligible studies and a total of 5,563 cows concluded that a positive effect on reproductive outcomes could not be shown.21 indeed, administration of either 1 or 2 pgf2α treatments before initiation of a double-ovsynch protocol had no effect on uterine health, p/ai, or maintenance of pregnancy in lactating holstein cows.22 318clinical theriogenology • volume 11, number 3 • september 2019 even though the presynch-ovsynch protocol was originally developed to increase p/ai of cows submitted to ftai, many farms inseminate cows to a detected estrus after the second pgf2α treatment of a presynch-ovsynch protocol, a practice commonly referred to as “cherry picking heats”, followed by submission of cows not detected in estrus to an ovsynch protocol. decreasing the interval between the second pgf2α treatment of presynch to initiation of the ovsynch protocol from 14 -11 days, however, increased ovulatory response to the first gnrh treatment and increased p/ai by ~ 7 percentage points when all cows were submitted to ftai.23 thus, if a presynch-ovsynch protocol is used for 100% ftai for first service, a shorter interval (i.e.10 12 days) between the second pgf2α treatment and initiation of the ovsynch protocol is better. when cows were inseminated to estrus after second pgf2α treatment of a presynch-ovsynch protocol, no difference in p/ai was reported when a 12 day versus a 14 day interval was compared,24 supporting the idea that inseminating cows to estrus during a presynch-ovsynch protocol negates the effect of presynchronization.25 further, anovular cows submitted to a presynchovsynch protocol have fewer p/ai than their cycling herd mates. because anovular cows lack a corpus luteum (cl) and therefore do not respond to the first 2 pgf2α treatments of a presynch-ovsynch protocol, ovsynch protocol is initiated in a low progesterone (p4) environment, resulting in fewer p/ai to ftai.4 because anovular cows represent 20 30% of cows submitted for first ftai,26,27presynchronization strategies using pgf2α alone, with or without inclusion of detection of estrus, do not yield high p/ai to ftai. presynchronization that combines gonadotropin releasing hormone and prostaglandin f2α two limitations of a presynchronization strategy that uses pgf2α alone are that: 1) pgf2α does not affect anovular cows or resolve the anovular condition before initiation of the ovsynch protocol, and 2) follicular growth is not tightly synchronized after 2 sequential pgf2α treatments administered 14 days apart. newer presynchronization strategies that combine gnrh and pgf2α overcome both of these limitations, thereby increasing p/ai to ftai. cows that were presynchronized using an ovsynch protocol (i.e. a double-ovsynch protocol) had more p/ai than cows submitted to a presynch-ovsynch protocol (50 versus 42%).28 in a subsequent study, there was a treatment by parity interaction in which the doubleovsynch protocol increased p/ai for primiparous, but not multiparous cows.29 we now know this parity effect is due to incomplete luteal regression, particularly for multiparous cows.30 presynchronization strategies used a combination of gnrh and pgf2α 6 7 days before ovsynch protocol (i.e. gnrh 6 days before and pgf2α 3 days before gnrh).31,32 presynchronization using a pgf2α 3 days before gnrh protocol yielded more p/ai than inseminating cows at estrus during cooler weather and was superior to a presynch-ovsynch 10 protocol during the summer.32 inclusion of gnrh into a presynchronization strategy increases p/ai to ftai by resolving the anovular condition before initiation of the ovsynch protocol, by more tightly controlling follicular development and luteal regression, and by presynchronizing cows so that the ovsynch protocol is initiated on either days 6 or 7 of the estrous cycle in a high proportion of cows, thereby optimizing the response of cows to each sequential treatment of the ovsynch protocol.4,33 synchronization methods for fixed time artificial insemination over time, there have been several variations of timing of treatments during an ovsynch protocol that were compared and used on dairy farms. for the purposes of this discussion, the first gnrh treatment of the ovsynch protocol will be referred to as g1, and the last gnrh treatment will be referred to as g2. several experiments have compared various timings of the treatments within the ovsynch protocol, as well as timing of ai relative to the last gnrh treatment of the protocol. these variations can lead to differences in p/ai, and a review of several key studies can help farms to determine which of the 4 variations may work best for a given situation. in the first published experiment using ovsynch,14 lactating cows were submitted to ftai ~ 24 hours after the last gnrh treatment of the protocol. all cows (n = 20) ovulated to the last gnrh treatment of the ovsynch protocol within 24 32 hours which is similar to the interval from the first standing event of estrus to ovulation of 27.6 hours.7 thus, from a physiologic perspective, timing of 319 clinical theriogenology • volume 11, number 3 • september 2019 ovulation is similar when comparing the interval from the first standing event of estrus or the last gnrh treatment of an ovsynch protocol to ovulation. to assess the effect of timing of ai relative to a synchronized ovulation, lactating dairy cows (n = 732) were randomly assigned to 5 treatments by stage of lactation and parity.34 ovulation was synchronized using an ovsynch 48 protocol, and ai varied from hours 0, 8, 16, 24, or 32 hours relative to g2. in this study, the 24-hour treatment is equivalent to the ovsynch 48 protocol. overall, cows in hours 0, 8, 16, and 24 treatments had more p/ai than cows in the 32 hours treatment (table 1). thus, although no statistical difference in fertility was detected when ai occurred from 0 24 hours after the last gnrh treatment of the ovsynch protocol, inseminating too late (i.e. at 32 hours) resulted in fewer p/ai.34 although this study included more than 700 cows, the number of experimental units in each treatment was less than 150 cows, thereby decreasing the statistical power necessary to detect differences among these treatments that may be physiologically relevant. to further evaluate timing of ai relative to g2, a field trial was conducted to compare 2 variations of a cosynch protocol (i.e. cosynch 48 and cosynch 72 compared in 2 earlier experiments,35,36 in which ftai occurred concomitant to g2, with a variation of the ovsynch protocol in which ai occurred 16 hours after g2.37 this third treatment is now referred to as an ovsynch 56 protocol. timing of ai in an ovsynch 56 protocol is supported by data in table 1, in which the 16 hour interval from the last gnrh treatment to ftai resulted in numerically (but not statistically) greater fertility than other treatments, as well as data reporting that optimal fertility should occur when cows are inseminated ~ 15 24 hours before ovulation.7,8 because timing of ovulation is similar when comparing the interval to ovulation from the first standing event of estrus or g2, timing of ai based on a cosynch protocol will not optimize timing of ai relative to an induced ovulation. table 1. effect of timing of ai relative to the last gnrh treatment of an ovsynch 48 protocol on pregnancies per artificial insemination (p/ai) in lactating holstein cows.a hours from second gnrh injection of ovsynch to ftai item 0 8 16 24 32 total n 149 148 149 143 143 732 p/ai (%) 37 41 45 41 32b 39 aadapted from pursley et al.34 bdiffers from other treatments within a row (p < 0.10). most farms using an ovsynch 56 protocol administer g1, the pgf2α treatment, and ftai in the morning, whereas g2 is administered in the afternoon to achieve a 56 hour interval from pgf2α treatment to the last gnrh treatment of the ovsynch protocol and a 16 hour interval from the last gnrh treatment to ftai. despite data in table 2 supporting that an ovsynch 56 protocol yields more p/ai, it is difficult for some farms to implement this timing of treatments, due to the inconvenience or inability to handle cows in the afternoon. most of these farms prefer the timing of the ovsynch 48 protocol or a cosynch 72 protocol. thus, these ovsynch variations are based on ease of implementation on farms rather than biology. because of the extended interval between the last gnrh treatment of the ovsynch protocol and ftai in a cosynch 72 protocol, many cows will display estrus more than 12 hours before scheduled ftai, thereby decreasing fertility to ftai.37 detection of estrus and ai from the pgf2α treatment to the last gnrh treatment of a cosynch 72 protocol can help to mitigate the decreased fertility to ftai when using this protocol variation. table 2. effect of treatment on pregnancies per artificial insemination (p/ai) and pregnancy loss in lactating holstein cows.a item cosynch 48 ovsynch 56 cosynch 72 p/ai 31-33 d, % (n) 27 (494) 36 (457) 27 (517) least squares estimate 29b 39c 25b p/ai 52-54 d, % (n) 25 (493) 33 (450) 25 (513) least squares estimate 27b 36c 23b pregnancy loss, % (n) 5 (131) 5 (158) 7 (137) aadapted from brusveen et al.37 b,cproportions with different superscripts differ (p < 0.05). 320clinical theriogenology • volume 11, number 3 • september 2019 the last option is a 5 day cosynch protocol in which the interval between g2 and the pgf2α treatment is decreased from 7 (7 day protocol) to 5 (5 day protocol). the 5 day cosynch protocol was first reported in a series of experiments in beef cows.38 although timing of ai after the pgf2α treatment differed between cows in 7 day versus 5 day protocol, cows submitted to the 5 day protocol has more p/ai than cows submitted to 7 day protocol in 2 experiments (80 versus 67%, respectively and 65 versus 56%, respectively). in 2010, the 5 day ovsynch protocol was compared to a 7 day cosynch 72 protocol in lactating holstein cows.39 in that study, cows submitted to the 5 day protocol received 2 pgf2α treatments, whereas cows submitted to the 7 day protocol received a single pgf2α treatment. overall, cows in the 5 day protocol had more p/ai than cows in the 7 day protocol (38 versus 31%). the authors conducted an analysis to control for a difference in luteal regression rates between cows receiving 1 versus 2 pgf2α treatments by analyzing only cows with p4 < 1 ng/ml on the day of ai, and cows submitted to the 5 day protocol has more p/ai than cows submitted to the 7 day protocol (39% versus 34%). the authors attributed this treatment effect to the decreased period of follicle dominance for cows in the 5 day cosynch protocol. colazo and ambrose40 also compared a 5 day cosynch protocol with 2 pgf2α treatments to a 7 day ovsynch protocol with 1 pgf2α treatment; however, p/ai did not differ between treatments in that study (39 versus 34%). a recent experiment directly tested the effect of addition of a second pgf2α treatment and the effect of decreasing the duration of the ovsynch protocol from 7 to 5 days in a resynch protocol.41 lactating holstein cows (n = 821) were randomly assigned at a nonpregnancy diagnosis (d 0 = 32 d after ai) to 1 of 3 resynch protocols: 1) 7d1pgf (gnrh, d 0; pgf2α, d 7; gnrh, d 9.5); 2) 7d2pgf (gnrh, d 0; pgf2α, d 7; pgf2α, d 8; gnrh, d 9.5); and 3) 5d2pgf (gnrh, d 2; pgf2α, d 7; pgf2α, d 8; gnrh, d 9.5). all cows received an intravaginal p4 insert (prid delta; ceva santé animale, libourne, france) at g1, which was removed at the first pgf2α treatment, and all cows received ai ~16 hours after g2. overall, there was no effect of treatment on p/ai (table 3). when these data were analyzed based on the presence or absence of a cl at g1, cows lacking a cl and receiving 2 pgf2α treatments had more (p = 0.03) p/ai than cows receiving 1 pgf2α treatment, regardless of protocol duration (i.e. 5 versus 7 days), whereas there was no effect of treatment for cows that had a cl at g1 (table 3). we concluded that addition of a second pgf2α treatment to a resynch protocol increased the proportion of cows undergoing complete luteal regression, thereby increasing p/ai, particularly for cows that have low p4 at g1, whereas decreasing the duration of the ovsynch protocol did not affect p/ai. nonetheless, the 5 day cosynch protocol is a good option for dairy farms that want to administer all protocol treatments and ai in the morning, thereby simplifying implementation of this protocol. table 3. effect of presence of a corpus luteum (cl) at day 0 on pregnancies per ai (p/ai) in holstein dairy cows 32 days after ftai.a treatment p valueb p/ai 7d1pgf 7d2pgf 5d2pgf t c1 c2 -------------------% (n) ------------------- overall 36 (266) 41 (268) 44 (265) 0.14 0.05 0.56 cows with a cl at g1 38 (196) 40 (191) 43 (189) 0.51 0.35 0.49 cows lacking a cl at g1 30 (70) 46 (77) 45 (76) 0.11 0.03 0.98 aadapted from santos et al.41 bc1: preplanned contrast between 7d1pgf (one pgf2α) and 7d2pgf + 5d2pgf (two pgf2α) treatments; c2: preplanned contrast between 7d2pgf (7 day protocol) and 5d2pgf (5 day protocol) treatments. inclusion of a second prostaglandin f2α treatment 24 hours after the first in ovsynch protocols a major modification to ovsynch protocols is the recommendation to include a second pgf2α treatment 24 hours after the first in the 7 day ovsynch protocol. inclusion of a second pgf2α treatment is absolutely necessary for 5 day cosynch protocol due a younger cl at pgf2α treatment that fail to regress after a single pgf2α treatment.42,43 addition of a second pgf2α treatment is highly recommended for all of the 7 day protocols, particularly when used for first ftai after a presynchronization strategy that incorporates both gnrh and pgf2α. lack of complete luteal regression, particularly for multiparous cows which is addressed by the addition of the second pgf2α treatment, was in fact the rate-limiting factor for 321 clinical theriogenology • volume 11, number 3 • september 2019 fertility to ftai.30 indeed, submission of lactating holstein cows to a double-ovsynch protocol and ftai for first insemination increased percentage of cows inseminated within 7 days after the end of the voluntary waiting period and increased p/ai at 33 and 63 days after first insemination, resulting in 64 and 58% more pregnant cows, respectively, than submission of cows for first ai after detection of estrus at a similar day in milk range.44 several experiments have been conducted to assess addition of a second pgf2α treatment on luteal regression and p/ai.30, 45-47 a recent meta-analysis of data from these experiments was conducted with the primary objective to evaluate the effect of an additional pgf2α treatment during the ovsynch protocol on luteal regression and p/ai.48 the meta analysis included 7 randomized controlled experiments from 6 published manuscripts including 5,356 cows and data regarding luteal regression at the end of the ovsynch protocol were available for 1,856 cows. including a second pgf2α treatment 24 hours after the first during the ovsynch protocol increased the relative risk (rr) of complete luteal regression at the end of the ovsynch protocol (rr = 1.14; 95% confidence interval = 1.10 to 1.17) using a fixed effects model and the rr for pregnancy (rr = 1.14; 95% confidence interval = 1.06 to 1.22) 32 days after ftai using a fixed effects model. no heterogeneity was observed among the 6 manuscripts regarding complete luteal regression and p/ai. the authors concluded that there was a clear benefit of including an additional pgf2α treatment during the ovsynch protocol on luteal regression (+11.6 percentage units) and on p/ai (+ 4.6 percentage units). inclusion of a second pgf2α treatment in 7 day ovsynch protocols is now recommended to increase fertility to ftai. although addition of a second pgf2α treatment to ovsynch protocols dramatically increases luteolysis and p/ai, it also increases the number of times cows have to be handled. a common question is whether increasing the dose of pgf2α at a single time can achieve a similar rate of luteolysis and/or p/ai as including a second pgf2α treatment. two prostaglandin products are available and approved for use in dairy cows in the us: dinoprost (i.e. native pgf2α) and cloprostenol (a pgf2α analog). doubling the dose of dinoprost from 25 to 50 mg does not appear to perform as well as two 25 mg dinoprost treatments administered 24 hours apart for first47 or resynch46 ftai. increasing the dose of cloprostenol from 500 to 750 µg increased the rate of luteal regression primarily in multiparous cows but tended to increase fertility (p = 0.05) only at the pregnancy diagnosis 39 days after ftai.49 finally, delaying a single dinoprost treatment by 24 h (i.e. from day 7 to day 8 of the protocol) without adjusting g2 and ai decreased luteal regression and p/ai.50 because of the complexity of much of the data generated thus far, more studies are needed to definitively answer this question using both prostaglandin products. at the present time, the new dcrc recommendation of adding a second pgf2α treatment 24 hour after the first to both 7 day and 5 day ovsynch protocols should be followed. resynchronization programs there are now 2 major options for resynchronization programs, based on timing of nonpregnancy diagnosis and initiation of the resynch protocol. although both options include detection of estrus and ai after an initial ai, some farms choose to minimize use of ai to estrus and submit nearly all cows to ftai. in this management scenario, the ai service rate is fixed based on the interval between inseminations, which is set by the timing of pregnancy diagnosis and the primary emphasis is focused on compliance to the protocols, a key element to their success. nonetheless, including detection of estrus after an initial ai can increase 21 day pregnancy rates by increasing the ai service rate. farm managers should keep in mind that they must manage t2 reproductive management systems in this scenario; one for the ftai protoco, and the other for the daily chore of detection of estrus and ai. nonetheless, most of the dcrc award-winning dairy herds in 2017, which all had annualized 21 day pregnancy rates between 30 and 40%, submitted all cows to ftai after a fertility program, inseminated any cows detected in estrus after first ftai and then submitted cows not detected in estrus and diagnosed not pregnant to a resynch protocol. 322clinical theriogenology • volume 11, number 3 • september 2019 return to estrus after ai accurate detection of cows failing to conceive to ai and returning to estrus from 18 32 days after ai is the earliest method for identifying and re-inseminating cows failing to conceive after ai. there are, however, several challenges for detection of estrus after ai. first, only, 52% of the eligible cows were detected in estrus and re-inseminated between ai and pregnancy diagnosis when detection of estrus was performed through continuous monitoring of activity after a previous ai until pregnancy diagnosis 32 days after ai51 second, estrous cycle duration varies widely with a high degree of variability among individual cows.52 finally, the high rate of early pregnancy losses in dairy cows increases the interval from insemination to return to estrus for cows that establish pregnancy, then undergo pregnancy loss.53 because of these issues with nonpregnant cows returning to estrus, implementation of a resynch strategy is critical for achieving high 21 day pregnancy rates. timing of pregnancy diagnosis and initiation of resynch in the first strategy for resynch, nonpregnancy diagnosis is conducted before initiation of the resynch protocol, whereas in the second strategy, the first gnrh treatment of a resynch protocol is initiated 7 days before nonpregnancy diagnosis. choosing between these 2 resynch variations depends on the reproductive management goals of the dairy farm. advantage of delaying g1 until pregnancy diagnosis is that more time is allowed for cows to show estrus for submission to ai, thereby decreasing the total number of cows submitted to a resynch protocol.54 for herds focused on detecting cows in estrus and minimizing cows submitted to ftai, this is a good option. the disadvantage of this approach is that the resynch protocol is delayed by 1 week, due to the need to identify nonpregnant cows before g1. the obvious disadvantage of administering g1 before pregnancy diagnosis is that all cows are treated with gnrh, regardless of their pregnancy status which is unknown at the time of treatment. herds that have excellent detection of estrus after an ai have a high proportion of cows diagnosed pregnant at the herd check, and these cows are unnecessarily treated with gnrh. by contrast, 1 advantage of administering g1 before pregnancy diagnosis is that ftai occurs 1 week earlier. overall, p/ai did not differ between cows submitted to a resynch protocol 32 or 39 days after ai,55 so the earlier resynch protocol decreases the interval between ftai services, thereby increasing the ai service rate. a second advantage of administering g1 before pregnancy diagnosis is that management decisions can be made based on the presence or absence of a cl at the pgf2α treatment of the ovsynch protocol (refer next section). presence or absence of a cl at initiation of the ovsynch protocol and fertility to resynch based on p4 profiles at each treatment during the ovsynch protocol, the best indicator of poor fertility to ftai is low p4 (i.e. cows lacking a cl) at the pgf2α treatment of the ovsynch protocol.4 one of the first strategies to increase p/ai to a resynch protocol attempted to determine the optimal interval after an initial ai to initiate g1, based on the physiology of the estrous cycle.56 assuming an estrous cycle duration of 21 23 days, administering g1 32 d after ai should correspond to initiating the resynch protocol around day 6 to 14 of the estrous cycle, a stage of the estrous cycle when a dominant follicle and a cl with midlevel p4 concentrations should be present. cows identified not pregnant 32 days after ai with a cl at g1 have more p/ai than cows without a cl.55,57 in several studies however, 16, 22 and 35% of cows diagnosed not pregnant 32 days after ai and that were not presynchronized with gnrh 7 days before pregnancy diagnosis lacked a cl at g1.51,56 when cows were synchronized for first ai and p4 profiles and cl diameter was measured until a pregnancy diagnosis 32 days later, 19% of cows diagnosed not pregnant lacked a cl > 10 mm in diameter.53 thus, resynch protocols are initiated in a low p4 environment in up to one-third of nonpregnant cows, which leads to a lack of complete luteal regression after treatment with pgf2α 7 days later, resulting in fewer p/ai. inclusion of a second pgf2α treatment 24 hours after the first into a resynch protocol, increases p/ai for cows initiating resynch in a low p4 environment.58 one strategy to treat nonpregnant cows without a cl at g1 is to supplement with exogenous p4 during the resynch protocol. cows without a cl at g1 and treated with a cidr insert for 7 days had more p/ai at first as well as resynch ai59,60,61 many veterinarians now use the presence or absence of a 323 clinical theriogenology • volume 11, number 3 • september 2019 cl at a nonpregnancy diagnosis to implement a strategy to increase fertility to resynch protocols or to increase the proportion of cows inseminated to a detected estrus after ai. based on this idea, a recent study assigned cows diagnosed not pregnant to various resynch strategies, based on ovarian structures62 the control treatment was a standard resynch protocol in which g1 was administered 32 days after ai and including a single pgf2α treatment. alternatively, cows diagnosed not pregnant 32 days after ai were assigned to a resynch strategy based on the presence or absence of a cl > 15 mm in diameter. nonpregnant cows with a cl received 2 pgf2α treatments 24 hours apart, followed by gnrh and ai (i.e. a resynch protocol without g1), whereas nonpregnant cows without a cl were submitted to a resynch protocol that included a second pgf2α treatment and a cidr insert. it is important to note that cows were detected in estrus and inseminated from initial ai to initiation of each of the 3 resynch treatments. the authors concluded that the shorter resynch program decreased time to pregnancy because of a decrease in the interval between ai services for nonpregnant cows with a cl and more p/ai in nonpregnant cows lacking a cl.62 this resynch strategy is a good option for herds that combine detection of estrus after first ai with a resynch strategy. herds that do not incorporate detection of estrus after an initial ai can implement a resynch strategy based on ovarian structures, as described.4 in this strategy, all cows are treated with gnrh 25 days after ai. pregnancy diagnosis is conducted using transrectal ultrasonography 32 days after ai and cows diagnosed not pregnant are classified as having or lacking a cl. nonpregnant cows with a cl continue an ovsynch 56 protocol by receiving a pgf2α treatment 32 days after ai, with the addition of a second pgf2α treatment 24 hours after the first. nonpregnant cows lacking a cl restart an ovsynch 56 protocol that includes a second pgf2α treatment 24 hours after the first (i.e. ggppg), as described.58 intravaginal p4 inserts (one cidr per cow) are included within the ovsynch protocol for cows without a cl, based on studies in which treatment with exogenous p4 increased p/ai for cows lacking a cl at initiation of an ovsynch protocol to that of cows with a cl at initiation of an ovsynch protocol.60,61 low progesterone, double ovulations, and twinning: a new problem with protocols low p4 during growth of an ovulatory follicle is associated an increased incidence of double ovulation.63 cows in which the preovulatory follicle develops in the absence of p4 from a cl have a greater incidence of codominant follicles resulting in double ovulations.64,65 all dairy cows experience a low p4 environment during the postpartum anovular period from calving to first ovulation. double ovulation rate after a spontaneous estrus was greater for anovular cows (i.e. low p4) than for cycling cows.66 incidence of double ovulation to g1 was greater for anovular than for ovular cows; however, incidence of double ovulation to g2 was similar between ovular and anovular cows.67 thus, the first postpartum ovulation results in a high double ovulation rate due to the lack of p4 during growth of the preovulatory follicle, whereas the first exposure to p4 during the postpartum anovular period decreases the incidence of double ovulation. to test the effect of p4 during growth of the ovulatory follicle on the incidence of double ovulation, holstein cows were randomly assigned to 2 presynchronization protocols that manipulated cows into either a high or a low p4 environment during an ovsynch protocol68 (table 4). cows in the high p4 treatment were submitted to a double-ovsynch protocol28 and had more p4 at the first gnrh treatment of the ovsynch protocol and at the pgf2α treatment of the ovsynch protocol than cows in the low p4 treatment. ovulatory response to the last gnrh treatment of the ovsynch protocol was similar between treatments; however, cows in the low p4 treatment, had more double ovulations than cows in the high p4 treatment. furthermore, fertility was greater and pregnancy loss was less for cows in the high versus the low p4 treatment. thus, cows with high p4 during growth of the ovulatory follicle had fewer double ovulations, more p/ai and fewer pregnancy losses than cows with low p4. 324clinical theriogenology • volume 11, number 3 • september 2019 table 4. effect of progesterone (p4) during growth of the preovulatory follicle on incidence of double ovulation in holstein dairy cows.a item low p4 (n = 259) high p4 (n = 255) p value p4 at 1st gnrh (ng/ml) 0.28 1.84 p4 at pgf2α (ng/ml) 2.23 4.40 ovulation to g2 (%) 95 95 ns double ovulation (%) 21 7 < 0.05 p/ai day 29 (%) 33 48 < 0.01 pregnancy loss 29 57 days (%) 16 4 < 0.05 aadapted from cunha et al.68 it is important to note that the study by cunha et al.68 was conducted before the second pgf2α treatment was included in the ovsynch protocol. therefore, we must now interpret these data based on a current understating of the physiology associated with these protocols in which a lack of complete luteal regression decreases p/ai. thus, in this study,68 cows in the low p4 treatment had high double ovulation rates, but low conception rates due to incomplete luteal regression. for cows that initiate an ovsynch protocol in a low-p4 environment, if you eliminated the luteal regression problem by adding a second pgf2α treatment, p/ai could increase dramatically due to increased double ovulations69 followed by increased pregnancy losses for cows that conceive unilateral twins,70 followed by an increase in twins for cows that maintain the twin pregnancy. thus, a new problem has arisen concurrent with the recommendation to add the second pgf2α treatment to ovsynch protocols, particularly when cows initiate the protocol in a low p4 environment. to further evaluate the effect of manipulating p4 before ai, lactating holstein cows (n = 80) were synchronized for first ai using a double-ovsynch protocol that included a second pgf2α treatment 24 hours after the first and were randomly assigned to receive 25 mg pgf2α 1 day after the first gnrh treatment of the breeding ovsynch protocol that included a used cidr insert (low p4), or to receive 2 new cidr inserts during the breeding ovsynch protocol (high p4). results of this experiment are shown in table 5.71 incidence of double ovulation was 3-fold greater for low p4 than for high p4 cows. overall, p/ai at 32 days did not differ between treatments; however, low p4 cows had more twin pregnancies than high p4 cows. we concluded that low p4 concentrations before ai increased incidence of double ovulations and twin pregnancies. data in table 5 agreed with a larger study in which cows were manipulated into high versus low p4 environments during growth of the ovulatory follicle.72 in that study, cows that were maintained in a low p4 environment during growth of the ovulatory follicle had a double ovulation rate of 49%, p/ai of 66.4% and pregnancy loss from 23% to calving of 33%.72 table 5. effect of progesterone (p4) during growth of the preovulatory follicle on follicle size, incidence of double ovulation, pregnancies per artificial insemination (p/ai), and twin pregnancies in holstein dairy cows.a item low p4 (n = 40) high p4 (n = 40) p value follicle size at g2 (mm) 16.4 ± 5 14.8 ± 0.3 < 0.01 double ovulations (%) 33 10 < 0.01 p/ai at 32 days (%) 45 53 0.97 twins at 32 days (%) 0 29 < 0.01 acarvalho et al.71 to summarize, the problem with the increased risk of double ovulation and twinning occurs when cows are submitted to an ovsynch protocol that includes a second pgf2α treatment and initiate the protocol in a low p4 environment. this scenario also leads to increased pregnancy losses due to bilateral twins72 and may explain a significant proportion of pregnancy losses that occur in dairy herds. there are 2 primary management scenarios under which this scenario arises. the first scenario is when herds that use a presynch-ovsynch protocol for first ai include detection of estrus after the second pgf2α treatment of the protocol. when an activity-monitoring system was used, ~ 70% of cows were inseminated to increased activity after the second pgf2α treatment of a presynch-ovsynch protocol and ~ half of the 325 clinical theriogenology • volume 11, number 3 • september 2019 cows not detected with increased activity had low p4 at the first gnrh treatment of the ovsynch protocol.25 this scenario can be avoided by using a presynchronization strategy that combines both gnrh and pgf2α, as these presynchronization strategies result in a high proportion of cows to have a cl at g1. a second scenario arises when herds submit cows without a cl either knowingly or unknowingly to a resynch protocol that includes a second pgf2α treatment. this scenario can be avoided by submitting cows to the resynch protocol based on ovarian structures with the nonpregnant cows lacking a cl treated with a cidr insert, which should increase p4 during the protocol and decrease double ovulation rate. conclusion development and optimization of fertility programs for first and resynch ftai remains an active area of research that has advanced dramatically over the past 20 years and will most certainly change in the future. it takes time for researchers to sift and winnow ideas and data to reach a consensus on protocols to recommend for use on commercial dairy farms and scientific progress has potential to change longstanding recommendations. an excellent and up to date source of information on synchronization protocols is on the dcrc web site: http://www.dcrcouncil.org/. conflict of interest there are no conflicts of interest to declare. references 1. caraviello dz, weigel ka, fricke pm, et al: survey of management practices on reproductive performance of dairy cattle on large us commercial farms. j dairy sci 2006:89:4723-4735. 2. norman hd, wright jr, hubbard sm, et al: reproductive status of holstein and jersey cows in the united states. j dairy sci 2009;92:3517-3528. 3. ferguson jd, skidmore a: reproductive performance in a select sample of dairy herds. j dairy sci 2013;96: 4. 1269-1289. 5. carvalho pd, santos vg, giordano jo, et al: development of fertility programs to achieve high 21-day pregnancy 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of artificial insemination on pregnancy rates, calving rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows. j dairy sci 1998;81:2139. 35. portaluppi ma, stevenson js: pregnancy rates in lactating dairy cows after presynchronization of estrous cycles and variations of the ovsynch protocol. j dairy sci 2005;88:914-921. 36. sterry ra, jardon pw, fricke pm: effect of timing of cosynch on fertility of lactating holstein cows after first postpartum and resynch timed ai services. theriogenology 2007;67:1211-1216. 37. brusveen dj, cunha ap, silva cd, et al: altering the time of the second gonadotropin-releasing hormone injection and artificial insemination (ai) during ovsynch affects pregnancies per ai in lactating dairy cows. j dairy sci 2008;91:1044-1052. 38. bridges ga, helser la, grum de, et al: decreasing the interval between gnrh and pgf2α from 7 to 5 days and lengthening proestrus increases timed-ai pregnancy rates in beef cows. 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resynchronized inseminations in lactating dairy cows. j dairy sci 2013;96:3788-3798. 56. fricke pm, caraviello dz, weigel ka, et al: fertility of dairy cows after resynchronization of ovulation at three intervals after first timed insemination. j dairy sci 2003;86:3941-3950. 57. giordano jo, wiltbank mc, guenther jn, et al: increased fertility in lactating dairy cows resynchronized with doubleovsynch when compared to ovsynch initiated 32 d after timed artificial insemination. j dairy sci 2012;95:639-653. 58. carvalho pd, fuenzalida mj, ricci a, et al: modifications to ovsynch improve fertility during resynchronization: evaluation of presynchronization with gnrh 6 days before ovsynch and addition of a second prostaglandin f2α treatment. j dairy sci 2015;98:8741-8752. 59. chebel rc, al-hassan mj, fricke pm, et al: supplementation of progesterone via internal drug release inserts during ovulation synchronization protocols in lactating dairy cows. j dairy sci 2010;93:922-931. 60. bilby tr, bruno rgs, lager kj, et al: supplemental progesterone and timing of resynchronization on pregnancy outcomes in lactating dairy cows. j dairy sci 2013;96:7032-7042. 61. bisinotto rs, castro lo, pansani mb, et al: progesterone supplementation to lactating dairy cows without a corpus luteum at initiation of the ovsynch protocol. j dairy sci 2015;98:2515-2528. 62. wijma r, perez mm, masello m, et al: 2018. a resynchronization of ovulation program based on ovarian structures present at nonpregnancy diagnosis reduced time to pregnancy in lactating dairy cows. j dairy sci 2018;101:1697-1707. 63. wiltbank mc, fricke pm, sangritasvong s, et al: mechanisms that prevent and produce double ovulations in dairy cattle. j dairy sci 2000;83:2998-3007. 64. stevenson, js, portaluppi ma, tenhouse de: factors influencing upfront singleand multiple-ovulation incidence, progesterone, and luteolysis before a timed insemination resynchronization protocol. j dairy sci 2007;90:5542-5551. 65. hayashi kg, matsui m, shimizu t, et al: 2008. the absence of corpus luteum formation alters the endocrine profile and affects follicular development during the first follicular wave in cattle. reproduction 2008;136:787-797. 66. lopez h, caraviello dz, satter ld, et al: relationship between level of milk production and multiple ovulations in lactating dairy cows. j dairy sci 2005;88:2783-93 67. gümen a, guenther jn, wiltbank mc: follicular size and response to ovsynch versus detection of estrus in anovular and ovular lactating dairy cows. j dairy sci 2003;86:3184-3194. 68. cunha ap, guenther jn, maroney mj, et al: 2008. effects of high vs. low progesterone concentrations during ovsynch on double ovulation rate and pregnancies per ai in high producing dairy cows. j dairy sci 2008;91(e-suppl 1):246. 69. fricke pm, wiltbank mc: effect of milk production on the incidence of double ovulation in dairy cows. theriogenology 1999;52:1133-1143. 70. lópez-gatius f, hunter rhf: spontaneous reduction of advanced twin embryos: its occurrence and clinical relevance in dairy cattle. theriogenology 2005;63:118-125. 71. carvalho pd, santos vg, fricke hp, et al: effect of manipulating progesterone before timed artificial insemination on reproductive and endocrine outcomes in high-producing multiparous holstein cows. j dairy sci 2019 (in press). 72. martins jpn, wang d, mu n, et al: level of circulating concentrations of progesterone during ovulatory follicle development affects timing of pregnancy loss in lactating dairy cows. j dairy sci 2018;101:10505-10525. 328clinical theriogenology • volume 11, number 3 • september 2019 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true 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prostaglandin failed to improve pharmacologically induced ejaculation in stallions oxytocin or prostaglandin failed to improve pharmacologically induced ejaculation in stallions alison mickelson, james graham, paula moffett, christian bisiau, patrick mccue college of veterinary medicine and biomedical sciences colorado state university, fort collins, co abstract pharmacologically induced ejaculation is utilized in stallions when traditional semen collection procedures are not appropriate or not effective. most common protocol includes oral imipramine hydrochloride, with intravenous xylazine hydrochloride given 1 2 hours later. goal was to document success rate from past clinical experience (historical data) and to determine whether addition of oxytocin along with xylazine or a prostaglandin analogue prior to xylazine treatment would enhance success rate in aged stallions in a clinical setting. semen was successfully collected in 3 of 7 aged stallions using the standard protocol. in the modified protocol, 22 times (out of 50 attempts) semen was successfully collected from 12 stallions. however, addition of oxytocin or a prostaglandin analog had a deleterious effect on passive emission of semen. keywords: stallion, pharmacologically induced ejaculation, imipramine, xylazine introduction semen is traditionally collected from stallions in an artificial vagina while stallions mount a mare or breeding phantom.1-3 traditional semen collection method may not be appropriate for stallions with severe musculoskeletal issues, neurologic defects, penile paralysis, or behavioral issues.4-6 alternatively, other techniques such as ground collection using an artificial vagina,7-9 manual stimulation,10,11 and pharmacologically induced ex copula ejaculation12,13 have been developed for stallions. pharmacologically induced ex copula ejaculation (‘chemical ejaculation’) can be accomplished by administration of imipramine hydrochloride alone14 or xylazine hydrochloride alone15 or a combination of imipramine followed by xylazine.12,16 imipramine is a tricyclic antidepressant drug used in human medicine in the management of depression,17 nocturnal enuresis,18 retrograde ejaculation,19 and premature ejaculation.20 it was hypothesized that imipramine lowers the ‘ejaculation threshold’ of stallions.4,6 xylazine hydrochloride is an α2 adrenergic receptor agonist used primarily for sedation in horses. xylazine hydrochloride or other α2 agonists such as detomidine hydrochloride3,21 is occasionally associated with a side effect of inducing passive emission of semen during sedation. other chemical ejaculation protocols have used various combinations of detomidine, butorphanol tartrate, imipramine, xylazine, clomipramine, and/or prostaglandin f2α.4,6,16,21-23 semen collected via chemical ejaculation was lower in volume and higher in sperm concentration;6,12,16,23 however, it has been used for fresh insemination, cool-transport, and cryopreservation.23,24 mares have been successfully bred.24,25 success rate of inducing ejaculation with imipramine and/or xylazine ranged from 33 to 68%,12,13,16 and varied between stallions and between studies. objectives were to: 1) document success rate of pharmacologically induced ejaculation of stallions from past clinical experience (historical data); and 2) determine if oxytocin treatment at the same time as xylazine or a prostaglandin analogue prior to xylazine would enhance ejaculation success rate of a standard imipramine-xylazine protocol in aged stallions. materials and methods all procedures were approved by institutional animal care and use committee. for the first part of the study, reproductive records of 12 stallions in which chemical ejaculation was attempted 50 times were reviewed. a standard protocol consisting of a single oral dose of imipramine hydrochloride (3.0 mg/kg) followed ~ 1 hour later by intravenous xylazine hydrochloride (0.33 0.5 mg/kg) was used. semen parameters from 22 normal stallions collected without medication during the same time period were reviewed for comparative purposes. clinical theriogenology • volume 12 number 4 • december 2020481 for the second part of the study, 7 quarter horse stallions (17 27 years; mean 22.7 ± 3.5) were used. stallions’ weight ranged from 430 to 530 kg with a body condition score between 4 and 6 (based on score index of 1 [poor] 9 [extremely fat]).26 three stallions were housed indoors in box stalls with daily turn out into a paddock and 4 stallions were housed outside in a paddock (brought inside into a box stall on treatment days). ‘cleanout’ collections were performed on all stallions prior to experiment. the chemical ejaculation procedure was managed by only 1 person in 5 x 5 meter box stalls with minimal outside disturbance. standard chemical ejaculation a standard protocol was used, consisting of a single oral dose of imipramine hydrochloride (3.0 mg/kg) followed 2 hours later with intravenous xylazine hydrochloride (0.5 mg/kg). semen was collected in a funnel held beneath the stallion using a long pole. funnel was lined with a disposable artificial vagina liner attached to a collection bottle fitted with a gel filter. stallions were quietly observed ~ for 70 minutes for behavioral responses to treatment until either ejaculation occurred or until they recovered from sedation without ejaculation. traditional semen ejaculation stallions were allowed a minimum of 2 days rest after chemical ejaculation attempt prior to traditional semen ejaculation. stallions were teased to a mare in estrus and then allowed to mount a breeding phantom. semen was collected in a colorado model artificial vagina (animal reproduction systems, chino, ca) fitted with a collection bottle and disposable inline nylon micromesh gel filter (animal reproduction systems). oxytocin added chemical ejaculation stallions were allowed a minimum of 2 days rest prior to chemical ejaculation attempt incorporating oxytocin in a standard protocol. standardized dosages of imipramine hydrochloride and xylazine hydrochloride were used, with the addition of intravenous oxytocin (20 units) treatment immediately after xylazine hydrochloride. prostaglandin added chemical ejaculation stallions were rested for a minimum of 7 days prior to chemical ejaculation attempts incorporating prostaglandins in the protocol. stallions were treated with standardized dose of imipramine hydrochloride and 2 hours later by either: a) 250 µg of cloprostenol sodium intramuscularly, followed 5 minutes later by 0.5 mg/kg xylazine intravenously (n = 2); or b) cloprostenol treatment without xylazine (n = 2). semen evaluation semen samples (traditional and chemical ejaculation) were evaluated for gel free volume and sperm concentration, motility, and morphology. volume was measured in a warmed graduated cylinder. sperm concentration was determined using a densimeter 591b (animal reproduction systems) or a nucleocounter® sp-100™ (chemometec, allerod, denmark). total and progressive sperm motility were evaluated by computer assisted semen analysis (spermvision®, minitube of america, inc., verona, wi) attached to a zeissax10 microscope. sperm morphology was evaluated using a differential interference contrast microscope (olympusex51) at 1,000 x magnification and high viscosity microscope immersion oil (resolve™,thermo scientific, waltham, ma). data analyses semen parameters (volume, concentration, total sperm number, total motility, and progressive motility) of chemical and standard collections were compared by anova. percentage of stallions that ejaculated using 3 protocols was compared by chi square. semen parameters (volume, concentration, clinical theriogenology • volume 12 number 4 • december 2020 482 total motility, progressive motility, and percentage of morphologically normal sperm) were compared by anova. data are presented as mean ± sd. values were considered significant at p < 0.05. results a total of 50 attempts at chemical ejaculation were performed on a total of 12 client-owned stallions over a 6 year period (2013 2018). semen was collected successfully in 22 (44%) attempts. four stallions were presented with musculoskeletal issues, 4 presented for ejaculatory dysfunction, 2 had neurological issues, 1 had squamous cell carcinoma lesions on his penis and 1 had a history of hemospermia. four of 12 stallions did not respond to chemical ejaculation; however, the remaining 8 stallions ejaculated at least once. only 1 stallion ejaculated to every chemical ejaculation attempt. semen parameters for ejaculates from the 22 successful traditional (without medication) and 22 medicated (imipramine-xylazine) collection methods are presented (table 1). total motility and progressive motility were the only semen parameters that were different (p < 0.05). gel free volume, sperm concentration, and total sperm numbers were not statistically different. table 1. semen parameters for ejaculates collected by traditional (without medication) and chemical (imipramine-xylazine) methods parameter without medication (n = 22) mean ± sd imipramine-xylazine (n = 22) mean ± sd p value interval from xylazine to emission of semen (minutes) n/a 13.4 ± 10.6 (3 34) n/a gel free volume (ml) 39.9 ± 20.6 (17 100) 37.7 ± 26.8 (8.2 130) 0.765 sperm concentration (x 106/ml) 318.3 ± 282.3 (71 1270) 443.3 ± 401.7 (141 873) 0.239 total sperm number 10,579 ± 7,718 (3,060 22,625) 12,512 ± 7,375 (3,240 33,150) 0.400 total sperm motility (%) 74.1 ± 13.2 (46 92) 60.6 ± 22.3 (15 89) 0.019 progressive sperm motility (%) 69.2 ± 14.5 (37 90) 52.8 ± 23.2 (10 84) 0.007 all stallions ejaculated when collected off a breeding phantom using an artificial vagina. success rate of semen collection was different (p < 0.05) between traditional collection and chemical ejaculation procedures. oral treatment of 3.0 mg/kg of imipramine hydrochloride, followed 2 hours later by intravenous treatment of 0.5 mg/kg of xylazine hydrochloride resulted in passive emission of semen in 3 of the 7 stallions (42.9%) after only 1 attempt. average interval between xylazine treatment and ejaculation was 33.1 minutes (range; 31 35 minutes). stallions’ ages for which chemical ejaculation was successful were 22, 22, and 27 years, whereas stallions’ ages for which chemical ejaculation was not successful were 17, 22, 22, and 27 years. none of the stallions, that were treated with standard imipramine/xylazine protocol, ejaculated when 20 units of oxytocin was given concurrently with xylazine hydrochloride. stallions did exhibit a greater degree of spasmodic muscle contractions in the dorsal scrotal region when oxytocin was included in the protocol compared to standard protocol. none of the stallions, previously treated with imipramine-xylazine, ejaculated when treated with a prostaglandin analogue. all stallions exhibited muscular contractions in the dorsal scrotal region and dropped their penis and gained an erection within 5 minutes after prostaglandin treatment. sweating and mild diarrhea were observed in 2 stallions. there were no differences (p > 0.05) in gel free volume, sperm concentration, total sperm motility, progressive sperm motility, and total sperm number between ejaculates collected from 3 stallions using imipramine-xylazine protocol and ejaculates collected from all 7 stallions without medication (table 2). there were also no differences (p > 0.05) in semen parameters when only comparing ejaculates from the same 3 stallions collected using the imipramine xylazine protocol or without medication. clinical theriogenology • volume 12 number 4 • december 2020483 table 2. semen parameters for ejaculates collected by traditional (without medication) and chemical (imipramine-xylazine) methods parameter without medication (n = 7) mean ± sd imipramine-xylazine (n = 3) mean ± sd p value gel free volume (ml) 41.1 ± 18.8 44 ± 40.7 0.92 sperm concentration (x 106/ml) 342.2 ± 253.4 795 ± 617.4 0.26 total sperm motility (%) 66.9 ± 22.5 89.7 ± 2.1 0.28 progressive sperm motility (%) 59.9 ± 23.8 85 ± 4.6 0.29 total sperm number (x 109) 11.2 ± 8.9 19.7 ± 7.2 0.97 discussion success rate of chemical ejaculation in our clinical program (44%) was similar to what has been reported (33 68%).12,13,16 success for individual stallions ranged from 100% (i.e. 5 for 5 in 1 stallion) to 0% (a combined 0 for 9 attempts for 4 stallions). the rationale for the prospective part of the study was to determine if addition of oxytocin or prostaglandins to standard protocol would improve success of chemical ejaculation in a population of aged stallions. the standard imipramine-xylazine protocol success rate for aged stallions in the current study (42.9%) was in the same range as in our clinical practice. specific titrated doses modified for each individual stallion significantly improved the success rate of chemical ejaculation. 15 limited anecdotal data have been reported on the effects of oxytocin or prostaglandins on pharmacologically induced emission of semen in stallions.4 oxytocin is a peptide hormone synthesized in the paraventricular nuclei of the hypothalamus and secreted from the posterior pituitary gland. oxytocin administration to stallions stimulates contraction of smooth muscles of the ductus deferens and epididymis and has been used clinically to alleviate blockage of plugged ampullae.4 it was hypothesized in the current study that oxytocin would enhance the success of imipramine-xylazine induced emission of semen by stimulation of smooth muscle contraction. however, none of the stallions ejaculated when oxytocin was added to the protocol. prostaglandin f2α is a fatty acid hormone which can induce smooth muscle contraction. a previous report noted that spontaneous ejaculations were observed within 10 minutes after intramuscular treatment of an analogue of prostaglandin f2α.4 in the current study, none of the stallions ejaculated when a dose of prostaglandin was incorporated in the protocol. type of prostaglandin, dose used, and timing of treatment could all have affected the outcome. semen collected via chemical ejaculation had lower volume and higher sperm concentration compared to traditional ejaculates.16 however, in the present study, all semen parameters were similar between the 7 traditional ejaculates and all 3 chemically induced ejaculates in the aged research stallions. passive emission of semen with chemical ejaculation, if successful at all, usually occurs within 3 5 minutes after xylazine treatment as the stallion is becoming sedated or less commonly when the stallion is recovering from sedation.12,27 in the present study, passive emission of semen occurred in all 3 stallions ~ 30 minutes after xylazine treatment. none of the stallions emitted semen in the early period after xylazine treatment. conclusion chemical ejaculation was successful in aged stallions using a standard protocol of imipramine and xylazine. addition of oxytocin or cloprostenol to the standard protocol at the dosages and times used apparently had deleterious effects on passive emission of semen in aged stallions, as all attempts were unsuccessful. conflict of interest none to declare. clinical theriogenology • volume 12 number 4 • december 2020 484 references 1. pickett bw, voss jl, squires el, et al: collection, preparation and insemination of stallion semen. animal reproduction and biotechnology laboratory bulletin no. 10: 2000. p. 17-54. 2. love cc: semen collection techniques. vet clin north am: equine pract 1992;8:111-128. 3. hurtgen jp: semen collection in stallions. in: samper jc, equine breeding management and artificial insemination. 2nd edition, st. louis; saunders/elsevier: 2009. p. 33-39. 4. mcdonnell sm: ejaculation physiology and dysfunction. vet clin north amer: equine pract 1992;8:57-70. 5. mcdonnell sm: techniques for extending the breeding career of aging and disabled stallions. clin tech equine pract 2005;4:269-276. 6. mcdonnell sm: pharmacological manipulation of ejaculation. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 1413-1414. 7. forney bd, mcdonnell sm: how to collect from stallions while they are standing on ground. proc am assoc equine pract 1999; p. 142-144. 8. meroni g, sieme h, burger d: efficiency of ground semen collection in the stallion. j equine vet sci 2012;32: 497-498. 9. schumacher j, riddell mg: collection of stallion semen without a mount. theriogenology 1986;26:245-250. 10. crump jr j, crump j: stallion ejaculation induced by manual stimulation of the penis. theriogenology 1989;31: 341-346. 11. mcdonnell sm, love cc: manual stimulation collection of semen from stallions: training time, sexual behavior and semen. theriogenology 1990;33:1201-1210. 12. mcdonnell sm, odian mj: imipramine and xylazine-induced ex copula ejaculation in stallions. theriogenology 1994;41:1005-1010. 13. johnston pf, deluca jl: chemical ejaculation of stallions after the administration of oral imipramine followed by intravenous xylazine. proc am assoc equine pract 1998; p. 12-15. 14. mcdonnell sm, garcia mc, kenney rm, et al: imipramine-induced erection, masturbation, and ejaculation in male horses. pharmacol biochem behav 1987;27:187-191. 15. mcdonnell sm, love cc: xylazine-induced ex copula ejaculation in stallions. theriogenology 1991;36:73-76. 16. mcdonnell sm: oral imipramine and intravenous xylazine for pharmacologically-induced ex copula ejaculation in stallions. anim repro sci 2001;68:153-159. 17. azima h, vispo rh: effects of imipramine (tofranil) on depressive states: a clinical and psychodynamic study. ama arch neurol psychiat 1959;81:658-664. 18. banerjee s, srivastav a, palan bm: hypnosis and self-hypnosis in the management of nocturnal enuresis: a comparative study with imipramine therapy. am j clin hypn 1993;36:113-119. 19. ochsenkuhn r, kamischke a, nieschlag e: imipramine for successful treatment of retrograde ejaculation caused by retroperitoneal surgery. international j androl 1999;22:173-177. 20. balon r: antidepressants in the treatment of premature ejaculation. j sex marital therapy 1996;22:85-96. 21. rowley dd, lock tf, shipley cf: fertility of detomidine hcl-induced ex copula-ejaculated stallion semen after storage at 5°c. proc am assoc equine pract 1999; p. 221-223. 22. josson a, whitacre m: does the addition of butorphanol have an effect on pharmacologically induced ejaculation in severely injured stallions? j equine vet sci 2012;32:490. 23. mcdonnell sm, turner rm: post-thaw motility and longevity of motility of imipramine-induced ejaculates of pony stallions. theriogenoiogy 1994;42:475-481. 24. feary dj, moffett pd, bruemmer je, et al: chemical ejaculation and cryopreservation of semen from a breeding stallion with paraphimosis secondary to priapism and haemorrhagic colitis. equine vet edu 2005;17:299-304. 25. card ce, manning st, bowman p, et al: pregnancies from imipramine and xylazine-induced ex copula ejaculation in a disabled stallion. can vet j 1997;38:171. 26. henneke dr, potter gd, kreider jl, et al: relationship between condition score, physical measurements and body fat percentage in mares. equine vet j 1983;15:371-372. 27. mccue pm, ferris ra: formulary and protocols in equine reproduction. 2nd edition, fort collins, colorado state university: 2018; p. 48. clinical theriogenology • volume 12 number 4 • december 2020485 18 contact chance l. armstrong armstcl@auburn.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9644, http://dx.doi.org/10.58292/ct.v15.9644 review report review of surgical procedures for creating bovine estrus detector males chance l. armstrong department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa abstract this article reviews the most common, effective, and currently recommended surgical techniques for preparing teaser bulls that maintain libido. it is the intent of this article to arm practitioners with a brief description of the surgical procedures and practices for the best surgical and long-term outcomes. keywords: estrus detector males, teaser bulls, gomer, sidewinder introduction estrus detection plays a vital role in the success of any artificial insemination program. there are multiple methods of heat detection employed on beef and dairy operations. the intact bull that has been rendered sterile is often described as a teaser or gomer bull.1 these animals are useful aids for detection of estrus in females that may not display strong external cues that they are ready for breeding. the teaser animal has demonstrated superior ability to detect estrus in comparison to human observation with reports of detection rates of 97–100 percent compared to 74 percent with commonly employed human detection schedules.2 this article reviews the most common, effective, and currently recommended surgical techniques for preparing teaser bulls that maintain libido. the surgical options to that prevent penetration of the genital tract of the female are desirable to limit infectious disease transmission. the true venereal diseases campylobacter fetus subspecies venerealis and tritrichomonas foetus are of particular concern when considering a procedure to avoid intromission. the vast number of techniques described in the literature are evidence that there is no perfect procedure. the bull selected for these procedures should be young, healthy and have a strong libido, although the latter is not capable of being measured chute-side. it is also recommended that the candidate for procedures should exhibit docile behavior because of frequent human interactions. pre-anesthetic preparation the bull should be fasted for 24–48 hours prior to deep sedation or general anesthesia to prevent bloat and the possibility of regurgitation with subsequent aspiration of ruminal contents.3 feed should be withheld for 48 hours, and water removed 12 hours prior to the procedure.4 pre-operative antimicrobials are highly encouraged if the procedure is not performed in a sterile environment but may also be useful with more invasive procedures. the author prefers to utilize beta-lactam antibiotics because of the spectrum of activity and common pathogens discovered in the urogenital tract of the bull.5 these antimicrobials should be administered prior to surgery for optimal efficacy. 6 for those procedures that require the bull to be in lateral recumbency for extended periods of time the surgeon must be mindful of providing adequate padding to prevent the development of peripheral neuropathies. utilization of inflated innertubes of different sizes to pad the shoulder and protect the radial nerve can be useful.3 surgical procedures epididymectomy the bilateral caudal epididymectomy is a preferred procedure to render the bull sterile. this procedure has the advantage of being performed in the standing position. the distal one-third of scrotum is aseptically prepped and a local block in administered. the most prominent area of the cauda epididymis is identified, and the scrotum is incised over this area. the incision is extended through the scrotal fascia and tunica vaginalis. the cauda epididymis will protrude through the skin incision when the adequate depth has been achieved. mailto:armstcl@auburn.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9644 19 citation line: clinical theriogenology 2023, 15, 9644, http://dx.doi.org/10.58292/ct.v15.9644 apply towel clamps and gentle traction to further exteriorize the epididymal tissue. hemostatic forceps are applied at the base of the cauda epididymis following exteriorization.5 the tissue is excised using a scalpel blade. the surgeon should be careful not to incise the tunica albuginea to avoid significant hemorrhage. the tissue is left to heal by second intention and the same procedure is performed on the contralateral testicle. the owner should wait 30 days to utilize this animal for estrus detection. it is the advice of the author to evaluate the ejaculate of the male for the observation of live spermatozoa prior to use. this will avoid any negative outcomes should the bull remain fertile after failure to render him sterile. this procedure does not prevent the bull from achieving intromission and other procedures are warranted in combination with removal of the cauda epididymis to prevent the possible transmission of infectious pathogens through coitus. vasectomy the bull may also be rendered sterile by transection of the ductus deferens as an isolated procedure or in conjunction with an epididymectomy. the surgical approach to the ductus deferens is on the caudal aspect of the scrotal neck. the bull is placed in lateral recumbency under light sedation. the author prefers using xylazine hydrochloride intramuscularly approximately ten minutes prior to tabling the bull at a dose of 0.05 to 0.1 mg/kg.3 the area should be clipped and aseptically prepped. local anesthetic is administered in the scrotal skin and extending into the spermatic cord proximal to the incision. the surgeon should make a 2 to 3 cm longitudinal incision through the skin and underlying tunica dartos muscle to expose the spermatic cord. a blunt instrument like a kelly forcep is placed under the spermatic cord to allow for exteriorization through the incision. the vaginal tunic is sharply incised, and the surgeon should avoid the cremaster muscle to prevent excessive hemorrhage. the fibrous, cord like, ductus deferens are carefully isolated from the testicular artery and vein. ligatures are placed proximally and distally 5 cm apart using absorbable suture.5 the tissue is transected and the tunica vaginalis is closed primarily. the dartos muscle and scrotal skin are then closed in a routine fashion. this procedure alone does not render the bull unable to achieve intromission and another procedure may be indicated to prevent intromission. the producer is advised to wait 30 days prior to utilizing this bull as described above. this procedure is most used in small ruminants to render the male sterile. penile translocation (sidewinder technique) this technique repositions the prepuce into a lateral orientation to prevent intromission with the female during estrus. the author as well as others prefer this method as it has been noted that teaser males made by this procedure have maintenance of libido following the procedure.1,7 a drawback to the procedure is that it requires the bull to be in lateral recumbency either under general anesthesia or deep sedation as it is more invasive than other procedures. the author prefers to relocate the prepuce into the fold of the flank. to prep the male for surgery the ventral abdomen is clipped and surgically prepared from the preputial orifice to the neck of the scrotum. the prepared area should extend two large hand widths dorsally to the fold of the flank. the preputial hairs should remain intact to provide urine egress and avoid urine scald in the flank region. to begin a 7 cm circular incision is made in the fold of the flank extending through the skin and cutaneous trunci into the muscular layer located in the fold of the flank. the surgeon should achieve adequate hemostasis and place a moistened 4×4 sponge over the area once proper dissection and hemostasis have been obtained.8 next, a marker suture is then placed on the dorsum of the external preputial orifice to give the surgeon orientation after translocation to the flank.7,8 this is followed by a circumferential incision through the skin 5 cm proximal to the external preputial orifice.8 next, the surgeon should make a longitudinal incision that connects with the circumferential incision cranially and extends 40–50 cm caudally along the sheath. blunt and sharp dissection is utilized to free the penis and prepuce away from the abdominal wall. it should be stressed that the surgical plane of dissection should remain close to abdominal wall to avoid large vessels in the elastic tissues. the dissection is complete when the penis and prepuce are free from the abdominal wall for the length of the longitudinal skin incision. this step is followed by blunt dissection through the tissue from the flank fold medially to form a tunnel for transposition of the penis and prepuce from abdominal midline to the flank. blunt dissection can be accomplished by utilizing long cervical forceps. the author prefers to start the blunt dissection from the circular incision in the flank and extend medial to the abdomen rather than to start from the abdominal midline and work laterally towards the flank fold. sterile obstetrical sleeves can be utilized to prevent contamination of the undermined tissue from the preputial hairs during the relocation process.8 warm saline applied to the lumen of the sterile sleeve can aid in the translocation of the penis and prepuce through the sleeve to the new opening in the flank fold. the sterile sleeve should be removed following transposition of the penis and prepuce to the flank fold. the marker suture previously placed allows the surgeon to position the external preputial orifice in the proper orientation in the flank. the subcutaneous tissue of the prepuce is then sutured in quadrants to the cutaneous trunci muscle. interrupted sutures using #1 absorbable suture are placed in each quadrant to secure subcutaneous tissue to the cutaneous trunci muscle. the skin is closed primarily using #3 monofilament non-absorbable sutures in an interrupted pattern. prophylactic antibiotics should be administered for 5 days post-operatively. oral non-steroidal anti-inflammatory drugs should be administered for 7–10 days to prevent excessive swelling and discomfort associated with the surgery. the bull should not be used for 30 days following the procedure. induced corpus cavernosal thrombosis the aim of this procedure is to prevent straightening of the sigmoid flexure by inciting a thrombus in the corpus cavernosum proximal to the sigmoid flexure which ultimately prevents ability of teaser animal to make intromission. malleable dental acrylic is often utilized for this procedure to create an artificial thrombus that prevents the corpus cavernosum from filling and achieving an erection.4,8 this type of acrylic sets up slow (12–24 hours) and does not cause a high-temperature exothermic reaction.9 this procedure has the advantage of being performed in a standing position. the bull is restrained and a caudal epidural with 2% lidocaine is applied for regional analgesia. the area between the rectum and the scrotal neck is prepared aseptically. the surgeon should make a midline incision 15–20 cm in length that ends approximately 6–8 cm http://dx.doi.org/10.58292/ct.v15.9644 citation line: clinical theriogenology 2023, 15, 9644, http://dx.doi.org/10.58292/ct.v15.9644 20 proximal to base of scrotum. the underlying thick fascia is dissected sharply, followed by blunt dissection of the elastic layers surrounding the penis. a loop of penis is exteriorized and the distal sigmoid flexure is identified as the site of attachment of the retractor penis muscles. following exteriorization and identification of the distal sigmoid flexure the surgeon should identify the ventral groove of the urethra as a landmark to avoid when placing the needle for insertion of the acrylic. a 14 gauge by 1.5-inch needle is placed into the dorsal aspect of the corpus cavernosum penis and 10–20 ml of saline is injected to ensure proper positioning of the needle prior to placement of the acrylc.4,8,9 the saline can be palpated as it is injected adjacent to the needle if in the proper position. it is a sign of improper placement of the needle if the solution is difficult to inject. the next step is to mix the dental acrylic and inject 5–10 ml through the needle that remains in the cavernous space.9 following placement of the acrylic, a penile suture is placed with #3 non-absorbable sutures. sutures are placed from the tunica albuginea on the lateral aspects of the penis at the level of the distal sigmoid flexure to the subcutaneous fascia.8,9 this will prevent penile prolapse when the bull is stimulated and relaxes the retractor penile muscles. the skin is closed with non-absorbable suture in routine fashion. urination following the procedure should be noted to ensure that the acrylic is not impeding the urethra. skin sutures can be removed at 10–14 days and the bull can return to use at this point. retrospective data suggests this procedure is a good option for prevention of coitus with most bulls (15/18) maintaining libido yet unable to achieve intromission following this procedure.9 penis tie-down technique the objective of this surgery is to create a permanent adhesion between the tunica albuginea of the penis and the ventral body wall. the bull is placed in right lateral recumbency following heavy sedation and the lateral aspect of the sheath is aseptically prepared. local anesthetic is injected halfway between the preputial orifice and the base of the scrotum on the lateral aspect of the sheath. a 15 cm skin incision is made midway between the preputial orifice and the scrotum, approximately 2 cm lateral to the midline.8 the incision is continued through the subcutaneous tissue, followed by blunt dissection that exposes the ventral abdominal wall and the tunica albuginea. multiple interrupted sutures utilizing non-absorbable suture material are placed in the incision in order to appose the tunica albuginea to the ventral body wall. the dead space should be minimized to avoid seroma formation between the skin and deep subcutaneous tissues. this can be accomplished utilizing absorbable suture material in two layers this is followed by closure of the skin in a routine manner with non-absorbable suture material. the bull should stay isolated and away from cycling females for 45 days to allow for fibrous adhesions to form between the body wall and the tunica albuginea.8 the bull should be observed by the owner for success or failure of the procedure prior to use for estrus detection. conflict of interest none to declare. references 1. morgan gl, dawson lj. development of teaser bulls under field conditions. vet clin north am food anim pract 2008;24:443–453. doi: 10.1016/j.cvfa.2008.06.004 2. mcdonald tj, foote rh, drost m, et al. preparation of teaser bulls  and steroid-implanted steers and their effectiveness in detecting estrus. theriogenology 1976;6:51–60. doi: 10.1016/ 0093-691x(76)90188-6 3. lin h, passler t, clark-price s, editors. farm animal anesthesia: cattle, small ruminants, camelids, and pigs. 2nd edition, hoboken; john wiley & sons: 2022. 4. gill, ms. surgical techniques for preparation of teaser bulls. vet clin north am food anim pract 1995;11:123–136. doi: 10.1016/ s0749-0720(15)30511-9 5. wolfe df: forward. in: wolfe df, moll hd: editors. large animal urogenital surgery. baltimore, md; williams and wilkins: 1998. 6. halleran jl, papich mg, li m, et al. update on withdrawal intervals following extralabel use of procaine penicillin g in cattle and swine. j am vet med assoc 2022;260:50–55. doi: 10.2460/ javma.21.05.0268 7. grissett g: preparation of teaser bulls. in: hopper r: editor. bovine reproduction. 1st edition, hoboken; wiley-blackwell: 2014: 181–186. 8. riddell mg. prevention of intromission by estrus-detector males. in: wolfe df, moll hd: editors. large animal urogenital surgery. baltimore, md; williams and williams: 1998:335–343. 9. davern aj, baird an, koziol jh. occlusion of the corpus cavernosum penis in bulls intended for estrus detection: 18 cases (2002–2016). j am vet med assoc 2019;254:512–519. doi: 10.2460/javma.254.4.512 http://dx.doi.org/10.58292/ct.v15.9644 https://doi.org/10.1016/j.cvfa.2008.06.004 https://doi.org/10.1016/​0093-691x(76)90188-6 https://doi.org/10.1016/​0093-691x(76)90188-6 https://doi.org/10.1016/s0749-0720(15)30511-9 https://doi.org/10.1016/s0749-0720(15)30511-9 https://doi.org/10.2460/javma.21.05.0268 https://doi.org/10.2460/javma.21.05.0268 https://doi.org/10.2460/javma.254.4.512 clinical theriogenology 2022; 14: 110 disorders of sexual development: a case of xx sex reversal in a mixed breed dog negative for sry (sex-determining region on the y chromosome) gene kelsey martin,a agata parsons,b greg burns,a gerrit bouma,b fiona hollinsheada adepartment of clinical sciences, bdepartment of biomedical sciences college of veterinary medicine and biomedical sciences colorado state university, fort collins, co abstract an 8-month mixed breed dog (from shelter) was evaluated for its abnormal external genital (enlarged os clitoris protruding externally from the vulva) condition. dog had phenotypic female appearance. testicular-like tissue was removed via laparoscopy-assisted gonadectomy. genotype was determined using blood and gonadal tissue (via histology and immunohistochemistry). dog had xx chromosomes and was negative for sry (sex-determining region on the y chromosome) gene. keywords: phenotypic sex, antimüllerian hormone, sex reversal background normal mammalian sexual development involves chromosomal sex (xx or xy [determined at fertilization]), followed by development of gonadal sex (ovary or testis), and finally expression of phenotypic sex (internal reproductive tract and external genitalia).1 errors in these 3 stages resulted in disorders of sexual development (dsds), manifesting in a variety of anatomical and physiological abnormalities in affected individuals.2 generally, dsds are classified based on the incongruencies that exist among the stages of sexual development. individuals that demonstrate a discrepancy between their chromosomal and gonadal sex development had a sex reversal (sr) phenotype and are considered either xx male or xy female.3 these sr individuals can be either positive or negative for sex-determining region on the y chromosome (sry) gene that regulates gonadal differentiation. absence of sry gene causes development of ovaries and a female phenotype. presence of sry gene results in activation of genes, including sox9, triggering ‘male’ development pathway toward formation of testes with internal and external masculinization.4 reports of xy sex reversal in dogs are rare.5,6 conversely, reports of xx sex reversal in dogs are more common.7 regardless, it is important for practitioners to be aware of this clinical presentation in addition to understanding the development of dsds and use proper nomenclature associated with these syndromes to systematically diagnose and appropriately manage sr clinical cases. case presentation history an 8-month intact mixed breed dog was presented to the small animal reproduction service at the colorado state university veterinary teaching hospital for evaluation of abnormal external genitalia. referring veterinarian identified the abnormality while managing the dog for a urinary tract infection after hematuria (observed by the owners shortly after adoption). besides hematuria, other presenting clinical signs included vulvar discharge and perivulvar dermatitis. current owners reported absence of estrous or intact male behaviors. previous owners confirmed that the dog never had any surgery (reproductive alteration or otherwise). there was no evidence of underlying endocrinopathy (e.g. hyperadrenocorticism leading to clitoral hypertrophy 8) nor was exposure to androgens or progestins9,10 during pregnancy or thereafter. physical examination, vaginal cytology, hematology, serum biochemistry dog had female phenotype appearance with normal vulvar conformation. however, had an enlarged clitoris (figure 1) with a palpable boney structure consistent with os clitoris. clitoral enlargement was noticed along the ventral aspect of vulva that protruded ~ 2 cm from the vulvar cleft when the patient was in standing position. under sedation, it was determined via urethral catheterization that urination occurred from within the vulva just cranial to the base of the clitoris, and not from the protruding os clitoris. there were no palpaclinical theriogenology 2022; 14: 111 ble testes nor scrotal structures. internal reproductive structures were not identifiable via abdominal ultrasonography. figure 1. externally protruding hyperplastic os clitoris a cotton tipped plastic handle swab was used to obtain a vaginal smear. swab was rolled onto a glass microscope slide that was then air dried and stained using a simple modified wrights-giemsa stain (diff-quik). vaginal smear was evaluated under light microscopy (100 200 x magnification). predominant (> 90%) cells were parabasal cells, indicating lack of estrogenic influence. complete blood count and serum biochemistry profiles were within normal canine reference ranges. owners declined further tests (e.g. serum antimüllerian hormone) and surgical options for os clitoris removal, but elected laparoscopic-assisted gonadectomy. gonadectomy patient was premedicated with 0.02 mg/kg atropine sulfate (atropine sulfate injection, west ward, eatontown, nj), 0.03 mg/kg acepromazine (acepromazine maleate injection, vetone, boise, id) and 0.05 mg/kg hydromorphone (hospira inc., lake forest, il); all were given intramuscularly. anesthesia was induced with 2 mg/kg ketamine (ketaset®, zoetis inc, florham park, nj) and 2 mg/kg propofol (zoetis, kalamazoo, mi) given intravenously. general anesthesia was maintained with isoflurane (akorn animal health, lake forest, il) in oxygen. a 2-cm ventral midline incision was made just caudal to the umbilicus and a single incision laparoscopic surgical port was placed into the abdomen to allow insufflation with co2 to a pressure of 10 mm hg. a zero-degree laparoscope was inserted into 1 of the ports and the interior of the abdomen was explored. left gonad was first visualized and grossly appeared like testis. using forceps and ligasure™, the tubular structure associated with the gonad (presumed to be the ductus deferens and pampiniform plexus) were ligated. vasculature and presumed ductus deferens on the right-hand side was followed into the inguinal ring and the remaining gonad was identified via deep external palpation. using electrocautery, a 2 cm incision above the inguinal ring (where the testis-like gonad was located) was made and closed castration was performed. abdominal cavity was examined before closure to confirm absence of ovaries, ovotestes, uterine tubular structure(s) or prostate. all incisions were closed in a standard, multiple layer fashion and there were no complications. patient’s recovery from anesthesia was uneventful and 2.2 mg/kg carprofen (rimadyl®, zoetis) was given subcutaneously for postoperative pain management. six months following surgery, a follow up phone consultation with the owner revealed that the clitoral enlargement had not reduced in size. there was no vulvar discharge or perivulvar dermatitis nor clitoral enlargement causing any irritation or distraction to the patient. additionally, urination was reported to be normal. diagnosis a case of dsd was suspected11 and appropriate tests were conducted (e.g. immunochemical localization of antimüllerian hormone [amh]12). histology of gonadal tissue (figure 2) revealed that the architecture of the tissue was consistent with hypoplastic testicular tissue (figure 3). empty seminiferous tubules lined by sertoli cells were noticed in both testes; however, testes had no maturing germ cells nor obvious signs of active spermatogenesis. interstitial tissue of testes had leydig cells and epididymides had empty tubules lined with columnar epithelium. gondal tissue was positive for amh (figure 4), confirming functional sertoli cells (low testosterone concentrations observed in hypogonadism13). figure 2. gross image of the gonadal tissue removed at surgery figure 3. hematoxylin and eosin-stained sections of testis (a) and epididymis (b); note absence of maturing germ cells in seminiferous tubules and normal epididymal epithelium with no sperm (200 x magnification). figure 4. immunolabeling for amh (black arrows) within the seminiferous tubules containing sertoli cells; note negative control (small insert) without primary antibody. chromosomal sex (i.e., xx versus xy) pcr was determined via amplifying14 amelogenin (amel) gene from genomic dna isolated from blood (qiagen dneasy blood & tissue kit). both x and y chromosomes were positive for amel and their gene length polymorphism allowed for genetic sex identification. pcr was performed using platinumtm ii taq hot-start dna polymerase (invitrogen), with 0.5 μl of the amelx/amely primers (5 μm), and cycle conditions 95°c for 4 minutes, 95°c for 30 seconds, 55 °c for 30 seconds, and 72°c for 20 seconds, with the last 3 steps repeated 35 times, followed by a final step of 72°c for 10 minutes. pcr amplification using amelx (on the x chromosome) and amely (on the y chromosome) specific primers yielded a 215 bp amplification product for amelx and 247 bp for amely (figure 5). furthermore, in a separate pcr assay, 2 pcr primer sets were designed to amplify a 350 or 480 bp fragment of canine sry gene (table) to determine if sry gene was present in the dna. pcr analysis of dna isolated from a known male dog was positive for sry (figure 6). pcr cycle conditions for amplification of sry were: 95°c for 4 minutes, 95°c for 30 seconds, 60°c for 30 seconds, and 72°c for 15 seconds, with the last steps repeated 40 times, followed by a final step of 72°c for 1 minute. sry was not detected in the genomic dna isolated from the patient. this finding suggested that alternative and unknown nonsry-mediated mechanism(s) might have occurred, leading to testes development.15 a b figure 3. hematoxylin and eosin-stained sections of testis (a) and epididymis (b); note absence of maturing germ cells in seminiferous tubules and normal epididymal epithelium with no sperm (200 x magnification). clinical theriogenology 2022; 14: 112 figure 4. immunolabeling for amh (black arrows) within the seminiferous tubules containing sertoli cells; note negative control (small insert) without primary antibody. chromosomal sex (i.e. xx versus xy) pcr was determined via amplifying14 amelogenin (amel) gene from genomic dna isolated from blood (qiagen dneasy blood & tissue kit). both x and y chromosomes were positive for amel and their gene length polymorphism allowed for genetic sex identification. pcr was performed using platinumtm ii taq hot-start dna polymerase (invitrogen), with 0.5 μl of the amelx/amely primers (5 μm), and cycle conditions 95°c for 4 minutes, 95°c for 30 seconds, 55 °c for 30 seconds, and 72°c for 20 seconds, with the last 3 steps repeated 35 times, followed by a final step of 72°c for 10 minutes. pcr amplification using amelx (on the x chromosome) and amely (on the y chromosome) specific primers yielded a 215 bp amplification product for amelx and 247 bp for amely (figure 5). furthermore, in a separate pcr assay, 2 pcr primer sets were designed to amplify a 350 or 480 bp fragment of canine sry gene (table) to determine if sry gene was present in the dna. pcr analysis of dna isolated from a known male dog was positive for sry (figure 6). pcr cycle conditions for amplification of sry were: 95°c for 4 minutes, 95°c for 30 seconds, 60°c for 30 seconds, and 72°c for 15 seconds, with the last steps repeated 40 times, followed by a final step of 72°c for 1 minute. sry was not detected in the genomic dna isolated from the patient. this finding suggested that alternative and unknown nonsry-mediated mechanism(s) might have occurred, leading to testes development.15 figure 5. amplified bands represent amelogenin (amel); bottom band is amel present on the x chromosome (215 bp), and the top band is amel present on the y chromosome (247 bp). lane 1: h20 (negative control). lane 2 & 3: genomic dna from the hermaphrodite dog. lanes 4 & 5: genomic dna from a known male (xy) dog. l = 100 bp ladder. table. primer sequences and amplicon sizes generated by pcr amplification name primer seq (5’ to 3’) amplicon size (bp) amelx ataatgacaaagaaaacatgac 215 (amelx) amely ctgctgagctggcaccat 247 (amely) dogsry1-f ggtgcagcggtacaacaaaa 350 dogsry1-r ttccgacgaggtcggtattt dogsry2-f ggtgcagcggtacaacaaaa 482 dogsry2-r cgtgtgtgtgcagccctact figure 6. amplified bands represent sry clinical theriogenology 2022; 14: 113 discussion diagnosis (sry-negative xx male with testes) was confirmed by histomorphological evaluation of the gonads, determination of the chromosomal sex and absence of the sry gene by pcr analysis. cases of sry-negative xx sr were most commonly reported in purebred dogs, suggesting heritability most often as an autosomal recessive trait.16–18 however, in this case of a mixed breed dog, the etiology was most likely a spontaneous event, due to either a genetic gain-of-function mutation or loss-of-function mutation that altered expression of key regulatory genes necessary for female or male sexual development. it is possible that overexpression of the sox9 gene had a key role in testicular differentiation and development in the formation of testes in this case. duplications within the sox9 gene, often in the promoter region, was the most common mutation leading to sry-negative xx sr phenotypes in dogs.4,19 therefore, overexpression of the sox9 gene might have been a key factor for testes induction in the absence of sry. however, mutations in this gene are not always the cause of sry-negative xx sr, and inactivating mutations of ovarian determining genes such as rspo1 or ctnnb1 could be responsible for the observed external female phenotype in this case. further genomic analyses are required to better understand what genetic factors for testes induction may exist.3 clinically, the challenge with dsd cases is that individuals can present with a wide range of anatomical abnormalities including ambiguous external genitalia, clitoral enlargement, hypospadias, and cryptorchidism. these anatomical abnormalities can then cause secondary clinical complications such as vaginitis, vulvar discharge, and urinary tract infections. anatomical and clinical developmental abnormality type depends on the degree of phenotypic masculinization. this is based on the amount of functional testis that developed and may vary depending on the quantity and timing of testicular secretions necessary to masculinize internal and external genitalia.20 furthermore, testosterone concentrations produced are insufficient in quantity or its conversion to dihydrotestosterone by 5α-reductase is impaired during the critical period for androgen-dependent masculinization, internal and external genitalia will fail to masculinize in whole or in part, as with this case. approximately 90% of xx sex reversal cases occur with bilateral ovotestes and the remainder (10%) with bilateral testes. however, there were some cases that occasionally presented with an ovary and ovotestis or an ovotestis paired with a testis.3 in this case, despite having 2 testes, the patient had phenotypic female appearance with no anatomical evidence of a scrotum or prepuce, an unusual presentation. primary clinical concern was the exposed (presumed hypertrophied) clitoris and secondary vaginitis associated with a vulvar discharge. normal sexual development is a process that is highly dependent on the timing of events plus the molecular and hormonal expression of karyotype in adequate and appropriate amounts. delayed or insufficient quantity of amh secretion in xy males during the critical period (regression of mullerian duct) result in persistence of all or part of the mullerian duct derivatives (oviducts, uterus, cervix, cranial vagina).18 this xx patient developed testes with functional (able to produce amh) sertoli cells that apparently suppressed the emergence of mullerian duct derivatives. learning points • determine the chromosomal, gonadal, and phenotypic sex of a suspected dsd patient. • assess the effect abnormal reproductive anatomy on the dog’s health and welfare. • implement a welfare-orientated management strategy that would alleviate the secondary clinical signs caused by clitoral hyperplasia. conflict of interest none to declare. acknowledgement authors thank kelsey tofany for excellent technical assistance (clinical examination and blood withdrawal) and patient management. references 1. meyers-wallen vn: inherited abnormalities of sexual development in dogs and cats. in: concannon pw, england g, verstegen iii j, et al: editors. recent advances in small animal reproduction. 1st edition, united states; international veterinary information service: 2001. 2. poth t, breuer w, walter b, et al: disorders of sex development in the dog-adoption of a new nomenclature and reclassification of reported cases. anim reprod sci 2010;121:197-207. 3. parma p, veyrunes f, pailhoux e: sex reversal in non-human placental mammals. sex dev 2016;10:326-344. 4. albarella s, de lorenzi l, rossi e, et al: analysis of xx sry-negative sex reversal dogs. animals 2020;10:1-13. 5. chaffaux s, cribiu ep: clinical, histological and cytogenetic observations on nine intersex dogs. genet sel evol 1991;23:81-84. 6. kang jt, kim hj, oh hj, et al: sry-positive 78, xy ovotesticular disorder of sex development in a wolf cloned by nuclear transfer. j vet sci 2012;13:211-213. 7. yoon h, han sh, kim j, et al: urogenital anomalies and urinary incontinence in an english cocker spaniel dog with xx sex reversal. j vet intern med. 2018;32:1166-1171. 8. purswell bj, kolster ka: surgical disease of the vulva and vagina. in: bojrab mj, monnet e: editors. mechanisms of disease in small animal surgery. 3rd edition, jackson; teton newmedia: 2015. 9. curtis em, grant rp: masculinization of female pups by progestogens. j am vet med assoc 1964;144:395-398. 10. sokolowski jh, zimbelman rg: canine reproduction: effects of multiple treatments of medroxyprogesterone acetate on reproductive organs of the bitch. am j vet res 1974; 35:1285-1287. 11. sumner sm, grimes ja, wallace ml, et al: os clitoris in dogs: 17 cases (2009-2017). can vet j 2018;59:606-610. 12. alm h, bodil sh: identifying ovarian tissue in the bitch using anti-müllerian hormone (amh) or luteinizing hormone (lh). theriogenology 2018;106:15-20. clinical theriogenology 2022; 14: 114 13. grinspon rp, bedecarrás p, ballerini mg, et al: early onset of primary hypogonadism revealed by serum anti-müllerian hormone determination during infancy and childhood in trisomy 21. int j androl 2011;34:e487-e498. 14.yan s, bai c, li y, et al: sex identification of dog by pcr based on the differences in the amelx and amely genes. anim genet 2013;44:604-607. 15. meyers-wallen vn: gonadal and sex differentiation abnormalities of dogs and cats. sex dev 2012;6:46-60. 16. melniczek jr, dambach d, prociuk u, et al: sry-negative xx sex reversal in a family of norwegian elkhounds. j vet intern med 1999;13:564-569. 17. campos m, moreno-manzano v, garcía-roselló m, et al: srynegative xx sex reversal in a french bulldog. reprod dom anim 2011;46:185-188. 18. meyers-wallen vn, schlafer d, barr i, et al: sry-negative xx sex reversal in purebred dogs. mol reprod dev 1991;53:266-273. 19. rossi e, radi o, de lorenzi l, et al: sox9 duplications are a relevant cause of sry-negative xx sex reversal dogs. plos one 2014;9:e101244. 20. schlafer dh, valentine b, fahnestock g, et al: a case of srypositive 38,xy true hermaphroditism (xy sex reversal) in a cat. vet pathol 2011;48:817-822. practical considerations for implementation of artificial insemination programs for beef cattle practical considerations for implementation of artificial insemination programs for beef cattle sandy johnson northwest research and extension center, kansas state university, colby, ks abstract the beef industry is increasingly taking advantage of effective methods available to synchronize ovulation in beef females. in addition to wide genetic choices possible with artificial insemination, estrus synchronization consolidates calving, increases calf age at weaning and with each season, increases average days postpartum at the start of breeding season. variability in artificial insemination pregnancy rates occur from year to year, but should not prompt substantial changes in procedures. inadequate nutrition limits reproductive success. skillful managers balance reproductive output and feed expenditures. adequate weight gain during the third trimester is critical to reproductive performance and timely reminders from a tool called management minder can ensure important dates are not missed. the estrus synchronization planner converts a diagram of an estrus synchronization protocol to a calendar with specific dates and times for action. attention to detail is important from nutritional management and execution of the estrus synchronization protocol to deposition of semen. economic efficiency of a breeding program increases if number of bulls needed for natural service are reduced following artificial insemination. balance number of bulls needed post artificial insemination with knowledge and experience of the artificial insemination program and relative risk aversion. early pregnancy detection allows staging of pregnancies and adds value for marketing, management and assessment of reproductive response. minimize stress of activity and animal movement to reduce embryonic loss. estrus synchronization systems can be adapted to a variety of production environments, enabling increased opportunities to pursue value-added genetics. keywords: artificial insemination, estrus synchronization, management minder, beef cattle, pregnancy rate, estrus synchronization planner introduction fixed time artificial insemination (ftai) works and the beef industry is increasingly taking advantage of effective methods available to synchronize ovulation in beef females. more and better information (e.g. genomically enhanced expected progeny difference) is available from breed associations to make profitable selection decisions. furthermore, marketing options are available to capture investments made in genetics. economic advantages of estrus synchronization (es) and artificial insemination (ai) programs were demonstrated in multiple settings.1-3 challenges come from applying known systems to a wide range of production environments. portable corrals and turn-key breeding services are available and can overcome some limitations. however, es and ai do require additional animal handling and may influence time in drylots or trap pastures and subsequent movement to grazing resources. a variety of stressors can increase embryonic loss and management must understand and minimize potential for problems. need for a sound year-round nutritional program to support optimal reproduction is recognized; however, sometimes it is difficult to achieve with adverse weather and other real-world challenges. this manuscript will outline several tools that can help implement es and ai programs and aid in year-round management. application of es and ai require attention to details from semen acquisition to clean up with natural service sires. broader production system impacts will be outlined that may be influenced by reproductive management choices. expectations it is important to start an ai program with sound expectations of results. variability in ai pregnancy rates will occur from year to year. sticking with a basic plan and working on refinement of details over several years is a good path towards improvement, rather than large changes each year. 285 clinical theriogenology • volume 11, number 3 • september 2019 reviews of embryonic loss place fertilization rate at 85 100%4,5 serving as an upper limit to pregnancy expectations. embryonic mortality data5 in low-yielding dairy cows and heifers were summarized; for every 100 head inseminated, 90 fertilized ova were present on day 4, 60 were pregnant on day 28, 56 remained pregnant on day 90, and 54 calved. evidence that beef cattle experience less embryo loss than lactating dairy cattle can be drawn from benchmark calving distribution data. summary of 2010 2107 chapstm database reported that 60% of mature beef cows calved in the first 21 days of the calving season (https://www.ag.ndsu.edu/dickinsonrec/chaps-software-1). proportion of multiparous beef cows that calved in the first 21 days of the 2017 spring season to natural service sires (18 populations, 2,372 calvings) at the meat animal research center was 72.4%, where cow pre-calving body condition averaged 6.3 (bob cushman, personal communication). a field trial with 34 missouri herds that used ftai after a cosynch + cidr protocol had ai pregnancy rates that averaged 65% (1,963/3,015). pregnancy rate to ai ranged from 60 69% in herds ≥ 100 head (n = 15) and 63 86% in herds < 50 (n = 10).6 there was no difference between pregnancy rate to ai (n = 13,942 first inseminations) and natural service (n = 6310 first inseminations) based on nonreturn rates (mean ± sd, 57.5 ± 9.2; 58.0 ± 13.1, respectively);7 however, rates were more variable with natural service. focus of an es and ai program is often on female management to improve pregnancy rate, but variation in sires was also a factor. standard deviation of service sires on conception rate was ± 3.68% and over ± 8% for herd years.8 pregnancy rate per natural serviced female (n = 2,316) averaged 53.7% and was lower for yearling bulls than those > 2 years.9 cow management when working with producers to incorporate ai for the first time, best case scenario is to start a year in advance to ensure nutrition and health foundations are in place. evidence of good nutritional management includes pregnancy rates of > 88 90% in a 60-day breeding period and > 50% calving in the first 21 days. identifying subgroups of animals for ai that calved early and have appropriate body condition is another option for starting an ai program. some herds may benefit from an intermediate step of using synchronization with natural service sires before shifting to ai. factors that influence postpartum interval to estrus were reviewed extensively.10,11 inadequate nutrition is generally the first limiter in common production settings. body condition at calving reflects energy status during late gestation and when inadequate, postpartum interval is extended, even if energy is adequate during lactation.12 a body condition score (bcs) ≥ 5 (1 = emaciated; 9 = obese) at calving is considered a good target. under a nutrient-rich environment, average postpartum interval to estrus was 55 67 days, depending on sire bred.13 in a summary of studies conducted in more typical commercial settings, proportions of cows cycling prior to estrus synchronization were 64% for multiparous and 55% for primiparous cows and increased 7.5% for every 10-day interval from ≤ 30 and > 90 day post calving.14 body condition score assessed at initiation of synchronization reflected a curvilinear pattern, with peak ai pregnancy rates in cows with condition scores of 5 and 6 and lower rates for cows with bcs < 5 and > 7.15 if selecting a subgroup of cows for ai, use cows with a bcs ≥ 5 at calving, 40 days or more postpartum at ai and in a positive energy balance. a synchronization protocol that contains a progestin may induce noncycling (late calving and marginal bcs, bcs ≈ 4) cows to cycle and conceive earlier. if a progestin insert is used, cows should be > 20 days postpartum at insertion. cows that are anestrus before synchronization often have similar pregnancy rates as cycling cows16 although few, if any, cows in these studies would be considered to be in deep anestrus. primiparous cows with bcs < 5 are less likely to be induced to ovulate compared to multiparous cows.14 lower-cost semen could be targeted for thin or late calving cows, as pregnancy response will be lower. planning for timely year-round management herds with good reproductive responses have good year-round management. the optimal time to cost-effectively put condition back on cows, if needed and account for fetal growth, is between weaning and calving. unfortunately, the start of the third trimester often passes without notice and more importantly action. third trimester is the period of greatest fetal growth and cows that do not gain weight 286clinical theriogenology • volume 11, number 3 • september 2019 during this time are losing body condition. an earlier than normal weaning date may help retain cow condition and conserve forage resources when limited. more days available to add fetal weight gain lower requirements for rate of gain and energy density of diet; however, if delayed too long, even a highconcentrate diet may not achieve needed performance. a web-based tool (management minder), available at www.ksubeef.org/managementminder or www.iowabeefcenter.org/software.html, was developed to remind producers of key dates related to management, e.g. start of third trimester. the management minder can act as a personal assistant that helps organize and manage a cattle operation. many annual activities occur in association with the start of key events: breeding soundness examinations need to be scheduled and completed before the start of the breeding season, high-magnesium mineral needs to be started before grazing and vaccine must be on hand before preconditioning. timely action can increase productivity and profit. when hours of sleep are short, such as during calving, the management minder provides an easy reference for upcoming action items. this program will create a list of activities and dates that can be imported into electronic calendar systems such as outlook, google or yahoo and be available on a smart phone.17 table 1 provides examples of activities that can be added to a calendar. calendars can be made for clients that include recommendations for health and reproductive management. getting details right numerous systems studied could be used for synchronization of estrus and ovulation in beef heifers and cows; however, a short list of protocols has been developed that are efficacious and minimize handling with the goal of increased likelihood of producer success.18 these recommended protocols are listed in major us semen company sire catalogs, at www.beefrepro.info or covered in online media coverage of an annual symposium dedicated to application of reproductive technology at www.appliedreprostrategies.com. the estrus synchronization planner was designed to help users select an estrus synchronization protocol from the short list of recommended protocols and administer it correctly. to use the planner, ms excel or open office software is necessary. the tool can be downloaded free of charge at the iowa beef center at http://www.iowabeefcenter.org/estrussynch.html and detailed help in using the tool is available on that website. a favorite feature of the tool is the calendar created that indicates dates, times, and products to be given based on selections of the user. many estrus synchronization projects require coordination of a number of individuals and the calendar produced can serve as a communication and planning tool with everyone involved. the calendar is particularly useful when planning longer-term progestin systems to ensure days are counted correctly. a common problem with implementing protocols is getting the various commercial products for gonadotrophin releasing hormone (gnrh) and prostaglandin f2α (pgf2α) confused. specific commercial products to be used can be selected so that the calendar shows the trade name, as well as the date and time of day of administration. for ftai systems, there is a recommended 3 6 hours window when insemination should occur. planning should include the time it takes to gather and work cattle, daylight hours and working temperatures to set a target insemination time for the group. based on the time indicated to start ai, the planner calculates when pgf2α should be given. if the planner shows this to be in the middle of the night, the time to give pgf2α can be reevaluated. an alert is given if the number of females in the group cannot be inseminated in a 3 hour interval, based on the estimate of number inseminated in 1 hour. do not synchronize any more females than can be inseminated in a given setting within a window of 3 4 hours. a new multigroup version of the planner was released in 2018 that allows users to incorporate up to 12 groups of cattle over a 12 month period. for example, replacement heifers might be scheduled for ai on may 1, followed by 2 year olds on may 15th and mature cows on may 16. this version also includes a supply sheet that can summarize quantity of supplies such as vials of gnrh, insemination sleeves or semen. first time users of this version will benefit from watching the short instructional video to understand the process that allows plans for the multiple groups to be developed and saved. this tool is also free and can be found at the same iowa beef center website as the original planner. 287 clinical theriogenology • volume 11, number 3 • september 2019 table 1. example of activities that can be added to electronic calendars from the management minder date item 1/11/2019 replacement heifer check weight #1 1/15/2019 check calving supplies and facilities 1/22/2019 get started on shopping for new sires 1/30/2019 begin evening feeding 2 year olds 2/13/2019 calving begins 2 year olds (+280 d) 2/16/2019 begin evening feeding mature cows 2/22/2019 record calving body condition score mature cows 3/1/2019 calving begins mature cows 3/10/2019 replacement heifer check weight # 2 3/10/2019 plan ai protocol yearling heifers 3/23/2019 body condition score sires 3/23/2019 semen and trich test bulls, vaccinate and deworm 3/23/2019 order products for spring vaccinations and parasite control 3/23/2019 get supplies and semen ordered for ai 3/23/2019 plan ai protocol 3/25/2019 pre-breeding vaccinations yearling heifers 3/25/2019 reproductive tract score/pelvic measurements 4/1/2019 start high mg mineral 4/1/2019 pre-breeding vaccinations mature cows 4/1/2019 test calves for bvd-pi 4/1/2019 implant non-replacement calves 4/15/2019 grass turnout 5/9/2019 ai begins yearling heifers 5/22/2019 turn in herd sires mature cows 5/22/2019 record breeding body condition score cows while not as robust as the excel versions, a mobile friendly version of the planner can be accessed at http://www.estrussynch.com/. it does require an internet connection to use it. in the mobile version, drop down menus provide choices and when the inputs are complete, the program will send an email with dates of activities as outlined. this tool works particularly well for starting discussions on estrus synchronization planning and a first look at calendar dates. 288clinical theriogenology • volume 11, number 3 • september 2019 progestin treatment two commercially available progestins are available for use in heifers; melengesterol acetate (mga®, zoetis, madison, nj) and eazi-breed cidr® insert (cidr, 1.38 g progesterone, zoetis). if uniform, consistent daily intake of mga can be achieved, it is less expensive and requires less handling to administer. because of intake variation, some prefer feeding mga in a portion of a limit-fed diet; however, there are successful operations that feed mga in a total mixed ration. longer term estrus synchronization protocols with either mga or cidr lend themselves to combining palpation for pregnancy, reproductive tract scores and/or pelvic area with the start of progestin treatment. vaccination for reproductive diseases can also be done at this time without negative impacts on fertility. the advantage to an intravaginal progestin device is that it is much easier to know that each animal received progestin. a relatively low percentage of vaginal inserts may be lost, particularly in heifers and drylot settings. shortening the tail of the insert is used to reduce loss from curious pen mates playing with tail. however, the tip of a shortened tail can cause irritation. a vaginal discharge can occur in association with vaginal inserts and tails on the inserts contribute to severity of discharge.19 various pathogens were identified at insert removal20 and they remained at insemination, even though the vaginal discharge was reduced.19 presence of organisms alone may not reduce fertility; however, producers should strive for cleanliness during insertion and recognize that excessive contamination can be harmful. producers ever vigilant on cost control have attempted to reuse cidr inserts. autoclaves, more common in laboratory settings, use heat and pressure to sterilize items. compared to new or used but not autoclaved cidr inserts, used autoclaved cidr inserts produced higher concentrations of progesterone in cows for the first 24 hours after insertion.21 progesterone concentrations in heifers from on farm “sterilization” methods of used inserts were compared to new and autoclaved used cidr inserts.22 on farm methods were dishwasher, microwave, toaster oven, dryer, boiling, and 60 days outdoor exposure to environment. none of the cleaning methods produced a release pattern of progesterone similar to new cidr inserts. inserts stored outside were more discolored and produced the lowest concentrations of progesterone. cleaning and storage can have negative impacts on the integrity of components of the cidr and its tail. time needed for an initial cleaning (before sterilization) and storage space and location between uses are additional complicating factors. injections for synchronization protocols to work, treatments should be given as prescribed and also should comply with beef quality assurance guidelines. newer working chutes provide better control and access to preferred injection sites in the neck in contrast to many older models. contrary to some assertions, injecting prostaglandin in the top rump (closer to the ovary) did not improve response.23 in the 2016 2017 beef quality audit, injection site lesions in the round continued to decrease in dairy and beef cows but were still at 15 and 7% of samples, respectively.24 survey information from the dairy industry indicated 32 70% of injections were given in hindquarters.25,26 semen quality and handling each step from collection of semen to deposition in the female must ensure semen quality and viability for best pregnancy success. a thorough breeding soundness exam assessing concentration, motility, and morphology of sperm should be performed before semen is frozen. small, independent collection operations may not offer a complete semen analysis as part of the freezing process. use semen processed at a certified semen services (css) lab. when receiving semen, do not expose canes or canisters above the frost line in the neck of the tank for longer than 8 seconds. start the ai season with a full tank of liquid nitrogen to raise the frost line and minimize semen exposure to damaging temperatures during high-use periods. implement a semen inventory system to minimize risk of exposure to elevated temperatures when locating straws. if using an electronic thaw unit, test the temperature with a calibrated thermometer before the season begins. replace card thermometers annually. thaw only as much semen as can be inseminated in 10 -15 minutes.27 do not allow straws to touch while thawing. follow provider recommendations for semen thawing temperature and time. use a timer when thawing semen and do not 289 clinical theriogenology • volume 11, number 3 • september 2019 guess. after thawing, protect semen from cold and heat shock. keep the thaw box, ai gun, scissors, and equipment clean. straw cutters can be difficult to clean and should be replaced rather than using a dirty cutter. semen packaged in 0.25 ml straws are more susceptible to handling damage than 0.5 ml straws, because of the greater surface to volume ratio. to avoid damage, movement from tank to tank or from tank to thaw unit should not exceed 3 seconds.28 although the uterine body is considered the optimal site of deposition of semen, correct placement was lower in several studies in dairy cattle and further evaluation indicated placement in the uterine horn resulted in higher conception rates in some cases. when conception rates from body insemination were > 60%, little advantage resulted from deposition in the uterine horn.27 this could be a reflection that uterine horn insemination is not easily confused with uterine body insemination, but the cervix can be confused with the uterine body. cervical deposition of semen does reduce conception rate and should be avoided. deposition of semen in the uterine horn did not improve conception rate with sexed semen.29,30 bull management post ai in many instances, a single ftai or some ai period is followed by the use of natural service sires to complete the breeding season. this raises the issue of how many natural service sires are needed to ensure adequate coverage at the estrus following synchronization. a number of factors will influence the number and timing of females in estrus following es and ai and subsequent need for bull power. a low ai pregnancy rate increases the number of females returning to estrus. the reason for a poor response could affect the distribution pattern of return to estrus. if females were anestrus and the synchronization protocol did not induce ovulation, the depth of anestrus would determine when they might show estrus. in contrast, if response of females to synchronization was good but semen quality and fertilization rate were poor, returns to estrus would likely be more synchronous. nonpregnant heifers returned to estrus in a biphasic pattern following a 5 day cosynch + cidr protocol. first peak was at 7 days after ftai and the second at 17 18 days.31 maximum percentage of heifers returning to estrus on any 1 day was 3.5% of the original group. pregnancy rate to ftai was 51%. a maximum of 18% of nonpregnant cows (3.1% of total group) returned to estrus on any given day following a cosynch + cidr timed ai protocol (pregnancy rate 55%), with 13% in estrus the day before peak and 16% in estrus the day after peak.32 a pressure-sensing mount detector (heatwatch) fitted 13 days after ftai identified 86% of nonpregnant heifers in estrus from days 15 through 26 with a maximum of 10% of the original group in estrus on any 1 day33 and pregnancy rate to ftai was 56%. some information is available regarding bull to female ratios when natural service sires are used on synchronized females. in single-sire mating groups of estrus-synchronized females (n = 2,316), pregnancy rates did not differ with bull to female ratios of 1:7 to 1:51.9 an optimal bull to heifer ratio of 1:25 was identified when comparing 1:50, 1:25 and 1:16 for females synchronized with mga. more variation between replicates was noted with the 1:50 group34 which agrees with the wide variation between bulls in number of offspring sired in multi-sire groups.35 information on bull to female ratios is summarized36 when a final pregnancy rate was reported with ai pregnancy rate. a normal ratio was defined as 1:20 1:30 and half the normal ratio was 1:50 1:60. final pregnancy rates were similar when half the normal rate was used after es and ai compared to the normal ratio and to normal ratios with total natural service.36 the 2007 2008 nahms survey reported an average of 23.7 females per mature bull.37 inability to reduce the number of natural service sires following ai increases costs and may negate economic benefits of ai.38 the importance of breeding soundness examinations and monitoring mating activity is heightened for use of natural service with synchronization or for cleanup natural service following es. yearling bulls have lower pregnancy rates when breeding estrus-synchronized females than bulls 2 years and older9 and should be used sparingly. use of a single sire was more efficient than multi-sire pastures for bulls mated to estrus-synchronized heifers, based on the lack of difference in pregnancy rates between the two groups.39 however, it is potentially riskier, since practical systems to evaluate libido prior to the breeding season are lacking. when bull workload is expected to be high, the use of bulls ≥ 2 years old in a 290clinical theriogenology • volume 11, number 3 • september 2019 relatively smaller pasture is advisable. the ability to rotate bulls in and out after a few days of breeding would be advantageous, but may not be practical. pregnancy rates to ai were 27% higher in females that displayed estrus at or before ftai.40 estrus detection or an estrus detection aid could monitor estrus response before ftai to inform decisions of bull numbers needed post ai when conditions warrant. experienced ai technicians may note when estrus response is low in females they are inseminating, but may have a difficult time quantifying to what degree without deliberate accounting. timing of bull exposure post ai depends on priorities that could range from maximizing early pregnancy regardless of sire, to marketing of ai-bred females. if marketing ai-sired seedstock females, accurate identification becomes increasingly important with increasing dna testing for paternity. as noted previously, return to estrus can be scattered from days to weeks after ai. for research purposes, natural service sires are typically withheld for 10 14 days after the last ai followed by early pregnancy detection with ultrasonography from 30 90 days after ai. when using ultrasonography between days 35 and 100 of pregnancy, calving date was predicted ± 15 days, 95% of the time when scanning under field conditions and measuring the first image of the following: crown rump, head biparietal, head length or truck diameter.41 the authors suggested that confirming breeding date should be limited to females with only 1 breeding before ultrasonography or that breedings are separated by > 1 estrous cycle. earlier diagnosis of pregnancy may require follow up due to embryonic or fetal loss. this can add cost, but there is more value in pregnancy diagnosis if stage of pregnancy and or sex is determined. early pregnancy diagnosis can be part of routine management to reduce stocking rate in yearling heifer pastures by removing nonpregnant heifers. regardless of whether individuals pregnant to ai sires will be marketed as such, early pregnancy diagnosis monitors success of an ai program or the first natural service cycle. in the case of an unusually low pregnancy response, the manager has more time and options to minimize the negative impact of this result and make needed changes to correct the issue. based on stage of pregnancy, females can be grouped for more precise feed or calving management and administration of precalving vaccinations that affect colostrum quality. avoiding stressors nutrition stressors from heat, sudden diet changes, transportation and disease can affect embryonic loss. good global nutrition is paramount to reproductive success; however, sometimes management choices contribute to short-term or unintended reductions in intake and or energy. weaned replacement heifer calves are often placed in a drylot and remain there until after an ai period. several studies support the concept that increased embryonic loss may occur in heifers that have not had grazing experience post weaning and subsequently have negative or reduced gains when first introduced to summer pasture.40 compared to heifers with grazing experience during development, naïve heifers took significantly more steps as measured by pedometers on the first few days after grass turnout and had lower adg.42 either because of lack of grazing experience or youthful interest in exploring new surroundings that does not leave time for grazing, average daily gain is reduced early in the grazing period. abrupt nutritional changes can influence embryo survival. heifers were assigned to a pasture allowance of either twice maintenance requirements (h) or 80% of maintenance (l) for 10 days before ai. the day after ai, using the same pasture allowances as pre-breeding, heifers were allocated to 4 treatments: ll, hh, lh, and hl. embryo survival was lowest in the hl group and did not differ among the other treatments.43 as variable weather conditions influence forage availability before and during the breeding season, overestimating forage availability could reduce reproductive response. use of historical stocking rates based on number of cows as compared to actual mature cow weight could contribute to errors in estimating available forage. 291 clinical theriogenology • volume 11, number 3 • september 2019 transportation limited information is available on effects of transportation stress following breeding; however, transportation increases concentrations of cortisol,44,45 an indicator of stress. heifers transported 350 miles on days 1 4 after ai had earlier conception dates and higher ai pregnancy rates than heifers moved on days 8 9 and 28 29.46 flunixin meglumine given 13 days after ai and before 4 6 hours of transportation reduced pregnancy loss in cows.45 data are not available that addressed the effect of distance or method of moving on level of stress. realities of weather and nutritional availability at pre and post-move sites can force timing of movement. weaned heifer calves acclimatized to handling had reduced concentrations of cortisol and were older at puberty compared to controls.47 there were no beneficial effects of acclimatization in mature cows; however, higher pregnancy rates were associated with lower cortisol concentrations.48 perhaps cows with favorable temperament scores are less susceptible to stress in general, including transportation. if movement must occur after insemination, utilize low-stress handling techniques and try to either move before day 5 when the embryo is still in oviduct or after complete attachment of embryo to uterus on day 42. heat overtime, most beef herds adapted breeding periods that fit their environment and avoid heat stress issues during breeding. however, variable weather conditions can result in unusually warm weather around the time of ai. heat stress has the biggest negative impacts during the final stages of ovulatory follicle development and until the embryo reaches the uterus.49 animals adjust to warmer temperatures over time, so early uncharacteristically warm days can cause more problems than if the same temperatures occurred later in the year. some producers moved the calving season so that peak forage quality more closely matches peak cow lactation requirements. however, in these programs, reproductive responses are vulnerable to weather variability. short-term management options are limited if natural shade is not available. vaginal temperature peaked 1 hour after peak environmental temperature and recovered after daylight hours passed.50 therefore, during periods of heat stress, cattle should be worked as early as possible in the morning and avoid work in the late afternoon or evening when heat loads are still high. systems impacts of adding es and ai programs calving management choice of timing of the calving season involves many considerations51 especially weather since newborn calves are vulnerable to weather extremes. a common concern for producers trying ftai for the first time is that all the calves will be born on a day when the weather is dangerous. calving from a ftai occurred over a 16 21 day interval, depending on sire. 52 maximum number of calves born on any day ranged from 15 20% of those calving to the ftai.52 another report of calving distribution following ftai in cows from 12 locations and multiple sires reported a range in duration of pregnancy from 258 296 days.53 producers that incorporate ai programs generally report less calf death loss at calving.3,54 preparation and focus on calving, in addition to use of high-accuracy calving ease sires may explain this result. regardless, planning for calving a synchronized group of females should include plans for shelter and management in response to common local weather variability and labor availability. postpartum interval in an ideal estrus synchronization program, more calves are born early in the calving season and are older and heavier at weaning. this is true for ai1 or natural service sires.55 the advantage for subsequent breeding seasons is that more cows will have a longer interval between calving and start of the next breeding season. as a result, more females should be cycling at the start of the breeding season and be more likely to conceive early. depending on initial calving distribution, there is often an incremental improvement in cows conceiving earlier in the breeding season for several years. this enables use of a shorter breeding period, while maintaining or improving final pregnancy rate.2 a short breeding period (45 60 days) even for the extremely well managed herd, risks a stressor (i.e. disease, heat, or nutrition) 292clinical theriogenology • volume 11, number 3 • september 2019 that could affect embryo survival not evident until after bulls are pulled. this emphasizes the importance of continuing to identify cows returning to estrus late in the breeding season or after season. leaving bulls longer, combined with early pregnancy diagnosis, is a good risk-management tool. cows pregnant beyond a targeted calving window should be marketed. if late breeding cows are retained as part of the main herd, the advantage of es on increasing the days postpartum at breeding can disappear. health application of es protocols and ai in cows involves gathering and sorting of cow/calf pairs typically 3 times in 8 12 days. whether it was because they were observing the calves more closely or activities associated with es increased exposure of calves to pathogens, es was one of a number of factors determined to have an association with incidence of respiratory disease in calves.56 this observation should serve to remind us that when we incorporate new management practices such as es that a broader system view of potential impacts should be taken into consideration to avoid unintended consequences. breeding season length breeding season length is among many reproductive management choices producers make. rebreeding 2 year old females is a challenge for many operations and consequently the use of es and ai avoided. a short breeding season for replacement heifers (< 40 days or less) can position heifers for better reproductive success as 2 year olds. table 2 illustrates how many 21 day estrous cycles could occur in 2 year old females during their second breeding season, based on the date of conception as a yearling heifer and a range of postpartum intervals to first estrus of 60, 90, and 120 days. in a high-nutrient availability setting (body condition score 6.1 before breeding), postpartum interval for 2 year old females averaged 55 days.13 however, in nutrient-restricted situations, the interval could be > 120 days.57 even with a 60-day postpartum interval, heifers that conceive late their first breeding season may only cycle once in a 63-day breeding season. assuming a 60% conception rate, 40% of late bred heifers are at risk to be not pregnant. with longer postpartum intervals, even a heifer that conceives by day 21 the first season is at great risk to be not pregnant the second season. leaving bulls out longer may be a good management decision, because bred females are typically worth more than nonpregnant females. however, those that conceive in the first 21 days as yearling heifers have greater longevity in the herd and produce almost 2 calves more in weaning weight over a lifetime.58 if nonpregnant yearling heifers can be profitable, then breeding enough yearling heifers to achieve the desired number of replacements after 20 30 days of breeding is advantageous. excess late bred pregnant or nonpregnant heifers are marketed. table 2. influence of postpartum interval to first estrus and the first conception date as a yearling heifer on number of possible estrus cycles during second breeding season. conception date as yearling heifer relative to mature cow breeding season days postpartum at start of mature cow breeding season postpartum interval 60 days 90 days 120 days estrous cycles in 63 days day -14 94 3 3 1.8 day 1 80 3 2.5 1.1 day 21 59 2.9 1.5 0.1 day 42 38 2 0.5 0 day 63 17 1 0 0 293 clinical theriogenology • volume 11, number 3 • september 2019 conclusion all available technology will be needed to meet the world demand for protein in the next 30 years. estrus synchronization and ai, although increasing in use, are still underutilized in us beef operations. the growing list of genetic selection tools will provide pathways to address production challenges and help reach value-added markets. as the dairy industry uses beef sires, data collection mechanisms in place at dairies may provide sire fertility data previously unavailable to the beef industry. improved understanding of sire to sire variation in fertility and factors influencing early embryonic loss represent areas that will likely improve outcomes of future ai programs. technology that makes it easier to collect and share data will help unravel the mechanisms. a variety of stressors can cause embryonic loss and management must understand the potential culprits and minimize. selection increased growth and performance of the beef herd, and may have changed tolerance to stress. when the environment is less than optimal, low stress-tolerance may increase embryonic loss or reduce pregnancy rates. no one questions the need for a sound year-round nutritional program to support optimal reproduction. the challenge in implementing ai programs is maintaining adequate nutrition to support reproduction in a cost-effective fashion in the face of droughts, floods and world trade issues with impacts on commodity prices. good animal husbandry and a sharp pencil or detailed spreadsheet remain important tools. acknowledgement sincere appreciation to mike smith, university of missouri for thoughtful review of this manuscript. conflict of interest there are no conflicts of interest to declare. references 1. rodgers jc, bird sl, larson je, et al: an economic evaluation of estrous synchronization and timed artificial insemination in suckled beef cows. j anim sci 2012;90:4055-4062. 2. lamb g: economics of ai versus natural service: using decision-aid tools. in proceedings, applied reproductive strategies in beef cattle; 2015; davis, ca. p. 198-207. 3. kastens m: facilitating the production of high quality beef with estrus synchronization and fixed-time ai. in proceedings, applied reproductive strategies in beef cattle; 2011; joplin, mo. p. 79-84. 4. inskeep, ek, dailey, ra: embryonic death in cattle. vet clin food anim 2005;21:437-461. 5. diskin mg, waters sm, parr mh, et al: pregnancy losses in cattle: potential for improvement. reprod fertil and dev 2016; 28:83-93. 6. patterson dj, schafer dj, busch dc, et al: review of estrus synchronization systems: mga. in proceedings, applied reproductive strategies in beef cattle; 2006; rapid city, sd. p. 61-101. 7. williamson nb, morris rs, anderson ga: pregnancy rates and non-return rates following artificial and natural breeding in dairy herds. aust vet j 1978;54:111-114. 8. everett rw, bean b: semen fertility an evaluation system for artificial insemination sires, technicians, herds and systematic fixed effects. j dairy sci 1986;69:1630-1641. 9. pexton je, farin w, rupp gp, et al: factors affecting mating activity and pregnancy rates with beef bulls mated to estrus synchronized females. theriogenology 1990;34:1059-1070. 10. short re, bellows ra, staigmiller rb, et al: physiological mechanisms controlling anestrus and infertility in postpartum beef cattle. j anim sci 1990;68:799-816. 11. yavas y, walton js: postpartum acyclicity in suckled beef cows: a review. theriogenology 2000;54:25-55. 12. hess bw, lake sl, scholljegerdes ej, et al. nutritional controls of beef cow reproduction. j anim sci 2005;83 (suppl 1): p. e90-e106. 13. cushman ra, allan mf, thallman rm, et al: characterization of biological types of cattle (cycle vii): influence of postpartum interval and estrous cycle length on fertility. j anim sci 2007;85:2156-2162. 14. stevenson, js, johnson, sk, milliken, ga: incidence of postpartum anestrus in suckled beef cattle:treatments to induce estrus, ovulation and conception. prof anim sci 2003;19:124-134. 15. kasimanickam r, firth p, asay m, et al: impact of body condition change post-breeding on reproductive performance of beef cows. clinical theriogenology 2012;4:469-476. 16. patterson dj, thomas jm, abel jm, et al: control of estrus and ovulation in beef cows. in proceedings, applied reproductive strategies in beef cattle; 2016; des moines, ia. p. 46-85. 294clinical theriogenology • volume 11, number 3 • september 2019 17. johnson, s, dahlke, g: tools for timeliness: estrus synchronization planner and management minder. in proceedings, applied reproductive strategies in beef cattle; 2018; ruidoso, nm. 18. johnson, sk, funston, rn, hall, et al: multi-state beef reproduction task force provides science-based recommendations for the application of reproductive technologies. j anim sci 2011; 89:2950-2954. 19. fischer-tenhagen c, von krueger x, heuwieser w: short communication: evaluation of vaginal discharge following treatment with a progesterone insert. j dairy sci 2012;95:4447-4451. 20. padula am, macmillan kl. effect of treatment with two intravaginal inserts on the uterine and vaginal microflora of early postpartum beef cows. aust vet j 2006;84:204-208. 21. colazo mg, kastelic jp, whittaker pr, et al: fertility in beef cattle given a new or previously used cidr insert and estradiol, with or without progesterone. anim reprod sci 2004; 81:25-34. 22. dahlen cr, klein si, lamb gc, et al: technical note: physiological response of beef heifers after receiving a reused controlled internal drug release insert processed with different heat-treating methods. j anim sci 2014;92:2275-2279. 23. pickworth cl, bell lm, greene, w, et al: site of pgf2α injection does not alter effectiveness of the select synch + controlled internal drug release and timed artificial insemination protocol. theriogenology 2017;87:161-166. 24. pfeiffer mm, gretchen gm, ramanathan r, et al: frequencies and severity of injection site lesions in muscles from rounds of cow carcasses. transl anim sci 2019;3:txy094. 25. glaze jr, jb, chahine, m. assessment of management and basic beef quality assurance practices on idaho dairies. j dairy sci 2009;92:1265-1271. 26. tozer tr, varga va, henning wr, et al: do dairy producers use effective management practices to improve the value of market cows? prof anim sci 2005;21:272-277. 27. diskin m. review: semen handling, time of insemination and insemination. animal 2018;12:75-84. 28. seidel g. profitable uses of sex-sorted semen. in proceedings applied reproductive strategies in beef cattle; 2001; joplin, mo. p. 349-352. 29. seidel ge, schenk jl, herickhoff la, et al: insemination of heifers with sexed semen. theriogenology 1999;52: 1407-1420. 30. seidel ge, schenk jl: pregnancy rates in cattle with cryopreserved sexed sperm: effects of sperm numbers per inseminate and site of sperm deposition. anim repro sci 2008;105:129-138. 31. liles pollock ag, greiner sp, whittier jw, et al: the effects of resynchronization of estrus using a progestin-based system. prof anim sci 2016;32:627-635. 32. larson je, thielen kn, funnell bj, et al: influence of a controlled internal drug release after fixed-time artificial insemination on pregnancy rates and returns to estrus of nonpregnant cows. j anim sci 2009;87:914-921. 33. colazo mg, kastelic jp, mainar-jaime rc, et al: resynchronization of previously timed inseminated beef heifers with progestins. theriogenology 2006;65:557-572. 34. healy vm, boyd gw, gutierrez ph, et al: investigating optimal bull:heifer ratios required for estrus-synchronzied heifers. j anim sci 1993;71:291-297. 35. van eenennaam al, weber kl, drake dj: evaluation of bull prolificacy on commercial beef cattle ranches using dna paternity analysis. j anim sci 2014;92:2693-2701. 36. nielson hr, funston rn: how many clean-up bulls are needed after estrus synchronization and artificial insemination? beef report. lincoln: university of nebraska; 2016. 37. beef 2007-08 part ii: reference of beef cow-calf management practices in the united states, 2001-08. national animal health monitoring system; 2009. 38. johnson, sk, jones rd: a stochastic model to compare breeding system costs for synchronization of estrus and artificial insemination to natural service. prof anim sci 2008;24:588-595. 39. farin pw, chenoweth pj, mateos er, et al: beef bulls mated to estrus synchronized heifers: singlevs multi-sire breeding groups. theriogenology 1982;17:365-372. 40. perry, ga, smith mf: management factors that impact the efficiency of applied reproductive technologies. in proceedings, applied reproductive strategies in beef cattle; 2015; davis,ca. p. 208-232. 41. kramer rw, smith dr, rupp gr, et al: estimation of calving date in beef cattle with realtime ultrasound. prof ani sci 2016;32:322-327. 42. perry ga, larimore el, perry bl, et al: grazing behavior of drylot-developed beef heifers and the influence of postinsemination supplementation on artificial insemination pregnancy success. prof anim sci 2015;31:264-269. 43. dunne ld, diskin mg, boland mp, et al: the effect of preand post-insemination plane of nutrition on embryo survival in beef heifers. j anim sci 1999;69:411-417. 44. crookshank hr, elissalde mh, white rg, et al: effect of transportation and handling of calves upon blood serum composition. j anim sci 1979;48:430-435. 45. merrill, ml, ansotegui, rp, burns, et al: effects of flunixin meglumine and transportation on establishment of pregnancy in beef cows. j anim sci 2007;85:1547-1554. 46. harrington te, king me, mihura he, et al: effect of transportation time on pregnancy rates of synchronizied yearling beef heifers. colorado state university beef program report 1995; p 81-86 47. cooke rf, arthington jd, austin br, et al: effects of acclimation to handling on performance, reproductive, and physiological responses of brahman-crossbred heifers. j anim sci 2009;87:3403-3412. 295 clinical theriogenology • volume 11, number 3 • september 2019 48. cooke rf, arthington jd, araujo bd, et al: effects of acclimation to human interaction on performance, temperament, physiological responses, and pregnancy rates of brahman crossbred cows. j anim sci 2009;87:4125-4132. 49. wolfenson, d, roth, z: impact of heat stress on cow reproduction and fertility. anim front 2019;9:32-38. 50. hamblen h, hansen pj, zolini am, et al: thermoregulatory response of brangus heifers to naturally occurring heat exposure on pasture. j anim sci 2018;96:3131-3137. 51. sprott lr, selk ge, adams dc: review: factors affecting decisions on when to calve beef females. prof animal sci 2001;17:238-246. 52. bader jf, kojima fn, schafer dj, et al: a comparison of progestin-based protocols to synchronize ovulation and facilitate fixed-time artificial insemination in postpartum beef cows. j anim sci 2005; 83:136-143. 53. larson je, lamb gc, stevenson, js et al: synchronization of estrus in suckled beef cows for detected estrus and artificial insemination and timed artificial insemination using gonadotropin-releasing hormone, prostaglandin f2alpha, and progesterone. j anim sci 2006;84:332-342. 54. slusher t: how do i profit from improved reproduction. in proceedings: applied reproductive strategies in beef cattle; 2013; stanton, va. p. 9-10. 55. whittier jc, caldwell rw, anthony rv, et al: effect of a prostaglandin f2α injection 96 hours after introduction of intact bulls on estrus and calving distribution of beef cows. j anim sci 991;69:4670-4677. 56. woolums ar, berghaus rd, smith dr, et al: producer survey of herd-level risk factors for nursing beef calf respiratory disease. j am vet med assn 2013;243:538-547. 57. lalman dl, keisler dh, williams je, et al: influence of postpartum weight and body condition change on duration of anestrus by undernourished suckled beef heifers. j anim sci 1997;75:2003-2008. 58. cushman ra, kill lk, funston rn, et al: heifer calving date positively influences calf weaning weights through six parturitions. j anim sci 2013;91:4486-4491. 296clinical theriogenology • volume 11, number 3 • september 2019 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 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/presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice hypertrophic osteopathy in a pregnant doe with bronchopneumonia   hypertrophic osteopathy in a pregnant doe with bronchopneumonia elisa heacock,a anabelle bates,b kate wulster,c timothy manzi,c daniela luethy,b michelle abrahamb aveterinary clinical sciences, school of veterinary medicine, philadelphia bdepartment of large animal internal medicine, school of veterinary medicine, kennett square cdepartment of radiology, school of veterinary medicine, kennett square university of pennsylvania, pa abstract an 18 month old boer doe in late pregnancy was presented for severe distal limb swelling and lameness of all 4 limbs, hyporexia, and tachypnea. doe was hyporexic for several days prior to presentation and was ~ 137 141 days pregnant. radiography showed that the doe had bronchopneumonia, and palisading periosteal proliferation of her metacarpal bones and proximal phalanges. a diagnosis of hypertrophic osteopathy secondary to bronchopneumonia, or pregnancy, or the combined effects of bronchopneumonia and pregnancy, was made. three months following parturition and treatment for pneumonia, doe was re-evaluated, at that time, both pneumonia and orthopedic changes were almost completely resolved. this represents the first reported case of hypertrophic osteopathy in a domestic goat. it is uncertain what role bronchopneumonia and advanced pregnancy had in the development of hypertrophic osteopathy. keywords: hypertrophic osteopathy, caprine, bronchopneumonia, pregnancy background hypertrophic osteopathy (ho) is a rare skeletal disease that is characterized by palisading osteoproliferation beneath the periosteum of long bones secondary to thoracic or less commonly abdominal pathology.1 it is most commonly reported in humans as hypertrophic osteoarthropathy that causes severe distal limb swelling and subperiosteal proliferations, but can also result in symmetrical digital clubbing of fingers and toes and arthropathy.1 in humans, it can rarely be a primary congenital disease, but is most often a secondary disease process.1 in animals, it has only been documented as a secondary disease, most commonly secondary to pulmonary disease; however, reports have also demonstrated intraabdominal disease, intrapelvic disease and pregnancy to have a strong association with ho,2-4 with no sex predilection in humans.2 most case reports in veterinary medicine documented ho in dogs; however, it has also been described in horses, deer, cats, cows, fowl, and takin, a wild bovid species.5,6 there are multiple theories as to the pathophysiology of musculoskeletal changes associated with ho, including both neurogenic and vascular pathways that are suspected to result in an elevation of circulating growth factors causing limb swelling and osteoproliferation.2 however, more recent literature has demonstrated a strong relationship between elevations in vascular endothelial growth factor (vegf) and platelet-derived growth factor (pdgf) with ho.1,4 pulmonary disease often results in pulmonary vessel shunting, thus decreased fragmentation of megakaryocytes into platelets that normally occurs in pulmonary vessels.1 these megakaryocytes and thrombocytes then lodge in distal vasculature causing occlusion of vessels and local hypoxia.1 this is the proposed mechanism for elevations in pdgf and vegf in distal capillaries. these then induce mesenchymal growth and periosteal proliferation of the long bones of the distal limbs.1 these growth factors are also elevated in systemic inflammatory conditions, hypoxia, and aberrant vascularization that can be associated with a variety of other disease processes as the primary disease causing secondary ho.1 clinically, veterinary patients ultimately diagnosed with hypertrophic osteopathy typically present with a stiff gait, reluctance to rise, or lameness characterized by painful and diffusely swollen distal limbs.7 radiographically, ho is a palisading pattern of periosteal reaction along long bones of distal extremities, particularly the proximal phalanges, metacarpal and metatarsal bones.2 we report the first documented case of presumptive hypertrophic osteopathy in a domestic goat. this report documents clinical theriogenology • volume 12 number 4 • december 2020516   the disease progression and resolution of suspected ho, secondary to the diagnosis of severe pneumonia and/or parturition. case presentation an 18-month old, 79.1 kg boer doe, ~ 137 141 days in pregnancy, was presented on an emergency basis to the university of pennsylvania’s new bolton center in march of 2019 for evaluation of suspected pregnancy toxemia. owner reported that the doe had a history of pain in all 4 limbs and reluctance to stand for extended intervals that had progressed to complete recumbency and lethargy in the week prior to presentation. in the days immediately prior to presentation, the doe had become anorexic, increasingly tachypneic and dull. on physical examination, doe appeared depressed and moderately painful with a heart rate of 112 beats per minute (normal: 70 90), a respiratory rate of 64 breaths per minute (normal: 15 40) and a rectal temperature of 38.5°c (normal: 37.8 39.7°). no jugular pulses were visible or palpable and cardiac auscultation was unremarkable. harsh bronchovesicular sounds were present in all lung fields with an increased respiratory effort, but no adventitious lung sounds were noted. doe was ambulatory but had a stiff gait and was reluctant to stand for extended intervals. doe had diffuse swelling distal to the mid radius or mid tibia, heat, and an overt pain response on palpation of each limb. doe had a markedly distended abdomen and an edematous vulva, consistent with her stage of pregnancy. a transabdominal ultrasonographic examination revealed 2 fetal heart beats and fetal fluids of normal echogenicity; however, no spontaneous fetal movements were noted. reproductive department recommended supportive care until parturition to allow for full fetal development. the remainder of the physical examination was unremarkable. initial blood chemistry revealed hyperglycemia (142 mmol/l; reference range: 54.0 93.0 mmol/l), hyperlactatemia (3.3 mmol/l; reference range: 0.6 1.4 mmol/l), and elevated betahydroxybutyrate (1.7 mmol/l: reference range; < 0.9 mmol/l). the packed cell volume (pcv) (29%; reference range: 28 44%) and total protein (6.4 mg/dl; reference range: 6.2 8.0 mg/dl) were within normal limits. doe was admitted to the hospital and a jugular catheter was placed. initial treatment consisted of a fluid bolus of a balanced electrolyte solution (1 liter, isolyte®, b. braun medical inc., bethlehem, pa) followed by a continuous intravenous infusion of a balanced electrolyte solution (3 ml/kg/hour, isolyte®, b. braun medical inc.) mixed with dextrose (1.7 mg/kg/min, dextrose 50% solution, covetrus, dublin, oh) and thiamine (2.4 mg/kg/hour, thiamine hcl, henry schein, dublin, oh). intravenous fluids were continued for the first 6 days of hospitalization. plasma fibrinogen concentrations were evaluated at admission and discharge (647 mg/dl decreased to 560 mg/dl; reference range: 200 400 mg/dl). blood chemistry panel performed at admission revealed mild abnormalities including sodium of 142 mmol/l (reference range: 143 149 mmol/dl), total protein of 6.3 g/dl (reference range: 6.4 7.4 g/dl), albumin of 2.6 g/dl (reference range: 2.8 3.8 g/dl), ck of 136 u/l (reference range: 28130 u/l), and ggt of 61 u/l (reference range:10 60 u/l). doe was treated with pantoprazole (westward, eatontown, nj) 1 mg/kg iv, every 24 hours, florfenicol (nuflor®, merck, madison, nj) 40 mg/kg sq, every 72 hours, and flunixin meglumine (covetrus, dublin, oh), 1.1 mg/kg iv every 24 hours. a rumen transfaunation was performed. during her first night in hospital, the doe became febrile with a rectal temperature of 40.6°c and was anorexic despite grain, alfalfa hay, and water being directly offered. treatment doe remained tachypneic and sternally recumbent in a straw-bedded stall during the 3 days prior to parturition. manual assistance was necessary for the doe to stand and to change from left to right sternal recumbency every 4 hours. despite regular flunixin meglumine treatment, the doe remained painful in all 4 limbs. distal forelimb radiographs were taken on second day of hospitalization to investigate swelling, heat and pain evident on palpation of these limbs that had palisading periosteal proliferations on the lateral and medial aspects of both metacarpal bones and proximal phalanges. radiographs revealed a clinical theriogenology • volume 12 number 4 • december 2020 517   moderate spiculated, palisading periosteal reaction affecting the proximal aspect of the third and fourth metacarpal bones bilaterally (left > right). the lateral aspect was more affected than the medial. a smooth periosteal reaction (figure 1) was also evident on both left and right radius. there was diffuse moderate soft tissue swelling and thickening of the antebrachium and distal soft tissues of both thoracic limbs. shortly after the distal limb radiographs were obtained, percutaneous perineural blocks of the median, ulnar, tibial and peroneal nerves were performed using liposomal bupivacaine (2.0 ml per limb, nocita®, aratana, leawood, ks) for extended local analgesia. however, no significant improvement in doe’s comfort level was noted. a brief transcutaneous thoracic ultrasonographic examination was also performed at this time that demonstrated evidence of pulmonary disease including coalescing comet tails visible cranioventrally in both lungs. there was no evidence of pleural or pericardial effusion as well as no evidence of obvious mediastinal masses. thoracic radiographs were acquired the following day (day 3). thoracic radiographs demonstrated a multilobar, moderate interstitial to alveolar pulmonary pattern, predominantly affecting the perihilar region, but also cranioventrally and superimposed over the heart-base with prominent air bronchograms (figure 2). a few pleural fissure lines were noted. these findings were most consistent with bronchopneumonia and parapneumonic pleural effusion. oxytetracycline (5 mg/kg iv, every 12 hours, liquamycin la-200®, zoetis, kalamazoo, mi) therapy was initiated to provide additional antibiotic therapy targeted specifically at the most common agents causing pneumonia in goats. all other treatments were continued. a second reproductive examination (day 3) revealed 2 fetal heartbeats. however, there were concerns that fetal viability may be compromised if doe’s health continued to deteriorate; therefore, parturition was induced with cloprostenol (1.5 ug/kg im, estrumate®, merck, madison, nj) and dexamethasone (0.25 mg/kg iv, phoenix, st. joseph, mo). doe was monitored frequently for kidding. the following night (day 4), 3 kids were delivered via assisted vaginal delivery. the next day (day 5), the patient appeared significantly brighter and more comfortable, however, was still mildly reluctant to stand and allow the kids to nurse. to provide additional pain relief a morphine treatment protocol was instituted (0.2 mg/kg im, every 8 hours, hospira inc., lake forest, il). the warmth that was previously palpable in all 4 limbs had substantially decreased; however, the diffuse limb swelling remained. doe’s appetite slowly returned over a 24 hour period. after almost 2 days of ambulating independently and allowing her kids to nurse appropriately the doe was tapered off intravenous fluid therapy and injectable flunixin meglumine was discontinued (day 6). with the doe’s continued improvement, injectable antibiotics, morphine and pantoprazole were discontinued (day 7). doe was treated with oral meloxicam beginning postpartum (2.8 mg/kg loading dose, then 1.0 mg/kg every 48 hours, carlsbad technology inc., carlsbad, ca) and discharged from the hospital (day 8). nine weeks after discharge, the doe returned for re-evaluation of the bronchopneumonia and ho. owner reported that the doe was doing well at home, but still had a mildly stiff gait. on presentation, the doe was bright, alert, and responsive with a physical examination within normal limits. the diffuse limb swelling had resolved in all 4 limbs and the doe was not painful on palpation. repeat radiographs were obtained of her distal forelimbs and thorax. limb radiographs revealed marked improvement of the periosteal reaction of the metacarpi (figure 3) and a persistent mild amount of irregular periosteal reaction proximally. thoracic radiographs revealed that the previously noted pulmonary pattern had resolved (figure 4). it was determined that the doe’s bronchopneumonia and ho had resolved. differential diagnosis hypertrophic osteopathy secondary to bronchopneumonia versus hypertrophic osteopathy secondary to pregnancy versus hypertrophic osteopathy secondary to both bronchopneumonia and pregnancy clinical theriogenology • volume 12 number 4 • december 2020518     figure 1. radiograph of right forelimb showing periosteal reaction suggestive of hypertrophic osteopathy figure 2. thoracic radiograph showing a multilobar interstitial to alveolar pattern, most suggestive of bronchopneumonia with secondary pleural effusion. figure 3. repeat radiograph of right forelimb showing near complete resolution of hypertrophic osteopathy figure 4. repeat radiograph of thorax showing resolution of diffuse alveolar pattern, suggesting resolution of bronchopneumonia. clinical theriogenology • volume 12 number 4 • december 2020 519   discussion this report documents a case of a periparturient boer doe with severe pneumonia and suspected secondary hypertrophic osteopathy to either the pneumonia, pregnancy, or a combination of both. although the pathophysiology of ho is becoming better understood, deficits remain. it is well documented that vegf and pdgf lead to the phenotypic changes associated with ho.1 both pulmonary disease and less commonly pregnancy have been implicated in causing secondary ho and this case may give further evidence to support that primary diseases can be associated with secondary ho.4 in most cases, ho develops secondary to severe thoracic disease such as neoplasia, granulomatous disease, or suppurative pneumonia as the primary lesion.8 in this case, the patient was initially afebrile and the pneumonia did not cause a moderately elevated plasma fibrinogen concentration, so it is uncertain whether the severity of the thoracic changes are comparable to the severe pulmonary disease reported in cases of ho described in the literature. we cannot conclude that pneumonia was not ultimately the cause of ho in this case; however, it challenges the current theories solely implicating thoracic disease as the inciting factor in the pathogenesis of ho and whether pregnancy may exacerbate pulmonary disease. furthermore, a recent study revealed 5 cases of presumptive ho in a takin that were associated with pregnancy.4 the takin in that study had clinical signs similar to the doe in the present report, including shifting leg lameness, a stiff gait, and distal limb swelling that were confirmed as ho with radiographs and one ct scan.4 clinical signs subsided and recurred with subsequent pregnancies up to 6 times in 5 takin.4 these investigators hypothesized that a genetic predisposition for the development of ho during pregnancy existed within the relatively small population of takin from which these animals were derived.4 the mechanism by which ho could be secondary to pregnancy is unknown; however, it has been documented that vegf is increased locally in the uterine environment during pregnancy associated with the proinflammatory environment that exists during the third trimester of pregnancy.9,10 released in response to elevations in vegf, uterine nk cell–derived ifn-γ causes alterations in the uterine vasculature and stroma.9 vascular endothelial growth factor is a potent angiogenic factor that has a partially causative role in ho, and is also involved in many physiological processes during pregnancy.9 it has been recognized as a paracrine and autocrine modulator of angiogenesis, remodeling, and vascular permeability in the endometrium, decidua, and trophoblast,9 but in cases of ho in periparturient animals, these authors question whether it has a more systemic role as well, at least in some animals. in the human literature, increases in serum vegf during the first trimester of pregnancy have been reported as normal and have been recognized as being positively correlated with stage of pregnancy, beta-hcg, estradiol, and progesterone concentrations.11 clinical signs associated with ho in the case reported herein were most severe during the days just prior to parturition, yet much improved postpartum. administration of nonsteroidal antiinflammatories and performing perineural blocks did not alleviate this patient’s pain, consistent with cases reported in humans.2 following parturition, the doe would stand and was willing to eat; however, it is difficult to conclude that pregnancy was the primary cause of ho in this patient, as the improvement in clinical status could have been due to decreased weight and relief from other metabolic strains that accompanied carrying three kids in utero. also lending support to pulmonary disease as the primary cause of ho, was that clinical improvement may have been due to an increase in pulmonary capacity achieved after delivering the kids, as well as treatment of bronchopneumonia with antibiotics. in this case, pregnancy may have exacerbated the pulmonary disease or been the primary cause of ho. however, it is uncertain whether the presence of bronchopneumonia, or pregnancy, or both led to the development of secondary ho in this doe. learning points  although very uncommon, hypertrophic osteopathy should be considered a differential in goats with painful, diffusely swollen legs, especially when thoracic disease or pregnancy is concurrent.  pain medications and supportive care may be helpful for mitigating the detrimental effects of hypertrophic osteopathy; however, treatment of the initiating cause often results in resolution of the disease. clinical theriogenology • volume 12 number 4 • december 2020520    there is potentially a genetic basis in the development hypertrophic osteopathy that requires further exploration. conflict of interest none to declare. references 1. callemeyn j, van haecke p, peetermans w, et al: clubbing and hypertrophic osteoarthropathy: insights in diagnosis, pathophysiology, and clinical significance. acta clin belg 2016;71:123-130. 2. chakraborty rk, sharma s: secondary hypertrophic osteoarthropathy. statpearls. treasure island; statpearls publishing llc:2020. 3. lavoie j, carlson g, george l: hypertrophic osteopathy in three horses and a pony. j am vet med assoc 1992;201:1900-1904. 4. marinkovich m, wisner e, brenner d: distal limb swelling and periosteal productive reaction in periparturient sichuan takin (budorcas taxicolortibetana): five cases of presumptive hypertrophic osteopathy. j zoo wildl med 2019;50:437-446. 5. chaffin m, ruoff w, schmitz d, et al: regression of hypertrophic osteopathy in a filly following successful management of an intrathoracic abscess. equine j 1990;22:62-65. 6. guyot h., sandersen c., rollin f: a case of hypertrophic osteoarthropathy in a belgian blue cow. can vet j 2011;52:1308-1311. 7. brodey rs: hypertrophic osteoarthropathy in the dog: a clinicopathologic survey of 60 cases. j am vet med assoc 1971;159:1242-1256. 8. mair t, dyson s, fraser j, et al: hypertrophic osteopathy (marie's disease) in equidae: a review of twenty‐four cases. equine vet j 1996;28:256-262. 9. ashkar a, di santo j, croy b: interferon gamma contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy. j exp med 2000;192:259-270. 10. mor g, cardenas i, abrahams v, et al: inflammation and pregnancy: the role of the immune system at the implantation site. ann n y acad sci 2011;1221:80-87. 11. daniel y, geva e, lerner-geva l, et al: levels of vascular endothelial growth factor are elevated in patients with ectopic pregnancy: is this a novel marker? fertil steril 1999;72:1013-1017. clinical theriogenology • volume 12 number 4 • december 2020 521 initial assessment of the postpartum mare and foal in clinical practice, the first examination of a normal mare and foal is typically performed from 8 24 hours postpartum. this allows adequate time for normal postfoaling events to proceed uninterrupted, including neonatal adaptation to ex utero life, mare-foal bonding, and passive immune transfer. mare owners should be advised to adhere to the 1-2-3 rule: by 1 hour the foal should be standing, by 2 hours the foal should be nursing and by 3 hours the mare should have passed fetal membranes. if there is a deviation from the 1-2-3 rule or other complications arise, earlier intervention is often warranted. during initial examination, the focus is on the general health and wellness of the mare and the foal and the assessment of the pair’s readiness for turnout. a complete physical examination is performed for both mare and foal. in addition, to routine parameters assessed during the physical examination, the mare’s mammary gland should be assessed to ensure that mammary development and milk production are adequate. the perineum is assessed for any evidence of foaling trauma. fetal membranes should be routinely examined for completeness of chorioallantois and for evidence of pathology. healthy fetal membranes weigh ~ 10 11% of the foal’s bodyweight. if fetal membranes are not completely presented, the mare’s reproductive tract should be examined for retention. identifying signs of placental pathology may aid in management of the mare and foal. a through physical examination of the neonate foal is necessary to identify any signs of illness or congenital disorders. postpartum mare and foal: management and considerations justin mcnaughten hanover shoe farms, inc., hanover, pa abstract veterinarians are often responsible for ensuring that the general health and wellbeing of the mare and foal while simultaneously overseeing the mare’s breeding. management of mare and foal is often tailored to suit the individual needs of the breeder. expectations and experience level for a commercial breeding operation may differ substantially from the first-time boutique hobby breeder. although differences among clients may exist, the clinical approach should be supported by scientific data and an accepted standard of practice. this paper reviews considerations for breeding the postpartum mare and discusses management of common conditions affecting the mare and foal. keywords: mare, foal, postpartum, breeding management, pregnancy careful observation of the unrestrained foal ensures that the foal appears physically mature and mentally appropriate. immature foals may exhibit a silky coat, lack mane and tail hair, have a domed forehead, display significant tendon and ligament laxity, and are often small.1 foals are assessed while walking and standing on a firm surface to identify any lameness or the presence of angular or flexural limb deformities. umbilicus is examined for signs of herniation, infection or evidence of a patent urachus. scrotum of male foal is palpated to determine if both testes are descended and for evidence of an inguinal or scrotal hernia. additional common neonatal abnormalities include cleft palate, entropion, scleral hemorrhage, petechiation, and rib fracture. at this time, routine screening blood samples taken for hematology, proteins, igg, plasma fibrinogen, and serum amyloid a may provide information on efficacy of passive transfer and early signs of disease such as neonatal isoerythrolysis or sepsis.1 if physical examinations do not reveal any medical or orthopedic abnormalities, the pair may be turned out into a small paddock. regular turnout is beneficial for both, as exercise improves uterine clearance in the mare and aids in skeletal, muscular and orthopedic development of the foal. management and considerations colostrum quality of a mare’s colostrum can be evaluated immediately postfoaling prior to foal nursing, using quantitative and qualitative methods. quantitative measurement of igg concentrations in colostrum can be obtained by a radial immunodiffusion assay clinical theriogenology 2021; 13: 265 at a diagnostic laboratory.2 brix refractometers offer a mareside qualitative measurement that correlates optical density of the sample with igg antibody content. measuring colostrum quality is not only a useful method to predict the likelihood of successful passive transfer but also to determine whether the colostrum is suitable for harvest and storage. the practice of banking colostrum is especially useful in the treatment of failure of passive transfer before gut closure and prevention of neonatal isoerythrolysis in at risk foals. colostrum donors should be screened to ensure that they do carry antibodies against equine red blood cells.3 even though immunoglobulin activity of frozen colostrum decreases over time, the nutrient value of older colostrum can be useful for neonates. postpartum complications and fertility postpartum complications may arise following a dystocia or the normal delivery of a foal. in these cases, accurate diagnosis is based on a comprehensive workup and rapid initiation of an appropriate therapeutic plan improves outcomes. the scope of these proceedings does not allow the necessary attention to the management of these conditions; therefore, the author directs the reader to an in-depth review of common postpartum problems.4 these conditions may not only be life-threatening but they can also be detrimental to future fertility.4,5 delay in uterine involution is appreciable in cases of dystocia, trauma to the reproductive tract and retained fetal membranes.6 alternations in the normal involution process include an increased influx of neutrophils into the endometrium and a delay in the glandular redifferentiation to the pregravid state.7 consequently, these compromised mares have lower pregnancy rates when compared to normal foaling mares.7 regardless of the inciting cause, mares should be given adequate time to allow for uterine involution to proceed prior to rebreeding. uterine involution understanding the process of uterine involution is of clinical importance in the management of postpartum mare. involution is a multifaceted process involving tissue remodeling, clearance of debris, contraction of endometrial glands, and transformation of the endometrium.8 mares are unique in that uterine involution occurs rapidly after parturition. in the normal mare, histological remodeling is complete by day 15 postpartum. size of rapidly decreases during the first week postpartum and may reach the pregravid state within 23 days postpartum9,10 in normal mares, transrectal ultrasonography reveals an echogenic fluid that begins to decrease at ~ day 5 and should be undetectable by day 15.10 uterine contractions aid in the expulsion of these luminal contents consisting of cellular debris and bacterial contaminates. normal lochia is nonfetid and may vary from red-tinge to a yellowish mucopurulent. in mares with complications during pregnancy or parturition, uterine involution may be delayed.7 although involution is functionally complete by day 7 and histologically complete by day 14, this process coincides with the first postpartum estrous cycle (foal heat) occurring between days 5 20.8,11,12 authors have reported up to a 20% decrease in pregnancy rates and an increased rate of early embryonic loss for foal heat breeding.12,13 day of ovulation, and the presence of intrauterine fluid have been implicated as factors negatively affecting reproductive outcomes associated with foal heat breeding. mares ovulating at or beyond 10 days postpartum had higher pregnancy rates than those ovulating prior to day 10 postpartum even though uterine involution was not complete.13 if ovulation and fertilization occur on day 10 postpartum, the embryo is sequestered in the oviduct for 5.5 days until uterine involution is complete.14 by contrast, the uterine environment may not be able to support embryonic development when mares conceive prior to day 10 postpartum.14 presence of intrauterine fluid at the time of foal heat breeding was predictive of lower pregnancy rates.10,15 researchers have also demonstrated that advancing age had a significant effect on pregnancy rates and embryo loss for mare bred on foal heat.12,15 researchers in brazil concluded that a mare older than 10 years of age should not be bred on foal heat if embryo loss is to be reduced and reproductive performance optimized.15 reproductive examination reproductive examination of the mare is typically scheduled within first 10 days postfoaling. a visual and functional evaluation of perineal conformation and the vestibulo-vaginal sphincter may reveal evidence of urine pooling, pneumovagina, or foaling trauma affecting the caudal reproductive tract.14 routine vaginoscopy is an essential part of the examination as it aids in identification of cranial lesions and small rectovaginal fistulas. manual palpation of the cervix may identify gross lesions, and if there is concern of cervical compromise, a follow-up examination should be performed during diestrus. mares with poor perineal conformation or a prepartum episiotomy should have caslick’s surgery performed. a breeding stitch is warranted to protect integrity of the surgical site during breeding or while reproductive procedures are ongoing. transrectal palpation of the postpartum uterus allows assessment of uterine size and tone, whereas ultrasonography allows for evaluation of uterine lumen. ultrasonography may reveal excess uterine fluid, flocculent debris, and also the presence of fetal membranes. in the normal postpartum mare, uterine tone is increased and > 90% of the uterus is palpable by day 5.9 in contrast, a poorly involuted uterus is often large, atonic, and lacks the presence of palpable rugae. presence of intrauterine fluid during uterine involution is a normal phenomenon.10 repeat examinations are performed to monitor the depth and character of uterine fluid and therapeutic treatment, including ecbolic agents and uterine lavage may be initiated to aid in uterine clearance, based on clinician preference.14 exercise and access to turnout should also be encouraged to promote uterine clearance. in a normal mare, presence of intrauterine clinical theriogenology 2021; 13: 266 fluid should resolve by day 15 postpartum and urine pooling should cease once uterine involution is complete.10,16 follicular activity is assessed whether the mares is destined to be bred during foal heat or the second postpartum estrous cycle. endometrial cultures obtained during the first postpartum estrus will often be positive as the uterus is readily contaminated during parturition. mixed bacterial growth is common with streptococcus equi zooepidemicus being the most commonly identified organism.17 accompanying endometrial cytology is useful to differentiate between infectious endometritis and postpartum bacterial contamination and can help confirm normally progressing uterine involution.11 recently, the endometrial cytological changes of mares with normal and abnormal parturitions were evaluated.18 uterine neutrophil counts increased until day 4 postpartum, followed by a constant decline, and can thus be readily used to evaluate progress in uterine involution. postpartum uterine treatments higher pregnancy rates are reported in mares that are bred after day 10.13 therefore, strategies to improve fertility for the first postpartum estrous cycle focus on methods to delay first ovulation or hasten uterine involution. regardless of the strategy, mares should have access to turnout as exercise will aid in uterine evacuation and improve gross involution.19 multiple exogenous steroid regimens have been investigated, with varying results. estradiol-17β alone did not delay the first postpartum ovulation.20 however, a combination of estradiol-17β and progesterone was an effective means to delay and synchronize first postpartum ovulation.21,22 progestin supplementation effectively delayed ovulation and pregnancy rates were higher in mares that ovulated after day 15.10,23 although progestin supplementation may be an effective method to delay ovulation, there was no ultrasonographic evidence that it hastens uterine involution.10 in mares with delayed uterine clearance, progestin treatment may be contraindicated.14 myriad treatment protocols aimed at hastening uterine involution and improving pregnancy rates have been tested, including postfoaling uterine lavage and various ecbolic agents. in the normal mare, postfoaling uterine lavage substantially decreased endometrial polymononuclear cells but did not improve pregnancy rates or hasten uterine involution.24-26 repeated ecbolic treatment failed to reduce uterine size or improve reproductive outcomes.27,28 recently, researchers at the university of gluck assessed effects of mycobacterium cell wall fraction (mcwf) on uterine involution; it decreased the interval to bacterial clearance and increased expression of proinflammatory cytokines.29 these effects may reduce tissue inflammation, ultimately hastening tissue repair. therefore, mcwf may be of value in the management of the foaling mare although effects on fertility are yet to be investigated. alternatively, the simplest approach to improve fertility after foaling is to forego foal heat. prostaglandin treatment on day 5 postovulation will hasten the return to estrus. researchers demonstrated favorable outcomes when comparing mares bred on the first postpartum cycle (foal heat) to mares bred on a prostaglandin-induced second postpartum estrus cycle, including a decreased rate of early embryonic loss and increased first cycle, seasonal, and per cycle pregnancy rates for mares bred on the prostaglandin-induced cycle.30 considerations for breeding on foal heat breeding on foal heat is often a controversial topic amongst veterinarians and breeders. the decision is often based on anecdotal experiences and validated by citing selected literature supporting or condemning the practice. increasing mare age, day of ovulation, and disturbances during the postpartum period delaying uterine involution are all associated with decreased reproductive outcomes.6,10,13,15 to improve outcomes, breeding on foal heat should be avoided for mares with any of the aforementioned factors or any other conditions that may delay uterine involution. nevertheless, foal heat breeding can be successfully implemented into breeding programs by abiding data-driven guidelines and strict selection criteria. pregnancy rates can be > 70% for mares bred on foal heat;12,31 free range management of these mares may be an important factor contributing to the success of foal heat breeding.12 as such, conditions affecting either the mare or foal that require stall confinement should preclude the mare from breeding on foal heat. as mare age has a substantial effect on reproductive outcomes, it is a key factor to be considered prior to breeding on foal heat.12,32 authors have suggested that mares > 10 years of age are not suitable for foal heat breeding.15 presence of intrauterine fluid is associated with lower pregnancy rates, regardless of reproductive status.33 although reports of early initiation of postbreeding uterine lavage may decrease early embryo loss for mares bred on foal heat, conflicting reports have demonstrated that the presence of intrauterine fluid, even with appropriate treatment at foal heat breeding, is associated with decreased reproductive outcomes.12,34 intrauterine fluid is a normal finding up to day 15 postpartum.10 however, if intrauterine fluid is present at foal heat breeding, the author suggests that the mare should not be bred, as reproductive outcomes will be decreased. ovulations occurring > 10 days postpartum are associated with improved foal heat breeding outcomes; thus, breeding on foal heat is not recommended for mares expected to ovulate prior to day 10 postpartum as histological involution will likely be insufficient to support pregnancy. initiation of protocols designed to delay the first postpartum ovulation may be warranted for mares foaling late in the breeding season.10 positive endometrial cultures are common amongst normal postparturient mares.17 they are often associated with contamination during parturition and exhibit minimal effect with foal heat fertility.35 an increased concentration of neutrophils clinical theriogenology 2021; 13: 267 in endometrial biopsy was more indicative of poor foal heat breeding outcomes; however, logistical constraints preclude their use in clinical practice.35 serial endometrial cytology in combination with clinical findings may be a valuable method for determining suitability for foal heat breeding.18 suitable candidates for breeding on foal heat include mares < 10 years of age, with free access to turnout, no periparturient disturbances and an absence of intrauterine fluid at the time of breeding with ovulation coinciding at ≥ day 10 postpartum. breeding mares that do not satisfy these criteria should be delayed until at least the second postpartum estrous cycle to improve reproductive outcomes. management of postpartum anestrus postpartum anestrus occurs in mares foaling early in the season that are not exposed to supplemental light during late pregnancy. this phenomenon is most commonly observed in mares foaling in the northern hemisphere before april 1st or in the southern hemisphere before october 1st.36 often a normal foal heat and ovulation are observed, but ovaries subsequently become hard and inactive with a typical follicle measuring < 15 20 mm in diameter.12,36 several hormonal regimes including gnrh agonists, progestins, the combination of progestins with estradiol, and dopamine antagonists have been utilized to restore cyclicity in the postpartum mare.37,38 the author’s preferred approach is based on firsthand experience using gnrh agonists or dopamine antagonists to successfully treat postpartum anestrus. buserelin (gnrh agonist) treatment resulted in adequate ovulatory, pregnancy, and return to estrus responses.38 buserelin (12.5 µg, intramuscular twice daily) is recommended. ovulation is induced with hcg once a dominant follicle ≥ 35 mm is identified. buserelin is discontinued once ovulation is detected. treatment protocols using dopamine antagonists, sulpiride (1 mg/kg, intramuscular twice daily) and domperidone (1.1 mg/kg, oral once daily) may also stimulate follicular development, ovulation, and luteinization of follicles in anestrus mares.36,39 ovulation can be induced with hcg or a gnrh agonist. a lack of follicular response within 21 days of treatment represents a failure of the mare to respond and treatment should be discontinued. to reduce the incidence of postpartum anestrus, mares destined to foal early in the season or mares that have a history of postpartum anestrus should be under supplemental lighting during late pregnancy.40 lighting regimes should be initiated by december 15th for the northern hemisphere or june 15th for the southern hemisphere.36 positive effects of supplemental light are not limited to decreasing the incidence of postpartum anestrus; use of artificial blue light delivered via a mask worn by mares in late pregnancy improved the average birth weight of foals and decreased pregnancy length compared to controls.41 common foal issues substantial time and effort are dedicated to the breeding process and the production of a foal, whether it is a destined to be an elite athlete or beloved companion. unfortunately, numerous congenital and pathological conditions may affect the foal. therefore, it is extremely important to provide proper veterinary care and attention during foal’s growth and development. the following are commonly recognized conditions that may affect the foal. failure of passive transfer an igg concentration < 400 mg/dl is indicative of failure of passive transfer. igg concentrations can be increased through the ingestion of good quality colostrum (> 23% brix) prior to gut closure, which occurs between 18 24 hours of age, or treatment of hyperimmune intravenous plasma transfer. plasma may increase igg concentrations by at least 200 mg/dl per liter. however, many insurance companies will require an igg concentration > 800 ng/dl. plasma transfusion reactions may occur that are evident by clinical signs including tachypnea, tachycardia, dyspnea, sweating, muscle fasciculations, fever, and sudden death.42 therefore, transfusions should be started slowly while patients are monitored. if clinical signs develop, transfusion should be stopped immediately.43 corticosteroid administration is helpful in decreasing inflammation. neonatal isoerythrolysis neonatal isoerythrolysis (ni) is an immunogenetic disease in which the maternal antibodies are directed against the foal’s red cell antigens.44 although more common in multiparous mares, ni can occur with first pregnancy.44 ingestion and absorption of these antibodies causes hemolysis. clinical signs appear between 24 36 hours postnursing and vary based on the severity and degree of anemia. pale to icteric mucus membranes, lethargy, rapid to labored breathing, tachycardia, and seizure like activity may occur. in severe cases, death may ensue. the condition is preventable. mares at risk of producing an ni foal may identified by screening the pregnant mare’s serum for anti-red cell antibodies or red cell typing based on the absence of factors aa, qa and ua. jaundiced foal agglutination (jfa) test may be utilized to detect the presence of maternal antibodies in the colostrum directed against red cell antigens.45 if the mare is at risk or jfa test is positive, the mare’s colostrum should be stripped and discarded. during this time the foal should muzzled to prevent suckling from that mare. the foal should be given colostrum from a suitable donor. feeding of the foal is supplemented for 24 48 hours or until a jfa test has determined that it is safe for the foal to begin nursing. clinical theriogenology 2021; 13: 268 rib fractures during parturition it is not uncommon for rib fractures to occur. rib fracture may involve a single or multiple ribs, unilateral or bilateral. fractures most commonly occur at or near the costochondral junction involving ribs 2 7. if rib fractures are suspected based on auscultation or palpation, ultrasonography should be performed to confirm diagnosis and to determine the number and degree of rib displacement, and any evidence of hemorrhage.46 initiation of medical or surgical management is based on the number of ribs involved, degree of displacement and presence of pneumothorax or hemothorax. conservative approach involving stall rest for up to 2 4 weeks and supportive care are often successful. one advantage of surgery is rapid stabilization of the fracture mitigating the risk further displacement and trauma to the lungs, heart, and diaphragm. flexural limb deformity a musculoskeletal condition as a result of excessive tendon or ligament laxity or contracture affecting the normal position of the joint. laxity and contracture may occur simultaneously affecting multiple limbs. diagnosis is by observing the foal’s walk and stand on a firm surface. treatment is based on the severity of the condition. analgesics, splinting the affected limb, toe extensions and oxytetracycline treatment have been utilized for tendon contracture. however, oxytetracycline may be nephrotoxic and is contradicted in a compromised foal.47 tendon laxity will often improve with controlled periods of exercise as the foal gains muscle tone. moderate to severe cases are recognized by the toe being elevated off the ground or the fetlocks being dropped. in these cases, heel extensions will support the fetlock joint.48 tendon laxity may be exacerbated by excessive turnout leading to muscle fatigue and administration of oxytetracycline. umbilical lesions umbilical infections, patent urachus, and umbilical hernia are common amongst foals. ultrasonographic examination of the umbilical remnants is useful in diagnosis and management of suspected omphalitis.49 the majority of cases will resolve within 3 weeks with medical management. a patent urachus can be congenital or occur secondary to omphalitis. in uncomplicated cases a patent urachus will often spontaneously resolve. topical application of silver nitrate may chemically cauterize and hasten the closure of the umbilical remnant. surgery is indicated when umbilical infections and patent urachus do not respond to medical management. small umbilical hernias will often resolve; however, if the hernia has not resolved by 4 to 6 months, surgery or banding is often warranted.50 septic joints joint infections should be the primary differential for a foal with an acute onset of lameness. careful palpation of the affected limb may identify synovial distension. foals with suspected septic joint require immediate veterinary attention and referral for intensive management that may improve outcome. antimicrobial selection should be based on culture and sensitivity results of the synovial fluid. broad spectrum antimicrobials should be initiated pending culture results. hematology, serum amyloid a, fibrinogen and total nucleated cell count of the synovial fluid should be monitored to assess response to treatment. intraarticular injection, reginal limb perfusion, and therapeutic lavage of the joint space may improve outcomes. management of the foal parasite control foal may invariably become infected with a number of helminths but parascaris spp., commonly-known as ascarids or roundworms, are the most common and of clinical importance.51,52 in addition to environmental exposure, the mare is another potential source of infestation for the foal. ingestion of parasites may occur while suckling as the larvae may be present in the dam’s milk, or due to coprophagy. deworming the pregnant mare with an ivermectin-containing product 4 6 weeks prefoaling and 48 hours postfoaling is recommended to decrease the potential worm burden. heavy ascarid burdens may cause weight loss, poor body condition, lethargy, colic, and the risk of life-threatening small intestinal impactions.52 migration of larvae is associated with signs of upper airway disease, including cough, and nasal discharge.53 heavy worm burdens of strongyloides westeri (> 2000 eggs per gram) have been associated with severe foal diarrhea.54 deworming programs should include anthelminthic treatments that are effective against adult, migrating, and encysted larvae with the intent of decreasing the potential overall worm burden. the following information is based on recommendations from the american association of equine practitioner’s parasite control guidelines. a minimum of 4 treatments during the first year of life, ~ every 3 4 months. targeted therapy based on fecal egg counts is not recommended in foals. the first treatment given between 2 3 months of age should be efficacious against ascarids. dewormers should subsequently be given every 3 4 months. at weaning, fecal egg counts may be utilized to differentiate between primarily strongyles or ascarids worm burdens. at ~ 9 months, use a product that is efficacious against tapeworms and ascarids. vaccination goal of prefoaling mare vaccination is to increase antibody concentrations in the mare’s colostrum aiding in development of the foal’s passive immunity. absorption of immunoglobulins clinical theriogenology 2021; 13: 269 offers protection against infections. as maternal antibodies may interfere with a foal’s response to vaccination, the foal’s vaccine schedule is based on whether or not a mare was vaccinated prepartum. the american association of equine practitioner’s vaccination guideline is available online and offers additional information and a comprehensive schedule for core and riskbased foal vaccinations.55 conflict of interest there are no conflicts of interest to declare. references 1.stoneham sj: the normal postpartum foal. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p.63-68. 2. mccue pm: evaluation of colostrum quality: brix refractometry. in: dascanio jj, mccue pm: editors. equine reproductive procedures. new york city; john wiley & sons: 2014. p. 297-298. 3. mccue pm: colostrum banking. in: dascanio jj, mccue pm: editors. equine reproductive procedures. new york city; john wiley & sons: 2014. p. 299-301. 4. turner rm: post-partum problems: the top ten list. proc am assoc equine pract 2007;53:305-319. 5. vandeplassche m, bouters r, spincemaille j, et al: observations on involution and puerperal endometritis in the mare. irish vet j 1983;37:126–32 6. steiger k, kersten f, aupperle h, et al: puerperal involution in the mare – morphological studies in correlation with the course of birth. theriogenology 2002;58:783-786. 7. belz jp, glatzel ps: fertility in mares after a disturbed as well as an undisturbed puerperium. significance of histological and cytological examinations of the uterus. tierärztliche praxis 1995;23:267-272. 8. gygax ap, ganjam vk, kenney rm: clinical, microbiological and histological changes associated with uterine involution in the mare. j reprod fertil suppl1979;27:571-578. 9. katila t, koskinen e, o’ijala m: evaluation of the postpartum mare in relation to foal heat breeding. i. rectal palpation, vaginoscopy, and ultrasound scanning. j vet med 1987;35:92-100. 10. mckinnon ao, squires el, harrison la, et al: ultrasonographic studies on the reproductive tract of mares after parturition: effect of involution and uterine fluid on pregnancy rates in mares with normal and delayed post partum ovulatory cycles. j am vet med assoc 1988;192:350-353. 11. stanton me: uterine involution. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition. ames; wileyblackwell: 2011. p. 2291-2293. 12. blanchard tl, thompson ja, brinsko sp, et al: mating mares on foal heat: a five-year retrospective study. proc ann conv am assoc equine pract 2004;50:525-530. 13. loy rg: characteristics of post partum reproduction in the mare. vet clin n amer: large anim practice 1980;2:345-59. 14. blanchard tl, macpherson ml: breeding mares on foal heat. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition. ames; wiley-blackwell: 2011. p. 2294-2301. 15. malheiros de souza jr, gonçalves pb, bertolin k, et al: age-dependent effect of foal heat breeding on pregnancy and embryo mortality rates in thoroughbred mares. j equine vet sci 2020;90:1-5. 16. easley kj: diagnosis and treatment of vesicovaginal reflux in the mare. vet clin north am equine practice 1988;4:407-416. 17. card c, lopate c: infectious diseases of the puerperal period. in: youngquist rs, threlfall wr: editors. current therapy in large animal theriogenology, 2nd edition. st louis: saunders, elsevier, 2007; pp. 138-143. 18. krohn j, eilenberg rd, gajewski z, et al: lochial and endometrial cytological changes during the first 10 days post-partum with special reference to the nature of foaling and puerperium in equine. theriogenology 2019;139:43-48. 19. hooper rn, blanchard tl, taylor ts, et al: identifying and treating uterine prolapse and invagination of the uterine horn. vet med 1993;88:60-65. 20. arrott c, macpherson m, blanchard t, et al: biodegradable estradiol microspheres do not affect uterine involution or characteristics of post partum estrus in mares. theriogenology 1994;42:371-384. 21. loy rg, evans mj, pemstein r, et al: effects of injected ovarian steroid on reproductive patterns and performance in post-partum mares. j reprod fertil 1982;suppl32:199-204. 22. bristol f, jacobs ka, pawyshyn v: synchronization of estrus in postpartum mares with progesterone and estradiol-17β. theriogenology 1983;19:779-785. 23. loy rg, hughes jp, richards wp, et al: effects of progesterone on reproductive function in mares after parturition. j reprod fertil 1975;suppl 23:291-295. 24. blanchard tl, varner dd, brinsko sp, et al: effects of postparturient uterine lavage on involution in the mare. theriogenology 1989;32:527-535. 25. mitchell ar, scott cj, cheong sh, et al: the effect of routine postpartum uterine lavage on endometrial cytology, culture, and pregnancy rates in thoroughbred broodmares. j equine vet sci 2019;80:5-9. 26. mccue pm, hughes jp: the effect of postpartum uterine lavage on foal heat pregnancy rate. theriogenology 1990;33:1121-1129. 27. blanchard tl, varner dd, brinsko sp, et al: effects of ecbolic agents on measurements of uterine involution in the mare. theriogenology1991;36:559-571. 28. gunduz mc, kasickci g, kaya hh: the effect of oxytocin and pgf2α on the uterine involution and pregnancy rates in postpartum arabian mares. anim reprod sci 2008;104:257-263. 29. fedorka ce, murase h, loux sc, et al: the effect of mycobacterium cell wall fraction on histologic, immunologic, and clinical parameters of postpartum involution in the mare. j equine vet sci 2020;90. 30. lowis tc, hyland jh: analysis of post-partum fertility in mares on a thoroughbred farm in southern victoria. aust vet j 1991;68:304-306. 31. camillo f, marmorini p, romagnoli s, et al: fertility at the first post partum estrous compared with fertility at the following estrous cycles in foaling mares and with fertility of nonfoaling mares. j equine vet sci 1997;17:612-616. 32. lane ea, mlj bijnen ml, osborne m: key factors affecting reproductive success of thoroughbred mares and stallions on a commercial stud farm. reprod dom anim 2016;51:181-187. 33. pycock jf, newcombe jr: the relationship between intraluminal uterine fluid, endometritis, and pregnancy rate in the mare. eq practice 1996;18:19-22. 34. malschitzky e, schilela a, mattos alg, et al: effect of intra-uterine clinical theriogenology 2021; 13: 270 fluid accumulation during and after foal-heat and of different management techniques on the post partum fertility of thoroughbred mares. theriogenology 2002;58:495-498. 35. kattila t, koskinen e, oijala m, et al: evaluation of the postpartum mare in relation to foal heat breeding. ii. uterus swabbing and biopsies. j am vet med assoc 1988;35:331-339. 36. leblanc mm, mckinnon ao: breeding the problem mare. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition. ames; wiley-blackwell: 2011. p. 2620-2642. 37. card c: hormone therapy in the mare. in: samper jc: editor. equine breeding management and artificial insemination. st louis; saunders elsevier; 2009; p. 89-97. 38. wolfsdorf ke, fedorka ce, lu kj: the effect of buserelin on reproductive performance in the transitional and anestrous mare. j equine vet sci 2018;66:98. 39. besognet b, hansen bs, daels pf: induction of reproductive function in anestrous mares using a dopamine antagonist. theriogenology 1997;47:467-480. 40. palmer e, driancourt ma: some interactions of season of foaling, photoperiod and ovarian activity in the equine. livest prod sci 1983;110:197-210. 41. nolan mb, walsh cm, duff n: artificially extended photoperiod administered to pre-partum mares via blue light to a single eye: observations on gestation length, foal birth weight and foal hair coat at birth. theriogenology 2017;100:126-133. 42. morris dd: the haemolymphatic system: blood and plasma therapy. in: higgins aj, snyder jr: editors. the equine manual, 2nd edition. london; elsevier saunders:1995. p. 525-527. 43. fennell lc, anderson ga, savage cj, et al: use of colostrum banking and immunoglobulin status of foals on a commercial thoroughbred stud farm. j am vet med assoc 2010;236:1085-1090. 44. johnson jr: neonatal isoerythrolysis. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition. ames; wiley-blackwell: 2011. p. 353-360. 45. blackmer jm, costa lr, koch c: the jaundiced foal agglutination test. vet technician 2002;23:577-579. 46. jean d, picandet v, macieira g, et al: detection of rib trauma in newborn foals in an equine critical care unit: a comparison of ultrasonography, radiography and physical examination. equine vet j 2007;39:158-163. 47. vivrette s, cowgill ld, pascoe j, et al: hemodialysis for treatment of oxytetracycline-induced acute renal failure in a neonatal foal. j am vet med assoc 1993;203:105-107. 48. lescun tb, admas s: tendon and ligament disorders. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition. ames; wiley-blackwell: 2011. p. 468-474. 49. reef vb. pediatric abdominal ultrasonography. in: reef vb: editor. equine diagnostic ultrasound. philadelphia; wb saunders: 1998. p. 364-403. 50. stick ja: abdominal hernias. in: auer ja, stick ja: editors. equine surgery. 3rd edition. st louis, mo; saunders elsevier: 2006. p. 491-499. 51. austin s, dipietro ja, foreman jh, et al: parascaris equorum infections in horses. comp contin educ pract vet 1990:12;1110-1118. 52. cribb nc, cote nm, boure lp, et al: acute small intestinal obstruction associated with parascaris equorum infection in young horses: 25 cases (1985–2004). new zeal vet 2006;54:338-343. 53. clayton hm: ascarids. recent advances. vet clin north am equine pract 1986;2:313-328. 54. netherwood t, wood jl, towsend hg, et al: foal diarrhea between 1991 and 1994 in the united kingdom associated with clostridium perfringens, rotavirus, strongyloides westeri, and clostridium spp. epidemiol infect 1996;117:375-383. 55. vaccination guidelines aaep http://www.aaep.org/info/ vaccination-guidelines-265 clinical theriogenology 2021; 13: 271 postpartum mare and foal: management andconsiderations stem cells: what, how, and why? stem cells: what, how, and why? diane carlisle department of neurological surgery, university of pittsburgh, pittsburgh, pa abstract the use of stem cells clinically has a solid history and also broad undeveloped potential. as we learn more about these cells and their properties, our ability to harness their potential for future benefit will grow. however, there is also significant confusion surrounding discussions of stem cells, due to vague definitions regarding the types of stem cells and controversies that are inherent in the uses of some types of stem cells. this review discusses the types of stem cells, their advantages and disadvantages, and how they can be used clinically and for research. specifically, the differences between various/common types of pluripotent stem cells is discussed. the utility of pluripotent stem cells compared to lineage restricted stem cells is also considered. keywords: pluripotent stem cells, embryonic stem cells, tissue-specific stem cells, directed differentiation introduction although there are several kinds of stem cells, in scientific and lay conversations, often the term ‘stem cells’ is used without specificity, which can lead to confusion and misunderstanding. this review focuses on the major types of stem cells, including the types of pluripotent stem cells, as well as lineagerestricted stem cells, their advantages and disadvantages, and their uses in the clinic and in the laboratory. types of stem cells before discussion of the characteristics of stem cells, their advantages and disadvantages, and their clinical and research uses, it is important to clarify the types of stem cells. in general, there are 3 major types of stem cells: pluripotent stem cells, lineage restricted or somatic stem cells, and totipotent stem cells. pluripotent stem cells are the most versatile of stem cells. these cells are named as such because they have the potential to become any cell type that develops from an embryo. indeed, the classic type of pluripotent stem cell is the embryonic stem cell. other sources of pluripotent stem cells are somatic cell nuclear transfer stem cells and induced pluripotent stem cells. beyond the ability to differentiate into any cell type in an embryo, pluripotent stem cell lines are immortal, with unlimited proliferation potential. embryonic stem cells embryonic stem cells are derived by dissecting and growing the cells that would become the embryo from a blastocyst stage embryo, before any differentiation into germ layers begins.1-3 human embryonic stem cells cells drew significant public attention upon publication in 1998,3 because they are generated from human embryos after in vitro fertilization. however, embryonic stem cells generated from other animals were in existence for more than a decade before the generation of human embryonic stem cells by the thomson group. mouse embryonic stem cells were first published in 1981,1 and other animal species followed.3-6 somatic cell nuclear transfer stem cells somatic cell nuclear transfer can also be used to generate embryos for the production of pluripotent stem cells. these cells are generated by removing the haploid nucleus from an oocyte, transferring in the diploid nucleus from a somatic cell, followed by chemical activation of the oocyte.7-11 like embryonic stem cells, human somatic cell nuclear transfer cells are highly controversial. first, they are controversial because they can also be used for reproductive cloning, as first publicized by dolly the sheep.12 second, much like human embryonic stem cells, the method involves the production of an early blastocyst from which the pluripotent stem cells are obtained.13 although human reproductive cloning has clinical theriogenology • volume 12 number 3 • september 2020 183 not been achieved for ethical reasons, cloned nonhuman primates have been made14 and thus it is theoretically possible, although inefficient. induced pluripotent stem cells induced pluripotent stem cells are somatic cells that are induced, through the re-expression of developmental genes, to become pluripotent. as with other types of pluripotent stem cells, this was first achieved in rodent cells,15 followed by other animals including humans.15 because they do not require the use of an embryo or oocyte during their production, their generation and use is less controversial than other types of pluripotent stem cells. uses of pluripotent stem cells pluripotent stem cells are used for many purposes, some of which were mentioned above. the most well-known uses are the generation of genetically manipulated animals, the production of somatic cells for transplantation, and the production of somatic cells for research. to generate genetically manipulated animals, pluripotent stem cells are isolated from embryos of the animal of interest, e.g. a mouse. those cells are genetically manipulated, typically using crispr/cas9 technology. the manipulated cells can then be injected into a blastocyst, which results in the generation of a chimeric animal. if any of the genetically altered cells contribute to the germline, then breeding the animal results in offspring that are genetically altered.16 this is commonly used to generate research mice to model diseases in the laboratory.16 one of the most powerful uses of pluripotent stem cells is the ability to direct differentiation into any cell type in the body. in this context, pluripotent stem cells can be expanded into large batches and differentiated into the cell type of interest, which is then used for research purposes. this strategy is particularly important for cells that are postmitotic in adults, such as neurons. proliferative cells can be obtained from a patient, grown in culture, and investigated; however, if the cell type of interest in the disease is postmitotic, obtaining a sufficient quantity of cells for experimental use is impossible. examples include neurodegenerative diseases such as alzheimer’s disease or amyotrophic lateral sclerosis (als), where the cell type of interest is the neuron. live human neurons generally cannot be studied, except through the generation of neurons from pluripotent stem cells. research in human neuroscience has accelerated exponentially since human pluripotent stem cells were discovered. the same directed differentiation procedure can be used to generate somatic cells for transplantation, with the caveat that the pluripotent stem cell generation, growth, and subsequent differentiation are done under general manufacturing procedure conditions, which are the practices required to make clinical grade products. however, a significant caveat to the translation of this technology to the clinic is that it is extremely difficult to ensure that differentiation efficiency is 100% before transplantation. less than perfect efficiency could result in the transfer of undifferentiated cells that result in teratoma formation in the patient. pluripotent stem cell characteristics pluripotent stem cells have incredible potential, but they are not easy to work with, for several reasons. they require a full knowledge of the starting cell source and ongoing quality control to be sure that they are and remain pluripotent. until this point, all the information above has applied to pluripotent stem cells from any source. this implies that embryonic stem cells, somatic cell nuclear transfer cells, and induced pluripotent stem cells are equivalent. however, there are data demonstrating that this is not true. when embryonic stem cells, somatic cell transfer stem cells, and induced pluripotent stem cells are examined side-by-side, there are phenotypic differences among them. although the exact consequences of these differences are unclear, we cannot assume that all sources of pluripotent cells will perform equivalently in the generation of somatic cells, regardless of whether the final use is research or transplantation. a key point that needs to be satisfied before using any pluripotent stem cell is to ensure that it is truly pluripotent. there are several levels of stringency that need to be met to prove pluripotency.18 the clinical theriogenology • volume 12 number 3 • september 2020184 easiest is to look at molecular criteria. for example, cells can be analyzed for the expression of pluripotent genes and the absence of expression of somatic genes.19 similar protein profiles can also be examined.19 however, molecular markers are not perfect and analysis of pluripotent functionality is always preferred. more rigorous is to look at the ability to differentiate into all 3 germ layers using in vitro differentiation protocols.18 the most rigorous method is to truly test pluripotentiality by producing chimeric animals and demonstrating that the pluripotent stem cell contributed to each germ layer plus germ cells.2 immortality is a key characteristic of pluripotent stem cells; however, this does not imply stability in culture. in fact, karyotypic instability was recognized as a concern early in the use of the cells.20 improved culture methods have decreased the occurrence of large chromosomal aberrations, but careful analysis demonstrates that with passage, pluripotent stem cell lines change over time, with numerous chromosomal differences, most less than 100 kilobases of dna in size.21 furthermore, these changes can alter the ability to differentiate into certain somatic cell types.21 thus, careful and constant quality control of pluripotent stem cells is required. advantages and disadvantages in summary, pluripotent stem cells are a powerful tool for medicine and research. their potency can be harnessed to generate somatic cells for research and for the clinic, as well as to advance our understanding of developmental biology. however, they have some key disadvantages. most importantly, anything less than completely efficient differentiation could result in development of subsequent cancer in a patient after transplantation.22 other disadvantages for clinical use include that differentiated pluripotent stem cells for transplantation are not immune matched to recipients and would be rejected without immune system suppression in the transplant recipient. lineage restricted stem cells the other major stem cell type is the lineage restricted stem cell, also referred to as somatic stem cell, tissue stem cell, or multipotent stem cell. these cells are obtained from mature tissues and are the natural source of cell regeneration in tissues during healing or other physiological cell expansions. these cells are typically restricted to differentiation into cells within their own germ layer, or even within their own tissue. advantages and disadvantages of lineage restricted stem cells: the major disadvantage of lineage restricted stem cells is that they are not pluripotent. they can become some cell types in the body, but not all. however, for clinical purposes, they have some significant advantages. first, since they are not pluripotent, they will not form teratomas upon transplantation. second, they have a long history of clinical use, demonstrating safety and efficacy. some examples include hematopoietic stem cells, mesenchymal stem cells, and tissue-specific stem cells. hematopoietic stem cells have been in use in the clinical setting for decades, particularly in the treatment of cancer. for this purpose, a person’s natural hematopoietic stem cells are destroyed and replaced using donated bone marrow, a major source of hematopoietic stem cells.23 mesenchymal stem cells are highly versatile, although their differentiation capabilities are controversial. initial studies demonstrated increased proliferation of mature cell types in many organs, including from other germ layers, after the injection of mesenchymal stem cells, leading to speculation that these cells could be pluripotent.24 other studies that traced the source of increased cell proliferation after mesenchymal stem cell transplantation demonstrated that the new cells generated do not come from the transplant, but from the tissue, suggesting that mesenchymal stem cells are powerful endocrine modulators that activate endogenous tissue stem cells without directly contributing to the tissue.25 other tissue specific stem cells are known to exist through their properties: the ability to proliferate to maintain the tissue stem cell reservoir while at the same time providing cells for differentiation into mature cell types. an example is spermatogonial stem cells, which have been clinical theriogenology • volume 12 number 3 • september 2020 185 characterized by their properties in the testes, although they are difficult to specifically isolate for study.26,27 totipotent stem cells totipotent stem cells are the least understood and also the least studied, but they are known to exist by their function. these cells distinguish themselves from pluripotent stem cells because they can become any cell type in the body (as pluripotent cells can) as well as the placenta (pluripotent cells cannot).2 these cells exist in the very earliest stages of development before the blastocyst forms; however, because they have not been isolated or grown in culture, relatively little is known about their biology.28 cell fate specification given that the uses of stem cells that generate the most interest are for regeneration of somatic cells for clinical and research uses, understanding the tenets of cell fate specification are key to utility of stem cells. two approaches to cell fate specification are direct reprogramming of somatic cells and use of developmental biology signaling programs for specification. in direct reprogramming, somatic cells of 1 type are treated with cocktails (genetic, protein, or small molecules) to directly turn 1 cell type into another. this approach skips the intermediate step of creating a stem cell, which makes it relatively fast. the biological methods for this are still being refined, but it has been accomplished for some applications, such as turning fibroblasts into neurons.29 however, the methods are experimental, and it has yet to be done under gmp conditions. additionally, because it eliminates the intermediate step of creating an immortal stem cell that allows for rapid expansion, it is not ideal when starting material is limited. the more established approach is to use the known signaling pathways of developmental biology to specify cell fate. this technique can be used starting from pluripotent stem cells, by initiating primitive streak specification, or it can be used on lineage restricted stem cells by initiating maturation of tissue specific progenitors into mature cell types. pluripotent stem cells can be guided through specification to endoderm, mesoderm, or ectoderm then to mature cells within each germ layer using developmental growth factors or with small molecule analogues that evoke the equivalent cellular signaling response.30 bioengineered matrices can also contribute to cellular signaling to direct differentiation. confirmatory assays similar to the confirmation of pluripotency, there are multiple tests of varying rigor for verifying the effectiveness of directed differentiation. the least rigorous is analysis of the gene expression profile, i.e. mrna from differentiated cells can be analyzed to determine if the expression profile matches that of a somatic cell of the same type that is obtained directly from the tissue of interest. similarly, but more rigorous is the analysis of the protein profile, demonstrating that the cell not only generated appropriate mrna, but translated that mrna to the appropriate protein profile. however, as previously mentioned, functional testing is the gold standard for confirmation of cell specification, demonstrating that the cell functions as and, ultimately, can integrate into the desired tissue.30 conclusion stem cells are fascinating with a solid history of providing clinical benefit and broad applicability for new uses as our understanding of how to harness their potential grows. the applications continue to expand as we learn how to tap into the potential of all that stem cells have to offer. conflict of interest there are no conflicts of interest to declare. clinical theriogenology • volume 12 number 3 • september 2020186 references 1. evans mj, kaufman mh: establishment in culture of pluripotential cells from mouse embryos. nature 1981;292: 154-156. 2. simerly cr, navara cs, castro ca, et al: establishment and characterization of baboon embryonic stem cell lines: an old world primate model for regeneration and transplantation research. stem cell res 2009. doi: s18735061(09)00022-1 [pii]10.1016/j.scr.2009.02.004. pubmed pmid: 19393591. 3. thomson ja, itskovitz-eldor j, shapiro ss, et al: embryonic stem cell lines derived from human blastocysts. science 1998;282:1145-1147. pubmed pmid: 9804556. 4. cibelli jb, stice sl, golueke pj, et al: transgenic bovine chimeric offspring produced from somatic cell-derived stemlike cells. nat biotechnol 1998;16:642-646. pubmed pmid: 9661197. 5. saito s, sawai k, ugai h, et al: generation of cloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells. biochem biophys res commun 2003;309:104-113. pubmed pmid: 12943670. 6. mitalipova m, calhoun j, shin s, et al: human embryonic stem cell lines derived from discarded embryos. stem cells 2003;21:521-526. epub 2003/09/12. pubmed pmid: 12968106. 7. ohkoshi k, takahashi s, koyama s, et al: in vitro oocyte culture and somatic cell nuclear transfer used to produce a l live-born cloned goat. cloning stem cells 2003;5:109-115. pubmed pmid: 12930622. 8. hyun s, lee g, kim d, et al: production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein. biol reprod 2003;69:1060-1068. pubmed pmid: 12773429. 9. simerly c, navara c, hyun sh, et al: embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction. dev biol 2004;276:237-252. pubmed pmid: 15581862. 10. mitalipov sm, wolf dp: nuclear transfer in nonhuman primates. methods mol biol 2006;348:151-168. pubmed pmid: 16988378. 11. thongphakdee a, numchaisrika p, omsongkram s, et al: in vitro development of marbled cat embryos derived from interspecies somatic cell nuclear transfer. reprod domest anim 2006;41:219-226. pubmed pmid: 16689885. 12. campbell kh, mcwhir j, ritchie wa, et al: sheep cloned by nuclear transfer from a cultured cell line. nature 1996;380:64-66. pubmed pmid: 8598906. 13. rao ms: are there morally acceptable alternatives to blastocyst derived esc? j cell biochem 2006;98:1054-1061. pubmed pmid: 16598787. 14. liu z, cai y, wang y, et al: cloning of macaque monkeys by somatic cell nuclear transfer. cell 2018;174:245. epub 2018/06/30. doi: 10.1016/j.cell.2018.01.036. pubmed pmid: 29958110. 15. yamanaka s: induction of pluripotent stem cells from mouse fibroblasts by four transcription factors. cell prolif 2008;41 suppl 1:51-56. doi: cpr493 [pii]10.1111/j.1365-2184.2008.00493.x. pubmed pmid: 18181945. 16. jax mice cars: generating mouse models with crispr/cas9 bar harbor md: jackson laboratories; 2016 [cited 2 020]. lt0071:[available from: https://jackson.jax.org/rs/444-buh-304/images/whitepaper_crispr.pdf. 17. zhao mt, chen h, liu q, et al: molecular and functional resemblance of differentiated cells derived from isogenic human ipscs and scnt-derived escs. proc natl acad sci u s a. 2017;114:e11111-e20. epub 2017/12/06. doi: 10.1073/pnas.1708991114. pubmed pmid: 29203658; pubmed central pmcid: pmcpmc5748177. 18. lin k, xiao az: quality control towards the application of induced pluripotent stem cells. curr opin genet dev 2017;46:164-169. epub 2017/08/22. doi: 10.1016/j.gde.2017.07.006. pubmed pmid: 28823985. 19. schatten g, smith j, navara c, et al: culture of human embryonic stem cells. nat methods. 2005;2:455-463. pubmed pmid: 16170868. 20. mitalipova mm, rao rr, hoyer dm, et al: preserving the genetic integrity of human embryonic stem cells. nat biotechnol 2005;23:19-20. epub 2005/01/08. doi: nbt0105-19 [pii]10.1038/nbt0105-19. pubmed pmid: 15637610. 21. tosca l, feraud o, magniez a, et al: genomic instability of human embryonic stem cell lines using different passaging culture methods. mol cytogenet. 2015;8:30. epub 2015/06/09. doi: 10.1186/s13039-015-0133-8. pubmed pmid: 26052346; pubmed central pmcid: pmcpmc4456787. 22. sosa e, chen d, rojas ej, et al: differentiation of primate primordial germ cell-like cells following transplantation into the adult gonadal niche. nature communications. 2018;9:5339. epub 2018/12/19. doi: 10.1038/s41467-018-07740-7. pubmed pmid: 30559363; pubmed central pmcid: pmcpmc6297357. 23. spangrude gj, heimfeld s, weissman il: purification and characterization of mouse hematopoietic stem cells. science 1988;241:58-62. epub 1988/07/01. doi: 10.1126/science.2898810. pubmed pmid: 2898810. 24. krabbe c, zimmer j, meyer m: neural transdifferentiation of mesenchymal stem cells--a critical review. apmis. 2005;113:831-844. epub 2006/02/17. doi: 10.1111/j.1600-0463.2005.apm_3061.x. pubmed pmid: 16480453. 25. gnecchi m, danieli p, malpasso g, et al: paracrine mechanisms of mesenchymal stem cells in tissue repair. methods mol biol 2016;1416:123-146. epub 2016/05/30. doi: 10.1007/978-1-4939-3584-0_7. pubmed pmid: 27236669. 26. valli h, phillips bt, shetty g, et al: germline stem cells: toward the regeneration of spermatogenesis. fertil steril. 2014;101:3-13. epub 2013/12/10. doi: 10.1016/j.fertnstert.2013.10.052. pubmed pmid: 24314923; pubmed central pmcid: pmcpmc3880407. 27. hill jr, dobrinski i: male germ cell transplantation in livestock. reprod fertil dev. 2006;18:13-18. epub 2006/02/16. doi: 10.1071/rd05123. pubmed pmid: 16478598. clinical theriogenology • volume 12 number 3 • september 2020 187 28. baker cl, pera mf: capturing totipotent stem cells. cell stem cell 2018;22:25-34. epub 2018/01/06. doi: 1 0.1016/j.stem.2017.12.011. pubmed pmid: 29304340. 29. treutlein b, lee qy, camp jg, et al: dissecting direct reprogramming from fibroblast to neuron using single-cell rna-seq. nature 2016;534:391-395. epub 2016/06/10. doi: 10.1038/nature18323. pubmed pmid: 27281220; pubmed central pmcid: pmcpmc4928860. 30. carlisle dl: directed differentiation of pluripotent stem cells in vitro. in: gardiner d, editor. regenerative engineering and developmental biology. boca raton, fl: crc press; 2017. clinical theriogenology • volume 12 number 3 • september 2020188 008_ms-8 carlisle clinical theriogenology 2022; 14: 91 serum anti-müllerian hormone concentrations in female alpacas: variations during the reproductive cycle and correlation with ovarian superstimulation response cristian patino,a eduardo arroyo,a michela ciccarelli,a,b jacobo rodriguez,c alan conley,d ahmed tibarya,b adepartment of veterinary clinical sciences, college of veterinary medicine and bcenter for reproductive biology, washington state university, pullman, wa cvista equine colorado clinic, fort collins, co ddepartment of population health and reproduction, school of veterinary medicine university of california, davis, ca abstract earlier, we validated an anti-müllerian hormone (amh) enzyme-linked immunosorbent assay kit for male alpacas. first, we compared the validation data with another kit. there was a high correlation (r2 = 0.94) between these 2 kits. second, we used the latter kit to determine serum amh concentrations during follicular and luteal phases of the reproductive cycle in female alpacas. there were no differences (p = 0.39) in serum amh concentrations in alpacas (n = 11) between peak follicular and luteal phases (mean ± sem, 1.33 ± 0.35 versus 1.18 ± 0.34 ng/ml, respectively). third, we treated female alpacas (n = 13; 5 11 years) after 14day treatment with decreasing doses of porcine follicle-stimulating hormone. there was no effect (p > 0.05) of day of treatment on serum amh concentrations. number of follicles (7 10 mm; mean ± sd [as determined via transrectal ultrasonography]) at end of treatment (12.69 ± 5.25; range: 6 24) was positively correlated (r2 = 0.7; p < 0.01) with serum amh concentrations. to conclude, the kit tested is usable for female alpacas; serum amh concentrations were not affected by the cycle stage nor by ovarian superstimulation treatment. furthermore, a significant correlation between serum amh serum concentrations and response to superstimulation suggested that estimation of serum amh concentrations may be valuable in determining ovarian follicular reserve. keywords: camelid, anti-müllerian hormone, enzyme-linked immunosorbent assay, follicular reserve introduction anti-müllerian hormone (amh), also known as müllerian inhibiting substance, is a homodimeric disulfide-linked glycoprotein member of the transforming growth factor ß superfamily.1 in male mammals, amh is produced by sertoli cells during fetal development.2,3 in contrast, in females, amh is expressed for the first time in the neonatal ovary exclusively by healthy granulosa cells of small primary growing follicles after recruitment from the primordial follicle pool.4,5 anti-müllerian hormone binds to specific receptors to activate intracellular signal transduction mechanisms.6-8 in females, amh has 2 functions: it inhibits primordial follicular growth (to prevent premature exhaustion of ovarian follicular reserve) and reduces fsh responses of preantral and small antral follicles (to suppress the development of nondominant follicles).9 serum concentrations of amh were determined in cows,10,11 ewes,12,13 mares,14 and women.9,15,16 serum concentrations increased a few months after birth, declined until the onset of puberty, then increased again and remained relatively stable throughout the cycle until ovarian senescence/ menopause.11,17,18 in cattle,19-22 women,7,9,23 mice,17 and small ruminants,10,24 serum amh concentrations remained constant during cycles and were positively correlated with the number of antral follicles in women.9 serum amh serum concentrations are considered an indirect measure of the number of morphologically healthy ovarian follicles. it is speculated that a single measurement of amh can predict follicular reserve. there was a good correlation between amh serum concentrations and quality of embryos produced after ovarian superstimulation in cattle,19,25-27 goats,28 and sheep.11,12,29 additionally, in cattle, serum amh concentrations were highly correlated with pregnancy rate and early embryo survival.22,30 there was a positive correlation between amh concentrations (measured just before fsh treatment) and the number of gonadotropin-responsive follicles that developed to the preovulatory stage in several ruminant species (cattle,20,25-27 goats,28 and sheep 11,12,29). although assisted reproductive technologies in camels31 and south american camelids32,33 have increased since the 1990s, studies on amh serum concentrations in female camelids are lacking. female camels present overlapping follicular waves in anovulation that makes ovarian superstimulation more difficult (reviewed by34). ovarian superstimulation protocol for cameclinical theriogenology 2022; 14: 92 lids has been extrapolated from other species, despite many differences in reproductive physiology. in camelids, however, embryo yield after superstimulation is highly variable and often poor due to lack of response, overstimulation, or poor fertilization.31-34 studies on amh in female camelids may shed some light on reasons for this variability in response to superstimulation and ovarian follicular dynamic abnormalities. furthermore, like several mammalian species, estimation of serum amh concentrations in camelids may be a good indicator of ovarian follicular reserve, fertility, and ovarian response to superstimulation. most studies used either human or species-specific enzyme-linked immunosorbent assay (elisa) commercial kits for amh serum concentration determination. validated elisa kits are available for cattle, dogs, horses, humans, mice, and sheep.35 earlier, we validated an amh enzyme-linked immunosorbent assay kit for male alpacas36 and determined that serum amh concentrations remained relatively stable in prepubertal and post pubertal males, but declined in adult males after castration.36 our aims were to validate a commercial elisa kit for female alpacas, characterize serum concentrations of amh in cycling alpacas, and to determine whether there was a correlation between amh serum concentrations and number of antral follicles after fsh ovarian superstimulation. materials and methods animals and sample collection healthy, multiparous, and reproductively sound huacaya female alpacas (5 11 years) were used. animals were housed and evaluated at the washington state university veterinary teaching hospital. animals were fed alfalfa and grass hay at a maintenance level to sustain a body condition score between 2.5 and 3 out of 5 (1 = emaciated, 5 = obese). water and mineral salt were provided ad libitum. animals were on regular internal and external parasite control and vaccination program. animals were not subjected to prior ovarian superstimulation. procedures were approved by the institutional animal care and use committee of washington state university. validation of anti-müllerian hormone enzyme-linked immunosorbent assay commercial kit unavailability of a validated36 commercial amh assay (amh elisa test kit cat # kamh-01; preventia diagnositcs, siasconset, ma) prompted us to validate an alternate commercial kit (amh elisa al-113 kit [ansh labs, webster, tx al-113]). sera collected from 5 male alpacas (before and after castration)36 were used. serum anti-müllerian hormone concentrations during cycle female alpacas (n = 11) were evaluated via transrectal ultrasonography (exago®, universal imaging, new york) using a 7.5 mhz linear array transducer mounted on a polyvinyl chloride extension handle. alpacas were monitored every other day until they developed a mature follicle (7 10 mm in diameter) and had uterine edema. ovulation was induced with intramuscular gnrh (50 μg/animal, [cystorelin®, merial, duluth, ga]) and verified via ultrasonography (corpus luteum) and serum progesterone concentrations. blood samples were collected via jugular venipuncture at ovulation induction (day 0) and during the luteal phase (7 days after ovulation induction). blood samples were allowed to clot, centrifuged (1,500 g for 10 minutes), and harvested sera were stored at – 20°c until analysis. ovarian superstimulation and serum anti-müllerian hormone concentrations females (n = 13) were synchronized with intramuscular gnrh (50 µg/animal [cystorelin®) and progesterone vaginal implants (controlled internal drug release [cidr, easy breed®, 0.3 mg progesterone, zoetis, kalamazoo, mi]), were inserted. ovaries were superstimulated with intramuscular porcine fsh (folltropin®, vetoquinol inc, fort worth, tx), twice daily at 12-hour intervals (day 12 16) in decreasing doses (30, 25, 20, 15, and 10 mg). vaginal implant was removed on day 14, and animals were given intramuscular cloprostenol (250 µg). transrectal ultrasonography was performed on days 0, 7, 12, 17, and 24; ovarian follicular status was determined on days of examination and mature follicles (7 10 mm) were counted at the end of porcine follicle-stimulating hormone (pfsh) treatment. blood samples were collected via jugular venipuncture for amh and progesterone assays on days 0, 3, 7, 12, 17, and 24 (figure 1). ovulation was induced with gnrh when most follicles reached 8 mm diameter. blood samples were allowed to clot, centrifuged (1,500 g for 10 minutes), and harvested sera were stored at – 20°c until essayed. hormone analyses serum amh concentrations were determined from 50 µl duplicate aliquots. monoclonal antibody pairs bind to the noncovalent amh complex that do not detect other related members of the tgf-ß superfamily. samples from each female were included in the same analytical run. intra-assay coefficients of variation for serum pools with high (13.5 ng/ml, n = 14), medium (6.2 ng/ml, n = 14), and low concentrations (2.3 ng/ml, n = 14) were 2.6, 5.5, and 4.1%, respectively. lowest calibrator was 0.41 ng/ ml and values below were estimated by extrapolation. figure 1. synchronization, ovarian superstimulation, and sampling protocol; porcine folliclestimulating hormone (pfsh), controlled internal drug release (cidr), transrectal ultrasonography (us), progesterone (p4) anti-müllerian hormone (amh), and cloprostenol (clp) progesterone concentrations were determined using a competitive enzyme immunoassay.37 briefly, 100 µl of serum was extracted with 2 ml of petroleum ether, the solvent was decanted and dried. extracts were reconstituted with 150 µl of horseradish peroxidase-conjugated progesterone in buffer, and 50 µl aliquots (in duplicate) were transferred to 96-well plates precoated with antisera raised against 11α-hemi-succinate-conjugated progesterone. progesterone analysis was completed in 2 analytical runs. the average intra-assay coefficient of variation of low (0.5 ng/ml) and high (4.9 ng/ml) pools of samples included in each run was 11.4%, with an assay sensitivity of 5 pg/well.37 data analysis serum amh concentrations obtained using preventia® kit were compared to those obtained with ansh (al-113) using pearson’s correlation coefficient. serum amh concentration at ovulation induction (day 0) and luteal phase (day 7) was compared by paired student’s t-test. effect of superstimulation on amh serum concentrations was evaluated using repeated measurement analysis. correlation between the number of antral follicles in response to fsh treatment and serum amh concentrations were determined by linear regression analysis. statistical analysis was performed using commercial software (statistix 10®, analytical software, tallahassee, fl). significance was set at p < 0.05. figure 1. synchronization, ovarian superstimulation, and sampling protocol; porcine follicle-stimulating hormone (pfsh), controlled internal drug release (cidr), transrectal ultrasonography (us), progesterone (p4) anti-müllerian hormone (amh), and cloprostenol (clp) clinical theriogenology 2022; 14: 93 progesterone concentrations were determined using a competitive enzyme immunoassay.37 briefly, 100 µl of serum was extracted with 2 ml of petroleum ether, the solvent was decanted and dried. extracts were reconstituted with 150 µl of horseradish peroxidase-conjugated progesterone in buffer, and 50 µl aliquots (in duplicate) were transferred to 96-well plates precoated with antisera raised against 11α-hemi-succinate-conjugated progesterone. progesterone analysis was completed in 2 analytical runs. the average intra-assay coefficient of variation of low (0.5 ng/ml) and high (4.9 ng/ml) pools of samples included in each run was 11.4%, with an assay sensitivity of 5 pg/well.37 data analysis serum amh concentrations obtained using preventia® kit were compared to those obtained with ansh (al-113) using pearson’s correlation coefficient. serum amh concentration at ovulation induction (day 0) and luteal phase (day 7) was compared by paired student’s t-test. effect of superstimulation on amh serum concentrations was evaluated using repeated measurement analysis. correlation between the number of antral follicles in response to fsh treatment and serum amh concentrations were determined by linear regression analysis. statistical analysis was performed using commercial software (statistix 10®, analytical software, tallahassee, fl). significance was set at p < 0.05. results validation of anti-müllerian hormone enzyme-linked immunosorbent assay commercial kit serum concentrations of amh from 5 male alpacas, before and after castration, as measured by the ansh assay, were closely associated with the previously published results using the preventia kit (r = 0.94, n = 10, p < 0.01) (figure 2). results validation of anti-müllerian hormone enzyme-linked immunosorbent assay commercial kit serum concentrations of amh from 5 male alpacas, before and after castration, as measured by the ansh assay, were closely associated with the previously published results using the preventia kit (r = 0.94, n = 10, p < 0.01) (figure 2). figure 2. correlation between serum anti-müllerian hormone concentrations measured via 2 kits in samples from male (n = 5) alpacas (before and after castration) serum anti-müllerian hormone concentrations during cycle serum amh concentrations (mean ± sem) on the day of ovulation induction (day 0) and during luteal phase (day 7) were 1.33 ± 0.35 ng/ml and 1.14 ± 0.34 ng/ml, respectively (figure 3). luteal phase (occurrence of ovulation in response to gnrh treatment) was verified on day 7 by visible corpus luteum via transrectal ultrasonography and by serum progesterone concentrations (samples had > 1.5 ng/ml; 3.26 ± 1.26 ng/ml [mean ± sem]). there was no difference (p = 0.39) in serum amh concentrations between estrus and diestrus. however, there was a significant variation in serum amh concentrations among females. minimum and maximum serum amh concentrations were 0.027 and 4.28 ng/ml on day 0 and 0.01 and 3.72 ng/ml on day 7, respectively. figure 2. correlation between serum anti-müllerian hormone concentrations measured via 2 kits in samples from male (n = 5) alpacas (before and after castration) serum anti-müllerian hormone concentrations during cycle serum amh concentrations (mean ± sem) on the day of ovulation induction (day 0) and during luteal phase (day 7) were 1.33 ± 0.35 ng/ml and 1.14 ± 0.34 ng/ml, respectively (figure 3). luteal phase (occurrence of ovulation in response to gnrh treatment) was verified on day 7 by visible corpus luteum via transrectal ultrasonography and by serum progesterone concentrations (samples had > 1.5 ng/ml; 3.26 ± 1.26 ng/ml [mean ± sem]). there was no difference (p = 0.39) in serum amh concentrations between estrus and diestrus. however, there was a significant variation in serum amh concentrations among females. minimum and maximum serum amh concentrations were 0.027 and 4.28 ng/ml on day 0 and 0.01 and 3.72 ng/ml on day 7, respectively. figure 3. serum anti-müllerian hormone concentrations on day 0 (estrus) and day 7 after ovulation induction in alpacas (n = 11) ovarian superstimulation and serum anti-müllerian hormone concentrations number of antral follicles (> 7 mm) after fsh treatment (day 17) was variable (12.69 ± 5.25 [mean ± sd]; range 6 24). there were no differences (p > 0.005) in serum amh concentrations within animals across sampling dates (table). however, there was a difference (p > 0.005) among female alpacas. correlation between mean amh serum concentration for each female alpaca and the number of follicles at the end of superstimulation treatment (day 17) was determined. there was a correlation (r = 0.46, p < 0.01) between the number of follicles after fsh treatment and serum amh concentrations (figure 4). further analysis after excluding 1 outlier had higher positive correlation (r = 0.7, p < 0.001; [figure 5]). correlation between amh serum concentrations and the number of corpora lutea could not be assessed as there was ovulation failure in several animals. serum progesterone concentrations increased in animals immediately after cidr insertion and then dropped; however, they remained above 1.5 ng/ml until cidr removal (figure 6). table. serum anti-müllerian hormone concentration (ng/ml) on various days of ovarian superstimulation female day 3 day 7 day 12 day 17 day 24 mean (sem) 1 0.525 0.612 0.564 0.564 0.653 0.58 (0.022) 2 1.337 1.298 0.791 0.736 0.887 1.01 (0.12) 3 3.193 3.123 3.698 2.497 3.917 3.29 (0.24) 4 3.193 3.123 3.698 2.497 3.917 3.29 (0.25) 5 3.213 2.529 2.771 2.04 2.404 2.59 (0.19) 6 4.282 3.719 4.314 7.719 4.582 4.92 (0.71) 7 0.741 0.972 0.802 0.828 0.775 0.82 (0.04) 8 2.961 2.785 2.498 2.131 3.429 2.76 (0.22) 9 0.491 0.491 0.491 1.769 2.286 1.11 (0.39) 10 1.627 4.785 2.454 1.576 2.506 2.59 (0.58) 11 1.292 1.535 1.439 2.052 1.708 1.61 (0.13) 12 0.027 0.01 0.01 0.01 0.061 0.02 (0.01) 13 0.541 0.32 0.313 0.357 0.515 0.41 (0.05) figure 3. serum anti-müllerian hormone concentrations on day 0 (estrus) and day 7 after ovulation induction in alpacas (n = 11) clinical theriogenology 2022; 14: 94 ovarian superstimulation and serum anti-müllerian hormone concentrations number of antral follicles (> 7 mm) after fsh treatment (day 17) was variable (12.69 ± 5.25 [mean ± sd]; range 6 24). there were no differences (p > 0.005) in serum amh concentrations within animals across sampling dates (table). however, there was a difference (p > 0.005) among female alpacas. correlation between mean amh serum concentration for each female alpaca and the number of follicles at the end of superstimulation treatment (day 17) was determined. there was a correlation (r = 0.46, p < 0.01) between the number of follicles after fsh treatment and serum amh concentrations (figure 4). further analysis after excluding 1 outlier had higher positive correlation (r = 0.7, p < 0.001; [figure 5]). correlation between amh serum concentrations and the number of corpora lutea could not be assessed as there was ovulation failure in several animals. serum progesterone concentrations increased in animals immediately after cidr insertion and then dropped; however, they remained above 1.5 ng/ml until cidr removal (figure 6). table. serum anti-müllerian hormone concentration (ng/ml) on various days of ovarian superstimulation female day 3 day 7 day 12 day 17 day 24 mean (sem) 1 0.525 0.612 0.564 0.564 0.653 0.58 (0.022) 2 1.337 1.298 0.791 0.736 0.887 1.01 (0.12) 3 3.193 3.123 3.698 2.497 3.917 3.29 (0.24) 4 3.193 3.123 3.698 2.497 3.917 3.29 (0.25) 5 3.213 2.529 2.771 2.04 2.404 2.59 (0.19) 6 4.282 3.719 4.314 7.719 4.582 4.92 (0.71) 7 0.741 0.972 0.802 0.828 0.775 0.82 (0.04) 8 2.961 2.785 2.498 2.131 3.429 2.76 (0.22) 9 0.491 0.491 0.491 1.769 2.286 1.11 (0.39) 10 1.627 4.785 2.454 1.576 2.506 2.59 (0.58) 11 1.292 1.535 1.439 2.052 1.708 1.61 (0.13) 12 0.027 0.01 0.01 0.01 0.061 0.02 (0.01) 13 0.541 0.32 0.313 0.357 0.515 0.41 (0.05) figure 4. correlation between serum anti-müllerian hormone concentrations and number of follicles after fsh treatment in alpacas (n = 13) figure 5. correlation between serum anti-müllerian hormone concentrations and number of follicles after fsh treatment in alpacas (n = 12) after removal of an outlier figure 4. correlation between serum anti-müllerian hormone concentrations and number of follicles after fsh treatment in alpacas (n = 13) clinical theriogenology 2022; 14: 95 figure 4. correlation between serum anti-müllerian hormone concentrations and number of follicles after fsh treatment in alpacas (n = 13) figure 5. correlation between serum anti-müllerian hormone concentrations and number of follicles after fsh treatment in alpacas (n = 12) after removal of an outlier figure 5. correlation between serum anti-müllerian hormone concentrations and number of follicles after fsh treatment in alpacas (n = 12) after removal of an outlier figure 6. serum progesterone concentrations (mean ± sem) on days 3, 7, and 12 after cidr insertion (day 0) discussion anti-müllerian hormone is a well-conserved glycoprotein among mammalian species. commercial amh assays kits are available for humans, cattle, small ruminants, horses, dogs, and rodents.35 recently, we validated36 a commercial test kit (amh elisa test kit cat # kamh-01; preventia diagnostics) for serum amh in male alpacas. however, the unavailability of this kit necessitated validation of an alternate kit. we used male alpaca serum collected earlier36 to validate the alternate commercial kit (amh elisa, al-113®, ansh lab). there was a high correlation among data obtained with these 2 commercial assay kits. both kits utilized monoclonal antibody pairs in a sandwich assay format. determination of the effect of cycle stage on amh serum concentration has diagnostic applications. if the cycle stage had no effect, a single random test could be used for prognostication of ovarian reserves. serum amh concentrations (with repeatability across multiple estrous cycles) were not influenced by cycle stage (estrus versus diestrus) in cows,11,21,38 goats,24 and mares.39,40 these patterns are expected since amh is expressed only in follicles that have been recruited from primordial follicle pool and not selected for dominance. neither dominant follicles nor corpora lutea are believed to contribute to circulating amh concentrations in these species.7,41,42 only in pigs is amh is expressed in preovulatory follicles and corpora lutea.43 serum amh concentrations during peak follicular activity (follicle diameter > 8 with uterine edema) and luteal phase were not different in our study, similar to other species (cattle,11,44 goats,24 and humans7,42). hence a random determination of serum amh concentration can be used (as in other species) to evaluate ovarian follicular reserve in alpacas. figure 6. serum progesterone concentrations (mean ± sem) on days 3, 7, and 12 after cidr insertion (day 0) discussion anti-müllerian hormone is a well-conserved glycoprotein among mammalian species. commercial amh assays kits are available for humans, cattle, small ruminants, horses, dogs, and rodents.35 recently, we validated36 a commercial test kit (amh elisa test kit cat # kamh-01; preventia diagnostics) for serum amh in male alpacas. however, the unavailability of this kit necessitated validation of an alternate kit. we used male alpaca serum collected earlier36 to validate the alternate commercial kit (amh elisa, al-113®, ansh lab). there was a high correlation among data obtained with these 2 commercial assay kits. both kits utilized monoclonal antibody pairs in a sandwich assay format. determination of the effect of cycle stage on amh serum concentration has diagnostic applications. if the cycle stage had no effect, a single random test could be used for prognostication of ovarian reserves. serum amh concentrations (with repeatability across multiple estrous cycles) were not influenced by cycle stage (estrus versus diestrus) in cows,11,21,38 goats,24 and mares.39,40 these patterns are expected since amh is expressed only in follicles that have been recruited from primordial follicle pool and not selected for dominance. neither dominant follicles nor corpora lutea are believed to contribute to circulating amh concentrations in these species.7,41,42 only in pigs is amh is expressed in preovulatory follicles and corpora lutea.43 serum amh concentrations during peak follicular activity (follicle diameter > 8 with uterine edema) and luteal phase were not different in our study, similar to other species (cattle,11,44 goats,24 and humans7,42). hence a random determination of serum amh concentration can be used (as in other species) to evaluate ovarian follicular reserve in alpacas. the potential for amh serum concentrations as a biomarker for fertility was established for cattle.11,21,30,44 lower amh serum concentrations reflected suboptimal fertility, lower pregnancy rate, and higher incidence of pregnancy loss compared to higher amh serum concentrations. poor reproductive efficiency, a major problem in the camelid industry, is primarily clinical theriogenology 2022; 14: 96 due to ‘repeat breeding’ (75.6%) and recurrent early pregnancy loss (18.3%).45 determination of amh concentrations could help to define the reproductive potential of a female alpaca, prevent excessive mating, and delay diagnosing follicular dynamics disorders. multiple ovulation and embryo transfer programs’ are important tools for genetic improvement in camelids. ovarian superstimulation treatments have often been extrapolated from ruminant studies. such treatments (e.g. pfsh and ecg alone or in combination) were used during the luteal phase and follicular wave emergence with variable results. initiation of treatment in relation to follicular dynamics, dose, protocol, and individual variation was considered for individual variability.31-34 a better understanding of camelids’ ovarian follicular dynamics and physiology of ovulation is needed to develop species-specific protocols. we used pfsh daily in decreasing doses over 5 days after a 14-day course of progesterone.34,46,47 despite a good response (number of follicles), number of ovulations (as estimated by corpora lutea) was very low, a common problem in alpacas.34,47,48 response to hormonal ovarian stimulation treatments in buffalo,49 cattle,19,21,22,25,26 goats,24,28 sheep,12,29 and woman41 was predicted using amh blood concentrations. in our study, serum amh concentrations were highly correlated with the number of follicles developed after ovarian superstimulation treatment. however, further studies using a larger number of females are warranted in order to determine whether alpacas with higher circulating amh concentrations will consistently develop a higher number of follicles in response to fsh ovarian superstimulation. consistent with research in cattle,19,20,25-27 in our study, there were no differences in serum amh concentrations before and after fsh treatment. conclusion we determined serum amh concentrations in male and female alpacas using a commercial assay kit. consistent with other species, serum amh concentrations in female alpacas remained constant during peak follicular and luteal phases. hence a single sample to estimate female ovarian follicular reserve and potentially predict their reproductive performance. serum amh concentrations in female alpacas were positively correlated with the number of dominant follicles that developed following ovarian superstimulation. as demonstrated in ruminants, future experiments should investigate whether there are correlations among amh serum concentrations, oocyte quality, and embryo survival in alpacas. funding partial funding from northwest camelid research foundation. conflict of interest the authors do not have any 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hormone concentration and the relationship with superovulatory response in japanese black cattle. j reprod dev 2017;63:95-100. 28. karakas alkan k, alkan h, kaymaz m. the effect of anti-müllerian hormone and progesterone concentrations on superovulation response and embryo yield in goats. theriogenology 2020;143:1-9. 29. torres-rovira l, gonzalez-bulnes a, succu s, et al. predictive value of antral follicle count and anti-müllerian hormone for follicle and oocyte developmental competence during the early prepubertal period in a sheep model. reprod fertil dev 2014;26:1094-1106. 30. ribeiro es, bisinotto rs, lima fs, et al. plasma anti-müllerian hormone in adult dairy cows and associations with fertility. j dairy sci 2014;97:6888-6900. 31. anouassi a, tibary a: development of a large commercial camel embryo transfer program: 20 years of scientific research. anim reprod sci 2013;136:211-221. 32. vaughan j, mihm m, wittek t: factors influencing embryo transfer success in alpacas: a retrospective study. anim reprod sci 2013;136:194-204. 33. campbell aj, tibary a: embryo transfer in camelids. clinical theriogenology 2017;9:321-327. 34. tibary a: monitoring and controlling follicular activity in camelids. theriogenology. 2018;109:22-30. 35. kumar a, kalra b, patel a, et al: new sensitive anti-müllerian hormone (amh) elisas for non-human primate, rodent, equine, bovine, canine and other species. poster, american  association for clinical chemistry; 2013; houston, tx. 36. ciccarelli m, tibary a, campbell aj, et al: effect of age and castration on serum anti-müllerian hormone concentration in male alpacas. theriogenology 2018;105:174-177. 37. munro c, stabenfeldt g: development of a microtitre plate enzyme immunoassay for the determination of progesterone. j endocrinol 1984;101:41-49. 38. pfeiffer ke, jury lj, larson je: determination of anti-müllerian hormone at estrus during a synchronized and a natural bovine estrous cycle. domest anim endocrinol 2014;46:58-64. 39. ball ba, el-sheikh ali h, scoggin ke, et al: relationship between anti-müllerian hormone and fertility in the mare. theriogenology 2019;125:335-341. 40. almeida j, ball ba, conley aj, et al. biological and clinical significance of anti-müllerian hormone determination in blood serum of the mare. theriogenology 2011;76:1393-1403. 41. hehenkamp wj, looman cw, themmen ap, et al. anti-müllerian hormone levels in the spontaneous menstrual cycle do not show substantial fluctuation. j clin endocrinol metab 2006;91:4057-4063. 42. tsepelidis s, devreker f, demeestere i, et al: stable serum levels of anti-müllerian hormone during the menstrual cycle: a prospective study in normo-ovulatory women. hum reprod 2007;22:1837-1840. 43. almeida f, costermans ngj, soede nm, et al. presence of antimüllerian hormone (amh) during follicular development in the porcine ovary. plos one. 2018;13:e0197894. 44. burns ds, jimenez-krassel f, ireland jl, et al: numbers of antral follicles during follicular waves in cattle: evidence for high variation among animals, very high repeatability in individuals, and an inverse association with serum follicle-stimulating hormone concentrations. biol reprod 2005;73:54-62. 45. tibary a, campbell a, rodriguez js, et al: investigation of male and female infertility in llamas and alpacas. reprod fertil dev 2021;33:20-30. 46. correa je, ratto mh, gatica r: superovulation in llamas (lama glama) with pfsh and equine chorionic gonadotrophin used individually or in combination. anim reprod sci 1997;46:289-296. 47. ratto mh, silva me, huanca w, et al: induction of superovulation in south american camelids. anim reprod sci 2013;136:164-169. 48. ratto mh, gatica r, correa je: timing of mating and ovarian response in llamas (lama glama) treated with pfsh. anim reprod sci 1997;48:325-330. 49. liang a, salzano a, d’esposito m, et al: anti-mullerian hormone (amh) concentration in follicular fluid and mrna expression of amh receptor type ii and lh receptor in granulosa cells as predictive markers of good buffalo (bubalus bubalis) donors. theriogenology 2016;86:963-970. contact agustin j. ruiz agustin.ruiz@gvequine.com.au © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9641, http://dx.doi.org/10.58292/ct.v15.9641 review report perineal urethrostomy in the male horse: a review of the indications, surgical technique, outcome, and complications agustin j. ruiz,a eduardo arroyob agoulburn valley equine hospital, congupna, victoria, australia bcollege of veterinary medicine, washington state university, pullman, wa, usa abstract perineal urethrostomy is a surgical technique performed for temporary or permanent urine diversion from the penile urethra in male patients with obstructive urinary outflow disease, extensive trauma or invasive tumor affecting penis and prepuce, to access the urethra or bladder to remove uroliths, and to treat horses affected with hemospermia or hematuria secondary to urethral rents. perineal urethrostomy has been advocated as the treatment of choice in geldings where hematuria does not resolve with conservative treatment and in stallions with hemospermia caused by urethral defects. most cases of geldings with hematuria are resolved with a single perineal urethrostomy surgery; however, stallions with hemospermia might need a second surgery. perineal urethrostomy also enables access to the urinary bladder in male horses with cystoliths following minimal dissection and without accessing the abdominal cavity. the most common complications directly related to this surgery are hemorrhage, urine scalding, and urethral stricture at the surgical site. the aim of this article is to review comprehensively the indications for perineal urethrostomy surgery, illustrate the surgical technique approach, and discuss outcomes and complications. keywords: perineal urethrostomy, corpus spongiotomy, urethral rent, hemospermia, hematuria introduction perineal urethrostomy (pu) is a surgical technique that entails the creation of a temporary or permanent opening of the urethral lumen most commonly at the level of the ischial arch in male horses. the surgery is indicated in several disorders of the equine penis, prepuce, urethra, and urinary bladder. the most common indication of pu in male horses is for resolution of hemospermia in the breeding stallion and hematuria in geldings due to urethral rents. the aim of this article is to review comprehensively the indications for pu surgery, illustrate the surgical technique approach, and discuss outcomes and complications. indications temporary pu at the level of the ischial arch is indicated for temporary urine diversion from the penile urethra in male patients with obstructive urinary outflow disease, to access the urethra or bladder to remove uroliths, and to treat horses affected with hemospermia or hematuria secondary to urethral rents. permanent pu is indicated in patients with extensive trauma or invasive squamous cell carcinoma affecting the penis and prepuce requiring permanent urinary diversion. one of the most common primary lesions of the equine urethra are longitudinally oriented urethral rents that extend from the surface of the urethra to the corpus spongiosum penis (csp) at the level of the ischial arch.1,2 males of any age and breed are affected, although quarter horses have been proposed to be more predisposed.3 a retrospective study evaluated 24 years of clinical cases presented to a referral facility and found that 33 male horses presenting with hematuria or hemospermia were diagnosed with a urethral rent on urethroscopy (0.08% of the total gelding and stallion population admitted during the study).4 the cause of urethral rents is idiopathic; however, it has been hypothesized to be associated with a combination of factors, occurring simultaneously, favoring the onset of this lesion. these include the abrupt reduction of the urethral diameter (from 3.5–5cm to 1–1.5cm) at the level of the ischial arch and the increased hydrodynamic forces occurring during erection, ejaculation, and final stages of urination that the urethra at the level of the ischial arch is exposed to.2,5 commonly, urethral rents clinically present as hemospermia in stallions and terminal hematuria in geldings mailto:agustin.ruiz@gvequine.com.au http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9641 citation line: clinical theriogenology 2023, 15, 9641, http://dx.doi.org/10.58292/ct.v15.9641 59 because of the communication between the csp and the urethral lumen through the mucosal defect.6,7 in geldings, presence of bright red blood exiting the urethral process at the end of urination apparently occurs when intraluminal urethral pressure decreases suddenly, secondary to forceful contractions of the bulbospongiosus penis (bsp) muscle while the pressure in the csp remains high.2 dysuria, pollakiuria, or pain are not commonly present. in stallions, hemospermia as a result of hemorrhage during or at the end of ejaculation occurs when contraction of the bsp during ejaculation and coitus causes a 45-fold pressure increase within the csp.2,4,8 hemospermia can have severe economic impacts in a breeding program mostly associated with variable degrees of fertility reduction in sires. in stallions on live-cover breeding programs, the first indication of hemospermia is reduced fertility rates and possibly evidence of blood on the mare’s perineal region or stallion’s glans penis following dismount. in stallions that are collected with an artificial vagina, hemospermia will be observed with a variable degree of bloodtinged ejaculate (figure 1). the presence of significant amount of blood in the ejaculate (>50% of whole blood) is associated with severe sperm damage due to the extensive peroxidative damage induced on sperm membranes as a consequence of the iron released by the red blood cells.9,10 a lower degree of blood contamination on the ejaculate (between 5–20% of whole blood per volume) has been associated with decrease in fertility11 and no effect on fertility,10 respectively. in some geldings, hematuria resolves spontaneously. while sexual rest alone seems not to resolve hemospermia in stallions, it is imperative to alleviate the severity of the clinical signs as an adjunct to non-invasive treatment.1,3 a variety of medical treatments have been attempted on affected horses. they are aimed to stop the bleeding and promote healing. these include intravenous administration of formaldehyde solution, oral administration of methenamine, systemic administration of antimicrobial drugs and more recently topical treatment with 4% policresulen solution.12–14 on the other hand, surgical treatment is aimed at decreasing pressure within the csp to minimize or prevent hemorrhage through the rent so that it can heal.1 the most common surgical approaches include pu and corpus spongiotomy (cs). alternative surgical options reported are direct suturing of the urethral defect via a pu with urethroscopy guidance, laser application to the edges of the defect combined with pu and buccal mucosal urethroplasty combined with pu.4,15,16 diagnostic approach to horses presenting with hematuria or hemospermia is based on the identification of different degrees of blood in the ejaculate or urine, followed by visualization of a urethral rent by urethroscopy.8,17 in horses affected with hematuria/hemospermia, clinical examination is generally unremarkable with the occasional presence of mild anemia on laboratory analysis of blood. urine samples collected mid-stream or by bladder catheterization may appear grossly normal. sediment examination on urinalysis or urine reagent strip may show presence of red blood cells. a thorough endoscopic evaluation of the urethra should be performed to examine the urethra, colliculus seminalis, seminal vesicles, urinary bladder, and ureteral openings to identify any evidence of trauma, presence of blood, or discontinuation of the urethral mucosa.8,18 almost exclusively, urethral rents appear as a 5–10 mm long, linear defect on the convex surface of the urethra at the level of the ischial arch (figure 2).1 the exact location can be confirmed by inserting percutaneously a hypodermic needle shaft through the perineal area at the level of the ischial arch by an assistant, while simultaneously performing the endoscopic evaluation of the urethra. the pressure applied by the needle shaft through the perineal area will deform the urethral lumen allowing to identify the exact location of the urethral rent by visualization by endoscopic camera. in some cases, urethral rents are undetected due to the collapse of the urethra in the sedated patient and small size of the tear.4 in these cases, performing the endoscopic figure 1. presence of blood in the ejaculate (hemospermia) of a stallion with an urethral rent. figure 2. urethral rent located at the level of the ischial arch in an adult stallion. http://dx.doi.org/10.58292/ct.v15.9641 60 citation line: clinical theriogenology 2023, 15, 9641, http://dx.doi.org/10.58292/ct.v15.9641 examination immediately after urination (geldings) or ejaculation (stallions) can aid to observe small traces of blood emanating from an otherwise undetected urethral rent.19 further diagnostic approaches involve the elimination of other causes of bleeding, such as vesicular adenitis, penile lesions (trauma, squamous cell carcinoma of urethral process or glans penis, infection, and habronemiasis), presence of calculi within any portion of the uri nary tract, and midline cysts of the colliculus seminalis.14,20,21 the ruling out process starts with a thorough physical examination of the penis and prepuce, particularly the glans of the penis and urethral opening, followed by transrectal palpation and ultrasonography of accessory sex glands (particularly seminal vesicles), bladder and kidneys. urolithiasis in horses accounts for approximately 8% of the diagnosis of equine urinary tract disease.3 clinical examination findings depend on location of the uroliths. affected horses typically present with anuria, stranguria, pollakiuria, and/or hematuria.3 in horses with urethral uroliths, respiratory and cardiac distress, general discomfort and straining to urinate without urine passage are commonly seen.22 diagnosis is based on transrectal palpation of the bladder, ultrasonography of kidneys, bladder, urethra, and urethroscopy/cystoscopy.23,24 cystoscopy allows direct visualization of uroliths in most cases; however, horses that have a history of urolithiasis may show urethral bruising consistent with recent calculus passage. two types of equine cystoliths are recognized, both primarily composed of calcium carbonate.24 type i cystoliths account for approximately 90% of the cases, are yellow-greening appearance, have a rough surface, and variable resistance to fragmentation. type ii cystoliths contain variable amounts of phosphorus, are gray-white in appearance, have a smooth surface and are more difficult to fragment.24 complete calculi removal is the treatment of choice for cystolithiasis.6,25 in male horses, described surgical approaches for cystoliths removal include pu, pararectal cystotomy (gokel’s approach), cystoscopy-guided techniques, and laparoscopy techniques (standing or under general anesthesia).23,26,27 temporary pu combined with daily topical treatment with antimicrobials and anti-inflammatory drugs has been advocated as the treatment of choice in geldings where hematuria does not resolve with conservative treatment and in stallions with hemospermia caused by urethral defects.1 temporary pu enables access to the urinary bladder in male horses with cystoliths following minimal dissection and without accessing the abdominal cavity.28 moreover, pu can be performed standing thereby avoiding the risks and expenses of general anesthesia. small cystoliths can be removed “en bloc” through a pu, although lithotripsy is required for removal of larger cystoliths.29 cystolith fragmentation through a pu has been performed using long-handled bone forceps, an osteotome and a mallet, laser, shockwave, and a customized scaler.27 surgical technique preparation of the patient pu is best approached with the horse standing in stock under sedation and analgesia.12 jugular vein catheter placement is recommended. sedatives options include the combination of an α2-agonists (detomidine hydrochloride [0.01 mg/kg iv]) or xylazine hydrochlo ride (0.2 to 0.5 mg/kg iv) with butorphanol tartrate (0.01 mg/kg iv) in bolus to effect. in some cases, an initial bolus can be followed by a continuous detomidine hydrochloride drip infusion at 0.01–0.04 mg/ kg/h.30 the rectum is evacuated. a sterile stallion urinary catheter is advanced using sterile technique from the urethral process into the urinary bladder to assist as a landmark for identification of the urethra during surgery. caudal epidural anesthesia is achieved by administration of 2% lidocaine (0.22 mg/kg) either alone or in combination with 10% xylazine hydrochloride (0.15–0.2 mg/kg) diluted to a volume of 7–10ml with sterile preservative free saline (0.9% nacl) solution is used to desensitize the perineal region. the tail is wrapped in an obstetric sleeve or bandaged, using cohesive or support bandage material, and tied laterally. the perineal region is prepared surgically. skin and subcutaneous tissues can be infused with local anesthetic if there is presence of sensitivity in the perineal area after placing the epidural or if preferred by the surgeon. good-quality surgical head lights are recommended to assist on visualization of the surgical field and assist on maintaining a midline orientation throughout the surgery. surgical approach an approximately 7–8 cm longitudinal midline skin incision is made in the perineum extending from 4–5 cm distal to the anus ventrally to the to the level of the palpable ischial tuberosities (figure 3).12 the longitudinal midline incision is continued dividing the subcutaneous tissues, the paired retractor penis muscles and the bsp muscles (figure 4). careful dissection technique while palpating the urinary catheter is recommended to maintain the correct midline orientation23,31 and figure 3. midline skin incision starting approximately 4–5 cm ventral to the anal sphincter. http://dx.doi.org/10.58292/ct.v15.9641 citation line: clinical theriogenology 2023, 15, 9641, http://dx.doi.org/10.58292/ct.v15.9641 61 (weitlaner muscle retractors) to maintain dissected muscle layers out of the visual field while reducing the excessive tissue manipulation and risk of subsequent edema (figure 4c). the dissection is continued through the csp that envelops the urethra allowing exposure of the urethra by retraction of these muscles (figure 5). increased bleeding from the csp is expected and commonly subsides spontaneously; however, it can be controlled by ligation and digital pressure if a situation warrants such measures. the length of the perineal incision should progressively decrease toward the deeper layers to avoid postoperative tissue pocketing of urine. the urethra is identified and stabilized by palpation of the urinary catheter. an approximately 3 cm longitudinal incision is made along the caudal surface of the urethra, and the mucosa is reflected abaxially (figure 6). in horses with intraluminal obstructions such as uroliths or cystoliths, stay sutures using 0polydioxanone (pds) are placed on each edge to allow manipulation by either retrograde hydropulsion using urinary catheter forceps inserted through the pu incision, or urethral dilators.29,33 for temporary (6 to 10 weeks) diversion of urine (temporary pu), a foley urinary catheter is placed through the temporary urethrostomy incision, and its balloon filled with sterile saline solution once enters the urinary bladder. the catheter is secured with stay sutures for 48 to 96 hours.12 for permanent diversion of urine (permanent pu) the csp, bsp, and retractor penis muscles are sutured along their cut edges with a 3-0 synthetic absorbable suture in a continuous pattern. the urethral mucosa and skin are approximated (marsupialized) using a 2-0 or 3-0 synthetic monofilament absorbable or nonabsorbable suture material in an interrupted pattern.12 apposition of the perineal skin and urethral mucosa is important to avoid excessive tension and minimize post-operative complications. stallions with hemospermia and geldings with hematuria secondary to urethral rents can be initially treated alternatively with incision of the csp without incision of the urethra, called corpus spongiotomy (cs).1,3,31 this surgery dissipates the a b c d figure 4. (a) skin and subcutaneous tissues dissected. (b) blunt dissection over the retractor penis muscle. (c) exposure of the bulbospongiosum penis (bsp) muscle – the use of muscle retractors (weitlaner) assists in improving visualization and avoid excessive tissue manipulation. (d) blunt dissection over the bulbospongiosum penis (bsp) muscle. figure 5. corpus spongiosum penis muscle dissected with exposure of the catheterized urethra. avoid large branches of the external pudendal artery with profuse hemorrhage and potential periurethral hematoma formation.32 this can be assisted by a pair of muscle retractors http://dx.doi.org/10.58292/ct.v15.9641 62 citation line: clinical theriogenology 2023, 15, 9641, http://dx.doi.org/10.58292/ct.v15.9641 pressure from the csp into the urethra to the subischial incision allowing the urethral rent to heal.1 primary closure of a urethral rent at the level of the ischial arch is performed when temporary pu or cs have not resolved the hematuria or hemospermia secondary to the urethral rent.12 post-surgical care temporary pu and cs are allowed to heal by second intention and are commonly healed within 2–3 weeks with minimal complications.2,33 the patient should be closely monitored to ensure that urination occurs without difficulty, particularly after pu for removal of urethral or cystic calculi. broad spectrum antibiotics and nonsteroidal anti-inflammatory drugs are commonly administered following surgery. the surgical site should be cleaned and disinfected daily with care not to disturb the sutures (if in place). the skin of the ventral perineum, inguinal region, and adductor surfaces of the hindlimbs should be cleansed daily and protective emollients applied due to high risk of urine soilage and urine scalding. in stallions with urethral rents, strict sexual rest for 6–8 weeks is very important because erection and contractions of the bulbospongiosus muscle can prevent the rent from healing.4,34 outcome in geldings presenting with hematuria, pu was shown to effectively resolve the vast majority of the cases after 1 surgery. in one study, pu resolved hematuria in 6 geldings after 1 surgical procedure.1 in another study, 13 out of 17 geldings underwent pu to resolve hematuria with successful results.4 additionally on the same study, 4 more male horses underwent cs which successfully resolved hematuria. an alternative to surgery is transendoscopic use of a neodymium:yttrium-aluminum-garnet (nd:yag laser) or diode laser to fuse the edges of the rent. this was successfully performed as sole treatment in one gelding and in combination with cs in another gelding.15 in stallions presenting with hemospermia, pu alone restored fertility in 11 out of 15 stallions after 1 surgical procedure in one study.3 in another study, pu alone resolved hemospermia in 1 out of 2 stallions.1 in another study, 6 stallions underwent pu with complete resolution and no complications in 4 of them. the remaining 2 stallions with recurrent hemospermia required a second pu with subsequent primary closure of the urethral rent with direct suturing guided by urethroscopic guidance.4 a stallion with recurrent hemospermia secondary to a urethral rent was treated with a pu and application of a buccal mucosal graft to the urethral rent successfully.16 laser  treatment alone failed to resolve hemospermia in one stallion; however, successfully resolved hemospermia recurrence in 4 stallions when combined with cs.15 chemical cauterization using 4% policresulen, a polycondensation product of meta-cresolsulfonic acid and phenol used as a topical hemostatic  and  antiseptic (albocresile 360 mg/g, takeda pharma figure 6. exposure of urinary catheter after incision of the urethral mucosa. figure 7. mild hemorrhage presented 2hs after corpus spongiotomy surgery in a stallion. http://dx.doi.org/10.58292/ct.v15.9641 citation line: clinical theriogenology 2023, 15, 9641, http://dx.doi.org/10.58292/ct.v15.9641 63 ltda, jaguariuna, sp, brazil) daily infused (for up to 4 days) topically in the urethra efficiently resolved hemospermia in 4 stallions.13 pu is an effective technique for uroliths removal in the male horse. recent advances on the uroliths manipulation and retrieval techniques have increased the success and significantly reduced complications and recurrence.34,35 complications previously reported complications with pu include rectal tear during cystic calculus manipulation, fistula formation, septic peritonitis, orchitis, postoperative tenesmus, excessive hemorrhage, urethral stricture, and dysuria.17,36,37 most of these complications appear to be closely associated with the underlying disease process (large uroliths that require manipulation) rather than with the surgical procedure.37 the most common complications directly related to the pu are hemorrhage, urine scalding and urethral stricture at the surgical site. mild, self-limiting hemorrhage caused by increased csp pressure secondary to contraction of the bsp occurs at the end of urination for up to 2 weeks (figure 7).37 hemorrhage from the urethral orifice and evidence of pain during urination are not commonly observed after surgery.1 severe hemorrhage from the pu is an uncommon complication.37 urine scalding has been reported as a delayed complication of permanent pu and associated with extension of the pu incision ventral to the ischium in one report38 but not in a different study.37 the use of appropriate regular cleaning and emollients is recommended in mild cases while surgical drains might be recommended for severe cases where there is leakage of urine from the surgical site into surrounding tissues.12 the use of indwelling catheters left in placed postoperatively has been regularly used with the aim to ensure patency of urine voiding; however, they have been recently associated with increased risk of additional complications including severe cystitis, urine scald, urethral stricture, and ruptured bladder when meticulous care and frequent monitoring have not been performed.37,39 for horses undergoing temporary pu, it is currently recommended to limit the duration of catheterization and remove it within 2–3 days.39 additionally, chances of stricture formation postoperatively may be reduced by appropriate hemostasis, tissue repair, and tissue drainage.40 stricture formation has been reported in up to 30% of the horses undergoing perineal urethrostomy for calculi removal.36,40 it has been postulated that urethral stricture formation depends on the degree of trauma induced during calculi extraction rather than the surgical approach itself.29,41 recent advances in cystoliths fragmentation and prevention of recurrence following cystolith removal through perineal urethrostomy in male horses has been made.34,35 the use of urethral mechanical dilators and laparoscopic pouch retrieval has shown to reduce complications by protecting the urethral mucosa from mechanic trauma during instrument insertion/ manipulation and urolith removal.35,36,41 for horses with urethral rents, most of the previously described complications can be avoided by changing the surgical approach from a pu to a cs, which has been successfully used for resolving hematuria in geldings with urethral rents in one surgical procedure. however, stallions presenting with hemospermia may require more than 1 pu, cs, or primary closure of the rent to resolve hemospermia.4 acknowledgments the authors are grateful to drs. ahmed tibary and allan gunn for critical review of the manuscript. conflict of interest none of the authors have any conflict of interest to declare. references 1. schumacher j, varner dd, schmitz dg, et al: urethral defects in geldings with hematuria and stallions with hemospermia. vet surg 1995;24:250–254. doi: 10.1111/j.1532-950x.1995.tb01326.x 2. taintor j, schumacher j, schumacher j, et al: comparison of pressure within the corpus spongiosum penis during urination between geldings and stallions. equine vet j 2004;36:362–364. doi: 10.2746/0425164044890571 3. sullins ke, bertone jj, voss jl, et al: treatment of hemospermia in stallions: a discussion of 18 cases. compend contin educ pract vet 1988;10:1396–1403. 4. glass kg, arnold ce, varner dd, et al: signalment, clinical features, 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https://doi.org/10.2746/0425164044890571 https://doi.org/10.2460/javma.249.12.1421 https://doi.org/10.1095/biolreprod12.2.289 https://doi.org/10.1095/biolreprod12.2.289 https://doi.org/10.1016/s0093-691x(02)00663-5 https://doi.org/10.1016/s0093-691x(02)00663-5 https://doi.org/10.1016/j.theriogenology.2016.04.084 https://doi.org/10.1016/b978-1-4377-0867-7.00066-1 https://doi.org/10.1016/b978-1-4377-0867-7.00066-1 https://doi.org/10.1016/j.jevs.2018.02.014 64 citation line: clinical theriogenology 2023, 15, 9641, http://dx.doi.org/10.58292/ct.v15.9641 15. madron m, schleining j, caston s, et al: laser treatment of urethral defects in geldings and stallions used as the primary treatment or in combination with a temporary subischial incision: eight cases (2003–2011). equine vet educ 2013;25:368–373. doi: 10.1111/eve.12050 16. hackett es, bruemmer j, hendrickson da, et al: buccal mucosal urethroplasty for treatment of recurrent hemospermia in a stallion. j am vet med assoc 2009;235:1212–1215. doi: 10.2460/ javma.235.10.1212 17. ball ba: diagnostic methods for evaluation of stallion subfertility: a review. j equine vet sci 2008;28:650–665. doi: 10.1016/j. jevs.2008.10.003 18. tibary a. endoscopy of the reproductive tract in the stallion. in: samper jc, pycock j, mckinnon ao: editors. current therapy in equine reproduction. st louis, mo; saunders: 2007:214–219. 19. varner dd, blanchard tl, brinsko sp, et al: techniques for evaluating selected reproductive disorders of stallions. anim reprod sci 2000;60:493–509. doi: 10.1016/s0378-4320(00)00115-9 20. pinto mr, neild dm, benegas d, et al: successful treatment of seminal vesiculitis with imipenem-cilastatin in a stallion. j equine vet sci 2014;34:544–548. doi: 10.1016/j.jevs.2013.10.001 21. bedford sj, mcdonnell sm, tulleners e, et al: squamous cell carcinoma of the urethral process in a horse with hemospermia and self-mutilation behavior. j am vet med assoc 2000;216:551–553. doi: 10.2460/javma.2000.216.551 22. trela jm, dechant je, culp wt, et al: use of an absorbable urethral stent for the management of a urethral stricture in a stallion. vet surg 2016;45:41–48. doi: 10.1111/vsu.12530 23. fubini sl, delco m: surgery of the equine urinary tract. vet clin n am equine pract 2022;38:141–153. doi: 10.1016/j.cveq.2021.11.010 24. diaz-espiñeira m, escolar e, bellanato j, et al: structure and composition of equine uroliths. j equine vet sci 1995;15:27–34. doi: 10.1016/s0737-0806(07)80577-6 25. duesterdieck-zellmer kf: equine urolithiasis. vet clin n am equine pract 2007;23:613–629. doi: 10.1016/j.cveq.2007.09.003 26. schumacher j, vaughan jt: surgery of the penis and prepuce. vet clin n am equine pract 1988;4:473–491. doi: 10.1016/s07490739(17) 30624-7 27. hawkins jf: surgical treatment of urolithiasis in male horses. equine vet educ 2013;25:60–62. doi: 10.1111/j.2042-3292. 2012.00449.x 28. laverty s, pascoe jr, ling gv, et al: urolithiasis in 68 horses. vet surg 1992;21:56–62. doi: 10.1111/j.1532-950x.1992.tb00011.x 29. abutarbush sm: diseases of the urinary system. ames, ia; john  wiley & sons: 2015:559–577. doi: 10.1002/978111926 5399.ch11 30. vigani a, garcia-pereira f: anesthesia and analgesia for standing equine surgery. vet clin n am equine pract 2014;30:1–17. doi: 10.1016/j.cveq.2013.11.008 31. schumacher j, schumacher j, schmitz d: macroscopic haematuria of horses. equine vet educ 2002;14:201–210. doi: 10.1111/j.20423292.2002.tb00173.x 32. schumacher j, varner dd: abnormalities of the penis and prepuce. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames, ia; wiley-blackwell: 2011:1130–1144. 33. debowes rm: surgical management of urolithiasis. vet clin n am equine pract 1988;4:461–471. doi: 10.1016/s0749-0739(17) 30623-5 34. de bernardis np, seabaugh ka, ismay j, et al: the use of pneumatic impact lithotripsy and a retrieval pouch to create a closed system for removal of cystic calculi in standing male horses. equine vet educ 2019;31:659–665. doi: 10.1111/eve.12843 35. katzman sa, vaughan b, nieto je, et al: use of a laparoscopic specimen retrieval pouch to facilitate removal of intact or fragmented cystic calculi from standing sedated horses: 8 cases (2012–2015). j  am vet med assoc 2016;249:304–310. doi: 10.2460/javma. 249.3.304 36. trotter gw, bennett dg, behm rj: urethral calculi in five horses. vet surg 1981;10:159–162. doi: 10.1111/j.1532-950x.1981. tb00652.x 37. kilcoyne i, dechant je: complications associated with perineal urethrotomy in 27 equids. vet surg 2014;43:691–696. doi: 10.1111/ j.1532-950x.2014.12169.x 38. wilson da, kramer j, constantinescu gm, et al: perineal urethrotomy in males. in: manual of equine field surgery. st. louis, mo; saunders elsevier: 2006:211–214. 39. lees ge: use and misuse of indwelling urethral catheters. vet clin n am small anim prac 1996;26:499–505. doi: 10.1016/ s0195-5616(96)50080-x 40. schott hc. hematuria. in: sprayberry ka, robinson ne: editors. robinson’s current therapy in equine medicine. 7th edition, st louis, mo; saunders elsevier: 2015:456–459. 41. menendez mi, fitch g: use of a laparoscopic retrieval device for  urolith removal through a perineal urethrotomy. vet surg 2012;41:629–633. doi: 10.1111/j.1532-950x.2012.00967.x http://dx.doi.org/10.58292/ct.v15.9641 https://doi.org/10.1111/eve.12050 https://doi.org/10.2460/javma.235.10.1212 https://doi.org/10.2460/javma.235.10.1212 https://doi.org/10.1016/j.jevs.2008.10.003 https://doi.org/10.1016/j.jevs.2008.10.003 https://doi.org/10.1016/s0378-4320(00)00115-9 https://doi.org/10.1016/j.jevs.2013.10.001 https://doi.org/10.2460/javma.2000.216.551 https://doi.org/10.1111/vsu.12530 https://doi.org/10.1016/j.cveq.2021.11.010 https://doi.org/10.1016/s0737-0806(07)80577-6 https://doi.org/10.1016/j.cveq.2007.09.003 https://doi.org/10.1016/s0749-0739(17)30624-7 https://doi.org/10.1016/s0749-0739(17)30624-7 https://doi.org/10.1111/j.2042-3292.2012.00449.x 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school of veterinary medicine department of veterinary clinical sciences, baton rouge, la abstract placenta is a pregnancy-specific organ responsible for maintenance of gestation and offspring survival in utero. similar to other immune-privileged sites, fetoplacental unit was considered sterile. however, there is now strong evidence in humans that placenta harbors a unique microbiome. although this work is in its infancy in veterinary medicine, there are accumulating metagenetic data to support the role of maternal reproductive tract microbiome in pregnancy success and adverse outcomes via placenta. this paper will review and highlight the emerging field of metagenetics in canine reproductive medicine. it will summarize what is known in humans, as well as veterinary species, with an emphasis on future metagenetic characterization of a canine placental microbiome and its potential role in pregnancy outcomes. keywords: dog, placenta, uterus, vagina, microbiome introduction placentation mammalian placenta is a unique and transient organ of pregnancy composed of fetal membranes in intimate association with the mother’s uterus. in dog, embryo first enters uterus approximately 11 days after luteinizing hormone (lh) surge.1 embryos then traverse the uterine body and horns for several days before attaching to endometrium at approximately 22 days after lh surge and the trophoblast invasion process begins to establish an uteroplacental interface and subsequently a fetoplacental unit. resulting canine placenta is grossly defined as having a zonary shape, due to an interdigitating zone of contact between endometrium and chorionic sac.2 microscopically, this contact has a degree of intimacy that places trophoblast cells in direct proximity to the interstitium of endometrium, but not endometrial blood vessels.2 therefore dogs have an endotheliochorial type of placentation, which is different to hemochorial type in humans that have trophoblast cells exposed to maternal blood. horse placenta is diffusely villous and an epitheliochorial type,2 which has 6 cell layers between maternal and fetal blood, giving it even less intimacy than dog. these macroscopic and microscopic differences in placentation are important to keep in mind when making translations between human microbiome data and that of veterinary species, including dogs. metagenetics, metagenomics, and microbiome due to the emergence of this exciting and novel field in veterinary medicine, academicians and clinicians alike are rapidly learning microbiome terms and concepts. entire microbiota community is considered when assessing the microbiome of any given environment. microbiota consists of resident bacteria, archaea, fungi, protists, and viruses in the environment of interest. microbiome is therefore the collective genomes of the microbiota community members and the study of this has been termed metagenomics. study of an environment’s metagenome, where scientists utilize the principles of molecular biology and genetics, is referred to as metagenetics. sequencing of metagenome results in read lengths of significant depth that bacterial genus and species can be accurately identified and organized into operational taxonomic units (otus) that are mapped to previously generated bacterial taxonomic databases. herein, bacterial microbiota of the female reproductive tract (environment) will be considered in the context of pregnancy outcomes where the host is the fetus and dam. 633 clinical theriogenology • volume 11, number 4 • december 2019     female reproductive tract microbiome role of vaginal and uterine microbiome interactions between host and bacteria it supports has long been recognized; however, many questions remain regarding how this microbial environment varies within and among individuals in both healthy and diseased states.3 human vagina is colonized with commensal bacteria, predominantly lactobacilli. therefore, a vaginal microbiota has been proposed to have a role in female reproductive physiology, pathogen defense, and function.4 acidic environment created by these lactobacillus spp. is thought to provide the first barrier of defense protecting upper reproductive tract from opportunistic pathogens in women.3,4 a meta-analysis provided compelling data in women to support a uterine microbiome that may function to modulate the local immune system in preparation for embryo implantation and placenta formation.5 when comparing endometrial microbiota with vaginal and cervical microbiota in women, endometrial microbiota was in general lower in relative abundance, but with an overall higher microbial diversity and richness.4,6 recently, canine vaginal and uterine microbiome was published.7 metagenetic characterization of canine vagina and uterus was obtained from vaginal swabs and endometrial tissue samples, respectively, at elective ovariohysterectomy. interestingly, top 5 genera identified in the vagina consisted of hydrotalea, ralstonia, mycoplasma, fusobacterium, and streptococcus while top 5 genera identified in the endometrium included pseudomonas, staphylococcus, corynebacterium, anaplasma, and dermacoccus. vagina of bitch is higher in richness in contrast to canine endometrial tissue being higher in diversity. vagina of estrual bitch had a distinct microbiome, whereas all other samples did not distinctly form clusters, regardless of stage of the estrous cycle. a study using multiple sampling techniques revealed that equine endometrium during estrus has a proteobacteria driven microbiome, with no significant difference between vaginal and endometrial microbiomes.8 also, there were no significant differences between sampling techniques: endometrial double-guarded swab, low volume lavage, or endometrial biopsy.8 in contrast, in another study, proteobacteria, firmicutes, bacteroidetes, and actinobacteria in cycling equine endometrium was in contrast to vaginal microbiome.9 however, equine endometrial microbiome does appear to be different in noncycling mares when compared to cycling mares during the physiologic breeding season (unpublished data). when making conclusions regarding female reproductive tract microbiome it is important to consider the stage of estrous cycle, which has unique sex steroid hormone influences, or lack thereof, on the immune milieu and potentially resident microbiome. further studies are necessary to determine a “core” vaginal and uterine microbiome that is advantageous for pregnancy success in all species, including dogs. with identification of canine reproductive tract microbiome, small shifts in microbiomes can be identified that may not be clinically apparent or previously detectable using conventional culture techniques, but could be clinically important. evidence for a canine placental microbiome in both women and veterinary species, placenta has been regarded as a sterile organ during pregnancy. placenta is responsible for maternal-fetal exchanges of nutrients and waste products, as well as tolerance of semi-allogeneic fetus that is composed of maternal and paternal antigens.2 successful placental development and fetal survival relies on immunotolerance by maternal uterine environment, which we are learning is more diverse and richer than previously thought. it was recently (2014) reported that, based on metagenetic techniques, human placenta harbors a unique microbiome.3 since then, nearly 100 new studies have been published on the human placental microbiome and metagenetic data are being generated in several veterinary species, including cows, dogs, and horses, that support a unique placental microbiome similar to that in women. taxonomic profile of human placental microbiome was discovered to be most similar to oral microbiome, followed closely by vaginal microbiome and further from fecal microbiome. bacterial dna was detected in only 50% of human placental tissue samples.10 based on these positive samples, healthy human placental microbiome has an abundance of firmicutes, tenericutes, proteobacteria, 634clinical theriogenology • volume 11, number 4 • december 2019     bacteroidetes, and fusobacteria.11 despite profound differences in placentation, this finding was consistent in horses.1 metagenetic analyses of the equine placenta, as done in humans, demonstrated 3 main phyla in the gravid horn (firmicutes, proteobacteria, bacteroidetes) and those same 3 phyla plus actinobacteria in the nongravid horn.12 most abundant bacterial phyla in gravid and nongravid chorioallantois share significant overlap, suggesting similar, but not identical, environments within different compartments of the chorioallantois. many of the same bacterial populations that characterized gravid chorioallantois described by the cornell university group were also identified in allantoic fluid of pregnant mares by investigators at oklahoma state university.9 figure. potential extra-placental bacterial sources (oral, fecal, vaginal microbiota) for canine placental microbiome; canine fetoplacental units (pink). it has been well established that there is an association between bacterial infection of reproductive tract and fertility.13 intense research efforts are focused on investigating bacterial endometritis in mare to improve fertility and pregnancy rates.14 in dogs, relationship between endometritis and conception and/or pregnancy failure remains unclear.15 transcervical catheterization by vaginal endoscopy can be performed to evaluate both cytology and bacteriology. in a study where infertile bitches were examined using transcervical catheterization, 70% had heavy bacterial growth when samples were cultured in vitro. the most common bacteria include pasteurella multocida, group g streptococcus, staphylococcus intermedius, escherichia coli and proteus mirabilis.15 these pathogens could all potentially contribute to a subclinical endometritis and even pyometra that may alter fertility as well as adverse pregnancy outcomes including abortion, stillbirth of preterm puppies, and neonatal loss. reason why some bitches become pregnant and others do not is hypothesized to be due to the immune milieu of uterus, which may be influenced by a resident microbiome (figure). importantly, associations between placental microbiome communities and adverse pregnancy outcomes in women, including preterm birth and preeclampsia, are rapidly being confirmed.3,6 these new data can serve to prevent maternal and fetal morbidity and mortality via a novel mechanism, i.e. restoring a “healthy” microbiota prior to pregnancy. preterm birth and neonatal death has been associated with intrauterine infections ascending from the lower genital tract.7 in dogs, reproductive tract consists of a rich and diverse microbial environment with > 300 otus of organisms identified.16 similar to humans, presence of certain microbiota are either positively or negatively associated with pregnancy outcome. presence of the any of the genera biberstinia, staphylococcus, pasteurella, corynebacterium, or methylobacterium in vagina of bitch significantly increases the chance of a stillborn puppy in a litter.17 thus, this emerging and exciting new field supports a role for a symbiotic microbiome at maternal-fetal interface that contributes to pregnancy maintenance and success. novel metagenetic analyses of the canine placenta will be important to enhance our understanding of placental function during pregnancy in the bitch. establishment of a “healthy” 635 clinical theriogenology • volume 11, number 4 • december 2019     reproductive tract microbiome could improve pregnancy outcomes in sub/infertile bitches. furthermore, analysis of canine placental microbiome in relation to other extra-placental body sites in healthy bitches, similar to what has been investigated in women and mares, will provide foundation for a better understanding of the relationship between canine placenta and resident bacterial populations during uncomplicated healthy and complicated pregnancies. methods to characterize canine placental microbiome by metagenetics to properly assess the canine placental microbiome, samples should be collected sterilely at the time of caesarian section. collection of fetal membranes after a vaginal whelping will have contamination from the vagina. canine endometrium and vagina have distinctly different microbiomes, collecting conceptuses that traverse vaginal vault will likely result in both microbiomes being represented. genomic dna can be extracted from various types of samples, such as swabs, fluid or tissue using commercial dna isolation kits. after dna extraction and purification, sample can be analyzed using multiple molecular genetic techniques. these involve isolation of 16s ribosomal dna and sequencing of amplified bacterial dna. therefore, metagenetic techniques yield live and dead bacteria populations. although dead and fragmented dna is not replicating within a host, they still represent ligands for host cells to recognize and act upon. therefore, these inactive bacterial fragments could potentially still contribute to a physiologic interaction with the host.5 a second pitfall includes quantification of an organism present, despite available read counts.18 it is important to know that while traditional identification of bacteria utilizes cultivating methods (i.e. aerobic and anaerobic culture), metagenetic techniques utilize non-cultivating methods. metagenetic analysis of placenta in horses should therefore be superior to cultivating methods that may fail to identify dormant bacteria that may reside at the maternal-fetal interface in pregnant mares. however, until these new methodologies are robustly tested and validated, the clinician should perform cultivating until noncultivating methods are routinely available and used. an outline of a suggested method to prospectively collect, process, and analyze canine placenta and extra-placental sites by metagenetic techniques is as follows: sample collection to characterize the canine placental microbiome, vaginal swab samples as well as collection of transcervical catheters for endometrial metagenetic sequencing will be first collected during routine breeding management. in addition, swabs of the buccal mucosa of the oral cavity and a fresh fecal sample will be collected and stored at -80°c. at the time of pregnancy confirmation, repeat swabs of the vaginal vault and from the buccal mucosa of the oral cavity, and a fresh fecal sample will be collected and stored at -80°c. at the time caesarean section delivery, placenta including marginal hematomas and amnion will be collected, dissected and stored at -80°c. dna isolation and amplification of 16s rrna frozen samples can be thawed and individually processed for genomic dna extraction. this will be performed according to standard protocols. samples homogenized, centrifuged and resuspended in nuclease-free water. an equal volume of lysozyme added and incubated for 12 hours at 56°c to maximize bacterial dna extraction. isolation of dna performed using a commercial dna isolation kit powersoil® (mo-bio laboratories inc) according to the manufacturer’s instructions. dna concentration determined using the qubit® 2.0 flurometer (life technologies, grand island, ny) and dna integrity assessed by electrophoresis. the v3 and v4 domain of bacterial 16s rrna8 amplified by pcr. primer sequence for 16s amplicon pcr is the 341f and 785r primers as previously described9 and optimized for the illumina miseq platform. earth microbiome project (http://www.earthmicrobiome.org)10 can be used to select 150 different 12 bp error-correcting golay barcodes for 16s rrna, as described.9 the pcr reaction performed in triplicate containing: microbial genomic dna (25 ng), 1x gotaq green master mix (promega, madison, wi), 1 mm magnesium chloride, and 10 µm of each primer. the pcr conditions for 16s rrna consist of an initial denaturation step of 3 minutes at 94°c; followed by 35 cycles of 94°c for 636clinical theriogenology • volume 11, number 4 • december 2019     45 seconds, 50°c for 1 minute, and 72°c for 90 seconds; and a final elongation step of 72°c for 10 minutes. no template controls and 2 positive controls (mock microbial communities) should be added to each pcr plate. replicate amplicons pooled and purified with a qiaquick pcr purification kit (qiagen, valencia, ca) and visualized by electrophoresis. amplicons are quantified using qubit® 2.0 flurometer then the amplicon aliquots are standardized to the same concentration. miseq sequencing final equimolar libraries will be sequenced using the miseq reagent kit v3 (600 cycles) on the miseq platform (illumina inc., san diego, ca). sequence reads analyzed using the quantitative insights into microbial ecology (qiime) program and assigned to otus. discriminant analysis is used to evaluate the correlation between bacterial taxa and the prevalence of each bacteria identified in the placental samples against the potential source of bacteria from extra-placental body sites. different prevalence of bacteria in each sample will be used as a covariate in a stepwise discriminant analysis model. variables removed in a stepwise manner until the only variable with a p < 0.005 is retained in the final model. significance preterm birth of stillborn puppies is not an uncommon outcome of pregnancy in dogs. diagnosing the cause of canine abortion is best done through necropsy of fetuses and fetal membranes. placental infection during canine pregnancy can be extremely difficult to diagnose, as outward clinical signs are often not present or subtle, consisting of restlessness, vulvar discharge, and lethargy. understanding relationship between canine placenta and resident bacterial populations of uterus and vagina could provide opportunity to establish symbiosis prior to pregnancy in order to aid in pregnancy success. furthermore, diagnosing dysbiosis in uterus before pregnancy or even extra-placental sites (oral, gastrointestinal and/or vaginal) could provide evidence for predicting associated adverse outcomes, such as abortion, in breeding bitches. identification of bacterial targets in extra-placental body sites and appropriate pre/probiotic therapies to improve adverse pregnancy outcomes would greatly enhance breeding management and perinatal care in our canine patients. authors advise caution and judicious usage of interventions to alter the microbiome of canine reproductive tract to improve fertility and pregnancy outcomes until an exhaustive microbiota characterization has been performed. conflict of interest no conflict of interest to declare. references 1. concannon pw: canine pregnancy: predicting parturition and timing events of gestation. ivis 2000. 2. furukawa s, kuroda y, sugiyama a. a comparison of the histological structure of the placenta in experimental animals. j toxicol pathol 2014;27:11-18. 3. green ka, zarek sm, catherino wh: gynecologic health and disease in relation to the microbiome of the female reproductive tract. fertil steril 2015;104:1351-1357. 4. wee ba, thomas m, sweeney el, et al: a retrospective pilot study to determine whether the reproductive tract microbiota differs between women with a history of infertility and fertile women. aust n z j obstet gynaecol 2018;58:341-348. 5. benner m, ferwerda g, joosten i, et al: how uterine microbiota might be responsible for a receptive, fertile endometrium. hum reprod update 2018;24:393-415. 6. verstraelen h, vilchez-vargas r, desimpel f, et al: characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the v1-2 region of the 16s rrna gene. peerj 2016;4:e1602. 7. lyman cc, holyoak gr, meinkoth k, et al: canine endometrial and vaginal microbiomes reveal distinct and complex ecosystems. plos one 2019;14:e0210157. 8. heil ba, thompson sk, kearns ta, et al: metagenetic characterization of the resident equine uterine microbiome using multiple techniques. j equine vet sci 2018;66:111. 9. holyoak gr, lyman cc, wieneke x, et al: the equine endometrial microbiome. clinical theriogenology 2018;10:273. 637 clinical theriogenology • volume 11, number 4 • december 2019     10. doyle rm, harris k, kamiza s, et al: bacterial communities found in placental tissues are associated with severe chorioamnionitis and adverse birth outcomes. plos one 2017;12:e0180167. 11. aagaard k, ma j, antony km, et al: the placenta harbors a unique microbiome. sci transl med 2014;6:237ra65. 12. xia yx, cornelius aj, donnelly cg, et al: metagenomic analysis of the equine placental microbiome. clinical theriogenology 2017;9:452. 13. canisso if, stewart j, coutinho da silva ma: endometritis: managing persistent post-breeding endometritis. vet clin north am equine pract 2016;32:465-480. 14. ferris ra: endometritis: diagnostic tools for infectious endometritis. vet clin north am equine pract 2016; 32:481-498. 15. fontaine e, levy x, grellet a, et al: diagnosis of endometritis in the bitch: a new approach. reprod domest anim 2009;44 suppl 2:196-199. 16. lyman cc, meinkoth k, desilva u, et al: 16s rdna-based profiling of canine reproductive tract microbiota reveals a complex microbial ecosystem. clinical theriogenology 2016;8:305. 17. cornelius aj, bichalho rc, cheong sh: the canine vaginal microbiome and associations with puppy survival. clinical theriogenology 2017;9:424. 18. bracewell-milnes t, saso s, nikolaou d, et al: investigating the effect of an abnormal cervico-vaginal and endometrial microbiome on assisted reproductive technologies: a systematic review. am j reprod immunol 2018;80:e13037. 638clinical theriogenology • volume 11, number 4 • december 2019 untitled traditional surgical and laparoscopic vasectomy in dogs and cats traditional surgical and laparoscopic vasectomy in dogs and cats robert mccarthy tufts cummings school of veterinary medicine, north grafton ma abstract loss of male reproductive hormones is associated with increased risk of several important clinical conditions in dogs and cats and alters normal behaviors. vasectomy provides sterilization without loss of hormones and is being requested by pet owners with increasing frequency. this review is designed to introduce surgical vasectomy by both traditional and laparoscopic methods. keywords: laparoscopic, vasectomy introduction sterilization of male dogs by gonadectomy has been considered standard of care in the united states for many years. proponents argue that loss of reproductive hormones after gonadectomy is advantageous in that several disorders, including benign prostatic hypertrophy, prostatic abscess, perineal hernia, testicular tumors and perianal adenomas are seen with decreased frequency or eliminated. recently, however, consequences of loss of reproductive hormones have been met with increased scrutiny. elimination of reproductive hormones is associated with increased risk of several important clinical conditions, including various neoplasia (hemangiosarcoma, osteosarcoma, transitional cell carcinoma of the bladder and urethra, prostate cancer), orthopedic diseases (torn cranial cruciate ligament, hip dysplasia) and cognitive impairment.1 gonadectomy predisposes animals to obesity, likely the most important clinical entity seen in small animal practice.2 effect of gonadectomy on behavior of male dogs is debated and somewhat poorly understood. despite common belief, male dogs do not necessarily behave better after castration. loss of testosterone is only expected to potentially decrease sexually dimorphic behaviors such as roaming, mounting and urine marking. aggression may or may not be affected by gonadectomy, and in certain circumstances may actually be increased.3 furthermore, dogs that have been castrated tend to be more fearful and sensitive to handling.4 vasectomy provides guaranteed infertility, while maintaining reproductive hormones. vasectomy in dogs using traditional surgical procedure vasectomy in male dogs is a simple, quick and atraumatic procedure easily performed by general practitioners. in most dogs, spermatic cords can be palpated as they extend from the scrotum towards the inguinal rings and aids in orientation. a 3 5 cm prescrotal midline skin incision is made just cranial to the location of a routine castration. careful dissection in a lateral direction exposes the spermatic cord. if localization is difficult, it is sometimes useful to manipulate the testicle caudally and cranially to tense and relax the cord. visualization is improved by separating the incision with gelpi or weitlaner retractors. once spermatic cord is identified it is isolated by circumferential passage of a hemostat. a small incision is then made in the vaginal tunic with a tenotomy scissor or performed bluntly with fine hemostats. extreme care should be taken to avoid any disruption of the pampiniform plexus. ductus deferens with its associated deferent artery is easily identified as a turgid white structure. circumferential ligatures are placed 1 2 cm apart and a short section of ductus deferens excised between them. closure of the vaginal tunic is not necessary. several interrupted absorbable sutures are placed to eliminate dead space, and the subcutaneous tissue closed in a continuous pattern. an intradermal pattern with fine absorbable suture is used to close the skin. 243 clinical theriogenology • volume 11, number 3 • september 2019 complications of vasectomy are rare and generally limited to incisional problems similar to routine gonadectomy. sperm granulomas have been reported histologically, but clinical sequelae of these changes appear extremely rare if the procedure is performed correctly.5 interval from vasectomy to azospermia in the dog is variable but is generally reported to be 2 4 weeks. flushing the ductus deferens at the time of surgery may decrease time to azoospermia to ~ 6 days.6 vasectomy in dogs by laparoscopic surgery general laparoscopic methods have been described in depth and many training courses and laboratories are available.7 in clinical practice, laparoscopic (as opposed to routine surgical) vasectomy is most commonly indicated when combined with a separate laparoscopic abdominal surgery such as prophylactic gastropexy. briefly, the urinary bladder is first emptied by either manual expression or catheterization. patient is placed in a dorsal slightly head-down (trendelenburg) position with the optical tower located towards the feet. abdomen is distended with co2 using either a closed (veress needle) or open (hasson) method. intraabdominal pressure between 10 and 13 mm hg maximizes working space while minimizing detrimental physiologic effects. after distension is obtained, either a single or double port method can be employed. for the single port method, a specially designed access port allowing passage of up to three separate devices is utilized (sils port, medtronic, minneapolis, mn). the port is placed just caudal to the umbilicus. after camera insertion, the ductus deferens is easily identified as it passes towards the inguinal ring on the affected side(s). unlike after passage through the inguinal ring, the ductus deferens is distinctly separate from other structures of the spermatic cord in this location. a vessel-sealing device (ligasure dolphin tip, medtronic) is passed through a second opening in the port and is used to simultaneously cut and seal the structure. if a vessel-sealing device is not available, endoscopic cautery (valleylab laparoscopic electrode, medtronic) can used in a similar fashion. a final option is to place two endoscopic vascular clips (endo clip, medtronic) and use endoscopic scissors for cutting the ductus. in any of these situations, graspers can be placed through the third port opening to grasp and stabilize the ductus if necessary. for the double port method standard single access laparoscopic ports are utilized. the camera port is placed just caudal to the umbilicus. after identification of the ductus deferens, a second port (versaone, medtronic) is placed under direct visualization 4 8 cm lateral to the camera port. a vessel sealing device, endoscopic cautery or vascular clips/scissors are then used to transect the ductus, as with the single port method. if additional stabilization of the ductus is necessary, a grasper can be passed through a third port 4 8 cm lateral to the camera port on the opposite side. vasectomy in cats vasectomy likely has limited application in house cats, but may be indicated in certain situations for barn cats, zoo cats and in research settings. vasectomy may also be useful for management of feral cat populations. despite application of substantial resources over prolonged intervals, there is little evidence that current methods of feral cat control are effective at eliminating feral cat populations. trap neuter release (tnr) is frequently recommended, but often fails because cats have high intrinsic growth rates and it is difficult or impossible to capture all resident cats. tnr programs may increase both adult and kitten survival rates, which further counteracts attempts at population control.8,9 in addition, immigration of new cats, usually be abandonment is common.10 lethal control methods can cause rapid temporary depopulation but have rarely proven effective in the long term, and are unacceptable to a large proportion of people.11 244clinical theriogenology • volume 11, number 3 • september 2019 management of feral cat colonies by sterilization with maintenance of reproductive hormones (trap vasectomy hysterectomy release, tvhr) has been predicted by computer simulation to cause more rapid population decline, because normal social behavior is maintained.12 vasectomized dominant male cats still compete for females, use up females receptive time, fight off fertile males attempting to copulate and may better prevent immigration of intruding males into the colony. following copulation with a vasectomized male, fertile females enter a prolonged period of sexual non-receptivity (pseudopregnancy), reducing the chance for fertile breeding. after hysterectomy, females continue to attract males and compete with fertile females for male courting and breeding time. vasectomy and hysterectomy do not increase survival of either adults or kittens, as with tnr. vasectomy in cats is quite simple and can easily be performed by general practitioners without advanced training or major equipment needs. a bilateral inguinal approach has been reported,13 but the author prefers to perform vasectomy in cats through a small caudal abdominal incision. a 2 3 cm skin incision is made such that its caudal extent is about 1 cm cranial to the pubic brim. for vasectomy, it is critical that the urinary bladder is completely empty. cystocentesis should be performed at surgery if preoperative bladder expression was inadequate. the bladder is retracted caudally, and both ductus deferens are visualized entering the bladder neck. these are bright white structures curving into the bladder neck, and are easily differentiated from the ureters. both ductus deferens are double ligated with 4-0 absorbable suture material and divided in the middle. alternately, they can simply by cauterized. cautery with an inexpensive hand held battery powered instrument is acceptable (argent, mckesson, san francisco, ca). closure is routine. complications after vasectomy in cats are rare, and generally limited to incisional problems typical of any short-duration surgery. time from performing vasectomy to azospermia in the cat is likely quite a bit longer than in dogs, with reported duration of ~ 49 days.6 conclusion traditional surgical and laparoscopic vasectomy are safe, relatively simple procedures in dogs and cats for both clinical and research situations where the goal is guaranteed sterilization with preservation of reproductive hormones. conflict of interest none to declare. references 1. mckenzie b: evaluating the benefits and risks of neutering dogs and cats. cab rev perspect agric vet sci nutr nat resour 2010;(045). 2. lefebvre sl, yang m, wang m, et al: effect of age at gonadectomy on the probability of dogs becoming overweight. j am vet med assoc 2013;243:236-243. 3. farhoody p, mallawaarachchi i, tarwater pm, et al: aggression toward familiar people, strangers, and conspecifics in gonadectomized and intact dogs. front vet sci 2018;(february):1-13. 4. duffy dl, serpell ja: non-reproductive effects of spaying and neutering on behavior in dogs. proc third int symp non-surgical contracept methods pet popul control 2003. 5. mayenco aa, garcia fp, sanchez m: sperm granuloma in the dog : complication of vasectomy. j small anim pract 1996;37:392-393. 6. frenette md, dooley mp, pineda mh: effect of flushing the vasa deferentia at the time of vasectomy on the rate of clearance of spermatozoa from the ejaculates of dogs and cats. am j vet res 1986;47:463-470. 7. fransson b, mayhew p: editors, small animal laparoscopy and thoracoscopy. ames: wiley blackwell; 2015. 8. finkler h, gunther i, terkel j: behavioral differences between urban feeding groups of neutered and sexually intact free-roaming cats following a trap-neuter-return procedure. j am vet med assoc 2011;238:1141-1149. 9. nutter fb: evaluation of a trap-neuter-return management program for feral cat colonies: population dynamics, home ranges, and potentially zoonotic diseases.phd thesis, north carolina state university raleigh, nc, 2005. 245 clinical theriogenology • volume 11, number 3 • september 2019 10. natoli e, maragliano l, cariola g, et al: management of feral domestic cats in the urban environment of rome (italy). prev vet med 2006;77:180-185. 11. slater mr: understanding issues and solutions for unowned, free-roaming cat populations. j am vet med assoc 2004;225:1350-1354. 12. mccarthy rj, levine sh, reed m: estimation of effectiveness of three methods of feral cat population control by use of a simulation model. j am vet med assoc 2013;243:502-511. 13. pineda mh, dooley mp: surgical and chemical vasectomy in the cat. am j vet res 1984;45:291-300. 246clinical theriogenology • volume 11, number 3 • september 2019 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 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/presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice introduction currently, there are 39 extant species within the family felidae, including the domestic cat (felis catus). due to ongoing habitat destruction and persecution, half of wild felid species are listed as vulnerable or ‘endangered’ by the international union for conservation of nature (table 1).1 therefore, ex situ management has become increasingly important in the conservation of several wild felid species, such as the cheetah (acinonyx jubatus), clouded leopard (neofelis nebulosa), and iberian lynx (lynx pardinus). ex situ individuals serve as a ‘genetic repository’ for retaining all existing heterozygosity in the case of an unexpected catastrophic event impacting wild counterparts, as well as a source population for future reintroduction. furthermore, these individuals also are invaluable resources for basic and applied research to generate information that can be challenging to obtain from free-ranging populations. finally, zoo animals also serve as ambassadors for raising public awareness and garnering financial and political supports for conservation of wild populations.2 reproductive science has had pivotal roles in genetic management of ex situ wildlife.3 for felids, reproductive studies began in the early 1980s due to the interest in the potential benefit of assisted reproductive technologies (arts) in maintaining heterozygosity of small, isolated captive populations, and to advance the understanding about species-specificity in reproductive mechanisms.4 to date, reproductive biology has been described in several felids, and patterns of ovarian steroid hormones have been reported for more than half of the 39 species. studies to date indicate that there are variations in reproductive characteristics among felid species, including age at sexual maturation, reproductive biology and assisted reproductive technologies in felids nucharin songsasen center for species survival, smithsonian conservation biology institute national zoological park, front royal, virginia abstract reproductive biology has been thoroughly described in several felid species. specifically, patterns of ovarian steroid hormones have been reported for more than half of 39 felids, and it has been observed that there is a high degree in reproductive diversity among species. for example, although females of most felids are polyestrous with induced ovulation, some species such as the clouded leopard (neofelis nebulosa) and fishing cat (prionailus viverrinus) spontaneously ovulate. to date, assisted reproductive technologies, including sperm cryopreservation, artificial insemination and in vitro embryo production have been developed in both domestic and wild felids that result in live offspring. this paper reviews the diversity of reproductive mechanisms and discusses the status of reproductive technologies that have been applied to wild felid conservation. keywords: felids, reproductive cycle, seasonality, assisted reproductive technologies reproductive seasonality, seminal traits, and ovulatory pattern.5 despite these differences, arts developed in domestic cats have been successfully applied to several wild felids.6 this paper summarizes the diversity of reproductive mechanisms within the family felidae and discusses the status of arts that have been applied to wild felid conservation. felid reproductive biology reproductive cycle of felids presents a great variation in terms of seasonality and ovulation mechanisms (table 1).7-23 the reproductive cycle consists of 4 stages: proestrus, estrus, diestrus, and anestrus.5 unlike canids,24 proestrus is rather short in felids, lasting < 1 day. estrus is characterized by the presence of an estrogen peak from preovulatory follicles. after ovulation (induced or spontaneous), female felids enter diestrus, characterized by elevated progesterone concentrations lasting throughout pregnancy or nonpregnant luteal phase. anestrus is the period of ovarian quiescence when circulating estrogens remain at basal concentrations. duration of interestrous interval varies among species, ranging from 1 145 days.25 age at sexual maturation of male and female felids varies among species depending on body size. specifically, small felids such as leopardus spp. reach sexual maturity between 1 2 years of age, whereas larger felids, such as lions and tigers reproduce at 3 4 years of age.5,26 reproductive biology (i.e. reproductive cycle, folliculogenesis, oogenesis, and spermatogenesis) has been thoroughly studied clinical theriogenology 2021; 13: 130 in the domestic cat.27-30 development of noninvasive methods to assess gonadal hormone metabolites in feces31 has allowed studies to determine reproductive cycles of wild felids. through these studies, reproductive cycles of 24 wild felid species have been described.5,25,32 we now know that there are differences in reproductive seasonality and ovulatory patterns among species or individuals within the same species. for example, reproductive seasonality of domestic cat is influenced by the duration of daylight. under natural light, cats living in areas from equator to tropic of cancer can produce litters throughout the year, whereas those living from tropics toward the polar circle breed seasonally (between january and july).33,34 female table 1. wild felids species reproductive characteristics and ‘red list’ status genetic lineage common name scientific name red list status reproductive seasnonality ovulatory pattern panthera tiger5 snow leopard7 leopard5 lion5,8 jaguar9 clouded leopard5 sunda clouded leopard panthera tigris p. uncia p. pardus p. leo p. onca neofelis nebulosa n. diardi en vu vu vu nt vu vu seasonal seasonal year-round year-round seasonal in temperate zone, but year-round if housed under long-day light cycle d.d induced induced occasionally spontaneous occasionally spontaneous induced spontaneous dd bay cat borneo bay cat asian golden cat73 marble cat catopuma badia pardofelis temminckii p.marmorata en nt nt d.d. year-round d.d. d.d. d.d. d.d. caracal caracal10 african golden cat serval caracal caracal c. aurata leptailurus serval lc vu lc year-round d.d. d.d. d.d. d.d. d.d. ocelot ocelot5 margay5 pampus cat11 andean cat northern tiger cat5 southern tiger cat5 geoffroy’s cat12 guina leopardus pardalis l. wiedii l. colocolo l. jacobita l. tigrinus l. guttulus l. geoffroyi l. guigna lc nt nt en vu vu lc vu year-round year-round year-round d.d. year-round year-round year-round d.d. induced spontaneous d.d. d.d. induced induced d.d. d.d. lynx bobcat13 canadian lynx13,14 iberian lynx13 eurasia lynx13 lynx rufus l. canadensis l. pardinus l. lynx lc lc en lc seasonal seasonal seasonal seasonal d.d. occasional spontaneous induced induced puma cheetah15 puma16,17 jaguarundi acinonyx jubatus puma concolor herpailurus yagouaroundi vu lc lc year-round year-round d.d. induced induced d.d. leopard cat pallas’s cat18,19 rusty-spotted cat20 leopard cat fishing cat21 flat headed cat otocolobus manul prionailus rubiginosus p. bengalensis p. viverrinus p. planiceps lc nt lc vu en seasonal year-round d.d. year-round d.d. induced d.d. d.d. spontaneous d.d. domestic cat domestic cat5 wild cat22 chinese mountain cat sand cat23 black footed cat jungle cat felis catus f. silvestris f. bieti f. margarita f. nigripes f. chaus na lc vu lc vu lc seasonal seasonal d.d seasonal d.d. d.d. occasionally spontaneous d.d. d.d. d.d. d.d. d.d. en: endangered; vu: vulnerable; nt: near threatened; lc: least concern; na: not assessed; d.d.: data deficiency clinical theriogenology 2021; 13: 131 cats can cycle year-round when housed indoors under 12 14 hours light cycle.5 furthermore, the ability of cat oocytes to complete nuclear maturation and develop into embryos after in vitro fertilization (ivf) is lower during august to october compared to other months.35 however, seasonal variations in seminal characteristics have not been consistently demonstrated in domestic cat.34-38 specifically, epididymal sperm obtained from cats castrated during increasing light (winter and spring) tended to have higher motility and total sperm cells than samples obtained during decreasing light (summer and fall), although variations in testosterone concentration were not observed.36 furthermore, motility and the proportion of morphologically intact epididymal sperm were higher in samples obtained in spring than in winter and testicular testosterone concentrations significantly reduced in autumn compared to spring.34 however, there were no differences in sperm concentration, motility, morphology, acrosome integrity, and ability to penetrate an oocyte among samples obtained throughout the year. 35 finally, there is no clear seasonal effect on the quality of ejaculated samples, although variations in plasma testosterone and luteinizing hormone (lh) concentrations between breeding versus nonbreeding season are observed.37 influence of seasonality on seminal quality is more pronounced in some wild felids, including pallas’s cat (otocolobus manual),18,39 ocelot (leopardus pardalis), margay (l. wiedii), northern tiger cat (l. tigrinus),40 eurasian lynx (lynx lynx),41 and snow leopard (panthera uncia).42 for example, serum lh and seminal characteristics were higher during breeding than nonbreeding season in pallas’s cat, although there is no seasonal effect on circulating testosterone concentrations.39 furthermore, reproductive season can be simulated when housing male pallas’s cats indoors under artificial light, although mating attempts do not result in offspring production.18 conversely, there is no seasonal effect on seminal characteristics and testosterone concentrations in some wild felids, including tiger (panthera tigris).43 some nonseasonal breeders, including the leopard, can breed year-round,44,45 despite reductions in seminal quality and fertility during some months of the year.46 generally, female felids are polyestrous animals. however, studies in eurasian and iberian lynx have demonstrated a ‘noncat-like’ ovarian cycle in these species.47 specifically, eurasian and iberian lynx (l. pardinus) exhibit seasonal monoestrus47,48 that differs from their cousins, bobcat (l. rufus)47 and canadian lynx (l. canadensis).14 another unique aspect of eurasian and iberian lynx is the presence of persistent corpora lutea (cls) that remain active (producing progesterone) for at least 2 years, and cooccur with new cls of the next cycle.13,48 to date, mechanisms supporting persistent cls are unknown. however, it has been suggested that persistent cls stimulate negative feedback to suppress folliculogenesis outside the breeding season.48 traditionally, domestic cat has historically been described as an induced ovulator and ovulation is induced by multiple mating events. specifically, mating stimulates the release of gonadotropin releasing hormone that, in turn, triggers the release of lh from the anterior pituitary that causes final follicle maturation and ovulation.44 however, physical contact through mating may not be the only mechanism that stimulates ovulation in felids. specifically, ovulation can be induced via visual, olfactory and/or auditory cues in the jaguar (p. onca).49 there is also evidence that noncontact mechanisms can induce ovulation in pallas’s cat, as ovulations are observed in females housed adjacent to a male.19 spontaneous ovulation has been observed occasionally in the lion (p. leo) and leopard (p. pardus), especially when females are housed together.44 in the clouded leopard, margay and domestic cat, spontaneous ovulation occurs more regularly.5 therefore, the ovulatory pattern in the family felidae appears to be speciesand/or individual-specific response to physical, visual, auditory, chemical and/or social stimuli.5 seminal characteristics have been thoroughly assessed in domestic cat. typically, domestic cat ejaculates obtained by electroejaculation (eej) are 100 to 738 μl in volume with 60.3 190 x 106 sperm/ml and 44 85% motility. samples obtained via artificial vagina are smaller (< 10 μl) in volume with higher concentration (541 1,730 x 106 sperm/ml) than via eej, but with comparable motility.50 to date, seminal traits have been characterized in 25 wild felid species demonstrating large variations in seminal quality, especially, morphologically abnormal sperm percentage within an ejaculate ranging 30 85% (table 2). teratospermia (i.e. > 60% morphologically abnormal sperm in an ejaculate) is common in felids, and this phenomenon is linked to reduced genetic variability. specifically, species lacking heterozygosity tend to produce more malformed sperm than genetically diverse counterparts. a clear example is the case of the florida panther (puma concolor coryi), a subspecies of puma concolor. due to human encroachment and agricultural expansion, the florida panther has experienced severe population declines that resulted in significant reductions in heterozygosity compared to other puma subspecies.51 consequently, florida panthers’ reproduction is severely compromised.51,52 florida panther ejaculates are smaller (< 1 versus 3 ml) and contain fewer morphologically normal sperm (6 versus 40%) than their counterparts in latin america.52 teratospermia was also observed in some domestic cats.53 teratospermic cats have a higher percent of morphologically abnormal sperm and lower circulating testosterone concentrations than normospermic individuals.53 sperm from teratospermic donors are compromised in metabolic function,54 and ability to undergo capacitation and acrosome reaction, that in turn, reduce fertilizing ability.55 furthermore, teratospermic samples are sensitive to osmotic stress, and thus are highly susceptible to cryopreservation compared to normospermic counterparts.56 finally, a recent study has demonstrated that cgmp and kinase phosphorylation pathways are downregulated in cat sperm from teratospermic donors, findings that further explain the mechanisms underlying reduced metabolic function and fertilization capacity.57 clinical theriogenology 2021; 13: 132 assisted reproductive technologies assisted reproductive technologies such as artificial insemination (ai) and in vitro embryo production developed for the domestic cat has been successfully applied to many wild felid species,6 with pregnancy and/or live offspring after ai with fresh or frozen-thawed sperm reported in 14 felids.6 furthermore, embryos have been produced by ivf in 16 wild felid species, nine of them resulted in births of live offspring.6 excellent reviews on reproductive technologies, including somatic cell nuclear transfer and stem cell technologies in felids have been published recently.6,74 therefore, this section will focus only on technologies that have been applied to wild felid conservation. table 2. seminal traits (mean ± sem) of wild felids species no. ejaculates volume (ml) concentration (x 106 sperm/ml) motility (%) morphologically abnormal sperm (%) lion*58 7 0.423 ± 0.112 1,940 ± 606.0 84.1 ± 7.7 54.0 ± 9.7 tiger59 13 7.0 ± 1.3 31.9 ± 8.6 81.5 ± 3.7 37.5 ± 6.9 tiger**60 2 na 52.3 ± 12.4# 38.3 ± 13.2 48.8 ± 13.9 leopard59 13 5.1 ± 0.6 46.2 ± 9.8 43.8 ± 5.7 79.5 ± 2.0 amur leopard*61 0.0067 ± 0.0037 1,698 ± 758 84.1 ± 9.7 na jaguar*62 11 0.35 ± 0.09 2,635.2 ± 482.8 77.0 ± 3.44 snow leopard42 36 1.54 ± 0.1 29.2 ± 5.7 76.5 ± 2.4 65.0 clouded leopard63 48 0.64 ± 0.03 27.5 ± 2.3 71.0 ± 2.1 38.9 ± 1.7 cheetah59 15 1.8 ± 0.3 27.3 ± 5.8 69.0 ± 5.8 64.6 ± 4.9 iberian lynx64 5 0.483 ± 63.0 7.6 ± 2.2 73.5 ± 5.6 82.3 iberian lynx**65 4 na 10.2 ± 1.78# 47.5 ± 2.5 69.3 eurasian lynx41 3 0.34 ± 0.06 8.86 ± 4.6 60.0 ± 30.0 na canadian lynx66 9 na 13.0 ± 3.6 46 ± 12.0 71.0 bobcat67 13 0.36 ± 0.08 24.4 ± 7.8 55.7 ±5.7 85.3 ocelot40 42 1.4 ± 0.1 101.2 ± 10.6 81.4 ± 1.2 16.7 ± 1.5 margay40 41 0.5 ± 0.01 75.6 ± 11.0 73.5 ± 1.3 42.6 ± 4.8 northern tiger cat40 52 0.3 ± 0.1 411.9 ± 46.3 71.4 ± 2.3 40.8 ± 4.5 serval66 6 na 46.0 ± 14.7 72 ± 8.2 44.0 pallas’s cat18 4 0.164 ± 0.030 51.3 ± 30.7 70.8 ± 2.6 48.0 leopard cat68 24 0.143 ± 0.015 37.0 ± 5.4 73.8 ± 2.6 34.6 ± 9.4 fishing cat69 8 0.5 ± 0.1 108 ± 29 73.0 ± 4.0 66.7 ± 15.9 flat headed cat70 0.121 ± 0.072 56.7 ± 18.7 56.3 ± 19.0 63.2 jungle cat*71 4 0.069 ± 0.016 75.13 ± 9.84 77.13 ± 8.16 26.2 ± 3.51 black footed cat72 12 na na 85.0 ± 1.21 52.8 sand cat72 18 na na 78.6 ± 1.61 60 asian golden cat*73 2 0.089 88.4 62.5 62 *samples were collected via urethral catheterization; **samples were collected via epididymal slicing; #total sperm; na: not assessed clinical theriogenology 2021; 13: 133 semen collection with the exception of the domestic cat where semen can be obtained via artificial vagina,50 sperm recovery from wild felids has been traditionally done with eej (table 2).25,75,76 however, during the past decade, urethral catheterization after medetomidine treatment has been widely applied to recover semen from domestic and wild felids, including lions,58 jaguars,62 amur leopards (p. pardus orientalis),61 jungle cats (felis chaus),71 and asian golden cats (pardofelis temminckii).73 comparisons between ejj and urethral catheterization had varying results. specifically, there were no differences in fertilizing ability and cryosurvival between samples obtained via urethral catheterization and eej, although the former had lower volume and higher concentration than latter.77 however, among urethral catheterization, eej, and epididymal slicing, seminal quality was lowest in samples obtained via urethral catheterization.78 difference between these 2 studies may be due to the variation in the interval between the sample collection methods, as ejaculation frequency impacts seminal quality.50 specifically, in the first study, there was a 4-day interval between urethral catherization and eej,77 whereas in the second study, all 3 methods were performed once on the same day with urethral catherization being conducted prior to eej and then epididymal slicing.78 despite the inconsistent results in seminal quality, urethral catheterization is simple and requires minimum equipment, and thereby can be applied to free-ranging felids living in a remote area.62 the ability to recover sperm postmortem allows preservation of valuable genetics of individuals that die unexpectedly. postmortem recovery of sperm has been attempted for at least 73 individuals across 16 species of wild felids.60,65,79 there were no differences between eej and epididymal slicing in seminal quality of domestic cat samples recovered from the same individual.78 such direct comparison is not feasible in wild felids as most samples were obtained after death,79 yet, information available to date has indicated that the quality of epididymal sperm of tigers60 and iberian lynx65 obtained postmortem appears to be slightly lower than those obtained via eej as reported before.59,64 finally, a recent study79 collected42 spermic samples from 67 gamete rescue attempts in 15 wild felid species. of the 42 samples, 14 (33.3%), 28 (66.6%) and 35 (83.3) were suitable for ai, ivf, and intracytoplasmic sperm injection (icsi), respectively.79 sperm cryopreservation and artificial insemination sperm cryopreservation has been broadly applied to preserve valuable genetic of several wild felids living both ex situ and in situ.60,74,79-83 egg yolk-based extenders containing glycerol as a cryoprotectant have been commonly used to cryopreserve sperm from both domestic and wild felids.79,80,82,84 however, egg yolk-based extenders are not chemically defined and can vary from batch to batch. furthermore, commercial egg yolk-based extenders had frequent bacterial or mycoplasma contamination.85 therefore, studies were conducted to explore the value of soy lecithin-based extender (soy) in cryopreservation of felid sperm. domestic cat sperm cryopreserved in soy fertilized cat oocytes at a rate similar to those frozen in egg yolk-based extender.86 furthermore, in a study in the black-footed cat (f. nigripes), sand cat (f. margarita), fishing cat, and pallas’s cat, there were no differences in postthaw motility and acrosome status between sperm cryopreserved in soy versus in egg yolk-based medium.81 furthermore, cryopreserved sperm from both treatments were able to fertilize in vivo matured domestic cat oocytes at a similar rate, although fertilization rates varied among species.81 to date, ai with fresh or frozen semen has been applied to both domestic and wild felids, resulting in pregnancies and live births.6 like other mammalian species, there are several factors that influence ai success. these include responses to ovarian stimulation, seminal quality, and time and site of insemination.6 induction of estrus and ovulation in felids commonly involves equine chorionic gonadotropin (ecg) treatment to stimulate ovarian follicle growth, followed by human chorionic gonadotropin (hcg) treatment 80 84 hours later to induce ovulation.75 an advantage of this ovarian stimulation regimen is that it minimizes stress associated with animal handling. however, ecg and hcg are large, foreign glycoproteins that persist in the circulation and, thereby induce production of gonadotropin-neutralizing antibodies.75 because of the refractory effect of ecg/hcg, it has been recommended that this hormone regimen is given to the same animal not more than once every 6 12 months.75 porcine follicle stimulating hormone (pfsh) also has been used to stimulate ovarian response in felids.75,87 however, this gonadotropin stimulation protocol requires multiple injections that present logistical challenge when applied to wild felids.75 furthermore, although pfsh treatment has been successfully used for recovery of mature oocytes and embryos in domestic cats and wild felids,87 pregnancies after ai have not been achieved in cheetahs, leopards, lions, and tigers.75 some felid species are insensitive to ecg/hcg treatment.75 interestingly, the variation in the response to exogenous gonadotropin is independent of body size. for example, domestic cat, leopard cat and northern tiger cat are the same size; yet, the latter species require twice the dosages of ecg compared to the 2 former felids.75 although intravaginal insemination with fresh and frozen semen has resulted in live births in the domestic cat, this ai method has not resulted in pregnancies when applied to wild felids, including cheetahs, tigers and clouded leopards.75 to date, successful pregnancies after ai with fresh or frozen semen in wild felids have been achieved by transcervical, intrauterine (either laparotomy or laparoscopy) or laparoscopic oviductal insemination.6,88 ejaculates of many small felids contain < 50 x 106 motile sperm, and thus limit the use of cervical or uterine ai.88 however, the recent development of laparoscopic oviductal insemination has allowed ai to be successfully applied to small felids, including pallas’s cat, fishing cat, sand cat, and ocelot.88 to date, live offspring has been produced after ai with fresh or frozen sperm in these species. oviductal ai also has been applied to larger felids, including tigers and clouded leopard.88 clinical theriogenology 2021; 13: 134 in vitro embryo production extrapolation of reproductive technologies developed in the domestic cat has yielded encouraging outcomes in several wild felids. specifically, currently, in vitro embryo production from in vivo or in vitro matured oocytes has been reported in 16 wild felids, of which 9 resulted in live offpring.6,76 intracytoplasmic sperm injection with cryopreserved ejaculated or epididymal sperm has also been attempted in the jaguarundi, lion and fishing cat, yielding 56 70% cleavage rate.76 however, transfer of preimplantation stage embryos did not result in pregnancy.76 although live offspring have been produced in a number of wild felids, in vitro embryo production has not been widely used in captive breeding programs for several reasons. first, there is limited information on species-specific reproductive endocrinology, gamete biology and embryogenesis. second, the complexity of the procedure and the need for specialized equipment and facility to recover oocyte, perform ivf or icsi and culture resulting embryos. third reason is the limited availability of developmentally competent oocytes, especially in aging females or those with poor health. recently, live births have been reported after transferring in vitro derived embryos produced from a 6 year old female cheetah into a younger recipient (3 year old).89 uterine pathologies can be frequently observed in cheetah when females reach 6 years of age resulting in infertility.90 nevertheless, these older females can produce developmentally competent oocytes that can be fertilized and developed into embryos in vitro.90 therefore, recent success in cheetah clearly demonstrates the potential benefit of ivf technology in preserving fertility of genetically valuable, and underrepresented females whose genetic would be lost otherwise. conclusion domestic cat serves as a valuable model for establishing reproductive technologies in wild felids. yet, due to the large diversity in reproductive mechanisms within the family felidae, there are still needs for species-specific research, especially on how to effectively manipulate female reproductive cycles for ai, oocyte retrieval or embryo transfer. although applied 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oocyte production, blastocyst development, and embryo transfer in the cat. theriogenology 2014;81:126-137. 88. swanson wf: practical application of laparoscopic oviductal artificial insemination for the propagation of domestic cats and wild felids. reprod fert dev 2019;31:27-39. 89. crosier ae, lamy j, bapodra p, et al: first birth of cheetah cubs from in vitro fertilization and embryo transfer. animals (basel) 2020;10. doi: 10.3390/ani10101811. 90. crosier ae, comizzoli p, baker t, et al: increasing age influences uterine integrity, but not ovarian function or oocyte quality, in the cheetah (acinonyx jubatus). biol reprod 2011;85:243-253. clinical theriogenology 2021; 13: 137 reproductive biology and assistedreproductive technologies in felids extracellular vesicles and assisted reproductive technology extracellular vesicles and assisted reproductive technology jennifer nagashima, marcia ferraz, nucharin songsasen center for species survival, smithsonian conservation biology institute national zoological park, front royal, va abstract research in biogenesis, composition, uptake, and functionality of extracellular vehicles (evs) has dramatically expanded in recent decades. these lipid membrane bound particles secreted from all cells contain regulatory molecules (proteins, peptides, rnas, dna fragments, and lipids) that are transferred to target cells upon binding to the plasma membrane. roles of evs were studied in cell to cell communication, mediation of cancer development and immune response, as biomarkers for disease and potential drug delivery systems. more recently, studies were conducted to investigate roles of evs in reproduction, ranging from gamete maturation, fertilization, and embryonic development to implantation. as such, potential use of evs for assisted reproductive technologies (art) has garnered substantial interest. objectives of this review are to present current understanding of evs in reproduction, with particular regard to recent research in understanding influence of reproductive cycles on ev production and cargo composition, and to discuss therapeutic potential of evs in reproductive technologies, including cryopreservation and areas of research that remain to be explored for efficient application of evs in art. keywords: extracellular vesicles, gametes, cryopreservation, organs on a chip, synthetic vesicle introduction extracellular vehicles (evs) are lipid membrane bound vesicles secreted by all cells.1 evs include exosomes (30 100 nm in size), released via fusion of a multivesicle body with a cell’s plasma membrane; microvesicles (mv, also known as ectosomes, 100 1000 nm), shed via budding from plasma membrane;2 and apoptotic bodies (up to 5000 nm), formed through contraction and membrane blebbing of dying cells3 (figure 1). evs contain regulatory molecules, including proteins, peptides, rna species, lipids, and dna fragments which may be taken up by nearby cells via either fusion or endocytosis of vesicles, contributing to cell to cell communication.4 owing to size overlap between exosomes and mv, it is difficult to separate 1 population from another and the 2 groups are often studied together as extracellular vesicles, or “evs” in healthy cells (i.e. excluding apoptotic bodies). multiple methods have been evaluated for ev isolation (reviewed5), including ultracentrifugation (most commonly utilized method6), density gradient centrifugation,7 size-exclusion chromatography,8 ultrafiltration,9 and sequential filtration.10 differences in isolation method become significant in that subpopulations of evs (i.e. more exosomes versus mv, or ev with specific surface markers) may be preferentially purified, based on isolation technique. for example, biological activity of endometrial evs isolated via a commercially available system were compared with those purified via ultracentrifugation and ultrafiltration.11 although ultracentrifugation had highest ev yield, evs isolated via total exosome isolation kit were more readily taken up by trophoblast cells, indicating better preservation of biological activity. further, regardless of purification method, copurified protein aggregates can confound the results of 2 most commonly utilized ev quantification methods, namely, colorimetric protein assays (e.g. bca) and nanoparticle tracking analysis (nta).12 additionally, our ability to fully characterize evs (both in terms of their composition and functionality) is challenged by the heterogenous nature of isolated evs. nevertheless, some characteristic features of ev subpopulations have been described. for example, owing to differing methods of release, proteins related to endosomal sorting complexes required for transport pathway are common to exosomes.13 exosomes are also enriched in tetraspanins (involved in cell adhesion and signaling14) and heat shock proteins.15,16 mv contain higher concentrations of proteins associated with plasma membrane.1 although lipid characterization has been particularly understudied in evs field as a whole,17 lipid clinical theriogenology • volume 12 number 2 • june 2020 89 composition and high cholesterol/phospholipid ratio of evs results in notable membrane stability, which presumably allows for greater protection of cargo and evs in vitro longevity.18 in response to confusion associated with the heterogeneity of evs and diversity of purification and quantification methods, in 2018, the international society for extracellular vesicles published updated standards for minimal information for ev studies.19 these serve as a guide for ev isolation, classification, and nomenclature, and are important to reduce variability in research results among laboratories which are due to inconsistent appellations, or attribution of functions specifically to mv/exosomes when isolation protocols produce heterogenous populations. improved understanding of ev sub-population compositions are also poised to hasten elucidation of the mechanisms of ev uptake into ‘recipient’ cells. it is currently understood that evs are taken up by cells through a variety of pathways, including clathrin-mediated endocytosis, caveolin-mediated uptake, and lipid raft-mediated internalization (reviewed20). whereas ev cargo transfer to recipient cells is documented,21 understanding molecular mechanisms of delivery is an area necessitating further study. still, evs are well studied for their role in cell to cell communication, including in mediating cancer development (reviewed5) and immune response (reviewed22). release of evs by cancer cells and immune system stimulated interest in them as a source of biomarkers for early disease detection (reviewed23). as a result of their natural in vivo stability, cargo carrying capacity, and ability to be taken up by cells, there is also growing interest in utilizing evs for in vivo drug and therapeutic protein delivery.24,25 although the field of reproduction-specific evs is still relatively new, several excellent reviews published in recent years highlighted growing interest and understanding of roles of evs in successful gamete functions, fertilization, embryo development, and pregnancy.26-30 objectives are to outline current understanding of evs in reproduction, including recent evaluations of reproductive stage on ev production and composition, and to discuss potential practical/therapeutic applications of evs in assisted reproduction, with a primary focus on cryopreservation. figure 1. schematic of extracellular vesicle (ev) biogenesis and composition, with exosomes secreted via fusion of multivesicle bodies from early endosome fusing with cell’s plasma membrane, microvesicles secreted via budding, and apoptotic bodies released via blebbing. collectively termed evs, these membrane-bound vesicles carry lipid, rna, dna, and protein cargo from their cells of origin. clinical theriogenology • volume 12 number 2 • june 202090 extracellular vesicles in reproduction overview of male reproductive tract extracellular vesicles to date, studies on male reproductive tract evs have focused on vesicles derived from epididymis (epididymosomes), and prostate gland (prostasomes). epididymosomes are released into lumen from large blebs that form and detach from parent epithelial cells.31 epididymosomes fuse with sperm during their transit through epididymis and deliver small rna,32,33 and protein cargo,34,35 and alter lipid composition of sperm’s plasma membrane,36 essential for sperm maturation process (reviewed37). specifically, epididymosomes reduce cholesterol/phospholipid ratio of bovine caput epididymal sperm,36 that results in increased membrane fluidity necessary for the sperm’s future ability to capacitate. gene ontology analyses of bovine epididymosomes classified proteins into 6 functional groups, including adhesion molecules, chaperones, enzymes, structural proteins, signal transducers, and transporters.37 many of these proteins are involved in sperm egg interactions, motility, plasma membrane modification, and elimination of defective gametes.38 transfer of small rnas (e.g. mirna and tsrna both small, noncoding rna that primarily function in rna silencing and posttranscriptional regulation39) from epididymosomes to sperm is a mechanism for transgenerational epigenetic inheritance.32,40 coincubation of immature sperm with epididymosomes in domestic cats sustained in vitro motility.41 delivered mirna from epididymal evs is linked to pregnancy success in mice, as intracytoplasmic sperm injection with sperm from caput epididymis displayed significant implantation failure following embryo transfer; a phenotype rescued by injection of cauda-specific mirna into embryos.42 it is apparent that these vesicles have a profound influence on not only male gametes, but also pregnancy success and, potentially, offspring/subsequent generation traits via epigenetic inheritance. prostasomes (reviewed43) are challenging to characterize, as they are mixed in with evs from other sources in seminal fluids. proteomic analyses of human prostasomes identified proteins in broad categories (enzymes, transport or structural integrity, gtp proteins, chaperones, signal transduction, and unannotated).44 supplementation of sperm with prostasomes improved sperm motility, attributed to delivery of calcium signaling45 and atp-generating46 machinery to the gamete. human prostasome membranes are high in cholesterol, sphingomyelin and glycosphingolipids.47,48 fusion with prostasomes enriched sperm’s plasma membranes in key lipids, including cholesterol.49 this, in turn, is apparently responsible for the prevention of premature acrosomal exocytosis in sperm exposed to prostasomes in cattle50 and humans.51,52 prostasomes also protect sperm against female immune system, including decreasing natural killer cell activity in vitro,53 indicating prostasomes function both to maintain sperm function and support survival. overview of female reproductive tract extracellular vesicles extracellular vesicles derived from vaginal (vaginosomes54), uterine (uterosomes, reviewed 55,56), oviductal (oviductosomes or oevs, reviewed27) and follicular (ffevs, reviewed30,57,58), fluids, and secretions from in vitro cultured embryos (reviewed59) are characterized in a variety of species. supplementation of evs from these sources benefit gamete function and/or embryo development. for example, coincubation of vaginal evs with murine sperm increased rates of progesterone-induced acrosome exocytosis compared to unsupplemented controls.54 this effect was attributed to transfer of tyrosine phosphorylated proteins and plasma membrane ca2+ atpase ([pmca], which removes calcium from cells) from evs to sperm, essentially priming cells for capacitation while preventing premature acrosome reaction. endometrial mesenchymal stem cell evs improved blastomere count and hatching rates in murine embryos.60 coincubation with evs also stimulated embryos to release vascular endothelial growth factor and platelet derived growth factor, which in turn may modulate endometrial receptivity to implantation.60 our laboratory recently demonstrated that domestic cat oviductal extracellular vesicles (oevs) bound to acrosomal region and midpiece of sperm and prevented premature acrosomal exocytosis (in vitro), while improving sperm motility and fertilizing capacity61 (figure 2). we postulated that enrichment in proteins identified in oevs was related to energy metabolism, calcium transport and clinical theriogenology • volume 12 number 2 • june 2020 91 membrane function, may have regulated this effect. in cattle, oevs modulate sperm calcium concentrations62 and they also modulated sperm capacitation in cattle and mice.62,63 porcine oevs reduced rates of polyspermy in vitro,64 and murine oev supplementation during in vitro fertilization and embryo culture improved live birth rates after embryo transfer.65 hence, recently, oevs have been utilized to improve in vitro embryo production technologies.27 follicular fluid evs, apparently produced by granulosa and cumulus cells, are taken up into cumulus cell layer of domestic cat oocytes (figure 2) and, in cattle, transferred to an oocyte via transzonal projections.66 bovine ffevs stimulated granulosa cell proliferation67 and cumulus cell expansion,68 and improved blastocyst production.68 bovine embryos produced in vitro in the presence of ffevs exhibited varying levels of global dna methylation compared to nonffev counterparts.69 taken together, it is clear that supplementation of evs to in vitro embryo production systems promises to improve not only production rates, but also embryo quality and potentially live birth success. extracellular vesicles and reproductive cycles despite strong evidence of beneficial actions of evs on reproduction, our understanding of how ev biogenesis and composition are modulated by reproductive cycle is limited. nevertheless, there is evidence that changes in secreted evs are under hormonal control.66,70-73 for example, ovariectomized sheep treated with progesterone for 14 days had higher ev count (based on nta) and mode diameter in their uterine fluid compared to untreated cohorts, indicating biogenesis and secretion of endometrial evs were modulated by progesterone.71 in an in vitro study with primary cell cultures of human endometrial epithelial cell lines, there were no differences in ev amounts (based on protein concentrations) from cells cultured only with estrogen (i.e. proliferative phase-mimetic) versus estrogen and progesterone (i.e. secretory phase mimetic).72 however, there were 380 differentially expressed proteins between evs of 2 treatment groups, many of which were involved in cytoskeletal organization, cell migration, cell adhesion, and extracellular matrix organization. these studies indicated that uterine ev production and their protein compositions are under hormonal control. figure 2. schematic of female reproductive tract evs and interaction with gametes/embryos, including pre-release uterosomes in a multivesicular body in ovine endometrial luminal epithelium (top left, tem image from burns et al 2016), oviductosomes or oev from domestic cats (lower middle, tem image fusing with sperm tail from ferraz et al 2019), and feline follicular fluid (top middle, fluorescent image with ev lipids labeled in green (dio) and ev proteins in blue (ghost dye) taken up by cumulus cells, manuscript under review) clinical theriogenology • volume 12 number 2 • june 202092 recently, counts, transcriptomes, and proteomes of oevs recovered during various stages of reproductive cycles have been characterized in several mammalian species. in murine oev, very few differences in mirna content were observed among reproductive stages (metestrus/diestrus versus proestrus/estrus).73 in domestic cats, we observed an increase in concentration, but not size distribution, in oevs recovered during midluteal phase compared to early luteal and late follicular phases (manuscript under review). conversely, protein and mrna/small rna content of bovine oev isolated during postovulatory and early luteal phases were enriched in proteins related to protein translation and transport, whereas those obtained during late luteal phase contained proteins related to vesicles, cytoskeleton, metalloexopeptidase activity, and innate immune system.70 furthermore, mirna with high abundance in recently ovulated and late luteal phase included those involved in fatty acid biosynthesis and metabolism, cell cycle, oocyte meiosis, and multiple signaling pathways.70 these results were further evidence for potential targeted messaging to gametes and/or embryos to promote fertilization and development by reproductive tract. also in a bovine model, mirna from ffev of 3 6 mm diameter follicles at various stages of the estrous cycle were compared.66 follicular fluid evs from early luteal ovaries (with evidence of recent ovulation, low follicular fluid progesterone concentration) were enriched in mirna involved in multiple signaling pathways, including foxo, hippo, mapk, and tgfβ compared to ffevs from midluteal phase ovaries (tan corpus luteum, high progesterone).66 when early luteal phase ffevs were supplemented to culture media during 24 hour in vitro oocyte maturation, expression of genes associated with pathways related to oocyte development, ovulation, and notch signaling were upregulated in cumulus cells, indicating that reproductive stage specific cargo of ffevs may have key roles in regulating gamete function. more tangentially, there is also evidence linking metabolic status as result of lactation to mirna composition of bovine follicular fluid evs.74 namely, cows in negative energy balance had ffev with downregulation of mirna associated with metabolic processes and oocyte growth compared to metabolically normal lactating heifers, which has implications for folliculogenesis during lactational anestrus. although in the earlier bovine ffev study, supplementing ffevs during ivm upregulated gene expression in the cumulus cell, no differences in oocyte maturation rates were observed between ffevs gametes versus controls.66 it is possible that 24 hour time frame of ivm was not long enough to facilitate transfer of ev cargo into the oocyte and influence meiotic maturation outcome. in another bovine study, ffevs were detected in cumulus cells via confocal microscopy 16 hours after incubation.60 we have similarly observed uptake of ffev lipids and proteins only in cumulus cells of feline oocytes after 18 hours coincubation (manuscript under review). however, in ova from domestic dogs, oev contents were observed in the oocyte after 72 hours incubation.75 it is clear, therefore, that an improved understanding of the kinetics of ev uptake into oocytes is necessary to further our understanding of their functional effects and any subsequent reproductive stage-specific differences. potential for dramatic differences in ev composition by reproductive stage/status are not limited to female reproductive tract. differences in protein composition of seminal plasma during breeding versus nonbreeding seasons have been characterized in sheep76,77 and goats.78,79 notably, there is some evidence that certain protein components of breeding season (autumn/winter) seminal plasma improved motility of frozen-thawed ram sperm; however, this effect was not observed in nonbreeding season or unsupplemented sperm.80 while focus of these studies was on seminal plasma, rather than isolated evs, it is possible that some proteins identified as interacting with sperm membranes are derived from evs. this assertion is supported by the finding that there were no quantitative differences in abundance of most metabolic enzymes isolated from ram seminal plasma extracellular vesicles versus whole seminal plasma.81 extracellular vesicles and assisted reproductive technologies of cryopreservation potential of ev for assisted reproductive technologies, especially in vitro embryo production, has been elegantly reviewed recently.28 here, we focus on new research into potential beneficial effects of evs in cryopreservation. recent reports of ev supplementation to gamete and/or embryo cryopreservation yielded varying results. significantly improved survival and pregnancy success were clinical theriogenology • volume 12 number 2 • june 2020 93 observed with cryopreserved and thawed in vitro-derived bovine embryos cultured in amniotic mv (from days 5 7 following ivf), compared to unsupplemented controls.82 zygotes coincubated with oevs from either the isthmus or ampulla of the oviduct were compared to unsupplemented or fetal calf serum supplemented controls.83 blastocysts developing from isthmic oev-supplemented cultures displayed improved post-thaw survival rate (determined via reexpansion of blastocoel cavity) compared to control and fbs supplemented groups. conversely, bovine embryos cultured with varying concentrations (based on protein content) of postovulatory oevs had improved day 7 blastocyst rates, but supplementation had no effect on cryotolerance of blastocysts, as evaluated by hatching rates.84 further, postthaw survival of bovine blastocysts sequentially cultured in oevs and uterus-derived evs was not significantly improved compared to controls.85 as each study utilized different ev sources (amniotic, oviductal and uterine fluid alone or in combination), isolation methods, and concentrations, as well as coculture timings and metrics of success, the full influence of reproductive evs on bovine embryo cryopreservation is not yet clear. in our laboratory, ffev supplementation during cryopreservation and thawing of domestic cat oocytes did not improve survival based on morphology, but did enhance ability of frozen and thawed oocytes to resume meiosis in vitro (manuscript under revision). still, proteomic analyses of cat ffevs identified proteins with known cryomodulating effects, including heat shock proteins (hsp), cytoskeletal elements, and oxidoreductases. for example, treatment of oocytes with cytoskeleton stabilizing agents improved cryopreservation success in mice86 and cows.87 significant loss of chaperone proteins like hsps and oxidoreductases have been observed following sperm cryopreservation in sheep88 and carp,89 respectively. transfer of these and other potentially cryoprotective proteins may have supported postthaw recovery of gametes; however, much more work is necessary in this area as well. for sperm, the addition of bovine oevs postthaw maintains viability while supporting capacitation during incubation, compared to sperm incubated under capacitating conditions in oev absence.62 cryopreservation significantly alters sperm membrane’s lipid compositions (reviewed90), including reducing sterols, phosphatidyl choline, and unsaturated fatty acids in caprine sperm membranes.91 as previously mentioned, evs contain high concentrations of lipids and proteins associated with lipid-rafts, which likely supports their membrane fusion and stability in vitro,92 but also could support recovery of cryodamaged sperm membranes upon fusion. lipid component is part of rationale behind addition of egg yolk to sperm cryopreservation media.93 more recently, a liposome-based medium (optixcell) was evaluated for cryopreservation of ram,94 buffalo,95 bull,96,97 deer,98 and rhino99 sperm, with promising results overall, even when compared to egg yolk, the current ‘gold-standard’. similar to evs, liposomes are phospholipid membrane vesicles, though they lack protein cargo. it is feasible that combining our understanding proteins present in ev membranes, particularly those likely to target gametes and promote uptake, we may be able to engineer vesicles with specific lipid and/or protein compositions to support cryosurvival while maintaining high affinity for gametes. future extracellular vesicles production for therapeutics applications of evs to assisted reproduction is faced with several challenges, including the aforementioned need to improve our ability to isolate, purify and characterize evs. however, even once we have a better understanding of evs and optimal (and standardized) methods to purify populations of interest, evs with consistent characteristics with low variability must be producible in large quantities. this is not feasible with in vivo sources. beyond inherent variability, flushing both oviducts (figure 3a) of a single individual with the aforementioned total exosome isolation kit, we isolate on average 3.1 x 1010 and 4.5 x 1010 ev/ml (in 1 ml) from domestic cats and dogs, respectively. in contrast, for our experimental purposes, we typically utilize ~ 3 x 107 evs/ml. though this is sufficient in a laboratory setting, high throughput production of evs is necessary for clinical applications. in vitro extracellular vesicles production to improve our ability to consistently increase ev production, much work on evs utilize in vitro produced vesicles from cell lines. impact of isolation and culture conditions on ev production has been reviewed,6 yet much is still unknown, in large part because our understanding of biogenesis of these clinical theriogenology • volume 12 number 2 • june 202094 vesicles is not fully elucidated. different cell types secreted different amounts of evs in vitro,6 but whether this is intrinsic to cells or perhaps in response to pre-established culture methods is not certain. presence or absence of serum in culture medium for neuroblastoma cells has been demonstrated to alter both quantity of produced evs as well as their protein expression levels.100alterations in lipid composition are also apparent from cell lines from same organ, as lipid profiling of prostate-derived cell line evs identified differences in molecular lipids between cell lines with different tumorgenicity.101 although culture conditions, once optimized, should allow for production of evs with definable characteristics, high throughput production remains challenging.102 owing to broad interest in evs in regenerative medicine and cancer research, this area has already begun to be explored. ev production from prostate cancer cells is significantly improved when cells are cultured in a bioreactor compared to a conventional system,103 with ev morphology and select surface markers not different between these 2 conditions. however, metabolic analyses indicated differences in ev composition between conventional versus bioreactor derived evs, particularly with regard to lipid contents. a 3d printer fabricated bioreactor has also been shown to enhance ev production for human endothelial cells.104 still, as trauma and cell stress have also been demonstrated to increase ev secretion105 and mrna/protein compositions,106 respectively. mechanical forces on cells are known to promote exocytosis,107 therefore it is also possible that shear forces on cells grown in bioreactor systems may be stimulating mechanical, stress-driven ev production. under these conditions, ev composition is likely altered and, potentially, less efficacious. more studies are needed to assess: 1) if dynamic culture systems consistently result in improved ev production across cell types, compared to conventional culture; and 2) how quality/composition/functionality of these evs compare with in vivo-derived counterparts and/or those produced by conventional culture systems. in a sense, platforms for improved in vitro production of reproductive ev are already in progress. organs on a chip technology, which has been applied to recreate in vitro microenvironment in more naturalistic conditions by mimicking tissue architecture, cell heterogeneity, and dynamic exposure to nutrients, growth factors and hormones, represent microfluidic bioreactors. automated and controlled flow with specifically timed hormone supplementations can mimic normal pulsatile hormone pattern of reproductive cycle, and flow-through containing evs readily collected (figure 3b). a bovine oviduct-on-a-chip, mimicking in vivo conditions in this organ, demonstrated epigenetic patterns of chip derived embryos to be more similar to fresh controls than those produced via conventional ivf.108 an artificial uterus has similarly been produced via microfluidic chip, consisting of figure 3. in vivo and vitro alternatives for collection of evs, with: a) flush of fluids containing ev from domestic cat oviducts, versus b) dynamic fluidic culture system where tissues/cells of interest can be maintained in channels (inset) with culture medium delivered via syringe pump and flow through containing ev collected in tubes. clinical theriogenology • volume 12 number 2 • june 2020 95 two channels separated with a porous membrane, with cell culture (endometrial and embryos) on apical side and perfusion on basolateral.109 higher rates of blastocyst development were obtained in chip compared to a conventional petri dish. no microfluidic systems for vaginal cells have been reported, though a human vaginal epithelial cell model with 3d organoid culture has been described.110 a combination ovary, fallopian tube, uterus, cervix, and liver reproductive system on a chip with human and murine tissues/cells has been produced (termed evatar), that mimics hormonal profiles of human menstrual cycle in vitro.111 we have previously described an ovary-on-a-chip for culture of domestic dog and cat ovarian tissues,112 and substantial strides have been made in development of in vitro spermatogenesis with testis on chip in recent years.113-115 conversely, microfluidic or bioreactor systems for accessory sex glands and/or epididymis are lacking. still, 3d prostate organoid cultures (human and mouse sources) have been described116 for application primarily to prostate cancer research. moreover, coculture of human cauda epididymal epithelial cells (spheroids) with caput epididymal sperm improved sperm motility and zp-binding compared to non-cocultured controls.117 although ev production has not been specifically compared with regards to culture system in any of described dynamic and/or 3d culture systems, we are optimistic that tools being developed to produce improved in vitro models of reproductive organs have potential to serve as controllable sources of reproductive evs for art as well. synthetic or manufactured extracellular vesicles several methods to manufacture evs have also been reported outside of those secreted ‘naturally’ in cell culture. these have been of particular interest for their potential as nanoscale drug delivery systems, taking advantage of their inherent cargo transfer (reviewed118-121) and gamete/embryo (reviewed29) abilities. most nanovesicles or “exosome mimetics” have been produced via disruption of cell membranes. for example, filter-based extrusion of natural killer cells were shown to maintain antitumor effects of parental/source cells, in both in vitro and in vivo mouse models.122 ev mimetics have been produced by treating cells with cytochalasin b, by which dissociated cell membranes roughly size of mv can be obtained.123,124 these ‘nanovesicles’ have similar morphology to natural evs (based on tem) and reportedly have similar content and functionality as parental cells.124 membrane extrusion has also been utilized to engineer ‘hybrid’ evs. for example, evs secreted from 3t3 fibroblast and a549 tumor cell line were fused with synthetic lipids.125 this method allowed for a significant increase in number of controlled size vesicles compared to evs purified from cell line secretions. further, engineered evs were demonstrated to be effective at drug delivery, as small interfering rna (sirna) loaded by electroporation into ev induced gene silencing in target cells comparable to commercially available kits. fusion of evs secreted from murine macrophages with engineered liposomes increased yield while maintaining targeted binding functionality of evs.126,127 these fusion evs also had demonstrable success as drug delivery vehicles when tested in vitro. getting further away from in vivo derived evs and cell sources, synthetic vesicles have been produced (reviewed92), including liposomes (previously discussed in cryopreservation section), composed of natural lipids and fully synthetic ‘polymersomes’, composed of amphiphilic block copolymers.128 though cargo of naturally secreted evs is not replicated in these manufactured vesicles, above studies demonstrated their ability to be loaded with specific cargos of interest. as a result, engineered evs have potential for drug delivery or other clinical and art applications. conclusion extracellular vesicle research represents a rapidly evolving field with diverse potential clinical applications. although work on reproductive evs is relatively new, epididymosomes, prostasomes, vaginasomes, uterosomes and oviductosomes have been characterized in numerous studies. this interest is owed in no small part to important effects observed on gamete maturation and function observed thus far. substantially more work is needed to fully understand biogenesis, uptake, and downstream effects of varying ev, particularly within context of reproductive tract. however, if new methods for in vitro production can consistently generate high quality and quantity vesicles, there is broad clinical potential of evs in assisted reproduction. clinical theriogenology • volume 12 number 2 • june 202096 conflict of interest there are no conflicts of interest to declare. acknowledgement authors thank national institute of health 1f32hd090854-01a1 (j.n.) and smithsonian institution for support. references 1. doyle lm, wang mz: overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. cells 2019;8:727-751. 2. abels er, breakefield xo: introduction to extracellular vesicles: biogenesis, rna cargo selection, content, release, and uptake. cell 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delivery. j extracell vesicles 2018;7:1440131. 26. machtinger r, laurent lc, baccarelli aa: extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation. hum reprod update 2016;22:182-193. clinical theriogenology • volume 12 number 2 • june 2020 97 27. almiñana c, bauersachs s: extracellular vesicles in the oviduct: progress, challenges and implications for the reproductive success. bioengineering 2019;6:32-57. 28. andrade gm, bridi a, gimenes lu, et al: extracellular vesicles and its advances in female reproduction. anim reprod 2019;16:31-38. 29. barkalina n, jones c, wood mja, et al: extracellular vesicle-mediated delivery of molecular compounds into gametes and embryos: learning from nature. hum reprod update 2015;21:627-639. 30. de ávila acfcm, da silveira jc: role of extracellular vesicles during oocyte maturation and early embryo development. reprod fertil dev 2020;32:56-64. 31. hermo l, jacks d: nature’s ingenuity: bypassing the classical secretory route via apocrine secretion. mol reprod dev 2002;63:394-410. 32. reilly jn, mclaughlin ea, stanger sj, et al: characterisation of mouse epididymosomes reveals a complex profile of micrornas and a potential mechanism for modification of the sperm epigenome. sci rep 2016;6:1-15. 33. trigg na, eamens al, nixon b: the contribution of epididymosomes to the sperm small rna profile. reproduction 2019;157:r209-r223. 34. sullivan r, frenette g, girouard j: epididymosomes are involved in the acquisition of new sperm proteins during epididymal transit. asian j androl 2007;9:483-491. 35. nixon b, de iuliis gn, hart hm, et al: proteomic profiling of mouse epididymosomes reveals their contributions to post-testicular sperm maturation. mol cell proteomics 2019;18:s91-s108. 36. schwarz a, wennemuth g, post h, et al: vesicular transfer of membrane components to bovine epididymal spermatozoa. cell tissue res 2013;353:549-561. 37. sullivan r: epididymosomes: role of extracellular microvesicles in sperm maturation. front biosci (schol ed) 2016;8:106-114. 38. girouard j, frenette g, sullivan r: comparative proteome and lipid profiles of bovine epididymosomes collected in the intraluminal compartment of the caput and cauda epididymidis. int j androl 2011;34:e475-e486. 39. cai y, yu x, hu s, et al: a brief review on the mechanisms of mirna regulation. genom proteom bioinf 2009;7: 147-154. 40. rompala gr, mounier a, wolfe cm, et al: heavy chronic intermittent ethanol exposure alters small noncoding rnas in mouse sperm and epididymosomes. front genet 2018;9:32. 41. rowlison t, ottinger ma, comizzoli p: key factors enhancing sperm fertilizing ability are transferred from the epididymis to the spermatozoa via epididymosomes in the domestic cat model. j assist reprod genet 2018; 35:221-228. 42. conine c, sun f, song l, et al: small rnas gained during epididymal transit of sperm are essential for embryonic development in mice. dev cell 2018;46:470-480.e473. 43. aalberts m, stout ta, stoorvogel w: prostasomes: extracellular vesicles from the prostate. reproduction 2014;147:r1-r14. 44. utleg ag, yi ec, xie t, et al: proteomic analysis of human prostasomes. prostate 2003;56:150-161. 45. park k-h, kim b-j, kang j, et al: ca2+ signaling tools acquired from prostasomes are required for progesteroneinduced sperm motility. sci singal 2011;4 ra31. doi: 10.1126/scisignal.2001595. 46. ronquist kg, ek b, morrell j, et al: prostasomes from four different species are able to produce extracellular adenosine triphosphate (atp). bba gen subjects 2013;1830:4604-4610. 47. brouwers jf, aalberts m, jansen jw, et al: distinct lipid compositions of two types of human prostasomes. proteomics 2013;13:1660-1666. 48. arvidson g, ronquist g, wikander g, et al: human prostasome membranes exhibit very high cholesterol/phospholipid ratios yielding high molecular ordering. bba biomembranes 1989;984:167-173. 49. burden hp, holmes ch, persad r, et al: prostasomes—their effects on human male reproduction and fertility. hum reprod update 2005;12:283-292. 50. frenette g, sullivan r: prostasome‐like particles are involved in the transfer of p25b from the bovine epididymal fluid to the sperm surface. mol reprod dev 2001;59:115-121. 51. pons‐rejraji h, artonne c, sion b, et al: prostasomes: inhibitors of capacitation and modulators of cellular signalling in human sperm. int j androl 2011;34:568-580. 52. carlini e, palmerini ca, cosmi ev, et al: fusion of sperm with prostasomes: effects on membrane fluidity. arch biochem biophys 1997;343:6-12. 53. tarazona r, delgado e, guarnizo m, et al: human prostasomes express cd48 and interfere with nk cell function. immunobiology 2011;216:41-46. 54. fereshteh z, bathala p, galileo ds, et al: detection of extracellular vesicles in the mouse vaginal fluid: their delivery of sperm proteins that stimulate capacitation and modulate fertility. j cell physiol 2019;234:12745-12756. 55. o'neil ev, burns gw, spencer te: extracellular vesicles: novel regulators of conceptus-uterine interactions? theriogenology 2020; in press. 56. kurian nk, modi d: extracellular vesicle mediated embryo-endometrial cross talk during implantation and in pregnancy. j assist reprod genet 2019;36:189-198. 57. tesfaye d, hailay t, salilew-wondim d, et al: extracellular vesicle mediated molecular signaling in ovarian follicle: implication for oocyte developmental competence. theriogenology 2020; in press. clinical theriogenology • volume 12 number 2 • june 202098 58. di pietro c: exosome-mediated communication in the ovarian follicle. j assist reprod genet 2016;33:303-311. 59. giacomini e, alleva e, fornelli g, et al: embryonic extracellular vesicles as informers to the immune cells at the maternal–fetal interface. clin exp immunol 2019;198:15-23. 60. blázquez r, sánchez-margallo fm, álvarez v, et al: murine embryos exposed to human endometrial mscs-derived extracellular vesicles exhibit higher vegf/pdgf aa release, increased blastomere count and hatching rates. plos one 2018; 13: e0196080. 61. de amm ferraz m, carothers a, dahal r, et al: oviductal extracellular vesicles interact with the spermatozoon’s head and mid-piece and improves its motility and fertilizing ability in the domestic cat. sci rep 2019;9:1-12. 62. franchi a, moreno-irusta a, domínguez em, et al: extracellular vesicles from oviductal isthmus and ampulla stimulate the induced acrosome reaction and signaling events associated with capacitation in bovine spermatozoa. j cell biochem 2020;121:2877-2888. 63. bathala p, fereshteh z, li k, et al: oviductal extracellular vesicles (oviductosomes, ovs) are conserved in humans: murine ovs play a pivotal role in sperm capacitation and fertility. mol hum reprod 2018;24:143-157. 64. alcântara-neto a, fernandez-rufete m, corbin e, et al: oviduct fluid extracellular vesicles regulate polyspermy during porcine in vitro fertilisation. reprod fertil dev 2019;32:409-418. 65. qu p, zhao y, wang r, et al: extracellular vesicles derived from donor oviduct fluid improved birth rates after embryo transfer in mice. reprod fertil dev 2019;31:324-332. 66. de ávila acfcm, bridi a, andrade gm, et al: estrous cycle impacts microrna content in extracellular vesicles that modulate bovine cumulus cell transcripts during in vitro maturation†. biol reprod 2019;102:362-375. 67. hung w-t, navakanitworakul r, khan t, et al: stage-specific follicular extracellular vesicle uptake and regulation of bovine granulosa cell proliferation. biol reprod 2017;97:644-655. 68. hung w-t, hong x, christenson lk, et al: extracellular vesicles from bovine follicular fluid support cumulus expansion. biol reprod 2015;93:117,111-119. 69. da silveira jc, andrade gm, del collado m, et al: supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development. plos one 2017; 12: e0179451. 70. almiñana c, tsikis g, labas v, et al: deciphering the oviductal extracellular vesicles content across the estrous cycle: implications for the gametes-oviduct interactions and the environment of the potential embryo. bmc genomics 2018;19:622-649. 71. burns gw, brooks ke, o’neil ev, et al: progesterone effects on extracellular vesicles in the sheep uterus. biol reprod 2018;98:612-622. 72. greening dw, nguyen hpt, elgass k, et al: human endometrial exosomes contain hormone-specific cargo modulating trophoblast adhesive capacity: insights into endometrial-embryo interactions. biol reprod 2016 feb;94(2):38. doi: 10.1095/biolreprod.115.134890. 73. fereshteh z, schmidt sa, al-dossary aa, et al: murine oviductosomes (ovs) microrna profiling during the estrous cycle: delivery of ovs-borne micrornas to sperm where mir-34c-5p localizes at the centrosome. sci rep 2018;8:16094. 74. hailay t, hoelker m, poirier m, et al: extracellular vesicle-coupled mirna profiles in follicular fluid of cows with divergent post-calving metabolic status. sci rep 2019;9:1-14. 75. lange-consiglio a, perrini c, albini g, et al: oviductal microvesicles and their effect on in vitro maturation of canine oocytes. reproduction 2017;154:167-180. 76. gündoğan m: some reproductive parameters and seminal plasma constituents in relation to season in akkaraman and awassi rams. turk j vet anim sci 2006;30:95-100. 77. cardozo j, fernández-juan m, forcada f, et al: monthly variations in ovine seminal plasma proteins analyzed by twodimensional polyacrylamide gel electrophoresis. theriogenology 2006;66:841-850. 78. la falci vsn, tortorella h, rodrigues jl, et al: seasonal variation of goat seminal plasma proteins. theriogenology 2002;57:1035-1048. 79. aguiar gv, van tilburg mf, catunda agv, et al: sperm parameters and biochemical components of goat seminal plasma in the rainy and dry seasons in the brazilian northeast: the season's influence on the cooling of semen. arq bras med vet zootec 2013;65:6-12. 80. domínguez m, falcinelli a, hozbor f, et al: seasonal variations in the composition of ram seminal plasma and its effect on frozen-thawed ram sperm. theriogenology 2008;69:564-573. 81. leahy t, rickard jp, bernecic nc, et al: ram seminal plasma and its functional proteomic assessment 2019;157:r243r256. 82. lange-consiglio a, lazzari b, pizzi f, et al: amniotic microvesicles impact hatching and pregnancy percentages of in vitro bovine embryos and blastocyst microrna expression versus in vi vo controls. sci rep 2020;10:1-10. 83. lopera-vasquez r, hamdi m, maillo v, et al: effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro. reproduction 2017;153:461-470. 84. le bourhis d, janati idrissi s, mermillod p, et al: effect of oviductal fluid extracellular vesicle supplementation during in vitro culture on development and quality of bovine embryos. reprod fertil dev 2020;32:158-158. 85. leal c, cañon-beltrán k, cajas y, et al: extracellular vesicles from oviduct and uterus in sequential culture improve the quality of bovine embryos produced in vitro. reprod fertil dev 2020;32:164-164. clinical theriogenology • volume 12 number 2 • june 2020 99 86. park se, chung hm, cha ky, et al: cryopreservation of icr mouse oocytes: improved post-thawed preimplantation development after vitrification using taxol™, a cytoskeleton stabilizer. fertil steril 2001;75:1177-1184. 87. morató r, izquierdo d, albarracín jl, et al: effects of pre‐treating in vitro‐matured bovine oocytes with the cytoskeleton stabilizing agent taxol prior to vitrification. mol reprod dev 2008;75:191-201. 88. pini t, rickard jp, leahy t, et al: cryopreservation and egg yolk medium alter the proteome of ram spermatozoa. proteomics 2018;181:73-82. 89. li p, hulak m, koubek p, et al: ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, cyprinus carpio l. theriogenology 2010;74:413-423. 90. mandal r, badyakar d, chakrabarty j: role of membrane lipid fatty acids in sperm cryopreservation. advances in andrology 2014;2014:1-9. 91. chakrabarty j, banerjee d, pal d, et al: shedding off specific lipid constituents from sperm cell membrane during cryopreservation. cryobiology 2007;54:27-35. 92. fernandez-trillo f, grover lm, stephenson-brown a, et al: vesicles in nature and the laboratory: elucidation of their biological properties and synthesis of increasingly complex synthetic vesicles. agnew chem 2017;56:3142-3160. 93. manjunath p: new insights into the understanding of the mechanism of sperm protection by extender components. anim reprod 2012;9:809-815. 94. luna-orozco jr, gonzález-ramos ma, calderón-leyva g, et al: comparison of different diluents based on liposomes and egg yolk for ram semen cooling and cryopreservation. iran j vet res 2019; 20: 126-130. 95. ansari ms, rakha ba, akhter s, et al: optixcell improves the postthaw quality and fertility of buffalo bull sperm. theriogenology 2016;85:528-532. 96. singh a, bhakat m, mohanty tk, et al: effect of tris-egg yolk, soya milk, and liposome-based extenders on sahiwal (bos indicus) sperm quality during preand post-cryopreservation stages. cryoletters 2019;40:94-102. 97. röpke t, oldenhof h, leiding c, et al: liposomes for cryopreservation of bovine sperm. theriogenology 2011;76:1465-1472. 98. stewart jl, shipley cf, katich as, et al: cryopreservation of white-tailed deer (odocoileus virginianus) semen using soybean-, liposome-, and egg yolk-based extenders. anim reprod sci 2016;171:7-16. 99. wojtusik j, stoops ma, roth tl: animal protein-free optixcell and shortened equilibration periods can replace egg yolk-based extender and slow cooling for rhinoceros semen cryopreservation. cryobiology 2019;89:21-25. 100. li j, lee y, johansson hj, et al: serum-free culture alters the quantity and protein composition of neuroblastomaderived extracellular vesicles. j extracell vesicles 2015;4:26883. 101. brzozowski js, jankowski h, bond dr, et al: lipidomic profiling of extracellular vesicles derived from prostate and prostate cancer cell lines. lipids in health and disease 2018;17:211. 102. davies og, rafiq qa: considerations for the bioprocessing, manufacture and translation of extracellular vesicles for therapeutic and diagnostic applications. cell gene ther insights 2017;3:683-694. 103. palviainen m, saari h, kärkkäinen o, et al: metabolic signature of extracellular vesicles depends on the cell culture conditions. journal of extracellular vesicles 2019;8:1596669. 104. patel db, santoro m, born lj, et al: towards rationally designed biomanufacturing of therapeutic extracellular vesicles: impact of the bioproduction microenvironment. biotechnol adv 2018;36:2051-2059. 105. kuravi sj, yates cm, foster m, et al: changes in the pattern of plasma extracellular vesicles after severe trauma. plos one 2017;12:e0183640. 106. de jong og, verhaar mc, chen y, et al: cellular stress conditions are reflected in the protein and rna content of endothelial cell-derived exosomes. j extracell vesicles 2012;1:18396. 107. apodaca g: modulation of membrane traffic by mechanical stimuli. am j physiol-renal 2002;282:f179-f190. 108. ferraz ma, henning hh, costa pf, et al: improved bovine embryo production in an oviduct-on-a-chip system: prevention of poly-spermic fertilization and parthenogenic activation. lab chip 201717:905-916. 109. li w-x, liang g-t, yan w, et al: artificial uterus on a microfluidic chip. chinese j anal chem 2013 41:467-472. 110. hjelm be, berta an, nickerson ca, et al: development and characterization of a three-dimensional organotypic human vaginal epithelial cell model. biol reprod 2010;82:617-627. 111. xiao s, coppeta jr, rogers hb, et al: a microfluidic culture model of the human reproductive tract and 28-day menstrual cycle. nat commun 2017;8:14584-14584. 112. nagashima jb, el assal r, songsasen n, et al: evaluation of an ovary‐on‐a‐chip in large mammalian models: species specificity and influence of follicle isolation status. j tissue eng regen m 2018;12:e1926-e1935. 113. komeya m, hayashi k, nakamura h, et al: pumpless microfluidic system driven by hydrostatic pressure induces and maintains mouse spermatogenesis in vitro. sci rep 2017;7:1-8. 114. komeya m, kimura h, nakamura h, et al: long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device. sci rep 2016;6:1-10. 115. yamanaka h, komeya m, nakamura h, et al: a monolayer microfluidic device supporting mouse spermatogenesis with improved visibility. biochem biophys res commun 2018;500:885-891. 116. drost j, karthaus wr, gao d, et al: organoid culture systems for prostate epithelial and cancer tissue. nat protoc 2016;11:347-358. 117. moore hd, curry mr, penfold lm, et al: the culture of human epididymal epithelium and in vitro maturation of epididymal spermatozoa. fertil steril 1992;58:776-783. clinical theriogenology • volume 12 number 2 • june 2020100 118. vader p, mol ea, pasterkamp g, et al: extracellular vesicles for drug delivery. adv drug delivery rev 2016;106:148-156. 119. batrakova ev, kim ms: using exosomes, naturally-equipped nanocarriers, for drug delivery. j control release 2015;219:396-405. 120. ohno s-i, drummen gp, kuroda m: focus on extracellular vesicles: development of extracellular vesicle-based therapeutic systems. int j mol sci 2016;17:172-191. 121. agrahari v, agrahari v, burnouf p-a, et al: extracellular microvesicles as new industrial therapeutic frontiers. trends biotechnol 2019;37:707-729. 122. zhu l, gangadaran p, kalimuthu s, et al: novel alternatives to extracellular vesicle-based immunotherapy–exosome mimetics derived from natural killer cells. artif cells nanomed biotechnol 2018;46:s166-s179. 123. gomzikova mo, zhuravleva mn, miftakhova rr, et al: cytochalasin b-induced membrane vesicles convey angiogenic activity of parental cells. oncotarget 2017;8:70496. 124. gomzikova mo, zhuravleva mn, vorobev vv, et al: angiogenic activity of cytochalasin b-induced membrane vesicles of human mesenchymal stem cells. cells 2019;9:95-111. 125. jhan y-y, prasca-chamorro d, zuniga gp, et al: engineered extracellular vesicles with synthetic lipids via membrane fusion to establish efficient gene delivery. int j pharm 2020;573:118802. 126. sato yt, umezaki k, sawada s, et al: engineering hybrid exosomes by membrane fusion with liposomes. sci rep 2016;6:21933. 127. rayamajhi s, nguyen tdt, marasini r, et al: macrophage-derived exosome-mimetic hybrid vesicles for tumor targeted drug delivery. acta biomater 2019;94:482-494. 128. rideau e, dimova r, schwille p, et al: liposomes and polymersomes: a comparative review towards cell mimicking. chem soc rev 2018;47:8572-8610. clinical theriogenology • volume 12 number 2 • june 2020 101 sample handling has minimal effect on blood progesterone concentrations in jennies sample handling has minimal effect on blood progesterone concentrations in jennies rachael ambrosia, robert gilbert, donald bergfelt, juan samper, erik peterson, hilari french ross university school of veterinary medicine, st. kitts, west indies abstract consistency of assay results is important for practitioners and investigators. in this context, for progesterone assays, appropriate handling and processing of blood samples is critical. objective was to compare progesterone assay results from jennies on the island of st. kitts in eastern caribbean from samples subjected to a variety of time and temperature treatments. five blood samples drawn via jugular venipuncture from 7 jennies at various stages of estrous cycle were randomly assigned to various treatments prior to storage at -80°c and subsequently thawed and assayed. handling methods were: immediate placement of blood in plain vacutainer tubes on ice for 1 hour (tr1); 1 hour of clot time before placing on ice for 24 hours (tr2); 1 hour of clot time at room temperature followed by serum separation and freezing (tr3); allowing 24 hours at room temperature before serum separation (tr4); and 1 hour at room temperature for blood collected in an edta tube before plasma separation (tr5). time and temperature treatment of samples used in this study did not influence progesterone assay results. keywords: progesterone, assay results, time, temperature, jennies introduction progesterone concentrations in serum or plasma are often utilized in reproductive medicine as basis for clinical decision-making and are equally important in research. reproducible results are important in both situations. evidence exists that sample handling before analysis may influence quantifiable concentrations of progesterone. for example, in bovids, there was appreciable loss of progesterone concentrations if whole blood was left to incubate at ambient temperature prior to analysis.1,2 although the rate of decline in detectable progesterone concentrations was different between bovine serum and plasma, immediate refrigeration and incubation at 4°c was recommended to slow the decline.3 in contrast, progesterone concentrations in canine serum declined when blood was refrigerated at 4°c following collection and processed after 3 hours, whereas concentrations remained relatively stable when kept at room temperature (22°c).4 however, plasma progesterone concentrations remained stable throughout 5 hours of storage at 4 or 22°c.4 hence, at least 2 hours of clotting time was suggested prior to centrifugation for blood samples used for progesterone assays to determine luteinizing hormone surge in bitches.4 for equine serum samples, a decline in detectable progesterone concentrations was noticed when samples were held at room temperature for 2 or 5 days.5 no significant differences in progesterone concentrations were reported in equine serum/plasma samples kept at 4 and 22°c.3 it was noteworthy that type of collection tube selected for storage of whole blood influenced assay results, with significantly higher progesterone concentrations in serum compared to plasma.6 objective of the study was to compare progesterone assay results from jennies on the island of st. kitts in the eastern caribbean from samples subjected to a variety of time and temperature treatments. goal was to ascertain whether immediate handling of samples was necessary. materials and methods animals seven nonpregnant (3 12 years, ~ 250 kg), jennies in estrus and diestrus (as determined by daily transrectal ultrasonographic examination of ovarian structures) were used for each treatment. research protocol was approved by ross university school of veterinary medicine iacuc. all jennies were housed together in paddocks from february 2016 to july 2017. they were fed fresh cut guinea grass ad libitum daily, had free access to fresh water and mineral blocks, and were given grain when handled for procedures. clinical theriogenology • volume 12 number 2 • june 2020 121 experimental design jennies were subjected to jugular venipuncture with 18 gauge 1.5’’ needle and 7 ml of blood was collected in 4 plain glass tubes and in 1 glass tube with edta anticoagulant. in treatment 1 (tr1), blood was immediately placed on ice for 1 hour. in treatment 2 (tr2), blood was allowed to clot at room temperature (~ 21°c) for 24 hours. in treatment 3 (tr3), blood was allowed to clot outside (~ 32°c) for 1 hour. in treatment 4 (tr4), blood was allowed to clot for 1 hour at ambient temperature and then placed on ice for 24 hours. in treatment 5 (tr5), blood drawn in edta anticoagulant tube was kept outside (~ 32°c) for 1 hour. tubes were centrifuged at 3,000 x g for 3 minutes at 4°c and aspirated serum/plasma was stored into labeled microcentrifuge tubes in an -80°c freezer until thawed for progesterone analysis. progesterone concentrations were determined using a commercial enzyme immunoassay kit (arbor assays k025-h5, ann arbor, mi). partial validation was conducted to assess possible matrix effects. a serum pool was prepared and diluted in a serial manner to determine parallelism with reference standard curve. four of the 6 dilutions were within the linear range of standard curve, with a coefficient of variation of 26%, indicative of parallelism (european medical agency, 2011).7 assay was conducted as per manufacture’s protocol using 50 µl of sample diluted 1:16 with assay buffer. progesterone concentrations that were outside the range of reference standard were further diluted and reanalyzed as necessary. within assay coefficient of variation was 17% and sensitivity was 0.05 ng/ml. data analyses effects of time and temperature handling of samples on progesterone concentrations were examined using mixed effects linear regression, with treatments (sample handling) regarded as fixed effects. jenny was treated as random effect to account for repeated measures. when this approach indicated no significant differences, all possible pairwise comparisons were made by paired student’s t-tests to ensure that no between-group differences were overlooked, although not strictly appropriate for this experimental design. stata®/ic version 11.1 (statacorp, college station, tx) was used for data analyses. results progesterone measured ranged from 1.81 to 33.60 ng/ml (figure). there was no difference (p = 0.26) in assay results between sample treatment protocols and there was no difference (p > 0.20) in all possible pairwise comparisons. discussion although some variation was present across progesterone concentrations, in each group, differences were not significant. samples from diestrus jennies had expected progesterone concentrations, whereas jenny’s e and g progesterone concentrations correlated to their estrous cycle stage (estrus or entering into estrus). findings on effects of time and temperature handling (up to 24 hours) agreed with an earlier study involving equine serum and plasma.3 in field conditions, if whole blood cannot be immediately refrigerated nor put on ice, samples can be kept at room temperature up to 24 hours. conclusion with no clinically relevant depreciation of serum or plasma progesterone obtained from jennies, whole blood can be drawn and allowed to clot at room temperature safely when indicated for progesterone assay. it is suggested that clinicians, owners, and researchers who prepare samples for progesterone assay in jennies need not handle whole blood differently within first 24 hours after collection. clinical theriogenology • volume 12 number 2 • june 2020122 conflict of interest authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. references 1. owens re, atkins dt, rahe ch, et al: time-dependent loss of radioimmunoassayable levels of progesterone following ambient temperature incubation of heparinized bovine blood. theriogenology 1980;13:305-309. 2. vahdat of, hurtgen jp, whitmore hl, et al: effect of time and temperature on bovine serum and plasma progesterone concentration. theriogenology 1979;12:371-374. 3. wiseman bs, vincent dl, scheffrahn ns, et al: changes in porcine, ovine, bovine, and equine blood progesterone concentrations between collection and centrifugation. anim reprod sci 1983;5:157-165. 4. volkmann dh: the effects of storage time and temperature and anticoagulant on laboratory measurements of canine blood progesterone concentrations. theriogenology 2006;66:1583-1586. 5. oltner r, edqvist le: changes in plasma progesterone levels during storage of heparinized whole blood from cow, horse, dog and pig. acta vet scandn 1982;23:1-8. 6. thuroczy j, wolfling a, tibold a, et al: effect of anticoagulants and sampling time on results of progesterone determination in canine blood samples. reprod domest anim 2003;38:386-389. 7. european medical agency. guideline on bioanalytical method validation 2011. figure. progesterone concentrations within 5 treatment groups for each of the 7 jennies sampled. immediate placement of blood in plain vacutainer tubes on ice for 1 hour (tr1); 1 hour of clot time before placement on ice for 24 hours (tr2); 1 hour of clot time at room temperature followed by serum separation and freezing (tr3); 24 hours at room temperature before serum separation (tr4); and 1 hour at room temperature for blood collected in an edta tube before plasma separation (tr5). clinical theriogenology • volume 12 number 2 • june 2020 123 clinical theriogenology 2022; 14: 356 sucrose and bovine serum albumin in association with dimethylformamide improved sperm parameters of frozen stallion semen madeline kurpita,a jose lena,b auniversity of adelaide school of animal and veterinary sciences, australia bnorth carolina state university college of veterinary medicine, raleigh, nc abstract postthaw stallion sperm integrity was determined after freezing semen in an extender supplemented with several sucrose concentrations, bovine serum albumin (bsa), and with and without dimethylformamide (dmf). two ejaculates from 6 stallions (n =12) were diluted and aliquots (n = 7) were made for treatment groups (25 mm sucrose + bsa [s25], s25 + dmf [s25dmf], 50 mm sucrose + bsa [s50], s50 + dmf [s50dmf], 100 mm sucrose + bsa [s100], s100 + dmf [s100dmf], and control (only dmf). semen was frozen in a computer-controlled freezer. sperm postthaw motility (total and progressive) and kinetics were assessed using casa. postthaw sperm plasma membrane and acrosomal integrity were evaluated using sybr-14/pi and fitc-pna, respectively. sperm motility was higher in s100dmf and s50dmf. plasma membrane integrity was higher in s100dmf, s50dmf, and s100. as sucrose concentration increased, plasma membrane integrity increased. treatment groups with sucrose and bsa, regardless of dmf, had higher acrosome integrity than control. sucrose and bsa in association with dmf in a freezing extender protected sperm integrity during freezing and thawing. keywords: stallion sperm, freezing, sucrose, dimethylformamide, plasma membrane introduction successful semen cryopreservation enhances benefits (long-distance semen transport, international trade, and extended use of superior genetic material) of artificial insemination (ai) over natural breeding.1,2 glycerol (gly) is commonly used as a permeable cryoprotectant for stallion semen cryopreservation; however, gly reduced sperm fertility.3 dimethylformamide (dmf) was as effective as gly in protecting stallion sperm from cryodamage.4,5 furthermore, dmf improved postthaw sperm motility and plasma membrane integrity. nonpermeable cryoprotectants (e.g., sucrose and trehalose) had beneficial effects during freezing in several species (rodents,7,8 rabbits,9 cattle,10 sheep,11,12 and pigs13). in stallions, sucrose14-16 and trehalose16,17 were used. a skim-milk egg yolk (smey) extender with sucrose and bovine serum albumin (bsa) instead of gly14 improved stallion sperm postthaw sperm motility and plasma membrane integrity compared to sperm frozen in a smey-gly freezing extender. furthermore, donkey semen frozen in a smey freezing extender with sucrose and bsa without gly had similar sperm postthaw motility and higher plasma membrane integrity compared to semen frozen with a smeygly freezing extender.15 improved sperm postthaw motility and plasma membrane were attributed to sucrose effects in reducing osmotic stress9 and bsa preventing lipid peroxidation.18 cryopreservation of stallion semen with sucrose and bsa in combination with dmf has not been reported. we investigated sperm postthaw integrity of semen frozen: a) in extenders containing dmf, several sucrose concentrations, and 1% bovine serum albumin (bsa); b) in extenders without dmf, several sucrose concentrations, and 1% bovine serum albumin (bsa); and c) extender with only dmf. materials and methods animals during summer in the southern hemisphere, semen was collected from stallions (n = 6) of known fertility, ranging in age from 3 to 16 (10.8 ± 3.5) years. except for 1 stallion (semen was frozen earlier using dmf) semen freezing capability was not known. animal use and handling procedures were approved (university of adelaide office of research ethics, compliance, and integrity). semen collection stallions that were not actively breeding had 3 semen collections prior to use.19 two days after the last semen collection, stallions had their semen collected for freezing, a day apart between the first and second semen collection. semen was collected using a hannover or missouri-model artificial vagina with an inline gel filter (minitube australia, australia). stallion had clinical theriogenology 2022; 14: 357 their penis washed with warm water and collection was performed over a phantom mount. semen preparation immediately after collection, total volume of gel-free semen was determined using a 50 ml conical tube (corning®, ma), and sperm concentration (106/ml) was determined using an automated cell counter (nucleocounter sp-100, chemometec, denmark). sperm motility and membrane integrity were assessed (described later). semen from ejaculates (n = 12) was extended 1:1 with equiplus (minutube australia) and centrifuged for 10 minutes at 1,000 x g. supernatant was aspirated with a vacuum, sperm pellet was resuspended and diluted with equiplus to a concentration of 400 x 106 sperm/ml. resuspended semen was transferred to semen freezing room (18°c) and divided into 7 equal aliquots. experimental design treatment groups (table 1) were: s25 (equiplus, 1% [w/v] bsa and 25 mm of sucrose [sigma-aldrich, australia]); s25dmf (s25 with 2% dmf); s50 (equiplus, 1% bsa and 50 mm of sucrose); s50dmf (s50 with 2% dmf); s100 (equiplus, 1% bsa and 100 mm of sucrose); s100dmf (s100 with 2% dmf); and control (equiplus with 2% (v/v) dmf (sigma-aldrich). freezing extenders were prepared in advance, divided into aliquots and stored at – 80°c until used. because semen samples were diluted (1:1) with treatment groups before freezing, freezing extenders were prepared at double the cryoprotectants concentration to reach the final concentration (200 x 106 sperm/ml) of treatment groups after dilution. after dilution, samples’ osmolality was assessed using an osmometer (advancedtm micro-osmometer, model 3 mo plus, thermofisher scientific, waltham, ma) that ranged from 379 to 769 mosm kg-1 (table 1). table 1. freezing extenders and osmolality (mosm kg-1) of each extender treatment freezing extender mosm kg-1 control equiplus + dmf 635 s25 equiplus + sucrose (25 mm) + bsa 379 s25dmf equiplus + sucrose (25 mm) + bsa + dmf 556 s50 equiplus + sucrose (50 mm) + bsa 401 s50dmf equiplus + sucrose (50 mm) + bsa + dmf 657 s100 equiplus + sucrose (100 mm) + bsa 447 s100dmf equiplus + sucrose (100 mm) + bsa + dmf 769 dmf = 2% v/v (0.26 m) dimethylformamide bsa = 1% w/v bovine serum albumin semen freezing semen was frozen using a computer-controlled rate freezer (icecube 14 s-a 230v, minitube australia). semen samples were then drawn into 0.5 ml plastic straws (minitube australia) and sealed with a sealing ball. straws were placed horizontally into the computer-controlled rate freezer. cooling rate from 18°c to 4°c was 0.3°c/minute and from 4°c to –160°c was 60°c/minute. straws were then plunged into liquid nitrogen (–196°c) and stored in liquid nitrogen tanks. after at least a week after freezing, 2 straws from each treatment group were thawed in a water bath at 37°c for 30 seconds. sperm motility after collection, an aliquot (1 ml) of fresh semen was extended with warm (37°c) equiplus to a concentration of 50 x 106 sperm/ml before motility assessment. postthaw sperm motility was assessed immediately after thawing at (0,15, and 30) minutes. before sperm motility assessment of frozen-thawed semen and to maintain osmolality consistency among treatments, each sample was diluted with its corresponding treatment to a concentration of 50 x 106 sperm/ml. five μl of fresh and/or frozen-thawed semen were placed over a warm glass slide (25 x 75 mm), covered with a coverslip (22 x 22 mm) and 3 fields were evaluated at 200 x magnification using casa (androvision®, minitube australia). analysis was performed using an image capture of 60 frames/second. sperm motility and kinematics assessed were: total motility (tm %, vcl < 35 μm/s and vsl < 15 μm/s), progressive motility (pm %, vcl ≥ 35 μm/s and vsl ≥ 15 μm/s), sperm curvilinear velocity (vcl, μm/s), sperm linear velocity (vsl, μm/s), average path velocity (vap, μm/s), amplitude of lateral head displacement (alh, μm), and beatcross frequency (bcf, hz/s). plasma and acrosomal membrane integrity fresh and thawed semen were diluted to a 1 x 106 sperm/ml with phosphate buffered saline (osmolality adjusted to match treatment groups). for sperm plasma membrane integrity (pmi), diluted semen was mixed with 1 μl of the sybr-14 and propidium iodide (sybr-14/pi) solution (reference [15407/0001], minitube australia) and incubated in dark for 10 minutes. fluorescent probes were excited with 488 nm (sybr-14) and 561 nm (pi) lasers. sperm emitting in red wavelength were considclinical theriogenology 2022; 14: 358 ered plasma membrane ‘damaged’ and sperm emitting in green wavelength were considered ‘intact’. for acrosome membrane integrity (acr), an aliquot of semen was diluted to a 1 x 106 sperm/ml, mixed with 1 μl of the fluorescein isothiocyanate (fitc) – peanut agglutinin (pna) stain (sigma-aldrich) and incubated in the dark for 10 minutes. fluorescent probe was excited with a 488 nm laser. sperm emitting in green wavelength were considered ‘damaged’ and unstained sperm were considered ‘intact’. for plasma and acrosomal membrane, assessment was made in duplicates from each sample using an attune nxt flow cytometer (thermofisher, australia). at least 10,000 sperm were evaluated before and after cryopreservation. data analyses equality of variance was assessed using levene’s test. square root of vcl (0 and 30 minutes) and alh (0 minute) were used for analysis. effect of treatment on the mean (± sem) postthaw tm (%), pm (%), vcl, vsl, vap, alh, and bcf at 0, 15, and 30 minutes were evaluated using analysis of variance (anova; ibm spss statistics 26, us). effect of treatment on the mean (± sem) percentage postthaw pmi and acr was also assessed using anova. level of significance was set at p < 0.05. when significance was observed, a pairwise comparison using tukey’s method was performed to assess differences among treatments. results ejaculates (n = 12) had the following (mean ± sem) characteristics before freezing: gel-free volume = 58.9 ± 7.8 ml, concentration = 205.1 ± 30.3 x 106/ml, tm = 73.4 ± 2.3%, pm = 72.2 ± 2.3%, intact pmi = 71.1 ± 2.3%, and intact acr = 91.3 ± 0.7%. there was no difference (p < 0.05) among ejaculates for the parameters evaluated. there was an effect (p < 0.05) of treatment on postthaw sperm motility and kinematic parameters. postthaw sperm motility (tm and pm) and sperm kinematic parameters (vcl, vsl, vap, bcf, and alh) at 0, 15, and 30 minutes in treatment groups s50dmf, s100dmf, and control were the highest (table 2 and 3). stallion sperm frozen in treatment groups s25 and s100 had the lowest postthaw sperm motility (tm and pm) and kinematics in most of the measured time points (figures 1 and 2). table 2. postthaw (mean ± sem) tm and pm at 0, 15, and 30 minutes of stallion sperm frozen with different sucrose concentrations +/dmf (2%) and bsa (1%) treatment time (minutes) tm (%) pm (%) dmf 0 32.1 ± 4.3ab 31.0 ± 4.2ab 15 29.1 ± 4.0ab 29.5 ± 4.0abc 30 24.6 ± 4.6ab 23.6 ± 4.5ab s25 0 14.7 ± 4.3c 14.0 ± 4.2c 15 16.7 ± 4.0c 15.8 ± 4.0d 30 15.1 ± 4.6c 14.5 ± 4.5b s25dmf 0 23.0 ± 4.3abc 22.3 ± 4.2abc 15 27.2 ± 4.0abc 25.9 ± 4.0abcd 30 22.5 ± 4.6ab 21.7 ± 4.5ab s50 0 19.3 ± 4.3bc 18.4 ± 4.2bc 15 21.3 ± 4.0bc 20.5 ± 4.0bcd 30 23.2 ± 4.6ab 22.2 ± 4.5ab s50dmf 0 33.3 ± 4.3a 32.1 ± 4.2a 15 36.1 ± 4.0a 34.8 ± 4.0a 30 33.8 ± 4.6a 32.8 ± 4.5a s100 0 16.8 ± 4.3c 16.0 ± 4.2c 15 19.8 ± 4.0c 18.8 ± 4.0cd 30 19.2 ± 4.6b 18.4 ± 4.5b s100dmf 0 33.5 ± 4.3a 32.0 ± 4.2a 15 34.4 ± 4.0a 32.1 ± 4.2ab 30 35.2 ± 4.6a 33.9 ± 4.5a rows and columns without common superscripts differed (p < 0.05) clinical theriogenology 2022; 14: 359 table 3. postthaw (mean ± sem) vcl, vsl, vap um/s, alh, bcg and hac at 0, 15, and 30 minutes of stallion sperm frozen with several sucrose concentrations +/dmf (2%) and bsa (1%) treatment time (minutes) vcl (µm/s) vsl (µm/s) vap (µm/s) alh um bcf (hz) hac (rad) dmf 0 27.3 ± 3.2a 20.0 ± 2.3a 20.7 ± 2.4a 0.7 ± 0.1a 1.6 ± 0.2 0.2 ± 0.1a 15 23.3 ± 3.0bc 17.4 ± 2.5bc 18.0 ± 2.6abc 0.6 ± 0.1ab 1.6 ± 0.2ab 0.2 ± 0.0ab 30 22 ± 3.3ab 17.0 ± 2.7abc 17.5 ± 2.7ab 0.5 ± 0.1ab 1.6 ± 0.2 0.1 ± 0.0abc s25 0 13.9 ± 3.2b 10.4 ± 2.3b 10.7 ± 2.4b 0.4 ± 0.1b 1.5 ± 0.2 0.1 ± 0.1b 15 13.9 ± 3.0d 10.2 ± 2.5c 10.6 ± 2.6c 0.4 ± 0.1c 1.1 ± 0.2b 0.1 ± 0.0c 30 14.2 ± 3.3c 11.1 ± 2.7c 11.3 ± 2.7c 0.4 ± 0.1b 1.4 ± 0.2 0.1 ± 0.0c s25dmf 0 20.9 ± 3.2ab 15.5 ± 2.3ab 16.0 ± 2.4ab 0.5 ± 0.1ab 1.6 ± 0.2 0.1 ± 0.1ab 15 23.6 ± 3.0abc 17.3 ± 2.5bc 18.0 ± 2.6abc 0.6 ± 0.1ab 1.5 ± 0.2ab 0.2 ± 0.0abc 30 18.3 ± 3.3ab 11.1 ± 2.7abc 14.5 ± 2.7bc 0.5 ± 0.1ab 1.6 ± 0.2 0.1 ± 0.0bc s50 0 14.0 ± 3.2b 10.1 ± 2.3b 10.5 ± 2.4b 0.4 ± 0.1b 1.3 ± 0.2 0.1 ± 0.1b 15 19.4 ± 3.0bcd 15.5 ± 2.5bc 15.8 ± 2.6bc 0.5 ± 0.1bc 1.5 ± 0.2ab 0.1 ± 0.0bc 30 27.3 ± 3.3a 20.7 ± 2.7ab 21.0 ± 2.7a 0.6 ± 0.1a 1.7 ± 0.2 0.2 ± 0.0ab s50dmf 0 27.1 ± 3.2a 19.8 ± 2.3a 20.5 ± 2.4a 0.7 ± 0.1a 1.7 ± 0.2 0.2 ± 0.1a 15 32.4 ± 3.0a 24.9 ± 2.5a 25.6 ± 2.6a 0.7 ± 0.1a 1.8 ± 0.2a 0.2 ± 0.0a 30 16.6 ± 3.3c 12.7 ± 2.7bc 12.4 ± 2.7c 0.5 ± 0.1ab 1.4 ± 0.2 0.1 ± 0.0c s100 0 13.8 ± 3.2b 9.3 ± 2.3b 9.4 ± 2.4b 0.4 ± 0.1b 1.2 ± 0.2 0.1 ± 0.1b 15 16.4 ± 3.0cd 11.7 ± 2.5c 11.2 ± 2.6c 0.5 ± 0.1bc 1.4 ± 0.2ab 0.1 ± 0.0bc 30 16 ± 3.3c 11.2 ± 2.7c 11.6 ± 2.7c 0.4 ± 0.1ab 1.3 ± 0.2 0.1 ± 0.0c s100dmf 0 27.8 ± 3.2a 18.0 ± 2.3a 19.0 ± 2.4a 0.8 ± 0.1a 1.6 ± 0.2 0.2 ± 0.1a 15 27.6 ± 3.0ab 20.2 ± 2.5ab 20.8 ± 2.5ab 0.7 ± 0.1a 1.7 ± 0.2a 0.2 ± 0.0a 30 29 ± 3.3a 21.4 ± 2.7a 22.1 ± 2.7a 0.6 ± 0.1a 1.6 ± 0.2 0.2 ± 0.0a rows and columns without common superscripts differed (p < 0.05) 0 5 10 15 20 25 30 35 40 45 50 control s100 s100dmf s25 s25dmf s50 s50dmf m ot ili ty (% ) treatment progressive motility 0 min 15 min 30 min a a ab abc bc c c a ab abc abcd bcd cd d a a ab abab b b figure 1. postthaw (mean ± sem) tm at 0, 15, and 30 minutes of stallion sperm frozen with several sucrose concentrations, +/dmf and bsa; bars without common letters within treatment groups differed (p < 0.05). 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 dmf s100 s100dmf s25 s25dmf s50 s50dmf in ta ct treatment plasma membrane a ababc bccc c figure 2. postthaw (mean ± sem) pm at 0, 15, and 30 minutes of stallion sperm frozen with several sucrose concentrations, +/dmf and bsa; bars without common letters differed (p < 0.05) among treatment groups. there was an effect (p < 0.05) of treatment on postthaw sperm pmi and acr integrity. postthaw pmi integrity of sperm frozen in treatment groups s100dmf, s50dmf, and s100 were higher than control, s25, s25dmf, and s50 (figure 3). as sucrose concentrations increased, postthaw intact pmi integrity increased, regardless of dmf presence (figure 3). postthaw sperm acr integrity in treatment groups s100, s50, s25, s100dmf, and s50dmf was higher than sperm frozen in control (figure 4). clinical theriogenology 2022; 14: 360 0 5 10 15 20 25 30 35 40 45 50 control s100 s100dmf s25 s25dmf s50 s50dmf m ot ili ty (% ) treatments total motility 0 min 15 min 30 min ab a a bc c c a a ab abc bc bc c a a bc bcbc c c abc figure 3. mean (± sem) postthaw intact plasma membrane (pmi) of stallion sperm frozen with different sucrose concentrations +/dmf (2%) and bsa (1%); bars without common letters within treatments groups differed (p < 0.05). 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 dmf s100 s100dmf s25 s25dmf s50 s50dmf in ta ct treatment acrosome a a a ab ab bc c figure 4. mean (± sem) postthaw intact acrosome (acr) of equine sperm frozen with different sucrose concentrations +/dmf and bsa; bars without common letters differed (p < 0.05) among treatment groups discussion freezing extender containing sucrose (50 and 100 mm), bsa, and dmf improved postthaw sperm integrity compared to sperm frozen in a freezing extender containing dmf only (control). postthaw sperm total and progressive motility, and kinematics of semen frozen in extenders containing sucrose and bsa in association with dmf (s50dmf and s100dmf) were either higher or similar to control. osmolality of the extenders in treatment groups s50dmf (657 mosm kg-1), s100dmf (769 mosm kg-1) and dmf (635 mosm kg-1) were higher than the 500 mosm kg-1 reported osmotic tolerance threshold for stallion sperm to maintain motility.20 nevertheless, the reported20 osmotic tolerance threshold gly and use of dmf in our study may have reduced the osmotic stress suffered by the sperm due to its lower molecular weight (mw = 73.10 g/mol).6 there was no interaction between sucrose and permeable cryoprotectants (dimethylacetamide, dmf or gly) preserving pmi sperm integrity during freezing stallion semen,16 possibly by the presence of egg yolk (10%) in the freezing extender. we did not use egg yolk and higher concentrations of sucrose (s100dmf and s50dmf) better preserved plasma membrane integrity. furthermore, that s50dmf treatment group pmi integrity was higher than the s50 treatment and not different than s100dmf, suggested that sucrose positively interacted with dmf. beneficial effects of this association may be due to the following. sucrose associated with membrane phospholipids head, stabilized membrane bilayer during freezing21 via electrostatic binding of saccharide hydroxyl groups to phosphate groups on the membrane lipid head,22 reduced ice crystal formation23-25 and provided an energy substrate.26 additionally, dmf stabilized the membrane bilayer,21 modulated cell dehydration,24 and decreased osmotic tension of the unfrozen fraction.27 furthermore, bsa antioxidant properties prevented lipid peroxidation that is involved in motility loss following cryopreservation.28 during fertilization, viable sperm undergo a morphological change known as capacitation.1 freezing-thawing stallion sperm induce capacitation-like changes, increasing the proportion of sperm with reacted acrosomes.29 in our study, postthaw acrosome integrity was better preserved with sucrose. similar to results of pmi integrity, as sucrose concentrations increased, postthaw acrosome integrity preservation improved. however, our results did not appear to suggest that sucrose and dmf interacted to improve acrosome protection during freezing-thawing of stallion sperm. minimum acceptable postthaw motility of frozen semen for commercial use is 30%.2,30 extenders s50dmf and s100dmf achieved a postthaw sperm total and progressive motility > 30% immediately and it was maintained until 30 minutes after thawing. additionally, s50dmf, and s100dmf extenders had higher postthaw intact pmi and acr. this suggested that freezing extenders containing sucrose and bsa in association with dmf may be an alternative for freezing stallion semen. our study had some limitations. freezing extender containing sucrose (50 and 100 mm), bsa, and dmf appeared acceptable protecting stallion sperm during freezing-thawing; however, the optimal concentration of sucrose for stallion sperm cryopreservation must be determined. although extenders with sucrose, bsa, and dmf, achieved a postthaw motility above the recommended 30%, the fertilization capacity of the thawed sperm is unknown. further research must investigate the optimal concentration of sucrose and fertilizing capacity after cryopreservation of stallion sperm with sucrose extenders. combining sucrose bsa and dmf in the freezing extender may be an alternative for cryopreservation of stallion semen. as concentrations of sucrose increased in combination with dmf, the postthaw pmi and acr integrity improved. acknowledgement authors thank dr. karen kind and staff at university of adelaide equine health and performance centre for their assistance. clinical theriogenology 2022; 14: 361 funding university of adelaide school of animal and veterinary sciences. conflict of interest none to declare. references 1. abou-haila a, tulsiani drp: mammalian sperm acrosome: formation, contents, and function. arch biochem biophys 2000;379:173-182. 2. loomis pr, squires el: frozen semen management in equine breeding programs. theriogenology 2005;64:480-491. 3. vidament m, vincent p, martin fx, et al: differences in ability of jennies and mares to conceive with cooled and frozen semen containing glycerol or not. anim reprod sci 2009;112:22-35. 4. squires el, keith sl, graham jk: evaluation of alternative cryoprotectants for preserving stallion spermatozoa. theriogenology 2004;62:1056-1065. 5. álvarez c, gil l, gonzález n, et al: equine sperm post-thaw evaluation after the addition of different cryoprotectants added to inra 96® extender. cryobiology 2014;69:144-148. 6. alvarenga ma, papa fo, landim-alvarenga fc, et al: amides as cryoprotectants for freezing stallion semen: a review. anim reprod sci 2005;89:105-113. 7. sztein jm, noble k, farley js, et al: comparison of permeating and nonpermeating cryoprotectants for mouse sperm cryopreservation. cryobiology 2001;42:28-39. 8. koshimoto c, mazur p: the effect of the osmolality of sugarcontaining media, the type of sugar, and the mass and molar concentration of sugar on the survival of frozen-thawed mouse sperm. cryobiology 2002;45:80-90. 9. rosato mp, iaffaldano n: cryopreservation of rabbit semen: comparing the effects of different cryoprotectants, cryoprotectant-free vitrification, and the use of albumin plus osmoprotectants on sperm survival and fertility after standard vapor freezing and vitrification. theriogenology 2013;79:508-516. 10. woelders h, matthijs a, engel b: effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing. cryobiology 1997;35:93-105. 11. aisen eg, medina vh, venturino a: cryopreservation and post-thawed fertility of ram semen frozen in different trehalose concentrations. theriogenology 2002;57:1801-1808. 12. jafaroghli m, khalili b, farshad a, et al: the effect of supplementation of cryopreservation diluents with sugars on the post-thawing fertility of ram semen. small rumin res 2011;96:58-63. 13. gómez-fernández j, gómez-izquierdo e, tomás c, et al: effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation. anim reprod sci 2012;133:109-116. 14. consuegra c, crespo f, bottrel m, et al: stallion sperm freezing with sucrose extenders: a strategy to avoid permeable cryoprotectants. anim reprod sci 2018;191:85-91. 15. diaz-jimenez m, dorado j, ortiz i, et al: cryopreservation of donkey sperm using non-permeable cryoprotectants. anim reprod sci 2018;189:103-109. 16. vafaei f, kohram h, zareh-shahne a, et al: influence of different combinations of permeable and nonpermeable cryoprotectants on the freezing capacity of equine sperm. j equine vet sci 2019;75:69-73. 17. el-badry da, abo el-maaty am, el sisy ga: the effect of trehalose supplementation of inra-82 extender on quality and fertility of cooled and frozen-thawed stallion spermatozoa. j equine vet sci 2017;48:86-92. 18. alvarez jg, storey bt: differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa. mol reprod dev 1995;42:334-346. 19. len ja, beehan d, eilts b, et al: sperm quality during extragonadal sperm reserve depletion in stallions. reprod domest anim 2019;54:412-414. 20. ball ba, vo a: osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential. j androl 2001;22:1061-1069. 21. anchordoguy tj, rudolph as, carpenter jf, et al: modes of interaction of cryoprotectants with membrane phospholipids during freezing. cryobiology 1987;24:324-331. 22. chen y, foote rh, brockett cc: effect of sucrose, trehalose, hypotaurine, taurine, and blood serum on survival of frozen bull sperm. cryobiology 1993;30:423-431. 23. uchida t, nagayama m, shibayama t, et al: morphological investigations of disaccharide molecules for growth inhibition of ice crystals. j cryst growth 2007;299:125-135. 24. oldenhof h, gojowsky m, wang s, et al: osmotic stress and membrane phase changes during freezing of stallion sperm: mode of action of cryoprotective agents. biol reprod 2013;88. 25. sieme h, oldenhof h, wolkers wf: mode of action of cryoprotectants for sperm preservation. anim reprod sci 2016;169:25. 26. leibo sp, songsasen n: cryopreservation of gametes and embryos of non-domestic species. theriogenology 2002;57:303-326. 27. mazur p: freezing of living cells: mechanisms and implications. am j physiol 1984;247. 28. alvarez jg, storey bt: taurine, hypotaurine, epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa and protect against loss of motility. biol reprod 1983;29:548-555. 29. schembri ma, major da, suttie jj, et al: capacitation-like changes in equine spermatozoa throughout the cryopreservation process. reprod fertil dev 2002;14:225-233. 30. brinsko sp: insemination doses: how low can we go? theriogenology 2006;66:543-550. clinical theriogenology 2022; 14: 50 introduction equine theriogenology is focused on the production of a live foal. infertility in horses is common and only ~ 67% of mares produce a live foal.1 number of mares becoming pregnant and producing a live foal is increasing;2,3 however, failure of pregnancy (fop) has been an important impediment to produce a live foal. in camelids, estimates are not readily available. investigations of fop in horses is a most rewarding diagnostic experience because success rate is very high (83 93%),47 higher than other species. camelids, in contrast, like other species, have a success rate of up to 50%.8 major difference between horses and other species is the commonality of noninfectious causes in horses that can be identified by lesions visible at gross/macroscopic evaluation. this will be the focus in this review. failure of pregnancy in horses and camelids is described in detail in review articles, textbook chapters, symposium proceedings, and diagnostic laboratory pamphlets. most diagnostic laboratories have a procedure, protocol, and/or list of diagnostic samples to aid in identifying infectious fop in horses and to a lesser extent, in camelids. most reviews and descriptions follow a familiar theme; separation of causes of fop into infectious causes and everything else is based on the ease with which diagnostic laboratories can process samples to identify microbial agents. in camelids, most fop cases are considered idiopathic; they have no infectious causes and have no lesions. many reports failure of pregnancy in horses and camelids robert foster department of pathobiology, ontario veterinary college, university of guelph, guelph, ontario, canada abstract determining pregnancy failures in horses and camelids is approached as a diagnostic investigation of an individual animal. it begins with the preliminary discussion around expectations of the investigation and apparently, diagnostic success rate is high in horses. however, diagnostic success in camelids is low. general approach from a pathology point of view is to identify several of noninfectious fetal and fetal membranes’ lesions and then determine the potential infectious causes; agents include viruses, eubacteria, protozoa, and fungi. bacterial and fungal infections are mostly ascending infections whereas viral and protozoal are systemic infections. when an infectious cause is excluded, the focus is directed on potential noninfectious causes and particularly those with detectible lesions. maternal evaluation, specifically, via endometrial biopsy and examination of fetus and fetal membranes (including umbilical cord) in horses, normally provide an explanation for noninfectious failure of pregnancy in several circumstances. special attention should be given to fetal thyroid gland, tracheal contents, and musculoskeletal system, particularly, the medulla of long bones. in horses, noninfectious failure of pregnancy with no lesions (idiopathic abortion) is observed in a small percentage of cases. in camelids, it is a common ‘diagnosis.’ keywords: horses, camelids, pregnancy, abortion, stillbirth, embryonic mortality on causes of fop are based on sample submissions to diagnostic laboratories.4,5,6,9 this approach is logical and practical. overlying this is the division of causes into those with identifiable lesions or those with no lesions. fortunately, the number of cases in horses where there are neither infectious agents nor lesions identified (idiopathic fop) is very low. furthermore, causes are classified (based on origin) into maternal, fetal, or placental. paternal causes possibility is also included in this review. the intention is not to replicate work already presented, but rather to highlight some important aspects and lesions that are easy to miss at macroscopic and microscopic evaluation. additionally, ‘idiopathic’ failure of pregnancies is considered. general approach to failure of pregnancy there are 2 parts to the general approach to fop. preliminary discussions with owners to ensure safety, reasonable expectations, and thorough evaluation. second, the diagnostic process. preliminary discussions begin before the diagnostic process and normally, includes discussing public health and safety issues, identifying the problem, performing a cost-benefit analysis of investigation and diagnosis, and discussing quarantine efforts of affected dams. in horses and camelids in north america, public health issues are minimal and in general, equine fetal membranes are not regarded as biohazardous above level 2. because horses and camelids are treated as clinical theriogenology 2022; 14: 51 individuals, attempts for them to become pregnant and give birth are costly, there is usually a benefit for evaluation particularly, in horses where the diagnosis rate is so high. highly infectious nature of equid alphaherpesvirus 1 (commonly known as equine herpesvirus 1; ehv-1) in horses means that every case should be evaluated for viral presence. this alone is a major focus in fop investigation in horses even though the prevalence of ehv-1 in diagnostic material is relatively low and is ~ 3 or 4% in several large studies.4-7,10,11 diagnostic process is like any other disease investigation and involves determining signalment, history, macroscopic evaluation, and collection of samples from the mare, fetus and fetal membranes, ancillary testing, and arriving at a diagnosis. careful evaluation of fetus and fetal membranes and the collection of appropriate samples as outlined by a particular diagnostic laboratory must be carried out for complete evaluation. during this process of sample collection, it is very important to have complete evaluation of all parts of fetal membranes and umbilical cord. when performing a postmortem examination on an equine fetus, evaluate thyroid gland, examine tracheal and bronchial contents, include skeletal muscle and sectioning of long bones. reporting findings is now greatly enhanced by taking photographs with a mobile phone; this is sufficiently helpful when seeking opinions from diagnostic and or reproductive pathologists or equine theriogenologists. careful macroscopical evaluation is very useful in identifying several noninfectious causes of fop that in the horse represent 30 – 40% of overall causes.4-7,10,11 infectious causes of failure of pregnancy infectious causes in horses account ~ 40% of the causes of fop.4-7,10,11stud owners give greater importance to equid alphaherpesvirus 1 because it is believed to have high infectiveness and has a propensity to move rapidly through a group of horses. however, it has not been a major infectious cause of fop.4-7,10,11 placentitis, either in the region of the ‘cervical star’ or in the body portion of fetal membranes (in nocardioform placentitis12), is a common lesion that when present is highly likely the cause of fop; amnionitis is also a distinct lesion. approximately 70% of cases of placentitis have a microbe identified as the potential cause.12,13 majority of infectious causes of fop are bacteria, and most common bacterial species are streptococcus equi zooepidemicus or gram-negative organisms. these usually infect fetal membranes by ascending through the cervix. there are several potential bacteria that can cause fop by the transcervical route. other bacteria (e.g. nocardioform actinomycetes) are usually sporadic.14 bacteria that infect fetus and fetal membranes include leptospira interrogans, neorickettsia risticii, borrelia burgdorferi, chlamydia psittaci, mycobacteria, and less commonly mycoplasma and coxiella burnetii. horses and camelids can be infected with these bacteria. fungal, protozoal, and viruses (other than equid alphaherpesvirus 1) are sporadic causes that can be recovered from diagnostic specimens with modern techniques particularly, molecular types (e.g. polymerase chain reaction-based assays). endometritis, including infectious endometritis, postbreeding endometritis, and postpartum endometritis all potentially contribute to fop and infertility in mares. it results in embryonic mortality, particularly, early embryonic mortality.15,16 important components in the development of endometritis include variations in physical clearance and uterine tone, nonadaptive molecular immune response, and nonadaptive cellular immune response; these are areas of active research. uterine clearance and tone are especially important and systems to measure clearance and tone are being developed. active inflammation of the endometrium is best detected by cytology and the effects of inflammation of the endometrium via endometrial biopsy. endometrial biopsy continues to be an important method of prognosis for fertility in mares.17-20 evaluation of the mare is an important component of evaluating fop; however, in middle or last third of pregnancy losses, the mare is often overlooked as a source of information about the fetomaternal interface and potential infectious agents. as with mares, camelids can be sampled for endometrial biopsy and this is a valuable adjunct to evaluation of infertility and fop. amnionitis and funisitis is a unique presentation of infection in horses. whereas a variety of bacteria can be recovered, the underlying process in the cause of disease, variously known as ‘mare reproductive loss syndrome’ or equine amnionitis, and fetal loss, was determined to be from the transmigration of setae of processionary caterpillars.21-27 noninfectious causes of failure of pregnancy noninfectious causes of fop represent a substantial component of the overall causes, ~ 50%. these causes can be classified into fetal, placental, maternal, fetomaternal incompatibility, and paternal components. several of these have identifiable lesions whereas some lack. noninfectious causes are classified into those with identifiable lesions and those without lesions. this aids in the investigation of the problem at the herd level. noninfectious causes with lesions noninfectious lesions of the mare, fetus, and fetal membranes are often identifiable at gross or macroscopic evaluation; however, some require careful evaluation of fetus and fetal membranes. noninfectious fetal lesions some lesions are highly visible congenital defects, and include hydrocephalus, craniofacial anomalies, schistosomus reflexus, and various musculoskeletal defects including arthrogryposis and angular limb deformities. fetal neoplasia such as hepatoblastoma,28,29 although extremely rare, is often very remarkable and do not present a challenge. clinical theriogenology 2022; 14: 52 dystocia or birth trauma have fetal abnormalities without routine histological changes and can only be determined at postmortem. twinning once was a major cause of fop in mares10 but is now uncommon. it was diagnosed as a cause of fop based on gross examination of the fetuses and fetal membranes. causes with a histologic lesion but no gross lesion requires collection of the appropriate sample. approximately 3% of foals with glycogen branching enzyme deficiency are aborted and histological evaluation of muscle is required.30 thyroid hyperplasia observed with and without musculoskeletal disease, and thyroid hyperplasia/congenital hypothyroidism-dysmaturity syndrome requires microscopic evaluation of the thyroid gland for diagnosis. thyroid histology must be combined with evaluation of long bones to detect osteopetrosis in those cases that do not have skeletal abnormalities such as mandibular prognathia, inappropriately ossified carpal and tarsal bones, flexural deformities of the forelimbs and ruptured tendons of the common digital extensor muscles.31-34 giant cell hepatopathy35 is a lesion of unknown cause identified in aborted fetuses that have no other identifiable causes of fop. in camelids, anomalies of the head are the most common, especially choanal stenosis. cardiac anomalies are also reported. noninfectious umbilical and fetal membranes’ lesions examination of fetal membranes and umbilicus in equine fop is a rewarding and reassuring experience once the investigator is familiar with normal fetal membranes and umbilical cord.36, 37 macroscopic evaluation of fetal membranes and umbilical cord in horses should always begin with measurement of the cord for length, as excessively long or excessively short cords cause fop. the length of cord varies among ponies, thoroughbreds and standardbreds, and draft breeds. for thoroughbreds and standardbreds there is some variation in what is considered the normal reference range and the general figures38 of 36 83 cm is a good guide. cords that are too short can potentially rupture or pull fetal membranes from the endometrium at the insertion of the cord.39 cords that are too long may have greater resistance to blood flow, become strangulated and wrap around a limb, the neck or the body, develop excessive torsion or develop an infarct of the placenta.6,39,40 excessive twists that are substantial may have evidence of torsion, particularly edema of the umbilical cord and dilation of the urachus. other abnormalities of the umbilical cord include a single umbilical artery9,41 strangulation(s) by the yolk sac remnant, a knot in the umbilical cord, umbilical hernia into the umbilical cord or the lesions of amnionitis and funisitis as occurs in mare reproductive loss syndrome/ equine amnionitis and fetal loss. camelids normally have a relatively long cord, and torsion and strangulation(s) occur sporadically.42 ingestion by mares of processionary caterpillars begins as a noninfectious process however the setae of the caterpillars move through the intestines into the uterus and placenta carrying with them various types of microbes.26,27 limited information is available regarding pathogenesis of hydrallantois and hydramnios in mares for clinically obvious reasons.43 an abnormal site of attachment of the embryo to the uterus resulting in an umbilical cord attached to the body of the uterus results in body pregnancy and fetal loss. formation of body pregnancy is an anomaly (limited information is known) and research in equine embryo attachment to the endometrium may provide insights in this area.44 currently, placental edema and prolonged pregnancy following ingestion of mycotoxins as part of fescue endophyte toxicoses,45,46 occurs sporadically. nutrition of the fetus and fetal membranes via endometrium occurs through a combination of histotrophic and hemotrophic means.47 secretions of endometrial glands are required for the uterine ‘milk’ that is taken up by trophoblasts at the base of the microcotyledons. adequate microcotyledonary surface area is required for hemotrophic transfer. endometrial disease, particularly, fibrosis and alteration of endometrial glands, is likely to alter placental development and the resulting reduced gland secretion, transfer of nutrients and surface area. endometrial biopsy is the only effective way to measure alteration to endometrial structure that could influence placental surface area. quantitative means to measure surface area in a diagnostic setting is not established. endometrial biopsy therefore remains an important component of investigation of fop and infertility in horses and camelids. twinning reduces the surface area of each placenta and is responsible for fop. twinning is now an unusual finding because of early detection and clinical intervention. it is more common in those animals that were not subjected to pregnancy testing. the prevalence of twinning is ~ 0.1% of pregnancies.48 noninfectious causes without lesions the most difficult group to identify as the cause of fop are those that are noninfectious, and which do not have lesions identifiable either macroscopically or microscopically. this is the group that includes maternal causes, fetomaternal incompatibilities and those causes that have toxic, nutritional, genetic or molecular anomalies. maternal causes may be readily identifiable and as such, fetal and placental examination may be done on farm but not sent to diagnostic laboratories. thus, maternal illness seldom is included in laboratory statistics. colic is a recognized predisposing factor for fop.49-51 age of mare,10,52 failure of a previous pregnancy,10 endometritis,53 endometrial fibrosis,18,54 uterine neoplasia,55 and hormonal failure 2,56 are all reported. it is generally considered that the majority of genetic or molecular causes of fop are responsible for embryonic mortality and return to estrus. it is variously called repeat early embryonic loss, equine early embryonic loss or early pregnancy loss.57,58 cases lacking macroscopic or microscopic abnormalities have no known cause, partially because routine evaluation of chromosomal or molecular abnormalities is not available. clinical theriogenology 2022; 14: 53 there are some reports of chromosomal abnormalities in the fetus including mosaicism 51 or chromosomal or molecular abnormalities in mares, including single nucleotide polymorphism60 or chromosomal translocations.61 this is a field where ongoing research is necessary. paternal causes of fop have received limited attention. it has long been recognized that some stallions have an abortion rate that is higher than others10 but there is limited research in this area. conclusion diagnosis of fop, especially in the mare, is a rewarding process with a very high rate of diagnostic success. a search for noninfectious causes including placental and umbilical cord lesions, and evaluation for unique diseases in mares and fetuses, particularly, bone, muscle, and thyroid tissues, increases the diagnostic success rate even further. conflict of interest author has no conflict of interest and no funding was received for this publication. references 1. lane ea, bijnen m, osborne m, et al: key factors affecting reproductive success of thoroughbred mares and stallions on  a commercial stud farm. reprod domest anim 2016;51:181-187. 2. allen wr, wilsher s: half a century of equine reproduction research and application: a veterinary tour de force. equine vet j. 2018;50:1021. 3. rose bv, firth m, morris b, et al: descriptive study of current therapeutic practices, clinical reproductive findings and incidence of pregnancy loss in intensively managed thoroughbred mares. anim reprod sci 2018;188:74-84. 4. giles rc, donahue jm, hong cb, et al: causes of abortion, stillbirth, and perinatal death in horses: 3,527 cases (1986-1991). j am vet med assoc 1993;203:1170-1175. 5. hong cb, donahue jm, giles rc jr, et al: equine abortion and stillbirth in central kentucky during 1988 and 1989 foaling seasons. j vet diagn invest 1993;5:560-566. 6. smith kc, blunden as, whitwell ke, et al: a survey of equine 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1993;5:56-63. 13. canisso i, ball ba, squires el, et al: comprehensive review on equine placentitis. proc am assoc equine pract 2015; p. 490-509. 14. tibary a, fite c, anouassi a, et al: infectious causes of reproductive loss in camelids. theriogenology 2006;66:633-647. 15. woodward em, troedsson mh: inflammatory mechanisms of endometritis. equine vet j 2015;47:384-389. 16. morris lha, mccue pm, aurich c: equine endometritis: a review of challenges and new approaches. reproduction 2020;160:r95-r110. 17. gordon lr, sartin em: endometrial biopsy as an aid to diagnosis and prognosis in equine infertility. j equine med surg 1978;2:328336. 18. kenney rm, doig pa: equine endometrial biopsy, in: current therapy in theriogenology 2. philadelphia; wb saunders: 1986. p.723-729. 19. schlafer dh: equine endometrial biopsy: enhancement of clinical value by more extensive histopathology and application of new diagnostic techniques? theriogenology 2007;68:413-422. 20.snider ta, sepoy c, holyoak gr: equine endometrial biopsy reviewed: observation, interpretation, and application of histopathologic data. theriogenology 2011;75:1567-1581. 21. sebastian mm, bernard wv, riddle tw, et al: review paper: mare reproductive loss syndrome. vet pathol 2008;45:710-722. 22. todhunter kh, perkins nr, wylie rm, et al: equine amnionitis and fetal loss: the case definition for an unrecognised cause of abortion in mares. aust vet j 2009;87:35-38. 23. mcdowell kj, webb ba, williams nm, et al: invited review: the role of caterpillars in mare reproductive loss syndrome: a model for environmental causes of abortion. j anim sci 2010;88: 1379-1387. 24. cawdell-smith aj, todhunter kh, anderson st, et al: equine amnionitis and fetal loss: mare abortion following experimental exposure to processionary caterpillars (ochrogaster lunifer). equine vet j 2012;44:282-288. 25. cawdell-smith aj, todhunter kh, perkins nr, et al: exposure of mares to processionary caterpillars (ochrogaster lunifer) in early pregnancy: an additional dimension to equine amnionitis and fetal loss. equine vet j 2013;45:755-760. 26. todhunter kh, cawdell-smith aj, et al: processionary caterpillar setae and equine fetal loss: histopathology of the fetal-placental unit from experimentally exposed mares. vet pathol 2014;51:1131-1142. 27. de vries c, vanhaesebrouck e, govaere j, et al: congenital ascites due to hepatoblastoma with extensive peritoneal implantation metastases in a premature equine fetus. j comp pathol 2013;148:214219. 28. neu sm: hepatoblastoma in an equine fetus. j vet diagn invest. 1993; 5: 634-637. 29. render ja, common rs, kennedy fa, et al: amylopectinosis in fetal and neonatal quarter horses. vet pathol 1999;36:157-160. 30. allen a: hyperplasia of the thyroid gland and musculoskeletal deformities in two equine abortuses. can vet j 1995;36:234-236. clinical theriogenology 2022; 14: 54 31. allen al, doige ce, fretz pb, et al: hyperplasia of the thyroid gland and concurrent musculoskeletal deformities in western canadian foals: reexamination of a previously described syndrome. can vet j 1994;35:31-38. 32. allen al, townsend hg, doige ce, et al: a case-control study of the congenital hypothyroidism and dysmaturity syndrome of foals. can vet j 1996;37:349-351;354-358. 33. koikkalainen k, knuuttila a, karikoski n, et al: congenital hypothyroidism and dysmaturity syndrome in foals: first reported cases in europe. equine vet educ 2014;26:181-189. 34. car bd, anderson wi. giant cell hepatopathy in three aborted midterm equine fetuses. vet pathol 1988;25:389-391. 35. pozor m: equine placenta – a clinician’s perspective. part 2: abnormalities. equine vet educ 2016;28:394-404. 36. prickett me: abortion and placental lesions in the mare. j am vet med assoc 1970;157:1465-1470. 37. whitwell ke, jeffcott lb: morphological studies on the fetal membranes of the normal singleton foal at term. res vet sci 1975;19:44-55. 38. whitwell ke: morphology and pathology of the equine umbilical cord. j reprod fertil 1975;23(suppl):599-603. 39. foote ak, ricketts sw, whitwell ke: a racing start in life? the hurdles of equine feto-placental pathology. equine vet j 2012;41(suppl):120-129. 40. girodroux m, lores m, vilaregut l, et al: a single umbilical artery and omphalophlebitis in an arabian foal. equine vet educ 2019;31:612. 41. johnson lw: llama reproduction. vet clin north am food anim pract 1989;5:159-182. 42. mitchell arm, delvescovo b, tse m, et al: successful management of hydrallantois in a standardbred mare at term resulting in the birth of a live foal. can vet j 2019;60:495-501. 43. quinn ba, hayes ma, waelchli ro, et al: changes in major proteins in the embryonic capsule during immobilization (fixation) of the conceptus in the third week of pregnancy in the mare. reproduction 2007;134:161-170. 44. putnam mr, bransby di, schumacher j, et al: effects of the fungal endophyte acremonium coenophialum in fescue on pregnant mares and foal viability. am j vet res 1991;52:2071-2074. 45. riet-correa f, rivero r, odriozola e, et al: mycotoxicoses of ruminants and horses. j vet diagn invest 2013;25:692-708. 46. bastos hba, martinez mn, camozzato gc, et al: proteomic profile of histotroph during early embryo development in mares. theriogenology 2019;125: 224-235. 47. anaya g, fernández me, valera m, et al: prevalence of twin foaling and blood chimaerism in purebred spanish horses. vet j 2018;234:142-144. 48. santschi em, slone de, gronwall r, et al: types of colic and frequency of postcolic abortion in pregnant mares: 105 cases (19841988). j am vet med assoc 1991;199:374-377. 49. boening kj, leendertse ip: review of 115 cases of colic in the pregnant mares. equine vet j 1993;25:518-521. 50. chenier ts, whitehead ae: foaling rates and risk factors for abortion in pregnant mares presented for medical or surgical treatment of colic: 153 cases (1993-2005). can vet j 2009;50:481-485. 51. bain am: foetal losses during pregnancy in the thoroughbred mare: a record of 2,562 pregnancies. n z vet j 1969;17:155-158. 52. darenius k: early foetal death in the mare. histological, bacteriological and cytological findings in the endometrium. acta vet scand 1992;33:147-160. 53. waelchli ro: endometrial biopsy in mares under nonuniform breeding management conditions: prognostic value and relationship with age. can vet j 1990;31:379-384. 54. canisso if1, pinn tl, gerdin ja, et al: b-cell multicentric lymphoma as a probable cause of abortion in a quarter horse broodmare. can vet j 2013;54:288-291. 55. allen wr, s. wilsher s: persistent endometrial cups in the same mare in two successive pregnancies. equine vet educ 2012;24:247250. 56. wilsher s: the uterus and early pregnancy failure in the mare. equine vet educ 2019;31:214-224. 57. kahler a, mcgonnell im, smart h, et al: fetal morphological features and abnormalities associated with equine early pregnancy loss. equine vet j 2020;53:530-541. 58. whitwell ke: investigations into fetal and neonatal losses in the horse. vet clin north am large anim pract 1980;2:313-331. 59. leon pm, campos vf, thurow hs, et al: association between single nucleotide polymorphisms in p53 and abortion in thoroughbred mares. vet j 2012;193:573-575. 60. ghosh s, das pj, avila f, et al: a non-reciprocal autosomal translocation 64,xx, t(4;10)(q21;p15) in an arabian mare with repeated early embryonic loss. reprod domest anim 2016;51:171174. clinical theriogenology 2022; 14: 44 pregnancy and perinatal losses: general concepts christopher premanandan college of veterinary medicine, the ohio state university, columbus, oh abstract determining and, by extension, preventing loss of pregnancy is of utmost importance to a reproductive medicine practioner. however, determining the cause of fetal loss can be a frustrating experience to the clinician and the diagnostician. in general, diagnostic rates are very low amongst any domestic animal species and the phrase ‘the cause of fetal loss was not apparent’ is considered a failure of the diagnostic approach. however, the owner, clinician and diagnostician should consider the value of negative findings in any abortion investigation in light of the clinical circumstances, condition of fetal tissues, diagnostic tests performed, and the gross and histologic findings. in contrast to pregnancy loss, performing postmortem diagnostics to perinatal mortality cases can be a rewarding endeavor depending on the species and the circumstances. whereas complete postmortem examination can be intimidating to the clinician, it is a technical skill that can be easily mastered with practice. a general practitioner should be aware that portions of postmortem diagnostics (primarily the postmortem examination) can be performed ‘in house’ and may provide benefits such as timely results and increased revenue to the practice. keywords: abortion, fetal loss, perinatal mortality, necropsy, diagnostic pathology introduction and definitions pregnancy loss is roughly categorized into: 1. embryonic death 2. midpregnancy fetal death followed by resorption 3. fetal death characterized by maceration, mummification or abortion 4. stillbirth definition of embryonic death varies among species and this variation is justified given the variable period of pregnancy in domestic animals. species embryonic death equine < 42 days1 bovine < 42 days2 canine < 15 17 days feline < 21 days ovine < 45 days3 caprine < 45 days4 definition of the perinatal period varies in the veterinary literature. in fact, multiple definitions are accepted in human pediatric medicine. these include infant deaths that occur prior to an interval from 7 to 28 days of age and fetal deaths > 20 28 weeks of pregnancy.5 definitions of perinatal death in veterinary medicine are inconsistent. some refer to deaths occurring only during midpregnancy (more traditionally referred to as abortion) whereas others align more with human definitions. for the purpose of this review, perinatal death is defined as a fully formed term-animal that was delivered dead or an animal that died within 48 hours after delivery. approach to recurrent fetal loss and perinatal loss in humans as it relates to veterinary medicine recurrent fetal loss (rpl) is defined as the loss of > 2 pregnancies. since majority of pregnancy loss in humans occurs before 10 weeks, the diagnostic focus is on classifying causes of miscarriage. the classification of potential causes is well described; however, there are variations in these guidelines among international standard organizations.6 a critical feature in the generation of these guidelines is the discussion of all conditions that may cause recurrent fetal loss, with recommendations for follow up additional testing or treatment. the value of standardizing criteria for rfl is clear. standardization has generated diagnostic algorithms for clinicians to follow to advise and perhaps optimize fertility in patients. the criterion of most comparable importance in veterinary medicine is arguably the anatomic category, similar lesions in the endometrium of domestic animals. adenomyosis, chronic endometritis and endometriosis are anatomic risk factors for rfl in humans.7 endometriosis is only observed in primates. adenomyosis is a common lesion of canine uterus; however, it is typically has little clinical relevance. for example, adenomyosis was an associated finding in only 1.5% of 399 infertile bitches9 whereas, chronic inflammation is a finding associated with infertility in the mare8 and the bitch.9 clinical theriogenology 2022; 14: 45 efforts have been taken to understand the categories and pathogenesis of rfl (table 1). epigenetic modifications such as dna methylation have a role and abnormal occurrences of dna methylation are associated with rfl and fetal developmental abnormalities.10 inflammatory mediators such as tnf-α are implicated in rpl when there is an inappropriate shift towards th-1 mediated inflammatory responses.11 table 1. commonly accepted categories associated with recurrent fetal loss category additional notes cytogenetic robertsonian translocation, trisomy anti-phospholipid syndrome associated with vascular thrombosis, lupus anticoagulant, anticardiolipin or antiꞵ2 glycoprotein antibody anatomic mullerian uterine abnormalities, uterotubal patency, uterine fibroids hormonal or metabolic hypothyroidism, hyperprolactinemia, hereditary thrombocytopenia investigation only recommended if a family history is present male factors dna fragmentation, sperm aneuploidy women with a higher incidence of miscarriage do have a higher incidence of uterine malformations;12 however, there is little evidence that corrective surgery results in a reduction of infertility.6 most efforts in veterinary medicine revolve around identification of infectious agents and more effort should be directed at establishing specific categories as documented with human rfl. diagnostic modalities to consider during postmortem evaluation of adult animals, it is not uncommon to recognize entities that render further diagnostic unnecessary. examples of this include gastric dilatation volvulus (dogs), strangulating mesenteric lipomas (horses), and hepatic lipidosis (cats). however, pathognomonic gross lesions are uncommon in abortion cases and a uniform approach to sampling and test selection is critical to maximize diagnostic yield. infectious disease is often of primary concern in the evaluation of fetal tissues and fetal membranes and perinatal losses. detection of infectious disease relies on appropriate selection, sampling and storage of tissue during this examination. a few entities causing fetal or neonatal loss can be recognized grossly. for example, canine herpesvirus-1 is often associated with multifocal renal cortical hemorrhage and hemorrhage in the liver and lungs. however, bacterial septicemia can also be associated with this lesions and polymerase chain reaction (pcr) detection of viral nucleic acid is a useful method to confirm the diagnosis. similarly, cotyledonary mineralization and necrosis have been associated with ovine toxoplasmosis whereas round target-shaped regions of hepatic necrosis are associated with campylobacter fetus ssp. fetus. in general, pathognomonic lesions are not common and should not be relied on exclusively for a diagnosis. postmortem examination the technical expertise required to perform a postmortem examination should be separated from the expertise required to interpret gross tissue changes. whereas it can take years to master interpretation of gross tissue changes, the good news is that utilizing a uniform technical approach will negate the need for meaningful gross interpretation and maximize diagnostic yield. equipment should be the first consideration. regardless of species, some items will be used in common. most of these items are related to tissue collection and storage for additional diagnostic testing. tissue collected for histopathology are stored in 10% neutral buffered formaldehyde (formalin). in most circumstances, this tissue can be stored indefinitely; however, this can impede the results of immunohistochemical (ihc) stains. it should be noted that ihc is rarely of use in these types of cases. clinical theriogenology 2022; 14: 46 figure 1. whirl-pak bag and convenient configurations to prepare them for tissue collection sterile collection containers are critical for fresh tissue collection. whirl-pak bags are an ideal container. they are light weight, inexpensive, and easy to use. bags should be labeled prior to starting the postmortem and as the postmortem is being performed, the perforated top is torn off and the bag can be propped open with wire supports at the edge of the opening or by opening the base so the bag stands on its own (figure 1). note that there are tags on the outside of the bag that are used to open the whirl-pak bag without contaminating the inner surface. these bags should not be physically opened by inserting fingers or instruments into the inside of the bag. prosection equipment varies depending on the species examined. for small animals, a scalpel blade, mayo scissors and forceps are typically sufficient. an older pair of scissors is preferable as they can be used to cut costochondral junction as well as sutures of the calvarium. for ruminant and equine fetuses, a sharp knife and cutters are necessary. a skilled prosector can perform a majority of the postmortem examination with these implements. a cleaver, handsaw or stryker saw is typically necessary for brain removal. culture swabs and collection of fluids is seldom helpful for microbiology and molecular diagnostics in postmortem diagnostics. tissues collected in a semisterile or sterile fashion is ideal. collection of the required solid organs as soon as the thoracic and abdominal cavities are open: 1. minimizes the chance of contamination and 2. reminds the prosector to collect tissue in the first place. the only exception to this is stomach or abomasal contents. in many species, this material represents an uncontaminated source of amniotic fluid contents. this material is best stored in a small glass or plastic cylindrical container such as a red top blood tube in a sterile fashion near the end of the prosection. all containers should be sealed properly prior to shipment. packages containing formalin jars should also contain absorbent material in case of leakage and whirl-pak bags should be contained in a second sealed plastic bag or container. actual postmortem procedure varies among prosectors; however, in general, are performed in either dorsal or left lateral recumbency, with the former being practical for fetal dogs and cats. skin incisions are made from the ventral aspect of the mandible to the pubis and the skin is reflected dorsally (and bilaterally if a ventral incision is made). at this point, the abdominal wall is incised at the linea alba and additional incisions are made at the body wall just caudal to the ribcage, allowing the abdominal contents to be exposed completely (figure 2). depending on the approach, the thoracic cavity is opened by cutting the sternum and the dorsal aspect of the ribs in a linear fashion (with a lateral approach) or the dorsal aspect of the ribs in a linear fashion bilaterally. at this point in the postmortem, it is most prudent to collect the fresh tissues required for ancillary diagnostic testing. a set of clean scissors and forceps are helpful at this point. immersing the tips of the instruments in ethanol is helpful prior to tissue collection; however, this step can be omitted if the prosector uses careful technique. most microbiology laboratories will heat sterilize the outside of the tissue prior to plating the sample. liver, lung, kidney and spleen are ideal for collection at this point and should be collected without puncturing the gastrointestinal tract. although contamination is less of an issue with a stillborn animals and aborted fetuses, this guideline should be adhered to for animals that have nursed. stomach content collection can be reserved until the gastrointestinal tract is examined and should be stored in a separate container than the other fresh tissues. in general, liver, lung, kidney, and spleen can be pooled together in 1 container for submission. fresh tissue can be frozen until testing is performed. tissue should be frozen at 70˚c if being stored for long intervals but can be stored at 20˚c if testing will take place soon after collection. clinical theriogenology 2022; 14: 47 figure 2. sampling of fresh tissue and storage of pooled tissue into a whirl-pak immediately after opening the peritoneum and thorax following collection of fresh specimens, tissue collection for histopathology (formalin fixation) can take place. most diagnostic laboratories will collect an exhaustive set of tissues for histopathology. this may be difficult to perform out in the field with equine and food animal perinatal deaths; however, it is important to remember that a very small percentage of the formalin fixed tissue is evaluated histologically (~ 3 μm tissue thickness). large amounts of tissue are not necessary for histologic assessment. it is more effective to provide tissue representative of lesions and multiple small representative areas of organs. all tissue submitted in formalin should be < 0.5 cm in thickness. eye and brain are the exceptions and can be submitted whole. if the organs are already < 0.5 cm in width (or close to this thickness), then they can be submitted whole. most canine or feline tissues can be submitted whole and it is unnecessary to extensively dissect most tissues in these species (heart, kidney, etc.) aside from sectioning them to a thinner width. the order in which organs are collected is less important, although generally, the gastrointestinal tract should be reserved until after solid organs have been sampled. fetal and perinatal tissues can exhibit different physical characteristics than those observed in adult animals, leading to misinterpretation of nonlesions. the most common of these is the appearance of fetal brain. due to incomplete myelination and increased water content, the brain will be soft and difficult to handle without traumatizing the tissue. in aborted fetuses, the brain can be poured out of the open calvarium. the soft nature of the tissue does not impede histologic evaluation as long as it is adequately formalin fixed. fetal pulmonary parenchyma tends to be reddish-purple and will often sink in formalin when obtained from stillborn animals or aborted fetuses. this is due to fetal atelectasis. if lungs consistently sink from perinates that died after parturition, this may be an abnormal finding. fetuses examined after an extended postmortem interval may have an accumulation of red opaque watery fluid in the peritoneal and pleural cavity. this change should generally be ignored particularly if the animal has been frozen and thawed before examination. due to the number of misleading postmortem findings, a uniform and consistent collection of tissues in the manner outlined above should take priority over identification of potential causes during the examination. abortion/fading neonate panels one of the more vital tools in the approach to these cases are standardized panels utilized for the detection of infectious diseases. these panels employ a variety of diagnostic modalities to detect pathogens. in general, panels directed towards food animal and equine abortion/perinatal disease cases are more commonly provided by state diagnostic laboratories. a panel submission brings the advantage of a simpler submission process without the need to select individual tests. however, it is a straightforward process to order the appropriate tests for a panel ‘a la carte’ that is often the case for canine and feline cases (table 2). table 2. common ancillary diagnostic tests ordered in canine and feline investigations species tests canine aerobic culture, salmonella spp. culture, brucella canis culture, leptospirosis spp. darkfield microscopy, leptospirosis spp. pcr, brucella canis pcr, toxoplasma pcr, canine herpesvirus-1 fluorescence antibody (fa), canine distemper virus fa, canine adenovirus fa feline aerobic culture, feline panleukopenia pcr, toxoplasma pcr, feline leukemia virus indirect fa, feline herpes pcr, feline calicivirus fa clinical theriogenology 2022; 14: 48 this does require some working knowledge of testing protocols and the limitations of these modalities. in either circumstance, it is critical to pay attention to the requirements for tissue preservation. for instance, certain tests cannot be performed on formalin fixed tissue (culture and reverse transcriptase pcr). this requirement outlines the need to save a set of fresh tissue separately from the formalin fixed tissue. the most common testing modalities used include: bacteriologic assays, fluorescence antigen assays, viral isolation, and molecular diagnostics. 1. bacteriologic assays in general, standard aerobic culture is acceptable in most cases. in most circumstances, tissues are preferable to swabs or fluid collection. culture for specific pathogens that may require specific bacteriological assays include salmonella spp. and brucella spp.9 however, the diagnostician must keep in mind that confirmation of brucellosis is often more effectively accomplished through serologic assays. 2. fluorescence antibody testing also referred to as ‘direct fluorescence antibody testing,’ this testing modality utilizes an antibody labeled with a fluorochrome directed at a specific antigen associated with a pathogen. this needs to be distinguished from ‘indirect fluorescence antibody testing,’ a test that utilizes a secondary fluorochrome labeled antibody directed at an unconjugated antibody directed at the antigen. these methods can be used to detect a variety of pathogens in research settings but in the context of this discussion, fa is primary utilized to detect viral pathogens. figure 3. illustration of fluorescent antibody testing in tissue 3. viral isolation viral isolation is the general term used for cultivation of virus particles as methods vary depending on the type of virus. cell cultures, embryonated eggs or live animal subjects can be utilized but, in most circumstances, cell culture is used. cell cultures can be primary (derived directly from an animal and propagated in media) or continuous immortalized cell culture lines derived from neoplasms. either can serve as a suitable media to propagate and identify viral organisms. the type of cell line chosen for cell culture largely depends on the tropism of the virus. cell lines of epithelial, mesenchymal and hematopoietic origin are available for this purpose. following propagation of a virus, multiple subsequent steps can be taken to assist in identification (electron microscopy, hemagglutinin inhibition assays, complement fixation assays, and immunofluorescence assays). viral isolation from tissue samples can be more complex that from a swab stored in viral isolation media, so it is critical that as soon as possible fresh tissue be prevented from degrading. either immediate submission for testing or freezing tissue as an intermediate step should take place. 4. molecular diagnostics this category covers a wide variety of testing modalities but pcr is the most cited in this discussion. a wide variety of pcr modalities are available, but an exhaustive discussion is beyond the scope of this work. in general, dna viruses are best detected using standard pcr assay whereas rna viruses are detected using reverse transcriptase pcr. additional modalities such as multiplex pcr and nested pcr can maximize the amplification yield if there is less viral nucleic acid in the specimen. techniques such as quantitative pcr or rt-pcr can determine the viral load in given sample but may not be critical in the diagnostic process. it is important to recall that any viral nucleic acid can be detected in pcr assay, even if intact viral particles are not present or are not the primary cause of the disease. clinical theriogenology 2022; 14: 49 serology whereas fetal serology can be performed, it is of little use diagnostically unless the fetus is immunocompetent. fetal serum can be collected in circumstances with mild postmortem degradation; however, it will be difficult in smaller species. maternal serology is more convenient to obtain and more likely to provide usable diagnostic information. paired serum samples are ideal and a necessity when certain pathogens are suspected (e.g. leptospira spp.). the first sample is ideally collected at abortion and the second is collected after 2 3 weeks. toxins and nutritional deficiencies exposure to toxins is certainly associated with teratogenesis, abortion and stillbirth. any toxin that induces severe maternal illness can result in secondary fetal loss. however, when referring to toxins that primarily have teratogenic effects or induce fetal loss, ruminants and horses are primarily affected. toxins range from plant based (ponderosa pine, locoweed), plant associated fungal products (ergot alkaloids) to pharmaceutical agents. the ideal tissues for toxicology testing can vary depending on the toxin; however, the general tissues that fulfill most test requirements include liver, spleen, and kidney. conclusion fetal and perinatal loss is a difficult scenario for most veterinarians to resolve for multiple reasons. in general, we have limited information regarding the specific causes of genetically related embryonic loss in domestic animals.14 in ruminants, horses, and pigs, at least nutritional deficiencies, toxicities and select infectious agents are well established causes.15,16 however, more work is necessary to identify specific causes of chromosomal abnormalities and dna damage, particularly in dogs and cats. in general, investigating fetal and perinatal loss may not be rewarding in all cases but the technical aspect of investigating these cases can be handled in an adequate manner by most practitioners with equipment that is available in most veterinary practices. whereas submission of a whole fetus to a diagnostic laboratory is optimal in most cases, the cost associated with the postmortem examination and ancillary diagnostic testing can be prohibitive. the option to perform the diagnostic examination ‘in house’ can provide a lower cost option to the client and increase the number of investigations in these scenarios. conflict of interest the author has no conflict of interest. no funding was received for this publication. references 1. vanderwall, d: early embryonic loss in the mare. j equine vet sci 2008 28. 691-702. 2. santos je, thatcher ww, chebel rc, et al: the effect of embryonic death rates in cattle on the efficacy of estrus synchronization programs. anim reprod sci 2004 jul;82-83:513-35. 3. dixon ab, knights m, winkler jl, et al: patterns of late embryonic and fetal mortality and association with several factors in sheep. j anim sci 2007;85:1274-84. 4. dantas af, riet-correa f, medeiros rm, et al: embryonic death in goats caused by the ingestion of mimosa tenuiflora. toxicon 2012;59:555-557. 5. barfield wd: committee on fetus and newborn. standard terminology for fetal, infant, and perinatal deaths. pediatrics 2016;137. 6. bender atik r, christiansen ob, elson j, et al: eshre guideline: recurrent pregnancy loss. hum reprod open 2018. 7. pirtea p, cicinelli e, de nola r, et al: endometrial causes of recurrent pregnancy losses endometriosis, adenomyosis, and chronic endometritis [published online ahead of print, 2021 feb 10]. fertil steril 2021;s0015-0282(20)32752-7. doi:10.1016/j. fertnstert.2020.12.010. 8. van camp sd: endometrial biopsy of the mare. a review and update. vet clin north am equine pract 1988;4:229-245. 9. gifford at, scarlett jm, schlafer dh: histopathologic findings in uterine biopsy samples from subfertile bitches: 399 cases (19902005). j am vet med assoc 2014;244:180-186. 10. pei cz, kim yj, baek kh: pathogenetic factors involved in recurrent pregnancy loss from multiple aspects. obstet gynecol sci 2019;62:212-223. 11. saito s, nakashima a, shima t, et al: th1/th2/th17 and regulatory t-cell paradigm in pregnancy. am j reprod immunol 2010;63:601610. 12. saravelos sh, cocksedge ka, li tc: prevalence and diagnosis of congenital uterine anomalies in women with reproductive failure: a critical appraisal. hum reprod update 2008;14:415-429. 13. lamm cg, njaa bl: clinical approach to abortion, stillbirth, and neonatal death in dogs and cats. vet clin north am small anim pract 2012;42:501-505. 14. khatib h, gross n: symposium review: embryo survival-a genomic perspective of the other side of fertility. j dairy sci 2019;102:37443753. 15. mee, jf: investigation of bovine abortion and stillbirth/perinatal mortality similar diagnostic challenges, different approaches. ir vet j 2020;73:20. 16. givens md, marley ms: infectious causes of embryonic and fetal mortality. theriogenology 2008;70:270-285. a note of appreciation despite their busy schedules, the following scholars graciously accepted invitations to review manuscripts submitted to clinical theriogenology and provided timely and thoughtful evaluations. the editor, copy editor (john kastelic), design editor (ahmed tibary), editorial board members, theriogenology associations (society for theriogenology, american college of theriogenologists, and theriogenology foundation), and franz management thank them for their time and effort. dalen agnew divakar ambrose* cheryl asa etta bradecamp* walter bravo janine brown margret casal sherrie clark-deener* gail colebern* jill colloton* bronwyn crane robyn ellerbrock yedra feltrer allan gunn tara harrison jennifer hatzel jill heatley camilo hernández-avilés richard hopper vanmathy kasimanickam* audrey a. kelleman kara kolster* michelle kutzler james kumi-diaka jen langan anne lichtenwalner bret mcnabb lisa metcalf lara metrione lynda miller* anneke moresco javier nevarez cynthia o’connor colin palmer* christopher premanandan bonnie raphael jan ramer jen roberts juan samper charlie scoggin scott stahl nucharin songasen cyril stephen kim thompson dickson varner brian whitlock* caitlin wiley karen wolfsdorf* enrique yarto *editorial board member list is not complete for 2021; some scholars helped after the december issue had gone to press (their names will appear in 2022 note). clinical theriogenology 2021; 13: 351 elective caesarean in bitch elective caesarean in bitch stuart mason, nicole rous monash veterinary clinic, oakleigh east, victoria, australia abstract elective caesarean in the bitch is a common procedure that is performed for a number of reasons, including but not limited to: fetopelvic disproportion (notably in brachycephalic breeds), poor whelping history, previous caesarean, inherent uterine disease, high-risk pregnancy, highly valuable puppies/bitches, frozen semen pregnancies, or owner convenience. to determine the best time for the caesarean, it is important to accurately identify expected whelping date, most commonly done by accurate ovulation timing, gestational ultrasound staging and progesterone assay of the bitch near term. to improve puppy viability, preterm corticosteroid administration stimulates surfactant production in fetal lung. one of the largest problems with elective caesarean, aside from fetal viability, is mismothering by the bitch. the risk of mismothering may be reduced by the use of prostaglandins to induce luteolysis and intranasal oxytocin administration to the bitch. ovariohysterectomy at the time of caesarean section is not recommended. keywords: elective, caesarean, bitch, oxytocin, intranasal introduction. elective caesarean in the bitch is a common procedure that is performed for a number of reasons, including but not limited to: foetopelvic disproportion (notably in brachycephalic breeds), poor whelping history, previous caesarean, inherent uterine disease, high risk pregnancy, highly valuable puppies/bitches, frozen semen pregnancies, or owner convenience. determination of the correct time to perform an elective caesarean is a balance of identifying the correct time without removing the puppies too soon or too late, minimising distress on the bitch and ensuring optimal mothering and milk production after surgery. in order to determine the best time for the caesarean, it is important to accurately identify the expected whelping date and using as many tools as available to determine the best time to perform the procedure to maximise probability of a good outcome. pregnancy length in the bitch is defined as 65 days from the lh surge, 63 days from ovulation and 57 days from the onset of cytological diestrus. in order to determine pregnancy duration, it is important to understand and stage the estrous cycle. timing of the estrous cycle of the bitch bitches spontaneously ovulate a primary oocyte arrested in prophase i of the second stage of meiosis.1,2 the luteinizing hormone (lh) surge occurs ~ 2 days before ovulation,3,4 stimulating the granulosa cells of the follicles to luteinize and produce progesterone, which may aid in resumption of meiosis.1 after ovulation, the corpus luteum forms from luteinized thecal and granulosa cells. the corpus luteum secretes progesterone, with progesterone being solely responsible for pregnancy maintenance in the bitch. six days after ovulation, the bitch enters diestrus.5 laboratory tests for staging the estrous cycle lh assay lh concentrations are undetectable until the point of the lh surge. daily serum analysis of lh is required due to the short, varied, length of the lh surge in the bitch (24 60 hours). there is a rising phase of up to 12 24 hours and then a decline over 12 36 hours.6 the only readily available lh tests are qualitative lh tests, which give a positive result for concentrations > 1 ng/ml. even with daily lh testing, it is possible to not detect the lh surge, since lh concentrations vary (4 14 ng/ml6,7) at the peak of the lh surge, and there is often a slow rise and fall. lh assays on their own are not recommended, since the lh surge occurs 3 8 days before oocyte fertilisation,7 coupled with the possibility of anovulatory cycles. 211 clinical theriogenology • volume 11, number 3 • september 2019 progesterone assay progesterone concentrations rise from the lh surge and they can be used as an indirect indicator of ovulation. progesterone continues to rise through estrus and early diestrus, with the cervix closing under the influence of increasing progesterone. progesterone is most commonly measured today by chemoluminescence, with serum being recommended rather than plasma.8 collection of blood for progesterone is best done on a fasted sample. refrigeration of samples within the first 2 hours after collection will reduce the measured progesterone concentration.8 the lh surge occurs at ~ 6 nmol/l (2 ng/ml) and ovulation at 15 25 nmol/l (4 8 ng/ml), with ovulation being deemed complete with values > 30 nmol/l (10 ng/ml).9 in the authors’ opinion, there is no ‘magical’ value of progesterone at which fertility is maximal; changes in values are more important to help identify ovulation. consistency of progesterone analysis is important, as different analytical machines will give different results. vaginal cytology the cytology of the vagina changes as a result of changing estrogen concentrations. rising estrogen concentrations from developing ovarian follicles result in hyperplasia of the vagina, represented by increasing cornification of vaginal cytology samples. as the vagina undergoes hypertrophy, the surface cells suffer effects of reduced oxygenation which presents as increasing enlargement of the cytoplasm, nuclear pyknosis and cornification of surface cells.10 percentage of anuclear cells increases to 50% of cornified cells by the start of estrus. six days after ovulation, diestrus ensues, characterised by an influx of neutrophils and parabasal cells reducing the percentage of cornified vaginal epithelial cells.5 vaginal cytology alone is a poor indicator for the ideal time to breed;5 however, in conjunction with progesterone assays and vaginoscopy, it aids in determination of the phase of the cycle (proestrus, estrus, diestrus or anestrus) and appropriateness of insemination.11,12 vaginoscopy vaginoscopy is performed using a 25 cm long, 11 mm diameter welch allyn sigmoidoscope. under the influence of rising progesterone and increasing estrogen after the lh surge, the deep edematous folds of proestrus begin to flatten out and reduce. as estrogen concentrations continue to reduce and progesterone continues to rise through estrus, hypertrophy and hyperplasia of the vagina reduces, resulting in a cobblestone appearance of the vagina (crenulation) indicating ovulation is complete and the bitch is in the fertile period. by the start of diestrus, the vagina returns to a flat state with no significant folds in the mucosa.13 to determine the lh surge, ovulation time and/or the 1st day of cytological diestrus, a combination of progesterone assay, lh assay, vaginal cytology and vaginoscopy are used.11 ultrasound determination of fetal age careful ultrasound examination of the pregnant bitch can aid in ascertaining an approximate due date, and also help to correlate due date with estrous cycle staging. the inner chorionic cavity provides good determination of pregnancy stage up to 37 days of pregnancy,14-17 whereas measurement of biparietal diameter is most accurate during the last 3 weeks of pregnancy.15,17,18 the presence of good renal development and fetal peristalsis help to indicate fetuses are mature enough to be removed from the uterus. ultrasound of the fetal heart rates in the near-term fetus will also help to determine the urgency of a caesarean procedure. heart rates > 180 bpm indicate stable fetuses, whereas heart rates < 180 bpm indicate fetal distress19 and the need to proceed quickly to a caesarean section. progesterone assay to assess luteal presence in the whelping bitch, fetal distress will result in a release of oetal acth, a rise in fetal cortisol concentrations and in turn a decrease in maternal progesterone due to luteolytic effects of cortisol. maternal progesterone concentrations will decrease to < 2 ng/ml (6 nmol/l) 24 36 hours before whelping; therefore, daily analysis of progesterone to detect a decrease to below this level is often used to indicate the time for an elective caesarean.20 212clinical theriogenology • volume 11, number 3 • september 2019 medications indicated in an elective caesarean in many cases of elective caesarean, fetuses are removed before normal endocrine changes associated with whelping occur. often to avoid fetal distress, puppies are removed before luteolysis. this process may have detrimental effects on the puppies and mother, as a result of no progesterone drop to stimulate a rise in prolactin for mothering and milk production, and no fetal cortisol release resulting in no stimulation of fetal lung surfactant. to help avoid these issues, corticosteroid administration, prostaglandin f2α and intranasal oxytocin may be used. corticosteroid therapy short-acting cortisol is given 2 12 hours before the scheduled elective caesarean. shortacting cortisol solutions are too short acting to induce luteolysis and labour. from day 57 of pregnancy, the fetal lung is capable of life outside of the uterus, although pulmonary surfactant is likely limited at this point.21 betamethasone stimulates production in fetal lung surfactant22 and additionally in dogs, is believed to improve fetal survivability after surgery.23 therefore, it is recommended to be used in elective caesareans wherein luteolysis has not occurred. prostaglandin f2α a low dose of prostaglandin f2α at the end of the elective caesarean procedure (5 10 µg/kg) is used to aid luteolysis, reduce progesterone concentrations and thereby increase prolactin concentrations to promote milk production and mothering. intranasal oxytocin issues of poor maternal bonding in the bitch are commonplace in small animal theriogenology practices. whilst any new mother can have issues accepting their newborn puppies, the problem presents itself most commonly in nulliparous bitches, more so those that have undergone caesarean section and more so those that have undergone elective caesarean section (observed, unpublished data by author). whilst prolactin concentrations rise in late diestrus, there is a marked rise subsequent to the progesterone decline from luteolysis induced by a rise in acth from fetuses. thus, there is a delayed rise in prolactin in bitches undergoing elective caesarean due to the procedure being undertaken before luteolysis has begun. some breeds appear to have more of a problem accepting puppies and not uncommonly will savage their puppies, notably american pit bull terriers, american staffordshire terriers, rottweilers, and german shepherds (observed, unpublished data). dealing with a bitch that is not accepting her puppies and trying to harm them is a difficult situation for both the veterinarian and the breeder; suggested treatments are limited and often lead to frustration, poor compliance and detrimental neonatal care. traditional therapies traditionally breeders would be recommended to sedate affected bitches, use muzzles and physical restraint whilst an assistant would place the puppies on the mammary glands to try and feed. the most commonly used sedatives are diazepam (0.5 mg/kg bid or tid po) or acetylpromazine (1 3 mg/kg bid po). diazepam, a benzodiazepine, whilst being a good anxiolytic agent, is poorly sedative, coupled with potential ante grade amnesia (inability to create memories after an amnesia effect) which may mean that when used to treat maternal aggression, the bitch may in fact not learn to accept the puppies as she is less likely to remember the feeding event clearly. acetylpromazine (acp), a phenothiazine derivative, when used orally has a good sedative effect, and through stimulation of prolactin will improve milk production (like metoclopramide). acp can have profound side effects including seizures (by reducing the seizure threshold), profound hypotension and should be used in caution in breeds known to carry the mutated multidrug resistant 1 (mdr1) gene mdr1-1δ (notably australian shepherds, border collies, collies, white swiss shepherds, german shepherds, old english sheepdogs, and sighthounds). animals homozygous for the mdr1-1δ gene have increased susceptibility to many drugs, including ivermectin, moxidectin, milbemycin oxime, digoxin, opioids, acp, vincristine and other chemotherapeutics. whilst acp can result in profound sedation, its use to combat mismothering must be questioned. any medication given to a nursing mother will pass on to the puppies and additionally there is little positive encouragement of the bitch to accept the puppies, rather merely hoping she will eventually accept them. 213 clinical theriogenology • volume 11, number 3 • september 2019 muzzling and physical restraint of the bitch is questionable as a long-term therapy to aid in mothering is difficult. not only does it require multiple assistants to feed the puppies every 2 6 hours, it is likely very stressful for the bitch. the increased anxiety and cortisol concentrations associated with this would be expected to have detrimental effects on both milk let down and milk production. whilst in some cases it may be necessary in the short term, this is not a long-term therapy. many breeders will often resort to supplementary feeding in cases wherein nursing is difficult or not possible. this itself, whilst adequate, is not ideal for the puppies. there are many supplementary milk’s for puppies sold on the market. most are poorly correlated to bitches milk. many breeders create their own formulas, or follow those of their forefathers, most of which are in no way correlated to bitch milk. handfeeding can create problems of overfeeding, underfeeding, diarrhoea or nutritional deficiencies whilst creating more work for the breeder. emerging therapies the causative agents of maternal bonding have been long shown to be oxytocin 24-28 and prolactin. effects of oxytocin on maternal bonding has been investigated in rats, voles and sheep. extrapolations have been made to humans, with reports of oxytocin concentrations related to postnatal depression in new mothers.24,29 oxytocin, a neuropeptide consisting of 9 amino acids,30 is synthesized in the paraventricular and supraoptic nuclei.28 oxytocin is transported and stored in the posterior pituitary, or via specific projections from the paraventricular nucleus, transported to other structures in the brain including the amyglada, hypothalamus and hippocampus.28 oxytocin is well known for its functions on milk let down and uterine contractions, subsequent to release from the posterior pituitary. oxytocin receptors are present throughout many tissues of the body, not just the uterus and mammary gland, including but not limited to the heart, liver and brain. oxytocin receptors are a g coupled protein receptor. binding of oxytocin to the g coupled protein receptor leads to a g qα coupling, leading to an increase in intracellular calcium concentration which leads to muscle contractions (this is important for uterine tissue and mammary tissue related to milk let down). furthermore, oxytocin receptors located on neurons will lead to release or inhibition of other hormonal neurotransmitters and modulators, e.g. serotonin, endogenous opioids and corticotrophin-releasing factor.24 oxytocin has a number of believed functions other than uterine contraction and milk let down. these include, but are not limited to, love and pair bonding, sexual behaviour, maternal bonding, increasing trust, pro-social behaviours and reduction in anxiety.28,31 a syndrome in humans, williams syndrome is characterised by increased trust, increased chance of approaching strangers, reduced social fear and love of music. the syndrome is characterised by deletion of 28 genes and affected individuals have significantly higher plasma oxytocin concentrations, implicating important behavioural effects of oxytocin.32 in rats, increased intracerebral concentrations of oxytocin and prolactin subsequent to lactating reduces effectiveness of the hpa and reduces cortisol concentrations when subsequently exposed to a stressful stimuli. this has also been shown in humans indirectly, wherein mothers breastfed their babies then were subsequently exposed to a stressful experience; mothers that breastfed first had lower cortisol concentrations, suggesting in humans, too, that the hpa is desensitised by oxytocin and prolactin.33 the release of oxytocin both peripherally and centrally has occurs in response to labour, parturition, lactation and vaginocervical stimulation in sheep34 via multi-synaptic neural pathways.35 other stimuli for central oxytocin release includes emotional stress, social interaction and sexual activity/mating.33 increased estrogen and reducing progesterone during late pregnancy not only stimulates an increase in prolactin, but stimulates an increase in oxytocin receptor expression +/ sensitivity of brain sites.35 subsequently, with stimulation of central oxytocin release from the paraventricular nucleus and upregulation of central oxytocin receptors, there is a positive effect on recognition of offspring odour, increased attractive perception of offspring odour, mobilisation of active components of maternal behaviour, reduced maternal aggression, decreased anxiety and fear and acquisition of maternal memory.33 results on oxytocin related to maternal behaviour in rats is contradictory. in early reports, intracerebroventricular injection of oxytocin improved maternal responses, whereas in subsequent work, rats with the oxytocin gene removed showed immediate maternal instincts postpartum without supplementation. this suggests in rats the effects of oxytocin may be facilitatory rather than the 214clinical theriogenology • volume 11, number 3 • september 2019 mainstay of maternal bonding.27 in estrogen-primed ewes, oxytocin induces full maternal responses in non-pregnant ewes in < 30 seconds following intracerebroventricular infusion of oxytocin.36 maternal experience improves responsiveness to oxytocin infusion in multiparous versus nulliparous ewes.37 research in voles shows good evidence of oxytocin related to social behaviour and bonding. prairie voles form long-term exclusive bonding with their male counterparts following mating; this same bonding occurs following intracerebroventricular infusion of oxytocin in the female, but no effect if given to the male without mating. in the male if vasopressin is infused then the bonding will occur, showing some sex differences.27 intranasal infusion of many molecules has been investigated. insulin, melanocyte stimulating hormone, vasopressin, igf1, neuropeptides, cytokines (erythropoietin) and carbamazepine have all cross the blood brain barrier following intranasal infusion.38 conversely, systemic administration of oxytocin intravenously resulted in < 0.1% of oxytocin crossing the blood brain barrier of sheep.34 the actual mode of transport, if any, of oxytocin into the brain subsequent to intranasal infusion is the topic of current research. suggested routes of transport include extra neural/peri neural routes along the trigeminal or olfactory nerve pathways, lymphatic transport, intraneuronal transport or active or passive transport from vasculature.38 there are also suggestions that intranasal administration stimulates the endogenous oxytocin system, which results in both peripheral and central oxytocin secretion. transport along the olfactory nerve is unlikely, as transport speeds are faster than the transport speed of the olfactory nerve. endogenous activation or transport via the trigeminal or subarachnoid routes are most likely. transport into the plasma is likely via the heavily vascularised nasal mucosa and several facial veins into peripheral circulation. in rats, prior administration of adrenaline intranasally before administration of neuropeptides reduced blood concentrations of neuropeptides without reducing csf concentrations.39 indicating it is traversing through vasculature. in humans, intranasal infusion of oxytocin will result in peak plasma concentrations 15 minutes after administration, with return to baseline by 75 minutes. in the same study, peak csf concentrations occurred at 75 minutes; however, peak brain concentrations are likely to be earlier, as many samples were first taken at 75 minutes, and additionally csf samples were collected via lumbar puncture which would delay peak concentrations 5 10 minutes (assuming 5 mm/min csf transport in humans).38 this is concurred via work in non-human primates, wherein peak csf concentrations occured by 35 minutes, based on cervical puncture. furthermore, higher doses have earlier peaks (10 minutes, 80 iu) compared to lower doses (60 minutes, 40 iu),40 creating questions about dose, effect and concentration gradients. the use of oxytocin in humans shows promise for diseases associated with persistent fear, repetitive behaviour, reduced trust and avoidance of social interactions, with promise for diseases associated with reduced oxytocin concentrations and altered oxytocin metabolism such as autism and schizophrenia.28 mothers diagnosed with postnatal depression, then given intranasal oxytocin and subsequently exposed to an intrusive stranger, had greater protective behaviour of offspring than those given a placebo.25 for 6 years, the author has administered 10 iu of oxytocin intranasally either every 2 hours or before each feed (2 6 hours) to all nulliparous bitches after elective caesarean until good maternal behaviour is noted by the owner. the solution is compounded by a compounding pharmacist to a dilution rate of 10 iu oxytocin/0.1 ml and administered via a human nasal spray bottle (0.1 ml/spray). the dose is extrapolated from that used in experimental studies of pigs (24 iu oxytocin intranasally).41 the dose frequency has been extrapolated from work in other species showing the lifespan of intranasally administered oxytocin to be at least 2 hours and maybe up to 7 hours.25 in total, there have been 127 subjects treated with the aforementioned treatment and it has been subjectively assessed that maternal behaviours of nulliparous mothers developed more hastily compared to those that received no treatment in the past or sedation and restraint therapy. it must be noted that this is not a controlled scientific study, only a clinical assessment and many variables may lead to perception of improvement due to chance. in the 2 years since the use of this therapy, the author has not had any nulliparous bitch after elective caesarean harm a puppy. in 1 case, a bernese mountain dog bitch undergoing therapy began showing obsessive maternal behaviours (overstimulating and cleaning of pups) which resolved after cessation of therapy. it must be noted that the therapy is off label, experimental and still anecdotal; however, from clinical experience it appears to have good promise. currently the author and others are having good success with administration of 10 iu in alternating 215 clinical theriogenology • volume 11, number 3 • september 2019 nostrils every 2 hours (lopate c, personal communication) until the bitch is mothering well, usually within 12 24 hours. there is scope for a controlled clinical trial observing maternal behaviours of nulliparous bitches after elective caesarean receiving either oxytocin or a placebo intranasally. whilst there is no clinical or scientific study of intranasal oxytocin therapy in bitches, there is a good body of literature that increased csf concentrations of oxytocin after intranasal administration in multiple species. additionally, with studies showing reduced anxiety, fear and improved maternal behaviour related to higher csf levels of oxytocin in multiple species, it is not clear why the same would not concur in the bitch. whether intranasal application of oxytocin leads to direct transit to the csf or activation of the endogenous oxytocin is not relevant in a clinical situation, as a cause and effect has been developed. management of the nulliparous bitch after elective caesarean is a challenging case for the both the veterinarian and the breeder, and subjectively on a clinical level, the use of intranasal oxytocin shows promise for aiding in appropriate therapy to improve welfare of breeding bitches and their puppies. ovariohysterectomy at time of caesarean section whilst advocated by many, ovariohysterectomy at the time of caesarean section is not recommended by the author. although not directly studied in bitches, the probability of a poor outcome of the bitch after surgery is increased dramatically by ovariohysterectomy at time of caesarean section (observed, unpublished data by author). during pregnancy, there is an expansion of fluid volume of the bitch, and cardiac adaptions to cope with this, notably increased fractional shortening, increased cardiac output and cardiac hypertrophy, all believed to improve blood flow to the gravid uterus.42 during the puerperium, there is gradual reduction in uterine size and blood flow43 along with a gradual return of the maternal cardiac changes to the non-pregnant state.44 a rapid change in hemodynamics with ovariohysterectomy is likely to have detrimental effects on the maternal heart function which may have difficulty adapting quickly. additionally, the enlarged uterus after removal of the foetuses contains a large volume of blood and its removal is likely to have detrimental effects on thermoregulation of the bitch. conflict of interest none to declare. references 1. tsutsui t: gamete physiology and timing of ovulation and fertilization in dogs. j reprod fertil suppl 1989;39:269-275. 2. reynaud k, fontbonne a, marseloo n, et al: in vivo meiotic resumption, fertilization and early embryonic development in the bitch. reproduction 2005;130:193-201. 3. phemister rd, holst pa, spano js, et al: time of ovulation in the beagle bitch. biol reprod 1973;8:74-82. 4. concannon pw, mccann jp, temple m: biology and endocrinology of ovulation, pregnancy and parturition in the dog. j reprod fertil suppl 1989;39:3-25. 5. root kustritz mv: managing the reproductive cycle in the bitch. vet clin north am small anim pract 2012;42:423-437. 6. concannon pw: endocrinologic control of normal canine ovarian function. reprod domest anim 2009;44 suppl 2:3-15. 7. concannon pw, hansel w, visek wj: the ovarian cycle of the bitch: plasma estrogen, lh and progesterone. biol reprod 1975;13:112-121. 8. volkmann dh: the effects of storage time and temperature and anticoagulant on laboratory measurements of canine blood progesterone concentrations. theriogenology 2006;66:1583-1586. 9. jeffcoate ia, lindsay fe: ovulation detection and timing of insemination based on hormone concentrations, vaginal cytology and the endoscopic appearance of the vagina in domestic bitches. j reprod fertil suppl 1989;39:277-287. 10. olson pn tm, wykes pm, nett tm: vaginal cytology. part i. a useful tool for staging the canine estrous cycle. compend contin educ pract vet 1984;6:288-298. 11. goodman mf: canine ovulation timing. probl vet med 1992;4:433-444. 12. silva ld, onclin k, verstegen jp: cervical opening in relation to progesterone and oestradiol during heat in beagle bitches. j reprod fertil 1995;104:85-90. 13. lindsay fef: the normal endoscopic appearance of the caudal reproductive-tract of the cyclic and non-cyclic titch post-uterine endoscopy. j small anim pract 1983;24:1-15. 14. beccaglia m, luvoni gc: comparison of the accuracy of two ultrasonographic measurements in predicting the parturition date in the bitch. j small anim pract 2006;47:670-673. 15. luvoni gc, beccaglia m: the prediction of parturition date in canine pregnancy. reprod domest anim 2006;41:27-32. 16. luvoni gc, grioni a: determination of gestational age in medium and small size bitches using ultrasonographic fetal measurements. j small anim pract 2000;41:292-294. 17. son ch, jeong ka, kim jh, et al: establishment of the prediction table of parturition day with ultrasonography in small pet dogs. j vet med sci 2001;63:715-721. 216clinical theriogenology • volume 11, number 3 • september 2019 18. kutzler ma, yeager ae, mohammed ho, et al.: accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography. theriogenology 2003;60:1309-1317. 19. zone ma, wanke mm: diagnosis of canine fetal health by ultrasonography. j reprod fertil suppl 2001;57:215-219. 20. smith fo: challenges in small animal parturition--timing elective and emergency cesarian sections. theriogenology 2007;68:348-353. 21. sipriani tm, grandi f, da silva lc, et al: pulmonary maturation in canine foetuses from early pregnancy to parturition. reprod domest anim 2009;44 suppl 2:137-140. 22. vannucchi ci, silva lc, lucio cf, et al: prenatal and neonatal adaptations with a focus on the respiratory system. reprod domest anim 2012;47 suppl 6:177-181. 23. regazzi fm, silva lcg, lucio cf, et al.: influence of prenatal maternal corticosteroid therapy on clinical and metabolic features and pulmonary function of preterm newborn puppies. theriogenology 2017;97:179-185. 24. bell af, erickson en, carter cs: beyond labor: the role of natural and synthetic oxytocin in the transition to motherhood. j. of midwifery & women's health 2014;59:35-42. 25. mah bl, bakermans-kranenburg mj, van ijzendoorn mh, et al: oxytocin promotes protective behavior in eepressed mothers: a pilot study with the enthusiastic stranger paradigm. depression and anxiety 2014. 26. galbally m, lewis aj, ijzendoorn m, et al.: the role of oxytocin in mother-infant relations: a systematic review of human studies. harvard review of psychiatry 2011;19:1-14. 27. kendrick km: oxytocin, motherhood and bonding. exper physiol 2000;85:111-124. 28. ishak ww, kahloon m, fakhry h: oxytocin role in enhancing well-being: a literature review. j affect disord 2011;130:1-9. 29. feldman r, weller a, zagoory-sharon o, et al: evidence for a neuroendocrinological foundation of human affiliation: plasma oxytocin levels across pregnancy and the postpartum period predict mother-infant bonding. psychological sci 2007;18:965-970. 30. gimpl g, fahrenholz f: the oxytocin receptor system: structure, function, and regulation. physiol rev 2001;81:629-683. 31. windle rj, shanks n, lightman sl, et al: central oxytocin administration reduces stress-induced corticosterone release and anxiety behavior in rats. endocrinology 1997;138:2829-2834. 32. dai l, carter cs, ying j, et al.: oxytocin and vasopressin are dysregulated in williams syndrome, a genetic disorder affecting social behavior. plos one 2012;7:e38513. 33. neumann id. brain oxytocin mediates beneficial consequences of close social interactions: from maternal love and sex. in: pfaff dw, kordon c, chanson p, christen y: editors. hormones and social behaviour. research and perspectives in endocrine interactions: springer berlin heidelberg; 2008. p. 81-101. 34. kendrick km, keverne eb, baldwin ba, et al: cerebrospinal fluid levels of acetylcholinesterase, monoamines and oxytocin during labour, parturition, vaginocervical stimulation, lamb separation and suckling in sheep. neuroendocrinology 1986;44:149-156. 35. pedersen ca: oxytocin regulation of maternal behavior from rodents to humans. oxytocin, vasopressin and related peptides in the regulation of behavior 2013:148-182. 36. kendrick km, keverne eb, baldwin ba: intracerebroventricular oxytocin stimulates maternal behaviour in the sheep. neuroendocrinology 1987;46:56-61. 37. keverne eb, levy f, guevara-guzman r, et al: influence of birth and maternal experience on olfactory bulb neurotransmitter release. neuroscience 1993;56:557-565. 38. striepens n, kendrick km, hanking v, et al.: elevated cerebrospinal fluid and blood concentrations of oxytocin following its intranasal administration in humans. sci rep 2013;3:3440. 39. dhuria sv, hanson lr, frey wh, 2nd: novel vasoconstrictor formulation to enhance intranasal targeting of neuropeptide therapeutics to the central nervous system. the j of pharmacol and exper therapeutics 2009;328:312-320. 40. born j, lange t, kern w, et al.: sniffing neuropeptides: a transnasal approach to the human brain. nature neurosci 2002;5:514-516. 41. rault j-l, mack la, carter cs, et al: prenatal stress puzzle, the oxytocin piece: prenatal stress alters the behaviour and autonomic regulation in piglets, insights from oxytocin. appl anim behav scie 2013;148:99-107. 42. blanco pg, tortora m, rodriguez r, et al: ultrasonographic assessment of maternal cardiac function and peripheral circulation during normal gestation in dogs. vet j 2011;190:154-159. 43. batista pr, gobello c, corrada y, et al.: doppler ultrasonographic assessment of uterine arteries during normal canine puerperium. anim reprod sci 2013;141:172-176. 44. almeida vt, uscategui rar, camacho aa, et al: influence of estrous cycle and gestation on cardiovascular system of bitches. anim reprod sci 2018;192:35-43. 217 clinical theriogenology • volume 11, number 3 • september 2019 218clinical theriogenology • volume 11, number 3 • september 2019 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages 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monitoring microbiome health using cytology and histopathology monitoring microbiome health using cytology and histopathology christopher premanandan, erin runcan college of veterinary medicine, the ohio state university, columbus, oh abstract microscopic evaluation of canine female reproductive tract tissues can provide a wealth of information regarding reproductive health of a bitch, including potential issues with fertility. a thorough understanding of procedures used to obtain samples, as well as microscopic anatomy of canine female reproductive tract, is important in order to understand strengths and limitations of these modalities. appropriate evaluation of canine female reproductive tract using cytology and histopathology requires not only a solid understanding of basic microscopic structure, but also an understanding of how reproductive tract, specifically cranial vagina and endometrium, undergo striking changes during stages of estrous cycle. this review will cover microscopic endometrial structure of bitch, including a review of endometrial histology, sampling techniques, application of microscopic evaluation, and relationship of these methods to microbiome health. keywords: microbiome, canine vagina, canine endometrium, cytology, histopathology microscopic endometrial structure during canine estrous cycle most clinicians are very familiar with physical changes within uterine tissue during estrous cycle, particularly those that routinely handle this organ during surgical manipulation. however, fewer may be familiar with microscopic changes. endometrial changes that occur during stages of estrous cycle are well documented.1,2,3 during mid anestrus, endometrial glands are shorter and of a smaller diameter while superficial epithelium is typically cuboidal or low columnar.4 during late anestrus, endometrium increases slightly in thickness and superficial endometrial epithelium is primarily cuboidal. as a bitch progresses into proestrus, epithelium comprising glands undergo a metaplastic change into a mucussecreting phenotype with a marked increase in cell size with a lesser degree of hyperplasia.4,5 glands increase in length during early proestrus. overall endometrial thickness and glandular appearance largely remain the same during remainder of proestrus and early part of estrus; however, there is a noticeable increase in extravasated erythrocytes and hemosiderin-laden macrophages present in interstitium during this stage.4 next noticeable change occurs during mid-estrus, characterized by a further increase in endometrial thickness, an increase in glandular length and hypertrophy of glandular epithelium.4 endometrial gland coiling becomes prominent. proliferation of these endometrial components progresses until early diestrus (~ days 6 or 7 after lh surge) where peak occurs. in nonpregnant bitch, endometrial glandular morphology largely remains static until third week of diestrus.5 during the interval extending to initiation of anestrus, individual glandular epithelium reduces in size relatively quickly, while glandular height reduces in a much more gradual fashion. endometrial edema is also most prominent from mid to late diestrus.5 most striking change in late diestrus is accumulation of small vacuoles in the cytoplasm of luminal and crypt epithelial cells that have been established to be accumulations of lipid.4,5 literature has conflicting information regarding timing of disappearance of these lipid vacuoles; however, the author has observed them frequently persisting into early anestrus. microscopic evaluation of endometrium in relation to fertility and microbiome assessments evaluation of endometrium utilizing various microscopic viewing modalities can be a helpful adjunct test in assessing canine fertility. in terms of biopsy procedures, most common methods of obtaining endometrial tissue are excisional or punch biopsies typically obtained during a caesarian section. these biopsies are optimal in terms of evaluation of representative tissue and provide ideal tissue for a pathologist to evaluate. this type of biopsy is typically taken during laparotomy, during laparoscopic surgery, or during a caesarian section when a small litter size or documented fetal resorption has occurred. abortion is another common indication for this type of biopsy. 619 clinical theriogenology • volume 11, number 4 • december 2019 although there are numerous advantages to an excisional biopsy taken in late diestrus/early anestrus, there are also some disadvantages. it is generally most convenient to take this biopsy during caesarian section. due to important changes in endometrial structure that occur during gestation, accurate histologic evaluation requires familiarity with these changes. in addition, some degree of acute inflammation is generally present at or near whelping that can complicate evaluation. another common method of endometrial biopsy is transcervical endometrial biopsy (teb). this procedure is helpful when obtaining endometrial samples in stages other than diestrus/early anestrus and a laparotomy is not ideal for a particular circumstance. it has been established that teb is comparable in terms of sensitivity to endometrial lesions as a full thickness biopsy.6 however, these specimens are prone to crush artifact that can render many specimens uninterpretable. in order to mitigate this, ideal submission consists of multiple endometrial specimens. another disadvantage to this method is that technical requirements are higher for this procedure. whereas the skill set is similar to that required for transcervical insemination, obtaining multiple specimens can be difficult, due to the need to repeatedly catheterize a nonestrous cervix. methods of specimen preservation most common methods of tissue preservation for microscopic evaluation utilize chemical fixation by protein cross linking or protein coagulation. these methods were reviewed by the author.7 briefly, the most common preservatives utilized for reproductive tissues include 10% neutral buffered formalin, bouin’s and modified davidson’s media.7 all 3 fixatives are adequate for preservation of uterine biopsies, but certain precautions should be taken. in particular, bouin’s can result in excessively brittle tissue with prolonged exposure. most tissues should be transferred to 70% ethanol after 24 48 hours of immersion in bouin’s solution. these 3 preservatives are acceptable for standard hematoxylin and eosin (h & e) sections and most histochemical applications, as well as techniques such as fluorescent in situ hybridization. formalin-fixed paraffin embedded tissue may be acceptable for immunohistochemical and immunofluorescence staining, although this is antibody dependent. careful selection of antibodies as well as antigen retrieval methods have a role in these methods.8 microbiome and endometrial histopathology standard histology (h & e stained sections) is a rather insensitive method of detecting microorganisms, whether these are part of a pathologic processes or a normal inhabitant of microbiome. microbiome refers to an array of microorganisms residing in a particular location of body (intestinal tract, skin, etc.). since the definition of microbiome includes bacteria, viruses, fungi and even single-cell eukaryotes, numerous microscopic imaging modalities are necessary to characterize it. regarding bacteria, it is common to not observe organisms even in pathologic conditions such as cystic endometrial hyperplasia/pyometra complex. standard histology should therefore not be considered a useful single modality to study uterine microbiome. standard endometrial histology is more useful as a first step in assessing endometrial architecture, determining if lesions are present and stage of estrous cycle, if possible. some histochemical staining techniques may be more useful as they can highlight microorganisms if present in the tissue. gram stains are helpful for detecting bacterial organisms, whereas periodic acid schiff and silver stains are helpful for identifying fungal organisms. in addition, immunohistochemical or immunofluorescent assays can detect specific species of organisms in histologic sections, as can nucleic acid-based assays (in situ hybridization). digital microscopic slide scanning also adds a valuable method of analyzing staining patterns in tissue that can be difficult to quantify visually. digital microscope slides involve scanning and digitizing visual information of any standard light microscopic preparation at several magnifications. resulting file can be viewed on a computer screen as one would view a glass slide on a microscope. although this has obvious applications for clinical diagnostics (ready access to remote second opinions for example), there are also applications for research in endometrial microbiome. these applications include quantification of microorganisms using algorithms that quantify positive pixels stained by immunohistochemical markers. 620clinical theriogenology • volume 11, number 4 • december 2019 whereas these approaches can be considered similar to traditional methods of assessing microbiome from histology, more recent methods are also available. if localizing presence of microorganisms within microanatomy of a structure is not required, detecting nucleic acids of various microorganisms can be performed by isolating dna from “scrolls” i.e. shaved-off portions of paraffinembedded tissue. this technique lends itself to assays such as polymerase chain reaction (pcr) or realtime pcr. detection and quantification of rna have also been described using this technique. if microanatomic localization is a necessity, laser capture microdissection (lcm) can be employed. lcm involves removal of specific regions or elements of a tissue section to procure a uniform population of cells or tissue region. once removed and isolated, this region can be subjected to dna or rna isolation techniques and subsequent pcr or reverse transcriptase pcr assays. this approach has been described regarding characterization of gastrointestinal microbiota, but has not yet been used to explore endometrial microbiome.9 conclusions monitoring microbiome utilizing microscopic methods can be a challenge and successfully implementation for experimental purposes requires a solid understanding of traditional and advanced microscopic imaging modalities. in addition, particularly in dogs, cycle-based changes in endometrial morphology can complicate evaluation. however, histologic evaluation of tissue has a role in endometrial microbiome monitoring, in conjunction with more traditional microbiological and molecular diagnostic modalities. references 1. van cruchten s, van den broeck w, d'haeseleer m, et al: proliferation patterns in the canine endometrium during the estrous cycle. theriogenology 2004;62:631-641. 2. gossler vsa, santos fag, azevedo ar, et al: evaluation of cell proliferation and endometrial thickness of bitches in different periods of diestrus. an acad bras cienc 2017;89:1719-1727. 3. sato j, nasu m, tsuchitani m: comparative histopathology of the estrous or menstrual cycle in laboratory animals. j toxicol pathol 2016;29:155-162. 4. barrau md, abel jh jr, verhage hg, et al: development of the endometrium during the estrous cycle in the bitch. am j anat 1975;142:47-65. 5. galabova g, egerbacher m, aurich je, et al: morphological changes of the endometrial epithelium in the bitch during metoestrus and anoestrus. reprod domest anim 2003;38:415-420. 6. christensen bw, schlafer dh, agnew dw, et al: diagnostic value of transcervical endometrial biopsies in domestic dogs compared with full-thickness uterine sections. reprod domest anim 2012;47 suppl 6:342-346. 7. premanandan c, runcan ee, coutinho da silva ma: preservation, handling, and common artifacts of the endometrial and testicular biopsy. clinical theriogenology 2017;9:159-166. 8. ramos-vara ja, miller ma: when tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry--the red, brown, and blue technique. vet pathol 2014;51:42-87. 9. wang y, antonopoulos da, zhu x, et al: laser capture microdissection and metagenomic analysis of intact mucosaassociated microbial communities of human colon. appl microbiol biotechnol 2010;88:1333-1342. 621 clinical theriogenology • volume 11, number 4 • december 2019 622clinical theriogenology • volume 11, number 4 • december 2019 untitled omniblank: clinical theriogenology 2022; 14: 362 a case of unilateral hydrocele in a dog secondary to inapparent inguinal hernia peyton draheim,a katelyn hlusko,a robert cole,a jamie douglas,a cornelius withers,b emily graff,b,c robyn wilborna adepartment of clinical sciences, bdepartment of pathobiology, cscott-ritchey research center auburn university college of veterinary medicine, auburn, al abstract a 6-year, male intact coonhound, was presented for unilateral scrotal enlargement of roughly 3 months’ duration. his left side scrotum was markedly enlarged and edematous and both testes palpated normal. rapid slide agglutination test (for brucellosis) and snap 4dx (idexx laboratories, inc., westbrook, me) test (for tick-borne disease) were negative. initial scrotal ultrasonography revealed accumulation of anechoic fluid with hyperechoic flecks and swirling surrounding the left testis. on further ultrasonographic examination, omentum appeared extending through the left inguinal ring and into the scrotum. a case of scrotal hernia with secondary hydrocele was diagnosed. hydrocele was resolved surgically and the dog returned to full athletic function. keywords: dog, scrotal enlargement, hydrocele, brucellosis, hernia background scrotal enlargement in any species could be indicative of multiple pathologies, including primary orchitis, epididymitis, testicular or scrotal neoplasia, testicular torsion, hernia, or hydrocele.1 a thorough scrotal and testicular examination is required to reach a diagnosis. with a breeding male, not only are there concerns regarding the health of the animal, but future reproductive capability may also be a priority for the owner. it is important to take a multi-modal approach in working up cases of scrotal enlargement to maximize the chances of an accurate diagnosis and successful outcome. case presentation a 6-year, 34 kg (75 lb) intact male treeing-walker coonhound, was presented for a unilateral scrotal enlargement. swelling was first noticed ~ 3 months prior to presentation. patient also had a history of aspermia (lack of sperm in the ejaculate) of unknown etiology that was diagnosed 15 months prior to presentation. on general physical examination, the dog appeared bright, alert, and responsive. rectal temperature was 102.1°f (38.9°c; reference range 100 102.6°f or 37.8 39°c), heart rate was 120 beats/minute (reference range 60 160), and respiratory rate was 30 breaths/minute (reference range 10 30). dermal irritation and hyperemia were noted along the dorsum and ventrum, and fleas presence were confirmed during physical examination. marked scrotal asymmetry was observed (figure 1). both testes were fully descended into the scrotum. on palpation, the left side of the scrotum felt edematous; left testis figure 1. scrotum on presentation, note marked asymmetry and left side enlargement felt normal and was easily isolated within the fluid-filled scrotum. right testis and right part of the scrotum palpated normal. dog did not appear uncomfortable, did not react to palpation, and no additional signs of inflammation were noted on either side of the scrotum. prepuce and penis appeared normal. prostate felt normal on digital transrectal examination. blood was submitted for a brucella canis rapid slide agglutination test (rsat), a complete blood count (cbc), and a chemistry panel. given the dog’s signalment and environment, a snap 4dx (idexx laboratories, inc.) test and a direct blood smear were performed to rule out tick-borne disease and blood parasites. the snap 4dx (idexx laboratories, inc.) was negative for heartworm, ehrlichia, lyme disease, and anaplasma; however, microfilaria were observed on the blood smear, so a modified knotts test was performed. microfilaria were from a benign parasite (acanthocheilonema reconditum) clinical theriogenology 2022; 14: 363 rather than from dirofilaria imitis. dog tested negative for brucellosis, and the 4dx panel was also negative. red blood cell density was mildly decreased (5.96 x 106/µl; reference range 6.02 8.64x106/µl). this is most likely a normal variation or may reflect a mild anemia. all other values were within the reference interval. abnormalities on the chemistry panel included hypoalbuminemia (2.47 g/dl; reference range 3.00 4.30 g/dl), a decreased albumin/globulin ratio at 0.60 (reference interval 0.70 1.90), hypocholesterolemia at 116 mg/ dl (reference interval 132 335 mg/dl), and a low anion gap (11.1; reference range 14 24). a free-catch urine sample was obtained and submitted for urinalysis and a urine protein/ creatinine ratio. urinalysis revealed a urine protein level of 283.2 mg/dl with a specific gravity of 1.031. urine protein: creatinine ratio was 1.2 (normal value is < 0.5 in dogs) and the sediment had 1 5 white blood cells per high-power field (hpf) with 0 3 squamous epithelial cells per hpf. a cursory ultrasonography of the scrotum was performed that revealed a large amount of anechoic fluid with some swirling material (sediment) evident within the left side of the scrotum. right side of the scrotum had no fluid accumulation, and the parenchyma of both testes imaged normally. aspiration of the scrotal fluid was considered at this time, but upon discussion with the owner, it was decided to perform complete abdominal imaging (radiographs and a more detailed ultrasonographic examination under sedation) and aspiration of the scrotal fluid on a subsequent day. differential diagnosis differential diagnosis for unilateral scrotal enlargement include orchitis, epididymitis, testicular or scrotal neoplasia, testicular torsion, hernia, and hydrocele.1 in this case, given the minimum database results and the preliminary imaging, hydrocele (the accumulation of serous fluid in a body cavity; in this case, the scrotum) was the most likely diagnosis. however, further imaging and fluid analysis were indicated to investigate an underlying cause for the hydrocele and a treatment plan. treatment dog returned 4 days later for further diagnostics. he remained bright, alert, and responsive with all physical examination parameters were within normal limits. patient was sedated with dexmedetomidine and butorphanol to facilitate diagnostics that included abdominal radiographs, abdominal ultrasonography, and aseptic aspiration of the scrotal fluid. three-view abdominal radiographs were obtained. radiographs  revealed an enlarged prostate that suggested benign prostatic hyperplasia (bph), and the enlarged scrotum that was grossly notable. otherwise, radiographs were unremarkable. abdominal ultrasonography also concluded that prostatic changes were consistent with bph. no free abdominal fluid was noted; however, there was fluid within the left side of the scrotum (figures 2 and 3) extending up to the level of the inguinal ring. presumed omental fat was noted to be extending through the left inguinal ring and into the scrotum. some echogenic particles were suspended within the scrotal fluid. right testis and scrotum appeared normal via ultrasonographic examination. compared to right testis, left testis and pampiniform plexus had decreased vascularity in color doppler ultrasonography.  abdominal ultrasonography revealed no signs of a testicular mass, testicular torsion, or orchitis. it was concluded that omental fat was extending through the left inguinal ring, suggestive of a left-sided scrotal hernia with secondary fluid accumulation, or hydrocele; this was nonreducible. fine needle aspiration (fna) of the fluid was performed that revealed mild suppurative inflammation with evidence of chronicity (figure 4). given these collective findings, the dog was diagnosed with a left-sided scrotal inguinal hernia with secondary hydrocele. at that time, there did not appear to be small intestinal herniation, though it was communicated to the owner that this could still occur. decision was made for the dog to undergo surgical correction of the hernia 3 weeks later. figure 2. ultrasonogram depicting fluid surrounding left testis (lt) compared to normal right testis figure 3. ultrasonogram depicting the flocculent nature of the fluid surrounding left testis figure 4. fine needle aspirate from scrotal swelling (x 100 objective). primary neutrophilic (blue arrow) inflammation with a mild lymphocytic (green arrow) and eosinophilic (red arrow) component. note evidence of macrophagic cleanup (orange arrow). clinical theriogenology 2022; 14: 364 patient underwent a caudal abdominal laparotomy with exposure of the left inguinal ring. greater omentum was determined to be herniated through the left inguinal ring within the vaginal process that extended into the scrotal cavity (figures 5 and 6). these findings were consistent with a diagnosis of a left-sided indirect inguinal hernia with an associated scrotal hernia. herniated contents were reduced back into abdominal cavity through the left inguinal ring. left inguinal ring defect was noted to be ~ 4.5 cm in length. after reduction of the herniated contents, the hernial sac was incised, revealing a large volume of serosanguinous fluid, presumably from the scrotum that was suctioned. hernial sac was partially amputated using electrosurgery, taking care to avoid the ductus deferens and pampiniform plexus. remainder of the hernial sac was then reduced into abdomen and the inguinal defect was repaired using nonabsorbable suture material (prolene) in a combination of horizontal mattress and simple interrupted suture patterns placed along the cranial aspect of the defect. approximately 1cm of the caudal-most aspect of the inguinal ring was left open to allow passage of the external pudendal vessels, ductus deferens, pampiniform plexus, and genitofemoral vessels and nerve. unilateral castration was considered but ultimately deemed unnecessary as the ductus deferens and pampiniform plexus were preserved. postsurgically, there was a marked difference in gross appearance of the scrotum. there were no complications and the dog recovered from anesthesia uneventfully. bilateral castration was discussed/recommended to the owner due to the risk of recurrence and the likelihood of this condition being a hereditary defect. figure 5. herniated contents before reduction. body wall (a) delineates abdominal cavity from the spermatic cord (b) and herniated omentum (c) passing through the inguinal canal into scrotum. figure 6: omentum (a) herniation through the inguinal canal and extending into the scrotal cavity; cranial (b) and caudal (c). ten days later, the dog returned on an emergency basis for evaluation of serosanguineous drainage from the surgical site. dog had removed the e-collar and owner has observed the dog licking the surgical site since the previous day. after examination, a surgical site seroma was diagnosed and all sutures were noted to be intact. dog was prescribed carprofen, trazodone, codeine sulfate, and silver sulfadiazine cream to be applied topically to the wound. he returned 12 days later with the presenting complaint of severe nonspecific pain, lethargy, a decreased appetite, and a reluctance to rise or ambulate. surgical site was unremarkable and complete healing was noted. after extensive diagnostics including a neurologic examination and spinal radiographs, the dog was diagnosed with discospondylitis at l7 s1. urine and blood samples were submitted for culture. rast was repeated and was negative similar to initial test (conducted 6 weeks before) results. urine culture revealed enterococcus faecium whereas blood culture was negative for any growth. dog was prescribed chloramphenicol. a repeat urine culture was performed 1 month later and that was negative for any growth. outcome after herniorrhaphy surgery and medical treatment for discospondylitis, dog returned to full athletic function. no further reproductive examinations were performed. discussion given the presence of scrotal enlargement and discospondylitis in this case, brucellosis should be the primary differential diagnosis. however, this dog had 2 rsat and a negative blood culture. brucella rsat can have false positive results but is very efficient in identifying dogs that are negative, making it a valuable screening test for brucellosis in dogs. there was no evidence of discospondylitis in the initial radiographs; it was only observed on the radiographs that were taken when the dog was presented on emergency 6 weeks later. given the pathophysiology of discospondylitis, it is highly unlikely that it would have been the result of the surgical repair of the hernia. since the specific inciting cause of the hernia itself could not be determined, it is impossible to know whether the subsequent discospondylitis was related to the hernia’s pathogenesis, but the timeline of presentation make this an unlikely scenario. a 1983 case study2 described a similar situation, in which a male intact dog presented clinical theriogenology 2022; 14: 365 with unilateral scrotal enlargement, and later with weight loss and spinal pain. brucellosis was diagnosed as determined by both rsat and mercaptoethanol tube agglutination test titers and discospondylitis was observed on radiographs. however, this dog exhibited pain on palpation of the enlarged scrotum and was diagnosed with both orchitis and epididymitis with lesions consistent with brucella canis on histopathological evaluation of the testes and epididymides after castration. similarities and differences between these cases highlighted the importance of digital palpation and ultrasonography of an enlarged scrotum to differentiate between orchitis and hydrocele. with orchitis, whether or not the underlying cause is brucella canis, one would expect the affected testis/es to exhibit evidence of inflammation, characterized by warmth on palpation and varying degrees of pain in response to digital palpation of the scrotum. ultrasonogram of normal testis should have a uniform texture with similar echogenicity as the spleen throughout the testicular parenchyma.3 in cases of orchitis, the testicular parenchyma will have irregular areas of varying echogenicity, and as observed in this case, a hydrocele will have substantial amount of anechoic fluid with or without echogenic particles within the fluid surrounding the testis/es. there are 2 reported subcategories of inguinal hernias: direct and indirect. in indirect inguinal hernia in males, abdominal viscera pass through vaginal process cavity and into scrotum, resulting in scrotal hernia as observed in this case. direct inguinal hernias, however, involve the passage of abdominal viscera through the inguinal ring adjacent to the vaginal process.4 dog in this case was nonpainful on palpation of the scrotum during every examination. possible explanations for this include a lack of bowel incarceration in the hernia, inflammation that was not yet severe enough to cause overt pain, or the dog may have been remarkably stoic. although scrotal aspiration is rarely performed in any species due to the risk of inoculating bacteria into the area, it was deemed necessary in this case to further evaluate the cause of fluid accumulation. in this case, scrotal hydrocele was presumably either as the result of obstruction (pressure caused by the herniated omentum) to venous outflow within the spermatic cord or reactive/inflammatory in nature from the herniated contents. although the dog was used frequently for competitive hunting, there were no known injuries that could have explained the inciting cause of the hernia. learning points • imaging is an invaluable tool when diagnosing the cause of scrotal enlargement in any species. although scrotal fluid aspiration is rarely performed due to associated risks, in some cases it becomes necessary to characterize the type of fluid that has accumulated. • scrotal pathology does not always necessitate castration or hemicastration. if 1 or both testis/es is/are determined to be free of innate pathology, dog may be left at least partially intact and a breeding future can be preserved. • it is paramount to rule out infectious causes of scrotal enlargement, especially brucellosis, as evidenced in this case. treating dogs for brucellosis is generally not recommended. due to high incidence of relapse and the uncertainty of antibiotic therapy resulting in a true cure, the risk of further transmission to humans and other animals remains high.5 conflict of interest no conflict of interest to declare. references 1. davidson ap: female and male infertility and subfertility. in: nelson rw, couto c: editors. small animal internal medicine. 5th edition, st. louis; mosby: 2014. p. 957. 2. anderson gi, binnington ag: discospondylitis and orchitis associated with high brucella titre in a dog. can vet j 1983;24:249252. 3. davidson ap: clinical conditions of the dog and tom. in: nelson rw, couto cg. small animal internal medicine. 5th edition, st. louis; mosby: 2014; p. 949. 4. smeak dd: abdominal hernias. in: slatter d: editor. in: textbook of small animal surgery, volume 1. 2nd edition, philadelphia; wb saunders:1993:433-454. 5. santos rl, souza td, mol jps, et al: canine brucellosis: an update. front vet sci 2021;8. clinical theriogenology 2022; 14: 366 cervical duplication in dogs david mahoney, amber nebel, michael whitacre, sara lyle north carolina state university college of veterinary medicine, raleigh, nc abstract two maiden bulldogs with cervical duplication were presented for breeding management. dogs were successfully impregnated via endoscope-assisted transcervical insemination (tci) and had their litters via cesarean surgery. a common uterine body between 2 cervical openings and 2 uterine horns was noticed (with no other reproductive abnormalities) at surgery. duplication of the cervix has apparently not been previously described in dogs. with tci becoming a more frequently used method of breeding, it is probable that defects involving failed or incomplete fusion of the paramesonephric duct during embryological development will be more frequently observed by clinicians. keywords: dogs, cervical duplication, endoscopy, hereditary, paramesonephric duct background during early embryonic sexual development, female reproductive tract of the dog starts to develop cranially from paramesonephric ducts. as these ducts extend caudally within the developing conceptus, they fuse to form a single uterine body, cervix, and cranial vagina.1-3 incomplete, inappropriate, or failure of fusion during this process leads to abnormalities of the cranial reproductive tract including, but not limited to, uterus unicornis, vaginal septae, cervical duplication, didelphis uteri, and simplex uterus.1-3 cervical duplication and external os have been described extensively in cattle and are believed to be inherited conditions.4-6 despite cervical duplication’s heritability and relevance to propagation, this condition is not a common cause of secondary complications with breeding, pregnancy, or parturition in cattle.4-6 to our knowledge this condition has not been described in dogs. in dogs, dystocia and cesarean surgeries are more common in brachiocephalic breeds (especially english bulldogs, french bulldogs, and boston terriers).7-9 to promote better animal welfare, the european union currently has animal welfare best practices in place. as per this policy dogs must be able to breed, whelp, and rear their litters naturally and dogs that had 1 or more cesarean surgeries cannot be used for breeding.10 abnormalities of the female reproductive tract may affect the ability to whelp naturally, and so it would be prudent not to breed affected animals.4-6,11-14 case presentations case 1 a 3-year, maiden female olde english bulldogge, was presented for breeding management and subsequent artificial insemination with fresh canine semen. complete clinical examination was conducted. chilled semen (from an unproven stud) was deposited in the cranial vagina using a 9-inch artificial insemination [ai] pipette (k-9 ai pipette jorgenson laboratories inc., loveland, co) 4 days after lh surge15,16 (determined via serum progesterone concentrations [between 2.0 2.5 ng/ ml] and vaginal cytology). progesterone concentrations were determined via established assays (tosoh bioscience aia 360, san francisco, ca). plastic sterile cotton-tipped swabs were used to collect vaginal epithelial cells and were rolled on to microscope slides. slides were stained with a commercial romanowsky stain variant (diff quik, jorgensen labs, loveland, co). pregnancy diagnosis was negative. for the subsequent breeding, 2 tcis (fresh semen from 1 stud dog) were used. during the initial tci, a vertical vaginal septum was noted to extend caudally 5 10 cm from the fornix. a cervical os was noticed on both sides of the septum. after careful probing, catheterization of the uterus was possible through each cervical os using a flexible catheter (ch-5 tci catheter minitube usa, verona, wi). one-half of the insemination dose was deposited through each cervical os. it was unclear during the procedure whether the 2 cervical openings communicated via a common cervical canal or uterine body. prior to insemination, the owners were contacted regarding potential complications with fertility, pregnancy, and whelping. after discussion, they elected to continue the procedure. clinical theriogenology 2022; 14: 367 a second tci procedure was performed 2 days later with fresh semen and all previous endoscopic findings were confirmed. case 2 a 2-year, female maiden english bulldog, was presented for her first breeding management. she was managed with similar protocols and recommendations as case 1, and after discussion with the owners, they elected to breed the dog via 2 fresh semen tcis. initial tci was performed successfully without complications; no septae or cervical os abnormalities were noted. a second tci was performed a day later and findings (a vertical septum extending caudally 5 10 cm from the fornix and 2 separate unobstructed external cervical ora) were similar to those observed in case 1. prior to insemination, owners were contacted regarding potential complications. after discussion and since initial insemination was already successfully performed, owners elected to continue with the procedure. differential diagnosis uterus didelphis, duplication of the cervix and external cervical os, and complete duplication of the reproductive tract were all potential differential diagnoses for these dogs. due to suspected higher frequency of bulldogs to pool fluid in the cranial vagina, it was difficult to assess whether the fluid noticed near contralateral cervical os after catheterization and insemination of the first cervical os arose from normal backflow and pooling or through a common uterine body. without performing additional endoscopy, contrast radiography, or advanced radiology including computed tomography and magnetic resonance imaging, a caudal paramesonephric duct fusion abnormality was the tentative diagnosis. advanced diagnostics would require general anesthesia or sedation; and therefore, for dogs’ and their potential litters safety, it was recommended to pursue further diagnostics after pregnancy if a diagnosis could not be elucidated at the time of elective cesarean surgery. outcome case 1 dog was confirmed pregnant (3 viable fetuses) via abdominal ultrasonography (s9 us-diagnostic system; c611, 13 3.9 mhz micro-convex transducer; sonoscape medical corp, shenzhen, china) on day 48 postlh surge. no abnormalities of the uterus or cervix were noted. a communication between the 2 cervical openings could not be confirmed. it was recommended to perform an elective caesarean surgery on or near her due date based on daily progesterone concentrations and assessment of pups’ development via transabdominal ultrasonography;17-19 however, the owners elected not to have this assessment. on day 67 postlh surge, case 1 was presented as an emergency (signs of stage i and/or ii labor > 24 hours, a green to black colored vaginal discharge, and uterine contractions without production of a fetus).9,20 a stillborn pup was palpated vaginally and, subsequently, delivered via digital vaginal extraction. more viable pups were observed via transabdominal ultrasonography and the dog was prepared for cesarean surgery. remaining pups were delivered via cesarean surgery and survived through discharge from the hospital. during surgery, a common uterine body between 2 cervical openings and 2 uterine horns was confirmed and no other reproductive abnormalities noted. concurrent ovariohysterectomy was performed as per owners’ decision. case 2 dog was confirmed pregnant via transabdominal ultrasonography on day 31 postlh surge. three fetal vesicles and 1 resorption site21,22 were observed. it was recommended that the dog be scheduled for an elective cesarean surgery. optimal timing was determined via daily serum progesterone concentrations from day 63 postlh surge and daily transabdominal ultrasonography to assess fetal heart rates and development using renal corticomedullary distinction, gastrointestinal layering, and gastrointestinal motility.17-19 on day 65 postlh surge, progesterone concentrations were < 2 ng/ml, and pups had excellent renal corticomedullary distinction, gastrointestinal layering, and gi motility; therefore, it was determined to be the ideal timing for elective cesarean surgery.17-19 three pups were delivered and they survived through weaning. during surgery, a common uterine body between 2 internal cervical ora and the uterine horns was confirmed, and no other reproductive abnormalities were observed. it was recommended that ovariohysterectomy be performed following weaning of the litter.18 discussion cervical duplication is a disorder that could be speculated to have consequences similar to those observed, although infrequently, in cattle including difficulty or inability to conceive and deliver live offspring.4-6 function of the cervix is to facilitate sperm movement in the uterus during fertile periods, maintain sterility of the cranial reproductive tract during pregnancy and nonfertile periods, and facilitate passage of offspring and fetomaternal membranes out of uterus during parturition.12-14 any abnormal orientation, structure, or cervical patency could obstruct or affect normal cervical function, thereby increasing the incidence of other reproductive issues including, but not limited to, unsuccessful breeding, ascending uterine infections, fetal resorptions, and abortions.4-6,12-14 besides reproductive complications associated with cervical duplication and/or the external cervical os, it would be prudent to be concerned that the condition may be inherited in dogs as it is in cattle.4-6 with any condition believed to be hereditary in nature, pedigree analysis is ideally performed to investigate the propagation of these conditions.23 for these 2 cases, due to owner compliance, we were only able to obtain a pedigree for 1 dog, and therefore, we were unable to complete a pedigree analysis. although these are apparently the first cases of cervical duplication in dogs to be published to the knowledge of the authors, in personal communications with experienced colleagues involved in canine reproduction, a number of them had observed this condition firsthand in bulldogs and other breeds. this suggests there is likely an inherited pattern for cervical duplication in dogs. one should question the ethics and animal welfare complications of continuing to breed with affected animals as occlinical theriogenology 2022; 14: 368 curred in these case studies. in these cases, it was our recommendation for both dogs to be sterilized following weaning of their litters18 and their progeny to be withheld from any future breeding programs.8 from an ethical standpoint, it is likely not in the owners’ or breeding community’s best ethical interest to continue to breed similarly affected animals.8 one could argue in the case of bulldogs, it might not change the outcome of these cases, as a majority of bulldog litters are delivered via cesarean surgery;7,9,17,19,24 however, with increasing changes to the breeding guidelines of brachiocephalic breeds, such as banning elective cesarean surgery in such breeds in european union and banning the breeding of any english bulldogs and cavalier king charles spaniels in norway, female animals with abnormalities of the reproductive tract should not be bred.8,10 one could also argue that as we do not have enough information on müllerian duct fusion abnormalities in dogs to make such claims; however, this might be an inherited condition that, with further gene expression or mutation, progresses to more diffuse fusion defects that could have detrimental effects on fertility, pregnancy rates, litter sizes, or animal welfare.23 any increase in the risk for dystocia or pyometra could be detrimental to an animal’s welfare.8 most common causes for dystocia in dogs are primary uterine inertia, fetomaternal disproportion, and fetal malposition.9,19,24 most english bulldogs have delivery via cesarean surgery due to higher prevalence of dystocia in this breed;7,9 therefore, caudal paramesonephric duct abnormalities might not commonly be detected in these breeds when inseminated without endoscopy. this might raise concern that the true prevalence of these abnormalities remains under-detected. the expectation would be that the true prevalence will be better elucidated as the use of tci and/or vaginal endoscopy increases. it is probable that use of tci will continue to increase over time due to pregnancy rates and litter sizes being substantially higher for tci in comparison to other methods of insemination including surgical or vaginal ai.25,26 with the increase in tci as a method of insemination, we would expect to have an increased incidence of abnormal findings in the caudal urogenital tract; although, as seen with the second case example, these abnormalities can still go undetected even when endoscopy is used. potential causes for the failure to detect the cervical duplication during the first tci could be lack of appropriate insufflation during the procedure; user experience; knowledge of endoscopy or tci practices; patient temperament, attitude, cooperation, and movement during the procedure; or endoscope-related technical issues including lens clarity. in the cases presented, cervical duplication was an incidental finding at the time of breeding. although 1 case resulted in dystocia and emergency cesarean surgery, it is heavily suspected that this outcome was more likely due to breed predisposition7,9,24 and owner compliance, rather than from pathology of the cervix. our reasoning is this is not a typical outcome for this condition in cows4-6 and the stillborn pup was completely within the vaginal canal at presentation; though since we do not have any information on this condition in dogs, it cannot be completely ruled out as a cause. the potential for more case reports of this condition will likely increase as endoscopic inseminations increase, thereby giving us more information about this abnormality. with more information about this condition, increased promotion of ethical breeding and animal welfare,8,10 and better breeding practices,8,10 cervical duplication could remain an anomaly instead of becoming another increasingly observable heritable condition. learning points • cervical and external cervical os duplication have been described as a hereditary disorder in cattle, but not in dogs • cervical and external cervical os duplication are disorders of sexual development involving incomplete fusion of the paramesonephric ducts that has the potential for increasing dystocia in animals • external cervical os duplication may become a more commonly observed disorder of sexual development in clinical practice due to the increased frequency of endoscope-assisted breeding • ethics of breeding should always be considered when potentially harmful (in terms of animal welfare) hereditary disorders are present conflict of interest none to report references 1. senger pl: embryogenesis of the pituitary gland and the male or female reproductive system. in: senger pl: editor. pathways to pregnancy and parturition. 2nd revised edition, pullman; current conceptions: 2005. p. 80-101. 2. spencer te, hayashi k, hu j, et al: comparative developmental biology of the mammalian uterus. curr top dev biol 2005;68:85-122. 3. pretzer sd: canine embryonic and fetal development: a review. theriogenology 2008;70:300-303. 4. roberts sj: infertility in the cow: hereditary or congenital anatomic defects of the reproductive tract. in: roberts sj: editor. veterinary obstetrics and genital diseases. 3rd edition, woodstock; david and charles: 1986. p. 524-527. 5. sittmann k, rollins wc, kendrick jw: a genetic analysis of the double cervix condition in cattle. j hered 1961;52:26-33. 6. ishiyama d, nakamura y, maeda k, et al: severe incomplete fusion of the mullerian ducts influences reproduction in holstein cattle. theriogenology 2019;123:209-215. 7. evans km, adams vj: proportion of litters of purebred dogs born by caesarean section. j small anim pract 2010;51:113-118. 8. farstad w: ethics in animal breeding. reprod domest anim 2018;53:4-13. 9. jutkowitz la: reproductive emergencies. vet clin small anim 2005;35:397-420. 10. european commission on health and food safety: responsible dog breeding guidelines: platform conclusions. eu platform on animal welfare 2020; p. 11. available from: https://ec.europa.eu/food/ system/files/2020-11/aw_platform_plat-conc_guide_dog-breeding. pdf 11. park ch, son ch: segmental agenesis of the uterine body, cervix and vagina in a bitch. veterinární medicína 2019;64:134-137. clinical theriogenology 2022; 14: 369 12. goericke-pesch s, schmidt b, wehrend a et al: changes in the histomorphology of the canine cervix through the oestrous cycle. theriogenology 2010;74:1075-1081. 13. senger pl: spermatozoa in the female tract: transport, capacitation, and fertilization. in: senger pl: editor. pathways to pregnancy and parturition. 2nd revised edition, pullman; current conceptions: 2005. p. 267-275. 14. senger pl: placentation, gestation, and parturition; parturition is a complex cascade of physiologic events. in: senger pl: editor. pathways to pregnancy and parturition. 2nd revised edition, pullman; current conceptions: 2005. p. 318-320. 15. mason sj: current review of artificial insemination in dogs. vet clin small anim 2018;48:576-580. 16. christenson bw: the physiology of ovulation timing in the bitch. clinical theriogenology 2011;3:3. 17. bailey cs: it’s time, past time, or is it? managed whelping and cesarean section in the dog. clinical theriogenology 2016;8:188-190. 18. mason sj, rous nr: elective caesarian in bitch. clinical theriogenology 2019;11:211-217. 19. runcan ee, coutinho da silva ma: whelping and dystocia: maximizing success of medical management. top comp anim med 2018;33:12-16. 20. arlt sp: the bitch around parturition. theriogenology 2020;150:452-457. 21. england gcw, russo m: ultrasonographic characteristics of early pregnancy failure in bitches. theriogenology 2006;66:1694-1698. 22. england gcw: ultrasound evaluation of pregnancy and spontaneous embryonic resorption in the bitch. j small anim pract 1992;33:430-436. 23. casal ml, meyers-wallen vn: pedigree analysis and inherited canine & feline reproductive diseases. clinical theriogenology 2012;4:255-269. 24. o’neill dg, o’sullivan am, brodbelt dc et al: canine dystocia in 50 uk first-opinion emergency care veterinary practices: clinical management and outcomes. vet rec 2019;184:409. 25. eilits be: fertility in a canine breeding colony using vaginal artificial insemination or transcervical insemination of fresh semen. anzcvs, science week july 2013. 26. werhahn f, urhausen c, günzel-apel a-r et al: pregnancy rates of bitches after intravaginal and transcervical insemination with fresh semen. reprod dom anim 2015;50:29. introduction as the american college of theriogenologists (act) celebrates 50 years since its inception in 1971, it is appropriate to pause and reflect on the events that lead to its creation and to honor the vision and the efforts of those involved. fortunately, some of the early letters and documents associated with the formation of the act have been preserved in a 500+ page tome entitled “early files of the american college of theriogenologists.” affectionately referred to as the “red book,” it is a compilation of early records, photocopied and bound into a book. in the copy that is in the act archives, there is the hand-written inscription: “no. 4 of ten. for: dr. fayne oberst. it is my hope that this volume will serve as a reminder of a worthy task. dave bartlett 5 november 1985.” as only 10 copies were produced, and the whereabouts of some is unknown, it has been a large inaccessible source of the college’s history. until now! in a quest to write a narrative about the formation of the act, i became aware of the existence of the red book and accessed the copy that is in the act archives. most of the pages were photocopies of correspondence or documents, with the majority being typed pages, and a few hand-written letters. although some of the documents were rather mundane, it was obvious that there were also several ‘nuggets of gold,’ although one had to look carefully to find them! as the contents of the red book chronicle the formation of the act, it is an important part of our heritage and deserves the american college of theriogenologists, from its pedigree to the neonatal period john kastelic professor of theriogenology, head of the department of production animal health university of calgary, faculty of veterinary medicine abstract early letters and documents associated with the formation of the american college of theriogenologists (act) were photocopied and bound to become the so-called act “red book,” with more than 500 pages. as only 10 of these books were produced, this important source of our history is relatively inaccessible. recently, this book was professionally scanned into a single, searchable file (.pdf format) and a table of contents produced, with both files available on the act website (www.theriogenology.org). this article, based exclusively on information from that book, briefly reviews some of the key events in 19th century animal reproduction, the roots of the society for theriogenology (sft) and efforts to create the act. keywords: american college of theriogenologists, charter diplomate, history to be more readily available. i had the entire book professionally scanned, creating a single, large .pdf file, that can be electronically searched. furthermore, i perused all pages and created a table of contents. both the .pdf of the book and the table of contents are available on the act website (www.theriogenology.org). this article is based exclusively on information derived from the red book; the pages cited correspond to the page numbers of the scanned .pdf file (the red book is a compilation and does not include page numbers). the objectives of this article were to: 1) draw attention to the digitized copy of the red book and its accompanying table of contents (available on www.theriogenology.org); 2) highlight some of the key events in animal reproduction in the 19th century, the pedigree of the sft and the creation of the act; and 3) convey some of the challenges and the amusing anecdotes recorded within the covers of the red book. animal reproduction prior to the act the history preceding the formation of the act was reviewed by dr. dave bartlett in the inaugural david e. bartlett lecture award, presented at the annual fall conference of the society for theriogenology in denver, colorado, in september, 1984, and the penultimate article in the red book.476-489 in this article, dr. bartlett summarized many of the highlights of the history of the act’s formation. in addition, the last article of the red book is the second bartlett address, 490-512 given by dr. clinical theriogenology 2021; 13: 121 steve roberts at the annual fall conference in sacramento, california, in 1985, just before the red book was bound. in this article, there is a brief summary of animal reproduction in the 20th century, highlighting key persons and events. both articles provide an interesting background regarding theriogenology and formation of the act. one of the most influential veterinarians in animal reproduction in the usa was dr. walter l. williams (1856-1945).492 he attended the illinois industrial university (which subsequently became the university of illinois) and the montreal veterinary college, graduating from the latter in 1879. he practiced in bloomington, illinois for 12 years, taught for 1 year at purdue university and 3 years at the montana agricultural college, before serving as professor of surgery and obstetrics at cornell university from 1896 to 1921; thereafter, he was professor emeritus at cornell for the remainder of his life.492 he was the author of classic books in our discipline, including “veterinary obstetrics” (published in 1909 and revised in 1917, 1931, 1940 and 1943) as well as “diseases of the genital organs of domestic animals” (published in 1921 and revised in 1939 and 1943). these books were translated into spanish and italian and were widely used in the usa and throughout the world.492 the next generation and direct descendent, dr. w.w. williams (the son of dr. w.l. williams, born in 1892) received his dvm from cornell in 1915.494 in the mid-1920’s, he collaborated with alfred savage (of the manitoba agricultural college) and undertook a study in bull fertility evaluation, regarded as the first large study of infertility in animals or humans.494-496 dr. w.w. williams subsequently earned his medical degree (1935) and established a human infertility clinic and practice in springfield massachusetts in 1936.496 erik blom, the famous danish veterinary andrologist, remarked that “this scientist never forgot his veterinary past.”496 he was a founding member of the american society for the study of infertility (essentially a “human counterpart” of the society for theriogenology) and the international fertility association, with their corresponding journals. furthermore, he appreciated the value of comparative reproductive biology and invited many veterinarians to present at meetings of these associations. in recognition of his contributions to theriogenology, dr. w.w. williams was designated an honorary diplomate of the act.496 in the era immediately after the second world war, horses as a means of transportation or pulling power were being increasingly replaced by automobiles and tractors.476 however, there were rising numbers of dairy and beef cattle being raised, and some research in bovine reproduction, mainly infertility and reproductive losses (primarily brucellosis).476 in addition, cryopreservation of bull semen was introduced and there were studies to improve and expand its utilization. most of the research in animal reproduction was being conducted by animal scientists and physiologists, but very few were veterinarians.476-477 origins of the society for theriogenology there was no formal association of veterinarians in the usa interested in animal reproduction until 1954, when the “rocky mountain society for the study of breeding soundness in bulls” (rmssbsb) was formed in colorado. the impetus for the creation of this group were severe blizzards in northern colorado and wyoming in 1949-50, killing thousands of farm animals and causing severe scrotal frostbite in beef bulls.501 prior to the breeding season in 1950, colorado state university veterinarians, including harold hill and lloyd faulkner, were asked to assist private practitioners to evaluate bull breeding soundness.501 the objectives of the rmssbsb were to standardize and share the essentials for evaluation of beef bulls for fertility. development of the w.g.r. marden electroejaculator in the late 1950’s facilitated the collection of bull semen.502 in 1966, the rmssbsb changed its name to become the “american society for the study of breeding soundness” (assbs), with increasing membership, broader geographical distribution, and inclusion of the bovine female.477 by 1967, it had become the american veterinary society for the study of breeding soundness (avssbs), and in 1974, it changed its name to the society for theriogenology, although during a transitional phase of 4 years, both names were used.495 in the early years, the society periodically produced a journal, published “when the spirit moved and the time permitted.”502 early attempts to create the act in the mid-1960’s, the avma formed their advisory board on veterinary specialities to promote formation of specialty groups.477 after unsuccessful attempts by the assbs to get reproduction established as a specialty group, in 1967, the avssbs believed that “formation of a specialty board in animal reproduction is appropriate and timely” and indicated that a proposed constitution, by-laws, and certification procedure for the proposed board had been created.6 the speciality group was originally termed “the american veterinary board of animal reproduction” but subsequently “board” was replaced with “college,” likely in 1967.29,32 furthermore, it was noted that the proposed board would allow a practitioner in the field to become certified, making “this board unique among the boards already in existance within the frame-work of the a.v.m.a., in that there is a definite area with which the practitioner can become associated and identified.”6 it was noted that a meeting with representatives of the a.v.m.a. had been scheduled for 22 february 1967, prior to a meeting with the specialty advisory board.6 the avssbs had written a first draft of a proposed constitution and by-laws for a proposed specialty group, the american college of veterinary reproduction; these documents were received by the a.v.m.a. advisory board on veterinary specialties on 10 february 1967, prior to a scheduled meeting of the reproduction group on 21-23 february 1967, at which their proposal would be further developed.7 the a.v.m.a. expressed their willingness to support these efforts, but they also indicated that they “suggest careful consideration before an application is submitted.”7 the american association clinical theriogenology 2021; 13: 122 of equine practitioners (a.a.e.p.) expressed their support for formation of a specialty board of animal reproduction, noting that “the a.a.e.p. believes that there is a definite need for such a board and that it would serve to advance the knowledge of this complex field.”8 in a letter to several colleagues dated 16 march 1967, fayne oberst noted that the constitution and bylaws for the specialty group had been revised and there was an initial decision to petition for specialty recognition on 1 april 1967, although based on comments from the a.v.m.a, perhaps it was prudent to wait until 1968.9-10 it was subsequently reported that the request for recognition was only to be a discussion item at the meeting on 1 april 1967.26 spring of 1970 and a new initiative the red book contains no record of what happened over the next 3 years. however, it was noted that “activities of 1970 began with a phone call from faulkner to bartlett with request that the latter undertake negotiations with the avma’s advisory committee on veterinary specialties.”57 in a letter (24 march 1970) from lloyd faulkner to jim scott, it was stated that dave bartlett would be the logical contact with the avma to complete negotiations with the advisory committee on specialties.58 furthermore, the work of jim scott and the avssr to bring recognition to the group was acknowledged.58 in that letter, it was referred to as the “college-to-be.” in the spring of 1970, dr. lloyd faulkner (colorado state university) initiated a new organizing committee that included: raimunds zemjanis (university of minnesota), stephen roberts (cornell university), fayne oberst (michigan state university), john kendrick (university of california-davis) and david bartlett (american breeders service, deforest, wi).477-478 drafts of previous constitutions for an “american veterinary board of animal reproduction” and an “american veterinary college of animal reproduction,” originally drafted by the avssbs, were modified to better match the expectations of the advisory board on veterinary specialties. regardless, when the organizing committee met with board of veterinary specialties in march, 1970, they faced considerable scepticism.478 at that time, many topics and procedures related to reproduction, in both large and small animals, were managed by surgeons or internists, and there were very limited numbers of practitioners or university faculty who had a reproduction focus. in 1971, dr. bartlett “shared his disappointment during visits to large herds of dairy cattle that tracks of dinosaurs were more evident than those of veterinarians.”169 furthermore, much of the research on animal reproduction was being done by non-veterinarians in animal science or dairy science faculties. in that regard, the american society of animal science had periodic symposia on animal reproduction and the society for the study of reproduction was very active.478 it was noted (18 may 1970) that a meeting had been held with the avma on 7 may 1970 and that the constitution had been changed as a result of that meeting, with the major change being deletion of the category “fellow.”68 soon thereafter, a meeting with the avma’s advisory board on veterinary specialties was arranged for 24 june 1970 at the international hotel in las vegas, nevada.86,89 at that meeting, kendricks, oberst, carroll, faulkner, and bartlett represented the act, with wagner as an interested guest.94 following that meeting, bartlett noted that the constitution had been further refined.106 origins of theriogenology and theriogenologist although the term ‘theriogenology’ is now widely known and accepted, that was not always the case. typical historical names for our discipline included veterinary obstetrics, genital diseases, animal reproduction, veterinary reproduction or breeding soundness, none of which were all-encompassing. although veterinary gynecology and veterinary andrology were established in europe, these terms were misleading, as according to their greek origins, “gynos” means woman and not female and “andros” means man and not male.375 when he was a faculty member at the university of minnesota in the 1950’s, dave bartlett had a discussion with a scholar of classical languages and the term ‘theriogonlogy’ was derived.479 on 2 april 1951, dave bartlett was lecturing to veterinary students and les larson recorded in his lecture notes the use of the term «theriogonology.»447 in a letter (14 may 1970) dave bartlett stated that “i do hear a bit of static on the term american veterinary college of animal reproduction. this actually means the american college pertaining to animals of animal reproduction. at best this is redundant. at least it is awkward.”61 in addition, he stated that about 20 years earlier, he had consulted with a greek scholar and noted that animal reproduction had gone beyond the available terminology.62 furthermore, he stated that “this week, i consulted with professor howe, university of wisconsin, who teaches their course in latin and greek medical terminology.”62 he reminded me that theatrics and theriotheraphy referred to veterinary medicine and treatment of the diseases of lower animals, respectively. furthermore, he also noted that “notice how in therianthropic the root was used to hold distinct animal from man. therio is an interesting possibility.”62 furthermore, he remarked that “the root word gen means: the coming into being of anything.” thereafter, he stated “it is quite acceptable to combine the two greek roots into the term theriogenology, meaning, very neatly, animal reproduction.” at the close of the letter, he stated: “what do you think about an american college of theriogenologists or an american board of theriogenology?”63 a few days later, steve roberts noted that he had carefully considered bartlett’s letter and that both he and his colleague k. mcentee approved the title “american college of theriogenologists.”85 on 28 september 1970, bartlett sent professor howe a note, indicating that “the word theriogenology seems to be having further results.” in addition, he stated that “i wanted you to know that your suggestion and counsel have clinical theriogenology 2021; 13: 123 been valuable and very genuinely appreciated.”108 howe replied: “i am glad my brain child, theriogenology, was acceptable to the brethren. surely, the only sufferers from such a name would be the animals, who won’t understand it anyway.”113 in the inaugural bartlett address (1984), dave bartlett commented: “in retrospect, the limited amount of derision and ridicule elicited upon introduction of two new terms, theriogenology and theriogenologist, was more amusing than significant. it was short lasting. students, who were already assimilating and digesting hundreds, if not thousands, of new words each year, were unmoved. for older veterinarians, whose professional vocabularies already included many thousands of words, addition of two more words should be of no consequence. theriogenology was soon found to be no more difficult to say than otolaryngology or anesthesiology.”483 one person, a greek veterinarian who had been a graduate student with steve roberts, wrote to his former supervisor and called into question the use of the term theriogenology.450-464 in turn, roberts sent a note to dave bartlett, who rebutted those concerns. bartlett stated: “steve, in no way do i feel qualified to debate the meaning of ancient or modern greek words with a contemporary greek. i must recognize the qualities of myself: although i’m reaching the age of disqualification as a contemporary, i’m somewhat short of qualifying as ancient! especially, as a greek.”457 in 1984, bartlett arranged another meeting with professor howe (who was retired) and who reiterated that the word theriogenology was still relevant and appropriate.462 fall of 1970, progress continues another critical step in the formation of the act was the conference of north american educators in animal reproduction and infertility (theriogenology), held at brainard, minnesota on 30 august to 3 september 1970, hosted by zemjanis.104-105 bartlett was unable to attend, but prepared an audio tape98 that was transcribed.99-103 there were 41 actively participating educators at the conference, from 23 of the 24 veterinary schools/ colleges in the usa and canada.105 this was the first time that many of these veterinary educators in reproduction had met in person.481 in the conference summary, the first 2 items were: “1) theriogenology or animal reproduction is a speciality that is an integral part of the veterinary curriculum; and 2) the word theriogenology should replace the phrase animal reproduction and obstetrics in the veterinary vocabulary.”481 it was noteworthy that at this conference, zemjanis was reported to have stated that “theriogenology just can’t make it as a liberal art.”486 soon after the educator’s conference in the fall of 1970, the organizing committee changed its name to the “organizing committee for the american college of theriogenology.”481 a list of potential charter diplomates was prepared, and those persons were invited to submit cvs and publications to the organizing committee and the advisory board on veterinary specialties. in september 1970, the organizing committee elected a panel of charter diplomates; they were asked to forward their cv and those documents, plus a revised copy of the constitution, were sent to the board on veterinary specialties in early 1971.212 as would be expected, creating the list of charter diplomates resulted in some regrets. in the inaugural bartlett address, dave bartlett stated: “the only regrettable occurrences that marred act’s otherwise smooth emergence into the mainstream of veterinary medicine were the expressed disappointments – some very bitter – of a few, well qualified and prominent veterinarians not included among the necessarily limited number of charter diplomates.”483 on 8 december 1970, bartlett sent a formal request to the advisory board on veterinary specialties, for probational recognition of the act as a veterinary specialty organization.114-116 it was noted that there was an organizing committee of 7 persons, with another 22 invited to submit their cv to be considered as charter diplomates. finally, it was noted that the first examination for new diplomates would be held in the fall of 1971.116 soon thereafter (16 december 1970), bartlett wrote to the organizing committee, indicating the need for having “the most dignified and handsome certificate of membership that can be created.” he emphasized the importance of this and stated “that this is not the time to save money or to search for the lowest bidder.”117-118 remarkably, it was subsequently noted (november 1972) that “cliff barker expressed his revulsion for the diploma of the act and the half-wit who designed it. several other members nodded their accord and brother john williams gave the sentiment his blessing, but the secretary offered to whip ‘em all. john explained that the quality of the paper, not the design, was the problem. president bartlett saved the demise of the secretary by announcing that the executive board would consider the matter and make its recommendation to the college at its annual meeting in philadelphia in july 1973”361-362 he also noted that the avma had announced the act and an explanation of “theriogenology;” this appeared in the 15 december 1970 issue of the journal of the american veterinary medical association.120,481 bartlett, faulkner, and oberst met with the advisory board on veterinary specialties on 12 march 1971. there was broad support for the petition to form the act, only minor changes suggested and assurance that there was an expectation of probational approval by the board and house of delegates in 1971.129 in a subsequent letter (3 may 1971), it was noted that the avma council on education had met on 23-25 april, reviewed the recommendations of the advisory board and recommended that the executive board and house of delegates grant probationary approval to the act.137 the act is born! the organizing meeting of the act was on 19 july 1971 at the detroit hilton hotel,158 as part of the 1971 joint annual meeting of the canadian and american veterinary medical associations.162-163 in the minutes of that meeting,164-171 it was noted that 17 of clinical theriogenology 2021; 13: 124 28 charter diplomates were present. the president, secretary and treasurer were bartlett, faulkner and oberst, respectively, whereas roberts, barker and zemjanis were board members with 1-, 2and 3-year terms. it was subsequently noted (1984) that none of the 6 members of the organizing committee had held office after 1976.484 there were 2 subcommittees, 1 for developing standards and procedures for certification and another for developing the scope of the examination.158 it was noted that charter diplomates were chosen by ballot from a list of approximately 70 and that all had a minimum of 10 years of post-dvm experience.169 furthermore, it was recommended that the avssbs change their name to include the term “theriogenology.”170 the final draft of the constitution was adopted on 19 july 1971 and submitted to the avma for filing with the articles of incorporation in illinois on 3 december 1971.142156 in august 1971 at detroit, the act received probationary approval by the avma’s house of delegates.212-213 the first invitation to veterinarians interested in candidacy for diplomate status was written on 16 august 1971196 and a petition to candidacy was prepared.197-199 this was advertised in javma; it was noted that applications were due on 15 march 1972, with a written examination of approved candidates to be held at the avma meeting in new orleans, with successful candidates then proceeding to an oral examination at the avssbs meeting in east lansing in the fall of 1972. to sustain the momentum, an act executive board meeting was planned for the red fox inn, kennett square, pennsylvania on 6 october 1971. minutes of this meeting included a decision to limit the first examination to the 10 most qualified candidates.216-219 furthermore, the avssbs and act planned to jointly sponsor a half-day program devoted to theriogenology at the avma meeting in new orleans (18-21 july 1972). finally, the minutes also mentioned that: “roberts was gung-ho to arrange an informal, unofficial liar’s seminar on bourbon street. zemjanis suggested that roberts should be in a geriatrics society.”218 the first class of diplomates by examination the first written examination of the act was given in new orleans on 17 july 1972. there were 8 petitions for candidacy, of which 6 were accepted, 1 was denied and 1 subsequently withdrew. a list of study materials (selected articles in theriogenology 1971) was prepared and distributed to candidates.266,286-316 although obviously now very dated, this list provides an overview of the foundational knowledge current at that time. of the 6 candidates for the written examination in new orleans, 5 passed274 and were subjected to an oral examination in east lansing on 5 november 1972.280,357 it was subsequently noted that were an additional 5 successful candidates in 1973.378 at the act meeting on 5 november 1972, myron fincher, walter gibbons and willard boyd were accepted as honorary members of the act.361 it was noted (1972) that some prospective candidates failed to petition because the objective of the examination were unclear.280-281 furthermore, it was also stated that the inaugural examination was “heavily oriented to the clinical application of knowledge in theriogenology. an excellent suggestion was made that the examining committee should develop an examination which allows the examinee some choice in the area of emphasis in which he has particular expertise. the examination could cover one optional area of emphasis and a mandatory general section.”281 further activities and developments at a meeting of the executive board on 19 july 1972 in new orleans, there was a discussion regarding financial matters, including the amount of funds that should be held in reserve. in the minutes, which were signed by secretary faulkner, it was stated that: “the meeting adjourned without further consideration of designation of amount to be held in reserve for exigency and ceiling on this operating budget. your secretary assumes that this lack of diligence can be attributed to the lewd ambitions on the part of the board to get to the riverboat blast. the secretary retired to the city library.”283 the act liaison committee was formed during the annual meeting in new orleans in july 1972.327 the purpose of this committee was to ensure a smooth working relationship with the avssbs and “to develop useful professional relationships with other organizations as circumstances may dictate and your judgement see fit.”327 at the act executive board meeting on 6 october 1971, the commitment of the act to work with the avssbs was emphasized and it was noted that the latter organization would revise its constitution and by-laws to include the word theriogenology or theriogenologists.218 in presentation to the avssbs in 1971,209-215 bartlett noted that “it is the intention that this college will be very closely related to the society and it is the intention that the members of the college will maintain dual membership.”214 furthermore, he also stated that a liaison committee between the college and the society had been appointed and that a working relationship was developing.214-215 in 1972, there were concerns that the “college is presently too heavily oriented toward the bovine; our program must be broadened to provide consistent interest in theriogenology in horses, swine, dogs, cats and other domestic and laboratory animal species.”360 furthermore, in 1972, it was noted that the american animal hospital association (aaha) had invited the act to participate in its 40th annual meeting in san antonio. “this emphasizes the profound need for the college to broaden its scope of interest in theriogenology to include the small animals and laboratory animals.”363 bartlett subsequently addressed the aaha, explaining the process of establishing the act, activities in the early days of the college and the desire to encourage more small animal theriogenologists.373-379 in 1972, it was stated that: “it is most desirable to achieve a balance of disciplinary and species interests within the college.”276 clinical theriogenology 2021; 13: 125 consequently, the act committee on horses was also formed during the annual meeting in new orleans in july 1972321 and the following was recorded: “it is recognized in the deliberations to date of act, personalities involved with theriogenology of cattle have predominated. it is intent of this organization to avoid unilateralness. in consequence, a committee on horses has been established in order to consider and advise as to what needs to be done to further this speciality within the equine species.”321 the act also formed a committee on practitioner education.358 “raising the level of competence of veterinary practitioners in the field of theriogenology has been a primary goal of the college during its organization and since its recognition. initial efforts to include a category of membership for practitioners in the college were not acceptable to the avma’s advisory board on specialty groups. our dedication to serving the needs of the practitioner of theriogenology abides, however.”358 getting ‘theriogenology’ into the dictionary and beyond bartlett contacted colleagues at a number of veterinary schools, asking them to alert him once the term “theriogenology” began to appear in the descriptions of university course calendars.397-400 once the word started to appear in print, bartlett contacted a number of dictionaries, alerting them of the terms “theriogenology” and “theriogenologist,” explaining the origins of the words and providing evidence of use (including photocopies of university course catalogs and other printed materials where the word was being used). presumably, he was aware that the name dr. murray was associated with the oxford dictionary and so he sent a letter directed to him. in a reply from the oxford dictionary on 15 august 1973, it was stated that “dr. murray died in 1915 and it falls to me to reply to your letter of 23 july.”426 in a letter to dr. john williams, bartlett commented on an announcement that had appeared in the university of georgia newsletter, stating: “i was glad to see that at some university a little originality, dignity, and class have been left with a college permitting it to develop an attractive announcement of their program. from most institutions today the college announcements appear as if they had been created by an army of occupation for an occupied country.”401 theriogenology textbooks and a journal it was noteworthy that in the second edition of his book (1971), roberts entitled it “veterinary obstetrics and genital diseases (theriogenology).”262-263 in a post-script of a letter sent to bartlett (5 june 1972), roberts indicated that he was leaving cornell and heading to vermont.262 in a cornell report, it was noted that after 30 years as a professor at cornell, roberts will become emeritus as of july 1, 1972.264 the third edition of this book, “genital diseases and obstetrics (theriogenology),” was published in 1984. in 1980, “current therapy in theriogenology,” a multi-author book edited by diplomate david morrow, was published.485 furthermore, the journal “theriogenology: an international journal of animal reproduction” was launched in 1974, edited by charter act diplomate john kendrick,485 with diplomate victor shille taking over in 1980 (and continuing until 2003). foreshadowing future challenges it is noteworthy that some of the current challenges faced by the act were already present in the early years! for example, even in the first year of the act, there were already some charter diplomates who were slow to pay their dues,221 prompting a subsequent query if any had to be removed from the roster due to nonpayment.228 regarding the difficulties to get persons to commit, in a letter to the executive board on 9 august 1973, secretary faulkner admonished his colleagues to “grit your teeth and resolve to do what is necessary for the good of the college, or admit that you don’t care much for theriogenology by your absence.”436 although we are perhaps inclined to think of it as a recent matter, maintenance of certification was part of the original by-laws, requiring diplomates to retain records of professional activities and continuing education.41 summary the objectives of this article were to: 1) draw attention to the digitized copy of the red book and its accompanying table of contents (available on www.theriogenology.org); 2) highlight some of the key events in animal reproduction in the 19th century, the pedigree of the sft and the creation of the act; and 3) convey some of the challenges and the amusing anecdotes recorded within the covers of the red book. although this article does not summarize all information included in the red book, it provides many key dates and activities in the formation of the act. having the .pdf file and the table of contents online and the ability to search the .pdf for key words, will make it easy to access and retrieve information. clinical theriogenology 2021; 13: 126 the american college of theriogenologists,from its pedigree to the neonatal period clinical theriogenology 2022; 14: 382 a note of appreciation despite their busy schedules, the following scholars graciously accepted invitations to review manuscripts submitted to clinical theriogenology and provided timely and thoughtful evaluations. the editor, copy editor (john kastelic), editorial board members, theriogenology associations (society for theriogenology, american college of theriogenologists, theriogenology foundation), and hannah steet consulting, thank them for their time and effort. divakar ambrose* annett annandale henry annandale orsolya balogh theresa beachler margret casal peter chenoweth gail colbern* (2) jill colloton* mariana diel de amorim marthina greer tiago guimarães allan gunn* (2) camilo hernández-avilés jason herrick milan hess fiona hollinshead reed holyoak vanmathy kasimanickam* (5) dale kelley kara kolster michelle kutzler* (5) réjean lefebvre* catharina linde forsberg charles love sara lyle candace lyman herris maxwell pat mccue justin mcnaughten lynda miller jim morehead jennifer nagashima jen roberts* xavier schneider lorenzo segabinazzi jack smith jenny sones cyril stephen* (2) riley thompson lindsey vansandt dawna voelki karen von dollen william whitler brain whitlock* *editorial board member numbers within parentheses indicate manuscripts reviewed 1 contact ramanathan kasimanickam ramkasi@wsu.edu ‡current address: animal health clinic, blackfoot, id 83221, usa © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9425, http://dx.doi.org/10.58292/ct.v15.9425 research report comparison of bull sperm morphology evaluation methods under field conditions rachel hanson‡, sadie reddick, sabrina thuerauf, katie webb, vanmathy kasimanickam, ramanathan kasimanickam college of veterinary medicine, washington state university, pullman, wa, usa abstract sperm morphological assessment is a critical component in bull breeding soundness evaluations. although sperm morphology is an important parameter to identify subfertile from fertile bulls, the evaluation can be biased because sperm staining methods used under field conditions may not be exact due to various factors, including artifacts. objective was to compare 2 sperm morphological evaluation methods. prebreeding season ejaculates of 1,216 angus cross bulls collected via electroejaculation were evaluated. for each bull, an unstained (uns) and an eosin-nigrosin stained (ens) semen smear were viewed under a phase contrast microscope and a brightfield microscope with an oil immersion lens, both at 1,000 × magnification. normal and percentage of abnormal sperm were identified by counting 200 sperm. inter-rater agreements between 2 clinicians for the percentage of abnormal sperm determination and its categories were very good (ens method, r = 0.84 – 0.96; uns method, r = 0.76 – 0.96; p < 0.01). no differences (p > 0.1) were observed for abnormal sperm percentage determination and its categories between 2 methods. correlation was very good between 2 methods for total abnormal sperm percentage determination (r = 0.91; p < 0.01) and its categories (r = 0.84 – 0.96; p < 0.05). additionally, 60 ejaculates were evaluated by triple stain (ts), ens, and uns methods. agreements between ts (percentage of sperm with damaged membrane) and ens (percentage of abnormal sperm) and between ts and uns methods were moderate (r = 0.58; p < 0.05) and fair (r = 0.43; p < 0.05), respectively. based on our findings, either technique can be used for bull sperm morphological evaluation under field conditions. considering the ease of semen smear preparation, the uns method can be a viable alternative to the ens method. keywords: bull, sperm morphology, eosin-nigrosin, triple stain, microscope, phase contrast introduction spermiogenesis includes reorganization of sperm nucleus, development and positioning of acrosome from the golgi apparatus, organization of tail structures, and restructuring of cytoplasm.1 transformation of spermatids during spermiogenesis is a key postmeiotic event contributing to major sperm morphological reorganizations.2 an exact evaluation of sperm parameters is necessary to predict bull fertility for natural breeding and for assisted reproductive technology (art).3,4 evaluation of bull’s breeding potential includes analysis of basic sperm characteristics (motility and morphology).5,6 sperm use in art procedures is expanding, especially for semen cryopreservation, semen sexing, and in vivo and in vitro embryo production, validating the need for diagnosing subfertile and infertile bulls.7–10 sperm morphological characteristics are the most distinguishing sperm parameter of subfertile and fertile bulls.11–13 additionally, sperm morphology has a high prognostic potential for evaluating bull fertility, and thus morphological assessment is critical for selecting bulls for breeding purposes.6,11–13 sperm morphological evaluation determines the percentage of normal and abnormal sperm.5,6 microscopic examination of ejaculates indicated that sperm morphological assessment has discrepancies, even within the same ejaculate, and these discrepancies create difficulties in determining bull fertility potential.6,14 various stains and methods were used for sperm morphological analysis, resulting in ambiguous outcomes.2,15 in this regard, eosin-nigrosin (ens) staining has remained the most commonly used technique for detecting sperm morphological abnormalities.5,6 however, it should be noted that sperm morphological evaluation methods can critically affect sperm morphology outcomes.16–18 veterinary practitioners also perform sperm morphological evaluation using unstained (uns) mailto:ramkasi@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9425 2 citation line: clinical theriogenology 2023, 15, 9425, http://dx.doi.org/10.58292/ct.v15.9425 semen smears under a phase contrast microscope under an oil immersion lens. easy-to-use inexpensive standard sperm morphological evaluation technique is important for exact, unambiguous analysis of sperm.19 therefore, we compared 2 methods (ens versus uns) for evaluating sperm morphological characteristics of bulls. additionally, we also evaluated ejaculates from 60 bulls by the triple stain (ts), ens, and uns methods to determine the agreement among these 3 methods to determine their field application. we hypothesized that bull sperm morphological evaluation using these methods is similar. materials and methods animals our prospective study used ejaculates (n = 1,216; angus cross; yearling to 5 years) collected from bulls (2017 to 2020) presented for routine bull breeding soundness evaluation.5 bulls were reared by private commercial beef cattle producers and were chosen without considering their fertility potential. ejaculates were collected from bulls via electroejaculation (pulsator iv, lane manufacturing, inc., denver, co, usa) applying minimal electrostimulation to obtain ejaculates. since it is a routine theriogenology procedure (with no extra manipulation of bulls involved), institutional animal care and use committee approval is not required, and the study is exempt. sperm morphological examination once sperm motility was assessed, sperm morphology slides were prepared using both methods for each bull. for the uns method, 10 μl of semen was placed on one end of a preheated (36°c) microscopic slide, and a thin smear was prepared by feathering technique. for the ens method, a drop of semen (5 μl) was placed on a preheated (36°c) microscopic slide, mixed with an equal volume of eosin-nigrosin dye, and a thin smear was prepared using feathering technique. slides were airdried, and 1 clinician prepared slides for sperm morphology. for the ens method, sperm morphology slides were viewed under a brightfield microscope at 1,000 × magnification under an oil immersion lens. for the uns method, sperm morphology slides were evaluated at 1,000 × under a phase contrast microscope under an oil immersion lens. sperm morphology slides were evaluated by 2 clinicians, on the same or on the following day, at the laboratory. for both techniques,  slides were scored after counting a minimum of 200 sperm per sample and categorized based on morphological abnormalities. sperm abnormality classification morphological abnormalities5,6 were calculated as a percentage of the total number of counted sperm. morphological categories used were abnormal heads (pear-shaped, narrow at the base, abnormal contour, acrosome defects, vacuoles and craters, diadem, and undeveloped, detached, narrow, big, little-normal, and short-broad heads), abnormal midpieces (bent, short, and rough edge), proximal cytoplasmic droplets, dag defect, bent tail, and coiled tail. comparison of sperm plasma, acrosomal, and mitochondrial membrane assessment was by the triple stain (ts) method with sperm abnormality assessment by ens and uns methods. semen samples from 60 bulls were used. immediately after semen collection, semen smears were prepared for ens and uns methods. additionally, an aliquot of the ejaculate was extended with a semen extender and was transported to the laboratory. semen samples were washed in pbs, diluted in talp sperm medium to reach the final concentration of 25 × 106 sperm per ml, and then stained using the ts staining method. triple staining procedure was performed according to the method described.20–22 for each bull, 12 μl of propidium iodide (pi) (0.5 mg/ml), 2 μl of mitochondrial (jc-1) (153 μm), and 50 μl of pisum sativum agglutinin (fitc-psa) (100 μg/ml) fluorescent probes were added to 150 μl of the diluted semen sample. the mixture was incubated at 37°c for 8 minutes, then placed on a warmed slide, covered by a coverslip, and evaluated using fluorescence microscopy (leitz, laborlux s) at 1,000 × magnification. two hundred sperm were examined per slide and were classified into 6 classes (table 1) according to the fluorescence emission from each dye. plasma (pi), acrosomal (fitc/psa), and mitochondrial (jc-1) membranes were simultaneously assessed.20–22 percentage of sperm with low membrane integrity (sperm exhibiting damaged plasma membrane), damaged acrosome, and low mitochondrial potential on triple stain was considered for comparison with the percentage of sperm abnormalities by ens and uns methods. furthermore, comparisons were made for sperm organelles among staining methods, and parameters compared are provided (table 2). data analyses data were analyzed using a statistical program (sas, version 9.4; sas institute, cary, nc, usa), and p value was set at ≤ 0.05 for significance. concordance correlation coefficient of lin was used to analyze inter-rater agreement for determining the percentage of abnormal sperm.23,24 the shapiro-wilk test was used to verify the normality of the data and levene’s test to verify the homoscedasticity of the variance. when necessary, data were transformed with log10 or arcsine for analysis, and nontransformed values were reported. mean abnormal sperm percentage by the 2 techniques was compared using the student’s t-test. correlation of abnormal sperm percentage by ens and uns methods was determined using the lin correlation coefficient method. correlation between the percentage of low sperm membrane integrity by the ts method and the percentage of abnormal sperm by the ens method, and the percentage of abnormal sperm by uns method were determined using the lin correlation coefficient method. furthermore, the percentage of plasma membrane integrity by ts versus the percentage of live sperm by ens or head defect by uns; intact acrosome membrane by ts versus normal acrosome by ens or uns methods; and high mitochondrial potential by ts versus normal midpiece by ens or uns methods were also determined using the lin correlation coefficient method. results images of morphologically abnormal sperm by ens (figure 1) and uns (figure 2) methods are provided. images of 6 classes (figure 3) by simultaneous assessment of plasma, acrosomal, and mitochondrial membranes are given. agreement (concordance correlation of lin, r) between 2 clinicians for determining various types of abnormal sperm percentage (table 3) by the ens method was very strong (r = 0.85 – 0.96; p < 0.01), whereas by the uns method, the agreement was moderate to very strong (r = 0.76 – 0.96; p < 0.01). http://dx.doi.org/10.58292/ct.v15.9425 citation line: clinical theriogenology 2023, 15, 9425, http://dx.doi.org/10.58292/ct.v15.9425 3 percentage abnormal sperm did not differ (p > 0.1) between ens and uns methods (table 4). the percentage of various sperm abnormalities by 2 methods was positively correlated (r = 0.84 – 0.96; p < 0.01). both methods detected acrosome defects and cytoplasmic droplets; however, nuclear vacuoles were detected most readily by the uns method. the percentage of total sperm defect did not differ between ts and ens but differed between ts and uns (table 5). the percentages of head, mid-piece, and tail sperm defects did not differ among methods (table 5). correlation between the percentage of abnormal sperm with damaged membranes by the ts method and the percentage of abnormal sperm by the ens method was moderate (r = 0.58; p < 0.05). similarly, the correlation between the percentage of sperm with damaged membranes by the ts method and the percentage of abnormal sperm by the uns method was fair (r = 0.43; p < 0.05) (table 5). correlations between ts and ens methods for sperm head, mid-piece, and tail defects were fair to very good, 0.73, 0.49, and 0.64, respectively (p > 0.05); furthermore, correlations between ts and uns methods were also fair to very good, 0.80, 0.40, and 0.61, respectively (p > 0.05) (table 5). plasma membrane integrity by ts versus live sperm percentage by ens or head defect by uns had a very good correlation (p < 0.01; table 6). correlation between intact acrosome membrane by ts versus normal acrosome by ens was moderate (p < 0.05; table 6) and by ts versus uns was good (p < 0.01; table 5). correlation between high mitochondrial potential by ts versus normal midpiece by ens was moderate (p < 0.05; table 5) or by ts versus uns was poor (p > 0.1; table 6). discussion sperm morphology in bulls was compared using the uns method and the commonly used ens method. both methods evaluated sperm morphology similarly. although similar studies16,17 have been conducted, the current study investigated sperm samples from a larger population of bulls to determine the repeatability of earlier studies’ outcomes. table 1. sperm classification for simultaneous assessment of plasma (propidium iodide – pi), acrosomal (pisum sativum agglutinin – fitc/psa), and mitochondrial (jc-1) membranes by triple stain fluorescent probes sperm classification pi fitc-psa jc-1 a. intact plasma membrane, damaged acrosome, and high mitochondrial potential (class iii) + red b. intact plasma membrane, intact acrosome, and high mitochondrial potential (ipiah – class i) red c. damaged plasma membrane, damaged acrosome, and high mitochondrial potential (class vii) + + red d. damaged plasma membrane, damaged acrosome, and low mitochondrial potential (class viii) + + green e. damaged plasma membrane, intact acrosome, and low mitochondrial potential (class vi) + green f. damaged plasma membrane, intact acrosome, and high mitochondrial potential (class v) + red pi positive (+) = red stained nucleus; fitc-psa positive (+) = green acrosome region; pi negative (-) = unstained nucleus; fitc-psa negative (-) = unstained acrosome. table 2. categories for comparison of sperm organelles categories triple stained eosin-nigrosin stain unstained plasma membrane integrity intact plasma membrane (pi, %)) % live sperm‡ % normal heads† intact acrosome % intact acrosome membrane (fitc) % normal acrosome* % normal acrosome* mitochondrial potential % high mitochondrial potential (jc-1) % normal midpiece§ % normal midpiece§ ‡sperm stained in pink/red color (sperm with structurally intact cell membranes, live sperm, are not stained, while dead sperm, with disintegrating cell membranes, are stained) were excluded; †abnormal heads (including pear-shaped, narrow at the base, abnormal contour, vacuoles and craters, diadem, and undeveloped, detached, narrow, big, little-normal, and short-broad heads) were excluded; *acrosomal defects (knobbed acrosome and acrosome reacted) were excluded; §abnormal midpieces (bent, short, rough edge, and dag defect) were excluded. http://dx.doi.org/10.58292/ct.v15.9425 4 citation line: clinical theriogenology 2023, 15, 9425, http://dx.doi.org/10.58292/ct.v15.9425 diadem/acrosome defects crater – head defect proximal droplet pyriform head midpiece reflex defect vacuoles -head defect detached head coiled tail dag defect acrosome defect figure 1. sperm with abnormal morphology (eosin-nigrosin stain); images were captured by iphone 12 under brightfield microscopy. coiled tail midpiece reflex defect figure 2. sperm with abnormal morphology (unstained); images were captured by iphone 12 under phase contrast microscopy. a b dc e f figure 3. images for simultaneous assessment of sperm plasma (propidium iodide – pi), acrosome (pisum sativum agglutinin – fitc/psa), and mitochondrial (jc-1) membranes by triple stain fluorescent probes in bulls. (a) intact plasma membrane, damaged acrosome membrane, and high mitochondrial potential; (b) intact plasma membrane, intact acrosome membrane, and high mitochondrial potential; (c) damaged plasma membrane, damaged acrosome, and high mitochondrial potential; (d) damaged plasma membrane, damaged acrosome membrane, and low mitochondrial potential; (e) damaged plasma membrane, intact acrosome membrane, and low mitochondrial potential; (f) damaged plasma membrane, intact acrosome, and high mitochondrial potential. http://dx.doi.org/10.58292/ct.v15.9425 citation line: clinical theriogenology 2023, 15, 9425, http://dx.doi.org/10.58292/ct.v15.9425 5 table 4. differences in sperm morphological abnormalities (means ± sd) relative to techniques sperm abnormalities (%) eosin-nigrosin stain unstained p value r p value head abnormalities 4.54 ± 3.19 5.42 ± 3.52 0.58 0.85 0.01 detached head 3.17 ± 2.35 4.11 ± 3.19 0.43 0.91 0.01 mid piece abnormalities 5.13 ± 3.31 6.12 ± 3.07 0.55 0.84 0.01 proximal droplet 6.36 ± 6.10 7.20 ± 8.54 0.49 0.91 0.01 distal droplet* 4.47 ± 4.81 4.99 ± 5.12 0.73 0.94 0.01 bent tail 5.41 ± 5.08 5.03 ± 5.73 0.67 0.94 0.01 coiled tail 2.90 ± 3.10 3.21 ± 4.12 0.84 0.96 0.01 total abnormalities 25.61 ± 13.31 28.42 ± 15.52 0.31 0.91 0.01 sd: standard deviation; r: lin’s concordance correlation coefficient; significant difference if p ≤ 0.05); *not classified as sperm abnormalities. table 3. degree of inter-rater agreement among independent observers of sperm abnormalities sperm abnormalities (%) eosin-nigrosin stain unstained r p value r p value head abnormalities 0.85 0.01 0.79 0.01 detached head 0.94 0.01 0.94 0.01 mid piece abnormalities 0.84 0.01 0.76 0.01 proximal droplet 0.91 0.01 0.92 0.01 distal droplet* 0.94 0.01 0.93 0.01 bent tail 0.94 0.01 0.96 0.01 coiled tail 0.96 0.01 0.96 0.01 total abnormalities 0.91 0.01 0.92 0.01 r: lin’s concordance correlation coefficient, the degree of agreement among independent observers who assessed sperm abnormalities; significant difference if p ≤ 0.05; *not classified as sperm abnormalities and not included to calculate percentages of morphologically normal sperm. table 5. mean ± sd % abnormal sperm by triple staining (ts), eosin-nigrosin stained (ens) and unstained (uns) methods, and association among 3 staining methods sperm defect (%) ts ens uns ts versus ens ts versus uns (%) p value ts versus ens (%) p value ts versus uns r p value r p value total 22.61 ± 16.31† 25.61 ±13.31 > 0.1 28.42 ± 15.52 0.05 0.58 0.05 0.43 0.05 head§ 9.10 ± 8.23 8.44 ± 6.91 > 0.1 9.55 ± 6.67 >0.1 0.73 0.01 0.80 0.01 midpiece* 12.16 ± 9.89 11.08 ± 9.34 > 0.1 13.12 ± 11.02 >0.1 0.49 0.05 0.40 0.05 tail 9.22 ± 9.01 7.96 ± 8.11 > 0.1 9.01 ± 8.98 >0.1 0.64 >0.01 0.61 > 0.01 †damaged plasma membrane, damaged acrosome, and low mitochondrial potential (ts) versus total abnormal sperm defect (ens or uns); §damaged plasma membrane and damaged acrosome (ts) versus head defect (ens or uns); *low mitochondrial potential (ts) versus midpiece defect (ens or uns); r: lin’s concordance correlation coefficient; significant difference if p ≤ 0.05. http://dx.doi.org/10.58292/ct.v15.9425 6 citation line: clinical theriogenology 2023, 15, 9425, http://dx.doi.org/10.58292/ct.v15.9425 additionally, sperm evaluation by the triple staining method used in this study can be considered as an unbiased quantitative assessment. using fluorescence microscopy to assess organelles functions with simultaneous acquisition of brightfield microscopic images of sperm morphology can identify subtle sperm features to predict negative impacts on fertility. sperm morphology is widely regarded as the most accurate and exact assessment of fertility under field conditions. moreover, sperm morphological characteristics can be used in diagnosing male reproductive disorders and studying the effect of exposure to reproductive toxins and hazards on male fertility.25–27 hence, standardized techniques for evaluating sperm morphology including morphometry are required. sperm morphological characteristics have historically been evaluated using a variety of staining methods – papanicolaou, eosin-nigrosin, trypan blue, giemsa, and diff-quick. nevertheless, a specific method of sperm staining for various species of livestock animals is lacking. stains used for sperm morphological analysis might induce morphologic or morphometric alterations to bull sperm.17 furthermore, dyes used have differing ph and osmolarity, and lengthy procedures may affect sperm shape and size, and thus the outcome of the sperm morphological evaluation.28–30 a study17 compared bull sperm morphology evaluated by brightfield microscopy of eosin-nigrosin stained dry-mount semen smears and differential interference phase contrast microscopy of wet-mounted semen, both at 1,000× magnification. differential interference phase contrast method was more effective in visualizing major defects. in contrast, brightfield microscopy was considered to cause more minor defects. differences (p < 0.05) were generally noted for defects in acrosome, midpiece, tail, and cytoplasmic droplets but not for sperm head defects. though some studies17,18 observed differences in identifying percentage of head, mid-piece, and tail sperm defects, those defects did not differ between the 2 methods in the current study. in the current study, bulls that failed to meet the normal sperm morphology criteria set forth by the society for theriogenology (70% normal sperm) were 12.4% (151/1,216) and 12.9% (157/1,216) by the ens and uns methods, respectively. both methods identified 151 bulls producing abnormal sperm, and the uns method identified an additional 6 bulls producing abnormal sperm. correlation between the 2 methods in identifying the total percentage of abnormal sperm was very good. further correlations for identifying percentage abnormal sperm categories (percentage of head, midpiece, and tail sperm) defects were also very good. the presence of a sperm defect may not always depict an artifact; main sources of variation in sperm morphometry are not only related to the staining technique but also due to heterogeneity in the sample preparation, the classification systems used, and evaluator competency for the assessment that is necessarily subjective.16,18 therefore, it is important to homogenize semen smear preparations, reading techniques, and classifications, and stress the importance of conducting quality controls and training programs to maintain operator competency.30–34 current study observed very good agreement between interpreters. in the current study, the 2 clinicians who evaluated the sperm morphology were experienced, and 1 clinician prepared sperm morphology slides for all bulls evaluated in the study. this substantiates the importance of qualified and experienced evaluators interpreting sperm morphology. in the present study, the percentage of head, midpiece, and tail defects among the 3 staining methods (ts, ens, and uns) was the same; however, the percentage of total abnormal sperm defects was different between ts and uns methods. the detection of the percentage of acrosome defects and nuclear vacuoles by the uns and ts methods in the current study was comparable and consistent with earlier studies.17,18 comparisons between the efficiency of these methods (ts versus ens and ts versus uns methods) identified the correlations between these methods were fair to very good. an explanation for the result observed could have been due to fresh semen used for the preparation. earlier studies demonstrated the effect of cooling/freezing and the duration of storage on membrane permeability as the procedure itself could have altered sperm lipid fraction, increased membrane permeability, reduced enzyme activities, changed membrane proteins, or reduced sperm cell membrane stability.35–37 therefore, sperm morphology evaluation after cooling and postthaw processes may require a different technique. both ens and uns techniques can be used for different purposes. the stain used in the ens method is a negative differential dye that recognizes intact live and dead sperm. the uns method is simple since it includes single-step preparation, it is fast, and it is easy to learn and perform consistently by everyone, which is a primary criterion for robustness and repeatability in the results. furthermore, the changes in ph and osmolarity during storage may affect sperm’s shape and size. table 6. correlation among normal sperm morphology parameters identified by triple staining (ts), eosin-nigrosin stained, and unstained methods. triple stain sperm parameter ens uns r p r p ipiah (triple stain) 0.85 0.01 0.82 0.01 plasma membrane integrity 0.88 0.01 0.43 0.05 acrosome integrity 0.52 0.05 0.67 0.01 high mitochondrial potential 0.49 0.05 0.34 0.22 ens (eosin-nigrosin stain); uns (unstained); ipiah (intact sperm plasma membrane, intact acrosome, and high mitochondrial potential). http://dx.doi.org/10.58292/ct.v15.9425 citation line: clinical theriogenology 2023, 15, 9425, http://dx.doi.org/10.58292/ct.v15.9425 7 the triple stain method is effective because it simultaneously detects damage to plasma membrane, acrosome status, and inner mitochondrial membrane potential, but it is not possible to perform under field conditions.12 this method was used in the current study as this offered a different perspective in terms of membrane integrity comparison with the ens and uns methods. a positive correlation was observed between the sperm viability test by pi and ens methods for identifying sperm plasma membrane integrity in dogs (r = 0.88),38 boars (r = 0.71),39 and bulls (r = 0.7840 and r = 0.8339), which is consistent with the current study. percentages of sperm with normal and abnormal acrosome membranes that were identified by ts and ens methods had a positive correlation. however, the correlation between ts and uns methods was better than the correlation between ts and ens methods for identifying acrosome-reacted sperm. this supports the findings of studies16,17 in which the sperm acrosome was visualized by phase contrast. the acrosome reaction is a time-dependent phenomenon that should not take place prematurely or too late.41 premature acrosome reaction and the inability of the sperm to release the acrosomal contents in response to proper stimuli (acrosome reaction insufficiency) were associated with male infertility.42 although the cause of premature acrosome reaction is unknown, the stimulus of independent premature initiation of acrosomal exocytosis appears to be related to a perturbation of the plasma membrane integrity. in this situation, the acrosome reaction may not involve a premature activation of the receptor-mediated process but reflect an inherent fragility of the sperm membrane, leading to a receptor-independent acrosomal loss.43 in the current study, the sperm characteristics were evaluated in fresh semen, and all necessary precautions were taken to avoid premature acrosome reaction. a study44 conducted on marmoset sperm observed that ens had a marked differentiation between sperm with intact and reacted acrosomes, with and without ionophore treatment, and closely correlated to the results obtained with fitc-psa. a study45 observed no significant correlations between the ens and jc-1 methods for the determination of total sperm defects and high mitochondrial membrane potential (mmp) (r = -0.22) and between total sperm defects and low mmp (r = 0.11). similarly, correlations between head defect and high mmp (r = -0.24), head defect and low mmp (r = 0.17), tail defect and high mmp (r = -0.35), and tail defect and low mmp (r = 0.28) were not significant between the ens and jc-1 methods. it should be noted that a structurally deficient mitochondrial sheath (swollen; gaps) may structurally damage the axoneme, resulting in a wide variety of midpiece defects.46 furthermore, sperm with low mmp or midpiece defects with mitochondrial aberrations may exhibit mitochondrial dysfunction, leading to high ros production since mitochondria are the main source of sperm-produced ros via the electron transport chain.47,48 in the current study, positive correlations between the ts and ens methods for the identification of percentage of normal and abnormal sperm mitochondrial membrane defects were identified; however, no correlation was observed between the ts and uns methods. though the fluoroprobe staining methods have varying levels of correlation with conventional methods used to evaluate sperm morphology, the benefits of the detailed assessment of sperm structure and functions by fluoroprobes should be taken into consideration when advanced methods are warranted to diagnose male infertility. results support the conclusion that either ens or uns technique can be used for bull sperm morphological evaluation under field conditions. considering the ease of semen smear preparation, the uns method can be a suitable alternative to the staining method. acknowledgments authors acknowledge the beef cattle producers and the college of veterinary medicine, washington state university, pullman, wa, for their support. conflict of interest authors declare that they have no conflict of interest. authors’ contribution rachel hanson: investigation, methodology, data collection, writing – original draft; final approval. sadie reddick: investigation, data collection; image acquisition; final approval. sabrina thuerauf: investigation, data collection; image acquisition; final approval. katie webb: investigation, data collection; image acquisition; final approval. vanmathy kasimanickam: conceptualization, investigation and supervision, methodology, writing – original draft, final approval. ramanathan kasimanickam: conceptualization, methodology, formal analysis, writing – review & editing, final approval. references 1. moreno rd, ramalho-santos j, chan ek, et al: the golgi apparatus segregates from the 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poultry sci 2007;9:143–149. doi: 10.1590/ s1516-635x2007000300001 21. angrimani dsr, bicudo lc, luceno nl, et al: a triple stain method in conjunction with an in-depth screening of cryopreservation effects on post-thaw sperm in dogs. cryobiology 2022;105:56–62. doi: 10.1016/j.cryobiol.2021.12.001 22. oliveira bm, arruda rp, thomé he, et al: fertility and uterine hemodynamic in cows after artificial insemination with semen assessed by fluorescent probes. theriogenology 2014;82:767–772. doi: 10.1016/j.theriogenology.2014.06.007 23. lin li: a concordance correlation coefficient to evaluate reproducibility. biometrics 1989;45:255–268. 24. altman dg, altman e: practical statistics for medical research. 1st ed. boca raton, fl, usa: chapman & hall/crc; 1999. 25. hatamoto-zervoudakis lk, duarte júnior mf, zervoudakis jt, et al: free gossypol supplementation frequency and reproductive toxicity in young bulls. theriogenology 2018;110:153–157. doi: 10.1016/j.theriogenology.2018.01.003 26. komsky-elbaz a, kalo d, roth z: effect of aflatoxin b1 on bovine spermatozoa’s proteome and embryo’s transcriptome. reproduction 2020;160:709–723. doi: 10.1530/rep-20-0286 27. silva efsjd, missio d, martinez cs, et al: mercury at environmental relevant levels affects spermatozoa function and fertility capacity in bovine sperm. j toxicol environ health a 2019;82:268–278. doi: 10.1080/15287394.2019.1589608 28. van den hoven l, hendriks jc, verbeet jg, et al: status of sperm morphology assessment: an evaluation of methodology and clinical value. fertil steril 2015;103:53–58. doi: 10.1016/j. fertnstert.2014.09.036. 29. natali i, muratori m, sarli v, et al: scoring human sperm morphology using testsimplets and diff-quik slides. fertil steril 2013;99:1227.e2–1232.e2. doi: 10.1016/j.fertnstert.2012.11.047. 30. henkel r, schreiber g, sturmhoefel a, et al: comparison of three staining methods for the morphological evaluation of human spermatozoa. fertil steril 2008;89:449–455. doi: 10.1016/j. fertnstert.2007.03.027 31. davis ro, gravance cg: standardization of specimen preparation, staining, and sampling methods improves automated sperm-head morphometry analysis. fertil steril 1993;59:412–417. doi: 10.1016/s0015-0282(16)55686-6. 32. mallidis c, cooper tg, hellenkemper b, et al: ten years’ experience with an external quality control program for semen analysis. fertil steril 2012;98:611.e4–616.e4. doi: 10.1016/j.fertnstert.2012.05.006 33. menkveld r: sperm morphology assessment using strict (tygerberg) criteria. methods mol biol 2013;927:39–50. doi: 10.1007/978-1-62703-038-0_5 34. eustache f, auger j: inter-individual variability in the morphological assessment of human sperm: effect of the level of experience and the use of standard methods. hum reprod 2003;18:1018–1022. doi: 10.1093/humrep/deg197 35. barthelemy c, royere d, hammahah s, et al: ultrastructural changes in membranes and acrosome of human sperm during  cryopreservation. arch androl 1990;25:29–40. doi: 10.3109/01485019008987591 36. aalseth ep, saacke rg: morphological change of the acrosome on motile bovine spermatozoa due to storage at 4 degrees c. j reprod fertil 1985;74:473–478. doi: 10.1530/jrf.0.0740473 37. kasimanickam r, kasimanickam v, pelzer kd, et al: effect of breed and sperm concentration on the changes in structural, functional and motility parameters of ram-lamb spermatozoa during storage at 4 degrees c. anim reprod sci 2007;101:60–73. doi: 10.1016/j.anireprosci.2006.09.001 38. graham jk, kunze e, hammerstedt rh: analysis of sperm cell viability, acrosomal integrity, and mitochondrial function using flow cytometry. biol reprod 1990;43:55–64. doi: 10.1095/ biolreprod43.1.55. http://dx.doi.org/10.58292/ct.v15.9425 https://doi.org/10.1016/s0015-0282(16)57981-3 https://doi.org/10.1073/pnas.2001754117 https://doi.org/10.1038/aja.2008.58 https://doi.org/10.1016/j.theriogenology.​2008.04.049 https://doi.org/10.1016/j.theriogenology.​2008.04.049 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https://doi.org/10.1016/j.fertnstert.2007.03.027 https://doi.org/10.1016/s0015-0282(16)55686-6. https://doi.org/10.1016/j.fertnstert.2012.05.006 https://doi.org/10.1007/978-1-62703-038-0_5 https://doi.org/10.1093/humrep/deg197 https://doi.org/10.3109/01485019008987591 https://doi.org/10.1530/jrf.0.0740473 https://doi.org/10.1016/j.anireprosci.2006.09.001 https://doi.org/10.1095/biolreprod43.1.55. https://doi.org/10.1095/biolreprod43.1.55. citation line: clinical theriogenology 2023, 15, 9425, http://dx.doi.org/10.58292/ct.v15.9425 9 39. peña ai, quintela la, herradon pg: flow cytometric assessment of acrosomal status and viability of dog spermatozoa. reprod dom anim 1999;34:495–502. 40. pintado b, de la fuente j, roldan er: permeability of boar and bull spermatozoa to the nucleic acid stains propidium iodide or hoechst 33258, or to eosin: accuracy in the assessment of cell viability. j reprod fertil 2000;118:145–152. 41. cummins jm, fleming ad, crozet n, et al: labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (mb): immobilization upon exposure to ultraviolet light and analysis of acrosomal status. j exp zool 1986;237:375–382. doi: 10.1002/jez.1402370310 42. tesarik j, mendoza c: alleviation of acrosome reaction prematurity by sperm treatment with egg yolk. fertil steril 1995;63:153–157. doi: 10.1016/s0015-0282(16)57311-7 43. esteves sc, sharma rk, thomas aj jr, et al: effect of in vitro incubation on spontaneous acrosome reaction in fresh and cryopreserved human spermatozoa. int j fertil womens med 1998;43:235–242. 44. valle rr, valle cm, nichi m, et al: validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (callithrix jacchus) sperm. theriogenology 2008;70:115–120. doi: 10.1016/j.theriogenology. 2008.03.011 45. bruinjé tc, ponce-barajas p, a. dourey a, et al: morphology, membrane integrity, and mitochondrial function in sperm of crossbred beef bulls selected for residual feed intake. can j anim sci 2019;99:456–464. doi: 10.1139/cjas-2018-0103 46. veeramachaneni r: spermatozoal morphology. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, oxford; wiley: 2011:1297–1307. 47. koppers aj, de iuliis gn, finnie jm, et al: significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. j clin endocrinol metab 2008; 93:3199–3207. doi: 10.1210/jc.2007-2616 48. amaral a, lourenço b, marques m, et al: mitochondria functionality and sperm quality. reproduction 2013;146:r163–r174. doi: 10.1530/rep-13-0178 http://dx.doi.org/10.58292/ct.v15.9425 https://doi.org/10.1002/jez.1402370310 https://doi.org/10.1016/s0015-0282(16)57311-7 https://doi.org/10.1016/j.theriogenology.​2008.03.011 https://doi.org/10.1016/j.theriogenology.​2008.03.011 https://doi.org/10.1139/cjas-2018-0103 https://doi.org/10.1210/jc.2007-2616 https://doi.org/10.1530/rep-13-0178 introduction when confronted with a case of feline infertility, male or female, a complete history including vaccination and disease status is essential. many issues can be contributed to improper management (see next section on history). a good general physical examination (observing for comorbidities that could affect reproduction) is performed next. body condition of the cat, overall appearance, grooming, and attitude should be observed in both males and females. in the male, special attention should be placed on examining the mouth and gums. mouth pain (e.g., gingivitis or tooth pain) can affect the ability of the male to bite the scruff of the female when mating and therefore hinder the breeding attempt. any back or rear limb pain in the male can also affect the breeding process or his willingness to mate. an external reproductive examination should follow the general physical examination and will be discussed later for the female and male separately. blood analyses (e.g., complete blood count, serum chemistry, and possibly hormonal testing) may be indicated depending on the findings of the physical and reproductive examinations. ultrasonography should be performed to evaluate both testes in the male, and uterus and ovaries in the female. sedation may be required depending on the cooperative behavior of the patient. if external or internal reproductive abnormalities are present, or if there is no other obvious cause for the infertility, a karyotype of one or both members of the breeding pair may be necessary to confirm whether the genetic makeup of the animal is normal. history specific questions to gain a baseline knowledge of the cattery management include.1 clinical approach to infertility in the cat aime johnson auburn university college of veterinary medicine auburn, al abstract causes of infertility in the cat are often multifactorial. cattery management should be addressed prior to evaluation of individual animals. factors such as housing/lighting, preventive care, and disease status of the colony should be evaluated. a complete physical examination is vital to rule in or out anatomical abnormalities leading to infertility. in the tom and queen, good breeding management is vital for success. in queens, infertility can be caused by persistent receptivity/estrous behavior, refusal of the male, and ovulation without pregnancy. when evaluating a tom, failure to mate or failure to achieve pregnancies are the most common categories. a detailed history and complete breeding soundness examination is important to differentiate and identify potential causes of infertility. keywords: cat, infertility, queen, tom • how many animals are housed in the facility (male and female, breeding and nonbreeding)? • what are their ages? • what is the source of each animal and how long has each been with the colony? • what is the diet, water source, and preventative health policy? • what is the supplemental lighting program? • what is the current disinfection policy for floors, litterboxes, bowls, etc.? • what is the procedure for bringing male and female together? • what is the procedure for pregnancy confirmation? • where are pregnant queens housed during pregnancy, delivery, and lactation? • what bedding or boxes are provided to the animals at each stage (breeding, pregnancy, queening, and lactation)? • is breeding or queening observed, or does it occur with no one present? • how are kittens managed from birth to weaning? • can a detailed breeding and queening report for each animal be provided? • what are the specific issues the cattery is experiencing? additional questions may be necessary depending on the answers received. cattery management vaccination status of all animals in the colony or household should be evaluated to ensure adequate disease control. although a closed colony is preferable with no new animals entering, such a policy may also limit the introduction of new genetics. a quarantine period with adequate testing procedures should be mandatory for all new additions. a quarantine process should also be in place for any cats that leave and return to the colony for breeding, exhibition, etc. cats in different stages should be housed separately. breeding animals, pregnant, and nursing mothers should be kept away from all other cats. late pregnant and lactating mothers should be kept in a quiet comfortable space to reduce stress and minimize stress-induced cannibalization of their young. in catteries, strict sanitation and adequate ventilation are essential to reduce or eliminate disease. poor sanitation often leads to clinical signs of disease and neonatal mortality from sepsis. standard operating procedures should be followed to prevent hygiene errors. litter boxes, bedding, toys, food, and water bowls should be cleaned and disinfected regularly. frequency will depend on the density of the cattery, but in general, cleaning should be performed daily with complete disinfectant done at least weekly. caution is needed to ensure that the disinfectant used is at an appropriate concentration, is sufficient for the major pathogens, and is safe for cats. readers are encouraged to consult guidelines2 for appropriate disinfectants for a variety of pathogens. lighting the queen is a long day breeder and cycles when the day length increases (february october in the northern hemisphere). during shorter days of the year, cyclicity ceases and queens enter an anestrous stage. toms have an overall decrease in hormone production and sperm quality during the nonbreeding season; however, it is not substantial enough to affect fertility.3 when exposed to artificial lighting for 12 14 hours per day, cats will continue to maintain cyclicity. most catteries use a 14:10 hours light:dark schedule. it is noteworthy that in this author’s experience, even cats maintained under this lighting system may experience a slight decrease in pregnancy rates during the shorter days of the year. a queen will typically start cyclicity within 1 2 months after initiating an artificial lighting period. if cats are exposed to light 24 hours a day, cyclicity will be maintained; however, the number of ‘heat cycles’ was reduced from 2 per month to only 1.4 if a group of breeding animals has been maintained under long-term artificial lighting (years) and the overall fertility has declined, allowing the queen to experience a season of anestrus by shortening the lighting period (8 hours of light) for 2 3 months may be beneficial before restarting the extended day lighting. cats housed in a home setting are often exposed to a combination of natural and artificial light. however, animals have the freedom to hide under objects or in dark rooms so the light they are exposed to is variable. because of this, queens in a house setting will often display seasonality and not exhibit cyclicity during the short daylight period. queen causes of infertility in the queen are often classified into broad categories and this discussion will focus on those causes that fail to produce a pregnancy despite appropriate breeding management. this discussion will not cover early pregnancy loss that may appear as infertility and must be differentiated from true infertility. persistent receptivity/estrous behavior, refusal of the male, and ovulation without pregnancy are 3 main categories to explore. a good history can determine which category applies to the patient. persistent estrus/receptivity persistent estrus and/or receptivity can be identified when a queen either continually displays behavioral signs of estrus, vaginal cytology is > 70% cornified, or she accepts the male continuously. persistent estrus may appear to occur in queens that naturally display a shorter interestrus period (siamese) and owners reporting that she is ‘in heat all the time’. daily records of behavior by the owner and a vaginal cytology every 2 3 days would determine if the queen is exhibiting normal cyclicity. in a natural breeding situation where the queen is presented to the male, queens that exhibit persistent receptivity are likely failing to ovulate. failure to ovulate can be suspected based on a history of a female exposed to the male, may or may not have been mated, refuse the male only to begin estrous behavior again 8 10 days later. in short, the queen is having normal cyclicity. ovulation failure may result from lack of adequate stimulation to produce lh surge or a primary ovarian dysfunction (e.g., ovarian follicular cysts). evaluation of the breeding records, number of witnessed matings, and presence or absence of the ‘after reaction’ of the queen following coitus will assist in assessing if adequate stimulation has occurred. the ‘after reaction’ follows intromission and is identified when the female rolls on her back, rubs her face on objects, and licks her perineum. this reaction lasts several minutes while the male usually watches from a distance. a survey of purebred catteries in sweden determined that only 35% of matings occurred when owners were present.5 wireless cameras are a helpful tool to evaluate matings that occurred unobserved. number and timing of matings that occur when queen is placed with the male will affect ovulation success. only 1 of 12 cats ovulated with a single mating on day 1 of estrus, whereas 4 of 12 ovulated after a single mating on day 4 of estrus.6 when 3 matings on a single day were allowed, 10 of 12 cats ovulated on days 1, 2, and 3 of the estrus period.6 it is generally recommended that 4 or more matings are needed to provide sufficient stimulation and lh release for ovulation to occur. confirmation of ovulation can be made by evaluating serum progesterone concentrations one week after observed matings. if progesterone concentrations are <less than2 ng/ ml, ovulation did not occur and the queen should be back in estrus within a few days. if there is inadequate copulatory stimulation, ovulation may be induced pharmacologically and followed by either natural mating or artificial insemination. a second cause of persistent estrus is ovarian (follicular) cysts. ovarian cysts are fluid filled structures producing estrogens that lead to prolonged expression of estrus. incidence of ovarian cysts increases with age. presence of cysts is best confirmed by transabdominal ultrasonography.7 follicular structures on the ovary measuring greater than 5 mm in diameter are suspected to be cystic as normal preovulatory follicles measure 2 3 mm in diameter. follicular cysts can be as large as 1 2 cm in diameter. treatment options include attempts to induce luteinization of the cyst(s) by either 500 iu hcg or 25 µg gnrh given intramuscularly. surgical aspiration or ablation of the cystic structure through laparotomy or laparoscopically have also been reported, with a return to fertility.8 granulosa cell tumors have been reported in the cat and may present as persistent estrus. on ultrasonography, they appear as a mixed echogenic mass. determination of serum anti-müllerian hormone concentrations can be helpful in determining the presence of a granulosa cell tumors. some overly aggressive males will continuously breed females even after they ovulate. this happens more often when the queen is submissive. often, the males that overbreed will leave a breeding injury/wound on the back of the queen’s neck from the excessive biting. if this is observed, the queen should be removed from the male. pregnancy can be checked around 30 days later, or serum progesterone can be evaluated one week later to confirm ovulation. refusal of a male when introducing a breeding pair, it is more appropriate to take the queen to the tom, as toms are very territorial. however, if the queen is shy or overly aggressive, she may not allow mating to occur either by fighting or laying on her back when the male approaches. confirming that the queen is in estrus may make introduction easier. vaginal cytology will have at least 60% cornified epithelium with small dark nuclei; however, intermediate cells may remain visible and total cornification may not reach 100% as in the dog. the nucleus of the cells become pyknotic, but typically will not disappear completely. if a queen is in estrus but refusing the male, there may be an incompatibility between the pair. queens in confirmed estrus accepted the male only 39% (15/38) of times.9 some queens will be more receptive to the male on day 3 of estrus. other conditions in the female that may lead to rejection of coitus are vaginal anomalies making breeding excessively painful. although queens are induced ovulators, spontaneous ovulation occurs more frequently than originally believed. incidence of spontaneous ovulations in a group of 15 young queens was 87% over 4.5 months.10 when a tom was added to the females’ room in a separate cage, the incidence of spontaneous ovulations over 10 days increased from 0 22% to 33 57%. the study concluded that queens housed together can spontaneously ovulate and that frequency increased in the presence of a male. if a female continues to refuse a male, serum progesterone concentrations should be assessed to determine if luteal tissue is present. if progesterone concentrations are > 2 ng/ml, luteal tissue is present. with pairs that are not compatible, artificial insemination should be performed. ovulation induction should be followed by semen collection and insemination 24 30 hours later. if spontaneous ovulation has occurred, the nonpregnant luteal phase lasts 35-40 days. queens should be monitored every 1 2 weeks utilizing vaginal cytology, behavior, or progesterone assay to determine when the luteal phase has ended. reintroduction to the male can occur after this period. ovulation without pregnancy queens that are mated successfully but fail to become pregnant will undergo a ‘pseudopregnancy’ stage. this is defined as a shortened luteal phase lasting 35 40 days instead of the full length of pregnancy. following mating, ovulation should be confirmed by testing the queen’s serum progesterone (> 2 ng/ml) concentrations. pregnancy diagnosis can be performed via ultrasonography ~ 30 days after mating. successful mating without pregnancy can be due to either the male or female. a full breeding soundness examination with semen analysis should be performed on the male. causes in the female are usually due to uterine pathology. a common primary uterine problem observed in the queen is cystic endometrial hyperplasia (ceh) with or without concurrent pyometra. in the author’s research cattery, ~ 75% of queens diagnosed with infertility (failure to produce offspring following appropriately managed breeding[s]) had histological evidence of ceh within their uterus. clinical signs in this ceh affected group of queens were first noted at 3 4 years of age (decreased litter size or failure to produce kittens consistently). intact female cats over 5 years of age have a greater risk of developing clinical disease associated with ceh/pyometra.11 an 88% incidence of ceh was observed in cats > 5 years old compared to 30% in cats aged 2 4, with a much greater incidence of the disease in colony raised cats compared to feral cats.12 diagnosis is suspected based on history of reduced fertility. a thickened uterine wall with prominent endometrial folds and small cystic areas within the uterus may be observed on ultrasound. a uterine biopsy provides definitive diagnosis and may be required for less severe cases. currently, uterine biopsy is best obtained surgically. if ceh is severe, prognosis for fertility is guarded and ovariohysterectomy is recommended. pyometra often occurs secondary to ceh and may result in ovariohysterectomy (preferred treatment for older queens not needed for future breeding). however, unlike observed in the bitch, queens are often not systemically ill and the only clinical sign may be a purulent vaginal discharge. treatment with a fluroquinolone, native prostaglandin, and cabergoline can provide appropriate successful medical treatment. medical management of pyometra in the queen should be performed on a case-by-case basis. underlying ceh cannot be corrected. subclinical endometritis may also cause infertility in the cat. it has become a leading cause of infertility in the bitch. among biopsy samples from 399 intact bitches, the most common lesion observed was endometritis (42.6%).13 although there is less information describing this lesion in the queen, it is likely that this pathology is also a substantial cause of infertility in this species. miscellaneous causes there are countless other potential causes of infertility or early pregnancy loss in the queen. infectious (bacterial and viral) should be ruled out with serology and culture. vaginal culture may be obtained but should be interpreted with caution due to high likelihood of contamination when swabbing the caudal vaginal canal. a cranial vaginal sample would be ideal; however, when in estrus, the queen’s cranial vagina measures only 1 2 mm in diameter when compared to 4 mm of the caudal vagina, making sampling difficult.14 if a pure culture is obtained, treatment would be indicated based on the susceptibility pattern. pure or heavy growth of ß-hemolytic streptococci or escherichia coli has been associated with endometritis and pyometra. mycoplasma sp., ureaplasma sp., coxiella burnetti, and chlamydia sp. have all been implicated in infertility. a pcr test is the most sensitive assay for these bacteria and diagnosis should be based on results and clinical signs. treatment with antibiotics should be reserved for animals exhibiting clinical signs and based on susceptibility of the positive culture. tom when evaluating infertility in a pair, the tom is the easier to evaluate. a complete breeding soundness examination should be performed starting with history, general physical examination, and external reproductive examination. puberty occurs between 7 12 months. prior to puberty, adhesion of the balanopreputial fold prevents full exposure of the penis. breakdown is androgen dependent and therefore makes this an initial reference point for pre versus post pubertal animals.15 androgen dependent penile spines are present on the proximal ⅔ of penis indicating the presence of circulating testosterone. testes should be located within the scrotum and be freely movable. scrotal skin should be free from dermatitis or wounds. both testes should palpate slightly firm and of similar size. ultrasonography of the testes should be performed after palpation. testes should be round to slightly oval. literature relating size of the testes to fertility is lacking in the tom but can be inferred based on other species. average daily sperm output for a standard tom has been estimated at 32 x 106.16 via ultrasonography, length, width, and height of each testis should be measured and the testicular volume (tv) calculated using the formula for an ellipse simplified to tv = 0.2533 x length x width x height). the process for placement of the ultrasound probe for measuring has been described.17 these measurements are most helpful when evaluating testicular size in a single tom over time rather than a comparison among males. semen collection in the tom is more difficult than other domestic species. collection using a home-made artificial vagina was first reported in the 1970s.18 in a clinic setting, collection using an artificial vagina is not practical unless the tom has been previously trained. the most effective method of semen collection is electroejaculation. the process this author uses has been described.19 although electroejaculation is the most consistent in obtaining sperm from the tom, it requires specialized equipment that may not be available in all clinics. the most practical method of obtaining a semen sample in the clinic setting is urethral catheterization after pharmacological induction. this technique was described using a high dose of medetomidine (120 μg/kg).20 since medetomidine is no longer available in the us, dexmedetomidine can be used at a comparable dose (60 μg/kg). lower doses of both medications were studied (50 μg/kg medetomidine and 25 μg/kg dexmedetomidine) but the sedation level and ejaculate collection were inadequate.21 once full sedation is achieved (cat in lateral and unresponsive), an open ended tomcat catheter should be inserted into the urethra 8 9 cm. measuring and marking the catheter prior to insertion will assist with proper placement. care must be taken not to enter the bladder. a single catheterization inserted and immediately removed following full sedation was preferrable to multiple catheterization attempts and provided an adequate sperm sample without damaging the urethra.22 the sample can be flushed from the catheter using an appropriate extender. this author prefers making extender using ham’s f-10 with 25 mm hepes, 1mm pyruvate and glutamine, and 5% fetal bovine serum. an alternative that is commercially available is test yolk buffer (refrigeration media, irving scientific). however, analysis of the motion characteristics may be difficult due to the interface egg yolk droplets if the extender is not filtered prior to use. once the sample is obtained, a complete sperm analysis should be performed including motility, morphology, and cytology to evaluate for inflammatory cells. causes of infertility in the male can be classified into unable/ refusal to mate and mating but not achieving pregnancies. if the male displays reduced libido, it could be due to stress, an overly aggressive female, or systemic disease. apart from history and physical examination, routine blood work should be performed. female should be removed from the male to allow the male to rest. a second female or the same female after confirming that she is in estrus can be returned several days later. if male is refusing to breed a female, physical limitations should be addressed (e.g. sore back or mouth). primary penile problems (e.g. persistent frenulum, hair ring, priapism, or paraphimosis) should also be evaluated. observation of the interaction between the male and female is ideal to assess behavioral problems with the male alone, or the pair. failure to produce litters for toms that are breeding naturally, inducing ovulation, but not achieving pregnancies, a semen analysis is vital. teratospermia, defined as > 40% abnormal sperm, is observed in small populations of cats where inbreeding or excessive line breeding has occurred.23 a single generation of inbreeding (offspring bred to parent) produced male offspring with < 15% morphologically normal sperm compared to 55% morphologically normal sperm in control animals, indicating that loss of genetic diversity leads to increased teratospermia in as short as 1 generation.23 unlike in other species, many teratospermic ejaculates in the tom demonstrate adequate motility (> 70%), in spite of low numbers of morphologically normal sperm. because the tom breeds multiple times a day, even toms that are classified as teratospermic may have normal fertility. azoospermia is the complete absence of sperm in the ejaculate. to differentiate this from ejaculation failure, seminal plasma alkaline phosphatase concentrations can be determined. a higher alkaline phosphatase (typically > 5,000 iu/l) concentration confirms that the sample has originated from the epididymis and a complete ejaculate was obtained. if azoospermia is confirmed, prognosis for return to fertility is poor unless a treatable underlaying disease is diagnosed. the tom should have a second semen evaluation 2 3 months later to detect improvement. culture of the ejaculate may be indicated if inflammatory cells are identified in the ejaculate. a pure culture should be a cause for concern and should be treated based on susceptibility. conclusion infertility in the queen and tom can be multifactorial. history and examination can help to prepare a differential list that leads to diagnosis of the specific problem. treatment varies and should be directed at the specific cause, if identified. conflict of interest none to declare. references 1. johnson ak: normal feline reproduction: the queen. j feline med surg 2022;24:204-211. 2. stull jw, bjorvik e, bub j, et al: 2018 aaha infection control, prevention, and biosecurity guidelines. j am anim hosp assoc 2018;54:297-326. 3. tsutsui t, onodera f, oba h, et al: plasma hormone levels and semen quality in male cats during non-breeding and breeding seasons. reprod domest anim 2009;44(suppl 2):291-293. 4. leyva h, madley t, stabenfeldt gh: effect of light manipulation on ovarian activity and melatonin and prolactin secretion in the domestic cat. j reprod fertil suppl 1989;39:125-133. 5. strom holst b, frossling j: the swedish breeding cat: population description, infectious diseases and reproductive performance evaluated by a questionnaire. j feline med surg 2009;11:793-802. 6. wildt de, seager sw, chakraborty pk: effect of copulatory stimuli on incidence of ovulation and on serum luteinizing hormone in the cat. endocrinology 1980;107:1212-1217. 7. davidson ap, baker tw: reproductive ultrasound of the bitch and queen. top companion anim med 2009;24:55-63. 8. johnston sd rkm, olson ps: canine and feline theriogenology. philadelphia, pa; saunders: 2001. 9. root mv, johnston sd, olson pn: estrous length, pregnancy rate, gestation and parturition lengths, litter size, and juvenile mortality in the domestic cat. j am anim hosp assoc 1995;31:429-433. 10. gudermuth df, newton l, daels p, et al: incidence of spontaneous ovulation in young, group-housed cats based on serum and faecal concentrations of progesterone. j reprod fertil 1997;51(suppl):177-184. 11. potter k, hancock dh, gallina am: clinical and pathologic features of endometrial hyperplasia, pyometra, and endometritis in cats: 79 cases (1980-1985). j am vet med assoc 1991;198:1427-1431. 12. perez jf, conley aj, dieter ja, et al: studies on the origin of ovarian interstitial tissue and the incidence of endometrial hyperplasia in domestic and feral cats. gen comp endocrinol 1999;116:10-20. 13. gifford at, scarlett jm, schlafer dh: histopathologic findings in uterine biopsy samples from subfertile bitches: 399 cases (19902005). j am vet med assoc 2014;244:180-186. 14. zambelli d, cunto m: vaginal and cervical modifications during the estrus cycle in the domestic cat. theriogenology 2005;64:679-684. 15. margaret v. root sdj, gary r. johnston, patricia n. olson: the effect of prepubertal and postpubertal gonadectomy on penile extrusion and urethral diameter in the domestic cat. vet radiol ultrasound 1996;37:363-366. 16. franca lr, godinho cl: testis morphometry, seminiferous epithelium cycle length, and daily sperm production in domestic cats (felis catus). biol reprod 2003;68:1554-1561. 17. johnson ak: normal feline reproduction: the tom. j feline med surg 2022;24:212-220. 18. sojka nj, jennings ll, hamner ce: artificial insemination in the cat (felis catus l.). lab anim care 1970;20:198-204. 19. johnson ak: evaluation in the tom: collection procedures, evaluation of sperm, and subsequent use. clinical theriogenology 2014;3:219-223. 20. zambelli d, prati f, cunto m, et al: quality and in vitro fertilizing ability of cryopreserved cat spermatozoa obtained by urethral catheterization after medetomidine administration. theriogenology 2008;69:485-490. 21. cunto m age, ballotta g, and zambelli d: effect of medetomidine and dexmedetomidine administration at different dosages on cat semen quality using urethral catheterization after pharmacological induction (ur.ca.p.i.). reproduction in domestic animals 2019;54:96-97. 22. cunto m, kuster dg, bini c, et al: influence of different protocols of urethral catheterization after pharmacological induction (ur. ca.p.i.) on semen quality in the domestic cat. reprod domest anim 2015;50:999-1002. 23. pukazhenthi bs, neubauer k, jewgenow k, et al: the impact and potential etiology of teratospermia in the domestic cat and its wild relatives. theriogenology 2006;66:112-121. approach 1 contact jessica looman jlooman@ttu.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 9801, http://dx.doi.org/10.58292/ct.v16.9801 research report effects of bovine ova density and culture supplements on cleavage and blastocyst development rates of in vitro embryos jessica looman,a zowie rodriguez,b lyric waugh,a sydney hickerson,a john gibbonsa atexas tech school of veterinary medicine, amarillo, tx, usa beastern new mexico university, portales, nm, usa abstract an optimized in vitro culture system is important to maximize bovine blastocyst development. four experiments were conducted to explore manipulation of culture environments and investigate the benefits of culturing embryos in groups. follicles were aspirated from abattoir-derived ovaries and selected oocytes were matured, fertilized, vortexed, and randomly placed in culture groups. experiment 1 evaluated the effects of ova culture density on blastocyst development among embryos cultured individually or in groups of 5, 10, 20, or 50 embryos. experiment 2 evaluated the effects of conditioned media from previous replicates on embryo development of the current culture. in experiment 3, cleaved embryos were grouped together after being in culture for 24 hours. lastly, in experiment 4, grouped embryos were placed in 10 μl culture drops to evaluate the effect of high density/low media volume on development. cleavage and blastocyst rates analyzed via chi-square indicated that although cleavage rates were similar among culture groups (and experiments), blastocyst development was lower (p < 0.05) in 1 embryo culture group compared to other groups. when conditioned culture media from previous replicates were added to original culture media, blastocyst development was similar among original and conditioned culture groups of 20 and 50 embryos. when cleaved embryos were amalgamated and cultured, blastocyst development was higher (p < 0.05) in culture groups of 10 than 1 cleaved embryo groups but similar to controls for both culture groups. when embryos were cultured in 10 μl drops, embryos cultured in groups of 5 had lower development to blastocysts compared to groups of 2, 10, 25, and control. in conclusion, these data indicated an apparent ‘helper effect’ expressed in culture environments of groups, and this effect apparently occurred after cleavage but before blastocyst development. direct or indirect role(s) that additional cells have on in vitro culture and the mechanism of this helper effect requires further investigation. keywords: single embryo culture, group embryo culture, cleavage, blastocyst introduction in vitro fertilization (ivf) is an important tool in the cattle industry. it enables producers to potentially improve reproductive performance, efficiency, and genetic gains while maintaining their financial goals. according to 2020 data reported by practitioners in the international embryo technology society, usa in vitro embryo production surpassed the number of in vivo produced embryos from multiple ovulation embryo transfer in 2016.1 with the embrace of this technology by the industry, comes a necessity to improve and advance the techniques to increase blastocyst developmental rates. achievement of high maturation and cleavage rates but low development to blastocysts could be due to incomplete cytoplasmic oocyte maturation and inadequate culture conditions, leading to impaired embryonic genome activation, or dna fragmentation.1 embryo culture is an important component in ivf and suboptimal conditions can compromise embryo development. absence of maternal signaling cues in in vitro media leads to metabolic dysfunction for embryos.2 with increasing demand for atp for expansion and proliferation, it is vital for embryos to absorb available nutrients and avoid potential toxins from external environment.2 still, there is much work to be done to optimize culture systems for bovine embryos. a positive relationship was observed among bovine embryos cultured together in groups improving development rates.3–7 murine embryos cultured in groups also performed better than mailto:jlooman@ttu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.9801 2 citation line: clinical theriogenology 2024, 16, 9801, http://dx.doi.org/10.58292/ct.v16.9801 embryos cultured alone8–11 and cooperative effect has also been witnessed in human in vitro produced embryos.12–15 however, interactions of embryos among each other are not fully understood. growth factors secreted by embryos were suggested to act in an autocrine fashion to stimulate development of other embryos in the same culture. embryonic microrna also were observed in the successful culture media of bovine embryos further suggesting that beneficial factors are shared among embryos in the same environment.16 others have concern that embryos cultured in groups may be exposed to negative effects of dying or delayed embryos and favor 1 embryo culture.17 however, it was speculated that in 1 embryo culture, beneficial factors would be too dilute to be effective and may need to be supplemented into the media.8 embryo density of a culture environment may affect the impacts of autocrine or paracrine interactions between embryos. a universally accepted ideal embryo density has not been established.18 embryo density is dependent on the culture drop volume and the number of cultured embryos; alteration of either can change embryo density. utilization of the culture media and its effectiveness is affected by embryo density; nutrients in the culture media are absorbed and utilized by embryos. toxic metabolites and secreted waste accumulate in the culture environment; however, various growth-promoting factors are secreted by embryos. appropriate balance between having sufficient nutrients for embryos, reducing concentrations of toxic metabolites, and having the appropriate dilution and uptake to benefit from secreted growth-promoting factors remains elusive.18 practically, in many cases number of ova that can be grouped together is dependent on the recovery from the female donor or sire selection. it is challenging when embryos need to maintain their identity and therefore cannot be pooled together with other embryos. in order to improve 1 embryo culture development, a better understanding of embryo interactions should be evaluated. four experiments were conducted to further elucidate how group culture and supplements benefit embryo development. specific aspects of group culture were spent embryo media, embryo density, and timing of the helper effect. at present, there is limited research on reutilization of spent media in the new media for embryo culture. furthermore, previous culture density studies had mixed results. objectives were to: 1. determine the optimal number of presumptive embryos per culture environment, 2. evaluate effects of conditioned media on blastocyst development, 3. investigate the stage of development at which the effect impacts blastocyst development and determine the effect of noncleaved embryos on developing embryos, and 4. examine the effect of embryo density on embryo cleavage and development. materials and methods oocyte collection and maturation all wash, maturation, fertilization, and culture media were supplied by ivf limited t/a ivf bioscience (cornwall, uk). bovine ovaries were obtained from abattoir (caviness beef packers, hereford, tx) and transported within 1 hour to laboratory at room temperature suspended in physiological saline solution. cumulus-oocyte-complexes were aspirated from 2-8 mm follicles using an 18-gauge needle and a vacuum pump (18-20 ml/minute) at a pressure of 100 mm hg. selected oocytes with compact cumulus cells and homogeneous cytoplasm were washed twice in wash media. groups of 10 cumulus-oocyte-complexes were placed in 50 μl drops of maturation media and incubated for 18-20 hours under a 10 ml oil overlay at 38.5°c in a fully humidified (5% co2, 5% o2, and 90% nitrogen) environment. sperm preparation and in vitro fertilization matured oocytes were washed twice in wash media and once in fertilization media. they were then placed in groups of 50 oocytes per well in 4-well plates containing 500 μl of fertilization media per well with a 400 μl oil overlay. sperm from 1 bull were obtained by centrifugation of frozen-thawed semen. prior to use in these experiments, the selected bull semen was fully characterized for motility, morphology, and concentration for in vitro fertilization. bovipure (bp-100) and bovidilute (bd-100) were obtained from nicadon laboratory (nidacon, mölndal, sweden). bovipure solution was diluted with bovidilute solution to obtain a bottom layer medium at 80% concentration and top layer medium at 40%. bovipure density gradient centrifugation columns were prepared in 15 ml conical tubes with 2 ml of 40% media layered over 2 ml of 80% media. frozen semen straws were placed in a water bath at 37°c for 30 seconds and semen was gently layered on top of bovipure top media. columns were centrifuged at 1,507 x g for 13 minutes. after centrifugation, fluid above the sperm pellet was carefully removed and 7 μl sperm pellet was added to each fertilization well (~ 500,000 sperm/ml final concentration). oocytes were co-incubated with semen in fertilizations well for 18-20 hours at 38.5°c in a fully humidified (5% co2, 5% o2, 90% nitrogen) environment. in vitro culture after fertilization, cumulus cells were removed from presumptive zygotes by vortexing in 250 μl of wash media for 2 minutes and 25 seconds. presumptive zygotes were randomly allocated to each group and placed in culture drops on a 60 x 15 mm cell culture dish under a 10 ml oil overlay. in experiment 1, to examine the effect of number of embryos per 50 μl drop of culture media, presumptive zygotes were randomly allocated to groups of 1, 5, 10, 20, or 50 presumptive zygotes per 50 μl drop of culture media. experiment 1 was replicated thrice using oocytes matured on the same day to remove any batch effect of oocytes. in experiment 2, to examine the effect of conditioned media, 25 μl of conditioned media was removed from previous replicates and added to 25 μl of fresh culture media to create a 50% concentrated 50 μl drop of conditioned media. conditioned media was pipetted directly out of a previous 7-day old culture drop and directly deposited into a drop of fresh culture media. presumptive zygotes were than randomly allocated to control or conditioned culture media drops in groups of 20 and 50 embryos per drop. experiment 2 was replicated twice using oocytes matured on the same day for each replicate. in experiment 3, to examine the effect of cleaved and noncleaved embryos in groups, presumptive zygotes were placed in culture in groups of 10. after 24 hours, embryos were evaluated and embryos that had cleaved were amalgamated into new groups of 10 or 1. in experiment 4, to create a high-density culture environment, embryos were place in 10 μl culture drops in groups of 2, 5, 10, and 25 embryos per drop. a control group of 10 embryos in 50 μl drops were also included. experiment 4 was replicated 6 times using oocytes matured on the same day for each replicate. data analyses contingency tables were constructed for cleavage outcomes for experiment 1 and 4 and blastocyst development outcomes http://dx.doi.org/10.58292/ct.v16.9801 citation line: clinical theriogenology 2024, 16, 9801, http://dx.doi.org/10.58292/ct.v16.9801 3 for experiments 1-4. cleavage and development to blastocysts were evaluated on the 7th or 8th day of culture. embryo grown alone was the observational unit with a binary outcome recorded (achieved developmental stage or not). data were analyzed via chi-square. pairwise comparison was performed using a 2-proportion z-tests. significance level was set at p < 0.05. in order to confirm that there were no differences in development from batches of oocytes on different days, cleavage and blastocyst development outcomes were regressed as a function of culture group and batch of ovaries using a logistical regression model. batches were not statistically different at predicting development of cleavage or blastocysts in the logistic regression model. because no batch effect was detected, a chi-square was performed to assess treatment differences. sample size calculations were performed for medium effect size and all sample sizes were adequate for 80% power and 0.05 significance level. all analyses were performed on r software, version 3.1. results in experiment 1 (table 1), although cleavage rates were similar among groups, embryos in groups of at least 5 had higher (p < 0.05) blastocyst development than 1 embryo cultured alone but there was no developmental benefit of more ova (5, 10, 20, or 50/culture). in experiment 2 (table 2), there was no significant difference in blastocyst development among groups that were cultured in conditioned media compared to original culture media. in experiment 3 (table 3), there was no significant difference in development rates among selected, cleaved embryos, and controls. again, control and original embryos in groups of 10 had higher (p < 0.05) developmental rates than 1 embryo cultured alone. in experiment 4 (table 4), cleavage rates were similar among groups. the group of 5 embryos per 10 μl drop had significantly lower blastocyst development than the groups of 2, 10, 25, and control. in all 4 experiments, there were no significant differences among batches of oocytes. discussion in experiment 1, embryos grouped in 5 or more had significantly higher blastocyst development but similar cleavage compared to embryos cultured alone that provided a baseline to initiate further exploration of group culture and helper effect concept. in groups of 5 or higher, various aspects (secreted factors in spent media, embryo density, and timing of group interaction) might have contributed to increased development of blastocysts. embryo density influences substances in the media and the quantity of factors that may be secreted or depleted by the embryos.19 similar results were reported when number of embryos were compared on development.20 frequencies of table 1. effects of number of embryos per 50 μl in vitro culture drop on cleavage and blastocyst development number of embryos per 50 μl drop total number of embryos cultured percent cleaved ± sem percent blastocysts ± sem 1 125 56.8 ± 4.4 7.2a ± 2.3 5 150 61.3 ± 3.9 19.3b ± 3.2 10 170 56.5 ± 3.8 20.0b ± 3.1 20 160 58.2 ± 3.9 19.4b ± 3.1 50 150 66.7 ± 3.9 21.3b ± 3.4 a,bwithin a column, means without a common superscript differed (p < 0.05) table 2. effects of conditioned media on blastocyst development number of embryos per 50 μl drop media total number of embryos cultured percent blastocysts ± sem 20 control 40 20.0 ± 6.4 20 conditioned 40 22.5 ± 6.7 50 control 50 20.0 ± 5.7 50 conditioned 50 20.0 ± 5.7 table 3. effects of grouping cleaved embryos on blastocyst development number of embryos per 50 μl drop treatment group total number of embryos cultured percent blastocysts ± sem 10 control 40 27.5a ± 7.2 10 cleaved 30 30.0a ± 8.5 1 cleaved 32 3.1b ± 3.1 1 control 65 7.7b ± 3.3 a,bwithin a column, means without a common superscript differed (p < 0.05) http://dx.doi.org/10.58292/ct.v16.9801 4 citation line: clinical theriogenology 2024, 16, 9801, http://dx.doi.org/10.58292/ct.v16.9801 blastocyst development in groups with 25, 50, and 100 embryos per 50 μl drop were higher than groups of 1, 5, 10, and 250.20 still, groups of 5 and 10 embryos were significantly higher than embryos cultured alone.20 a negative effect was also observed once embryo density reached 250 embryos per 50 μl drop.20 however, when embryos were cultured embryos in groups of 5, 10, 20, and 30 embryos per 100 μl drop, it was reported that the group of 30 embryos had significantly lower cleavage and blastocyst development rates than groups of 5, 10, and 20, suggesting a negative effect of high density.21 in contrast, experiment 4 results had no apparent negative effect of culturing 25 embryos in 10 μl drops. the group of 25 embryos had similar blastocyst development rates to groups of 2, 10, and control and the group of 5 embryos had significantly lower blastocyst development than other groups. interestingly, the group of 2 embryos performed similar to groups of 10, 25, and control. apparently, only a single other occupant in the cohort restored blastocyst development to that of the controls (10 embryos per 50 μl drop). further, it may not matter if the second occupant was not exposed to sperm.22 size of the culture environment appears to be a factor of embryo density that impacts development. some studies have reported that a reduced volume of culture media improved embryo quality and development.21,23 however, there was an increase in development with decreased volume in mice embryos.7 nine murine embryos cultured in 50 μl drops had significantly higher blastocyst development compared to 9 embryos cultured in 25, 12.5, or 6.25 μl drops.7 similarly, there was an increase in embryo density negatively impacted developmental competence and expression of heat shock protein 70.1 gene in bovine embryos.21 conversely, when microfluidic platforms were used to achieve submicroliter culture systems for murine embryos, embryos placed in groups of 2 per 100 nl culture chamber achieved 81.8% blastocyst development, whereas embryos grouped in 2 embryos per 5 μl conventional culture drop had a 3.3% blastocyst rate.11 this study suggested that surface area to volume ratios of the culture environment might be important. in our study, adding conditioned media from previous replicates in experiment 2 had no beneficial effect on blastocyst development compared to controls. when epidermal growth factor and transforming growth factor-131 were added to their culture media for bovine embryos, there was an improvement in blastocyst development.4 overall, there was a significant difference in embryo development between 1 embryo and embryos cultured in groups of 5.4 addition of bovine oviductal epithelial or other cells to produce an undefined media positively influenced development.2 bovine oviductal epithelial cells secrete many beneficial nutritional factors and their composition evolves throughout the estrous cycle.1 oviduct fluid has a crucial role in protecting the developing embryo against oxidative stress by secreting free radical scavenging proteins and modulating antioxidant enzyme levels. dna fragmentation that is a major contributor to cell death and the formation of low-quality embryos, is primarily caused by an imbalanced redox state and inadequate levels of antioxidants.2 despite the cells’ positive influence on development, the protocol is inefficient, costly, and unsanitary for commercial production.24 similar pitfalls, combined with the potential for large offspring syndrome, are observed in a popular strategy of adding fetal serum to culture media. although it may improve development, there is concern for its potential to spread pathogens and there are regulatory restrictions placed on embryos cultured in animal-based sera.1 although metabolic waste is excreted into culture media, beneficial factors were also identified.25 spent media from embryo culture was used for noninvasive preimplantation genetic testing for aneuploidy.25 the quantity of cell-free dna obtained from spent dna can vary depending on laboratory procedure. depending on whether a single-step media, or sequential media that is changed once or twice between fertilization and blastulation is utilized, impact components are left in the spent media. through the ability to capture the beneficial factors secreted by embryos themselves and using the conditioned media to enable or embryos to develop, costly and unsanitary addition of sera and oviductal cells could be avoided. growing evidence supports various paracrine factors (e.g. epidermal growth factor,26 platelet-activating factors,27 and insulin-like growth factors [igfs]) are secreted into spent media by preimplantation embryos.28 various cytokines positively influenced embryo viability.29 a combination of fibroblast growth factor-2, leukemia inhibitory factor, and igf1 supplemented in culture media of bovine embryos improved development and quality.29 micrornas and dna are other factors that may be secreted by embryos in the culture media.16 small rna sequencing and live embryo imaging demonstrated that embryonic mirnas were essential for bovine embryos transitioning from morula to blastocyst.16 exosomes may also have an important role in the communication among embryos in the same culture environment. exosomes are tetraspanin cd9-positive membranous microvesicles that package and transport these paracrine factors.28 exosomes mediated cell to cell communication by selectively delivering a wide range of proteins, lipids, mrna, dna fragments, and nucleic acid that modify cellular function of the recipient cells.2,30 exosomes also establish communication between embryo and maternal interactions.2 exosomes and conditioned media from bovine oviduct epithelial cells increased blastocyst production of bovine embryos.2 recently, embryos that developed to the blastocyst stage had substantially table 4. effects of number of embryos per 10 μl in vitro culture drop on cleavage and blastocyst development compared to control (10 embryos per 50 μl drop) number of embryos per drop size of drop (μl) total number of embryos cultured percent cleaved ± sem percent blastocysts ± sem control 50 110 59.1 ± 4.7 20.0a ± 3.8 2 10 88 73.9 ± 4.7 15.9a ± 3.9 5 10 90 70.0 ± 4.8 4.4b ± 2.2 10 10 110 68.2 ± 4.4 16.4a ± 3.5 25 10 75 69.3 ± 5.3 16.0a ± 4.2 a, a,bwithin a column, means without a common superscript differed (p < 0.05) http://dx.doi.org/10.58292/ct.v16.9801 citation line: clinical theriogenology 2024, 16, 9801, http://dx.doi.org/10.58292/ct.v16.9801 5 elevated levels of fatty acid binding protein 3 compared to embryos that stopped developing at the 8-16 cell stage in the same culture environment, suggesting lipid management may impact embryo development (unpublished data). a multi-step culture embryo culture process and a continuous culture media system was evaluated. nutritional requirements of embryo’s change as they progress from zygote to morula to blastocyst that likely mimics in vivo environment as embryo progresses from oviduct to uterus.31 morulae’s prefer energy substrates (e.g. pyruvate, lactate, and amino acids). bovine embryos have limited utilization of glucose until after compaction. embryo’s utilization and uptake of glucose increases and by the time a blastocyst is established, glucose is utilized more than any other available nutrient.31 in a continuous culture system, maintaining the appropriate balance between sufficient nutrients to meet embryonic demand and breakdown of embryo metabolic waste products. ammonium is a factor of concern. amino acids in culture medium are easily broken down into ammonium at 37°c.32 a build-up of ammonium has negative effects on physiology, viability, and fetal development of mouse and human embryos.32 embryos may secrete beneficial factors that are important for embryos around them or they may remove detrimental factors from embryos of marginal quality. in order to maintain identity of 1 embryo, while sharing culture media, certain well of well systems were developed.7 in these types of dishes, 1 embryo is cultured in a microwell and culture drop covered multiple microwells thus embryos shared culture media. embryos grown alone in microwells with shared culture media had no improvement in blastocyst development.7 this study suggested that shared media alone was not able to overcome the problem of 1 embryo culture or possibly ineffective sharing of factors.7 in experiment 3, no difference in development was detected between embryos selected out after cleavage and controls. cleavage rate was evaluated 45 hours after insemination and noncleaved embryos were either removed or left in the dish as controls.6 cleaved embryos were either left in their original drop or combined into a new group. these researchers reported results similar to experiment 3; noncleaved embryos had no negative effect on the potential of embryos in the same group to become blastocysts.6 human embryos were grouped after day 3 in groups of 2-5 embryos per drop based on embryo quality grades and they were also either grouped randomly regardless of quality or grouped together based on quality.19 selecting embryos on day 3 based on quality, significantly promoted higher blastocyst development compared to controls.19 further research is needed to determine whether poor quality or undeveloped embryos have a positive or negative impact on development of neighboring embryos. in our experiments, a decision was made to utilize 1 bull, to control potential variations that might arise among different bulls. it is important to recognize that sires exhibit distinct capacities in producing blastocysts, potentially influencing experiments’ outcome.33 this variability among bulls may be attributed to differences in sperm capacitation during fertilization process, wherein the kinetics of sperm penetration has a pivotal role.34 sires with varying capacities for sperm capacitation result in differences in fertilizing capacity, impacting sperm penetration timing.34 this consideration underscores the importance of controlling for bull-specific effects. in summary, our results demonstrated that culturing bovine embryos in groups significantly improved development to blastocyst stage. in all situations, 1 embryo cultured alone performed significantly lower than embryos cultured in groups of > 5. high-density culture environments had no negative effect on blastocyst development. conditioned media from previous replicates had not enhanced development of new embryos in culture. lastly, grouping cleaved embryos together had not improved development. further research is required to identify differences in chemical composition of group culture media and individual culture media to better understand the exchange among grouped embryos. conflict of interest none to report. references 1. ferré lb, kjelland me, strøbech lb, et al: review: recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods. animal 2020;14:991-1004. doi: 10.1017/ s1751731119002775 2. sidrat t, khan aa, joo md, et al: bovine oviduct epithelial cell-derived culture media and exosomes improve mitochondrial health by restoring metabolic flux during pre-implantation development. int j biochem mol biol 2020;21:7589. doi: 10.3390/ ijms21207589 3. ferry l, mermillod p, massip a, et al: bovine embryos cultured in serum-poor oviduct-conditioned medium need cooperation to reach the blastocyst stage. theriogenology 1994;42:445-453. doi: 10.1016/0093-691x(94)90682-9 4. keefer cl, stice sl, paprocki am, et al: in vitro culture of bovine ivm-ivf embryos: cooperative interaction among embryos and the role of growth factors. theriogenology 1994;41:1323-1331. doi: 10.1016/0093-691x(94)90491-z 5. o’doherty em, wade mg, hill jl, et al: effects of culturing bovine oocytes either singly or in groups on development to blastocysts. theriogenology 1997;48:161-169. doi: 10.1016/s0093-691x (97)00199-4 6. wydooghe e, vandaele l, piepers s, et al: individual commitment to a group effect: strengths and weaknesses of bovine embryo group culture. reproduction 2014;148:519-529. doi: 10.1530/ rep-14-0213. 7. dai sj, xu cl, wang j, et al: effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo. j assist reprod genet 2012;29:617-623. doi: 10.1007/s10815-012-9744-8 8. paria bc, dey sk: preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors. proc natl acad sci usa 1990;87:4756-4760. doi: 10.1073/ pnas.87.12.4756 9. lane m, gardner k: effects of incubation volume and embryo density on the development and viability of mouse embryos in vitro. hum reprod 1992;17:558-562. doi: 10.1093/oxfordjournals.humrep.a137690 10. kato y, tusnoda y: effects of the culture density of mouse zygotes on the development in vitro and in vivo. theriogenology 1994;41:1315-1322. doi: 10.1016/0093-691x(94)90490-a 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single or group culture of mammalian embryos: the verdict of the literature. j reprod stem cell biotechnol 2011;2:77-87. 18. shi c, sun tc, chen sw, et al: effects of embryo density on cell number of day 3 embryos cultured in a 30-μl drop: a retrospective cohort study. zygote 2022;30:487-494. doi: 10.1017/s0967199 421000812 19. tao t, robichaud a, mercier j, et al: influence of group embryo culture strategies on the blastocyst development and pregnancy outcome. j assist reprod genet 2013;30:63-68. doi: 10.1007/ s10815-012-9892-x 20. nagao y, iijima r, saeki k: interaction between embryos and culture conditions during in vitro development of bovine early embryos. zygote 2008;16:127-133. doi: 10.1017/s09671994 08004644 21. de oliveira atd, lopes rff, rodrigues jl: gene expression and developmental competence of bovine embryos produced in vitro under varying embryo density conditions. theriogenology 2005;64:1559-1572. doi: 10.1016/j.theriogenology.2005.03.019 22. looman j, hickerson s, gibbons j: effects of unfertilized oocytes and blastocysts on bovine in vitro embryo development. in 2023 aeta and ceta/acte joint convention proc am em tran assoc 2023. 23. gil ma, abeydeera lr, day bn, et al: effect of the volume of medium and number of oocytes during in vitro fertilization on embryo development in pigs. theriogenology 2003;60:767-776. doi: 10.1016/s0093-691x(03)00051-7 24. carvalho av, canon e, jouneau l, et al: different co-culture systems have the same impact on bovine embryo transcriptome. reproduction 2017;154:695-710. doi: 10.1530/rep-17-0449 25. belandres d, shamonki m, arrach n: current status of spent embryo media research for preimplantation genetic testing. j assist reprod genet 2019;36:819-826. doi: 10.1007/ s10815-019-01437-6 26. thongkittidilok c, tharasanit t, songsasen n, et al: epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos. j reprod dev 2015;61:269-276. doi: 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1998;49:83102. doi: 10.1016/s0093-691x(97)00404-4 32. gardner dk, hamilton r, mccallie b, et al: human and mouse embryonic development, metabolism and gene expression are altered by an ammonium gradient in vitro. reproduction 2013;146:49-61. doi: 10.1530/rep-12-0348 33. lockhart kn, drum jn, balboula az, et al: sire modulates developmental kinetics and transcriptome of the bovine embryo. reproduction 2023;166:337-348. doi: 10.1530/rep-23-0030 34. barceló-fimbres m, campos-chillón l, seidel jr g: in vitro fertilization using non-sexed and sexed bovine sperm: sperm concentration, sorter pressure, and bull effects. reprod domest anim 2011;46:495-502. doi: 10.1111/j.1439-0531.2010.01696.x http://dx.doi.org/10.58292/ct.v16.9801 https://doi.org/10.1002/dvdy.21918 https://doi.org/10.1016/s0015-0282(16)57925-4 https://doi.org/10.1016/s0015-0282(16)57925-4 https://doi.org/10.1016/s0015-0282(16)58507-0 https://doi.org/10.1093/humrep/14.9.2333 https://doi.org/10.1093/humrep/14.9.2333 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ovulation in beef heifers and cows control of estrus and ovulation in beef heifers and cows david patterson, jordan thomas division of animal sciences, animal science research center university of missouri, columbia, mo abstract until recently, labor required to detect estrus for individual cows or heifers in a herd was the primary reason beef producers viewed artificial insemination as impractical. however, improvements in methods to control estrus and ovulation in beef heifers and cows are now expanding the use of artificial insemination by reducing the time required to detect estrus or eliminating estrus detection entirely. these methods evolved as a result of expanded understanding of the bovine estrous cycle and improved ability to effectively manipulate both follicular waves and luteal lifespan. modern protocols are now capable of facilitating fixed time artificial insemination in beef cattle through orchestrated use of progestins, gonadotropin releasing hormone, and prostaglandin. these strategies offer effective control of estrus and ovulation in estrous cycling females and are also capable of inducing an ovulatory estrus in high percentages of peripubertal heifers and anestrous postpartum cows. additionally, alternative breeding management strategies have been developed to achieve higher fertility while still reducing labor associated with estrus detection, such as combined estrus detection and fixed time artificial insemination approaches, or more recent approaches such as split time artificial insemination. advancements in applied reproductive technologies now afford beef producers flexibility in matching specific breeding management protocols to defined management systems. this review focuses on systems currently recommended to facilitate fixed time artificial insemination among beef heifers and cows in the united states, where these technologies offer beef producers a clear opportunity to simultaneously improve genetic merit and reproductive management of their herds. keywords: estrus synchronization, fixed time artificial insemination, beef heifer, beef cow introduction methods to control estrus and ovulation among beef heifers and cows have improved greatly over the past 25 years. cow-calf producers can now successfully manage groups of females of mixed estrous cycling status and successfully perform fixed time artificial insemination (ftai). these strategies offer more precise control of estrus and ovulation in estrous cycling females, while inducing an ovulatory estrus in high percentages of peripubertal heifers and anestrous postpartum cows. collectively, improvements in methods to control estrus and ovulation in beef heifers and cows are expanding the use of artificial insemination (ai) by reducing the time required to detect estrus or eliminating estrus detection entirely. breeding management strategies now offer the potential to effectively manage reproduction and expedite genetic progress, ultimately enhancing efficiencies of production and adding value to beef cattle produced and marketed in the us.1-4 in total, these are important accomplishments considering the fact that estrus synchronization and artificial ai remain the most significant and widely applicable reproductive biotechnologies available for cattle.5 development of the various systems currently in use by industry began with the initial discovery that progesterone inhibited ovulation6 and preovulatory follicular maturation.7-9 subsequent research led to the finding that prostaglandin f2 (pg) and its analogs were luteolytic in the bovine.10-15 this led to the combined use of progestational agents and pg to control the luteal phase of the estrous cycle. with expanded understanding of changes that occur during a follicular wave,16 it became clear that precise control of estrous cycles in cattle would require manipulation of both follicular waves and luteal lifespan. assembling protocols that were ultimately capable of facilitating ftai then hinged on orchestrated use of progestins, gnrh, and pg to enable beef producers to successfully breed cattle by appointment. review articles published over the past nearly 60 years provide a sequential overview of research that subsequently led to development of these protocols and strategies that would enhance reproductive management of our nation’s beef herds.17-38 265 clinical theriogenology • volume 11, number 3 • september 2019 the beef reproduction task force (brtf) was formed during the recent period of evolving science in the us that resulted in systems to allow producer-acceptable results with a single ftai.39 the brtf has provided scientific based recommendations for the application of reproductive technologies to the us beef industry since the turn of the century. in 2004, the brtf was joined by leaders in the ai and pharmaceutical industries, along with bovine veterinary practitioners to form the us beef reproduction leadership team (brlt). the group annually sponsors a 2 day workshop entitled “applied reproductive strategies in beef cattle (arsbc)”, a program targeted at beef producers, ai industry personnel, veterinarians, allied industry representatives, and academicians. in addition, each year beginning in 2005, the brtf publishes a list of recommended protocols for beef heifers and cows. this recommendation provides a detailed listing of protocols best suited to ai performed on the basis of: 1) detected estrus; 2) a combination of estrus detection followed by ftai; and 3) ftai.39 one important consideration related to review of protocols recommended for use in the us is that no estrogen product is currently approved or legally available for use in estrus synchronization of cattle.40 estrogen products are available and in use with estrus synchronization protocols in other countries, such as brazil and canada; however, based on criteria used by the brtf and brlt and in compliance with fda guidelines, no recommended estrus synchronization protocol uses an estrogen. this review will focus on systems currently recommended and used in the us to facilitate ftat in beef heifers and cows; comprehensive reviews detailing development of these protocols was published in the most recent proceedings from arsbc.36-38 estrus synchronization and artificial insemination contribute to heifer development programs lifetime reproductive performance, longevity in the herd, and cumulative pounds of calf weaned over a heifer’s productive lifespan are highly correlated with the date on which a heifer conceives during the first breeding season.1,41-43 the investment in time and resources in heifers from weaning to breeding requires that management efforts be made to facilitate puberty onset and maximize the likelihood of early pregnancy. estrus synchronization programs create an opportunity for heifers to conceive early in the breeding period, which is a significant opportunity given the relationship between early conception and long term reproductive success. in addition, estrus synchronization improves time management on farms and ranches by concentrating the breeding and resulting calving periods.44-45 beef producers are now able to utilize treatments that offer the potential to effectively synchronize estrus in pubertal heifers, while at the same time induce puberty among preor peripubertal heifers. this benefits groups of heifers with mixed cyclicity status, particularly breeds or biological types that are late-maturing, but of sufficient age and weight at the time of treatment to permit successful application.46 progestins were first reported to induce estrus in peripubertal heifers47 and were originally combined with estrogen to mimic changes that occur in concentrations of blood hormones around the time of puberty. increased progesterone is thought to be a prerequisite for development of normal estrous cycles. progesterone increases during the initiation of puberty in the heifer48 and before resumption of normal ovarian cyclicity in postpartum suckled beef cows.49-50 progestins stimulate an increase in follicular growth that results in increased production of estrogen by ovarian follicles.51-54 progestin treatment results in increased lh pulse frequency during the treatment period55-57 and initiation of estrous cyclicity in peripubertal beef heifers.46-47,58 studies suggest that stimulatory effects of progestins on lh secretion59 are greatest after removal of the steroid56-57,60 and increase with heifer age.60 long term progestin-based protocols for heifers melengestrol acetate prostaglandin f2 melengestrol acetate (mga) is an orally active progestin approved for use to synchronize estrus in replacement heifers when fed at a rate of 0.5/mg/head/day.61 when consumed on a daily basis, mga will suppress estrus and prevent ovulation.57 it may be fed in a single daily feeding with a grain or protein carrier. heifers that fail to consume the required amount of mga on a daily basis may return to estrus prematurely during the feeding period, which will reduce estrous response during the synchronized 266clinical theriogenology • volume 11, number 3 • september 2019 period. therefore, adequate bunk space (60 linear cm/head) must be available so that all animals consume feed simultaneously.27-28 this practice ensures that all females receive adequate intake. heifers should be observed for behavioral signs of estrus each day of the feeding period, which may be done as animals approach the feeding area and before feed distribution. heifers will exhibit estrus beginning 48 hours after mga withdrawal, and this will continue for 6 7 days. it is generally recommended that heifers that exhibit estrus during this period not be inseminated or exposed for natural service, due to reduced fertility at the first estrus after mga withdrawal. therefore, pg should be administered 19 days after the last feeding day of mga, with ftai performed 72 hours after pg (figure 1). it is important to note that use of mga as part of any estrus synchronization protocol in beef cows constitutes an extra-label use of medicated feed that is prohibited by the animal medicinal drug use and clarification act and regulation 21 cfr 530.11(b). the feeding of mga is specifically approved for estrus suppression in heifers only.61 although 35 years of feeding mga to beef cows and beef heifers demonstrated that mga is safe, effective and economical,20,45,56,62-81 feeding mga to adult cows is not an fda approved label claim and therefore strictly prohibited by the fda. figure 1. melengestrol acetate (mga) prostaglandin f2α (pg) with ftai. mga-pg protocol consists of mga feeding for 14 days at a rate of 0.5 mg/head/day, pg administration on day 19 and ftai at 72 hours after pg, concurrent with administration of gonadotropin-releasing hormone (gnrh).62-64 14 day controlled internal drug release + prostaglandin f2 a comprehensive series of studies in beef heifers led to development of the 14 day controlled internal drug release (cidr)-pg protocol (figure 2), followed by comparisons of the protocol to the 7 day cosynch + cidr and mga-pg protocols.82-89 these studies carefully characterized estrus distribution patterns following removal or withdrawal of the progestins and subsequent distribution of estrus following the administration of pg. results from these studies highlighted differences in variance for interval to estrus, which ultimately explain the degree of estrus synchrony that is achieved during the synchronized period. in every case, the degree of synchrony achieved was greatest for heifers assigned to the 14 day cidr-pg protocol. in addition, results from these experiments noted similarities in ftai pregnancy rates comparing heifers that were peripubertal or estrous cycling prior to treatment initiation. mallory et al.88 reported that conception to ai and ai pregnancy rates did not differ between 14 day cidr-pg and mga-pg treated heifers following inseminations performed on the basis of detected estrus. in addition, pregnancy rates after ftai were later compared90 among heifers assigned to the 2 protocols, with 62 and 61% of heifers conceiving to the mga and cidr treated groups, respectively. short term progestin-based protocols for heifers 7 day cosynch + controlled internal drug release lucy et al.58 summarized results from initial studies conducted in the us involving cidr-based protocols for use in synchronizing estrus in beef heifers. these data were submitted to fda in support of the original approval for the cidr in beef heifers. heifers with a cidr inserted for 7 days with pg 267 clinical theriogenology • volume 11, number 3 • september 2019 figure 2. 14 day cidr-pg with ftai. the 14 day cidr-pg protocol involves cidr insertion for 14 days, waiting 16 days to administer prostaglandin f2 (pg) and ftai at 66 hours after pg with gonadotropin-releasing hormone (gnrh) administered at ai.89 administered on day 6 of cidr treatment had higher pregnancy rates compared to untreated control or pg treated heifers. treatment with a cidr increased synchronization rates within the first 3 days following pg, resulting in enhanced pregnancy rates. the improved pregnancy rate in prepubertal beef heifers treated with cidr was noteworthy because control or pg treated heifers that were prepubertal prior to treatment never attained pregnancy rates that were comparable to those that received a cidr. later, lamb et al.91 (figure 3) lead a multi-state effort involving beef heifers assigned to a 7 day cosynch + cidr protocol to determine whether: 1) an injection of gnrh at cidr insertion would enhance pregnancy rates in beef heifers; and 2) administration of an estrus synchronization protocol followed by ftai could yield pregnancy rates similar to a protocol requiring detection of estrus. lamb et al.91 concluded that: 1) gnrh at cidr insertion did not improve pregnancy rates after ftai; 2) gnrh at cidr insertion did not alter percentage of heifers detected in estrus or distribution of estrus after pg; and 3) a combination of detecting estrus and ai before clean-up ai enhanced pregnancy rates over ftai. figure 3. 7 day cosynch + cidr with ftai. the 7 day cosynch + cidr protocol consists of a 7 day controlled internal drug release intravaginal insert (cidr; 1.38 g progesterone), gonadotropin-releasing hormone (gnrh) administration at cidr insertion and prostaglandin f2α (pg) administered at cidr removal on day 7. ftai is performed 54 hours following pg, concurrent with gnrh administration.91 5 day cosynch + controlled internal drug release the 5 day cosynch + cidr protocol (figure 4) was added most recently by the brtf to the list of recommended protocols for ftai in beef heifers. development of this protocol was based on the hypothesis92-93 that reducing the length of cidr treatment from 7 to 5 days in the cosynch + cidr protocol would increase secretion of estradiol by the ovulatory follicle, decrease the incidence of induced ovulation of follicles with reduced estrogenic activity and potentially result in improvements in ftai pregnancy rates. the hypothesis was based on the premise that day 4 dominant follicles have higher intrafollicular concentrations of estradiol-17β (e2) and a greater ability to produce e2 compared to older follicles.94 bridges et al.95 reported that maximum preovulatory concentrations of e2 tended to be greater in 5 day compared to 7 day cidr treated cows that failed to respond to gnrh at cidr insertion and that postovulatory circulating concentrations of progesterone were greater among 5 day compared to 7 day treated cows. increased follicular concentrations of e2 and elevated postovulatory concentrations of progesterone are believed to reflect greater physiological maturity of the dominant follicle and to result in higher pregnancy rates resulting from ai.96-97 therefore, bridges et al.92 proposed that if cidr removal 268clinical theriogenology • volume 11, number 3 • september 2019 and ai are more optimally timed with the 5 day protocol to coincide with follicular development, higher ai pregnancy rates may be achieved. regrettably there are no published studies illustrating estrus distribution patterns for the 5 day cosynch + cidr protocol in beef heifers on the basis of estrous cyclicity status of heifers at the time treatments are imposed.98 recently, however, pregnancy rates after ftai were compared in heifers assigned to the 5 day and 7 day cosynch + cidr protocols99,100 and the 5 day cosynch + cidr and 14 day cidr-pg protocols.101-103 the only reported difference between protocols from any of these studies was an increase in pregnancy rate that resulted after ftai among 5 day compared to 7 day cosynch + cidr treated heifers.99 figure 4. 5 day cosynch + cidr with ftai. the 5 day cosynch + cidr protocol consists of a 5 day cidr insert (1.38 g progesterone), gonadotropin-releasing hormone (gnrh) administration at cidr insertion and prostaglandin f2α (pg) administered at cidr removal and again 8 ± 2 hours later. ftai is performed 60 hours following the first injection of pg, concurrent with gnrh administration.93,98 comparing pregnancy rates resulting from fixed time artificial insemination on the basis of reproductive tract score and protocol an on-farm beef heifer development and marketing program was initiated in missouri in 1996 (show-me-selecttm replacement heifer program)104 to enable beef producers to improve breeding performance of heifers during their first breeding season and during subsequent calving and rebreeding periods as 2 year olds. the intent of the program was to ensure that heifers that enter the herd as raised or purchased replacements contribute to the sustained increase in performance and productivity of cowherds for the long term.105 the program has improved reproductive management of beef herds in missouri through increased use of reproductive technologies.106-107 field data collected in support of this program include an array of reproductive evaluations unique to the beef industry. producers that enroll heifers in the program are required to follow detailed management guidelines and adhere to approved animal health protocols. prebreeding evaluations are performed on all heifers 4 6 weeks prior to breeding by licensed veterinarians. as part of the evaluation, each heifer is assigned a reproductive tract score (rts), which is used to characterize pubertal status and resulting fertility.108-110 the scores range from 1 to 5 (1 = infantile; 2 and 3 = noncycling/prepubertal; 4 and 5 = cycling/pubertal) and are determined using transrectal ultrasonography or palpation. in recent years, participants in the program have relied heavily on ftat to breed heifers. heifers enrolled in the program that receive ai may not be exposed for natural service for a minimum of 14 days after ai and pregnancy diagnoses must be performed within 90 days after the start of the breeding period. these requirements, when considered in total, provide a unique opportunity to evaluate reproductive data on large numbers of beef heifers from the perspective of: 1) pretreatment estrous cyclicity status; 2) the various protocols used to synchronize estrus; and 3) pregnancy rates resulting from ftai. field data collected from 2010 to 2018 were used to evaluate relationships between rts and pregnancy outcome after ftai. a summary of rts and ftai pregnancy rate is provided in table 1 for 39,938 heifers that were evaluated from 2010-2018.111 these data support establishing prebreeding criteria that may be used to identify heifers that are good candidates for a ftai program. pregnancy rates resulting from ftai are compared in table 2 on the basis of rts prior to the time the various protocols to synchronize estrus were initiated. data for heifers 269 clinical theriogenology • volume 11, number 3 • september 2019 assigned rts score of 1 were excluded from this summary, as the majority of these heifers are culled by producers prior to breeding. these data indicate that evaluation of reproductive status of heifers prior to the first breeding season is useful in determining success of the development period and in determining which protocol to use to synchronize estrus prior to ftai.111-112 the 14 day cidr-pg protocol has gained widespread use in heifer programs across missouri, as seen from numbers presented in table 2. pregnancy rates resulting from ftai suggest more favorable field results are obtained on-farm using the 14 day cidr-pg compared to the 7 day cosynch + cidr and mga-pg protocols. additionally, the 14 day cidr-pg protocol has gained widespread acceptance among veterinarians involved in the program, as prebreeding booster vaccinations and reproductive exams are performed coincident with cidr insertion.111 table 1. pregnancy rates resulting from ftai among heifers in the missouri show-me-selecttm replacement heifer program, based on pretreatment rts (n = 39,938). reproductive tract score 1 2 3 4 5 number of heifers 237 1,155 11,104 14,340 13,102 number pregnant 11 313 5,260 7,269 6,698 ftai pregnancy rate (%) 5a 27b 47c 51d 51d *pregnancy rates resulting from ftai based on rts. these data include pregnancy rates for 39,938 heifers that were inseminated beginning during the fall of 2010 through fall of 2018. a-dpercentages without a common superscript differ (p < 0.05). the missouri show-me-selecttm replacement heifer program continues to see an increase in the overall percentage of heifers exposed for ai, with the majority of these heifers are being inseminated using a ftai approach rather than detection of estrus. an important consideration related to the success of these ai programs and the expanded use of ai in missouri is that all heifers are required to undergo a prebreeding examination to determine estrous cyclicity status. this practice allows for better determination of the appropriate estrus synchronization protocol and also provides critical information in situations where troubleshooting may be required.111-112 these conclusions are supported by studies reported by hall113 and gutierrez et al.114 which involved comparison of heifers that were inseminated on the basis of ftai followed by a natural service clean-up period versus natural service only. gutierrez et al.114 reported that rts influenced both the number of beef heifers that became pregnant during the breeding season and the time at which heifers became pregnant. table 2. pregnancy rates resulting from ftai among heifers in the missouri show-me-selecttm replacement heifer program, based on pretreatment estrous cyclicity status based on rts and protocol used to synchronize estrus (n = 39,695). ftai protocol non-cycling+ cycling++ combined total no. % no. % no. % 7 day cosynch + cidr 205/616 33a,x 526/1,193 44b,x 731/1,809 40x mga – pg 81/230 35a,x 274/576 48b,x 355/806 44x 14 day cidr – pg 5,287/11,413 46a,y 13,163/25,667 51b,y 18,450/37,080 50y +,++non-cycling heifers were assigned a rts of 2 or 3; cycling heifers were assigned rts of 4 or 5 (anderson et al. 1991). a,bpercentages within rows with different superscripts differ p < 0.01. x,ypercentages within columns with different superscripts differ p < 0.01. short term progestin-based protocols for postpartum beef cows 7 day cosynch + controlled internal drug release lucy et al.58 smmarized results from initial studies conducted in the us involving cidr based protocols for use in synchronizing estrus in postpartum beef cows. these data were submitted to fda in support of the original approval for the cidr. three treatments were involved in the study and included: 270clinical theriogenology • volume 11, number 3 • september 2019 1) untreated control; 2) pg only; and 3) 7 day cidr-pg with pg administered on day 6 of cidr treatment. the 7 day cidr-pg protocol yielded greater pregnancy rates compared to control or pg treatments. treatment with cidr increased synchronization rates within the first 3 days following pg, resulting in enhanced pregnancy rates. the improved pregnancy rate in anestrous cows treated with the cidr was noteworthy, because anestrous cows in the control or pg treatments never attained pregnancy rates that were similar to those of the 7 day cidr-pg treated group. the drawback of the protocol was that pg was administered on day 6 after cidr insertion, which required an additional day of animal handling. a series of studies were then designed to determine whether inclusion of a cidr to a gnrh-pg (cosynch)115-116 based protocol would increase pregnancy rates resulting from ai.117-118 larson et al.118 reported an improvement in ftai pregnancy rates in postpartum beef cows with addition of a cidr to the cosynch protocol, noting that differences resulted from the potential added benefit of induced cyclicity among anestrous cows that occurred following cidr removal. furthermore, addition of a cidr to the cosynch protocol prevented premature expression of estrus that occurs between gnrh and pg among cows that do not receive a cidr; in the absence of a cidr, it is estimated that 5 20% of the total number of cows treated will exhibit estrus prior to or immediately after pg injection82,119-121 therefore, addition of the cidr to the cosynch protocol successfully prevented the premature expression of estrus prior to or following pg (figure 5). figure 5. cows in the 7 day cosynch + cidr treatment received gnrh and cidr inserts on day 0. pg was administered and cidr inserts were removed on day 7. all 7 day treated cows were inseminated (ftat) 66 hours following treatment, with gnrh administered at ai.80,118,122 does timing of insemination affect pregnancy rates resulting from ftai following administration of the cosynch + controlled internal drug release protocol? several studies involving the cosynch + cidr protocol evaluated effect of timing of ftai on subsequent pregnancy rates. timing of insemination following the cosynch + cidr protocol was based on recommendations from pharmaceutical and ai industries (54 66 hours) and other reports where the timing of ai included 48, 54, 56, 60, 64, 66, and 72 hours post pg80, 117-118,122-124 therefore, busch et al.122 compared ftai pregnancy rates among lactating beef cows synchronized with the cosynch + cidr protocol that were inseminated at 54 or 66 hours after pg and characterized estrous response of cows in each treatment prior to ftai (figure 6). busch et al.122 reported a significant effect of time of insemination on pregnancy rates resulting from ftai; cows inseminated at 66 hours after pg were 1.32 times more likely to conceive to ftai than cows inseminated at 54 hours following pg. busch et al.122 reported that estrous response following pg and prior to ftai was greater among cows inseminated at 66 hours (50%) than cows inseminated at 54 hours (26%). cows that exhibited estrus prior to ftai had significantly higher pregnancy rates (76%) than cows that failed to exhibit estrus before ai (56%). perhaps the most interesting observation from the study, however, was the fact that pregnancy rates among cows that exhibited estrus prior to insemination were higher for cows inseminated at 66 hours (81%) compared to those inseminated at 54 hours (65%), highlighting importance of properly timed inseminations when appointment breeding is performed. 271 clinical theriogenology • volume 11, number 3 • september 2019 figure 6. busch et al.122 compared ftai pregnancy rates among lactating beef cows synchronized with the cosynch + cidr protocol in which case cows were inseminated at 54 or 66 hours after cidr removal and pg. the onset of estrus prior to ftai in beef cows improved pregnancy rates when compared to cows that fail to exhibit estrus.125 busch et al.122 reported that cows that exhibited estrus following administration of the cosynch protocol had greater serum e2 concentrations during the 2 days prior to insemination compared to cows that were induced to ovulate. busch et al.122 concluded that cows that exhibited estrus may have attained concentrations of e2 necessary to effectively prepare follicular cells for luteinization and (or) induced an adequate number of uterine progesterone receptors,126 thereby providing an adequate uterine environment for pregnancy establishment and maintenance. based on these data, higher estrous response rates prior to ftai in beef cows should result in greater pregnancy rates resulting from ftai, provided that ai is performed within an acceptable interval after the peak estrous period. mirando et al.127 reviewed efforts in missouri to integrate fundamental aspects of control of the estrous cycle in beef cattle, with wide-scale application of the technology in the field, both of which are required to enhance competitiveness of the us livestock industry. justification for this approach centered on the concern that continued low adoption rates of these technologies in the us will ultimately erode the competitive position of the us beef cattle industry. on-farm field demonstrations were conducted in missouri (figure 7) involving 73 herds and 7,028 cows. ftai was performed on postpartum beef cows using the 7 day cosynch + cidr protocol, with ftai performed 66 hours after cidr removal and pg administration. pregnancy rates resulting from ftai averaged 62% for the 73 herds. pregnancy rates resulting from ftai ranged from 38 to 86%, and only 7 of the 73 herds had pregnancy rates that were below 50%. field demonstrations in missouri127 and florida4 illustrate that increased profits can be achieved through changes in calving distribution patterns of herds by increasing percentage of cows that calve over a more concentrated interval and earlier in the calving period. 5 day cosynch + controlled internal drug release the rationale for and development of the 5 day cosynch + cidr protocol developed by bridges et al.92,95 (figure 8) was presented in the heifer portion of this review. bridges et al. 92,95 concluded that reducing the interval from gnrh and cidr insertion from 7 to 5 days, administering 2 injections of pg at cidr removal and again 12 hours later and extending the interval to 72 hours from cidr removal to ftai was an effective estrus synchronization protocol for use in facilitating ftai in postpartum beef cows. 272clinical theriogenology • volume 11, number 3 • september 2019 figure 7. locations of on-farm field demonstrations in missouri involving 7,028 cows in 73 herds. cows at these various locations were synchronized using the 7 day cosynch + cidr protocol and were inseminated 66 hours after cidr removal and pg. pregnancy rates resulting from ftai averaged 62%.127 it is important to note that 2 injections of pg are required with the 5 day protocol to effectively regress accessory corpora lutea that form as a result of gnrh-induced ovulations at the initiation of treatment. therefore, the current recommendation for use of this protocol to facilitate ftai in beef cows is to administer 2 doses of pg 8 hour apart, the first coincident with the time of cidr removal and the second 8 hours later95,97,128-129 figure 8. the 5 day cosynch + cidr protocol for cows consists of a 5 day cidr insert (1.38 g progesterone), gonadotropinreleasing hormone (gnrh) administration at cidr insertion, and pg administered at cidr removal and again 8 ± 2 hours later. ftai is performed 72 hours after the first injection of pg, concurrent with gnrh administration.92, 95 how do the 7 day and 5 day cosynch + controlled internal drug release protocols compare in postpartum beef cows? wilson et al.130 (figure 9) and whittier et al.131 compared the 5 day and 7 day cosynch + cidr protocols on the basis of pregnancy outcome and practical application in the field. wilson et al.130 reported comparable pregnancy rates resulting from ftai among cows assigned to the 2 protocols, although the study reported by whittier et al.131 showed a 3% improvement in pregnancy rate resulting from ftai among cows assigned to the 5 day protocol. the 5 day protocol provides an effective alternative to the 7 day protocol for use in facilitating ftai; however, beef producers must consider the increased labor and treatment costs associated with its use. although bridges et al.95 reported higher pregnancy rates among cows assigned to the 5 day versus 7 day cosynch + cidr protocol, it is important to contrast those results with the study by wilson et al.130 cows assigned to the 7 day protocol 273 clinical theriogenology • volume 11, number 3 • september 2019 figure 9. cows in the 5 day cosynch + cidr treatment received gnrh and a cidr insert on day 0. pg was administered and cidr inserts were removed on day 5. a second injection of pg was administered 12 hours after the first pg injection. all 5 day treated cows were inseminated (ftat) 72 hours following treatment with gnrh administered at ai. cows in the 7 day cosynch + cidr treatment received gnrh and cidr inserts on day 0. pg was administered and cidr inserts were removed on day 7. all 7 day treated cows were inseminated (ftat) 66 hours following treatment with gnrh administered at ai.128 in wilson’s experiment received a single injection of pg and were inseminated 66 hours after pg; whereas, 7 day treated cows in bridges’ study received 2 injections of pg, with ai performed 60 hours after the first pg injection. a possible explanation for differences in results between the 2 studies130 related to differences in timing of ai for the 7 day treated cows in each study. timing of insemination for the respective protocols paralleled the timing of peak estrus for each protocol based on the results from wilson et al.130 for cows assigned to the 5 day protocol, the mean time of ai (72.6 hours), paralleled the mean interval to estrus (71.2 hours) reported by wilson et al.130 from a separate experiment. likewise, for the 7 day treated cows, the mean time of ai (66.5 hours) paralleled the mean interval to estrus (64.8 hours). bridges et al.95 hypothesized that shortening the duration of cidr treatment from 7 to 5 days would better time cidr removal coincident with optimal follicular development, and that lengthening the time interval between pg and ai would result in higher pregnancy rates following ftai. arguably, lengthening this time period between pg and ai to 66 hours for cows assigned to the 7 day protocol may explain the similarity in ftai pregnancy rates between treatment groups, versus those reported by bridges et al.95 collectively, the results from busch et al.122 and wilson et al.130 suggest that pregnancy rates resulting from ftai are perhaps more a function of properly timed ai, rather than timing cidr removal and follicular development. how do the 14 day controlled internal drug release prostaglandin f2 and 7 day cosynch + controlled internal drug release protocols compare in primiparous 2 year old beef cows? reproductive management of primiparous 2 year old cows presents a unique challenge in managing beef herds, since this age group of females typically experiences the highest incidence of reproductive failure.132 abel et al.133 designed a study to compare short and long term cidr-based protocols to synchronize estrus and ovulation in primiparous 2 year old beef cows (figure 10). abel et al.133 proposed that extended progesterone exposure using the 14 day cidr-pg protocol would successfully overcome problems related to protracted postpartum intervals in younger age females, improve pregnancy rates to ftai and increase final pregnancy rates at the end of the breeding season. 274clinical theriogenology • volume 11, number 3 • september 2019 figure 10. abel et al.131 compared short and long term cidr based protocols to synchronize estrus and ovulation in primiparous 2 year old beef cows. the 14 day cidr-pg and 7 day cosynch + cidr protocols were compared on the basis of estrous response following pg up to the point of ftai, pregnancy rates after ftai, and final pregnancy rate. the study133 compared the 14 day cidr-pg and 7 day cosynch + cidr protocols on the basis of estrous response following pg up to the point of ftai, pregnancy rates after ftai and final pregnancy rate. estrous response prior to ftai was higher for 7 day compared to 14 day treated cows (74 versus 43%, respectively); however, pregnancy rates resulting from ftai were similar (7 day, 64%; 14 day, 63%). despite the significant difference in estrous response between cows assigned to each protocol, there was no difference in pregnancy rate between groups after ftai. low estrous response rates were observed in previous studies that evaluated long term cidr-based protocols in mixed-age groups of beef cows; however, despite lower numbers of cows that exhibited estrus, pregnancy rates resulting from ftai were acceptable.134-136 it is worth noting that, among a subset of cows in abel’s study, circulating concentrations of e2 at ftai were nearly 2 fold higher among 14 day cidr-pg treated cows, irrespective of estrous status, compared to 7 day treated cows. higher concentrations of e2 among 14 day treated cows, despite smaller dominant follicle diameter, suggest that dominant follicles were in an active growth stage among cows within this treatment group, compared to 7 day treated cows for which growth of the dominant follicle already plateaued.133 this may be a logical conclusion, as aromatase activity decreases in granulosa cells when growth of the dominant follicle plateaus.94 abel’s results support the concept that preovulatory follicles among cows assigned to the 14 day protocol were physiologically mature at the time gnrh was administered and ftai performed. this may help to explain the similarity in pregnancy rate in the face of reduced estrous response among cows assigned to the 14 day cidr-pg protocol. managing anestrus with progestin-based protocols herd owners are faced with uncertainty in knowing the number of cows that have returned to estrus following calving and the number that remain anestrus prior to each breeding period. one of the overlooked advantages in administering a cidr based protocol in postpartum beef cows lies in the fact that these protocols can effectively induce estrous cyclicity among a high proportion of cows that were anestrus prior to treatment initiation. figure 11 illustrates results from 6 published studies conducted by our laboratory at the university of missouri comparing pregnancy rates after ftai. blood samples were collected from cows in each of these studies at 2 time points before assignment to an estrus synchronization protocol to determine pretreatment estrous cyclicity status, based on concentrations of progesterone. pregnancy rates following ftai were then compared between cows that were anestrus prior to treatment and cows that were estrous cycling. the combined results from these 6 studies clearly 275 clinical theriogenology • volume 11, number 3 • september 2019 demonstrate the significant benefit that results from using progestins in protocols designed to facilitate ftai programs. by advancing the date during the breeding season on which anestrous cows have a first opportunity to conceive, these protocols support improved reproductive management of the entire cow herd, regardless of cyclicity status. figure 11. pregnancy rates among postpartum beef cows resulting from ftat and based on pretreatment estrous cyclicity status. estrous cyclicity status was determined based on 2 blood samples for progesterone 8 10 days before and on the day treatments to synchronize estrus were initiated. the graph illustrates results from 6 published studies involving 3,074 cows, of which 54 and 46% of the cows were estrous cycling or anestrus, respectively, prior to initiation of treatment.79,80,122,130,134,136 protocols for bos indicus influenced females efforts to improve the reproductive and genetic management of beef cattle operations in the us should encompass the full complement of breeds and biological types of cattle that comprise our nation’s cowherd. in total, a plethora of research has been designed and conducted specifically for bos indicus influenced females; however, as indicated in the introduction to this review, many of these protocols involve estrogen products that cannot be used legally in the us there are however, limited numbers of published reports specifically focused on development and evaluation of protocols to synchronize estrus and ovulation prior to ftat for bos indicus influenced females in the us this point is unfortunate, given the fact that breeds or breed crosses in gulf coast states that comprise this biological type represent nearly 25% of the entire us cowherd.137 bos indicus influenced cattle are known to exhibit increased susceptibility to stress and are unique from bos taurus females based on their distinct reproductive physiology.138-144 these differences have influenced development of systems to effectively control estrous cycles of bos indicus influenced cattle and limited the use of ai in regions of the us where these breeds or biological types are ideally better adapted to tropical and subtropical environments. thomas and locke38 recently reviewed current strategies designed to synchronize estrus in bos indicus influenced females and noted that there are currently no protocols recommended by the brtf for use in synchronizing estrus in heifers of these breeds or biological types. inherent genetic differences in age at puberty between bos indicus and bos taurus breeds are apparent and affect the outcome of treatments designed to synchronize estrus and ovulation. management as it relates to development of these heifers during the postweaning to prebreeding period is critical143,145-147, and implementation of an ai program among a group of mixed-cyclicity bos indicus influenced heifers is a challenge. recent studies148-149 suggest that long term progestin-based (mga-pg and 14 day cidr-pg) protocols may be used effectively in synchronizing estrus in bos indicus-influenced heifers prior to natural service or ftai. these protocols appear to perform similarly among bos indicus-influenced heifers compared to bos taurus heifers, when results are considered on the basis of pubertal status of heifers at the time treatments are initiated. there is evidence to suggest that short term progestin-based protocols (5 and 7 day cosynch + cidr) currently used in bos taurus heifers perform poorly when used to synchronize estrus in bos 276clinical theriogenology • volume 11, number 3 • september 2019 indicus-influenced heifers.150-151 however, results with short term protocols also seem to be highly influenced by pubertal status of heifers prior to treatment initiation.144, 152 although short term protocols may effectively induce estrous cyclicity in pre or peripubertal heifers,46-47 an advantage of long term protocols may be that heifers are not exposed for breeding or inseminated on the first pubertal estrus.153-154 currently, only 1 protocol is recommended by the brtf for bos indicus-influenced cows: the pg 5 day cosynch + cidr or “bee-synch” protocol151 (figure 12). this protocol requires that pg and gnrh be administered concurrent with cidr insertion to induce luteolysis among cyclic cows, reduce circulating concentrations of progesterone during treatment and enhance follicular development. administering pg at cidr insertion alters the timing of estrous response compared to the standard 5 day cosynch + cidr protocol, so ftai is performed at 66 hours following cidr removal. split time artificial insemination thomas et al.155-156 tested the hypothesis that pregnancy rates in beef heifers and cows to timed ai may be improved by delaying insemination of females that fail to express estrus prior to the time ftai is scheduled to be performed. this approach, termed split time artificial insemination (stai), improved pregnancy rates compared to ftai following administration of the 14 day cidr-pg protocol for heifers and the 7 day cosynch + cidr protocol for cows. when stai is performed, activation status of an estrus detection aid at the time of ftai determines whether ai is performed at the standard time or delayed until 20 24 hours later. heifers and cows were administered gnrh at 66 hours, irrespective of estrous status in the original field trials that compared stai and ftai. the working hypothesis in those studies155-156 was that delayed insemination of nonestrous females would better align the timing of insemination with the timing of gnrh-induced ovulations. this approach to breeding management increased overall pregnancy rates. figure 12. the pg 5 day cosynch + cidr® (bee synch) protocol for bos indicus influenced beef cows consists of: 5 day cidr insert (1.38 g progesterone); prostaglandin f2α (pg) and gonadotropin releasing hormone (gnrh) administration at cidr insertion; and pg administered at cidr removal and again 8±2 hours later. ftai is performed 66 hours following the first injection of pg, concurrent with gnrh administration.151 later experiments157-161 (figure 13) evaluated the optimal timing of gnrh administration when using stai. results from these studies clearly demonstrated that administration of gnrh was not required for heifers or cows that exhibit estrus prior to ai and that administration of gnrh to females that fail to exhibit estrus could occur concurrent with insemination at the delayed time point. this work confirmed that higher pregnancy rates resulting from stai compared to ftai are due primarily to higher total rates of estrous response prior to insemination, in contrast to the theory that timing of insemination is more optimally aligned relative to gnrh-induced ovulations. despite the high overall estrous response that is observed in heifers and cows when split time ai is practiced following administration of 14 day cidr-pg and 7 day cosynch + cidr protocols, a percentage of females still fail to exhibit estrus by the delayed time point.157-159 pregnancy rates resulting from ai are generally reduced among females that fail to exhibit estrus,125 despite the fact gnrh is routinely administered concurrent with ai. 277 clinical theriogenology • volume 11, number 3 • september 2019 knickmeyer et al.162 designed experiments to evaluate pregnancy rates resulting from ftai or stai among beef heifers following administration of the mga-pg and 7 day cosynch + cidr protocols in heifers. total estrous response increased among beef heifers assigned to stai versus ftai treatments for mga-pg (stai 88%; ftai 72%) and 7-day cosynch + cidr (stai 74%; ftai 47%) protocols, respectively. the increased estrous response resulting from stai was associated with a corresponding increase in pregnancy rates to ai following the mga-pg protocol; however, similar improvements in pregnancy rate were not observed following the 7 day cosynch + cidr protocol. differences in results between protocols may have occurred as a result of differences following treatment with long versus short term progestin-based protocols (figure 13). although short term treatment with a progestin was reported to effectively induce estrous cyclicity in pre or peripubertal heifers46-47,60 there may be an advantage to long term treatments when considering resulting pregnancy outcomes.153-154 this point is worth considering based on the observation that fertility associated with the first pubertal ovulation is typically reduced compared to subsequent ovulations.153-154 long term progestin-based protocols may offer an advantage over short term protocols in that inseminations are not performed on the pubertal estrus and improvements obtained using a split time ai approach have been observed among heifers only following long term protocols. figure 13. the 14 day cidr-pg and mga-pg protocols with split time ai for heifers and 7-day cosynch + cidr protocol with stai for cows.157-159;162-163 estrotect patches are applied at pg treatment. females with activated patches at 66 hours are inseminated without administering gnrh at ai. nonestrous females at 66 hours are inseminated 20 24 hours later and gnrh is only administered to females at this point that failed to exhibit estrus. conclusion ensuring long term sustainability of us beef cattle operations will require continued research of new technologies in reproduction and effective integration of these technologies with other disciplines, including genomics, health, and economics. given the anticipated growth in global population and declining availability of natural resources, application of effective technologies will be critical as 278clinical theriogenology • volume 11, number 3 • september 2019 agriculture seeks ways to improve efficiencies of production. outcomes stemming from use of the various protocols and breeding strategies outlined in this review are remarkable and exemplify high-impact transfer of fundamental research to industry end-users. as innovations in reproductive technologies continue to evolve, the need for additional applied research efforts is without question. moving forward, research efforts should focus on developing: 1) improved methods of estrous cycle control for bos indicus influenced females; 2) improved strategies to expand use of sexed semen; 3) ways in which to distinguish sires based on fertility differences when used in conjunction with ftai; and 4) ways in which to more effectively translate improvements in technology to industry partners. conflict of interest there are no conflicts of interest to declare. references 1. dziuk pj, bellows ra: management of reproduction in beef cattle, sheep and pigs. j anim sci 1983;57 suppl 2:355. 2. patterson dj, mallory da, nash jm, et al: strategies to optimize use of ai in cow/calf production systems: focus on fixedtime ai protocols for heifers. proc applied reproductive strategies in beef cattle 2011; p.11-40. 3. patterson dj, da mallory, 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setpagedevice 1 contact michela ciccarelli michela.ciccarelli@wsu.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9319, http://dx.doi.org/10.58292/ct.v15.9319 case report diaphragmatic hernia involving gravid uterus in a dog michela ciccarelli,a,b tania perez,a william dernella adepartment of veterinary clinical science, college of veterinary medicine, washington state university, pullman, wa; bschool of molecular bioscience, college of veterinary medicine, washington state university, pullman, wa abstract a 4-year german shorthair pointer pregnant dog was presented for severe tachypnea and depression. she had 1 natural breeding 52 days ago. diaphragmatic hernia with intrathoracic displacement of the uterus was diagnosed via radiography and ultrasonography. emergency herniorrhaphy was elected, and the owner requested to save the litter if possible. right uterine horn tip, spleen, part of liver, pancreas, and part of greater omentum were in the thorax. intraoperative transuterine ultrasonography confirmed live fetuses, and pregnancy was permitted to continue. patient received supportive medical care for 3 days. based on ultrasonography findings (eminent danger to litter), a decision was made to perform an emergency cesarean surgery (56 days after breeding). six of 11 pups were alive, and 1 mummified fetus had an elongated neck (perhaps due to entrapment in the diaphragmatic hernia). keywords: dog, pregnancy, herniorrhaphy, cesarean surgery, ultrasonography background diaphragmatic hernia is the passage of abdominal organs into thoracic space through a diaphragmatic defect. it can occur as a congenital defect or secondary to trauma.1 traumatic diaphragmatic hernias are most commonly caused by car accidents (62–81%)2; less common causes are bite wounds and falls from a height. traumatic muscular rupture or avulsion of diaphragm permits abdominal contents to enter the pleural cavity, resulting in reduced lung volume and subsequent dyspnea.3 liver is the most common organ to herniate (82–88%),4,5 and its pressure on lungs further compromises lung expansion, leading to atelectasis and pleural effusion.3 entrapped intestines or stomach may become gas distended, obstructed, or ischemic, resulting in metabolic disturbances, decreased cardiac return, and sepsis.1 animals with acute traumatic diaphragmatic hernias may be presented in severe respiratory distress and shock and may require emergency surgery. concurrent traumatic lesions are present in 25–38% of the cases.6 in chronic diaphragmatic hernias, clinical signs may be nonspecific (e.g. vomiting or weight loss), requiring a complete evaluation to reach a definitive diagnosis.5 abdominal radiographs often provide a ready diagnosis of herniation, especially in an emergency setting. animals with a traumatic diaphragmatic hernia may have an indistinct diaphragmatic line, pleural effusion, dorsal displacement of lungs, and abnormal soft tissue opacities within the pleural space.7 right side hernias were reported to be more common (68%) and usually characterized by more than 1 organ herniated in the pleural space.7 erect dorsoventral radiographic images may improve the shift of the organ and visualization. if the gastrointestinal tract is displaced in the chest, gastrointestinal contrast study may be helpful to confirm the diagnosis.8 wholebody computed tomography, although costly, has the ability to diagnose herniation and possible concurrent orthopedic, neurologic, and intra-abdominal injuries.9 as an alternative to computed tomography, transabdominal ultrasonography may aid the diagnosis. abdominal organs may be visualized in the thoracic cavity close to heart.8 surgical repair is often the chosen treatment; however, it must be delayed until the patient is stable. high mortality rate (33%) was reported when the herniorrhaphy was performed within 24 hours after presentation.1 animals with chronic traumatic hernias are often referred to experienced surgeons for treatment, particularly when the liver is involved, because *presented (abstract) at the society for theriogenology 2022 conference; authors acknowledge that right uterine horn was herniated (not left uterine horn as published in the conference proceedings), and error is regretted. mailto:michela.ciccarelli@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9319 2 citation line: clinical theriogenology 2023, 15, 9319, http://dx.doi.org/10.58292/ct.v15.9319 of extensive adhesions. in some of these patients, a prosthetic implant, transversus abdominal muscle flap, or diaphragmatic advancement may be required to close the hernia.5 to our knowledge, apparently, no previous reports documented herniation of pregnant uterus into thoracic space, pregnancy continuation, and delivery of viable pups after herniorrhaphy. case presentation a 4-year 34 kg pregnant female german shorthair pointer was referred for severe respiratory distress that started 2 days before presentation. she had 1 natural breeding 52 days ago. owner reported trauma of unknown origin a year previously that led  to pleural effusion and respiratory distress that were treated medically. at presentation, the patient was quiet and alert and had hyperemic mucous membranes. abdomen felt soft and nonpainful, and fetuses were palpable in the caudal abdomen. her heart rate was 100 beats/minute, and normal sinus arrhythmia was noted. there was no enlargement of her peripheral lymph nodes, and her rectal temperature was 38°c. there were muffled lung sounds of the ventral lung lobes on auscultation. she was ~ 5% dehydrated. radiographs revealed substantial amount of soft tissue presence in the pleural space. right lung lobes appeared dorsally displaced, and an abnormally mineralized fetus was superimposed over the caudal thorax. several other normal fetuses were in the cranial abdomen (figure 1). all above findings confirmed a suspected diaphragmatic hernia involving pregnant uterus. intravenous lactated ringer’s solution (vetivex® [veterinary phylyte injection 7.4; balanced electrolyte solution] 140 ml/kg/24 hours) was given for hypovolemia. oral tramadol (2 mg/kg), intravenous acepromazine (0.02 mg/kg), and intravenous butorphanol (0.4 mg/kg) were given for pain control and tranquilization. it was estimated that the patient had at least 1 week to whelp. this was based on breeding history (1 breeding based on 1 progesterone concentrations [5.6 ng/ml] determination), calculation of biparietal diameter (based on average [24 mm] of 2 pups), and a lack of fetal intestinal peristalsis evaluated via ultrasonography. due to the large litter size (11–12 pups, determined via radiographs) and fetal rapid growth during the final weeks of pregnancy, increased compression on the lungs and additional patient compromise was predicted. treatment surgery for hernia repair was deemed necessary. blood was drawn to evaluate progesterone concentrations before herniorrhaphy to aid in deciding on whether to maintain pregnancy. progesterone concentrations were 6.37 ng/ml, which is sufficient to maintain pregnancy.10 after discussing options, owner elected herniorrhaphy and pregnancy maintenance. preoperative blood work revealed the following: anemia (packed cell volume 30% [reference range{rr}: 36–56%]), hypocalcemia (8.4 mg/dl [rr: 9–11.3 mg/dl]), hyponatremia (147 meq/l [rr: 149–158 meq/l]), hypochloremia (110 meq/l [rr: 112–119 meq/l]), slightly lower creatinine (0.6 mg/dl [rr: 0.8–1.6 mg/dl]), albumin (2.1 g/dl [rr: 2.9–3.8 g/dl]), and total proteins (5.1 g/dl [rr: 5.5–7.5 g/dl]). chest tubes were placed bilaterally for aspiration of thoracic fluid. preoperative medications included intravenous methadone (0.5 mg/kg) and cefazolin (22 mg/kg). general anesthesia was induced with intravenous propofol (0.88 mg/kg) and maintained with sevoflurane in 100% oxygen. patient was kept on mechanical ventilation (5 breaths/minute). a ventral midline celiotomy was performed. left uterine horn was exteriorized, a 7 cm tear was noted on the right side extending from the tendinous portion of the diaphragm into the pars sternalis. thoracic cavity had right uterine horn tip, spleen, right medial, and lateral lobes of liver, pancreas, adjacent small intestine, and part of omentum. herniorrhaphy was performed using lembert and simple continuous patterns. a rotational flap of the internal oblique muscle aided in the closure of the most ventral portion of the hernia. porcine submucosa was sutured over the herniorrhaphy for additional reinforcement. bronchodilators, inhalation albuterol (1 puff [90 µg]), and intravenous terbutaline (0.005 mg/kg) were given to improve oxygenation. before closure, intraoperative transuterine ultrasonography was performed. four fetuses were visualized; fetal heart rates ranged from 60 to 70 beats/minute that indicated fetal stress (attributed to uterine circulation compromise and anesthesia). routine closure of the linea alba was performed. postoperative analgesia included a continuous rate infusion of intravenous fentanyl (3 µg/kg/1 hour) and intravenous methadone (0.25 mg/kg) as needed. intravenous lactated ringers (vetivex® [140 ml/kg/2 hours]) and 6% hydroxyethyl starch 130/0.4 in 0.9% sodium chloride (vetstarch™ zoetis [31 ml/kg]) for 1 hour were given to maintain normal volemia. antibiotic therapy consisted of intravenous ampicillin (22  mg/kg, once every 6 hours) and amoxicillin-clavulanic acid (20 mg/kg, once every 12 hours). a day after surgery, 1 ml (0.088 mg/kg) of oral altrenogest was empirically given to aid in maintaining uterine quiescence and prevent premature delivery. thoracic fluid was continuously drained. temperature, heart rate, respiratory rate, packed cell volume, total proteins, blood glucose, blood urea nitrogen, lactate, and fetal well-being were monitored every 2 hours. fetal heart rates returned to normal, which ranged from 204 and 260 beats/minute 2 hours after recovery. however, 36 hours after surgery, 3 fetuses had no heartbeat, and 3 others had heart rates between 204 and 275 beats/minute. blood chemistry and complete blood count (cbc) revealed hypoalbuminemia (2.1 g/dl [rr: 2.9–3.8 g/dl]), leukocytosis (28.1 × 103/μl [rr: 4.5–16 × 103/μl]) with a left shift neutrophilia (segmented neutrophils 22.480 × 103/μl [rr: 2.8–13.4 × 103/μl]), and elevated liver enzymes (alanine aminotransferase 878 u/l [rr: 0–100 u/l] and alkaline phosphatase 107  u/l [rr: 0–96 u/l]). due to inflammatory figure 1. lateral thoracic radiograph. note displaced lung lobes and mineralized fetal skeletons (arrows) in thorax. http://dx.doi.org/10.58292/ct.v15.9319 citation line: clinical theriogenology 2023, 15, 9319, http://dx.doi.org/10.58292/ct.v15.9319 3 leukogram associated probably with dead fetuses and previous herniorrhaphy, an emergency cesarian surgery was elected. due to urgency to have the procedure and to avoid fetal depression, premedication was avoided, and general anesthesia was induced with intravenous propofol (4 mg/kg) and maintained with sevoflurane in 100% oxygen. celiotomy through the previous incision was performed. uterus was exteriorized, fetuses were delivered, and ovariohysterectomy was performed. six pups were resuscitated with mild effort and immediately placed in a pup incubator with controlled temperature (29.4–32.2°c), oxygen (40–60%), and humidity (55–65%). plasma (1.5 ml/100 g body weight) typed for the patient was given subcutaneously to prevent passive transfer failure. pups were fed by an orogastric tube using a milk replacer (1–2 μl/100 g body weight). average body weight was 70 g. patient was given intravenous methadone (0.25 mg/kg) for pain control and intravenous cefazolin (22 mg/kg). patient also received intravenous lactated ringers (vetivex® [140 ml/ kg/24 hours]) and intravenous vetstarch™ (31 μl/kg/1 hour). a blood transfusion (150 ml whole blood) was given to restore packed cell volume and total protein concentrations. patient recovered uneventfully from the second surgery; however, she was not producing milk. over the 24 hours after cesarean surgery, an increase in the mammary gland development and milk production was observed, so she was weaned off intravenous medications and reunited with her pups. outcome six of 11 pups were successfully resuscitated; 2 were severely autolyzed, and 1 was mummified with neck incarceration in the hernia (figure 2). other 3 nonviable pups were in good postmortem condition, consistent with recent death. apgar score of pups delivered alive was 4–5. patient remained stable and began to eat the day after cesarean surgery, and she was discharged from the hospital 48 hours later. dog and pups were doing well at examination on day 15. discussion diaphragmatic hernia in dogs is usually caused by trauma and  may be life-threatening if not diagnosed immediately. however, in this case, there are situations in which less severe herniation becomes chronic and remains undiagnosed.5 it was speculated that incarceration of 1 of the growing fetuses in the diaphragmatic lesion occluded the defect, allowing initially normal respiration. however, with pregnancy advancement, additional pressure from the enlarging uterus forced more abdominal organs into the thoracic cavity, jeopardizing the lives of dog and pups. pregnancy complicated anesthesia induction, maintenance, and monitoring. however, for both surgeries performed in this patient, the anesthetic protocol was chosen carefully considering the anesthetized patient’s stability and the survival of the near-term fetuses. general anesthesia in pregnant dogs is best induced with injectable anesthetics that allow smooth induction and rapid intubation. in this report, the patient was induced with propofol because of its rapid onset and minimal residual effect on pups.11–13 although propofol crosses the placenta and is eliminated slower by pups than dog, it stimulated lesser neurologic depression than pups delivered from dogs induced with ketamine and midazolam.14 compared to other drugs, propofol had no negative effect on pup breathing, movement, or vocalization after delivery via cesarean surgery.15 in this case, sevoflurane was used to maintain anesthesia because it has a very rapid induction and recovery, and it is rapidly eliminated by the neonate. isoflurane can be used if sevoflurane is not available since it has been associated with better neonatal survival at 7 days compared to methoxyflurane, xylazine, and ketamine.16 however, it may have dosedependent side-effects like vasodilation, resulting in hypotension and respiratory depression. in general, inhaled anesthetics has been associated with decreased breathing in pups after cesarean surgery.15 local epidural would have been helpful in this case to decrease the amount of anesthetics necessary and reduce fetal absorption. it has been demonstrated that epidural anesthesia produced the least neurologic and respiratory depression in neonates after cesarean surgeries compared to injectable or inhaled anesthetics.14 to reduce pain associated with surgery, opioids are frequently used because they minimally affect cardiac output, systemic blood pressure, and oxygen delivery. however, they can induce bradycardia in pups after delivery requiring an opioid antagonist (e.g. naloxone) treatment.17 as the patient was experiencing moderate pain in the present report, methadone was given after pups delivery. most common complication after diaphragmatic hernias repair is pneumothorax, and the recurrence of pulmonary edema was preemptively avoided with bronchodilators treatment. in dogs, the extremely variable estrous cycle length and the prolonged time that sperm can remain viable in the female reproductive tract lead to whelping from 57 to 72 days after breeding.18 serum progesterone concentrations measured during proestrus to determine luteinizing hormone (lh) surge allow for the most accurate estimate of the whelping day. parturition is expected 65 ± 2 days after lh surge.19,20 stillbirth and perinatal death are common in preterm delivery; in fact, just before parturition, the physiologic increases in cortisol concentrations are fundamental for final pulmonary maturation. thus, premature pups present with respiratory distress due to lung structure immaturity and lack of pulmonary surfactant leading to hypoxia and death. the highest mortality rate is if they are delivered ≥ 5 days before the due date.21 in this case, the only information available from the history was the breeding date, and figure 2. mummified fetus. note the elongation of neck and incarceration. http://dx.doi.org/10.58292/ct.v15.9319 4 citation line: clinical theriogenology 2023, 15, 9319, http://dx.doi.org/10.58292/ct.v15.9319 determination of 1 progesterone concentrations (5.6 ng/ml) on day 2 before breeding made it impossible to determine the exact due date. pregnancy length was determined via radiography and ultrasonography. radiographs have mineralization of some fetal structures that can help determine a range of pregnancy length. however, the fetuses may be completely mineralized as early as 58 days after lh surge, at which time, they would not survive extrauterine life.19,22 ultrasonography is considered a better tool to assess pregnancy length and fetal maturity. in late pregnancy, measurement of biparietal diameter of fetuses is considered accurate, together with assessment of body diameter.23,24 in the present case, biparietal diameter of the fetuses was, on average 24 mm, suggested that the patient was 8 days from parturition.25 ultrasonography is also used to evaluate organ development and estimate pregnancy length. best organs to evaluate pregnancy end are fetal gastrointestinal tracts and kidneys. intestines will start having peristalsis at 62–64 days of pregnancy that persisted near term.25 kidneys have a clear differentiation of the cortex and medulla closer to term.26 in the present case, repeated ultrasonographic evaluations were performed, primarily to appreciate fetal stress and the presence or absence of heartbeat. more attention should have been given to fetal organ development to better estimate pregnancy length. the presence of live fetuses observed intraoperatively during the herniorrhaphy procedure allowed us to decide whether pregnancy can be maintained. normal heart rate of a canine fetus is between 220 and 240 beats/minute.27 heart rates < 140–160 beats/minute indicate stress due to hypoxia. in the present case, the fetal heart rates were lower during the hernia repair; however, they returned to normal immediately after surgery. ultrasonographic evaluation 2 days after surgery identified the presence of dead fetuses that led to the emergency cesarean surgery. an important consideration of this case is the risk taken in performing the hernia repair while maintaining pregnancy. pregnant animals are at a higher risk of death and abortion when undergoing surgery. this is due to many physiologic changes that pregnancy induces in the body, including increased plasma volume with a resulting decrease in packed cell volume and total proteins, leading to decreased oncotic pressure, decreased protein binding drugs, and increased cardiac output. increased abdominal pressure leads to decreased venous return and less uterine perfusion. change in gastrointestinal tract ph leads to vomiting, reflux, and aspiration under anesthesia. another factor exacerbating abdominal surgery risk is the dorsal recumbency of the patient that induces vena cava compression with an additional decrease in venous return, maternal hypotension, and decreased perfusion of the placenta. in the present case, the patient was already severely compromised due to diaphragmatic hernia. therefore, some pups may have died during the hernia repair for reasons discussed above. additionally, pregnant uterus was manipulated and kept outside the abdomen for some time during herniorrhaphy that could have caused uterine contractions (reason for giving altrenogest to the patient after surgery). apparently, there are only a few case reports describing diaphragmatic herniation of pregnant uterus in dogs, and they reported either negative or unknown outcomes.28 first report described a 2-year maltese dog with dyspnea and cyanosis that had 1 gravid horn herniating into thoracic cavity. dog made a complete recovery after diaphragmatic hernia repair; however, there was no information regarding fetal outcome.29 a second case described a 17-month afghan dog, bred 59 days before presentation, that was a victim of an accident that led to episodes of acute respiratory distress. lateral radiograph revealed fetal skeleton in the chest. one viable pup was delivered via cesarean surgery. pup attempted to suckle after surgery; however, it was euthanized shortly thereafter due to a cleft palate, although the dog made a complete recovery.30 another report involved a 3-year intact female pointer that was examined for anorexia, lethargy, and green vaginal discharge. cranial abdominal and thoracic radiographs revealed a peritoneal-pericardial diaphragmatic herniation with 1 mineralized fetus in the pericardial sac. dog died shortly after radiographs were taken, and the diagnosis was confirmed on postmortem examination.31 a recent report32 documented a mixed breed pregnant dog that was adopted and presented for a 10-day duration of breathing effort. radiographs revealed that 4 pups were in the chest and 3 in the abdomen. dog underwent an emergency herniorrhaphy and ovariohysterectomy and had 6 live pups.32 the dog also had chronic diaphragmatic hernia with herniation of the gravid uterus.28 dog was hit by a car 1 year before and was presented for severe dyspnea. pups were unwanted, so the dog underwent emergency herniorrhaphy and ovariohysterectomy.28 apparently, there are no reports of herniation of pregnant uterus into the thoracic space and pregnancy continuation, and delivery of healthy pups after herniorrhaphy. learning points • day of ovulation is estimated by detecting lh surge; however, the determination of exact day of ovulation or day 1 of diestrus is the most accurate method to ascertain fetal readiness for birth. • if day of ovulation was not determined, ultrasonographic fetal measurements and organ development coupled with radiographic findings may aid in estimating the whelping date and to time cesarean surgery to deliver viable pups. • anesthetized dog’s stability and the well-being of nearterm fetuses have to be considered in formulating anesthetic protocols. • intraoperative transuterine ultrasonography can be safely performed to assess fetal viability. conflict of interest authors disclose no conflicts of interest. references 1. boudrieau rj, muir ww: pathophysiology of traumatic diaphragmatic hernia in dogs. compend contin educ pract vet 1987;9:379–385, 386. 2. gibson twg, brisson ba, sears w: perioperative survival rates after surgery for diaphragmatic hernia in dogs and cats: 92 cases (1990–2002). j am vet med assoc 2005;227:105–109. doi: 10.2460/javma.2005.227.105 3. worth aj, machon rg: traumatic diaphragmatic herniation: pathophysiology and management. compend contin educ pract vet 2005;12:178–191. http://dx.doi.org/10.58292/ct.v15.9319 http://dx.doi.org/10..2460/javma.2005.227.105 citation line: clinical theriogenology 2023, 15, 9319, http://dx.doi.org/10.58292/ct.v15.9319 5 4. wilson gp, newton cd, burt jk: a review of 116 diaphragmatic hernias in dogs and cats. j am vet med assoc 1971;159:1142–1145. 5. minihan ac, berg 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comparison of real-time ultrasound, palpation and radiography in pregnancy detection and litter size determination in the bitch. vet radiol 1986;27:102–108. doi: 10.1111/j.1740-8261.1986.tb00013.x 23. kutzler ma, yeager ae, mohammed ho, et al: accuracy of canine parturition date prediction using fetal measurements obtained by  ultrasonography. theriogenology 2003;60:1309–1317. doi: 10.1016/s0093-691x(03)00146-8 24. beccaglia m, luvoni gc: comparison of the accuracy of two ultrasonographic measurements in predicting the parturition date in the bitch. j small anim pract 2006;47:670–673. doi: 10.1111/j.1748-5827.2006.00108.x 25. lopate c: estimation of gestational age and assessment of canine fetal maturation using radiology and ultrasonography: a review. theriogenology 2008;70:397–402. doi: 10.1016/j.theriogenology. 2008.05.034 26. nyland tg, mattoon js: small animal diagnostic ultrasound. elsevier health sciences; new york, 2002. 27. gil emu, garcia daa, giannico at, et al: canine fetal heart rate: do accelerations or decelerations predict the parturition day in bitches? theriogenology 2014;82:933–941. doi: 10.1016/j. theriogenology.2014.04.025 28. deschamps jy, kolb h, descol c, et al: chronic diaphragmatic hernia with herniation of the gravid uterus in a female dog – a case report and a review. rev med veterinaire 2012;163:299–301. 29. sullivan jl, puckett jr, sevedge jp: ruptured diaphragm with herniation of the gravid uterus and abdominal viscera. j am vet med assoc 1969;155:941–942. 30. bellenger cr, milstein m, mcdonell w: herniation of gravid uterus into the thorax of a dog. mod vet pract 1975;56: 553–555. 31. iwasaki m, sterman fda, fonseca ac: what is your diagnosis? peritoneopericardial diaphragmatic hernia with herniation of mineralized fetuses. j am vet med assoc 1999;214:1775–1776. 32. merbl y, kelmer e, shipov a, et al: resolution of persistent pneumothorax by use of blood pleurodesis in a dog after surgical correction of a diaphragmatic hernia. j am vet med assoc 2010;237:299–303. doi: 10.2460/javma.237.3.299 http://dx.doi.org/10.58292/ct.v15.9319 http://dx.doi.org/10..2460/javma.2005.227.105 http://dx.doi.org/10..2460/javma.2003.222.1237 http://dx.doi.org/10..4142/jvs.2004.5.2.157 http://dx.doi.org/10..1016/j.theriogenology.2008.04.038 http://dx.doi.org/10..1111/j.1365-2044.1989.tb09263.x http://dx.doi.org/10..1111/j.1365-2044.1989.tb09264.x http://dx.doi.org/10..1111/j.1365-2044.1989.tb09264.x http://dx.doi.org/10..1136/vr.154.13.387 http://dx.doi.org/10..5326/0380090 http://dx.doi.org/10..5326/15473317-36-4-359 http://dx.doi.org/10..1016/j.cvsm.2004.10.012 http://dx.doi.org/10..1111/j.1740-8261.1984.tb01924.x http://dx.doi.org/10..1111/rda.13669 http://dx.doi.org/10..1111/j.1740-8261.1986.tb00013.x http://dx.doi.org/10..1016/s0093-691x(03)00146-8 http://dx.doi.org/10..1111/j.1748-5827.2006.00108.x http://dx.doi.org/10..1016/j.theriogenology.​2008.05.034 http://dx.doi.org/10..1016/j.theriogenology.​2008.05.034 http://dx.doi.org/10..1016/j.theriogenology.2014.04.025 http://dx.doi.org/10..1016/j.theriogenology.2014.04.025 http://dx.doi.org/10.2460/javma.237.3.299 11 contact jennifer koziol jkoziol@ttu.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9642, http://dx.doi.org/10.58292/ct.v15.9642 review report practical review of diagnosis, treatment, and prognostic indicators of acquired conditions of the penis and prepuce in the bull jennifer koziol texas tech school of veterinary medicine, amarillo, tx, usa abstract this review will discuss the most common acquired conditions of the penis and prepuce in the bull along with diagnosis, treatment, and prognosis for each of those conditions. conditions discussed will include preputial lacerations, penile hematomas, retropreputial abscesses, phimosis/paraphimosis, and deviations of the penis. it is the intent of this manuscript to arm practitioners with clinically important knowledge regarding medical treatment and prognosis so that they may assist producers in the decision-making process following the identification of a reproductive injury or defect in a bull. this manuscript will not discuss surgical interventions in depth but will discuss when surgical intervention is warranted. keywords: bovine, penis, prepuce, preputial trauma introduction breeding bulls can present to the veterinarian for a variety of issues and concerns involving the penis and prepuce. however, some conditions of the penis and prepuce may not be readily evident to the owner and may only be noted by the veterinarian during a routine bull breeding soundness exam. regardless of the presentation this review will highlight the most noted acquired conditions of the penis and prepuce. it is the intent of this review to be helpful to the practitioner in the diagnosis, medical treatment and communicating a future prognosis for breeding bulls that are diagnosed with one of the following acquired conditions. anatomy of the sheath, prepuce, and penis the sheath of the bull is considered a double invagination of skin that contains the nonerect penis.1 the diameter and length of the prepuce vary from bull-to-bull and more significantly from breed to breed with bos indicus and bos indicus-influenced bulls having significantly more sheath and prepuce length as compared to bos taurus bulls. the average length of the prepuce in bulls is 35–40 cm and ~4 cm in diameter. there is a gradual transition from the haired sheath to the nonhaired prepuce. the prepuce terminates at the area of the preputial ring where it joins the glans penis. there are many interdigitating layers of elastic tissue between the tunica albuginea of the penis and the epithelium of the prepuce which allows the penis to be freely movable within the sheath and go from the retracted state to fully extended and erect.1 anatomy and physiology of erection in the fibroelastic penis of ruminants, erectile tissues of the corpus cavernosum penis (ccp) are contained within a strong, relatively inelastic tunica albuginea. most of the blood contained within the ccp enters through the crura of the penis and all blood contained within the ccp must exit through the crura. following sexual stimulation, reflex dilation of arteries supplying the penis increases blood flow through the deep artery of the penis into the crura and subsequently into the ccp. the stimuli which increase blood flow through the crura also relax the retractor penis muscles, thereby removing traction from the sigmoid flexure and allowing the tip of the mildly engorged free portion of the penis to extend from the sheath. with continued sexual stimulation, the paired ischiocavernosus muscles overlying the crura initiate rhythmic contractions, compressing the crura against the ischium to occlude vascular flow into and out of the ccp, thereby creating a closed hydraulic tube. additional rhythmic contractions of the ischiocavernosus muscles increase the pressure of the blood contained within the crura and ccp to generate the high pressures required for full erection and extension. following ejaculation, the ischiocavernosus muscles relax and intrapenile pressures return to baseline as blood exits the ccp through the crura.2,3 mailto:jkoziol@ttu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9642 12 citation line: clinical theriogenology 2023, 15, 9642, http://dx.doi.org/10.58292/ct.v15.9642 preputial trauma eversion of the prepuce in bos taurus bulls carrying the polled gene, some degree of eversion of the prepuce is common when they are relaxed, although it is uncommon in horned bulls. bulls that are heterozygous or homozygous for the polled gene lack the paired retractor prepuce muscles which normally function to retain the internal lamina of the prepuce within the preputial orifice.4–6 in bos indicus and bos indicus-influenced breeds with a pendulous sheath conformation, the length of the internal lamina of the prepuce and the size of external os of the prepuce predispose bulls to eversion of the prepuce or preputial prolapse.7 in some affected bulls, 1.27 to 10.16 cm of preputial skin is commonly everted.7 however, the length of everted preputial skin demonstrated by some bulls may exceed what has been previously reported in literature. bulls with a high degree of preputial eversion are more subject to preputial lacerations, frostbite, and other trauma to the prepuce.8 if the exposed prepuce is free of lesions and the bull can retract the prepuce when stimulated, the everted prepuce should not affect the potential to breed.9 preputial lacerations bulls with a pendulous sheath and excessive preputial skin may traumatize preputial tissues separate from the breeding act. regardless, most preputial lacerations in bulls occur at the time of the ejaculatory lunge. as the free portion of the penis enters the vagina during coitus, preputial skin slides caudally up the shaft of the penis and folds of redundant skin gather at the preputial orifice. this bunching of preputial skin usually occurs without incident, but if the redundant preputial skin is entrapped between the bull’s abdomen and the bony pelvis of the female, it can be traumatized by the compressive forces during the ejaculatory lunge. in mild cases, the prepu tial epithelium remains intact and subsequent edema in the damaged tissues results in an uncomplicated preputial prolapse. however, if compression of the entrapped prepuce results in sufficient pressure, it can cause bursting of the epithelium and damage to underlying elastic tissues.10 lacerations of the prepuce with subsequent preputial prolapse are more common in bos indicus breeds and their crosses than in bos taurus bulls, due to the pendulous sheath, excessive length of prepuce, and/ or large preputial orifices in those breeds. even serious preputial injuries in bos taurus bulls often do not result in preputial prolapse.11 rupture of the epithelium of the prepuce at the time of the ejaculatory lunge predictably occurs longitudinal to the long axis of the ventral aspect of the prepuce, but the wound assumes a transverse orientation as the bull attempts to retract the penis into the preputial cavity. the resulting transverse orientation of the laceration effectively shortens the length of the traumatized tissue.11 following preputial injury, bulls of bos taurus breeds are usually able to retract the damaged prepuce into the preputial cavity and the injury goes unnoticed until cellulitis, abscessation, or stenosis occurs. in contrast, edema in the traumatized tissue frequently causes preputial prolapse in bos indicus type bulls. the dependent edema increases the size and weight of the prolapsed tissue, leading to prolapse of an additional portion of the prepuce, thereby worsening the condition.3 preputial prolapse is often more severe in naturally polled bulls, due to the lack of retractor prepuce muscles, as these muscles serve to elevate the prepuce and this elevation can minimize edema formation in damaged tissues.4–6 wolfe and carson constructed a useful 4-point classification scale that incorporates severity of the preputial injury to derive a prognosis for return to function and guide treatment decisions11 (table 1 and figure 1). goals of medical management of preputial lacerations and subsequent prolapse are to control sepsis, reduce edema, and eventually return damaged tissues to the preputial cavity. begin therapy by thoroughly cleaning and flushing the wound with dilute antiseptic, combined with gentle debridement of devitalized, necrotic tissue. following cleansing and debridement, an application of emollients and topical antibiotics can be applied to the preputial tissue. the purpose of this is to prevent further desiccation and reduce infection within tissues. ‘petercillin’ also known as ‘petermycin’ is a mixture of anhydrous lanolin, oxytetracycline, and scarlet oil and often used as an emollient and topical antibiotic treatment.11 however, scarlet oil is not an u.s. food and drug administration (fda) approved product and amduca only allows for extra label drug use for fda approved products. other topical treatments reported are the use of betadine sugar mixture as well as the use of silver sulfadiazine (ssd) cream. following application of topical preparation of choice, the injured tissues should be covered with bandaging. to begin preputial bandage, insert a latex tube into the preputial orifice to allow urine to egress from the prepuce. once the tube is correctly placed such that the proximal end is proximal to the proposed bandage, place an orthopedic stocking over table. preputial injury classifications by wolfe and carson.11 category description i simple preputial prolapse with slight-tomoderate edema without laceration, necrosis, or fibrosis. respond well to medical or surgical treatment. good prognosis for return to service. ii prolapsed prepuce has moderate-to-severe edema, may have superficial lacerations or slight necrosis, but no evidence of fibrosis. surgery is usual course of therapy. good to guarded prognosis. iii severe edema of the prolapsed prepuce with deep lacerations, moderate necrosis, and slight fibrosis. surgery is indicated with guarded prognosis. iv prolapsed prepuce has been exposed for an extended interval and there is severe edema, deep lacerations, deep necrosis, fibro sis, and often abscessation. culling of bull usual recommendation. guarded to poor prognosis follows surgery. http://dx.doi.org/10.58292/ct.v15.9642 citation line: clinical theriogenology 2023, 15, 9642, http://dx.doi.org/10.58292/ct.v15.9642 13 the exposed tissue. then apply a snug elastic tape bandage beginning at the distal end of the prolapsed tissue and working proximally, overlapping the tape as it advances up the prolapsed tissue and preputial orifice to the sheath where it can be adhered to haired skin3,11 (figure 2). ensure that the bandage applies sufficient compression to reduce edema but does not strangulate tissues. for bulls with a very pendulous prolapse and severe edema, utilization of a sling made of net or burlap allows the prepuce to be held close to the abdomen to encourage lymphatic flow and reduction of edema (figure 3). frequent bandage changes are necessary, and when bandages are changed, perform 15–20 minutes of hydrotherapy to the wound to assist in edema reduction and removal of devitalized tissue. some bulls with minor injuries may return to service without surgery, although recurrent injuries are common.11 surgical resection and anastomosis of the preputial scar can improve outcomes and are indicated when the bull’s value warrants the investment.12–14 surgical outcomes are improved by preoperative wound management, as described above. retropreputial abscesses retropreputial abscess formation following preputial laceration occurs more commonly in bulls of bos taurus breeds than in bos indicus breeds. following preputial injury, bos taurus bulls often retract the penis into the sheath so that prolapse of the prepuce does not occur. consequently, the injury commonly goes unnoticed until the laceration becomes contaminated and cellulitis and abscessation ensue (figure 4). needles should never be introduced transdermally into the abscess. phlegmon may extend from the preputial orifice to the scrotum, or the swelling may be confined to a small, well-defined area along the sheath. retropreputial abscesses can be differentiated from penile hematomas as retropreputial abscesses are located more distally along the shaft of the penis closer to the preputial orifice and are seldom symmetrical as compared to a penile hematoma. careful physical examination of the swelling is warranted as abdominal abscesses may occur near the sheath but not directly communicate with the sheath and prepuce giving the bull a much better prognosis for recover. ultrasound may be useful in these cases to determine extent of damage and associated anatomical landmarks. treatment options are limited to systemic antibiotic therapy and local treatment with cold-water hydrotherapy to reduce edema. occasionally drainage into the preputial cavity can be established at the site of the disruption of the preputial skin. figure 1. grade 3 preputial prolapse. figure 2. bandaging of prepuce: a urine egress tube has been placed within the preputial cavity. the tube and sheath have been covered with an orthopedic stocking and covered in an elastic type bandage material. figure 3. burlap cloth is used to elevate the pendulous prepuce. the burlap cloth can be secured with elastic bungee cords. http://dx.doi.org/10.58292/ct.v15.9642 14 citation line: clinical theriogenology 2023, 15, 9642, http://dx.doi.org/10.58292/ct.v15.9642 efforts to use endoscopy to induce drainage into the preputial cavity have met limited success (personal communication, c. armstrong). retropreputial abscesses should never be drained through the skin of the sheath as the procedure will likely extend the infection through the peripenile elastic layers and increase subsequent adhesion formation. prognosis for return to breeding soundness is always guarded for bulls with a retropreputial abscess, as adhesions within the elastic tissues or between the elastic layers and the skin of the sheath can interfere with penile extension. only a small percentage of bulls heal sufficiently to allow normal penile extension and return to service. penile hematoma in bulls, the term hematoma of the penis is used to indicate the condition which follows rupture of the tunica albuginea of the penis. this condition is also referred to as a broken or fractured penis. during normal erection, venous outflow to the ccp is obstructed and contraction of the ischiocavernosus muscles increases pressure within the penis to approximately 14,200 mmhg.2,15 during the normal coital act, the bull mounts, makes one or two searching motions with the penis to locate the vulva, and achieves intromission and ejaculation with a single forceful lunge. if the penis is outside the vulva during the ejaculatory lunge and is thrust against the escutcheon of the female or if the female collapses during coitus, severe angulation of the penile shaft drastically increases the intracorporeal pressure to greater than 70,000 mmhg, a level sufficient to rupture the tunica albuginea.16 the most common site for rupture is on the dorsal surface of the distal sigmoid flexure, just above the point of insertion of the paired retractor muscles, although in rare instances it may occur at other locations.17,18 the erect penis contains less than 250 ml of blood at the time of rupture of the tunica albuginea. this blood is under pressure at the time of rupture and forcefully enters the peripenile elastic tissue, creating a symmetrical swelling in the sheath immediately cranial to the base of the scrotum. the hematoma may grow if repeated sexual stimulation induces additional attempts at erection due to continued leakage of blood through the rent in the tunica albuginea. occasionally, the swelling may extend caudally along the retractor penis muscles. hematoma of the penis is readily diagnosed by physical examination and ultrasound and can be differentiated from a retropreputial abscess by the typical location and symmetry of the swelling. mild to moderate prolapse of the prepuce, which may have a distinct bluish tint secondary to the subcutaneous blood, is often the first sign of injury that an owner notices, albeit it may not be present in every bull. this prolapse of the prepuce occurs secondary to edema. subcutaneous hemorrhage may be evident on the sheath near the scrotum in light-colored bulls. palpation of the symmetrical swelling on the dorsum of the distal bend of the sigmoid flexure of the penis confirms rupture of the tunica albuginea. the penis will not extend due to swelling within elastic tissues. do not attempt to manually extend the penis or use an electroejaculator in cases of rupture of the tunica albuginea, as such efforts may exaggerate damage to the already damaged elastic tissues. rupture of the tunica albuginea may result in loss of reproductive function. multiple complications following rupture of the tunica albuginea may occur and include: abscess formation at the site of the hematoma, adhesions between the penis and peri-penile tissues, development of vascular shunts between the ccp and the surrounding vasculature, injury to the prolapsed prepuce, and damaged or destruction of the dorsal nerves of the penis.3 approximately 50% of bulls with rupture of the tunica albuginea of the penis resume breeding following conservative (non-surgical) therapy consisting of at least 60 days of sexual rest, prophylactic systemic antibiotic therapy to prevent abscess formation following hematoma, hydrotherapy of the sheath to promote circulation multiple times a day, and treatment of the preputial prolapse if warranted.11 conservative therapy alone may be indicated when diagnosis is delayed, or the economic value of the bull does not justify the expense of surgery. the bull should undergo a complete breeding soundness examination prior to being returned to service and the owner should be advised of all potential complications. owners should also be advised to monitor the bull during the figure 4. retropreputial abscess characterized by the location more distally along the shaft of the penis closer to the preputial orifice and asymmetrical appearance between left and right side of the animal. http://dx.doi.org/10.58292/ct.v15.9642 citation line: clinical theriogenology 2023, 15, 9642, http://dx.doi.org/10.58292/ct.v15.9642 15 breeding season to ensure that intromission is occurring as loss of sensation of the glans penis is a sequela that has been reported following injury and consequent return to service in bulls that suffered a penile hematoma. adhesions formed following the rupture and consequently the tearing of adhesions during breeding are thought to damage the nerves at the distal bend of the sigmoid flexure. the adhesions are not severe enough to inhibit full extension of the penis.19 surgical treatment to remove the blood clot and suture the rent in the tunica albuginea, when performed 3–7 days after the injury, can result in approximately 80% of bulls returning to breeding soundness.11 owners should be advised that even following successful management and resolution, recurrence is possible during subsequent breeding. phimosis and paraphimosis phimosis or the inability to extend the penis is most common secondary to preputial stenosis resulting from scar tissue11 (figure 5). other causes of phimosis include a short penis that is simply too short to extend beyond the preputial orifice when erect, adhesions within the elastic layers of the prepuce and surrounding epithelium, or large penile fibropapillomas. extension of the penis is dependent upon the gliding of the elastic tissues and preputial epithelium to allow the free portion of the penis and internal lamina of the prepuce to exit the preputial orifice. formation of adhesions between the penis, elastic tissue, and skin following trauma may effectively inhibit the movement of the penis, resulting in complete or partial impairment of penile extension. the site of the adhesion may be identified by palpation or visualization of skin gathering or wrinkling over the adhesion during an attempted erection. stenosis of the preputial lumen may occur after preputial injury, and subsequent management (either medical or surgical). contraction of scar tissue that replaces damaged elastic tissue leads to constriction of the preputial lumen and resulting preputial stenosis can prevent extension of the penis. stenosis may be severe enough to interfere with the evacuation of urine from the preputial cavity. large penile fibropapillomas may also result in phimosis if the diameter of the fibropapilloma is larger than the preputial orifice. paraphimosis, the inability to retract the penis, may occur following preputial laceration or preputial trauma (figure 6). preputial contusions or lacerations with subsequent edema prevent retraction of the penis into the sheath. rarely bulls with balanoposthitis caused by herpes virus infection extend the inflamed penis and prepuce, which then becomes edematous, preventing penile retraction. regardless of the reason for the paraphimosis, the exposed skin of the penis and prepuce will desiccate and the superficial layers of the skin become necrotic and slough. to protect the penile skin, apply an emollient ointment containing an antibacterial agent on the exposed penis and prepuce and cover it with orthopedic stockinette then apply a pressure bandage as described above. daily bandage changes and cold water hydrotherapy may reduce edema. continue daily bandage changes until the bull can retract the penis then continue to infuse the emollient and antibiotic ointment for 7–10 days in attempt to reduce the risk of preputial stenosis. paraphimosis secondary to preputial lacerations has only a fair-to-guarded prognosis for return to breeding soundness, depending on the extent of the injury and the timeliness of treatment. bulls with paraphimosis caused by herpes viral infections have a good prognosis for return to breeding soundness, unless there is deep necrosis of the peripenile elastic tissues. bulls severely affected with infectious pustular balanoposthitis should be given a minimum of a 30 days sexual rest.11 figure 5. severe preputial stenosis secondary to injury leading to phimosis. figure 6. paraphimosis secondary to preputial trauma. severe necrosis of the prepuce and penis offer a grave prognosis. http://dx.doi.org/10.58292/ct.v15.9642 16 citation line: clinical theriogenology 2023, 15, 9642, http://dx.doi.org/10.58292/ct.v15.9642 inability to make intromission erection failure penile engorgement begins prior to mounting in the normal bull and full erection should occur as the bull mounts to make intromission. occasionally, painful conditions of the spine, rear limbs, or pelvis prevent the animal from achieving erection and mounting and pathology of the lumbar or sacral nerves may prevent normal function of the muscles of erection.11 determining the cause of erection failure begins with a thorough history, physical examination, and an observed test mating. an accurate breeding history is important to distinguish between congenital or acquired abnormalities. erection in the bull depends on the penis being a closed vascular system and in the normal penis there are no venous outlets from the ccp along the body or shaft of the penis. an electroejaculator may be useful as an aid in determining if a bull is able to achieve and/or maintain an erection, but the electroejaculator does not stimulate a physiologic erection and observation of the animal’s performance during an observed test mating is more reliable for diagnosis of erection failure than electroejaculation.18 when erection failure is present, contrast cavernosography is useful to determine if failure is caused by vascular abnormalities such as vascular shunts or to thrombosis of the ccp. review and diagnosis of cavernosal shunts are discussed in other chapters within this special edition. deviations of the penis deviation of the normal alignment of the erect penis will inhibit the ability to copulate. three types of penile deviations are described. in decreasing order of occurrence, they are the spiral or corkscrew deviation, the ventral or rainbow deviation, and s-shaped deviation. spiral deviation of the penis spiral deviation, the most common of the penile deviations, is the result of deficiencies in function of the dorsal apical ligament of the penis that normally serves to support the erect penis and maintain the normal alignment necessary for intromission. the dorsal apical ligament originates from the tunica albuginea proximal to the base of the free portion of the penis and runs beneath the penile skin, along the dorsum of the free portion. the terminal fibers of the dorsal apical ligament rejoin the tunica albuginea near the distal end of the ccp and insert centrally with a broad set of fibers. the terminal fibers of the dorsal apical ligament attaching to the left side of the distal penis are heavier and less pliable than those of the center or on the right side.20,21 observations of a bull penis during simulated coitus using a transparent artificial vagina demonstrated that following intromission, the central portion of the dorsal apical ligament often slips to the left side of the glans near the time of the ejaculatory lunge and the penis assumes a corkscrew shape and spirals within the vagina. most bulls exhibited spiraling within the artificial vagina to some extent, but not at every service.22 ashdown and smith speculated that spiraling of the penis within the vagina following intromission might increase tactile stimulation and promote ejaculation.23 pathologic spiral deviation of the penis occurs when the dorsal apical ligament slips to the left side of the free portion of the penis prior to intromission. affected bulls often have a history of one or more successful breeding seasons before the onset of impotence due to penile deviation, with most cases being diagnosed between 2.5 and 5 years of age.8 histories rarely indicate previous trauma or penile injury. abnormal spiraling usually occurs after penile protrusion; however, in some bulls, spiraling may occur within the preputial cavity.24 premature spiraling of the penis can also occur in bulls that have suffered denervation of the glans penis following injury. this phenomenon will be discussed in other chapters within this special edition. elements leading to the premature occurrence of this otherwise normal phenomenon are poorly understood.20,21,23 malfunction of the dorsal apical ligament due to shortening of the ligament or lengthening of the penis as the bull ages was once regarded as the etiology, but this remains unproven. it is speculated that since spiraling of the penis within the vagina in normal bulls occurs concurrent with peak intra-penile pressure, pathologic spiraling of the distal penis may be the result of premature achievement of peak erectile pressure prior to intromission.8 irrespective of the cause, bulls with premature spiral deviation of the penis are unable to complete the copulatory act. diagnosis is often apparent, but can be difficult if the pathologic deviation does not occur at every breeding attempt, especially early in development of the condition.11 diagnosis of spiral deviation should only be made after observation of the breeding act or during a test mating. development of spiraling during electroejaculation is frequently observed and has not been predictive of a tendency to spiral during natural service. spiraling during attempted electroejacu lation should not be the sole diagnostic criterion. correction of spiral deviation of the penis requires surgical intervention. several techniques for placing a fascia lata or synthetic graft between the apical ligament and the tunica albuginea of the penis to reinforce the dorsal apical ligament attachment have been described. these and other techniques are used to strengthen the attachment of the dorsal apical ligament to the dorsum of the penis and prevent premature spiral deviation of the penis.11 ventral deviation of the penis ventral deviation of the penis occurs less commonly than spiral deviation. the exact etiology is unknown; however, there is speculation that the deviation results from a combination of altered architecture of the tunica albuginea of the penis or apical ligament and altered blood flow through the ventral portion of the ccp. both suspected causes are likely the result of chronic traumatic injury.11 the penile body assumes a long gradual ventral curvature as erection progresses, with the deviation frequently originating proximal to the junction of the sheath and prepuce. the degree of deviation becomes progressively more evident as erection progresses. this condition is often referred to as a ‘rainbow’, due to the arc formed by the erect penis. unlike http://dx.doi.org/10.58292/ct.v15.9642 citation line: clinical theriogenology 2023, 15, 9642, http://dx.doi.org/10.58292/ct.v15.9642 17 spiral deviation, ventral deviation can be suspected based on observation during elec troejaculation, but confirmation by an observed test breeding is recommended. surgical correction of ventral deviation of the penis is less likely to be successful than correction of the spiral deviation and should only be attempted when the ventral deviation is limited to the free portion of the penis.11 s-shaped deviation of the penis the s-shaped curvature of the penis is the least common of the reported penile deviations. this condition usually occurs in bulls 4 years of age or older and is thought to be caused by inade quate apical ligament length or excessive penile length with a normal apical ligament. the condition may develop following trauma to the apical ligament with subsequent contracture and shortening of the ligament. some affected bulls may be able to breed successfully.8 no successful therapy for correction of it has been described, but in bulls of high genetic merit, semen can be collected with use of an artificial vagina or electroejaculation and used for artificial insemination. references 1. beckett s, wolfe d: anatomy of the penis, prepuce, and sheath. in: wolfe df, moll hd: editors. large animal urogenital surgery. 2nd edition, baltimore, md; willians & wilkens: 1998. p. 201. 2. beckett sd: physiology of erection and ejaculation. in: wolfe d, moll hd: editors. large animal urogenital surgery. 2nd edition, baltimore, md; williams & wilkens: 1998. p. 211. 3. maxwell h: inability to breed due to injury or abnormality of the  external genitalia of bulls. in: hopper r: editor. bovine reproduction. 1st edition, ames, ia; wiley-blackwell: 2015. p 113–127. 4. long se, hignett pg: preputial eversion in the bull. a comparative study of prepuces from bulls which evert and those which do not. vet rec 1970;86:161–164. doi: 10.1136/vr.86.6.161 5. long se, rodriguez dubra c: incidence and relative clinical significance of preputial eversion in bulls. vet rec 1972;91:165–169. doi: 10.1136/vr.91.7.165 6. long se, hignett pg, lee r: preputial eversion in the bull: relationship to penile movement. vet rec 1970;86:192–194. doi: 10.1136/vr.86.7.192 7. venter ha, maree c: factors affecting prolapse of the prepuce in bulls. j s afr vet assoc 1978;49:309–311. 8. roberts sj: veterinary obstetrics and genital diseases (theriogenology). 3rd edition, woodstock, vt; published by author: 1986. 9. carson r, hudson r: diseases of the penis and prepuce. in: howard j, smith r: editors. current veterinary therapy: food animal practice. 3rd edition, philadelphia, pa; saunder: 1993. p. 796–797. 10. wolfe d, hudson r, walker d: common penile and preputial problems of bulls. comp contin educ vet 1983;5:s447–s456. 11. wolfe d, beckett s, carson r: acquired conditions of the penis and prepuce. in: wolfe df, moll hd: editors. large animal urogenital surgery. 2nd edition, baltimore, md; willians & wilkens: 1998. p. 237–272. 12. memon ma, dawson lj, usenik ea, et al: preputial injuries in beef bulls: 172 cases (1980–1985). j am vet med assoc 1988;193:481–485. 13. desrochers a, st-jean g, anderson de: surgical management of preputial injuries in bulls: 51 cases (1986–1994). can vet j 1995;36:553. 14. kasari t, mcgrann j, hooper r: cost-effectiveness analysis of treatment alternatives for beef bulls with preputial prolapse. j am vet med assoc 1997;211:856–859. 15. lewis je, walker df, beckett sd, et al: blood pressure within the corpus cavernosum penis of the bull. j reprod fertil 1968;17: 155–156. doi: 10.1530/jrf.0.0170155 16. beckett sd, reynolds tm, walker df, et al: experimentally induced rupture of corpus cavernosum penis of the bull. am j vet res 1974;35:765–767. 17. wolfe df, mysinger pw, hudson rs, et al: ventral rupture of the penile tunica albuginea and urethra distal to the sigmoid flexure in a bull. j am vet med assoc 1987;190:1313–1314. 18. ashdown rr, glossop ce: impotence in the bull: rupture of the corpus cavernosum penis proximal to the sigmoid flexure. vet rec 1983;113:30–37. doi: 10.1136/vr.113.2.30 19. hudson rs. the effect of penile injuries on the breeding ability of bulls. in proceedings of the annual meeting of the society for theriogenology. oklahoma city (ok); 1978, pp. 83–86. 20. ashdown r, coombs m: spiral deviation of the bovine penis. vet rec 1967;80:737–738. doi: 10.1136/vr.80.25.737 21. ashdown rr, ricketts sw, wardley rc: the fibrous architecture of  the integumentary coverings of the bovine penis. j anat 1968;103:567–572. 22. seidel ge, foote rh: motion picture analysis of ejaculation in the bull. j reprod fertil 1969;20:313–317. doi: 10.1530/jrf.0. 0200313 23. ashdown rr, smith ja: the anatomy of the corpus cavernosum penis of the bull and its relationship to spiral deviation of the penis. j anat 1969;104:153–160. 24. barth ad: bull breeding soundness. 3rd edition, saskatoon; western canadian association of bovine practitioners: 2013. http://dx.doi.org/10.58292/ct.v15.9642 https://doi.org/10.1136/vr.86.6.161 https://doi.org/10.1136/vr.91.7.165 https://doi.org/10.1136/vr.86.7.192 https://doi.org/10.1530/jrf.0.0170155 https://doi.org/10.1136/vr.113.2.30 https://doi.org/10.1136/vr.80.25.737 https://doi.org/10.1530/jrf.0.0200313 bovine sexed semen production and utilization bovine sexed semen production and utilization leonardo brito, ramakrishnan vishwanath, claas heuer, kenneth evans stgenetics, navasota, texas abstract it has been 30 years since the first publication describing the birth of live offspring resulting from sexed mammalian sperm and ~ another 15 years have passed since the technology first became commercially available to cattle producers. significant research investments on flow cytometry engineering and sperm biology ensured continuous improvements of the technology and current processing methods now allow producers access to sexedultratm, sexed semen with 90% gender purity and fertility comparable to that obtained with conventional semen. sexed semen is a product now offered by all major bovine genetic companies around the world and is an important tool in capturing additional genetic gain through its strategic use in breeding programs. current state of the art in the fundamentals of sperm sex sorting, the biology of the process, industrial production and field results are described in this review. keywords: cattle, artificial insemination, sexed semen, fertility, sexedultratm introduction the world’s population is expected to grow to ~ 10 billion by 2050. population growth, combined with increased urbanization and per capita increases in income, is expected to increase food demand by 50% from 2012 levels. satisfying increased food demands with existing production practices will result in more intense competition for natural resources, increased greenhouse gas emissions, and further deforestation and land degradation.1,2 the sustainability, even the very existence, of the world’s cattle industry relies on strategies and initiatives to meet the protein needs of 10 billion people in a way that is economical, healthy and good for the environment. production efficiency (productivity per animal unit and land unit) relates to sustainability through its effects on economics and environmental impacts. use of artificial insemination enabled large scale genetic selection programs in cattle and those have been the major contributors to increases in animal productivity, efficiency, product quality and environmental and economic advancements in the last half century. as an example, milk production in us increased by 59% with 64% fewer cows in 2007 compared to 1944. production of the same volume of milk produced in 1944 required only 21% of the cows, 23% of the feedstuff, 10% of the land and 35% of the water in 2007. as a consequence, greenhouse gas production also decreased by 41%.3 in the last 15 years, cattle industry was revolutionized by development and adoption of new genetics and breeding technologies, namely the use of genomics for animal selection and commercial use of sexed semen for artificial insemination. genomic selection reduced generation intervals and accelerated rates of genetic gain in extraordinary fashion in dairy cattle and new programs in beef cattle offer promising results. although use of sexed semen for artificial insemination is rightfully considered a reproductive biotechnology, it could be argued that it should also be considered genetic selection, since gender is a genetic trait. most genetic traits can be manipulated through selection, but before sexed semen was available, producers had to accept the probability that births would result in calf ratios of ~ 50:50 female to male. due to impact of gender on animal production systems, it is described as “the most important genetic trait.”4 as such, sexed semen will continue to be one of the main drivers of cattle production efficiency and sustainability. sexed semen production greatly improved since the beginning of commercial application, but still continuous to evolve rapidly. incorporation of the most recent advancements into production of sexed semen resulted in a differentiated product, sexedultra 4mtm, that now allows producers to obtain > 90% calves of the desired gender with fertility rates comparable to those obtained with conventional semen. 297 clinical theriogenology • volume 11, number 3 • september 2019 brief history of sexed semen technology sexed semen technology was initially developed at us government research centers. in studies started at the lawrence livermore national laboratory in the 1970s, scientists studying health effects of radiation using mouse sperm as a model to indicate damage to the germline developed flow cytometer techniques that allowed precise measurement of sperm dna content; this lead to the breakthrough demonstration of the potential use of these techniques to identify x and y sperm populations, based on dna content differences.5 further development of the technology occurred at the usda beltsville agricultural research center in the 1980s and 1990s, when changes to sperm staining methods and further advancements in flow cytometry not only lead to the major breakthrough of live births of rabbits produced with sexed semen,6 but also supported potential commercial application of the technology. following encouraging results using low-dose insemination with fresh semen in cattle, in the mid-1990s usda granted a license to xy inc., a company funded by the colorado state university research foundation, cytomation inc. and private investors, to commercialize the beltsville sexed semen technology for nonhuman mammalian sperm.5 further developments in rapid speed flow cytometry lead to a leap in production from a few hundred sperm/second to ~ 3000 sperm per second at ~ 90% accuracy.7 development of methods for sexed semen cryopreservation8 and demonstration of acceptable pregnancy rates obtained with frozen-thawed sexed semen 9 further opened doors for commercial application of the technology. commercial licenses were granted to bull studs in the early 2000s and commercial tests started being conducted around the world. development of the technology changed when sexing technologies (st/stgenetics) secured a sorting license in 2004 and started to establish a small number of sorting labs. in 2007, st acquired xy inc. and refocused the commercial approach to allow bull studs access to larger and consistently growing amounts of sexed semen of consistent quality at reasonable costs.10 today, the world’s largest bull studs use st technology to offer sexed semen from a diverse group of top bulls as an essential and important portion of their product portfolios. overview of sexed semen production sexed semen production is based on the difference in dna content between x and y sperm, resulting from the difference in size between x and y chromosomes. on average, the difference in dna content between bull x and y sperm is ~ 4%, although subtle differences occur among breeds (4.22% in jerseys, 4.07% in angus, 4.01% in holstein and 3.7% in brahman).11 hoechst 33342 (h33342) is a dye that permeates the intact cell membrane and binds selectively to a/t base pairs along the minor groove of dsdna. hoechst 33342 exhibits a relatively large stokes shift (excitation/emission maxima of ~ 350/460 nm), making it very useful in assessing precise amounts of dna in living cells.12,13 a flow cytometer is used to quantify sperm dna content. briefly, h33342 dye dna bound molecules are excited by a laser as sperm pass 2 fluorescence detectors that measure the intensity of fluorescence. the strength of the fluorescence signals depends on the number of fluorescing molecules bound to dna, thus allowing differentiation of x and y sperm. there are significant differences in the production processes of conventional and sexed semen. in contrast to conventional semen, which requires minimal intervention and handling, processing sexed semen involves ~ 30 steps (figure 1). after collection and evaluation, semen must be prepared for sorting. this involves extension with appropriate buffers, removal of seminal plasma, and adjustment of cell concentration to optimal range. the sample is then incubated with optimal concentrations of h33342 for a predetermined interval and an exclusive product, ultraseptm is used to reduce proportion of dead sperm in the sample prior to sorting. sperm that are not properly oriented, or that contain abnormal dna amount and therefore fall outside the ‘normal’ range of fluorescent intensity, are gated out and eliminated from the final population. use of a stain-quenching compound that is impermeable to intact plasmalemma allows identification, quantification and elimination of all sperm with disrupted membranes (figure 2). in addition, droplets with sperm that cannot be classified (e.g. signals too close together, > 1 cell in the droplet) also receive no charge and are directed to the discard stream. therefore, live sperm population is 298clinical theriogenology • volume 11, number 3 • september 2019 enriched throughout the process, starting with selection of ejaculates with high motility, removal of a large proportion of dead sperm by ultraseptm during the staining process, gating out non-oriented and dead sperm and eliminating all sperm with undetermined signals during the sort process. in contrast, other commercial sexed semen production technologies actually enrich the population with dead sperm, since no cell is removed from the sample during the process and undesired sperm are actively destroyed using a high-potency laser. figure 1. schematic representation of sexed semen processing. stained sperm are pumped in a stream in front of a laser beam, causing illuminated sperm to emit a very bright blue fluorescence. this fluorescence is measured as sperm flow single file in front of a photomultiplier tube (pmt). specialized software part of the cytome genesistm system is used to analyze relative fluorescence of the x and y sperm populations and select population(s) to be captured (figure 2). a crystal vibrator is used to break the fluid stream into individual droplets containing a single sperm. sperm are then sorted by placing opposite electrical charges on droplets containing x sperm from those containing y sperm. droplets fall past positive and negative electrical fields that separate droplets into 2 streams for collection, whereas a third stream of uncharged droplets is discarded (figure 3). sorted sperm are collected into tubes containing appropriate buffers to protect cells during sorting and cooling. after sorting, tubes are slowly cooled to 5oc, additional extenders containing cryoprotectants are added and tubes are centrifuged to obtain concentrated sperm pellets. the number of recovered cells is determined and extenders added to obtain the desired concentration. after a period of equilibration, semen is loaded into straws and frozen in a programmable freezer using the optimal freezing curve. 12,13 quality control quality control procedures are performed according to guidelines established by the national association of animal breeders/certified semen services,14 in addition to analyses used exclusively for sexed semen. quality control includes evaluation of raw ejaculates for obvious signs of contamination, including debris, urine, water, blood or white blood cells. any sign of contamination results in ejaculate discard. additionally, sperm motility and morphology are evaluated in each ejaculate using phase contrast and dic microscopy, respectively. sperm morphology defects are classified as primary (sperm acrosome and head defects) and secondary (sperm tail defects). only ejaculates that meet minimum requirements are processed further. after production, thawed straws are incubated for 3 hours at 36oc prior to evaluation of sperm motility and presence of sperm agglutination using phase-contract microscopy, and acrosome integrity 299 clinical theriogenology • volume 11, number 3 • september 2019 using dic microscopy. motility videos are recorded and archived with the batch records. all videos are also remotely reviewed by an independent reviewer before any batch is released. post-thaw motility and acrosome integrity obtained after a recent qc personnel training exercise and implementation of computer-assisted sperm analysis (casa) equipment at northern american laboratories are described in figure 4. enrichment of live sperm population throughout the process results in high post-thaw sperm motility and acrosome integrity even after 3 hours of post-thaw incubation. figure 2. flow cytometry histograms used to analyze the relative fluorescence of the x and y sperm populations and select the sorted population. in histograms (1), the dead and properly oriented sperm populations can be differentiated and gated. the degree of difference (peak-to-valley ratio or pvr) in fluorescence intensity between the x and y sperm in the oriented population can be visualized in histograms (2), whereas the population of interest (desired gender) is gated in histograms (3). high-productivity sorting (a) results in the maximum number of straws produced per allotted time and requires a high event rate to obtain a high sorting rate; in this example, > 40,000 sperm/second going through the sorter and >9,300 sperm/second sorted. high-efficiency (b) sorting results in the maximum number of straws produced from the allotted amount of ejaculate and requires adjusting the event rate to maximize the proportion of sperm sorted from the overall sperm population; in this example, over 29% of available sperm are sorted for the desired gender (> 5,000 sperm/second sorted for ~ 17,000 sperm/second going through the sorter). different sorting modes allow bull studs to strategically plan production according to bull age, availability of semen, and demand to adequately fulfill their customers’ needs for sexed semen.10 figure 3. high-voltage plates used to deflect electrically charged droplets containing the desired sperm. a thin stream of fluid can be observed as it is deflected by the plate on the left and into the collecting tube. droplets containing non-desired sperm, dead sperm, non-oriented sperm, and multiple sperm are not charged and are simply collected into the waste stream (metal tube in the middle). a common misconception is that x and y sperm are always simultaneously sorted, and that sperm of the non-preferred gender is commercialized as conventional semen. in fact, the vast majority of production involves production of only one type of sperm and the other type is completely discarded. 300clinical theriogenology • volume 11, number 3 • september 2019 figure 4. sexedultratm post-thaw sperm motility (top) and acrosome integrity (bottom) after 3-hours incubation at 36oc. results obtained using hamilton-thorne ivos ii casa and ident method (motility) and dic microscopy (acrosome integrity). data from north american laboratories for holstein and jersey bulls between february 1 and may 10, 2019. overall (mean ± sem) post-thaw motility = 57.2 ± 0.2% and acrosome integrity = 70.1 ± 0.1%. total sperm number in the straw is determined using the nucleocounter (chemometec®, denmark), using automatic quantification of propidium iodide fluorescently labeled sperm dna within a known volume and calculation of sperm concentration. because sperm identification is specific, there is no interference from seminal plasma composition and gel, lubricants, extenders, or debris, yielding very accurate and precise evaluations. aerobic bacterial count is determined on all batches by streaking processed semen on trypticase soy agar with 5% sheep blood (tsa) plates. samples are incubated at 37oc for 24 36 hours and number of colony forming units (cfu) determined. gender purity is evaluated using genesistm high-resolution flow cytometry, which allows precise targeting of purity. samples from sorting systems (i.e. production equipment) are collected and verified multiple times throughout each batch production to ensure equipment settings are properly setup and accurate so that the targeted gender purity is obtained, while maintaining optimal levels of productivity. gender purity is also evaluated on each produced batch as part of the quality control process (figure 5). before sample analysis, the genesistm high resolution flow cytometer is calibrated using standard, certified sperm nuclei to ensure pre-determined specifications are met, including percentage of oriented cells for the side fluorescence detector path and quality of resolution on dna content on the forward fluorescence detector path. after the instrument is properly calibrated, analyses are conducted at a low event rate, ensuring that percentage of oriented sperm is > 80%, and evaluating a large number of cells (3,000 to 5,000 sperm). images generated for purity analysis are recorded and archived with batch 301 clinical theriogenology • volume 11, number 3 • september 2019 records (figure 6). all images are also remotely reviewed by a qualified technical expert based in the us before any batch is released. figure 5. use of genesistm high-resolution flow cytometry allows precise targeting of sexed ultratm gender purity. highresolution flow cytometry is used throughout the production process to ensure sorting equipment are properly calibrated and setup (a: note genesis 1 analyzer on the left being used in a production laboratory). high-resolution flow cytometry is also used as part of the quality control process to ensure minimum gender purity on all approved semen batches (b). figure 6. genesistm high-resolution flow cytometry for analysis of semen gender purity. before analysis, the instrument is calibrated using certified sperm nuclei as standard to ensure predetermined specifications are met, including percentage of oriented cells for the side fluorescence detector path and quality of resolution on dna content on the forward fluorescence detector path; note the clear distinction of between the x and y sperm in the histogram in the middle of the figure (top). after the instrument is properly calibrated, analyses are conducted at low event rate, ensuring that gains are properly set, percentage of oriented sperm is greater than 80%, sperm plots are properly aligned, centered, rotated and gated. two distinct peaks are observed in the histogram in the middle of the figure and purity is determined by evaluating 3,000 to 5,000 sperm (bottom). 302clinical theriogenology • volume 11, number 3 • september 2019 flow cytometry analysis is the only well-validated method to evaluate semen gender purity. validation refers to the observation of gender skew of live offspring closely resembling the skew in x or y chromosome bearing sperm in the semen sample, as demonstrated in the first study describing the birth of live animals obtained with sexed semen.6 several studies describing results involving use of commercial sexed bovine semen have provided support for validity of flow cytometry analysis to evaluate batch purity, as gender skew rates after birth of hundreds of thousands of live calves have ranged from 89 to 93% (table 1). when calving data from individual bulls were cross-referenced with batch production records, proportion of female calves closely resembled the average batch gender purity determined during quality control. batches with average gender purity of 91% resulted in 8,306 female calves from 9,148 births or 91% female ratio (table 2). table 1. field reports of female calf ratio after artificial insemination using sexed semen. reference country inseminations female ratio borchersen & peacock15 denmark 1,588 91% dejarnette et al.16 usa 19,546 89% norman et al.17 usa 128,702 heifers 25,910 (cows) 91% 89% joezy-shekalgorabi et al.18 iran 1,154 91% table 2. association of sexed ultratm gender purity determined using genesistm high-resolution flow cytometry with field reports of female calf ratio. calving data from herd management programs of stgenetics collaborating dairies in the us. bull semen batches semen purity* calves female calves female ratio jersey a 52 92% 2,895 2,663 92% jersey b 49 91% 1,966 1,809 92% jersey c 110 91% 1,513 1,362 90% jersey d 87 91% 1,434 1,262 88% jersey e 18 92% 773 711 92% jersey f 42 90% 567 499 88% total 358 91% 9,148 8,306 91% *average across batches industrialization of sexed semen production progress in adoption of technologies requires several intersecting elements to converge, e.g. price, efficiency, performance, ease of use and acceptance. in the case of sexed semen, there were 2 critical elements to successful industrialization of the technology. the first was improvements in understanding and manipulating sperm biology, which were directly related to fertility performance (more on this below). the second element was development of flow cytometry engineering and technology. original equipment used for sexed semen production was adapted from medical research. these instruments were expensive, bulky, difficult to operate and with low throughput, which represented significant barriers for scaling-up production. modern equipment has been developed and manufactured by cytonme inc., a company of the st group (www.cytonome.com). cytonome is a biotechnology engineering and manufacturing company that designs and develops cutting-edge cell sorting platforms, offering powerful application-specific technology for industrial markets. equipment development included reduction of fluidic instability and pressure, laser noise, electronic and photodetector noise and acoustic vibration, while improving sperm orientation, light collection efficiency, resolution and signal processing. current equipment uses a solid-state laser for uv excitation, dual orthogonal detectors (at 0o and 90o to the laser), an orienting nozzle, and digital electronics to provide sorted subpopulations of x or y sperm at rates of ~ 8,000 sperm/second when operating at an input event rate of 40,000 sperm per second.19,20 cytome genesistm is the system developed on the hydris™ platform used for production of sexed semen (figure 7). the system is highly automated and designed around user needs, providing simplified operation, reduced instrument size and lower cost in a streamlined workstation layout. cytome genesistm utilizes a compact proprietary multisorter design. each of the 3 sort units can be operated 303 clinical theriogenology • volume 11, number 3 • september 2019 independently and dedicated sort electronics ensure stable, accurate, highspeed sorting across multiple sorting units. the system is operated via a touchscreen and can be remotely accessed and controlled over the internet. control settings (i.e. alignment, droplet setup and droplet delay) are highly automated and require minimal user intervention. the operator can easily control each independent sorting unit using the built-in sort monitoring functions and real time displays. since each sorting unit is independent, interruption of any individual sorting unit will not affect remaining sorting units. these capabilities provide ease of use in busy, high-throughput sorting facilities, critical in a 24/7 environment. figure 7. genesistm is the latest generation of sexed semen flow cytometry sorters manufactured by cytonome/st, inc. the system has a multi-sorter design and the fastest overall sort rate of any conventional high-speed cell sorter of its size. the cytonome genesistm is highly automated and designed around user needs, providing simplified operation, reduced instrument size and lower cost in a streamlined workstation layout. cytome genesistm is a technology marvel that ushered a new era of large-scale, industrial sexed semen production. in may 2019, sexedultratm technology is being utilized for production of sexed semen in 27 laboratories from all major bovine genetic companies across 15 countries. these include 5 stgenetics bull studs, 18 st managed service laboratories and 4 licensee laboratories that combined operate > 500 flow cytometry sorters and produce > 13 million sexed semen straws per year. sexedultratm and sexedultra 4mtm continuous research and development investment in sexed semen production technology resulted in significant improvements in semen quality and fertility, so much so that a new product label was created. although the sexedultratm label was officially launched in 2013, it is important to understand that the product is a culmination of a series of innovations that combined to create a product significantly different from that produced using xy inc. legacy technology. in addition to improvements to flow cytometry technology described above, other innovations included optimization of flow cytometry media (sheath fluid) and extenders, large scale media and extender production for global distribution, optimization of staining conditions and worldwide adoption of modern, standard equipment (figure 8). initial laboratory evaluations indicated that results from in vitro semen quality tests, including sperm motility and acrosome integrity, were superior when semen was processed using sexedultratm technology when compared to xy legacy technology (figure 9). in addition, use of sexedultratm semen for in vitro fertilization resulted in greater production of blastocysts and greater proportion of freezable embryos (table 3).21 further evaluations of sexedultratm post-thaw sperm motility, viability and acrosome integrity revealed those to be equal or better than conventional semen. also, the decline in semen quality after 3 hours of in vitro incubation was lower in sexedultratm compared to conventional semen (figure 10).22 interestingly, dna fragmentation in conventional semen was not high at the baseline (~ 2%), but dropped to nearly zero in sexed semen, indicating that sperm with damaged dna are removed during the sorting process. in addition, sperm dna was more stable in sexedultratm semen and fragmentation did not increase after several periods of in vitro incubation for both frozen-thawed and fresh semen, whereas fragmentation increased significantly in conventional semen (figure 11).22 304clinical theriogenology • volume 11, number 3 • september 2019 figure 8. sexedultratm technology was the culmination of a series of innovations that combined to create a superior product. substantial investments in research & development ensure the continuous cycle of innovations to improve product quality. figure 9. effect of sexedultratm technology on in vitro semen quality tests. sperm motility and progressive motility were determined using computer-assisted semen analysis (casa) and percentage intact acrosome (pia) was determined using dic microscopy (n = 12 bulls). **bars with superscripts differ (p < 0.001). adapted from gonzalez-marin et al.21 figure 10. comparison of post-thaw sexedultratm and conventional semen quality using contemporaneous ejaculates of the same bull (n = 10). after thawing, semen was incubated at 37oc for 3 h and at 18oc for 8 h and 24 h. sperm motility was evaluated visually and using hamilton-thorne ivos ii casa. sperm viability and acrosome integrity were determined using flow cytometry with hoechst 33342, propidium iodide, fitc-pna fluorescent stains. *bars with superscripts differ (p < 0.005). adapted from gonzalez-marin et al.22 305 clinical theriogenology • volume 11, number 3 • september 2019 figure 11. comparison of sexedultratm and conventional semen dna fragmentation index (dfi) using contemporaneous ejaculates of the same bull in frozen (n = 10) and fresh (n = 7) semen. after thawing, semen was incubated at 37oc for 3 hours and at 18oc for 8 and 24 hours. fresh semen was incubated at 37oc throughout the experimental period. dfi was determined using the bull sperm halomax® commercial kit (halotech dna, madrid, spain). *values with superscripts differ (p < 0.05). adapted from gonzalez-marin et al.22 table 3. effect of sexedultratm technology on in vitro embryo production. adapted from gonzalez-marin et al.21 no. of oocytes cleavage rate blastocyst rate freezable embryos* xy legacy 5,082 32.7% 18.4%a 9.2%a sexedultratm 5,081 34.8% 22.3%b 13.2%b *grades 1 and 2. a,brows with different superscripts differ (p < 0.05). in an initial field trial with a small number of inseminations involving industry partners, holstein and jersey heifer conception rates were 7.4% greater when sexedultratm was compared to xy legacy technology (table 4). a larger field trial in collaboration with select sires in 41 holstein commercial herds in the us indicated that heifer conception rates were 4.5% greater when sexedultratm technology was used (table 4).23 data compiled by researchers from the usda on sexed semen usage for holstein females in the united states demonstrated the positive effects of sexedultratm technology on conception rates. data on sexed semen inseminations in heifers and cows between 2007 and 2015 showed a consistent reduction in conceptional rate differences between sex sorted and conventional semen, coinciding with global introduction of sexedultratm in 2013 (figure 12).24 table 4. effect of sexedultratm technology on heifer conception rates. select sires trial results from vishwanath.23 no. of inseminations conception rate sexing technologies trial xy legacy 1,166 47.3%a sexedultratm 957 54.7%b cr improvement 7.4% select sires trial xy legacy 3,384 41.6%a sexedultratm 3,546 46.1%b cr improvement 4.5% a,brows with different superscripts differ (p < 0.01) within trial. 306clinical theriogenology • volume 11, number 3 • september 2019 figure 12. conception rates in holstein females in the united states. only inseminations from 2007 through 2015 with confirmed outcomes were included: 5,963,876 heifer inseminations (1,323,721 to sexed semen) and 42,232,502 cow inseminations (253,586 to sexed semen). mean conception rates for heifer sexed semen inseminations increased due to improved technology (42% in 2007 compared to 49% in 2015). comparable conception rates for heifer conventional inseminations were 56, and 59% for 2007, and 2015, respectively. conception rates for sexed-semen inseminations to cows were 26% in 2007, and 30% in 2015 compared to 30, and 32% for conventional inseminations during the same years. adapted from hutchison and bickhart.24 successful sexed semen production must address susceptibilities of sperm to staining, laser exposure, high dilution, elevated pressure and resistance to several changes in media composition that occur during the process. historically, compounding deleterious effects of these factors resulted in what could be described as uncompensable changes to sperm, as increasing insemination dosage from the 2.1 million sperm used as the industry standard resulted in little to no significant gain in conception rates. although some sire by dosage interactions were observed, across sires, sexed semen dosages of 2.1, 3.5 or 5 million sperm had no effect on conception rates in holstein heifers and cows.25,26 in another study comparing sexed and conventional semen dosages of 2.1 and 10 million sperm, sexed semen resulted in a decrease in conception rates by an almost identical magnitude within both sperm dosages. although sexed semen conception rates were improved by the 10 million sperm dosage, conception rates were not comparable to either dosage of conventional semen.27 one of the most interesting observations since implementation of sexedultratm technology is that not only have deleterious effects of semen processing been minimized, but also that resulting adverse biological changes to sperm became compensable. in a study conducted in collaboration with german genetics international, ejaculates from 5 bulls were split 4 ways and processed using xy legacy technology with 2.1 million sperm dosage or using sexedultratm technology with 2.1, 3 and 4 million sperm dosages; contemporaneously produced conventional semen with 15 million sperm dosage served as control. nonreturn rates (56 days) were evaluated after insemination of 7,855 heifers with sexed semen and 62398 heifers with conventional semen. as expected, xy 2.1 million resulted in lower conception rates when compared to both sexedultratm and conventional treatments. although sexedultratm 2.1 and 3 million sperm dosages produced results lower than conventional semen, increasing the dosage to 4 million sperm resulted in conception rates comparable to conventional semen (figure 13).28 these results were the first to demonstrate: (i) consistently improved conception rates with increased sexed semen dosage; and (ii) conception rates equivalent to conventional semen with sexedultra 4mtm sexed semen. stgenetics adopted sexedultra 4mtm as its standard product in 2015 and officially launched the product in 2017. some bull studs also conducted internal trails and have recently announced the release of similar products (see selected™ sexedultra™ 4m from select sires and genchoice™ 4m from genex). 307 clinical theriogenology • volume 11, number 3 • september 2019 figure 13. effect of sexedultra 4mtm on 56 days non-return rates (nrr) recorded at german genetics international (n = 5 bulls). a,bbars with different superscripts differ (p < 0.001). adapted from lenz et al.28 sexed semen field fertility field fertility data from stgenetics collaborating dairies across the us between 2012 and 2017 have been analyzed. records were obtained directly from dairy management programs (e.g. dairycomp, pcdart) and included 2,123,153 conventional semen inseminations and 1,105,969 sexed semen inseminations from 9,085 holstein and 1,682 jersey sires. a linear mixed model was fitted to the data that included an interaction term between year-month of insemination and semen type, the insemination number (1-3) and the age of the service sire at the time of insemination. random effects included the service sire and a herd-year-season of insemination effect. the model was fitted separately for heifers and cows, whereas the cow model included the lactation number (1 2) as an additional fixed effect. least square means (lsm) on the interaction between year-month and semen type were used to describe the changes in conception rates over time while averaging over the remaining fixed effects: y xβ zs wh ϵ where, y vector of insemination results 0,1 x fixed effect design matrix β solution vector for fixed effects z service sire design matrix s solution vector random sire effect that follows 𝑀𝑉𝑁 0, i𝜎 w herd year season hys design matrix h solution vector for random hys effect that follows 𝑀𝑉𝑁 0, i𝜎 ϵ vector of i. i. d. residuals ∼ 𝑀𝑉𝑁 0, i𝜎 models were fitted using restricted maximum likelihood as implemented in the lme4 package for r.29,30 least square means and multiple contrasts were estimated using the emmeans package.31 conventional and sexed conception rates in holstein and jersey are shown in figures 14 and 15. conception rates in cows had strong seasonality, consistent over years with any semen type. however, seasonality was not observed in heifers. conception rate for conventional semen in cows in january 2012 was 38%, whereas sexed semen reached 25%. in heifers the difference in conception rates was more 308clinical theriogenology • volume 11, number 3 • september 2019 pronounced, i.e. 58% for conventional and 38% for sexed semen. conception rates started to improve in 2013 with introduction of sexedultratm. in june 2014, conventional and sexed semen conception rates in cows were almost at the same level for the first time (34 and 33%, respectively). in 2015, conception rates of sexed semen were stable 88 and 86% of conventional semen conception rates in cows and heifers, respectively. since mid-2016, relative conception rates of sexed semen were consistently > 90% in cows. figure 14. least square means of conception rates for conventional (n = 1,880,094 inseminations) and sexed semen (n = 558,007 inseminations) from holstein bulls (n = 9,085) in cows (n = 1,496,740) and heifers (n = 941,361). data from herd management programs of stgenetics collaborating dairies across the us (n = 181). bands indicate standard errors. figure 15. least square means of conception rates for conventional (n = 243,059) and sexed semen (n = 547,962 inseminations) from jersey bulls (n = 1,682) in cows (n = 547,962) and heifers (n = 367,779). data from herd management programs of stgenetics collaborating dairies across the us (n = 115). bands indicate standard errors. field fertility data with the use of sexedultratm in beef cattle is somewhat limited. however, when used in conjunction with a variety of fixed time artificial insemination (ftai) strategies, relative conception rates of ~ 80 85% have been observed in heifers and lactating cows (table 5). 309 clinical theriogenology • volume 11, number 3 • september 2019 table 5. conception rates in beef cattle using conventional and sexedultratm semen and various fixed-time artificial insemination strategies. reference female category conventional sexedultratm relative thomas et al.32 heifers 59.9% (257/429) 51.7% (218/422) 86% baruselli et al.33 lactating zebu cows 50.3% (75/149) 45.0% (127/282) 89% crites et al. 34 heifers and lactating cows 56.7% (114/201) 49.2% (95/193) 87% colazo et al.35 heifers 59.1% (394/667) 47.6% (316/664) 81% bo et al.36 heifers 58.3% (247/424) 49.3% (210/426) 85% thomas et al.37 lactating cows 64.7% (525/812) 47.9% (387/808) 74% insemination timing closing the fertility gap in cattle, it has been estimated that a minimum of 6 hours is required for inseminated sperm to reach the oviducts and undergo the necessary physiological changes to acquire fertilization potential (i.e. capacitation), with the number of capacitated sperm increasing progressively 8 18 hours after insemination.38,39 conversely, although the oocyte may retain fertilization potential for up to 20 hours, the optimum period is much shorter and is estimated to be only 6 10 hours after ovulation.40 these physiological phenomena result in a quadratic association between insemination to ovulation interval and conception rate. conception rates increase as the interval decreases, until a point when conception starts to decrease in proximity to and after ovulation. early inseminations result in high levels of unfertilized ova due to inadequate sperm lifespan, whereas late inseminations result in poor embryo quality, most likely due to aging oocytes.41 in practice, optimum insemination timing is a compromise aimed to increase the probability of achieving the highest number of capacitated sperm in the oviduct from the time of ovulation to 6 10 hours later. functional in vitro studies demonstrated that sex sorting changes sperm protein tyrosine phosphorylation and ctc staining patterns that resemble capacitation.42 sorted ram sperm bind in fewer numbers to oviduct epithelial cell monolayers in vitro and detach more rapidly than unsorted sperm.43,44 in a more recent study, binding of sexed porcine sperm to oviduct cells was reduced by more than half compared to conventional controls, but the percentage of sperm that bound to purified soluble glycans and the location of binding was similar between control and sexed sperm.45 this changed ability of sexed sperm to bind to oviduct cells suggests that these sperm might be at more advanced stages of capacitation and require less time to complete capacitation in the oviduct than non-sexed sperm. another important aspect to consider in relation to differences in fertility between sexed and conventional semen is physiological semen heterogeneity. any given semen sample contains distinct sub populations of sperm that become ready for fertilization at different intervals post-insemination. this heterogeneity is directly related to the window of time that a given semen sample retains fertilizing potential. in fact, variation in fertility of an individual semen sample or amongst multiple semen samples from the same individual has been attributed to the heterogeneity of the sperm population within said sample.46 therefore, if semen heterogeneity is altered, it may lead to changes in fertility, depending on insemination timing. given that the entire sperm population is exposed to the same conditions (i.e. temperature, ph, lipoproteins, antioxidants, etc.) during the sorting process and that sex sorting is a highly selective process, it is likely that the sexed semen is more homogeneous than conventional semen, with most sperm within similar stages of capacitation. observations from studies using ftai and estrus detection systems have provided tacit evidence that sexed semen might contain a more homogeneous sperm population that require less time to complete capacitation. when using conventional semen, conception rates are optimal when females are inseminated from mid-estrus to the end of standing estrus. considering the interval between onset of estrus to ovulation as ~ 26 30 hours and duration of estrus as ~ 12 18 hours, optimum insemination timing is then ~ 12 22 hours prior to ovulation. although the same quadratic association between insemination to ovulation interval and conception rate is observed with sexed semen, conception rates seem optimal when inseminations are performed closer to ovulation (~ 6 hours prior to until 10 hours after ovulation). jersey heifers inseminated with sexed semen 6.5 hours before presumptive ovulation when using a cidr/estradiol benzoate/pgf2α ftai protocol had a conception rate 1.9 fold greater than heifers 310clinical theriogenology • volume 11, number 3 • september 2019 inseminated 12.5 hours before presumptive ovulation (i.e. insemination either 54 or 60 hours after cidr removal). timing of insemination (6.5 or 12.5 hours prior to ovulation) did not affect conception rate when heifers where inseminated with conventional semen.47 in lactating jersey cows fitted with heatrumination long-distance collars, conception rates with sexed semen were greatest when insemination was performed within 22 36 hours after reaching activity threshold. since ovulation was determined to have occurred ~ 26 hours after activity threshold, optimum insemination was therefore between 4 hours before to 10 hours after ovulation. conception rates obtained with optimum insemination timing were close to 50%, but dropped to < 30% when insemination was performed > 14 hours before ovulation.48 in beef heifers and cows, the approach of delaying insemination by 24 hours and administering gnrh at the time of insemination for females that had not shown signs of estrus 66 hours after pgf2α treatment in a 14 day cidr pg ftai protocol (split time insemination) seemed to improve overall conception rates.33,37 in beef cattle, there is an increasing body of evidence demonstrating the positive association of estrus expression with sexed semen fertility, using a variety of ftai strategies. estrus expression is associated with high circulating estradiol concentrations, known to regulate several physiological processes critical for establishment and maintenance of pregnancy, including effects on follicular cells, oocytes, gamete transport and preparation of the uterine environment. estrus expression is also a proxy for effectiveness of hormonal treatments in inducing synchronous ovulations. conception rates in females that expressed estrus inseminated with sexedultratm were similar to that observed using conventional semen, but were much lower in females that did not express estrus (table 6). based on these observations, recommendations have been made for incorporating estrus detection aids (e.g. chalk, scratch pads) into ftai strategies and selectively use sexed and conventional semen based on estrus expression by the time of insemination. with this approach, no decline is conception rate is expected and the overall resulting gender skew is directly related to efficacy of the program in result in estrus expression. for example, if the ftai strategy produces estrus in 75% of the females and the conception rates are 55% for both females with signs of estrus inseminated with sexedultratm and females not in estrus inseminated with conventional semen, than an overall conception rate of 55% and gender skew of 80% (semen gender purity 90%) would be expected. table 6. conception rates in beef cattle using conventional and sexedultratm semen and various fixed-time artificial insemination strategies according to expression of estrus by the time of insemination. conventional sexedultratm with estrus sexedultratm without estrus thomas et al.32 59.9% 54.7% 28.6% baruselli et al.33 50.3% 53.1% 26.7% crites et al. 34 56.7% 59.8% 27.3% colazo et al.35 59.1% 56.7% 27.8% bo et al.36 58.3% 53.2% 38.8% thomas et al.37 64.7% 52.3% 35.0% these observations clearly indicate that breeding management practices optimized for conventional semen might not necessarily be optimal for sexed semen. minor adjustments to management, especially insemination timing, significantly improve conception rates obtained with sexed semen and help close the fertility gap. sexed semen utilization data compiled by researchers from the usda on holstein breeding involving 5,963,876 heifer inseminations (1,323,721 to sexed semen) and 42,232,502 cow inseminations (253,586 to sexed semen) in the united states indicated that sexed semen utilization rate in heifers increased from 22.5% of total inseminations in 2013 to 30.7% in 2015. although sexed semen utilization was still low in cows, rates increased from 0.5% in 2013 to 1% in 2015.24 data obtained from stgenetics collaborating dairies across the us show that relative use of sexed semen increased from 7.6% in 2012 to 24% in 2017 in holstein and from 31 to 84% in jersey (table 7). there has been a steady increase of sexed semen usage across all lactations, most prominently in heifers, 311 clinical theriogenology • volume 11, number 3 • september 2019 for which sexed semen has been the dominant semen type since 2015, but also in firstand secondlactation cows for which use of sexed semen is becoming more common. it is also evident the trend of almost exclusive use of sexed semen in jersey; relative sexed semen usage has been > 80% in heifers and first and second lactation cows since 2016. the increase in sexed semen utilization is likely associated with the desire to increase selection pressure in heifers (see below) and improvements in fertility in both heifers and cows. table 7. absolute number of inseminations using sexed semen and relative proportion of overall inseminations across lactations and years in holstein and jersey. data from herd management programs of stgenetics collaborating dairies in the us (n = 296) including 1,105,696 sexed semen inseminations and 2,123,153 conventional semen inseminations. holstein jersey year heifers lactation 1 lactation 2 heifers lactation 1 lactation 2 2012 47,424 (30%) 2,508 (2%) 416 (0%) 17,620 (59%) 5,954 (26%) 4,236 (24%) 2013 66,301 (40%) 3,245 (2% 486 (0%) 19,648 (54%) 6,121 (24%) 3,740 (21%) 2014 71,358 (43%) 5,206 (4%) 548 (0%) 23,259 (49%) 12,043 (39%) 7,519 (35%) 2015 90,063 (55%) 11,627 (7%) 3,289 (3%) 64,410 (81%) 40,026 (70%) 18,560 (64%) 2016 106,162 (66%) 19,992 (12%) 5,972 (5%) 90,885 (90%) 64,686 (85%) 34,518 (82%) 2017 90,401 (71%) 23,361 (19%) 9,648 (11%) 68,696 (94%) 46,841 (86%) 19,200 (70%) sire availability genomic selection has had a tremendous impact on the age of dairy sires with available conventional and sexed semen (figure 16a). whereas sire age for conventional semen was ~ 50 60 months in 2012, it decreased to < 40 months by 2017. sire age for sexed semen 2012 was ~ 70 months, indicating that sexed semen from mostly old and proven sires were available at that time. since 2015, there has been no difference between the age of sires for conventional or sexed semen, as most bovine genetic companies offer sexed semen on a diversified lineup of proven and young genomic bulls. the genetic merit of sires with available sexed semen was relatively low when commercial application of the technology started in the 2000’s, a reality that is very different from today. evaluation of a core genetic index, ‘net merit $’, reveals that average genetic value of sires with sexed semen is now similar to sires with conventional semen. this trend is more pronounced in holsteins than in jerseys (figure 16b). figure 16. (a) smoothed sire age and (b) net merit $ according to breed (ho: holstein, je: jersey), semen type, and year. 312clinical theriogenology • volume 11, number 3 • september 2019 with increasing interest in beef sexed semen, including for use in beef on dairy breeding schemes, there has also been an increase in availability of beef sires. in may 2019, sexed female and male semen are available in the us for > 75 beef sires of several breeds, including angus, red angus, simmental, hereford, wagyu and brahman through stgenetics alone. genomic selection and sexed semen genetic progress for a given population can predicted using the breeder’s equation:49 δg = (i × r × σ)/gi, where δg is the progress in genetic standard deviations per year, i is the selection intensity, r is the accuracy of selection, σ is the genetic standard deviation in the population under selection, and gi is the generation interval. genomics and sexed semen allow producers to affect the breeder’s equation and genetic progress in multiple ways. genomic prediction increases prediction accuracy of selection (r) of candidates without progeny information from ~ 0.5 to 0.8 for most traits; however, ability to make accurate selection decisions very early in life decreases the generation interval (gi) for sires of bulls from 6.5 to 1.75 years.50 accordingly, genomic selection alone might double genetic progress per year across all pathways of selection. the biggest potential of sexed semen is evident in commercial dairies. historically, there was little to no selection on the female side on commercial dairies. with replacement rates of 40% and accounting for calf losses and a sex ratio of 50% females, dairy producers needed to keep every female simply to maintain herd size. however, by increasing the proportion of females to ~ 90%, sexed semen offers a way of increasing selection intensity (i) on replacement females. table 8 shows an example of how genomics and sexed semen can affect genetic progress on the female side on commercial dairies. given that everything else is equal, the use of sexed semen can increase δg for the example trait, milk yield, from 38.5 kg per year to 105.6 kg per year. sexed semen in conjunction with genomic selection can leverage that progress even further to 184.8 kg per year. making use of the available technologies can therefore enhance genetic progress in dams by a factor of 3 5. table 8. predicted genetic progress for milk yield in dams of dams using different breeding strategies, including traditional (parent average and conventional semen), incorporation of sexed semen, and incorporation of both sexed semen and genomic selection. strategy σ p i r gi δg traditional 1100 0.8 0.35 0.4 4 38.5 sexed semen 1100 0.4 0.96 0.4 4 105.6 sexed semen + genomic selection 1100 0.4 0.96 0.7 4 184.8 𝜎: genetic standard deviation in the population under selection; p: proportion of animals selected as replacements; r: accuracy of selection; i: selection intensity; gi: generation interval; 𝛥𝐺 is the progress in genetic standard deviations (milk kg per year). conclusion sexedultratm represents the ‘coming of age’ of sexed semen production technology. with 90% gender purity and fertility comparable to that obtained with conventional semen, this proven product now allows producers worldwide different opportunities to strategically manage breeding and genetic improvement programs to increase productivity and profitability. conflict of interest there are no conflicts of interest to declare. references 1. wu g, fanzo j, miller dd, et al: production and supply of high-quality food protein for human consumption: sustainability, challenges, and innovations. ann n y acad sci 2014;1321:1-19. 2. fao: the future of food and agriculture-trends and challenges. rome:fao; 2017. 3. national research council: critical role of animal science research in food security and sustainability. washington, dc: the national academies press; 2015. 313 clinical theriogenology • 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sex-sorted sperm dosage on conception rates in holstein heifers and lactating cows. j dairy sci 2008;91:1778-1785. 26. dejarnette jm, mccleary cr, leach ma, et al: effects of 2.1 and 3.5x10(6) sex-sorted sperm dosages on conception rates of holstein cows and heifers. j dairy sci 2010;93:4079-4085. 27. dejarnette jm, leach ma, nebel rl, et al: effects of sex-sorting and sperm dosage on conception rates of holstein heifers: is comparable fertility of sex-sorted and conventional semen plausible? j dairy sci 2011;94:3477-3483. 28. lenz rw, gonzalez-marin c, gilligan tb, et al: sexedultra™, a new method of processing sex-sorted bovine sperm improves conception rates. reprod fertil dev 2016;29:203-204. 29. bates d, machler m, bolker b, et al: fitting linear mixed-effects models using lme4. j statistical software 2015; 67:1-48. 30. r core team: r: a language and environment for statistical computing. vienna: r foundation for statistical computing; 2017. 31. length rv: least-squares means: the r package lsmeans. j statistical software 2016;69:1-33. 32. thomas jm, locke jwc, vishwanath r, et al: effective use of sexedultra™ sex-sorted semen for timed artificial insemination of beef heifers. theriogenology 2017;98:88-93. 33. baruselli ps, campos filho ep, crepaldi ga, et al: new strategies to improve pregnancy rate at tai using sex-sorted semen. anim reprod 2017;4:711 (abstract). 34. crites br, vishwanath r, arnett am, et al: conception risk of beef cattle after fixed-time artificial insemination using either sexedultra™ 4m sex-sorted semen or conventional semen. theriogenology 2018;118:126-129. 35. colazo mg, whittaker p, macmillan k, et al: evaluation of a modified gnrh-based timed-ai protocol associated with estrus detection in beef heifers inseminated with sex-selected or conventional semen. theriogenology 2018;118:90-95. 36. bo ga, huguenine e, de la mata jj, et al: programs for fixed-time artificial insemination in south american beef cattle. anim reprod 2018;15 (suppl.1): 952-962. 37. thomas jm, locke jwc, bonacker rc, et al: evaluation of sexedultra 4m™ sex-sorted semen in timed artificial insemination programs for mature beef cows. theriogenology 2019;123:100-107. 38. hawk hw: transport and fate of spermatozoa after insemination of cattle. j dairy sci 1987;70:1487-1503. 314clinical theriogenology • volume 11, number 3 • september 2019 39. wilmut i, hunter rh: sperm transport into the oviducts of heifers mated early in oestrus. reprod nutr dev 1984;24:461-468. 40. brackett bg, oh yk, evans jf, et al: fertilization and early development of cow ova. biol reprod 1980;23:189-205. 41. saacke rg. insemination factors related to timed ai in cattle. theriogenology 2008;70:479-84. 42. bucci d, galeati g, tamanini c, et al: effect of sex-sorting on ctc staining, actin cytoskeleton and tyrosine phosphorylation in bull and boar spermatozoa. theriogenology 2012;77:1206-1216. 43. de graaf sp, evans g, maxwell wmc, et al: in vitro characteristics of fresh and frozen-thawed ram spermatozoa after sex-sorting and re-freezing. reprod fertil dev 2006;18:867-874. 44. hollinshead fk, gillan l, o’brien jk, et al: in vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing. reprod fertil dev 2003;15:351-359. 45. winters ra, nettenstrom ln, lopez dg, et al: effect of sorting boar spermatozoa by sex chromosomes on oviduct cell binding. theriogenology 2017;108:22-28. 46. rodriguez-martinez h: can we increase the estimative value of semen assessment? reprod dom anim 2006;41(suppl 2):2-10. 47. sales jn, neves ka, souza ah, et al: timing of insemination and fertility in dairy and beef cattle receiving timed artificial insemination using sex-sorted sperm. theriogenology 2011;76:427-435. 48. bombardelli gd, soares hf, chebel rc: time of insemination relative to reaching activity threshold is associated with pregnancy risk when using sex-sorted semen for lactating jersey cows. theriogenology 2016;85:533-539. 49. rendel jm, robertson a: estimation of genetic gain in milk yield by selection in a closed herd of dairy cattle. j genet 1950;50:1-8. 50. schaeffer lr: strategy for applying genome-wide selection in dairy cattle. j anim breed genet 2006;123:218-223. 315 clinical theriogenology • volume 11, number 3 • september 2019 316clinical theriogenology • volume 11, number 3 • september 2019 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged 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christopher premanandan, erin runcan college of veterinary medicine, the ohio state university, columbus, oh abstract striking microscopic changes take place in canine endometrium morphology during placentation. gross structure (zonary placentation) and nature of maternal-fetal interface (endotheliochorial) is generally well understood. however, histological changes in endometrial tissue proper and how they impact fertility assessment utilizing histology may be less well understood. this review will cover evaluation of canine placenta (gross and microscopic), difficulties in correlating histologic evaluation to fertility and fetal loss, and implications in interpreting histopathology results. anatomy of canine placentation most veterinarians versed in reproductive medicine are familiar with gross structure of canine placenta. chorioallantois is composed of zonary and nonzonary regions, with zonary region being the circumferential site of attachment to canine endometrium. dark green peripheral regions of zonary region are referred to as marginal hematoma and are largely responsible for accumulation of uteroverdin, a dehydrogenated form of bilirubin.1 interestingly, this same compound responsible for green color has been observed to also create a pigmentary change in some avian egg shells. dispersion of uteroverdin results in a striking change in placentas that are submitted for histologic evaluation. while distinction formed by contrast between marginal hematomas and labyrinth proper are easy to recognize immediately after parturition, once in formalin, this uteroverdin diffuses out and stains all components of placentas a deep green color. microscopic structure of canine placenta has been covered extensively in literature.2,3 briefly, placenta is divided into glandular zone, junctional zone, and labyrinth. glandular zone is composed of remodeled pre-existing endometrium in which marked reorganization of remnants of endometrial glands take place.2,3 endometrial glands take on a linear arrangement along stratum basalis and become markedly dilated. labyrinth represents primary interface between fetal and maternal vascular tissue with intervening trophectoderm and periodic acid-schiff positive basement membrane.2 arrangement of fetal and maternal vascular structure with trophectoderm is lamellar in nature, comprising a complex branching network between allantoic surface and tissue layers closer to endometrium. in between labyrinth and glandular zone lies junctional zone, composed of long thin extensions of vascularized (maternal) connective tissue with an overlying covering of trophoblasts.3 during parturition, separation of chorioallantois from endometrium primarily occurs at this location and remnants of junctional zone can often be observed in biopsies taken from placentation sites. placental evaluation it is important to recall that evaluation of placentation involves both endometrium and fetal membranes. placental evaluation can be a critical step in investigating fetal loss in bitch. a majority of investigations into fetal loss or perinatal death are limited to fetus/neonate proper and a significant amount of diagnostic information can be lost if chorioallantois is prematurely discarded or simply not submitted. in addition to fetal tissue and placenta, a uterine biopsy can be very helpful to confirm preexisting endometrial disease that may have an impact on placentation. this, of course, may not be an option with natural delivery; however, it is a simple procedure that can be performed during caesarian section. obvious gross changes can be difficult to detect in canine placentas and are not reliable for diagnostic purposes. although certain infectious etiologies can have characteristic gross lesions (canine herpesvirus, brucella canis), it is best to treat cases in a consistent and uniform manner. complete evaluation of placenta includes histopathology and ancillary diagnostics, meaning a portion of tissue 623 clinical theriogenology • volume 11, number 4 • december 2019 should be preserved in fixative and another portion saved fresh or frozen. additional diagnostic testing for pathogens then can be pursued at a later date, if histopathology suggests that testing is indicated. both of these evaluations should be coordinated with the evaluation of fetal tissue in appropriate situation. microscopic evaluation of placenta by an anatomic pathologist should also be performed in a uniform manner. the author typically makes multiple cross sections from either side of marginal hematomas through zonary region in a perpendicular fashion to evaluate cut surface. typically, 2 regions of zonary chorioallantois are selected to evaluate that include a small portion of nonzonary chorioallantois. in addition, a portion of amnion is also submitted for histologic processing. by processing a uniform section of chorioallantois, anatomy can be consistently evaluated and artifacts associated with oblique sections can be avoided. common confounding features placental evaluation primarily refers to evaluation of chorioallantois. as mentioned previously, uniform dark green discoloration of chorioallantois and amnion are expected after prolonged immersion in formalin and very little can be easily determined with gross evaluation at this stage. microscopically, chorioallantois should be evaluated for presence of inflammation. in addition, certain pathogens, most notably brucella canis, can be detected by light microscopy in the cytoplasm in trophoblasts. it is important in these cases to also examine trophoblasts on nonzonary chorioallantois for presence of organisms. ancillary diagnostics (culture, molecular testing) should accompany light microscopy in cases where infectious disease is highly suspected. this is best approached in the form of an “abortion panel.” typical canine abortion panel consists of standard aerobic culture and testing for specific pathogens such as brucella canis, leptospira spp, canine herpesvirus-1, canine distemper, and canine adenovirus. in cases where placenta does not exhibit overt inflammation, infectious causes of fetal loss cannot be completely excluded. however, noninfectious/inflammatory causes should be considered more carefully. unfortunately, very little has been characterized in terms of noninfectious diseases of canine fetal membrane. the author has frequently observed regions of coagulation necrosis and hemorrhage within labyrinth. it is difficult to attribute this lesion to fetal loss in all cases without more literature characterizing spectrum of changes in fetal membranes associated with completely healthy litters. similar lesions observed in human placentas have more established guidelines in regards to fetal health.4,5 it has been proposed that infarctions occurring in central regions of chorionic plate that affect between 5 and 20% of the parenchyma are diagnostically significant.4 while significant differences in placental structure between human and canine have to be recognized, similar guidelines would be helpful in assessing the diagnostic significance of these lesions in canine placenta. since endometrial tissue is often sampled in cases of subfertility, maternal aspect of placenta is one aspect of evaluation that presents difficulty during evaluation. endometrial lesions associated with subfertility in bitch have been characterized. some of these lesions include lymphoplasmacytic inflammation, eosinophilic inflammation, endometrial fibrosis and cystic endometrial hyperplasia.6 significance of other lesions, such as endometrial glandular atrophy, has not been well characterized. if a full-thickness endometrial biopsy is taken at caesarian section, evaluation of endometrium is straightforward, as its appearance at this stage is uncomplicated. however, if fetal loss takes place prior to projected parturition date and an endometrial biopsy is taken between the time of fetal death and predicted date of parturition, then the pathologist must contend with endometrial changes associated with uterine involution. histological changes of uterine involution are well characterized.7,8 immediately after parturition, glands at basal aspect of endometrium are visible, however, typical appearance of glandular penetrations extending from luminal surface, as present in nongravid endometrium, will not be present.7,8 a week following parturition, decidual cells are present within endometrial interstitium and glands may reduce in diameter. endometrial glands typically return to a normal appearance ~ 7 weeks postpartum.7,8 however, very little information describing density of endometrial glands following parturition is available, which makes diagnosis of endometrial atrophy or glandular loss difficult. in addition, aggregates of collagen coalesce at superficial surface of endometrium starting 2 weeks postpartum and eventually slough off 624clinical theriogenology • volume 11, number 4 • december 2019 from 5 to 7 weeks postpartum.7,8 starting from 14 days postpartum, infiltration of lymphocytes, plasma cells and macrophages becomes progressively pronounced, peaking at 7 weeks postpartum, but is reduced to minimal infiltration during anestrus.8 because of this, examining pathologist should be aware of parturition date to account for normal leukocyte infiltration. minimal leukocyte infiltration should be present in normal endometrium if a biopsy is taken during caesarean section on the date of parturition. above stated changes describe normal involution and do not address endometrial changes that occur following mid-gestational fetal loss. there is little to no literature describing endometrial changes that progressively take place during this stage of gestation, but one can speculate that many changes are similar and perhaps less striking in nature. conclusions evaluating placenta can be a helpful adjunct diagnostic when investigating causes of subfertility in bitch. however, nuances of canine placentation should be kept in mind when investigating and interpreting results. further investigation into non-clinically significant lesions involving chorioallantois is important to establish which lesions may actually be variations of normal and should be ignored. in addition, myriad of changes that occur within canine endometrium during involution should be kept in mind when interpreting endometrial biopsies, especially when these biopsies are taken postpartum or following fetal loss. references 1. lemberg r: bile pigments. vi. biliverdin, uteroverdin and oocyan. biochem j 1934;28:978-987. 2. furukawa s, kuroda y, sugiyama a: a comparison of the histological structure of the placenta in experimental animals. j toxicol pathol 2014;27:11-18. 3. endotheliochorial placentation cat, dog, bat. in: wooding fbp, burton gj: editors. comparative placentation: structures, functions, and evolution. berlin; springer: 2008. p. 169-184. 4. stanek j: hypoxic patterns of placental injury: a review. arch pathol lab med 2013;137:706-720. 5. stanek j: comparison of placental pathology in preterm, late-preterm, near-term, and term births. am j obstet gynecol 2014;210:234.e1-6. 6. gifford at, scarlett jm, schlafer dh: histopathologic findings in uterine biopsy samples from subfertile bitches: 399 cases (1990-2005). j am vet med assoc 2014;244:180-186. 7. al-bassam ma, thomson rg, o'donnell l: normal postpartum involution of the uterus in the dog. can j comp med 1981;45:217-232. 8. orfanou dc, ververidis hn, pourlis a, et al: post-partum involution of the canine uterus gross anatomical and histological features. reprod domest anim 2009;44 suppl 2:152-155. 625 clinical theriogenology • volume 11, number 4 • december 2019 626clinical theriogenology • volume 11, number 4 • december 2019 untitled omniblank: metabolic and environmental effects on embryo epigenome metabolic and environmental effects on embryo epigenome juliano sangalli, pablo rossa university of são paulo, department of veterinary medicine, pirassununga, sp, brazil, auniversity of california davis, deparment of animal science, davis, ca abstract extensive epigenetic changes occur during early embryonic development, which are necessary to reset the gametic gene expression program to an embryonic totipotent state and ensure full developmental potential is achieved. epigenetic changes are catalized by enzymes, many of which use products of intermediate cell metabolism as cofactors or substrates. early development occurs in a fluid environment provided by various compartments as gametes/embryos develop and whose composition is sensitive to metabolic and physiological changes. therefore, nutritional and environmental factors can potentially alter the embryonic epigenome, influencing the trajectory of development, with long term effects on healthspan. in this manuscript, we review connections between metabolism and epigenetics and how environmental factors can affect gametes and embryos, with potential effects on the animal. keywords: epigenetics, preimplantation development, metabolism, healthspan, dna methylation, histone modifications introduction epigenetics (above genetics) refers to changes in phenotypes not caused by changes in the genetic code. at the molecular level, epigenetics is mediated by modifications of dna and chromatin that do not involve changes in the dna code. these modifications are mostly represented by dna methylation, posttranslational histone modifications (e.g. acetylation and methylation), and small noncoding rnas.1 there are many phenomena controlled by epigenetic mechanisms including, among others, cellular differentiation, genomic imprinting, and x chromosome inactivation (xci).2 in multicellular organisms, almost all cells contain the same dna sequence, but can vary widely in shapes and functions, due to differences in epigenetic information.3 genetic imprinting is a phenomenon in mammals and some other species, in which only 1 copy (allele) of certain genes is expressed in a parent of origin basis, irrespective of the actual allele sequence. for example, in female cells, an entire x chromosome is inactivated by epigenetic mechanisms to compensate for gene dosage differences with males carrying only 1 x chromosome.4 generally, epigenetic changes are inherited across cell divisions, so that once a differentiated cell divides, it gives rise to a similarly differentiated cell (i.e. skin cell give rise to skin cell); or in the case of genomic imprinting, the identity of the parental alleles is maintained in all cells of the individual; or for the case of x chromosome inactivation, the same x chromosome is silenced in all progeny that originated from the cell undergoing the initial xci event.5 although epigenetic marks are inherited during cell division, they are not permanent and are actively modified in situations such as cellular differentiation, allowing a single cell precursor to give rise to multiple types of differentiated cells; reactivation of x chromosomes during development; or erasure and re-establishment of imprinting in the germline to confer parental origin specificity to the next generation.6 one of the most dramatic changes in epigenetic information is observed during early development, as 2 highly differentiated cells, sperm and egg, are reprogrammed to the totipotent naïve state of early blastomeres (embryo cells).7 recently, there has been considerable interest in whether environmental factors (e.g. diet, temperature) modulate establishment and maintenance of epigenetic modifications and could thereby influence gene expression and phenotype in organisms.1 several recent studies documented that chemical pollutants, dietary components, temperature changes and other external stresses can have long-lasting effects on development, metabolism, health and production in livestock.8,9 given that 1) epigenetic changes can be affected by the environment, 2) there are dynamic changes in epigenetic information during early development, and 3) epigenetic states can be inherited during cell division, it is reasonable to expect that alterations to embryos or gametes, may compromise their epigenomes, with long-term consequences for the ensuing animal. current livestock production systems often expose animals to adverse metabolic and environmental stresses that could 187 clinical theriogenology • volume 11, number 3 • september 2019 hamper optimal healthspan and reproductive capacity. in addition, use of artificial reproductive technologies exposes gametes and/or embryos to suboptimal conditions that can affect embryo metabolism and animal health/productivity.10 the objective of this manuscript is to survey the connection between various environmental stressors and the livestock embryonic epigenome. early embryonic development and epigenetic reprogramming embryonic development starts with fusion of the spermatozoon with an oocyte. fertilization triggers a complex cellular programme that transforms 2 highly specialized germ cells, oocyte and sperm, into a totipotent zygote.6 the transition from 2 highly specialized cells containing unusual chromatin states to an early embryo state is among the most dramatic and complex reprogramming events in biology.11 detailed molecular changes in the epigenome of livestock embryos have been comprehensively reviewed elsewhere,12-14 so we provide a very general overview. after the sperm nucleus enters the oocyte cytoplasm, genome wide epigenetic remodeling occurs. the paternal genome exchanges protamines for histones, undergoes an active process of dna demethylation, which requires the activity of ten-eleven translocation (tet) enzymes and acquires a new set of histone modifications, necessary for proper gene expression and embryo development. concomitantly, maternal dna is passively demethylated. after zygotic demethylation, the embryonic genome continues to be demethylated during the following few cleavage divisions until the blastocyst stage. similarily, extensive changes in histone modifications ensue after fertilization, leading to an overall increase in histone acetylation and loss of repressive histone marks (e.g. h3k9me3 and h3k27me3). this loss of repressive dna methylation and histone modifications is thought to facilitate reactivation of the embryonic genome.15 also, global epigenetic remodeling during preimplantation development ensures that gametic genomes return to pluripotency.16 for example, loss of methylation in promoters of master regulators of pluripotency genes such as oct4 and nanog is required for establishment and maintenance of the inner cell mass lineage in the blastocyst. periconceptional period and its importance to proper embryonic development, animal growth and healthspan correct embryonic development is fundamental for animals to have a healthy life, grow properly and reproduce. among all events occurring during animal ontogenesis, the periconceptional period is critical.17 the periconceptional period usually comprises all events taking place during gamete maturation, fertilization and early embryonic development. in a manner somewhat arbitrary, we can also consider implantation and early stages of placenta formation part of the periconception period. it is now clear that early embryo development in mammals can be affected by parental diet, as well as environmental stress factors, including factors affecting either parent in the months and weeks that precede fertilization.18 gamete maturation and early embryonic development occur in a fluidic medium (i.e. epididymal, follicular and oviductal fluids) which is susceptible to alterations by animal diet and environment. during the periconceptional period, exposure to altered diets or environmental stressors can result in epigenetic alterations with potential effects on subsequent animal development and performance.17 there are epidemiological evidences and studies in laboratory animals that an inadequate or deficient diet, exposure to chemical toxicants and environmental stressors during the periconceptional period can affect embryo metabolism, development and even behavior of offspring.19 some classical examples demonstrating high sensitivity of maturing gametes and developing embryos to environmental insults include folic acid deficiency causing neural tube defects in humans 20 or altered coat color in mice,21 dysmelia caused by thalidomide exposure in humans 22 and reduced fertility of heat stressed dairy cows.23 however, for these and other observations, molecular mechanisms connecting stressors to altered phenotypes are not fully understood. 188clinical theriogenology • volume 11, number 3 • september 2019 connection between metabolites and alterations to the epigenome a model by which nutrients can regulate gene expression was elegantly proposed by francois jacob and jacques monod in 1960;24 however, precise molecular mechanisms linking nutrient availability to appropriate gene expression response remain poorly understood. recently, it has been demonstrated that virtually all components from intermediary metabolism (e.g. acetyl-coa, α-ketoglutarate, β-hydroxybutyrate, butyrate, lactate and succinate) act as cofactors or cosubstrates of chromatin-modifying enzymes.25 curiously, the epigenetic regulatory machinery is highly responsive to metabolic cues. for example, metabolites (e.g. s-adenosyl methionine (sam), acetyl-coa) are substrates for enzymes that catalyze deposition of covalent modifications on histones, dna and recently discovered to be present on rnas. these modifications, referred to as posttranslational modifications (ptms) of chromatin, have a major role in activation or repression of gene transcription. ptms include acetylation, methylation, phosphorylation, butyrilation, and crotonylation of histone proteins and methylation of dna. some of these chromatin modifications are involved in maintenance of stable patterns of gene expression (e.g. dna methylation and h3k9me3), usually referred to as epigenetic regulation.26 since intermediate metabolite availability is required for activity of histone-modifying enzymes, nutritional deficiencies or exesses by consumption of specific types of food potentially disrupt chromatin homeostasis and consequently alter gene expression patterns.27 epigenetic marks are controlled by 3 types of enzymes: “writers” add epigenetic modifications, which are recognized by “readers” (effector proteins). when a specific epigenetic mark is no longer necessary, or the cell needs to change the pattern of gene expression, these marks are removed by “erasers”.28 here we will exemplify the most common chromatin modifying enzymes and their cofactors or substrates (comprehensive reviews are available).25,26 histone acetyltransferases (hats) use acetyl-coa as an acetyl donor. histone acetylation is associated with active chromatin and is removed by histone deacetylases (hdacs) that are part of transcriptional repressor complexes. most hdacs require the micronutrient zn2+ as a cofactor.29 curiously, the ketone body beta-hydroxybutyrate was discovered to be an endogenous regulator of hdacs.30 because ketone bodies are an integral part of ruminant biology, connections between ketone bodies and epigenetic remodeling are crucial to understand reproduction and physiology in cattle, especially dairy. sirtuins are a special group among all hdacs, classified as class iii hdacs and require nad+ as a cosubstrate; they are a potential connection between lowcalorie situations and gene silencing.31 histone and dna methylation are mediated by lysine methyltransferases (kmts) and dna methyltransferases (dnmts), respectively. both classes of enzymes use s-adenosylmethionine (sam) as methyl donor. sam is the second most common enzymatic cofactor after atp and has a major role in epigenetics and embryo development. sam is part of 1-carbon metabolism, which has another crucial player, folate. because animals cannot synthesize folate, they must obtain it by dietary intake.32 paternal or maternal deficiency in folate causes severe fetal abnormalities, ranging from neural tube defects to abnormal spermatogenesis,33 emphasizing importance of understanding connections among diet, epigenetics and development. removal of methyl groups from lysine residues in histones involves 2 classes of lysine demethylases (kdms). demethylation by lsd1 (lysine-specific demethylase 1) requires reduction of flavin adenine dinucleotide (fad). however, the jumonji c (jmjc) domain-containing kdms catalyze a different demethylation reaction that requires α-ketoglutarate (αkg), oxygen and fe(ii). hydroxylation of 5mec in dna by tet enzymes, a crucial step for reprogramming parental genomes after fertilization, involves a similar reaction. collectively, these demethylases are known as αkg-dependent dioxygenases.34 fluctuation in oxygen concentrations can affect activity of kmds and tet enzymes, which can result in altered epigenetic remodeling and embryonic development. this is supported by the observation that increased oxygen during in vitro embryo culture can affect capacity of embryos to establish pregnancies and develop to term.35 lipids are also dietary components with regulatory activities on chromatin modifying enzymes. metabolites such as sam (described above) connect lipid metabolism to histone methylation. butyrate is produced in the rumen and has strong hdac inhibitory activity. other fatty acids produced in the rumen (e.g. propionate) or ruminal wall (e.g. beta-hydroxybutyrate) have regulatory and epigenetic roles. since their concentrations can be manipulated by ruminant diets 189 clinical theriogenology • volume 11, number 3 • september 2019 (i.e. concentrates versus forages), they provide an avenue to understand the connection between nutrition and epigenetic regulation in livestock.36 epigenetic maturation of gametes based on the observation that after fertilization there is a massive erasure of dna methylation and remodeling of histones and their modifications brought by gametes, mechanisms by which epigenetic information can be passed from parent to offspring through the germline remain difficult to rationalize.37 surprisingly, there are compelling evidences that some epigenetic marks can escape this reprogramming.38 in addition, it has been demonstrated that gametes undergo epigenetic remodeling during their maturation process.39 gametes gain cargos (i.e. small noncoding rnas, lipids, proteins) and acquire specific epigenetic marks during their final stages of maturation; these may contribute to epigenetic inheritance of parental environment to the offspring.40 epididymis in mammals, final maturation of sperm occurs during its transit through the the epididymis, a long and specialized convoluted tube that exchanges secretory factors required for sperm to gain motility and fertility.41 composition of epididymal luminal fluid is distinctly different from that of blood plasma, indicating potential mechanisms to protect sperm from undesired molecules. the blood epididymis barrier, responsible for this process, carefully controls the lumicrine microenvironment so that sperm are bathed in an appropriate fluid milieu at each stage of maturation as they travel through each segment of the epididymis (i.e. caput, corpus and cauda).42 highly specialized and region specific microenvironments are created along the epididymal lumen by active secretion and absorption of water, ions, organic solutes, and proteins. for instance, some molecules can be concentrated 10 100 fold (i.e. inositol, carnitine) in the lumen of the caput epididymidis, whereas others, such as glucose and albumin, are effectively excluded. additionally, there are variations in physical parameters, such as osmolarity, oxygen tension and extracellular ph (i.e. becomes acidic in the cauda region).42 in the past few years, there has been accumulating evidence that parents can transmit dietary and environmental information to their offspring via epididimal transport to sperm.40 from an evolutionary perspective, if parents can “inform” their progeny about prevailing environmental conditions, offspring would have a higher chance of surviving or coping with potential stressors.43 the paternal model of epigenetic inheritance is attractive for study, as it can be cleanly tested. using ivf or icsi, it was possible to demonstrate that environmental information was carried in the spermatozoon.40,44,45 on the maternal side, the uterus can affect the offspring phenotype, bringing a confounding factor.19 howewer, as discussed above, a plausible mechanism explaining epigenetic inheritance of phenotypic traits is still lacking and difficult to reconcile. difficulty arises from some observations: 1) large part of sperm dna methylation is erased following fertilization, and 2) sperm is largely devoid of nucleosomal histones.37 supporting the idea that sperm dna methylation is probably not a carrier of epigenetic information, mice eating several diets (control, low protein, high fat) had no consistent effects on sperm cytosine methylation.46 regarding histones, mammalian sperm exhibit a highly unusual chromatin state that is markedly different from other somatic cells,47 as the vast majority of histone proteins are lost during spermatogenesis. histones are first replaced by transition proteins and later by small basic proteins termed protamines; however, not all histones are lost, with murine sperm retaining up to ∼ 8% of their histones.47 recent studies of histone retention in human and mouse sperm suggest a bias for sperm histone retention as promoters of genes expressed during early development.48 conversely, in other studies, the majority of sperm histone retention preferentially occurred in gene-poor genomic regions, with only a small subset of nucleosomes being retained over promoters of developmental regulators.49 the genomic location of nucleosome retention is still a matter of debate, and it seems that protocols used to digest chromatin can affect results.50 currently there is some level of consensus that retained histones may carry epigenetic information, which could affect gene expression in early embryos and contribute to intergenerational epigenetic inheritance. indeed, using mass spectrometry, it was possible to identify multiple ptms on histones and protamines in adult mouse sperm.51 since ptms are responsive to the environment, they can be modified during spermatogenesis or epididymal maturation and contribute to transfer of epigenetic information from sire to offspring. 190clinical theriogenology • volume 11, number 3 • september 2019 another potential mechanism by which the lumicrine microenvironment from the epididymis can affect histone marks on sperm is changes in ph. recently it was demonstrated that cellular histone acetylation levels globally decrease as ph decreases.52 along the epididymis, luminal ph decreases and the cauda region has an acidic extracellular ph,53 which could be important for controlling histone acelytlation in sperm retained histones. among all potential carriers of epigenetic information (i.e. dna methylation, histone marks and small noncoding rnas) small rnas are gaining considerable interest as the main player. small rnas comprises several small molecules (i.e. < 40 nucleotides), including well-studied micrornas, sirnas, pirnas, as well as less understood molecules such as enhancer-derived rnas (ernas) and trna fragments (trfs).54 these molecules are implicated in various transgenerational epigenetic inheritance paradigms such as: 1) rna interference in caenorhabditis elegans; 2) paramutation in maize; 3) epigenetic silencing in arabidopsis and fission yeast; and 4) silencing of transposons in the drosophila germline. however, most of these cases occur in organisms whose genome encodes an rna-dependent rna polymerase, providing a mechanism by which an initiating signal can be maintained over multiple organismal generations. on the contrary, mammals lack these key enzymes, which makes small rna epigenetic inheritance far less robust and subtle.55 that said, a plausible mechanisms in mammals could be represented by small rnas delivered to the embryo at fertilization that could alter zygotic gene expression.43 since embryonic development progresses as a cascade of events, altering events at the beginning of development can affect cell differentiation or allocation, eventually causing placental problems. placental malfunction can lead to fetal growth restriction and thrifty phenotype in adulthood.19 supporting this view, in a model of chronic paternal stress, it was possible to identify 9 mirnas differentially expressed in sperm of stressed animals. injection of those micrornas to unexposed zygotes caused a remarkable recapitulation of the offspring stress dysregulation phenotype.56 in another example, causing traumatic stress in early life altered mouse microrna (mirna) expression, behavioral and metabolic responses in progeny. injection of sperm rnas from traumatized males into fertilized wild-type oocytes reproduced behavioral and metabolic alterations in resulting offspring.44 in 2 independent studies, male mice submitted to low-protein or chronic high-fat diets had different populations of trfs in mature sperm. injection of purified trfs fragments from males kept on diets into normal oocytes caused metabolic disorders and concomitant alteration of genes in metabolic pathways in the progeny.40,45 curiously, these trfs were gained during epididymal transit.40 extracellular vesicles secreted by the epididymal cells, also termed epididymosomes, shaped the sperm epigenome after testicular maturation.57 this is a remarkable example of soma to germline relationships in mammals. furthermore, the cargo secreted by the epididymal cells changes based on animal diet, offering some potential mechanisms to explain how dietary information can be transmitted from father to offspring. all of these observations and mechanisms of epigenetic inheritance gathered from laboratory animals open avenues to improve animal production in livestock. in the near future, it may be possible to screen for specific micrornas or other small noncoding rna entities in semen from boars, bulls, bucks, rams and stallions to identify candidates conferring higher fertility or desirable phenotypic characteristics. ovarian follicle the ovarian follicle is the basic unit of the female reproductive system. each follicle contains 1 oocyte and companion cells, which periodically grow to release a mature and competent oocyte. the oocyte growth phase is a protracted process in mammals, taking ~ 3 wk in mice and several months in humans and livestock.58 during growth, the oocyte increases > 100 fold in volume as it accumulates organelles and an enormous supply of molecules (e.g. messenger rnas and proteins), that are necessary to acquire meiotic competence and support early stages of embryogenesis. oocyte meiotic and developmental competence is gained in a gradual and sequential manner during folliculogenesis.59 epigenetic marks are also acquired gradually during this process. oocytes acquire dna methylation in a size-dependent manner.60 several histone modifications (e.g. h3k9ac, h4k12ac) increase steadily during oocyte growth and become globally deacetylated by the time of germinal vesicle breakdown.61 191 clinical theriogenology • volume 11, number 3 • september 2019 during its growth, the oocyte interacts with its companion somatic cells. communication between oocyte and the granulosa is vital for oocyte development and granulosa cell differentiation.62 additionally, oocytes depend on cumulus cells to gain metabolites and regulatory signals, which are fundamental for nuclear and cytoplasmic maturation.63 cumulus-oocyte complexes are surrounded by follicular fluid, composed of secretions from follicular cells and exudates from plasma.64 because oocyte growth and maturation are lengthy processes, oocytes are potentially exposed to changing environmental conditions that might induce epigenetic modifications affecting embryo development.65 some epigenetic modifications acquired during oocyte maturation are retained during early embryonic development. failure to establish the correct epigenetic pattern during oogenesis may disrupt embryonic development. given the crucial role of specific posttranslational modifications of histones in regulating gene activity, they also represent a potential mechanism for intergenerational transmission of epigenetically induced states.65 fine-tuning of the process of histone acetylation/deacetylation is crucial for regulation of gene expression during folliculogenesis and chromosome condensation during meiosis progression.61 deletion of the histone deacetylase genes, hdac1 and hdac2, leads to an arrest of oocyte growth and development, as well as a decrease in overall transcriptional activity. the ketone body beta-hydroxybutyrate (bhb) was discovered to be an endogenous regulator of hdacs activity.30 pioneer studies demonstrated that the follicular fluid mirrors plasma bhb concentrations.66 that said, this may be a mechanism by which folliculogenesis can be affected in early post-partum cows, linking a dietary-altered molecule (bhb) with a chromatin-modifying enzyme (hdac). in vitro models of oocyte exposure to bhb concentration in cows with subclinical/clinical ketosis failed to demonstrate a detrimental developmental effect or global effects on histone acetylation when oocytes were treated with bhb alone.67 however, when other molecules were also altered in the culture medium (e.g. glucose), some detrimental effects were observed.68 follicular fluid is a complex biochemical environment, making it difficult to mimic in vitro. additionally, every dietary component may cause only a minor affect on a specific pathway, although the sum of multiple alterations could undermine oocyte competence.69 several well-documented examples of diet causing intergenerational epigenetic effects on offspring have been described, mainly in mice, with a recent report of this epigenetic phenomenon in monkeys.70 in livestock, there is compelling epidemiological evidence that parental diets affect offspring metabolism.8,71 exposing females to various dietary regimens (i.e. high fat, low protein, micronutrient deficient) causes widespread effects on their offspring’s health.72 these outcomes are observed even when embryos at the blastocyst stage are transferred to pseudopregnant recipients, ruling out altered uterine milieu as the causative mechnism. several of the reported studies usually fed mice altered diets for various intervals. surprisingly, effects were observed after feeding a high-fat diet for as little as 4 days before mating. these experiments target oocytes during the final stages of growth and during meiotic maturation and demonstrate that this is a critical period during the lengthy process of oogenesis.65 interestingly, harmful effects of metabolism can be mitigated by pharmacological interventions. treatment with endoplasmic reticulum stress inhibitors for 4 days alleviated oocyte er stress and improved oocyte quality in genetically obese mice.73 a number of defects in oocytes have been identified that could underlie alterations in offspring health. for example: 1) mitochondria with reduced activity and altered ultrastructure; 2) excessive accumulation of lipids in the cytoplasm; and 3) meiotic spindle abnormalities, are features commonly observed on animals exposed to altered diets.72 granulosa cells of diabetic mice or from mice fed high-fat diets have impaired glucose uptake and increased apoptosis.65 similarly, cows in massive lipolysis caused by energy imbalance have elevated nefa concentrations in follicular fluid, which also causes apoptosis of follicular cells.74 communication between oocytes and surrounding granulosa cells is fundamental for acquisition of some regulatory rnas (e.g. micrornas) and various metabolites.75,76 these molecules can be transported to oocytes by trans-zonal projections and through gap junctions or can be packed into exosomes and released into the perivitelline space for oocyte uptake.39 as discussed above, since multiple metabolites can affect chromatin modifying enzymes activity,25,26 alterations to granulosa cells could impact the oocyte epigenome. while the mentioned studies and observations indicate 192clinical theriogenology • volume 11, number 3 • september 2019 direct influence of maternal nutritional state on multiple parameters of oocyte physiology and potential connections with epigenetic machinery, mechanisms by which diet or disease can influence quality of livestock oocytes remain to be fully elucidated. oviduct to creat a new organism from 2 completely distinct cells (i.e. oocyte and spermatozoon), massive reprogramming of the gametes’ epigenome is necessary. this reprogramming occurs shortly after fertilization, as the embryo transits through the oviduct.11 the mammalian oviduct is a complex conduit responsible for final maturation of oocyte and spermatozoon, providing an adequate environment for fertilization and nourishing the early embryo. embryos spend the first few days of development in the oviduct before they reach the uterus to undergo implantation. during this interval, the oviductal environment has an opportunity to modulate the embryonic epigenome and affect the trajectory of embryonic development.77 to support early embryonic development, the oviduct secretes molecules such as glycosaminoglycans, proteoglycans, carbohydrates, ions, hormones, growth factors, cytokines and microvesicles loaded with mirnas and proteins. also, the oviduct controls physiological parameters such as o2 and co2 concentrations, ph, temperature, osmotic pressure, water content, electrolyte concentration and viscosity.78 however, the exact oviductal medium composition and its modulation by the environmental, remain ill defined for most livestock species. early embryos progress quickly through the cell cycle, with chromatin-modifying enzymes working intensively during embryo transit through the oviduct. that said, these processess are likely sensitive to signals received from the oviduct, which can arrive via variousways of communication.79 on this basis, there are several mechanisms by which the oviduct can shape the embryo epigenome. for example: 1) fluctuations in oxygen concentration can alter activity of dioxygenases such as tets that oxidize 5-mc in the paternal pronuclei allowing its removal; 2) epigenetic remodeling enzymes can also be affected by variations in oviductal ph or alterations in luminal secreted substrate concentration, some of which are necessary for enzymatic activity; and 3) variations in ions such as ca2+, which may affect exocytotic processes involving secretion of extracellular vesicles (i.e. oviductosomes), were recently reported to contain small rnas that can be uptaken by embryos.77 recently, it was reported that embryos induce changes in oviductal gene expression and consequently modulate their own environment, demonstrating that communication is bidirectional.80 in vitro models are helping to elucidate roles of various oviductal constituents on embryos. exposing bovine oviduct epithelial cell (boec) to elevated non-esterified fatty acids (nefa) in vitro affected their metabolism and their barrier function, substantiating the idea that the oviduct acts as a gatekeeper and may actively alter early embryonic developmental conditions.81 using in vitro embryo culture systems, it was demonstrated that high oxygen concentrations deregulate epigenetic writers and erasers enzymes, affecting global levels of embryonic dna methylation.82 although full consequences of these observations needs to be determined, embryos cultured in high oxygen concentrations generally have more reactive oxygen species and lower developmental potential. two recent studies have shown direct correlations between presence of oviduct factors during embryo culture and dna methylation marks in the pig and cow.83,84 porcine blastocysts produced in vitro with oviduct fluids as additives in culture medium had global dna methylation and gene expression patterns closer to those of the in vivo-produced blastocysts. in addition, embryos produced in absence of reproductive fluids had methylation changes in critical genes controlling embryonic development, such as insulin-like growth factor 2 receptor igf2r and neuronatin nnat.85 bovine embryos cultured with oviductal fluid had higher methylation in cpg island of line-1 retrotransposons, suggesting that oviductal fluid components can protect against harmful retrotransposition, by maintaining retroelements silenced.86 these studies supported the view that the oviductal environment can modulate the epigenetic landscape of the embryo. the oviduct also secretes regulatory molecules known as embryokines, growth factors that modulate embryonic development by targeting various signaling pathways. bovine embryos treated with the embryokine colony stimulating factor-2 (csf-2) during in vitro culture resulted in animals with similar birthweight to controls but increased growth rate,87 supporting the notion that the preimplantation period is not only important for embryo survival, but also for programming healthspan of the resulting animal. 193 clinical theriogenology • volume 11, number 3 • september 2019 conclusion although mammalian gametes and embryos develop under a highly controlled internal medium, alterations to their environment are possible as a consequences of environmental, dietary, metabolic and physiological conditions.8,88,89 understanding interactions of the embryo with its environment and effects on changes of the embryonic epigenome on further development are fundamental to ensure that health of embryos/offpring is not compromised by production system interventions. also, improved understanding of effects of the epigenome on offspring health could be harnessed to introduce directed epigenome changes to the embryo with predictable results. epigenome editing in livestock using small rnas packed in extracellular vesicles,39,57 addition of molecules (i.e. metabolites, embryokines) to the culture system,67,87 or using crispr-dcas9 systems,90 could become a common practice, with potential to revolutionize the livestock industry. acknowledgement juliano sangalli was supported by são paulo research foundation (fapesp) grants #2016/13416-9 and #2018/09552-0. pablo ross was supported by nih r01hd095007 and usdanifa-afri w3171. conflict of interest authors declare no conflicts of interest. references 1. feil r, fraga mf: epigenetics and the environment: emerging patterns and implications. nat rev genet 2012;13:97-109. 2. jaenisch r, bird a: 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false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice comparison of two altrenogest preparations in mares: a pilot study comparison of two altrenogest preparations in mares: a pilot study jaymie loy,a scott norman,a-c heather ip,a and cyril stephen,a,b aschool of animal & veterinary sciences, charles sturt university, wagga wagga, nsw, australia bgraham centre for agricultural innovation, charles sturt university, wagga wagga, nsw, australia ckallangur veterinary surgery, kallangur, qld, australia abstract altrenogest is a synthetic progestin used in reproductive management of horses. aims were to characterize and compare plasma altrenogest concentrations associated with weekly injectable versus daily oral preparations of altrenogest treatments. we hypothesized that an injectable preparation would not maintain concentrations higher than an oral preparation given once weekly. two mares with no in vivo source of progesterone received either treatment a (150 mg intramuscular altrenogest on days 0, 7, and 14) or treatment b (0.044 mg/kg bodyweight of oral altrenogest daily for 3 weeks). after a 3-week washout period, mares received the opposite treatment for 3 weeks. blood was collected prior to treatments and on days 0, 1, 4, 7, 8, 11, 14, 15, 18, and 21. high-performance liquid chromatography-tandem mass spectrometry was used for altrenogest assays. mean minimum plasma concentrations after treatments a and b were 1.51 ± 0.65 and 2.38 ± 1.53 ng/ml, respectively. plasma altrenogest concentrations were significantly higher in treatment a than treatment b at 24 hours posttreatment (days 1, 8, and 15). no significant difference between plasma altrenogest concentrations was detected on days 4 and 7 of each week. finding that plasma altrenogest concentrations were higher for treatment a at all time points except days 7, 14, and 21 supported our hypothesis. an injectable altrenogest preparation may be considered a more practical and safer solution for long-term treatment; based on this study, it was comparable to a proven daily oral altrenogest treatment for a minimum of 4 days. keywords: altrenogest, equine, injectable, oral, plasma introduction altrenogest is a synthetic progestin commonly used in mare reproduction for behaviour modification, control of estrus, maintenance of pregnancy and uterine quiescence, and induction of lactation in nonparturient mares.1-4 such uses require altrenogest treatment ranging from 10 120 days. currently, only 1 oral altrenogest preparation is available in australia with a demonstrated progestational effect in mares. however, daily oral altrenogest treatment poses risks to personnel who provide treatment, since it is an oil-based preparation that is readily absorbed through skin.5 human risks from cutaneous exposure include disrupted menstrual cycles, prolonged pregnancy, decreased libido in men, headaches, fever, abdominal pain, nausea, diarrhea, and rashes.6 in addition to inherent safety risks to personnel, daily oral altrenogest treatment is logistically difficult and time consuming.7, 8 recent research has focused on determining efficacy of other routes of altrenogest treatment. although intrarectal and intravaginal treatments of altrenogest (0.044 mg/kg) resulted in absorption, plasma concentrations were significantly lower than oral treatment.5 doubling oral dose (0.088 mg/kg) was recommended for intrarectal treatment to mares unable or unwilling to receive oral treatment.9 this treatment resulted in plasma concentrations > 0.5 ng/ml that were maintained for 5.5 hours (range, 3 8 hours). although intrarectal treatment is potentially useful for mares that cannot receive oral treatment, increased handling of this hormone, every 4 8 hours, poses a greater health risks to personnel. consequently, there is a need to ensure a more convenient and safer delivery method. in recent years, despite previous studies supporting supplementation of > 225 mg,10 australian pesticides and veterinary medicines authority approved treatment of injectable preparations of altrenogest (150 mg/500 kg body weight every 5 7 days). this reduced frequency of handling may be a safer alternative for personnel. however, there is a need to assess its efficacy or bioequivalence compared to a proven daily oral treatment regimen. although pharmacokinetic studies that determined half-life and excretion rates of injectable altrenogest were limited to a single dose treatment, clinical cases traditionally require treatment beyond a single dose. additionally, there is apparently no documentation of plasma altrenogest clinical theriogenology • volume 12 number 4 • december 2020511 concentrations (pac) associated with oral altrenogest treatment at 0.044 mg/kg for the recommended treatment period of 15 days. objectives of this pilot study were to characterize plasma altrenogest concentrations after oral and injectable preparations over a 3-week period. secondly, to determine if an injectable preparation is equivalent to an oral preparation for the recommended 7-day treatment period, to ascertain if injectable altrenogest can be used as a safer substitute for oral altrenogest. material and methods this research was conducted at charles sturt university (csu), with institutional animal care and ethics committee approval (approval no. 15/037). animals two healthy mares aged 6 (horse 1; cleveland bay; 570 kg) and 8 (horse 2; stockhorse; 458 kg) years, with proven inability to produce progesterone from a luteal source (ovariectomized and hypoplastic ovaries, respectively), were used in this pilot study. treatment and blood sampling this study was conducted between june and august 2015 as a bioequivalence crossover design trial. both mares received either treatment a (3 ml of 50 mg/ml intramuscular altrenogest [ovu-mate®; randlab, chipping norton, nsw, australia]) on days 0, 7, and 14) or treatment b (0.044 mg/kg bodyweight daily oral altrenogest [regumate®; intervet, bendigo, vic, australia]) for 3 weeks. after a 3-week wash-out period, mares received the opposite treatment for a further 3 weeks. venous blood was collected into heparinised tubes (10 ml) for plasma altrenogest assay, prior to treatments (day 0) and on days 1, 4, 7, 8, 11, 14, 15, 18, and 21. assay and data analysis samples were analyzed for pac using high-performance liquid chromatography-tandem mass spectrometry (lcms-ms) in a commercial laboratory (pia pharma pty ltd), according to methods previously reported.11 quantitative analysis was based on an internal standardization using c21h24o2, gestrinone with a molecular weight of 308.421 g/mol. in comparison, altrenogest structure is c21h26o2 with a molecular weight of 310.437 g/mol. a calibration curve, prepared with concentrations ranging from 1 to 50 ng/ml altrenogest, was used for quantitation. correlation coefficient (r) was 0.9996 which met in-house criteria (r > 0.99). the analyst was blinded to treatments. differences in pac between treatment routes were analyzed for days 1, 4, and 7 of each week (days 1, 4, 7, 8, 11, 14, 15, 18 and 21) using a series of paired student’s t-tests. interactions between pac and time were tested by repeated measures analysis using mixed procedure of sas. results daily oral altrenogest treatment resulted in minimum pac between 0.87 and 4.17 ng/ml (table). weekly altrenogest injections resulted in minimum pac ranging from 0.64 to 2.79 ng/ml over the treatment period (table). table. plasma altrenogest concentrations (ng/ml) over 21-day treatment period treatment group day day day day day day day day day day 0 1 4 7 8 11 14 15 18 21 horse 1 a < 0.5 13.16 2.38 0.64 14.37 4.31 1.6 17.2 4.05 1.31 b < 0.5 1.79 3.36 3.8 3.57 3.36 3.89 3.25 4.17 3.63 horse 2 a < 0.5 13.29 3.86 1.2 10.91 3.21 1.51 7.52 3.09 2.79 b < 0.5 0.96 1.06 0.87 1.03 0.93 1.13 1.02 0.87 0.96 a. injectable altrenogest once weekly (days 0, 7, and 14) intramuscular; b. oral altrenogest once daily for 21 days clinical theriogenology • volume 12 number 4 • december 2020 512 mean minimum pac following treatment a and treatment b were 1.51 ± 0.65 and 2.38 ± 1.53 ng/ml, respectively (mean ± sd). mean pac for treatment a in horses 1 and 2 over the trial period were 6.56 ± 6.46 and 5.26 ± 4.32 ng/ml and for treatment b in horse 1 and 2 were 3.42 ± 0.68 and 0.98 ± 0.09 ng/ml. plasma altrenogest concentrations in treatment a was significantly higher than treatment b at 24 hours posttreatment (days 1, 8, and 15 of the trial period). posttreatment, with weekly altrenogest, pac markedly decreased between days 1 and 4, and days 4 and 7 over each treatment week (figure). treatment a treatment b -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 day -2 0 2 4 6 8 10 12 14 al tre no ge st (n g/ m l) injection injection injection figure. mean plasma altrenogest concentrations over the 3-week period. treatment a: intramuscular altrenogest treatment (injection) once weekly on days 0, 7, and 14. treatment b: oral altrenogest treatment once daily for 21 days. mean pac from treatment a was greater than that of treatment b on day 4 (3.48 ± 0.72 and 2.29 ± 1.5 ng/ml, respectively). by day 7 of each week, mean pac of treatment a were below treatment b (1.51 ± 0.65 and 2.38 ± 1.53 ng/ml). no significant difference between pac of selected preparations was detected on days 4 or 7 of each week. discussion injectable alternogest resulted in an immediate increase in pac > 10 ng/ml at 24 hours after treatment. by 4 days posttreatment, pac markedly decreased and were > 2 ng/ml. mean pac were higher for a minimum of 4 days in injectable altrenogest compared to oral altrenogest. on day 7 of each week, pac of injectable altrenogest were lower than oral altrenogest (1.51 ± 0.65 and 2.38 ± 1.53 ng/ml, respectively). similar results were observed when weekly injectable altrenogest (readyserve® 150 mg/week) was used.12 mean pac were highest ~ 24 hours after treatment and were > 1 ng/ml on day 4 and < 0.5 ng/ml at 6.2 days (148h). an in vivo assessment of readyserve® at the same dosage used in this study was not reliable in supressing estrus in all mares for the full 7-day treatment window. comparable to this, our findings indicated that concentrations associated with injectable altrenogest preparations were higher than oral altrenogest for 4 days, but may not effective between days 5 and 7 posttreatment in mares. other injectable altrenogest, not readily available in australia, have been successful in suppressing estrus and ovulation in mares treated for up to 33 clinical theriogenology • volume 12 number 4 • december 2020513 days.10 however, the recommended dosage is 1.5 3.3 times higher than the dosage used in our study. therefore, further investigation with increased dosing regimens would be beneficial, as it may result in higher pac, more effective suppression of estrus and reduced frequency of treatments, thereby minimising human contact and associated risks. for both treatments, horse 1 had higher pac than horse 2. interestingly, horse 1 was considered a body condition (fatness) score of 2 (thin), whereas horse 2 was considered a body condition score of 4 (fat) on a scale of 0 to 5. altrenogest was detected in liver, fat, kidneys and muscle after treatment. liver had the highest tissue concentrations of altrenogest posttreatment, followed by fat.13 to date, there are no studies in horses that indicated whether high body condition influenced circulating plasma concentrations of progestogens. however, an influence was noted in cattle, rats and humans.14-16 a horse’s body condition could influence circulating plasma concentrations, particularly if altrenogest is deposited in fat. as altrenogest is marketed with a weightdependent dosage, further research is warranted to ensure that body condition does not adversely influence altrenogest efficacy. during the 3-week trial period, mean minimum concentrations of altrenogest for both preparations had an increasing trend. at the end of the first full treatment period for each preparation, (7 days for injectable and 24 hours for oral), mean minimum concentrations for treatments a and b were 0.92 and 1.38 ng/ml, respectively. however, at the end of the 3-week trial period, mean minimum concentrations for treatment a and treatment b were 2.05 and 2.30 ng/ml, respectively. although our sample size was not large enough to identify a significant difference, this observation may be an indication of a cumulative effect worthy of further research. a cumulative effect following long-term altrenogest has not been reported in horses. however, during prolonged altrenogest treatment in pigs, an accumulation in blood plasma has been demonstrated.13 further research into the possibility of a cumulative effect of long-term altrenogest treatment could be useful to determine if minimum concentrations of a weekly injectable preparation would eventually maintain concentrations comparable to a daily oral preparation. there were several limitations associated with this pilot study. a decision was made to use mares with no in vivo source of progesterone, which are scarcely available, and resulted in a sample size of only 2 mares. a larger sample size would have provided a better indication of mean and minimum pac after treatment, allowing for significant differences to be discriminated. restrictions on sampling frequency did not allow documentation of peak concentrations. however, minimum concentrations associated with both treatments were characterized, as sampling occurred immediately prior to treatments. results from this preliminary study characterized pac during treatment and provided evidence that injectable altrenogest was comparable to daily oral altrenogest for a minimum of 4 days, but may not be comparable or effective between days 5 and 7 posttreatment. this should be taken into consideration if using injectable altrenogest as a substitute for oral altrenogest in mare reproductive management, as minimum target pac are yet to be determined for either pregnancy management or estrus suppression. further research with a larger number of animals is required to determine bioequivalence of these 2 preparations. conflict of interest none to declare. funding randlab pty ltd, chipping norton, new south wales, australia. acknowledgement authors thank joe pippia at pia pharma for hplc-ms analysis and alex tinning and kirsten volz for their assistance in handling and management of mares. references 1. squires el: use of progestins in open and pregnant mares. anim reprod sci 1993;33:183-193. 2. daels pf: induction of lactation and adoption of the orphan foal. proc am assoc equine pract 2006. 3. hodgson d, howe s, jeffcott l, et al: effect of prolonged use of altrenogest on behaviour in mares. vet j 2005;169:113-115. clinical theriogenology • volume 12 number 4 • december 2020 514 4. hinrichs k, sertich pl, kenney rm: use of altrenogest to prepare ovariectomized mares as embryo transfer recipients. theriogenology 1986;26:455-460. 5. hornberger k, lyman cc, baldrighi jm, et al: plasma altrenogest concentrations in mares after intrarectal or intravaginal administration. clinical theriogenology 2019;11:59-65. 6. fda. animal drug safety communication: fda highlights potential health risks to people exposed to altrenogest products for horses or pigs. 2018 [cited 2020 january]; available from: https://www.fda.gov/animalveterinary/cvm-updates/fda-animal-drug-safety-communication-fda-highlights-potential-health-risks-peopleexposed. 7. shideler rk, squires el, voss jl, et al: progestagen therapy of ovariectomized pregnant mares. j reprod fertil suppl 1982;32:459-464. 8. squires el, heesemann cp, webel sk, et al: relationship of altrenogest to ovarian activity, hormone concentrations and fertility of mares. j anim sci 1983;56:901-910. 9. ellis ke, council-troche rm, von dollen ka, et al: pharmacokinetics of intrarectal altrenogest in horses. j equine vet sci 2019;72:41-46. 10. storer wa, thompson jr dl, gilley rm, et al: evaluation of injectable sustained release progestin formulations for suppression of estrus and ovulation in mares. j equine vet sci 2009;29:33-36. 11. machnik m, hegger i, kietzmann m, et al: pharmacokinetics of altrenogest in horses. j vet pharmacol ther 2007;30:86-90. 12. mcconaghy ff, black l, colgan s, et al: studies of the pharmacokinetic profile, in vivo efficacy and safety of injectable altrenogest for the suppression of oestrus in mares. aust vet j 2016;94:248-255. 13. european medicines agency. committee for medicinal products for veterinary use. 2004. [cited 2019 november]; available from: http://www.ema.europa.eu/docs/en_gb/document_library/maximum_residue_limits__report/2009/11/wc500009996.pdf. 14. norman s: factors influencing the incidence of dystocia in beef heifers. vet sci;2002, university of queensland. https://espace.library.uq.edu.au/view/uq:157957 15. stirrat li, o’reilly jr, barr sm, et al: decreased maternal hypothalamic-pituitary-adrenal axis activity in very severely obese pregnancy: associations with birthweight and gestation at delivery. psychoneuroendocrinology 2016;63:135-143. 16. crew rc, mark pj, clarke mw, et al: obesity disrupts the rhythmic profiles of maternal and fetal progesterone in rat pregnancy. biol reprod 2016;95:55. clinical theriogenology • volume 12 number 4 • december 2020515 2019 editor™s note a note of appreciation the editor and editorial board members of clinical theriogenology, theriogenology associations (society for theriogenology, american college of theriogenologists, and theriogenology foundation), and franz management thank following scholars for their time and effort in reviewing submitted manuscripts in 2018 2019. angel abuelo glen almond marco alvarenga divakar ambrose* (2) scott bailey aubrey baird ursula bechert etta bradecamp* leonardo brito tony bruinjé teresa burns alexis campbell claire card julie card julie cecere peter chenoweth soon cheong bruce christensen sherrie clark-deener * (2) gail colebern* (2) jill colloton* (2) lionel dawson thomas doherty charles estill (2) allan gunn erik hofmeister fiona hollinshead richard hopper vanmathy kasimanickam* (2) firdous khan kara kolster* (2) michelle kutzler catharina lindeforsberg robert lofstedt sara lyle* david mastas patrick mccue sue mcdonnell lynda miller* marcelo miragaya peter morresey ali naqvi suppawiwat ponglowhapan christopher premanandan malgor pozor jennifer roberts (2) de reyes maria schnobrich jack smith jennifer sones cyril stephen mitsuhiro takagi masayasu taniguchi regina turner john verstegen william whitler brian whitlock* (2) karen wolfsdorf* *editorial board member number within parenthesis indicate manuscripts reviewed information provided in canine symposium is published in this issue. editor thanks drs. michelle kutzler and jennifer sones for formatting and dr. john kastelic for copy editing submitted manuscripts. 597 clinical theriogenology • volume 11, number 4 • december 2019 598clinical theriogenology • volume 11, number 4 • december 2019 untitled omniblank: introduction equids are seasonally polyestrus with most mares in anestrus during winter. melatonin, present at higher concentrations in long periods of uninterrupted darkness, inhibits gonadotropin releasing hormone (gnrh) secretion from hypothalamus. consequently, concentrations of luteinizing hormone and follicle stimulating hormone are reduced, resulting in anestrus.1 in late winter and early spring, ambient daylight lengthens in duration and intensity, initiating spring transition. with an average duration of 45 days, sufficient gonadotropin concentrations are reached at the end of spring transition, for the first ovulation of the year to occur. regular cyclicity resumes at this point, initiating the physiologic breeding season.2 hormone therapy and photoperiod adjustments are used to manipulate estrous cycle by hastening spring transition, with the goal for an earlier physiologic breeding season. a photoperiod length of at least 16 hours in 24 hours at an adequate intensity of light (> 100 lux), is required to advance spring transition.2 other reproductive effects of increased photoperiod length include: increased testicular size, sperm production and output in stallions, earlier onset of puberty, shorter pregnancy length, and increased foal weight.3,4 daylength has less influence on double ovulation and twinning rates than other factors (e.g. breed, age of mare, and use of reproductive medications).5 however, in a population of european thoroughbreds,6 increase in twin pregnancy rate effects of sunlight hours and hormones on double ovulation, and singleton and twin pregnancies in mares martha mellish, kasadee allan, bronwyn crane atlantic veterinary college, university of prince edward island charlottetown, pe, canada abstract equine twin pregnancies are almost exclusively dizygotic, without the application of advanced reproductive technologies, requiring 2 ovulations in 1 estrous cycle. breeding records were used to determine the effects of sunlight hours, prostaglandin f2α, human chorionic gonadotropin, deslorelin (a gonadotropin releasing hormone agonist), and progesterone and estradiol on double ovulation rates, and singleton and twin pregnancy rates. breeding records of mares (n = 267) and their estrous cycles (n = 914) were analysed. double ovulations occurred in 10.5% (96/914) of estrous cycles. twin pregnancies were observed in 42.7% (38/89) of mares that had double ovulations. overall, per estrous cycle pregnancy rate was 47.2% (405/858) and twin pregnancies was 4.4% (38/858). double ovulations had higher (p < 0.001) per cycle singleton pregnancy rate (69.7%; 62/89) than 1-ovulation cycles (44.6%; 343/769). deslorelin increased (p < 0.05; or =1.24 95% ci) double ovulations and human chorionic gonadotropin tended (p = 0.089; or =1.68; 95% ci) to increase double ovulations. deslorelin use resulted in an odds ratio of 2.47 for a positive pregnancy (either singleton or twin) diagnosis compared to cycles without deslorelin use. none of the factors examined had a substantial impact on twin pregnancy rates. keywords: equine twins, double ovulation, deslorelin, sunlight, pregnancy rate was observed in june/july (1.76%; 40/2157) compared to february/march (0.9%; 29/3229); however, double ovulation rate was not provided. increased double ovulation rates were also observed in august (16.5%) and september (26.5%) compared to other months in a population of predominantly american standardbreds located in finland.7 reported double ovulation rates ranged widely (0.83 42.8%), depending on the population studied.6,8 double ovulation is a prerequisite for dizygotic pregnancies.9 survival of sperm in uterine tubes up to 7 days is a reason to have embryonic vesicles that are substantially different in size.10 twin pregnancies that survive beyond 40 days of pregnancy often suffer from placental insufficiency. this can lead to abortion, fetal mummification, dystocia and birth of small, weak foals and negatively impact the mare’s foaling ability in the following breeding seasons.9-13 ovulation inducing agents increase the efficiency of equine reproduction programs.14 by inducing ovulation in a predictable time frame, typically within 48 hours of a well timed treatment, less inseminations per estrous cycle were needed and odds of achieving optimal timing of insemination relative to ovulation was higher.14 increases in pregnancy and twinning rates were observed with human chorionic gonadotropin (hcg) and deslorelin, and a gnrh analogue.9,13 in an australian study, pregnancy rates were 72.0% (147/204), 72.0% (1042/1447), and clinical theriogenology 2021; 13: 81 64.5% (527/817) for deslorelin, hcg, and control, respectively.5 use of hcg in a population of thoroughbred mares tended to increase pregnancy rates (or 1.21; p = 0.06) and twinning rates (or 2.18; p < 0.05).15 however, these observations could vary with the population of mares studied. in another study, hcg had no effect on double ovulation or twinning rates,7 but this same population had a substantial increase in double ovulation rates with prostaglandin f2α treatment compared to cycles managed without a hormone (16.2 versus 9.9%).7 increased twinning in a thoroughbred mare population with use of hcg alone was less remarkable than the combined use of hcg and prostaglandin f2α. combination of medications resulted in an odds ratio of 6.37 for twin pregnancies compared to controls.15 effects of a progesterone and estradiol 17ß (p&e) protocol on double ovulations and twin pregnancy rates were not extensively studied. reproductive hormones are extensively used in mares that have a short breeding season (delayed spring transition) due to their geographic location. combined impacts of reproductive hormones and daylight hours on double ovulation rates and pregnancy rates for these regions is not known. we hypothesized that both reproductive manipulations and increasing daylight hours would increase double ovulation rate. objective was to analyze the effects of daylight hours and use of prostaglandin f 2α, deslorelin, hcg, and p&e on the rates of double ovulation and resulting singleton and twin pregnancy rates materials and methods breeding records (2011 2019) maintained at the institution for client-owned mares (n = 267) and their estrous cycles (n = 914) were used. mares were located at 46.50n longitude and 63.40w latitude. over 50% of the broodmares were standardbreds, with warmbloods, quarter horses, and draft breeds represented to a lesser degree. mare age was not recorded in the records. mares in the study carried their own pregnancies. breeding records included: date of examination, ovarian activity, uterine abnormalities, presence of embryonic vesicle(s), endometrial cysts, cervical tone, stallion name, and reproductive treatments. fresh semen was the predominant type of semen used in these cycles in addition to cooled, shipped, and frozen semen. mares were examined at 12 16, 24 28, and 60 70 days postovulation. a mare was classified as pregnant if ultrasonographic evidence of pregnancy was observed. diagnosis of singleton or twin pregnancies was made at the initial pregnancy examination and this pregnancy status was used for analysis. twin pregnancies that were manually reduced to singleton pregnancies remained classified as twin pregnancies for the purpose of this study. pregnancy outcomes were unknown in 49 single ovulation cycles and seven double ovulation cycles. these cycles were therefore not included in the pregnancy rate calculations. values for daylight hours were rounded to the nearest half decimal to create uniformity throughout the dataset and an interval of a half hour within the statistical analyses. medications used include: dinoprost 5 10 mg im (lutalyze®, zoetis canada inc. kirkland, qc, canada), cloprostenol 0.25 0.5 mg im (estrumate®, intervet canada corp., kirkland, qc, canada), deslorelin 1.5 mg im (trutina pharmacy inc., ancaster, on, canada) and hcg 2500 5000 iu iv (chorulon®, intervet canada corp., kirkland, qc, canada). protocol for p&e (chiron compounding pharmacy inc, guelph, on, canada) treatment was: daily 150 mg progesterone and 10 mg estradiol 17ß im for 10 days, with 0.25 0.5 mg cloprostenol im on the 10th day. after cloprostenol treatment, 2500 5000 iu of hcg was given when a 35 mm follicle was present (~ 7 9 days). data analyses data were analyzed using a statistical software program (minitab 19, minitab llc, state college, pa). singleton pregnancy rates with single and double ovulations cycles were compared using a two-sample test of proportions. odds ratio was used to compare odds of a pregnancy in a cycle that used deslorelin with odds of a pregnancy that did not use deslorelin. sunlight hours, prostaglandin f2α, deslorelin, and hcg treatment in relation to double ovulation rates were evaluated using multivariable analysis to determine odds ratios with a high and low ci of 95%. use of p&e and twin pregnancy rate was evaluated using fisher’s exact test. significance was established at p ≤ 0.05. a tendency was established at p = 0.06 0.10. generalized estimating equation (gee) method (stata, statacorp) was used to account for dependence of multiple records per mare. results per cycle double ovulation rate was 10.5% (96/914), pregnancy rate was 47.2% (405/858), and twin pregnancy rate was 4.4% (38/858). double ovulations had a higher (p < 0.001) per cycle singleton pregnancy rate (69.7%; 62/89 [95% ci: 60.1 79.2%]) than single ovulations (44.6%; 343/769 [95% ci: 41.1 48.1%]). mares with double ovulations had a twin pregnancy rate of 42.7% (38/89). deslorelin used cycles (n = 330) had 2.47 times more positive pregnancy examination, either singleton or twin, ([95%; ci:1.85 3.32] p < 0.001) compared to cycles (n = 528) that did not use deslorelin. per cycle pregnancy rate of cycles that used deslorelin was 60.9% (201/330) and was 38.6% (204/528) in cycles that did not use deslorelin. results of logistic regression analysis (both for double ovulations and twin pregnancies) are presented (table). increasing daylight hours had no significant effect. deslorelin increased (p = 0.045; or = 1.57) the rate of double ovulation. a tendency (p = 0.089) towards increasing double ovulations occurred with hcg. prostaglandin f2α and p&e administration had no impact on the rate of double ovulation. none of the factors examined had substantial impact on twin pregnancy rates. clinical theriogenology 2021; 13: 82 individual mares repeated for several years and multiple cycles were evaluated in a single year. the assumption of independence could not be held true due to these factors. the mean gap between cycles of the same season was 21.3 days (standard deviation 11.5 days), which is accurate to the biology of equine estrous cycles. data were then evaluated using the gee method and results were similar. original multivariable logistic regression was used for analysis. discussion effect of daylight hours on double ovulation rates or twin pregnancy rates was not substantial. study location (eastern canada) has a large seasonal variation in daylength, with > 15 daylight hours during summer solstice and < 9 hours during winter solstice.16 since appreciable variation (northern location) in daylength has not impacted the rate of double ovulation it is reasonable to assume that daylight hours in equator areas might have minimal impact. further study with a larger number of cycles would be worthwhile to determine if significance could be established between daylight hours and multiple ovulations double ovulation twin pregnancies variable odds ratio (95% ci) p value odds ratio (95% ci) p value daylight 1.189 (0.956 1.478) 0.119 1.203 (0.815 1.774) 0.353 deslorelin 1.569 (1.010 2.439) 0.045 0.946 (0.465 1.925) 0.878 hcg 1.6848 (0.924 3.071) 0.089 1.288 (0.512 3.238) 0.590 p&e 0.5053 (0.117 2.191) 0.362 * * prostaglandin 1.242 (0.541 2.851) 0.609 1.826 (0.380 8.764) 0.452 table. odds ratio for variables listed for double ovulations and twin pregnancies *no significant difference between the use of p&e and the occurrence of twin pregnancies. or multiple pregnancies. rate of double ovulations (10.5%) was consistent with reports for standardbreds (13 -15%).5,7 deslorelin increased (p = 0.045) double ovulation rate and hcg use had a trend (p = 0.089). multiple ovulations are a requirement for dizygotic twins or multiple pregnancies. higher singleton pregnancy rate observed with double ovulations cycles (69.7%; 62/89) compared to single ovulation cycles (44.6%; 343/769) suggested that double ovulation cycles could be desirable, especially, in an embryo transfer program. however, proper management of twin pregnancies resulting from double ovulations, within populations of mares intended to carry their own pregnancy, is essential. early diagnosis of twins through prompt and repeated pregnancy examinations is essential, particularly, when a second dominant follicle is detected at insemination. records analyzed here reflect per cycle pregnancy rates for fresh semen, shipped cooled semen, and frozen semen. evaluation of combined pregnancy rates, rather than separating pregnancy rate according to type of semen is a limitation of the study. overall pregnancy rate of 47.2% in our study was lower than generally accepted pregnancy rates for fresh and shipped cooled semen of 50 -70%. similar mare populations had pregnancy rates of 51.7 56.7% for fresh and shipped cooled semen.7,17 we incorporated frozen semen cycles in our dataset which have generally lower reported per cycle pregnancy rates compared to fresh and cooled semen.7,17 combination of pregnancy results from several semen types may be the cause of a lower-than-expected overall pregnancy rate. higher singleton pregnancy rate with double ovulation cycles than single ovulation cycles reflects that the mare’s reproductive status can be as important as the semen type used on resulting pregnancy rates. use of deslorelin increased the odds of a pregnancy by 2.47 times. association of an ovulation induction agent with an increased chance of pregnancy can be carried into clinical practice. increased odds of pregnancy might be due to the predictable timing of ovulation from the use of deslorelin. decreased time from insemination to ovulation for fresh and shipped semen and insemination close to ovulation for frozen semen breedings improved the odds of pregnancy. less resulting inseminations per cycle improved stallion efficiency and minimized uterine inflammatory responses.15 deslorelin use increased rate of multiple ovulations that increased opportunities for fertilization in each estrous cycle. use of deslorelin did not increase twinning rates in this population; however, it might have the benefit of increasing the odds of embryo recovery. conclusion an increase in daylight hours and use of prostaglandin, and p&e had no influence on twin pregnancy rates. use of hcg had a tendency to increase double ovulation rate. deslorelin use increased rate of double ovulations per cycle and singleton pregnancy rate. double ovulation cycles had a higher singleton pregnancy rate than single ovulation cycles. clinical theriogenology 2021; 13: 83 acknowldgements authors thank henrik stryhn and jason stull for assistance with statistical analysis. conflict of interest none to declare. funding supported by ‘summer canada student jobs’ and a poster was presented at the 2020 national veterinary scholars symposium. references 1. dini p, ducheyne k, lemahieu i, et al: effect of environmental factors and changes in the body condition score on the onset of the breeding season in mares. reprod dom anim 2019;54:987-995. 2. mckinnon ao: vernal transition into the breeding season. in: mckinnon, ao, squires el, vaala, we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 1706-1714. 3. nolan mb, walsh cm, duff n, et al: artificially extended photoperiod administered to pre-partum mares via blue light to a single eye: observations on gestation length, foal birth weight and foal hair coat at birth theriogenology 2017:100;126-133. 4. clay cm, clay jn: endocrine and testicular changes associated with season, artificial photoperiod, and the peri-pubertal period in stallions. vet clin north am equine pract 1992:8;31-56. 5. mckinnon ao: origin and outcome of twin pregnancies. in: mckinnon, ao, squires el, vaala, we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 2350-2358. 6. merkt h, jochle w: more twin pregnancies as season progresses. j equine vet sci 1999:19;536-537. 7. katila t: effects of hormone treatments, season, age and type of mares on ovulation, twinning and pregnancy rates of mares inseminated with fresh and frozen semen. pferdeheilkunde 2003:6;619-624. 8. davies morel mcg, newcombe jr, lauber m: manual reduction of multiple embryos in the mare: the effect on subsequent pregnancy outcome. vet j 2012:192;322-325. 9. veronesi mc, battocchio m, faustini m, et al: relationship between pharmacological induction of estrous and/or ovulation and twin pregnancy in the thoroughbred mares. domest anim endocrinol 2003:25;133-140. 10. crabtree jr: management of twins in horses. brit vet assoc: in practice 2018:40;66-74. 11. klewitz j, krekeler n, ortgies f et al: evaluation of pregnancy and foaling rates after reduction of twin pregnancy via transvaginal ultrasound-guided aspiration in mares. j am vet med assoc 2013:242;527-532. 12. meshram bn, kamble mv, dalvi rs: detection and management of twin pregnancies in thoroughbred mares. intas polivet 2004:5;311-312. 13. ginther oj: twin embryos in mares i: from ovulation to fixation. equine vet j 1989:21;166-170. 14. finan s, lamkin e, mckinnon a: comparative efficacy of biorelease deslorelin® injection for induction of ovulation in oestrus mares: a field study. aust vet j 2016; 94:338-340. 15. perkins nr, grimmett jb: pregnancy and twinning rates in thoroughbred mares following the administration of human chorionic gonadotropin (hcg). nz vet j 2001:49;94-100. 16. time and date as. (n. d.). timeanddate.com. retrieved december 16, 2020 https://www.timeanddate.com/sun/canada/ charlottetown?month=6&year=2012 17. sieme h, schafer t, stout tae, et al: the effects of different insemination regimes on fertility in mares. theriogenology 2003:60;1153-1164. clinical theriogenology 2021; 13: 84 editor™s note a note of appreciation despite their busy schedules, the following scholars graciously accepted invitations to review manuscripts submitted to clinical theriogenology in 2020 and provided timely and thoughtful evaluations. the editor, copy editor (john kastelic), editorial board members, theriogenology associations (society for theriogenology, american college of theriogenologists, and theriogenology foundation), and franz management thank them for their time and effort. divakar ambrose* orsolya balogh don bergfeldt henkbert bertschinger gabriel bo etta bradecamp* soon hon cheong bruce christensen sherrie clark-deener* betsy coffman gail colebern* jill colloton* lionel dawson mariana diel de amorim natalie fraser sean finan ed garrett mohanathas gobikrushanth allan gunn ben hart aime johnson carolynne joone (2) vanmathy kasimanickam* (3) kara kolster* chris kuster kristina lu christianne magee lynda miller* (2) jane morrell colin palmer* suppawiwat ponglowhapan jennifer schleining maria schnobrich camilla scott cyril stephen dickson varner brian whitlock* karen wolfsdorf* (2) eleas wu     *editorial board member numbers within parentheses indicate manuscripts reviewed   clinical theriogenology • volume 12 number 4 • december 2020474 fetal programming: maternal-fetal interactions and postnatal performance fetal programming: maternal-fetal interactions and postnatal performance caleb lemley department of animal and dairy sciences, college of agriculture and life sciences mississippi state university, mississippi state, ms abstract placental and fetal development is sensitive to direct and indirect effects of maternal environment. environmental stimulus or insult during critical periods of development (e.g. fetal period) can program lifelong production characteristics of animal, irrespective of their genotype. several environmental factors negatively impact placental development and blood flow during pregnancy and hinder offspring vigor. timing of environmental insult or therapeutic intervention during pregnancy is critical, as developmental sensitivity of fetoplacental unit changes from early to late pregnancy. association between poor growth performance of offspring and decreased blood flow to uterus and placenta during pregnancy was established. nutrient restriction during early to midpregnancy decreased uterine artery blood flow, leading to compensatory growth of placental blood vessels during exponential fetal growth. although many studies investigated offspring phenotypes following maternal nutrient restriction or overfeeding during pregnancy, fewer concentrated on enhancing postnatal performance in livestock based on offspring body weight. survival rate of offspring born at average weight was higher than those with below-average birth weight. in this regard, dietary supplements promoting uterine blood flow increased postnatal growth and weaning weights. elucidating consequences of specific supplements on continual plasticity of placental functional capacity will allow us to determine important mediators of offspring growth and development. keywords: cattle, development, fetus, nutrition, placenta, sheep introduction irrespective of animal’s genotype, environmental stimulus or insult during a critical period of development can impact phenotype. exposure to environmental stimulus or insult may establish a permanent postnatal phenotype, resulting in adverse consequences for milk production, carcass yield, feed efficiency, and/or reproductive function.1,2 process of permanently altering animals’ phenotype through environmental stimuli is referred as ‘developmental programming hypothesis’.2 for example, 2 animals with similar genotype but raised in different environments are expected to have differing lifelong phenotypic characteristics, which is further explained by developmental plasticity. importantly, magnitude of phenotypic change is vastly different between these 2 animals with similar genotype, if their exposure to different environments occurred while they were embryos, fetuses, calves, weaned heifers, or mature cows. changes in animals’ developmental trajectory with lasting consequences were greatest in embryos and fetuses, with decreasing developmental plasticity with increasing animal age. study of developmental programming during fetal period, an age of high developmental plasticity, is known as ‘fetal programming’. support for fetal programming concept (strong association between birth weight and lifelong developmental consequences) is growing.3,4 for example, low birth weight offspring are at increased risk of morbidity and mortality, slowed postnatal growth, poor body composition (increased fat and reduced muscle growth), metabolic disorders, cardiovascular pathologies, and dysfunction of several organs (ovaries, testes, mammary gland and gastrointestinal tract).4,5 livestock are specifically at risk due to poor nutritional environments during pregnancy (e.g. breeding young, growing, peripubertal dams that are competing for nutrients with fetus). in addition, poor pasture conditions or environmental heat stress in relation to seasonal breeding can decrease nutrient availability for both dam and fetus during critical periods of development.6 although these initial fetal programming studies focused exclusively on offspring (fetal or birth) weight, we now understand that multiple measurements of offspring size at birth can predict developmental trajectory. therefore, phenotypic changes in livestock production as a result of clinical theriogenology • volume 12 number 3 • september 2020252 fetal programming may be independent of birth weight7-9 with environmental insults during early pregnancy altering phenotypic changes (production) despite no change in birth body weight.10 insufficiencies during pregnancy, resulting in reduced fetal growth and development, are detrimental to livestock, where newborns represent next generation of meat and milk producing animals. several animal models of fetal and placental growth restriction (e.g. maternal nutritional plane, maternal age, heat stress, hypoxic stress, and fetal number) were developed to better unravel relationships among uterine blood flow and offspring development.11-14 establishment of functional fetal and placental circulation is one of the earliest events during conceptus development15,16 and exponential increase in placental exchange is vital for maintaining remarkable growth and development of fetus during last half of pregnancy.17 therefore, understanding impacts of maternal environment on placental function is especially relevant to these proceedings, as majority of mammalian livestock raised for red meat production spend 30 40% of their life being nourished by placenta. percent time in each phase of beef production from conception to harvest is depicted in figure 1. in addition, amount of developmental plasticity of offspring varies during their lifespan, with maximal influences of developmental programming occurring during embryonic and fetal stages (figure 1). figure 1. percent time in each phase of beef production from conception to harvest (womb to tomb), note: nearly one-third of life is spent developing in utero when fetus is most vulnerable to environment, due to increased developmental plasticity. fetal growth and organ development embryonic period in cattle is defined as time from conception (single cell embryo, as zygote) to completion of organogenesis. this embryonic period typically extends from days 1 42 of pregnancy, with highest percent pregnancy wastage occurring during this interval. fetal period is defined as the remainder of pregnancy from days 42 280 at organ differentiation completion (figure 2).18 characterization of bovine fetal growth throughout pregnancy allowed researchers to hypothesize phenotypic changes to offspring that experience specific periods of environmental insults that may perturb normal development in utero. for example, nutrient deprivation or heat stress during days 60 120 of pregnancy will undoubtedly have different impacts on fetal development compared to similar environmental insults during days 180 240 of pregnancy. this is where timing becomes a critical component of fetal programming outcomes. in addition to extrinsic environmental effects altering fetal growth, several inherent intrinsic mechanisms are associated with fetal growth and development. during clinical theriogenology • volume 12 number 3 • september 2020 253 days 70 100 of pregnancy, fetal weight across several breeds of cattle increased ~ 10 grams per day.19 further along in pregnancy (days 200 250), rate of fetal growth increased to ~ 200 300 grams per day; however, absolute growth of late term fetus declined to 100 grams per day, which may be due to inherent function of fetus exceeding capabilities of uteroplacental exchange near term. alternatively, fetus may be secreting or altering its own hormone profiles near term, favoring proper maturation and differentiation of organs overgrowth. figure 2. timeline of bovine fetal development.18 bovine fetal growth typically lags allantoic fluid volume and expansion of chorioallantoic membrane.19,20 nutrient partitioning by uteroplacenta and continuous supply of nutrients is a prerequisite for fetal growth. chorioallantois expansion over endometrium allows for maximal surface area for nutrient exchange during development, which should support acceleration in fetal growth. there was significant positive correlation for total amniotic fluid volume and fetal weight, but no correlation for total allantois fluid volume and fetal weight.19 cloning of cattle by somatic cell nuclear transfer allowed researchers to further define this association between fetal fluid homeostasis and fetal and placental development.21 cloned bovine fetuses with abnormal placentation are usually associated with excessive fluid accumulation in fetal sacs (hydrops syndrome). in this experimental model, cloned embryos with clinical theriogenology • volume 12 number 3 • september 2020254 hydrops syndrome surviving until midpregnancy have overgrowth of placentomes, fetal liver, and fetal kidney compared to normal pregnancies.21 in addition, organomegaly of offspring’s liver, kidney, and heart is typically accompanied with polyhydramnios (in several mammalian species studied). moreover, underlying mechanisms of fluid sac homeostasis are not fully understood in bovine pregnancy and further research is needed to elucidate cause and effect relationships. failure of proper organ development occurs in cloned calves that die shortly after birth. using this experimental model, researchers have examined 8 developmentally important genes in 6 organs (heart, liver, spleen, lung, kidney, and brain). of these developmental genes, kidney was the least affected organ associated with calf mortality, whereas heart was most affected by gene dysregulation compared to normal offspring.22 of these developmental genes, vascular endothelial growth factor (vegf) was upregulated in cloned offspring associated with early neonatal mortality. vascular endothelial growth factor was implicated in stimulating vasculogenesis and angiogenesis to restore oxygen supply to tissues when blood circulation is inadequate. appropriate concentrations of vegf during organogenesis and early organ development are vital for proper establishment of fetal cardiovascular system. downregulation of vegf could decrease fetal organ angiogenesis, with fatal consequences to fetus (pregnancy wastage) or neonate (calf mortality); however, upregulation may result in organomegaly or fetal cardiac dysfunction, with similar fatal consequences to newborn. placental development and growth placenta has major role in fetal growth regulation. ruminant placenta is morphologically classified as cotyledonary and histologically as syndesmochorial. in nonpregnant ruminants, caruncles are organized in 2 dorsal and 2 ventral rows that run length wise along uterine horns. chorioallantois has flat surface that becomes irregular when it starts to cover caruncles, due to growth and expansion of conceptus within uterine lumen. this process is followed by recognition of cotyledons.23 caruncularcotyledonary unit is called a placentome and is formed from growth and interdigitation of fetal villi and caruncular crypt adopting a convex shape.24 contact surface area is enhanced as cotyledon’s finger-like projections enter crypts formed in caruncles. placentomes vary in size; however, they are bigger at uterine horn base and decrease in size close to tip.23 placentome is the primary functional area of physiological exchanges between mother and fetus. efficiency of placental nutrient transport is directly related to uteroplacental blood flow.25 all nutrients and wastes that are exchanged between maternal and fetal systems are transported via uteroplacenta.25-26 establishment of functional fetal and uteroplacental circulations is one of the earliest events during embryonic/placental development.15-16 to support exponential increase in fetal growth during last half of pregnancy, proper growth and development of uteroplacental vascular bed must occur during first half of pregnancy.25,27 understanding factors that impact uteroplacental blood flow will directly impact placental efficiency and thus fetal growth. however, despite much research in placentalfetal interactions area, regulators of placental growth and vascularization, including uteroplacental blood flow, are still largely unknown, particularly in cattle. our laboratory has been investigating blood perfusion and blood flow of reproductive tract using doppler ultrasonography (applies doppler effect principle, named after austrian physicist christian doppler; change in frequency of a wave for observer moving relative to source of respective wave). example of this principle is perception of sound or differences in pitch one hears from stationary siren as person driving past the siren. another example is that you are the stationary object and a car with a siren is driving past you. in this example, differences in pitch from moving siren will change as the car is driving towards you, the stationary object, and then away from you. in doppler ultrasonography, stationary object is the transducer of ultrasound machine, which is detecting shift in frequency of red blood cells moving past stationary object.28 color can be assigned to normal b-mode, brightness gray scale image, by selecting color function of doppler ultrasound machine. this will assign color based on directional flow (e.g. blood moving towards transducer probe is displayed in red and blood moving away from it is displayed in blue). clinical theriogenology • volume 12 number 3 • september 2020 255 good example of 2 vessels in close proximity and moving in opposite directions is illustrated in figure 3a. note the head and body of fetal sheep resting in amniotic sac at ~ day 40 of pregnancy. top red arrow is pointing to umbilical cord, which is surrounded by color box and is showing a difference in directional blood flow from umbilical vein and umbilical artery. at this stage of pregnancy, umbilical cord is very small; however, it is still large enough to measure vessel diameter. apart from color images, doppler ultrasonography can estimate velocity of blood moving through a vessel. figure 3b illustrates measurements taken while using doppler mode. sample gait cursor (green i) is placed on top of umbilical cord of a sheep fetus at day 90 of pregnancy (figure 3b). results are graphed in figure 3c, where pulsatile umbilical artery cardiac cycle waveforms are visible above green x-axis line. y-axis is detecting velocity (centimeters per second) of blood flow, whereas x-axis is depicting time in seconds. in addition, each pulsatile waveform of umbilical artery matches up with 1 fetal heart beat, enabling fetal heart rate calculation. in addition to umbilical cord blood flow examination, our laboratory has used doppler ultrasonography to examine uterine artery blood flow during mid to late pregnancy in cattle. figure 3d illustrates a doppler ultrasonography image of maternal uterine artery of a pregnant holstein heifer ~ 180 days of pregnancy. pulsatile cardiac cycles of uterine artery represent maternal heart rate. moreover, these doppler data allow calculation of velocity of blood flow during peak systolic and diastolic contractions of heart. numbers on y-axis are in centimeters per second and are negative as red blood cells are moving away from transducer. in comparison, we also examined blood flow through hepatic portal vein of lactating dairy cows (figure 3e). in this example, blood is flowing towards transducer, thereby giving a positive velocity on y-axis. in addition, nonpulsatile blood flowing through hepatic portal vein has an average velocity of ~ 50 centimeters per second. data generated using doppler ultrasonography are comparable to previous experiments estimating umbilical, uterine and hepatic blood flow with dye dilution techniques.7,8,18 data on development of bovine placentome capillary bed are limited. cotyledonary growth progressively increases throughout pregnancy in cattle.29,30 histological analysis of capillary bed development was performed in mid and late bovine pregnancies.30 during this period, capillary area density, a measure related to blood flow, decreases ~ 30% in caruncular tissue, but increases ~ 186% in cotyledonary tissue. also, number of capillaries increases ~ 150 and 80% in caruncular and cotyledonary tissue, respectively. capillary surface density, a measurement related to nutrient exchange, increased in both caruncular and cotyledonary tissues (32 and 172%, respectively), whereas capillary size decreases 67% in caruncular tissue and increases 71% in cotyledonary tissue from mid to late pregnancy. pattern of capillary development is very different between maternal and fetal portions of placentome, possibly due to energy demands of these independent tissues that share a similar function, delivery of nutrients to developing fetus. placenta has fundamental role in supporting metabolic fetal demands. although placental growth slows during last half of pregnancy, placental function increases dramatically to support exponential fetal growth rate.20,31 for example, in sheep and cattle, uterine blood flow increases ~ 3 4 fold from mid to late pregnancy.25,32-34 relationship between uteroplacental blood flow and conceptus size throughout pregnancy is further defined below. cardiovascular adaptations during pregnancy physiologic state of dam is associated with significant but reversible alterations to metabolic demand and alterations to endocrine and cardiovascular systems. maternal cardiovascular functional capacity changes dramatically during pregnancy, whereby systemic arterial blood pressure and vascular resistance decreases and cardiac output, heart rate, stroke volume and blood volume increase.35 although not all variables determined during bovine pregnancy, several mammalian species (including sheep) have decrease in mean arterial pressure in early pregnancy that persists throughout pregnancy. moreover, clinical theriogenology • volume 12 number 3 • september 2020256 figure 3. doppler ultrasonography images of ovine umbilical cord at ~ day 40 (a) and day 90 (b) of pregnancy. cardiac cycle waveforms of ovine day 90 umbilical artery (c) showing peak systolic blood velocity (psv) and end diastolic blood velocity (edv). doppler ultrasonography image of bovine uterine artery and corresponding cardiac cycle waveforms (d) at day 180 of pregnancy. doppler ultrasonography image of nonpulsatile bovine hepatic portal vein (e) of a lactating nonpregnant dairy cow. decrease in arterial pressure (~ 5 10% decrease) is minor compared to ~ 20 30% decrease in total peripheral vascular resistance. maternal cardiac output increases 30 40% in nonpregnant versus pregnant ruminants. therefore, increase in cardiac output is associated with dramatic decline in systemic vascular resistance, allowing researchers to characterize pregnancy as a state of systemic vasodilation, resulting in profound increases in total systemic flows to all vascular beds. most of these studies characterize this relationship by the equation; systemic vascular resistance = mean arterial pressure/cardiac output. using above equation, it is apparent that decrease in systemic vascular resistance during pregnancy and increase in cardiac output (total systemic blood flow) help to maintain arterial pressure. in addition, apart from this relationship, increases in blood volume and activation of renin-angiotensin system may also contribute to maintaining blood pressure during this physiological state of substantial vasodilation; however, limited data exists in cattle.35 to determine specific contributions to increase in cardiac output occurring during pregnancy, we must first examine cardiac output equation, which states that: cardiac output = heart rate x stroke volume, whereby stroke volume equals volume of blood pumped from 1 ventricle of heart with each beat. in majority of mammalian species, heart rate increases by ~ 15% during pregnancy which does not fully explain 30 40% increase in cardiac output. therefore, stroke volume may increase 30 35% during pregnancy and is one of the major contributors to this clinical theriogenology • volume 12 number 3 • september 2020 257 increase in cardiac output. in sheep treated chronically with estrogen, induced increase in left ventricular heart dimensions and enlargement were similar to pregnancy; therefore changes to endocrine system during pregnancy may help mediate temporal changes to maternal cardiovascular function.35 rise in maternal cardiac output during pregnancy is also associated with increased plasma and blood volume in cows.18 increased blood volume varies among species and depends on dam’s nutritional status and singleton versus twin pregnancies. bovine blood volume expands by 10 20% during pregnancy, whereas in litter-bearing species, blood volume may expand by 30 50% during pregnancy. with increase in plasma volume, dam must maintain a proper balance of water and electrolyte retention; therefore, similar to alterations in maternal arterial pressure, this increase in plasma volume will be integrated with the renin-angiotensin system, which can serve additional purpose as an extrinsic modulator of kidney function and urinary secretion. in addition, to dramatic changes in maternal cardiovascular system during pregnancy, it is even more noteworthy that most mammals return to nonpregnant levels of cardiovascular function within 2 5 weeks postpartum.18 although lactating, highproducing nonpregnant dairy cattle have a substantial increase in cardiac output compared to nonpregnant and nonlactating counterparts, this redistribution of blood flow during transition period from uteroplacental vasculature towards mammary gland is still a phenomenal physiological feat enabling peak lactation shortly after parturition. several animal models of fetal programming were extensively studied in ewes,14 although extrapolation to cattle should be minimal, due to drastic differences in placental development between sheep and cattle (figure 4). in ewe, placenta reaches its maximum size during first two-thirds of pregnancy, whereas ~ 90% of fetal growth occurs during last third of pregnancy.17 in contrast, bovine placenta continues to increase in size exponentially as pregnancy proceeds; however, bovine fetal growth is much greater compared to placental growth (figure 4).25 placenta is involved in transporting nutrients and wastes between maternal and fetal circulations and altered placental function was associated with abnormalities in fetal development. efficiency of placental nutrient transport is directly related to placental blood flow.25,26 key factors affecting placental nutrient transfer capacity are size, nutrient transporter abundance, nutrient synthesis and metabolism, and hormone synthesis and metabolism.36 large increases in blood flow to reproductive tract are necessary to support both nutrient and waste exchange between mother and offspring. several environmental factors negatively impact placental development and blood flow during pregnancy, all of which can hinder offspring health and vigor. regulators of placental nutrient transport and uteroplacental blood flow are still largely unknown, with most research efforts focusing on rodent models, which are different from livestock species. elucidating consequences of specific hormonal supplements on the continual plasticity of placental function will allow determination of important endogenous mediators of offspring growth and development. maternal nutrient restriction poor forage quality in grazing systems can negatively impact nutritional intake of beef cattle. pregnant beef cows grazing poor forage can alter fetal growth during increased periods of developmental plasticity. thus, provisions from environment can program these offspring to experience changes in mortality and morbidity rates, slowed postnatal growth, altered carcass weights, and meat quality characteristics.37 relationship between maternal nutritional plane during late pregnancy and calf mortality was examined as early as 1975, where maternal nutrient restriction for 100 days prepartum decreased calf birth weight by 7% and increased calf mortality rate by 10%, whereas an additional 20% of calves died between birth and weaning due to scours.38 direct effects of nutritional plane on offspring production characteristics are dependent on timing of insult and magnitude of nutrition deprivation in relation to fetal and placental development.39 in dealing with timing, it is also important to consider separation of prenatal clinical theriogenology • volume 12 number 3 • september 2020258 figure 4. fetal and placental growth in ewe and cow (redrawn).17,25 versus postnatal maternal factors that may influence these developmental programming responses. for example, changes in meat quality of offspring born to dams experiencing late pregnancy maternal nutrient restriction may carry over into early lactation of this dam. thus, researchers are prevented from identifying important time of maternal nutritional insult that led to negative offspring outcomes. this is vital when considering most economically feasible therapeutic interventions to mitigate negative developmental programming outcomes. adaptations of placenta during maternal nutrient restriction are incomplete and less is known about specific differences amongst breeds of cattle. normal physiology such as pregnancy length, fetal growth, placentome weight, even uterine blood flow can differ substantially between breeds of cattle.33,34 therefore, magnitude of fetal programming is expected to be breed dependent and further research is needed to identify when and with what breeds should interventions be sought. in a recent study, we examined the effect of early to mid pregnancy nutrient restriction on uterine blood flow and fetal development in brahman and angus heifers.40 heifers were restricted to 60% of net energy requirements for pregnant cattle from days 50 180 of pregnancy. this early to mid pregnancy nutrient restriction decreased uterine artery blood flow and fetal weight at day 180 of pregnancy irrespective of heifer breed (table 1). moreover, efficiency of uterine artery blood flow relative to fetal weight was improved in nutrient-restricted versus adequate-fed dams (table 1). a similar response was observed in brahman heifers, irrespective of nutrient restriction, signifying a lesser amount of uterine blood flow needed to clinical theriogenology • volume 12 number 3 • september 2020 259 grow similar weight fetus from a brahman versus angus dam (table 1). a portion of these responses have been associated with increased placental efficiency in nutrient-restricted dams and brahman dams.40 using a novel imaging technique to ascertain macroscopic blood vessel density of cotyledons (figure 5), we observed significant increases in blood vessel density in brahman versus angus dams. as noted in the figure 5 illustration, cotyledonary blood vessel density is heterogenous, with areas of high versus low vascularity within the same placentome. moreover, nutrient restriction and decreased uterine artery blood flow increased cotyledonary blood vessel density, which we believe to be a compensatory mechanism within fetal membranes.40 angus and brahman heifers subsets were allowed to calve and postnatal growth monitored though weaning (unpublished observations). most postnatal measurements of growth were unaffected by maternal nutrient restriction from days 50 180 of pregnancy. however, heart girth increased in calves born to nutrient restricted versus adequate-fed dams, which may have in utero overcompensation of fetal growth when nutrient-restricted dams are re-alimented to adequate nutrition during late pregnancy. table 1. uterine artery blood flow (bf) at day 175 of pregnancy and fetal flow at day 180 of pregnancy in nutrient-restricted (res) or control (con) fed angus and brahman heifers. nutritional treatments were applied from days 50 to 180 of pregnancy. data reported are main effect of nutritional plane and main effect of breed. results adapted from.40 p value item con res sem trt nutritional treatment uterine artery bf, liter/minute 5.04 2.33 0.73 0.035 fetal weight, kg 9.56 7.35 0.61 0.047 uterine artery bf by fetal weight, liter/minute/kg of fetus 0.52 0.29 0.07 0.058 p value item angus brahman sem breed breed uterine artery bf, liter/minute 4.66 2.71 0.76 0.119 fetal weight, kg 8.04 8.88 0.61 0.388 uterine artery bf by fetal weight, liter/minute/kg of fetus 0.53 0.28 0.07 0.054 apart from nutritional management during pregnancy, we also examined heifer development practices and season on uterine artery blood flow during mid to late pregnancy.41 for example, beef producers opting for low-input forage-based replacement heifer management programs have lighter weights at breeding, with some heifers reaching only 50 55% of expected mature body weight at breeding versus a traditionally recommended target weight of 60 65% of expected mature body weight. we concluded that heifers developed on low0input management schemes until confirmation of pregnancy (days 30 45) had no compromise in uterine blood flow or calf birth weights compared to conventionally developed heifers. moreover, volume of late pregnancy uterine artery blood flow relative to maternal body weight was significantly increased in low-input versus conventionally developed heifers, which may be a compensatory mechanism to safeguard fetal growth and development.41 in addition to low-input heifer development programs, we also examined effect of calving season on uterine artery blood flow, as differences in postpartum anestrus interval, conception rates, and weaning weights were reported between fall and spring calving herds.42,43 a portion of these responses could be programmed in utero via changes in nutrient and waste exchange between dam and fetus. in these initial studies, we observed an increase in uterine artery blood flow in the last third of pregnancy, consistent with exponential growth of fetus in springversus fall-calving heifers.41 although cattle are considered nonseasonal breeders, seasonal clinical theriogenology • volume 12 number 3 • september 2020260 figure 5. fluorescent detection of macroscopic cotyledonary blood vessel density in placentomes collected at day 180 of pregnancy. three representative images and one negative control placentome with cotyledonary surface up are shown.40 changes in photoperiod, thermal stress, and nutrient availability can influence numerous performance and reproductive traits. changes in hormone concentrations, as a result of photoperiod, may influence blood distribution to the reproductive tract, specifically changes in melatonin-modulated cardiovascular function.44 maternal melatonin supplementation amplitude of melatonin secretion has been associated with improved oxidative status and altered hormone metabolism in rats and sheep, and altered cardiovascular function in several mammalian species.45-47 in several studies, melatonin partially regulated blood pressure and blood flow.48 melatonin has both direct and indirect effects on the cardiovascular system and may cause either arterial vasodilation or vasoconstriction, depending on the origin of the blood vessel under investigation. taking into account the above physiological responses, which can be partially altered by peripheral concentrations of melatonin, our research team examined effects of melatonin supplementation on uteroplacental development and functional capacity. similar to other fetal programming models, our initial studies focused on pregnant ewe lambs. using this sheep model of intrauterine growth restriction, we supplemented dietary melatonin as a potential therapeutic during mid to late pregnancy.49 in our sheep model, ewes were supplemented with 5 mg of melatonin or no melatonin and allocated to receive 100% (adequate) or 60% (restricted) of nutrient requirements from days 50 130 of pregnancy. using doppler ultrasonography, we observed an increase in umbilical artery blood flow at day 130 in ewes supplemented with dietary melatonin, whereas uterine artery blood flow was unaffected by maternal melatonin supplementation.49 at day 130, uterine artery blood flow decreased in nutrient-restricted versus adequatefed ewes. although melatonin supplementation failed to rescue fetal weight in restricted-fed ewes, we clinical theriogenology • volume 12 number 3 • september 2020 261 observed similarities between fetal size and measurements of uterine and umbilical blood flow during mid to late pregnancy. recently, we examined uterine artery blood flow in holstein heifers supplemented with 20 mg of dietary melatonin from days 190 262 of pregnancy.7 uterine artery blood flow increased by 25% in melatonin-treated versus control heifers (table 2). surprisingly, calf birth weights were not different between treatments; however, calf body weight at 9 weeks of age increased in calves born to melatoninsupplemented dams versus control dams.8 therefore, similar to other pregnancy models, an increase in uteroplacental blood flow during mid to late pregnancy is associated with alterations in postnatal offspring growth and development. this is apparent in the dairy heifer study, as calves were removed from dams, managed identically and fed a similar milk replacer and starter diet prior to weaning at 8 weeks of age. therefore, postnatal maternal factors were removed from this dairy project, allowing us to propose direct fetal programming responses. due to observed differences in dairy calf body weights, we replicated a similar experiment in beef cows. in this follow-up study, heifers and cows were assigned to 1 of 2 treatments: melatonin implants (mel; n = 29) or no melatonin implant control (con; n = 28) starting on day 180 of pregnancy and ending on day 270.9 as expected, uterine artery blood flow increased in commercial beef heifers and cows supplemented with melatonin during the last third of pregnancy (table 2). similar to the dairy heifer study, beef calf birth weights were not different; however, a 26 kg increase in weaning weight was observed in calves born to melatonin-supplemented versus control dams.9 although results were similar to dairy study, it is important to note that postnatal maternal factors (e.g. postpartum cow health, colostrum composition, and lactational performance) could be contributing to increased weaning weights of calves born to melatonin-supplemented dams. table 2. uterine artery blood flow (bf), calf birth weight, and calf weaning weight at 9 weeks of age from dairy heifers treated with (mel) or without (con) dietary melatonin from days 190 262 of pregnancy.7,8 uterine artery bf, calf birth weight, and calf weaning weight at 28 weeks of age from beef cows treated with (mel) or without (con) dietary melatonin from days 180 270 of pregnancy.9 p value item con mel se trt dairy heifers uterine artery bf, liter/minute 5.73 7.16 0.35 0.02 birth weight, kg 37.4 35.8 2.4 0.63 weaning weight, kg 86.0 99.1 2.4 0.01 p value item con mel se trt beef cows uterine artery bf, liter/minute 5.76 7.87 0.57 0.01 birth weight, kg 31.1 31.8 1.8 0.77 weaning weight, kg 195.5 221.5 7.3 0.01 taken together, results we observed in both cattle studies following melatonin supplementation allowed our research group to speculate on potential circadian alterations of reproductive tract during pregnancy. for example, rhythms generated from circulating concentrations of melatonin could mediate circadian rhythms in placenta and developing fetus during pregnancy. potentially this could indicate a natural 24 hour rhythm in uterine artery blood flow, which may be influenced by exogenous melatonin supplementation. therefore, from a livestock production standpoint, alterations in circadian rhythms during specific windows of pregnancy may lead to changes in offspring body composition; however, these fetal programming data are lacking. nevertheless, the potential of establishing and/or disrupting these circadian rhythms during specific time points of pregnancy may alter production characteristics of offspring, which warrants further investigation. clinical theriogenology • volume 12 number 3 • september 2020262 epigenetic mechanisms of fetal programming nearly every cell within an organism retains the entire genetic information or genome; however, cellular differentiation selectively programs different expression rates of genetic code. in contrast to earlier definitions of epigenesis, more contemporary usage of epigenetics refers to “the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in dna sequence”.50 therefore, epigenetic processes can program gene activity or expression patterns without altering the genetic code and differentiated adult cells have the capacity to retain epigenetic modifications from fetal development or even previous generations. two primary mechanisms imparting epigenetic modifications to the genome are: 1) dna methylation and 2) histone modifications.51,52 these modifications were implicated as the primary mechanism of developmental programming responses that may lead to later life noncommunicable diseases. an interesting proposal of epigenetic modifications relies on key nutritional cofactors associated with one-carbon metabolism, such as folate, vitamin b12, vitamin b5, choline, s-adenosyl methionine, betaine, and homocysteine.4 this proposal was shown in rodent studies, where dietary provisions of choline, folate, and vitamin b12 during pregnancy prevented dna hypomethylating properties of bisphenol a, thereby altering gene expression and mitigating development of obesity in offspring.53 therefore, developmental origins of adult disease involve reprogramming the epigenome by environmental factors, e.g. maternal nutrition.54 postnatal outcomes animal models were developed and investigated to further our understanding of mechanisms underlying fetal origins of health and disease in humans.6 for example, maternal nutrient restriction during prenatal period is usually associated with low birth weights (intrauterine growth restriction) in offspring, as well as postnatal development of hypertension, obesity, and diabetes.55 lower birth weights were associated with increased postnatal morbidity and mortality,56 poor postnatal growth, and decreased weaning weight in sheep.57 however, a portion of these postnatal responses may be related to alterations in mammary gland development, which could lead to postnatal malnourishment and developmental programming responses occurring after the fetal period of development. differences in birth weight can modestly predict carcass weight irrespective of dietary influences. magnitude of growth restriction at birth in lambs, pigs, and steers was correlated with carcass yield leaving clear financial implications for intrauterine growth restriction in livestock species.58 specifically, in cattle, low birth weight was accompanied with slow postnatal growth, lower weaning weights and significant decreases in carcass weights at 30 months of age.59 some of these responses can be related to skeletal muscle development as improved nutritional plane during midpregnancy increased live weight and carcass weight and tenderness in crossbred steers.60 compromised pregnancies, via changes in maternal nutritional plane, were strongly associated with postnatal and later life changes to metabolic and endocrine functional capacity in offspring. insulin sensitivity was a primary target of fetal programming as epidemiological evidence associated birth weight with an increased risk of developing type ii diabetes.61 in an ovine maternal nutrient restriction model, malnourishment during late pregnancy decreased peripheral insulin sensitivity in young offspring.62 similarly, changes to maternal nutritional plane (over or under-feeding) have been associated with increased lipid accumulation in liver of 1 year old sheep, as well as changes in hepatic fatty acid oxidation.63,64 other endocrine changes in offspring born to compromised pregnancies were related to multiple hypothalamic-pituitary axes, e.g. those targeting thyroid and adrenal glands.65 since information related to adulthood and transgenerational consequences of fetal programming is limited, stringent environmental scenarios over several years are needed to fully relate these later life metabolic changes to fetal period treatments. in sheep, intergenerational programming of a metabolic syndrome phenotype was observed in grandsons and granddaughters of a maternal obesity model.66 moreover, these responses were sex specific, as granddaughters (f2 females) had greater insulin resistance compared to f2 males.66 although birth weight continues to be an indicator of potential adverse fetal programming events in offspring, it is important to note that birth weight provides limited information about body composition, morphometric size measurements, or individual organ size.65 this is especially relevant in clinical theriogenology • volume 12 number 3 • september 2020 263 early pregnancy maternal nutrient restriction studies, which usually do not alter birth weight or placental weight if dams are realimented to control diets prior to the exponential increase in fetal growth. crossbred beef heifers subjected to maternal nutrient restriction from 11 days prior to ai through day 110 of pregnancy had increased concentrations of testosterone during nutrient restriction compared to control-fed heifers.10 on day 94 of pregnancy, prior to end of nutrient restriction, fetal size was not different between treatment groups; however, the diameter of aortic root increased in fetuses from nutrient restricted dams versus control. in this study, pregnancy length, placental weight, and birth weight were not affected by maternal nutrient restriction.10 similarly, postnatal growth of offspring born to nutrient restricted dams was similar to controls until slaughter at 95 weeks of age. however, female offspring born to nutrient restricted dams had decreased ovarian reserves, enlarged aortas and increased blood pressure compared to their control counterparts.10 therefore, in cattle, changes in maternal nutrition during first trimester of pregnancy may program female offspring reproductive characteristics and hypertension, which may be related to increased testosterone concentrations during pregnancy.10 similar to these lack of changes in birth weight, we stimulated uterine artery blood flow via maternal melatonin supplementation in both dairy heifers and beef cows, with no significant impact on calf birth weight.7,9 however, postnatal growth and weaning weight increased in offspring of melatonin supplemented versus control dams.8,9 conclusion insufficiencies during pregnancy, resulting in reduced fetal growth and development, are detrimental to livestock production. we consistently observed positive associations with uterine and umbilical blood flow in sheep and cattle relative to fetal and postnatal offspring size. doppler ultrasonography increased our understanding of blood flow and blood perfusion during important reproductive events, enabling producers to apply specific strategies to improve reproductive efficiency of livestock. early to midpregnancy nutrient restriction increased placental efficiency in both angus and brahman heifers. however, late pregnancy nutrient restriction was associated with decreased birth weight, increased mortality and slowed postnatal growth of surviving offspring. low input heifer development programs resulting in 50% of mature body weight at breeding did not negatively impact uterine artery blood flow and calf birth weight. spring calving heifers had increased uterine blood flow compared to their fall-calving counterparts, which may be related to environmental differences and even hormonal changes with decreasing daytime length. furthermore, we consistently identified increased umbilical and uterine blood flows in sheep and cattle during melatonin supplementation. importance of this pathway in relation to development and transmission of 24 hour rhythms to offspring has not been elucidated in cattle. negative insults during pregnancy were associated with dysregulation of metabolic and endocrine function in the postnatal period. moreover, carcass yield and meat quality can be negatively affected by decreased or increased maternal nutrition during specific windows of pregnancy. studies are needed to further expand these fetal programming responses related to adult offspring and potential transgenerational carryover. conflict of interest there are no conflicts of interest to declare. acknowledgement this conference paper is a contribution of the mississippi agricultural and forestry experiment station. this material is based upon work that is supported by the national institute of food and agriculture, us department of agriculture, hatch project under accession number 1011100. additional funding was provided by the us department of agriculture, agricultural research service, biophotonic initiative number 58-6402-3-018. clinical theriogenology • volume 12 number 3 • september 2020264 references 1. 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rosenfeld cr, morriss fh, jr., makowski el, et al: circulatory changes in the reproductive tissues of ewes during pregnancy. gynecol obstet invest 1974;5:252-268. clinical theriogenology • volume 12 number 3 • september 2020 265 33. ferrell cl: maternal and fetal influences on uterine and conceptus development in the cow: i. growth of tissues of the gravid uterus. j anim sci 1991;69:1945-1953. 34. ferrell cl: maternal and fetal influences on uterine and conceptus development in the cow: ii. blood flow and nutrient flux. j anim sci 1991;69:1954-1965. 35. magness rr: maternal cardiovascular and other physiologic responses to the endocrinology of pregnancy. in: bazer fw: editor, endocrinology of pregnancy. totowa; humana press: 1998. p. 507-539. 36. fowden al, ward jw, wooding fpb, et al: programming placental nutrient transport capacity. j physiol 2006; 572:5-15. 37. robinson dl, café lm, greenwood pl: meat science and muscle biology symposium: developmental programming in cattle: consequences for growth, efficiency, carcass, muscle, and beef quality characteristics. j anim sci 2013; 91:1428-1442. 38. corah lr, dunn tg, kaltenbach cc: influence of prepartum nutrition on the reproductive performance of beef females and the performance of their progeny. j anim sci 1975;41:819-824. 39. funston rn, larson dm, vonnahme ka: effects of maternal nutrition on conceptus growth and offspring performance: implications for beef cattle production. j anim sci 2010;88(e. suppl.):e205-e215. 40. lemley co, hart cg, lemire rl, et al: maternal nutrient restriction alters uterine artery hemodynamics and placentome vascular density in bos indicus and bos taurus. j anim sci 2018;96:4823-4834. 41. cain aj, lemley co, walters fk, et al: pre-breeding beef heifer management and season affect mid to late gestation uterine artery hemodynamics. theriogenology 2017;87:9-15. 42. king gj, macleod gk: reproductive function in beef cows calving in the spring or fall. anim reprod sci 1984; 6:255-266. 43. gaertner sj, rouquette fm, long cr, et al: influence of calving season and stocking rate on birth weight and weaning weight of simmental-sired calves from brahman-hereford f1 dams. j anim sci 1992;70:2296-2303. 44. lemley co, vonnahme ka: physiology and endocrinology symposium: alterations in uteroplacental hemodynamics during melatonin supplementation in sheep and cattle. j anim sci 2017;95:2211-2221. 45. wallace jm, robenson jj, wigzell s et al: effects of melatonin on the peripheral concentrations of lh and progesterone after oestrus, and on conception rate in ewes. j endocrinol 1988;119:523-530. 46. forcada f, abecia ja, cebrian-perez ja et al: the effect of melatonin implants during the seasonal anestrus on embryo production after superovulation in aged high-prolificacy rasa aragonesa ewes. theriogenology 2006;65:356-365. 47. juaniaux e, poston l, burton gj. placental-related diseases of pregnancy: involvement of oxidative stress and implications in human evolution. hum reprod update 2006;12:747-755. 48. pandi-perumal sr, trakht i, spence dw, et al: the roles of melatonin and light in the pathophysiology and treatment of circadian rhythm sleep disorders. nat clin pract neurol 2008;4:436-447. 49. lemley co, meyer am, camacho le, et al: melatonin supplementation alters uteroplacental hemodynamics and fetal development in an ovine model of intrauterine growth restriction. am j physiol regul integr comp physiol 2012;302:r454-r467. 50. russo, vea, martienssen, ra, riggs ad: editors. epigenetic mechanisms of gene regulation. woodbury; cold spring harbor laboratory press: 1996. 51. hon gc, rajagopal n, shen y, et al: epigenetic memory at embryonic enhancers identified in dna methylation maps from adult mouse tissues. nat genet 2013;45:1198-1206. 52. sabin lr, delas mj, hannon gj: dogma derailed: the many influences of rna on the genome. mol cell 2013; 49:783-794. 53. dolinoy dc, huang d, jirtle rl: maternal nutrient supplementation counteracts bisphenol a-induced dna hypomethylation in early development. proc natl acad sci 2007;104:13056-13061. 54. ganu rs, harris ra, collins k, et al: 2012. maternal diet: a modulator for epigenomic regulation during development in non-human primates and humans. int j obes suppl 2012;2:s14-s18. 55. langley-evans sc: developmental programming of health and disease. proc nutr soc 2006;65:97-105. 56. gardner ds, buttery pj, daniel z, et al: factors affecting birth weight in sheep: maternal environment. reproduction 2007;133:297-307. 57. borwick sc, rae mt, brooks j, et al: undernutrition of ewe lambs in utero and in early post-natal life does not affect hypothalamic-pituitary function in adulthood. anim reprod sci 2003;77:61-70. 58. yates dt, petersen jl, schmidt tb et al: looking back and moving forward-how reproductive physiology has evolved: fetal origins of impaired muscle growth and metabolic dysfunction: lessons from the heat-stressed pregnant ewe. j anim sci 96:2987-3002. 59. greenwood p, café l, hearnshaw h, et al: long-term consequences of birth weight and growth to weaning on carcass, yield and beef quality characteristics of piedmontese-and wagyu-sired cattle. anim prod sci 2006;46:257-269. 60. underwood, kr, tong jf, price pl, et al: nutrition during mid to late gestation affects growth, adipose tissue deposition, and tenderness in cross-bred beef steers. meat sci 2010;86:588-593. 61. barker dj: the fetal origins of type 2 diabetes mellitus. annals of internal medicine 1999;130:322-324. 62. husted sm, nielsen mo, tygesen mp et al: programming of intermediate metabolism in young lambs affected by late gestational maternal undernourishment. am j physiol 2007;293:e548-e557. clinical theriogenology • volume 12 number 3 • september 2020266 63. hyatt ma, gardner ds, sebert s et al: suboptimal maternal nutrition, during early fetal liver development, promotes lipid accumulation in the liver of obese offspring. reproduction 2011;141:119-126. 64. nicholas lm, rattanatray l, morrison jl et al: maternal obesity or weight loss around conception impacts hepatic fatty acid metabolism in the offspring. obesity 2014;22:1685-1693. 65. khanal p, nielsen mo: impacts of prenatal nutrition on animal production and performance: a focus on growth and metabolic and endocrine function in sheep. j anim sci and biotech 2017;8:75. 66. pankey cl, walton mw, odhiambo jf et al: intergenerational impact of maternal overnutrition and obesity throughout pregnancy in sheep on metabolic syndrome in grandsons and granddaughters. dom anim endocrinol 2017;60:67-74. clinical theriogenology • volume 12 number 3 • september 2020 267 017_ms-017 lemley color only figure 5 3 contact margaret v. root kustritz rootk001@umn.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9589, http://dx.doi.org/10.58292/ct.v15.9589 review report year-one knowledge and skills in theriogenology: a preliminary study margaret root kustritz,a patricia sertich,b aime johnson,c scott madilld adepartment of veterinary clinical sciences, university of minnesota college of veterinary medicine, st. paul, mn, usa, bdepartment of clinical studies – new bolton center, university of pennsylvania school of veterinary medicine, philadelphia, pa, usa, cdepartment of clinical sciences, auburn university college of veterinary medicine, auburn, al, usa, ddepartment of veterinary population medicine, university of minnesota college of veterinary medicine, st. paul, mn, usa abstract veterinary practitioners who had just completed their first postgraduate year of work were surveyed to determine what knowledge and skills in theriogenology were used in that first year with the goal of helping veterinary schools prioritize components of the model core curriculum in their curricular offerings. the survey was based on a core curriculum created by the american college of theriogenologists. forty-eight surveys were returned; response rate could not be calculated. the majority of participants were in small animal practice but a wide range of employment experiences was represented. the only component listed as very commonly used in all practice types was ‘reproductive anatomy in any species.’ reproductive work in general was not managed by small animal practitioners, reflecting current caseload in most small animal primary care clinics in the united states, which focuses on sterilized small animal patients. reproductive work was more common among large animal practitioners, particularly those in mixed and food animal practices. the core curriculum is comparative and in this study many of the components rated as being used very commonly required input from disciplines outside of theriogenology for training, including anesthesia, surgery, and medical imaging. it is important for those teaching theriogenology to be an active part of all conversations about curriculum at academic institutions and that the comparative nature of theriogenology be stressed to ensure all necessary components are being taught to prepare graduates for theriogenology work in practice. keywords: curriculum, theriogenology, competencies introduction there is a growing interest in use of competencies to guide medical education. competency frameworks for veterinary medical education have been created locally and as part of an international consortium.1,2 the american association of veterinary medical colleges endorses the competency-based veterinary education framework of 32 competencies divided into 9 domains.a the domains are clinical reasoning and decision-making, individual animal care and management, animal population care and management, public health, communication, collaboration, professionalism and professional identity, financial and practice management, and scholarship. competencies of this type are general and do not address what specific content and skills should be included for each a. https://www.aavmc.org/programs/cbve/ discipline in veterinary medical training. individual educational researchers and colleges and organized medicine cooperatives have created lists of knowledge and skills required of veterinary students at graduation.b,3,4 often these are created with extensive input from practicing veterinarians. the american college of theriogenologists has developed a model core curriculum (appendix) and completed a survey of veterinarians across the united states to determine what knowledge and which skills in theriogenology were most commonly used in all types of veterinary practice.5 the model core curriculum was the basis of this study that surveyed veterinary school graduates who had just completed a year of veterinary practice. the survey attempted to determine what knowledge and skills in theriogenology were used in that first year with the goal of helping veterinary schools prioritize b. https://aaep.org/core-competencies mailto:rootk001@umn.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9589 https://www.aavmc.org/programs/cbve/ https://aaep.org/core-competencies 4 citation line: clinical theriogenology 2023, 15, 9589, http://dx.doi.org/10.58292/ct.v15.9589 components of the model core curriculum in their curricular offerings. materials and methods graduates from the veterinary class of 2021 from auburn university, university of minnesota, and university of pennsylvania were invited to complete a survey based on the model core curriculum. surveys were anonymous. graduates were asked from which school they had graduated and what kind of veterinary employment they had in their first year after graduation (may 2021–may 2022). for each component of the model core curriculum, participants were asked to identify how frequently they used that information or skill in their first year of practice with ratings of never, rarely (less than once a month), commonly (at least once a month), and very commonly (at least weekly). for basic science components, participants were asked to extrapolate how frequently they used that information as clinicians. for those who do reproductive work seasonally, they were asked to select the option that was most accurate during the breeding season. descriptive data are presented, with no statistical analysis. results forty-eight surveys were returned, with 40 from university of minnesota, 6 from university of pennsylvania, and 2 from auburn university. the survey was offered to multiple schools for distribution and the authors do not know the complete population to which this may have been distributed so response rate cannot be calculated. the majority of participants were in small animal practice, but a wide range of employment experiences was represented (table 1). frequency of use of information or skill by employment type is tabulated (tables 2–9). components are listed by category table 1. employment during the first year of veterinary practice designation number of responses small animal practice 17 internship – small animal 10 mixed animal practice with common domestic species 4 internship – equine 3 equine practice 2 food animal practice 2 residency 2 equine and food animal practice 1 internship – large animal 1 internship – small animal in shelter medicine 1 small animal, equine and food animal practice 1 small animal, equine, food animal, and exotics practice 1 small animal and exotics practice 1 small animal and food animal practice 1 small animal and mixed animal practice 1 table 2. small animal clinician* – general content very commonly used reproductive anatomy in any species commonly used — rarely used reproductive endocrinology in any species reproductive biology in any species (folliculogenesis, spermatogenesis, fertilization, placentation, mammary gland development) histology of reproductive tissues in any species * small animal = small animal practice, internship – small animal, internship – small animal in shelter medicine, small animal and mixed practice, small animal and exotics practice (n = 30). table 3. small animal clinician* – theriogenology content very commonly used small animal general anesthesia commonly used small animal sterilization – surgical small animal ovariohysterectomy/ovariectomy/ hysterectomy small animal castration (descended or cryptorchid) small animal mammary neoplasia small animal estrous cycle rarely used small animal pregnancy diagnosis/pregnancy management small animal dystocia management/obstetrics small animal infectious diseases of the reproductive tract small animal breeding management small animal mastitis small animal female neoplasia of the reproductive tract small animal male neoplasia of the reproductive tract small animal neonatal resuscitation small animal cesarean section small animal periparturient disorders (metritis, hypocalcemia/eclampsia, subinvolution of placental sites) small animal pregnancy termination small animal male infectious diseases of the reproductive tract small animal testing for heritable disorders, breeding planning * small animal = small animal practice, internship – small animal, internship – small animal in shelter medicine, small animal and mixed practice, small animal and exotics practice (n = 30). http://dx.doi.org/10.58292/ct.v15.9589 citation line: clinical theriogenology 2023, 15, 9589, http://dx.doi.org/10.58292/ct.v15.9589 5 table 4. equine clinician** – general content very commonly used reproductive anatomy in any species reproductive endocrinology in any species reproductive biology in any species (folliculogenesis, spermatogenesis, fertilization, placentation, mammary gland development) commonly used histology of reproductive tissues in any species ** equine = equine practice, internship – equine (n = 5). table 5. equine clinician** – theriogenology content very commonly used equine standing anesthesia equine breeding management equine manipulation of estrus (pharmacologic, lighting, etc.) equine pregnancy diagnosis/pregnancy management equine artificial insemination/embryo transfer equine transrectal palpation equine ultrasound equine passage of pipette/biopsy instrument/insemination pipette into uterus commonly used equine estrous cycle equine causes of abnormal cycling including anestrus equine periparturient disorders (metritis, retained fetal membranes) equine castration (descended or cryptorchid) equine caslick’s surgery equine endometritis equine breeding soundness examination/semen collection and evaluation equine recumbent (general) anesthesia rarely used equine abortion equine female infectious diseases of the reproductive tract equine dystocia management/obstetrics equine herd health/performance goals/economic / record-keeping equine mastitis equine male infertility equine neoplasia of the male reproductive tract equine perineal reconstruction equine male infectious diseases of the reproductive tract equine testing for heritable disorders, creating a breeding plan equine neoplasia of the female reproductive tract equine neonatal resuscitation equine cesarean section equine ovariectomy ** equine = equine practice, internship – equine (n = 5). table 6. food animal clinician† – theriogenology content very commonly used bovine estrous cycle bovine breeding management bovine pharmacologic manipulation of estrus bovine causes of abnormal cycling including anestrus bovine pregnancy diagnosis/pregnancy management bovine dystocia management (obstetrics, fetotomy) bovine periparturient disorders (hydrops, vaginal/uterine prolapse, metritis, retained fetal membranes, endometritis, hypocalcemia) bovine female infectious diseases of the reproductive tract bovine mastitis bovine male infertility bovine male infectious diseases of the reproductive tract bovine herd health / performance goals / economics / record-keeping bovine transrectal palpation bovine ultrasonography bovine breeding soundness examination / semen collection and evaluation bovine standing anesthesia bovine artificial insemination / embryo transfer bovine testing for heritable disease / breeding planning bovine neonatal resuscitation small ruminant parturition / obstetrics commonly used bovine teat surgery bovine castration small ruminant breeding management small ruminant pregnancy diagnosis / pregnancy management small ruminant abortion bovine neoplasia of the female reproductive tract bovine neoplasia of the male reproductive tract bovine cesarean section small ruminant estrous cycle small ruminant breeding soundness examination / semen collection and evaluation rarely used bovine passage of pipette / biopsy instrument / insemination pipette into uterus bovine caslick’s surgery bovine ovariectomy † food animal = food animal practice (n = 2). http://dx.doi.org/10.58292/ct.v15.9589 6 citation line: clinical theriogenology 2023, 15, 9589, http://dx.doi.org/10.58292/ct.v15.9589 table 7. food animal clinician – theriogenology content very commonly used bovine estrous cycle bovine breeding management bovine pharmacologic manipulation of estrus bovine causes of abnormal cycling including anestrus bovine pregnancy diagnosis/pregnancy management bovine dystocia management (obstetrics, fetotomy) bovine periparturient disorders (hydrops, vaginal/uterine eversions, metritis, retained fetal membranes, endometritis, hypocalcemia) bovine female infectious diseases of the reproductive tract bovine mastitis bovine male infertility bovine male infectious diseases of the reproductive tract bovine herd health/performance goals/economics/ record-keeping bovine transrectal palpation bovine ultrasonography bovine breeding soundness examination/semen collection and evaluation bovine standing anesthesia bovine artificial insemination/embryo transfer bovine testing for heritable disease/breeding planning bovine neonatal resuscitation small ruminant parturition/obstetrics commonly used bovine teat surgery bovine castration small ruminant breeding management small ruminant pregnancy diagnosis/pregnancy management small ruminant abortion bovine neoplasia of the female reproductive tract bovine neoplasia of the male reproductive tract bovine cesarean section small ruminant estrous cycle small ruminant breeding soundness examination/semen collection and evaluation rarely used bovine passage of pipette/biopsy instrument/insemination pipette into uterus bovine caslick’s surgery bovine ovariectomy † food animal = food animal practice (n = 2). table 8. mixed animal clinician†† – general content very commonly used reproductive anatomy in any species commonly used reproductive endocrinology in any species reproductive biology in any species (folliculogenesis, spermatogenesis, fertilization, placentation, mammary gland development) rarely used histology of reproductive tissues in any species †† mixed animal = mixed animal practice with common domestic species; small animal, equine and food animal practice; small animal, equine, food animal and exotics practice; small animal and food animal practice; equine and food animal practice; internship – large animal; residency (n = 11). components were listed as never used in any practice type; these were ‘small animal male infertility’, ‘small animal artificial insemination’, small animal contraception – pharmacologic’, ‘small animal breeding soundness examination/semen collection and evaluation’, ‘porcine breeding management’, ‘porcine breeding soundness examination/semen collection and evaluation’, and ‘porcine artificial insemination’. two or more participants suggested that more should be taught about ovariohysterectomy and castration in rabbits, reproductive issues including dystocia in small mammals and reptiles, egg binding in birds (including commercial poultry) and reptiles, and small animal ultrasonography. several small animal participants spontaneously noted that dystocia was the most common clinical presentation managed. discussion variation in use of knowledge and skills by employment type was similar to other studies. a study describing veterinarians’ perceptions of skills, knowledge, and attributes of new veterinary graduates suggested that large animal theriogenology was not considered valuable by small animal practitioners whereas canine and large animal theriogenology were considered valuable by large animal practitioners.6 this is likely because only a few small animal practitioners do any large animal work whereas many large animal practitioners do some small animal work. within small animal, only 1 participant noted that they worked in a practice that worked with dog breeders so the scoring in this study was reflective of the average small animal practitioner, with a much greater focus on sterilization and treatment of disease of spayed female dogs than on breeding work. that large animal practice was much more likely to have a significant focus on breeding work (only in some species) was reflected in the ratings provided by mixed animal and food animal practitioners. ratings for large animal topics may reflect ability of nonveterinarians to perform some of this work. a survey of over 1,000 veterinarians representing training at all but 3 schools in north america identified basic procedures as most valuable in training (e.g. transrectal palpation in cows and mares and interpretation of vaginal cytology specimens in dogs).5 in that survey, dystocia management and from highest to lowest weighted score within that category. the only component listed as very commonly used in all practice types was ‘reproductive anatomy in any species.’ seven http://dx.doi.org/10.58292/ct.v15.9589 citation line: clinical theriogenology 2023, 15, 9589, http://dx.doi.org/10.58292/ct.v15.9589 7 table 9. mixed animal clinician †† – theriogenology content very commonly used --commonly used bovine estrous cycle bovine pharmacologic manipulation of estrus bovine pregnancy diagnosis/pregnancy management bovine transrectal palpation bovine breeding management bovine dystocia management (obstetrics, fetotomy) bovine periparturient disorders (hydrops, vaginal/uterine eversions, metritis, retained fetal membranes, endometritis, hypocalcemia) bovine castration small animal general anesthesia small animal ovariohysterectomy/ovariectomy/ hysterectomy small animal castration (descended or cryptorchid) bovine causes of abnormal cycling including anestrus bovine ultrasonography bovine standing anesthesia small animal sterilization – surgical small ruminant parturition/obstetrics rarely used bovine female infectious diseases of the reproductive tract bovine mastitis small animal estrous cycle small animal mammary neoplasia bovine breeding soundness examination/semen collection and evaluation bovine neonatal resuscitation equine estrous cycle equine standing anesthesia small ruminant pregnancy diagnosis/pregnancy management bovine male infertility bovine cesarean section equine periparturient disorders (metritis, retained fetal membranes) small animal pregnancy diagnosis /pregnancy management bovine herd health/performance goals/economics/ record-keeping bovine artificial insemination/embryo transfer equine pregnancy diagnosis/pregnancy management small animal periparturient disorders (metritis, hypocalcemia/eclampsia, subinvolution of placental sites) small ruminant abortion bovine passage of pipette/biopsy instrument/insemination pipette into uterus equine transrectal palpation equine ultrasound equine castration (descended or cryptorchid) small animal dystocia management/obstetrics small animal male neoplasia of the reproductive tract small animal cesarean section small ruminant estrous cycle bovine male infectious diseases of the reproductive tract equine breeding management equine manipulation of estrus (pharmacologic, lighting, and others) small animal infectious diseases of the reproductive tract small animal mastitis small animal female neoplasia of the reproductive tract small animal neonatal resuscitation small ruminant breeding management equine artificial insemination/embryo transfer small animal breeding management small ruminant breeding soundness examination/semen collection and evaluation bovine testing for heritable disease / breeding planning bovine neoplasia of the female reproductive tract equine causes of abnormal cycling including anestrus equine endometritis small animal pregnancy termination small animal testing for heritable disorders, breeding planning equine dystocia management/obstetrics equine female infectious diseases of the reproductive tract equine passage of pipette/biopsy instrument/insemination pipette into uterus equine recumbent (general) anesthesia porcine parturition/obstetrics bovine neoplasia of the male reproductive tract bovine teat surgery bovine caslick’s surgery equine neoplasia of the female reproductive tract equine breeding soundness examination / semen collection and evaluation equine neonatal resuscitation equine caslick’s surgery small animal spontaneous abortion porcine estrous cycle porcine infectious causes of reproductive loss †† mixed animal = mixed animal practice with common domestic species; small animal, equine and food animal practice; small animal, equine, food animal and exotics practice; small animal and food animal practice; equine and food animal practice; internship – large animal; residency (n = 11). http://dx.doi.org/10.58292/ct.v15.9589 8 citation line: clinical theriogenology 2023, 15, 9589, http://dx.doi.org/10.58292/ct.v15.9589 was rated as highly important in all species and a paucity of training in small animal and in exotics was noted. the latter still appears to be true. among basic science topics, reproductive anatomy in any species was identified as very commonly used regardless of type of employment in the first year. reproductive biology and reproductive endocrinology were commonly or very commonly used in all large animal employment and were rarely used in small animal; this again reflects lack of regular breeding work in most small animal practice situations. knowledge of reproductive tissue histology was less common in all employment types. the core curriculum on which this was based (appendix) clearly demonstrated the comparative nature of theriogenology teaching. in a survey of dairy veterinarians, reproductive knowledge and skills for individual animals in theriogenology, surgery, and nutrition were noted as valuable.7 in this study, many of the components rated as being used very commonly required input from disciplines outside of theriogenology for training, including anesthesia, surgery, and medical imaging. it is important for those teaching theriogenology to be an active part of all conversations about curriculum at academic institutions and that the comparative nature of theriogenology is stressed to ensure all necessary components are taught to prepare graduates for theriogenology work in practice. a recent publication reported that 95% of students surveyed felt that their clinical development was negatively impacted due to restrictions imposed by the covid-19 pandemic.8 the students in the current study were in a cohort that completed their training under those restrictions and that may have altered results of this survey. the other obvious limitation of this study was the relative lack of response from dedicated large animal practitioners. further research must be conducted to fully understand the impacts of tracking on capabilities of new graduates and to provide a more accurate list of required knowledge and skills in the first year by practice type. competency frameworks include many aspects of veterinary medicine outside of clinical knowledge and skills, including teamwork, communications, and lifelong learning (footnote a). recent graduates in a study noted that a focus purely on veterinary knowledge was less important in practice than were nontechnical attributes, when comparing their employment to their clinical training.9 graduates must be employable at graduation and must also have skills that will permit them to be successful and satisfied as veterinary practitioners.10 data from this study can be used to guide curriculum development, with incorporation of work from other disciplines and nontechnical skill development to help create new graduates who can perform all the aspects of theriogenology required in their chosen employment. conflict of interest none. authors’ contribution dr. root kustritz conceptualized the study, created the survey and sent it out to graduates of the university of minnesota, drafted the paper, and completed revisions requested by the reviewer. drs. sertich and johnson sent out the survey to graduates at their respective schools. drs. sertich, johnson, and madill reviewed the draft and provided input for improvement of the manuscript. all authors have read and approved the final version of the manuscript and have agreed to the submission. references 1. bok hgj, jaarsma dadc, teunissen pw, et al: development and validation of a competency framework for veterinarians. j vet med educ 2011;38:262–268. doi: 10.3138/jvme.38.3.262 2. matthew sm, bok hgj, chaney kp, et al: collaborative development of a shared framework for competency-based veterinary education. j vet med educ 2020;47:579–593. doi: 10.3138/ jvme.2019-0082 3. duncan c, dale vhm, pead mj: clinical veterinary students’ perceptions of a ‘day one’ skills guide. vet rec 2011;169:13–18. doi: 10.1136/vr.d1437 4. welsh pjk, jones lm, may sa, et al: approaches to defining dayone competency: a framework for learning veterinary skills. rev sci tech off int epiz 2009;28:771–777. doi: 10.20506/ rst.28.2.1921 5. root kustritz mv, chenoweth pj, tibary a: efficacy of training in theriogenology as determined by a survey of veterinarians. j amer vet med assoc 2006;229:514–521. doi: 10.2460/ javma.229.4.514 6. kreisler re, stackhouse nl, graves tk: arizona veterinarians’ perceptions and consensus regarding skills, knowledge, and attributes of day one veterinary graduates. j vet med educ 2020;47:365–377. doi: 10.3138/jvme.1117-166r2 7. luby cd, mcintyre k, jelinski md: skills required of dairy veterinarians in western canada: a survey of practicing veterinarians. can vet j 2013;54:267–270. 8. thompson jl, mackay j, blacklock kb: veterinary students’ views on surgical entrustable professional activities and the impact of covid-19 on clinical competence development. vet rec 2022;191(9):e1978. doi: 10.1002/vetr.1978 9. rhind sm, baillie s, kinnison t, et al: the transition into veterinary practice: opinions of recent graduates and final year students. bmc med educ 2011;11:64–73. doi: 10.1186/1472-6920-11-64 10. bell ma, cake ma, mansfield cf: beyond competence: why we should talk about employability in veterinary education. j vet med educ 2018;45:27–37. doi: 10.3138/jvme.0616-013r1 http://dx.doi.org/10.58292/ct.v15.9589 https://doi.org/10.3138/jvme.38.3.262 https://doi.org/10.3138/jvme.2019-0082 https://doi.org/10.3138/jvme.2019-0082 https://doi.org/10.1136/vr.d1437 https://doi.org/10.20506/rst.28.2.1921 https://doi.org/10.20506/rst.28.2.1921 https://doi.org/10.2460/javma.229.4.514 https://doi.org/10.2460/javma.229.4.514 https://doi.org/10.3138/jvme.1117-166r2 https://doi.org/10.1002/vetr.1978 https://doi.org/10.1186/1472-6920-11-64 https://doi.org/10.3138/jvme.0616-013r1 citation line: clinical theriogenology 2023, 15, 9589, http://dx.doi.org/10.58292/ct.v15.9589 9 appendix theriogenology core curriculum for nontracking or mixed animal track students suggested prerequisites to veterinary school medical terminology endocrinology genetics topics in veterinary curriculum basic sciences anatomy male and female, representative large and small animal species histology gonads uterus uterine tubes mammary tissue endocrinology hypothalamus pituitary gonads uterus placenta thyroid adrenal  embryology and placentation  reproductive biology sex determination and differentiation estrous cycle, representative species (cow?) folliculogenesis/oogenesis/ovulation/luteinization seasonal, environmental, nutritional effects puberty pharmacologic manipulation of estrus introduction to advanced reproductive technologies (e.g. semen cryopreservation, cloning, and embryo manipulation) spermatogenesis/sperm maturation emission/ejaculation fertilization/embryo movement /maternal recognition of pregnancy pregnancy/parturition artificial insemination contraception – surgical, pharmacological, immunological mammary gland development/lactation/milk biology pharmacology use of reproductive hormones and analogues use of antibiotics/withdrawal times behavior clinical sciences bovine female estrous cycle/breeding management pharmacologic manipulation of estrus anestrus cyclical aberrations pregnancy diagnosis/pregnancy management dystocia management/obstetrics, fetotomy abortion periparturient disorders – hydrops, vaginal/uterine eversion, metritis, retained fetal membranes, endometritis http://dx.doi.org/10.58292/ct.v15.9589 10 citation line: clinical theriogenology 2023, 15, 9589, http://dx.doi.org/10.58292/ct.v15.9589 infectious diseases/disorders of the reproductive tract mastitis male infertility, male infectious diseases/disorders of the reproductive tract general herd health/reproductive performance goals/economics/record keeping artificial insemination, embryo transfer testing for heritable disorders/creation of breeding plans to minimize genetic disorders neoplasia of the reproductive tract techniques history taking/physical examination/creation of a diagnostic and treatment scheme transrectal reproductive examination ultrasonography breeding soundness examination/semen collection and evaluation passage of pipette/biopsy instrument/insemination pipette into uterus neonatal resuscitation anesthesia surgery cesarean surgery teat surgery caslick’s surgery ovariectomy castration  equine female estrous cycle/breeding management manipulation of estrus anestrus cyclical aberrations pregnancy diagnosis/pregnancy management dystocia management/obstetrics abortion periparturient disorders – metritis, retained fetal membranes endometritis infectious diseases/disorders of the reproductive tract mastitis male infertility, male infectious diseases/disorders of the reproductive tract general herd health/reproductive performance goals/economics/record keeping artificial insemination, embryo transfer testing for heritable disorders/creation of breeding plans to minimize genetic disorders neoplasia of the reproductive tract techniques history taking/physical examination/creation of a diagnostic and treatment scheme transrectal reproductive examination ultrasonography breeding soundness examination/semen collection and evaluation passage of pipette/biopsy instrument/insemination pipette into uterus neonatal resuscitation anesthesia surgery cesarean surgery http://dx.doi.org/10.58292/ct.v15.9589 citation line: clinical theriogenology 2023, 15, 9589, http://dx.doi.org/10.58292/ct.v15.9589 11 castration (descended and cryptorchid) caslick’s surgery perineal reconstruction ovariectomy small animal female estrous cycle/breeding management pregnancy diagnosis/pregnancy management dystocia management/obstetrics abortion periparturient disorders – metritis, sips, eclampsia infectious diseases/disorders of the reproductive tract mastitis mammary neoplasia male infertility, male infectious diseases/disorders of the reproductive tract general artificial insemination contraception testing for heritable disorders/creation of breeding plans to minimize genetic disorders neoplasia of the reproductive tract techniques history taking/physical examination/creation of a diagnostic and treatment scheme breeding soundness examination/semen collection and evaluation neonatal resuscitation anesthesia surgery cesarean section ovariohysterectomy/ovariectomy  castration (descended and cryptorchid) porcine estrous cycle/breeding management parturition/obstetrics infectious causes of reproductive loss breeding soundness examination, male artificial insemination ovine/caprine estrous cycle/breeding management pregnancy diagnosis/pregnancy management abortion parturition/obstetrics breeding soundness examination, male http://dx.doi.org/10.58292/ct.v15.9589 immune response to breeding inflammation and/or infection of the endometrium is the leading cause of subfertility in mares, resulting in persistent breeding-induced endometritis (pbie), affecting 10 15% of the broodmare population.1 deposition of sperm, seminal plasma, and/or bacteria in the reproductive tract elicits an inflammatory response that leads to activation of the innate arm of the immune system. this begins with the stimulation of pattern-recognition receptors,2 induction of the complement system,3-5 increases in proinflammatory signaling molecules,6-9 and recruitment of immune cells to the site of inflammation.5,10-12 release of prostaglandin f2α (pgf2α) from an inflamed endometrium and appearance of activated leukocytes coincide with an increase in myometrial contractility, leading to eventual expulsion of excess fluid and contaminants.13,14 in reproductively sound mares that are resistant to pbie, this inflammation is resolved within 24 36 hours, whereas a subset of mares that experience prolonged inflammation are considered susceptible to pbie.9,13 this leads to uterine fluid accumulation, extended neutrophilia within the uterine lumen, and persistent inflammation for > 96 hours, potentially impeding embryo viability upon migration from the oviduct. although both susceptible and resistant mares experience this activation of the innate immune response, susceptible mares fail to mount the antiinflammatory response required to mitigate the proinflammatory signaling, and this is speculated to be the primary cause of persistent inflammation in these mares. the critical period for the immune response is within 6 hours after insemination.8 in this period, resistant mares experienced an upregulation of various antiinflammatory and immunomodulator use in mares carleigh fedorka, mats troedsson department of veterinary sciences, university of kentucky, lexington, ky abstract a delicate balance exists between the immune system and the female reproductive tract, modulating numerous aspects of reproductive health. this includes, but is not limited to, ovulation, breeding-induced uterine inflammation, fertilization, pregnancy maintenance, response to placental infection, and postpartum involution. additionally, stimulation of innate and adaptive immune systems can assist in clearing pathogens from the reproductive tract, thereby diminishing inflammation and/or infection that may inhibit reproductive health. in this review, the mechanisms through which the immune system modulates reproductive tract will be elucidated and critically evaluated immunomodulators will be discussed. keywords: immunomodulators, persistent breeding-induced endometritis, placentitis immunomodulatory cytokines, including interleukin (il)-1 receptor antagonist, il-10, and il-6.8 in contrast, susceptible mares failed to upregulate these immunomodulating cytokines in this critical period, leading to prolonged inflammation. this was specifically noted in increased expression of proinflammatory cytokines (il-1β and il-8), both contributing to chemotaxis of various immune cells to sites of inflammation, and specifically in recruitment of polymorphonuclear neutrophils (pmn).15 therefore, increased expression of these cytokines coincides with the pronounced neutrophilia noted in susceptible mares. additionally, susceptible mares accumulated excessive nitric oxide (no) in the uterus after breeding.14 this has also been noted via an upregulation of mrna expression for inducible nitric oxide synthase (inos) in the endometrium of susceptible mares after breeding.16 accumulation of no in susceptible mares is believed to cause impaired myometrial contractions, resulting in delayed uterine clearance of inflammation from the uterus.17,18 differences in immune response between susceptible and resistant mares are summarized (figure 1). several therapeutics are utilized for the management of pbie, with many targeting the resolution of this persistent inflammation. deemed immunomodulators, these therapeutics modify the immune response or the function of the immune system through a variety of pathways, including stimulation or suppression of signaling molecules, recruitment of essential immune cell types, or activation of other aspects of immunity, including antigen recognition and processing. critically evaluated immunomodulators available for pbie treatment are reviewed. clinical theriogenology 2021; 13: 238 immunomodulation of persistent endometritis selection of an immunomodulator for the treatment of pbie must be considered carefully, as proinflammatory signaling is necessary for the clearance of excess sperm, debris, and pathogen recognition, whereas antiinflammatory signaling is crucial for a timely resolution of this inflammation. pivotal studies on the immune response to breeding indicate that activation of proinflammatory pathways occurs as rapidly as 30 minutes after deposition of semen and is necessary for the chemotaxis of pmns into the uterine lumen for proper degradation of nonviable sperm and bacteria.5,8,19 therefore, antiinflammatory or immunomodulating therapeutics either prior to breeding or immediately after breeding may impede a mare’s essential innate immune response. consequently, results from studies must be interpreted critically regarding the time and route of treatment and observed downstream effects. nonsteroidal antiinflammatory drugs nonsteroidal antiinflammatories (nsaids) are routinely used with the intent to diminish persistent inflammation noted in susceptible mares. most nsaids inhibit cyclooxygenases (cox-1 and cox-2), thereby decreasing production of prostaglandins. since the primary activity of pgf2α is activation of myometrial contractility, inhibition of this lipid can lead to insufficient uterine contractions and luminal fluid accumulation if mares are not concurrently treated with an ecbolic to stimulate uterine contractions. intravenous flunixin meglumine (1.1 mg/kg, 2 hours after insemination) treatment, substantially increased the number of pmns in the uterine lumen at 8 hours; however, they were decreased at 25 hours after insemination.20 in contrast, select cox-2 inhibitors (function solely through the inhibition of cox-2 with no effect on cox-1) had a positive effect on uterine clearance after breeding. when treated orally once in the periovulatory period, firocoxib (equioxx®, 0.2 mg/ kg) decreased the severity of neutrophilia. additionally, firocoxib treatment did not impact ovulation, embryo collection, or embryo mobility.21,22 vedaprofen, an additional selective cox-2 inhibitor, increased fertility in pbie mares.23 oral vedaprofen (2 mg/kg) administered twice during the periovulatory period improved pregnancy rates; however, only a limited number of mares were evaluated.23 it is emphasized that all nsaid treatments of pbie should be accompanied by ecbolic treatment to stimulate uterine contractions glucocorticoids glucocorticoids are a class of corticosteroids that modulate the immune system by activating downstream functions of the glucocorticoid receptor. although various glucocorticoids figure 1. immune response to breeding in resistant and susceptible mares to persistent breeding-induced endometritis clinical theriogenology 2021; 13: 239 are available to the practitioner, only a few have been critically evaluated for their efficacy in treating pbie. dexamethasone functioned as an antiinflammatory agent to both sperm and bacterial challenges.24,25 additionally, dexamethasone decreased endometrial expression of positive acute phase protein, serum amyloid a (saa),26 and similarly prednisolone treatment decreased expression of this protein.27 clinically, a single intramuscular treatment of 50 mg dexamethasone at breeding improved pregnancy rates in mares with > 3 risk factors associated with susceptibility to pbie.28 although limited research has been conducted on the antiinflammatory functions of corticosteroids, a subset was evaluated clinically. treatment with acetate-9-α-prednisolone (0.1 mg/ kg) twice daily also had a positive effect on pregnancy rates in mares with excessive inflammation.29 twice daily intramuscular treatment with isoflupredone acetate (predef® 2x, 10 20 mg) 24 hours prior to bacterial challenge in susceptible mares altered the immune response.27 responses included reduction in immunoglobulin g (igg) and increases in positive acute phase proteins’ (e.g. haptoglobin) concentrations. it is noteworthy that various glucocorticoids altered secretion of gnrh and lh, in addition to altering the function of hypothalamic-pituitary-gonadal axis.30 additionally, prolonged dexamethasone treatment has been associated with ovulation failure.31 therefore, treatment of pbie should be limited to a single dose of glucocorticoid. bacterial extracts extracts from nonpathogenic bacteria are utilized for their antiinflammatory, antimicrobial, and anticancer functions in a variety of diseases and disorders. of these bacterial extracts, only 2 have been evaluated for their efficacy in the treatment of equine pbie: mycobacterium cell wall fraction (mcwf; settle®, novavive; canada) and propionibacterium acnes (eqstim®, neogen; us). in the research setting, settle® has shown both antimicrobial and antiinflammatory actions in susceptible mares. settle® decreased endometrial expressions of il-1β, a proinflammatory cytokine and increased cytokine il-10, an antiinflammatory cytokine, after a bacterial challenge with gram positive streptococcus equi.32 additionally, settle® substantially reduced inflammation in several mares that cultured positive for bacteria. these antiinflammatory effects were also noted after a challenge with gram negative escherichia coli, wherein settle® substantially decreased the endometrial expression of the positive acute phase protein saa, decreased intrauterine fluid, and also decreased the number of mares that cultured positive for intrauterine bacterial growth.24 after a challenge with freeze-killed sperm in susceptible mares, settle® substantially reduced the endometrial expression of proinflammatory cytokines il-1β and ifnγ.25 settle® was effective at 1.5 mg dose, either given intravenously or infused into the uterine lumen.33 eqstim® treatment of endometritis along with other conventional therapies improved pregnancy rates in mares with clinical endometritis.1 however, effects of this therapeutic as a sole modality are unknown. biologics during the 1970s, naturally sourced biologics, were evaluated for the treatment of inflammation/infection of uterus. intrauterine infusion of equine colostrum was the first agent tested.34 due to its high immunoglobulin content and potential antimicrobial action, colostrum was collected from mares immediately after parturition and 120 ml infused into the uterus of subfertile mares. mares returned to normal cyclicity and uterine lumen had various immunoglobulins (igg, igm, and ige). however, intrauterine deficiency in igg and iga in susceptible mares was not likely the cause of susceptibility to persistent endometritis.4 a key factor believed to distinguish resistant from susceptible mares is the innate immune system’s ability to degrade and digest bacteria and excess sperm from the uterine lumen. the addition of blood serum substantially increased opsonization potential in both resistant and susceptible mares, indicating an antimicrobial effect of blood plasma. uterine-derived pmns from susceptible mares were able to migrate toward a chemical gradient and phagocytosis was fully functional if a healthy environment was provided.35 however, phagocytosis was impaired in uterine secretions from susceptible mares. it was concluded that dysfunctions of pmns from susceptible mares were due to a poor environment rather than the pmns of susceptible mares. nevertheless, it was suggested that addition of fresh blood-derived leucocytes to autologous plasma prior to post-breeding intrauterine infusions can improve pregnancy rates in barren mares.36 there was clinical improvement (24/29 mares) and pregnancy rate (15/29 mares) in mares experiencing uterine inflammation after intrauterine infusion of plasma.37 however, it was ineffective in the treatment of either lymphocytic or infectious endometritis.38 these findings were further supported by the observation that intrauterine treatment with autologous blood plasma in susceptible mares was less effective than antibiotics, ecbolics, or uterine lavage in mares with acute infectious endometritis.39 it is noteworthy that there was a subgroup of individual susceptible mares that responded adequately to autologous plasma treatment. decades later, plasma treatment protocols were modified with intrauterine treatment of platelet-rich plasma (prp). a concentrate of prp protein derived from whole blood, contained several growth factors and cytokines released from the intracytoplasmic granules when cells are activated.40 prp is utilized in several tissues and disorders for its antiinflammatory, antimicrobial, and angiogenic properties, including joint disease, soft tissue injury, and skin wounds.41-43 prp functioned as an antiinflammatory agent when infused within the uterine lumen by decreasing the expression of proinflammatory cytokines (cox-2, il-1β, and tnf).44-45 although a small number of mares were investigated, this study demonstrated an increase in fertility rates in susceptible mares receiving prp, without a detected change in intrauterine clinical theriogenology 2021; 13: 240 fluid. furthermore, time of treatment (24 hours preinsemination or 4 hours postinsemination) had similar effects. although prp did not increase antiinflammatory cytokine expression, it decreased mrna expression of proinflammatory cytokines.46 clinically, intrauterine prp treatment at insemination decreased the amount of intrauterine fluid and improved pregnancy rates, indicating that time of treatment may cause variable outcomes.47 intrauterine infusion of lactoferrin, expanded our understanding of its immunomodulation. lactoferrin, present in high concentrations in both milk and seminal plasma, has been described as both antiinflammatory and antimicrobial in a variety of species.48-52 however, its bactericidal properties have not been elucidated in the horse. in a periovulatory susceptible mare, 150 µg/ml human recombinant lactoferrin (re-lf) treatment, suppressed endometrial expression of pleiotropic tnf.52 lactoferrin treatment along with artificial insemination in healthy resistant mares also substantially decreased expression of various proinflammatory cytokines (including il-1β and il-8).53 in susceptible mares, re-lf treatment, 6 hours after insemination, decreased endometrial expression of proinflammatory ifnγ and simultaneously increased expression of antiinflammatory il-1rn.51 additionally, re-lf substantially decreased the number of neutrophils in the uterine lumen. further work is necessary to assess bactericidal properties of re-lf and its efficacy in treatment of infectious endometritis. mesenchymal stem cells (mscs) have also been investigated for their antiinflammatory and regenerative effects.54 for the past 2 decades, mscs have been utilized in a variety of diseases/ disorders in humans; however, research in their capabilities for horses is more limited. intrauterine infusion of bone marrow-derived mscs at 24 hours pre-insemination in normal mares decreased the number of neutrophils in the uterine lumen in addition to increasing the expression of antiinflammatory il-1rn.55 antiinflammatory effects of mscs in the treatment of endometritis were compared between adipose tissue and endometrium.56 both adipose tissue and endometrial-derived mscs decreased concentrations of pleiotropic il-6 and tnf in the luminal fluid. within the endometrium, adipose-derived mscs significantly decreased expression of proinflammatory il-1a and il-8 in addition to il-6 and tnf. in contrast, endometrial-derived mscs significantly decreased proinflammatory il-8 and simultaneously increased endometrial expression of antiinflammatory il-10. no cellular infiltration within the endometrium was noted 24 hours following infusion of mscs had no cellular infiltration, although there was a substantial decrease in luminal neutrophilia.57 this study concluded that mscs are a safe and effective antiinflammatory option for the uterine lumen without invading or implanting within the endometrium. immune response to pregnancy the immune system of pregnant females exists in a delicate balance between protection and recognition, as it must both tolerate the semi-allogeneic fetus while still defending the body from pathogens that can impede the health of both mother and fetus. it is now understood that this phenomenon is due to fluctuating adaptive immune system, specifically effector and regulatory t lymphocytes.58 these immune cells are both crucial for pregnancy maintenance and proper embryo development, and also essential for responses to pathogen detection.59-61 however, many of the signals necessary for pathogen recognition and elimination are similar to the proinflammatory mechanisms preceding parturition, leading to an increased risk of preterm labor or abortion after detection of microbes in the reproductive tract during pregnancy. involvement of regulatory t cell (treg) in pregnancy maintenance is a topic of interest in the development of therapeutic options within this field. in human medicine, several immunomodulators that heighten or mimic treg function are currently investigated for their ability to support pregnancy in addition to preventing placental infection and/or preterm labor. tregs are both antiinflammatory and antiapoptotic, and therefore critical in the prevention of effector-type immune responses to paternal antigens present in feto-placental tissues. limited research has been done in this field in equine reproduction, but it is a topic of interest for future research. we review the therapeutic options currently available for equine practitioners and identify potential future targets. immunomodulators for pregnancy maintenance immunomodulation for improvement of pregnancy maintenance has been attempted through a variety of avenues, including transplanting lymphocytes from donor stallions, in addition to stimulation of naïve lymphocytes towards treg development through various cytokines and biologics treatment.62 many of these immunomodulators (e.g. recombinant granulocyte-macrophage colony-stimulating factor [filgrastim]) or intravenous immunoglobulin (plasma) improved success of pregnancy in the human.62-67however, the authors are not aware of similar studies in the horse. in contrast, some effort has been made to determine the use of lymphocyte donation to improve pregnancy maintenance in women and mares. women experiencing repeated spontaneous abortion during early pregnancy had improved pregnancy success after receiving lymphocyte donation from either the sperm donor or a third-party donor.68 in 1 meta-analysis, women receiving lymphocytes from the sperm donor had improved pregnancy success by 68%, whereas women that received lymphocytes from a third-party donor improved pregnancy success by 36%. lymphocyte donation has been attempted in mares.69 autologous lymphocyte treatment improved pregnancy rates in thoroughbred mares that had previously experienced early embryonic loss.69 in a second study, lymphocytes had no effect on pregnancy outcome. however, neither study had a control group, so the results should be interpreted carefully. additionally, it is unknown what proportion of this lymphocyte population clinical theriogenology 2021; 13: 241 were effector or regulatory lymphocytes, and the responses noted could be mitigated or enhanced if these populations were altered. lower percentage of tregs during estrus strongly correlated with an increased risk of early embryonic loss,70 further indicating the crucial role of these cells on pregnancy maintenance. immune response to pregnancy-related complications in women, the balance between recognition of self and detection of pathogen becomes skewed preceding numerous pregnancy-related complications. this alteration in the adaptive immune response is most noted as an increase in activation/recruitment of the proinflammatory th17 cells alongside a diminished treg response, leading to rejection of the semi-allogeneic tissue and eventual abortion.59,71-74 in mares, the leading cause of infectious abortion is placental infection, and this contributes to 19% of all abortions in north america.75,76 we recently reported77 a comparable adaptive immune response to the experimental induction of ascending placentitis to what has been reported in women, with activation of effector functions of the adaptive immune system and suppression of the regulatory arm (figure 2). this was observed both locally to the feto-placental unit with an increase in th17related transcripts, alongside dysregulation of treg-related transcripts. additionally, induction of ascending placentitis led to immune alterations in circulation, including an increase in various proinflammatory (il-2, ifnγ), antiinflammatory (il-5, il-10) and pleiotropic cytokines (il-6, tnf), with il-6 having the highest fold change of among the cytokines investigated.78 further research conducted in our laboratory suggested that the fetus also had an active immune response to placentitis and it was primarily characterized by expression, production, and secretion of both antiinflammatory (il-10) and pleiotropic (il-6) cytokines.79 pleiotropic il-6 was recently observed to function through the classical il-6r signaling pathway during the pathophysiology of ascending placentitis, leading to various downstream antiinflammatory effects, including inhibition of apoptosis and proliferation of epithelial cells.80 further work in our laboratory had similar cytokines increase during both ascending and focal mucoid placentitis (the etiology included nocardioform placentitis.81) since all mares investigated within this study produced a viable neonate, additional research is necessary to determine the profile of these cytokines preceding abortion. immunomodulation of pregnancy-related complications a few experimental models exist for induction of pregnancy-related complications, forcing inferences made from the experimental induction of ascending placentitis towards various etiologies in equine reproduction. for ascending placentitis, treatment commonly includes an antimicrobial, progestin (altrenogest; regu-mate®), and antiinflammatory (pentoxifylline and/or nsaid) agents.82 pentoxifylline is considered both antiinflammatory and anticytokine, due to its dual ability to inhibit the nfkβ pathway while simultaneously acting as both antitnf and antiil-6, both of which have been found to elevate during the pathophysiology of ascending placentitis.83 however, no studies have investigated the role of pentoxifylline on the immune response of the horse. additionally, although the oral dosage utilized in the present study was 8.8 mg/kg,84,85 no study has assessed the appropriate dose to use in the horse, with figure 2. overview of dysregulation th17/treg response after equine placental infection. equine ascending placentitis begins by detection of extracellular s. zooepidemicus that is recognized by an antigen presenting cell. this combination stimulates naïve t cells to mature into th17 cells through activation of various cytokines (il-6 and il-23), cell surface markers (il-1r1, il-6r, and il-21r) and transcriptions factors (stat3 and irf4). additionally, cytokines (tgf-b), cell surface markers (lrrc32/garp), and transcription factors (foxp3) that assist in the maturation of tregs decreased after detection of pathogen, potentially dysregulating treg production. this imbalance of th17/treg maturation precedes abortion in a variety of species and may activate preterm parturition or abortion in the horse after ascending placental infection. clinical theriogenology 2021; 13: 242 extrapolations being made from other species with differing placentation. although other nsaids have been evaluated for their efficacy in treatment of placentitis, none has been assessed as a sole modality; therefore, results should be interpreted critically. clinically, twice daily intravenous flunixin (1 mg/kg) and oral phenylbutazone (2.2 4.4 mg/kg), and once daily oral firocoxib (57 mg) are routinely used, albeit with only limited data to support their treatment.84-88 flunixin meglumine has been investigated in conjunction with tms, altrenogest, and estradiol cypionate.88 while the addition of estradiol cypionate improved pregnancy outcomes, flunixin meglumine as a sole modality had limited positive effects. firocoxib has also been used along with altrenogest and tms.87 following treatment with firocoxib, a substantial decrease in both il-1β and il-6 secretion was observed. as il-6 has a 4-fold increase following the induction of ascending placentitis, this suppression is intriguing and deserves further work. immune response to postpartum involution equine pregnancy lasts 340 ± 20 days;89 therefore, subsequent pregnancy must be established within the first month postpartum to achieve continued commercial success. in the mare, first postpartum estrus occurs from 7 16 days after parturition and is referred to as 'foal heat'. this coincides with the process of uterine repair needed after parturition. uterine involution includes reduction of uterine size, removal of residual debris, contraction of endometrial glands, and elimination of lingering immune cell types. it takes approximately 13 15 days and is necessary to ensure a uterine environment that is not hostile to the impending embryo arrival. recent research from our laboratory described the role of the immune system in postpartum involution. a significant relationship was noted between the day postpartum and endometrial expression of pleiotropic (il-6 and gm-csf) and antiinflammatory cytokines (il-10).90 expression was highest immediately postpartum and then declined as involution progressed. this coincided with a decline in both bacterial growth and neutrophilia, indicating signaling mechanisms required to disallow persistent inflammation from occurring during this physiologic event. several therapeutics have been attempted to hasten this physiological process to ensure pregnancy success, but with minimal success. we review the use of immunomodulation to alter involution and improve 'foal heat' breeding success. immunomodulation of postpartum involution parturition enables immense bacterial invasion into the vaginal vault, through the relaxed cervical canal and eventual localization in the uterine lumen and endometrium. this leads to increased neutrophilia and the potential for persistent inflammation/ infection that may hinder future fertility postpartum. intravenous mcwf (1.5 mg ; settle®) treatment administered 24 hours after parturition improved 'foal heat' breeding success and substantially decreased both bacterial presence and magnitude of neutrophilia.91 additional studies (1.5 mg iv mcwf treatment at 24 hours and at 7 days postpartum) increased endometrial expression of proinflammatory cytokines (il-1β and ifnγ).90 as these cytokines are essential for the recruitment and chemotaxis of necessary immune cells for bacterial degradation and clearance, it was not surprising that this increase coincided with a reduction in the severity of bacterial growth, in addition to decreasing days required for a clean culture. conclusion reproductive immunology is constantly advancing our understanding of mare physiology. continued work is necessary to further elucidate the pathophysiology of several diseases and disorders associated with equine reproduction, including breeding-induced inflammation, immune tolerance of the semi-allogeneic fetus, immune responses to pregnancy-related complications, and immune-mediated response to parturition. as our understanding of this physiology broadens, inferences can be made towards several immunomodulators that may improve these processes, thereby advancing mare reproductive performance. conflict of interest authors have no affiliations, 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complications in the horse part i: cytokines. j equine vet sci 2020. 82. bailey cs, macpherson ml, pozor ma, et al: treatment efficacy of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest in experimentally induced equine placentitis. theriogenology 2010;74:402-412. 83. alijotas-reig j, esteve-valverde e, ferrer-oliveras r, et al: tumor necrosis factor-alpha and pregnancy: focus on biologics. an updated and comprehensive review. clin rev allergy immunol 2017;53:40-53. 84. macpherson ml: treatment strategies for mares with placentitis. theriogenology 2005;64:528-534. 85. macpherson ml, bailey cs: a clinical approach to managing the mare with placentitis. theriogenology 2008;70:435-440. 86. macpherson ml: diagnosis and treatment of equine placentitis. vet clin north am equine pract 2006;22:763-776. 87. macpherson ml, giguere s, pozor ma, et al: evidence for anti-inflammatory effects of firocoxib administered to mares with experimentally induced placentitis. am j reprod immunol 2021:e13396. 88. curcio br, canisso if, pazinato, et al: estradiol cypionate aided treatment for experimentally induced ascending placentitis in mares. theriogenology 2017;102:98-107. 89. rossdale pd, silver m: the concept of readiness for birth. j reprod fertil suppl 1982;32:507-510. 90. fedorka ce, murase h, loux sc, et al: the effect of mycobacterium cell wall fraction on histologic, immunologic, and clinical parameters of postpartum involution in the mare. j equine vet sci 2020;90:103013. 91. fumuso e, alvarez g, bruno s, et al: non-specific immunomodulation at post-partum improves uterine condition and fertility in mares. proc world equine vet association 2003. clinical theriogenology 2021; 13: 246 immunomodulator use in mares the 2020 therio conference will be held at the historic station square at the sheraton pittsburgh hotel in pittsburgh, pennsylvania on july 22 –25. pittsburgh is known as the city of steel, city of bridges, and the city of champions. when visiting this unique city, you are warmly greeted from the locals and instantly become a part of the community. join us to view the breathtaking skyline, enjoy trendy shops and eateries, venture in the outdoors, and explore pittsburgh’s 90 charming and distinctive neighborhoods. the 2020 therio conference will focus on the equine program, along with production animal and small animal tracks. the conference will begin wednesday with a limited number of veterinarians being given the opportunity to participate in the equine symposium. the opening session abstracts will be presented wednesday evening followed by the opening session abstract reception. thursday morning begins at 7:30 am with the presentations of the bartlett award and address and the theriogenologist of the year award and address. the dr. jerry rains memorial abstract competition and student case presentations will follow along with the society for theriogenology and american college of theriogenologists business meetings. students will also be given the opportunity to participate in an equine seminar and lab. the opening reception in the exhibit hall will be thursday evening with the opportunity to visit with our vendors, relax with a cocktail, and enjoy light hors d’oeuvres. the theriogenology foundation silent auction will open for browsing and bidding during the reception. the annual merck student quiz bowl will also be held this evening. friday morning will begin with the plenary session and feature a presentation on new technologies being applied to reproduction, including crispr-mediated gene editing by dr. katrin hinrichs and the latest on stem cell therapy, including applications to the reproductive system presented by dr. ashlee watts. abstract presentations will follow the opening session along with tracks in small animal, production animal and equine. friday evening will feature the awards dinner reception, followed by the annual awards dinner, veterinary story night and live auction. during the veterinary story night, attendees will be entertained with a story which will be paired with a unique item available for the live auction. attendees will also get to participate in the “textto donate” event to vote for their favorite storyteller. all proceeds will benefit the theriogenology foundation. saturday will begin with a presentation by current act diplomates for those interested in learning more about becoming a certified veterinarian in theriogenology and a roundtable discussion with “careers in theriogenology”. a full day of ce will also be offered saturday with tracks in small animal, production animal and equine. the conference will end with a group outing to be announced soon for saturday evening. group rates are available at the conference hotel until june 18, 2020 or the room block is sold out. reservations can be made through the link on the conference website or by contacting the hotel and asking for the society for theriogenology group rate. we hope that you will make plans to join us in the enticing city of pittsburgh. more information will follow in the near future regarding a full agenda, symposium details, and conference registration. please visit www.therio.org for additional conference information and or www.visitpittsburgh.com for things to do during your stay. 2020 therio conference and equine symposium preview https://www.therio.org/ http://www.visitpittsburgh.com 2019 therio conference recap the charming city of savannah provided the location for the 2019 annual therio conference. we had excellence attendance this year, with 489 total attendees for the conference plus 115 attendees for the symposium and student lab. the conference was held july 24-27 at the westin savannah harbor golf resort & spa. the week began with the much anticipated act exam and concluded with colleagues taking a private trolley ride with a tour of the haunted history of savannah. the small animal symposium kicked off the meeting wednesday with “a crash course in veterinary microbiomics” by dr. candace lyman, followed by her presentation in “vaginal microbiome: what not to do”. dr. christopher premanandan captured the audience with presentations on “monitoring reproductive biome health using cytology and histopathology” and “canine placentation normal gross and histologic structure as well as confounding features during evaluation”. the small animal symposium sponsor, idexx, held a lunch presentation with dr. elizabeth schooley on “the art of measuring progesterone: understanding immunoassays”. dr. jenny sones discussed “canine placental microbiome” and dr. michelle kutzler closed out the symposium with presentations on “reproductive microbiome alterations: clinical management of pyometra” and “reproductive microbiome alterations: clinical management of prostatitis”. the student small animal seminar and lab, sponsored by sonosite-a fujifilm company, was held on thursday with presentations by theriogenology residents, drs. luis de aguiar and viviane gomes. they also led the morning lab on reproductive ultrasonography along with drs. michelle kutzler and jenny sones. a roundtable lunch was held to discuss the future of companion animal theriogenology. the students ended the day with a lab focusing on progesterone, relaxin, and brucella canis assays. thursday morning began by honoring dr. rob lofstedt with the theriogenologist of the year award, sponsored by universal imaging, inc. and the david e. first place quiz bowl winners auburn university second place quiz bowl winners texas a&m university bartlett award for lifetime achievement in theriogenology honoring dr. ahmed tibary. eight presenters competed in the dr. jerry rains memorial abstract competition, sponsored by merck animal health. the sft and act business meetings were held in the afternoon. during the act business meeting, the new diplomates were introduced following the successful completion of this year’s act exam. the student case presentations and student/ mentor reception were also held that afternoon. the exhibit hall opening reception was held thursday evening in conjunction with the student quiz bowl, sponsored by merck animal health. the students at auburn university came in first place as the quiz bowl winners with texas a&m coming in second place. plenary sessions, sponsored by the american college of theriogenologists, featured “a primer to genome editing” by dr. claudia klein and “metabolic and environmental effects on embryo epigenome” by dr. pablo ross. case reports, mixed species, production animal, and equine abstract presentations were held in the morning followed by small animal, production animal, and equine tracks in the afternoon. the awards dinner reception was held in the grand ballroom pre-function, followed by the awards dinner and inaugural veterinary story night and live auction, sponsored by merck animal health. ron mcdaniel from merck animal health kept guests entertained as usual during the captivating stories that were paired with unique auction items. the inaugural storyteller of the year award went to dr. carol mcleod by audience voting in the text-to-donate fundraiser. dr. bob youngquist was presented the tf career excellence in theriogenology award. dr. jimmy alexander recognized the student case presentation winners: breanthony baker, texas a&m university (first place); katelyn kimble, texas a&m university (second place); colleen kutzler, university of minnesota (third place); nichole whitfill, auburn university (fourth place); lindsay hilburger, cornell university (fifth place); and gabrielle montone, auburn university (sixth place). winners of the tee shirt design contest were auburn university (first place) and texas a&m university (second place). dr. candace lyman presented the award for student chapter of the year to auburn university. lincoln memorial university came in second place for the scoty award. dr. juan romano, abstract committee chair for 2019, presented the dr. jerry rains david e. bartlett award recipient dr. ahmed tibary theriogenologist of the year dr. rob lofstedt storyteller of the year award dr. carol mcleod scoty award winner auburn university memorial abstract competition awards, sponsored by merck animal health. this year’s winners were: dr. carleigh fedorka, lexington, kentucky (first place); mackenzie roberts, corvallis, oregon (second place); dr. chun kuen mak, baton rouge, louisiana (third place); and dr. zachary turner, athens, georgia (fourth place). dr. robyn wilborn, 2019 sft president, announced dr. pete sheerin as the dr. john steiner award for excellence in practice recipient. dr. sheerin has exemplified standards of excellence in his leadership and promotion of theriogenology. the theriogenology foundation raised a total of $69,879.45. this year’s live auction generated $32,578.00 in addition to $25,000.00 in sponsorships for the evening’s storytellers and $4,416.45 for the text-to-donate voting for the stories. the silent auction, craft beer tasting tickets, and quiz bowl audience participation raised $7885.00 for the foundation. the final day of the conference began with a roundtable discussion focusing on “careers in theriogenology” led by drs. isaac bott, jill colloton, richard hopper, and paul mennick. those interested in becoming a certified theriogenologist attended the session “so you want to become a diplomate?” hosted by members of the act. the small animal track, production animal track, sponsored by lane manufacturing, inc., and equine track, sponsored by rood & riddle equine hospital and veterinary pharmacy, all presented a full day of innovative ce sessions. while some headed home after saturday’s sessions, many stayed to tour the historical city of savannah or embark on the therio group outing. two trolleys transported attendees on a private tour through the haunted history of savannah. the 2019 annual therio conference in savannah held fun for all and included great networking with colleagues, sight-seeing, and outdoor adventures. make plans to join us for the 2020 therio conference on july 22-25 in the spectacular city of pittsburgh, pennsylvania. abstract winners (l to r): dr. zachary turner (fourth place), dr. chun kuen mak (third place), mackenzie roberts (second place) and dr. carleigh fedorka (first place) congratulations 2019 act diplomates! (l to r): sicilia grady-newark, delaware; mary ontiveros-matamata, new zealand; mohammad ibrahim-athens, georgia; elena martinez de andino-college station, texas; babiche alida heil-scone, nsw, australia; julia baldrighi-stillwater, oklahoma; pouya dini-lexington, kentucky; and anna mitchell-ithaca, new york student case presentation winners (l to r): colleen kutzler-university of minnesota (third place), lindsay hilburger-cornell university (fifth place), nichole whitfill-auburn university (fourth place), breanthony baker-texas a&m university (first place), gabrielle montone-auburn university (sixth place), and katelyn kimble-texas a&m university (second place) dr. sheerin was born and raised in new york city. his first experience with horses was soon after his family moved to lagrangeville, a small-town north of the city. he began working on a thoroughbred breeding farm where he gained experience in all aspects of the thoroughbred industry. during high school, he also competed on the varsity crew team, and his love of rowing took him to syracuse university where he majored in biology. following graduation, dr. sheerin moved to florida to manage a small thoroughbred breeding farm in ocala. several years later, he began graduate research in equine reproductive physiology at the university of florida under mentor dr. dan sharp, while managing the endocrinology lab at the university of florida college of veterinary medicine. dr. sheerin then entered the veterinary school, where he expanded his interest in theriogenology, particularly large animal theriogenology. after graduating from the university of florida college of veterinary medicine in 1994, dr. sheerin joined a mixed practice in live oak, florida prior to moving to new freedom, pennsylvania to join nandi veterinary associates. dr. john hurtgen’s knowledge and teaching encouraged dr. sheerin to return to the university of florida for a theriogenology residency. dr. sheerin became board certified in 2001, and upon completion of his residency, dr. sheerin and his family returned to new freedom. the following year, the sheerins moved to lexington, kentucky to join rood and riddle equine hospital, where dr. sheerin joined one of his veterinary school mentors, dr. michelle leblanc, as a staff theriogenologist. the sheerins returned to nandi in 2011 to continue dr. hurtgen’s legacy. he is a member of the american association of equine practitioners, the american veterinary medical association and the society for theriogenology where he served as 2016-2017 president. dr. sheerin has lectured both nationally and internationally on reproductive aspects of the mare and stallion. dr. sheerin and his wife barbara have three daughters. pete and barb compete in combined driving with a homebred pony. dr. john steiner award for practitioner excellence dr. pete sheerin career excellence in theriogenology award on july 26 at the annual therio conference in savannah, georgia, the theriogenology foundation was proud to honor robert s. youngquist, dvm, dact with the career excellence in theriogenology award for his contributions to the field of reproductive veterinary medicine. dr. youngquist has made major and significant contributions to the field of theriogenology in research, teaching and service activities during his career at the university of missouri. he was the editor of current therapy in large animal theriogenology published in 1997 by w.b. saunders and has also served on the editorial boards of the american journal of veterinary research and theriogenology. dr. youngquist also served as the founding editor and editor for the past ten years of the clinical theriogenology journal. youngquist received his dvm in 1971 from iowa state university and earned the status of diplomate of the american college of theriogenologists in 1976. since earning his diplomate status, he has been heavily involved with act having served on the examination committee, and as the college’s secretary (1980-1983) and president (1984-1985). he received the award for theriogenologist of the year in 2001. in addition, dr. youngquist has been active in the society for theriogenology and was recognized in 2001 as the recipient of the dr. david e. bartlett award. his interests include all domestic species, but his research has focused on normal and abnormal ovarian function in cattle. youngquist had a keen interest in working with students and has served as chairperson and on the committee for numerous master’s students. he has twice received the “norden award for distinguished teaching in veterinary medicine” and in 1996 received the “dean’s impact award”. dr. bob youngquist silver gold bronze platinum a special thank you to our therio 2019 sponsors do you know someone who would make a great director of the sft board? in accordance with policy, the society of theriogenology requests your suggestions for the board of directors. names received from the general membership will be forwarded to the nominating committee for consideration. directors serve a term of three years, beginning at the annual therio conference. if you would like to suggest an active society member for service as a director on the board, please submit the following information to the executive office no later than march 2, 2020. please ensure the individual’s willingness to serve on the board (if elected) prior to submission. please direct any questions to dr. isaac bott, chair of the nominating committee at bottisaac@yahoo.com. when suggestions are submitted we will need the following contact information on both the person submitting the suggestion and also the suggested individual’s: name, address, phone, email, and practice type. mail, email, or fax information to: charles f. franz, dvm executive director society for theriogenology 761 tiger oak drive pike road, al 36064 fax: 334-270-3399 email: charles@franzmgt.com you may submit nominations online at http://therio.org/ boardnominationform you must be logged in to the sft website to access the board nomination form located under board and officers. thank you to all of our 2019 therio conference exhibitors all vet imaging/microvet diagnostics the american kennel club, inc. animal reproduction systems, inc. aurora pharmaceutical b.e.t. pharm/b.e.t. labs biomerieux botupharma usa, inc. breeder's choice cargosense, inc. chemometec a/s christian veterinary mission e.i. medical imaging, elsevier, inc. emberequine esaote north america fertility technology resources idexx imv technologies usa/imv imaging jorgensen laboratories karl storz lane manufacturing, inc. merck animal health mds incorporated minitube usa multimin usa, inc. neogen parsemus foundation platinum performance, inc. puppywarmer spectrum veterinary sonosite-a fujifilm company universal imaging, inc. veterinary books by success concepts zoetis mailto: https://www.therio.org/page/boardnominationform https://www.therio.org/page/boardnominationform thank you to all of the individuals, companies, and organizations that supported the theriogenology foundation by donating to the 2019 live auction, silent auction, and story night! a huge thank you to all the following individuals for all their efforts in planning the 2019 therio conference. annual conference program chair dr. colin palmer equine track program chair dr. sara lyle small animal track program chair drs. michelle kutzler and jenny sones production animal track program chair dr. leonardo brito live auction chair dr. jim floyd silent auction chairs drs. jim floyd and will schultz small animal symposium chairs drs. michelle kutzler and jenny sones student small animal seminar and lab chairs drs. michelle kutzler and jenny sones 2019 funds raised live auction $32,578.00 silent auction $5,585.00 beer tasting $2,040.00 quiz bowl audience participation $260.00 sponsorship of stories $25,000.00 text-to-donate voting for stories $4,416.45 total: $69,879.45 dr. morgan agnew dr. jane barber dr. steve brinsko dr. sara brunsden dr. gail colbern dr. jill colloton dr. marco coutinho da silva dr. brad fields dr. jim floyd dr. charles franz dr. natalie fraser dr. julie gard schnuelle dr. reed holyoak dr. kit kampschmidt dr. ira kaplan dr. audrey kelleman dr. jennifer koziol ms. colleen kutzler dr. charles love dr. dave matsas mr. ron mcdaniel dr. carol mcleod mrs. rebecca meyers dr. anita migday dr. anneke moresco dr. dale paccamonti dr. willis parker dr. and mrs. tom riddle dr. peter ryan mrs. ingrid schultz dr. mary sebzda dr. phil senger dr. katherine settle dr. cliff shipley dr. jack smith ms. melissa stagnaro dr. mike thompson dr. ahmed tibary dr. wendy vaala dr. dirk vanderwall ms. jenna ward dr. nichole whitfill mr. barney wilborn dr. dwight wolfe auburn university student chapter lane manufacturing, inc. minitube usa neogen puppywarmer theriogenology foundation 2019 in review research grants the theriogenology foundation offers research grants up to $5,000 for proposals that focus on all aspects of theriogenology including (infertility, endocrinology, neonatology, obstetrics, etc.). in exceptional circumstances, grants up to $10,000 may be considered. active diplomates, honorary members of the american college of theriogenologists, veterinarians who are society for theriogenology members in private practice, industry or on faculty, or residents enrolled in act approved residency programs (including the alternate route) and have a minimum of one year remaining in their training program are eligible to apply for these grants. • two research grants were awarded in 2019: dr. alvaro garcia guerra, the ohio state university “effect of anogenital distance on age at onset of puberty in beef heifers”; and dr. jamie l. stewart, virginia-maryland college of veterinary medicine, “efficacy of deslorelin acetate on induction of ovulation in does”. the working dog project the goal of the working dog project is to identify genetic explanations for why some dogs succeed in working dog programs while others do not, and to help better target individual dogs to different programs, such as assistance versus law enforcement work. this new project is based at the broad institute of mit and harvard, in cambridge, massachusetts, and is generously funded by the theriogenology foundation and akc reunite. act student outreach the act seeks to promote awareness of the college and to encourage veterinary students to aspire to diplomate status after graduation. the purpose of this fund is to facilitate travel and related expenses for non-local act diplomates invited by student groups to visit and teach at veterinary schools. act/tf hopes that these visits will stimulate student enthusiasm and interest in the college by broadening students’ awareness of the many opportunities open to our diplomates. this opportunity is open to all student groups. it is not required that the school has an active student chapter of the sft. one grant totaling $1,000 was awarded in 2019. travel grants in 2019 the tf also awarded $ 3,275 in student travel grants to assist veterinary students who were travelling to attend the therio conference in savannah, ga. these students were presenting abstracts, posters, student case presentations or taking part as a quiz bowl team member at the conference. akc, akc-chf, tf residency travel grants all of the residencies are made possible by generous grants from the american kennel club and the akc canine health foundation. each resident was awarded $750 towards their travel to the annual therio conference. student externship grants student externship grants are grants intended to support experiential educational advancement of students interested in theriogenology. one student externship grant totaling $1,000 has been awarded. student chapter grants the tf receives and considers requests for funding of student chapter grants intended to provide educational advancement of students interested in theriogenology. furthermore, primary consideration is given to funding requests which provide an educational experience in theriogenology that is beyond the scope of the basic curriculum. the tf may consider all or partial funding of a request based on the annual budget and the merit of the educational experience to students. funding to cover a speaker’s travel expense is also available. two student chapter grants have been awarded totaling $995. zemjanis outreach fund this grant is intended to aid veterinarians traveling from developing countries to the united states for educational purposes or for sending a trainer to a developing country to conduct training on-site. 2019 tf grants awarded as of 10/25/19 student travel 10 grants $3,275 student externship 1 grant $1,000 research-bovine 2 grants $10,953 akc, akc-chf, tf travel 3 grants $2,250 act student outreach 1 grant $1,000 student chapter 2 grants $995 2020 bartlett award nominations sought nominations are now being sought for the 2020 david e. bartlett award for lifetime achievement in theriogenology, presented annually to a distinguished individual who has made important contributions to the field of theriogenology. the award intended to cement the efforts of the society, college, and foundation toward common goals in animal reproduction, to reward and inspire excellence, improve the visibility of theriogenology, and to annually recognize the efforts of the founders particularly honors dr. david e. bartlett, first president of the college. among his many accomplishments, dr. bartlett was responsible for deriving the terms “theriogenology” and “theriogenologists,” and was instrumental in gaining hard-won avma recognition for the american college of theriogenologists in 1971. the 2020 david e. bartlett award for lifetime achievement in theriogenology will be presented during the annual therio conference in pittsburgh, pennsylvania. the recipient will be notified in advance, allowing family and friends to be on hand. the honoree will address those in attendance and will have the option of submitting a contribution to the conference proceedings. the honoree will receive $1,000 cash, an engraved statue of nandi, and complimentary registration, hotel accommodations and transportation to the 2020 conference. please submit the accompanying preliminary entry form and nominate an individual to add to the list of preeminent recipients. entry forms must be received no later than january 4, 2020. nominations may also be made online at http://www.therio.org/bartlettawardform. you must be signed in as a sft member to access the form. it is located under the 2020 therio conference and also under the bartlett award recipient list on the sft website. 2020 dr. david e. bartlett award for lifetime achievement in theriogenology preliminary entry form sponsored by the theriogenology foundation in cooperation with the society for theriogenology & american college of theriogenologists purpose: to reward a distinguished theriogenologist who has made important contributions to the field of theriogenology. nominee: full address: current employment and position: home phone: business phone: fax: email: provide a short description of nominee’s qualifications for this award. use a separate sheet of paper containing a maximum 350 word narrative bio, highlighting nominee’s key accomplishments. nominated by: full address: home phone: business phone: fax: email: nominations must be received by january 4, 2020 submit online at: http://www.therio.org/bartlettawardform or mail to: theriogenology foundation, bartlett award 761 tiger oak drive pike road, al 36064-3063 call for act nominations to board the nominating committee of the act and the executive board are calling for nominations for the following positions: 1. vice president – this is a four-year term beginning with the position of vice president, moving to president elect, president and then immediate past president. 2. treasurer three-year term 3. executive board member – director three-year term active diplomates may be considered for any of these positions. travel is required to board meetings in january and one held during the summer convention. travel costs for the january board meeting are reimbursed by the college. please forward nominations to dr. richard hopper no later than february 15, 2020. biographical sketches and information along with ballots will be included in the spring act newsletter. the following information should be included with submissions for nominations: 1. name, school, year of graduation and position running for, 2. year achieving diplomate status and route (residency vs practitioner), 3. species emphasis and main focus of work, 4. qualifications specific for the post you are seeking-include education, personal experience, vision etc. please restrict the above comments to 350 words or less. send submission by february 15, 2020 to: dr. richard hopper au-cvm clinical sciences vaughn teaching hospital 1500 wire road auburn, al 36849-5540 email: rmh0067@auburn.edu 2020 annual sft membership renewal it is that time of year again! make sure you do not miss out on any of the great benefits that the society has to offer: •hospital listing by species and procedures for search on web site business tool for referrals •reduced registration fees for the annual therio conference •access to the members-only area of the society website, including past proceedings and newsletters •easy access to proceedings of the society’s annual therio conference online •free webinars •clinical theriogenology; the official quarterly publication of the society for theriogenology •access to sft-sa & sft-la, small animal and large animal listservs for case discussion with specialists •networking among fellow veterinarians with varied discipline interests •resource information on web site for your convenience you can renew your dues at the sft web site, www.therio.org important dates act exam applications due……………december 1 call for abstracts due ……………………february 1 tf research grant pre-proposals due….february 15 call for act board nominations due…february 15 call for student case presentations and student research presentations due……………….march 10 sft board nominations due……………...march 21 act exam fees dues……………………….april 15 tf research grant full proposals due……...april 15 student travel grants applications due...……june 1 student quiz bowl applications due…………june 1 student scoty applications due………...….june 1 tf research grant final decisions………….june 30 call for student case presentations-2020 annual therio conference student case presentations call for student case presentations also available at www.therio.org attention to all sft student members! the sft program committee is pleased to announce the call for submission of student theriogenology case presentations at the annual therio conference in pittsburgh, pennsylvania. the competition will be held during the annual conference, july 22-25, 2020. the purpose of this competition is to promote student interest in sft, to encourage investigative and communication skills and allow students greater participation in our annual meeting. all sft student members, defined as those sft student members in the professional degree curriculum leading to a dvm, vmd or other equivalent degree, are qualified to enter the competition. the student theriogenology case presentations will give the sft membership a look at future leaders and participants of the sft. six presentations will be selected to be presented orally. you be notified no later than april 15, 2020 whether your presentation has been selected for presentation. further instructions will follow notification. each presentation will be 10 minutes in length. time has been allotted for questions after each presentation. those not selected for oral presentation may be selected for a poster presentation. a panel of academicians and private practitioners will review manuscripts. students will be responsible for making their own travel arrangements and accommodations. conference hotel information will be available on the sft website. a copy of the guidelines and submission instructions can be obtained from the sft website, your sft faculty liaison, or dr. alexander. please refer to the “guidelines for application submission” for preparation of your manuscript. abstracts must be submitted by march 10, 2020. please submit them in microsoft word format to tara beasley at the society office, therioconference@franzmgt.com, with a copy to your advisor. include the words "student case presentation" in subject line. an acknowledgement of your submission should be received within 24 hours. if you do not receive acknowledgement, please contact the office at 334-395-4666. please be aware that this is a competition. the contestants will be judged on both the quality of their investigation and the quality of the presentation. therefore, you must attend the meeting in pittsburgh to be eligible for the awards. the sft will award six cash prizes: first place $ 650 fourth place $ 375 second place $ 525 fifth place $ 300 third place $ 450 sixth place $ 200 more than one student may be involved with the presentation of a case or research project, however only one prize will be awarded per presentation. all students (presentations) who are accepted to present abstracts may be awarded a travel stipend. grant information is available on the theriogenology foundation website, www.theriofoundation.org. travel grants must be applied for via the website. please contact your sft faculty liaison for more information and for assistance in preparing your manuscript. you may also contact dr. james h. alexander at: jalexanderdvm@gmail.com. we hope you will participate in this event. sincerely, james h. alexander, dvm http://www.therio.org/ http://www.therio.org/displaycommon.cfm?an=1&subarticlenbr=42%20 mailto:therioconference@franzmgt.com http://www.theriofoundation.org/ guidelines for application submission society for theriogenology student case presentation competition annual therio conference, pittsburgh, pennsylvania, july 22-25, 2020 all applicants must be student members of the society for theriogenology. this includes student members graduating in 2020. an abstract of the student case report must be received by march 10, 2020. please include the following with your submission: 1. name, home address and school address 2. veterinary school; year of graduation 3. telephone number 4. email address 5. name, title, address, email address and phone number of the faculty member working with you. 6. abstract of your case (300 words or less in the body of the abstract). a) title and authors b) the importance of the case to theriogenology c) a discussion of the diagnostic approach and treatment options considered. d) results/discussion e) references 7. normal style, 12-point times new roman font, single spacing, 1” margins 8. presentations must be submitted in microsoft word format to tara beasley at the sft office at therioconference@franzmgt.com. please copy your submission email to your advisor. you will receive an email acknowledgement of your submission within 24 hours (during business hours). if you do not receive an acknowledgment, please call the office at 334-395-4666. we will not need any photos, graphs, or diagrams at this time. prior to the conference you will be required to submit a copy of your power point presentation to the sft office to the attention of tara beasley at the society office, therioconference@franzmgt.com, no later than june 25, 2020. you will also be required to bring a copy with you to the conference. you will be allowed to update your presentation after june 25, up until 4 hours before your presentation. questions can be submitted to: james h. alexander, dvm at jalexanderdvm@gmail.com new sft officers were installed on thursday, july 25 at the sft business meeting. officers installed: dr. colin palmer, saskatoon, sk, canada, president; dr. jack smith, starkville, mississippi, president elect; dr. ahmed tibary, pullman, washington, vice president; dr. robyn wilborn, auburn, alabama, immediate past president; and dr. candace lyman, stillwater, oklahoma, secretary/treasurer. newly elected members of the board of directors are drs. hilari french, miramar, florida; heath king, mississippi state, mississippi, and wendy vaala, alma, wisconsin. new sft officers installed for 2019-2020 dr. robyn wilborn, immediate past president is presented a replica nandi by incoming president dr. colin palmer mailto:therioconference@franzmgt.com mailto:therioconference@franzmgt.com mailto:jalexanderdvm@gmail.com call for student research presentations-2020 annual therio conference student research presentations call for student research presentations also available at www.therio.org attention to all sft student members! the sft program committee is pleased to announce the call for submission of student theriogenology research presentations at the annual therio conference in pittsburgh, pennsylvania. the competition will be held during the annual conference, july 22-25, 2020. the purpose of this competition is to promote student interest in sft, to encourage investigative and communication skills and allow students greater participation in our annual meeting. all sft student members, defined as those sft student members in the professional degree curriculum leading to a dvm, vmd or other equivalent degree, are qualified to enter the competition. the student theriogenology research presentations will give the sft membership a look at future leaders and participants of the sft. four presentations will be selected to be presented orally. you be notified no later than april 15, 2020 whether your presentation has been selected for presentation. further instructions will follow notification. each presentation will be 10 minutes in length. time has been allotted for questions after each presentation. those not selected for oral presentation may be selected for a poster presentation. a panel of academicians and private practitioners will review manuscripts. students will be responsible for making their own travel arrangements and accommodations. conference hotel information will be available on the sft website. a copy of the guidelines and submission instructions can be obtained from the sft website, your sft faculty liaison, or dr. alexander. please refer to the “guidelines for application submission” for preparation of your manuscript. abstracts must be submitted by march 10, 2020. please submit them in microsoft word format to tara beasley at the society office, therioconference@franzmgt.com, with a copy to your advisor. include the words "student research presentation" in subject line. an acknowledgement of your submission should be received within 24 hours. if you do not receive acknowledgement, please contact the office at 334-395-4666. please be aware that this is a competition. the contestants will be judged on both the quality of their investigation and the quality of their presentation. therefore, you must attend the meeting in pittsburgh to be eligible for the awards. the sft will award four cash prizes: first place $ 650 third place $ 450 second place $ 525 fourth place $ 375 more than one student may be involved with the presentation of a case or research project, however only one prize will be awarded per presentation. all students (presentations) who are accepted to present abstracts may be awarded a travel stipend. grant information is available on the theriogenology foundation website, www.theriofoundation.org. travel grants must be applied for via the website. please contact your sft faculty liaison for more information and for assistance in preparing your manuscript. you may also contact dr. james h. alexander at: jalexanderdvm@gmail.com. we hope you will participate in this event. sincerely, james h. alexander, dvm jalexanderdvm@gmail.com http://www.therio.org/ http://www.therio.org/displaycommon.cfm?an=1&subarticlenbr=42%20 mailto:therioconference@franzmgt.com http://www.theriofoundation.org/ mailto:jalexanderdvm@gmail.com guidelines for application submission society for theriogenology student research presentation competition annual therio conference, pittsburgh, pennsylvania, july 22-25, 2020 all applicants must be student members of the society for theriogenology. this includes student members graduating in 2020. an abstract of the student case report must be received by march 10, 2020. please include the following with your submission: 1. name, home address and school address 2. veterinary school; year of graduation 3. telephone number 4. email address 5. name, title, address, email address and phone number of the faculty member working with you. 6. abstract of your case (300 words or less in the body of the abstract). a) title and authors b) the importance of the research to theriogenology c) a description of the experimental design, and materials and methods d) results/discussion e) references 7. normal style, 12-point times new roman font, single spacing, 1” margins 8. presentations must be submitted in microsoft word format to tara beasley at the sft office at therioconference@franzmgt.com. please copy your submission email to your advisor. you will receive an email acknowledgement of your submission within 24 hours (during business hours). if you do not receive an acknowledgment, please call the office at 334-395-4666. we will not need any photos, graphs, or diagrams at this time. prior to the conference you will be required to submit a copy of your power point presentation to the sft office to the attention of tara beasley at the society office, therioconference@franzmgt.com, no later than june 25, 2020. you will also be required to bring a copy with you to the conference. you will be allowed to update your presentation after june 25, up until 4 hours before your presentation. questions can be submitted to: james h. alexander, dvm at jalexanderdvm@gmail.com new act officers were installed thursday, july 25 at the act business meeting. officer installed: dr. richard hopper, auburn, alabama, president; dr. dirk vanderwall, logan, utah, president elect; dr. john dascanio, harrogate, tennessee, vice president; dr. kara kolster, glen allen, virginia, secretary; and dr. reed holyoak, stillwater, oklahoma, immediate past president. new act officers installed for 2019-2020 new act president dr. richard hopper thanks immediate past president dr. reed holyoak for his service. mailto:therioconference@franzmgt.com mailto:therioconference@franzmgt.com mailto:jalexanderdvm@gmail.com call for abstracts 2020 each year the act sends out a call for abstracts to be presented and included in the proceedings for the therio conference in the upcoming year. below is the information relative to this process. the society for theriogenology and american college of theriogenologists issue a call for research and case abstracts to be presented at the annual therio conference july 22 25, 2020 in pittsburgh, pennsylvania. application deadline is february 1, 2020. abstracts will be considered in four categories: 1. competitive category: abstracts submitted for this category must have a graduate student, resident, intern, post-doctoral scholar, veterinary student, or undergraduate student as the first author (only one submission per first author will be accepted in this category). abstracts on material that has already been published or has been accepted for publication at the time of submission will not be considered. these abstracts will be judged on the basis of scientific merit (written). the eight abstracts with the highest written scores will be selected for presentation during the abstract competition. first authors chosen to present in the abstract competition will receive one complimentary registration for the meeting. the abstracts will be presented during a plenary session and judged for presentation quality (oral and understanding of scientific basis). financial awards will be given to the four presenters with the highest total score out of 50 points (written (25 points) plus oral (25 points)). the first author (graduate student, resident, intern, post-doctoral scholar, veterinary student, or undergraduate student) will be required to present the abstract during the abstract competition at the therio conference. oral presentations will be 10 minutes in length; an additional two to three minutes will be allowed for questions only. time limits will be strictly enforced for the abstract competition; exceeding the 10‐minute time limit will result in disqualification from the competition. other high‐scoring (15/25; 60%) abstracts not selected for the abstract competition will be chosen for presentation during a non‐ competitive session, and one of the authors will be required to present the abstract at the therio conference during a general session or educational session. presenters of these abstracts will not receive complimentary registration for the conference. if the appropriate author is not available to present the abstract at the therio conference, you may petition the chair of the scientific abstract committee to request permission for an alternative author to present the information. in the case of a competitive abstract for which the submitting graduate student/resident is not available to present the abstract but for which a suitable substitute presenter can be found, the abstract may still be included for presentation during the competitive session but it will not be scored and will not be eligible for awards. 2. non‐competitive category: all individuals, including practitioners, faculty, graduate students, residents, intern, post-doctoral scholars and veterinary students are encouraged to submit abstracts to be considered for presentation during a general session or an educational session (there may be more than one submission per first author in this category). abstracts on material that has already been published or has been accepted for publication at the time of submission will not be considered. presenters in this category will not receive a complimentary registration to the conference. oral presentations will be 10 minutes in length; an additional two to three minutes will be allowed for questions only. 3. case abstract category: all individuals, including practitioners, faculty, graduate students, residents, interns and postdoctoral scholars are encouraged to submit noteworthy case reports for consideration for presentation during a general or educational session. abstracts on material that has already been published or has been accepted for publication at the time of submission will not be considered. case reports will not be considered for inclusion in the competitive category. there may be more than one submission per first author in this category. presenters in this category will not receive a complimentary registration to the conference. 4. poster category: all individuals, including practitioners, faculty, graduate students, residents, interns and postdoctoral scholars are encouraged to submit abstracts/ case reports to be presented as posters. abstracts on material that has already been published or has been accepted for publication at the time of submission will not be considered. there may be more than one submission per first author in this category. presenters in this category will not receive a complimentary registration to the conference. posters are normally displayed in the exhibit hall. you are encouraged to be present as often as possible during exhibit hall breaks to answer questions about your poster. however, all poster presenters are expected to be present during the friday lunch hour when the conference will advertise your presence. abstracts and case abstracts not selected for oral presentation may be given the option to be presented as posters, based on scored ranking. posters will be limited to the 20 highest scoring submissions. general: presenters of research abstracts, posters, and case abstracts will not be reimbursed for travel or other expenses incurred in presenting an abstract, nor will they receive an honorarium. all accepted abstracts will be printed in the annual therio conference proceedings, clinical theriogenology. submission of an abstract for consideration in either the competitive or any noncompetitive sessions is considered a commitment that, should the abstract be accepted in any of the categories, the appropriate author (as described in the “call for abstracts”) will attend the therio conference to present the abstract in a formal oral or poster session. in the event that no suitable person is available to present an accepted abstract or poster at the therio conference, the submitting/presenting author will be barred from submission of scientific abstracts for consideration for presentation at the therio conference and will not be allowed to speak at the therio conference for a period of five (5) years. all co‐ authors will be barred from submission of scientific abstracts for consideration for presentation at the therio conference and will not be allowed to speak at the therio conference for one (1) year. penalized authors have the right to petition the executive board (eb) of the american college of theriogenologists for a reduction or waiver of the penalty. a written petition that includes the reason for the absence from the meeting should be submitted to the chair of the scientific abstract committee no later than one week before the therio conference. the chair and other members of the abstract committee will review the petition and present the case to the eb at the august board meeting. authors will be notified of the decision of the board by email after the conclusion of the therio conference. please note: veterinary student case reports and veterinary student research reports are separate submissions. details for those submissions can be found on the sft (therio.org) website. guidelines: what to send and how to send it electronically: submit via email (address on top of submission form) or on a cd (compatible with microsoft word) general: authors should read the guide for authors for the journal clinical theriogenology. specific instructions for submission of abstracts can be found on the act website (www.theriogenology.org) under “call for abstracts”. font: single spaced, 12-point times new roman length: no longer than one page paper: 8.5” x 11” margins: 1.5” top and bottom – 1” left and right header: the title, authors, and their affiliations must be centered at the top of the page. content: abstracts should state clearly the hypothesis and aims of the project, describe the methods used, and summarize the findings. keywords (up to five) should be listed at the bottom of the page. authors are encouraged to consider the criteria for judging provided on the website. form: you must complete and submit the submission form for your abstract. abstracts not adhering to these guidelines will not be considered for presentation. deadline: abstracts must be received no later than february 1, 2020 for consideration. if you have not received an acknowledgement that we have received your abstract submission by february 5, 2020, please call our office at 334.395.4666. the act/sft abstract submission author agreement form must be signed by all authors. a copy of the completed form must be submitted along with the abstract. the form is available on both the act and sft websites. access the required call for abstracts application form on either the act website at www.theriogenology.org or the sft website at www.therio.org under the 2020 therio conference heading, listed under education and events on both sites. those submitting abstracts for consideration will be notified by march 30, 2020 if their abstract(s) were accepted or declined. 2020 conference preview 2019 therio conference recap steiner-career in excellence 2019 sponsors (2) 2019 exhibitors-sft board noms 2019 auction donations 2019 tf review 2020 bartlett nomination act board noms-sft dues call for 2020 student case presentations final call for 2020 student research presentations final 2020 call for abstracts for ctj clinical theriogenology 2022; 14: 81 editor’s note dear theriogenology friends, ‘a desire accomplished is sweet to the soul.’ for me, it is a personal privilege to bring out yet another issue of our journal. forty colleagues have participated in preparing 6 manuscripts for this issue. it takes intentional collaboration and active participation in bringing a project to fruition. i thank our colleagues who worked on these projects and created manuscripts. mentors and team members of these manuscripts deserve our appreciation. i thank the corresponding authors of these manuscripts who worked with me patiently during the review process, editing, and copyediting. my sincere thanks to 12 colleagues who assisted me in the review process. current issue covers projects in domestic and exotic species. two articles describe anti-müllerian hormone involvement in reproduction. it remains to be an important topic in human infertility. for example, its role in the pathogenesis of polycystic ovary syndrome and disorders of male infertility continues to be studied. further clinical investigations in animals are necessary to understand the role of this hormone in fertility and infertility. to colleagues who have agreed to serve as guest editors for our electronic issues, please give me an update (petera@purdue.edu) as soon as possible. i look forward to publishing our electronic issues. please continue to submit quality and well-written manuscripts. we are not quite there to submit our journal credentials toward pubmed indexing. regards, augustine introduction large ovary syndrome (los) can manifest itself through a variety of clinical signs.1 various ovarian pathologies causing the los have been described with the following overall prevalence of reproductive disorders in cattle: ovarian cysts (oc) 6 19%,2 ovarian hematoma (oh) < 1.5%, ovarian abscess (oa) < 1.0%, oophoritis and perioophoritis (oo) < 1.0%,3 ovarian neoplasms such as granulosa-theca cell tumors (gct) 0.5%,4 ovarian adhesions (oad) 1.5%, developmental anomaly 0.2%, and periovarian tumor (pot) < 0.5%.3 most commonly diagnosed ovarian abnormality by a bovine practitioner is the ovarian follicular cyst (fc). a preliminary diagnosis of the condition is usually based on a transrectal palpation of the ovaries and a history of anestrus.5 only 2% of cows with oc present frequent and irregular signs of estrus with edematous swelling of the vulva and abundant clear mucus discharge, whereas 74% of cows present anestrus as the main sign.6 cows with an ovarian cyst are most often identified at first reproductive system examination in the postpartum period or at the first examination for pregnancy. generally, diagnosis of follicular cysts by transrectal examination is confirmed by ultrasonography. fc appears as a large fluctuating anechoic fluid-filled structure > 25 mm in diameter with a very thin outer wall of < 3 mm and in the absence of a corpus luteum.7 solitary cysts are more common than multiple cysts. in chronic cases, sinking of sacral ligaments, upward displacement of the coccygeal vertebrae, and thickening of the neck and brisket may large ovary syndrome in cattle: a retrospective study ali torabi,a angelika stock,a dario vallejo,b sylvain nichols,a réjean lefebvrea abovine theriogenology, department of clinical sciences, faculty of veterinary medicine university of montreal, st-hyacinthe, qc, canada bdepartment of theriogenology, school of veterinary medicine, faculty of agrarian sciences university of antioquia, medellin, colombia abstract similar to horses, large ovary syndrome in cattle (los) is characterized by an excessive size and deformity of 1 or both ovaries. most often, a bovine practitioner refers these cases to a theriogenologist to confirm diagnosis and provide treatment. objectives were to review the process of diagnosis and to assess factors impacting fertility after removal of the large ovary in cows with los. we present 20 los cases referred to the veterinary teaching hospital of the university of montreal (chuv). data were collected on calving history, cyclicity, previous treatments, results of general and reproductive examinations including transrectal palpation and ultrasonographic evaluation, and histopathological results after ovary removal. twelve los cases (60%) were identified as granulosa-theca cell tumors (gct), 4 (20%) as ovarian abscesses (oa), and 4 (20%) as ovarian hematomas (oh). anestrus was the most noticeable clinical sign reported by owners. on average, the ovarian volume (height x length x width) was 1,945, 1,187, and 524.2 cm3 for gct, oa, and oh respectively. concomitant conditions, such as endometritis and urovagina, had negative impacts on fertility. from these data, we concluded that cows presented with los postpartum warrant a complete reproductive examination. although 95% of the cows demonstrated a good outcome after surgical removal of the large ovary, untreated concomitant reproductive disorders resulted in culling. keywords: ovarian anomaly, infertility, diagnosis, prognosis be observed.8 cows diagnosed with oc have a dysfunctional hypothalamic-pituitary-ovarian feedback loop resulting in a basal peripheral progesterone (p4) concentrations and increased estradiol concentrations especially at the early stages of cystic development. aberrant lh pulse frequency and amplitude translate into an inadequate lh surge necessary for ovulation to occur. studies suggest that in early postpartum, follicular cyst development is associated with disrupted insulin signalling pathway due to a negative energy status of the newly lactating cow.9 various hormonal treatments are routinely used, either gnrh (gonadotropin-releasing hormone) or hcg (human chorionic gonadotrophin) alone or an additional prostaglandin treatment ~ 9 14 days later.10 a synchronization protocol (ovsynch) is frequently used to treat cows with oc. if treatment remains unsuccessful and the ovarian size continues to increase, a more detailed examination of the reproductive tract should be performed to rule out other ovarian anomalies.11 in contrast, oh and oa are uncommon conditions with unclear and ambiguous etiology and less defined clinical manifestations.12 because the animal may still cycle normally, the finding is often unexpected and difficult to diagnose by most veterinarians and supplementary diagnostic procedures should be carried out by a theriogenologist and a histopathologist to make a definitive diagnosis. limited information is available for oh in the literature. in cases of oa, an ascending infection of the uterus travelling via oviduct to the ovary is suspected. clinical theriogenology 2021; 13: 352 some earlier reports considered enucleation of the corpus luteum as a possible cause and more recent studies described follicular aspiration, a technique which has gained increased applications in the last decades, as a major culprit. inflammation of the ovary (oophoritis), including surrounding structures (perioophoritis) are rare conditions in farm animals and may be observed in connection with uterine infection and peritonitis.13 paraovarian cysts occur quite rarely compared to horses and are usually not included in the list of a differential diagnosis in cattle los cases. paraovarian cysts are cystic structures that occur in the broad ligament adjacent to the ovaries and can be mistaken for follicular cysts.14 ultrasonography alone may not be diagnostic for this type of cyst and meticulous transrectal palpation generally provides a better sense of cyst location, type, and mobility. ovarian tumors are rare in cattle and classified into 3 categories: surface epithelial tumors (e.g. cystadenomas and papillomas), sex cord-gonadal stromal tumors (e.g. granulosa-theca cell tumors and fibromas), and germ cell tumors (e.g. teratomas and dysgerminomas).15 sex cord-gonadal stromal tumors are the most common ovarian neoplasm in cattle.16 these are a heterogeneous group of benign and malignant tumors, including tumors composed of cells that produce ovarian hormones like granulosa cells and granulosa-theca cell tumors.17 sex cord-gonadal stromal tumors may produce steroid hormones that result in either nymphomania, mainly at the early stage of the condition followed by anestrus or virilism in long-standing cases.18 these tumors contain mixed areas of specialized gonadal stroma: cortical and medullary stromal cells, granulosa cells (gcs), and follicular theca internal cells. most ovarian tumors are diagnosed as a mass approximately the size of a fist inside the abdominal cavity; however, they can reach > 30 cm in diameter in severe cases.19 in sex cord-gonadal stromal tumors, the contralateral ovary is typically regressed and without follicular activity due to inhibin and amh production by the ovarian granulosa cell tumor.18,20 objectives were to review the process of diagnosis and to assess factors impacting fertility after removal of the large ovary. materials and methods terms for search and file data used for analysis we identified animal admissions with a ‘large ovary’ as the main cause for referral in vetview, (veterinary hospital & laboratory management system, athens, ga) and reviewed bovine hospital logbook and theriogenology service of the faculty of veterinary medicine at the university of montreal (chuv) for the years 2012 2020. all procedures were approved (protocol number ia120633) by comité d’éthique de l’utilisation des animaux of the university of montreal. individual files were thoroughly analyzed and the data compiled. to determine shortand long-term prognosis, we collected follow-up information by phone interviews with owners and referring veterinarians. a positive short-term outcome was defined as a return to normal milk production within 1 week after hospital discharge. a positive long-term outcome was defined as the cow becoming pregnant within 2 months of hospitalization discharge. initial diagnosis of los was made by the referring veterinarian in a routine reproductive visit of the herd. analyzed data included reason for referral, animal description (age, days in milk, and duration of clinical signs), previous treatments on the farm, and case history. description of the population chuv receives referrals from herds in the provinces of quebec and ontario. herds represent mainly holstein dairy cows, with an average of ~ 80 cows/farm in quebec and 100 cows per farm in ontario. clinical examination at chuv a complete physical examination (temperature, heart rate, and respiratory rate) was performed on all cows upon admission. transrectal palpation was performed to determine uterine size and to identify corpus luteum (≥ 20 mm diameter), follicles (≥ 10 mm diameter), and cysts. transrectal ultrasonography was performed with a 7.5 mhz linear probe (ibex, e.i. medical imaging, loveland, co). vulva was cleaned, dried, and wiped with an alcohol gauze and a disposable vaginoscope moistened with a minimal amount of sterile lubricant was inserted into the vagina. with a light source, the vaginal vault and the cervix were examined visually for discharge that was categorized using a 4-point classification system: 0 = no discharge, 1 = clear and translucent mucus, 2 = cloudy mucus with or without flecks of pus (< 50% pus), 3 = mucopurulent discharge (> 50% pus), and 4 = purulent discharge.21 clinical endometritis was defined as a purulent vaginal discharge (score of > 2) after 21 days postpartum.22 cytological endometritis or subclinical endometritis was defined based on > 5% neutrophils on endometrial cytology without vaginal discharge after 21 days postpartum.23 cervix was classified as grade 0 (normal) without abnormality; grade 1 (normal) with the second cervical fold swollen without redness and prolapsing through the first ring, and grade 2 with the second fold being swollen, red and prolapsing through the first ring without purulent vaginal discharge.23 clinical cervicitis was defined as a cervical grade of 2. sampling for uterine cytology and bacteriology cytobrush technique was used to collect samples of mucosal cells from the uterus to diagnose a possible subclinical endometritis. briefly, cytobrush samples were collected using a modified cassou gun. double-protected cassou containing the cytobrush was inserted in the vagina and placed in front of the external cervical ring, the double-pipette perforated and the cassou gun containing the cytobrush was passed through the cervix. once the uterine body was reached, the cytobrush was exposed and rolled over the mucosa to collect cells and then withdrawn into the cassou gun clinical theriogenology 2021; 13: 353 before removing it back out through the cervix and vagina in the double-pipette. cytobrush was then rolled on a sterile glass slide, air-dried, and stained. smears were examined under the microscope at 400 x magnification, and a total of 200 cells were counted and characterized. percentage of polymorphonuclear neutrophils (pmns) was determined for each cow. after rolling the cytobrush on a sterile slide, it was cut and packaged in a sterile plastic tube and transported to the diagnostic bacteriology laboratory of the faculty of the veterinary medicine. in the laboratory, bacteriological samples were cultured for aerobic and anaerobic bacteria using standard methods for bacteriological testing (api system: biomerieux, marcy-etoile, france). for microbiological analysis, the brushes were plated onto sheep blood agar with a sterile disposable plastic loop (soy agar with 5% sheep blood, becton dickinson, franklin lakes, nj). plates were incubated for 48 hours at 35°c under aerobic conditions and then examined. when growth was observed, colony types were identified based on morphology, pigmentation, and hemolytic patterns. organisms were further subcultured and identified using biochemical profiles and other standard methods. plates containing > 1 colony-forming units were considered positive for bacterial growth.24 blood sampling and analysis all laboratory tests were performed by the chuv diagnostic laboratory. blood was collected via venipuncture of the coccygeal vein to assess p4 and a complete blood panel was performed. hemiovariectomy via flank laparotomy feed was withheld for 36 hours prior to surgery. antibiotic (procaine penicillin g 22,000 iu/kg every 24 hours) was given preoperatively to all cows and for at least 72 hours after surgery. surgeries were performed in standing position by an ipsilateral caudal flank approach under a proximal paravertebral anesthesia of the last thoracic and the first 2 lumbar spinal nerves or by a line block at the incision line with 2% lidocaine (vetoquinol n.-a. inc., lavaltrie, qc, canada). after aseptically preparing the surgical site, a vertical incision below and cranial to the tuber coxae was performed. external oblique abdominal muscles were incised and the internal abdominal and transversus abdominis muscles were bluntly dissected before incising the peritoneum. ovarian pedicle was double ligated with 2 braided glycolide/ lactide copolymer (polysorb, covidien, mansfield, ma) before sharp transection. in some cases, the transection was performed using a ligature (covidien). peritoneum and transversus abdominis muscle, and the internal and external oblique abdominal muscles, were sutured together using 2 braided glycolide/ lactide copolymer (polysorb, covidien) in a simple continuous pattern. skin was closed with 2 polycaprolactam (supramid, serag-wiessner, naila, germany) in a ford interlocking pattern. hematocrit was measured immediately prior to and 24 hours after surgery to assess the risk of internal bleeding. owners were instructed to remove skin sutures ~ 14 days after surgery and 3 weeks before attempting artificial insemination. macroscopic and histopathological examinations representative samples of the ovary were preserved in 10% formalin for 24 hours before being dehydrated in alcohol and embedded in paraffin. thin tissue sections (4 μm) were made and placed onto slides for hematoxylin and eosin staining. after a general assessment, the pathologist randomly chose 5 areas under 400 x magnification for each slide before undertaking a complete evaluation. results animals a total of 24 cases of los were identified. four cases had to be excluded from the study because of incomplete medical history. of these 20 cases, 12 were diagnosed with gct, 4 with oa, and 4 with oh (table). all animals were holstein cattle with an average age of 3.5 years (6 months to 4.5 years). one animal was a heifer and the others were milking cows ranging from 90 to 240 days in milk (dim), with a median dim of 145 days. in total, 75% (3/4) of oh cases were considered to be in anestrus and 25% (1/4) were repeat breeder cases. in oa cases, 50% of animals were in anestrus and repeat breeders, respectively. however, in ovarian gct, 42% (5/12) were in anestrus, 50% (6/12) were presented as a repeat breeder and 8% (1/12) were nymphomanic at admission. approximately 95% of cows had a normal calving history. california mastitis test (cmt) and the leukosis test were negative for all cases, with the exception of 1 leukosis positive cow in the oa group. none of the cows had signs of systemic disease, such as fever, anorexia, nor lethargy. all animals were alert upon admission and in good body condition (3.0 3.75). heart and respiratory rates were within normal limits. the median length of days in milk (dim) for the oh, oa, and gct groups were similar: 4 months (3 6 months), 4.5 (4 5 months) and 4 months (3 8 months), respectively. reproductive system examination percentage of cows with an enlarged ovary on the left side was 50, 50, and 75, for the oh, oa, and gct groups, respectively (table). in all cases, the contralateral ovary was static without active structures. the uterus was < 3 cm diameter and symmetrical without adhesions, via transrectal ultrasonography. vaginoscopy examination via a speculum revealed a normal vagina with the exception of 1 oh cow with urovagina and pneumovagina, 1 oa cow with cervicitis25 and 2 gct cows with an urovagina and a pneumovagina, respectively (table). average size of the enlarged ovary for the oh, oa, and gct groups were 10 x 5 x 4 cm, 8 x 10 x 4 cm, and 12 x 9 x 7 cm, respectively. structures such as corpus luteum or dominant follicles could not be palpated on the contralateral ovary in cows with gct and no abnormal lymph nodes were identified. clinical theriogenology 2021; 13: 354 echogenicity of the oa varied from anechoic to hyperechoic and homogenous to heterogenous, and was surrounded or not by a capsule (thick wall). more circumscribed and mature was the core of the abscess, the more hyperechoic it became and more evident was the surrounding anechoic or hypoechoic halo and acoustic enhancements (figure 1c). in the case of a homogenous anechoic content, the thickness of the wall may be a distinctive characteristic compared to oh (figure 1b) and gct (figure 1d). abscesses are occasionally partitioned by echogenic bands, making differentiation from gct difficult. presence of adhesions around the ovary (oviduct, fimbriae, and surrounding tissues) on transrectal palpation should be expected in cases of oa because of localized inflammation. ultrasonographically, gct had multiple hypoechoic and hyperechoic regions and, unlike normal follicles that are distributed in the periphery of the ovary, hypoechoic fluid-like structures of varying sizes were distributed throughout the whole ovary and presented angular borders. some gct ovaries had a honeycomb appearance and others only few fluid structures and a more echogenic ovarian texture. surgical findings except for the oa cases, no adhesions were observed on the reproductive tract. in the 4 cases of oa, minor adhesions between the ipsilateral uterine horn, the rectum, and the abdominal wall were noticed. however, there was several pus-filled structures in the ovarian parenchyma that each one was encapsulated by a thick fibrotic capsule and in some case some pus was oozing during excision and exteriorization of the affected ovary (figure 2c). in gross examination, making a differential diagnosis between oh and gcts is not always feasible since in both cases the ovary looked like larger, more firm, lobulated and more spherical. it was only after dissection of the ovary following excision that this differentiation could be assessed. in oh, the presence of a large, single, blood-filled cavity in the center of the ovary that was surrounded by a thick capsule and also embedded by the ovarian echotexture (figure 2b) was distinguishable from that of gcts in which the surface of the ovary resembled more lobulated and more firm (figure 2d). after removal and dissection of the ovary in ohs fresh blood gushed out of the cavity. however, in gcts, depending on the cellularity type, the fluid inside the cavities had lower quantity although it was less sanguine. no evidence of uterine abnormalities was observed in any of the above cases. in most cases, the large ovary could not be exteriorized before excision complicating the hemostasis of large vessels of the mesovarium. laboratory analyses hematology and biochemistry analyses revealed mild changes in all groups, with a mild neutrophilia and lymphopenia. mean and range of serum p4 concentrations were 1.5 ng/ml (1.4 -1.7) for oh, 1.6 ng/ml (1.3 1.8) for oa, and 1.2 ng/ml (0.2 3.4) for gct respectively. trueperella pyogenes was isolated from the abscess of all cows with oa (no bacterial culture sample was ovarian anomaly ovarian hematoma ovarian abscess granulosa cell tumor number of cases 4 4 12 age (years) 4.3 2-4 0.5 4.5 reproductive signs 3 anestrus, 1 repeat breeder (urovagina) 2 anestrus, 2 repeat breeders 5 anestrus, 6 repeat breeders, 1 nymphomaniac calving history normal, 3 6 months postpartum 3 normal, 1 dystocia, 4 5 months postpartum normal, 3 8 months postpartum cmt normal normal normal leukosis test negative positive for 1 case negative transrectal examination 4 large left ovary (lo), well involuted uterus 2 large lo, 2 large right ovary (ro) 9 large lo and 3 large ro vaginal examination 3 normal, 1 pneumovagina and urovagina with no discharge 3 normal, 1 cervicitis with no discharge 10 normal, 1 urovagina and 1 pneumovagina transrectal ovarian echography 2 ro: static, lo: 10 x 5 x 4 cm 2 lo: static, ro: 9 x 5 x 4 cm 2 ro: static, lo: 7 x 10 x 4 cm 2 lo: static, ro: 8 x 11 x 3 cm 9 ro: static, lo: 12 x 8 x 5 cm 3 lo: static, ro: 10 x 7 x 5 cm hormonal assay (range) p4: 1.4 1.7 ng/ml p4: 1.3 1.8 ng/ml p4: 0.2 3.4 ng/ml biopsy a large proportion of the ovarian parenchyma is occupied by large coalescences of hemorrhage and necrosis no ovarian structure, strongly infiltrated fibrous tissue of lymphocytes, plasma cells, macrophages and a smaller number of neutrophils microand macro-follicular structures arranged in anarchic form in an abundant cellular stroma; anisocytosis and anisokaryosis are moderate bacterial culture no ovarian sample taken trueperella pyogenes (4 cases) no ovarian sample taken table. ovarian anomalies associated with large ovary syndrome and their characteristics clinical theriogenology 2021; 13: 355 taken from oh and gct groups). endometrial cytology results were within the normal range (< 5% neutrophils). histologically, a large proportion of oh cases had coalescence of hemorrhage and necrosis infiltrated by inflammatory cells. in oa and oh cases, there was scant ovarian tissue. in oa cases, the ovarian stroma was replaced by collagenous tissue infiltrated by lymphocytes, plasma cells, and neutrophils. in gct cases, the ovaries presented cell density of round or polygonal cells, often organized in superimposed and disorganized cellular layers forming a multitude of irregular lobules of variable sizes, and infiltrated with inflammatory cells. these irregular lobules resembled follicles with varying degrees of anisocytosis and anisokaryosis. in some cases, the cell infiltration was not capsulated. the round cells (granulosa cells) had formed micro and macro-follicular structures arranged in anarchic form in an figure 1. differential diagnosis of left ovary based on transrectal ultrasonography. a. follicular cyst; usually smaller than other anomalies and hypoechoic, b. ovarian hematoma; hemorrhagic cavity filled with heterogenous echogenic liquid containing fibrin materials, c. ovarian abscess; multiple hyperechoic structures within the ovarian context, and d. granulosa-theca cell tumor; very large multicompartmental and lobular structures throughout the ovary. figure 2. differential diagnosis of left ovary based on gross examination of 4 large ovaries after ovariectomy. a. follicular cyst; large fluid-filled structure at the surface of the ovary, b; ovarian hematoma; large red and hemorrhagic structure at the surface of the ovary, c. ovarian abscess; multiple encapsulated and suppurative structures localized to different parts of the ovarian tissue, and d. granulosatheca cell tumor; multilobular and multifollicular shape on the ovarian surface. clinical theriogenology 2021; 13: 356 abundant cellular stroma, showing anisocytosis, anisokaryosis, multiple nucleoli. rate of differentiation was high, and no necrosis was observed in histologic sections, an indication of benign tumors. mitotic index for 11 of 12 samples was below 10 mitoses per 10 high-power fields. one sample had a mitotic index above 19 mitoses per 10 high-power fields. shortand long-term outcomes all cows had a good short-term prognosis, returning to normal production within 1 week after being released from the hospital. as expected, the priority of farmers was to have these cows return to normal fertility. all cows became pregnant within 2 months after being released from the hospital (19/20 pregnant), with the exception of 1 cow with urovagina and pneumovagina (oh). in this particular cow, urovagina and pneumovagina were not treated during hospitalization. discussion based on our data, los occurred in 0.5% of dairy cow referrals to the chuv (24 of 4,500 total cases). in total, 60% of these cases of los were associated with gct, for an incidence rate of 0.03% per year. a similar incidence of ovarian tumors was obtained with abattoir-derived material (0.7%, 11/1,480 cases).26 although ovarian tumors have been reported as more prevalent in animals over the age of 11 years, animals of all ages can be affected.27 the dairy industry is currently culling cows on average in the third lactation and only 40% of culled cows have 3 lactations or more (canadian dairy information center, cdic). mean age of cows in the present study was 3 years (median: 3.5 years) and the mean number of lactations was 2. of the 12 cows with gct, there was only 1 heifer, 6-months old, without mammary gland development. none of the cows was pregnant at the time of the los diagnosis, although gct has been reported for various parities, pregnant and nonpregnant cows.28 such reported cases in pregnant cows reflected most likely an early form of the disease in which the tumor was small and not producing sufficient quantities of hormones to interfere with the normal reproductive cycle of the affected cow. in the present study, gct occurred on the left ovary in 75% of cases (9 of 12); however, it can occur on either side.29 incidence of oa and oh was < 0.1% (8/4,500). surveys of reproductive tract examinations of cows at abattoirs had very low incidences: 0 cases in a study of 8071 cows and a single case in a study of 1,300 cows.30 given the substantial increase in the use of new biotechnology such as follicular aspiration for in vitro fertilization or follicular wave synchronization, one would have expected a greater incidence of these conditions.³¹ most of the cases in the present study were diagnosed during a routine reproductive health visit, at a median dim of 145 days that represents a time in lactation when > 50% of cows in the herd should have been diagnosed pregnant.³² late diagnosis of los may be due to the use of systematic synchronization protocols as reproductive management tool associated with the absence of a complete reproductive examination in early postpartum (30 40 dim). no clear conclusion could be drawn from the present data. a late presumptive diagnosis of los can have negative consequences for the affected cow and the herd because finding the condition in late lactation increases the risk of culling. an early diagnosis would increase the chance of a quicker return to production and pregnancy. in addition, an earlier diagnosis offers an easier approach of hemiovariectomy via colpotomy, because of the smaller size of the affected ovary in early development of the condition. however, a smaller ovary would complicate the differential diagnosis. success of surgical approach for removal of a large ovary depends on the size and the location of the ovary. in order to safely remove the enlarged ovary in the case of oh and gct by colpotomy, the ovary should be < 8 10 cm in diameter (i.e. can fit in the palm of the hand). nonetheless, removal of large gct, oh, and oa via colpotomy can increase the risk of hemorrhage (oh, gct) and peritonitis (oa).³³ however, colpotomy can potentially be performed at the farm with less expense and sanitary risk. once the primary complaint, such as anestrus, or less frequently, nymphomania is presented to the veterinary practitioner, the most important step of the initial examination to be performed is transrectal palpation and ultrasonographic examination of the ovaries. in the case of los, a very systematic approach to assess the form, size, localization, consistency, mobility, and ultrasonographic appearance of the ovaries is imperative. presence of a large, single, circular, fluctuating, and anechoic fluid-filled structure ~ 25 mm in diameter with a very thin outer wall (figure 1a) in a normal size ovary allows a presumptive diagnosis of ovarian fc. repeated examinations, in the absence of a response to the common treatment of ovarian cysts, or the presence of an oversize ovary should suggest a more meticulous and exhaustive differential diagnosis examination. determining the size of the abnormal ovary is very important for the differential diagnosis. for oh, oa, and gct, the ovaries were > 8 cm long. in contrast to cattle, most of the ovaries with gct in human remain within normal size.34 in the present study, gct volume was more prominent than that of the oh or oa. with the increase of the ovarian mass, tissue metabolism and vascularization become more important. evaluation of the blood supply around the ovary is crucial in deciding on the surgical approach to avoid the risk of fatal hemorrhage. the presence of adhesions, in the case of oa, may help in the differential diagnosis but again will force the surgeon to a cautious, meticulous, and adopt a stepwise surgical approach. abscess may to be attached to surrounding structures, such as the rectum, the intestinal tract, or the uterine horn, reducing the ovary’s mobility. whereas this is an important observation for prognosis and surgical management, adhesions to the rectum are sometime difficult to assess by transrectal palpation. as a result of variation in the ultrasonographic appearance of los anomalies, ultrasonography alone cannot guarantee an accurate diagnosis, although it is still an important technique for clinical theriogenology 2021; 13: 357 establishing the right diagnosis. in humans with a larger variety and number of ovarian tumors, ultrasonography has high sensitivity (84 89%) and specificity (98.2%), but low positive predictive value (35.3%).35 ultrasonography allows the clinician to rule out additional anomalies in other organs or tissues, e.g. iliac and iliofemoral lymph nodes, the cervix, and the uterus with a higher degree of certainty. evidence of some follicular activity in a fairly normal sized contralateral ovary (static) on ultrasonography help differentiate oa and oh from gct in which contralateral ovary is atrophied. in the present cases, short-time hospitalization made the assessment of the follicular function difficult. unfortunately, there is no clear and definitive ultrasonographic pattern of gct. in mares, for example, its appearance varies from enlarged multicystic structures to a solid mass or single large fluid-filled cyst.36 sometimes, the affected ovary may have the typical honeycomb appearance on ultrasonography. thus, the ultrasonographic appearance of gct can be confusing with other types of ovarian tumors and los conditions. in contrast to manual transrectal palpation and ultrasonography of the ovary, hematological and biochemical analysis were unreliable for diagnosing los, even in the case of oa. our hematological results showed a simple pattern of stress, and the changes on the biochemistry panel were not significant. however, the hemato-biochemistry profiles of affected animals are still important for revealing underlying diseases that are not discovered during physical examination but could have an impact on the surgical management and prognosis as well as long-term reproductive efficiency. in the present study, all cases were negative for leukosis except for 1 animal with oa. however, studies in cattle had no clinical association between bovine leukosis virus infection and the incidence of the ovarian tumors or other conditions.37 hormonal tests could be useful diagnostic tools in the differential diagnosis of los and, more specifically, in cases of hormone-secreting tumours like gct. biomarker like amh for bovine gct diagnosis has the highest diagnostic accuracy and sensitivity. an amh concentration of ≥ 0.36 ng/ml had a sensitivity of 100% and specificity of 99.1%. the literature mentions that elevated serum inhibin (> 0.69 ng/ml) is a marker for ovarian gct. this finding supports the hypothesis that the secretion of follicle-stimulating hormone (fsh) from the anterior pituitary is suppressed, probably due to the overproduction of inhibin and androgens by neoplastic cells.38 similar studies in the mare had elevated serum inhibin and testosterone concentrations in 87 and 54%, respectively. like in the mare, sensitivity of amh for detecting gct was significantly higher than inhibin or testosterone. an amh profile could also be part of patient monitoring for disease remittance after removal of gct. after hemiovariectomy, amh declined rapidly to normal concentrations within 1 week postsurgery.39 in the present study, no amh measurement were performed to confirm the final diagnosis of gct before and after surgical removal of the los because of cost and time lag for the results. in the case where a gct is suspected in a smaller size ovary, assessing the amh would be an essential diagnostic tool. in cases of oa and oh, it is expected to have in normal physiological concentrations of amh. finally, histopathology remains the main definitive diagnostic test for los and, more specifically, for ovarian tumors with the highest specificity (99.8%). oh is more common in mares, and it can be differentiated from gct based on physical examination and profiles of hormones like amh.40 all los diagnostics of the present report were confirmed by histopathology. the mitotic index for all but 1 case of gct indicated no malignancy; however, malignancy does not necessarily indicate the invasive nature of the gct in the cow. histology of oh revealed that a large proportion of ovarian parenchyma samples were occupied by several large haematoma and necrotic coalescence, accompanied by extensive exudation of fibrin, red blood cells and degenerate neutrophils. conclusion in general, los remains a rare anomaly. large size ovary is more likely to be associated with gct than with oa or oh. meticulous transrectal palpation and ultrasonographic examination are essential to diagnose los in order to assess the form, size, localization, consistency, mobility, and the appearance of the ovaries. although the ultrasonographic appearance of los varies, ultrasonography still represents a very important diagnostic tool for ruling out and characterizing other anomalies of the reproductive tract. the amh assessment is a good tool to confirm the diagnosis of gct but the cost and time lag for the results remains a major concern. however, with a suspicion of gct with a small ovary on farms, assessment of amh concentration is recommended. hemiovariectomy of a large ovary (gct, oh, or oa) has a very good shortand long-term 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mare: a review of 52 cases. equine vet educ 2016;28:75-82. 37. el‐sheikh ali h, kitahara g, nibe k, et al: endocrinological characterization of an ovarian sex cord–stromal tumor with a sertoli cell pattern in a japanese black cow. reprod domest anim 2019;54:1501-1504. 38. ali he-s, kitahara g, nibe k, et al: plasma anti-müllerian hormone as a biomarker for bovine granulosa-theca cell tumors: comparison with immunoreactive inhibin and ovarian steroid concentrations. theriogenology 2013;80:940-949. 39. patrick dj, kiupel m, gerber v, et al: malignant granulosa-theca cell tumor in a two-year-old miniature horse. j vet diagn 2003;15:60-63. 40. curtin dje: ovarian hematoma in an 11-year-old thoroughbredhanovarian mare. can vet j 2003;44:589-591. clinical theriogenology 2021; 13: 359 introduction spermatogenesis occurs in seminiferous tubules of testes that produce millions of sperm daily.1 the foundation of this process is formed by activities of an undifferentiated spermatogonial population that contains a stem cell pool and resides at the basement membrane of seminiferous tubules.2 a foundational pool of spermatogonial stem cells (sscs) is established during neonatal development and sustained via self-renewal in adulthood.3,4 out of the ssc pool, transit amplifying progenitor spermatogonia arise that undergo a series of mitotic cell divisions to bolster the population prior to initiation of a differentiating transition in response to a pulse of retinoic acid.5 differentiating spermatogonia undergo further mitotic amplifying divisions while gaining competency for initiation of meiosis as preleptotene spermatocytes and progressively migrating through the seminiferous epithelium towards tubule lumen.5 after 2 meiotic divisions (primary and secondary spermatocytes) haploid spermatids are formed that undergo morphogenesis to form elongate spermatids and are subsequently released into seminiferous tubules. sertoli cells, the only somatic cell type in the seminiferous epithelium, nurture the entire spermatogenic process. interstitial tissue of testicular parenchyma that is present among seminiferous tubules includes leydig cells, macrophages, and blood vessels. leydig cells are the source of testicular androgens which are required for spermatogenesis via signaling within sertoli cells.6 outside seminiferous tubules, peritubular myoid cells are positioned to provide single cell transcriptomics to define germ cell function in livestock jon m. oatley, colton robbins, michela ciccarelli school of molecular bioscience, college of veterinary medicine, washington state university, pullman, wa abstract spermatogenesis is a specialized developmental process that produces millions of sperm each day from puberty to old age. crosstalk among testicular germ and somatic cells provides required direction for homeostasis of spermatogenesis and regeneration after an insult. understanding spermatogenesis and the importance of sperm are critical. sperm contribute genetic information that influences the traits of offspring, thus shaping performance, resiliency, and fitness of animal populations. recent emergence of single cell rna sequencing analysis (transcriptomics) opened a new avenue to explore reproductive biology by defining heterogeneity and crosstalk within complex cell lineages (e.g. male germline). new insights in the regulation of developmental processes at a molecular level provides invaluable information for researchers and clinicians to address underlying causes of infertility/subfertility and design novel advanced reproductive technologies. keywords: single cell rna sequencing, livestock, spermatogenesis, testis structural support and potentially contribute signals to the spermatogonial niche microenvironment. an orchestrated crosstalk among the testicular soma of leydig, myoid, and sertoli cells with spermatogenic germ cells is required for continuous and robust steady-state spermatogenesis, and for regeneration of spermatogenesis following cytotoxic insult. development of the spermatogenic lineage begins with primordial germ cells (pgcs) that arise from the inner lining of yolk sac during the pregastrulation embryonic period.7 after specification, pgcs undergo migration to genital ridges where they coalesce with somatic cells to form undifferentiated gonads. at this point, all cells populating genital ridges have the bipotential capacity to differentiate into either testicular or ovarian tissue. a delicate interplay of molecular signaling occurs within somatic cells of xx and xy embryos to drive the generation of either presertoli or pregranulosa populations that will form fetal testis or ovary, respectively.8 in xy embryos, expression of the y chromosome gene sry in cells migrating from the mesonephric mesenchyme to genital ridge leads to development of presertoli cells that create interdigitations around prospermatogonial germ cells to form seminiferous cords. the prospermatogonial population is the fetal precursor to the spermatogenic lineage and arises from pgcs at sex determination. during late fetal period, the prospermatogonial population undergoes waves of proliferation and apoptosis to set a developmentally pristine population that will enter a period of mitotic arrest.3,9 prospermatogonia remain in clinical theriogenology 2021; 13: 127 quiescence until after birth when reentry to the cell cycle occurs over a period that parallels transition to postnatal spermatogonial states.3 coincident with morphogenesis of seminiferous cords, interstitial compartment, connective tissues, blood vessels, and leydig cells also arise during fetal development. although origins of fetal leydig cell population are still not fully understood, it is their production of testosterone that drives development of wolffian ducts and masculinization of external genitalia. all of these embryonic and fetal events occur in orchestration to establish a male gonad that, at birth, is developmentally primed to support proper establishment of a continually cycling spermatogenic lineage during the prepubertal period. overview of single cell rna sequencing technology the ability to quantify rnas within cells on a large scale has been game changing for the field of biosciences for more than 2 decades. beginning with microarray analysis that profiled rnas of bulk populations of cells and evolving to single cell rna profiling, the transcriptome of specific cell populations that make up tissues during different developmental time points and circumstances can be defined in a precise way. a limitation of bulk rna profiling approaches has been an inability to uncover heterogeneity of what is seemingly homogenous populations and difficulty in parsing out transcriptomes of distinct cell populations that comprise a complex tissue. single cell rna sequencing (scrna-seq) was developed to overcome these limitations and provides scientists with a tool to define genes being expressed in individual cells that comprise a lineage or tissue. outcomes of these studies are leading to groundbreaking discoveries of new cellular states, uncovering of novel molecular mechanisms governing those states, and deepening our understanding disease etiology. a general workflow of scrna-seq analysis is presented (figure 1a) and key steps include: 1. generation of a single cell suspension via disassociation of a tissue using mechanical or enzymatic means. 2. emulsification of single cell suspension to suspend individual cells into droplets. 3. lysis of individual cells to liberate mrnas. 4. ligation of adapters and reverse transcription to create a cdna library that carries a molecular barcode that is unique for each cell. 5. sequencing of cdna libraries. a raw data matrix is created in which genes constitute row barcodes, samples constitute column barcodes, and values within the matrix are read counts representing the expression of a particular gene in an individual figure 1. overview of single cell rna-sequencing workflow: a. schematic of mrna collection, cdna amplification, barcoding and demultiplexing of reads for single cells. b. examples of outputs from computational analysis of single cell rna-sequencing data. clinical theriogenology 2021; 13: 128 sample. raw data are then processed for normalization and quality control. normalization provides scaling of counts to remove technical variations and determine gene expression based on mrna/cdna abundance. quality control measures allow for elimination of libraries derived from nonviable cells. 6. normalized and quality-controlled data are then analyzed using computational and bioinformatic programs such as cell ranger, seurat, and monocle. outputs of these analyses include clustering of cells into populations based on similarities in gene expression profiles, characterizations of cell clusters based on biomarkers or differentially expressed genes, and generation of fate trajectory maps to understand how cells advance along a developmental path from undifferentiated to terminally differentiated states (figure 1b). use of single cell rna sequencing technology to deepen the understanding of male germ cell biology and testicular function over the last few years, scrna-seq technology was used to generate advances in understanding of molecular pathways that control spermatogenesis and testis development in a variety of mammalian species.10-14 through comparison of single cell gene expression profiles of total testicular and germ cell populations from adult mice, monkeys, and humans, conserved and divergent pathways that govern sperm generation have been uncovered.14 interestingly, the outcomes of these studies have begun to reveal previously undescribed gene expression characteristics for specific cell types of spermatogenic lineage and testicular support cell populations. these observations suggest that subsets of cells with different functionalities may exist in what have historically been regarded as homogenous populations with similar biological properties. from a human health perspective, understanding the causes of fertility disorders and devising treatment strategies for them may be advanced by comparing single cell gene expression profiles among germ cells and testicular somatic cells of mice, monkeys, and men. additionally, the integration of information being generated from germ cells of domestic animal species is starting to uncover novel regulators of germ cell function that are conserved across mammalia.15 for livestock, utility of single cell gene expression profiling is in the discovery of new pathways regulating spermatogenesis that can be exploited for developing and refining advanced/assisted reproductive technologies. recently, we generated scrna-seq profiles for testicular tissue of prepubertal bulls and boars, including fetal and perinatal male piglets, and integrated this information with mouse and human profiles to compile a comprehensive comparative species database (unpublished). ongoing exploration of this extensive database is yielding new information about molecules expressed by germ cells that are conserved across species. importantly, we have identified previously undescribed cells surface molecules and transcription factors that are expressed by germ cells and/or testicular somatic support cells from livestock to man. currently, we are using these advances in knowledge to devise strategies for purification of germ cells from testicular tissue and optimize conditions to grow livestock spermatogonia in vitro. these strategies are being matched with advanced reproductive technologies such as spermatogonial stem cell transplantation to bolster the utility as a breeding tool in livestock production. conflict of interest jmo is the founder of apys biotechnology, llc, an animal biotechnology company specializing in the commercialization of advanced reproductive technologies and gene editing applications in livestock references 1. johnson l, petty cs, neaves wb: a comparative study of daily sperm production and testicular composition in humans and rats. biol reprod 1980;22:1233-1243. 2. oatley jm, brinster rl: regulation of spermatogonial stem cell self-renewal in mammals. annu rev cell dev biol 2008;24:263-286. 3. law nc, oatley mj, oatley jm: developmental kinetics and transcriptome dynamics of stem cell specification in the spermatogenic lineage. nat commun 2019;10:2787. 4. de rooij dg russell ld: all you wanted to know about spermatogonia but were afraid to ask. j androl 2000;21:776-798. 5. griswold md: spermatogenesis: the commitment to meiosis. physiol rev 2016;96:1-17. 6. willems a, roesl c, mitchell rt, et al: sertoli cell androgen receptor signalling in adulthood is essential for post-meiotic germ cell development. mol reprod dev 2015;82:626-627. 7. clark jm, eddy em: fine structural observations on the origin and associations of primordial germ cells of the mouse. dev biol 1975;47:136-155. 8. spiller c, koopman p, bowles j: sex determination in the mammalian germline. annu rev genet 2017;51:265-285. 9. hamer g, de rooij gd: mutations causing specific arrests in the development of mouse primordial germ cells and gonocytes. biol reprod 2018;99:75-86. 10. guo j, grow ej, yi c, et al: chromatin and single-cell rna-seq profiling reveal dynamic signaling and metabolic transitions during human spermatogonial stem cell development. cell stem cell 2017;21:533-546. 11. guo j, sosa e, chitiashvili t, et al: single-cell analysis of the developing human testis reveals somatic niche cell specification and fetal germline stem cell establishment. cell stem cell 2021;28:764-778. 12. guo j, grow ej, mlcochova h, et al: the adult human testis transcriptional cell atlas. cell res 2018;28:1141-1157. 13. hermann bp, chen k, singh k, et al: the mammalian spermatogenesis single-cell transcriptome, from spermatogonial stem cells to spermatids. cell rep 2018;25:1650-1667. 14. shami an, zheng x, munyoki sk, et al: single-cell rna sequencing of human, macaque, and mouse testes uncovers conserved and divergent features of mammalian spermatogenesis. dev cell 2020;54:529-547. 15. soto da, ross pj: similarities between bovine and human germline development revealed by single-cell rna sequencing. reproduction 2021;161:239-253. clinical theriogenology 2021; 13: 129 single cell transcriptomics to define germ cellfunction in livestock 29 contact john cavalieri john.cavalieri@jcu.edu.au © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9593, http://dx.doi.org/10.58292/ct.v15.9593 review report guidelines for the use and reuse of animals for teaching within veterinary medical education programs john cavalieri,a brad dowling,a leo foyle,a craig godfrey,b janice lloyd,a josephine penny,a william tranter,a sally watts,a jamie wearn,c graham willisd acollege of public health, medical and veterinary sciences, james cook university, townsville, qld, australia, bresearch and innovation services, james cook university, townsville, qld, australia, cu-vet werribee equine hospital, the university of melbourne, werribee, vic, australia, dpowermation, brisbane, qld, australia abstract use and reuse of animals for educational purposes could adversely affect animal welfare. guidelines for quantifying, monitoring, and planning the use and reuse of animals have been developed. within this framework, animals are assigned points for usage, with more points being allocated to procedures that may have a greater adverse effect on animal welfare. usage of individual animals is limited to a maximum of 8 points in a calendar week, 24 points in a month, or 60 points within a 16-week study period and any associated examination period. advantages and disadvantages of the system are discussed, while modifications are expected as knowledge emerges on the impacts of procedures on animal welfare. keywords: animal welfare, ethics, education, veterinary science, domestic animals, fish introduction education in veterinary science is reliant on the use of animals for students to develop a sufficient level of competence in a range of clinical and diagnostic procedures and an ability to maintain a safe work environment for them and others who may be in proximity. in the last 10 years, there has been a worldwide increase in the number of students enrolling in veterinary degrees to meet a combination of workforce demands and high demand for student enrolments. the united states (us) bureau of labour and statistics in 2021 projected that the number of people employed as veterinarians between 2020 and 2030 will increase by 16.8%.1 the number of students enrolled in us and australian colleges of veterinary medicine has more than doubled since 1980 with a steady average increase in enrolment per year in the us between 2000 and 2020 by about 3%.2 in australia, between 2007 and 2013, there was a 61% increase in the number of students who commenced veterinary degrees.3 this increase in student enrolments could increase the usage of individual animals for educational purposes that could reduce welfare outcomes for animals, particularly where there are budgetary constraints or limitations on animal numbers resulting in individual animals being used more often. given the need to maintain animal welfare and to satisfy increasing community concerns for the usage and quality of care of animals, it is imperative that educational institutions operate within frameworks that maintain appropriate standards of animal welfare.4 increasing use of alternatives to animal use through the use of models, mannequins, simulators, multimedia software, virtual reality, and cadavers has reduced the reliance on live animals.5,6 a systematic review of humane alternatives to the use of animals for education within life and health sciences identified that humane methods of teaching resulted in learning outcomes superior (30%), equivalent (60%), or inferior (10%) to those produced by traditional, more harmful methods of animal use.6 simulations can better prepare students for carrying out procedures on animals in some situations but not in every circumstance, which, therefore, requires educators to continue to use animals for educational purposes.6,7 when the use of animals is necessary for educational purposes, a tiered approach to training is advocated where students progress from using non-animal simulators to cadavers and finally live animals if necessary.7,8 simulations of varying fidelity have been used, for example, to train students in venipuncture, animal handling, intubation, bandaging, surgical and reproductive procedures, mailto:john.cavalieri@jcu.edu.au http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9593 30 citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9593 obstetrics, and imaging. the use of animals for teaching purposes, however, provides several educational advantages that cannot always be achieved through simulation.4,9–12 use of animals provides authentic learning opportunities directly mimicking procedures and scenarios that students will encounter in clinical practices. in some instances, the use of animals compared to alternatives for educational purposes was more appropriate for teaching complex, multifaceted skills, and it may increase the depth of understanding and retention of knowledge.4 use of animals offers kinesthetic learning experiences and provides valuable opportunities for learners to rapidly assess and respond to scenarios that involve multiple body systems. while working with animals, students may learn to develop empathy and improved moral awareness.13 this may assist students to better understand the impact of procedures on animals and the importance of how to ameliorate any adverse changes in behavior or animal welfare that might arise when undertaking procedures with animals. using animals in veterinary education, however, causes students to become desensitized and/or develop a utilitarian ethical standpoint on the use of animals and reduces empathy.14,15 this highlights the need for students to appreciate any potential implications for animal welfare when using animals and to be able to recognize and ameliorate activities that may have a detrimental effect on animal welfare. development of proficiency also requires some repetition to develop required standards of proficiency. use of animals is also necessary for students to develop competencies required for certification.16 any increased usage and reusage of animals for educational purposes could, however, present adverse outcomes for animal welfare unless animal usage is managed appropriately. there is relatively limited information available providing guidelines on how animals should be used for educational purposes, particularly, with regard to the welfare burden of repetitive use, although a number of reports describe physiological and behavioral responses to routine procedures conducted during veterinary education.17–20 upholding the principles of reduction, refinement, and replacement, the so-called ‘three r’s’ are embedded within the ethical framework that governs the conduct of the use of animals for research and education.21 principles are also provided through legislation, codes of practice, and guidelines and policies.22–29 a guide to optimize animal welfare, entitled the ‘five provisions and aligned animal welfare aims’ (table 1), has overarching aims to help animals not only to survive but also to thrive within the environment in which they are maintained.30 specific guidelines are lacking that would govern a range of procedures and learning activities that are commonly undertaken within veterinary training facilities and in a context where animals may be reused during an academic year for education and/or research. in response to the potential for increased animal usage and adverse effects on the health and welfare of animals during the education of veterinary students, guidelines and procedures were developed to monitor, quantify, and plan animal usage at james cook university. the guidelines inform how and when animals can be used for educational purposes and when periods of rest are required and provide a framework for quantitatively monitoring animal usage. these guidelines, although far from a definitive or perfect guide, give assertion to comments by dr bernard rolling that, “animals [are] were more than just resources, that they [are] were complex creatures that humans should treat with care and dignity.”31 animal usage guidelines these guidelines are used to assess the usage of large animals, fish, and laboratory animals owned by james cook university for practical classes for teaching veterinary students and, where necessary, to limit their usage in each duration. the guidelines represent an attempt to quantify the welfare burden on an animal from involvement in teaching activities and to set limits to animal usage for educational purposes. it should be noted that although usage scores have been assigned, the results of research studies that directly validate the scores or limits that have been assigned were not always available. while impacts of various procedures and usage are not fully known, there is also a risk that misdiagnoses of the effects of various procedures on animal welfare that could occur. as such, usage scores will be subject to review, and modifications are expected. this document is also not meant to provide a detailed description of the procedures undertaken or provide an ethical judgement on whether a procedure should be undertaken or not. rather, for procedures that are approved by an animal ethics committee, it aims to provide a reference guide to animal usage allowing for usage table 1. five provisions and aligned animal welfare aims26 provisions animal welfare aims 1. good nutrition: provide ready access to fresh water and a diet to maintain full health and vigor minimize thirst and hunger and enable eating to be a pleasurable experience 2. good environment: provide shade/shelter or suitable housing, good air quality and comfortable resting areas minimize discomfort and exposure and promote thermal, physical, and other comforts 3. good health: prevent or rapidly diagnose and treat disease and injury, and foster good muscle tone, posture and cardiorespiratory function minimize breathlessness, nausea, pain, and other aversive experiences and promote the pleasures of robustness, vigor, strength, and well-coordinated physical activity 4. appropriate behavior: provide sufficient space, proper facilities, congenial company, and appropriately varied conditions minimize threats and unpleasant restrictions on behavior and promote engagement in rewarding activities 5. positive mental experiences: provide safe, congenial, and species-appropriate opportunities to have pleasurable experiences promote various forms of comfort, pleasure, interest, confidence, and a sense of control http://dx.doi.org/10.58292/ct.v15.9593 citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9593 31 to be monitored and documented with the purpose of avoiding over-usage of animals to an extent that will adversely affect their health and welfare. recommendations on usage limits in developing the guidelines, points were initially applied on a scale from 1 to 8, with more points being applied with an increasing magnitude of the stress load expected to be imposed on animals (table 2). it is recommended that each individual animal not exceed a total score of 8 points in each calendar week, 24 points in a month, or 60 points within a 16-week study period and any associated examination period where animals are used for assessment of student competencies. points have been assigned to species that are frequently used within veterinary education programs and for procedures that are taught within the veterinary program at james cook university. consideration was given when assessing the magnitude of points assigned for various procedures that in an educational setting may take longer to apply and cause more stress (e.g. a procedure by an experienced clinician during a routine clinical procedure may impose less stress [table 3]). records should be kept for each individual animal use and the total cumulative score for each animal. where scores are likely to exceed recommended usage within the time frames suggested, these animals should not be used in further classes or used within this time frame. timetables and resources should be continually revised to determine ways of avoiding any potential overuse. when extenuating circumstances prevail, individual requirements should be discussed with a veterinarian or other appropriate expert and those who oversee or are managing the practical class to determine what other options are available, or whether further use can be considered. for example, illness of some animals may require them to be removed from a class. if replacements are not available, activities will need to be curtailed or modified, or consideration may need to be given if planned activities can be accommodated by the remaining animals. animals suffering from any illness that renders the animal with ongoing pain, lethargy, incoordination, or elevation of vital signs above normal limits will render the animal unsuitable for any practical class. minor illnesses that do not involve elevation of vital signs above normal values, where any pain or distress is minor or where the bodily region affected is remote from the region being used in a practical class and where involvement of the animal is unlikely to adversely affect the health and welfare of the animal use may be permitted following the approval of a veterinarian. where relevant, the opinion of others who have expertise in the care and use of the animal in question can also be sought. common sense should prevail regarding inclusion or exclusion of animals from practical teaching activities. the observations of the person in charge of the practical session should be taken into consideration, and the well-being of the animal is to be considered irrespective of the number of points it has accumulated. for example, if an animal has accumulated a total of 4 points for a given week (with 8 points being the maximum) but is expressing aversion to reuse, then it should be considered that an adequate rest period is recommended before considering further use or that further training of the animal and/or the user or adjustments to facilities may be needed before reuse is contemplated. there are examples of behaviors (table 4) that, when expressed, would be associated with a recommendation for immediate withdrawal from a learning activity. medication of animals is often required as part of a practical teaching activity (e.g. reproductive hormones, sedatives, and local anesthetic and analgesic medications). the use of these medications will be included in any ethics application and description of the practical teaching activity. if treatments are required that are directly associated with a teaching activity, table 2. description of categories related to levels of intervention that may impact upon animal welfare category description 1. minimal interference/ observation no or virtually no potential for stress. there is no pain or suffering involved. activities requiring only passive cooperation from the animal, not be expected to compromise the animal’s welfare any more than normal handling or feeding, and any restraint is well tolerated and of relatively short duration (< 2 hours). 2. minor conscious intervention little potential for stress. animal is subjected to procedures that would normally not require anesthesia or analgesia. any pain is minor, and analgesia is usually unnecessary. procedures do not normally require chemical restraint or are associated with significant aversion. 3. mild intervention moderate potential for stress. animal is subjected to procedures that would normally not require anesthesia or analgesia, but procedures are generally more invasive or longer in duration than a minor conscious intervention. any pain is mild, and analgesia is likely to be unnecessary, although some distress may occur. minor procedure with light sedation or local or topical anesthesia. 4. moderate intervention significant potential for stress. animal is subjected to a procedure that requires deep sedation and/or the short-term administration of analgesics. invasive surgery with the animal conscious. general anesthesia of short duration where any surgical procedure is minor. the number or duration of procedures is more than an animal usually experiences with a routine clinical procedure. 5. major intervention major potential for stress. animal may be rendered unconscious with as little pain or distress as possible. depending on the procedure, pain may be minor or moderate, and post-operative analgesia is likely to be appropriate. http://dx.doi.org/10.58292/ct.v15.9593 32 citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9593 ta b le 3 . a n im al u se p o in t sc o re a ll o ca ti o n sc o re c at eg o ry d es cr ip ti o n g en er al eq u in e b ov in e o vi n e/ ca p ri n e po rc in e fi sh la bo ra to ry an im al s/ ro de n ts c h ic ke n s 1 m in im al in te rf er en ce o b se rv at io n si n gl e ac ts , n ot re qu ir in g co n fin em en t o bs er va tio n g en er al a n im al h an dl in g ex te rn al p al pa tio n n on -in va si ve p hy si ca l ex am in at io n re ct al te m pe ra tu re b od y co n di tio n sc or in g o ra l a dm in is tr at io n o f a su bs ta n ce c on fin em en t i n s to ck s (< 6 0 m in ut es ) se m en c ol le ct io n w ith ar tifi ci al v ag in a (a v ) lu n gi n g (< 1 h o u r) te as in g (< 1 h o u r) a p p li ca ti o n o f h al te r c ar d ia c, p u lm o n ar y, o r ab d o m in al a u sc u lt at io n li m b b an d ag in g se m en c o ll ec ti o n (a v, 1 c o ll ec ti o n ) c o n fi n ed in s to ck s fo r p h ys ic al e xa m in at io n , ex te rn al p al p at io n ( < 60 m in u te s) a p p li ca ti o n o f es tr u s d et ec ti o n a id s. pa rt ia l c li n ic al ex am in at io n b lo o d s am p le (1  c o ll ec ti o n ) se m en c o ll ec ti o n (a v,  1 c o ll ec ti o n ) h an d li n g, r es tr ai n t, an d e xa m in at io n se ss io n w h ic h m ai n ly in vo lv e b o d y co n d it io n sc o ri n g, o ra l d re n ch in g, h o o f tr im m in g, n o n -i n va si ve p h ys ic al ex am in at io n , in cl u d in g re ct al te m p er at u re se m en c o ll ec ti o n (a v, 1 c o ll ec ti o n ) c at ch w it h m in o r re st ra in t (j u ve n il e) se m en co ll ec ti o n (1  c o ll ec ti o n ) m an u al re st ra in t fo r sh o rt d u ra ti o n (< 3 0 se co n d s) m an u al re st ra in t fo r sh o rt d u ra ti o n (< 2 m in u te s) 2 m in o r co n sc io u s in te rv en ti o n c o n fi n em en t in st o ck s/ cr u sh ( 60 -1 20 m in u te s) c o n fi n em en t o u t o f st o ck s (< 2 40 m in u te s) w it h f ee d , w at er , a n d s h ad e co n ti n u al ly a va il ab le in tr av en o u s, s u b cu ta n eo u s, o r in tr am u scu la r in je ct io n o f a lo w v o lu m e o f n o n -i rr it at in g su b st an ce ve n ip u n ct u re c o m p le te c li n ic al ex am in at io n fe ca l s am p le co ll ec ti o n f ro m re ct u m fl ex io n te st in g, h oo f t es tin g (< 1 h ou r) a pp lic at io n o f a n os e tw itc h (u n ex pe ri en ce d op er at or ) si n gl e n as og as tr ic in tu ba tio n by e xp er ie n ce d op er at or in cl ud in g ap pl ic at io n o f n os e tw itc h c on fin ed in s to ck s fo r ph ys ic al e xa m in at io n , ex te rn al p al pa tio n ( 60 –1 20 m in ut es ). c on fin ed o ut si de s to ck s (t ie d to fe n ce ) fo r b an da ge ap pl ic at io n , l im b lif te d, ca rd io re sp ir at or y au sc ul ta tio n , a n d h oo f t es te r ap pl ic at io n . a rt ifi ci al in se m in at io n n as ol ac ri m al d uc t i rr ig at io n a dm in is tr at io n o f a n in tr av ag in al in se rt . b re ed in g so un dn es s ex am in at io n ( b se ; 1 co lle ct io n ; e le ct ro ej ac ula tio n o r m an ua l) . pr eg n an cy d ia gn os is u p to 3 tr an sr ec ta l ex am in at io n s o ro ga st ri c in tu ba tio n . ep id ur al a n es th es ia . a bd om in op ar ac en te si s. r um en oc en te si s li ft in g of li m bs , la m en es s ex am in at io n , an d cl aw tr im m in g. a rt ifi ci al in se m in at io n c lin ic al e xa m in at io n h an d li n g, r es tr ai n t, an d e xa m in at io n se ss io n w h ic h m ay in vo lv e b o d y co n d it io n s co ri n g, o ra l d re n ch in g, h o o f tr im m in g, n o n -i n va si ve p h ys ic al e xa m in ati o n , i n cl u d in g re ct al t em p er at u re , ve n ip u n ct u re , b se , u ri n e co ll ec ti o n , an d r ec ta l f ec al co ll ec ti o n . c li n ic al ex am in at io n b se w it h s em en co ll ec ti o n b y ar ti fi ci al v ag in a p h ys ic al ex am in at io n w it h co n fi n em en t fo r re st ra in t. a p p ly n o se sn ar e <2 m in u te s. ea r n o tc h in g (n eo n at e) ta il /t ee th tr im m in g (n eo n at e) li gh t re st ra in t fo r a sh o rt d u ra ti o n (< 1 m in u te ) an d t an k tr an sf er . (c on tt in ue d) http://dx.doi.org/10.58292/ct.v15.9593 citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9593 33 ta b le 3 . a n im al u se p o in t sc o re a ll o ca ti o n sc o re c at eg o ry d es cr ip ti o n g en er al eq u in e b ov in e o vi n e/ ca p ri n e po rc in e fi sh la bo ra to ry an im al s/ ro de n ts c h ic ke n s 4 m il d in te rv en ti o n c on fin em en t i n st oc ks (2 –4 h ou rs ) d et ai le d ex am in at io n of a b od y sy st em re qu ir in g di ag no st ic te st s th at in vo lv e m ild in te rv en tio n an d su bc ut an eo us o r in tr am us cu la r i nj ec tio n of a lo w v ol um e of no nir ri ta tin g su bs ta nc e pr oc ed ur es m ay in vo lv e ve ni pu nc tu re fo r t he a dm in is tr at io n of s ed at iv es a nd /o r an xi ol yt ic s n eu ro lo gi ca l e xa m in at io n ( < 2 h ou rs ) d et ai le d la m en es s ev al ua tio n (< 3 h ou rs ) d et ai le d op h th al m ic ex am in at io n d et ai le d or al e xa m in at io n n as og as tr ic in tu ba tio n de m on st ra tio n b y ve te ri n ar ia n ( 1 at te m pt ) w ith s tu de n t pa lp at io n /a ss is ta n ce u pp er re sp ir at or y en do sc op y g as tr os co py re pr od uc tiv e ex am in at io n s (< 2 h ou rs ) pa lp at io n p er re ct um , u p to 8 e xa m in atio n s, w ith a m ax im um o f 5 m in ut es re co m m en de d af te r ev ac ua tio n o f t h e re ct um , tr an sr ec ta l u ltr as on og ra ph y of th e re pr od uc tiv e tr ac t a n d va gi n al c av ity e xa m in at io n in cl ud in g ut er in e la va ge a n d en do m et ri al /u te ri n e sw ab li m b re st ra in t w it h lig h t s ed at io n . c as ti n g. c on fin em en t 4 h ou rs . re pr od uc ti ve e xa m in ati on s (4 to 8 tr an sr ec ta l e xa m in at io n s, u p to 5 m in ut es in d ur at io n ) n as og as tr ic in tu ba ti on lo ca l a n es th es ia : h ea d (3 p oi n ts ) re gi on al in tr av en ou s an es th es ia pa ra ve rt eb ra l n er ve bl oc ks li n e bl oc k/ in ve rt ed lbl oc k su bc on ju n ct iv al in je ct io n li ve r bi op sy c as ti n g w it h s ed at io n in se rt io n o f a n in tr av en ou s ca th et er b se w it h s em en co ll ec ti o n b y el ec tr o ej ac u la ti o n h an dl in g, re st ra in t, an d ex am in at io n se ss io n , w h ic h m ay in vo lv e bo dy co n di tio n sc or in g, n on -in va si ve ph ys ic al ex am in at io n , ve n ip un ct ur e (o n e pr oc ed ur e) , w in g cl ip pi n g, a n d cl oa ca l s w ab . 6 m o d er at e in te rv en ti o n ex am in at io n of a b od y ca vi ty a n d sa m pl e co lle ct io n b y st ud en t m in or s ur ge ry n ot in vo lv in g ge n er al an es th es ia ( < 2 ho ur s) h ea vy s ed at io n a n d/ or ge n er al a n es th es ia w ith ou t s ur ge ry pr oc ed ur es m ay in vo lv e ve n ip un ct ur e fo r t h e ad m in is tr at io n of s ed at iv es a n d/ or an xi ol yt ic s d en ta l p ro ce du re s re qu ir in g h ea vy s ed at io n a bd om in oc en te si s. (m an da to ry 6 -m on th ex cl us io n b ef or e re pe at ed ) m in or s ur ge ry p er fo rm ed b y st ud en t u n de rt ak in g se da tio n an d an al ge si a – la ce ra tio n re pa ir, m as s re m ov al c as tr at io n ( < 3 m o n th s) – n o n -s u rg ic al va se ct o m y g en er al an es th es ia w it h ve n ip u n ctu re g en er al an es th esi a w ith ve n ipu n ct ur e an d/ or pa re n te ra l in je ct io n (c on tt in ue d) ta b le 3 . a n im al u se p o in t sc o re a ll o ca ti o n sc o re c at eg o ry d es cr ip ti o n g en er al eq u in e b ov in e o vi n e/ ca p ri n e po rc in e fi sh la bo ra to ry an im al s/ ro de n ts c h ic ke n s 1 m in im al in te rf er en ce o b se rv at io n si n gl e ac ts , n ot re qu ir in g co n fin em en t o bs er va tio n g en er al a n im al h an dl in g ex te rn al p al pa tio n n on -in va si ve p hy si ca l ex am in at io n re ct al te m pe ra tu re b od y co n di tio n sc or in g o ra l a dm in is tr at io n o f a su bs ta n ce c on fin em en t i n s to ck s (< 6 0 m in ut es ) se m en c ol le ct io n w ith ar tifi ci al v ag in a (a v ) lu n gi n g (< 1 h o u r) te as in g (< 1 h o u r) a p p li ca ti o n o f h al te r c ar d ia c, p u lm o n ar y, o r ab d o m in al a u sc u lt at io n li m b b an d ag in g se m en c o ll ec ti o n (a v, 1 c o ll ec ti o n ) c o n fi n ed in s to ck s fo r p h ys ic al e xa m in at io n , ex te rn al p al p at io n ( < 60 m in u te s) a p p li ca ti o n o f es tr u s d et ec ti o n a id s. pa rt ia l c li n ic al ex am in at io n b lo o d s am p le (1  c o ll ec ti o n ) se m en c o ll ec ti o n (a v,  1 c o ll ec ti o n ) h an d li n g, r es tr ai n t, an d e xa m in at io n se ss io n w h ic h m ai n ly in vo lv e b o d y co n d it io n sc o ri n g, o ra l d re n ch in g, h o o f tr im m in g, n o n -i n va si ve p h ys ic al ex am in at io n , in cl u d in g re ct al te m p er at u re se m en c o ll ec ti o n (a v, 1 c o ll ec ti o n ) c at ch w it h m in o r re st ra in t (j u ve n il e) se m en co ll ec ti o n (1  c o ll ec ti o n ) m an u al re st ra in t fo r sh o rt d u ra ti o n (< 3 0 se co n d s) m an u al re st ra in t fo r sh o rt d u ra ti o n (< 2 m in u te s) 2 m in o r co n sc io u s in te rv en ti o n c o n fi n em en t in st o ck s/ cr u sh ( 60 -1 20 m in u te s) c o n fi n em en t o u t o f st o ck s (< 2 40 m in u te s) w it h f ee d , w at er , a n d s h ad e co n ti n u al ly a va il ab le in tr av en o u s, s u b cu ta n eo u s, o r in tr am u scu la r in je ct io n o f a lo w v o lu m e o f n o n -i rr it at in g su b st an ce ve n ip u n ct u re c o m p le te c li n ic al ex am in at io n fe ca l s am p le co ll ec ti o n f ro m re ct u m fl ex io n te st in g, h oo f t es tin g (< 1 h ou r) a pp lic at io n o f a n os e tw itc h (u n ex pe ri en ce d op er at or ) si n gl e n as og as tr ic in tu ba tio n by e xp er ie n ce d op er at or in cl ud in g ap pl ic at io n o f n os e tw itc h c on fin ed in s to ck s fo r ph ys ic al e xa m in at io n , ex te rn al p al pa tio n ( 60 –1 20 m in ut es ). c on fin ed o ut si de s to ck s (t ie d to fe n ce ) fo r b an da ge ap pl ic at io n , l im b lif te d, ca rd io re sp ir at or y au sc ul ta tio n , a n d h oo f t es te r ap pl ic at io n . a rt ifi ci al in se m in at io n n as ol ac ri m al d uc t i rr ig at io n a dm in is tr at io n o f a n in tr av ag in al in se rt . b re ed in g so un dn es s ex am in at io n ( b se ; 1 co lle ct io n ; e le ct ro ej ac ula tio n o r m an ua l) . pr eg n an cy d ia gn os is u p to 3 tr an sr ec ta l ex am in at io n s o ro ga st ri c in tu ba tio n . ep id ur al a n es th es ia . a bd om in op ar ac en te si s. r um en oc en te si s li ft in g of li m bs , la m en es s ex am in at io n , an d cl aw tr im m in g. a rt ifi ci al in se m in at io n c lin ic al e xa m in at io n h an d li n g, r es tr ai n t, an d e xa m in at io n se ss io n w h ic h m ay in vo lv e b o d y co n d it io n s co ri n g, o ra l d re n ch in g, h o o f tr im m in g, n o n -i n va si ve p h ys ic al e xa m in ati o n , i n cl u d in g re ct al t em p er at u re , ve n ip u n ct u re , b se , u ri n e co ll ec ti o n , an d r ec ta l f ec al co ll ec ti o n . c li n ic al ex am in at io n b se w it h s em en co ll ec ti o n b y ar ti fi ci al v ag in a p h ys ic al ex am in at io n w it h co n fi n em en t fo r re st ra in t. a p p ly n o se sn ar e <2 m in u te s. ea r n o tc h in g (n eo n at e) ta il /t ee th tr im m in g (n eo n at e) li gh t re st ra in t fo r a sh o rt d u ra ti o n (< 1 m in u te ) an d t an k tr an sf er . (c on tt in ue d) http://dx.doi.org/10.58292/ct.v15.9593 34 citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9593 ta b le 3 . a n im al u se p o in t sc o re a ll o ca ti o n sc o re c at eg o ry d es cr ip ti o n g en er al eq u in e b ov in e o vi n e/ ca p ri n e po rc in e fi sh la bo ra to ry an im al s/ ro de n ts c h ic ke n s 8 m aj o r in te rv en ti o n g en er al a n es th es ia re q u ir in g p re an d /o r p o st o p er at iv e an al ge si a c as tr at io n n as o ga st ri c in tu b at io n b y st u d en ts ( 3 st u d en ts , 1 re p ea t p er s tu d en t) u n d er h ea vy s ed at io n ( in cl u d in g o p io id a n al ge si a) . m an d at o ry 6 -m o n th ex cl u si o n u n ti l p ro ce d u re re p ea te d . c as tr at io n – s u rg ic al c al f p ro ce ss in g (c as tr at io n , d eh o rn in g, b ra n d in g) c es ar ea n s ec ti o n (m in im u m 4 -w ee k re co ve ry ). g en er al an es th es ia w it h t ai l ve n ip u n ctu re a n d o ra l g av ag e ta b le 4 . e xa m p le s o f si gn s se en in a n im al s fr o m d if fe re n t sp ec ie s n ec es si ta ti n g w it h d ra w al f ro m t ea ch in g ac ti vi ti es a n im al r ea ct io n t o ac ti vi ty eq u in e b ov in e o vi n e/ ca p ri n e po rc in e fi sh la b o ra to ry an im al s/ ro d en ts c h ic ke n s a n im al s tr o n gl y re si st s ac ti vi ty , sh ow s si gn ifi ca n t si gn s o f st re ss a n d / o r ag gr es si o n , a n d b eh av es in a w ay th at p u ts it se lf o r p eo p le in d an ge r tr em b li n g, ab n o rm al s w ea ti n g, p er si st en t p aw in g o f gr o u n d , a b n o rm al e le va ti o n in h ea rt o r re sp ir at o ry ra te , a tt em p ts t o ju m p o u t o f st o ck s, re fu sa l t o g o in to st o ck s d if fi cu lt y am b u la tin g, r is in g, o p en m o u th p an ti n g, u n ex p ec te d ag gr es si o n , re p ea te d re cu m b en cy o p en m o u th b re at h in g, c h ar gi n g (r am s) , r ep ea te d vo ca li za ti o n , tr em b li n g o p en m o u th b re at h in g, s ki n d is co lo ra ti o n , m u sc le t re m o rs , o u t o f co n tr o l m ov em en t, w il li n g to r u n u n d er , o ve r, o r th ro u gh h an d le rs a n d o b st ac le s d is o ri en ta ti o n , in ab il it y to su b m er ge o r m ai n ta in o ri en ta ti o n . c h an ge in sk in p ig m en ta ti o n r u ffl ed h ai r co at , se p ar at io n , cr o u ch ed p o si ti o n , d is o ri en ta ti o n , p er si st en t ag it at io n , tr em o rs is o la ti o n , i n ap p ete n ce , m o u th b re at h in g, in ab il it y to m ai n ta in p o st u re o r b al an ce , p er si st en t ru ffl ed fe at h er s. a b n o rm al d is ch ar ge s http://dx.doi.org/10.58292/ct.v15.9593 citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9593 35 this does not incur additional usage points, as this has already been factored in the categorization of teaching activities. potential advantages and disadvantages of the guidelines these guidelines help provide clear limits of animal usage that can be followed by animal managers and users, providing an auditable trail for external assessment of animal usage. when planning learning activities, the number of animals required within a given period can be determined. when animal numbers are inadequate to service the educational needs, the guidelines provide objective data for justifying the sourcing of additional animals. in these circumstances, where educational needs cannot be met by existing animal resources, simulations to ease animal use should be considered, even if this is perceived as a lower quality learning experience. software has been developed to record animal usage, enabling rapid assessment of an animal’s eligibility to be enrolled within a learning activity and electronic capture of animal usage.32 this software was developed in response to the need to record, quantify, and monitor animal usage. it includes an exportable electronic database that tabulates data such as the date, animal identification, species, practical class group, procedure being conducted, weight, and body condition score. the database also stores information about treatments that have been applied, comments can be recorded, and the identification of the person who entered the data is recorded. data are entered as a report form that is viewable in a web browser, which includes a drop-down menu to select the procedures being conducted, with the points value being automatically assigned (although this can be manually overridden). data can be entered via an electronic mobile device facilitating data capture at the point and the time of usage with data being uploaded to a server when reception of a wireless fidelity service is available. reports for individual animals and their accumulated points over several set intervals, and reports for all activities and treatments can be produced. as with all data recording tasks, compliance remains a challenge. developing an interface between the software and a full clinical recording database would broaden the utility of the application in a clinical teaching facility, although provisions are available within the existing software to manually describe treatments and provide comments. this, however, currently remains beyond the budgetary priorities of our institution. other modifications include maintaining animals in groups with technical staff recording and monitoring the points accumulated for each group of animals, and the group being assigned to teaching activities that are permissible within their accumulated number of points. the guidelines are limited in that they do not consider that repetitive exposure may induce a degree of habituation and reduce stress responses over time.33 this is difficult to incorporate for individual animals as their responses may differ and the time taken to habituate may vary. nevertheless, with future iterations, it may well be that the level of points assigned for a particular procedure could be greater for an initial exposure or when student experience is low, whereas lesser points may be assigned to animals when habituation has occurred, and stress has lessened or student experience improves. in addition, realtime adjustment in point allocations could occur for individual animals that appear to be less tolerant of procedures. the guidelines also do not consider stressors that may exist in an animal’s environment and outside of specific learning sessions. examples include the quality of housing and husbandry, provision and quality of environmental enrichment, recent adverse weather events or illnesses, stage of pregnancy or interval postpartum, the quality of animal care, the training of individuals involved in the care and movement of animals, and the state of facilities that are used to house and handle animals. there is also a possibility that resources or the ability to express certain behaviors “might provide ‘relief’ by reducing the intensity of some negative effects or ‘benefit’ by increasing opportunities to experience positive affects,” thereby influencing an animal’s stress load or ability to manage stress.34 this acknowledges that stress may be imposed from the procedures conducted within a learning activity and imposed or ameliorated in association with the quality of the animals’ life and surrounding experience within its environment. this should be considered when assessing an animal’s short-term and cumulative stress load and suitability to be included within a learning activity.35 however, it is acknowledged that it is difficult to capture this ‘history’ within the current animal usage system as described. in time, it may be possible to assign points attempting to quantify stress loading that may occur outside of a learning activity or perhaps to reduce points where animals are suitably habituated, students are more experienced, or if activities or the environment outside of learning activities enhances or adversely affects an animal’s ability to manage stress. usage within research studies research studies may be undertaken in a context where it may be more difficult to manage animal usage within the recommended guidelines presented in this paper. for example, investigation into the development of a new vaccine may induce elevations in vital signs that, according to the guidelines, would render an animal unsuitable for usage but such a study would be necessary to document responses to treatment. some studies may require the intensive use of animals for short periods that would mean that the number of points could exceed limits that have been set in the guidelines. for example, animals may be intensively blood sampled and subjected to several examinations relevant to the context of a study. animals could also be given a general anesthetic and have surgery performed, and follow-up sampling could exceed the points system as outlined, but this may be necessary in the context of where the aims and methods for a research project have been approved by an animal ethics committee and meet any regulatory requirements. some studies might also have outcomes that are unexpected and require additional monitoring to fully evaluate responses. therefore, the use of these guidelines may be inappropriate for some research studies in the context of showing that the points have been exceeded, and that all work should immediately cease. preconsideration of the justification, purpose, and planned interventions and possible outcomes as part of the normal process for managing the ethical use of animals in scientific studies will help manage welfare outcomes in this context. mandatory reporting of adverse and unexpected events is also part of the normal process for managing research studies involving the use of animals.25 nevertheless, these guidelines could provide additional reference to ethics committees and researchers and serve as a guide as to when animal usage is exceeding limits that could adversely impact on animal welfare. application to fish there appear to be no specific guidelines on how to regulate and assess use and repeated use of fish from a welfare perspective, although fish have been included in these guidelines. there is little doubt that fish exhibit behaviors in response to http://dx.doi.org/10.58292/ct.v15.9593 36 citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9593 noxious stimuli that cause pain in other animals, and that they possess nociceptors and opiate receptors involved in pain information processing analogous to those in mammals.36 there are a number of publications that describe the welfare of recreational fishing and welfare indicators in fish.37–39 however, most of the published work on assessing the welfare of fish relates to research and farming rather than teaching. unlike terrestrial animals, the endpoint of most teaching exercises involving fish often results in euthanasia, precluding the need for methods to assess welfare based on repetitive use. international and national differences in legislation result in differing approaches to welfare. in australia, for example, south australia and western australia do not include fish in their animal welfare legislation, whereas the northern territories only cover the welfare of fish when they are held in captivity or dependent on a person for food, a position that would appear to cover teaching activities. the australian veterinary association’s position statement on fish welfare covers captivity, handling, illness, and euthanasia, but there is no reference to repeated use.40 fish are included in the australian code for the care and use of animals for scientific purposes, which includes teaching activities and provides guidance to animal ethics committees to consider the ratio of students to animals (fish).25 it also considers the number of times that each animal will be used in each class and/or handled per day and/or per week, and whether an animal (fish) is appropriate for reuse. at james cook university, fish are currently not reused for activities involving anesthesia, handling, examination, venipuncture, and parenteral medications including vaccinations. conclusion guidelines for quantifying, monitoring, and planning the use and reuse of animals have been developed and are being applied at james cook university to regulate animal usage. procedures associated with animal usage in each species are assigned points from 1 to 8, where 1 is assigned for minimal intervention and/or observation and 8 is where a procedure is regarded as a major intervention and is expected to be associated with relatively minor to moderate pain, normally requiring analgesia and may render the animal unconscious with subsequent recovery. points are assigned following known impacts on animal welfare from scientific literature or, when not available, by veterinarians and other suitable experts, experienced with the application of procedures to animals and expected outcomes. usage of individual animals is limited to a maximum of 8 points in a calendar week, 24 points in a month, or 60 points within a 16-week study period and any associated examination interval. limitations of the system are acknowledged, given that the impact of procedures on animal welfare may not have been subjected to controlled studies, and that the system may not be applicable in every scenario in which animals are used or take into consideration stressors that may exist in an animal’s environment or experience outside of a learning activity. the points system can be modified according to changes in knowledge or by consensual agreement based on knowledge of and potential adverse effects on animal welfare. the system as outlined provides guidelines for planning, monitoring, and limiting animal usage in veterinary education. conflict of interest authors, except graham willis, do not have any conflict of interest to declare. graham willis is a director of powermation that designed and sells the software zumon for recording animal usage. authors’ contributions john cavalieri: conceptualization, original draft writing, and manuscript review. brad dowling, leo foyle, craig godfrey, janice lloyd, josephine penny, william tranter, and sally watts: writing – review and editing jamie wearn: conceptualization, review, and editing. graham willis: software and manuscript review. all authors gave their final approval of the manuscript. references 1. u.s. bureau of labor statistics: employment projections program.  2021. available from: https://www.bls.gov/emp/tables/ emp-by-detailed-occupation.htm [cited 11 march 2022]. 2. american association of veterinary medical colleges: annual data report 2020–2021. available from: https://www.aavmc.org/ wp-content/uploads/2021/05/2021-aavmc-annual-data-report.pdf [cited 11 march 2022]. 3. thinkplus: the australian veterinary association report on projection modelling for the veterinarian workforce. australian veterinary association: 2014. available from: https://www.ava. com.au/siteassets/advocacy/veterinary-workforce/australian-veterinary-workforce-technical-paper.pdf [cited 11 march 2022]. 4. vemulapalli th, donkin ss, lescun tb, et al: considerations when writing and reviewing a higher education teaching protocol involving animals. j am assoc lab anim sci 2017;56:500–508. 5. noyes ja, carbonneau kj, matthew sm: comparative effectiveness of training with simulators versus traditional instruction in veterinary education: meta-analysis and systematic review. j vet med educ 2021;49:25–38. doi: 10.3138/jvme-2020-0026 6. zemanova ma, knight a: the educational efficacy of humane teaching methods: a systematic review of the evidence. animals 2021;11:114. doi: 10.3390/ani11010114 7. pereira gd, dieguez j, demirbas ys, et al: alternatives to animal use in veterinary education: a growing debate. ank univ vet fak derg 2017;64:235–239. doi: 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39. toni m, manciocco a, angiulli e, et al: review: assessing fish welfare in research and aquaculture, with a focus on european directives. animal 2019;13:161–170. doi: 10.1017/s1751731118000940 40. australian veterinary association: fish welfare. australian veterinary association: 2009. available from: https://www.ava. com.au/policy-advocacy/policies/hunting-and-fishing/fish-welfare/ [cited 11 march 2022]. http://dx.doi.org/10.58292/ct.v15.9593 https://doi.org/10.1080/03057240903528733 https://doi.org/10.1177/026119290403201s85 https://doi.org/10.1136/vr.146.10.269 https://animalowners.rcvs.org.uk/document-library/day-one-competences/ https://animalowners.rcvs.org.uk/document-library/day-one-competences/ https://doi.org/10.1016/j.applanim.2021.105371 https://doi.org/10.1016/j.theriogenology.2014.11.010 https://doi.org/10.1016/j.theriogenology.2014.11.010 https://doi.org/10.1016/j.jevs.2016.04.010 https://doi.org/10.1016/j.jevs.2017.08.013 https://caat.jhsph.edu/principles/the-principles-of-humane-experimental-technique https://caat.jhsph.edu/principles/the-principles-of-humane-experimental-technique https://eur-lex.europa.eu/legal-content/en/txt/?uri=celex%3a01998l0058-20191214 https://eur-lex.europa.eu/legal-content/en/txt/?uri=celex%3a01998l0058-20191214 https://www.legislation.qld.gov.au/view/pdf/2016-07-01/act-2001-064 https://www.legislation.qld.gov.au/view/pdf/2016-07-01/act-2001-064 https://www.aphis.usda.gov/animal_welfare/downloads/ac_bluebook_awa_508_comp_version.pdf https://www.aphis.usda.gov/animal_welfare/downloads/ac_bluebook_awa_508_comp_version.pdf https://www.nhmrc.gov.au/about-us/publications/australian-code-care-and-use-animals-scientific-purposes https://www.nhmrc.gov.au/about-us/publications/australian-code-care-and-use-animals-scientific-purposes https://www.nhmrc.gov.au/about-us/publications/australian-code-care-and-use-animals-scientific-purposes https://www.ava.com.au/policy-advocacy/policies/cattle-health-and-welfare/use-of-female-cattle-for-pregnancy-testing-and-artificial-insemination-schools/ https://www.ava.com.au/policy-advocacy/policies/cattle-health-and-welfare/use-of-female-cattle-for-pregnancy-testing-and-artificial-insemination-schools/ https://www.ava.com.au/policy-advocacy/policies/cattle-health-and-welfare/use-of-female-cattle-for-pregnancy-testing-and-artificial-insemination-schools/ https://www.ava.com.au/policy-advocacy/policies/cattle-health-and-welfare/use-of-female-cattle-for-pregnancy-testing-and-artificial-insemination-schools/ https://www.jcu.edu.au/__data/assets/pdf_file/0005/632948/animal-monitoring-and-humane-intervention-points-guideline.pdf https://www.jcu.edu.au/__data/assets/pdf_file/0005/632948/animal-monitoring-and-humane-intervention-points-guideline.pdf https://www.jcu.edu.au/__data/assets/pdf_file/0005/632948/animal-monitoring-and-humane-intervention-points-guideline.pdf https://anzccart.adelaide.edu.au/ua/media/9/aust-ethical-guide2013.pdf https://anzccart.adelaide.edu.au/ua/media/9/aust-ethical-guide2013.pdf https://www.animalethics.org.au/policies-and-guidelines/animals-in-teaching https://www.animalethics.org.au/policies-and-guidelines/animals-in-teaching https://doi.org/10.3390/ani6100059 https://www.avma.org/javma-news/2022-01-01/obituary-bernie-rollin-ethicist-who-wanted-better-world-animals-dies-78 https://www.avma.org/javma-news/2022-01-01/obituary-bernie-rollin-ethicist-who-wanted-better-world-animals-dies-78 https://www.avma.org/javma-news/2022-01-01/obituary-bernie-rollin-ethicist-who-wanted-better-world-animals-dies-78 http://www.zumon.net/ http://www.zumon.net/ https://doi.org/10.1016/j.applanim.2017.02.020 https://doi.org/10.1080/00480169.2014.966167 https://doi.org/10.3390/ani4040729 https://doi.org/10.1093/ilar.50.4.338 https://jukuri.luke.fi/bitstream/handle/10024/544503/welfare_published.pdf?sequence=1 https://jukuri.luke.fi/bitstream/handle/10024/544503/welfare_published.pdf?sequence=1 https://doi.org/10.1017/s1751731118000940 https://www.ava.com.au/policy-advocacy/policies/hunting-and-fishing/fish-welfare/ https://www.ava.com.au/policy-advocacy/policies/hunting-and-fishing/fish-welfare/ https://www.ava.com.au/policy-advocacy/policies/hunting-and-fishing/fish-welfare/ electrosurgical bipolar vessel sealing for a standing flank ovariectomy in beef heifers joe smith,a,b,c,e guillermo salazar,c♦ ciara peace,c♦ jeff olivarez,a,b,c katie vermeulen,a,b kelley black,a,b camille guss,a,b dane tatarniuk,d tyler dohlman,a,c caitlin wileya,c aveterinary diagnostic and production animal medicine, bbiomedical sciences cfood animal and camelid hospital dveterinary clinical sciences iowa state university, ames, ia elarge animal clinical sciences, university of tennessee, knoxville, tn ♦cosecond authors abstract traditional ovariectomy techniques for heifers (e.g. colpotomy) have adverse effects ranging from hemorrhage to mortality. electrosurgical bipolar vessel sealing devices offer a superior ligation of ovarian pedicle and associated structures. in ruminants, standing flank laparotomy has been used for abdominal visibility. we performed ovariectomy in 2 companion beef heifers via a unilateral flank approach, combined with electrosurgical ligation. surgeries were performed in standing animals, under local anesthesia, with no observed shortor long-term adverse effects. this technique is worth considering for cases where the security of electrosurgical ligation combined with the accessibility of a flank laparotomy are desired. additional research is needed to determine the viability of the technique for cattle of various ages and breeds. keywords: cattle, laparotomy, ligasure, ovariectomy, spay introduction various techniques are described for ovariectomy in bovine practice. ovariectomy is performed to suppress estrus in feedlot heifers, for brucellosis control, for research purposes, and for diseased ovary removal.1 while routine, surgical removal of bovine ovary is not without risks, as intraoperative and/or postoperative hemorrhage is a potential life threatening complication.2 vaginal approaches to ovariectomy, common in feedlot heifers, offer convenient access. however, care must be taken to avoid damaging viscera or uterine vessels by the ovariectomy instrument, as these approaches dictate that the technique is performed blind, albeit with guidance via transrectal palpation.1 flank approaches offer better visualization of the ovarian and uterine structures, but can still present challenges with hemostasis and suture security. mortality from the current described techniques ranged from 0.26 6%, with certain animal welfare concerns.3,4 in ovariectomy via colpotomy, as currently practiced, failure to confirm hemostasis after ovary removal increased risks of bleeding from the ovarian pedicle.5-7 electrosurgical bipolar vessel sealing devices are utilized in multiple areas of veterinary medicine for hemostasis, ranging from small intestinal resection and anastomoses in horses8 to enucleation in rabbits.9 ligasuretm (ls) is an electrosurgical bipolar vessel sealing device (covidien ls1020 ligasure atlas, medtronic, minneapolis, mn) used in many areas of veterinary medicine to minimize hemorrhage and to reduce surgery time. the ls device was successfully used to seal vessels of < 7 mm in diameter in equine models.8 use of this device had profoundly decreased hemorrhage in pet pig ovariohysterectomy procedures, wherein hemorrhage has been a complication associated with standard ligation technique.10,11 recently laparoscopic ovariectomy with a ls device has been described for cattle.7 although this technique has multiple benefits, including a minimally invasive incision and direct observation of vessel sealing, it requires many people to manage equipment and 1 more person to provide guidance via transrectal palpation.7 another recent unilateral ovariectomy technique via flank incision used an endoscopic articulating linear cutter instrument for ligation.12 although this technique provided excellent visualization of structures and hemostasis, drawbacks exist in staples cost and possibility of foreign body (stainless steel staple) occurrence within the abdomen of a food animal, poses a safety risk in the ‘food chain’. therefore, the goal was to demonstrate the feasibility of adopting ls for hemostasis in a standing bilateral laparoscopic ovariectomy via a unilateral standard flank incision. this amalgamation of existing clinical theriogenology • volume 13 number 1 • march 2021 60 techniques could allow for excellent hemostasis with the ease of a standard procedure performed through a conventional abdominal laparotomy approach. method ex vivo procedure was refined on a cadaver bovine uterus (figures 1 & 2). initially, the right ovary was identified and held in the left hand of the operator. ligation with the ls device started with the mesovarium and mesosalphinx, continuing with multiple bites until the right ovary was free from the broad ligament. distal aspect of the right uterine horn was then evaluated for cauterization and released. procedure was repeated for the left ovary. figure 1. ex vivo description of right ovariectomy with ligasure device. a. right ovary held by operator’s left hand, ligasure jaws encompassing mesovarium, mesosalphinx, and uterine tube. b. positioning of ligasure jaws. c & d. after first cautery ligation, demonstrating advancement of ligasure jaws through remaining mesovarium and broad ligament. figure 2. ex vivo description of left ovariectomy. a. left ovary held by operator's left hand and ligasure jaws encompassing mesovarium, mesosalphinx, and uterine tube. b. placement of jaws after initial ligation, to further advance through mesovarium and broad ligament. c. positioning for final advancement to ligate through remaining broad ligament d. uterus after bilateral ovariectomy e. ligated ovary, demonstrating ligation through pedicle and complete detachment of mesovarium and mesosalphinx. clinical theriogenology • volume 13 number 1 • march 202161 in vivo two 1-year old mixed breed intact beef heifers, owned by a local farm animal sanctuary, were presented for bilateral ovariectomy. client elected this procedure due to the desire to eliminate mounting behavior exhibited by the heifers during estrus, as 1 of them was blind and presented a safety risk. both heifers were on a grass hay diet and raised together in an ~ 2-acre paddock. they spent most of the day outside and went into an indoor stall at night. heifer 1 was bright, alert and responsive, weighting 365 kg with a body condition score of 5/9. physical examination findings were within normal limits. packed cell volume (pcv) and total protein (tp) were within reference range at 31% and 6.8 g/dl respectively. heifer 2 was also bright, alert and responsive, weighing 319 kg with a body condition score of 4/9. she had bilateral absence of menace, dazzle and presence of pupillary light response reflexes and palpebral reflex. both eyes also exhibited ventral strabismus, rotatory nystagmus, hypermature cataracts, uveitis, posterior synechiae, anterior synechiae, and retinal detachment. despite being nonvisual, the second heifer was able to ambulate, eat and drink well on her own. no blepharospasm was observed. through extensive previous workup and consultation with veterinary ophthalmology specialists, the heifer was determined to be in pain from her eye pathology and previous inflammation had been managed with oral meloxicam (meloxicam tablets usp, zydus pharmaceuticals, pennington, nj) and topical ketorolac (ketorolac ophthalmic solution, sandoz, princeton, nj). heifer 2 had a grade v/vi, right basilar systolic heart murmur that was associated with a small, previously diagnosed, congenital ventricular septal defect. pcv and tp were within reference range at 36% and 6.6 g/dl respectively. both animals were deemed acceptable candidates for the surgical procedure and fasted for 24 hours prior to surgery. prophylactic antibiotic coverage was provided subcutaneously with 2.5 mg/kg of tulathromycin (draxxin, zoetis, parasippany-hills, nj). preoperatively, 1.1 mg/kg of flunixin meglumine (banamine, merck, kenilworth, nj) was given intravenously and vaccinated with a booster vaccination for clostridium perfringens (types c and d) and tetanus toxoid (barvac cd/t, boerhinger ingelheim, ingelheim am rhein, germany). heifer 1 was premedicated intravenously with 0.05 mg/kg of butorphanol (butorphic, akorn inc., lake forest, il) and intramuscularly with 5 mg of xylazine (anased, lloyd labs, shenandoah, ia). heifer was placed in a mobile cattle restraint chute for the procedure. left flank was clipped and aseptically prepared, first with a preliminary scrub and then in an aseptic fashion using chlorhexidine and alcohol. a distal paravertebral block was performed with 2% lidocaine (lidocaine injectable, aspen pharmaceuticals, irvine, ca) on the spinous processes of the 1st lumbar, 2nd lumbar, and 4th lumbar vertebra, and a line block on the caudal flank area was performed, all with 2% lidocaine. the surgery site was finally aseptically prepared with alternating scrubs of chlorhexidine and alcohol. a 14 cm vertical incision was made using a #10 blade cranioventral to the tuber coxae. skin, subcutaneous tissue, and external abdominal oblique were incised and the remaining muscle layers and peritoneum were bluntly dissected by hand in a grid technique. while incising through the body wall, bleeding was controlled by applying pressure to soft tissues with 4 x 4 gauze. left and right ovaries were manually palpated and visually identified. although ovaries were visualized, we were not able to be completely exteriorize them to the level of the incision. a 37 mm ls (covidien ls1020 ligasure atlas, medtronic) was introduced into the abdomen (figure 3a) with 1 hand over the jaws, protecting surrounding soft tissues. with 1 hand in the abdomen, the right ovary was isolated and the ls was placed caudal to the ovary, over the uterine tube, ensuring no other tissue was incorporated. right ovarian pedicle was clamped, cauterized, severed, and the ovary was removed with the pedicle assessed with 4 x 4 gauze for bleeding prior to proceeding to the left ovary. left ovary was then palpated, identified, and ls was used in the same manner to remove the ovary. left pedicle was visually evaluated for any signs of hemorrhage and after examination was released back into the abdominal cavity. peritoneum and transversus abdominus were closed with 1 polydioxanone suture (pds ii, ethicon, us) in a simple continuous pattern. remaining muscle layers and subcutaneous tissues were closed with 1 pds in a simple continuous pattern. skin was closed with 2-0 polyamide suture having ‘s curved’ needle (braunamid, braun, tuttlingen, germany) using a ford clinical theriogenology • volume 13 number 1 • march 2021 62 interlocking pattern. a single simple interrupted suture was placed at the ventral aspect of the incision. the area was cleaned, and an aluminum spray (aluspray, neogen, france) bandage was applied. figure 3. a: position of the operator advancing the ligasure device through a flank incision for ovarian ligation. b: closeup view of the ovary after removal (note complete ligation through pedicle). heifer 2 was premedicated with butorphanol (butorphic, akorn inc.,). heifer was restrained, prepped and had a distal paravertebral nerve block as described for the first heifer. an additional inverted l block over the incision was performed with 2% lidocaine (lidocaine injectable, aspen pharmaceuticals). surgery site was finally aseptically prepped with alternating scrubs of alcohol and chlorhexidine. a 15 cm vertical incision was made using a #10 blade cranial and ventral to the tuber coxae. skin, subcutaneous tissue, and muscle layers were then dissected as described for the first heifer. ls was introduced, ovarian pedicles ligated, and incisions closed as described for the first heifer. outcome duration of surgeries were 80 and 77 minutes respectively and heifers tolerated surgeries well. ovaries appeared normal and had clear margins (figure 3b). day after surgery, heifer 1 had a pcv of 34% and tp of 6. 8 g/dl and heifer 2 had 33% and 6.6 g/dl respectively. transabdominal ultrasonography on day 2 postsurgery revealed no evidence of free fluid and peristalsis was noted in both heifers. skin sutures were removed on day 10 postsurgery by the referring veterinarians and no complications were evident. client reported at 8 months postovariectomy that there was no estrous behaviour in heifers. discussion in equine practice, ovariectomies are commonly performed for disease, performance, and behavioural reasons. we explored the feasibility of combining electrosurgical vessel sealing as described for laparoscopic ovariectomy in cattle and adopted a flank approach as described for unilateral ovariectomy.7,12 combination of these aspects formed a hybrid technique that was well tolerated by 2 heifers and allowed for a relatively quick ovariectomy with good vessel ligation. as anticipated, estrous behavior was absent and furthermore there were no postoperative complications. currently, specific effects of ls ovariectomy within the bovine abdomen or with respect to changes to the peritoneal environment are not known. stallions that underwent standing laparoscopic cryptorchidectomy with a ls device had higher total nucleated cells in peritoneal fluid at 24 hours postprocedure compared to stallions that had standard ligatures.13 however, at 72 hours, no differences were observed between groups for peritoneal nucleated cell count, red blood cell count, or total protein.13 although abdominocentesis was not performed in our cases, there was no clinical evidence of peritonitis as both heifers postsurgically had normal physical examination parameters and adequate appetite. a unilateral ovariectomy technique utilizing a linear stapler that could potentially be modified for bilateral ovariectomy procedures in cattle was described.12 however, a relative paucity of information exists regarding the migration of staples in cattle. in humans the migration and use of linear staples has led to multiple uncommon adverse effects, including volvulus, serosal laceration, and leakage.14-16 although beef heifers that had surgeries were companion animals, in the united states, companion food clinical theriogenology • volume 13 number 1 • march 202163 animal species are still considered food animals from a regulatory perspective.17-19 with this categorization leaving a metallic object in a food animal would be a food safety risk due to carcass contamination. our technique would alleviate this carcass risk as no foreign material was used for ovarian ligation. tulathromycin was utilized as a prophylactic antimicrobial due to broad spectrum action and ability to penetrate into tissues.20,21 often anecdotally discussed for abdominal surgeries in the bovine, intraabdominal treatment of penicillin was not considered, due to no empirical data suggesting the superiority of this route of treatment.22 in hindsight, antimicrobial prophylaxis may not have been necessary, as ovariectomies are generally clean procedures and both heifer surgeries were completed in < 90 minutes. prevention of nociceptive pain wind-up was provided with premedication with a nonsteroidal antiinflammatory drug (nsaid) flunixin meglumine. although not labelled for prevention or treatment of surgical pain, flunixin has been demonstrated to decrease cortisol concentrations in calves undergoing surgery.23 transitioning from 1 nsaid to another can potentially lead to complications, however there are several cases of ruminants being initially treated with a single dose of flunixin and then being transitioned to meloxicam without adverse effects.24,25 heifers in our case study did not demonstrate any evidence of abomasal ulceration and postoperatively were maintained on oral meloxicam (1 mg/kg) once daily for 5 days. treatment with nsaids (ketoprofen or meloxicam) in dairy cows after flank laparotomy (rumen fistula placement) improved outcomes of pain with no difference between them.26 a higher fasting time of at least 36 hours was recommended27 as a means to reduce rumen volume and increase visualization of the abdomen, although 24 hours may be sufficient.7 we fasted the animals for 24 hours prior to surgery. four authors of this report who performed surgeries were able to easily identify and manipulate both ovarian pedicles in each animal. transrectal guidance by an assistant, as described for manipulation of the ovaries for laparoscopic ovariectomy in cattle,7 was not necessary for our cases. however, this technique will be useful in cases difficult to identify and ligate an ovarian pedicle. if a unilateral approach is not sufficient for identification of the opposite ovary, a bilateral flank approach is warranted.27 clinicians should exercise caution when using a ls or other electrosurgical device, since electric sparks are possible with electrosurgical instruments.28 additionally, authors took care to identify ovarian pedicle with their hand to avoid thermal injury to adjacent tissues. thermal injury after ls procedures has been reported in people, with specific examples being anal stenosis after ls hemorrhoidectomy,29 and ureteral lesions after perirenal fat dissection.30 according to the manufacturer, the tissue sealed and cauterized with a ls can reach 100ºc that can spread laterally to up to 2 mm.30 additionally, care should be taken to not include too much tissue in the instrument jaws, as this could lead to unnecessarily repeated cycles, and increased lateral thermal damage. caution should be used to clearly ligate only the ovarian pedicle and no surrounding tissues. standing sedation was achieved with butorphanol in heifer 2 with additional xylazine (5 mg, total dose) in heifer 1. while both surgeries were completed in a timely fashion, future applications of this technique could evaluate other sedation techniques such as the butorphanol-ketamine-xylazine for standing sedation.31 while generally regarded as safe, clinicians should be aware that this combination has been associated with regurgitation in other large ruminant species, so care should be taken when assessing patients for this sedation technique.32 pain scores are not known for ls procedures compared to standard technique in cattle. in cats undergoing, ls versus open ovariectomy, no difference in pain scores were noted.33 similarly, in women undergoing hysterectomy via ls or robi bipolar forceps, no difference was noted between groups and postoperative pain scoring.34 in mares undergoing laparoscopic ovariectomy, pain was reduced by the mesovarian injection of lidocaine, when compared to intraovarian injection.35 epidural anaesthesia has also been of benefit for cattle undergoing laparotomy.36,37 future investigation of our technique could involve mesovarian lidocaine injection and epidural anaesthesia. in other species, primarily dogs and cats, there are reports of remnants of ovarian tissue revascularizing and contributing to signs of estrus and cyclicity. in previously mentioned techniques for ovariectomy in cattle, ovaries are generally dropped into abdomen after excision (as with the willis spay clinical theriogenology • volume 13 number 1 • march 2021 64 tool). there are little to no reported data that proves this is a potential complication in cattle. this was attributed to ovariectomies being primarily done in heifers that enter a feedlot where their lifespan is too short to observe any potential effects of revascularization. for large animals, options include colpotomy, laparoscopic, ventral, and flank incisions. in mares, standing laparoscopic ovariectomy may be performed for elective or pathologic conditions.38-41 standing laparoscopic ovariectomy in mares provided good access with low morbidity regardless of the method of hemostasis used (linear stapler, bipolar sealing device, modified roeder’s knot, vessel sealing system or laser).42 however, 11% of mares that developed some form of a postoperative complication, ranging from incisional seromas and infection to fever and abdominal discomfort.42 additionally, mares that underwent standing laparoscopic ovariectomy had similar cortisol concentrations, regardless of the method of mepivacaine application (topical or injection into ovarian pedicle).43 similar studies are needed in cattle. ‘dropped ovaries’ revascularizing in omentum is possible in mares, however, not confirmed when a mare was examined several months after a procedure.44 generally, mares live much longer than several months and the question of whether revascularization can occur may still remain. ovarian remnant revascularization and hormone production occurred in 8 of 9 cats.45 since these heifers reside at a farm sanctuary where they are likely to live for multiple years beyond the lifespan of a normal production animal, removal of ovaries as described reduces any risk of potential revascularization of the ovaries. we recognize the limitations (small number of animals and the controlled conditions in which the procedures were performed) of our report. while the heifers were clinically observed for 4 days postoperatively, they were not evaluated via abdominocentesis for inflammatory changes. future studies need to evaluate the effect of ls ovariectomy on inflammation in the abdominal environment, as well as pain from the technique. further exploration of the feasibility of the technique in cattle of various breeds and ages, as the heifers in our study were young and both of beef breeds. the ls is not the only electronic vessel sealing device in practice, additional investigations on the use of other devices (e.g. enseal® [surgrx, redwood city, ca] used for equine ovariectomy46) are warranted. conclusion ovariectomy using a ls device through flank laparotomy incision was well-tolerated in heifers with no adverse effects or complications. electrosurgical vessel-sealing (via a ls device) for standing flank ovariectomy was feasible. although not necessarily practical in a field setting (on farm), a left flank approach for bilateral ls ovariectomy is worth considering to minimize complications (reduced haemorrhage, no foreign material left in abdomen, and complete removal of ovaries). conflict of interest none to declare. references 1. prado tm, schumacher j, dawson lj: surgical procedures of the genital organs of cows. vet clin north am food anim 2016;32:727-752. 2. marchionatti e, nichols s, babkine m, et al: outcome following partial unilateral hysterectomy by standing flank approach in dairy cattle. vet surg 2016;45:943-948. 3. mccosker k, letchford p, petherick jc, et al: morbidity, mortality and body weight gain of surgically spayed, yearling brahman heifers. aust vet j 2010;88:497-503. 4. jubb tf, fordyce g, bolam mj, et al: trial introduction of the willis dropped ovary technique for spaying cattle in northern australia. aust vet j 2003;81:66-70. 5. habermehl nl: heifer ovariectomy using the willis spay instrument: technique, morbidity and mortality. can vet j 1993;34:664-667. 6. rupp gp, kimberling cv: a new approach for spaying heifers. vet med sm anim clin 1982;77:561-565. 7. sato r, kanai e, kitahara g, et al: transrectal guidance of the ovaries reduces operative time during bovine laparoscopic ovariectomy. j vet med sci 2017;79:2019-2022. 8. rumbaugh ml, burba dj, natalini c, et al: evaluation of a vessel-sealing device for small intestinal resection and anastomosis in normal horses. vet surg 2003;32:574-579. 9. moore ba, robertson j, tarbert dk, et al: a novel surgical technique for enucleation in rabbits to reduce the risk of intraand post-operative orbital hemorrhage. vet ophthalmol 2020;23:409-413. 10. biedrzycki a, brounts sh: a less invasive technique for spaying pet pigs. vet surg 2013;42:346-352. clinical theriogenology • volume 13 number 1 • march 202165   11. cypher e, videla r, pierce r, et al: clinical prevalence and associated intraoperative surgical complications of reproductive tract lesions in pot-bellied pigs undergoing ovariohysterectomy: 298 cases (2006-2016). vet rec 2017;181:685. 12. rizzo a, piccinno m, lacitignola l, et al: application of an innovative technique for unilateral ovariectomy in dairy cows. vet rec 2016;179:463. 13. seabaugh ka, goodrich lr, morley ps, et al: a comparison of peritoneal fluid values after laparoscopic cryptorchidectomy using a vessel-sealing device versus a ligating loop and removal of the descended testis. vet surg 2013;42:600-606. 14. kim r, moore r, schmidt l, et al: volvulus caused by a free intraperitoneal staple after laparoscopic appendectomy: a case report. int j surg case rep 2019;65:259-261. 15. matsuzawa f, homma s, yoshida t, et al: serosal laceration during firing of powered linear stapler is a predictor of staple malformation. surg innov 2017;24:590-597. 16. lee s, ahn b. the relationship between the number of intersections of staple lines and anastomotic leakage after the use of a double stapling technique in laparoscopic colorectal surgery. surg laparosc endosc percutan tech 2017;27:273281. 17. lord lk, wittum te: survey of humane organizations and slaughter plants regarding experiences with vietnamese potbellied pigs. j am vet med assoc 1997;211:562-565. 18. smith j, cuneo m, walton r, et al: spontaneous pneumothorax in a companion kunekune pig due to pulmonary bullae rupture. journal of exotic pet medicine 2020;34:6-9. 19. schleining ja, bergh ms: surgical correction of angular and torsional metatarsal deformity with cylindrical osteotomy and locking compression plates in a calf. vet surg 2014;43:563-568. 20. lin z, he c, magstadt dr, et al: tissue residue depletion and estimation of extralabel meat withdrawal intervals for tulathromycin in calves after pneumatic dart administration. j anim sci 2019;97:3714-3726. 21. smith js, mochel jp, borts dj, et al: effects of experimentally induced respiratory disease on the pharmacokinetics and tissue residues of tulathromycin in meat goats. j vet pharmacol ther 2019;42:420-429. 22. o'connor am, gould sa, coetzee jf, et al: what is the evidence? j vet clin north am food anim 2011;239:314-316. 23. kleinhenz m, van engen n, smith j, et al: the impact of transdermal flunixin meglumine on biomarkers of pain in calves when administered at the time of surgical castration without local anesthesia. livest sci 2018;212:1-6. 24. breuer r, merkatoris p, tepley s, et al: treatment of cerebrospinal nematodiasis in a boer buck. vet rec case rep 2019;7:e000706. 25. smith j, klostermann c, harm t, et al: abomasal hamartoma in a la mancha wether. vet rec case rep 2017;5:e000515. 26. newby nc, tucker cb, pearl dl, et al: short communication: a comparison of 2 nonsteroidal antiinflammatory drugs following the first stage of a 2-stage fistulation surgery in dry dairy cows. j dairy sci 2013;96:6514-6519. 27. bleul u, hollenstein k, kahn w: laparoscopic ovariectomy in standing cows. anim reprod sci 2005;90:193-200. 28. stormont g, deibert cm: surgical fire safety. statpearls. treasure island; statpearls publishing llc: 2020. 29. gravante g, venditti d: postoperative anal stenoses with ligasure hemorrhoidectomy. world j surg 2007;31:245-245. 30. selli c, turri fm, gabellieri c, et al: delayed-onset ureteral lesions due to thermal energy: an emerging condition. arch ital urol androl 2014;86:152-153. 31. abrahamsen ej: chemical restraint and injectable anesthesia of ruminants. vet clin north am food anim 2013;29:209-227. 32. smith js, sheley m, chigerwe m: aspiration pneumonia in two tibetan yak bulls (bos grunniens) as a complication of ketamine-xylazine-butorphanol anesthesia for recumbent castration. j zoo wildl med 2018;49:242-246. 33. coisman jg, case jb, shih a, et al: comparison of surgical variables in cats undergoing single-incision laparoscopic ovariectomy using a ligasure or extracorporeal suture versus open ovariectomy. vet surg 2014;43:38-44. 34. taskin s, sukur ye, altin d, et al: bipolar energy instruments in laparoscopic uterine cancer surgery: a randomized study. j laparoendosc adv surg tech a 2018;28:645-649. 35. farstvedt eg, hendrickson da: intraoperative pain responses following intraovarian versus mesovarian injection of lidocaine in mares undergoing laparoscopic ovariectomy. j am vet med assoc 2005;227:593-596. 36. baniadam a, afshar fs, ahmadian f: analgesic effects of tramadol hydrochloride administered via caudal epidural injection in healthy adult cattle. am j vet res 2010;71:720-725. 37. fierheller ee, caulkett na, bailey jv: a romifidine and morphine combination for epidural analgesia of the flank in cattle. can vet j 2004;45:917-923. 38. hubert jd, burba dj, moore rm: evaluation of a vessel-sealing device for laparoscopic granulosa cell tumor removal in standing mares. vet surg 2006;35:324-329. 39. petrizzi l, guerri g, straticò p, et al: laparoscopic ovariectomy in standing mule mares. j equine vet sci 2020;84:102857. 40. smith se, devine dv: hand-assisted laparoscopic ovariectomy and colpotomy in standing mares. vet surg 2013;42:586-590. 41. seabaugh ka, schumacher j: urogenital surgery performed with the mare standing. vet clin north am equine 2014;30:191-209. clinical theriogenology • volume 13 number 1 • march 2021 66   42. röcken m, mosel g, seyrek-intas k, et al: unilateral and bilateral laparoscopic ovariectomy in 157 mares: a retrospective multicenter study. vet surg 2011;40:1009-1014. 43. koch dw, easley jt, hatzel jn, et al: prospective randomized investigation of topical anesthesia during unilateral laparoscopic ovariectomy in horses. vet surg 2020;49 suppl 1:o54-o59. 44. shoemaker rw, read ek, duke t, et al: in situ coagulation and transection of the ovarian pedicle: an alternative to laparoscopic ovariectomy in juvenile horses. can j vet res 2004;68:27-32. 45. denardo ga, becker k, brown no, et al: ovarian remnant syndrome: revascularization of free-floating ovarian tissue in the feline abdominal cavity. j am anim hosp assoc 2001;37:290-296. 46. dunay mp, németh t, makra z, et al: laparoscopic cryptorchidectomy and ovariectomy in standing horses using the enseal® tissue-sealing device. acta vet hung 2012;60:41-53. clinical theriogenology • volume 13 number 1 • march 202167 1 contact katelyn waters kmw0030@auburn.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9643, http://dx.doi.org/10.58292/ct.v15.9643 review report diagnosis of penile denervation in the bull: a review katelyn waters,a richard hopper,a jamie m. douglasb adepartment of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa bdepartment of pathobiology and diagnostic investigation, college of veterinary medicine, michigan state university, east lansing, mi, usa abstract healthy bulls with satisfactory breeding potential are important to the beef cattle industry. erection, mounting, intromission, and ejaculation with deposition of semen into the cow’s reproductive tract define successful mating for a breeding bull. healthy bulls with satisfactory breeding potential are a vital part of the beef cattle industry. while injuries to the penile nerves are relatively uncommon, damage to the dorsal penile nerves is often a career ending injury for a bull. disease processes that commonly affect the dorsal nerve of the penis include penile hematomas (via rupture of the tunica albuginea) and complex preputial injuries extending into penile tissues. penile hematomas, cavernosal shunts, and complex preputial injuries can all cause failure of the penis to effectively achieve intromission. evidence of failed intromission is most commonly described as continual searching of the bull’s glans penis during mounting and absence of the ejaculatory lunge. disturbance of the dorsal nerve of the penis is strongly suspected following a test mating where no structural or musculoskeletal abnormalities are noted, yet the bull fails to achieve intromission. definitive diagnosis of denervation of the dorsal nerve of the penis requires advanced diagnostics. the sensory nerve conduction velocity test evaluates nerve functional integrity via measurements of amplitude and velocity of delivered electrical signals. an efficient and rapid diagnosis of dorsal penile nerve injury is important to provide the owner a useful prognosis so that they can make an appropriate management decision to diminish economic losses. keywords: bull, penile, denervation, nerve, conduction, velocity introduction the act of mating, which includes erection, mounting, intromission, and ejaculation in the bovine species is a rapid process that can be difficult to witness from afar as the entire process lasts approximately 15 seconds.1,2 when bulls present for infertility, whether that be due to a high percentage of open cows in the herd or inability to properly breed, the first step is to perform a bull breeding soundness examination (bbse). a bbse following the standards set by society for theriogenology includes, a physical exam, examination of the accessory sex glands (figure 1), scrotal circumference, and evaluation of a sperm motility and morphology. evaluation of libido or breeding ability is not included in a standard bbse in the united states. when a bull suspected of infertility successfully passes a bbse the next step in the infertility workup is to perform a test mating. a test mating will allow visualization of the act of coitus while allowing confirmation that the bull has the libido and structural soundness to achieve an erection, mount the female, and successfully achieve intromission and ejaculation. review of normal anatomy and normal mounting innervation to the penis is supplied by the paired pudendal nerves which eventually branches into the paired dorsal nerves of the penis (figure 2).1,3 injuries that result in the inability to successfully achieve intromission most commonly involve both of the paired dorsal nerves of the penis.2 this was proven by beckett et al. when bulls that underwent unilateral dorsal penile neurectomies were still able to achieve intromission and ejaculation; however, time from mounting to copulation and ejaculation was prolonged.4 unilateral neurectomy did result in the inability of one bull to complete copulation and ejaculation, though it was noted that the affected dorsal nerve contained over 75% of the innervation for the penis of that particular bull. this finding lead to speculation that if there is an uneven distribution of sensory neurons in the paired dorsal nerves, unilateral neurectomy can prevent copulation.5 in the aforementioned study, bilateral neurectomy of the dorsal nerves of the penis was performed and all affected bulls were unable to successfully achieve intromission and did not ejaculate.2,4 mailto:kmw0030@auburn.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9643 2 citation line: clinical theriogenology 2023, 15, 9643, http://dx.doi.org/10.58292/ct.v15.9643 etiology injuries such as rupture of the tunica albuginea with resultant hematoma formation can directly or indirectly cause insult to the dorsal nerve of the penis.5 direct insults to the penile nerves occur when the nerves are directly injured. for example, with laceration of the glans penis (figure 1) or tunica albuginea rupture, profuse hemorrhage from the ruptured corpus cavernosum penis could directly affect the penile nerves. indirect insult occurs in instances of scar tissue remodeling or entrapment of the penis by scar tissue.5 penile hematomas occur at the dorsal aspect of the distal bend of the sigmoid flexure (figure 1) when the penis is at peak erection.1 rupture of the tunica albuginea occurs following an acute, severe increase in the corpus cavernosum penis pressure due to a sudden bending of the penis. such bending can occur when the bull makes the ejaculatory lunge and fails to complete intromission, or because of similar bending associated with a sudden fall of either the bull or female during mating.1 clinically, rupture of the tunica albuginea is seen as a swelling directly cranial to the scrotum.1 penile hair rings associated with the homosexual riding of young bulls housed with other young bulls, along with masturbatory behavior are other examples that threaten to result in indirect insult to the penile nerves. injury in these instances often results from avascular necrosis of the penis and associated damage of the dorsal nerves of the penis due to the constriction of the penis from the entrapped hair.1 diagnostics diagnostics for accessing the function of the dorsal nerves of the penis include test mating, semen collection utilizing an artificial vagina, a direct noxious stimulus to the penile skin, and nerve conduction velocity testing. the ability to achieve intromission (assess nerve function) is not routinely performed during a standard bbse. if penile denervation is suspected, utilization of hemostats or fingers to manually pinch the skin on the free portion of the penis, in attempt to elicit a withdrawal response, can be performed.2 however, absence of a withdrawal response does not confirm lack of penile sensation. some normal bulls simply exhibit a decreased withdrawal reaction.2 the most commonly utilized diagnostic test for evaluating penile nerve innervation, ability to achieve a complete erection, and presence of any structural abnormalities, such as a ventral deviation of the penis is through the utilization of a test mating.1,4,5 while it is possible to perform a test mating with limited facilities, the test will always require the presence of a cow or heifer in standing heat. ideally, evaluations of mating ability should be performed in a quiet small area that ensures room for movement or, better, in larger facilities employing stanchions to restrain the mount animal. the test mating area should also allow adequate observation of the copulatory event and limit any distractions for the bull.2 the area should be level, providing for secure, well-maintained footing to ensure that adequate stabilization of the pelvic limbs can occur during mounting and the ejaculatory thrust. this area should also include viewing areas from all sides of the pen or enough room to safely observe the mating while in the pen along-side the animals. the following events describe what usually occurs in a normal test mating. initially, the bull will approach the female from the side, after which the bull will nuzzle the vulva once at her hindquarters. the bull then traditionally exhibits the flehmen response, ensuring the female is in estrus.1,2 once estrus is confirmed, the bull’s prepuce will drop while the erection process is occurring. just prior to mounting the pelvis of the cow the bull will chin press on the cow’s rump. provided the cow is standing still, the bull will then mount the cow. once mounted, the bull will quickly align himself directly behind the cow and make two to three searching motions with the glans of the penis near the vulva. once the vulva is located, a single ejaculatory lunge facilitates intromission and ejaculation, after which the bull will dismount.2 bulls experiencing penile denervation will elicit normal test mating behavior until they have mounted the cow, affected bull’s often place their penis alongside the tail head of the cow or on the udder ventral to the cow’s vulva, rather than on the vulva itself. the normally observed searching motions of the glans are not performed and intromission does not occur.2 (see video 1 for test mating). the use of a testing mating using an artificial vagina can also be utilized for penile nerve assessment. once the bull has figure 1. gross reproductive anatomy of the bull, courtesy of jamie m douglas. figure 2. bull in right laterally recumbency, penis is held in extension via the backhaus towel clamp secured by the dorsal apical ligament. associated cross section of the bull distal penis, courtesy of jamie m douglas. http://dx.doi.org/10.58292/ct.v15.9643 https://clinicaltheriogenology.net/index.php/ct/article/view/9643/15537 citation line: clinical theriogenology 2023, 15, 9643, http://dx.doi.org/10.58292/ct.v15.9643 3 been properly stimulated to cause protrusion and erection, an artificial vagina may be placed over the penis.2 normal bulls ejaculate soon after the placement of the artificial vagina. bulls with injury to the dorsal nerves of the penis fail to ejaculate.2 false negative test results can occur when performing test mating with an artificial vagina if the artificial vagina was not correctly prepared, the bull is not fully stimulated, or if the bull is experiencing musculoskeletal pain associated with mounting.2 the gold standard for definitive diagnosis of penile denervation in bulls is evaluation of the sensory ability of the dorsal nerves of the penis by evaluating the nerve conduction velocity of the suspected nerves, following a failed test mating. this technique described by mysinger et al. will be further discussed in the following section.6 sensory nerve conduction velocity testing while most bulls suffering from penile denervation injuries can be diagnosed via a test mating or via history of reproductive decline in the form of open females in the herd, an advanced diagnostic modality of sensory nerve conduction velocity testing (snct) is available.5 the electromyography, or the recording of electrical activity in muscle tissue is a relatively fast diagnostic procedure with almost instant results. while the test mating requires little to no specialized equipment, snct requires specific specialized equipment and trained personnel. equipment needed for an snct includes: a tilt table with restraint and an electromyography machine (emg). bulls being tested should be placed on the tilt table in right lateral recumbency, and their penis must be extended and remain extended for the duration of the procedure.2,6 after extension, 3 electrodes are placed along the penis through the skin to the level of the tunica albuginea as described by mysinger et al.2,6 (figure  3). for complete, detailed description of scnt in the bull and test interpretation, please refer to mysinger et al. and wolfe et al.2,6 bulls with normal penile nerve innervation exhibit the presence of nerve conductivity as seen in figure 4. while results can be skewed if the test is interpreted incorrectly or the electrodes are placed improperly, the snct provides for a definite diagnosis of penile denervation. in bulls experiencing penile sensory denervation, they exhibit lack of nerve conductivity as seen in figure 5. as with many further diagnostic testing modalities, cost is often a limiting factor for performance of an scnt. currently at our referral hospital, the cost associated with the scnt and the interpretation from a boarded neurologist is approximately 150 dollars. however, when discussing the diagnosis of a valuable bull, the cost associated with scnt maybe minute. an example of the use of the scnt from the authors’ experience is its use in a 4-year-old, shorthorn bull that presented to the clinic with history of a successful breeding as a 2-year-old, followed by low pregnancy rates observed 3-year-old breeding season. his previous bbse information was unknown. at presentation, a bbse was performed, and he was found to be a satisfactory potential breeder according to the standards set by the society for theriogenology. the bull had a scrotal circumference of 37.5 cm. parameters of his sperm morphology were 70% normal spermatozoa, 11% with head abnormalities, and 19% with mid-piece abnormalities. during the bbse, touching of the free portion of the penis elicited spiral deviation of the penis. of note, spiral deviations can occur during a bbse when utilizing electro-ejaculation and should not be figure 3. example of the restraint of the bull and placement of the proximal electrodes, photo courtesy of jessica cowley. figure 4. normal evoked potentials, photo courtesy of amy yanke. figure 5. example of an abnormal nerve conduction test on the electromyography machine, photo courtesy of jessica cowley. http://dx.doi.org/10.58292/ct.v15.9643 4 citation line: clinical theriogenology 2023, 15, 9643, http://dx.doi.org/10.58292/ct.v15.9643 diagnosed solely on the presence during the bbse.5 seidel and foote even demonstrated that a spiral deviation of the penis can occur normally once intromission has occurred by utilization of a transparent artificial vagina.5,7 after passing of the bbse by the shorthorn bull, two test matings were performed. during the first test mating, though erection of the penis was appreciated, neither protrusion of the penis nor mounting of the teaser cow in estrus was achieved. during the second test, mating, erection, protrusion of the penis, and mounting of the cow in estrus were observed. of note, the bull subsequently made several searching motions upon mounting the cow. however, despite these searching motions, the bull failed to achieve intromission. each time the bull’s penis came in contact with the cow’s perivulvar region, the penis would undergo spiral deviation (corkscrew), the deviation rapidly resolving moments after contact. several days following the test mating, the bull underwent scnt and was found to have a lack of nerve conductivity at the level of the free portion of the penis just proximal to the glans penis. this bull is an interesting case, as it is possible the bull was experiencing both a spiral deviation due to a laxity in the dorsal apical ligament as well as sensory denervation of the distal penis. in this case, the bull would be a candidate for semen collection and cryopreservation to preserve his genetics. conclusion penile denervation injuries are career ending for natural breeding, but genetics can be preserved with the utilization of semen collection and cryopreservation. the scnt serves as the testing modality for the definitive diagnosis of penile sensory denervation in bulls. benefits of performing scnt include, conclusion of a definitive diagnosis and confirmation that the penile nerves are intact, prior to surgical intervention for other penile or preputial injuries. downfalls for scnt are the need for specialized equipment and training, the tedious nature of performing and interpreting the test, along with the associated cost. the gold standard workup for evaluation of the sensory function of the dorsal nerves of the penis is the utilization of a test mating followed by definitive diagnosis of denervation of the sensory nerves of the penis via the sensory nerve conduction velocity test. acknowledgement thank you to drs. amy yanke and jessica cowley for the figures, and dr. misty edmondson for her management of the clinical case. conflict of interest and funding none to declare. references 1. wolfe df: abnormalities of the bull–occurrence, diagnosis and treatment of abnormalities of the bull, including structural soundness. animal 2018;12(s1):s148–s157. doi: 10.1017/ s1751731118000939 2. wolfe df, moll hd: examination and special diagnostic procedures of the penis and prepuce: bulls, rams, and bucks. in: wolfe df, moll hd: editors. large animal urogenital surgery. 2nd edition, media, baltimore, maryland; williams & watkins: 1999: 221–231. 3. nabors, b: anatomy of the reproductive system of the bull. in: hopper r: editor. bovine reproduction. 2nd edition, hoboken; wiley-blackwell: 2021:1–10. 4. beckett sd, hudson rs, walker df: effect of local anesthesia of the penis and dorsal penile neurectomy on the mating ability of bulls. j am vet med assoc 1978;173(7):838–839. 5. maxwell, h: inability to breed due to injury or abnormality of the  external genitalia of bulls. in: hopper r: editor. bovine reproduction. 2nd edition, hoboken; wiley-blackwell: 2021: 155–172. 6. mysinger pw, wolfe df, redding rw, et al: sensory nerve conduction velocity of the dorsal penile nerves of bulls. am j vet res 1994;55(7):898–900. 7. seidel ge, foote rf: motion picture analysis of ejaculation in  the bull. j reprod fertil 1969;20:313–317. doi: 10.1530/ jrf.0.0200313 http://dx.doi.org/10.58292/ct.v15.9643 https://doi.org/10.1017/s1751731118000939 https://doi.org/10.1017/s1751731118000939 https://doi.org/10.1530/jrf.0.0200313 https://doi.org/10.1530/jrf.0.0200313 introduction there are 3 main ways stallion semen is presented for artificial breeding after collection in an artificial vagina (av): fresh semen, cool-stored (cs) semen (extended and packaged in a container that cools the semen as it is transported), and frozen-thawed (ft) semen (processed in a freezing extender and frozen for later use). cool-stored stallion sperm was introduced to the warmblood industry in the 1980s to provide wider distribution of stallion sperm throughout north america.1,2 subsequently, the application was accepted in many breed registries, including standardbred and quarter horse breeds. this technique introduced the concept of preserving stallion sperm in a liquid-cooled form for a short-term (24 72 hours), thus facilitating widespread distribution without the necessity to move either the stallion or mare. this allowed mares and stallions to remain on their home farm while reducing potential disease exposure and travel stress. as with the introduction of any new technique, concerns arose regarding the maintenance of sperm quality and how best to maximize fertility. initially, semen was extended using traditional dilution ratios for fresh semen (i.e. 1:1 or 1:2, semen:extender). although this is effective in many cases, there are ‘poor cooler’ stallions. an initial cause of this phenomenon was related to a combination of high seminal plasma concentrations (50% for 1:1 or 33% for 1:2 dilution ratio) and inadequate extender components for the number of sperm in the sample. to address this problem, the concept of diluting semen to a cool-stored and frozen-thawed stallion semen: thoughts on collection, evaluation, processing, insemination, and fertility camilo hernández-avilés, charles love department of large animal clinical sciences, college of veterinary medicine and biomedical sciences texas a&m university, college station, tx abstract semen evaluation is a fundamental part of breeding soundness examination of males, including stallions. new technologies have been implemented in both the research arena and clinical practice for analysis of stallion sperm; therefore, theriogenologists and general equine practitioners should become familiar with principles, technical advantages, and limitations of each of these assays. more importantly is the interpretation of results and its translation to clinical situations. common methods used for storage of stallion sperm (e.g. cooled storage or freezing/thawing) can affect tests that should be performed when establishing quality and potential fertility of a semen sample. the present document describes routine semen analysis assays performed at the author’s laboratory when determining sperm quality and potential fertility of stallions. keywords: stallion, breeding soundness, testes, semen quality, fertility final sperm concentration (i.e. 30 x 106/ml of final volume) was introduced.3 this approach usually reduced effects of seminal plasma. nevertheless, there are 2 challenges regarding coolstored semen extension; providing enough metabolic nutrients to maintain longevity of sperm quality (motility, viability, and dna quality) and having an amount of seminal plasma that is not detrimental to sperm longevity. evaluation of stallion sperm quality is a fundamental part of the breeding soundness examination4 and includes the determination of sperm motility, viability, concentration, total sperm in the ejaculate, and sperm morphologic features. routinely, the evaluation of sperm quality is limited to sperm motility using a technique such as light or phase-contrast microscopy. additionally, sperm number is determined either by direct (e.g. hemocytometry that relies on identification of sperm) or indirect (e.g. spectrophotometry that uses light transmittance) methods.5 combining sperm concentration and motility information allow the practitioner to calculate a sperm ‘dose’ and the number of mares that can be bred from an individual ejaculate. in addition, these sperm parameters are used as part of a breeding soundness examination to determine the suitability of a stallion for breeding or aid in the diagnosis of subfertility.4 although these methodologies (motility and concentration determination) have their limitations, they were sufficient when the horse industry relied exclusively on fresh semen. however, as the introduction of cool-stored and frozen-thawed semen clinical theriogenology 2021; 13: 280 became popular and widespread, the horse industry was faced with more instances of judging stallions as ‘subfertile’ due to a change in the breeding method. this necessitated a more thorough understanding of sperm quality features in various processing techniques and how the results would be interpreted. one area of particular importance became the evaluation of semen handling technique (e.g. av preparation, handling, and cleaning; extender-type preparation and use) and its effects on sperm quality. the evaluation of sperm motility alone became insufficient and the introduction of additional sperm quality features became relevant.5 semen collection semen collection procedures for cooled and frozen are similar to those employed for fresh semen. these procedures include correct maintenance, storage, and cleaning of the av; proper extender handling and usage; and proper handling of the semen during and following collection. it is likely that what appear to be relatively minor unnoticed breaks in these procedures have limited noticeable impact when fresh semen is inseminated can have a substantial impact when semen is stored for an extended interval. semen processing processing semen after ejaculation should be a planned, efficient activity, realizing that any break in the system can result in a long-term decline in sperm quality. there are many commercial semen extenders, providing the practitioner with a variety of choices. in general, these extenders provide a similar level of protection to the sperm and there is little difference in sperm quality when they are compared. occasionally, an individual extender will impart a more beneficial effect on sperm quality than another. initially, after collection, a raw sample should immediately be evaluated using a phase-contrast microscope, to provide a general idea of sperm motility and sperm morphology. although this is not an ideal method to evaluate sperm motility because of the inconsistent appearance of sperm concentration, it is nevertheless important to be aware of any potential concerns regarding sperm quality as processing proceeds. if sperm motility is only evaluated following extension, the clinician will be unable to differentiate poor sperm quality due to an inherent stallion problem or collection procedure, or because of a potentially toxic semen extender. sperm in fresh semen do not face the same challenges as those in cs or ft semen. the primary difference is the potential reduction in sperm quality of the inseminate of ct or ft compared to fresh semen due to the storage conditions. although there are many instances when the fertility of cs and ft are good and may approach fresh semen, the variation in sperm quality within those methods is broader than with fresh. in effect, what may be very good when evaluated in the fresh state can be very different when cooled or frozen. this wide variation is a challenge to the practitioner because evaluation of sperm motility alone is often insufficient to render an adequate diagnosis and management plan. other differences among fresh, cs, and ft include the inability to determine sperm concentration of cs and ft semen using spectrophotometric techniques, due to interference by nonsperm components (e.g. milk products and egg yolk). this limits the practitioners’ ability to determine total sperm in a cs or ft sample received for breeding unless they use hemocytometry or a more expensive alternative (e.g. nucleocounter®sp-100tm [chemometec, allerød, denmark]) that can determine sperm concentration in extended samples.6 sperm motility and viability sperm motility can be determined microscopically or using a computer-assisted motility analysis (casa) system.7 although microscopy is more subjective, primarily due to the broad range in variation among readers, casa systems, while more objective, are not without their own limitations. the limitations of both techniques become more relevant when cs and ft sperm are evaluated since sperm quality and sperm numbers for insemination are often limited compared to fresh semen. recognizing a limitation in sperm quality becomes more important since ‘suitability’ for cooling and freezing is performed following a ‘test’ cool or freeze. addition of sperm viability (i.e. plasma membrane intactness) test has been an important adjunctive to evaluation of stallion sperm, especially for cs sperm. even very low sperm motility may not be diagnostic or predictive of poor fertility, especially when cooled sperm are evaluated. low sperm motility can occur in the presence of either high or low sperm viability.8 the former (low motility/high viability) may result in normal fertility, whereas the latter is associated with low fertility. it is unclear why the low motility/high viability circumstance occurs, but elevated seminal plasma concentrations (> 5 10%) in the stored sample may be a factor. seminal plasma is not a generic component of the semen and differs among stallions with respect to its ability to confer a positive or negative effect on the sperm quality of stored sperm. therefore, if the clinician is presented with a ‘poor’ cooler, an effort should be made to determine the viability to clarify the prognosis. the simplest method is the eosin-nigrosin stain commonly used for morphologic evaluation. this stain also ‘doubles’ as a viability stain rendering nonviable sperm a pink hue compared to white, viable sperm. in addition, the nucleocounter®sp-100tm can also determine sperm viability as well as concentration. a note on sperm motility parameters sperm motility, regardless of the method used, has long relied on total and progressive motility, with progressive motility considered the most ‘sensitive’ measure. as we move forward, we should consider that progressive motility is an archaic, outdated clinical theriogenology 2021; 13: 281 term that has no meaning or relevance to clinical theriogenology. why? the inability to clearly define this endpoint, either by definition or objective casa evaluation, is a limitation. nevertheless, even if there was consensus regarding a definition, what is the relevance of the value? for background, the initial intent of the term was to infer a population of sperm in a sample that had better motility because they were progressive rather than not. the lack of progressivity also suggested pathology was associated with those sperm. we now know that this is largely incorrect. the quality of sperm motion varies considerably, with the motility of some stallions characterized by a high percent of nonprogressive sperm; however, these individuals have other sperm characteristics (viability, dna quality, and morphology) that are associated with fertile stallions, and they themselves are very fertile. this should not be interpreted only as an academic point. rendering an incorrect interpretation of a stallion’s sperm quality based solely on a low percent of progressive motility may have substantial economic impact on a stallion’s value as a potential breeding prospect. the concept of ‘doing no harm’ is relevant! sperm concentration this sperm quality measure is often overlooked and furthermore substantial misinterpretations are possible due to the method of evaluation. as mentioned above, these methods include a hemocytometer, spectrophotometric-type machines, as well as cytometry, including the nucleocounter®sp-100tm and flow cytometry. hemocytometry is the only true direct method, in which sperm are visually identified, making it a readily available specific test. the limitation of hemocytometry is that only a limited number of sperm are counted (100’s) that may not represent an ejaculate with billions of sperm. an additional concern is reader variation in preparation and evaluation of the sample. these factors can have a substantial role and may result in inaccurate values. spectrophotometric methods provide an efficient technique to determine sperm concentration but are limited by a lack of sperm specificity, especially when the sample is diluted, in which case the opacity of the seminal plasma itself may cause an artificial increase of 10 20 x 106 sperm/ml in the final concentration. semen contaminants such as nonsperm debris (smegma) may also cause spurious values. a false high-value results in the perception that sperm numbers are adequate, when in fact subthreshold sperm numbers are inseminated. the practitioner should also be concerned about the concentration of the sperm sample they have received (either cooled or frozen). evaluation of sperm motility alone is an insufficient measure of sperm quality. excellent sperm motility associated with low sperm numbers can result in low fertility. morphologic features evaluation of stallion sperm morphologic features differentiates normal and abnormal sperm forms.9-11 various techniques to prepare sperm include a dry mount in which the sperm is combined with a background stain (e.g. eosin-nigrosin [en]) and allowed to dry on the slide. a wet-mount technique can be used in which the semen sample is fixed in a buffered-formol saline solution. the en technique has the advantage of also evaluating the sperm viability and being a quicker and simpler technique requiring only a light microscope and low magnification. however, the en technique can induce artifactual morphologic changes (e.g. bent tails and detached heads) due to composition changes in the stain or preparation error. in addition, the en technique, due to the lesser quality of the image, may result in the clinician missing subtle, but fertility-limiting abnormalities such as abnormal midpieces. the bfs technique is usually performed at a magnification of 1000 x using either phase-contrast or differential interference microscopy, techniques that provide enhanced resolution compared to light microscopy. evaluation of sperm morphology is the only test that reflects the stallion’s intrinsic sperm quality. our laboratory has maintained a similar classification system as the stallion breeding soundness evaluation manual. in addition to percent normal sperm, the types of abnormalities include: abnormal acrosomes, heads, and midpieces; proximal and distal droplets; detached normal heads; bent midpieces and tails; coiled tails, and premature germ cells. identifying specific abnormalities rather than combining abnormalities into large categories such as primary/ secondary or major/minor classification systems is preferred, since individual sperm morphogenetic features can aid in the diagnosis, prognosis, and treatment of certain conditions. some abnormalities are associated with transient conditions such as sperm accumulation (e.g. detached abnormal heads, hairpin tail, distal droplets) whereas others are associated with testicular dysfunction (abnormal heads and midpieces, coiled tails, and premature germ cells). additionally, certain abnormalities, when they occur together in sufficient numbers, tend to be associated with a reduction in fertility. sperm dna quality sperm dna quality can be determined by the sperm chromatin structure assay (scsa), a technique that uses a metachromatic dye, acridine orange, and a flow cytometer.12 this technique measures the susceptibility of sperm dna to be denatured after treatment with an acid-detergent solution. upon exposure of the stained sperm to a laser, the acridine orange signal will shift from green fluorescence (double-stranded dna), to red fluorescence (single-stranded dna), if damage to the dna backbone has been sustained. studies using scsa have determined the relationship between sperm dna integrity and stallion fertility.13 in stallions with poor sperm quality as consequence of testicular dysfunction, a higher extent of dna-susceptibility to denaturation is observed.14 such changes are similar to those observed in stallions with low fertility.13 furthermore, methods used for stallion sperm storage can have also a detrimental effect on sperm dna integrity. for clinical theriogenology 2021; 13: 282 example, studies conducted in our laboratory suggested that increased concentrations of seminal plasma (10 20%, v:v) in cooled stored semen15 or storage time and temperature16 increased the rate of sperm dna damage. these findings have profound clinical implications, particularly when very diluted ejaculates are processed by simple dilution prior to shipping. since the sperm concentration in the ejaculate is low, in theory less semen extender should be added to respect the ‘minimal’ sperm concentration that should be accommodated in a dose of cooled semen. also, since some stallions are considered ‘poor-coolers’, some practitioners ship semen doses at 15 25°c. we have observed that both scenarios have reduced sperm quality and potential fertility of samples sent to our laboratory for sperm quality analysis. our experience with the scsa have led us to consider this assay, in conjunction with sperm morphology, as fundamental semen quality tests, making them valuable to draw conclusions regarding the intrinsic sperm quality of stallions. semen centrifugation and processing it is in the best interest of the clinician to attempt to provide processing conditions that maintain sperm motility for the storage period of interest. semen centrifugation using a cushion media has become a common technique to ensure that sperm quality is maximized. semen centrifugation, like all techniques, must be performed correctly or complications can result in the ‘treatment being worse than the disease.’ centrifugation has several advantages including standardization of seminal plasma concentration (< 10%), facilitate storage of sperm at a higher sperm concentration (250 x 106 sperm/ml); use of a deep-horn insemination technique, i.e. insemination of small-volume, high-concentrate sperm deposited at the tip of the uterine horn ipsilateral to the site of anticipated ovulation. cushion centrifugation is not restricted to fresh semen that is to be cool-stored but can also be exploited with shipped semen that is received, especially in a large volume sample with sperm of average to below-average quality. the process will ‘freshen’ the sample by removing the seminal plasma/extender supernatant and replacing it with a fresh extender, in a small volume (5 10 ml), enabling deep-horn insemination. inseminating a smaller volume provides assurance that less semen volume will be refluxed out the vagina and more retained close to the site of ovulation, thus assuring that more sperm will colonize the oviduct. this effect may be particularly relevant for stallions with ‘marginal’ sperm quality. we have observed that centrifugation of stored semen can increase the longevity of stored sperm in circumstances when sperm motility is low, due to high concentrations of seminal plasma, while sperm viability remains high.17 a small volume of the extended sample should be retained for evaluation after 24 hours to assure sperm quality and to address any concerns the recipient of the sample may have after they receive the sample. it is also recommended that the sperm quality of the stallion be evaluated before the anticipated start of the breeding season to address potential concerns and modifications to routine semen processing. sperm quality evaluation a thorough semen quality evaluation can be performed before the breeding season, with particular attention to the ability of the stallion’s sperm to tolerate the cool-storage process. this evaluation should include total sperm motility, viability, and ensuring that an accurate determination of sperm concentration can be performed. seminal plasma can have multiple effects on the longevity of cool-stored sperm quality. historically, it was assumed that if a decline in sperm motility occurred after storage it was solely caused by a pathologic (i.e. toxic) effect of seminal plasma. when sperm viability was introduced as a recommended adjunct to the evaluation of sperm quality, it was recognized that sperm motility could decline dramatically after 24 hours of storage, yet sperm viability remained high.8,17 this contrasted with stored samples in which both motility and viability were low after storage. we also became aware that those samples in which viability was retained also resulted in good fertility compared to samples that had poor viability. the relevance to the clinician is that ‘poor’ sperm motility after storage should not be assumed to be ‘subfertile;’ rather, those samples should be also evaluated for viability. evaluation of sperm morphological features should be considered an essential component of a routine evaluation of cool-stored sperm. ‘poor’ cool-stored sperm quality commonly originates from features identified in a stallion’s morphology. for instance, sperm storage (sperm ‘accumulators’ or spermiostasis) can render low sperm motility in cool-stored samples even when initial motility appears good. sperm accumulation has a variety of morphological manifestations including detached heads, distal droplets, and distal midpiece reflexes (hairpin tails) in varying frequencies. the clinician should be aware that the presence of a limited number of these defects may signal a ‘subclinical’ manifestation of an accumulator, which may result in a stored sample of poor quality. although sperm are motile initially, they are ‘aged’, and may be more ‘fragile’ to cooling and freezing. interpretation (epicrisis) of sperm quality the evaluation of stallion sperm quality intends to render an opinion/interpretation of the results as they relate to the patient from which the sample was derived. the interpretation will consider the history of the stallion. additionally, history of the semen sample (e.g. farm of origin; who collected and processed the sample; conditions under which the sample was exposed prior to your evaluation; fertility history; the difference in fertility between on-farm breedings with fresh versus coolshipped sample) must also be considered in instances where the clinician is only presented with the semen and does not have first-hand contact with the stallion. ideally, the clinician would like to know the initial sperm quality (motility, viability, and dna quality) so that a comparison can be made between sperm clinical theriogenology 2021; 13: 283 quality at the time of collection and the cool-stored or frozen sample. if the quality of the stored sample is inadequate, then the question becomes whether the sperm quality is inherent to that stallion (i.e. it came out of the stallion in that condition) or whether it was acquired during storage. prognosis will tend to be worse if the condition is inherent to the stallion, although the possibility exists that short-term stress may have resulted in a transient decline in sperm quality (e.g. elevated ambient temperature during summer). in contrast, a precipitous decline in sperm quality due to storage may be ‘treated’ by a change in the conditions (e.g. collection technique, av preparation, semen extender composition, centrifugation, etc.) prior to storage. evaluation of the sperm morphologic features is helpful to determine the cause of poor sperm quality due to inherent versus acquired causes. a high percent of morphologically normal sperm should result in stored samples that exhibit good sperm motility and viability, whereas low motility and especially viability suggest sperm conditions prior to evaluation have been suboptimal. interpretation of sperm quality in our clinical practice is based on data in tables 1 6. these tables represent studies that have attempted to provide perspective regarding the relationship between sperm quality features and fertility. data regarding the evaluation of cool-stored stallion sperm and embryo recovery rate are in table 1.18 this study measured sperm quality features and the number of sperm that were received in the shipped sample. threshold values give the practitioner perspective as to embryo recovery rate2 sperm quality values3 sperm parameter threshold1 average high average high total sperm motility (%) ≥65 93/174 (53) 153/230 (67) 50±11 (5 60) 69±5 (65 85) progressively sperm motility (%) ≥45 57/107 (53) 189/287 (64) 36±11 (0 45) 59±6 (50 75) viable sperm (%) ≥71 70/138 (51) 178/269 (66) 56±18 (0 71) 80±5 (71 92) morphologically normal (%) ≥47 46/97 (47) 170/269 (63) 35±10 (5 46) 64±10 (47 88) total sperm number (x 109) ≥1.14 64/129 (50) 181/272 (67) 0.78±0.22 (0.20 1.12) 1.91±0.86 (1.14 6.67) total motile sperm (x 109) <0.60 53/114 (46) 189/283 (67) 0.4±0.1 (0.03 0.6) 1.2±0.6 (0.6 5.3) total morph. normal (x 109) <0.94 117/222 (53) 84/112 (75) 0.57±0.23(0.06 0.94) 1.4±0.63 (0.94 4.20) total progressively motile sperm (x 109) <0.55 61/129 (47) 181/268 (68) 0.4±0.2 (0.01 0.5) 1.1±0.5 (0.5 4.7) sperm concentration (x 106/ml) ≥31.6 72/141 (51) 182/277 (66) 22±6 (4 32) 60±48 (32 482) total viable sperm (x 106) <0.74 59/120 (49) 186/280 (66) 0.5±0.2 (0.07 0.7) 1.4±0.7 (0.8 5.6) total pmvs (x 109) <0.32 39/95 (41) 203/301 (67) 0.2±0.1 (0 0.3) 0.8±0.4 (0.3 3.8) total pmms (x 109) ≤0.37 65/121 (54) 134/210 (64) 0.2±0.1 (.01 0.4) 0.7±0.4 (0.4 3.4) % compαt ≥26.8 14/33 (42) 147/236 (62) 33±13 (27 72) 13±5 (4 27) meanαt ≤253 39/85 (46) 122/184 (66) 273±22 (254 374) 233±14 (186 254) modeαt >233 44/93 (47) 117/175 (67) 244±16 (233 374) 216±13 (165 232) table 1. sperm parameter, threshold value, the embryo recovery rate between 2 fertility groups (average and high) and sperm quality values between 2 fertility groups (mean ± sd (range) for cool-stored stallion sperm 1threshold value that separates the average and high fertility groups 2embryos recovered/total attempts (% recovery rate). between columns, all rates are different (p < 0.05) 3represents mean ± sd (range) sperm quality value for the average and high fertility groups. between columns, all rates are different. pmvs = progressively motile, viable sperm pmms = progressively motile, morphologically normal sperm reprinted from love cc, noble jk, standridge sa, et al: theriogenology 2015;84:1587-1593. clinical theriogenology 2021; 13: 284 what separates high from average fertility samples. as expected, samples with higher numbers of higher quality also result in an increased sperm recovery rate. information from a study that evaluated sperm motility, morphology, and dna quality, based on fresh sperm quality, are in tables 2-6.11,13 these data evaluated relationships among seasonal pregnancy rates, per cycle pregnancy rates, and sperm quality. stallions included in the study had no history of reduced fertility; therefore, they are assumed to represent differences in fertility among several levels of fertility. these tables are not intended to provide cutoffs or pass/fail conclusions, but rather to provide some perspective regarding interpretation of sperm quality. the reader can easily see that there is considerable variation within fertility levels. this should not be surprising, as mare and management factors have a large role in fertility outcomes.19 ‘fertile’ stallions can be ‘subfertile’ due to poor mare quality and/or poor semen handling/processing, etc. in contrast, ‘subfertile’ stallions can be ‘treated’ by breeding to mares of high reproductive quality and using enhanced breeding management and become ‘fertile’. these tables also highlight the ability of per cycle pregnancy rates to identify differences in fertility compared to seasonal pregnancy. seasonal pregnancy rate is an accumulation of breedings, whereas per cycle pregnancy rate determines the efficiency by which a stallion renders mares pregnant. two stallions can have the same seasonal pregnancy rate, with 1 being much more inefficient (i.e. less fertile) than the other. therefore, seasonal pregnancy rate is not as good at separating stallions of different fertility levels. this has led to the incorrect interpretations of sperm quality features, that stallions with different levels of sperm quality have the same fertility (due to similar seasonal pregnancy rates); thus, sperm quality is of little use in identifying those individuals of lesser fertility. 1 (n = 22) 2 (n = 11) seasonal pregnancy rate (%) 90 ± 6 (79 100) 58 ± 21 (12 77) pregnant/cycle (%) 62 ± 16 (36 100) 41 ± 17 (8 66) pregnant/first cycle (%) 62 ± 22 (0 100) 48 ± 17 (14 80) total motility (%)1 78 ± 12 (44 92) 68 ± 20 (18 88) progressive motility (%) 72 ± 12 (43 88) 63 ± 20 (16 84) rapid (%) 69 ± 12 (41 87) 60 ± 20 (13 84) moderate (%) 3 ± 2 (1 8) 3 ± 1 (2 6) slow (%) 5 ± 2 (1 11) 4 ± 1 (2 6) anticrit 0.81 ± 0.44 (0 2) 0.73 ± 0.7 (0 2.3) critical value 88 ± 5 (74 95) 87 ± 6 (73 94) linearity 77 ± 4 (69 84) 76 ± 5 (67 82) path velocity (µ/s) 193 ± 23 (157 241) 178 ± 27 (104 199) progressive velocity (µ/s)2 173 ± 19 (143 208) 157 ± 25 (88 183) table 2. the mean (+ sd) and (range) for stallion sperm motility variables for the seasonal pregnancy rate from mares bred with fresh semen seasonal pregnancy rate groupsgroup 1≥ 78% and ≤ 100%; group 2≥ 0% and < 78% n = number of stallions 1p = 0.08 2p = 0.06 reprinted from love cc: theriogenology 2011;76:547-557. clinical theriogenology 2021; 13: 285 morphology variables 1 (n = 56) 2 (n = 19) normal 51 ± 18 (11 85) 43 ± 23 (4 75) abnormal heads 12 ± 10 (0 45) 14 ± 10 (1 43) detached heads 2 ± 2 (0 10) 4 ± 10 (1 44) proximal droplets 19 ± 15 (2 60) 23 ± 17 (8 70) distal droplets 7 ± 6 (0 28) 5 ± 5 (0 17) bent midpieces 0.23 ± 0.62 (0 4) 1 ± 4 (0 17) general midpiece abnormality 7 ± 6 (1 29) 8 ± 6 (3 26) hairpin tail 4 ± 4 (0 16) 5 ± 6 (0 20) coiled tail1 2 ± 2 (0 11) 4 ± 4 (0 14) premature germ cell 1.5 ± 1.7 (0 8) 1.3 ± 2.1 (0 7) table 3. mean (± sd) and (range) for stallion sperm morphology variables for the seasonal pregnancy rate from mares seasonal pregnancy rate groups group 1≥ 78% and ≤ 100%; group 2≥ 0% and < 78% n = number of stallions 1-p = 0.02 reprinted from love cc: theriogenology 2011;76:547-557 1 (n = 8) 2 (n = 20) 3 (n = 4) seasonal pregnancy rate (%) 97 ± 4 (90 100) 86 ± 10 (50 100) 61 ± 29 (12 -100) pregnant / cycle (%) 91 ± 10 (75 100) 56 ± 6 (45 74) 32 ± 13 (8 45) pregnant / first cycle (%) 91 ± 10 (75 100) 58 ± 16 (0 88) 34 ± 14 (0 50) total motility (%)1 83 ± 5 (76 91)a 76 ± 12 (44 92)a 48 ± 21 (18 66)b progressive motility (%)2 77 ± 6 (64 85)a 71 ± 12 (43 88)a 44 ± 20 (16 63)b rapid (%)3 74 ± 9 (57 83)a 68 ± 12 (41 88)a 42 ± 20 (13 60)b critical value4 87 ± 7 (74 95)a 89 ± 4 (81 95)a 83 ± 7 (73 90)b linearity 77 ± 3 (74 84) 77 ± 4 (67 82) 74 ± 4 (69 78) path velocity (µ/s)5 196 ± 26 (163 230)a 190 ± 19 (157 214)a 162 ± 41 (104 194)b progressive velocity (µ/s)6 174 ± 20 (146 206)a 171 ± 17 (143 208)a 139 ± 34 (88 166)b percent pregnant / cycle groups – group 1≥ 76% and ≤ 100%; group 2≥ 46% and < 76%; group 3≥ 0% and < 46%. n = number of stallions 1-p < 0.0001; 2-p < 0.001; 3-p < 0.0002; 4-p < 0.04; 5-p < 0.04; 6-p < 0.008 reprinted from love cc: theriogenology 2011;76:547-557 table 4. the mean (± sd) and (range) for stallion sperm motility variables for the percent mares pregnant/cycle clinical theriogenology 2021; 13: 286 morphology variables 1 (n = 11) 2 (n = 49) 3 (n = 13) normal1 67 ± 8 (50 76)a 48 ± 15 (17 83)b 41 ± 27 (7 85)b abnormal heads 9 ± 6 (1 22) 12 ± 9 (0 45) 17 ± 13 (1 43) detached heads2 2 ± 2 (1 8)a 2 ± 2 (1 10)a 6 ± 11 (0 44)b proximal droplets3 8 ± 3 (5 14)a 20 ± 14 (2 51)b 25 ± 18 (4 60)b distal droplets4 6 ± 4 (2 16)a 8 ± 7 (0 28)a 2 ± 2 (1 7)b bent midpieces 1 ± 1 (0 4) 0.2 ± 0.4 (0 1) 1 ± 5 (0 -17) general midpiece abnormality5 6 ± 4 (1 14)a 7 ± 6 (1 29)ab 10 ± 6 (2 26)b hairpin tail 4 ± 3 (0 9) 4 ± 4 (0 16) 5 ± 6 (0 20) coiled tail6 1 ± 1 (0 3)a 2 ± 2 (0 7)a 5 ± 5 (0 14)b premature germ cell 1 ± 2 (0 6) 1 ± 2 (0 8) 2 ± 2 (0 7) table 5. the mean (± sd) and range () for stallion sperm morphology variables for the percent mares pregnant/cycle percent pregnant/cycle groups – group 1≥ 76% and ≤ 100%; group 2≥ 46% and < 76%; group 3≥ 0% and < 46% n = number of stallions 1-p < 0.002 2-p < 0.03 3-p < 0.0008 4-p < 0.03 5-p < 0.03 6-p < 0.04 reprinted from love cc. theriogenology 2011;76:547-557. fertility parameter scsa variable 1 2 3 spr 90 ± 6 (79 100) 58 ± 21 (12 77) na meanαt 5 232 ± 16 (203 -297) 241 ± 23 (222 296) na sdαt 85 ± 22 (50 149) 90 ± 26 (55-171) na compαt 3 16 ± 8 (4 38)a 23 ± 13 (9 52)b na fcp 89 ± 9 (78 100) 58 ± 7 (46 75) 25± 18 (0 45) meanαt 227 ± 9 (213 243) 233 ± 16 (208 297) 237± 21 (203 281) sdαt 4 73 ± 11 (54 96) 89 ± 21 (50 -149) 94 ± 30 (55 171) compαt 2 12± 5 (4 28)a 17 ± 7 (6 38)b 25 ± 13 (5 52)c pc 91± 10 (75 100) 56 ± 6 (45 74) 32± 13 (8 45) sdαt 4 229 ± 11 (213 243)a 233 ± 15 (206 297)a 248± 27 (203 296)b sdαt 76 ± 12 (62 96) 87 ± 22 (50 149) 97 ± 30 (55 171) compαt 1 13± 6 (7 28)a 17 ± 7 (4 38)a 27 ± 14 (5 52)b table 6. means ± sd and range () for sperm chromatin structure assay (scsa) values for each fertility group within each fertility parameter (spr, fcp and pc). means ± sd and range () for spr, fcp and pc are given in the same row as the heading fertility groups seasonal pregnancy rate (spr), group 1-spr > 80%, group 2-spr < 80%; first cycle pregnancy rate (fcp), group 1fcp > 75%, group 2-fcp < 75% and > 45%, group 3-fcp < 45%; overall cycles per pregnancy (pc), group 1pc > 75%, group 2-pc < 75% and > 45%, group 3pc <45%. letters within rows are different, based on the following significance levels: 1p < 0.0001; 2p < 0.001; 3p < 0.004; 4-p < 0.01; 5p < 0.06. reprinted from love cc & kenney rm. theriogenology 1998; 50:955-72. clinical theriogenology 2021; 13: 287 conflict of interest none funding legends premier stallion season auction, texas a&m university references 1. douglas-hamilton dh, osol r, osol g, et al: a field study of the fertility of transported equine semen. theriogenology 1984;22:291-304. 2. henderson sv, capewell v, johnson w: foal registration: transported versus non-transported semen. proc am assoc equine pract 1998;44:7-11. 3. varner dd, blanchard tl, love cl, et al: effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters. theriogenology 1987;28:709-723. 4. kenney rm, hurtgen j, pierson r, et al: manual for clinical fertility evaluation of the stallion. hastings, ne. society for theriogenology 1983. 5. rigby sl, varner dd, thompson ja, et al: measurement of sperm concentration in stallion ejaculates using photometric or direct sperm enumeration techniques. proc annu conv am assoc equine pract 2001;47:236-238. 6. comerford kl, love cc, brinsko sp, et al: validation of a commercially available fluorescence-based instrument to evaluate stallion spermatozoal concentration. proc annu conv am assoc equine pract 2008; 54:367-368. 7. jasko dj, little tv, lein dh, et al: comparison of spermatozoa movement and semen characteristics with fertility in stallions: 64 cases (1987-1988). j am vet med assoc 1992; 200:979-985. 8. kiser am, brinsko sp, love cc, et al: relationship of sperm quality to fertility after 4 days of cooled storage of equine sperm. j equine vet sci 2014; 32: 602-605. 9. jasko dj, lein dh, foote rh: determination of the relationship between sperm morphologic classifications and fertility in stallions: 66 cases (1987-1988). j am vet med assoc 1990;197:389-394. 10. love cc, varner dd, thompson ja: intraand inter-stallion variation in sperm morphology and their relationship with fertility. j reprod fertil, suppl 2000;56:93-100. 11. love cc: relationship between sperm motility, morphology and the fertility of stallions. theriogenology 2011;76:547-557. 12. evenson dp, darzynkiewicz z, melamed mr: relation of mammalian sperm chromatin heterogeneity to fertility. science 1980:210:1131. 13. love cc, kenney rm: the relationship of increased susceptibility of sperm dna to denaturation and fertility in the stallion. theriogenology 1998;50:955-972. 14. love cc, kenney rm: scrotal heat stress induces altered sperm chromatin structure associated with a decrease in protamine disulfide bonding in the stallion. biol reprod 1999;60:615-620. 15. love cc, brinsko sp, rigby sl, et al: relationship of seminal plasma level and extender type to sperm motility and dna integrity. theriogenology 2005;63:1584-1591. 16. love cc, thompson ja, lowry vk, et al: effect of storage time and temperature on sperm dna and fertility. theriogenology 2002;57:1135-1142. 17. love cc, blanchard tl, varner dd, et al: effect of daily semen centrifugation and resuspension on the longevity of equine sperm quality following cooled storage. theriogenology 2012;77:1911-1917. 18. love cc, noble jk, standridge sa, et al. the relationship between sperm quality in cool-shipped semen and embryo recovery rate in horses. theriogenology 2015;84:1587-1593. 19. brinkerhoff jm, love cc, thompson ja, et al. influence of mare age, pre-breeding mare status, breeding methods, and stallion on first cycle pregnancy rates on a large commercial breeding farm. anim reprod sci 2010;121s:s159. clinical theriogenology 2021; 13: 288 cool-stored and frozen-thawed stallion semen:thoughts on collection, evaluation, processing,insemination, and fertility 1 contact rebecca funk rafunk@vt.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9395, http://dx.doi.org/10.58292/ct.v15.9395 case report double cervices in a gypsy vanner mare joanna kania,a katie wilson,a terje raudsepp,b caitlin castaneda,b matthew jevit,b maria horteloup,c nadia saklou,a catherine jula,a rebecca funka adepartment of large animal clinical sciences, virginia college of veterinary medicine, blacksburg, va, usa; bdepartment of veterinary integrative biosciences, texas a&m university, school of veterinary medicine and biosciences, college station, tx, usa; cnutrep, llc, blacksburg, va, usa abstract a 7-year, purebred gypsy vanner, maiden mare was presented with abnormal cervical tissue that was detected during prebreeding examination. external vulvar examination, transrectal and ultrasonographic examination of uterus, and internal vaginal vault and cervical examination (via speculum and endoscopy) were conducted. karyotype analysis included sex chromosome study with dual-color fluorescence in situ hybridization and polymerase chain reaction for sry gene. mare had normal 64,xx karyotype with no evidence of mosaicism. two distinct, patent, cervical ora separated by a frenulum, leading to a single uterine body, were observed. it was recommended to avoid breeding because of dystocia risk. to the authors’ knowledge, this is the first reported case of this nature in a mare. keywords: mare, reproductive anomaly, double cervices, fluorescence in situ hybridization, karyotyping background any deviation from normal sexual development, whether chromosomal, gonadal, or anatomical, is referred to as disorders in sexual development (dsd).1 in horses, dsds are understudied but not uncommon and clinical manifestations are often associated with reproductive problems. normal development of cervix and uterus rely on appropriate differentiation of müllerian duct and urogenital sinus. in people, abnormalities of müllerian duct formation resulted in congenital uterine and cervical aberrations.2 congenital uterine and cervical abnormalities in mares were observed in draft breeds; however, breed associations of dsds were not fully investigated.3–5 although these structural abnormalities are not necessarily associated with infertility, chromosome analysis is useful in exploring fertility complications. compromised fertility was noted in an irish cob mare with congenital cervical abnormality and concurrent chromosomal aberration (mosaic aneuploidy of the x chromosome).5 this case report describes double cervices in a 7-year mare with no other reproductive tract, chromosomal, or karyotype abnormalities. case presentation a 7-year, gypsy vanner (also known as irish cob) maiden mare was referred for investigation of abnormal cervical anatomy detected during a prebreeding evaluation. mare had a history of recurrent colic but was otherwise apparently healthy. complete reproductive evaluation was warranted because the mare was intended for breeding. on presentation, mare was bright and alert. her external genitalia had upright vulvar orientation, ~ 80% vulvar opening was below pelvic brim with appropriate vulvar and vulvo-vaginal seals. transrectal palpation revealed symmetrical uterine horns and ovaries of adequate size, shape, tone, and texture for a cyclic, maiden, draft mare. transrectal ultrasonography revealed normal uterus with no cysts, fluid, or edema. mare had 1 uterine body with 2 distinct uterine horns visualized from the bifurcation of the uterus. both ovaries had a corpus luteum. two firm, taught cervices were felt and could be followed to caudal uterine body (figure 1). mare had adequate vulvo-vaginal seal when speculum was inserted. moderate amount of thick and white mucus was present on vaginal vault’s ventral aspect. two patent, cervical ora were identified at the cranial surface of the vault that had normal tone, size, and structure. a thin (~ 2 cm) frenulum was noticed between the 2 cervices. a sterile endoscope was initially inserted through the right cervix and her uterus was insufflated and visualized (figure 2). there was scant amount of thick and white mucus in the uterus. uterine lining, both uterine horns, mailto:rafunk@vt.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9395 2 citation line: clinical theriogenology 2023, 15, 9395, http://dx.doi.org/10.58292/ct.v15.9395 oviductal papillae, and uterine bifurcation were visualized in their entirety and were unremarkable. endoscope was removed from the right cervix and passed through the left cervical os into uterine body. scope was retroflexed to obtain an image of both cervical openings within uterine body lumen. blood sample was collected for short-term pokeweed-stimulated lymphocyte culture. thirty metaphase cells were analyzed; seven were karyotyped that identified a 64,xx female karyotype with no observed chromosomal abnormalities. polymerase chain reaction (pcr) for the y-linked male sex determination gene sry was negative, consistent with xx sex chromosomes. to verify nonmosaic 64,xx karyotype, dual-color fluorescence in situ hybridization (fish) was conducted with a combination of 2 probes: flow-sorted horse x chromosome painting probe and probe specific to equine testis-specific transcript 7 (etsty7) ampliconic array that is present in x and y chromosomes.6–8 probes were labeled by nick translation (i.e. x painting probe) with biotin using biotin-nick translation mix and etsty7 with digoxigenin using dig-nick translation mix [roche diagnostics, basel, switzerland], following manufacturer’s protocol. hybridization and signal detection followed standard protocols described.8 biotin-labeled probes were detected with alexa fluor® 488 streptavidin conjugate (molecular figure 1. ultrasonogram of caudal reproductive tract (note 2 analogous cervices [arrows]). figure 2. endoscopic images of two cervicies (a), frenelum between 2 cervices (b), uterine body lumen (c) http://dx.doi.org/10.58292/ct.v15.9395 citation line: clinical theriogenology 2023, 15, 9395, http://dx.doi.org/10.58292/ct.v15.9395 3 probes, life technologies, carlsbad, ca) and digoxigenin-labeled probes with dylight®594 antidigoxigenin  conjugate (vector laboratories, burlingame, ca). chromosomes were counterstained with 4’,6-diamidino-2-phenylindole. one hundred cells were analyzed using a motorized fluorescence microscope (axio imager m2p [zeiss]) equipped with a high-resolution progressive scan ccd camera coolcube 1 and isis v5.3.18 software (metasystems gmbh, altlußheim, germany). fish with probes specific for equine sex chromosomes (figure 3) was consistent with the results of karyotyping (all 100 cells had 64,xx karyotype). there was no evidence for mosaicism for xy cells or cells with a single x chromosome. mare was diagnosed with an isolated dsd, namely double cervical ora without other concurrent genetic, structural, or reproductive abnormalities. no further diagnostics or interventions were performed and the mare was discharged with recommendations against carrying a foal due to risk for dystocia. discussion dual cervical ora has been previously reported in a maiden gypsy vanner mare, with concurrent uterine didelphys and chromosomal abnormalities.5 although complete uterine didelphys (2 separate reproductive tracts from vaginal vault to uterine horn) is a normal anatomy in several mammals (e.g., marsupials and rodents),9,10 in the mare 1 vaginal vault and cervix leading to bicornate uterine body is the norm. reproductive tract abnormalities were documented in several species due to partial or full failure of fusion of the müllerian ducts.10 abnormalities documented in normally monodelphic (1 reproductive tract) animals were bands of tissue remaining within the cervical os and/or canal, double external cervical os joining into 1 cervical canal, or, rarely 2 external cervices each leading to a uterine horn separated by a complete septum (true didelphys).11 rarely, 2 complete and separate cervical canals opening into 1, structurally normal, uterus were observed. brown throated sloth (bradypus variegatus) has a simplex uterus with 2 complete cervical canals (considered normal anatomy).12 a case of rare dsd (cervical duplication) in the absence of chromosomal aberrations is documented. this was confirmed by an initial cytogenetic analysis of 30 cells, followed by fish analysis of sex chromosomes in 100 cells, ruling out low-level mosaicism for a chromosomally aberrant cell line. causative factor(s) and incidence of cervical  anomalies in horses have not been identified. documentation of 2 mares of the same breed with dsd should warrant further exploration into a possible breed disposition for these conditions.5 there was no evidence of infertility in this patient; however, parturition complications are possible. although there is limited information on parturition complications in patients with isolated double cervices, there have been studies that explored parturition complications in patients with vaginal septa. vaginal dystocia was common in these patients and often the reason for discovering the anomaly.13,14 although double cervices are different from vaginal septa, they both cause an obstruction of the egress from the uterus. during embryogenesis, sry gene differentiates gonads into testes. typically, this gene is on y chromosome; however, in some cases in people it was on x chromosomes that was associated with dsds.15 since this mare was sry-negative, an sry anomaly was not responsible for dsd. this case report highlighted the importance of thorough examination of potential breeding animals including vaginoscopy and hysteroscopy. karyotype analysis is not commonly performed; however, it can be helpful in identifying potential dsd. as demonstrated in this case, some forms of dsd are chromosomally normal, suggesting possible involvement of submicroscopic genetic changes. in addition, exposure to some endocrine-disrupting agents altered uterine development in other species.16 the broad range of possible disruptions to normal sexual development in horses is not as widely documented as. in other species, nor are they fully understood. further study of causative factors in horses will increase understanding and better handling of unusual dsd cases. learning points • double cervices can occur without other reproductive tract, chromosomal, or karyotype abnormalities • breeding animals should be examined throuughly including, if possible, endoscopy and hysterscopy examinations conflict of interest none to report. references 1. allen l: disorders of sexual development. obstet gynecol clin north am 2009;36:24–45. doi: 10.1016/j.ogc.2009.02.001 2. golan a, langer r, bukovsky i, et al: congenital anomalies of the müllerian system. fertil steril 1989;51:747–755. doi: 10.1016/ s0015-0282(16)60660-x figure 3. a typical 64,xx cell as revealed by fluorescence in situ hybridization (fish) with equine x chromosome painting probe (green) and a probe for etsty7 ampliconic array (arrows), the latter present in x and y chromosomes http://dx.doi.org/10.58292/ct.v15.9395 https://doi.org/10.1016/j.ogc.2009.02.001 https://doi.org/10.1016/s0015-0282(16)60660-x https://doi.org/10.1016/s0015-0282(16)60660-x 4 citation line: clinical theriogenology 2023, 15, 9395, http://dx.doi.org/10.58292/ct.v15.9395 3. volkmann d, gilbert r: uterus bicollis in a clydesdale mare. equine vet j 1989;21:71. doi: 10.1111/j.2042-3306.1989.tb02093.x 4. blue mg: a uterocervical anomaly (uterus bicorpor bicollis) in a mare and the manual disruption of early bilateral pregnancies. n z vet j 1985;33:17–19. doi: 10.1080/00480169.1985.35137 5. murcia-robayo ry: an unusual case of uterus didelphys in an infertile mare with mosaic x-chromosome aneuploidy. reprod biol endocrin 2018;2:22–25. 6. raudsepp t, chowdhary bp: construction of chromosome specific paints for metaand submeta-centric autosomes and the sex chromosomes in horse and their use to homologous chromosomal segments in donkey. chromosome res 1999;6:103–114. 7. janečka je, davis bw, ghosh s, et al: horse y chromosome assembly displays unique evolutionary features and putative stallion fertility genes. nat commun 2018;9:2945. doi: 10.1038/ s41467-018-05290-6 8. raudsepp t, chowdhary bp: fish for mapping single copy genes.  methods mol biol 2008;422:31–49. doi: 10.1007/ 978-1-59745-581-7_3 9. machado da, ontiveros ae, behringer rr: mammalian uterine morphogenesis and variations. in: gridley t, oxburgh l, eds. current topics in developmental biology. vol. 148. cambridge, ma: academic press; 2022, pp. 51–77. 10. spencer te, hayashi k, hu j, et al: comparative developmental biology of the mammalian uterus. in: schatten g, ed. current topics in developmental biology. vol. 68. cambridge, ma: academic press; 2005, pp. 85–122. 11. card c: congenital abnormalities of the cervix in mares. equine vet ed 2012;24:347–350. doi: 10.1111/ j. 2042-3292. 2011.00363.x 12. favoretto sm, dasilva eg, menezes j, et al: reproductive system of brownthroated sloth (bradypus variegatus, schinz 1825, pilosa, xenarthra): anatomy and histology. anat histol embryol 2016;45:249–259. doi: 10.1111/ahe.12193 13. heinonen pk: longitudinal vaginal septum. eur j obstet gynecol reprod biol 1982;13:253–258. doi: 10.1016/0028 -2243(82) 90106-x 14. gormley rk, crabtree jr: female caudal reproductive tract abnormalities. equine vet edu 2021;33:411–414. doi: 10.1111/ eve.13307 15. albu cc, albu df, muşat ar, et al: the crucial role of sry gene in the determination of human genetic sex: 46, xx disorder of sex development. rom j morphol 2019;60:1311–1316. 16. schimpf mg, milesi mm, luque eh et al: evaluation of development of the rat uterus as a toxicity biomarker. in: palmeira cmm, de oliveira dp, dorta dj, eds. toxicity assessment methods and protocols. methods in molecular biology, vol. 2240. clifton, n.j.: springer protocols; 2021, pp. 103–117. http://dx.doi.org/10.58292/ct.v15.9395 https://doi.org/10.1111/j.2042-3306.1989.tb02093.x https://doi.org/10.1080/00480169.1985.35137 https://doi.org/10.1038/s41467-018-05290-6 https://doi.org/10.1038/s41467-018-05290-6 https://doi.org/10.1007/​978-1-59745-581-7_3 https://doi.org/10.1007/​978-1-59745-581-7_3 https://doi.org/10.1111/​j.​2042-3292.​2011.00363.x https://doi.org/10.1111/ahe.12193 https://doi.org/10.1016/0028​-2243(82)​90106-x https://doi.org/10.1111/eve.13307 https://doi.org/10.1111/eve.13307 steps toward the generation of ovarian somatic cells from pluripotent stem cells           steps toward the generation of ovarian somatic cells from pluripotent stem cells alisha bothun, dori woods department of biology, northeastern university, boston, ma abstract the generation of functional gametes, both eggs and sperm, from murine pluripotent stem cell (psc) sources, has set the stage for the eventual use of this emerging technology in other species. with the field enthusiastically embracing this eventuality, in particular for animal conservation efforts, there are a number of key factors to consider regarding the applicability of these methods across species, particularly with regard to the generation of eggs. to date, published studies point to the need for fetal somatic tissue and primitive granulosa cells to serve as a niche for the growth and maturation of oocytes generated from pscs. in practice, the need for such tissue represents a major limitation when attempting to apply this to species in which access to fetal ovaries is limited or unethical. to circumvent this, we and others have derived methods to generate ovarian granulosa cells from pscs, albeit with low yield. herein we present an update on the status of generating early stage granulosa cells from pscs, and provide evidence for improvements based on a stepwise, 2-dimensional protocol for the directed differentiation of human pscs. keywords: granulosa cell, ovarian soma, granulosa precursor cell, pluripotent stem cell introduction the premise that oocytes, and potentially eggs, could be generated from pluripotent stem cells (pscs) was first established in 2003,1 with the initial characterization of oocyte-like cells that formed within primitive follicle-like structures. subsequent attempts at the maturation and utilization of psc-derived oocytes were largely incremental,2 and it was not until 2012 that functional oocytes capable of maturation, fertilization, and the ability to give rise to live offspring were realized.3 notably, the protocol that was used to accomplish this feat, as well as a subsequent report by the same group, relied on the innate ability of psc-derived primordial germ cell-like cells (pgclcs) to interact with endogenous fetal ovarian tissue, providing the microenvironment and granulosa cells that enabled follicle growth and maturation, and with it oocyte development.3-5 an additional recent report in which functional eggs were generated from induced pscs (ipscs) derived from granulosa cells similarly used fetal ovarian tissue.6 together, these reports highlighted the notion that the fetal ovarian somatic microenvironment is not only nurturing, but likely requisite for the development of functional eggs from psc sources. albeit exciting and groundbreaking for the fields of stem cell and reproductive biology, adaptation of this technology to species outside of the easily obtained and manipulated laboratory mouse must overcome the reliance on fetal tissue. this is particularly relevant in instances where obtaining fetal tissue is unethical, which pertains to humans, or unpractical, as is the case for endangered species. therefore, to circumvent this limitation, an alternative source of ovarian granulosa cells or their precursors that can be utilized in conjunction with psc-derived female gametes should be identified. although much research effort has understandably centered on the female germline, efforts to progress the derivation of ovarian somatic cells from pscs have not been entirely lacking. soon after the discovery of embryonic stem cells, it was possible to direct embryonic stem cells into a steroidogenic lineage by inducing expression of nuclear receptor steroidogenic factor 1 (sf-1),7 a protein involved in endocrine development and steroidogenesis. the steroidogenic cells produced only generated minimal amounts of progesterone, and were unresponsive to human chorionic gonadotropin; however, this work opened the door to the possibility of generating steroidogenic cells from pluripotent sources. later, other groups verified the production of steroidogenic cells by retrovirus-mediated transfection to transduce human mesenchymal stem cells with sf-1, resulting in cells expressing various steroidogenic genes and producing progesterone. additionally, mouse embryonic stem cells with inducible sf-1 also generated steroidogenic cells resembling adrenocortical cells.8,9 in a study investigating epigenetic memory, induced pluripotent stem cells (ipscs) were generated from mouse and human granulosa cells, and were then clinical theriogenology • volume 12 number 4 • december 2020529           subjected to undirected differentiation; these experiments yielded cells capable of producing more estrogen and progesterone than true embryonic stem cells or from fibroblast-derived ipscs.10 this work highlighted the many uses of pluripotent stem cells and could have useful implications in characterizing the differentiation stages required to obtain high efficiency for the differentiation of steroidogenic cells. generating large numbers of granulosa cells by this method that are capable of hormone synthesis would require healthy granulosa cells to generate ipscs, a lengthy and costly endeavor to complete on a per person basis. furthermore, those methods would be impossible when functional granulosa cells are absent or not obtainable. the production of steroidogenic cells provided proof of concept for the use of pluripotent stem cells to generate gonadal lineages. our previous studies built upon this and demonstrated that in mouse models, embryonic stem cells are capable of generating functional granulosa cells.11 mouse embryonic stem cells (mescs) containing a dual-fluorescence reporter for germ cell specific pou5f1-driven gfp reporter and forkhead box l2 (foxl2)-driven discosoma sp. red (dsred) to label granulosa cells were allowed to spontaneously differentiate. foxl2-dsred labeled cells localized nearly exclusively with pou5f1-gfp positive cells, and demonstrated a progressive differentiation profile, first expressing primitive markers associated with granulosa cell precursors (including foxl2; wingless-type mmtv integration site family, member 4, wnt4; follistatin, fst; and kit ligand kitl), followed by the acquisition of later granulosa cell makers, such as follicle stimulating hormone receptor (fshr), cyp19a1, and steroid acute regulatory protein (star) and were capable of producing progesterone and estradiol in vitro. when the mesc derived foxl2-dsred positive cells were injected into neonatal mouse ovaries, follicles were generated incorporating the labeled cells. this study not only showed that mescs are capable of generating steroidogenic cells, but that given the appropriate cues and environment, they are capable of participating in folliculogenesis and steroidogenesis. unfortunately, the spontaneous differentiation rate of mescs into foxl2 expressing cells is low and unpredictable, and more robust methods are needed for eventual utility as a substitute for fetal ovarian tissue. here, we propose and test a strategy for the directed differentiation of granulosa cells from human embryonic stem cells. by mimicking developmental pathways in a stepwise, 2-d differentiation utilizing small molecules and ligands to target specific pathways, we have established a framework for identifying human, granulosa-like cells from a pluripotent stem cell source. these cells will form the groundwork for establishing the hormone producing capabilities of stem cell derived granulosa cells, and will enable in vitro studies for human follicle formation, for which there is currently no model. this strategy might also help advise work in other species that have similar limitations in background knowledge regarding organogenesis of the ovary. materials and methods cell culture and differentiation human embryonic stem cells (hesc) lines wa09 (wicell) and esi051 (esibio) were maintained in feeder-free culture conditions in essential 8 media (thermofisher scientific, waltham, ma) on tissue culture grade plates coated with hesc-qualified matrigel (corning, corning, ny). cells were routinely passaged by edta (0.5 mm) dissociation and all cells were used prior to passage 55. prior to differentiation, cells were dispersed by brief incubation in 0.05% trypsin-edta and were washed prior to plating at 45,000 cells/well in 12-well plates in essential 8 media supplemented with revitacelltm supplement (thermofisher scientific) to aid in single cell seeding and consistent plating density. when cells reached 50% confluent, differentiations were begun by transition to differentiation media (dmem/f12 [thermofisher scientific], 1x nonessential amino acids [thermofisher scientific], 20% knockout tm serum replacement [thermofisher scientific], 0.11 mm beta mercaptoethanol) supplemented as described with the following compounds: 10 µm chir99021 (sigma, st. louis, mo), 1 µm retinoic acid (sigma), 100 ng/ml human recombinant bfgf (thermofisher scientific), 1 µg/ml jagged 1 (stemrd, burlingame, ca), or 50 µm resveratrol (mp biomedical, santa ana, ca). gene expression clinical theriogenology • volume 12 number 4 • december 2020 530           rna was isolated by reliaprep rna mini kit (promega, madison, wi) and reverse transcription was carried out using the revertaid first strand cdna synthesis kit (thermo, thermofisher scientific). quantitative rt-pcr was performed with fast sybr green (thermo, thermofisher scientific) on a steponeplus thermocycler (applied biosystems, foster city, ca), using the following primers: b2m f-ggcattcctgaagctgacag, r-tggatgacgtgagtaaacctg, foxl2 facggcctgtactcggtttac, rtccaccgatctttcaaacaa, and amhr2 f-tgtgtttctcccaggtaatccg, raatgtggtcgtgctgtaggc. fold change in gene expression was calculated using the ∆∆ct method, with b2m as the reference. cell sorting and analysis cells were dissociated in 0.25% trypsin-edta, washed in media, and blocked for 20 minutes on ice-cold blocking buffer (2% bovine serum albumin and 2% normal goat serum in pbs). cells were stained in mouse antiamhr2 (ab64762, 0.1 µg/ml) on ice for 30 minutes, washed, and labeled with goat antimouse alexa fluor 488 (1:500) on ice for 45 minutes. cells were washed and re-suspended in cold facs buffer (1% fetal bovine serum, 25 mm hepes, 1 mm edta in pbs). samples were analyzed on a bd facs aria iii special order flow cytometer equipped with the following lasers and filter sets: blue laser (488 nm) equipped with fsc and ssc detectors and a fitc filter (505lp; 530/40 nm) and a uv laser (355 nm) equipped with a dapi filter (450/50 nm). dapi (sigma, 0.2 µg/ml) was added to cells immediately prior to sorting. cells were gated from debris by fsc versus ssc plots, followed by gating for singlet events and lastly, gating for dapi negative events for dead cell exclusion. population gating was performed in flowjo (ashland, or) for statistical analyses. results we presented a method to differentiate cells from pscs with molecular signatures of human granulosa-like cells. to differentiate granulosa cells from hescs, undifferentiated cells were exposed to a stepwise differentiation via small molecules to induce signaling cascades proposed for each stage and rtqpcr was performed to detect increases in marker gene expression at each stage (figure 1). because human pre-granulosa cells are not well characterized, we analyzed our previously reported comprehensive figure 1. differentiation strategy for the production of human pregranulosa cells from pluripotent stem cells. human embryonic stem cells (wa09, wicell) were maintained in feeder free conditions and subjected to 2-d directed differentiation. cells were plated and treated with gsk3 inhibitor (gsk3i, chir99021, 10 µm) to induce mesendodermal lineage via wnt signaling. further treatment with fetal growth factor (bfgf, 100 ng/ml) and retinoic acid (1 µm) was utilized to induce cells of a gonadal/renal lineage, and subsequent treatment with notch activators, jagged 1 (1 µg/ml), a ligand for notch receptors, and resveratrol (50 µm), a compound that induces notch signaling, to increase the incidence of potential granulosa-like cells. cells were subject to quantitative rt-pcr to assess gene expression throughout differentiation (genes chosen for analysis shown in gray). quantitative mass spectrometric proteomics dataset of human fetal ovarian tissue across developmental time points12 to determine that forkhead box l2 (foxl2), a transcriptional regulator required for granulosa cell formation in mice, is expressed just prior to and during folliculogenesis in humans (figure 2a). to confirm that foxl2 expression was specific to pre-granulosa cells, immunohistochemistry (ihc) verified that foxl2 was not detected in tissue from 56 days of development prior to follicle formation, and as germ cell cyst breakdown occurred, was localized specifically to somatic cells surrounding germ cells and was primarily nuclear (figure 2b). clinical theriogenology • volume 12 number 4 • december 2020531           figure 2. foxl2 as a marker for human pre-granulosa cells based on proteomics and ihc confirmation of expression and localization. (a) quantitative mass spectrometry detection of pre-granulosa cell protein foxl2 at four stages of fetal development (days 47, 108, 122, and 137) and in a representative adult tissue sample. (b) representative ihc images of foxl2 immunostaining in human ovarian tissue from 56, 116, or 137 days of development. primary antibodies were labeled and detected with dab substrate (brown) and nuclei were counterstained with hematoxylin (blue). scale bar = 100 µm. during the first stage of in vitro differentiation, cells were treated with chir99021, a glycogen synthase kinase 3 (gsk3) inhibitor that activates wnt signaling, for 48 hours to induce differentiation to the mesendoderm. chir99021 treatment resulted in a 520-fold increase in brachyury (tbxt gene) expression at 48 hours (p = 0.0005) (figure 3a). following mesendoderm induction, cells were treated with bfgf and retinoic acid for 48 hours, followed by treatment with either jagged 1 or resveratrol to induce notch signaling (figure 1). in comparison to vehicle controls, foxl2 expression increased 3.1or 4.5-fold in response to bfgf and retinoic acid, followed by jagged 1 or resveratrol, respectively (p > 0.05) (figure 3b). figure 3. directed differentiation results in an increase in mesoderm marker t at 48 hours, followed by an increase in foxl2 at day 6. (a) inducing wnt signaling from 0 8 hours resulted in a 521-fold increase in t expression at 48 hours (p = 0.0005). (b) subsequent treatment with bfgf/retinoic acid followed by notch activators jagged 1 or resveratrol resulted in a 3.1or 4.5-fold increase in pregranulosa cell marker foxl2. fold change calculated against b2m and compared to vehicle controls at each timepoint. additionally, we assessed the expression of amhr2 gene expression to determine the eventual utility of using amhr2 as a live cell marker for facs sorting of granulosa-like cells, using the stepwise directed differentiation protocol. following the directed differentiation approach described and using resveratrol specifically for the final treatment, amhr2 expression increased at day 10 of differentiation in both wa09 (21.5-fold, p = 0.005) and esi051 cells (58.4-fold, p = 0.001) over vehicle control clinical theriogenology • volume 12 number 4 • december 2020 532           differentiations (figure 4a). when differentiated cells were labeled with antiamhr2 and analyzed by facs, there was an increase in amhr2+ cells from day 0 to day 10 (p = 0.005), and no difference in percent of amhr2+ cells at day 10 between vehicle and directed differentiations for wa09 (p > 0.05). however, there was an increase in amhr2+ cells with directed differentiation at day 10 for esi051 (2.6-fold increase, p = 0.008), indicating a difference between the 2 cell types with regards to surface marker expression (figure 4b). figure 4. directed differentiation results in an increase in granulosa cell marker amhr2 at day 10 of differentiation and live cells can be sorted by facs labeling for amhr2. (a) inducing wnt signaling from 0-48 hours, followed by subsequent treatment with bfgf/retinoic acid and resveratrol, resulted in a 21.5or 58.4-fold (wa09 p = 0.005 and esi051 p = 0.001) increase in granulosa cell marker amhr2 at day 10. fold change calculated against b2m and compared to vehicle controls at each timepoint, n = 3. (b) facs analysis of amhr2-labeled cells in undifferentiated cells and at day 10 of a differentiation under vehicle control conditions or under directed differentiation conditions. the population of amhr2+ cells was unchanged for wa09 cells at day 10 of differentiation (p > 0.05) and increased over vehicle for esi051 cells (p = 0.008). discussion the need for a psc-derived ovarian microenvironment containing granulosa cell precursors to complement strategies for the generation of functional eggs across species is apparent, as future applications cannot rely on a steady source of fetal gonads to serve in this capacity. although mice have served as an important model for understanding mammalian ovarian organogenesis, key differences in the timing of differentiation processes, including folliculogenesis, across species call for an assessment of the developmental timeline, when possible. to this end, we previously completed a comprehensive proteomic analysis of human fetal ovarian tissue12 to inform us in the selection of our differentiation pathway, including the significance of wnt signaling components.13 additionally, research has shed light on the origin of granulosa cells, using various animal model systems to lineage trace cells and estimate the dynamics of follicle formation. granulosa cells were once assumed to be derived from either the mesonephros, from which ovarian somatic tissues arise during development, or the ovarian surface epithelium.14-16 in mice, granulosa cells originate from the surface epithelium and may constitute several waves of differentiation.17 in bovine models, precursors to these cells, termed gonadal ridge epithelial (grel) cells, express lgr5 and appear to act similar to other epithelial stem cell niches, in which cells are differentiated and migrate into the ovary to form primordial follicles.18 although these experiments have begun to elucidate the origin and differentiation pathway of granulosa cells, none have identified whether these pathways are directly paralleled in human ovarian organogenesis, and efforts to find conserved pathways between species have been sporadic. here, building upon previously established protocols we demonstrated a protocol for the stepwise directed differentiation of granulosa precursor cells from human pscs. first, mesendoderm was differentiated from hescs by activating wnt signaling via gsk3 inhibition with the small molecule, chir99021, which dramatically increased the expression of mesendoderm marker t within 24 48 hours of culture. this was consistent with previously established protocols, in which treatment with chir99021 significantly and reliably induced t expression within the first 2 days of treatment.19 subsequently, bfgf clinical theriogenology • volume 12 number 4 • december 2020533           and retinoic acid were simultaneously applied20 to induce cells of the intermediate mesoderm, from which the gonads are derived and prior to formation of pregranulosa cells. following successful differentiation to intermediate mesoderm, induction of notch signaling, a requisite for the initiation of granulosa cell specification and the onset of folliculogenesis, was applied through addition of compounds jagged 1 ligand, or the compound resveratrol.21,22 this stepwise differentiation was successful in generating cells with increased expression of foxl2 and amhr2, markers of pregranulosa and early granulosa cells, respectively. additionally, amhr2 is a viable target for live cell antibody tagging and subsequent facs sorting for culture and analysis. this approach has several important benefits; primarily, the differentiation time is relatively brief (days) and does not require embryoid body formation, reducing the labor associated with embryoid bodies and spheroid formation; second, the sorting of amhr2-labeled cells does not require genetic manipulation to generate a reporter line for identification and isolation, making this strategy widely applicable. finally, whereas the mammalian ovary functions to develop and mature oocytes for reproduction, it is a vital component of the tightly regulated hypothalamic-pituitary-gonadal axis that maintains hormonal stasis throughout the female reproductive lifespan in mammals. granulosa cells are at the crux of steroid production within the ovary, and, in concert with theca cells, generate estradiol and progesterone in response to luteinizing hormone and follicle stimulating hormone surges during reproductive years. this finely tuned steroid biosynthesis pathway is disrupted due to follicle loss in the case of polycystic ovarian syndrome, in response to chemotherapeutic agents, exposure to toxicants, and inevitably with age. the development of synthetic sources of granulosa cells or their precursors from pluripotent stem cells is not only key to the reproductive success of these artificial gametes, but also represents a putative way to address the endocrine disruption associated with ovarian failure. conclusion we previously established a framework for identifying human, granulosa-like cells from a pluripotent stem cell source. building upon established protocols, we demonstrated a protocol for the stepwise directed differentiation of granulosa precursor cells from human pscs. we presented a method to differentiate cells from pluripotent stem cells with molecular signatures of human granulosa-like cells and we have provided evidence for improvements, based on a stepwise, 2-dimensional protocol for the directed differentiation of human pscs. conflict of interest none to declare. acknowledgement supported by the national science foundation (nsf) career award 1750996 to dw. references 1. hübner k, fuhrmann g, christenson lk, et al: derivation of oocytes from mouse embryonic stem cells. science 2003;300:1251-1256. 2. nicholas cr, haston km, grewall ak, et al: transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic strategy for female infertility. hum mol genet 2009;18:4376-4389. 3. hayashi k, ogushi s, kurimoto k, et al: offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. science 2012;338:971-975. 4. hayashi k, hikabe o, obata y, et al: reconstitution of mouse oogenesis in a dish from pluripotent stem cells. nat protoc 2017;12:1733-1744. 5. hikabe o, hamazaki n, nagamatsu g, et al: reconstitution in vitro of the entire cycle of the mouse female germ line. nature 2016;539:299-303. 6. tian c, liu l, ye x, et al: functional oocytes derived from granulosa cells. cell rep 2019;29:4256-4267.e9. 7. crawford pa, sadovsky y, milbrandt j: nuclear receptor steroidogenic factor 1 directs embryonic stem cells toward the steroidogenic lineage. mol cell biol 1997;17:3997-4006. 8. yazawa t, inanoka y, mizutani t, et al: liver receptor homolog-1 regulates the transcription of steroidogenic enzymes and induces the differentiation of mesenchymal stem cells into steroidogenic cells. endocrinology 2009;150: 3885-3893. clinical theriogenology • volume 12 number 4 • december 2020 534           9. yazawa t, kawabe s, inaoka y, et al: differentiation of mesenchymal stem cells and embryonic stem cells into s teroidogenic cells using steroidogenic factor-1 and liver receptor homolog-1. mol cell endocrinol 2011;336:127-132. 10. anchan r, gerami-naini b, lindsey js, et al: efficient differentiation of steroidogenic and germ-like cells from epigenetically-related ipscs derived from ovarian granulosa cells. plos one 2015;10(3):e0119275. 11. woods dc, white ya, niikura y, et al: embryonic stem cell-derived granulosa cells participate in ovarian follicle formation in vitro and in vivo. reprod sci 2013;20:524-535. 12. bothun am, gao y, takai y, et al: quantitative proteomic profiling of the human ovary from early to mid-gestation reveals protein expression dynamics of oogenesis and folliculogenesis. stem cells dev 2018;27:723-735. 13. bothun am, woods dc: dynamics of wnt signaling components in the human ovary from development to adulthood. histochem cell biol 2019;151:115-123. 14. motta pm, makabe s: development of the ovarian surface and associated germ cells in the human fetus. a correlated study by scanning and transmission electron microscopy. cell tissue res 1982;226:493-510. 15. wilhelm d, palmer s, koopman p: sex determination and gonadal development in mammals. physiol rev 2007; 87:1-28. 16. maheshwari a, fowler pa: primordial follicular assembly in humans--revisited. zygote 2008;16:285-296. 17. mork l, maatouk dm, mcmahon ja, et al: temporal differences in granulosa cell specification in the ovary reflect distinct follicle fates in mice. biol reprod 2012;86:37. 18. hummitzsch k, irving-rodgers hf, hatzirodos n, et al: a new model of development of the mammalian ovary and follicles. plos one 2013;8:e55578. 19. araoka t, mae s, kurose y, et al: efficient and rapid induction of human ipscs/escs into nephrogenic intermediate mesoderm using small molecule-based differentiation methods. plos one 2014;9(1):e84881. 20. lam aq, freedman bs, morizane r, et al: rapid and efficient differentiation of human pluripotent stem cells into intermediate mesoderm that forms tubules expressing kidney proximal tubular markers. j am soc nephrol 2014;25:1211-1225. 21. edson ma, nagaraja ak, matzuk mm: the mammalian ovary from genesis to revelation. endocr rev 2009; 30:624-712. 22. yu xm, jaskula-sztul r, ahmed k, et al: resveratrol induces differentiation markers expression in anaplastic thyroid carcinoma via activation of notch1 signaling and suppresses cell growth. mol cancer ther 2013;12:1276-1287. clinical theriogenology • volume 12 number 4 • december 2020535 2018: integrating canine semen freezing into your practice integrating canine semen freezing into your practice ana j. adams hamby road animal hospital, alpharetta, ga introduction as breedings using frozen canine semen have gained popularity among dog breeders, the need for reputable freezing centers has also increased. veterinarians are uniquely positioned and easily trained to be able to offer this service in a general practice setting with a relatively modest investment of time, finances, and personnel. this review will identify and discuss the principal aspects involved in successfully setting up and integrating canine semen freezing into practice. keywords: canine, semen, cryopreservation, artificial insemination, extender practice setting canine semen freezing is a technique that can easily be incorporated into a wide variety of practice settings ranging from a general mixed practice in a rural setting to an exclusive canine reproduction specialty practice in a metropolitan area. factors to consider in the evaluation of whether or not this service would contribute in a positive way to your current practice environment include having a clear understanding of the fact that providing clients with the opportunity to freeze their dog’s semen will likely increase the eventual need and demand for additional reproductive services. as a manager in our practice accurately stated recently, “with reproduction, if you’re not all in, this may not be for you.” although the techniques themselves may not require extensive training or certifications to be able to accurately perform, the timing precision required when breeding with canine frozen semen may require increased doctor/staff availability in order to be able to meet the client’s need in a timely fashion. because of the shortened longevity of frozen/thawed semen, weekend/holiday office hours are often necessary in order to obtain optimal conception rates and litter sizes. awareness of and sensitivity to the needs of clients investing large amounts of time, effort, and finances in order to achieve a particular breeding will result in minimized frustration for all parties involved. services offered canine semen freezing centers are found throughout the united states and abroad in a wide variety of settings ranging from compact mobile freezing stations temporarily set up at dog shows to large canine theriogenology specialty practices in metropolitan areas. having a good working relationship among colleagues offering similar services is critical to maintaining open lines of communication with clients, particularly due to the fact that shipping semen from one freezing center to another is often necessary in order to minimize travel and resulting stress for the bitch. veterinarians have the added benefit of being able to offer medical and surgical services when required, making their practices an appealing referral alternative for non-veterinarian staffed freezing centers for dogs needing further diagnostic or surgical options than they are equipped to provide. the actual methods used to freeze canine semen vary widely. the basic premise involves taking a fresh semen sample (initially at body temperature) gradually through progressive decreases in temperature until freezing in liquid nitrogen is finally achieved. semen can be frozen in pellets or straws and stored indefinitely in liquid nitrogen in tanks that are typically maintained at a constant temperature (independently of an exterior power supply) through a regular supply of nitrogen from an outside source. the most important difference among the more commonly used freezing methods resides in the extenders used, some of them with proprietary recipes and others compounded in-house by independent freezing centers. clinical theriogenology • volume 10, number 3 • september 2018193 costs involved the costs involved in setting up a canine semen freezing center will vary according to the freezing method chosen by a practitioner and will be limited only by the speed at which the center subsequently grows. initial investment costs can be divided up into four general categories: 1) semen collection/evaluation laboratory equipment and supplies; 2) semen freezing equipment; 3) liquid nitrogen dewars; and 4) semen shipping tanks. franchising fees if applicable will depend on the parent organization and may be individually determined. semen freezing the initial step in freezing canine semen involves obtaining a sample of acceptable quality in order to ensure adequate semen viability once thawed, as conception rates and litter sizes will be directly affected. semen collection and evaluation processes described in detail in earlier proceedings include basic assessments of progressive motility, sperm concentration, and sperm morphology, with recommended minimum standards for each parameter pre-established by the society for theriogenology. adherence to these standards is critical to the success of frozen semen breedings. accurate documentation of semen analysis results obtained when semen is initially processed and frozen allows owners to later make informed decisions regarding potential breedings using these same collections. semen freezing can be done in pellets or straws, with minor variations in methodology used to obtain the final breeding units. following evaluation of the ejaculate, the sample is taken through a series of steps that include refrigeration and eventual freezing using extenders that include cryoprotective agents to help buffer the extreme temperature changes to which the sperm will be exposed. proper identification of breeding units including stud dog identification (ideally via permanent identification such as a microchip or tattoo), breed, date of freezing, registration number(s), and freezing center identification is critical to ensure accuracy in parentage of offspring from litters produced by breedings using said semen. documentation of the semen collection process and inseminations using frozen semen is required by the american kennel club. registration of litters born from frozen canine semen will also require proper identification of breeding units used as well as dna identification of the stud dog. semen freezing contracts offer both freezing centers and semen owners the opportunity to clearly understand the terms and conditions under which semen is frozen and potentially stored and/or shipped to another destination. ownership or transfer of ownership of frozen semen is also a critical piece that requires definition in order to avoid potential mishandling of frozen semen and/or potentially litigious circumstances. inseminations with frozen canine semen due to the increased fragility and shortened lifespan of frozen semen compared to fresh or chilled semen, breedings involving frozen semen require increased precision regarding ovulation timing in order to obtain optimal results. commonly used methods for defining the most fertile period (discussed in previous editions of theriogenology) include vaginoscopy, vaginal cytology, and luteinizing hormone (lh) and progesterone testing. once the most fertile time frame is identified, intrauterine inseminations (as opposed to intravaginal inseminations) result in the highest conception rates and optimal litter sizes per breed. intrauterine insemination is achieved via transcervical or surgical insemination, with the former quickly gaining popularity among breeders in the united states, particularly when anesthesia or other surgical risks such as increased scar tissue or adhesions, or multiple inseminations on a particular heat cycle are considered. familiarity with and expertise in the different forms of artificial insemination are critical to the utilization of frozen semen. offering clients the ability to not only freeze and store canine semen will greatly enhance the rate at which this service becomes a significant profit center for a practice. transport of frozen semen frozen semen is often transferred from one facility to another in order to facilitate breedings without transporting the bitch long distances. semen release forms are typically used by storage facilities to grant semen owners the opportunity to move semen from one location or owner to another. specialized clinical theriogenology • volume 10, number 3 • september 2018 194 liquid nitrogen tanks known as “dry shippers” allow frozen semen to be maintained at the proper temperature during shipping over a longer period of time (up to 7-10 days if needed) through continued exposure to liquid nitrogen vapors. dry shippers range in size, configuration, and quality from inexpensive/disposable to heavy duty, rechargeable tanks that are made to withstand harsh handling conditions often encountered in air cargo travel. domestic and international transport is common and appropriate documentation is critical to ensure proper handling of semen in a timely fashion once it reaches the desired destination, particularly in the case of international shipping. importation regulations established by individual countries must be obtained prior to freezing in order to allow for special considerations regarding blood testing or proper semen identification. clear identification of all shipper contents (including thaw media) and accompanying health certificates based on the importation requirements of the recipient country is critical to ensure efficient processing through customs and timely arrival of semen shipments at their final destination. upon arrival, frozen semen is quickly transferred into semi-permanent storage tanks and dry shippers returned to their original location when applicable. insurance costs and coverage of frozen semen during transport vary depending on the stated value of the semen, insurance policies in place at each facility, or the shipping company involved, with limitations of coverage ideally defined and clearly communicated to all parties involved in advance. semen storage some freezing centers store frozen semen at their facility while others ship newly frozen semen to larger, more centrally located storage facilities for long-term storage and subsequent distribution. the wide variety of available options allows practitioners to custom design a system that works well for their particular organization and clientele. semen can be stored in larger liquid nitrogen tanks (available in different sizes) that provide a stable temperature of -196ºc independently of an external power source with regular replenishing of liquid nitrogen from a local supplier. proper maintenance and monitoring of tank integrity and liquid nitrogen supply is critical to avoid potential damage to stored semen that would result in devastating consequences for all parties involved. proper identification and accompanying documentation of all stored semen is also paramount to minimize confusion or errors once the semen is utilized. annual storage fees are usually required by semen storage facilities in order to cover the costs of liquid nitrogen and tank maintenance, and may vary among centers based on the number of stud dogs or the number of breeding units stored. risks/benefits when considering the possibility of integrating canine semen freezing into a practice setting, it is necessary to account for the benefits and risks this service can potentially create for both veterinarians and staff. increased demands on time required for training in the methodology selected will exist initially, added to the accompanying staff demands involved in ensuring that the required documentation for collection, distribution, and eventual utilization of frozen semen is executed accurately. registration of litters obtained from frozen semen breedings depends on information obtained at times decades earlier, with obviously devastating results for breeders when such information is unobtainable retroactively due to death of a stud dog or lack of clearly identifiable parentage. freezing centers that are equipped to receive and store semen from other facilities create an additional opportunity for increased revenue, both from storage fees once the semen is transferred from the shippers into the semi-permanent larger storage tanks, as well as for services required for ovulation timing and insemination services once the semen is eventually utilized. increased awareness by colleagues in the industry of the services offered will likely translate into increased demand by the tightly knit and well connected breeder community for freezing, storage, and breedings services that will translate into increased revenue for the practice. clinical theriogenology • volume 10, number 3 • september 2018195 references 1. mason sj: a retrospective clinical study of endoscopic-assisted transcervical insemination in the bitch with frozenthawed dog semen. reprod domest anim 2017; 52 suppl 2:275-280. 2. mason sj, rous nr: comparison of endoscopic-assisted transcervical and laparotomy insemination with frozenthawed dog semen: a retrospective clinical study. theriogenology 2014;82:844-850. 3. batista m, santana m, niño t, et al: sperm viability of canine and caprine semen samples preserved in a dry shipper. anim reprod sci 2012; 130:105-110. 4. macedo sp, malm c, henry mr, et al: endoscopic transcervical intrauterine artificial insemination in labrador retriever bitches. res vet sci 2012;92:494-500. 5. england gc, millar km: the ethics and role of ai with fresh and frozen semen in dogs. reprod domest anim 2008; 43 suppl 2:165-71. 6. peña fj1, núñez-martínez i, morán jm: semen technologies in dog breeding: an update. reprod domest anim 2006;41 suppl 2:21-29. 7. ponglowhapan s, chatdarong k, sirivaidyapong s, et al: freezing of epididymal spermatozoa from dogs after cool storage for 2 or 4 days. theriogenology 2006; 66:1633-1636. 8. thomassen r, sanson g, krogenaes a, et al: artificial insemination with frozen semen in dogs: a retrospective study of 10 years using a non-surgical approach. theriogenology 2006; 66:1645-1650. 9. schäfer-somi s, kluger s, knapp e, et al: effects of semen extender and semen processing on motility and viability of frozen-thawed dog spermatozoa. theriogenology 2006;66:173-182. 10. cremonesi f, salamon l, groppetti d, et al: results of a single transcervical endoscopic insemination using frozen semen in the bitch. vet res commun 2005; 29 suppl 2:187-189. 11. hermansson u, linde forsberg c: freezing of stored, chilled dog spermatozoa. theriogenology 2006; 65:584-593. 12. eilts be: theoretical aspects of canine semen cryopreservation.theriogenology. 2005; 64(3):692-7. review. 13. martínez ai: canine fresh and cryopreserved semen evaluation. anim reprod sci 2004;82-83:209-224. 14. burgess cm, bredl jc, plummer jm: vital and ultrastructural changes in dog spermatozoa during cyopreservation. j reprod fertil suppl 2001;57:357-363. 15. thomassen r, farstad w, krogenaes a, et al: artificial insemination with frozen semen in dogs: a retrospective study. j reprod fertil suppl 2001;57:341-346. 16. linde-forsberg c, ström holst b, govette g: comparison of fertility data from vaginal vs intrauterine insemination of frozen-thawed dog semen: a retrospective study. theriogenology 1999; 52:11-23. 17. linde-forsberg c, forsberg m: results of 527 controlled artificial inseminations in dogs. j reprod fertil suppl1993; 47:313-323. 18. england gc: cryopreservation of dog semen: a review. j reprod fertil suppl 1993; 47:243-255. 19. morton db, bruce sg: semen evaluation, cryopreservation and factors relevant to the use of frozen semen in dogs. j reprod fertil suppl 1989; 39:3 20. linde-forsberg c, forsberg m: fertility in dogs in relation to semen quality and the time and site of insemination with fresh and frozen semen. j reprod fertil suppl 1989; 39:299-310. 21. ferguson jm, renton jp, farstad w, et al: insemination of beagle bitches with frozen semen. j reprod fertil suppl 1989; 39:293-298. 22. farstad w, berg ka: factors influencing the success rate of artificial insemination with frozen semen in the dog. j reprod fertil suppl 1989; 39:289-92. 23. kolster ka: evaluation of canine sperm and management of semen disorders. vet clin north am small anim pract 2018; apr 17 pii: s0195-5616(18)30014-7. doi: 10.1016/j.cvsm.2018.02.003. [epub ahead of print]. 24. hesser a, darr c, gonzales k, et al: semen evaluation and fertility assessment in a purebred dog breeding facility. theriogenology 2017;87:115-123. 25. goodman m: demystifying ovulation timing. clin tech small anim pract 2002;17:97-103. 26. goodman m:ovulation timing. concepts and controversies. vet clin north am small anim pract 2001;31:219-235. clinical theriogenology • volume 10, number 3 • september 2018 196 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice canine nomograph evaluation improves puppy immunization canine nomograph evaluation improves puppy immunization laurie larson, bliss thiel, vanessa santana, ronald schultz companion animal vaccine and immuno diagnostics service laboratory department of pathobiological sciences university of wisconsin school of veterinary medicine madison, wi abstract immunization failure in puppies by modified live vaccines for canine distemper virus and canine parvovirus can occur due to interference from maternally derived antibody. quantitative measurement of specific antibody (via half-life degradation analysis; nomograph) is available to determine passive transfer from dam. nomograph enables vaccination timing and followup titer testing tailored for each litter. objective was to evaluate effectiveness of this approach and use of a nomograph. puppies (506 puppies < 1 year) that had nomograph completed for their dam were not different in protection rate compared to vaccinated adults and were proven immune at 15.9 weeks. a cohort of similar puppies at 21.6 weeks that did not have nomograph completed for their dam was more likely to have not responded to vaccination. keywords: canine, nomograph, titer, vaccination, distemper, parvovirus introduction maternally derived antibody (mda) interference with vaccination is considered as main cause for “failure to immunize” in puppies < 6 months old.1-4 determining breeding dam antibody titers and applying half life degradation analysis to litter (nomograph) improved timing for canine distemper virus (cdv) vaccination.5 this concept was not widely adopted, since very few diagnostic laboratories offered canine vaccinal antibody testing. beginning in late 1990’s, an alarming increase in adverse reactions to vaccines, most notably feline vaccine associated sarcoma,6 triggered veterinary profession to reevaluate annual vaccinations, standard practice at that time, for pet dogs and cats. american association of feline practitioners and later american animal hospital association developed vaccination guidelines for cats and dogs, respectively.7,8 world small animal veterinary association (wsava) also published similar vaccination guidelines.9 these guidelines described vaccines in 3 categories (core, noncore or not recommended). core vaccines are able to provide complete protection against widespread diseases that induce high morbidity and mortality. for canine, these vaccines are cdv, canine parvovirus (cpv2), canine adenovirus (cav1, cav2), and rabies. guidelines urged that all dogs receive cdv, cav1, cav2 and cpv2 vaccines to provide protection from infection for as long as 9 years.10-15as a result, early guidelines suggested administration of core vaccines to adult dogs at not more than 3 year intervals. sterile immunity is defined as stage of humoral immune response that “sterilizes” a specific virus, thus totally preventing infection. this applies both to vaccine and disease causing wildtype viruses. modified live viral vaccines ability to infect and replicate are blocked in face of high antibody titers. this blockage happens whether antibody is produced actively by dog or passively acquired in puppy. modified live viral vaccine neutralized by antibody provides no benefit to puppy or adult; actively immune dog usually has no increase in titer, whereas a puppy remains immunologically naïve.4 currently, canine vaccinal antibody testing is fast becoming part of standard veterinary care. multiple laboratories across north america offer quantitative antibody testing to determine immunity against cdv and cpv2, and qualitative, point of care antibody screening tests are also available to clinicians. improved availability of testing, coupled with an increased interest in appropriate vaccine use, contributed to growth in this area.16-18 although vaccine adverse reactions are rare, this risk is not offset by benefit in actively immune dog with titers above sterile immunity thresholds. most recent version of aaha canine vaccine guidelines suggest antibody testing helps clinicians balance risks and benefits of vaccination.8 puppies present a unique challenge regarding antibody testing, due to potential for residual maternally derived immunoglobulin and likelihood of immunization failure. most recent wsava clinical theriogenology • volume 12 number 3 • september 2020 215 vaccine guidelines recommend antibody testing of puppies at 6 months of age9 since all initial levels of mda will have dissipated. this is also the earliest age that qualitative point of care tests may be applied, according to manufacturer. testing pups at 24 weeks is a significant improvement over past practice of simply assuming vaccinated pups are immune. unfortunately, however, testing at 6 months of age still leaves a population of puppies at potential risk of disease, especially during this critical period. because a clinician usually does not know potential mda levels, multiple doses of vaccines are given to puppies over many weeks in an effort to both deliver an effective dose as early in puppy’s life as possible and to continue until such time as maternal antibody is believed to have dissipated. in some situations, litters may be vaccinated as frequently as every week, beginning as early as 5 weeks of age, potentially increasing chances of adverse events (e.g. immunosuppression or allergic reaction). although a definite link between puppy core vaccination and hypertrophic osteodystrophy was not established,19 producers of dog breeds at highest risk for hypertrophic osteodystrophy (weimaraners, irish setters, etc.) were among first to request nomograph service through our laboratory. although initial impetus was to avoid “shot gun” approach of administering multiple doses of vaccine over many weeks, it was soon realized that nomographs also allowed followup quantitative titer testing of puppies much younger than 24 weeks. based on our testing, average titers for breeding dams are approximately 1:640 for cpv2 and 1:64 for cdv. however, range of titers was quite large, from < 1:2 (negative) to 1:20,480. depending on antibody amount transferred at birth, age that a litter had no maternal antibody ranged from day of birth to 22 weeks of age. because of possible failure of passive transfer, nomograph should not be used to predict protection against wildtype viruses. breeders with known elevated risk of parvovirus in their kennels are urged to submit samples collected directly from selected pups at 3 4 weeks of age. this approach controls for failure of passive transfer and provides a direct measurement of maternal antibody for half life degradation analysis. because it can be difficult and stressful to collect serum from small puppies, naïve puppy baseline titer testing is only suggested in the face of high disease risk. rather than an evaluation of protection for a litter, nomograph analysis of breeding dam antibody level is intended to be a conservative estimate of duration of maternal antibody interference with modified live viral vaccines. reported percent transfer estimates of 60 70%1 was confirmed by our laboratory (data not shown). however, because we had transfer rates up to 100% in some excellent colostrum producing dams, nomograph is calculated based on conservative assumption of 100% transfer from dam to litter. best timing to collect bitch sera for nomograph analysis is 2 weeks before expected whelping date or 2 weeks after whelping. active colostrum production time is avoided, as circulating antibody titers are decreased due to sequestration of immunoglobulin g (igg) in mammary gland. ideally, nomograph should be completed for each litter in order to generate most accurate interpretation of puppy results followup testing. at minimum, a nomograph should be completed within 1 year before whelping. once dam’s titer is known and set at 100% transfer to her litter at day 0, half life degradation analysis is applied. rates of specific antibody decline for canine vary from 9 12 days.1,5 rate of 12 days was chosen to provide a more conservative estimate. standard deviation inherent to assays is graphed by showing titers 1 dilution above and 1 below reported titer. variability of mda titers between pups in a litter is within test variability parameters in majority of litters we tested for prevaccination baseline (complete data not shown). litters tested include submissions from private breeders concerned about parvovirus in their kennels, as well as purpose bred beagle litters for selection to vaccine research studies. vaccine administration is suggested when degradation analysis estimates indicate first successful immunization.1,5 a second dose of vaccine is suggested when interfering mda is very highly likely to be completely catabolized. these ages often vary between distemper and parvovirus; however, most often cpv2 titers are higher than those against cdv. in these litters, an additional dose of monovalent parvovirus vaccine is suggested at an older age. with high maternal titers against both cdv and cpv2, a dose of combination vaccine at 8 9 weeks is suggested as “optional”. although it is prudent to give a clinical theriogenology • volume 12 number 3 • september 2020216 dose of combination core vaccine to puppies before transfer to new homes, stakeholders will know that there is a good chance that this dose may not successfully immunize. quantitative titer testing is suggested 2 weeks (completion of mda degradation) after presumed final dose of vaccine. materials and methods serum samples submitted over a 3 year interval from across us and canada to companion animal vaccine and immuno diagnostics service (cavids) laboratory were used. samples were grouped based on whether nomograph had been completed for the dam or not. protection rates for sera from these groups were compared against those of a group of sera from > 5,000 individual adult dogs with history of vaccination. for all groups, including adult dogs, only first sample submitted for an individual was included for analysis. data (date of birth and vaccination history of commercial canine vaccinations) of dogs < 1 year were used. nomograph followup group included 506 individual puppies, with an average age of 15.9 weeks (range 8.7 50.9). this group included 202 distinct litters from 188 dams, with 49 breeds represented. golden retriever made up 38.3% of this group, followed by the labrador retriever at 11.2% (table 1). table 1. top 5 breeds’ percents in nomograph group group without nomograph included 235 individual puppies at average age of 21.6 weeks (range 7.7 49.4). this group was comprised of 90 breeds, including “mixed breed”. labrador retriever was 18.3% of this group, followed by golden retriever at 13.2% (table 2). table 2. top 5 breeds’ percents in without nomograph group serology methods included hemagglutination inhibition assay to determine antibody against cpv2 and serum virus neutralization assay to determine antibody against cdv. both assays are considered “gold standard,” directly test antibody function, and provide quantitative endpoint titers. test sera are doubly diluted in duplicate across a 96 well plate and incubated with a standardized amount of infectious cdv or cpv2. after 1 hour, indicator cells are added to all wells. plates are examined after further incubation. endpoint titer is reported as the last dilution at which antibody is able to neutralize viral activity.20,21 threshold of protection determined by challenge of immunity.10 both hi and svn assays detect igg and igm simultaneously and are well suited to test initial vaccine responses 2 3 weeks after presumed final dose of vaccine. data analyses chi square was used with significance of p < 0.05. protection rates of both groups of pups were compared against protection rates of a general population of vaccinated adult dogs. threshold of number of pups top 5 breeds nomograph group percent total 194 golden retriever 38.3 57 labrador retriever 11.2 24 nova scotia duck tolling retriever 4.7 21 rottweiler 4.1 21 soft-coated wheaten terrier 4.1 number of pups top 5 breeds without nomograph group percent total 43 labrador retriever 18.3 31 golden retriever 13.2 24 irish setter 10.2 15 mixed breed 6.4 12 poodle, standard and toy 5.1 clinical theriogenology • volume 12 number 3 • september 2020 217 protection for adult dogs was set at ≥1:8 (cdv) and ≥1:40 (cpv2). using this threshold, expected rate of protection is 97% for cdv and 91% for cpv2. to provide a more stringent definition of protection for puppies, and to be able to compare groups, threshold of protection for puppies was set 2 fold higher for both viruses with ≥ 1:32 for (cdv) and ≥ 1:160 for cpv2. results protection rate for the nomograph group was 95.7% (484/506) against cdv (95% ci [95.5, 95.9]) and 90.5% (458/506) against cpv2 (95% ci [90.3, 90.7]). average age of protected puppies was 15.9 weeks for both viruses. compared to rates of protection in adult population, p values were 0.0755 (cdv) and 0.7024 (cpv2), indicating no significant differences. protection rates in the group without nomograph were lower at 85.5% (201/235) against cdv (95% ci [85.2, 85.8]) and 81.7% (192/235) against cpv2 (95% ci [81.4, 82]). average age of protected puppies was 21.3 and 22.5 weeks of age for cdv and cpv2, respectively. compared to expected values in adult dogs, p values were 0.0001 for both viruses, indicating a highly significant proportion of these pups were unprotected. (figures 1, 2; table 3, 4) pe rc en ta ge o f s er um s am pl es clinical theriogenology • volume 12 number 3 • september 2020218 adult cdv puppies with nomograph puppies without nomograph 0 50 100 cdv percentage protected figure 2 pe rc en ta ge o f s er um s am pl es cdv protected cdv not protected **** table 3. percentage protected/not protected against cpv2 for groups of puppies cpv-2 ≥160 group percent protected (number) average age (weeks) percent not protected (number) average age (weeks) p value compared to adults nomograph 90.5 (458/506) 15.9 9.5 (48/506) 15.7 0.7024 without nomograph 81.7 (192/235) 22.5 18.3 (43/235) 17.8 0.0001** table 4. percentage protected/not protected against cdv for groups of puppies cdv ≥32 group percent protected (number) average age (weeks) percent not protected (number) average age (weeks) p value compared to adults nomograph 95.6 (484/506) 15.9 4.3 (22/506) 14.3 0.0755 without nomograph 85.5 (201/235) 21.3 14.5 (34/235) 23.5 0.0001** discussion maternally derived antibody is only 1 potential factor affecting vaccination failure. other factors include poor vaccine handling and storage, incorrect administration and genetic issues. however, current study confirmed that mda had a highly significant impact on vaccinal response in puppies. examining role of genetics as a risk factor for poor vaccine response was beyond the scope of this study; however, there were some noteworthy findings. inclusion of 6.4% “mixed breed” puppies in group without nomograph did not seem to have a significant positive impact on protection rates for this group. interestingly, in nomograph group, most puppies that failed to respond to vaccine were unique within their litters. in most cases, littermates of nomograph nonresponders responded well (data not shown). one litter of 10 labrador retrievers was the exception, with 7 pups that failed to respond to cpv2 clinical theriogenology • volume 12 number 3 • september 2020 219 vaccine. after a further dose of vaccine, 6 of 7 responded well. seventh pup required 1 additional dose before eventually having an active response at 20 weeks. mechanism of non-responsiveness in this litter is unknown, but the phenomenon emphasized importance of titer testing puppies to be certain of immunity. it is also interesting to note that this single litter (out of 202 litters tested) comprised 14.6% of 48 puppies that did not respond to parvovirus in the nomograph followup group. although nomograph report suggests tailored vaccination and follow-up testing schedules for each litter, veterinarians and puppy owners are free to interpret and implement nomograph data as they wish. in the current study, compliance was not determined precisely, but the nomograph group was generally in agreement with nomograph-generated schedules (data not shown). one notable exception was the single puppy in the nomograph group tested at 8.7 weeks of age. although testing at this age was not in accordance with nomograph based suggestions for the litter, this sample was retained in the nomograph group data set. this puppy was negative for cpv2 antibody. in another interesting finding, the youngest puppy in the group without a nomograph was protected against distemper, but not for parvovirus, at 7.7 weeks of age. this pup’s distemper titer was very strong at 1:4,096, clearly an active response to vaccine. for this titer to be attributable to mda, the dam would have had to have an extremely unlikely cdv antibody titer of 1:65,536 with a 100% transfer rate to her litter. in general, samples from very young pups are not submitted when dam’s titers are not known. this is reflected in the smaller size and older average age of the group without nomographs. however, in instances where the puppy has had an adverse reaction to vaccination or other health issue that may complicate further vaccination, quantitative testing and “reverse” degradation analysis can be applied, along with a stringent threshold of protection. response to vaccination and protection rates for all groups, regardless of age or nomograph status, was much higher for cdv than for cpv2. although mechanism responsible is not clearly understood, it possibly stems from the fact that parvovirus has more recently emerged into domestic canine population than distemper virus and has experienced relatively fewer generations of coevolution. current study confirmed that nomograph analysis of maternal antibody titers against cdv and cpv2 provides veterinarians and dog breeders with useful information to guide litter vaccination decisions and speed confirmation of immunity. in case of low maternal titers, much peace of mind can be gained by early proof of protection, especially for critical socialization experiences, e.g. puppy kindergarten. in the case of higher maternal titers, vaccination series will be extended, in some cases beyond standard final dose at 16 weeks of age. for all litters and stakeholders, overall outcomes are improved. dog breeders can provide added value to their puppy buyers by providing information about dam’s titers. veterinarians can make better informed vaccination decisions. most importantly, more families may be spared emotional and financial impacts of severe morbidity and mortality associated with cdv and cpv2 infections. future studies could examine factors that may influence nomograph, such as determining if active colostrum production induces change in circulating antibody titers for bitch; improving understanding of extent of influence of litter size on passive transfer rate; establishing if there are measurable differences in antibody degradation rate due to breed-specific body size, caloric restriction and/or metabolism; characterizing impacts of caesarian section on colostrum production and passive transfer; and determining effect of administration of supplemental antibody products (e.g. fresh frozen plasma) on passive antibody titers in recipient pups. longer-term collection of titer data of puppies vaccinated according to nomograph as they enter young adulthood and beyond is currently underway. conclusion current study demonstrated that breeding dams nomograph testing provided important information to improve core immunization outcomes for puppies < 1 year and facilitated earlier followup titer testing for immunity. regardless of whether nomograph was done for a dam, authors support wsava guidelines in urging that all puppies are tested for antibody titers by 6 months to detect failure of immunization during this highest risk period. methods to detect unprotected puppies are readily available to veterinary practitioner, either through point of care screening tests at 24 weeks, or clinical theriogenology • volume 12 number 3 • september 2020220 quantitative testing earlier when maternal titers are known. best medical practice should support applying this standard. presumption that vaccination is equivalent to immunization may lead to unfortunate, but avoidable surprises. acknowledgement dr. james baker, originator of canine nomograph in 1958, richly deserves appreciation. authors are grateful to avidog international, llc and their dog breeder members who submitted a large proportion of sera for nomograph analysis. conflict of interest authors are employed by cavids laboratory offers (fee for service) serology testing, including nomograph and nomograph follow-up testing of puppies. authors are grateful to cavids laboratory clients for supporting this entire study by their fees. references 1. pollock rv, carmichael le: maternally derived immunity to canine parvovirus infection: transfer, decline, and interference with vaccination. j am vet med assoc 1982;180:37-42. 2. pardo mc, tanner p, bauman j, et al : immunization of puppies in the presence of maternally derived antibodies against canine distemper virus. j comp pathol 2007;137:72-75. 3. roth ja: mechanistic bases for adverse vaccine reactions and vaccine failures. in: schultz rd, editor, advances in veterinary medicine: veterinary vaccines and diagnostics, academic press; cambridge: 1998. p. 681-700. 4. day mj, schultz rd: vaccination. in: veterinary immunology: principles and practice. academic press; cambridge :1999. p. 681-700. 5. baker, robson, gillespie, et al: a nomograph that predicts the age to vaccinate puppies against distemper. cornell veterinarian. 1958 aug: 158-167. 6. hartman k, day mj, thiry e, et al: feline injection-site sarcoma : abcd guidelines on prevention and management. j feline med surg. 2015;17:606-613. 7. scherk ma, ford rb, gaskell rm, et al: aafp feline vaccination advisory panel report. j feline med surg 2013;15:785-808. doi: 10.1177/1098612x13500429. erratum in: j feline med surg 2013;15:np2. j feline med surg 2014;16:66. 8. ford rb, larson lj, mcclure kd, et al: aaha canine vaccination guidelines. j am anim hosp assoc 2017; 53:243-251. 9. day mj, horzinek mc, schultz rd, et al: guidelines for the vaccination of dogs and cats. world small animal veterinary association, vaccine guidelines group. j sm anim pract 2016;57:1-45. 10. abdelmagid oy, larson l, payne l, et al: evaluation of the efficacy and duration of immunity of a canine combination vaccine against virulent parvovirus, infectious canine hepatitis virus, and distemper virus experimental challenges. vet ther 2004;5:173-186. 11. mouzin de, lorenzen mj, haworth jd, et al : duration of serologic response to five viral antigens in dogs. j am vet med assoc 2004;224:55-60. 12. gill m, srinivas j, morozov i, et al: three-year duration of immunity for canine distemper, adenovirus, and parvovirus after vaccination with a multivalent canine vaccine. int j appl res vet med 2004;2:227-234. 13. schultz rd: duration of immunity for canine and feline vaccines: a review. vet microbiol 2006;117:75-79. 14. larson lj, schultz r: three-year serologic immunity against canine parvovirus type 2 and canine adenovirus type 2 in dogs vaccinated with a canine combination vaccine. vet ther 2007;8:305-310. 15. larson lj, schultz rd: three-year duration of immunity in dogs vaccinated with a canarypox-vectored recombinant canine distemper virus vaccine. vet ther 2007;8:101-106. 16. schultz rd, ford rb, olsen j, et al: titer testing and vaccination: a new look at traditional practices. vet med 2002;97:1-13. 17. tizard i, ni y: use of serologic testing to assess immune status of companion animals. j am vet med assoc 1998;213:54-60. 18. jensen wa, totten js, lappin mr, et al: use of serologic tests to predict resistance to canine distemper virus-induced disease in vaccinated dogs. j vet diag invest 2015;27:576-580. 19. harrus s, waner t, aizenberg i, et al: development of hypertrophic osteodystrophy and antibody response in a litter of vaccinated weimaraner puppies. j small an prac 2002;43:27-31. 20. schultz rd, adams ls: immunologic methods for the detection of humoral and cellular immunity. vet clin north am small anim clin1978; 8:721-753. 21. carmichael le, joubert jc, pollock rv: hemagglutination by canine parvovirus: serologic studies and diagnostic applications. am j vet res 1980;41:784-791. clinical theriogenology • volume 12 number 3 • september 2020 221 012_ms-012 l.larson use of chain écraseur for removal of retained abdominal testis in a bull   use of chain écraseur for removal of retained abdominal testis in a bull pablo yepez,a sabrina hall,a agustina grippo,b tulio pradoa adepartment of veterinary clinical sciences, college of veterinary medicine university of tennessee, knoxville, tn bfaculty of agronomy and veterinary, national university of rio cuarto, córdoba, argentina abstract a 4 year old banteng bull was presented to the university of tennessee for a routine castration. on physical examination, it was determined that the bull’s right testis had not descended and thus was deemed cryptorchid. right testis was not palpable in the inguinal canal nor visible via ultrasonography of caudal abdomen. due to the likelihood that the testis was located in the abdomen, surgery under general anesthesia was elected. because of its location close to right kidney, manual removal became difficult. an écraseur was successfully utilized to remove the retained testis. keywords: banteng bull, castration, cryptorchid, écraseur introduction cryptorchidism is a rare condition in bulls where 1 or both testes have not descended into the scrotum.1 incidence of cryptorchidism in bulls is 0.17% in north america, with shorthorn and polled hereford the most represented breeds. there is an overall combined incidence of this condition of 1.4% in other breeds such as hereford, angus, and holstein-friesian.2,3 incidence of cryptorchidism in banteng breed is not reported. undescended testis may be located in the abdominal cavity, or underneath skin in the scrotal region, usually away from its normal pathway of descent.4 during fetal development, pathway of testicular descension is characterized by 3 stages. stages include nephric displacement, transabdominal passage, and inguinal passage that ends when the gubernaculum ceases to grow and testis migrate to inguinal opening. by week 22 of pregnancy, testis should reach the scrotum.5-7 in cases of cryptorchidism, spermatogenesis was negatively affected in retained testis due to exposure to elevated temperatures within the abdomen, whereas a descended testis remained normal.8 despite the difference in normal spermatogenesis, testosterone production was not affected in the retained testis.8 tumors have been reported in undescended testes in bulls.9 diagnosis is usually via transrectal palpation or ultrasonography and may be inconclusive in some cases.2 measurement of testosterone concentrations following hcg or gnrh treatment in cryptorchid bulls was not rewarding as in other species, since cryptorchid bulls had a near normal serum testosterone concentrations in the presence of elevated serum concentrations of lh and fsh.7,10 a better alternative for diagnosis of cryptorchidism in bulls would be determination of antimüllerian hormone and inhibin concentrations, reliable biomarkers for the presence of testicular tissue.11 this technique report describes the use of a chain écraseur, for the first time, in the removal of cryptorchid testis, as used for removal of ovaries in mares.12 method a 500 kg, 4-year old banteng bull was presented for routine castration. bull was immobilized with carfentanil (carfentanil, 3 mg/ml, zoopharm, laramie, wy) and xylazine (xylamed, 100 mg/ml, vet one, boise, id) in a squeeze chute for evaluation preceding routine castration. on physical examination, the right testis was not palpable within scrotum nor in the inguinal canal. using an aloka 1400 ultrasound scanner with a 3.5 mhz curvilinear abdominal probe, caudal abdomen and inguinal area were examined. retained testis could not be identified. due to bull’s disposition, it was not possible to perform transrectal palpation. due to the likelihood that the testis could be located in the abdomen, we requested the owner to bring the bull back for castration and for an exploratory laparotomy under general anesthesia. bull was held off feed for 24 hours and water for 12 hours prior to surgery. anesthesia was induced intramuscularly with 0.013 mg/kg carfentanil citrate in combination with 0.2 mg/kg of xylazine hcl and was maintained with isoflurane. bull received intravenously 2 mg/kg ketamine hcl (zetamine, clinical theriogenology • volume 12 number 4 • december 2020526   100 mg/ml, vet one) during surgery. bull was placed in left lateral recumbency. after sterile preparation of the surgical field, a 12 cm right parainguinal incision was made with a number 10 scalpel blade. right testis was palpable caudal to right kidney and an attempt was made to exteriorize it. due its location and attachment it could not be exteriorized. at this point a decision was made to use a chain écraseur (48 cm ratchet type écraseur [chassaignac], jorgensen laboratories, loveland, co) to separate the retained testis from its attachment prior to exteriorization. retained testis and its pedicle were identified by digital manipulation. four fingers were inserted into écraseur’s chain loop and slipped over the grasped testis to encircle the pedicle. the chain was tightened around the pedicle using the rachet, with verification that the chain did not encircle other structures. ratchetting continued until the pedicle was severed and the testis was extracted through the incision. the palm of the hand was held beneath the pedicle to check for bleeding (none was noted). peritoneum was closed with #1 polydioxanone suture (pds) in a simple continuous pattern. body wall and subcutaneous tissues were closed in a simple continuous pattern with #1 polydioxanone suture (pds). skin was closed in a ford interlocking pattern with #1 polydioxanone suture (pds). a closed castration technique was performed with a serra emasculator (35.5 cm, jorgensen laboratories, loveland, co) to remove the descended testis. a transfixing suture #1 polydioxanone suture (pds) was placed to ligate the cord above the emasculator. sedation was reversed with intravenous treatment of natrexone hydrochloride ([1.16 mg/kg] naltrexone, 50 mg/ml, zoopharm) and yohimbine hcl usp (0.13 mg/kg [yohimbine, 5 mg/ml, letco medical, decatur al]). bull was treated intramuscularly with penicillin g procaine 20,000 iu/kg (penone pro, vet one, madison, nj) and intravenously with 1.1 mg/kg of flunixin meglumine (banamine, 50 mg/ml, merck, madison, nj). in addition, a tetanus toxoid was given subcutaneously on the left side. outcome there were no complications and the animal appeared normal in a subsequent examination. discussion cryptorchidism (‘hidden testis’) is a postnatal phenotype that affects normal production of sperm1,13,14 and known to be hereditary.13 testis can be in various locations (ectopic testis).1,13 ectopic testes are commonly positioned on either side of the penis, cranial to the base of the scrotum and oriented with their long axis parallel to the penis.1 based on our observations, ectopic testis was lower in our differentials. testicular monorchidism (presence of 1 testis) is a rare condition that has to be considered as a differential in cases such as this when the hidden testis is not palpable or identifiable by ultrasonography. neoplasia9,15 may require removal of retained testis. furthermore, it is advisable also to remove the descended testis for ethical reasons. there are 5 typical laparotomy approaches for a cryptorchidectomy: flank, inguinal, parainguinal, suprapubic paramedian, and ventral midline.1,13,16,17 one of the options is a laparoscopic procedure if the testis is located in the abdomen. this procedure has less complications during and after surgery, but in this case, since the retained testis was not identified on palpation or ultrasonography, this technique was not an option. recumbent position that was required for this bull would have made it difficult to access the testis via a laparoscope and also the possibility of its location in the inguinal canal. abdominal testes are best removed via flank celiotomy.13 apparently, a flank celiotomy using a chain écraseur removal was the best option given the location of the testis. use of a chain écraseur provided a slow and controlled process for crushing in a steady state until transection. ligatures were therefore unnecessary, as reported.18 none of the possible complications such as hemorrhage, swelling, redness and infection13 occurred in this case. use of an écraseur is not a new idea, since this technique has been used in other species for removal of various structures (e.g. ovaries in cows or seminal vesicles in bulls, excision of a pharyngeal granuloma in a cow,17,19-22 transpalpebral enucleation, removal of the ovaries in mares,12,18 and castration in elephants23). however, for the first time, an écraseur was successfully utilized to remove a retained testis. clinical theriogenology • volume 12 number 4 • december 2020 527   conclusion use of chain écraseur can be a viable tool in the removal of a retained testis from a cryptorchid bull. this was the first time using this technique and it has shown excellent results. clinicians can consider the use of a chain écraseur in retained testis cases. conflict of interest none to declare. references 1. riddell mg: developmental anomalies of the scrotum and testis. in: wolfe df, moll hd, editors: large animal urogenital surgery. 2nd edition, baltimore; williams and williams: 1998. p. 283-294. 2. st jean g, gaughan em, constable pd: cryptorchidism in north american cattle: breed predisposition and clinical findings. theriogenology 1992;38:951-958. 3. godfrey rw, dodson re: breeding soundness evaluations of senepol bulls in the us virgin islands. theriogenology 2005;63:831-840. 4. kaneko y, torisu s, kitahara g, et al: laparoscopic cryptorchidectomy in standing bulls. j vet med sci 2015; 77:631-635. 5. hullinger rl, wensing cjg: descent of the testis in the fetal calf. cells tissues organs 1985; 121:63-68. 6. setchell bp: development of the testis. in: the mammalian testis, setchell, bp: editor. new york; cornell university press: 1978. p. 30-43. 7. kellaway rc, seamark rf, farrant rk: sterilisation of cattle by induced cryptorchidism. aust vet j 1971;47:547-550. 8. parkinson t j, mcgowan m: abnormalities affecting reproductive function of male animals. in: noakes de, parkinson tj, england acw: editors. veterinary reproduction and obstetrics. 10th edition, china; elsevier: 2019. p. 635-668. 9. osawa t, miura m, yamagishi n, et al: fibrolipoma of a cryptorchid testis in a young bull. j vet med sci 2011;73:1253. 10. schanbacher bd: testosterone secretion in cryptorchid and intact bulls injected with gonadotropin-releasing hormone and luteinizing hormone. endocrinology 1979;104:360-364. 11. scarlet d, aurich c, ille n, et al: anti-muellerian hormone, inhibin a, gonadotropins, and gonadotropin receptors in bull calves after partial scrotal resection, orchidectomy, and burdizzo castration. theriogenology 2017;87:242-249. 12. prado t, schumacher j: how to perform ovariectomy through a colpotomy. equine vet edu 2019; 31:209-213. 13. gilbert ro, cable c, fubini sl, steiner a: surgery of the bovine reproductive system and urinary tract. in: fubini sl, ducharme ng: editors. farm animal surgery. 2nd edition. maryland heights; elsevier: 2017. p. 359-360. 14. amann rp, veeramachaneni dnr: cryptorchidism in common eutherian mammals. reproduction 2007;133:541-561. 15. lópez a, ikede b, ogilvie t: unilateral interstitial (leydig) cell tumor in a neonatal cryptorchid calf. j vet diagn invest 1994;6:133-135. 16. brinsko sp, blanchard tl, varner dd, et al: surgery of the stallion reproductive tract. in: manual of equine reproduction. 3rd edition. maryland heights; elsevier: 2011. p. 242-275. 17. prado tm, dawson lj, schumacher j: surgical procedures of the genital organs of bulls. vet clin north am food anim pract 2016;32:701-725. 18. garcía-lópez jm, poulin ae, doran re, quinteros dd, abuja ga: transpalpebral enucleation using a chain écraseur. equine vet edu 2009;21:603-607. 19. hull bl, monke dr, rohde r: a new technique for seminal vesiculectomy. in: proceedings of the 14th technical conference on artificial insemination and reproduction. milwaukee 1992. p. 100-103. 20. blaser m, steiner a, hirsbrunner g: a novel technique for surgical removal of protruded perivaginal fat in cattle. vet rec 2013;173:220. 21. boileau mj, jann hw, confer aw: use of a chain écraseur for excision of a pharyngeal granuloma in a cow. j am vet med assoc 2009; 234:935-937. 22. al-eknah mm, noakes de: uterine activity in cows during the oestrous cycle, after ovariectomy and following exogenous oestradiol and progesterone. br vet j 1989;145:328-336. 23. foerner jj, houck ri, copeland jf, et al: surgical castration of the elephant (elephas maximus and loxodonta africana). j zoo wildlife med 1994;1:355-359. clinical theriogenology • volume 12 number 4 • december 2020528 clinical theriogenology 2022; 14: 370 ovarian remnant syndrome in small animals: case series kiki mullikin,a michael byron,b janice chen,c soon hon cheong,b cathy gartley,c mariana diel de amorimb alaurel highlands animal health, somerset, pa bcornell university college of veterinary medicine, ithaca, ny contario veterinary college, university of guelph, guelph, canada abstract our objective was to report the utility of various diagnostic tests in identifying ovarian remnant syndrome (ors) in dogs and cats. medical records from 2 referral teaching hospitals (health sciences centre at the ontario veterinary college and cornell university hospital for animals) were examined and 48 animals (31 dogs and 17 cats) were chosen. data included were based on sufficient clinical or diagnostic evidence of ors. histopathology was used as the confirmatory test for ors. there was no difference between the proportions of dogs versus cats diagnosed with ors. similarly, there was no difference in the proportions of ovarian remnants (or) between large, medium, or small dogs, or the side (right, left, or bilateral) in which or was diagnosed. vaginal cytology and transabdominal ultrasonography findings, and serum progesterone concentrations had the highest chance of correctly identifying an or prior to exploratory surgery. transabdominal ultrasonography had strong agreement with or location identified at surgery. presumptive intraoperative diagnosis of or was possible in 39/41 cases (95.1%). auxiliary diagnostic testing should be recommended to confirm functional ovarian tissue before surgery to reduce unnecessary surgery. additionally, transabdominal ultrasonography examination may reduce surgical time since ovarian remnant location has strong agreement with surgical findings. keywords: dogs, cats, ovarian remanent, anti-müllerian hormone, progesterone concentrations, transabdominal ultrasonography introduction ovarian remnant syndrome (ors) is defined as the presence of functional gonadal tissue in a bitch or queen that had undergone a previous ovariectomy or ovariohysterectomy (ohe).1,2 clinical presentations of ors include: mammary gland enlargement or masses, pollakiuria and stranguria, vulvar or vaginal masses, polyuria and polydipsia, recurrent urinary tract infection, and vaginal discharge and pseudopregnancy.3,4 presence of functional ovarian remnant tissue is most often the result of incomplete surgical removal. it can also be the result of dropping a tissue fragment into the surgical site or failing to identify and remove ectopic ovarian tissue.4-6 studies on surgical complications of postohe reported that 17 43% of the referral cases, presented for postoperative complications after ovariohysterectomy, were for ors.7,8 animal’s age, breed, body condition, whether the surgical procedure was elective or emergent, and the experience of the surgeon were not associated with a higher likelihood of ors.4,9 however, in a larger scale and in a more recent study that examined the medical records of 1,880 bitches, higher weight was associated with more postohe complications, including ors.10 relating to this aspect of surgical access, the right ovary (located more cranially) was the more common site for finding an ovarian remnant compared to left.11,12 additionally, reports vary on whether ors is more common in dogs or cats,4,9 with some authors suggesting a higher likelihood in dogs due to the presence of its unique ovarian bursa.3,13 the mean time to recurrence of estrous behavior following spay is reportedly 1.3 years in dogs and 3 years in cats.1,3,4 most commonly reported clinical signs are associated with proestrus and estrous signs.3 vaginal discharge may or may not be present depending on the amount of uterine stump left at ohe; other differentials for vaginal discharge such as coagulopathies, vaginitis, trauma, vaginal neoplasia, and vaginal foreign bodies should be ruled out.4 diagnosis of ors can be presumptive based on clinical signs or supported by ancillary testing such as, vaginal cytology, measuring estradiol and progesterone concentrations (before and after stimulation with gonadotropin releasing hormone [gnrh] or human chorionic gonadotropin [hcg]) or luteinizing hormone (lh) or anti-müllerian hormone (amh) or via ultrasonography.1,3,9,11,14 vaginal cytology (based on abundance of superficial epithelial cells) is the cheapest and clinical theriogenology 2022; 14: 371 fastest method for presumptive diagnosis of ors in dogs and cats presenting with proestrus or estrus clinical signs.1,4 vaginal cytology results are useful to determine the presence of functional ovarian tissue and endogenous estrogen secretions; however, exogenous and adrenal estrogen exposure should be ruled out.15 additionally, vaginal cytology in cat presents both a challenge to obtain and to interpret.16 serum estradiol concentrations > 20 pg/ml are indicative of ors in a ohe dog. however, reliability of estradiol as a marker of ors is limited because it is not produced solely by the ovaries; furthermore, it varies on a daily, weekly, and seasonal basis, and also there is variability among dogs.17 serum lh concentrations are expected to be elevated in spayed animals due to lack of negative feedback of estradiol;1 however, a false positive could occur if the test is performed on the day of an lh peak in dogs, or false negative if the test is performed during the period of shorter day length in cats.18 baseline progesterone concentrations are very reliable as it is an indication of active luteal tissue in the dog, and remain elevated for 4 months of the year in most dogs. however, in queens, progesterone concentrations are not useful indicator of the presence of functional ovarian tissue unless ovulation has been induced.9 anti-müllerian hormone in adult females is secreted exclusively by granulosa cells of the ovary and therefore has been used as a serum biomarker for ors.19 however, amh results can be inconclusive in remnant ovarian tissues that have corpora lutea. therefore, amh and progesterone tests should be performed together for higher accuracy. additionally, amh has not been thoroughly investigated in prepubertal bitches that could lead to false negative results. despite its limitations, amh and progesterone have become the gold standard diagnosis of ors prior to surgical exploration.14 use of transabdominal ultrasonography in detecting ovarian remnants continues to be debated; the successful diagnosis of ors depends on the skill of the examiner, the size of the remnant, and estrous cycle stage.1,19 a study reported that ultrasonography was more helpful as an ancillary test to history and clinical signs rather than a replacement to hormonal testing.3 additionally, in 32 dogs with ors transabdominal ultrasonography was able to identify a remnant in 30 out of 31 dogs (97%) that had a remnant confirmed via histopathology examination.11 exploratory laparotomy/laparoscopy is the treatment of choice for ors and histopathology is considered the definitive diagnosis.1,11 our objective was to describe the most common clinical features, signalment of animals diagnosed with ors and diagnostic tools utilized. materials and methods medical records from 2 referral veterinary teaching hospitals from 1984 to 2020 were reviewed to identify dogs and cats with a history of previous ohe that presented with clinical signs of estrus and/ or clinical signs that led to the presumptive diagnosis of ors. cases were included if sufficient clinical or diagnostic evidence of an or was present. diagnosis of ors was via vaginal cytology, serum estradiol concentrations, serum progesterone concentrations (> 0.5 ng/ml),14 measurement of progesterone concentrations after an gonadotropin stimulation test, amh concentrations, identification of ovarian remnant via transabdominal ultrasonography by a board-certified radiologist or radiology resident, identification of an ovary during an exploratory laparotomy/laparoscopy, histopathology, or any combination of these techniques. cases referred for ors but did not have confirmation of ovarian remnant via histopathology, at surgery, or via vaginal cytology in combination with progesterone analysis were excluded. following information, if available, was collected: species, breed, size of breed (small, medium or large), age at presentation, age at ohe, time from spay to clinical signs, presenting complaint, clinical signs observed by the owner and the attending veterinarian, results of vaginal cytology, serum estradiol, progesterone, lh, or amh concentrations, transabdominal ultrasonography findings, side of suspected ovarian remnant via ultrasonography (right, left or both), surgical approach for exploratory laparotomy (ventral midline or laparoscopy), side of the remnant at surgery (right, left or both), and histopathological diagnosis. data analyses statistical analysis was performed with jmp pro software version 16 (sas institute inc., cary, nc, usa), with a level of significance of p < 0.05. fischer’s exact test was performed to test the differences in the proportions of species, breed size, and ovarian remnant side at surgery. a kappa coefficient of agreement was used to determine the correlation between ovarian remnant side diagnosed via transabdominal ultrasonography to surgery findings. results forty-eight animals (31 dogs and 17 cats) were diagnosed with ors. dog population consisted of small, medium, and large breed dogs that included 10 labrador retrievers and 12 mixed breed dogs. remaining breeds were akita, boston terrier, brittany spaniel, brussels griffon, kerry blue terrier, pug, rottweiler, german short-haired pointer, and west highland white terrier. in the cat population, 13 were domestic shorthair, with the remainder comprised of a himalayan, a bengal, and 2 domestic longhair cats. the median and interquartile range age at presentation was 2.3 (1.4 5.7) years, whereas the age at presentation for dogs and cats respectively was 3.5 (1.6 5.9) years and 1.5 (1 3.5) years. the median and interquartile time between previous ovariohysterectomy and presentation of clinical signs resembling ors was 0.5 (0.3 2) years, whereas for dogs and cats the median and interquartile range were 1.0 (0.3 2.8) years and 0.5 (0.3 0.8) years, respectively. there was no difference in the proportion of dogs versus cats diagnosed with ors or between the proportion of large breed dogs (36.7%) compared to medium (36.7%) and small dogs (26.7%). there was no difference in the proportions of the location of the or, where 8.5% of the cases were identified in both locations, 40.4% were identified on the left side and the remaining 51.0% were on the right. as per owners, 45 of 48 animals (93.8%) exhibited classical signs of ors. these signs included bloody vaginal discharge, vulvar swelling, and attracting males in dogs and rolling, vocalizing, and attracting males in cats (table 1). of those animals presenting for classical signs of estrus, vaginal cytology clinical theriogenology 2022; 14: 372 was performed on 24 animals (50.0%), and 12/24 of these animals had a cytological finding consistent with estrous smear (50.0%). the remaining 12 vaginal cytology were classified as proestrus, diestrus, or inconclusive. seven of those cytology had concurrent progesterone concentrations consistent with ovarian function (> 0.5 ng/ml) and 4 had progesterone concentrations that were < 0.5 ng/ml. progesterone concentrations were not analyzed in 1 remaining dog. table 1. number of affected animals and percentage of affected cases out of total number of cases by presenting clinical signs clinical sign number affected percent affected dogs (n = 31) vulvar swelling 23 74.2 bloody vaginal discharge 20 64.5 cats (n = 17) vocalizing 13 76.5 dogs and cats combined (n = 48) mammary development 10 20.8 attracting males 6 12.5 abdominal/mammary neoplasia 3 6.3 surgical complications of previous ohe 2 4.2 serum estradiol concentrations were measured in 4 animals (8.5%); 2 of these had serum estradiol concentrations > 20 pg/ml. baseline serum progesterone concentrations were measured in 16 dogs (51.6%). of these 16 dogs, 10 had serum progesterone concentrations > 0.5 ng/ml (62.5%). measurement of serum progesterone concentrations after stimulation with hcg or gnrh was performed in 6 cats and 1 dog (14.6%). serum progesterone concentrations increased > 0.5 ng/ml in all 6 cats, but not in the dog in response to hcg or gnrh stimulation; however, this dog had positive estradiol concentrations and ovarian remnant was identified via surgery and histopathology. an in-house lh test was performed in 5 animals (3 dogs and 2 cats) that correctly identified ors in 4/5 (80%) of animals and subsequently confirmed via histopathology. in 1 case ors was not identifiable via lh concentrations and progesterone concentrations of this dog was < 0.2 ng/ml; however transabdominal ultrasonography and surgery correctly identified or that was confirmed via histopathology. amh assays were performed in 6 animals (5 dogs and 1 cat) (12.5%); 3/5 dogs tested positive, whereas the other 2 were inconclusive. however, progesterone concentrations on these were > 0.5 ng/ ml and surgery/histopathology confirmed the presence of an ovarian remnant. the single cat that had the amh performed was positive for amh and that was confirmed via histopathology. diagnostic examinations and clinical findings in 48 cases are listed (table 2). cases that were correctly diagnosed with various diagnostic tests compared to histopathology confirmation of or are identified (table 2). table 2. diagnostic examinations and clinical findings case species breed age at presentation (years) presenting complaint vaginal cytology* p4 † (ng/ ml) amh lh ultrasonography findings surgical approach or at surgery surgery/histology findings 1 dog labrador retriever 5.9 estrous signs ventral midline right (+) right ectopic or, bilateral paraovarian cyst (+) 2 dog mixed 7.3 caudal abdominal mass renal cyst (-) ventral midline both (+) ovarian tissues and uterine horn 3 dog akita 1.4 estrous signs inc 27.66 ventral midline right (+) right entire ovary normal (+) 4 dog mixed 9.4 estrous signs diestrus 0.13 uterine stump (-) ventral midline right (+) right granulosa cell tumor (+) 5 dog labrador retriever 4.3 estrous signs 0.38‡ ventral midline left (+) mass resembling ovarian tissue§ clinical theriogenology 2022; 14: 373 case species breed age at presentation (years) presenting complaint vaginal cytology* p4 † (ng/ ml) amh lh ultrasonography findings surgical approach or at surgery surgery/histology findings 6 dog labrador retriever 5.6 estrous signs right ovary (+) 7 dog rottweiler 6.8 postspay complication inconclusive (-) ventral midline left (+) left ovarian tissue (+) 8 dog labrador retriever 3.5 estrous signs ventral midline right (+) right or w/ cl and follicles (+) 9 dog mixed 2.2 estrous signs ventral midline right (+) right or w/ cl (+) 10 dog mixed 1.7 estrous signs right ovary w/ cl (+) ventral midline right (+) right ovary w/ cl, follicles (+) 11 dog mixed 2.0 estrous signs intact right ovary and uterine horn remnant (+) ventral midline right (+) right ovary, uterine tube 12 dog mixed 1.3 estrous signs estrus ventral midline right (+) or with cl, uterine tube (+) 13 dog mixed 13.4 estrous signs caudal abdominal mass (-) ventral midline left (+) left or with granulosa cell tumor (+) 14 dog brittany spaniel 0.8 estrous signs estrus 1.68 right ovary w/ cystic structures (+) ventral midline right (+) right entire ovary w/ cl and follicles (+) 15 dog boston terrier 6.1 estrous signs proestrus 0.03 + 16 dog labrador retriever 2.2 estrous signs proestrus/ diestrus 0.61 inc right or (+) laparoscopy right (+) right ovary 17 dog mixed 1.1 estrous signs right or (+) ventral midline right (-) right ovary w/ cl and follicles (+) 18 dog mixed 9.7 estrous signs proestrus/ diestrus 31.4 + left or (+) 19 dog pug 5.9 estrous signs diestrus large midabdominal mass (-) ventral midline left (+) left ovary w/ cyst (+) 20 dog labrador retriever 6.4 vomiting, diarrhea 5.1 right or (+) ventral midline right (+) right ovary w/ cl and paraovarian cyst (+) 21 dog mixed 3.7 estrous signs proestrus/ diestrus 3.5 + right or w/ cl (+) ventral midline right (+) right or w/ cl (+) 22 dog german shorthaired pointer 4.1 estrous signs proestrus/ diestrus 0.78 inc right or (+) ventral midline right (+) right or w/ multiple cl (+) 23 dog westie 3.6 estrous signs right or and oviduct (+) ventral midline right (+) entire ovary, uterine tube (+) 24 dog brussels griffon 5.7 estrous signs proestrus/ diestrus 0.34 uterine stump (-) ventral midline both (+) left granulosa cell hyperplasia and right hydrosalpinx (+) 25 dog labrador retriever 1.4 estrous signs diestrus 0.44 or (unknown location) (+) laparoscopy right (+) endometrial hyperplasia and focal hydrosalpinx (-) 26 dog mixed 2.3 estrous signs estrus 0.2 laparoscopy right (+) right ovarian remnant and hydrosalpinx (+) 27 dog kerry blue terrier 1.4 estrous signs diestrus 0.57 + right or (+) laparoscopy right (+) ovarian remnant and hydrosalpinx (+) clinical theriogenology 2022; 14: 374 case species breed age at presentation (years) presenting complaint vaginal cytology* p4 † (ng/ ml) amh lh ultrasonography findings surgical approach or at surgery surgery/histology findings 28 dog mixed 3.0 estrous signs estrus right ovary (+) laparoscopy right (+) or w/ cl (+) 29 dog mixed 2.0 pseudopregnancies bilateral ovaries and cyst-like oviductal structures (+) laparoscopy both (+) left and right ovarian remnant, cystic remnant of uterine horn (+) 30 dog softcoated wheaten terrier 1.0 estrous signs diestrus 20.0 + right ovary (+) ventral midline right (+) ovotestis w/ cl (+) 31 dog labrador retriever 1.6 estrous signs 0.57 left or (+) laparoscopy left (+) granulosa cell hyperplasia, cl, uterine tube (+) 32 cat dsh 10.3 mammary tumors; cyclicity ventral midline left (+) or (follicles and cl); mammary gland adenocarcinoma and ductal carcinoma (+) 33 cat himalayan 10.7 estrous signs large abdominal mass suspect of ovarian tumor (+) ventral midline both (+) granulosa cell tumor (+) 34 cat dsh 1.8 estrous signs estrus ventral midline right (+) follicles, granulosa cells (+) 35 cat dlh 3.4 estrous signs ventral midline left (+) left cystic ovary w/ luteinization (+) 36 cat dsh 1.5 estrous signs 7.34** left ovary (+) ventral midline left (+) left ovary w/ cl and follicles (+) 37 cat dsh 0.8 estrous signs uterine stump (-) ventral midline left (+) left ovary w/ follicles (+) 38 cat dsh 2.4 estrous signs left ovary w/ follicles or cl (+) ventral midline left (+) left ovary w/ follicles (+) 39 cat dlh 1.0 estrous signs ventral midline left (+) left entire ovary 40 cat dsh 0.7 estrous signs left or w/ follicles (+) ventral midline left (+) left ovary, uterine tube (+) 41 cat dsh 1.0 estrous signs estrus 0.93** no or (-) laparoscopy left (+) left ovary w/ cl and follicle (+) 42 cat dsh 3.5 estrous signs left ovary (+) laparoscopy left (+) left granulosa cell hyperplasia (+) 43 cat dsh 1.1 estrous signs estrus 4.87** + left or (+) laparoscopy left (+) entire normal ovary (+) 44 cat dsh 1.2 estrous signs estrus 7.58** laparoscopy left (+) normal ovarian tissue (+) 45 cat dsh 1.1 estrous signs estrus 6.23** laparoscopy right (+) normal ovarian tissue, oviduct (+) 46 cat dsh 4.8 estrous signs estrus 10.09** ventral midline right (+) normal ovarian tissue (+) 47 cat bengal 1.0 estrous signs estrus + left ovary w/ cysts (+) laparoscopy left (+) normal ovarian tissue (+) 48 cat dsh 1.5 estrous signs estrus + left or (+) laparoscopy left (+) ovary w/ cl and cystic rete ovarii (+) positive results 12 16 4 4 25 44 39 clinical theriogenology 2022; 14: 375 case species breed age at presentation (years) presenting complaint vaginal cytology* p4 † (ng/ ml) amh lh ultrasonography findings surgical approach or at surgery surgery/histology findings negative results 12 6 2 1 8 1 1 total tested 24 22 6 5 33 45 41 percentage of correctly identified via histopathology as final diagnos 61.9% 83.3% 66.6% 71.4% 95% estrous signs = vulva enlargement + vaginal bleeding in canines; rolling and vocalizing in felines p4 = progesterone; or = ovarian remnant; cl = corpora lutea; inc = inconclusive *vaginal cytology only marked positive if in estrus †progesterone concentrations positive if > 0.5 ng/ml ‡interpretation of hcg stimulation: progesterone baseline: 0.38; posthcg (1 hour): 0.18; posthcg (5 hours): 0.31; estradiol baseline: 37.31; posthcg (1 hour): 65.78; posthcg2 (5 hours): 31.75 surgery/histology report: (+) sign identifies cases which had histopathology performed and therefore description from pathology, otherwise description from surgery finding §histology report was lost **serum progesterone samples measured after hcg or gnrh stimulation transabdominal ultrasonography was performed by a boar-certified radiologist or a radiology resident in 33/48 animals (68.8%). an ovarian remnant was diagnosed ultrasonographically in 25/33 animals (75.8%). thirty-one animals that had transabdominal ultrasonography underwent an exploratory laparotomy to remove the remnant. there was no difference between species or size of dog in the probability of identifying an ors via transabdominal ultrasonography. ultrasonography identified the side on which the remnant was located in 23/25 cases (92.0%). the kappa coefficient of agreement of remnant side on transabdominal ultrasonography with the actual side determined at surgery was very strong at 0.7 (0.7, 1) (lower 95% ci, upper 95% ci) (p < 0.0001). forty-five animals underwent an exploratory laparotomy to remove the or (93.8%). a ventral midline approach was performed on 31/45 of the animals (68.9%); 14/45 animals underwent a laparoscopic procedure (31.1%). samples were submitted for histopathology in 41/45 (91.1%) of the animals. an or was identified in 39/41 (95.1%) of the samples submitted for histopathology; the histopathology report for 1 of the 2 unconfirmed samples could not be located and the other histopathology result was consistent with uterine and oviduct tissue. two of the 39 ovarian tissue samples were granulosa cell tumors. both samples belonged to dogs, and the time from ohe to clinical signs for each was 9.4 and 12.4 years, respectively. example of an or in a dog is provided (figure); or was on the right side of the abdomen (identified via transabdominal ultrasonography and confirmed via histopathology). figure. ultrasonogram of suspected right gonad (craniocaudal view); note: hypoechoic gonad with multifocal anechoic follicles, presumed to be or (a); histopathological (h&e stain) confirmation of the right or surrounded by a thin layer of adipose tissue, with 2 cystic structures surrounded by ovarian stroma and presence of a corpus luteum (arrow) (b), and higher magnification of the corpus luteum demonstrating luteal cells (c). discussion the mean age at presentation for dogs and cats with ors has been reported to be 2.8 years and the interval between ohe and presentation to be 1.4 years.3 in our study, the median age at presentation was 2.3 years and the interval between ohe and presentation was 0.5 years, with the owners reporting clinical theriogenology 2022; 14: 376 signs from immediately after ovariohysterectomy up to 12.4 years later. data were skewed towards younger animals similar to reports.3,4 furthermore, in the clinical history, several owners reported that their animals returned to estrus from very soon after ohe to several months following ohe, but they did not pursue further diagnostics or treatment until later in the animal’s life. weight may be associated with ors as a complication postohe;4,9,10” however, not all had similar finding. in our study, we did not evaluate animals’ weight as not all of them were available; however, when breed size (small, medium, or large) was evaluated, no difference in the proportion of ors were present among dog breed sizes. in this regard, some studies have demonstrated that cats are more likely to have ors,4,9 but others have demonstrated the opposite.3,13 in our study, no difference was detected between the proportions of cats and dogs with ors. however, the number of cats in our study was lower and therefore the data should be interpreted with caution. when vaginal cytology demonstrated predominantly superficial cells consistent with estrus, not surprisingly all animals were diagnosed on histopathology. of the 12 cytology that were suspected to be proestrus, diestrus, or undetermined, 7 had serum progesterone concentrations consistent with presence of luteal function (> 0.5 ng/ml) that enabled to confirm the diagnosis of ors. four animals had progesterone concentrations < 0.5 ng/ml, and 1 of these 4 cases had ors confirmed via amh whereas the other 3 had confirmation at surgery. it is important to note that in cats, progesterone was measured after ovulation induction. these findings supported the importance of multiple diagnostics, as a single cytology sample may not always be enough to determine the stage of the estrous cycle. however, vaginal cytology remains an easy, cheap and simple test to perform and when combining with progesterone measurements will increase the odds of correctly identifying ors.1 amh was only measured in 6 animals. the low number of cases is due to the fact that most of the cases included in this study were seen before amh testing was available,19 and because of the high expense to send samples to referral laboratories for amh testing. additionally, some of the cases were less complex and presented with more obvious signs of estrus concurrent with corroborating vaginal cytology and/or confirmation with elevated serum progesterone. that serum estradiol was measured only in 4 animals whereas vaginal cytology was performed in 24 cases was most likely due to vaginal cytology being a cheaper, easier, and faster method to diagnose endogenous estrogens with less chances for laboratory reference range, assay and diurnal variations.17 clinician preferences, advancement on diagnostic capabilities throughout the years and financial budget could have affected the diagnostic test performed. in this sample population, no difference in the proportions were noted between right and left or. this contradicts (right sided remnants were significantly higher than left) certain reports,3,11 suggesting surgical error. however, others have demonstrated that surgical experience of the veterinarian is not associated with increased chance of complication for ors.4,9 this study has not evaluated surgeon’s experience, as most of the or that were diagnosed were referrals, and therefore experience of the veterinarian that performed the original ohe was unavailable. a study with a larger population of dogs and cats would be required to determine if the side of or and if weight or size of the animals may influence the risk of ors. percentage of correctly identifying an or via transabdominal ultrasonography was consistent with what was reported.3 additionally, of those cases that we were able to identify an ors via transabdominal ultrasonography, there was a strong agreement of the location of the remnant with surgical findings. likewise, a study that used transabdominal ultrasonography for prelaparoscopy diagnosis of ors also demonstrated that transabdominal ultrasonography can be quite reliable in identifying the remnant location.11 species or breed size of dog were not associated with whether or not a remnant was identified in transabdominal ultrasonography. however, our study’s dog population lacked giant breeds that may be more difficult for the ultrasonographer and machine capabilities. the level of experience of the ultrasonographer performing the examination was not evaluated during this study and could be a confounding factor for the successful diagnosis of an ovarian remnant via transabdominal ultrasonography. we would expect more experienced ultrasonographers to have even higher detection rates. further studies regarding ultrasonographer’s experience (board-certified radiologist versus radiology resident) are warranted with a larger population of animals. of the 45 exploratory surgeries performed, 1 was negative for histopathology. in this case, a remnant was identified on the midline via transabdominal ultrasonography and grossly at surgery, but histopathology results revealed cystic endometrial hyperplasia and focal hydrosalpinx. tissues were not submitted for histopathology in 4 cases that underwent surgery. in all 4 cases, a remnant was identified grossly at surgery. in 3/4 cases, transabdominal ultrasonography and/or progesterone concentrations were used to confirm the presence of an or prior to surgery, and the remaining case had no diagnostics performed prior to surgery, but a complete ovary was identified at surgery. similar to our study, identification of ovarian remnants at surgery (gold standard) had a high sensitivity compared to histopathology.11,12 surgical removal of ovarian remnants remains the treatment of choice.1 in our study, vaginal cytology results, serum progesterone concentrations, and transabdominal ultrasonography findings had the highest chance of correctly identifying a or prior to exploratory surgery, and transabdominal ultrasonography was very effective in locating the remnant side that could help reduce surgical and anesthesia time. auxiliary diagnostic testing should be recommended to confirm functional ovarian tissue before surgery to reduce the chance of unnecessary surgery. additional studies are needed to determine which combination of diagnostic tests, in addition to clinical signs, is the most cost effective and accurate method of diagnosing an ovarian remnant prior to pursue an invasive exploratory abdominal surgery. acknowledgement authors thank stephen parry (cornell statistical consulting unit) for statistical analyses advice and sarah lyn milicic for editorial assistance. clinical theriogenology 2022; 14: 377 references 1. johnston sd, root-kustritz mv, olson pns: disorders of the canine ovary. in: johnston sd, root-kustritz mv, olson pns: editors. canine and feline theriogenology. philadelphia; saunders elsevier: 2001. p. 199-200. 2. ball ba, almeida j, conley aj: determination of serum antimüllerian hormone concentrations for the diagnosis of granulosa-cell tumours in mares. equine vet j 2013;45:199-203. 3. ball rl, birchard sj, may lr, et al: ovarian remnant syndrome in dogs and cats: 21 cases (2000-2007). j am vet med assoc 2010;236:548-553. 4. wallace ms. the ovarian remnant syndrome in the bitch and queen. vet clin north am small anim pract 1991;21:501-507. 5. diel de amorim m, lerer a, durzi t, et al: identification of ectopic ovotestis in a dog with xx ovotesticular, sry-negative, disorder of sexual development. reprod domest anim 2018;53. doi:10.1111/ rda.13174 6. fontes gs, mccarthy rj: ovarian remnant syndrome in a cat with ovarian tissue in theomentum. j am vet med assoc 2020;257:631634. 7. okkens ac, dieleman sj, v d gaag i: [gynaecological complications following ovariohysterectomy in dogs, due to: (1) partial removal of the ovaries. (2) inflammation of the uterocervical stump (author’s transl)]. tijdschr diergeneeskd 1981;106:1142-1158. 8. pearson h: the complications of ovariohysterectomy in the bitch. j small anim pract 1973;14:257-266. 9. miller dm: ovarian remnant syndrome in dogs and cats: 46 cases (1988-1992). j vet diagn invest 1995;7:572-574. 10. muraro l, white rs: complications of ovariohysterectomy procedures performed in 1880 dogs. tierarztl prax ausg k kleintiere heimtiere 2014;42:297-302. 11. van nimwegen sa, van goethem b, de gier j, kirpensteijn j: a laparoscopic approach or removal of ovarian remnant tissue in 32 dogs. bmc vet res 2018;14:333. doi:10.1186/s12917-018-1658-y 12. percival a, singh a, gartley c, et al: single-port laparoscopic treatment and outcome of dogs with ovarian remnant syndrome: 13 cases (2010-2018). j am anim hosp assoc 2020;56:114-119. 13. naiman jh, mayhew pd, steffey ma, et al: laparoscopic treatment of ovarian remnant syndrome in dogs and cats: 7 cases (2010-2013). j am vet med assoc 2014;245:1251-1257. 14. place nj, cheraskin j-l, hansen bs: evaluation of combined assessments of serum anti mullerian hormone and progesterone concentrations for the diagnosis of ovarian remnant syndrome in dogs. j am vet med assoc 2019;254:1067-1072. 15. berger dj, lewis tp, schick ae, et al: canine alopecia secondary to human topical hormone replacement therapy in six dogs. j am anim hosp assoc 2015;51:136-142. 16. johnston sd, root-kustritz m v, olson pns: disorders of the feline ovaries. in:johnston sd, root-kustritz m v, olson pns: editors. canine and feline theriogenology. philadelphia; saunders elsevier: 2001. p. 457-459. 17. frank la, mullins r, rohrbach bw: variability of estradiol concentration in normal dogs. vet dermatol 2010;21:490-493. 18. bateman hl, vansandt lm, newsom j, et al: temporal changes in serum luteinizing hormone following ovariohysterectomy and gonadotropin-releasing hormone vaccination in domestic cats. reprod domest anim 2017;52:332-335. 19. place nj, hansen bs, cheraskin j-l, et al: measurement of serum anti-mullerian hormone concentration in female dogs and cats before and after ovariohysterectomy. j vet diagn invest 2011;23:524-527. postpartum anestrus of beef cows: importance and evaluation postpartum anestrus of beef cows: importance and evaluation robert larson department of clinical sciences, college of veterinary medicine kansas state university, manhattan, ks abstract duration of anestrus period and infertility in postpartum suckled beef cows impact reproductive performance and efficiency of cow-calf operations. percent cows calving in first 21 days of calving season is higher for herds with shorter (average 50 days) rather than longer (average 80 days) length of postpartum anestrus. because primiparous cows may have a prolonged postpartum anestrus compared to similarly managed multiparous cows, breeding replacement heifers before mature cows, sometimes referred to as providing a heifer lead time, may increase likelihood that primiparous cows have resumed fertile estrous cycles and have opportunity to conceive during first 21 days of breeding season subsequent to first parturition. while having a 42 day heifer lead time resulted in greater pregnancy success and average weaning weight compared to no lead time, ~ two-thirds of improvement in outcomes was realized with a heifer lead time of only 21 days. determining length of postpartum anestrus following first and subsequent pregnancies can assist veterinarians and producers to develop optimal herd management strategies. keywords: postpartum, anestrus, infertility, bovine introduction postpartum period of infertility is time from calving until a cow resumes fertile estrous cycles and can become pregnant again. there are 2 important events place during postpartum period; uterus must return to a prepregnancy state and the hormones that control estrous cycle must resume normal function to allow fertile ovulations. during postpartum period, cow has highest nutritional demand of the year as lactation is initiated and daily milk production peaks at ~ 60 90 days postpartum, uterus undergoes involution, and fertile estrous cycles are resumed, enabling mating and conception for subsequent calving season. uterus decreases in weight from 20 25 pounds at parturition to < 1 pound by 25 days postpartum. during first few days after parturition, uterus is normally thick-walled and tonic, but if infection is present, it may have a thinner wall and atonic. uterine involution is not affected by suckling and is similar in beef and dairy cattle. normal involution process begins shortly after parturition, upper two-thirds of caruncles undergo necrosis due to loss of blood supply and necrotic tissue is sloughed and expelled as part of the uterine lochia.1 uterus usually is near nonpregnant size by 3 4 weeks postpartum, with previously pregnant horn still slightly larger.2 parturition is followed by an ~ 3 week interval when conception is not possible. estrus and ovulation seldom occur together during this period and, if fertilization occurred and embryo reached uterus, placentation would be virtually impossible. period of infertility is followed by 2 3 weeks when fertility is possible, but not optimal.2 uterine involution appears not to be a barrier to fertility after 5 6 weeks postpartum in cows, unless delayed by inflammation or infection.2 anestrus is major component of postpartum infertility and is affected by several minor factors: season, breed, parity, dystocia, presence of a bull, and carryover effects from previous pregnancy and 2 major factors (suckling and nutrition).3-6 as reported earlier,5 for spring-calving herds, duration of postpartum anestrus decreased with increasing julian day.7 season may truly have a role in modifying duration of postpartum anestrus through light stimulation of pineal gland.8 presumably, differences in duration of postpartum anestrus associated with season or julian day could also be related to quality and quantity of available forage. suckling has a dramatic effect on postpartum interval. in suckled cows, first ovulation is delayed to 30 days or more postpartum. cows that have their calves weaned at birth have shorter postpartum interval than do cows that are suckled. if calves are weaned at some time after birth but before estrous cycles begin, cows will return to estrus in a few days. postpartum intervals can be decreased by complete clinical theriogenology • volume 12 number 3 • september 2020 241 weaning and to a less predictable extent, by short-term weaning (48 hours) or restricted suckling.9-12 conception success is lower up to 40 days after parturition, as function of first corpus luteum as measured by serum progesterone concentrations is less than subsequent cycles, and with reduced estrus signs.3 normal corpus luteum function during an early postpartum estrous cycle can be obtained by pretreatment with a progestin.13 a cow’s nutritional demand is highest of production cycle during postpartum period primarily due to demands of lactation, which peaks at ~ 60 90 days after calving.14,15 quantity and quality of feed intake, nutrient reserves stored in the body and competition for nutrients from other body functions besides reproduction (lactation, growth, etc.) are all factors that affect length of time required to resume cycling. body weight and condition score, are good indicators of energy status and rebreeding performance after calving. inadequate precalving and/or postcalving energy or protein nutrition extends length of postpartum anestrus. postpartum anestrus lasts an average of 55 65 days for multiparous cows in good body condition (longer if low body condition).7,16-19 regardless, recognize that full consideration of average postpartum length means that ~ 50% of cows in good body condition have not resumed fertile cycles by 55 65 days. postpartum anestrus lasts an average of 80 100 days for primiparous cows in good body condition.20,21 dystocia will increase length of postpartum anestrus. adverse effects of dystocia can be overcome at least partially by providing early obstetrical assistance. importance of length of anestrus there are several reproductive constraints in beef cattle production. first is that when a fertile cow and a fertile bull mate, about 60 70% of time a viable calf will be detected at determination of pregnancy status during midpregnancy.22 however, ~ 30% of time following a fertile mating, early embryo dies within first 13 days. when an embryo is lost before maternal recognition of pregnancy (~ 13 days after ovulation), cow will display estrus and ovulate an oocyte about 21 days after her last ovulation and have another 60 70% probability of achieving and maintaining pregnancy.22 another constraint is that there are 283 days of pregnancy and 365 days in a year, which means that a cow must achieve a viable pregnancy within 82 days after calving in order to maintain a 365 day calving interval. based on these 2 constraints, best herd reproductive performance that can be expected would be depicted with a histogram where 65% of the herd becomes pregnant in first 21 days. this would indicate that nearly every cow has resumed fertile estrous cycles by the 21st day of breeding season and bulls are able to successfully mate cows. cows that are fertile during first 21 days of breeding season but fail to maintain a pregnancy from first mating, are expected to express estrus and ovulate approximately 21 days after their first ovulation and will have another 60 70% probability of establishing a viable pregnancy which results in another 23% of herd becoming pregnant in the second 21 days of the breeding season. finally, if cows fail to establish viable pregnancies after 2 fertile matings, ~ 60 70% of remaining cows become pregnant in the third 21 days of breeding season, leaving the herd with 5% nonpregnant cows after a 63 day breeding season (figure). in order to investigate effect of length of postpartum infertility on percentage of cows in a herd that conceive and maintain a viable pregnancy during first 21 days of breeding season, average age at weaning and total weight of calves weaned over a 10 year interval, a deterministic, dynamic systems model was developed23 to compare 22 combinations of multiparous and primiparous postpartum anestrus length. model compared herds that averaged 50, 60, 70, and 80 day postpartum anestrus for multiparous cows and 50, 60, 70, 80, 90, 100, and 110 day postpartum anestrus for primiparous cows. percent cows calving in first 21 days of calving season is higher for herds with shorter (average 50 days) rather than longer (average 80 days) length of postpartum anestrus.23 earlier calving cows produce heavier calves at weaning, as demonstrated by a model that calculated weaning weights that averaged 65 lbs heavier for herds with 50 day average duration of postpartum anestrus compared to herds with 80 day average duration of postpartum anestrus.23 in addition, cows with a 70 day or greater length postpartum anestrus were less likely to become pregnant in a 63 day breeding season.23 clinical theriogenology • volume 12 number 3 • september 2020242 figure. histogram of the percentage of cows that conceive and maintain a viable pregnancy if nearly all cows in the herd ovulate a fertile oocyte during the first 21st days of breeding season and are mated by fertile bulls and 60 70% of available (nonpregnant) cows conceive and maintain a viable pregnancy in each subsequent 21 day breeding period. because postpartum anestrus lasts longer in primiparous cows than similarly managed multiparous cows, nulliparous heifers must be bred to calve before multiparous cows to enable them to ovulate a fertile oocyte during first 21 days of their second breeding season. some cattle production consultants recommend that cow-calf producers breed nulliparous cows before primiparous and multiparous cows, sometimes referred to as providing a heifer lead time. a deterministic, dynamic systems model was used to evaluate herds with an average 60 day postpartum anestrus for multiparous cows and one of 42 combinations of 60, 70, 80, 90, 100, or 110 day average postpartum anestrus for primiparous cows and 0, 7, 14, 21, 28, 35, or 42 day heifer lead times.24 whereas having a 42 day heifer lead time resulted in greater pregnancy success and average weaning weight compared to shorter lead times, approximately two-thirds of improvement in outcomes was realized with a heifer lead time of only 21 days.24 in addition, primiparous cow cohorts with average length of postpartum anestrus of 70 days or longer should be bred to calve ahead of cows or they will contribute to negative annual reproductive momentum and an unacceptable percentage of primiparous cows will fail to become pregnant in a 63 day breeding season.24 evaluation of length of anestrus duration of postpartum anestrus can be a major contributing factor to herds with poor reproductive performance and low calf weight weaned per cow exposed over time and should be a ruleout when investigating lower than desired reproductive efficiency. it appears that maintaining an average duration of postpartum anestrus for multiparous cows to < 60 days results in improved production outcomes when compared to a longer postpartum anestrus. in addition, herds with a shorter duration of postpartum anestrus are more resilient to negative effects of various unpredictable and sporadically occurring adverse production events. quantifying a herd’s average length and range of postpartum anestrus could be useful to veterinarians seeking to improve reproductive management and performance of beef cow-calf herds. an estimate of average and range of anestrus duration could be determined by selecting a sample of cows in a herd that have calved at various time points in calving season and monitoring them for estrus signs and behavior to establish resumption of estrous cycles. determining duration of postpartum anestrus for agespecific cohorts such as primiparous versus multiparous cows may have additional value. several clinical theriogenology • volume 12 number 3 • september 2020 243 technological aids (heat-detection patches, chin-ball markers, and electronic, indirect monitoring technologies) were developed to assist with monitoring cattle for onset of estrus. many of these technologies are inexpensive and are easily applied to cattle. conflict of interest there are no conflicts of interest to declare. references 1. gier ht, marion gb: uterus of the cow after parturition: involutional changes. am j vet res 1968;29:1-23. 2. kiracofe gh: uterine involution: its role in regulating postpartum intervals. j anim sci 1980;51(suppl. 2):16-28. 3. short re, bellows ra, staigmiller rb, et al: physiological mechanisms controlling anestrus and infertility in postpartum beef cattle. j anim sci 1990;68:799-816. 4. viker sd, larson rl, kiracofe gh, et al: prolonged postpartum anovulation in mastectomized cows requires tactile stimulation by the calf. j anim sci 1993;71:999-1003. 5. yavas y, walton js:. postpartum acyclicity in suckled beef cows: a review. theriogenology 2000;54: 25-55. 6. crowe ma: resumption of ovarian cyclicity in post-partum beef and dairy cows. reprod dom anim 2008;43(suppl 5): 20-28. 7. cushman ra, allan mf, thallman rm, et al: characterization of biological types of cattle (cycle vii): influence of postpartum interval and estrous cycle length on fertility. j anim sci 2007;85:2156-2162. 8. sharpe ph, gifford dr, flavel pf, et al: 1986. effect of melatonin on postpartum anestrus in beef cows. theriogenology 1986;26:621-629. 9. shively te, williams gl: patterns of tonic luteinizing hormone release and ovulation frequency in suckled anestrous beef cows following varying intervals of temporary weaning. domest anim endocrinol 1989;6:379-387. 10. stagg k, diskin mg, sreenan jm et al: follicular development in long-term anoestrous suckler beef cows fed two levels of energy postpartum. anim reprod sci 1995;38:49-61. 11. quintans g, vazquez ai, weigel ka: effect of suckling restriction with nose plates and premature weaning on postpartum anestrous interval in primiparous cows under range conditions. anim reprod sci 2009;116:10-18. 12. vasconcelos jl, safilho og, perez gc, et al: intravaginal progesterone device and/or temporary weaning on reproductive performance of anestrous crossbred angus x nelore cows. anim reprod sci 2009;111:302-311. 13. ramirez-godinez ja, kiracofe gh, schalles rr, et al: endocrine patterns in the postpartum beef cow associated with weaning: a comparison of the short and subsequent normal cycles. j anim sci 1982;55:153-158. 14. grings ee, roberts aj, geary tw, et al: milk yield of primiparous beef cows from three calving systems and varied weaning ages. j anim sci. 2008;86:768-779. 15. tedeschi lo, fox dg: predicting milk and forage intake of nursing calves. j anim sci 2009;87:3380-3391. 16. rutter lm, randel rd: postpartum nutrient intake and body condition: effect on pituitary function and onset of estrus in beef cattle. j anim sci 1984;58:265-274. 17. houghton pl, lemenager rp, horstman la, et al: effects of body composition, preand postpartum energy level and early weaning on reproductive performance of beef cows and preweaning calf gain. j anim sci 1990;68:1438-1446. 18. perry rc, corah lr, cochran rc, et al: influence of dietary energy on follicular development, serum gonadotropins, and first postpartum ovulation in suckled beef cows. j anim sci 1991;69:3762-3773. 19. lents ca, white fj, ciccioli nh, et al: effects of body condition score at parturition and postpartum protein supplementation on estrous behavior and size of the dominant follicle in beef cows. j anim sci 2008;86:2549-2556. 20. ciccioli nh, wettemann rp, spicer lj, et al: influence of body condition at calving and postpartum nutrition on endocrine function and reproductive performance of primiparous beef cows. j anim sci 2003;81:3107-3120. 21. berardinelli jg, joshi ps: introduction of bulls at different days postpartum on resumption of ovarian cycling activity in primiparous beef cows. j anim sci 2006;83:2106-2110. 22. bondurant, rh: selected diseases and conditions associated with bovine conceptus loss in the first trimester. theriogenology 2007;68:461-473. 23. shane dd, larson rl, sanderson mw, et al: a deterministic, dynamic systems model of cow-calf production: the effects of the duration of postpartum anestrus on production parameters over a 10 year horizon. j anim sci 2017;95:1680-1695. 24. shane dd, larson rl, sanderson mw, et al: a deterministic, dynamic systems model of cow-calf production: the effects of breeding heifers before mature cows over a 10 year horizon. j anim sci 2017;95:4533-4532. clinical theriogenology • volume 12 number 3 • september 2020244 015_ms-015 r.larson 2 2019 reproductive microbiome alterations: canine prostatitis   reproductive microbiome alterations: canine prostatitis michelle kutzler oregon state university, department of animal and rangeland sciences, corvallis, or abstract prostate is the only accessory sex gland in dog. during periods of sexual rest, prostatic fluid is continuously and constitutively secreted into the proximal urethra where a small amount flows antegrade to exert bactericidal effects and to prevent ascent of lower urinary tract bacteria. pathologic conditions of the prostate gland are common in dogs and their incidence increases with age. bacterial prostatitis and benign prostatic hyperplasia account for ~ 85% of all prostatic disorders. bacterial prostatitis can develop as an acutely fulminating emergency condition or as a chronic disorder in conjunction with benign prostatic hyperplasia. although signalment, clinical signs and pathogenesis vary between these 2 disease forms, diagnosis and treatment are similar. all dogs with suspected prostatitis should have a complete physical examination, including transrectal palpation and abdominal ultrasonography, as well as a minimum database with complete blood count, chemistry panel, and prostatic fluid analysis and culture (with sensitivity). most common isolate in bacterial prostatitis is escherichia coli (there are various subpathotypes that promote prostatic virulence). in addition, a variety of other gram-negative and grampositive bacterial and fungal organisms can cause prostatitis in dogs. successful treatment of prostatitis depends upon which form of the disease is present. acute prostatitis requires aggressive systemic therapy with sensitive antimicrobials. for chronic prostatitis, treatment goals must include both reducing prostatic hyperplasia (to eliminate the predisposing cause) as well as antimicrobials (selected based upon sensitivity as well as pharmacokinetics). trimethoprim-sulfadiazine, erythromycin, chloramphenicol, and enrofloxacin are all suitable choices. keywords: acute prostatitis, benign prostatic hyperplasia, chronic prostatitis, escherichia coli introduction prostate is located in the retroperitoneal space and completely encircles the proximal urethra and the neck of the urinary bladder.1 numerous prostatic ducts open into the proximal urethra.2 age, androgenic stimulus, disease, and degree of urinary bladder distention affect its position. beginning at ~ 8 months of age, androgenic stimulation arising from sexual maturity results in prostatic enlargement. prostatic enlargement repositions prostate cranially over the pelvic brim as it increases in size, to a total or partial intra-abdominal position.3 prostatic size (volume in cm3) is also correlated with body weight (bw), and volume of a normal intact dog’s prostate can be accurately calculated from the equation 0.867 * bw (kg) + 1.885 * age (years) + 15.88.4 length, width, and depth of prostate gland can be determined ultrasonographically and followed over time.5 following castration, prostate atrophies and returns to intrapelvic position. degree of urinary bladder distention also affects prostate position such that a full bladder displaces the prostate intraabdominally, whereas an empty bladder facilitates an intrapelvic position.6 prostate gland is the only accessory sex gland in the dog. normal prostatic fluid is clear and slightly acidic (ph 6.15 6.5), owing to the presence of prostatic acid phosphatase.7 however, > 90% of t protein secreted in prostatic fluid is canine prostatic specific esterase (cpse).8-9 enzyme cpse is an arginine esterase present mainly on the apical regional of prostatic secretory epithelial cells. serum concentrations of cpse are elevated in all forms of prostate disease.10-11 during erection and ejaculation, parasympathetic stimulation increases rate of prostatic fluid production. sympathetic stimulation ejects prostatic secretions into urethra to become seminal plasma.12 prostatic secretions account for > 97% of total ejaculate volume and aid in sperm transport by stimulating uterine contractions.13-14 during rest, prostatic fluid is continuously and constitutively secreted into proximal urethra where majority of fluid refluxes retrograde into the bladder.15-16 a small amount of the prostatic secretion flows antegrade where it exerts bactericidal effects to prevent ascent of lower urinary tract bacteria.17 in humans, antimicrobial activity of prostatic fluid is primarily attributable to its high zinc concentration.18 649 clinical theriogenology • volume 11, number 4 • december 2019   however, unlike in men, zinc does not have an important role in resolving bacterial prostatitis in dogs.9,19 pathological alterations of glandular tissue may result in changes in prostatic fluid composition and loss of its physiological role.17 bacterial prostatitis occurs when host defense mechanisms are compromised by concurrent predisposing conditions (benign prostatic hyperplasia) or primary urinary tract infections (cystitis). these conditions allow virulent microorganisms to ascend into prostate, adhere and multiply.5,20 pathologic conditions of the prostate gland are common in dogs and their incidence increases with age. approximately 8% of all male dogs > 10 years old had prostatic disease, whereas only 0.6% of male dogs < 4 years old had prostatic disease. large breed dogs (over 20 kg) are overrepresented in all prostatic diseases except neoplasia.21 breeds most frequently identified to have prostatic disease include doberman pinscher, german shepherd dog, rottweiler, american staffordshire terrier, berger de beauce dog, and bernese mountain dog.21-22 bacterial prostatitis and benign prostatic hyperplasia account for ~ 85% of all prostatic disorders.21-22 bacterial prostatitis can develop as an acutely fulminating emergency condition or as a chronic disorder in conjunction with benign prostatic hyperplasia. although signalment, clinical signs, and pathogenesis vary between these 2 disease forms, diagnosis and treatment are similar. all dogs with suspected prostatitis should have a complete physical examination, including transrectal palpation and abdominal ultrasound, as well as a minimum database with complete blood count, chemistry panel, and prostatic fluid analysis and culture (with sensitivity).5 acute prostatitis acute prostatitis is rare in dog. acute prostatitis can occur in dogs of any age or sex status but young, intact males are overrepresented. clinical signs include anorexia, fever, depression, vomiting, and gait abnormalities. abdominal palpation and/or transrectal palpation may not be possible due to moderate to extreme pain emanating from inflamed prostate. a neutrophilia with a left shift will be present on a complete blood count.5 urinalysis will typically reveal pyuria and evidence of bacteria.5 acute prostatitis most likely develops secondary to a cystitis or from a hematogenous origin. in cases of acute prostatitis, collection of a prostatic fluid sample for culture is be made via a fine needle aspirate to avoid the uncertainty as to whether prostatic fluid has been expressed. ultrasonographic appearance of a prostate with acute prostatitis is a normal size with a hypoechoic parenchyma, instead of the normal homogeneous echodense appearance.23 because prostatic-blood barrier has been breached by infection, acute prostatitis can be treated with any appropriate antibiotic as determined by culture and sensitivity.22 chronic prostatitis chronic prostatitis is common in older, intact male dogs. benign prostatic hyperplasia has a key role in development of chronic prostatitis (refer below). clinical signs associated with chronic prostatitis are variable, depending on degree of prostatomegaly.5,24 unlike acute prostatitis, systemic signs (e.g. fever, depression) and pain are not present. most common clinical signs associated with chronic prostatitis include urethral discharge, hematuria, and tenesmus. urethral discharge can be clear, purulent or hemorrhagic.2,16 in a 14-year retrospective study of prostatic disease, hemorrhagic urethral discharge was the only clinical sign observed in 23% of cases, which could be exacerbated by sexual arousal.25 in addition, urinary symptoms (hematuria, pollakiuria, stranguria, dysuria, pyuria, polyuria) are exhibited in > 50% of dogs experiencing chronic prostatitis.21 although widely described as a clinical sign associated with prostatitis, tenesmus is only present when the prostate is morbidly enlarged. it is important to mention that many dogs with chronic prostatitis are asymptomatic.21 laboratory findings are generally within normal limits or inconclusive for prostatic disease. ultrasonographic appearance of a dog with chronic prostatitis is coarsely hyperechoic throughout the parenchyma with regions of heterogenous echotexture and dystrophic mineralization resulting from fibrosis and chronic inflammation.23,26 benign prostatic hyperplasia prostate undergoes continual, androgen-dependent growth, leading to both glandular epithelial cell hyperplasia and hypertrophy (referred to as benign prostatic hyperplasia (bph).17,27-28 ninety percent of dogs > 9 years old have bph. with increasing age, a modest decrease in serum testosterone 650clinical theriogenology • volume 11, number 4 • december 2019   concentrations combined with no change in serum estradiol-17 concentrations result in a relative decrease in the serum androgen:estrogen ratio.30 in addition, transiently proliferating/amplifying cells are more abundant in the prostate of older males. both of these conditions may contribute to the age-related increase in size.17 prostatic enlargement is both uniform and diffuse. although the condition is described as “benign”, hypervascularization of hypertrophic/hyperplastic tissue can result in vascular leakage or hemorrhage into the gland.13,31-32 blood in preputial discharge, urine and/or seminal plasma is the most frequently reported clinical sign of bph. additional clinical signs involving the urinary tract (e.g. dysuria, stranguria), gastrointestinal tract (e.g. constipation, tenesmus) or systemic (e.g. abdominal pain, limping) are infrequently reported.17,32-33 regardless, nearly half of histologically confirmed cases of bph were asymptomatic.22 within the prostatic parenchyma, testosterone of testicular origin is metabolized to dihydrotestosterone (dht) by the enzyme 5 -reductase.34 although both androgens stimulate prostate growth and secretions, dht is far more biologically active in that it binds to the androgen receptor with twice the affinity of testosterone and has a 5 fold slower rate of dissociation.34 in addition to glandular epithelial cell hyperplasia and hypertrophy, intraparenchymal cyst formation may result from obstruction of parenchymal ducts, causing accumulation of prostatic secretions.27-28 prevalence of intraparenchymal cysts in asymptomatic dogs with bph is 14%.4,35 in addition to glandular epithelial cell hyperplasia and hypertrophy, intraparenchymal cysts may predispose prostate to bacterial infection.27-28 diagnosis of prostatitis prostatic fluid collection and evaluation collection and analysis of prostatic fluid is an essential part of diagnosing prostatitis. prostatic fluid may be obtained by ejaculation, prostatic wash, or fine-needle aspiration.5 prostatic fluid can be collected aseptically by ejaculation as long as care is taken to change collection containers following collection of combined sperm-rich fraction and that the tip of the penis does not touch inside of sterile collection container. technique for prostatic massage has been previously described.36 unlike with ejaculation, there can be uncertainty as to whether a prostatic fluid has been collected using prostatic massage. technique of fine-needle aspiration for collecting prostatic fluid samples has been described.2 in many instances, fine-needle aspirate samples are nondiagnostic. to improve success with sample collection, aspiration should be performed as needle is redirected several times within the prostate gland. in addition, negative pressure should be slowly released before needle is withdrawn. complications associated with fine-needle aspiration include periprostatic haemorrhage and abscess formation.36-38 it is important to note that urine bacterial culture results are well correlated with prostatic fluid culture results, such that a cystocentesis may be a safer alternative to a fine needle aspirate of the prostate if prostatic fluid could not be collected by other methods.7 there is a strong correlation (r = 1) between urine and prostatic fluid culture when compared to culture of prostatic tissue (all dogs with a positive fluid culture had escherichia coli (e. coli) isolated from the prostatic tissue and all dogs with negative fluid cultures had negative prostatic tissue cultures).19 prostatic fluid evaluation is highly diagnostic for prostatitis. however, in contrast to humans, prostatic fluid ph, specific gravity and cholesterol zinc concentrations are not reliable indicators for chronic prostatitis.39 prostatic fluid should be assessed by culture and cytologic evaluation. recovered fluid should be centrifuged at 1000 x g for 10 minutes and the resultant pellet used for both culture and cytology.24 sample should be submitted for aerobic culture and sensitivity.20 it is important to isolate the type of bacteria present, as well as quantify bacterial count, in order to correctly interpret findings.40-41 quantitative bacterial cultures yielding > 100,000 bacteria of a single species/ml of prostatic fluid or > 2 log10 one or more bacterial species in prostatic fluid samples over the number of colonies of the same species in paired urethral or urine samples are indicative of prostatic infection.11,14,41 heavy growth of a pure culture of gram-negative bacteria occurs in > 70% of cases.2,5,40 651 clinical theriogenology • volume 11, number 4 • december 2019   remaining sample should be used for cytology.24 normal prostatic cytology will have parabasal epithelial cells usually with very low numbers of red blood cells and neutrophils (typically < 5 red blood cells or polymorphonuclear neutrophils per high-power field). in prostatitis with bph, cytologv evaluation of prostatic fluid reveals epithelial dysplasia with a significant number of neutrophils that may have degenerative changes and perhaps intracellular bacteria.24 however, a lack of neutrophils on prostatic cytology does not entirely rule out prostatitis, because neutrophils may be in a distinct segment of the prostate that is not communicating with the secretory ducts.24 in cases of chronic prostatitis, inflammatory component may be reduced or confined to predominately mononuclear cells (macrophages, lymphocytes, plasma cells).5,7,42-44 inflammatory changes in prostatic fluid are associated with histological inflammation in > 80% of cases.2,19,41,45-46 etiologic agents most common isolate in bacterial prostatitis is e. coli.5,20 however, a reliable discriminatory typing scheme to differentiate commensal nonvirulent strains commonly found in the lower urinary tract from specific pathotypes of e. coli does not exist for dogs.47 in humans, e. coli isolates that infect the prostate have several virulence attributes (e.g. a group i or group ii capsule synthesis gene and some of the iron uptake related genes (such as irp, fyua, or iuta), as well as at least 1 of the following adhesin related genes: pap (p fimbriae), sfa or foc (s/f1c fimbriae), afa or dra (dr binding adhesins).48 presence of adhesin-associated genes (fima, fimh, papc, and sfad) was positively associated with a hospital stay for 1 or more days at the time of infection.49 in addition, a uropathogenic e. coli subpathotype has been positively associated with development of prostatitis in men.49 to be classified as uropathogenic e. coli, isolates must contain the hemolysin gene hlya, cytotoxic necrotizing factor cnf1, uropathogenic-specific protein usp, and iron uptake related genes. investigations in dogs are needed to identify specific factors and/or genes that promote prostatic virulence. in addition to e. coli, any opportunistic bacteria ascending from the urethra can cause prostatitis. in decreasing order of incidence: staphylococcus sp., pseudomonas sp., klebsiella sp., proteus sp., enterobacter sp., pasteurella sp., and hemophilum sp., streptococcus sp., and ureaplasma sp. have all been isolated from cases of bacterial prostatitis in dogs.2,15-16,22,30,41 infections with anaerobic bacteria have not been reported.16,22,31 brucella canis has been isolated in cases of both acute and chronic prostatitis.5,15,50 a pure culture of mycoplasma canis has also been isolated from the ejaculate of a dog with chronic prostatitis.51 however, several mycoplasma species are commensal to the lower urinary tract of healthy dogs.52-54 borrelia burgdorferi has been detected by pcr in urine from dogs with prostatitis, but more research is needed to determine if this organism can contribute to prostatitis.55-56 additionally, histologic examination of a biopsy from a dog with chronic prostatitis yielded leishmania sp., although bacterial culture was negative.57 prostatitis from trueperella pyogenes has been usually described in cattle, goats, sheep, and horses, but not in dogs, which may be because these bacteria are not a normal flora of this species.58-61 although not bacterial, prostate infections with fungal organisms (blastomyces dermatitidis, cryptococcus neoformans, or coccidioides immitis) occur infrequently and usually part of a systemic fungal infection.15,61-63 treatment successful treatment of prostatitis depends upon which form of the disease is present. acute prostatitis requires aggressive therapy.17 intravenous fluid and nonsteroidal anti-inflammatory drug (nsaid) administration are usually used. in acute prostatitis, blood–prostate barrier is broken, resulting in an easy penetration of antibiotics and other drugs into the gland, independent of ph and oil solubility of the active compound.2 therefore, any antimicrobial selected based upon culture and sensitivity results would be appropriate.20 drugs such as broad-spectrum penicillin derivatives or a third-generation cephalosporin may initially be used to good effect.24 antimicrobials must be selected more carefully in cases of chronic prostatitis because the blood-prostate barrier is generally intact20 (refer below). castration is not recommended in the immediate management of acute prostatitis because of the increased 652clinical theriogenology • volume 11, number 4 • december 2019   risk for formation of scirrhous spermatic cords and therefore should be postponed for 4 weeks after clinical resolution.15 for chronic prostatitis, treatment goals must include both antimicrobials (to treat infection) as well as reducing prostatic hyperplasia (to eliminate the predisposing cause). for chronic prostatitis, antimicrobial selection must be based upon culture and sensitivity (or targeted to e. coli without culture results), in addition to the pharmacokinetics of the antimicrobial.5 most antimicrobial agents useful against uropathogenic bacteria diffuse poorly into prostatic tissue (blood-prostate barrier). blood-prostate barrier prevents diffusion of drugs that are highly protein bound in plasma from entering prostatic fluid in therapeutic concentrations.15 in addition, ability of an antimicrobial to cross an epithelial membrane depends on its lipid solubility and its pka (acid dissociation constant).16,64 blood-prostate barrier permits access only to lipophilic drugs and those not highly bound to proteins.24 drugs with low lipid solubility do not cross epithelial membranes (e.g. ampicillin, penicillin, cephalothin). the pka is the ph at which a drug exists equally in both ionized and non-ionized forms. only nonionized forms can cross epithelial membranes. basic drugs (e.g. trimethoprim-sulfamethoxazole, erythromycin) diffuse easily from blood (ph 7.4) to the more acidic prostatic environment (ph 6.1 6.5).5,7,65-68 these drugs become concentrated on the side with the greatest ionization (greatest charge; e.g. the acidic canine prostatic fluid).69 however, when surveyed, 30% of veterinarians said that they would prescribe amoxicillin-clavulanate, a drug with poor penetration into prostate tissues.70 both chloramphenicol and enrofloxacin (a fluorinated quinolone) can readily cross epithelial membranes regardless of their ph, due to their zwitterion characteristics.5,64,71 zwitterions are neither purely acidic nor basic because they have 2 ionizing groups, one positively charged and one negatively charged.24 zwitterions have 2 or more pka values and at their isoelectric point, least amount of the drug is uncharged; whereas at higher or lower ph values, more drug is ionized and is therefore less diffusible.72 trimethoprim has good broad-spectrum activity, but is not effective against anaerobic infections.17,24 pairing with a sulfa drug (trimethoprim-sulfadiazine 15 mg/kg every 12 hours orally) does not seem to affect prostate penetration.20,73 long-term treatment with trimethoprim, as is required for prostatitis cases, can lead to deleterious idiosyncratic and immune-mediated adverse effects, such as keratoconjunctivitis sicca, hepatopathy, anemia, folate deficiency, hypersensitivity, and skin eruptions.20,24 with prolonged therapy, a baseline schirmer tear testing is recommended with periodic reevaluation and owner monitoring for ocular discharge. erythromycin crosses the prostate epithelium similar to trimethoprim but has poor action against gram negative bacteria.74 erythromycin should not be used until a culture and sensitivity show that the pathogenic bacteria are gram-positive organisms and are sensitive to this drug.24 chloramphenicol (40 50 mg/kg every 8 hours orally) is often reserved for multidrug-resistant infections or anaerobic bacterial infections.20 not only can myelosuppression occur in dogs with long-term therapy, humans are at risk for developing idiosyncratic aplastic anemia from the chloramphenicol.20 in general, chloramphenicol is antagonistic with enrofloxacin and other fluoroquinolones.75 enrofloxacin has a low molecular weight, which favors tissue penetration. enrofloxacin penetrates prostate well and distributes not only into extracellular fluid but also into the intracellular space.76-79 enrofloxacin has an antimicrobial spectrum that includes most bacteria that cause prostatitis.80 in general, enrofloxacin has excellent activity against enterobacteriaceae sp. and pseudomonas sp.; good to moderate activity against staphylococci, mycobacteria sp., chlamydia sp., mycoplasma sp., and ureaplasma sp.; and little or no activity against streptococci sp., enterococci sp., and anaerobic bacteria.74 minimum inhibitory concentration (mic) of enrofloxacin is low (range: 0.01 to 2.0 µg/ml).81 enrofloxacin is approved for use in dogs for treatment of urinary tract infections, although it is used in an extralabel manner for treatment of prostatitis (e.g. doses of 10 20 mg/kg once daily orally).20 enrofloxacin achieves maximum prostatic fluid and tissue concentrations (1.4 pg/ml) that exceeded serum concentrations and remained above mic for at least 6 hours.81 enrofloxacin has anecdotally increased frequency and intensity of seizures in epileptic dogs.82 after repeated administration of enrofloxacin, hepatic clearance is decreased and elimination half-life of theophylline and caffeine are increased, reportedly by decreasing demethylation of theophylline by hepatic p450 enzymes, 4-oxoquinolone 653 clinical theriogenology • volume 11, number 4 • december 2019   metabolite.83-85 in addition, crystalluria can occur in dogs at high doses of enrofloxacin.75 noninflammatory, erosive arthropathies can be observed in growing animals treated with enrofloxacin or other fluoroquinolones.75 these erosions are preferentially located at weight-bearing joints.86 articular cartilage forms vesicles after several moderately large doses, which can then progressively rupture and produce cartilaginous erosions.75 this observation is due to an early phase burst in oxidative metabolism in immature (but not mature) chondrocytes that may precipitate cell death.87-88 for this reason, immature large breed dogs should not be treated with fluoroquinolones.75 it is of interest to point out that ciprofloxacin does not concentrate in prostate the same way enrofloxacin does.81,89 fluoroquinolones differ from each other by substitutions in their chemical ring structure, which can affect the particular isoelectric point and therefore, their tissue penetration.81 in addition, it is also possible that differences in serum protein binding of each quinolone may account for lack of tissue penetration.81 in dogs, a therapeutically equivalent dose of ciprofloxacin has been suggested to be 4 5 times the dose (on a mg/kg basis) of enrofloxacin. antimicrobial treatment of chronic prostatitis requires prolonged treatment for 6 12 weeks, regardless of earlier disappearance of clinical signs.5,24 potential side effects of a long-term antibiotic treatment include bacterial resistance, liver dysfunction, renal dysfunction, anemia (high risk for chloramphenicol, fluoroquinolones), arthropathy (fluoroquinolones) and some possible complications of trimethoprim administration (e.g. hypothyroidism), urolith formation or keratoconjunctivitis sicca.16,90 follow-up urine and prostatic fluid cultures should be performed prior to ending and 30 days after ending therapy to assure resolution of infection.5 prostatic hyperplasia can be reduced by castration in dogs without valuable breeding potential.24 prostatic size is significantly reduced (> 70%) within 7 14 days following castration and completely involuted in 4 months.5,16,91 as a result of rapid prostatic atrophy, clearance of infection from cases of chronic prostatitis occurs more than twice as fast compared to intact dogs.19 although medical management of bph is not as effective as castration in reducing prostatic size, it is a viable option for valuable breeding dogs.92 finasteride, a 5 -reductase inhibitor, prevents conversion of testosterone to dht93-94 and produces a dose-dependent regression in prostate size.95 at a daily oral dose of 0.1 0.5 mg/kg (maximum 5 mg per dog per day) for 16 weeks, finasteride decreases prostatic diameter, prostatic volume, and serum dht concentration by 20, 43, and 58%, respectively.16,91 a 50 70% decrease in prostatic volume has been noted after 16 53 weeks of treatment.92-93,96-98 this is enough of a reduction in size and function to cause a resolution of clinical signs in most cases.24 treatment can then be tapered to administration every 2 3 days, but must be continued as effects of finasteride are reversible in < 8 weeks after treatment is discontinued.91 therefore, the recommendation is to continue finasteride treatment continuously until breeding career of dog is over and castration can be performed. unlike gnrh agonist or immunotherapies available in other countries for treating canine bph, finasteride does not alter lh secretion, so semen quality or quantity and fertility are not adversely affected.5,98 semen volume, however, is significantly reduced. finasteride is teratogenic to male fetuses especially during early gestation so pregnant women should have someone else medicate their dogs. however, teratogenic effects should not be an issue in dogs because female dogs are not treated with finasteride and the half-life of finasteride in semen is low enough to not be a teratogenic concern at the time of breeding.24 ancillary therapies for treatment (or prevention) of prostatitis d-mannose is a sugar that competitively binds to mannose-fimbriae on uropathogenic strains of e. coli, thereby inhibiting adhesion to epithelium.99 it has been used to prevent recurrent urinary tract infections in humans, but there have been no studies of clinical efficacy in veterinary patients for urinary tract or prostatic infections.20 an extrapolated anecdotal dose for dogs is one-quarter teaspoon per 20 pounds of body weight orally 3 times daily.20 proanthocyanidin is the “active ingredient” in cranberry. it alters phenotypic expression of fimbriae on uropathogenic e. coli, which subsequently inhibits bacterial adherence to epithelial cells.100 a meta-analysis reviewed effects of supplementing with cranberry products in 1049 human subjects, and 654clinical theriogenology • volume 11, number 4 • december 2019   treated group had fewer urinary tract infections over 12-month observation period compared to placebo.101 however, there has not been adequate research using cranberry products in dogs.102 in addition, quality and potency among over-the-counter products is extremely variable, making it impossible to provide recommendations on safety or efficacy of this method. in humans, there is great interest in the use of probiotics. probiotics are a form of bacterial interference and recommended as a treatment and preventive measure for recurrent urinary tract infections in women. lactobacillus sp. create an acidic environment that inhibits uropathogenic e. coli colonization, modulates host immune function, and may also downregulate virulence factor expression in pathogenic bacteria.103 two studies in dogs evaluated efficacy of probiotics and reported no significant differences in vaginal microflora.104-105 however, prospective studies are needed in male dogs to evaluate potential role of probiotics for treatment and prevention of prostatitis. a biofilm is composed of organisms adhered together by a self-produced polysaccharide matrix.106 some bacteria have the capacity for biofilm formation, which facilitates colonization.106-107 it has been suggested that as bacteria within biofilm become sessile, they are protected from immune system. they are also immune to antimicrobial treatment and are inherently resistant to even shear force removal.106 strategies used to prevent biofilm formation have included silver coating, nanoparticles, iontophoresis, urease and other enzyme inhibitors, liposomes, bacteriophages, quorum sensing inhibitors, and vibroacoustic stimulation.20 research is needed in understanding how biofilms contribute to pathogenesis of canine prostatitis. references 1. evans h, christensen g: the urogenital system. in: evans h, editor. miller’s anatomy of the dog. 3rd edition, philadelphia; wb saunders:1993. p. 494. 2. barsanti ja, finco dr: canine bacterial prostatitis. vet clin north am small anim pract 1979;9:679. 3. gordon n: the position of the canine prostate gland. am j vet res 1961;22:142. 4. ruel y, barthez py, mailles a, et al: ultrasonographic evaluation of the prostate in healthy intact dogs. vet radiol ultrasound 1998;39:212-216. 5. smith j: canine prostatic disease: a review of anatomy, pathology, diagnosis, and treatment. theriogenology 2008;70:375-383. 6. ellenport cr: carnivore urogenital apparatus. in: getty r, editor. sisson and grossman’s anatomy of domestic animals. 5th edition, philadelphia; wb saunders:1975. p. 1576. 7. barsanti ja, prasse kw, crowell wa, et al: evaluation of various techniques for diagnosis of chronic bacterial prostatitis in the dog. j am vet med assoc 1983;183:219. 8. johnston sd: performing a complete canine semen evaluation in a small animal hospital. vet clin n am small anim 1991;21:545-551. 9. chapdelaine p, duby jy, frenette g, et al: identification of arginine esterase as the major androgen dependent secreted by dog prostate and preliminary molecular characterization. j androl 1984;5:206. 10. isaacs w, shaper j: isolation and characterization of the major androgen-dependent glycoprotein of canine prostatic fluid. j biol chem 1983;258:660-665. 11. bell fw, klausner js, hayden dw, et al: evaluation of serum and seminal plasma markers in the diagnosis of canine prostatic disorders. j vet int med 1995;9:149-153. 12. bruschini h, schmidt ra, tanagho ea: neurologic control of prostatic secretion in the dog. invest urol 1978;15:288290. 13. huggins c, massina mh, eichelberger l, et al: quantitative studies of prostatic secretion. i. characteristics of the normal secretion; the influence of thyroid, suprarenal ad testis extirpation and androgen substitution on the prostatic output. j exp med 1939;70:543. 14. england gcw, moxon r, freeman sl: stimulation of mating-induced uterine contractions in the bitch and their modification and enhancement of fertility by prostatic fluid. reprod domest anim 2012;47 suppl 6:1-5. 15. johnston sd, kamolpatan k, root-kustritz mv, et al: prostatic disorders in the dog. anim reprod sci 2000;6061:405-415. 16. johnston s, root-kustritz m, olson p: chapter 20. disorders of the canine prostate. in: johnston s, root-kustritz m, olson p, editors. canine and feline theriogenology. philadelphia; saunders: 2001. p. 337-355. 17. nizanski w, levy x, ochota m, et al: pharmacologic treatment for common prostatic conditions in dogs—benign prostatic hyperplasia and prostatis: an update. reprod dom anim 2014;49 suppl 2:8-15. 18. chevalier s, mckercher g, chapdelaine a, et al: serum and prostate growth-promoting factors for steroid-independent epithelial cells of the adult dog prostate. prostate 1991;19:207-220. 655 clinical theriogenology • volume 11, number 4 • december 2019   19. cowan la, barsanti ja, brown j, et al: effects of bacterial infection and castration on prostatic tissue zinc concentration in dogs. am j vet res 1991;52:1262-1264. 20. olin sj, bartges jw: urinary tract infections: treatment/comparative therapeutics. vet clin n am small anim pract 2015;45:721-746. 21. polisca a, troisi a, fontaine e, et al: a retrospective study of canine prostatic diseases from 2002 to 2009 at the alfort veterinary college in france. theriogenology. 2016;85:835-840. 22. krawiec da, heflin d: study of prostatic disease in dogs: 177 cases (1981-1986). j am vet med assoc 1992;200:1119-1122. 23. johnston gr, feeney da, rivers b, et al: diagnostic imaging of the male canine reproductive organs. vet clin north am small anim pract 1991;21:553-589. 24. christensen bw: canine prostate disease. vet clin north am small anim pract 2018;48:701-719. 25. read ra, bryden s: urethral bleeding as a presenting sign of benign prostatic hyperplasia in the dog: a retrospective study (1979-1993). j am anim hosp assoc 1995;31:261-267. 26. feeney da, johnston gr, walter pa, et al: canine prostatic ultrasonography --1989. semin vet med surg small anim 1989;4:44-57. 27. habenicht uf, schwarz k, schweikert hu, et al: development of a model for the induction of estrogen-related prostatic hyperplasia in the dog and its response to the aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione: preliminary results. prostate 1986;8:181-194. 28. rhodes l, ding vdh, kemp rk, et al: estradiol causes a dose-dependent stimulation of prostate growth in castrated beagle dogs. prostate 2000;44:8-18. 29. tsutsui t, hori t, shimizu m, et al: regression of prostatic hypertrophy by osaterone acetate in dogs. j vet med sci 2000;62:1115-1119. 30. brendler cb, berry sj, ewing ll, et al: spontaneous benign prostatic hyperplasia in the beagle. age associated changes in serum hormone levels, and the morphology and secretory function of the canine prostate. j clin invest 1983;71:1114-1123. 31. verstegen j: conditions of the male. in: simpson g, england gcw, harvey m: editors. bsava manual of small animal reproduction and neonatology. 1st edition, gloucester, uk; bsava: 2008. p. 71-82. 32. reichmann m, bruskewitz rc: benign prostatic hyperplasia: manifestations and intervention. in: lepor h, lawson rk, editors: prostate diseases. philadelphia: saunders; 1993. p. 108. 33. palmieri c, lean fz, akter sh, et al: a retrospective analysis of 111 canine prostatic samples: histopathological findings and classification. res vet sci 2014;97:568-573. 34. grino pb, griffin je, wilson jd: testosterone at high concentrations interacts with the human androgen receptor similarly to dihydrotestosterone. endocrinology 1990;126:1165-1172. 35. black gm, ling gv, nyland tg, et al: prevalence of prostatic cysts in adult, large-breed dogs. j am anim hosp assoc 1998;34:177-180. 36. barsanti ja, shotts eb jr, prasse k, et al: evaluation of diagnostic techniques for canine prostatic diseases. j am vet med assoc 1980;177:160-163. 37. barr f: ultrasound-guided biopsy techniques. in: barr f, editor. diagnostic ultrasound in the dog and cat. oxford; blackwell scientific publications:1990. p. 178. 38. barr f: percutaneous biopsy of abdominal organs under ultrasound guidance. j small anim pract 1995:36:105-113. 39. branam je, keen cl, ling gv, et al: selected physical and chemical characteristics of prostatic fluid collected by ejaculation from healthy dogs and from dogs with bacterial prostatitis. am j vet rec 1984;45:825-829. 40. kustritz mvr: collection of tissue and culture samples from the canine reproductive tract. theriogenology 2006;66:567-574. 41. ling gv, branam je, ruby al, et al: canine prostatic fluid: techniques of collection, quantitative bacterial culture, and interpretation of results. j am vet med assoc 1983;183:201-206. 42. kraft m, brown m, leroy e: cytology of the canine prostate. ir vet j 2008;61:320-324. 43. dorfmann m, bartsanti ja: diseases of the canine prostate gland. compend contin educ vet 1995;17:791-810. 44. mahapokai w, van den ingh ts, van mil f, et al: immune response in hormonally-induced prostatic hyperplasia in the dog. vet immunol immunopathol 2001;78:297-303. 45. barsanti ja, prasse kw, crowell wa, et al: evaluation of various techniques for diagnosis of chronic bacterial prostatitis in the dog. j am vet med assoc 1983;183;219-224. 46. ling gv, nyland tg, kennedy pc: comparison of two sample methods for quantitative bacteriologic culture of canine prostatic fluid. j am vet med assoc 1990;196:1479-1482. 47. kohler cd, dobrindt u: what defines extraintestinal pathogenic escherichia coli? int j med microbiol 2011; 301:642647 48. hamelin k, bruant g, el-shaarawi a, et al: occurrence of virulence and antimicrobial resistance genes in escherichia coli isolates from different aquatic ecosystems within the st. clair river and detroit river areas. appl environ microbiol 2007;73:477-484. 49. hutton ta, innes gk, harel j, et al: phylogroup and virulence gene association with clinical characteristics of escherichia coli urinary tract infections from dogs and cats. j vet diagn invest 2018;30:64-70. 656clinical theriogenology • volume 11, number 4 • december 2019   50. corrente m, franchini d, decaro n: detection of brucella canis in a dog in italy. new microbiol 2010;33:337-341. 51. l’abee-lund tm, heiene r, friis nf: mycoplasma canis and urogenital disease in dogs in norway. vet rec 2003;153:231-235. 52. doig pa, ruhnke hl, bosu wtk: the genital mycoplasma and ureaplasma flora of healthy and diseased dogs. can j comp med 1981;45:233-238. 53. greene ce: mycoplasmal, ureaplasmal, and l-form infections. in: infectious diseases of the dog and cat. philadelphia; saunders:1998. p. 174-178. 54. lein dh: mycoplasma infertility in the dog: diagnoses and treatment. proc 9th annual conf soc theriogenology 1989; p. 307-313. 55. demaerschalck i, ben messaoud a, de kesel m, et al: simultaneous presence of different borrelia burgdorferi genospecies in biological fluids of lyme disease patients. j clin microbiol 1995;33:602-608. 56. bauerfeind r, kreis u, weiss r, et al: detection of borrelia burgdorferi in urine specimens from dogs by a nested polymerase chain reaction. zentralbl bakteriol 1998;287:347-361. 57. mir f, fontaine e, reyes-gomez e, et al: subclinical leishmaniasis associated with infertility and chronic prostatitis in a dog. j small anim pract 2012;53:419-422. 58. ribeiro mg, risseti rm, bolanos cad, et al: trueperella pyogenes multispecies infections in domestic animals: a retrospective study of 144 cases (2002 to 2012). vet q 2015;35:82-87. 59. greene ce: infectious diseases of the dog and cat. 4th edition, st. louis; elsevier: 2012. p. 484-485. 60. radostits om, gay cc, hinchcliff kw, et al: veterinary medicine: a textbook of the diseases of cattle, horses, sheep, pigs, and goats. philadelphia; saunders elsevier: 2007. p. 722-724. 61. caswell jl, williams kj: prostate and bulbourethral glands. in: maxie mg, editor. jubb, kennedy and palmer’s pathology of domestic animals, vol 3. 5th edition, st louis; elsevier: 2007. p. 604-609. 62. reed lt, balog ka, boes km, et al: pathology in practice. j am vet med assoc 2010;236:411-413. 63. klausner js, johnston sd, bell fw: canine prostatic diseases. in: kirk rw, editor. current veterinary therapy xii. philadelphia; saunders:1995. p. 1103-1108. 64. barsanti ja, finco dr: canine prostatic disease. vet clin north am 1986;16:587-599. 65. fair wr, cordonnier jj: the ph of prostatic fluid: a reappraisal and therapeutic implications. j urol 1978;120:695698. 66. meares em: prostatitis. a review. urol clin north am 1975;2:3-27. 67. meares em: prostatitis: review of pharmacokinetics and therapy. rev infect dis 1982;4:475-83. 68. baumueller a, kjaer tb, madsen po, et al: prostatic tissue and secretion concentrations of rosamicin and erythromycin. experimental studies in the dog. invest urol 1977;15:158-160. 69. wagenlehner fme, weidner w, sorgel f, et al: the role of antibiotics in chronic bacterial prostatitis. int j antimicrob ag 2005;26:1-7. 70. barzelai id, whittem t: survey of systemic antimicrobial prescribing for dogs by victorian veterinarians. austral vet j 2017;95:375-385 71. memon ma: common causes of male dog infertility. theriogenology 2007;68:322-328. 72. aagaard j, gasser t, rhodes p, et al: mics of ciprofloxacin and trimethoprim for escherichia coli: influence of ph, inoculum size and various body fluids. infection 1991;19 suppl. 3:s167-169. 73. fowler je: antimicrobial therapy for bacterial and nonbacterial prostatitis. urology 2002;60:24-26. 74. stamey ta, meares em, winningham g: chronic bacterial prostatitis and the diffusion of drugs into the prostatic fluid. j urol 1970;103:187-194. 75. brown sa: fluoroquinolones in animal health. j vet pharmacol therap 1996;19:1-14. 76. wolfson js, hooper dc: fluoroquinolone antimicrobial agents. clin microbiol rev 1991;324:382-394. 77. green ce, budsberg sc: veterinary use of quinolones. in: hooper dc, wolfson js, editors. quinolone antimicrobial agents. 2nd edition, washington, dc; american society for microbiology:1993. p. 473-488. 78. paton jh, reeves ds: fluoroquinolone antibiotics: microbiology, pharmacokinetics and clinical use. drugs 1988;36:193-228. 79. scheer m: concentrations of active ingredient in the serum and in tissues after oral and parenteral administration of baytril. vet med rev 1987;2:104-118. 80. o’shea jd: studies on the canine prostate gland. i. factors influencing its size and weight. j comp pathol 1962;72:321-331. 81. dorfman m, barsanti j, budsberg sc: enrofloxacin concentrations in dogs with normal prostate and dogs with chronic bacterial prostatitis. am j vet res 1995;56:386-390. 82. van cutsem pm, babish jg, schwark ws: the fluoroquinolone antimicrobials: structure, antimicrobial activity, pharmacokinetics. clinical use in domestic animals and toxicity. cornell vet 1990;80:173-186. 83. bowles sk, popovski z, rybak. m.j: et al; effect of norfloxacin on theophylline pharmacokinetics at steady state. antimicrob agents chemother 1988;32;510-513. 84. rybak mj, bowles sk, chandrasekar ph: et al: increased theophylline concentration secondary to ciprofloxacin. drug intell clin pharm 1987;21:879-881. 85. harder s, staib ah, beer c, et al: 4-quinolones inhibit biotransformation of caffeine. eur j clin pharmacol 1988;35:651-656 657 clinical theriogenology • volume 11, number 4 • december 2019   86. neu hc: quinolones: a new class of antimicrobial agents with wide potential uses. med clin north am 1988;72:623636. 87. hayem g, petit px, levacher m, et al: cytofluorometric analysis of chondrotoxicity of fluoroquinolone antimicrobial agents. antimicrob agents chemother 1994;38:243-247. 88. thuong-guyot m, domarle 0, pocidalo jj, et al: effects of fluoroquinolones on cultured articular chondrocytes flow cytometric analysis of free radical production. j pharmacol exp ther 1994;271:1544-1549. 89. dorflinger t, larsen eh, gasser tc, et al: the concentration of various quinolone derivatives in the dog prostate. in: weidner w, editor. therapy of prostatitis, experimental and clinical data. munich; zuckschwerdt: 1986. p. 35-39. 90. rubin si: managing dogs with bacterial prostatic diseases. vet med 1990;85:387–394. 91. sirinarumitr kj, johnston sd, root kustritz mv, et al: effects of finasteride on size of the prostate gland and semen quality in dogs with benign prostatic hypertrophy. j am vet med assoc 2001;218:1275-1280. 92. cohen sm, werrmann jg, rasmusson gh, et al: comparison of the effects of new specific azasteroid inhibitors of steroid 5a-reductase on canine hyperplastic prostate: suppression of prostatic dht correlated with prostate regression. prostate 1995;26:55-71. 93. lange k, cordes ek, hoppen ho, et al: determination of concentrations of sex steroids in blood plasma and semen of male dogs treated with delmadinone acetate or finasteride. j reprod fertil suppl 2001;57:83-91. 94. shibata y, fukabori y, ito k, et al: comparison of histological compositions and apoptosis in canine spontaneous benign prostatic hyperplasia treated with androgen suppressive agents chlormadinone acetate and finasteride. j urol 2001;165:289-293. 95. sirinarumitr k, sirinarumitr t, johnston sd, et al: finasteride-induced prostatic involution by apoptosis in dogs with benign prostatic hypertrophy. am j vet res 2002;63:495-498. 96. laroque pa, prahalada s, gordon lr, et al: effects of chronic oral administration of a selective 5a-reductase inhibitor, finasteride, on the dog prostate. prostate 1994;24:93-100. 97. laroque pa, prahalada s, molon-noblot s, et al: quantitative evaluation of glandular and stromal compartments in hyperplastic dog prostates: effect of 5-alpha reductase inhibitors. prostate 1995;27:121-128. 98. iguer-ouada m, verstegen jp: effect of finasteride (proscar msd) on seminal composition, prostate function and fertility in male dogs. j reprod fertil suppl 1997;51:139-149. 99. kranjčec b, papeš d, altarac s: d-mannose powder for prophylaxis of recurrent urinary tract infections in women: a randomized clinical trial. world j urol 2014; 32:79-84. 100. gupta k, chou m, howell a, et al: cranberry products inhibit adherence of p-fimbriated escherichia coli to primary cultured bladder and vaginal epithelial cells. j urol 2007;177:2357-2360. 101. wang ch, fang cc, chen nc, et al: cranberry-containing products for prevention of urinary tract infections in susceptible populations: a systematic review and meta-analysis of randomized controlled trials. arch intern med 2012;172:988-996. 102. weese js, blondeau jm, boothe d, et al: antimicrobial use guidelines for treatment of urinary tract disease in dogs and cats: antimicrobial guidelines working group of the international society for companion animal infectious diseases. vet med int 2011;2011:263768. doi: 10.4061/2011/263768. 103. darouiche ro, hull ra: bacterial interference for prevention of urinary tract infection. clin infect dis 2012;55:14001407. 104. hutchins r, vaden s, jacob m, et al: vaginal microbiota of spayed dogs with or without recurrent urinary tract infections. j vet intern med 2014;28:300-304. 105. hutchins r, bailey c, jacob m, et al: the effect of an oral probiotic containing lactobacillus, bifidobacterium, and bacillus species on the vaginal microbiota of spayed female dogs. j vet intern med 2013;27:1368-1371. 106. dicicco m, neethirajan s, singh a, et al: efficacy of clarithromycin on biofilm formation of methicillin-resistant staphylococcus pseudintermedius. bmc vet res 2012;8:225. doi: 10.1186/1746-6148-8-225. 107. nam eh, chae js, whang cy: characterization and zoonotic potential of uropathogenic escherichia coli isolated from dogs. j microbiol biotechnol 2013;23:422-429. 658clinical theriogenology • volume 11, number 4 • december 2019 untitled introduction in bulls, antibiotics are used for numerous reproductive clinical disorders.1-3 seminal adenitis syndrome represents 1 of the most frequent reproductive diseases in young and old bulls3,4 and is usually treated with antibiotics given locally or systemically.3,5,6 antibiotic selection for this clinical condition and other genital infections (e.g. orchitis) is based on personal experience, anecdotal evidence, extrapolation from other species, or based on the results of microbiological culture and sensitivity tests. the chosen antibiotic has to be used at the correct dose, route, and frequency for an acceptable period.7 most of the information regarding pharmacokinetics of antibiotics in the male genital tract is derived from human and dog models.8-10 however, the anatomy and physiology of these 2 species are different from ruminants.11 pharmacokinetics of oxytetracycline and tulathromycin in plasma and semen of beef bulls juan romano,a andrea barbarossa,b,c giampiero pagliuca,b,c graciela villadóniga,dteresa gazotti,b,c beatrice mislei,e elisa zironi,b,c gaetano marib,e alarge animal clinical sciences, college of veterinary medicine & biomedical sciences, texas a&m university, college station, tx bdepartment of veterinary medical sciences chealth sciences and technologies interdepartmental centre for industrial research university of bologna ozzano dell’emilia, bologna, italy dst. joseph regional health center, pediatric services, bryan, tx enational institute of artificial insemination, university of bologna, cadriano, italy abstract objectives were to evaluate the pharmacokinetic parameters of tulathromycin and long-acting oxytetracycline in bulls’ plasma and semen. in the first experiment, long-acting oxytetracycline (10 or 20 mg/kg) were given to bulls either subcutaneously or intramuscularly. in the second experiment, tulathromycin (2.5 mg/kg) was given subcutaneously. liquid chromatography coupled with tandem mass spectrometry was used to measure antibiotic concentrations in plasma and semen samples. plasma maximum concentration (cmax) for 10 mg/kg oxytetracycline was 2,841 ng/ml at 12 hours (tmax) with a half-life of 20.1 ± 5.9 hours. semen cmax was 11,515 ng/ml at 24 hours (tmax) with a half-life of 23.7 ± 4.1 hours. for 20 mg/kg, the plasma cmax was 5,269 ng/ml at 12 hours (tmax) with a half-life of 18.1 ± 0.4 hours. semen cmax was 55,040 ng/ml at 24 hours (tmax) with a half-life of 15.7 ± 1.2 hours. plasma cmax of tulathromycin was 160 ng/ml at 21 ± 6 hours (tmax). semen cmax was 1,539 ng/ml at 33.0 ± 18.0 hours (tmax). the cmax between plasma and semen was different (p = 0.008). plasma terminal half-life (81.4 ± 27.6 hours) of tulathromycin had a tendency (p = 0.10) to be shorter than semen (114.7 ± 21.7). it was concluded that for genital infections of bulls either tulathromycin or long-acting oxytetracycline are adequate antibiotics based on their pharmacokinetic properties. keywords: ruminants, abortion, diseases, pregnancy loss all tetracyclines are equally active and typically have similar broad spectrum for both aerobic and anaerobic gram-positive and gram-negative bacteria, mycoplasmas, rickettsia, chlamydia, and even some protozoa, e.g. amoeba.12 oxytetracycline povidone is a broad-spectrum antibiotic with a long half-life and is used for wide range of diseases. tetracyclines inhibits binding of the bacterial 30s ribosomal subunit, specifically at the aminoacyl-trna acceptor («a») site on the mrna ribosomal complex, thus preventing ribosomal translation.12 tulathromycin is a macrolide triamilide antibiotic approved for the treatment and prevention of respiratory diseases in cattle, pigs, and other animals.13,14 like other macrolides, it binds to the 50s subunit of bacterial ribosomes and consequently inhibits protein synthesis, leading to inhibition of cell division and cell death. tulathromycin’s spectrum of activity includes gram-negative, clinical theriogenology 2021; 13: 206 gram-positive, and mycoplasma microorganisms.13,15 in cattle, tulathromycin presents unique pharmacokinetic characteristics such as rapid absorption from the injection site, extensive tissue and high-volume distribution, elevated and sustained drug concentration in lungs, and slow elimination.13 although oxytetracycline and tulathromycin are used in bulls for genital infections, their pharmacokinetics in bulls’ semen has not been investigated. the objective was to investigate the pharmacokinetics of oxytetracycline and tulathromycin in plasma and semen after a single parenteral route treatment in beef bulls. materials and methods the study was approved by the university of bologna animal welfare committee and conducted at the university national institute of artificial insemination. oxytetracycline animals four simmental bulls were randomly selected and subjected to physical and breeding soundness examinations.16 average age was 15.3 ± 0.3 months (range; 15 16 months), weight was 648.9 ± 25.9 kg (range; 645 680 kg), and body condition score17 was 6.0 ± 0.4 (range; 5.5 6.5). bulls were maintained in individual pens and received a ration of corn silage, mixed hay, and alfalfa with water ad libitum. additionally, each bull received once a day 2.5 kg of concentrate (pellets with 14% crude protein). experimental design bulls had no history of oxytetracycline treatment. a single dose (10 or 20 mg/kg) of long-acting oxytetracycline (terramicina, zoetis italy, rome) was given. two bulls received 10 mg/ kg and 2 bulls received 20 mg/kg, either subcutaneously or intramuscularly on the right-side neck. each bull received < 10 ml per injection site. plasma and semen samples were collected at 0, 12, 24, 36, 48, 72, and 96 hours after treatment. blood (10 ml) was collected from caudal vessels in vacuum tubes containing lithium heparin. semen was collected via electroejaculation using an automatic electroejaculator (60 mm 2 electrode; pulsator v, lane manufacturing, denver, co). samples were immediately refrigerated, centrifuged (600 × g for 30 minutes) within 1 hour after collection, and stored at 80˚c. oxytetracycline analysis oxytetracycline concentrations in plasma and semen were measured18 using liquid chromatography coupled with tandem mass spectrometry. tulathromycin animals four simmental bulls were randomly selected and subjected to a physical examination and a breeding soundness examination.16 average age was 15 months ± 0.2 months (range; 15 16 months), average weight was 639.3 ± 32.9 kg (604 681 kg), and average body condition score17 was 6.1 ± 0.5 (5.5 6.5). bulls were maintained in individual pens and received a ration of corn silage, mixed hay, and alfalfa with water ad libitum. additionally, each bull received once a day 2.5 kg of concentrate (pellets with 14% crude protein). experimental design bulls had no history of tulathromycin treatment. a single dose (2.5 mg/kg) of tulathromycin (draxxin, zoetis italy, rome) was given subcutaneously either at left ear base (n = 2) or on the left side neck (n = 2). plasma and semen were collected at 0, 12, 24, 48, 72, 96, 144, 192, and 240 hours after treatment, as described above for oxytetracycline treatment. tulathromycin analysis liquid chromatography coupled with tandem mass spectrometry was used to measure19 tulathromycin concentrations in plasma and semen. pharmacokinetic parameters noncompartmental analysis was performed to estimate the pharmacokinetic parameters in plasma and semen for each individual animal. a standard software, pk-solver add-in for excel,20 was used to estimate the pharmacokinetic parameters. following variables were calculated for plasma and semen of each animal: time of peak drug concentration (tmax), peak drug concentration (cmax), apparent elimination half-life (t1/2), calculated as ln (2)/λz, λz being the first order rate constant associated with the terminal portion of the time-concentration curve as estimated by linear regression of time versus log concentration, and area under the time-concentration curve from time zero to the last observed concentration (auc0-last), calculated by the linear trapezoidal rule. data analyses statistical software21 was used to determine parameters (mean, standard deviation, and range). a paired student’s t-test and a software program (pk-solver) for pharmacokinetics parameters20 were used. an alpha error of 5% was used to accept the alternative hypothesis. results oxytetracycline all bulls had a mild swelling at the injection site. plasma clinical theriogenology 2021; 13: 207 oxytetracycline concentrations in bulls treated with 10 mg/kg of oxytetracycline either subcutaneously (1,470 ± 1,090 ng/ml) or intramuscularly (1,330 ± 990 ng/ml) were not different (p = 0.82). similarly, mean semen concentrations (5,710 ± 4,640 ng/ml and 5,390 ± 3,160 ng/ml) were not different (p = 0.88). plasma oxytetracycline concentrations in bulls treated with 20 mg/kg of oxytetracycline either subcutaneously (2,540 ± 1,970 ng/ml) or intramuscularly (2,590 ± 2,030 ng/ml) were not different (p = 0.96). similarly, mean semen concentrations (25,600 ± 22,900 ng/ml and 19,400 ± 17,200 ng/ml) were not different (p = 0.58). because there were no significant differences for the 2 sites of treatment, the means for plasma and semen were analyzed together. plasma and semen concentrations for the 10 mg/kg dose (1,400 ± 990 and 6,480 ± 3,520 ng/ml) and 20 mg/kg dose (2,570 ± 1,910 and 26,200 ± 18,700 ng/ml) were different (p = 0.001 and p = 0.001). oxytetracycline doses of 10 mg/kg and 20 mg/kg resulted in different (p = 0.07) mean plasma concentrations (1,400 ± 990 and 2,570 ± 1,910 ng/ml) and in different (p = 0.004) mean semen concentrations (6,480 ± 3,520 ng/ml and 26,200 ± 18,700 ng/ml). oxytetracycline plasma concentrations above 1,000 ng/ml persisted (p = 0.001) for 48 hours for the 10 mg/kg dose compared to 66 hours for the 20 mg/kg dose. oxytetracycline semen concentrations above 1,000 ng/ml remained elevated for over 96 hours for both the 10 and 20 mg/kg dose and remained above that threshold longer (p = 0.0001) compared with plasma levels (96 versus 57 hours). at 96 hours, the oxytetracycline mean semen concentrations for 10 mg/kg dose were 1,700 ± 260 ng/ml and for the 20 mg/kg dose were 3,340 ± 260 ng/ml. for the 10 mg/kg dose, the plasma cmax was 2,841 ± 401 ng/ ml at 12 hours (tmax) with a half-life of 20.1 ± 5.9 hours. the plasma auc0-last was 112,560 ± 8,067 ng/ml/hour. the semen cmax was 11,515 ± 2,445 ng/ml at 24 hours (tmax) with a halflife of 23.7 ± 4.1 hours. the semen auc0-last was 550,387 ± 13,081 ng/ml/hour. for the 20 mg/ml dose, the plasma cmax of 5,269 ± 111 ng/ml was achieved at 12 hours (tmax) with a half-life of 18.1 ± 0.4 hours. the semen cmax was 55,040 ± 10,605 ng/ml at 24 hours (tmax) with a half-life of 15.7 ± 1.2 hours. the semen auc0-last was 2,153,942 ± 384,669 ng/ml/hour. tulathromycin mean plasma concentrations of tulathromycin for 2 subcutaneous injection sites (77.9 ± 43.3 and 73.7 ± 39.7 ng/ml) were not different (p = 0.84). mean semen concentrations of tulathromycin (608 ± 374 ng/ml and 867 ± 599 ng/ml) were also not different (p = 0.29). in the absence of significant differences between the 2 injection sites, means for plasma and semen were analyzed together. mean plasma tulathromycin was lower (p = 0.001) than semen (75.8 ± 40.2 and 781 ± 482 ng/ml). the plasma cmax was 160 ± 26 ng/ml at 21.0 ± 6.0 hours (tmax). the semen cmax was 1,539 ± 444 ng/ml at 33.00 ± 18.00 hours (tmax). the cmax between plasma and semen was different (p = 0.008) with no differences (p = 0.35) in tmax between plasma and semen. the terminal half-life for plasma (81.4 ± 27.6 hours) had a tendency (p = 0.10) to be shorter than in semen (114.7 ± 21.7 hours). the plasma auc0–last (15,440 ± 1,717 ng/ ml/hour) was lower (p = 0.01) compared to semen (171,071 ± 58,556 ng/ml/hour). discussion oxytetracycline oxytetracycline treatment has been reported to produce local irritation and extensive tissue damage.22 therefore, subcutaneous treatment may be a better option than intramuscular. nevertheless, pharmacokinetics of the drug should be considered before an alternative route of treatment is recommended. similar to reports23-27 for 5, 10, or 20% oxytetracycline formulations, mean plasma or semen concentrations for both doses of oxytetracycline were not different for 2 routes of treatment. the area around the injection site was larger in the subcutaneous route than in the intramuscular route. inflammation after subcutaneous treatment could have contributed to more absorption of oxytetracycline, resulting in similar pharmacokinetics. plasma cmax at 12 hours (tmax) for 10 mg/kg was lower than reported.28 first blood sample collected at 12 hours (plasma oxytetracycline concentration was already in the descending phase) in the present study could explain the difference. plasma cmax for 20 mg/kg was lower than reported25,29,30 that also had different tmax (4.7 6.2,25 6,29 and 3.9 hours30). first blood sample collected at 12 hours in the present study could explain the difference. plasma auc0-last for the 20 mg/kg dose was in agreement with a previous report.25 to the authors’ knowledge, this is the first study that determined pharmacokinetics of long-acting oxytetracycline in bull semen for 2 doses. successful treatment requires selection of an antibiotic based on the microbiological results and pharmacokinetic properties. higher concentrations in semen than plasma were attributed to a lipophilic drug with a high volume of distribution and high tissue concentrations.30 when bacteriostatic antibiotics such as long-acting oxytetracycline are used, the plasma or semen concentrations should not decrease below the effective minimum inhibitory concentrations during treatment. therefore, the selection of dosing intervals and the desired minimum semen concentrations require basic pharmacokinetic information as a guideline. in general, a serum concentration between 500 and 1,000 ng/ml has been suggested as the effective therapeutic level. the minimum inhibitory concentrations for clinical theriogenology 2021; 13: 208 tetracyclines against most susceptible pathogenic microorganisms in cattle (bacillus anthracis, mycoplasma spp., pasteurella spp., staphylococcus aureus, streptococcus pyogenes, and streptococcus pneumoniae) ranged from 120 1,000 ng/ml.31 tulathromycin only a mild swelling at the site of injection was detected, especially at the base of the ear, that had disappeared at 5 days after treatment. the average plasma cmax (160 ng/ml) was lower than reported (277 ng/ml,32 300 ng/ml,33 and 500 ng/ml).13,15 one possible explanation for this difference could be due to the timing (12 hours after treatment) of first blood sample, a time the plasma concentrations of tulathromycin must have been already in a descending phase. this is supported by 2 reasons. first, in those studies, it was determined at 0.25 hour,34 < 1 hour,13 and 0.7 hour;33 second, 24 240 hours concentrations were similar to reported.13,15 the terminal half-life of 81.4 ± 27. 6 hours (range; 71 96 hours) was in agreement with earlier reports.13,15,32,33 the plasma auc0-last in the present study (18,382 ng/ml/hour) was comparable to earlier reports13,34 (17,885 and 16,700 ng/ ml/hour) but higher than others.15,32,33,35 the efficacy of any antimicrobial is determined by both its pharmacokinetic and pharmacodynamic properties. antibiotics have been classified into 2 major groups: those with bacteriostatic antimicrobial action that exhibit time-dependent killing action or those with bactericidal antimicrobial action that behave with either time-dependent or concentration-dependent killing, or both.15 tulathromycin has both bacteriostatic antimicrobial and bactericidal antimicrobial time-dependent action.13 to the authors’ knowledge, this is the first study that determined pharmacokinetics of tulathromycin in bull semen using a standard recommended dose for cattle. semen cmax of tulathromycin was higher than plasma with a tendency of longer half-life compared to plasma. apparently, tulathromycin elimination from male’s genital tract was slower, probably because of delayed exposure in the organs of elimination, and this can be considered an advantage for male reproductive treatments. furthermore, the semen auc0-last was higher than plasma. acknowledgments supported by funds from the department of veterinary medical sciences, university of bologna, italy (prot. n. 0005783). authors thank giulia cristoni, angelo ferrari, and fabrizio lollini for their assistance. conflict of interest all authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. references 1. welles eg, tyler jw, wolfe df, et al: eperythrozoon infection in young bulls with scrotal and hindlimb edema, a herd outbreak. theriogenology 1995;43:557-567. 2. montes aj, wolfe df, welles eg, et al: infertility associated with eperythrozoon wenyonii infection in a bull. j am vet med assoc 1994;204:261-263. 3. romano je, brinsko sp, blanchard tl, et al : male reproductive disorders. in: smith bp, van metre d, pusterla n, editors. large animal internal medicine. 6th edition, st. louis, mo; elsevier: 2020. p. 1505-1519. 4. ball l, griner la, carroll ej: the bovine seminal vesiculitis syndrome. amer j vet res 1964;25:291-302. 5. martínez mf, arteaga aa, barth ad: intraglandular injection of antibiotics for the treatment of vesicular adenitis in bulls. anim reprod sci 2008;104:201-211. 6. martínez mf, barth ad: early detection and treatment of vesicular adenitis in bulls. anim reprod sci 2007;101:252-256. 7. srinivasan a: antibiotic stewardship: why we must, how we can. cleve clin j med 2017;84:673-679. 8. bulitta jb, kinzig m, naber ck, et al: population pharmacokinetics and penetration into prostatic, seminal, and vaginal fluid for ciprofloxacin, levofloxacin, and their combination. chemotherapy 2011;57:402-416. 9. frimodt-møller pc, dørflinger t, madsen po: distribution of ciprofloxacin in the dog prostate and various tissues. urol res 1984;12:283-286. 10. naber ck, steghafner m, kinzig-schippers m, et al: concentrations of gatifloxacin in plasma and urine and penetration into prostatic and seminal fluid, ejaculate, and sperm cells after single oral administrations of 400 milligrams to volunteers. antimicrob agents chemother 2001;45:293-297. 11. romano je, brinsko s: reproductive physiology of the male. in: bradley g. klein, editor. cunningham’s textbook of veterinary physiology. 6th edition, st. louis, mo; saunders & elsevier: 2020. p. 471-479. 12. baxter p, mckellar q: plasma and lung concentrations of oxytetracycline after its intramuscular administration in rats. lab anim sci 1995;45:107-109. 13. evans na: tulathromycin: an overview of a new triamilide antimicrobial for livestock respiratory disease. vet ther 2005;6:83-95. 14. villarino n, brown sa, martín-jiménez t: the role of the macrolide tulathromycin in veterinary medicine. vet j 2013;198:352-357. 15. nowakowski m, inskeep p, risk j, et al: pharmacokinetics and lung tissue concentrations of tulathromycin, a new triamilide antibiotic in cattle. vet ther 2004;5:1-7. 16. koziol jh, armstrong cl: manual for breeding soundness examination of bulls. society for theriogenology: 2nd edition, 2018. p. 147. 17. richards mw, spitzer jc, warner mb: effect of varying levels of postpartum nutrition and body condition at calving on subsequent reproductive performance in beef cattle. j anim sci 1986; 62:300-306. 18. bayliss maj, rigdova k, kyriakides m, et al: development, validation and application of a novel hplc-ms/ms method for the measurement of minocycline in human plasma and urine. j pharm biomed anal 2019;30:90-98. 19. zhou q, zhang g, wang q, et al: pharmacokinetic/pharmacodynamic modelling of tulathromycin against pasteurella multocida in a porcine tissue cage model. front pharmacol 2017;28;8:392. 20. zhang y, huo m, zhou j, et al: pk-solver: an add-in program for clinical theriogenology 2021; 13: 209 pharmacokinetic and pharmacodynamic data analysis in microsoft excel. comput methods programs biomed 2010;99:306-314. 21. minitab 17: minitab inc. state college, pa, usa. 22. rasmussen f, ladefoged o: tissue damage at the injection site after intramuscular injection of drug preparations formulated by addition of polyvinyl-pyrrolidone. acta vet scand 1974;15:636-638. 23. nouws jfm, vree tb: effect of injection site on the bioavailability of an oxytetracycline formulation in ruminant calves. vet quart 1983;5:165-170. 24. de laistre banting a, fanneau de la horie gc: subcutaneous and intramuscular injection of oxytetracycline in calves: comparison of serum concentration and local tolerance. j vet pharmacol therap 1987;10:184-186. 25. clarke cr, wang z, burrows ge, et al: pharmacokinetics of two long-acting oxytetracycline products administered subcutaneously and intramuscularly. j vet pharmacol therap 1999;22:65-67. 26. ames tr, larson vl, stowe cm: oxytetracycline concentrations in healthy and diseased calves. am j vet res 1985;44:1353-1357. 27. davey la, ferber mt, kaye b: comparison of the serum pharmacokinetics of a long acting and a conventional oxytetracycline injection. vet rec 1985;117:426-429. 28. mevius dj, nouws jfm, breukink hj, et al: comparative pharmacokinetics, bioavailability and renal clearance of five parenteral oxytetracycline‐20% formulations in dairy cows, vet quart 1986;8:285-294. 29. breeze r, gay c: plasma levels of a long-acting oxytetracycline in cattle. bov pract 1981;16:22-23. 30. mestorino ne. mariño hernández e, marchetti l, et al: pharmacokinetics and tissue residues of an oxytetracycline/diclofenac combination in cattle. rev sci tech off int epiz 2007:26:679-690. 31. de boer m, heuer c, hussein h, et al: minimum inhibitory concentrations of selected antimicrobials against escherichia coli and truperella pyogenes of bovine uterine origin. j dairy sci 2015;98:4427-4438. 32. cox sr, mclaughlin c, fielder ae, et al: rapid and prolonged distribution of tulathromycin into lung homogenate and pulmonary epithelial lining fluid of holstein calves following a single subcutaneous administration of 2.5 mg/kg body weight. intern j appl res vet med 2010;8:129-137. 33. gáler d, hessong s, beato b, et al: an analytical method for the analysis of tulathromycin, an equilibrating triamilide, in bovine and porcine plasma and lung. j agric food chem 2004;52:2179-2191. 34. rivera jd, woolums ar, giguère s, et al: pharmacokinetics of tulathromycin following administration to stocker cattle with remote delivery devices. j anim sci 2019;97:4482-4487. 35. coetzee jf, kleinhenz md, magstadt dr, et al: pneumatic dart delivery of tulathromycin in calves results in lower antimicrobial concentrations and increased biomarkers of stress and injection site inflammation compared with subcutaneous injection. j anim sci 2018; 96:3089-3101. clinical theriogenology 2021; 13: 210 pharmacokinetics of oxytetracycline andtulathromycin in plasma and semen ofbeef bulls introduction many progestins have been investigated in equine veterinary medicine, including medroxyprogesterone acetate, hydroxyprogesterone hexanoate, norgestomet, megesteral acetate, melengestrol acetate, and proligestone (table). several studies1-5 investigated the progestogenic efficacy of these progestins and determined only altrenogest to be effective. altrenogest maintained pregnancy after a luteolytic dose of prostaglandin f2α (pgf2α), whereas medroxyprogesterone acetate (mpa), hydroxyprogesterone hexanoate, norgestomet, and megesteral acetate treatment were unable to prevent abortion.1 furthermore, the ability of proligestone, mpa, and melengestrol acetate to bring mares out of seasonal transition was not consistent.2-4 in contrast, both progesterone in oil2 and altrenogest5 effectively hastened estrus and conception in both early and late seasonally transitional mares. therefore, this review will focus on altrenogest, with minimal inferences to other progestins. pharmacokinetics of altrenogest altrenogest (17α-allyl-17β-hydroxy-estra-4, 9, 11-trien-3-one) is also known as allyltrenbolone. although classified as a progestin, altrenogest structurally resembles androgens and binds to the androgen receptor with 75% affinity of dihydrotestosterone,6 although minimal anabolic effects have been noted, including no increase in body mass in either intact stallions or mares.7 however, mares treated with altrenogest in late pregnancy had fillies born with an enlarged clitoris (figure 1).8 trace concentrations of androgen and anabolic steroid trenbolone were noted in horses receiving altrenogest, leading to restrictions of its use in many equine organizations, including irish horseracing regulatory board, british horseracing association, australian progestin use in mares carleigh fedorka department of veterinary science, university of kentucky, lexington, ky abstract progesterone is in constant fluctuation throughout the lifespan of the horse. as a key regulator of the hypothalamic-pituitary-gonadal axis, progesterone is involved in numerous aspects of reproduction. synthetic analogs of progesterone, deemed progestins, are widely available to industry to act as progestogenic within the reproductive tract, although few are bioactive in the horse. utilized to suppress estrous, delay ovulation, maintain pregnancy, and for behavioral modifications, progestins are one of the most common class of pharmaceuticals in veterinary medicine. in this review, we discuss the progestins available to equine industry, their efficacy, pharmacokinetics, and potential side effects following treatment. keywords: progestin, estrus synchronization, delay of ovulation, pregnancy maintenance racing board, and federation equestre internationale. to predict withdrawal times for altrenogest, its pharmacokinetics were determined after oral, injectable, and rectal treatments. figure 1. anabolic effects (enlarged clitoris) of altrenogest treatment during late pregnancy clinical theriogenology 2021; 13: 247 adult mares (n = 10) were treated with oral altrenogest (0.044 mg/kg).9 in most mares, maximum concentration (cmax) was reached within 15 30 minutes. mean cmax was 35 ng/ml on day 1 and 31 ng/ml on day 5. in contrast, mean cmax in urine was 1720 ng/ml on day 1 and 2107 ng/ml on day 5. time for total clearance (with a limit of detection of 2 ng/ml) was 12 days. due to risk of human interaction with oral altrenogest, injectable forms have gained popularity within industry, and the pharmacokinetics of which have been investigated.10 after intramuscular treatment (0.3 mg/kg), mean circulating concentrations were 33.52 ng/ml. additionally, the area under the curve for intramuscular treatment was 5.6-fold higher than the oral product. it was hypothesized that intramuscular treatment formed a depot that slowed absorption, prolonging clearance compared to oral treatment. several pregnant mares that are admitted to the hospital for medical or surgical events may be prohibited from oral medications. therefore, pharmacokinetics of intrarectal treatment of altrenogest have also been assessed. altrenogest was detected in circulation as early as 15 minutes after oral and rectal treatment (0.088 mg/kg).11 however, the cmax for per rectal altrenogest was considerably lower than that of oral treatment (2.54 versus 16.00 respectively), and clearance of altrenogest was more rapid in the rectal treatment group. although rectal treatment was effective in delivering altrenogest, the bioavailability was only 5.47% compared to oral treatment. despite the option that rectal treatment may be a viable alternative for hospitalized mares, a dosage of 0.088 mg/kg every 4 8 hours would be necessary to maintain therapeutic concentrations. use of progestins suppressing estrus suppression of estrus in sport and race mares is desirable and progestins are commonly used to accomplish this goal. progestin treatment suppresses estrus in cycling mares through its high affinity for progesterone receptor (pr) resulting in endometrial and myometrial environments similar to a pregnant mare. progesterone in oil (100 mg) effectively suppressed estrus and mares returned to estrus within 3 4 days after cessation of treatment.2 repeated treatment with progesterone (either intramuscular or repositol) led to its accumulation within the system that persisted for > 10 days after the last treatment.12 daily oral treatments with 0.044 mg/kg altrenogest was also sufficient to bind to the pr to suppress estrus.13 similarly, compounded long-acting injectable altrenogest formulations suppressed behavioral estrus when used as recommended by the manufacturers.10,14 furthermore, long-term altrenogest treatment did not affect future fertility potential15 and highdose and prolonged altrenogest treatment did not affect overall hematologic or biochemical properties of the mare.16 in contrast, norgestomet, mpa, hydroxyprogesterone caproate, and megesterol acetate had no effect on estrus suppression17-20 to our knowledge, no other synthetic progestins have been critically evaluated for their ability to suppress estrus-like behavior. delaying ovulation although select progestins successfully suppressed estrus in the mare, conflicting results exist for the ability to delay ovulation, and success may depend on the phase of the estrous cycle in which treatment is initiated. in one study, neither oral altrenogest nor a progesterone implant (controlled intravaginal releasing device) were effective in preventing ovulation when short-term treatment began in the preovulatory phase.21 in contrast, shortterm treatment of 0.044 mg/kg altrenogest successfully delayed ovulation when treated at the detection of a 35 mm follicle.22 when a double dose of altrenogest (0.088 mg/kg) was administered, the delay of ovulation was noted in a preovulatory mare.23 however, this was not repeatable in a larger field setting.1 interestingly, use of an injectable sustained-release vehicle with varying doses of altrenogest delayed ovulation substantially.24 the effect was profound when lactide-glycolide microparticles were added, delaying ovulation by 33.5 days after pgf2α treatment. however, mpa had no effect on ovulation when combined with the same vehicle or microparticle formulation. hastening transition mares are seasonally polyestrous long-day breeders and are therefore anovulatory in the months surrounding the winter equinox. a transitionary phase exists between anestrus and proper cyclicity; and shortening this phase has been attempted through the administration of exogenous steroids. the proposed rational for progestin treatment is believed to inhibit the release of luteinizing hormone (lh) from the pituitary, allowing for a repositol of the gonadotropin to form, thereby allowing for ovulation to be induced. progestin treatment has been shown to hasten the first ovulation of the year dependent on the stage of transition and the specific progestin used. altrenogest hastened the occurrence of first estrus and reduced the interval to conception in late transitional mares (> march 15, 20 mm follicle) with no impact on early transitional mares (< march 15th, 14 mm follicle).5 however, when a higher dose (0.22 mg/kg) was used, results were not repeatable.25 other progestins (1500 mg of proligestone) induced comparable ovulation and pregnancy rates to altrenogest treatment in transitional mares.13 it should be noted that only a minimal number of control mares were used in this study, and pretreatment follicular status was not assessed. controlled release of intramuscular progesterone (la p4; biorelease) was effective in shortening the duration to first ovulation in late transitional mares; however, it was ineffective in the early transitional mare.26 studies using intravaginal progesterone-releasing devices to hasten first ovulation had mixed results. although controlled intravaginal releasing devices combined with progesterone were ineffective in improving pregnancy rates in the first estrous cycle,27 89% of anestrous mares ovulated within 10 days after progesterone-releasing clinical theriogenology 2021; 13: 248 intravaginal devices were remove.28 it should be noted that mild to moderate vaginitis was observed in a subset of mares after implantation of the progesterone-releasing devices. these devices are not approved for equine use in most countries (except for cue-mare® in new zealand). overall, the efficacy of progestin treatment in shortening the duration to first ovulation is inconsistent and depends on the reproductive status of the mare in addition to the progestin used. synchronizing estrus for simplicity of breeding in addition to timing embryo transfer, progestins are commonly used to regulate both estrus and diestrus phases of the cycle. although progestins suppress the release of lh, their effect on fsh is less evident, resulting in normal follicular development.29 hence, progestin treatment results in a reduction of ovulations, but does not synchronize follicular growth in treated mares. in order to effectively synchronize estrus in mares, a combination of progestins and estrogens can be used. this ‘p&e’ treatment consists of 10 days of intramuscular treatment of 150 mg progesterone in oil alongside 10 mg estradiol-17β (figure 2). this was followed by a luteolytic dose of pgf2α on the 10th day of treatment.30 it is expected that mares will have a preovulatory follicle 8 days after cessation of treatment and respond to an ovulatory-inducing agent. this has been repeated by replacing progesterone in oil with altrenogest, and responses were comparable.31 supporting early pregnancy progesterone is produced in ovaries (primary and secondary corpora lutea) after ovulation until 120 150 days of pregnancy when pregnancy becomes fully dependent on 5α-reduced progestogens from the feto-placental unit, including 5α-dhp and 20α-5p.32-35 secondary luteal deficiency, as a result of endogenous release of pgf2α, is associated with systemic or uterine inflammation and endotoxemia. this premature release is detrimental to pregnancy if it occurs prior to this shift in progestogen release. although low systemic progesterone concentrations are incompatible with maintenance of pregnancy, there are limited scientific data to support a primary failure of the corpus luteum (cl) as a cause of embryonic loss.36 measuring circulating progesterone concentrations in a single blood sample to determine if pregnancy is at risk is not accurate unless it is less than 1 ng/ml, due to variations in secretion of this hormone during the day. studies have indicated the progestogenic potential of altrenogest, while no other synthetic progestin has been found as effective. either 150 mg progesterone in oil or 0.044 mg/kg altrenogest maintained pregnancy after a luteolytic dose of pgf2α in ovariectomized mares.37 interestingly, a double dose of altrenogest (0.088 mg/kg) was more effective in pregnancy maintenance in ovariectomized recipient mares after embryo transfer.38 in this study, it was determined that the standard dose of 0.044 mg/kg produced poor uterine tone and decreased cervical competence. the authors recommended the double dose to be utilized during embryo transfer procedures. several other synthetic progestins (1000 mg hydroxyprogesterone caproate, 1000 mg mpa, 500 mg hydroxyprogesterone hexanoate, 15 mg norgestomet, and 500 mg megestrol acetate) were unable to maintain pregnancy.39 mares treated with altrenogest maintained pregnancy after pgf2α treatment whereas other progestins were not able to prevent abortion.1 in addition, long-acting progesterone (150 mg) compounds were effective in preventing early pregnancy loss after a luteolytic dose of pgf2α and are worth considering.40 there is no cross reactivity between altrenogest and progesterone in commercial progesterone assays; therefore, progesterone assays are helpful in determining whether a cl has undergone luteolysis (p4 < 1 ng/ml) or is still active during treatment. assay results can guide the clinician to make a decision (discontinue or continue treatment) until the feto-placental unit provides necessary progestogen support to maintain pregnancy. however, altrenogest treatment can suppress endogenous production of progesterone and may interfere with formation of secondary cl.41,42 one study found altrenogest supplementation to decrease the production of equine chorionic gonadotropin (ecg) in mares greater than 8 years of age, but also found altrenogest treatment to maximize embryo size in the aged mare population.43 figure 2: standard progesterone and estrogen ‘p&e’ treatment protocol (daily 150 mg progesterone in oil or 0.044 mg/kg altrenogest with 10 mg estradiol-17β). treatment is initiated when a 25 mm follicle is detected. a luteolytic dose of pgf2α is given on day 10 and transrectal ultrasonography is used to assess follicular development. when a 35 mm follicle (6 9 days after cessation of treatment) is detected, hcg is given and mares are inseminated 24 36 hours later. clinical theriogenology 2021; 13: 249 supporting late pregnancy treatment with a double dose of altrenogest (0.088 mg/kg) prevented late-term abortion in mares after abortion was attempted with a synthetic prostaglandin from 90 115 days of pregnancy.44 progestins have been suggested to maintain pregnancy in late pregnancy by inducing myometrial quiescence and suppressing pgf2α secretion, presumably by modulation of cytokines. therefore, altrenogest is routinely used during pregnancy for the prevention or treatment of ascending placentitis, a leading cause of infectious abortion in north america. combined treatment of antimicrobial (trimethoprim sulfamethoxazole), immunomodulator (pentoxifylline), and progestin (altrenogest) was effective in treating experimentally induced disease (transcervical inoculation with streptococcus zooepidemicus).45 although none of the untreated mares carried to term, 10/12 (83%) of the treated mares produced a viable foal. since treatments were not assessed individually, it is unclear if altrenogest treatment alone prevented abortion. this confers with recent research that demonstrated a withdrawal of pr preceded premature parturition in experimentally induced disease, indicating that altrenogest supplementation may be of no benefit.46 this was further supported by a study47 that demonstrated that addition of altrenogest treatment had no effect on pregnancy length, interval from inoculation to delivery, or neonatal outcome in comparison to mares solely treated with antimicrobials and antiinflammatories. in that study, the addition of estradiol cypionate to antimicrobial/antiinflammatory treatment increased pregnancy length from induction of disease, in addition to improving neonatal viability. furthermore, altrenogest treatment has been shown to shorten pregnancy length.48 altrenogest supplementation was also unnecessary in pregnancy maintenance after late ovariectomy (> 100 days), regardless of concentration or time of treatment.49 there are some discrepancies with regard to safety of altrenogest treatment in midto late-pregnancy. mares treated with altrenogest in late pregnancy had shorter gestation, poorer neonatal outcomes,48 and fillies were born with an enlarged clitoris.8 additionally, after treatment in late pregnant mares, altrenogest was detectable within both fetal fluid compartments and in grossly higher concentrations in fetal compared to maternal serum.50 yet, foals born to mares receiving altrenogest in late pregnancy had normal onset of puberty, comparable fertility, and reproductive function to controls.51,52 based on these findings, the efficacy and necessity of progestin supplementation in mid to late-pregnancy needs to be further investigated. hastening postpartum involution in mares, first postpartum estrus occurs within 5 20 days53,54 while the uterus is still undergoing repair from tissue alterations during pregnancy and foaling. uterine involution is not complete until 15 days postpartum and mares ovulating > 10 days postpartum are more likely to achieve and maintain pregnancy compared to those that ovulated earlier.55 this is attributed to the interval of time (5.5 days) that the embryo resides within the oviduct after fertilization before migrating to uterine lumen, at which point involution should be complete. therefore, progestins were used56-58 in an attempt to delay ovulation in order to improve fertility at first postpartum estrus, with mixed results. one study found altrenogest to delay ovulation and increased pregnancy rates in postpartum mares when administered for the initial 8 days.56 additionally, altrenogest had no effect on other aspects of uterine involution, including uterine tone, size, or retention of fluid. a second study found altrenogest treatment to delay first ovulation by 10 days but did not improve pregnancy rates when fed for 15 days postpartum.57 progesterone (100 mg) in oil treatment from days 5 to 14 postpartum successfully inhibited ovulation in 6/9 mares.58 additionally, the researchers noted no histological changes in treated mares, and pregnancy was achieved in 66% of treated mares, comparable to other estrous cycles. addition of estradiol-17β to this treatment was tried.59 there was no delay in ovulation when mares were treated with either 150 mg progesterone in oil + 10 mg estradiol-17β or 300 mg progesterone in oil + 20 mg estradiol-17β, and pregnancy rates were comparable among the groups and controls.59 it was noted that none of the p&e treated mares ovulated prior to 10 days postpartum, whereas 20% of control mares ovulated early. additionally, less variability from time to ovulation was noted in treated mares. overall, progestin treatment appears to successfully delay ovulation postpartum; however, there are conflicting data on its ability to improve first estrous cycle pregnancy rates. potential side effects of progestins to immune system progestins affect the immune system in a variety of species.60 in humans, similar synthetic progestins to those used in mares are available for contraception, many of which have detrimental effect on the immune system. in most species, progesterone is highly antiinflammatory and enacts this function on the immune system through signaling on the pr.61-64 in contrast, synthetic progestins such as mpa and levonorgestrel (lng) increase the expression or production of a variety of proinflammatory mediators.60,65-67 additionally, the specific function of the individual progestin appears to be affected by the receptor through which it enacts, as mpa activates both pr and glucocorticoid receptors, whereas lng solely activates the pr.68,69 through this increase in proinflammatory cytokine activation, in addition to recruitment of varying immune cells, women on synthetic progestins were found to be at higher risk for a variety of diseases, including hiv,70-75 hsv-2,76-78 chlamydia,79,80 gonorrhea,81 and even influenza.60,82 it is believed that the increased risk of viral infection is due to proinflammatory activation of nfκβ that binds to the terminal repeat of viral dna, thereby promoting viral replication. a recent study investigated effects of altrenogest on the immune system of nonpregnant mares, with similar results to those reported in humans.83 in vitro, altrenogest caused a dose-dependent clinical theriogenology 2021; 13: 250 increase in the expression of the proinflammatory cytokine il-1β in addition to the pleiotropic cytokine il-6, in peripheral blood mononuclear cells. this was also noted in vivo, wherein under the diestrus influence of endogenous progesterone, control mares had decreased expression of proinflammatory cytokines (ifnγ, il-1β, and il-8) while the peripheral blood mononuclear cells of altrenogest-treated mares did not decrease in the expression of either il-1β or il-8. additionally, altrenogest altered expression of a variety of cytokines within the endometrium, including ifnγ, il-10, and il-1rn, indicating both local and systemic effects of altrenogest on the immune system of the mare. this may be clinically relevant, as altrenogest treatment in nonpregnant mares has been associated with vaginal candidiasis84 and uterine inflammation.85 interestingly, it was determined that altrenogest alters the immune system of the nonpregnant mare without activating glucocorticoid receptors. as it is structurally a 19-nortestosterone, and more comparable to lng, this was not surprising. in early pregnancy, altrenogest treatment decreased endometrial expression of the pr.86 additionally, an increase in the number of polymorphonuclear neutrophils was noted within the endometrium after treatment. in late pregnancy, minimal changes were observed in endometrial gene expression of il-6, il-8, or cox-2 after altrenogest treatment.85 in this same study, altrenogest treatment had no effect on the expression of the progesterone, estrogen, or oxytocin receptors, conflicting with early pregnant mare data. this is interesting, as progesterone itself inhibited the nfκβ pathway, including downstream cox-2 and il-1β,87 again indicating a differing pathway of effect for altrenogest. foals born to mares that were treated with altrenogest during late pregnancy up to the day of foaling were affected by treatment. this included an imbalanced neutrophil to lymphocyte ratio, increased cortisol, decreased potassium, and decreased calcium within hours after birth.88 in an additional study, neonates born to mares receiving late-term altrenogest, had decreased respiratory function and substantially more problems in the perinatal period.48 conclusion progestins are valuable clinical tools in managing several aspects of equine reproduction. although only natural and synthetic (altrenogest) progestins are currently available, both compounds are efficacious in suppressing estrus, hastening transition, and maintaining early pregnancy. in the past, progestin treatment has been considered to be without side effects and perhaps overused as an insurance policy for pregnancy maintenance. based on recent studies, long-term use of altrenogest, particularly during late pregnancy, may be associated with immune-related side effects in mares and foals. this possibility should be considered when benefits are weighed against risks in clinical situations (figure 3). figure 3. risk to benefit ratio of altrenogest treatment clinical theriogenology 2021; 13: 251 conflict of interest author has no affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. references 1. bruemmer je, coy rc, olson a, et al: efficacy of altrenogest administration to postpone ovulation and subsequent fertility in mares. j eq vet sci 2000;20:450-453. 2. loy rg, swan sm: effects of exogenous progestogens on reproductive phenomena in mares. j anim sci 1966;25:821-826. 3. van der holst w, van laar ph, oldenkamp ep: prolonged spring oestrus in mares: the use of progestogens with specific reference to proligestone. theriogenology 1985;24:609-617. 4. lopez-bayghen c, zozaya h, ocampo l, et al: melengestrol acetate as a tool for inducing early ovulation in transitional mares. acta vet hung 2008;56:125-131. 5. webel sk, squires el: control of the oestrous cycle in mares with altrenogest. j reprod fertil suppl 1982;32:193-198. 6. bauer er, 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effect of nonsurgical embryo transfer procedure and/or altrenogest therapy on endogenous progesterone concentration in mares. j equine vet sci 2011;31:57-62. 43. willmann c, schuler g, hoffmann b, et al: effects of age and altrenogest treatment on conceptus development and secretion of lh, progesterone and ecg in early-pregnant mares. theriogenology 2011;75:421-428. 44. daels pf, besognet b, hansen b, et al: effect of progesterone on prostaglandin f2 alpha secretion and outcome of pregnancy during cloprostenol-induced abortion in mares. am j vet res 1996;57:1331-1337. 45. bailey cs, macpherson ml, pozor ma, et al: treatment efficacy of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest in experimentally induced equine placentitis. theriogenology 2010;74:402-412. 46. el-sheikh ali h, legacki el, loux sc, et al: equine placentitis is associated with a downregulation in myometrial progestin signalingdagger. biol reprod 2019. 47. curcio br, canisso if, pazinato fm, et al: estradiol cypionate aided treatment for experimentally induced ascending placentitis in mares. theriogenology 2017;102:98-107. 48. neuhauser s, palm f, ambuehl f, et al: effects of altrenogest treatment of mares in late pregnancy on parturition and on neonatal viability of their foals. exp clin endocrinol diabetes 2008;116:423-428. 49. knowles je, squires el, shideler rk, et al: progestins in midto latepregnant mares. j equine vet sci 1994;14:659-63. 50. palm fm, schenk i, neuhauser s, et al: concentrations of altrenogest in plasma of mares and foals and in allantoic and amniotic fluid at parturition. theriogenology 2010;74:229-235. 51. squires el, shideler rk, mckinnon ao: reproductive performance of offspring from mares administered altrenogest during gestation. j equine vet sci 1989;9:73-76. 52. naden j, squires el, nett tm, et al: effect of maternal treatment with altrenogest on pituitary response to exogenous gnrh in pubertal stallions. j reprod fertil 1990;88:177-83. 53. gygax ap, ganjam vk, kenney rm: clinical, microbiological and histological changes associated with uterine involution in the mare. j reprod fertil suppl 1979:571-578. 54. blanchard tl, thompson ja, brinsko sp, et al: mating mares on foal heat: a five-year retrospective study. proc am assoc equine pract 2004. p. 525-530. 55. loy rg: characteristics of postpartum reproduction in mares. vet clin north am large anim pract 1980;2:345-349. 56. mckinnon ao, squires el, harrison la, et al: ultrasonographic studies on the reproductive tract of mares after parturition: effect of involution and uterine fluid on pregnancy rates in mares with normal and delayed first postpartum ovulatory cycles. j am vet med assoc 1988;192:350-353. 57. sigler dh, ericson de, gibbs pg, et al: reproductive traits, lactation and foal growth in mares fed altrenogest. j anim sci 1989;67:1154-1159. 58. loy rg, hughes jp, richards wp et al: effects of progesterone on reproductive function in mares after parturition. j reprod fertil suppl 1975:291-295. 59. bruemmer je, brady ha, blanchard tl: uterine involution, day and variance of first postpartum ovulation in mares treated with progesterone and estradiol-17beta for 1 or 2 days postpartum. theriogenology. 2002;57:989-995. 60. hall oj, nachbagauer r, vermillion ms, et al: progesterone-based contraceptives reduce adaptive immune responses and protection against sequential influenza a virus infections. j virol 2017;91. 61. hardy db, janowski ba, corey dr, et al: progesterone receptor plays a major antiinflammatory role in human myometrial cells by antagonism of nuclear factor-kappab activation of cyclooxygenase 2 expression. mol endocrinol 2006;20:2724-733. 62. jones la, kreem s, shweash m, et al: differential modulation of tlr3and tlr4-mediated dendritic cell maturation and function by progesterone. j immunol 2010;185:4525-4534. 63. lei k, chen l, georgiou ex, et al: progesterone acts via the nuclear glucocorticoid receptor to suppress il-1beta-induced cox-2 expression in human term myometrial cells. plos one. 2012;7:e50167. 64. butts cl, shukair sa, duncan km, et al: progesterone inhibits mature rat dendritic cells in a receptor-mediated fashion. int immunol 2007;19:287-296. 65. deese j, masson l, miller w, et al: injectable progestin-only contraception is associated with increased levels of pro-inflammatory cytokines in the female genital tract. am j reprod immunol 2015;74:357-367. 66. coleman js, mwachari c, balkus j, et al: effect of the levonorgestrel intrauterine device on genital hiv-1 rna shedding among hiv-1infected women not taking antiretroviral therapy in nairobi, kenya. j acquir immune defic syndr 2013;63:245-248. 67. eastman aj, bergin il, chai d, et al: impact of the levonorgestrelreleasing intrauterine system on the progression of chlamydia trachomatis infection to pelvic inflammatory disease in a baboon model. j infect dis 2018;217:656-666. 68. attardi bj, zeleznik a, simhan h, et al: comparison of progesterone and glucocorticoid receptor binding and stimulation of gene expression by progesterone, 17-alpha hydroxyprogesterone caproate, and related progestins. am j obstet gynecol 2007;197:599 e1-7. 69. kontula k, paavonen t, luukkainen t, et al: binding of progestins to the glucocorticoid receptor. correlation to their glucocorticoid-like effects on in vitro functions of human mononuclear leukocytes. biochem pharmacol 1983;32:1511-1518. 70. huijbregts rp, helton es, michel kg, et al: hormonal contraception and hiv-1 infection: medroxyprogesterone acetate suppresses innate and adaptive immune mechanisms. endocrinology. 2013;154:1282-1295. 71. lavreys l, chohan v, overbaugh j, et al: hormonal contraception and risk of cervical infections among hiv-1-seropositive kenyan women. aids 2004;18:2179-2184. 72. lavreys l, baeten jm, martin hl, jr., et al: hormonal contraception and risk of hiv-1 acquisition: results of a 10-year prospective study. aids 2004;18:695-697. 73. lavreys l, baeten jm, kreiss jk, et al: injectable contraceptive use and genital ulcer disease during the early phase of hiv-1 infection increase plasma virus load in women. j infect dis 2004;189:303-311. 74. mcclelland rs, lavreys l, katingima c, et al: contribution of hiv-1 infection to acquisition of sexually transmitted disease: a 10-year prospective study. j infect dis 2005;191:333-338. clinical theriogenology 2021; 13: 253 75. ralph lj, mccoy si, shiu k, et al: hormonal contraceptive use and women’s risk of hiv acquisition: a meta-analysis of observational studies. lancet infect dis 2015;15:181-189. 76. quispe calla ne, vicetti miguel rd, boyaka pn, et al: medroxyprogesterone acetate and levonorgestrel increase genital mucosal permeability and enhance susceptibility to genital herpes simplex virus type 2 infection. mucosal immunol 2016;9:1571-1583. 77. kaushic c, ashkar aa, reid la, et al: progesterone increases susceptibility and decreases immune responses to genital herpes infection. j virol. 2003;77:4558-4565. 78. gillgrass ae, fernandez sa, rosenthal kl, et al: estradiol regulates susceptibility following primary exposure to genital herpes simplex virus type 2, while progesterone induces inflammation. j virol 2005;79:3107-3116. 79. kaushic c, zhou f, murdin ad, et al: effects of estradiol and progesterone on susceptibility and early immune responses to chlamydia trachomatis infection in the female reproductive tract. infect immun 2000;68:4207-4216. 80. kaushic c, murdin ad, underdown bj, et al: chlamydia trachomatis infection in the female reproductive tract of the rat: influence of progesterone on infectivity and immune response. infect immun 1998;66:893-898. 81. xu l, dong b, wang h, et al: progesterone suppresses th17 cell responses, and enhances the development of regulatory t cells, through thymic stromal lymphopoietin-dependent mechanisms in experimental gonococcal genital tract infection. microbes infect 2013;15:796-805. 82. hall oj, limjunyawong n, vermillion ms, et al: progesterone-based therapy protects against influenza by promoting lung repair and recovery in females. plos pathog 2016;12:e1005840. 83. fedorka ce, ball ba, walker of, et al: alteration of the mare’s immune system by the synthetic progestin, altrenogest. am j reprod immunol 2019:e13145. 84. montes aj, montes lf, vaughan jt, et al: vulvo vaginal candidiasis in thoroughbred mares following progestogen administration intravaginal treatment with clotrimazole. j equine vet sci. 2001;21:68-70. 85. palm f, walter i, nowotny n, et al: progestin treatment does not affect expression of cytokines, steroid receptors, oxytocin receptor, and cyclooxygenase 2 in fetal membranes and endometrium from pony mares at parturition. theriogenology 2013;79:59-68. 86. willmann c, budik s, walter i, et al: influences of treatment of early pregnant mares with the progestin altrenogest on embryonic development and gene expression in the endometrium and conceptus. theriogenology 2011;76:61-73. 87. lei k, georgiou ex, chen l, et al: progesterone and the repression of myometrial inflammation: the roles of mkp-1 and the ap-1 system. mol endocrinol 2015;29:1454-1467. 88. neuhauser s, palm f, ambuehl f, et al: effect of altrenogest-treatment of mares in late gestation on adrenocortical function, blood count and plasma electrolytes in their foals. equine vet j 2009;41:572-577. clinical theriogenology 2021; 13: 254 progestin use in mares david e. bartlett award for lifetime achievement in theriogenology the 2020 bartlett address terry blanchard professor emeritus, department of large animal clinical sciences college of veterinary medicine & biomedical sciences texas a&m university, college station, tx i wish to thank the american college of theriogenologists, society for theriogenology, and theriogenology foundation for bestowing this award on me. as i remember, this award was originated to honor our forefathers responsible for creating our specialty college. the members that founded the rocky mountain society for the study of breeding soundness comprised most of this group. their vision and perseverance are legendary to our members. they set the bar high. as such, i can't help but feel that my professional career doesn't approach this standard. my interest in theriogenology was peaked when i was in the us peace corps in the philippines. i was recruited to help develop artificial breeding strategies in the native water buffalo (carabao). funds available for this project were delayed for some time, and i was asked to teach animal science and preveterinary college students. i discovered that i really enjoyed teaching. i am reminded that no one has a meaningful career uninfluenced by others. i was fortunate to be selected for a large animal reproduction residency with bob kenney at new bolton center. while at new bolton center, i was also mentored by wendell cooper, john hurtgen, and marolo garcia. these amazing mentors were the beginning of many that suffered through my presence and instructed me patiently and tirelessly. other mentors that greatly influenced my career include my graduate advisor ron elmore, bob youngquist, and clarence bierschwal at the university of missouri. on my return to new bolton center, i was fortunate to work with dickson varner, katrin hinrichs, and stan brown. while earlier in their academic careers, i believe i learned more from them than they from me. i later joined dickson at texas a&m university. this was the beginning of a long and storied career shared with dickson. he has always been the visionary that brought me along for an amazing ride. i also would not have enjoyed a meaningful career without the enduring support of ron elmore, ron martens, and bill moyer at texas a&m university. finally, veterinary students and residents i taught that have become lifelong friends as well as colleagues include jim moorehead, charlie love, steve brinsko, and margo macpherson and numerous others. again, i reiterate my gratitude to the american college of theriogenologists, society for theriogenology, and theriogenology foundation for this award. clinical theriogenology • volume 12 number 3 • september 2020168 004_ms-004 blanchard clinical theriogenology 2022; 14: 10 editor’s note dear theriogenology friends, our journal is entering its 14th year. another year of progress. for the last 4 years, i along with you all, were privileged to build on the foundation laid by the founding editor, dr. bob youngquist, who has been an important force for the discipline over the years. i made more theriogenology friends in the last 4 years than my 40 years of academic life! three issues are worth mentioning. changing of the guards! i express my profound appreciation to the members of the editorial board for their insights and contributions to the journal. five of them (drs. etta bradecamp, sara lyle, lynda miller, colin palmer, and karen wolfsdorf) have completed their terms and they deserve our gratitude for their time and effort. furthermore, 5 colleagues (drs. allan gunn, michelle kutzler, réjean lefebvre, jennifer roberts, and cyril stephen) will be taking their places and have already contributed to our journal (abstracts and manuscripts and worked with me in reviewing manuscripts). all incoming members will submit manuscripts. additionally, our international members (allan, cyril, and réjean) will help our journal to publish more manuscripts by overseas colleagues. electronic issues four years ago, copy editor dr. john kastelic and i, discussed on ways to improve our journal. colleagues were approached to create reviews in their areas of expertise, keeping a focus on applied aspects of theriogenology. recently, dr. ahmed tibary, our design editor, also suggested to the boards that we ought to create a ‘rubric of timely topics’ in comparative and species-specific topics in theriogenology. time has come to accomplish this goal! dr. igor canisso, a wonderful colleague, organized 15 electronic issues with variety of manuscripts. our goal is to have them published within next 3 years. we are thankful to igor and colleagues who will serve as guest editors and will also contribute. we will continue to have our regular 4 print issues per year. let me stress again, in addition to case reports, reviews, please also submit research manuscripts! last issue pertains to our recent journal survey. i am in touch with colleagues who volunteered to create reviews in their areas of expertise. i look forward to receiving their reviews. our leadership (society for theriogenology, american college of theriogenologists, and theriogenology foundation) deserve our appreciation for their vision to promote scholarship in our discipline. thanks to omnipress and franz management for continuing to refine our journal archives. i appreciate the support and friendship of drs. john kastelic, ahmed tibary, charles franz, and mrs. tara beasley (our management office). purdue university has been a great help. i am very grateful to my wife damayanthy peter for devoted daily prayer, encouragement, and tea. regards, augustine introduction the control of bovine trichomoniasis has proven to be challenging despite aspects of the disease that make it amenable to control and eradication. there are several reasons for the lack of control in some areas of the us. one of the primary challenges is in not having an educated and organized approach to keep trichomoniasis out of a herd or eliminate it should the herd become infected. the following narrative describes the steps to create an overall control plan for trichomoniasis, explains the use of a web-based assessment tool, and offers suggestions for further research that may aid in the control of this disease. control the following 4 steps (education, risk evaluation, plan development, and execution), outline a coordinated series of actions necessary to effectively control this devastating disease. education to quote h.p. harding, “in conclusion, may i say that it is my opinion that if more care was taken in the sale and purchase of barren cows, in the purchase of bulls of breeding age, and if the farmers could be sufficiently educated to the unique opportunities for control that this disease offers, then its total elimination should be practicable in the quite near future.”1 educating producers and veterinarians to distinctive features of t. foetus that include venereal transmission, typical transient infection in females, and a predilection for the prepuce in chronically infected older bulls. this will provide a basic management and control of bovine trichomoniasis jeff ondrak 6u ranch, steele city, ne abstract bovine trichomoniasis has been recognized as a cause for bovine reproductive failure since the late 1800’s. description of the causative organism, tritrichomonas foetus, led to its control. however, effective control has not occurred in many parts of the us. the purpose of this discussion is to provide steps to have an effective control plan for this disease, present a tool to develop a plan, and outline future research opportunities that may strengthen our knowledge of this disease and aid in its control. keywords: tritrichomonas foetus, biosecurity, microbiome understanding of trichomoniasis so that a practical and a sound plan for control is recognized as essential, developed, and implemented. risk evaluation evaluating a cattle herd’s risk of trichomoniasis exposure is necessary to determine the level of preparedness an operation should achieve. knowing the prevalence in a specific locale is important in determining if the herd is at no, low, or high risk for trichomoniasis. however, determining accurate and current prevalence estimates is difficult, as many of the prevalence estimates in the literature are quite dated and most are specific to a region or state that may not be comparable to most producer’s location. plan development after assessing the risk of trichomoniasis in a cattle herd, an informed, practical plan should be developed. the following 6 aspects of a biosecurity program should be addressed to ensure there are no gaps. recordkeeping accurately tracking reproductive performance can provide indications of reproductive failure that may be an early indication of trichomoniasis and allow an organized investigation into the cause. records of animal location and movement may also allow strategic testing of specific management groups in multi-unit operations that may also clinical theriogenology 2021; 13: 235 lead to an early diagnosis of the disease before it spreads into uninfected units within the operation.2 isolation isolate cattle if unplanned commingling with neighboring herds has occurred. females should be isolated from the rest of the herd until end of breeding season and their pregnancy status can be confirmed. bulls should be isolated and tested to ensure that they are t. foetus-negative before being allowed to return to breeding. any new additions, whose t. foetus status is questionable, should be isolated until they are confirmed negative. testing routine, systematic testing of bulls for t. foetus can provide early detection of a disease incursion and is a key component of a trichomoniasis prevention program for herds in trichomoniasis-endemic areas, herds neighboring t. foetus-positive herds, or herds recovering from a recent outbreak. the decision to test and which testing protocol to utilize should be guided by the local prevalence of trichomoniasis, the herd owner’s aversion to the risk of a disease incursion, and balancing the cost and benefits of testing. generally, surveillance testing occurs sometime between the end of a breeding season and the beginning of the next and includes a single test of all bulls in the herd. the advantage of surveillance testing closely after the breeding season is early detection of infection that allows time to develop and implement a complete trichomoniasis control program before the next breeding season. bulls tested under this program must not be exposed to cows prior to the next breeding season. alternatively, all bulls may be tested immediately before the breeding season, as that has the advantage to time the test to coincide with an annual breeding soundness examination, thereby reducing the number of times the bulls must be handled. however, this may not allow sufficient time for appropriate management of the disease before the start of the breeding season. before any surveillance testing is undertaken, the issue of false-positives should be fully explored. in populations of bulls with a very low prevalence of trichomoniasis (e.g. virgin and yearling bulls) there can be occasional unexpected positive test results. owners and veterinarians should be prepared to interpret these results in a way that minimizes the impact of these false-positive tests. vaccination a commercial t. foetus vaccine (trichguard®, boehringer ingelheim vetmedica, inc., ingelheim, germany) is licensed and available for use in the us. the vaccine is a killed product that requires 2 initial doses, with the second dose given ~ 4 weeks prior to breeding. an annual booster dose, 4 weeks prior to breeding, is required. the vaccine may increase cervical vaginal mucus trichomonal antibodies, increase calving rates, and decrease duration of infection for vaccinated versus controls. however, there is no evidence that the vaccine can eliminate carrier cows, and it does not prevent infection and subsequent establishment of the organism in the herd. even with vaccine use, t. foetus can become established in a herd without implementation of other biosecurity measures.3 sanitation there is evidence that t. foetus can be mechanically transmitted from infected to uninfected animals.4 although this is not a common form of transmission, it is important to practice good sanitation to avoid transmitting the organism through obstetrical instruments, artificial insemination equipment, or semen collection devices. traffic control fences should be monitored closely to rapidly identify when unplanned commingling with another herd has occurred so immediate steps can be taken to address the problem and reduce the risk of introducing trichomoniasis. execution for a plan to be effective it must be executed correctly. an investigation5 into reproductive failure in a cattle operation identified t. foetus to be an underlying cause. after a plan was created and implemented the herd returned to reproductive success indicating the value of not only identifying the problem but also following appropriate steps to correct the problem.5 trich consult creating an effective, herd-specific management plan for trichomoniasis utilizing steps outlined previously may appear overwhelming, especially, in light of the variations among herds regarding t. foetus status, risk of exposure, herd owner willingness to accept risk, and ability to implement appropriate management practices. to facilitate such a process trich consult (collaborative, online, novel, science-based, user-friendly, learning, tool) was developed as a web-based trichomoniasis assessment tool that incorporates science-based recommendations into an interactive format that mimics a conversation with an expert. trich consult uses a series of questions to assess the t. foetus status and management practices of a herd and uses the responses to the questions to provide feedback that allows the user to evaluate the impact of their decision. each question also includes a link to additional information and references to aid the individual in making informed decisions. after all questions have been answered, an optional, customized report may be generated that summarizes the questions and responses with comments suggesting prudent choices and warnings for clinical theriogenology 2021; 13: 236 responses that may have negative consequences. trich consult is currently available for use free of charge at the kansas state university beef cattle institute website. future opportunities there are several aspects of bovine trichomoniasis that are open to further investigation including improved local and regional prevalence estimates, improved sampling protocols, and treatment options. however, metagenomics offers the possibility of understanding the interaction of t. foetus within the microenvironment of the bovine reproductive tract, which may provide insights into prevention and treatment strategies. trichomonas vaginalis is a human reproductive tract parasite that produces symptoms in humans similar to clinical signs of bovine trichomoniasis caused by t. foetus. a human study identified an interaction between lactobacillus acidophius and t vaginalis in vitro6 whereas others have demonstrated an association between the vaginal microbiome of women and their risk for developing sexually transmitted diseases including trichomoniasis, suggesting that the microbiome of the reproductive tract may provide opportunities for prevention and treatment.7-10 with development of next generation sequencing technology, the microbiome of the bovine reproductive tract has been described11 in an attempt to understand the host-microbiome relationship and its influence on the reproductive efficiency of cattle.12 additional studies have explored the specific relationship between the vaginal microbiome and reproductive outcomes and disease that hold the potential for a better understanding of the pathogenesis of reproductive tract disease in cows.13-15 although culture-dependent studies have reported routine isolates from the preputial cavity of bulls, there is a lack of information regarding the preputial microbiome utilizing next generation sequencing technology.16 the path to bovine trichomoniasis infection in males and females is most likely multifactorial. however, the findings of the previously mentioned studies in human and bovine females and lack of microbiome information of the bovine prepuce suggest for an exploration of the reproductive tract microflora in cows and bulls in association with t. foetus infection, a reasonable step to understand this disease. conclusion trichomoniasis has long been recognized as a disease with substantial impact on reproductive performance in infected herds and severe financial implications for the farm or ranch. characteristics of the disease lend itself to control; however, effective control will only be achieved through education, risk assessment, careful planning and meticulous execution of the plan. use of a web-based trichomoniasis assessment tool, trich consult, may be a valuable way to facilitate development of a trichomoniasis control plan. future investigation into multiple facets of this disease may also provide valuable insights into additional opportunities for its control. conflict of interest none to report. references 1. harding hp: infertility in cattle with special reference to trichomoiniasis: i. the clinical aspect and diagnosis of trichomoniasis, with particular reference to the bull. vet rec 1950;62:539-540. 2. ondrak jd, keen je, rupp gp, et al: repeated sampling and testing by culture and pcr to detect tritrichomonas foetus carrier bulls in an infected nebraska herd. j am vet med assoc 2010;237:1068-1073. 3. hall mr, kvasnicka wg, hanks d, et al: improved control of trichomoniasis with trichomonas foetus vaccine. agri pract 1993;14:29-34. 4. goodger wj, skirrow sz: epidemiologic and economic analysis of an unusually long epizootic of trichomoniasis in a large california dairy herd. j am vet med assoc 1986;189:772-776. 5. barling ks, field rw, snowden kf, et al: acute trichomoniasis and sub-optimal fertility in a cow/calf herd: an investigation and case management. bov pract 2005;39:1-5. 6. mcgrory t, meysick k, lemchuk-favel lt, et al: the interaction of lactobacillus acidophilus and trichomonas viaginalis in vitro. j parasitol 1994;80:50-54. 7. martin dh, zozaya m, lillis ra, et al: unique vaginal microbiota that includes an unknown mycoplasma-like organism is associated with trichomonas vaginalis infection. j infect dis 2013;207:1922-1931. 8. brotman rm, bradford ll, conrad m, et al: association between trichomonas vaginalis and vaginal bacterial community composition among reproductive-age women. sex transm dis 2012;39:807-812. 9. brotman rm, klebanoff ma, nansel tr, et al: bacterial vaginosis assessed by gram stain and diminished colonization resistance to incident gonococcal, chlamydial, and trichomonal genital infection. j infect dis 2010;202:1907-1915. 10. martin hl, richardson ba, nyange pm, et al: vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type i and sexually transmitted disease acquisition. j infect dis 1999;180:1863-1868. 11. laguardia-nascimento m, branco kmgr, gasparini mr, et al: vaginal microbiome characterization of nellore cattle using metagenomics analysis. plos one 2015;10:11. 12. clemmons ba, reese st, dantas fg, et al: vaginal and uterine bacterial communities in postpartum lactating cows. front microbiol 2017;8:1047. 13. bicalho mls, santin t, rodrigues mx, et al: dynamics of the microbiota found in the vaginas of dairy cows during the transition period: associations with uterine diseases and reproductive outcome. j dairy sci 2017;100:3043-3058. 14. shpigel ny, adler-ashkenazy l, scheinin s, et al: characterization and identification of microbial communities in bovine necrotic vulvovaginitis. vet j 2017;219:34-39. 15. gonzalez moreno c, fontata c, cocconcelli ps, et al: vaginal microbial communities from synchronized heifers and cows with reproductive disorders. j app microbiol 2016;121:1232-1241. 16. garcia guerra a, chaban b, hill je, et al: clinical sensitivity and specificity of a real-time pcr assay for campylobacter fetus subsp venerealis in preputial samples from bulls. am j vet res 2014;75:851-860. clinical theriogenology 2021; 13: 237 management and control of bovine trichomoniasis introduction assisted reproductive techniques aim to disseminate the genetics of an animal by maximizing production of live offspring compared to natural breeding. for that to occur, sperm survival, fertilization, implantation of the embryo, and delivery of a live offspring should be controlled to prevent losses. artificial insemination (ai) allows the ejaculate to be evaluated, to use a minimum number of progressive motile sperm, and to breed at an optimal time frame to maximize fertilization. additionally, ai allows the use of several breeding doses from an ejaculate, maximizing stallion use. appropriate nutrients and/or stimulatory proteins in association with antibiotics are used to preserve sperm during shipment and/or storage. breeding success is influenced by semen quality and the mare’s immunologic response to semen. therefore, compounds that improve semen quality and reduce post mating endometritis, are of great value. sperm motility and viability can be improved by semen extenders. uterine inflammatory response can be reduced by decreasing contamination and supporting uterine clearance by using uterine lavage, ecbolic agents, and antiinflammatory compounds. new techniques to improve semen quality in conjunction with the control of post breeding uterine inflammation would maximize sperm survival in the female reproductive tract and provide an optimal uterine environment for the embryo to establish pregnancy. recently, platelet rich plasma (prp) was used to reduce effect of platelet rich plasma lysate and fibroblast growth factor 2 on stallion sperm motility fabio pinaffi,a,b robyn wilborn,a lindsey boone,a aime johnsona a department of clinical sciences, college of veterinary medicine, auburn university, auburn, al bdepartment of veterinary clinical sciences, college of veterinary medicine, oklahoma state university, stillwater, ok abstract growth factors (gfs) are known to modulate cell function and their presence in semen could be advantageous to sperm. in humans and mice, fibroblast growth factor 2 (fgf2) improved semen motility. platelet rich plasma (prp), rich in gfs including fgf2, reduced post mating inflammatory response within the uterus; however, its effects on sperm are not known. prp lysate (prpl) is much purer than prp and contains higher concentrations of gfs. hence, the objective of the present study was to evaluate the effect of supplementing fresh and cool-stored stallion sperm with either prpl at 1, 2.5, 5, and 10% (also containing 1 iu/ml of heparin), or fgf2 at 0.1, 1, 10, and 100 ng/ml. motility parameters were evaluated using computer assisted semen analysis at 0, 0.5, 1, 1.5, 6, and 24 hours after treatment. for both prpl and fgf2 treatments, there were no differences in total and progressive motility among groups. concentrations of prpl > 5% induced sperm agglutination via head-to-head attachment, starting at hour 1 and was more pronounced for 10% prpl than 5% prpl, suggesting a dose-dependent characteristic. direct addition of prpl to semen extender at doses < 5% might not substantially affect sperm motility, whereas doses > 5% might affect sperm motility due to head-to-head attachments. addition of fgf2 at the concentrations studied may not affect sperm characteristics. keywords: stallion semen, platelet-rich plasma lysate, fibroblast growth factor 2, sperm motility inflammation in mares susceptible to persistent mating-induced endometritis (pmie)1 and in mares with chronic degenerative endometritis (cde).2 mares are defined as susceptible to pmie when they are unable to overcome the inflammatory process within 12 24 hours after breeding3 and cde is a degenerative process in the endometrium characterized by fluid retention and a reduced ability to clear inflammation. in pmie susceptible mares, uterine infusion of prp 24 hours prior to ai substantially reduced intrauterine inflammatory cells and when infused either 24 hours prior to or 4 hours after ai, lowered cox-2 labeling.1 additionally, prp treatments increased pregnancy rates in pmie susceptible mares (30% without prp infusion versus 60% with prp infusion). in cde mares, prp treatment (4 hours after ai) decreased intrauterine fluid accumulation and decreased neutrophil numbers (24 hours after ai) in the uterine lumen; however, it did not decrease nitric oxide release.2 prp treatment before ai had a more pronounced reduction in inflammatory cell influx within the uterus,1 suggesting better efficacy in controlling inflammation since it acts prior to the inflammatory insult. however, direct effects of prp on sperm characteristics remain unknown. platelet rich plasma contains more platelets compared to whole blood.4,5 platelets are a natural reservoir of multiple proteins, cytokines, growth factors, adhesive proteins, clotting factors, fibrinolytic factors, proteases and antiproteases, and clinical theriogenology 2021; 13: 89 membrane glycoproteins.6,7 platelet lysate (pl) is an acellular serum substitute obtained after lysis of platelet concentrates and is comprised of growth and chemotactic factors that promote cell proliferation and recruitment.8 the higher purity of pl and acellularity imply higher gf concentrations compared to prp.9 the acellularity characteristic of pl is suggested to have a low potential for immune reaction when used autologously; however, immune reactions caused by heterologous versus autologous pl have not yet been characterized. additionally, studies comparing pl with prp regarding their growth factor contents and their stimulatory potential on cell function are lacking. platelet pheresis in horses10 resulted in 1-2.3 fold (1.8fold average) higher concentration of platelets in prp lysate than whole-blood platelet count. furthermore, platelet lysates can be stored at -20°c up to 5 months and still maintain growth factor content stability.11 fibroblast growth factor (fgf2), one of the key growth factors in prp,12 improved characteristics of human and mouse sperm13-15 and increased percent motile sperm with higher kinematics after incubation with recombinant human fgf2.14,15 the rhfgf2 concentration (0.1-1 ng/ml) used was similar to reported doses used with human prp (0.15 0.30 ng/ml).12 the concentration of fgf2 in human ejaculates was positively correlated with total sperm number and motility. ejaculates exposed to exogenous fgf2 prior to sperm recovery (swim up technique) increased recovery of motile sperm.13 objectives of this study were to determine if equine prpl or fgf2 treatments would enhance sperm motility (sperm motion characteristics) and improve sperm survival in uterus by reducing semen-induced uterine inflammation. material and methods animals number of animals used was based on a power analysis of 80% and by applying preliminary data from humans,13,14 wherein sperm motility increased from 42 ± 4 to 55 ± 4% after treatment with 100 ng/ml of rcfgf2 in 5 subjects. selection of mature stallions was based on breeding soundness examination, presence of a normal external and internal reproductive tracts via visual examination and transrectal palpation, and fertility based on previous breeding season performance when a minimum of 30% total motility and > 1 x 109 progressively motile sperm were used. all procedures were performed in accordance with the institutional animal care and use committee at auburn university. semen collection two separate ejaculates were collected from each stallion (n = 7). collection was performed using a missouri style artificial vagina with stallion exposed to an estrus or ovariectomized mare and mounting a breeding phantom. semen analysis was performed by the same person within 30 minutes after collection. after first analysis and immediate dilution with semen extender, semen samples were maintained at 37°c until assigned to a treatment group (< 45 minutes from collection) platelet rich plasma lysate. the prpl was prepared as described.10 briefly, platelet concentrates (from 5 donor horses) were obtained following plateletpheresis (cobe spectra dual-needle). platelets were fractured using 2 freeze-thaw cycles followed by 3 centrifugation cycles. the prpl was then filtered through a 40-μm cellulose acetate membrane (emd millipore, billerica, ma) to remove cellular debris. an equal portion of lysate from each horse was combined to obtain a pooled product. samples of prpl (aliquots of 1 ml) were stored in a freezer at -80°c for further use. recombinant fibroblast growth factor 2 equine rcfgf2 was produced by bacterial expression (r&d systems, minneapolis, mn). as recommended by the manufacturer, fgf2 was reconstituted to a maximum concentration of 250 μg/ml in water containing at least 0.1% bovine serum albumin. after reconstitution, diluted fgf2 was added to the ejaculate to achieve the desired concentrations of 0, 0.1, 1, 10, and 100 ng/ml. treatment doses were formulated based on recombinant fgf2 treatments of human and mouse semen.14,15 reconstituted fgf2 was stored under sterile conditions between -20 to -70°c and used within 3 months (expiration date). semen sample preparation sperm concentration was measured using a nucleocounter® sp-100™ (chemometec, allerod, denmark). a volume containing 600 x 106 sperm was placed in 50 ml conical tubes, diluted 1 part semen to 4 parts of standard native phosphocaseinate-based extender (inra 96®) and centrifuged at 900 x g for 10 minutes. the supernatant was removed and the sperm pellet re-suspended to a final concentration of 50 x 106 sperm per ml (confirmed via nucleocounter). the prpl treatment groups consisted of the following: extended semen (ctrl), extended semen with 1 iu/ml of heparin (ctrl hep), extended semen with 1 iu/ml of heparin and 1% prpl (1% prpl, extended semen with 1 iu/ml of heparin and 2.5% prpl (2.5% prpl), extended semen with 1 iu/ml of heparin and 5% prpl (5% prpl), and extended semen with 1 iu/ ml of heparin and 10% prpl (10% prpl). the fgf2 groups consisted of the following: extended semen (ctrl), extended semen with 0.1 ng/ml of fgf2 (fgf2-0.1), extended semen with 1 ng/ml of fgf2 (fgf2-1), extended semen with 10 ng/ ml of fgf2 (fgf2-10), and extended semen with 100 ng/ml of fgf2 (fgf2-100). clinical theriogenology 2021; 13: 90 semen analysis semen analysis was performed at 0, 0.5, 1, 1.5, 6, and 24 hours. samples were cooled to 5°c and stored in a standard semen shipping container after hour 1 analysis. motion characteristics of sperm were assessed objectively after warming using a computer assisted sperm analysis (sperm vision® sar, casa software with pc monitor, minitube, verona, wi) with light threshold between 180 255, field-of-view depth of 20 μm, pixel ratio of 16 100 μm,2 and assessment requirements of 5000 sperm or 7 fields. warmed aliquots of each treatment were placed in a 20 μm standard count analysis chamber (sc20.01. fa; leja®, nieuw-vennep, the netherlands). parameters selected for analysis14 were total (tmot %) and progressive (pmot %) motility, average path (vap μm/s), straight-line (vsl μm/s) and curvilinear (vcl μm/s) velocities, amplitude of lateral head displacement (alh μm); beat/cross frequency (bcf hz); straightness (str %), and linearity (lin %). additional measurements were performed including the total number of cells counted (totcell number), distance curved line (dcl μm), distance average path (dap μm), distance straight line (dsl μm), wobbling (wob, vap/vcl), slow motility (slowmot %), nonprogressive motility (npmot %), average orientation change (aoc degree), and concentration (conc cells/ml). sperm morphology was subjectively assessed in the initial ejaculate by evaluating 100 sperm stained with eosin/nigrosin stain. the following abnormalities were assessed: abnormal head shape, abnormal midpiece, detached head, proximal droplet, distal droplet, bent tail, coiled tail, and premature germ cells.16 data analyses data were analyzed separately for each experiment (prpl and fgf2). differences among groups, time, and interaction between groups and time were tested. data were examined for normality using the shapiro-wilk test and if data were not normally distributed, they were transformed into natural logarithm or rank prior to each analysis. the sas mixed procedure (version 9.4; sas institute, cary, nc) was used with a repeated statement to account for correlation between sequential measurements and differences were detected by tukey’s test. a probability of p ≤ 0.05 indicated that the difference was significant. results heparin effect on semen to monitor effects of heparin on prpl-treated samples, one heparin-only treated group was compared to a nontreated control. there was no effect of group, time, or interaction between group and time, and between heparin treated and control groups for all motility variables. platelet rich plasma lysate for totcells and conc, there were differences among groups, time, and interaction between group and time. both variables had a decrease starting at hour 1 in groups 5 and 10% prpl. values for groups 5 and 10% prpl remained lower than all other groups starting at hour 1.5, and values for group 10% prpl were lower than group 5% prpl starting at hour 1.5. in both groups, head-to-head attachment (hha) was observed on the screen; however, it was not possible to objectively quantify. significant differences over time were noted for tmot, pmot, dcl, dap, vcl, vap, alh, aoc, dsl, vsl, bcf, str, wob, lin, and slowmot; however, there were no differences among groups nor interaction between groups and time. therefore, groups were combined for each variable to evaluate changes over time. tmot and pmot gradually decreased from 0 1.5 hours, followed by a plateau in tmot and pmot after hour 1.5 (figure 1). distance straight line (dsl) and vsl had a gradual decrease from hours 0 1, a plateau from hours 1 1.5, and a gradual decrease until hour 24. bcf, str, wob, lin, and figure 1. total motility (tmot), progressive motility (pmot), and concentration (conc) at 0, 0.5, 1, 1.5, 6, and 24 hours in the control group and with 0, 1, 2.5, 5, and 10% of prpl in 1 iu/ml of heparin. significant time changes in direction (increase or decrease) for groups combined are represented by a circle and the first significant change on direction for time in the groups combined is indicated by an asterisk (*). group differences within a time point are represented by lowercase letters (a, b, and c). groups with statistically similar values at a specific time point are grouped by a square and represented by a lowercase letter (a, b, and c). clinical theriogenology 2021; 13: 91 slowmot had an increase from hour 0 0.5, a decrease from hours 0.5 and 1, a plateau from hours 1 1.5, and a gradual decrease until hour 24. fibroblast growth factor 2 there were no differences between prpl and fgf2, and no effect of group or interaction between groups and time for all fgf2 treated groups. however, a significant effect of time was noticed for all variables, except for totcells and conc that had constant values throughout the experiment. therefore, all groups were combined and changes among times had a similar pattern to prpl experiment. the variables tmot and pmot had a gradual decrease from hours 0 6 followed by motility maintenance until hour 24 (figure 2). the variables dcl, dap, vcl, vap, and alh decreased from hours 0 1, and aoc decreased from hours 0 6. there was an increase was on dcl, vcl, and alh starting at hour 0.5, and with dap, vap, and aoc starting at hour 6. the variables dsl and vsl had a gradual decrease starting at hour 0 until hour 24. a decrease was present with bcf, str, and lin, starting at hour 0.5 and on wob and slowmot starting at hours 1 and 1.5, respectively. there were no significant effects of group, time or interaction between group and time between prpl or fgf2 treatments on any sperm motility variables. discussion treatment with prpl at concentrations > 5% resulted in sperm hha, identified on gross microscopic examination by decreased totcells and conc values (note: casa does not identify agglutinated cells as individual motile sperm). head to head agglutination could be considered an artifactual reaction to components of prpl (i.e. fibrin) rather than an important physiological event. it is possible that prpl contributed to slight amounts of gel formation entrapping sperm and resulting in hha. regardless of the doses of prpl, hha was observed. since the heparin dose remained constant in this study, further work may evaluate increasing amounts of heparin and observe the incidence of hha. use of serum in bull sperm resulted in hha in immotile sperm.17 several compounds (heparin, serum or follicular fluid in monkeys,18 bulls,19,20 and pigs21) induced hha. in contrast, hha was not due to heparin addition in our study. in rabbits, hha was detected (in utero) when motile sperm came in contact with uterine luminal secretions.22 furthermore, hha was induced by dibutyril-camp and due to dilution, washing, cold shock, extreme ph, or certain osmotic conditions.23,24 however, these conditions were controlled across groups in the current study. besides the hha after prpl treatment, and in contrast with reports in human and mouse,13,14,25 there was no effect on motility by either prpl or fgf2 treatments. however, these studies used various media and commercially available semen extenders (with no mention of components for patent purposes). it is reasonable to consider that factors present in semen extenders could potentially interfere or even mask effects of additional prpl and fgf2 on sperm motility improvement. future studies may determine the effect of a nonnative phosphocaseinate-based extender (could be beneficial for stallions that do not have an adequate sperm preservation with native phosphocaseinate-based extenders). however, the complete absence of extender would compromise the lifespan and viability of sperm during storage due to lack of energy source, buffer, and antibiotics. the hypothesis that prpl treatment is not deleterious for sperm survival was supported at concentrations < 10%. however, hha occurred in concentrations > 5%. sperm survival can be measured by the maintenance of sperm motility over a period that decreased at a similar rate in all groups. since there was no difference in sperm motility between treated versus control groups, it could be suggested that direct prpl addition to the figure 2. total motility (tmot), progressive motility (pmot), and concentration (conc) during 0, 0.5, 1, 1.5, 6, and 24 hours in the control group and after treatments with recombinant equine fgf2 at concentrations 0.1, 1, 10, and 100 ng/ml. significant time changes in direction (increase or decrease) for groups combined are represented by a circle. the first significant change on direction for time in the groups combined are indicated by an asterisk (*) clinical theriogenology 2021; 13: 92 ejaculate does not negatively affect sperm motility parameters. addition of heparin to sperm has been shown to induce sperm capacitation at higher doses than was used in this study.26 evaluation of sperm kinetic analysis showed no differences between groups in lateral/side to side head movement and tail beat frequency (indicating hyperactivity). hyperactivation can be an indicator of sperm capacitation.27 as in bulls,8 both hyperactivation and acrosome reaction during capacitation are processes that can be independently stimulated. although addition of prpl had no apparent benefit to sperm, it may be beneficial in modulating post-mating endometritis, since the dense alpha granules of platelets secrete a multitude of chemokines, cytokines, and bioactive molecules with regenerative capacity, rather than just growth factors.29 in pmie mares, there was a down-regulation of mrna expression in endometrial proinflammatory cytokines (il-1b, il-6, il-8, and inos)30 when autologous prp was infused 24 36 hours before ai. furthermore, fluid retention was reduced and pregnancy rates improved.31 however, there was no study that used prpl instead of prp to evaluate the overall volume and concentration of prpl necessary to provide uterine immunomodulation after breeding in mares. infusion of 20 ml of prp either 24 hours before or 4 hours after ai in pmie mares was successful in controlling the inflammatory response and increased pregnancy rates.2 however, use of prpl has to be tested in the uterus for its immunomodulatory effect and immune responses after autologous versus heterologous origin. the hypotheses that fgf2 added to semen extender enhances sperm motility was not supported, but the hypothesis that sperm motility would be maintained was supported. there were no significant changes in sperm motility after fgf2 treatment. sperm motility was maintained throughout the experiment and the decrease of motility over 24 hours was similar in all fgf2-treated groups. therefore, there was no beneficial or detrimental effect of fgf2 on sperm motility. this finding was contrary to human studies where recombinant human fgf2 treatment (incubation with 10 ng/ml of fgf2) increased both total number and motile sperm recovery and improved sperm vcl, vsl, and lin values.13 this suggests that the fgf2/fgfrs system is involved in motility regulation similar to results observed in mice.14,32 in the present experiment, there was no significant effect on sperm characteristics. there was also no effect of fgf2 at the concentrations tested that would encourage the clinical use of fgf2 for improvement of stallion sperm motility. one important difference from the present experiment and others performed in humans and mice is the media used for the analysis.13,14,32 in the present experiment, the extender used has a broad variety of factors that are beneficial to sperm. therefore, as suggested in the prpl experiment, the presence of the extender could interfere with fgf2 actions or even mask positive effects on sperm motility. additionally, sperm capacitation effect of fgf2 should be considered, as in the murine model (fgf2 treatment increased sperm motility, velocity, enhanced intracellular ca2+ concentrations, and acrosomal exocytosis).32 in conclusion, use of prpl or fgf2, at the concentrations studied, did not have apparent detrimental effects on sperm over 24 hours. doses of prpl < 10% did not interfere with sperm survival and prpl doses > 5% induced hha. the physiological relevance hha observed after treatment with prpl is uncertain and is more suggestive of an artifactual reaction between sperm and components of prpl and the semen extender. for further investigation, the use of raw semen would be preferred to test the effect of both compounds without extender interference. for sperm capacitation, the addition of prpl and fgf2 without the extender component should be tested with the molecular evaluation of either acrosomal exocytosis, ca2+ intracellular influx, and sperm hyperactivation. clinically, prpl could be added to extended semen prior to breeding, focusing on uterine inflammatory control. however, studies are needed to control hha and to characterize the immunomodulatory effect of prpl within the uterus. acknowledgements authors thank dr. john f. peroni (university of georgia) for prpl samples and marti mccoy for assistance in stallion management and handling. research supported by the theriogenology foundation and the department of clinical sciences (auburn university). conflict of interest no conflict of interest to declare. references 1. segabinazzi lg, friso am, correal sb, et al: uterine clinical findings, fertility rate, leucocyte migration, and cox-2 protein levels in the endometrial tissue of susceptible mares treated with platelet-rich plasma before and after ai. theriogenology 2017;104:120-126. 2. reghini mf, ramires neto c, segabinazzi lg, et al: inflammatory response in chronic degenerative endometritis mares treated with platelet-rich plasma. theriogenology 2016;86:516-522. 3. troedsson mht, loset k, alghamdi am, et al: interaction between equine semen and the endometrium: the inflammatory response to semen. anim reprod sci 2001;68:273-278. 4. marx re: platelet-rich plasma (prp): what is prp and what is not prp? implant dent 2001;10:225-228. 5. wasterlain as, braun hj, dragoo jl: contents and formulations of platelet-rich plasma. oper tech in orthop 2012;22:33-42. 6. santos s, sigurjonsson oe, custodio ca, et al: blood plasma derivatives for tissue engineering and regenerative medicine therapies. tissue eng part b rev 2018;24:454-462. 7. salamanna f, veronesi f, maglio m, et al: new and emerging strategies in platelet-rich plasma application in musculoskeletal regenerative procedures: general overview on still open questions and outlook. biomed res int 2015;2015:846045. 8. burnouf t, strunk d, koh mb, et al: human platelet lysate: replacing fetal bovine serum as a gold standard for human cell propagation? biomaterials 2016;76:371-387. clinical theriogenology 2021; 13: 93 9. klatte-schulz f, schmidt t, uckert m, et al: comparative analysis of different platelet lysates and platelet rich preparations to stimulate tendon cell biology: an in vitro study. int j mol sci 2018;19:1-18. 10. sumner sm, naskou mc, thoresen m, et al: platelet lysate obtained via plateletpheresis performed in standing and awake equine donors. transfusion 2017;57:1755-1762. 11. rauch c, feifel e, amann e, et al: alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media. altex 2011;28:305-316. 12. xiong g, lingampalli n, koltsov jcb, et al: men and women differ in the biochemical composition of platelet-rich plasma. am j sports med 2018;46:409-419. 13. garbarino azua dj, saucedo l, giordana s, et al: fibroblast growth factor 2 (fgf2) is present in human spermatozoa and is related with sperm motility. the use of recombinant fgf2 to improve motile sperm recovery. andrology 2017;5:990-998. 14. saucedo l, buffa gn, rosso m, et al: fibroblast growth factor receptors (fgfrs) in human sperm: expression, functionality and involvement in motility regulation. plos one 2015;10:e0127297. 15. saucedo l, rumpel r, sobarzo c, et al: deficiency of fibroblast growth factor 2 (fgf-2) leads to abnormal spermatogenesis and altered sperm physiology. j cell physiol 2018;233:9640-9651. 16. card c: cellular associations and the differential spermiogram: making sense of stallion spermatozoal morphology. theriogenology 2005;64:558-567. 17. brown dv, senger pl: influence of homologous blood serum and motility and head-to-head agglutination in nonmotile ejaculated bovine spermatozoa. biol reprod 1980;23:271-275. 18. boatman de, bavister bd: stimulation of rhesus monkey sperm capacitation by cyclic nucleotide mediators. reprod fertil 1984;71:357-366. 19. ehrenwald e, foote rh, parks je: bovine oviductal fluid components and their potential role in sperm cholesterol efflux. mol reprod devel 1990;25:195-204. 20. lefebvre r, suarez ss: effect of capacitation on bull sperm binding to homologous oviductal epithelium. biol reprod 1996;54:575-582. 21. funahashi h, day bn: effects of follicular fluid at fertilization in vitro on sperm penetration in pig oocytes. j reprod fertil 1993;99:97-103. 22. brown dv, senger pl: influence of incubation in utero on motility and head-to-head agglutination of ejaculated rabbit spermatozoa. j reprod fertil 1982;66:283-289. 23. leahy t, rickard jp, aitken rj, et al: penicillamine prevents ram sperm agglutination in media that support capacitation. reproduction 2016;151:167-177. 24. mann t: biochemistry of stallion semen. j reprod fertil 1975:47-52. 25. bader r, ibrahim jn, moussa m, et al: in vitro effect of autologous platelet-rich plasma on h2o2 -induced oxidative stress in human spermatozoa. andrology 2020;8:191-200. 26. varner dd, bowen ja, johnson l: effect of heparin on capacitation/ acrosome reaction of equine sperm. arch androl 1993;31:199-207. 27. loux sc, crawford kr, ing nh, et al: catsper and the relationship of hyperactivated motility to intracellular calcium and ph kinetics in equine sperm. biol reprod 2013;89:1-15. 28. marquez b, suarez ss: different signaling pathways in bovine sperm regulate capacitation and hyperactivation. biol reprod 2004;70:1626-1633. 29. maynard dm, heijnen hfg, horne mk, et al: proteomic analysis of platelet α-granules using mass spectrometry.j thromb haemost 2007;5:1945-1955. 30. woodward em, metcalf es, scoggin cf, et al: the effect of immune modulators on endometrial cytokine expression in mares susceptible to persistent breeding induced endometritis. clinical theriogenology 2013;5:364. 31. metcalf es, scoggin k, troedsson mht: the effect of platelet-rich plasma on endometrial pro-inflammatory cytokines in susceptible mares following semen deposition. j eq vet sci 2012;32:498. 32. saucedo l, sobarzo c, brukman ng, et al: involvement of fibroblast growth factor 2 (fgf2) and its receptors in the regulation of mouse sperm physiology. reproduction 2018;156:163-172 clinical theriogenology 2021; 13: 94 ablation of endometrial cysts using endometrial biopsy forceps ablation of endometrial cysts using endometrial biopsy forceps betty radny, tulio prado, jessica klabnik-bradford department of veterinary clinical sciences college of veterinary medicine, university of tennessee, knoxville, tn abstract eight mares varying in breed and age were presented for breeding soundness examination and infertility evaluation. transrectal ultrasonographic examination of uterus revealed the presence of 1 or more endometrial cysts. cysts were ablated by removing a portion of cyst wall using endometrial biopsy forceps. all mares became pregnant following artificial insemination performed 1 or 2 estrous cycles following ablation. keywords: mare, endometrium, cyst, pregnancy, biopsy forceps background pregnancy failure can be due to stallion and management factors, failure of conception, early embryonic death, and failure of maternal recognition of pregnancy due to an endometrial cyst(s). endometrial cysts can induce pregnancy loss by interfering with embryo mobility.1,2 cysts can be ablated by surgical excision, hysteroscopic biopsy forceps, snare, laser (diode, co2, or nd/yag laser), electrocautery or by injection of 70% ethanol (ethanol sclerotherapy technique3). most methods are expensive and can be uneconomical for many mare owners. use of endometrial biopsy forceps as a manual ablation method has apparently not been reported. benefits are low cost, minimal sedation, mild endometrial scarring and improved fertility. case presentation history eight mares varying in breed and age (6 22 years) were examined for infertility during 2017 2019 breeding seasons. mares failed to establish pregnancy despite multiple artificial inseminations with fertile stallion semen. a detailed breeding soundness examination was performed (table 1). table 1. breeding soundness examination findings # breed year examined age uterine cytology findings culture result kenney scale biospy grade 1 arab 2017 22 na na iia 2 aqh 2017 17 within normal limits na na 3 stb 2017 6 within normal limits no growth na 4 pony 2018 12 within normal limits na na 5 appaloosa 2018 14 na na na 6 aqh 2018 13 within normal limits no growth iib 7 aqh 2018 13 lymphocytic endometritis no growth iib 8 stb 2019 22 na na na mares had either a large single or multiple endometrial cyst(s) located in the uterine lumen ranging from 15 55 mm in diameter (figure 1). presence of endometrial cysts was suspected to be the cause of infertility in all 8 mares, possibly due to interference in embryo mobility resulting in early pregnancy loss.1,2 clinical theriogenology • volume 12 number 1 • march 202047 figure 1. examples of ultrasonographic images of endometrial cysts treatment mares were sedated with 0.4 ml of detomidine hydrochloride (dormosedan, zoetis inc., kalamazoo, mi) given intravenously. location of cyst(s) within the uterus was identified by transrectal ultrasonographic examination. tail was bandaged with rolled gauze, elevated, and secured to the dorsal cross-bar of the stock. feces were removed manually from the rectum and the perineum scrubbed with povidone-iodine soap and rinsed with water. a 30-cm long endometrial biopsy forceps (equine alligator forceps, meditools.com.au) was inserted into vagina and cervix was dilated digitally to introduce forceps into uterine lumen. cyst was stabilized transrectally between index and middle finger, as the forceps were pressed against the cyst. a 5-mm portion of cyst wall (figure 2) was removed. if multiple cysts were present, procedure was repeated until a small portion of each cyst was removed. transrectal ultraonography confirmed complete deflation of cyst(s). figure 2. portion of a wall of a cyst removed by endometrial biopsy forceps after cyst ablation, the uterus was infused with 2 g of ampicillin (ampicillin sodium, g.c. hanford manufacturing co., syracuse, ny). ovulation was induced in mares that were in estrus at the time of cyst ablation with 1.8 mg deslorelin acetate given intramuscularly (sucromatetm, thorn bioscience llc, louisville, ky) when the follicle reached > 35 mm, as determined by transcrectal ultrasonographic examination. mares were bred with chilled semen either at 24 hours after ovulation induction or at next estrus (table 2). mares (regardless of estrous cycle stage) were treated with 20 units of oxytocin (bimeda mtc animal health inc. cambridge, on, canada) 4 6 hours after post-ablation ampicillin treatment. mares in estrus during ablation were given a second oxytocin treatment 8 hours following insemination with chilled semen, to prevent fluid accumulation in uterus. clinical theriogenology • volume 12 number 1 • march 2020 48 table 2: endometrial cysts’ sizes, breeding information, and ablation outcomes # breed year examined estrous cycle stage1 endometrial cysts size in mm interval between ablation and breeding mrop2 1 arab 2017 estrus 20 22 days yes 2 aqh 2017 estrus 16 22 days yes 3 stb 2017 diestrus 55 1 year later yes 4 pony 2018 estrus 15 5 days yes 5 appaloosa 2018 estrus 20 1 day yes 6 aqh 2018 estrus 26, 16 2 days yes 7 aqh 2018 estrus 18 40 days3 yes 8 stb 2019 estrus 30 x 3 1 day yes 1stage of estrous cycle (based on transrectal ultrasonographic examination of ovaries) at ablation 2designates if mare became pregnant and carried embryo past maternal recognition of pregnancy (mrop) 3mare #7 failed to ovulate at first post-ablation estrus (~ 20 days after ablation) three mares were bred at first estrus following ablation, became pregnant and maintained pregnancy past maternal recognition of pregnancy (mrop). four mares were bred at concurrent estrus following ablation and were able to maintain pregnancy past mrop. one mare had the largest cyst (figure 3) that completely blocked the left uterine horn. this mare was in diestrus with no signs of edema, making it difficult to dilate cervix. cervix was dilated by applying 400 µg of misoprostol (greenstone llc, peapack, nj). misoprostol was crushed and mixed in sterile lubricant and applied to cervix. cervix was digitally dilated over 2 3 hours after misoprostol application. following cyst ablation, transrectal ultrasonographic examination was performed to confirm ablation (figure 4). mare was bred artificially at the following season after confirming cyst absence (figure 5) and became pregnant. figure 5. ultrasonogram of left uterine horn of a mare 1 year after cyst ablation figure 3. ultrasonogram of left uterine horn of a mare; note the 55-mm cyst that completely blocked the lumen figure 4. ultrasonogram of left uterine horn of a mare following cyst ablation clinical theriogenology • volume 12 number 1 • march 202049 outcome all 8 mares became pregnant, despite varying intervals between ablation and insemination. discussion endometrial cysts are often observed in mares > 10 years of age. mares with 1 or more cysts of < 2 5 mm in diameter can become pregnant; however, 1 large or multiple small cysts may interfere with embryo mobility, a critical component of maternal recognition of pregnancy (mrop).4,5 cysts located at the base of uterine horns may be especially problematic.6 an endometrial cyst may mimic an embryonic vesicle during ultrasonographic or transrectal examination of uterus.4,7 uterine mapping prior to breeding is often included in the medical record to help distinguish between embryo and cyst. endometrial cysts typically arise from lymphatic lacunae8-11 and can range from microscopic to centimeters in diameter. as the cyst increases in size, it protrudes further into the uterine lumen and is more likely to interfere with mrop.1 regardless of mare’s age, if embryo attaches adjacent to an endometrial cyst, the cyst can act as a space occupying mass, altering embryonic vesicle configuration. this may interfere with development of embryonic vesicle and may lead to embryo loss. an embryo that attaches directly adjacent to an endometrial cyst may suffer deprivation of nutrients, leading to its death.11 cyst can be excised surgically or ablated transendoscopically using endoscopic biopsy forceps or snare, a laser (diode, co2, or nd/yag laser) or by electrocautery.11-13 however, cyst removal with these techniques can be financially difficult for some owners. endometrial biopsy forceps introduced blindly into uterus allows clinician to perform transrectal ablation of cyst in field conditions. this process is financially more feasible than traditional techniques. removing a section of each cyst wall has prevented cyst from refilling and is assumed to create minimal scaring. it is possible that there may be less endometrial scarring, facilitating embryo attachment. given the simple and quick operation of the procedure, endometrial cyst ablation with endometrial biopsy forceps followed by transrectal ultrasonographic conformation is feasible both in hospital and ambulatory settings. learning points • endometrial cysts can be mistaken for an embryo during pregnancy diagnosis. • endometrial cysts can interfere with mobility of embryo and cause its death. • endometrial cysts can be ablated with endometrial biopsy forceps. references 1. ginther oj, pierson ra: ultrasonic anatomy and pathology of the equine uterus. theriogenology 1984;21:505-516. 2. brook d, frankel k: electrocoagulative removal of endometrial cysts in the mare. equine vet sci 1987;7:77-81. 3. carluccio a, gloria a, mariotti f, et al: ethanol sclerotherapy for the treatment of uterine cysts in the mare. j equine vet sci 2018;63;27-29. 4. stout tae: the early pregnancy. in: breeding management and artificial insemination. 2nd edition. st. louis: elsevier saunders; 2009. p. 223-239. 5. eilts be, scholl dt, paccamonti dl, et al: prevalence of endometrial cysts and their effect on fertility. biol reprod 1995;5:527-532. 6. pycock jf: breeding management of the problem mare. in: breeding management and artificial insemination. 2nd edition. st. louis: elsevier saunders; 2009. p. 141-142. 7. leblanc mm, lopate c, knottenbelt dc, et al: the mare. in: equine stud farm medicine and surgery. 1st edition. london: elsevier saunders; 2003. p. 203-204. 8. ferreira jc, gastal el, ginther oj: uterine blood flow and perfusion in mares with uterine cysts: effect of the size of the cystic area and age. reproduction 2008;135:541-550. 9. stanton mb, steiner jv, pugh dg: endometrial cysts in the mare: j equine vet sci 2004;24:14-19. 10. tannus rj, thun r: influence of endometrial cysts on conception rate of mares. j vet med assoc 1995;42:275-283. 11. brisko sp, blanchard tl, varner dd, et al: pregnancy loss: in: manual of equine reproduction. 3rd edition. maryland heights: elsevier mosby; 2011. p. 94-113. 12. pinto c, frazer gs: reproduction. in: equine medicine, surgery and reproduction. 2nd edition, philadelphia: elsevier saunders; 2013. p. 290. 13. ley wb, higbee rg, holyoak gr: laser ablation of endometrial and lymphatic cysts. clin tech equine pract 2002;1:28-31. clinical theriogenology • volume 12 number 1 • march 2020 50 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (adobe rgb \0501998\051) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype true /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize 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/monoimagedownsampletype /bicubic /monoimageresolution 1200 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects false /checkcompliance [ /none ] /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile (none) /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname () /pdfxtrapped /false /createjdffile false /description << /enu ([based on 'hi-res'] use these settings to create high quality adobe pdf documents suitable for a delightful viewing experience and printing of business documents. created pdf documents can be opened with acrobat and adobe reader 7.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads 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/convertcolors /converttocmyk /destinationprofilename (u.s. web coated \(swop\) v2) /destinationprofileselector /usename /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 0.864000 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice 2019 vaginal microbiome: what not to do vaginal microbiome: what not to do candace lyman oklahoma state university abstract little advancement has been made over the years in defining normal canine reproductive tract microbiota. existing survey data were obtained using the practical, but flawed, culture-based technique. many of our clinical decisions pertaining to canine vagina are impacted by vaginal culture swab results; however, based on literature, culture results significantly underestimate bacterial diversity and concurrently overestimate role of isolated bacteria as they are more “visible.” as clinicians practicing in “omics era” we must be prepared to interpret data from 16s rdna gene sequencing for bacterial identification. with recently published information paving the path for characterizing a resident microbiome in the canine reproductive tract, we can begin to integrate this information into clinical practices, particularly those that pertain to ever-frustrating cases of canine vaginitis. with this information, our clinical practices should aim to identify an accurate cause and to do no (further) harm to the canine vaginal microbiome. keywords: microbiome, 16s rdna, vaginitis, vaginal culture introduction based on various veterinary resources, it is clear that the vagina of the canine is far from sterile. however, general consensus, with a few outliers, tend to accept the paradigm that the uterus is a sterile environment and needs to remain as such in order to establish and sustain a successful pregnancy. owners of breeding bitches and stud dogs frequently take this even one step further in presuming that both the uterus and vagina of a fertile bitch is sterile and, in efforts to assess prospective fertility of a breeding bitch, will request that vaginal cultures be obtained by the veterinarian providing cycle management services for the bitch. this then subjects the veterinarian to the responsibility of interpreting the results from vaginal culture swabs. what bacterial species are “acceptable” to the practitioner when assessing relevance to a positive culture result? where is the literature to support “lack of pathology” in the presence of specific species of bacteria? how is the practitioner to determine if culture results are indicative of dysbiosis present in the bitch’s reproductive tract or not? the “sterile uterine environment” concept was recently challenged by the observation that human placenta harbors a diverse microbiome of viruses, bacteria and fungi.1-2 therefore, if a placenta containing such organisms can result in delivery of a healthy infant, then should the presence of these organisms not be considered normal to the uterus? this is not a new area to be investigated in theriogenology studies reporting bacterial species identified in the canine uterus and vagina were published 40 years ago.3 still, little advancement has been made and minimal information exists that attempts to define “normal canine reproductive tract microbiota” because nearly all published data were obtained using practical, but flawed, culture-based techniques. unfortunately, these techniques fail to detect > 90% of microflora present in the sampled environment.4 in essence, culture vastly underestimates bacterial diversity present in any area of the body, while at the same time likely overestimates the role of the bacteria cultured simply because they are more “visible.” following content is intended to be useful in assisting clinicians as they navigate through the changing world of microbiomics as it relates to the canine reproductive tract, and more specifically, the ever-accessible canine vagina. avoid subscribing to “sterile vagina” or “sterile uterus” concepts is there a normal microflora within a bitch’s reproductive tract? our group at oklahoma state university has proposed that results contained within our 2019 publication “canine endometrial and vaginal microbiomes reveal distinct and complex ecosystems” is a first step in describing normal flora within canine female reproductive tract. results obtained from vaginal swabs and endometrial tissue samples are reported by phyla and genera, as the silva database does not allow taxonomic classification at species level. top 5 genera identified in vagina included hydrotalea, ralstonia, mycoplasma, 615 clinical theriogenology • volume 11, number 4 • december 2019 fusobacterium and streptococcus. top 5 genera identified in endometrium included pseudomonas, staphylococcus, corynebacterium, anaplasma and dermacoccus. when comparing 2 different regions of the reproductive tract, vagina is higher in “richness” (number of bacteria present in a given area), but endometrium is higher in “diversity” (variety of organisms identified in a given area). what phyla or genera do bacteria commonly cultured from the vagina and endometrium represent? as reported in various literature sources, common bacteria including, but not limited to, enterococcus spp., streptococcus spp., escherichia spp., and staphylococcus spp. would all belong to one of the top 5 phyla represented in the canine reproductive tract.3,5-8 one large theme was apparent while comparing the 25 samples compared in our project and that theme was “individual diversity.” it seems that whereas there were phyla and genera most heavily represented within our population of bitches evaluated in our study, none of them closely resembled one another when comparing bacterial richness and diversity for bacteria present. obviously, commitment to further defining the canine reproductive microbiome needs to be maintained if information obtained thus far will have any chance of impacting clinical practice with development of effective probiotics or infusion treatment regimens. resist your startle reflex when viewing culture results or 16s rdna data by comparing previously compiled lists of “commonly isolated bacteria in culture” to our “top 5 phyla/genera reported via 16s rdna” from vaginal and endometrial samples, we realized that today’s emerging information failed to confirm culture-based results obtained from previous years; in addition, formerly obtained culture-based results are failing to support today’s discoveries.3,5-8 previously published results revealed that bacteria isolated with highest frequency from vaginal swabs from a group of bitches included enterococcus, streptococcus, pasteurella, and escherichia genera.8 however, if comparing those culture results to our 16s rdna data, none of those organisms were represented in top 5 genera most represented in canine vagina.9 so then, how do we interpret the significance, or even reliability, of either result? are bacteria isolated on petri dishes responsible for subfertility or infertility in the bitch or not? are bacteria truly representative of the environment from which the sample was taken, or were the presence of these bacterial species exaggerated after media enrichment? in order to fully answer these questions in a way that will impact clinical practice, we will need to be on the lookout for progress updates coming our way that includes data of samples from infertile or infected bitches as analyzed via metagenomic methods. however, in spite of the incongruency in the results obtained from 2 distinct diagnostic methods, it would appear that we do have enough information at our fingertips to troubleshoot some commonly encountered clinical scenarios. for example, if a bitch owner expresses anxiety over a mycoplasma spp. isolated from a routine vaginal swab, we can subdue their concerns in an evidence-based fashion rather than solely from a practice-based approach. alas, this would be a prime opportunity to communicate progress of information being shared at continuing education events today! resist utilizing antibiotics in the absence of careful case (i.e. vagina) selection vaginitis can be frustrating; key to accurately troubleshooting this feminine issue is to carefully define the cause for owner concern and pet presentation. the entirety of canine vagina (reaching up to cervix) is difficult to characterize in terms of appearance and characteristics in most clinical settings, due to equipment or cost limitations. we may become frustrated and, therefore, tempted to reach for anything that would be effective for treating a supposed infection; in other words, we feel at a loss and partake in a response to treatment approach. nevertheless, impact of antibiotic consumption reaches much farther than most of us consider with each prescription dispensed. many of us have worked with cases, not to mention owners, that have tried to force use to prescribe an antibiotic despite little justification based on diagnostic or clinical evidence. what is even more perplexing is when this reported approach appears to result in pregnancy when previous infertility was unexplained. what should a veterinarian do? there are significant incentives to exercise the sagacious use of antibiotics in our patients including, but not limited to, an antibiotic’s ability to disturb host metabolism and absorption of vitamins, alteration of susceptibility to 616clinical theriogenology • volume 11, number 4 • december 2019 host infections, and overgrowth of yeast and/or clostridium difficile.10 broad-spectrum antibiotics that primarily target anaerobic bacteria, for example clindamycin, have a known negative impact on intestinal microbiota which results in reduced host protection against gut colonization by bacterial pathogens. even use of amoxicillin can result in increased resistant enterobacteria.10 whereas some antibiotic regimens will only alter gut microbiota from baseline for a short interval (e.g. days or weeks), other regimens have been documented to alter gut microbiota in humans for up to 4 years.10,11 a prospective pilot study reported that of 54 women administered a short course of oral antibiotics for nonreproductive issues, treated patients had an increased prevalence of asymptomatic vaginal colonization of candida species compared to nontreated and age-matched controls (10 of 27 versus 3 of 27, respectively). similarly, an increase in incidence of symptomatic vulvovaginal candidiasis was also documented when treated patients were compared to nontreated patients (6 of 27 versus 0 of 27, respectively).12 although microbiomes of human and canine reproductive tracts cannot be described as similar, and therefore a predicted response to commonly utilized antibiotics should not be extrapolated from one species to the other, veterinarians and owners likely underestimate the impact of antibiotics on systemic microbiome; therefore, this impact should be consciously discussed, weighed, and clinical decisions managed accordingly. to improve case selection for antibiotic use in our daily practices, we can be cognizant of other causes of subfertility or infertility in the bitch and perform appropriate case work-ups following client education. other than a presumed bacterial vaginitis or endometritis, other causes of subfertility or infertility such as inappropriate timing, suboptimal semen quality, or even intrauterine fluid accumulation around the time of ovulation, should be evaluated.13,14 with increased accessibility of ultrasound equipment, perhaps more of us should begin performing more detailed examinations of the bitch’s reproductive tract as is done by our more equine-focused colleagues during estrous cycle management prior to breeding. avoid unwise douching practices once again, vaginitis can be frustrating. while it may be tempting to lavage (i.e. douche) vagina or vestibule with fluids spiked with small amounts of antiseptic, it will behoove clinicians to avoid this practice. however, in the case of a primary noninfectious vaginitis, with the intent of clearing tenacious mucoid secretions, physiologic saline solution alone may be safely utilized sparingly.15 in women, lactobacillus bacteria that inhabit vagina are thought to maintain an appropriate ph environment that makes it inhospitable for other bacterial species that can induce a symptomatic bacterial vaginitis to survive; in contrast, utilizing antiseptics or a high-frequency douching schedule will inarguably impact normal vaginal flora and make the patient more susceptible to a bacterial vaginosis by upsetting vaginal flora. although a step by step description regarding how to work up a canine vaginitis case is outside the scope of these proceedings, this should be a priority when presented with a case of suspected vaginitis. appropriate diagnostic procedures will prevent inappropriate treatment from being implemented and avert secondary problems from arising. conclusion more information pertaining to vaginal (and uterine) dysbiosis is forthcoming in veterinary species. be proactive in understanding and seeking information that, while currently in its infancy, will impact the way we approach cases in the clinic. in the meantime, resist practices that can weaken, or even obliterate, normal microbiome of the canine reproductive tract. when possible, rededicate yourself to pursuit of a diagnosis and be accurate in interpretation of results from diagnostic tests. 617 clinical theriogenology • volume 11, number 4 • december 2019 references 1. aagaard k, ma j, antony km, et al: the placenta harbors a unique microbiome. sci transl med 2014;6:237ra65. 2. wassenaar tm, panigrahi p: is a foetus developing in a sterile environment? lett appl microbiol 2014;59:572-9. 3. olson pns, mather ec: canine vaginal and uterine bacterial-flora. j am vet med assoc 1978;172:708-711. 4. theron j, cloete te: molecular techniques for determining microbial diversity and community structure in natural environments. crit rev microbiol 2000;26:37-57. 5. baba e, hata h, fukata t, et al: vaginal and uterine microflora of adult dogs. am j vet res 1983;44:606-609. 6. schultheiss pc, jones rl, kesel ml, et al: normal bacterial flora in canine and feline uteri. j vet diagn invest 1999;11:560-562. 7. maksimovic a, maksimovic z, filipovic s, et al: vaginal and uterine bacteria of healthy bitches during different stages of their reproductive cycle. vet rec 2012;171:375. doi: 10.1136/vr.100886 8. groppetti d, pecile a, barbero c, et al: vaginal bacterial flora and cytology in proestrous bitches: role on fertility. theriogenology 2012;77:1549-1556. 9. lyman, c, holyoak gr, meinkoth k, et al: canine endometrial and vaginal microbiomes reveal distinct and complex ecosystems. plos one 2019;14:e0210157. doi: 10.1371/journal.pone.0210157 ecollection 2019. 10. jernberg c, lofmark s, edlund c, et al: long-term impacts of antibiotic exposure on the human intestinal microbiota. microbiol 2010;156:3216-3223. 11. ferguson km, jacob me, theriot cm, et al: dosing regimen of enrofloxacin impacts intestinal pharmacokinetics and the fecal microbiota in steers. front microbiol 2018;19:2190. doi: 10.3389/fmicb.2018.02190 12. xu j, schwartz k, bartoces m, et al. effect of antibiotics on vulvovaginal candidiasis: a metronet study. j am board fam med 2008;21:261-268. 13. barstow c, wilborn r, johnson ak: breeding soundness examination of the bitch. vet clin north am small anim pract 2018;48:547-566 14. freeman sl, green mj, england gcw: prevalence and effect of uterine luminal free fluid on pregnancy and litter size in bitches. theriogenology 2013;80:73-76. 15. lopate c: reproductive disorders of the spayed bitch. clinical theriogenology 2014;6:205-218. 16. aslan e, bechelaghem n: to douche or not to douche; hygiene habits may have detrimental effects on vaginal microbiota. j obstet gynaecol 2018;38:678-681. 618clinical theriogenology • volume 11, number 4 • december 2019 untitled 2019 use of a new artificial vagina without a liner reduced sperm motility in a stallion use of a new artificial vagina without a liner reduced sperm motility in a stallion ana sanchez-rodriguez,a sabrina hall,a page mauk,b jessica klabnik-bradford,a carlos pinto,c shelby hayden,d brian whitlock,a tulio pradoa adepartment of large animal clinical sciences, university of tennessee, knoxville, tn bdon alberto corporation farm, lexington, ky, cdepartment of veterinary clinical sciences louisiana state university, baton rouge, la, dheartland equine hospital, tonganoxie, ks abstract a 24 year old tennessee walking horse stallion was presented at beginning of breeding season for a sudden decrease in sperm motility noticed 1 hour after semen was collected with a new artificial vagina. stallion was healthy and previous breeding soundness examinations indicated no signs of subfertility. collection procedure, processing temperature, semen extender addition, and seminal plasma exclusion were examined as possible causes. none identified the reason for reduced motility. when the same artificial vagina, with or without a liner, was used to collect semen from this stallion and 2 other stallions, sperm motility increased for all 3 stallions when a liner was used. therefore, the new artificial vagina was regarded as the cause of decreased sperm motility. keywords: semen, stallion, artificial vagina, sperm motility background decreased sperm motility in stallions has been attributed to disease, age,1 season,2 hot weather,3 or stress induced by physical activity.3 many other factors can contribute to a sudden decline in sperm motility, e.g. freezing or cooling procedures for preservation.3 we investigated the reason for an abrupt decrease in sperm motility in a heathy stallion that had normal sperm motility in the previous season. case presentation a 24 year old tennessee walking horse stallion with no signs of disease and with normal fertility parameters in the previous breeding season had a sudden decrease in sperm motility (table 1). semen was collected in a new artificial vagina (av) using an established (18 years) protocol. breeding soundness examination did not identify any anomalies related to decreased motility. sperm concentration, motility, and morphology were assessed immediately after collection. all parameters were within normal values (concentration: 150 190 x 106/ml, motility 50 70%, morphology 60 71% normal sperm); however, 1 hour after sperm collection, sperm were immotile. table 1. semen parameters in a stallion during 2012 and 2013 breeding seasons. date volume (ml) concentration (millions/ml) total sperm/ejaculate (billions) morphology (%) motility (%) 2/15/2012 60 280 16.8 55 82 2/17/2012 60 250 15 72 94 2/20/2012 50 273 13.5 74 82 2/22/2012 60 355 21.3 60 94 3/11/2013 75 190 14.2 60 70 3/13/2013 90 162 14.6 70 50 3/14/2013 50 173 8.7 60 50 3/15/2013 90 150 13.5 71 50 causes for a decrease in sperm motility were investigated. temperature was measured with an infrared thermometer (irt207, general) in every step of semen processing and no changes occurred that could be related to a decrease in motility. various semen extenders were used [(e-z mixing original formula (of), e-z mixing cool-store-transport (cst), and e-z freezing modified french (mfr5)] 607 clinical theriogenology • volume 11, number 4 • december 2019 with no improvement in sperm motility. semen was extended 1:1 with e-z freezing modified french (mfr5); then, seminal plasma was removed by centrifugation (600 x g for 6 minutes) and the pellet resuspended with extender to eliminate possible detrimental effects of seminal plasma on sperm viability.4 this procedure did not improve sperm motility. finally, it was decided to collect the stallion with and without a disposable plastic av liner to determine whether direct contact of semen with the av was deleterious. sperm motility was maintained when a liner was used, but a sharp decrease occurred when a liner was not used. to verify if these results were specific to this stallion or were repeatable in other stallions, the same av was tested, with and without a liner, in 2 other stallions (a 4 year old appaloosa and a 11 year old american paint horse). sperm from stallion 2 were immotile even at 0 hours and stallion 3 sperm were immotile at 1 hour (figure). presence of a spermicidal component in the newly purchased av was suspected. figure. sperm motility over time (from 0 to 60 minutes) of ejaculates from 3 stallions collected with and without a liner in the artificial vagina. differential diagnosis chronic motility reduction has been reported in both young (< 3 years) and old (> 14 years), stallions,1 during warmer seasons,2,3 or due to stress caused by physical activity.3 infectious diseases (e.g. equine arteritis virus disease)5 or testicular trauma6 can adversely affect sperm motility. iatrogenic spermicidal toxins or various handling methods can cause a sharp decrease in quality of semen parameters. based on differences in semen motility when collecting with versus without a plastic av liner, we inferred that the av had component(s) that adversely affected sperm motility. 608clinical theriogenology • volume 11, number 4 • december 2019 treatment samples were collected from 3 stallions using the same av previously described,7 both with and without a liner, and were evaluated for motility over time. the same extender was added to each sample following initial evaluation. after 1 hour, motility of each sample, collected using a liner was not affected. in samples collected without a liner, motility decreased to 0% within 1 hour following collection. when collected with a liner, semen motility was unaffected. therefore, we inferred that the newly purchased av caused decreased sperm motility. outcome when a liner was used in av, collected semen had expected sperm motility in all 3 stallions (table 2). after 24 and 48 hours of cooling, sperm were immotile in semen collected without a liner (table 2). table 2. sperm motility of cooled extend semen 0, 24, and 48 hours after collection in 3 stallions. stallion 1 stallion 2 stallion 3 time (hours) liner no liner liner no liner liner no liner 0 85 40 70 0 70 50 24 70 0 45 0 50 0 48 60 0 10 0 15 0 discussion sperm motility can be affected by season, cleaning protocol of av and penis, poor management and semen handling practices, noninfectious and infectious diseases, and chemical residues in equipment used.2-4, 8-13 reproductive pathology was not noted in these 3 stallions. sperm motility was similar when tested without seminal plasma in these 3 stallions. effect of season was unlikely to be a factor, as this apparent problem was noticed during the breeding season. in the present case, the av was suspected to be the reason for sudden change in sperm motility. semen collection from 2 other stallions with and without a liner in av confirmed that the av was the most likely cause. liners are not commonly used when a single av is used for a particular stallion during a breeding season. changes in materials to make a new av by manufacturers could be detrimental to sperm and could go unnoticed until multiple mares are diagnosed nonpregnant. testing a new av is necessary to avoid misdiagnosis. to our knowledge, this is the first case report of a spermicidal effect caused by an av. a limitation of the study was not being able to determine the specific cause of reduced motility, whether it was due to a cleaning procedure or some toxic material in the rubber. ideally, a semen sample collected with a different av (routinely used for collection and proven nontoxic) without a liner would have made this argument (new av as the cause) stronger. if both avs had a similar problem with these stallions, then it could be due to some toxic residue in the av after cleaning. however, if another av yielded a normal sample compared to the new av in question, then it could be attributed to some toxic material in the rubber. this approach is suggested for future investigation. learning points • artificial vaginal surface can be toxic to sperm. • surface of a new av may contain toxic material that can render sperm immotile. • verify safety of a newly procured av by testing semen quality immediately and 1 hour after collection. 609 clinical theriogenology • volume 11, number 4 • december 2019 references 1. dowsett kf, knott lm: the influence of age and breed on stallion semen. theriogenology 1996;46:397-412. 2. janett f, thun r, niederer k, et al: seasonal changes in semen quality and freezability in the warmblood stallion. theriogenology 2003;60:453-461. 3. katila t, nivola k, reilas t, et al: factors affecting reproductive performance of horses. pferdeheilkunde 2010;26:6-9. 4. brinsko sp, blanchard tl, varner dd, et al: semen preservation. in: manual of equine reproduction. 3rd edition, maryland heights; elsevier mosby: 2011. p. 207-227. 5. neu sm, timoney pj, lowry sr: changes in semen quality in the stallion following experimental infection with equine arteritis virus. theriogenology 1992;37:407-431. 6. zhang j, ricketts sw, tanner sk: antisperm antibodies in the semen of the stallion following testicular trauma. equine vet j 1990;22:138-141. 7. love cc: semen collection techniques. veterinary clinics of north america: equine practice 1992;8:111-128. 8. ley wb, slusher sh: infertility and diseases of the reproductive tract of stallions. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. e-book. elsevier health sciences; 2006:15-23. 9. betsch jm, hunt pr, spalart m, et al: effects of chlorhexidine penile washing on stallion semen parameters and sperm chromatin structure assay. j reprod fertility 1991;suppl 44:655-656. 10. knottenbelt dc, pascoe rr, lopate c, et al: feeding and exercise. in: equine stud farm medicine and surgery. 1st edition, philadelphia; elsevier saunders: 2003. p. 21-24. 11. knottenbelt dc, pascoe rr, lopate c, et al: the stallion. in: equine stud farm medicine and surgery. 1st edition, philadelphia; elsevier saunders: 2003. p. 43-112. 12. dascanio jj, kasimanickam r: breeding the mare with frozen semen. equine vet educ 2008;ae/december 2008: 667-672. 13. limone le, shaughnessy dw, gómez-ibáñez s, et al: the effect of artificial vagina lubricants on stallion sperm motion measures and semen ph during cooled storage. theriogenology 2002;58:333-336. 610clinical theriogenology • volume 11, number 4 • december 2019 untitled 2018: abnormalities of the estrous cycle abnormalities of the estrous cycle frances o. smith, john c. lawrence smith veterinary hospital, inc., burnsville, mn abstract veterinarians and dog breeders expect that all bitches will follow a typical estrous cycle of proestrus with serosanguinous vaginal discharge (nine days),estrus standing behavior (nine days) and diestrus (60 days). breeders often assume that the bitch should be bred on the tenth and twelfth day of her estrous cycle. in reality proestrus can last up to 17 days, estrus can last up to 17 days and diestrus can last 56-58 days after ovulation. beyond the typical (normal) estrous cycles a bitch may exhibit an anovulatory cycles, slow preovulatory progesterone rise, a cycle with an insufficient luteal phase, a split heat, persistent estrus or may fail to cycle. keywords: anovulation, progesterone, split heat, luteal insufficiency, slow rise in preovulatory progesterone, failure to cycle introduction breeding management of the bitch requires measurement of preovulatory serum progesterone in order to determine the proper time for insemination. progesterone values are used to estimate the day of the luteinizing hormone (lh) peak (day 0) so that both timing insemination and predicting parturition dates can be accomplished.1 it is generally accepted that a progesterone value of 2.0 ng/ml is associated with the lh surge. in theory a typical bitch should ovulate two days after the lh surge and should have fertilizable secondary oocytes 2-3 days later. progesterone values at ovulation vary from laboratory to laboratory but generally are in the 4.0-10.0ng/ml range. split heats a split heat is a cycle in which proestrus signs of vaginal swelling and serosanguinous vaginal discharge occur without progression to ovulation followed by a brief window of apparent anestrus followed by a normal estrous cycle one to two months later. split heats are most commonly seen with a first puberal heat cycle. the bitch is fertile during the normal portion of the cycle. some bitches may demonstrate split heats as a manifestation of an abnormal interestrus interval. silent heats a silent heat is a cycle in which the external signs of the estrous cycle are not visible – minimal vulvar swelling or vaginal discharge but normal follicular development and ovulations. the most common cause of silent heats is owner failure to observe. silent heats may be seen in bitches under heavy physical activity and/or training stress. this author recommends that the owner of a bitch with suspected silent heat blot the vulvar area daily for any signs of discharge and began vaginal cytology and progesterone measurement as soon as any signs are recognized. alternatively progesterone measurements can be performed monthly to detect an unrecognized cycle. the stud dog typically will demonstrate interest in a bitch that is experiencing a silent heat. slow preovulatory progesterone rise this is the most common unusual cycle that is seen in my practice. in this type of cycle, the bitch exhibits a plateau in her progesterone rise at about 2ng/ml which persists for a number of days (three or more) and then progresses with a rapid rise consistent with ovulation. these cycles are very frustrating for both the clinician and the client who wants to know “just how many of these progesterones am i going to have to run”. the slow rise indicates that the cycle is unusual but is not necessarily an infertile cycle. to achieve good fertility these cycles often require multiple inseminations. parturition date is more difficult to predict from these cycles because it is difficult to associate the lh peak with the progesterone value. clinical theriogenology • volume 10, number 3 • september 2018181 anovulation anovulation is defined as the failure of serum progesterone concentrations to exceed 2ng/ml during estrus. vaginal cytology progresses from cornified to non-cornified epithelial cells but progesterone concentration characteristic of diestrus fails to occur and the bitch enters anestrus. anovulation is detected by a combination of vaginal cytology and every other day quantitative progesterone measurements beginning within the first five days of an estrous cycle. anovulation can occur due to failure of the ovary to deliver enough estrogen to elicit an lh surge, insufficient gonadltropin releasing hormone (gnrh) from the hypothalamus, failure of the pituitary to secrete sufficient lh or failure of the ovary to respond to a normal lh surge. treatment is not recommended as many bitches will cycle normally at the next estrous cycle. some authors recommend treatment with human chorionic gonadotropin (hcg) at the subsequent cycle however that can result in persistent estrus and or pyometra.2 insufficient luteal phase luteal insufficiency is the decrease in serum progesterone concentration to<2ng/ml prior to the end of diestrus. this decrease can occur from shortly after ovulation to late in diestrus whether the bitch is pregnant or nonpregnant. without measuring serum progesterone on a routine basis, luteal insufficiency will not be identified. this author recommends measuring progesterone levels at the time of pregnancy ultrasonography if the bitch is not pregnant or if the bitch has a history of previous pregnancy loss. luteal insufficiency is poorly documented as the cause of canine embryonic resorption or abortion although it is often investigated and suspected. progesterone levels <2ng/ml for greater than 24 hours typically results in pregnancy loss. progesterone replacement therapy can be administered. progesterone in oil intramuscularly has been used to supplement progesterone but has been difficult to obtain as of this writing. regumate® (altrenogest) at 0.088mg.kg daily can be given to support the pregnancy. this is extra –label use and should be accompanied by a signed client consent form that outlines the usage and informs the client of the risk of masculinization of female fetuses. administration of exogenous progesterone may have less impact on the fetus when administered at day 50 or later. quantitative progesterone assays using chemiluminnescence or radioimmunoassy do not measure altrenogest so progesterone measurements obtained while the bitch is being treated will reflect only endogenous progesterone. exogenous progesterone administration must cease by day 60-61. the client should expect decreased lactation in bitches supplemented with progesterone. persistent estrus persistent estrus can be the result of exogenous or endogenous estrogen production. persistent estrus as the result of endogenous estrogen is abnormal. the estrogen source is typically due to ovarian dysfunction but granulosa cell tumors should be considered in the differential diagnosis. ultrasonography of the ovary may be useful but it is a challenge to confirm abnormal follicles or follicular cysts. estrogen assays are difficult and difficult to interpret so the diagnosis of persistent estrus is based on the presence of fully cornified vaginal epithelial cells for three to six weeks or longer. bitches with persistent estrus typically have progesterone levels in the preovulatory range<2.0ng/ml. estrogen levels when measured in these bitches were in the normal range. in addition to the negative effect on fertility, persistent estrus can result in irreversible bone marrow suppression. the definite therapy for persistent estrus is ovariohysterectomy however most clients will ask for alternative options. once a definite diagnosis is reached treatment may be attempted by using hcg (500-1000units im – repeated in 48 hours) or gonadorelin at 3.3mcg/kg once daily for three days. responses to therapy should be assessed using vaginal cytology and progesterone measurement. the bitch should be monitored for the development of pyometra after successful treatment. treatment failure should prompt the clinician to examine the ovaries surgically as ultrasonography may fail to identify the ovarian mass. clinical theriogenology • volume 10, number 3 • september 2018 182 failure to cycle the puberal estrous cycle occurs at variable ages in bitches. in breeds such as the basenji that cycle only annually or in in sighthounds first estrus may occur up to 24 months of age. bitches that have not cycled by 24 months of age should be examined for evidence of previous ovariohysterectomy or evidence of an abnormality of sexual development including turner’s syndrome and trisomy x. summary there is a wide timeline for the normal estrous cycle in the bitch. in order to assess true abnormalities of the cycle, history taking will be a vital tool. the client will need to commit to extensive hormonal testing in any bitch with either an unusual or abnormal cycle. references 1. meyers-wallen,v.n. unusual and abnormal estrous cycles. theriogenology 2007;68:1205-1210 2. root-kustritz,m.v. small animal theriogenology. st.louis: butterworth heinemann; 2003. p. 563-569. clinical theriogenology • volume 10, number 3 • september 2018183 clinical theriogenology • volume 10, number 3 • september 2018 184 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype true /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice theriogenologist of the year award theriogenologist of the year in the sars-cov-2 pandemic claire card department of large animal clinical sciences, western college of veterinary medicine university of saskatchewan, saskatoon, canada to be nominated and chosen as the theriogenologist of the year (toy) is a great honor and i am deeply humbled by this award. i am also deeply saddened that my colorful big apple new yorker friend peter brunelli of universal ultrasound, who died of covid19, and who has sponsored this award, is not here today for me to thank. peter used to chid me about being “the theriogenologist who practices what she preaches,” reminding everyone within ear shot that i had 4 children and 2 stepchildren! peter was an incredible supporter of the american college of theriogenologists and the society for theriogenology, and i will miss him dearly. this has been quite a year to reflect upon in the backdrop of the coronavirus severe acute respiratory syndrome coronavirus-2 (sars-cov-2) pandemic. a word cloud of the pandemic would be filled with: zoonosis. pangolins. bats. cats. ferrets. mink. sars. mers. emerging disease. public health emergency. air born. respiratory droplet spread. pneumonia. ventilator. borders closed. social distancing. quarantine. n95. facemasks. ppe. community spread. shelter in place. vulnerable populations. doubling rates. flattening the curve. fomites. contact tracing. self-quarantine. supershedder. reproduction number r0, case fatality rate. crysis. reviewing the past awardees of toys is impactful. my thoughts included: what an incredible list, how fortunate i am to have known these people, how our profession has been advanced by their scientific discoveries, how we need to encourage the nomination of more women, and how the heck did my name get on the list? this is my opportunity to thank those who illuminated my path. i want to acknowledge my formative years at cornell university with mentors: ron gorewit, bruce currie, peter nathanielsz, mary smith, joanne fortune, my phd supervisor ron butler, and my committee members don schlafer, and bill hansel; members of the theriogenology section: drs. bob hillman, rob gilbert, barry ball, tom little, peter daels, and vicki myers wallen; and my fellow grad students lisa freeman, george haluska, rick canfield, charlie elrod, and susan huyler. your help and support were invaluable. i owe a great deal to the dedication and iron work ethics of the students i supervised at the western college of veterinary medicine’s combined residency and graduate program, including drs: shawn haas, stephen manning, theresa burns, natalie bragg, farshad maloufi, alejandro rey, alexandra rauch, farhad ghasemi, tal raz, sarah eaton, dawn nairne, mariana diel de amorim, and maria lopez rodriguez, and the other graduate students i had the opportunity to mentor. you have all been an inspiration, and watching you become talented and sought-after clinicians and scientists has been a highlight for me. similarly, thanks go out to my many collaborators over the years: drs. ed squires, jason bruemmer, irwin liu, don thompson, claudia klein, elemir simko, bernard laarveld, nadia cymbaluk, tasha epp, greggory starrak, and robert foster along with the sea of undergraduate students, now veterinarians: drs. mark corrigan, jodyne green, sylvia carly, barb hunter, allister gray, ilse dedden, ellie ripley, jasmine paulson, patrick roberts, lyndsay rogers, megan jurasek, kayla nielsen, brad mckell, mikayla swirski, brandi bakken and ashlyn ketterer. thanks to tammy kimmel and veterinary technicans: mikhaela thasher, and rebecca johnston, along with the mason family’s hilltopper clydesdales, ron schreiner of d and r ranch, and jack and linda iveson of jaclyn quarter horses. a big thank you to all of our clients and referring veterinarians who have shown great trust in us over the years. lastly all of the members of the act, sft, and tf family who are my friends and whom i have had the pleasure of knowing and serving with over many years. you have made our veterinary speciality theriogenology vibrant, relevant and progressive. clinical theriogenology • volume 12 number 3 • september 2020 169 the award is often given to acknowledge significant recent research contributions. our research contributions from dr. diel de amorim’s phd work have centered on the role of oxytocin (oxt) and oxytocinase,a an enzyme that metabolizes oxytocin, in the maternal recognition of pregnancy (mrp) in mares, most of that is under review. if i had to describe the foundation for our research work, it was built on the work of those who came before. ground breaking work was done in the early days by dan sharp on prostaglandin,1,2 pat sertich on embryonic and endometrial prostaglandin secretion,3 elaine watson4,5 on endometrial and luteal oxytocin, gordon woods and jim weber on embryonic pge section,6 dirk vanderwall on pge and the effects of oxytocin on the corpus luteum,7 claudia klein and mats troedsson8 on molecular aspects of mrp, bob douglas and ollie ginther on prostaglandin,9 christine aurich and sven budik on interferons and oxytocin receptors,10 tom stout and twink allen on embryo migration and the endometrial luteolytic pathway,11,12 along with many others. we have expended considerable effort to understand some of the complexities of mrp in the mare. we confirmed that oxytocinase was present in many equine tissues, including luteal, uterine and trophoblast tissue. oxtocin is produced as a preprohormone bound to neurophysin -1 and is then processed into the active hormone oxt. we showed that whereas luteal tissue expresses oxt, there is little hormone produced, making luteal origin oxytocin a possible intraluteal paracrine regulator, but an unlikely regulator of endometrial prostaglandin secretion. there is more work to be done to understand the role of oxt gene expression and posttranslational processing of the oxytocin/neurophysin prohormone oxt protein in mrp. we have also another research focus from the thesis work of dr. maria lopez rodriguez, that is related to equine thyroid function and fetal development. in the early north american veterinary literature, the birth of contracted legged abnormal foals was identified.13 gross and histologic thyroid abnormalities were reported and associated with low concentrations of iodine in the prairie soil.14,15 outbreaks of disease16 and foals with severe musculoskeletal abnormalities including: dysmaturity, hypothermia, severe limb contracture, rupture of the common digital extensor tendons, mandibular prognathism, abnormal umbilicus, thyroid metaplasia and carpal/tarsal boney dysgenesis were identified17. this collection of abnormalities was termed congenital hypothyroidism dysmaturity syndrome (chds).18 equine fetal studies including thyroidectomy19 proved that fetal thyroid hormone was actively secreted in the last third of gestation20. equine fetal thyroidectomy showed the critical importance of the thyroid in equine fetal development, and recreated many of the clinical signs of chd.19 research work by others highlighted the epigenetic effects of thyroid hormone on fetal development.21,22 investigators identified that no trace mineral supplementation17 and exposure to mustard plants23,24 were a risk factor for chds. mustard plants contain compounds called glucosinolates that are metabolized into goitrogens and alter thyroid function25. our studies showed that feeding glucosinolates combined with a low iodine diet resulted in reduced serum iodine concentrations similar to the effects on other species2.5-27 we demonstrated that broodmares often have insufficient concentrations of iodine, in spite of access to salt blocks and mineral supplements.28 depending on the voluntary consumption of salt blocks or mineral products by broodmares to meet their iodine and other trace mineral needs may lead to insufficiency. we also reported that equine mammary tissue concentrates iodine and is a source of iodine for the foal.28 equine neonates have very high concentrations of serum iodine, total thyroxine and total triiodothyronine, all of that decrease rapidly over the first 10 days of life and play a key role in neonatal adaptation. however, questions still remain and further studies are needed with known and confirmed intakes of iodine to confirm the appropriate reference concentrations. what have my years in research taught me? what would that word cloud look like? team. determination. hard work. repeat. redo. redesign. focus. literature. hypothesis. gene. protein. elisa. western blot. mass spec. immunohistochemistry. resilience. my family, my husband peter barnacle, daughters kirstin, ursula, johnna and son ryan, step-children shawn, tricia and grandchildren a oxytocinase is also called leucyl-cystinyl-aminopeptidase (lnpep), insulin responsive aminopeptidase (irap) and placenta leucine aminopeptidase (plap) clinical theriogenology • volume 12 number 3 • september 2020170 owen, jake, nicholas, and samantha, along with my good friends helped me survive. much of my success is due to their support. it was not easy being a female large animal veterinarian and i hope that my efforts have made it easier for those who follow. key messages i wish to give to others is that the amount of funding for equine and small animal research remains woefully inadequate and needs to improve. this will rely on our profession’s advocacy for changes in government policy and continued private and industry support for foundations such as tf and other granting agencies. my partnership with rural women farmers in uganda has taught me that the true measure of one’s life is not about the things money can buy, but in how much we give to others. as investigators, i encourage everyone to continue to ask questions, and to keep checking your sources. additionally, to members of the public, respect science, it is the pathway out of ignorance, out of the pandemic and out of the pernicious challenges that lie ahead, including rapid species extinction and climate breakdown. references 1. sharp dc, mcdowell kj, weithenauer j et al: the continuum of events leading to maternal recognition of pregnancy in mares. j reprod fertil suppl 1989;37;101-107. 2. sharp c, thatcher m, salute me et al: relationship between endometrial oxytocin receptors and oxytocin induced prostaglandin f2 alpha release during the oestrous cycle and early pregnancy in pony mares. j reprod fertil 1997;109:137-144. 3. watson ed, sertich pl: prostaglandin production by horse embryos and the effect of co-culture of embryos with endometrium from pregnant mares. j reprod fertil 1989;87:331-336. 4. watson ed, buckingham j, björkstén ts: immunolocalisation of oxytocin in the equine ovary. equine vet j 1999;31:174-175. 5. watson ed, buckingham j, björkstén t et al: immunolocalization of oxytocin and neurophysin in the mare uterus. j reprod fertil suppl 2000;56;289-296. 6. weber ja, freeman da, vanderwall dk et al: prostaglandin e2 secretion by oviductal transport-stage equine embryos. biol reprod 1991;45:540-543. 7. vanderwall dk, woods gl, weber ja et al: corpus luteum function in non-pregnant mares following intrauterine admnistration of prostaglandin e2 or estradiol 17-beta. theriogenology 1994:42;1069-1083. 8. klein c, troedsson mht: maternal recognition of pregnancy in the horse: a mystery still to be solved. reprod fertil dev 2011;23:952-963. 9. douglas rh, ginther oj: concentration of prostaglandins f in uterine venous plasma of anesthetized mares during the estrous cycle and early pregnancy. prostaglandins 1976;11:251-260. 10. aurich c, budik s: early pregnancy in the horse revisited – does exception prove the rule? j anim sci biotechnol 2015;6:1-8. 11. stout tae, allen wr: prostaglandin e(2) and f(2 alpha) production by equine conceptuses and concentrations in conceptus fluids and uterine flushings recovered from early pregnant and dioestrous mares. reproduction 2002;123:261-268. 12. de ruijter-villani m, van tol hta, stout tae: effect of pregnancy on endometrial expression of luteolytic pathway components in the mare. reprod fertil dev 2015;27:834-845. 13. rodenwald bw, simms b: iodine in brood mares. proc american society of animal production 1935; p. 89-92. 14. abbott c, prendergast j: histological variations in animal thyroids in western canada. can med assoc j 1934;31: 465-473. 15. schlotthauer cf: incidence and types of disease of the thyroid gland of adult horses. javma 1931;78:211-218. 16. doige ce, mclaughlin bg: hyperplastic goitre in newborn foals in western canada. can vet j 1981;22:42-45. 17. allen al, townsend hgg, doige ce et al: a case-control study of the congenital hypothyroidism and dysmaturity syndrome of foals. can vet j 1996;37:349-358. 18. allen al: congenital hypothyroidism in horses: looking back and looking ahead. equine vet educ 2014;26:190-193. 19. allen al, fretz pb, card ce, et al: the effects of partial thyroidectomy on the development of the equine fetus. equine vet j 1998;30:53-59. 20. allen al, doige ce, haas sd et al: concentrations of triiodothyronine and thyroxine in equine fetal serum during gestation. biol reprod mono 1995;1:49-52. 21. peugnet p, robles m, wimel l, et al: management of the pregnant mare and long-term consequences on the offspring. theriogenology 2016;86:99-109. 22. chavatte-palmer p, peugnet p, robles m: developmental programming in equine species: relevance for the horse industry. anim front 2017;7:48-54. clinical theriogenology • volume 12 number 3 • september 2020 171 23. hines m, gay c, talcott c: congenital hypothyroidism and dysmaturity syndrome of foals: diagnosis and possible risk factors. proc 15th american college of veterinary internal medicine 1997; p. 363-364. 24. lopez-rodriguez mf, rhodes m, diel de amorim m, et al: thyroid dysfunction and iodine deficiency in a miniature jenny associated with neonatal congenital hypothyroidism dysmaturity syndrome. clinical theriogenology 2019;11:143-148. 25. tripathi mk, mishra as. glucosinolates in animal nutrition: a review. animal feed science and technology 2007;132:1-27. 26. kursa j, trávníček j, rambeck w, et al: goitrogenic effects of extracted rapeseed meal and nitrates in sheep and their progeny. vet med (praha) 2000;45:129-140. 27. lopez-rodriguez mf, cymbaluk n, epp t, et al: effects of the glucosinolate sinigrin in combination with a noniodine supplemented diet on serum iodine and thyroid hormone concentrations in nonpregnant mares. j equine vet sci 2020; 91:doi:10.1016/j.jevs.2020.103110 28. lopez-rodriguez mf, cymbaluk nf, epp t, et al: a field study of serum, colostrum, milk iodine, and thyroid hormone concentrations in postpartum draft mares and foals. j equine vet sci 2020;90:doi:10.1016/j.jevs.2020.103018 clinical theriogenology • volume 12 number 3 • september 2020172 005_ms-005 card current state of spay/neuter programs in united states and effect on overall animal numbers current state of spay/neuter programs in united states and effect on overall animal numbers david haworth, two dogs veterinary consulting, llc abstract surgically altering (spaying/ovariohysterectomy and neutering/orchidectomy) of companion dogs and cats is a culturally enforced phenomenon in the united states. this is unusual, almost unique to our culture and represents the largest experiment of long term health consequences of a surgical procedure ever performed. over the past 5 decades, spaying and neutering pets has become a normal, expected part of responsible pet ownership. veterinarians have achieved remarkable market penetration of this technique with an estimated 80% of cats and 69% of dogs altered.1 spay/neuter programs defined as intentional, formalized efforts to have large numbers of dogs and cats altered – have historically been shelter-based and performed on institutionally owned animals, but there is a shift underway towards organizations providing these services to privately owned animals in under-resourced communities. these programs have unquestionably had an impact on dog populations, although evidence in cats is less compelling. limitations of existing population estimates for dogs and cats are substantial. nonetheless, impacts are directionally consistent and indicate reductions in unwanted dogs and cats in the us. while this is positive, there have also been some unexpected negative consequences of widespread spay/neuter programs, including impacts on availability of adoptable animals available through rescue groups and shelters, as well as impacts on proportions of breeds available in certain geographic regions and cities. keywords: spaying, neutering, ovariohysterectomy, canine population, shelter introduction and history surgical removal of organs required for reproduction has been done since the earliest days of animal domestication. however, large scale surgical interventions as a means of population control is a relatively new approach and there is some evidence that it may not be effective in all species and in all environments. in the us, widespread surgical neutering of male and female companion animals has been occurring for the past 50 years. the first high volume, low cost spay and neuter clinic opened in los angeles in 1969, although individual veterinary practices have offered the service for much longer, with modern techniques becoming prevalent in the 1930s. in fact, the overwhelming majority of surgical neutering happens at the ~ 26,000 private practices in the us, almost all of which offer spaying and neutering as a routine service. this presentation will focus, however, on the role of large-scale spaying and neutering programs in overall control of dog and cat populations. an interesting aspect is the degree to which individual veterinarians or organized veterinary groups resist establishment of low cost spay/neuter facilities. they argue that low cost clinics, especially those with tax exempt status, unfairly capture revenue that would otherwise go to private veterinary practices. this has not been shown to be the case and in fact most spay/neuter clinics encourage their clients to follow up with established veterinarians when they can afford to. today, most mainstream veterinary practice consultants encourage private veterinarians to collaborate with local spay/neuter facilities, benefitting from the stream of new clients and not having to perform a surgery that generally has low profit margins for clients who would otherwise choose low cost options. another factor relevant to this discussion is that the past 50 years also corresponds to an extraordinary rise in the strength and recognition of the human-animal bond. the role of companion animals in society has dramatically changed over this interval, moving rapidly from the backyard to the bedroom and ultimately to the bed. driving this trend are numerous societal factors, such as scattering of the nuclear family, greater wealth in general, greater societal acceptance of pets in more places and several key technologies that have made pet ownership easier and safer – specifically 1) safe, convenient flea and tick medication, 2) easily stored and prepared food for pets, and 3) safe vaccines for most communicable diseases. 219 clinical theriogenology • volume 11, number 3 • september 2019 this trend does not appear to be slowing. nationally, 56.8% of households own a dog or a cat. this is 15% greater than the 41.1% of us households that have children under the age of 18, which means there are 19 million more households with pets than with children. pets are a huge business, with us spending estimated at $72.1b in 2018 ($30b on food, $15b on supplies/over the counter pharmaceuticals, $17b spent on veterinary care, $6b on services and $2b on live animal purchases).2 importantly, 85% of dog owners and 76% of cat owners consider their pets to be family members, which drive many behaviors like humanization (e.g. clothing for pets), indulgence in toys and movement towards purchasing premium food. spay/neuter programs today most organized spay and neuter programs are paid for in whole or in part by philanthropy or municipal governments. over the past decade, due to lower intake volumes at shelters and the perceived remaining areas of need, many programs have moved away from being exclusively offered to institutionowned animals and towards being offered to members of the public. not without controversy, both fully and partially subsidized procedures are offered in most communities and often the price paid by the client is determined on a sliding scale relative to their income. this change in targeted animal populations is based on the recognition that whereas most communities have ready access to veterinary services, there are many communities that do not, and these communities are where the largest remaining need appears to be. in many ways, this can be seen as a victory for the animal welfare community, since people who can afford to spay or neuter their animals do so. it is expected (and increasingly demanded by statute) that pets adopted from agencies will either be spayed or neutered at the time of adoption, or that the promise to spay or neuter them in the near future be a condition of adoption (usually in the case of very young or ill animals). since 28% of dogs and 31% of cats are obtained through rescue groups or shelters, this guarantees a large number of animals are altered every year. because costs associated with spay/neuter programs come from a wide array of sources (local, regional or national philanthropies, municipal governments, special projects within an organization and even individual volunteer teams) and that most shelters incorporate costs of their internal programs into operating costs, the total amount spent on programs is impossible to accurately ascertain. however, the 2 largest animal welfare charities, petsmart charities and the petco foundation, spend a combined $20 million per year on these programs, so it is not unreasonable to assume that the actual number being spent by all charities and municipalities exceeds $200 million. it is also clear that even with such a high level of investment, the vast majority of spay/neuter surgeries are not performed as part of these programs, but rather they are performed at private veterinary clinics. it is, however, logical to argue that free or low cost programs and clinics: 1) disproportionally impact families that would otherwise not be able to spay or neuter their animals due to economic limitations; and 2) contribute to the general culture of spaying and neutering being an expected part of pet ownership. this cultural expectation is very real. there are hundreds of social cues that remind people that spaying and neutering pets is normal and expected. in veterinary medicine, we have expounded the health benefits for decades, and whereas positive impacts for population health are (probably) clear, those for individual health are more questionable. there is no doubt that a dog cannot develop uterine cancer without a uterus, but that same argument could be made about any number of nonvital organs. furthermore, this is a unique cultural phenomenon in the world. for example, in sweden only 3% of dogs are sterilized3 and it is illegal to do so without a medical reason. but there the cultural pressure is much lower and the responsibilities associated with pet ownership very different. after all, bob barker (and later drew carey) have been signing off the “price is right” game show with the signature phrase “…help control the pet population, have your animals spayed and neutered” since 1981.4 while only a single example, it is representative of the overall cultural expectations that pet owners face in the us. 220clinical theriogenology • volume 11, number 3 • september 2019 how many dogs and cats are there? according to the american veterinary medical association (avma), there were ~ 77 million dogs and 58 million cats in the us in 2016. these numbers that are in sharp contrast to the american pet products association (appa) estimates of 89.7 million dogs and 94.2 million cats, although they are the 2 most widely cited sources. both estimates are based on surveys, and inherently over-represent populations that can participate (i.e. own a computer and/or phone, are willing to participate and reachable), although the avma survey is much larger – 50,000 completed questionnaires compared to about 2500 for appa – so we will generally rely on the avma numbers. the trend is that there has been a steady increase in the number of pets in the us since the survey began in 1991. determining the number of animals that enter us shelters is also a challenging task. estimates vary widely, from 6.5 million animals according to the american society for the prevention of cruelty to animals (aspca)5 to 2.7 million animals according to the most definitive web-based data collection site, shelters animals count.6 even with that relatively large discrepancy, the number of animals entering shelters over the past several decades has been on a steady and dramatic decline, which can be at least partially attributed to spay/neuter programs. rowan and kartel performed a thorough review of both shelter intake and euthanasia data from the 1970s to the present.7 while earlier data are not nearly as robust as current data, trends are clearly positive, both in an absolute sense and relative to human population. in 1973, the humane society of the united states (hsus) estimated that 13.5 million animals were euthanized in the us. in 2019, that number is ~ 1.3 million, a decrease of 90%. this is even more dramatic when it is recognized that municipal and private shelters are often the euthanasia-center-of-last-resort for parts of the population who cannot access private veterinarians, and therefore there is a portion of those 1.3 million animals for which euthanasia is the most appropriate and humane option. for dogs, we have reached a point where there are not enough adoptable animals in some regions and some groups are having to stretch the definition of an “adoptable” animal in order to meet demand. while these supply discrepancies are solvable through transport programs – bringing animals from areas with too many adoptable animals to areas that have too few – in the short term, the issue could have dramatic consequences in the future supply of dogs if not addressed soon. how much of that decline in animals going into shelters is due to spaying and neutering programs is a matter of debate. while it is logical to conclude that having fewer intact animals should result in fewer animals entering shelters and fewer animals being euthanized, there are confounding factors that should not be ignored. the first is that the fecundity of dogs and cats means that a small number of intact adults can produce large numbers of offspring, potentially enough to make up for those spayed and neutered. a second factor is the “no kill” movement, which has become a driving force in the animal welfare community. the no kill movement began in the early 1970s in san francisco by richard avenzino when he led the san francisco society for the prevention of cruelty to animals (spca). it has since been propagated and driven by others, most notably nathan winograd and the best friends animal society, although as a philosophy and catch phrase it enjoys broad support. at its essence, it is a series of programs and incentives to make individual shelters (or by extension, communities, states, and regions) release 90% of the animals that enter the shelters alive, presumably into loving homes. this emphasis on a single metric – the live release rate (lrr) – has undoubtedly driven many organizations to be better managed and saved millions of lives. but there have also been unintended consequences that have confounded understanding of how many animals enter shelters and therefore how spay/neuter programs have impacted dog and cat populations. people who are attracted to animal welfare want to maximize the number of animals’ lives saved, and thanks to social media and political campaigns calling those organizations that do not meet the 90% lrr threshold “kill shelters” or “high kill shelters”, groups are highly motivated to achieve “no kill” status. unfortunately, this can result in less than ideal management techniques. for instance, some organizations now limit animals that they accept to only those they feel can be adopted. if a group wants 100% lrr, they can achieve it readily if they only accept highly adoptable puppies. it is likely not 221 clinical theriogenology • volume 11, number 3 • september 2019 helping the unwanted dog population significantly, but they will have very impressive statistics. other techniques include making it difficult to relinquish animals, forcing unadoptable animals to other groups by not taking on municipal contracts, or partnering with other groups to remove difficult animals, thereby transferring the problem around the community. this makes understanding the exact impact of spay/neuter programs on the homeless animal population even more difficult, because these incentives lead some to misrepresent or ignore a portion of the animals in the general population. the last point to be made in regard to the impact of spay/neuter programs on the dog population concerns overrepresentation of some breeds. blocky-headed, square-jawed dogs – labeled with various names including pit bulls, bully breeds or bully crosses – comprise ~ 6% of the general us dog population, but between 45 and 90% of shelter populations, albeit with strong regional differences. similar proportions exist for chihuahua and chihuahua-mixes, although the logistical challenges associated with too many chihuahuas are very different than those with too many pit bulls. the point is not about the relative merits or challenges with these breeds, it is that clearly there are too many of these animals having too many unwanted litters, despite targeted programs being in place for decades, including breed-specific bans in certain communities and various breed-specific spay/neuter programs. it appears that numbers in certain areas are worse than ever and represent a problem with ready access to minimum veterinary care, including spaying and neutering services. that is the story with dogs – spaying and neutering encouraged as a cultural norm, adoption being an acceptable (often preferable) way to acquire a dog, readily accessible surgical offerings with some major exceptions and some interesting impacts on breed selection – all of which have created the current situation of declining numbers of animals being relinquished to shelters, with a concomitant reduction in euthanasia, albeit with remaining challenges. what about cats? the situation with cats is less clear. in most areas of the country, there is still an abundance of cats in shelters and whereas numbers in some areas have declined, there are some disturbing trends that indicate that the problem is still as large, but because of several factors – the no kill movement being a significant one – the community is just not counting those cats as unwanted. there are millions of dollars being spent for trap neuter release programs (where community-owned cats are trapped, surgically altered and then released to where they were originally sourced) without conclusive evidence that they are effective in all situations. obviously, cats that are altered can’t reproduce, but because: 1) cats are highly fecund when environmental conditions are right; 2) it is rare for the individual animals in colonies of cats to be stable over time; and 3) not all cats in a colony can be captured and altered, there is an argument that removing some individuals, even a majority of the individuals, from the breeding pool does not appreciably alter the population of that ecosystem of cats over time. this is not to imply that trap neuter release programs are not effective solutions for some issues related to community-owned cats, but the evidence is not there that it is an effective sole method of population control. conclusion spaying and neutering programs have been used for population control and responsible pet ownership for close to 80 years in the us, with an upsurge in their popularity during the past 4 or 5 decades. for dogs, these programs have had an effect on limiting the overall population in both direct and indirect ways. directly, there are millions of animals that have been altered in these programs. the overall number is likely a small fraction of the number of privatelyowned animals surgically sterilized by veterinarians in private practice, but the cultural expectation that all pets should be spayed or neutered unless they are going to be responsibly bred has some basis and reinforcement in these programs. getting an accurate picture of population impacts of these programs with robust data is difficult if not impossible. verifiable numbers simply do not exist, but extrapolations are possible and impact trends are clear. there are fewer animals coming into shelters than ever before and spay/neuter programs have had a role in that, although there are some confounding factors. lastly, cat populations, while declining in some areas of the country, are much slower to move than dogs and overall trends are much less clear. 222clinical theriogenology • volume 11, number 3 • september 2019 conflict of interest the author has no business relationship with any of the manufacturers or corporations referenced in the manuscript. the author is the former president of petsmart charities and morris animal foundation, both of which actively support organized spay and neuter programs. references 1. american veterinary medical association. avma pet ownership and demographics sourcebook. 2nd edition, schaumburg: avma;2018. 2. americans are spending more on their pets than ever before: $72 billion. www.americanpetproducts.org (accessed 10 february 2019). 3. von heimendahl a. neutering in the uk and europe. veterinary practice 2011;43:9. 4. cbs biography for bob barker. www.cbs.com (accessed 24 september 2012). 5. aspca fast facts. www.aspca.org (accessed 10 february 2019). 6. 2016 animal sheltering statistics. www.shelteranimalscount.org (accessed 10 february 2019). 7. rowan a, kartel t: dog population and sheltering trends in the united states of america. animals. 2018;8:68. 223 clinical theriogenology • volume 11, number 3 • september 2019 224clinical theriogenology • volume 11, number 3 • september 2019 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false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice seeking stem cell efficacy œ immunogenicity matters! seeking stem cell efficacy – immunogenicity matters! ashlee watts department of large animal clinical sciences college of veterinary medicine & biomedical sciences texas a&m university, college station, tx abstract despite hundreds of publications and registered clinical trials, definitive evidence of mesenchymal stem cell (msc) efficacy does not exist. the notion that mesenchymal stromal cells (mscs) are immune privileged is changing: mscs constitutively express mhci, can be induced to express mhcii and allo-immunization against allogeneic mscs has been documented. despite the clinical safety of allo-mscs, which has been shown exhaustively without evidence for acute rejection reactions or local tissue inflammation, immune recognition and rejection occurs with a reduction in msc efficacy, we have demonstrated localized tissue inflammation secondary to immune recognition of allomscs. we have also demonstrated localized tissue inflammation secondary to immune recognition of fetal bovine serum contaminants to mscs. this localized tissue inflammation supports the clinical relevance of allo-recognition of mhc mismatched mscs and fbs contaminated mscs, despite the lack of clinically apparent adverse reaction. these findings may explain why definitive proof of msc efficacy has not been achieved. keywords: equine, bone marrow, mesenchymal, stem, stromal, regenerative lameness is one of the leading reasons for suffering and ill health in the horse, and it is estimated that approximately 60% of lameness is due to osteoarthritis. mesenchymal stem cells (mscs) are a regenerative therapy used to mitigate chronic pain and slow the progression of osteoarthritis. in people, there are fda non-cross matched allogenic (non-self) and autologous (self) msc trials for joint disease and approximately half of all current msc clinical trials in people are allogenic.1 this is because allogenic mscs have been long considered non-immunogenic, and are more easily commercialized. this remains true in horses as well, and several groups maintain the feasibility of allogenic msc use.2,3 however, as clinical data accumulate, it appears there is greater variability in efficacy of allogeneic therapies as compared to autologous, causing the immune privileged status of allo-mscs to be questioned.4 in horse, published reports document a regenerative effect when autologous mscs are used to treat joint disease, but anecdotal reports (where allogeneic mscs are often used) are of minimal treatment effect. the lack of consistent lameness reduction is because allogeneic mscs are recognized by the immune system, thus precluding the beneficial effects. this is in agreement with our previous work that showed in horse a significant adverse response when allogenic-mscs were confined to the intraarticular environment compared to auto-mscs.5 long considered immune privileged, allogeneic (non-self, from one horse to another) mesenchymal stem cells (mscs) have enhanced commercialization potential.6,7 the immune privileged status of mscs stems from absent mhcii expression, low mhci expression, and immunomodulatory action by mscs. despite low expression of mhci, we have recently demonstrated immune recognition of non-self mscs in horses. this immune recognition may explain the greater variability in efficacy of allogeneic therapies that has been recognized in people and suggested in the horse.3,4,8,9 our recent work, along with data from equine researchers at ncsu demonstrating cytotoxic t cell responses in vitro after allogeneic msc administration, provides sufficient evidence to question not only the efficacy but also the safety of allogeneic mscs in the horse.10,11 despite decades of mesenchymal stromal cell (msc) therapy, definitive clinical evidence of efficacy does not exist, and it has been postulated that lack of efficacy is due to msc preparation technique.12,13 supplementation of mesenchymal stromal cells (mscs) with fetal bovine serum (fbs) has been a standard practice since mscs were first described in 1970.14 since then, numerous veterinary and human medical patients have received mscs supplemented with fbs for various medical conditions, clinical theriogenology • volume 12 number 3 • september 2020180 including osteoarthritis. although clinical detection of adverse reactions appears to be rare, xenogen exposure to the msc recipient because of fbs supplementation can cause local reactions,15 anaphlyaxis,16 and anamnestic responses,17 which have been associated with msc-non response.18 while immune recognition and resultant adverse reactions are concerning, immune recognition and resultant cytotoxicity against mscs may be a key factor in the elusive proof of msc efficacy for most applications including osteoarthritis.13,19 many blood derivatives including platelet products,20 umbilical cord blood serum21 and chemically defined media have been investigated to replace fbs supplementation of mscs. based on the ever-decreasing proportion of clinical.trials.gov registered msc trials, with fbs supplementation of 80% of regulatory submissions in 20141 to 40% in 2019, it appears that the human regenerative medicine field agrees that fbs should be replaced. surprisingly, the veterinary regenerative medicine community has not adopted this stance and fbs supplementation of mscs remains the industry standard.2,3 similarly, fbs supplementation remains well-accepted in many preclinical msc investigations in laboratory animals.22 this may be because definitive evidence for immune targeting and cytotoxicity of fbs supplemented mscs does not exist. regardless of this disparity in human, veterinary and preclinical fields, it appears that no autologous supplement has replaced fbs with unaltered growth, characterization or clinical efficacy of mscs. we have previously shown that 48 hours of fbs depletion is sufficient to mitigate the mild local adverse reaction after repeated intra-articular injection of mscs supplemented with fbs in horses.5 we sought to develop an autologous, xenogen-free method for supplementation of mscs that would obviate immune targeting of transplanted mscs by the recipient, by removing fbs for the entire culture period. our efforts with platelet lysate and platelet releasate, both autologous and pooled, were disappointing as has been previously reported.23 with platelet products, isolation of mscs was generally successful, but growth kinetics, cell surface markers, cell morphology and differentiation ability were more variable when compared to the response to fbs supplementation by the same donors. by developing a replacement to fetal bovine serum supplementation of msc culture, immune mediated targeting of culture expanded cells will be abolished. by evaluating immune reaction of matched and mismatched recipients we are developing a hierarchy of stem cell immunogenicity, which is a necessary step in pursuit of an acceptable allogeneic donor. conflict of interest: none to report. references 1. mendicino m, bailey am, wonnacott k: msc-based product characterization for clinical trials: an fda perspective. cell stem cell 2014;14:141-145. 2. colbath ac, dow sw, hopkins ls, et al: allogeneic vs. autologous intra-articular mesenchymal stem cell injection within normal horses: clinical and cytological comparisons suggest safety. equine vet j 2019 may 23, 3. owens sd, kol a, walker nj, et al: allogeneic mesenchymal stem cell treatment induces specific alloantibodies in horses. stem cells int 2016;2016:5830103. 4. nauta aj, westerhuis g, kruisselbrink ab, et al: donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. blood 2006;108:2114-2120. 5. joswig a, mitchell a, cummings kj, et al: repeated intra-articular injection of allogeneic mesenchymal stem cells causes an adverse response compared to autologous cells in the equine model. stem cell res ther 2017;8:42. 6. ankrum ja, ong jf, karp jm: mesenchymal stem cells: immune evasive, not immune privileged. nat biotechnol 2014 32:252-260. 7. paterson yz, rash n, garvican er, et al: equine mesenchymal stromal cells and embryo-derived stem cells are immune privileged in vitro. stem cell res ther 2014;5:90. 8. pigott jh, ishihara a, wellman ml, et al: inflammatory effects of autologous, genetically modified autologous, allogeneic, and xenogeneic mesenchymal stem cells after intra-articular injection in horses. vet comp orthop traumatol 2013;26:453-460. 9. ferris dj, frisbie dd, kisiday jd, et al: clinical outcome after intra-articular administration of bone marrow derived mesenchymal stem cells in 33 horses with stifle injury. vet surg 2014:43:255-265. 10. pezzanite lm, fortier la, antczak df, et al: equine allogeneic bone marrow-derived mesenchymal stromal cells elicit antibody responses in vivo. stem cell res ther 2015;6:54. clinical theriogenology • volume 12 number 3 • september 2020 181 11. berglund ak, schnabel lv: allogeneic major histocompatibility complex-mismatched equine bone marrow-derived mesenchymal stem cells are targeted for death by cytotoxic anti-major histocompatibility complex antibodies. equine vet j 2017;49:539-544. 12. jevotovsky ds, alfonso ar, einhorn ta, et al: osteoarthritis and stem cell therapy in humans: a systematic review. osteoarthr cartil 2018;26:711-729. 13. barry f: msc therapy for osteoarthritis: an unfinished story. j orthop res 2019;37:1229-1235. 14. friedenstein aj, chailakhjan rk, lalykina ks: the development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. cell tissue kinet 1970;3:393-403. 15. selvaggi ta, walker re, fleisher ta: development of antibodies to fetal calf serum with arthus-like reactions in human immunodeficiency virus-infected patients given syngeneic lymphocyte infusions. blood 1997;89:776-779. 16. macy e, bulpitt k, champlin re, et al: anaphylaxis to infusion of autologous bone marrow: an apparent reaction to self, mediated by ige antibody to bovine serum albumin. j allergy clin immunol 1989;83:871-875. 17. sundin m, ringdén o, sundberg b, et al: no alloantibodies against mesenchymal stromal cells, but presence of antifetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients. haematologica 2007;92:1208-1215. 18. horwitz em, gordon pl, koo wk, et al: isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. proc natl acad sci u s a. 2002;99:8932-8937. 19. galipeau j, sensébé l: mesenchymal stromal cells: clinical challenges and therapeutic opportunities. cell stem cell 2018;22:824-833. 20. jonsdottir-buch sm, lieder r, sigurjonsson oe: platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells. plos one 2013;8:e68984. 21. shetty p, bharucha k, tanavde v: human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells. cell biol int 2007;3:293-298. 22. abbuehl j, tatarova z, held w: long-term engraftment of primary bone marrow stromal cells repairs niche damage and improves hematopoietic stem cell transplantation. cell stem cell 2017 21:255.e6. 23. longhini alf, salazar te, vieira c, et al: peripheral blood-derived mesenchymal stem cells demonstrate immunomodulatory potential for therapeutic use in horses. plos one 2019;14:e0212642. clinical theriogenology • volume 12 number 3 • september 2020182 007_ms-007 watts fiboys in a boxflchilled and frozen canine semen shipments “boys in a box”chilled and frozen canine semen shipments joann randall animal hospital of woodstock woodstock, il abstract canine semen used for shipping or cryopreservation should meet minimal semen evaluation standards for concentration, motility and morphology to maximize artificial insemination success. samples with increased morphological abnormalities may benefit from filtration or gradient centrifugation; however, total numbers will be much lower. extenders are added to sperm-rich fraction to supply energy source, buffer the environment, decrease bacterial growth and protect sperm membranes during chilling and freezing. many commercial and homemade extenders are available, along with various shipping box options, to successfully transport semen virtually anywhere in world. additional cryoprotectants are added to frozen semen extenders to prevent ice crystal formation within sperm. standardized protocols and routine maintenance of nitrogen tanks, and vapor shipper dewars increase chances of successfully protecting, storing and shipping canine semen. keywords: chilled semen, cryopreservation, semen extenders, canine, dog introduction it has become very common to use chilled and frozen semen for canine artificial insemination, due to convenience and advantages of shipping semen instead of costs and risks of shipping bitches or dogs to be bred, or driving long distances for a natural breeding. frozen semen can store genetic material of breed lines for later use and also helps expand genetic diversity. protocols must be followed when shipping or receiving chilled or frozen semen to ensure highest semen quality at insemination. stud dog goal of breeding is to supply healthy puppies for companionship, service, etc. therefore, all dogs collected for chilled or frozen semen use should have a normal physical examination, be current on health clearances and vaccinations and be brucella canis negative. a dna number is required by akc in order to register puppies born using chilled or frozen semen. this is easily obtained by mailing a cheek swab. to maximize chances for a litter, only semen that meets minimal evaluation parameters in regards to concentration, motility and morphology should be chilled or frozen. for international shipments, collecting veterinarian should check with usda regarding stud dog mandatory health testing required prior to collecting semen to meet importation requirements for destination country. current recommendations are available online (www.aphis.usda.gov). semen preparation presence of first or third fractions of ejaculate may adversely affect semen motility and morphology.1 these 2 fractions, along with urine, can be removed with supernatant after centrifugation. regardless, very effort should be made to capture only second (sperm rich) fraction during collection. filtration procedures or centrifugation gradients may improve semen quality. with filtration, ejaculate is passed through a filter (e.g. glass wool). using a centrifugation gradient, sample is carefully pipetted on top of a layer of specialized media in a test tube and centrifuged. normal sperm move through media and remain on tube bottom as a pellet while media binds abnormal sperm. some media may aid in blood removal from sample, particularly digested blood, and centrifugation media can also be used to remove urine. these 2 procedures may improve percent morphologically normal sperm; however, drastic reduction in total numbers, especially with glass wool filtration, may preclude shipping or freezing that sample.2,3 clinical theriogenology • volume 12 number 3 • september 2020210 extenders canine semen can be shipped at refrigerator temperatures (4 5ºc) and used within a few days or frozen and stored in liquid nitrogen (-195ºc) to be used until years later. regardless, sperm-rich portion will need to be mixed with an extender, a liquid preparation supplying multiple benefits. semen extenders contain energy source (e.g. glucose or fructose4) to support motility and contain buffers to help stabilize ph and osmolarity of environment offsetting acid-base changes of metabolism during storage.5 they also contain a protein source (e.g. egg yolk or milk) to protect sperm membrane integrity during temperature fluctuations during chilling, freezing and thawing. percent progressively motile sperm declines gradually with time in extended samples chilled to 4 5ºc, with slower decline in extenders containing 20% egg yolk compared to skim-milk based extenders.5 commercial preparations have ingredients that are often proprietary and vary with their composition (camelot farms, college station, tx; international canine semen bank, sandy, or; synbiotics, kansas city, mo; clone, doylestown, pa; and minitube, verona, wi). many formulas available for homemade extenders; however, these have increased chances for more variability between batches. preparing and shipping chilled semen prior to shipping, inseminating veterinarian should inform collecting veterinarian about type of insemination being done and preferred volume desired. proper labeling of all plastic or glass tubes containing semen with permanent marker is paramount! the society for theriogenology has guidelines on their webpage (www.therio.org) regarding tube identification and paperwork recommendations to ensure correct identification. records annotating semen evaluation results, extender used, dilution, volume, etc. should be in duplicate, with clinic retaining a copy. an established clinic protocol should be followed for every shipment.6 collected ejaculate is centrifuged (300 700 x g for 5 6 minutes) if it contains any of first or third fractions, after which supernatant is withdrawn and extender added (at similar temperature as semen). most commercial extenders recommend a dilution of 1 part semen to 4 parts extender. usually, 2 6 ml for intrauterine or a transcervical insemination and 1 10 ml for vaginal inseminations are shipped, depending on bitch size and amount requested by inseminating veterinarian. a small aliquot should be removed and refrigerated until shipped sample is evaluated upon arrival. this saved sample can be used to evaluate semen if it arrives in poor condition at insemination facility to determine if semen did not chill well or if something happened to the box after leaving collection facility (i.e. temperature extremes). after cap tube is secured, tube top is wrapped with parafilm® (bemis company, neenah, wi). scotch tape, strapping tape, and duct tape should not be used as they do not provide a watertight seal. plumber’s tape, if used, should not have any contact with semen as it may be spermicidal (depending on brand). to maintain a cool temperature, most commercial semen transport boxes are lined with extruded polystyrene foam and use 1 or 2 plastic liquid bricks that will need to be frozen prior to shipping. to ensure that extended semen does not come into direct contact with frozen brick, tube and frozen brick(s) should be thoroughly wrapped with either several layers of newspaper or some form of packing material, e.g. bubble wrap. chilled canine semen shipped in equitainer® (hamilton research inc, south hamilton, ma), a plastic shipping container that is primarily used to ship stallion semen, had highest percent motility 48 hours post-chilling compared to a styrofoam box or a thermos flask7. this container is also approved for multiple uses.a new thermos system called ship-mate® (my breeder supply, elk city, ok), is an electronic-chilled vacuum flask that uses a battery inside a thermos to maintain temperature at 5 cº for duration of transport and can record temperature. research is needed to verify accuracy. chilled semen should be shipped as quickly as possible and maintained between 4 5ºc to ensure optimal quality upon delivery. fedex and ups are both reliable for pickup and overnight deliveries. when shipping on a friday for a saturday delivery, be sure to mark appropriate box on shipping label to avoid delivery on monday. when shipping on saturday for a monday delivery, be sure to mark the waybill as an overnight shipment; otherwise, it will be delivered on tuesday if marked as 2 day shipment. clinical theriogenology • volume 12 number 3 • september 2020 211 receiving a chilled semen shipment a chilled semen shipment should be opened upon arrival and frozen bricks inspected. if they are warm, semen temperature is most likely too warm and could influence semen quality and bacterial growth. after 48 hours, extended canine semen maintained at 4 5ºc with antibiotics effectively controlled aerobic bacterial growth, but not when held at room temperature.8 a drop of extended semen should be warmed on a 37ºc slide warmer and examined for motility, morphology and a general estimate of total numbers to compare to data on shipping form. any large discrepancies should be reported to collecting veterinarian to allow for changes in future planned shipments, e.g. using a different extender. stud dogs that have never had semen chilled should have a “chill test” done several weeks prior to shipping where sperm rich fraction is extended using various extenders, refrigerated overnight and motility and morphology verified 24 hours later to determine the extender that maintained best quality overnight. if insemination cannot be performed immediately, extended semen should be refrigerated and used as soon as possible. extended chilled semen is best used within 48 hours of collection.5 canine semen tends to tolerate chilling very well and may often maintain excellent motility for many days (sometimes up to 8 10) regardless of extender used. you do not have to use a specific 5 or 10 day extender to expect similar results; most semen that ships well in a 5 or 10 day extender will ship just as well in many other extenders. some of 5 and 10 day extenders seem to promote agglutination of sperm to coverslip upon warming; although this does not appear to affect fertility, it makes it difficult to accurately assess motility. frozen semen principles frozen semen extenders are very similar in makeup to chilled semen extenders, except most contain glycerol and egg yolk as cryoprotectants to stabilize and protect sperm exposed to the damaging temperatures of liquid nitrogen. during cryopreservation, glycerol, ethylene glycol, or dimethylsulfoxide9 permeates through sperm plasma membrane, interacts with water molecules, and inhibits formation of hydrogen bonds necessary for formation of damaging ice crystals. egg yolk contains lipids lecithin and cephalen5 that are too large a molecular weight to cross cell membranes; therefore, ice crystal formation is inhibited by causing cellular dehydration, freezing point depression and increasing media viscosity at lower temperatures.9,10 semen is slowly cooled to 4 5ºc and then frozen in either straws or pellets. prelabeled polyvinylchloride straws, 0.25 or 0.50 milliliters in volume, are filled with cooled semen and an air bubble located centrally in straw. straws are then sealed and frozen by suspending them over liquid nitrogen vapor and then plunging them directly into liquid nitrogen.5 straws are placed in canes and stored vertically in goblets in liquid nitrogen cryotanks. pellets are made by placing individual 50 100 µl drops of cooled semen into small depressions made in dry ice and then immersing pellets into liquid nitrogen and storing them in pre-labeled cryovials. decision to freeze semen in pellets versus straws is personal choice. pellets are convenient in that the entire vial, usually a calculated amount to breed 1 bitch, is thawed, whereas straws are thawed individually. pellets take less room to store, as vials are packaged in a box and several boxes fit on a cane; each goblet can only hold ~ 6 8 canes of straws. straws, however, are more accepted internationally. straws also prevent cross contamination with anything that may be in the liquid nitrogen and make it impossible to combine or mix semen from 2 animals together. vapor shipper dewars frozen semen is shipped in a “dry shipper” dewar, a vacuum-insulated canister lined with a hydrophobic material that absorbs liquid nitrogen, thereby keeping the frozen semen stable in nitrogen vapor during transport. liquid nitrogen itself is labeled as a hazardous material by us department of transportation and cannot be used for shipment, making it necessary to use a dry shipper to transport semen.11 canister is housed in an outer heavily padded, plastic container that helps keep the canister upright during transport. clinical theriogenology • volume 12 number 3 • september 2020212 a face shield and protective insulated gloves should be worn when working with liquid nitrogen. canister will need to be filled, or “charged” 24 36 hours prior to a shipment, by slowly adding liquid nitrogen to canister using a funnel until level reaches bottom of the neck. care must be taken not to spill liquid on vacuum cap as this may cause shrinkage of seal allowing air to leak into vacuum space. neck plug and lid are replaced and canister is allowed to sit dormant so nitrogen can permeate absorbent. this process is repeated until level of liquid nitrogen no longer falls. weigh shipper and check manufacturer’s manual as there are suggested full weights to indicate normal absorbent filling12 and holding times. evaporation rate of a vapor shipper is calculated to monitor charging capability. vapor shipper is charged, liquid nitrogen dumped out and tank weight recorded. canister is weighed again after sitting dormant for 24 hours and second weight recorded. evaporative loss per day is calculated by multiplying difference between 2 weights by 0.5606. if this number is > 1.9 (liters/day), vapor shipper should either be replaced or sent to manufacturer to be revacuumed. vapor shippers should also be weighed prior to shipping and when they return, to confirm that evaporative rate/day is within acceptable limits. frozen semen shipment just prior to shipping, tank needs to be inverted over an insulated bucket and held until liquid no longer runs out. canister neck plug and lid should be replaced and canister watched for 1 hour to be certain no condensation or frost collects around top that could indicate a leaking tank. canes loaded with straws or cryovials are then transferred to goblet inside canister, and neck plug and lid inserted. lid is then anchored shut with a zip tie. all paperwork (copy of freeze report, instructions for thawing, and return fedex label) is placed in a manila envelope next to canister, along with any necessary supplies for inseminating veterinarian and outer container is closed and anchored with a zip tie, or left unanchored if canister needs to be sealed by us department of agriculture for an international shipment. like chilled semen, fedex or ups are preferred carriers and tracking numbers should be sent to receiving veterinarian. receiving frozen semen when you receive a frozen semen shipment, container should first be inspected for damage. it is then opened and inner canister lid carefully removed watching for a vapor “puff,” confirming that canister is still charged. contents should be quickly transferred to a bucket of liquid nitrogen, carefully inventoried to verify shipment of correct semen and then transferred into a regular liquid nitrogen storage tank, as shipper stability is unknown. if you do not have any cryostorage tanks and need to hold frozen semen a few days before using, it is best to either add liquid nitrogen to dry shipper daily or arrange for arrival of frozen semen on insemination day. before returning canister, remove goblet and invert it over cryobucket to ensure that a cryovial or straw did not dislodge from cane during transit. closely examine cryovials containing pellets to be sure that vials themselves are not fractured into tiny shards and that semen is still in a round, pelleted form and not a thawed glob on vial bottom. straws should also be examined for damage such as cracking or extrusion of end plugs. any signs of tank failure or damage should be immediately reported to vapor shipper facility. maintenance of storage canisters liquid nitrogen level in storage tanks should be measured weekly using a calibrated cryostick and recorded in a log to monitor evaporation loss prior to topping them off. any tank evaporating at a faster rate than previously noted should be removed from service. conclusion canine semen is routinely being shipped everywhere in us as well as internationally, allowing ease of breeding and increasing availability of diverse genetic material. there are many extenders and shipping box options available for chilled semen to preserve semen quality during transport. frozen semen can be stored indefinitely, allowing access to several genetic lines. a great deal of paperwork and maintenance is involved when dealing with semen shipping and freezing to ensure quality and accuracy. clinical theriogenology • volume 12 number 3 • september 2020 213 hopefully, evidence-based research will provide standardized protocols for canine semen shipping, cryopreservation and insemination in near future to increase chances of producing healthy, normal-sized litters. conflict of interest there are no conflicts of interest to declare. references 1. england cw, allen we: factors affecting the viability of canine spermatozoa. ii. effects of seminal plasma and blood. theriogenology 1992;37:373-381. 2. robert ma, jayaprakash g, pawshe m, et al: collection and evaluation of canine semen-a review. int j environ sci technol 2016;5:1586-1595. 3. root kustritz mv: applied small animal andrology. in: chenoweth pj, lorton sp, editors. animal andrology theories and applications. wallingford (uk); cab international: 2014. p 177-196. 4. ponglowhapen s, essen-gustavsson b, forsberg cl: influence of glucose and fructose in the extender during longterm storage of chilled canine semen. theriogenology 2004;62:1498-1517. 5. johnston sd, root kustritz mvr, olson pns: semen collection, evaluation and preservation. in: canine and feline theriogenology. w.b. saunders; philadelphia: 2001. p. 298-304. 6. escobar sr: adding reproduction to your practice: the good, the bad and the ugly. proc society for theriogenology annual conference and sft/act symposium 2004; p. 237-239. 7. lopes g, simoes a, ferreira p, et al: differences in preservation of canine chilled semen using different transport containers. anim reprod sci 2009;112:158-163. 8. barstow c, root kustritz mv: effect of antibacterial agents in semen extender on bacterial growth in extended canine semen held at 5 degrees c and 20 degrees c for up to 48 hours. clinical theriogenology 2014;6:231-238. 9. eilts be: theoretical aspects of canine semen cryopreservation. theriogenology 2005;64:692-697. 10. holt wv, penfold lm: fundamental and practical aspects of semen cryopreservation. in: chenoweth pj, lorton sp, editors. animal andrology theories and applications. wallingford (uk): cab international; 2014; p. 76-99. 11. dry shippers. university of washington environmental health and safety pamphlet. 2002. 12. mve operating instruction manual for vapor shipper dewars; ref 11562640 rev d;6/05. p. 2-9. clinical theriogenology • volume 12 number 3 • september 2020214 011_ms-11 randall introduction much progress has been made in our understanding of tritrichomonas foetus (t. foetus) and trichomoniasis since the organism was implicated in causing reproductive failure in cattle. this review will cover the following topics: preputial distribution, bull susceptibility, sampling devices, culture, polymerase chain reaction (pcr) assay, immune response and vaccines, and treatment. preputial distribution distribution of t. foetus in the preputial cavity of bulls was documented in 1943.1 highest number of organisms were isolated in the midshaft and caudal portion of free penis, preputial area surrounding penis, galea glandis, and near preputial opening, in that order.1 limited effort was made to reexamine these findings until 1999, when the presence of t. foetus in the midshaft and caudal penis and to a lesser degree in the prepuce was evident in paraffin-embedded sections of preputial tissues.2 bull susceptibility it has long been recognized that bull age affected susceptibility to t. foetus. older bulls had increased risk of becoming t. foetus carriers by contracting infection through breeding activity, their longevity in the herd, and their hierarchical dominance.3 this age-related phenomenon of longevity and dominance in bulls was partially substantiated in 1970 reports from australia.4,5 as a control measure, bulls > 8 years of age were replaced by young bulls, negative for t. foetus. prevalence of infection remained significantly lower in replacement bulls compared to older bulls (at their removal from the herd) suggesting that young bulls were less likely to be carriers. bovine trichomoniasis jeff ondrak 6u ranch, steele city, ne abstract the association of tritrichomonas foetus with bovine reproductive failure was first identified ~ 130 years ago, and it became apparent in the us cattle population ~ 100 years ago. research has increased our understanding of this disease. current knowledge regarding clinical signs, diagnosis, and prevention is reviewed. keywords: tritrichomonas foetus, bovine, diagnosis, treatment, vaccine several studies assessed the correlation between bull’s age and risk of t. foetus infection. although these studies suggested that the risk for t. foetus carrier bull status increased in aged bulls, this conclusion was questionable due to limited bull numbers and potential for bias due to uneven distribution of age groups. furthermore, these studies lacked controls. many detailed studies6-8 attempted to account for these factors. in 1 study, 2% of bulls < 3 years of age had t. foetus infection (p < 0.025) compared to 6.7% in bulls > 4 years.6 subsequently, 2 larger epidemiologic studies7,8 had similar trends. mean age of infected bulls was 5.5 ± 1.6 years (p < 0.001) and mean age of uninfected bulls was 3.9 ± 2.3 years.7 bulls > 5 years of age were 2.2 times (or = 2.2, 95% ci; 1.1 4.3: p = 0.022) more likely to be t. foetus positive than bulls < 5 years of age.8 a proposed explanation for this relationship between age and t. foetus carrier status is the development of crypts in the epithelium of penis and prepuce in aged bulls. a similar relationship was established for bovine vibriosis.9 susceptibility increased substantially at > 5 years of age and was associated with and possibly linked to an increase in size and number of penile epithelial crypts.9 trichomoniasis investigators likewise implicated the development of crypts in aged bulls as a cause for age-related susceptibility to t. foetus10,11 and immunohistochemical staining of paraffin embedded sections of preputial tissues apparently supported this explanation.12 however, more recent work questioned the validity of this viewpoint.13 sampling devices various sample collection techniques were proposed, including preputial swabbing using a cotton swab,14 preputial scraping clinical theriogenology 2021; 13: 221 with a specially designed instrument,15 preputial lavage,14 and preputial aspiration via a pipette.16 swabbing consisted of passing a cotton swab into preputial cavity to the fornix area (via a speculum in the preputial orifice), moving the swab back and forth and rotating it around the glans penis, and then removing the swab.14 pipette aspiration was more reliable for direct examination of specimens than swabs if lower numbers of t. foetus were present in the prepuce.17 although the number of organisms present in the prepuce dictated pipette aspiration versus preputial swabs, growth of organisms in an enrichment medium (rather than direct examination) minimized this difference.14 a long, thin metal rod with shallow grooves (perpendicular to the shaft) at 1 end was used to scrape samples from the bovine preputial cavity.15,18 specimen collection with these devices was achieved by passing the grooved end of the rod into the preputial cavity, thrusting the head forward into the fornix area and drawing it back repeatedly (20 30 cycles) then withdrawing it from the prepuce and flushing the material from the instrument. this device was superior to preputial lavage to identify t. foetus in preputial specimens; however, supporting data and statistical analyses were not clear.15 this device was also superior to pipette aspiration for direct examination of the specimen for t. foetus identification. placing the specimen in a culture medium within 2 hours after collection resulted in 3 times more prolonged t. foetus survival in culture compared to specimens aspirated by pipette.18 there were no significant differences between techniques (sensitivity for pipette aspiration was 91.6% [95% ci, 84.3 95.7%] and the scraping device was 93.3% (95% ci, 87.2 96.7%).19 details regarding preputial lavage or douche technique vary among investigators. in general, a volume of sterile normal or phosphate buffered saline is instilled into preputial cavity with a syringe or rubber bulb via a pipette whose free end is positioned in the fornix area, the prepuce is massaged over the penis while holding the preputial orifice closed to prevent loss of saline, and lavage fluid is collected by aspirating with an instillation apparatus. fluid retrieved by this method is typically placed in a test tube and centrifuged or preputial debris allowed to settle to form a pellet at the bottom of the container. the pellet is examined directly for t. foetus or inoculated into an enrichment medium and analysed.14,20 a comparison of the efficacy of preputial lavage and pipette aspiration for t. foetus recovery for direct examination concluded that lavage was more efficient for recovery of t. foetus at lower preputial concentrations.14 pipette aspiration had 65 positive specimens out of 83 (78.3% sensitivity [95% ci, 67.6 86.3%]), whereas lavage had 69 positive specimens out of 84 (82.1% sensitivity [95% ci, 71.9 89.3%]).20 authors concluded that these 2 collection methods were comparable as sensitivities were not significantly different. preputial lavage and pipette aspiration specimens for culture and pcr analysis were compared. 21 sensitivities for these 2 methods for pcr were not significantly different, although the difference approached significance when dna extraction from the specimen was delayed for 5 days.21 a modified glass vaginal pipette and a rubber bulb (used by earlier investigators to collect t. foetus samples from female cattle) were used for aspirating samples from bulls’ preputial cavities.16 the free end of the pipette is passed into the preputial cavity to the fornix, aspirated with the rubber bulb as the free end of the pipette is moved back and forth over the surface of the penis and prepuce multiple times, and then the pipette is removed from the preputial cavity after gently releasing the suction from the rubber bulb. this device, adapted to use a plastic infusion pipette and a disposable syringe, has become the primary sampling technique in the us, due to its ease of use and lack of a better alternative. culture and pcr prior to pcr culture, microscopic examination was the diagnostic test of choice for bovine trichomoniasis and sensitivity estimates ranged from 67.7 to 98.4%.22,23 although multiple media and solutions were used for the transport and enrichment of samples, a proprietary medium in a specially designed in vitro cultivation envelope (inpouch™tf, biomed diagnostics, inc., white city, or) improved the ease of sample handling and is widely used throughout the us. examination of the inoculated inpouch™tf (tf) is done by fixing the lower portion of the pouch in a plastic clip (size similar to a microscope slide). the clip is systematically scanned for several minutes for motile organisms (morphologically consistent with t. foetus), daily for 6 days, as recommended by the manufacturer.22,24 sensitivity of tf cultures was 98.4% (150 bulls each sampled once). cultures were examined once daily for 5 days maintained at 37˚c.23 culture sensitivities were 91.6% (95 ci, 84.3 95.7%)19 and 95.8% (95% ci, 89.6 98.5%).24 furthermore, specimen handling must be impeccable. influence of various preanalytical factors on t. foetus detection in inoculated tf with equal numbers of preputial lavage and pipette aspirated specimens from bulls known to be naturally infected was determined. sensitivities were not significantly different for preputial lavage versus pipette aspiration; therefore, data were combined to determine sensitivity. total number of positive specimens by both collection methods in tf (n = 73) was divided by the total number of specimens collected from known positive bulls (n = 83) and the overall sensitivity was 88%.20 a large beef cattle herd, investigated for infertility, had a sensitivity of 73%.7 authors concluded that low culture sensitivities was likely due to reduced specimen quality due to various preanalytical conditions, including: extensive and remote cattle working facilities that made handling and transport of large numbers of specimens difficult; fractious bulls; harsh clinical theriogenology 2021; 13: 222 environmental conditions; inconsistent bull identification; contamination of specimens with dirt and feces; and a large number (n = 750) of specimens.7 similarly, the overall sensitivity of culture was 70.4% in a herd investigated for infertility. however, sensitivity for specimens collected during weeks 1 and 3 was 83.3% and for week 2 was 44.4%. investigators concluded preanalytical factors (e.g. variations in collection, handling, culture techniques) and fluctuating preputial t. foetus populations might have decreased week-2 sensitivity. this study highlighted the significance of preanalytical factors over analytical factors when reasonable analytical techniques are used.25 lowest reported sensitivity (67.8%, 95% ci; 51.1 84.1) for tf culture was from experimentally infected bulls sampled weekly for 6 weeks via pipette aspiration, with the tf incubated at 3˚c for 7 days and examined on days 1, 3, 5, and 7 postinoculation. sensitivity was calculated as the number of positive culture samples out of the total number of samples from experimentally infected bulls. this was probably an underestimation of sensitivity, as 4 bulls produced no positive specimens by culture over the 6-week study and were most likely not t. foetus infected.22 although the sensitivity of t. foetus culture has a wide range due to preanalytical and analytical factors that varied among studies, until recently, diagnostic specificity of t. foetus culture had been assumed to be nearly 100%. based on brightfield microscopy (100 400 x), other trichomonads in specimens from virgin bulls had morphological and motility characteristics similar to t. foetus. however, further testing of these trichomonads, including staining, scanning electron microscopy, and pcr revealed 4 anterior flagellae. it was speculated that these organisms were commensal (intestinal) trichomonads (tetratrichomonas pavlovi or tetratrichomonas buttreyi), likely present in feces and transferred to the prepuce during sodomy.26 to overcome t. foetus culture sensitivity and specificity concerns, investigators examined the value of pcr as a diagnostic assay. the assumptions were: amplification of dna segments specific to t. foetus would reduce or eliminate false positives, thereby increasing specificity and identification of positive specimens without the presence of a living organism; or when specimens contained fewer organisms, that would increase testing sensitivity by decreasing false negatives. a pcr assay was developed utilizing primers tf1 and tf2 to amplify a 162-base pair (bp) product from an unspecified region of t. foetus dna. assay sensitivity (88.6%) was comparable to traditional culture, despite its ability to detect as few as 1 organism in pure medium and 10 in medium with smegma. no false positives were detected from 8 t. foetus negative bulls.27 primers tfr3 and tfr4 were developed to target the 5.8s rrna, its1, and its2 regions for pcr amplification while incorporating an uracil dna glycosylase system to prevent dna carryover from previous reactions and a dna enzyme immunoassay (deia) for amplicon detection. the 347 bp amplification product was obtained from 8 isolates of t. foetus, tritrichomonas suis, and tritrichomonas mobilensis, but no amplification product was produced from pcr assays of specimens containing other trichomonads, bacterial dna, or bovine dna. the assay was able to detect quantities of dna equivalent to a single t. foetus organism in pure media and as few as 50 organisms per ml in specimens containing smegma, bacteria, and other debris. false positive specimens that had been an issue with the tf1-2 pcr were not observed with the tfr3-4 assay, indicating higher test specificity.28 a pcr (tfr3-4 pcr) with a reported detection limit of 2 organisms per ml of specimen had a sensitivity of 31 90% and a specificity of 98%.21 for a detection limit of 5 organisms per ml, the diagnostic sensitivity was 98.3% and the diagnostic specificity was 93.7%.29 real-time pcr (rtpcr) was used for t. foetus diagnosis using primers tff2 and tfr2 and a fluorescent probe after employing a heat lysis method for crude cell lysate preparation. the sequence of this tfr2 differed from the previously mentioned tfr2. the detection limit for rtpcr was a single cell equivalent for laboratory-spiked preputial smegma specimens with less than a cell equivalent per assay reliably detected from several heat-lysed specimens; this was a 2500-fold higher analytical sensitivity than culture and similar to tfr3-4 pcr in analytical sensitivity. a field-based comparison30 suggested that rtpcr was superior to culture. specimens from 159 animals in known t. foetus infected herds had 3 positive specimens for culture and 14 positive specimens for rtpcr. however, no repeat testing of test positive animals was undertaken to confirm their tf status, leaving open the possibility of rtpcr false positive results. no rtpcr diagnostic sensitivity or specificity estimates were provided.30 immune response and vaccines precise immunological mechanism for t. foetus clearance from the female bovine reproductive tract is unknown. antibodies and complement activated by t. foetus surface antigens conferred protection from the organism.31 elimination occurred when trichomonads were opsonized with antibodies and complement before exposure to neutrophils.32 repeated t. foetus infections appeared to cause an anamnestic response. the interval cows remained infected decreased in subsequent exposures, when mean durations of infection for first, second, and third exposures were 20.3, 9.8, and 11 weeks respectively.33 after a previous infection with t. foetus, trichomonads were cleared from heifers’ reproductive tracts within 3 weeks after reinfection.34 other researchers reported that 6 cows were resistant to reinfection when exposed to t. foetus 4 months after recovery from a previous infection.35 however, this immunologic memory appeared to be short lived. 36 estimated clinical theriogenology 2021; 13: 223 length of partially protective immunity was < 15 months, based on cow fertility following t. foetus exposure.36 although clearance of the organism from the female reproductive tract is typical, infections persisted for up to 300 days37 or up to 22 months postbreeding.38 carrier cows remained infected through a normal pregnancy with t. foetus isolated up to 9 weeks39 or 63 97 days40 after delivering an apparently normal calf. despite not knowing the exact immune mechanism, a vaccine was reported to reduce losses associated with t. foetus infection in heifers by lowering the rate of t. foetus infection, decreasing the estimated duration of infection, and interfering with the ability of t. foetus to cause early embryonic death.41 a commercial vaccine (trichguard®, boehringer ingelheim vetmedica, inc., st. joseph, mo) substantially increased pregnancy and calving rates in heifers and reduced losses associated with t. foetus infection.42 vaccination led to resistance to infection up to 5 years of age43 and resistance to t. foetus colonization.44 however, for bulls, a critical review and meta-analysis of the whole-cell killed vaccine reported a likely overestimation of the benefits of vaccination and a low to very low quality of evidence.45 treatment although various substances can clear t. foetus from infected animals, nitroimidazole derivates are commonly used.46 single ipronidazole treatment was effective in 92.8% bulls and 3 daily treatments in 100% of bulls.47 regardless of their effectiveness, toxicity and resistance concerns remain unknown.46 furthermore, the use of nitroimidazole derivates in food producing animals is currently not approved in the us. use of a commercially available vaccine as a therapeutic agent did not significantly decrease infection prevalence.48 currently, no effective legal treatment for bovine trichomoniasis is available in the us. conclusion ever since tritrichomonas foetus was first described as a cause for reproductive failure in cattle, a substantial effort has been made to better understand the organism’s interaction with the bovine reproductive tract and to improve diagnosis, prevention, and treatment. despite great strides, there are many opportunities for further research in pathogenesis, immunology, diagnostics, prevention, and treatment. conflict of interest none to report. references 1. hammond dm, bartlett de: the distribution of trichomonas foetus in the preputial cavity of infected bulls. am j vet res 1943;4:143-149. 2. rhyan jc, willson kl, wagner b, et al: demonstration of tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally infected bulls. vet pathol 1999;36:406-411. 3. van eenennaam al, weaber rl, drake dj, et al: dna-based paternity analysis and genetic evaluation in a large, commercial cattle ranch setting. j anim sci 2007;85:3159-3169. 4. christensen hr, clark bl, parsonson im: incidence of tritrichomonas foetus in young replacement bulls following introduction into an infected herd. aust vet j 1977;53:132-134. 5. christensen hr, clark bl: spread of tritrichomonas foetus in beef bulls in an infected herd. aust vet j 1979;55:205. 6. bondurant rh, anderson ml, blanchard p, et al: prevalence of trichomoniasis among california beef herds. j am vet med assoc 1990;196:1590-1593. 7. rae do, chenoweth pj, genho pc, et al: prevalence of tritrichomonas foetus in a bull population and effect on production in a large cow-calf enterprise. j am vet med assoc 1999;214:1051-1055. 8. rae do, crews je, greiner ec, et al: epidemiology of tritrichomonas foetus in beef bull populations in florida. theriogenology 2004;61:605-618. 9. samuelson jd, winter aj: bovine vibriosis: the nature of the carrier state in the bull. j infect dis 1966;116:581-592. 10. ball l, mortimer rg, cheney jm: trichomoniasis: diagnosis, pathogenesis, treatment and control. proc annu conf am assoc bov pract 1984;16:163-165. 11. bondurant rh, honiberg bm: trichomonads of veterinary importance. in: kreier jp: editor. parasitic protozoa. 9th edition, san diego; academia press: 1994. p. 112-188. 12. rhyan jc, wilson kl, wagner b, et al: demonstration of tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally infected bulls. vet pathol 1999;36:406-411. 13. strickland l, edmondson m, maxwell h, et al: surface architectural anatomy of the penile and preputial epithelium of bulls. clin theriogenology 2014;6:445-451. 14. fitzgerald pr, hammond dm, miner ml, et al: relative efficacy of various methods of obtaining preputial samples for diagnosis of trichomoniasis in bulls. am j vet res 1952;13:452-457. 15. sutka p, katai pl: rapid demonstration of bull trichomonadosis in unstained smear preparations from preputial scrapings. acta vet hung 1969;19:385-389. 16. hammond dm, bartlett de: establishment of infection with trichomonas foetus in bulls by experimental exposure. am j vet res 1943;4:61-65. 17. hammond dm, bishop vr, jeffs g, et al: a quantitative study of trichomonas foetus in preputial samples from infected bulls. am j vet res 1950;11:308-314. 18. tedesco lf, errico f, del baglivi lp: diagnosis of tritrichomonas foetus infection in bulls using two sampling methods and a transport medium. aust vet j 1979;55:322-324. 19. parker s, campbell j, ribble c, et al: comparison of two sampling tools for diagnosis of tritrichomonas foetus in bulls and clinical interpretation of culture results. j am vet med assoc 1999;215;231-235. 20. schonmann mj, bondurant rh, garner ia, et al: comparison of sampling and culture methods for the diagnosis of tritrichomonas foetus infection in bulls. vet rec 1994;134:620-622. 21. mukjufhi n, irons pc, michel a, et al: evaluation of a pcr test for the diagnosis of tritrichomonas foetus infection in bulls: effects of sample collection method, storage and transport medium on the test, theriogenology 2003;60:1269-1278. clinical theriogenology 2021; 13: 224 22. cobo er, favetto ph, lane vm, et al: sensitivity and specificity of culture and pcr of smegma samples of bulls experimentally infected with tritrichomonas foetus. theriogenology 2007;68:853-860. 23. appell lh, mickelsen wd, thomas mw, et al: a comparison of technique used for the diagnosis of tritrichomonas foetus infections in beef bulls. agri pract 1993;14:30-34. 24. parker s, campbell j, gajadhar a: comparison of the diagnostic sensitivity of a commercially available culture kit and a diagnostic culture test using diamond’s media for diagnosing tritrichomonas foetus in bulls. j vet diagn invest;15:460-466. 25. peter da, fales wh, miller rb: tritrichomonas foetus infection in a herd of missouri cattle. j vet diagn invest 1995;7:278-280. 26. bondurant rh, gajadhar a, campero cm, et al: preliminary characterization of a tritrichomonas foetus-like protozoan isolated from preputial smegma of virgin bulls. bov pract 1999;33:124-127. 27. ho msy, conrad pa, conrad ph, et al: detection of bovine trichomoniasis with a specific dna probe and pcr amplification system. j clin micro 1994;32:98-104. 28. felleisen rsj, lambelet n, bachmannp, et al: detection of tritrichomonas foetus by pcr and dna enzyme immunoassay based on rrna gene unit sequences. j clin micro 1998;36:513-519. 29. mutto aa, giambiaggi s, angel so: pcr detection of tritrichomonas foetus in preputial bull fluid without prior dna isolation. vet parasitol 2006;136:357-361. 30. mcmillen l, lew ae: improved detection of tritrichomonas foetus in bovine diagnostic specimens using a novel probe-based real time pcr assay. vet parasitol 2006;141:204-215. 31. aydintug mk, leid rw, widders pr: antibiody enhances killing of tritrichomonas foetus by the alternative bovine complement pathway. infect and immun 1990;58:944-948. 32. aydintug mk, widders pr, leid rw: bovine polymorphonuclear leukocyte killing of tritrichomonas foetus. infect and immun 1993;61:2995-3002. 33. clark bl, dufty jh, parsonson im: the frequency of infertility and abortion in cows infected with tritrichomonas foetus var. brisbane. aust vet j 1986;63:31-32. 34. skirrow sz, bondurant rh: induced tritrichomonas foetus infection in beef heifers. j am vet med assoc 1990;196:885-889. 35. murname d: field and laboratory observations on trichomoniasis of dairy cattle in victoria. aust vet j 1959;35:80-83. 36. clark bl, dufty jh, parsonson im: the effect of tritrichomonas foetus infection on calving rates in beef cattle. aust vet j 1983;60:71-74. 37. mancebo oa, russo am, carabajal ll, et al: persistence of tritrichomonas foetus in naturally infected cows and heifers in argentina. vet parasitol 1995;59:7-11. 38. alexander gi: an outbreak of bovine trichomoniasis in queensland and its control. aust vet j 1953;29:61-66. 39. skirrow sz: identification of trichomonad-carrier cows. j am vet med assoc 1987;191:553-554. 40. goodger wj, skirrow sz: epidemiologic and economic analysis of an unusually long epizootic of trichomoniasis in a large california dairy herd. j am vet med assoc 1986;189:772-776. 41. kvasnicka wg, hanks d, huang jc, et al: clinical evaluation of the efficacy of inoculating cattle with a vaccine containing tritrichomonas foetus. am j vet res 1992;53:2023-2027. 42. edmondson ma, joiner ks, spencer ja, et al: impact of a killed tritrichomonas foetus vaccine on clearance of the organism and subsequent fertility of heifers following experimental inoculation. theriogenology 2017;90:245-251. 43. clark bl, dufty jh, parsonson im: immunisation of bulls against trichomoniasis. aust vet j 1983;60:178-179. 44. cobo er, corbeil lb, gershwin lj, et al: preputial cellular and antibody responses of bulls vaccinated and/or challenged with tritrichomonas foetus. vaccine 2010;28:361-370. 45. baltzell p, newton h, o’connor am: a critical review and meta-analysis of the efficacy of whole-cell killed tritrichomonas foetus vaccines in beef cattle. j vet intern med 20013;27:760-770. 46. skirrow sz, bondurant rh: bovine trichomoniasis. vet bull 1988;58:591-603. 47. skirrow sz, bondurant rh, farley j, et al: efficacy of ipronidazole against trichomoniasis in beef bulls. j am vet med assoc 1985;187:405-407. 48. alling c, rae do, ma x, et al: systemic humoral immunity in beef bulls following therapeutic vaccination against tritrichomonas foetus. vet parasitol 2018;255:69-73. clinical theriogenology 2021; 13: 225 bovine trichomoniasis background a multiparous, american quarter horse mare in late pregnancy was presented for a mass protruding from the vagina. mare was bred naturally the previous summer. based on breeding dates, the mare was 340 days in pregnancy and foaling was anticipated in 31 days. mare had multiple normal pregnancies and foalings in the past 10 years. mare was housed on pasture, was up to date on vaccinations, and had received routine anthelmintics. case presentation mare was mildly unconfortable with no evidence of active straining. mare had a temperature of 37.1°c, a pulse of 60 beats/ minute, and a respiratory rate of 20 breaths/minute. mucous membranes were pink and moist and all peripheral lymph nodes were within normal limits. mammary glands appeared enlarged, with slightly larger right gland, and waxing of teats was noticed. gut sounds and digital pulses were within normal limits. left front fetlock appeared broken forward at a steep angle, and the margin of the hoof was irregular, with longer medial aspect. mare had adequate flesh with a body condition score of 5/9, weighing ~ 500 kg. normal urination and defecation were noticed after physical examination. transrectal palpation revealed the presence of a live normally positioned fetus. due to late pregnancy with appreciable fetal movement, ultrasonography was not performed. examination of the perineal area revealed the presence of a large pink to red colored mass of apparent vaginal tissue protruding from the vaginal prolapse in an american quarter horse mare carolyn chisholm,a kevin walters,a david christiansen,a heath king,a richard hopper,b darcie sidelingera acollege of veterinary medicine, mississippi state university, mississippi state, ms bcollege of veterinary medicine, auburn university, auburn, al abstract a 15 year old, multiparous, american quarter horse mare in late pregnancy, was presented for a mass protruding from the vagina that was noticed 12 hours ago. physical examination revealed a large, 25 x 12.5 x 12.5 cm, pink to red vaginal protrusion with moderate amounts of mucoid discharge. on transrectal palpation, it was evident that the mare was carrying a live fetus. reminder of the physical examination was unremarkable. vaginal prolapse was diagnosed that was manually reduced and an episioplasty procedure was performed. mare was discharged with instructions to be monitored closely for 14 days and then re-evaluated, unless there were signs of impending parturition. owner was instructed to seek immediate veterinary care if signs of parturition were noted. foaling occurred without the knowledge of the owner, resulting in labial tear. mare and a premature foal were presented at the hospital. foal died during treatment. mare was euthanized months later due to unrelated lameness. to authors’ knowledge, this is the first case report of a vaginal prolapse in a mare. keywords: mare, vaginal prolapse, episioplasty vulva (figure). there was moderate amount of mucoid discharge without apparent changes to the cervix. mare’s hindquarters and perineum appeared relaxed and normal. after a thorough digital examination and further inspection of the protruding tissue and adjacent structures, vaginal prolapse without cervical involvement, was diagnosed treatment mare was restrained in stocks and was given intravenously 200 mg of xylazine and 10 mg of acepromazine. vaginal tissue and surrounding perineal region were thoroughly cleaned using dilute betadine solution, cotton, and warm water. vaginal tissue was manually reduced using gentle pressure and manipulation. nitrofurazone ointment was liberally applied to vaginal tissue and labia. after reduction of the prolapsed tissue, episioplasty was performed to reduce vulvar opening length.1 labial tissue was desensitized with 20 ml of 2% lidocaine, given subcutaneously near the mucocutaneous junction starting at the dorsal commissure and extending ventrally ~ 3 5 cm. mucocutaneous junction of labia was incised, using a # 10 scalpel blade, starting at the dorsal commissure and extending ventrally until ~ 1 2 cm below the ischiatic arch,1 and labial tissue was not removed. after incision, a modified vertical mattress suture pattern (modified caslick’s) was performed using catgut # 1 absorbable suture to oppose labial edges to minimize recurrence of vaginal prolapse.1,2 mare was given intravenously clinical theriogenology 2021; 13: 95 1.1 mg/kg of flunixin meglumine for pain and inflammation. additionally, 2 liters of mineral oil and 1 liter of water were given via a nasogastric tube as a laxative. mare was treated orally with trimethoprim and sulfamethoxazole tablets (32 mg/kg, every 12 hours for 14 days) to prevent ascending placentitis. outcome mare was scheduled for 14 days re-examination, suture removal, and for possible episiotomy. four days prior to scheduled examination, mare foaled in the night without owner’s presence and placenta was not found. mare and foal were immediately transported to the hospital. mare had a temperature of 37.8°c, a pulse of 60 beats/minute, and a respiratory rate of 28 breaths/ minute. abdominal gut sounds and digital pulses were within normal limits and mare’s body condition score was 5/9. softness of hindquarters suggested recent foaling. mammary gland appeared enlarged with waxing of teats with no secretions. due to foal’s condition, colostrum was not collected. mare had a tear involving the dorsal two-thirds of right labia. vaginal tissue appeared normal with no evidence of prolapse. sutures were intact and the labial tear was repaired by reconstruction with suture (catgut # 1) applied in an interrupted suture pattern after routine surgical preparation and local infusion of lidocaine. a laterally recumbent filly foal was presented. foal was hypovolemic, hypothermic, and hypoglycemic. foal appeared premature with a silky hair coat, bulging forehead, limb laxity, poor muscle tone, and floppy/curled ears. complete blood count revealed leukopenia with moderate neutrophilia and lymphopenia. a bear hugger was used to correct hypothermia and the foal was given intravenously isotonic crystalloid fluids with dextrose. plasma transfusion was later given and throughout treatment her respiratory rate was fluctuant. foal experienced multiple episodes of regurgitation and it was elected to pass a nasogastric tube. shortly after an indwelling nasogastric tube was placed, foal experienced respiratory arrest, became agonal and died. necropsy suggested fetal/neonatal sepsis secondary to ascending placentitis. mare was kept in hospital for a night. post foaling vaginal examination or culture were not performed to allow healing of reduced prolapsed tissue. mare was continued on oral trimethoprim and sulfamethoxazole (32 mg/kg every 12 hours) for an additional 7 days. mare had normal defecation and urination through the night with normal intake of water and was discharged the next day. discussion vaginal prolapse has been documented in cattle, goats, sheep, camels, dogs, cats, and women.1-16 to authors’ knowledge, it has not been reported in mares. a mare had cervical hyperplasia and mass that led to vaginal protrusion.17 a vaginocervical prolapse was reported in a nonpregnant mare.18 the present case had a true vaginal prolapse. possible etiologies for this condition in mares include: increased abdominal pressure in late pregnancy leading to increased straining, abnormal changes in hormone concentrations, or weakened tissue from previous pregnancies. in this case, the exact etiopathogenesis is unknown. combination of vaginal tissue laxity and concurrent increased abdominal pressure experienced due to late pregnancy might have contributed to this condition. mare had no history of urinary problems or gastrointestinal irregularities. there were also no physical evidence or history of urine scalding, loose stool, or abnormal conformation that would cause the mare to figure. gross image of vaginal prolapse clinical theriogenology 2021; 13: 96 have a pneumovagina. differential diagnoses for this condition include: uterine and urinary bladder prolapses, and vaginal, cervical and vulvar masses. numerous factors in other species were implicated in contributing to vaginal prolapse. imbalance of hormonal concentrations such as lower progesterone with higher estrogen in dogs have been observed to cause vaginal hyperplasia and subsequent prolapse.3,5 superstimulation of ovaries using follicle stimulating hormone might lead to vaginal prolapse in sheep and cattle.4,6 in ruminants, concentrations of calcium and phosphorus imbalances, poorly digestible roughage, extreme cold weather, and internal gastrointestinal parasites (increases in abdominal pressure) can predispose to vaginal prolapse.4,6 other factors include, excess perivaginal fat, prior perivaginal tissue injury, poor conformation, and incompetence of the constrictor vestibule or vulvae musculature.4,6,8,9 vaginal prolapse most commonly occurs in multiparous women among the ages of 40 70.7-9 in most species, it occurs during the last few weeks of pregnancy when intraabdominal pressure is maximum.6 a hereditary component has also been suggested in cattle and sheep.1,4,6 treatment of vaginal prolapse varies among species. in smaller companion species (dogs and cats), ovariohysterectomy or ovariectomy was suggested to remove the effect of ovarian hormones.3,5 in livestock species, affected animals can be treated with a temporary fixation method such as a buhner’s suture or bootlace stitch, prolapse pins, or an ewe savor as used in ewes and does. it is also recommended that livestock be culled after parturition due to the hereditary component of the condition and the probability of reoccurrence.1 some owners elect to cull prior to parturition due to case management. if elected to cull after parturition, the animal must be monitored closely multiple times a day to determine whether the temporary fixation needs removal to prevent peripartum complications. due to the possibility of reoccurrence and the potential hereditary component, we recommended no future breedings for this mare. although this mare was placed on antibiotics after vaginal prolapse reduction, she delivered a septic foal likely as a result of ascending infection due to cervical and vaginal exposure, although the cervix appeared normal on presentation. daily monitoring for ascending placentitis might have been beneficial. it is also not known whether existing placentitis predisposed this mare to vaginal prolapse. uterine ultrasongraphy could have helped to rule out placentitis. additionally, after treatment, daily ultrasonography, determination of serum progesterone and estrone sulfate concentrations, and calcium concentrations in mammary secretions could have been used to monitor for ascending placentitis. however, hospitalization and daily monitoring was not a practical economic option for the client. for mares with ascending placentitis, a multi-modal approach is typically required to control infection, inflammation and to promote uterine quiescence. management options for such an approach include: induction of parturition to deliver the foal from the infectious environment, tocolytics to suppress labor, progestogens, pentoxyfyline, and antiprostaglandin treatment. in retrospect, at discharge from the hospital after episioplasty procedure, we could have emphasized the importance of frequent observation of the mare for signs of parturition and for vulvar discharge. mare was euthanized months later due to unrelated lameness issue. learning points • multiparous mares are at low risk of developing vaginal prolapse. • vaginal prolapse should be included in differential diagnoses for a mare in late pregnancy presented with tissue protruding through the labia. • to prevent peripartum complications, client education is important after episioplasty procedure in a late pregnant mare. • if mare is not hospitalized, client is expected to monitor the mare frequently for signs of parturition and for vulvar discharge. conflict of interest no conflict of interest to declare. references 1. braun w: periparturient infections and structural abnormalities, in: youngquist rs, threlfall wr: editors. current therapy in large animal theriogenology. 2nd edition, philadelphia; elsevier saunders: 1986. p. 572-574. 2. hopper rm: surgical correction of abnormalities of genital organs of cows. in: youngquist rs, threlfall wr: editors. current therapy in large animal theriogenology. 2nd edition, philadelphia; elsevier saunders: 1986. p. 463-472. 3. darbaz i, ergene o, canooglu e, et al: the assessment of diagnostic and ultrasonographic findings in a bitch with true vaginal prolapse. kafkas universitesi veteriner fakultesi dergisi journal: 2017;23:843-846. 4. munibullah, zulqarnain, ghaffar a, et al: rectal and vaginal prolapse in sheep: a case report. fuuast j biol 2019;9:171-173. 5. antonov a, karadaev m: a rare case of true vaginal prolapse in a bitch after abortion due to hypoluteoidism. bulgarian j of vet med 2019; 22:501-505. 6. borakhatariya d, gadara a, kasundra a: surgical management of recurrent cervico-vaginal prolapse by ovario-hysterectomy in stray cows: a report of 12 cases. indian j vet sci biotech 2017;12:146-149. 7. shrestha r, singh a, pradhan b, et al: five years audit on vaginal hysterectomy for utero vaginal prolapse. j inst of med 2018;40:70-74. 8. nkwabong e, fomulu jn: urethrovaginal fistula following vaginal prolapse of a pedunculated uterine myoma: a case report. j med case reports 2017;11:292. 9. calvert h, kapurubandara s, nikam y, et al: a vaginal angiomyofibroblastoma as a rare cause of a prolapsing vaginal mass: a case report and review of literature. case rep obs gyn 2018;8579026: 1-5. 10. senger pl: pathways to pregnancy and parturition 2nd edition. idaho; current conceptions: 2005. p.10-40. 11. lakshmikantan u, murugan m, ganesan a, et al: surgical management of perineal laceration in a cow affected with cervico vaginal clinical theriogenology 2021; 13: 97 prolapse. indian j anim res 2018;52:934-935. 12. oo zm, nayzar m, soe at, et al: surgical treatment of a cervico-vaginal prolapse in an elephant in myanmar. gajah 2016;44:36-39. 13. nayak s, samantara s: surgical correction of chronic cervico-vaginal prolapse in a red sindhi cow through intravaginal approach. vet world 2010;3:427-428. 14. minchev p: the use of a new surgical method in eversion and prolapse of vagina in animals. j am vet med assoc 1957;130:344 15. winkler jk: repair of the bovine vagina prolapse by cervical fixation. j am vet med assoc 1966;149:768. 16. miesner md, anderson de: management of uterine and vaginal prolapse in the bovine. vet clin food anim 2008;24:409-419. 17. riera fl, hinrichs k, hunt pr, et al: cervical hyperplasia with prolapse in a mare. j am vet med assoc 1989;195:1393-1394. 18. richardson gf, honey pg, karns pa, et al: vaginocervical prolapse in a mare. vet med small anim clin 1983;78:398-401. 19. wilborn rr, pugh dg: donkey reproduction. in: mckinnon ao, squires el, vaala we: editors. equine reproduction, 2nd edition. ames; wiley-blackwell: 2011. p. 2837. clinical theriogenology 2021; 13: 98 2019 prostaglandin f2? induced luteolysis is delayed but not prevented by acute administration of luteotropic drugs in lactating nonpregnant holstein cows prostaglandin f2α induced luteolysis is delayed but not prevented by acute administration of luteotropic drugs in lactating nonpregnant holstein cows divakar ambrose,a,b marcos colazo,a mohanathas gobikrushanthb alivestock and crops research branch, alberta agriculture and forestry, edmonton, ab, canada bdepartment of agricultural, food and nutritional science university of alberta, edmonton, ab, canada abstract inadvertent use of prostaglandin f2α (pgf2α) in pregnant cattle could result in luteolysis and pregnancy loss. using a nonpregnant cow model, we determined if luteotropic agents could counteract luteolytic effects of pgf2α. ovarian status of 20 lactating nonpregnant holstein cows was synchronized using an ovsynch protocol and ovulation confirmed by transrectal ultrasonography 48 hours after the second gonadotropin releasing hormone (gnrh). eight days after ovulation, 25 mg of dinoprost (native pgf2α) was administered im to induce luteolysis. five minutes after pgf2α, cows were treated im with gonadorelin (gnrh, 100 μg; n = 5), human chorionic gonadotropin (hcg, 1,000 iu; n = 6), porcine pituitary luteinizing hormone (plh, 25 mg; n = 5), or saline (control, 2 ml; n = 4). blood samples were collected before pgf2α (0 hour) and at 1, 6, 12, 18, 24, 30, 36, 42, 48, 60, 72, and 84 hours after pgf2α to monitor subsequent luteal activity by measuring plasma progesterone concentrations. although none of the treatments counteracted the luteolytic action of pgf2α, the rate of decline in plasma progesterone within 1 hour after pgf2α was greater (p = 0.04) in control than in gnrh, hcg, and plh (2.2 versus 0.2, 0.3, and 0.1 ng/ml/hour, respectively). in addition, plh-treated cows tended (p = 0.07) to have greater overall mean plasma progesterone for up to 84 hours (1.6 ± 0.2 ng/ml) than other treatments combined (1.1 ± 0.1 ng/ml). in the present study, giving gnrh, hcg or plh 5 minutes after pgf2α administration was not effective in countering luteolytic effects of pgf2α. nevertheless, plh treatment tended to delay pgf2α induced luteolysis, which warrants further investigation. keywords: bovine pregnancy, abortion, corpus luteum, porcine pituitary lh, progesterone introduction corpus luteum (cl), a transient endocrine gland, undergoes dynamic changes to regulate reproductive cyclicity and thereby fertility of mammals. it produces the steroid hormone progesterone (p4) that has a key role in establishment and maintenance of pregnancy. in the absence of a conceptus, cl undergoes luteolysis due to prostaglandin f2α (pgf2α) released from the uterus.1 in case of pregnancy, uterine release of pgf2α is blocked by bovine interferon-tau, the signal of maternal recognition of pregnancy from placental trophoblast cells in ruminants.2 as approved products for reproductive management, both native (dinoprost) and synthetic (cloprostenol) preparations of pgf2α are used routinely in dairy cattle, especially in estrus and ovulation synchronization protocols, integral to herd breeding programs. as pregnancy maintenance in cattle, at least up to 150 days of gestation is dependent on luteal p4,3 inadvertent use of pgf2α, especially during the first 5 months of pregnancy, usually results in abortion. under such a circumstance, an immediate remedial action that either prevents or delays pgf2α-induced luteolysis would be beneficial, at least until exogenous p4 therapy could be initiated. that bovine luteinizing hormone (lh) and human chorionic gonadotropin (hcg) have antiluteolytic properties in cattle was first demonstrated in holstein heifers subjected to oxytocininduced luteolysis.4 in early studies in ewes, ovine lh,5 and gonadotropin releasing hormone (gnrh),6 extended cl lifespan of, possibly by delaying or countering action of pgf2α. in rats, antiluteolytic effect of hcg was evident by pregnancy maintenance in 70% of pgf2α-treated pregnant rats.7 in studies from our laboratory, in cattle treated with exogenous porcine lh (plh), plasma lh concentrations remained higher than that of controls for up to 20 hours after 599 clinical theriogenology • volume 11, number 4 • december 2019 treatment.8-10 higher than basal concentrations of lh for a prolonged interval in plh-treated cattle may protect the cl from luteolytic action of pgf2α by saturation of luteal membrane lh receptors, as hypothesized.11 all of the above products (hcg, gnrh, and plh) are approved for use in dairy cattle in canada. however, efficacy of these products as luteotropic agents to counteract pgf2α-induced luteolysis in cattle has not been adequately investigated. we hypothesized that a luteotropic agent given to cows soon after pgf2α administration prevents or delays luteolysis, as indicated by greater p4 concentrations compared to cows not given a luteotropic treatment. therefore, our objective was to determine if luteolytic effects of pgf2α in dairy cattle could be counteracted by gnrh, hcg or plh given 5 minutes after pgf2α administration. materials and methods cattle and housing the study was conducted at the dairy research and technology centre of the university of alberta. cattle were handled and cared for in accordance with the canadian council on animal care guidelines (2009)12 and experimental procedures were approved by the animal policy and welfare committee, department of agricultural, food and nutritional science, university of alberta. twenty-four lactating nonpregnant holstein cows (7 primiparous and 17 multiparous) were enrolled in the study. cows were individually fed a total mixed ration (primary ingredients were barley silage, alfalfa silage, alfalfa hay, and concentrates) and housed in tie stalls and let out for approximately 2 hours of exercise during weekdays. diets were formulated according to nrc (2001)13 to meet requirements of a 650 kg lactating cow producing 30 kg of milk/day and delivered individually by a data ranger (american calan inc., northwood, nh), once daily at 0800 hour. cows had ad libitum access to fresh water. cows averaged (mean ± standard deviation) 3.0 ± 0.4 lactation and 266 ± 25 day postpartum at the beginning of the experiment. average 305 days milk yield was 9,540 kg. experimental design and treatments ovarian status of cows was synchronized using an ovsynch protocol.14 in brief, the protocol consisted of gnrh (100 μg gonadorelin acetate, im; fertiline®, vetoquinol n. a. inc. lavaltrie qc, canada), followed 7 days later by pgf2α (25 mg dinoprost tromethamine, im; lutalyse®, zoetis canada inc. kirkland, qc, canada), and a second gnrh (100 μg gonadorelin acetate, im) given 48 hours after pgf2α. transrectal ultrasonography (aloka 500, aloka co ltd., tokyo, japan) using a 7.5 mhz linear-array transducer, was first conducted at the time of the second gnrh of ovsynch to confirm the presence of putative ovulatory follicle(s) (≥ 10 mm in diameter). ovulation was confirmed 48 hours later by absence of a follicle previously detected. eight days after ovulation, pgf2α (25 mg dinoprost tromethamine, im) was administered to induce luteolysis in all cows. luteotropic treatments were randomly administered im (n = 6 per treatment), exactly 5 minutes after pgf2α administration. interval between pgf2α and luteotropic treatments was an arbitrary yet realistic time frame under field conditions, to take remedial action, if pgf2α was administered inadvertently to a pregnant animal and the mistake quickly discovered. treatments were: gnrh (100 µg gonadorelin acetate, im; fertiline®); hcg (1,000 iu human chorionic gonadotropin, im; chorulon®, intervet, kirkland, qc, canada); plh (25 mg porcine pituitary lh, im, lutropin-v, bioniche animal health, belleville, on, canada); or control (2 ml sterile saline, im). blood sampling and progesterone assay blood samples were collected into evacuated tubes containing sodium heparin (vacutainer, beckton dickinson and co., franklin lakes, nj) from an indwelling jugular 600clinical theriogenology • volume 11, number 4 • december 2019 catheter. blood samples were collected immediately before pgf2α (0 hour), 1 and 6 hours after pgf2α, then every 6 hours until 48 hours, and thereafter every 12 hours for the next 36 hours, up to 86 hours after pgf2α treatment. samples were placed on ice immediately after collection and centrifuged at 1500 x g for 20 minutes at 4°c, plasma harvested and frozen at -20 °c until assayed for p4, in duplicate, using a direct enzyme immunoassay (quanticheck , faculty of veterinary science, budapest, hungary). this assay uses an antip4 monoclonal antibody and horseradish peroxidase as the enzyme label, with a sensitivity (lowest detection limit) of 0.5 ng/ml and has been compared to established assays.15 the intra-assay coefficient of variation was 6.5%. statistical analyses differences in plasma p4 concentrations by luteotropic treatment (gnrh, hcg, plh, or saline), time of sampling (0 hour before pgf2α and 1, 6, 12, 18, 24, 30, 36, 42, 48, 60, 72, and 84 hours after pgf2α), parity (primiparous and multiparous), and interactions between luteotropic treatment and time were tested by repeated measures analysis using mixed procedure of sas. four cows (gnrh, n = 2; hcg and plh, n = 1 each) with p4 concentrations < 1 ng/ml at 0 hour (before pgf2α), indicative of either delayed or poor luteal function, were excluded from the analysis. in the remaining 20 cows, p4 concentration at 0 hour was used as a covariate to account for potential influence of p4 at time of pgf2α administration on subsequent p4 concentrations. time of sampling was used as a repeated measurement nested within cow. covariance structure of the repeated measurement and appropriate final model were chosen based on the lowest akaike’s information criteria. since plh-treated cows had greater mean plasma p4 concentration than gnrh-treated cows, a pairwise comparison between plh and other groups combined (control, gnrh, and hcg) was also performed using an orthogonal contrast statement. in addition, differences among luteotropic treatments in rate of decline in mean plasma p4 concentrations from 0 to 1 hour after giving pgf2α, were tested using mixed procedure. differences in least square means were tested using pdiff option. significant differences were reported if p ≤ 0.05 and considered a tendency if p > 0.05 and ≤ 0.10. results changes in mean plasma p4 concentrations by luteotropic treatment, time and their interactions are presented in figure 1. none of the luteotropic treatments countered the luteolytic effect of pgf2α (p = 0.24), as evident from declines in p4 concentrations. the rate of decline in plasma p4 concentrations (ng/ml/hour) within the first hour after pgf2α administration, however, differed (p = 0.04) between luteotropic treatments. in this regard, cows treated with gnrh (0.2 ± 0.2), hcg (0.3 ± 0.2), or plh (0.1 ± 0.2) had slower rates of decline than cows treated with saline (2.2 ± 0.2 ng/ml/hour; figure 2). moreover, orthogonal contrast analysis revealed that plh-treated cows tended to have greater overall mean p4 concentration from both 0 to 84 hours (1.6 ± 0.2 versus 1.1 ± 0.1 ng/ml; p = 0.07) and from 12 to 84 hours (1.0 ± 0.2 versus 0.5 ± 0.1 ng/ml; p = 0.10) after pgf2α administration, compared to mean p4 concentrations for other treatments combined. regardless of luteotropic treatment, mean (± sem) plasma p4 concentrations (ng/ml) differed by time (p < 0.01), declining from 0 to 12 hours after pgf2α (4.1 ± 0.2 [0 hour], 3.6 ± 0.2 [1 hour], 2.3 ± 0.2 [6 hour] and 0.8 ± 0.2 [12 hour]) and then remained unchanged from 12 to 84 hours after pgf2α, ranging from 0.4 to 0.7 ng/ml. discussion bovine lh and hcg in heifers,4 ovine lh and gnrh in ewes,5,6 and hcg in rats,7 were reported to have antilutelolytic properties against exogenous oxytocin, endogenous pgf2α, exogenous estradiol and pgf2α, respectively. on the contrary, in the present study, none of the agents, at the administered dose, prevented luteolysis in the nonpregnant cow model, as evident from declines in p4 concentrations from 0 to 84 hours after pgf2α administration. 601 clinical theriogenology • volume 11, number 4 • december 2019 few studies have evaluated efficacy of luteotropic agents in preventing or reversing luteolysis in ruminants. in a pioneering study4 in cyclic holstein heifers, antiluteolytic effects of bovine lh (30 mg daily), hcg (2,000 iu), urea-treated hcg (2,000 iu) and bovine prolactin (60 mg daily) on oxytocin (0.33 usp units/kg bw) induced luteolysis were evaluated. both bovine lh and hcg when given concurrently with oxytocin treatment not only overcame luteolytic effects of oxytocin, but also increased cl weight and luteal progesterone concentration, indicative of luteotropic effects. when the lh component of hcg was destroyed by urea treatment before administration, the preparation was no longer luteotropic, indicating that lh is the luteotropin in cattle. likewise, prolactin also had no luteotropic effect. in a contrasting study,16 continuous iv infusions of purified bovine lh (10 μg/minute) for 10 hours, starting 4 hours prior to giving 25 mg pgf2α, at days 10 12 of the estrous cycle in beef cattle, failed to prevent pgf2α-induced luteolysis. intravenous infusions of ovine lh (4 μg/minute), ovine prolactin (42 μg/minute), or lh + prolactin for 12 hours, failed to prevent luteolysis in ewes following im injections of 6.66 mg pgf2α given 2 and 6 hours after infusions began.17 on the contrary, prolactin had an antiluteolytic effect in pregnant rats when given on day 4 of gestation,18 and maintained pregnancy in 90% of rats when given on day 10 of gestation.7 prolactin and prolactin receptor mrna are expressed in bovine cl throughout the estrous cycle, suggesting a role for prolactin in luteal function in cattle.19 however, as pioneering studies in cattle4 and sheep17 reported prolactin not to prevent luteolysis, it may not be a suitable candidate for further consideration as an antiluteolytic agent. conversely, plh is a potential candidate for further research as an antiluteolytic agent for two reasons. first, the rate of p4 decline in the first hour after pgf2α administration was the smallest and second, the overall mean plasma p4 concentration up to 84 hours was highest in plh-treated cows in the present study. although the overall mean p4 concentration of 1.6 ng/ml in plh-treated cows was greater than that of all other treatments, such a low p4 concentration during diestrous is likely insufficient to sustain pregnancy, particularly in lactating dairy cows that have lower plasma p4 concentrations, likely due to increased p4 metabolism.20 small and repeated doses of hcg prevented estradiol-induced luteolysis in macaques (macaca fascicularis).21 therefore, using a conceptually similar approach, it would be worth investigating whether repeated doses of plh treatment would effectively counteract luteolysis in cattle. inducing formation of a new cl could be another strategy to increase endogenous p4 to maintain pregnancy. however, this process takes time and cannot be implemented quickly when pgf2α is inadvertently administered to pregnant cattle under field conditions. in this regard, when an induced cl formed on the ovary ipsilateral to the pregnant horn after forced lysis of the original cl (while the pregnancy was temporarily sustained by exogenous p4), was capable of maintaining the pregnancy even after exogenous p4 supplementation ended.22,23 more recently, it was reported24 that 72% (18 of 25) of pregnant beef cows and heifers maintained their pregnancies for at least 7 days after induced luteolysis, when exogenous p4 treatments (100 mg sc daily for 7 days) were given, with the first p4 injection starting 2, 6, 10, 12, or 18 hours after pgf2α was given during days 30 90 of pregnancy. when an accessory cl was successfully induced using hcg in a subset of those cattle (mean ± sd, 38 ± 5.5 days after pgf2α), 80% carried the pregnancy to term.24 authors concluded that pregnancies could be maintained following an accidental administration of pgf2α without the need for extended p4 supplementation, when an accessory cl was successfully induced. an accessory cl could be induced with gnrh or plh, perhaps as effectively as hcg. however, to prevent p4 concentrations from plummeting to basal concentrations after accidental pgf2α administration, a quick remedial measure may be to concurrently insert two intravaginal p4 devices to maintain higher circulating p4 concentration (2.7 ng/ml)25 than attainable with a single p4 device (1.0 ng/ml),26 thereby protecting the embryo from exposure to very low p4 concentrations due to pgf2α-induced decline in luteal p4. as many dairy farms and bovine practitioners are likely to have intravaginal progesterone devices readily available, this approach 602clinical theriogenology • volume 11, number 4 • december 2019 would be more practical than progesterone injections. thus, future studies should investigate a combined treatment approach of repeated injections of plh to maximize lh support to the cl exposed to pgf2α and insertion of two intravaginal progesterone devices to maintain p4 concentrations that could sustain a pregnancy. if a pregnancy could be sustained temporarily with this combined treatment strategy, inducing an accessory cl at the earliest opportunity should be actively considered as a permanent solution to sustain the pregnancy to term, as demonstrated in cattle.24 although plh is available in the canadian market, to the authors’ knowledge there is no fda approved plh that is currently available in the united states. the treatment strategies proposed in this paper would be extra label use in many jurisdictions; therefore, any such treatment must occur under the order and discretion of a veterinarian. conclusion in the present study, giving gnrh, hcg or plh 5 minutes after pgf2α administration were not effective in countering luteolytic effects of pgf2α in a nonpregnant lactating cow model. nevertheless, plh treatment tended to delay pgf2α-induced luteolysis, which warrants further investigation. annotation using any of the luteotropic drugs or exogenous progesterone for the purpose described in the manuscript may constitute extra label use and should only be used at the discretion of a practitioner who has an established veterinarian-patient-client-relationship. acknowledgement authors acknowledge technical assistance of govindarajan thangavelu (university of alberta) during this study. product donations by bioniche animal health (lutropin-v) and vetoquinol canada inc. (fertiline) are also gratefully acknowledged. authors’ contribution da: study concept, experimental design, data collection, manuscript writing; mc: experimental design, data collection, progesterone assays, manuscript review; mg: data analysis, manuscript writing. conflict of interest authors declare no conflicts of interest. references 1. thatcher ww, wolfenson d, curl js, et al: prostaglandin dynamics associated with development of the bovine conceptus. anim reprod sci 1984;7:149-176. 2. bazer fw, song g, thatcher ww: roles of conceptus secretory proteins in establishment and maintenance of pregnancy in ruminants. asian australas j anim sci 2012;25:1-16. 3. gross ts, williams wf: in vitro steroid synthesis by the placenta of cows in late gestation and parturition. j reprod fertil 1988;83:565-573. 4. donaldson le, hansel w, van vleck ld: luteotropic properties of luteinizing hormone and nature of oxytocin induced luteal inhibition in cattle. j dairy sci 1965;48:331-337. 5. karsch fj, roche jf, noveroske jw, et al: prolonged maintenance of the corpus luteum of the ewe by continuous infusion of luteinizing hormone. boil reprod 1971;4:129-136. 6. adams te, kinder je, chakraborty pk, et al: ewe luteal function influenced by pulsatile administration of synthetic lhrh/fshrh. endocrinol 1976;97:1460-1467. 7. chatterjee a: the possible mode of action of prostaglandins: x. antagonism between prostaglandin f2a and prolactin or human chorionic gonadotropin: a comparative study. prostaglandins 1976;12:525-534. 8. ambrose jd, kastelic jp, rajamahendran r, et al: progesterone (cidr)-based timed ai protocols using gnrh, porcine lh or estradiol cypionate for dairy heifers: ovarian and endocrine responses and pregnancy rates. theriogenology 2005;64:1457-1474. 603 clinical theriogenology • volume 11, number 4 • december 2019 9. ree to, colazo mg, lamont aga, et al: the effect of porcine luteinizing hormone in the synchronization of ovulation and corpus luteum development in nonlacatating cows. theriogenology 2009;72:120-128. 10. behrouzi a, colazo mg, ambrose dj: alterations in bone morphogenetic protein 15, growth differentiation factor 9, and gene expression in granulosa cells in preovulatory follicles of dairy cows given porcine lh. theriogenology 2016;85:1249-1257. 11. henderson km, mcnatty kp: a biochemical hypothesis to explain the mechanisms of luteal regression. prostaglandins 1975;9:779-797. 12. canadian council on animal care guidelines on the care and use of farm animals in research, teaching and testing. 2009. http://ccac.ca/documents/standards/guidelines/farm_animals.pdf 13. national research council. nutrient requirements of dairy cattle. 7th edition, national academic press, washington, dc. 2009. 14. pursley jr, mee mo, wiltbank, mc: synchronization of ovulation in dairy cows using pgf2α and gnrh. theriogenology 1995;44:915-923. 15. colazo mg, ambrose dj, kastelic jp, et al: comparison of 2 enzyme immunoassays and a radioimmunoassay for measurement of progesterone concentrations in bovine plasma, skim milk, and whole milk. can j vet res 2008;72:32-36. 16. gonzález-menció f, murphy bd, manns j:failure of exogenous lh to prevent pgf2α-induced luteolysis in beef cows. prostaglandins 1977;14:535-542. 17. sasser rg, niswender gd, nett tm: failure of lh and/or prolactin to prevent pgf2α-induced luteolysis of ovine corpora lutea. prostaglandins 1977;13:1201-1208. 18. fuchs a, mok e, sundaram k: luteolytic effects of prostaglandins in rat pregnancy, and reversal by luteinizing hormone. eur j endocrinol 1974;76:583-596. 19. shibaya m, murakami s, tatsukawa y, et al: bovine corpus luteum is an extrapituitary site of prolactin production. mol reprod dev 2006;73:512-519. 20. sangsritavong s, combs dk, sartori rf, et al: high feed intake increases liver blood flow and metabolism of progesterone and estradiol 17b in dairy cattle. j dairy sci 2002;85:2831-2842. 21. westfahl pk, horton le, stadelman hl, et al: small doses of human chorionic gonadotropin prevent estradiol-induced luteolysis. endocrinol.metab 1984;10:e84-e87. 22. lulai c, dobrinski i, kastelic jp, et al: induction of luteal regression, ovulation and development of new luteal tissue during early pregnancy in heifers. anim reprod sci 1994;35:163-172. 23. bridges pj, wright dj, buford wi, et al: ability of induced corpora lutea to maintain pregnancy in beef cows. j anim sci 2000;78:2942-2949. 24. ferguson ce, kesler dj, godke ra: maintenance of pregnancy in beef cattle after a luteolytic dose of prostaglandin f2α. vet med anim sci 2014;2:1. 25. bisinotto rs, ribeiro es, lima f, et al: targeted progesterone supplementation improves fertility in lactating dairy cows without a corpus luteum at the initiation of the timed artificial insemination protocol. j dairy sci 2013;96:2214-2225. 26. cerri rl, rutigliano hm, chebel rc, et al: progesterone concentration, follicular development and induction of cyclicity in dairy cows receiving intravaginal progesterone inserts. anim reprod sci 2009;110:56-70. 604clinical theriogenology • volume 11, number 4 • december 2019 figure 1. plasma progesterone (p4) concentrations at administration of pgf2α (25 mg dinoprost; 0 hour) and at 1, 6, 12, 18, 24, 30, 36, 42, 48, 60, 72 and 84 hours after either a luteotropic or saline (control) treatment given 5 minutes after pgf2α in dairy cows. luteotropic treatments were gonadotropin releasing hormone (gnrh; 100 μg im; n = 5), human chorionic gonadotropin (hcg; 1,000 iu; n = 6), porcine pituitary luteinizing hormone (plh; 25 mg im; n = 5) or control (sterile saline; 2 ml im; n = 4). there was no overall counteracting effect of luteotropic treatment on pgf2αinduced luteolysis (p = 0.24); however, orthogonal contrast analysis revealed that mean p4 concentration tended to be greater in plh treated cows up to 84 hours after pgf2α administration compared to the overall mean for other treatments (1.6 ± 0.2 versus 1.1 ± 0.1 ng/ml; p = 0.07). irrespective of the luteotropic treatment given, plasma p4 concentrations differed (p < 0.01) by time, with the time effect being most evident between 0 and 12 hours. concentrations of p4 remained unchanged between 12 and 84 hours after pgf2α treatment. however, there was no interaction between categories of luteotropic treatment and time (p = 0.80). 0 1 2 3 4 5 6 0 1 6 12 18 24 30 36 42 48 60 72 84 pl as m a pr og es te ro ne (n g/ m l) hours relative to pgf2α administration control gnrh hcg plh treatment; p = 0.24 time; p < 0.01 treatment x time; p = 0.80 605 clinical theriogenology • volume 11, number 4 • december 2019 figure 2. mean (± sem) rate of decline in plasma progesterone (p4) concentrations between 0 and 1 hour after pgf2α (25 mg dinoprost) administration in lactating dairy cows. a luteotropic agent or control treatment was given 5 minutes after pgf2α (0 hour). luteotropic treatments were gonadotropin releasing hormone (gnrh; 100 μg im; n = 5), human chorionic gonadotropin (hcg; 1,000 iu; n = 6), porcine pituitary luteinizing hormone (plh; 25 mg im; n = 5) or control (sterile saline; 2 ml im; n = 4). cows treated with gnrh (0.2 ± 0.2), hcg (0.3 ± 0.2) or plh (0.1 ± 0.2) had a smaller rate of decline than those treated with saline (2.2 ± 0.2 ng/ml/hour). a,bcolumns without a common superscript differed (p = 0.04). 2.2 0.2 0.3 0.1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 control gnrh hcg plh ra te o f d ec lin e in p la sm a p 4 (n g/ m l/h ou r) be tw ee n 0 an d 1 ho ur a fte r p g f 2 α control / luteotropic treatments p = 0.04 a b b b 606clinical theriogenology • volume 11, number 4 • december 2019 untitled clinical theriogenology 2022; 14: 11 introduction reproduction is economically important to production animal species and removing subfertile individuals is of high priority as 1 bull may breed many cows. the breeding potential of an individual bull can be determined by performing a bull breeding soundness evaluation (bbse). it consist of a brief physical examination, assessment of reproductive organs, transrectal palpation of the accessory sex glands, collection of an ejaculate, and evaluation of sperm motility and morphology.1,2 to be classified as a ‘satisfactory potential breeder’, a bull should be physically and reproductively sound and at least 70% of sperm evaluated must be morphologically normal.2 abnormal sperm morphology is the most common reason that bulls are classified as a category other than ‘satisfactory’ (> 75% of bulls were classified as ‘unsatisfactory’ due to morphology alone or in combination with other measurements1). normal morphology is important for sperm motility within the reproductive tract and ability to fertilize oocytes.3 furthermore, the assessment of agreement among veterinarians performing bbse becomes beneficial to develop future training standards. stallion and man sperm analysis studies4-6 determined the effects of evaluator experience, sperm morphology slide preparation methods, and methodology by which sperm morphology slides were evaluated on the outcome of sperm morphology classification. values reported in a subsequent human sperm morphology study7 (by experienced laboratory technicians, biologists, and physicians) were comparable to reference values of andrology laboratories that had highly trained staff members. authors concluded that following recommended methods coupled with experience are critical to achieve relevant and comparable results.6 although substantial differences among evaluators and assessment methods were observed in stallion and man sperm morphology studies, to date, there have been no studies evaluating effects of these variables on the classifications of bull sperm morphology. therefore, the objectives of this study were to compare: intra-evaluator variation for bull sperm morphology assessment; variation among veterinary students in their fourth year (clinical year) with strong interest in theriogenology; variation among theriogenologists; agreement between theriogenologists and fourth year veterinary students; effect of time to complete the analysis of bull sperm morphology slides on evaluator type and cell number assessed; effect of number of sperm assessed per morphological slide evaluation on the percent normal morphology. bull sperm morphology assessment varied by evaluator ashley reeves,a jessica klabnik,c lew strickland,b,c tulio prado,b,c pablo jarrin-yepez,b cheyenne dingemans,d liesel schneider,c brian whitlockb,c acomparative and experimental medicine, bdepartment of large animal clinical sciences cdepartment of animal science, university of tennessee, knoxville, tn ddepartment of field services and theriogenology, university of georgia, athens, ga abstract assessment of sperm morphology is an important part of bull breeding soundness evaluation (bbse). whereas the effects of evaluator experience and evaluation method on the sperm morphology estimation were assessed in other species, no such study was conducted for bulls. our objectives were to assess the effects of evaluator experience and number of sperm assessed on bbse outcomes. a single eosin-nigrosin sperm morphology slide from individual semen samples, collected from 35 yearling bulls was used. in experiment i, 6 individuals (3 board-certified theriogenologists [dact] and 3 fourth-year veterinary students [vs]) evaluated 100 sperm from 35 slides twice (at least 1 week between evaluations). in experiment 2, 3 dact evaluated 100, 200, and 400 sperm from the same 5 sperm morphology slides to determine if assessing a higher number of sperm would increase the agreement of morphologic characteristics. in experiment 1, there was a difference (p < 0.0001) in the percent of sperm classified as morphologically normal between evaluator types (vs versus dact). furthermore, variation among evaluators affected sperm morphology assessments and bull breeding soundness evaluation classifications. whereas the time needed to evaluate slides increased (p = 0.96) with increasing number of sperm assessed, there was no effect (experiment 2) of number of sperm evaluated on percent normal sperm, indicating that evaluating more than 100 sperm may not be justifiable. further investigation on slide preparation, microscope use, assessor experience, and continuing education/training is important to ensure the repeatability and validity of evaluating bovine sperm morphology. keywords: bull, sperm, morphology, assessment clinical theriogenology 2022; 14: 12 materials and methods animals and slide preparation thirty-five semen samples from 35 consigned black angus bulls (14 18 months of age; 1 sample per bull) were used in this study. bulls were housed at the university of tennessee bull test station (spring hill, tn). all bulls received ad libitum access to pelleted feed, hay, and water. semen was collected (as ‘convenience samples’; breeding soundness evaluation was conducted on each bull for reasons unrelated to this study) by electroejaculation using a lane pulsator v (lane manufacturing, denver, co). a single eosin-nigrosin (morphology stain, society for theriogenology, mathews, al) stained semen smear slide was prepared from the semen of each bull by 1 theriogenologist. after a cursory view of each slide to ensure each slide had an acceptable concentration and distribution of appropriately stained sperm, slides were coded (1 35) to eliminate any identification of the bull and date of collection. evaluators and experimental design evaluators reviewed bull sperm morphology classification criteria defined by the manual for breeding soundness examination of bulls (2018 edition).2 objective of this initiative was to ensure that evaluators were using the same criteria to classify sperm morphology to minimize the variation between and among individuals. sperm morphology was evaluated under 1,000 x (oil immersion; cargile nondrying immersion oil for microscopy, type b code 1248, lot 110193) magnification and bright field microscopy using the same microscope (omax lab biological binocular compound microscope 40 2,000 x w halogen light) in the same room. experiment 1 six individuals evaluated all 35 slides. three were diplomates of american college of theriogenologists (dact) and 3 were fourth-year veterinary students (vs) who completed an advanced veterinary reproductive elective and self-identified as interested in theriogenology. for each slide, 100 sperm were evaluated and classified according to the 2018 society for theriogenology, manual for breeding soundness examination of bulls.2 each individual evaluated all 35 slides in the same order (1 35) in 1 ‘timed sitting’. after 1 week, second assessment of all slides was completed by each individual. experiment 2 three dact evaluated 5 morphology slides using similar methods described before. sperm (100, 200, and 400) were evaluated in 1 ‘timed sitting’ for each slide twice with at least 1 week between evaluations. data analyses to calculate the intraclass correlation coefficient (icc) for each evaluator, counts of normal sperm for each of the 35 slides were analyzed within the icc_sas macro.8 the 2-way mixed effects icc calculation was used to assess the consistency of 1 evaluator among multiple measures of the same slide.9 the agreement among dact of grading a slide as pass or fail was analyzed using cohen’s kappa agreement. data on percent normal sperm were analyzed using separate mixed model anova (proc glimmix, sas 9.4); fixed effects of evaluator, evaluator type, slide number (to determine effect of order in which the slides were evaluated) or number of sperm assessed, and interactions (as appropriate) were tested. simple linear regression (proc reg) was utilized to assess the effect of the number of sperm read on the time required to complete the assessment, and separately, the effect of the number of sperm evaluated on the time to complete the evaluation of 5 slides. data from experiment 2 were analyzed using logistic regression (proc glimmix) with a binary distribution. effect of total number of sperm assessed impacted the probability for a bull to fail the morphology assessment was tested. statistical significance was set at α = 0.05. results experiment 1 intraclass correlation coefficients were calculated for each evaluator based on 2 readings of normal sperm for 35 slides (table 1). each evaluator had varying degrees of consistency (p < 0.001; table 1). there was an interaction (p < 0.0001) of evaluator type (dact versus vs) and slide number on the percentage or proportion of sperm classified as morphologically normal, indicating that the effect of evaluator type depended on the slides that were evaluated. among slides that differed between reviewer type, vs had a higher (p = 0.0001) percent normal sperm recorded compared to dact (table 2). mean percent normal sperm recorded for vs was numerically higher for most slides, changing the outcome of a potential bbse from ‘deferred’ or ‘unsatisfactory’ to ‘satisfactory’ for 7 of the 35 (20%) slides. furthermore, there was an effect (p < 0.0001) of evaluator on coefficient of variation among individuals, but no effect (p = 0.78) of evaluator type. the agreements among dact when examining 35 of the same morphology slides, differed among individuals (table 3). number of bulls that were classified as ‘unsatisfactory’/’deferred’ based on morphological assessment differed numerically among evaluators. there was no effect (p = 0.53) of evaluator type on the time needed to complete evaluations of slides; the average time to evaluate 100 sperm for 35 slides was 134.6 minutes for dact and 145.5 minutes for vs with a sem of 11.43 minutes. however, there was an effect of individual within evaluator type (p = 0.04; figure 1), such that some evaluators took 30 minutes longer to complete their assessments compared to the fastest evaluators within an evaluator type. table 1. intraclass correlation coefficients (icc) for each evaluator (35 slides in 2 separate duplicate sessions) evaluator icc 95% ci dact1 0.94 0.89, 0.97 dact2 0.91 0.83, 0.95 dact3 0.95 0.90, 0.97 vs1 0.95 0.90, 0.97 vs2 0.75 0.56, 0.86 vs3 0.86 0.74, 0.93 clinical theriogenology 2022; 14: 13 table 2. mean percent of morphologically normal sperm as determined by theriogenologists (dact) and veterinary students (vs) in experiment 1. grey highlighted numbers represent slides for which the dact and vs results would change the bse outcome from ‘unsatisfactory’ to ‘satisfactory’. asterisks (*) denote significant differences between dact and vs. slide number dact vs sem 1 41.33 41.50 5.52 2 66.83 69.33 5.52 3 62.16 72.00 5.52 4 38.16 48.00 5.52 5 82.00 91.16 5.52 6 78.66 85.33 5.52 7 67.83 68.83 5.52 8 80.00 73.16 5.52 9 39.66 46.66 5.52 10 67.00 81.83 5.52 11 76.00 77.16 5.52 12 72.33 80.16 5.52 13 84.16 80.83 5.52 14* 26.16* 45.66* 5.52 15 62.66 74.00 5.52 16 78.83 81.00 5.52 17 82.83 84.50 5.52 18 81.00 88.33 5.52 19 74.33 81.16 5.52 20 78.33 79.33 5.52 21 64.50 71.00 5.52 22 82.83 78.66 5.52 23 66.33 72.50 5.52 24 81.00 81.33 5.52 25 78.66 78.33 5.52 26 61.16 75.66 5.52 27 86.66 83.50 5.52 28 72.66 83.83 5.52 29 67.50 68.83 5.52 30* 43.16* 62.16* 5.52 31 4.33 10.33 5.52 32 73.00 78.83 5.52 33* 58.33* 83.16* 5.52 34* 8.00* 30.83* 5.52 35 58.33 65.50 5.52 clinical theriogenology 2022; 14: 14 table 3. agreement among theriogenologists (dact) using the fleiss agreement guidelines with multiple evaluators examining the same slides. evaluator 1 evalutor 2 kappa coefficient agreement dact1 dact2 0.3158 poor dact2 dact3 0.4944 fair dact1 dact3 0.6441 good a ab a a a b 0 25 50 75 100 125 150 175 200 225 dact vs ti m e (m in ut es ) evaluator type 1 2 3 figure 1. time (in minutes) to evaluate 100 sperm for 35 slides consecutively per individual. least square means and standard error bars are represented. each colored bar denotes individual evaluator for each evaluator type. lower case letters above bars denote differences (p < 0.05) among dact evaluators; capital letters denote differences among vs evaluators; and similar letters within evaluator type did not differ. experiment 2 for dact evaluators, there was no effect (p = 0.96) of increasing the number of sperm evaluated on percent of sperm classified as morphologically normal, and the total number of sperm assessed did not impact the probability of that bull to fail the morphology assessment (p = 0.95), indicating that evaluating > 100 sperm is unlikely to have an effect on the bbse classification for a particular bull (figure 2). there was an effect of number of sperm assessed on time needed to complete the evaluation (p = < 0.0001) such that the variation in the number of sperm read explained 73% of the variation in time taken for the assessments (r2 = 0.73). additionally, there was an interaction (p < 0.0001) between evaluator and number of sperm assessed on time to completion; for some evaluators, as the total number of sperm evaluated per slide increased, time taken for completion also increased (figure 3). clinical theriogenology 2022; 14: 15 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 m ea n pe rc en ta ge n or m al sp er m slide number 100 200 400 figure 2. percentage normal sperm by increasing cell count: performed by dact. least square means and standard error bars are represented. note: no change in ‘satisfactory’ versus ‘unsatisfactory’ outcome. 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 m ea n pe rc en ta ge n or m al sp er m slide number 100 200 400 figure 3. time (in minutes) taken for dact to assess 100, 200, and 400 sperm in 5 morphology slides. least square means and standard error bars are represented. similar letters did not differ (p < 0.05). clinical theriogenology 2022; 14: 16 discussion effect of evaluator experience on the morphologic classification of bull sperm was determined for the first time. increasing the number of sperm evaluated per slide during a bbse, from 100 (the current accepted standard) up to 400, is unlikely to alter the percent of sperm classified as morphologically normal and therefore unlikely to affect the outcome of a bbse. therefore, for bulls presented for a bbse that are just below the acceptable percent of normal sperm morphology for a ‘satisfactory’ classification, continuing to assess more sperm is unlikely to yield a different outcome. although standard cutoffs for adequate normal sperm morphology have been used for decades, most bulls not classified as ‘satisfactory potential breeders’ are a result of too few morphologically normal sperm10-13 and results reported here indicate that variation among evaluators may affect the proportion of sperm classified as morphologically normal and ultimately the outcome of the bbse. an interesting finding was the effect of individual evaluator on intra-evaluator variation of sperm morphology, indicating that an evaluator is not necessarily consistent with their own classification criteria. this should be considered when performing multiple breeding soundness examinations in 1 sitting. this effect was observed when the evaluator assessed 35 slides in 1 sitting, but would it occur for an occasional bbse not at a high-volume testing station or veterinary hospital? another interesting finding was that for some slides there were differences between dact and vs in the percent normal sperm, and in this case, vs always reported a higher percentage of sperm as morphologically normal. we concluded that occasionally vs are less discriminating than dact in morphological evaluation. other studies involving the assessment of stallion sperm morphology reported variation among veterinarians for all morphology classification categories.4 similarly, a second investigation attributed the differences of sperm abnormalities to the education level of the evaluator, years of experience evaluating sperm morphology slides, and the differences among evaluators on classifications of abnormalities.6 our results indicate a variation among evaluators for morphology classifications (normal versus abnormal). however, the classification of specific morphological abnormalities was not recorded in our experiments to further investigate where those differences may lie. furthermore, our results indicate a difference between evaluator types (dact versus vs) that could be due to a difference in quality or quantity of training or experience. the individuals within the dact evaluators had received more extensive/ exhaustive training in the assessment of sperm morphology of multiple species and they had performed more bbse than vs evaluators, meaning that they had more experience evaluating bull spermiograms. whereas the dact evaluators differed from the vs evaluators, agreement among dact evaluators was not consistent suggesting variation within the same evaluator type should be considered. the studies conducted in man sperm morphology assessment included laboratory technicians, physicians, and biologists.5,7 their results indicate that participants who do not routinely use the recommended procedures were less strict and gave varied results compared to participants who followed the guidelines and reference values established by andrologists.5 their various training methods/guidelines included attending training sessions in semen analysis at a central standardizing/validating laboratory with proficiency assessment biannually using blind coded sperm suspensions and videotapes, following criteria set forth in the world health organization laboratory manual for the examination of man semen and/or standardization workshops on semen morphology assessment prior to the study.7,14,15 this study demonstrated the imperative need for standardization, training, and quality control for man sperm morphology assessment.5 similarly, a manual is available for the recommended criteria for bbse;2 however, recommended training and quality controls have not been established. this information should encourage discussions directed toward improvement of continuing education opportunities and quality controls for veterinarians to remain current in their sperm morphology examination skills. the differences between the dact and vs evaluators on percent of normal sperm would have changed the classification of the bbse (‘satisfactory,’ ‘deferred,’ or ‘unsatisfactory’) for multiple evaluators (7 out of 35 evaluators (20%) associated with the various sperm morphology slides. for example, using only sperm morphology as a criterion, the bull associated with microscope slide 10 would have been classified as ‘satisfactory’ by the vs evaluators (81% of sperm were classified as morphologically normal) but ‘unsatisfactory’ or deferred by the dact evaluators (67% of sperm were classified as morphologically normal). since current standards were determined from a study by an unknown number of evaluators with undescribed experience, the true fertility status of ‘borderline’ bull in a natural service scenario may or may not be congruent with their classification.16 further investigation into the natural breeding success of the bulls in this study can determine if the differences in the evaluations of the dact and vs had clinically relevant effects on fertility. although studies have reported an effect of evaluators and their experience on sperm morphology evaluation, those studies did not determine evaluators with minimal experience or training in morphology assessment for a comparison. further investigation would include more evaluators of varying experience and training with a larger sample size of evaluators to confirm if this difference is repeatable. for decades, the standard number of sperm assessed for morphology examination has been 100. this was most likely chosen for ease of calculation conversion to percent, but to our knowledge there are no reported data to validate this as the ideal number of sperm to be evaluated in bulls or other species. it is logical to expect that assessing a higher number of sperm would mean the estimated sperm morphology would more accurately represent the true sperm morphology. therefore, evaluating a higher number of sperm during a morphological assessment would more accurately estimate the true percentage of normal sperm, thereby improving the accuracy of bbse classifications (‘satisfactory’, ‘unsatisfactory’, or ‘deferred’) since sperm morphology has the single greatest effect on the bbse classification.1 however, results reported here indicate that increasing the number of sperm evaluated (up to 400), did not improve or change the result of classifying a bull as ‘satisfactory based on morphology assessment. based clinical theriogenology 2022; 14: 17 on results reported, additional time needed to evaluate more than 100 sperm during a routine bbse would be difficult to justify, in part, because evaluating additional sperm did not result in higher agreement among assessments. further investigation with increased slide numbers would be beneficial to validate our findings. the final suggestions and results of the previously mentioned studies were centered on investments in training, continuing education, proficiency testing, and other quality control measures to minimize this variation in evaluation.4,5 another aspect of the bbse that may affect the ability of the evaluator to assess sperm morphology is the equipment used to complete the evaluation. the same microscope, room, and oil were used for this study to help eliminate bias and most veterinarians will use a traditional compound light microscope similar to the one used here. however, if a veterinarian performs a high volume of bbse, they may invest in a microscope with fluorescent capabilities, phase contrast, or the ability to increase magnification. a fluorescent stain with subsequent microscopy would allow evaluation of sperm membrane integrity, acrosome status, dna integrity and more.17 additionally, using phase contrast allows the sperm to appear on a darker background to better highlight the sperm and eliminates the need for staining. increasing magnification would increase the resolution of individual sperm allowing for more precise evaluation. these alternate microscopic techniques could prove useful for breeding programs in further assessing sperm function. in this study, the evaluators utilized the same room for the entirety of the investigation, whereas in practice, facilities may vary from indoor to outdoor, lighting, and temperatures possibly affecting the results. sample preparation methods and setting recommendations must be strictly followed to obtain credible results and the species, extender, hardware/software, and operator can all affect the output values.18,19 although casa was not used, visual sperm evaluation in men sperm is comparable to casa to parameters assessed (including morphology).18 despite our smaller sample size, findings emphasized the importance of further investigation into slide preparation, equipment used, evaluator experience, and continued training and practice. conflict of interest none to declare. references 1. carson rl, wenzel jgw: observations using the new bull-breeding soundness evaluation forms in adult and young bulls. vet clin north am food anim pract 1997;13:305-311. 2. koziol jh, armstrong cl: manual for breeding soundness examination of bulls, 2nd edition, mathews, society for theriogenology, 2018. 3. barth ad, oko rj: abnormal morphology of bovine spermatozoa. iowa state university press. 1st edition, ames, iowa 1989:3-7;19-89. 4. brito lfc, greene lm, kelleman a, et al: effect of method and clinician on stallion sperm morphology evaluation. theriogenology 2011;76:745-750. 5. eustache f, auger j: interindividual variability in the morphological assessment of human sperm: effect of the level of experience and the use of standard methods. hum reprod 2003;18:1018-1022. 6. murcia-robayo ry, jouanisson e, beauchamp g, et al: effects of staining method and clinician experience on the evaluation of stallion sperm morphology. anim reprod sci 2018;188:165-169. 7. guzick ds, overstreet jw, factor-litvak p, et al: sperm morphology, motility, and concentration in fertile and infertile men. n engl j med 2001;345:1388-1393. 8. lu l: reliability analysis: calculate and compare intra-class correlation coefficients (icc) in sas. nesug statistics and data analysis 2007;1-4. https://lexjansen.com/nesug/nesug07/sa/sa13.pdf. 9. koo wk and my li: a guideline of selecting and reporting intraclass correlation coefficients for reliability research. j chiropr med 2016;15:155-163. 10. monday jd, larson rl, theurer me, et al: factors associated with yearling bulls passing subsequent breeding soundness evaluations after failing an initial evaluation. j am vet med assoc 2018;253:16171622. 11. conkey n, okafor c, strickland l, et al: factors associated with bulls not classified as satisfactory potential breeders. clinical theriogenology 2019;11:447. 12. bruner ka, mccraw rl, whitacre md, et al: breeding soundness examination of 1,952 yearling beef bulls in north carolina. theriogenology 1995;44:129-145. 13. roberts jn, grooms dl, thompson er, et al: evaluation of bull breeding soundness examination. clinical theriogenology 2018;10:397-408. 14. kruger tf, menkveld r, stander fs, et al: sperm morphologic features as a prognostic factor in in vitro fertilization. fertil steril 1986;46:1118-1123. 15. who laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th edition, cambridge, england: cambridge university press 1999. 16. wiltbank jn, parish nr: pregnancy rate in cows and heifers bred to bulls selected for semen quality. theriogenology 1986;25:779-783. 17. farah oi, cuiling l, jiaojiao w, et al: use of fluorescent dyes for readily recognizing sperm damage. j reprod infertil 2013;14:120-125. 18. talarczyk-desole j, berger a, taszarek-hauke g, et al: manual vs. computer-assisted sperm analysis: can casa replace manual assessment of human sperm in clinical practice? ginekologia polska 2017;88:56-60. 19. amann rp, waberski w: computer-assisted sperm analysis (casa): capabilities and potential developments. theriogenology 2014;81:5-17. 65 contact peyton draheim pfdraheim@gmail.com © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9619, http://dx.doi.org/10.58292/ct.v15.9619 case report utilization of colpotomy in the mare peyton draheim,a richard hopperb ahagyard equine medical institute, lexington, ky, usa bcollege of veterinary medicine, auburn university, auburn, al, usa abstract colpotomy, or a surgical incision of the vagina, has been used as an approach to urogenital surgery in the mare since the early 20th century. the most common use of this approach is for ovariectomy, though it has also been reported as an approach to the urinary bladder for repair of tears and removal of cystoliths as well as to the uterus to facilitate reduction of a twin fetus via transuterine cranio-cervical dislocation. advantages of the colpotomy approach include the ability to perform standing surgery (no need for general anesthesia), shortened surgical time, and a reduced postsurgical recovery time. disadvantages are the blind nature of some procedures performed via colpotomy (i.e., ovariectomy and associated potential complications such as hemorrhage and postoperative evisceration, with the most serious being death of the mare). this manuscript reviews the various reported uses of colpotomy in the mare as well as potential complications. keywords: mare, colpotomy, ovariectomy, bladder, twin reduction, urogenital surgery introduction as early as 1903, colpotomy has been reported as a surgical approach in the mare.1 colpotomy can be used to gain access to the mare abdomen without the need for general anesthesia. this results in a shortened surgical time and reduced postoperative recovery time,2 which translates into fewer anesthesia-associated risks to the mare and also a lower financial burden to the client. there are significant potential complications associated with colpotomy reported in the literature, including hemorrhage, evisceration, and peritonitis, though these may be minimized with proper surgical techniques and postoperative care.3 ovariectomy via colpotomy indications for an ovariectomy include a desire to diminish estrus behavior in performance mares, create a teaser mare for stallion semen collection, or sterilize a recipient mare to be used in an embryo transfer program.4 additionally, neoplastic ovaries smaller than 10 cm or those that can easily be grasped and held in the surgeon’s hand may be removed via colpotomy.4 other than colpotomy, surgical approaches for ovariectomy include celiotomy via the flank, ventral midline, or vaginal approach (natural orifice transluminal endoscopic surgery; notes), and the flank laparoscopic approach. the reduced surgical time compared to celiotomy and the lack of general anesthesia lowers the financial cost to the client, making colpotomy an attractive surgical option in some cases. while transvaginal notes and flank laparoscopy are also performed in the standing mare, the key difference between these approaches and conventional colpotomy is that the only specialized instrumentation required for conventional colpotomy is a chain ecraseur, while notes and laparoscopy require more advanced instrumentation.5 additionally, external scarring may be avoided with the use of the notes or colpotomy approaches. ovariectomy via colpotomy has been previously described in the literature.4,6,7 briefly, feed can be withheld from the mare for at least 24 hours prior to surgery with the goal of reducing the number of fecal balls present within the small colon that could be mistaken for an ovary during the procedure. preoperative antibiotics and an anti-inflammatory (i.e., flunixin meglumine at 1.1 mg/kg iv) should be administered. the mare should be restrained in stocks with the tail wrapped and secured to one side. sedation and analgesia may be achieved via administration of detomidine hcl (0.002–0.022 mg/kg iv) and butorphanol (0.002–0.022 mg/kg iv). sedation may be re-dosed as needed throughout the procedure, or a continuous rate infusion (cri) of detomidine or detomidine with butorphanol can be used. a caudal epidural should be administered with 2% lidocaine (0.22 mg/kg) alone or in combination with xylazine (0.17 mg/kg); this serves to desensitize a portion of the caudal reproductive tract, but its main benefit is prevention of straining by the mare during surgery. a mailto:pfdraheim@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9619 66 citation line: clinical theriogenology 2023, 15, 9619, http://dx.doi.org/10.58292/ct.v15.9619 caudal epidural may be administered in either the first or second intercoccygeal space in the mare (figures 1 and 2). the angle of needle introduction should be 30° with horizontal rather than perpendicular to the spinal canal if the second intercoccygeal space is chosen.8 manure should be evacuated from the rectum and the perineum should be cleansed with water and a mild soap, as is standard for any vaginal procedure in the mare. the vagina is prepared for surgery by introducing betadine™ scrub on cotton pledgets followed by lavage with dilute betadine™ solution. all of the solution is then evacuated, and the lips of the vulva are separated manually with the goal of creating a pneumovagina. introduction of a sterile vaginal speculum will also facilitate this. the creation of a pneumovagina is one of the most important parts of ensuring a successful colpotomy. this results in a tight drum-like dilation of the cranial vaginal vault, making it easier to identify and avoid vaginal arteries and create a safe incision. conversely, when the vaginal wall is flaccid, the stabbing motions required to make the vaginal incision can result in inadvertently nicking the internal iliac artery. the surgeon should at minimum wear sterile surgical gloves over sterile palpation sleeves. once the surgeon has identified via palpation the site on the cranial vaginal wall where the incision will be made (usually at the 10 o’clock or 2 o’clock position, with the cervix being the center of the clockface6), a sterile laparotomy sponge soaked in 2% lidocaine is held up against this site for several minutes to provide topical anesthesia. a piece of suture or umbilical tape is then secured to a number 10 scalpel blade (to allow easy localization should the blade be accidentally dropped within the vagina), which the surgeon introduces into the vagina, taking care to keep the blade guarded until ready to make the incision (figure 3). a small incision (approximately 1 cm, enough to just fit the tip of the surgeon’s finger) is made through the vaginal wall and the scalpel is removed from the vagina. the surgeon first introduces one finger into the incision, bluntly dilating with increased numbers of fingers until the incision can accommodate the entire hand. at this point, the peritoneum is bluntly entered with either a quick forward stabbing motion with the forefinger or a tearing action with the thumb and forefinger (figure 4). the hole in the peritoneum is then dilated until the hand and forearm of the surgeon are within the abdominal cavity. the ovaries are identified in situ and the ovarian pedicles are palpated for any abnormalities. another lidocaine-soaked laparotomy sponge, this time with attached suture or umbilical tape, is taken into the abdomen and held against the ovarian pedicle for several minutes to provide some topical analgesia. the surgeon then obtains the chain ecraseur, with the loop of chain encircling the four fingers (figure 5). the surgeon introduces the ecraseur into the vagina, through the colpotomy incision, and into the abdomen. the first ovary (usually the ovary that is contralateral to the colpotomy incision; i.e., remove the left ovary first if the colpotomy is on the right side of the vagina and vice versa) is identified and the chain loop is placed around the ovarian pedicle. once certain that the chain encircles the ovarian pedicle and nothing else, the surgeon or an assistant begins slowly tightening the ecraseur (figure 6). continuous careful palpation of the chain and pedicle by the surgeon is necessary while the chain tightens to ensure that no bowel or mesentery slips into the loop and is inadvertently transected. most mares will react to some extent while the chain tightens but should relax once the ovary is removed. it is important that there not be tension on the figure 1. palpation of the space between the first and second coccygeal vertebrae (first intercoccygeal space) for administration of a caudal epidural in the mare. figure 2. palpation of the space between the second and third coccygeal vertebrae (second intercoccygeal space) for administration of a caudal epidural in the mare. figure 3. the scalpel blade must be carefully guarded while taken through the vestibule and vagina to avoid inadvertently damaging the mucosa. this can be done by grasping the tip of the blade between the flexed thumb and forefinger (a); the tip of the #10 blade is exposed by extending the thumb and forefinger (b). http://dx.doi.org/10.58292/ct.v15.9619 citation line: clinical theriogenology 2023, 15, 9619, http://dx.doi.org/10.58292/ct.v15.9619 67 pedicle while the chain is tightened, as this may increase the risk of hemorrhage from the ovarian artery via a recoiling action of the vessel once the ovary is removed.4 the second ovary is removed in the same fashion as the first ovary, unless a unilateral ovariectomy is being performed (i.e., in the case of neoplasia). the colpotomy incision is not sutured, as this may lead to vaginitis and straining by the mare.4 a caslicks vulvoplasty should be performed to reduce the risk of ascending contamination of the vagina and abdomen, as well as to prevent catastrophic evisceration. cross-tying the mare for 2–3 days postoperatively is optional. the purported value of cross-tying is to prevent recumbency and the associated intraabdominal pressure changes when the mare rises to reduce the chances of evisceration4, this has been suggested to be unnecessary6 and is not performed by the authors. antimicrobial and analgesic therapy should be continued for 3–5 days postoperatively.4 the colpotomy incision contracts rapidly (to one finger’s diameter within 3 days4) and should be completely healed within 3 weeks.6 utilization of colpotomy for twin reduction via cranio-cervical dislocation cranio-cervical dislocation (ccd) refers to the method of twin reduction commonly performed between days 55–150 of gestation whereby the first cervical vertebrae is manually dislocated from the cranium, severing the spinal cord and allowing the surviving twin to obtain nourishment from the entirety of the endometrium.9 various approaches have been described for ccd in the mare, including transrectal and flank laparotomy. a disadvantage of the transrectal approach is the potential for damage to the rectal mucosa, including a risk of rectal tearing due to the excessive manipulation inherent with the procedure. like the flank laparotomy, the use of the colpotomy approach obviates that concern and it has recently been successfully used for the reduction of twins via ccd.10,11 bassett et al. describe a success rate of 71% (n = 29) when using ccd via colpotomy to reduce dizygotic twins and a success rate of 50% (n = 2) when reducing monozygotic twins.11 one reason offered as a potential explanation for the difficulty in successfully reducing monozygotic twins is the intimate association of such twin fetuses within a single allantochorion.11 hall et al. successfully reduced dizygotic twins in a mare approximately 60 days pregnant, one being located within the uterine body, via ccd through colpotomy.10 neither the transrectal nor the laparotomy approach could be used in this case due to the position of the fetus within the uterine body. the colpotomy was performed as previously described for ovariectomy4 and once the uterus was identified the fetus within the uterine body underwent ccd via transuterine manipulation. immediately after the procedure, transrectal ultrasonography revealed a slow, faint heartbeat in the reduced twin, which completely subsided within the following 2 days. in this case, the mare went on to have a healthy singleton foal at approximately 342 days gestation.10 it was concluded that transuterine ccd via colpotomy is a viable option for twin reduction. utilization of colpotomy to access the urinary bladder the most common surgical disorder of the bladder in horses is cystolithiasis.12 type i (yellow-green and spiculated) and type ii (gray-white and smooth) stones should be manually removed from the bladder, while horses with sabulous urolithiasis, which is secondary to bladder paralysis, are not surgical candidates and should be medically managed.12 rupture of the urinary bladder, or cystorrhexis, is more common in foals than in adult horses; in the adult cystorrhexis is most figure 4. the surgeon’s forefinger can be seen entering the abdominal cavity through the colpotomy. this image was taken with the aid of laparoscopy. for orientation, the uterus is labeled (a). figure 5. a standard chain ecraseur commonly used for ovariectomy via colpotomy. figure 6. the surgeon’s hand is grasping the ovary and the chain of the ecraseur encircles the ovarian pedicle. this image was taken prior to tightening the ecraseur. as the ecraseur tightens, the surgeon should continually palpate to ensure that the ovarian pedicle is the only structure within the chain loop and that no portion of the ovary is left behind. http://dx.doi.org/10.58292/ct.v15.9619 68 citation line: clinical theriogenology 2023, 15, 9619, http://dx.doi.org/10.58292/ct.v15.9619 commonly associated with urolithiasis, trauma, and parturition.13 during parturition, the bladder may either become trapped between the pelvic brim of the mare and a bony prominence of the foal, or the urethra may become occluded during a dystocia, resulting in bladder rupture.14 single tears less than 5 cm in length may be medically managed via maintenance of a urinary catheter and abdominal drainage,15 though primary surgical closure of defects is generally preferred.14 surgical approaches to the bladder reported in the literature include the traditional caudal ventral midline laparotomy, eversion of the bladder through a vaginal or urethral incision, direct eversion of the bladder through the urethral orifice, laparoscopy, and a transurethral endoscope-guided approach.16 approaches to the bladder that incorporate a colpotomy include incising the cranial vaginal wall, the urethra, or the urethral sphincter (urethral sphincterotomy). in one reported case of a postpartum mare with a ventral bladder tear, a 1–2 cm incision was made through the vaginal floor, 5–10 cm caudal to the cervix17; this approach will be described below. a 1–2 cm incision was made through the vaginal floor and digitally enlarged until the peritoneal cavity could be entered, at which point the bladder was identified via palpation and everted through the colpotomy. an assistant surgeon kept the bladder everted in an appropriate position with stay sutures, while the primary surgeon repaired the defect in two layers. the bladder was then returned to the abdomen and the colpotomy sutured in a single simple continuous layer.17 rodgerson et al. describe the use of a 24 f foley catheter to facilitate identification of the optimal colpotomy site.17 in this case, a postpartum mare was diagnosed with a ventral bladder tear via digital examination through the urethral orifice. the mare was sedated and restrained routinely for standing urogenital surgery. the foley catheter was passed through the urethra and into the bladder and was digitally palpated to identify the site of colpotomy, which was approximately 5 cm cranial to the urethral orifice and 3 cm in length and extended into the urethra. the bladder tear was identified via digital palpation through the incision into the urethra, and traction was applied with an index finger through the tear to prolapse the bladder through the colpotomy. stay sutures were used to maintain the bladder in position while the tear was closed in two layers. the bladder was returned to the abdomen and the urethral wall and vaginal floor were closed separately in a simple continuous pattern.17 urethral sphincterotomy to exteriorize the bladder has been described.13,18 in this approach, an approximately 5-cm-long colpotomy is created through the urethra and external urethral sphincter. the bladder is grasped, everted into the vagina, and repaired as previously described. while it has been reported that care should be taken to ensure that suture material does not enter the bladder lumen to prevent the formation of cystic calculi,12 there are reports of full-thickness bladder repair in adult horses that did not result in any post-operative calculi formation.13 in one case report, none of the mares that underwent urethral sphincterotomy experienced any complications, such as incontinence, urine pooling, or infertility.17 however, there is evidence in the literature of postoperative urine dribbling and urine pooling after urethral sphincterotomy.18 advantages of the standing vaginal approaches to the bladder are the decreased surgical time and lack of general anesthesia, the lack of external scarring (improved cosmesis), and the ability to access defects in the bladder that are unable to be exteriorized through a ventral midline laparotomy due to either their location or the extensive damage to the bladder that precludes the significant traction required for sufficient exteriorization. the above-described methods, while discussed in the context of repair of cystorrhexis, may also be used for removal of bladder stones. alternative surgical approaches include laparoscopic and transurethral techniques, though as these procedures do not involve a colpotomy, they are beyond the scope of this manuscript. the reader is directed to other sources for information on these surgical techniques.14,15,16,19 potential complications potential complications of the colpotomy approach in the mare include laceration of the vaginal artery during incision, hemorrhage from the ovarian pedicle post-ovariectomy, accidental dissection into the rectum or urinary bladder, postoperative evisceration, peritonitis, surgical site abscess or hematoma formation, and formation of vaginal adhesions.3 careful and intentional identification of the planned incision site via digital palpation and creation of a pneumovagina are ways to avoid laceration of major vessels during creation of the colpotomy. the transvaginal use of a 7.5 mhz linear array ultrasound probe with doppler to identify the vaginal branch of the internal pudendal artery and the uterine branch of the urogenital artery prior to creation of the colpotomy has also been reported.20 during ovariectomy, once the chain ecraseur has been released and the ovary removed, it can be difficult to identify excessive hemorrhage from the transected pedicle.3 further, as a decrease in serum total protein (tp) and packed cell volume (pcv) may not be evident for 6–24 hours postoperatively, using these parameters to monitor acute blood loss in the recently ovariectomized mare is difficult.4 clinical signs such as heart rate, respiratory rate, pulse quality, and mucous membrane color should be monitored in all postoperative mares, and fluid therapy and/or a whole blood transfusion may be considered. placement of a caslicks suture following the colpotomy procedure may prevent catastrophic evisceration of abdominal viscera out of the mare but will not prevent the viscera from gaining access to the vagina and vestibule.3 restraint of the mare in cross-ties to prevent recumbency for several days postoperatively may also be used to prevent evisceration from intraabdominal pressure changes, but there are conflicting opinions on this tactic.4,6 finally, as with any procedure that enters the abdomen, there is a risk of postoperative peritonitis. this risk, along with that of abscess and hematoma formation, can be minimized with meticulous surgical technique and maintenance of sterility. conclusion colpotomy is a useful approach for standing urogenital surgical procedures in the mare. ovariectomy, twin reduction via ccd, and repair of cystorrhexis via colpotomy have all been reported in the literature. potential complications such as hemorrhage and evisceration may be avoided with careful attention to detail and the use of appropriate precautions before, during, and after the procedure. with the advantages of both a shorter surgical time and a shorter postoperative recovery time, the lack of general anesthesia, and improved cosmesis, colpotomy should be considered a viable option in the experienced veterinarian’s hands when planning urogenital procedures in the mare. http://dx.doi.org/10.58292/ct.v15.9619 citation line: clinical theriogenology 2023, 15, 9619, http://dx.doi.org/10.58292/ct.v15.9619 69 references 1. williams wl: surgical and obstetrical operations. ithaca: cornell university libraries; 1903. 2. rodgerson dh, loesch da: ovariectomy. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edtion, ames; wiley-blackwell: 2011:2564–2573. 3. embertson rm: selected urogenital surgery concerns and 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reproductive procedures. 1st edition, hoboken; john wiley & sons: 2014: 222–225. doi: 10.1002/9781118904398.ch67 10. hall s, klabnik j, yepez p, et al: uterine body fetal reduction in a mare: cranio-cervical dislocation via colpotomy. clin theriogenol 2020;12:522–525. doi: 10.58292/ct.v12.9452 11. bassett a, rock k, propp c, et al: reduction of equine monozygotic twins using cranio-cervical dislocation via colpotomy. clin theriogenol 2021;13:319. 12. schott hc, woodie jb: bladder. in: auer ja, stick ja: editors. equine surgery. 4th edition, st. louis, mo; elsevier/saunders; 2012: 840–866. doi: 10.1016/b978-1-4377-0867-7.00065-x 13. higuchi t, nanao y, senba h: repair of urinary bladder rupture through a urethrotomy and urethral sphincterotomy in four postpartum mares. vet surg 2002;31:344–348. doi: 10.1053/jvet.2002.33593 14. rijkenhuizen abm, van loon tjam, boswinkel m: laparoscopic repair of a ruptured bladder in an adult mare. equine vet educ 2008;20:183–189. doi: 10.2746/095777308x292137 15. pye jl, collins nm, adkins ar: transurethral endoscopic-guided intraluminal closure of multiple urinary bladder tears in a standing mare. equine vet educ 2018;30:127–131. doi: 10.1111/eve.12622 16. hall md, rodgerson dh: transurethral intraluminal closure of a caudally located bladder neck tear in a standing mare. equine vet educ 2020;32:111–115. doi: 10.1111/eve.13064 17. rodgerson dh, spirito ma, thorpe pe, et al: standing surgical repair of cystorrhexis in two mares. vet surg 1999;28:113–116. doi: 10.1053/jvet.1999.0113 18. white kk: urethral sphincterotomy as an approach to repair of rupture of the urinary bladder: a case report. j equine med surg 1977;1:250–253. 19. stephen jo, harty ms, hollis ar, et al: a non-invasive technique for standing surgical repair of urinary bladder rupture in a post-partum mare: a case report. irish vet j 2009;62:734–736. doi: 10.1186/2046-0481-62-11-734 20. alvarez av, boone l, horzmann k, et al: hybrid natural orifice transluminal endoscopy surgery (notes) to perform bilateral ovariectomy in mares. vet surg 2021;51:174–182. doi: 10.1111/vsu.13687 http://dx.doi.org/10.58292/ct.v15.9619 https://doi.org/10.1016/j.cveq.2008.10.007 https://doi.org/10.1111/eve.12801 https://doi.org/10.1111/j.1532-950x.2011.00871.x https://doi.org/10.1002/9781118904398.ch67 https://doi.org/10.58292/ct.v12.9452 https://doi.org/10.1016/b978-1-4377-0867-7.00065-x https://doi.org/10.1053/jvet.2002.33593 https://doi.org/10.2746/095777308x292137 https://doi.org/10.1111/eve.12622 https://doi.org/10.1111/eve.13064 https://doi.org/10.1053/jvet.1999.0113 https://doi.org/10.1186/2046-0481-62-11-734 https://doi.org/10.1111/vsu.13687 testicular hypoplasia and azoospermia in a crossbred boar testicular hypoplasia and azoospermia in a crossbred boar edgar garrett,a jamie stewart,b igor canisso a adepartment of veterinary clinical medicine, college of veterinary medicine university of illinois, urbana il bdepartment of large animal clinical sciences virginia-maryland college of veterinary medicine, blacksburg, va abstract an 8 month old boar was examined for absence of sperm in semen at initial semen collection in a boar stud. clinical examination revealed a clinically healthy boar with marginally acceptable testicular size and normal libido. ejaculation was confirmed by measuring seminal plasma alkaline phosphatase concentrations. histologic examination of biopsies from both testes revealed testicular hypoplasia. historical semen production characteristics of close relatives implied a possible genetic cause, and boar was removed from breeding herd. keywords: azoospermia, crossbred boar background azoospermia is an extreme example of testicular dysfunction. causes are genetic hypoplasia, congenital or acquired obstructions to sperm outflow, and testicular degeneration secondary to disease, injury and temperature extremes. reason for azoospermia is diagnosed by concentrations of semen alkaline phosphatase (ejaculation failure) and by testicular biopsy findings (seminiferous tubule dysfunction). testicular hypoplasia and segmental aplasia of epididymis are associated with genetic defects in pigs.1 in these instances, cytogenetic analysis of animals and their close relatives are performed to identify carriers of genetic defects for decreased fertility. critical evaluation of semen parameters of related boars is also recommended to identify congenital causes of subfertile or infertile animals. case presentation an 8 month old, crossbred boar, was presented to university of illinois veterinary teaching hospital (vth) when attempts to collect semen on boar resulted in samples with no sperm. boar was housed in a well-managed breeding facility that sold semen from crossbred boars used primarily for exhibition. owners reported that members of this boar’s family line tended to have small testes and low semen production. expected sperm number is 10 100 billion per collection when semen collection is performed twice per week.2 boar’s sire, also a crossbred boar, produced good motile sperm with low proportion of morphologic defects; however, sperm output was poor (~ 30 billion sperm in once a week collection). collecting more than once weekly resulted in considerable reduction in sperm number. boar’s sire sired 6 other boars from 6 different sows, of which 3 boars had small testes and 2 of these had adequate sperm motility with low number (~ 30 billion sperm per collection even if semen collection was once per week) and other 3 boars were normal. a daughter of boar’s sire bred to him, produced 2 boars (1 had normal semen production, and other had oligospermia and asthenospermia) from different litters. boar’s grandsire had normal semen parameters; however, a hemicastration was performed when he was 6 years old. details of medical condition requiring hemicastration were not known. boar’s dam produced 12 litters and mating to boars other than her sire yielded boars with normal semen parameters. boar’s sire was also seen at vth on 8 occasions over 2 years for idiopathic peritoneal effusion. inflammatory, infectious, neoplastic, cardiac and hepatic etiologies were ruled out. approximately 20 liters of peritoneal fluid was drained from abdomen at each visit. peritoneal effusion stopped without specific treatment. at presentation, boar had a normal demeanor and gait. he weighed 150 kg and rectal temperature was 37.8°c (reference range 38.6 40°c). no abnormalities were detected on physical examination. boar displayed an acceptable libido and mounted a dummy readily. penis was examined during semen collection and appeared anatomically normal. semen was collected using gloved-hand technique into an insulated collection bottle with a filter to separate gel from semen. approximately 15 ml of clear, gel-free clinical theriogenology • volume 12 number 2 • june 2020134 ejaculate was obtained. multiple aliquots of ejaculate were examined using a phase contrast microscope (olympus bx50, olympus scientific solutions americas corporation, waltham, ma) at 100 x and 400 x magnification, and no sperm were observed. lack of sperm in an ejaculate can be due to ejaculation failure, hypogonadotropic hypogonadism, defective spermatogenesis, obstruction of sperm outflow, and testicular degeneration. in this case, ejaculation failure was suspected due to low volume of seminal plasma collected. to test for ejaculation failure, an aliquot of gel-free fraction was submitted to clinical pathology laboratory of vth for measurement of seminal alkaline phosphatase (alp) concentration. sample contained 32,500 u/l alp (reference range: 24,276 55,678 u/l), consistent with a successful ejaculation.3 hypogonadotropic hypogonadism is very rare in domestic animals. in a finnish study reporting results of semen collection on 2,048 boars, 1 animal was diagnosed with hypogonadotropic hypogonadism and had a testicular weight of only 26 g.1 this condition was not suspected in this case given its rarity and the boar’s subjectively near-normal size testes. due to low probability of this condition and budget constraints, diagnostic efforts were focused on structural and functional problems of testes, including developmental defects. boar was sedated with tiletamine and zolazepam (telazol, zoetis, parsippany, nj) with 250 mg of ketamine (zetamine, vet one, boise, id) and 250 mg of xylazine (anased la, vet one, boise, id) to yield a solution containing 50 mg/ml of each drug (telazol, zetamine, and anased la) given at a dose of 1.6 mg/kg im of each drug in combination. palpation of scrotum and its contents revealed both testes and epididymides free from adhesions, with no palpable abnormalities and normal testicular tone. ultrasonographic examination was performed with a curved array 5.0 2.0 mhz transducer (sonoscape s8exp, universal imaging inc., bedford hills, ny). both testes had normal echogenic pattern, and left testis measured 8.2 (length) x 5.3 (width) x 4.8 (height) cm and right testis measured 8.8 (length) x 6.0 (width) x 4.9 (height) cm (figure 1). segmental aplasia of ductus deferens was suspected; however, surgery or necropsy was necessary for visual inspection to confirm. therefore, testicular biopsy was elected. biopsies of each testis were performed using a 14 gauge biopsy instrument (tru-cut, merit medical, south jordan, ut). biopsy samples were fixed in 10% buffered formalin and submitted to university of illinois veterinary diagnostic laboratory for histologic examination. biopsy samples were embedded in paraffin and stained with hematoxylin-eosin. testicular parenchyma of both testes was predominantly composed of interstitial cells with rare seminiferous tubules. seminiferous tubules appeared atrophied and contained few sertoli cells and rare sperm, consistent with congenital testicular hypoplasia (figure 2). figure 1. ultrasonographic images of left (a) and right testis (b) a b clinical theriogenology • volume 12 number 2 • june 2020 135 figure 2. representative histologic appearance of testicular biopsy samples fixed in formalin and stained with hematoxylin and eosin. (a) a predominance of interstitial cells with rare seminiferous tubules (200 x). (b) seminiferous tubule with cellular vacuolization (400 x). (c) seminiferous tubule with rare sperm (400 x). outcome cause of azoospermia was determined to be due to testicular hypoplasia. based on our findings, removal of boar from breeding facility was recommended and no further followup was possible. discussion azoospermia in a young boar could be due to delayed puberty, ejaculation failure, obstruction of sperm outflow, testicular hypoplasia and testicular degeneration. puberty is loosely defined in males as onset of sexual behavior. throughout puberty, there is an increase in penis size that results in sigmoid flexure formation. this increase in size, coupled with onset of mounting activity, helps to break prepuce attachment (frenulum) to penis. time to onset of puberty in boars varies considerably by breed but sufficient pubertal development for breeding activity is not expected until after 5 months of age.4 pubertal development could be delayed due to poor nutrition, chronic disease, primary hypogonadism (e.g. klinefelter’s syndrome) or secondary hypogonadism (e.g. pituitary disorder). in this case, boar exhibited appropriate sexual response, exteriorized his penis and ejaculated completely, indicating attainment of puberty by an acceptable age. postpubertal, young boars are expected to have an ejaculate volume of 100 300 ml.5 in this case, low volume of seminal plasma was possibly due to ejaculation failure or bilateral obstruction of mesonephric ducts’ derivatives. various congenital defects in epididymis and ductus deferens reported in boars are believed to be of genetic origin.1,6 high alp concentrations in ejaculate in this boar confirmed a b c clinical theriogenology • volume 12 number 2 • june 2020136 epididymis and ductus deferens patency. this diagnostic method developed in dogs and stallions (where major sources of alp include epididymis and testes) has been used in boars.3,6 findings in this boar are consistent with those of other species, demonstrating that semen alp is a useful parameter for ruling out ejaculation failure or outflow obstruction as a cause of azoospermia in swine. testicular hypoplasia in domestic animals is due to genetic, endocrine, developmental defects. in boars, hypogonadotropic hypogonadism, chromosomal defects, and arrested spermatogenesis are causes of testicular hypoplasia.1,7 in testicular hypoplasia, testes do not grow to a normal size because of reduced mass of seminiferous epithelium, but otherwise appear normal. microscopic abnormalities seen with testicular hypoplasia may include a total lack of germ cells, uniformly arrested spermatogenesis or spermatogenic arrest that varies between tubules.7 in boars, there is a proliferation of sertoli cells between birth and 1 month of age and then another significant proliferation between 3 4 months of age. proliferation of sertoli cells coincides with significant increase in seminiferous tubule length.8 boars are recognized to have rapid testicular growth at 4 6 months of age, followed by slow growth to 8 months of age. testicular diameter only increases by 2 cm from 8 months of age to 2 years of age.4 thus, testicular dimensions at 8 months of age are a good representation of a boar’s mature testicular size. while generally accepted standards for boar breeding soundness evaluation are lacking, similar to current case, suggested minimum acceptable testicular dimensions for a boar at 8 months of age are 8 x 5 cm.5 a survey of 431 boars stratified by breed reported a range of average testicular lengths of 9.6 11.9 cm and average testicular widths of 5.0 5.7 cm in boars 6 11 months of age.9 measurements in this current case were ~ 1 cm shorter than reported average. given low ejaculate volume and marginal testicular size, spermatogenic arrest was considered. azoospermia associated with spermatogenic arrest was reported in pigs, with testicular weight ~ half of that in normal boars.1 several reports linked this condition to genetics, which appears likely in this case, given familial history. in finnish yorkshire pigs, a defect in tex14 gene was linked to spermatogenic arrest.10 another report described translocation of a segment of chromosome 13 to y chromosome resulting in spermatogenic arrest and azoospermia.11 although these defects are considered rare in general population, spermatogenic arrest accounted from 9 of 16 cases of azoospermia in a large population of finnish yorkshire and landrace boars.1 patient was discharged while awaiting testicular biopsy results and owner was not willing to pursue further. limitation of this report was lack of genetic analysis. similar to testicular hypoplasia, testicular degeneration results in small testicular size, decreased semen production, and histologic abnormalities in seminiferous tubules. testicular degeneration can be caused by extreme heat or cold, systemic disease, nutritional deficiencies, and trauma.7 a boar suffering from testicular degeneration would likely have had evidence of reproductive success followed by a decrease in fertility leading to a clinical examination, whereas an animal with testicular hypoplasia will never achieve normal fertility. in this case, problem was recognized at first attempt of semen collection. boar was presented in november, making exposure to extremes of heat or cold in preceding 3 months unlikely. in some species, testicular degeneration causes changes in testes that appear as hyperechogenic areas in testicular parenchyma,12 which presumably is due to fibrosis and mineralization.7 although ultrasonographic changes in boars with testicular degeneration have not been described, echogenicity of testicular parenchyma in this boar was subjectively normal during ultrasound examination based on comparison to published images.13 given boar’s age, good management at breeding facility, and a lack of prior disease or trauma based on history, testicular degeneration was unlikely. given familial history of reproductive problems, testicular hypoplasia was most plausible explanation for clinical findings. in this case, azoospermia was due to testicular hypoplasia. familial history was highly suggestive of a genetic defect. histologic appearance of testes in this case with sertoli cells only populating some seminiferous tubules and only a few sperm in other tubules was consistent with hypoplasia; however, predominance of interstitial cells and near lack of seminiferous tubules makes histologic appearance in this case distinctly different than other cases of hypoplasia. clinical theriogenology • volume 12 number 2 • june 2020 137 learning points  measuring alkaline phosphatase concentrations in semen is a logical first step to rule out ejaculation failure or aplasia of epididymis or ductus deferens in cases of azoospermia.  testicular biopsy is next logical step in azoospermic boars with normal seminal plasma concentration of alp to determine if testicular hypoplasia or degeneration is present.  familial history should be gathered in detail for boars with poor sperm production to identify related animals for semen evaluation and genetic testing to identify genetic defects that could affect fertility of future generations. conflict of interest none to report. references 1. kopp c, ijas r, flyckt a, et al: morphometric evaluations of testicular tissues from azoospermic boars in finnish yorkshire and landrace breeds. theriogenology 2008;70:1129-1135. 2. larson, k: evaluation of boar semen. in: morrow da: editor. current therapy in theriogenology. 2nd edition, philadelphia; wb saunders: 1986. p. 972-975. 3. clark sc, clements km, shipley cf: alkaline phosphatase activity as an ancillary test for characterizing infertility in the boar. abstract, clinical theriogenology 2003:5. 4. kuster c, althouse g: reproductive physiology and endocrinology of boars. in: youngquist r and threlfall w: editors. current therapy in large animal theriogenology. 2nd edition, philadelphia; saunders: 2007. p. 717-721. 5. shipley, cf: breeding soundness examination of the boar. j swine health prod 1997;7:117-120. 6. clements km, shipley cf, coleman da, et al: azoospermia in an 8 month old boar due to bilateral obstruction at the testis/epididymis interface. can vet j 2010;51:1130-1134. 7. foster ra: male genital system. in: maxie, mg: editor. jubb, kennedy & palmer’s pathology of domestic animals: volume 3. 6th edition, philadelphia; wb saunders: 2016. p. 465-510. 8. franca lr, avelar gf, almeida ffl: spermatogenesis and sperm transit through the epididymis in mammals with emphasis on pigs. theriogenology 2005;63:300-318. 9. chacon j, castillo g, vargas b: testicular dimensions in boars. anim reprod sci 2018;194:e11 10. sironen a, uimari p, venhoranta h, et al: an exonic insertion within tex14 gene causes spermatogenic arrest in pigs. bmc genomics 2011;12:591-599. 11. villagomez daf, revay t, donaldson b, et al: azoospermia and testicular hypoplasia in a boar carrier of a novel y-autosome translocation. sex dev 2017;11:46-45. 12. gouletsou pg: ultrasonographic examination of the scrotal contents in rams. small rumin res 2017;152:100-106. 13. pinho ro, camilo bs, lima dma, et al: the use of ultrasonography in the reproductive evaluation of boars. reprod dom anim 2018;53:393-400. clinical theriogenology • volume 12 number 2 • june 2020138 clinical theriogenology 2022; 14: 67 failure of pregnancy in dogs and cats robert foster department of pathobiology, ontario veterinary college, university of guelph guelph, ontario, canada abstract failure of pregnancy in dogs and cats is approached as a diagnostic investigation of an individual animal. it begins with a preliminary discussion on zoonotic disease and expectations (seldom is the case) of evaluation. general approach from a pathology point of view is to identify potential infectious causes of pregnancy failure and to correlate these observations with lesions. potential infectious agents include eubacteria, fungi, viruses and protists. when infectious causes have been ruled out, the focus is then on noninfectious causes and particularly those with lesions. maternal evaluation, including endometrial biopsy after uterine involution, is part of the investigation. special effort should be made to collect umbilical arteries and lungs of fetus, and fetal membranes around the marginal hematoma. failure of pregnancy due to noninfectious causes without lesions contributes to a large percentage of cases, suggesting maternal, paternal, molecular, nutritional, or toxic causes. keywords: dogs and cats, pregnancy, abortion, stillbirth, embryonic mortality introduction there are 2 parts to the general approach to investigate failure of pregnancy in any species, particularly, in dogs and cats: a preliminary discussion with owners to ensure safety, expectations, and thoroughness of the evaluation and second, the diagnostic process. preliminary discussion begin discussions before the diagnostic process and include a discussion on public health and safety issues, identifying the problem, performing a cost-benefit analysis of investigation and diagnosis, and considering isolation of affected dams. zoonotic pathogens in dogs and cats include the eubacteria salmonella spp. and campylobacter spp. and in dogs brucella canis. appropriate precautions and personal protective equipment are essential. in cats, coxiella burnetii is an important consideration.1-4 abortion and stillbirth are rare; however, placenta may harbor large numbers of organisms that spread during cesarian section or parturition. a thorough risk assessment should therefore be conducted along with appropriate precautions. because dogs and cat submissions are not usually subsidized in government laboratories, failure of pregnancy investigation can be a costly process and is usually only attempted when there is a public health risk in breeding kennels and catteries, or when the owner is prepared for the costs involved. reproductive performance and kitten mortality rates are known5 but the diagnostic success rate for failure of pregnancy investigation is unknown. often postmortem and subsequent histological evaluation for lesions are performed before additional testing is used to verify the presence of an infectious agent or to verify that the organism recovered is responsible for pregnancy failure. diagnostic process it is similar to other disease investigation and involves determining signalment, history, macroscopic evaluation and collection and laboratory evaluation of samples from dam, fetus and fetal membranes, a tentative diagnosis. complete investigation includes careful examination of fetus and fetal membranes, umbilical cord, and collection of appropriate samples as outlined by a particular diagnostic laboratory. it is preferential to submit whole puppies and placentas, however to reduce costs, sampling of the fetal membranes to include the labyrinth, marginal hematoma and nonlabyrinthine portion is critical. reporting findings and lesions is made easier now by taking photographs with a mobile phone. there are few normal structures in dog and cat fetal membranes that may be mistaken for lesions. most are aware of the presence of a marginal hematoma. puppies in particular, are often autolyzed and thus have watery bloody fluid in their subcutis and body cavities. true hemorrhage had blood clots or is a fluid with a normal or near normal erythrocyte content. infectious causes of failure of pregnancy various infectious agents are reported to cause pregnancy failure. they include viruses, bacteria, and protozoa. many case reports, reviews and conference proceedings listed bacteria recovered from cases of infectious abortion or stillbirth.6-8 clinical theriogenology 2022; 14: 68 many eubacteria are responsible for failure of pregnancy6 including streptococci9 but there are some that are of greater importance, particularly, those with public health implications. salmonella spp. and campylobacter spp. are particularly important in dogs and cats.7,10-14 brucella canis is increasing its geographical range and continues to be important in causing failure of pregnancy.15-17 as with many bacteria, there are usually few or no postmortem changes either in the fetus or placenta. diagnosis is suspected when histopathology identifies neutrophilic placentitis or when salmonella spp. or campylobacter spp. is identified by culture of the placenta and/ or fetus. although there is initially a maternal infection, clinical signs in the mother may not be apparent. case studies of infection in a kennel and kennel workers demonstrates the public health importance.10 failure of pregnancy caused by coxiella burnetii in dogs and cats is extremely unusual. infection of the placenta of cats, however, is particularly important because of a lack of awareness of infection in outdoor cats. from a public health perspective, it is very important, particularly for veterinarians and veterinary practices.1-4 a risk assessment should always be performed when dealing with a cat that is queening or undergoing a cesarian section. viruses responsible for pregnancy failure in dogs and cats are the classical and well-known viruses of each particular species7 including; in dogs, canid alphaherpesvirus 1,18,19 carnivore bocaparvovirus one (canine minute virus),20 and canine distemper virus;21,22 and in cats, felid alphaherpesvirus18 feline immunodeficiency virus,23,24 alphacoronavirus 1 (feline infectious peritonitis virus), feline leukemia virus,25 and carnivore protoparvovirus 1 (feline parvovirus).22 fortunately, molecular diagnostic tests make identifying these agents much easier. protozoa responsible for failure of pregnancy are toxoplasma gondii in cats26 and neospora caninum and leishmania infantum in dogs.27 noninfectious causes of failure of pregnancy noninfectious causes of failure of pregnancy represent a substantial number of the overall causes. these causes can be classified into fetal, placental, maternal, fetal – maternal incompatibility and paternal components. some have identifiable lesions and whereas some do not. noninfectious causes are classified into those with identifiable lesions and those without lesions. this classification aids in the investigation of the problem at the kennel or cattery level. noninfectious fetal lesions. among the reported noninfectious fetal lesions, anomaly rates were very high in dogs and observed up to 10% of puppies born.28,29 some lesions are highly visible congenital defects, and include omphalocele, gastroschisis, amelia, anencephaly, anal atresia, fetal hydrops and cleft palate. these do not present a challenge. more challenging is those with a histologic lesion but no gross lesion. pulmonary immaturity and dysplasia are frequent, particularly in stillborn pups. careful evaluation of all organs is thus required for identification. noninfectious placental lesions. identification of noninfectious placental lesions is not reported; careful examination of fetal membranes, umbilical cords, and the fetus is necessary. awareness of conditions such as a single umbilical artery is necessary to find such a lesion! noninfectious causes without lesions. a diagnosis of ‘idiopathic’ failure of pregnancy is all too common in dogs and cats. considering the reported high prevalence of congenital abnormalities, it is likely that chromosomal and genetic abnormalities exist. routine evaluation for these is not available. maternal causes may be readily identifiable and therefore, samples not sent to diagnostic laboratories. failure of pregnancy with a maternal basis is seldom included in laboratory statistics as materials are not usually submitted. dystocia,5 endometritis, endometrial fibrosis, uterine neoplasia, hypoluteoidism, and hypothyroidism30 are all possible contributors. endometrial biopsy to identify endometrial fibrosis or other changes that may result in failure of pregnancy (as opposed to failure to become pregnant) is seldom routinely done and is in its infancy. fetal maternal incompatibility is an area of investigation that is not pursued in practice. presence of excessively aggressive trophoblasts31 and subinvolution of placental sites are reported as postpartum complications in bitches but the influence of the interaction of invasive trophoblasts and the endometrium and maternal fetal immunologic incompatibility awaits further study. paternal causes of failure of pregnancy have received little attention but are likely present. pedigree analysis and trial breeding are required to identify such causes. conclusion not much has changed in the last 14 years since what was written8 ‘knowledge of the causes of canine or feline pregnancy loss is limited and the success rate for making a definitive diagnosis is disappointingly low.’ financial constraints have not changed and until there is a greater emphasis on investigating the failure of pregnancy in dogs and cats, further progress is unlikely. microbes of the classic diseases of dogs and cats continue to be evaluated in pregnancy failures in these species. conflict of interest author has no conflict of interest. no funding was received for this publication. references 1. langley jm, marrie tj, covert a, et al: poker players’ pneumonia. new engl j med 1988;319:354-356. 2. kopecny l, bosward kl, shapiro a, et al: investigating coxiella burnetii infection in a breeding cattery at the centre of a q fever outbreak. j feline med surg 2013;15:1037-1045. clinical theriogenology 2022; 14: 69 3. fujishiro ma, scorza av, gookin jl, et al. evaluation of associations among coxiella burnetii and reproductive abnormalities in cats. j feline med surg 2016;18:344-347. 4. stefanetti v, compagnone a, sordini c, et al: retrospective biomolecular investigation of coxiella burnetii and leptospira spp. dna in cases of abortion, stillbirth and neonatal mortality in dogs and cats. top companion anim med 2018;33:122-125. 5. fournier a, masson m, corbière f, et al: epidemiological analysis of reproductive performances and kitten mortality rates in 5,303 purebred queens of 45 different breeds and 28,065 kittens in france. reprod domest anim 2017;52(suppl 2):153-157. 6. givens md, marley ms: infectious causes of embryonic and fetal mortality. theriogenology 2008;70:270-285. 7. pretzer sd: bacterial and protozoal causes of pregnancy loss in the bitch and queen. theriogenology 2008;70:320-326. 8. schlafer dh: canine and feline abortion diagnostics. theriogenology 2008;70:327-331. 9. lamm cg, ferguson ac, lehenbauer tw, et al: streptococcal infection in dogs: a retrospective study of 393 cases. vet pathol 2010;7:387-395. 10. morse ev, duncan ma. canine salmonellosis: prevalence, epizootiology, signs, and public health significance. j am vet med assoc 1975;67:817-820. 11. redwood dw, bell da: salmonella panama: isolation from aborted and newborn canine fetuses. vet rec 1983;112:362. 12. bulgin ms, ward ac, sriranganathan n, et al: abortion in the dog due to campylobacter species. am j vet res 1984;45:555-556. 13. odendaal mw, de cramer kg, van der walt ml, et al: first isolation of campylobacter jejuni from the vaginal discharge of three bitches after abortion in south africa. onderstepoort j vet res 1994;61:193-195. 14. sahin o, burrough er, pavlovic n, et al: campylobacter jejuni as a cause of canine abortions in the united states. j vet diagn invest 2014;6:699-704. 15. carmichael le, kenney rm: canine abortion caused by brucella canis. j am vet med assoc 1968;152:605-616. 16. brennan sj, ngeleka m, philibert hm, et al: canine brucellosis in a saskatchewan kennel. can vet j 2008;49:703-708. 17. gyuranecz m, szeredi l, ronai z, et al: detection of brucella canis–induced reproductive diseases in a kennel. j vet diagn invest 2011:23:143-147. 18. smith kc: herpesviral abortion in domestic animals. vet j 1997;153:253-268. 19. pratelli a, colao v, losurdo m: serological and virological detection of canine herpesvirus-1 in adult dogs with and without reproductive disorders. the vet j 2014;200:257-260. 20. decaro n, amorisco f, lenoci d, et al. molecular characterization of canine minute virus associated with neonatal mortality in a litter of jack russell terrier dogs. j vet diagn invest 2012;24:755-758. 21. krakowka s, hoover ea, koestner a, et al: experimental and naturally occurring transplacental transmission of canine distemper virus. am j vet res 1977;38:919-922. 22. decaro n, carmichael le, buonavoglia c: viral reproductive pathogens of dogs and cats. vet clin north am small anim pract 2012;42:583-598. 23. coats ks: the feline immunodeficiency virus-infected cat: a model for lentivirus-induced placental immunopathology and reproductive failure (mini-review). am j reprod immunol 2005;54:169-185. 24. coats ks, boudreaux ce, clay bt, et al: placental immunopathology in the fiv-infected cat: a role for inflammation in compromised pregnancy? vet immunol immunopathol 2010;134:39-47. 25. powers ja, chiu es, kraberger sj, et al: feline leukemia virus (felv) disease outcomes in a domestic cat breeding colony: relationship to endogenous felv and other chronic viral infections. j virol 2018;92: e00649-18. 26. dubey jp: a review of sarcocystis of domestic animals and of other coccidia of cats and dogs. j am vet med assoc 1976;169:1061-1078. 27. dubey jp, rosypal ac, pierce v, et al: placentitis associated with leishmaniasis in a dog. j am vet med assoc 2005;227:1266-1269. 28. groppetti d, martino pa, ravasio g, et al: prognostic potential of amniotic fluid analysis at birth on canine neonatal outcomes. vet j 2015;206:423-425. 29. ortega-pacheco a, lezama-garcía ma, colín-flores r, et al: presence of congenital anomalies in three dog litters. reprod domest anim 2020;55:652-655. 30. panciera dl, purswell bj, kolster ka, et al: reproductive effects of prolonged experimentally induced hypothyroidism in bitches. j vet intern med 2012;26:326-333. 31. rosenberg lm, marinoff j, crouch ee, et al: uterine perforation secondary to metritis and placenta percreta in a postpartum bitch. can vet j 2020;61:584-588. introduction common prostatic diseases of dogs include benign prostatic hyperplasia (bph), prostatitis, prostatic cysts, prostatic abscesses, and less commonly neoplasia such as squamous metaplasia.1,2 the most prevalent prostatic disease is bph3 and it is diagnosed in almost every intact male dog.3,4 prevalence of bph is 80% in dogs > 6 years and 95% in dogs > 9 years old.4 the etiology and factors that increase severity of clinical signs are still not fully understood in dogs with bph. previous studies have retrospectively examined histologic features of canine prostatic disease and characterized affected populations.3,5 however, many studies focus solely on prostate size as an end point and contrary to previous belief, there may be weak correlation between prostate size and severity of clinical signs.6,7 bph in dogs consists of diffuse, symmetrical prostatic enlargement growing outward, away from the urethra, due to both hyperplastic and hypertrophic processes.8 symmetrical growth away from the urethra is a feature unique to canine bph. in men, bph growth occurs in a nodular fashion and can be both outward and inward toward the urethra. it is this variation in prostatic growth (suggestive of differences in the pathogenesis of prostatic hyperplasia in people and dogs) that lead some a retrospective review of canine benign prostatic hyperplasia with and without prostatitis hannah ruetten,a* marlyse wehber,a* mary murphy,a* clara cole,a* simran sandhu,a steven oakes,a dale bjorling,b kenneth waller iii,b katrina viviano,c chad vezinaa *authors contributed equally adepartment of comparative biosciences; bdepartment of surgical sciences; cdepartment of medical sciences school of veterinary medicine, university of wisconsin-madison, madison, wi abstract benign prostatic hyperplasia (bph) is the most common prostatic disorder in older intact male dogs, but despite its prevalence, there are inconsistencies in clinical diagnosis and treatment. although prostate size was historically considered the hallmark feature of bph in men, currently, there is only a weak correlation between prostate size and clinical severity. we performed a retrospective cohort study with the primary objective of assessing clinical signs, ultrasonographic findings, treatments, and outcomes in dogs diagnosed with bph, with and without concurrent prostatitis. we reviewed medical records and obtained data on presenting signs, prostatic imaging, and prevalence of concurrent bacteriuria. prostate size was determined by ultrasonography and compared to the calculated expected size based on patient age and weight. treatment and outcome were described for the cases with a minimum 2 months follow-up. median age of dogs diagnosed with bph was 8 years. clinical signs were present in 16/25 dogs and scored as mild to moderate (median zambelli’s symptom index for bph score 12). the median prostatic volume to body mass ratio was 1.60 mm3/kg. prostate size did not correlate with the symptom severity. concurrent bacteriuria was confirmed in 4/25 cases via bacterial culture and/or cytology. treatments pursued and responses were only available in a subpopulation of dogs (n = 9) and were highly variable. studies are needed to determine if current treatment options for bph in dogs resolve associated clinical signs in addition to reducing prostate size. keywords: dogs, benign prostatic hyperplasia, prostate size, urinalysis, clinical signs scientists and veterinarians to the false conclusion that dogs do not develop clinical signs in response to bph like their human counterparts. available literature reports that clinical signs of bph are limited to prostatic enlargement and that most dogs do not manifest additional signs.9 however, some veterinarians have noted, anecdotally, that the ability of older intact male dogs to urinate improves dramatically after castration. diagnosis of bph in dogs is usually presumptive, based on physical examination, patient history, ultrasonographic findings, clinical signs, fine needle aspirate, prostatic wash, or monitored response to therapy.2 a diagnostic procedure often performed by veterinarians offering reproductive services is collection of prostatic fluid by manual stimulation into a sterile tube, providing a large sample for analysis. definitive diagnosis is based on prostatic biopsy that is invasive and therefore uncommon.2 however, prostatic enlargement is listed as a criterion of all canine prostatic diseases, making additional diagnostics beyond digital rectal examination (dre) critical for an accurate diagnosis.1,2,8 treatment for canine bph often exclusively focuses on reducing the size of the prostate gland. castration is currently the most effective way to reduce prostate size in canines.10 however, in clinical theriogenology 2021; 13: 360 breeding animals or dogs with preexisting prostatic bacterial infection, other modalities may be considered. medical management includes the 5α-reductase type ii inhibitor, finasteride, that prevents conversion of testosterone (t) to the more potent androgen dihydrotestosterone (dht).11 finasteride reduced prostate size in humans and dogs.12 finasteride is given for the remainder of life, but does not impair fertility,11,13 making it an option for breeding animals. however, limited information is available on the efficacy of finasteride in reducing clinical signs associated with bph (other than prostatic enlargement). the purpose of this retrospective medical record review was to describe canine bph in a clinical population of client-owned dogs. the study’s primary objectives were to report the clinical signs, prostatic ultrasonographic findings and prevalence of concurrent bacteriuria in dogs diagnosed with bph, as well as assess if ultrasonographic prostatic size correlated with severity of clinical signs. a secondary objective was to describe the treatments and outcomes of dogs diagnosed with bph including clinical signs. we hypothesized that bph is often asymptomatic in intact male dogs, prostatic size does not correlate with clinical signs, and concurrent bacteriuria is an uncommon clinical complication of bph. materials and methods animals a computerized medical record search was performed for discharge reports containing the search term ‘benign prostatic hyperplasia’ from january 1, 2014 through december 31, 2018 at the university of wisconsin veterinary care. all discharge reports from intact male dogs were reviewed. client-owned intact male dogs clinically diagnosed with bph and that underwent ultrasonographic prostatic imaging were considered for study inclusion. included dogs also needed to have prostatic sampling for evaluation of prostate/urinary tract bacterial colonization (urine or prostatic fluid bacterial culture performed and/or prostatic fine needle aspirate for cytologic evaluation). dogs did not need to have a complete urinalysis performed to be included, but it was recorded when available. dogs diagnosed with prostatic abscesses or neoplasia, protein losing nephropathy, chronic kidney disease, bladder neoplasia, penile neoplasia, ureteral obstruction, or newly diagnosed systemic comorbidities were excluded. study design a retrospective cohort design was used. the medical records were read in detail and age, sex, weight, breed, presence of preexisting medical conditions, and whether bph was the main differential diagnosis or an incidental finding were recorded. using the information within the medical record including clinical signs, bph severity was scored according to the published zambelli’s symptom index for bph (zsi-bph) score, 11 categories scored from 1 to 4 based on severity, for a total minimum of 11 and maximum of 44.14 the criteria not specifically defined for zsi-bph scoring, weight loss and total score binning, were defined in this study as follows. weight loss categories were scored as none (no change in body weight in kg), light (< 1 kg), moderate (1 5 kg), and severe (> 5 kg). zsi-bph total scores were binned into 4 categories: asymptomatic (11), mild (12 22), moderate (23 33), and severe (34 44). this scoring paradigm was selected because it takes an unbiased approach and rates each clinical sign equally for evaluating severity. additionally, the ultrasonographic prostatic changes were recorded that included the tissue’s echotexture and any additional changes noted (e.g. cysts or mineralization). diagnostic laboratory tests performed were recorded that included cytologic findings, microbiology results, including specimen cultured, and bacterial species isolated. finally, details of treatment performed, if any, were recorded. prostate size was estimated both by expected size using body weight (in kg, bw) and age (in years, a) as previously reported15 and calculated size (p-cs) based on prostatic ultrasonography. briefly, expected prostatic length ((0.055 x bw) + (0.143 x a) + 3.3), width (0.047 x bw) + (0.089 x a) + 3.45), height (sagittal (0.046 x bw) + (0.069 x a) + 2.68 and transverse (0.044 x bw) + (0.083 x a) + 2.25), and volume ((0.867 x bw) + (1.885 x a) + 15.88).15 using prostatic ultrasonographic images, each dog’s prostatic size was calculated. electronic calipers were used to measure length, width, and height along a sagittal and, when available, transverse plane in ultrasonographic still images (figure 1). ultrasonographic measurements were used to calculate prostate volume (length x width x (height sagittal + height transverse)/2) x 0.523).15 in the absence of transverse views, the sagittal height was used in calculations in place of both the width and the mean of the sagittal and transverse heights. prostate volume expressed as volume per kg body weight was calculated as p-cs/ bw. data analyses all numerical data is reported as a median and range. a spearman correlation was used to determine correlations between the calculated ultrasonographic prostatic size and clinical severity scoring (zsi-bph). statistical analyses were performed using graphpad prism (version 8.4.0 for windows, graphpad software, san diego, ca, www.graphpad.com). differences were considered significant when p < 0.05. results two hundred and seventy-five discharge reports were examined and after duplicates (n = 90) were removed, a total of 185 canine cases were screened. twenty-five intact male dogs met the study’s inclusion criteria. cases were excluded for following reasons: neutered males (n = 52), ‘puppy discharges’ describing the benefits of neutering (n = 3), no imaging pursued (n = 29), no prostatic sampling performed (n = 69), documented concurrent lower urinary tract disease (n=6), and concurrent comorbidities (n = 1). clinical theriogenology 2021; 13: 361 the median age at diagnosis of bph was 8 years, ranging from 1 to 14 years in this population of dogs. dogs weighed 3 68 kg with a median of 34 kg. eight of the cases were labrador retrievers, 2 were bernese mountain dogs, and remaining breeds were represented by a single case: border collie, rough coated collie, pomeranian, poodle, field spaniel, flat coated retriever, german shorthaired pointer, golden retriever, spinone, greater swiss mountain dog, leonberger, mastiff, newfoundland, small musterlander, and a labrador/german shepherd mix. clinical signs nine of 25 dogs with bph were asymptomatic (zsi-bph score 11). fourteen dogs diagnosed with bph had mild clinical signs figure 1. ultrasonographic measurement of the prostate using electronic calipers in a (a) sagittal view and (b) transverse view (median zsi-bph score 14.5, range; 12 22). two dogs had moderate clinical signs (zsi-bph score 26). the mild clinical signs included anorexia, < 1 day (n = 2), anorexia < 1 week (n = 2), weight loss light (n = 1), weight loss moderate (n = 2), regular, but difficult and painful defecation (n = 2), urination with weaker or interrupted flow (n = 1), urination with very weak or interrupted flow (n = 1), urinary retention with urinary drip or paradoxal urinary incontinence (n = 1), urinary incontinence (n = 4), urethral leakage (n = 1), and hematuria (n = 7). the moderate clinical signs included anorexia for > 1 week (n = 1), urinary retention with urinary drip or paradoxal urinary incontinence (n = 2), urinary incontinence (n = 2), urethral leakage (n = 2), hematuria (n = 1). overall, the median zsibph score was 12 (range; 11 26). results of all zsi-bph categories are summarized (table 1). prostatic palpation, ultrasonographic findings, and size the results of dre and prostatic ultrasonographic echotextures are summarized (table 2) for this group of dogs clinically diagnosed with bph. only 17/25 dogs had dre performed and the prostate was not assessed during rectal in 1 dog and prostates were not palpable in 5 dogs. when dre was performed and prostate was described, the majority of descriptions were incomplete. all dogs had ultrasonographic performed; however, many were described as ‘normal bph’ rather than descriptions of echotexture, and many echotexture descriptions were incomplete. the p-cs expressed as volume per kg body weight ranged from 0.21 to 3.31 cm3/kg with a median volume of 1.60 cm3/kg. compared to p-es, 16 had p-cs smaller than their expected volume. only 9 cases had prostates that were > p-es. also, there was no correlation between prostate volume per kg body weight and total zsi-bph score (r = – 0.0016. 95% confidence interval – 0.4068 to – 0.4042, p = 0.9940, figure 2). medical records were reviewed for dogs with bph and total zsi-bph calculated based on scoring 11 criteria from 1 to 4 with possible total zsi-bph scores of 11 44.14 p-cs was calculated using ultrasonographic imaging of each dog’s prostate. electronic calipers were used to measure length, width, and height along a sagittal and, when available, transverse plane in ultrasonographic still images (figure 1). ultrasonographic measurements were used to calculate prostate volume (length x width x (height sagittal + height transverse)/2) x 0.523).15 in the absence of transverse views, the sagittal height was used in our calculations in place of both the width and the mean of the sagittal and transverse heights. prostate volume expressed as volume per kg body weight was calculated as p-cs/ bw. relationship (r = – 0.0016, 95% confidence interval – 0.4068 to – 0.4042, p = 0.9940) between prostate volume per kg body weight and clinical sign severity (zsi-bph) is depicted (figure 2). prostatic cytology, bacterial culture, and urinalysis cytology and/or bacterial culture was a selection criterion for clinical theriogenology 2021; 13: 362 our study. one dog had cytology alone performed, 18 had bacterial culture alone performed, and 6 had both types of sampling performed. complete urinalysis was not a selection criterion for our study but was done in a subset of dogs (n = 19). cytological evaluation of the prostate, aspirates (n = 3) and wash (n = 4), were described and reviewed by a board-certified clinical pathologist. aspirates were moderately to highly cellular and consisted of a homogeneous population of prostatic cells in a background of blood cells. cells were arranged in flat sheets in all 3 specimens with no evidence of neoplasia, inflammation, or infection. the cytologic diagnosis was consistent with benign prostatic hyperplasia. prostatic wash specimens were acellular to poorly cellular on direct smear. a cytocentrifuged specimen in all 4 cases described epithelial cells consistent with urogenital or transitional epithelium. none of the prostatic wash samples identified signs of neoplasia or infection. only 1/4 cases had inflammation noted and it was characterized as lymphocytic and plasmacytic inflammation. twenty-four dogs had bacterial cultures performed. samples cultured included urine collected via cystocentesis (n = 5), catheter (n = 4), or void (n = 11), and/or prostatic wash fluid (n = 6). two dogs had both urine and prostatic fluid cultures performed. positive bacterial urine cultures occurred in 4 dogs with escherichia coli (e. coli) (> 100,000 cfu/ml) isolated in all 4 cases. all 4 cultures were susceptible to nitrofurantoin, tetracycline, and tmp-sulfa antibiotics. one was resistant to ciprofloxacin and another resistant to amoxicillin/clavulanate. three of 4 dogs that had a culture confirmed bacteriuria also had urinalysis, and interestingly, only 2 dogs had bacteria on the urine sediment. nineteen dogs had urinalysis. urine was collected via catheter (n = 3), cystocentesis (n = 6), and void (n = 10). results of the urinalysis including color, turbidity, specific gravity, ph, wbcs, rbcs, transitional cells, protein, casts, crystals, bilirubin, ketones, glucose, and sperm are summarized (table 3). treatment and outcome nine of our 25 cases had a minimum of 2-month follow-up. three dogs were asymptomatic at the time of initial clinical evaluation and never developed clinical disease requiring treatment over 1 2 years of follow-up available. remaining 6 dogs anorexia absent (20) 1 day (2) 1 day to 1 week (2) > 1 week (1) weight loss absent (22) light (1) moderate (2) severe (0) dyschezia /tenesmus (severity) absent (23) regular, but difficult and painful defecation (2) irregular, difficult and painful defecation (0) defection impossible or absent in some days (0) dysuria (severity) absent (20) urination with weaker or interrupted flow (1) urination with very weak or interrupted flow (1) urinary retention with urinary drip or paradoxal urinary incontinence (3) urinary incontinence absent (19) present (6) urethral leakage (amount) absent (22) few drops (2) many drops (1) stains on floor (0) urethral leakage (frequency) absent (22) since 1 to 7 days (1) since 7 to 15 days (1) since > 15 days (1) hematuria (amount) absent (17) sometimes pinkish urine (3) pinkish urine (2) red urine (3) hematuria (frequency) absent (17) 1 episode per year (4) 2 to 3 episodes per year (3) >3 episodes per year (1) hematuria (duration) absent (18) since 1 to 7 days (5) since 7 to 15 days (1) since > 15 days (1) total severity score asymptomatic (9) mild (14) moderate (2) severe (0) table 1. zambelli symptom index at diagnosis of dogs with a clinical diagnosis of benign prostatic hyperplasia figure 2. volume calculated size (p-cs) per kg body weight (bw) and zambelli’s symptom index for benign prostatic hyperplasia (zsi-bph); each dog represents 1 dog clinical theriogenology 2021; 13: 363 digital rectal examination of prostate (n = 11)† ultrasonographic prostatic echotextures (n = 25) shape symmetrical (6) asymmetrical (2) not described (3) shape symmetrical (11) asymmetrical (1) not described (13) margin smooth (1) irregular (1) not described (9) diffuse echopattern homogenous (1) heterogenous (13) not described (11) discomfort painful (0) nonpainful (6) not described (5) echotexture hyperechoic (10) hypoechoic (0) not described (15) intra-prostatic structures hyperechoic structures (3)* hypoechoic structures (3)* anaechonic structures (6)* absent/not described (16) table 2. description of prostate palpation based on digital rectal examination (dre) and prostatic ultrasonographic echotextures of dogs with a clinical diagnosis of benign prostatic hyperplasia table 3. urinalysis of dogs clinically diagnosed with benign prostatic hyperplasia *note: some had structures of multiple echogenicity †no rectal examination was performed in 8 dogs, prostate was not assessed during rectal examination in 1 dog, prostate was not palpable in 5 dogs urine color slightly yellow (2), yellow (9), dark yellow (3), amber (3), red tinged (1), dark red (1) turbidity clear (5), slightly hazy (3), hazy (3), cloudy (8) specific gravity range: 1.014 1.054, median: 1.027 ph range: 6 8.5, median: 7.5 wbcs absent (1), rare (6), 1 5/hpf (6), 5 10/hpf (1), 10 25/hpf (1), 25 50/hpf (1), 50 100/hpf (2), tntc (1) rbcs absent (5), rare (2), 1 5/hpf (4), 5 10/hpf (1), 10 25/hpf (0), 25 50/hpf (1), 0 100/hpf (4), tntc (2) protein absent (1), trace (2), 1+ (5), 2+ (5), 3+ (3), 4+ (3) transitional epithelial cells present (16), absent (3) casts absent (15), granular (4), hyaline (1), cellular (1) sperm present (13), present (13), absent (6) bilirubin positive (8), negative (11) ketones positive (0), negative (19) crystals absent (13), amorphous (3), bilirubin (1), triple phosphate (2) glucose absent (18), trace (1)† *four dogs with proteinuria had a follow-up urine protein: creatinine ratio (median: 2.055; range: 0.04 19.78) † no evidence of hyperglycemia hpf (high power field); tntc (too numerous to count) clinical theriogenology 2021; 13: 364 with bph had clinical disease (e.g. hematuria, stranguria, dysuria, pollakiuria, or urinary accidents) at initial clinical evaluation and were treated with oral antibiotics (n = 5), prazosin 2 mg (0.1 mg/kg) once every 8 hours (n = 1), finasteride 5 mg (0.2 mg/kg) once daily (n = 1), and/or megestrol 20 mg (0.6 mg/kg) once daily (n = 1). oral antibiotic treatments for confirmed or suspected secondary prostatitis included amoxicillin (500 mg [15 mg/kg] once every 12 hours [n = 1]), doxycycline (300 mg [9 mg/kg] twice daily (n =1), trimethoprim/sulfamethoxazole (480 mg [14 mg/kg]) twice daily (n = 1), enrofloxacin (272 mg [10 mg/kg] once daily [n = 1]), and ciprofloxacin (750 mg [24 mg/kg] once daily [n =1]). the dog treated with finasteride alone had resolution of clinical signs within 2 months before case was lost to follow-up. the 5 remaining dogs with clinical signs at initial presentation failed medical treatment and did not have resolution of clinical signs at any point during follow-up despite incremental decreases in their prostate size. after failing medical treatment, 2 dogs were castrated. both dogs experienced incomplete prostatic involution, based on follow-up ultrasonographic, and had persistent lower urinary tract signs that were treated symptomatically with intermittent antibiotics based on documented bacteriuria (n = 2). one of the 2 dogs treated for bacteriuria responded well to antibiotics and had a negative urine culture after treatment, whereas the other dog had persistent bacteriuria despite antibiotic treatment. additional supportive medications prescribed chronically in 1 dog including oral prazosin 2 mg (0.1 mg/kg) and bethanechol 20 25 mg (0.6 0.7 mg/kg) once every 8 hours. discussion purpose of this retrospective medical record review was to describe canine bph in a clinical population of client-owned dogs. it is important to note that this review took place at an institution that treats bph patients through the internal medicine department and does not routinely perform semen collection in dogs. therefore, semen and/or prostatic fluid analysis, that can be a helpful diagnostic tool for bph, were not included in this study. it is also important to note that we used a diagnostic tool for clinical signs of bph retrospectively rather than prospectively. it is possible that not all clinical signs we scored were noted in the record. it is also possible that not all clinical signs were assessed in each patient. therefore, our zsi-bph scores may be low estimates of true clinical sign severity. as expected, most dogs with bph experienced only mild clinical signs or remained asymptomatic. when mild to moderate clinical signs were present, they included: urinary incontinence, urethral leakage, dysuria, hematuria, tenesmus, and dyschezia. prostatomegaly is considered the hallmark feature of canine bph and is often the sole measure for determining efficacy of treatments. however, we observed that there was no correlation between prostate size and severity of clinical signs in our population. further, a comparison of p-es to p-cs suggests that the majority of cases (18/25) had prostates that were equal to or smaller than expected rather than enlarged. these findings suggest that prostate enlargement alone should not be used to identify patients with bph. cpse assays may be a more predictive diagnostic tool as they are highly predictive of a cytologic diagnosis of bph in dogs.16 our finding that the majority of cases (18/25) had prostates that were equal to or smaller than expected rather than enlarged suggests that subjective prostatic enlargement may be over diagnosed in intact male dogs. this may be in part due to the fact that in the us most dogs are neutered at a young age and many practitioners are not accustomed to examining prostates in intact male dogs, therefore they may perceive the prostate of an aged intact male as enlarged when it is of an appropriate size for that dog’s age and weight. implementing a standard practice of measuring the prostate volume using ultrasonographic and comparing it to the calculated expected prostatic size based on body weight and age, as done in this study and a previous study,16 may be a more objective measure to use to establish an ultrasonographic diagnosis of prostatic enlargement in intact male dogs. we determined the prevalence of concurrent bacterial infection in our population as an indicator of concurrent prostatitis and potential etiology of urinary dysfunction in dogs with bph. only 4 of the dogs with bacterial urine cultures and/or cytology performed cases had a positive bacterial culture. in all 4 cases e. coli was isolated in high numbers (> 100,000 cfu/ ml). interestingly, 18/19 cases that had urinalysis performed had pyuria and 11/19 had pyuria with ≥ 1 5 wbc per hpf (high power field) suggesting the presence of lower urinary tract inflammation in majority of cases, despite only 3 of these dogs, having confirmed and documented bacteriuria via bacterial culture. prostate inflammation is strongly correlated with symptom severity in men and is associated with relapse of symptoms following treatment.17-20 prostatic inflammation is also correlated with prostatic fibrosis in men20-22 and we have previously identified an increase in prostatic collagen with age in intact male dogs23 and a shift in the cell type producing collagen 1a1 with age in intact male dogs.24 the clinical finding of wbcs in the urine of dogs with bph in the absence of bacteriuria may indicate an area for further study to determine if prostate inflammation predicts zsi-bph severity. the majority of cases with follow-up at this tertiary veterinary care facility were comprised of complicated cases that failed standard bph treatments. only 2 cases with follow-up elected castration and both cases had persistent utis and urinary signs. in fact, only 1 of the 6 cases with follow-up that presented with urinary signs improved following treatment, pointing to a need for further study of treatments’ efficacy in reducing clinical signs for bph and for bph with concurrent uti in dogs. we recently characterized a uropathogenic e. coli mouse model of prostate infection and inflammation25 that holds promise for testing of optimal treatments clinical theriogenology 2021; 13: 365 for dogs and men with both bph and concurrent uropathogenic e. coli infection. overall, this study demonstrated the presence of clinical signs other than prostatomegaly in dogs with bph. clinical signs of bph using zsi-bph scoring did not correlate with prostatic size. objective determinations of prostatomegaly are lacking in intact male dogs and prostatomegaly associated with bph may be over diagnosed in dogs. concurrent bacterial infections of the prostate were infrequent in this population of dogs with bph but based on urinalysis findings clinical lower urinary tract inflammation may be present in majority of patients. there is a need for future studies to develop additional diagnostic tests for bph in dogs that do not rely so heavily on prostatic size, determine the influence of prostatic inflammation on severity of clinical signs, and determine treatments that not only reduce prostate size but also alleviate concurrent clinical signs including urinary dysfunction. acknowledgement funded by national institutes of health grants: tl1 tr002375, t35 od011078, f30 dk122686, u54 dk104310 and university of wisconsin-madison, school of veterinary medicine. the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. conflict of interest none to declare. references 1. johnston sd, kamolpatana k, root-kustritz mv, et al: prostatic disorders in the dog. anim reprod sci 2000;60-61:405-415. 2. smith j: canine prostatic disease: a review of anatomy, pathology, diagnosis, and treatment. theriogenology 2008;70:375-383. 3. polisca a, troisi a, fontaine e, et al: a retrospective study of canine prostatic diseases from 2002 to 2009 at the alfort veterinary college in france. theriogenology 2016;85:835-840. 4. berry sj, strandberg jd, saunders wj, et al: development of canine benign prostatic hyperplasia with age. prostate 1986;9:363-373. 5. palmieri c, lean fz, akter 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11. sirinarumitr k, johnston sd, kustritz mv, et al: effects of finasteride on size of the prostate gland and semen quality in dogs with benign prostatic hypertrophy. j am vet med assoc 2001;218:1275-1280. 12. angrimani dsr, francischini mcp, brito mm, et al: prostatic hyperplasia: vascularization, hemodynamic and hormonal analysis of dogs treated with finasteride or orchiectomy. plos one 2020;15:e0234714. 13. angrimani dsr, bicudo lc, llamas luceño n, et al: does finasteride treatment for benign prostatic hyperplasia influence sperm dna integrity in dogs? basic clin androl 2020;30:39. 14. zambelli d, cunto m, gentilini f: validation of a model to develop a symptom index for benign prostatic hyperplasia in dogs. reprod domest anim 2012;47(suppl 6):229-231. 15. ruel y, barthez py, mailles a, et al: ultrasonographic evaluation of the prostate in healthy intact dogs. vet radiol ultrasound1998;39:212-216. 16. pinheiro d, machado j, viegas c, et al: evaluation of biomarker canine-prostate specific arginine esterase (cpse) for the diagnosis of benign prostatic hyperplasia. bmc vet res 2017;13:76. doi 10.1186/ s12917-017-0996-5. 17. bushman wa, jerde tj: the role of prostate inflammation and fibrosis in lower urinary tract symptoms. am j physiol renal physiol 2016;311:f817-f21. 18. roehrborn c: the impact of acute or chronic inflammation in baseline biopsy on the risk of progression in the mtops study (abstract). eur urol suppl 2005;4:5. 19. torkko kc, wilson rs, smith ee, et al: prostate biopsy markers of inflammation are associated with risk of clinical progression of benign prostatic hyperplasia: findings from the mtops study. j urol 2015;194:454-461. 20. macoska ja, uchtmann ks, leverson ge, et al: prostate transition zone fibrosis is associated with clinical progression in the mtops study. j urol 2019;202:1240-1247. 21. ma j, gharaee-kermani m, kunju l, et al: prostatic fibrosis is associated with lower urinary tract symptoms. j urol 2012;188:1375-1381. 22. rodriguez-nieves ja, macoska ja: prostatic fibrosis, lower urinary tract symptoms, and bph. nat rev urol 2013;10:546-550. 23. ruetten h, wegner ka, romero mf, et al: prostatic collagen architecture in neutered and intact canines. prostate 2018;78:839-848. 24. ruetten h, cole c, wehber m, et al: an immunohistochemical prostate cell identification key indicates that aging shifts procollagen 1a1 production from myofibroblasts to fibroblasts in dogs prone to prostate-related urinary dysfunction. plos one 2020;15:e0232564. 25. ruetten h, sandhu jk, mueller b, et al: a uropathogenic e. coli uti89 model of prostatic inflammation and collagen accumulation for use in studying aberrant collagen production in the prostate. am j physiol renal physiol 2020;320:f31-f46. clinical theriogenology 2021; 13: 366 introduction domestic cats are seasonal polyestrous species and long-day breeders with a positive photoperiod influence. queens cycle repeatedly during the breeding season when melatonin concentrations are lower, unless interrupted by pregnancy or pseudopregnancy. effect of seasonality is not very substantial for tomcat. pathway to contraception is to reduce concentrations of gonadotropins (lh and fsh) leading to reduced concentrations of steroid hormones with an ultimate goal to prevent reproduction. considering the reproductive physiology of domestic cat, to achieve contraception, there are 3 main possible approaches: • progestin: a prescription drug that induces anestrus, inhibits ovulation, and to a certain extent, interferes with spermatogenesis. • melatonin: ‘off-label’ implants that mimic the release of endogenous melatonin during short photoperiods causing suppression of sexual activity (anestrus) and has very limited effect on ovulation and spermatogenesis. • gnrh agonist: disrupts reproductive functions in female and male. however, the only implant available (deslorelin – suprelorin 4.7 mg® virbac) is used ‘off-label’ in cats. another method is to induce ovulation and therefore pseudopregnancy resulting in lack of estrous signs due to high concentrations of progesterone for ~ 45 days. ovulation is induced by hcg or mechanical vaginal stimulation and/or teaser male (vasectomized or even neutered tomcats). medical contraception in cat: new and old drugs cindy maenhoudt, alain fontbonne ecole nationale vétérinaire d’alfort, centre d’etude en reproduction des carnivores maisons-alfort (paris), france abstract control of cat overpopulation is up until now has been a lost battle. the search for a cheap, reliable and easily applicable contraceptive covers a wide range of possibilities, but lately, very limited advances have been made. main objective of drug-mediated fertility control in cats is to prevent reproduction (special concern to overpopulation) and to prevent estrus and male sexual behaviors in ownedcats and ultimately help management of catteries. there are 3 forms of delivery of contraceptive drugs in cats: tablets, injections, and implants. all contraceptive control discussed here is hormone-based, with a wide variation of possible side effects. currently, there are no contraceptive methods that are 100% effective and each should be considered a temporary solution. keywords: medical contraception, cats, progestin, melatonin, gnrh agonist currently, different hormonal approaches (long-term implants of gnrh agonist, melatonin implant, and gnrh vaccine) are possible as alternatives to progestin. these preparations appear to exhibit adequate contraceptive effect with less unfavorable effects than progestins. progestins currently, progestins are the most used contraceptive agents to prevent estrus in the queen. the mechanism of contraception remains controversial and pathways (negative feedback and suppression of gnrh, interference with gamete transportation, and blockage of implantation) have been suggested. there are drugs with oral or injectable formulations. the oral formulation is known as the ‘cat contraceptive pill.’ the most widely used active substance is megestrol acetate (ma). however, there are also 2 other progestins, medroxyprogesterone and proligestone. the major limitations of progestins are the intensity and severity of side effects that vary in relation to the drug, dose, frequency and phase of the estrous cycle in which given and pre-existing medical conditions. cases of mammary cancer, mammary hyperplasia, cystic endometrial hyperplasia/pyometra complex, ovarian cysts, and hormonal disorders like diabetes mellitus were observed in animals treated with progestins (reviewed1). affinity of progestin to the progesterone receptor and to steroid hormone receptors (e.g. androgens) and/or glucocorticoids will also impact its side effects. however, the dose appears to overcome the affinity, as lower dosages of ma seem to guarantee a higher level of effectiveness while reducing the risk of side effects clinical theriogenology 2021; 13: 156 to a minimum, both in frequency and in severity.2 during the covid-19 pandemic, due to the reduction in spaying program, ma became a good alternative.3 low doses of ma (0.625 0.1 0.2 mg/kg weekly), can be given safely; in practical terms, an oral dose of 2.5 mg of ma per female once a week is adequate. in case the queen has signs of estrus, the dose should be 5 mg per cat per day for 3 days, followed by once weekly treatment schedule. however, this protocol should be applied with care due to an increased risk of pyometra. in italy,2 a lower dose (0.01 0.03 mg/kg once a day) of ma (estropill, msd) is currently available, but needs further investigation. it is important to adhere to the duration of treatment (maximum of 2 weeks) to prevent the queen returning to cyclicity. progestin treatment should not be extended beyond 2 years, ideally ≤ 1 year. melatonin suppression of estrus in female domestic cats by melatonin mimics the physiological events of seasonality observed in many animals (e.g. horse, sheep, and hamsters). during dark periods, the pineal gland secretes melatonin (n-acetyl-5-methoxytryptamine). in queen, melatonin suppresses hypothalamic secretion of gnrh, thereby this neuromodulator has an inhibitory effect on the female reproductive cyclicity.4 melatonin is available as oral preparation, but the need to medicate cats every day limits this formulation. our group used oral melatonin and the results were not very remarkable and the reluctance of the queen to ingest the tablet was a limitation.5 nevertheless, estrus was suppressed (~ 63 days) from the beginning of treatment when melatonin (4 mg tablets/cat/day [melatol®, elisium s.a., buenos aires, argentina]) was given 2 hours prior to lights off.6 currently, the best option is the melatonin implant (melovin, ceva, marseille, france). its use in domestic cats is ‘off-label,’ but it is still a good alternative to inhibit estrous behavior for short periods. the success rate is > 80% and the duration of suppression is variable. suppression of estrus was observed on an average for 103.9 days (56 156 days) in 81.4% (22/27) of implanted queens (reviewed7). in a survey by our group,8 prevention of estrus as reported by owners was 79% (33/42 queens). no side effects were reported in neither studies nor in clinical setting. the selection of queens is very important. melatonin implants will have a better chance to induce anestrus in females having marked seasonality. another aspect to improve the response is in choosing the best time of estrous cycle for implantation. if implanted during the early part of interestrous interval, longer suppression of estrus is observed. in practical terms, before using melatonin implants, the anamnesis will guide the likelihood of a positive response. it appears that some breeds are more susceptible to the photoperiod. before implantation, progesterone concentrations should be measured (to avoid implanting females that had ovulated) and a vaginal smear done to evaluate whether the queen is in estrus. in case of an estrous smear, it is recommended to reexamine the queen 1 week later, if possible, to maximize the duration of suppression by implantation during interestrus. the effect of the melatonin implant is not instantaneous, some queens may have estrus up to 10 days postimplantation,7,8 therefore treated animals should be separated from a tomcat for at least 2 weeks to avoid unwanted mating. due to the lack of side effects and success of estrus prevention (up to 3 months), melatonin implant is a good option as a short-acting contraceptive for privately owned queens. deslorelin gnrh agonist deslorelin, is a slow-release implant (suprelorin 4.7 mg, virbac) that can be used to suppress estrus and to inhibit spermatogenesis in queens and tomcats, respectively. after a short interval of hyperstimulation (‘flare-up’ effect), both females and males have a downregulation phase of gnrh secretion and consequently have lower concentrations of fsh and lh. side effects are not often observed, and in case of any undesirable effect, the implant can be removed. reversible weight gain appears to be the main side effect reported in males and females.8 the limitation is the duration of action. although most of the studies had an efficacy on average for 13 (males) or 16 (females) 22 months,8,9 some animals may be suppressed for up to 3 years. therefore, this choice for breeding animals should be made with caution. the only formulation used by our group is the 4.7 mg implant. the contraceptive effects are reversible with normal pregnancy rates and litter sizes with good maternal behavior.8,9 in prepubertal queens, the implant allows the postponement of onset of puberty to an age close to 2 years.10 implantation the procedure by our group is to insert the implant (of deslorelin or melatonin) in an area posterior to the umbilicus (for ease of placement and removal if necessary). rarely the animal (male or female) needs sedation, the cat is placed on dorsal recumbency with mild restraint. local anesthesia (lidocaine 10%) is applied at the site of injection. suprelorin® has an applicator and for the melovin®, an applicator is adapted. the site of implantation is scrubbed and the implant is injected subcutaneously (sc). it is easy to verify the location of these implants (melovin® is ± 3 mm and suprelorin® is ±10 mm). induction of ovulation and pseudopregnancy for shortterm suppression estrus in the queen can also been interrupted by inducing ovulation. as mentioned above, a particularity of domestic cats is that mechanical stimulation of sensory receptors of the skin of the perineal region, vulva, vagina, and cervix induces ovulation. during natural breeding, the penile spines are the source of stimulation, and the number of days in estrus and the frequency of mating are highly associated with the intensity and frequency of the lh surge11 and thereby ovulation. ovulation without fertilization induces a prolonged state of luteal activity and therefore absence of estrus known as pseudopregnancy. the duration of pseudopregnancy is variable from 35 to 76 days (average 45 days)12 by a sterile tomcat, hormonal treatment or mechanical stimulation of the vagina. clinical theriogenology 2021; 13: 157 vaginal stimulation with a cotton swab can be used to mimic the stimulus caused by penile spines during copulation and therefore induce the lh surge. the procedure needs to be repeated to achieve the goal (induce ovulation). there are 2 limitations to this method of contraception. first, the technique is not always well tolerated by the female cat. second, the use of the procedure repeatedly can increase the risk of pathologies related to progesterone exposure. immunocontraception immunocontraception is a subject of research and is still not available in the clinical setting. although results of gnrh vaccine and zona pellucida vaccine have not been very successful in cats,13-15 a study using a single-dose gnrh immunocontraceptive vaccine (gonacon™) had promising results.16 limitations of immunocontraceptives are, the need of booster vaccinations to improve response, return to fertility once antibody concentrations decrease and severe lesions at the site of the injection due to adjuvants in the vaccines to improve efficacy. general consideration the best contraceptive choice depends on availability, safety, security and efficiency. the request for a contraceptive for domestic cats is a high demand amongst our clientele. although progestin is not used frequently, we strongly believe that the adaptation of the dose and the selection of animals can substantially decrease side effects. currently, for short duration of estrus suppression, the melatonin implant is normally chosen. the success rate is encouraging and some females have been repeatedly treated over more than 1 breeding season. for long-term suppression, deslorelin is a good tool for animals with conditions that preclude anesthesia. in case of breeding stock, as mentioned before, the lack of control over the duration of suppression should be extensively discussed with the owners to avoid disappointment and regrets due to the loss of animals for reproductive purposes. in some situations, it may be judicious to remove the implant after 4 months of implantation to reduce the duration of downregulation. return of fertility has been well documented in both male and female cats. however, the quality of semen in some tomcats may not be enough to guarantee pregnancy after deslorelin implant. the age of animals at implantation is an important consideration. selection of contraceptive for pet owned cats should always consider the breeding value of the animal, age, health, and duration of suppression. there is still a need for a more practical solution to control cat overpopulation than the current methods. conflict of interest none to report. references 1. borges p, fontbonne a, payan-carreira r: new approaches for hormonal contraception in female cats. in: colleen kevins: editor. common diseases, clinical outcomes and developments in veterinary healthcare, hauppauge, new york: nova science publishers inc., 2015. p. 47-68. available from: https://www.researchgate.net/publication/298034535 new approaches for hormonal contraception in female cats [accessed apr 15 2021]. 2. romagnoli s: progestins to control feline reproduction: historical abuse of high doses and potentially safe use of low doses. j. feline med surg 2015;17:743-752. 3. alliance for contraception in cats and dogs covid-19 response page:https://accd.org/available-products/progestin-contraceptives/ covid-19-updates. [accessed apr 15 2021]. 4. leyva h, addiego l, stabenfeldt g: the effect of different photoperiods on plasma concentrations of melatonin, prolactin and cortisol in the domestic cat. endocrinology 1984;115:1729-1736. 5. furthner e, maenhoudt c, amiriantz s, et al: oral melatonin supplementation in female domestic cats, a double blinded prospective study. proceedings of the 22nd european veterinary society for small animal reproduction congress 2019;103-104. 6. faya m, carranza a, priotto m, et al: long-term melatonin treatment prolongs interestrus, but does not delay puberty, in domestic cats. theriogenology 2011;75:1750-1754. 7. schäfer-somi s: effect of melatonin on the reproductive cycle in female cats: a review of clinical experiences and previous studies. j. feline med surg 2017;19:5-12. 8. furthner e, roos j, niewiadomska z, et al: contraceptive implants used by cat breeders in france: a study of 140 purebred cats. j. feline med surg 2020;22:984-992. doi: 10.1177/1098612x19901023. 9. goericke-pesch s, georgiev p, atanasov a, et al: treatment of queens in estrus and after estrus with a gnrh-agonist implant containing 4.7 mg deslorelin; hormonal response, duration of efficacy, and reversibility. theriogenology 2013a;79:640-646. doi:10.1016/j.theriogenology.2012.11.018 10. cecchetto m, salata p, baldan a, et al: postponement of puberty in queens treated with deslorelin. j feline med surg 2017;19:1224-1230. doi: 10.1177/1098612x16688406. 11. concannon p, hodgson b, lein d: reflex lh release in estrous cats following single and multiple copulations. biol reprod 1980;23:111-117. 12. wildt de, seager swj, chakraborty pk: effect of copulatory stimuli on incidence of ovulation and on serum luteinizing hormone in the cat. endocrinology 1980;107:1212-1217. 13. gorman sp, levy jk, hampton al, et al: evaluation of a porcine zona pellucida vaccine for the immunocontraception of domestic kittens (felis catus). theriogenology 2002;58:135-149. 14. levy jk, mansour m, crawford pc, et al: survey of zona pellucida antigens for immunocontraception of cats. theriogenology 2005;63:1334-1341. 15. fischer a, benka va, briggs jr, et al: effectiveness of gonacon as an immunocontraceptive in colony-housed cats. j feline med surg 2018;20:786-792. doi:10.1177/1098612x18758549 16. levy jk, friary ja, miller la, et al: long-term fertility control in female cats with gonacon™, a gnrh immunocontraceptive. theriogenology 2011;76:1517-1525. clinical theriogenology 2021; 13: 158 medical contraception in cat: new and old drugs factors affecting bull sexual development factors affecting bull sexual development leonardo brito, stgenetics, navasota, tx abstract this review describes some of the physiological events associated with bull sexual development and factors that affect this development. keywords: bull, puberty, testis, scrotal circumference, semen introduction sexual development is a major determinant of cattle production efficiency. the ability to breed animals at younger ages reduces generation intervals and increases genetic gains. however, reduced sperm production and poor semen quality due to immaturity are common causes of poor reproductive performance of young bulls, representing a serious loss of superior genetic stock. the ability to collect and freeze semen from younger bulls is also desired to reduce the time required for progeny testing and to hasten artificial insemination and sire selection. therefore, an understanding of pubertal changes and factors that affect sexual development is required to promote successful use of young bulls for reproductive purposes. the process of testicular development that leads to initiation of spermatogenesis in bulls involves complex maturation mechanisms of the hypothalamus-pituitary-testes axis. sexual development can be divided into 3 periods according to changes in gonadotropins and testosterone concentrations, namely the infantile, prepubertal and pubertal periods. these changes are accompanied by changes in testicular cell proliferation and differentiation. infantile period is characterized by low gonadotropin and testosterone secretion and relatively few changes in testicular cellular composition. this period extends from birth to ~ 2 months of age in bos taurus bulls. gonadotropin secretion during the infantile period is low, due to reduced gnrh secretion; however, maturation of the hypothalamus increases gnrh pulse secretion and drives the transition out of the infantile period.1-3 during this period, the testicular parenchyma is composed by seminiferous cords without a lumen lined by undifferentiated sertoli cells and gonocytes. mesenchymallike cells are the majority of the cells in testicular interstitial tissue.4-7 prepubertal period is characterized by a temporary increase in gonadotropins secretion, the “early gonadotropin rise.”1-3 this is a critical event in sexual development of bulls. it is not only associated with dramatic changes in testicular cellular composition, initial increases in testosterone secretion and timing of attainment of puberty, but also has long-lasting effects on testicular growth and sperm production. this period extends from ~ 2 6 months of age in bos taurus bulls. the early gonadotropin rise is driven by increased gonsadotropin releasing hormone (gnrh) pulse secretion, as demonstrated by a dramatic increase in luteinizing hormone (lh) pulse frequency (figure 1). the lh secretion pattern during the prepubertal period is also associated with age at puberty in bulls raised in contemporary groups, suggesting that this is the physiological mechanism by which genetics affect sexual development.8-9 during the prepubertal period, there is a progressive increase in proportion of the testicular parenchyma occupied by seminiferous tubules, with increasing tubule diameter. adult-type sertoli cells develop, creating extended junctional complexes above newly formed spermatogonia. furthermore, there is differentiation of testicular interstitial mesenchymal-like cells into leydig cells and onset of testosterone secretion.4-7 pubertal period is characterized by reduced gonadotropin secretion, increased testosterone secretion, initiation of spermatogenesis and eventual appearance of sperm in the ejaculate.1-3 this period also coincides with the start of rapid testicular growth and extends from ~ 6 to 12 months of age in bos taurus bulls. testicular growth is associated with increasing seminiferous tubule diameter and length. most sertoli cells complete their morphological differentiation and attain adult structure, forming a functional blood-testis barrier. germ cell proliferation is maximal between 4 and 8 months of age, 347 clinical theriogenology • volume 11, number 3 • september 2019 representing expansion of the spermatogonial stem cells, with mature sperm appearing in seminiferous tubules at ~ 8 10 months of age.4-7 puberty after spermatogenesis is established, there is a gradual increase in number of testicular germ cells supported by each sertoli cell and an increase in efficiency of the spermatogenesis that results in the production of a number of sperm sufficient for those to appear in the ejaculate. in general, puberty is defined as the process of changes by which a bull becomes capable of reproducing. this process involves development of gonads and secondary sexual organs and acquiring the ability to breed. for research purposes, however, puberty in bulls is usually defined as an event instead of a process. most researchers define attainment of puberty by production of an ejaculate containing ≥ 50 million sperm with ≥ 10% motile sperm.10 age at puberty determined experimentally can be affected by the age that semen collection attempts are performed, interval between attempted collections, method of semen collection (artificial vagina or electroejaculator), response of the bull to the specific semen collection method and experience of the collectors. moreover, age at puberty is affected by genetics, management and nutrition. although data from large trials comparing bulls of various breeds raised as contemporary groups are scarce, some liberties could be taken to make some generalizations. dairy bulls usually mature faster and attain puberty earlier than beef bulls. bulls from continental beef breeds (with the exception of charolais) usually attain puberty later than bulls from british beef breeds, especially angus bulls. bulls from double-muscled breeds are notorious for being late maturing. puberty is delayed in bulls from tropically adapted bos taurus breeds and in unadapted bulls raised in the tropics. in general, bos indicus bulls attain puberty at considerably older ages than bos taurus bulls. there is large variation in age and body weight at puberty across and within breeds. although on average bos taurus bulls attain puberty with sc between 28 and 30 cm, regardless of the breed,11 that there is still considerable variation in sc at puberty is sometimes overlooked. interesting observations have been reported in studies evaluating differences between earlyand late-maturing bulls.8,9,12 bulls that attain puberty earlier were generally heavier and had greater sc than bulls that attained puberty later; however, both weight and sc were smaller at puberty in early-maturing bulls. these observations not only indicate that precocious bulls develop faster, but also suggest that sexual precocity is not simply related to earlier attainment of a threshold of body or testicular development. in fact, these thresholds seem to be lower in early-maturing bulls, whereas late-maturing bulls must reach a more advanced stage of body and testicular development before puberty is attained. scrotal circumference is a moderately heritable trait in cattle; therefore, direct selection can have a very significant impact on sc. studies have also demonstrated moderate to high phenotypical correlations between sc and growth traits and estimates of genetic correlations with growth traits are generally positive.13-14 either the combination of direct selection for sc and/or indirect selection for growth traits is likely responsible for the general trend of increasing sc over the years in certain breeds. increased sc is expected to be associated with decreased age at puberty. effects of nutrition on sexual development very few studies have evaluated the effect of nutrition from birth to maturity on sexual development and reproductive function in bulls. a series of experiments have shown that the most pronounced effects of nutrition occur during the prepubertal period. these studies demonstrated that adverse effects of low nutrition during the prepubertal period cannot be compensated by improved nutrition during the pubertal period and that the beneficial effects of high nutrition during the prepubertal period are sustained, even if maintenance diets are fed thereafter. low nutrition during the peripubertal period in beef bulls reduced gonadotropin secretion, delayed increases in circulating testosterone concentrations, delayed puberty and decreased testicular size at 16 months of age, whereas high nutrition produced opposite results. 348clinical theriogenology • volume 11, number 3 • september 2019 in 1 study in which bulls received different nutrition from 2 to 16 months of age, reduced lh pulse frequency during the prepubertal period resulted in delayed puberty in bulls receiving low nutrition, whereas a more sustained increase in lh pulse frequency in bulls receiving high nutrition was associated with hastened testosterone production and larger testes at 16 months of age compared to bulls receiving low or medium (control) nutrition.15 these observations were corroborated by additional studies designed to investigate effects of nutrition specifically during the prepubertal period. bulls that received high nutrition only from 2 7 months of age also had a more sustained increase in lh pulse frequency, greater testosterone secretion, were 2 weeks younger at puberty and had greater testes weight at 16.5 months of age compared to bulls receiving control nutrition.16 conversely, reduced lh secretion resulting from low nutrition from 2 7 months of age was associated with increased age at puberty and smaller testes at 16 months of age, even when these bulls received control or high nutrition after 7 months of age and lh and testosterone secretion were not different after the change in nutrition.17 differences in yearling sc due to age of the dam in beef bulls could also be interpreted as an indication that nutrition during the pre-weaning period affects sexual development, although possible in utero effects cannot be completely excluded. scrotal circumference in bos taurus beef bulls increases as age of the dam increases until 5 9 years of age and subsequently decreases as dams get older. inclusion of weight as a covariate in the models describing sc resulted in decreased effects of age of the dam, indicating that effect of age of the dam on testicular growth seem to be primarily due to age of dam effects on bull’s body weight, likely related to differences in milk production.14,18,19 this theory is also supported by reports that, similar to that in bulls receiving low nutrition, lh secretion after gnrh challenge was greater from 3.5 6 months of age in bulls raised by multiparous versus primiparous females.20 several studies described effects of nutrition only during the pubertal period, i.e. after initial hormonal changes that regulate sexual development have occurred. in general, these studies indicate that low nutrition has adverse effects on growth and sexual development. in 1 study, bulls receiving one-third of the amount supplied to their twin controls had lower body and vesicular gland weights, vesicular gland fructose and citric acid contents and circulating and testicular testosterone concentrations, whereas circulating androstenedione concentrations were increased.21 in another series of experiments, beef bulls 8 -12 months old receiving diets with low levels of crude protein (8, 5 and 1.5%) for 3 6 months had markedly reduced testes, epididymis and seminal glands weights when compared to control bulls fed diets containing 14% crude protein.22 although low nutrition during the pubertal period has adverse effects on reproductive function, potential beneficial effects of high nutrition after weaning are questionable at best. under field conditions, postweaning high energy diets are frequently associated with impaired reproductive function in bulls, likely related to altered testicular thermoregulation due to excessive fat deposition above and around the testes. in 1 report, sperm motility decreased and proportion of sperm defects increased with age in hereford bulls fed to gain > 1.75 kg/day, which was significantly different from bulls fed to gain ~ 1 kg/day (control). even after the high-nutrition diet was changed to a control diet, bulls previously fed high nutrition continued to have lower semen quality. there was greater deposition of fat around the testicular vascular cone in the scrotal neck in bulls in the high-nutrition group and the difference between body and testes temperature was reduced in this group compared to bulls in the control group. this difference was still present after the high-energy diet was changed and bulls had lost considerable weight, indicating that fat in the scrotum is more difficult to lose than other body fat.23 in another series of experiments, angus, hereford, and simmental bulls were fed high nutrition (80% grain and 20% forage) or medium nutrition (forage only) from ~ 6.5 to 12 24 months of age. in general, bulls receiving high nutrition had greater body weight and backfat, but paired testes weight was not affected by diet. moreover, bulls receiving high nutrition had lower daily sperm production and epididymal sperm reserves and greater proportion of sperm abnormalities. the authors indicated that increased dietary energy may adversely affect sperm production and semen quality due to fat deposition in the scrotum, which reduced the amount of heat that can be radiated from the scrotal skin, thereby increasing scrotal and testicular temperatures.24-27 observations from another study indicated that bulls 349 clinical theriogenology • volume 11, number 3 • september 2019 fed high-nutrition diets had greater sc and scrotal weight than bulls fed medium-nutrition diets, but paired testes weight was not different between the 2 groups, indicating that greater sc was likely associated with accumulation of scrotal fat.28 conclusion studies have supported the intuitive assumption that low nutrition has adverse effects on sexual development and reproductive function, regardless of the bull’s age. however, most research seems to indicate that high nutrition is only beneficial during the first 6 months of life, which presents a challenge to bull producers. beef bull calves are usually nursing until 6 8 months of age and very little attention is paid to their nutrition, whereas nutrition offered to young dairy bull calves is often suboptimal. efforts to obtain maximum weight gain during the first months after birth by offering high-nutrition diets and adopting management practices like creep feeding will be compensated by reduced age at puberty and greater sperm production capacity in adult bulls. it is also clear that, although high-nutrition diets after 6 months of age may be associated with greater sc, this effect is likely the result of fat accumulation in the scrotum and not actually greater testicular size. moreover, sperm production, semen quality and serving capacity are all compromised in bulls receiving excessive nutrition after this age. adjusting diets accordingly to maximize growth, but prevent over conditioning after the peripubertal period, is advisable. conflict of interest there are no conflicts of interest to declare. references 1. rawlings n, fletcher p, henricks d, et al: plasma luteinizing hormone (lh) and testosterone levels during sexual maturation in beef bull calves. biol reprod 1978;19:1108-1112. 2. amann r, walker o: changes in the pituitary-gonadal axis associated with puberty in holstein bulls. j anim sci 1983;57:433-442. 3. amann r, wise m, glass j, et al: prepubertal changes in the hypothalamic-pituitary axis of holstein bulls. biol reprod 1986;34:71-80. 4. abdel-raouf m: the postnatal development of the reproductive organs in bulls with special reference to puberty (including growth of the hypophysis and the adrenals). acta endocrinol (copenh) 1960;34(suppl 49):1-109. 5. sinowatz f, amselgruber w: postnatal development of bovine sertoli cells. anat embryol 1986;174:413-423. 6. wrobel k: the postnatal development of the bovine leydig cell population. reprod domest anim 1990;25:51-60. 7. wrobel k: prespermatogenesis and spermatogoniogenesis in the bovine testis. anat embryol 2000;202:209-222. 8. evans a, davies f, nasser l, et al: differences in early patterns of gonadotrophin secretion between early and late maturing bulls, and changes in semen characteristics at puberty. theriogenology 1995;43:569-578. 9. aravindakshan j, honaramooz a, bartlewski p, et al: pattern of gonadotropin secretion and ultrasonographic evaluation of developmental changes in the testis of early and late maturing bull calves. theriogenology 2000;54:339-354. 10. wolf f, almquist j, hale e: prepubertal behavior and pubertal characteristics of beef bulls on high nutrient allowance. j anim sci 1965;24:761-765. 11. lunstra d, ford j, echternkamp s: puberty in beef bulls: hormone concentrations, growth, testicular development, sperm production and sexual aggressiveness in bulls of different breeds. j anim sci 1978;46:1054-1062. 12. brito l, silva a, unanian m, et al: sexual development in earlyand late-maturing bos indicus and bos indicus × bos taurus crossbred bulls in brazil. theriogenology 2004;62:1198-1217. 13. knights s, baker r, gianola d, et al: estimates of heritabilities and of genetic and phenotypic correlations among growth and reproductive traits in yearling angus bulls. j anim sci 1984;58:887-893. 14. lunstra d, gregory k, cundiff l: heritability estimates and adjustment factors for the effects of bull age and age of dam on yearling testicular size in breeds of bulls. theriogenology 1988;30:127-136. 15. brito l, barth a, rawlings n, et al: effect of nutrition during calfhood and peripubertal period on serum metabolic hormones, gonadotropins and testosterone concentrations, and on sexual development in bulls domcst anim endocrinol 2007;33:1-18. 16. brito l, barth a, rawlings n, et al: effect of improved nutrition during calfhood on serum metabolic hormones, gonadotropins, and testosterone concentrations, and on testicular development in bulls. domest anim endocrinol 2007;33: 460-469. 17. brito l, barth a, rawlings n, et al: effect of feed restriction during calfhood on serum concentrations of metabolic hormones, gonadotropins, testosterone, and on sexual development in bulls. reproduction 2007;134:171-181. 350clinical theriogenology • volume 11, number 3 • september 2019 18. kriese l, bertrand j, benyshek l: age adjustment factors, heritabilities and genetic correlations for scrotal circumference and related growth traits in hereford and brangus bulls journal of animal science 69 478-489. 19. crews d jr, porteous d: age of dam and age at measurement adjustments and genetic parameters for scrotal circumference of canadian hereford bulls. can j anim sci 2003;83:183-188. 20. bagu e, davies k, epp t, et al: the effect of parity of the dam on sexual maturation, serum concentrations of metabolic hormones and the response to luteinizing hormone releasing hormone in bull calves. reprod domest anim 2010;45:803-810. 21. mann t, rowson l, short r, et al: the relationship between nutrition and androgenic activity in pubescent twin calves, and the effect of orchitis. j endocrinol 1967;38:455-468. 22. meacham t, cunha t, warnick a, et al: influence of low protein rations on growth and semen characteristics of young beef bulls. j anim sci 1963;22:115-120. 23. skinner j: nutrition and fertility in pedigree bulls. in: gilmore d, cook b, editors. environmental factors in mammals reproduction. macmillan publishers; 1981, p. 160-168. 24. coulter g, kozub g: testicular development, epididymal sperm reserves and seminal quality in two-year-old hereford and angus bulls: effects of two levels of dietary energy. j anim sci 1984;59:432-440. 25. coulter g, carruthers t, amann r, et al: testicular development, daily sperm production and epididymal sperm reserves in 15-mo-old angus and hereford bulls: effects of bull strain plus dietary energy. j anim sci 1987;64:254260. 26. coulter g, bailey d: epididymal sperm reserves in 12-month-old angus and hereford bulls: effects of bull strain plus dietary energy. anim reprod sci 1988;16:169-175. 27. coulter g, cook r, kastelic j: effects of dietary energy on scrotal surface temperature, seminal quality, and sperm production in young beef bulls. j anim sci 1997;75:1048-1052. 28. seidel g jr, pickett b, wilsey c, et al: effect of high level of nutrition on reproductive characteristics of angus bulls. proc int congress anim reprod and ai 1980; p. 359. 351 clinical theriogenology • volume 11, number 3 • september 2019 352clinical theriogenology • volume 11, number 3 • september 2019 omniblank: editor’s note dear theriogenology friends, we thank the scientific and abstract committee of the american college of theriogenologists for their hard work. abstracts truly represent an enormous amount of time, effort and resources invested by the investigators and their institutions. we are impressed with the broad range of topics, techniques, and abstract quality! there is always a possibility of creating manuscripts from abstracts. although some abstracts will need further work before a manuscript can be created, many have sufficient information to become either a case report or original paper. please consider expanding your abstract into a manuscript and when doing so, keep clinical theriogenology in mind as a potential target journal. we really appreciate the willingness of speakers to create very thoughtful and complete reviews for our journal that enrich these pages. for me, it was a privilege and pleasure to work with these scholars. we thank you all for your contributions to theriogenology. in addition, editorial members deserve our appreciation for reviewing and refining the reviews. our time spent in editing, copyediting, and formatting are well worth the effort. we are fortunate to have colleagues to help me in these aspects. they are supporting me in my goal to improve the quality of our journal. please join with me in thanking drs. john kastelic and ahmed tibary. ms. tara beasley’s help in organizing and managing the timely arrival of abstracts and manuscripts, and working with the committee, authors and me is greatly appreciated. on another related topic, we are thankful to dr. riley thompson for creating a survey regarding our journal and for our franz management to complete and compile the results. thank you for your encouraging comments and by the way, i look forward to receiving reviews (10 of you have agreed to create reviews for the journal). finally, i express my deep appreciation to the leadership of our 3 organizations and dr. charles franz for their support and assistance. regards, augustine 1. editor award plenary.indd 11. editor award plenary.indd 1 7/28/2021 11:22:11 am7/28/2021 11:22:11 am clinical theriogenology 2021; 13: 113 clinical theriogenology 2021; 13: 114 editor’s note clinical theriogenology 2022; 14: 98 breeding soundness examination in a clouded leopard taylor lashlee,a adrienne difoggio,a pablo jarrin-yepez,a heather schwartz,b margarita colburn,b andrew cushing,a tulio pradoa acollege of veterinary medicine, university of tennessee, knoxville, tn bnashville zoo, nashville, tn abstract a 3-year male clouded leopard was presented to the university of tennessee college of veterinary medicine theriogenology services for a breeding soundness examination. on physical examination, the left testis felt abnormally small. on ultrasonography, no abnormalities were observed; testis and prostate were of normal size. semen was collected with a pulsator electroejaculator and a ram electroejaculator probe (commonly used to collect semen in small ruminants). the purpose of using this electroejaculator and probe was to determine if they could be used to collect semen from a clouded leopard. using manual settings with this device we collected ~ 0.2 0.3 ml of semen. motility and morphology of sperm were acceptable. we concluded that the pulsator iv electroejaculator and ram probe can be used to collect semen as part of breeding soundness evaluation in a clouded leopard. keywords: nondomestic feline, electroejaculator, manual settings, semen evaluation background clouded leopards (neofelis genus) are medium-sized nondomestic felines, and the genus consists of 2 species.1 one species, neofelis nebulosa, is present in southeast asia, and the other, neofelis diardi, is in the islands of borneo and sumatra.2 under human care, the average lifespan of clouded leopards is 13 to 15 years, and they reach sexual maturity between 20 and 30 months.2-4 reproduction is highest between 2 and 4 years in males and between 1 and 5 years in females. reproduction usually starts to decline after 6 years of age.4 clouded leopards have long estrous cycles, varying from 15 to 40 days and estrus typically lasts for 3 6 days.3 additionally, > 40% of females display spontaneous ovulation.3,6 however, unlike domestic cats, they rarely exhibit obvious signs of sexual receptivity under human care.6 a seasonal influence has also been observed in clouded leopards. in males, motile sperm number peaked in june and july.7 in western zoos, short periods of anestrus may occur in late summer and early fall.3 pregnancy lasts for ~ 90 days.3 clouded leopards are considered vulnerable in the wild, and their numbers are continuing to decrease.1,3 habitat destruction and illegal hunting are big contributors to their declining numbers.4,5 therefore, breeding is important to maintain their survival and genetic diversity. however, breeding clouded leopards under human care is challenging, even in exhibits that mimic their natural environment.3 in addition to an inconsistent ovulation pattern in females, males exhibit high levels of aggression that can potentially lead to serious injury and female’s death.2,8 successful breeding had occurred with pairs that have been introduced before puberty.3,7additionally, male clouded leopards have a high percentage of abnormal sperm. teratospermia and pleiomorphism are common findings in clouded leopards, and this is likely related to low genetic diversity in the breeding population under human care.23,6-8 clouded leopard sperm have abnormal acrosomes, coiled tail, and midpiece defects2,7,9 motility ranged from 55 to 80% and normal morphology ranged from 20 to 35%.2,5,9 electroejaculation under general anesthesia is a method adopted to collect semen. this procedure is relatively safe, with anesthesia usually being the biggest concern.10 ketamine hydrochloride has been used to anesthetize these animals.2,5,11,12 with ketamine, one must carefully monitor for increased heart rate, increased blood pressure, apneustic breathing, and bronchodilation, as these are common side effects.13 a disadvantage of electroejaculation in cats has been urine contamination. this has occurred with high voltage or when the electrode is positioned cranially.11 historically, electroejaculation probes used in wild cats have varied. in general, the probe has had 3 longitudinal strips of copper that are 0.4 cm apart and positioned ventrally, and the diameter varied among species.11 stimulations and voltage have also varied. a probe (2.5 cm in diameter and 7 cm in length) with electrodes (4.6 mm diameter and 56.8 mm length) was used in clouded leopards, and it was inserted into the rectum ~ to 10 12 cm. stimulation consisted of 2 3 series that were divided into 2 3 sets of 10 stimuli each, using 15 volts with 4 5 seconds of rest between each stimulus.2 furthermore, a 1.6 cm diameter probe was used and stimuli were divided into 3 series of 2 6 volts.12 a 1.6 cm in diameter and 23 cm in length probe also was used and stimuli were divided into 3 series of 5 8 volts.7 clinical theriogenology 2022; 14: 99 currently, a nondomestic feline semen collection procedure has been developed by the smithsonian conservation biology institute that is used in clouded leopards. personal communication (dr. adrienne crosier from the smithsonian conservation biology institute) provided us with the 2018 updated procedures used by the institute to conduct felid semen collection, evaluation, and freezing. this procedure uses either a 1.6 or 1.9 cm diameter probe, depending on the size of the animal. the probe and the rectum should be well lubricated with a nonspermicidal gel, and the probe should be gently inserted into the rectum. electrodes should be positioned over the accessory sex glands, with the central electrode positioned ventrally. this procedure consists of 3 series and with each series consisting of 30 stimulations. for the first series, 10 stimulations are given at 2 volts, 10 at 3 volts, and 10 at 4 volts. for the second and third series, 20 stimulations are given at 3 volts and 10 at 4 volts. two seconds rest should be given between each stimulus. however, each electroejaculation attempt should be modified to that specific patient, since it can vary among species, individuals, and the individual’s response on various days.10 collected semen should be evaluated for volume, ph, appearance, and consistency and sperm motility, morphology, and concentration.2,5,10 none of these factors alone sufficiently predicts fertility, so they should be assessed together to determine a more accurate prediction of male fertility.10 fresh semen may be used for artificial insemination or can potentially be cryopreserved for later use. however, motility is lower in postthaw semen with possible lower fertility rates.5 we performed a breeding soundness examination on a clouded leopard imported from thailand to determine his fertility. electroejaculation was performed with a pulsator electroejaculator (pulsator iv, lane manufacturing inc., denver, co) and ram probe (12 cm long, 2 cm diameter, 2 electrodes [figure 1]), traditionally used in small ruminants. this electroejaculator and probe, figure 1. pulsator iv electroejaculator and ram probe commonly used by large animal theriogenologists, were used to determine if a more widely available electroejaculator and probe could be used to collect a clouded leopard. semen volume, motility, and morphology were analyzed after collection. case presentation a 3-year 33 kg male clouded leopard (neofelis nebulosa) at the nashville zoo (recently brought from thailand) was presented to the university of tennessee college of veterinary medicine theriogenology services for a breeding soundness examination on september 15, 2020. he was fasted for 8 16 hours and was moderately active prior to anesthesia. he was anesthetized intramuscularly with a total of 280 mg of ketamine hydrochloride (zetamine 100 mg/ml, vetone, boise, id) and 1,680 µg of dexmedetomidine hydrochloride (dexdomitor 0.5 mg/ml, zoeitis inc, kalamazoo, mi). he was initially given 100 mg of ketamine (3 mg/kg) and 690 µg of dexmedetomidine (20 µg/kg). however, he was not sedated enough and was given another 100 mg of ketamine and 690 µg of dexmedetomidine. since he was not fully sedated another 50 mg of ketamine and 300 µg of dexmedetomidine was given. subsequently, another 30 mg of ketamine was given. once he was fully sedated, he was maintained on 3% of isoflurane via facemask. animal was placed in right lateral recumbency and appeared healthy on physical examination. he was initially thought to be a cryptorchid. the zoo medicine team also collected urine samples (using a retrograde urinary catheter coated with nonspermicidal sterile lube) for pheochromocytomas, the most common neoplasia in clouded leopards.14 pheochromocytomas were identified in 45% of clouded leopards that were necropsied.15 however, pheochromocytomas only consist of 2% of mortality in clouded leopards.14 urethral catherization was performed prior to electroejaculation to avoid semen contamination. penis was exteriorized and cleaned with saline and gauze. scrotum was palpated and testes were measured with calipers. transcutaneous ultrasonography of testes was performed using a 3.5 mhz convex transducer. prostate was examined via transrectal ultrasonography using a 5 mhz rectal transducer. approximately, 60 ml of nonspermicidal sterile lube was applied to the probe. more nonspermicidal sterile lube was applied into the rectum prior to electroejaculation. manual settings on the ejaculator were used to avoid applying too high voltages. voltages applied were manually manipulated (quarter to half-way up to the maximum volts on each step) that have been previously established by the manufacturer. with this approach, voltages were able to stay similar to voltages used in a standardized nondomestic feline procedure. each step was held for 5 seconds, and steps were repeated 3 5 times, based on twitching response by the animal. a 2 second rest at 0 volts was given in between steps. frequency (hertz) remained at 15.02 +/0.02. semen was analyzed for motility and morphology. for motility analysis, a drop of semen was placed on a prewarmed microscope slide, and a cover-slip was placed on top. motility was subjectively evaluated under a phase contrast microscope at 10, 20, and 40 x objectives. for morphology analysis, a drop of eosin-nigrosin stain was placed on a prewarmed microscope slide, and then a drop of semen was added to the slide. this slide was mixed with another slide to make a thin layer to avoid sperm overlapping. morphology was evaluated clinical theriogenology 2022; 14: 100 by counting 100 individual sperm under oil objective (1,000 x). semen was not sufficient for extending to have further examinations. outcome penis appeared normal and healthy (figure 2), and had spines indicating sexual maturity. both testes were palpable. however, the right testis was bigger compared to the left (2.3 cm in height, 4.1 cm in length, and 2.6 cm in width.) on ultrasonography, right testis measured 4.2 cm in length and calculated3 scrotal volume was 14.5 cm. a consistent measurement of left testis with calipers was not possible (unable to maintain a consistent grip). however, it was measured (1.5 cm in height and 3 cm in length) via ultrasonogram. testes and prostate appeared normal. figure 2. extended penis electroejaculation was accomplished (~ 0.2 0.3 ml of ejaculate) using the pulsator iv electroejaculator probe (2.5 cm diameter and 12 cm in length with 2 electrodes). a phase contrast microscope was used for semen analysis. forty percent of sperm had normal motility (400 x objective) and 66% had normal morphology (eosin-nigrosin stain under 1000 x oil immersion objective). primary abnormalities were acrosome defects (e.g. knobbed acrosomes), narrow heads, tightly coiled tails, and bent midpieces (figure 3). semen appeared viable for reproduction. figure 3. abnormal sperm discussion objective was to determine if this genetically valuable clouded leopard would be fertile producing viable sperm. a breeding soundness examination was conducted that included semen collection and evaluation. an electroejaculator probe, commonly utilized in small ruminants (widely available), was used to collect semen. clouded leopards are categorized as vulnerable and their numbers are on the decline.1,3 therefore, zoos have important roles in clouded leopard conservation. however, breeding clouded leopards presents several challenges. inconsistent ovulation pattern, high incidence of male aggression, and high percentage of abnormal sperm were observed.2,8 therefore, sperm collection and evaluation are important. clouded leopards have a high percentage of abnormal sperm (acrosomal defects, midpiece defects, and coiled tail defects).2-3,5-9 therefore, sperm morphology and motility are usually lower. morphology of normal sperm has ranged from 20 to 35%, and motility ranged from 55 to 80%.2,5,9 these defects are possibly due to a low genetic diversity in the clouded leopard population under human care.2,6,7 in this case, normal morphology was 66% and motility was 40%. higher percent of normal morphology in this clouded leopard could be due to its genetically diverse (thailand) nature, supporting the theory that the lower genetic variation in the breeding population under human care could be causing low normal morphology. more studies are needed to confirm this observation. abnormal sperm in this clouded leopard’s ejaculate include acrosome defects, narrow heads, tightly coiled tail defects, and bent midpieces. similar to this case, 40% of clouded leopard sperm had abnormal acrosomes (increased to 70% with coomassie blue).5 we did not use coomassie blue, so it is possible that this clouded leopard had a higher percent of abnormal acrosomes that was not obvious under the phase contrast microscope. a variety of techniques (probe size and stimulation procedures) have been used.2,7,10-12 a nondomestic felid semen collection, evaluation, and freezing procedure was developed by the smithsonian conservation biology institute that used a 1.6 or a 1.9 cm diameter probe. however, we used a 2.5 cm diameter and 12 cm length probe with 2 electrodes, a probe, commonly used in small ruminants, and therefore, more widely available to veterinarians than a probe specifically used for wild felids. with this probe, we collected ~ 0.2 0.3 ml of ejaculate, demonstrating that this commonly available probe can be used to collect semen in clouded leopards. semen volume, motility, and morphology were analyzed in this case. however, since the objective of this case was primarily to determine if this male could be fertile producing viable sperm, the semen was not cryopreserved. although this clouded leopard had lower percent of normal and motile sperm, percent normal sperm was higher compared to earlier observations. additionally, it had an abnormally small testis and apparently the other testis was able to compensate to produce adequate sperm. furthermore, his penis and prostate were normal based on physical examination and ultrasonographic observations. in conclusion, this cloudclinical theriogenology 2022; 14: 101 ed leopard was considered fertile and capable of producing viable sperm. learning points • pulsator iv electroejaculator with the ram probe (12 cm long and 2 cm diameter with 2 electrodes) can be used to collect semen in male clouded leopards. • pulsator iv electroejaculator and ram probe are easily available, and they can be used as an alternate method to collect semen in clouded leopards for data collection, semen evaluation, and potential artificial insemination. references 1. grassman l, lynam a, mohamad s, et al: neofelis nebulosa. the iucn red list of threatened species 2016:e.t1451a97215090. 2. zainuddin z, tarmizi mm, yap k, et al: first evaluations and cryopreservation of semen samples from sunda clouded leopards (neofelis diardi). animals 2020;10:1072. 3. brown jl: female reproductive cycles of wild female felids. animal reprod sci 2011;124:155-162. 4. yamada jk, durrant bs: reproductive parameters of clouded leopards (neofelisnebulosa). zoo bio l 1989:8:223-231. 5. pukazhenthi b, laroe d, crosier ae, et al: challenges in cryopreservation of clouded leopard (neofelis nebulosa) spermatozoa. theriogenology 2006;66:1790-1796. 6. thongphakdee a, sukparangsi w, comizzoli p, et al: reproductive biology and biotechnologies in wild felids. theriogenology 2020:150;360-373. 7. wildt de, howard jg, hall, ll, et al: reproductive physiology of the clouded leopard: i. electroejaculates contain high proportions of pleiomorphic spermatozoa throughout the year. biol reprod 1986:34;937-947. 8. tipkantha w, thuwanut p, maikeaw u, et al: successful laparoscopic oviductal artificial insemination in the clouded leopard (neofelis nebulosa) in thailand. j zoo wildl med 2017;48:804-812. 9. tipkantha w, thuwanut p, siriaroonrat b, et al: mitigation of sperm tail abnormalities using demembranation approach in the clouded leopard (neofelis nebulosa). reprod domest anim 2016;52:214-218. 10. durrant bs: semen collection, evaluation, and cryopreservation in exotic animal species: maximizing reproductive potential. ilar journal 1990;32:2-10. 11. paz rc: wildlife cats reproductive biotechnology. cur front cryobiol 2012. 12. howard jg, roth tl, byers, et al: sensitivity to exogenous gonadotropins for ovulation induction and laparoscopic artificial insemination in the cheetah and clouded leopard. biol reprod 1997:56:1059-1068. 13. rosenbaum sb, gupta v, palacios jl: ketamine. in: statpearls. treasure island, fl. 14. thorel m, pignon c, arne p, et al: clouded leopard (neofelis nebulosa) morbidity and mortality in captive-bred populations: a comprehensive retrospective study of medical data from 271 individuals in european, asian, and australian zoos. j zoo wildl med 2020;51:150. 15. corner s, walsh t, padilla l et al: histologic and immunohistochemical characterization of pheochromocytomas in 20 clouded leopards (neofelis nebulosa). vet pathol 2016:54;269-276. introduction reproductive science has critical roles in species conservation and management.1,2 specifically, advances in understanding species’ reproductive biology facilitate development of reproductive technologies that are useful for ensuring genetic and demographic viability of ex situ wildlife populations and assist in development of strategies to control overpopulated species.1 of the 37 canidae family (includes domestic dog [canis familiaris]) species, 5 are listed as ‘endangered’ or ‘critically endangered’ by the international union of conservation of nature. therefore, development of assisted reproductive technologies (arts) would certainly be useful for conservation and management of these threatened canids. currently, existing knowledge on canid reproductive biology is mostly gleaned from domestic dog studies.3-7 it is well recognized that reproductive biology of female canids is unique compared to other mammalian species. specifically, female reproductive cycle is characterized by an extended proestrus followed by protracted estrus with each period lasting on average of 1 week.8 estrous period is characterized by an estrogen peak that coincides with rising progesterone concentrations before ovulation.8 estrus is followed by diestrus, a luteal phase averaging 2 months in duration irrespective of pregnancy. diestrus is succeeded by anestrus, an extended interval of ovarian quiescence.8 whereas domestic dogs exhibit nonseasonal monoestrus once or twice a year,8 most wild canids breed seasonally. the onset of breeding season in wild canids varies among species current and future perspectives of reproductive technologies in domestic and wild canids nucharin songsasen center for species survival, smithsonian conservation biology institute national zoological park, front royal, virginia abstract due to their unique reproduction, reproductive technology advances in canids have lagged behind compared to other mammalian species. currently, semen cryopreservation and artificial insemination have been widely used in domestic dog. however, artificial insemination in wild canids is still limited due largely to poor semen quality, high susceptibility of sperm to cryopreservation procedures, and inability to noninvasively predict ovulation and timed insemination. for female canids, inability to consistently mature oocytes in vitro has limited the use of embryo technology in these species. however, birth of the first domestic dog puppies produced via in vitro fertilization has reinvigorated research interests in this area. this presentation will summarize the status of assisted reproductive technologies in domestic and wild canids and will discuss new, exciting research in fertility preservation and application of reproductive technologies in wild canid conservation. keywords: canids, artificial insemination, in vitro maturation and fertilization, in vitro folliculogenesis, somatic cell nuclear transfer and is dependent on environmental factors, such as latitudes and rainfalls.9,10 furthermore, although most canids are spontaneous ovulators, there has been evidence of induced estrus or ovulation in the island fox (urocyon littoralis), maned wolf (chrysocyon brachyurus),11,12 and seasonal polyestrus in dholes (cuon alpinus) and bush dog (speothos venaticus), indicating diversity in reproductive mechanisms within canidae family. canid oocyte is also unique compared to that of other mammalian species. specifically, domestic dog ovaries contain a higher proportion (7 11%) of polyovular follicles than those (4%) of the domestic cat.13,14 although polyovular follicles release multiple oocytes, there is evidence that only 1 gamete is capable of undergoing maturation and fertilization.14 another unique feature of canid oocytes is that they contain large amount of cytoplasmic lipids compared to other mammalian species, including cat and pig.15 to date, the extent to which cytoplasmic lipids have roles in oocyte development is unknown. nevertheless, it has been suggested that the challenges in applying conventional in vitro oocyte maturation systems to the dog is partly associated with the unusually large amount of cytoplasmic lipid in this species.16 finally, the most striking feature of dog gamete biology is that the oocyte ovulates in an immature stage requiring up to 48 72 hours to complete nuclear maturation within the oviduct,6,15,16 and this characteristic undoubtedly contributes to challenges in developing in vitro maturation (ivm) system for dog oocytes. clinical theriogenology 2021; 13: 138 due to inability to reliably mature dog oocytes in vitro, arts that require the use of in vitro matured gametes have lagged behind compared to other species. nevertheless, a handful of live offspring have been produced from in vitro derived embryos produced from in vivo matured oocytes via in vitro fertilization (ivf) or somatic nuclear transfer (scnt),17-19 and thousands of pups were born from ai with fresh and frozen-thawed sperm.20,21 this review will summarize the status of reproductive technologies in domestic and wild canids and will discuss exciting research in fertility preservation and the application of reproductive technologies in wild canid conservation. semen collection and artificial insemination apart from domestic dog and farmed fox species, semen collection is normally performed by electroejaculation (eej) in wild canids.22-27 maned wolf,28 gray wolf (canis lupus),29,30 and crab-eating fox (cerdocyon thous)31 ejaculates also have been obtained using digital stimulation, although this is not a routine method. the limitation of digital stimulation technique is that it requires preconditioning of animals to physical restraint, and thereby, relies on the availability of animal trainers. however, this method does not require anesthesia and can be performed more frequently than eej (multiple collections per week versus once or twice during breeding season).28 recently, urethral catherization (after medetomidine treatment) developed for felids32,33 has been successfully applied to the domestic dog34 and red wolf (canis rufus).27 like eej, this method requires anesthesia; however, it does not require specialized equipment, and therefore, can be applied to individuals living in situ or under conditions where eej is not feasible. semen characteristics of domestic and wild canid ejaculates collected using various techniques are summarized (table). a major challenge in semen collection in canids, especially via eej and urethral catheterization is urine contamination.24,27,34 the prevalence of urine contamination varies among species, collection method and time of the year. specifically, this author has observed urine contamination as a common feature in the maned wolf, regardless of collection methods. for red wolf, urethral catheterization often results in urine contamination compared to eej.27 as a result, it is recommended that urethral catheterization semen collection method should not be used in red wolf for cryopreservation of sperm.27 urine alters osmolarity and ph of semen samples that, in turn, increases the proportions of sperm with bent and coiled tail, decreases motility,27,35 and increases the susceptibility of sperm to osmotic stress. for the african wild dog (lycaon pictus), urine contamination in semen samples collected via eej occurred more often when samples were collected from subordinate males during prebreeding season species volume (ml) concentration (x 106 sperm/ml) motility (%) morphologically normal sperm (%) citations digital manipulation domestic dog maned wolfa breeding season non-breeding season gray wolfb blue foxb crab-eating foxa 1-30 1.3 ± 1.2 0.4 ± 0.6 1.7 ± 0.2 0.39 ± 0.26 0.39 ±0.18 300 1000 73.9 ± 87.2* 6.1 ± 4.9* 290.8 ± 53.5 491.8 ± 594.4 463.7 ±84.3 70 76.1 ± 23.9 80 ±14.5 91.7 ± 1.5 n/a 86.0 ±16.9 70 36.5 ± 24.0 20.8 ±19.8 n/a 89.9 ± 4.4 2.0 ±1.0 36 28 30 37 31 electroejaculation domestic dog coyoteb red wolfa red wolfb african wild dogb african wild dogb breeding season non-breeding season maned wolfb 1.8 1.67 ± 0.4 6.15 ± 5.6 4.7 ± 0.7 0.6 ± 0.1 na na 2.0 ± 0.6 129.6 549.2 ± 297.7 96.7 ± 178.7 146.5 ± 25.7 212.3 ± 87.3 32.3 ± 9.2 27.4 ± 11.5 43.4 ± 18.2 30.1 90.4 ± 4.5 80.8 ±16.9 71.2 69.5 ± 3.3 47.4 ± 6.7 17.3 ± 10.2 59.8 ± 4.9 n/a 78.0 ± 13.5 46.5 ±14.1 73.6 ± 3.2 76.2 ±6.2 50.9 ± 5.2 40.6 ± 9.8 28.1 ± 4.4 38 26 27 39 25 24 22 urethral catheterization domestic dogb red wolfa 0.09 ± 0.03 0.36 ± 0.08 1,186.67 ± 304.66 50.4 ± 23.5 58.3 ± 8.7 ~40% 53.2 ± 5.6 na 34 27 table. seminal traits of domestic dog and wild canids *total sperm per ejaculate amean ± standard deviation bmean ± standard error of mean clinical theriogenology 2021; 13: 139 than breeding season.24 to circumvent this issue, a common practice for semen collection in wild canids is to remove urine and flush the urinary bladder with saline prior to eej. to date, ai with fresh, chilled and frozen-thawed sperm has been widely used in the domestic dog21 and farmed foxes.40 offspring were produced from ai with fresh or frozen-thawed sperm in gray wolves,41,42 mexican gray wolves,40 and red wolves.39 however, ai has not been routinely applied to genetic management of threatened and endangered canids, likely due to the lack of knowledge on species’ reproductive biology, the challenges in predicting ovulation onset and effectively manipulating female reproduction as well as poor seminal quality (table 1).40 therefore, future research should focus on developing a noninvasive method to predict ovulation in endangered canids. for example, the ability to assess luteinizing hormone in urine samples would be extremely useful for ovulation prediction and timed insemination when frequent blood sampling is not feasible. sperm cryopreservation first records of live birth after ai with cryopreserved sperm were reported in 196943 for domestic dog and in 1975 for gray wolf.42 since these first successes, canid sperm cryopreservation has been widely studied in both domestic dog21,44-46 and wild canids, including gray wolf,47,48 red wolf,27,39,49 maned wolf,22 african wild dog,25 red fox,50 and blue fox.50 glycerol has been generally used as a cryoprotectant for canid sperm.21,25,27 however, in 1 study, dimethyl sulfoxide was superior to glycerol for cryopreservation of maned wolf sperm.22 interestingly, dimethyl sulfoxide was toxic for dog sperm,51 indicating species differences in the response to cryoprotective additives. like pig52,53 and horse,54 there appears to be male-to-male variations in the susceptibility of sperm to cryopreservation that is independent of the quality of fresh semen and normal fertility at natural mating.21,46,55 whelping rate and litter size obtained from frozen-thawed dog sperm are about 23 30% less than fresh sample.21 nevertheless, ai with frozen-thawed sperm has been applied in domestic dog breeding and has resulted in thousands of puppies.21 unlike domestic dog, studies in wild canids, including african wild dog25 and red wolf have demonstrated precipitous decreases in motility and viability of frozen-thawed sperm following incubation despite acceptable viability immediately after postthaw.49 our laboratory recently evaluated the effects of extracellular vesicles (evs) from domestic dog oviducts on postthaw survival of red wolf sperm. evs contain proteins, rna, and dna messages that deliver to neighboring cells, that in turn regulate recipient cells’ function.56 we reported that thawing red wolf sperm in medium containing dog oviductal evs supports sperm motility and acrosomal membrane integrity after 2 hours of incubation compared to nonevs control.57 proteomic analysis also revealed that dog oviductal evs contain several proteins that influence mitochondria function, plasma and acrosomal membrane integrity, and stress responses,57 indicating the useful potential of oviductal evs in improving posthaw survival and longevity of wild canid sperm. in vitro oocyte maturation and fertilization in vitro oocyte maturation (ivm) due to their unique reproductive and gamete biology, development of ivm systems for canids has been far from successful.16 investigations have included impacts of stage of reproductive cycle, culture medium and protein, and hormone (gonadotropins and gonadal steroids) and growth factor supplementation (see review16). nevertheless, on average, only 15 20% of cultured oocytes achieve the metaphase ii (mii) stage after 48 72 hours of in vitro culture.16 supplementing the culture medium with 10 mm caffeine during the first 24 of 72 hours of incubation increased maturation rates compared to unsupplemented controls (42.2 versus 25.5%) with small proportion (4%) of gametes from the former treatment developing to morula stage after ivf.58 interestingly, incubating dog oocytes with caffeine during 24 48 or 72 hours of culture did not improve maturation rate. the beneficial effect of caffeine on dog ivm is likely due to activation of maturation-promoting factor and mitogen-activated protein kinase (mapk),59 2 kinases with critical roles in chromatin reconfiguration during oocyte maturation.60 supplementation of insulin like growth factor-1,61 growth differentiation factor-9 and bone morphogenetic protein1562 also enhanced nuclear maturation of dog oocytes compared to unsupplemented control. yet, overall mii rates in those studies were still < 20%. during the past several years, interest in examining the roles of reproductive evs in regulating gamete function has substantially increased.56 oviductal exosomes (the smallest evs) stimulated cumulus cell proliferation by activating epidermal growth factor receptor (egfr)/mapk signaling pathway.63 this indicates the potential role of oviductal evs in regulating dog oocyte maturation, as cumulus cells are known to induce meiotic resumption and support cytoplasmic maturation in several mammalian species.64 dog’s oviductal and cumulus cells recovered during estrus had higher levels of mapk1 than cells recovered during anestrus and diestrus.65 yet, coincubation of dog oocytes with oviductal cells from estrus resulted only in 10% of the cultured gametes developing to mii.65 coincubation of estrous oocytes with oviductal cells significantly improved mii rate after 72 hours ivm compared to controls (47 versus 11%).66 furthermore, in vitro matured estrous oocytes developed to 8-cell stages (66%) following parthenogenetic activation and in vitro culture at a rate comparable (85%) to in vivo matured gametes.66 despite the discrepancy in the above studies, findings to date are encouraging and emphasize the need to further explore the roles of reproductive evs (from follicle fluid and/or oviduct) in development and maturation of dog oocytes. most ivm studies recover dog oocytes from tissues obtained during routine ovariohysterectomy. follicle size significantly influenced developmental competence of the dog oocyte.67 specifically, ~ 80% of oocytes from follicles > 2 mm in diameter complete nuclear maturation in vitro compared to only 16 to 38% of those from smaller (0.5 < 2 mm) source follicles.67 because > 2 mm diameter follicles only appear during proestrus and estrus, it is hypothesized clinical theriogenology 2021; 13: 140 that the overall low ivm success in canids is likely due to that the oocytes from smaller follicles have not fully acquired developmental competence and therefore, are not able to mature under culture conditions developed for fully grown gametes.67 exposure of dog oocytes for 48 hours to meiotic inhibitor compounds (e.g., roscovitine and butyrolactone) inhibited meiotic resumption of dog oocytes in vitro.68 such oocytes were able to resume meiosis at a higher rate than those not been exposed to meiotic inhibitor compounds.68 therefore, it may be useful to explore the influence of short-term inhibition of meiotic resumption followed by coincubation with oviductal cells or evs on dog oocyte development. such studies may provide insights in mechanisms regulating gamete maturation, information that is critical for the development of an effective ivm system for this species. in vitro fertilization the initial report of ivf using in vitro matured dog oocytes was published more than 40 years69ago, although embryonic development was not reported. since then, several investigators have attempted to perform ivf of incubated oocytes, albeit with limited success.16,58,70-77 thus far, there is only a single report demonstrating the production of 1 blastocyst from ivf from in vitro matured oocytes,72 and 1 non-term pregnancy after transferring in vitro derived presumptive zygotes into recipient females.73 in vitro maturation and fertilization have also been conducted in silver fox; however, embryonic development was not reported.78 to date, there have been a few studies on dog and fox ivf using in vivo matured gametes. only 1 oocyte reached morula (144 hours after ivf) when blue fox oocytes (n = 36) were inseminated with frozen-thawed sperm.79 approximately, 12% of oocytes collected 4 days after ovulation developed to 2-cell stage post ivf; 5 embryos developed further but arrested at the 4-cell stage. only recently was the first litter of pups produced from cryopreserved, in vitro derived embryos.17 two factors contributing to that success included supplementation of magnesium to the sperm capacitation medium and use of day 6 (post lh surge) oocyte for ivf.17,80 interestingly, the presence of progesterone during ivf did not impact fertilization and embryonic development.17 oocyte and embryo cryopreservation large amounts of intracellular lipid within the canid oocyte presents an additional challenge in developing arts. to date, a handful of studies have been conducted on canid oocyte cryopreservation.81-84 due to the lack of effective ivm systems to assess developmental competence of cryopreserved gametes, morphological assessment or vital staining have been used to determine cryopreservation success in most studies. by using the open-pull straw technique, the percentage of vitrified-warmed dog oocytes completing nuclear maturation was similar to that of fresh control, although more cryopreserved gametes were arrested at the gv stage than those of fresh counterparts.83 approximately, 90% of blue fox oocytes vitrified using the two-step open-pulled straw method exhibited normal morphology post-warming and 11% of these gametes developed to the mii stage, comparable to the fresh control.84 finally, 60% of dog81 and mexican gray wolf oocytes82 maintained viability (based on vital staining) after vitrification using the cryotop technique. successful embryo cryopreservation in canids either by vitrification85 or slow freezing method86 has been reported in the domestic dog, demonstrating a stage-dependency in the susceptibility to cryopreservation. specifically, blastocysts are more sensitive to vitrification than those at the earlier stages of development (1-cell to morula stages).85 although dog blastocysts cryopreserved using the slow freezing method were able to re-expand during in vitro culture, transferring these embryos did not result in offspring production.86 to date, live births have been produced from embryos that had been frozen during cleavage stages (2 16 cells). for example, transfer of 77 vitrified-warmed 4 16-cell dog embryos resulted in the birth of 7 live offspring (9.1%).85 furthermore, 16% birth rate were achieved after transferring 6 embryos vitrified using a closed vitrification system (vit kit).87 this same vitrification method has been used to cryopreserve in vitro derived embryos resulting in live birth.17 in vitro folliculogenesis each ovary contains thousands of immature follicles enclosing oocytes that are never ovulated and thus never contribute to reproduction. the ability to activate and grow immature follicles to a mature stage producing a competent oocyte would help preserve genetically valuable dog models of human diseases and endangered canids.88 during the past decade, advances have been made in development of in vitro culture system for dog ovarian tissue and isolated follicles.89-96 compared to domestic cat, dog ovarian tissues are highly susceptible to in vitro culture, likely due to the highly rigid cortex that limits nutrient supply to the enclosed follicles.97 studies have examined effects of growth factors, including epidermal growth factors and vascular endothelial growth factors,92,98as well as an anti-apoptotic agent, z-vad-fmk99 on the activation and survival of enclosed follicles, with varying results. the current in vitro culture protocol can maintain the viability of enclosed follicles for only 7 days.92 nevertheless, the culture system developed for dog has been applied to the maned wolf with comparable results. these findings underscore the importance of the dog model for developing arts for threatened and endangered canids. follicle stimulating hormone (fsh) is essential for in vitro growth of isolated dog follicles;90,93although they increase in size and produce steroids in the presence of fsh, gonadotropin supplementation does not support the growth and survival of the resident gamete.90 this was attributed to disruption of the communication between the oocyte and the surrounding granulosa cells.90 our laboratory has demonstrated that supplementing culture medium with 100 ng/ml activin promotes dog follicle growth and antral cavity expansion and supports oocyte’s chromatin integrity by maintaining the transzonal projection for 12 days.100 most recently, it was reported that supplementing culture media with 2 camp modulators, cilostamide and forskolin can sustain viability of cultured oocytes by promoting camp production and gap junction activity.101 because clinical theriogenology 2021; 13: 141 the communication between the oocyte and surrounding granulosa cells is critical for folliculogenesis and oogenesis, future work on in vitro culture of isolated follicles should focus on developing approaches that support the maintenance of the cell-cell communication during long-term incubation. somatic cell nuclear transfer and transgenesis since the first report on live birth in 2005, there have been several studies on scnt in domestic dogs.19,102,103 the protocol developed for the dog has also been applied to gray wolves104,105 and coyotes,106 resulting in production of live offspring. due to the inability to in vitro mature dog oocytes, studies to date have utilized in vivo matured gametes as the recipient cells for a variety of donor cell types, including fetal and adult fibroblasts as well as adipose derived mesenchymal stem cells.103,107 despite the low success rate (<5% live birth from numbers of transferred embryos), cloned individuals appear to have normal health and reproductive competence.103 specifically, health and reproductive assessment of cloned individuals (n = 3 dogs) revealed that age-related hematological and serum biochemical parameters as well as circulating hormone concentrations of cloned dogs are similar to non-cloned counterparts.103 furthermore, cloned dog ovaries exhibit morphological changes in the same manner as non-cloned individuals. finally, live puppies have been produced after ai of cloned females with fresh semen from a cloned male dog.108 the successful production of scnt dog embryos has facilitated the application of transgenesis technology in this species.109-111 the first transgenic dog that expressed a red fluorescent protein gene was produced in 2009.18 since then, there have been several reports on the production of transgenic offspring as models for studying human disorders, including type 2 diabetes112 and alzheimer’s disease.113 summary and future perspectives due to their unique female reproductive characteristics, the development of arts in the domestic dog and their wild cousins has proven to be extremely challenging. nevertheless, substantial progress has been made during the past decade, including the first production of ivf puppies and numerous offspring from scnt and transgenesis. recent studies on the influence of reproductive evs on the function and cryosurvival of dog and wild canid gametes, and utilization of organ-on-a-chip technology in growing dog ovarian cortices and isolated follicles in vitro114 have provided encouraging results. such studies will likely provide insights into mechanisms regulating gamete formation and function and generate information useful for development of effective tools to preserve/extend fertility of domestic and wild canids. so far, the domestic dog has served as a valuable model for establishing reproductive technologies in wild canids and dog protocols have been successfully applied to their endangered cousins. yet, there are still needs for species-specific research due to the enormous diversity in reproductive biology within the family canidae.10 conflict of interest there are no conflicts of interest to declare. references 1. iucn. the iucn red list of threatened species. version 2020-3. https://ww.iucnredlist.org 2021.ž 2wildt de, wemmer c: sex and wildlife: the role of reproductive science in conservation. biodiversity and conserv 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sp: nuclear maturation of canine oocytes cultured in protein-free media. mol reprod dev 2002;62:407-415. 78. feng hl, liu jm, wen xh, et al: follicular oocyte maturation and sperm penetration in vitro in the silver fox (vulpes vulpes). anim reprod sci 1994;36:163-170. 79. farstad w, hyttel p, grondahl c, et al: fertilization in vitro of oocytes matured in vivo in the blue fox (alopex lagopus). j reprod fertil (suppl) 1993;47:219-226. 80. nagashima jb, travis aj, songsasen n: the domestic dog embryo: in vitro fertilization, culture, and transfer. in herrick jr: editor. comparative embryo culture: methods and protocols. new york, springer; new york: 2019. p. 247-267. 81. abe y, lee d-s, kim s-k, et al: vitrification of canine oocytes. j mamm ova res 2008;28:32-36. 82. boutelle s, lenahan k, krisher r, et al: vitrification of oocytes from endangered mexican gray wolves (canis lupus baileyi). theriogenology 2011;75:647-654. 83. turatham b, saikhun k, sangsuwan p, et al: effects of vitrification on nuclear maturation, ultrastructural changes and gene expression of canine oocytes. reprod biol endocrinol 2010;8:70. 84. zhou g-b, ma c-b, liu g-s, et al: vitrification of farmed blue fox oocytes in ethylene glycol and dmso-based solutions using open-pulled straw (ops). cryoletters 2009;30: 112-118. 85. abe y, suwa y, asano, t. et al: cryopreservation of canine embryos. biol reprod 2011;84:363-368. 86. guaitolini crf, taffarel mo, teixeira ns, et al: post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing. theriogenology 2012;78:576-582. 87. nagashima j, kim yh, hollinshead f, et al: ivf in the dog: recent advancements and future directions. clin theriogenology 2017;9:261-266. 88. comizzoli p, songsasen n, wildt de: protecting and extending fertility options for females of wild and endangered species. cancer treat res 2010;156:87-100. 89. fujihara m, comizzoli p, wildt de, et al: cat and dog primordial follicles enclosed in ovarian cortex sustain viability after in vitro culture on agarose gel in a protein-free medium. reprod domest anim 2012;47:102-108. 90. nagashima j, wildt de, travis aj, et al: follicular size and stage and gonadotropin concentration affect alginate-encapsulated in vitro growth and survival of preand early antral dog follicles. reprod fertil dev 2015;29:262-273. 91. songsasen n, woodruff tk ,wildt de: in vitro growth and steroidogenesis of dog follicles as influenced by the physical and hormonal microenvironment. reproduction 2011;142:113-122. 92. thongkittidilok c, wildt de, songsasen n: responsiveness of intraovarian dog follicles in vitro to epidermal growth factor and vascular endothelial growth factor depends on ovarian donor age. reprod domest anim 2017;52:114-122. 93. serafim mk, araujo vr, silva gm, et al: canine preantral follicles cultured with various concentrations of follicle-stimulating hormone clinical theriogenology 2021; 13: 144 (fsh). theriogenology 2010;74:749-755. 94. serafim mk, duarte ab, silva gm, et al: impact of growth hormone (gh) and follicle stimulating hormone (fsh) on in vitro canine preantral follicle development and estradiol production. growth horm igf res 2015;25:85-89. 95. serafim mk, silva gm, duarte ab, et al: high insulin concentrations promote the in vitro growth and viability of canine preantral follicles. reprod fertil dev 2013;25:927-934. 96. pereira lmc, lopes md, songsasen n: effect of oxygen tension on in vitro viability and development of dog follicles enclosed within the ovarian cortex. reprod domest anim 2019;54:1139-1144. 97. songsasen n, comizzoli p, nagashima j, et al: the domestic dog and cat as models for understanding the regulation of ovarian follicle development in vitro. reprod domest anim 2012;6:13-18. 98. abdel-ghani ma, shimizu t, suzuki h: expression pattern of vascular endothelial growth factor in canine folliculogenesis and its effect on the growth and development of follicles after ovarian organ culture. reprod domest anim 2014;49:734-739. 99. costa pereira lm, thongkittidilok c, lopes md, et al: effect of anti-apoptotic drug z-vad-fmk on in vitro viability of dog follicles. theriogenology 2018;122:124-129. 100. nagashima jb, wildt de, travis aj, et al: activin promotes growth and antral cavity expansion in the dog ovarian follicle. theriogenology 2019;129:168-177. 101. thongkittidilok c, doriguzzi n, nagashima j, et al: cilostamide and forskolin maintain gap junction function of incubated dog follicles. theriogenology 2020;142:222-228. 102. jeong yw, kim jj, hossein ms, et al: influence of somatic cell donor breed on reproductive performance and comparison of prenatal growth in cloned canines. theriogenology 2014;81:1207-1213. 103. lee sh, oh hj, kim mj, et al: dog cloning-no longer science fiction. reprod domest anim 2018;53 (suppl 3):133-138. 104. oh hj, kim mk, jang g, et al: cloning endangered gray wolves (canis lupus) from somatic cells collected postmortem. theriogenology 2008;70:638-647. 105. kim mk, jang g, oh hj, et al: endangered wolves cloned from adult somatic cells. cloning stem cells 2007;9:130-137. 106. hwang i, jeong yw, kim jj, et al: successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes. reprod fertil dev 2013;25:1142-1148. 107. kim ga, oh hj, park, je et al: species-specific challenges in dog cloning. reprod domest anim 2012;47 (suppl 6):80-83. 108. park je, hong sg, kang jt, et al: birth of viable puppies derived from breeding cloned female dogs with a cloned male. theriogenology 2009;72:721-730. 109. eun k, hong n, jeong yw, et al: transcriptional activities of human elongation factor-1α and cytomegalovirus promoter in transgenic dogs generated by somatic cell nuclear transfer. plos one 2020;15:e0233784. 110. kim mj, oh hj, park je, et al: generation of transgenic dogs that conditionally express green fluorescent protein. genesis 2011;49:472-478. 111. zou q, wang x, liu y, et al: generation of gene-target dogs using crispr/cas9 system. j mol cell biol 2015;7:580-583. 112. jeong yw, lee gc, kim jj, et al: establishment of a canine model of human type 2 diabetes mellitus by overexpressing phosphoenolypyruvate carboxykinase. inter j mol med 2012;30:321-329. 113. lee gs, jeong yw, kim jj, et al: a canine model of alzheimer’s disease generated by overexpressing a mutated human amyloid precursor protein. inter j mol med 2014;33:1003-1012. 114. nagashima j, el assal r, songsasen n, et al: evaluation of an ovaryon-a-chip in large mammalian models: species specificity and influence of follicle isolation status. j tissue eng regen med 2018;12:e1926-e1935. anireprosci.2021.106698. clinical theriogenology 2021; 13: 145 current and future perspectives of reproductivetechnologies in domestic and wild canids news from society for theriogenology and american college of theriogenologists from the president of the society for theriogenology for most of us the beauty of spring has been with us for at least a few weeks and has been a pleasant distraction from the drastic changes we have witnessed as a result of the covid-19 pandemic. spring also brings many reminders that life goes on and that we adapt to change. for my own family, we are nearly at the end of one of the most enjoyable calving seasons we have ever had. despite all that is going on in the world, calves are going to be born and animals need to be fed. no one complained about not being able to get off of the farm and every teenager or young adult living under my roof pitched in. i would say that the 2020 calf crop will be the quietist, most handled group of calves that we have ever raised. at the time of this writing we are still not sure that we will be able to hold our annual conference; at least not in the traditional sense. thank you to all who completed our online survey sharing your thoughts regarding postponing verses cancelling the conference and letting us know how you feel about trying a web-based format. your responses will help the board decide how best to proceed. many organizations are cancelling or postponing their events and many universities have banned or advised against travel for their staff. we must first clarify issues surrounding our contract with the conference hotel in pittsburgh and with that information we will be determining our next steps. jack smith and the rest of the 2020 conference team have done a wonderful job of putting together an excellent program. from the board’s strategic planning sessions held in savannah, january 2019 we set a course to develop more web-based learning opportunities including capturing conference seminars. before the pandemic our goal was to capture seminars at the 2020 conference. my mother always said “necessity is the mother of invention!” i don’t think we will be able to claim to have invented anything related to webinars or the internet, but certainly our current situation might just be the catalyst that will help us launch something great for our organization. charles, tara and i have already had a web-based meeting with an organization that can help us produce an outstanding product; and charles and tara are exercising due diligence to ensure that the ce is r.a.c.e. approved. on may 20th our board will meet to decide what our next steps will be. you will know soon after. i am proud of the society for theriogenology and the outstanding group of people on the board and in the sft office. i am honoured to be part of an organization that will ensure that a high quality conference is offered one way and/or another. sincerely, colin palmer sft president 2019-2020 clinical theriogenology • volume 12 number 2 • june 202068 from the president of the american college of theriogenologists as i prepare this missive, i realize that it is not to inform, but instead to share uncertainty. an annual conference, with a schedule of wonderful topics to be delivered by an incredible cast of speakers, at a great venue; that may or may not occur. a certifying exam that may be administered to the candidates over the internet or a combination with some on-site. additionally, some candidates may choose to wait until next year. like the rest of the country or more correctly the world, we have to wait. wait to see if the wave of illness and death subsides. wait to see if things return to normal or at least normal enough to allow travel and some semblance of our previous routines. all of this said, important work for the college is continuing. at our last board meeting the maintenance of certification and training and credentialing committees both provided reports. board members continue to meet with respective subcommittees dealing with issues such as outreach and engagement, updates needed for the gig, evaluation and tracking of theriogenology sub-competencies, and work to enhance the quality of the clinical theriogenology journal. with respect to our conference, our executive director, dr. charles franz is continuing preparation for the meeting. the executive board discussed contingencies relating to the administration of the exam. specifically, dr. brian whitlock, the chairperson for the examination committee reported on options with respect to provision of a means to offer this year’s exam to those that choose to sit for it this year, but will be unable to travel to pittsburgh. this alone will represent an enormous effort. considerations include the timeline at which an exam is delivered and the identification of on-site exam proctors. by-words for us all during this time are obviously patience, flexibility, and perhaps optimism. we also, need to consider the challenges and problems of our organization in the context of what so many others are dealing with; remembering that many are facing the loss of loved ones, not to mention the economic hardships that many are and will face. that pretty much summarizes what i can report, so i am going to change tact and share some of my thoughts on springtime and getting older. i’m what is termed an academic clinician. i am currently working at a veterinary college that is strangely missing all or at least most of its students. my wife used to repeat the oft-quoted statement that college towns would be the very best place to live – except for all the students. these words haunt me now, as it seems very sad without them. we are still seeing emergencies and in fact continuing a fair amount of farm work as it seems much of what theriogenologists consider routine is in fact crucial to the agricultural community. so, here at auburn, we continue to ply our trade and do so with some great residents, technicians and a small group of student residents (4th years that were on salary for night technician duties providing a “loophole” that allows them to stay). i was called out late a couple weeks back and unable to go back to sleep, i penned the following. perhaps it will provide a diversion from thoughts of this very strange time we are experiencing. its springtime and i feel older. to many i know that sounds odd, antithetical, and just plain backwards from our usual thoughts of this wonderful season filled with conception, newness, birth. so, i guess i’m a contrarian. perhaps, not by nature, but instead from experience. after fifteen years of practice i joined the faculty at mississippi state and i remember thinking of the old adage; “if you want to stay young, surround yourself with young people”. well i have to tell you that for me, being around veterinary students made me feel old, in fact, ancient, and certainly “behind the times”. as time went on, i reached first the age of the average students’ parents, and then of late the age of their grandparents fully realizing this when approached by a student to tell me that i had “taught her dad”. well you get the point. i think for me the newness; the hustle and bustle of springtime seems to have that same effect; that of feeling my age. clinical theriogenology • volume 12 number 2 • june 2020 69 and so this, my 42nd spring as a veterinarian and i move very slowly at 4:30 in the morning and i’m awful tired when suppertime rolls around. i don’t have as much “night call” as i used to, but when i do roll out of the bed to answer the phone, it feels as though i am coming out of some kind of coma, perhaps the kind that resulted from a bad automobile accident in which a lot of your bones were crushed. it is usually best to make some coffee while i dress and wisest still to pre-medicate (your nsaid of choice). even down south (auburn, alabama), it is cool late at night and that helps. the uncertainty that i felt decades ago as i would drive toward the clinic or a farm, has been replaced by the certainty that over the years, i have seen pretty much everything. i arrive and everything goes well. in fact, i am really just an observer as students and a resident handle the work. toward the end of that late night emergency, when after finally becoming completely awake, awake to the point that you realize that it will be hard to go back to sleep (when you finally can) and when you think about how tired you will be tomorrow (actually today). i stop for a moment and look for a minute at the product of our work. i am guessing that if you have read this far, an image comes to mind. perhaps it is the tired cow, a heifer only moments ago, reaching down to lick the flank of a still wet calf. maybe it is a mare softly nickering to a wobbly foal attempting to rise for the first time or that basket full of puppies. that is when i smile, a smile that emanates from my very being and i realize how very blessed that i am to have the wonderful life i’ve had and still be able to enjoy this wonderful profession. of course, i still feel very tired and very, very old. all the best and please stay safe, sincerely, richard hopper act president 2019-2020 clinical theriogenology • volume 12 number 2 • june 202070 from the president of the theriogenology foundation the global strategy for 2020 has become survival. in february, while china was already facing hundreds of fatalities from a coronavirus infection-associated pneumonia, eight of us were eagerly packing our bags to head to detroit for the tf strategic planning meeting. one day before departure, a midwestern snowstorm bore down on michigan, grounding our group. drs. hopper and floyd arranged a plan b at auburn, where a zoom meeting brought the great thinkers from michigan, alabama, mississippi and boston together through technology. almost. my lack of preparedness to work remotely became evident. as displayed in the photo, i was the keeper of all the easel-paper charts and talking points which had been neatly packaged in a fedex tube for travel. camera on my computer for zoom hookup? not a chance. audio through the pc? nope. powerpoint for the team? negative. nestled in the mecca of boston high tech, i instead sent cellphone shots of the papers that i had scotch-taped around my home office, fought with the ge speaker phone button on my landline, and connected to our patiently waiting strategists. after lamenting how weather had forced us to shelter-in-place, we spent the next 7 hours glued to our chairs discussing the survival of the theriogenology foundation. truly. a few key events required us to change the course of the theriogenology foundation. dr. charles franz gave notice of his resignation as tf executive director in december. in january at the combined board meeting, a proposal for act, sft, and tf to equally share the funding of a new ed position was rejected. since the tf alone could not financially sustain this position, the tf had to be restructured. ultimately, the strategic planning committee recommended a foundation which will primarily fund and administer the core student support functions of travel, research, and education. all the great student opportunities such as the student chapter grants, externships and residencies are still in place. the theriogenology foundation will cease to administer the working dog project, and has transferred ownership, assets, and control to the darwin’s ark foundation (www.darwinsark.org). by march, it was a virus, not terrorism or the weather, that was grounding planes and halting businesses. years ago, we defined the theriogenology foundation through our mission statement as “a global resource that supports education and research in reproductive medicine; ensuring that future generations of animals continue to enrich our lives through service, companionship, and food for a growing human population while conserving our natural resources.” i still believe that veterinarians are central to the immense challenges of animal productivity, food safety/security and sustainability. but for now, a smaller and more narrowly focused foundation will hunker down to protect the ability of our students to learn and expand their knowledge and competency in theriogenology. our proposed new mission statement is: the theriogenology foundation supports education and research in animal reproductive medicine. period. as for me, i either need to sign up at the local community college for computer education or finally retire to the rocking chair on the front porch. i’ll let you know in july, either in person or via zoom. stay well. anita migday clinical theriogenology • volume 12 number 2 • june 2020 71 http://www.darwinsark.org/ dr. david e. bartlett award for lifetime achievement in theriogenology dr. terry blanchard dr. terry blanchard studied at k-state and received his doctorate in veterinary medicine in 1976. he was in a mixed animal practice at smith animal clinic, inc., crown, point, indiana, for a year after graduation. dr. blanchard joined the u.s. peace corps in the philippines where he taught pre-veterinary students and performed research on reproduction in water buffaloes from 1977-1979. dr. blanchard wrote a thesis on the effects of anabolic steroids on stallion testicular function while working on his master’s degree. he received his master’s degree from the university of missouri in 1983. he completed his residency in large animal reproduction at the university of pennsylvania, new bolton center in 1981, and then joined the faculty at the university of missouri-columbia. he became certified as a diplomate in the american college of theriogenologists in 1982. he rejoined the faculty at the university of pennsylvania from 19841986. dr. blanchard then joined the faculty at texas a&m university from 1986-2005. he moved to lexington, kentucky, as a resident veterinarian at hill ‘n’ dale farm from 2005-2007, and rejoined the faculty at texas a&m university in 2008. he has been a faculty advisor to approximately 40 resident/graduate students. dr. blanchard is the co-author and editor of two textbooks on stallion reproduction and one textbook on equine reproduction, 18 book chapters, and over 130 referenced journal publications. dr. blanchard has served as secretary of the act examining committee and was recognized in 2006 as the act as the theriogenologist of the year. dr. blanchard is currently a member of american veterinary medical association, american association of equine practitioners, texas equine veterinary association, kansas veterinary medical association, society for theriogenology and american college of theriogenologists. dr. blanchard is married to kathleen. he has four daughters: shanna, holly, katie and angela; and two sons: john and michael. 2020 proficiency awards the "proficiency in theriogenology award”, sponsored by the society for theriogenology, is a student award to be given annually to one senior at each veterinary school/college in the united states and canada. congratulations to all award recipients (as of 4/24/2020)! jenna ward (texas a&m university) morgan rea (virginia-maryland college) samantha nieves (university of california-davis) chet mcclure (university of illinois) jessica bottger (oregon state university) sarah stanley (university of georgia) anum ahmed (kansas state university) brooke unruh (purdue university) rachel wilson (mississippi state university) ava kent (ross university) tiffany mavromatis (louisiana state university) sarah randell (university of florida) clinical theriogenology • volume 12 number 2 • june 202072 theriogenologist of the year dr. claire card dr. claire card is a graduate of cornell university where she earned 3 degrees (bsc, dvm, and phd) and was a post doctoral fellow. she is a full professor and clinician scientist at the wcvm, university of saskatchewan. she is an equine theriogenologist at the vet med centre and has an active research program. she instructs veterinary students in equine and companion animal reproduction, physiology and sustainable development. she educates veterinary students to be “woke” to the challenges of racism, inequality and climate change and the role the veterinary profession has in the solutions. she has held many leadership positions in veterinary medicine and is a past -president of the act, past board member of the saskatchewan vma, sft and tf. she is currently a board member of and volunteers for veterinarians without borders (vwb) canada and for the past 12 years has been working in a project in rural uganda focussed on impoverished hiv aids effected women, to improve their livelihoods. she has a wonderful partner peter barnacle who has supported her in being an outlier, having no boundaries and in loving her large blended family of 6 children, 4 grandchildren and 3 ugandans. in her spare time she is a 4th dan black belt in tae kwondo and enjoys running, reading, cooking, nature and travel. call for nominations 2021 theriogenologist of the year award nomination due date november 15, 2020. the american college of theriogenologists is seeking nominations for “theriogenologist of the year,” an award which recognizes a theriogenologist for recent, outstanding contribution(s) to the discipline of theriogenology. the award is accompanied by a plaque and monetary honorarium of $1,500. 2021 nomination form qualifications include: a. act diplomate in good standing b. has made recent, outstanding contribution(s) to the discipline of theriogenology. 1. in most cases the contribution(s) will have been made within the past 5 year period. 2. outstanding contribution(s) may take many forms and will be judged in part on furthering awareness and understanding of the discipline of theriogenology and theriogenologists, including mentorship and training. 3. note: the award is not intended to recognize career achievement. clinical theriogenology • volume 12 number 2 • june 2020 73 https://www.theriogenology.org/page/toynomination 2021 bartlett award nominations sought nominations are now being sought for the 2021 david e. bartlett award for lifetime achievement in theriogenology, presented annually to a distinguished individual who has made important contributions to the field of theriogenology. the award intended to cement the efforts of the society and college toward common goals in animal reproduction, to reward and inspire excellence, improve the visibility of theriogenology, and to annually recognize the efforts of the founders particularly honors dr. david e. bartlett, first president of the college. among his many accomplishments, dr. bartlett was responsible for deriving the terms “theriogenology” and “theriogenologists,” and was instrumental in gaining hard-won avma recognition for the american college of theriogenologists in 1971. the 2021 david e. bartlett award for lifetime achievement in theriogenology will be presented during the annual therio conference in omaha, nebraska. the recipient will be notified in advance, allowing family and friends to be on hand. the honoree will address those in attendance and will have the option of submitting a contribution to the conference proceedings. the honoree will receive $1,000 cash, an engraved statue of nandi, and complimentary registration, hotel accommodations and transportation to the 2021 conference. please submit the accompanying preliminary entry form and nominate an individual to add to the list of preeminent recipients. entry forms must be received no later than january 4, 2021. nominations may also be made online at http://www.therio.org/bartlettawardform. you must be signed in as an sft member to access the form. it is located under the therio conference page and also under the bartlett award recipient list on the sft website. 2021 dr. david e. bartlett award for lifetime achievement in theriogenology preliminary entry form sponsored by the theriogenology foundation in cooperation with the society for theriogenology & american college of theriogenologists purpose: to reward a distinguished theriogenologist who has made important contributions to the field of theriogenology. nominee: full address: current employment and position: home phone: business phone: fax: email: provide a short description of nominee’s qualifications for this award. use a separate sheet of paper containing a maximum 350-word narrative bio, highlighting nominee’s key accomplishments. nominated by: full address: home phone: business phone: fax: email: nominations must be received by january 4, 2021 submit online at: http://www.therio.org/bartlettawardform or mail to: theriogenology foundation, bartlett award 761 tiger oak drive pike road, al 36064-3063 clinical theriogenology • volume 12 number 2 • june 202074 http://www.therio.org/bartlettawardform http://www.therio.org/bartlettawardform therio foundation 2020 conference fundraising events tf silent auction 25.0 the passing along of boutique items from colleague-tocolleague through the silent auction has become a signature event of our annual conference. we are limiting the number of items to 25 again this year as it worked out well last year! here are some suggestions for items: *educational items (text books, etc.) *arts and crafts *liquor/wine *sporting events *veterinary equipment once you decide on what your priceless creation or generous gift will be, please e-mail the attached form along with pictures of the item(s) by june 15th, 2020 to: jgfloydjr@gmail.com. get your donation into the spotlight by submitting early…….the final item list will be posted on the website by july 1 so the membership has a month to start saving their dollars to bid! after 25 items have been selected, we will respectfully ask you to hold your gift until 2021! no on-site donations can be accepted this year. thank you for your continued support of the theriogenology foundation silent auction!!! we look forward to seeing you in pittsburgh!! awards dinner and story night this year’s live auction will be held in conjunction with the first annual veterinarian story night. purchase your ticket at: therio.org. sealed bids for live and silent auction items are accepted until july 23. items will be posted online and updated as submitted. please complete the sealed form on the tf website at: http:// www.theriofoundation.org/? page=sealedbidform. craft beer tasting fundraiser for the theriogenology foundation during the opening session reception wednesday evening, july 22 or the awards dinner and story night reception, july 24, attendees will have the chance to experience some locally brewed craft beer and help the theriogenology foundation at the same time. for $40 you will receive four samples of beer. once you have sampled and chosen your favorite, you will get a full serving of that beer. proceeds will benefit the theriogenology foundation. tickets may be used at the event of your choice. clinical theriogenology • volume 12 number 2 • june 2020 75 mailto:jgfloydjr@gmail.com http://www.theriofoundation.org/?page=sealedbidform http://www.theriofoundation.org/?page=sealedbidform http://www.theriofoundation.org/?page=sealedbidform https://www.therio.org/page/2020therioconf thank you! a huge thank you to the following individuals for all their efforts in planning the 2020 conference! annual conference program chair dr. jack smith equine track program chair dr. marco coutinho da silva production animal track program chair dr. heath king small animal track program chair dr. cheryl lopate equine symposium chairs drs. marco coutinho da silva and pat mccue student equine wet lab chair dr. marco coutinho da silva educators’ forum chair dr. hilari french silent auction chairs drs. jim floyd and will schultz live auction chair dr. jim floyd cbest chair dr. marty greer thank you to our 2020 sponsors! (as of 4/24/20) platinum plus sponsor idexx platinum sponsors lane manufacturing, inc. merck animal health rood & riddle equine hospital and veterinary pharmacy universal imaging, inc. gold sponsors sonosite-a fujifilm company silver sponsors american college of theriogenologists theriogenology foundation bronze sponsors michelson prize & grants the american kennel club, inc. breeder's choice christian veterinary mission e.i. medical imaging elsevier fertility technology resources idexx jorgensen laboratories juniper systems karl storz veterinary endoscopy america, inc. lane manufacturing, inc. merck animal health mila international, inc. multimin usa minitube usa professional embryo transfer supply, inc. sonosite-a fujifilm company universal imaging, inc. puppywarmer thank you to our 2020 exhibitors! (as of 4/24/2020) clinical theriogenology • volume 12 number 2 • june 202076 wednesday, july 22 act educators’ forum opening session abstracts opening session reception equine symposium and art workshop (additional registration required) opening session abstracts identifying risk factors for mastitis in postpartum bitches jennine lection fungal growth is more likely to be affected by hormones in equine uterine isolates compared with isolates from non-reproductive sites jennine lection a novel method to reduce egg-laying in companion avian species using the hen as a model sandra ayres sperm-bound antisperm antibodies are associated with poor cryosurvival of stallion spermatozoa maria ferrer effect of energy substrates on cool-stored stallion spermatozoa camilo hernandez-aviles effects of recent feeding on canine serum progesterone william whitler the effect of slide type to evaluate motility parameters of frozen-thawed equine spermatozoa using casa paul loomis effect of different glycerol concentrations on a new extender for dog freezing semen rogerio souza thursday, july 23 bartlett award and address terry blanchard theriogenologist of the year award and address claire card dr. jerry rains memorial abstract competition sft business meeting act business meeting student case and research presentations 2020 annual conference agenda clinical theriogenology • volume 12 number 2 • june 2020 77 student/mentor reception opening reception in the exhibit hall student quiz bowl student equine wet lab dr. jerry rains memorial abstract competition sponsored by merck animal health sperm protein reactive with anti-sperm antibody (sprasa) is immunoexpressed in equine primordial, primary, secondary, and tertiary follicles brynley cozzi a comparison of two methods to induce the acrosome reaction in stallion sperm camilo hernandez-aviles effects of uterine-sperm contact time on the degree of endometrial inflammation in mares bred by deep horn rectally guided artificial insemination sofia kovacsy investigation of inflammatory proteins as novel diagnostic biomarkers for endometritis in the mare jennine lection oxytocin-induced secretion of 13,14-dihydro-15-keto-prostaglandin f2α (pgfm) in mares with prolonged corpus luteum (cl) function brendan sarnecky pharmacodynamics of clomiphene citrate in cyclic mares cory anderson the effect of mycobacterium cell wall fraction on histological, immunological, and clinical parameters of equine postpartum involution carleigh fedorka transcriptomic analysis of equine chorioallantois reveals key regulators and pathways involved in ascending placentitis hossam el-sheikh ali student case and research presentations monozygotic twins in a thoroughbred mare bred by live-cover katelyn kimble pregnancy toxemia in the bitch kalie beckers dystocia due to a congenital hydrocephalus in a miniature dachshund colleen kutzler mummification of fetus and abortion secondary to umbilical cord torsion rachel doenges left testicular rupture in a red angus bull sara dietz successful treatment of ascending placentitis in a thoroughbred mare hannah neer clinical theriogenology • volume 12 number 2 • june 202078 alfaxalone cross-reactivity affecting progesterone concentrations in cats joshua trumble influence of extender, temperature, and equilibration time on post-thaw sperm motility in ram semen (ovis aries) peri pelletier sonographic appearance of the late gestation sheep fetal intestine and kidney elizabeth frieden assessing the efficacy of deslorelin acetate in advancing ovulation for use in timed artificial insemination protocols in goats sarah legg friday, july 24 scientific abstract presentations awards dinner reception awards dinner, story night, and live auction plenary session the future is coming! new technologies being applied to reproduction in animals and man katrin hinrichs in search of stem cell efficacy – immunogenicity matters ashlee watts mixed species abstracts comparison of glucose and lactate concentrations between healthy equine and mule foals and of an automated laboratory analyzer and the accutrend® plus system yatta boakari effect of ejaculation frequency, prostaglandin f-2α, and cold storage on canine semen yield and post-thaw quality kendra zelachowski the seroprevalence of brucellosis in hog hunting dogs, imported dogs, and dogs presenting for routine spay and neuter in the southeastern united states alyssa helms effect of injection of synamid® (polyacrylamide hydrogel) into the reproductive tract of mares stephanie walbornn uterine expression of leptin, rhoa and rho-associated kinases in bitches with primary uterine inertia and with obstructive dystocia orsolya balogh evaluating the efficacy of a silicone y-design intrauterine device as a horse contraceptive in a captive breeding trial cory anderson description of submissions diagnosed serologically positive for canine brucellosis at the cornell animal health diagnostic center (2014-2019) soon hon cheong clinical theriogenology • volume 12 number 2 • june 2020 79 the association of intravaginal progesterone-releasing device and estradiol-17β is equivalent to the combination of estradiol-17β and long-acting progesterone to synchronize acyclic embryo surrogate mares lorenzo segabinazzi production animal abstracts effect of age on follicle stimulating hormone receptor expression in ovine endometrium hayder habeeb acupuncture reduces milk n-acetyl beta-d-glucosaminidase in dairy cows with mastitis elise ryan dominant follicle removal prior to superovulation taylor mittleider the effects of zinc on the maturation and fertilization of bovine oocytes brianna price bovine sperm morphology analysis varies greatly by reader ashley reeves effects of motility activation and cryopreservation on the glycocalyx of fish sperm bryan blawut platelet-rich plasma reduces endometrial macrophages in postpartum beef heifers michelle kutzler ultra-fast cooling reduced oxidative stress in vitrified bovine oocytes soon hon cheong equine abstracts cooled-transported epididymides for donkey semen cryopreservation yamilka lago-alvarez intrauterine infusion of platelet-rich or -poor plasma to modulate persistent breeding-induced endometritis (pbie) in embryo donor mares lorenzo segabinazzi effect of pyruvate on the rate of lactate-induced spontaneous acrosome reaction of stallion sperm luisa ramirez-agamez streptococcus equi subspecies zooepidemicus endometritis in mares: culture, cytology and antimicrobial susceptibility tests christina divine comparison of pregnancy rates in mares inseminated with frozen semen preplus post-ovulation versus postovulation only in a commercial breeding program christian bisiau use of serum amyloid a and other inflammatory markers to monitor the inflammatory response in mares with periparturient complications mette christoffersen clinical effects of pre-breeding intrauterine prp in the mare lauren pasch clinical theriogenology • volume 12 number 2 • june 202080 expression of prostaglandin e2 and oxytocin receptors in the stallion accessory sex glands robyn ellerbrock case reports fungal endometritis in a mare diagnosed during the mid-embryo period of gestation allan gunn pyometra associated with hyperammonemia in a mare daniela orellana gestational ultrasound diagnosis of omphalocele in a toggenburg goat fetus bret mcnabb presence of tritrichomonas foetus in the urethra of a chronically infected bull jessica rush hypertestosteronism in an intact female alpaca secondary to an interstitial cell tumor katelyn waters suspected spermatic cord torsion with fertility testing and post-surgical findings in a dog audrey kelleman repair of a full thickness uterine tear via iatrogenic uterine prolapse in an anesthetized thoroughbred broodmare justin mcnaughten therapy and evaluation of early embryonic loss in a subfertile bitch hannah smith abstract poster presentations generation of hormone-responsive organoids from fresh and cryopreserved equine endometrium: a comparison between domestic and endangered przewalski’s mares riley thompson semen parameters and fertility of cooled stallion semen extended with sodium caseinate and phosphocaseinate based extenders giorgia podico sperm parameters after cushion centrifugation of stallion cooled-stored semen claire kaplan evaluating the impact of a systemic treatment protocol on uterine biopsy grade and uterine microbiome in mares with pre-treatment kenney grades of iib-iii adam bassett effect of platelet rich plasma lysate (prpl) and fibroblast growth factor 2 (fgf2) on sperm motility in stallions fabio pinaffi prevalence of and potential impact on fertility of pars pituitary intermedia dysfunction in a thoroughbred broodmare population in england sarah moore breeding characteristics of stallions standing at a commercial thoroughbred stud in central kentucky charles scoggin clinical theriogenology • volume 12 number 2 • june 2020 81 kisspeptin (kp) and rfamide-related peptide 3 (rfrp-3) neurons in the hypothalamus of adult and juvenile dairy cattle; expression of oestrogen receptor alpha (erα), and inputs to gonadotrophin releasing hormone (gnrh) neurons allan gunn oviductal insemination by hysteroscopic hydrotubation in mares: a preliminary investigation yuji inoue effect of oxytocin administration during mid diestrus on early pregnancy in the thoroughbred mare justin mcnaughten using a combination of estradiol cypionate (ecp) and altrenogest to control the timing of ovulation in mares william whitler prevalence of malignancy in canine mammary masses in a population of shelter dogs sharon pindar use of intravaginal progesterone releasing device (prid™ delta) to hasten first ovulation in mares: side effects and pregnancy rates on one commercial thoroughbred farm david trundell individual variation of frozen-thawed sperm from stallions to survive dilution and cooling after thawing kristin klohonatz comparison of pregnancy rates using a new versus a once-used cidr in a 7-day estrus synchronization protocol during the breeding season in ewes dinesh dadarwal retrospective review of uterine prolapse in mares ghislaine dujovne small animal track introduction to stem sells – what they are, how to grow them, how to make them do what we want them to diane carlisle stem cells and female infertility jonathan tilly using pluripotent stem cells to treat male factor infertility charles easley production animal track postpartum anestrus of beef cows: importance and evaluation bob larson using the pregnancy analytics app to add value to pregnancy diagnosis of beef herds bob larson bull reproductive development and sperm production enhancement ahmed tibary clinical theriogenology • volume 12 number 2 • june 202082 equine track michelle leblanc memorial equine lectures embryo-maternal communication during the establishment of equine pregnancy; the role of mare age in early pregnancy loss tom stout reproductive endocrinology and immunology of the pregnant mare barry ball saturday, july 25 so you want to become a diplomate? career paths in theriogenology student case poster presentations small animal track current state of embryo transfer in dogs karine reynaud current state of ivm/ivf in dogs karine reynaud “this stud's a dud!”canine semen evaluation protocols and pitfalls joann randall “boys in a box”-chilled and frozen canine semen shipments joann randall evaluation of canine nomograph as a tool to improve puppy immunization outcomes laurie larson pedigree analysis and genetic counseling cynthia o'connor genetic testing and inbreeding coefficients cynthia o'connor production animal track fetal programming: maternal-fetal interactions caleb lemley fetal programming: postnatal performance caleb lemley physiology and reproductive techniques for the whitetail doe jason coe advanced reproduction and semen collection in whitetail deer jason coe urogenital surgery in camelids ahmed tibary postpartum factors influencing fertility james ferguson clinical theriogenology • volume 12 number 2 • june 2020 83 dairy reproductive management programs james ferguson equine track genomics and transcriptomics of the normal and abnormal equine placenta: a better understanding of late gestational function and dysfunction barry ball cervix and myometrium: role in preterm and normal term birth in the mare barry ball managing recipient mares to optimize pregnancy rates in an equine art program tom stout factors that threaten late pregnancy: how can we detect them? margo macpherson what do we know about medication in equine pregnancy? margo macpherson management of dystocia pat mccue clinical cases discussion margo macpherson, pat mccue, tom stout student case and research poster presentations anasarca in a neonatal bulldog puppy secondary to a ventricular septal defect claudia acevedo halter stallion anejaculation due to dorsal displacement of the soft palate breanthony baker priapism in a quarter horse gelding hannah carter evaluating changes in milk ph, milk protein, and udder development in the late pregnant ewe and effects on lamb serum total protein concentrations mary clapham canine herpes virus in a breeding kennel of wiredhaired dachshund jordan farrell a retrospective analysis on determinants of litter size in a colony of working dogs jordan farrell foal born using intracytoplasmic sperm injection to a friesian mare with unexplained subfertility alexandra grillos granulosa cell tumor and prolonged estrus in a boxer bitch christina havrila detection of tritrichomonas foetus in a beef herd cecilia hernandez clinical theriogenology • volume 12 number 2 • june 202084 genetic causes of golden retriever congenital hypomyelinating polyneuropathy blair hooser delayed uterine prolapse in a thoroughbred mare with retained fetal membranes joanna kania the importance of breeding management in abnormal estrous cycles: a case study tokaj kozak sertoli cell tumor in an alpaca samantha mccarter scrotal hydrocele in an angus bull maryanna mcclure bilateral ovarian adenocarcinoma in a geriatric shih tzu miranda senft bilateral cystadenoma with a granulosa cell tumor in a bernese mountain dog miranda senft a third degree rectovaginal joshua trumble cervical adenoma and excisional biopsy in a spayed labrador retriever bitch julia zuercher please note that schedule is subject to change. (titles may appear different in the conference issue of clinical theriogenology.) student equine wet lab thursday, july 23, 2020 sponsored by sonosite-a fujifilm company the goal of this one-day course is to provide veterinary students with an opportunity to learn about common procedures performed in equine clinical practice. laboratory sessions will provide hands-on opportunities for semen collection, semen evaluation and processing, palpation and ultrasonography of the mare reproductive tract, and collection and interpretation of common uterine diagnostics. semen collection and evaluation palpation and ultrasound uterine culture, cytology, and artificial insemination evaluation of uterine cytology and biopsy clinical theriogenology • volume 12 number 2 • june 2020 85 special events equine symposium & art workshop sponsored by universal imaging, inc. act educators’ forum sponsored by theriogenology foundation opening session abstracts student equine wet lab sponsored by sonosite-a fujifilm company dr. david e. bartlett award for lifetime achievement in theriogenology theriogenologist of the year award sponsored by universal imaging, inc. dr. jerry rains memorial abstract competition sponsored by merck animal health sft business meeting act business meeting student case presentations exhibit hall opening reception theriogenology foundation craft beer tasting student quiz bowl sponsored by merck animal health student t-shirt contest scoty award plenary speakers drs. katrin hinrichs and ashlee watts sponsored by american college of theriogenologists canine breeder excellence seminar track theriogenology foundation silent auction annual awards dinner reception annual awards dinner sponsored by merck animal health dr. john steiner award for practitioner excellence theriogenology foundation veterinarian story night and live auction equine symposium and art workshop wednesday, july 22, 2020 sponsored by universal imaging, inc. the workshop will include didactic sessions in the morning and a wet-lab in the afternoon. didactic sessions will provide participants with the most relevant and practical information on equine in-vitro embryo production. experts will discuss factors that affect oocyte yield, quality and developmental potential during collection, transport and preservation. in the laboratory participants will have hands-on opportunities in all the steps involved in oocyte collection, evaluation and transport. participants will practice transvaginal follicular aspirations in live mares, evaluation and handling of oocytes and packaging of oocytes for shipment. participants must be familiar with equine ovarian palpation to participate. collection and transport of equine oocytes for in vitro embryo production tom stout opu and oocyte transport: clinical considerations rob foss diet and another influencers of oocyte developmental potential elaine carnevale embryo and oocyte preservation katrin hinrichs art workshop oocyte aspiration oocyte evaluation and transport additional registration required. clinical theriogenology • volume 12 number 2 • june 202086 dr. kristina baltutis dr. kristina baltutis is a small animal practitioner in central north carolina. she has served on the sft board of directors since 2017, when she graduated from north carolina state university's college of veterinary medicine. during this time, dr. baltutis has worked diligently on expanding and coordinating student initiatives including sft's presence at the annual savma symposium, leading discussion sessions for sft students and faculty at the sft conference, coordinating the annual student-mentor discussion at the sft conference, and starting a quarterly newsletter for sft students. recognizing that our students are our future, she is passionate about enabling opportunities for our students so they may fulfill their goals as members of the society for theriogenology. dr. baltutis has worked in general practice, emergency practice, and as a relief vet. she owns a small animal mobile practice with an emphasis on canine theriogenology. outside of veterinary medicine, dr. baltutis enjoys spending time with her border collie, golden retrievers, cats, chinchilla, and spouse. dr. g. reed holyoak g. reed holyoak is a professor, holds the bullock equine reproduction endowed professorship in the veterinary clinical sciences department of the center for veterinary health sciences at oklahoma state university. he received his b.s. in animal science in 1983 and m.s. in animal reproduction in 1984 from brigham young university before earning his dvm from washington state university in 1988 and phd from university of kentucky in 1992. he became a diplomate of the act in 2000 via the alternate route, served on the act examination committee from 2005 to 2011 (being chair in 2010) and served as secretary to the act board 2012 to 2016, act vice-president 2017, president elect 2018, president 2019 and is currently past-president of the act. holyoak’s current research interests include reproductive infectious diseases, including the reproductive tract normal microbiota, the development and implementation of an equine intrauterine device for use in feral horses, and the integration of acupuncture within theriogenology. he has published on equine, bovine, and canine reproduction topics. reed’s aspiration within the sft is to be of useful service. dr. riley thompson dr. thompson received her bs in agriculture from tennessee tech university in 2012 and graduated with her dvm from the university of tennessee in 2017. during her dvm education, dr. thompson served as the scaaep co-president, theriogenology club secretary, and class of 2017 treasurer. she also was inducted into the phi zeta veterinary honor society and was awarded the utcvm outstanding senior theriogenology student award. she is graduating with her phd in a co-appointment between the university of tennessee and smithsonian conservation biology institute (scbi) in may 2020 where she focused on cryopreservation and organoid culture of domestic and endangered equid endometrial tissue. she will begin a post-doctoral fellowship in june 2020 at colorado state university with dr. fiona hollinshead developing in vitro technologies for evaluating endometrium and generating extracellular vesicles. outside of work, dr. thompson enjoys reading, baking, and hiking with her dachshund, sherlock. 2020 board of directors candidates clinical theriogenology • volume 12 number 2 • june 2020 87 neoplastic considerations for spaying and neutering dogs neoplastic considerations for spaying and neutering dogs evan sones animal cancer care clinic, ft. lauderdale, fl abstract neutering of companion animals is common practice in the united states and many other parts of the world. there is growing concern that the uniform recommendation of neutering is detrimental to the pet population by affecting normal functions of other body systems. this may predispose these animals to disease, including, but not limited to, neoplastic conditions. although the mechanisms of these changes are largely unknown, there is a growing number of studies showing a correlation between cancer and neuter status. a review of these articles, as provided here, can be beneficial to inform practitioners, enabling a more thorough discussion of spay and neuter with their clients. keywords: spay, neuter, neoplasia, cancer introduction spay and neuter have long been the standard of care recommendations for pet dogs and cats in the us. two common reasons given for this instruction are behavioral modification and population control. however, gonadectomy and the subsequent lack of sex hormones can impact many other processes in the body and may influence development of other diseases, including cancer. the mechanism by which sex hormones, or lack thereof, influence development or progression of cancer is not always clear. hormone receptors are present in a number of canine tumors, including mammary tumors,1-6 perianal gland tumors7and meningiomas.8 the level of hormone receptors may also influence outcome in treatment of meningiomas.9 removing the sex hormone influence can help to improve outcome in perianal gland tumors10 and tumors of the reproductive tract.11,12 the opposite effect can be seen when administering exogenous hormones, that can lead to development of tumors in dogs and cats.13,14 the influence of sex hormone exposure and development of mammary tumors is well known. if a dog is spayed prior to the first estrous cycle, the risk of mammary cancer is only 0.5%. this risk increases to 8% if spayed after the first estrus, then increasing to 26% after the second.15 there was not a significant decrease in risk of developing mammary cancer if spayed after the second estrus in this original study, although there was some benefit found in later studies.15-18 spay after development of mammary carcinoma does not seem to influence the outcome for dogs, except in certain subsets.19 other cancers of the reproductive tract can be reduced significantly by spay and neuter. removing the gonad eliminates the risk of cancer development for uterine, ovarian, and testicular tumors. vaginal leiomyomas are an uncommon, benign tumor of intact female dogs, and rarely occurs in spayed dogs. spay at the time of leiomyoma removal decreases the risk of recurrence after even incomplete removal.11,12 prostatic carcinoma is an uncommon cancer in male dogs.20-23 there has been an increase in the risk of developing prostatic cancer in neutered dogs. this increase in risk ranges from 2 to 8 times that of intact stud dogs.24,25 lymphoma is a very common cancer in dogs.26,27 a large, multi-institutional retrospective study using the veterinary medical database compared ~ 15,000 canine lymphoma patients with 1.2 million dogs from the general population. this study identified a decreased risk of developing lymphoma in intact female dogs, about half the likelihood of spayed females, intact males, or neutered males.28 transitional cell carcinoma of the urinary bladder has a higher incidence in female dogs, with a risk ratio ranging from 1.71 to 1.91:1 of females to males.29-31 the underlying cause of this 239 clinical theriogenology • volume 11, number 3 • september 2019 increased risk in females is not known to be hormonally influenced. however, 1 study reported increased risk in both sexes with gonadectomy.26 also, the risk of transitional cell carcinoma of the prostate is higher in neutered dogs.25 early, smaller studies on canine osteosarcoma showed some variation in sex predilection, with some indicating males have an increased risk and some indicating females.31-36 however, a much larger review of 1,775 patients did not show any sex predilection.37 neutering does carry some increased risk of developing osteosarcoma in 2 studies.8 the first found a 1.9 times increased risk in spayed females and 1.4 times in neutered males.38 the second found an increased risk of 1.3 fold in neutered compared to intact male dogs.39 hemangiosarcoma was more likely in spayed females, which were about 2 times more likely to develop hemangiosarcoma compared to intact females.40 this association was also present for cardiac tumors, with spayed females having a 4 times higher relative risk compared to intact females.41 this study also found diagnosis of cardiac hemangiosarcoma was 5 times greater in spayed females.41 although not as robust, there was also an increased risk of 1.6 for heart tumors in neutered males when compared to their intact counterparts.41 there have been a number of studies attempting to determine any relationship between gonadectomy and the development of cancer in specific breeds. one study reviewed 683 rottweilers and found an increased risk of osteosarcoma if the dog was neutered prior to 12 months of age. this held true for both male and female rottweilers.41 golden retrievers have been evaluated due to the high incidence of cancer in this breed. in a group of 759 golden retrievers, females spayed after 12 months of age had an increased risk of hemangiosarcoma, 4 times greater than females spayed before 12 months or intact females.42 this was not the case for males in the same study.42 lymphoma is also more common in spayed and neutered golden retrievers, with an increased risk of 3 4 times.42 in male golden retrievers castrated before 1 year of age, the risk for developing lymphoma was increased by 3 times.42 lastly, golden retrievers were evaluated for the risk of developing mast cell tumor in relation to reproductive status. there was an increased incidence of mast cell tumor in female golden retrievers that were spayed prior to first year at 2.3% and after first year at 5.7%, when compared to intact females at 0%.42 there was no difference in the incidence between castrated and intact males in this same study.42 a final breed specific study evaluated risk and age of onset of cancer in vizslas. this study found a similar increased risk to golden retrievers of developing lymphoma, with an increase of 3 4 times in castrated or spayed vizslas.42,43 the risk of developing a mast cell tumor was also higher, increased by ~ 2 4 times than that of intact vizslas.43 these mast cell tumors also developed at an earlier age.43 gonadectomy potentially has some influence on lifespan in dogs. in reviewing > 40,000 dogs in the us, there was an increase in average lifespan from 7.9 to 9.4 years with gonadectomy. the benefit was higher in females with an increase in life expectancy of 26%, and only 14% in males.44 in sterilized dogs, in this study, transitional cell carcinoma, osteosarcoma, mast cell tumors, and lymphoma were all more common; mammary tumors were less common; and there was no difference in the incidence of melanoma, squamous cell carcinoma, and prostate cancer.44 discussion information presented here must be taken with the proverbial “grain of salt”. these studies show an association between gonadectomy and a change in the risk for certain cancers. however, the cause is generally not known, although certainly theories exist. development of cancer is generally multifactorial and involves many environmental and exogenous factors, as well as endogenous factors, such as genetics and exposure to sex hormones. there are also considerations when interpreting the studies presented here. these studies are often breed specific and we must be careful in extrapolating this to the general population of dogs. there is also likely a case selection bias in that many of these studies are performed in 240clinical theriogenology • volume 11, number 3 • september 2019 referral institutions. client factors may influence which dogs are presented to a referral institution and this patient group may not represent a true general population of pets. retrospective studies also have inherent bias, which is the nature of most of these studies. conclusion the idea of individualized medicine is becoming more prevalent in human and veterinary medicine. this is the approach that should be utilized when determining the need for spay and neuter in companion dogs. there are many factors to consider when making this decision, which may not be readily apparent to the average pet owner. the american college of theriogenologists and the society for theriogenology hold the position that “the decision to spay or neuter…should be made solely by the pet’s owner with the direct input of their veterinarian and will be dependent on each particular animal’s situation.”45 this statement should be upheld as often as possible to continue to practice the highest quality of medicine that is expected by current-day clients. conflict of interest the author has no conflict of interest to report. references 1. macewen eg, patnaik ak, harvey hj, et al: estrogen receptors in canine mammary tumors. cancer res 1982;42:2255-2259. 2. rutteman gr, misdorp w, blankenstein ma, et al: oestrogen (er) and progestin receptors (pr) in mammary tissue of the female dog: different receptor profile in non-malignant and malignant states. br j cancer 1988;58:594-599. 3. illera jc, perez-alenza md, nieto a, et al: steroids and receptors in canine mammary cancer. steroids 2006;71:541-548. 4. millanta f, calandrella m, bari g, et al: comparison of steroid receptor expression in normal, dysplastic, and neoplastic canine and feline mammary tissues. res vet sci 2005;79:225-232. 5. donnay i, rauis j, devleeschouwer n, et al: comparison of estrogen and progesterone receptor expression in normal and tumor mammary tissues from dogs. am j vet res 1995;56:1188-1194. 6. geraldes m, gartner f, scmitt f: immunohistochemical study of hormonal receptors and cell proliferation in normal canine mammary glands and spontaneous mammary tumours. vet rec 2000;146:403-406. 7. pisani g, millanta f, lorenzi d, et al: androgen receptor expression in normal, hyperplastic, and neoplastic hepatoid glands in the dog. res vet sci 2006;81:231-236. 8. adamo pf, cantile c, steinberg h: evaluation of progesterone and estrogen receptor expression in 15 meningiomas of dogs and cats. am j vet res 2003;64,1310-1318. 9. theon ap, lecouteur ra, carr ea, et al: influence of tumor cell proliferation and sex-hormone receptors on effectiveness of radiation therapy for dogs with incompletely resected meningiomas. j am vet med assoc 2000;216:701-707. 10. wilson gp, hayes hm: castration for treatment of perianal gland neoplasms in the dog. j am vet med assoc 1979;174:1301-1303. 11. thacher c, bradley rl: vulvar and vaginal tumors in the dog: a retrospective study. j am vet med assoc 1983;183:690-692. 12. herron ma: tumors of the canine genital system. j am anim hosp assoc 1983;19:981-994. 13. stovring m, moe l, glattre e: a population-based case-control study of canine mammary tumours and clinical use of medroxyprogesterone acetate. apmis 1997;105:590-596. 14. misdorp w, romijn a, hart aa: feline mammary tumors: a 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man, baltimore: williams & williams. 1981. 27. moulton je, harvey jw: tumors of lymphoid and hematopoietic tissue. in: moulton je, editor: tumors of domestic animals, 3rd edition, university of california press; 1990. 28. villamil ja, henry cj, hahn aw, et al: hormonal and sex impact on the epidemiology of canine lymphoma. j cancer epidemiol 2009:591753, 10.1155/2009/591753 29. glickman lt, raghavan m, knapp dw, et al. herbicide exposure and the risk of transitional cell carcinoma of the urinary bladder in scottish terriers. j am vet med assoc 2004;224:1290-1297. 30. norris am, laing ej, valli, ve et al: canine bladder and urethral tumors: a retrospective study of 115 cases (1980-1985). j vet intern med 1992;6:145-153. 31. mutsauers aj, widmer wr, knapp dw: canine transitional cell carcinoma. j vet intern med 2003; 17;136-144. 32. brodey rs, riser wh: canine osteosarcoma: a clinicopathologic study of 194 cases. clin orthop relat res 1969;62:54-64. 33. brodey rs, sauer rm, medway w: canine bone neoplasms. j am vet med assoc 1963;143:471-495. 34. brodey rs, abt da: results of surgical treatment in 65 dogs with osteosarcoma. j am vet med assoc 1976;168:1032-1035. 35. misdorp w, hart aa: some prognostic and epidemiologic factors in canine osteosarcoma. j natl cancer inst 1979;62:537-545. 36. spodnick gj, berg j, rand wm, et al: prognosis for dogs with appendicular osteosarcoma treated by amputation alone: 162 cases (1978-1988). j am vet med assoc 1992;200:995-999. 37. ehrhart np, ryan sd, fan tm: tumors of the skeletal system. withrow sj, vail dm, page rl, editors, withrow & macewen’s small animal clinical oncology 5th edition, st. louis (m0): elsevier; p. 463. 38. ru g, terracini b, glickman lt: host related risk factors for canine osteosarcoma. vet j 1998;156:31-39. 39. priester wa, mckay fw: the occurrence of tumors in domestic animals. natl canc inst monogr 1980; 54:1-210. 40. prymak c, mckee lj, goldschmidt mh, et al: epidemiologic, clinical, pathologic, and prognostic characteristics of splenic hemangiosarcoma and splenic hematoma in dogs: 217 cases (1985). j am vet med assoc 1988;193:706-712. 41. ware wa, hopper dl: cardiac tumors in dogs: 1982-1995. j vet intern med 1999;13:95-103. 41. cooley dm, beranek bc, schittler dl: endogenous gonadal hormone exposure and bone sarcoma risk. cancer epidemiol biomarkers prev 2002;11:1434-1440. 42. de la riva gt, hart bl, rarver tb, et al: neutering dogs: effects on joint disorders and cancers in golden retrievers. plos one 2013;8:e55937, 10.1371/journal.pone.0055937. 43. zink mc, farhoody p, elser se, et al: evaluation of the risk and age of onset of cancer and behavioral disorders in gonadectomized vizslas. j am vet med assoc 2014;244:309-319. 44. hoffman jm, creevy ke, promislow de: reproductive capability is associated with lifespan and cause of death in companion dogs. plos one 2013;8:e61082. https://www.theriogenology.org/page/positionstatements 242clinical theriogenology • volume 11, number 3 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/multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice follicle stimulating hormone receptors are expressed in ewe™s reproductive and nonreproductive tissues follicle stimulating hormone receptors are expressed in ewe’s reproductive and nonreproductive tissues logan kleditz,a hayder habeeb,b,c michelle kutzlerb adepartment of electrical engineering and computer science bdepartment of animal and rangeland sciences, oregon state university, corvallis, or cdepartment of animal production, al-qasim green university, babylon, iraq abstract follicle stimulating hormone receptors (fshr) are present in granulosa cells, sertoli cells, and other sites within the reproductive tract (uterine tube, uterus, placenta, and cervix). they are also present in many nonreproductive tissues (skin and adipose tissue, bone and cartilage, thyroid tissue, bladder and urethra, monocytes and endothelial cells within colon, pancreas, kidney, lung, liver, and in stomach cancer tissues of various species). however, the expression of fshr outside of the reproductive tract in sheep, has not been reported. objective was to determine fshr expression in ovine ovary, uterus, liver, kidney, heart, and skeletal muscle. tissue samples were collected at slaughter and fshr expression was determined via immunohistochemistry in formalin-fixed and paraffin-embedded tissue sections. within the reproductive tract, fshr were expressed in granulosa cells, luteal cells, and endometrial glandular epithelial cells. in liver, fshr were expressed within hepatocytes and vascular endothelial cells. expression of fshr in cardiac myocytes was limited to cell membranes. kidneys had fshr expression on the renal tubular epithelial cells and the capillary endothelial cells. lastly, in the skeletal muscle, fshr were expressed in the endomysium. in human cancers, fshr activation in nonreproductive tissues is involved in vascular remodeling, angiogenesis, cell proliferation, migration, and invasion. effects of fshr activation on ovine nonreproductive tissues remains unknown and warrants investigation into risks of frequent use of fsh agonists in sheep for nonseasonal estrous cycle manipulation. keywords: heart, kidney, liver, skeletal muscle, ovary, uterus introduction follicle stimulating hormone is secreted from the anterior pituitary gland and its secretion is regulated by estradiol, inhibin, and activin.1-3 receptors for follicle stimulating hormone are present in granulosa cells and sertoli cells.4,5 in addition, follicle stimulating hormone receptors (fshr) are present in the bovine uterine tube and ovine uterus, placenta, and cervix.6-8 besides the reproductive tract, fshr are present in the skin and adipose tissue, bone and cartilage, thyroid tissue, bladder and urethra, monocytes and endothelial cells within colon, pancreas, kidney, lung, liver, and stomach cancer.9-17 expression of fshr is well documented outside of the reproductive tract in other species, but not in sheep. follicle stimulating hormone agonists are routinely used for nonseasonal estrous cycle manipulation in ewes. if fshr are expressed outside the reproductive tissues, this could result in nonspecific side effects (e.g. vascular remodeling, angiogenesis).17 objective was to determine whether fshr are expressed in tissues outside of the reproductive tract in sheep. we hypothesized that in addition to ovary and uterus, fshr is expressed in liver, skeletal muscle, heart, and kidney. materials and methods tissues (ovarian follicle, ovarian corpus luteum, endometrium, heart, liver, thigh muscle, and kidney) were collected from a single crossbred ewe at slaughter. this ewe had not received any treatment prior to slaughter. all procedures were approved by the institutional animal care and use committee. tissues were fixed in 10% formalin, embedded in paraffin, and sectioned (5 μm) onto charged slides. sections were incubated at 60ºc for 45 minutes, deparaffinized in xylene, and rehydrated in a graded ethanol series (100, 75, and 50%). sections were washed in a diluted buffer (wash buffer 10x #s3006, dako, carpinteria, ca) and then exposed to heat-induced antigen retrieval with diluted sodium citrate buffer (target retrieval solution 10x concentration #s1699, dako) in a pressure cooker (nordicware® tender cooker, minneapolis, mn) boiled for 10 minutes and cooled for 20 minutes. slides were washed clinical theriogenology • volume 12 number 4 • december 2020507 with deionized water and tissue-specific endogenous peroxidase activity was blocked with 3% hydrogen peroxide (diluted from 30% hydrogen peroxide solution #5240-05, macron, pa). nonspecific binding was inhibited using a serum-free protein block for 20 minutes at room temperature (protein block serumfree ready to use #x0909, dako). for fshr, a polyclonal antirabbit fsh receptor (f3929, sigmaaldrich, st. louis, mo) was applied at a 1:200 dilution and incubated for 105 minutes at room temperature. universal negative control rabbit igg (nc495h, biocare medical, pacheco, ca) was applied to all adjacent sections to serve as negative controls. all slides were thoroughly washed in wash buffer prior to applying a secondary antibody (ih-8064 immunobioscience, mukilteo, wa) for 30 minutes at room temperature. slides were then thoroughly washed in wash buffer and then incubated with a chromogen (novared, vector laboratories, burlingame, ca) for 7.5 minutes at room temperature to detect the peroxidase activity. slides were then washed in wash buffer, counterstained with hematoxylin, washed in tap water, dehydrated in a graded ethanol series (50, 75, and 100%), passed through 3 successive xylene baths, and coverslipped. slides were evaluated at 400 x magnification with a leica dm4000b microscope using bright field microscopy. representative images from each tissue were digitally captured using a qimaging camera (qicam 12-bit, #qic-f-m-12-c, qimaging, surrey, bc, canada) and qcapturepro image capturing software (qimaging). evidence of fshr expression in the tissues was determined by the presence of red staining specific to individual cells. results within the reproductive tract, fshr were expressed in ovarian granulosa cells and endothelial cells (figure 1a), ovarian luteal cells (figure 1b), and endometrial glandular epithelial cells and endothelial cells (figure 1c). within liver, fshr were expressed in hepatocytes (liver epithelial cells) and endothelial cells (figure 1d). follicle stimulating hormone receptors were expressed on the cell membrane of cardiac myocytes (figure 1e). in the kidney, fshr were expressed on the renal tubular epithelial cells and the endothelial cells (figure 1f). lastly, in the skeletal muscle, fshr were expressed in the endomysium (figure 1g). negative controls showed no positive staining (data not shown). discussion similar to findings in humans and mice,17,18 in our study, fshr were expressed in vascular endothelial cells within the liver and kidney in sheep. in human cancer, endothelial cell fshr respond to fsh stimulation by vascular remodeling and angiogenesis.17,19 stimulation of vascular endothelial cell fshr in menopausal women results in vascular cell adhesion molecule 1 (vcam-1) synthesis that recruits monocytes in the pathogenesis of atherosclerosis.20,21 in addition, epithelial cell fshr stimulation in humans induces proliferation, migration, and cancer cell invasion.22-24 effect of fshr activation on ovine nonreproductive tissues remains unknown and warrants investigation into risks of frequent use of fsh agonists in sheep for nonseasonal estrous cycle manipulation. in addition to fshr expression in the epithelium, they are also expressed in muscle. similar to what was observed in the present study, atlantic salmon also expressed fshr in cardiac myocytes.25 with respect to skeletal muscle, fshr expression in the current study was limited to the endomysium. in human adipocytes, fshr activation opposes β3 adrenergic signaling by interacting with gαi-coupled receptor and the camp response element-binding protein (creb)-mediated pathway.26,27 beta-3 adrenergic receptor stimulation of skeletal muscle induced hypertrophy in mice.28 beta-3 adrenergic receptor stimulation of cardiac muscle induced vasodilation and relaxation to contractility.29,30 it is not known if stimulation of fshr in skeletal and/or cardiac muscle would alter β3 adrenergic signaling. however, this could substantially impact overall animal health. in conclusion this is the first report of fshr expression outside the reproductive tract in sheep. however, replication is necessary involving more animals. frequent use of fsh agonists in sheep in nonseasonal estrous cycle manipulation may be accompanied by health risks associated with fshr activation (e.g. stimulation of vascular remodeling, angiogenesis, vcam-1 synthesis, clinical theriogenology • volume 12 number 4 • december 2020 508 epithelial cell proliferation, and alterations in cardiac and/or skeletal β3 adrenergic signaling). figure 1. representative images (scale bar = 50 µm) of tissues with expression (arrows) of follicle stimulating hormone receptor a: granulosa cells and endothelial cells of a preovulatory follicle b: luteal cells of corpus luteum c: glandular epithelium and endothelial cells of endometrium d: hepatocytes and endothelial cells in liver e: cardiac myocytes of heart f: renal tubular cells and endothelial cells in kidney g: endomysium of skeletal muscle acknowledgment authors thank brynley cozzi for assistance with image preparation. conflict of interest authors disclose that there were no actual or potential conflicts of interest regarding the research that affected their ability to objectively present or review the research or data. funding oregon sheep commission and the ministry of higher education and scientific research in iraq (graduate student assistantship). references 1. demyashkin ga: inhibin b in seminiferous tubules of human testes in normal spermatogenesis and in idiopathic infertility. syst biol reprod med 2018;65:1-9. 2. ying sy: inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone. endocr rev 1988;9:267-293. 3. christensen a, bentley ge, cabrera r, et al: hormonal regulation of female reproduction. horm metab res 2012; 44:587-591. a  g  f e d  c b  clinical theriogenology • volume 12 number 4 • december 2020509 4. simoni m, gromoll j, nieschlag e: the follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. endocr rev 1997;18:739-773. 5. swider-al-amawi m, kolasa a, sikorski a, et al: the immunoexpression of fsh-r in the ductuli efferentes and the epididymis of men and rat: effect of fsh on the morphology and steroidogenic activity of rat epididymal epithelial cells in vitro. j biomed biotechnol 2010; doi:10.1155/2010/506762. 6. li c, ma y, yi k, et al. the interactions between nerve growth factor and gonadotrophins in bovine oviduct. anim reprod sci 2014;149:117-123. 7. grazul-bilska at, reyaz a, valkov v, et al: et al: follicle stimulating hormone receptor protein is expressed in ovine uterus during the estrous cycle and utero-placenta during early pregnancy. an immunohistochemical study. acta histochemica 2018;120:420-428. 8. habeeb hmh, hazzard tm, stormshak f, et al: effect of different dosages of pg-600 on ovulation and pregnancy rates in ewes during the breeding season. transl anim sci 2019;3:429-432. 9. welle mm, reichler, et al: immunohistochemical localization and quantitative assessment of gnrh-, fsh-, and lhreceptor mrna expression in canine skin: a powerful tool to study the pathogenesis of side effects after spaying. histochem cell biol 2006;126:527-535. 10. cui h, zhao g, liu r, et al: fsh stimulates lipid biosynthesis in chicken adipose tissue by upregulating the expression of its receptor fshr. j lipid res 2012;53:909-917. 11. ji y, liu p, yuen t, et al: epitope-specific monoclonal antibodies to fsh beta increase bone mass. proc natl acad sci usa 2018;115:2192-2197. 12. kong d, guan q, li g, et al: expression of fshr in chondrocytes and the effect of fsh on chondrocytes. biochem biophys res commun 2018;495:587-593. 13. liu j, chen g, meng xy, et al: serum levels of sex hormones and expression of their receptors in thyroid tissue in female patients with various types of thyroid neoplasms. pathol res pract 2014;210:830-835. 14. coit va, dowell fj, evans np: neutering affects mrna expression levels for the lhand gnrh-receptors in the canine urinary bladder. theriogenology 2009;71:239-247. 15. ponglowhapan s, church db, khalid m: differences in the expression of luteinizing hormone and follicle-stimulating hormone receptors in the lower urinary tract between intact and gonadectomised male and female dogs. domest anim endocrinol 2008;34:339-351. 16. robinson lj, tourkova i, wang y, et al: fsh-receptor isoforms and fsh-dependent gene transcription in human monocytes and osteoclasts. biochem biophys res commun 2010;394:12-17. 17. radu a, pichon c, camparo p, et al: expression of follicle-stimulating hormone receptor in tumor blood vessels. n engl j med 2010;363:1621-1630. 18. crawford ed, schally av, pinthus jh, et al: the potential role of follicle-stimulating hormone in the cardiovascular, metabolic, skeletal, and cognitive effects associated with androgen deprivation therapy. urol oncol 2017;35:183-191. 19. planeix f, siraj ma, bidard fc, et al: endothelial follicle-stimulating hormone receptor expression in invasive breast cancer and vascular remodeling at tumor periphery. j exp clin cancer res 2015;34:12. doi:10.1186/s13046-015-0128-7. 20. el khoudary sr, santoro n, chen hy, et al: trajectories of estradiol and follicle-stimulating hormone over the menopause transition and early markers of atherosclerosis after menopause. eur j prev cardiol 2016;23:694-703. 21. el khoudary sr, wildman rp, matthews k, et al: endogenous sex hormones impact the progression of subclinical atherosclerosis in women during the menopausal transition. atherosclerosis 2012;225:180-186. 22. ben-josef e, yang sy, ji th, et al: hormone-refractory prostate cancer cells express functional follicle-stimulating hormone receptor (fshr). j urol 1999;161:970-976. 23. zheng w, lu jj, luo f, et al: ovarian epithelial tumor growth promotion by follicle-stimulating hormone and inhibition of the effect by luteinizing hormone. gynecol oncol 2000;76:80-88. 24. sanchez am, flamini mi, russo e, et al: lh and fsh promote migration and invasion properties of a breast cancer cell line through regulatory actions on the actin cytoskeleton. mol cell endocrinol 2016;437:22-34. 25. mikalsen ab, haugland o, rode m, et al: atlantic salmon reovirus infection causes a cd8 t cell myocarditis in atlantic salmon (salmo salar l.). plos one 2012;7:e37269. 26. liu p, ji y, yuen t, et al: blocking fsh induces thermogenic adipose tissue and reduces body fat. nature 2017; 546:107-112. 27. liu xm, chan hc, ding gl, et al: fsh regulates fat accumulation and redistribution in aging through the galphai/ca(2+)/creb pathway. aging cell 2015;14:409-420. 28. puzzo d, raiteri r, castaldo c, et al: cl316,243, a β3-adrenergic receptor agonist, induces muscle hypertrophy and increased strength. sci rep 2016;6:37504. doi:org/10.1038/srep37504. 29. dessy c, balligand jl: beta3-adrenergic receptors in cardiac and vascular tissues: emerging concepts and therapeutic perspectives. adv pharmacol 2010;59:135-163. 30. cannavo a, koch wj: targeting β-3 adrenergic receptors in the heart-selective agonism and β-blockade. j cardiovasc pharmacol 2017;69:1-78.   clinical theriogenology • volume 12 number 4 • december 2020 510 introduction antibiotic use in human and animal medicines has rapidly accelerated since the discovery of penicillin in 1928. consequently, microorganisms resistant to particular antimicrobials or classes of antibiotics have emerged. growing trends in antimicrobial resistance underscore the urgency of directed, rational, and prudent antibiotic usage. antibiotics are indispensable in treating endometritis. however, not all causes of uterine inflammation call for antibiotics, and many other therapeutic approaches can be used to decrease reliance only on antibiotics. endometritis is not a foe that will be conquered by an ever-escalating arms race of antibiotics and chemical infusions. rather, endometritis should be respected as a condition with which every reproductive clinician must coexist, and only through appreciating its origins and accordingly modifying its natural course success (pregnancy) can be achieved. at the forefront of this approach is a focus on diagnostic precision to ensure that all underlying and contributing causes are addressed. endometrial resistance has historically been attributed to physical clearance of irritants in response to appropriate local inflammation. physical clearance requires an appropriate disposition of the genital tract,1 competence and function of anatomical barriers,2,3 myometrial contractility,4,5,6 lymphatic drainage,7and mucociliary clearance.8,9 additionally, local cellular and humoral aspects of the immune system are important.5 treatment of persistent endometritis includes restoration of appropriate external genital anatomy,2 augmentation of uterine clearance,4 and overcoming deficiencies in function of humoral and cellular immune response to endometrial contamination.10 excellent reviews discussing alternatives to primary antimicrobials for treatment of endometritis are available.11,12 nonantibiotic therapies for endometritis: what, when, and why kristina lu, karen von dollen hagyard equine medical institute, lexington, ky abstract endometritis, inflammation of the inner lining of uterus, can be caused by several insults. the simplicity of this definition belies the havoc it can wreak on a mare’s reproductive efficiency. potential inciting causes include a physiologic response to foreign invasion (breeding-induced endometritis), bacterial infection, fungal infection, and an iatrogenic interference in uterus. regardless of its source, inflammation creates an inhospitable embryonic environment. keywords: equine endometritis, mare, antibiotics since reproductive efficiency declines in the face of poor systemic health, general physical examination and establishing an overall health are important components of initial approaches to treat endometritis. age and immunosuppressive diseases (e.g. pituitary pars intermedia dysfunction) have potential impacts on body’s resistance to infection and endometrial resistance to disease.13,14 anatomic considerations a mare’s uterus is protected from external environment by 3 distinct caudal to cranial anatomic barriers: vulva, vestibulovaginal fold, and cervix. a compromise of 1 or more of these barriers will predispose a mare to uterine contamination and to subsequent endometritis. any amount or type of antibiotic cannot effectively resolve this type of endometritis unless the anatomical cause is rectified. importance of mare’s vulva, as the frontline barricade between environmental contaminants and reproductive tract, cannot be overemphasized. functionality is compromised when vulva deviates from its ideal position of being vertically oriented, in the same plane as anus with > 70% positioned below the pelvic brim. caslick’s vulvoplasty is commonly performed to correct imperfect vulvar anatomy. mares with more severe anatomical deficits may be good candidates for perineal body reconstruction (common approaches include the gadd and pouret techniques). moving inward, the next anatomical barrier is the vestibulovaginal fold. assessment of vestibulovaginal fold can be made by parting the vulvar labia. mares with an incompetent clinical theriogenology 2021; 13: 260 vestibulovaginal fold will aspirate air that causes audible ‘windsucking’ and pneumovagina. cervix is the most cranial anatomical defense for the uterus. longitudinal folds of cervix are contiguous with endometrial folds, thereby providing direct access from vagina to uterus. cervix is dynamic throughout a mare’s reproductive career. its elasticity is illustrated by its ability to stretch to accommodate 50+ kg of foal at parturition and constrict to a neck that can sometimes be difficult to navigate even a small diameter instrument during diestrus. this plasticity is not to be taken for granted, as each extreme can pose challenges for managing endometritis. cervix that is damaged during foaling trauma is vulnerable to lose its function to serve as a barrier to opportunistic ascending infections. in contrast, cervix that fails to appropriately dilate during estrus promotes debris accumulation within the uterus and exacerbates endometritis. beyond these major anatomic defenses of the uterus, description of endometritis would be incomplete if additional anatomic concerns were not discussed. uterus that is substantially pendulous over the pelvic brim is prone to poor uterine clearance and subsequent accumulation of fluid and debris that will hamper efforts to achieve pregnancy. mares that had trauma to the reproductive tract (e.g. dystocia), surgery (e.g. cesarean section or mass removal), or iatrogenic exposure to irritating substances may be affected by adhesion formation or other mechanical disruption within the uterus and/or in caudal reproductive tract. vesicovaginal reflux (‘urine pooling’) can have many contributing causes, including anatomic variables. downstream effects of urine pooling include the potential for direct transport of pathogens into uterus, and for generalized vaginitis and endometritis. uterine lavage uterine lavage is the keystone of endometritis treatment. uterine lavage provides a method of removing detritus and inflammatory products from the uterus and rinsing away planktonic bacteria. uterine irrigation should be considered fundamental in treating endometritis without disregarding its inherent inflammatory effects. autologous plasma, potassium penicillin, prostaglandin f2α treatments reduced uterine neutrophils and cleared streptococcus equi subspecies zooepidemicus; however, large volume lavage alone had similar effects.15 postbreeding uterine lavage 4 6 hours after breeding can be useful in managing postbreeding inflammation and to increase pregnancy rates.16-18 effective management of postbreeding inflammation may mitigate progression to chronic endometritis.11 ecbolic therapy endometritis is classically (but not necessarily) accompanied by fluid accumulation within the uterine lumen. evacuation of this fluid is the goal in therapeutic approaches. ecbolic therapy can take on several forms that may be also used in combination. these include oxytocin, carbetocin, cloprostenol, acupuncture, and exercise. small, frequent doses of oxytocin have substantial impact whereas larger doses can lead to tetanic uterine contractions that are ineffective. hormonal status of the mare influences the recommended dose of oxytocin required to have a desired effect. smaller doses of oxytocin (10 iu) are effective prior to ovulation, whereas after ovulation, larger doses (25 iu) may be necessary.19 carbetocin, a synthetic analog of oxytocin, has prolonged half-life (17.2 versus 6.8 minutes) and requires a smaller dose (1 mg carbetocin equivalent to 50 iu oxytocin).20 cloprostenol, a synthetic analog of prostaglandin f2α is an effective ecbolic agent for clearing intrauterine fluid.21 concern regarding its postovulation use is warranted; however, cloprostenol could be used until the second day after ovulation without negatively impacting pregnancy rates, despite having lower serum progesterone concentrations in mares receiving cloprostenol versus oxytocin.22 acupuncture is an attractive adjunct therapy to increase uterine contractility and fluid clearance in mares.23,24 lastly, exercise represents extreme value in promoting fluid clearance, is economical, effective,25 and easy to use. intrauterine treatments several nonantibiotic intrauterine therapies are available and considerable variation exists among clinicians regarding preferred treatment regimens. commonly employed nonantibiotic intrauterine products include tris-edta, tricide™ (molecular therapies, llc, athens, ga), dimethyl sulfoxide (dmso), hydrogen peroxide, n-acetylcysteine, kerosene, ceragyn® (csa biotech, spanish fork, ut), and bactivatetm (bojesen and petersen biotech aps, copenhagen, denmark). recent and ongoing research has suggested amniotic microvesicle infusion,26 lactoferrin,27 and stem cells28 as potential aids in combating endometritis, although these are not yet as widespread in clinical use as those mentioned above. tris-edta and its third-generation cousin tricide® are chelators that presumably act to destabilize bacterial structural integrity, especially useful for those bacteria existing in a biofilm mass.29 tris-edta is safe in an equine uterus30 and its activity against bacterial biofilms has been demonstrated in vitro.31 chelators are often instilled into the uterus following lavage and in conjunction with antimicrobials to potentiate their action.32 despite a dearth of peer-reviewed resources to support its use in treating endometritis in mares, dmso is utilized widely for this purpose. at increasing concentrations, dmso induces membrane thinning, followed by transient membrane water pores, and then disintegration of membrane bilayer structure.33 it is recognized as an antiinflammatory agent and has the ability to reduce biofilm biomass in vitro.34 intrauterine use of solutions containing up to 30% dmso was safe.35 its economy, accessibility, and versatility of use make it an clinical theriogenology 2021; 13: 261 attractive option for approaching endometritis. hydrogen peroxide, the household and medicine cabinet staple, exerts its antibacterial effects through free radical generation and subsequent oxidation. as with dmso, controlled studies evaluating its use in the equine uterus are lacking; however, in vitro work suggests that it is an effective antibiofilm disrupting agent.31 anecdotally, 3 5% hydrogen peroxide infusion can induce palpable adhesions and serosanguineous discharge in some mares. povidone iodine is an antimicrobial commonly added to lavage fluids. after disassociation, iodine penetrates microbial cell membranes to oxidize proteins, nucleotides and fatty acids. dilute preparations may be more rapidly bactericidal than full strength solutions.36 uterine lavage with 0.05% povidone iodine (5 ml of 10% betadine solution in a liter) was not associated with deleterious effects; however, infusion of 1% povidone iodine caused chronic endometrial inflammatory changes.16,37 the mucolytic n-acetylcysteine improved reproductive performance in mares and resulted in decreased cox-2 presence in endometrial biopsy samples.38,39 we have observed enhanced sensitivity in diagnosis of bacterial endometritis when performing uterine lavage for culture and cytology following n-acetylcysteine infusion (lu et al: unpublished data). the proprietary commercial products ceragyn® and bactivatetm are labeled for use in managing subfertility in mares. ceragyn® mimics antimicrobial peptide, is intended to penetrate and destroy biofilms, and is available in both lavage and as infusion formulations for use within the equine uterus. bactivatetm is not a treatment for endometritis per se, but rather a method to complement diagnosis of subclinical endometritis due to dormant beta streptococcus sp.40 intrauterine infusion of kerosene is intended as a chemical curettage of the endometrium. anecdotes attributed to success of kerosene are abundant; however, practitioners are cautioned that kerosene is not a panacea it may seem to be, and that inherent risks to mare and personnel exist with its use.41 case selection and client communication are important when using kerosene and for treatments that have no peer-reviewed clinical trials. the idea that the equine endometrium has the ability to support a robust mucus layer that can aid and abet nefarious microorganisms is not new. however, the past decade’s work42 has promoted a resurgence of discussion regarding the potential for bacterial biofilm formation in the equine uterus and what can be done to diagnose and address this condition. it is challenging to definitively diagnose and characterize bacterial biofilm in vivo, leaving extrapolation from in vitro work as our current best resource for combating tenacious bacterial endometritis due to suspected biofilm presence. a project has evaluated 3.3% n-acetylcysteine, edta, and hydrogen peroxide as antibiofilm treatments against isolates of escherichia coli, klebsiella pneumoniae, and pseudomonas aeruginosa. of treatments evaluated, no single treatment was active against all bacterial isolates, highlighting an important point about the individuality of bacterial isolates.31 this challenge has been addressed by the development of an in vitro biofilm assay that is currently available in clinical practice to target bacteria in a more bespoke fashion than empiric treatment selection. immune modulation and antiinflammatories use of glucocorticoids prednisolone acetate and dexamethasone around breeding in mares have been described to control inflammation. oral prednisolone acetate (0.1 mg/kg, every 12 hours) improved pregnancy rates in a limited group of mares bred with frozen semen.43 similarly, intravenous dexamethasone (0.1 mg/kg) at breeding improved pregnancy rates in mares classified as at-risk for susceptibility to persistent mating induced endometritis.44 furthermore, in mares challenged by intrauterine inoculation of a known quantity of escherichia coli, intravenous dexamethasone (0.1 mg/kg) resulted in modulation of the innate uterine immune response.45 potential interactions between systemic corticosteroid treatment and pituitary/ovarian function should not be overlooked. quarter horse mares (n = 18) received either placebo, intravenous dexamethasone (0.05 mg/kg, twice a day) or oral prednisolone (0.5 mg/kg, twice a day) for 5 days in early estrus. mares treated with dexamethasone had a 40% ovulation rate (2/5 mares), those treated with prednisolone had 83% ovulation rate (5/6 mares) and those receiving placebo had 100% ovulation rate (6/6 mares).46 these results suggest case selection for using glucocorticoids should carefully weigh risks and benefits, and the lowest doses and length of therapy should be prescribed. nonsteroidal antiinflammatories can also be harnessed to control endometrial inflammation. caution is necessary since a substantial increase in anovulatory follicle development was observed in mares treated with flunixin meglumine (2 mg/kg, twice a day, a supratherapeutic dose for standard antiinflammatory purposes),47 and at therapeutic doses of meloxicam (0.6 mg/kg, once a day) and phenylbutazone (4.4 mg/kg, once a day).48 in contrast, other projects involving flunixin meglumine at therapeutic doses (1.1 mg/kg, once a day)49 and phenylbutazone (4.4 mg/kg, once a day)50 observed substantial differences in ovulation rates compared to placebo treated mares. the cyclooxygenase-2 (cox-2) inhibitor firocoxib is an attractive choice given its more selective systemic effects and reduced postbreeding uterine inflammation (as assessed by polymorphonuclear cell presence on cytology and biopsy as well as endometrial cox-2 expression) while not negatively impacting ovulation.51 similar promising work from europe has provided support for the use of the cox-2 inhibitor vedaprofen to improve pregnancy rates in mares.52 although platelet rich plasma (prp) has been considered as a more recent therapy in equine reproductive medicine, the clinical theriogenology 2021; 13: 262 concept originated from a 1940 study with frog legs and it was observed that some unknown property of platelets controlled the formation of edema.53 from these beginnings, prp has evolved, and used widely in human and veterinary medicine for a wide range of purposes, from musculoskeletal and wound healing to dentistry and reproduction. intrauterine infusion of prp has modulated the immune response in mares previously diagnosed with chronic degenerative endometritis54 and improved pregnancy rates in mares classified as susceptible to persistent mating induced endometritis.55 measured exposure to emasculated bacterial products has been demonstrated to have positive immune effects. immune stimulation through systemic treatment of propionibacterium acnes (eqstim®, immunovet, inc., tampa, fl) improved endometrial cytology scores and pregnancy rates in barren mares.56,57 recently, mares were inseminated and treated intravenously with either dexamethasone (50 mg at insemination) or mycobacterial cell wall extract (mcwe, settle®, novavive, ontario, canada, 1.5 mg, at 24 hours prior to insemination). nitric oxide concentrations in uterine secretions and inflammatory cytokine presence in uterine biopsy samples (collected 6 hours after insemination) were used as end points for assessing uterine inflammatory response. mares treated with mcwe had lower nitric oxide concentrations in uterine secretions than untreated mares or mares treated with dexamethasone.58 conclusion historical and ongoing research have identified several products and techniques that have the potential to aid in the resolution of endometritis. when approaching this ever-expanding ‘pantry of options’, a strategic approach rooted in fundamentals is advised. critical consideration of why a mare is ‘dirty’ or ‘problematic’ will provide a blueprint for addressing underlying factors and primary endometritis. conflict of interest there are no conflicts of interest to declare. references 1. leblanc mm, neuwirth l, jones l, et al: differences in uterine position of reproductively normal mares and those with delayed uterine clearance detected by scintigraphy. theriogenology 1998;50:49-54 . 2. caslick ea: the vulva and the vulvo-vaginal orifice and its relation to genital health of the thoroughbred mare. cornell vet 1937;27:178-187. 3. watson ed: post-breeding endometritis in the mare: anim reprod sci 2000;60-61:221-232. 4. leblanc m, neuwirth l, mauragis d, et al: oxytocin enhances clearance of radiocolloid from the uterine lumen of reproductively normal mares and mares susceptible to endometritis. equine vet j 1994;26:279-282. 5. troedsson mh, liu ik, ing m, et al: multiple site electromyography recordings of uterine activity following an intrauterine bacterial challenge in mares susceptible and resistant to chronic uterine infection. j reprod fertil 1993;99:307-313. 6. evans mj, hamer jm, gason lm, et al: clearance of bacteria and non-antigenic markers following intra-uterine inoculation into maiden mares: effect of steroid hormone environment. theriogenology 1986;26:37-50. 7. leblanc m, johnson r, calderwood mays m, et al: lymphatic clearance of india ink in reproductively normal mares and mares susceptible to endometritis. biol of reprod monograph 1995;1:501-506. 8. morresey p, causey r, leblanc m: scanning electron microscopy of ciliated endometrial cells from reproductively normal mares and from mares with delayed uterine clearance. anim reprod sci 2010;121(1-2, supplement):94-95. 9. causey rc: mucus and the mare: how little we know. theriogenology 2007;68:386-394. 10. watson ed, stokes cr, bourne fj: cellular and humoral defence mechanisms in mares susceptible and resistant to persistent endometritis. vet immunol immunopathol 1987;16:107-121. 11. morris lha, mccue pm, aurich c: equine endometritis: a review of challenges and new approaches. reproduction 2020;160:r95-110. 12. troedsson mht, nielsen jm: non-antibiotic treatment of equine endometritis. pferdeheilkunde 2018;34:17-22. 13. mcfarlane d, hill k, anton j: neutrophil function in healthy aged horses and horses with pituitary dysfunction. vet immunol immunopathol 2015;165:99-106. 14. schöniger s, schoon ha: the healthy and diseased equine endometrium: a review of morphological features and molecular analyses. animals 2020;10:1-2. 15. troedsson mh, scott ma, liu ik: comparative treatment of mares susceptible to chronic uterine infection. am j vet res 1995;56:468-472. 16. brinsko sp, varner dd, blanchard tl, et al: the effect of postbreeding uterine lavage on pregnancy rate in mares. theriogenology 1990;33:465-475 17. brinsko sp, varner dd, blanchard tl: the effect of uterine lavage performed four hours post insemination on pregnancy rate in mares. theriogenology 1991;35:1111-1119. 18. knutti b, pycock jf, van der weijden gc, et al: the influence of early postbreeding uterine lavage on pregnancy rate in mares with intrauterine fluid accumulations after breeding. equine vet educ 2000;12:267-270. 19. gutjahr s, paccamonti dl, pycock jf, et al: effect of dose and day of treatment on uterine response to oxytocin in mares. theriogenology 2000;54:447-456. 20. schramme ar, pinto crf, davis j, et al: pharmacokinetics of carbetocin, a long-acting oxytocin analogue, following intravenous administration in horses. equine vet j 2008;40:658-661 21. combs gb, leblanc mm, neuwirth l, et al: effects of prostaglandin f2α, cloprostenol and fenprostalene on uterine clearance of radiocolloid in the mare. theriogenology 1996;45:1449-1455. 22. nie gj, johnson ke, wenzel jg, et al: effect of administering oxytocin or cloprostenol in the periovulatory period on pregnancy outcome and luteal function in mares. theriogenology 2003;60:1111-1118. 23. rathegeber r: acupuncture therapies for equine reproductive disorders. proc bluegrass reproductive symposium 2000. 24. schofield wa: use of acupuncture in equine reproduction. theriogenology 2008;70:430-434. 25. swift la, christensen bw, samocha mb, et al: randomized comparative trial of acupuncture and exercise versus uterine ecbolics in the clinical theriogenology 2021; 13: 263 treatment of persistent postbreeding endometritis in mares. j equine vet sci 2020;86:102821. 26. lange-consiglio a, funghi f, cantile c, et al: case report: use of amniotic microvesicles for regenerative medicine treatment of a mare with chronic endometritis. front vet sci 2020;7:1-6. 27. fedorka ce, scoggin ke, boakari yl, et al: the anti-inflammatory effect of exogenous lactoferrin on breeding-induced endometritis when administered post-breeding in susceptible mares. theriogenology 2018;114:63-69. 28. navarrete f, saravia f, cisterna g, et al: assessment of the anti-inflammatory and engraftment potential of horse endometrial and adipose mesenchymal stem cells in an in vivo model of post breeding induced endometritis. theriogenology 2020;155:33-42. 29. vaara m: agents that increase the permeability of the outer membrane. microbiol rev 1992;56:395-411. 30. youngquist rs, blanchard tl, lapin d, et al: the effects of edta-tris infusion on the equine endometrium. theriogenology 1984;22:593-599. 31. ferris ra, mccue pm, borlee gi, et al: in vitro efficacy of nonantibiotic treatments on biofilm disruption of gram-negative pathogens and an in vivo model of infectious endometritis utilizing isolates from the equine uterus. j clin microbiol 2016;54:631-639. 32. lyle sk, leblanc mm, staempfli sa, et al: how to use a buffered chelator solution for mares with chronic endometritis. proc am assoc equine pract 2011; p. 16-18. 33. gurtovenko aa, anwar j: modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulfoxide. j phys chem b 2007;111:10453-10460. 34. yahya mf, alias z, karsani sa: antibiofilm activity and mode of action of dmso alone and its combination with afatinib against gramnegative pathogens. folia microbiol 2018;63:23-30. 35. ley wb, bowen jm, sponenberg dp, et al: dimethyl sulfoxide intrauterine therapy in the mare: effects upon endometrial histological features and biopsy classification. theriogenology 1989;32:263-276. 36. berkelman rl, holland bw, anderson rl: increased bactericidal activity of dilute preparations of povidone-iodine solutions. j clin microbiol 1982;15:635-639. 37. olsen lm, al-bagdadi fk, richardson gf, et al: a histological study of the effect of saline and povidone-iodine infusions on the equine endometrium. theriogenology 1992;37:1311-1325. 38. gores-lindholm ar, leblanc mm, causey r, et al: relationships between intrauterine infusion of n-acetylcysteine, equine endometrial pathology, neutrophil function, post-breeding therapy, and reproductive performance. theriogenology 2013;80:218-227. 39. melkus e, witte t, walter i, et al: investigations on the endometrial response to intrauterine administration of n‐acetylcysteine in oestrous mares. reprod domest anim 2013;48:591-597. 40. petersen mr, skive b, christoffersen m, et al: activation of persistent streptococcus equi subspecies zooepidemicus in mares with subclinical endometritis. vet microbiol 2015;179:119-125. 41. coyle j, ferrer m, fishman h, et al: side effects and occupational risks of using intrauterine kerosene in a mare. clinical theriogenology 2017;9:97-104. 42. ferris ra, mccue pm, borlee gi, et al: model of chronic equine endometritis involving a pseudomonas aeruginosa biofilm. infect immun 2017;85:1-14. 43. papa fo, dell’aqua jaj, alvarenga ma, et al: use of corticosteroid therapy on the modulation of uterine inflammatory response in mares after artificial insemination with frozen semen. pferdeheilkunde 2008;24:79-82. 44. bucca s, carli a, buckley t, et al: the use of dexamethasone administered to mares at breeding time in the modulation of persistent mating induced endometritis. theriogenology 2008;70:1093-1100. 45. christoffersen m, woodward em, bojesen am, et al: effect of immunomodulatory therapy on the endometrial inflammatory response to induced infectious endometritis in susceptible mares. theriogenology 2012;78:991-1004. 46. ferris ra, mccue pm: the effects of dexamethasone and prednisolone on pituitary and ovarian function in the mare. equine vet j 2010;42:438-443. 47. cuervo-arango j, domingo-ortiz r: systemic treatment with high dose of flunixin-meglumine is able to block ovulation in mares by inducing hemorrhage and luteinisation of follicles. theriogenology 2011;75:707-714. 48. lima ag, costa lc, alvarenga ma, et al: does clinical treatment with phenylbutazone and meloxicam in the pre‐ovulatory period influence the ovulation rate in mares? reprod domest anim 2015;50:771-775. 49. donnelly cg, sones jl, dockweiler jc, et al: effects of flunixin meglumine on postponement of ovulation in mares. am j vet res 2019;80:306-310. 50. brickner km, ferris ra, trundell da, et al: effect of chronic nsaid administration on ovulation in mares. clinical theriogenology 2017;9:456. 51. friso am, segabinazzi lg, cyrino m, et al: periovulatory administration of firocoxib did not alter ovulation rates and mitigated post-breeding inflammatory response in mares. theriogenology 2019;138:24-30. 52. rojer h, aurich c: treatment of persistent mating‐induced endometritis in mares with the non‐steroid anti‐inflammatory drug vedaprofen. reprod domest anim 2010;45:e458-460. 53. danielli jfl: capillary permeability and oedema in the perfused frog. j physiol 1940;98:109-129. 54. reghini mf, neto cr, segabinazzi lg, et al: inflammatory response in chronic degenerative endometritis mares treated with platelet-rich plasma. theriogenology 2016;86:516-522. 55. metcalf es: the effect of platelet-rich plasma (prp) on intraluminal fluid and pregnancy rates in mares susceptible to persistent mating-induced endometritis (pmie). j equine vet sci 2014;1:128. 56. rohrbach bw, sheerin pc, cantrell ck, et al: effect of adjunctive treatment with intravenously administered propionibacterium acnes on reproductive performance in mares with persistent endometritis. j am vet med assoc 2007;231:107-113. 57. zingher ac: effects of immunostimulation with propionibacterium acnes (eqstim®) in mares cytologically positive for endometritis. j equine vet sci 1996;16:100-103. 58. woodward em, metcalf es, scoggin ke, et al: the effect of immune modulators on endometrial cytokine expression in mares susceptible to persistent breeding induced endometritis. clinical theriogenology 2013;5:364. 59. molina nm, sola-leyva a, saez-lara mj, et al: now opportunities for endometrial health by modifying uterine microbial composition: present or future? biomolecules 2020;10:1-21. clinical theriogenology 2021; 13: 264 nonantibiotic therapies for endometritis:what, when, and why 2019 a crash course in veterinary microbiomics a crash course in veterinary microbiomics candace lyman oklahoma state university abstract veterinary microbiomics has arrived and is thriving in today’s “omics era.” investigating microbial diversity within veterinary species will allow researchers to determine the physiologic state of the microbiome in healthy animals as a first step in developing treatments for animals with ailments resulting from dysbiosis. what impacts on clinical practices will arise as a result of the genomic information steadily accumulating in laboratory settings? as those answers come to fruition, it is our job as medical professionals to be capable of interpreting data and results provided to us. in order to develop and utilize a skill set, veterinarians should become acquainted with basic foundational concepts of veterinary microbiomics. building on these terms and definitions, we will explore, compare and contrast the clinical relevance of currently available information from those species for which this work has been performed. understanding these concepts will ultimately provide veterinarians with tools to analyze data and integrate information into clinical practices when appropriate. keywords: metagenomics, 16s rdna, 16s rrna, microbiota, next generation sequencing introduction not long ago we entered an interval coined “omics era” due to our improved ability to collect large amounts of information on organisms at the molecular and protein levels and filter that information utilizing newer computational and statistical tools. previously, utilization of next-generation sequencing techniques was cost prohibitive. however, with improvements in equipment and automated efficiency, this burden has been lifted and information pertaining to the bacterial population in various areas of the body is now accumulating with impressive momentum. investigating microbial diversity within veterinary species will allow researchers to determine the physiologic state of the microbiome in healthy animals as a first step in developing treatments for animals with ailments resulting from dysbiosis. whereas the day of veterinary microbiomics has successfully arrived, we as clinical veterinarians must now catch up by delving into a subject matter that has yet to even be integrated into veterinary school curriculums. in order to develop and utilize that skill set, veterinarians will need to become acquainted with basic foundational concepts of veterinary microbiomics. building on these terms and definitions, we will explore, compare and contrast clinical relevance of currently available information from those species for which this work has been performed. understanding these concepts will ultimately provide veterinarians with tools to analyze data and integrate information into clinical practices when appropriate. what viable treatments or practices will arise as a result of the stockpiles of information being shared with veterinarians? with a basic understanding of veterinary microbiomics, veterinarians will be able to answer this question for themselves in clinical settings. terms to define, understand, and utilize metagenomics: study of genetic material recovered directly from environmental samples. microbiome: microorganisms in a particular environment (including body or a part); combined genetic material of microorganisms (not limited to only bacteria) in a particular environment. microbiomics: this term is used frequently in lay press but it is not frequently utilized in the microbiome community. refers to the study of microbiome, the totality of microbes in (particular) environments; a study of microbes by considering all constituents collectively. 611 clinical theriogenology • volume 11, number 4 • december 2019 microbiota: commonly misused to refer to only bacteria, this is an ecological community of commensal, symbiotic, and pathogenic microorganisms (including bacteria, archaea, protists, fungi and viruses) present in and on all multicellular organisms. human genome project: an international scientific research project with the goal of determining the sequence of nucleotide base pairs that comprise human dna, and of identifying and mapping all genes of the human genome from both a physical and a functional standpoint; although it started in 1990 and took 13 years and $1 billion to complete, today the human genome can be sequenced in 1-2 days for ~ $3,0005,000.1,2,3 operational taxonomic units (otus): an operational definition used to classify groups of closely related individuals; refers to clusters of organisms grouped by dna sequence similarity (usually defined as 97% similar). sequencing results are often reported in otus (e.g. “firmicutes had 74 otus”). phylum: a principal taxonomic category (e.g. proteobacteria). genus: a principal taxonomic category, denoted by a capitalized latin name in italics (e.g. escherichia). dna sequencing: a process to determine the order of the 4 chemical building blocks (i.e. “bases”) that make up a dna molecule; these sequences tell scientists what kind of information is carried in a particular dna segment and changes in a gene sequence can highlight a gene that may cause disease. next generation sequencing: a fast and efficient dna sequencing technology capable of deciphering entire genomes (e.g. human genome). a read: in dna sequencing, an inferred sequence of base pairs corresponding to all or part of a single dna fragment (e.g. over 4 million reads of the v4 region of the 16s rdna gene were obtained). silva or greengenes database: comprehensive web resources for up-to-date information, qualitycontrolled databases of aligned ribosomal rna (rrna) gene sequences from bacteria, eukaryota, and archaea domains. when comparing sequenced results, it is imperative that identified sequences are not compared against a bank of known pathogens for a particular species, since these banks will not consider all possible normal, nonculturable bacteria and therefore are inherently biased and limiting. 16s rrna vs 16s rdna: rdna, or “ribosomal dna”: dna sequence that codes for ribosomal rna. rrna is the transcribed product of rdna whereas rrnas combined with proteins make up ribosomes. pcr 16s rdna sequencing process: isolation of an unknown bacteria, bacterial dna extraction, amplification of the 16s rrna gene (i.e. pcr), sequencing of a portion of the 16s rrna gene, comparison of sequenced gene with previously logged database sequences to identify a match (and therefore, bacterial identity). richness: a measure of the number of species represented in an ecological community; does not take into account abundance of individual species. diversity: takes into account bacterial species richness and bacterial species evenness. metagenomics vs 16s rdna profiling: “shotgun metagenomics” surveys the entire genomes of all the organisms present in a sample rather than only those genomes in the 16s sequences; therefore, all organisms (bacteria, viruses, fungi) will be captured.4 612clinical theriogenology • volume 11, number 4 • december 2019 conclusion understanding these terms and concepts will enable veterinarians to better grasp forthcoming literature and presentations reporting results pertaining to veterinary microbiomes, whether it be describing the canine gut or reproductive tract. having provided explanations and definitions of these commonly utilized terms, veterinarians can now make practical and effective use of this knowledge in a way that will allow them to analyze data and integrate information into daily clinical practice. references 1. lewis t 2013, human genome project marks 10th anniversary, live science health, viewed 12 feb 2019, <https://www.livescience.com/28708-human-genome-project-anniversary.html>. 2. www.genome.gov 2010, national human genome research institute: frequently asked questions, viewed 20 feb 2019, <https://www.genome.gov/11006943/human-genome-project-completion-frequently-asked-questions/>. 3. www.genome.gov 2015, national human genome research institute: dna sequencing, viewed cited 20 feb 2019, < https://www.genome.gov/10001177/dna-sequencing-fact-sheet/>. 4. www.blog.genohub.com, 2018, 16s sequencing vs. shotgun metagenomics: which one to use when it comes to microbiome studies, viewed 20 feb 2019, <https://blog.genohub.com/2018/04/12/16s-sequencing-vs-shotgunmetagenomics-which-one-to-use-when-it-comes-to-microbiome-studie/> 613 clinical theriogenology • volume 11, number 4 • december 2019 614clinical theriogenology • volume 11, number 4 • december 2019 untitled omniblank: 25 contact allan gunn algunn@csu.edu.au © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9592, http://dx.doi.org/10.58292/ct.v15.9592 research report use of abstract reflection to enhance student learning from theriogenology practical classes allan gunn,a,b john harper,a jason condona,b aschool of agricultural, environmental and veterinary sciences, charles sturt university, wagga wagga, australia bgulbali institute, charles sturt university, wagga wagga, australia abstract veterinary training is a resource-consuming process. practical sessions throughout the curriculum are considered vital aspects of professional training and consume large amounts of time, space, and human resources and are not without risks to human and animal health. it is, therefore, important that the educational experience is optimized during practical sessions. mentee dialogue with mentors, as part of institutional peer teaching support, aimed to optimize the practical sessions to develop professional expertise (knowledge and skills) of veterinary theriogenology students. we used the theory of experiential learning and reflective observation as a means of enhancing learning during the practical sessions. we attempted to do this by discussing abstract concepts associated with authentic learning tasks covered in each practical session. anonymous end-of-course student feed-back revealed that the process encouraged in-depth and alternative critical thinking and discussion in the groups, which was a fun way for them to embed the knowledge and develop the skills being taught. the use of ‘abstract reflection’ appears to be a useful and efficient way of enhancing the value of laboratory practical teaching and learning resources within the veterinary theriogenology curriculum. the vibrancy associated with collegiate interactions between academic staff members and educational designers results in a more enthusiastic and beneficial teaching culture and learning environment, and the development of students to become better, agentic, and more deliberate professionals. keywords: abstract reflection, pedagogy, practicals, experiential learning, andragogy introduction veterinary training is a resource-consuming process. practical, or laboratory, sessions throughout the curriculum are considered vital aspects of professional training and are required for accreditation of veterinary degrees by registration authorities. they consume large amounts of time, space, and human resources, and are not without risks to human and animal health.1 this requirement is particularly true for theriogenology where student knowledge and skills are required in several domestic species with similar but sufficiently different attributes to require multiple species exposure. as a result, the number of practical sessions scaffolded within the didactic lectures for reproduction subjects is relatively high to expose students to the basic and more advanced skills pertinent to each species. the practical classes are inherently resource, animal, personnel, and materials intensive. assessments designed to appraise theoretical knowledge that had been reinforced by the authentic practical sessions were not being answered as if the students had attended the aligned lecture and practical sessions. disappointingly, students did not appear to be able to immerse themselves in the holistic teaching experience when responding to the assessment tasks. to overcome this pedagogical gap, it was decided to make the practical sessions a more effective part of the intrinsic learning experience by encouraging transactive cognitivism and constructivism by the students. at a mentorship of teaching program, ideas were brainstormed that could, or might, be useful to enhance the pedagogical experience using appropriate epistemology. a process was added to the practical sessions that were already structured and relevant to the didactically delivered material, to try and induce student-relevant reflection at practical mailto: algunn@csu.edu.au http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9592 26 citation line: clinical theriogenology 2023, 15, 9592, http://dx.doi.org/10.58292/ct.v15.9592 sessions. abstract questioning of ideas and principles pertinent to that laboratory session became part of the concluding process of each practical session. development and implementation of concepts and activities on reflection of teaching, and student learning, it became clear that just because something is taught, it does not mean it will be learned. in a voluntary university mentorship program, we took up the challenge to develop student metacognitive skills by encouraging internal dialogue in their learning as an important part of their, and a teacher’s, role in developing the link between knowledge and use of that knowledge,2 thereby bringing ‘order to chaos’ while learning and understanding new concepts. we brainstormed ideas and used scholarship for our learning and teaching development, with the intention to transform pedagogy to andragogy. the aim was to use kolb’s experiential learning model3–5 where students undertake iterative processes using previous knowledge, new knowledge, that of their peers, and potentially that of teachers. the intention was to induce some mental dissonance in a safe academic environment, as well as the sharing of ideas and thoughts, to increase awareness of what they were doing in the practical session. it was planned that, by creating a mental picture, and linking it to pertinent factual information, the memory of the procedures and processes would be authentic, resulting in more durable and pertinent understanding by the students, thereby using hermeneutics, the theory and study of interpretation, to develop a heuristic, or learning by discovery, culture in the students’ activities. this was to increase student agency,6 creating deliberate, professional-enabling, productive learning in complex environments resulting in thoughtful, courageous, and morally responsible learners.7 reflective practice occurred only with assertive students, and it was our aim to encourage reflection in all students and to use the opportunity for students to develop autonomy8 and self-authorship.9 as a result we had to foster an environment of ‘relaxed professional conduct’ where enquiry and research were fostered within the laboratory setting. after deliberation, literature searching, the benefit of experience, and many iterations and alterations of plans, it was decided that a series of 5 ‘abstract questions’ pertinent to the practical/laboratory session would be handed out to groups of students at the beginning of the practical session. the practical session would continue as it would normally occur with active student participation. once the practical session was concluded, the whole class would informally gather to address the questions set to each group. this gave the opportunity for the group to reflect on their question and make their suggestions as to possible reasons why they had concluded as they did. the rest of the class were encouraged to challenge those conclusions and to offer alternative suggestions. at the beginning of this process, we considered it important not to be too confrontational, as this was a challenge of ideas, and not personal affronts for those thoughts or ideas. it was important that we developed and behaved as experiential educators. we had to be more than just facilitators to balance attention to the learner with the subject matter in a complex relational process where the students could reflect on the meaning of the ideas and develop the skill of applying those ideas.5 examples of questions male anatomy practical session (near the beginning of the subject): explain why or how the blood pressure in the corpus cavernosum of ruminants has been reported to be as high as 15,000 mm hg when the systolic blood pressure of most animals is 120 mm hg. reproductive examination of the live cow laboratory session (near the end of the subject): explain how and why a cow stands to be mounted by another cow when in estrus. note that it was not always obvious if there was a clear answer, but the point was to highlight to the students that sometimes things just are, with our current knowledge base. results the process was not originally intended to be a formal study; as a result, the outcomes are observational and subjective, based on personal interpretations and informal voluntary anonymous student responses to the process at the end of the subject. fifty-seven students submitted responses (out of a possible 59 in the year), of which, 3 did not answer the question, and responded to the practicals in general. fifty-four of the respondents gave positive responses to the question as to whether the end of session questions were beneficial or not by the students, and most mentioned that this should continue in the future. some of the comments included: • assisted with catching up points of the practical that were either unclear or they had missed during the practical session. • it assisted them in active engagement rather than passive participation (summarizednot written as such). • they brought in practical aspects that were not necessarily directly associated with the practical session. • it allowed us to use our brains more and to stimulate learning. • they helped to answer questions that had arisen during the practical session. • i loved the reflection questions…… also done in a relaxed and inclusive way. • good, but maybe should be given beforehand so they could be researched. • sometimes the questions/answers seemed a bit beyond my knowledge……. (making them simpler might not have benefitted the advanced students). • they made me think outside the box… and made us question why we do things the way we do. • ……… made me realize areas i needed to spend more time studying. • they involved critical thinking and encouraged us to challenge previous understanding and perspectives. http://dx.doi.org/10.58292/ct.v15.9592 citation line: clinical theriogenology 2023, 15, 9592, http://dx.doi.org/10.58292/ct.v15.9592 27 • it is good they are done in a group… so someone else might know the answer (and so they can be discussed). • they made practicals more interactive. keywords from the student responses are represented as a word cloud (figure). discussion development of abstract questioning after practical sessions fits with the role of reflection to develop agency, self-authorship, confidence, maturity, and authority.9 encouraging all students to reflect on their practice, is part of the educators’ responsibility to facilitate students to develop autonomy and learning responsibility, and not just in the assertive students.10 the primary aim of this academic reflective intervention was to increase the efficiency in the use of resources in the practical sessions to enhance student learning. the rationale was ultimately to move from pedagogy to andragogy where the students take control of their learning. the principles of the reflective process enhancing learning are based on experiential learning where deeper learning and understanding occurs collaboratively. however, students do not always openly engage in collaborative learning as it is a more complex and difficult task.11 the aim of the abstract questioning was to stimulate the conversational framework, with the learners modulating between practice and concepts, and initiating the exchange of ideas, thereby inducing debate and dialogue. then, developing a shared focus and an output after challenging each other with appropriate conflict, in the knowledge that it is not a personal affront but an opportunity to develop intellectually and professionally. as new experiential educators,5 we had to work on developing the appropriate safe and nonjudgmental environment that could result in shared inputs and outputs and practice modelling the environment for collaborative learning to occur.11 the learning and teaching environment allowed feedback loops to occur between facilitator, students, and other staff, for the growth and cementing of knowledge to occur in the students. interestingly, most students enjoyed the challenge of the abstract questioning. they appeared to take more away from the practical sessions than having a teacher orientated wrap-up of the contents, without encouraging thinking about processes by using abstract discussion topics. the point of encouraging understanding, rather than rote learning, is the key to higher order thinking and results in understanding of processes rather than reproducing information regardless of the context. mostly, the students began to look forward to the questions, and became actively involved in the process of linking theory with praxis, fitting with our pedagogical aim of developing student agency in a safe and reflective learning environment. students were always encouraged, by both staff and peers, to engage in the learning process in a disciplined, fun and interesting way, so that end of subject assessment was part of a journey of learning to become, and to create, a deliberate and consummate professional.8 however, there were some students that wanted the questions prior to the practical so they could research the topics, or became very frustrated with the dissonance and that there was not always an answer to the questions. ultimately, the aim was to induce some uncertainty, thereby induce deep thinking to enhance learning and the value of the practical sessions. the hope from this reflective experience was not only to increase the value of laboratory sessions to student learning, but to set the stage for professional lifelong learning. understanding the uncertainties during professional training involving the complexities of learning clinical skills will enable the students to develop appropriate skills, study techniques, and resilience, to be proactive and manage feelings of uncertainty during clinical training and professional practice.12 from a teacher, facilitator or experiential educator perspective, the process was an interesting pedagogical and teaching collegiality exercise. it is difficult or impossible to demonstrate success as determined by better responses to examination questions and overall marks. subjectively, there appeared to be greater engagement in the practical sessions by the students undertaking abstract reflections after practical classes, compared to previous years. figure. word cloud representation of words used by the students in their unofficial anonymous feedback at the end of the subject, having undertaken the abstract reflection process http://dx.doi.org/10.58292/ct.v15.9592 28 citation line: clinical theriogenology 2023, 15, 9592, http://dx.doi.org/10.58292/ct.v15.9592 it is interesting that whereas we saw the questions as a way to foster reflection, ‘reflection’ was a minor word in the word cloud. it appears that the learners were very much outcome-focused: ‘beneficial,’ ‘understanding,’ ‘consolidate’ and ‘knowledge’. the words relating to the process were mentioned less: ‘reflection’, ‘critical thought’, ‘connection’. it is possible that the students have not actually reflected on the process of their learning, and it might be that the questioning process has a legacy of the students shaping their learning accordingly. undertaking thematic assessment of the words used by the students might result in a different interpretation of the word cloud where there was use of words having a similar reflective meaning (e.g. reflection, cementing theory, critical thought, solidification, connection, think of information, and reinforce). regardless, the development of comfort in questioning, will serve them well in their future learning and professional practice. conclusion reflecting on teaching outcomes and using scholarship for learning and teaching in the presence of vibrant mentors can be an enlightening experience. using appropriate affordances and encouraging the use of practical situations to induce self and peer discovery in a safe and vibrant reflective learning environment can be an efficient way of making students better, agentic, and more deliberate professionals. acknowledgement allan gunn thanks denise wood for her pedagogical guidance and input in the successful fellow of higher education academy application that shares much of this material. wilma pfitzner and the faculty of science at charles sturt university are acknowledged for the opportunity offered to the authors in the teaching mentorship program. jason condon and allan gunn acknowledge the friendship and mentorship of their late colleague and friend john harper, for his enthusiastic encouragement for us to always ‘light the fire’ of learning in students. conflict of interest none authors’ contribution all authors conceived the ideas. allan gunn wrote most of the article, jason condon reviewed. john harper was unable to be part of the article submission process but was integral in the design of the process and supported the aim to submit it for publication. references 1. root kustritz mv, chenoweth pj, tibary a: efficacy of training in theriogenology as determined by a survey of veterinarians. j am vet med assoc 2006;229:514–521. doi: 10.2460/javma.229.4.514 2. national research council. how people learn: brain, mind, experience, and school: expanded edition. washington, dc; the national academies press: 2000. 3. kolb ay, kolb da: the learning way: meta-cognitive aspects of experiential learning. simul gaming 2009;40:297–327. doi: 10.1177/1046878108325713 4. kolb ay, kolb da: learning styles and learning spaces: enhancing experiential learning in higher education. acad manag learn educ 2005;4:193–212. doi: 10.5465/amle.2005.17268566 5. kolb ay, kolb da, passarelli a, et al: on becoming an experiential educator: the educator role profile. simul gaming 2014;45: 204–234. doi: 10.1177/1046878114534383 6. reeve j, tseng cm: agency as a fourth aspect of students’ engagement during learning activities. contemp educ psychol 2011;36:257–267. doi: 10.1016/j.cedpsych.2011.05.002 7. trede f, mcewen c: carving out the territory for educating the deliberate professional. in: trede f, mcewen c: editors. educating the deliberate professional. professional and practice-based learning. new york; springer: 2016:15–28. 8. trede f, smith m: teaching reflective practice in practice settings: students’ perceptions of their clinical educators. teach high educ 2012;17:615–627. doi: 10.1080/13562517.2012.658558 9. jackson d, trede f: the role of reflection after placement experiences to develop self-authorship among higher education students. in: billett s, orrell j, jackson d, valencia-forrester f: editors. enriching higher education students’ learning through post-work placement interventions. professional and practice-based learning. new york; springer: 2020:189–208. 10. trede f: role of work-integrated learning in developing professionalism and professional identity. asia pac j educ 2012;13:159–167. available from: https://www.scopus.com/inward/record.uri?eid= 2-s2.0-84873884299&partnerid=40&md5=eb67b243428f7c24e12b6e8e7d54fcc1 [cited 16 december 2022]. 11. laurillard d: teaching as a design science. building pedagogical patterns for learning and technology. in: teaching as a design science. new york; taylor & francis: 2012:187–208. 12. cripps sj, vinten cek: veterinary undergraduates’ attitudes to and understanding of clinical skills learning. vet rec 2022;191:e1320. doi: 10.1002/vetr.1320 http://dx.doi.org/10.58292/ct.v15.9592 https://doi.org/10.2460/javma.229.4.514 https://doi.org/10.1177/1046878108325713 https://doi.org/10.5465/amle.2005.17268566 https://doi.org/10.1177/1046878114534383 https://doi.org/10.1016/j.cedpsych.2011.05.002 https://doi.org/10.1080/13562517.2012.658558 https://www.scopus.com/inward/record.uri?eid=​2-s2.0-84873884299&partnerid=40&md5=eb67b243428f7c24e12b6e8e7d54fcc1 https://www.scopus.com/inward/record.uri?eid=​2-s2.0-84873884299&partnerid=40&md5=eb67b243428f7c24e12b6e8e7d54fcc1 https://www.scopus.com/inward/record.uri?eid=​2-s2.0-84873884299&partnerid=40&md5=eb67b243428f7c24e12b6e8e7d54fcc1 https://doi.org/10.1002/vetr.1320 adenoviral-vectored gonadotropin releasing hormone vaccine for estrus suppression in mares adenoviral-vectored gonadotropin releasing hormone vaccine for estrus suppression in mares rochelle jensen,a aime johnson,a robyn wilborn,a timothy braden,b michelle kutzler,c scot roberts,d kent van kampen,e joshua trumble,a henry bakerf acollege of veterinary medicine, auburn university, auburn, al bdepartment of anatomy physiology and pharmacology, auburn university, auburn, al cdepartment of animal and rangeland sciences, oregon state university, corvallis, or daltimmune inc. gaithersburg, md ethe van kampen group, inc., payson, ut fscott ritchey research center, auburn university, auburn, al abstract objective of this study was to evaluate an adenoviral-vectored gonadotropin releasing hormone vaccine’s ability to temporarily suppress reproductive cyclicity in mares. five mares were vaccinated twice, 30 days apart and 5 mares served as unvaccinated controls. following year, 4 vaccinated mares and 2 naïve mares (adjuvant controls) were given a gonadotropin releasing hormone peptide-adjuvant vaccine containing 100 µg of gonadotropin releasing hormone. antibody responses, ovarian follicular dynamics and serum progesterone concentrations were determined during 2 ovulatory seasons. during first year, vaccinated mares developed gonadotropin releasing hormone antibodies (p < 0.05), but there was no significant effect on estrous cyclicity. during second year, antibody responses increased (p < 0.05), accompanied by suppression of estrous cyclicity (determined by progesterone concentrations and transrectal palpation and ultrasonography). three of 4 treated mares returned to cyclicity between 154 and 326 days, whereas the fourth mare was still in anestrus at study completion. adjuvant control mares developed a short-lived, minimal antibody response and cycled normally. in conclusion, mares developed an immune response to homologous immunization using an adenoviral-vectored gonadotropin releasing hormone vaccine. additional heterologous vaccination with a gonadotropin releasing hormone peptide-adjuvant vaccine demonstrated establishment of immune memory and achieved estrus suppression. keywords: estrous cycle, horse, immunocontraception, interestrus interval introduction during estrus, mares can become difficult to train and ride. this unpredictable behavior may endanger handlers and riders. progesterone and other progestogens are used to suppress estrus in mares.1-3 in united states, altrenogest is the most commonly used synthetic progestin product labeled for estrus suppression in mares.1 although effective for estrus suppression, clinical use of these products is limited due to high cost and need for daily administration. other progestogens available in injectable forms can induce local swelling and muscle pain that can interfere with training and performance.1,4 intrauterine devices such as glass marbles prolong luteal phase in mares, but are unreliable and have a low success rate.5-7 hence there is a need for a reliable, cost-effective, and practical method for temporary suppression of cyclicity in mares. blocking gonadotropin releasing hormone (gnrh) receptor binding is effective in controlling reproduction and sex-related behavior. due to its high degree of homology among mammals and its control of both male and female gonadal function, a method that suppresses gnrh can be used in many species in both sexes.8-12 synthetic agonists and antagonists suppressed reproductive function.8,13-17 however, field use of gnrh agonists (such as deslorelin) for estrus suppression in mares resulted in variable response rates that are short-lived, requiring expensive repeated or continuous administration.18-21 antignrh antibodies inhibit binding of gnrh to its receptor, preventing receptor activation and release of gonadotropins from anterior pituitary. this induces infertility and prevents reproductive behavior in many species.11 one of the greatest challenges in formulating a gnrh vaccine that can induce a physiologically effective immune response is limited antigenicity of gnrh. gonadotropin releasing hormone is weakly immunogenic, due to its small size (10 peptides) and recognition as ‘self’ by the immune system.22 clinical theriogenology • volume 12 number 2 • june 2020124 gnrh peptide-adjuvant vaccine formulations effectively suppressed ovarian function and cyclicity in mares.1,23-28 these vaccines caused a rapid increase in antignrh antibodies, reduced circulating progesterone and suppressed follicular activity.23,26,27 equitytm (zoetis, australia) is a commercial, protein-based, antignrh vaccine labeled for use in horses and licensed in australia, but not in united states. initial and booster doses of equitytm (200 μg of gnrh in each dose) given 30 days apart resulted in estrus suppression from 3 months to > 2 years.25-29 in addition to high variability in interestrus interval, there was a high incidence of adverse effects. some effects were mild (e.g. local injection site swelling) or more severe, to include transient fever.27,29 these adverse reactions limited field use of gnrh peptide-adjuvant vaccines in high-performance mares. mares that suffer skin swelling, neck pain, or fever may not perform optimally. viruses engineered to express antigenic proteins can induce an immune response and improve vaccine safety and efficacy. for example, the human adenovirus variant 5 (ad5) vector induced potent humoral and cell-mediated immune responses, had intrinsic adjuvant properties, induced innate immunity, had effective memory response, provided a natural presentation of immunogens and had broad host tropism.30,31 nonreplicating ad5-vectored vaccines are adaptable for induction of protective immunity and did not vary significantly as a function of either antigen expressed or species immunized.30,32-35 prime-boost immunizations can be given with unmatched delivery vectors (heterologous vaccination) and in many cases, were more effective than homologous prime-boost delivery modality.36 objective of this study was to determine if an adenovirus vector engineered to express gnrh antigen (ad-gnrh) could induce antignrh antibodies in mares following homologous prime-boost vaccinations, sufficient to suppress cyclicity. it was hypothesized that this vaccine is antigenic with no adverse reactions in immunized mares. second objective was to utilize a heterologous vaccination strategy in which administration of a subtherapeutic dose of a protein-antigen based gnrh (p-gnrh) vaccine could be used to determine if homologous prime-boost vaccination of mares with ad-gnrh elicited immune memory. prime-boost immunizations can be given with unmatched delivery vectors (heterologous vaccination) and in many cases, were more effective than homologous prime-boost delivery modality.36 the p-gnrh dose used was less than total prime-boost dose recommended by the manufacturer (2 x 1.0 ml injections each containing 200 µg of gnrh, given 4 weeks apart (equitytm, zoetis).29 it was hypothesized that vaccination of mares using a protein-antigen construct containing 100 µg (0.5 ml) of gnrh elicits sufficient gnrh antibody production to suppress estrous cycle only in mares primed with ad-gnrh. materials and methods experimental design twelve, nonpregnant light-horse cycling mares (14 23 years) that belonged to auburn university equine reproduction center teaching herd were used. mares were housed by groups in large pens and fed free-choice bermuda hay supplemented with grain. study was conducted over 2 consecutive ovulatory seasons, starting may 2015. during first season, 5 mares were randomly assigned to control (ad-gnrh control) and 5 mares to treatment (ad-gnrh) groups. treatment consisted of 2 intramuscular injections of ad-gnrh vaccine. each tr mare received 1 ml dose containing 4.64 x 1010 infectious units (ifu) into the left cervical musculature, followed by a booster after 30 days. vaccine effects were measured for 12 months. the ad-gnrh control mares were monitored for cyclicity and seasonality. because antibody response to this immunization schedule was ineffective to suppress cyclicity, a heterologous vaccination strategy was utilized during the subsequent ovulatory season.37 following spring, 4 of 5 ad-gnrh mares were given a single 100 µg (0.5 ml) intramuscular injection of a p-gnrh vaccine. to determine effects of a single administration of this vaccine at this low dose, 2 unvaccinated mares were given similar single dose (100 µg; p-gnrh control). effects of p-gnrh vaccination were measured for 12 months. data collection for this project ended 23 months after initial vaccination with ad-gnrh. clinical theriogenology • volume 12 number 2 • june 2020 125 vaccine the ad-gnrh vaccine vector consisted of a thoroughly characterized e1/e3 deleted ad5 human adenovirus produced for use in animal immunization.30,31,38,39 this vector expressed a nucleotide sequence consisting of a tissue plasminogen activator (tpa) leader sequence followed by multimers of gnrh (ehwsyglrpg) linked to the leukotoxin a1 gene of pasteurella haemolytica (lkt) and t-helper epitopes. the ad5 vector vaccine was synthesized by genscript (piscataway, nj), and cloned into the ad5 vector to generate adenovirus 5-pasteurella haemolytica-gonadotropin releasing hormone (adlktgnrh) vaccine, as described.40 adenovirus 5-pasteurella haemolytica-gonadotropin releasing hormone vaccine virus was propagated on hek293 cells, purified on a cesium chloride gradient, sterilized using 0.22 μm filtration and stored at -80oc in a formulation buffer.41 viral titer was determined by adeno-xtm rapid titer kit (bd biosciences, palo alto, ca) on hek293 cells. correct structure of the antigen was verified by dna sequencing (genewiz, germantown, md). each 1 ml dose of vaccine contained 4.64 x 1010 ifu of vector. assays antignrh antibodies and serum progesterone concentrations were monitored throughout the study. a blood sample was collected by jugular venipuncture from each mare immediately prior to initial vaccination with ad-gnrh vaccine, then monthly thereafter. serum was separated, aliquoted, and frozen (-80oc) until analyzed. serum antignrh antibody was determined with 125i-gnrh (l8008, sigma-aldrich, st louis, mo) using a radioimmunoprecipitation technique. samples were assayed in duplicate using 100 µl of 125i-gnrh added to 100 µl of test serum diluted 1:100 in 200 µl buffer. after overnight incubation at 4oc, 100 µl of bovine igg (250 µg) was added (sigma-aldrich) and bound 125i-gnrh-ab complexes precipitated from unbound hormone by adding 500 µl of a 24% solution of polyethylene glycol (carbowax™ peg 8000, p156500, thermo fisher scientific, waltham, ma). reaction tubes were centrifuged at 1400 x g for 15 minutes and radioactivity in the precipitate was measured using a gamma counter (packard, cobra ll, ramsey, mn). nonspecific binding (nsb) of 125i-gnrh was determined from the mean of duplicate tubes in which diluted serum was replaced by pbs/bsa buffer. mean nsb was subtracted from individual sample measurements. serum antignrh antibody bound to 125i-gnrh was expressed as a percentage of a known internal standard positive control of rabbit antignrh antisera used in this laboratory. internal standard negative control serum was also of rabbit origin. serum progesterone concentrations were determined using validated chemiluminescence immunoassay (immulite® diagnostic products corporation, los angeles, ca). lower detection limit was 0.2 ng/ml and interand intra-assay cvs were < 10%.27 ovarian and uterine activity transrectal palpation and ultrasonography (5 mhz linear-array transducer, micromaxx, sonosite inc, bothell, wa) of reproductive tracts were performed. presence of corpus luteum (cl) and nature of uterine edema (none, slight, moderate, or heavy) were recorded. diameter of largest follicle on each ovary was measured. during ovulatory season, ultrasonography was performed twice weekly (months 1 7 during year 1 and months 13 19 during year 2). during nonovulatory season, ultrasonography was performed once weekly months 8 12 during year 1 and months 13 20 during year 2). interestrus interval interestrus interval (iei) was measured from ovulation of 1 or more dominant estrus follicles to ovulation of the succeeding estrus dominant follicle/s. diestrus was determined based on ultrasonographic evidence of a cl and serum progesterone concentration > 2 ng/ml and anestrus based on no or minimal uterine edema observed concurrent with no or minimal ovarian follicle growth, no ultrasonographic evidence of a cl and serum progesterone concentration < 1 ng/ml.23,28 suppression of cyclicity was defined as presence of anestrus during the breeding season. a return to cyclicity was defined by a period of estrus, growth and clinical theriogenology • volume 12 number 2 • june 2020126 ovulation of > 1 dominant follicles and subsequent establishment of a cl that maintained serum progesterone concentrations > 1 ng/ml for ~ 7 18 days. vaccine-induced adverse effects vaccine-induced adverse effects were monitored by twice-daily physical examination and inspection of injection site for 3 consecutive days, or until vaccine-induced adverse reaction resolved, whichever was the longest. for the first 3 days after vaccination, mares continued to be monitored by twice daily observations only (inspection of injection site and overall demeanor). all animal procedures were approved by the institutional animal care and use committee, office of animal resources, auburn university. data analyses data were analyzed using minitab®18 statistical software (state college, pa) and summarized using microsoft excel (1808, santa rosa, ca). presence of antignrh antibodies over both ovulatory seasons was analyzed using anova and was measured based on an upper 99% confidence interval of 3.32%. interestrus interval was examined using wilcoxon rank sum test. significance was set at p < 0.05. results antignrh antibody all mares were seronegative for antignrh antibody prior to first vaccination (month 1). effects of both ad-gnrh and p-gnrh vaccination are summarized in figure 1. three of 5 ad-gnrh mares responded to the initial ad-gnrh vaccination with antignrh antibody production by day 30, on the day of ad-gnrh booster. all ad-gnrh mares produced antignrh antibody following the ad-gnrh booster vaccination. maximum antibody response for within ad-gnrh mares varied but were within the range of 3.59 to 36.01% relative to the internal standard. these responses occurred at different time points for individual mares but ranged from july (month 2) to november (month 6). duration of continued antibody production also varied between individual mares and ranged from 2 to 11 months after initial ad-gnrh vaccination. all ad-gnrh mares responded to p-gnrh vaccination with production of antignrh antibody, with no response from ad-gnrh control mares. there was an effect of p-gnrh vaccination (f [1,106] = 147.17, p < 0.05). maximum individual antignrh antibody production following p-gnrh vaccination within ad-gnrh mares ranged from 35.54 to 89.72%. maximum antibody production for individual mares occurred at various time points, but occurred from july (month 14) to february (month 21). antignrh antibody was still measurable in all ad-gnrh mares at study completion (24 months). antignrh antibody was not measurable in sera collected from p-gnrh control mares prior to p-gnrh vaccination. maximum antignrh production following p-gnrh vaccination for the 2 p-gnrh control mares was 7.72 and 6.45%, which occurred during june (month 13) and july (month 14), respectively. interestrus interval following homologous prime and boost ad-gnrh vaccinations, 4 of the 5 ad-gnrh mares displayed normal iei (mean iei: 23 ± 2 days), not different from ad-gnrh control mares (mean iei: 22 ± 2 days) (p > 0.05). one ad-gnrh mare (mare #5) experienced 2 prolonged luteal phases (70 and 91 days, respectively). data for this mare were excluded for statistical analysis of iei because interestrus intervals were > 33 days, 1.5 times the interquartile range above the third quartile of all data. immediately prior to p-gnrh vaccination, 3 of the 4 remaining treatment mares displayed normal ieis (25 ± 4 days). the same mare that had experienced prolonged luteal activity during the first ovulatory season (ad-gnrh mare #5) also experienced prolonged luteal activity that extended into the second ovulatory season, but data for this mare were also not considered for analysis of iei prior to p-gnrh booster vaccination. by 30 days post p-gnrh vaccination, cyclicity was suppressed in all treatment mares. three of 4 ad-gnrh mares returned to cyclicity between 154 and 326 days following vaccination. mare ad-gnrh #5 was still in anestrus at study completion. serum progesterone concentrations and antignrh antibody responses were not clinical theriogenology • volume 12 number 2 • june 2020 127 evaluated after study completion, but continuation of twice-weekly transrectal ultrasonographic examinations revealed that anestrus persisted until 1088 days from the time that the mare received the p-gnrh vaccination. two p-gnrh control mares exhibited normal iei (27 ± 3 days). figure 1. antignrh antibody responses of individual mares following homologous ad-gnrh initial and booster vaccination and p-gnrh vaccination. time of initial and booster ad-gnrh vaccination (↑) and the p-gnrh vaccination (*). data for ad-gnrh control mares not shown. ad-gnrh mare #4 was removed from the study prior to p-gnrh vaccination, due to a chronic injury requiring euthanasia. progesterone concentrations following initial and booster ad-gnrh vaccinations, serum progesterone concentrations for 4 of the 5 ad-gnrh mares (figure 2), all of which experienced normal iei, reflected normal cyclicity. based on oncemonthly progesterone samples, there was minimal effect of seasonality on cyclicity. during months 7 and 9, only 2 of the 5 mares had baseline serum progesterone concentrations were also consistent with anestrus, based on transrectal ultrasonographic examinations. during month 8, 4 of 5 treatment mares had baseline serum progesterone concentrations. by 30 days after p-gnrh vaccination, serum progesterone concentrations of all treatment mares had declined to baseline (< 2 ng/ml). serum progesterone concentrations for the p-gnrh control mares reflected normal cyclicity throughout the study period. 0 10 20 30 40 50 60 70 80 90 100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 an tib od y r es po ns e (% o f i nt er na l s ta nd ar d) month ad-gnrh #1 ad-gnrh #2 ad-gnrh #3 ad-gnrh #4 ad-gnrh #5 p-gnrh control #1 p-gnrh control #2 positive antibody response ↑ ↑ clinical theriogenology • volume 12 number 2 • june 2020128 figure 2. progesterone concentrations for individual treatment mares (ad-gnrh) following homologous prime and boost ad-gnrh vaccination (arrows). progesterone concentrations for both ad-gnrh and p-gnrh control mares (p-gnrh control) following administration of the p-gnrh vaccine (*) during the subsequent ovulatory season (month 12). adverse vaccine reactions there were no systemic vaccine reactions observed after any vaccination. there were no vaccine site reactions observed following the initial ad-gnrh vaccination. one day following ad-gnrh booster vaccination, 3 of 5 treatment mares developed a nonpainful 1 3 cm raised nodule at the injection site that resolved within 3 days without treatment. following p-gnrh vaccination of ad-gnrh mares, 2 of 4 mares developed a small (< 2 cm) raised, nonpainful nodule that resolved without treatment within 3 days. p-gnrh control mares did not experience any adverse, vaccine-associated reactions. discussion two immunizations 30 days apart with an ad-gnrh vaccine stimulated production of anti-gnrh antibody in all treated mares, but estrous cyclicity was not suppressed. the antignrh antibody response that occurred in ad-gnrh treatment mares following p-gnrh vaccination effectively suppressed cyclicity. it is already known that p-gnrh vaccination at the labeled dose results in antignrh antibody production and suppression of cyclicity.29 at the dose used in the current study (25% of labeled initial dose), transient lowlevel anti-gnrh antibodies were produced in control mares, which had no effect on cyclicity. this confirms that the dose used in the current study was able to elicit immune memory induced by ad-gnrh priming. vaccine-mediated immune responses require not only an antigen-specific increase in antibodies, but also production of antibodies with high avidity and persistence of vaccine antibodies and/or generation of immune memory cells capable of rapid and effective expansion upon subsequent exposure.42 duration of estrus suppression was highly variable between mares, ranging from 154 days to > 35 months, consistent with other studies evaluating effects and duration of gnrh immunization in mares.25,27,28 duration of effect of the same gnrh peptide-adjuvant vaccine utilized for a heterologous vaccination strategy (similar to current study) was from 6 weeks to > 2 years.29 cyclicity was suppressed up to 2 years or more with youngest mares (≤ 4 years) experienced a longer interval from vaccination to returned cyclicity when compared to older mares (≥ 11 years), with no change in the titer.25 all mares used in the current study were ≥ 14 years of age. further research on ad-gnrh vaccination of mares warrants incorporation of evaluation of age effect on both period of anestrus following vaccination, and gnrh antibody response. heterologous vaccination strategy including administration of a p-gnrh vaccination resulted in temporary estrus suppression in 3 of 4 treated mares, whereas mare ad-gnrh #5 was still anestrus at study completion. 0 2 4 6 8 10 12 14 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 se ru m p ro ge st er on e (n g/ m l) month ad-gnrh #1 ad-gnrh #2 ad-gnrh #3 ad-gnrh #4 ad-gnrh #5 p-gnrh control #1 p-gnrh control #2 ↑ ↑ * clinical theriogenology • volume 12 number 2 • june 2020 129 evaluation of duration of estrus suppression cannot be discussed without consideration of reproductive seasonality of mares. mares naturally enter anestrus during the fall and winter. at a latitude of 38° north, the mean date of anestrus onset in young mares (< 5 years of age) is november 6th (range october to january), with an average duration of anestrus being 176 ± 30 days.43 mature mares at this same latitude (> 10 years of age) generally continue to exhibit normal cyclicity at the time that younger mares become seasonally anestrus, with the mean time of onset of anestrus occurring during the first week of january. as a result, mature mares experience a relatively shorter duration of anestrus (75 ± 18 days). all mares used in this experiment were mature (> 10 years of age); therefore, induction of estrus suppression was attributed to vaccine effects and not seasonality. additionally, all control mares continued to cycle at the time of year that treatment mares first experienced a cessation in reproductive cyclicity. the highest magnitude of antibody response was measured from treatment mares during the interval when mares may become seasonally anestrus, and a return to cyclicity was observed at a time of the season that mares would be expected to return to cyclicity as a result of seasonality, in the absence of gnrh immunization. while this makes objective measure of duration of vaccine effect challenging, all treatment mares remained anestrus as a direct result of vaccine-induced effects for least 154 ± 3 days. in a clinical setting, this interval spans the majority and in some mares the entire, ovulatory season. one of the major challenges to gnrh immunization is to overcome the phenomenon of tolerance to the gnrh antigen. a variety of carrier proteins have been used successfully to overcome tolerance and enhance induction of immunity against gnrh.9,12,23,26 the ad-gnrh vaccine used in this study contained the carrier protein bacterial leukotoxin (leukotoxin a1 gene pasteurella haemolytica). similar carrier protein used in the study was used in an earlier vaccine.45 this vaccine consisted of 8 tandem repeats of gnrh fused to each terminus of a 52 kda fragment of leukotoxin a (pasteurella haemolytica). cats and mice immunized with an ad5 vector and protein vaccines expressing multiple copies of the gnrh antigen ligated to this leukotoxin carrier peptide experienced an antibody response that inhibited gonadal development.45 vaccine used in this study had the same structure as that used safely in cats and mice. estrus suppression was not achieved by 2 immunizations with the ad-gnrh vaccine in this study. there was an apparent induction of immune memory. this is evidenced by the fact that the anti-gnrh antibody response in treatment mares was significantly different from that in p-gnrh control mare and was therefore a result of ad-gnrh priming. in addition to the construct of vaccine carrier, dose of an adenoviral vector can influence consistency and strength of the immune response to an expressed antigen.46 because there was an observed antignrh antibody response to ad-gnrh vaccination that did not effectively suppress cyclicity, it is possible that the ad5 vector dose used may have been inappropriate to elicit enough antignrh antibodies to inhibit gnrh activity. it has been shown that antibody production must reach a threshold to impede gnrh activity.23,44 immunogenicity of an ad5 vector encoding mycobacterial antigens using a heterologous prime-boost vaccination regime was evaluated in cattle.46 cattle were primed with live attenuated mycobacterium bovis vaccine (bacillus calmette–guérin vaccine) and ad5 vectored vaccine served as the heterologous vaccine. an optimum dose of 2.0 x 109 ifu of the ad5 vector given by the intradermal route conferred the most consistent and strongest immune response compared to lower doses.46 infectious unit dose used to vaccinate mares in this experiment was based on testing in laboratory mice using the accepted biopharmacological calculation-based method of determining body surface area. based on this calculation, the dose required was anywhere from 4.64 x 1010 2 x 1011 ifu. additional studies in mares are required to determine an optimal dose or route of administration that would provide an enhanced immune response to 2 immunizations with an ad-gnrh vaccine. all mares experienced minimal side effects following vaccination with ad-gnrh and again following vaccination with p-gnrh. minor reaction occurred at the injection site following ad-gnrh and p-gnrh vaccinations. following second ad-gnrh vaccination and p-gnrh vaccination, 3 of 5 and 2 of 4 mares, respectively, developed a small (< 1 cm) injection site nodule that resolved within 3 days without treatment. this finding was interesting, due to reported high incidence of reactions following vaccination with other protein-based gnrh vaccines. reported effects include raised nodules, swelling and pain at the injection site,29 stiffness of the neck, pyrexia, and apathy.23 these effects are often due to adjuvants. development of a viral vectored vaccine therefore has promise for immunizing mares against gnrh without a need for adjuvants. side effects observed in this study were not severe and would not be expected to interfere with athletic clinical theriogenology • volume 12 number 2 • june 2020130 performance. based on previous work, adenoviral vectors seemed to be able to prime and boost b cell responses more effectively than other vectors.47 this may be due to prolonged, high-level antigen expression following adenoviral vector vaccination, which favors b cell priming. since modification of cyclicity was a primary objective of this study, a nonreplicating ad-gnrh vaccine was a primary choice for suppression of cyclicity. results of our study provided “proof of concept” that mares can mount an immune response and establish immune memory following ad-gnrh prime-boost vaccination. moreover, this immune response was enhanced with a p-gnrh vaccination administered at a fraction of labeled dose. conclusion homologous ad-gnrh prime-boost vaccination using a replication-defective adenovirus vector encoding multimers of gnrh, bacterial leukotoxin and t-helper epitopes induced antignrh antibody in vaccinated mares. the dose, construct and/or frequency used in this study did not result in suppression of cyclicity. despite this, mares developed immune memory that led to suppression of cyclicity when p-gnrh was administered 12 months later. vaccinated mares experienced minimal, clinically insignificant local effects following vaccination with either ad-gnrh or p-gnrh. these results provided a promising gateway for further studies with larger numbers and to develop a cost-effective and safe ad-gnrh vaccine that can be used for reversible suppression of estrous cycle in mares. conflict of interest scot roberts is an employee of altimmune inc. and has received company stock. source of funding birmingham racing commission. acknowledgement we thank jianfeng zhang for preparation of ad-gnrh vaccine and zoetis for providing equity®. references 1. mccue p: estrus suppression in performance horses. j equine vet sci 2003;23:342-344. 2. wiepz g, squires e, chapman p: effects of norgestomet, altrenogest, and/or estradiol on follicular and hormonal characteristics of late transitional mares. theriogenology 1988;30:181-193. 3. hodgson d, howe s, jeffcott l, et al: effect of prolonged use of altrenogest on behaviour in mares. vet j 2005;169:322-325. 4. vanderwall d, marquardt j, woods g: use of a compounded long-acting progesterone formulation for equine pregnancy maintenance. j equine vet sci 2007;27:62-66. 5. rivera del alamo m, reilas t, kindahl h, et al: mechanisms behind intrauterine device-induced luteal persistence in mares. anim reproduction sci 2008;107:94-106. 6. rivera del alamo m, reilas t, kindahl h, et al: the luteostatic effect of intrauterine devices in mares is associated with endometrial expression of cox-2. anim repro sci 2010;121:227-228. 7. nie g, johnson k, braden t, et al: use of an intra-uterine glass ball protocol to extend luteal function in mares. j equine vet sci 2003;23:266-273. 8. toydemir t, kilicarslan m, olgac v: effects of the gnrh analogue deslorelin implants on reproduction in female domestic cats. theriogenology 2012;77:662-674. 9. ghoneim i, waheed m, al-eknah m, et al: immunization against gnrh in the male camel (camelus dromedarius): effects on sexual behavior, testicular volume, semen characteristics and serum testosterone concentrations. theriogenology 2012;78:1102-1109. 10. wenzinger b, kahn w, bleul u: the use of a gnrh vaccine in mares and stallions to influence undesirable behavior: a retrospective study of 31 cases. schweizer archiv fur tierheilkunde 2010;152:373-377. 11. baker h, griffin b: immunization of cats and dogs with an anti-gnrh protein vaccine with molecular adjuvantation. proceedings of the symposium on nonsurgical methods for pet population control, breckenridge, colorado, june 24-27, 2004. 12. powers j, baker d, davis t, et al: effects of gonadotropin-releasing hormone immunization on reproductive function and behavior in captive female rocky mountain elk (cervus elaphus nelsoni). biol reprod 2011;85:1152-1160. 13. finch a, caunt c, armstrong s, et al: agonist-induced internalization and downregulation of gonadotropin-releasing hormone receptors. am j physiol cell physiol 2009;297:c591-600. clinical theriogenology • volume 12 number 2 • june 2020 131 14. van zeeland y, pabon m, roest j, et al: use of a gnrh agonist implant as alternative for surgical neutering in pet ferrets. vet rec 2014;175:166. 15. grosset c, peters s, peron f, et al: contraceptive effect and potential side-effects of deslorelin acetate implants in rats (rattus norvegicus): preliminary observations. can j vet res 2012;76:209-214. 16. geyer a, daub l, otzdorff c, et al: reversible estrous cycle suppression in prepubertal female rabbits treated with slowrelease deslorelin implants. theriogenology 2016;85:282-287. 17. marino g, rizzo s, quartuccio m, et al: deslorelin implants in pre-pubertal female dogs: shortand long-term effects on the genital tract. reprod domet anim 2014;49:297-301. 18. johnson c, thompson d, cartmill j: pituitary responsiveness to gnrh in mares following deslorelin acetate implantation to hasten ovulation. j animal sci 2002;80:2681-2687. 19. johnson c, thompson d, kulinski k, et al: prolonged interovulatory interval and hormonal changes in mares f ollowing the use of ovuplant™ to hasten ovulation. j equine vet sci 2000;20:331-336. 20. fitzgerald b, meyer s, affleck k, et al: effect of constant administration of a gonadotropin-releasing hormone agonist on reproductive activity in mares: induction of ovulation during seasonal anestrus. am j vet res 1993;54:1735-1745. 21. briant c, ottogalli m, morel m, et al: use of a gnrh antagonist, antarelix, associated or not with hcg, to control ovulation in cyclic pony mares. domest anim endocrinol 2003;24:305-322. 22. samoylov a, cochran a, schemera b, et al: humoral immune responses against gonadotropin releasing hormone elicited by immunization with phage-peptide constructs obtained via phage display. j biotechnol 2015;216:20-28. 23. imboden i, janett f, burger d, et al: influence of immunization against gnrh on reproductive cyclicity and estrous behavior in the mare. theriogenology 2006;66:1866-1875. 24. pryor p, tibary a: management of estrus in the performance mare. clin tech in equine pract 2005;4:197-209. 25. schulman m, botha a, muenscher s, et al: reversibility of the effects of gnrh-vaccination used to suppress reproductive function in mares. equine vet j 2013;45:111-113. 26. tshewang u, dowsett k, knott l, et al: preliminary study of ovarian activity in fillies treated with a gnrh vaccine. aust vet j 1997;75:663-667. 27. dalin a, andresen o, malmgren l: immunization against gnrh in mature mares: antibody titres, ovarian function, hormonal levels and oestrous behaviour. j vet med a physiol pathol clin med 2002;49:125-131. 28. donovan c, hazzard t, schmidt a, et al: effects of a commercial canine gonadotropin releasing hormone vaccine on estrus suppression and estrous behavior in mares. anim reprod sci 2013;142:42-47. 29. elhay m, newbold a, britton a, et al: suppression of behavioural and physiological oestrus in the mare by vaccination against gnrh. aust vet j 2007;85:39-45. 30. van kampen k, shi z, gao p, et al: safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans. vaccine 2005;23:1029-1036. 31. tang d, devit m, johnston s: genetic immunization is a simple method for eliciting an immune response. nature 1992;356:152-154. 32. brun a, albina e, barret t, et al: antigen delivery systems for veterinary vaccine development. viral-vector based delivery systems. vaccine 2008;26:6508-6528. 33. toro h, ginkel f, tang d, et al: avian influenza vaccination in chickens and pigs with replication-competent adenovirus– free human recombinant adenovirus 5: bioone: 2010. 224-231, 34. okamba f, arella m, music n, et al: potential use of a recombinant replication-defective adenovirus vector carrying the cterminal portion of the p97 adhesin protein as a vaccine against mycoplasma hyopneumoniae in swine. vaccine 2010;28:4802-4809. 35. pacheco j, brum m, moraes m, et al: rapid protection of cattle from direct challenge with foot-and-mouth disease virus (fmdv) by a single inoculation with an adenovirus-vectored fmdv subunit vaccine. virology 2005;337:205-209. 36. lu s: heterologous prime–boost vaccination. curr opin immunol 2009;21:346-351. 37. chmielewska a, naddeo m, capone s, et al: combined adenovirus vector and hepatitis c virus envelope protein prime-boost regimen elicits t cell and neutralizing antibody immune responses. j virol 2014;88:5502-5510. 38. tang dc, zhang j, toro h, et al: adenovirus as a carrier for the development of influenza virus-free avian influenza vaccines. expert rev vaccines 2009;8:469-481. 39. toro h, van ginkel fw, tang dc, et al: avian influenza vaccination in chickens and pigs with replication-competent adenovirus-free human recombinant adenovirus 5. avian diseases 2010;54:224-231. 40. tang d, zhang j, toro h, et al: adenovirus as a carrier for the development of influenza virus-free avian influenza vaccines. expert rev vaccines 2009;8:469-481. 41. evans r, nawrocki d, isopi la, williams d, et al: development of stable liquid formulations for adenovirus-based vaccines. j pharm sci 2004;93:2458-2475. 42. siegrist c: vaccine immunology. vaccines 2008;5:1725. 43. fitzgerald b, mcmanus c: photoperiodic versus metabolic signals as determinants of seasonal anestrus in the mare. biol reprod 2000;63:335-340. 44. janett f, stump r, burger d, et al: suppression of testicular function and sexual behavior by vaccination against gnrh (equity) in the adult stallion. anim reprod sci 2009;115:88102. 45. robbins s, jelinski m, stotish r: assessment of the immunological and biological efficacy of two different doses of a recombinant gnrh vaccine in domestic male and female cats (felis catus). j reprod immunol 2004;64:107-119. clinical theriogenology • volume 12 number 2 • june 2020132 46. dean g, clifford d, gilbert s, et al: effect of dose and route of immunisation on the immune response induced in cattle by heterologous bacille calmette-guerin priming and recombinant adenoviral vector boosting. vet immunol immunopathol 2014;158:208-213. 47. geiben-lynn r, greenland j, frimpong-boateng k, et al: kinetics of recombinant adenovirus type 5, vaccinia virus, modified vaccinia ankara virus, and dna antigen expression in vivo and the induction of memory t-lymphocyte responses. clin vaccine immunol 2008;15:691-696. clinical theriogenology • volume 12 number 2 • june 2020 133   herd level factors associated with pregnancy success and distribution in beef cow-calf herds tara fountain,a robert larson,b,c brad whiteb,c atallgrass veterinary hospital, concordia, ks bbeef cattle institute, kansas state university, manhattan, ks cdepartment of clinical sciences, college of veterinary medicine kansas state university, manhattan, ks abstract identifying factors that influence pregnancy success is important in order to optimize beef cowcalf herd management. a logistic regression model was used to evaluate the effect of relevant factors (categories were: herd size, body condition score, breeding season length, and timing of start of breeding season) on the probability of pregnancy status at 2 time-points (day 21 and end of the breeding season). data were collected by convenience sampling from herds (n = 241) consisting of 8,217 head located in the midwest and great plains regions of the us from 2012 to 2017. herds with < 50 head had reduced probability of pregnancy (51 and 78%) compared to medium-sized (50 99 head) herds (63 and 84%). cows in thin body condition (score ≤ 4 out of 9) at pregnancy diagnosis had reduced probability of becoming pregnant (44 and 64%) compared to cows in moderate body condition ([score 5 6 out of 9] 62 and 86%) and fleshy condition ([score ≥ 7 out of 9] 66 and 91%). herds with a short (< 63 days) or medium (63 84 days) breeding season had increased probability of pregnancy (65 and 58%) for the first 21-day interval compared to herds with a long (> 84 days) breeding season (49%). herds that started the breeding season in the fall (september 30 december 31) had higher probability of pregnancy at the end of the breeding season (88%) compared to spring (april 1 june 30) start dates (82%). management factors (herd size, body condition score, breeding season length, and timing of start of breeding season) had substantial impacts on the probability of pregnancy. keywords: beef cow, reproductive efficiency, histogram introduction managing herds to calve in a front-loaded scenario, where 60% or more of the cows become pregnant in the first 21-day interval of the breeding season, can lead to increased pounds of calf weaned per cow exposed for breeding.1 front-loaded herds enable a larger percentage of the herd to finish the postpartum anestrus period before the breeding season begins. this creates reproductive momentum, allowing a greater percentage of the herd to be cycling and becoming pregnant early in the breeding season. in turn, this allows for more pounds weaned over a cow’s lifetime.2 progeny born in the first 21 days of the calving season will weigh more at weaning, and subsequently weigh more at later economically important times of life, heifers will have greater success during breeding, and steers will have more marbling and increased carcass value.3 herds that can attain these management goals can have economic success in both short and long terms, due to increased efficiency. veterinarians who estimate fetal age by 21-day intervals can use the resulting herd pregnancy distribution to help differentiate between potential causes of reproductive failure due to nutrition, male fertility, female fertility, or pregnancy loss.1 objective of this study was to determine if factors such as herd size, breeding season length, body condition of cow at mid pregnancy, or the time of year when breeding season starts, affected the likelihood of pregnancy at the end of the first 21-day interval and at the end of breeding season. materials and methods herd description pregnancy status data were collected from veterinarians and producers throughout the midwest. data entry methods included hand-notation on paper, computer spreadsheet, and mobile device application (pregnancy analytics app [beef cattle institute, kansas state university, manhattan, ks]). clinical theriogenology • volume 13 number 1 • march 202145   herds were included based on the availability and completeness of datasets. to be included, fetal age estimation had to be collected in a manner that allowed categorizing individual cows into 20or 21-day intervals.4,5,6,7 if a herd was presented with fetal age estimated by month or trimester, the entire herd was excluded. the initial dataset included 625 herds with pregnancy diagnosis staged into 20or 21-day intervals. only herds with maximum estimated fetal age of 140 days or fewer, and herd size > 10 head were included in the final model (figure 1). final dataset included 241 herds representing 8,217 cattle from herds ranging from 11 to 233 animals. a total of 384 herds were removed due to the exclusion criterion. each dataset included an individual cow identification number, date of pregnancy diagnosis, fetal age estimation, and a total animal count for herd size. figure 1. flow chart depicting the number of herds retained after applying each exclusion criterion. pregnancy intervals pregnancy examination data collected by transrectal palpation or ultrasonographic evaluation at a single herd examination no more than 140 days after the start of the breeding season were categorized in either 20or 21-day intervals of fetal age estimates. herds that categorized fetal age estimate data in 20-day intervals were adjusted by multiplying the percentage identified as becoming pregnant in the first 20 days of the breeding season by 1.05 (5% increase) to equilibrate to a standard percentage becoming pregnant during the first 21 days of the breeding season for all herds. percent of cows bred were evaluated for 2 endpoints: those cows that were categorized as becoming pregnant in the first 21 days of the breeding season, and those categorized as becoming pregnant by the end of the breeding season, regardless of breeding season length. categories based on factors evaluated herds were categorized based on number of cows examined for pregnancy diagnosis into 3 categories: small (10 49 heads), medium (50 99 heads), and large (100 233 heads). breeding season length was calculated across all data sources by the difference between the earliest estimated conception date and the latest estimated conception date. herds were categorized based on breeding season length into 3 categories: short (< 63 days), medium (63 84 days), and long (> 84 days). body condition score data collected at the time of pregnancy diagnosis no more than 140 days after the start of the breeding season were based on a 9-point scale, where classification 1 is associated with extremely thin cattle, and classification 9 is associated with extremely obese cattle.8 the 9-point scale was collapsed into 3 categories for the data set, with the thin category including cows classified as having a score less than or equal to 4, the moderate category including cows classified as having a score of 5 or 6, and cows classified as having a score of 7 or greater being categorized as being fleshy. the start of each breeding season was determined by finding the earliest estimated conception date in each herd. the start of the breeding season was then categorized into quarters: winter, spring, clinical theriogenology • volume 13 number 1 • march 2021 46   summer and fall. winter breeding was january 1 march 31, spring breeding was april 1 june 30, summer breeding was july 1 september 29 and fall breeding was september 30 december 31. statistical analyses before analyzing the data, only herds with > 10 head and with individual cattle diagnosed with pregnancies 140 days or fewer were included. the criterion to include only pregnancies 140 days or fewer was established to minimize inaccuracy when staging the pregnancies to 21-day intervals. all statistical analyses were conducted in rstudio (rstudio, version 3.3.3, boston, ma). a logistic regression model was used to evaluate the effect of relevant factors (herd size category, breeding season length category, body condition score category, and timing of breeding season category) on the probability of pregnancy status at 2 time-points (21 days and end of breeding season) for herds with data for all the variable categories (241 herds, 8,217 cattle). interactions were evaluated using a type iii wald (chi-square) test. results pregnancy intervals on average, 85.3% of each herd was pregnant at the time of pregnancy diagnosis, with 53.6% becoming pregnant by the end of the first 21-day interval (table and figure 2). for the first (21-day) interval model, factors that were associated (p < 0.05) with the model-adjusted probability of pregnancy included: herd size, cow body condition, and breeding season length. for the model-adjusted probability of pregnancy for the entire breeding season, factors that were associated (p < 0.05) included: herd size, body condition, and timing of the start of the breeding season. table. raw data from 241 herds (8,271 cows) mean sd minimum/maximum % pregnant in first 21 days of breeding season 53.6 18.5 5.2/100 % pregnant at end of breeding season 85.3 14.8 26.4/100 figure 2. overall raw average percent pregnant by 21-day interval throughout the breeding season for beef cow-calf herds included in the study (n = 8,217 cows). clinical theriogenology • volume 13 number 1 • march 202147   herd size herds ranged in size from 10 to 233 cows, with a median of 45. herd size was associated with the probability of pregnancy diagnosis for the first 21-day interval (figure 3a, p = 0.03, and the end of the breeding season (figure 3b, p < 0.01). for both the first 21-day interval and the overall breeding season, small herds had reduced probability of pregnancy (51 and 78%, respectively) compared to medium (63 and 84%) herds. figure 3. model-adjusted probability of pregnancy +/2 sem for the first 21-day interval (figure 3a: left chart – black columns) and overall breeding season (figure 3b: right chart – grey columns) by herd size (n=241 herds). bars without a common superscript have different model-adjusted probabilities (p < 0.05). body condition score majority (87.5%) of cows in the data set were described as having moderate body condition, whereas 4.6% were classified as thin, and 7.8% were classified as fleshy. body condition had substantial effects on first 21-day model (p < 0.01) and overall breeding season (p < 0.01). thin cows had reduced probability of pregnancy (44%) as compared to moderate (62%) and fleshy (66%) cows for the first 21-day outcome. for the end of breeding season, thin cows had the lowest probability of pregnancy (64%), whereas fleshy cows had the highest probability of pregnancy (91%). breeding season length over half the herds (65.2%) had a breeding season that was classified as short (< 63 days). breeding season length was associated (p < 0.01) with the probability of pregnancy in the first 21-day interval, with short-season length herds having an increased probability of pregnancy (65%) compared to long-season herds (49%; figure 4). there was no association (p = 0.14) between breeding season length and probability of pregnancy diagnosis at the end of the breeding season. start of breeding season the start of the breeding season was associated with the probability of pregnancy at the end of breeding season (p < 0.01), but was not associated with the first 21-day interval (p = 0.26). herds starting the breeding season in spring had reduced probability of pregnancy (82%) compared to herds starting the breeding season in the fall (88%). herds starting the breeding season in the winter and summer had numerically (but not statistically) reduced probabilities of pregnancy (81 and 78%, respectively) as compared to spring and fall. discussion although this dataset is not representative of the entire cow-calf industry, it does describe a relevant sub-set of us beef herds. similar to industry averages, the majority of the herds in this dataset clinical theriogenology • volume 13 number 1 • march 2021 48   figure 4. model-adjusted probability of pregnancy +/2 sem for the first 21-day interval by breeding season length (n = 241 herds). bars without a common superscript have different model-adjusted probabilities (p < 0.05). were < 50 head, with herds categorized as “small” herds (10 49 head) accounting for 80% of the population. cattle producers rely on cow herd productivity in order to be economically sustainable. herd size can affect profitability and efficiency, as larger herds tend to have decreased overhead costs and feed costs per head.9 in this dataset, smaller herds had reduced probability of pregnancy during the first 21 days of breeding and for the entire breeding season compared to medium-sized herds; perhaps this was a result of the larger comparative importance of off-farm income and subsequent reduced time and management directed to the cow herds’ productivity in smaller compared to larger herds. factors such as body condition at various times during the annual production cycle influence both the length of the postpartum anestrus interval10,11 and fertility,12 and should be managed accordingly. cows with lower bcs at calving have longer postpartum anestrus when compared to cows with a higher bcs.10 the current dataset indicates that cows classified as having thin body condition at pregnancy diagnosis were less likely to have become pregnant in the first 21-day interval, and by the end of the preceding breeding season. the reduction in pregnancies for thin cows could be attributed to the previously mentioned prolonged anestrus period, or as an indication of health constraints during the breeding season or early gestation that negatively impact both body condition and fertility.10,11,12 however, little research has been conducted to investigate the effect of body condition at pregnancy diagnosis on fertility during the preceding breeding season. management techniques can be implemented in order to change the reproductive momentum of beef herds. cow-calf production is limited by the 365 days in a year and a 283-day pregnancy, requiring that cows conceive within 82 days after calving to maintain a 365-day calving interval or fewer. a herd’s breeding season length can have important impacts on subsequent and future calf crops. over 50% of operations in the us (34.1% of the us cow herd) do not have a defined breeding season.13 of the operations that had defined breeding seasons, nearly 70% calved in a 3-month period.13 according to the usda nahms survey, the length of the breeding season for herds identifying as having a single defined breeding season averaged 110 days, which is nearly twice as long as the median breeding season length (60 days) of this dataset.13 based on this dataset, herds with a short breeding season length tended to have increased probability of pregnancy at 21 days compared to herds with long breeding seasons. herds with reduced time allotment to become pregnant, may have indirectly selected for cows that are capable of completing the postpartum anestrous period prior to the start of breeding or early in the breeding season. an extended breeding season results in an extended calving season that will result in an increased proportion of cows having few days available after calving to complete postpartum anestrus prior to the start of the subsequent breeding season.1 in this dataset, there was no difference between season length and probability of becoming pregnant by the end of the breeding season, most likely due to the increased clinical theriogenology • volume 13 number 1 • march 202149   mating opportunities in the long breeding season herds. the shorter season length herds had already attained 65% pregnant by the first 21-day interval, thus fewer cows were available for breeding in the remaining 21-day periods of the breeding season. considering environmental constraints, particularly periods of peak and nadir forage availability, the date to start the breeding season can impact the reproductive success of beef cow herds. improved reproductive success is associated with improved postpartum plane of nutrition, and subsequent short postpartum anestrous interval. results from this study indicate that cows exposed to bulls starting in the spring season had reduced probability of pregnancy as compared to fall breeding seasons. however, both spring and fall breeding season herds had numerically increased probability of pregnancy as compared to winter and summer breeding season herds. the impact of day length at the start of the breeding season or the change in day length post-calving and/or available forage quantity and quality at various times of the year may influence the length of postpartum anestrus and fertility of beef cows.14 herds that start their breeding season in the winter or summer may have reduced probability of pregnancy due to environmental challenges (e.g. extreme temperatures, poor forage availability) or other environmental factors. these results provided additional considerations when determining the optimum breeding season start date. conclusion within this dataset, management factors such as breeding season length, timing of the start of the breeding season, herd size, and body condition score at pregnancy diagnosis impacted pregnancy success. because of the positive association between a short breeding season and a large proportion of the herd becoming pregnant in the first 21 days of the breeding season, managing a beef cow-calf herd’s breeding season length may be useful to improve subsequent calving distributions, thereby impacting the potential calf weight weaned. optimizing herd nutrition and resulting body condition score is crucial for reproduction success. in the future, more research needs to be conducted to evaluate the economics of these management choices. conflict of interest none to declare. acknowledgement authors thank drs. bolinger, goehl, hullman, meyer, and robért, veterinarians who contributed data to this project and jiena gu for assistance with data management. references 1. larson rl, white bj: reproductive systems for north american beef cattle herds. vet clin north am food anim pract 2016;32:249-266. 2. cushman ra, kill lk, funston rn, et al: heifer calving date positively influences calf weaning weights through six parturitions. j anim sci 2013;91:4486-4491. 3. funston rn, musgrave ja, meyer tl et al: effect of calving distribution on beef cattle progeny performance. j anim sci 2012;90:5118-5121. 4. spire mf. breeding season evaluation of beef herds. in: howard jf: editor. current veterinary therapy, 2nd edition. philadelphia; wb saunders:1986; p. 808-811. 5. bretzlaff ka. pictorial guide to bovine pregnancy diagnosis. veterinary medicine 1987;82:295-304. 6. larson rl. evaluating information obtained from pregnancy examination in beef herds. veterinary medicine 1999;94:566-576. 7. larson rl, white bj: evaluating information obtained from diagnosis of pregnancy status of beef herds. vet clin north am food anim pract 2016;32:319-224. 8. wagner jj, lusby ks, oltjen jw, et al: carcass composition in mature hereford cows: estimation and effect on daily metabolizable energy requirement during winter. j anim sci 1988;66:603-612. 9. miller aj, falkner db, knipe rk, et al: critical control points for profitability in the cow-calf enterprise. app anim sci 2001;17:295-302. 10. houghton pl, lemenager rp, hendrix ks, et al: effects of body composition, preand postpartum energy intake and stage of production of energy utilization by beef cows. j anim sci 1990;68:1447-1456. clinical theriogenology • volume 13 number 1 • march 2021 50 11. perry rc, corah lr, kiracofe gh, et al: endocrine changes and ultrasonography of ovaries in suckled beef cows during resumption of postpartum estrous cycles. j anim sci 1991;69:2548-2555. 12. waldner cl, garcia guerra a: cow attributes, herd management, and reproductive history events associated with the risk of nonpregnancy in cow-calf herds in western canada. theriogenology 2013;79:1083-1094. 13. usda, part ii: reference of beef cow-calf management practices in the united states, 2007-2008. 2009:13-17. 14. cushman ra, allan mf, thallman rm, et al: characterization of biological types of cattle (cycle vii): influence of postpartum interval and estrous cycle length on fertility. j anim sci 2007;85:2156-2162. clinical theriogenology • volume 13 number 1 • march 202151 clinical theriogenology 2022; 14: 26 premature germ cells in the ejaculate of a 4-year-old male labrador retriever james barrett,a jamie douglas,b christopher premanandan,c robyn wilbornb abaytree animal hospital, valdosta, ga bdepartment of clinical sciences, college of veterinary medicine, auburn university, auburn, al cdepartment of veterinary biosciences, college of veterinary medicine, the ohio state university, columbus, oh abstract a healthy, 4-year-old, labrador retriever was presented for routine semen collection for cryopreservation. ejaculate had an abundance of atypical round cells along with substantial morphologically abnormal sperm. atypical round cells were identified as premature germ cells, indicating testicular insult or damage of unknown etiology. after multiple visits and a period (3 months) of rest from heavy training, premature germ cells were not observed and noticeable improvement in sperm morphology was evident. apparently, changes observed were induced by heat stress given the location and intensity of training, season of the year, history and signalment of the patient, and the fact that all parameters improved following temperature management changes and rest from training. this case illustrated the importance of recognizing an unusual cell type during a routine semen analysis and to arrive at a possible etiology and resolution of the clinical problem. keywords: canine, semen analysis, spermatogenesis, premature germ cells case presentation a 4-year-old, 30.6 kg (67.3 lb), black, intact male labrador retriever was presented to the auburn university college of veterinary medicine bailey small animal teaching hospital for semen collection and cryopreservation. the patient, a working field trial retriever, had returned from several months of heavy training 2 weeks before presentation. semen had been collected 6 months earlier, and the spermiogram had abnormal sperm morphology and low total sperm numbers. additionally, during an earlier visit for semen collection, patient exhibited apprehension and had decreased libido. patient was bright, alert, and responsive. vital signs were within normal limits. penis, prepuce, testes, and scrotum had no obvious abnormalities. prostate was not digitally (transrectal) palpated in an effort to reduce negative influences on libido and semen collection. an ejaculate was collected using manual stimulation and teasing was accomplished using preserved vaginal scent swabs collected from an estrous female. adequate libido was observed, and the patient readily ejaculated 2 ml (normal range: 1 30 ml1) of semen. ejaculate was white and total sperm motility, determined via subjective analysis, was 60%, with 50% progressive motility. concentration, quantified by a hemocytometer, was 67 x 106/ml with a total sperm number (tsn) of 134 x 106. ideal tsn range is 300 x 106 2 x 109, with at least 70% progressively motile and morphologically normal sperm.1,2 sperm morphology was examined using eosin-nigrosin stain and only 8% of sperm had normal shape/ structure (table 1). on wet-mount motility examination, multiple atypical round cells were identified (figure 1). table 1. sperm morphology evaluation on initial presentation for evaluation normal morphology abnormal heads abnormal midpieces tailless proximal droplets distal droplets bent tails coiled tails 8% 63% 32% 14% 2% 0% 0% 10% clinical theriogenology 2022; 14: 27 figure 1. photomicrograph of wet-mount preparation admixed with sperm (200 x magnification). note atypical round cells (premature round cells). atypical round cells identified on cytology are considered premature germ cells (pgcs) or sperm cell precursors. these cells are released prematurely from seminiferous tubules of spermatogenic epithelium and are commonly referred to as spheroids due to their characteristic round shape. their presence in semen indicated aberrations in spermatogenesis.3 patient was discharged with instructions to return in 3 weeks for further diagnostic evaluation and semen analysis. treatment after ~ 3 weeks, the patient returned for evaluation and was bright, alert, and responsive. physical examination findings were again unremarkable except for an elevated rectal temperature of 103.2ºf (normal range 100.5 -102.5ºf). semen was collected as before. libido was adequate and an ejaculate was easily collected. a low volume (0.5 ml) white ejaculate was obtained. based on subjective analysis total and progressive sperm motility were 40 and 30%, respectively. sperm concentration was 30 x 106/ml and tsn was 15 x 106. morphological evaluation using eosin-nigrosin stain revealed that only 6% of sperm had normal shape/structure (table 2). similar round cells were again observed in the spermiogram (figure 2); however, rest of the cytology was unremarkable with no neutrophils or other evidence of inflammation. the round cells were interpreted to be pgcs based on their small size and higher nuclear to cytoplasmic ratio. further diagnostic tests at this visit included the following: complete blood count and serum biochemistry profile, urinalysis, abdominal, and testicular ultrasonography, and semen culture. laboratory results were within normal limits. abdominal and testicular ultrasonographic examinations were unremarkable. semen culture had no bacterial growth. earlier, the patient had tested (rapid slide agglutination test) negative for brucella canis. results of diagnostic tests and lack of signs of systemic illness confirmed that the patient was in good overall health. atypical round cells, decreased tsn, and abnormal sperm suggested that the patient had suffered a testicular damage or insult. whereas this damage or insult had no apparent adverse impact on general health, it disrupted spermatogenesis and reduced fertility. theriogenologist overseeing the patient recommend rest from all training and vigorous exercise for 3 months, with monthly semen evaluations to determine whether resumption of normal spermatogenesis was possible. return of normal spermatogenesis would be implied by improvement of sperm quality and disappearance of pgcs in ejaculate. by brief discontinuation of training, potential thermal or psychological stressors could be removed. unfortunately, the patient was actively campaigning as a trialing retriever, and it was not feasible to stop work. continuation of training activities associated with upcoming trials superseded the need to cryopreserve semen at that period. once the current competition season ended, semen collection and reevaluation could be performed following a minimum rest period of at least 60 days. clinical theriogenology 2022; 14: 28 table 2. sperm morphology evaluation on follow-up examination 3 weeks after initial visit normal morphology abnormal meads abnormal midpieces tailless proximal droplets distal droplets bent tails coiled tails 6% 46% 32% 14% 2% 4% 14% 22% figure 2. photomicrograph of eosin-nigrosin stained preparation (200 x magnification). note 1 deeply stained round cell (premature germ cell). outcome on initial semen evaluation following a designated rest period of 3 months, the patient’s ejaculate was assessed again for possible cryopreservation. two ejaculates were collected and assessed, and improvement in sperm quality was noted. first ejaculate had 60% total motility and 50% progressive motility, and the second ejaculate had 70% total motility and 60% progressive motility. although pgcs were present, the number had substantially reduced, based on change in percentage (4 to 1% pgcs). despite improved motility parameters, sperm morphology and concentration (tsn 444 x 106; 41% normal morphology) were still less than ideal, and semen cryopreservation was not attempted. patient’s rest from training was extended by 1 more month. there was an improvement in normal sperm morphology and concentration (tsn 978 x 106; 42% normal morphology). a bitch in estrus accompanied the patient at this appointment and served as the tease source for semen collection. increased libido was observed for this collection and attributed to the presence of the female; thus future semen collections for this patient were performed with a bitch in estrus. patient returned after 1 week for semen collection. a total of 11 ml of ejaculate was collected. total sperm motility was 80% and progressive sperm motility was 70%. sperm concentration was 80 x 106/ml and tsn was 880 x 106. although the semen had only 42% morphologically normal sperm (table 3), this was a substantial improvement from previous visits and a total of 6 straws of semen were frozen, and 3 4 straws were recommended per breeding. two subsequent semen collections in the following month yielded adequate quality ejaculates and 2 additional doses of straws were cryopreserved. percentage of pgcs in serial ejaculates from the patient is summarized (table 4). quantification of pgcs was performed by a dual boarded (theriogenology and anatomic pathology) person, blinded to the case progression. counts were performed in triplicate and then averaged. results are reported as % of pgcs compared to mature sperm after counting 100 cells on the slide (e.g. out of 100 cells counted, 24 were pgcs and 76 were sperm, yielding a result of 24% pgcs). table 3. sperm morphology after the designated rest period of 4 months normal morphology abnormal heads abnormal midpieces tailless proximal droplets distal droplets bent tails coiled tails 42% 5% 5% 18% 15% 0% 18% 1% clinical theriogenology 2022; 14: 29 table 4. average percent of premature germ cells (pgcs) in serial ejaculates date pgcs 6/27/2018 24 7/19/2018 4 1/3/2019 1 2/7/2019 1 2/8/2019 0 2/14/2019 0 5/29/2019 5 6/21/2019 5 although the patient’s spermiogram never entirely normalized, pgc counts, total sperm number, and morphology parameters improved considerably after removal from training activities. even though it cannot be confirmed, heat stress during training is strongly suspected to have been the cause of testicular insult in this case. scheduling semen collection during the patient’s noncompetitive season (~ 90 days after the last competition) allowed improved sperm health, facilitated cryopreservation, and yielded a positive outcome for this patient and client. discussion testis has endocrine and exocrine roles. interstitial (leydig) cells provide endocrine function in the testis synthesizing testosterone from progesterone in response to luteinizing hormone from the anterior pituitary. sustentacular (sertoli) cells support seminiferous tubules’ germinal epithelium, wherein the exocrine product (sperm) is produced. process of sperm production is known as spermatogenesis. spermatogenesis consists of spermatocytogenesis (differentiation of spermatogonia into spermatids) and spermiogenesis (further differentiation of spermatids into sperm). during these processes, spermatogonia mitotically divide to produce primary spermatocytes that undergo meiosis to eventually produce round spermatids within the seminiferous tubules. spermatids then mature into sperm along the seminiferous tubules undergoing final maturation and storage within epididymis (figure 3). within seminiferous tubules, there is a cycle of seminiferous epithelium and spermatogenic wave. the cycle of the seminiferous epithelium refers to synchronous development of a stage of the cycle, as defined by cellular associations present at a distinct location of the seminiferous tubule. figure 3. schematic gross anatomy and microscopic representation of the process of spermatogenesis within testis (drawn by jamie douglas) clinical theriogenology 2022; 14: 30 cross-section of a particular stage of the seminiferous epithelium has multiple layers of germ cells, with each layer composed of sperm cell precursors of a distinct level of maturity. as these sperm cells mature, each successive stage of the cycle of the seminiferous epithelium results in maturing cells migrating toward the lumen of the seminiferous tubule. spermatogenic wave refers to the dynamic change of a given stage of seminiferous epithelium along the length seminiferous tubule. this results in a constantly changing site for spermiation, or release of sperm, along the seminiferous tubule. importance of cycle of the seminiferous epithelium and spermatogenic wave is that they provide continual sperm production that remains uninterrupted regardless of the frequency of ejaculation. issues (e.g. heat stress) that result in injury to spermatogenic epithelium affect spermatogenesis via disruption in succession of sperm cell lines. this disruption in succession takes time to manifest within the ejaculate and is often the reason for evidence of insult not immediately visualized in the ejaculate. similarly, effects of insult to seminiferous epithelium take time to be cleared from an ejaculate. spermatogenesis is a process that occurs over 62 days in the dog,1,4 needing a minimum of 62 days for all the effects of an injury to be cleared from the ejaculate. patient was presented for evaluation of fertility. on successive semen evaluations, the patient produced sperm with many head and midpiece defects in addition to the presence of unidentified round cells. cytologic evaluation of the ejaculate can be completed by performing a simple smear (similar to a blood smear) using undiluted semen and wright’s giemsa stain.1,4 the smear should be assessed for white blood cells, red blood cells, bacteria, and for abnormal cells. semen is not a sterile fluid, but excessive accumulation of white blood cells, blood, or bacteria can be indicative of inflammation or infection within the reproductive tract. if inflammation or infection is suspected, culture and sensitivity of the seminal fluid would be warranted. blood, inflammatory cells, or bacteria in semen warrants for additional diagnostics to further assess health and fertility. unusual type of round cells identified in this case were later confirmed to be an abnormal sperm cell precursor, or pgc. round cells identified in the ejaculate have 2 possible origins: nonspermatogenic or spermatogenic. nonspermatogenic round cells include various epithelial cells, polymorphonuclear granulocytes, macrophages, and lymphocytes. round cells of spermatogenic origin, as in this case, indicate severe disruption of spermatogenesis and appear in the ejaculate following damage of the seminiferous epithelium. migration of germ cells into the lumen of the seminiferous epithelium is a highly ordered and complex event. damage to seminiferous epithelium disrupts interconnecting bridges between developing germ cells. these disruptions result in premature release of excessive numbers of germ cells into the lumen of the seminiferous tubules, and ultimately, their release into the collecting ducts of the testis and later in the ejaculate.3 lack of any known illness at presentation of a stud dog with suspected fertility issues warrants diagnostics beyond the standard breeding soundness examination. in this patient, diagnostic tests indicated that disruption of spermatogenesis had occurred, but provided no explanation for underlying cause for the disruption. of interest, this patient’s physical examination was unremarkable except for an isolated incident of a slightly elevated rectal temperature during clinical examination. no history of previous illness was reported. for these reasons, an alternate explanation was pursued. following the veterinarian’s recommendation, the owner purchased a thermometer for surveillance of rectal temperature while working. this allowed identification of rectal temperatures of 107 108ºf in the patient following only 5 7 minutes of work via 2 controlled retrieves (light work). for perspective, a working retriever would be conditioned to perform for longer periods (20 30 minutes) of time without substantial increases in rectal temperature following short-duration exercise. confirmation of those temperatures leads us to believe that severe hyperthermia was occurring during routine training contributing to testicular thermal injury. additionally, the patient’s geographical location of training (southeastern us) and high drive to perform required tasks contributed to the likelihood of hyperthermic episodes. geographical location and increased environmental temperature can be further supported by reviewing the percentage of pgcs in the ejaculate. months with higher ambient temperatures correlated with increased pgc counts in the ejaculate. in bulls, prolonged scrotal hyperthermia resulted in severe disruption of spermatogenesis.5 bulls that experienced scrotal hyperthermia had a high number of sperm defects resulting in head and midpiece abnormalities. short-term scrotal insulation in dog had no major effects on spermatogenesis; however, long-term exposure to increased testicular temperature has not been evaluated.6 because this patient is a working dog in a humid, subtropical climate, it is likely that the patient sustained periods of elevated body temperature that resulted in testicular hyperthermia and disrupted spermatogenesis. due to the training and activities performed, the patient may have overwhelmed the process by which the body thermoregulates testicular temperature, thus sustaining thermal damage of the seminiferous epithelium. damage to vulnerable cells of the germinal epithelium within the testis would result in the severe defects observed in the patient’s spermiograms. learning points • a thorough understanding of the process and duration of spermatogenesis is required to make appropriate clinical decisions in cases of suspected male infertility. • this case illustrated that routine spermiogram allows the practitioner to identify atypical cells and critically evaluate an abnormality associated with spermatogenesis. • evaluation of disruption in spermatogenesis based on spermiograms enables clinicians to make appropriate decisions to solve the clinical problem. conflict of interest authors have no conflict of interests to disclose. contact annett annandale annett.annandale@murdoch.edu.au © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9590, http://dx.doi.org/10.58292/ct.v15.9590 research report the bovine pregnancy diagnosis challenge: evidence-based evolution of a teaching intervention over 8 years of use annett annandale,a,b henry annandale,b kate may,a dietmar holma afaculty of veterinary science, university of pretoria, pretoria, south africa, bschool of veterinary medicine, murdoch university, perth, australia abstract this article describes the implementation of an educational intervention, ‘bovine pregnancy diagnosis challenge’ over an 8-year period, and how evidence-based changes were applied to ensure the best learning outcome for bovine pregnancy diagnosis (pd) via transrectal palpation. with the worldwide shift from mainly knowledge-based to competency-based veterinary courses, many new and innovative teaching interventions have been introduced into veterinary education. these teaching interventions include but are not limited to simulators, models, virtual realities, applications, and computer-assisted learning tools; of which, many have been implemented into various veterinary training programs. while these advances are noteworthy, the question still remains as to whether or not the implementation of these teaching interventions is optimal. do we know the ‘when,’ ‘who’, and ‘how’ of a specific intervention that will optimize the educational outcome? the described evolution of the bovine pd challenge shows how these questions were investigated for transrectal palpation skills, and how changes were implemented to optimize student training. this is an example of an approach that could be applied widely for validation and skills training investigations within veterinary education to optimize learning outcomes. keywords: teaching intervention, bovine pregnancy diagnosis, transrectal palpation, clinical hands-on skills introduction by definition, a teaching intervention is ‘a specific program or set of steps to address an academic need.’ in educational settings, these interventions are often focused teaching sessions that are different from existing or conventional teaching practices. with the worldwide shift from mainly  knowledge-based to competency-based veterinary courses,1–8 many new and innovative teaching interventions have been introduced into veterinary education in the past 20 years. these changes have resulted in veterinary schools graduating entry-level veterinarians with advanced skills.9,10 the increased expectations of veterinary graduates, as highlighted by the ‘day one competency and year one skills’ lists published by veterinary governing bodies, include not only theoretical knowledge but also professional attitudes and clinical hands-on skills.1–4,9 traditionally, hands-on experience was ensured through live animal and cadaver exposure, but more recently, skills acquisition opportunities have become limited by welfare and ethical concerns about the use of live animals for training purposes, large student cohort sizes, budget constraints, and difficulty sourcing cadaver materials.6,9,11–21 the development of a variety of teaching interventions of varying fidelity levels was inspired by balancing the demands of a strongly skills-oriented veterinary education with respecting societal expectations of higher ethical standards. these teaching interventions include but are not limited to simulators, models, skills laboratories, virtual realities, applications, online programs, and computer-assisted learning tools9,11–13,15,22,23; of which, many have been implemented into various veterinary training programs.24–33 while these advances are noteworthy, the question still remains as to whether or not the implementation of these teaching interventions is optimal. do we know the ‘when,’ ‘who,’ and ‘how’ of a specific intervention that will optimize the educational outcome? this article describes the implementation of an educational intervention over an 8-year period, and how evidence-based changes were applied to ensure the best learning outcome for bovine pregnancy diagnosis (pd) via transrectal palpation (trp). pd via trp is an important competency for graduating veterinarians planning to work in large animal practice. this skill is, however, challenging to teach, and the competence level of students after trp training is difficult to assess. therefore, mailto:annett.annandale@murdoch.edu.au http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9590 citation line: clinical theriogenology 2023, 15, 9590, http://dx.doi.org/10.58292/ct.v15.9590 13 the  ‘pregnancy diagnosis challenge’ (pd challenge) was developed. the aims of this teaching intervention were to establish a way of accurately measuring students’ trp competence and to incentivize student learning by identifying the pd challenge champion who, based on the pd assessments done on live cows, had the highest pd accuracy (sensitivity and specificity).34,35 at the same time, a longitudinal research project was implemented to enable the evaluation  of the learning outcome as well as various teaching and  training interventions aimed at fast-tracking pd competency.34,36–39 design and evidence-based evolution of the pregnancy diagnosis challenge and results the pd challenge was set up to consist of pd via trp training, followed by a knock-out competition where students were graded on their overall pd accuracy, sensitivity, and specificity in live cow trps. the setup of the pd assessment has been described.34–36 the best participants of the first round took part in the final round of the pd challenge, where they performed more pds in both beef and dairy cattle within a set time limit, which made it possible to identify the most accurate student under time pressure and crown the pd challenge champion. the university of pretoria’s 6-year bachelor of veterinary science (bvsc) program includes 9 semesters of didactic preclinical training and 3 semesters of clinical, work-integrated learning during the last 18 months of study.40 the pd challenge was part of the preclinical training in 2014 and 2016 and was moved to later stages of the curriculum as part of the work-integrated learning from 2018 onward. the first 2 pd challenges (2014 and 2016) were designed to determine if pd accuracy could be reliably and efficiently measured in a large group of students, and if so, to establish baseline values for student pd accuracy.34,35,36 the 2nd to 4th pd challenges (2016, 2018, and 2019) were used to answer the questions of ‘when’ the pd challenge should be done (curricular implementation), ‘who’ (which students should participate), and ‘what’ in terms of training was best to optimize the learning outcomes by adjusting student cohorts, training methods, and student exposure.36,37 more recent iterations of the pd challenge (5th and 6th versions, 2021 and 2022, respectively) included the implementation of known methods to improve pd accuracy based on our previous research, and the use of an objective structured clinical examination (osce)like assessment on trp of non-pregnant cows,35 to tailor 2 parallel programs within the pd challenge for students who are either already competent in bovine trp or who still need improvement. the first pd challenge in 2014 was done within 1 semester of the didactic 4th year veterinary reproduction module, which included the initial student trp and pd training for the entire 4th year cohort (n = 138).34 until 2014, students’ posttraining trp skill level and pd accuracy had not been measured, and the outcome of the training was unknown. the initial trp training consisted of palpation of bovine abattoir-derived reproductive organs and university-owned non-pregnant cattle and was followed up by pd training on either breed’n betsy®a rectal examination simulators or on beef cows at a university satellite facility.34 the first pd challenge therefore gave a baseline and insight into our 4th year students’ pd capabilities and allowed for the evaluation of simulator versus live cow pd training.34 while the 4th year student trp competence is not expected to be at the same level as that of experienced large animal practitioners who routinely perform pds, a specificity (defined as students’ ability to correctly identify non-pregnant cows) of 41% indicated the priority area that needed improvement (table). in order to expose students to additional ‘refresher’ training on rectal examination simulators and trp in live, non-pregnant cows, the 2016 pd challenge was moved to the 5th year student cohort (n = 128) after the initial trp and pd training in their 4th year.35,36 interestingly, this additional training did not appear to improve student performance or improve pd accuracy (table). from 2018 onward, the pd challenge was organized as a 2-week block. the first week entailed an intense training program dedicated to trp training and consisted of a mandatory day of supervised bovine trp and pd training at the teaching institution, supervised and executed by experienced faculty. the training included inspection and palpation of abattoir-derived specimens of bovine female reproductive organs, skills laboratory exposure to rectal examination simulators (breed’n betsy®a and haptic cowb), and exposure to theoretical information on bovine trp and pd for self-study. students were then exposed to 2 days of live cow pds in large animal practices, which was supervised by experienced veterinarians. the training was then followed by the first and final round of knock-out competitions during the second week.37 the pd challenge was also moved from 5th to final year and was made available only to students selecting the production elective (n = 59) during their 3 semesters of workplace-based training. during the 2018 pd challenge, students were given focused access to live cow palpations during this later stage of their curriculum. results of pd assessments performed during the 2014, 2016, 2018, and 2019 pd challenges are summarized (table). table. overall student pd accuracy, sensitivity, and specificity for 4th, 5th, and final year student cohorts 2014 2016 2018 2019 4th year student cohort 5th year student cohort final year student cohort final year student cohort pregnancy rate (%) 62 49 64 66 cows ≤ 6 months pregnant (%) 46 70 69 64 overall student pd accuracy (%) 71 61 82 85 sensitivity (%) 87 79 88 89 specificity (%) 41 42 67 79 http://dx.doi.org/10.58292/ct.v15.9590 14 citation line: clinical theriogenology 2023, 15, 9590, http://dx.doi.org/10.58292/ct.v15.9590 discussion results demonstrated that the various training interventions and advancing from preclinical to clinical student cohorts for trp and pd training improved students’ pd accuracy. improvement in specificity is particularly encouraging and shows that in 2014, students would only have recognized 1 out of 3 (33%) non-pregnant cows correctly,34 in 2018, this had advanced to 2 out of 3 (66%),37 and to 4 out of 5 (80%) in 2019. the differing pregnancy rate and range of pregnancy stages in 2014 and 2016 can account for the discrepancies in student pd accuracy and sensitivity across these 2 years. in 2014, the pregnancy rate was 62% compared to 49% in 2016, with 46% and 70% of pregnant cows < 6 months pregnant for 2014 and 2016, respectively (table). both sets of results showed that students’ pd sensitivity is higher in cows > 6 months pregnant,34,36 so the higher percentage of cows < 6 months pregnant in combination with the lower pregnancy rate in general in the 2016 study made a correct diagnosis more difficult and explains the lower student pd accuracy and sensitivity. the stage-corrected sensitivity (defined as the proportion of pregnant cows in which pregnancy stage was correctly identified by the student) was 43% for final-year students who had received a 1-week intense trp and pd training program (2018).37 this was higher than the reported 31% for a 5th student cohort (2016)36 but is an area that could still be improved. from 2018 onward, only students who had chosen a production animal as elective as part of their 3 final year work-integrated learning semesters were able to enroll in the 1 week intense bovine pd training.37 the student cohorts were therefore smaller (n = 59) and were composed of students with a specific interest in food animal practice, usually with additional hands-on clinical experience in production animals. the majority of these student participants had been exposed to bovine trps and pds during their clinical rotations in reproduction, herd health, as well as private practice placements. another change from 2018 onward was to provide students with access to live cow palpation opportunities in large animal private practices and to take this training load away from the university.37 participating veterinary practices were identified and contacted by the pd challenge organizers to ensure real-life workplace-based exposure for pd challenge students. financial support for student travel was secured through the teaching institution and allocated on an individual student basis. placing the pd challenge into the final-year of veterinary training has subsequently been proven successful, and the overall pd accuracy, sensitivity, and specificity were higher in 2018 and 2019 compared to previous student cohorts (table). the advancement of pd specificity from 41% in 2014 to 79% in 2019 was a major improvement in this important variable and highlighted the importance of ‘when,’ ‘who,’ and ‘what’ for enhancing training outcomes. some changes in 2018 included the need for students to pass a validated trp osce35 prior to being allowed to proceed to the allocated 2 days of live-cow palpation practice with a private practitioner before the pd challenge. in 2019, the live-cow trp exposure at private practices was increased from a 2 day to a 4 day period. in 2020, the pd challenge was cancelled due to the disruptions in the clinical training program brought about by the covid-19 pandemic; however, in 2021, the pd challenge was resumed in a similar format to that of 2019. over the years, the small number of finalists identified during the op pd challenge received additional exposure when they qualified for the final round of the challenge, during which, the pd champion was determined. after the 2021 pd challenge, the organizers realized, however, that those students who did not qualify for the final round are in fact the ones who would benefit most from additional exposure, and the program was therefore again revised for the 2022 season by making 2 pertinent changes. the first change was that students were allowed to spend 1 week in private practice for trp training prior to the pd challenge week, and this placement week could be from 4 months prior to the pd challenge. this was done in response to comments received from private veterinarians, indicating that the placements did not always happen at a convenient time in their herd health programs, and this change made it possible for students to attend practices during weeks when they could benefit the most from the exposure provided by the practitioner. the second important change made in 2022 was that the 1 week program of the pd challenge was started with an osce-type assessment on a live non-pregnant cow, where students were assessed on their trp skills. this osce-type assessment was previously validated and is known to correlate with pd accuracy.35 based on the outcome of this osce-type assessment, students were assigned to 2 groups: ‘challengers’ and ‘qualifiers’. the latter group was those with lower assessment scores, and an alternate program was designed for them to ensure more structured and intensively guided trp training via ultrasonography on non-pregnant cows to provide immediate and self-driven feedback, and intensive one-on-one pd training on cows in the third trimester of pregnancy in one of the institution’s training herds. the ‘qualifiers’ then had a pd challenge on their own but on the same herd as those palpated by the ‘challengers.’ some of the challenges encountered during implementation of this teaching intervention included how to accurately measure student skill level for pd via trp after training, how to collect research data while teaching and how to motivate students to perform at their best to gather good and meaningful research data. to increase student motivation and incentivize students, through industry partner involvement, prestigious prizes were offered to the pd champion. summary the pd challenge was initially set up to include entire student cohorts (4th and 5th year student cohorts in 2014 and 2016 with 138 and 128 students, respectively) and moved subsequently to only enable students with an active interest in production animal practice as a career choice as part of the elective program to enroll. this shift allowed for more intense training and exposure of these students and allowed the organizers to overcome the challenge of limited training opportunities outside the veterinary training institution. an iterative process of continuously assessing learning outcomes and modifying our training approach allowed us to answer the following questions for this specific teaching intervention: ‘when’ (curricular implementation): as part of the elective during the final 3 semesters of workplace-based experience; ‘who:’ final year students with an interest in large animal practice; http://dx.doi.org/10.58292/ct.v15.9590 citation line: clinical theriogenology 2023, 15, 9590, http://dx.doi.org/10.58292/ct.v15.9590 15 ‘what:’ an intense 1 week training program including live animal trp and pd exposure as well as voluntary repeat/refresher model and simulator training. the pd challenge is now an annual elective event and shows encouraging results in assisting students to become competent in bovine pd via trp while overcoming some of the previously reported challenges in live animal exposure. the development of this teaching intervention and the changes implemented over a number of years in response to varying challenges could be used as an example for other scenarios to optimize learning outcomes.39 furthermore, the concept of ‘blending a fun student project with research while teaching’ makes best use of both time and effort in collecting research data while also allowing for teaching and motivating students to perform at their best.39 this approach could be implemented widely for validation and skills training investigations within veterinary education. conflict of interest authors have no conflict of interest to report. authors’ contributions annett annandale: conceptualization, methodology, investigation, resources, writing, review, and editing. henry annandale: initial teaching intervention idea, conceptualization, methodology, writing, review, and editing. kate may: methodology, investigation, writing, review, and editing. dietmar holm: conceptualization, methodology, investigation, resources, writing, review, and editing. references 1. bok hgj, jaarsma dadc, teunissen pw, et al: development and validation of a competency framework for veterinarians. j vet med educ 2011;38:262–269. doi: 10.3138/jvme.38.3.262 2. bok hgj, teunissen pw, boerboom tbb, et al: international survey of veterinarians to assess the importance of competencies in 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citation line: clinical theriogenology 2023, 15, 9590, http://dx.doi.org/10.58292/ct.v15.9590 25. baillie s, mellor dj, brewster sa, et al: integrating a bovine rectal palpation simulator into an undergraduate veterinary curriculum. j vet med educ 2005;32:79–85. doi: 10.3138/jvme.32.1.79 26. langebaek r, toft n, eriksen t: the simspay-student perceptions of a low-cost build-it-yourself model for novice training of surgical skills in canine ovariohysterectomy. j vet med educ 2015;42:166–171. doi: 10.3138/jvme.1014-105 27. read ek, vallevand a, farrell rm: evaluation of veterinary student surgical skills preparation for ovariohysterectomy using simulators: a pilot study. j vet med educ 2016;43:190–213. doi: 10.3138/ jvme.0815-138r1 28. greenfield cl, johnson al, schaeffer dj, et al: comparison of surgical skills of veterinary students trained using models or live animals. j am vet med assoc 1995;206:1840–1845. 29. gopinath d, mcgreevy pd, zuber rm, et al: developments in undergraduate teaching of small-animal soft-tissue surgical skills at the university of sydney. j vet med educ 2012;39:21–29. doi: 10.3138/jvme.0411.044r 30. griffon dj, cronin p, kirby b, et al: evaluation of a hemostasis model for teaching ovariohysterectomy in veterinary surgery. vet surg 2000;29:309–316. doi: 10.1053/jvet.2000.7541 31. holmberg dl, cockshutt jr, basher awp: use of a dog abdominal surrogate for teaching surgery. j vet med educ 1993; 20:61–62. 32. fahie m, cloke a, lagman m, et al: training veterinary students to perform ovariectomy using the moose spay model with traditional method versus the dowling spay retractor. j vet med educ 2016;43:176–183. doi: 10.3138/jvme.0915-150r 33. annandale a, scheepers e, fosgate gt: the effect of ovariohysterectomy model practice on surgical times for final-year veterinary students’ first live-animal ovariohysterectomies. j vet med educ 2020;47:44–55. doi: 10.3138/jvme.1217-181r1 34. annandale a, annandale ch, fosgate gt, et al: training method and other factors affecting student accuracy in bovine pregnancy diagnosis. j vet med educ 2018;45:224–231. doi: 10.3138/ jvme.1016-166r1 35. annandale a, fosgate gt, bok h, et al: ability of a bovine transrectal palpation objective structured clinical examination to predict veterinary students’ pregnancy diagnosis accuracy.  vet rec 2019;185:171–171. doi: 10.1136/vr.105022 36. annandale a, fosgate gt, eksteen ca, et al: influence of an exercise program, muscle strength, proprioception, and arm length on veterinary students’ bovine pregnancy diagnosis accuracy. j vet med educ 2020;e20190043. 37. annandale a, may ce, van der leek ml, et al: effect of a high-intensity one-week training programme and student-level variables on the bovine transrectal palpation and pregnancy diagnosis skills of final-year veterinary students. vet rec 2020;187:e99. doi: 10.1136/vr.105909 38. annandale a, fosgate gt, eksteen ca, et al: electromyographic analysis of muscle activation patterns during bovine transrectal palpation and the development of the bovine pregnancy diagnosis improvement exercise program. j vet  med  educ 2021;48:686–697. doi: 10.3138/jvme-2020 0039 39. annandale a: thinking differently about clinical skills training: the bovine pregnancy diagnosis via transrectal palpation showcase. utrecht university: 2020. [cited 2020 july 6] available from: https://dspace.library.uu.nl/handle/1874/397523. 40. irons pc, holm de, annandale ch: curricular renewal at the southern tip of africa: the 2016 veterinary curriculum at the university of pretoria. j vet med educ 2017;44:440–449. doi: 10.3138/jvme.0316-062r http://dx.doi.org/10.58292/ct.v15.9590 https://doi.org/10.3138/jvme.32.1.79 https://doi.org/10.3138/jvme.1014-105 https://doi.org/10.3138/jvme.0815-138r1 https://doi.org/10.3138/jvme.0815-138r1 https://doi.org/10.3138/jvme.0411.044r https://doi.org/10.1053/jvet.2000.7541 https://doi.org/10.3138/jvme.0915-150r https://doi.org/10.3138/jvme.1217-181r1 https://doi.org/10.3138/jvme.1016-166r1 https://doi.org/10.3138/jvme.1016-166r1 https://doi.org/10.1136/vr.105022 https://doi.org/10.1136/vr.105909 https://doi.org/10.3138/jvme-2020-0039 https://doi.org/10.3138/jvme-2020-0039 https://dspace.library.uu.nl/handle/1874/397523 https://doi.org/10.3138/jvme.0316-062r 1 contact carlos pinto cpinto@lsu.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9437, http://dx.doi.org/10.58292/ct.v15.9437 case report canine sertoli cell tumor: anti-müllerian hormone, inhibin b, and estrone sulphate fiona herzog,a daniella adams,a alissa st. blanc,a jeongha lee,b ingeborg langohr,b carlos pinto,a adepartment of veterinary clinical sciences, bdepartment of pathobiological sciences and louisiana animal disease diagnostic laboratory, louisiana state university school of veterinary medicine, baton rouge, la, usa abstract a unilaterally castrated male boxer was referred for chronic generalized alopecia and pruritus. physical examination revealed symmetrical alopecia, pendulous prepuce, lichenification of the scrotum, and enlargement of mammary papillae. penile/preputial cytology revealed superficial epithelial cells. transabdominal ultrasonographic examination revealed a globoid mass with heterogenous echogenicity in the left caudal abdomen. the presumptive diagnosis was sertoli cell tumor (sct) associated with cryptorchidism. exploratory laparotomy and histology of the removed mass confirmed the diagnosis. three weeks after surgery, serum anti-müllerian hormone (amh) concentrations decreased from 8,435 to 56 ng/ml and inhibin b decreased from 805 to < 6 pg/ml. two months after surgery dermatoses subsided and there was substantial regression of enlarged nipples. this report highlights the diagnostic value of practical procedures (penile/preputial cytology, transabdominal ultrasonography, and measurement of serum amh and inhibin b concentrations) to aid in the diagnosis of cryptorchidism and sct, especially in patients with generalized skin conditions. keywords: cryptorchidism, feminization, paraneoplastic syndrome background canine sertoli cell tumor (sct) is the most common testicular neoplasm associated with feminization syndromes. the prevalence of feminization in cases of sct has been reported as 241 and 39%,2 with an increased frequency of the syndrome occurring in cryptorchid testes.2 clinical signs of feminization are consequent to hyperestrogenism associated with a secretory tumor. these include bilateral symmetrical alopecia of the trunk and flanks, hyperpigmentation of inguinal skin, gynecomastia, pendulous prepuce, squamous metaplasia of the prostate, and attraction by male dogs. preputial cytology is a useful diagnostic aid in cases of sct to determine the effects of estrogens on preputial epithelial cells; a bioassay for hyperestrogenism particularly, for dogs with feminizing signs. additional methods of diagnosis include imaging, exploratory surgery, and histology. limited studies have evaluated the endocrine characteristics of canine sct. serum estradiol concentrations had variable elevation,3–6 whereas inhibin concentrations increased in sct dogs compared to controls.3,5 serum anti-müllerian hormone (amh) concentrations were determined in patients with testicular and other tumors.6 these findings suggested that canine scts secrete several hormones that have diagnostic potential. this report describes a case of canine sct with feminization syndrome and evaluation of serum amh, inhibin b, and estrone sulphate concentrations before and after tumor removal. case presentation a 6-year unilaterally castrated (at 6 months of age) male boxer was presented with a history of chronic alopecia and pruritus. approximately 18 months after castration, the patient developed dermatoses and was treated for presumed atopic dermatitis for several years until referral. at presentation, the patient’s dermatologic lesions included diffuse alopecia, moderate to severe lichenification and scaling along the sternum, axillary region, and on medial thighs (bilateral). the scrotal skin had lichenification and edema, and the prepuce was moderately pendulous and edematous. mammary papillae were enlarged, elongated, and mildly edematous (figures 1 and 2). the owner reported that changes to the prepuce and mammary papillae had developed concurrent with signs of alopecia. the patient was receiving 6 mailto:cpinto@lsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9437 2 citation line: clinical theriogenology 2023, 15, 9437, http://dx.doi.org/10.58292/ct.v15.9437 monthly injections of lokivetmab (a monoclonal antibody that acts against interleukin 31) for suspected atopic dermatitis. preputial cytology revealed superficial epithelial cells. transabdominal ultrasonography revealed an oval to globoid mass (4.2 × 3.3 cm) with irregular contour (suspected left testis), located ventro-cranially and slightly left to the urinary bladder. the mass appeared moderately cystic and heterogenous in echogenicity, with no distinct mediastinum testis (figure 3). mild to moderate iliac lymphadenopathy and splenic nodules were observed. prostate measurements (35 × 32 × 40 mm) were within normal parameters for an intact dog of the patient’s size and age.7,8 fine needle aspirates of the left medal iliac lymph node and spleen were consistent with reactive lymphoid hyperplasia and splenic nodular hyperplasia with low-grade extramedullary hematopoiesis, respectively. thoracic radiographs had no evidence of pulmonary metastases. complete blood cell count and serum biochemistry revealed no abnormal findings. treatment an exploratory laparotomy was performed. the suspected retained left testis was lateral and adjacent to the left aspect of the urinary bladder. the left testis and a section of the spermatic cord with which it was associated, and the enlarged left medial iliac lymph node were surgically removed. tissues were submitted for histological evaluation. pathology the left testis had a multinodular appearance and measured 5.0 × 4.0 × 3.5 cm (figure 4). the cut surface had a tan, soft, figure 3. oblique ultrasonographic image of the irregular mass (33.2 mm between calipers a and 41.8 mm between calipers b) of suspected retained left testis (note moderate cystic and heterogenous echogenicity appearance) figure 1. sternum, mammary gland, and prepuce (note diffuse alopecia across sternum, enlargement of mammary papillae, and pendulous prepuce) figure 2. mammary gland and prepuce (note diffuse ventral alopecia, enlarged mammary papillae, and pendulous prepuce) http://dx.doi.org/10.58292/ct.v15.9437 citation line: clinical theriogenology 2023, 15, 9437, http://dx.doi.org/10.58292/ct.v15.9437 3 multinodular mass with multiple cysts containing brown fluid. microscopic examination of the abdominal mass confirmed its testicular origin. structures identified included seminiferous tubules, epididymis, and pampiniform plexus. the testis appeared enlarged and partially replaced by a neoplastic mass consistent with a sct of diffuse growth pattern (figure 5a). the neoplasm was partially encapsulated, infiltrative, multilobulated, composed of polygonal to elongated cells (figure 5b) forming variably sized, coalescing lobules separated by a moderate amount of fibrous stroma and palisading along the connective tissue trabeculae. neoplastic cells had distinct cell borders, eosinophilic to amphophilic, finely granular or clear vacuolated cytoplasm, and round to oval nuclei with finely stippled chromatin and a variably distinct nucleolus. mild anisocytosis and anisokaryosis were present, with a mitotic count of 15 per 2.37 mm2 (equivalent to 10 fn22/400x fields or 10 high-power fields) (figure 5d). frequent neoplastic cells within blood vessels (tumor emboli) were also noted, including a small cluster of neoplastic cells in the pampiniform plexus (figure 5c). seminiferous tubules that remained at the periphery of the neoplasm were small and devoid of germ cells, denoting marked atrophy (figure 5d) and the epididymis was similarly observed that was devoid of sperm. no evidence of metastasis was detected in the left medial iliac lymph node. endocrinology serum samples were sent to uc davis endocrinology laboratory for amh, inhibin b, and estrone sulphate analysis by enzyme-linked immunosorbent assay (elisa). serum was collected prior to and 3 weeks after surgery. serum amh concentrations decreased from 8,435 to 56 ng/ml after tumor removal (reference interval reported as < 0.15 ng/ml for castrated dogs; ansh labs, webster texas). serum inhibin b concentrations similarly decreased from 807 to < 6 pg/ml. estrone sulphate concentrations were 1.5 ng/ml before surgery and 1.3 ng/ml after surgery (reference ranges for estrone sulphate and inhibin b were not reported by the laboratory). outcome dermatoses resolved 6–8 weeks after surgery. the enlargement of the mammary papillae was largely resolved by 6 months after surgery. cranial thoracic mammary papillae were still mildly enlarged; however, the remaining papillae had returned to normal size. the patient’s signs of pruritus were satisfactorily controlled with biannual injections of lokivetmab. consultation with the oncology service for tumor surveillance was recommended 3 months after tumor removal but was declined by the owner. discussion the patient was presented with a history of chronic alopecia that poorly responded to long-term treatment for allergic dermatitis. an estrogen-secreting sct was the cause of the patient’s hair loss. hyperestrogenism due to exogenous or endogenous sources of estrogen is a known cause of noninflammatory alopecia.9 clinical signs of alopecia associated with hyperestrogenism are derived from the binding of estrogen-to-estrogen receptor α that is involved in the regulation of hair follicle cycle. this binding alters the anagen-telogen phase transition of the hair follicle cycle by inducing premature catagen (regression stage) and prolonging telogen (resting phase).10 this results in a failure of hair growth that can mimic inflammatory and other noninflammatory causes of alopecia. additional signs of feminization, including elongation of the mammary papillae and  pendulous prepuce during clinical examination, prompted assessment for potential exogenous or endogenous sources of estrogen. this highlighted the importance of a thorough and problem-based approach to the clinical investigation of alopecia in male dogs. the persistence of mild pruritus after resolution of alopecia suggested that an unrelated source of inflammatory dermatitis was possible. superficial epithelial cells comprised the dominant cell type identified on preputial cytology, consistent with hyperestrogenism. preputial cytology has previously been demonstrated to be a useful investigative tool in cases of sct, in which the presence of > 20% superficial epithelial cells, as a predictor of elevated serum estradiol concentrations, had a sensitivity and specificity of 80 and 90%, respectively.11 in addition to preputial cytology, abdominal ultrasonography was an important diagnostic modality employed to determine the cause of feminization in the current patient. abdominal ultrasonography permitted identification and localization of the retained testis, facilitating a rapid diagnosis and preparation for surgical removal. anti-müllerian hormone amh is a glycoprotein hormone that belongs to the transforming growth factor (tgf) β-family.12 secretion of amh from sertoli cells is most pronounced during fetal figure 4. photograph of retained left testis (note multinodular appearance and irregular contour) and pampiniform plexus to its right http://dx.doi.org/10.58292/ct.v15.9437 4 citation line: clinical theriogenology 2023, 15, 9437, http://dx.doi.org/10.58292/ct.v15.9437 development and prior to puberty. in male fetus, amh signals mullerian duct regression, preventing formation of uterine tubes, uterus, and cranial vagina.13 amh may have a role in regulating leydig cell proliferation, function, and testosterone production.14 during fetal development and prior to puberty, follicle-stimulating hormone (fsh) promoted the production of amh whereas gonadal testosterone had an inhibitory effect on amh secretion after puberty onset.15 however, the basal expression of amh throughout life is independent of gonadotropins.16 amh has been a useful predictor for the presence of testicular tissue in dogs,17,18 horses19 and calves20 for scrotal and cryptorchid testes. limited studies have also investigated its value as a clinical biomarker for canine sct. immunohistochemical analysis of 24 sct expressed amh.21 in the same study, the sertoli cells of unaffected fetuses and pups < 45 days also expressed amh whereas the sertoli cells of older pups and adult dogs did not. the contrast in amh expression between normal adult and sct testes may attest to the diagnostic potential of amh in canine sct. serum amh concentrations in dogs with scts were > 22 ng/ml compared to control dogs that returned to normal concentrations (< 10 ng/ml).6 in this study, concentrations of serum amh in csct patients were significantly elevated compared to dogs in both control and other tumor groups. serum amh concentration declined precipitously after surgery. a similar trend was observed22 where a patient that was investigated for nonpruritic alopecia had markedly elevated serum amh concentrations compared to 2 intact control dogs. a sct affected cryptorchid testis was identified, and 3 months following surgical removal, serum amh concentrations declined in this patient to concentrations similar to 2 castrated control dogs. serum amh concentrations after surgery were 56 ng/ml. serum amh concentrations reference ranges (ansh labs, webster, texas) for intact and castrated male dogs are 0.2–73.4 ng/ml and < 0.15 ng/ml, respectively. in this patient, amh concentrations therefore did not decline to baseline concentrations 3 weeks after surgery. there are no reports on the halflife of amh in dogs, although a relatively prolonged half-life of 1.5 days has been described in stallions.19 persistence of amh above baseline concentrations 3 weeks after surgical removal of the sct may reflect a similarly long half-life in figure 5. representative photomicrographs of histology of cryptorchid left testis. (a) sertoli cell tumor compressing the markedly atrophied testicular parenchyma; bar = 1 mm. (b) polygonal to elongated neoplastic cells palisading along the connective tissue trabeculae. a mitotic figure is in the center (arrow); bar = 50 µm. (c) small clusters of tumor emboli (arrows) present within a vein in the pampiniform plexus; bar = 100 µm. (d) atrophic testicular parenchyma with sertoli cells in shrunken seminiferous tubules devoid of germ cells; bar = 200 µm. http://dx.doi.org/10.58292/ct.v15.9437 citation line: clinical theriogenology 2023, 15, 9437, http://dx.doi.org/10.58292/ct.v15.9437 5 dogs, the marked elevation in serum amh prior to surgery, or the presence of a secondary tumor. inhibin b inhibin is a glycoprotein hormone that contains an α and 1 of 2 possible β subunits designated βa (inhibin a) or βb (inhibin b).23 inhibin b is the predominant isoform in the adult male of several species including boar,24 stallion,25 rat,26 and human.27 inhibin exerts a regulatory role on fsh secretion from the anterior pituitary gland. production of inhibin is stimulated by fsh, whereas inhibin has a conversely inhibitory effect on fsh release, completing a negative feedback loop.28 the principal source of inhibin has historically been considered to be sertoli cells.28 however, inhibin subunits were expressed in leydig or germ cells and the cellular source of inhibin varied with species and changed during transitions from fetal, neonatal, and adult life.29–33 inhibin α expression was only detected in sertoli cells of neonates,34,35 whereas inhibin was expressed in leydig cells of both neonate and adult testes.35,36 these studies indicated a possible shift of inhibin production from sertoli to leydig cell origin from neonatal to adult life in dogs. reports on inhibin expression in canine scts have been inconsistent. inhibin α was expressed in 13 of 21 canine sct34 and none of the 5 canine scts.36 serum inhibin b in this case markedly declined from 805 ng/ml prior to surgery to nondetectable concentrations (< 6 pg/ml) at 3 weeks after tumor removal. concentrations of serum inhibin b have not been reported in dogs. inhibin detected by radioimmunoassay (ria) has been reported for normal intact and castrated dogs as 0.51–2.50, and 0.05– 0.11 ng/ml, respectively.37 antibody utilized in this assay is known to detect many forms of inhibin, including the free α subunit and inhibin α-β dimers (inhibin a and b).38 therefore, it is not possible to make a direct comparison between inhibin b concentrations determined by elisa in the present report inhibin concentrations were determined via ria. given the rapid decline in inhibin b to noneditable concentrations after tumor removal, it would seem reasonable to suspect that the neoplastic testis to be the origin of this hormone in the patient prior to surgery. in the absence of reference ranges for normal intact males, it cannot be determined if concentrations of inhibin b in patient’s presurgical serum sample were due to the presence of normal or neoplastic testicular tissue. the hypothesis that a sct may be a source of peripheral elevations in serum inhibin concentrations is supported.3,5 an increase in serum inhibin-like immunoreactivity was (determined by ria) detected in 95 and 53 dogs with sct compared to controls. further studies to investigate the potential of inhibin b as a marker of gonadal status or neoplastic conditions of the testis may be warranted. estrone sulphate estrogen is produced in the male gonad by the aromatization of androgens through the action of cytochrome p450 aromatase.39 in mammalian species testes, aromatase expression has been predominantly localized to leydig cells. however, species variations in the cellular distribution of this expression across leydig, sertoli, germ cells, and sperm exist.39 cellular expression of aromatase has also been documented in canine scts.40 estrogens in circulation are comprised of 3 forms: estradiol, estrone, and estriol. conjugation of estrone by estrone sulfotransferase resulted in the formation of estrone sulphate.41 estrone sulphate is produced in substantial quantities in stallion42 and boar43 testis and by the feto-placental unit of many domestic species including mare,44 ewe,45 doe,46 and sow.47 estradiol concentrations have been variably elevated in cases of sct3–6 and did not always correlate with feminization.3 dogs with sct had significantly higher estradiol concentrations compared to controls, although estradiol was only above the reference range for some patients.4,5 it has been suggested that clinical signs of feminization with normal estradiol reflect secretion of other forms of estrogen by scts.4 in the present case, serum estrone sulphate concentrations were 1.5 and 1.3 ng/ml in pre and postsurgical samples, respectively. minimal changes in serum estrone sulphate concentrations after tumor removal in this patient may reflect extra-gonadal sources. however, in the absence of established reference ranges for estrone sulphate in male castrated or intact dogs, the interpretation of this result is largely speculative. the clinical significance of this finding remains undetermined. histological examination of the neoplastic testis and spermatic cord revealed tumor emboli within testis and pampiniform plexus blood vessels, implicating a risk of metastatic disease. metastatic spread to the medial iliac lymph node has been described.48 however, in the present case, histological evaluation of the enlarged left medial iliac lymph node revealed no neoplastic cells. preoperative thoracic radiographs also had no evidence of pulmonary metastasis. based on limited literature reports, the rate of metastatic disease in sct appears to be 2–8%.1,49 however, metastatic spread of sct has manifested up to 4 years after removal of the primary tumor.50 for cases with metastatic disease, the prognosis should be considered poor.48,51–54 for these reasons, and in light of significant concentrations of serum amh concentrations remaining 3 weeks after  surgical removal of the neoplastic testis, an appointment for tumor surveillance 3 months after surgery was recommended. learning points • amh is a useful endocrine diagnostic marker for identification of retained testis in canine patients and may assist in identifying sct • a precipitous decline in inhibin b to undetectable concentrations was observed after surgical removal of sertoli cell-affected retained testis • effects of hyperestrogenism on hair follicle growth can mimic other more common conditions of alopecia. additional diagnostic tests (preputial cytology, ultrasonography, and hormonal assays) may assist in timely and accurate diagnosis of sct • the spermatic cord along with the affected testis should be submitted for histologic analysis. identification of tumor emboli in cord vessels may guide prognosis and follow-up tumor surveillance conflict of interest authors have none to declare. http://dx.doi.org/10.58292/ct.v15.9437 6 citation line: clinical theriogenology 2023, 15, 9437, http://dx.doi.org/10.58292/ct.v15.9437 references 1. lipowitze aj, schwartz a, wilson gp, et al: testicular neoplasms and concomitant clinical changes in the dog. j am vet med assoc 1973;163:1364–1368. 2. reif js: the relationship between cryptorchidism and canine 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veterinary medicine, louisiana state university, la bgoulburn valley equine hospital, congupna, victoria, australia clarge animal clinical sciences, college of veterinary medicine, michigan state university, mi abstract although parturition, even unattended, may proceed without difficulty, acute postpartum complications in mares can be life threatening. timely attention to complicated postpartum cases is crucial for mare’s survival, especially first 24 hours after foaling. these mares may present with adverse clinical signs, due to gastrointestinal and/or urogenital system disorders, such as displacements and hemorrhage, respectively, which can have substantial effects on health and fertility. this case report discusses 2 mares referred to the same hospital for signs of colic within 24 hours postpartum: 1) large colon volvulus and broad ligament hematoma; and 2) right dorsal colonic displacement with uterine artery rupture. this report emphasizes concomitant occurrence of 2 disease processes, which may be omitted when assessed individually. therefore, it is imperative that postpartum mare is fully assessed, as foaling associated hemorrhage may be masked by alimentary anomalies or vice versa. keywords: parturition, postpartum, colic, hematoma, uterus, colon background broodmares are at an increased risk of developing abdominal disease, such as gastrointestinal pathology and uterine artery hemorrhage1 in the peripartum period.2 in part, this was attributed to changes in dietary composition,3 violent movement of foal’s extremities during parturition,4 decompression and movement of internal organs post foaling,5 and/or inflammatory responses related to stress. a large study reviewed 163 postpartum hospital referrals and reported that ⅓ of these cases had gastrointestinal disease, with colon volvulus being responsible for 16.6% of those cases.2 uterine arterial hemorrhage was responsible for approximately 40% of fatalities reported in postpartum mares, with majority occurring in the immediate postpartum period.2 cases described herein provide a topical and relevant review of complications frequently seen in a postpartum mare with signs of colic. clinical presentations of these conditions can pose a diagnostic dilemma, due to difficulty in differentiating them6 and treatment for gastrointestinal displacements/volvulus can be different than that for uterine artery associated hemorrhage.4 it is necessary for clinicians to consider that these concurrent issues may be present. case 1 presentation a 10 year old primiparous, 502 kg thoroughbred mare delivered a live colt foal on the farm with vaginal assistance. mare demonstrated mild abdominal discomfort and retained fetal membranes. fetal membranes were removed manually by referring veterinarian after 12 hours, following ecbolic therapy. however, mare continued to display persistent signs of abdominal discomfort despite analgesics (detomidine hydrochloride at 0.01 mg/kg iv; dormosedan, zoetis, kalamazoo, mi) and butorphanol tartrate (0.01 mg/kg iv; torbugesic, zoetis), and was referred 16 hours after foaling for signs of colic. on presentation, mare was quiet, alert, and responsive but tachycardic at 60 beats per minute (bpm), pink mucous membranes with capillary refill time (crt) of < 2 seconds with decreased borborygmi on abdominal auscultation. reminder of mare’s physical examination findings were within limits. nasogastric intubation yielded no net reflux and transrectal palpation in addition to transabdominal ultrasonography (2 3.5 mhz convex transducer) revealed no significant findings. a complete blood count revealed a normal packed cell volume (pcv) of 35% (reference range: 30 46%) and total protein (tp) of 8 g/dl (reference range: 5.8 8.7 g/dl), with a mild neutrophilia of 13 x 103/l (reference range: 2.6 6.8 x 103/l) that was consistent with potential inflammation, infection or stress. although peripheral blood lactate was very mildly increased (2.13 mmol/l, reference range: < 2 clinical theriogenology • volume 12 number 2 • june 2020 139 mmol/l), no abdominocentesis was performed, precluding a comparison between these 2 values. a markedly increased concentration of creatine kinase (2,715 u/l, reference range: 119 287 u/l) and aspartate aminotransferase (ast 538 u/l, reference range: 168 494 u/l) were noted on the serum chemistry. combined plasma muscle enzymes concentrations were used as prognostic indicators in colic cases and substantially associated with nonsurvival and intestinal ischemia cases. however, further research is indicated to understand the etiology of increased activity and relevance as a true preoperative indicator of severity and prognosis.7 treatment initial management to attempt to stabilize the patient included: iv fluid therapy (lactated ringer’s solution at 1.5 liters/hour, supplemented with 29 grams calcium gluconate and 20 milliequivalents of potassium chloride per liter) and n-butylscopolammonium bromide (0.3 mg/kg iv; buscopan, boehringer ingelheim, st joseph, mo) as a spasmolytic. however, mare’s mentation continued to decline and persistent signs of colic were displayed, despite 2 more repeated administrations of xylazine (0.25 mg/kg iv; xylamed, vetone, boise, id) and butorphanol tartrate (0.01 mg/kg iv). due to her worsening clinical status, exploratory celiotomy was recommended. perioperative treatment included: penicillin g potassium (22,000 iu/kg iv, every 6 hours; sandoz, princeton, nj), gentamicin sulfate (6.6 mg/kg iv once daily; vetone, boise, id) and flunixin meglumine (1.1 mg/kg iv, once daily; prevail, vetone, boise, id). intraoperatively, a 180 degree volvulus of ascending colon was identified and corrected. both small and large intestines were determined to be viable; however, an ~ 23 cm long, hematoma on the dorsal aspect of left broad ligament was also discovered. care was taken during surgical correction of volvulus to limit manipulation within the caudal abdomen so as not to disrupt the hematoma within broad ligament. mare recovered without complication from surgery and general anesthesia. except for mild tachycardia 24 hours postsurgery, mare’s postoperative physical examination parameters and the hematology findings returned to normal. postfoaling reproductive tract examination was performed 24 hours following surgery. on transrectal ultrasonography (linear 5 10.0 mhz transducer) examination, revealed normal ovaries and large pendulous and involuting uterus with intraluminal fluid accumulation (10 x 11 cm in depth; figure 1). a low dose of oxytocin treatment was initiated (2 iu im; oxytocin, vetone, boise, id) as an ecbolic therapy, every 2 hours for 24 hours, then, every 4 hours, for 3 days. authors prefer smaller more frequent doses of oxytocin (compared to accepted 20 iu) to have reduced exacerbation of colic signs. in addition to exogenous supply of oxytocin, mare releases oxytocin from posterior pituitary gland during nursing and bonding to facilitate uterine involution that incidentally has an endogenous effect on uterine contractility.8 reproductive tract transrectal examination, 2 days following treatment, revealed substantial reduction in the amount of intraluminal uterine fluid to a 1 x 3 cm pocket (figure 2). figure 1. transrectal ultrasonogram showing intrauterine fluid accumulation (3 days postpartum). intrauterine fluid accumulation, flocculent fluid with mixed echogenicity within uterine lumen (measured by dotted line [10.59 cm]) uterine endometrium clinical theriogenology • volume 12 number 2 • june 2020140 figure 2. transrectal ultrasonogram (5 days postpartum, 2 days after ecbolic therapy initiation) showing decreased intrauterine anechoic fluid accumulation. outcome mare and foal were discharged 9 days after surgery with following treatment regimen: trimethoprim sulfamethoxazole (20 mg/kg, every 12 hours orally; rising health, saddlebrook, in) for 7 days and phenylbutazone (1 gram, every 12 hours orally; vetone, boise, id) for 3 days. foal was normal at 3 months physical examination and the owners elected to delay breeding for financial reasons. case 2 presentation a 17 year old multiparous, 418 kg thoroughbred mare, that delivered a live colt foal unassisted (born at 366 days of pregnancy), had fetal membrane expulsion within 2 hours, was presented for acute signs of colic. mare was reluctant to stand at 3 hours postpartum. mare was referred following treatment with 10 liters of iv bolus, flunixin meglumine (1.1 mg/kg iv, once daily), butorphanol tartrate (0.01 mg/kg iv) and a sedative dose of detomidine hydrochloride (0.01 mg/kg iv). mare had a history of a previous surgically corrected large colon displacement 3 years prior to this event. this was followed 1 year later with hospitalization also in our facilities for an unknown cause of abdominal discomfort that was treated medically. on presentation, mare appeared was and depressed with tachycardia (80 bpm), had pale and dry mucous membranes with a crt of 2 seconds, decreased borborygmi, and was continually trying to lie in lateral recumbency. remainder of her vitals were within normal limits. nasogastric intubation yielded no net reflux, and transrectal palpation findings were within normal limits for a < 24 hour postpartum mare. transabdominal ultrasonography revealed swirling free fluid with mixed echogenicity surrounding intestine within the peritoneal cavity, indicative of a hemoabdomen (figure 3). abdominocentesis confirmed the presence of hemorrhagic fluid with a marked elevation in lactate at 10.2 mmol/l (reference range: < 2 mmol/l) and total protein of 5.2 g/dl (reference range: > 4 g/dl). the abnormalities from the mare’s cbc revealed an anemia with a pcv of 24% (reference range: 30 46%), hypoproteinemia; total protein 4 g/dl (reference range: 5.8 8.7 g/dl) and hyperlactatemia 7.9 mmol/l (reference range: < 2 mmol/l), as well as a leukocytosis 12.1 x 103/l (reference range: 5 11.6 x 103/l) with a mature neutrophilia 9.7 x 103/l (reference range: 2.6 6.8 x 103/l). mare was stabilized with following intravenous medications: 1 liter of frozen plasma (protein volume replacement 7100, mg biologics, st. ames, ia), lactated ringers solution (1.5 liters/hour), and intrauterine fluid accumulation (measured by dotted line [92.99 cm]) uterine endometrium clinical theriogenology • volume 12 number 2 • june 2020 141 aminocaproic acid (40 mg/kg, hospira, lake forest, il) in 1 liter of 0.9% hypertonic saline given slowly. aminocaproic acid inhibits fibrinolysis by inhibiting plasminogen activation and increasing alpha-2antiplasmin activity, helping to maintain clot formation once it has occurred.9 figure 3. transabdominal ultrasonogram on admission revealing echogenic swirling free fluid, consistent with blood, in ventral abdomen (bottom arrow). treatment within 6 hours after admission, mare’s clinical presentation continued to decline, including a decrease in pcv to 15% and abdominal discomfort that did not improve despite an additional 2 doses of xylazine (0.25 mg/kg iv) and butorphanol tartrate (0.01 mg/kg iv). a presumptive diagnosis of uterine rupture or uterine-associated arterial hemorrhage was made; however, gastrointestinal causes of colic could not be ruled out. decision was made to perform an exploratory celiotomy. perioperative treatment included: penicillin g potassium (22,000 iu/kg iv, every 6 hours), gentamicin sulfate (6.6 mg/kg iv, once daily), flunixin meglumine (1.1 mg/kg iv, once daily), and a tetanus toxoid (1 ml im; zoetis, kalamazoo, mi). during surgical preparation, 2 liters of 6% hetastarch (hospira, lake forest, il) iv and 6 l of whole blood was collected from a hospital donor and administered. at exploratory celiotomy, a right dorsal colon displacement and a 32 x 25 x 9 cm hematoma within right broad ligament (occupying most of the pelvic canal and extended cranially beyond the pelvic inlet) was identified. significant contusions and bruising to the nongravid uterine horn were also noted (figure 4). manipulation of the caudal abdomen was minimized as much as possible to avoid any potential disruption of the hematoma. colonic displacement was corrected and, due to the mare’s previous history, a colopexy was performed. echogenic flocculent fluid, consistent with blood in the abdominal cavity skin and subcutaneous tissue clinical theriogenology • volume 12 number 2 • june 2020142 figure 4. case 2 intraoperative image of the uterus demonstrating hemorrhagic clot and substantial contusions and bruising to the nongravid horn of the uterus recovery from surgery and anesthesia was unremarkable and the foal was reunited with mare. however, concern of continuing hemorrhage remained presumably from the original insult and a second dose of aminocaproic acid (40 mg/kg slowly iv in 1.0 l of 7.2 % hypertonic saline) and yunnan baiyao, a chinese herbal medication, (13 mg/kg, every 12 hours orally, yunnan baiyao group, yunnan, china) was administered. yunnan baiyao had no effects on hemostatic function in healthy horses;10 however, it decreased surgical blood loss in human patients, apparently by increased expression of platelet surface receptors11 and in ponies.12 mare was stable after 24 hours and physical examination findings were within normal limits. pcv stabilized and increased to 25% at 72 hours. postoperative transrectal ultrasonographic examination (linear 5 10 mhz transducer) revealed a moderate volume of grade 3 intrauterine fluid, therefore, an ecbolic regime (oxytocin 2 iu, every 4 hours, im for 3 days) was implemented. it was noted that foal was not gaining weight appropriately so offerings of milk replacer via bucket feeding was initiated. foal’s nutrition was maintained during the remainder of hospitalization. daily transrectal and transabdominal ultrasonographic findings revealed less free fluid in uterus than before (small volume and grade 4), and a more organized hematoma structure formation in broad ligament suggesting resolution of active uterine artery bleed. mare’s colic signs returned within 48 hours postsurgery. despite medical management and analgesics treatment, colic signs of tachycardia, tachypnea, flank watching, and laying in lateral recumbency persisted. a repeat celiotomy through initial ventral midline incision was performed, and a large colon impaction was diagnosed. an enterotomy was performed, the large colon was evacuated and the colopexy was reversed. mare continued on perioperative antibiotics, flunixin meglumine, iv fluid therapy and was administered lidocaine hydrochloride (cri 0.05 mg/kg/min iv; lidocaine 2%, vetone, boise, id) for pain and the prevention of postoperative ileus. although mare remained comfortable, showing no signs of abdominal pain, sudden copious purulent serosanguineous drainage from the abdominal incision occurred 24 hours after second surgery. ultrasonographic evaluation of incision suggested partial incisional dehiscence. mare returned to surgery where abdominal cavity lavage and drain placement was followed by an incisional revision. mare was managed postoperatively with an abdominal bandage and twice daily abdominal lavage with 10 l of sterile saline through a drain. metronidazole (15 mg/kg, every 8 hours orally; alembie, bridgewater, nj) was added to antibiotic treatment regimen, as abdominal fluid findings were consistent with peritonitis. although no further complications were noted with the incision, mare began to display intermittent mild colic signs, predominantly tachycardia and laying in lateral recumbency 5 days following drain placement (11 days from initial presentation). mare was unresponsive to aggressive pain management (cri of lidocaine [0.05 left uterine horn substantial bruising and contusions to uterine horn broad ligament hematoma uterine bifurcation right uterine horn clinical theriogenology • volume 12 number 2 • june 2020 143 mg/kg], ketamine hydrochloride [0.6 mg/kg/hr, zetamine, vet one, boise, id], and detomidine hydrochloride [0.0044 mg/kg/hour]), along with butorphanol tartrate (0.01 mg/kg im, every 4 hours) and flunixin meglumine (1.1 mg/kg, iv, every 12 hours). after 36 hours of therapy, owner elected for euthanasia. outcome postmortem examination revealed previously discovered hematoma within broad ligament, now with a fibrinous adhesion to distal small colon. fibrinous adhesion had created a 180 degree twist in small colon, partially occluding the lumen. discussion although largely based on anecdotal information, mare breed, age and parity may be predisposing factors for colic in postpartum mare.4 as veterinarians, being aware of these potential factors, may be able to differentiate probable causes of abdominal pain in postfoaling period in a more timely manner. attended parturition and regular postpartum monitoring, can result in earlier identification of signs indicative of postpartum complications. this allows veterinarian to institute aggressive therapies and/or rapid referral if necessary. early identification of problems and rapid decision making in postpartum period can be critical to overall prognosis for survival. poorer outcomes tend to be in cases where there is delayed diagnosis, often related to complications such as systemic compromise or gross abdominal contamination.13 most common causes for referral in postpartum mare are large colon volvulus or displacement and urogenital hemorrhages.13 two cases presented in this report were determined to have conditions associated with both gastrointestinal and reproductive systems, yet each case presented in unique ways. a large us study illustrated that gastrointestinal disease accounted for > ⅓ of clinical cases within first 30 days postpartum, with nearly 17% being due to a large colon volvulus.2 a uk study also demonstrated the increased prevalence of large colon volvulus in postpartum mares, which were 13 times more likely to be diagnosed as such.5 while 88% of postpartum mares with gastrointestinal complications survived,14 in locations where mares had increase distances to travel for referral treatment, prognosis can be much worse. although presenting signs are variable, majority mares, with either or both gastrointestinal or urogenital disease processes, will present similarly with moderate to severe abdominal discomfort unresponsive to analgesics. further diagnostic evaluation such as transrectal palpation, transabdominal ultrasonography and abdominocentesis, can often be very helpful in providing further insight into severity and the nature of pathology, and provide suggestive information to help recommend next steps in treatment for a rewarding outcome.13 delayed referral or clinical decisions can ultimately lead to poorer prognosis and/or further complications. many theories were proposed for higher risk of postpartum mares developing a large colon displacement or volvulus compared to remainder of equine population. one possibility is large ‘abandoned space’ in caudal abdomen previously inhabited by fetus that permits more freedom for gastrointestinal organs to relocate or twist.5 during stages i and ii of labor, foal may cause trauma to extrauterine organs with its limbs movements, despite protective covering over hooves and being encapsulated within uterus. management changes often occur in late pregnancy and postpartum period for these mares, including altered and enhanced feeding practices, which is a consistent risk factor for colic.3, 5 these diet changes can disturb normal gastrointestinal motility, and potentially change the microbiome and volatile organic compounds within large colon. a study reported that mares that did not experience colic, had very little change in their hindgut bacteria postfoaling; however, mares that displayed signs of colic postpartum had significant alterations in their microbiome, particularly associated with presence of firmicutes and proteobacteria.15 these findings suggest that evaluation of fecal microbiome in postpartum mare could allow for identification of at risk mares, thereby allowing caretakers to change management practices, eliminate mare’s fecal microbiota dysbiosis and possibly minimize chances of colic. clinical theriogenology • volume 12 number 2 • june 2020144 urogenital extrauterine hemorrhages are most frequently related to middle uterine, uteroovarian or external iliac arteries, with uterine artery being the most common.13 majority of literature suggests that risks substantially increase with age, with 78% of reported cases being mares 15 years of age or older.1 this age predilection has been attributed to atrophy of smooth muscle cells in vessel wall, accompanied by arterial wall fibrosis.2 due to repeated loading and excessive fatigue of arterial wall from multiple pregnancies, ‘elastica interna’ is disrupted, thinned and can fragment, thereby also increasing chances of rupture.6,16 depending on vessel location, large volumes of blood can either be contained within uterine lumen, trapped between layers of broad ligament, or escape into abdominal cavity (hemoabdomen).13 in severe cases of uterine artery rupture, there may be overt signs of hypovolemic shock, including pale mucous membranes and delayed crt, and these cases can progress rapidly to acute fatalities. however, often, clinical signs observed with urogenital hemorrhages after 24 hours are dull mentation, tachycardia, and muscle fasciculations. when hemorrhage is contained within broad ligament, an organized hematoma can form, limiting blood loss. this is consistent with findings from a postmortem study in which hematomas in broad ligaments presumed to be from the uterine artery, were confirmed as the source of hemorrhage in 8 out of 11 mares.17 although there is no definite predilection to 1 particular side of uterus being more affected, it has been suggested the right side is more prone to such injury, attributable to displacement by cecum.13 management of mares with hemorrhage can vary depending on severity of blood loss and clinical signs. supportive therapy such as iv fluids, blood transfusion, and medications such as aminocaproic acid to help improve blood clot stability can be important to utilize when managing these cases. surgery may or may not be indicated, depending on diagnosed or suspected cause of the discomfort. manipulation of uterus and surrounding structures during diagnostic examinations or intraoperatively should be minimized to limit potential disruption of organizing hematomas. postsurgery oxytocin use in mares with hematomas is controversial and clinician dependent due to concern of uterine contraction disturbing a hematoma. low dose oxytocin therapy, which was used in these cases, is reported to promote uterine involution and is indicated even in cases with suspected uterine lacerations, as long as the mare remains comfortable.18,19 administration of oxytocin, as compared to other ecbolics, has no effect on heart rate, blood pressure or hematological parameters in early postpartum mares.18 both mares of this report displayed colic signs that are associated with gastrointestinal disease and had concurrent findings of broad ligament hematomas. as previously discussed, postpartum hemorrhage is widely considered to be age related, however probability also increases with each succeeding pregnancy beyond 10 years of age.20 while diagnosis of a hemorrhagic event in case 2 was based on physical examination and diagnostic findings, exact origin of hemorrhage could not be determined prior to surgery. although her anesthetic risk increased due to a compromised cardiovascular state, surgery was deemed necessary due to uncontrolled pain and suspicion of a concurrent intestinal lesion. with this information in mind, it is important to remember that any mare is at potential risk of hemorrhage.17 prognosis for survival is ultimately important for production of future offspring; however, fertility may be compromised by postpartum complications. uterine laceration cases typically have an 80% chance of survival, whereas with colic, it can be as low as 40% after a large colon volvulus.2,21 therefore, there is a variable survival range and prognosis for postpartum complications, depending on the underlying cause. postpartum conception rates are not affected in ~ 84% of mares, depending on the site and severity of hematoma.13 in cases with severe hemorrhage into abdomen or uterus, there is a poorer prognosis for survival and therefore fertility.22 additionally, when hemorrhage is contained in broad ligament, it may take several weeks for hematoma to resolve,13 potentially causing a mare to skip a breeding season or having a shorter broodmare career. this will depend on when insult occurred relative to timing of breeding season, size of hematoma and how owners are advised by veterinarian (for example live breeding may not be recommended, due to concern of clot disruption). however, during this peripartum period, coagulation reference ranges may be inappropriate, and there are ongoing studies to determine if they hinder diagnosis or treatment of a hemostatic disorders during pregnancy.23 mares that survive may still be fertile, and these mares are likely to become pregnant when rebred.6 they are, clinical theriogenology • volume 12 number 2 • june 2020 145 however, considered a high risk mare for potential hemorrhage at subsequent parturition, due to increased fibrosis and lost elasticity of vessels.8 for mares, however, that present for colic, their prognosis for potential reproductive success should not be compromised, assuming the reproductive tract remains normal. broodmares have had successful reproductive careers following surgical correction of large colon volvulus, even after multiple surgeries.24 recurrent colic, however, remains a hazard and techniques such as colopexy or large colon resection and anastomosis, should be considered in such cases. learning points  importance of obtaining a full and complete history, including signalment  reduced time to referral is critical in postpartum mares  any postpartum mare is at an increased risk of either urogenital hemorrhage or gastrointestinal disease conflict of interest authors have no conflicts of interest to declare. references 1. williams b, bryant u: periparturient arterial rupture in mares: a postmortem study. j equine vet sci 2012;32:281-284. 2. dolente ba, sullivan ek, boston r, et al: mares admitted to a referral hospital for post-partum emergencies: 163 cases (1992–2002). j vet emerg crit care 2005;15:193-200. 3. hudson jm, noah bs, cohen d, et al: feeding practices associated with colic in horses. j am vet med assoc 2001;219:1419-1425. 4. turner rm: post-partum problems: the top ten list. proc am ass equine pract 2007; p. 305-319. 5. suthers jm, pinchbeck gl, proudman cj, et al: risk factors for large colon volvulus in the uk. equine vet j 2012;45:558-563. 6. frazer gs: post-partum complications in the mare. part 1: conditions affecting the uterus. equine vet educ 2003;15:36-44. 7. krueger cr, ruple-czerniak a, hackett es: evaluation of plasma muscle enzyme activity as an indicator of lesion characteristics and prognosis in horses undergoing celiotomy for acute gastrointestinal pain. bmc vet res 2014;10:s7. 8. knottenbelt dc, pascoe rr, lopate c, et al: equine stud farm medicine and surgery. 1st edition, edinburgh; saunders: 2003. p. 282, 345. 9. heidmann p, tornquist sj, qu a, et al: laboratory measures of hemostasis and fibrinolysis after intravenous administration of epsilon-aminocaproic acid in clinically normal horses and ponies. am j vet res 2005;66: 313-318. 10. ness sl, frye ah, divers tj, et al: randomized placebo-controlled study of the effects of yunnan baiyao on hemostasis in horses. am j vet res 2017;78:969-976. 11. tang zl, wang x, yi b, et al: effects of the preoperative administration of yunnan baiyao capsules on intraoperative blood loss in bimaxillary orthognathic surgery: a prospective, randomized, double-blind, placebo-controlled study. int j oral maxillofac surg 2009;38:261-266. 12. graham l, farnsworth k, cary j: the effect of yunnan baiyao on the template bleeding time and activated clotting time in healthy halothane anesthetized ponies. j vet em crit care 2002;12:279. 13. tibary a, pearson lk: medical problems in the immediate post-partum period. proc am ass equine pract 2012; p. 362-369. 14. hackett es, embertson rm, hopper sa, et al: duration of disease influences survival to discharge of thoroughbred mares with surgically treated large colon volvulus. equine vet j 2015;47:650-654. 15. weese js, holcombe sj, embertson rm, et al. changes in the faecal microbiota of mares precede the development of post partum colic. equine vet j 2015;47:641-649. 16. ueno t, nambo y, tajima y, et al: pathology of lethal peripartum broad ligament haematoma in 31 thoroughbred mares. equine vet j 2010;42:529-533. 17. arnold ce, payne m, thompson ja, et al: periparturient hemorrhage in mares: 73 cases (1998–2005). j am vet med assoc 2008;232:1345-1351. 18. nagel c, trenk l, wulf m, et al: oxytocin treatment does not change cardiovascular parameters, hematology and plasma electrolytes in parturient horse mares. theriogenology 2017;91:69-76. 19. canisso if, rodriguea js: a clinical approach to the diagnosis and treatment of retained fetal membranes with an emphasis placed on the critically ill mare. j equine sci 2013;33:570-579. 20. oikawa m, nambo y, miyamoto m, et al: post-partum massive hematoma within the broad ligament of the uterus in a broodmare possibly caused by rupture of the uterine artery. j equine sci 2009;20:41-46. clinical theriogenology • volume 12 number 2 • june 2020146 21. santschi em: colic and pregnant mares. in: blikslager at, white na, moore jn et al: editors. the equine acute abdomen. 3rd edition, hoboken; john wiley & sons inc.: 2017. p. 819-830. 22. scoggin cf, mccue pm: how to assess and stabilize a mare suspected of periparturient hemorrhage in the field. am ass equine pract 2007; p. 342-348. 23. bazzano m, giannetto c, et al: hemostatic profile during late pregnancy and early postpartum period in mares. theriogenology 2014; 81:629-642 24. leahy er, holcombe sj, hackett es, et al: reproductive careers of thoroughbred broodmares before and after surgical correction of >/=360 degree large colon volvulus. equine vet j 2018;50:208-212. clinical theriogenology • volume 12 number 2 • june 2020 147 ms-1 formatter ms-2 table of contents blank page ms-3 news section sft president's letter-june 2020 act presidents letter-june 2020 tf president's letter-june 2020 ctj news section june 2020_final bartlett-proficiency awards 2020 toy and bartlett 2020 2021 bartlett award nomination form tf fundraising events 2020 sponsors and exhibitors 2020 2020 conference agenda for ctj special events and symposium 2020 board of directors nominees ms-4 editor's note ms-5 figure 2 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/colorimageresolution 150 /colorimagedepth -1 /colorimagemindownsampledepth 1 /colorimagedownsamplethreshold 1.50000 /encodecolorimages true /colorimagefilter /dctencode /autofiltercolorimages true /colorimageautofilterstrategy /jpeg /coloracsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /colorimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000coloracsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000colorimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasgrayimages false /cropgrayimages false /grayimageminresolution 150 /grayimageminresolutionpolicy /ok /downsamplegrayimages true /grayimagedownsampletype /bicubic /grayimageresolution 150 /grayimagedepth -1 /grayimagemindownsampledepth 2 /grayimagedownsamplethreshold 1.50000 /encodegrayimages true /grayimagefilter /dctencode /autofiltergrayimages true /grayimageautofilterstrategy /jpeg /grayacsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /grayimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000grayacsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasmonoimages false /cropmonoimages false /monoimageminresolution 300 /monoimageminresolutionpolicy /ok /downsamplemonoimages true /monoimagedownsampletype /bicubic /monoimageresolution 600 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects true /checkcompliance [ /none ] /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname (http://www.color.org) /pdfxtrapped /false /createjdffile false /description << /enu () >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 524288 /lockdistillerparams false /maxsubsetpct 100 /optimize false /opm 0 /parsedsccomments true /parsedsccommentsfordocinfo false /preservecopypage true /preservedicmykvalues true /preserveepsinfo false /preserveflatness false /preservehalftoneinfo false /preserveopicomments false /preserveoverprintsettings true /startpage 1 /subsetfonts true /transferfunctioninfo /preserve /ucrandbginfo /preserve /useprologue false /colorsettingsfile () /alwaysembed [ true ] /neverembed [ true ] /antialiascolorimages false /cropcolorimages false /colorimageminresolution 150 /colorimageminresolutionpolicy /ok /downsamplecolorimages true /colorimagedownsampletype /bicubic /colorimageresolution 150 /colorimagedepth -1 /colorimagemindownsampledepth 1 /colorimagedownsamplethreshold 1.50000 /encodecolorimages true /colorimagefilter /dctencode /autofiltercolorimages true /colorimageautofilterstrategy /jpeg /coloracsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /colorimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000coloracsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000colorimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasgrayimages false /cropgrayimages false /grayimageminresolution 150 /grayimageminresolutionpolicy /ok /downsamplegrayimages true /grayimagedownsampletype /bicubic /grayimageresolution 150 /grayimagedepth -1 /grayimagemindownsampledepth 2 /grayimagedownsamplethreshold 1.50000 /encodegrayimages true /grayimagefilter /dctencode /autofiltergrayimages true /grayimageautofilterstrategy /jpeg /grayacsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /grayimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000grayacsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasmonoimages false /cropmonoimages false /monoimageminresolution 300 /monoimageminresolutionpolicy /ok /downsamplemonoimages true /monoimagedownsampletype /bicubic /monoimageresolution 600 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects true /checkcompliance [ /none ] /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname (http://www.color.org) /pdfxtrapped /false /createjdffile false /description << /enu () >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 524288 /lockdistillerparams false /maxsubsetpct 100 /optimize false /opm 0 /parsedsccomments true /parsedsccommentsfordocinfo false /preservecopypage true /preservedicmykvalues true /preserveepsinfo false /preserveflatness false /preservehalftoneinfo false /preserveopicomments false /preserveoverprintsettings true /startpage 1 /subsetfonts true /transferfunctioninfo /preserve /ucrandbginfo /preserve /useprologue false /colorsettingsfile () /alwaysembed [ true ] /neverembed [ true ] /antialiascolorimages false /cropcolorimages false /colorimageminresolution 150 /colorimageminresolutionpolicy /ok /downsamplecolorimages true /colorimagedownsampletype /bicubic /colorimageresolution 150 /colorimagedepth -1 /colorimagemindownsampledepth 1 /colorimagedownsamplethreshold 1.50000 /encodecolorimages true /colorimagefilter /dctencode /autofiltercolorimages true /colorimageautofilterstrategy /jpeg /coloracsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /colorimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000coloracsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000colorimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasgrayimages false /cropgrayimages false /grayimageminresolution 150 /grayimageminresolutionpolicy /ok /downsamplegrayimages true /grayimagedownsampletype /bicubic /grayimageresolution 150 /grayimagedepth -1 /grayimagemindownsampledepth 2 /grayimagedownsamplethreshold 1.50000 /encodegrayimages true /grayimagefilter /dctencode /autofiltergrayimages true /grayimageautofilterstrategy /jpeg /grayacsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /grayimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000grayacsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasmonoimages false /cropmonoimages false /monoimageminresolution 300 /monoimageminresolutionpolicy /ok /downsamplemonoimages true /monoimagedownsampletype /bicubic /monoimageresolution 600 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects true /checkcompliance [ /none ] /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname (http://www.color.org) /pdfxtrapped /false /createjdffile false /description << /enu () >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 524288 /lockdistillerparams false /maxsubsetpct 100 /optimize false /opm 0 /parsedsccomments true /parsedsccommentsfordocinfo false /preservecopypage true /preservedicmykvalues true /preserveepsinfo false /preserveflatness false /preservehalftoneinfo false /preserveopicomments false /preserveoverprintsettings true /startpage 1 /subsetfonts true /transferfunctioninfo /preserve /ucrandbginfo /preserve /useprologue false /colorsettingsfile () /alwaysembed [ true ] /neverembed [ true ] /antialiascolorimages false /cropcolorimages false /colorimageminresolution 150 /colorimageminresolutionpolicy /ok /downsamplecolorimages true /colorimagedownsampletype /bicubic /colorimageresolution 150 /colorimagedepth -1 /colorimagemindownsampledepth 1 /colorimagedownsamplethreshold 1.50000 /encodecolorimages true /colorimagefilter /dctencode /autofiltercolorimages true /colorimageautofilterstrategy /jpeg /coloracsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /colorimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000coloracsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000colorimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasgrayimages false /cropgrayimages false /grayimageminresolution 150 /grayimageminresolutionpolicy /ok /downsamplegrayimages true /grayimagedownsampletype /bicubic /grayimageresolution 150 /grayimagedepth -1 /grayimagemindownsampledepth 2 /grayimagedownsamplethreshold 1.50000 /encodegrayimages true /grayimagefilter /dctencode /autofiltergrayimages true /grayimageautofilterstrategy /jpeg /grayacsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /grayimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000grayacsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasmonoimages false /cropmonoimages false /monoimageminresolution 300 /monoimageminresolutionpolicy /ok /downsamplemonoimages true /monoimagedownsampletype /bicubic /monoimageresolution 600 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects true /checkcompliance [ /none ] /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname (http://www.color.org) /pdfxtrapped /false /createjdffile false /description << /enu () >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 524288 /lockdistillerparams false /maxsubsetpct 100 /optimize false /opm 0 /parsedsccomments true /parsedsccommentsfordocinfo false /preservecopypage true /preservedicmykvalues true /preserveepsinfo false /preserveflatness false /preservehalftoneinfo false /preserveopicomments false /preserveoverprintsettings true /startpage 1 /subsetfonts true /transferfunctioninfo /preserve /ucrandbginfo /preserve /useprologue false /colorsettingsfile () /alwaysembed [ true ] /neverembed [ true ] /antialiascolorimages false /cropcolorimages false /colorimageminresolution 150 /colorimageminresolutionpolicy /ok /downsamplecolorimages true /colorimagedownsampletype /bicubic /colorimageresolution 150 /colorimagedepth -1 /colorimagemindownsampledepth 1 /colorimagedownsamplethreshold 1.50000 /encodecolorimages true /colorimagefilter /dctencode /autofiltercolorimages true /colorimageautofilterstrategy /jpeg /coloracsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /colorimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000coloracsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000colorimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasgrayimages false /cropgrayimages false /grayimageminresolution 150 /grayimageminresolutionpolicy /ok /downsamplegrayimages true /grayimagedownsampletype /bicubic /grayimageresolution 150 /grayimagedepth -1 /grayimagemindownsampledepth 2 /grayimagedownsamplethreshold 1.50000 /encodegrayimages true /grayimagefilter /dctencode /autofiltergrayimages true /grayimageautofilterstrategy /jpeg /grayacsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /grayimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000grayacsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasmonoimages false /cropmonoimages false /monoimageminresolution 300 /monoimageminresolutionpolicy /ok /downsamplemonoimages true /monoimagedownsampletype /bicubic /monoimageresolution 600 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects true /checkcompliance [ /none ] /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname (http://www.color.org) /pdfxtrapped /false /createjdffile false /description << /enu () >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice historyitem_v1 trimandshift range: current page trim: none shift: move up by 18.00 points normalise (advanced option): 'improved' keep bleed margin: no d:20200513061559 32 1 0 0 full 1388 388 fixed up 18.0000 -20.2536 both 4 currentpage 6 currentavdoc none 0.0000 top qite_quiteimposingplus4 quite imposing plus 4.0m quite imposing plus 4 1 6 20 6 1 1 historyitem_v1 trimandshift range: current page trim: none shift: move up by 13.50 points normalise (advanced option): 'improved' keep bleed margin: no d:20200513061630 32 1 0 0 full 1388 388 fixed up 13.5000 -20.2536 both 4 currentpage 6 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full 1388 388 fixed up 9.0000 -20.2536 both 11 subdoc 20 currentavdoc none 0.0000 top qite_quiteimposingplus4 quite imposing plus 4.0m quite imposing plus 4 1 10 20 19 10 1 historylist_v1 qi2base << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 524288 /lockdistillerparams false /maxsubsetpct 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/omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true 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[1 1 1 1] >> /grayimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000grayacsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasmonoimages false /cropmonoimages false /monoimageminresolution 300 /monoimageminresolutionpolicy /ok /downsamplemonoimages true /monoimagedownsampletype /bicubic /monoimageresolution 600 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects true /checkcompliance [ /none ] /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname (http://www.color.org) /pdfxtrapped /false /createjdffile false /description << /enu () >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 524288 /lockdistillerparams false /maxsubsetpct 100 /optimize false /opm 0 /parsedsccomments true /parsedsccommentsfordocinfo false /preservecopypage true /preservedicmykvalues true /preserveepsinfo false /preserveflatness false /preservehalftoneinfo false /preserveopicomments false 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/grayimageminresolutionpolicy /ok /downsamplegrayimages true /grayimagedownsampletype /bicubic /grayimageresolution 150 /grayimagedepth -1 /grayimagemindownsampledepth 2 /grayimagedownsamplethreshold 1.50000 /encodegrayimages true /grayimagefilter /dctencode /autofiltergrayimages true /grayimageautofilterstrategy /jpeg /grayacsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /grayimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000grayacsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasmonoimages false /cropmonoimages false /monoimageminresolution 300 /monoimageminresolutionpolicy /ok /downsamplemonoimages true /monoimagedownsampletype /bicubic /monoimageresolution 600 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects true /checkcompliance [ 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/includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 524288 /lockdistillerparams false /maxsubsetpct 100 /optimize false /opm 0 /parsedsccomments true /parsedsccommentsfordocinfo false /preservecopypage true /preservedicmykvalues true /preserveepsinfo false /preserveflatness false /preservehalftoneinfo false /preserveopicomments false /preserveoverprintsettings true /startpage 1 /subsetfonts true /transferfunctioninfo /preserve /ucrandbginfo /preserve /useprologue false /colorsettingsfile () /alwaysembed [ true ] /neverembed [ true ] /antialiascolorimages false /cropcolorimages false /colorimageminresolution 150 /colorimageminresolutionpolicy /ok /downsamplecolorimages true /colorimagedownsampletype /bicubic /colorimageresolution 150 /colorimagedepth -1 /colorimagemindownsampledepth 1 /colorimagedownsamplethreshold 1.50000 /encodecolorimages true /colorimagefilter /dctencode /autofiltercolorimages true /colorimageautofilterstrategy /jpeg /coloracsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /colorimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000coloracsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000colorimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasgrayimages false /cropgrayimages false /grayimageminresolution 150 /grayimageminresolutionpolicy /ok /downsamplegrayimages true /grayimagedownsampletype /bicubic /grayimageresolution 150 /grayimagedepth -1 /grayimagemindownsampledepth 2 /grayimagedownsamplethreshold 1.50000 /encodegrayimages true /grayimagefilter /dctencode /autofiltergrayimages true /grayimageautofilterstrategy /jpeg /grayacsimagedict << /qfactor 0.40 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /grayimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /jpeg2000grayacsimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 15 >> /antialiasmonoimages false /cropmonoimages false /monoimageminresolution 300 /monoimageminresolutionpolicy /ok /downsamplemonoimages true /monoimagedownsampletype /bicubic /monoimageresolution 600 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages true /monoimagefilter /ccittfaxencode /monoimagedict << /k -1 >> /allowpsxobjects true /checkcompliance [ /none ] /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname (http://www.color.org) /pdfxtrapped /false /createjdffile false /description << /enu () >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice 2020 news preparation has begun as the therio student chapters gear up to compete in the annual student chapter of the year competition happening in july at the 2020 therio conference in pittsburgh, pennsylvania. each student chapter will be judged on participation throughout the year, attendance at the conference, presentation of abstracts, and the merck student quiz bowl. teams earn points for paid sft student memberships, chapter events (wet labs, speakers, meeting attendance), and member participation at school (i.e., savma palpation team) and entering the t-shirt design competition. they also earn points for the number of students who attend the annual therio conference each year. this includes extra points for those students who submitted an abstract or case presentation and additional points for selection of oral or poster presentation. each team will also receive an extra point for submitting a basket of their school items for the silent auction. student sft clubs are becoming famous for their shirts. does your sft student chapter have a fun tshirt design? bring them to pittsburgh for the annual sft t-shirt design competition. shirts will be on display in a central area at the meeting and all attendees will be given a ballot for voting. the chapter with the winning t-shirt design will receive a cash award ($300 for 1st place, $100 for 2nd) as well as points toward winning the 2020 scoty award. the merck student quiz bowl will be held thursday evening during the exhibit hall opening reception. teams will consist of three members and the winning teams will receive funds for their chapter and prizes provided by merck animal health. mr. ron mcdaniel will once again host the competition. the first place team will be awarded $300 and the second place team will be awarded $100. the winning chapters will be announced at the awards dinner on friday evening, july 24. the quiz bowl application can be found at: https:// www.therio.org/page/studentquizbowlapp. the winning scoty will be announced during the therio awards dinner on friday night, july 24. prizes include a traveling banner and a prize of $1,000 to the first place team. the team coming in second place will receive a prize of $500. entries are due in to the sft office by june 1, 2020. the scoty application can be found at: https://www.therio.org/page/scotyapplication. a student equine seminar and lab will be held thursday (additional registration required). the seminar will focus on semen collection, evaluation, palpation and ultrasound. the lab will focus on uterine culture, cytology, and artificial insemination as well as evaluation of uterine cytology and biopsy. support your fellow classmates by attending the student case presentations thursday afternoon. all students that are registered for the conference will be paired with a mentor. the student/mentor reception will take place immediately following the presentations. register now at: www.therio.org. student events in pittsburgh clinical theriogenology • volume 12 number 1 • march 202017 https://www.therio.org/page/studentquizbowlapp https://www.therio.org/page/studentquizbowlapp https://www.therio.org/page/scotyapplication https://www.therio.org/ 2020 annual conference agenda wednesday, july 22 act educators’ forum opening session abstracts opening session reception equine symposium (additional registration required) equine symposium collection and transport of equine oocytes for in vitro embryo production tom stout opu and oocyte transport: clinical considerations rob foss diet and another influencers of oocyte developmental potential elaine carnevale embryo and oocyte preservation katrin hinrichs art wet lab group 1: oocyte aspiration; group 2: oocyte evaluation and transport group 1: oocyte evaluation and transport; group 2: oocyte aspiration thursday, july 23 bartlett award and address theriogenologist of the year award and address dr. jerry rains memorial abstract competition student equine seminar and lab (additional registration required) sft business meeting act business meeting student case presentations student research presentations student/mentor reception opening reception in the exhibit hall merck student quiz bowl clinical theriogenology • volume 12 number 1 • march 2020 18 student equine seminar and lab group 1: semen collection and evaluation; group 2: palpation and ultrasound group 2: semen collection and evaluation; group 1: palpation and ultrasound group 1: uterine culture, cytology and artificial insemination; group 2: evaluation of uterine cytology and biopsy group 2: uterine culture, cytology and artificial insemination; group 1: evaluation of uterine cytology and biopsy friday, july 24 plenary session scientific abstract presentations scientific abstract poster presentations species tracks awards dinner reception awards dinner and veterinarian story night and live auction plenary session the future is coming! – new technologies being applied to reproduction in animals and man katrin hinrichs in search of stem cell efficacy – immunogenicity matters ashlee watts small animal track introduction to stem sells – what they are, how to grow them, how to make them do what we want them to diane carlisle stem cells and female infertility jonathan tilly stem cells and male infertility kyle orwig production animal track postpartum anestrus of beef cows: importance and evaluation bob larson using the pregnancy analytics app to add value to pregnancy diagnosis of beef herds bob larson bull development and sperm production enhancement ahmed tibary equine track embryo-maternal communication during the establishment of equine pregnancy tom stout the role of mare age in early pregnancy loss tom stout reproductive endocrinology and immunology of the pregnant mare barry ball clinical theriogenology • volume 12 number 1 • march 202019 saturday, july 25 so you want to become a diplomate? career paths in theriogenology species tracks student case poster presentations canine breeder excellence seminar track (additional registration required) therio group outing pittsburgh riverhounds sc vs. charlotte independence small animal track current state of embryo transfer in dogs karine reynaud current state of ivm/ivf in dogs karine reynaud how to perform a rapid, accurate semen eval in a busy day-to-day practice joann randall how to perform a rapid, accurate semen eval in a busy day-to-day practice (continued) joann randall nomograph and titer testing prior to breeding or during pregnancy laurie larson pedigree analysis and genetic counseling cynthia o’connor genetic testing and inbreeding coefficients cynthia o’connor production animal track fetal programming: maternal-fetal interactions caleb lemley fetal programming: postnatal performance caleb lemley physiology and reproductive techniques for the whitetail doe jason coe advanced reproduction and semen collection in whitetail deer jason coe urogenital surgery in camelids ahmed tibary postpartum factors influencing fertility james ferguson dairy reproductive management programs james ferguson clinical theriogenology • volume 12 number 1 • march 2020 20 equine track genomics and transcriptomics of the normal and abnormal equine placenta: a better understanding of late gestational function and dysfunction barry ball cervix and myometrium: role in preterm and normal term birth in the mare barry ball managing recipient mares to optimize pregnancy rates in an equine art program tom stout factors that threaten late pregnancy: how can we detect them? margo macpherson what do we know about medication in equine pregnancy? margo macpherson management of dystocia pat mccue clinical cases discussion margo macpherson, pat mccue, tom stout clinical theriogenology • volume 12 number 1 • march 202021 do you know someone who would make a great director of the sft board? in accordance with policy, the society of theriogenology requests your suggestions for the board of directors. names received from the general membership will be forwarded to the nominating committee for consideration. directors serve a term of three years, beginning at the annual therio conference. if you would like to suggest an active society member for service as a director on the board, please submit the following information to the executive office no later than march 21, 2020. please ensure the individual’s willingness to serve on the board (if elected) prior to submission. please direct any questions to dr. robyn wilborn chair of the nominating committee at wilborr@auburn.edu. when suggestions are submitted we will need the following contact information on both the person submitting the suggestion and also the suggested individual’s: name, address, phone, email, and practice type. mail, email, or fax information to: charles f. franz, dvm executive director society for theriogenology 761 tiger oak drive pike road, al 36064 fax: 334-270-3399 email: charles@franzmgt.com you may submit nominations online at http://therio.org/ boardnominationform you must be logged in to the sft website to access the board nomination form located under board and officers. important dates call for abstracts due……………………………………….….february 1 tf research grant pre-proposal deadline………………….….february 15 call for act board nominations……………………….………….march 1 call for student case presentations due……………….………....march 10 call for student research presentations due……………………..march 10 sft board nominations due……………………………………....march 21 act exam fees due………………………………………………..april 15 tf research grant full proposal deadline………………………....april 15 student travel grant application deadline…………………………...june 1 student quiz bowl application deadline………………………….….june 1 scoty application deadline………………………………………...june 1 tf research grant final decisions…….……………………..……..june 30 therio conference early bird registration ends……………………....june 7 therio conference hotel registration discount deadline……………june 18 act exam application deadline…………………...…………..december 1 thank you! a huge thank you to the following individuals for all their efforts in planning the 2020 conference! annual conference program chair dr. jack smith equine track program chair dr. marco coutinho da silva production animal track program chair dr. heath king small animal track program chair dr. cheryl lopate equine symposium chair dr. marco coutinho da silva student equine seminar & lab chairs drs. marco coutinho da silva and pat mccue educators’ forum chair dr. hilari french silent auction chairs drs. jim floyd and will schultz clinical theriogenology • volume 12 number 1 • march 2020 22 mailto: http://therio.org/boardnominationform http://therio.org/boardnominationform 2020 student events 2020 conference agenda for ctj important dates best age for spay and neuter: a new paradigm best age for spay and neuter: a new paradigm benjamin hart, lynette hart, abbey thigpen, neil willits school of veterinary medicine and department of statistics, university of california, davis, ca abstract in small animal practice, neutering (including spaying) males and females at about 6 months has become standard. using our extensive computerized case record system of the veterinary hospital, with currently about 60,000 cases per year, we launched a 10 year project to provide breed-specific and gender-specific, data-based information on joint disorders and certain cancers in dogs associated with neutering at ≤ 6 months, 6 11 months, 1 ≤ 2 years and 2 8 years. our 3 published studies on the golden retriever, labrador retriever, and german shepherd dog reveal an increase in the incidence of a joint disorder with neutering done within the first year to 2 4 times that of the 3 5% incidence of intact dogs. in golden retriever females, neutering at any age was associated with an occurrence of one of the cancers followed to 3 4 times that of the 3 percent incidence of intact females. the overall project, just completed, includes data and analyses on 35 breeds and 5 weight categories of mixed breed dogs. considering joint disorders, it is evident that the vulnerability to early neutering similar to the retrievers is related to body size, with small dog breeds showing no vulnerability. the increased risks of cancers with neutering, is breed specific, except that small dog breeds are generally not affected. the rather striking differences among breeds associated with neutering provides a strong rationale for taking into account breed and gender in advising clients about the appropriate age for spay or neuter of a puppy. keywords: neutering, spaying, joint disorders, cancers in the us1 and increasingly in much of europe, the practice of neutering male and spaying female dogs (referred to as neutering) has become routine and is increasingly being performed at ~ 6 months of age. in fact, in veterinary small animal practice, one could argue that neutering at 6 months has become a standard, with an abundance of low-cost clinics offering this service to dog owners in general. however, during the past decade, investigations have shown that the debilitating joint disorders of hip or elbow dysplasia and cranial cruciate ligament tears or ruptures may increase in association with neutering of male and/or female dogs.2,3 other studies have shown that some cancers, particularly hemangiosarcoma,4,5 lymphoma,6 and mast cell tumors,7 have increased in association with neutering in some male and female dogs. other studies, focusing on occurrence of multiple cancers, revealed that the reported incidence of cancers in vizslas was higher in neutered than intact dogs,8 whereas a study utilizing the veterinary medical database concluded neutered males and females were more likely to die of a cancer than intact dogs.9 however, none of these studies focused on age of neutering. given major morphological and physiological differences between breeds, it is only logical that if neutering would have an effect on the likelihood of a disease occurrence that some breeds would be affected but not others. however, the investigations on neutering and certain diseases cited above have generally pooled data from several breeds, the 2 genders, and/or the age of neutering, and thus offer no useful information with regard to determining the best age for neutering a dog of a particular breed to avoid increasing the likelihood of a serious or debilitating disease. the project at our center set out to provide breed-specific, gender-specific, data-based information on joint disorders, the cancers mentioned in the above studies, mammary cancer, pyometra and urinary incontinence, associated with neutering at various ages (≤ 6 months, 6 11 months, 1 ≤ 2 years and 2 8 years). we utilized the extensive computerized case record system going back to 2010. with a single case record system, the same diagnostic criteria were applied to all analyses. published findings include 3 popular breeds: the golden retriever (n = 1,247),10,11 labrador retriever (n = 1,933)11 and german shepherd dog (n = 1,257).12 in all 3 breeds, there was an increase in the incidence of 1 or more of the joint disorders associated with neutering done in the first year in males and females to 2 4 times that of the 3 5 percent incidence of intact dogs. the increase in risks in 1 or more of the cancers followed was quite pronounced in female golden retrievers, where neutering at any 235 clinical theriogenology • volume 11, number 3 • september 2019 age was associated with an occurrence of 1 or more of the cancers followed that was 3 4 times that of the 3 percent incidence of intact females. in contrast, with male golden retrievers, and male and female labrador retrievers and german shepherd dogs, there was no increase in cancers above that of the dogs left intact. results in these 3 breeds are a representation of the variability in the occurrence of joint disorders and cancers that we found in many of the other breeds subsequently examined. our large project just completed, and pending publication, includes data and analyses on 32 additional breeds (including 3 varieties of poodles) and 5 weight categories of mixed breed dogs. the final list of breeds are: australian cattle dog, australian shepherd, beagle, bernese mountain dog, border collie, boston terrier, boxer, bulldog, cavalier king charles spaniel, chihuahua, cocker spaniel, collie, corgi (both breeds combined), dachshund, doberman pinscher, english springer spaniel, german shepherd dog, golden retriever, great dane, irish wolfhound, jack russell terrier, labrador retriever, maltese, miniature schnauzer, pomeranian, poodle-toy, poodle-miniature, poodlestandard, pug, rottweiler, saint bernard, shetland sheepdog, shih tzu, west highland white terrier, and yorkshire terrier. for the mixed breed dogs, weight categories were: small < 10 kg (< 22 lbs), medium 10 19 kg (22 42 lbs), standard 20 29 kg (43 64 lbs), large 30 39 kg (65 86 lbs) and giant 40+ kg (87+ lbs). joint disorders examined were: hip dysplasia, cranial cruciate ligament tear or rupture, and elbow dysplasia and cancers were lymphoma (lymphosarcoma), hemangiosarcoma and mast cell tumor. mammary cancer, pyometra and urinary incontinence were examined in female dogs. diseases were tracked in dogs left intact and in those neutered in these age ranges: < 6 months, 6 11 months, 1 year (12 23 months), and 2 8 years. the diseases were tracked through 11 years of age or until last seen at the hospital. the computerized hospital record system, with currently > 60,000 cases admitted per year, provided the dataset. the hospital serves as a primary care facility, as well as being a secondary and tertiary facility. the statistical evaluations, with standardized diagnostic criteria applied to various diseases, required a large database with a computerized record system. the study focused on proportional differences in disease occurrence between neuter age groups and intact dogs. survival analysis was used to test for differences with respect to the hazard of a disease in the neutered and intact groups, adjusting for the differences in time at risk. a two-tailed statistical level of significance was set at p < 0.05. although each breed was analyzed separately, with no statistical comparisons between breeds, overall findings with each breed or mixed-breed group allow for some general comparisons. considering the occurrence of joint disorders among all the breeds, it is evident that the vulnerability to early neutering is related to body size, as seen when comparing small-dog breeds, such as the chihuahua, pomeranian, or yorkshire terrier to dogs of larger breed size. the generalization about body size also applies to dogs of the mixed-breed categories. another general finding was that the increased risks of cancers with early neutering, such as reported in the golden retriever, and seen in some other breeds, including the border collie, boston terrier, and boxer, appear to apply to purebred dogs and not to mixed-breed dogs, where there was no significant relationship between neutering and cancer occurrence. in the small-dog breeds, with the exception of the shih tzu, there was no association between cancer incidence and spaying at any age. while only a proportion of akc registered dog breeds could be covered in our project, the complete data set will soon be available and clinicians then can, in many instances, extrapolate some guidelines for a breed not covered by reviewing results with related breeds and mixed breed weight categories. this rule will apply most aptly to debilitating joint disorders. the findings can also be used for shelters that wish to practice neutering on the principle that it is best for the long-term health of the dogs being adopted, where with small dogs the age of neutering makes no difference, but with large dogs some consideration could be given to allowing delaying of neutering until 1 (or sometimes 2) years of age. conclusion the rather striking differences among breeds with regard to vulnerability in the increase in joint disorders and some cancers associated with neutering of male and female dogs provides a strong rationale for taking into account breed and even the gender, in advising clients about the appropriate age for spay 236clinical theriogenology • volume 11, number 3 • september 2019 or neuter of a puppy. in conclusion, the time has come to adopt a new paradigm for spaying and neutering dogs that is based on benefiting the long-term health and welfare of the specific canine companion in question. acknowledgement supported by american kennel club, canine health foundation, and center for companion health, uc davis school of veterinary medicine; versatility in poodles. conflict of interest authors have no conflict of interest to declare. references 1. trevejo r, yang m, lund em: epidemiology of surgical castration of dogs and cats in the united states. j am vet med assoc 2011;238:898-904. 2. duerr fm, duncan cg, savicky rs, et al: 2007. risk factors for excessive tibial plateau angle in large-breed dogs with cranial cruciate disease. j am vet med assoc 2007;231:1688-16912. 3. witsberger th, villamil ja, schultz lg, et al: prevalence of, and risk factors for, hip dysplasia and cranial cruciate ligament deficiency in dogs. j am vet med assoc 2008;232:1818-1824. 4. ware wa, hopper dl: cardiac tumors in dogs: 1982–1995. j vet intern med 1999;13:95-103. 5. prymak c, mckee lj, goldschmidt mh, et al: 1988. epidemiologic, clinical, pathologic, and prognostic characteristics of splenic hemangiosarcoma and splenic hematoma in dogs: 217 cases. j am vet med assoc 1988;193:706-712. 6. villamil ja, henry cj, hahn aw, et al: hormonal and sex impact on the epidemiology of canine lymphoma. j cancer epidemiol 2009: 1-7. doi:10.1155/2009/591753 7. white cr, hohenhaus ae, kelsey j, et al: cutaneous mcts: associations with spay/neuter status, breed, body size, and phylogenetic cluster. j am anim hosp assoc 2011;47:210-216. 8. zink mc, farhoody p, elser se, et al: 13. evaluation of the risk and age of onset of cancer and behavioral disorders in gonadectomized vizslas. j am vet med assoc 2013:244;309-319. 9. hoffman jm, creevy ke, promislow del: reproductive capability is associated with lifespan and cause of death in companion dogs. plos one 2013:8:4. doi:10.1371. 10. torres de la riva g, hart bl, farver tb, et al: neutering dogs: effects on joint disorders and cancers in golden retrievers. plos one 2013:8,2. doi:10.1371/journal.pone.0055937 11. hart bl, hart la, thigpen, ap, et al: long-term health effects of neutering dogs: comparison of labrador retrievers with golden retrievers. plos one 2014:9:7. doi:10.1371/journal.pone.0102241. 12. hart bl, hart la, thigpen ap, et al: neutering of german shepherd dogs: associated joint disorders, cancers and urinary incontinence. vet med sci 2016;2:191-199. 237 clinical theriogenology • volume 11, number 3 • september 2019 238clinical theriogenology • volume 11, number 3 • september 2019 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web 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/untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice evolution of synchronization protocols and use of fixed time artificial insemination in beef cattle in south america evolution of synchronization protocols and use of fixed time artificial insemination in beef cattle in south america gabriel bó,1 pietro baruselli,2 alejo menchaca,3 reuben mapletoft4 1instituto de reproducción animal córdoba and universidad nacional de villa maria cordoba, argentina 2department of animal reproduction, university of são paulo, são paulo, brazil 3instituto de reproducción animal uruguay fundación irauy, montevideo, uruguay 4wcvm, university of saskatchewan, saskatoon, sk, canada abstract as our understanding of ovarian function in cattle improved, our ability to control it also increased. although prostaglandin f2α (pgf2α) has been used to synchronize estrus for several years, development of fixed time artificial insemination (ftai) protocols permitted widespread application of ai for breeding management and genetic improvement in beef herds in south america. over the last 20 years, a 10 fold increase in ai was largely due to use of ftai. main reasons for this growth were the possibility of obtaining acceptable pregnancy rates without estrus detection and induction of cyclicity in lactating cows that were anestrus and heifers that were prepubertal at the beginning of the breeding season. most ftai treatments for beef cattle in south america are based on use of progesterone (p4) releasing devices and estradiol to synchronize follicle wave emergence and ovulation, with pregnancies per ai (p/ai) ranging from 40 60%. these protocols are adapted for use with sexed semen with acceptable p/ai and several protocols have been developed for re-synchronization of ovulation for a second ftai in nonpregnant animals. most of these protocols are implemented on a regular basis, allowing producers to access high quality genetics through bull selection and to increase overall pregnancy rates during a breeding season. furthermore, it provided practitioners involved in those programs a new source of income and diversification of their practices to activities other than regular clinical work. keywords: estrus synchronization, ovulation, fixed time artificial insemination, beef cattle, pregnancy rates introduction increasing knowledge of ovarian physiology in cattle, mainly through application of real time ultrasonography in the late 1980’s, provided approaches for effective manipulation of ovarian function. protocols designed to control both luteal and follicular function permitted synchronization of ovulation and efficient implementation of fixed time artificial insemination (ftai).1 bovine practitioners around the world are now using these protocols, especially in south america.2 over the last 20 years, use of artificial insemination (ai) in south america increased 10 fold, due largely to the use of ftai.2 this presentation will briefly review the ftai protocols currently used and describe evolution of this technology in south america. synchronization of ovulation and fixed time artificial insemination protocols in beef cattle estradiol/progesterone based treatments for fixed time artificial insemination estradiol and progesterone (p4) treatments involve insertion of a p4 releasing device and administration of 2 mg of estradiol benzoate (eb) on random days of the cycle (day 0; to induce follicle atresia and synchronize follicular wave emergence 4 days later), pgf2α at the time of p4 device removal on days 7, 8 or 9 (to ensure luteolysis) and the subsequent application of 1 mg eb 24 hours later, gnrh or plh 54 hours later, or 0.5 or 1 mg of estradiol cypionate (ecp) at the time of p4 device removal to synchronize ovulation.1 another treatment instrumental in successful application of ftai in south america is administration of equine chorionic gonadotropin (ecg) at the time of removal the p4 device,1,3 as this improves growth of dominant follicle and increases ovulation rate4,5 and circulating p4 concentrations in the subsequent luteal 255 clinical theriogenology • volume 11, number 3 • september 2019 phase in cows with postpartum anestrus.5,6 in south america, 80% of suckled cows and 50% of yearling heifers are not cycling at the beginning of breeding season in most commercial herds.7 therefore, use of ecg has permitted increases in p/ai from 30 35% to 45 60% in bos indicus1,4 and bos taurus herds5,7 with high incidences of postpartum anestrus. gnrh based treatments gnrh based protocols are used widely for beef cattle in north america and europe. although gnrh use is limited in south america because of cost and availability of less expensive estradiol esters, there is increased interest in gnrh based protocols among practitioners, due to political pressure from the european union to avoid estradiol treatments in eu certified herds. most commonly used gnrh protocol is called cosynch; gnrh is administered at the time of ftai to synchronize ovulation.8 in general, cosynch protocols have included insertion of a p4 device to overcome poor ovulation rates after first gnrh in heifers9 and in anestrus postpartum suckled beef cows.10 data on the addition of ecg to gnrh/p4 based treatment protocols are more controversial, with reported improvements in p/ai in bos indicus11 and bos taurus cows in postpartum anestrus12 and in primiparous bos taurus cows that were not presynchronized.13 however, no improvement in p/ai has been reported in bos taurus cows with low incidences of postpartum anestrus and moderate to high body condition scores (bcs).14 extending the proestrus period in ftai protocols protocols for ftai that prolong the period from p4 device removal to ovulation were developed with the objective of lengthening the interval of preovulatory estradiol exposure and improving uterine function and early embryo development.15,16 this protocol is referred to as the 5 day cosynch + p4 and reported to result in higher p/ai than 7 day cosynch + p4 in beef cows.15,17 main changes in this protocol are a reduced interval of presence of p4 device from 7 to 5 days, to avoid adverse effects of persistent follicles on fertility in cows not ovulating to first gnrh, and a prolonged interval from p4 device removal to gnrh treatment to lengthen proestrus and increase exposure to elevated circulating estradiol concentrations prior to ovulation.18 higher estradiol concentrations in the preovulatory period have been associated with an increased ability of the uterus to support conceptus development19,20 and lower embryonic losses in the interval between maternal recognition of pregnancy and placental attachment.21 because of the shorter interval between the first gnrh and induction of luteolysis in the 5 day cosynch + p4 protocol, a single treatment of pgf2α was not effective in inducing luteolysis in beef cows that ovulated in response to gnrh;22 2 doses of pgf2α 8 -12 hours apart were required for high p/ai.23 in a large field trial with 2465 postpartum beef cows, p/ai was greater (p < 0.05) in cows receiving two pgf2α treatments, 8 hours apart (55%) than those receiving only one pgf2α (48%), with those receiving two pgf2α at the same time being intermediate (51%).16 hence, double pgf2α given 8 24 hours apart seemed necessary to maximize fertility with the 5 day protocol. if farm conditions do not permit the extra handling, a double dose of pgf2α given at device removal would be an acceptable alternative, or the first gnrh may not be administered, especially in heifers (see below). use of the 5 day cosynch + p4 protocol has also been investigated in bos indicus breeds in south america, with lower p/ai in suckled nelore cows than with the conventional 8 day estradiol/p4 based protocol24. however, an important difference in this study was that 400 iu ecg was used in the estradiol/p4 based protocol but it was not used in the 5 day cosynch + p4 protocol. to confirm the importance of ecg, we found no differences in p/ai in cycling cows treated with either 5 day cosynch + p4 or estradiol/p4 based protocol without the use of ecg, but p/ai was higher in cows in postpartum anestrus that received 400 iu ecg at p4 device removal (5 day cosynch + p4: 46.3%, 120/259; estradiol/p4 based: 54.5%, 151/277) than in cows treated with 5 day cosynch + p4 but without ecg (26.8%, 71/265; p < 0.05).12 the 5 day cosynch + p4 protocol was examined in heifers,25 with some modifications; for example, colazo and ambrose26 and cruppe et al.27 reported that p/ai did not differ in heifers that did not receive gnrh at the time of insertion of a p4 device. the important issue with this 256clinical theriogenology • volume 11, number 3 • september 2019 approach is that heifers are not highly responsive to gnrh administered at random stages of the follicular wave and the 5 days of p4 are not sufficient for development of a persistent follicle; by not administering the first gnrh, a single injection of pgf2α is all that is required to induce luteal regression. controversy still exists concerning the necessity of using one or two doses of pgf2α, when the first gnrh is administered in the 5 day protocol, with no reported differences28 and higher pregnancy rates when two doses of pgf2α were used, with intervals ranging from 6 24 hours.25,29 optimal timing of ftai remains controversial; kasimanickam et al.28 reported higher p/ai in heifers inseminated at 56 hours after p4 device removal than at 72 hours, whereas day25 suggested ftai 60 66 hours after p4 device removal or insemination 12 hours after estrus observed using tail patches or tail paint and ftai/gnrh to all those not in estrus by 72 hours. expression of estrus influences p/ai in cows30 and colazo et al.31 have reported similar findings in heifers inseminated with sexed semen, suggesting the possibility of splitting insemination times based on expression of estrus (i.e. delaying insemination in animals not detected in estrus by 72 hours after removal of a p4 device). estradiol/p4 based protocols with extended proestrus we recently conducted a series of experiments to evaluate an estradiol/p4 based protocol with a prolonged proestrus interval, which has been named jsynch.32 the treatment consists of administration of 2 mg eb at the time of insertion of a p4 device that is removed 6 days later. a single dose of pgf2α and sometimes 300 (heifers) or 400 (cows) iu of ecg are given at device removal. all cattle receive gnrh and are ftai, without estrus detection, 72 hours later (day 9). if estrus detection aids, such as tail paint or patches are used, the time of ai in heifers in estrus can be advanced to 60 or 66 hours after p4 device removal and only those not displaying estrus by 72 hours receive gnrh and ftai at that time.33 studies using real time ultrasonography and hormone determinations have shown that the growth rate of the ovulatory follicle and serum p4 concentrations in the ensuing luteal phase were greater in heifers treated with the 6-day jsynch protocol than in those treated with the conventional 7 day estradiol/p4 based protocol. furthermore, immunohistochemistry and real-time pcr of biopsies taken on day 6 after ovulation suggested that heifers treated with the prolonged proestrus protocol had a more mature uterine environment for embryo development.34 in most studies conducted in beef bos taurus heifers, p/ai was higher with the jsynch protocol than with the conventional estradiol/p4 protocol (table 1).33,35 although p/ai did not differ in a study in bos indicus heifers,36 p/ai was higher in nelore heifers treated with the jsynch protocol than those treated with the conventional protocol in a more recent study (table 1).37 table 1. pregnancy rates in beef heifers treated with the jsynch or with the conventional estradiol based treatment protocols. experiments reference jsynch conventional p value 1 bó et al. 2016 (n = 583) 59.7% (175/293) 53.1% (154/290) 0.12 2 bó et al. 2016 (n = 208) 67.9% (70/103) 46.6% (49/105) 0.01 3 bó et al. 2016 (n = 555) 37.8% (104/275) 49.3% (138/280) 0.01 4 bó et al. 2016 (n = 2.349) 56.1% (631/1,125) 50.7% (620/1,224) 0.01 5 motta et al. 2016 (n = 785) 52.9% (207/391) 53.6% (211/394) 0.8 6 pincinato et al. 2018 (241) 62.9% (68/108) 48.1% (64/133) 0.01 total 54.7% (1,255/2,295) 50.1% (1,236/2,426) 0.01 in experiments 1, 2 and 3, heifers did not receive ecg at device removal. in experiments 4, 5 and 6, all heifers received 200 (experiment 4) or 300 iu ecg at p4 device removal. in experiment 2, heifers were in low body condition score (4 6 in a 1 9 scale) and losing weight, whereas in all other studies, heifers were in good body condition score (6 7) and gaining weight. 257 clinical theriogenology • volume 11, number 3 • september 2019 fixed time artificial insemination using sexed semen improvements in sorting and freezing procedures with sexed semen and increasing number of sperm from 2.1 x 106 4 x 106 sperm per straw has created interest in the application of this technology in beef herds. the new product is called sexedultratm.38 experiments with sexedultratm semen have been performed in beef heifers and suckled cows in south america. the overall recommendation with the conventional estradiol/p4 based protocols is to delay insemination time with sexed semen in both heifers and cows by 12 hours and to inseminate only those that have shown estrus by the time of ftai.33,39 we performed 2 studies to evaluate p/ai in beef heifers (n = 850) and suckled cows (n = 877) treated with the jsynch protocol and inseminated with sexedultratm or nonsorted (conventional) semen from split ejaculates of the same angus sires.33,40 all cattle were treated with the jsynch protocol as described above and were tail painted for estrus determination. cattle with the paint rubbed off by 60 or 72 hours after device removal were inseminated at 72 hours, whereas those not displaying estrus by 72 hours received gnrh at that time and were inseminated at 84 hours. in heifers, the overall p/ai were 49.3% for those inseminated with sexedultratm and 58.3% for those inseminated with conventional semen (p < 0.05, table 2).33 in suckled cows, the overall p/ai was 45.3% for those receiving sexedultratm semen and 68.3% for those inseminated with conventional semen (p < 0.05, table 2).40 in summary, protocols designed for ftai in cows and heifers can be adapted for sexed semen. although, p/ai are lower than those obtained with conventional semen, delaying the time of ai or limiting ai to those showing estrus would result in p/ai ranging from 40 and 50% and often higher. table 2. pregnancy rates in angus heifers and suckled cows inseminated with sexed (sexedultratm) or conventional semen according to the expression of estrus (tail paint reading) and time of insemination (ai) after removal of the progesterone releasing device. n estrus 60 hours ai 72 hours estrus 72 hours ai 72 hours no estrus at 60 or 72 hours ai 84 hours total beef heifers sexedultra (4 x 106 sperm) 426 59.0% (104/176) 45.5%a (61/134) 38.8% (45/116) 49.3%a (210/426) conventional (25 x 106 sperm) 424 69.2% (119/172) 58.0%b (79/136) 42.2% (49/116) 58.3%b (247/424) beef suckled cows sexedultra (4 x106 sperm) 435 40.8%a (93/228) 43.0%a (34/79) 56.5%a (70/124) 45.3%a (197/435) conventional (25 x 106 sperm) 442 65.8%b (154/234) 67.9%b (57/84) 74.2%b (92/124) 68.6%b (303/442) ab denotes differences in p/ai between sexed or conventional semen (p < 0.05). resynchronization treatments with p/ai ranging from 40 60% after the first ai, producers are asking researchers and practitioners for development of protocols that would achieve higher p/ai in a breeding season or even the possibility of developing protocols in which 85 95% of the cows get pregnant only by ai, without the necessity of cleanup bulls. several resynchronization protocols were investigated over the years; however, most require estrus observations or an interval of approximately 40 days between the first and second ftai.35,41 in order to be able to inseminate nonpregnant cows as early as possible, resynchronization treatments must start earlier than pregnancy diagnosis. one protocol developed involves insertion of a used p4 device (i.e. previously used in the first synchronization) on days 14 16 after the first ai and its removal on day 23 when gnrh is administered. ultrasonographic pregnancy diagnosis is performed on day 258clinical theriogenology • volume 11, number 3 • september 2019 30 and those that are non-pregnant receive pgf2α combined with either 0.5 mg ecp at that time or gnrh at the time of ftai on day 32. p/ai for the first and second ftai at an interval of 32 days and the overall p/ai in a field trial involving 6431 beef cows and heifers were 57, 51 and 79%, respectively.35 two other protocols for resynchronization are called resynch 22 and resynch 14.41 in the resynch 22, cows receive 2 mg eb and heifers 1 mg eb at the time of p4 device insertion on day 22 after ftai. pregnancy diagnosis is performed at device removal on day 30 when nonpregnant cattle receive pgf2α and ecp and are inseminated on day 32. in a recent field trial, the use of three consecutive ftai with the resynch 22 protocol had a similar overall pregnancy rate (87.8%, 663/755) as achieved using cleanup bulls after two ftai using resynch 22 (87.7%, 263/300) and greater pregnancy rate than one ftai followed by bull exposure (77.1%, 347/450).42 in resynch 14, the initial treatment starts 14 days after ftai with reinsertion of a used p4 device and administration of 100 mg p4 im.41 pregnancy assessment is done by the evaluation of the vascularity of the cl using doppler ultrasonography at the time of p4 device removal on day 22. nonpregnant cattle receive pgf2α and ecp at that time and are inseminated on day 24. in a recent study, similar p/ai were observed for resynch 22 and resynch 14 groups following the first ftai (48% versus 53%; p = 0.57) and resynchronization (56 versus 51%; p = 0.37), respectively. however, resynch 14 reduced the interval between ftai, which resulted in a 21-day pregnancy rate of 87.5% compared to 66% with resynch 22.43 in conclusion, with the existing resynchronization programs it is now possible to breed beef cows exclusively with ftai, eliminating the need for estrus detection and cleanup bulls. application of fixed time artificial insemination in south america south american countries represent the largest beef herd in the world. in 2016, latin american countries had approximately 359 m cattle (accounting for 24% of the world's cattle population) and produced 15.1 m tons of beef.2 of the main beef producing countries in south america, brazil (60.8% of the total), argentina (14.7%) and uruguay (3.3%) are also prominent in development and application of assisted reproductive biotechnologies. through training and continuing education programs, many of which one or more of the authors have organized or participated in, these technologies are being used increasingly in other south american countries. it is through these efforts, in partnership with the pharmaceutical industry, that the ruminant reproductive revolution in south america has become widespread. brazil and argentina, in particular, are not only very active in research but also in continuing education and postgraduate student training in reproductive biotechnology. in just one program, the specialization in bovine reproduction at the institute of animal reproduction in cordoba argentina (irac), more than 1200 practitioners from most south american countries have taken postgraduate training since 2002. most beef herds in brazil and paraguay are composed of bos indicus or bos indicus crosses, whereas argentina and uruguay often have more bos taurus breeds. bos indicus-influenced synthetic breeds have increased in argentina due to the recent expansion of agriculture that has pushed cattle breeding to less favorable environments. it is noteworthy that bos indicus breeds tend to have prolonged postpartum anestrus and low body condition scores on pasture, with an increased interval from calving to conception and low pregnancy rates.45 however, in a study involving 21,329 suckled bos taurus cows and heifers examined by ultrasonography at the beginning of the breeding season in commercial beef farms in uruguay, 80 90% of cows and 40% of heifers are not cycling (i.e. without a cl) at the beginning of the breeding season.7 thus, in these pasture-based cow calf production systems, synchronization protocols are not only necessary to produce pregnancies by ai during a short breeding season, but also to induce cyclicity in cows in anestrus that would likely not get pregnant if not treated.46 these are the main reasons why ftai has been incorporated into the breeding schemes of most progressive producers.1,35 breeding objectives have been to inseminate early in the breeding season, early ultrasonographic pregnancy diagnosis and re-insemination of nonpregnant cattle as soon as possible.41 as was mentioned previously, because estradiol preparations have been available in south america, most ftai protocols include estradiol as a means of synchronizing follicle 259 clinical theriogenology • volume 11, number 3 • september 2019 wave emergence and ovulation.1 as herd cyclicity and body condition scores are usually low, progestin devices and ecg are usually included in the synchronization protocols.1,3,6,35 protocols of 7, 8 or 9 days progestin treatment have resulted in similar pregnancy rates, and shortened progestin protocols with a lengthened proestrus involving either estradiol or gnrh have been used with success.41 ftai is increasing the use of ai, especially with bos taurus sires on bos indicus cows in brazil, paraguay and the northern part of argentina. in brazil, the use of ftai increased from ~ 1 m protocols in 2005 (11% of all ai) to 10.5 m protocols in 2015 (77% of all ai), and a further increase to > 11 m ftai in 2016 (figure 1).2 this represents more than a 10 fold increase in the use of ai in the brazilian beef herd. in argentina, there were < 200 x 103 ftai protocols in 2002 and this has increased to ~ 2.5 m in 2015 and > 3 m in the last breeding season. this accounts for > 90% of inseminations done in beef cattle in argentina, which is about 15% of the beef breeding females in the country (figure 1). uruguay and paraguay have experienced similar trends, with 300 x 103 ftai in uruguay and 500 x 103 ftai in paraguay, representing ~ 10% of their breeding females. in total, > 15 m cattle were inseminated by ftai in these four countries over the past year. the adoption of this technology has been suggested to be an excellent example of a technological change in the production sector emerging from scientific developments in the academic sector.2 figure 1. evolution of the application of ftai in uruguay, brazil and argentina. data were calculated based on official semen and hormone sales in the three countries. although the evolution figures for paraguay are not available, the number of cows ftai in the 2017 breeding season is included for reference and comparison. impacts of use of fixed time artificial insemination in commercial beef herds one of the main advantages of implementing ftai programs in a beef herd is that more cows can be impregnated earlier in the breeding season to genetically improved bulls, resulting in heavier weaning weights (reviewed46). between 40 and 60% of the cows could become pregnant on the first day of the breeding season, resulting in a higher number of cows calving at the beginning of the calving season. their calves will be older and heavier at weaning, and the use of genetically improved bulls will also result in heavier calves at weaning. impact of ftai is equally effective in various management systems in argentina and brazil.45,46 a recent study47 compared weaning weights and kilograms of beef per cow using estradiol/p4 based protocols with ftai or bull exposure versus traditional natural service. at the beginning of the breeding season, cows were randomly allocated to three treatment groups. cows in two groups received the conventional estradiol/p4 based treatment with ecg at device removal and then half of those cows were ftai and the other half directly exposed to 5% bulls for 90 days immediately after p4 device removal. cows in the control group were exposed directly to 5% bulls without synchronization treatments at the same time that devices were removed in two synchronized groups. cows in ftai group were also exposed to 5% bulls 10 days after ftai for the remainder of the breeding season. pregnancy diagnosis was performed by four sequential ultrasonographic examinations every 30 days. pregnancy rates after the first 25 days and at the end of the breeding season were significantly higher in cows in 260clinical theriogenology • volume 11, number 3 • september 2019 the two synchronized groups than in those in natural service (p < 0.05; table 3). however, the most important finding was the difference in weaning weights. calves in ftai group were 27.6 kg heavier and those in the synchronized group that were directly exposed to bulls were 19.1 kg heavier than those in the natural service group. furthermore, cows in the ftai group produced 69 kg more of beef at weaning than those exposed to natural service (table 3). changes in calving patterns and weaning weights have been considered the main reason why the most progressive cow/calf producers in south america have adopted this technology to improve profits.2,45,46 table 3. pregnancy rates during the first 25 days and at the end of a 90 day breeding season and weaning weights of calves produced, in beef cows that were synchronized with an estradiol/p4 protocol and exposed to bulls (synch with bulls), or ftai and then exposed to bulls for the remainder of the breeding season (ftai plus bulls), or directly exposed to bulls without any synchronization treatment (natural service). 25 day pregnancy rate (%) overall pregnancy rate (%) calf weaning weights (kg) synch with bulls 54.1% (60/111)b 94.6% (105/111)b 204.8 ± 28.8b ftai plus bulls 58.1% (61/105)b 96.2% (101/105)b 216.8 ± 19.8c natural service 25.5% (35/137)a 83.9% (115/137)a 191.2 ± 18.2a abc denotes differences within pregnancy rates or weaning weights (p < 0.05). conclusion ability to control follicular wave emergence and ovulation in cycling and noncycling cattle has allowed successful implementation of ftai in beef herds in south america. treatments with gnrh or estradiol and p4 releasing devices made ftai possible in beef cattle. these treatments are practical and easy to perform by the farm staff and are efficient and useful for inducing cyclicity at the beginning of the breeding season. practitioners in most south american countries are using these protocols, which has facilitated the use of ai in beef herds and application of other assisted reproductive technologies. currently, ftai procedures account for > 85% of all ai done in beef cattle in argentina, brazil, uruguay, and paraguay. successful application of ftai in south america is resulting in the dissemination of new and improved genetics and increased reproductive performance in all classes of cattle, with a corresponding increase in economic activity. conflict of interest there are no conflicts of interest to declare. acknowledgement research was supported by foncyt (pict 2017-4550), universidad nacional de villa maría, zoetis and sexing technologies from argentina, and anii, syntex uruguaya sa and fundaciba from uruguay. references 1. bó ga, baruselli ps, mapletoft rj: synchronization techniques to increase the utilization of artificial insemination in beef and dairy cattle. anim reprod 2013;10:137-142. 2. mapletoft rj, bó ga, baruselli ps et al: evolution of knowledge on ovarian physiology and its contribution to the widespread application of reproductive biotechnologies in south american cattle. anim reprod 2018;15 (suppl 1):1003-1014. 3. baruselli ps, reis el, marques mo, et al: the use of treatments to improve reproductive performance of anestrus beef cattle in tropical climates. anim reprod sci 2004;82-83:479-486. 4. sá filho mf, ayres h, ferreira rm, et al: equine chorionic gonadotropin and gonadotropin-releasing hormone enhance fertility in a norgestomet-based, timed artificial insemination protocol in suckled nelore (bos indicus) cows. theriogenology 2010;73:651-658. 261 clinical theriogenology • volume 11, number 3 • september 2019 5. núñez-olivera r, de castro t, garcía-pintos c, et al: ovulatory response and luteal function after ecg administration at the end of a progesterone and estradiol-based treatment in postpartum anestrous beef cattle. anim reprod sci 2014;146:111-116. 6. baruselli ps, sá filho mf, ferreira rm, et al: manipulation of follicle development to ensure optimal oocyte quality and conception rates in cattle. reprod dom anim 2012;47 (suppl 4):134-141. 7. menchaca a, núñez r, wijma r, et al: how fertility can be improved in fixed-time ai programs in beef cattle. proc x international symposium on animal reproduction, irac, cordoba, argentina, 2013; p. 103134. 8. geary tw, whittier jc, hallford dm, et al: calf removal improves conception rates to the ovsynch and cosynch protocols. j anim sci 2001;79:1-4. 9. martinez mf, kastelic jp, adams gp, et al: the use of progestins in regimens for fixed-time artificial insemination in beef cattle. theriogenology 2002;57:1049-1059. 10. lamb gc, stevenson js, kesler dj, et al: inclusion of an intravaginal progesterone insert plus gnrh and prostaglandin f2α for ovulation control in postpartum suckled beef cows. j anim sci 2001;79:2253-2259. 11. pincinato d: 2012. follicular dynamics and fertility in beef suckled cows synchronized with progesterone releasing devices and gnrh. master of science thesis. faculty of agriculture sciences, national university of cordoba, cordoba, argentina 2012; p. 45. 12. huguenine e, peracchia s, benitez r, et al: effect of the utilization of 5-day co-synch protocols combined or not with ecg in suckled cows in postpartum anoestrus. proc x symposium on animal reproduction, irac, córdoba, argentina, 2013; p. 313 (abstract). 13. small ja, colazo mg, kastelic jp, et al: effects of progesterone presynchronization and ecg on pregnancy rates to gnrh-based, timed-ai in beef cattle. theriogenology 2009;71:698-706. 14. marquezini ghl, mercadante vrg, olson kc, et al: effects of equine chorionic gonadotropin on follicle development and pregnancy rates in suckled beef cows with or without calf removal. j anim sci 2013;91:1216-1224. 15. bridges ga, helser la, grum de, et al: decreasing the interval between gnrh and pgf2α from 7 to 5 days and lengthening proestrus increases timed-ai pregnancy rates in 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progesterone insert nor initial gnrh treatment affected pregnancy per timed-insemination in dairy heifers subjected to a co-synch protocol. theriogenology 2011:76:578-588. 27. cruppe lh, day ml, abreu fm, et al: the requirement of gnrh at the beginning of the five-day co-synch + controlled internal drug release protocol in beef heifers. j anim sci 2014;92:4198-4203. 28. kasimanickam r, asay m, firth p, et al: artificial insemination at 56 h after intravaginal progesterone device removal improved ai pregnancy rate in beef heifers synchronized with five-day co-synch controlled internal drug release (cidr) protocol. theriogenology 2012:77:1624-1631. 29. peterson c, alkar a, smith s, et al: effects of one versus two doses of prostaglandin f2alpha on ai pregnancy rates in a 5-day progesterone-based, co-synch protocol in crossbred beef heifers. theriogenology 2011;75:1536-1542. 30. richardson bn, hill sl, stevenson js, et al: expression of estrus before fixed-time ai affects conception rates and factors that impact expression of estrus and the repeatability of expression of estrus in sequential breeding seasons. anim reprod sci 2016;166:133-140. 262clinical theriogenology • volume 11, number 3 • september 2019 31. colazo mg, macmillan k, mapletoft rj. modifications of a 5-d gnrh-based timed-ai protocol to optimize fertility in holstein heifers inseminated with sex-selected semen. anim reprod 2018;15(suppl 1):1111 (abstract). 32. de la mata jj, bó ga. estrus synchronization and ovulation using protocols with estradiol benzoate and gnrh and reduced periods of insertion of a progesterone releasing device in beef heifers. taurus 2012;55:17-23. 33. bó ga, huguenine e. de la mata jj, et al: programs for fixed-time artificial insemination in south american beef cattle. anim reprod 2018; 15 (suppl 1):952-962. 34. de la mata jj, núñez-olivera f, cuadro f, et al: impact of extending the proestrus length in an oestradiol and progesterone-based oestrus synchronization program on follicular and luteal development, uterine gene expression, and protein localization, and pregnancy rates in beef heifers. reprod fertil dev 2018; 30:1541–1552 35. bó ga, de la mata jj, baruselli ps, et al: alternative programs for synchronizing and resynchronizing ovulation in beef cattle. theriogenology 2016;86:388-396. 36. motta jcl, colli mha, penteado l, et al: pregnancy rate to ftai in nelore and crossbreed heifers submitted to j-synch protocol (6 days). anim reprod 2016;13:401. (abstract). 37. pincinato d, peres lc, lorentz l, et al: pregnancy rates in nelore heifers using a shortened estradiol/progesterone-based protocol that provides for a lengthened proestrus (j-synch). anim reprod 2018;15:350 (abstract) 38. vishwanath r: sexed sperm vs conventional sperm – a comparative discussion. proceedings, applied reproductive strategies in beef cattle 2015; p. 250-256. 39. baruselli ps, campos filho ep, crepaldi ga, et al: new strategies to improve pregnancy rate at tai using sex-sorted semen. anim reprod 2017;14:711 (abstract). 40. bó ga, huguenine e, de la mata jj, et al: pregnancy rates in suckled beef cows synchronized with a shortened progesterone/estradiol-based protocol (j-synch) and inseminated with conventional or sexedsorted semen. reprod fertil dev 2019;31:129 (abstract). 41. baruselli ps, ferreira rm, colli mha, et al: timed artificial insemination: current challenges and recent advances in reproductive efficiency in beef and dairy herds in brazil. anim reprod 2017;14:558-571. 42. crepaldi ga, freitas bg, mingoti rd, et al: reproductive efficiency of nelore cows submitted to three different reproductive strategies in a 64 days breeding season. anim reprod 2017;14:698 (abstract). 43. penteado l, rezende rg, mingoti rd, et al: pregnancy rate of nelore cows submitted to resynchronization starting 14 or 22 days after prior ftai. anim reprod 2016;13:450. (abstract). 44. bó ga, baruselli ps, martinez mf. pattern and manipulation of follicular development in bos indicus cattle. anim reprod sci 2003;78:307-326. 45. baruselli ps, ferreira , sa filho m, et al: review: using artificial insemination v. natural service in beef herds. animal 2018;12 (suppl 1): s45-s52. 46. bó ga, baruselli ps: synchronization of ovulation and fixed-time artificial insemination in beef cattle. animal 2014;8 (suppl 1):144-150. 47. burtre c, pérez wallace s: evaluation of ftai, the synchronization and natural service in the reproductive and productive performance in beef cows in the north of patagonia. proc xii international symposium on animal reproduction, irac, cordoba, argentina 2017; p. 366 (abstract). 263 clinical theriogenology • volume 11, number 3 • september 2019 264clinical theriogenology • volume 11, number 3 • september 2019 264clinical theriogenology • volume 11, number 3 • september 2019 introduction synchronization of ovulation and fixed time artificial insemination protocols in beef cattle omniblank: growing interest in hormone sparing dog sterilization and recommendations for standard identification methods growing interest in hormone sparing dog sterilization and recommendations for standard identification methods linda brent parsemus foundation, san francisco, ca abstract sterilization methods for pets have been around for more than a century, but the practice of spaying and neutering dogs varies globally, from being considered a standard of responsible care in some countries to an infringement of animal welfare in others. in the us, advocacy for spay/neuter programs became widespread in the 1970s to address canine overpopulation. more recently, research on the impact of canine neutering has identified potentially serious health and behavior consequences of removal of the gonads and associated sex hormones that appear to be influenced by sex, breed, age and environment. an alternative is hormone preserving sterilization, including hysterectomy and vasectomy, which allows population control while maintaining natural hormone concentrations. informal analyses regarding alternatives to traditional spay/neuter indicate that interest from the public and veterinarians has grown in the last 2 years, public demand for veterinarians who offer alternatives is increasing and although most veterinarians acknowledge the pros and cons of gonadectomy, the number providing hormone preserving sterilization is very low. given current trends toward individualized medicine and increasing public demand, it is likely that the number of practitioners who offer vasectomy, hysterectomy or other hormone reserving sterilization procedures will grow. now is the time to develop standard methods of identifying dogs who have received such procedures, so that they do not unnecessarily undergo a second surgery. following an analysis of current practice and available identification methods, we recommend that simple green tattoos be applied to the inguinal area (“x” for hysterectomy and “v” for vasectomy) to identify sterilized dogs. keywords: spay/neuter, hysterectomy, vasectomy, identification, tattoo, hormone sparing introduction and history sterilization of cats and dogs as a standard part of pet care is a relatively new concept and not practiced widely on a global scale. neutering a dog or cat usually refers to gonadectomy: castration of males (orchiectomy) and spaying in females (ovariohysterectomy). however, several other methods for ensuring that pets do not reproduce have been developed, which are of increasing interest as we better understand impacts of gonadectomy on health and welfare. one of the earliest accounts of pet sterilization is an 1893 description of neutering an unanesthetized male cat.1 during the early 20th century, killing a female’s kittens shortly after birth was a customary method of birth control. it was not until the 1930s that spaying and castrating cats was recommended.1 lack of safe anesthetics caused greater risk of surgery and pet sterilization remained infrequent until the latter half of the century. over time, owning dogs and cats changed from a utilitarian practice to one of companionship. however, increases in number of intact, free-roaming dogs and cats in the us. ultimately resulted in the shelters being overrun with stray animals by the 1960s. in 1973, 13.5 million dogs and cats were euthanized annually by us shelters, equating to ~ 20% of the owned dog and cat populations (65 million).2 the number of animal shelters continued to grow over time, from a few hundred in the 1960s to 3,500 by 2015.2 sterilizing animals was an obvious method to reduce pet overpopulation; in the us, spay/neuter clinics opened in the 1970s to provide low cost/high volume service. animal protection organizations supported widespread spay/neuter programs and lobbied for stricter regulations on licensing, control and sterilization of pets. by 2010, a majority of states in us had mandatory spay/neuter laws to address the homeless pet population. in 32 states, animal shelters were required to sterilize dogs and cats prior to releasing them (except animals that were too young or medically unfit for the surgery).3 247 clinical theriogenology • volume 11, number 3 • september 2019 spay/neuter programs, along with other efforts to address pet overpopulation in the us, successfully reduced dog and cat euthanasia rates as well as shelter intakes, which declined rapidly in the 1970s. by the mid 1980s, euthanasia rates of cats and dogs had decreased to 7.6 10 million (or 10% of owned dogs and cats) and shelter intakes were reduced by 50%.2 gonadectomy has become the standard in the us, with over 80% of owned dogs being neutered. veterinary schools train their students to perform orchiectomy and ovariohysterectomy as common surgical procedures in dogs and cats. however, the practice of neutering pets varies greatly across the world, and in some cultures, it may be considered cruel or harmful. for example, by 2011 as many as 80% of male and female dogs were neutered in the uk but in germany and scandinavia it is less common and the practice is often regulated.4 yet, a lack of standard sterilization does not always result in serious pet overpopulation. in norway and sweden, for example, there are virtually no stray dogs even though neutering is very uncommon, usually only allowable due to an individual health concern.5 "responsible pet ownership" instead of spay/neuter is the standard. in the us, animal cruelty laws derive from an agrarian tradition of considering animals as property (to be protected) and contain many exemptions when an action is of utility to the owner.6 european companion animal legislation, by contrast, aims to protect the animal for its own sake, including provisions not to cause unnecessary harm.6,7 implications of gonadectomy and hormone preserving options removal of gonads and associated sex hormones, is known to have significant health and welfare impacts on dogs beyond halting reproduction. on the positive side, gonadectomized dogs are not as likely to have diseases associated with the sex organs, such as mammary, ovarian and testicular cancers, pyometra and prostate disorders.8 yet a growing body of research on the outcomes of canine gonadectomy indicate that serious health complications unrelated to sex organs may occur. obesity, urinary incontinence, various cancers, immune-mediated diseases, musculoskeletal disorders and cognitive and behavior problems are more common in gonadectomized dogs.8-13 natural hormone feedback mechanisms become unregulated in neutered dogs. emerging research indicates that the high levels of unopposed luteinizing hormone likely influence development of diverse health disorders.14 the relationships between sex hormones, health and wellness are not simple and may be influenced by many factors, including sex, breed, age and environment. although additional studies will help to elucidate these interactions, enough information has emerged that dog owners are increasingly seeking other options to control reproduction without impacting natural hormones. such gonad sparing sterilization options have been known for some time, but are not commonly practiced by veterinarians in the us. for female dogs, the first publication on hysterectomy (also called ovary-sparing spay or partial spay) was an elegant and succinct treatise published in 197215 and tubal ligation surgery was reported in 1973.16 both surgical procedures preserve the ovary and associated hormones, but complete hysterectomy is usually preferred, since it eliminates the risk of pyometra by removing both the uterus and cervix. for male dogs, the surgical option is vasectomy. nonsurgical hormone-preserving options are also of great research interest, especially for resource-limited environments. a recently published study reported successful canine sterilization while preserving testosterone using calcium chloride dihydrate in alcohol injected into the epididymis with ultrasound guidance;17 dogs retained normal hormone profiles, yet were sterile. other experimental approaches, such as epididymal ligation or therapeutic ultrasound, have promising results.18,19 whereas nonsurgical gonad sparing options will likely become available in the future, highly-informed dog owners are increasingly becoming aware of existing surgical techniques to sterilize while preserving hormones. interest in alternative canine contraception as part of its mission to create meaningful improvements in human and animal health and welfare by advancing innovative and neglected medical research, the parsemus foundation has supported research on new sterilization techniques for pets and its website serves as a resource for veterinarians and the public regarding hormone sparing alternatives to traditional spay and neuter.20 additionally, the site 248clinical theriogenology • volume 11, number 3 • september 2019 maintains a free listing service of veterinarians who offer contraceptive options for dogs beyond ovariohysterectomy (spay) and orchiectomy (neuter).21 the organization is regularly contacted by pet owners looking for a veterinarian in their area who offers hormone sparing methods and by veterinarians who have questions or wish to join the listing service. enumeration of these contacts from september, 2013 through december, 2018 provided information on the recent trend in interest in options beyond traditional spay/neuter. total quarterly contacts increased 4 fold by 2017 2018 (from 2.9 contacts/quarter in 2013 2016 to 12.5 contacts/quarter in 2017 2018; figure 1). this increase likely reflects how the growing number of scientific publications, media placements and internet posts on the topic are translating into public demand and veterinary interest in hormone-sparing methods. figure 1. contacts to the parsemus foundation regarding alternatives to traditional spay/neuter from the last quarter of 2013 through the last quarter of 2018. the contacts were via email and included the public seeking a veterinarian or asking questions (grey line) and veterinarians or others registering or updating information on the listing of veterinarians maintained at the website (black line).21 contacts increased 427% from 2013 2016 to 2017 2018. a social media group (ovary sparing spay and vasectomy info facebook group) was launched in may 2013 to discuss alternatives to traditional spay and neuter. as of january 28, 2019, the group had 13,553 members (8,500 active), with 72% of the members from the us followed by canada (10%), uk (8%) and australia (5%). the group also maintains a listing of veterinary clinics around the world that offer these procedures (170 clinics as of february 7, 2019).22 clearly the awareness and interest in sterilization options beyond hysterectomy and orchiectomy has grown in recent years. however, the experience of the parsemus foundation indicates that there are not enough veterinary practitioners who offer alternative services to meet client demand. the american veterinary medicine association estimates that there are > 68,000 companion animal veterinarians working in the us.23 yet only 195 veterinarians are listed as offering hysterectomy or vasectomy on the lists maintained by parsemus foundation or the ovary sparing spay and vasectomy info facebook group. the low proportion of practitioners offering hormone sparing alternatives was confirmed in a survey of 81 veterinarians at the 2017 avma conference. whereas 73.4% reported discussing long-term health risks of traditional spay and neuter with dog owners prior to surgery, only 7.4% percent reported performing hysterectomies or vasectomies in dogs.24 we are also not aware of any animal shelters offering the option of hormone-preserving sterilization, which may dissuade some individuals from adopting from shelters that require gonadectomy before release. looking forward and the need to address identification some may feel that the data available on negative sequelae from ovariohysterectomy and orchiectomy are not consistent or significant enough to warrant a change from the current practice. with any complex system, though, numerous individual and external factors influence outcomes. the recognition of this fact in human medicine has resulted in a move away from a model in which all patients receive the same care based on average responses to clinical trials, toward personalized (or precision) 249 clinical theriogenology • volume 11, number 3 • september 2019 medicine, in which care is matched to an individual patient based on genetics, environment, lifestyle and other factors. veterinary oncology is beginning to adopt this perspective.25 sterilization method similarly requires an individualized approach, with the veterinarian and client evaluating risk factors specific to each dog. with the movement toward personalized medicine and increasing public demand, it is likely that over time, more veterinarians will begin to offer a variety of canine sterilization options, including hormone-preserving methods. standardizing methods to identify dogs that have undergone sterilization is needed now. apart from castration, which is usually visually identifiable, other methods (including nonsurgical sterilization) may not be obvious unless the dog is marked in some way. appropriate identification of sterilized dogs is important so that the dog does not undergo surgery a second time unnecessarily. identification methods have been used in domestic animals for centuries and include various attributes: inherent features (visibility, duration and information conveyed) as well as practical features (ease and expense of administration and detection). the dog’s situation (stray or owned) and aspects of the sterilization procedure (whether sedation is required) are also important components of choosing appropriate identification methods. for a coherent review of the pros and cons of dog marking procedures, see the alliance for contraception of cats and dogs.26 for example, collars are highly visible, can convey different types of information, do not require sedation, are inexpensive, and are easy to apply, but they are not permanent and are not well-suited to stray dog populations since they must be monitored for replacement. in evaluating the best identification option for hormone-preserving sterilization, the following considerations are relevant:  dogs receiving hormone preserving sterilization are usually owned  permanent identification is important, since identifying sterilization status becomes relevant when there is a lack of prior medical information (if dog is rehomed, lost or abandoned)  hormone preserving methods, including hysterectomy, vasectomy and intraepididymal chemical sterilization, require sedation or general anesthesia of the dog the most common identification methods for owned dogs are collar, microchip or tattoo. collars are not appropriate for identification in this situation; they are not considered permanent and even if a tag were created indicating the dog had been sterilized, the veterinary practitioner has little influence on whether the dog will retain the identification. microchips are becoming more common for dog identification. the microchip is inserted under the skin and a code can be read with a scanning device using radio frequency signaling. the code must be registered with a service linking it to the owner’s information. other information, such as sterilization status, can be maintained with the owner’s data. microchipping has been shown to assist with reuniting lost pets with their owners, but the inaccuracy of the owner’s data can hamper the usefulness of microchips.27,28 additionally, there is added expense to implant a microchip, as well as the need to have a microchip reader and access to the microchip registry. while some countries now mandate microchip dog identification and iso standards, the us has not adopted a standard radio frequency, making microchip reading more complicated.29 microchips are gaining in popularity for pet identification, but tattoos have been commonly used to identify dogs since the mid 20th century in the us. a tattoo is visible, so there is no need for additional sensing equipment. unlike collars and microchips, which rely on the dog owner’s continued diligence for effectiveness, tattoos can be fully completed by the veterinarian at the time of sterilization. tattoos can be applied using several types of equipment: a tattoo clamp device which usually creates a number inside the ear; a tattoo pen which can be used to create freehand marks; or a needle filled with tattoo ink, which creates simple straight line marks (figure 2). 250clinical theriogenology • volume 11, number 3 • september 2019 figure 2. inguinal area of dog two years after tattoo using a needle to inject green tattoo ink to identify sterilization (arrow indicating tattoo). photo credit: ruth steinberger, spayfirst. the ink is inserted into the dermis layer of the skin and the mark is considered permanent, although the quality of the mark can degrade over time making it difficult to read. microneedle tattoo patches, based on the microneedle vaccine delivery patch technology30, is a new concept being studied that may provide a permanent mark without the need for equipment or sedation of the dog.31 tattoos appear to be the most commonly used identifier for sterilized dogs, although a variety of marks are used. the aspca provides instruction for standardized marking of dogs that have been spayed or neutered, creating a single straight line of green ink.32 however, there is a dearth of information available on what practitioners are adopting to identify dogs with nontraditional sterilization techniques. to gain information on what identification, if any, veterinary practitioners use to mark dogs receiving hormone preserving sterilization, a survey was sent to 102 veterinarians who offer sterilization procedures beyond traditional spay and neuter. the brief survey asked if dogs receiving hysterectomy or vasectomy were marked, and if so, how. twenty four responses were received. results indicated that a majority of veterinarians do not provide any identification of dogs receiving a hysterectomy (85.7%) or a vasectomy (68.2%). of the few who did mark dogs following hysterectomy, a microchip was used by 1 respondent, a green line on abdomen by another, and internal staple by the third respondent. to identify dogs receiving a vasectomy, 1 respondent used microchip and 6 used green tattoos (four used a green “v”-shaped tattoo and 2 used a green straight line tattoo). clearly, greater use of marking is required to prevent unnecessary repeat surgeries. however, lack of standardization has been a barrier to greater use. after review of common identification methods for owned dogs and methods being used currently by veterinarians, as well as consultation with theriogenologist michelle kutzler who has advocated for hormone-sparing dog sterilization, we recommend a simple tattoo in the inguinal area as the standard for hormone-preserving sterilization methods:  hysterectomy: green “x” slightly lateral to midline near umbilicus  vasectomy: green “v” lateral and cranial to scrotal region the simplicity of the marks does not require dedicated tattoo equipment or special skill, so that most veterinarians could provide the identification. tattoos can be created using a needle or scalpel to apply the tattoo ink to the dermis. the procedure is completed while the dog is sedated for a sterilization procedure, and no additional input from the owner is required and no additional equipment is needed for detection. although clarity of a tattoo may decrease over time, in this case it is not necessary that it is legible, but just visible enough to inform a practitioner that a sterilization procedure had already been completed. conclusion gonadectomy became a standard of responsible veterinary care to address pet overpopulation issues in the late 20th century in the united states. ovariohysterectomy and orchiectomy have been aggressively advocated for both owned and stray dog populations, with low cost/high volume spay/neuter clinics augmenting services provided by companion animal veterinary clinics, and animal shelters often mandating gonadectomy prior to release of adoptable dogs. 251 clinical theriogenology • volume 11, number 3 • september 2019 after decades of clinical experience with large numbers of gonadectomized dogs, researchers have begun to identify negative impacts on health and welfare after loss of natural hormones.8,14 it has been only recently that potential negative outcomes of spay/neuter have gained the attention of the public, and our analysis indicates an increase in public interest in hormone-sparing sterilization methods. hysterectomy, vasectomy and nonsurgical epididymal approaches are options to sterilize without impacting hormones, but are not widely practiced by veterinarians. public demand is likely to grow, and the trend toward individualized, precision medicine will further influence the decision making process used by veterinarians and clients to determine the best sterilization method for a dog. thus, hormonesparing sterilization methods are expected to take a place as one of several available canine contraception options. with the expected growth in the number of dogs receiving hormone-sparing sterilization surgery, it is important that identification is standardized so that dogs do not undergo unnecessary surgery if they are no longer with their original owners. following analysis of markings currently in use, we recommend simple, green ink tattoos in the inguinal area, "x" for hysterectomy and "v" for vasectomy. conflict of interest the author claims no conflict of interest. acknowledgement author acknowledges the pioneering efforts of elaine lissner, founder and trustee of parsemus foundation, in supporting and advocating for more contraceptive options for pets and thank her for review of an earlier version of this manuscript. the concepts in this manuscript have been improved by discussions with dr. michelle kutzler of oregon state university and joyce briggs of the alliance for contraception in cats and dogs. references 1. hartwell s: cats and cat care – 1940s-1960s: neutering & population control. 1996 -2004 [cited 2019 feb 4]. in: cats and cat care through the ages [internet]. available from http://messybeast.com/retro1940-neutering.htm 2. rowan a, kartal t: dog population & dog sheltering trends in the united states of america. animals 2018;8:3-20. 3. hodges c: brief summary of state spay and neuter laws. 2010. 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[internet]: new york: american society for prevention of cruelty; c2019 [cited 2019 feb 12]. spay/neuter tattoo application. available from: https://www.aspcapro.org/resource/spayneuter-tattoo-application 253 clinical theriogenology • volume 11, number 3 • september 2019 254clinical theriogenology • volume 11, number 3 • september 2019 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.7 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false 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/destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice introduction cryopreservation of ejaculated sperm has been used for years to permit the dissemination of genetic material in many species.1-5 in alpacas, most cryopreservation extenders used for ejaculated sperm contain tris, citrate, glycerol, and egg yolk.3-7 however, various extender components (such as skim milk) have been tested in attempts to improve postthaw sperm motility.4 cryopreservation of epididymal sperm facilitates saving genetic material in cases of unexpected death.8 for male alpacas that are being castrated to facilitate animal management and fleece production, epididymal sperm can also be cryopreserved at castration.9 in addition, collection of epididymal sperm avoids handling viscous seminal plasma that allows for a more refined cryopreservation method.6,9 similar to cryopreservation of ejaculated sperm, various extender components (such as lactose) have been investigated in alpaca epididymal sperm in attempts to improve postthaw motility.3 general practitioners who work with alpacas often also work with horses and have access to commercial equine semen freezing extenders. objective of this preliminary study was to evaluate alpaca epididymal sperm viability and total and progressive sperm motility following cryopreservation using 3 commercially available equine semen extenders. we hypothesized that equine semen extenders yield postthaw results comparable to reported for alpaca epididymal sperm cryopreservation. cryopreservation of alpaca epididymal sperm using commercial equine semen extenders: preliminary investigation anna mcallister,a michelle kutzlerb adepartment of integrative biology, oregon state university, corvallis, or bdepartment of animal and rangeland sciences, oregon state university, corvallis, or abstract cryopreservation of epididymal sperm enables saving genetic material in cases of unexpected death or castration. objective of this preliminary study was to evaluate alpaca epididymal sperm viability and motility following cryopreservation using 3 commercially available equine semen extenders. we hypothesized that equine semen extenders yield postthaw results comparable to those reported for alpaca epididymal sperm cryopreservation. epididymal sperm samples were collected from 4 adult intact male alpacas at castration. sperm samples were extended at a 1:1 ratio with 3 commercial equine semen extenders. extended semen was loaded into 0.5-ml straws and frozen in liquid nitrogen vapor. straws were immediately thawed in a circulating 38°c water bath. following thawing, viability and total and progressive motility were evaluated. postthaw viability and motility was less than what has been reported for alpaca epididymal sperm cryopreservation. additional research is needed to determine if poor results observed are related to the semen extender or the freezing/thawing method used. keywords: camelid, castration, freezing, motility, straw, viability materials and methods four adult intact male alpacas (mean ± sd: 7.5 ± 2.1 years) that were formerly herd sires and recently sired crias were used in this preliminary study. males were routinely castrated under general anesthesia using intravenous tiletamine and zolazepam (telazol®, zoetis). briefly, a scrotal incision was made over each testis and the fascia surrounding the testis was stripped. next the spermatic cord was crushed using an emasculator such that a section of vas deferens could be obtained. the skin incisions were allowed to heal by second intention. immediately following castration, vas deferens and cauda epididymides were dissected free from each testis (figure 1). for each side, the ductal lumen was slowly flushed with 0.5 ml of sterile saline (0.9%) in retrograde direction and the sperm-containing effluent was collected into an eppendorf tube. epididymal sperm sample was evenly aliquoted into 3 groups (gent extender [ge], lactose edta extender [le], and modified french equine extender [mf5]). each group was extended at a 1:1 ratio with 1 of 3 commercially available equine semen extenders (table). extended semen was loaded into labeled 0.5-ml cryopreservation straws, sealed, and frozen in liquid nitrogen vapor for 10 minutes before plunging into liquid nitrogen for storage. semen was thawed at 38°c for 60 seconds and examined under phase microscopy at 400 x magnification to determine postthaw total and progressive motility. in addition, semen was stained with clinical theriogenology 2021; 13: 367 eosin and nigrosin to determine postthaw viability. all procedures were approved by an institutional animal care and use committee [protocol #3664]. mean ± sd postthaw viability, total motility, and progressive motility were determined for each extender and compared among extender groups using a one-way analysis of variance (graphpad prism 9, san diego, ca). significance was defined as p < 0.05. results dissecting and catheterizing vas deferens and cauda epididymides for flushing sperm cells was not technically difficult. three of the 4 alpacas had viable sperm after freezing and thawing. there were no differences in viability, total motility, or progressive motility among extender groups (p = 0.3873, p = 0.1573, and p = 0.1496, respectively; figure 2). there was a variable amount of red blood cells in each epididymal sample; however, this did not affect the viability or motility. hyperactivated motility was observed in 1 postthaw sample frozen with the le extender. extender ingredients gent extender for frozen stallion semen (minitube of america, inc.) glucose, glycerol, egg yolk, skim milk, lactose, sodium, potassium citrate, gentamicin e-z freezin™ ‘lactose edta’ equine semen extender (animal reproduction systems) glucose, glycerol, egg yolk, lactose, equex stm, dehydrated sodium citrate, disodium edta, sodium bicarbonate, ticarcillin disodium e-z freezin™ ‘modified french’ equine semen extender (animal reproduction systems) glucose, glycerol, egg yolk, nonfat dry milk, lactose, sodium citrate dihydrate, potassium citrate, monohydrate, hepes free acid, ticarcillin disodium table. equine cryopreservation semen extenders and their ingredients figure 1. vas deferens (below bar; bar = 2 cm) and cauda epididymides (arrow) figure 2. mean ± sd postthaw viability (left y-axis), total motility (right y-axis) and progressive motility (right y-axis) for 3 alpaca epididymal semen extenders: gent extender (ge) for frozen stallion semen, e-z freezin™ ‘lactose edta’ (le) equine semen extender, and e-z freezin™ ‘modified french’ (mf5) equine semen extender for alpaca epididymal sperm. alpaca epididymal semen extender clinical theriogenology 2021; 13: 368 discussion alpaca cryopreserved epididymal sperm can be stored as pellets or in straws. sperm pellets are frozen on the surface of dry ice (78.5°c), whereas sperm packed in straws are frozen in liquid nitrogen vapor (-140°c to -180°c). research regarding postthaw motility between the 2 freezing methods had conflicting results. pelleted frozen alpaca epididymal sperm had higher postthaw motility compared to straws frozen in liquid nitrogen vapor.3 postthaw motility when pooled across time was significantly higher when alpaca sperm was frozen in straws compared to frozen in pellets.10 it is also important to consider the artificial insemination technique when selecting a method for cryopreservation.3,11 sperm cryopreserved in straws allows for insemination directly from the straw that can be performed in seconds after thawing.11 for these reasons, the current research was focused on alpaca epididymal sperm frozen in straws. selection of an appropriate semen extender is essential to the success of sperm cryopreservation. alpaca epididymal sperm has been evaluated for sperm motility following cryopreservation.2,3,9,12,13 ejaculated alpaca sperm in skim milk-based extenders yield higher postthaw total motility compared to tris-based extenders.4,13 lactose-based extenders yielded higher postthaw total motility (18.2 ± 5.7%) compared to tris-based extenders (11.3 ± 3.0%).3 based on this research, we sought to compare postthaw motility in 3 lactose-based extenders, with 2 having skim milk. interestingly, postthaw motility for all 3 extenders were lower (< 5%). however, extender that had no skim milk (le) yielded a numerically higher postthaw total and progressive motility compared to skim milk-based extenders. the main limitation present in this investigation was lower postthaw total and progressive motility. the 4 alpacas used for this preliminary study had previously sired cria and were assumed to have normal sperm motility, but this was not tested prior to castration and epididymal sperm cryopreservation. future research in alpacas’ epididymal sperm should only be conducted after a complete breeding soundness examination. conclusion epididymal alpaca sperm can be cryopreserved using commercial equine semen extenders. however, postthaw motility in this preliminary study was less than what has been reported for alpaca epididymal sperm cryopreservation. conflict of interest none to report. acknowldgements study was supported by the morris animal foundation (do7la007). authors thank tessa fiamengo and justine gullaba for their assistance. references 1. ruiz j, santayana rp, mendoza mj, et al: effect of oocyte maturation time, sperm selection method and oxygen tension on in vitro embryo development in alpacas. theriogenology 2017;95:127-132. 2. santiani a, ugarelli a, evangelista-vargas s: characterization of functional variables in epididymal alpaca (vicugna pacos) sperm using imaging flow cytometry. anim reprod sci 2016;173:49-55. 3. morton km, bathgate r, evans g, et al: cryopreservation of epididymal alpaca (vicugna pacos) sperm: a comparison of citrate-, trisand lactose-based diluents and pellets and straws. reprod fertil dev 2007;19:792-796. 4. santiani a, huanca w, sapana r, et al: effects on the quality of frozen-thawed alpaca (lama pacos) semen using two different cryoprotectants and extenders. asian j androl 2005;7:303-309. 5. acosta as, vargas se, cuya mv, et al: effect of the addition of two superoxide dismutase analogues (tempo and tempol) to alpaca semen extender for cryopreservation. theriogenology 2013;79:842-846. 6. morton km, evans g, maxwell wm: effect of glycerol concentration, equex stm supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen-thawed epididymal alpaca (vicugna pacos) sperm. theriogenology 2010;74:311-316. 7. mamani-mango g, gonzales mm, hidalgo mr, et al: effect of extender and freezing rate on quality parameters and in vitro fertilization capacity of alpaca spermatozoa recovered from cauda epididymis. biopreserv biobank 2019;17:39-45. 8. neuhauser s, bollwein h, siuda m, et al: comparison of the effects of five semen extenders on the quality of frozen-thawed equine epididymal sperm. j equine vet sci 2019;79:1-8. 9. al-essawe em, abraham c, kunkitti p, et al: extenders for alpaca epididymal spermatozoa: comparsion of inra96 and andromed. anim reprod sci 2020;223:106629. 10. stuart cc, vaughan jl, kershaw cm, et al: effect of diluent type, cryoprotectant concentration, storage method and freeze/thaw rates on the post-thaw quality and fertility of cryopreserved alpaca spermatozoa. sci rep 2019:9:12826. 11. holt wv: basic aspects of frozen storage of semen. anim reprod sci 2000;62:3-22. 12. kershaw-young cm, maxwell w: the effect of seminal plasma on alpaca sperm function. theriogenology 2011;76:1197-1206. 13. banda rj, evangelista vs, ruiz gl, et al: effect of extenders based on tris, tes and skim milk on cryopreservation of epididymal alpaca sperm. revista de investigaciones veterinarias del perú 2010;21:145-153. clinical theriogenology 2021; 13: 369 introduction most encounters between bacteria and the equine endometrium result in an acute period of subclinical infection and occasionally clinical symptoms. after an acute infection, in a majority of mares, invading bacteria are eliminated and infection is resolved. however, in a minority of cases, small numbers of bacteria survive and cause persistent infections that can be difficult to eliminate. development of acute and chronic cases of endometritis is the result of deficiencies in the mare’s ability to eliminate an infection and causative bacterium’s unique pathogenic properties. mare’s uterine defense mechanisms to bacterial infection are well understood and consist of physical, immunological, and mechanical barriers.1 bacteria utilize several methods to survive degradation by the host immune system and antibiotic therapy. one survival tool utilized by bacteria is the production of a biofilm. biofilms allow bacteria to remain undetected by the host immune system, prevent exposure to antibiotics, and allow for exchange of genetic material leading to antibiotic resistance.2 pathophysiology presence of bacteria within the uterine lumen results in a rapid influx of neutrophils, immunoglobulins, and serum proteins. neutrophils from susceptible mares have reduced in vitro ability to phagocytize bacteria compared to resistant mares. inflammation associated with the innate immune system results in fluid production into uterine lumen.3-5 final defense mechanism against bacterial endometritis is mechanical uterine clearance of bacteria and inflammatory products. mares susceptible to uterine infections have decreased clearance of uterine fluid compared to resistant mares. after intrauterine inoculation with bacteria, susceptible and resistant mares had similar uterine myometrial contractions for biofilm and latent bacteria in equine uterus ryan ferris summit equine, inc., gervais, or abstract subfertility in the mare can be frustrating to the clinician. recent data suggested that bacteria in either a biofilm or latent state may be responsible for certain cases of subfertility. recent work has identified the pathophysiology for these types of chronic infections in the mare and best methods for therapy. the goal is to elucidate the pathophysiology of biofilm and latent infections and the diagnosis, and treatment of these types of infections in clinical practice. keywords: mare, endometritis, biofilm, latent bacteria 6 8 hours postinoculation; however, the contractile activity decreased in susceptible mares after 8 hours.6-8 failure to clear bacteria and inflammatory products from the uterus, results in continued activation of the innate immune system, resulting in a further increase in inflammatory cells, immunoglobulins, and serum proteins reaching the uterus that continue to activate the innate immune system. bacterial lifestyle-planktonic versus biofilm bacteria are capable of living in 2 lifestyles (planktonic or biofilm state). planktonic bacteria are single bacterial cells free flowing in suspension. bacteria in this lifestyle are utilizing available nutrients for procreation. these individual cells are relatively susceptible to recognition and degradation by the host immune system, susceptible to changes in environment (e.g., desiccation, lack of nutrients, and others), and sensitive to antibiotics. however, the planktonic cell paradigm does not accurately reflect the growth of bacteria in nature associated with a biofilm.2 in the last several decades the biofilm state has been considered as more prevalent lifestyle with ~ 99% of the overall world bacterial biomass living in a biofilm. in natural environments these biofilms are invariably a multispecies of microbial community harboring bacteria that stay and leave with purpose, share their genetic material at high rates, and fill distinct niches within the biofilm. first step in biofilm formation is migration and adherence to a surface. individual bacteria will migrate (if capable) until other bacteria (same species or other) are encountered and micro-colonies start to form. at this point, planktonic and biofilm lifestyles start to diverge, genes associated with flagella are down regulated and genes associated with polysaccharide production increase. this exopolysaccaride (eps) matrix forms the scaffold for the biofilm community. clinically, biofilms can cause substantial difficulty for clinicians to eliminate once these chronic infections are established. bacteria within a biofilm are protected from the host immune system as white blood cells have reduced ability for movement and function, and the thick layer of eps prevents antibodies from reaching bacteria deep within the biofilm. biofilms protect bacteria from antibiotics by providing a diffusion barrier that decreases the amount of antibiotics that reach the protected bacterial colonies and creates a microenvironment that slows down the metabolism and therefore the replication rate of bacteria that also makes them more resistant to antimicrobial agents. ultimately, biofilms are associated with development and maintenance of subpopulations of ‘persister cells.’9-14 as antimicrobial agents come in contact with biofilm, agents must traverse through a thick layer of eps, dna, rna, lipids and proteins in order to reach bacteria buried deep within this protective barrier. bacteria in the outer region may be killed, but a decrease in the concentrations of antibiotics reaching the inner layer bacteria contributes to the formation of a nidus for chronic infection. thick layer of eps in biofilms not only prevents antibiotics from penetrating, but limits diffusion of oxygen and nutrients. oxygen and nutrient deprivation consequently result in a decrease in metabolic rate compared to planktonic or free individual bacteria. this reduction in metabolic rate provides additional antimicrobial resistance as antibiotics typically only act upon rapidly multiplying bacteria.10,14-16 it has been proposed that biofilms have an important role in chronic uterine infections resistant to antimicrobials due to biofilm production. additionally, acute and chronic nonhealing wounds on the distal equine limb contained a significantly greater incidence of biofilm producing bacteria compared to a skin sample near the wound.17 biofilms in the horse evaluation of bacteria isolated from the equine uterus suggests that the majority of isolates of streptococcus equi subsp. zooepidemicus, escherichia coli (e.coli), pseudomonas aeruginosa (p. aeruginosa), and klebsiella pneumonia are capable of producing a biofilm in vitro.18 in mares, using a model of infectious endometritis, a biofilm involved infection has been clearly identified. the adherent biofilm material is multi-focal with the greatest adherence occurring between the tissue folds and in uterine horns. bacteria are at greater numbers deep in the endometrial glands compared to the luminal surface. treatment options may need to penetrate deeper into the glands and tissue to effectively clear these infections. there is an alteration in the host immune response with reduced pmns surrounding areas of adherent biofilm compared to areas free of bacteria. unfortunately, no clinical diagnostic tests are available for detection of a biofilm-related infection.19 in human medicine, a biofilm is suspected if appropriate antibiotic therapy is given and the infection is unable to be eliminated. treatment options for biofilms bacteria residing in a biofilm can be up to 1,000 times more resistant to treatment with antibiotics compared to free-living (i.e., planktonic) bacteria. simple treatment of antibiotics has been unable to eliminate chronic infections suspected of involving a biofilm in both human and veterinary medicine. the goal in treating a biofilm associated infection is to remove the biofilm material and kill the bacteria residing within the biofilm. a series of in vitro and in vivo studies were conducted to assess biofilm dispersal and/or bacterial killing for antibiotics and nonantibiotic agents alone or in combination against gram-negative bacteria.19 data indicate that antibiotics and nonantibiotic agents are more effective against biofilm if treated concurrently (i.e., in the same syringe). uterine infusions (based on the in vitro data) are provided (table). amount of either antibiotic or nonantibiotic agent for each infusion is the minimum effective concentrations against e. coli, k. pneumoniae, and p. aeruginosa. duration of treatment must be at least 72 hours, repeated every 24 hours (i.e., a uterine infusion of the selected combination once every 24 hours for 3 consecutive days). this treatment protocol resulted in complete biofilm dispersal and bacterial killing in vitro. it is important to note that some nonantibiotic agents and antibiotics should not be combined in the same syringe. for example, in vitro data indicated that mixing acetylcysteine with antibiotics in the same syringe reduced antibiotics activity. we recommend antibiotic sensitivity testing for all gram-negative organisms. bacteria inherently resistant to an antibiotic will still be resistant when that antibiotic is used in combination with a nonantibiotic agent. latent bacteria or persister cells and infections persister cells, representing ~ 1% of all bacteria in a free-floating state, are characterized by tolerance to antibiotics with no change in genetic expression. it is often believed that these bacteria are potentially dormant and metabolically inactive. this phenomenon was originally described in the 1940’s in that cultures of staphylococcus aureus exposed to lethal doses of penicillin resulted in < 1% of the original cfus surviving penicillin exposure.20 although this work was conducted before genetic sequencing was available, authors did not feel the acquired antibiotic resistance was due to a mutation in the bacteria as subsequent culturing and exposure to antibiotics resulted in continued susceptibility of these previous tolerant colonies. latent bacteria in the horse it has been clearly identified that some mares can have a population of dormant streptococcus equi subsp. zooepidemicus deep in the uterine glands.21 this population of bacteria would not be identified on routine culture (not actively dividing bacteria) or cause substantial inflammation or infection. however, if these bacteria were to leave this dormant stage after the establishment of pregnancy, the resulting bacterial growth will induce inflammation and infection leading to pregnancy loss. table. antibiotic and nonantibiotic combinations for the treatment of biofilm associated bacterial endometritis in mares tris edta: final concentration in the syringe should be 50 mm tris and 3.5 mm edta note: tris-edta and tricide are similar; however, tricide is not equivalent to tris-edta in regards to bacterial killing to make tris-edta: 16oz bottle of dechra triz-edta crystals; add 8 oz of sterile water (this is different than the bottle instructions). two x concentration of tris-edta solution will be further diluted by the antibiotics below to the proper final concentration. antibiotic drug amount tris edta expansion volume final volume notes: amikacin (250 mg/ml) 4 mls (1 gram) 30 mls 16 mls sterile fluid (saline, lrs, sterile h2o) 60 mls 10 mls of 8.4% sodium bicarbonate should be added to the amikacin ceftiofur (1 gram reconstituted in 20 mls) 20 mls (1 gram) 30 mls 10 mls sterile fluid (sterile h2o) 60 mls ciprofloxacin (10 mg/ml) 40 mls (400 mg) 40 mls 0 80 mls split between 2 syringes h2o21% final concentration in the syringe a 3% stock solution is available at many drug stores and veterinary distributors antibiotic drug amount h2o2 expansion volume final volume notes: amikacin (250 mg/ml) 4 mls (1 gram) 20 mls 26 mls sterile fluid (saline, lrs, sterile h2o) 60 mls 10 mls of 8.4% sodium bicarbonate should be added to the amikacin ciprofloxacin (10 mg/ml) 40 mls (400 mg) 20 mls 0 60 mls dmso30% final concentration in the syringe 99% stock solution is used for calculations below antibiotic drug amount dmso expansion volume final volume notes: ceftiofur (1 gram reconstituted in 20 mls) 20 mls (1 gram) 20 mls 20 mls sterile fluid (sterile h2o) 60 mls ciprofloxacin (10 mg/ml) 40 mls (400 mg) 20 mls 0 60 mls treatment for latent bacteria goal for treating mares with latent or dormant bacteria is to force the bacteria to move from the dormant state into a metabolically active state in which identification and treatment can be performed. dormant streptococcus zooepidemicus can be activated by infusing a proprietary medium (bactivate) into uterus.22 at 24 hours after infusion, 64% (15/25) mares were positive for streptococcus zooepidemicus compared to 8% (1/12) mares infused with pbs. proprietary medium forced bacteria to move from the dormant state to a metabolically active state to initiate treatment.22 interestingly, breeding may also activate dormant state bacteria, as 55% (16 of 29) of mares with a negative culture prior to breeding that retained fluid postbreeding were positive for streptococcus zooepidemicus.23 authors concluded that it was more likely to be dormant streptococcus zooepidemicus that was reactivated compared to introduction at breeding.23 development of post-mating fluid in barren mares could be due to inflammation from breeding and reactivation of dormant bacteria. conclusion overall, the incidence of biofilm or latent bacteria is unknown in the broodmare population. with relatively higher per cycle overall pregnancy rates in broodmares, one can suspect that the incidence rate of biofilm or latent bacteria is low. biofilm and latent bacteria must be considered as a cause of subfertility in individual mares failing to become pregnant. research has helped to understand the development of infections, improve diagnostic techniques, and provide effective treatment strategies for biofilm or latent bacterial endometritis. conflict of interest none to declare. references 1. causey rc, leblanc mm: clinical and subclinical endometritis in the mare: both threats to fertility. reprod dom anim 2009;44 suppl. 3:10-22. 2. donlan rm costerton jw: biofilms: survival mechanisms of clinically relevant microorganisms. clin microbiol rev 2002;15:167193. 3. troedsson mht, liu ikm, thurmond m: function of uterine and blood-derived polymorphonuclear neutrophils in mares susceptible and resistant to chronic uterine infection: phagocytosis and chemotaxis. biol reprod 1993;49:507-514. 4. ohman t, klein c, doty a, et al: the phagocytic function of bloodderived polymorphonuclear neutrophils after administration of dexamethasone for the modulation of post-breeding endometritis in the mare. pferdeheilkunde 2011;27:267-270. 5. troedsson mh, liu ik, thurmond m: immunoglobulin (igg and iga) and complement (c3) concentrations in uterine secretion following an intrauterine challenge of streptococcus zooepidemicus in mares susceptible to versus resistant to chronic uterine infection. biol reprod 1993;49:502-506. 6. leblanc mm, asbury ac, lyle, sk: uterine clearance mechanisms during the early postovulatory period in mares. am j vet res 1989;50:864-867. 7. troedsson mh, liu ik, ing m, et al: multiple site electromyography recordings of uterine activity following an intrauterine bacterial challenge in mares susceptible and resistant to chronic uterine infection. j reprod fert 1993;99:307-313. 8. troedsson mht, wistrom aog, liu ikm, et al: registration of myometrial activity using multiple-site electromyography in cyclic mares. j reprod fert 1993;99:299-306. 9. jefferson kk, goldmann da, pier gb: use of confocal microscopy to analyze the rate of vancomycin penetration through staphylococcus aureus biofilms. antimicrob agents chemother 2005;49:2467-2473. 10. brown mr, allison dg, gilbert p: resistance of bacterial biofilms to antibiotics: a growth rate related effect? j antimicrob chemother 1998;22:777-780. 11. anwar h, strap jl, costerton jw: establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy. antimicrob agents chemother 1992;36:347-351. 12. stewart ps, costerton jw: antibiotic resistance of bacteria in biofilms. lancet 2001;358:135-138. 13. shah d, zhang z, khodursky a, et al: persisters: a distinct physiological state of e. coli.bmc microbiology 2006;6:53. 14. chiang wc, pamp sj, nilsson m, et al: the metabolically active subpopulation inpseudomonas aeruginosa biofilms survives exposure to membrane-targeting antimicrobials via distinct molecular mechanisms. fems immunol med microbiol 2012;65:245-256. 15. williamson ks, richards la, perez-osorio ac, et al: heterogeneity in pseudomonas aeruginosa biofilms includes expression of ribosome hibernation factors in the antibiotic-tolerant subpopulation and hypoxia-induced stress response in the metabolically active population. j bacteriol 2012;194:2062-2073. 16. walters mc, roe f, bugnicourt a, et al: contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. antimicrob agents chemother 2003;47:317-323. 17. pezzanite l, hendrickson da: controlling wound bacteria and biofilm. proc am assoc equine pract 2021; p. 58-63. 18. loncar kd, ferris ra, mccue pm, et al: in vitro biofilm disruption and bacterial killing using non-antibiotics compounds against gramnegative equine uterine pathogens. j equine vet sci 2016;53:94-99. 19. ferris ra, mccue pm, borlee gi, et al: in vitro efficacy of nonantibiotic treatments onbiofilm disruption of gram-negative pathogens and an in vivo model of infectious endometritis utilizing isolates from the equine uterus. j clin micro 2016;54:631-639. 20. hobby gl, meyer k, chaffee e: observations on the mechanism of action of penicillin. proc soc exp biol med 1942;50:281-285. 21. petersen mr, nielsen jm, lehn-jensen h, et al: streptococcus equi subspecies zooepidemicus resides deep in the chronically infected endometrium of mares. clinical theriogenology 2009;1:161-165. 22. petersen mr, skive b, christoffersen m, et al: activation of persistent streptococcus equi subspecies zooepidemicus in mares with subclinical endometritis. vet microbiol 2015;179:119-125. 23. christoffersen m, söderlind m, rudefalk sr, et al: risk factors associated with uterine fluid after breeding caused by streptococcus zooepidemicus. theriogenology 2015;84:1283-1290. biofilm and latent doxycycline diffused to fetoplacental unit and with no apparent complications to resulting foal doxycycline diffused to fetoplacental unit and with no apparent complications to resulting foal fernand dantas,a igor canisso,b zhong li,c lorena feijó,a carlos nogueira,a augusto postal,a josiane feijó,a bruna r curcioa adepartamento de clínicas veterinárias, faculdade de medicina veterinária universidade federal de pelotas, pelotas, rs, brazil bdepartment of veterinary clinical medicine, college of veterinary medicine croy j carvey biotechnology center, university of illinois urbana-champaign, urbana, il antimicrobial therapy is used in broodmare practice to treat various bacterial infections (e.g. pneumonia, septic arthritis, enterocolitis, and placentitis). aminoglycosides, ß lactams, and potentiated sulfonamides are all considered safe for use in pregnant animals and metronidazole is assumed to be safe, based on anecdotal use in clinical practice. however, there is limited knowledge about putative toxicity and ability of tetracyclines to diffuse to the fetoplacental unit. due to evolving antimicrobial resistance, expansion of veterinary armamentarium is warranted. doxycycline is a broad spectrum antimicrobial frequently used to treat intracellular microorganisms due to its high cellular penetration. this study aimed to assess doxycycline diffusion to fetoplacental unit during late pregnancy and potential toxicity to resulting foal. twelve healthy light horse breed mares were enrolled in this study at 320 days of pregnancy. six mares received compounded doxycycline (10 mg/ kg, po, q12h, rood and riddle pharmacy, lexington, ky) until delivery, whereas 6 mares were untreated controls. all mares had daily physical examinations and ph assessments of mammary gland secretions for detection of impending foaling. blood samples were collected before commencement of study and then every 7 days until foaling for determination of complete blood cell counts, blood chemistry and plasma concentrations of doxycycline. foaling was induced with oxytocin (10 units, im) when mammary secretion ph was d 6.4. during second stage of labor, allantoic fluid was collected by free catch and amniotic fluid by puncture. blood samples from mares and foals were collected immediately after parturition and 7 days postpartum. foals were examined twice a day during their first 7 days. concentrations of doxycycline in plasma (mare and foal) and fetal fluids were assessed with lc ms/ms. after foaling, mares and foals were subjected to standard postpartum care for 60 days. blood biochemistry and blood cell counts were assessed for normal distributions with shapiro-wilk’s test and then compared with anova (statistix 10.0). significance was set at p < 0.05. on average, mares received 15 ± 2 days of doxycycline. doxycycline concentrations in allantoic fluid were significantly higher than in amniotic fluid (73.55 ± 13.93 versus 8.32 ± 3.31 ng/ml, respectively). doxycycline was also detected in plasma of foals immediately after birth (35.52 ± 4.28 ng/ml). none of treated mares or their respective foals developed any clinical signs suggestive of doxycycline toxicity (e.g. diarrhea and jaundice). there were no differences in results of complete blood cell counts or blood chemistry between treated versus untreated control foals. in conclusion, administration of doxycycline to late term pregnant mares did not result in apparent clinical abnormalities in mares or respective foals. this was apparently the first study to demonstrate that doxycycline crosses the equine placenta and achieves high concentrations in the fetoplacental unit. although longitudinal studies assessing the potential side effects of doxycycline to foals born from treated mares are necessary, based on our findings, doxycycline can be used to treat various infections in pregnant mares. keywords: antimicrobial, pregnancy, equine 417 clinical theriogenology • volume 11, number 3 • september 2019 418clinical theriogenology • volume 11, number 3 • september 2019 omniblank: embryo-maternal communication during the establishment of equine pregnancy embryo-maternal communication during the establishment of equine pregnancy tom stout department of clinical sciences, faculty of veterinary medicine utrecht university, utrecht, the netherlands abstract over the last 30 years, improvements in veterinary management have resulted in higher per cycle pregnancy rates in commercial horse breeding programmes. however, the incidence of early pregnancy loss has not diminished and ~ 10% of pregnancies detected on day 15 fail to survive beyond day 42 of pregnancy. to develop strategies to combat pregnancy loss, it is first necessary to understand why these pregnancies fail. in this respect, a sizeable proportion probably results from intrinsic embryonic abnormalities that cannot be remedied; another significant subset is, however, likely to result from an ‘inadequately receptive’ uterus or failure of the uterus to physiologically adapt to the developing conceptus’ changing needs, as a result of inadequate embryo-maternal communication. communication between embryo and dam starts during the oviductal period, as evidenced by selective transport of viable embryos into the uterus on day 6 postovulation, whereas unfertilized oocytes remain in the oviduct. conversely, retarded development exhibited by embryos produced in vitro demonstrates the importance of the oviductal environment to the early embryo. once in the uterus, the embryo must steer maternal physiology to ensure adequate nutrient provision and prepare the endometrium for implantation. this communication includes ‘maternal recognition of pregnancy’ signalling to extend the lifespan of the primary corpus luteum and ensure continued secretion of progesterone. although the identity of the embryonic pregnancy recognition signal remains unknown, our understanding of how endometrial function is altered to avert luteolytic prostaglandin f2α release and become receptive to implantation has improved. keywords: equine embryo, endometrium, maternal recognition of pregnancy, implantation introduction over the last 30 years, a range of technological and pharmacological breakthroughs, allied to a better understanding of factors that compromise fertility, has led to a marked rise in the per cycle pregnancy rate in intensively managed horses.1 during the same period, assisted reproductive technologies (e.g. embryo and oocyte transfer) and more recently ovum pick up and intracytoplasmic sperm injection (icsi) for in vitro embryo production,2 have been adopted commercially either to circumvent specific forms of subfertility or to aid genetic selection. despite successful introduction of these techniques to overcome subfertility, and general improvement in reproductive efficiency, there has been little progress in combating one of the major sources of economic loss within horse breeding, namely early pregnancy loss.3 after initial detection of pregnancy at around day 15 postovulation, ~ 5 15% of all pregnancies will fail to reach term in young healthy mares, often without any obvious explanation for why the pregnancy was lost. pregnancy losses are even more common in older (> 18 years) mares,4 or following the transfer of in vitro produced embryos,5 where > 25% of day 15 pregnancies are reported to fail. interestingly, more than half of all pregnancy losses occur in the first 6 weeks of pregnancy.6 this strong bias to early loss is presumably in large part because conceptuses with serious intrinsic defects, such as gross abnormalities of chromosome number (aneuploidy), are most likely to fail during early development.7 moreover, the equine conceptus undergoes an incredible transformation during the first 7 weeks of life, from a microscopic ball of undifferentiated cells that imbibes nutrients from the surrounding oviductal fluids, to a fetus with recognisable body parts and precursors of all of the major organs required for extra uterine life, that is provided with nutrients and respiratory gasses by a dedicated placenta. to enable this remarkable growth and development, uterine physiology needs to evolve in parallel with conceptus development. one of the defining events in the conceptus directed adaptation of maternal physiology is the so called ‘maternal recognition of pregnancy’ (mrp), a process by which the day 10 14 equine conceptus suppresses cyclical luteolysis, and thereby extends the lifespan of the primary corpus luteum. this safeguards the supply of maternal progesterone required to stimulate endometrial production of histotroph on which the conceptus depends for nutrition before the development of a stable placental attachment from clinical theriogenology • volume 12 number 4 • december 2020536 around day 40.8 conceptus derived signals also combine with luteal progesterone to modify endometrial physiology in preparation for implantation. clearly, therefore, the way in which the early embryo and its dam communicate to ensure that uterine physiology is coordinated with embryonic development is critical to the successful maintenance of pregnancy. this paper discusses some of the important elements of embryo-maternal communication during the first 6 weeks of equine pregnancy. embryo development in the oviduct and descent into the uterus the equine embryo remains in the oviduct for an unusually long interval and finally descends into the uterus on day 6 postovulation.9 during its sojourn in the oviduct, the embryo’s genome is activated and it progresses to the late morula stage of development. although little is known about embryo and oviduct interaction, the microenvironment of the oviductal ampulla clearly plays an active role in supporting early development; indeed, although in vitro produced (ivp) equine embryos reach the blastocyst stage at a similar time to in vivo embryos (7 8 days postfertilization), at this stage they contain many fewer cells,10 a higher percentage of which are apoptotic11,12 and, following transfer to a recipient mare, ivp embryos lag 2 3 days behind in development and are more prone to early embryonic death.5 a positive influence of the oviduct on equine embryo development is also illustrated by the more rapid development and increased likelihood of reaching the blastocyst stage of 2 4 cell embryos transferred into the oviduct of a recipient mare soon after icsi, compared to embryos maintained in vitro.12 although it also appears that the developing embryo induces changes in oviductal epithelial cell gene and protein expression,13,14 the presence of an embryo in the oviduct does not appear to affect the mare’s subsequent ability to support pregnancy, as demonstrated by the high initial and ongoing pregnancy rates in well synchronised embryo transfer recipient mares.15 conversely, the early equine embryo has been reported to influence maternal physiology beyond the confines of the oviduct, in the form of the ‘early pregnancy factor’ (epf) reported to be detectable in the serum of pregnant mares from day 2 postovulation.16 although it is unclear whether epf plays any further role in pregnancy recognition or maintenance, a reliable assay for epf would be invaluable for investigating the scale and timing of embryonic loss and potentially for improving the efficiency of et programmes, (e.g. by ensuring that only mares ‘guaranteed’ to deliver an embryo are flushed). unfortunately, field trials of an ‘early conception factor’ assay were disappointing, with a high rate of false positives limiting the ability of the test to discriminate between pregnant and nonpregnant mares.17 the equine embryo’s oviductal phase is brought to an end by an enigmatic form of embryonic signalling, referred to as selective oviductal transport. only developing embryos are able to reliably descend into the uterus, whereas unfertilized oocytes remain trapped in the oviductal ampulla.18 this selective transport is mediated by prostaglandins, in particular prostaglandin e2 (pge2) that viable embryos begin to secrete from around day 4 postfertilization.19 the early postfertilization embryo is retained in the ampulla of the oviduct by a tightly closed ampullary-isthmic junction; the pge2 secreted by the developing morula relaxes the circular smooth muscle of the isthmus, causing it to dilate and allow the embryo to pass through into the uterus.20 failure of viable embryos to initiate oviductal transport does not seem to occur, since tubal pregnancy has not been reported as a complication in mares. glycoprotein capsule another unusual feature of equine pregnancy that both depends on and, almost certainly plays a role in, early embryo-maternal communication, is the formation and subsequent disappearance of the acellular glycoprotein ‘capsule’. the capsule forms between the trophectoderm and zona pellucida very soon after the embryo enters the uterus, roughly coincident with blastocyst formation.21 the mucin-like glycoproteins that initially make up the capsule are secreted by the trophectoderm;22,23 however, they are unable to cross-link to form a confluent structure in vitro.10 it therefore appears that capsule coalescence requires input from the endometrium, a supposition strengthened by the report of increased capsular glycoprotein production by ivp embryos exposed to uterocalin,24 a progesterone dependent endometrial protein that associates strongly with the capsule of early uterine stage conceptuses.25 nevertheless, it is not entirely clear whether the primary uterine contribution to capsule formation is in the form of structural elements, or by providing a microenvironment that facilitates cross-linking and assembly of the trophectoderm-derived glycoproteins into a confluent structure. clinical theriogenology • volume 12 number 4 • december 2020 537 soon after capsule formation is completed, the day 7 embryo ‘hatches’ from its zona pellucida; however, its newly formed ‘tertiary embryo coat’ remains as a physical barrier between the trophectoderm and the endometrium. the capsule subsequently increases in thickness until ~ day 17, developing a bilaminar appearance that may reflect an additional (presumably endometrial) source of structural components.25,26 soon after embryo fixation (cessation of migration), however, dissolution of the capsule begins, with a rapid decline in dry weight and loss of continuity somewhere between days 208 and 23;27 by day 30 the capsule has disintegrated completely.8 although it is not known how the capsule is dissolved, the process is likely to involve both trophectodermal and endometrial enzymes. moreover, there appears to be a central role for progesterone in stimulating enzymatic dissolution, since induction of luteolysis shortly before conceptus fixation (day 16) prevents capsule dissolution and the desialylation and degradation of capsule-associated proteins associated with normal degradation.27,28 although the functions of the capsule are largely speculative, that it is interposed between the trophectoderm and endometrium means it almost certainly plays some role in mediating embryomaternal interaction and communication. moreover, the capsule is essential for conceptus survival in utero, since removing it from early day 7 embryos by micromanipulation prior to embryo transfer prevented the embryos from developing into ultrasonographically visible pregnancies.29 conversely, although ivp embryos have no confluent capsule when they reach the blastocyst stage, they have accumulated capsular glycoproteins within the perivitelline space. subsequently, they develop an apparently normal capsule following transfer to the uterus of a suitable recipient mare. physically, the capsule is both tough and elastic and it has been proposed to help the blastocyst maintain its spherical shape, and to provide mechanical protection during the ‘mobile phase’ when the relatively delicate conceptus vesicle is ‘squeezed’ around the uterine lumen by myometrial contractions.30 in addition, the capsular glycoproteins are rich in negatively charged sialic acid residues that have been postulated to confer antiadhesive properties and thereby facilitate conceptus migration;27 indeed, the end of the conceptus mobile phase is associated with extensive desialylation of the capsule.31 in this way, the capsule plays at the very least an indirect role in embryonic maternal recognition of pregnancy signalling (i.e. by promoting migration). more intriguingly, the capsule has been proposed to play an active role in embryo-maternal dialogue by acting as a ‘mail-box’32 to store, modify or transfer endometrial proteins involved in nutrient transport (e.g. uterocalin) or in stimulating trophectoderm cell proliferation. similarly, the capsule may act as a repository for trophectodermal proteins that modulate endometrial function, or act in a paracrine fashion to regulate trophectoderm cell migration or proliferation (e.g. igfbp333). maternal recognition of pregnancy signalling the archetypal embryo-maternal signalling process during early intrauterine development is mrp, during which the developing embryo biochemically signal(s) its presence to its dam to ensure that she undergoes the physiological adjustments necessary to maintain a uterine environment conducive to embryonic survival.34 since the pivotal determinant of this ‘supportive environment’ is a continuing supply of luteal progesterone, mrp is usually taken to refer specifically to the conceptus initiated events that prolong the lifespan of the primary corpus luteum. we now know that mrp in the mare involves suppression of the endometrium’s ability to release large pulses of the luteolytic hormone, prostaglandin f2α (pgf2α), in response to hypothalamic-pituitary35 (and/or endometrial)36 oxytocin, during days 10 16 postovulation.37 to ensure sufficient delivery of the presumed conceptus antiluteolytic signal to the endometrium to adequately suppress pgf2α release and thereby avert luteolysis, the spherical equine conceptus must migrate throughout the entire uterine lumen during the day 10 16 period when luteolysis would otherwise occur. indeed, when equine conceptuses were surgically restricted to a single uterine horn, luteolysis was not blocked. 38 somewhat paradoxically, conceptus vesicle migration appears itself to be driven by myometrial contractions induced by prostaglandins produced either directly by the conceptus or locally in the uterine wall in response to other conceptus derived mediators.30 one of the reasons why it has not been possible to definitively identify the conceptus factor responsible for initiating mrp in the mare may be that the process, as in other species, involves multiple signals and more than one phase. even though the identity of the mrp signal(s) remains a mystery, it has been established that the initial suppression of endometrial pgf2α secretion involves a clinical theriogenology • volume 12 number 4 • december 2020538 conceptus directed down regulation of endometrial prostaglandin endoperoxide synthase 2 (ptgs2) activity39,40 accompanied by a reduction in endometrial oxytocin receptor (oxtr) expression.40 however, following the cessation of conceptus mobility at around day 16, the endometrium does develop the ability to secrete pgf2α in response to oxytocin,41,42 a change that is reflected by contemporaneous upregulations in ptgs2 and oxtr expression.40 this implies that prevention of luteolysis beyond the time of conceptus fixation depends either on the disappearance of the oxytocin ‘trigger’ (e.g. hypothalamic-pituitary ot release only occurs in a defined time window) and/or on disabling of another link in the luteolytic cascade. in this latter respect, expression of the endometrial pgf2α receptor (ptgfr), that is thought to play an important role in establishing the local feedback (pgf2α oxt) and feedforward (pgf2α pgf2α) loops essential to generating the large pulses of pgf2α release required to ensure luteal destruction, is down regulated until at least day 21 of pregnancy;40 this may explain why inadvertent luteolysis is not very common in the early postfixation period, despite the presence of sufficient oxtrs and ptgs2. given that the oxytocin responsiveness central to cyclical luteolysis develops at around day 10 postovulation,37,43 it is logical to assume that conceptus signalling required for mrp must also begin at or before day 10. frustratingly, the conceptus signal(s) responsible for down regulating ptgs2 and otxr expression, and thereby effecting the first phase of mrp, remains elusive.44 it has been reported that the equine conceptus product that inhibits endometrial pgf2α secretion must be small, with a molecular weight estimated at ~ 1 6 kda45 or 3 10 kda;46 however, these studies did not identify specific candidates. similarly, while changes in the transcriptome of conceptuses during the mrp period revealed various conceptus derived molecules likely to play roles in embryo-maternal interaction,47 it did not unearth a putative antiluteolytic signal. nevertheless, the transcriptomic data adds to the list of hormones, cytokines and growth factors, such as pge2,42 igf-1,48 and estrogens,49 secreted by mrp stage equine conceptuses and likely to play important roles in driving other events critical to pregnancy maintenance, such as conceptus migration,30 increased uterine vascularity,50 and changes in uterine gland secretions that modify the composition of histotroph.51 indeed, transcriptomic studies have indicated a significant number of genes differentially regulated in the endometrium of pregnant and nonpregnant mares between days 8 -14 postovulation. these genes are regulated by estrogen or pge2.52,53 recently, we examined the effect of asynchronous embryo transfer on the transcriptome of both conceptus and endometrium; one of the most striking findings was that hundreds of genes relating to ‘extracellular exosome’ were differentially regulated in either the conceptus membranes or the endometrium as a result of asynchrony between the two, suggesting that extracellular vesicles play an important role in trafficking molecules important to conceptus endometrial communication.54 preparing endometrium for implantation and placentation implantation involves the establishment of a stable attachment to facilitate more efficient, hemotrophic exchange between the conceptus and endometrium. a normally developing embryo and an appropriately ‘primed’ uterus are prerequisites for successful implantation; even then, carefully coordinated communication between the embryonic trophectoderm and maternal endometrial epithelium is required to further modify the endometrial surface and thereby ‘permit’ attachment and stimulate the trophectoderm cells to attach and subsequently either invade into (chorionic girdle cells), or interdigitate with (other chorionic cells), the endometrium.55,56 initial attachment takes place during the ‘window of receptivity to implantation’,57 when the previously nonreceptive endometrial surface is modified to allow conceptus adhesion. an important aspect of endometrial preparation is a period of exposure to progesterone that results in a down regulation of progesterone receptors (prs) in the luminal and glandular epithelial cells but, possibly crucially, not in the stromal cells.55 these stromal cells are proposed to produce ‘progestamedins’ that allow progesterone to fine tune the preparations of the endometrial epithelium for its role in implantation, despite the ‘switching off’ of their own prs.55 one of the first steps in implantation is intimate apposition of the trophectoderm to the endometrium, an event that can only occur once the capsule starts to disintegrate at around day 23 of pregnancy.27 in most species, attachment of the trophectoderm to the endometrium requires removal of surface glycocalyx components that previously inhibited this process and, in particular, mucins that coat the endometrial luminal epithelium (e.g. muc158). in the mare, there does not appear clinical theriogenology • volume 12 number 4 • december 2020 539 to be a reduction in endometrial muc1 expression in preparation for attachment,59 but it may be significant that the blastocyst capsule is itself rich in mucin-like glycoproteins. as discussed previously, these capsular glycoproteins are desialylated at the time of conceptus fixation26 by a process that must be progesterone dependent, since it fails if luteolysis is induced prior to fixation.27 in short, desialylation of the capsular glycoproteins may be an equine specific equivalent to the progesterone primed dismantling of endometrial surface mucins in the preparation for trophectodermendometrium attachment. whether degradation of the capsule alone is sufficient to permit attachment is not known, and it seems more likely that luteal progesterone and conceptus derived signals combine to further promote and stabilize trophectoderm-endometrium attachment. the need for coordinated interaction between the progesterone primed endometrium and the developing conceptus if pregnancy is to proceed normally is clearly illustrated by the delayed development of equine embryos transferred into the uterus of a recipient mare that ovulated 5 days after the donor.60 indeed, it is likely that a species specific minimum period of progesterone priming is required for the endometrium to be able to both provide adequate histotrophic nutrition and become receptive for implantation. moreover, recent transcriptomic studies identified a range of cytokines, growth factors and corresponding receptors that are upregulated either in the trophectoderm, the endometrium or both, in response to luteal progesterone and/or local trophectodermal hormones including estrogens and pge2, in the third and fourth weeks of pregnancy.52,61 there also appear to be roles for molecules known to be instrumental to implantation in other species, such as leukaemia inhibitory factor,62 osteopontin, integrins and various members, receptors and binding factors from the insulin-like and fibroblast growth factor families.47,61,63 however, it is not known how the endometrial surface is modified to play its contrasting roles in the two distinct implantation events seen during equine pregnancy, namely invasion of the highly proliferative chorionic girdle cells versus interdigitation of the remaining, noninvasive chorionic cells.8 nevertheless, deficiencies in endometrial receptivity and/or aberrations in the embryo-maternal signalling processes required for implantation almost certainly contribute to the high incidence of pregnancy loss between the end of the conceptus mobile phase (day 16) and the onset of definitive placental formation (day 40).8 conclusion during the first 6 weeks of pregnancy, the equine conceptus develops from a single cell ‘zygote’ to a fetus with a functional circulation and precursors of all of the organs required for postnatal life. to ensure the successful establishment and maintenance of pregnancy, embryomaternal signalling plays critical roles in ensuring luteal maintenance and, together with luteal progesterone, in preparing the endometrium for its role in implantation and establishment of a more stable platform for nutrient and gaseous exchange. conflict of interest none to declare. references 1. morris lh, allen wr: reproductive efficiency of intensively managed thoroughbred mares in newmarket. equine vet j 2002;34:51-60. 2. galli c, duchi r, colleoni s, et al: ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: from the research laboratory to clinical practice. theriogenology 2014;81:138-151. 3. rose bv, firth m, morris b, et al: descriptive study of current therapeutic practices, clinical reproductive findings and incidence of pregnancy loss in intensively managed thoroughbred mares. anim reprod sci 2018;188:74-84. 4. allen wr, brown l, wright m, et al: reproductive efficiency of flatrace and national hunt thoroughbred mares and stallions in england. equine vet j 2007;39:438-445. 5. morris lha: the development of in vitro embryo production in the horse. equine vet j 2018;59:712-720. 6. allen wr. luteal deficiency and embryo mortality in the mare: reprod domest anim 2001;36:121-131. 7. shilton ca, kahler a, davis bw, et al: whole genome analysis reveals aneuploidies in early pregnancy loss in the horse. sci rep 2020;10:13314. 8. allen wr, wilsher s: a review of implantation and early placentation in the mare. placenta 2009;30:1005-1015. 9. freeman da, weber ja, geary rt, et al: time of embryo transport through the mare's oviduct. theriogenology 1991;36:823-830. 10. tremoleda jl, stout ta, lagutina i, et al: effects of in vitro production on horse embryo morphology, cytoskeletal 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secrete insulin-like growth factor-binding protein 3. biol reprod 2000;62:1804-1811. 34. short rv: implantation and the maternal recognition of pregnancy. in: wolstenholme gew, o'connor m, editors. ciba foundation symposium on foetal autonomy, london, uk. j and a churchill ltd; 1966, p. 2-26. 35. vanderwall dk, silvia wj, fitzgerald bp: concentrations of oxytocin in the cavernous sinus of mares during luteolysis: temporal relationship with concentrations of 13, 14-dihydro-15-ketoprostaglandin f2alpha. j reprod fertil 1998;112:337-346. 36. stout tae, lamming ge, allen wr: the uterus as a source of oxytocin in the cycling mare. j reprod fert 2000;suppl 56:281-287. 37. goff ak, pontbriand d, sirois j: oxytocin stimulation of plasma 15-keto-13,14-dihydro prostaglandin f2 during the oestrous cycle and early pregnancy in the mare. j reprod fertil 1987;suppl 35:253-260. 38. mcdowell kj, sharp dc, peck ls, et al: effect of restricted conceptus mobility on maternal recognition of pregnancy in mares. equine vet j 1985;suppl 3:23-24. 39. boerboom d, brown ka, vaillancourt d, et al: expression of key prostaglandin synthases in equine endometrium during late diestrus and early pregnancy. biol reprod 2004;70:391-399. 40. de ruijter-villani m, van tol ht, stout ta: effect of pregnancy on endometrial expression of luteolytic pathway components in the mare. reprod fertil develop 2015;27:834-845. 41. starbuck gr, stout tae, lamming ge, et al: endometrial oxytocin receptor and uterine prostaglandin secretion in mares during the oestrous cycle and early pregnancy. j reprod fertil 1998;113:173-179. 42. stout tae, allen wr: prostaglandin e2 and f2α production by equine conceptuses and concentrations in conceptus fluids and uterine flushings recovered from early pregnant and dioestrous mares. reproduction 2002;123:261-268. clinical theriogenology • volume 12 number 4 • december 2020 541 43. stout tae, lamming ge, allen wr: oxytocin administration prolongs luteal function in cyclic mares. j reprod fertil 1999;116:315-320. 44. klein c, troedsson mh: maternal recognition of pregnancy in the horse: a mystery still to be solved. reprod fertil dev 2011;23:952-963. 45. weithenauer j, sharp dc, mcdowell kj, et al: characterisation of the equine conceptus prostaglandin inhibitory product. biol reprod 1987;36 suppl 1:abstract 329. 46. ababneh mm, troedsson mht: michelson jr, et al: partial characterization of an equine conceptus prostaglandin inhibitory factor. j reprod fertil 2000;suppl 56:607-613. 47. klein c, troedsson mh: transcriptional profiling of equine conceptuses reveals new aspects of embryo-maternal communication in the horse. biol reprod 2011;84:872-885. 48. walters kw, roser jf: anderson gb: maternal-conceptus signaling during early pregnancy in mares: oestrogen and insulin-like growth factor i. reproduction 2001;121:331-338. 49. zavy mt, mayer r, vernon mw, et al: an investigation of the uterine luminal environment of non-pregnant and pregnant pony mares. j reprod fertil 1979;suppl 27:403-411. 50. silva la, gastal el, beg ma, et al: changes in vascular perfusion of the endometrium in association with changes in location of the embryonic vesicle in mares. biol reprod 2005;72:755-761. 51. zavy mt, sharp dc, bazer fw, et al: identification of stage-specific hormonally induced polypeptides in the uterine protein secretions of the mare during the oestrous cycle and pregnancy. j reprod fertil 1982;64:199-207. 52. klein c, scoggin ke, ealy ad, et al: transcriptional profiling of equine endometrium during the time of maternal recognition of pregnancy. biol reprod 2010;83:102-113. 53. merkl m, ulbrich se, otzdorff c, et al: microarray analysis of equine endometrium at days 8 and 12 of pregnancy. biol reprod 2010;83:874-886. 54. gibson c, de ruijter-villan m, bauersachs s, et al: asynchronous embryo transfer followed by comparative analysis of conceptus membranes and endometrium identifies processes important to the establishment of equine pregnancy. int j mol sci 2020;21:2562. 55. bazer fw, spencer te, johnson ga, et al: comparative aspects of implantation. reproduction 2009;138:195-209. 56. de ruijter-villani m, stout t: the role of conceptus-maternal signalling in the acquisition of uterine receptivity to implantation in mammals. reprod domest anim 2015;50 suppl 3:7-14. 57. dey sk, lim h, das sk, et al: molecular cues to implantation. endocr rev 2004;25:341-373. 58. carson dd, desouza mm, kardon r, et al: mucin expression and function in the female reproductive tract. hum reprod update 1998;4:459-464. 59. wilsher s, gower s, allen wr: persistence of an immunoreactive muc1 protein at the feto-maternal interface throughout pregnancy in the mare. reprod fertil dev 2013;25:753-761. 60. gibson c, de ruijter-villani m, stout tae: negative uterine asynchrony retards early equine conceptus development and upregulation of placental imprinted genes. placenta 2017;57:175-182. 61. klein c. novel equine conceptus/endometrial interactions on day 16 of pregnancy based on rna sequencing. reprod fertil dev 2015;28:1712-1720. 62. de ruijter-villani m, deelen c, stout ta: expression of leukaemia inhibitory factor at the conceptus/maternal interface during preimplantation development and in the endometrium during the oestrous cycle in the mare. reprod fertil dev 2015;28:1642-1651. 63. de ruijter-villani m, van boxtel pr, stout ta: fibroblast growth factor-2 expression in the preimplantation equine conceptus and endometrium of pregnant and cyclic mares. theriogenology 2013;80:979-989. clinical theriogenology • volume 12 number 4 • december 2020542 1 contact theresa beachler beachler@iastate.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 9400, http://dx.doi.org/10.58292/ct.v16.9400 review report embryonic diapause: a unique stage of development theresa beachler department of veterinary diagnostic and production animal medicine, college of veterinary medicine, iowa state university, ames, ia, usa abstract embryonic diapause is a unique stage of development that occurs in some mammalian species. diapause serves as an evolutionary reproductive strategy to maximize the survivability of dam and offspring. although individual mechanisms of control vary among species, the overarching physiology of diapause extends the length of pregnancy to delay parturition until either seasonal, environmental, nutritional, or internal physiologic constraints are optimal for parturition and rearing of young. this review discusses concepts and highlights control mechanisms unique to rodents, mink, and tammar wallabies. keywords: embryonic diapause, development, blastocyst, pregnancy introduction embryonic diapause, also known as delayed implantation, is a temporary state of arrested development that occurs in some mammals at the blastocyst stage. embryonic diapause is an evolutionary strategy to delay parturition to ensure the optimal seasonal, nutritional, environmental, and physiologic conditions occur for both survivability and the rearing of offspring. diapause was first noted in roe deer (capreolus capreolus) in the mid-19th century.1 subsequently, it was observed in over 130 species across various orders ranging from rodents, marsupials, numerous carnivores, including minks, weasels, skunks, bears, seals, armadillos, bats, and some species of deer.2,3 the defining characteristic of diapause is the dramatic reduction or cessation of cellular activity in the embryo at the g0/g1 stage.3 in some species (e.g. mice and marsupials) cellular activity stops in its entirety with cessation of all cell divisions, transcription, and translation, whereas in others, such as the roe deer and some carnivores (mink, weasels, and bears), growth and activity remains but at an incredibly slow rate.3,4 in roe deer, for example, ~ 10% of cells proliferate where the embryo expands from 300 to 20,000 cells over a period of 4 months, with the majority of growth occurring within the trophectoderm compared to the inner cell mass.4 maintenance or length of delayed development may range from a brief halt lasting for a few days, as observed in mice and rats (4-10 days), to much longer extended period of ~ 10 months in the badger and up to 11 months in the tammar wallaby.2 types of diapause diapause is classified into 2 categories, either facultative or obligate diapause, with most species, excluding the tammar wallaby, experiencing 1 or the other.1-3,5 facultative diapause occurs in response to a limiting internal physiological factor.2,3,5 facultative diapause is also sometimes referred to as lactational diapause and is best understood in mice and some marsupials, where development is delayed in the presence of suckling young until weaning occurs.4 obligate diapause or seasonal diapause has been best studied in carnivores, where delayed development occurs in every pregnancy under a seasonal photoperiod control.5 for example, in many carnivores residing in the northern hemisphere, such as the mink or skunk, and in bears, development is delayed until decreased melatonin is produced from the pineal gland in response to the onset of long days to support the birth and rearing of young during the most favorable environmental and nutritional conditions.2,5 therefore, the hormone prolactin is a key player in both varieties of diapause with marked interspecies variability. species-specific examples of lactational and seasonal control mice rodents such as rats and mice experience the first postpartum estrus within 24 hours of parturition.6,7 in mice mailto:beachler@iastate.edu http://dx.doi.org/10.58292/ct.v16.9400 2 citation line: clinical theriogenology 2024, 16, 9400, http://dx.doi.org/10.58292/ct.v16.9400 (mus muscularis) that were successfully mated, lactation in the presence of suckling young results in elevated concentrations of prolactin.6-8 high concentrations of prolactin prevent the estrogen surge that occurs at 3.5 days postovulation, initiating a facultative or lactational diapause for a period of 1 day to several weeks.5,8 larger litter sizes additionally have been associated with longer periods of delayed development.7 following weaning and the removal of the inhibitory effects of prolactin, embryos are reactivated by the estrogen surge and subsequent elevations in progesterone concentrations that prepare the endometrium for implantation.5 this has been supported experimentally by antagonist/dopamine agonist (bromocriptine) treatment, which terminated diapause and induced reactivation and implantation.9 embryo transfer in ovariectomized female rats that were treated with progesterone prior to the natural estrogen surge, entered into an arrested state with reactivation occurring after estradiol treatment.10 mink american mink (neovison vison) is 1 of the best studied obligate or seasonal diapause family members. in american mink residing in the northern hemisphere, embryo development is delayed until decreased melatonin production occurs from the pineal gland in response to the onset of long days or > 12 hours of daylight after the spring equinox.11-13 mink breed during spring, with matings occurring between february and april. subsequently, prolactin concentrations remain low in response to high nocturnal melatonin production (< 12 hours of daylight). regardless of the time of mating and ovulation, lengthening photoperiod and concurrent reduction in melatonin increases circulating prolactin concentrations, resulting in ovarian (corpora lutea) produced progesterone increase.11,12 on the contrary in rodents, experimental treatment with the dopamine agonist bromocriptine extended diapause, whereas treatment with the dopamine antagonist pimozide terminated diapause.14,15 therefore, prolactin is directly luteotrophic, ending diapause and resuming embryonic development in mink. wallabies tammar wallabies (macropus eugenii) are unique as they may undergo both a facultative and obligate diapause in response to elevated concentrations of prolactin for a total period lasting up to 11 months.5 in the southern hemisphere, tammar wallabies typically give birth to a single joey in late january to early february and enter a postpartum estrus shortly after, with mating occurring as early as 1 hour after birth.16,17 in successfully bred females, high concentrations of prolactin from the suckling young contribute to the initiation of lactational diapause. if the joey is lost during the breeding season (january through may), embryonic development resumes in the absence of high prolactin concentrations.18 after the winter solstice (june), only the seasonal photoperiod is sufficient to cause reactivation, with no response if joey is lost after this period. after the subsequent summer solstice (december), prolactin concentrations decrease in response to decreased photoperiod and increase in melatonin, allowing for blastocyst reactivation.18,19 tammar wallabies, therefore, are unique species as they may display either a single or a combination of a facultative or obligate diapause depending on the time of year and if a suckling joey is present during periods of pregnancy. additional mechanisms for reactivation or cessation of diapause at the blastocyst stage, embryos of species that undergo diapause have not yet initiated implantation and remain within the uterine lumen either within (marsupials and carnivores) or hatched from the zona pellucida (rodents, roe deer, and armadillos). in addition to varied hormonal changes as discussed above under lactational or seasonal control, reactivation or the cessation of the period of diapause appears to also be mediated by a combination of cytokines, growth factors, and transcriptional factors produced from the uterine endometrium within uterine secretions and the embryo itself, all of which is not completely understood.2 at the arrested blastocyst stage, embryos have yet to initiate implantation and may not begin the process until multiple days after reactivation. factors identified with altered patterns of expression during reactivation are conserved across many species include epidermal growth factors (egf and hb-egf), leukemia inhibitory factor (lif), insulin-like growth factor (igf), platelet-derived growth factor (pdgf), fibroblast growth factor (fgf), platelet-activating factor (paf), transforming growth factor (tgf-β), interleukin 1β (il1β), bone morphogenic protein-2 (bmp2), and prostaglandin synthetase ptgs2 (cox2).1,5,20-22 in mice, leukemia inhibitory factor (lif) has a critical molecule in reactivation and the onset of implantation as its expression is substantially upregulated within the endometrial glands of pregnant mice on days 4 and 5 of pregnancy.21,22 female mice lacking functional lif genes produced viable embryos that failed to implant at the blastocyst stage in vivo but underwent appropriate development when transferred to wild-type recipients.22 similarly, epidermal growth factor (egf) has been critical for implantation in mice and rats with knockout receptor dams failing all embryonic development beyond the blastocyst stage with degradation of the inner cell mass.23 additionally, signaling molecules or transcription factors encoded by the wingless family (wnt) and muscle segment homeobox genes (msx1 and msx2) were involved in activation and implantation in mice, mink, and wallabies.1,2,24-27 wnt signaling may also contribute to the altered spacing of embryos which in some species occurs simultaneously with embryo reactivation.24 muscle segment homeobox genes (msx1 and msx2) are further mediated by wnt5a signaling with marked downregulation of these genes and their products occurring at embryo reactivation and implantation in mice, mink, and wallabies.24,25 conclusion mammalian embryonic diapause is a phenomenon defined by a temporary arrest in blastocyst development as a reproductive strategy to promote both the survival of the dam and neonate until either environmental or physiologic factors are favorable. although a whole host of variability is demonstrated, many of the hormonal and signaling factors are highly conserved. conflict of interest none. references 1. fenelon jc, renfree mb: the history of the discovery of embryonic diapause in mammals. biol reprod 2018;99:242-251. doi: 10.1093/biolre/ioy112 http://dx.doi.org/10.58292/ct.v16.9400 https://doi.org/10.1093/biolre/ioy112 citation line: clinical theriogenology 2024, 16, 9400, http://dx.doi.org/10.58292/ct.v16.9400 3 2. renfree mb, shaw g: diapause. annu rev physiol 2000;62:353375. doi: 10.1146/annurev.physiol.62.1.353 3. lopes fl, desmarais ja, murphy bd: embryonic diapause and its regulation. reproduction 2004;128:669-678. doi: 10.1530/rep.1.00444 4. rüegg ab, bernal s, moser fn, et al: trophectoderm and embryoblast proliferate at slow pace in the course of embryonic diapause in the roe deer (capreolus capreolus). biosci proc 2020;10:181-196. doi: 10.1530/biosciprocs.10.013 5. renfree mb, fenelon jc: the enigma of embryonic diapause. development 2017;144:3199-3210. doi: 10.1242/dev.148213 6. long ja, evans hm: the oestrous cycle in the rat and its associated phenomena. mem univ calif 1922;6:1-148. 7. weichert ck: the experimental shortening of delayed pregnancy in the albino rat. anat rec 1940;77:31-47. doi: 10.1002/ar.1090770105 8. mantalenakis sj, ketchel mm: frequency and extent of delayed implantation in lactating rats and mice. j reprod fertil 1966;12:391-394. doi: 10.1530/jrf.0.0120391 9. flint ap, renfree mb: bromocriptine-induced implantation during lactation in the rat. j reprod fertil 1981;62:181-183. doi: 10.1530/jrf.0.0620181 10. yoshinaga k, adams ce: delayed implantation in the spayed, progesterone treated adult mouse. j reprod fertil 1966;12:593-595. doi: 10.1530/jrf.0.0120593 11. murphy bd, james da: the effects of light and sympathetic innervation to the head on nidation in mink. j exp zool 1974;187:267276. doi: 10.1002/jez.1401870210 12. murphy bd, concannon pw, travis hf, et al: prolactin: the hypophyseal factor that terminates embryonic diapause in mink. biol reprod 1981;25:487-491. doi: 10.1095/biolreprod25.3.487 13. deng l, chunjin l, chen l, et al: research advances on embryonic diapause in mammals. anim reprod sci 2018;198:1-10. doi: 10.1016/j.anireprosci.2018.09.009 14. papke rl, concannon pw, travis hf, et al: control of luteal function and implantation in the mink by prolactin. j anim sci 1980;50:1102-1107. doi: 10.2527/jas1980.5061102x 15. murphy bd: precocious induction of luteal activation and termination of delayed implantation in mink with the dopamine antagonist pimozide. biol reprod 1983;29:658-662. doi: 10.1095/ biolreprod29.3.658 16. renfree mb, tyndale-biscoe ch: intrauterine development after diapause in the marsupial macropus eugenii. dev biol 1973;32:2840. doi: 10.1016/0012-1606(73)90217-0 17. rudd cd: sexual behavior of the male and female tammar wallabies (macropus eugenii) at post-partum oestrus. j zool 1994;232:151-162. doi: 10.1111/j.1469-7998.1994.tb01565.x 18. hinds la, tyndale-biscoe ch: plasma progesterone levels in the pregnant and non-pregnant tammar, macropus eugenii. j endocrinol 1982;93:99-107. doi: 10.1677/joe.0.0930099 19. tyndale-biscoe ch, hearn jp: pituitary and ovarian factors associated with seasonal quiescence of the tammar wallaby, macropus eugenii. j reprod fertil 1981;62:225-230. doi: 10.1530/ jrf.0.0630225 20. hardy k, spanos s: growth factor expression and function in the human and mouse preimplantation embryo. j endocrinol 2002;172:221-236. doi: 10.1677/joe.0.1720221 21. bhatt h, brunet lj, stewart cl: uterine expression of leukemia inhibitory factor coincides with the onset of blastocyst implantation. proc natl acad sci usa 1991;88:11408-11412. doi: 10.1073/ pnas.88.24.11408 22. stewart cl, kaspar p, brunet lj, et al: blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. nature 1992;359:76-79. doi: 10.1038/359076a0 23. threadgill dw, dlugosz aa, hansen la, et al: targeted disruption of mouse egf receptor: effect of genetic background on mutant phenotype. science 1995;269:230-234. doi: 10.1126/science.7618084 24. mohamed oa, jonnaert c, labelle-dumais k, et al: uterine wnt/beta-catenin signaling is required for implantation. proc natl acad sci usa 2005;102:8579-8584. doi: 10.1073/ pnas.0500612102 25. cha j, sun x, bartos a, et al: a new role for muscle segment homeobox genes in mammalian embryonic diapause. open biol 2013;3:130035. doi: 10.1098/rsob.130035 26. fenelon jc, banerjee a, murphy bd: embryonic diapause: development on hold. int j dev biol 2014;58:163-174. doi: 10.1387/ ijdb.140074bm 27. cha j, dey sk: cadence of procreation: orchestrating embryo-uterine interactions. semin cell dev biol 2014;34:56-64. doi: 10.1016/j.semcdb.2014.05.005 http://dx.doi.org/10.58292/ct.v16.9400 https://doi.org/10.1146/annurev.physiol.62.1.353 https://doi.org/10.1530/rep.1.00444 https://doi.org/10.1530/biosciprocs.10.013 https://doi.org/10.1242/dev.148213 https://doi.org/10.1002/ar.1090770105 https://doi.org/10.1530/jrf.0.0120391 https://doi.org/10.1530/jrf.0.0620181 https://doi.org/10.1530/jrf.0.0120593 https://doi.org/10.1002/jez.1401870210 https://doi.org/10.1095/biolreprod25.3.487 https://doi.org/10.1016/j.anireprosci.2018.09.009 https://doi.org/10.2527/jas1980.5061102x https://doi.org/10.1095/biolreprod29.3.658 https://doi.org/10.1095/biolreprod29.3.658 https://doi.org/10.1016/0012-1606(73)90217-0 https://doi.org/10.1111/j.1469-7998.1994.tb01565.x https://doi.org/10.1677/joe.0.0930099 https://doi.org/10.1530/jrf.0.0630225 https://doi.org/10.1530/jrf.0.0630225 https://doi.org/10.1677/joe.0.1720221 https://doi.org/10.1073/pnas.88.24.11408 https://doi.org/10.1073/pnas.88.24.11408 https://doi.org/10.1038/359076a0 https://doi.org/10.1126/science.7618084 https://doi.org/10.1073/pnas.0500612102 https://doi.org/10.1073/pnas.0500612102 https://doi.org/10.1098/rsob.130035 https://doi.org/10.1387/ijdb.140074bm https://doi.org/10.1387/ijdb.140074bm https://doi.org/10.1016/j.semcdb.2014.05.005 introduction since 2007, the release of subcutaneous implants containing a gnrh superagonist (deslorelin) into european market has led to several changes in veterinary practice regarding approaches to fertility control in the male dog. for example, france is a country with a ‘latin spirit’, where many owners do not like the idea of having their male dog castrated. more and more owners are also reluctant to seek a surgical procedure for their pet that is perceived as mutilation, a source of suffering, and considered as potentially risky because it requires general anesthesia. in some northern european countries or in germany, there are now many male dogs that are first ‘implanted’ to test the effect of a potential surgical orchidectomy that could be subsequently performed. in this article, we review the potential uses of deslorelin subcutaneous implants in male dogs and share our experience at alfort national veterinary college, paris. mode of action of slow-release deslorelin implants gnrh, a decapeptide with a highly conserved structure in vertebrates, is the key hormone in mammalian reproductive function. secreted in a pulsatile manner in the hypothalamo-pituitary portal system and transported to the pituitary gland where it releases gonadotropins (fsh and lh) into systemic circulation. gonadotropins stimulate production of steroids medical control of reproduction in the male dog with deslorelin subcutaneous implants alain fontbonne, cindy maenhoudt ecole nationale vétérinaire d’alfort, centre d’etude en reproduction des carnivores maisons-alfort (paris), france abstract slow-release subcutaneous implants containing gnrh agonist deslorelin were released into the european market in 2007. since then, they are widely used per label by european veterinarians for the prevention of fertility in adult male dogs. additionally, they are also used ‘off-label’ to postpone puberty, reduce unwanted behavior potentially linked to testosterone, eliminate clinical signs induced by benign prostatic hyperplasia, reduce the size of perianal adenomas, or reverse hair loss due to alopecia x in intact male dogs. after an initial ‘flare-up’ effect (short-duration increases in blood testosterone concentrations immediately after implantation) there is a perceptible decline in testosterone concentrations within a few weeks, due to pituitary downregulation. the dog becomes ‘medically castrated’ and infertile, with a reduction in testes size and spermatogenic arrest. normal plasma testosterone concentrations are recovered in 18 months in 98% of dogs and reimplantations are possible for life. preferably, reimplantation should occur prior to disappearance of previous implant action to avoid another ‘flare-up’ effect of reimplantation. after loss of implant function, it usually takes 1 2 months for complete sperm quality restoration before dog is potentially fertile again. keywords: male dog, fertility control, prostate, unwanted behavior, hair loss, deslorelin by the gonads, mainly testosterone in males, and estrogen in females and in males. the pulsatile secretion of gnrh is the real conductor of reproductive function. more than 2000 gnrh agonist and antagonist molecules have been synthesized in the last 30 years. these compounds are used in human medicine and in veterinary medicine, but mostly in large animals (rarely in carnivores). whereas the use of gnrh antagonists in veterinary medicine is complicated and costly,1 the agonist forms have been widely used. their mode of action depends on treatment conditions: a single injection stimulates pituitary gonadotropins and, consequently, sex steroid hormones, whereas treatment over several days leads to secondary desensitization of gonadotropic cells that prevents secretion of fsh and lh and ‘silences’ the pituitary-gonadal axis. this desensitization is due to a postreceptor mechanism that induces an arrest of the synthesis of the subunits lhβ and fshβ.2 regarding deslorelin, modifications in the initial sequence of the amino acids of gnrh make this molecule a super-agonist; its stability is increased and its binding affinity for specific gnrh receptors is increased 7-fold.3 the implant, the vector of the molecule, is composed of a lipid matrix (triglyceride) that does not pose any biocompatibility problem. subcutaneous implantation using an implanter the size of a microchip allows continuous release of deslorelin. clinical theriogenology 2021; 13: 151 two implants are sold to veterinarians in europe for veterinarians, with label indications for the suppression of fertility in male dogs: suprelorin 4.7 mg® and suprelorin 9.4 mg® (the latter is also authorized for male ferrets).4 these implants have a minimum duration of efficacy of 6 months for the former and 1 year for the latter. however, the initial release kinetics of the product from the implant matrix are different in these 2 products, and the ‘castrating’ effect is more rapidly obtained with the 4.7 mg implant than with the 9.4 mg implant. therefore, we recommend an initial implantation with a 4.7 mg implant, followed by an optional reimplantation 6 months later with either a 4.7 or a 9.4 mg implant. the dog can thus be reimplanted, for example every year, and thus ‘chemically castrated’ throughout its life.4 action of deslorelin implants on testosterone in male dogs often, the implants are easily placed under the skin in the inter-scapular area, without clipping. after implantation, deslorelin stimulates the pituitary secretions of lh and fsh; this initial ‘flare-up’ effect causes a transient increase in testosterone concentrations that may be accompanied by a slight increase in the dog’s activity and/or urinary marking.3 this initial ‘flare-up’ effect is often mentioned and feared by veterinarians and owners, but it seems to be extremely short with deslorelin implants. in a study with frequent sampling, testerosterone concentrations increased 20 minutes after implant (6 mg deslorelin), reached peak concentrations at 40 minutes, and at 5 hours the concentrations were not significantly different from before treatment or from control dogs treated with a placebo.5 the castration effect occurs in a second phase; testosterone concentrations become basal in ~ 80% of dogs < 20 days after implant.6 after an initial ‘flare-up’ for 2 -3 days, testosterone concentrations decreased substantially and remained closer to 0 for 200 400 days, whereas the dog with a placebo implant had normal testosterone concentrations (figure). diminishing effectiveness of the implant is evident by the progressive resumption of testosterone secretion. normal plasma concentrations recovered in 18 months after implantation of a 4.7 mg implant in 98% of dogs. however, the inhibitory effect lasts an average of 10 months in medium and large dogs and 13 months in small breeds.6 miniature breeds (chihuahua, miniature pinscher, pomeranian, toy poodle, etc.) may have more prolonged inhibition, occassionaly almost definitive. therefore, owners should be advised of this in case they wish to breed these dogs in the future (virbac, unpublished data). clinical modifications after implantation after implantation, most studies observed a decrease in the volume and consistency of testes. in general, testicular volume decreased ~ 3 times 5 weeks after implantation.5 regarding eventual behavioral changes, it has similar effects as surgical castration. in particular, some dogs may experience an increase in appetite that presents a risk of weight gain. nutritional advice is therefore essential. also, as with surgical castration, the effect on certain behaviors is uncertain, such as roaming, attraction to estrous bitches, mounting people’s legs or other dogs and urinary marking.7 in 50 60% of cases, these annoying behaviors are improved but rarely completely eliminated. aggression towards other males is most often substantially reduced. treatment with deslorelin implants improved 4 behavior problems in male dogs (roaming, dominance, urine marking, and mounting) in 50% of male dogs.8 these authors suggested that sterilization figure. testosterone concentrations in 10 dogs (pw 285 to pw 311) that had a 4.7 mg deslorelin implant (time 0); data from virbac corporation, used with authorization. time (days) clinical theriogenology 2021; 13: 152 obtained with these implants might accurately predict changes that can be expected after surgical castration in individual dogs that was confirmed in an another study by the same group.9 behavioral disorders in male dogs improved after treatment with implants containing azagly-nafarelin (another gnrh agonist that has not been commercialized).10 however, some dogs had temporary increases in 1 of 4 behavior problems during the first few days after implantation,8 probably due to the initial rise in testosterone concentrations. as no clear protocol is currently described to avoid potential side effects linked to the initial rise in testosterone concentrations, this indication should be applied with great care. a recent study11 was conducted in swiss military dogs to assess their working abilities compared to intact males via a standard behavioal test series for swiss military dogs (obedience, protection of the handler against an attacker, searching for a hidden person in a building, and reaction to social environment during a city walk). treatment with deslorelin in these military dogs had no measurable effect on any of the test situations compared to intact males. in order to obtain continuous medical castration, a new implant must be placed, ideally while the current implant is active. it is not advisable to wait to reimplant until testes increased in size.4 waiting too long to reimplant will generate a new transient stimulating effect of ‘flare-up’ and the increase in testosterone concentrations may lead to undesirable male behavior for 1 2 weeks. if testes have already increased in size, use a 4.7 mg implant. to avoid owners forgetting reimplantation, many veterinarians use software to automatically send reminder letters or e-mails regarding reimplantation. note that the matrix of the implant is biocompatible and it dissolves very slowly.therefore, it is not necessary to remove the implant and furthermore reimplantion is required only after several months. effects on fertility there is a decrease in the number of sperm in the ejaculate. after an average of 4 6 weeks of treatment, the dogs become azoospermic. the transition to azoospermic state is however progressive with increased teratozoospermia. after a deslorelin implant, percentage of sperm with abnormal morphology, mainly tail abnormalities, may increase from an average of 7 70% in 35 days.6 however, teratospermia was not observed12 and veterinarians should be aware of the risk of fertile matings within 1 month after implantation. after 5 weeks, dogs usually have atrophic and aspermatogenic seminiferous tubules, with spermatogenesis blocked at the spermatogonial stage.13 at the end of action of the implant, testicular histological architecture is restored and it takes 7 9 weeks, the duration of spermatogenesis, for sperm to appear. after initial implantation, it usually takes another 1 3 months for sperm quality to be completely restored before the dog is potentially fertile again.6 however, to the best of our knowledge, there are no published studies on potential for complete recovery after successive implantations. ‘off-label’ potential indications of deslorelin implants postponement of puberty early-age neutering is an effective technique for canine population control; however, surgical neutering may not be possible in various situations. eleven dogs, 4-month-old, were implanted with 4.7 or 9.4 mg deslorelin, or placebo14 and their sexual behavior and testicular size monitored every 2 months. ejaculates were collected and evaluated at 8, 12, 15, 18, 24, 30, 32, 34, and 36 months of age. most dogs (3/4) implanted with 4.7 mg deslorelin had normal male sexual behavior at 34 months. from this group, 2 dogs had normal semen quality whereas semen could not be collected from the other dog, and after castration, sperm were not obtained after epididymal flushing. one dog implanted with 4.7 mg deslorelin and 4 dogs implanted with 9.4 mg deslorelin remained in a prepubertal reproductive status at 30 34 months. the delay in puberty was longer in dogs implanted with a higher dose of deslorelin. however, to date, implantation of young male dogs before puberty does not seem to be frequent in europe. behavioral disorders potentially linked with testosterone ‘off-label’ treatment of deslorelin implants may be of interest for management of some real behavioral disorders.15 to address this question, a test course16 was set up in 9 pedigree dogs. animals were included according to 5 criteria, including intra-species aggression or inter-species aggression. each dog received a 4.7 mg deslorelin implant and was monitored for 6 months. success was defined as at least a 50% decrease in the occurrence of the primary problem and a case progress rating of 6 -10 was given by the dog owner at the end of the study. nine cases were classified as successful. in 8 cases, the number of occurrences of the primary pattern was zero. the remaining cases had a reduction of up to 90%. the best scores were for intra-species aggression. however, castration in male dogs may increase some agressive behaviors towards humans, especially strangers.17 to date, it is still impossible to know if treatments with deslorelin implants may be beneficial in case of real behavioral problems, such as separation anxiety or aggression. prostatic problems in dog, prostate diseases are very frequent. nearly 80% of elderly dogs have benign prostate hyperplasia (bph). one of the main principles guiding the treatment of these conditions is to reduce prostate size. this is generally achieved subsequent to decline in serum testosterone concentrations after surgical castration. however, some owners are reluctant to castrate their dogs, or some older animals may be at-risk if general anaesthesia is performed. long-term treatment based on gnrh agonists results in 40 60% reduction in prostate volume. this reduction is achieved in 4 6 weeks,18,19 regardless of the agonist used. a deslorelin implant (4.7 mg) were used in 14 dogs that had bph.20 authors observed a decrease in the prostatic volume from day 14 after implantation, with ~ 60% decrease in prostatic volume at 1 month, with reduction of prostatic cysts (determined by ultrasonography). moreover, clinical theriogenology 2021; 13: 153 there was a quick suppression of clinical signs in all dogs and no apparent increase in the clinical signs during the ‘flare-up’ period after implantation. other authors, using doppler ultrasound to estimate the degree of blood supply of the prostate, observed a rapid decrease beginning as early as 5 days postimplantation, a quicker response to treatment.21,22 a recent study23 observed a minimum efficacy of 36 weeks after deslorelin implantation in suppressing clinical signs of bph. it remains controversial, however, due to the initial ‘flare-up’ effect, if it is recommended to use deslorelin implants as a first-line treatment in dogs presenting clinical signs related to bph, or to first use an antiandrogenic drug to suppress these signs and implant the dog 3 or 4 months later after clinical resolution in order to avoid recurrence. of course, the dog will have to be reimplanted regularly thereafter. perianal adenomas due to the role of testosterone in development of perianal adenomas, some authors have tried deslorelin implant to decrease the size of these tumors to facilitate further surgical treatment. nine dogs with perianal adenomas diagnosed cytologically or histologically were followed several months by measuring with a caliper the largest diameter of the tumor, and testosterone concentrations were measured on days 1, 45, 90, and 180. eight of 9 dogs had a 30% reduction in tumor size on day 30, and in 6/9 dogs there was complete resolution on day 180.24 dermatological problems deslorelin implants have also proved to be beneficial in dermatology. ten pomeranian dogs (including 8 intact males), 4 italian spitz (3 intact males, 1 neutered female), 3 miniature poodles (2 intact males, 1 neutered female), 2 siberian huskies (intact males) and 1 intact male chow chow dog with confirmed hair cycle arrest (alopecia x) were included in a study.25 each dog was treated with a subcutaneous implant containing 4.7 mg deslorelin. responder dogs were reimplanted 6 months after the first implant to obtain a 1-year pharmacological exposure and to have 1-year follow-up. hair regrowth was visible within 3 months in 12 of 16 intact male dogs (75%); no hair regrowth was observed in any neutered female dogs. the overall response to therapy was 60%. no adverse effects were noted. based on these findings, deslorelin may be a treatment option for intact male dogs with idiopathic hair cycle arrest. final considerations when using implants ‘off-label’, due to potential side effects, we recommend to implant the dog under the abdominal skin, posterior to the umbilical area.26 in case of any further problem, it will be easy, with a small skin incision, to remove the implant under slight sedation. however, others prefer implanting under the skin on the medial part of the rear leg.27 conflict of interest none to report. references 1. gobello c: effects of gnrh antagonists vs agonists in domestic carnivores, a review. reprod domest anim 2012;47:373-376. 2. navarro c, schober pa: pharmacodynamics and pharmacokinetics of a sustained-release implant of deslorelin in companion animals. proceedings of 7th international symposium on canine and feline reproduction 2012. p. 177-178. 3. fontaine e, fontbonne a: clinical use of gnrh agonists in canine and feline species. reprod domest anim 2011;6:344-353. 4. fontbonne a, maenhoudt c, borges p, et al: utilisation d’implants sous-cutanés d’agonistes de la gnrh chez les carnivores domestiques pour maîtriser la reproduction. bulletin de l’académie vétérinaire bulletin de l’académie vétérinaire de france 2014;167:165-170. 5. junaidi a, williamson pe, cummins jm, et al: use of a new drug delivery formulation of the gonadotrophin-releasing hormone analogue deslorelin for reversible long-term contraception in male dogs. reprod fertil dev 2003;15:317-322. 6. trigg te, doyle ag, walsh jd, et al: a review of advances in the use of the gnrh agonist deslorelin in control of reproduction. theriogenology 2006;66:1507-1512. 7. maarschalkerweerd rj, endenburg n, kirpensteijn j, et al: influence of orchiectomy on canine behaviour. vet rec 1997;140:617-619. 8. de gier j, winke c: use of deslorelin to control hypersexuality in male dogs. in symposium deslorelin: deslorelin in practice, proceedings of 7th european veterinary society for small animal reproduction congress 2010. p. 9-10. 9. de gier j, okkens ac, oei chy, et al: behaviour and the pituitary axis in dogs before and after surgical or chemical castration with the gnrh agonists deslorelin. proceedings of 7th international symposium on canine and feline reproduction 2012. p. 54-55. 10. goericke-pesch s, wilhelm e, ludwig c, et al: evaluation of the clinical efficacy of gonazon implants in the treatment of reproductive pathologies, behavioral problems and suppression of reproductive function in the male dog. theriogenology 210;73;920-926. 11. gfrerer n, taborsky m, würbel h: no evidence for detrimental effect of chemical castration on working ability in swiss military dogs. appl anim behav sci 2019;211:84-87. 12. romagnoli s, siminica a, sontas bh, et al: semen quality and onset of sterility following administration of a 4.7-mg deslorelin implant in adult male dogs. reprod domest anim 2012;47:389-392. 13. junaidi a, williamson pe, trigg te, et al: morphological study of the effects of the gnrh superagonist deslorelin on the canine testis and prostate gland. reprod dom anim 2009;44:757-763. 14. sirivaidyapong s, mehl ns, trigg te: delay of puberty and reproductive performance in male dogs following the implantation of 4.7 and 9.4 mg gnrh-agonist deslorelin at an early pre-pubertal age. reprod domest anim 2012;47:400-402. 15. driancourt ma, briggs jr: gonadotropin-releasing hormone (gnrh) agonist implants for male dog fertility suppression: a review of mode of action, efficacy, safety, and uses. front vet sci 2020;7:483:1-11. 16. beata c, marion m, massal n, et al: la desloréline (suprelorin®) pourrait-elle être utilisée dans certaines affections comportementales: une étude préliminaire. could deslorelin (suprelorin®) be used in the management of some bahavioural disorders: a preliminary study. revue vétérinaire clinique 2016;51:49-54. clinical theriogenology 2021; 13: 154 17. farhoody p, mallawaarachchi i, tarwater pm, et al: aggression toward familiar people, strangers, and conspecifics in gonadectomized and intact dogs. front vet sci 2018 26;5:18:1-13. 18. vickery b, mcrae g, bonasch h: effect of chronic administration of a highly potent lhrh agonist on prostate size and secretory function in geriatric dogs. prostate 1982;3:123-130. 19. ponglowhapan s, lohachit c, swangchan-uthai t, et al: the effect of the gnrh agonist deslorelin on prostatic volume in dogs. 3rd european veterinary society for small animal reproduction congress european congress 2002. p.150. 20. jurczak a, domosławska a, janowski t, et al: treatment of benign prostatic hyperplasia (bph) in dogs using gnrh agonist implant suprelorin®preliminary results. proceedings of 7th european veterinary society for small animal reproduction congress 2010. p. 118. 21. polisca a, orlandi r, troisi a, et al: clinical efficacy of the gnrh agonist (deslorelin) in dogs affected by benign prostatic hyperplasia and evaluation of prostatic blood flow by doppler ultrasound. reprod domest anim 2013;48:673-680. 22. nižañski w, levy x, ochota m, et al: pharmacological treatment for common prostatic conditions in dogs benign prostatic hyperplasia and prostatitis: an update. reprod domest anim 2014;49:8-15. 23. nižañski w, ochota m, fontaine c, et al: comparison of clinical effectiveness of deslorelin acetate and osaterone acetate in dogs with benign prostatic hyperplasia. animals 2020;10:1936:1-16. 24. domingo v, javier de andres f, rollon e, et al: clinical response of canine perianal adenomas to subcutaneous implants of deslorelin proceedings of the european society of veterinary oncology, spring congres, glasgow (scotland);2011. 25. albanese f, malerba e, abramo f, et al: deslorelin for the treatment of hair cycle arrest in intact male dogs. vet dermatol 2014;25:519-522. 26. fontaine e, mir f, vannier f, et al: induction of fertile oestrus in the bitch using deslorelin, a gnrh agonist. theriogenology 2011;76:1561-1566. 27. walter b, otzdorff c, brugger n, et al: estrus induction in beagle bitches with the gnrh-agonist implant containing 4.7 mg deslorelin. theriogenology 2011;75:1125-1129. clinical theriogenology 2021; 13: 155 medical control of reproduction in the maledog with deslorelin subcutaneous implants introduction development and application of reproductive technology in nondomestic species relies heavily on information gained from domestic model species.1 rhinoceros are a member of the order perissodactyla, and their closest living domestic relative is the horse. similar to other livestock species, horses are used in commercial enterprise where monetary gains and losses are directly tied to reproductive performance. as such, equine reproduction has become a highly focused area of study with results generated from sample sizes unparalleled to those working in nondomestic species field.2 research findings in the horse have proved an invaluable resource to those working to conserve rhinoceros. however, as with all model systems that include a domestic and wild counterpart, substantial differences exist between the reproductive physiology of the rhinoceros overcoming reproductive disorders in female greater one-horned rhinoceros (rhinoceros unicornis) to improve artificial insemination and natural breeding success monica stoops,a,b anneke moresco,c,d justine o’brien,e,f linda penfold,g james gillis,g jessye wojtusik,b lara metrioneg aomaha’s henry doorly zoo and aquarium, omaha, ne bcincinnati zoo & botanical garden, center for conservation and research of endangered wildlife, cincinnati, oh creproductive health surveillance program, morrison, co ddenver zoo, denver, co einstitute of science and learning, taronga conservation society australia, new south wales, australia fseaworld parks & entertainment, san diego, ca gsouth-east zoo alliance for reproduction and conservation, yulee, fl abstract reproductive disorders of female greater one-horned rhinoceros (gohr [rhinoceros unicornis]) contribute to relatively lower (50%) conception success from artificial insemination (ai) with frozen-thawed sperm, in comparison to rhino’s closest domestic relative, the mare. determining the causes of subfertility or infertility can be especially challenging in nondomestic species like rhinoceros. formative research identified anovulation and intraluminal uterine fluid accumulation as primary factors associated with the established 50% conception rate from gohr ai. we present an integrative approach taken to identify and treat female gohr reproductive dysfunctions to improve ai outcome to 66%. recommended diagnostics for the gohr practitioner include, but are not limited to, a breeding soundness examination, transrectal ultrasonography of the reproductive tract and ovaries, and urinary hormone analysis. as similar reproductive dysfunctions affect breeding success in their closest living domestic relative, the mare, comparable therapeutic treatment options were explored in female gohr. consequently, substantial advancement in gohr hormone therapies were used to ensure ovulation and resolve uterine clearance issues with sucromate® and oxytocin treatments, respectively. managed breeding programs, like that for the gohr, serve a noteworthy role in the strategy to conserve threatened species. in seeking strategies to address reproductive dysfunction in a cohort of females, we improved the procedural rate of success in ai. reported strategies can also assist gohr natural breeding efforts and collectively support the long-term resilience and conservation role of this managed population. keywords: assisted reproductive techniques, ovulation induction, endometritis, anovulation, intraluminal fluid and horse that preclude making sweeping generalizations. in fact, remarkable differences exist even among various rhinoceros species.3-9 it is, therefore, imperative to use appropriate methodologies and to draw interpretations based on established species-specific norms (i.e. estrous cycle length, follicular growth pattern, and preovulatory follicle size). the current size of the north american managed population of greater one-horned rhinoceros (gohr [rhinoceros unicornis]) is 110 animals distributed across 39 facilities. substantial genetic skew in the founder gohr population served as the impetus for development and application of semen collection/cryopreservation and artificial insemination (ai) in this species.10 gohr calves have been produced from ai using frozen-thawed sperm clinical theriogenology 2021; 13: 394 with an ai success rate of 50% achieved using a standing sedation approach.10 quality of frozen-thawed sperm is generally not a problem in gohr.11 therefore, comparisons to equine ai are made to studies utilizing semen from stallions classified as good for freezing, meaning a postthaw sperm progressive motility of 40 60%.2 conception rates of 60 75% per cycle have been achieved for mares inseminated with > 300 x 106 motile frozen-thawed sperm within 24 hours before to 6 hours after ovulation.12-14 previous successful gohr ai procedures utilized semen exhibiting a 60% postthaw motility to inseminate a minimum of 500 x 106 motile sperm 24 48 hours before ovulation.10 through retrospective analysis of ultrasonography and urinary hormone data it was observed that some of these cycles were nonovulatory, decreasing the potential success rate of the ais.10 since that formative work, the authors have developed a comprehensive strategy to identify and treat common reproductive disorders encountered in gohr to further advance the application of ai technology to more individuals and improve conception success from a single insemination attempt (figure 1). female breeding soundness examination subfertility has been identified as a major issue affecting natural and assisted rhinoceros breeding efforts.4-10,15-23 its impact has been directly felt by those developing nondomestic assisted reproductive techniques (art), as young, proven females are rarely afforded for study.1 instead, available subjects are frequently those that have failed to succeed via a natural breeding approach.1 subfertility of the mare can result in substantial economic losses to equine industry, and equine practitioners implement breeding soundness examination (bse) as a means to identify and subsequently treat subfertile mares to improve reproductive outcome.24 it is recommended that gohr females undergoing ai be subjected to bse. at minimum, past reproductive history should be reviewed along with recent and concurrently collected ultrasonography information, endocrine and behavior data ahead of any ai attempt. given an average estrous cycle length of 45 days in gohr,8 a 3-month collection timeframe ahead of planned ai is desired. as rhinoceros are nonseasonal breeders, examinations can occur irrespective of time of year. ultrasonography transrectal examination of the reproductive tract of domestic livestock is routine practice and the authors propose it should be a trained voluntary behavior for all rhino in managed care.5,7-8,25 the degree of stress caused by operant conditioning and the act of routinely conducting transrectal ultrasonography on gohr has been studied via fecal glucocorticoid metabolite and behavioral analysis.26 results indicate ultrasonographic reproductive assessments conducted using operant conditioning are considered nonstressful events.26 in general, rhinoceros are very tolerant of transrectal examination if appropriately conditioned. given the normal follicular growth pattern and size in this species, it is recommended that a low frequency curved array transducer be used for transrectal examination. the left ovary is located more cranial than the right ovary, sometimes out of the practitioner’s reach, and an extension tool is needed to facilitate transducer reach to the left ovary and distal horn. it is important that examinations are conducted by one who is familiar with normal ultrasonographic findings at various stages of the gohr estrous cycle. notable figure 1. flowchart of the process used to identify and, when possible, treat reproductive disorders contributing to failure to establish pregnancy in greater one-horned rhinoceros (gohr) after ai or natural breeding. clinical theriogenology 2021; 13: 395 differences as compared to the mare include, but are not limited to, the substantial size of the gohr preovulatory follicle, development of corpora hemorrhagica (ch) versus corpora lutea (cl) and lack of edematous changes in the uterus leading up to ovulation.8 from the perspective of establishing ai candidacy, it is important to identify any potential barriers for sperm transport and embryo mobility throughout the reproductive tract, including intact hymen. pathologic conditions such as tumors, cysts and intraluminal uterine fluid can be identified via ultrasonography. gohr are prone to the development of leiomyomas20 so their presence should not be unexpected, especially in older nulliparous females (figures 2a, b). size and location of cysts within the uterine horns are important to document especially for future referral when conducting postai examinations to determine early pregnancy. similar to the mare, gohr embryonic vesicle movement throughout the uterus is needed for maternal recognition of pregnancy.27 depending on the size and location of pathology could prevent signaling and thus release of pgf2α and regression of the ch. endocrinology in comparison to other rhinoceros species, the authors’ experience is that gohr are not as behaviorally compliant to routine blood collection. therefore, urine has become the gold standard biological matrix for gohr hormone evaluation as it correlates to systemic hormone dynamics more closely than feces.8,10 additionally, urine is easy to collect as female gohr habitually urinate in specific areas in the enclosure. gohr are the only rhinoceros species in which estrogens and estrogen metabolites can be reliably measured and accurately reflect ovarian follicle development;8-9 therefore, the gohr practitioner has the unique ability to track hormone dynamics throughout the follicular phase. specifically, urinary estrogen conjugate (ec) and pregnanediol glucuronide (pdg) concentrations and their pattern of excretion can be used to monitor follicular and luteal phases, respectively (figures 3a, b).3,8 the gohr follicular phase averages 14 days and is followed by a 17 21 day luteal phase.8 mean estrous cycle length is 45 days, but can vary substantially.8 however, longer estrous cycles in nonpregnant female gohr are typically due to a delay in emergence of the next dominant follicle,8 whereas a longer cycle figure 2. ultrasonogram of cervix and uterus in a 31-year multiparous greater one-horned rhinoceros (gohr). note: multiple a) cervical and b) uterine leiomyomas (arrows). figure 3. urinary a) estrogen conjugate (ec) and b) pregnanediol glucuronide (pdg) concentrations and pattern of excretion in greater one-horned rhinoceros (gohr) that ovulated after deslorelin in saline ( ) or sucromate® ( ). data are aligned by day from ovulation induction agent treatment. ultrasonograms of c) a preovulatory follicle 47 hours postsucromate® injection and d) resulting ch on day 18 postovulation during a 21-day luteal phase. clinical theriogenology 2021; 13: 396 length in nonpregnant female african rhinoceros is associated with an extended luteal phase.6-7 collecting urinary hormone data permits the gohr practitioner, through an iterative process, to determine what constitutes baseline ec and pdg concentrations for each female. this information is critical to accurately time ai events that are scheduled according to days after urinary pdg reaches baseline and ec is above baseline. behavior the female gohr typically exhibits overt estrous behavior. similar to mares, estrual gohr have reduced appetite and frequent urination, urine squirting, vulva winking, increased locomotion, and vocalization (whistling and snorting).8 peak estrus lasts for 12 24 hours; however, gohr can exhibit cycles with multiple days of some estrual behavior.8 during these ‘split’ estrus cycles there are typically several days of quiet in between estrual events making it difficult to determine whether a ‘pre’ or ‘true’ estrus has taken place. it is only ‘true’ estrus that is associated with the lh surge.8 however, silent estrus has been observed in some older gohr.10,21 estrus intensity can vary among females. therefore, accurate assessment of estrous behaviors relies on the astute observational skills of rhinoceros care staff and the knowledge they possess regarding ‘sometimes-subtle nuances’ associated with an individual animal.10 subfertility anovulation all rhinoceros species in managed care have been documented to experience anovulatory cycles on occasion.5,7,8 this form of ovarian dysfunction has been a major impediment to breeding in the mare28 and gohr. similar to domestic mares, there remains no definitive means to predict early in the cycle whether a given gohr follicle will be destined for anovulation. in mares the incidence of anovulation increases with age and several causative factors such as insufficient gonadotropin production and elevated estrogen production from the preovulatory follicle have been documented.28 timing of previous gohr ai procedures were based on natural cycles of each specific female.10 a downfall to this approach was that over a quarter of ai attempts were conducted on anovulatory cycles.10 most anovulatory cycles in gohr are associated with follicles that subsequently undergo luteinization.8,10 the resulting progestogen production occurs over the course of a normal luteal phase length thus mandating that ultrasonography be performed to confirm ovulation (figure 4a).8,10 gohr practitioners cannot rely solely on urinary pdg concentrations postestrus or postgnrh to serve as confirmation of ovulation. similar to the mare, only a limited number of anovulation cases in gohr occur without any significant luteinization following estrogen decline.8,10 the previously published gohr ai protocol relied on behavioral cues from females to time sedation/insemination, that only permitted 24 hour advanced notice for staff, and an ovulation induction agent was not used.10 as a result, several procedures were not timed correctly due to cycles in which there was a split-estrus, whereas others were hindered by subsequent ovulation failure.10 given anovulation was the most frequently encountered disorder during prior gohr ai efforts, an effective ovulation induction protocol was sought. preliminary attempts to induce ovulation using gonadorelin diacetate tetrahydrate (cystorelin™), a gnrh agonist, were unsuccessful due to mistimed injections.8 whereas follicle size is the key determinant for timing of ovulation induction in mare29-30 and white rhino (ceratotherium simum simum),18-19,22,31 gohr develop and sustain a large follicle size throughout the majority of their follicular phase,8 thereby limiting similar application. to correct the timing of injections to induce ovulation, treatment trials using 2 deslorelin synthetic gnrh analogue formulations (table) were used during natural estrous cycles (n = 15 cycles) in 7 adult female gohrs (6 31 years). as substantial intraand inter-individual variability exists in gohr preovulatory follicle size, timing of all but 1 treatment was based on days urinary ec concentrations were above baseline for each female (figure 3a). in 1 case, deslorelin treatment timing was based on the number of days from prior estrus and knowledge of behavioral estrus dates for the female’s earlier 3 estrous cycles. deslorelin dosages were extrapolated from those used in domestic mares to induce ovulation during the periovulatory period. unlike mare that exhibit multiple days of estrus and an extended lh surge, the gohr estrus lasts only 24 hours and has a narrow lh surge. compounded deslorelin in saline29,32 was used earlier but became unavailable and was treatment (year[s] treated) dosage (mg) number of females number of cycles age at treatment number of days urine ec elevated at treatment follicle size (cm) at treatment ovulation success rate deslorelin in saline32 (2009) 3.75 n = 2 n = 4 15.8 ± 1.3 14.3 ± 0.9 14.9 ± 0.4 75% 3/4 sucromate® (2011 2018) 3.15 4.95 n = 5 n = 11 16.6 ± 2.5 13 ± 1.1 13.7 ± 0.8 100% 11/11 table. ovulation induction summary in female greater one-horned rhinoceros (gohr) treated with 2 intramuscular deslorelin formulations *verified via ultrasonography and urinary hormone analysis or conception following ai clinical theriogenology 2021; 13: 397 replaced with another deslorelin product, extended release30 sucromate®. fourteen of 15 cycles in gohr treated with deslorelin resulted in ovulation induction by 48 hours after treatment and was followed by a normal luteal phase length (table, figure 3a d). retrospective urinary ec analysis of the single unsuccessful ovulation induction trial revealed peak ec concentrations were measured 2 days before treatment with deslorelin in saline and suggests that this treatment was given too early (figure 4a). the anovulatory follicle (figure 4b, 4c) subsequently luteinized, resulting in elevated urinary pdg concentration over a ~17 day timeframe (figure 4a). to date, 2 of 3 gohr ais performed in conjunction with sucromate® treatment at ai resulted in conception and birth of healthy calves. retrospective urinary pdg analysis confirmed an extended luteal phase was associated with a single unsuccessful induced ovulation ai procedure. this female was not conditioned for routine transrectal ultrasonography and so imaging of the reproductive tract was not possible. following this event, the female was conditioned for routine ultrasonography that resulted in diagnosis and treatment with a separate disorder (intraluminal uterine fluid accumulation [ifa]) that likely inhibited establishment of viable pregnancy during the aforementioned failed ai. by monitoring urinary ec concentrations to determine initiation of the follicular phase, currently, ai procedures can be timed 1 week ahead and conception rate may be improved with sucromate® treatment to ensure ovulation, however larger sample size studies are needed. intraluminal uterine fluid the inability to effectively clear uterine fluid that accumulates during the periovulatory period and/or postbreeding may be associated with preor post-conception failure events (figure 1). instances of early pregnancy loss have been reported in all managed rhinoceros species following natural breeding.4-5,21 it has also been observed after several conceptive ai procedures in gohr10 and white rhinoceros.33 occurrences thereof were revealed during ultrasonography by documenting disappearance of a previously positively identified embryonic vesicle/fetus.45,10,21,33 in 3 cases, examination frequency permitted visualization of ifa before and after embryonic demise.5-10 in each circumstance, ifa was detected during the late diestrus period (figure 5a). a strong association exists between the identification of ifa in late diestrus and impending early embryonic death (eed) in mares.34 in mares, breeding induced uterine inflammation is a natural physiologic reaction to semen and generally a short-lived process.35-36 however, in a subset of individuals the initial inflammation becomes pathologic and results in an inhospitable uterine environment for the developing embryo.37 mares become clinically classified with this disorder if unable to clear inflammation by 48 hours postbreeding.37 susceptibility increases with mare age, uterine position, cervical tightness or fibrosis and poor perineal conformation.37 the authors hypothesize that these same factors may also contribute to development of uterine inflammation in gohr, although further research is needed to elucidate this association. in mares, delays in uterine clearance translate to persistence of inflammation that can either directly or indirectly result in eed. therefore, the gohr practitioner needs to pay close attention to individuals identified via ultrasonography as accumulating intrauterine fluid during the periovulatory period. analyzing urinary pdg profiles before an ai attempt will also help detect luteal phase figure 4. concentrations of urinary steroid metabolites (a) estrogen conjugate (ec, ) and pregnanediol glucuronide (pdg, ) during a failed ovulation induction attempt in a greater one-horned rhinoceros (gohr) using deslorelin in saline. data are aligned by day from ovulation induction agent treatment. corresponding ultrasound images of the follicle on (b) day 0 and (c) day 12. clinical theriogenology 2021; 13: 398 lengths of shorter than normal duration and therefore indicative of premature luteolysis linked to inflammatory release of pgf2α and reproductive failure (i.e. eed).38 mares inseminated with frozen-thawed semen had an increased incidence of postbreeding intraluminal uterine fluid accumulation and lower pregnancy rates per cycle compared to fresh and chilled semen.13 paradoxically, it is standard practice to remove seminal plasma during processing of stallion semen for cryopreservation,39 but the lack of seminal plasma in extended equine semen has been linked to higher polymorphonuclear neutrophils (pmn) recovered from the uterus postinsemination compared to inseminates that contain this biological fluid.40-41 in addition, components of seminal plasma have an active role in sperm transport and survival in the reproductive tract of the mare.42 given presence of seminal plasma may influence ai success, the authors recommend continued adherence to the practice of maintaining this biological fluid during processing of gohr ejaculates for cryopreservation.11 in addition to maintaining seminal plasma in the inseminate, intervening therapies have helped overcome mechanical aspects of the uterine defense system that fail in mares with clearance issues. favorable results have been achieved in mares by treating with oxytocin43-46 that promotes strong, but short-lived myometrial contractions to eliminate intraluminal uterine fluid accumulation via cervical and lymphatic drainage.43-45 given oxytocin’s short half-life, intramuscular treatment is preferred (supports prolonged systemic circulation) compared to intravenous treatment. we report application of oxytocin treatment to resolve uterine fluid accumulation in a gohr. hormone analysis revealed the affected female experienced short luteal phases (< 17 days) during several estrous cycles in which breeding did not occur. a longer than normal luteal phase (23 day) followed her first induced ovulation ai attempt, prompting speculation that eed may have occurred. ultrasonography was used to confirm occurrence of ifa during the periovulatory period of an unbred cycle. for the second induced ovulation ai attempt (and ultimately conceptive) in this female, intramuscular oxytocin was given (25 iu) a day before and a day after ai. this timeframe correlated to days 3 and 1 before ovulation. in mares, uterine response to oxytocin is greatest on day 2 before ovulation and lowest on day 2 after ovulation.43,46 in addition to oxytocin treatment, given the potential for frozen-thawed sperm to induce endometritis,47-49 fresh semen was collected from a proven gohr bull at another facility, chilled overnight, transported and used for fresh-chilled ai. many traditional semen extenders, including those used for rhinoceros sperm cryopreservation, contain an animal protein source such as egg yolk or skim milk.11 introducing these constituents into a compromised uterine environment has potential to exacerbate an already existing inflammatory process. modifications were made to the traditional gohr semen extender that included lowering the percentage of egg yolk down to 2% from the standard 10% volume and adding supplemental antioxidants (st genetics, novasota, tx) to help maintain sperm quality and reduce oxidative stress during overnight transport. efficacy of oxytocin treatment was demonstrated by observation of vaginal discharge, ultrasound evidence demonstrating the absence of ifa at ai, presence of an embryonic vesicle after ai, continued absence of ifa, and measured spikes in urinary creatinine (figure 5b). oxytocin has antidiuretic properties50-52 and activity is reflected in urine creatinine concentration. creatinine is routinely measured in gohr urine samples to accurately report reproductive hormone results by accounting for variation in hydration status. significantly higher urine creatinine figure 5. a) ultrasonogram of intraluminal uterine fluid accumulation (asterisk) during late diestrus in a greater onehorned rhinoceros (gohr) that experienced early embryonic death (eed) following a conceptive artificial insemination (ai) procedure. b) urinary creatinine concentrations (ng/ml) before and after oxytocin treatment in a female gohr. data are aligned by day from ai. the dashed line reflects mean creatinine concentration before and after oxytocin treatments. oxytocin (25 iu) was given intramuscularly on days -1 and +1 from ai. circled data points correspond to creatinine concentrations measured in the first urine collected on the day after each oxytocin treatment. clinical theriogenology 2021; 13: 399 concentrations were measured the day after each oxytocin treatment in gohr (figure 5b). specifically, creatinine increased 3 and 6 times over baseline (1.36 ± 0.05 ng/ml) following the first and second oxytocin injection, respectively (figure 5b). conclusion reproductive disorders of female gohr decrease the likelihood that a calf is produced via natural and/or assisted breeding. only a limited number gohr exist in the north american managed population and the reproductive contribution of each individual is fundamental to maintaining adequate genetic diversity in this population. unequal gohr founder representation was the incentive for ai development in this species and its application has expanded the breeding management tools available to help ensure population sustainability. reproductive dysfunctions identified during formative gohr ai work limited the conception rate to 50%. by addressing and treating disorders that otherwise result in a failure to conceive or the inability to establish a pregnancy following ai, conception rates have improved to upwards of 66%. currently, the approach relies on a better understanding of the fundamental species-specific reproductive physiology of the female gohr and the similarities with that of its closest living domestic relative, the horse. conducting a bse that includes a comprehensive reproductive history and the collection of contemporary ultrasonography, endocrine and behavior data permits the gohr practitioner to assess the probability of a female being able to conceive and carry a calf to term. it enables improved diagnosis of causes of infertility and subfertility and development of an effective treatment plan to optimize ai outcome. anovulation emerged as the primary reproductive problem throughout original ai trials that led to a large-scale ovulation induction study establishing sustained release deslorelin, sucromate®, as a highly effective ovulation induction agent in gohr. previous successful ai procedures relied on behavioral cues from the female to time sedation/insemination and permitted < 24 hours of advanced notice for staff. currently, procedures can be timed a week in advance by using urinary hormone analysis to determine initiation of the follicular phase, and ovulation is ensured by treatment with deslorelin. intraluminal uterine fluid accumulation and/or postbreeding endometritis may be additional disorders that require additional treatments. ultrasonography of the uterus and urinary hormone dynamics of the luteal phase(s) are key to diagnosing these disorders in gohr and help delineate whether a pre or postconception failure event has occurred. the first application of oxytocin treatment in a gohr to resolve intraluminal uterine fluid accumulation was successful and was associated with a healthy full-term pregnancy, when earlier efforts failed. the systematic approach taken to identify and treat reproductive disorders in female gohr have not only broadened the application of ai to more individuals, many of those of high genetic value for population sustainability, but improved the chance of successful conception following a single insemination attempt. conflict of interest there are no conflicts of interest to declare. acknowledgement supported by grants from the institute of museum and library services (imls; mg-30-14-0072-14 and mg-246037-oms-20). authors extend extreme gratitude to the many veterinary, managerial, and rhino care staff at the facilities (center for conservation of tropical ungulates, cincinnati zoo & botanical garden, denver zoo, fort worth zoo, oklahoma city zoo, omaha’s henry doorly zoo and aquarium, zoo miami) where this work was conducted. their support, knowledge and participation have substantially contributed to advancement in assisted reproductive technology for the gohr. appreciation is also extended to the aza gohr species survival plan coordinator and genetic advisor for their input into sperm sires for the ai procedures. references 1. herrick jr: assisted reproductive technologies for endangered species conservation: developing sophisticated protocols with limited access to animals with unique reproductive mechanisms. biol reprod 2019;100:1158-1170. 2. allen wr: the development and application of the modern reproductive technologies to horse breeding. reprod dom anim 2005;40:310-329. 3. kasman lh, ramsay ec, lasley bl: urinary steroid evaluations to monitor ovarian function in exotic ungulates: iii. estrone sulfate and pregnanediol-3-glucuronide excretion in the indian rhinoceros (rhinoceros unicornis). zoo biol 1986;5:355-361. 4.berkeley ev, kirkpatrick jf, schaffer ne, et al: serum and fecal steroid analysis of ovulation, pregnancy and parturition in the black rhinoceros (diceros bicornis). zoo biol 1997;16:121-132. 5.radcliffe rw, czekala nm, osofsky sa: combined serial ultrasonography and fecal progestin analysis for reproductive evaluation of the female white rhinoceros (ceratotherium simum simum): preliminary results. zoo biol 1997;16:445-456. 6. patton ml, swaisgood rr, czekala nm, et al: reproductive cycle length and pregnancy in the southern white rhinoceros (ceratotherium simum simum) as determined by fecal pregnane analysis and observations of mating behavior. zoo biol 1999;18:111-27. 7. radcliffe rw, eyres ai, patton ml, et al: ultrasonographic characterization of ovarian events and fetal gestational parameters in two southern black rhinoceros (diceros bicornis minor) and correlation to fecal progesterone. theriogenology 2001;55:1033-1049. 8.stoops ma, pairan rd, roth tl: follicular, endocrine and behavioural dynamics of the indian rhinoceros (rhinoceros unicornis) oestrous cycle. reproduction 2004;128:842-856. 9.roth tl: a review of the reproductive physiology of rhinoceros species in captivity. int zoo yearb 2006;40:130-143. 10. stoops ma, campbell mk, dechant cj, et al: enhancing captive indian rhinoceros genetics via artificial insemination of cryopreserved sperm. anim reprod sci 2016;172:65-70. 11.stoops ma, atkinson mw, blumer es, et al: semen cryopreservation in the indian rhinoceros (rhinoceros unicornis). theriogenology 2010;73:1104-1115. 12. cristanelli mj, squires el, amann rp, et al: fertility of stallion semen clinical theriogenology 2021; 13: 400 processed, frozen and thawed by a new procedure. theriogenology 1984;22:39-45. 13.jasko dj, moran dm, farlin me, et al: pregnancy rates utilizing fresh, cooled and frozen-thawed stallion semen. proc am assoc equine pract 1992; p. 649-660. 14. thomassen r: insemination with stallion semen frozen in 0.5ml straws. reprod dom anim 1993;28:289-293. 15. hermes r, hildebrandt tb, goritz f: reproductive problems directly attributable to long-term captivity -asymmetric reproductive aging. anim reprod sci 2004;82/83:49-60. 16. hermes r, hildebrandt tb, walzer c, et al: the effect of long non-reproductive periods on the genital health in captive female white rhinoceroses (ceratotherium simum simum, c.s. cottoni). theriogenology 2006;65:1492-1525. 17. hermes r, goritz f, streich wj, et al: assisted reproduction in female rhinoceros and elephants current status and future perspective. reprod domes anim 2007;42:33-44. 18. hildebrandt tb, hermes r, walzer c, et al: artificial insemination in the anoestrous and the postpartum white rhinoceros using gnrh analogue to induce ovulation. theriogenology 2007;67:1473-1484. 19. hermes r, hildebrandt tb, walzer c, et al: estrus induction in white rhinoceros (ceratotherium simum). theriogenology 2012;76:1217-1223. 20. hermes r, goritz f, saragusty j, et al: reproductive tract tumours: the scourge of woman reproduction ails indian rhinoceroses. plos one 2014;9:92595. 21. stoops ma, west gd, roth tl, et al: use of urinary biomarkers of ovarian function and altrenogest supplementation to enhance captive breeding success in the indian rhinoceros (rhinoceros unicornis). zoo biol 2014;33:83-88. 22. stoops ma, o’brien jk, niederlander j, et al: administration of biorelease progesterone and estradiol or gnrh to induce estrous cycles and breeding in anestrous african white rhinoceroses (ceratotherium simum simum). proc 6th international society of wildlife endocrinology conference, 2017. p. 111. 23. stoops m, moresco a, larsen rs: clinical assessment and treatment of infertility in a greater one-horned rhinoceros (rhinoceros unicornis). clinical theriogenology 2019;11:497. 24. mccue pm: the problem mare: management, philosophy, diagnostic procedures, and therapeutic options. j equine vet sci 2008;28:619-626. 25. mccue pm: ultrasound evaluation of the non-pregnant mare. in: dascanio j, mccue p: editors. equine reproductive procedures. ist edition, hoboken; john wiley & sons: 2014. p. 26-31. 26. capiro jm, stoops ma, freeman ew, et al: effects of management strategies on glucocorticoids and behavior in indian rhinoceros (rhinoceros unicornis): translocation and operant conditioning. zoo biol 2014;33:131-143. 27. ginther oj: equine pregnancy: physical interactions between the uterus and conceptus. proc am assoc equine pract 1998; p. 73-104. 28. ginther oj, gastal el, gastal mo, et al: incidence, endocrinology, vascularity, and morphology of hemorrhagic anovulatory follicles in mares. j equine vet sci 2007;27:130-139. 29.mccue pm, magee c, gee ek: comparison of compounded deslorelin and hcg for induction of ovulation in mares. j equine vet sci 2007;27:58-61. 30. ferris ra, hatzel jn, lindholm arg, et al: efficacy of deslorelin acetate (sucromate) on induction of ovulation in american quarter horse mares. j equine vet sci 2012;32:285-288. 31. pennington pm, marshall kl, capiro jm, et al: ovulation induction in anovulatory southern white rhinoceros (ceratotherium simum simum) without altrenogest. conserv physiol 2019;7:coz033. 32. roth tl, schook mw, stoops ma: monitoring and controlling ovarian function in the rhinoceros. theriogenology 2018;109:48-57. 33. pennington pm, marshall kl, capiro jm, et al: pregnancies following long luteal phases in southern white rhinoceros (ceratotherium simum simum). zoo biol 2020,39:141-144. 34. ginther oj: embryonic loss in mares: incidence, time of occurrence and hormonal involvement. theriogenology 1985;23:77-89. 35. katila t: post-mating inflammatory responses of the uterus. reprod dom anim 2012;47:31.41. 36.troedsson mht, loset k, alghamdi am, et al: interaction between equine semen and the endometrium: the inflammatory response to semen. anim reprod sci 2001;3-4:273-278. 37. troedsson mht: breeding-induced endometritis in mares. vet clin north am equine pract 2006;22:705-712 38. daels pf, stabenfeldt gh, kindahl h, et al: prostaglandin release and luteolysis associated with physiological and pathological conditions of the reproductive cycle of the mare: a review. equine vet j 1989;21(suppl 8):29-34. 39. jasko dj, moran sm, farlin me, et al: effect of seminal plasma dilution or removal on spermatozoal motion characteristics of cooled semen. theriogenology 1991;35:1059-1067. 40. alghamdi as, foster dn, troedsson mht: equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (pmns) and improves the fertility of fresh semen inseminated into inflamed uteri. reproduction 2004;127:593-600. 41. palm f, walter i, budik, et al: influence of different semen extenders and seminal plasma on pmn migration and on expression of il-1ß, il-6, tnf-α and cox-2 mrna in the equine endometrium. theriogenology 2008;70:843-851. 42. troedsson mht, desvousges a, alghamdi as, et al: components in seminal plasma regulating sperm transport and elimination. anim reprod sci 2005;89:171-186. 43. stull cl, evans jw: oxytocin binding in the uterus of the cycling mare. j equine vet sci 1986;6:114-119. 44. pycock jf, newcombe jr: assessment of the effect of three treatments to remove intrauterine fluid on pregnancy rate in the mare. vet rec 1996;138:320-323. 45. cadario me, merritt am, archbald lf, et al: changes in intrauterine pressure after oxytocin administration in reproductively normal mares and those with a delay in uterine clearance. theriogenology 1999;51:1017-1025. 46. gutjahr s, paccamonti dl, pycock jf, et al: effect of dose and day of treatment on uterine response to oxytocin in mares. theriogenology 2000;54:447-456. 47. kotilainen t, huhtinen m, katila t: sperm-induced leukocytosis in the equine uterus. theriogenology 1994;41:629-636. 48. sieme h, bonk a, hamann h, et al: effects of different artificial insemination techniques and sperm doses on fertility of normal mares and mares with abnormal reproductive history. theriogenology 2004;62:915-928 49. samper j: breeding the problem mare by artificial insemination. proc am assoc equine pract 2008; p. 408-413. 50. cho cl, digiovanni sr, luter a, et al: oxytocin as an antidiuretic hormone ii. role of v2 vasopressin receptor. am j physiol 1995;269:f78-85. 51. joon kw, jeon us, kim gh, et al: antidiuretic action of oxytocin is associated with increased urinary excretion of aquaporin-2. nephrol dial transplant 2004;19:2480-2486. 52. li c, wang w, summer sn, et al: molecular mechanisms of antidiuretic effect of oxytocin. j am soc nephrol 2008;19:225-232. clinical theriogenology 2021; 13: 401 contact jessica cowley j.cowley@auburn.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 review report a review of small ruminant cesarean section: case selection, surgical techniques, care of the neonates, and postoperative care of the dam jessica cowley, jenna stockler, herris maxwell department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa abstract cesarean section is an important life-saving surgical procedure frequently performed by small ruminant practitioners. knowledge of normal parturition and indications for obstetrical intervention are necessary for successful outcomes. if a doe or ewe is observed in active labor for more than an hour without signs of progression toward delivery of a fetus, obstetrical examination is indicated to guide appropriate interventions. it is often possible to relieve a dystocia without surgical intervention if the cervix is adequately dilated and the dam’s pelvis is of sufficient size to allow manipulation of the fetus. if vaginal delivery cannot be performed safely, a cesarean section or fetotomy is indicated. the surgery is generally performed with sedation and local anesthesia, with a lumbosacral epidural preferred by the authors. the dam may be positioned in right lateral recumbency, standing, or dorsal recumbency, based on the surgeon’s preference and specifics of the case. the abdomen is entered routinely, the gravid uterus identified, and gently exteriorized through the abdominal incision. a hysterotomy incision is made over the greater curvature of the uterus to allow removal of the fetus(es). the uterus is closed with absorbable suture using an inverting pattern. the abdominal incision is closed routinely. systemic antibiotics, ecbolic agents, and analgesic agents are recommended for the dam postoperatively. survival of the dam is reported at 81–99% with post-surgical complications encountered in 33–77% of cases. females may be retained for future breeding following surgery, with 84–100% of females reported to become pregnant in the subsequent breeding season. keywords: hysterotomy, ewe, doe, dystocia, c-section parturition parturition is divided into three stages. stage one, the preparatory stage, is characterized by the initiation of myometrial contractions, and lasts from 2 to 12 hours in small ruminants.1 during this stage, the female will usually leave the group, appear restless, and may urinate frequently. cervical relaxation occurs during stage one and passage of the cervical plug may be observed. stage two labor begins when a fetus enters the pelvic portion of the reproductive tract and is generally accompanied by forceful abdominal contractions, rupture of the chorioallantoic membranes, and expulsion of the allantoic fluid. stage two ends when the last fetus is expelled by forceful abdominal muscle contractions of the dam.2 during stage two, if the fetus is in cranial longitudinal presentation with forelimbs extended, the amniotic membrane will protrude from the vulva and rupture, followed shortly by the appearance of fetal forelimbs and head. caudal longitudinal presentation is not uncommon and may be considered normal if both hind limbs are extended and delivery progresses normally.3 when multiple fetuses are present, deliveries may occur in quick succession or the female may rest briefly in between delivery of subsequent fetuses.4 the third stage of labor begins immediately after delivery of the final fetus and concludes with passage of the fetal membranes.2 expulsion of the membranes usually occurs within four hours of normal parturition.2 if delivery was uncomplicated, retention of fetal membranes is of little concern until 12–18 hours post-partum.4 lochia, the brown-red discharge from the involuting postpartum uterus, may be present for up to three weeks.5,6 dystocia dystocia is defined as slow or difficult labor.7 dystocia is present when labor is prolonged, interrupted, or when delivery of the fetus(es) cannot be completed in a timely manner without assistance. any female that fails to progress normally through the stages of parturition, either from stage one to stage two or mailto:j.cowley@auburn.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9620 citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 71 during stage two labor, should be examined. if a doe or ewe is in active labor, without producing a fetus, for longer than thirty minutes to one hour, assessment and obstetrical intervention is indicated.8 dystocia in small ruminants is relatively uncommon. historically, in well managed herds less than 5% of births require assistance.3 current normal dystocia rates in large herds has not been reported. the primary causes of dystocia differ between sheep and goats. in ewes, insufficient cervical dilation is reported as the cause of dystocia in 14–44% of cases presented.9–12 in does, feto-pelvic disproportions reportedly account for 23–52% of dystocia cases.11,13,14 other factors identified as causes of dystocia include uterine torsion, vaginal prolapse, fetal malformation or malposition, and metabolic disorders.9 a significant increase in the occurrence of dystocia has been reported in ewes when diagnosed with pregnancy toxemia.15 dystocia can often be relieved by trans-vaginal manipulation of the fetus. manipulation requires that the dam’s pelvis be large enough to admit a hand for obstetrical assistance and the cervix be sufficiently dilated to allow for correction of errors in presentation, position, or posture.16 epidural anesthesia administration prior to vaginal palpation in small ruminants is likely to decrease the likelihood of uterine, cervical, or vaginal tears occurring by reducing straining by the dam.17–19 successful resolution of dystocia and delivery of the fetus(es) occur frequently on farm settings. cases not easily resolved on farm require further interventions such as fetotomy, cesarean section (c-section), or euthanasia. all methods can be performed on farm, or preferentially in a hospital setting.4 in the cases of small ruminant dystocia presented to referral facilities, 43–95% required c-section.12–14,20,21 if a fetotomy is to be attempted, it is recommended to perform a subcutaneous fetotomy instead of a percutaneous approach.3 a subcutaneous fetotomy is safer for the dam since instruments are not inserted into the birth canal potentially damaging friable maternal tissues.2,3 this type of fetotomy is performed by exteriorizing a fetal limb and making a skin incision around the limb, preferably above the carpus.22 the skin incision is continued up the medial side of the fetal limb but it is not necessary to extend the incision so far as to insert the blade into the vagina.22 the practitioner’s hand is used to undermine the skin to the level of the shoulder blade; the muscular and tendinous attachments are broken down, and the limb is pulled free.2 decapitation and neck amputation are easily performed in a dead and emphysematous fetus to allow for further extraction of the fetal body.4 it is an important consideration when performing small ruminant obstetrical intervention that many causes of abortion may be zoonotic and personal protective equipment is recommended.4 anesthesia anesthesia for c-section procedures is case and surgeon dependent, but generally utilizes a combination of sedation and local or regional anesthesia. sacrococcygeal or lumbosacral epidural anesthesia is advantageous as it can induce recumbency while providing anesthesia to the paralumbar fossa or abdominal midline.19 the proximal paravertebral, distal paravertebral, line block, or inverted l block can also be used for anesthetizing the paralumbar fossa. a line block is the most appropriate technique for the ventral midline celiotomy approach of performing a c-section. patient condition, demeanor, and surgical approach dictates the choices for sedative agents or uncommonly the need for general anesthesia. at the authors’ hospital, administration of a lumbosacral epidural is commonly the first step in surgical preparation for small ruminants undergoing cesarean section. epidurals in sheep and goats can be performed as in cattle at the sacrococcygeal space (s5-co1) but are also easily performed and reliably effective when administered at the lumbosacral space.19 following sterile preparation, the depression overlying the lumbosacral space is identified by palpation on midline from along a transverse line drawn from the wings of the ilium and identifying the dorsal spinous processes of the last lumbar vertebra and the first sacral vertebra. an 18or 20-gauge 25 millimeter (mm) needle is usually sufficient to administer 1–2  milliliters (ml) per 10 kilograms (kg) of 2% lidocaine (vetone®, mwi, boise, id) into the epidural space using the hanging drop technique (figure 1).23 a longer needle, such as figure 1. palpation of the landmarks for a lumbosacral epidural. http://dx.doi.org/10.58292/ct.v15.9620 72 citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 a 20-gauge 75 mm or spinal needle, may be required in larger does and ewes.24 epidural administration of 2% lidocaine at this dose and volume will provide analgesia of the caudal abdomen to the level of the umbilicus sufficient for either the flank or ventral midline approach; the animal will become recumbent.24–26 should the subarachnoid space be penetrated while attempting to place the needle for administration of the lumbosacral epidural, flow of spinal fluid from the needle will be observed. in that instance, the needle may be either repositioned, or half of the dose originally planned for epidural anesthesia can be safely administered into the subarachnoid space.19 other reports describe using xylazine (anased® injection, akorn, lake forest, il) at 0.07 milligram/ kilogram (mg/ kg) with or without lidocaine to extend the duration of epidural anesthesia.24 if the lumbosacral epidural fails to provide adequate analgesia for surgery, paralumbar fossa anesthesia can be supplemented with either an inverted l block or a line block. alternatively, distal and proximal paravertebral blocks have been described for small ruminants using smaller volumes of lidocaine compared to cattle.25 two percent lidocaine can be diluted 1:1 using normal saline to increase the volume for distribution of the local anesthetic agent. a line block is commonly chosen by some practitioners for the ventral midline approach and inhalant isoflurane can be used to maintain anesthetic depth if necessary. small ruminant practitioners should be aware that sheep and goats are more sensitive to lidocaine toxicity compared to cattle. the reported safe doses of lidocaine range from 4 to 10 mg/kg.2,24,25,27,28 at the author’s hospital, maximum levels of 2% lidocaine used in small ruminants is 6 mg/kg.4 convulsions due to iv administration of lidocaine have been reported to occur as low as 3.7 mg/kg in adult ewes, but toxic effects were not apparent when administered subcutaneously until an average of 12 mg/kg in goat kids.29,30 an example case of a local anesthetic protocol for a 25 kg doe, of which the maximum safe dose of lidocaine would be 7.5 ml (150 mg), would be to administer 2.5 ml of 2% lidocaine as a lumbosacral epidural and use 5 ml of 2% lidocaine diluted 1:1 with normal saline to allow 10 ml of solution to be used as a line block or regional block. in addition to local and regional anesthesia, some level of sedation is advised. midazolam (midazolam hydrochloride, west-ward, eatontown, nj) is commonly used beginning at 0.2 mg/kg and increasing if necessary to 0.4 mg/kg.31 xylazine is usually not recommended as a first choice because fetal hypoxemia, respiratory acidosis, increased uterine contractions, and decreased uterine blood flow have been associated with its use.32 if necessary, xylazine may be used at low doses (0.01–0.05 mg/kg) after removing any live fetuses.31 placement of a jugular catheter for venous access facilitates drug re-dosing and allows for intravenous fluid support during and following surgery. surgical procedure c-section in the small ruminant is performed utilizing a left paralumbar incision or a ventral midline approach. the surgical approach chosen must include consideration of the health of the dam, the risk to the fetus(es), and the availability of support personnel. the best visualization and exposure of the uterus is possible with a ventral midline approach and is preferred when uterine contents may be contaminated.24 when ruminants are positioned in dorsal recumbency, respiratory compromise from abdominal pressure on the diaphragm can occur. some clinicians prefer general anesthesia and endotracheal intubation when performing a ventral midline approach to allow for controlled ventilation.16 a paralumbar fossa approach allows adequate visualization for most cases with less compromise to patient ventilation.2 mild respiratory compromise can be minimized by providing flow-by nasal oxygen and monitoring for cyanosis of the mucous membranes. flank approach the left paralumbar fossa is the most common surgical approach chosen.4,33 in this approach, the patient is positioned in modified right lateral recumbency tilted at a ~30 degree angle toward the surgeon. in a field setting, if patient demeanor and systemic stability allows, the patient can be restrained in a standing position. often, a show trimming stand placed against a wall is used to restrain the dam and allows a convenient operating height for the surgeon.34 the left flank is surgically clipped and prepped. a vertical or slightly oblique 15–25 centimeter (cm) skin incision is made beginning approximately 10 cm below the transverse processes of the lumbar vertebrae centered in the paralumbar fossa (figure 2).24 the skin and the cutaneous trunci muscle are incised sharply. occasionally, if a small fetus is expected, the muscles can be ‘gridded’ or parted along the fiber plane, but this approach reduces the degree of surgical exposure and is seldom used by these authors.35 in routine cases, the external and internal abdominal oblique muscles are incised with either a scalpel blade or scissors (figure 3). caution must be utilized on incision of the transversus abdominis muscle and peritoneum to avoid accidental incision of the rumen due to its proximity to the incision line. any accidental incision into figure 2. left flank approach paralumbar fossa incision. http://dx.doi.org/10.58292/ct.v15.9620 citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 73 the rumen should be closed first with a simple continuous pattern.35 second, after copious lavage of the region and changing of surgical gloves, the rumen incision is oversewn utilizing absorbable suture in a cushing pattern.35 opening and entering of the peritoneum is associated with the sound of air being drawn into the abdomen due to the negative pressure in the abdomen. sheep and large-breed goats may have abundant retroperitoneal fat that complicates the surgical approach.4 after entering the abdomen, the rumen is moved cranially to facilitate access to the uterus (figure 4). carefully grasping a fetal limb through the uterine wall allows the surgeon to draw the uterus to the incision and gently exteriorize a uterine horn containing a fetus (figure 5).16 exteriorization of the uterus can be facilitated with a tocolytic dose of epinephrine (epinephrine 1:1000, vetone, boise, id) administered at 0.5–1 mg (0.5–1 ml of a 1:1000 solution) intramuscularly depending on the size of the ewe or doe.2 following exteriorization of the uterus, the surgeon can isolate the organ using sterile towels or lap sponges and protect the abdominal cavity from contamination following hysterotomy and extraction of the fetus(es) (figure 6). when possible, the hysterotomy incision is best made over the greater curvature of the uterus to minimize hemorrhage (figures 7 & 8).36 the incision can be made directly over a fetal limb or a fetal head.4 to minimize uncontrolled uterine tears, the hysterotomy incision should be long enough to allow for delivery of the largest portion of the fetus. following removal of the first fetus, the surgeon’s hand is then inserted into the hysterotomy site to retrieve the remaining fetuses. the surgeon’s hand can be directed caudally toward the uterine bifurcation and into the contralateral horn to allow retrieval of all figure 3. incision of the transversus abdominis muscle and peritoneum. figure 4. after the rumen has been moved cranially, the uterus is grasped and brought to the abdominal incision. figure 5. a fetal limb is grasped through the uterine wall to aid in exteriorization of the organ. http://dx.doi.org/10.58292/ct.v15.9620 74 citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 other fetuses.16 in some cases, a second hysterotomy may be required to reach a fetus in the opposite uterine horn. abdominal contamination is inevitable in many c-section cases but all precautions should be taken to mitigate. the hysterotomy site should be kept fully exteriorized and isolated with sterile towels or lap sponges to ensure uterine fluid does not drain back into the peritoneal cavity. this is especially important if a dead fetus was removed at surgery (figure 9). unattached portions of the fetal membranes may be trimmed to facilitate uterine closure, but cotyledonary figure 6. a fetal limb is grasped through the uterine wall to aid in exteriorization of the organ. figure 7. hysterotomy is made over the greater curvature of the uterine horn. figure 8. the incision is made long enough for the fetus to be extracted without uncontrolled uterine tearing occurring. figure 9. maintenance of exteriorization of the hysterotomy site to prevent abdominal contamination. http://dx.doi.org/10.58292/ct.v15.9620 citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 75 attachments should not be forcefully detached.2 the hysterotomy site is closed using a partial thickness inverting suture pattern, such as the utrecht, cushing, or lembert to achieve a tight serosa to serosa seal (figures 10–20).37 sutures should not penetrate the uterine lumen. absorbable suture of size 0 or 1 on an atraumatic taper needle should  be  used; poliglecaprone 25 (monocryl™, ethicon), polydioxanone (pds™, ethicon), or chromic gut (chromic gut, ethicon) are common choices (figure 21). a double-layer closure (“over-sew”) of the hysterotomy site is indicated if the uterine contents were severely contaminated or if the uterine wall is friable (figure 22). the use of intrauterine boluses or antibiotics is controversial and not recommended due to the risk of endometrial damage occurring.4 following initial closure, the uterus should be carefully examined for previously undetected uterine tears and excessive hemorrhage. after uterine closure, the uterine serosal surface should be lavaged with sterile isotonic fluid to remove large blood clots and debris. repeated abdominal lavage and suction may be indicated if extrauterine contamination of the peritoneal cavity occurred. sodium carboxymethylcellulose in a 1% solution (carboxymethylcellulose sodium salt, high viscosity, mp biomedical llc, solon, oh) is sometimes instilled into the abdomen to decrease the formation of adhesions.38,39 infusion doses range from 30 ml total up to 14 ml/kg.38,39 an alternative to sodium carboxymethylcellulose is to use 30 ml of glycerol (glycerin u.s.p. 99.5%, humco labs, texarkana, tx) in 1 liter (l) of saline to make a 3% solution.37 the abdominal incision through the paralumbar fossa should be closed in three layers: 1) the peritoneum and transversus abdominis muscle, 2) the internal and external abdominal figure 10. beginning knot of utrecht pattern as described by roberson, 2004.48 figure 11. beginning knot of utrecht pattern as described by roberson, 2004.48 figure 12. beginning knot of utrecht pattern as described by roberson, 2004.48 http://dx.doi.org/10.58292/ct.v15.9620 76 citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 oblique muscles with the subcutaneous fat, and 3) skin.16 size 1 or 2 absorbable suture, such as coated polyglactin 910 (vicryl, ethicon), polydioxanone (pds™, ethicon), or chromic gut (chromic gut, ethicon), in a continuous pattern is used for the peritoneum and muscle layers. size 0 or 1 non-absorbable suture in a ford-interlocking pattern is frequently used for skin closure. ventral midline approach the ventral midline surgical approach provides better uterine exposure than the more commonly used left paralumbar fossa approach.16 this approach is used most when severe contamination of uterine contents is suspected. the patient is positioned in dorsal recumbency and the ventral abdomen from the umbilicus to cranial udder is surgically clipped and prepped. a skin incision is made just cranial to the udder and extended cranially 20–25 cm.2,24 identification of the subcutaneous abdominal veins (milk vein) is imperative as these veins are large in late pregnancy.24,40 the subcutaneous tissue is bluntly dissected until the linea alba is identified. the abdominal wall is tented, and a stab incision is made through the linea alba taking care to avoid incising any underlying viscera. the incision can then be safely extended using mayo scissors. upon entering the abdomen, the greater omentum and abdominal viscera will need to be moved cranially to allow access to the uterus.24 a fetal limb can be gently grasped through the uterine wall and the entire organ exteriorized from the abdomen and isolated using lap sponges or sterile towels. as discussed above, the use of a tocolytic dose of epinephrine will facilitate uterine exteriorization. after the uterus is packed off to prevent abdominal contamination, an incision is made over the greater curvature of the uterus to minimize hemorrhage. the uterus should be kept exteriorized and uterine contents drained away from the incision line if contaminated uterine contents are present. the uterus is closed as described for the flank approach utilizing an inverting pattern with absorbable suture on an atraumatic needle. once closed, the uterus is lavaged, cleaned of any blood clots and debris, and examined extensively for tears. abdominal lavage as described for the paralumbar approach is often advisable if labor has been protracted or peritoneal contamination is suspected. the ventral abdomen is closed using size 0, 1, or 2 absorbable suture with an atraumatic needle in a routine method preferred by the surgeon. commonly, a simple continuous pattern utilizing polyglactin 910 (vicryl, ethicon) is chosen. closure must be secure enough to allow healing of the linea alba to prevent herniation of abdominal contents. the subcutaneous space is closed using absorbable suture in a continuous pattern. size 1 or 3 non-absorbable suture in a ford interlocking pattern or simple interrupted suture pattern is used for skin closure. post-partum care postoperatively, patient status should be assessed, and dehydration and electrolyte abnormalities addressed. postsurgical antibiotics, especially if there was a dead fetus, are figure 13. beginning knot of utrecht pattern as described by roberson, 2004.48 figure 14. utrecht suture pattern for uterine closure. http://dx.doi.org/10.58292/ct.v15.9620 citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 77 frequently necessary. currently no antibiotics are labeled for the post-operative treatment for food producing animals and regulations for extra-label drug use must be followed. commonly used antibiotics at the author’s hospital include ceftiofur sodium (naxcel®, zoetis, kalamazoo, mi) at 2–4 mg/kg intravenously (iv) or intramuscularly (im) every 12–24 hours, oxytetracycline (liquamycin®, zoetis, kalamazoo, mi) at 20 mg/kg im or subcutaneously (sq) figure 15. utrecht suture pattern for uterine closure. figure 16. utrecht suture pattern for uterine closure. figure 17. utrecht suture pattern for uterine closure. figure 18. ending knot of utrecht as described by roberson, 2004.48 http://dx.doi.org/10.58292/ct.v15.9620 78 citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 every 24–48 hours, or florfenicol (nuflor®, merck animal health, rahway, nj) at 40 mg/kg sq once.41 post-partum care of the dam should include pain management. as mentioned for post-operative antibiotics, no drugs have food and drug administration approval for this use, and appropriate precautions for extra-label drug use must be a consideration. non-steroidal anti-inflammatories (nsaid) used include flunixin meglumine (banamine®, intervet/ merck, madison, nj) at 1.1 mg/kg iv every 12–24 hours for three days or meloxicam at 1 mg/kg orally every 24 hours.41 meloxicam may be useful if the patient is not maintained in hospital.41 opioids such as butorphanol (torbugesic®, zoetis, kalamazoo, mi) at 0.1 mg/kg sq every 6–8 hours or morphine (morphine sulphate injection, hikma, eatontown, nj) at 0.1–0.5 mg/kg sq every 6–8 hours may be administered for additional pain management for up to 48 hours.41 most patients are standing and eating quickly after surgical recovery and non-steroidal anti-inflammatories are often discontinued three days after surgery, depending on assessment of the dam’s pain level. passage of fetal membranes should occur within 12 hours after a normal delivery. after a c-section is performed, passage of the necrotic membranes is imperative for hysterotomy site healing. oxytocin (vetone, boise, id) is used as an ecbolic agent at 5–10 international units (iu) im, sq, or iv up to every 3 hours until the fetal membranes have passed.2,16 prompt management of retained fetal membranes will reduce the likelihood of other more severe complications occurring. surgical complications following emergency c-section postsurgical complications reportedly occur in 33–77% of cases.9,12,13 of all figure 19. ending knot of utrecht as described by roberson, 2004.48 figure 20. ending knot of utrecht as described by roberson, 2004.48 figure 21. uterus after a utrecht closure has been placed. http://dx.doi.org/10.58292/ct.v15.9620 citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 79 complications, retained fetal membranes is reported most commonly at 13–45%.9,13 other postoperative complications include delayed incisional healing, incisional dehiscence, vaginal prolapse, anorexia, and anemia.13 the preoperative state of the dam following prolonged dystocia impacts the likelihood of developing complications.9 in one study comparing the outcome of ewes following vaginal delivery or elective c-section, only 8% of the ewes subjected to c-section developed postoperative complications.34 another study reported that the development of complications was higher in cases that did not receive postoperative antibiotics.13 reports of dam survival postoperatively are high at 81–99%.9,12–14,20,34 historically, a difference has been observed in dam survivability if live or autolyzed fetuses were present at the time of surgery. dams with live fetuses had a 98% survival rate and dams with autolyzed fetuses present had a survival rate of 57%.10 this difference in survival rate has not been duplicated in other reports.9,12,20,34 ewes that present with a history of concurrent disorders, such as vaginal prolapse or pregnancy toxemia, are reported to have a c-section survival rate of 68.5% compared to ewes with no concurrent disorders which had a survival rate of 96.2%.9 in one report, all females presenting with pregnancy toxemia died.13 other reports revealed a 70%–75% mortality rate for does diagnosed with pregnancy toxemia.42,43 for ewes that underwent c-section, if presenting with a history of repeated vaginal prolapse, or had a vaginal prolapse at the time of presentation, there was a 10-fold increased likelihood of a c-section having to be performed.10 for ewes undergoing c-section, there was a significant association with survival when presenting with no vaginal prolapse.10 future fertility of interest to producers is the future fertility of the dam after a c-section. in retrospective studies, small numbers have been followed to re-breeding with reports of 84–100% of postsurgical cases achieving pregnancy in the next breeding season.9,13 in a report comparing vaginal delivery and elective c-section, there was no difference in pregnancy rates between ewes that vaginally delivered or underwent c-section the prior year, with a pregnancy rate of 89% in both groups.34 neonatal care reported neonate survival rates range from 42 to 82% postoperatively.9,12–14 duration of labor for greater than 12 hours was significantly associated with fetal mortality.9 in a study comparing vaginally delivered lambs versus elective c-section delivered lambs, there was no postoperative or long term differences between survivability of the lambs, emphasizing the need for timely intervention of dystocia incidences.34 in a recent study comparing kid survival rates after delivery from a c-section performed under sedation or under general anesthesia, kid survival rate was significantly higher in the sedation (90.4%) group compared to the general anesthesia group (66.7%).26 when neonates are first delivered from the uterus, they may need resuscitation depending on the duration of labor and the amount or type of anesthetic administered to the dam. oxygen supplementation should be available. depending on the dose and type of sedation administered to the dam, a reversal agent can be given to the neonate. midazolam is reversed using flumazenil (baxter healthcare, deerfield, il) at 0.01 to 0.02 mg/kg intravenously.44 the effects of xylazine are reversed using atipamazole hydrochloride (antisedan®, zoetis inc, kalamazoo, mi) at 0.1 mg/kg intramuscularly.31 neonates should receive 50 ml/kg of colostrum within the first two hours after birth and a total of 200 ml/kg in the first 24 hours.4 commercial small ruminant colostrum replacer products are preferred if available, but bovine colostrum replacers may be used for adequate igg transfer.45 total protein levels are assessed one to two days after birth for diagnosis in transfer of passive immunity.46 total protein values of 5.1 to 5.4 grams/ deciliter or below have been correlated to failure in the transfer of passive immunity in kids.46,47 conclusion cesarean section is commonly performed in small ruminants and can be necessary to ensure the viability of both the dam and neonate(s). a preoperative assessment of the dam is always necessary, and the welfare of the ewe/doe and the lamb(s)/kid(s) must guide the choice of technique and post-operative management. mastery of this procedure is important for any veterinarian that treats small ruminants. acknowledgements the authors would like to thank jennie c. hill for her creation of the drawings used in the publication. figure 22. second layer uterine closure utilizing a modified cushing pattern. http://dx.doi.org/10.58292/ct.v15.9620 80 citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 references 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2017:561–567. 36. wallace c: a technique for performing cesarean section in goats. vet med small anil clin 1982;77:791–793. 37. baird an: surgery of the uterus. cattle, sheep, and goats. in: wolfe df, moll hd: editors. large animal urogenital surgery. baltimore, md; williams & wilkins: 1999. p. 417–420. http://dx.doi.org/10.58292/ct.v15.9620 https://doi.org/10.1111/j.1751-0813.1990.tb07404.x https://doi.org/10.1111/rda.13855 https://doi.org/10.1016/0007-1935(89)90118-8 https://doi.org/10.1136/vr.101370 https://doi.org/10.2460/javma.2004.224.275 https://doi.org/10.1016/0921-4488(92)90059-d https://doi.org/10.1016/j.smallrumres.​2015.10.017 https://doi.org/10.1016/j.smallrumres.​2015.10.017 https://doi.org/10.1136/inpract.22.9.546 https://doi.org/10.21423/aabppro20064668 https://doi.org/10.1136/inpract.21.1.2 https://doi.org/10.2460/javma.22.05.0191 https://doi.org/10.2460/javma.22.05.0191 https://doi.org/10.1016/j.cvfa.2012.11.006 https://doi.org/10.1016/j.cvfa.2012.11.006 https://doi.org/10.1213/00000539-198909000-00002 https://doi.org/10.1213/00000539-198909000-00002 https://doi.org/10.1292/jvms.58.211 https://doi.org/10.1016/j.theriogenology.2012.11.001 citation line: clinical theriogenology 2023, 15, 9620, http://dx.doi.org/10.58292/ct.v15.9620 81 38. ewoldt jm, anderson de, hardy j, et al: evaluation of a sheep laparoscopic uterine trauma model and repeat laparoscopy for evaluation of adhesion formation and prevention with sodium carboxymethylcellulose. vet surg 2004;33:668–672. doi: 10.1111/j.1532-950x.2004.04090.x 39. moll hd, wolfe df, schumacher j, et al: evaluation of sodium carboxymethylcellulose for prevention of adhesions after uterine trauma in ewes. am j vet res 1992;53:1454–1456. 40. mansour m, wilhite r, rowe j: the pelvis and reproductive organs. in: mansour m, wilhite r, rowe j: editors. guide to ruminant anatomy: dissection and clinical aspects. hoboken; john wiley & sons: 2017:168. 41. fajt v, brown kr, pugh dg: appendix 1 – commonly used drugs and veterinary feed directive in sheep, goats, and cervids. in: pugh dg, baird an, edmondson ma, et al: editors. sheep, goat, and cervid medicine. 3rd edition, oxford; elsevier: 2021:517–538. 42. simpson km, taylor jd, streeter rn: evaluation of prgonostic indicators for goats with pregnancy toxemia. j am vet med assoc 2019;254:859–868. doi: 10.2460/javma.254.7.859 43. lima ms, silveira jm, carolino l, et al: usefulness of clinical observations and blood chemistry values for predicting clinical outcomes in dairy goats with pregnancy toxaemia. ir vet j 2016;69:16. doi: 10.1186/s13620-016-0075-4 44. seddighi r, doherty tj: field sedation and anesthesia of ruminants. vet clin north am food anim pract 2016;32:553–570. doi: 10.1016/j.cvfa.2016.05.002 45. berge a, hassid g, leibovich h, et al: a field trial evaluating the health and performance of lambs fed a bovine colostrum replacement. j anim res nutr 2018;3:1:6. 46. batmaz h, kaçar y, topal o, et al: evaluation of passive transfer in goat kids with brix refractometer and comparison with other semiquantitative tests. turk j vet anim sci 2019;43:596–602. doi: 10.3906/vet-1905-31 47. o’brien j, sherman d: field methods for estimating serum immunoglobulin concentrations in newborn kids. small rumin res 1993;11:79–84. doi: 10.1016/0921-4488(93)90120-7 48. roberson jr: surgical considerations. in: fubini sl, ducharme ng: editors. farm animal surgery. 1st edition, st. louis, mo; saunders: 2004:58–74. http://dx.doi.org/10.58292/ct.v15.9620 https://doi.org/10.1111/j.1532-950x.2004.04090.x https://doi.org/10.2460/javma.254.7.859 https://doi.org/10.1186/s13620-016-0075-4 https://doi.org/10.1016/j.cvfa.2016.05.002 https://doi.org/10.3906/vet-1905-31 https://doi.org/10.1016/0921-4488(93)90120-7 introduction sperm acrosome associated 3 (spaca3, also known as sperm protein reactive with antisperm antibody (sprasa) and sperm lysosome-like protein 1 (sllp1), is a unique, intra-acrosomal, nonbacteriolytic, conventional-type lysozyme-like protein of mammalian sperm.1,2 sperm acrosome associated 3 is expressed on the inner acrosomal membrane of human, cattle, sheep, and deer sperm.3 in addition to other testis specific lysozyme-like proteins, spaca3 has been identified as a biomarker for male fertility.4-7 sperm acrosome associated 3 is also reported to have a role in sperm-egg plasma membrane adhesion and fusion during fertilization in humans1 and mice.8 sperm acrosome associated 3 might have a role in female reproduction. women experiencing infertility have higher concentrations of spaca3 antibodies than fertile women.9 additionally, female mice immunized against spaca3 had profound infertility.9 in cattle, dogs, and cats, spaca3 is expressed in ovarian follicles at all stages of development and localized to the ooplasm and granulosa cells with weak staining in theca cells.9 in a preliminary study, spaca3 expression was examined in the equine ovary of 3 horses.10 the objective of the current study was to confirm spaca3 expression in the equine ovary from a larger sample size consisting of both domesticated and feral mares materials and methods both domesticated mares (n = 8) and feral mares (n = 8) were used in this study. domesticated mares (3 14 years old) were pastured on a ranch located in stayton, oregon. each pasture contained a 4.9 x 7.3 meter, 3-sided shelter. feral mares (3 years sperm acrosome associated 3 protein expression in equine primordial, primary, secondary, and tertiary follicles brynley cozzi, zahra kiesler, michelle kutzler department of animal and rangeland sciences, college of agricultural science oregon state university, corvallis, or abstract the objective of this study was to characterize the protein expression of sperm acrosome associated 3 (spaca3) in the equine ovary. formalin-fixed and paraffin-embedded ovarian sections from 16 horses were processed for routine immunohistochemistry for spaca3. representative images were digitally captured at 400 x magnification. in all mares, spaca3 was expressed in granulosa cells of all stages of follicles. expression of spaca3 in all equine follicular stages suggests that this may be a permanent immunosterilant target for the management of feral horse herds. additional research is needed to determine if horses can produce a robust humoral response to a spaca3 vaccine to induce sustained infertility. keywords: granulosa cell, horse, ovary, sperm acrosome associated 3 protein old) were maintained at the warm springs herd management area located in harney county, oregon. the procedures were conducted under a protocol approved by the institutional animal care and use committee of oregon state university (protocol #3924). ovaries were obtained via a standing colpotomy performed by an experienced veterinarian. briefly, feed was withheld for 36 hours prior to surgery. for sedation and analgesia, detomidine hydrochloride (0.02 0.04 mg/kg), butorphanol tartrate (0.01 0.04 mg/kg), and xylazine hydrochloride (0.9 1.2 mg/ kg) were given intravenously. surgical preparation included wrapping the tail with gauze and tying it up to keep it out of the surgical field, transrectal palpation to manually evacuate the feces, scrubbing the perineal area with chlorhexidine (vet solutions, inc., bedford, tx), and flushing the vagina with very dilute povidone iodine. an incision in the anterior dorsolateral wall of the vagina was made, 2% lidocaine was injected into each ovarian pedicle for local analgesia, and the ovaries were removed using a chain ecrasure. the vaginal incision was left to heal by second intention. for postsurgical analgesia, flunixin meglumine was given intravenously (1.2 -1.5 mg/kg) and buprenorphine hydrochloride (10 mg) was given subcutaneously. additionally, each mare received intramuscularly the long-acting antibiotic ceftiofur crystalline-free acid (6.6 mg/kg). ovaries were hemi-sectioned, fixed in 10% neutral buffered formalin, and paraffin embedded. sections were serially sectioned (4 µm) on charged slides for routine immunohistochemistry for spaca3. slides from all mares were processed in 1 experiment clinical theriogenology 2021; 13: 85 to avoid variations. briefly, slides were deparaffinized in xylene and rehydrated in a graded ethanol series (100, 75, and 50%). antigen retrieval was conducted by incubating sections in a citrate buffer, target retrieval solution #s1699 (dako north america inc., carpinteria, ca) in a microwave for 10 minutes and cooled for 20 minutes. slides were then washed in buffer (dako north america inc., wash buffer #s3006) and tissue-specific endogenous peroxidases were inhibited by incubating slides in 3% hydrogen peroxide. slides were washed again and nonspecific binding was blocked with serum-free protein (protein block serum-free ready to use, #x0909, dako north america inc.) for 20 minutes at room temperature. subsequently, slides were tapped off and the primary antibody (#21137-1ap, proteintech, rosemont, ca) diluted 1:200 (background reducing components, #s3022, dako north america inc.) was applied to sections for 105 minutes at room temperature. a fusion protein with the following sequence was used to produce primary antibodypyagvclayftsgfnaaaldyeadgstnngifqinsrrwcsnltpnvpnvcrmycsdllnpnlkdtvicamkitqepqglgyweawrhhcqgkdltewvdgcdf. primary antibody reactivity was confirmed by the manufacturer in the mouse testis and was also confirmed in a preliminary experiment in the equine testis. specificity of immunostaining was verified by replacing the primary antibody with negative control rabbit serum (negative control rabbit igg, #nc495h, biocare medical, pacheco, ca). slides were washed in buffer and a secondary antibody (one step horseradish peroxidase-conjugated polymer antirabbit igg, #ih-8064-osu-15, immuno bioscience, mukilteo, wa) was applied to sections for 30 minutes at room temperature. the secondary antibody was washed off and novared (#sk4800, vector laboratories inc., burlingame, ca) was applied to sections for 5 minutes at room temperature. sections were then counter stained in hematoxylin, dehydrated in a graded series of ethanol (50, 75, and 100%), moved through a series of 3 xylene baths and cover slipped. slides were evaluated by a single observer at 400 x magnification with a bright-field microscope (leica dm4000b, leica microsystems inc. buffalo grove, il). representative images from each ovary were electronically captured using a digital camera (qimaging, qicam 12-bit, #qic-f-m-12-c, surrey, bc, canada) and image capture software (qcapturepro, surrey). the cellular expression on spaca3 was recorded for primordial, primary, secondary, and tertiary follicles. results spaca3 was localized (figure 1) to the sperm acrosomes in the equine testis, and to the pregranulosa cells of primordial follicles (figure 2), and to the granulosa cells of primary (figure 2a), secondary (figure 2b) and tertiary follicles (figure 2c) of all equine ovaries examined. there was no positive staining in any other cell type. in addition, slides stained with the universal negative did not have any positive staining (figures 1 and 2). figure 1. spaca3 immunoexpression (arrow) in the equine testis is localized to the sperm acrosome (scale bar = 20 µm). negative control in upper left inset. figure 2a. spaca3 immunoexpression in equine ovarian follicles. a: pregranulosa cells in primordial follicles (arrow) and granulosa cells in primary follicles (arrowhead) express spaca3 (scale bar = 25 µm). figure 2b. spaca3 immunoexpression in equine ovarian follicles. granulosa cells in secondary follicles (arrow) express spaca3 (scale bar = 100 µm). clinical theriogenology 2021; 13: 86 figure 2c. spaca3 immunoexpression in equine ovarian follicles. granulosa cells in tertiary follicles (arrow) express spaca3 (scale bar = 350 µm). negative controls in upper left insets discussion as in cattle, dogs, and cats, spaca3 was present in equine pregranulosa cells of primordial follicles and granulosa cells of primary, secondary, and tertiary follicles. however, unlike in dogs and cats, the equine oolemma did not appear to express spaca3. the equine oolemma appears to have a lower capacity for spermatozoa-oolemma fusion and penetration rates compared to other species that could explain the lack of spaca3 expression in horses.11 in the us, the feral horse population has drastically exceeded the carrying capacity of the public lands where they are managed.12 immunization of horses against porcine zona pellucida (pzp) results in an antibody-based block to sperm-oocyte binding and subsequent fertilization, with a variable efficacy of immunocontraception.13-17 current pzp vaccines have a duration of efficacy for up to 2 years and need to be given again to provide continued immunocontraception.14,17-18 because oocytes in the primordial follicles lack a zona pellucida, the ovarian reserve follicles remains unaffected by pzp vaccines. gonadotropin releasing hormone (gnrh) vaccines have been used in horses. vaccines against gnrh stimulate antignrh antibody production that inhibit the release of fsh and lh and therefore prevent estrous cyclicity.19 similar to pzp, as the gnrh antibody titers decline over time, normal estrous cyclicity returns in mares. this work has provided a foundation for future research in the development of a permanent immunosterilant for the management of feral horse herds. a permanent nonsurgical sterilization method for feral horses is highly desired by public land managers. an ideal immunocontraceptive for feral horses would target primordial follicles and developing follicles. spaca3 is strongly expressed in the pregranulosa cells of equine primordial follicles; therefore, development of a spaca3 vaccine for horses might result in permanent sterilization. additional research is needed to determine if horses can produce a robust humoral response to a spaca3 vaccine to in order to induce sustained infertility. acknowldgements authors thank dr. l. pielstick for collection of the ovaries. conflict of interest authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding funded by the united states department of agriculturenational institute of food and agriculture (#1021276), oregon state university agricultural research foundation (#9258a), undergraduate research scholarship and arts program, and college of agricultural sciences continuing researchers grant. references 1. mandal a, klotz kl, shetty j, et al: sllp1, a unique, intraacrosomal, non-bacteriolytic, c lysozyme-like protein of human spermatozoa. biol reprod 2003;68:1525-1537. 2. zheng h, mandal a, shumilin ia, et al: sperm lysozyme-like protein 1 (sllp1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form. andrology 2015;3:756-771. 3. chiu wwc, erikson ekl, sole ca, et al: sprasa, a novel sperm protein involved in immune-mediated infertility. eur soc human reprod embryol 2004;19:243-249. 4. kovac jr, pastuszak aw, lamb dj: the use of genomics, proteomics, and metabolomics in identifying biomarkers of male infertility. fertil steril 2013;99:998-1007. 5. mcreynolds s, dzieciatkowska m, stevens j, et al: toward the identification of a subset of unexplained infertility: a sperm proteomic approach. fertil steril 2014;102:692-699. 6. agarwal a, sharma r, durairajanayagam d, et al: major protein alterations in spermatozoa from infertile men with unilateral varicocele. reprod biol endocrinol 2015;13:1-22. 7. kwon ws, rahman ms, lee js, et al: discovery of predictive biomarkers for litter size in boar spermatozoa. mol cell proteomics 2015;14:1230-1240. 8. herrero mb, mandal a, digilio lc, et al: mouse sllp1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization. dev biol 2005;284:126-142. 9. wagner a, holland oj, tong m, et al: the role of sprasa in female fertility. reprod sci 2015;22:452-461. 10. cozzi b, habeeb h, kutzler m: sperm protein reactive with antisperm antibody is immunoexpressed in equine primordial, primary, secondary, and tertiary follicles. clinical theriogenology 2020;12:362. 11. mugnier s, dell’aquila me, pelaez j, et al: new insights into the clinical theriogenology 2021; 13: 87 mechanisms of fertilization: comparison of the fertilization steps, composition, and structure of the zona pellucida between horses and pigs. biol reprod 2009;81:856-870. 12. bureau of land management. wild horse and burro on-range population estimates. bureau of land management. accessed on february 5, 2021 from https://www.blm.gov/programs/wild-horse-and-burro/ about-the-program/program-data. 13. kirkpatrick j, liu i, turner j, et al: long-term effects of porcine zonae pellucidae immunocontraception on ovarian function in feral horses (equus caballus). j reprod fert 1992;94:437-444. 14. kirkpatrick j, naugle r, liu ikm, et al: effects of seven consecutive years of porcine zona pellucida contraception on ovarian function in feral mares. biol reprod 1995;52 (monograph series1):411-418. 15. powell dm, monfort sl: assessment: effects of porcine zona pellucida immunocontraception on estrous cyclicity in feral horses. j appl anim welf sci. 2001;4:271-284. 16. barber mr, fayrer-hosken ra: possible mechanisms of mammalian immunocontraception. j reprod immunol 2000;46:103-124. 17. ransom ji, cade bs, hobbs nt: influences of immunocontraceptives on time budgets, social behavior, and body condition in feral horses. appl anim behav sci 2010;124:51-60. 18. turner jw jr, liu ik, flanagan dr, et al: porcine zona pellucida (pzp) immunocontraception of wild horses (equus caballus) in nevada: a 10-year study. reprod suppl 2002;60:177-186. 19. miller la, johns be, killian gj: immunocontraception of white-tailed deer with gnrh vaccine. am j reprod immunol 2000;44:266-274. clinical theriogenology 2021; 13: 88 duration of pregnancy is shorter in cavalier king charles spaniels duration of pregnancy is shorter in cavalier king charles spaniels kristina baltutis, katherine settle, theresa beachler, sara lyle, scott bailey department of clinical sciences, college of veterinary medicine north carolina state university, raleigh, nc abstract duration of pregnancy was evaluated in 17 cavalier king charles spaniel bitches, based on timing of lh surge (progesterone concentrations between 2.0 and 3.0 ng/ml, with continued rise in subsequent 48 hours) and compared to 17 bitches of other breeds. duration of pregnancy was shorter (p < 0.05) in cavalier king charles spaniel bitches compared to others (mean ± sd, 62.8 ± 2.0 days [range; 60 66 days] versus 64.5 ± 1.4 days [range; 62 68 days]). this observation has clinical implications for pregnancy management of this breed, including recommendations for scheduling a timed caesarean section and approaches to managing late-term complications. keywords: cavalier king charles spaniel, duration of pregnancy, parturition introduction exact prediction of duration of pregnancy has important clinical implications for management of canine pregnancy. correct prediction can aid clients and veterinarians in the decision-making process surrounding periparturient interventions, thereby avoiding dystocia and improving neonatal survival. perinatal mortality is relatively higher in dogs compared to other species.1-4 perinatal mortality, defined as the sum of stillborn puppies and puppies that died by day 7 after parturition, was 24.6%, whereas mortality from 8 days to 8 weeks in the same group was only 1%.4 respiratory distress following dystocia and septicemia are 2 common causes of perinatal mortality.5 this is especially important in bitches with high-risk pregnancies, including those with a singleton pregnancy, history of pyometra, abortion, embryonic reabsorption, insufficient luteal phase, or dystocia.6 in ideal circumstances, parturition or caesarean section (c-section) would be performed after final fetal lung and organ maturation, but before fetal/neonatal distress occurs due to dystocia. in cases of high-risk pregnancy, elective c-section is frequently planned to occur shortly before predicted parturition. determination of duration of pregnancy and accurate estimation of date of parturition are critical to develop a treatment plan. due to difficulty and variability of assessing accurately during pregnancy, prediction of parturition date is frequently based on events during breeding.7 when measured from first of multiple matings, duration of pregnancy, can vary anywhere from 57 to 72 days attributed to long canine behavioral estrus and prolonged survival of canine semen within the reproductive tract.8 duration of pregnancy can also be measured from the first day of a diestrual vaginal smear (56 58 days), from the estimated day of ovulation (62 64 days), or from the day of lh surge or estimated lh surge (64 66 days).7-12 use of this latter method for prediction of parturition date is well-characterized in literature, with multiple studies demonstrating a high degree of accuracy and repeatability both when lh is measured directly and when lh surge is identified indirectly as the period of early progesterone (p4) rise.10-12 however, while this method works well for “dogs in general”, several studies have demonstrated evidence for breed specific duration of pregnancy.9,14-16 cavalier king charles spaniel (ckcs), a toy breed ranking as the 19th most popular dog breed by the american kennel club, is anecdotally reported by ckcs breeders to consistently whelp earlier than their predicted parturition date, based on a duration of pregnancy of 65 days from lh surge. this was supported by a previous study that specifically included ckcs.9 in that population of 25 dogs, parturition was noted on average on day 61 ± 1.5 after estimated ovulation. these findings are clinically significant, because animals with a shorter natural duration of pregnancy would experience increased risk of dystocia prior to a traditionally timed c-section compared to another animal. we therefore aimed to confirm the clinical impressions of ckcs owners and the findings reported from european lines of dogs in our clinical population. to accomplish this, we retrospectively analyzed breeding management and parturition data from 2 facilities in central north carolina. we hypothesized that this population of ckcs would have a significantly shorter duration of pregnancy than bitches of other breeds. clinical theriogenology • volume 12 number 4 • december 2020475 materials and methods selection of ckcs litters records from 2 facilities (a small animal private practice and a university theriogenology service) in central north carolina were reviewed to identify client owned litters of ckcs to be included in this retrospective case series. litters were included if data were available on preinsemination p4 concentrations and parturition date. litters were excluded, if no live puppies were produced from the pregnancy, if puppies were delivered via scheduled c-section, or if medical intervention took place before delivery of first pup but were not excluded for undergoing emergency c-section if bitch delivered 1 or more pups prior to surgery. a total of 17 litters from 10 bitches were included in the case series. selection of nonckcs litters records from both facilities were reviewed to identify 17 litters of breeds other than ckcs to be included in the control group. same criteria as described for ckcs were used to include or exclude nonckcs litters. cases were selected for inclusion randomly and stratified based on the bitch’s weight, in 10 kg increments. litters were randomly selected for inclusion in the study from these categories. no more than 2 litters were selected from any 1 weight group, and only 1 litter per breed was included in the control group. duration of pregnancy of the litters was not calculated prior to their selection for inclusion in the study. breeds included in the study were lucas terrier, tibetan terrier, french bulldog, english springer spaniel, border collie, airedale terrier, nova scotia duck tolling retriever, labrador retriever, polish lowland sheepdog, german shorthaired pointer, briard, black and tan coonhound, german shepherd dog, gordon setter, doberman pinscher, rottweiler, and great dane. calculation of duration of pregnancy duration of pregnancy was defined as the number of days between estimated lh surge and delivery of the first puppy. day of lh surge was defined as the day when p4 was between 2.0 and 3.0 ng/ml, with a continued rise recorded in subsequent 48 hours. per cycle, 2 7 blood samples (3.76 ± 1.1) were collected in untreated glass red top tubes for p4 assay, based on patient presentation and clinician preference. samples were analyzed at 2 clinical diagnostic laboratories (antech diagnostics [fountain valley, ca] and north carolina state university clinical pathology laboratory). at both locations, p4 was determined using chemiluminescence assays. data analyses data were evaluated for normalcy with a shapiro-wilk test and two-sample student’s t-tests were subsequently used to compare between groups, attending clinician and laboratory performing the p4 assays. linear regression was performed to characterize the relationship between duration of pregnancy and bodyweight (nonckcs) and the relationship between duration of pregnancy and litter size in ckcs. all statistical analyses were performed with statistix 10 (statistix 10, tallahassee, fl). results mean (± sd) serum p4 concentration at first presentation was 1.2 ± 1.1 ng/ml and at final presentation was 9.9 ± 4.6 ng/ml. there were no differences between groups. average duration of pregnancy for the ckcs group, 62.8 ± 2.0 days with a range of 60 66 days (figure 1) was shorter (p < 0.05) compared to the average duration of pregnancy for the nonckcs group (64.5 ± 1.4 days with a range of 62 68 days; figure 2). linear regression analysis revealed no relationship between body weight and duration of pregnancy in the control group (r2 = 0.02, range 14.8 125 pounds). data on litter size were not available for all animals. data were not available for nonckcs animals and available only for 11 ckcs litters. for litters with reported pregnancy outcomes (11 of 17 ckcs litters), there was no relationship between litter size and pregnancy length (r2 = 0.06, median number of puppies 5, range 1 7). clinical theriogenology • volume 12 number 4 • december 2020 476 figure 1. mean duration of pregnancy (62.8 ± 2.0 days) in 17 litters (range; 1-7 pups) from 10 ckcs bitches figure 2. mean duration of pregnancy (64.5 ± 1.4 days) in 17 nonckcs litters discussion accurate prediction of duration of pregnancy in bitches enables better periparturient decisions, including decision to perform a c-section.7 in evaluating data from breed clubs in uk, the rate of c-sections in ckcs was determined to be 13.1%,18 a moderate rate as compared to other breeds evaluated. c-sections may be performed as an emergency procedure after onset of stage ii of labor, or may be performed as a timed elective procedure in singleton pregnancy, historic dystocia or inability of the owner to reliably detect and supervise parturition. in such cases, timing of c-section (after final lung and organ maturation is complete, but prior to onset of fetal distress or mortality) is vital to shortand long-term survival of puppies. traditionally, timed c-sections are performed on days 63 65 of pregnancy (61 63 days postovulation). however, this time-range may not be ideal for ckcs. in the present study, average duration of pregnancy in ckcs was 2 days shorter than bitches of various other breeds, suggesting that, in this breed, initiation of prepartum monitoring protocols and timed c-section should be advanced to days 59 60 and 61 63 after the lh surge, respectively. physiological relationship between lh surge and parturition date was used to define the duration of pregnancy in beagle dogs as 65 ± 0.1 days from lh surge.8 subsequent work from numerous laboratories determined the average duration of pregnancy for numerous breeds based on lh surge, preovulatory rise in p4, estimated ovulation dates, and breeding dates (table 1). comparison of preovulatory p4 rise among golden retrievers, german shepherd dogs, and labrador retrievers revealed that german shepherd dogs were 3 times more likely, and golden retrievers 4 times more likely, to have a clinical theriogenology • volume 12 number 4 • december 2020477 longer duration of pregnancy compared to labrador retrievers.15 this study also compared duration of pregnancy as determined by cytologic diestrus in hounds to that of labrador retrievers, and reported that hounds were 8 times more likely to have a longer pregnancy. in another study, duration of pregnancy, defined as the period from mating to parturition, was determined in 113 dogs of 6 breeds (31 dobermans, 31 labrador retrievers, 14 german shepherd dogs, 13 bernese mountain dogs,12 golden retrievers, and 12 west highland white terriers). breeding was scheduled immediately if p4 exceeded 7.9 ng/ml (25 nmol/l), within 24 hours if p4 was 5.0 7.9 ng/ml (16 25 nmol/l), and within 24 48 hours if p4 was 4.1 5.0 ng/ml (13 16 nmol/l).14 duration of pregnancy of west highland white terriers was determined to be significantly longer than that of german shepherds, labrador retrievers, and dobermans that did not differ significantly from each other.14 recently, data on 1203 inseminations representing 1146 individual bitches of 84 different breeds were evaluated.16 of these breeds, only greyhounds had significantly different (longer) duration pregnancy (table 1) that was measured from preovulatory rise in p4 > 2 ng/ml. to date, only 1 study specifically investigated duration of pregnancy in ckcs. in that study, data from 162 pregnancies in 151 bitches of 53 breeds were used to identify factors influencing duration of pregnancy.9 duration of pregnancy was defined as the interval between ovulation and parturition, and ovulation was defined as the day when plasma concentrations of p4 reached 6 ng/ml and subsequently rose the following day. this study included 25 ckcs and duration of pregnancy in ckcs was 61 ± 1.5 days from estimated day of ovulation. in the present study, duration of pregnancy was 62.8 ± 2.0 days when calculated from lh surge, similar to the above study that used ovulation as day 1 of pregnancy. comparisons between studies was difficult, since varying determinants were applied to determine onset of pregnancy using varying hormonal assay methods. whereas there was no difference between ria and chemiluminescence assay,11 interassay variability between laboratories is well-known and can complicate interpretation of research and clinical data. in this retrospective analysis, our clinical estimation of lh surge was used to define day 0. there were no differences in duration of pregnancy between clinicians or between laboratories that performed the p4 assays. a shorter duration of pregnancy of cavalier king charles spaniels in these 2 practices was confirmed in comparison to other clinical cases and also by an earlier study.9 a specific cause for shortened duration of pregnancy in ckcs was not elucidated in this retrospective study, nor in prior studies from other groups. in addition to breed, other factors that were characterized and compared to duration of pregnancy in this study included nonpregnant body weight of bitch and litter size. however, linear regression analysis revealed no relationship between body weight or litter size and duration of pregnancy in the present study.9,19 these findings were consistent with several other large studies that demonstrated no effect of either litter size or body weight on pregnancy length.14,16 irrespective of whether the differences are genetic or have an alternate underlying cause, clinical consequences of differences in pregnancy length are considerable. clinicians facing a high-risk pregnancy, elective c-section or potential dystocia must weigh the effects of medical intervention on both maternal and fetal health. data from this study suggested that medical therapy to prolong pregnancy may have to be terminated earlier in this breed than others and that surgical removal of puppies from an at-risk pregnancy may be achieved with good neonatal outcomes earlier than in some other breeds. in this breed, bitches may experience life-threatening dystocia prior to elective c-sections that are routinely scheduled clinical theriogenology • volume 12 number 4 • december 2020 478 table 1. breed-specific studies on duration of pregnancy breed duration of pregnancy (days) factor used to define the beginning of pregnancy reference beagle 65.1 ± 0.1 lh surge, measured directly by ria concannon, et al: 1983 cavalier king charles spaniel 61 ± 1.5 ovulation, defined as occurring when p4 = 6 ng/ml mir, et al: 2011 doberman 61.4 ± 1.0 date of breeding was timed by determination of p4 concentrations okkens, et al: 2001 german shepherd dog 60.4 ± 1.7 date of breeding was timed by determination of p4 concentrations okkens, et al: 2001 63.6 ± 2.1 preovulatory rise in p4 to estimate lh surge eilts, et al: 2005 63.2 ± 1.8 ovulation, defined as occurring when p4 = 6 ng/ml mir, et al: 2011 golden retriever 64.7 ± 1.5 preovulatory rise in p4 to estimate lh surge eilts, et al: 2005 64.3 ± 1.3 ovulation, defined as occurring when p4 = 6 ng/ml mir, et al: 2011 hound 66.0 ± 2.8 8 days prior to onset of cytologic diestrus day 1 eilts, et al: 2005 labrador retriever 60.9 ± 1.5 date of breeding was timed by determination of p4 concentrations okkens, et al: 2001 62.9 ±1.3 preovulatory rise in p4 to determine lh eilts, et al: 2005 west highland white terrier 62.8 ± 1.2 date of breeding was timed by determination of p4 concentrations okkens, et al: 2001 greyhound 68.0 ± 1.5 lh surge, estimated by rise in p4 > 2 ng/ml hollinshead, hanlon: 2017 rottweiler 65.6 ± 1.6 ovulation, defined as p4 = 6 ng/ml mir, et al: 2011 for 63 or 64 days after lh surge, whereas ckcs bitches that present with signs of “premature labor” at 59 or 60 days of pregnancy may in fact deliver fully mature viable puppies with no evidence of disease. recognition of the variability in duration of pregnancy in this and other breeds, along with adjunctive diagnostic tools to estimate fetal maturity, can help clinical decision-making and improve outcomes in cases of complications of pregnancy in ckcs. clinical theriogenology • volume 12 number 4 • december 2020479 conclusion duration of pregnancy in ckcs bitches was 62.8 ± 2.0 days from estimated lh surge. parturition dates several days earlier than otherwise expected can be a normal finding in this breed and should be considered when determining the best option for periparturient interventions. consideration of breed related differences in duration of pregnancy may be critical to improving neonatal survival in ckcs by reducing inherent biases in clinicians hesitant to attempt c-section too early and encouraging timely diagnostic and treatment interventions in both normal and compromised pregnancies in this breed. conflict of interest none to declare. funding supported through donations from raleigh kennel club. references 1. brickell js, mcgowan mm, pfeiffer du, et al: mortality in holstein-friesian calves and replacement heifers, in relation to body weight and igf-i concentration, on 19 farms in england. animal 2009;2:1175-1182. 2. macdorman mf, kirmeyer s: national vital statistics reports 2009;57:1-11 3. mccue pm, ferris ra: parturition, dystocia and foal survival: a retrospective study of 1047 births. eq vet j 2012;44:22-25. 4. tønnessen r, borge ks, nødtvedt a, et al: canine perinatal mortality: a cohort study of 224 breeds. theriogenology 2012;77:1788-1801. 5. grundy sa: clinically relevant physiology of the neonate. vet clin n am small anim pract 2006;36:443-459. 6. johnson c: pregnancy management in the bitch. theriogenology 2008;70:1412-1417. 7. kim y, travis aj, meyers-wallen vn: parturition prediction and timing of canine pregnancy. theriogenology 2007;68:1177-1182. 8. concannon p, whaley s, lein d, et al: canine gestation length: variation related to time of mating and fertile life of sperm. am j vet res 1983;44:1819-1821. 9. mir f, billault c, fontaine e, et al: estimated pregnancy length from ovulation to parturition in the bitch and its influencing factors: a retrospective study in 162 pregnancies. reprod domest anim 2011;46:994-998. 10. holst pa, phemister rd: onset of diestrus in the beagle bitch: definition and significance. am j vet res 1974;35:401406. 11. kutzler ma, mohammed ho, lamb sv, et al: accuracy of canine parturition date prediction from the initial rise in preovulatory progesterone concentration. theriogenology 2003;60:1187-1196. 12. tsutsui t, hori t, kirihara n, et al: relation between mating or ovulation and the duration of gestation in dogs. theriogenology 2006;66:1706-1708. 13. cohen ja, holle dm, meyers-wallen vn: accuracy of canine parturition prediction from lh peak. clinical theriogenology 2009;1:570. 14. okkens ac, teunissen jm, van osch w, et al: influence of litter size and breed on the duration of gestation in dogs. j reprod fertil suppl 2001;57:193-197. 15. eilts be, davidson ap, hosgood g, et al: factors affecting gestation duration in the bitch. theriogenology 2005;64:242-251. 16. hollinshead f, hanlon d: factors affecting the reproductive performance of bitches: a prospective cohort study involving 1203 inseminations with fresh and frozen semen. theriogenology 2017;101:62-72. 17. concannon pw, hansel w, visek wj: the ovarian cycle of the bitch: plasma estrogen, lh and progesterone. biol reprod 1975;13:112-121. 18. evans km, adams vj: proportion of litters of purebred dogs born by caesarean section. j small anim pract 2010;51:113-118. 19. gavrilovic bb, andersson k, forsberg cl: reproductive patterns in the domestic dog—a retrospective study of the drever breed. theriogenology 2008;70:783-794. clinical theriogenology • volume 12 number 4 • december 2020 480 revisiting subcutaneous fetotomy for removal of forelimb revisiting subcutaneous fetotomy for removal of forelimb glennon mays, steven brinsko department of large animal clinical sciences college of veterinary medicine and biomedical sciences texas a&m university, college station, tx abstract removal of a forelimb is often all that is necessary to allow vaginal delivery of an oversized fetus. although the percutaneous utrecht method is efficient, it requires expensive equipment, as well as adequate training and experience to be safely employed. this manuscript describes using a modified chisel, an older, simpler and less expensive method of subcutaneous fetotomy for forelimb removal. keywords: fetotomy, subcutaneous, forelimb, chisel introduction fetal-maternal oversize is the most common cause of dystocia in cattle and may also occur, albeit much less commonly, in mares. when a fetus is no longer viable, oftentimes, removal of 1 forelimb in its entirety will reduce fetal size sufficiently to allow safe and efficient removal by forced extraction. use of a fetatome and employment of percutaneous utrecht method is the procedure most practitioners are currently familiar with to accomplish this task. however, many practitioners, even those who frequently provide obstetrical assistance, do not possess a fetatome. a quality fetatome, complete with all necessary ancillary equipment, can cost in excess of $500.00, which can be a substantial financial outlay, especially for new practitioners. in addition, proper training and proficiency in the use of fetatome is imperative for a successful outcome. a much simpler and inexpensive method for removing fetal forelimbs (applicable but more difficult for hindlimbs) is the subcutaneous method using an obstetric chisel (figure 1). according to professor dr. c.h.w debois,1 development of subcutaneous method of fetotomy occurred early in the 18th century and by the 19th century, several books were written in the netherlands describing this technique, with improvements continuously described until the 1930s, after which it was gradually replaced by percutaneous fetotomy. early veterinary text books describe advantages of subcutaneous fetotomy in calves and foals. unfortunately, many us veterinary schools either no longer teach fetotomy or provide only token instruction. described below is the subcutaneous method preferred and used for many years by the first author, which is a modification of the technique described by williams2 and keller-benesch’s modification of de bruin’s technique.3 method as with any obstetrical procedure, adequate restraint of dam and employing basic principles of cleanliness and lubrication are paramount. when a fetotomy is undertaken, an epidural should be administered and pelvic girdle should be devoid of competing fetal body parts as much as possible, to facilitate manipulation and dissection of the limb. removal of head or placing the head and/or the opposite limb anterior to cervix, followed by repulsion of fetus out of birth canal and into the uterine body, will greatly enhance available space for further mutation and fetotomy (figure 2). ideally, fetotomy should be done with dam standing. however, if dam is laterally recumbent, removal of the uppermost limb is the most advantageous approach. after mutating the fetus to provide adequate space, initial traction applied to the limb of focus is essential, but the limb should be minimally exteriorized before proceeding. further exteriorization places more shoulder into the narrow space of birth canal. ideally, one must be able to advance one’s hand (with knife) proximal to the calf’s scapulohumeral joint, to make the initial incision. without mild to moderate traction on the limb, advancing the operator’s hand/arm simply repels the calf’s limb away from the clinician. moreover, tension on the limb facilitates incisional direction and depth. traction is maintained throughout the entire process. clinical theriogenology • volume 12 number 1 • march 202051 figure 1. three fabricated fetotomy chisels. note that points extending on either side of the blade should be blunt to avoid penetrating skin. instrument at the bottom was designed for use in small ruminants. figure 2. fetus with head and opposite limb reflected to provide more room for removal of extended limb. figure 4. type of pocket knife and blade preferred by dr. mays for incising the fetal skin in preparation for subcutaneous fetotomy. figure 5. linear incision made from scapulohumeral joint to the level of circular cut at mid-metacarpal region. figure 6. sharp and digital dissection is performed on the skin proximal to circular cut. figure 3. encircling cut made at mid metacarpal region. care is taken not to sever flexor or extensor tendons. clinical theriogenology • volume 12 number 1 • march 2020 52 figure 7. finger holes made by stab incisions near each skin corners where longitudinal and encircling cuts join. figure 8. application of traction to a finger hole as the fetal chisel is inserted under reflected skin to initiate skinning process. figure 9. after skin is circumferentially reflected at the level of forearm, chisel is inserted to facilitate separation of skin from underlying tissues. figure 10. freeing skin above scapulohumeral joint using blunt digital dissection, note: traction applied to skin distally, facilitates dissection. figure 11a. after disruption of the pectoral muscles, leg can now be removed by applying traction, note: entire leg should slide out of birth canal, leaving skin attached to torso. figure 11b. entire forelimb, from hoof to scapula, removed via subcutaneous fetotomy. figure 12. after removal of one forelimb, extraction of the fetus is accomplished by applying traction to the finger hole in the skin of the removed forelimb, the fetal head and the remaining forelimb. clinical theriogenology • volume 12 number 1 • march 202053 a circular cut is made (through the skin) at the distal aspect of the initial incision in the midmetacarpal region (figure 3). carefully avoid severing extensor or flexor tendons; they must be intact to facilitate final limb removal by traction. once the circular cut is completed, the two ‘corners’ of skin at the union of the longitudinal and encircling cuts are lifted away from the subcutis by sharp dissection. it is helpful to exteriorize (if possible) this portion of the limb at this point for visualization and improved working space. although further exteriorization of the limb is advantageous at this point, in some cases, disparity in fetal and birth canal sizes precludes this luxury. first author prefers a sharp pocket knife (figure 4) with a rounded or blunted point, a blade many stockmen use when castrating calves. scalpel blades are too short, too thin for adequate control and commonly break during the procedure. moreover, introducing a scalpel into the deeper recesses of birth canal between fetus and vaginal wall has a very high probability of creating maternal damage. knife is then positioned proximally over the anterior (cranial) surface of the shoulder joint; a linear incision is made, progressing distally on the limb to the level of the circular cut made at mid metacarpal region (figure 5). it is imperative that the incision extends through the thickness of skin. depending on degree of sharpness of the cutting instrument and one’s ability to apply consistent and substantial pressure while making the cut, repeated passes are often required (at least for certain locations) before complete skin thickness is incised. if inadvertent cutting into muscle occurs with this first incision, it presents no additional challenge. typically, difficulty is encountered in positioning one’s hand within the birth canal to apply sufficient pressure with the cutting edge to incise the skin full thickness. hence, there is often necessity for swift repeated passes (2 or 3) to completely cut through the skin the entire length of the incision. continuing with sharp and digital dissection, skin proximal to the circular cut is reflected circumferentially and proximally to the carpal joint (figure 6). at this point, a ‘finger hole’ is made by stab incision near each of the two skin corners where the longitudinal and encircling cuts join (figure 7). applying mild traction via these holes facilitates separation of the skin during dissection to reflect skin from carpus. moreover, finger holes allow application of traction as the fetal chisel is inserted under the reflected skin to initiate the undermining process (figure 8). finger hole(s) may rip during the dissection process with chisel use. therefore, subsequent stab incisions may be required when selecting sites for the original locations. a combination of sharp and blunt dissection is preferable for skinning the carpus. use of chisel at this point is neither productive nor necessary. manipulating fingers and thumbs to produce separation of skin is generally sufficient. a sharp knife also can be used as necessary to complete the separation. remember, the idea is to skin the leg; therefore, avoid cutting through the skin once the initial incisions are made, except when making the stab incisions for finger holds. once skin is circumferentially reflected at the level of the forearm, insertion of the chisel facilitates separation of skin from underlying tissues, as the proximal skinning process continues (figure 9). ultimately, the chisel advances to ‘unsleeve’ the shoulder with each successive guided thrust of the instrument. the intention is to direct the advance of chisel along the subcutaneous plane of the entire length of leg to effectively skin the leg within the birth canal/uterine body. careful understanding of the chisel location at all times prevents chisel from inadvertently slipping from beneath the skin along the longitudinal cut. separation of the skin is better accomplished along this line by digital dissection until enough distance from the margin is gained to safely begin chisel insertion under the skin. in the live cow, freeing skin above the scapulohumeral joint with the chisel may not be possible due to anatomical constraints; therefore, this is better accomplished using blunt digital dissection (figure 10). when separation of skin and subcutis is complete, reassess to locate any isolated areas of attachment that may have been missed. most tightly connected areas can easily be separated with digital exploration and dissection, or the chisel can be reinserted for more difficult attachments. pectoral muscles will offer greatest impediment to limb removal. these can be separated digitally or with the use of either a linde’s or guenther’s embryotomy [sic] knife. leg removal can be accomplished by applying traction. clinical theriogenology • volume 12 number 1 • march 2020 54 note that forelimb removal can also be accomplished using a traction device, such as a hand-operated winch or fetal extractor, obviating the need for dissection of the pectoral muscles. with successful preparation, the entire leg should slide out of birth canal, leaving skin attached to the torso (figure 11 a, b). after the limb has been removed, remainder of the fetus is extracted via traction on the remaining limb, fetal head and skin of the amputated limb (figure 12). this technique is more difficult to perform with recent intrauterine fetal death, whereas necrosis and early fetal decomposition ease skin separation. this procedure was implemented numerous times during many careers in a multi-doctor mixed animal practice. although this fetotomy process enabled vaginal removal of decomposing fetal remains countless times, the authors have never experienced chisel penetration through fetal skin, resulting in damaged maternal tissues. if the fetus has been dead for more than 16 18 hours, subcutaneous emphysema may be present. in these cases, after the encircling cut is made and a short longitudinal incision is extended proximally from this circumferential incision, removal of the limb can sometimes be accomplished with traction and minimal dissection. this simple technique provides limb removal including the shoulder thereby creating substantial reduction in size of remaining fetus to allow vaginal delivery and avoiding caesarian section. acknowledgement authors thank dr. leon scrutchfield for encouraging them to write this manuscript. references 1. bierschwal c, de bois c. the technique of fetotomy in large animals. bonner springs; v.m. publishing: 1972. p. 6-50. 2. williams wl. veterinary obstetrics. ithaca; published by the author: 1909. p. 645-648. 3. benesch f, translated by wright og. veterinary obstetrics. london; bailliè, tindall, and cox: 1938. p. 202-205. (editor’s note: online edition of the manuscript has color photographs) clinical theriogenology • volume 12 number 1 • march 202055 clinical theriogenology • volume 12 number 1 • march 2020 56 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (adobe rgb \0501998\051) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype true /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false 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/convertcolors /converttocmyk /destinationprofilename (u.s. web coated \(swop\) v2) /destinationprofileselector /usename /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 0.864000 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice 27 contact steven brinsko sbrinsko55@gmail.com © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9625, http://dx.doi.org/10.58292/ct.v15.9625 case report amputation of the mare’s cervix for the treatment of pyometra steven brinsko department of large animal clinical sciences, college of veterinary medicine, texas a&m university, college station, tx, usa abstract pyometra in the mare is commonly accompanied by or results from occlusion of the lumen of the cervix. formation of excessive transluminal adhesions, cicatrices, and tortuosity not only inhibits natural evacuation of uterine contents, but often impedes or even prevents attempts to establish therapeutic drainage. this paper describes a simple, efficient technique to facilitate access to the uterus and establish drainage. it can be used prior to cervical wedge resection for removal of excessive distorted tissue or as a standalone procedure. keywords: mare, cervix, amputation, pyometra introduction the cervix of the mare is very susceptible to damage and the formation of adhesions. common traumatic events resulting in adhesions include lacerations sustained during parturition, and dystocia, or repeated efforts at cervical manipulation that occur with intrauterine therapy, artificial insemination or embryo transfer.1 pyometra is defined as the accumulation of mucopurulent material within the uterus.2 mares with distorted cervical anatomy or transluminal cervical adhesions are at risk for the development of pyometra due to mechanical impairment of uterine clearance mechanisms. dilating the cervix to enable uterine lavage and intra-uterine therapy is essential in the treatment of pyometra.3 until the cervical wedge resection (figure 1) described by arnold et al.,4 ovariohysterectomy had been the only reported option for treatment of pyometra cases resulting from severe intra-luminal cervical adhesions where patency could not be maintained and pyometra continued to recur.5 6 -7 for mares with pyometra secondary to transluminal cervical adhesions, cervical wedge resection enables treatment of pyometra and has allowed for production of foals via embryo transfer in at least one mare.4 it is important to note that when performing cervical wedge resections which extend cranially to the internal cervical os, inadvertent penetration of the anterior vagina can result in communication with the abdominal cavity, creating the potential for contamination of the peritoneal cavity with uterine contents.4 because of this potential for contamination of the abdomen with uterine contents during surgery, uterine lavage is recommended to be performed prior to surgery. unfortunately, due to their status as embryo donors and repeated manipulation of the cervix during diestrus, cervical adhesions commonly develop in these mares, and their cervices can be significantly distorted by fibrosis and the formation of diverticulae. in mares with such a tortuous cervix, it can be much more difficult to identify the cervical lumen and the location of the internal os. this makes the manual breakdown of cervical adhesions, the dilation and canulation of the cervix to facilitate uterine lavage, and the subsequent wedge resection of the cervix much more difficult. this manuscript describes a simple and effective procedure for amputation of the mare’s cervix that removes abnormal and redundant cervical tissue to enable further treatment of pyometra. this technique can be used to facilitate uterine lavage and cervical wedge resection or as a stand-alone procedure. materials and methods prior to surgery, the mare is restrained in a set of stocks. flunixin meglumine (1.1 mg/kg [0.5 mg/lb], iv; banamine®, merk animal health, madison, nj) is administered, and sedation is provided with detomidine hydrochloride (0.01 mg/kg [0.0045 mg/lb], iv; dormosedan®, zoetis, parsipanny, nj) and butorphanol tartrate (0.01 mg/kg [0.0045 mg/lb], iv; torbugesic®; fort dodge animal health, ft. dodge, ia). caudal epidural may be administered if desired but, generally, is not necessary. the mare’s tail is wrapped, reflected toward the side or dorsally, and the perineum is aseptically prepared. stay sutures or vulsellum-type forceps are placed caudally near the mailto:sbrinsko55@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9625 28 citation line: clinical theriogenology 2023, 15, 9625, http://dx.doi.org/10.58292/ct.v15.9625 external os and the cervix is retracted caudally. a 1.5 – 2.0 meter length of fetotomy wire (jorgenson labs, loveland, colorado) is threaded through a double tubed instrument, 50 cm in length (cervitome), modeled after the utrecht fetotome (figure 2). the loop of fetotomy wire is passed over the retraction implement(s) and around the cervix. while an assistant holds mild tension on the fetotomy wire via the wire handles and maintains traction on the cervix, the operator advances the wire loop and the head of the instrument to the desired point of amputation (figure 3). the assistant is then instructed to gently increase tension on the wire and the operator ensures that the head of the cervitome is snug against the cervix at the amputation site, caudal to the internal cervical os. (note that if the amputation is performed at or anterior to the internal cervical os, as can occur with excessive traction and tenting of the vaginal fornix, there is a risk of entering the peritoneal cavity.) the cervix is then amputated using the sawing motion of the wire, mimicking decapitation during fetotomy. once the wire and instrument are placed properly, the amputation procedure takes less than a minute to perform. the cervical tissue is then removed via the stay sutures or forceps (figure 4). figure 3. placement of “cervitome” firmly against the mare’s cervix with wire looped around the cervix for amputation. (modified from brinsko sp, et al.: manual of equine reproduction, third edition, maryland heights, mo, mosby, inc/elsevier, 2010). figure 1. cervical wedge resection. (modified from brinsko sp, et al.: manual of equine reproduction, third edition, maryland heights, mo, mosby, inc/elsevier, 2010). figure 2. “cervitomes” used for amputation of the mare’s cervix. the instrument above the ruler is a pair of stainless-steel tubes that were taped together for the first application of this procedure. the instrument below the ruler used for subsequent procedures was fabricated with a hardened head to mimic the utrecht model fetotome. figure 4. grossly abnormal cervical tissue removed with vulsellum forceps after amputation. figure 5. view of cervical stump, immediately after amputation. note that there is minimal hemorrhage. http://dx.doi.org/10.58292/ct.v15.9625 citation line: clinical theriogenology 2023, 15, 9625, http://dx.doi.org/10.58292/ct.v15.9625 29 as with cervical wedge resection, post-operative management of mares following cervical amputation is important in maintaining cervical patency. daily application of a compounded ointment consisting of dexamethasone (27 mg), oxytetracycline (3.6 grams) in 151 g of lanolin or vitamin a&d ointment for 7–14 days in and around the cervical stump helps promote re-epithelialization and minimize adhesion formation. additionally, manual palpation of the defect and dilation of the lumen during application of the ointment help ensure the cervix heals and remains patent. a foleyor bivonatype catheter with an inflatable cuff and a heimlich valve attached distally can also be used to help maintain luminal patency. results there is minimal transient hemorrhage associated with the procedure and the provision of hemostasis is unnecessary (figure 5). the procedure has been performed on five mares that were not intended to be future breeding prospects. the establishment of a patent cervical lumen or spontaneous uterine drainage was obtained in all cases, and the results reported from the referring veterinarians and owners were very satisfactory. in all cases, sufficient uterine drainage was established, which either resolved or prevented further recurrence of pyometra. discussion the cervical wedge resection procedure is an effective method of treating pyometra in the mare. prior to surgery, it is important to achieve temporary patency of the cervix in order to remove the mucopurulent contents of the uterus.4 however, in many cases, significant cervical abnormalities such as extensive luminal adhesions, redundant tissue, luminal tortuosity and/or diverticulae make canulation of the cervix to establish uterine drainage extremely difficult if not impossible. because the mare’s cervix is so susceptible to trauma, repeated aggressive attempts to establish cervical patency often results in additional edema and adhesion formation, thus perpetuating the problem. in some cases, even when luminal patency can be established, the amount of abnormal tissue present makes the wedge resection tedious and time consuming. since this tissue is neither functional nor needed, it makes sense to remove it either as an adjunct to, or as an alternative to the wedge resection procedure. in addition to facilitating the wedge resection procedure, removal of this excess tissue enables more efficient canulation of the cervix to establish uterine drainage and can in some cases, obviate the need for the wedge resection. the excised cervical tissue can at times appear grossly normal while having mural and luminal pathology (figure 6a,b) or  grossly abnormal as seen in figure 4. since the internal cervical os remains intact and varying amounts of normal cervical tissue can be preserved, mares undergoing this procedure could retain the potential to be embryo donors. if at least 1/3 to 2/3 the length of normal cervix can be preserved and the uterus/endometrium has not been irreparably damaged due to a long-standing pyometra or other pathology, it may even be possible for such mares to carry a foal to term. the cervical amputation procedure described here is a rapid, simple technique that can be performed by most practitioners having basic surgical training, and without the need for expensive instrumentation. acknowledgements the author has adhered to the principles of veterinary medical ethics of the avma. conflict of interest the author has no conflicts of interest. references 1. tibary, a: failure to dilate. in: mckinnon ao, squires el, vaala we, et  al: editors. equine reproduction. 2nd edition, ames; blackwell publishing: 2011:2724–2730. 2. asbury ac, lyle sk: infectious causes of infertility. in: mckinnon ao, voss jl, editors. equine reproduction. ames; blackwell publishing: 1993:381–391. 3. brinsko sp: endometritis. in: brinsko sp, blanchard tl, varner dd, et  al: editors. manual of equine reproduction. 3rd edition, maryland heights; mosby elsevier: 2011:73–84. doi: 10.1016/ b978-0-323-06482-8.00015-6 4. arnold ce, brinsko sp, varner dd: cervical wedge resection for treatment of pyometra secondary to transluminal a b figure 6. relatively normal externally appearing, amputated cervix (a). internally, there was significant mural scarring and luminal cicatrix formation (b). http://dx.doi.org/10.58292/ct.v15.9625 https://doi.org/10.1016/b978-0-323-06482-8.00015-6 https://doi.org/10.1016/b978-0-323-06482-8.00015-6 30 citation line: clinical theriogenology 2023, 15, 9625, http://dx.doi.org/10.58292/ct.v15.9625 cervical adhesions in six mares. j am vet med assoc 2015;246:1354–1357. doi: 10.2460/javma.246. 12.1354 5. santschi em, adams sb, robertson jb, et al: ovariohysterectomy in six mares. vet surg 1995;24:165–171. doi: 10.1111/j.1532950x.1995.tb01311.x 6. rotting ak, freeman de, doyle aj, et al: total and partial ovariohysterectomy in seven mares. eq vet j 2003;35:29–33. doi: 10.2746/0425164044864633 7. freeman de, rotting ak, kollman m, et al: ovariohysterectomy in mares: 17 cases (1988–2007). in proc am assoc equine pract 2007:370–373. http://dx.doi.org/10.58292/ct.v15.9625 https://doi.org/10.2460/javma.246.12.1354 https://doi.org/10.2460/javma.246.12.1354 https://doi.org/10.1111/j.1532-950x.1995.tb01311.x https://doi.org/10.1111/j.1532-950x.1995.tb01311.x https://doi.org/10.2746/0425164044864633 _hlk64379504 1 contact amber nebel-karp amber.nebel@gmail.com © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9837, http://dx.doi.org/10.58292/ct.v15.9837 case report staphylococcus pseudintermedius causing presumptive ascending canine abortion amber nebel-karp department of clinical sciences, college of veterinary medicine, north carolina state university, raleigh, nc, usa abstract despite growing literature regarding evidence of commensal and sometimes pathogenic bacteria in the uterine and vaginal microbiome of dogs, no direct association with reproductive disease has been linked to staphylococcus pseudintermedius, an opportunistic vaginal commensal (commonly cultured). a multiparous dog had an infectious abortion during late pregnancy over the course of a week. the infectious agent identified on fetal autopsy via culture of the stomach contents and spleen was staphylococcus pseudintermedius. keywords: dog, staphylococcus pseudintermedius, abortion, ascending infection background during the past few decades, more attention is given to the vaginal and uterine microbiome across domestic species and in human medicine. the dogma that the genital tract is a sterile environment is being replaced as evidence for commensal and pathogenic bacteria cultured from the genital tract is catalogued. based on several studies, some of the most common bacterial isolates from the vagina are escherichia coli, staphylococcus pseudintermedius (s. pseudintermedius), streptococcus canis, and mycoplasma in ~ 60–80% of clinically healthy dogs.1–4 most of these commensals have the potential of becoming opportunistic pathogens resulting in reproductive disease aside from s. pseudintermedius2 that has been suggested to serve more of a protective function along with streptococcus species against pathogenic strains via competition.1 as a result of further research, we are recognizing that there is a urogenital microbiome in veterinary species that differs from women. in spayed dogs, lactobacillus is not a common isolate as in humans to protect the vaginal microbiome from pathologic infection.5,6 study on the effects of sterilization on the urogenital microbiome suggested a blunting of microbiome diversity with pasteurella canis and proteus as the most common organisms cultured from healthy spayed females.7 stage of the estrous cycle in dogs has also been documented to cause a shift in the vaginal microbiome with s. pseudintermedius and mycoplasma as more common in estrus and e. coli in anestrus.1 top 3 bacterial isolates from the canine endometrium were pseudomonas, staphylococcus, and corynebacterium.8–12 this case report serves as a direct association between s. pseudintermedius and a presumptive ascending abortion in a dog where fetal sepsis with s. pseudintermedius was confirmed on fetal autopsy. possible theories on how the ascending infection occurred, the clinical presentation, and diagnostic approach to the case are discussed. case presentation a 3-year, multiparous australian labradoodle, was presented on 29 october 2021, on day 55 postlh for diagnostic work-up and treatment for premature labor. although coowned and housed in a ‘guardian home’, the dog was kept throughout her reproductive cycle and pregnancy by a single experienced breeder. dog was current on vaccination, flea/tick/heartworm prevention, and maintained on a dry kibble diet with no raw additives. she had been bred once previously in 2020 and had 8 healthy pups. on the most recent cycle, breeding management (consisting of repeated examinations, brucella canis testing via rapid card agglutination test [rcat; zoetis, parsippany, nj]) progesterone assay, and vaginal cytology) was provided by the referral practice. progesterone concentrations were: 0.74 on 31 august 2021, 0.95 on 2 september 2021, 7.76 on 6 september 2021, and 11.8 ng/ml on 7 september 2021. dog was bred via a single tci (transcervical insemination; karl storz, el saegundo, ca) to a proven 3-year stud on 7 september 2021 with good-quality fresh semen per analysis via nucleocounter® nc-100tm (chemometec, allerod, denmark), computer-assisted sperm analysis (casa; sperm vision® minitube, delevan, wi, usa), and manual wet-mount morphology (652.96 × 106 progressively motile sperm [82.6 progressive motility, 85.73 viable, and 70% normal morphology]). on the same day, she was mated (unintentional) by a 7-year male (also proven, possibility of dual-sire litter) at the 2 citation line: clinical theriogenology 2023, 15, 9837, http://dx.doi.org/10.58292/ct.v15.9837 breeder’s home where she was housed during breeding management. she was confirmed pregnant via transabdominal ultrasonography on 11 october 2021 and was isolated from other dogs. on 28 october 2021 (day 54 postlh), the dog went into premature labor and aborted 4 fetuses in a span of 2.5 hours (between 4:00 and 6:30 pm) with none surviving. she was taken to a local emergency facility, where radiography and transabdominal ultrasonography were performed. five remaining fetuses had normal heartrates (200–250 beats per minute [bpm]), and the dog was clinically stable with no abnormalities on physical examination without signs of distress aside from panting. bloodwork was not performed and the dog was discharged after subcutaneous fluid (plasmolyte, 100 ml) treatment. breeder was advised to monitor overnight and to have a follow-up examination the next morning. on presentation on 29 october 2021 (55 days postlh), another transabdominal ultrasonography was performed (s9 us-diagnostic system; c611, 13–3.9 mhz micro-convex transducer; sonoscape medical corp, shenzhen, china) that revealed 5 viable fetuses with heartrates >190 bpm, some intestinal layering, and no free fluid in the uterus or overt fetal abnormalities. based on the partial abortion of her litter, blood was drawn for progesterone, complete blood count, and serum chemistry. a vaginal swab was obtained for cytology and a swab from a day-54 aborted fetus that was kept in the refrigerator after delivery for autopsy. her serum progesterone concentrations were lower (1.37 ng/ml) (tosoh bioscience aia 360, san fransico, ca), and other bloodwork had a moderate leukocytosis (16,670 white blood cells [reference range 4,360–11,900]) characterized by a mature neutrophilia (13,669 cells [reference range 2,841–9,112]) and a reticulocytosis (129,000). her serum chemistry had minor protein and electrolyte imbalances that were attributed to aborting part of the litter and uterine contractions (calcium, albumin, sodium, and chloride). vaginal cytology revealed multiple neutrophils, lymphocytes, and plasma cells with intracellular cocci bacteria. based on these diagnostic results, an infectious cause of abortion causing a secondary hypoluteoidism was suspected. differential diagnoses considered included ascending infection from opportunistic vaginal microflora (staphylococcus, streptococcus, or escherichia coli), primary hypoluteoidism, and brucella canis. brucellosis was lower on the differential list as the dog and stud dogs had a repeated history of negative rcats, including ones obtained at the time of breeding. a brucella rcat was not repeated for the dog at abortion. treatment oral augmentin® (amoxicillin/clavulanic acid, at a dose of 17 mg/kg twice daily for 10 days) and regumate® (0.088 mg/kg altrenogest once daily until 2 days before her estimated whelping window of 7 november 2021 ± 1 day13,14) treatment were initiated on day 55 postlh, and the dog was scheduled for reevaluation on the following monday (1 november 2021, day 59 postlh) after the weekend. altrenogest, a synthetic progestin, was selected to combat the effects of low progesterone. the goal for progestin therapy was to increase cervical tone and myometrial relaxation to prevent further abortion of remaining fetuses in hopes the remaining litter would survive to maturity. progestogens inhibit gap junction formation in the uterus by preventing the expression of contraction associated proteins via estrogen receptor binding on myometrial cells.15–18 as altrenogest is a synthetic progestin, it has some affinity to progesterone receptors in the endometrium allowing it to have a similar effect. a recent review on altrenogest19 reported that altrenogest has a molecular structure similar to androgen, allowing it to also bind to androgen receptors. this would explain reports of masculinization of female fetuses and hypospadias of male fetuses in dogs if exposed before day 45 of pregnancy with only clitoral hypertrophy noted after day 45 of pregnancy exposure in female fetuses.13 amoxicillin and clavulanic acid were selected as broad-spectrum therapy due to the history and the identification of numerous intracellular cocci on vaginal cytologic examination in order to limit the spread of an ascending or hematogenous infection to the other fetuses.20 empirical antibiotic therapy without culture and sensitivity results was considered vital for fetal outcome due to the potential for rapid spread of infection and fetal compromise. augmentin® was selected due to its broad-spectrum nature, as well as its high margin of safety for pregnancy as a class b fda labelled drug.21,22 it is a hydrophilic penicillin with low protein binding (~ 25%) allowing it to have a high volume of distribution into extracellular fluid such as amniotic fluid. it also freely crosses the placenta via passive diffusion making it an ideal antibiotic to treat uterine and fetal infections.22,23 in women, maternal serum, placental tissue, and umbilical tissue had concentrations of amoxicillin above the minimum inhibitory concentration (mic) for streptococcus agalactiae 2 hours after oral treatment.24 the only compartment tested that was barely below mic concentrations in between doses was the amniotic fluid at 72 compared to > 75%.24 unfortunately, the autopsy report for the fetus was not available (including culture and sensitivity results) until 21 days after the last pup whelping. outcome when the dog was presented for her reevaluation on the following monday (1 november 2021 day 59 postlh), the breeder reported that she had aborted 2 more fetuses over the weekend (fetus # 5 and # 6) leaving only 3 fetuses left in the pregnancy. on ultrasonography, fetuses appeared viable with heartrates ranging from 195 to 243 bpm with intestinal layering and renal corticomedullary distinction noted in all fetuses. at this point, all prescribed medications were continued to try to have at least 1 viable fetus make it to her expected whelping window. she was sent home to be monitored with the understanding that if she aborts anymore fetuses, they would be premature and would not survive. dog aborted another fetus (fetus # 7) 3 days after her first reevaluation (day 62 postlh) and was examined again on friday 5 november 2021 (day 63 postlh). at this reexamination (day 63 post lh), there was only 1 viable fetus (heart rate 230 bpm) out of 2. at this time, progestin therapy was discontinued and her antimicrobials were continued until completed on 8 november 2021. the next evening (day 64 postlh), the breeder reported that the dog had whelped the remaining 2 pups with only the viable pup surviving. despite partially aborting her pregnancy over the course of a week, the dog remained bright, active, and had a normal appetite throughout. a few weeks after the dog aborted her litter, the final autopsy report had been uploaded to her medical record. based on the autopsy report, the submitted fetus had moderate acute sepsis and mild pulmonary hemorrhage with presence of multiple citation line: clinical theriogenology 2023, 15, 9837, http://dx.doi.org/10.58292/ct.v15.9837 3 gram-positive cocci on the lung and renal surfaces. stomach contents and spleen were cultured that had growth of s. pseudintermedius confirming sepsis to be the cause of abortion. based on sensitivity test, s. pseudintermedius was susceptible to all antimicrobials on the panel including amoxicillin/clavulanic acid. a secondary isolate of pseudomonas aeruginosa was also cultured from the spleen with growth only in thioglucholate; on sensitivity, this was resistant to amoxicillin/clavulanic acid. antibiotics that the pseudomonas were susceptible to were aminoglycosides and ceftazidime. pathology results were discussed with the breeder on 30 november 2021 (24 days after the dog whelped her last pup), breeder reported that the sole surviving pup was doing well and thriving. discussion to the author’s knowledge, this is the first published report of a highly suspected ascending cause of abortion from s. pseudintermedius based on fetal autopsy. there have been previous reports on canine abortions resulting from ascending streptococcal, salmonella, and e. coli4,8,10,12 infections and others regarding vaginal microbiome altering between proestrus, estrus, and diestrus.1–3 due to the high number of gram-positive cocci on the pleural surface, renal surface, and cultured from both the stomach and spleen of the fetus submitted for autopsy, the author and the pathologist believe that the cause of the fetal sepsis was s. pseudintermedius instead of the p. aeruginosa. the autopsy report was prepared by a board-certified pathologist who also did not mention any gram-negative rods noted on any surface via cytology, and p. aeruginosa was only discovered on splenic culture only in thioglucholate. the inciting cause allowing s. pseudintermedius access to the uterus and fetuses determined, as is the outcome for most clinical abortions that present to the hospital. primary hypoluteoidism allowing relaxation of the cervix and ascending infection is a possibility but unlikely since primary hypoluteoidism is uncommon in dogs. the most likely progression of disease was an ascending infection from poor cervical tone leading to secondary hypoluteoidism.25 another possible cause could be vaginal trauma from the natural mating that could have led to a break in the vaginal mucosa allowing ascension of infection during immune suppression in diestrus. a potential reason for why the dog aborted over the course of the week could be from a delay caused by progestin therapy or from the next fetus becoming nonviable closest to the cervix. the latter reason is more likely due to her aborting despite being on regumate®. serum progesterone concentrations were not reevaluated as the initial value was 1.37 ng/ml and the likelihood that it would not have increased from endogenous sources, the corpus luteum. also, it is known in equine medicine, progestins do not cross react with progesterone on most analyzers, so her progesterone concentration would not artificially increase due to regumate®.26 although this was not a sterile abortion, the dog was systemically and clinically stable having no signs of sepsis. this suggested that her leukocytosis was in response to the local inflammation and local infection of the abortion. another area of discussion would have been monitoring fetal heart rates at home by the breeder between evaluations. this addition to the dog’s treatment plan was not pursued, as the breeder did not have a doppler ultrasound unit nor the university (potential renting to breeders). regarding the treatment initiated in this case, there are many ways this could have been modified. other methods of uterine quiescence could have been used in adjunction to regumate® or in place of, the most common being terbutaline that has a tocolytic effect on the canine uterus. this was not chosen due to ease of access to regumate® in the hospital and the need to call terbutaline into a local human pharmacy, thereby delaying treatment. however, some may argue that a transition from regumate® to terbutaline would have been advised to reduce the risk of genital abnormalities and possibly better control of premature uterine contractions. due to the late availability (27 november 2021) of autopsy report that included culture and sensitivity of both bacterial isolates, antibiotic adjustments were not possible prior to last pup whelping (6 november 2021). if this was known sooner, addition of gentamicin sulfate or transitioning to ceftazidime (a cephalosporin) could have been initiated to cover both the s. pseudintermedius and p. aeruginosa. human literature has conflicting reports of cephalosporins potentially causing congenital malformations in a small percentage of newborns exposed in the first trimester are made.22 there are also reports regarding aminoglycosides causing nephrotoxicity in newborns exposed during pregnancy due to renal immaturity causing lowered clearance with gentamicin.27 both amikacin and streptomycin have been reported to cause congenital deafness from ototoxicity.27 despite these reports, though coverage of the p. aeruginosa would have been done with either ceftazidime or gentamicin sulfate. a second point of discussion in antimicrobials is extended coverage to any surviving pups with clavamox® drops after delivery to treat neonatal sepsis. this was not done in this case but is a valid point that was overlooked during the time. brucellosis was not highly suspected as a cause of this abortion due to both studs had negative brucellosis results within 7 months from breeding, as did the dog. a repeat rcat was not performed as she had no direct interaction with any other dog outside of 2 males at breeding. however, there are other ways that brucellosis can be transmitted aside from breeding such as urine contamination, mucosal-oral contact, vaginal discharge, and aborted materials.12 lastly, human studies have reported success in ‘rescuing fetuses’ via cesarean surgery before term. this could have been attempted in this case, but conservative management was elected as not all fetuses were fully mature on ultrasonography with intestinal peristalsis on day 59 postlh. if surgical rescue was attempted on day 62 postlh, the last 3 remaining pups may have survived if a glucocorticosteroid (e.g. solumedrol) was given to hasten final maturation and surfactant production by type ii pneumocytes. as it was reported by the breeder when fetus # 7 was aborted on day 62 postlh, the pup was unable to sustain respiration and passed away after a few minutes. this is most likely due to not having produced enough surfactant to maintain alveolar  opening; in dogs, surfactant is produced in the last 12–24 hours before parturition. although it is commonly cultured from vaginal samples, ascending infection and abortion from s. pseudintermedius are uncommon,4,8,10 compared to s. canis or e. coli. we encourage practitioners to gain client consent to submit neonates aborted for diagnostic autopsy as this may be the most clinically relevant diagnostic and lead to a confirmatory or presumptive diagnosis as in this case. open discussion on management of premature labor and abortion in dogs is necessary between clinicians. 4 citation line: clinical theriogenology 2023, 15, 9837, http://dx.doi.org/10.58292/ct.v15.9837 learning points • s. pseudintermedius is a commensal bacterium of the skin that shares the same embryologic origin as the vestibule of the vulva12 • s. pseudintermedius is a commensal of the endometrium in healthy dogs • although s. pseudintermedius is a commensal caudal vaginal microflora, for the first time, its association with canine abortion is reported conflict of interest none to report. references 1. golińska e, sowińska n, tomusiak-plebanek, et al: the vaginal microflora changes in various stages of the estrous cycle of healthy female dogs and the ones with genital tract  infections. bmc vet res 2021;17:8. doi: 10.1186/ s12917-020-02710-y 2. groppetti d, pecile a, barbero c, et al: vaginal bacterial flora and cytology in proestrous bitches: role on fertility. theriogenology 2012;77:1549–1556. https://doi.org/10.1016/j. theriogenology.2011.11.022 3. stockner pk, baker d: canine vaginal flora: a technique for sampling and clinical observation. canine pract 1979;6:18–22. 4. lynch sa, helbig kj: the complex diseases of staphylococcus pseudintermedius in canines: where to next? vet sci 2021;8:1–7. https:// doi.org/10.3390/vetsci8010011 5. hutchins rg, vaden sl, jacob me, et al: vaginal microbiota of spayed dogs with or without recurrent urinary tract infections. j vet intern med 2014;28:300–304. https://doi.org/10.1111/ jvim.12299 6. hutchins rg, bailey cs, jacob me, et al: the effect of oral probiotic supplementation on the vaginal microbiota of spayed female dogs. j vet intern med 2013;27:1368–1371. https://doi. org/10.1111/jvim.12174 7. rota a, corro m, patuzzi i, et al: effect of sterilization on the canine vaginal microbiota: a pilot study. bmc vet res 2020;16:1–10. https://doi.org/10.1186/s12917-020-02670-3 8. shambulingappa be, anand manegar g, ananda kj: study on aerobic bacterial flora in canine abortions. vet world 2010;3:111–112. 9. lyman cc, holyoak gr, meinkoth k, et al: canine endometrial and vaginal microbiomes reveal distinct and complex ecosystems. plos one 2019;14:1–17. https://doi.org/10.1371/journal. pone.0210157 10. khandekar gs, gaikwad sv, choudhari ke, et al: bacterial isolation after ovariohysterectomy for selection of antibacterials in canine pyometra. intas polivet 2015;16:235–237. 11. sanť anna mc, fabretti ak, martins mim: clinical approach to canine vaginitis. semina: londrina 2012;33:1544. https://doi. org/10.5433/1679-0359.2012v33n4p1543 12. bjurström l: aerobic bacteria occurring in the vagina of bitches with reproductive disorders. acta vet scand 1993;34:29–34. https://doi.org/10.1186/bf03548220 13. johnston sd, root kustritz mv, olson pns: canine and feline theriogenology. 1st ed. philadelphia, pa: saunders; 2001, p. 43, p. 66–69, 87–95. 14. gunzel-apel a, urhausen c, wolf k, et al: serum progesterone in pregnant bitches supplemented with progestin because of expected or suspected luteal insufficiency. reprod domest anim 2012;47:55–60. https://doi.org/10.1111/rda.12029 15. gruyter de, carp hja: progestogens in the prevention of miscarriage. horm mol biol clin invest 2016;27:55–62. https:// doi.org/10.1515/hmbci-2015-0058 16. gruyter de, carp hja: progestins in luteal support. horm mol bio clin invest 2020;42:143–148. https://doi.org/10.1515/ hmbci-2019-0067 17. carp hja: progestogens and pregnancy loss. climacteric 2018; 21:380–384. https://doi.org/10.1080/13697137.2018.1436166 18. graubner fr, tavares pereira m, boos a, et al: canine decidualization in vitro: extracellular matrix modification, progesterone mediated effects and selective blocking of prostaglandin e2 receptors. j reprod dev 2020;66:319–329. https://doi.org/10.1262/jrd.2019-157 19. fedorka c: progestin use in mares. clinical theriogenology 2021:13;247–254. 20. sperling s, mitchell a, cheong sh, et al: singleton pregnancy with concurrent pyometra in the contralateral horn in a bitch with a live puppy outcome. reprod domest anim 2018;53:1609–1612. https://doi.org/10.1111/rda.13290 21. glaxosmithkline. augmentin® prescribing info [fact sheet]. u.s. department of food & drug association; n.d. [cited 2023 october 14]. available from chrome extension://efaidnbmnnnibpcajpcglclefindmkaj/https://www.accessdata.fda.gov/drugsatfda_docs/label/2008/0 50575s037550597s044050725s025050726s019lbl.pdf 22. sa del fiol f, gerenutti m, groppo fc: antibiotics and pregnancy. pharmazie 2005;60:483–493. https://doi.org/10.1002/ chin.200540250 23. daniel s, doron m, fishman b, et al: the safety of amoxicillin and clavulanic acid use during the first trimester of pregnancy. br j clin pharmacol 2019;85:2856–2863. https://doi.org/10.1111/bcp.14118 24. zareba-szczudlik j, romejko-wolniewicz e, lewandowski z, et al: evaluation of the amoxicillin concentrations in amniotic fluid, placenta, umbilical cord blood and maternal serum two hours after oral administration. neuroendocrinol lett 2017;38:502–508. 25. uchańska o, ochota m, eberhardt m, et al: dead or alive? a review of perinatal factors that determine canine neonatal viability. animals 2020;12:5–6. https://doi.org/10.3390/ani12111402 26. wynn maa, esteller-vico a, legacki el, et al: a comparison of progesterone assays for determination of peripheral pregnane concentrations in the late pregnant mare. theriogenology 2018;106:127–133. https://doi.org/10.1016/j.theriogenology.2017.10.002 27. app. gentamicin injection, usp [fact sheet]. u.s. department of food & drug association; 2013. [cited 2023 october  14]. available from: chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://www.accessdata.fda.gov/drugsatfda_docs/ label/2014/062366s033lbl.pdf 1 contact celeste morris c.morris@missouri.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9806, http://dx.doi.org/10.58292/ct.v15.9806 case report nonpuerperal chronic endometritis with pyometra causing systemic illness in a production sow celeste morris,a,* katherine watson,b diana schwartz,c richard pereirad awilliam r. pritchard veterinary medical teaching hospital, school of veterinary medicine, university of california, davis, ca, usa bcalifornia animal health and food safety laboratory system, school of veterinary medicine, university of california, davis, ca, usa cveterinary diagnostics and preclinical research services, davis, ca, usa ddepartment of population health and reproduction, school of veterinary medicine, university of california, davis, ca, usa *current address: veterinary health center, university of missouri college of veterinary medicine, columbia, mo, usa abstract a second parity, late pregnant, commercial mixed-breed sow was examined on a farm with a 5-day history of inappetence and lethargy that was unresponsive to treatment with flunixin meglumine. clinical signs were suspected due to an enlarged, fluid-filled hollow abdominal organ identified via transabdominal ultrasonography. further characterization of abdominal viscera and fluid was limited by the sensitivity of the imaging equipment. flunixin meglumine was discontinued and a dietary supplement was added to the feed to encourage intake. repeat transabdominal ultrasonography was performed 4 days later due to a lack of improvement with worsening lethargy, continued inappetence, and fever. the sow was euthanized because of poor prognosis, chronicity of disease, and lack of pregnancy. on necropsy, the uterine horns measured 20 cm in diameter and occupied ~ 80% of the abdominal cavity, compressing the intestines and liver. the uterine lumen contained watery, turbid fluid. the left ovary had multiple grossly appreciable corpora lutea, and right ovary had multiple corpora albicantia. although uterine disease is poorly defined in swine, based on the gross and histologic findings this sow was diagnosed with nonpuerperal chronic endometritis with intrauterine accumulation of purulent fluid (consistent with pyometra). additionally, the sow was diagnosed with salpingitis, interstitial nephritis, lymphadenitis, and gastric ulceration, all likely contributing to her declining health and systemic illness. keywords: sow, pyometra, endometritis, transabdominal ultrasonography background reproductive diseases are one of the most frequently reported reasons for culling in the swine industry. early recognition and treatment of urogenital disorders are essential to optimize sow reproductive performance; however, to maintain economic efficiency, sow culling for decreased performance is a widely recognized herd health management strategy. thus, uterine diseases in sows, including pyometra, endometritis, and metritis, remain poorly characterized. this case demonstrates the need for a more concrete classification and characterization of uterine diseases in swine. our goal is to review the current literature and compare uterine inflammatory conditions in sows with other common domestic species. case presentation a second parity, commercial, mixed breed sow was examined on a farm with a 5-day history of decreased appetite, lethargy, and lack of response to treatment with flunixin meglumine. the sow was bred 88 days before presentation and was due to farrow in 28 days. she was diagnosed pregnant (transabdominal ultrasonographic visualization of fluid-filled uterine cross sections) between 30 and 40 days of pregnancy. she exhibited no outward signs of return to estrus and had no other abnormal systemic health signs or concerns until this time. the sow was lethargic and reluctant to move but was responsive to environmental stimuli. her heart rate and temperature were within normal limits (80 beats per minute and 100°f, mailto:c.morris@missouri.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9806 2 citation line: clinical theriogenology 2023, 15, 9806, http://dx.doi.org/10.58292/ct.v15.9806 respectively). she was eupneic. cardiac and thoracic auscultation were within normal limits. her eyes, ears, nose, and throat were symmetrical. no ocular or nasal discharge was noted. brief neurologic, musculoskeletal, and integumentary examinations were within normal limits. her abdominal contour was normal. she had a body condition score of 3/5. her vulva appeared normal with no discharge. no feces or urine were visualized. peripheral lymph nodes were not visually enlarged. a brief transabdominal ultrasound performed with a handheld, on-farm ultrasound (keebovet wed-2000av, mount prospect, il) used for routine pregnancy diagnosis revealed a hollow viscera distended with fluid. no other abnormalities were noted. although fetal parts were not observable (due to ultrasound equipment with limited diagnostic imaging capability), it was suspected that sow was pregnant given the large fluid-filled loops (5–7 cm in diameter). at this stage of pregnancy, with a machine of better imaging capability, visualization of bony structures (ribs, spine, long bone, or skull) and/ or heartbeats is possible. top differential for inappetence and lethargy were gastric ulceration resulting from stress, systemic effects, nonsteroidal antiinflammatory drug treatment, and/or  discomfort from an enlarged, late pregnant uterus. recommendations after this visit included monitoring for progression of clinical signs and alterations in overall condition, including fever, lameness, depression, vomiting, or inappetence, in addition to giving ensure® (abbott laboratories, chicago, il) to increase caloric intake. other treatments for gastric ulceration were declined due to logistical challenges, financial constraints, and meat withdrawal concerns. the sow was examined 4 days later because of a decline in her overall condition. she remained lethargic and refused her normal commercial diets despite consuming the recommended ensure®. the sow was dull and more reluctant to move than at the previous visit. her temperature was 103.2°f, pulse was 100 beats per minute, and breaths were normal (12 breaths per minute). she had developed moderate abdominal distention, was lame on her left rear limb, and had brownish mucoid feces. all other systems were unchanged from the previous visit. transabdominal ultrasonography performed with a multi-species machine (ibex pro, loveland, co) revealed numerous distended loops of hollow viscera varying in diameter from ~ 4 to 10 cm, containing homogenous hyperechoic fluid (figure 1). this ultrasound equipment allowed clear visualization of the fluid and definitive determination that that there were no detectable fetuses, fetal parts, or fetal remains. it was undetermined whether this hollow abdominal organ was part of the gastrointestinal tract or uterus. differential diagnoses for multiple loops of distended bowel included lower gastrointestinal blockage (partial or full), torsion, or intussusception. top differential diagnosis for a distended uterus lacking fetuses was uterine infection, including endometritis, metritis, or pyometra. a transcutaneous fluid sample was taken that yielded a yellow to white turbid fluid (figure 2b). ovaries could not be visualized during an ultrasonographic examination. outcome the sow was euthanized due to poor prognosis, chronicity of disease without improvement, and a lack of viable pregnancy. prostaglandins used to evacuate uterine fluid and/or antibiotics were discussed, but due to chronicity of disease, uncertainty surrounding the fluid-filled viscous organ, and unlikely return to full reproductive productivity, these therapies were not elected. postmortem findings uterus: closed pyometra with severe plasmocytic and lymphocytic endometritis and distal salpingitis with marked intraluminal purulent fluid and neutrophilic exocytosis left ovary: multiple corpora lutea and albicantia right ovary: multiple corpora albicantia lumbar lymph nodes: lymphadenomegaly kidney: mild, multifocal, lymphoplasmacytic interstitial nephritis with tubular ectasia, red blood cell casts, and tubular thyroidization stomach (pars esophagea): regionally extensive erosion uterine horns measured 20 cm (~ 7.87 cm in diameter) and occupied ~ 80% of the abdominal cavity, compressing the figure 1. transabdominal ultrasonographic images of the sow’s uterus on second examination. note multiple loops of homogenous hyperechoic fluid. http://dx.doi.org/10.58292/ct.v15.9806 citation line: clinical theriogenology 2023, 15, 9806, http://dx.doi.org/10.58292/ct.v15.9806 3 intestines and liver. the lumen contained a watery, off-white, turbid, slightly sour-smelling fluid (figure 2b). the mucosal surface of the uterus was diffusely dark red with prominent vessels (figure 2a). the oviducts were moderately thickened. the left ovary had multiple corpora lutea (cls) (figure 2a) and the right ovary had multiple corpora albicantia (cas). the sublumbar and pelvic lymph nodes were markedly enlarged, lobulated, and measured 2 × 3 × 2 cm. the lymph node immediately adjacent to uterus within pelvis contained green, desiccated creamy material on cut section. the stomach contained watery, bright yellow contents. mucosa around the pars esophageal region was irregular and flaky. bacteriology histology specimen type culture type results uterine fluid aerobic actinomyces hyovaginalis, moderate numbers anaerobic none detected lymph node swab aerobic proteus swarming immunohistochemistry a porcine serology-based abortion panel was negative for antibodies to leptospira bratislava, l. canicola, l. grippotyphosa, l. hardjo, l. icterohaemorrhagiae, and l. pomona, porcine parvovirus (ppv), pseudorabies virus (prv), and brucella abortus. cytology interpretation: marked neutrophilic and lymphoplasmacytic inflammation with polymicrobial bacterial sepsis and unremarkable to hyperplastic uterine epithelium figure 2. gross photo of the sow’s uterus and uterine fluid. a: note red mucosal surface (bracket) with prominent, congested vasculature (blue arrow) and numerous corpora lutea on the left ovary (black arrow). b: after incising along the greater curvature, a copious amount of opaque, pale-yellow, watery fluid was released. tissue histologic findings stomach pars esophageal region of the stomach was covered by amphophilic material (interpreted as mucin) and hemorrhage. a regional defect of the mucosa was noted with the surrounding lamina propria being expanded by lymphocytes and plasma cells admixed with a smaller number of neutrophils. uterus endometrial glands were variably dilated by clear space and scant to moderate amounts of granular, eosinophilic fluid and/or viable and degenerate epithelial cells. submucosa was markedly expanded by large numbers of plasma cells admixed with fewer lymphocytes and neutrophils. traversing the mucosa were moderate numbers of neutrophils. submucosal and myometrial vessels were markedly congested (figure 3). kidney tubules were variably ectatic and empty or filled with hyaline casts. interstitium was multifocally infiltrated by small numbers of lymphocytes and plasma cells. one glomerulus had increased mesangium and vessels containing globular eosinophilic material. left ovary multiple corpora lutea, corpora albicantia, developing follicles (figure 4). http://dx.doi.org/10.58292/ct.v15.9806 4 citation line: clinical theriogenology 2023, 15, 9806, http://dx.doi.org/10.58292/ct.v15.9806 description: wright geimsa stained smear was highly cellular with a patchy pale blue background, many free cell nuclei, and numerous heterogeneous bacteria of mixed morphology (rods and cocci). highly degenerate neutrophils were observed, often containing bacteria similar to that noted in the background, admixed with plasma cells, sometimes in small aggregates, small lymphocytes, and occasionally larger lymphocytes (figure 5). several mildly disrupted clusters of cuboidal to columnar epithelial cells were also appreciated. these cells had a moderate amount of mid-blue cytoplasm with distinct cell margins. nuclei were ovoid, often polarized, with coarsely stippled chromatin and single small nucleoli. nuclear to cytoplasmic ratios were moderate to high with mild anisocytosis and anisokaryosis. discussion definitions of uterine disease are not well established or widely accepted in sows compared to other species, which makes clinical diagnosis and differentiation of uterine disease challenging. regardless, impaired fertility from reproductive disorders (including any uterine, ovarian, endocrine, vaginal, or vulvar condition resulting in an inability to conceive and maintain a pregnancy to term), accounts for up to  one-third or more of all swine culling indications.1–7 outwardly, reproductive disease usually presents as failure to conceive, increased returns to estrus, anestrus, or abortion. diagnosis of reproductive disorders can be aided by transabdominal ultrasonography, but this also presents many challenges and limitations because it is normal to visualize fluid within the uterus after breeding, during estrus, or associated with pregnancy; however, any fluid present in the uterus outside of these times is considered abnormal.8 in many species, it is common to classify uterine disease into categories such as pyometra, metritis, and endometritis based on the presence of fluid, evaluation of fluid via transabdominal ultrasonography and/or cytology, vaginal and/or vulvar discharge and ovarian structures. defining characteristics of pyometra in any species are an accumulation of purulent fluid within the uterine lumen9 and usually the presence of a functional corpus luteum (cl) that maintains a functionally closed cervix and prevents expulsion of the suppurative material.10 clinically, pyometra can be differentiated from pregnancy fluid based on b-mode real-time ultrasonography and fluid echogenicity.11 in dairy cows, pyometra is often the result of ovulation occurring before uterine involution is complete that results in the formation of a corpus luteum and closure of the cervix before the uterus is cleared of all pathogens.12 in both dairy and beef cows, pyometra can also result from pathogen introduction during insemination or breeding, as can be the case with tritrichomonas foetus infections.12 in canines, pyometra occurs frequently in conjunction with cystic endometrial hyperplasia due to the effects of progesterone on the uterine lining during the diestrus stage of the female cycle.13,14 female dogs have an extended luteal phase, in comparison to livestock species, as it is similar in length to that of pregnancy;13 as a result, the pyometra is proposed to be a result of bacterial infection of the progesterone-primed endometrium.10 in mares, pyometra is also defined as the presence of purulent fluid in the uterus with or without the presence of a cl. this condition can be further complicated by endogenous or exogenous mediated closure of the cervix, the presence of endometrial cups, or poor physical abilities of the uterus to drain in this species.15 in sows, pyometra is often a sequela to endometritis or metritis9 and can be differentiated from other uterine diseases by cytologic fluid analysis, but this condition is uncommon in this species9,10, therefore cytological definitions of pyometra are not well established. metritis is defined as inflammation of all layers of the uterus.9,10,16,17 in cows, metritis is characterized by malodorous fluid and myometrial edema, occurring within 2–3 weeks after calving, and usually does not cause systemic disease. systemically affected cows with metritis are usually termed as having septic and/or toxic metritis, which has different treatments and implications than a metritis localized to the uterus.12 in female dogs, metritis is a disease of the puerperal period and occurs 0–7 days after whelping frequently resulting in systemic illness.18 in mares, metritis also extends through the uterine wall, making equids more prone to developing perimetritis and peritonitis which can lead to laminitis and septicemia.19 diagnosis of metritis in sows is based on the accumulation of uterine fluid and increased uterine size during the involution process.20 it is usually observed within 24–48 hours after parturition16 with fetid, malodorous vaginal discharge. examination of the uterus postmortem often reveals a uterus distended by purulent fluid mixed with necrotic material. sows are often systemically affected, presenting with vague signs such as inappetence and fever. histologically, edema and bacterial infiltration of the uterine wall may be present along with edema and purulent exudate within the myometrium itself.16 deep infiltration into the myometrium in sows resulting in metritis is rare.21 endometritis is defined as inflammation that is limited to the endometrium.10 in cows, endometritis is limited to the mucosal and submucosal layers of the uterus and does not cause systemic illness. classifications of clinical and subclinical endometritis in cows have recently been proposed, depending on the presence of discharge or lack thereof, respectively, and is limited to diagnosis in animals > 21 days after calving, as cows have an extended uterine involution period.12 in female dogs, endometritis may present as part of a cystic endometrial hyperplasia-pyometra complex14 or on its own as a source of infertility.22 in sows, endometritis is associated with infertility that can be either temporary or permanent.23 in mares, a transient postcoital endometritis is commonly observed after breeding,10 that depending on the severity and persistence of fluid, may require intervention to resolve. endometritis can also be observed after foaling, after artificial insemination, or due to poor conformation.19 endometritis has been cited as the most common inflammatory process affecting the porcine uterus. sows can present with and without vaginal or vulvar discharge and with or without return to estrus.1,24,25 inconsistent presentation and lack of systemic illness in this species are challenges for the diagnostician.16 in addition to classification based on clinical appearance as discussed above, endometritis can also be defined and differentiated based on the cellular infiltration of the uterus on histological examination and timing in relation to parturition and breeding. endometritis in sows can be classified as puerperal or nonpuerperal,8,25 and further into acute, subacute, and chronic.10,21,26–29 before these classifications were available, endometritis in the sow was classified based on the presence of uterine fluid but not defined histologically.1,24 in addition to uterine fluid classifications, acute and subacute http://dx.doi.org/10.58292/ct.v15.9806 citation line: clinical theriogenology 2023, 15, 9806, http://dx.doi.org/10.58292/ct.v15.9806 5 endometritis are more likely to present with a vulvar discharge, whereas chronic endometritis may be more difficult to diagnose outwardly other than failure to conceive or maintain a pregnancy which manifests clinically as increased returns to estrus.2,21,25,30 an additional scale consisting of mild, mild to moderate, moderate, and severe has been published, taking into consideration leukocyte infiltration and endometrial changes.30 no standard definitions of acute, subacute, and chronic nonpuerperal endometritis have been established; however, several criteria have been published. cell types and numbers present in the endometrium as well as damage to normal structures have been proposed as a system to differentiate acute, subacute, or chronic endometritis (table 1).1,21,26,27,29,30 when evaluating cellular infiltration in sows, stage of the estrous cycle must also be considered. pathologic cellular infiltration versus normal cellular infiltration at different phases of the estrous cycle will vary. depending on the phase of the estrous cycle, it can be normal for fluid to be present; so, if the information is available, histologic, visual, and/or ultrasonographic evaluation should consider ovarian structures.26,27 unlike diestrus, estrus presents a more active type of cellular migration with an open cervix due to the effects of estrogen. it is normal to observe increased numbers of neutrophils and lymphocytes. despite the cell types, focal accumulations of cells and destruction of normal architecture (e.g. uterine glandular epithelium) are considered pathological.7,29 general guidelines on cell types observed normally during different stages of the sow estrous cycle are presented (table 2). in addition to outward clinical signs and cytologic examination to classify endometritis in sows, transabdominal table 1. proposed histologic definitions of acute, subacute, and chronic endometritis in the sow. disease definition cell types normal maximum of 15 inflammatory cells per field (400 x) during estrus or metestrus26 acute endometritis no further classification within acute endometritis • > 100 neutrophils per 20 high power fields (hpfs [400 x])27 • > 20 neutrophils for estrous females and > 5 for diestrous females in subepithelial layer and/or any neutrophils present in the stromal layer2 • > 20 neutrophilic granulocytes per hpf 7 moderate • number of neutrophilic granulocytes substantially higher than normally observed without other abnormalities29 • >20 neutrophilic granulocytes per hpf with no alterations to the endometrium26 severe • number of neutrophilic granulocytes substantially higher than normally observed in conjunction with endometrial damage29 • > 40 neutrophilic granulocytes cells per hpf with alterations to the endometrium26 subacute endometritis • increased neutrophilic granulocytes and lymphocytes26,29 • 40–100 neutrophils with > 10 plasma cells per 20 hpfs27 acute-chronic endometritis simultaneous appearance of: • > 15 lymphocytes within the subepithelial later and/or > 5 lymphocytes within the stromal layer and/or > 5 plasma cells within one or both layers regardless of the stage of the cycle2 • > 20 neutrophils for estrous females and > 5 for diestrous females in subepithelial layer and/or any neutrophils present in the stromal layer2 chronic endometritis no further classification within chronic endometritis • lymphocytes, plasma cells, and histiocytes substantially higher than normal29 • > 20 lymphocytes, plasma cells, histiocytes per hpf 26 • > 40 lymphocytes, > 10 plasma cells, and < 20 neutrophils per 20 hpfs in subepithelial layers27 • > 15 lymphocytes within the subepithelial later and/or >5 lymphocytes within the stromal layer and/or > 5 plasma cells within one or both layers regardless of the stage of the cycle2 mild • > 5 lymphocytes per hpf in subepithelial layer and/or > 15 lymphocytes per hpf in lamina propria21 severe • dense infiltration of lymphocytes and plasma cells21 http://dx.doi.org/10.58292/ct.v15.9806 6 citation line: clinical theriogenology 2023, 15, 9806, http://dx.doi.org/10.58292/ct.v15.9806 ultrasonography has also been used, but there is no clear definition of the ultrasonographic appearance of endometritis in this species. in diestrus, when the cervix is closed and the uterus is under the influence of progesterone (quiescent), there should not be any intraluminal fluid and uterine wall should appear homogenous.28 if fluid is present with endometritis, there are many different documented descriptions ranging from anechoic with no definitive form24 to a heterogenous echogenic fluid resembling a snow flurry.8 some differentiate the types of endometritis (acute, acute-chronic, and chronic) based on the type of fluid that is visible, indicating that fluid can be observed ultrasonographically if endometritis is either acute or acute-chronic.26,27 severe acute endometritis is described as more of the snow flurry, flocculent fluid type,25,28 whereas chronic endometritis can have a wide range of appearances. because of this, it has been argued that there is no relationship between chronic endometritis and ultrasonographic uterine size or echotexture.2 further, chronic endometritis cannot be definitively diagnosed by ultrasonographically because it can appear similar to normally involuted uterus.2,8,28 because there is no consensus, interpretation of uterine imaging findings should be performed in conjunction with ovarian findings to help interpret whether ovarian findings are consistent with normal variations in uterine echotexture based on circulating hormones or if the two are in opposition.8,25 in terms of systemic manifestations of uterine disease, elevated core temperatures in conjunction with other signs of systemic illness (lethargy, inappetence), as observed in this case, have been associated with endometritis in sows.7,32,33 in addition to endometritis, this sow had gross and histologic evidence of salpingitis. this is not surprising given that uterine infections are a commonly reported source of oviductal disease.25 urinary bladder in this case was unremarkable; however, pigs with cystitis are 3.5 times more likely to develop endometritis due to the urethral connection between the urinary and genital tracts.7 interstitial nephritis was observed histologically, but this is a nonspecific finding and was not associated with pyelonephritis to indicate an ascending infection from the grossly normal urinary bladder. regionally extensive erosion of the stomach likely contributed to the sow’s decline but is likely secondary to other disease processes. in terms of specific pathogens of endometrial disease in swine, most are bacterial in origin. viral pathogens causing uterine disease in sows are rare. bacterial pathogens in endometritis include gram-negative pathogens (e.g. escherichia coli, klebsiella spp., chlamydia spp., or proteus spp.) and gram-positive pathogens (e.g. staphylococcus spp., trueperella pyogenes, streptococcus spp., and enterococcus table 2. normal cell types observed in porcine uterus by phase of the estrous cycle. cell type phase of estrous cycle proestrus, estrus (follicular) diestrus (luteal) neutrophils increased number of neutrophilic granulocytes26,29 high infiltration of neutrophils at proestrus and estrus in the subepithelial layer31 neutrophilic granulocytes almost absent26,29 rarely observed in the glandular layer31 substantially lower neutrophils during the luteal phase27 lymphocytes increased numbers26,29 substantially increased numbers of intraepithelial lymphocytes with round nucleus31 largest number of lymphocytes in the subepithelial layer31 substantially increased numbers of lymphocytes27 high numbers during early diestrus and low numbers during late diestrus31 low numbers26,29 plasma cells substantially increased numbers of plasma cells in subepithelial and connective tissue layers27 almost absent29 macrophages most macrophages were observed at estrus31 macrophages observed in early diestrus in subepithelial and glandular layer31 substantially higher in the subepithelial and the glandular tissue layers of the endometrium27 other cell types mast cells mast cells in the subepithelial layer31 eosinophils low numbers of eosinophilic granulocytes26,29 largest number of eosinophils during diestrus compared to other stages in the subepithelial layer31 rarely observed in the glandular layer31 substantially higher in the subepithelial and the glandular tissue layers of the endometrium27 http://dx.doi.org/10.58292/ct.v15.9806 citation line: clinical theriogenology 2023, 15, 9806, http://dx.doi.org/10.58292/ct.v15.9806 7 spp.). specifically, staphylococcus aureus, staphylococcus hyicus, streptococcus suis, and streptococcus equisimilis1,7,25–27 have been implicated as causes of endometrial disease in sows. in this case, culture of actinomyces hyovaginalis from uterine fluid is unusual, as this is not considered a major pathogen of endometritis in sows or gilts. actinomyces spp. are facultatively anaerobic, gram positive, slow growing bacteria that have been isolated as normal flora from a variety of anatomical sites in both human and veterinary medicine, including skin and oral mucosa.25,34 in veterinary medicine, actinomyces hyovaginalis specifically has been cultured from swine necrotic lung abscesses35 as well as a tracheobronchial lymph node in a goat.36 other species of actinomyces have been implicated in a variety of conditions in veterinary medicine. for example, actinomyces bovis is a normal inhabitant of the bovine oral microflora and is the causative agent of mandibular osteomyelitis (‘lumpy jaw’) in cattle.10 in swine, actinomyces hyovaginalis has been associated with sporadic abortions and embolic pneumonia.25 it is thought to be a normal inhabitant of the vaginal flora of pigs (specifically biovar type ii) and has been isolated in vaginal discharge and aborted fetal cultures. it is likely that actinomyces hyovaginalis associated disease is likely underreported.25 in a study where gilts underwent intrauterine inoculation of actinomyces spp., 1 developed no clinical signs and another developed severe acute endometritis.26 because this organism is a normal inhabitant of the vagina, its presence alone in vaginal swabs or on aborted fetuses does not imply that it is the causative agent of disease; however, culture from uterine fluid in conjunction with the uterine disease is more relevant. this case was classified as chronic endometritis complicated by pyometra after taking into consideration existing proposed definitions of uterine disease, histological and cytological evaluations, and most likely estrous cycle stage. grossly and histologically observed cls (figure 4) likely resulted in cervical closure and intrauterine fluid accumulation, consistent with pyometra (figure 2). accumulation of uterine fluid along with lymphoplasmacytic infiltration and lack of vulvar discharge is consistent with definitions of chronic endometritis. with this classification, it is important to take into consideration both the histologic and ovarian findings in addition to ultrasonographic findings. it is unknown in this case whether the cls would have persisted pathologically if the animal was not euthanized or if the sow was in the middle of a normal luteal phase that would have resulted in luteolysis of the grossly observed cls. additionally, another challenge for interpretation in this case is whether a bacterial infection during copulation/insemination resulted in endometritis and early embryonic loss, or whether the animal had an underlying, clinically silent endometritis and then ovulated with an already compromised endometrium, causing closure of the cervix and accumulation of intrauterine fluid. histologic examination of the tissues demonstrated a dense infiltrate of predominantly plasma cells, consistent with chronic endometritis (figure 3, table 1); however, the clinical presentation figure 3. histologic images of the sow’s uterine horns. (a) subgross hematoxylin and eosin (h&e) image of a uterine horn in which the submucosa and overlying hyperplastic mucosa form multiple folds resembled papillary projections. (b) at higher magnification, the submucosa appeared expanded by sheets of inflammatory cells (asterisk) with markedly congested blood vessels. (c) a higher magnification of the mucosal surface demonstrated a dense infiltration of plasma cells (asterisk) with neutrophils traversing the epithelial surface (black arrow) and marginating within blood vessels (blue arrow). http://dx.doi.org/10.58292/ct.v15.9806 8 citation line: clinical theriogenology 2023, 15, 9806, http://dx.doi.org/10.58292/ct.v15.9806 figure 4. gross and histologic images of the sow’s left ovary. a: gross image of the medially incised ovary demonstrating numerous corpora lutea (white *) and a corpora albicans (blue arrow). subgross (b) and higher magnification (c) images of a h&estained section of ovary with corpora lutea (black *), developing follicles (+), and a corpora albicans (#). d: high magnification image of luteal cells with foamy cells containing steroid hormones. e: high magnification image of a an involuted corpus luteum (corpus albicans), that appeared white due to the presence of fibroblasts and collagen. f: scattered amongst the normal fibromuscular ovarian stroma are various developing follicles. black arrows designate primordial follicles consisting of a primary oocyte with a single layer of squamous cells. larger follicle is a secondary follicle (blue arrow) with a primary oocyte surrounded by multiple layers of granulosa cells that are variably supported by theca folliculi (encasement of rich blood supply and connective tissue). http://dx.doi.org/10.58292/ct.v15.9806 citation line: clinical theriogenology 2023, 15, 9806, http://dx.doi.org/10.58292/ct.v15.9806 9 and purulent fluid also fulfill the definition of pyometra (figure 5). therefore, the classification of uterine disease in this sow is chronic endometritis (figure 3) and pyometra. systemically, considering the elevated temperature and adjacent lymph node enlargement, it is possible that the sow was septic and bacterial showering resulted in nephritis, increased temperature, and lymphadenopathy, all contributing to her outward clinical decline. learning points • there are no standard definitions of different types of uterine disease in swine • reproductive disease is a substantial cause of culling in sows • classification of endometritis in sows is challenging due to differences in immune cells during different stages of the estrous cycle and inconsistent uterine appearance on ultrasonography conflict of interest none to report. references 1. muirhead m: epidemiology and control of vaginal discharges in the sow after service. vet rec 1986;119:233–235. doi: 10.1136/ vr.119.10.233 2. kauffold j, rautenberg t, hoffmann g, et al: a field study into the  appropriateness of transcutaneous ultrasonography in the diagnoses of uterine disorders in reproductively failed pigs. theriogenology 2005;64:1546–1558. doi: 10.1016/j.theriogenology. 2005.03.022 3. heinonen m, leppavuori a, pyorala s: evaluation of reproductive failure of female pigs based on slaughterhouse material and herd record survey. anim reprod sci 1998;52:235–244. doi: 10.1016/ 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reproduction. st. louis, missouri: mosby/elsevier; 2011. 20. björkman s, oliviero c, kauffold j, et al: prolonged parturition and impaired placenta expulsion increase the risk of postpartum metritis and delay uterine involution in sows. theriogenology 2018;106:87–92. doi: 10.1016/j.theriogenology.2017.10.003 21. dalin am, kaeoket k, persson e: immune cell infiltration of normal and impaired sow endometrium. anim reprod sci 2004;82– 83:401–413. doi: 10.1016/j.anireprosci.2004.04.012 22. angela t. gifford, janet m, et al: histopathologic findings in uterine biopsy samples from subfertile bitches: 399 cases (1990– 2005). j am vet med assoc 2014;244:180–186. doi: 10.2460/ javma.244.2.180 23. dial gd, maclachlan nj: urogenital infections of swine. part i. clinical manifestations and pathogenesis. compendium food animal 1988;10:63–70. 24. martinez e, vazquez jm, roca j, et al: use of real-time ultrasonic scanning for the detection of reproductive failure in pig herds. anim reprod sci 1992;29:53–59. doi: 10.1016/0378-4320(92) 90019 25. althouse gc, kauffold j, rossow s, et al: diseases of the reproductive system. in: zimmerman jj, karriker la, ramirez a, et al: editors. swine reproduction. 11th edition, hoboken, nj: john wiley & sons, inc; 2019, p. 373–393. 26. de winter p, verdonck m, de kruif a, et al: bacterial endometritis and vaginal discharge in the sow: prevalence of different bacterial species and experimental reproduction of the syndrome. anim reprod sci 1995;37:325–335. doi: 10.1016/0378-4320 (94)01342 27. tummaruk p, kesdangsakonwut s, prapasarakul n, et al: endometritis in gilts: reproductive data, bacterial culture, histopathology, and infiltration of immune cells in the endometrium. comp clin path 2010;19:575–584. doi: 10.1007/s00580-009 0929-1 28. kauffold j, peltoniemi o, wehrend a, et al: principles and clinical uses of real-time ultrasonography in female swine reproduction. animals 2019;9:1–17. doi: 10.3390/ani9110950 29. de winter pjj, verdonck m, de kruif a: endometritis and vaginal discharge in the sow. anim reprod sci 1992;28:51–58. doi: 10.1016/0378-4320(92)90091 30. dalin am, gidlund k, eliasson-selling l: post-mortem examination of genital organs from sows with reproductive disturbances in a sow-pool. acta vet scand 1997;38:253–262. doi: 10.1186/ bf03548488 31. kaeoket k, persson e, dalin am: the sow endometrium at different stages of the oestrous cycle: studies on morphological changes and infiltration by cells of the immune system. anim reprod sci 2001;65:95–114. doi: 10.1016/s0378-4320(00)00211-6 32. meile a, nathues h, kauffold j, et al: ultrasonographic examination of postpartum uterine involution in sows. anim reprod sci 2020;219:1–7. doi: 10.1016/j.anireprosci.2020.106540 33. stiehler t, heuwieser w, pfutzner a, et al: the course of rectal and vaginal temperature in early postpartum sows. j swine health prod 2015;23:72–83. 34. valour f, sénéchal a, dupieux c, et al: actinomycosis: etiology, clinical features, diagnosis, treatment, and management. infect drug resist 2014;7:183–197. doi: 10.2147/idr.s39601 35. aalbaek b, christensen h, bisgaard m, et al: actinomyces hyovaginalis associated with disseminated necrotic lung lesions and slaughter pigs. j comp pathol 2003;129:70–77. doi: 10.1016/ s0021-9975(03)00005-7 36. schumacher vl, hinckley l, gilbert k, et al: actinomyces hyovaginalis-associated lymphadenitis in a nubian goat. j vet diagn invest 2009;21:380–384. doi: 10.1177/104063870902100315 http://dx.doi.org/10.58292/ct.v15.9806 https://doi.org/10.1016/j.anireprosci.2010.08.028 https://doi.org/10.1016/j.anireprosci.2010.08.028 https://doi.org/10.1016/b978-0-7216-9323-1.x5001-6 https://doi.org/10.3168/jds.2021-21203 https://doi.org/10.1016/j.theriogenology.2017.10.003 https://doi.org/10.1016/j.anireprosci.2004.04.012 https://doi.org/10.2460/javma.244.2.180 https://doi.org/10.2460/javma.244.2.180 https://doi.org/10.1016/0378-4320(92)90019 https://doi.org/10.1016/0378-4320(92)90019 https://doi.org/10.1016/0378-4320(94)01342 https://doi.org/10.1016/0378-4320(94)01342 https://doi.org/10.1007/s00580-009-0929-1 https://doi.org/10.1007/s00580-009-0929-1 https://doi.org/10.3390/ani9110950 https://doi.org/10.1016/0378-4320(92)90091 https://doi.org/10.1186/bf03548488 https://doi.org/10.1186/bf03548488 https://doi.org/10.1016/s0378-4320(00)00211-6 https://doi.org/10.1016/j.anireprosci.2020.106540 https://doi.org/10.2147/idr.s39601 https://doi.org/10.1016/s0021-9975(03)00005-7 https://doi.org/10.1016/s0021-9975(03)00005-7 https://doi.org/10.1177/104063870902100315 introduction there are distinct differences in elephant reproduction compared to other animals. elephants have a long estrous cycle (~ 14 17 weeks) divided into luteal (8 10 weeks) and follicular (4 7 weeks) phases. follicular phase has 2 luteinizing hormone (lh) surges. first lh surge (nonovulatory) occurs ~ 3 weeks after progesterone (p4) reaches baseline concentrations, and the dominant follicle becomes the primary corpus luteum (cl) after the second lh surge (ovulatory) that occurs 19 22 days after first surge.1 some of the nonovulatory follicles from the first lh surge are recruited to become secondary cls. primary progestins in elephants are 5.α-reduced pregnanes, whereas in most other mammals it is progesterone (4-pregnene-3, 20-dione). although p4 is a minor progestin in elephants, it is present at lower concentrations that are measurable by enzyme-linked immunosorbent assays (elisas), useful for monitoring cyclicity and pregnancy. normal serum p4 concentrations during estrous cycle are in ng/ml and during pregnancy rise substantially over estrous cycle concentrations,1 although the values reported vary among laboratories and with the antibody used. bulls have intrabdominal testis near the kidneys, with no pampiniform plexus; however, normal body temperature is 35 36°c with normal sperm production. bulls have all 4 secondary sex glands. bulls exhibit musth (peripheral testosterone concentrations increase 8 100 times) and challenge bigger, older bulls for access to females in estrus. musth in elephants may occur any time of the year, but poor body condition may delay or prevent musth.2 semen from bulls for artificial insemination (ai) or frozen storage may be collected in a sedated/anesthetized bull via electroejaculation, or in a nonsedated bull by manual rectal massage of the ampullae glands.3 female reproductive anatomy the vulvar opening is not located just beneath the anus, as current understanding of elephant reproduction dennis schmitt,a,b wendy kisoa awhite oak conservation foundation, yulee, fl; bw.h. darr college of agriculture, missouri state university, springfield, mo abstract elephants have many unique reproductive characteristics, including a prolonged estrous cycle and pregnancy, possible embryonic diapause, musth in male elephants, and unique reproductive anatomy of both sexes. most of what is currently known regarding elephant reproduction is from studies conducted from elephants under human care. elephant reproduction continues to be explored and new information is vital for conservation of elephants under human care or in free-ranging habitats. keywords: elephants, musth, estrous cycle, semen, pregnancy, fetal development in other mammals, but is between the back legs, ~ 1 meter below the anus, in an adult elephant. this location facilitates the calf to be delivered horizontally just above the ground below the abdomen. nulliparous cows have a strong hymen present between the vagina and the urogenital tract with 2 or 3 openings; however, usually only 1 is patent, and the others might be remnants of wolffian ducts,2 and are present until the birth of the first calf. the opening is ~ 0.3 cm in diameter. the vagina in multiparous females may be ~ 50 cm long and the cervix has longitudinal folds that resemble equids. the uterus has a small uterine body with long horns 0.5 0.7 meter. the oviduct is relatively short, and the ovary is present close to the uterine tip.2 although the penis does not reach into the vagina, the mobility of the penis and flexure of the glans penis allows penetration of the vertical urogenital canal. ejaculation is into the cranial urogenital canal just anterior to the caudal pelvic brim. a substantial volume of ejaculate is usually expelled at dismount. the hymen remains intact in nulliparous cows after breeding and can rendering artificial insemination challenging. the ejaculate must travel through the opening in the hymen to enter the vagina, and then navigate through the vagina to the cervical os, through the cervix and into the uterus. for sperm to reach the oocyte, they travel over 1 meter from deposition in a nulliparous cow to the site of fertilization. although ovulation is ~ 12 24 hours after second lh surge, the state of meiosis for the oocyte is unknown at ovulation or the interval from ovulation until it is ready for fertilization. additionally, the lifespan of sperm in the reproductive tract of elephants after natural breeding or artificial insemination is unknown.2 the minimum insemination dose is unknown as not enough inseminations have been performed with limited numbers of sperm. fresh inseminations are performed using semen from 13 bulls with shipped, cooled semen and utilizing all samples deemed suitable for clinical theriogenology 2021; 13: 390 insemination at the receiving elephant facility. estrous cycle ovaries are approximately twice as large as in cattle. ovulatory follicles and cls are 1.5 3 cm in diameter. the follicular phase of the estrous cycle is prolonged with 2 lh surges during the 6 8 weeks follicular phase. there are 2 3 nonovulatory follicles on each ovary that partially luteinize following lh1 and remain dormant until lh2, when 1 dominant follicle ovulates and becomes a cl and the nonovulatory partially luteinized cls are thought to activate with lh2.1-7 approximately 3 weeks after progesterone concentrations reach baseline, an lh1 surge occurs with several of nonovulatory follicles partially luteinizing. on average 21 (range 19 23) days after lh1, an lh2 surge stimulates ovulation 12 24 hours later. progesterone concentrations in the us are usually monitored from weekly serum or fecal samples via elisa assays. serum p4 concentrations often briefly increase during 2 3 days prior to lh2 surge, then decrease on the day of ovulation and subsequently increase dramatically within 1 2 days following the lh2 surge. progesterone concentrations are elevated for ~ 10 14 weeks and then decrease to baseline concentrations. estrus begins at ~ 3 4 years in elephants in human care in western countries, and at 6 7 years in range countries. freeranging asian elephants in range countries begin cycling at 5 7 years and at 8 10 years in african elephants. they usually become pregnant after 2 or 3 cycles and have a pregnancy interval of 4 6 years.5 uterine pathologies develop in many nulliparous cycling elephants starting during their mid to late 20’s and early 30’s. asian elephants primarily develop leiomyomas and african elephants primarily develop endometrial cysts; both are identified throughout the uterus.7 it is assumed that these are the result of repeated estrous cycles due to elevated estrogen concentrations, as they are not observed in multiparous elephants that first became pregnant at 6 10 years and have calves every 4 6 years. pregnancy after a successful breeding with fertilization, p4 concentrations remain similar to nonpregnant elephants in their luteal phase until they begin to decrease at 8 9 weeks before recovering at weeks 10 12 postbreeding equivalent to and higher than normal luteal phase concentrations.3 p4 concentrations during first two-thirds of pregnancy are often 2 3 times higher than nonpregnant estrous cycles. during the last third of pregnancy, most elephants’ p4 concentrations begin to decrease, but remain higher than nonpregnant estrous cycles.8-12 prior to 8 9 weeks of pregnancy an embryonic vesicle is not visible via ultrasonography. at 8 9 weeks, the embryo is ~ 1 3 cm in diameter and reaching ~ 5 cm in diameter. over the next 2 3 weeks the embryo grows dramatically until ~ week 12 14 when the head, leg buds, and trunk can be identified via transrectal ultrasonography. during this time, the zonary placenta and umbilical cord become evident. the placenta is endothelial zonary, similar to dogs.2 the possibility that this is a fixed embryonic diapause has been proposed, but it could be slow development of the early embryo and embryonic vesicle rather than a true embryonic diapause. at week 16, the calf is well formed and independent movement of the fetus is observed. crown rump length can be used to determine gestational age in a growing fetus after weeks 10 12 of pregnancy. most detectable embryonic loss occurs after 9 weeks (when the embryo can be detected via transrectal ultrasonography) during the first 14 16 weeks of pregnancy.8 however, fetal loss/abortion can occur throughout pregnancy. in most pregnant cows, the ability to visualize the fetus transrectally is lost due to the pregnant uterus moving below the depth of ultrasound penetration in a standing elephant. however, the ability to visualize the growing fetus can be extended if the pregnant cow is examined in lateral recumbency. the pregnant uterus can often be visualized after ~ 40 weeks of pregnancy while standing as it is usually heavier than the gastrointestinal tract and descends to the floor of the abdomen. this allows transabdominal monitoring of the growing fetus; however, that may not be possible during all examinations.13 the gender of an elephant fetus is not evident in ultrasonography due to similar locations of external genitalia, ovaries, and testes in both sexes. at 12 months the fetus is ~ 30 cm in length and height. the calf continues to grow until term at ~ 93 weeks (~ 660 days) ± 4 weeks. at term, the fetus is 90 cm in height and weighs ~ 100 150 kg in western countries and is 70 80 cm in height and weighs 50 75 kg in range countries. parturition pregnant elephants in the us are usually monitored weekly with p4 elisa assays. as parturition nears, the frequency of serum sampling increases. usually, daily serum sampling begins by day 630 of a known breeding date and is followed closely until parturition. in most elephants, p4 concentrations decrease to baseline 3 5 days prior to onset of phase 1 labor, although phase 2 labor can occur as early as day 2 of baseline or as long as 2 weeks following p4 going to baseline. phase 1 labor is noted by behaviors associated with impending parturition. they may include agitation, frequent urination and defecation, slapping vulva with tail, sudden freezing of stance, etc. the transition of phase 1 to phase 2 labor can be monitored via transrectal ultrasonography. relaxation and dilation of the cervix is visible and the progression of the fetal membranes and fetus into the birth canal can be easily determined.11 calves are presented approximately equally cranially or caudally into the birth canal. prior to the observation of active labor, passing of a cervical plug may be observed. assisted delivery of a calf is limited to manual stimulation of labor contractions by massage of the rectum over the vagina and cranial urogenital canal and/or exogenous oxytocin. prior to treatment, the position of the calf in the birth canal with clinical theriogenology 2021; 13: 391 both feet in position should be confirmed. manual massage of the rectum is performed with a closed fist and substantial amounts of good-quality lubricant. often 3 4 attendants are needed in rotation to initiate productive contractions by the cow. if contractions are not continued, the cow can be treated with low doses of oxytocin. elephants are sensitive to oxytocin and it should be used with discretion. one of the authors uses 40 iu of oxytocin as an initial dose intramuscularly. if after 1 hour, contractions are not maintained, he considers giving 50 60 iu of oxytocin. if the cow has 1 or 2 contractions after either dose and stops immediately, no further stimulation is given to eliminate the risk of uterine rupture. as the placenta and calf pass out of the pelvis it creates a bulge under the anus. often the bulge will disappear from view as contractions ebb and flow. next to appear are the feet of the calf. if the calf is presented caudally, the legs protrude much further compared to cranial presentations. then, the calf turns downward into the vertical urogenital canal. it is during this turn that the chorioallantois ruptures or as often described, when the ‘water breaks’. this occurrence can be difficult to differentiate visually from urination, as there is not much fluid in this compartment. as the umbilical cord is relatively short, time for delivery of the calf after turning downward is critical. it still has almost 2 meters to travel before being expelled. delivery of the calf occurs as the calf is expelled from the vulvar opening, as the calf becomes located just caudal of the umbilicus of the cow due to extension of the vertical urogenital canal forward as the calf passes through. dystocia in elephants occurs primarily because of obesity of the cow and oversized calves. however, occasionally dystocia occurs as the result of malformed calves, or malpositioned limbs. no cow has successfully survived a cesarian surgery.2 however, if the allantochorial membrane has not ruptured and the feet have not passed through the pelvis, the cow will usually survive. the calf may be resorbed, become semi-mummified, or be passed many months later. the cow often begins cycling again in a year or 2 and may deliver the calf later. one of the authors observed a cow beginning to cycle again 2 years later and trying to deliver the calf 5.5 years after term. at term the calf was presented cranially, but when delivered 5.5 half years later, the calf was presented caudally. the calf was extracted via a vestulotomy and weighed > 450 pounds without head and 1 leg that apparently had liquified. there was no indication of an infection upon delivery. the calf was intact except for the parts that had liquified. a few cows have become pregnant subsequent to delivering a retained calf.9 phase 3 of parturition involves expulsion of fetal membranes and usually occurs within a few hours after delivery of the calf, often subsequent to the calf’s first nursing bout. occasionally, elephants experience retained fetal membranes. removal is complicated by the weight (15 20 kg) and the inability to reach it manually. elephants transfer most of their antibodies transplacentally, and there is no true colostrum excreted by the cow. if it is necessary to supplement calves that are not effectively nursing, milk can be extracted from the cow’s mammary glands either by hand milking or by using a human breast pump. elephant nipples have 10 13 milk ducts on each breast. mastitis is rarely noted in elephants. male reproductive anatomy as mentioned earlier, the elephant penis is very mobile and has strong levator penis muscles. the bull has large seminal vesicles that produce most of the ejaculate. ampullae store a substantial amount of sperm and the success of semen collection can be gauged on the size of seminal vesicles and ampullae. however, manual stimulation often results in urine contamination. prostate glands vary in each species. african elephant prostate glands are larger (~ 5 cm) and the lobes are filled with irregular glandular tissue. the asian elephant prostate gland is small (2 cm) and the glandular tissue is homogenous. all of these glands can be reached transrectally with a hand-held ultrasound transducer. the bulbourethral glands are located caudal to the root of the penis and dorso-laterally to the urethra; they can be examined transcutaneously on either side of the penis. testes are located just caudal and medial to kidneys. they can be examined ultrasonographically by using a transducer holder, extending the operator’s reach by 35 45 cm. bulls with testis > 12 cm in diameter usually are considered capable of producing viable sperm.14 testes do not have a pampiniform plexus to cool them as the normal body temperature of elephants is in the range of scrotal testes in other species. additionally, epididymides of the elephant are not adhered to the testes but are coiled ducts that extend to the vas deferens that connect to the ampullae. reproductive evaluation of the bull is hindered by the fact that most collections without an electroejaculator do not yield a complete ejaculate, although a few bulls will respond to manual massage with complete ejaculation and very high-quality semen samples. attempted use of an electroejaculator without heavy sedation or anesthesia is not recommended.3 if the ejaculate does not contain seminal vesicle fluid the sperm are usually immotile. manual massage often results in urine contamination. semen storage high quality semen can be used for fresh cooled insemination. african elephant semen is very tolerant to a range of extenders. a common extender used for fresh cooled insemination with short-term storage can be as simple as hepes buffered saline or as complex as test or homemade egg yolk-based extender.2 the complete ejaculate is extended depending on the sperm concentration 2 4 times and cooled, and put into an equine semen shipper to be sent to the cow’s facility. successful pregnancies occurred in the last few years using african elephant frozen semen. asian elephant semen is not tolerant of simple extenders, and for fresh cooled semen test extender is recommended for cooling with the same protocol as noted for african semen. however, there were no successful asian elephant pregnancies clinical theriogenology 2021; 13: 392 from frozen semen to date, although a conception was reported with early embryonic loss. musth musth is a unique physiological state in elephants. musth bulls become aggressive and their testosterone concentrations are highly elevated. beyond behavioral and endocrine changes, it is manifested by drainage from temporal glands (a modified salivary gland) located between their eye and their ear, excessive urine dribbling, and loss of appetite. during musth, the smell is similar to that of cattle with ketosis, but much more potent. this communicates to other elephants their status, to discourage competition for estrous females. bulls in this state of musth often begin to lose weight and go out of musth.2 however, bulls in human care with high-quality feedstuffs can be obese and may stay in musth for several months, whereas bulls in free-ranging conditions have much shorter musth. it is thought that the dominant bull in free-ranging conditions will cause subordinate bulls to delay their musth around the dominant bull in the region. control of musth of bulls in human care has been a concern for many years. castration was performed in > 20 bulls in the 70’s and early 80’s. however, they were not castrated until they were 4 6 years. several of them had already developed an aggressive attitude and castration did not alter their personalities. there are currently 3 castrated bulls in the us and all are of mild disposition. recently, with the availability of a gnrh vaccine, a few bulls were vaccinated and with subsequent boosters have not displayed musth.2 the success of this vaccine will depend on whether it can be reversed if needed for the bull to resume sperm production, or if vaccinated prior to puberty, whether it will be permanent, or can go through puberty at an older age. conflict of interest there are no conflicts of interest to declare. references 1. brown jl: reproductive endocrinology. in: fowler me, mikota sk: editors. biology, medicine, and surgery of elephants. ames; blackwell: 2006. p. 377-388. 2. schmitt d: reproductive system. in: fowler me, mikota sk: editors. biology, medicine, and surgery of elephants. ames; blackwell: 2006. p. 347-355. 3. hildebrandt, t, lueders, i, hermes, r, et al: reproductive cycle of the elephant. anim reprod sci 2011;124:176-183. 4. hermes r, olson d, goritz f, et al: ultrasonography of the estrous cycle in female african elephants loxodonta africana. zoo biol 2000;19:369-382. 5. lueders i, niemuller c, rich p, et al: gestating for 22 months: luteal development and pregnancy maintenance in elephants. proc r soc b 2012; doi:10.1098/rspb.1038 6. allen wr: ovulation, pregnancy, placentation and husbandry in the african elephant loxodonta africana. philos trans r soc lond b biol sci 2006;29:361:821-834. 7. thitaram, chatchote and brown, janine l. monitoring and controlling ovarian activity in elephants. theriogenology 2018;109:42-47. 8. brown, jl, schmitt, dl, bellem a, et al: hormone secretion in the asian elephant (elephas maximus): characterization of ovulatory and anovulatory luteinizing hormone surges. biol reprod 1999;61:1294-1299. 9. lueders i, niemuller c, gray c, et al: luteogenesis during the estrous cycle in asian elephants (elephas maximus). reproduction 2010;140:777-786. 10. stansfield fj, allen wr: luteal maintenance of pregnancy in the african elephant loxodonta africana. reproduction 2012;143:845-854. 11. hermes r, hildebrandt tb, goritz f: reproductive problems directly attributable to long-term captivity – assymetric reproductive aging. anim reprod sci 2004;82-83:49-60. 12. howard j, bush m, de vos v, et al: electroejaculation, semen characteristics and serum testosterone concentrations of free-ranging african elephants loxodonta africana. j reprod fertil 1984;72:187-195 13. hildebrandt tb: reproductive and diagnostic ultrasonography. in: fowler me, mikota sk: editors. biology, medicine, and surgery of elephants. ames blackwell: 2006. p. 357-355. 14. drews b, hermes r. goritz f, et al: early embryo development in the elephant assessed by serial ultrasound examinations. theriogenology 2008;69:1120-1128. 15. hildebrandt t, hermes r, pratt n, et al: ultrasonography of the urogenital tract in elephants loxodonta africana and elephas maximus: an important tool for assessing female reproduction function. zoo biol 2000;19:321-332. 16. hildebrandt t, hermes r, pratt n, et al: ultrasonography of the urogenital tract in elephants loxodonta africans and elephas maximus: an important tool for assessing male reproduction function. zoo biol 2000;19:333-245. clinical theriogenology 2021; 13: 393 1 contact joe smith animal197@gmail.com © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9609, http://dx.doi.org/10.58292/ct.v15.9609 case report schistosomus reflexus with another fetus in a beef heifer lyndsee miniard,a cailey nichols,a joe smith,a pablo jarrin-yepez,a remigiusz grzeskowiak,a kim newkirkb alarge animal sciences, university of tennessee college of veterinary medicine, knoxville, tn, usa bbiomedical and diagnostic sciences, university of tennessee college of veterinary medicine, knoxville, tn, usa abstract schistosomus reflexus (sr) is a rare, fatal fetal disorder observed in ruminants and other species. defining features include spinal inversion, exposure of abdominal viscera, and limb abnormalities. apparently, this is the first report documenting a case of sr with another fetus in a red aberdeen-angus beef heifer. furthermore, a ventricular septal defect in a case of sr has not been documented before. keywords: beef cattle, heifer, schistosomus reflexus, twins, cesarean surgery background schistosomus reflexus (sr) is a rare, fatal congenital disorder characterized by spinal inversion, exposure of the abdominal organs, and limb abnormalities in fetuses.1 this condition has been reported in cattle, sheep, goats, buffalo, camels, rhinoceros, donkeys, cats, and nonmammalian species (e.g. sea turtles).2–9 abnormalities displayed in cases of sr suggested that the defect started early in embryos, possibly right after gastrulation, and possibly has a heritable component.1 sr is typically observed at parturition, most commonly presenting as dystocia. abnormalities associated with the condition rendered vaginal delivery challenging or impossible, and often a cesarean surgery or fetotomy was necessary.10 because of poor prognosis in some cases, slaughter or euthanasia of the dam was considered. multiple fetuses complicated handling sr cases. multiple fetuses with 1 as sr and the other apparently normal were reported in dairy cattle, buffalo, sheep, and goats, but not in beef cattle.11–14 in cattle, only 4.4% of twin fetuses were associated with a sr fetus.10,11 information on sr involving twins in beef cattle is limited. a beef cow presented for dystocia that had twins, with 1 displaying sr, is described below. case presentation a 2-year red aberdeen-angus heifer (with ~ 6-hour history of dystocia) was presented. parturition was on the expected day (based on the day of successful artificial insemination). heifer was allowed to labor at owner’s farm for several hours; owner palpated (transvaginal) the heifer and felt an abnormal calf and possibly viscera in the uterus. primary veterinarian was called who referred the case. heifer was stable on presentation; however, her vital parameters were higher (rectal temperature: 40.2°c; pulse rate: 100 beats per minute, and respiratory rate: 50 breaths per minute), presumably from labor stress and ambient environmental conditions. small intestinal loops were noticed protruding from her vulva and heifer had a bloody vulval discharge. her ventral abdomen was covered with a mixture of mud and sweat, and she was excessively salivating. fetal viscera were palpable (transrectally) within the vaginal canal and uterus. case management a decision was made to perform cesarean surgery as the heifer had been in active labor for an extended period without progress. fetotomy was not an option because of uncertainty (extent of fetal and possibly uterine pathology). left paralumbar flank approach was adopted (heifer standing). an area behind the last rib to tuber coxae was clipped and prepped. after local anesthesia (inverted l block) with 2% lidocaine, abdomen was opened. an incision (~ 40 cm) was made and additional topical lidocaine blocks (with 2% lidocaine) were used on the musculature. the incision was 10 cm below the transverse processes of the lumbar vertebrae and 10 cm cranial to tuber coxae. abdominal muscle layers were bluntly separated longitudinally from the muscle fiber orientation in a mailto:animal197@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9609 2 citation line: clinical theriogenology 2023, 15, 9609, http://dx.doi.org/10.58292/ct.v15.9609 grid-like pattern. peritoneum was then sharply incised for abdominal cavity entry. uterus was located and exteriorized. a forelimb of a fetus was palpated through the uterine wall and was used as a grip. gentle traction was applied to bring the uterus to the abdominal incision. approximately 30 cm full-thickness incision was made in the uterine wall parallel to palpated fetal limb. this allowed identification of fetal hindlimb. obstetrical chains were placed on the hindlimbs and gentle traction was applied to the dead fetus. a second fetus was felt on further uterine palpation. fetal hindlimbs were exteriorized and the fetus was removed from the uterus in a similar manner to first fetus. gross abnormalities noted were consistent with sr. uterus had 2 separate small, full-thickness lacerations (4 – 5 cm) dorsal and ventral to the uterine incision. uterine tears and the primary incision were closed in 2 layers after the uterus was flushed copiously with sterile saline. first layer of each incision was closed (utrecht pattern) using # 2 polyglactin 910 suture. second layer was closed (cushing inverted pattern) using # 2 polyglactin 910 suture and the uterus was placed back in the abdomen. muscular layer was closed (simple continuous pattern) with # 3 polyglactin 910. subcutaneous tissue was closed (simple continuous pattern) using # 2 polyglactin 910. skin was closed (ford interlocking pattern) with # 2 polymerized caprolactam. after closure, the incision was sprayed with an aluminum spray bandage and the heifer was given antiinflammatory (intravenous flunixin meglumine, 1.1 mg/kg) and perioperative antibiotic (intramuscular procaine penicillin g, 22,000 iu/kg). heifer recovered uneventfully and walked to her stall unassisted. outcome calf 1 was a female red aberdeen-angus, appeared grossly normal, and had no outward abnormalities (identified prior to submitting for necropsy). calf 2 was also a female red aberdeen-angus. this calf’s appearance was consistent with sr and the small intestinal loops protruding from the heifer’s vulva at presentation belonged to this calf. calf appeared grossly normal, and the stillborn female calf was in good condition (body condition score [bcs] of 3 out of 5) and weighed 27.9 kg. postmortem examination revealed abnormalities in heart and lungs, including hemorrhage in the right atrium, a small amount of red fluid in the pericardial sac. abnormally dark and rubbery lungs indicated physiologic atelectasis. findings confirmed that calf’s death was due to dystocia (second fetus was lodged in the vagina preventing normal parturition). calf 2 was grossly abnormal (figure 1); however, was in good condition (bcs 3 out of 5) and weighed 25.9 kg. calf had prominent spinal curvature (lordosis) with failure of fusion of the body wall (noted along the thorax and abdomen). due to failure of body wall closure, both the thoracic and abdominal viscera were exposed. all 4 limbs appeared abnormally angled with apparent fusion of the joints (ankylosis). further examination of the viscera revealed a small, abnormal opening in the interventricular septum of the heart just below the aortic  valve (referred as ventricular septal defect [figure 2]). diagnosis was sr (lordosis, exposure of thoracic and abdominal viscera, and limb). since a definitive cause of sr remained unknown, heifer samples were submitted for neospora caninum, bovine respiratory syncytial virus, toxoplasmosis, infectious bovine rhinotracheitis, bovine viral diarrhea, and for selenium concentrations (table 1). after discharge, the cow was transitioned to oral meloxicam (0.5 mg/kg, once a day for 5 days) for pain control and intramuscular procaine penicillin g (22,000 iu/kg, twice a day for 3 days) for antibacterial coverage during initial recovery. cow was allowed to rest (recovery from uterine surgery and healing of reproductive tract) for 4 – 5 months by her owner. after recovery interval, the cow was allowed to return to normal activity on pasture, where she was placed with a bull. after her first estrous cycle, she was examined table 1. results of serological testing for the heifer sample test result serum neospora caninum – ifa negative serum bovine respiratory syncytial virus – ifa positive serum toxoplasmosis – ifa negative serum infectious bovine rhinotracheitis (ibr) bovine viral diarrhea (bvd) ibr 1:32; bvd 1:64 serum selenium 0.06 ppm – marginal figure 1. schistosomus reflexus calf (calf 2) figure 2. ventricular septal defect (1 x 1 cm) noted in schistosomus reflexus calf (calf 2) http://dx.doi.org/10.58292/ct.v15.9609 citation line: clinical theriogenology 2023, 15, 9609, http://dx.doi.org/10.58292/ct.v15.9609 3 (transrectal palpation) for pregnancy and was determined to be pregnant. cow’s pregnancy progressed normally for the first 4 – 5 months and was sold to another beef cattle producer. at term, the cow delivered a normal calf; further reproductive information is not available (inability to contact the owner). discussion in bovine practice, sr is not a common occurrence, with limited numbers suggesting that it accounted for < 1% of dystocias in certain cattle populations.10 cases of sr involving twins or multiple fetuses are more limited, with some examples in dairy cattle,1,11 lambs,13,15 kids,14,16,17 buffalo,18 camels,5 cats,19 dogs,19 and foals.20 although sr has occurred in ruminants with twins (table 2), this is the first report documenting a case of sr in a beef cattle heifer with twins and a ventricular septal defect (vsd). defining features of sr in ruminants include spinal inversion, exposure of abdominal viscera, limb ankylosis, positioning of the legs adjacent to the skull, and lung and diaphragm hypoplasia.1 in this case, exposure of abdominal viscera and severe spinal inversion and a membranous (vsd) were present. latter defect was not a common finding in other ruminant sr cases.1 although not previously reported in conjunction with sr cases, vsd remains the most common congenital cardiac defect in ruminants, commonly, in the region below the aortic valve (as observed in this case) in cattle.25,26 prevalence of sr in cattle has been reported (0.0127 – 1.3%10), encompassing both dairy (80%) and beef cattle breeds. to authors’ knowledge no such prevalence report exists for sr in red angus cattle. dystocia requiring intervention is a common occurrence with sr cases in bovine practice.28 intervention selection is largely dependent on presentation and position of the fetus,21 and the duration of stage 1 of parturition. factors (e.g. fetal size in relation to vaginal canal) and the expertise of available clinicians cannot be overlooked.29 sr prevalence rate was 0.93% (16 cases out of 1,716 dystocias).27 twin births had a higher incidence of dystocia due to malpresentation of 1 fetus or simultaneous presentation of both fetuses.30 most common methods of correcting dystocias include manual manipulation and delivery of fetus, fetotomy, or via cesarean surgery. cesarean surgery was conducted in 25.6% of cases, fetotomy was performed in 56.7%, and manual traction was successful in only 3.3% of cases.10 mild traction and manual manipulation of the fetus, whether by use of obstetrical chains or other means, is generally pursued first and is a less invasive option.10,31 this method of manipulation was more often reported successful in small ruminant cases of sr than in cattle, unless the sr fetus was smaller than a normal full-term calf.4 if manual manipulation fails, the secondary method is dependent on whether the fetus is viable and whether sufficient space in the uterus and vaginal canal is available for introduction and use of a fetotome. if fetotomy is an option based on space and the fetus has been confirmed dead, a partial or full fetotomy can be performed. in cases of sr, fetotomies are commonly performed when the degree of spinal curvature prevents passage of fetus through the birth canal.21 cesarean surgeries are frequently performed due to inadequate space in the uterus or birth canal and the inability to otherwise reposition the fetus or manipulate a fetotome safely.9,21 surgical management of dystocia allows for a more thorough examination of the reproductive tract of the cow and is the method of choice if presentation of the dystocia was delayed from onset of parturition.32 in each method described, table 2. cases of twin ruminants with sr species findings twin outcome bovine (holstein cross)22 exposed abdominal viscera, marked retroflexion of spine, anterior and lateral positioning of the pelvic bones, sacrum, and hind limbs normal caprine14 mummified fetus, marked ventral spinal curvature, lateral bending of fetal body and chest wall, incomplete closure of abdominal wall with exposed viscera stillborn water buffalo21 thoracic and abdominal viscera exposed, spinal inversion, enlarged liver and rumen conjoined caprine16 ventral spinal curvature, exposed abdominal viscera, incomplete closure of abdominal wall conjoined ovine15 thoracic and abdominal viscera exposed, mummified fetuses, ventral spinal curvature conjoined bovine (holstein)11 prognathism, wry nose, lordosis, atresia ani, bifurcated scrotum, dorsally deviated sternum, total eventration of abdominal and thoracic viscera freemartin ovine13 kyphoscoliosis of thoracic vertebrae, exposed abdominal viscera due to failure of abdominal wall closure conjoined ovine23 thoracic and abdominal viscera exposed, incomplete closure of chest wall, lateral reflection and ankylosis in lumbar and thoracic regions normal caprine24 thoracic and abdominal viscera exposed, acute angulation and ankylosis of lumbar vertebrae normal http://dx.doi.org/10.58292/ct.v15.9609 4 citation line: clinical theriogenology 2023, 15, 9609, http://dx.doi.org/10.58292/ct.v15.9609 medical management (e.g. caudal epidural) and providing adequate sedation and pain control should be executed. although currently it is believed that sr is heritable condition in ruminants,1 recent work in sea turtle has suggested a toxicity or epigenetic etiology.33 in olive ridley sea turtle (lepidochelys olivacea) embryos with sr malformations and without malformations, a positive correlation was identified with mercury concentrations and dna methylation in embryos with sr abnormalities.22 multiple drugs and compounds caused changes in dna methylation and presented with epigenetic or quasiepigenetic responses, including beta-lactam antibiotics, lithium, and opioids.34 currently, the association is between sr in cattle and any toxicities or epigenetic changes is unknown. future studies should investigate the role of either of these potential influences on sr in ruminants. learning points • sr is a rare and fatal condition observed in cattle. defining features of the condition include spinal inversion, exposure of abdominal viscera of fetus. • a case of sr (with ventricular septal defect) with a normal twin fetus in a red angus beef heifer is documented. • although fetotomies are the most frequently utilized method of extracting a single sr fetus, in cases of twins, cesarean surgery may be necessary due to inadequate space (uterus and birth canal) for manipulation. conflict of interest none to declare. acknowledgement authors thank dr. pierre-yves mulon for assistance in management of the case after surgery. references 1. laughton kw, fisher kr, halina wg, et al: schistosomus reflexus syndrome: a heritable defect in ruminants. anat histol embryol 2005;34:312–318. doi: 10.1111/ j. 1439-0264. 2005.00624.x 2. tharp vl: bovine dystocia caused by schistosomus reflexus. cornell vet 1947;37:394–396. 3. tsuma v, abuom t: a case report of schistosomus reflexus in a lamb. kenya vet 2008;32:41–43. doi: 10.4314/kenvet.v32i1.45298 4. suthar d, sharma v, dabas v, et al: per-vaginal handling of schistosomus reflexus as a cause of dystocia in a goat. vet world 2011;4:330–331. doi: 10.5455/vetworld.4.330 5. elias e: left ventrolateral cesarean section in three dromedary camels (camelus dromedarius). vet surg 1991;20:323–325. doi: 10.1111/j.1532-950x.1991.tb01277.x 6. dubbin es, welker fh, veit hp, et al: dystocia attributable to a fetal monster resembling schistosomus reflexus in a donkey. j am vet med assoc 1990;197:605–607. 7. kawata k, tiba t: a rare case of schistosomus reflexus in the cat. j vet res 1961;9:179–181. 8. bárcenas-ibarra a, rojas-lleonart i, lozano-guzmán ri, et al: schistosomus reflexus syndrome in olive ridley sea turtles (lepidochelys olivacea). vet pathol 2017;54:171–177. doi: 10.1177/0300985816651682 9. dutt r, singh g, chandolia r: delivery of a schistosomus reflexus monster through caesarean section in a murrah buffalo. buffalo bull 2019;38:165–168. 10. knight rp: the occurrence of schistosomus reflexus in bovine dystocia. aust vet j 1996;73:105–107. doi: 10.1111/j.1751-0813.1996. tb09988.x 11. cavalieri j, farin p: birth of a holstein freemartin calf co-twinned to a schistosomus reflexus fetus. theriogenology 1999;52:815–826. doi: 10.1016/s0093-691x(99)00174-0 12. sitali m, mwaanga e, zulu v, et al: schistosomus reflexus from a holstein-friesian cow-case report. theriogenol insight 2014;4:65. doi: 10.5958/2277-3371.2014.00735.9 13. dennis s: schistosomus reflexus in conjoined twin lambs. vet rec 1972;90:509–510. doi: 10.1136/vr.90.18.509 14. bawaskar m, sahatpure s, sheetal s, et al: mummified schistosomus reflexus co-twinned with normal goat foetus-a rare case report. int j environ sci technol 2018;7:1479–1482. 15. motunrayo ah, ogechi n, bire gd, et al: dystocia due to schistosomus reflexus (cojoined twins) in a yankassa ewe. j coast life med 2015;3:333–335. 16. olaogun s, abiola j, jeremiah o: case report: dystocia due to schistosomus reflexus (co-joined twins) in a west african dwarf doe (female goat) in ibadan, nigeria. trop vet 2017;35:117–122. 17. singh d, kumar p, et al: dystocia due to schistosomus reflexus (co-twin) in a marwari goat-a case report. life sci leafl 2017;94:10–12. doi: 10.21887/ijvsbt.v12i4.7690 18. rao a, sreemannarayana o, rao a: schistosomus reflexus in a buffalo conjoined twin-a case report. indian vet j 1984;61:80. 19. bertolo phl, de aguirra lrvm, martins dm, et al: schistosomus reflexus in a dog and a cat. acta sci vet 2017;45:4. doi: 10.22456/1679-9216.86180 20. addo p, cook j, dennis s: schistocoelia in a twin foal. equine vet j 1984;16:69–71. doi: 10.1111/j.2042-3306.1984.tb01858.x 21. pandey ak, kumar s, gunwant p, et al: schistosomus reflexus monster fetus in bovine and its successful management. res j vet pract 2017;5:25–27. 22. pugazharasi c, jaganath v, sarath t, et al: per-vaginal delivery of schistosomus reflexus foetal monster co-twined with live calf in a holstein-friesian crossbred cow at field level. haryana vet 2022;61:123–124. 23. smith, id, schistosomus reflexus in conjoined twin lambs. vet rec 1969;85:138–139. doi: 10.1136/vr.85.6.138 24. bedford, pgc: schistosomus reflexus in a goat: a case report. vet rec 1967;80:326. 25. streeter rn, step dl: diagnostic ultrasonography in ruminants. vet clin north am food anim pract 2007;23:541–574. doi: 10.1016/j.cvfa.2007.07.008 26. harmon mw: ruminant and camelid cardiology. in: durham he, ed. cardiology for veterinary technicians and nurses, hoboken, nj: wiley-blackwell; 2017;443–458. http://dx.doi.org/10.58292/ct.v15.9609 https://doi.org/10.1111/​j.​1439-0264.​2005.00624.x https://doi.org/10.4314/kenvet.v32i1.45298 https://doi.org/10.5455/vetworld.4.330 https://doi.org/10.1111/j.1532-950x.1991.tb01277.x https://doi.org/10.1177/0300985816651682 https://doi.org/10.1111/j.1751-0813.1996.tb09988.x https://doi.org/10.1111/j.1751-0813.1996.tb09988.x https://doi.org/10.1016/s0093-691x(99)00174-0 https://doi.org/10.5958/2277-3371.2014.00735.9 https://doi.org/10.1136/vr.90.18.509 https://doi.org/10.21887/ijvsbt.v12i4.7690 https://doi.org/10.22456/1679-9216.86180 https://doi.org/10.1111/j.2042-3306.1984.tb01858.x https://doi.org/10.1136/vr.85.6.138 https://doi.org/10.1016/j.cvfa.2007.07.008 citation line: clinical theriogenology 2023, 15, 9609, http://dx.doi.org/10.58292/ct.v15.9609 5 27. sloss ve, johnston de: the cause and treatment of dystocia in beef cattle in western victoria. aust vet j 1967;43:13–21. doi: 10.1111/j.1751-0813.1967.tb04757.x 28. kipouridis k, karagiannidis a: the occurrence of schistosomus reflexus in bovine dystocia. j hell vet med soc 2001;52:264–266. doi: 10.12681/jhvms.15455 29. purohit gn, kumar p, solanki k, et al: perspectives of fetal dystocia in cattle and buffalo. vet sci dev 2012;2:e8:31–42. doi: 10.4081/vsd.2012.3712 30. gregory ke, echternkamp se, cundiff lv: effects of twinning on dystocia, calf survival, calf growth, carcass traits and cow productivity. j anim sci 1996;74:1223–1233. doi: 10.2527/ 1996.7461223x 31. funnell bj, hilton wm: management and prevention of dystocia. vet clin north am food anim pract 2016;32:511–522. doi: 10.1016/j.cvfa.2016.01.016 32. purohit gn, mehta js: dystocia in cattle and buffaloes: a retrospective analysis of 156 cases. vet pract 2006;7:31–34. 33. martín-del-campo r, bárcenas-ibarra a, lund g, et al: mercury concentration, dna methylation, and mitochondrial dna damage in olive ridley sea turtle embryos with schistosomus feflexus syndrome. vet pathol 2019;56:940–949. doi: 10.1177/ 030098 5819868649 34. anderson sj, feye km, schmidt-mccormack gr, et al: off-target drug effects resulting in altered gene expression events with epigenetic and ‘quasi-epigenetic’ origins. pharmacol res 2016;107:229–233. doi: 10.1016/j.phrs.2016.03.028 http://dx.doi.org/10.58292/ct.v15.9609 https://doi.org/10.1111/j.1751-0813.1967.tb04757.x https://doi.org/10.12681/jhvms.15455 https://doi.org/10.4081/vsd.2012.3712 https://doi.org/10.2527/​1996.7461223x https://doi.org/10.2527/​1996.7461223x https://doi.org/10.1016/j.cvfa.2016.01.016 https://doi.org/10.1177/​030098​5819868649 https://doi.org/10.1016/j.phrs.2016.03.028 38 contact jennifer koziol jkoziol@ttu.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9594, http://dx.doi.org/10.58292/ct.v15.9594 research report a survey of the use of theriogenology related models across veterinary curricula jennifer koziol, philippa gibbons, john dascanio texas tech school of veterinary medicine, amarillo, tx, usa abstract the use of models within theriogenology curricula has become increasingly more common over the last few years. currently, there are no compiled data regarding the types of theriogenology related models used at veterinary institutions across the world. a 55-question survey regarding the use of models in the theriogenology curricula was distributed via multiple listservs. a total of 31 responses were garnered representing 24 veterinary institutions from 5 continents. data collected in the survey indicated that large animal transrectal palpation models, dystocia models, and small animal ovariohysterectomy and castration models are the most used models. commercial models are widely used; however, multiple models are built at institutions. only 1 model listed by respondents has been validated in the literature. institutionally created models are most often built by faculty and most of the models described are used only for developing technical skills. a large percentage of respondents that listed models believed that the models improved students’ technical skills, but for the most part are not a substitute for live animal experiential learning. keywords: model, theriogenology, veterinary medicine, clinical skills introduction in the past 2 decades, model use in veterinary curricula has become widespread.1 veterinary institutions have moved toward a competency-based curriculum.2 this trend has necessitated the need for clinical and professional skills courses in the curriculum to be developed or to become more robust. traditionally, cadaver tissue was used before students were introduced to live animals, particularly for surgical skills, whereas other hands-on skills were performed first on live animals.3 however, challenges in sourcing cadaver tissue, increasing class sizes, costs of animals and cadavers, and institutional animal care and use committees (iacuc) and the united states department of agriculture (usda) regulations led to efforts to reduce, refine, or replace (3 rs) direct animal use.4 this, combined with identified gaps between traditional approaches to training veterinary professionals and current demands of the modern veterinary practice to hire veterinarians who are practice-ready, has increased the need for the use of models and clinical skills simulators within veterinary medical education.5–8 the use of models in the curriculum can also increase the time dedicated to practicing and enhancing skill development by the student. model use has been demonstrated to be effective in teaching clinical skills in veterinary medicine.5 models can come in a variety of forms, from low fidelity to high fidelity, commercially produced or made at institutions. highfidelity models are characterized as being life-like and mimic a high level of realism, often using technology. comparatively, low-fidelity models use minimal to no technology, do not interact with the student, are not life-like, and are not intended to hone specific technical skills.9,10 veterinary simulation models commonly would be considered mediumto low-fidelity with a small number of high-fidelity models available for use in the veterinary curriculum. nonetheless, even low-fidelity models were sufficient for teaching veterinary clinical skills.11–13 cadaver tissue may be used in models to mimic realism or better simulate experience with live-animals.3 high-fidelity models often are associated with higher cost, potentially limiting the number of models available for instruction. the level of fidelity chosen by an institution should provide the end user with the simulation needed to achieve the skill desired, thus fidelity level of the model should be validated. models of varying fidelity can be used to provide users with the means to learn a new skill, to refine a current skill, or to expand current skill repertoire. the use of models has meant that clinical skills can be introduced earlier in the curriculum than previously, due to the decreased chance in causing harm to an animal secondary to lack of knowledge or experience. models allow for multiple reiterations/practice sessions that ultimately allows for greater development of technical skills.1 introduction to technical skill is key in developing mastery.1 models can also be available 24 hours per day and 7 days a week, enabling students to practice technical skills without direct veterinary supervision but with clinical use direction. simulations and models can also be used throughout the curriculum when there is the desire to add more skills than animal or case availability mailto:jkoziol@ttu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9594 citation line: clinical theriogenology 2023, 15, 9594, http://dx.doi.org/10.58292/ct.v15.9594 39 allows (e.g. learning fracture repair or surgical procedures). clinical skills can be combined with professional skills, allowing students to integrate hard and soft skills.6,14 models offer many advantages in the curriculum. one of the top advantages is that it creates a ‘safe space to fail’ for the student, without the pressure of harming a live animal.15–18 in addition, the challenge of time or repeated use is not an issue compared to live animal.18 models can also be useful to simulate situations when it is not possible to have large numbers of live animals, there are a large number of students that need experience in a certain procedure that far surpasses the number of animals a university has, or a certain procedure (e.g. cesarian surgery) that is hard to recreate on demand. multiple studies across species have investigated what skills and other competencies are required of new veterinary graduates. common reproductive skills and competencies that appear in these studies include, but are not limited to, bovine castrations, bovine pregnancy transrectal palpation and ultrasonography, bovine dystocias including the use of fetotomy, bovine cesarian surgery, bovine and equine artificial inseminations, bull breeding soundness examinations, equine pregnancy transrectal palpation and ultrasonography, small animal abdominal ultrasonography, small animal castrations and ovariohysterectomies, and swine reproductive examinations.19–31 these studies have focused on what species-specific veterinarians do for the most part, leaving individual institutions to adapt their skills lists for core and elective courses as they reflect on their specific curricula and regional veterinary needs. as mentioned, models can be bought or developed intra-institutionally. purchased models are often expensive, limiting the number potentially purchased, whereas models created in-house require a space to develop and build the model, labor and expertise in construction, and purchase of construction equipment and building materials. many types of equipment can be potentially used to create models, varying from simple metal and woodworking machinery to more complex equipment such as 3-d printers, vacuum formers, and computer numerical control machines. a search of veterinary models that pertain to the field of theriogenology, and that were published as validated models in the peer reviewed literature, include bovine castration,32 canine ovariohysterectomy,10,33,34 bovine transrectal palpation,6,35–39 canine castration,40,41 and an ovine cesarian surgery model.42 commercially available theriogenology related models include bovine and equine palpation (veterinary simulation industries (vsi) calgary, ab, canada, breed’n betsy byaduk, victoria, australia), dystocia (vsi), artificial insemination (breed’n betsy), uterine prolapse (breed’n betsy), and canine spay (vsi, syndaver tampa, fl, rescue critters simi valley, ca). the objective of this paper is to describe models that are used in veterinary theriogenology curricula worldwide, and how they are integrated into the curriculum. materials and methods an online survey was created using survey monkey® and distributed on the following listservs: society for theriogenology large animal, society for theriogenology small animal, american college of theriogenologists, and veterinary clinical skills + simulation, allowing for a world-wide audience. a 55-question survey asked respondents geographic information, general information about their veterinary curriculum, which skills they used theriogenology models for, and in what context they most frequently used the models. additionally, respondents could choose to give details on 2 of the main models used, and check a list of all models that they used in their program. an ‘other’ category was available for models not included on list. respondents could choose to remain anonymous and choose not to answer questions (appendix). descriptive analyses of the survey results were performed. results a total of 31 responses were collected from a minimum of 24 veterinary institutions representing 5 continents (all but antarctica and south america). the response rate cannot be calculated as the number of people that accessed the survey is unknown. some respondents chose to not identify their home institutions. six respondents chose to remain anonymous. full completion rate for this survey was 55% including description of at least 1 model; respondents finished in an average of 5 minutes. six of the 31 respondents reported using a validated model. twenty-six various types of models were further described by the survey takers that included: 6 large animal dystocia, 5 canine ovariohysterectomy, 4 bovine transrectal palpation, 4 equine transrectal palpation, and 1 each of equine female perineum, small ruminant vaginal suture, feline ovariohysterectomy, canine transcervical insemination, feline castration, canine castration, and small ruminant dystocia. models were most commonly used in year 3, followed by years 4, 2, 5, and 1. models were most frequently used only for technical skills with 1 model used for professional skills/communication training and 4 models used in clinical scenarios. those models used in clinical scenarios include canine ovariohysterectomy, canine transcervical insemination, bovine dystocia, and bovine transrectal palpation. of the models further described and built at institutions, most were built by faculty members. these models included: transrectal palpation boxes, and models for large animal dystocia, canine ovariohysterectomy, equine female perineum, bovine dystocia, small ruminant vaginal suture, feline castration, canine transcervical artificial insemination, canine abdomen, canine castration, and feline ovariohysterectomy. eight of the described models were built by faculty, with 2 involving students. five were built by staff/veterinary nurses and 1 respondent reported using a model builder associated with the university. nine models were obtained from commercial vendors. the most frequently named vendors and models were vsi, syndaver, and breed’n betsy and breed’n bonnie. cadaver tissue was reported to be used with 3 models including 2 transrectal palpation models and a large animal dystocia model. tissue was most commonly sourced from an abattoir, followed by sourcing from the institutional necropsy service. http://dx.doi.org/10.58292/ct.v15.9594 40 citation line: clinical theriogenology 2023, 15, 9594, http://dx.doi.org/10.58292/ct.v15.9594 types of theriogenology models (figure) dystocia and fetotomy models large animal dystocia models were the most common type of model reported by respondents with 74% stating that they use 1 or more types of models within the curriculum. a variety of models were described from commercially available sources including bovine models from vsi to bovine models made in-house from plywood and uterine bags. one lambing model and 1 farrowing model were also reported; these were made within their respective institutions. cadaver tissue may or may not have been used in these models. cadaver and live animal experiences usually followed the use of dystocia models, depending on the institution. respondents reported that dystocia models on average improved students’ skills and were a moderate to good replacement for live animals. the 6 respondents who gave further information regarding dystocia models stated that they owned between 1 and 5 dystocia models, with an average of 3. they also reported a ratio of students to faculty ranging from 4:1 to 24:1 with an average of 13:2. one small ruminant dystocia model was described. two models were used in that particular curriculum. the model was used at a student to faculty ratio of 5:1 and was thought by the respondent to greatly improve students’ technical skills and was a very acceptable substitute for live animals. fetotomy models are also frequently used with 45% of participants marking that they used that model in their curriculum. no respondents gave further details. transrectal palpation models equine and bovine transrectal palpation models are the second most commonly reported models. commercially available models were most frequently used with vsi, breed’n betsy, and breed’n bonnie being named. homemade bovine transrectal palpation boxes were also mentioned by some respondents. four respondents gave more information regarding bovine transrectal palpation models. they reported an average of 4 models per institution with a range of 1 to 8. student to faculty ratios ranged from 6:1 to 35:3 with an average of 8:1. most respondents felt that models greatly improved student skills but were not adequate replacements for transrectal palpation of live animals. models were primarily used as an introduction to transrectal palpation and were followed by palpation of abattoir-derived reproductive tracts and live animals. no respondents further elaborated on the use of equine transrectal palpation models in the curriculum. small animal ovariohysterectomy and castration models thirty-five percent of respondents reported that canine ovariohysterectomy models were used in their curriculum, whereas 19% reported using feline ovariohysterectomy models and 26% reported the use of both canine and feline castration models. both commercial (syndaver) and intra-institutionally derived models were described, with 1 validated canine castration model.40 use of canine ovariohysterectomy models was further described by 5 respondents. it was reported that institutions owned between 2 and 50 ovariohysterectomy models with an average of 26. student to faculty ratios for the use of the models ranged from 4:1 to 21:2, with an average ratio of 19:2. model use was commonly followed by live animal surgeries, or cadaver use was followed by live surgeries. respondents believed that use of models improved the students’ skills but were not an adequate substitute for live animals. one feline ovariohysterectomy model was further described. the respondent indicated that the institution owns 50 of these models and that the model was used with a student to faculty ratio of 15:2. exposure to the model was followed by cadaver and live animal experiences. the respondent reported that the model improved students’ skills but was not a replacement for live animals. a feline castration model was also described by 1 respondent. only 1 model was owned by the institution, and it was used with a student to faculty ratio of 12:1. this model was the only experience for this activity within the institution and was thought to greatly improve technical skills, but not a replacement for live animals. cesarian surgery models nineteen percent of respondents reported the use of large animal cesarian surgery models; only 1 respondent noted the use of small animal cesarian surgery models. no further description of these models was given by respondents. artificial insemination and advanced reproductive techniques models multiple models representing artificial insemination in various species were utilized by respondents. eleven respondents reported the use of models for large animal artificial insemination. additionally, there was 1 report of the use of a porcine artificial insemination model, 1 report on the use of a small ruminant laparoscopic artificial insemination model, and 1 report on the use of a canine transcervical insemination model. regarding the transcervical insemination model, the respondent reported that the model was made intra-institutionally and its use was followed by procedures on live animals. the model was used with a student to faculty ratio of 1:1. the respondent reported that the canine transcervical insemination model slightly improved students’ skills and was not a replacement for live animals. a bovine embryo transfer model was reported by 1 respondent, but no further information was reported. large animal castration models twelve percent reported the use of bovine castration models, and 9% of respondents reported the use of equine castration models. no further descriptions were given. caslick’s surgery models nine percent used equine caslick’s surgery models. no further descriptions were given. http://dx.doi.org/10.58292/ct.v15.9594 citation line: clinical theriogenology 2023, 15, 9594, http://dx.doi.org/10.58292/ct.v15.9594 41 large animal vaginal prolapse models nine per cent of respondents reported the use of large animal vaginal prolapse models. one respondent described the use of a small ruminant vaginal suture model for teaching technical skills associated with large animal vaginal prolapse. two of these models were available for use with a student to faculty ratio of 8:1. the respondent believed that the model improved students’ skills and was not an acceptable replacement for live animals. other models multiple other models were noted by respondents including 1 canine neonatal care model, 1 male dog urethral catheterization model, 1 pregnancy ultrasonography model, and 1 equine perineal preparation model. the equine perineal preparation model was used in a veterinary nurse program with a student to faculty ratio of 15:1. use of this model was the only experience with the technical skill and the survey respondent believed that it greatly improved students’ skills and was an adequate replacement for live animals. discussion a total of 31 respondents representing 5 continents and both 4and 5year veterinary education programs were studied. large animal transrectal palpation, large animal dystocia, and small animal ovariohysterectomy and castration models were the most prevalent models used by the respondents that provided feedback. it is not surprising that these models were the most prevalent considering the reduced number of teaching animals available at some veterinary institutions, decreased use-limits per animal, increasing class sizes, and increased controversy surrounding the use of cadavers in veterinary medical education.6 all of the respondents felt that models improved students’ skills to some degree. respondents reported that dystocia models could replace live animal, whereas other model types (specifically transrectal palpation and surgical) could not entirely replace live animals. this is important as it is not possible to replace training with live animals for all skills performed on a model. as an example, use of models as preparation for progression to live surgery is highly valuable for sequential development of skill level and confidence. commercially available full species models, while widely used, are expensive to buy, with some costing over $10,000 usd for a single model. they may also be costly to replace if damaged during use or if one must replace parts due to single-use or wear-and-tear. to reduce expenses and allow for a smaller student to model ratio it is often necessary to build lower-fidelity models at an institution. based on the findings of this survey, that job primarily falls to faculty who may have other academic responsibilities and may not have expertise in model building. additionally, it could not be elucidated from our survey if model building was the primary duty of the staff/veterinary nurses that are creating models or if this was an additional duty. the price of intra institutionally derived models was also highly variable and was reportedly inconsistent and therefore was not included in the results. other factors to consider are the expertise needed for model building, the cost per square foot of space allocated for construction equipment, the cost of construction equipment, maintenance of equipment, licenses and fees associated with software, and numerous other expenses depending on the complexity of the model-building space and needs of the institution. the above-mentioned factors can weigh into the decision about where and how models are created by each institution. as the use of models and the increased demand to teach clinical skills grows, faculty and staff alike may find it very useful to turn to validated models and methods to aid in the development and refinement of courses. the authors believe that continuing to foster a culture of collaboration and sharing of model design is important to improve student competency. for those unfamiliar with model validation, validation of a model is an endeavor by the creator to associate data such as scores produced by a grading rubric with the skill that the model is claimed to teach. validation methodology is highly varied amongst published reports, but current dogma would suggest that a validation argument should be constituted as 5 sources of validity evidence.10,43–45 a validated, effective model would assemble evidence from several but not all 5 sources. these 5 sources include: content evidence, response process evidence, internal structure (reliability) evidence, evidence of relationship with other variables, and consequences evidence.10,43 validation of veterinary models has taken the form of experts on the subject using the models and then providing their opinion on the realism, fidelity, and effectiveness of the model for skill development. alternatively, validity studies have taken the form of students using a model versus another accepted training model (e.g. cadaver or training video) with final assessment on a traditional teaching method (e.g. live animal). the associated rubric along with the model may then be used by other institutions. the process of validation of a model and creation of an associated grading rubric can be very time consuming.10 nonetheless, use of shared resources may help many educators as they develop and build their institutions’ clinical skills program. another point of discussion is the student to faculty ratio. the ideal ratio likely varies depending on the educational level of the student, type of task, and the type of prelaboratory learning that is expected, but most tasks are guessed to be in the range of 4:1 to 12:1 for effective teaching.46 a ratio of 10:1 student to faculty ratio was efficient and effective for teaching suturing in one study.46 teaching ratios reported for many of the models far exceeded this ratio and it would be of interest to determine many of the models noted far exceeded this ratio and it would be of interest to determine if this is an appropriate ratio for those models as noted by faculty and students. authors recognize several survey limitations. the survey was taken by participants on multiple continents in which the veterinary curriculum differs greatly in the number of years spent in a program and the definition of preclinical versus clinical training. another limitation was that the survey was lengthy and further questions were not added in efforts to prevent survey fatigue and decreased response rate. a final limitation was the relatively low response rate to the survey, likely associated with inability of this study to capture all reproductive models that are in use at veterinary colleges across the world. conclusion models in the theriogenology curriculum do not replace live animals but do allow veterinary students to gain key technical skills prior to performing skills on live animals. this allows students to gain mastery of a skill, to practice in a safe situation, and have repetitive practice without detriment to an animal while embracing the 3 rs of animal use. models do not http://dx.doi.org/10.58292/ct.v15.9594 42 citation line: clinical theriogenology 2023, 15, 9594, http://dx.doi.org/10.58292/ct.v15.9594 have to be high-fidelity to be effective teaching models and many models may be built intra-institutionally. a cost-analysis should be considered when trying to decide if models should be bought or built in-house. dedicated faculty and staff are often needed by a program to effectively provide clinical skills opportunities and develop models. many iterations of a model may need to be developed and tested before an effective model is produced. continued validation of theriogenology models followed by publication may be an aid to help faculty and staff across institutions provide effective learning opportunities for their students. there is a need amongst veterinary institutions to create a repository of model building plans, how the specific model is used, and how the model can be altered for other purposes. in conclusion, the use of models and simulators are advantageous for augmentation of theriogenology curriculum, and can be used to improve the reproductive skill levels of veterinary graduates. conflict of interest none authors’ contribution the corresponding author declares that everyone who made significant contributions to the study or manuscript appear as coauthors. furthermore, all authors contributed to design and analysis of the survey and construction of the manuscript. references 1. rösch t, schaper e, tipold a, et al: clinical skills of veterinary students – a cross-sectional study of the self-concept and exposure to skills training in hannover, germany. bmc vet res 2014;10:969. doi: 10.1186/s12917-014-0302-8 2. competency-based veterinary education (cbve). available from: https://cbve.org/framework [cited 18 december 2022]. 3. hart la, wood mw, weng hy: mainstreaming alternatives in veterinary medical 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enter the name of your institution 2. please enter the name of your institution 3. if you would like to share information about how you built your model(s) please enter your name and email 4. please enter your continent of origin a. north america b. south america c. europe d. australia/oceania e. asia f. africa 5. please check all models that you use in your program. add additional in the other option a. canine ovariohysterectomy b. canine castration c. feline ovariohysterectomy d. feline castration e. bovine castration f. equine castration g. equine caslicks h. large animal dystocia i. large animal fetotomy j. bovine palpation per rectum k. equine palpation per rectum l. large animal vaginal prolapse m. large animal uterine torsion n. large animal ai o. small animal cesarian section p. large animal cesarian section q. other (please specify) 6. you will have the opportunity to add up to 2 of the major models you use. a comment box at the end of the survey will allow you to list all models you use. please enter the name of the reproductive model you most commonly use 7. what technical skill is this model for a. canine ovariohysterectomy b. canine castration c. feline ovariohysterectomy d. feline castration e. bovine castration f. equine castration g. equine caslicks h. large animal dystocia i. large animal fetotomy j. bovine palpation per rectum k. equine palpation per rectum l. large animal vaginal prolapse m. large animal uterine torsion n. large animal ai o. small animal cesarian section p. large animal cesarian section q. other (please specify) 8. which year of the curriculum is this model primarily used a. year one b. year two c. year three d. year four e. year five 9. choose all years that the model is used in the curriculum a. year one b. year two c. year three d. year four e. year five 10. what is the course title for the course where this model is primarily used 11. is this course core (required) or elective 12. is the model used for technical skills only or in combination with a clinical scenario or professional skills (communication training) a. only technical skills b. used with clinical scenarios c. used with professional skills (communication training) d. other (please specify) 13. where is the model obtained a. commercial model vendor b. another university’s workshop c. created within your institution d. other (please specify) 14. please enter the name of the commercial vendor 15. is the model you are using validated in the literature a. yes/no 16. please enter the citation for the model validation 17. does the model use cadaver tissues a. yes/no 18. what is the source of the cadaver tissue a. in house pathology service/euthanasia of in-house case b. local abattoir/slaughterhouse c. local shelter d. other (please specify) 19. how many of this exact model do you use in the primary course at one time 20. how many students are in the class where this model is used 21. how many students are in the instructional laboratory where this model is primarily used (i.e., 50 students per lab group) 22. how many instructors are in the laboratory where this model is primarily used 23. what is the cost to create/purchase this model (in usd) 24. what is the cost to maintain this model (in usd) 25. do you use this model as the only experience prior to students going to clinics (clinical rotations) with live animals a. yes, the model is the only experience for this activity b. no, a live animal experience follows the use of the model c. no, a cadaver experience follows the use of the model http://dx.doi.org/10.58292/ct.v15.9594 citation line: clinical theriogenology 2023, 15, 9594, http://dx.doi.org/10.58292/ct.v15.9594 45 d. no, a cadaver then a live animal experience follows the use of this model e. no, another model follows the use of this model f. other (please specify) 26. do you feel this model has improved students’ skills a. a great deal b. a lot c. a moderate amount d. a little e. none at all 27. do you feel the use of this model can replace use of live animals a. a great deal b. a lot c. a moderate amount d. a little e. none at all 28. do you feel you have the expertise to create this model in house, whether by you or others at your institution a. a great deal b. a lot c. a moderate amount d. a little e. none at all 29. do you have another model to enter (if yes, questionnaire repeats) figure. percentage of different types of theriogenology models used by respondents. http://dx.doi.org/10.58292/ct.v15.9594 21 contact darcie sidelinger darcie.sidelinger@msstate.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9640, http://dx.doi.org/10.58292/ct.v15.9640 review report surgical management of bovine inguinal herniation darcie sidelinger,a richard hopper,b heath kinga adepartment of pathobiology and population medicine, college of veterinary medicine, mississippi state university, starkville, ms, usa bdepartment of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa abstract inguinal herniation is not a rare occurrence in cattle, there are on average one to two cases per year in the authors’ experience, and surgical management is required if the goal is correction of the problem. bovine inguinal hernias are classified as either congenital or acquired. acquired hernias are further differentiated as direct or indirect depending on pathogenesis and whether or not there is damage to the tunic. two surgical approaches are described below and can be successfully utilized for repair. following surgery and recovery, bulls often return to breeding without incident. keywords: bovine, bull, hernia, surgical repair introduction bovine inguinal hernias are classified as either congenital or acquired based on when they occur in the life of a bull. congenital inguinal hernias occur in neonatal calves and are considered hereditary. surgical repair can be performed; however, bilateral castration is recommended at the time of the procedure. acquired inguinal hernias occur in adult bulls and are classified based on their potential pathogenesis or what structures they disrupt. indirect inguinal hernia is the most common and occurs when abdominal contents exit through the normal, but dilated opening of the inguinal ring. conversely, direct inguinal hernias occur when normal anatomy is disrupted, and abdominal contents enter the scrotum through an acquired defect in the peritoneum or body wall.1–3 herniation tends to occur more frequently on the left side. this is hypothesized to be related to the weight of the rumen and the propensity of cattle to lie in right sternal recumbency with the left rear leg abducted, according to wolfe and rodning.4 presentation of the patient varies based on the nature of the hernia. because indirect inguinal hernias technically occur through a normal, but dilated opening, they often do not present with urgent and life-threatening clinical signs. the most common complaint is swelling of the scrotum often with a characteristic hourglass appearance.4 due to the lack of clinical signs, indirect inguinal hernias often go unnoticed and can become chronic leading to adhesion formation which can result in compromise of the bowel and abdominal pain. direct inguinal hernias more often present acutely with signs of abdominal pain or hypovolemia.4 this is due to the nature of direct inguinal hernias occurring through a traumatic rupture of the peritoneum. the peritoneum can be damaged and torn by the bull fighting with other bulls or attempting to jump over a fence resulting in the viscera escaping retroperitoneally into the neck of the scrotum.5 direct inguinal hernias are emergent as incarceration of the intestine is a common sequela.4 diagnosis of inguinal hernias can be achieved through transrectal palpation of the dilated inguinal ring or body wall defect while appreciating intestine exiting through the ring or body wall. more accurate clinical diagnosis can be made through ultrasonography of the swelling and identifying loops of bowel within the scrotum.1,2,5 once diagnosed, an approach to repair can be considered based on the value of the bull and plans of the producer. the following are discussions of the methods of surgical correction of this condition in the bull. inguinal approach the patient fasts 36–48 hours prior to surgery with water removed in the last 12 hours preceding surgery. an iv catheter is placed for induction of anesthesia as this procedure is performed under general anesthesia. the patient is placed in right lateral recumbency, and the left rear leg is hoisted mailto:darcie.sidelinger@msstate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9640 22 citation line: clinical theriogenology 2023, 15, 9640, http://dx.doi.org/10.58292/ct.v15.9640 figure 1. curvilinear incision made to approach inguinal hernia repair. figure 2. isolated hernia sac after careful dissection. vertically. the inguinal area and scrotum are then clipped and aseptically prepared. a curvilinear incision is made in the inguinal region (figure 1) to allow access to the hernia contents and expose the inguinal ring for partial or complete closure. dissection continues until the hernia sac is identified (figure 2). once encountered, the sac should be opened, and its contents examined for evidence of incarceration and adhesions (figure 3). if no intestinal compromise is identified, then the herniated bowel is reduced through the inguinal ring into the abdomen (figure 4). if the herniated bowel is strangulated, a resection and anastomosis must be performed to remove the affected tissues. if a resection is required, the prognosis decreases substantially. following reduction of the hernia, the surgery can proceed in one of two different approaches; hemicastration of the affected side or partial closure of the inguinal ring to spare the testicle.1,2,5 when performing hemicastration of the affected testicle along with inguinal hernia repair, the inguinal incision is extended over the spermatic cord and an elliptical-shaped portion of the skin is removed from the lateral aspect of the scrotum. after isolating the testicle and placing reimer emasculators, two circumferential ligatures (miller’s knots) and one transfixing ligature are placed, the testicle is removed, and the stump is examined for hemorrhage before allowing it to retract into the abdomen (figure 5). the inguinal ring can then be closed by pre-placing #4 non-absorbable polyamide (braunamid, b. braun, bethlehem, pa) or polyglactin 910 (vicryl®, ethicon, bridgewater, nj) suture in either simple interrupted or cruciate pattern from the medial to lateral border. primary closure typically begins by tightening the suture in the center and working outward.1 when attempting to spare the testicle, the incision in the parietal tunic must be closed after inspection of the herniated contents. the fascia of the internal abdominal oblique muscle is then identified and secured to the caudal aspect of the inguinal ring over and under the spermatic cord for added security of the closure (figure 6). the inguinal ring is only partially closed as described above leaving room for the spermatic cord and 3–4 cm of additional space. the sutures are again pre-placed in either a simple interrupted or cruciate pattern and secured oneby-one starting near the center of the closure (figure 7). care must be taken while closing to ensure that the cord and two fingers (3–4 cm) are capable of passage through the ring.1 following either of the above approaches, the subcutaneous layer is closed using #2 absorbable suture (catgut, b. braun) in a simple continuous pattern. the skin is closed in a ford interlocking pattern using #4 non-absorbable polyamide suture (braunamid). standing flank approach this approach is most utilized for treatment of an indirect inguinal hernia, which is often not life-threatening and thus can be performed on an elective basis (figure 8).5 however, it is also an option for direct inguinal hernias. advantages for this approach are obvious. it does not require surgical level anesthesia or the surgical theater necessary for the inguinal approach. additionally, because the inguinal ring is only partially closed from inside the abdomen, it may be easier to preserve the involved testicle. following the pre-surgical preparation of the left paralumbar fossa either an inverted-l or paralumbar block is used for local http://dx.doi.org/10.58292/ct.v15.9640 citation line: clinical theriogenology 2023, 15, 9640, http://dx.doi.org/10.58292/ct.v15.9640 23 figure 3. opened hernia sac examination of intestinal contents before replacing into abdomen. figure 4. inguinal ring with herniated contents reduced. figure 5. hemicastration of the affected testicle. a circumferential and transfixing ligature are placed above the site for placement of the emasculator. figure 6. the fascia of the internal abdominal oblique muscle is secured to the caudal aspect of the inguinal ring over and under the spermatic cord for added security of the closure. http://dx.doi.org/10.58292/ct.v15.9640 24 citation line: clinical theriogenology 2023, 15, 9640, http://dx.doi.org/10.58292/ct.v15.9640 figure 7. preplaced sutures through the medial and lateral borders of the inguinal ring. figure 8. classic appearance of indirect inguinal hernia in the bull. video 1. this video demonstrates the application of traction to the herniated contents through the standing flank approach. resistance was felt likely due to adhesions. anesthesia.5 adequate sedation can be accomplished by utilization of the commonly used ‘standing’ or ‘k-state’ stun (xylazine 0.02 mg/kg, butorphanol 0.01 mg/kg, ketamine 0.05–0.1 mg/kg). this is administered via an intravenous route or alternatively the dosage can be doubled and given intramuscularly. the abdomen is then entered in a routine manner and the inguinal ring is identified. the herniated bowel is grasped, and traction is applied to reduce the hernia. resistance is likely due to adhesions and a decision should be made as to how much traction is employed (video 1). in spite of efforts to avoid trauma to incarcerated intestines, tearing of the intestine can occur, but can be repaired following reduction and exposure (figures 9 and 10). failure to reduce the hernia by traction at this stage will require transition to the inguinal approach. authors recommend once the hernia is reduced, a small tube (1/4” i.d., 3/8” o.d., 7’ length), gas sterilized, (portex equine stomach tube, foal, jorgensen laboratories, llc, loveland, co) is introduced at least 8–10 cm (or as far as can be advanced atraumatically) into the inguinal ring. this is done so that 40–60 ml of a carboxymethylcellulose 1% solution and 15 grams oxytetracycline hydrochloride soluble http://dx.doi.org/10.58292/ct.v15.9640 https://clinicaltheriogenology.net/index.php/ct/article/view/9640/15766 citation line: clinical theriogenology 2023, 15, 9640, http://dx.doi.org/10.58292/ct.v15.9640 25 powder (tetroxyò hca-280, bimeda, inc., le sueur, mn) mixture can be delivered into the space formally occupied by the herniated viscera. the purpose of this is to decrease the potential for fibrosis. it also serves to fill what is a potential space, thus decreasing the possibility of hematoma formation. this is followed by closure of the inguinal ring, which is performed by blindly placing 2–3 interrupted sutures (a vertical mattress pattern is recommended by the authors) or by utilizing the continuous blind suture technique with #2 or #3 non-absorbable suture (vicryl or braunamid). if a short continuous pattern is utilized, then it is recommended that the final pass of suture exits ipsilateral to the initial tie to avoid laxity in the suture line that results when a knot with tension is made across an opening. also, care must be taken during closure of the inguinal ring to avoid the vasculature entering and exiting the abdomen through the ring. the flank is then closed in a routine manner.2,5 post-op care parental antibiotics and nsaids are recommended preand post-operatively. the authors recommend 6.6 mg/kg ceftiofur crystalline free acid (excedeò, zoetis, kalamazoo, mi) administered once subcutaneously in the posterior aspect of the ear and 1.1 mg/kg flunixin meglumine (banamineò, merck animal health, madison, nj) administered once intravenously pre-operatively. following surgery, the authors recommend meloxicam tablets (meloxicam tablets, zydus pharmaceuticals inc., pennington, nj) 1.1 mg/kg administered orally once daily for three consecutive days followed by 0.55 mg/kg every other day as needed post-surgically. also, the authors suggest the snug, but not tight bandaging of the scrotum with elasticon™ (figure 11). this helps decrease post-operative swelling and/or control local post-surgical hemorrhage (figure 12). the bandage should be removed in 24–48 hours, and it is beneficial to carefully move the single testis or, following procedures in which a hemi-castration is not performed, both of the testes up and down within the scrotum at this time and once daily for 3–5 days. the authors recommend isolation from other cattle and sexual rest for 60 days. choosing a patient and an approach the authors contend that bulls presenting with indirect inguinal hernia make reasonable surgical candidates if scrotal swelling is the only complaint and intestinal peristalsis can be appreciated on ultrasonography of the herniated abdominal contents. prognosis is good for return to breeding soundness. if the testicle on the herniated side has any evidence of degeneration or fluid surrounding the testicle, it is the authors’ recommendation to perform hemicastration of that testicle while completing the hernia repair. if a hemicastration is to be performed, an inguinal approach would be appropriate. for sparing of the testicle, either the inguinal approach with partial closure of the inguinal ring or the standing flank approach can be utilized. the standing flank approach is most appropriate for indirect inguinal hernia. if the patient is presenting with a possible direct inguinal hernia, an inguinal approach should be considered for better exposure of the defect in the peritoneum and access to the intestines in case a resection and anastomosis are required. for patient selection, it is the authors opinion that bulls presenting with indirect inguinal hernia not intended for sale as figure 9. identification of incarcerated intestines on standing flank approach. figure 10. resection and anastomosis of the incarcerated intestine. http://dx.doi.org/10.58292/ct.v15.9640 26 citation line: clinical theriogenology 2023, 15, 9640, http://dx.doi.org/10.58292/ct.v15.9640 a breeding bull can be successfully treated with surgery and return to breeding. if hemicastration is necessary, it is found that the remaining testicle can produce up to 75% of the production by both testicles by compensatory hypertrophy.6 advantages of the inguinal approach are that it provides better exposure of the inguinal ring and visualization of closure of the ring. it also affords the ability to extend the incision and hemicastrate if necessary. disadvantages of the inguinal approach are that it requires general anesthesia and lateral recumbency which carry risks of other complications such as aspiration pneumonia and radial nerve paralysis. advantages of the standing flank approach are that the bull does not require general anesthesia or recumbency, the incision is in the flank instead of on the ventrum decreasing incisional complications and dehiscence. disadvantages of the standing flank are that visualization of the inguinal ring is not possible and closure is therefore done blindly. it may also be difficult to reduce the herniated viscera depending on the extent of herniation. the standing flank approach would also make a hemicastration difficult to perform during the same surgical procedure. conflict of interest and funding the authors have no conflicts of interest to declare. this publication did not receive any specific grants from funding agencies (public, commercial, or not-for-profit sectors). author’s contributions richard hopper describes the standing flank approach. darcie sidelinger describes the inguinal approach and wrote the introduction and post-op care sections. all authors edited and verified the articles accuracy. references 1. king eh: surgery of the scrotum and its contents. in: hopper rm: editor. bovine reproduction. 2nd edition, hoboken, nj; wileyblackwell: 2021. p. 200–209. 2. hopper r, king h, walters k, et al: management of urogenital injury and disease in the bull: the scrotum and its contents. clin theriogenol 2012;4:332–338. 3. wolfe df: unilateral castration for acquired conditions of the scrotum. in: wolfe df, moll hd: editors. large animal urogenital surgery. 2nd edition, baltimore, md; williams and wilkins: 1998. p. 313–320. 4. wolfe df, rodning sp: diagnosis and management of inguinal hernia in bulls. in: anderson de, rings dm: editors. food animal practice. 5th edition, st. louis, mo; saunders, 2009, p. 356–359. 5. riddell mg, powe ta, pugh dg: developmental anomalies of the scrotum and testes. in: wolfe df, moll hd: editors. large animal urogenital surgery. 2nd edition, baltimore, md; williams and wilkins: 1998. p. 283–294. 6. wolfe df: surgery of the scrotum and its contents. in: hopper rm:  editor. bovine reproduction. 1st edition, wiley: 2015. p. 136–141. figure 11. bull with elastikon wrap following hemicastration and inguinal hernia repair. figure 12. postoperative swelling in a bull following inguinal hernia repair. http://dx.doi.org/10.58292/ct.v15.9640 1 contact jessica klabnik jlk0066@auburn.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9265, http://dx.doi.org/10.58292/ct.v15.9265 review report when the plan goes awry: how to negotiate estrus synchronization errors in beef cattle jessica klabnik,a emma hornb adepartment of veterinary clinical sciences, auburn university college of veterinary medicine, auburn, al, usa, bgeneuity clinical research services, maryville, tn, usa abstract estrus synchronization protocols vary widely, and errors during implementation are not uncommon. when an error is made, recommendations for resolution must consider the initial purpose of implementing the synchronization protocol in the herd. the ideal solution is to convert the protocol to a different published protocol. when this is not possible, the safest solution for maintaining an acceptable pregnancy rate is often either restarting a synchronization program or potentially foregoing artificial insemination for the season. knowledge of estrous cycle physiology and how hormone treatments manipulate the cycle are foundational when attempting to modify a protocol based on the specific error that is made. errors made early in a synchronization protocol tend to be more manageable than those made toward the end of the protocol. the use of less valuable semen and immediate introduction of a clean-up bull should be considered to maximize pregnancy outcomes when attempting to correct an error during estrus synchronization. keywords: artificial insemination, bovine, estrus synchronization introduction the goal of estrus synchronization is to create a herd of fertile females that will either express estrus and/or ovulate within an ideal timeframe. if breeding is based on estrus detection, a larger variability of timeframe may be acceptable. however, when fixed-timed artificial insemination (ai) is planned, a tighter synchrony of the group is critical.1 ovulation occurs ~ 30 hours after the onset of estrus in bos taurus, providing a very narrow window in which successful pregnancy can occur as a result of insemination.2 success of synchronization is practically determined by pregnancy outcome, rather than estrus expression or ovulation rates. the determination of an ovulation rate is not practical, and neither the ovulation rate nor estrus expression is directly predictive of pregnancy outcome. numerous factors affect success of estrus synchronization, including the selection of a protocol that fits the herd type.3 protocols often differ for b. taurus versus b. indicus, dairy versus beef, and conventional versus sex-sorted semen. in the presynchronization period, animal health, animal nutrition, liquid-nitrogen tank maintenance, and pharmaceutical storage and date of expiration should be evaluated. critical factors include proper dosing, route of treatment, and timing of pharmaceuticals during synchronization, and semen handling, ai technique, and ai timing. the ability of the producer to comply with drug treatment and ai timings should be considered when selecting a protocol. after synchronization, animal handling, stress, transport, and nutrition can also impact success.3 errors during synchronization related to pharmaceutical treatment are not uncommon.4 there are numerous published protocols that can be quite complex, particularly if presynchronization or resynchronization protocols are included. most protocols use several pharmaceuticals, with varying frequency and/or dosage. unfortunately, this results in seemingly infinite opportunities for error during implementation of a synchronization protocol. ultimately, this also means that a ‘one-size fits most’ solution is not realistic or reliable. recommendations regarding solutions must be made carefully since a previous bad experience may negatively impact perception of estrus synchronization or ai.5,6 the purpose of this review is to provide useful information for recommending a feasible solution after an error has occurred during estrus synchronization in a herd. this includes the following: mailto:jlk0066@auburn.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9265 2 citation line: clinical theriogenology 2023, 15, 9265, http://dx.doi.org/10.58292/ct.v15.9265 1. a brief review of the purposes of implementing estrus synchronization; 2. four general categories of solutions to consider; and 3. core synchronization theory and the underlying estrous cycle physiology. a strong understanding of synchronization mechanisms and estrous physiology is vital to successfully modify an existing protocol. superstimulation, superovulation, and estrogen-based protocols are outside the scope of the presented information. purposes of implementing estrus synchronization the intended purpose of estrus synchronization must be considered when deciding on a solution for errors in protocol execution. cost-prohibitive or time-consuming corrections may jeopardize the intended benefits of synchronization. estrus synchronization protocols are used to increase economic impact, genetic selection, and overall efficiency of production in cattle herds.7 synchronization can increase pregnancy rates to fixed-time ai, embryo transfer, or live cover insemination,8,9 which, in turn, increases profitability and decreases cost of rebreeding. more uniform calf crops of superior genetics can be produced when synchronization is combined with ai. this enhances the marketability of calves and increases their market value.10–12 some synchronization protocols can also induce cyclicity in noncycling individuals. however, pregnancy rates were significantly lower in females that were not cycling prior to synchronization/ai compared to those that were cycling.13 critical evaluation of errors and determining solutions with the purpose of estrus synchronization in mind, consideration can be given to 4 general solutions: restart the protocol, forgo ai for the season, convert to a different published protocol, or modify the protocol based on knowledge of synchronization theory and estrous cycle physiology. restarting a synchronization protocol is a straightforward option for the producer. if time in the breeding season allows, and the operation can cover the cost of restarting the synchronization protocol, this may be a feasible solution to circumvent errors made in the initial synchronization attempt. it may reduce the risk of another error resulting from lack of familiarity with a new or modified protocol.14 however, the cost of restarting may render this option less desirable. costs may include additional pharmaceuticals, extra labor, and loss of production associated with delayed breeding (i.e. lighter calf crop at weaning and shorter or shifted breeding season). these costs are quite considerable in a commercial beef operation but may be overcome in certain seed stock beef operations.15 restarting the protocol allows for a fresh start with an already known and prepared-for protocol. a second general solution is to forgo ai and solely use clean up bull(s) for that breeding season. ideally, the bull(s) must be able to breed multiple times while exposed to the herd over a window of time, minimizing negative effects if the error resulted in less synchronous ovulation. bulls with high libido may achieve multiple services in a routine estrus synchronization program.16,17 this option may reduce labor and pharmaceutical costs. one concern with this option is ensuring sufficient bull power for the synchrony of expected estrus. in a non-synchronized herd, the serving capacity of a fertile bull is expected to be 1 bull to 25 to 60 cows.18 in a study of synchronized females, pregnancy rates were not affected by either the serving capacity ratio (ranging from 1:7 to 1:51) or the number of females exhibiting estrus when exposed to bulls classified as high or medium libido.19 however, serving capacity may still be a concern as libido is not routinely tested,20 and newer synchronization protocols may have tighter estrus expression compared to more simple protocols (i.e. syncro-mate b or ‘two-shot’ prostaglandin f2α [pgf2α]).19 therefore, when considering this solution, the type of protocol in use and the stage of the protocol in which the error occurred must be considered. in some cases, errors during estrus synchronization can be corrected. in an ideal situation, the correction changes the planned protocol to another published protocol. a good starting place to find additional protocols is a local extension website. insemination, semen sexing, and embryo transfer companies have synchronization protocols listed and diagramed for producer use; these companies may also provide consulting, services, and products useful for executing protocols. the multistate beef reproduction task force is aimed at bringing research and extension together, and their website (https://beefrepro.org/) contains links for downloadable resources and protocol timelines.21 because protocols should be chosen based on the producer’s needs and capabilities, the decision to transform an existing protocol once an error has occurred to a different published protocol requires evaluation of both the producer’s abilities and the timing of the error in the initial protocol.22 less ideal situations (i.e. unable to switch to another published protocol) are much more common, and modifications of the protocol in these instances are likely to result in poorer synchrony.23 if breeding is based on estrus detection, lesser synchrony resulting from normal physiologic variability of the estrous cycle may not be of concern. estrous behavior may be expressed earlier and for a shorter duration in bos indicus cattle compared to bos taurus.24,25 ideally, ai should be performed 6 24 hours prior to ovulation, which is ~ 2 14 hours after the onset of standing heat.26,27 in a fixed-time ai protocol in which the window of insemination is narrow (± 2 4 hours), variability in the synchronization of ovulation is more detrimental. regardless of the program type, poorer synchrony that results from either a poorly timed luteinizing hormone (lh) surge or prolonged periods of follicular dominance is of particular concern. a narrow window of oocyte viability occurs after oocyte maturation is triggered by the lh surge.28 prolonged periods of follicular dominance reduce embryonic development and quality due to prolonged exposure of the oocyte to follicular estradiol, which has been correlated with delayed meiotic progression and blastocyst rate.29–31 because the lh surge and follicular dominance occur toward the end of the protocol, it is easier to correct errors occurring at the beginning of the protocol before these events are underway. errors toward the end of synchronization tend to be more challenging to overcome. fertility of the oocyte within a late atretic dominant follicle declines rapidly as crucial estradiol production declines and progesterone production slowly increases with granulosa cell aging.32 the ovulated oocyte, arrested at metaphase ii, is highly susceptible to postovulatory aging, resulting in decreased viability.33,34 decreased developmental competence, or the oocyte’s ability to be fertilized and give rise to a healthy embryo, is significantly decreased due to postovulatory oocyte aging.35 decreasing estradiol concentrations http://dx.doi.org/10.58292/ct.v15.9265 https://beefrepro.org/ citation line: clinical theriogenology 2023, 15, 9265, http://dx.doi.org/10.58292/ct.v15.9265 3 due to the decline of the dominant follicle, and potential postovulatory oocyte aging can both compromise fertility near the end of the estrous cycle. even females displaying estrus may have aged oocytes and be subfertile.36 in these cases, modification of synchronization is highly situational, depending on the protocol implemented and the error that was made. critical evaluation of what was given, not given, and the timing of these events in light of their physiologic purpose is crucial and complex. therefore, the authors highly recommend use of less valuable semen and immediate introduction of a clean-up bull to attempt to maximize pregnancy outcomes if restarting synchronization and foregoing ai are not options. the only relatively simple error to overcome is when gonadotropin releasing hormone (gnrh) treatment at ai is forgotten. gnrh should be given as soon as possible to help ensure ovulation as close to insemination as possible, but it is likely that the majority of well-synchronized females will ovulate in an acceptable time frame, even if ovulation is less synchronous than initially desired.37 integration of estrous cycle physiology and synchronization protocol a strong knowledge base of synchronization theory and underlying estrous cycle physiology is critical in cases where modification of the protocol is the reasonable solution. estrous cycle reviews are available that detail information beyond core concepts related to synchronization.24,38 as the bovid has a continuous, monoovulatory, and nonseasonal estrous cycle,24,38 synchronization protocols are designed to manipulate a group of females at variable stages of their cycle into 1 particular part of the cycle (estrus) at the same time. in a synchronization protocol, all individuals receive the same pharmaceutical at the same time, regardless of their status in the estrous cycle. producers do not keep track of each animal’s stage in the estrous cycle for practical reasons, and therefore, the entire herd receives uniform treatment protocol to minimize labor for producers and increase herd synchrony. because the bovid is continually cycling, certain synchronization protocols may alter hormonal events midcycle and initiate the next cycle in which breeding will occur in an optimal timeframe.39,40 at synchronization, the ‘ideal’ cow (figure. cow a: panels c and d) would already be cycling and nearing the end of her 21-day cycle, such that she would ovulate 6 to 24 hours26,27 after planned insemination without manipulation. this cow would be ‘ideal’ because her cycle already aligns with the scheduled breeding at the end of synchronization. she would arrive at the planned breeding date and ovulate, without need for interference. it is impossible, however, to determine which cows, if any, are the ‘ideal’ member of the herd before synchronization. this means that the ‘ideal’ cow still received synchronization drugs along with her herdmates. however, most individuals in the herd will not be in this ‘ideal’ category, and their cycle will need to be manipulated to ensure that they ovulate at the appropriate time. when progesterone is low, ovulation results from a lh surge acting on the dominant follicle.41 synchronization of herd-mates (figure. cows b through e) is aimed at manipulating cycles to align 3 key requirements: a low progesterone environment, lh surge, and the existence of a dominant follicle containing a fertile cumulus-oocyte-complex.42,43 low progesterone a low progesterone environment is required for the dominant follicle to ovulate.41 this environment is necessary for ovulation because high progesterone environments prevent an lh surge from occurring. progesterone inhibits episodic gnrh secretion from the hypothalamus. conversely, gnrh exerts a positive effect on the pulsatile release of lh from the anterior pituitary, eventually culminating in a preovulatory lh surge and subsequent ovulation. under a high progesterone environment, the dominant follicle can become atretic, regress, and a new follicular wave begins.41 progesterone is produced by the corpus luteum.38 circulating progesterone concentrations in cattle with a functional corpus luteum must reach serum concentrations > 1 ng/ml to be considered physiologically relevant.44 in the natural estrous cycle, progesterone concentrations decrease (< 1 ng/ml) due to lysis of the corpus luteum that results from the luteolytic activity of pgf2α as it binds to its receptors. the number of pgf2α receptors on the corpus luteum increases from the early to late luteal phase of the cycle, peaking around day 16 when luteolysis typically occurs.45 pgf2α is produced by the uterus.38 to synchronize a low progesterone environment, pgf2α treatment may be given to cattle. if the number of pgf2α receptors is sufficient, lysis will occur and progesterone concentrations will decline.46,47 concentrations of serum progesterone decline to < 2 ng/ml within 12 hours after 30 mg of intramuscular pgf2α. 48 typically, a corpus luteum will be capable of responding to a single dose of exogenous pgf2α by days 5 7 of the cycle (if day 0 is the day when ovulation last occurred).49,50 although the majority of females in an unsynchronized herd will have a corpus luteum capable of responding (figure. panel e and f), a significant remainder will not (figure panel g).51 therefore, pgf2α may also be used to presynchronize the herd in some protocols. the simplest example of this is a ‘two-shot’ pgf2α protocol. in this protocol, the first pgf2α treatment synchronizes the majority of females. after enough time has passed to allow their new corpus luteum to mature and be responsive to pgf2α, a second pgf2α treatment is given. females with a less mature corpus luteum at the first treatment likely did not respond. it is expected that these individuals’ corpus luteum will be mature enough by the second pgf2α treatment to respond and lyse or be close to natural lysis.51,52 another way to create a low progesterone environment is the timely removal of progesterone treatment. progesterone treatment for a short period prevents the female bovid from ovulating and subsequently developing a young corpus luteum. this period in which luteolysis is expected to occur in the protocol is depicted (figure, panel h). the source of progesterone is via a controlled internal drug release device (cidr) and removal at pgf2α treatment with the intent of creating a low progesterone environment in all animals at the same time.53 notably, bos indicus-influenced cattle may be more sensitive to progesterone’s effects during earlier aspects of the protocols. improved pregnancy rates to fixed-time ai result when a functional corpus luteum is eliminated at the beginning of the protocol.25 luteinizing hormone surge the hypothalamic-pituitary-gonadal axis is differentially regulated by progesterone and estrogen. the hypothalamic surge center is responsible for inducing a lh surge in low progesterone environments when progesterone’s negative inhibition on gnrh release is absent. progesterone also negatively inhibits follicle stimulating hormone production, yet cohorts of small follicles are recruited due to transient rises in circulating http://dx.doi.org/10.58292/ct.v15.9265 4 citation line: clinical theriogenology 2023, 15, 9265, http://dx.doi.org/10.58292/ct.v15.9265 follicle stimulating hormone, even in a high progesterone environment. eventually, 1 follicle is selected, and the growing dominant follicle produces increasing concentrations of estradiol during proestrus. after corpus luteum regression, and without the negative feedback of progesterone on gnrh, lh then increases in a pulsatile fashion.54 these changes culminate in the lh surge and subsequently ovulation.55 after ovulation, the estradiol producing follicle becomes a figure. use of estrus synchronization to modify cows at various stages of the estrous cycle in an example herd. a herd of unsynchronized females (panels a, c, e, g, i, and k) may be at different stages of their estrous cycle at the start of a given protocol. cow a is considered the ideal cow because no modification of her cycle is needed for her to ovulate near the planned breeding date (panels c and d). in a herd of cycling females, the stage of the cycle in all cows is unknown, and therefore, all cows will be enrolled in an estrus synchronization program. implementing an estrus synchronization program (e.g. 7-day co-synch + cidr68,79 [panel b]), will modify the cycles of herd mates (panels f, h, j, and l) to eventually coincide with the ideal cow by the time of the planned breeding date (panel d). note timeline presented is approximate, and cycles are not exactly to scale. http://dx.doi.org/10.58292/ct.v15.9265 citation line: clinical theriogenology 2023, 15, 9265, http://dx.doi.org/10.58292/ct.v15.9265 5 progesterone producing corpus luteum.56 detailed information on the hypothalamic-pituitary-gonadal axis and effects of progesterone are available.25,53,57–59 although an lh surge may occur within estrus synchronization programs, the timing may be too variable for fixed-time ai programs (figure. panel i).23,28 theoretically, ovulation of a dominant follicle could be induced by stimulation of various aspects of the hypothalamic-pituitary-gonadal axis. this may include treatment with estrogens, gnrh, lh, or their analogues.60 as estrogens are illegal in food producing species in the united states, a gnrh analogue is typically given.  bos indicus-influenced cattle have a highly variable and  relatively low rate of ovulation to exogenous gnrh compared to bos taurus.61,62 an lh surge is expected to occur ~ 2 hours after exogenous gnrh treatment.63,64 ovulation occurs ~ 22 34 hours (in bos taurus63,64) or 26 28 hours (in bos indicus65) after the lh surge. sperm must travel from the site of deposition (i.e. uterus during ai) to the site of fertilization (i.e. ampullary-isthmic junction of the uterine tube). to coordinate travel efforts of both gametes, gnrh is commonly given 16 24 hours prior to ai.64,66,67 however, to reduce labor costs, gnrh is given at ai in some protocols (figure. panel j).21,68,69 the majority of inseminations in north america utilize conventionally frozen semen. protocols designed for insemination using other semen types (i.e. fresh, chilled, or sex-sorted), number of sperm (i.e. sexsorted), or  in other anatomic locations (i.e. uterine horn breeding)  impact the timing of insemination and gnrh treatment. dominant follicle in the natural estrous cycle, bos taurus cattle can have either 2 or 3 follicular waves. the 2 wave cycle produces a follicular wave ~ every 10 days, whereas the 3 wave cycle produces a wave ~ every 7 9 days.70 the average length of the estrous cycle is 21 days ± 2 days.71 however, cattle with 3 wave estrous cycles tend to have longer cycles, landing in the 22+ day range overall.70 bos indicus cattle estrous cycle average length is 21 days for 2 wave and 22 days for 3 wave cycles. four and 5 wave cycles have also been reported.24 each wave contains 3 stages: emergence/recruitment, selection, and dominance.38 a dominant follicle is lined with mural granulosa cells possessing sufficient lh receptors to respond to lh surge.72 dominant follicles also produce inhibin, a hormone that prevents progression of other follicles.32 as previously reviewed, a dominant follicle will become atretic in a high progesterone environment, as it is unable to ovulate.53 in a low progesterone environment, the growing dominant follicle produces increasing concentrations of estradiol during proestrus, triggering the lh surge, and eventual ovulation.55 the natural variability in wave length24,70 results in considerable challenges when attempting to synchronize a group of females. if wave timing is left unmanaged, some cattle may have significantly delayed ovulation.73 this could result in subfertility due to aging of the sperm.27 currently, no therapies exist to change the rate of follicle development (i.e. speed it up or slow it down). ovulation of the first wave dominant follicle results in reduced pregnancy per ai compared to the second wave. however, when progesterone was given, similar pregnancies per ai resulted.74 for practical purposes, wave number is not a consideration related to  estrus synchronization, particularly when the protocol includes progesterone treatment. altogether, current synchronization protocols rely on inducing a wave at the same time in all herd members, so that dominant follicles, capable of responding to lh surge, will be present at the same time.75 variability in ovulation timing due to wavelength can be managed by utilizing an induced lh surge. induction of lh surge was described in the previous section (figure. panel j). it is worth reiterating that only a dominant follicle, with sufficient lh receptors, will ovulate in response to induced lh surge.72 if the present follicle is much younger and, therefore, has not acquired dominance or the appropriate number of lh receptors, it is not capable of ovulation (figure. panel k). eliminating a large, inhibin-producing follicle helps to synchronize the next follicular wave in cattle.32 initially, this was performed by follicle ablation,76 but most protocols now use a gnrh analogue for this purpose. gnrh exposure results in the induction of an lh surge and the regression (if high progesterone) or ovulation (if low progesterone) of any existing follicles.64,77,78 after a new wave begins, sufficient time is allowed for the follicle to progress toward dominance and any young corpus luteum to mature. the corpus luteum is then lysed, followed by a second dose of a gnrh analogue treatment near the time of ai to cause ovulation of the dominant follicle (figure. panel l). because exogenous gnrh induction of a new wave may result in a new corpus luteum,64,77 subsequent use of pgf2α is mandatory if the cow is to be bred before natural luteolysis would occur.80 a female bovid will not respond to the initial dose of gnrh analogue if a dominant follicle is not present at treatment. therefore, some protocols include initial measures to create a persistent dominant follicle prior to this initial gnrh dose. long-term (i.e. ~ 14 day) progesterone treatment is used.81 during this period, the female will lyse her own corpus luteum but may not ovulate due to high concentrations of progesterone. lysis of the mature corpus luteum may be induced by uterine release of pgf2α. 31,49 under natural circumstances, the lysis of the corpus luteum will cease progesterone production and remove the feedback of progesterone on hypothalamic gnrh release, allowing for eventual ovulation and subsequent lh surge.82 in cases where progesterone is given and a corpus luteum is not present, circulating progesterone concentrations are at physiologically relevant concentrations that are high enough to prevent lh surge but lower than the concentrations produced by a mature corpus luteum. these lower progesterone concentrations result in persistence of a dominant follicle, rather than follicle turnover. in the absence of lh surge (natural or induced), the dominant follicle persists.83 when progesterone treatment is terminated, ovulation will occur, a corpus luteum will form, and a new wave will start. synchrony of later waves is then used for breeding purposes. with these protocols, the initial oocyte in the persistent dominant follicle ages, and the herd will express unfertile estrus after the termination of progesterone treatment. producers must wait to breed according to protocol for acceptable pregnancy outcomes.14 a similar concept is used in the relatively new 7 & 7 protocol. initially, exogenous progesterone and prostaglandin are given to mimic natural corpus luteum lysis and stalling of a dominant follicle for up to 7 days. the gnrh analogue in the middle of the 14 days of progesterone treatment ultimately results in a new wave that is then utilized for breeding.84 conclusion errors during synchronization related to pharmaceuticals’ treatment are not uncommon. the opportunities for error during implementation of a synchronization protocol can http://dx.doi.org/10.58292/ct.v15.9265 6 citation line: clinical theriogenology 2023, 15, 9265, http://dx.doi.org/10.58292/ct.v15.9265 appear quite infinite and ultimately suggest that a ‘one-size fits most’ solution is not realistic. recommendations for solutions must be made carefully, considering both the producer and the affected herd. with the purpose of estrus synchronization in mind, 4 general solutions can be considered: restart the protocol, forgo ai for the season, convert to a different published protocol, or modify the protocol. a strong understanding of synchronization theory and estrous cycle physiology is vital if the chosen solution is to modify an existing protocol. synchronization protocols manipulate cycles to align 3 key requirements: low progesterone environment, lh surge, and the existence of a dominant follicle containing a fertile cumulus-oocyte-complex. acknowledgment authors thank herris maxwell and chance armstrong for intellectual discussions regarding manuscript content and j. lannett edwards for the use of her dominant follicle image. conflict of interest none to declare. references 1. pursley jr, silcox rw, wiltbank mc: effect of time of artificial insemination on pregnancy rates, calving rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows. j dairy sci 1998;81:2139–2144. doi: 10.3168/jds. s0022-0302(98)75790-x 2. roelofs jb, van eerdenburg fj, soede nm, et al: various behavioral signs of estrous and their relationship with time of ovulation in dairy cattle. theriogenology 2005;63:1366–1377. doi: 10.1016/j. theriogenology.2004.07.009 3. lamb gc: overcoming compliance issues to 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hartmann w: regulation of the hypothalamic-pituitary-gonadal axis and the manipulation of the estrous cycle of bovine females. in: bergstein-galan tg: editor. reproduction biotechnology in farm animals. hyderabad; avid science: 2018. p. 75–96. 61. saldarriaga jp, cooper da, cartmill ja, et al: ovarian, hormonal, and reproductive events associated with synchronization of ovulation and timed appointment breeding of bos indicus-influenced cattle using intravaginal progesterone, gonadotropin-releasing hormone, and prostaglandin f2α1. j anim sci 2007;85:151–162. doi: 10.2527/jas.2006-335 62. zuluaga jf, saldarriaga jp, cooper da, et al: presynchronization with gonadotropin-releasing hormone increases the proportion of bos indicus-influenced females ovulating at initiation of synchronization but fails to improve synchronized new follicular wave emergence or fixed-time artificial insemination conception rates using intravaginal progesterone, gonadotropin-releasing hormone, and prostaglandin f2α1. j anim sci 2010;88:1663– 1671. doi: 10.2527/jas.2009-2480 63. martinez mf, adams gp, bergfelt dr, et al: effect of lh or gnrh on the dominant follicle of the first follicular wave in beef heifers. anim reprod sci 1999;57:23–33. doi: 10.1016/s0378-4320(99)00057-3 64. pursley jr, mee mo, wiltbank mc: synchronization of ovulation in dairy cows using pgf2α and gnrh. theriogenology 1995;44:915–923. doi: 10.1016/0093-691x(95)00279-h 65. pinheiro ol, barros cm, figueiredo ra, et al: estrous behavior and the estrus-to-ovulation interval in nelore cattle (bos indicus) with natural estrus or estrus induced with prostaglandin f2α or norgestomet and estradiol valerate. theriogenology 1998; 49:667–681. doi: 10.1016/s0093-691x(98)00017-x 66. binversie j, pfeiffer k, larson j: modifying the double-ovsynch protocol to include human chorionic gonadotropin to synchronize ovulation in dairy cattle. theriogenology 2012;78:2095–2104. doi: 10.1016/j.theriogenology.2012.08.004 67. stevenson js, pursley jr, garverick ha, et al: treatment of cycling and noncycling lactating dairy cows with progesterone during ovsynch1. j dairy sci 2006;89:2567–2578. doi: 10.3168/jds. s0022-0302(06)72333-5 68. geary t, whittier j, hallford d, et al: calf removal improves conception rates to the ovsynch and co-synch protocols. j anim sci 2001;79:1–4. doi: 10.2527/2001.7911 69. geary tw, whittier jc, thrift fa, et al: effects of a timed insemination following synchronization of ovulation using the ovsynch or co-synch protocol in beef cows. prof anim sci 1998;14:217– 220. doi: 10.15232/s1080-7446(15)31832-5 70. ginther o, knopf l, kastelic j: temporal associations among ovarian events in cattle during oestrous cycles with two and three follicular waves. reproduction 1989;87:223–230. doi: 10.1530/ jrf.0.0870223 71. crowe ma: reproduction, events and management| estrous cycles: characteristics. in: mcsweeney p, mcnamara j: editors. encyclopedia of dairy sciences. 3rd edition, oxford; academic press: 2011. p. 948–953. 72. colazo mg, kastelic jp, davis h, et al: effects of plasma progesterone concentrations on lh release and ovulation in beef cattle given gnrh. domest anim endocrinol 2008;34:109–117. doi: 10.1016/j.domaniend.2006.11.004 73. taponen j, katila t, rodrıguez-martınez h: induction of ovulation with gonadotropin-releasing hormone during proestrus in cattle: influence on subsequent follicular growth and luteal function.  anim reprod sci 1999;55:91–105. doi: 10.1016/ s0378-4320(99)00011-1 74. denicol ac, lopes g, mendonça lgd, et al: low progesterone concentration during the development of the first follicular wave reduces pregnancy per insemination of lactating dairy cows. j dairy sci 2012;95:1794–1806. doi: 10.3168/jds.2011-4650 75. wolfenson d, thatcher ww, savio jd, et al: the effect of a gnrh analogue on the dynamics of follicular development and synchronization of estrus in lactating cyclic dairy cows. theriogenology 1994;42:633–644. doi: 10.1016/0093-691x(94)90380-2 76. bergfelt dr, lightfoot kc, adams gp: ovarian synchronization following ultrasound-guided transvaginal follicle ablation in heifers. theriogenology 1994;42:895–907. doi: 10.1016/0093691x(94) 90113-w 77. macmillan k, thatcher w: effects of an agonist of gonadotropin-releasing hormone on ovarian follicles in cattle. biol reprod 1991;45:883–889. doi: 10.1095/biolreprod45.6.883 78. thatcher w, drost m, savio j, et al: new clinical uses of gnrh and its analogues in cattle. anim reprod sci 1993;33:27–49. doi: 10.1016/0378-4320(93)90105-z 79. lamb g, stevenson j, kesler d, et al: inclusion of an intravaginal progesterone insert plus gnrh and prostaglandin f2α for ovulation control in postpartum suckled beef cows. j anim sci 2001;79:2253–2259. doi: 10.2527/2001.7992253x 80. diskin mg, austin ej, roche jf: exogenous hormonal manipulation of ovarian activity in cattle. domest anim endocrinol 2002;23:211–228. doi: 10.1016/s0739-7240(02)00158-3 81. mcdowell cm, anderson lh, lemenager rp, et al: development of a progestin-based estrus synchronization program: ii. reproductive response of cows fed melengestrol acetate for 14 days with injections of progesterone and prostaglandin f2α. j anim sci 1998;76:1273–1279. doi: 10.2527/1998.7651273x http://dx.doi.org/10.58292/ct.v15.9265 https://doi.org/10.2527/1999.7771823x https://doi.org/10.1016/j.yfrne.2012.08.004 https://doi.org/10.2527/jas1980.514949x https://doi.org/10.2527/jas1980.514949x https://doi.org/10.1111/asj.12056 https://doi.org/10.2527/jas.2006-335 https://doi.org/10.2527/jas.2009-2480 https://doi.org/10.1016/s0378-4320(99)00057-3 https://doi.org/10.1016/0093-691x(95)00279-h https://doi.org/10.1016/s0093-691x(98)00017-x https://doi.org/10.1016/j.theriogenology.2012.08.004 https://doi.org/10.3168/jds.s0022-0302(06)72333-5 https://doi.org/10.3168/jds.s0022-0302(06)72333-5 https://doi.org/10.2527/2001.7911 https://doi.org/10.15232/s1080-7446(15)31832-5 https://doi.org/10.1530/jrf.0.0870223 https://doi.org/10.1530/jrf.0.0870223 https://doi.org/10.1016/j.domaniend.2006.11.004 https://doi.org/10.1016/s0378-4320(99)00011-1 https://doi.org/10.1016/s0378-4320(99)00011-1 https://doi.org/10.3168/jds.2011-4650 https://doi.org/10.1016/0093-691x(94)90380-2 https://doi.org/10.1016/​0093-691x(94)90113-w https://doi.org/10.1016/​0093-691x(94)90113-w https://doi.org/10.1095/biolreprod45.6.883 https://doi.org/10.1016/0378-4320(93)90105-z https://doi.org/10.2527/2001.7992253x https://doi.org/10.1016/s0739-7240(02)00158-3 https://doi.org/10.2527/1998.7651273x citation line: clinical theriogenology 2023, 15, 9265, http://dx.doi.org/10.58292/ct.v15.9265 9 82. christensen a, bentley ge, cabrera r, et al: hormonal regulation of female reproduction. horm metab res 2012;44:587–591. doi: 10.1055/s-0032-1306301 83. custer e, beal w, wilson s, et al: effect of melengestrol acetate (mga) or progesterone-releasing intravaginal device (prid) on follicular development, concentrations of estradiol-17 β and progesterone, and luteinizing hormone release during an artificially lengthened bovine estrous cycle. j anim sci 1994;72:1282–1289. doi: 10.2527/1994.7251282x 84. andersen cm, bonacker rc, smith eg, et al: evaluation of the 7 & 7 synch and 7-day co-synch plus cidr treatment regimens for control of the estrous cycle among beef cows prior to fixed-time artificial insemination with conventional or sex-sorted semen. anim reprod sci 2021;235:106892. doi: 10.1016/j.anireprosci.2021. 106892 http://dx.doi.org/10.58292/ct.v15.9265 https://doi.org/10.1055/s-0032-1306301 https://doi.org/10.2527/1994.7251282x https://doi.org/10.1016/j.anireprosci.2021.106892 unnatural selection? considerations in canine and feline genetic counseling unnatural selection? considerations in canine and feline genetic counseling cynthia o’connor east bridgewater veterinary hospital, east bridgewater, ma abstract molecular genetics and growing availability of molecular technologies are expanding exponentially, pushing boundaries of modern medicine. these bring continued challenges for veterinary genetic counseling which often falls behind human counterparts. there is an increasing need for veterinarians to not only understand this technology, but also to counsel clients to use it appropriately and be an informed voice in current political and socioeconomic debates. a basic understanding is important for veterinarians to use available technologies to control genetic diseases while maintaining genetic diversity in population. veterinarians, particularly reproductive veterinarians, are in a unique position to bridge the knowledge gap between advancements in genetic science and clinical applications in small dog and cat breeding operations. veterinarians can help breeders utilize tools such as estimated breeding values, inbreeding or heterozygosity estimates and mutation based genetic disease tests optimally in their breeding programs. there is a critical need to understand that mistakes of past should be replaced by exciting technological advances of future to implement breeding strategies that will more effectively reach small animal breeder’s goals of producing dogs and cats that not only are what is desired for appearance, temperament, and workability, but also live long and healthy lives. keywords: genetic counseling, genetic testing, estimated breeding values, population diversity, inbreeding coefficient, heterozygosity introduction with recent advances in molecular genetics and growing availability of molecular technology, we witnessed emergence of genomics into our everyday lives. as we still struggle with bioethics, this technology is expanding at an exponential pace. we live not only in a world where we are constantly pushing boundaries of modern medicine, but also where this technology is readily available, even to public. this brings continued challenges for veterinary profession, which has often fallen behind human counterparts in veterinary genetic counseling. there is an increasing need for veterinarians to not only understand this technology, but to counsel our clients regarding implementation of this technology, as well as to have a voice in current political and socioeconomic debates. currently, there are > 700 known hereditary diseases in dogs and ~ half as many in cats, with a growing number of new diseases reported every year.1 this led to development and growing need for incorporation of clinical genetics into veterinary practice, with small animal practitioners having an ever growing and vital role in both genetic counseling and detection of potentially new genetic diseases. hereditary disease is caused by a dna mutation that can be passed from parent to offspring, whereas a congenital disease is present at birth. with this distinction, congenital diseases can be genetic, but not all congenital diseases are genetic.2 for example, an autosomal recessive gene mutation in portuguese water dogs results in early-age dilated cardiomyopathy and sudden death (portuguese water dog juvenile dilated cardiomyopathy); however, perinatal infection with parvovirus can result in myocarditis with resultant myocardial damage, heart failure, and sudden death in young puppies as well.3,4 onset of clinical signs can vary for hereditary diseases. some hereditary diseases may be apparent at early ages where most common presentations are embryonic/fetal death, stillbirth, or fading puppies/kittens. unfortunately, many are undiagnosed as breeders and veterinarians may not pursue additional diagnostics in these prenatal and neonatal cases. traditionally, hereditary disorder clinical signs may not be recognized until after weaning, as musculoskeletal, ocular, digestive and other anomalies may not be readily identifiable during neonatal phase of development. some diseases may have a much later onset. one example is progressive retinal atrophy; affected animals suffer from retinal atrophy that leads to eventual blindness, with clinical signs rarely apparent before 3 5 years of age.5 clinical theriogenology • volume 12 number 3 • september 2020222 number and variety of genetic diseases is extremely large and many are very rare, with new diseases recognized at an exponential rate.6 as such, it is important for a practicing veterinarian to consult reference sources (listed in table) to obtain knowledge about a known genetic disorder, breed distributions, and distinguishing characteristics regarding diagnosis, treatment, and control. when previously undefined disorder is encountered, evidence of its genetic etiology should be ruled out.7  does the disorder occur in a greater frequency within a line or breed than in general population?  is the disease more common in animals with a higher degree of inbreeding (you need more than a typical 3 5 generation pedigree to reveal a more accurate degree of inbreeding)?  does the disease have a characteristic age of onset and clinical course, especially when seen in young animals?  is the same syndrome found in another species and is it known to be genetic?  is there a specific phenotypic defect or syndrome that is associated with a specific chromosomal abnormality?  can the disease process be related to a molecular defect such as a defect in an enzyme pathway, structural protein, or molecular receptor? it is noteworthy that genetic diseases are not limited to purebred dogs and cats. although mixedbreed dogs generally have a lower degree of inbreeding, many populations (e.g. local stray cats) may have a higher than expected degree of inbreeding. in general, autosomal recessive diseases are more likely to be expressed when there is a higher degree of inbreeding. however, dominant disease and polygenetic diseases may be just as likely in mixed breed populations as they are in more inbred populations, depending upon the disease and the population.2 diagnostic approach to identifying genetic disorders clinical approach to identify genetic disease begins with a thorough history and physical examination. additional queries regarding littermates and relatives as well as in some cases, a population medicine approach when dealing with kennels and catteries, will assist with collection of infectious disease, toxin, nutritional, and other important data to be considered in investigation of new diseases. diagnostic tests generally are required to further support a genetic disorder in a diseased animal.8 for example, radiology and other imaging techniques may reveal skeletal malformations, echocardiogram may reveal cardiac anomalies and ophthalmologic examination may further define an inherited eye disease. routine tests such as a complete blood cell count, chemistry screen and urinalysis may suggest specific hematologic or metabolic disorders and may help rule out many acquired disorders. based on these findings, additional clinical function testing may more clearly define a gastrointestinal, hepatic, renal, or endocrine problem.10,11 histopathology of a tissue biopsy or a necropsy evaluation from an affected animal are often required for a complete evaluation and definitive diagnosis for animals with a genetic defect. the latter is particularly important when faced with a fading neonatal puppy or kitten, as this may give information vital to surviving littermates as well as future planned breeding; however, this important diagnostic tool is often underused. few laboratories provide special diagnostic tests that allow for investigation into a possible inborn error of metabolism (table). inborn errors of metabolism include biochemical disorders due to a genetic defect in structure and/or function of a protein or receptor. for example, a deficiency in enzyme β glucuronidase resulting in the lysosomal storage disorder mucopolysaccharidosis vii has been reported in german shepherd dogs as well as a mixed-breed dog.10 most useful specimen to detect biochemical derangements is urine, as abnormal metabolites are filtered but not resorbed by kidneys. clinical theriogenology • volume 12 number 3 • september 2020 223 table. some useful websites relating to canine and feline genetic diseases listings of available tests and testing center information:  http://www.akcchf.org/  https://embarkvet.com/  https://www.optimal-selection.com  https://www.betterbred.com/  http://research.vet.upenn.edu/default.aspx?alias=research.vet.upenn.edu/penngen  http://www.vmdb.org/cerf.html  ttp://www.vetgen.com/  http://www.vgl.ucdavis.edu/  http://www.vetdnacenter.com/  http://www.caninegeneticdiseases.net/  http://www.healthgene.com/  http://www.labradorcnm.com/  http://www.vdl.umn.edu/  http://www.vetmed.wsu.edu/deptsvcgl/  http://www.aht.org.uk/genetics.html  http://vetmed.tamu.edu/labs/cytogenics-genomics  http://www.babs.unsw.edu.au/canine_genetics_facility.php  http://www.medigenomix.de/zuechterservice_hund.html  http://www.catgenes.org/  http://www.dogenes.com/  http://www.animalsdna.com/ databases and recommendations for health screening:  http://omima.angis.org.au/  http://sydney.edu.au/vetscience/lida/  http://ic.upei.ca/cidd/  http://www.vet.cam.ac.uk/idid/  http://www.caninehealthinfo.org/  http://www.gdcinstitute.org/  http://www.rvc.ac.uk/vectar/ metabolic screening laboratory:  http://research.vet.upenn.edu/default.aspx?alias=research.vet.upenn.edu/penngen karyotyping/cytogenetic services:  http://vetmed.tamu.edu/labs/cytogenics-genomics/karyotyping  http://www.vgl.ucdavis.edu/services/index.php  http://www.vet.upenn.edu/ryanvhupforsmallanimalpatients/specialtycareservices/medicalgenetics/rese archfacilities/cytogenicslab/tabid/708/default.aspx selected parentage testing services:  http://www.vgl.ucdavis.edu/services/index.php  http://www.vetgen.com/canine-profiling-parentage.html  http://www.vetdnacenter.com/canine-parentage-test.html  http://www.dnares.in/canine-veterinary-genetic-dna-parentage-testing-kits-laboratory.php  http://www.uq.edu.au/vetschool/agl  https://www.pawprintgenetics.com/parentage/product-select/  https://dnaproofofparentage.com/  https://www.happydogdna.com/proof-parentage/  https://www.homednadirect.com/animal-dna-tests/ selected dna storage services:  https://www.ofa.org/about/dna-repository  http://www.animalsdna.com/www.animalsdna.com/web/page/canine/dna_banking/index.html  https://www.vetgen.com/services-other.html  https://www.antagene.com/en/antagene/dna-storage-service in breeding/heterozygosity evaluations  https://embarkvet.com/  https://www.optimal-selection.com  https://www.betterbred.com/ clinical theriogenology • volume 12 number 3 • september 2020224 once identified, defect can be further investigated with more specific protein assays. section of medical genetics at university of pennsylvania school of veterinary medicine is one of few places that perform such tests to diagnose as well as to discover novel hereditary disorders www.vet.upenn.edu/penngen.9 in addition, few laboratories offer cytogenetic studies to evaluate for potential abnormalities in chromosomes (table). any cell capable of dividing can be used for this purpose; however, most commonly, blood lymphocytes or skin fibroblasts are used. for lymphocyte culture, blood is collected into sodium heparin, cultured in media and stimulated to divide. cells are arrested in mitosis during metaphase where chromosomes are compacted. chromosomes can then be stained to yield a typical banding pattern of chromosomes used in traditional karyotyping, or fluorescent probes can be used in a technique known as fluorescence in situ hybridization. a thorough investigation into the family history of a patient with a suspected genetic disease is also important to determine a potential mode of inheritance. knowing how a disease is passed from generation to generation is critical to plan a breeding program to manage genetic diseases, as well as starting investigation into a genetic cause of a new disease presentation. inheritance patterns reported in veterinary medicine include autosomal recessive, autosomal dominant, x-linked recessive, x-linked dominant and complex (polygenetic) diseases.2 recessive diseases account for a majority of diseases for which a known inheritance pattern and a genetic defect are not identified.12 however, with continued advances in molecular biology and technology, this is also becoming true for complex (polygenetic) disorders. types of genetic disorders autosomal recessive diseases are identified most commonly at presentation of affected animals with both sexes equally represented born to clinically normal parents. typically, clinically normal parents have a common ancestor. these animals that are phenotypically normal are referred to as carriers (heterozygous for the disease-causing allele).2 common theories for increased prevalence of expression of autosomal diseases in purebred dog and cat populations include higher degree of inbreeding related to popular sire effects, selective inbreeding, and bottlenecks in their populations. autosomal dominant diseases are often seen with an affected individual produced from at least one affected parent, since carrying either one (heterozygous) or both (homozygous) copies of mutant allele will result in disease. however, not uncommonly, new mutations can occur which result in an affected animal that is produced by 2 clinically normal parents.2 in some cases, diseases are referred to as being incompletely dominant. traditionally, incomplete dominance occurs when expression of disease with a heterozygous genotype (one copy of the mutant allele) is an intermediate or has variable disease expression. in these cases, parents with the disease-causing allele may not exhibit any clinical signs and appear normal, yet they may pass that disease causing allele to their offspring. some theorize that interactions with other modifying genes and in some cases, environment, affect disease expression and severity, making some believe that a proportion of these incompletely dominant diseases may have inheritance patterns more similar to complex modes of inheritance. x-linked recessive diseases are distinguished mainly by males being predominantly affected. females are far less likely to be affected based on the presence of 2 x chromosomes and requirement for an affected male to survive long enough to reproduce with a carrier female in order to produce an affected female offspring.8 first canine mutation discovered was x-linked recessive disease, hemophilia b.13 xlinked dominant diseases are extremely rare, e.g. x-linked alport syndrome in samoyed dogs.14 y-linked disorders are caused by y chromosome mutations. since males inherit a y chromosome from their fathers, every son of an affected father will be affected. however, since females only inherit an x chromosome from fathers, female offspring of affected fathers are always normal with y-linked disorders. since y chromosome is relatively small and contains very few genes, there are relatively few y-linked disorders and none reported in small animals. another rare mode of inheritance in veterinary medicine is mitochondrial inheritance. this type of inheritance, also known as maternal inheritance, clinical theriogenology • volume 12 number 3 • september 2020 225 applies to mutations of genes in mitochondrial dna. since only oocytes contribute mitochondria to the developing embryo, only mothers can pass on mitochondrial conditions to their offspring.8 complex disorders are more difficult to identify as they result from combination of effects of multiple genes (polygenetic) as well as environmental influences that result in an expressed phenotype. although complex disorders often cluster in breed or family lines, they do not have a clear-cut pattern of inheritance as seen with single-gene disorders.2 this non-mendelian inheritance pattern, as is often used to describe complex disorders, makes it difficult to determine an animal’s risk of inheriting or passing on these diseases. complex disorders are also more difficult to study and identify all factors leading to expression of disease. however, common veterinary diseases are increasingly recognized as having a genetic, heritable component. in fact, some common diseases recognized in veterinary medicine meet these criteria, including hip dysplasia, hypothyroidism, cancer and atopy.15 some argue that everything has a genetic component and it is how these genes are expressed by a multitude of factors that determines rate of disease. with increasing knowledge of gene involvement in disease, clinical veterinary genetics becomes increasingly important in diagnosis, management and prevention of disease in our patients. identification of unknown genetic diseases: genetic tests advantages and limitations veterinarians are part of an important team involved in identification and control of genetic diseases. breeders, pet owners, primary care veterinarians, veterinary specialists, veterinary researchers, genetic databases and research funding institutions comprise vital pieces of this team. when a new genetic disease is suspected, team members need to work together to compile information and resources required to determine gene defect(s) involved. sometimes this is not straightforward and easy. in general, researchers often modify and combine multiple techniques in uncovering a genetic mutation process, most common being genome-wide association study and candidate gene approach. genome-wide association studies compare dna of 2 groups of participants: affected animals and similar animals without disease (normal controls). dna is collected from these individuals and gene chips along with computer technology are used to read millions of dna sequences. however, rather than reading entire dna sequence, single nucleotide polymorphisms (snps) are used. snps are variations in a single nucleotide of a dna sequence, used as markers for evidence of dna variation. different variations are then identified along with their association with different traits. if genetic variations are more frequent in diseased animals as compared to normal controls, variations are considered associated with the disease. associated genetic variations are then considered linked-markers to the region of the genome where the disease-causing problem is likely to reside. most snp variations associated with disease are not in dna region that codes for a protein. instead, they are usually in large noncoding regions on the chromosome between genes that are edited out of the dna sequence when proteins are processed. however, once these markers are linked to a disease, further molecular techniques can be utilized to narrow down the region and sequence potential genes, thus identifying mutations.16 another method utilized in genetic mutation investigation is candidate gene approach. this approach requires researchers to investigate validity of an educated guess about genetic basis of a disorder, as opposed to genome-wide association studies that are predicated on an unbiased search of entire genome without preconceptions about role of a certain gene. similar to genome-wide association studies, candidate gene approach involves comparison of affected individuals with normal controls; however, since 1 gene is the focus, large populations are not required for an association with disease to be detected. major difficulty with this approach is that to choose a potential candidate gene, researchers must already have an understanding of disease pathophysiology and potential genes that may influence the mechanism of that specific disease, e.g. gene mutation known to cause same disease in another species.17,18 often, linkage to a disease is known before mutation is identified. linked marker testing can then be utilized to assist breeders in breeding choices before a mutation-based test is established. it is important for veterinarians and breeders to understand advantages and limitations for a particular genetic test to achieve their goals of controlling genetic diseases while maintaining genetic diversity in population as a whole. several types of inherited disease screening and genetic tests are described in veterinary clinical theriogenology • volume 12 number 3 • september 2020226 medicine, including phenotypic testing, linked-marker testing and mutation-based tests. in short, not all genetic tests are created equally and understanding various types of tests along with mode of inheritance of a disease is vital to use tests appropriately. for example, linked marker testing may have 2 potentials for errors. first error can occur from a recombination event where the marker is no longer linked to mutant allele, resulting in either a false positive or false negative test.2 in general, closer the marker is to mutant allele, more likely they will remain together, or linked, and less likely recombination will result in their separation. another error occurs if the marker is not linked to mutant allele, but is present in a high enough frequency in the population that it may initially appear linked, resulting in a false positive test.2 although caution must be used when interpreting test results, it is important to recognize that a linked marker test is extremely useful when dealing with a disease for which the mutation is not yet known. once a gene mutation is identified, it is important to note that these mutations are very specific. small animals within same or a closely related breed may likely have same disease-causing mutation for a particular disease. however, small animals of other breeds, particularly unrelated breeds, with same disorder, may also have different mutations that may not be detected with a mutation-based test.8 there may also be > 1 genetic mutation within a breed that may result in similar clinical signs and in these cases, all mutations need to be evaluated. dna tests have several advantages. test can be performed at any age and long before clinical signs become apparent, detecting affected, normal and carrier animals. dna can be extracted from any nucleated cell, such as white blood cells, cheek cells, hair follicles, semen and even formalinized tissue. cheek swabs should be used very cautiously or avoided in nursing animals due to potential contamination of oral cavity with maternal nucleated cells.8 since dna is very stable and small quantities are required, it can be banked for long-term storage and utilized in future genetic studies. there are several veterinary dna storage facilities for this purpose. key factor in usefulness of dna for future studies is determined by complete and thorough records on that animal. an animal suffering from an inherited disease needs to have an accurate diagnosis of the cause of that disease in order to prevent false associations when utilizing that animal’s dna for a potential gene mutation study. for example, a cat with suspected liver disease due to amyloidosis needs to have histopathological conformation of that disorder, or there is a risk that an animal with hepatic adenocarcinoma may confuse and invalidate future genetic studies. although identifying genes causing disease is of valuable assistance, that alone is insufficient. understanding how to implement testing and educating clients to responsible use of these technologies is vital. so, knowledge of molecular genetics and current biotechnology is only 1 piece of the puzzle. genetic diversity development of pedigree animals are due to selection for a particular set of physical and/or behavioral characteristics. this can be considered both a “blessing and a curse”, because the result has been obtained from inbreeding within close family lines resulting in both preservation of desired features as well as undesirable genetic traits becoming fixed within breeds.19,24 closed populations, such as purebred animals, with high levels of selection pressure, suffer from a loss of genetic diversity. for example, in dogs, 2 major events resulted in a significant loss in genetic diversity which only continues in these closed populations. these 2 major events include domestication and development of breeds. in managed populations, genetic diversity can be maintained by careful selection in order to maximize optimal contributions of each breeding animal.20.26 however, in most purebred organizations, e.g. dog breed clubs, there is no single organization to assist with directing breeding strategies.24 this is why veterinary genetic counseling is so vital to the future of dog breeding. individual breeders should have insight into population genetics and methods to preserve as much genetic diversity in these closed breeds as possible.23 veterinarians offer a great way to improve that insight and bridge the knowledge gap. evaluating genetic diversity one of the oldest observations of population genetics is that inbreeding reduces fitness in offspring compared to offspring of unrelated individuals. consequently, most human populations prohibit marriage of related individuals and many animal breeders discuss measures of inbreeding. inbred clinical theriogenology • volume 12 number 3 • september 2020 227 individuals have a lower degree of genome wide diversity or heterozygosity because a fraction of their genome is identical by descent inherited by a common ancestor. all measures of inbreeding attempt to predict the proportion of the genome that is identical by descent.21,22 classical measure of this is the pedigree in breeding coefficient.28,29 calculated inbreeding coefficient predicts proportion of the genome that is inherited by descent due to known common ancestor of parents and the assumption that the pedigree founders are unrelated and not inbred. while traditionally, the coefficient of inbreeding has been considered the best measure for an individual’s inbreeding, it has been suggested to be imprecise due to several factors, including inability to account for inbreeding caused by distant ancestors not included in the pedigree. with development of molecular technologies, genetic evaluations of inbreeding have been replacing traditional probability calculations.27 several techniques have been described to evaluate genetic diversity being reported as an evaluation of heterozygosity rather than the traditionally reported inbreeding coefficient designed by probability calculations of pedigrees.25,26 there are 3 main companies offering genetic testing services that includes an evaluation of heterozygosity. these companies are utilizing either short tandem repeats (strs) or single nucleotide polymorphisms (snps). a short tandem repeat (str) in dna occurs when a pattern of 2 or more nucleotides is repeated and repeated sequences are directly adjacent to each other. an str is also known as a microsatellite. pattern can range from 2 6 base pairs and is typically in a noncoding region of dna. as noted above, single nucleotide polymorphisms (snps) are genome regions in which 2 strains differ by a single base pair. both methods of genome scanning have application-dependent advantages and disadvantages. microsatellies can arise through replication slippage, unequal crossing over, or mutations extending or interrupting a series of repeats, whereas snps generally arise via point mutations. as a result, new microsatellite variations arise more frequently than new snp variations. however, the absolute number of snp differences is about a thousand-fold higher than microsatellite differences. thus, snp and microsatellite analyses can provide complementary information, with each is better suited for some tasks than others.28 microsatellites have been the genetic markers of choice for > 2 decades. they are informative and interspersed throughout the entire genome. however, microsatellites can be more time consuming for trained personnel to analyze, even with appropriate software or automated methods. recent advances in high-throughput dna sequencing, computer software and bioinformatics have increased popularity of snps. they have promising advantages, including greater abundance, genetic stability, simpler nomenclature and suitability to automated analysis and data interpretation. furthermore, snps have been used in discovery of quantitative trait loci (qtl), the association of phenotypic traits and genetic markers allowing for evaluation of specific productive traits and identification of individuals and breeds.23 consolidation of tests: current trends currently, there is a move toward consolidation of many available genetic tests from small businesses and universities to larger companies offering analysis of genetic diversity (based on snps or microsatellites to evaluate in-breeding). sudden availability of large amounts of genetic data for breeders is frequently misinterpreted and is an opportunity for veterinarians to bridge the knowledge gap. some of these companies include embark, optimal selection and betterbred. optimal selection currently offers similar testing for cats. embark and optimal selection are utilizing snps, whereas betterbred, which is partnered with the university of california davis veterinary genetics laboratory, utilizes microsatellites and is available only for certain breeds. in addition to offering tools for evaluating in-breeding of individual dogs, both optimal selection and embark have begun offering a “match making tool” that compares genetic diversity information between individuals of a proposed mating. estimated breeding values currently, the vast majority of genetic tests available are for single-gene disorders. many dog and cat breeders continue to rely solely on phenotypic data when dealing with complex disorders such as hip dysplasia. they are mostly screening prospective breeding animals and selecting breeding pairs with this clinical theriogenology • volume 12 number 3 • september 2020228 limited information; their goal is to reduce disease incidence, but the approach has flaws and progress is slow. in this area, we can learn more from large-animal producers who have utilized estimated breeding values (ebv) for decades to manage diseases with more complex inheritance and improve production parameters. estimated breeding values (ebvs) are the value of an individual as a genetic parent. they utilize all that is known about a trait, in an effort to predict the potential that the trait will be expressed in the offspring. the more that is known about the trait, including the environmental conditions associated with individuals as well as heritability percentage, the greater accuracy.30 large-animal producers have used ebvs to do everything from increase volume, butterfat and protein in milk to improve production of lean meat from beef animals, to increase feed efficiency and improve reproduction.30 in us beef industry, various breed associations are responsible for calculating ebv’s. most associations, however, contract with a university that specializes in advancing technology for doing the (inherently complex) ebv calculations.30 major universities where contract computing is done include iowa state university, cornell university, colorado state university and university of georgia. computing ebv's for dairy industry is performed by animal improvement programs laboratory, beltsville agricultural research center, agriculture research service, us department of agriculture where staff work year-round to improve and implement process of calculating ebv’s for dairy cattle.30 estimated breeding values change, as data used to calculate them is constantly evolving. understanding their use in breeding programs for those unfamiliar with genetics can be overwhelming. for the large-animal industry, the cooperative extension service was very successful in educating farmers on the use of ebvs. one of the most successful tools for teaching beef cattle breeders how to use ebv’s was the computer cow game.30 this created real-world scenarios where producers would make breeding choices based on ebvs. typical of the real-world, a certain percentage of cows in every herd would fail to conceive, calves would die and other calamities would strike. out of all this mayhem and information, however, emerged new calves with new records that became the basis for a new round of ebv’s.30 selection decisions could be made and the process repeated. cattle breeders enjoyed competing with each other to see who could produce the greatest genetic improvement in their herd in 5 generations of selection. such a tool was suggested by prominent canine geneticist, dr. elton leighton, specifically written for dog breeding and implemented online, could be used to teach dog breeders about ebv’s.30 dr. leighton and the team at the seeing eye, have been utilizing estimated breeding values for years to improve genetic selection for dogs to succeed in their program as well as to improve dogs’ health, in particular, hip health. they continue to publish successful demonstrations of their research showing how dog breeders can incorporate these techniques to improve selection for these more complex inherited diseases.31 the task of calculating ebv’s is generally complex and relies on diverse sharing of a large amounts of data, with accuracy of the ebv’s based on accuracy of information reported, or lack thereof. to be successfully implemented with dogs, groups of dog breeders, e.g. national parent clubs or the american kennel club, need to decide that routinely computing ebv’s is an important task to be undertaken. any of these dog breeder organizations can turn to u.s. beef or dairy industries or seeing eye for examples of how to organize data collection and calculation processes. recent advances in computing algorithms and computer software to implement these algorithms now facilitate simultaneously calculating ebv’s on several traits measured on thousands of dogs.30 conclusion with growing advancements in molecular genetics and genetic tests being developed at an exponential rate, it is important for veterinarians and breeders to have a basic understanding of how to utilize these techniques in order to control genetic diseases while maintaining genetic diversity of the population as a whole. emergence of genomics into clinical veterinary practice has led to the need for development of a team-based approach to control and identify genetic disorders in small animals.8 hard work and cooperation among breeders, pet owners, primary-care veterinarians, veterinary specialists, veterinary researchers, genetic databases and funding institutions has resulted in development of nearly 200 genetic tests.6 this research has not only benefited the lives of our small animal patients and their clinical theriogenology • volume 12 number 3 • september 2020 229 families, but has also increased understanding of genetic diseases in other species, including humans. going forward, we all need to continue our vital roles in this process so that we can further unlock mysteries behind some very common diseases in veterinary medicine. veterinarians, particularly reproductive veterinarians, are in a unique position to bridge the knowledge gap between advancements in genetic science and clinical application in small dog and cat breeding operations. we can assist breeders in understanding current genetic status of their own breed so they can work to preserve population size of their breed. we can help breeders utilize tools such as estimated breeding values, inbreeding or heterozygosity estimates, and mutation-based genetic disease tests optimally in their breeding programs. we can help them to understand that the mistakes of past should be replaced by exciting technological advances of future to implement breeding strategies that will more effectively reach their goal of producing dogs and cats that not only are what is desired for appearance, temperament and workability, but also live long, healthy lives. conflict of interest there are no conflicts of interest to declare. references 1. nicholas fw: online mendelian inheritance in animals (omia): a comparative knowledgebase of genetic disorders and other familial traits in non-laboratory animals. nucleic acids res 31:275-277 (http://omima.angis.org.au/). 2. traas am, casal m, haskins m, et al: genetic counseling in the era of molecular diagnostics. theriogenology 2006;66:599-605. 3. werner p, raducha mg, prociuk u, et al: a novel locus for dilated cardiomyopathy maps to canine chromosome 8. genomics. 2008;6:517-521. 4. van vleet jf, ferrans vj: myocardial diseases of animals. am j pathol 1986;124;98-178. 5. ackerman, l: the genetic connection: a guide to health problems in purebred dogs. aaha press, 2000. 6. giger u. clinical tools to diagnose hereditary disorders. proceedings of the 34th world small animal veterinary congress 2009 são paulo, brazil. 7. patterson df, aguirre ga, fyfe jc, et al: is this a genetic disease? j small animal pract 1989;30:127-139. 8. giger u: clinical genetics. in textbook of veterinary internal medicine. ettinger s, feldman e. saunders, philadelphia, 2005. 9. giger u, jezyk pf: diagnosis of inborn errors of metabolism in small animals. in: current veterinary therapy xi. kirk rw, editor. wb saunders; philadelphia: 1992. p. 18-22. 10. giger u. new insights into hereditary diseases and genetic predisposition to disease in dogs. proceedings of the 34th world small animal veterinary congress 2009 são paulo, brazil. 11. giger u. peculiarities of feline hereditary disorders. proceedings of the 34th world small animal veterinary congress 2009 são paulo, brazil. 12. meyers-wallen vn: ethics and genetic selection in purebred dogs. reprod domest anim 2003;38:73-76. 13. evans jp, brinkhouse km, brayer gd, et al: canine hemophilia b resulting from a point mutation with unusual consequences. proc natl acad sci usa 1989;86:10095-10099. 14. cox ml, lees ge, kashtan ce, et al: genetic cause of x-linked alport syndrome in a family of domestic dogs. mamm genome 2003;14:396-403. 15. suter nb, ostrander ea: dog star rising: the canine genetic system. nat rev genet 2004;5:900-910. 16. pearson ta, manolio ta: how to interpret a genome-wide association study j amer vet assoc 2008;299: 1335-1344. 17. aguirre gd, ray k, acland gm: candidate gene studies in canine progressive retinal atrophy. digital j ophthalmol 1998;4:3 www.djo.harvard.edu/site.php?url=/physicians/oa/381logy 18. kwon jm, goate am: the candidate gene approach. alcohol research & health 2000;24:164-168. 19. farrell ll, schoenbeck jj, wiener p, et al: the challenges of pedigree dog health: approaches to combating inherited disease. canine genet epidemiol 2:3.doi: 10.1186/s40575-015-0014-9.ecollection 2015 20. leroy g, abitbol m: breeding strategies against genetic disorders in dog breeds. anim genet 2009;23:323-332. 21. mellersh c: dna testing and domestic dogs. mamm genome 2012;23:109-123. 22. ostrander ea: both ends of the leash the human links to good dogs with bad genes. n engl j med 2012;367: 636-646. 23. padgett ga: control of canine genetic diseases. howell book house, ny.1998. 24. rooney n, sargan d: pedigree dog breeding in the uk: a major welfare concern? a report commissioned by the royal society for the prevention of cruelty to animals. 2009. 25. ives ar, whitlock mc: inbreeding and metapopulations. science 2002;295:454-455. clinical theriogenology • volume 12 number 3 • september 2020230 26. allendorf fw, hohenlohe pa, luikart g., genomics and the future of conservation genetics. nat rev genet 2010;11: 697-709. 27. balloux f, amos w, coulson t: does heterozygosity estimate inbreeding in real populations? mol ecol 2004;13: 3021-3031. 28. keller mc, visscher pm, goddard me: quantification of inbreeding due to distant ancestors and its detection using dense single nucleotide polymorphism data. genetics 2011;189:237-249. 29. wright s: coefficients of inbreeding and relationship. am nat 1922;56:330-338. 30. leighton, ea: using estimated breeding values to reduce the incidence of genetic diseases in dogs. presented at a meeting of the international elbow working group. 1999. 31. leighton ea, holle d, biery dn, et al: genetic improvement of hip-extended scores in 3 breeds of guide dogs using estimated breeding values: notable progress but more improvement is needed. plos one. 2019;14(2):e0212544. published 2019 feb 22. doi:10.1371/journal.pone.0212544. clinical theriogenology • volume 12 number 3 • september 2020 231 013_ms-013 o'connor     successful pregnancies in a mare after laparoscopic unilateral ovariohysterectomy jennifer clulow, samuel nugent, angus adkins scone equine group, liverpool street, scone, nsw, australia abstract a 6 year old thoroughbred mare was presented for investigation of infertility. over 3 consecutive seasons, she had been bred naturally (14 controlled estrous cycles) with 3 thoroughbred stallions and did not become pregnant. the mare was bred artificially over 3 estrous cycles with semen from a quarter horse stallion (known for excellent fertility). both ovaries appeared functional. during embryo collection on day 7 postovulation, failure of appropriate dilation of left uterine horn was noticed after embryo collecting medium was infused. diagnostic hysteroscopy was performed and partial patency defect of the left uterine horn was identified. after sedation and local anesthesia, a unilateral laparoscopic ovariohysterectomy was performed while the mare was standing. approximately 80% of the left uterine horn was removed using a vasculature sealing device to provide hemostasis and intracorporeal suturing technique was used for uterine closure. subsequently, the mare became pregnant and foaled twice. keywords: mare, infertility, uterus, hysteroscopy, laparoscopy, unilateral ovariohysterectomy background successful pregnancy and birth of a healthy foal are possible in mares with only 1 functional uterine horn.1 however, infertility may become an issue in such cases, depending on the physiological capability of the remaining functional uterine horn. furthermore, inability of the embryo to move between uterine horns can cause failure of maternal recognition.2 in certain cases, it is not the absence of a uterine horn, but rather limited function of a uterine horn that may cause infertility. these cases, not only complicate infertility diagnosis but also may be difficult to solve. we used hysteroscopy to diagnose a uterine patency defect of a uterine horn that had been the cause of infertility and applied laparoscopic hysterectomy technique to correct this problem. case presentation history a 6 year old maiden thoroughbred mare was presented for investigation of infertility. the mare failed to become pregnant over 3 consecutive breeding seasons. during this time, she had been bred naturally (3 thoroughbred stallions over 14 managed estrous cycles). her endometrial score was iia (mild lymphocytic endometrium). oral treatment with altrenogest (regumate® merck animal health, madison, nj) was implemented once during her first breeding season (0.088 mg/kg), starting on day 5 postovulation. during second and third breeding seasons, pge2 gel (prostin e2 vaginal gel, pfizer, new york, ny) was laparoscopically applied to uterine tubes for suspected uterine tube obstruction as a cause of infertility. during first laparoscopic procedure, both uterine tubes appeared dilated. mare was bred naturally in 3 estrous cycles (twice with 1 of the initial stallions and with a new stallion on the third cycle) with no resultant pregnancy. a year later, a second laparoscopic procedure was performed. the left uterine tube appeared dilated at its cranial half and there was an obvious area of scarring and constriction in the middle of the uterine tube. visible bands constricting the uterine tube were transected, in addition to pge2 gel application.3 the right uterine tube appeared normal as observed during initial laparoscopy and pge2 gel application was repeated. clinical examination late in the third breeding season, the mare was presented for assessment and was in good physical condition. initial examination (transrectal palpation and ultrasonography) identified 2 functioning ovaries, with follicular activity on both ovaries and evidence of a recent ovulation (corpus hemorrhagicum) on the left ovary. a low volume uterine lavage was performed and a clitoral swab was submitted for culture and sensitivity. intrauterine treatment was initiated for 3 consecutive days with ceftiofur (1 g, accent®, zamira, australia) for escherichia coli and β haemolytic streptococcus spp (group c). a controlled (for embryo collection on day 7 postovulation) breeding cycle was clinical theriogenology • volume 13 number 1 • march 2021 52 conducted. the mare was inseminated with fresh semen (> 500 x 106 progressively motile sperm) from a quarter horse stallion (history of 100% first cycle conception rates that breeding season). breeding was 1 day before ovulation, based on transrectal ultrasonography findings (table). table. ovulation, artificial insemination, embryo recovery details, and pregnancy details ovary ovulated date of insemination date of ovulation embryo recovery/pregnancy left march 14/15 march 16 negative right april 4 april 5 positive 7 days right and left may 14/15 may 15 positive 14 days embryo collection was attempted on day 7 post ovulation in first 2 estrous cycles. the left uterine horn’s inability to dilate appropriately was observed after infusion of embryo collecting medium. in the third estrous cycle, embryo collection was not performed, and mare was diagnosed pregnant on day 14 postovulation. the conceptus was attached to the base of the left uterine horn and grew normally until ~ 25 days. an embryo proper could be visualized at this time; however, heartbeat could not be identified, and conceptus regressed by day 30, despite oral altrenogest (0.044 mg/kg) and 3 g of aspirin (ace chemical company, australia) treatments daily, starting day 5 postovulation. a hysteroscopy was performed using a 1.5 m endoscope with a 9.2 mm diameter to examine the endometrium and determine uterine horn patency. examination identified a normal right uterine horn and endometrium. however, ~ one-third along the length of the left uterine horn, a blind end appeared, preventing left utero-tubal papillae visualization. further instillation of air was required, and a small opening appeared, allowing passage of the video endoscope with difficulty. remainder of the uterine horn appeared normal and the utero-tubal papillae was visible. treatment removal of the affected uterine segment was recommended, and surgery was scheduled for the nonbreeding season. a unilateral laparoscopic ovariohysterectomy was performed while the mare was standing, using standard flank laparoscopic technique with the mare sedated and restrained in stocks. an indentation in uterine contour was noticed in the left uterine horn, just cranial to uterine body and corresponding to the location identified in hysteroscopic examination. approximately 80% of the left uterine horn was removed. ovarian pedicle and broad ligament were desensitized with 50 ml of 2% mepivacaine (troy laboratories, australia). a vasculature sealing device (covidien ligasure, ls1037, medtronic, minneapolis, mn) was used to transect and seal vasculature of ovarian pedicle and broad ligament to the point of obstruction. it was also used to transect the uterine horn immediately caudal to the uterine obstruction before intracorporal suturing of the uterus, using a double layer of a barbed suture (covidien v loc™, vloca008l, medtronic) placed with an endo stitch (173027, medtronic). the ovary and uterine horn were removed from the abdomen with the aid of a retrieval bag (covidien endo catch ii specimen pouch, 15 mm, medtronic). surgery time was 52 minutes. the mare was treated orally with phenylbutazone (bute paste®, ranvet, australia) 1 g, once daily for 5 days. outcome one month later, hysteroscopy was performed, and endometrium appeared to be healing well and the mare was returned to a stud farm for routine breeding management. one week later, a prebreeding examination was performed by the farm veterinarian and transrectal ultrasonography identified a palpable mass with mixed echogenicity at the surgical site on the left uterine horn stump. serum amyloid a (saa) concentrations were 2707 µg/ml and white cell count was normal. no other sources of acute inflammation were identified. treatment was initiated orally with doxycycline (bova compounding, australia) 10 mg/kg twice daily for 3 weeks) because of its oral bioavailability, required duration of treatment, and the antimicrobial susceptibilities in this farm. a reduction in the abscess was noted over time and doxycycline was discontinued based on saa concentrations (0 µg/ml). clinical theriogenology • volume 13 number 1 • march 202153   in the subsequent breeding season, the mare failed to conceive and maintain pregnancy following her first breeding attempt. after second unsuccessful breeding of the season, klebsiella pneumoniae infection of the clitoris was identified. clitoris was treated topically with amikacin sulphate (50 mg/ml) ointment (bova compounding) for 3 days. pregnancy was detected after the third breeding of the season and the mare commenced orally treatment with altrenogest (0.088 mg/kg), following the detection of embryo at 14 days and treatment continued throughout pregnancy. a healthy 50 kg colt was foaled on 363 days of pregnancy and continued to grow and develop normally, similar to his cohort. the mare was subsequently bred again and became pregnant following her first breeding at 34 days postpartum. a healthy 53 kg filly foaled at 359 days gestation. discussion prior to admission, the mare was bred during 14 estrous cycles (3 consecutive breeding seasons) by 3 stallions. during this period, mare had 5 ovulations from the right ovary and was bred to 1 specific stallion for 4 of these 5 ovulations. infertility continued, despite attempts to correct the suspected uterine tube blockade via laparoscopy. however, these procedures enabled visualization of scarring and constriction of left uterine tube and allowed transection of the fibrous material that caused constriction. it was not known whether infertility was due to functional inability of the left ovary or a left uterine horn problem. the contribution of the left uterine horn became partially evident in 1 controlled breeding cycle, as the conceptus remained in situ. however, it failed to thrive when attached to the base of the left uterine horn, despite hormonal and anti-inflammatory treatments to support pregnancy. this case demonstrated the value of hysteroscopy in the identification and diagnosis of uterine disease and abnormalities. a uterine problem that we diagnosed via hysteroscopy had been the cause of this barren maiden mare’s 3 year history of infertility. although the majority of ovulations were from the left ovary, the defect identified in the left uterine horn had contributed to the apparent infertility of this mare, likely due to restricted conceptus mobility and resulted in the failure of maternal recognition of pregnancy.2 the defect caused complete closure of the left uterine horn; incidentally, a substantial amount of air was required to insufflate the uterus to allow passage of the scope. furthermore, fluid could not be palpated in the left uterine horn during manual transrectal manipulation of the uterus during embryo retrieval. this defect might have restricted the entry of semen and possibly embryo migration.2 although altrenogest supplementation allowed embryo to grow, restriction of embryo mobility might have resulted in pregnancy loss. total and partial ovariohysterectomy have been performed to remove uterine tumors4-7 or to resolve pyometra8-9 in mares that were unresponsive to medical therapy. in the present case, a partial ovariohysterectomy was used to correct a uterine patency defect. a laparoscopic approach was undertaken to allow standing surgery, reduce patient morbidity, improve visualization of structures, improve hemostasis, and to shorten the convalescent period. although hand assisted laparoscopic techniques were reported, 5,8,9 exteriorization of uterus was necessary in such cases, rather than performing partial hysterectomy within the abdomen, as described herein. we adopted more advanced laparoscopic skills and equipment to complete the procedure in a relatively short interval. an enlarged structure identified at the site of uterine closure after hysteroscopy was possibly an abscess due to uterine stump infection.8 elevated saa concentrations suggested this possibility. hysteroscopy procedure might have damaged the tissue, resulting in a hematoma and abscess formation. mare was treated with doxycycline, due to concerns of abscess rupture and peritonitis and the enlarged structure resolved in 3 weeks. prolonged pregnancies (363 and 359 days) were likely due to physically restricted placental surface area. this restricted placentation resembled type a twin placentae situation10 wherein 1 twin occupied uterine body and 1 uterine horn (68% total uterine surface area) or in a case of uterine unicornis.1 although ~ 80% of the left uterine horn was removed, the remaining right uterine horn was sufficient in size (noted at the time of hysteroscopy) to support the growth of foal to maturity by providing adequate microscopic area of fetomaternal placental contact.11 it is important to recognize that foal birth weights (50 and 53 kg) were within normal limits for the mare’s age and parity.12 clinical theriogenology • volume 13 number 1 • march 2021 54 learning points  hysteroscopy is a useful tool in the diagnosis of infertility and in the treatment of blocked uterine tubes.  partial hysterectomy can be performed efficiently with advanced laparoscopic skills and equipment.  birth of a normal foal is possible after partial hysterectomy. conflict of interest none to report references 1. gallacher k, gilbert ro: case report: successful foaling in a warmblood mare with uterus unicornis. clinical theriogenology 2018;10:51-58. 2. mcdowell kj, sharpe dc, grubaugh w, et al: restricted conceptus mobility results in failure of pregnancy maintenance in mares. biol reprod 1988:39:340-348. 3. pye j, clulow jr, adkins a: laparoscopic transection of restrictive bands of the mesosalpinx as adjunct to the use of prostaglandin e2 for mares with suspected uterine tubal blockage. aust vet j 2018:96:252-256. 4. muurlink t, walmsley j, whitton c: successful laparoscopic surgery for a uterine leiomyoma in a mare. equine vet educ 2008;20:508-511. 5. heijltjes lm, rijkenhuizen abm, hendriks wk, et al: removal by laparoscopic partial ovariohysterectomy of a uterine leiomyoma assumed to have cause fetal death in a mare. equine vet educ 2009;21:198-203. 6. janicek jc, rodgerson dh, boone bl: use of hand assisted laparoscopic technique for removal of a uterine leiomyoma in a standing mare. j am vet med assoc 2004;225:911-914. 7. berezowski c: diagnosis of a uterine leiomyoma using hysteroscopy and a partial ovariohysterectomy in a mare. can vet j 2002;42:968-970. 8. rottin ak, freeman de, doyle aj, et al: total and partial ovariohysterectomy in seven mares. equine vet j 2003;35:29-33. 9. santschi em, adams sb, robertson jt, et al: ovariohysterectomy in six mares. vet surg 1995;25:165-171. 10. delling u, howard rd, pleasant rs, et al: hand-assisted laparoscopic ovariohysterectomy in the mare. vet surg 2004;33:487-494. 11. jeffcott lb, whitwell ke: twinning as a cause of fetal and neonatal loss in the thoroughbred mare. j comp pathol 1973:83:91-106. 12. allen wr, wilsher s, turnbull c, et al: influence of maternal size on placental, fetal and postnatal growth in the horse. i. development in utero. reproduction 2002;123:445-453 13. elliot c, morten j, chopin j: factors affecting foal birth weight in thoroughbred horses. theriogenology 2009;71:683-689. clinical theriogenology • volume 13 number 1 • march 202155 98 contact muhammad-salman waqas salman.waqas@wsu.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9638, http://dx.doi.org/10.58292/ct.v15.9638 review report laparoscopic-assisted cryptorchidectomy in south american camelids and nigerian dwarf goats dane schwartz, muhammad-salman waqas, eduardo arroyo, agustin ruiz and ahmed tibary comparative theriogenology service, department of veterinary clinical sciences, college of veterinary medicine, washington state university, wa, usa abstract cryptorchidism is a common sexual development disorder in males with a potential hereditary origin in goats and camelids. cryptorchid animals should be removed from breeding and castrated to eliminate male behavior. the objective of this paper is to describe the presentation and procedure of laparoscopic-assisted cryptorchidectomy in goats and south american camelids. records of 18 nigerian dwarf goats and 16 camelid cases that were presented to the wsu theriogenology services were included. goats ranged in age from 2 months to 1 year and camelids (14 alpacas and 2 llamas) were between 15 months and 5 years of age. in goats, 15/18 (83%) were unilateral cryptorchids while 3/18 (17%) were bilateral. the right testicle was retained abdominally in 73.3% (11/15) of the unilaterally cryptorchid goats. all the camelids presented as unilateral cryptorchids with abdominally retained testis. the left testis was retained in 9/16 (56.25%) cases while the right was retained in 7/17 (43.75%). laparoscopic-assisted cryptorchidectomy was performed under general anesthesia in all cases. the remaining scrotal testicle was castrated using a closed technique. there were no surgical complications. ability to confirm the presence of cryptorchid testis without laparotomy, smaller incision, lesser intraabdominal manipulation, and lesser-no post/surgical complications advocate the use of this technique for cryptorchidectomy. keywords: goats, alpacas, llamas, testis, surgery introduction cryptorchidism is one of the most common congenital disorder of sexual development in males.1 in alpacas, an abattoir study on 792 animals reported a 3% incidence of unilateral cryptorchidism (58.3% left and 41.7% right).2 bilateral cryptorchism is rare in camelids and has been described in an srynegative xx, sex reversal case in a llama with multiple congenital abnormalities.3 monorchism, absence of one testicle, has been reported in alpacas and is accompanied by agenesis of the ipsilateral kidney.2 in goats, a 3% incidence of cryptorchidism has been reported in slaughtered goats in india.4 the incidence of cryptorchidism is higher in west african dwarf goats and ranges from 33 to 66% in some regions.5–7 nigerian dwarf goats are a popular breed as companion animals in the usa and one of the most common breeds to visit hospital for cryptorchidism (a tibary, clinical observation). cryptorchid animals produce normal levels of testosterone and display normal male behavior and mating ability.8 in one case of bilateral cryptorchidism in a dromedary camel, rutting, mating, ejaculation, and induction of ovulation were like normal camels although the ejaculates from the affected camel were azoospermic (a tibary, clinical observation). unilaterally affected males can be fertile if the descended testicle is not removed. unilateral cryptorchid nigerian dwarf goats are fertile but show an increased percentage of sperm abnormalities.6,7 diagnosis of cryptorchidism is based on history and clinical examination of the scrotal and inguinal regions. however, in animals displaying male-like behavior with unclear history of castration and no testicles in the scrotun or inguinal region, ultrasonographic examination and endocrine evaluation are  necessary to detect the presence of testicular tissue. ultrasonography of the suspected cryptorchid goat or camelid has not been described thoroughly in the literature but it does allow the clinician to determine the location, size, and morphology of the retained testes. endocrine diagnosis has been used mainly in camelids and is based on the response to a human chorionic gonadotropin (hcg) stimulation test. mailto:salman.waqas@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9638 citation line: clinical theriogenology 2023, 15, 9638, http://dx.doi.org/10.58292/ct.v15.9638 99 at  least a twofold increase in serum testosterone concentration is observed 8 h following administration of hcg (3,000 to 5,000 iu, iv) to confirm presence of testicular tissue.9–11 cryptorchidism is suspected to be hereditary in south american camelids2,12 and nigerian dwarf goats.7 three cases of cryptorchidism were described in closely related males from a herd of captive vicunas.13 a high incidence of cryptorchidism (66%) has been reported in nigerian dwarf bucks where cryptorchid animals are used for breeding.5 therefore, cryptorchid animals should be eliminated from breeding due to hereditary concerns. castration also has the benefit of eliminating male behavior and the risk for induction of ovulation in camelids8 along with the undesirable buck smell found in intact companion goats. several surgical techniques for removal of the retained testis via laparotomy have been described.14–16 in camelids and small size goats such as nigerian dwarfs, these techniques can be challenging and are associated with increased post-surgical complications. the objectives of this paper are to describe an ultrasonographic approach to diagnosis of cryptorchidism and a minimally invasive laparoscopic-assisted approach for cryptorchidectomy in south american camelids and goats. material and methods records of south american camelids (14 alpacas and 2 llamas) and nigerian dwarf goats (n = 18) that presented for cryptorchidectomy to the comparative theriogenology service at washington state university veterinary teaching hospital were included in this retrospective study. camelids ranged in age from 15 months to 5 years of age and goats were between 2 months and 1 year of age. all goats were acquired by owners for the purpose of serving as companion animals. two goats were purchased as wethers and the owners became concerned when the males began to display male-like behavior and buck odor at 6 months of age. a bilaterally cryptorchid goat also presented which was presumed to have been castrated by elastic banding by the breeder at 1 week of age. all animals were in good health upon presentation. after physical examination, males were examined by transcutaneous ultrasonography (sonoscape® with a 5 mhz frequency linear or curvilinear transducer) to determine the location of the retained testis prior to surgery. the animals were examined while in dorsal recumbency. the transducer was placed over the external inguinal ring and the entire region from the inguinal ring to midline was imaged on each side. endocrine diagnosis (hcg stimulation test) was performed in only one alpaca that had a history of castration. total protein concentration and hematocrit were determined in all patients prior to surgery. for laparoscopic-assisted cryptorchidectomy, camelids were fasted for 24 h, and water was withheld for 12 h prior to surgery. total protein concentration and hematocrit were determined in all patients prior to surgery. anesthesia was induced with an intramuscular injection of ketamine (4 mg/ kg; ketaset®, zoetis, kalamazoo, mi), xylazine (0.4 mg/kg; anased®, akorn, lake forest, il) and butorphanol (0.04 mg/ kg; torbugesic®, zoetis, kalamazoo, mi) in combination, intubated and maintained with isoflurane (piramel, talangana, india) in oxygen as needed. the patient was positioned in dorsal recumbency. the abdominal area from the xyphoid process to the inguinal region was clipped and aseptically prepared and draped for surgery. the patient was placed in trendelenburg position to allow cranial displacement of the abdominal viscera, reduce the risk of damage to underlying organs, and aid in visualization of the retained testis. a 5 mm skin incision was made on the umbilical scar and a teat cannula was inserted into the abdomen for insufflation with carbon dioxide (co2) to a pressure of 15 mm hg. the same incision site was used to create a portal for the endoscope using a 10 mm diameter trocar and cannula. a second skin incision was made in the parainguinal region on the same side as the retained testis to create a new portal for insertion of an atraumatic forceps. once the testis was located and isolated with the forceps, it was grasped at the level of the gubernaculum and brought in proximity to the body wall. the parainguinal incision was enlarged to allow exteriorization of the testis. the testis, its respective vascular cone and the gubernaculum were exteriorized. the vascular cone was transfixed with absorbable material (polyglactin 910, #0; vicryl®, ethicon, guaynabo, puerto rico) and the testicle was excised (figure 1). the body wall and skin incision were closed in routine fashion with resorbable material. post-surgical management includes procaine penicillin g (penone pro®, vetone®, mwi, boise, id) 22,000 iu/kg sc sid for 3 days or ceftiofur crystallin-free acid 6.6 mg/kg sc once) and non-steroidal anti-inflammatories (flunixin meglumine 1.1 mg/kg iv sid for 2 days; banamine®, merck, madison, nj). figure 1. laparoscopic-assisted cryptorchidectomy, (a) placement of the endoscope portal over the umbilical scar, (b) placement of the forceps portal, (c) parainguinal incision for exteriorization of the testicle, (d) exteriorization of the retained testicle, (e) transfixation and removal, (f) incision site for exteriorization of the testis. http://dx.doi.org/10.58292/ct.v15.9638 100 citation line: clinical theriogenology 2023, 15, 9638, http://dx.doi.org/10.58292/ct.v15.9638 goats were fasted for 18 to 24 h. anesthesia was induced either with ketamine (1 mg/kg im; ketaset®, zoetis) xylazine (0.1 mg/kg im; anased®, akorn) and butorphanol (0.01 mg/ kg; torbugesic®, zoetis) in combination or with propofol (4 mg/kg iv; propoflo®, zoetis, kalamazoo, mi ) after sedation with butorphanol (0.2 mg/kg iv; torbugesic®, zoetis) and midazolam (0.1 mg/kg iv; almaject, morristown, nj). anesthesia was maintained with isoflurane in oxygen after endotracheal intubation and draped as previously described. animals were placed in dorsal recumbency, and the scrotum and ventral abdomen prepared aseptically. a 1 cm skin incision was made about 4 to 6 cm lateral to midline, contralateral to the side of the retained testis. the abdominal cavity was insufflated with medical grade co2 through a teat cannula prior to insertion of a 6 mm trocar and canula which served as  the laparoscope portal. the patient was placed in trendelenburg position. a second incision was made parallel to the inguinal ring ipsilateral to the retained testis. babcock forceps were introduced into the second portal and the testis was grasped at the level of the gubernaculum (figure 2). the incision length was extended to allow exteriorization of the testicle. the spermatic cord was transfixed with 0 pds suture  (ethicon, guaynabo, puerto rico), transected with metzenbaum scissors and the testis was removed. the incision was sutured in two planes with 2-0 pds (ethicon, guaynabo, puerto rico) to close the body wall with a simple continuous pattern and skin with an intradermal pattern. a single cruciate suture was used to close the laparoscope portal incision. the scrotal testis was castrated using a standard closed technique. for bilaterally cryptorchid cases, both testes were removed from a single paramedian incision instead of a parainguinal incision. post-surgical management includes antibiotics (procaine penicillin g; penone pro®, vetone®; 22,000 iu/kg sc sid for 3 days or ceftiofur crystalline-free acid; excede®, zoetis, kalamazoo, mi; 6.6 mg/kg sc once) and non-steroidal anti-inflammatories (flunixin meglumine; banamine®, merck; 1.1 mg/kg iv sid for 2 days). results all camelids in this case series presented with a unilateral abdominally retained testis. testicular identification by transabdominal inguinal ultrasonography was possible in all cases. all testes were in the abdominal position near the inguinal region or close to the bladder (figure 3). the left testis was retained in nine cases (56.25%), while the right was retained in seven cases (43.75%). the surgical procedure was completed in 35 ± 12 min (mean ± sd). the animals recovered uneventfully, and no complications were reported after discharge from the hospital. in goats, 15 (83%) cases were unilateral and 3 (17%) were bilateral cryptorchids. of the 15 unilateral cryptorchid cases, 11 were right (73.33%) and four were left (26.67%). all unilaterally affected males were identified by the owner as suspect cryptorchid before presentation and diagnosis was confirmed by ultrasonography (figure 4). in this case series there were three full brothers (2 bilateral cryptorchid, 1 unilateral cryptorchid) and five half-brothers (all unilateral cryptorchid with figure 2. laparoscopic-assisted cryptorchidectomy, (a) site of placement of the endoscope portal (1) and forceps portal (2), (b) abdominal testis visualized by laparoscopy, (c) abdominal testis grasped by the gubernaculum, (d) exteriorization of the retained testicle. (e) transfixation of spermatic cord and removal. figure 3. transcutaneous ultrasonographic localization of the retained testis in a cryptorchid alpacas. arrow indicated the retained testis, ub = urinary bladder. http://dx.doi.org/10.58292/ct.v15.9638 citation line: clinical theriogenology 2023, 15, 9638, http://dx.doi.org/10.58292/ct.v15.9638 101 same sire). the retained testes were located intra-abdominally near the bladder and identified by transabdominal inguinal ultrasonography in all cases. the retained testis length and width were 21.8 ±2.1 mm and 18.3 ± 1.5 mm (mean ± sd), respectively. all animals recovered uneventfully from surgery and were discharged 1 day post-operatively from the hospital. no complications were reported on follow-up calls to the owners. discussion cryptorchidism is not an infrequent complaint in veterinary practice. although unilaterally cryptorchid animals may have some fertility, the consensus is to eliminate these animals from breeding population as the disorder is suspected to be heritable. diagnosis of cryptorchidism is based on history and clinical examination in most cases with or without the use of transabdominal ultrasound. in alpacas, diagnosis of cryptorchidism may be challenging as male-like behavior and mating are possible in males that have been castrated at an older age. in these cases, endocrine evaluation helps in differentiating between geldings displaying male-like behavior and cryptorchid animals. the diagnosis is based on determination of serum testosterone concentration before and 8 h after administration of hcg (3000 iu).9,10,13 recently, it has been shown that determination of anti-müllerian hormone on a single serum sample allows diagnosis of cryptorchidism in hemicastrated alpacas.10,17 in the present case series, endocrine testing was performed in only one alpaca as in all the other cases cryptorchidism was evident from the history (lack of castration) and confirmed by percutaneous ultrasonography. in the present study, unilateral cryptorchidism in goats affected predominately the right testicle. this agrees with other reports on west african dwarf goats.5,6,14 a study of 29 small ruminant cases (20 sheep and 9 goats) found cryptorchidism was bilateral in 27.6% of cases and unilateral in 72.4%. among unilateral cases, the right testis was affected in 85.7% and the left testis in 14.3%.14 the study included boer (77.8%), la mancha (11.1%), and mixed breed (11.1%). it is not clear why cryptorchidism is predominantly right-sided. several surgical techniques have been described for cryptorchidectomy in camelids and goats including a parainguinal, paramedian and caudal paramedian approaches. in camelids, the parainguinal approach for cryptorchidectomy is common.15,16 caudal paramedian approach for cryptorchidectomy was also described in small ruminants less than 4 months old with 6.9 and 3.4% minor and major complications respectively. finding and exteriorizing the testis using the laparotomy technique can be challenging and require larger incisions. additionally, a second incision is often necessary in bilaterally cryptorchid cases.14 this results in increased surgical time and risks for complications. the laparoscopic-assisted technique described here was not associated with any complications. conclusion laparoscopic-assisted cryptorchidectomy is a safe, quick, and effective method for cryptorchidectomy in south american camelids and nigerian dwarf goats. it allows proper localization, manipulation, and removal of retained testicles while reducing surgical time and post-operative complications. clients breeding nigerian dwarf goats should be educated about the high prevalence of cryptorchidism in this breed and its potential hereditary component. cryptorchidism may be missed by some owners during early castration by elastic banding. references 1. amann rp, veeramachaneni dn: cryptorchidism in common eutherian mammals. reproduction 2007;133:541–561. doi: 10.1530/rep-06-0272 2. sumar j: studies on reproductive rathology in alpacas. ms thesis, department of obstetrics and gynaecology, college of veterinary medicine, swedish, university of agricultural sciences, uppsala, 1983. 3. drew ml, meyers-wallen vn, acland gm, et al: presumptive srynegative xx sex reversal in a llama with multiple congenital anomalies. j am vet med assoc 1999;215:1134–1139. 4. khan la, rghuwanshi ds, sirsat pr, et al: incidence of cryptorchidism in goats. ind j f vet 2008;3:74. 5. oguejiofor cf, ochiogu is, okoro ol, et al: consequences of unilateral cryptorchidism on semen and sperm characteristics in west african dwarf goats. asian pac j reprod 2018;7:172–177. doi: 10.4103/2305-0500.237055 figure 4. transcutaneous ultrasonographic localization of the retained testis in a cryptorchid goats. arrow indicated the retained testis. http://dx.doi.org/10.58292/ct.v15.9638 https://doi.org/10.1530/rep-06-0272 https://doi.org/10.4103/2305-0500.237055 102 citation line: clinical theriogenology 2023, 15, 9638, http://dx.doi.org/10.58292/ct.v15.9638 6. igbokwe ic, ezeasor dn, onwuka os: comparative study of the morphology of the testes and excurrent ducts system in the unilateral cryptorchid west african dwarf goats. niger vet j 2007;28:40–47. doi: 10.4314/nvj.v28i3.3565 7. igbokwe io, abba y, geidam su, et al: sperm output from unilateral cryptorchid sahel goats. comp clin path 2014;23:819–822. doi: 10.1007/s00580-013-1695-7 8. tibary a, pearson lk, anouassi a: applied andrology in camelids. in: chenoweth p, lorton s: editors. animal andrology: theories and applications. wallingford; cabi: 2014. p. 418–449. 9. fite c, tibary a, reeves j, et al: testosterone response in male alpacas (lama pacos) following hcg administration and correlation with testicular weight. theriogenology 2005;64:782. 10. tibary a, campbell a, rodriguez j, et al: urogenital surgery in camelids. clin theriogenol 2020;12:271–291. 11. el-zawam a, tibary a, patino c: basal levels and hcg responses of serum testosterone and estrogen in male alpacas. front vet sci 2020;7:595856. doi: 10.3389/fvets.2020.595856 12. rietschel w: kryptorchismus beim vicunja (lama vicugna). tierarztliche praxis 1990;18:459–461. 13. perkins nr, frazer gs, hull bl: endocrine diagnosis of cryptorchidism in a llama. aust vet j 1996;74:275–277. doi: 10.1111/ j.1751-0813.1996.tb13773.x 14. haig fm, baird an, hawkins jf, et al: caudal paramedian approach to cryptorchidectomy in 29 small ruminants (2011–2019). vet surg 2020;50:170–176. doi: 10.1111/vsu.13546 15. parker je, semevolos sa: use of a parainguinal approach for cryptorchidectomy in alpacas. j am vet med assoc 2002;220: 1835–1836. doi: 10.2460/javma.2002.220.1835 16. garcia pereira fl, allen a, anouassi a, et al: parainguinal cryptorchidectomy under general anaesthesia in a bactrian camel (camelus bactrianus). j camel pract res 2004;11:103–107. 17. ciccarelli m, tibary a, campbell aj, et al: effect of age and castration on serum anti-müllerian hormone concentration in male alpacas. theriogenology 2018;105:174–177. doi: 10.1016/j. theriogenology.2017.09.032 http://dx.doi.org/10.58292/ct.v15.9638 https://doi.org/10.4314/nvj.v28i3.3565 https://doi.org/10.1007/s00580-013-1695-7 https://doi.org/10.3389/fvets.2020.595856 https://doi.org/10.1111/j.1751-0813.1996.tb13773.x https://doi.org/10.1111/j.1751-0813.1996.tb13773.x https://doi.org/10.1111/vsu.13546 https://doi.org/10.2460/javma.2002.220.1835 https://doi.org/10.1016/j.theriogenology.2017.09.032 https://doi.org/10.1016/j.theriogenology.2017.09.032 clinical theriogenology 2022; 14: 110 disorders of sexual development: a case of xx sex reversal in a mixed breed dog negative for sry (sex-determining region on the y chromosome) gene kelsey martin,a agata parsons,b greg burns,a gerrit bouma,b fiona hollinsheada adepartment of clinical sciences, bdepartment of biomedical sciences college of veterinary medicine and biomedical sciences colorado state university, fort collins, co abstract an 8-month mixed breed dog (from shelter) was evaluated for its abnormal external genital (enlarged os clitoris protruding externally from the vulva) condition. dog had phenotypic female appearance. testicular-like tissue was removed via laparoscopy-assisted gonadectomy. genotype was determined using blood and gonadal tissue (via histology and immunohistochemistry). dog had xx chromosomes and was negative for sry (sex-determining region on the y chromosome) gene. keywords: phenotypic sex, antimüllerian hormone, sex reversal background normal mammalian sexual development involves chromosomal sex (xx or xy [determined at fertilization]), followed by development of gonadal sex (ovary or testis), and finally expression of phenotypic sex (internal reproductive tract and external genitalia).1 errors in these 3 stages resulted in disorders of sexual development (dsds), manifesting in a variety of anatomical and physiological abnormalities in affected individuals.2 generally, dsds are classified based on the incongruencies that exist among the stages of sexual development. individuals that demonstrate a discrepancy between their chromosomal and gonadal sex development had a sex reversal (sr) phenotype and are considered either xx male or xy female.3 these sr individuals can be either positive or negative for sex-determining region on the y chromosome (sry) gene that regulates gonadal differentiation. absence of sry gene causes development of ovaries and a female phenotype. presence of sry gene results in activation of genes, including sox9, triggering ‘male’ development pathway toward formation of testes with internal and external masculinization.4 reports of xy sex reversal in dogs are rare.5,6 conversely, reports of xx sex reversal in dogs are more common.7 regardless, it is important for practitioners to be aware of this clinical presentation in addition to understanding the development of dsds and use proper nomenclature associated with these syndromes to systematically diagnose and appropriately manage sr clinical cases. case presentation history an 8-month intact mixed breed dog was presented to the small animal reproduction service at the colorado state university veterinary teaching hospital for evaluation of abnormal external genitalia. referring veterinarian identified the abnormality while managing the dog for a urinary tract infection after hematuria (observed by the owners shortly after adoption). besides hematuria, other presenting clinical signs included vulvar discharge and perivulvar dermatitis. current owners reported absence of estrous or intact male behaviors. previous owners confirmed that the dog never had any surgery (reproductive alteration or otherwise). there was no evidence of underlying endocrinopathy (e.g. hyperadrenocorticism leading to clitoral hypertrophy 8) nor was exposure to androgens or progestins9,10 during pregnancy or thereafter. physical examination, vaginal cytology, hematology, serum biochemistry dog had female phenotype appearance with normal vulvar conformation. however, had an enlarged clitoris (figure 1) with a palpable boney structure consistent with os clitoris. clitoral enlargement was noticed along the ventral aspect of vulva that protruded ~ 2 cm from the vulvar cleft when the patient was in standing position. under sedation, it was determined via urethral catheterization that urination occurred from within the vulva just cranial to the base of the clitoris, and not from the protruding os clitoris. there were no palpaclinical theriogenology 2022; 14: 111 ble testes nor scrotal structures. internal reproductive structures were not identifiable via abdominal ultrasonography. figure 1. externally protruding hyperplastic os clitoris a cotton tipped plastic handle swab was used to obtain a vaginal smear. swab was rolled onto a glass microscope slide that was then air dried and stained using a simple modified wrights-giemsa stain (diff-quik). vaginal smear was evaluated under light microscopy (100 200 x magnification). predominant (> 90%) cells were parabasal cells, indicating lack of estrogenic influence. complete blood count and serum biochemistry profiles were within normal canine reference ranges. owners declined further tests (e.g. serum antimüllerian hormone) and surgical options for os clitoris removal, but elected laparoscopic-assisted gonadectomy. gonadectomy patient was premedicated with 0.02 mg/kg atropine sulfate (atropine sulfate injection, west ward, eatontown, nj), 0.03 mg/kg acepromazine (acepromazine maleate injection, vetone, boise, id) and 0.05 mg/kg hydromorphone (hospira inc., lake forest, il); all were given intramuscularly. anesthesia was induced with 2 mg/kg ketamine (ketaset®, zoetis inc, florham park, nj) and 2 mg/kg propofol (zoetis, kalamazoo, mi) given intravenously. general anesthesia was maintained with isoflurane (akorn animal health, lake forest, il) in oxygen. a 2-cm ventral midline incision was made just caudal to the umbilicus and a single incision laparoscopic surgical port was placed into the abdomen to allow insufflation with co2 to a pressure of 10 mm hg. a zero-degree laparoscope was inserted into 1 of the ports and the interior of the abdomen was explored. left gonad was first visualized and grossly appeared like testis. using forceps and ligasure™, the tubular structure associated with the gonad (presumed to be the ductus deferens and pampiniform plexus) were ligated. vasculature and presumed ductus deferens on the right-hand side was followed into the inguinal ring and the remaining gonad was identified via deep external palpation. using electrocautery, a 2 cm incision above the inguinal ring (where the testis-like gonad was located) was made and closed castration was performed. abdominal cavity was examined before closure to confirm absence of ovaries, ovotestes, uterine tubular structure(s) or prostate. all incisions were closed in a standard, multiple layer fashion and there were no complications. patient’s recovery from anesthesia was uneventful and 2.2 mg/kg carprofen (rimadyl®, zoetis) was given subcutaneously for postoperative pain management. six months following surgery, a follow up phone consultation with the owner revealed that the clitoral enlargement had not reduced in size. there was no vulvar discharge or perivulvar dermatitis nor clitoral enlargement causing any irritation or distraction to the patient. additionally, urination was reported to be normal. diagnosis a case of dsd was suspected11 and appropriate tests were conducted (e.g. immunochemical localization of antimüllerian hormone [amh]12). histology of gonadal tissue (figure 2) revealed that the architecture of the tissue was consistent with hypoplastic testicular tissue (figure 3). empty seminiferous tubules lined by sertoli cells were noticed in both testes; however, testes had no maturing germ cells nor obvious signs of active spermatogenesis. interstitial tissue of testes had leydig cells and epididymides had empty tubules lined with columnar epithelium. gondal tissue was positive for amh (figure 4), confirming functional sertoli cells (low testosterone concentrations observed in hypogonadism13). figure 2. gross image of the gonadal tissue removed at surgery figure 3. hematoxylin and eosin-stained sections of testis (a) and epididymis (b); note absence of maturing germ cells in seminiferous tubules and normal epididymal epithelium with no sperm (200 x magnification). figure 4. immunolabeling for amh (black arrows) within the seminiferous tubules containing sertoli cells; note negative control (small insert) without primary antibody. chromosomal sex (i.e., xx versus xy) pcr was determined via amplifying14 amelogenin (amel) gene from genomic dna isolated from blood (qiagen dneasy blood & tissue kit). both x and y chromosomes were positive for amel and their gene length polymorphism allowed for genetic sex identification. pcr was performed using platinumtm ii taq hot-start dna polymerase (invitrogen), with 0.5 μl of the amelx/amely primers (5 μm), and cycle conditions 95°c for 4 minutes, 95°c for 30 seconds, 55 °c for 30 seconds, and 72°c for 20 seconds, with the last 3 steps repeated 35 times, followed by a final step of 72°c for 10 minutes. pcr amplification using amelx (on the x chromosome) and amely (on the y chromosome) specific primers yielded a 215 bp amplification product for amelx and 247 bp for amely (figure 5). furthermore, in a separate pcr assay, 2 pcr primer sets were designed to amplify a 350 or 480 bp fragment of canine sry gene (table) to determine if sry gene was present in the dna. pcr analysis of dna isolated from a known male dog was positive for sry (figure 6). pcr cycle conditions for amplification of sry were: 95°c for 4 minutes, 95°c for 30 seconds, 60°c for 30 seconds, and 72°c for 15 seconds, with the last steps repeated 40 times, followed by a final step of 72°c for 1 minute. sry was not detected in the genomic dna isolated from the patient. this finding suggested that alternative and unknown nonsry-mediated mechanism(s) might have occurred, leading to testes development.15 a b figure 3. hematoxylin and eosin-stained sections of testis (a) and epididymis (b); note absence of maturing germ cells in seminiferous tubules and normal epididymal epithelium with no sperm (200 x magnification). clinical theriogenology 2022; 14: 112 figure 4. immunolabeling for amh (black arrows) within the seminiferous tubules containing sertoli cells; note negative control (small insert) without primary antibody. chromosomal sex (i.e. xx versus xy) pcr was determined via amplifying14 amelogenin (amel) gene from genomic dna isolated from blood (qiagen dneasy blood & tissue kit). both x and y chromosomes were positive for amel and their gene length polymorphism allowed for genetic sex identification. pcr was performed using platinumtm ii taq hot-start dna polymerase (invitrogen), with 0.5 μl of the amelx/amely primers (5 μm), and cycle conditions 95°c for 4 minutes, 95°c for 30 seconds, 55 °c for 30 seconds, and 72°c for 20 seconds, with the last 3 steps repeated 35 times, followed by a final step of 72°c for 10 minutes. pcr amplification using amelx (on the x chromosome) and amely (on the y chromosome) specific primers yielded a 215 bp amplification product for amelx and 247 bp for amely (figure 5). furthermore, in a separate pcr assay, 2 pcr primer sets were designed to amplify a 350 or 480 bp fragment of canine sry gene (table) to determine if sry gene was present in the dna. pcr analysis of dna isolated from a known male dog was positive for sry (figure 6). pcr cycle conditions for amplification of sry were: 95°c for 4 minutes, 95°c for 30 seconds, 60°c for 30 seconds, and 72°c for 15 seconds, with the last steps repeated 40 times, followed by a final step of 72°c for 1 minute. sry was not detected in the genomic dna isolated from the patient. this finding suggested that alternative and unknown nonsry-mediated mechanism(s) might have occurred, leading to testes development.15 figure 5. amplified bands represent amelogenin (amel); bottom band is amel present on the x chromosome (215 bp), and the top band is amel present on the y chromosome (247 bp). lane 1: h20 (negative control). lane 2 & 3: genomic dna from the hermaphrodite dog. lanes 4 & 5: genomic dna from a known male (xy) dog. l = 100 bp ladder. table. primer sequences and amplicon sizes generated by pcr amplification name primer seq (5’ to 3’) amplicon size (bp) amelx ataatgacaaagaaaacatgac 215 (amelx) amely ctgctgagctggcaccat 247 (amely) dogsry1-f ggtgcagcggtacaacaaaa 350 dogsry1-r ttccgacgaggtcggtattt dogsry2-f ggtgcagcggtacaacaaaa 482 dogsry2-r cgtgtgtgtgcagccctact figure 6. amplified bands represent sry clinical theriogenology 2022; 14: 113 discussion diagnosis (sry-negative xx male with testes) was confirmed by histomorphological evaluation of the gonads, determination of the chromosomal sex and absence of the sry gene by pcr analysis. cases of sry-negative xx sr were most commonly reported in purebred dogs, suggesting heritability most often as an autosomal recessive trait.16–18 however, in this case of a mixed breed dog, the etiology was most likely a spontaneous event, due to either a genetic gain-of-function mutation or loss-of-function mutation that altered expression of key regulatory genes necessary for female or male sexual development. it is possible that overexpression of the sox9 gene had a key role in testicular differentiation and development in the formation of testes in this case. duplications within the sox9 gene, often in the promoter region, was the most common mutation leading to sry-negative xx sr phenotypes in dogs.4,19 therefore, overexpression of the sox9 gene might have been a key factor for testes induction in the absence of sry. however, mutations in this gene are not always the cause of sry-negative xx sr, and inactivating mutations of ovarian determining genes such as rspo1 or ctnnb1 could be responsible for the observed external female phenotype in this case. further genomic analyses are required to better understand what genetic factors for testes induction may exist.3 clinically, the challenge with dsd cases is that individuals can present with a wide range of anatomical abnormalities including ambiguous external genitalia, clitoral enlargement, hypospadias, and cryptorchidism. these anatomical abnormalities can then cause secondary clinical complications such as vaginitis, vulvar discharge, and urinary tract infections. anatomical and clinical developmental abnormality type depends on the degree of phenotypic masculinization. this is based on the amount of functional testis that developed and may vary depending on the quantity and timing of testicular secretions necessary to masculinize internal and external genitalia.20 furthermore, testosterone concentrations produced are insufficient in quantity or its conversion to dihydrotestosterone by 5α-reductase is impaired during the critical period for androgen-dependent masculinization, internal and external genitalia will fail to masculinize in whole or in part, as with this case. approximately 90% of xx sex reversal cases occur with bilateral ovotestes and the remainder (10%) with bilateral testes. however, there were some cases that occasionally presented with an ovary and ovotestis or an ovotestis paired with a testis.3 in this case, despite having 2 testes, the patient had phenotypic female appearance with no anatomical evidence of a scrotum or prepuce, an unusual presentation. primary clinical concern was the exposed (presumed hypertrophied) clitoris and secondary vaginitis associated with a vulvar discharge. normal sexual development is a process that is highly dependent on the timing of events plus the molecular and hormonal expression of karyotype in adequate and appropriate amounts. delayed or insufficient quantity of amh secretion in xy males during the critical period (regression of mullerian duct) result in persistence of all or part of the mullerian duct derivatives (oviducts, uterus, cervix, cranial vagina).18 this xx patient developed testes with functional (able to produce amh) sertoli cells that apparently suppressed the emergence of mullerian duct derivatives. learning points • determine the chromosomal, gonadal, and phenotypic sex of a suspected dsd patient. • assess the effect abnormal reproductive anatomy on the dog’s health and welfare. • implement a welfare-orientated management strategy that would alleviate the secondary clinical signs caused by clitoral hyperplasia. conflict of interest none to declare. acknowledgement authors thank kelsey tofany for excellent technical assistance (clinical examination and blood withdrawal) and patient management. references 1. meyers-wallen vn: inherited abnormalities of sexual development in dogs and cats. in: concannon pw, england g, verstegen iii j, et al: editors. recent advances in small animal reproduction. 1st edition, united states; international veterinary information service: 2001. 2. poth t, breuer w, walter b, et al: disorders of sex development in the dog-adoption of a new nomenclature and reclassification of reported cases. anim reprod sci 2010;121:197-207. 3. parma p, veyrunes f, pailhoux e: sex reversal in non-human placental mammals. sex dev 2016;10:326-344. 4. albarella s, de lorenzi l, rossi e, et al: analysis of xx sry-negative sex reversal dogs. animals 2020;10:1-13. 5. chaffaux s, cribiu ep: clinical, histological and cytogenetic observations on nine intersex dogs. genet sel evol 1991;23:81-84. 6. kang jt, kim hj, oh hj, et al: sry-positive 78, xy ovotesticular disorder of sex development in a wolf cloned by nuclear transfer. j vet sci 2012;13:211-213. 7. yoon h, han sh, kim j, et al: urogenital anomalies and urinary incontinence in an english cocker spaniel dog with xx sex reversal. j vet intern med. 2018;32:1166-1171. 8. purswell bj, kolster ka: surgical disease of the vulva and vagina. in: bojrab mj, monnet e: editors. mechanisms of disease in small animal surgery. 3rd edition, jackson; teton newmedia: 2015. 9. curtis em, grant rp: masculinization of female pups by progestogens. j am vet med assoc 1964;144:395-398. 10. sokolowski jh, zimbelman rg: canine reproduction: effects of multiple treatments of medroxyprogesterone acetate on reproductive organs of the bitch. am j vet res 1974; 35:1285-1287. 11. sumner sm, grimes ja, wallace ml, et al: os clitoris in dogs: 17 cases (2009-2017). can vet j 2018;59:606-610. 12. alm h, bodil sh: identifying ovarian tissue in the bitch using anti-müllerian hormone (amh) or luteinizing hormone (lh). theriogenology 2018;106:15-20. clinical theriogenology 2022; 14: 114 13. grinspon rp, bedecarrás p, ballerini mg, et al: early onset of primary hypogonadism revealed by serum anti-müllerian hormone determination during infancy and childhood in trisomy 21. int j androl 2011;34:e487-e498. 14.yan s, bai c, li y, et al: sex identification of dog by pcr based on the differences in the amelx and amely genes. anim genet 2013;44:604-607. 15. meyers-wallen vn: gonadal and sex differentiation abnormalities of dogs and cats. sex dev 2012;6:46-60. 16. melniczek jr, dambach d, prociuk u, et al: sry-negative xx sex reversal in a family of norwegian elkhounds. j vet intern med 1999;13:564-569. 17. campos m, moreno-manzano v, garcía-roselló m, et al: srynegative xx sex reversal in a french bulldog. reprod dom anim 2011;46:185-188. 18. meyers-wallen vn, schlafer d, barr i, et al: sry-negative xx sex reversal in purebred dogs. mol reprod dev 1991;53:266-273. 19. rossi e, radi o, de lorenzi l, et al: sox9 duplications are a relevant cause of sry-negative xx sex reversal dogs. plos one 2014;9:e101244. 20. schlafer dh, valentine b, fahnestock g, et al: a case of srypositive 38,xy true hermaphroditism (xy sex reversal) in a cat. vet pathol 2011;48:817-822. 2020 ovulation, pregnancy, and lambing rates during nonbreeding season with or without exogenous gonadotropin stimulation ovulation, pregnancy, and lambing rates during nonbreeding season with or without exogenous gonadotropin stimulation hayder habeeb,a,b timothy hazzard,a fred stormshak,a michelle kutzlera adepartment of animal and rangeland sciences, oregon state university, corvallis, or bdepartment of animal resources, al-qasim green university, babylon, iraq abstract our objective was to evaluate reproductive effects of varying gonadotropin dosages on anestrous ewes pretreated with progesterone and then exposed to a novel ram. hypothesis was that a lower dosage of p.g. 600® (equine chorionic gonadotropin 80 iu/ml and human chorionic gonadotropin 40 iu/ml) induces estrus in anestrous ewes. twenty-four anestrous ewes were treated with intravaginal progesterone-releasing devices for 9 days and given prostaglandin f2α 2 days prior to device withdrawal. on the day of progesterone withdrawal (day 0), ewes were given 5 ml of p.g. 600® (t1; n = 8), 1.5 ml of p.g. 600® (t2; n = 8) or 5 ml saline (control group, c; n = 8). three rams were rotated every 4 hours through each group of ewes for 4 days. venous blood samples were collected on day 0 prior to treatment (0 hour) and at 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 336 hours. serum estradiol-17β and progesterone concentrations were determined by chemiluminescence. ovulation and pregnancy rates were determined using transrectal ultrasonography on days 9 11 and on days 21 and 28. serum progesterone concentrations increased (p < 0.00001) in t1 compared to t2 and c groups. serum estradiol concentrations, ovulation, pregnancy, and fecundity rates, and weaning weights were not significantly different among groups. we concluded that gonadotropin treatment neither enhanced nor diminished reproductive productivity. keywords: cidr, corpora lutea, estrus induction, anestrus, progesterone introduction sheep are seasonally polyestrus, cycling during shortened daylight.1 breeding management practices that adhere to the ewe’s natural breeding season limit ewe productivity (average of 1 lamb crop/ewe/year). to increase ewe’s productivity to 3 lamb crops for every 2 years or 5 lamb crops for every 3 years, ewes must be bred outside of the natural breeding season.2 as hours of daylight increase, decreased secretory pattern of melatonin results in dopamine increase in a15 dopamine neurons in the retrochiasmatic area in the hypothalamus3 that gradually inhibits kisspeptin from the arcuate nucleus, resulting in inhibition of gonadotropin releasing hormone (gnrh) secretion.4 reduced secretion of gnrh results in lower luteinizing hormone (lh) and follicle stimulating hormone (fsh) secretion until ovarian follicular activity becomes static. with static follicular activity, estradiol-17β concentrations remain low, activating the estrogen responsive inhibitory neural system in the brain. increasing brain sensitivity to estrogen makes estradiol the primary inhibitor for gnrh and lh pulse frequency.5,6 introduction of a novel ram to anestrous ewes increases lh secretion7,8 that induces ovulation in approximately half of anestrous ewes without any other treatment.9,10 the mechanism of this phenomenon is not clear, but it is believed to be mediated through pheromones from the ram’s sudoriferous gland. pheromones act through ewe’s olfactory bulb on the ventromedial nucleus and the preoptic area of the hypothalamus, leading to increased kisspeptin release11 that increases gnrh and lh pulse frequency.12 in ruminants, prior exposure to progesterone is necessary for both expression of estrous behavior and normal luteal lifespan.13 therefore, using progesterone priming prior to novel ram exposure and breeding should result in a fertile estrus with ovulation and normal luteal function. this strategy has resulted in pregnancy in approximately 50% of ewes.14 gonadotrophin treatment increases percentage of ewes ovulating following progesterone pretreatment and ram exposure. multiple injections of porcine fsh increase rates of ovulation15 and pregnancy in ewes bred outside the breeding season.16 however, gonadotropin treatment decreases embryo clinical theriogenology • volume 12 number 1 • march 202023 viability, based on embryo collection studies.17 similar to multiple injections of fsh, a single injection of equine chorionic gonadotropin (ecg) increases ovulation rate,18,19 but appears to decrease embryo viability.20 it is noteworthy that anestrous ewes treated with progesterone ovulate when they are also treated with gonadotropins or exposed to rams, but not after progesterone treatment alone.21 p.g. 600® (intervet/merck animal health, madison, nj) is approved for estrus induction in swine. it contains a combination of equine chorionic gonadotropin (ecg; 80 iu/ml) and human chorionic gonadotropin (hcg; 40 iu/ml). in united states, p.g. 600® is commonly used off-label to induce estrus and ovulation in anestrous ewes.22-25 ewes are commonly given 5 ml p.g. 600®, containing 400 iu ecg and 200 iu hcg.22,24 however, this amount of gonadotropins to ewes during breeding season has overstimulated ovaries and reduced fertilization rates, as well as increased estradiol-17β concentrations.26 therefore, we hypothesized that a lower dosage of p.g. 600® induces estrus in anestrous ewes without overstimulating ovaries. objective of this study was to evaluate reproductive effects of varying gonadotropin dosages in anestrous ewes pretreated with progesterone and then exposed to a novel ram. materials and methods twenty-four multiparous polypay ewes, 2 6 years old, were used for this experiment. the fecundity rate for these ewes prior to this experiment was 1.7 ± 0.4. animal use was approved by the oregon state university institutional animal care and use committee (acup #4865). estrus induction regimen used for this experiment was the same protocol that was used in the previous experiment conducted during breeding season.26 briefly, during late spring (may in the northern hemisphere), an intravaginal progesterone-releasing device (0.3 g progesterone; eazi-breed™ cidr, zoetis, kalamazoo, mi) was inserted and removed after 9 days. cloprostenol (125 μg; estrumate®, intervet/merck animal health, madison, nj) was injected intramuscularly 2 days prior to progesterone withdrawal. at progesterone withdrawal (day 0), ewes were treated with p.g. 600® (intervet/merck animal health, madison, nj) or saline. treatment group 1 (t1) received 5 ml (400 iu ecg and 200 iu hcg) intramuscularly (n = 8) and treatment group 2 (t2) received 1.5 ml (120 iu ecg and 60 iu hcg) intramuscularly (n = 8). control group (c) received an injection of 5 ml saline, equivalent volume of the higher dose of p.g. 600® (n = 8). rams used were novel to ewes prior to their first exposure. three rams were used and each remained for 4 hours in a given group of ewes over 4 days. rams were rotated between ewe groups to eliminate any effect of ram preference on pregnancy rates. however, it is understood that this experimental design of rotating rams every 4 hours is not a practical management tool for sheep producers. prior to treatment and immediately following progesterone withdrawal, jugular venous blood samples were collected (day 0). additionally, blood samples were collected at 2, 4, 6, 8, 12, 24 (day 1), 48 (day 2), 72 (day 3), 96 (day 4), 120 (day 5), 144 (day 6), 168 (day 7), and 336 (day 14) hours after treatment. blood samples were centrifuged at 1,620 × g after overnight storage at 4ºc. sera were separated and stored at -20ºc until analyzed for progesterone and estradiol-17β. serum progesterone and estradiol-17β concentrations were determined using chemiluminescence (immulite®1000, siemens healthcare diagnostics, tarrytown, ny) in a single assay for each steroid. progesterone and estradiol-17β intra-assay coefficient of variation was 4.6 and 10.9%, respectively. assay detection limits for progesterone and estradiol-17β were 0.2 ng/ml and 20 pg/ml, respectively. transrectal ultrasonography (7.5-mhz linear array, mindray model #50l60eav, shenzhen, china) was used on days 9 11 after treatment, to determine number of ovulations per ewe by counting corpus luteum (cl) present on each ovary, as described.27 mean ± standard deviation (sd) ovulation rate was calculated for each group. in addition, height and width of each cl was recorded and the mean ± sd corpora lutea diameter were calculated for each group. transrectal ultrasonography was used on days 21 and 28 after treatment injection to image both uterine horns to determine pregnancy status. pregnancy rate was calculated for each group. following lambing, number of lambs per ewe (fecundity rate) and birth weights were recorded and mean ± sd fecundity rate and birth weight was calculated for each group. in clinical theriogenology • volume 12 number 1 • march 2020 24 addition, total litter weight was calculated for each ewe and then the mean ± sd litter weight calculated for each group. at weaning, weight was record and mean ± sd weaning weight was calculated for each group. a repeated measure analysis of covariance (ancova) was used to analyze estradiol-17β and progesterone concentrations over time and among treatment groups. a one-way analysis of variance (anova) was used to analyze data on ovulation rate, corpora lutea diameter, fecundity rate, birth weight, litter weight, and weaning weight among treatment groups. a chi-square test was used to compare pregnancy rates among treatment groups. significance was defined as p < 0.05. results there was no effect of treatment on serum estradiol-17β concentrations (figure 1). however, serum progesterone concentration was greater in t1 compared to t2 and c ewes at 168 and 360 hours (p < 0.00001; figure 2). ovulation rate was not significantly different among groups (table). however, cl diameters were significantly greater in t1 (16.1 ± 3.6 mm) compared to t2 (14.2 ± 3.5 mm), but not compared to c (14.9 ± 3.4 mm). fecundity rate, birth weight, litter weight, and weaning weight did not significantly differ among treatments (table). pregnancy rate was numerically lower in t1 (50%) and t2 (62.5%) compared to c (87.5%) (p = 0.1056). figure 1. ewes treated with 5 ml p.g. 600® (group t1; n = 8), 1.5 ml p.g. 600® (group t2; n = 8) or 5 ml saline (group c; n = 8), were sampled hourly from the treatment injection time (0 hour) and at (2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 336 hours) post treatment. there were no effects of treatment on estradiol-17β concentrations over time. clinical theriogenology • volume 12 number 1 • march 202025 figure 2. ewes treated with 5 ml p.g. 600® (group t1; n = 8), 1.5 ml p.g. 600® (group t2; n = 8) or 5 ml saline (group c; n = 8), were sampled hourly from the treatment injection time (0 hour) and at (2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 336 hours) post treatment. serum progesterone concentrations were higher in t1 compared to t2 and c ewes at 168 and 360 hours (*p < 0.00001). table. results of p.g. 600® administration on reproductive characteristics of ewes during the non-breeding season. ewes were treated with 5 ml p.g. 600® (group t1; n = 8), 1.5 ml p.g. 600® (group t2; n = 8) or 5 ml saline (group c; n = 8). no significant differences among groups in all parameters. discussion ovulation rate in anestrous ewes in our study did not differ between treatment groups receiving progesterone and gonadotropins or progesterone alone, consistent with a previous study.28 that ovulation rate in anestrous ewes was not affected by gonadotropin dose was similar to an earlier report.15 it is important to note that in both studies,15,28 multiple injections of fsh were used instead of p.g. 600® and fsh was administered until 24 or 36 hours prior to progesterone removal instead of concurrent with progesterone removal (as in present study). item treatment groups group t1 t2 c ovulation rate (mean ± sd) 2.23 ± 0.47 2.0 ± 0.26 2.0 ± 0.24 pregnancy rate 50 % 62.5 % 87.5 % fecundity rate (mean ± sd) 2.0 ± 0 2.25 ± 1.25 1.85 ± 0.37 birth weight, kg (mean ± sd) 4.0 ± 0.53 3.85 ± 1.02 3.96 ± 0.94 litter weight, kg (mean ± sd) 7.49 ± 1.02 8.66 ± 3.92 7.36 ± 1.56 weaning weight, kg (mean ± sd) 20.45 ± 3.1 19.92 ± 5.04 21.2 ± 5.45 * * clinical theriogenology • volume 12 number 1 • march 2020 26 pregnancy rate was not significantly different among groups (control = 87.5%, 1.5 ml p.g. 600® = 62.5%, and 5 ml p.g. 600® = 50%). similar findings were reported earlier.22,29 anestrous ewes treated with melengestrol acetate (mga) and 5 ml of p.g. 600® had pregnancy rates not different from mga treatment alone. furthermore, treatment of anestrous ewes with intravaginal progesterone followed by 3 ml p.g. 600® resulted in pregnancy rates that were not different from treatment with intravaginal progesterone alone.25 however, a higher dose of p.g. 600® (> 3.49 ml) decreased lambing rate,30 a finding that served as the basis for our hypothesis. p.g. 600® (400 iu of ecg and 200 iu of hcg) increased serum progesterone concentrations (p < 0.00001) compared to controls (figure 2), similar to an earlier report31 wherein ewes were treated with 300 iu ecg after progesterone withdrawal. however, the finding that higher progesterone concentration during early gestation increased embryo survival32 was not supported by our findings. increases in serum progesterone concentration were likely due to increased cl diameters in the 5 ml p.g. 600® treatment group.33-35 similar to earlier findings,31 fecundity rate, birth weight, litter weight, and weaning weight of lambs did not significantly differ among treatment groups (table 1). in conclusion, during nonbreeding season, administration of p.g. 600® did not affect ovulation rate, fecundity rate, lambing rate, birth, or weaning weights but did increase circulating progesterone concentrations (and cl diameter at 5 ml dosage). furthermore, there was a trend for the higher dose of p.g. 600® to be detrimental to pregnancy rate in the polypay breed. acknowledgment authors thank the oregon sheep commission for funding this research and the ministry of higher education and scientific research in iraq for graduate student financial assistance. conflict of interest authors disclose that there was no actual or potential conflict of interest in conducting this research and with their ability to objectively present/review the research or data. references 1. posbergh cj, murphy rj, thonney ml: further testing of melatonin receptor 1a for out-of-season reproduction in the cornell flock and allelic frequencies compared with romney sheep. j anim sci 2017;9:1939-1944. 2. keisler dh: sheep breeding strategies. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. st. louis, missouri: saunders; 2007. p. 659. 3. thiéry jc, gayrard s, le corre s, et al: dopaminergic control of lh secretion by the a15 nucleus in anoestrous ewes. j reprod fertil suppl 1995;49:285-296. 4. weems p, jeremy s, iain j, et al: effects of season and estradiol on kndy neuron peptides, colocalization with d2 dopamine receptors, and dopaminergic inputs in the ewe. endocrinology 2017;158:831-841. 5. barrell gk, moenter sm, caraty a, et al: seasonal changes of gonadotropin-releasing hormone secretion in the ewe. biol reprod 1992;46:1130-1135. 6. goodman rl, bittman el, foster dl, et al: alterations in the control of luteinizing hormone pulse frequency underlie the seasonal variation in estradiol negative feedback in the ewe. biol reprod 1982;27:580-589. 7. martin gb, scaramuzzi rj, lindsay dr: effect of the introduction of rams during the anoestrous season on the pulsatile secretion of lh in ovariectomized ewes. j reprod fertil 1983;67:47-55. 8. martin gb, cognie y, schirar a, et al: diurnal variation in the response of anoestrous ewes to the ram effect. j reprod fertil 1985;75:275-284. 9. oldham cm, martin gb, knight tw: stimulation of seasonally anovular merino ewes by rams. i. time from introduction of the rams to the preovulatory lh surge and ovulation. anim reprod sci 1979;1:283-290. 10. minton je, coppinger tr, spaeth cw, et al: poor reproductive response of anestrous suffolk ewes to ram exposure is not due to failure to secrete luteinizing hormone acutely. j anim sci 1991;69:3314-3320. 11. fabre-nys c, cognie j, dufourny l, et al: the two populations of kisspeptin neurons are involved in therram-induced lh pulsatile secretion and lh surge in anestrous ewes. endocrinology 2017;158:3914-3928. 12. martin gb, oldham cm, cognié y, et al: the physiological responses of anovulatory ewes to the introduction of rams a review. livest prod sci 1986;15:219-247. 13. wheaton je, windels hf, johnston lj: accelerated lambing using exogenous progesterone and the ram effect. j anim sci 1992;70:2628-2635. clinical theriogenology • volume 12 number 1 • march 202027 14. knights m, hoehn t, marsh d, et al: reproductive management in the ewe flock by induction or synchronization of estus. 2003 west virginia agric for exp sta bul 726, morgantown, west virginia. 15. knights m, baptistea qs, dixona ab: effects of dosage of fsh, vehicle and time of treatment on ovulation rate and prolificacy in ewes during the anestrous season. small rumin res 2003;50:1-9. 16. knights m, maze td, bridges pj, et al: short-term treatment with a controlled internal drug releasing (cidr) device and fsh to induce fertile estrus and increase prolificacy in anestrous ewes. theriogenology 2001;55:1181-1191. 17. torrès s, cognié y, colas g: transfer of superovulated sheep embryos obtained with different fsh-p. theriogenology 1987;27:407-419. 18. cognie y: current technologies for synchronisation and artificial insemination of sheep. in: oldham cm, martin gb, purvis iw, editors, reproductive physiology of merino sheep concept and consequences. perth: school of agriculture, university of western australia;1990. p. 211. 19. barrett dmw, bartlewski pm, batista-arteaga m, et al: ultrasound and endocrine evaluation of the ovarian response to a single dose of 500 iu of ecg following a 12-day treatment with progestogen-releasing intravaginal sponges in the breeding and nonbreeding seasons in ewes. theriogenology 2004;61:311-327. 20. ryan jp, hunton jr, maxwell wmc: increased production of sheep embryos following superovulation of merino ewes with a combination of pregnant mare serum gonadotrophin and follicle stimulating hormone. reprod fertil dev1991;3:551-560. 21. umberger sh, jabbar g, lewis gs: seasonally anovulatory ewes fail to respond to progestogen treatment in the absence of gonadotropin stimulation. theriogenology 1994;42:1329-1336. 22. safranski tj, lamberson wr, keisler dh: use of melengestrol acetate and gonadotropins to induce fertile estrus in seasonally anestrous ewes. j anim sci 1992;70:2935-2942. 23. cline ma, ralston jn, seals rc, et al: intervals from norgestomet withdrawal and injection of equine chorionic gonadotropin or p.g. 600 to estrus and ovulation in ewes. j anim sci 2001;79:589-594. 24. windorski ej, schauer cs, wurst ak, et al: effects of melengestrol acetate and p.g. 600 on fertility in rambouillet ewes outside the natural breeding season. theriogenology 2008;70:227-232. 25. d’souza kn, rastle-simpson sl, redhead ak, et al: gonadotropin stimulation using p.g. 600® on reproductive success of non-lactating anestrous ewes. anim reprod sci 2014;148:115-120. 26. habeeb hmh, hazzard tm, stormshak f, et al: effect of different dosages of pg-600 on ovulation and pregnancy rates in ewes during the breeding season. transl anim sci 2019;3:429-432. 27. schrick fn, surface ra, pritchard jy, et al: ovarian structures during the estrous cycle and early pregnancy in ewes. biol reprod 1993;49:1133-1140. 28. knights m, hoehn t, lewis pe, et al: effectiveness of intravaginal progesterone inserts and fsh for inducing synchronized estrus and increasing lambing rate in anestrous ewes. j anim sci 2001;79:1120-1131. 29. jabbar g, umberger sh, lewis gs: melengestrol acetate and norgestomet for the induction of synchronized estrus in seasonally anovular ewes. j anim sci 1994;72:3049-3054. 30. cross lj, cross rm, stormshak f: optimal dose of pg600 when given to progestogen-synchronized ewes during anestrus as affected by day of the year and temperature. transl anim sci 2019;3:172-180. 31. rekik m, haile a, abebe a, et al: gnrh and prostaglandin-based synchronization protocols as alternatives to progestogen-based treatments in sheep. reprod domest anim 2016;51:924-929. 32. fermin lm, pain sj, gedye kr, et al: timing of exogenous progesterone administration is critical for embryo development and uterine gene expression in an ovine model of maternal constraint. reprod fertil dev 2018;30:1699-1712. 33. o’shea jd, rodgers rj, wright pj: morphometric analysis and function in vivo and in vitro of corpora lutea from ewes treated with lhrh during seasonal anoestrus. j reprod fertil 1984;72:75-85. 34. duggavathi r, bartlewski pm, pierson ra, et al: luteogenesis in cyclic ewes: echotextural, histological, and functional correlates. biol reprod 2003;69:634-639. 35. kaulfuss kh, giucci e, süss r, et al: an ultrasonographic method to study reproductive seasonality in ewes isolated from rams. reprod domest anim 2006;41:416-422. clinical theriogenology • volume 12 number 1 • march 2020 28 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (adobe rgb \0501998\051) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy 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/pagesize [612.000 792.000] >> setpagedevice clinical theriogenology 2022; 14: 106 thromboembolic disorder in a dog after cesarean surgery yamilka lago-alvarez,a hailey rose,a denae campanale,a ian porter,a kenneth simpson,a soon hon cheong,a mariana diel de amorima adepartment of clinical science, college of veterinary medicine, cornell university, ithaca, ny abstract a 3-year female dog (intact primiparous) overweight labrador retriever was presented as an emergency (fever, lethargy, and anorexia) patient. four days earlier, this dog had a dystocia (secondary uterine inertia with stillborn fetus) that was relieved via cesarean surgery. disseminated intravascular coagulation panel indicated marked increases in d-dimers, increases in partial prothrombin time, and decreases in antithrombin iii activity. abdominal ultrasonography revealed a severely mottled spleen with multifocal infarction. arterial blood gas analysis indicated increases in alveolar-arterial oxygen gradient. pulmonary thromboembolism was suspected. based on the history (peripartum obesity and cesarean delivery) and clinical manifestation, it was hypothesized that the pregnancy-related hypercoagulable state and postpartum period led to the development of venous thromboembolism. to authors’ knowledge, this is the first case report of a suspected thromboembolic disorder in a postcesarean dog. keywords: overweight, peripartum, pulmonary embolism, deep vein thrombosis, spleen infarction background venous thromboembolism (vte) is a complex disorder that can manifest as pulmonary embolism, deep vein thrombosis1 or cerebral venous thrombosis.2 pregnancy and postpartum period in women are highly associated with increased risk of vte.1-3 incidence of vte is higher (5-fold) in pregnant women compared to nonpregnant women.4,5 although pregnancy alone is a risk factor for vte in women, additional risk factors include age (> 35 years), cesarean delivery, hypertension, heart disease, obesity, and postpartum infection.1,5,6 furthermore, hypercoagulable state of pregnancy in women is another contributing factor; characterized by increases in coagulation factors (ii, vii, viii, ix, and x), and increases in von willebrand factor and fibrinogen.1,5,7 additionally, hormones (estrogen and progesterone) are indirectly responsible for venous thrombosis.2,5,8,9 hormonal therapy (estrogen and progesterone) may place women at increased risk for blood clots. estrogen therapy increases synthesis of coagulation proteins (high risk for venous thrombosis).2,5,8,9 progesterone therapy has a relaxing effect on the muscle, and apparent vasodilation leading to disorders of veins (increased capacity) and valves (insufficiency). apparently, decreased blood vessel tone and an enlarged uterus cause obstructions in iliac vessels leading to venous stasis.2,5,8,9 dogs with immune-mediated hemolytic anemia, sepsis, neoplasia, protein-losing nephropathies, hyperadrenocorticism, or cardiac diseases had vte.10 clinical signs associated with pulmonary embolism included acute onset of hypoxemia and dyspnea.11 although deep vein thrombosis present as sudden paralysis and pain associated with rear limbs,11 in a peripartum patient, exclusion of other conditions (metritis, mastitis, and eclampsia) is critical. furthermore, diagnosis of laboratory tests (coagulation profiles, d-dimer, and thromboelastography) and diagnostic imaging (thoracic radiography, computed tomography, and abdominal ultrasonography) are necessary for a definitive diagnosis.12 this case (without sepsis and with no history of cancer or vte) had cesarean surgery and developed vte. case presentation a 3-year, overweight female (intact primiparous) labrador retriever presented for a dystocia because of secondary uterine inertia. prior to presentation, 1 stillborn and 3 viable fetuses were delivered. transabdominal ultrasonography revealed a nonviable fetus (no heartbeat) and all remaining fetuses had normal heart rates (> 220 beats per minute). there was no fetal obstruction (digital examination) within the birth canal and lack of a ferguson reflex. emergency cesarean surgery was performed, and 5 puppies (4 viable and 1 stillborn) were delivered. fetal membranes were removed at surgery and patient was discharged on the same day. four days after surgery, the patient was admitted through the emergency service for fever (104.3°f), lethargy, and anorexia. on physical examination (day 4 postpartum), the patient appeared quiet, alert, and responsive, had a heart rate of 136 beats per minute (normal: 80 160), a mildly increased respiratory rate of 32 breaths per minute (normal: 15 30), a rectal temperature of 102.6 f (normal: 100.5 102.5), and a body condition score of 8 out of 9 (43.50 kg). bronchovesicular sounds were normal in the entire lung field with an increased respiratory effort. cardiac auscultations were unremarkable. on abdominal palpation, tenderness was noted. midline incision clinical theriogenology 2022; 14: 107 from the surgery appeared to be healing appropriately with no signs of infection (e.g. redness, swelling, or discharge). mammary glands palpated normally and had normal milk secretion in every teat without signs of redness, discoloration, or pain. cytology and culture of mammary secretions were not performed. dog had moderate quantity of normal lochia without foul odor. vaginal cytology revealed parabasal and intermediate cells with occasional cocci without inflammatory cells. uterine horns had small quantity of anechoic fluid and mild to moderately thickened walls with normal wall layering (figure 1) that was interpreted as normal postpartum involution. blood gas analysis revealed metabolic acidosis (ph 7.27, reference interval (ri) 7.32 7.38), hyperchloremia (121 meq/l, ri 110 119), normoglycemia (70 mg/dl, ri 60 120), and anemia (25%, ri 42 57). after triage assessment and fluid treatment, patient remained febrile (103.2 f). considering the clinical condition, hospitalization for further diagnostic testing was recommended. neonatal puppies were with the dam (for nursing) and remained healthy during hospitalization. additional bloodwork (day 5 postpartum) revealed a normocytic, normochromic, regenerative anemia (hematocrit of 27%, absolute reticulocytes 99.6 x 103/μl), leukocytosis with a neutrophilia characterized by a left shift and monocytosis (wbc 43.6 x 103/ μl, ri 5.7 14.2; segmented neutrophils 35.8 x 103/μl, ri 2.7 9.4; band neutrophils 0.4 x 103/μl, ri 0.0 0.1; monocytes 2.6 x 103/μl, ri 0.1 1.3), normal thrombocytes (197 x 103/μl, ri 186 545), hyperproteinemia (8.0 g/dl, ri 5.9 7.8) and hypoalbuminemia (1.9 g/dl, ri 3.2 4.1). ionized calcium concentrations were within normal limits (1.31 meq/l, ri 1.18 1.37). abdominal ultrasonography revealed a mottled spleen with well-demarcated hypoechoic regions that had a ‘lacey’ appearance (figure 2a) and had no detectable blood flow on color doppler examination (figure 2b). mild pneumoperitoneum and mild uteromegaly (interpreted as normal findings [recent laparotomy and postpartum uterus]) were noted. no other abnormalities (e.g. ischemic lesions [expected with a systemic issue]) were detected. vascular changes noted via ultrasonography prompted a disseminated intravascular coagulation panel. figure 1. longitudinal image of left uterine horn (arrowheads) containing small quantity of intraluminal anechoic fluid; note descending colon (asterisks) dorsal to uterus figure 2. longitudinal/crossectional images of spleen. a. severely mottled spleen with innumerable hypoechoic regions (arrowheads) suggestive of acute, multifocal splenic infarcts. b. decreased blood flow on doppler. there were marked increases in d-dimers (4,965 ng/ml, ri 0 575), decreases in antithrombin iii activity (50%, ri 65 145), and increases in activated prothrombin time (19.6 secs, ri 8.5 15.5), consistent with a consumptive coagulopathy. during the second night, patient developed increased respiratory rate and effort (36 breaths per minute) with a corresponding hemoglobin saturation oxygenation of 93% (ri > 95%). an arterial blood gas evaluation revealed an alveolar-arterial oxygen gradient of 36 mm hg (ri 10 25), partial pressure of oxygen 79.3 mm hg (ri 95 -109), and partial pressure of carbon dioxide 29 mm hg (ri 34 40). patient was started on oxygen supplementation. thoracic radiography revealed mild bronchial wall mineralization with no evidence of pneumonia. increased alveolar-arterial oxygen gradient with normal thoracic radiographs and consumptive coagulopathy condition, pulmonary thromboembolism was suspected. a focused echocardiogram was performed that revealed trace regurgitation of the mitral and tricuspid valve. there was no evidence of pulmonary hypertension. however, it was apparent that type iv pulmonary hypertension secondary to systemic inflammation and pyrexia was resolving. absence of high velocity and tricuspid regurgitation and improving clinical signs clinical theriogenology 2022; 14: 108 were interpreted as consistent with a resolving pulmonary thromboembolic event. treatment at hospitalization (day 4 postpartum), the patient was given intravenously a balanced electrolyte solution bolus ([5 ml/ kg/hour]; plasma-lyte a®, baxter healthcare, deerfield, il). subsequently, intravenous fluid (60 ml/kg/day) therapy was initiated and continued. additional treatment included intravenous ampicillin/sulbactam (unasyn®, pfizer, new york, ny) and maropitant citrate ([1 mg/kg every 24 hours]; cerenia®, zoetis, kalamazoo, mi) for an infection of unknown origin and to prevent emesis. anticoagulant therapy was strongly considered; however, not instituted. differential diagnosis thromboembolic disease (secondary to postpartum hypercoagulability), infection (following surgery) leading to sepsis, and rickettsial spleenopathy with emboli were considered. mastitis was excluded since mammary glands appeared normal (via inspection and palpation) and because teats expressed milk. retained fetal membranes and metritis were also excluded as entire fetal membranes were removed at surgery. furthermore, vaginal discharge and cytology were consistent with normal lochia. additionally, ionized calcium concentrations were within normal limits throughout hospitalization. outcome patient was discharged on day 6 postpartum with oral amoxicillin/clavulanate ([13.75 mg/kg every 12 hours for 14 days]; clavamox®, zoetis) to control fever and leukocytosis. antimicrobial therapy started during hospitalization and continued. a week later, primary veterinarian evaluated the patient and conducted bloodwork. results revealed a normocytic, normochromic, regenerative anemia (hematocrit 23.7%, absolute reticulocytes 85.7 x 103/μl), leukocytosis with a neutrophilia characterized by a left shift and monocytosis (wbc 46.53 x 103/μl [ri 5.05 16.76]; segmented neutrophils 32.79 x 103/μl [ri 2.95 11.64]; monocytes 2.91 x 103/μl [ri 0.16 1.23], and normal thrombocyte (209 x 103/μl [ri 186 – 545]). patient was continued on oral amoxicillin/clavulanate ([13.75 mg/kg every 12 hours]; clavamox®, zoetis) for an additional month. despite persistent leukocytosis, initial antimicrobial therapy was continued because of marked improvement in clinical signs and for the safety of nursing neonates. patient was doing well (as per the owner) at 4 months after discharge. discussion cesarean surgery is performed in small animal obstetrics to prevent or to treat dystocia.13,14 surgical intervention was necessary in ~ 60 80% of dystocia cases (58% occurred on an emergency basis).15,16 cesarean surgery was needed in 48.6% (341/701) dystocia cases.17 postcesarean problems included systemic disturbances, hemorrhage, and incisional and infectious complications.15 this case was presented 4 days after cesarian surgery for fever and lethargy. most common postcesarean complications (e.g. peritonitis, incisional infection, metritis, mastitis, eclampsia) were excluded and the animal was suspected to have vte. occurrence of vte has not been previously reported in a postcesarean dog. possible causes of vte include immune-mediated hemolytic anemia, sepsis, neoplasia, protein-losing nephropathies, hyperadrenocorticism, and cardiac diseases.10 this case apparently developed vte without underlying disease (e.g. primary cardiac or respiratory problems). furthermore, there was no evidence of peritonitis or sepsis. blood cultures were not performed and therefore peritonitis or sepsis should be considered as risk factors and included in differential diagnosis. in companion animals, the correlation of antemortem diagnosis with postmortem confirmation for vte remains poor.10,11 alternative diagnostic tests in the investigation for patients presenting with acute onset of tachypnea, hypoxemia (determined by arterial blood gas or pulse oximeter) and absence of evidence of other causes of respiratory distress on thoracic radiographs via an echocardiogram).12 in this case, a disseminated intravascular coagulation panel suggested consumptive coagulopathy. splenic infarction was incidental and may be unrelated to this case; however, an increase alveolar-arterial gradient with normal thoracic radiographs suggested multiple thromboembolisms that responded to oxygen supplementation. in many cases, animals were treated based on clinical signs compatible with a vte rather than documentation of the clot.10,11 despite advanced diagnostic techniques, diagnosis of vte remains difficult, in human and veterinary medicine. advanced imaging (e.g. computer tomography angiography) was not pursued in this case because of cost constraints. risk factors among women are age (> 35 years), pregnancy, cesarean delivery, hypertension, heart disease, obesity, and postpartum infection.1,5,6 similarly, this case had increased risk because of cesarean surgery, obesity, and suspected postpartum infection. possibly, vte developed because of a pregnancy-related hypercoagulable state. increased d-dimer concentrations and multifocal infarction in the spleen were noteworthy. estimation of d-dimers concentrations,18 clinical signs, thoracic radiographs, and arterial blood gas analysis were helpful in reaching a tentative diagnosis. in women, d-dimer was used as an exclusion criteria biomarker for vte disease; however, its usefulness during pregnancy had limitations because d-dimer concentrations were higher during pregnancy, preeclampsia, infection, malignancy, and in postoperative patients.18 hypercoagulable state is considered a protective mechanism against pregnancy-related complications. in late pregnant women, marked increases in maternal blood procoagulant activity were characterized by elevation of vii, x, vii, fibrinogen, and von willebrand factor.1,5,7 similarly, in pregnant dogs changes in coagulation factors (e.g. increased platelets, increased activity of specific clotting factors vii, viii, ix, and xi, and increased concentrations of fibrinogen degradation products) were observed.19-21 additionally, pregnancy-related hypercoagulable state in women has been associated with hormonal changes that protect against premature placental separation and secure hemostasis at parturition.22,23 however, these may not be the case in dogs. for example, increased production of acute-phase reactant proteins and plasma fibrinclinical theriogenology 2022; 14: 109 ogen concentrations were caused by a local reaction of the coagulation system, possibly, because of alterations in uterine epithelium and endothelium induced by placentation.20,24 in this patient, fibrinogen concentrations were within normal limits during postcesarean hospitalization. in conclusion, a case of apparent thromboembolic disorder was diagnosed in an overweight postcesarean dog. it is suggested that postcesarean dogs with potential risk factors should be closely monitored for clinical signs related to vte. conflict of interest no conflict of interest or funding sources to report. learning points • although not common, vte following cesarean section should be considered as a differential in an overweight postpartum bitch that develops respiratory distress. • studies on changes in coagulation factors in dogs at various stages of pregnancy are warranted. references 1. devis p, knuttinen mg: deep venous thrombosis in pregnancy: incidence, pathogenesis andendovascular management. cardiovasc diagn ther 2017;7:300-319. 2. bates sm: pregnancy-associated venous thromboembolism: prevention and treatment. semin hematol 2011;48:271-284. 3. mclean k, cushman m: venous thromboembolism and stroke in pregnancy. hematology 2016;2016:243-250. 4. phillippe hm: overview of venous thromboembolism. am j manag care 2017;23:376-382. 5. biron-andréani c: venous thromboembolic risk in postpartum. phlebolymophology 2013;20:167-173. 6. james ah, jamison mg, brancazio lr, et al: venous thromboembolism during pregnancy and the postpartum period: incidence, risk factors, and mortality. am j obstet gynecol 2006;194:1311-1315. 7. galambosi pj, gissler m, kaaja rj, et al: incidence and risk factors of venous thromboembolism during postpartum period: a populationbased cohort-study. acta obstet gynecol scand 2017;96:852-861. 8. friedman am, ananth cv: obstetrical venous thromboembolism: epidemiology and strategies for prophylaxis. semin perinatol 2016;40:81-86. 9. ropacka-lesiak m, kasperczak j, breborowicz gh: risk factors for the development of venous insufficiency of the lower limbs during pregnancy. ginekologia polska 2012;83:939-942. 10. epstein se, hopper k, mellema ms, et al: diagnostic utility of d-dimer concentrations indogs with pulmonary embolism. j vet intern 2013;27:1646-1649. 11. johnson lr, lappin mr, baker dc: pulmonary thromboembolism in 29 dogs: 1985-1995. j vet intern med 1999;13:338-345. 12. goggs r, benigni l, fuentes vl, et al: pulmonary thromboembolism. j vet crit care 2009;19:30-52. 13. pretzer sd: medical management of canine and feline dystocia. theriogenology2008;70:332-336. 14. kutzler m: dystocia and obstetric crises. in: silverstein d, hopper k: editors. small animal critical care medicine. philadelphia; wb saunders: 2009. p.611-615. 15. van goethem b: cesarean section. in: complications in small animal surgery. hoboken; john wiley & sons: 2017. p.522-527. 16. sophia x, jayakumar c, vidya v, et al: outcome and complications of repeat cesarean deliveries in dogs. j pharma innov 2019;8:456-459. 17. o’neill dg, o’sullivan am, manson ea, et al: canine dystocia in 50 uk first-opinion emergency care veterinary practices: clinical management and outcomes; canine dystocia in 50 uk first-opinion emergency care veterinary practices: clinical management and outcomes. doi:10.1136/vr.104944 18. gutiérrez garcía i, pérez cañadas p, martínez uriarte j, et al: d-dimer during pregnancy: establishing trimester-specific reference intervals. scand j clin lab invest 2018;78:439-442. 19. gerbasi fr, bottoms s, farag a, et al: increased intravascular coagulation associated with pregnancy. obstet gynecol 1990;75:385389. 20. gentry pa, liptap rm: influence of progesterone and pregnancy on canine fibrinogen values. j small anim pract 1981;22:185-194. 21. bunck cf, mischke r, günzel-apel ar: investigation of the fibrinolytic system during nonpregnant and pregnant oestrous cycles of bitches. reprod fert suppl 2001;57:207-214. 22. brenner b. haemostatic changes in pregnancy. thromb j 2004;114:409-414. 23. åstedt b, lindoff c, lecander i: significance of the plasminogen activator inhibitor ofplacental type (pai-2) in pregnancy. semin thromb hemost1998;24:431-435. 24. concannon pw, gimpel t, newton l, et al: postimplantation increase in plasma fibrinogen concentration with increase in relaxin concentration in pregnant dogs. am j vet res 1996;57:1382-1385. 2018: severe fibrosis and squamous metaplasia in a 14-year-old thoroughbred mare severe fibrosis and squamous metaplasia in a 14-year-old thoroughbred mare leatrice w. elliman,a maria r. schnobrich,a laura a. kennedyb arood and riddle equine hospital, lexington, ky; bveterinary diagnostic laboratory, university of kentucky, lexington, ky hysteroscopy is a useful diagnostic tool in evaluation of the subfertile mare. in a clinical study of hysteroscopic examinations for breeding soundness, 39% (45/108) revealed uterine pathology.1 this abstract describes the history and clinical findings in a mare that hysteroscopy was integral in determining the mare’s future prognosis for breeding. a 14-year-old, barren thoroughbred mare was presented for reproductive evaluation with a history of chronic endometritis (3 years) that had been treated by various methods, including intrauterine kerosene. in 2016 the mare became pregnant on the third cover and foaled a very small foal after prolonged gestation (377 days). the mare was not bred in 2017. a reproductive evaluation was requested to determine the mare’s future prognosis for breeding. transrectal palpation and ultrasonography revealed inactive (anestrus) ovaries, no uterine edema, 3 cm of echogenic fluid in the uterine lumen, and a moderately toned cervix. vaginoscopy revealed hyperemic vaginal mucosa and viscous yellow to white fluid on the vaginal floor. the vaginal fluid did not have evidence of urine contamination (creatinine concentration (0.5mg/dl). visual and digital examination of the cervix revealed no abnormalities. a small volume uterine lavage was performed and cytological examination of the recovered efflux revealed severe neutrophilic inflammation and cocci were seen. aerobic culture of the efflux recovered a beta-hemolytic streptococcus spp. hysteroscopic examination revealed numerous (>15) multifocal, small (3-10mm), white sub-epithelial structures distributed throughout the endometrium. at the base of the horns circumferential, white-tan, spider-web lesions covered the endometrium and several were surrounded by small aggregates (1cm) of multiple black pinpoint lesions. biopsy of the most normal appearing endometrium revealed a category iii endometrium due to extensive neutrophilic, lymphocytic and eosinophilic endometritis with moderate fibrosis and gland atrophy. notable was the periglandular inflammation and diffuse presence of hemosiderophages. histologic evaluation of a single white focal lesion revealed a markedly abnormal endometrium. there was near total loss of glandular structures, with replacement by dense fibrous connective tissue. the overlying epithelium demonstrated marked squamous metaplasia, without evidence of normal, pseudostratified columnar endometrial epithelium. immediately subjacent to the abnormal epithelium the tissue was infiltrated by large numbers of lymphocytes, plasma cells and neutrophils, with transmigration of inflammatory cells through the epithelium. the gross appearance of the mare’s endometrium, in addition to the mare’s history gave the mare a poor to grave prognosis for producing commercially viable offspring and the owner decided to retire the mare from breeding. this case of severe uterine fibrosis and endometrial squamous metaplasia highlights the benefit of hysteroscopy as a diagnostic and prognostic tool in determining the extent of uterine damage and recommendations for further breeding management. keywords: mare, hysteroscopy, endometritis, fibrosis, metaplasia reference 1. bradecamp ea, schnobrich mr, sheerin pc, et al: results of hysteroscopic examinations performed as part of breeding soundness evaluations on mares presented for infertility-a retrospective study. clin therio 2016;8:331. clinical theriogenology • volume 10, number 3 • september 2018 332 practical clinical genetics kathryn meurs department of clinical sciences, college of veterinary medicine north carolina state university, raleigh, nc abstract there is increasing evidence that many forms of congenital and acquired disease in veterinary medicine are of a familial origin. a veterinary practioner may be consulted on genetic issues of breeding animals to assist in breeding decisions and to provide pet owners information regarding etiology of diseases. knowledge of mode of inheritance of a disease and information on possible genetic markers will help the practioner to arrive at recommendations for an individual animal and for breeding. keywords: genetics, mutation, inherited, breeding introduction there is increasing evidence that several forms of congenital and acquired disease in veterinary medicine are of familial origin. as of 2005, 430 canine diseases and 180 feline diseases had been identified as familial. it has been estimated that 5 10 new familial diseases are identified every year. many familial veterinary diseases are assumed to be associated with improved control of diseases caused by environmental factors and the desire to breed animals to maintain an appearance, and selecting animals from a small group of popular founders (founder effect). it is reasonable to consider a familial etiology for common feline and canine heart diseases, particularly those with strong breed predispositions. many forms of congenital and acquired disease have a strong breed predisposition suggesting a familial etiology. a small animal practioner may be requested to provide consultation on genetic issues to provide information to owners of breeding animals to aid them in breeding decisions and to provide pet owners about etiologies of disease. breed specific lists of known and presumed inherited diseases for dogs and cats are available at the following web sites: cats: http://www.fabcats.org/breeders/inherited_disorders/ index.php dogs: http://www.vet.cam.ac.uk/idid/ and http://www.upei. ca/~cidd/intro.htm although the number of genetic diseases in small animals is substantial, the actual genetic cause is not commonly known. even in the absence of knowledge of a molecular cause substantial amount of information can be provided from understanding the pattern of inheritance. genetic diseases are most commonly observed in purebred animals. breed organizations have intentionally limited their gene pool by preventing breeding to other breeds. therefore, the gene pool is limited and closed size, and aggressive removal of breeding animals because of the identification of a defect will make the gene pool even smaller. consequently, one must carefully recommend removal of breeding animals based on severity of defect, mode of inheritance, and importance of a particular animal to the breed. genetic mutations rarely have an all or nothing effect. for some diseases, a genetic cause has now been identified and a genetic test can be done. presence of the mutation does not mean that all animals will exhibit the trait, or similar severity of the trait; this depends on disease ‘penetrance,’ a poorly understood phenomenon that likely involves genetic modifiers. modes of inheritance knowledge on the mode of inheritance of a specific trait can be used to provide guidance about reducing the prevalence of a trait within a particular line of animals. determination of the pattern of inheritance will be useful for determining the best recommendations for breeders and will allow development of plans to exclude or include affected animals in breeding programs. common conditions include autosomal recessive, autosomal dominant, x-linked recessive, and x-linked dominant. x-linked x-linked traits are almost always recessive and should have the following criteria: more affected males than females, an affected male crossed with a normal (noncarrier) female should produce silent carriers, silent carrier females should have a 50:50 chance of passing the trait on to male offspring, and affected females are the result of a cross between a silent carrier female and an affected male (not a frequent occurrence). example: progressive retinal atrophy in the akita. recommendation: if animals with an x-linked trait are used for breeding, they should always be bred to an unrelated line, as this will decrease the presence of the trait in the line. autosomal recessive autosomal recessive traits should have the following criteria: disease should appear to ‘skip’ a generation (parents do not usually exhibit the trait), males and females should equally exhibit the trait, the mating of 2 silent carriers (heterozygotes, carry 1 copy of the abnormal gene) should produce offspring that exhibit the trait in a 3:1 ratio, and if both parents exhibit the trait, all offspring should exhibit the trait. example: diabetes mellitus in keeshond recommendation: if animals with an autosomal recessive trait are used for breeding, they should always be bred to an unrelated line, as this will decrease the presence of the trait in the line. autosomal dominant autosomal dominant traits should have the following criteria: males and females should equally exhibit the trait, every affected individual should have at least 1 affected parent, and all heterozygotes (1 copy of the abnormal gene) are affected and transmit a mutant gene to half of their offspring. affected animals may carry the genetic mutation on 1 or both copies of the gene. if it is on both copies of the gene (1 inherited from each parent) they are considered homozygous for the mutation and will pass 1 copy of the mutation to every offspring. example: dilated cardiomyopathy in doberman pinscher recommendation: if animals with an autosomal dominant trait are used for breeding, they have a 50 100% chance of passing on the trait (depending on if they are heterozygous or homozygous for the trait). therefore, only those with the mildest form of the disease and the most positive attributes should be selected for use. utilization of molecular information for screening and therapeutic issues in some cases, a molecular genetic cause for a specific trait has been identified and a genetic test is available. genetic tests are generally a pcr test that identifies either a marker for the disease or identifies the actual genetic mutation. for pcr, a small dna sample is provided by the clinician or owner and a region of interest is amplified so it can be carefully inspected. the dna is usually provided in a blood sample in an edta tube, a buccal swab or even a semen sample. the dna is inspected for the abnormality by the laboratory and for the presence or absence of the marker or mutation. however, breeders and owners should be cautioned and advised how to best use the information. results should be carefully considered and should be weighed against the severity of the trait, the size of the breed’s gene pool, the mode of inheritance of the trait and the positive traits that this individual animal brings to a breed. in some cases, strict screening and removal programs may be very detrimental to small gene pools in specific breeds; therefore, breeding recommendations should be carefully designed. general guidelines for counseling owners about genetic diseases. genetic test results negative/wildtype: this indicates that the individual animal does not carry any copies of the known disease genetic variant (mutation). considerations for the individual animal and breeding population: no special considerations, since this animal should neither develop the inherited disease nor have the ability to spread disease within the population. positive heterozygous: this indicates that the animal has 1 copy of the normal gene (wild type) and 1 copy of the disease genetic variant. considerations for the individual animal: if the disease is autosomal recessive, this animal should never develop the inherited disease, so no special considerations are needed. however, if the disease is autosomal dominant, this animal is at risk of developing disease. since this animal carries the disease variant and is at risk of disease development, a patient management strategy that includes annual monitoring for signs of disease and considers dietary, medical or other options. if the disease is xlinked recessive, a male with the disease variant on his x chromosome is likely to develop disease, whereas a female with the disease variant on 1 x chromosome is likely to be a silent carrier. considerations for breeding: if autosomal recessive, this animal will not develop disease and can be bred to an animal that is negative. this strategy will likely produce both negative and positive heterozygous animals but neither will develop disease. if the disease is autosomal dominant, a similar strategy for breeding could be considered and one could breed a positive heterozygous animal to a genotype negative animal. the offspring of this mating (positive heterozygous to a negative) will ideally produce at least some genotype negative offspring and 1 of these with the desirable traits of the parents could be selected to replace the positive heterozygous parent in future breedings. this breeding will also produce a few positive heterozygous animals. hence this has a risk of producing animals that may suffer from disease. therefore, this strategy should be considered with regard to the type of disease that may develop in the offspring. if the animal is exceptional due to personality, health, intelligence or other characteristics, one may more likely to try this approach once or twice in hopes of producing a genotype negative replacement animal. if the disease is xlinked recessive, a male with the mutation on the x chromosome could be bred to a negative female. this will produce both male and females that do not have disease. the male offspring of this mating will not carry the mutation since males cannot pass on their x chromosome to their sons. female offspring will also be clear of disease, they would need to carry it on both x chromosomes to actually demonstrate the disease. however, importantly, females with a disease variant on 1 x chromosome will be silent carriers of the trait. positive homozygous: this animal has 2 copies of the disease variant. considerations for the individual animal: positive homozygous animals have the highest risk of developing the disease. a patient management strategy should be developed that may include annual monitoring for signs of disease and consideration to dietary, medical, or other options. recommendations for breeding animals: since positive homozygous animals carry 2 copies of the mutation disease they will certainly pass on the mutation even when bred to a negative animal. finally, since these canine and feline familial diseases are complicated with issues that include incomplete penetrance, variable expression, closed gene pools and variable phenotypes. it is ideal to use a testing service that can provide the most expertise in genetic counseling for pet owners. practical clinical introduction aglepristone is a competitive progesterone antagonist that binds to progesterone receptors and thereby impairs the biological effects of progesterone. it is an antiprogestin, with similar molecular structure to mifepristone used in human medicine. its affinity to progesterone receptor is 3.12 and 9.26 times higher than progesterone for the bitch and queen, respectively.1 an aglepristone progesterone compound (alizin® [virbac, france]) is approved in europe as a subcutaneous (sc) injection. it is also available in australia, new zealand, canada and in the us under a controlled use drug prescription. it is used it in situations wherein the objective is to block the progesterone action (reviewed1,2). applications include, pregnancy termination in bitches (only approved indication) and queen, medical treatment of pyometra, induction of parturition, in deciding elective cesarean section (c-section), and treatment of feline mammary fibroadenomatosis. pregnancy termination a case of ‘misalliance’ (unwanted mating) can be treated in 2 ways under the supervision of a licenced veterinarinan. either a dose of alizin® (10 mg/kg, sc) is given after end of estrus (confirmed by vaginal cytology) not knowing if the mating resulted in a pregnancy, or terminating pregnancy once confirmed by ultrasonography. in both cases, a second dose (24 hours later) is necessary. however, in our experience, a third dose (1 week after the first injection) is necessary for most pregnant dogs. although the efficacy at the end of estrus is higher than 95%, it is worthwhile to examine for pregnancy. additionally, cases of pregnancy termination should be followed antiprogestin use in europe cindy maenhoudt, alain fontbonne ecole nationale vétérinaire d’alfort, centre d’etude en reproduction des carnivores maisons-alfort (paris), france abstract aglepristone is a competitive progesterone antagonist that binds to progesterone receptors and is used in small animal reproductive management. this article is a brief review of the following aspects: approved use in pregnancy termination in bitches and queens; medical treatment of pyometra; induction of parturition; planning of an elective cesarean section; and treatment of feline mammary fibroadenomatosis. keywords: aglepristone, antiprogesterone, small animals, reproductive management (1 week later) by abdominal ultrasonography to ensure that pregnancy is arrested (no viable embryo/fetuses remaining) and evaluate the amount of fluid in the uterine lumen. although the efficacy of the product is closer to 100%, in late pregnancy (> 38 days) it may require repeated treatment for total efficacy. treatment failure is rare. pregnancy termination (before 45 days) is the only indication for approved use. therefore, alizin® use in pregnancies over 45 days is not recommended and is also ‘off-label.’ side effects vary and seem to be associated with the onset of the treatment in relation to the stage of pregnancy. following signs were observed: anorexia and/or depression (with weight loss), vomiting, diarrhea, and polydipsia. vaginal discharge, if observed, started within 2 days after the onset of treatment and lasted 1 2 weeks in bitches that were at least 30 days pregnant. its use in a late stage of pregnancy (~ 40 days) can lead to fetal expulsion. it must be noted that alizin® shortens the interestrus interval. terminating unwanted pregnancies in the queen with alizin® is ‘off-label.’ when used as early as days 5 6 after mating, the success was closer to 100%,3 termination rates decreased to ~ 88 % between days 25 35. the efficacy is as low as 67% in queens treated after 45 days of pregnancy2 and therefore it is not advised for a late stage. pyometra management alizin® (10 mg/kg, sc) can be used in breeding animals that have no systemic complications. absence of ovarian pathologies (e.g. ovarian cysts) is important before beginning of treatment. two initial injections are given 24 hours apart, followed by clinical theriogenology 2021; 13: 159 weekly injections until complete recovery. a modified protocol (injections at day 0, 2, 5, and 8) had high efficacy and there was no recurrence within 24 months.4 vaginal discharge is observed within a few hours after the first injection. it is recommended that treated animals do not ingest the vulvar discharge and an elizabethan collar is recommended. additionally, it is important to combine the treatment with other therapies such as antibiotics, fluid therapy, and correction of gastrointestinal signs. the success rate is over 80%. although, the combination of aglepristone with cloprostenol is described, it is not used frequently in our clinic. subsequent fertility appears to be not affected by alizin® treatment. parturition induction inducing parturition and more frequently, scheduling a c-section, are indications for alizin®. since induction of parturition is not a current request of clientele, limited data are available in clinical setting. in the studies performed to evaluate the efficacy of aglepristone to induce parturition, the results were similar to normal parturition. timing of induction used for beagle bitches was 60 days after estimated lh surge5 and in other breeds it was 59 61 days postovulation.6 oxytocin was also used in the above studies. induction in bitches over 20 kg was less effective. for the elective planned c-section cases, the protocol is to follow the bitch after 60 days from a well-estimated ovulation date to decide the time of alizin® injection (15 mg/kg sc). serum progesterone concentrations are evaluated to ensure that the bitch has not started parturition (i.e. it is not at term) and the injection is given no more than 18 hours before the surgery to avoid onset of parturition. ultrasonography is necessary to evaluate fetal viability and maturation.7 in our experience, verifying milk secretion before the procedure was necessary to decrease the risk of agalactia/hypogalactia in the first 24 hours after c-section. our collective data from the last 10 years had a neonatal mortality (in the first 2 weeks of life) after planned c-section was 9.89%. dead puppies were not further examined, but no obvious signs of prematurity were observed at the time of c-section.8 this is a safe procedure to avoid dystocia. for feline mammary hyperplasia although this tumor can regress spontaneously, the hormonal component of the disease (exposure to progesterone) supports alizin® as a treatment option. the protocol is injections (15 mg/kg, sc) on days 1 and 2, then once per week until complete regression of mammary lesions. the success rate is over 90%, but in some cases, more than 5 injections are necessary to reach full recovery. if clinical signs are related to exogenous long-acting progesterone use, treatment should be repeated weekly for at least 5 weeks, even if regression of lesions occurred.9 final considerations since the product is oil-alcohol based, the injection needs to be subcutaneous. after injection, a slight massage of the injection site is necessary. if the volume of the injected solution exceeds 5 ml, the drug must be injected at multiple sites. local pain at the site of injection (sometimes edema, skin thickening, ulceration, and localized lymph node enlargement) can be observed. in case of show dogs and dark color-coated animals, we prefer less exposed areas as injection sites. in our experience, systemic adverse effects as anorexia, excitation, depression, vomiting and diarrhea are infrequently observed. alizin® is a welcome addition in the therapy and management of small animal reproductive problems. conflict of interest none to report. references 1. gogny a, fieni f: aglepristone: a review on its clinical use in animals. theriogenology 2016;85:555-566. 2. kowalewski m, pereira mt, papa p et al: progesterone receptor blockers: historical perspective, mode of function and insights into clinical and scientific applications. tierarztliche praxis ausgabe k: kleintiere – heimtiere. 2020;48:433-440. 3. goericke-pesch s, georgiev p, wehrend a: prevention of pregnancy in cats using aglepristone on days 5 and 6 after mating. theriogenology 2010;74:304-310. 4. contri a, gloria a, carluccio a, et al: effectiveness of a modified administration protocol for the medical treatment of canine pyometra. vet res comm 2015;39:1-5. 5. fieni f, gogny a: clinical evaluation of the use of aglepristone associated with oxytocin to induce parturition in bitch. reprod domest anim 2009;44:167-169. 6. fontbonne a, fontaine e, lévy x, et al: induction of parturition with aglepristone in various sized bitches of different breeds. reprod domest anim 2009;44:170-173. 7. lopate c: gestational aging and determination of parturition date in the bitch and queen using ultrasonography and radiography. vet clin north am small anim pract 2018;48:617-638. 8. roos j, maenhoudt c, zilberstein l, et al: neonatal puppy survival after planned caesarean section in the bitch using aglepristone as a primer: a retrospective study on 74 cases. reprod dom anim 2018;53 (suppl 3):85-95. 9. jurka p, max a: treatment of fibroadenomatosis in 14 cats with aglepristone – changes in blood parameters and follow‐up. vet record case reports 2013;1(1) e657rep https://doi.org/10.1136/vetreccr.165.22.657rep clinical theriogenology 2021; 13: 160 antiprogestin use in europe 2018: in-vitro equine embryo production: an adventure in multi-unit collaboration in-vitro equine embryo production: an adventure in multi-unit collaboration c.c. lyman,a r. coy,b j.w. bailey,b g.r. holyoakb acenter for veterinary clinical sciences, oklahoma state university, stillwater, ok; broyal vista southwest, purcell, ok the accessibility of assisted reproductive techniques related to oocyte collection and in vitro fertilization in the mare is increasingly becoming more widespread for private equine practitioners and their clients. the necessity of hauling a valuable mare long distances to the closest facility for transvaginal aspiration (tva) of oocytes and on site intracytoplasmic sperm injection (icsi) over a period of potential weeks before obtaining a pregnancy no longer exists. instead, a mare’s primary veterinarian can manage the patient’s cycle and briefly transport her to a nearby facility where tva can be performed. with tva services being offered with increasing frequency in densely equine populated areas it is increasingly common for tva to be offered as an outpatient procedure even if icsi laboratories are not present at that location. the majority of reports associated with tva and icsi are associated with benchtop research and typically do not continue beyond blastocyst development to report pregnancy rates. a cumulative pregnancy rate after tva and icsi were performed on immature oocytes, with shipment of blastocysts to an off-site recipient herd has been reported at 72% with a 52% foaling rate.1 in 2017, in collaboration with an exclusively equine reproduction veterinary practice, we worked together to provide tva services for client-owned mares. mares were referred from the veterinary practice to our university setting where outpatient tva procedures were performed; 330 follicles were aspirated and 212 oocytes were recovered (mare age range from 4-26 years) and shipped overnight at room temperature in an equitainer® to an icsi laboratory via fedex™. a total of 43 (26.1%) blastocysts resulting from 165 icsi procedures were obtained; 29 embryos were vitrified for future use and 14 embryos were transported to the referring reproductive veterinary practice via courier for fresh transfer to recipient mares on day 7 or 8 after icsi. a 14 day pregnancy rate of 71.4% (10/14) and a 50% (7/14) pregnancy rate at heartbeat check were recorded. three recipient mares have already successfully foaled and four are due to foal later in the spring of 2018. in light of these results sending a mare to a local practice specializing in tva, as opposed to a distant, self-contained facility that has both tva and icsi capabilities has benefits for referring equine practitioners, their clients and patients. being a key element in the management of the mare’s cycle prior to tva has allowed the primary veterinarian to remain an integral partner in providing these specialty services. shorter duration stays at referral facilities translates into less expense for the mare owner. finally, as collaborators in this working partnership we have found working as a team to offer such services has enhanced the working relationship between practitioners and specialists. keywords: in-vitro embryo production, oocyte, icsi, tva, mare reference 1. rader k, choi yh, hinrichs k: intracytoplasmic sperm injection, embryo culture, and transfer of in-vitro produced blastocysts. vet clin north am equine pract 2016;32:401-413. clinical theriogenology • volume 10, number 3 • september 2018333 87 contact michela ciccarelli michela.ciccarelli@wsu.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9645, http://dx.doi.org/10.58292/ct.v15.9645 review report common swine field reproductive surgeries michela ciccarelli,a,b giuseppe catone,c cecilia vullod acollege of veterinary medicine, washington state university, pullman, wa, usa bschool of molecular bioscience, washington state university, pullman, wa, usa cdepartment of veterinary medicine, messina university, messina, italy ddepartment of chemical, biological, pharmaceutical and environmental sciences, messina university, messina, italy abstract reproductive surgery in commercial swine is limited by the relative limited financial value of animals with limited market value. in herds containing animals with high genetic value, biosecurity concerns often preclude the movement of animals to a hospital facility. performing some procedures on the farm may be economically feasible and reduce culling rates. growing concerns for animal welfare and the increasing popularity of pigs as pets dictates a larger involvement of veterinarians in surgical procedures in swine.1 this manuscript describes common reproductive surgeries for male swine, practical anesthetic protocols, and the post operative treatments that a veterinary practitioner should be able to perform. the most frequently requested surgery in boars is castration, which is usually performed by management during the first week of age as a portion of on-farm processing. in adult boars, castration is occasionally requested to improve the quality of the meat and reduce the boar taint in animals destined for slaughter. orchiectomy of adult boars is increasingly requested for pot-bellied pigs, and other breeds maintained as pets and rescued by “sanctuaries” to avoid unwanted breedings and reduce aggressive behavior. inguinal herniation, a common disorder in intact boars, often requires surgical correction in both production settings and pet pigs. because of the presumed heritable basis, surgery in animals intended for breeding should be discouraged. swine have one of the highest incidences of cryptorchidism among common livestock species.2 surgical correction of cryptorchidism in breeding animals is strongly discouraged due to the likelihood of a heritable basis for the failure of testicular descent. cryptorchidectomy as a portion of bilateral castration is often requested for animals being shown in livestock exhibitions and occasionally in commercial facilities for animals intended for slaughter. keywords: porcine, castration, anesthesia, inguinal hernia, cryptorchidism field anesthesia for swine field anesthesia of pigs is complicated by factors uncommon to other species: swine are easily stressed by handling and restraint, increasing the metabolic clearance of many medications, including sedatives and anesthetics; obtaining accurate body weights is often very difficult on farm: venous access is limited, and swine can be difficult to restrain sufficiently to appropriately administer anesthetic agents. to facilitate anesthetic administration, it is important to isolate the pig in a quiet area before administering premedication. for adult animals, it can be useful to restrain the patient in a calf chute to limit motion and, at the same time, obtain an accurate weight. younger pigs can be held in dorsal or lateral recumbency.3 swine have thick skin and, like humans, are fixed-skin animals with too little subcutaneous space to accept large-volume injections. few peripheral vessels are accessible, and swine are prone to vasospasm during phlebotomy. the most common site for intravenous (iv) injections are the lateral or medial auricular veins (figure 1) which can be accessed with small-diameter intravenous catheters by an experienced phlebotomist if the animal is sufficiently restrained. sedation before attempting auricular vein catheterization for induction of anesthesia is advised.4 the muscles of the dorsolateral neck are a good site for small-volume intramuscular (im) injections. for larger volume im injections, either semimembranosus or semitendinosus muscles just above the hock or the caudal portion of the biceps femoris muscle using an inchand-a-half long needle are suggested.5 in swine intended for food, intramuscular injection into the gluteal (ham) muscles is discouraged because of the potential for muscle inflammation and fibrosis in a high-value carcass area.6 in the author’s experience, the easiest way to administer intramuscular injections for sedatives to a pig is the use of an extension tube connected to an 18 g 1-inch and half needle. this can be mailto:michela.ciccarelli@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9645 88 citation line: clinical theriogenology 2023, 15, 9645, http://dx.doi.org/10.58292/ct.v15.9645 done by only one operator, with the pig only minimally restrained (figure 2). to reduce the risk of regurgitation or aspiration, healthy swine should be fasted for at least 12 hours, and access to the water should be eliminated 6 hours before general anesthesia. longer fasting is not necessary because swine are monogastrics, and fasting longer than 24 hours may contribute to gastritis and post operative vomiting. swine benefit from other pre-anesthetic agents prior to anesthetic induction.7 anticholinergic agents like atropine or glycopyrrolate administered preoperatively will dry the upper respiratory tract, reduce oral secretions, and counter the bradycardia associated with general anesthesia. a variety of sedative agents have been used as premedications. acepromazine (0.11 – 1.1 mg/kg im, iv, sc) is commonly used but may cause alpha-adrenergic blockade at high doses. benzodiazepines are often advocated. diazepam (0.5–10 mg/ kg sq; 0.44–2 mg/kg iv administered slowly) or midazolam (0.1–0.5 mg/kg im, sq, or iv) have frequently been employed as pre-anesthetic tranquilizers. midazolam is preferred over diazepam for im use because it has more consistent absorption from muscles. figure 1. catheterization of an ear vein for intravenous drug administration. figure 2. intramuscular administration of sedatives via an extension tube to an unrestrained pig. figure 3. endotracheal intubation of an adult pig utilizing a laryngoscope. figure 4. administration of inhalation anesthesia using a homemade mask. http://dx.doi.org/10.58292/ct.v15.9645 citation line: clinical theriogenology 2023, 15, 9645, http://dx.doi.org/10.58292/ct.v15.9645 89 figure 5. castration of an adult boar. scrotal technique. one incision is made over each testis in the ventral aspect of the scrotum. figure 6. open castration technique. the skin, scrotal fascia, and parietal visceral tunic are incised to allow visualization of the albuginea of the testis. visualization and identification of the spermatic cord facilitate ligation and emasculation. figure 7. pre-scrotal technique. the testes are pushed cranially and to the midline of the pig. figure 8. pre-scrotal castration. the incision is made on the midline and extended cranially. http://dx.doi.org/10.58292/ct.v15.9645 90 citation line: clinical theriogenology 2023, 15, 9645, http://dx.doi.org/10.58292/ct.v15.9645 benzodiazepines likely increase the length of the anesthetic recovery period, especially if used intraoperatively. for the induction of anesthesia, the authors prefer the combination of ketamine (5–10 mg/kg) and xylazine (2.5 mg/kg). ketamine is a dissociative anesthetic agent that produces rapid immobilization with minimal cardiovascular depression but does not provide good visceral analgesia and provides very little muscle relaxation. a combination of ketamine with xylazine improves performance in those areas. following induction, the endotracheal tube is placed utilizing a laryngoscope, as shown in figure 3, and inhalation anesthesia is used for maintenance. in some instances, endotracheal intubation of adult animals may be difficult or impossible due to the presence of the pharyngeal diverticulum, and a mask may be used to deliver anesthetic gases and maintain oxygenation (figure 4). either isoflurane or sevoflurane may be utilized safely in pigs. isoflurane has a low blood gas solubility resulting in rapid anesthetic recoveries and allowing rapid alteration of the depth of anesthesia. although isoflurane produces a dose-dependent depression of the cardiovascular system, it has the greatest margin of safety of inhalation anesthetics currently in use and is considered the inhalation agent of choice in swine. sevoflurane has similar physiologic effects and may be used instead of isoflurane. the author performs castration of adult boars under general anesthesia. the animal is sedated with a combination of xylazine (2.5 mg/kg) and ketamine (5–10 mg/kg) intramuscularly in the neck behind the ear. when the animal is sufficiently sedated and immobilized, an intravenous catheter (22 g) is placed in one of the ear veins, and an additional figure 9. pre-scrotal castration. with the help of an assistant, tension is applied to the testis while increasing the incision length. figure 10. pre-scrotal castration. ligation of the spermatic cord with a miller’s knot. figure 11. pre-scrotal castration. excision of the spermatic cord following ligation. figure 12. pre-scrotal technique. a 5-mm scud may be used to seal and cut the vessels of the spermatic cord. http://dx.doi.org/10.58292/ct.v15.9645 citation line: clinical theriogenology 2023, 15, 9645, http://dx.doi.org/10.58292/ct.v15.9645 91 dose of ketamine (1–2 mg/kg) is given iv. when the animal is deeply sedated, it is moved to the surgery table and intubated. lidocaine (20 mg/ml) is injected directly into the testicular parenchyma (4–10 ml depending on the size of the testes) or into the spermatic cord (2–3 ml) of both testes. it is important to remember that the toxic dose for lidocaine in swine is 5mg/kg, and care is taken not to exceed that amount.2 castration of adult boars in this chapter, we will describe only options to neuter weaned pigs and adult boars. commercial pigs are normally castrated during their first week of life without the use of sedatives or anti-inflammatories. increasing attention to animal welfare figure 13. pre-scrotal technique. closure of the skin with a simple continuous pattern using absorbable suture material. figure 14. laparoscopic view of an inguinal hernia with intestines passing through the internal inguinal ring. figure 15. oblique incision over the affected inguinal ring in a piglet with an indirect inguinal hernia. figure 16. twisting the spermatic cord to reduce the herniated organs through the inguinal canal. note that the parietal vaginal tunic is intact and the intestines never leave the peritoneal cavity. http://dx.doi.org/10.58292/ct.v15.9645 92 citation line: clinical theriogenology 2023, 15, 9645, http://dx.doi.org/10.58292/ct.v15.9645 has led to recommendations for sedation or general anesthesia for pigs older than one month of age. in europe, using sedatives and analgesics during castration of all pigs is mandatory, regardless of age.8 castration is often delayed in pot-bellied pigs, and other pigs maintained as pets. in these cases, the surgery is primarily done to render the animal sterile and reduce male behavior. pot-bellied pigs that live in the household should be castrated before 12 weeks of age to reduce the development of the preputial diverticulum,5 which results in less accumulation of malodorous smegma. castration of adult boars in commercial herds is not common. still, mastery of the technique and the knowledge of possible challenges are important in those occasional  cases and increasingly frequent requests for the castration of mature pet pigs, rescued animals, or research animals. the size of the spermatic cord and associated vasculature is the major challenge in castrating adult boars. in neonatal piglets, the spermatic cord is very thin, and the testicles can be pulled away from the scrotum with minimal hemorrhage. in the post-pubertal boars, the pampiniform plexus is extremely large, and failure to ligate or emasculate could lead to death from blood loss. in heavy mature boars, appropriate surgical positioning during surgery and anesthesia is critical to avoid compressive myopathy. boars must be maintained on a soft surface when positioned in dorsal or lateral recumbency. figure 17. miller’s knot placed on the spermatic cord following reduction of the hernial contents. figure 18. ultrasonographic images of pig cryptorchid testes. (arrows) http://dx.doi.org/10.58292/ct.v15.9645 citation line: clinical theriogenology 2023, 15, 9645, http://dx.doi.org/10.58292/ct.v15.9645 93 preparation castration of the older boar is best performed under deep sedation or general anesthesia. the pig may be placed in lateral or dorsal recumbency, and the surgical site prepped sterilely. scrotal technique a 5–10 cm incision is made through the skin at the caudo-ventral aspect of the scrotum while tensing the scrotal skin over the testis (figure 5). either an open or closed castration can be performed. the open castration provides better visualization of the spermatic cord and facilitates the placement of ligatures on the vessels for hemostasis (figure 6). an open castration is more time-consuming and may require resectioning a portion of the vaginal tunic with the closure of the tunics to minimize the chance of evisceration. following the skin incision, in an open castration, the common vaginal tunic is incised, and the testicular tunica albuginea is exposed. the testis is exteriorized, and the ligament of the tail of the epididymis identified; the fascia surrounding the spermatic cord is stripped using dry gauzes to allow direct visualization of the pampiniform plexus and deferent duct. depending on the size of the spermatic cord, simple ligation plus or minus emasculation can be performed to provide hemostasis. the authors prefer to place two ligations as close as possible to the inguinal region and then emasculate the spermatic vessels distal to the sutures. a miller’s knot and a transfixing suture or two transfixing ligatures using absorbable suture material size #0 or 1 are recommended. attaching a hemostat to the suture leads before emasculation or transection of the spermatic cord is useful to allow retrieval of the spermatic cord stump to check for hemorrhage. following the removal of the first gonad, the procedure is repeated on the opposite side. closure of the skin is seldom done when using the scrotal approach; leaving the surgical wound open promotes drainage. pre-scrotal technique following induction of anesthesia, the boar is positioned in dorsal recumbency, and the inguinal area is prepped for sterile surgery. pressure through the scrotal skin is applied to one testis, and the gonad is pushed cranially into a position on the midline just cranial to the scrotum (figure 7). with one testis held in this position, a skin incision is made through the skin over the testis and continued for 3–8 cm cranially, depending on the pig’s size and age. the incision must be sufficient to allow the testicles to be exteriorized (figures 8 and 9). with this approach, a closed castration is more commonly chosen, and the parietal tunic is left intact. the first testicle is delivered through the prescrotal incision following dissection of the subcutaneous connective tissues, and the fascia is stripped away from the testis and spermatic cord to allow exteriorization. two curved kelly hemostats or similar instruments are placed across the spermatic cord proximal to the testis and used to crush the tissues of the spermatic cord. the spermatic cord is ligated with one transfixing suture above the hemostats and a second ligature with a miller’s knot figure 19. laparoscopic-assisted technique. placement of the first trocar at the level of the umbilical scar. figure 20. laparoscopic-assisted technique. the second port is placed to allow the introduction of babcock forceps to grasp the retained testis. http://dx.doi.org/10.58292/ct.v15.9645 94 citation line: clinical theriogenology 2023, 15, 9645, http://dx.doi.org/10.58292/ct.v15.9645 between the transfixion ligature and the testis (figure 10). the spermatic cord is sharply excised (figure 11), the hemostats removed, and the spermatic cord checked for evidence of hemorrhage. alternatively, a sonicisiontm, a cordless ultrasonic dissector (scud), can be used for emasculation to produce coagulation and transection of vessels (figure 12). the remainder of the spermatic cord is placed back into the incision after ensuring adequate hemostasis. the procedure is repeated, with the second testis being manipulated through the same skin incision. the skin incision is closed with the surgeon’s preferred pattern using absorbable suture material (figure 13). medications systemic antibiotic coverage should be provided for 2–3 days before the surgery and continuing for 2–3 days post-surgery. anti-inflammatories and analgesics such as flunixin meglumine can be administered intraoperatively to decrease inflammation and pain post-operatively. fly spray is applied to the wound. the animal should be kept in a clean, dry stall for at least a week. complications the most common complications are hemorrhage, abscess formation, evisceration through an undetected inguinal hernia, and seroma formation. applying ice packs to the scrotum as the boar recovers from anesthesia helps decrease the swelling. anti-inflammatories such as meloxicam (0.4 mg/kg) per os may be administered to decrease inflammation and pain. a dose of ceftiofur crystalline free acid (6.6 mg/kg sq) may be indicated in cases of likely post-operative infections when daily therapy is difficult. post-op it is common to observe an enlarged scrotum post-surgery for a week following surgery; care during the surgical approach to locate the incisions very ventrally will allow good drainage and minimize post operative edema. the boar should not be allowed to be with females for at least ten days post-surgery, as unwanted pregnancies could theoretically occur from the sperm left in the ampullae and ductus deferens. inguinal hernia repair inguinal hernias occur when abdominal organs, most commonly intestines, pass through a defect of the abdominal wall adjacent to the inguinal ring or pass through the inguinal canal.9 in domestic swine, herniation through a congenitally enlarged inguinal ring is a common developmental defect and is thought to have a heritable basis.10 pigs with inguinal herniation may sometimes become anorexic if intestinal strangulation occurs but more commonly have few signs other than scrotal enlargement and perhaps retarded growth rates, which contribute to economic losses for the producers and unwanted pain and stress for the animals.9–11 routine castration of animals with an undetected inguinal hernia will often be followed by evisceration. there are two classifications of inguinal hernia, direct and indirect. direct herniation occurs when abdominal organs pass through acquired defects of the posterior wall of the inguinal canal and do not communicate with the vaginal cavity. indirect herniation occurs when the intestines or omentum pass through the inguinal canal and into the space between the common and parietal vaginal tunics. contents of an inguinal hernia may become ‘scrotal’ when the organs descend completely into the scrotal portion of the vaginal cavity between the two portions of the vaginal tunics (figure 14). indirect inguinal hernias in swine appear to have a genetic link, and boars affected by this disorder should not be considered for use as breeding stock. the frequency of the defect in commercial pigs varies from 1.7% to 6.7%, depending on the breed, line, and management practices.12 figure 21. laparoscopic-assisted technique. exteriorization of the cryptorchid testis visualized by laparoscopy. figure 22. open cryptorchidectomy. local block of the skin using 2% lidocaine. http://dx.doi.org/10.58292/ct.v15.9645 citation line: clinical theriogenology 2023, 15, 9645, http://dx.doi.org/10.58292/ct.v15.9645 95 palpation of the inguinal area and scrotal contents aids the  evaluation of animals with an enlarged scrotum and coupled with an ultrasonographic evaluation, it helps in differentiating inguinal herniation from hydrocele, scirrhous cord, testicular hematoma, and other causes of scrotal  enlargement. potential consequences of a chronic inguinal hernia include intestinal incarceration and strangulation which are more likely in cases of direct herniation. inguinal herniorrhaphy surgical repair of indirect inguinal hernias is best accomplished before the pig is castrated, and the procedure is generally performed on young animals. the patient is positioned in dorsal recumbency with its rear legs slightly elevated. in most uncomplicated cases, injectable agents supplemented with local infiltration of the surgical site with lidocaine are appropriate. in older animals, inhalation anesthesia might be justified. the inguinal area is prepped for sterile surgery, and an oblique incision is made over the affected inguinal ring (figure 15). if possible, the hernia contents should be reduced or partially reduced before making the skin incision. like a closed castration, subcutaneous tissue is bluntly dissected until the parietal vaginal tunic is encountered. maintaining the integrity of the parietal vaginal tunic prevents intestinal exteriorization and facilitates replacement into the abdominal cavity. the attachment of the ligament of to the tail of the epididymis is manually disrupted, and the testis and spermatic cord are isolated. the intact tunic containing the testis and herniated organs is gently twisted to force the intestines through the inguinal canal and into the abdomen (figure 16). a transfixing suture and a miller’s knot are then placed on the spermatic cord as close to the inguinal ring as possible (figure 17) to ligate the testicular vessels and close the peritoneal cavity. the spermatic cord is transected, and the testis removed. the inguinal ring is closed with additional interrupted absorbable sutures. to assess for complete strong closure of the inguinal ring, manual pressure is applied to the abdominal wall. additional sutures may be placed in the subcutaneous tissues. the skin is closed with an absorbable suture using an intradermal pattern. the contralateral testis is then removed routinely. it is important to evaluate the contralateral inguinal ring and rule out bilateral inguinal herniation before the completion of the castration. in cases of chronic herniation, post-operative severe scrotal serum accumulation can occur. for this reason it is suggested to make the incision as ventral as possible to improve post surgical drainage. in long standing inguinal hernias, adhesions may have formed between the intestines and the walls of the vaginal cavity. in this situation, the parietal vaginal tunic will need to be carefully incised, the intestinal adhesions broken down and the herniated intestines reduced into in the abdomen prior to closing the inguinal canal. cryptochidectomy cryptorchidism is a congenital abnormality in which one or both testicles fail to descend into the scrotum. in most livestock species, the testes descended before birth. cryptorchid testes may be located in the abdomen, usually caudal to the ipsilateral kidney, in the inguinal canal, taking the shape of a cigar due to the narrow space, or subcutaneously in the inguinal region. cryptorchidism is common in pigs, with a reported prevalence of 2.2%13 in older literature, with no recent estimates available. like most other species, unilateral cryptorchidism is more common than bilateral in pigs. either testis may be retained, but the right testis is the most commonly affected. failure of testicular descent is believed to be a heritable homozygous recessive trait in some breeds of pigs,14 but there are no recent breeding studies to confirm this. inbreeding likely increases the incidence of cryptrchididm.2 figure 24. open cryptorchidectomy. ligation of the spermatic cord and excision of the testis. figure 23. open cryptorchidectomy. digital palpation of the abdominal testis based on ultrasonographic identification and exteriorization. http://dx.doi.org/10.58292/ct.v15.9645 96 citation line: clinical theriogenology 2023, 15, 9645, http://dx.doi.org/10.58292/ct.v15.9645 for spermatogenesis to occur, the testes must be at a temperature lower than the body. in cryptorchid testes, due to the higher temperatures, the parenchyma degenerates leading to seminiferous tubules containing almost exclusively spermatogonia and sertoli cells. insufficient proliferation, excessive apoptosis, and autophagy were found to be the major factors involved in the regulation of the decrease in spermatogonia in cryptorchid boar testes.15 for this reason, the cryptorchid boar fertility is compromised. even if there is enough sperm production from the descended testis, the unilateral cryptorchid pigs should be castrated to avoid perpetuating this genetic trait in the progeny. the most common presentation is young boars with unilateral cryptorchidism that need to be castrated before puberty to avoid the undesirable effects of “boar taint” on meat quality. alternatively, boars thought to be castrated are presented to the veterinarian because they exhibit male traits. cryptorchidism is often seen in miniature and pet pigs.16 when one scrotal testis is present, scrotal palpation helps identify which of the testes is retained. if the animal has been hemicastrated, and the surgical wound has healed, it may be difficult to define if the retained gonad is the left or right. ultrasonographic evaluation is useful for the identification of the retained testicle and to determine if it is located abdominally or inguinally, guiding the selection of the surgical approach and reducing the duration of anesthesia. ultrasonographic evaluation is better performed when the boar is under general anesthesia. a linear or convex transducer can be used. the exam should start from the inguinal ring and continue toward the cranial abdomen. abdominal testes are found between the ipsilateral kidney and the inguinal ring, lateral to the bladder. after the testicle is visualized (figure 18), the location can be marked on the skin with a surgical pen. we will describe two surgical techniques to remove the ectopic testicles, a laparoscopic-assisted and cryptorchidectomy via laparotomy. laparoscopic-assisted cryptorchidectomy the pig is placed under general anesthesia and positioned in dorsal recumbency. the abdomen is prepped for sterile surgery. following a local block with 2% lidocaine at the site of the umbilical scar, a 1.5 cm skin incision is made cranial to the external preputial orifice. a laparoscopic cannula and trocar are tunnelled caudally subcutaneously for 2 cm and then used to penetrate the abdominal wall and peritoneum to enter the abdominal cavity, taking care to avoid penetrating any underlying viscera (figure 19). the trocar is then removed from the cannula, and a co2 line is attached to it. the abdomen is distended with co2 to 12 mm/hg pressure to facilitate visualization of abdominal organs. the surgical table is tilted to  elevate the hindquarters and place the boar in the trendelenburg position, displacing the abdominal viscera cranially and facilitating identification of the retained testis. a 10 mm, 0° operative scope is inserted through the cannula, and the abdomen is searched to locate the retained testis. following the application of a lidocaine block, a second laparoscopic port is created by inserting a cannula and trocar into the abdomen in the area overlying the testis. when the trocar is removed, and babcock forceps are introduced to grasp the testis and elevate it to the body wall (figure 20). the incision at the site of the second port is extended, and the testis is exteriorized (figure 21). the spermatic cord is ligated as previously described for open castration. alternatively, a radiofrequency-driven bipolar electrosurgical device (ligasure™ ) may be used intra-abdominally to transect the spermatic cord and seal the associated vessels before removal. the portal sites are closed with cruciate or simple continuous sutures using appropriate absorbable suture material. cryptorchidectomy via laparotomy following induction of general anesthesia, the boar is placed in dorsal recumbency, and the abdomen is prepped for sterile surgery. the abdomen is prepped sterilely, and a local block with 2% lidocaine is placed subcutaneously over the area of the proposed inguinal incision (figure 22). when the retained testis is in the inguinal canal, an incision is made on the skin overlying the external inguinal ring, and blunt dissection of the underlying connective tissue is pursued until the vaginal process of the peritoneum is identified. a small incision of the vaginal process allows for exploration to identify the ligament of the tail of the epididymis. when traction is applied to the ligament, the testis can be exteriorized. if the retained testis is abdominal, a paramedian incision is made lateral to and parallel to the prepuce. the underlying muscle is dissected bluntly to expose the peritoneum. the peritoneum is then elevated with thumb forceps and incised to gain access to the abdominal cavity. inclining the surgical table to position the head lower than the hind legs will displace abdominal viscera cranially, helping find the retained testis. introducing a finger in the incision is often sufficient to allow exploration and identification of the retained testis or ductus deferens and exteriorization of the retained gonad (figure 23). occasionally location of the gonadal structures is more difficult, and the abdominal incision may need to be enlarged to permit a more thorough search. once the testis is located and brought out of the incision, the spermatic cord is isolated and ligated using a transfixing suture figure 25. open cryptorchidectomy. castration of the descended testis and suture of the skin incisions. http://dx.doi.org/10.58292/ct.v15.9645 citation line: clinical theriogenology 2023, 15, 9645, http://dx.doi.org/10.58292/ct.v15.9645 97 and miller’s knot. following secure placement of the ligatures, the spermatic cord is transected distal to the sutures, and the testis removed. the pedicle is inspected, and if the hemorrhage is controlled replaced in the abdomen (figure 24). the abdominal incision and skin are sutured routinely, and the scrotal testis is removed through a separate incision (figure 25). complications exteriorization of the retained testis can be difficult if the spermatic cord and pampiniform plexus are very short or closely adhered to the inguinal canal. it is important not to apply too much force to induce rupture of the vessels; in those situations, enlarging the incision site sufficiently to allow intra abdominal ligation of the spermatic cords is required to avoid hemorrhage. post-op anti-inflammatories and antibiotics are administered as described for the other procedures above. references 1. anderson de, mulon py: anesthesia and surgical procedures in swine. in: zimmerman jj, karriker la, ramirez a, schwartz kj, stevenson gw, zhang j, eds. diseases of swine. hoboken, new jersey: john wiley & sons, ltd, 2019:171–196. 2. amann rp, veeramachaneni dnr: cryptorchidism in common eutherian mammals. reproduction 2007;133:541–561. doi: 10.1530/rep-06-0272 3. hodgkinson o: practical sedation and anaesthesia in pigs. in pract 2007;29:34–39. doi: 10.1136/inpract.29.1.34 4. smith ac, ehler wj, swindle mm: chapter 14 – anesthesia and analgesia in swine. in: kohn df, wixson sk, white wj, et al: editors. anesthesia and analgesia in laboratory animals. san diego, ca; american college of laboratory animal medicine: academic press; 1997:313–336. 5. ko jch, thurmon jc, tranquilli wa, et al: problems encountered when anesthetizing potbellied pigs. vet med 1993; 88:435–437. 6. fubini sl, ducharme n. in: farm animal surgery. 2nd edition. nx amsterdam, nx: elvesier; 2017. 7. huichu lin. comparative anesthesia and analgesia of ruminants and swine. in: veterinary anesthesia and analgesia. hoboken, new jersey: wiley online library; 2015. 8. eu monitor. directive 2008/120. minimum standards for the protection of pigs (codified version). 9. clarnette td, hutson jm: is the ascending testis actually ‘stationary’? normal elongation of the spermatic cord is prevented by a fibrous remnant of the processus vaginalis. pediatr surg int 1997;12:155–157. doi: 10.1007/bf01349987 10. du z-q, zhao x, vukasinovic n, et al: association and haplotype analyses of positional candidate genes in five genomic regions linked to scrotal hernia in commercial pig lines. plos one 2009;4:e4837. doi: 10.1371/journal.pone.0004837 11. searcy-bernal r, gardner ia, hird dw: effects of and factors associated with umbilical hernias in a swine herd. j am vet med assoc 1994;204:1660–1664. 12. thaller g, dempfle l, hoeschele i: maximum likelihood analysis of rare binary traits under different modes of inheritance. genetics 1996;143:1819–1829. doi: 10.1093/genetics/ 143.4.1819 13. dolf g, gaillard c, schelling c, et al: cryptorchidism and sex ratio are associated in dogs and pigs. j anim sci 2008;86: 2480–2485. doi: 10.2527/jas.2007-0607 14. rothschild mf, christian ll, blanchard w: evidence for multigene control of cryptorchidism in swine. j hered 1988;79:313–314. 15. fan x, liu y, yue m, et al: effect of cryptorchidism on the histomorphometry, proliferation, apoptosis, and autophagy in boar testes. animals 2021;11:1379. doi: 10.3390/ ani11051379 16. woods al, tynes vv, mozzachio k: special considerations for show and pet pigs. in: zimmerman jj, karriker la, ramirez a, schwartz kj, stevenson gw, zhang j, eds. diseases of swine. hoboken, new jersey: john wiley & sons, ltd, 2019:211–220. doi: 10.1002/9781119350927.ch13 http://dx.doi.org/10.58292/ct.v15.9645 https://doi.org/10.1530/rep-06-0272 https://doi.org/10.1136/inpract.29.1.34 https://doi.org/10.1007/bf01349987 https://doi.org/10.1371/journal.pone.0004837 https://doi.org/10.1093/genetics/​143.4.1819 https://doi.org/10.2527/jas.2007-0607 https://doi.org/10.3390/ani11051379 https://doi.org/10.3390/ani11051379 https://doi.org/10.1002/9781119350927.ch13 editor’s note dear theriogenology friends, it has been a privilege and pleasure to work with the leaders of our 3 organizations (society for theriogenology, american college of theriogenologists, and theriogenology foundation) and our journal is able to grow under the watchful eyes and support of our leaders. dr. charles franz, executive director, invites me to participate in the executive board meetings, places the suggestions of the editorial board members of clinical theriogenology for consideration, and implements them after approval by the leaders. thanks charles! i do appreciate dr. john kastelic’s help in copy editing. thanks john! starting from this issue, we will have manuscripts published in a reader-friendly layout. dr. ahmed tibary, not only created this new layout for us but freely gave his time to work with me to have them ready. i am well pleased with this new layout. thanks ahmed! we were very careful to catch errors that might have crept in because of the conversion from 1 software to another. despite our best efforts to correct them, we would have missed some. our sincere apologies! this new layout calls for high-quality figures. in this regard, there is an important change regarding how figures are prepared and submitted with manuscripts (please refer to the updated authors’ instructions). we will have a similar layout for reviews and abstracts for the conference issue. please feel free to write to me (petera@purdue.edu) or contact members of the editorial board, if you have concerns about this new layout or if you have suggestions to improve the quality of our journal. regards, augustine clinical theriogenology 2021; 13: 76 1 contact jennifer koziol jkoziol@ttu.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9271, http://dx.doi.org/10.58292/ct.v15.9271 review report pathophysiology, diagnosis, and management of testicular degeneration in the bull jennifer koziol,a colin palmerb atexas tech school of veterinary medicine, amarillo, usa bwestern college of veterinary medicine, saskatoon, sk, canada abstract testicular degeneration in the bull is described as diffuse disintegration of seminiferous tubule structure and function causing a profound reduction in sperm concentration, scrotal circumference, and an increase in sperm morphologic abnormalities resulting in subfertility or permanent infertility. degenerative changes within the seminiferous epithelium occur most commonly after an insult to spermatogenesis or may be idiopathic. common insults include trauma, stress (environmental, heat, nutritional), or toxins. idiopathic is most often associated with age-related changes. clinical signs of testicular degeneration are discussed along with approaches to treatment and potential prognoses as related to treatment and management practices that can be employed by the general practitioner and specialist alike. this review will also encompass a discussion on the pathophysiology of this disease so that the practitioner has a detailed understanding of the disease, which improves their ability to employ evidence-based medicine and sound clinical reasoning. keywords: testicular degeneration, bull, infertility, subfertility, spermatogenesis, spermiogram introduction testicular degeneration can be described as a diffuse disintegration of seminiferous tubule structure and function causing profound, subfertility of varying duration and in some cases permanent infertility. in bulls, most cases of testicular degeneration involve both testes; unilateral degeneration does occur but is less common. both literature reports and the authors’ clinical impressions suggest that the odds of full recovery are favorable depending on the severity of the degenerative process; however, at diagnosis, the inciting cause is most often speculative warranting at best a guarded prognosis. softening of the testes, loss of scrotal circumference and sperm quality and quantity are consistently reported clinical signs. watery, low sperm-concentrated ejaculates may be noticed; particularly in cases of bilateral testicular degeneration with high proportions of morphologically abnormal sperm, often greater than 80%.1 testicular degeneration is an acquired condition usually limited to the seminiferous tubules and rarely extending beyond the tubular basement membrane to affect the interstitial tissue. testes undergoing degeneration may have predisposing congenital anomalies; however, to enable differentiation from other conditions a diagnosis of testicular degeneration should be reserved for cases where the release of fully formed sperm has been previously evident indicative of postpubertal, seminiferous tubule function. demonstratable decreases in sperm production, sperm quality, and testicular size are fundamental diagnostic requirements for testicular degeneration. small testis, oligospermia/azoospermia, and teratozoospermia are also observed in cases of testicular hypoplasia. usually considered to be a congenital condition, hypoplastic testis(es) do not develop normal functional capacity and never reach mature size; whereas in cases of degeneration the testis has reached mature size and full function prior to reduction in size.2 testicular degeneration most commonly occurs secondary to an insult to the testis. potential insults include trauma, stress, abnormal testicular thermoregulation, toxins, ischemia, nutritional deficiencies or excess, infection, sperm outflow obstructions, and neoplasia. the extent of testicular degeneration depends on the severity and duration of the testicular insult. mild and short duration testicular insults may cause a transient increase in sperm morphologic abnormalities evident in the spermiogram with a return to normal sperm production within days to a few weeks.3,4 in these cases, seminiferous tubule function remains intact and degeneration does not occur. longer duration disturbances and severe insults that result in substantial tissue disruption may mailto:jkoziol@ttu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9271 2 citation line: clinical theriogenology 2023, 15, 9271, http://dx.doi.org/10.58292/ct.v15.9271 progress to degeneration.5 testis may be able to partially or fully regenerate after degeneration depending on the extent of the damage and the quality of the repair process. return to normal seminiferous epithelial function without fibrosis and scarring obviously favors regeneration and return to acceptable sperm quality. cases of testicular degeneration where no inciting cause can be identified are often classified as idiopathic. most cases of idiopathic testicular degeneration have been observed in middle to older aged bulls; therefore, it is also referred to as age-related testicular degeneration. as the testes age, production of gametes and the production of hormones are adversely affected. idiopathic testicular degeneration is usually progressive with a steady decline in fertility that persists indefinitely. pathophysiology normal testicular function is dependent on appropriate endocrine, autocrine, and paracrine control. in the male, gonadotropin releasing hormone (gnrh) is secreted from the hypothalamus in a pulsatile nature resulting in waves of luteinizing hormone (lh) and follicle stimulating hormone (fsh) release. lh acts on the leydig cells within the testes that are responsible for synthesizing progesterone – the majority of which is converted to testosterone. this pulsatile nature of lh secretion is necessary for normal testicular function as sustained lh can lead to a refractory response from leydig cells secondary to a downregulation in the number of receptors. this culminates in reduced secretion of testosterone from leydig cells. high concentrations of testosterone must be maintained for normal spermatogenesis to occur. the pulsatile release of lh and consequently testosterone prevents negative inhibition of fsh that is needed for the maintenance of sertoli cells. sertoli cells convert testosterone to dihydrotestosterone and estradiol along with secreting androgen binding protein (abp). approximately 80% of the secreted abp goes into the lumen of the seminiferous tubule and travels to epididymis whereas the remainder is secreted into the interstitial compartment and is absorbed into the systemic circulation. abp binds testosterone and is responsible for not only maintaining a high concentration of testosterone within the testes but also transporting testosterone to epididymis. although a number of causal factors are identified here it is plausible that most have similar effect, or root cause, which is a disturbance in testosterone release. hence, a decline in testosterone secretion leads to a disturbance in spermatogenesis that may progress to degeneration. most common causal factors associated with abnormal spermatogenesis are: abnormal testicular thermoregulation; hormonal imbalances, particularly those associated with stress; and the effect(s) of toxins or expression of deleterious genes.4 intratesticular injection with zinc acetate caused varying degrees of testicular degeneration in 10 bos indicus bull calves. the mechanism of action of zinc acetate was unknown but was proposed to be either a direct toxic effect on testicular cells or an autoimmune effect caused by widespread disruption of the blood–testis barrier. vacuolization of sertoli cells was also observed in this study, which often precedes a loss of germ cells.6 at least in this example, the overall significance of disturbed testosterone release likely had a minor role in the pathophysiology of testicular degeneration compared with overwhelming cellular damage leading to what appeared to be permanent degeneration. stress typically elevates systemic cortisol concentrations, profoundly decreasing release of lh and ultimately testosterone.7–9 stress has many origins, including environment (weather, heat, humidity, nutrition, and social hierarchy) illness, or injury; all causing changes in the spermiogram similar to those induced by disruption of testes thermoregulation. cells in early meiosis including the primary spermatocyte along with cells in spermiogenesis are extremely sensitive to alterations in the hormonal milieu secondary to stress, illness, or trauma. however, earlier germ cells, including spermatogonial stem cells, along with testicular somatic cells, which include leydig and sertoli cells, appear to be more resistant to adverse changes.10,11 alterations in the hormonal milieu within testis affect seminiferous tubules. lh, fsh, and testosterone concentrations are all affected after insults, which in turn, impairs seminiferous tubular function and consequently disrupting spermatogenesis. in some cases, seminiferous tubules may be severely damaged, resulting in sloughing of differentiated cells into the tubular lumen, leaving only sertoli cells and spermatogonia. when empty tubules in the testes lose turgidity, testes become noticeably softer, and the change is recognized as testicular degeneration. testicular degeneration may be permanent but is most often temporary or transitory. after resolution of the insult spermatogonia may repopulate the tubules allowing testes to return to normal size and sperm production. within a histologic section, there may be some normal tubules and also tubules with cellular damage accounting for varying proportions of normal and abnormal sperm in an ejaculate. although there are no large-scale reports to support their belief, the authors of the current article conjecture that testicular degeneration may be a more frequent occurrence in bulls in hotter and more humid climates. disruption of testes thermoregulation is the most understood cause of testicular degeneration.12 the most common cause of transitory testicular degeneration is ‘summer infertility’ associated with seasonally elevated ambient temperatures and humidity.13 a long duration scrotal insulation study using a 20-day treatment period proved to be very effective in disrupting the testicular thermoregulatory processes sufficiently to cause degeneration in 3 of 5 treated bulls.14 somewhat surprisingly testicular degeneration did not occur, or was at least temporary, in 2 bulls suggesting that considerable individual variation did exist. accumulation of fat in the scrotum also limits testicular thermoregulation. overconditioning bulls has been recognized as a major factor leading to testicular degeneration in cooler climates.5 sustained elevated body temperatures associated with fever can lead to testicular degeneration; however, it appears that the body’s stress response may confound the effect of heat. localized inflammation associated with trauma may result in unilateral testicular degeneration. a number of other localized inflammatory conditions have the potential to  disrupt thermoregulatory processes sufficiently to cause testicular degeneration; however, few if any cases clearly indentifying the cause and effect have been documented. examples include, diffuse scrotal dermatitis, severe diffuse frostbite, and orchitis. feeding of a high starch diet to growing bulls was associated with inflammation within the testes, disrupted spermatogenesis and evidence of testicular degeneration in some bulls. a suggested cause of the testicular insult was the release of bacterial toxins after ruminal acidosis that could have caused a local inflammatory reaction within the gonadal tissue.15 rare, congenital conditions such as congenitally short scrotum and incomplete testes descent into the http://dx.doi.org/10.58292/ct.v15.9271 citation line: clinical theriogenology 2023, 15, 9271, http://dx.doi.org/10.58292/ct.v15.9271 3 scrotum, and acquired lesions limiting free movement of these testes may impair thermoregulatory mechanisms enough to cause degeneration.5 higher than normal testicular temperatures in one testis may impede thermoregulatory mechanisms in the contralateral testis causing a disruption in spermatogenesis or in severe cases, degeneration of both testes.13 the examiner may observe that while one testis feels firm, fibrotic, and clearly shrunken the other testis is larger, but soft. these clinical findings coupled with a watery ejaculate and teratozoospermia suggest that both testes are undergoing degeneration but are at different stages of the degenerative process. a detailed review of the effects of heat stress on bull fertility suggested the need for heightened awareness of the detrimental effects of heat stress on bull fertility in the light of increases in global temperature due to climate change.16 age, genetics, and husbandry practices (e.g. access to shade and water) are important considerations for mitigating the effect of heat on testicular function. older bulls may be more susceptible to heat stress than their younger counterparts and bos indicus bulls are more suited to maintaining fertility in hotter and more humid environments than bos taurus bulls. without a means of cooling, higher environmental temperatures cause heat stress within the testis. the ensuing hypoxia leads to increased reactive oxygen species production, which when in excess have a negative effect on spermatogenesis.17 differences between breeds, individual animals, and the intensity and duration of the heat insult influence the effect on spermatogenesis and whether the disruption in testicular function progresses to testicular degeneration. scrotal insulation for 8 days was associated with sertoli and leydig cell dysfunction.12 testosterone concentrations increased initially after scrotal insulation and then declined and remained low for at least 96 days; beyond when normospermia returned. this longer duration scrotal insulation study was the first to demonstrate that scrotal insulation was associated with a decline in testosterone concentrations. idiopathic, or age-related testicular degeneration in the bull largely remains an enigma with little evidence that it occurs in bulls in the scientific literature. an age-related decline in daily sperm production caused by a loss of spermatocytes during meiosis has been reported in men. this condition has been associated with a reduction in the number of leydig cells, interstitial cells, myoid cells, and sertoli cells.18 heightened sensitivity of older bulls to hotter environmental temperatures16 combined with individual variation in the response to thermoregulatory insult14 may have a role at least in some cases. cases of testicular degeneration with no apparent cause, most often observed in mid-aged to older stallions, are often labelled as idiopathic testicular degeneration.19 studies of subfertile stallions suggested that a defect in the steroidogenic pathway, likely at the level of the testes, is probably responsible. xenograft studies using diseased equine testicular tissue cografted with pig testis revealed that the equine tissue continued to degenerate unlike the pig tissue following treatment with exogenous gonadotropins or endogenous hormones.19 clinical assessment history testicular degeneration is most often identified during a breeding soundness examination (bse) as an incidental, often unexpected, cause of subfertility. bulls may also be presented for a perceived fertility issue. astute individuals may notice an obvious decline in scrotal circumference, severe asymmetry of the scrotum, or a failure to establish pregnancy in a cycling female(s). bulls in use at a stud are likely to exhibit a decline in both quantity and quality of sperm in the ejaculate before other physical signs are evident. a recommended management practice is that all natural service sires should have a bse within a month or 2 of the impending breeding season. sires used yearround, as may be the case in bull studs or in dairies employing natural service, should be examined regularly. a properly conducted bse comprises a physical examination incorporating an examination of the scrotum and its contents including a measurement of the scrotal circumference, and an assessment of semen quality – most importantly sperm morphology. a complete history is useful in determining a possible inciting reason for testicular degeneration. for example, if the bull experienced a particularly stressful or painful situation (e.g. lameness, severe weight loss, or illness). bulls that were overconditioned on high-energy diets may experience substantial weight loss when placed in a new environment. all too often, bulls must transition from a high-grain diet to pasture or adapt to a low-energy diet while at the same time facing social dominance or hierarchical challenges. the quality of the feed is of little relevance if the bull cannot eat enough. a decline in scrotal circumference often occurs with a substantial decline in body weight; especially, in bulls that were overconditioned. generally, this is thought to reflect a loss of fat in the scrotal and testicular interstitial tissue. feeding high energy rations during the postweaning development period was associated with the deposition of fat in the scrotum causing erroneously larger scrotal circumferences, palpably softer testes, greater difficulty thermoregulating the testes and poor semen quality.20 increased fat deposition in the scrotum and testes can effectively insulate the testes severely compromising spermatogenesis and causing testicular degeneration.20 high ambient temperatures and high humidity for sustained periods have also been linked to the occurrence of testicular degeneration. the clinician should always note the age and breed of the bull. older bulls may be more susceptible to thermoregulatory challenges or age-related changes.16 young bulls with very small testes developed testicular degeneration at 2–3 years of age.21 small testes believed to be the result of hypoplasia and poor semen quality is a common finding in bulls of the double-muscled, belgian blue breed. a greater amount of interstitial connective tissue postulated to be interfering with internal blood flow within the testes was associated with an increased occurrence of testicular degeneration within the breed.22 finally, a review of the bull’s previous bse results can be valuable. quantifiable changes in scrotal circumference and a reliable assessment of sperm morphology are much better than owner speculation. recent and timely bses can also point to a potential cause of the degeneration. testicular degeneration may be confused with testicular hypoplasia. evidence of declining scrotal circumference coupled with decreasing sperm quality are important initial clues for distinguishing degeneration from hypoplasia. physical examination testicular degeneration in the bull may present as a unilateral or bilateral condition. unilateral testicular degeneration is often associated with trauma; local infection; sperm outflow obstruction; or neoplasia. causal factors for bilateral testicular degeneration are more apt to be: trauma; stress (weather, http://dx.doi.org/10.58292/ct.v15.9271 4 citation line: clinical theriogenology 2023, 15, 9271, http://dx.doi.org/10.58292/ct.v15.9271 nutritional, pain, and social hierarchy); systemic illness associated with fever; insulation of the scrotum (hydrocele, inguinal hernia, fat, etc.); toxins; or age-related changes. a decrease in testicular volume quantified by a decrease in scrotal circumference coupled with palpable change in the texture of the testes are hallmarks of the disease. a loss of palpable resilience within the testis, commonly referred to as softness, is most evident early in the disease during an increase in testicular temperature.13 testicular turgidity or resilience is created by functioning fluid-filled seminiferous tubules, comprising 80% of the volume of a healthy sexually mature testis, surrounded by soft tissue. in long-standing cases the testes may become palpably firm; however, palpable resilience will not be evident. testicular degeneration and testicular hypoplasia may be difficult to distinguish; especially, in young bulls with an uncertain history as small, undersized testes are common to both conditions.23,24 differentiation may be facilitated by comparing the size of the affected testis to corresponding epididymis. visual assessment and careful palpation of the epididymides are usually sufficient, but the examiner may also want to compare bulls of similar age. if the epididymis is small and proportional in size to the undersized testis, testicular hypoplasia is likely.25 conversely, if the epididymis is well developed and near normal size, one can assume that testicular and epididymal development were normal and that testicular degeneration and corresponding testicular atrophy has occurred. it is also important to remember that testicular hypoplasia is a congenital condition.2 severe cases of degeneration can progress to testicular fibrosis. damage to the seminiferous tubule lining may not be repairable resulting in scarification and calcification of the tissue. fibrosis appears on ultrasonography as echogenic densities. reports from western canada and argentina indicate that fibrotic lesions in testicular tissue are quite common and not necessarily associated with a tangible decline in fertility.26 the underlying etiology of testicular changes resulting in testicular fibrosis remains speculative and congenital malformation of the tubule-to-rete connection, infectious disease processes resulting in tubule destruction, and trauma to the testes have all been proposed. testicular fibrosis may range from a few small areas with no effect on fertility to severe fibrosis throughout the testes. the presence of many fibrotic lesions may not preclude production of an ejaculate with normal morphology, as some bulls with severe fibrosis produced semen with up to 94% morphologically normal sperm in a report. the authors suggested that large amounts of scar tissue would be expected to reduce sperm production, but that changes in semen production by bulls with varying degrees of severity of testicular fibrosis has not been investigated.26,27 ultrasonographic evaluation of the testis will not allow for differentiation between normal testicular tissue and that which has lost germinal epithelium.28 ultrasonography after induced testicular degeneration by scrotal insultation failed to demonstrate a correlation between pixel intensity and semen quality during bse.29,30 furthermore, pixel intensity for prediction of future fertility had limited correlation with bse outcomes.31–33 echogenic areas varying in number and size are frequently detected in the testis and associated with fibrosis, but they do not have substantial effect on the percentage of normal sperm within the ejaculate.26 summarizing these reports, ultrasonography can be useful for determining that fibrosis is indeed present but is not useful for predicting the quality of sperm produced or the outcome of a bse. semen assessment testicular degeneration follows an insult to spermatogenesis. decreased sperm production and the appearance of teratozoospermia in the ejaculate occur soon after the insult and if monitored regularly over a few weeks, a predictable series of defects will appear sequentially. the seminiferous epithelium is lined with 5-7 layers of germinal cells supported and nourished by sertoli cells. from a1 spermatogonia at the basement layer of the tubule epithelium to fully formed sperm ready to be released into the tubule lumen requires ~ 61 days in the bull. an additional 10 days is needed for transport to and through the epididymis during which further maturation of sperm occurs. cells in meiosis and spermatids undergoing the final metamorphic changes to become fully formed sperm, known as spermiogenesis, are the most sensitive to adversity. cells lost in meiosis simply degenerate, the process of spermatogenesis ends, and no subsequent generations of cells are produced leading to reduced sperm output. disruption of critical maturation processes for spermatids, and recently released sperm in the testes and epididymis leads to the appearance of morphologic abnormalities in the spermiogram.3,11 mild disturbances may only affect spermatids or epididymal sperm with a short-lived period of increased numbers of morphological abnormalities. a study characterizing the sequential appearance of morphologic abnormalities reported that there were differences between animals in the overall degree of response to the negative stimulus reflected in the proportion of morphologically abnormal sperm and the proportion of certain types of defects.4 more severe or prolonged disturbances cause destruction of the spermatocytes and spermatogonia. not all tubules will be affected in the same way; however, it is widespread degeneration in the tubules that causes the noteworthy changes that define the condition. spermatocytes that are disrupted during meiosis and survive the remainder of the spermatogenic process may have chromatin abnormalities leading to reduced fertility and poor embryo survival.16 because testicular degeneration is the outcome of a progressive process, changes in semen characteristics become evident before identifiable changes in scrotal circumference or palpable texture of testis. oftentimes, an assessment of semen quality does not occur until sometime after the insult when widespread seminiferous tubule disruption has already progressed to degeneration. physical examination of testes may initiate suspicions of testicular degeneration in these cases and subsequent examination of sperm morphology will help confirm the diagnosis. the appearance of teratozoospermia is commonly observed in cases of testicular degeneration. no single morphological aberration is exclusive to degeneration. small numbers of spheroids, which are believed to be immature spermatogenic cells may be seen as well as teratoid forms and medusa cells. distal midpiece reflexes, proximal cytoplasmic droplets, daglike and other midpiece disruptions, coiled principal pieces, detached heads, tapered and pyriform heads, knobbed acrosomes, all types of nuclear vacuoles, microcephalic sperm and sperm with abnormal dna condensation were evident following known insults to spermatogenesis.3,4 the sperm head or nucleus is largely made up of chromatin. variations in nuclear shape within a spermiogram may reflect varying levels of chromatin stability. using conventional microscopic techniques even normal appearing sperm may have poor chromatin and reduced fertility.34 bulls recovering from a severe disturbance to spermatogenesis and testicular degeneration often will produce sperm with more variation in nuclear size and shape. in the unstained semen sample immature spermatogenic cells may be confused with white blood cells. different stages of immature spermatogenic cells typically http://dx.doi.org/10.58292/ct.v15.9271 citation line: clinical theriogenology 2023, 15, 9271, http://dx.doi.org/10.58292/ct.v15.9271 5 appear in an ejaculate that allow for a variation in size of the cells as compared with white blood cells, which are more uniform in size. to differentiate between an immature spermatogenic cell and a white blood cell a semen smear can be air dried and stained in diff-quik® (available from numerous suppliers) or very similar wright-giemsa stain. neutrophils and lymphocytes will be stained by diff-quik® whereas immatures spermatogenic cells will not be stained sufficiently to allow the practitioner to easily differentiate.24,35 the duration of the insult and the time that passed from the initial insult, assuming that it was acute in action, will dictate the sperm morphologic abnormalities that will be observed, and the sequence of the abnormalities noticed. oligospermia and asthenozoospermia may also be noticed in ejaculates. the majority of beef bulls in north america are natural service sires and are not trained to an artificial vagina for semen collection. semen samples collected by other means (e.g. electroejacualtion or transrectal massage) are not representative of a physiologic ejaculation. while sperm concentration can be calculated samples may be more or less concentrated than a natural ejaculation and therefore, not truly representative of what the bull can produce. it is also important to remember that sperm motility can be altered by environmental conditions. samples obtained by massage are especially prone to chilling. nevertheless, effectively performed electroejaculation and transrectal massage are both very suitable methods for clinical assessments of quantity and quality of sperm production.36 endocrinologic evaluation the relationship between plasma hormone concentrations and testicular degeneration in bulls has not been adequately studied. in other species (e.g. the stallion) estimation of circulating hormone concentrations is an unsatisfactory diagnostic tool for testicular degeneration.24 due to daily and seasonal fluctuations in hormone, detecting changes between normal and pathological concentrations is difficult for diagnosing testicular degeneration preemptively or during the early stages of disease. by the time that hormone concentrations become consistently inappropriate, the diagnosis is readily apparent by physical examination.24 histopathologic evaluation histopathologic evaluation of a testis affected with testicular degeneration will reveal cytoplasmic vacuolization, absence of later stage germ cells and loss of architecture of the seminiferous epithelium.21 although histopathologic findings can help to define testicular degeneration and testicular biopsy would provide definitive evidence, it is rarely justified. once the practitioner has obtained a satisfactory history and performed a proper bse a diagnosis of testicular degeneration can be made with confidence. it should also be considered that a single biopsy may not be representative of the testes as a whole.24 testicular biopsies can be safely performed with no subsequent decrease in sperm quality appreciated after biopsy.37 this study was performed on normal bulls and no information exists regarding the use of this procedure on the already degenerated or compromised testicle.37 although a testicular biopsy may be useful in some cases often the quality of the sperm ejaculated will give a similar insight into the health of the seminiferous epithelium without the potential risks associated with a testicular biopsy. effects of feeding a high energy feed were studied. a starch rich diet was fed to angus x charolais bulls (n = 40) for 94 days with a mean age of 14 ± 1.7 months at the beginning of the trial.15 after a 10-day period of adaptation, body weight, an assessment of testes size, and ultrasonography of the testes were performed every 28 days from the beginning of the measurement period until day 84. at the end of the experiment each bull underwent electroejaculation and semen analysis. the testes were harvested at slaughter and submitted for an assessment of histological architecture with evidence of degeneration and tissue healing, seminiferous tubule atrophy, and evidence of disruption of spermatogenesis being scored. mean scrotal circumference and testes volume increased as expected throughout the feeding period yet lesions consistent with degeneration were observed including sperm with vacuoles, tubules with thickened basement membranes, interstitial tissue edema, multinucleated giant cells in the lumen of tubules and tubular atrophy. there was also evidence of permanent tissue damage including fibrosis, microcalcification, and complete loss of seminiferous tubules; although, the frequency of these lesions was low amongst analyzed samples. however, evidence of an insult to spermatogenesis was present on 100% of the histograms.15 although examination of testes histopathology is not practical in most settings the occurrence of degenerative changes, including permanent lesions, varied between individuals and areas within the testic.15 treatments, management, and prognosis recovery from testicular degeneration is possible in most cases if the initiating cause is removed. three, 4 or even several months may be required for the seminiferous epithelium to recover to preinsult sperm production. no treatment has been successful in treating testicular degeneration other than removal of the initial trauma or stressor. partly due to the usual uncertainty in determining the cause of the degeneration compounded by the individual variation that is evident following any insult to spermatogenesis predicting when a satisfactory bse should be expected is a risky, if not impossible, undertaking. when breeding season is imminent it is usually best to cull and replace all but the most valuable bulls. valuable bulls may be worth feeding and housing for several months provided a worthwhile return to normal sperm production is plausible. the use of gnrh treatment in a belgium blue bull with testicular degeneration has been described. in that report an osmotic pump was inserted subcutaneously, and the bull received frequent doses (2.5 µl/h) of buserelin for 9 weeks. after treatment an increase in sperm concentration, volume and quality was noticed. the treatment did allow the authors to determine that the bull suffered from hypothalamic dysfunction. one could surmise that once treatment stopped the bull would return to a state of testicular degeneration.38 no other studies reported on the use of buserelin. in stallions gnrh has not been shown to be effective as a treatment for testicular degeneration in controlled studies.24,39 in cases of unilateral testicular degeneration or damage, removal of the compromised testis has been advocated in hopes of salvaging valuable genetics. especially in those instances where infammation persists; for example, an abscess, sperm granuloma or tumor; heat radiating from the damaged testicle may overwhelm the physiological mechanisms maintaining the contralateral testis 4–5°c below body temperature leading to secondary testicular degeneration.40 aside from weighing the likelihood for recovery, the deciding http://dx.doi.org/10.58292/ct.v15.9271 6 citation line: clinical theriogenology 2023, 15, 9271, http://dx.doi.org/10.58292/ct.v15.9271 factor to perform a hemicastration should be that the inflammation associated with postoperative tissue healing will be short-lived compared with leaving the compromised testis in situ. another reason to consider hemicastration, is that the blood–testis barrier within the compromised testis may have sustained damage leading to the production of fertility limiting antisperm antibodies.24,41 removal of 1 testis often results in hypertrophy of the remaining testicle. the remaining testicle will produce 1.5 x the sperm production of 1 testis leaving a bull at ~ 75% of his original sperm production ability.42 it should be noted that the hypertrophy only occurs in normal testes and a contralateral testis damaged by excessive heat for prolonged periods or has exposure to antisperm antibodies cannot be expected to achieve such results. conclusion testicular degeneration in the bull is often discovered on a bull bse; therefore, determining a cause may be difficult. an insult to the testis(es) resulting in a disturbance of spermatogenesis that progresses to a disruption of the seminiferous epithelium is the most common sequence of events although poorly understood idiopathic or age-related testicular degeneration can be observed in some bulls. disruption of testes thermoregulatory mechanisms is the most studied model for inducing testicular degeneration and may be the most common cause of spontaneously occurring testicular degeneration with overfeeding, obesity, age, and ambient heat/humidity being substantial risk factors. although not definite, it appears that both thermoregulatory and stress-induced testicular degeneration involve a disturbance in the hypothalamic–pituitary–gonadal axis resulting in insufficient testosterone concentrations to maintain seminiferous tubule function; however, an endocrine disturbance may not be evident when the animal is presented for a clinical examination. evaluation of the quality and quantity of sperm combined with examination of testes often supports a definitive diagnosis. histopathology and ultrasonography may be of benefit in some cases but are often not warranted. prognosis is dependent on severity and duration of the insult yet often the identity of the cause is speculative warranting a guarded prognosis. serial semen 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domest anim 2012;47 suppl 3:45–51. doi: 10.1111/j.1439-0531.2012.02042.x 33. tomlinson m, jennings a, macrae a, et al: the value of trans-scrotal ultrasonography at bull breeding soundness evaluation (bbse): the relationship between testicular parenchymal pixel intensity and semen quality. theriogenology 2017;89:169–177. doi: 10.1016/j.theriogenology.2016.10.020 34. ostermeier gc, sargeant ga, yandell bs, et al: relatiosnhip of bull fertility to sperm nuclear shape. j androl 2001;22:595–603. 35. koziol jh, armstrong cl: sperm morphology of domestic animals. 1st edition. hoboken, nj; john wiley & sons: 2021. 36. palmer cw: welfare aspects of theriogenology: investigating alternatives to electroejaculation of bulls. theriogenology 2005; 64: 469–479. doi: 10.1016/j.theriogenology.2005.05.032 37. heath am, carson rl, purohit rc, et al: effects of testicular biopsy in clinically normal bulls. j am vet med assoc 2002;220:507–512. doi: 10.2460/javma.2002.220.507 38. contri a, gloria a, wegher l, et al: successful use of a gonadotropin-releasing hormone (gnrh) analog for the treatment of tertiary hypogonadism (gnrh deficiency) in a 5-year-old belgian blue bull. vet q 2012;32:51–54. doi: 10.1080/ 01652176. 2012.677866 39. blanchard t, varner d, bretzlaff k, et al: testicular degeneration in large animals: identification and treatment. vet med 1991; 86:537–542. 40. brito lf, silva ae, barbosa rt, et al: testicular thermoregulation in bos indicus, crossbred and bos taurus bulls: relationship with scrotal, testicular vascular cone and testicular morphology, and effects on semen quality and sperm production. theriogenology 2004;61:511–528. doi: 10.1016/s0093-691x(03)00231-0 41. zhang j, ricketts s, tanner s: antisperm antibodies in the semen of a stallion following testicular trauma. equine vet j 1990;22:138– 141. doi: 10.1111/j.2042-3306.1990.tb04228.x 42. wolfe d, hudson r, carson, r, et al: effect of unilateral orichectomy on semen quality in bulls. j am vet med assoc 1985;186:1291–1293. http://dx.doi.org/10.58292/ct.v15.9271 http://dx.doi.org/10.1016/j.theriogenology.​2007.09.034 http://dx.doi.org/10.1016/j.theriogenology.​2007.09.034 http://dx.doi.org/10.1111/j.2042-3306.1994.tb04060.x http://dx.doi.org/10.1111/j.2042-3306.1994.tb04060.x http://dx.doi.org/10.1016/j.anireprosci.2019.06.009 http://dx.doi.org/10.1016/j.anireprosci.2019.06.009 http://dx.doi.org/10.1016/j.anireprosci.2007.05.002 http://dx.doi.org/10.2527/jas1982.5361645x http://dx.doi.org/10.1016/0093-691x​(87)90231-7 http://dx.doi.org/10.1016/0093-691x​(87)90231-7 http://dx.doi.org/10.1016/s0378-4320​(03)00082-4 http://dx.doi.org/10.1016/s0378-4320​(03)00082-4 http://dx.doi.org/10.1016/j.theriogenology.2012.01.022 http://dx.doi.org/10.1111/j.1439-0531.2012.02042.x http://dx.doi.org/10.1016/j.theriogenology.2016.http http://dx.doi.org/10.020 http://dx.doi.org/10.1016/j.theriogenology.2005.05.032 http://dx.doi.org/10.2460/javma.2002.220.507 http://dx.doi.org/10.1080/ 01652176.2012.677866 http://dx.doi.org/10.1016/s0093-691x(03)00231-0 http://dx.doi.org/10.1111/j.2042-3306.1990.tb04228.x pilot studies on mare reproductive loss syndrome pilot studies on mare reproductive loss syndrome stuart burns,a lenn harrison,b peter sheerinc aburns equine veterinary service, paris, ky buniversity of kentucky veterinary diagnostic laboratory, lexington, ky crood and riddle equine hospital, lexington, ky abstract past investigations utilized administration of either whole eastern tent caterpillars or their integument, to pregnant mares to study mare reproductive loss syndrome. five pilot studies using various approaches were conducted to reproduce clinical signs of this syndrome. it is suggested that a stable ester toxin, mandelonitrile benzoate, present in cuticle (formed after ingestion of leaves with prunasin) is implicated. keywords: caterpillar, mandelonitrile benzoate, prunasin, abortion introduction an unusual abortion storm in pregnant mares in ohio river valley region of us in 2001 and 2002 was recognized as mare reproductive loss syndrome (mrls). clinical signs were early and late abortions, pericarditis, and panophthalmitis. approximately 9% of 2001 foal crop was lost to late pregnancy abortions and 26% of 2002 crop was lost to early gestation abortions. by september 2002, jockey club reported a 15% decline in foal registrations compared to same interval in 2001.1 economic loss to equine industry was estimated ~ $500m.2 there were no pathognomonic lesions identified in late pregnancy abortions and tissue autolysis at time of conceptus expulsion in early pregnancy losses prevented detection of significant lesions.2 a total of $2,313,552 was provided over 2002 2003 by multiple equine industry agencies, including kentucky thoroughbred association, kentucky thoroughbred owners and breeders association, agriculture development board, grayson-jockey club research foundation, university of kentucky and usda agriculture research service.3 researchers from university of kentucky gluck center, university of kentucky veterinary science department, university of kentucky veterinary diagnostic laboratory (ukvdl) alone, or in cooperation with local equine veterinary practitioners, published research findings. collectively, research identified that eastern tent caterpillar (etc; malacosoma americanum) had a central role in causing mrls and 2 hypotheses were advanced to explain pathogenesis. these hypotheses suggested either consumption of etcs with bacteria-carrying setae (septic setae) or a possible toxin, were responsible for abortion. since feeding sterilized (irradiated4 or soaked in 95% ethanol for 2 hours5) caterpillars induced abortion, alimentary tract bacteria were considered responsible by colonizing affected tissues to cause abortion.6,7 however, other researchers8 suggested that bacteria that were isolated had little tendency to induce inflammation in colonized tissue. when fed irradiated etcs, 3 of 6 pregnant mares aborted at longer than expected time, without clinical signs, suggesting absence of an infectious agent in this syndrome.2,9 thus, septic setae and toxin hypotheses both remain unproven2 and exact cause of mrls is undetermined.10 overall objective was to determine the right hypotheses using 5 pilot studies. each study was designed to answer a single primary question of relevance, keeping animal numbers in perspective,11 and using evidence-based principles.12,13 experiments 1 and 2 were approved by university of kentucky institutional animal care use committee and experiments 3 to 5 were conducted in compliance with usda guidelines (refer previous paper in this issue). an independent ultrasonographer was engaged to eliminate any potential investigator bias and provide outside confirmation that horses participating in these protocols were in good health and receiving appropriate care. experiment 1 transmission of toxic irritants produced by lepidopteran caterpillars via aerosol exposure has been reported.14,15 chemical agents responsible for “urticating hairs” associated with these caterpillars clinical theriogenology • volume 12 number 2 • june 2020 111 may vary within 12 common families associated with caterpillar toxins. usual signs of toxicity include urticating dermatitis, renal failure, asthma, intracerebral hemorrhage and osteochondritis.14 caterpillar envenoming is associated with specialized setae, spines or urticating hairs,14 which by themselves, are inert. these setae may be covered with, or contain within a hollow shaft, the offending toxic chemical(s) (diaz 2006, personal communication). objective was to determine if an unidentified toxic agent associated with etc could reproduce abortions, clinical signs and/or tissue changes associated with mrls following exposure to air containing an etc toxin and/or setae. materials and methods four pregnant (180 300 days) mares were maintained in a paddock that provided free choice grazing and an open water source. each mare was exposed to vapors produced by live, freshly fed etc placed in a metal strainer with a mesh size of 1.7 mm, covered with a double layer of wire screen of 1.5 mm mesh size. these containers were held in a canvas nosebag feeder (well tolerated by mares) placed over the mare’s muzzle, thus ensuring that most inhaled air would also have been in contact with etc (figure 1). freshly fed, live etc were used for each exposure, which lasted 6 hours per day for 10 days. each mare was examined by transrectal ultrasonography every 48 hours to confirm pregnancy. temperatures, heart rates and respiration rates were recorded once a day. figure 1. canvas nose bag containing etcs wire mesh container results three of 4 mares were unaffected by exposure to any vapors or aerosolized setae associated with etc. all remained normal and all 3 mares delivered live, normal foals. an unexpected variable was introduced on day 7, when the 4th mare immersed her entire nosebag containing etc in an open water source to drink. this behavior was repeated on day 8 and the mare aborted 72 hours later. enterobacter spp. were isolated from placenta and all fetal organs. expelled placenta was normal in size and weight, but had tan coloration, suggesting placental separation before abortion2 and/or diffuse intracapillary thrombosis caused reduction in placental perfusion. discussion findings suggested that etcs did not provide aerosolized setae or a vapor containing a chemical toxin and setae and/or toxin associated with their cuticle did not volatilize sufficiently to allow aerosol transmission. abortion in 1 mare was caused by consumption of water contaminated by etc, suggesting that setae and any bacteria associated with etc, or a toxin associated with cuticle of etc, were stable in water and it could have been a transfer medium that moved setae, bacteria or toxin from etc cuticle to mare. open water system might have been a risk factor in this abortion.16 water transmission in this protocol did not eliminate the possibility of setae involvement, as pore size of the container screen containing etc setae would permit transfer of setae through screens to allow contact with oral or nasal mucosa of test animal. bacterial isolates were associated with 90% of all mrls abortions.8 clinical theriogenology • volume 12 number 2 • june 2020112 histopathological findings in this aborted fetus were limited to a fetal pneumonia and mild placentitis, reported in 65 and 10% of mrls abortions, respectively.8 these findings are commonly associated with late pregnancy fetal loss in mrls. conclusion abortion, bacterial isolates and tissue changes associated with mrls can be induced by consumption of water contaminated by etcs. transmission of the causative agent of mrls via contaminated aerosols could not be demonstrated or did not occur, so attention should be directed to water as a transmitting medium. experiment 2 inhalation of possible vapors and/or setae produced by etcs did not induce mrls, but water contaminated by etcs induced mrls. protocol used in experiment 1 could not determine whether abortifacient was a water-stable toxin or due to exposure to etcs.17 to determine if water contaminated by etcs, but not containing setae, could induce mrls abortion, clinical signs and/or tissue changes associated with mrls. objective was to determine if setae were a necessary component of mrls, or if syndrome could be reproduced in their absence by exposure to water containing an unidentified toxin, but no setae. materials and methods five pregnant (273 310 days) mares were maintained together in a paddock similar to that in experiment 1. freshly fed live etcs (500 grams) were placed at room temperature in 2.5 liters of distilled water for 30 minutes. water was filtered through 6 layers of sterile gauze to remove any particulates, including setae, that may have originated from intact etcs. each day water was prepared fresh and examined microscopically for setae. mares were gavaged once daily for 10 days with 500 ml of water and examined by transrectal ultrasonography every 48 hours. temperatures, heart rates and respiration rates were recorded once a day. results four mares remained unaffected and had normal temperature, pulse, and respiration. on day 9 of exposure, 1 mare developed tachypnea that persisted for 4 days. three days after the 10th exposure, this mare aborted with an uncorrectable dystocia and she was euthanized. necropsy findings included congestion of all tissues, plus 300 ml of amber and slightly cloudy fluid in pericardial sac. fluid analysis (total protein: 300 mg/dl, wbc: 1320, specific gravity: 1.024, ph: 8.0, increased glucose and low viscosity) suggested vascular permeability and leakage of peripheral blood components into pericardial space. placental surface was a brownish color, similar to that observed in experiment 1 abortion. there were no gross lesions on the amnion or allantois. histopathological findings in fetus were a mild neutrophilic fetal pneumonia and a mild amnionitis. no bacteria were isolated from fetal tissues. discussion dystocia,18 prepartum placental separation, tachypnea and pericarditis in this mare together with fetal pneumonia (observed in 65% of mrls abortions) and amnionitis (observed in 80% of mrls abortions), were reported earlier in mrls.8 this is the only reported experimentally induced mrls abortion accompanied by pericarditis. conclusion mrls can be induced by a water stable toxin passing from etc cuticle into water in contact with its surface. this protocol indicated that gastrointestinal exposure of setae-free etc contaminated water could induce mrls, thereby suggesting etc setae are not obligatory to cause mrls. clinical theriogenology • volume 12 number 2 • june 2020 113 experiment 3 experiments 1 and 2 demonstrated that water contaminated by etcs could induce mrls. experiment i protocol could not eliminate the possibility of setae contamination with possible tissue penetration. experiment 2 provided a challenge dose of water contaminated by etcs, but filtered to remove all particulates, including setae. setae-free challenge caused 1 of 5 mares to suffer an mrls abortion accompanied by pericarditis. objective was to confirm that exposure of oral mucosa alone to fluid contaminated by contact with etcs allows transmission of an unknown toxin in sufficient quantities to induce abortion, clinical signs and/or tissue changes associated with mrls. materials and methods five pregnant (45 65 days) mares were maintained similar to earlier experiments. mares were challenged with 100 ml of etc contaminated saline sprayed into their oral cavity once daily for 5 consecutive days. this challenge fluid was prepared by placing 35 grams of dead etcs into 35 ml of sterile saline, followed by centrifugation at 1600 x g for 10 minutes. to increase concentration of an unknown cuticle toxin, resulting supernatant was filtered through waterman quantitative filter paper and frozen in 15 ml aliquots in plastic vials. mares were then examined every 48 hours for 20 days by transrectal ultrasonography for changes in fetal fluid echogenicity and fetal heartbeat. fluid echogenicity was scored 1 to 5 as described,19 with higher numbers indicating increased echogenicity. results two of 5 mares developed echogenicity score 3 in their fetal fluids on day 13, remaining 3 pregnancies maintained anechoic fluids. however, none of 5 mares aborted within 50 days after last exposure and all subsequently delivered normal foals. discussion anechoic fetal fluids are normal in equine pregnancies from days 10 85 of pregnancy, with any echogenicity during this interval regarded as an indication of fetal insult.19 there was a 100% correlation between detection of increased echogenicity of fetal fluid preceding early pregnancy losses during 2001 and 2002 mrls outbreak. not all mares exhibiting fetal fluid echogenicity aborted (73% maintained their pregnancies).20 nevertheless, observation of fetal fluid echogenicity was considered an indicator for presence mrls etiological agent.4,2 two pregnancies sharing this change in echogenicity of fetal fluids suggest that causative agent can cross oral mucosa without penetration by setae and initiate an inflammatory response in amnion, causing increased vascular permeability, allowing leakage of thrombin and fibrinogen to form insoluble strands of fibrin, followed by sloughing of some amniotic epithelium into the amniotic fluid, increasing echogenicity. there is a dose-response relationship between tissue response and abortion.2 failure to proceed to abortion was likely related to dose and duration of exposure. limited supply of challenge fluid resulted in only 5 days of treatment. also, use of dead etcs may have reduced amount of toxin recovered. body motion of live etcs may be a significant factor in moving toxic agent from its storage site at setae base (figure 2) to cuticle surface and getting washed from surface to enter challenge fluid. figure 2. cross section of tent caterpillar cuticle; (a) setae and (b) storage site. clinical theriogenology • volume 12 number 2 • june 2020114 experiment 4 experiment 3 suggested that agent causing mrls was present in supernatant collected after etcs were centrifuged with an equal weight of water. based on university of kentucky veterinary diagnostic laboratory records, at least 10 species of bacteria were recovered from 682 late pregnancy abortions and only 1.6% of these isolates accounted for oral commensals, alpha streptococci and actinobacillus.21 source of nonhemolytic streptococci22 and actinobacillus23 in aborted fetuses was from their dam’s oral mucosa. unique dna of cultured isolates from each mare’s oral mucosa matched identically to similar organisms retrieved from her aborted foal. type of bacteria isolated from late pregnancy aborted fetuses is dependent upon types of bacteria colonizing mucosal surface exposed to toxic agent. consuming contaminated forage or water results in highest level of exposure to streptococci and actinobacilli, considered normal, commensal organisms in oral mucosa. when this mucosa is bypassed by delivering challenge dose of whole etcs via a nasogastric tube, enteric bacteria are more readily involved, as intestinal mucosa becomes location of primary contact with toxin. when delivery of challenge agent via intravenous infusion resulting abortion could be only due to agent delivered. objective was to determine if sterile abortion would result when contact exposure of the oral/intestinal mucosal is bypassed by intravenous presentation of the toxin associated with mrls. material and methods challenge agent was prepared by placing 100 g of live etcs in 100 ml of sterile water; these caterpillars had been denied access to cherry tree leaves for 24 hours, as fitzgerald24 reported that etc foregut will be clear of mandelonitrile, a degradation product of prunasin, within 7 hours after last ingestion of cherry tree leaves. thus, by 24 hours, there would be little or no contamination of the challenge fluid by foregut regurgitation, so that source of any persisting toxin would likely be from surface of etc’s cuticle. mixture of etcs and water was agitated gently for 10 minutes to stimulate activity of live etcs and to wash off any material on cuticle surface. this contaminated water was then poured off, leaving etcs in mixing vessel. this supernatant was filtered through 6 layers of sterile gauze to remove all particulate matter and frozen immediately in 25 ml aliquots in glass vials. a thawed sample was cultured and very few colonies of mixed growth bacteria were observed (considered nonpathogenic bacillus spp. based on morphology and biochemical analyses). intradermal injection (0.2 ml) of this challenge fluid was administered to a gelding to assess its ability to initiate an inflammatory response. the injection site was biopsied 96 hours later; there was inflammation and mild collagen necrosis within upper and middle dermis that included infiltrating neutrophils and macrophages, many of which were necrotic. adjacent blood vessels had mild perivascular accumulations of neutrophils, macrophages and eosinophils. we planned to send samples of etc contaminated challenge fluid and sterile amniotic and allantoic fluid samples recovered from aborted conceptus (if any) to institute of biological chemistry at washington state university for identification of possible etiological agents.25 four pregnant (days 60, 65, 85, and 100) mares were maintained on pasture, similar to earlier experiments. last 2 pregnant mares (controls) received intravenously 100 ml sterile saline. first 2 mares received challenge agent intravenously (50 ml on day 1 to evaluate their tolerance to infusion, followed by 100 ml per day for 5 more days). mares were scanned transrectally at 48 hour intervals for fetal fluid echogenicity grading19 and fetal heartbeat. results two control mares were unaffected. their fetuses maintained a heartbeat and fetal fluids appeared normal. both treated mares (60 and 65 days of pregnancy), tolerated intravenous administration of challenge agent, with no indications of discomfort; they maintained normal body temperatures, pulse, and respiration rates during 6 days of treatment. fetal fluids remained anechoic for first 5 days of challenge. on day 6, 1 mare developed uterine edema in nongravid uterine horn and had grade 2 fetal fluid echogenicity. she aborted between days 6 and 8 while at pasture and abortus was not found. second treated mare was then examined at 24 hour intervals. her fetal fluids remained anechoic for 8 days after clinical theriogenology • volume 12 number 2 • june 2020 115 following first treatment. uterine edema was observed in nongravid uterine horn on days 9 and 10. on day 11, fetal fluid became grade 2 echogenic and fetus died. six hours after a fetal heartbeat could no longer be detected, mare was anesthetized to harvest conceptus, fetal membranes and uterus, as “fresh tissues” for bacterial culture and gross and histopathological examinations; however, fetus and its membranes delivered spontaneously during administration of general anesthetic (figure 3). no gross lesions were observed in fetus or membranes and no bacteria were cultured from either source. sloughed squamous cells from inner surface of amnion and clotted strands of fibrin were observed during cytological examination of amniotic fluid and amniotic portion of umbilical cord was inflamed (funisitis). fibrin and sloughed amniotic cells caused fetal fluid echogenicity. endometrial stroma had hemorrhage, as noted in 60% of mrls abortions in 2001 and 2002.8 base of the endometrial cups was infiltrated with activated neutrophils (figure 4) demonstrating an inflammatory response to a systemic irritant. edema was observed in nongravid uterine horns both aborted mares. figure 3. aborted fetus 6 days after intravenous treatment figure 4. activated neutrophils at base of endometrial cup (antczak df and allen wr [published with permission]) chromatographic analysis revealed that challenge agent, amniotic and allantoic fluids contained 358, 438 and 236 components, respectively; challenge agent and amniotic fluid shared 23 compounds in common, challenge agent and allantoic fluid shared 30 components and allantoic and amniotic fluids shared 72 components. discussion sixteen early-stage pregnant aborting mares did not undergo luteolysis and had plasma progesterone concentrations of 4 5.9 ng/ml at time of fetal death.20 plasma progesterone was not measured in our mares. fetal fluid echogenicity preceding abortion in treated mares occurred only 24 48 hours before fetal death or abortion compared to 72 hours or more observed in many clinical cases of mrls. this suggests that toxic agent was lethal before greater echogenicity could develop in fetal fluids. clinical theriogenology • volume 12 number 2 • june 2020116 however, histological signs of inflammation were similar to clinical cases of mrls. although revealed by chemical signature, the identity of each molecule remains unknown and it is tempting to speculate that the 23 compounds shared by the challenge fluid and the amniotic fluids may have included the toxin that caused mrls. experiment 5 some degree of tissue inflammation is a consistent feature of mrls. however, most frequent isolates of oral commensal bacteria isolated from mrls aborted fetuses are not capable of initiating such an inflammatory response or they would do so while in contact with oral mucosa. a previously unidentified toxic agent that can be washed from cuticle of etc is able to initiate an inflammatory response and early pregnancy loss resembling mrls. this agent is related to diet of etcs which feed on black cherry (prunus serotina) leaves that contain prunasin that degrades into glucose and mandelonitrile, an intense, but relatively unstable, tissue irritant. mandelonitrile degrades to hydrogen cyanide and benzaldehyde. when 4 mares were given 3 mg/kg of mandelonitrile orally, a peak of cyanide in the blood, derived from the mandelonitrile degradation, was as high as 2025 ng/ml which occurred within 3 minutes after ingestion, but decreased to 250 ng/ml within 30 minutes. seven pregnant mares fed 2 mg/kg of mandelonitrile in apple sauce twice daily for 14 days had peak blood concentrations of cyanide immediately after ingestion of mandelonitrile that stabilized at 315 ng/ml (range, 215 to 594 ng/ml) within 30 minutes. this dose and duration of exposure to mandelonitrile in circulation was unable to induce mrls.26 mandelonitrile is present in etc foregut after feeding.27 it combines with mandelonitrile radicals, benzyl nitrile or possibly benzoic acid, to form a remarkably stable ester, mandelonitrile benzoate (mb), present on etc cuticle surface.28 freezing etcs produced a crystalline substance on their cuticle that could be shaken off the surface. these crystals were suspended in methylene chloride and the resulting solution injected directly into a gc/ms apparatus which confirmed the presence of mb. mb is an intensely irritating cyanogenic compound. its stability does not allow release of a sufficient quantity of free cyanide to be detectable by colorimetric methods. this stability is useful to etc, as it prevents absorption of free cyanide that would be toxic to etc while retaining repellant properties of benzaldehyde that protects etc from insect and vertebrate predation. because of this stability, plasma cyanide concentrations in early pregnant mares could not be detected following their exposure to gavaged etcs, even though many mares aborted. objective was to determine if pregnant mares would develop clinical signs of mrls (e.g. fetal fluid echogenicity and/or abortion) after oral exposure to mandelonitrile benzoate. this agent is produced by etc and is derived from stabilization of mandelonitrile present in the etc foregut after ingestion of prunasin-rich black cherry tree leaves. materials and methods mb was prepared using a modified method of the procedure described.29 this preparation would be expected to contain equal amounts of r and s isomers of mandelonitrile. it is unknown whether both isomers have equal biological activity. however, the r isomer is the only form in black cherry tree leaves20 so that the preparation of mb may have only half the biological activity of naturally produced mandelonitrile benzoate. it was examined for its potential to initiate an inflammatory response by injecting 0.2 ml of mb solution intradermally into a test gelding. mandelonitrile benzoate is insoluble in water and 95% ethanol, but is soluble in dmso. three 0.2 ml intradermal injections each of water, dmso alone and mb dissolved in dmso were administered to a gelding and injection sites were biopsied 96 hours later. water and dmso alone injection sites shared a tissue response that was essentially normal for a skin section. mb/dmso injection site, however, had inflammation and necrosis within midand deep-dermis; this included infiltrating neutrophils and macrophages, many of which were necrotic. adjacent and deeper blood vessels had moderate perivascular accumulations of neutrophils, macrophages and eosinophils nearly identical to those observed in the tissue response to intradermal injections of washings from the etc cuticle in experiment 4. three pregnant (days 45, 65, and 180) were maintained on pasture in similar conditions to other experiments. they were exposed by oral spraying of 10 ml of a solution containing 1 g of mb twice daily clinical theriogenology • volume 12 number 2 • june 2020 117 for 14 days, similar to dose of mandelonitrile fed in applesauce.26 mares were examined by transrectal ultrasonography every 48 hours to assess fetal fluid echogenicity and fetal health (heartbeat); last mare (day 180) was examined transrectally with a 5.0 mhz transducer and transabdominally with a 3.5 mhz transducer every 48 hours, all beginning on first day of exposure to mb oral spray. results mare 1 (45 days of pregnancy) fetal fluids became grade 1 echogenic on day 13. this level of echogenicity remained unchanged for next 12 days and no further changes were observed. this mare did not abort. mare 2 (65 days of pregnancy) fetal fluid echogenicity became grade 2 on 6th day of exposure. this progressed to grade 3 by day 13 and grade 4 by day 20 after first exposure. on day 24 (still at grade 4 echogenicity) fetus was observed to be at internal cervical os, through which fetal membranes were protruding. fetus still had a heartbeat, but based on dilated cervix, position of fetus and membranes and prolonged interval of increasing fetal fluid echogenicity, abortion was imminent. fetus and placenta were removed by vaginal delivery rather than risk losing or contaminating specimen. there were no gross lesions on fetus or placenta. fetal liver and spleen had marked diffuse echymotic hemorrhages, as observed in 60% of mrls abortions and focal, minimal, endodermal, squamous metaplasia was observed on amniotic portion of umbilical cord, indicating early funisitis, observed in 80% of mrls abortions. no other histological lesions were noted. no bacteria were isolated from any fetal tissue or fluid and tests for leptospirosis and ehv1 virus were negative. cytological examination of allantoic fluid revealed abundant rbcs, sloughed chorionic cells and a few neutrophils, consistent with increased vascular permeability. in mare 3 (180 days of pregnancy), fetal fluid echogenicity was at grade 2 (prior to first exposure to mb), regarded as normal for mares beyond 85 days of pregnancy. echogenicity increased to grade 3 on day 7 and to grade 4 by day 9. fetal activity decreased by 50% within 48 hours after first exposure and continued to decline until day 4 of treatment, but then began to recover by day 6. fetal heart rate was 124 on first day of exposure, decreased to 112 at 48 hours after first treatment and declined further to 104 by day 7. it increased again to 112 by day 11 and remained stable thereafter for the duration of treatment. conceptus was not aborted, although fetus appeared to have incurred some stress. discussion mandelonitrile benzoate caused increased fetal fluid echogenicity, considered indicative od the presence of the agent that causes mrls.2,20 mb caused histological and cytological changes in day 65 conceptus that were identical to those in mrls abortions in 2001 and 2002. hence, tissue response to exposure to mb and etc cuticle washings was almost identical, as was response to intradermal exposure to both agents. based on these findings, we inferred that mb may be the etiological agent of mrls. conclusion collectively, these experiments supported the suggestion that mrls can be induced without any contribution from etc setae or bacteria. it is possibly a chemical toxin derived from prunasin which is ingested by etcs and in which its metabolites are processed in the foregut of the etc to form the very stable toxin, mandelonitrile benzoate. this toxin is ingested by mares consuming contaminated forage or water. it acts as a tissue irritant, initiating inflammatory response that eventually induces abortion. however, findings need to be replicated in a larger population of test mares using a mb challenge containing only the r isomer of mandelonitrile. acknowledgment facilities, staff, and laboratory support were donated by park equine veterinary hospital, rood and riddle equine hospital and robert henthorn’s bluegrass veterinary laboratory. funding of animals, their care and miscellaneous expenses were provided by the burns family trust. clinical theriogenology • volume 12 number 2 • june 2020118 conflict of interest authors have no conflicts of interest to report. references 1. powell d: mrls continues to mystify scientists in kentucky. equine health repost by the national institute for animal agriculture. spring 2003; p. 125. 2. sebastian mm, bernard wv, riddle tw, et al: review paper: mare reproductive loss syndrome. vet pathol 2008; 45:710-722. 3. brown k: mrls research funding exceds $2 million: more needed. http//www.bloodhorse.com/horseracing/articles/179878/mrls-research-funding-exceeds-2-million-more-needed 2003. 4. sebastian mm, bernard wv, riddle tw, et al: review paper: mare reproductive loss syndrome. vet pathol 2008;45:710-722. 5. bernard wv, leblanc mm, and webb ba, et al: evaluation of early fetal loss induced by gavage with eastern tent caterpillars in pregnant mares. j am vet med assoc 2004;225:717-721. 6. tobin t, harkins jp, roberts jf: the mare reproductive loss syndrome and the eastern tent caterpillar ii: a toxicokinetic/clinical evaluation and a proposed pathogenesis: septic penetrating setae. int j appl res vet med 2004;2:142-158. 7. mcdowell kj, webb ba, williams nm, et al: invited review: the role of caterpillars in mare reproductive loss syndrome: a model for environmental causes of abortion. j anim sci 2010;88:1379-1387. 8. williams nm, bolin dc, donahue jm, et al: gross and histopathological correlates of mare reproductive loss syndrome. first workshop on mare reproductive loss syndrome (mrls) 2003; p. 24-25. 9. sebastian mm: pathology of late term aspects of mare reproductive loss syndrome. phd dissertation 2004; veterinary science, university of kentucky, lexington, ky. 10. duckworth a: health zone abortions in broodmares. the blood-horse january 3, 2019;60-66. 11. guiffrida wa: defining the primary research question in veterinary research studies. j am vet med assoc 2016;249:547-550. 12. rossdale pd: objectivity versus subjectivity in medical progress. equine vet j 2003;35:331-332. 13. muir ww: is evidence-based medicine our only choice? equine vet j 2003;35:337-338. 14. diaz jh: the evolving global epidemiology, syndromic classification, management, and prevention of caterpillar envenoming. am j trop med hyg 2005;72:347-357. 15. maier h, spiegel w, kinaciyan t, et al: the oak processionary caterpillar as the cause of an epidemic of airborne disease: survey and analysis. br j dermatol 2003;149:990-991. 16. choen nd, carey vj, donahue sg, et al: case-control study of late term abortions associated with the mare reproductive loss syndrome in central kentucky. j am vet med assoc 2003;222:199-209. 17. harrison lr, burns sj, sebastian mm, et al: ingestion of a soluble factor, but not inhalation of volatile chemicals, from eastern tent caterpillars induces abortion (mrls) in pregnant mares. tenth international syndrome on equine reproduction 2010; p. 339-340. 18. brown s: field and clinical observations related to late fetal loss in mare reproductive loss syndrome. first workshop on mare reproductive loss syndrome 2003; p. 14-15. 19. vince k, riddle w, leblanc m, et al: ultrasonographic appearance of fetal fluids between 55 and 176 days of gestation in the mare: effect of mare reproductive loss syndrome. proc annu conv am assoc. equine pract 2002; p. 350-352. 20. riddle wt, leblanc mm: update on mare reproductive loss syndrome. proceedings of the society for theriogenology 2004; p. 85-99. 21. giles rc, donahue jm, hong cb, et al: causes of abortions, stillbirths and perinatal deaths: 3527 cases (1986-1991). j am vet med assoc 1993;203:1170-1175. 22. newman k, zaffarano j: microbiology of mrls. kentucky thoroughbred owners and breeders foundation mrls update. keeneland sales pavilion 2005. 23. donahue jm, sells sf, bolin, dc: classification of actinobacillus spp isolates from horses involved in mare reproductive loss syndrome. am j vet research 2006;67:1426-1432. 24. fitzgerald td, jeffers pm, and mantella d: depletion of host-derived cyanide in the gut of the eastern tent caterpillar, malacosoma americanum. j chem ecol 2002;28:257-268. 25. plumb rs, johnson ka, rainville p, et al: rapid comm mass spectrum 2006;20:1989-1994. 26. harkins jb, dirikolu l, sebastian mm, et al: cherry trees, plant cyanogens, caterpillars and mare reproductive loss syndrome: toxicological evaluation of a working hypothesis. first workshop on mare reproductive loss syndrome 2003; p. 68-74. 27. peterson sc: breakdown products of cyanogenesis: repellency and toxicity to predatory ants. naturwissenschaften 1986;73:627-628. 28. harrison jr, trail c: unpublished data, assays of eastern tent caterpillars, fayette and surrounding counties, veterinary diagnostic laboratory, university of kentucky. 2003. clinical theriogenology • volume 12 number 2 • june 2020 119 29. francis f, davis cm: preparation of the acyl derivatives of the aldehydecyanohydrins part 1. j chem soc 1909;95:1403-1405. 30. conn, ee. in: conn ee: editor. the biochemistry of plants, a comprehensive treatise. volume 9, new york; academic press: 1981. p. 483-484. clinical theriogenology • volume 12 number 2 • june 2020120 1 contact dirk vanderwall dirk.vanderwall@usu.edu this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http://creativecommons.org/ licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9270, http://dx.doi.org/10.58292/ct.v15.9270 research report effect of human chorionic gonadotropin treatment on the duration of oxytocin-induced prolonged corpus luteum function in mares dirk vanderwall,a dustin kinney,b holly clement,a johanna rigas,c david wilsona adepartment of veterinary clinical and life sciences, college of veterinary medicine, utah state university, logan, ut; buinta veterinary services, ballard, ut; caltasciences, everett, wa abstract oxytocin is used increasingly in mares to prolong corpus luteum (cl) function to suppress estrus. when mares develop prolonged cl function in response to oxytocin treatment, the cl generally remains functional for 60–90 days. however, a longer duration of cl function can make this method of estrus suppression even more clinically useful. we hypothesized that human chorionic gonadotropin (hcg) treatment during the period of oxytocin-induced prolonged cl function would extend the duration of cl function (i.e. beyond 90 days). prolonged cl function was induced in mares (n = 14) by treating them with 60 units of intramuscular oxytocin once daily on days 7–14 after ovulation. mares were then randomly assigned equally to a control group that received no additional treatment and an hcg-treated group that received 2,500 units of intramuscular hcg on days 30, 45, 60, 75, and 90 after ovulation. jugular blood samples were collected for progesterone concentration determination on the day of ovulation and then 3 times (m, w, and f) weekly for 120 days. duration of cl function was not different (p > 0.05) and it was 78.0 ± 7.5 and 91.4 ± 20.4 days (mean ± standard deviation [sd]) in control and hcg-treated mares, respectively. therefore, hcg treatment (during the period of oxytocin-induced prolonged cl function) did not extend cl function, hence alternative methods should be explored. keywords: mare, oxytocin, corpus luteum, hcg introduction oxytocin treatment is used increasingly to prolong corpus luteum (cl) function as a means of suppressing estrous behavior in performance mares by allowing continued secretion of progesterone to keep mares out of estrus naturally.1 oxytocin treatment of mares beginning in mid diestrus prolonged cl function by inhibiting the expression of endometrial cyclooxygenase-2,2,3 the inducible form of the rate-limiting enzyme necessary for endometrial secretion of prostaglandin f2α (pgf2α) at luteolysis. as a result, the luteolytic process (i.e. regression of the cl) was blocked causing cl function to continue beyond its typical duration of 2 weeks. this is in sharp contrast to the stimulatory effect of oxytocin, both endogenous and exogenous, on endometrial secretion of pgf2α during late diestrous in mares.4–6 importantly, therefore, oxytocin can have a proluteolytic or an antiluteolytic effect in mares, and these diametrically opposed responses occur in a tightly controlled, time-dependent manner during the estrous cycle. the most widely used oxytocin treatment protocol for estrus suppression utilizes 60 units of intramuscular oxytocin once daily on days 7–14 after ovulation and it generally induces prolonged cl function in >70% of treated mares.7 an alternative method that does not require detection of ovulation utilizes 60 units of intramuscular oxytocin once daily for 29 consecutive days.8 estrous behavior rather than ovulation as the basis for initiating oxytocin treatment has also been described.9 additional work is underway to develop a formulation of oxytocin that does not require daily treatment.10 when mares develop prolonged cl function in response to oxytocin treatment, the cl generally remains functional for 60–90 days. however, when oxytocin treatment is used for estrus suppression, a longer duration of cl function is preferable. we hypothesized that human chorionic gonadotropin (hcg) treatment during the period of oxytocin-induced prolonged cl function would further extend cl function (i.e. beyond 90 days) via two potential mechanisms: (1) a mailto:dirk.vanderwall@usu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9270 2 citation line: clinical theriogenology 2023, 15, 9270, http://dx.doi.org/10.58292/ct.v15.9270 direct luteotrophic effect and/or (2) by inducing ovulation of a diestrous follicle(s) resulting in the formation of new cl(s) that remain functional for an additional 60–90 days. therefore, the objective was to determine whether repeated hcg treatment during the period of oxytocin-induced prolonged cl function further extends cl function. materials and methods animals this study was conducted in the northern hemisphere (41.7°n) under natural photoperiod using quarter horse-type mares (n =  14) between 6 and 15 years of age that weighed 300–500 kg. animal procedures were approved and conducted following the guidelines of the utah state university institutional animal care and use committee (protocol #2249). experimental protocol beginning in may, the reproductive tract of each mare was examined via transrectal palpation and ultrasonography, and when mares developed an ovarian follicle ≥35 mm in diameter in conjunction with prominent endometrial edema, they were examined daily until ovulation was detected (day 0). jugular blood samples were collected on day 0 and then every m, w, and f through day 120. blood samples were allowed to clot at room temperature, then serum was recovered and kept frozen at –20°c until progesterone concentrations were determined. after ovulation, prolonged cl function was induced in mares (n = 14) by treating with 60 units of intramuscular oxytocin (bimeda – mtc animal health, inc., cambridge, ontario, canada) once daily on days 7–14. mares were then randomly assigned equally to a control group that received no additional treatment and an hcg-treated group that received 2,500 units of intramuscular hcg (chorulon®, merck animal health, summit, nj) on days 30, 45, 60, 75, and 90 after ovulation. after reconstituting each vial of lyophilized hcg with the sterile diluent provided by the manufacturer, individual doses were drawn into syringes and kept frozen at –20°c until use. mares that received hcg treatment were examined (via transrectal palpation and ultrasonography) on days of treatment to assess their ovarian follicular status. mares that had ovarian follicle(s) ≥ 30 mm in diameter were reexamined 3  days later to determine whether ovulation(s) had occurred after hcg treatment. progesterone assay progesterone was measured using a commercially available kit (immulite progesterone, siemens, malvern, pa) designed for an enzyme-amplified chemiluminescence assay system (immulite 1000, diagnostic products corporation, los angeles, ca) and performed according to the manufacturer’s protocol. intraassay coefficient of variation was 3.9% and the interassay coefficient of variation was 5.8%. the sensitivity of the assay was 0.2 ng/ ml, and values below the assay sensitivity were assigned a value equal to the sensitivity. data analyses duration of cl function (number of days the progesterone concentration was > 1.0 ng/ml), a continuous outcome variable, was compared between control and hcg-treated mares, an input categorical variable, with a two-tailed t-test (graphpad, san diego, ca). means of all daily blood progesterone concentrations (continuous outcome variable with repeated measures) from day 0 through the day progesterone concentrations dropped below 1.0 ng/ml (or until day 120) were compared between control and hcg-treated groups using a two-tailed t-test. alpha value for determination of significance was set at p < 0.05. results control and hcg-treated mares’ progesterone profiles are depicted (figure 1). the duration of cl function was not different (p > 0.05) and it was 78.0 ± 7.5 and 91.4 ± 20.4 days (mean ± standard deviation [sd]) in control and hcg-treated mares, respectively. two hcg-treated mares had blood progesterone concentrations >1.0 ng/ml through day 120; however, in the other 5 mares progesterone concentrations returned to baseline between days 74 and 91. although there was no difference in the duration of cl function, daily blood progesterone concentrations were higher (p < 0.0001) in hcg-treated mares compared to control mares (figure 1; 7.8 ± 4.1 vs. 5.1 ± 2.7 ng/ml [mean ± sd] respectively). six diestrous ovulations occurred within 3 days of hcg treatment in 5 mares. three ovulations occurred after hcg treatment on day 30, two ovulations occurred after treatment on day 60 and 1 ovulation occurred after treatment on day 75. diestrous ovulations occurred in two mares that maintained cl function through day 120 (after hcg treatment on day 30 in 1 mare and day 75 in the other mare), but in the other 3 mares with diestrous ovulations progesterone concentrations dropped to baseline between days 74 and 91 (ovulation occurred after hcg treatment on day 30 in 1 mare; day 60 in 1 mare; and day 30 and 60 in 1 mare). quite notably in 1 mare, following a diestrous ovulation after hcg treatment on day 60 (i.e. ovulation detected on day 63), blood progesterone dropped precipitously to basal concentrations within 2 weeks despite the presence of the newly formed cl (figure 2). discussion despite the fact that hcg treatment of mares with oxytocin-induced prolonged cl function resulted in diestrous ovulations and higher concentrations of progesterone, it did not successfully extend the duration of cl function. as evidenced by the precipitous fall in progesterone concentration that occurred within 2 weeks after an hcg-induced diestrous ovulation on day 63 in 1 mare (and a similarly precipitous drop in progesterone concentrations in mares in control and treated groups), it appeared that it was the return of the endogenous luteolytic process in that period that was responsible for the inability of hcg treatment to extend the duration of cl function. the postulate11 that a return of the luteolytic mechanism was responsible for the cessation of luteal function after ~60  days was supported in the present study. return of the luteolytic mechanism between days 60 and 70 of oxytocin-induced prolonged cl function has now been demonstrated based upon the ability of exogenous oxytocin to stimulate endometrial secretion of pgf2α in the same manner that it does at the expected time of luteolysis in cycling mares (i.e. through its proluteolytic effect).12 in light of the return of the luteolytic process after ~day 60 during the period of prolonged cl function, it is plausible that retreatment with oxytocin before the luteolytic process is http://dx.doi.org/10.58292/ct.v15.9270 citation line: clinical theriogenology 2023, 15, 9270, http://dx.doi.org/10.58292/ct.v15.9270 3 reestablished could be used to extend the duration of cl function. if retreatment with oxytocin can block the return of the luteolytic process, it could plausibly be combined with the hcg treatment protocol described here. for example, had the return of the luteolytic process been blocked (by retreatment with oxytocin) in the mare that ovulated after hcg treatment on day 60, the newly formed cl should have remained functional for an additional 60 days or longer, resulting in a total duration of prolonged cl function of >120 days. based on the results mentioned above,12 the optimal time to readminister oxytocin would be between days 50 and 60 of prolonged cl function, because treatment of oxytocin in that interval did not appreciably stimulate endometrial secretion of pgf2α, and all of the mares maintained luteal function after treatment. in contrast, oxytocin treatment further and beyond day 60 rendered a more robust pgf2α secretory response and cl function was more likely to decline rapidly after treatment.12 considering that prolonged cl function occurs spontaneously in mares,11 return of the luteolytic process may have developed naturally as a means of ensuring that these mares return to a cyclic state allowing them to have an opportunity for breeding and establishment of pregnancy, which is otherwise precluded during the period of prolonged cl function. even with the return of the luteolytic process, a 60–90 day period of prolonged cl function represents a considerable portion of the physiological breeding season that is not available for breeding. therefore, it seems likely there has been selection pressure for a process that reestablishes the luteolytic mechanism in mares with prolonged cl function, so they can return to a cyclic state. in conclusion, although 2,500 units of intramuscular hcg treatment on days 30, 45, 60, 75, and 90 of oxytocin-induced prolonged cl function resulted in diestrous ovulations and higher concentrations of progesterone, it did not extend the duration of luteal function. the dose (2,500 units) of hcg used in this study is commonly used to induce ovulation in mares; however, it is not known whether a different dose (higher or lower) and/or different treatment schedule may figure 1. (a) serum progesterone concentrations from day of ovulation (day 0) through day 120 in 7 control mares with oxytocin-induced prolonged cl function. for clarity, lines are truncated once progesterone concentrations dropped below 1.0 ng/ml. (b) serum progesterone concentrations from day of ovulation through day 120 in 7 mares with oxytocin-induced prolonged cl function that were treated with 2,500 units of hcg im on days 30, 45, 60, 75, and 90 after ovulation. for clarity, lines are truncated once progesterone concentrations dropped below 1.0 ng/ml. (c) mean serum progesterone concentrations from day of ovulation through day 120 in 7 control mares with oxytocin-induced prolonged cl function (broken line) and 7 mares with oxytocin-induced prolonged cl function that were treated with 2,500 units of intramuscular hcg on days 30, 45, 60, 75, and 90 after ovulation (solid line). daily blood progesterone concentrations were higher in hcg-treated mares compared to control mares (p < 0.0001). for clarity, lines are truncated once progesterone concentrations dropped below 1.0 ng/ml. variability in the mean values was due to the m, w, f bleeding schedule that resulted in a sub-set of mares being represented on any given day after ovulation. http://dx.doi.org/10.58292/ct.v15.9270 4 citation line: clinical theriogenology 2023, 15, 9270, http://dx.doi.org/10.58292/ct.v15.9270 have impacted the outcome. because there is now compelling evidence that it is a return of the luteolytic process after 60 days that is responsible for the cessation of prolonged cl function, it seems plausible that retreatment of oxytocin (potentially in conjunction with hcg) prior to day 60 could inhibit the return of the luteolytic process leading to an extension of the period of prolonged cl function. acknowledgement authors thank bettina conrad and sherrie petty for technical assistance. conflict of interest and funding authors have no conflict of interest to declare. supported by utah agricultural experiment station. references 1. holyoak r, lyman c, hornberger k: when it comes to estrus suppression, is mare a four-letter word? clin theriogenol 2022;14:233–239. 2. keith l, ball ba, scoggin k, et al: diestrus administration of oxytocin prolongs luteal maintenance and reduces plasma pgfm concentrations and endometrial cox-2 expression in mares. theriogenology 2013;79:616–624. doi: 10.1016/j.theriogenology. 2012.11.015 3. rebordao mr, galvao a, pinto-bravo p, et al: endometrial prostaglandin synthases, ovarian steroids, and oxytocin receptors in mares with oxytocin-induced luteal maintenance. theriogenology 2017;87:193–204. doi: 10.1016/j.theriogenology.2016.08.028 4. vanderwall dk, silvia wj, fitzgerald bp: concentrations of oxytocin in the intercavernous sinus of mares during luteolysis: temporal relationship with concentrations of 13,14-dihydro-15keto-prostaglandin f2α. j reprod fertil 1998;112:337–346. doi: 10.1530/jrf.0.1120337 5. shand n, irvine chg, turner je, et al: a detailed study of hormonal profiles in mares at luteolysis. j reprod fertil 2000;suppl 56:271–279. 6. goff ak, pontbriand d, sirois j: oxytocin stimulation of plasma 15-keto-13,14dihydro prostaglandin f-2α during the oestrous cycle and early pregnancy in the mare. j reprod fertil 1987;suppl 35:253–260. 7. vanderwall dk, parkinson k, rigas j: how to use oxytocin treatment to prolong corpus luteum function for suppressing estrus in  mares. j equine vet sci 2016;36:1–4. doi: 10.1016/j. jevs.2015.09.007 8. parkinson kc, vanderwall dk, rigas j, et al: effect of chronic administration of oxytocin on corpus luteum function in cycling mares. j equine vet sci 2020;90:102991. doi: 10.1016/j.jevs.2020. 102991 9. manning hs, runcan ee, dias de moraes cr, et al: using estrous behavior to time initiation of oxytocin administration to prolong luteal function in mares. j equine vet sci 2019;75:78–81. doi: 10.1016/j.jevs.2019.01.012 10. sarnecky ba, vanderwall dk, mason hm, et al: evaluation of a proprietary slow-release oxytocin formulation on corpus luteum function in mares. j equine vet sci 2019;77:28–30. doi: 10.1016/j. jevs.2019.01.004 11. stabenfeldt gh, hughes jp, evans jw, et al. spontaneous prolongation of luteal activity in the mare. equine vet j 1974;6:158–163. doi: 10.1111/j.2042-3306.1974.tb03952.x 12. sarnecky ba, vanderwall dk, mason h, et al: oxytocin-induced secretion of 13,14-dihydro-15-keto-prostaglandin f2α in mares with prolonged corpus luteum function. clin theriogenol. 2020;12:366. figure 2. serum progesterone concentrations from day of ovulation (day 0) through day 120 in 1 mare with oxytocin-induced prolonged cl function that was treated with 2,500 units of intramuscular hcg on days 30, 45, 60, 75, and 90 after ovulation. at hcg treatment on day 60 (black arrow), the mare had a 44 mm follicle on the right ovary, and on day 63 (grey arrow) that follicle had ovulated as evidenced by the presence of a new cl. despite having a newly formed cl on day 63, mare’s progesterone concentrations dropped precipitously from 9.9 ng/ml on day 72 to < 1.0 ng/ml on day 74. for clarity, the line has been truncated at the point when the progesterone concentration dropped below 1.0 ng/ml. http://dx.doi.org/10.58292/ct.v15.9270 http://dx.doi.org/10.1016/j.theriogenology.​2012.11.015 http://dx.doi.org/10.1016/j.theriogenology.​2012.11.015 http://dx.doi.org/10.1016/j.theriogenology.2016.08.028 http://dx.doi.org/10.1530/jrf.0.1120337 http://dx.doi.org/10.1016/j.jevs.2015.09.007 http://dx.doi.org/10.1016/j.jevs.2015.09.007 http://dx.doi.org/10.1016/j.jevs.2020.102991 http://dx.doi.org/10.1016/j.jevs.2020.102991 http://dx.doi.org/10.1016/j.jevs.2019.01.012 http://dx.doi.org/10.1016/j.jevs.2019.01.004 http://dx.doi.org/10.1016/j.jevs.2019.01.004 http://dx.doi.org/10.1111/j.2042-3306.1974.tb03952.x 2018: medical and surgical management of a case of chronic mastitisin a mare medical and surgical management of a case of chronic mastitis in a mare college of veterinary medicine, auburn university, auburn, al a 9-year old american paint mare was presented with an enlarged right mammary gland of greater than one year’s duration. the owner reported that the mare had been lactating since her last foaling three years previously. during examination, a thick cream-colored substance was expressed from the right mammary gland. culture and gram stain were negative for bacteria but a large number of degenerative neutrophils were present on cytology. the mare was diagnosed with galactorrhea and mastitis. treatment was initiated with daily milk removal and instillation of intramammary ceftiofur (spectra-mast®) for four days. the mare responded to treatment with resolution of mammary enlargement. nine months later, the mare returned with a large plaque of ventral edema. ultrasonography of the ventral abdomen revealed enlarged inguinal lymph nodes and fluid accumulation with diverse echogenicity within the mammary gland. cytology of fluid from the mammary gland showed suppurative inflammation and bacterial culture revealed nocardia organisms. bilateral mastectomy was performed under general anesthesia due to the history of chronic mastitis and failure of medical management alone. an hourglass-shaped incision was made, and blunt dissection was used to separate the gland at the body wall. postoperative care included systemic antibiotics, flunixin meglumine, and hydrotherapy. despite careful management, complete dehiscence occurred seven days later and the wound healed via second intention. at recheck examination seven weeks later, the surgical site was free of drainage and healing was adequate. this case documents an atypical case of mastitis in a mare. generally, mammary gland problems in the mare are uncommon.1 the most common signs are swelling, heat and ventral edema, and the most common organisms isolated are streptococcus spp.2,3 cytology and bacteriological testing are recommended to rule out other causes, such as trauma and neoplasia.4 medical management may include milking, intramammary or systemic antibiotics, hydrotherapy, or surgical excision as a last resort. keywords: equine, galactorrhea, mastectomy, mastitis references 1. mcgladdery aj: differential diagnoses and treatment of diseases of the equine mammary gland. equine vet educ 1998;21:266-268. 2. mccue pm, wilson wd: equine mastitisa review of 28 cases. equine vet j 1989;21:351-353. 3. smiet e, grinwis gc, van den top jg, et al: equine mammary gland disease with a focus on botryomycosis: a review and case study. equine vet educ 2012;24:357-366. 4. brendemuehl jp: mammary gland enlargement in the mare. equine vet educ 2008;20:8-9. clinical theriogenology • volume 10, number 3 • september 2018349 l.m. benningfield, r.r. wilborn, a.k. johnson, r.l. jensen, l.h. boone 1 contact anum ahmed soclean13@ufl.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2023, 15, 9379, http://dx.doi.org/10.58292/ct.v15.9379 case report pregnancy and spontaneous partial abortion associated with salmonella in a dog anum ahmed,a connor ellis,b audrey kellemana adepartment of large animal clinical sciences, bdepartment of small animal clinical sciences, university of florida college of veterinary medicine, gainesville, fl, usa abstract a 2-year mixed breed dog was presented on day 50 after breeding for an emergency condition (greenish brown vulvar discharge and abortion). the patient was underweight (lower body condition score), had nonfetid vulvar discharge, and was negative for ferguson reflux. patient had 10 premature fetuses (determined via transabdominal ultrasonography and radiography). serum progesterone concentration was 1.25 ng/ml. overnight hospitalization therapies included intravenous fluids, terbutaline, fenbendazole, altrenogest, progesterone, and antibiotics. based on vaginal discharge culture results (salmonella), antibiotics treatment was altered. three days later, neutrophilia with left shift and a fetal death were observed. because cesarean surgery with ovariohysterectomy was declined by the owner, altrenogest and terbutaline treatments were discontinued to permit whelping. patient was in dystocia after delivering 2 stillborn and 2 live pups. one pup was delivered medically and 1 necrotic pup via cesarean surgery. intrauterine culture (collected during surgery) was negative. on day 7 postpartum, the patient and 3 remaining pups were healthy. this case demonstrated the difficulties of managing fetal-placental infections. keywords: dog, pregnancy, fetal death, infection, dystocia background abortion is defined as the expulsion of a dead or a living fetus that is incapable of having independent life.1 abortions typically occur during the second half of pregnancy. other categories of pregnancy loss include early embryonic death up to 35  days (prior to fetal organogenesis) of pregnancy or stillbirth of mature dead fetus at term.1,2 causes of abortion are infectious (bacterial, viral, protozoal, or fungal) and noninfectious categories.3 duration of canine pregnancy can be defined in a number of ways, with the least accurate based on the number of days after a single mating (ranged from 57 to 72 days).2 this wide range is due to breeding early in estrus or late at the start of diestrus. final fetal maturation only occurs in the last 1 – 2 days of pregnancy. delivery of premature fetuses generally results in decline and/or neonatal death.2 challenges due to salmonella infection with the management of fetal-placental infections are described. case presentation a 2-year-old bernese mountain/standard poodle mixed breed (bernedoodle) pregnant dog was presented at a veterinary clinic on day 50 after a single natural breeding, for a brownish green vulvar discharge noticed earlier in the day. serum progesterone concentrations were 2.1 ng/ml. the dog was discharged, presuming that labor was imminent. later that evening, the dog aborted a premature fetus, 8 hours later, and was brought to the university emergency service. prior history included a normal pregnancy and whelping, neither travels nor contact with wildlife, and the dog had no predation behaviors. sire (bernedoodle) of this pregnancy was reportedly healthy with unknown brucellosis status. physical examination findings were underweight (body condition score of 3/9 [royal canin© scale])4 and a moderate amount of nonfetid dark brown vulvar discharge. a large number of fetuses (fetal heart rates ranged between 200 and 250/beats per minute [bpm]) were observed via focal transabdominal ultrasonography. serum progesterone concentration was 1.25 ng/ml. bloodwork findings included mild normocytic normochromic anemia (categorized regenerative due to anisocytosis and polychromasia), mild monocytosis, mild hypocalcemia, and mild hyponatremia. additional tests included leptospirosis witness test (witness® lepto, zoetis, parsippany, nj), brucella canis rapid slide agglutination test (d-tec® cb, zoetis), canine herpesvirus titer (colorado state university, fort collins, co), and snap 4dx testing soclean13@ufl.edu mailto:soclean13@ufl.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9379 2 citation line: clinical theriogenology 2023, 15, 9379, http://dx.doi.org/10.58292/ct.v15.9379 (anaplasma, ehrlichia, lyme, and heartworm) (snap® 4dx plus test, idexx, westbrook, me); all were negative. ten premature fetuses were counted (abdominal radiography), and their immaturity was determined based on the level of calcification, without evidence of fetal death (figure 1). an immediate cesarean surgery with ovariohysterectomy was recommended but was declined by the owner. as the patient appeared stable, outpatient therapy was discussed initially; however, another fetus aborted necessitating overnight hospitalization. therapies included intravenous lactated ringer’s solution (90 ml/kg/day [excel®, b. braun medical inc., bethlehem, pa]), subcutaneous terbutaline (0.03 mg/kg three times a day [brethine®, hikma pharmaceuticals plc, london, united kingdom]), oral altrenogest (0.088 mg/kg once a day [regu-mate®, merck animal health, rahway, nj]), oral amoxicillin and clavulanate potassium (14 mg/kg twice a day [clavamox®, zoetis, parsippany, nj]), oral clindamycin (11.8 mg/kg twice a day [cleocin®, lannett company, inc., philadelphia, pa]), and oral fenbendazole (50 mg/kg once a day for 3 days [panacur®, merck animal health, rahway, nj]). both aborted fetuses were submitted for necropsy, and were premature (lack of hair growth). next day physical examination findings remained unchanged. reexamination with targeted transabdominal ultrasonography confirmed many fetuses with heart rates above 200/bpm and poor development of fetal kidney parenchyma and gastrointestinal tract that indicated prematurity. manual digital examination of the vagina did not elicit either a visible or palpable ferguson reflex, and neither fetuses nor associated fetal membranes were present. moderate amount of brown mucoid discharge in the vagina prevented viewing of the cervix during vaginoscopy. there was neutrophilic inflammation without intracellular bacteria (figure 2) in the vaginal discharge cytology. a sample of the discharge obtained from the inside of the cranial portion of the vaginoscope (to decrease the risk for contamination) was submitted for aerobic culture. intramuscular progesterone in oil (9.8 mg/kg [progesterone injection usp, hikma pharmaceuticals plc, london, uk]) was given once. it was recommended to increase the caloric intake. due to financial constraints, the patient was discharged from the emergency service for continued outpatient care. outpatient reexamination was performed 2 days later (on day 53 after breeding). nonfetid brown vulvar discharge continued (the rest of the examination was unremarkable). upon further inquiry, the dog, reportedly, had been hyporexic throughout pregnancy, and the diet was supplemented with boiled chicken breast and fried eggs. patient also had an increase in thirst. a free-catch urine specific gravity was 1.008 (> 1.030).5 due to borderline hyposthenuria that can be identified in pyometra, a renal panel was performed, which revealed that the dog was not azotemic (table). progesterone concentrations were 6.24 ng/ml. multiple viable fetuses and 1 fetus without a heartbeat were observed via targeted transabdominal ultrasonography. air shadowing surrounding the fetus (figure 3) indicated fetal demise. final aerobic culture of the vaginal discharge grew salmonella sp., with subspeciation and serotype pending. based on culture and sensitivity results, clindamycin (cleocin®, lannett company, inc.) was discontinued, and oral ciprofloxacin (30 mg/kg once a day [cipro®, bayer healthcare pharmaceuticals inc., wayne, nj]) was prescribed. in spite of fetal demise, as the patient was clinically stable, further outpatient management continued. the dog was presented for an outpatient reexamination once more (on day 56 after breeding). the dog had 2 episodes of vomiting since prior visit and was trembling in the car for an hour prior to the appointment. the dog had lost 0.5 kg since prior visit, was tachycardic (160/bpm), had mild erythema of the skin and ear pinnae, and was mildly hyperthermic (39.4°c). there was continued vulvar discharge and initial mammary secretions from a few teats, consistent with colostrum. due to the concern for systemic inflammatory response syndrome (sirs)6 on clinical examination (elevated temperature and tachycardia), bloodwork was performed. patient had moderate normocytic normochromic anemia of pregnancy, with a marked neutrophilia and a left shift, and mild monocytosis. a mild hypoglycemia and mild hypocalcemia were also noted (table). progesterone concentrations were lower (1.10 ng/ml). targeted transabdominal ultrasonography revealed similar findings as before (multiple normal viable fetuses and 1 dead fetus [figure 4]). further maturation of the live fetuses was evident by slight distinction of the cortical and medullary parenchyma of fetal kidneys (figure 5) and a more thickening of gastrointestinal tract layers in two of the fetuses7 (figure 6). given the patient’s deterioration and concerning laboratory findings, immediate cesarean surgery with ovariohysterectomy figure 1. right lateral abdominal radiograph (50 days after breeding), note the 10 fetuses and poor calcification (immaturity). figure 2. vaginal discharge cytology (note degenerate neutrophils), 1000× total magnification. http://dx.doi.org/10.58292/ct.v15.9379 citation line: clinical theriogenology 2023, 15, 9379, http://dx.doi.org/10.58292/ct.v15.9379 3 was again recommended; however, surgery was declined. recommendation was made to discontinue terbutaline (brethine®, hikma pharmaceuticals plc) and altrenogest (regu-mate®, merck animal health), to allow the dog to whelp at home. amoxicillin and clavulanate potassium (clavamox®, zoetis) and ciprofloxacin (cipro®, bayer healthcare pharmaceuticals inc.) were continued. whelping, dystocia, and cesarean surgery a last dose of altrenogest (regu-mate®, merck animal health) was given at home the following morning (day 57 after breeding). later that night, the dog delivered a stillborn fetus. the dog then went into spontaneous labor ~ 60 hours after the last dose of altrenogest (regu-mate®, merck animal health) and delivered 1 live pup. subsequently, the dog was given an unknown dose of oral calcium (oral cal plus™ gel, breeder’s edge®, orange city, ia) at home by the owners. three hours passed prior to delivery of another live pup. forty-five minutes later, a stillborn fetus was delivered. contractions failed to progress. the dog was presented for emergency management of dystocia (day 59 after breeding). the dog had lost 2.5 kg, moderate amount of nonfetid brown vulvar discharge, and colostrum secretions in all teats. a mild ferguson reflex was present on digital vaginal examination, and fetal membranes were visualized at the cervix via vaginoscopy. targeted transabdominal ultrasonography was performed; there was 1 dead fetus, 1 with a normal heart rate (240/bpm), and one with a critically lower heart rate (120/bpm). blood gas analysis revealed mild anemia and normal ionized calcium concentrations (table). cesarean surgery was recommended due to evidence for severe fetal distress. owner, however, declined surgery due to purported concern of the current welfare of the patient than of the fetuses and requested medical therapy for dystocia. monitoring fetal heart rates (via ultrasonography) and ensuring no obstruction (via vaginal examination prior to each injection), 3 serial doses (starting at 2 iu, then table. complete blood count, serum chemistry, venous blood gas panel, and progesterone concentrations on days 50, 53, 56, and 59 after mating test day 50 after mating day 53 after mating day 56 after mating day 59 after mating reference range hematocrit (calculated) 34 n/a 26.4 30.6 40.0–56.0% neutrophils n/a n/a 19.64 n/a 2.7–8.9 k/µl band neutrophils n/a n/a 5.86 n/a 0 k/µl monocytes n/a n/a 2.9 n/a 0.1–0.8 k/µl glucose 87 73 73 n/a 78–124 mg/dl calcium (total) n/a n/a 8.2 n/a 8.7–10.4 mg/dl calcium (ionized) 1.17 n/a n/a 1.37 1.18–1.35 mmol/l blood urea nitrogen n/a 11 n/a n/a 7–27 mg/dl creatinine n/a 0.58 n/a n/a 0.6–1.5 mg/dl progesterone 1.25 6.24 1.10 n/a 0.0–0.4 ng/ml figure 3. transabdominal ultrasonographic image of fetal demise, note the hyperechoic foci around fetus (air shadowing), and fetus is poorly defined due to autolysis. figure 4. transabdominal ultrasonographic image of live (star) and autolyzed (triangle) fetuses. http://dx.doi.org/10.58292/ct.v15.9379 4 citation line: clinical theriogenology 2023, 15, 9379, http://dx.doi.org/10.58292/ct.v15.9379 increasing to 3 iu and 5 iu with each subsequent dose) of  intramuscular oxytocin (oxytocin, vetone, ontario, canada) were given every 20 – 30 minutes. doses were increased with each injection until a notable clinical response of contractions was observed. subcutaneous 2 ml calcium gluconate 10% (fresenius kabi, lake zurich, il) diluted with sterile saline was given every 2 hours to aid uterine contractions. one live pup with meconium staining was delivered. a second round of medical management with oxytocin (oxytocin, vetone) injections every 20 – 30 minutes was performed starting at a 5 iu dose, but contractions failed to continue. as a result, cesarean surgery was approved by the owner. one live pup and one necrotic fetus were delivered via hysterotomy. uterus contained a large amount of thick black fluid, and a sample was submitted for aerobic culture. uterus was copiously lavaged prior to closure. nine fetuses were accounted for (2 aborted, 4 whelped at home, one delivered vaginally at hospital, and two delivered via cesarean surgery) out of an initial total of 11 (10 observed via abdominal radiology and one aborted prior to presentation). there was a large amount of gas in the abdomen (introduced intraoperatively) with no evidence of fetal calcification in the reproductive tract or the gastrointestinal tract (radiography) that would have suggested fetal ingestion by the dog at home. pups nursed, urinated, and defecated prior to discharge. amoxicillin and clavulanate potassium (clavamox®, zoetis) and ciprofloxacin (cipro®, bayer healthcare pharmaceuticals inc.) were continued for 2 weeks. additionally, the dog was prescribed medications as needed: oral tramadol (4.4 mg/ kg twice a day for 5 days [amneal pharmaceuticals llc, glasgow, ky]), subcutaneous oxytocin (three times a day for 10 days [vetone]), and oral metoclopramide (0.22 mg/kg three times a day for 5 days [teva pharmaceuticals, parsippany, nj]). due to risk of fetal infection, the pups were prescribed oral trimethoprim sulfa (10 mg/kg twice a day for 2 weeks [aurobindo pharma, dayton, nj]), subcutaneous ceftiofur sodium (2.5 mg/kg twice a day for 2 weeks [naxcel®, zoetis, parsippany, nj]), and subcutaneous 0.9% nacl (0.9% sodium chloride injection usp, b. braun medical inc., bethlehem, pa) 3 – 5 ml per pup as needed. outcome dam and litter were presented 1 week postpartum. one neonate died 2 days postpartum. the dam was eating well, there were no concerns with incision site, and pups were nursing well and were gaining weight. physical examination was unremarkable. intraoperative aerobic culture (uterine fluid) had no growth. cornell abortion panel had lower titer (1:50) suggesting exposure to toxoplasma gondii. second titer was recommended to confirm active infection but was declined. a free-catch urine was concentrated (specific gravity >1.050). fetal necropsies and placental histopathology were consistent with neutrophilic chorioplacentitis, associated with gram negative rods (figure 7). partial abortion was attributed to salmonella enterica subsp. houtenae serotype iv 50:z4,z23:(flint). three live pups were recovered from a total of 11  fetuses. the dam was healthy and cleared of the infection based on clinical signs and negative uterine culture. figure 5. transabdominal ultrasonographic image of a canine fetal kidney (outlined with caliper), note the degree of architectural detail indicates progression of fetal maturity. figure 7. histopathology of fetal chorionic membrane obtained at abortion, note the rod-shaped (bacilli), 1000× total magnification. figure 6. transabdominal ultrasound image of a fetal gastrointestinal tract, note the architectural detail indicating progression of fetal maturity. http://dx.doi.org/10.58292/ct.v15.9379 citation line: clinical theriogenology 2023, 15, 9379, http://dx.doi.org/10.58292/ct.v15.9379 5 discussion this case demonstrated the challenges associated with the management of severe canine pregnancy infections. escherichia coli is the most common bacterial organism to cause uterine infection in dogs.3 though uncommon, salmonella sp. can also cause uterine infections leading to abortions. salmonella is a mobile, nonspore forming gram negative bacteria of the family enterobacteriaceae. there are multiple subspecies and serovars present worldwide: s. panama was isolated from aborted pups8 and s. typhimurium from newborn kittens.8 mode of transmission is most commonly by fecal-oral route, as the bacteria are shed through feces and through contact with fomites or vertical transmission in avian and reptile species.8 this bacteria is of public health importance, as it is a zoonotic pathogen.8,9 dogs are typically asymptomatic when infected or have gastrointestinal signs of diarrhea, vomiting, and/or anorexia. rare, but severe, in-utero infections in dogs result in fever, abortion, stillbirth, weak neonates, or death.8,9 historically, antibiotics used successfully for therapy for salmonellosis included amoxicillin, enrofloxacin, trimethoprim-sulfonamide, and chloramphenicol. it is a good practice to decide antibiotic choice based on culture and sensitivity.9 salmonella enterica was recovered in 11.9% of environmental isolates, 5% of raw meat feeds, 14% of raw meat-fed dog fecal samples, and 10.5% and 4.5% from vacuum cleaner waste samples from households where dogs were and were not fed raw meat, respectively.9 salmonella enterica subsp. houtenae (salmonella subspecies iv) was initially isolated from cockatiels and now is prevalent in many reptile species, raw poultry, and racoons.10 infection in children can lead to meningitis.10 in another recent case (university of florida) with the same outcome of a dog abortion, an organism from salmonella subspecies iv was isolated in a dog that consumed raw beef and lizards.11 the bacteria was not further speciated in that case. dog in that prior study did not have gastrointestinal signs as in this case, but instead was febrile and hyporexic. to our knowledge, this is only the second reported canine abortion caused specifically by salmonella subspecies iv. in this case, the owner reported that the dog frequently drank water from a well in the backyard, inhabited with lizards and other reptiles, that could have served as a potential salmonella reservoir. another possible source of infection was from improperly cooked chicken and eggs that were fed during pregnancy. poultry kept at temperatures between 7 and 47°c allowed growth of salmonella sp.9 although a final etiology of salmonella was determined in this case, the definitive source of the organism remains unclear. diagnostics and early treatment for infections in pregnant dogs are crucial in efforts to provide a positive case outcome. aerobic culture revealed salmonella sp., and paired with sensitivity results, allowed for appropriate antibiotic choice. during pregnancy, certain antibiotics are harmful to the dog and/or fetuses. fluoroquinolone class caused cartilage damage in canine fetuses;12,13 however, cartilage lesions in equine fetuses were not identified after fluoroquinolone treatment between 46 and 60 days of pregnancy.12 in this case, ciprofloxacin was prescribed as the bacteria was susceptible to fluoroquinolones. although ovariectomy was indicated (welfare of the dog), owner declined. amoxicillin and clavulanate potassium (a beta lactam) and clindamycin (a lincosamide) used were safe during pregnancy.13 apart from antimicrobial therapy, additional medications were given to the dog during pregnancy. fenbendazole was given as an anthelmintic. terbutaline was given in an effort to prevent uterine contractions during pregnancy. as there was systemic infection present, it was assumed that the fetuses would have congenital neonatal sepsis, and thus, treatment was initiated at birth. ceftiofur sodium is a beta-lactam antibiotic that is labeled for dogs with a wide margin of safety in neonates.14 this salmonella sp. was also susceptible to trimethoprim based on the antibiogram; however, a lower dose was used for the neonates than what is labeled for dogs due to underestimated pup birth weights. additionally, risk with use of this antibiotic included transient hypothermia and bone marrow suppression.14–16 maintenance of pregnancy is dependent on progesterone production and secretion from the corpora lutea (cl). systemic inflammation caused cl lysis and decreased progesterone concentrations.2 progesterone or progestogens can be given in these cases to maintain pregnancy and prevent abortion. altrenogest is an oral, short-acting, synthetic progestogen given daily.17 this medication can be given at home by owners. altrenogest maintained pregnancy in ovariectomized dogs.18 progesterone in oil is an injectable, more rapid, and longer acting formulation that is typically given every 72 hours in hospital when there is an imminent risk for pregnancy loss due to hypoluteoidism.17 altrenogest does not cross-react with progesterone assays, whereas progesterone in oil is readily measured by the assay.18 on day 5 prepartum, progesterone concentrations (determined via radioimmunoassay) in seven normal dogs ranged between 2.6 and 7.8 ng/ml.19 progesterone concentrations in the morning diestrual dogs’ blood samples were typically double compared to afternoon samples.20 progesterone concentrations are measured either via radioimmunoassay or chemiluminescence; the latter concentrations were ~ 85% of those of the radioimmunoassay.21 in the present case, progesterone concentrations were measured in the early afternoon via chemiluminescence; both may account for the lower concentrations identified around 1 week prepartum. additionally, the dog was receiving altrenogest supplementation, and it is unknown if a diurnal pattern occurs in dogs given exogenous progesterone or progestogen. differences between pre and postprandial progesterone concentrations in dogs may also affect these results, as a substantial decrease in concentrations was observed 1 hour after feeding in pregnant dairy cows.22 progesterone in oil treatment provided quick hormone replacement initially and then was maintained with oral altrenogest. risk factors for salmonellosis include a weakened immune system, improper diet including raw foods, and poor hygienic conditions.8 canine pregnancy is maintained by elevated progesterone concentrations.2 progesterone in women created an immunosuppressed state that made pregnant women more susceptible to infections.23 similarly, this is speculated to occur in pregnant dogs, with steroid hormones suppressing neutrophilic activity.24 client education is important to implement hygiene protocols in the household and prevent zoonosis in pets with known infections. best practices include wearing gloves, frequent hand washing, and isolation from those in the household. having a designated area in the yard for the infected animal to urinate and defecate is recommended. people, especially pregnant women, should avoid contact with feces from infected animals at home to prevent zoonotic spread.25 learning points • salmonella is not a common organism identified in utero-placental infections in dogs. • salmonella is a gram-negative, zoonotic bacteria prevalent worldwide that typically causes asymptomatic http://dx.doi.org/10.58292/ct.v15.9379 6 citation line: clinical theriogenology 2023, 15, 9379, http://dx.doi.org/10.58292/ct.v15.9379 infections in dogs; however, treatment is required in severe cases. • culture and sensitivity testing is important in guiding appropriate antibiotic therapy; however, in the case of salmonellosis, fluoroquinolones use during pregnancy may be associated with neonatal arthropathy. • progesterone or a synthetic progestogen (e.g. altrenogest) treatment in cases of hypoluteoidism can maintain pregnancy. • in pregnancy infections, establishing a priority to manage either dam or fetal health is necessary, if a positive outcome is not expected for both. acknowledgment authors thank dr. randall walker (department of veterinary pathology) for gross necropsy and histopathological examinations. conflict of interest and funding anum ahmed’s residency program was funded by the american kennel club/canine health foundation/ theriogenology foundation grant. authors report no conflict of interest. references 1. johnson ca: high-risk pregnancy and hypoluteoidism in the bitch. theriogenology 2008;70:1424–1430. doi: 10.1016/j. theriogenology.2008.09.010 2. johnston sd, root kustritz mv, olson pn: canine pregnancy. in: kersey r, lemelledo d, eds. canine and feline theriogenology. philadelphia, pa: saunders; 2001, pp. 66–104. 3. pretzer sd: bacterial and protozoal causes of pregnancy loss in the bitch and queen. theriogenology 2008;70:320–326. doi: 10.1016/j.theriogenology.2008.04.035 4. purina. body condition system. purina. available from: https:// www.purina.com/sites/g/files/auxxlc196/files/2021-01/body condition_dog_122120.pdf [cited 26 april 2022]. 5. iris international renal interest society. urine specific gravity. iris international renal interest society. available from: http:// www.iriskidney.com/education/urine_specific_gravity.html#:~:text=what%20usg%20values%20are%20considered,1.035%20 to%201.060%20for%20cats [cited 29 april 2022]. 6. pierini a, gori e, lippi i, et al: neutrophil-to-lymphocyte ratio, nucleated red blood cells and erythrocyte abnormalities in canine  systemic inflammatory response syndrome. res vet sci. 2019;126:150–154. doi: 10.1016/j.rvsc.2019.08.028 7. nyland tg, mattoon js: ovaries and uterus. in: callen pw, ed. small animal diagnostic ultrasound. saint louis, mo: wb saunders co.; 2002, pp. 231–249. 8. dróżdż m, małaszczuk m, paluch e, et al: zoonotic potential and prevalence of salmonella serovars isolated from pets. infect ecol epidemiol 2021;11:1–20. doi: 10.1080/20008686.2021.1975530 9. cobb ma, stavisky j: salmonella infections in dogs and cats. in: barrow pa, methner u, eds. salmonella in domestic animals. 2nd ed. loughborough: cab international; 2013, pp. 318–336. 10. andruzzi mn, krath ml, lawhon sd, et al: salmonella enterica subspecies houtenae as an opportunistic pathogen in a case of meningoencephalomyelitis and bacteriuria in a dog. bmc vet res 2020;16:1–5. doi: 10.1186/s12917-020-02652-5 11. allen-durrance a, mazzaccari km, woliver cl: bacteremia and late-term abortion secondary to salmonellosis in a dog. j am anim hosp assoc 2022;58:262–264. doi: 10.5326/ jaaha-ms-7237 12. ellerbrock re, canisso if, podico g, et al: diffusion of fluoroquinolones into equine fetal fluids did not induce fetal lesions  after enrofloxacin treatment in early gestation. vet j 2019;253:1–7. doi: 10.1016/j.tvjl.2019.105376 13. rebuelto m, loza me: antibiotic treatment of dogs and cats during pregnancy. vet med int 2010;2010:1–8. doi: 10.4061/2010/385640 14. mata j, papich mg: pharmacologic considerations in the young patient. in: peterson me, kutzler ma, eds. small animal pediatrics: the first 12 months of life. saint louis, mo: saunders; 2011, pp. 232–243. 15. frank la, hnilica ka, may er, et al: effects of sulfamethoxazole-trimethoprim on thyroid function in dogs. am j vet res 2005;66:256–259. doi: 10.2460/ajvr.2005.66.256 16. mcmichael m, dhupa n: pediatric critical care medicine: physiological considerations. compend contin educ vet 2000;22: 206–214. 17. root kustritz mv: use of supplemental progesterone in management of canine pregnancy. in: concannon pw, england g, verstegen j, iii, et al: eds. recent advances in small animal reproduction. ithaca, ny: international veterinary information service; 2001. 18. eilts be, paccamonti dl, hosgood g, et al: the use of ally-trenbolone as a progestational agent to maintain pregnancy in ovariectomized bitches. theriogenology 1994;42:1237–1245. doi: 10.1016/0093-691x(94)90872-9 19. concannon pw, butler wr, hansel w, et al: parturition and lactation in the bitch: serum progesterone, cortisol and prolactin. biol reprod 1978;19:1113–1118. doi: 10.1095/biolreprod19.5.1113 20. steinetz bg, goldsmith lt, hasan sh, et al: diurnal variation of serum progesterone, but not relaxin, prolactin, or estradiol-17β in the pregnant bitch. endocrinology 1990;127:1057–1063. doi: 10.1210/endo-127-3-1057 21. nothling jo, de cramer kgm: comparing the values of progesterone in the blood of bitches as measured with a chemiluminescence immunoassay and a radioimmunoassay. reprod domest anim 2018;53:1136–1141. doi: 10.1111/rda.13216 22. vasconcelos jlm, sangsritavong s, tsai sj, et al: acute reduction in serum progesterone concentrations after feed intake in dairy cows. theriogenology 2003;60:795–807. doi: 10.1016/ s0093-691x(03)00102-x 23. lin vh, chen j, liao c, et al: the rapid immunosuppression in phytohemagglutinin-activated human t cells is inhibited by the proliferative ca2+ influx induced by progesterone and analogs. steroids 2016;111:71–78. doi: 10.1016/j.steroids.2016.01.010 24. bartoskova a, ondrackova p, leva l, et al: the effects of in vitro exposure to progesterone and estradiol-17β on the activity of canine neutrophils. vet med (praha) 2014;59:202–209. doi: 10.17221/7481-vetmed 25. hertzer jn, fujishiro m, lawhon sd, et al: treatment and management of salmonella prostatitis in a heartworm-positive intact male dog: a case report. bmc vet res 2021;17:1–6. doi: 10.1186/ s12917-021-02836-7 http://dx.doi.org/10.58292/ct.v15.9379 https://doi.org/10.1016/j.theriogenology.2008.09.010 https://doi.org/10.1016/j.theriogenology.2008.09.010 https://doi.org/10.1016/j.theriogenology.2008.04.035 https://www.purina.com/sites/g/files/auxxlc196/files/2021-01/bodycondition_dog_122120.pdf https://www.purina.com/sites/g/files/auxxlc196/files/2021-01/bodycondition_dog_122120.pdf https://www.purina.com/sites/g/files/auxxlc196/files/2021-01/bodycondition_dog_122120.pdf www.iriskidney.com/education/urine_specific_gravity.html#:~:text=what%20usg%20values%20are%20considered,1.035%20to%201.060%20for%20cats www.iriskidney.com/education/urine_specific_gravity.html#:~:text=what%20usg%20values%20are%20considered,1.035%20to%201.060%20for%20cats www.iriskidney.com/education/urine_specific_gravity.html#:~:text=what%20usg%20values%20are%20considered,1.035%20to%201.060%20for%20cats https://doi.org/10.1016/j.rvsc.2019.08.028 https://doi.org/10.1080/20008686.2021.1975530 https://doi.org/10.1186/s12917-020-02652-5 https://doi.org/10.5326/jaaha-ms-7237 https://doi.org/10.5326/jaaha-ms-7237 https://doi.org/10.1016/j.tvjl.2019.105376 https://doi.org/10.4061/2010/385640 https://doi.org/10.2460/ajvr.2005.66.256 https://doi.org/10.1016/0093-691x(94)90872-9 https://doi.org/10.1095/biolreprod19.5.1113 https://doi.org/10.1210/endo-127-3-1057 https://doi.org/10.1111/rda.13216 https://doi.org/10.1016/s0093-691x(03)00102-x https://doi.org/10.1016/s0093-691x(03)00102-x https://doi.org/10.1016/j.steroids.2016.01.010 https://doi.org/10.17221/7481-vetmed https://doi.org/10.1186/s12917-021-02836-7 https://doi.org/10.1186/s12917-021-02836-7 introduction improvement of offspring genetic health begins with an understanding of the selection tools that are available and the ethical responsibilities to use these tools in assisting with breeding decisions.1 discovery of genetic markers of disease in companion animals and the advent of readily available at-home testing kits increased testing for genetic disease in companion animals.2 dog’s breed identification testing is performed to fulfill owner’s interest and curiosity. however, knowing that their pet carries a genetic mutation for a specific medical issue might have important clinical implications for the future care of the pet. although the ability to estimate an individual animal’s risk of genetic disease has become increasingly available,3,4 accessible resources to navigate test interpretation and clinical implications of the findings might be inadequate, leaving owners and veterinarians with many unanswered questions.5,6 veterinarians, especially those working in general practice or having an interest in theriogenology, are often the first line of contact for evaluation of an animal suspected for a familial disease or a genetic problem. genetic diseases are of significant concern for veterinarians and their patients. a genetic online veterinary counseling service was created to provide a forum so that the complex nature of genetic disease could be better navigated and discussed. to the authors’ knowledge, this is the first database of its kind providing a free, readily available counseling service. services are provided by a university veterinary genetics laboratory in conjunction with the theriogenology services of 2 veterinary colleges. goals are to provide information regarding familial diseases, specific mutations, and genetic test interpretation to veterinarians, pet breeders and owners. veterinary genetic counseling service: a preliminary report katie withowski,a alyssa helms,b victor stora,c kathryn meursa adepartment of veterinary clinical sciences, north carolina state university, raleigh, nc bdepartment of small animal clinical science, virginia-maryland college of veterinary medicine, blacksburg, va creferral center for animal models of human genetic disease, university of pennsylvania, philadelphia, pa abstract data generated from an online veterinary genetics counseling service from november 1, 2018 to october 31, 2019 were reviewed. data were tabulated regarding the individual submitting the inquiry and relationship to the pet, species, breed, and medical issue(s) thought to be familial. inquiries were assigned to 1 or more of the following: risk assessment and heritability, pre-breeding testing and pedigree review, and information regarding a specific mutation or minor health concern. this study demonstrated that clients are desirous for information that could help promote health in canine companions. furthermore, there is an interest and need for additional genetic resources for both veterinarians and owners. keywords: canine, genetic counseling, heritability, genetic testing objective of this paper was to review 1 year data generated from this service and provide a summary of the types of questions and areas of concerns that veterinarians and owners submitted regarding familial diseases. materials and methods consultations were provided by a team including a veterinarian with a phd in genetics, a veterinarian who had completed a medical genetics residency, and 2 theriogenology residents in combined residency/medical genetics programs. the service was promoted both through its location on the genetic testing website (https://cvm.ncsu.edu/nc-state-vet-hospital/small-animal/ genetics/) and through the american kennel club-canine health foundation. consultation requests from november 1, 2018 until october 31, 2019 were gathered and tabulated. information on the individual submitting the inquiry and their relationship to the pet was obtained. the term breeder was defined as any person who owns or plans to own an animal intended for breeding, or has an animal already used to produce progeny. pet owner was defined as any person who owns or plans to own an animal not intended for breeding purposes. a veterinarian acted on behalf a client where a valid veterinary-client-patient relationship existed. patient demographics including species, breed, and medical issue(s) recognized as familial were collected. each inquiry was assigned to 1 or more of the following: risk assessment and heritability estimates, pre-breeding testing, pedigree record review, information regarding a specific mutation (either to determine presence or have results analyzed), and minor clinical theriogenology 2021; 13: 77 health concern (including coat color, breed make-up, and age). results thirty-five submissions (table 1) were received and evaluated through the online genetic testing portal during the 12-month interval. over half (63 %; 22/35) of the submissions were from pet breeders with 2 of those indicating breed club affiliation with the intent to distribute knowledge to their respective breed club. of the remaining submissions, 20% (7/35) were pet owners with questions regarding an individual animal and 17% (6/35) were veterinarians submitting on behalf of a client with questions regarding an individual animal, unrelated to breeding. although the service was provided for all species, all submissions were for dogs. inquires represented dogs in 23 american kennel club recognized breeds, and 4 mixed breed, 2 non-american kennel club breeds, and 1 foundation stock service breed. overall, 75% (26/35) of the inquiries were related to breeding an animal and the impact of a familial trait on the breed overall and 25% (9/35) were inquiries regarding the impact of a trait on an individual pet animal not intended for breeding. inquiries regarding risk assessment and heritability were most common (17 inquiries), followed by pre-breeding testing and pedigree record review (9), information regarding a specific mutation (either to determine presence or have results analyzed (6), and of minor health concern (6 [including coat color, breed makeup, and age]). distribution of inquiries is summarized (figure). genetic consultations are categorized by organ system (table 2). largest number of inquiries included questions regarding neurologic, musculoskeletal, and ophthalmic diseases or disorders. although kinked tails and umbilical hernias are seemingly relatively benign issues, kinked tails could be indicative of other spinal abnormalities and could be disqualifications in conformation showing for certain breeds. similarly, umbilical hernias breeds represented number of inquiries topic of inquiry labrador retriever 4 ocular certification; hepatitis with copper accumulation; hemangiosarcoma; coat color mixed breed 4 coat color; age testing; breed identification & dilated cardiomyopathy; deafness australian shepherd 2 breed screening pre-breeding & coat color; degenerative myelopathy golden retriever 2 cataracts american bully 1 breed screening pre-breeding beagle 1 degenerative myelopathy bearded collie 1 coat color black russian terrier 1 epilepsy boston terrier 1 deafness cairn terrier 1 breed screening pre-breeding cavalier king charles spaniel 1 midline defect (cleft palate), kinked tail, umbilical hernia & hip dysplasia chinese shar-pei 1 persistent right aortic arch & megaesophagus clumber spaniel 1 midline defect (bifid nose) & umbilical hernia doberman pinscher 1 dilated cardiomyopathy & von willebrand’s disease giant schnauzer 1 hypothyroidism great dane 1 polycythemia vera labradoodle 1 hypoadrenocorticism leon berger 1 hip dysplasia miniature schnauzer 1 breed screening pre-breeding norwich terrier 1 norwich terrier pembroke welsh corgi 1 degenerative myelopathy & cystic endometrial hyperplasia-pyometra complex portuguese water dog 1 gm1-gangliosidosis gm1-gangliosidosis 1 breed screening pre-breeding standard poodle 1 keratoconjunctivitis sicca & hypoadrenocorticism vizsla 1 cataracts whippet 1 lung lobe torsion, skin allergy & hemangiosarcoma table 1. number of inquiries for each breed and topic of inquiry clinical theriogenology 2021; 13: 78 might have a hereditary basis and could have larger implications regarding a dam’s ability to carry a litter to term successfully. discussion the majority of inquiries to the genetics consultation service were from individuals self-identified as dog breeders; however, almost 40% of the inquiries came from dog owners or veterinarians requesting information regarding an individual pet indicating increased interest in familial disease beyond that needed for breeding purposes. although inquiries were made most commonly regarding purebred dogs, mixed breed dogs were also represented. there were zero inquiries regarding familial health issues in cats or other species. this could simply be a reflection of promotion of the program through the american kennel club-canine health foundation. however, it could also be indicative of a lesser understanding or interest in familial disease in the cat. promotion of this tool to cat breeders and cat owners might be beneficial to draw attention to feline familial health concerns. there were 4 inquiries regarding mixed breed dogs indicating an interest in genetics and familial disease, even in mixed breed dog populations. the most common disease or disorder inquiries appeared to parallel the commonality of diseases that occur across canine population and included: cataracts, degenerative myelopathy, deafness, endocrine disease, cancer (hemangiosarcoma), and dilated cardiomyopathy.8,9 however, some inquiries were in diseases to be familial (lung lobe torsion) or without a well-known breed predilection for an issue. this might be associated with an increased interest and curiosity regarding genetics as a cause of medical issues even in situations where it is not clearly a familial issue. questions regarding risk assessment, heritability and pre-breeding screening were most common. however, questions regarding genetic test interpretation were also frequent and illustrate the important need for increased availability and promotion for additional guidance in this area. this study demonstrated the interest and need for additional genetic resources for veterinarians and owners who have access to genetic tests. careful use of genetic information (refer ‘selected genetic resources for veterinarians and clients’) might help to prevent or decrease disease occurrence in companion animals. however, the impact of genetic testing on animal health is likely dependent on the owner and veterinarian understanding the genetic information obtained from the test and its possible applications.10 as these records imply, clients are interested in information that could help promote the familial health of their canine companions. as concerted efforts are made in implementing canine genetic counseling recommendations, a shift in the health of dogs might be incrementally improved with both time and knowledge.1 conflict of interest none to report. figure. distribution of inquires organ system disease, disorder or condition of concern neurologic degenerative myelopathy3 deafness2 gm1-gangliosidosis epilepsy ophthalmic cataracts3 keratoconjunctivitis sicca digestive hepatic fibrosis megaesophagus hepatitis with copper accumulation respiratory lung lobe torsion urogenital / reproductive cystic renal dysplasia ectopic ureter cystic endometrial hyperplasia-pyometra complex endocrine hypoadrenocorticism2 hypothyroidism musculoskeletal midline defects2 umbilical hernia kinked tail hip dysplasia cardiovascular dilated cardiomyopathy2 persistent right aortic arch integumentary skin allergies other cancer risk – hemangiosarcoma2 von willebrand’s disease polycythemia vera table 2. genetic consultations categorized by organ system (number within parenthesis indicates total consults) clinical theriogenology 2021; 13: 79 acknowldgements authors thank dr. diane brown for her vision of a greater understanding of familial disease in the dog and the application of this knowledge to the improvement of canine health. selected genetic resources for veterinarians and clients orthopedic foundation for animals/ canine health information center database, statistics by breed, disease overview, dna tests, research, health screening clinic search https://www.ofa.org/ canine health foundation review of the current state of genetic testing in dogs, a living resource https://www.akcchf.org/educationalresources/library/articles/review-of-thecurrent-state.html the international partnership for dogs (in collaboration with chf, wasava and others) harmonization of genetic testing for dogs by breed, disease or test, labs https://dogwellnet.com/ctp/ uc davis veterinary genetics laboratory dna testing https://vgl.ucdavis.edu/ upei cidd database canine inherited disorders database https://cidd.discoveryspace.ca/index.html upenn penngen dna testing https://www.vet.upenn.edu/research/ academic-departments/clinical-sciencesadvanced-medicine/research-labs-centers/ penngen references 1. bell, js: genetics based selection pressure to breed better, healthier dogs. clinical theriogenology 2012;527-534. 2. traas am, casal m, haskins m, et al: genetic counseling in the era of molecular diagnostics. theriogenology 2006;66:599-605. 3. karlsson ek, lindbald-toh k: leader of the pack: gene mapping in dogs and other model organisms. nat rev genet 2008;9:713-725. 4. plassais j, kim j, davis bw, et al: whole genome sequencing of canids reveals genomic regions under selection and variants influencing morphology. nat commun 2019;10:1489. 5. fowler kj, sahhar m, tassicker r: genetic counseling for cat and dog owners and breeders – managing the emotional impact. j am vet med assoc 2000;216:498-501. 6. shaffer l: special issue on canine genetics: animal models for human disease and gene therapies, new discoveries for canine inherited diseases, and standards and guidelines for clinical genetic testing for domestic dogs. hum genet 2019;38:437-440. 7. akc staff: may 2020. the most popular dog breeds of 2019. american kennel club. 05 april 2020. https://www.akc.org/expert-advice/ dog-breeds/2020-popular-breeds-2019. 8. bellumori tp, famula tr, bannasch dl: prevalence of inherited disorders among mixed-breed and purebred dogs: 27,254 cases (1995–2010). j am vet med assoc 2013;242:1549-1555. 9. ostrander ea, wayne rk, freedman ah: demographic history, selection and functional diversity of the canine genome. nat rev 2017;18:705-720. 10. metallinos, dl: canine molecular genetic testing. vet clin north am small anim pract 2001;31:421-431 clinical theriogenology 2021; 13: 80 single layer density centrifugation improved semen quality in a stallion with hydrocele sónia macedo,a,b ana filipe,a tiago guimarães,a,b antónio rochaa,b ainstituto de ciências biomédicas abel salazar, universidade do porto, porto, portugal bcentro de estudos de ciência animal, porto, portugal abstract hydrocele was diagnosed in a 21 year old stallion with a history of subfertility. subjective progressive motility and normal sperm from ejaculates (n = 12) were (mean ± sem) 25 ± 11% and 24 ± 7%, respectively. from each ejaculate,1 sample was centrifuged through a silica colloid and refrigerated for 72 hours. an uncentrifuged sample served as control. morphologically normal sperm was higher (p < 0.05) in semen centrifuged through the silica colloid. sperm motility in cool-stored semen was higher (p < 0.05) in silica colloid centrifuged semen compared to control at 24, 48, and 72 hours postrefrigeration. percent viable sperm and sperm with high mitochondria membrane potential were higher (p < 0.05) at 24 hours of postrefrigeration for semen centrifuged through silica colloid. three mares were inseminated at alternate estrus, either with semen centrifuged through the silica colloid or with control. inseminations (n = 3) with uncentrifuged semen (≥ 500 x 106 motile sperm) did not result in pregnancy. however, inseminations with fresh semen (n = 2) or 24 hours chilled semen (n = 2) obtained after centrifugation with single layer colloid (≥ 170 x 106 motile sperm) resulted in pregnancies. we concluded that hydrocele had apparently contributed to decreased semen quality and fertility in this stallion, and centrifugation of the ejaculate through a silica colloid improved semen quality. keywords: stallion, semen, hydrocele, single layer centrifugation introduction hydrocele refers to accumulation of serous fluid between the visceral and parietal layers of tunica vaginalis. it can be either permanent or may appear in periods of high ambient temperature, resolving after temperature decreases. it is usually bilateral and may be accompanied by scrotal edema.1,2 relationship between hydrocele and fertility is known.2,3 impact on spermatogenesis (decline in semen quantity and quality) was apparently due to fluid accumulation, resulting in heat-induced testicular degeneration.2,4 treatment of hydrocele may at best provide only transient improvement.2 however, it is possible to improve the quality and fertilizing ability of some poor quality ejaculates through density centrifugation with silica colloids.5 a 21 year old lusitano dressage stallion with a history of subfertility was presented. the stallion was intensively utilized in a dressage school. detailed information on his reproductive use could not be obtained. stallion produced 11 registered foals: 9 in 1999, 1 in 2013, and 1 (last one) in 2014. existence of a subfertility problem and a genital “swelling” was recognized by the referring veterinarian. stallion appeared normal on physical examination except for the enlarged scrotum (figure 1). procedures conducted adhered to the ethical principles of equine semen collection centers (eu directive 92/65/eec). materials used were obtained with the knowledge and authorization of stallion owner. figure 1. bilateral diffused enlargement of the scrotum (s) figure 2. ultrasonographic images of left (l) and right (r) testes; note the presence of hypoechogenic fluid (f) l  r  f  s  clinical theriogenology • volume 13 number 1 • march 2021 56 figure 3. ultrasonographic image of left (l) and right (r) epididymides; note the presence of hypoechogenic fluid (f) on palpation, thickened scrotal wall, and presence of scrotal and preputial edema were felt. scrotal width, as measured with a caliper, was 12 cm. the fluid-filled scrotum made palpation of the freely movable testis difficult. it was not possible to differentiate head and body of the epididymides, and only the epididymal tails could be felt. the stallion showed no sign of discomfort during palpation. ultrasonography (aloka prosound 2 with 5 7.5 mhz probes, aloka holding europe, steinhausertrasse, switzerland) revealed hypoechoic fluid accumulation within the vaginal cavity (figures 2 and 3) leading to a clinical diagnosis of bilateral hydrocele. there were no noticeable changes observed in relation to the amount or echogenicity of the fluid throughout the study. the stallion demonstrated good libido, displaying an immediate full erection of penis when teased in the presence of a mare in estrus. the erect penis appeared normal and there was no evidence of pathology on palpation. method twelve ejaculates were collected using a missouri artificial vagina (imv technologies, l’aigle, france) at biweekly intervals, using a phantom as a mount and a mare in estrus. subjective semen progressive motility was assessed by examining 10 µl of semen placed on a prewarmed (37ºc) slide covered by a cover slip, using a light microscope with a built-in stage warmer at 200 x magnification. sperm concentration was estimated using a spectrophotometer (spermacue®, minitub ibérica, tarragona, spain) and sperm morphology was evaluated by examining 100 unstained sperm in wet mounts, using a phase-contrast light microscope (axiostar 4x105tar, carl zeiss microscopy gmbh, jena, germany) at 1000 x magnification. raw semen had the following values (mean ± sem) for sperm parameters: subjective sperm progressive motility 25 ± 11% (range; 15 50); gel-free semen volume 99 ± 38 ml (range; 40 150); sperm concentration, 184 ± 50 x 106/ml (range; 112 310); and morphologically normal sperm, 24 ± 7% (range; 13 38). ejaculates (n = 12) were filtered and gel-free semen was diluted to a concentration of 100 x 106 sperm/ml in a prewarmed (37ºc) antibiotic-free extender (equiplus ref. #13570/0201; minitub ibérica; tarragona, spain). processed ejaculates were used either as control (c) or centrifuged through single layer silica colloid (slc). group c ejaculates were further diluted in antibiotic-free equiplus to a final concentration of 50 x 106 sperm/ml and refrigerated for 72 hours in an equitainer® (minitub ibérica; tarragona, spain). group slc ejaculates were prepared for centrifugation by carefully pipetting 15 ml of extended ejaculate (100 x 106 sperm/ml) over 15 ml of androcoll-e® (minitub ibérica) in a 50 ml sterile falcon centrifuge tube. after centrifugation at 600 x g for 20 minutes, the supernatant was removed using a sterile pasteur pipette. sperm remaining in the pellet were resuspended in antibiotic-free equiplus to a final concentration of 50 x 106 sperm/ml and refrigerated for 72 hours in an equitainer®. sperm kinetics of c and slc samples were evaluated using a computer-assisted sperm analysis system (isas®; proiser, valencia, spain) before refrigeration and at 24, 48, and 72 hours postrefrigeration. for each sample, a 5 chambered semen slide (isas-d4c20, proiser, valencia, spain) was filled with extended semen and 5 fields were analysed for percentage of total motility (tm), curvilinear velocity (vcl), average path velocity (vap), straight line velocity (vsl), linearity index (lin) and straightness index (str). the settings used for the computer-assisted semen analysis were as follows: 25 frames/second, cell size 4 µm to 75 mm, velocity of rapid cells > 90 µm/second, and straightness 35%. at 1 and 24 hours postrefrigeration, sperm (10,000 per sample) were subjected to dye-sperm coincubation and evaluated for membrane and acrosome integrity6 and mitochondrial membrane potential utilizing an epics xl flow cytometer (beckman coulter, brea, ca), equipped with the expo32adc software (beckman coulter). samples (50 µl) were stained with 5 µl of arachis hypogaea lectin conjugate (fitc/pna; 100 µg/ml; (catalog # l 7381, sigmaaldrich, munich, germany) and incubated with propidium iodide (pi; 2.99 mm; catalog # p 4170, sigma-aldrich). this stain combination allowed for identification of 4 sperm populations: minimal green and red fluorescence (sperm with intact plasma and intact acrosomal membranes); minimal green and enhanced red fluorescence (sperm with damaged plasma membrane and intact acrosomal membrane); minimal red and enhanced green fluorescence (sperm with intact plasma membrane and damaged acrosomal membrane); and enhanced red and green fluorescence (sperm with damaged plasma membrane and acrosomal membranes). propidium-iodide-negative sperm with either intact or damaged/reacted acrosome were considered viable (viability). mitochondrial membrane potential was assessed by flow cytometry after staining with 0.5 µl of jc-1 (5,5', 6,6'–tetrachloro-1,1', 3,3' tetraethylbenzymidazolyl l f fr clinical theriogenology • volume 13 number 1 • march 202157 carbocianyne iodide). stock solution had 3 mm jc-1 (catalog # t4069, sigma-aldrich) in dmso (catalog # d8418, sigma-aldrich)7 with a final concentration of 1.5 µm. dmso in the semen samples at analysis was ~ 0.05%. data analyses levene’s test was applied to test for equality of variances) and independent 2-sample student’s t-tests were applied to compare semen parameters between groups, using spss version 24 for windows (ibm® spss® statistics, new york, ny). significance was set at p < 0.05. breeding management three mares with endometrial biopsies graded as iia8 were artificially inseminated at 24 hours after intravenous treatment with 1500 iu of hcg (chorulon®, msd animal health, paço de arcos, portugal) on detection of a follicle ≥ 35 mm. three mares were randomly assigned to receive their first insemination with either c semen or slc semen. insemination dose of c semen contained ≥ 500 x 106 motile sperm (tm ranged from 22 to 33% and normal sperm ranged from 13 to 38%). insemination dose of slc semen (either fresh [n = 2] or 24 hours refrigerated [n = 2]) contained ≥ 170 x 106 motile sperm, (tm ranged from 30 to 55% and normal sperm ranged from 36 to 50%). two mares were inseminated twice with fresh and refrigerated slc semen. a third mare was inseminated with fresh slc semen, with c semen and with 24 hours refrigerated slc semen at subsequent estruses. pregnancy diagnosis was performed on days 12 14 postovulation via transrectal ultrasonography and fetal heartbeat was assessed on day 25 postovulation. luteolysis was induced in nonpregnant mares (on days 14 16 days postovulation) and in pregnant mares (on day 25 postovulation) with intramuscular treatment of 10 mg of prostaglandin f2α analog (dinoprost; dinolytic®, zoetis, porto salvo, portugal). results morphologically normal sperm were higher (p < 0.05) in slc group compared to c group (44 ± 4 versus 24 ± 2%). before refrigeration, differences (p > 0.05) were observed in tm (40% ± 5 versus 33% ± 3%). tm was higher (p < 0.05) in slc group for all duration of refrigeration (table 1). higher (p < 0.05) lin, vsl, and str were observed at 24 hours after refrigeration in slc group compared to c group (40 ± 1 versus 34 ± 1%, 32 ± 2 versus 25 ± 2 µm/second, and 74% ± 2 versus 64 ± 2%, for slc and c groups, respectively). at 48 hours and 72 hours after refrigeration, str was higher (p < 0.05) in slc group (68 ± 3 versus 58 ± 2% and 70 ± 3 versus 59 ± 4%, respectively) compared to c group. there were no differences (p > 0.05) between 2 groups for the remaining kinetic parameters analyzed. table 1. total motility (tm) in percent for control (c) and androcoll-e® centrifuged (slc) semen at 24, 48, and 72 hours of refrigeration duration of refrigeration in hours c slc tm tm 24 15 ± 2a 30 ± 3b 48 10 ± 2a 25 ± 3b 72 13 ± 1a 30 ± 4b a,wwithin row, means without a common superscript differed (p < 0.05) cytometry analyses revealed viability of slc semen to be higher (p < 0.05) compared to c semen after 24 hours of refrigeration (74% ± 3 versus 57% ± 5%, respectively). acrosome reaction (pna+pi-) was not different. after 24 hours of refrigeration, percentage of sperm with higher mitochondrial membrane potential was 59 ± 2% in slc samples and 47 ± 4% in the c group (p < 0.05). none of the inseminations with c semen resulted in pregnancies. all inseminations with slc semen resulted in pregnancies. fetal heartbeat was detected in 3 pregnancies, and 1 embryo was lost between days 14 25 postovulation. discussion although hydrocele is not always associated with decreased fertility2 it can affect spermatogenesis.2,4 in this case, apparently, age-related testicular degeneration contributed to the low quality of the ejaculates. however, no evidence of testicular degeneration was noted 3 months before, during or after the study. higher incidence of abnormal sperm and poor semen motility are compatible with disrupted spermatogenesis, possibly due to testicular heat stress. 2,44improvement of semen quality is possible after centrifugation with single layer colloid centrifugation.9,10,11 to authors’ best knowledge, this is the first report of the use of this technique in poor quality ejaculates collected from a stallion with hydrocele. although the number of inseminations was limited, pregnancies were established in mares inseminated with treated semen that had a lower number of motile sperm. clinical theriogenology • volume 13 number 1 • march 2021 58 conclusion hydrocele possibly contributed to poor sperm quality in this stallion. single layer density centrifugation of the ejaculate improved semen quality by decreasing the number of abnormal sperm and increasing the number of sperm with high mitochondrial membrane potential. apparently, improved semen quality resulted in higher pregnancy rate. acknowledgement supported by sónia macedo’s doctoral grant sfrh/bd/123416/2016, from foundation for science and technology (fct), ministry for science, technology and higher education, portugal. conflict of interest no conflict of interest to declare. references 1. ball ba: diagnostic methods for evaluation of stallion subfertility: a review. j equine vet sci 2008;28:650-665. 2. varner dd, schumacher j, blanchard tl, et al: in: pratt pw: editor. diseases and management of breeding stallions. goleta, california; american veterinary publications: 1991. p. 242-244. 3. henry m, amaral d, tavares ff, et al: hydrocele of the vaginal cavity of stallions. j. reprod fertil suppl 2000;56:13-21. 4. schumacher j, varner dd: abnormalities of the spermatic cord. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 1145-1155. 5. morrell jm, mari g, kútvölgyi g, et al: pregnancies following artificial insemination with spermatozoa from problem stallion ejaculates processed by single layer centrifugation with androcoll-e reprod dom anim 2011;46:642-645. 6. ortgies f, klewitz j, görgens a, et al: effect of procaine, pentoxifylline and trolox on capacitation and hyperactivation of stallion spermatozoa. andrologia 2012;44:130-138. 7. morillo-rodriguez a, ortega-ferrusola c, macías-garcia b, et al: freezing stallion semen with the new cáceres extender improves post thaw sperm quality and diminishes stallion-to-stallion variability. anim reprod sci 2011;127:78-82. 8. kenney rm, doig pa: equine endometrial biopsy. in: youngquist rs, threlfall wr: editors. current therapy in theriogenology. 2nd edition, philadelphia; elsevier saunders: 1986. p. 723-729. 9. costa al, martins-bessa a, rebello de andrade a, et al: single layer centrifugation with androcoll-e™ improved progressive motility and percentage of live spermatozoa with intact acrosome of chilled stallion semen but did not have an effect on dna integrity. open j anim sci 2012;2:159-165. 10. morrell jm, rodriguez-martinez h: biomimetic techniques for improving sperm quality in animal breeding: a review. open androl j 2009;1:1-9. 11. johannisson a, morrell jm, thorén j, et al: colloidal centrifugation with androcoll-e prolongs stallion sperm motility, viability and chromatin integrity, anim reprod sci 2009;116:119-128. clinical theriogenology • volume 13 number 1 • march 202159 exposure to environmental contaminants and the impact on reproductive health imogen harris,a richard lea,b rebecca sumnerb ahartpury university and hartpury college, gloucester, united kingdom bschool of veterinary medicine and science the university of nottingham, sutton bonington, united kingdom abstract reports are illustrating increasing evidence of perturbed reproductive health in a variety of species. given the rate of change and the widespread occurrence amongst a variety of species, such observations allude to an environmental drive opposed to a natural genetic change. extensive use and dissemination of plastics that contain anthropogenic organic chemicals is suggested as a plausible etiology of such adverse fertility trends given the drastic increase in global plastic production. direct industrial emission and chemical migration from plastic product matrices, in which such chemicals originate, leads to environmental deposition. once ubiquitous within the environment, such environmental chemicals, otherwise known as xenobiotics that are known to modulate endocrine signalling, are consistently available for uptake by humans and animals on a global scale. a variety of species are proposed as sentinel models to further explore the impact of a polluted ecosystem on reproductive health. pregnant animal exposure to xenobiotics, during the key developmental programming window, is of great concern due to the potential for epigenetic modifications on the developing fetus. this review aims to discuss such concepts and routes of exposure, to highlight areas for further research within the field. keywords: xenobiotics, reproduction, dog, fertility, environment introduction global plastic production reportedly stands at 320 x 106 tons per annum. around 40% of such plastics are single use and contain a variety of anthropogengic organic chemicals. direct industrial emission and migration from product matrices, in which such chemicals originate, leads to environmental deposition.1 exposure to environmental chemicals (ecs), often endocrine disruptive in nature, have been suggested in the etiology of adverse fertility trends.2 common anthropogenic organic chemical classes include; bisphenols, dioxins, phthalate ethers, parabens, polycyclic aromatic hydrocarbons, and perfluorinated compounds (pfcs), with existing overlap between some chemical congeners.3,4 such chemicals are typically produced through or utilized in a range of industrial and agricultural processes.5 these include uses as plasticisers, flame-retardants, solvents, preservatives, additives, coatings, pesticides, herbicides, fungicides, and fertilizers. given the ability of ecs to leach from products into nearby surroundings, chemicals remain ubiquitous within the environment; present in air, water, soil, and sediment.6,7 here, ecs are consistently available for uptake by humans and animals on a global scale, through a variety of means.8 with global increases reported in the occurrence of obesity, metabolic syndrome, and associated diseases (e.g., pcos and diabetes) it has been postulated that this is also linked to exposure to chemicals, giving rise to the term ‘metabolism disrupting chemicals’.9 in humans, reports suggest that the etiology and pathophysiology of metabolic diseases could be due to environmental chemical exposure.10 many chemicals that appear to impact on metabolic function, also have endocrine disrupting activity and thus may adversely affect both reproductive function and metabolic disease.11 exposure routes of environmental chemicals one of the main deposition pathways for ec contamination is via a carnivorous diet. consumption of contaminated meat, particularly when fat content is high, is considered as a main exposure route in carnivorous and omnivorous species, given the lipophilic nature of many ecs. this leads to biomagnification across trophic levels, with apex predators incurring the highest bodily burdens. considering 3 aquatic species with high blubber content but differential dietary sources, the herbivorous dugongs has polybrominated diethyl ester biological burdens of 120 ng/g lipid weight.12 this level, whilst high, is still 8 times less than the apex predator species, the killer whale.13,14 similar biomagnification of ecs has been reported in terrestrial ecosystems and agricultural food chains,15 as part of human consumption.16 dog is a close companion to man and shares the same habitat. for this reason, dog is exposed to the same environmental conditions, including environmental chemicals present in home. for this reason, dog is considered, an ideal sentinel model to investigate human exposure to environmental pollutants.14 canine diet (commercially available pet foods) has common environmental chemicals.17 since similar chemical types were detected in dog semen and testes (collected from routine neuters), effects of environmental/gonadal contaminants on sperm quality parameters were tested. deleterious chemical effects were reported on the quality of dna and sperm motility in dog and human.18 food assessed for environmental chemicals contained meat sources from grazing animals (consumed by man who have a meat-based diet).17 pasture contamination through the routine use of sewage sludge fertilizers (biosolids) is therefore of primary concern.19 such fertilizers promote the deposition of a broad range of toxic chemicals onto agricultural land, in addition to complex mixtures of microplastics.20 the ability of chemicals originating from their polymer matrices, to leach into the surrounding environment or digestive tract, if ingested, provides an initial exposure route to biota. fetuses from pregnant ewes grazed on such pastures, as well as their offspring, exhibit perturbations in in both female and male reproductive development.17,21,22 highly chlorinated congeners, including polychlorinated biphenyls (pcbs), bond strongly to soil organic matter, reducing the uptake into plants through root structures, and instead are readily absorbed from the surrounding air.8 however, when pasture concentrations are lower, direct consumption of contaminated soil may result in an additional exposure route to grazing species. around 21,000 tonnes of surface soil are reported to contain pcbs23 that are known reproductive toxicants.17,18 although banned in the 1970’s, these ecs are reported to be present within uk soil at an average concentration of 2.52 mg/kg, with the highest concentrations reaching 80.6 mg/kg.24 as alluded to above, biomagnification within the human can then ensue from a meat-based diet, by consuming animal species that have grazed on such treated lands. theoretically, this would mean that a plant-based diet would result in lower biomagnification of contaminants. unpublished, preliminary data in the horse actually showcases concentrations of certain contaminants to be higher than sentinel models (e.g., dog) fed on meat-based diets (harris, unpublished data). this is potentially indicative of alternative environmental exposure (e.g., water or plant-based feedstuffs). a common approach to breaking down contaminated materials in soil is a process called bioremediation. using fungi or bacteria alongside plant material, ecs can be drawn from the soil into plant matter. reports illustrate the ability of plant ‘alfalfa’ to remove pcbs from contaminated soil by uptake into roots and leaves.25 although this is a beneficial aspect for clearing contaminated soil, due to the digestible energy content of alfalfa, this feedstuff is suggested for pregnant herbivores and breeding stallions.26 giving rise to a potential exposure route for herbivorous diets. a further risk factor is xenobiotic run off into water systems. although a range of xenobiotics are reported present in water, 27,28 di-ethyl hexyl phthalate (dehp) is a common plasticizer and a reported carcinogen,29 known to perturb reproductive health at lower exposure concentrations.17 this phthalate is present in tap water, bottled water and barrelled water supplies. concentrations of dehp were initially greater in tap water, but increased concentrations of dehp were observed in plastic bottled water that was heated to 60°c, a finding expected due to the ability of chemicals to leach from the product matrices.30 this increase in chemical pollutants is not restricted to plastic products. it is estimated that with every 1°c increase in environmental temperature, the volatility of polychlorinated bi-phenyls would rise by around 10 15%, increasing pollutant mobility and promoting further uptake within the ecosystem.31 such change could be an addition to the concept of biomagnification (figure 1). figure 1. biomagnification of xenobiotics within the aquatic ecosystem. industrial processes, agricultural deposition and waste leaching gives rise to biomagnification within the ecosystem, starting from consumption of phytoplankton, magnifying within endangered apex predators. a similar process occurs on land. figure not to scale. figure is authors own (i.t.h). fetal environment environmental chemical exposure is likely to occur throughout life, beginning in utero, continuing postpartum, throughout adolescence, adulthood and gametogenesis. placenta is a dynamic endocrine organ and has incorporating roles (e.g., homeostasis, fetal growth and sustaining pregnancy).32 perturbed placental function can impact fetal development and growth, contributing to chronic health issues in adult life.33 mono (2-ethylhexyl) phthalate, the primary metabolite of dehp, is reported to perturb trophoblast differentiation, thereby acting as an endocrine disruptor to the very early developing placenta.34 intrauterine environment is where the fetus is most susceptible to the exposure of external ecs due to endocrine mediated developmental period.35 it is thus concerning that ecs (e.g., phthalates36 and bisphenols [bp]37) have the ability to cross the placental barrier, exposing the developing fetus to a range of toxic chemicals. in an in vivo murine model, bp-a and bp-s altered 13 sets of identical placental genes, causing morphological defects within the midpregnancy placenta that persisted until parturition.38 such exposure may lead to the indirect disruption of essential developmental processes of the gonads and reproductive system, leading to chronic reproductive perturbations in an adult life. fetal transfer is complex, although placenta works to protect the fetus against exposure to xenobiotics there is the common consensus that ecs have an accumulatory nature towards fetal compartment due to cross talk of signalling pathways.39 analogue lipophilicity, polarity and hydrogen-bonding are reported to impact placental transfer efficiency.37,40 relatively lower concentrations of bisphenol congeners are actively transported to the fetal compartment.41 aryl hydrocarbon receptor is highly expressed within the placenta and a key receptor that works to protect the maternal-fetal interface and placental barrier from xenobiotic exposure. it is hypothesized that pollutants (e.g., bisphenol-a), interfere with the activity of the aryl hydrocarbon receptor, reducing the typical endocrinological function and metabolic activities of the placenta.42 due to the physiochemical specificities of chemical metabolites and their inability to be removed from the fetal compartment in their glucurono-conjugated forms, a back-metabolism cycle is initiated by which the bioactive forms are resynthesized. this cycle increases fetal exposure substantially.43 for bp-s, although the placental transfer in the materno-fetal direction was only 0.4%, this back-metabolism increases fetal exposure to the bioactive form by 87%.41 additionally, it has been suggested that amniotic fluid acts as a reservoir by which the fetus is reexposed to bps through swallowing and dermal adsorption, raising further concern over fetal exposure.37,41 ecs are also reported to have reprotoxic effects through endocrinological interactions.44,45 depending on specific chemical composition, steroidogenic perturbations are a result of affinities for different receptors. this is an area that certainly needs to be furthered within an appropriate sentinel model. preliminary data assessing the addition of pcb-153 (2,2’,4,4’,5,5’-hexachlorobiphenyl) and dehp on lh-induced testosterone secretion in the canine sentinel, to determine the impact of toxicants on endocrine function, did not appear to inhibit endocrine function.17 male reproductive health there is an increasing body of published evidence indicating that human male fertility and reproductive health has declined over the last 40 60 years. geographically dependant temporal declines in human semen quality are becoming an increasing concern, with meta-analytical studies suggesting an approximate 50% decline in sperm concentration over the past 70 years.46,47 results from these meta-analytical studies remain heavily scrutinized, a result of developments in semen analysis methodologies, heterogeneity and the inclusion of historical data sets. such limitations were suggested to be substantial factors influencing the adverse trends reported, thus questioning the true declines in semen quality.48 following the application of stricter inclusion criteria, in addition to completion in accordance with standardized protocols (meta-analysis of observational studies in epidemiology)49 more recent meta-analyses continue to suggest a decline in fertility.50 this rate of decline appears to have no plateau, raising substantial concerns for future male fertility. temporal declines are specific to the western world, including europe, north america, australia and new zealand, with trends failing to prevail in south africa, asia and south america.50 such trends, with distinct geographical variation would suggest the influence of environmental factors, although socio-economic contexts could pose as additional contributors. parallel trends in sperm quality parameters to that in humans have been reported in a dog sentinel model, with an overall decline of 30% in progressive sperm motility.17 geographical variation is also evident within exposure to environmental chemicals. dog sentinel model showcases how testicular chemical profiles vary regionally, alongside varying testicular pathological profiles. testis collected from finland had reduced pathologies compared to testis collected from denmark and the uk. such findings provide additional support to the concept that the environment likely influences reproductive function.51 many socio-economic interactions do not retain relevance in such species. additionally, data were collated from a single lab with consistent analytical methods, thereby adding to the weight of evidence exhibited in the human and limiting the criticism over semen quality assessments. decline in semen quality is proposed to be associated with bioaccumulation from meat-based diets, supported by the fact that a meta-analysis of temporal trends in the herbivorous stallion sperm quality does not appear to be overly altered. 52 however, collecting sperm quality data from a singular lab, preliminary data do actually have similar patterns of declining sperm quality within the herbivorous breeding stallion (harris, unpublished). as mentioned, dog, given the association to human lifestyle, is proposed as a useful sentinel model to assess the deleterious impact of environmental chemicals on human reproductive health. undertaking an updated analysis of temporal trends in canine sperm quality, originally assessing sperm quality over a 26-year time frame,17 continued to have a decline in sperm motility over time, yet not to as a substantial degree (figure 2). figure 2. updated analysis of temporal trends in canine sperm quality over a 32-year timeframe. percent normal sperm motility. data expanded utilising part of published data.17 scientific report articles are published under a cc by license allowing for maximum dissemination where users are free to adapt data. error bars represent ± 1 sem. vertical dotted lines represent time point within the programme where dogs with poor semen quality were removed from the programme. the cause of this is unknown. diagonal lines showing declining sperm quality are plotted for graphical purposes only. temporal trends in sperm quality are also linked with global increases in reproductive perturbations; including testicular cancer (tca) and genitourinary abnormalities, such as cryptorchidism and hypospadias.2,53 this is evident within both the primate and dog. the reproductive trends present today are collectively termed testicular dysgenesis syndrome.2 globally, tca incidences in humans have increased 2-fold over the past few decades, with most cases apparent in younger generations. although a variety of factors could give rise to such changes, these trends are suggested to be a result of toxicant exposure at vital periods of sexual development.54,55 over a similar time frame, increases in cryptorchidism have been reported in male pups from the same population of stud dogs that exhibited a decrease in sperm motility.17 preliminary evidence within the canine additionally suggests an increased incidence of testicular tumours over a 40-year period.56 geographical variation in human reproductive perturbations are also heavily reported, with higher incidence rates in industrialised and agricultural areas, indicative of interactions between chemicals utilized within these industries.19,57 female reproductive health a further sensitive window of exposure is during the complex process of follicle development from the primordial follicle pool to mature preovulatory graafian follicles. ovarian somatic cells are specialised, multidisciplinary cells that are paramount for optimum reproductive function and follicle differentiation; with roles in germ cell support steroidogenesis and growth.58 in canine sentinel model studies, certain environmental chemicals were present at higher concentrations in the dog ovary then in the testis (van der mescht, unpublished data). in addition, when coculturing murine ovarian tissue with the chemicals present within the canine ovary, there is an enhanced sensitivity to of the earlier follicle types (primordial and primary follicles). with the follicular population formed during development determining the reproductive lifespan of an individual, ensuring a balance between apoptosis, proliferation and differentiation is crucial. primordial follicles are those that define the ovarian reserve, therefore modifications to such follicles could be detriment to fertility. understanding how environmental chemicals affect the molecular and biochemical signalling of the granulosa cell, to support the oocyte, is an area that requires further study. key programming window and transgenerational impact of ecs it has been suggested that ec exposure may manifest long after initial exposure due to epigenetic modifications originating from exposure at the critical window of genitourinary development in the foetus and new-born.59,60 modifications include dna methylation, microrna or histone alterations.59 as discussed above, placenta is a dynamic organ that supports fetal development. studies are beginning to indicate how exposure to phthalates during pregnancy is associated with genome wide modifications of placental dna methylation, impacting fetal development.61 focussing on reproductive development, the bipotential gonad during early pregnancy incorporates many signalling pathways and molecules that instigate, and control, crucial embryological developmental pathways. utilization of mouse knockout models provides insight into such genetic determinants of the bipotential gonad development. gata binding protein 4 (gata4) remains as 1 of the earliest markers crucial for formation and development of the gonadal ridge.62 loss of gata4 gene expression is reported to inhibit formation of gonadal ridge.62 further development and maintenance of the gonadal ridge is determined by genes (e.g., wilms’ tumour suppressor 1 gene and steroidogenic factor 1) essential for early gonadal development.63 under the influence of wnt signalling, the binary fate decision of gonadal formation is chosen. a recent review discusses how the observed human male reproductive disorders might have a fetal origin, due to an androgen dependant programming window during early gestation.64 one of the major signalling pathways throughout ovarian differentiation is that of wnt4/rspo1 signalling.65 in the absence of the male sex determinant gene, the cascade of genetic pathways to promote female development sees wnt mediated stimulation, like that of the ligand wnt4 induce the expression of downstream effectors,66 such as follistatin and β-catenin.67 β-catenin is a pro-ovarian signalling molecule which induces expression of the pivotal female development transcriptional target, foxl2.68-70 should a toxicant impair the expression of male sex determining genes in an xy embryo, then it is plausible for wnt mediated stimulation to ensue, to follow the feminisation pathway.71 epigenetic mechanisms, with adverse effects on reproductive potential, are considered to be transgenerational.72,73 in killer whale populations, ec concentrations in calves are higher than in their lactating mothers.74 comparably, in suckling polar bear cubs, pcb concentrations surpassed that of maternal contamination.75 such research demonstrates the accumulatory nature of toxic ecs within the developing neonate. the subsequent exposure of such chemicals to the suckling offspring is likely to perturb reproductive development and future fertility, as reported in other species,76 having future transgenerational impact. the transgenerational impact of contaminants has been shown to induce delayed pubertal onset, impaired gametogenesis and impaired steroidogenic gene expression. furthermore, maternal behaviours have also been shown to be impaired following transgenerational studies of toxicant mixtures, raising concern of not only reproductive health, but also wellbeing of future generations.77 within the sheep model, exposure to environmental contaminants has been shown to induce testis transcriptome modifications, which authors report, if not corrected by or during puberty, would likely have adverse outcomes for future generations adult life.22 reports discuss how epigenetic mechanisms are the means by which xenobiotics mediate such transgenerational effects.78 figure 3. avenues for xenobiotic perturbations within testicular tissue. as the housing unit of sperm development, testicular health has a significant impact on sperm quality and reproductive success, from pathological perturbations to epigenetic modifications. figure is authors own (i.t.h). conclusion the sentinel has historically been exploited as a model for biomonitoring environmental conditions in addition to health responses to toxic and infectious agents in other populations of species, including humans.79 common biomonitor species include ants,1 birds,80,81 sheep,82 dogs,14 and aquatic species (oysters, fish, and killer whales).83-85 many socio-economic interactions do not retain relevance in such species, although exception is given to species undergoing artificial reproductive techniques, which may still influence trends in reproductive health. canids and felines share close environmental conditions with that of their owners, thus representing important models for bio-monitoring human health.14 although the precise drivers behind these reproductive temporal trends remain uncertain, there is increasing evidence that anthropogenic environmental change may be a key factor.86 there is a distinct need to increase public understanding in order to promote a sustainable future. although microorganisms are reported to be able to degrade toxic environmental compounds,87 the use of the dog as a sentinel model, or additional sentinel models, would add substantial information in order to further or dispute previous research, and add to the weight of evidence that showcases how xenobiotics are perturbing the environment and future health of individuals. only with further research can we work to drive change within a polluted environment. conflict of interest there are no conflicts of interest to disclose. references 1. wania f: assessing the potential of persistent organic chemicals for long-range transport and accumulation in polar 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laparoscopic artificial insemination to your small ruminant practice–a practical review swanand r. sathe college of veterinary medicine, iowa state university, ames ia abstract laparoscopic artificial insemination (lai) is an intrauterine method of insemination, especially utilized in the small ruminant species to bypass their unique anatomically tortuous cervix. there are several advantages of lai that include efficient use of processed semen leading to higher pregnancy rates. success of lai programs depends on proper implementation of estrus synchronization programs, patient selection and thorough knowledge of the reproductive physiology. in addition, proper equipment and surgical expertise help in reducing patient morbidity and mortality rates. laparoscopic artificial insemination can be associated with several complications as a result of inadequate patient preparation, poor technique or equipment failure. hence, a through planning is essential to carry out the procedure safely and with consistent success rates. addition of lai to a small ruminant/food animal practice can be quite profitable and professionally fulfilling, with appropriate investment and training of the veterinarian and technical staff. keywords: laparoscopic, ai, ewes, does, insemination introduction and background there is a growing demand for use of frozen and processed semen in united states, especially in the show lamb industry, where owners are willing to pay a premium to introduce newer genetics in their flocks. however, fertility rates with frozen semen using traditional breeding approaches like vaginal and trans-cervical artificial insemination (tcai) are low. this is due to the long and tortuous nature of the cervix in small ruminants and the presence of four to seven cervical rings. these cervical rings point caudally into the lumen thus a providing a physical barrier1-5 to attempts at passing an insemination pipette during tcai. there is evidence that pregnancy6 and lambing rates7 improve as the depth of semen deposition in the cervix increases. the degree of penetration of the insemination pipette through the cervix depends on the breed, age and stage of the estrous cycle especially in ewes.8 deeper semen deposition results in greater numbers of motile spermatozoa available for fertilization thus leading to higher pregnancy rates.9 laparoscopic artificial insemination is an advanced assisted reproductive technique that enables such deep intrauterine deposition of semen and helps bypass the physical barriers of the caudal reproductive tract in small ruminants. pregnancy rates using frozen semen deposited intra-uterine via laparoscopy have yielded higher pregnancy rates (60-80%) consistently when compared to vaginal and tcai methods.10-12 in addition to higher pregnancy rates, it is possible to use lower concentrations of spermatozoa per breeding thus leading to more animals bred per ejaculate. the average dose required for breeding a ewe using frozen semen can be as less as 20 to 25 million live spermatozoa, when compared to higher doses required via the vaginal (400 million live spermatozoa) and trans-cervical route (100-200 million live spermatozoa).13 thus a single entire frozen ejaculate can be used to inseminate as many as 50 to 100 ewes, thus leading to a more efficient use of semen. the main disadvantage of providing lai service to producers has been the high equipment cost and the relatively steep curve of surgical expertise required to perform to perform the procedure safely. however, with the availability of newer and more portable laparoscopy equipment, it is now possible to offer these services cost-effectively at a hospital and field setting. the following paper will discuss the lai procedure in detail with emphasis on surgical techniques, equipment needed, and useful practice tips, based on our set up at the theriogenology service at iowa state university, college of veterinary medicine. selection of patients laparoscopic artificial insemination though minimally invasive is still a surgical procedure nonetheless and hence young, healthy ewes/does in appropriate body condition scores (bcs) are ideal clinical theriogenology • volume 10, number 3 • september 2018211 candidates for the surgery. it has been shown that an optimum bcs results in higher ovulation as well as pregnancy rates.14,15 fat/obese animals not only prove to be a surgical risk, but also may not respond appropriately to synchronization protocols (artificial insemination or embryo transfer), thus increasing the net surgical procedure time. a decrease in embryonic viability and subsequently lower pregnancy rates have been observed in animals maintained on a body score of below 2 and above 4.16 the proposed mechanism behind pathogenesis in overly conditioned animals has been attributed to lower progesterone levels possibly because of increased liver blood flow, leading to increased clearance of progesterone from the circulation.17 the preparation of animals for estrus synchronization and lai begins several weeks prior to actual date of the procedure. this involves vaccinations, deworming and increasing the plane of nutrition (flushing) to achieve an ideal bcs. however, in a recent report18 the pregnancy rates declined in ewes handled four to six weeks prior to lai for various managemental procedures such as deworming, vaccinations and feet trimming. a possible explanation to this decreased pregnancy rate was attributed to the stress experienced during such procedures. hence, special care in terms of minimizing stress should be undertaken when handling animals for routine preventive managemental procedures. in addition, attention should be paid to detect the presence of systemic diseases in the flock such as infections of the respiratory system which increases the surgical risk to the affected animal. the estrus synchronization protocols are sent out several weeks prior to the day of the procedure. a thorough knowledge of reproductive physiology, seasonal and breed variations, appropriate duration of protocols and drug dosages are essential for adequate response from patients. older animals that have undergone surgical embryo transfers or multiple lai procedures in the past can have extensive adhesions of the omentum to parietal peritoneum thus forming a curtain/barrier or, in some cases adhesions to the reproductive tract. this can lead to difficulties in visualization of the reproductive tract and increase the duration of surgery or the need to abandon the procedure. based on the reproductive history, animals suspected of being pregnant should be checked via trans-abdominal ultrasonography examination before initiating the synchronization protocols. since the lai procedure involves restraining animals in a trendelenburg position, it is recommended to keep the animals off-feed for at least 16 to 20 hours and off-water for at least 12 hours to prevent abdominal fill and minimize chances of regurgitation and aspiration. equipment costs laparoscopic ai most equipment and associated costs quoted here are from the manufacturer karl storz germany, though there are other manufacturers available in the market as well. a) trocars and cannulasthere are two suitable sizes available i) 10 mm and ii) 5 mm. estimated cost $500. we prefer using the smaller size as there are several advantages: i) enables creating smaller incisions and ports, ii) lesser chances of abdominal perforation due to lesser effort required during insertion iii) lesser wear and tear of instruments, iv) no need for trocar adaptors for ai guns v) rnables use of smaller rigid 5 mm telescopes (laparoscopes). b) telescope/laparoscope (available options are 0 and 30 degrees): we prefer using a 0 degree scope, but a 30 degree oblique telescope can also be used effectively. estimated cost $7000 (new) $ 3500 (refurbished). c) light source with halogen/xenon bulbs and cables: approximate cost around $7000. there are handheld portable endoscope light sources available now which are better suited for field work and just as effective costing around $500. d) video camera and screen (optional, since many practitioners in a field setting prefer looking through the lens of the laparoscope instead of using a camera and screen). approximate cost $3500-4000. a new mobile video-endoscopy unit available from karl storz known as the tele pack vet x led® is multifunctional and contains a camera, light source; air insufflation unit and image capture capabilities. this unit is light, portable and can be carried out to the field in a carry case (provided). it can clinical theriogenology • volume 10, number 3 • september 2018 212 also be adapted for concurrent use in small animal and equine clinical practice. approximate cost $25,000. e) air insufflation unit: a medical air insufflation unit with carbon dioxide tank can be used to ensure an ideal abdominal pressure. however the equipment is expensive and cumbersome to move around especially when performing this procedure at a field level. also the rate of insufflation is relatively slower leading to increased surgical time and increased duration of the patient in a trendelenburg position. instead of a traditional air insufflation unit, a regulator can be attached directly to the co2 tank which enables faster insufflation. the disadvantage is that the degree of insufflation is subjective and based on abdominal percussion and degree of distention. yet another alternative is to use a commercial vacuum pump system (eg. gast® vacuum pump) and attach an inline filter. the advantage of using such filter systems is that besides co2, other alternative gases such as medical grade air or room air can also be used for insufflation. disadvantages of use room air are that it is risky to use in a dusty environment due to the danger of causing peritonitis. a recent research study19 has shown that there is no danger of hypoxia or infections in using medical grade air vs co2 nor were there any differences in pregnancy rates observed between the two groups. in a hospital setting we prefer to use the vacuum pump and insufflate with room air. medical grade air insufflation unit: $8000, co2 tank with regulator:$ 250, commercial vacuum pump (eg: gast® mfg.): $500. d) laparotomy surgical pack: we recommend having ready access to a laparotomy surgical pack, for the purpose of clamping and suturing any subcutaneous bleeding vessels after the lai procedure or for performing an emergency laparotomy in case of an abdominal organ perforation. e) laparoscopy ai cradle: these are specialized cradles that can be tilted almost up to 90 degrees to position animals in a trendelenburg position. we recommend buying cradles with aluminum frames as they are sturdy yet light to transport in case of field work (sydell® iowa, minitube® usa,etc.). approximate cost: $1500. semen processing a) ai guns and sheaths: i) transcap with guide (imv®, france): $550; aspics for semen straws: $70/25 pcs. ii) robertson’s ai gun with sheaths (minitube® usa): $500. b) semen processing equipment: semen tube holders, semen straw thawing equipment, slide warmer, portable microscope, 0.25 cc straws, straw cutters etc. (total cost around $3000 including the microscope). sedation and premedication light sedation is usually recommended since the total duration of the procedure (preparation and surgery) takes roughly about 10 to 15 minutes only. the goal is to have the patient stand up on their feet after they are loaded off the cradle and start searching for food. two classes of drugs that we usually use for sedation are alpha-2 agonists (inj. xylazine @ 0.05 to 0.1 mg/kg of the 20 mg/ml large animal formulation i.v. or i.m.) and tranquillizers (acepromazine @ 0.05 to 0.1 mg/kg i.v. or i.m.). opioids such as butorphanol (0.05-0.2 mg/kg iv, im, sq) may also be used 5 to 10 minutes before handling animals for restraint. the advantage is that it affords some degree of analgesia but personal experience has shown that it also leads to lesser degree of sedation than alpha-2 agonists, causing more struggling among patients when they are restrained in a trendelenburg position. other medication such as anti-inflammatories (flunixin meglumine @ 1.1 mg/kg i.v.) and in some cases long acting antibiotics (ceftiofur crystalline free acid or long acting pxytetracyclineextra label usage) can also be administered during restraint and surgical preparation. local analgesia in form of lidocaine hydrochloride is administered at each proposed incision site (2 ml of 2% lidocaine hydrochloride subcutaneously) and each site is scored with a hypodermic needle to mark the area for future reference. an appropriate withdrawal time for meat and milk should be relayed to the producer when using any of the above drugs (www.farad.org). clinical theriogenology • volume 10, number 3 • september 2018213 pre-surgical preparation the patient (ewe/doe) is restrained and sedated with the appropriate sedative agent and observed for clinical effects. once adequate sedation is confirmed, the animal is then lifted and restrained on a special, custom made laparoscopic ai cradle. the foreand hind-feet (at level of hocks) are restrained securely, a face mask or a towel is used to cover the eyes and the animal moved to the surgical preparation station. the wool on the ventral abdomen is clipped from the level of the mammary glands and extending cranially up to the umbilicus. the area is surgically scrubbed with 2% chlorhexidine scrub alternating with 70% isopropyl alcohol. special attention is to be paid to the inguinal gutters as they accumulate loose dirt, dried feces, natural sebaceous secretion, and which tend to contaminate the surgical site when the animal is suspended in a trendelenburg position. two sites about a hand’s width cranial to mammary glands and adjacent to the left and right mammary/superficial epigastric veins are identified. the site can be medial or lateral to the veins and the choice is dependent on the preference of the surgeon and size of the patient. for larger patients (>120 lbs.) we recommend a more medial approach since the abdomen is wide and tends to get wider when insufflated with co2/air. this prevents the laparoscopic instruments from reaching the reproductive tract or aligning with each other during the insemination process. on smaller patients (<120 lbs.), a more lateral approach is recommended to afford adequate insufflation and avoid more medially placed abdominal organs. the surgical sites thus selected are superficially scored with a 20g-1 inch needle, and 2 ml. of 2% lidocaine hydrochloride infiltrated in the subcutaneous tissues and musculature. the skin scoring is performed to identify the surgical sites during surgical procedure, since the local anesthetic tends to dissipate quickly. a final surgical scrub is performed before wheeling the patient to the surgical station. surgical procedure the lai cradle is elevated up to 45 degrees, to position the ewe in a trendelenburg position. a number 11 scalpel blade is used to create 0.5 inch incisions through the skin and fascia up to the level of muscle over the proposed pre-scored incision sites. a 1.5 inch blunt teat cannula attached to a flexible insufflation hose is inserted through the muscle layers and intra-abdominally, via the farther incision from the surgeon with a firm, calculated push. air insufflation is carried out untill the ventral abdomen feels adequately tense. with practice it is possible to carry out lai with lesser amounts of air safely. the advantage of insufflating lesser amount of air is to reduce the degree of hypoxia to the patient while in trendelenburg position. a 5 mm trocar and cannula are inserted in the abdomen through the near incision with calculated pressure, the trocar withdrawn and a 5 mm telescope/laparoscope inserted via the cannula to visualize the interior of the caudal abdomen. the reproductive tract (uterine body and horns) are usually located ventral (from a surgeons’ point of view) to the urinary bladder. in animals that have responded adequately to estrus synchronization protocols, a distinct tone and hyperemia can be identified affecting the reproductive tract. the tract appears pale to dark pink and responds by curling when it is touched by the laparoscopic instruments. the location of the tract and its relation to surrounding structures is noted to ascertain the ease with which an intra-uterine injection can be performed safely. on some occasions, a distended bladder can hide the reproductive tract partially or completely. decompression of the bladder during premedication and surgical preparation is strongly recommended to prevent this from happening. the caudal sac of the rumen, distended cecum or loops of small intestine can sometimes prevent visualization of the reproductive tract. this can be minimized by keeping the patient off feed and water as recommended. once the reproductive tract is visualized, another similar sized trocar and cannula is inserted adjacent to the teat cannula. a loaded laparoscopic ai gun with an external sheath (imv®, france) is then inserted through this port and aligned opposite the greater curvature of uterine horns under laparoscopic guidance. the external sleeve/guide can be used to manipulate the uterine horns from underneath overlying structures such as the bladder of omentum to the desired angle. the aspic and needle apparatus is then exposed keeping them as close to the uterine horns as possible and with a quick jab the needle is seated at the level of the mid-horn. the semen is then injected, the needle withdrawn and a second 0.25 cc semen straw injected in other uterine horn in the same fashion. care is to be taken to ensure proper depth of the needle placement while inseminating to clinical theriogenology • volume 10, number 3 • september 2018 214 prevent semen leaking in the abdomen. there are no reported advantages of injecting both horns, and the entire dose can be deposited in one horn only, resulting in similar pregnancy rates.20 however, when performing lai procedures in animals superovulated for embryo recovery, it might be beneficial to inject both horns so that adequate numbers of spermatozoa are available on both sides for migration and fertilization in presence of increased intrauterine mucus due to higher circulating estrogen levels. once the uterine horns are injected, a quick assessment is made to ascertain that there is no excessive bleeding or uterine horn lacerations. the laparoscope and the ai gun are withdrawn from the cannulas. the spring loaded or side valves are decompressed to deflate the abdomen and the animal is lowered to a horizontal plane after removing all instruments from the abdominal ports. the skin incisions are closed with the help of non-absorbable suture material (2-0 polypropylene) in a cruciate suture pattern after ensuring that there is no excessive bleeding. on occasion, subcutaneous branches of the mammary/superficial epigastric veins may bleed excessively. the bleeding vessels are clamped and ligated individually and then the skin incisions closed in an interrupted suture pattern. skin staplers can also be used and are faster to apply, but can be costlier than suture material. we prefer to cover the abdominal incisions with a water resistant antibiotic free aluminum based wound spray (aluspray®, neogen animal safety, lexington, ky). we also prefer administering a reversal agent in case alpha-2 agonists were used for sedation (eg. tolazoline, yohimbine) as soon as the animal returns to a horizontal plane. most animals stand up immediately or at least assume a sternal recumbency after being placed on the ground. in cases of prolonged lateral recumbency it is advised to monitor the animal’s vital signs closely and prop the animal in a sternal position to avoid aspiration pneumonia. if there are adequate numbers of personnel available we recommend running two to three lai cradles in succession especially when there are large numbers of animals synchronized for timed breeding. complications arising during laparoscopic ai procedure as with any surgery, there are numerous complications that can arise during the lai procedure. complication arising during insemination of systemically unhealthy animals one of the major concerns when carrying out lai is patient mortality due to underlying systemic disease conditions such as respiratory tract infections. a thorough history and physical examination of preferably each animal (when dealing with small groups) is crucial in order to avoid identifying these unhealthy patients. when the ewes/does walk in our hospital through the chute system we observe each individual for signs of coughing and wheezing, especially evident when handling or segregating animals in different groups or during premedication. despite taking due care, there may be animals that are missed, raising the percent morbidity and mortality rates. minimizing the surgical procedure time greatly helps in reducing chances of such complications, and is largely dependent on surgical speed and efficient teamwork which is gained through practice. judging the tone and color of the reproductive tract greatly helps in assessing whether the patients have responded adequately to estrus synchronization protocols. for patients that have poorly responded or not responded at all (flaccid, pale tract) to the hormonal protocols, a decision can thus be reached quickly to abandon the insemination process early enough during surgery. it is always important to constantly monitor vital parameters and signs for regurgitation from the nose/mouth to prevent aspiration pneumonia. flow-by oxygen via face mask can also help in lessening the degree of hypoxia experienced during the procedure. in animals that are obese or metabolically compensated, we advise the owner against performing the procedure, or decreasing angle of elevation from 45 to 30 degrees after insufflating the abdomen. this reduces pressure on the diaphragm and lessens the degree of hypoxia experienced during the surgical procedure. rupture/puncture of abdominal viscera the abdominal organs in danger of being punctured are the urinary bladder in the caudal abdomen, small intestinal loops and cecum in the mid-abdomen and caudal sac of the rumen in the cranial abdomen. to minimize the risk of perforation there are a few things to be kept in mind before and during the surgical procedure: clinical theriogenology • volume 10, number 3 • september 2018215 i) ensure that the animals are adequately fasted and kept off-water ii) ensure that the urinary bladder is emptied/decompressed to minimize chances of perforation iii) proper and adequate insufflation of the abdominal cavity iv) proper placement of the trocars: the surgical sites for trocar placement (as described above) should be meticulously selected. too cranial a site can result in perforation of the caudal sac of the rumen. too median a placement can result in mammary vein punctures, urinary bladder ruptures, cecal and small bowel perforations. while placing the trocars through the proposed surgical sites it is also important to angle them laterally. using smaller trocars (5 mm) also enables for easier insertions through the body wall. the larger 10 mm trocars can create a considerable drag leading to greater force required for insertion and subsequently greater risk of perforation of abdominal organs. one of the early signs of a possible gastrointestinal tract perforation is methane-like odor evident from the cannula immediately after removal of the trocar. the trocar may have greenish-yellow fecal contents stuck on its end and along the shaft. bladder ruptures should be suspected with presence of a sudden increase in blood tinged fluid evident in the abdominal cavity. most ruptures can be confirmed on direct visualization of the organ. however, on some occasions the sudden deflation of a distended viscus can change shape and orientation of the affected organ thus preventing confirmation by direct visualization. on rare occasions, rupture of a major blood vessel such as the aorta or its branches can lead to excessive bleeding and sudden death. subcutaneous emphysema in larger animals having a broad abdominal girth there is risk of subcutaneous emphysema developing if the trocars are placed too laterally. this disrupts the normal tissue planes and increases risk of the trocar getting caught between different layers of the abdominal wall, making it difficult to enter the abdominal cavity. in cases of subcutaneous emphysema, the air insufflation needs to be stopped immediately and the leaked air gently massaged out of the incision site. if addressed early, the same site can be utilized for trocar placement. but if a significant amount of subcutaneous air is present, it is best to select another surgical site after suturing the earlier port. abscess formation/peritonitis/sepsis a small percentage of animals in the flock may develop peritonitis, external or internal abscesses, sepsis and death. this usually happens when contaminated instruments are used without cleaning them between animals. we recommend dipping and thoroughly wiping off the instruments using 70% alcohol or dilute 2% chlorhexidine solution, after each surgery and before they can be used for another animal. hematoma/bleeding due to the proximity of surgical site to branches of the mammary/superficial epigastric veins, it is possible to accidently incise a collateral branch leading to excessive subcutaneous bleeding. the blood can leak into abdominal cavity coating laparoscopic instruments and blurring the visual field. presence of blood can also lead to formation of intra-abdominal adhesions in thefuture. after surgery, these vessels can continue bleeding in the subcutaneous tissue leading to formation of a localized hematoma. since the clotted blood and fibrin are a good media for bacteria to thrive, this can lead to formation of an abscess. after the abdomen is deflated and laparoscopic instruments withdrawn, the surgical sites should be meticulously checked for presence of small bleeding vessels. these should be ligated before suturing/stapling the incision sites. intra-abdominal adhesions in animals having undergone lai procedures or surgical embryo flushes in the past, intraabdominal adhesions of the omentum to the body wall can form. these result in a partial or complete division of the abdominal cavity thus making it difficult to align laparoscopy instruments through the two opposite ports. efforts to maneuver the instruments around the adhesions or sometimes injecting through the omental barrier are the only options available. on occasions creating three abdominal ports or creating clinical theriogenology • volume 10, number 3 • september 2018 216 ports on the same side of the body can help circumvent around these adhesions. sometimes incomplete insufflation can lead to the omentum getting caught on end of the laparoscopic instruments. adequate insufflation of the abdominal cavity usually results in the omentum falling off instruments affording a clear view of the abdomen. ai gun failure sometimes the ai guns may get stuck due to faulty ‘o’ rings or due to faulty batch of semen straws. on occasion, because of ‘o’ ring failure, the injecting straw can disengage and fall in to abdomen (in case of aspic straws, imv®). hence, a laparoscopic grasping forceps should be included in the lai surgery pack to address such complications. it is always a good idea to load an ai gun with a mock straw filled with saline before start of the procedure to ensure that all parts of the instrument are working well. inability to seat the injection needle in the uterine lumen in cases where the animals fail to respond adequately to synchronization protocols, the ai gun needle may slip out of the uterine musculature because of the lack of uterine tone. owners should be informed about such lack of response, the ai process stopped and the animal resynchronized. we document the color and tone observed for each animal during the surgical process. this provides us and the owner a valuable feedback when observing for pregnancy rates and infertility problems in the flock. the uterine tone is graded as: grade 0-no response; grade 1medium degree of response; grade 2 adequate/good response. the color can vary from pale to different shades of pink depending on stage of estrus. the uterine horn increases in thickness during superovulation protocols as compared to conventional ai protocols, and hence a marginally longer aspic needle should be used to ensure the proper intraluminal deposition of semen. uterine bleeding the uterine tract in estrus has increased blood supply; hence a little oozing from the injection sites is expected. to minimize extensive bleeding or prevent uterine vessel lacerations, it is advisable to inject along the greater curvature of the uterine horns. the lesser curvature which is close to the broad ligament has a rich vasculature and is prone to bleed extensively if injected. in our practice, the most common complications we have observed are hypoxia in over conditioned animals, subcutaneous bleeding, omental adhesions from prior surgeries and localized hematomas. on rare occasions, we have observed perforations of the gastrointestinal organs (in unfasted animals) and the urinary bladder.21 these usually occur during trocar placements, thus highlighting the importance of feed/water withdrawal and bladder decompression prior to conducting the procedure. conclusions laparoscopic artificial insemination involves a substantial investment in form of buying equipment and training for the procedure. the success of lai program requires good teamwork and coordination. a trained veterinarian performs the lai, a trained technician/assistant handles semen processing and other personnel take part in handling, sedating and preparing animals for surgery. on occasion, producers and owners can be trained adequately to prepare animals for the surgery. it takes a few attempts for the team to get used to the flow of the procedure. however, once the operator and the team become proficient with the process, it is possible to inseminate as many as 100 to 200 animals per day. addition of lai to your small ruminant private practice can thus be an economically feasible option that can turn profitable quickly, once producers see an increased pregnancy rate even with use of processed semen. conflict of interest statement the author claims no direct or indirect affiliation with any of the manufacturer’s listed in the text. the information regarding the equipment and various manufacturers is solely based on experience and is for the reader’s reference only. clinical theriogenology • volume 10, number 3 • september 2018217 references 1. dun r: the cervix of the ewe. its importance in artificial insemination of sheep. aust vet j 1955;101-103. 2. evans g, maxwell w: salamon’s artificial insemination of sheep and goats. sydney: butterworths; 1987. p. 154-159. 3. fair s, lonergan p, duffy d et al: ewe breed differences in fertilisation rates and embryo quality after cervical or laparoscopic artificial insemination. proc 15 int cong anim reprod 2004;2:375. 4. fukui y, roberts e: further studies on non-surgical intrauterine technique for artificial insemination in the ewe. theriogenology 1978;10:381-393. 5. more j: anatomy and histology of the cervix uteri of the ewe: new insights. acta anat 1984;120:156-159. 6. halbert g, dobson h, walton j, et al: field evaluation of a technique for transcervical intrauterine insemination of ewes. theriogenology 1990;33:1231-1243. 7. salamon s, maxwell: frozen storage of ram semen. ii. causes of low fertility after cervical insemination and methods of improvement. anim reprod sci 1995;38:1-36. 8. kershaw mc, khalid m, mcgowan mr, et al: the anatomy of the sheep cervix and its influence on the transcervical passage of an inseminating pipette into the uterine lumen. theriogenology 2005;64:1225-1235. 9. eppleston j, salamon s, moore nw et al: the depth of cervical insemination and site of intrauterine insemination and their relationship to the fertility of frozen-thawed ram semen. anim reprod sci 1994;36:211-225. 10. shipley cfb, buckrell bc, mylne mja, et al: artificial insemination and embryo transfer in sheep. in: youngquist rs, threlfall wr editors. current therapy in large animal theriogenology. 2nd ed. st. louis: saunders-elsevier; 2007. p. 629-641. 11. anel l, kaabi m, abroug b, et al: factors influencing the success of vaginal and laparoscopic artificial insemination in churra ewes: a field assay. theriogenology 2005;63:1235-1247. 12. mckusick bc, thomas dl, gottfredson rg, et al: a comparison of transcervical and laparoscopic intrauterine artificial insemination techniques on reproductive performance of ewes. proc 46th annual spooner sheep day, department of animal science, university of wisconsin-madison; 2000. p. 32-39. 13. cseh s, faigl v, amiridis gs: semen processing and artificial insemination in health management of small ruminants. anim reprod sci 2012;130:187-192. 14. kleemann do, walker sk: fertility in south australian commercial merino flocks: relationships between reproductive traits and environmental cues. theriogenology 2005;63: 2416-2433. 15. kenyon pr, morel pch, morris st: the effect of individual liveweight and condition scores of ewes at mating on reproductive and scanning performance. n z vet j 2004;52:230-235. 16. abdel-mageed i: body condition scoring of local ossimi ewes at mating and its impact on fertility and prolificacy. egyptian j sheep goat sci 2009;4:37-44. 17. parr ra: nutrition-progesterone interactions during early pregnancy in sheep. reprod fertil dev 1992;4:297-300. 18. mccappin n, murray rd: some factors affecting pregnancy rate in ewes following laparoscopic artificial insemination. vet rec 2011;168:99. 19. haan jd, hay kraus bl, sathe sr: a comparison of the effects of carbon dioxide and medical air for abdominal insufflation on respiratory parameters in xylazine-sedated sheep undergoing laparoscopic artificial insemination. n z vet j. 2018 mar 28:1-12. doi: 10.1080/00480169.2018.1458661. 20. perkins nr, hill jr, pedrana rg: laparoscopic insemination of frozen-thawed semen into one or both uterine horns without regard to ovulation site in synchronized merino ewes. theriogenology 1996;46:541-545. sathe sr: sudden death secondary to bladder rupture in an ewe undergoing laparoscopic insemination procedure-a case report. j vet med surg2018; 2:19. doi: 10.4172/2574-2868.100019. clinical theriogenology • volume 10, number 3 • september 2018 218 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice clinical theriogenology 2022; 14: 18 introduction for most livestock species, there is an economical influence to have a certain sex of offspring. livestock producers may increase the probability for progeny of the most desired sex by various means of manipulating the breeding processes. there are many options to preselect sex of offspring, including embryo transfer, sex-sorted sperm, and preimplantation genetic testing.1,2 in equine industry, desired sex of offspring is dependent on the intended sport. for example, majority of top performing (polo) mares can be inseminated and capable of producing embryos concurrent to their sport careers for transfer to broodmare recipients, thereby maintaining financial productivity.3 colts are more desirable among racing thoroughbreds because of higher sale prices and earning potential, and ability to be used as a breeding stallion after a successful career; a thoroughbred mare can only produce 1 foal per year, whereas a stallion can breed many mares in a year.4 breeding restrictions placed on thoroughbreds registered in jockey club also influence the desire for colts. jockey club does not approve artificial insemination (ai), cloning, and embryo transfer for thoroughbreds.5 registered thoroughbreds are required to be conceived only by natural breeding (‘live cover’), making it difficult to manipulate the chances for a particular sex of a resulting foal. given breeding restrictions like those placed on thoroughbreds, breeders consider other methods to increase the probability of desired sex offspring. period between breeding and ovulation (pbbo) had an influence on resulting foal sex.6 this approach can be a useful tool for breeders of horses such as influence of period between breeding and ovulation on foal sex ratio sabrina hall,a pablo jarrin-yepez,a liesel schneider,b jessica klabnik-bradford,a william riddle,c woodrow friend,c maria schnobrich,c tulio pradoa acollege of veterinary medicine, bdepartment of animal science university of tennessee, knoxville, tn; crood and riddle equine hospital, lexington, ky abstract objective was to determine the effect of period between breeding and ovulation (pbbo) on the sex of the foal. foaling records (n = 288) from 2015 2018 breeding seasons and from 2 veterinary hospitals were used. there were 122 (42%) fillies and 166 (58%) colts. foal sex records were categorized into 3 groups based on pbbo < 24 hours, 24 48 hours, and > 48 hours. there was an effect (p < 0.0001) of pbbo on the probability of the foal being a colt. if the pbbo was < 24 hours, the odds for a colt were 23.3 times higher compared to > 48 hours group (or = 23.6, 95% ci: 3.0, 185, p = 0.003). additionally, if the pbbo was < 24 hours, the odds for a colt were 4.6 times higher compared to 24 48 hours group (or = 4.6, 95% ci: 2.0, 10.5, p = 0.0003). there were no differences (p = 0.14) between odds for a colt between 24 48 and > 48 hours groups. confounding variables such as sire, age of dam, size of ovulated primary follicle, and type of semen (fresh or chilled) were not significant. results indicated that the closer a mare is bred to ovulation with fresh semen, it is more likely that she will have a colt. keywords: ovulation, sex-ratio, mare, stallion, natural breeding the thoroughbred where natural breeding is necessary. this can also be useful as a more economical method in preselecting foal sex. this retrospective study tested the merit behind the theory that the pbbo has an influence on resulting foal’s sex. we hypothesized that closer a mare is bred to ovulation, the more likely that she will have a colt. materials and methods data from 2 veterinary hospitals for 2015 2018 breeding seasons were used. electronic medical records included name of the mare, breed, date of birth, reproductive status, information regarding ovulation induction medications (type and date), hormones used, date of breeding, date ovulation detected, estimated period in hours from breeding to ovulation, number of ovulations, size of largest primary follicle, type of semen (fresh or chilled) used, stallion name, postbreeding treatments, fetal sex, sex of foal, and veterinarian. breeding records of twin pregnancies were not used. animals two-hundred-eighty-eight foaling records were analyzed. there were 279 thoroughbreds, 5 american quarter horses, 2 paso finos, 1 arabian, and 1 appaloosa mare. mares bred were between 3 and 22 years. every thoroughbred mare was inseminated by natural breeding. nine mares represented other breeds and were artificially inseminated (fresh or chilled semen). one hundred seven stallions were used to breed 288 clinical theriogenology 2022; 14: 19 mares. stallions were between 3 and 24 years. there were 122 (42%) fillies and 166 (58%) colts. information selected periods between ovulation and breeding, sire, semen type (fresh or chilled), size of primary follicle, and mare’s age. pbbo was categorized into 3 groups: mares that ovulated < 24 hours after breeding (group 1), mares that ovulated 24 48 hours after breeding (group 2), and mares that ovulated > 48 hours after breeding (group 3). sires were compared within each group regardless of breeding method (natural breeding or ai). data analyses freq procedure was utilized to descriptively analyze frequencies of foal sex in relation to other possible explanatory variables. additionally, freq procedure was utilized to assess number of each stallion’s breeding. each stallion was assigned to groups to test if frequently used stallions tended to yield more male or female foals. finally, for inferential statistical analyses, separate, single variable generalized linear mixed models with binary distribution and logit link were used in the glimmix procedure of sas 9.4 (sas institute, cary, nc) to model the probability of a foal being a colt. if variables were individually associated with foal sex, they were added into a model and manually eliminated in a backward fashion to elicit the most parsimonious explanatory model. the ilink function was used to convert logits to mean probabilities. model adjusted probability to yield a colt and standard errors are presented. significance was set at alpha = 0.05. records for mares that conceived twins were not used for statistical analysis due to difficulty in identifying those that were fertilized first. results period between breeding to ovulation descriptive statistics for the number of colts and fillies born to dams with each pbbo are provided (table). there was an effect (p < 0.0001) of pbbo on the probability of having a foal colt. if the pbbo was < 24 hours, the odds for having a colt were 23.6 times higher compared to the odds for a colt in the > 48 hours group (or=23.6, 95% ci: 3.0, 185, p = 0.003). additionally, if the pbbo was < 24 hours, the odds for a colt birth were 4.7 times higher compared to the odds for a colt in the 24 48 hours group (or = 4.7, 95% ci: 2.0, 10.5, p = 0.0003). there was no difference (p = 0.14) between odds for a colt in 24 48 hours and > 48 hours pbbo groups (figure). table. descriptive statistics reporting the number (percentages) of colts and fillies born to dams bred within pbbo categories pbbo (hours) colts fillies total < 24 156 (64%) 65 (36%) 242 24 48 9 (28%) 23 (72%) 32 > 48 1 (7%) 13 (93%) 14 figure. model-adjusted probabilities for the outcome of a colt foal were different (p < 0.0001) for pbbos of < 24 hours, 24 48 hours, and > 48 hours. error bars represent 1 se of the mean, and bars with similar letter are not different at α = 0.05. clinical theriogenology 2022; 14: 20 sire for the 288 foaling records, there were 107 exceptional stallions used as sires. of the 107 sires, 19 were utilized to sire 5 or more foals. these 19 stallions were evaluated to determine if there was an association between sire and foal sex. sire was not associated (p = 0.87) with the probability for a foal to be a colt. age of dam age of dam ranged from 3 to 22 years (mean = 8.56, median = 7.98). age was considered a possible predictor of foal sex. for the purpose of analysis, mares’ age was categorized into 4 groups: mares ≤ 4 years (category 1); mares 4.1 7.9 years (category 2); 8 11.9 years (category 3); and > 12 years (category 4). there were 90, 93, 62, and 45 foals born to mares within the age categories 1, 2, 3, and 4, respectively. there was no relationship (p = 0.76) between mare age and foal sex outcomes. type of semen/method of insemination there were 2 types of semen utilized in the dataset: fresh and chilled. there were 9 foals conceived using chilled semen compared to 279 conceived with fresh semen via natural breeding. there was no effect (p = 0.15) of semen type on foal sex. although there was likely insufficient power to detect a difference in proportion of male or female foals to semen type, numerically 6 of 9 (67%) foals conceived with chilled semen were fillies, compared to 116 (42%) fillies of 279 conceived via natural breeding. size of ovulated primary follicle size of primary follicle ranged from 30 to 52 mm (mean = 40.08 mm, median = 40.0 mm). for analyses, follicle size was categorized into 3 groups based upon distribution of values. categories 1, 2, and 3 corresponded to sizes 30 35, 35.1 42.9, > 43 mm, respectively. there was no association (p = 0.28) between primary follicle size category and foal sex. use of ovulation inducing hormones ovulation inducing hormones were utilized in 68 conceptions. these hormones included human chorionic gonadotropin (hcg), (2,500 iu; iv hcg, chorulon: intervetinc, unterschleissheim, germany), deslorelin acetate (1ml im; sucromate, thorn bioscience llc, louisville, ky) and the combination of hcg and deslorelin acetate (sucromate, thorn bioscience llc) at the same dose. number of pregnancies after use of each hormone or hormone combination was 8, 58, and 2, respectively. we analyzed the binary variable (yes or no) of hormone used with regard to foal sex outcomes. there was no association (p = 0.50) of hormone use and foal sex. full model although the only variable associated with foal sex was pbbo, we ran the full model including age of dam, size of primary follicle, use of ovulation inducing hormones, and pbbo to determine if there were any major confounding variables. in this full model, only pbbo was importantly associated (p < 0.0001) with foal sex. other variables did not affect foal sex, nor did they largely impact or confound the effect of pbbo. discussion influence of pbbo on foal sex support research conducted in humans and many animal species. other factors tested, such as the sire and age of dam, did not influence sex ratio, although some studies have reported otherwise. data from several studies in humans have indicated that if pbbo is < 24 hours, there was either an equal sex ratio or a skew towards males.7 compared to y, x sperm are more resilient, making it more likely for them to succeed in achieving fertilization under stressful environmental conditions.6 vulnerability of y sperm is attributed to the act of early capacitation and consequently, due to prolonged stay in uterus and oviduct.6 in humans, y-bearing sperm had a substantially higher motility and swam through cervical mucus more efficiently than x-bearing sperm, and possibly provided more opportunities to fertilize ova around ovulation.7 in deer, decreases and increases in sex ratio were observed based on early and late natural breeding, respectively. mating that occurred within 36 hours of the onset of estrus (early), there was a decrease (27%) in number of male fawns. in mating that occurred within 37 96 hours after the onset of estrus (late), there was an increase (70%) in number of male fawns. pbbo effect in cattle was inconsistent.6,8 in dairy cattle, there was an insignificant difference in sex ratio in various insemination-ovulation periods; early: 36 20 hours prior to ovulation; intermediate: 20 28 hours before ovulation; late: 8 12 hours after ovulation resulted in 50, 50, and 55% females respectively.9 this effect has rarely been studied in the mare, furthermore the sample was not adequate.10 however, sex ratio was not different even when a larger sample size (n = 433) was used.11 embryos (n = 16) derived from ovulations before 48 hours from ai resulted in more male offspring (57% males versus 43% females) than those (n = 10) that ovulated 48 hours after ai (30% males versus 70% females).12 there was a significant difference among those embryos that were fertilized after 48 hours from ai to those that were fertilized before 24 hours12 and no significant difference between those that ovulated between 24 and 48 hours. this was speculated to be due to unequal amount of resilience among x and y sperm postbreeding, a possible reason for conflicting results on the impact of length of pbbo on sex ratios, particularly, in cattle that are synchronized for breeding. result was also not significant in horses that tested this hypothes.1 we suggest that pbbo has significant impact on the sex of the resulting foal, possibly due to the early capacitation of the y sperm. in horses, there may be an association among individual sires and sire’s breed on secondary sex ratio.13 foaling records (n = 4,491) from 92 stallions (thoroughbred, arabian, and akhal-teke) indicated that sire’s breed had an influence on offspring sex ratio. arabian and thoroughbred stallions were reported to have higher (63%) or lower (46.1%) offspring sex ratio than the expected 50%. akhal-teke stallions had 53.9%, not significantly different from the hypothetical expected 50%.13 results of the aforementioned study indicated that the probability of conceiving a certain foal sex can be increased clinical theriogenology 2022; 14: 21 with the use of semen from stallions that have a proven skewed ratio of progenies, although our results did not suggest that individual stallions are more likely to produce a specific sex of foal. this was possibly due to the lower number of stallions with multiple breeding records used in this analysis. age of the mare did not influence foal sex ratio. however, in a larger a study (n = 59,950) sex ratio decreased with an increase in mare’s age.14 more fillies were born to mares that were > 15 years compared to younger mares.14 it was suggested that mare’s age had a marked influence on offspring sex ratio.14 sex ratio of the offspring of the mare have been known to markedly deviate from the 1:1 ratio in situations of decreased body condition of the mare at conception.10 age of the mare could be correlated with a decreased condition or nutritional status that would skew resulting sex ratios at conception.10 there were not enough numbers of ai in our study to compare among methods of natural breeding and ai on the impact of the resulting sex ratio in horses. however, it has been suggested that sex ratio may not be different between frozen and fresh sperm use in women, and overall, ai appeared to favor male offspring (60%).7 evidence is accumulating that estrus synchronization or ovulation induction treatments in cattle may influence sex ratio.6,8 similar reports are not available for mares. conclusion closer a mare is bred to ovulation with fresh semen, the more likely that she will have a colt supporting the theory of resiliency of the x sperm and the long-term vulnerability of the y sperm within the mare reproductive tract. other possible factors (sire, age of dam, method of insemination, size of ovulated primary follicle, or use of ovulation inducing hormones) may not impact foal sex ratio. conflict of interest none to declare. references 1. panarace m, pellegrini ro, basualdo mo, et al: first field results on the use of stallion sex sorted semen in a large-scale embryo transfer program. theriogenology 2014;81:520-525. 2. herrera c: clinical applications of preimplantation genetic testing in equine, bovine, and human embryos. j equine vet sci 2016;41:2934. 3. pashen rl, lascombes fa, darrow md: the application of embryo transfer to polo ponies in argentina. equine vet j 1993 (suppl15);119-121. 4. chezum b, wimmer b: roses or lemons: adverse selection in the market for thoroughbred yearlings. rev econ stat 1997;79:521-526. 5. the american stud book principal rules and requirements. jockey club interactive registration, 2021. (accessed 7 may 2021). 6. rorie rw: effect of timing of artificial insemination on sex ratio. theriogenology 1999;52:1273-1280. 7. sampson jh, alexander nj, fulgham dl, et al: gender after artificial induction of ovulationand artificial insemination. the american fertility society 1983;40:481-484. 8. rorie rw, lester td, lindsey br, et al: effect of timing of artificial insemination on genderratio in beef cattle. theriogenology 1999;52:1035-1041. 9. roelofs jb, bouwman eb, pedersen hg, et al: effect of time of artificial insemination on embryo sex ratio in dairy cattle. anim reprod sci 2006;93:366-371. 10. aurich c, schneider j: sex determination in horses current status and future perspectives. anim reprod sci 2014;146:34-41. 11. davies morel mcg, newcombe, jr, holland sj: factors affecting gestation length in the thoroughbred mare, anim reprod sci 2002;74:175-185. 12. drexler ap, prado tm, amelse ll, et al: effects of time of insemination relative to time of ovulation on embryonic sex ratio in mares. clinical theriogenology 2016;8:357. 13. gharagozlou f, akbarinejad v, youssefi r, et al: effect of sireassociated factors on secondary sex ratio of offspring in equine. j equine vet sci 2014;34:926-929. 14. santos mm, maia ll, nobre dm, et al: sex ratio of equine offspring is affected by the ages of the mare and stallion. theriogenology 2015;84:1238-1245. clinical theriogenology 2022; 14: 31 acknowledgement dr. douglas’ theriogenology residency training program is sponsored by the american kennel club canine health foundation and the theriogenology foundation. references 1. johnston sd: performing a complete canine semen evaluation in a small animal hospital. vet clin of north am. small anim pract 1991;21:545-550. 2. eilts be, paccamonti dl, pinto c: artificial insemination in the dog. in: root kustritz mv, editor. the practical veterinarian: small animal theriogenology. st. louis: butterworth-heineman; 2003. p. 61-95. 3. johanisson e, campana a, luthi r, de agostini a: evaluation of ‘round cells’ in semen analysis: a comparative study. hum reprod update 2000;6:404-412. 4. johnston sd, root kustritz mv, olson pns: canine and feline theriogenology. philadelphia: wb saunders: 2001. p. 287-306. 5. barth ad, bowman pa: the sequential appearance of sperm abnormalities after scrotal insulation or dexamethasone treatment in bulls. can vet j 1994;35:93-102. 6. henning h, masal c, herr a, et al: effect of short-term scrotal hyperthermia on spermatological parameters, testicular blood flow and gonadal tissue in dogs. reprod dom anim 2014;49:145-157. validation of an in-clinic immunoassay for measurement of canine progesterone validation of an in-clinic immunoassay for measurement of canine progesterone elizabeth schooley, edward obare, maha yerramilli, jun li, tiffany glavan idexx laboratories, inc., westbrook, me idexx recently introduced an immunoassay to measure canine progesterone on the catalyst® suite of chemistry analyzers. objectives were to: (1) validate lc ms for measurement of canine plasma progesterone concentrations; (2) evaluate accuracy of the in-clinic catalyst® progesterone test compared to results obtained by lc ms; and (3) evaluate repeatability of the catalyst® progesterone test across multiple analyzers. the lc ms progesterone assay was validated in accordance with fda guidelines for linearity, carryover, sensitivity, selectivity, accuracy, and precision. chromatographic separation was performed on a shimadzu nexera ultra high-performance liquid chromatograph using acquity beh 300 c4 1.7 µm, 2.1 x 100 mm analytical column with analyte detection by a sciex api 4000 mass spectrometer with multiple reaction monitoring with progesterone-[13c3] as an internal standard. the dynamic range of the lc-ms assay is 0.2 40 ng/ml and accuracy is traceable to the nist standard (standard reference material 971). the assay exceeded fda criteria of intra/inter day accuracy/precision, demonstrating imprecision of less than 7% cv and accuracy within 95 108% of the expected value over the analyte range. lower limit of quantification and linearity studies support a dynamic range of 0.2 40 ng/ml (s/n e 10; r2 = 0.99). the lower limit of quantitation was 0.2 ng/ml and had a linearity with an r2 value of 0.99 or better. there was no evidence of carryover or interferences of progesterone/progesterone[2,3,4-13c3] is m/z transitions observed. to evaluate the catalyst® progesterone test compared to lc ms, 107 blood samples were collected from peri ovulatory bitches presented to 3 veterinary hospitals for breeding management. within 30 minutes after collection, lithium heparin plasma was separated from erythrocytes. plasma samples were analyzed both on the catalyst® progesterone assay (within 48 hours after collection) and lc ms at idexx r&d (within 1 week after collection). precision was assessed by repeated analysis (80 total replicates each) of 3 control fluids in the range of clinical interest. statistical analyses were performed using jmp® 14.0.0. results are reported with 95% confidence limits in parentheses. mean progesterone obtained by lc ms was 5.6 ng/ml (range: 0.2 to 19.4 ng/ml). catalyst® progesterone assay mean progesterone was 5.9 ng/ml (range: 0.2 to 19.3 ng/ml). passing bablok regression analysis had an intercept -0.07 ng/ml (-0.2 0.02), slope 1.07 (1.02 1.12), and tau 0.89. pearson’s correlation coefficient was 0.98. the mean difference (bland altman plot) was 0.27 ng/ml (0.06 0.47; sem 0.1 ng/ml). increased variation at the higher end of the dynamic range (> 16 ng/ml), where unlikely to impact clinical decisions. in the precision study, mean concentrations of 0.43 ng/ml (± sd 0.07), 3.5 ng/ml (± sd 0.28) and 16.0 ng/ml (± sd 1.35). catalyst® progesterone test can be used for in clinic measurement of canine plasma progesterone concentrations, due to its good correlation to the reference method, lc ms, and good precision in the range of clinical interest. keywords: progesterone, canine, lc ms, immunoassay 413 clinical theriogenology • volume 11, number 3 • september 2019 414clinical theriogenology • volume 11, number 3 • september 2019 omniblank: uterine body fetal reduction in a mare: cranio-cervical dislocation via colpotomy   uterine body fetal reduction in a mare: cranio-cervical dislocation via colpotomy sabrina hall, jessica klabnik, pablo yepez, tulio prado department of large animal clinical sciences, college of veterinary medicine university of tennessee, knoxville, tn abstract a 15-year old american saddle horse mare, pregnant (~ 60 days) with twins, was referred. transrectal ultrasonography revealed the presence of 1 fetus in uterine body and the other in right uterine horn. inconvenient location (uterine body) of the fetus made it difficult to apply established techniques for twin reduction. cranio-cervical dislocation via colpotomy was performed on the twin located in the uterine body. heartbeat was not detected at 48 hours postprocedural transrectal ultrasonography. remaining twin was delivered at term without complications. keywords: mare, colpotomy, cranio-cervical dislocation, twin, body pregnancy background early (< 30 days) diagnosis of twin pregnancy is of utmost importance for successful reduction. late diagnosis necessitates the use of more invasive procedures (transvaginal or transcutaneous ultrasound-guided fine needle aspiration).1 furthermore, these techniques are not designed for uterine body fetal (> 60 days) reduction. most twin reduction techniques were optimal for fetuses located unilaterally or bilaterally at the base of uterine horns.1 for the first time, via colpotomy,2 manual transuterine cranio-cervical dislocation technique was applied to successfully reduce a uterine body pregnancy. case presentation a 15-year old american saddle horse mare was referred for the reduction of a 60-day twin pregnancy. mare was pasture bred for 5 days and 24 days after the last breeding, presence of twins were detected via transrectal ultrasonography. one embryo (~ 23 24 days) was present in the right uterine horn and the other (18 19 days) in the caudal uterine body. in the past, mare had multiple ovulations and twin pregnancies and the previous season’s twins were both lost as a complication to ‘vesicle pinching’ technique. knowing mare’s last year outcome, it was decided not to intervene, with the possibility that the twin located in uterine body would resorb or die on its own, since singleton pregnancies established in the caudal uterine body have an extremely low chance of survival (17%).3 mare was examined on days 32 and 45 of pregnancy by transrectal ultrasonography. at day 32 examination, normal growth and heartbeats of both twins were observed. at day 45 examination, both twins had normal growth and heart rates. however, uterine body twin had grown into right uterine horn, closer to the other fetus. at day 55 of pregnancy, an attempt was made to interfere with the growth of uterine body twin by applying pressure to the fetus with an ultrasound probe. mare was then given intravenously 1.1 mg/kg flunixin meglumine (prevail™, vet one, boise, id) and orally 0.044 mg/kg altrenogest (regu-mate™, merck animal health, summit, nj). in the days following the procedure, the mare was examined by transrectal ultrasonography and it was apparent that the uterine body twin had grown and it had normal heart rate, hence the mare was referred. one fetus was located in the caudal uterine body and the other another was at the base of right uterine horn. although cranio-cervical dislocation (ccd) of fetus can be performed transrectally or intraabdominally,4 neither approach could be used for a body pregnancy, because of fetal location. a colpotomy approach, typically used for ovariectomy, was performed to facilitate the ccd of a fetus located in the uterine body. treatment mare was initially sedated with intravenous treatment of 0.5 ml butorphanol (torbugesic®, fort dodge animal health a division of wyeth, new york, ny) and 0.5 ml detomidine (dormosedan, clinical theriogenology • volume 12 number 4 • december 2020522   kalamazoo, mi). a presurgical dose of 15 ml of ceftiofur (excede, zoetis, kalamazoo, mi) was given intramuscularly in the left pectoral muscle. vulva was cleaned using a betadine scrub. a caudal epidural was performed using a combination of 3 ml lidocaine (lidocaine, vet one, boise, id) and 0.8 ml (80 mg) xylazine (vet one, boise, id). buscopan ([3.5 ml] boehringer ingelheim vetmedica, inc., st. joseph, mo) was given intravenously for muscle relaxation, and 1 ml detomidine was given intravenously for a longer duration of sedation. vaginal branch of the internal pudendal artery was palpated,2 and a lidocainesoaked gauze was applied cranial and dorsal to the artery2 for several minutes to provide a topical block at the selected incision site. next, a short, full-thickness incision was made with a number 10 scalpel blade through the dorsolateral aspect of fornix of vagina, at 9.0 or 10.0 hour position, ~ 2 cm dorsolateral to external cervical os and dorsal and cranial to vaginal artery.2 incision was stretched by blunt dissection until a hand could pass through it to grasp the uterus. twin fetus located in the uterine body was transuterinely identified and its cervical spine was manually dislocated. subsequently, transrectal ultrasonography was performed; heartbeat was slow and faint after the procedure and completely subsided within the next 2 days. fetus located in the right uterine horn, was also examined via transrectal ultrasonography immediately after surgery and had normal heartrate. vaginal wall incision was not closed. caslick’s procedure was performed to prevent pneumovagina and contamination of the vestibule, vagina, and abdomen. after surgery, the patient was treated intravenously with 10 ml flunixin meglumine and orally with 10 ml altrenogest once daily for 4 days. mare was kept in the hospital for 3 days; it was not allowed to lie down, to decrease the likelihood of evisceration through the incision and mare was evaluated (transrectal ulrasonography) daily until she was discharged. outcome twin located in the right uterine horn continued to grow through full duration of pregnancy and was born without complications at an estimated 342 days of pregnancy. fetus that had undergone ccd was not recovered at the time of birth of the surviving twin. in conclusion, colpotomy approach, typically used for ovariectomy, was successfully applied to facilitate the ccd of a fetus located in the caudal most aspect of uterine body. this case provided evidence that a ccd via colpotomy can be successful and is a viable option for safe reduction of a fetus located in the uterine body, a location that is difficult to reach by conventional methods of twin reduction. discussion twin pregnancies are an important cause of abortion in mares5 and accounted for 6 30% of pregnancy loss.4 extensive application of ultrasonography in pregnancy diagnosis and suitable twin reduction methods have reduced pregnancy loss due to twins to 5.4%.6 twin pregnancies most commonly result from double ovulations that are either synchronous or asynchronous.7 asynchronous ovulations are possibly due to prolonged lh peak.7 twin pregnancies resulted in early fetal loss, late term abortions, and birth of small or growth retarded foals and rendered rebreeding difficult.8 damage to the reproductive tract of the mare can occur from late term abortions that often resulted in foaling difficulties.8 a twin foal that is born alive is likely to be smaller than normal as a result of intrauterine growth retardation8 and have a poor survival rate without immediate critical care.8 therefore, a twin pregnancy must be reduced early in pregnancy to avoid late term abortion. twins are typically located unilaterally or bilaterally in the base of either uterine horn.1 in rare cases, as in the present case, a uterine body fixation is possible in a twin pregnancy. uterine body pregnancies and twin pregnancies are undesirable in mares. uterine body pregnancies in the mare have seldom been reported and often resulted in loss of pregnancy due to inability of placenta to expand.3 however, fetus located in the cranial aspect of uterine body had a higher likelihood of survival (possible placental expansion within the bifurcation) than fetus located in the caudal aspect.3 although prostaglandins have been used successfully to eliminate twin pregnancies before endometrial cup formation to facilitate mare’s return to estrus,3 other methods were developed to reduce 1 of the twins. most twin reduction procedures for mares are ideal for unilateral or bilateral twins in the base of the uterine horn(s).1 later in pregnancy (> 45 days), it is possible to apply transvaginal clinical theriogenology • volume 12 number 4 • december 2020 523   ultrasound-guided method to perform ‘fetal stabbing’ or yolk sac or allantoic fluid aspiration.9 however, these methods had very low success rate (0 25%).9 injection of kcl into fetal heart is another procedure carried out transabdominally for twin reduction,1,4,10 with good success rate (49%), if performed between 115 -130 days of pregnancy1 and was used successfully at 200 days of pregnancy.10 transvaginal ultrasound-guided fine needle aspiration of allanto-amniotic fluid or injection of kcl into fetal heart was considered for this case. however, inability to properly place the ultrasound probe to effectively perform injection procedure precluded this approach. possibility of applying ccd technique was contemplated in this case. depending on the stage of pregnancy, it has been used either transrectally or intraabdominally via standing flank incision.4 transrectal approach was feasible between days 55 and 90 of pregnancy when the fetus can still be reached and identified. an intraabdominal approach was possible between days 58 and 110 of pregnancy after the development of fetal bones and muscles.4 transrectal and intraabdominal ccd were successful in 63 and 63.10% of cases respectively.4 these routes of performing ccd were not considered in this case due to inability to firmly grasp the fetus transrectally and increased risk of peritonitis due to distance required to reach the uterine body through laparotomy. colpotomy approach has been used to successfully for ovariectomies as a safer, more rapid alternative to other methods of open and laparoscopic ovariectomy.11 colpotomy was performed in a standing animal, under sedation, in a shorter time, requiring least instrumentation and ideal for cosmetic reasons.10 primary disadvantages to this approach is that the surgeon is operating blindly and a surgeon with a small arm diameter is required when operating on mares with a narrow pelvis, ponies, and mules.2,11 there is also the risk of evisceration and peritonitis.12 in this case, ccd technique was used in twin reduction due to its high success rate for pregnancies of 60 days and colpotomy approach was successfully applied, given the difficulty in reaching uterine body by other methods. colpotomy has been mentioned13 as a potential approach for twin reduction using ccd technique and we successfully carried out these principles. learning points  colpotomy is an alternative approach to reach uterine body.  colpotomy approach can be successfully used to reduce a twin in the uterine body by cranio-cervical dislocation of the fetus.  cranio-cervical dislocation via colpotomy approach is ideal to reduce a 60-day old twin located in the uterine body. references 1. macpherson ml, reimer jm: twin reduction in the mare: current options. anim reprod sci 2000;60-61:233-244. 2. prado tm, schumacher j: how to perform ovariectomy through a colpotomy. equine vet educ 2017;31:209-213. 3. jobert ml, leblanc mm, pierce sw: pregnancy loss in equine uterine body pregnancies. equine vet educ 2005;17:161-165. 4. wolfsdorf ke, rodgerson d, holder r, et al: success rate of post-fixation twin reduction using cranio-cervical dislocation. proc am assoc equine pract 2009;55:257-261. 5. miller a, woods gl: diagnosis and correction of twin pregnancy in the mare. vet clin north am equine pract 1988;4:215-220. 6. laugier c, foucher n, sevin c, et al: a 24-year retrospective study of equine abortion in normandy (france). j equine vet sci 2011;31:116-123. 7. davies morel mcg, newcombe jr, reynolds n: asynchronous ovulation in mares: seasonal variations in frequency. vet rec 2015;176:310-313. 8. mckinnon ao: twin reduction techniques. in: samper jc, pycock jf, mckinnon ao: editors. current therapy in equine reproduction. 1st edition, philadelphia; saunders: 2007. p. 357-373. 9. journée sl, de ruijter-villani m, hendriks wk, et al: efficacy of transvaginal ultrasound-guided twin reduction in the mare by embryonic or fetal stabbing compared with yolk sac or allantoic fluid aspiration. theriogenology 2013;80:346349. 10. bass l, wilkins j: birth of a live foal after transabdominal ultrasound-guided cardiac injection of a 200-day equine twin fetus. j equine vet sci 2014;34:549-555. clinical theriogenology • volume 12 number 4 • december 2020524   11. tate lp jr, fogle ca, bailey cs, et al: laparoscopic-assisted colpotomy for ovariectomy in the mare. vet surg 2012;41:625-628. 12. smith se, devine dv: hand-assisted laparoscopic ovariectomy and colpotomy in standing mares. vet surg 2013;42:586-590. 13. sitters s, wolfsdorf k: twin reduction: cranio-cervical dislocation. in: dascanio jj, mccue pm: editors. equine reproductive procedures. 1st edition, hoboken; john wiley & sons: 2014. p. 222-225. clinical theriogenology • volume 12 number 4 • december 2020 525 introduction reproductive surgeries are generally elective but no surgery is without risk. as such, a complete breeding soundness evaluation (bse) and a thorough review of the mare’s breeding history are warranted prior to surgery. the bse should include transrectal palpation and ultrasonography of the reproductive tract, vaginal speculum examination, digital cervical palpation, endometrial culture and cytology, and an endometrial biopsy. a bse not only aids in identifying factors associated with infertility, but results of endometrial biopsy may also offer a prognosis for future fertility. this is an important consideration, as preexisting degenerative changes to the endometrium are irreversible and associated with a poor prognosis of overall fertility, making the reproductive surgery futile. reviewing a mare’s reproductive history helps to ensure that the cause of infertility cannot be attributed to stallion effect, inappropriate breeding management, endometritis or degenerative changes in the endometrium. in cases of unexplained infertility, additional diagnostic techniques including hysteroscopy, laparoscopy, and hormone assays may be considered to ensure that further reproductive surgical procedures are not performed unnecessarily. the aim is to describe common clinical conditions unresponsive to medical management and further highlight diagnostic approaches and surgical treatment options. caudal reproductive tract pneumovagina the 3 natural physical barriers from the caudal reproductive reproductive surgeries: why necessary and what to perform for specific problems justin mcnaughten hanover shoe farms, inc., hanover, pa abstract in mares, causes of infertility and subfertility are often multifactorial. numerous congenital, pathological, and neoplastic conditions affecting the reproductive tract have been associated with decreased fertility. despite a clinician’s best attempt to resolve or improve fertility through medical management, incorporation of an appropriate surgical technique is often indicated. reproductive surgeries are often necessary to address underlying anatomical abnormalities or eliminate pathological conditions negatively affecting mare fertility. the main goal of any reproductive surgery should be to improve, restore, or protect a mare’s future fertility. this review will focus on the importance of surgical intervention in the chronically infertile mare. keywords: reproductive surgery, pregnancy, uterus, mare, infertility tract to the uterus are the vulva, vestibulo-vaginal sphincter, and cervix. the vulva and vestibulo-vaginal sphincter represent the primary anatomical protection from uterine infection.1 any alternations in perineal conformation or disturbances in the natural anatomical uterine defense mechanisms can be detrimental to reproductive performance. incompetence or a breach in 1 or more of these barriers may arise via effects of genetics, age, body condition, parity, and trauma.2 loss of the airtight vulvar seal or compromise of the vulva or vestibulo-vaginal sphincter predispose the mare to pneumovagina. consequently, pneumometra, inflammation, and infection resulting in acute or chronic endometritis are common sequelae that are unlikely to respond to medical treatment unless the underlying anatomical problem is corrected. diagnosis of pneumovagina involves visual and functional evaluation of the perineum and vestibulo-vaginal sphincter. perineum includes anus, vulva, and adjacent skin including perineal body. normal labia are full and firm and meet evenly on the midline, with 80% or more of the vulvar opening positioned below the pelvic brim.3 excessive vulvar length above the pelvic brim and an increased angle of declination decreases coaptation of the vulvar labia. if separation of the labia elicits an audible sound there is evidence of incompetence. observation for aspiration of air and transrectal ultrasonography can be used to further confirm the presence of air within the vaginal cavity and potentially within the uterine lumen. poor perineal conformation may be exacerbated during estrus or pregnancy as the weight of the gravid uterus may alter the external anatomy. clinical theriogenology 2021; 13: 272 multiple surgical approaches have been described to treat pneumovagina, based on the severity of the condition. vulvoplasty or the caslick’s operation, is the oldest and most common reproductive surgery performed to prevent air aspiration and contamination of the reproductive tract.4,5 caslick’s procedure involves removal of a mucosal strip from the labial margin and suturing the apposed tissues, resulting in formation of a permanent airtight seal.2 temporary approaches utilizing staples, super glue, and suturing the vulvar labia without the removal of a mucosal strip have also been described but are often ineffective. a breeding suture may be placed to protect the integrity of the caslick’s during reproductive procedures. fistula formation and dehiscence may occur, especially in older mares with tissue fibrosis.2 perineal body reconstruction, also referred to as an episioplasty or a gadd’s procedure, is utilized when caslick’s fails to resolve the pneumovagina due to insufficient perineal body tissue or vestibulo-vaginal sphincter incompetence.2 perineal body is the musculofibrous shelf of tissue between the floor of the anus and the roof of the vestibule; its integrity can be compromised via foaling trauma or repeated stretching of the vulvar and vestibular constrictor muscles in older multiparous mares.6 diagnosis of a weak or torn perineal body is by digital palpation. although 4 8 weeks’ sexual rest following episioplasty is recommended to allow for adequate healing, the procedure can be successfully performed on the day of ovulation in the at-risk breeding mare.7 perineal body transection, or the pouret’s operation, is reserved for mares with extremely poor perineal conformation and in which pneumovagina fails to resolve following vulvoplasty or episioplasty.2 advanced age and increased parity exacerbate the degree to which the anus sinks, pulling the vagina cranially and the vulvar labia above the pelvic brim.2 perineal body transection frees the attachments between the rectum and the dorsal vaginal vault, minimizing the pull of the sunken anus on the vagina and restoring the vulva to a more normal vertical position.8 modification of pouret’s technique incorporates a horizontal suture to correct pneumovagina; 3 this modified technique substantially improved vulvar coaptation, perineal position, and reproductive outcomes in 14 of 18 mares. authors suggest that artificial insemination may be performed immediately postoperatively, whereas natural breeding may occur within 3 weeks after surgery.3,8 unfortunately, a caslick’s or gadd’s procedure often require the surgery to be repeated for the duration of the broodmare’s career. severe perineal tearing may arise if an episiotomy or the dorsal vestibule are not opened prior to foaling.2 vesicovaginal reflux failure of the mare to completely void urine results in vesicovaginal reflux (vvr) or urine pooling. urovagina or urometra is accumulation of urine in the cranial vagina or uterus, respectively. normal outflow of urine is inhibited when the cranial vagina slopes cranioventrally. age-related changes, poor body condition, foaling trauma, excessive caslick’s length, and incorrect vulvoplasty have been identified as predisposing factors.9,10 detrimental effects on fertility are multifaceted and persistent urine contamination may reduce the mare’s ability to conceive or carry to term. continual contact with urine can lead to local tissue inflammation, endometritis, or have a noxious effect on sperm.11-13 a definitive diagnosis is easily achieved when urine is visualized in the vaginal fornix via vaginoscopy. however, urine pooling may be intermittent in some mares and depends on the tone of the reproductive tract that is cyclically altered under the influence of estrogen.14 although repeat examinations during estrus have been suggested to aid in the diagnosis, measuring intrauterine fluid creatinine concentration is an objective means of diagnosing urometra.14,15 in the postpartum mare, urine pooling may be transient and self-limiting for up to 30 days postfoaling.14 in cases of infertility in which urine pooling has been diagnosed, or in postpartum mares that continue to experience urometra, a urethral extension surgery may be indicated.14 a presurgical reproductive evaluation that includes endometrial biopsy is recommended, as chronic urine pooling is associated with periglandular fibrosis.14 the goal of any of the described surgeries to manage vvr is to reduce the potential for cranial movement of urine. in mild cases of vvr, caudal relocation of the transverse fold has been described and may be indicated for mares with minor conformational deficiencies or estrogen-induced transient relaxation of the reproductive tract.9 however, relocation of the transverse fold is often only temporary and may be contraindicated in mares with significant conformational issues.13 a recent technique16 involves injecting polyacrylamide hydrogel into the transverse fold, thus creating a bulge that remained palpable for up to 60 days postinjection. authors suggest that bulking up the surrounding tissue may serve as an alternative to surgery in mild cases of vvr.16 the more traditional approach to surgically correct vvr is creation of a urethral extension to transport urine further caudally before expulsion from the urethra. there are numerous surgical techniques describing formation of a tubular tract from the vestibular mucosa.6 regardless of the technique, caudal urethral extensions are prone to the development of fistulae and mares should be assessed approximately 2 weeks postoperatively13,17 although surgery is often delayed to the end of the breeding season, mares may be bred in the same estrous cycle as surgery and have adequate pregnancy rates.17 cervical trauma cervix is the third and most cranial anatomical barrier to the uterus. in diestrus and in pregnancy, under the influence of progesterone the cervix forms a seal protecting the uterus from the external environment and ascending infection. cervical clinical theriogenology 2021; 13: 273 lacerations may occur when cervix fails to sufficiently dilate during foaling, abortion, or dystocia. excessive stretching of the muscular layer of the cervix during foaling may result in cervical incompetence.18 cervical trauma may predispose the mare to chronic endometritis and infertility.18 mares able to achieve pregnancy early may fail to carry the foal to term as cervical incompetence increases the risk of ascending placentitis or abortion.19 when a cervical laceration is suspected, evaluation should be performed while the mare is in diestrus with maximal cervical tone. visual assessment is often unrewarding and manual palpation is required to make an accurate diagnosis. circumferential palpation of the cervix is performed by inserting the index finger into the cervical lumen and the opposing thumb on the vaginal side of the cervix.20 palpation of each lateral half of the cervix may identify adhesions, thinning of the fibromuscular layer, or full-thickness defects in the cervical wall, and allow for estimation of defect length.18,20,21 not all cervical lesions require surgical repair. small lesions may be incidental allowing mares to conceive and proceed with an uncomplicated pregnancy. surgical intervention is indicated in mares with a history of infertility and an appreciable cervical defect or lacerations involving 50% or more of the vaginal cervix.20 in a retrospective analysis of surgical outcomes of mares diagnosed with cervical lesions, 61.9% of mares that underwent surgical repair delivered a live foal compared to 48.4% of mares that did not have surgery. this study also demonstrated that rapid diagnosis and earlier surgical intervention improved live foal outcome.22 surgical approach to cervical defects is based on surgeon preference and lesion location.18,23 if multiple defects exist, individual repairs should be staged 3 4 weeks apart.21 surgery is performed during diestrus to improve visualization of the defect margins.18 alternatively, surgery may be performed under the influence of supplemental progesterone or several days post-breeding in an effort to maintain breeding efficiency.18 postoperative complications are rare, but adhesions within the cervical lumen, stenosis, uterine infections, and cervical incompetence have been reported.18,24 four weeks of sexual rest is recommended before rebreeding. artificial insemination is favored if permitted by the breed registry; otherwise, a breeding roll should be incorporated for natural cover to minimize possible trauma.24 although surgical correction improves cervical integrity, lesions heal by fibrosis and as such, the elasticity of the tissue is permanently reduced.25 consequently, an estimated 20 50% of surgically corrected cervical tears may require a subsequent surgery postfoaling.18,20,26 a recent report26 identified decreased foaling rates with each successive surgery.26 in cases of cervical incompetence, a cervical cerclage suture in late pregnancy improved cervical function, decreased the risk of ascending placentitis and strengthened the cervix against mechanical forces.19 although no complications were reported, cervical damage may occur if the mare delivers with the suture in place.19 oviductal lesions salpingitis, hydrosalpinx, neoplasia, adhesions, cysts, and oviductal blockages have been described in the mare. postmortem studies have revealed an array of lesions affecting the oviduct. in most cases, however, the presence of a lesion could not be related to a distinct negative effect on fertility.27 oviductal abnormalities in the mare are rare but should be considered when all other potential causes of subfertility or infertility have been excluded. confidently diagnosing oviductal pathology is often difficult, as most lesions affecting the region go undetected using conventional methods. although transrectal palpation and ultrasonography offer limited diagnostic value for most oviductal abnormalities, they have aided in the diagnosis and monitoring of fimbrial, paraovarian, or uterotubal cysts.27 an investigation should include a thorough review of the breeding history. failure to achieve pregnancy associated with 1 ovary would support a presumptive diagnosis, and laparoscopic investigation may then aid in the definitive diagnosis of oviductal abnormalities. at present, the majority of cases of suspected oviductal pathology are based on diagnosis by exclusion. in the past decade, credence has been given to oviductal blockages as a cause of mare infertility. although experimental methods have been described to assess oviductal patency, in a clinical setting, suspected oviductal blockage is often a diagnosis of exclusion.28,29 techniques described to resolve the presumptive condition include catheterization of the oviduct via laparotomy, laparoscopic application of prostaglandin e2 (pge2), hysteroscopic hydrotubation, and deep-horn application of misoprostol (pge1) to the oviductal papillae.30-34 laparoscopic application of pge2, hysteroscopic hydrotubation, and deep-horn application of pge1 are the most widely accepted techniques. in a study, 93% (14/15) of mares with unexplained infertility conceived in the same or subsequent breeding season following laparoscopic application of pge2 to the oviduct.31 hysteroscopic hydrotubation technique involves insertion of a catheter into oviductal papilla and flushing them with sterile saline in a retrograde direction;32 92.8% (26/28) of previously barren mares conceived after treatment during estrus or diestrus.35 application of pge1 to the tip of the uterine horn lumen using a deep-horn insemination pipette, during diestrus in a subset of barren mares resulted in 68% (15/22) conception within 2 estrous cycles after treatment.33 there was no significant difference in pregnancy rates between laparoscopic pge2 application and oviduct hydrotubation.36 although pregnancy outcomes attained after these treatments suggest an improvement in fertility, each technique has associated risks and variable costs. laparoscopic approaches carry the inherent risks and increased costs of surgery, as well as the cost of the pge2. in addition, postoperative stall rest may be a clinical theriogenology 2021; 13: 274 limitation for some clients. although hydrotubation avoids surgery, iatrogenic damage to the oviduct and endometritis secondary to hysteroscopy are important considerations. despite increased risks and potentially higher costs, endoscopic approaches may offer some diagnostic advantages over the minimally invasive deep-horn application of pge1. the hysteroscopic hydrotubation approach enables visualization of the uterus and may identify uterine pathologies that were previously undiagnosed.37 a major advantage of the laparoscopic approach is that it allows for direct visualization of the oviductal region.37,38 this approach may aid in the diagnosis of adhesions, neoplasia, cysts, or other lesions that may be the suspected cause of infertility. adhesions, large cysts, and acquired abnormalities may disrupt the normal anatomy and function of the oviduct. surgery can be attempted to restore anatomy and improve function. in a small subset of mares, after transection of restrictive bands of the mesosalpinx and laparoscopic pge2 application, 89% (8/9) of previously barren mares conceived and 78% (7/9) of these mares also conceived the following year.39 if a lesion is inoperable or neoplasia is suspected, unilateral ovariectomy may be warranted to improve breeding efficiency.27 in breeds in which ovum pick-up is permitted, the affected ovary may be left in place and this technique considered as a method to obtain embryos. uterine pathologies endometrial cysts endometrial cysts are generally considered nonpathological and simply a nuisance complicating pregnancy diagnosis. however, cysts may be associated with decreased reproductive performance. a significant difference was observed comparing seasonal pregnancy rates at days 14 and 40 for mares with cysts (77.6 and 71.4%) to a cohort without cysts (91.5 and 88.0%).40 the presence of cystic lesions represents underlying degenerative endometrial changes. the physical effects of endometrial cysts may impede uterine clearance, interfere with embryo migration, and inhibit normal embryonic development and placentation.41 early embryo loss has occurred when the conceptus was fixed adjacent to a cyst and uterine contact was limited.42 cystic regions inhibited microcotyledon development resulting in avillous lesions on the chorioallantois.43 the most common method of diagnosing endometrial cysts is transrectal palpation and ultrasonography. ultrasonographic appearance of cysts ranges from individual, discreet, solitary lesions to multiple, irregular-bordered, multicompartmental structures. infusion of a sterile solution into the uterus creates a contrast enabling transrectal ultrasonography to differentiate between luminal and mural cysts.41 hysteroscopy can be used to ascertain the specific location of the cysts. endometrial cyst removal has improved fertility in some mares.44-47 several techniques have been described including manual ablation, puncture, fine-needle aspiration, mechanical rupture, hysteroscopic guided electrocautery, and laser therapy.48 however, no clinical comparisons of treatment options and outcomes have been reported. manual ablation or mechanical rupture of cysts is performed during estrus or postpartum. cervical dilation must be sufficient to facilitate passage of the instrumentation and the operator’s hand. manual rupture is performed by placing the cyst between 2 fingers and tearing or crushing the cyst off the uterine wall.48 mechanical rupture is accomplished by ensnaring the cyst with suture or embryotomy wire and sawing through the base of the tissue.49 alternatively, the stalk of pedunculated cysts may be transected with endometrial biopsy forceps. hysteroscopic guided electrocautery and laser therapy have been described.44,45,50,51 this approach offers the benefit of direct visualization of cysts and possible identification of concurrent pathology.37,50 it is suggested that electrocautery is utilized for larger pedunculated cysts whereas laser therapy is reserved for smaller or nonpedunculated cysts.41 the hysteroscopic procedure is best performed during diestrus as a tight cervical seal improves uterine distension. based on clinician preference, the uterus is insufflated with air or distended with sterile fluid to allow passage of the endoscope through the uterine lumen. recently, a technique involving ethanol sclerotherapy as a potential alternative was described. endometrial cysts were emptied endoscopically, and 70% ethanol in saline solution was injected to fill 40% of the original volume.52 the suggested mechanism of action is that ethanol strips the inner layer of the cyst, inducing thrombosis of small vessels and coagulative necrosis followed by fibrosis.52 following this treatment, 87.5% (6/7) of chronically infertile mares achieved a pregnancy and delivered a foal. regardless of the technique utilized, any residual cystic tissue should be removed, and serial uterine lavage should be performed until the efflux is clear. concurrent use of antibiotics is warranted in select cases. although hemorrhage and endometritis have occasionally been associated with the procedure, recovery is rapid and pregnancy can be achieved on the same cycle as cyst removal. it is important to note that preexisting underlying uterine pathology increases the incidence of new cyst development.41 therefore, the author suggests that the procedure should be performed in preparation for the season; otherwise, owners should be made aware of the potential for recurrence. pendulous uterus the normal orientation of the mare’s uterus is horizontal and above or level with the brim of the pelvis. with increased age and parity, the mesometrium is repeatedly stretched resulting in an increasingly pendulous uterus with a ventral slope in relation to the pelvis. often, as the uterus slopes ventrally, the rectum and vagina are pulled cranially resulting in a sunken anus and poor perineal conformation. as a consequence, the clinical theriogenology 2021; 13: 275 ventrally oriented uterus lends itself to an increased risk of contamination, delayed uterine clearance, and chronic endometritis.53 as a recognized cause of infertility in the older mare, a ventrally sloping uterus can be problematic and challenging for clinicians.54 the workup for these mares is relatively straightforward; detection of a pendulous, ventrally sloped uterus via transrectal palpation should confirm the diagnosis. clinical indications include the lack of fluid resolution despite appropriate intensive management. an endometrial biopsy should be performed as a prognostic indicator. uteropexy, laparoscopic imbrication of the mesometrium, was attempted to improve uterine clearance.55 the surgical technique confirmed that the orientation of the pendulous, ventrally sloping uterus could be restored to a more horizontal position.55 in addition, uterine assessment via transrectal palpation and ultrasonography demonstrated a subjective reduction in the overall size of the uterus postoperatively. laparoscopic assessment revealed that newly formed adhesions between the mesometrium and uterus supported the horizontal uterine position.55 an improvement in perineal conformation was also noted. use of barbed suture for mesometrial imbrication resulted in similar postoperative changes; however, the study did not confirm whether adhesion formation occurred.56 laparoscopic uteropexy appears to produce favorable outcomes in chronically infertile mares. pregnancy rates from cases with available follow-up data from 2 studies were 47% (9/19) and 67% (2/3), respectively.55,56 unfortunately, effects on future fertility are unclear, due to the lack of published information and case-controlled studies. the recommended interval from surgery to rebreeding is variable amongst reports. in many cases, the chronicity of the disease would suggest that prolonged sexual rest is warranted and may optimize outcomes. this hypothesis was supported;56 2 mares that became pregnant after uteropexy conceived on their first cycle after sexual rest for 6 and 7 months, respectively.56 pioneers of uteropexy caution that despite improving uterine clearance, preexisting endometrial fibrosis may impair the ability of the uterus to support and maintain a pregnancy to term.57 in cases with poor endometrial biopsy scores, uteropexy may be beneficial to restore fertility through embryo transfer, if permitted by the breed registry. despite minimal to no intraoperative or postoperative complications being reported, the author has experienced a case with significant post-operative complications. the mare was presented for an abscess draining from the paralumbar fossa after uteropexy. additional investigations revealed that the abscess extended medially, involving the reproductive tract. stenosis of the right uterine horn, luminal intrauterine adhesions, adhesions between the uterine horn, ovary and body wall and paraovarian abscessation of the right ovary were all appreciable. uterine anomalies complete ovariohysterectomy is generally reserved as a salvage procedure to improve a mare’s quality of life when reproductive conditions fail to respond to conservative medical management. the most commonly reported indication is chronic pyometra.58 unfortunately, ovariohysterectomy results in sterilization. congenital anomalies, focal uterine tumors, uterine hematomas, uterine adhesions and ovarian masses with associated uterine horn adhesions are all causes of mare infertility. it is hypothesized that the negative effects on fertility of these conditions may be the result of alterations in uterine environment, uterine horn obstruction, or their combinations. endometritis is often associated with uterine tumors, and uterine horn obstruction may interfere with fertilization, embryo migration or embryo implantation.59 in many cases a presumptive diagnosis is made via transrectal palpation and ultrasonography. some authors have suggested that hysteroscopy may be a valuable tool to aid in the diagnosis of these conditions.59,60 in select cases, partial hysterectomy may be an option to improve fertility. although the exact amount of uterine tissue required to maintain an equine pregnancy is unknown, in rare cases of uterus unicornis, pregnancy was attainable and maintained, resulting in successful delivery of foals.61-63 authors have reported pregnancies in chronically infertile mares following the removal of uterine masses via partial ovariohysterectomy.64-68 successful pregnancy and live foaling were reported60 after a partial ovariohysterectomy that addressed a congenital abnormality, suspected as a cause of infertility.60 the standing laparoscopic approach to partial ovariohysterectomy is minimally invasive with few reported complications. the time to rebreeding was variable amongst case reports. one mare conceived 60 days after surgery, whereas the majority of mares were bred the following season. in a mare, pregnancy was not identified over 3 estrous cycles and she subsequently became an embryo donor.68 in addition, authors have reported that fertility may be permanently restored and several mares have produced multiple foals following surgery.60,65 cesarean cesarean section or c-section is most commonly performed in an emergency situation to resolve a dystocia when vaginal delivery cannot be achieved. it is a true surgical emergency that should proceed rapidly and efficiently. the longer the interval between the rupture of the chorioallantois and resolution, the lower the probability of foal survival.69 c-section may also be successfully utilized as an elective procedure when natural delivery is deemed undesirable. candidates for an elective c-section include conditions that may compromise the birth canal or when natural delivery is life-threatening or endangers the future fertility of the mare. chronic pelvic fractures, clinical theriogenology 2021; 13: 276 cervical adhesions, vaginal fibrosis, previous surgical repair of the cervix, partially ruptured prepubic tendon, hepatic lipidosis, previous history of dystocia, and uterine artery hemorrhage have all been indications for elective c-section.69-73 authors have reported favorable outcomes for both mare and foal survivability. of the reports reviewed, 100% of mares undergoing going elective c-sections survived to discharge and some have undergone subsequent successful elective c-sections.69-73 the author has experience with a mare having successfully undergone 5 elective c-sections. more than 90% of foals survived to discharge after elective c-section compared to < 30% of foals surviving after emergency c-section.69-73 in addition to complications associated with abdominal surgery, retained fetal membranes is the most commonly reported complication following elective c-section.70 timing of the elective surgery is crucial to ensure that a viable foal is produced. it has been well documented that pregnancy length cannot be used to determine fetal viability.74 the author has reviewed clinical cases in which 8 elective c-sections were performed with concurrent gastro-intestinal disease; in these instances, mammary secretions were not assessed and despite all mares being at least day 330 of pregnancy, foal survival to discharge was 0% (mcnaughten, unpublished data). early reports based the timing of surgery on common signs of impending parturition such as physical status, udder development, softening of perineal tissues, and behavior.75 however, the author feels that these measures are subjective and may be misinterpreted. fetal readiness prior to elective c-section was determined72 according to the presence of colostrum in the udder and all mares and foals survived to discharge. in a normal mare, changes in the composition of mammary secretions (such as calcium concentration and ph) remain the most reliable predictor of foal readiness and impending parturition, can be readily measured and should be utilized prior to elective c-section to improve foal survival.76-79 conclusion causes of infertility and subfertility tend to be multifaceted and are often unresponsive to medical management alone. reproductive surgeries are often necessary to improve reproductive outcomes in chronically infertile mares. complete breeding soundness evaluation is warranted prior to surgery to ensure an accurate diagnosis and favorable postoperative prognosis for future fertility. early diagnosis and rapid implementation of the appropriate surgical procedure offer an immediate benefit and improve the future prognosis of the mare. conflict of interest there are no conflicts of interest to declare. references 1. tibary a, pearson lk, fite cl: reproductive tract infections. in: sellon dc, long mt: editors. equine infectious diseases. 2nd edition, st. louis; w.b. saunders: 2014. p. 106-115. 2. bradecamp ea: pneumovagina. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition. ames; wileyblackwell: 2011. p. 2537-2544. 3. papa fo, melo cm, monteiro ga, et al: equine perineal and vulvar conformation correction using a modification of pouret’s technique. j equine vet sci 2014;34:459-464. 4. caslick ea: the vulva and the vulvo-vaginal orifice and its relation to genital health of the thoroughbred mare. cornell vet 1937;27:178-187. 5. pascoe rr: vulvar conformation. in: samper jc, pycock jf, mckinnon ao: editors. current therapy in equine reproduction. st louis: saunders, 2007; p. 140-145. 6. mckinnon ao, jalim sl: surgery of the caudal reproductive tract. in: mckinnon ao, squires el, vaala we, et al: editors. equine 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in mares. vet rec 2012;170:618-619. 14. easley kj: diagnosis and treatment of vesicovaginal reflux in the mare. vet clin north am equine practice 1988;4:407-416. 15. schnobrich mr, gordon dl, scoggin cf, et al: creatinine concentrations of accumulated intrauterine fluid to confirm the clinical diagnosis of urometra in mares. vet rec 2017;180:304. 16. walbornn s, kazcor j, bartley c, et al: effect of polyacrylamide hydrogel injection into reproductive tract of mares. clinical theriogenology 2020;12;373. 17. jalim sl, mckinnon ao: surgical results and fertility following correction of vesico-vaginal reflux in mares. aust vet j 2010;88:182-185. 18. pollock pj, russell tm: cervical surgery. in: mckinnon ao, squires el,vaala we, et al: editors. equine reproduction. 2nd edition. ames; wiley-blackwell: 2011. p. 2559-2563. 19. bucca s: how to manage cervical incompetence by application of a cervical cerclage suture in the pregnant mare. proc am assoc equine pract 2013; p. 28-33. 20. brown js, varner dd, hinricks k, et al: surgical repair of the lacerated cervix in the mare. theriogenology 1984;22:351-359. 21. embertson rm, henderson ce: cervical tears. in: samper jc, pyock jf, mckinnon ao: editors. current therapy in equine reproduction. st louis; saunders: 2007. p.130-133. 22. makloski-cohorn cl, embertson rm, payton me, et al: post-operative fertility in mares with cervical defects. j equine vet sci 2014;34:137. clinical theriogenology 2021; 13: 277 23. o’leary jm, rodgerson dh: how to repair cervical tears using trendelenberg position. proc am assoc equine pract 2009; p. 269-271. 24. mckinnon ao, vasey jr: selected reproductive surgery of the broodmare. in: samper jc, pyock jf, mckinnon ao: editors. current therapy in equine reproduction. st louis; saunders: 2007. p. 146-160. 25. embertson rm: selected urogenital surgery concerns and complications. vet clin north am equine pract 2009;24:643-661. 26. scoggin cf, makloski-cohorn c, embertson et al: recurrence of cervical defects and live-foal rates in thoroughbred broodmares undergoing one or more surgeries for defects of the cervix. j equine vet sci 2018;66:92-93. 27. schnobrich mr: a review of the equine oviduct: pathology, evaluation, and current treatments. clinical theriogenology 2019;11:379-385. 28. arnold ce, love cc: laparoscopic evaluation of oviductal patency in the standing mare. theriogenology. 2013;79:905-910. 29. sitters s, dascanio jj: starch granule test for the evaluation of oviductal patency. in: dascanio jj, mccue pm: editors. equine reproductive procedures. john wiley & sons: 2014. p. 93-94. 30. zent ww, liu km, spirito ma: oviduct flushing as a treatment for infertility in the mare. equine vet j 1993;15:47-48. 31. allen wr, wilsher s, morris l, et al: laparoscopic application of pge2 to re-establish oviducal patency and fertility in infertile mares: a preliminary study. equine vet j 2006;38:454-459. 32. inoue y: hysteroscopic hydrotubation of the equine oviduct. equine vet j 2013;45:761-765. 33. alvarenga ma, segabinazzi lg: application of misoprostol as a treatment of unexplained infertility in mares. j equine vet sci 2018;71:46-50. 34. bennet s, griffin r, rhoads w: surgical evaluation of the oviduct disease and patency in the mare. proc am assoc equine pract 2002; p. 347-349. 35. inoue y, sekiguchi m: clinical application of hysteroscopic hydrotubation for unexplained infertility in the mare. equine vet j 2018;50:470-473. 36. walbornn rs, schnobrich mr, bradecamp ea, et al: pregnancy rates after laparoscopic application of pge2 or hysteroscopic hydrotubation of the uterine tubes. proc am assoc equine pract 2018; p. 245. 37. schnobrich mr: how to use endoscopy (hysteroscopy and laparoscopy) to manage the sub-fertile mare. proc am assoc equine pract 2016; p.169-174. 38. kollman m, rottig a, heberling a, et al: laparoscopic techniques for investigating the equine oviduct. equine vet j 2011;43:106-111. 39. pye j, clulow j, adkins a: laparoscopic transection of restrictive bands of the mesosalpinx as adjunct to the use of prostaglandin e2 for mares with suspected uterine tubal blockage. aust vet j 2018;96:252-256. 40. tannus rj, thun r: influence of endometrial cysts on conception rate of mares. zentralbl veterinairmed a 1995;42:275-283. 41. stanton mb: uterine cysts. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition. ames; wiley-blackwell: 2011. p. 2665-2668. 42. mcdowell kj, sharp dc, grubagh w, et al: restricted conceptus mobility results in failure of pregnancy maintenance. biol reprod 1988;39:340-348. 43. bracher v, matthias s, allen wr: influence of chronic degenerative endometritis (endometrosis) on placental development in the mare. equine vet j 1996;28:180-188. 44. brook d, frankel k: electrocoagulative removal of endometrial cysts in the mare. j equine vet sci 1987;7:77-80. 45. bilkslager at, tate lp, weinstock d: effects of neodymium:yttrium aluminum garnet laser irradiation on endometrium and on endometrial cysts in six mares. vet surg 1993;22:351-356. 46. garrett c, johnson ak, wilborn rr: endometrial cyst ablation in a 23-year old dutch warmblood mare. clinical theriogenology 2015;7:317. 47. miller lm, ferrer sm: conception following endoscopic removal of endometrial cysts in a mare. clinical theriogenology 2014;6:41-45. 48. mckinnon ao: uterine abnormalities. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition. ames; wiley-blackwell: 2011. p. 2137-2161. 49. deluca ca, gee ek, mccue pm: how to remove large endometrial cysts with an improvised snare: a simple technique for practitioners. proc am assoc equine pract 2009; p. 328-330. 50. ley wb, higbee rg, holyoak gr: laser ablation of endometrial and lymphatic cysts. cli tech equine pract 2002;1:28-31. 51. griffin rl, bennett sd: nd:yag laser photoablation of endometrial cysts: a review of 55 cases (2000-2001). proc am assoc equine pract 2002; p. 58-60. 52. carluccio a, gloria a, mariotti f, et al: ethanol sclerotherapy for the treatment of uterine cysts in the mare. j equine vet sci 2018;63:27-29. 53. troedsson, mht: uterine clearance and resistance to persistent endometritis in the mare. theriogenology 1999:52;461-471. 54. leblanc mm: advances in the diagnosis and treatment of chronic infectious and post-mating-induced endometritis in the mare. reprod domest anim 2010;45:21-27. 55. brink p, schumacher j, schumacher j: elevating the uterus (uteropexy) of five mares by laparoscopically imbricating the mesometrium. equine vet j 2010;42:675-679. 56. corsalini j, gialletti r, lotto e, et al: laparoscopic uteropexy (mesometrium imbrication) in three mares using a barbed suture. j equine vet sci 2016;40:102-105. 57. brink p, schumacher j: results of laparoscopic uteropexy (imbrication of mesometria) in standing horses. ecvs proceeding 2013; p. 79-80. 58. roetting ak, freeman de: hysterectomy. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition. ames; wiley-blackwell: 2011. p. 2574-2577. 59. berezowski c: diagnosis of a uterine leiomyoma using hysteroscopy and a partial ovariohysterectomy in a mare. can vet j 2002;43:968-970. 60. clulow jr, nugent sj, adkins ar: successful pregnancy in a mare following hysteroscopy and partial hysterectomy for a uterine patency defect. proceedings for the 40th bain fallon memorial lectures 2018. 61. gallacher k, gilbert ro: successful foaling in a warmblood mare with uterus unicornis. clinical theriogenology 2018;10:51-58. 62. brown ja, hodder ad, benak j, et al: uterus unicornis in two mares. aust vet j 2007;85:371-374. 63. newcombe jr: uterus unicornis in two mares. vet rec 1997;141:21. 64. lofstedt rm, spurlock g, willimas r, et al: leiomyosarcoma in the uterus of a mare. comp cont educ practicing vet 1987;9:93. 65. santschi em, slone de: successful pregnancy after partial hysterectomy in two mares. j am vet med assoc 1994;205:1180-1182. 66. janicek jc, rodgerson dh, boone bl: use of a hand-assisted laparoscopic technique for removal of a uterine leiomyoma in a standing mare. j am vet med assoc 2004;225:911. 67. muurlink, t, walmsley j, whitton c: successful laparoscopic surgery for a uterine leiomyoma in a mare. equine vet educ 2008;20:508-511. clinical theriogenology 2021; 13: 278 68. rotting ak, freeman de, doyle aj, et al: total and partial ovariohysterectomy in seven mares. equine vet j 2003;35:29. 69. hodder ad, atkins ar, adams pl, et al: cesarean section in the mare: 82 cases (2001-2011). aust vet j 2012;3:66-66. 70. abernathy-young kk, leblanc, mm, embertson rm, et al: survival rates of mares and foals and postoperative complications and fertility of mares after cesarean section: 95 cases (1986–2000). j am vet med assoc 2012;241:927-934. 71. juswiak js, slone de, santschi em, et al: cesarean section in 19 mares: results and postoperative fertility. vet surg 1990;19:50-52. 72. watkins jp, taylor ts, day wc, et al: elective cesarean section in mares: eight cases (1980-1989). j am vet med assoc 1990;197:1639-1645. 73. freeman de, hungerford ll, schaeffer d, et al: caesarean section and other methods for assisted delivery: comparison of effects on mare mortality and complications. equine vet j 1999;31:203. 74. nagel c, jörg aurich j, aurich c: prediction of the onset of parturition in horses and cattle. theriogenology 2020;150:308-312. 75. embertson rm: ovaries and uterus. in: auer ja, stick ja: editors. equine surgery. 3rd edition. st louis, w.b. saunders. 2006 p. 855-864. 76. ousey jc, dudan f, rossdale pd: preliminary studies of mammary secretions in the mare to assess foetal readiness for birth. equine vet j 1984;16:259-263. 77. ley wb, bowen jm, purswell bj, et al: the sensitivity, specificity and predictive value of measuring calcium carbonate in mares’ prepartum mammary secretions. theriogenology, 1993;40:189-198. 78. canisso i, ellerbrock r: how to interpret ph profiles of mammary gland secretions to predict imminent parturition in mares. proc am assoc equine pract 2016; p.187-192. 79. de amorim md, montanholi y, morrison m, et al: comparison of foaling prediction technologies in periparturient standardbred mares. j equine vet sci 2019;77:86-92. clinical theriogenology 2021; 13: 279 reproductive surgeries: why necessary and whatto perform for specific problems introduction pregnancy loss is an important cause of economic loss to livestock producers. additionally, many infectious causes of pregnancy loss in ruminants are zoonotic agents posing a health risk for livestock owners, animal handlers, and veterinarians.1-3 the investigation goal in any animal disease outbreak is to identify the cause rapidly and accurately. determining the cause of pregnancy loss or neonatal death in ruminants is often unsuccessful.4 aborted fetuses often die in utero and are autolyzed when received at the laboratory, hampering interpretation of postmortem findings.5 consequently, a specific cause was identified in only ~ 30% of midterm and late-term pregnancy losses in cattle and 57% in sheep.1,2,6 however, this should not be considered a diagnostic failure. in a majority of cases, the diagnostic emphasis is towards ruling out infectious causes of abortion. when results are negative, noninfectious causes of abortion may be suspected; however, infectious causes cannot be completely ruled out. veterinary diagnostic laboratories have an important role in the identification of causes of pregnancy loss and in surveillance of infectious diseases that cause reproductive failure. additionally, they also support interstate and international livestock trade by providing testing to ensure compliance with the conditions and measures necessary to minimize potential risks associated with animals and animal products.7,8 laboratory use in pregnancy loss diagnosis andrés de la concha-bermejillo,a juan romanob atexas a&m veterinary medical diagnostic laboratory blarge animal clinical sciences, college of veterinary medicine & biomedical sciences texas a&m university, college station, tx abstract pregnancy loss and perinatal mortality are important economic and public health issues for the livestock industry. to minimize losses and prevent human infections, rapid and accurate identification of the cause is critical. veterinary diagnostic laboratories not only have an important role in identifying causes of pregnancy loss but also in monitoring (surveillance) infectious diseases that cause reproductive failure. appropriate specimen collection and handling are essential prerequisites for reliable test results. consequently, laboratory test procedures and their value may be compromised by using specimens that are not collected, labelled, handled or stored properly prior to testing. principles of biosecurity and biocontainment must be strictly followed during sample collection. for accurate determination of the cause of pregnancy loss in ruminants, it is recommended to use a battery of diagnostic tests done in parallel. these tests include histopathology, real time polymerase chain reaction (rtpcr) assay, bacterial and viral isolation, detection of fetal antibodies in serum or fluid from thorax or abdomen, and maternal serology. veterinary laboratory professional staff can provide consultation services to clients regarding test selection and testing strategies, interpretation of results, surveillance, disease prevention and control programs. this review provides essential information on collection of diagnostic samples and interpretation of test results for the diagnosis of most common infectious causes of pregnancy loss in ruminants. keywords: ruminants, abortion, diagnosis, pregnancy loss, laboratory appropriate specimen collection and handling are essential prerequisites for accurate test results. the value of a laboratory test procedure may be compromised by using specimens that are not collected, labelled, handled or stored properly.8 this requires a combined effort among various people (owner, animal caretaker, and veterinarian) and the veterinary diagnostic laboratory. in this process, the owner and the animal caretaker need to provide an adequate clinical history that includes clinical signs, nutrition, breeding program, history of vaccination, time and type of vaccines, medications, and other pertinent information. based on the information provided, the veterinarian has to establish a preliminary diagnosis and obtain adequate samples for the diagnostic laboratory.9 samples collected without clear directions often do not yield meaningful results. ruling out all possible causes of pregnancy loss may be costly. most diagnostic laboratories prioritize tests for the most common causes of infectious abortion and those with zoonotic potential.4 the diagnostic samples should preferably be submitted using an overnight delivery service. most diagnostic laboratories do not accept routine shipments on weekends or holidays. all veterinary laboratory diagnostic tests must be validated following the guidelines of the american association of veterinary laboratory diagnosticians requirements for an accredited veterinary medical diagnostic laboratory and the world organization for animal health.10 clinical theriogenology 2021; 13: 211 principles of biosecurity and biocontainment must be strictly followed during sample collection. biosecurity refers to programs for infectious disease control that reduce/prevent the introduction of new diseases into an operation from outside sources. biocontainment refers to programs that reduce/prevent the movement of infectious diseases within the operation or animal groups.11 this review provides essential information on the collection of diagnostic samples and the interpretation of test results for the diagnosis of most common infectious causes of pregnancy loss in ruminants. a more detailed description of the cause, pathogenesis and risk of human infection of these diseases is reviewed in the proceedings.1-3 collection and submission of laboratory samples procedures for submission of diagnostic samples to the veterinary diagnostic laboratory are complex and must follow multiple regulatory shipping requirements enforced by multiple entities.12 along with laboratory samples, the veterinarian should submit a detailed clinical history that includes owner’s name, animal species, breed, sex, age, animal identification, nutritional program, clinical signs, percent animals aborting, stage of pregnancy when abortion occurred, details of any fetal malformation, treatments and vaccinations, and environmental influences. diseases suspected and tests requested should be clearly stated in the submission form.9 providing more information to the laboratory usually results in faster and more accurate test results. laboratory professional staff can provide consultation services to clients in areas of test selection and testing strategies, interpretation of results, and disease prevention, surveillance, and control programs. many diagnostic laboratories now offer cattle and small ruminant abortion diagnostic plans. tests included in diagnostic panels vary among laboratories and animal species but in general include histopathology, bacteriology, serology, real time polymerase chain reaction (rtpcr) assay, the latter for bovine viral diarrhea virus (bvdv)/border disease virus (bdv), bovine herpes virus-1 (bhv-1), leptospira spp., chlamydia abortus, coxiella burnetii, neospora caninum, toxoplasma gondii, in addition to determining vitamin a levels and performing a trace mineral panel.13,14 the success of determining the cause of abortion in ruminants largely depends on the type and quality of samples submitted to the diagnostic laboratory. submission of fetal membranes increases the probability of making a diagnosis by 2.3 times.15 whenever handling biological material, from either live or dead animals, the risk of zoonotic disease should be considered and precautions taken to avoid human infection.16 it is strongly recommended that debilitated older individuals, pregnant women, immunocompromised people and children are not involved in collecting samples and visiting or cleaning birthing areas. proper personnel protective equipment including coveralls, disposable rubber gloves, rubber boots, safety goggles or masks, and when possible n95 masks or ‘powered air purifying respirators’ should be worn when handling samples. when a zoonotic disease is suspected, this should be clearly indicated on the submission form. in small ruminants, all abortions should be considered potentially zoonotic, and principles of biosecurity and biocontainment strictly followed.17,18 samples must be packaged properly to prevent leakage of blood or tissue fluids during shipment and ensuring that they arrive at the laboratory in good condition. shipments must comply with all applicable laws (local, state, and federal) governing packing, marking, and labeling.12 submission of most laboratory samples requires a watertight inner primary container, a watertight inner secondary container and a sturdy outer shipping box (tertiary container). inclusion of absorbent materials inside the secondary container is recommended to minimize contamination of other packages in case a container breaks or leaks. tissue samples for histopathology should be submitted in 10% buffered formalin. if formalin is not available, tissue samples must be sent chilled with cold packs but not frozen. collection and submission of laboratory samples from aborted fetuses in abortion cases, particularly in small ruminants, it is recommended to submit the entire fetus and fetal membranes to the diagnostic laboratory to prevent exposure of personnel to zoonotic agents and to avoid contamination of the premises. fetal membranes are very important to identify the cause of abortion and when available should be submitted along with the fetus. examination of fetal membranes can yield information that may be important in the selection of ancillary tests and interpretation of laboratory results. fetal membrane lesions of diagnostic significance can be recognized for many economically important infectious abortifacient diseases.19 poor viability of cloned bovine fetuses during days 35 60 has been associated with poor chorioallantoic development.20 concurrently, along with fetal membranes and fetus, submission of serum samples from animals that have aborted and from those that had normal deliveries in the same herd/flock is recommended. when submission of the entire fetus is not possible, a field necropsy should be performed, and complete sets of tissues chilled on ice or fixed in 10% buffered formalin submitted to the laboratory. necropsies should be performed as soon as possible after abortion. necropsies of aborted fetuses and examination of fetal membranes can provide a wealth of information that can help to establish animal health prevention and control programs.21 safety during field necropsies is of paramount importance to avoid exposure of personnel and contamination of premises. personnel protective equipment should be used during necropsy procedure. at the end of necropsy, all remaining tissue should be properly disposed, and instruments and equipment should be thoroughly disinfected. fetus and fetal membranes should be shipped to the laboratory, as indicated above. adequate cooling packages should be included within clinical theriogenology 2021; 13: 212 the secondary container but should not come in direct contact with samples.11 preparation of tissue samples for histopathology tissues for histopathology should be collected as soon as possible during necropsy, but not frozen to avoid autolysis. with the exception of brain, these tissues should be < 1 cm thick (to promote fixation) but can be 3 5 cm in length and width. they should be submitted in 10% phosphate buffered formalin (10 volumes of formalin per volume of tissue) in plastic jars with properly sealing lids.8,22 tissues for histopathology should include at least samples from 3 cotyledons and 3 sections of intercotyledonary area, brain (entire half of the brain including brain stem and cerebellum), heart, lung, thymus, liver, spleen, kidney, adrenal gland, and skeletal muscle. in general, tissues from the gastrointestinal tract of aborted fetuses are of limited diagnostic value, although they are important in cases of neonatal death. a second set of tissues and stomach contents should be collected, chilled and sent in individual whirl-pak or ziploc bags for microbiology, molecular analyses, virology and/or toxicology. fresh tissues should be larger than fixed tissues and be ~ 3 6 cm thick.8. preparation of tissue samples for bacteriology and mycology fetal tissues and fluids for culture should be aseptically collected individually in sealable whirl-pak or ziploc bags or sterile tubes and sent chilled with cold packs. saline, broth or formalin should not be added to the samples.22 preferred samples for microbiology diagnosis in abortion cases include fetal membranes, fetal stomach content, fetal tissues including lung, liver, and brain. preparation of tissue samples for molecular analyses in the last 2 decades, real time rtpcr has been the pillar in the diagnosis of infectious abortion in ruminants. tissue samples for detection of microbial nucleic acid by pcr can be submitted refrigerated in individual whirl-pak or ziploc bags or sterile tubes chilled with cold packs. blood should be collected and thoroughly mixed in edta tubes (purple tops) and not in heparin tubes (green tops). clotted blood is not suitable for molecular testing. bacterial culture swabs submitted in bacterial culture media (gel, charcoal) are not appropriate for rtpcr testing. swabs for rtpcr testing should be moistened with 1 2 drops of sterile saline and submitted in sterile leak-proof containers (e.g. red top tubes).14 amount of nucleic acid (dna or rna) of specific pathogens present in a given sample is determined in rtpcr assays that amplify specific sections of microbial nucleic acid using fluorescent-labelled oligonucleotide probes, with results given as cycle threshold (ct) values. the cutoff value provides classification of results as either ‘detected’ or ‘not detected’ and may also include an indeterminate range. using fluorescent probes, these assays monitor the increase in target nucleic acid as it is amplified over a total of 40 45 rtpcr cycles. the cycle number at which this threshold is crossed is termed the ‘cycle threshold’ or ‘ct.’ the fewer the cycles needed to reach the ct, the more nucleic acid of that specific microbial agent is in the sample. for example, ct values < 28.0, 28.1 35.0, and 35.1 to 40 45.0 indicate abundant, moderate or minimal amounts, respectively, of target nucleic acid in the sample. in veterinary diagnostic laboratories, dilution series of the target nucleic acid are not included. generally, only a single positive amplification control concentration is routinely included for rtpcr assays, thereby limiting the precision of quantification and making most of the assays semiquantitative rather than absolute. furthermore, rtpcr assays should be validated and incorporate either an exogenous or endogenous internal control.23,24 preparation of tissue samples for analytical chemistry samples of fetal tissue, in particular liver and kidney, should be placed in sealable containers and chilled. when vitamins or mineral deficiencies are suspected, 10 grams of fetal liver should be placed in additive-free transport containers and chilled with cold packs. for vitamin a determination, samples should be protected from light immediately after collection and sent to the laboratory on ice by an overnight courier. since abortions can be the result of nutritional deficiencies or toxins, it is recommended to submit both feed and water for analysis. maternal blood collected in purple and red top-top tubes (edta and serum; both samples are required) can be submitted for metal and mineral panel determination by inductively-couple plasma/mass spectrometry.25 preparation of samples for serology fetal fluids, fetal blood (from the heart) and maternal blood for serology should be collected in sterile red-top glass tubes or separator tubes and placed into padded, protective containers for shipping. placing glass blood/serum tubes between frozen ice packs without protective padding will often result in tubes breaking and leaking. interpretation of maternal serology testing for the diagnosis of infectious causes of abortion can be difficult. use of threshold serum titers or paired serum samples collected at abortion and 2 3 weeks later is a common practice. however, these practices may not be accurate with some infectious agents such as bvdv, infectious bovine rhinotracheitis, neosporosis, and leptospirosis, due to the time lag between infection and abortion. lack of change in serum antibody titer in acute and convalescent samples has been reported for leptospirosis, bvdv, and infectious bovine rhinotracheitis. interpretation of results is further complicated in animals that have been vaccinated, since serologic tests do not differentiate between vaccineand infection-induced antibodies. an alternative diagnostic approach suggested is the collection of blood samples from groups of clinical theriogenology 2021; 13: 213 animals that have aborted and from herd mates that maintain pregnancy or had normal parturition.26 a 2-by-2 table (see below) is used to classify pregnancy loss and nonpregnancy loss females according to their exposure to the risk factor being evaluated. the degree of association between the risk factor and pregnancy loss is measured by calculating the odds ratio (or), i.e. the ratio of the odds of pregnancy loss for females exposed to the risk factor to the odds of pregnancy loss for females not exposed to the risk factor. in the case of antibodies, titers are necessary to establish a threshold level of risk factor present or absent. an or of 1 implies that there is no association between the risk factor and pregnancy loss. an or > 1 implies that the risk factor is associated with an increased risk of pregnancy loss. an or < 1 suggest that females with the risk factor have a decreased risk of pregnancy loss. in general, a risk factor with an or of > 3 may be considered significant and they may act to eliminate or alter that risk factor. this odd ratio depends on an adequate number of samples and the use of correct statistical analysis (fisher’s exact test or chi-square analysis) to arrive at a sound conclusion. an or calculated using a single 2-by-2 table may not be accurate in herd situations in which more than 1 risk factor directly influences the same production parameter.27 to increase the diagnostic value of the maternal serum agglutination test (sat) for the diagnosis of salmonella dublin culture‐positive bovine abortion and stillbirth, the predicted probability values, rather than the traditional arbitrary breakpoints of negative, inconclusive and positive, increased the diagnostic value of the maternal sat. information previously considered inconclusive can be derived from test results by using predicted probability values.28 placentitis placenta is the organ most frequently affected in cases of infectious abortion in ruminants. the etiological agent is often identified during histologic examination of fetal membranes or can be detected with ancillary tests. many bacteria and fungi that cause abortion in ruminants cause placentitis including brucellosis, campylobacteriosis, chlamidiosis, coxiellosis, listeriosis, yersinosis, and aspergillosis. apicomplexan parasites such as neospora caninum and toxoplasma gondii also cause placentitis and the organisms can be associated with fetal membrane lesions in some cases.29 inflammation of fetal membranes is often not uniform. entire fetal membranes can be submitted double-bagged and chilled on ice, or multiple portions of cotyledonary and intercotyledonary areas, fixed in 10% formalin for histopathology and chilled on ice for bacterial/fungal culture and molecular analysis, should be submitted. fetal malformation causes of congenital fetal malformations can be genetic, toxic, nutritional, and infectious. in cattle, bvdv fetal infection can cause fetal malformation.30,31 infection with bdv in ruminants during the second or last trimester of pregnancy causes fetal malformation, fetal mummification, abortion, stillbirth, and birth of weak or normal lambs.32 in north america, cache valley virus (cvv) is a common cause of fetal malformation in small ruminants and less frequently cattle.33,34 experimental infection of pregnant sheep with 2 california serogroup bunyaviruses, lacrosse virus and san angelo virus, and a bunyamwera serogroup member, main drain virus, induced a range of lesions similar to those induced by cvv including arthrogryposis, hydrocephalus, fetal death, axial skeletal deviations, anasarca, and oligohydramnios.35 fetal teratogenesis in sheep, goats and cattle have been described in natural and experimental infections with exotic bunyaviruses including akabane virus, schmallenberg virus, rift valley fever virus, and wesselsbron disease virus.36,37 vertical transmission of bluetongue virus (btv) from dam to fetus in enzootic areas is considered negligible. in these areas (including the us), btv-induced pregnancy loss and congenital malformations have been associated with btv strains from modified live virus vaccine strains.38 infection of cattle, sheep and goats with the european btv-8 wild-type strain demonstrated a high incidence of transplacental transmission and pregnancy loss in natural circumstances.39 veratrum californicum in sheep, astragalus pubentissimus (locoweed) and benzimidazoles are toxic causes of fetal malformation in sheep and cattle.40-42 genetic causes of fetal malformation include abnormalities of the chromosomes or genes. bovine pregnancy loss nonpregnancy loss risk factor present (+) pregnancy loss with risk factor (a) nonpregnancy loss with risk factor (b) risk factor absent (-) pregnancy loss without risk factor (c) nonpregnancy loss without risk factor (d) the or is calculated from the 2-by-2 table using the following equation: or = a x d / b x c clinical theriogenology 2021; 13: 214 fetal abnormalities have been reviewed in detail.43 fetal mummification in cattle, fetal mummification occurs after formation of placenta and fetal ossification. fetal mummification must be differentiated from fetal maceration, in which the fetus putrefies due to bacteria and oxygen entering the uterus when the cervix is open.44 mummification of bovine fetuses is uncommon and cows do not always respond to treatment with prostaglandin f2α. most often, mummification occurs between 3rd and 8th months of pregnancy without lysis of the corpus luteum and the cervix remaining closed.45 mummification of a fetus occurs when it dies in utero and undergoes subsequent autolysis in the absence of bacterial infection and putrefaction. a definitive etiology is rarely determined, due to tissue degeneration and autolysis. mummification process usually renders worthless the analysis of bacteria, protozoa and viruses by standard methods, but nucleic acid of some abortifacient infectious organisms can be detected by rtpcr in fetal tissues. proposed causes of fetal mummification in cattle include bovine viral diarrhea, leptospirosis, and neosporosis.45 in a study, 25% of bovine mummified fetuses were positive for neospora caninum dna by pcr.46 in small ruminants, fetal mummification is often the result of infection with border disease virus, chlamydia abortus, coxiella burnetii, or toxoplasma gondii.44 chlamydiosis chlamydiosis caused for chlamydia abortus is an important cause of late-term abortion in small ruminants. the complement fixation test is used by many veterinary diagnostic laboratories to detect ch. abortus antibodies in maternal serum. complement fixation titers of 1:16 1:32 are considered positive for most laboratories. the titer may increase to 1:80 or higher 2 3 weeks after abortion.47 the complement fixations test is not ch. abortus specific and positive titers can arise from cross reactivity to ch. pecorum. several enzyme-linked immunosorbent assay (elisa) tests with varied specificity and sensitivity have been used for the diagnosis of chlamydiosis.48 a fluorescent antibody test is used by some veterinary diagnostic laboratories to detect ch. abortus in fetal membranes, liver or spleen. an rtpcr assay is used for the detection of chlamydia spp nucleic acid, but this test detects ch. psittaci, ch. felis and ch. abortus and does not distinguish among them.13,14 coxiellosis coxiellosis, caused by the intracellular bacterium coxiella burnetii, is an important zoonotic disease that causes pregnancy loss in small ruminants and less commonly in cattle and other species. in the us, c. burnetti is a class b bioterrorism select agent subject to the select agent external icon regulations (42 cfr part 73 of the electronic code of federal regulations). in some veterinary diagnostic laboratories, in all cases of small ruminant abortions, the standard operating procedure is to first rule out c. burnetii by rtpcr before proceeding with other diagnostic tests.14 when a case is positive for c burnetii, all tissues and samples are appropriately disposed and no further testing is done. detection of c. burnetii antibodies in maternal serum is by elisa, immunofluorescence assay or complement fixation. antibodies in maternal serum indicate exposure to c. burnetii but do not confirm c. burnetii as the cause of abortion. however, lack of antibodies in maternal serum rules out coxiellosis as the cause of abortion.16 c. burnetii igm and igg phase ii specific antibodies in adult animals can be detected 2 weeks post-infection and remain increased for up to 13 weeks. antibodies directed against c. burnetii phase i increase generally 4 weeks later compared to the phase ii antibodies. serum antibodies in infected animals can be detected for several months up to years. all rtpcr positive results in aborted fetuses should be corroborated by testing maternal serum for c. burnetii antibodies.19,49 leptospirosis leptospirosis is an important zoonotic disease caused by > 260 antigenically distinct serovars belonging to 25 serogroups grouped in 9 pathogenic species, 5 intermediate and 6 saprophytic species of leptospira, a gram-negative bacterium belonging to the order spirochetales.50 leptospirosis can cause abortion, fetal mummification, stillbirths, the birth of weak calves, and infertility in cattle. several species of leptospira cause abortion in cattle. leptospira species can be classified as host-adapted, infecting maintenance host animals, or nonhost-adapted, infecting accidental host animals. most common leptospira species causing bovine abortions and infertility in north america is leptospira borgpetersenii serovar hardjo.51 cattle that become chronically infected with serovar hardjo serve as reservoirs for infection to other cattle and humans. some cattle with chronic hardjo infections have low or no agglutinating antibody titers.52,53 diagnostic tests for leptospirosis include detection of serum antibodies and demonstration of the organism or its dna in tissues or body fluids of aborted fetuses by rtpcr or other tests. due to its low cost and wide availability, a microscopic agglutination test (mat) is the most common method used for demonstration of antibodies in animal serum. with mat however, determining the stage of infection can be difficult in cases of bovine abortion or stillbirth, as infection usually occurs 1 4 weeks before the expulsion of the fetus and by this time, mat titers would have stabilized. some consider the presence of high serum antibody titers satisfactory to establish a diagnosis. in some serovars such as pomona, grippotyphosa, icterohaemorrhagiae and canicola, maternal serum antibody titers > 1,600 appear to correlate with abortion. however, maternal antibodies in abortion caused by serovar hardjo are often lower or negative at abortion; therefore, a low antibody titer does not rule out leptospirosis. antibodies to leptospira clinical theriogenology 2021; 13: 215 spp. can be demonstrated in serum of some aborted fetuses at dilutions of 1:10, but some fetuses can be positive by rtpcr without antibodies in pleural fluid.54 the mat does not differentiate between vaccine-associated antibodies and those from an active infection. in general, vaccinated animals develop low agglutination titers that last 1 3 months. however, some outliers develop high antibody titers that persists for several months after vaccination. additionally, cross reaction between serovars occurs with mat, complicating interpretation of results.52 new methods for the detection of leptospira serum antibodies have been developed but are not widely used by veterinary diagnostic laboratories. the microsphere immunoassay (mia), which uses luminex xmap technology, had better sensitivity compared to mat and it determined reactive from nonreactive antibodies and differentiated igm from igg responses.55 currently, the most common test for the diagnosis of leptospira abortion in cattle is rtpcr done on tissues of aborted fetuses. the preferred fetal tissue is kidney, although other fetal tissues can be used. veterinary diagnostic laboratory rtpcr assays will amplify leptospira dna of all serotypes, but will not differentiate between serotypes. other tests used for the diagnosis of leptospirosis in aborted fetuses are immunofluorescence for demonstration of the organism in urine and tissues and immunohistochemistry or special stains for demonstration of the organism in formalin-fixed, paraffin-embedded tissue sections. however, these methods are considered to have low sensitivity and are infrequently used. cache valley virus cache valley virus is an arbovirus of the family bunyaviridae that is endemic in north america and infects a wide range of domestic and wild animals, and humans. viral infection is usually asymptomatic in adult sheep, goats, and cattle. viral infection during pregnancy may result in embryonic mortality, fetal mummification, fetal malformation, and pregnancy loss in sheep and goats, and less often in cattle.33,34 during natural cvv infection of pregnant ruminants, cvv may cross the placenta, infect the fetus and cause congenital arthrogryposis and hydranencephaly.56 the congenital malformations induced by cvv during spontaneous infections in sheep have been reproduced experimentally by intrauterine inoculation of the virus between days 27 54 of pregnancy.57 experimentally infected ovine fetuses mount an innate and adaptive immune response that presumably contributes to viral clearance in infected animals.58 virus isolation from full-term aborted fetuses with cvv-induced malformations is unsuccessful. some diagnostic laboratories offer a gel-based pcr for the demonstration of cvv-rna in tissues of aborted fetuses. however, as cross contamination in gel-based pcr platforms is common, their use is discouraged. an rtpcr assay has been developed for the detection of california serogroup viruses and cvv, for use in human surveillance, but is not routinely used for cvv in small ruminants.59 until rtpcr methodology is validated in small ruminants for detection of cvv-rna in late-term malformed ruminant fetuses, the results of gel-based pcr are difficult to interpret. determination of cvv neutralizing antibodies in fetal serum from blood clots in the heart or in fetal fluids is the most reliable test to diagnose cvv fetal infection in late-term malformed fetuses. the presence of cvv antibodies in maternal serum is an indication of exposure but not proof that fetal malformation was caused by cvv. the lack of antibodies in maternal serum rules out cvv as the cause of fetal malformation. the presence of serum cvv antibodies before breeding in ewes and goats is protective of transplacental fetal infection with cvv but not against other related bunyaviruses.33,34 bluetongue bluetongue is an insect-transmitted, noncontagious disease of ruminants caused by bluetongue virus (btv), the prototype virus of the genus orbivirus in the family reoviridae. in endemic areas, abortion, stillbirth, and fetal deformities have been attributed to tissue culture adapted vaccine strains strains of btv in sheep and cattle, and less often goats.38 during the 2006 european btv outbreak, the ability of btv-8 field strain to cross the placenta and infect the fetus was a major concern because of transplacental transmission and increased abortions, stillbirths, and fetal deformities in cattle.60 severe brain lesions were observed in the brain of bovine fetuses 12 or 20 days after btv-11 experimental inoculation at 120 days of pregnancy. virus was recovered from brain, blood and a pool of lung and liver from all experimentally infected animals. viral group-specific antibodies were detected in 2 of 8 inoculated fetuses by the agar gel immunodiffusion test.61 serological assays available for demonstration of btv antibodies include complement fixation, virus neutralization, agar gel immunodiffusion test, and several elisa formats. virus isolation is done by inoculation of susceptible sheep or embryonated chicken eggs with heparinized blood or homogenized lymph nodes, spleen, or lung of infected animals. blood and tissue samples for diagnostics need to be preserved at 4˚c, but not frozen. the rtpcr assay is a commonly used diagnostic test with high sensitivity and specificity. a positive rtpcr result does not necessarily indicate the presence of infectious virus, as it can detect btv nucleic acids after the virus is no longer viable. antibodies can be demonstrated in precolostral serum samples of lambs infected in utero and the virus also can be isolated in some of these lambs due to immune tolerance and persistent infection.62 bovine viral diarrhea bovine viral diarrhea (bvd) is an important infectious disease in most cattle-producing countries worldwide and is caused clinical theriogenology 2021; 13: 216 by bovine viral diarrhea virus (bvdv), a member of the genus pestivirus in the flaviviridae family.63 diagnosis of bvdv infection is challenging, due to numerous virus types and subtypes and the variety of clinical syndromes induced by bvdv, including animals that are persistently infected (pis).64 currently, phylogenetic analysis has identified 21 pestivirus subtypes (bvdv 1a-u) and 4 pestivirus subtypes 2 (bvdv 2a-2d). four pestivirus h subtypes (hobi a-d) have been identified in europe. although not currently reported in the us, pestivirus h subtypes are a concern since they are not routinely detected by current diagnostic tests, and current vaccines may not fully protect against these subtypes.65 although of minor clinical importance, infections of cattle with bdv, a small ruminant pestivirus, can interfere with bvdv eradication programs.66 current laboratory techniques for bvdv testing in cattle samples have not been critically validated for their use to test for bvdv in heterologous species.64 virus neutralization tests for bvdv 1a, 1b, and 2 are used for demonstration of bvdv antibodies in animal serum. interpretation of the results should be in conjunction with vaccination history, including type of vaccine and interval from vaccination to sample collection. presence of neutralizing antibodies in nonvaccinated calves 6 12 months of age is highly correlated with virus circulation in the herd. after administration of killed or modified-live vaccines, titers range from 32 256 and from 512 1536, respectively, several months after vaccination. however, pi animals generally have no or low antibody titers. bvdv naïve animals that become infected generally have titers in the low to mid thousands, but will decrease to levels similar to those observed with mlv vaccines. determining acute and convalescent serum samples may provide definitive proof of recent infection.13 acute infection can be detected by virus isolation or rtpcr from serum, blood, nasal swabs, tracheal wash, semen or from lung or lymphoid tissue recovered at necropsy from adult animals and aborted fetuses. blood for rtpcr should be collected in edta tubes. clotted blood or blood collected in green tops are not suitable for rtpcr testing. bvdv persistent infection can be diagnosed on paired samples taken 3 weeks apart by virus isolation, antigen capture elisa and rtpcr in serum, whole blood, milk or skin or by immunohistochemistry on ear notch biopsies. up to 24 ear notches can be pooled and tested by rtpcr initially. when a pool of ear notches gives a positive result, individual samples are tested for identification of individual infected animals.14 control and eradication of bvdv infection in cattle herds involves a multidimensional approach that includes the identification and culling of persistently infected animals, vaccination, and biosecurity.64 sentinel serology a critical step in the control and eradication of bvd is identification and elimination of pi calves. determining the bvdv infection status in large herds by testing every animal may be labor intensive and costly. a system that has been used to overcome this problem consists of sentinel serology. in this approach, usually a small group of seronegative, nonvaccinated calves are introduced in the herd and subsequently tested for the presence of bvdv neutralizing antibodies. calves used as sentinels have to be ~ 6 12 months old with no bvdv colostrum antibodies that could interfere with serological testing and also that are not vaccinated against bvdv.67 if there has been no vaccination (sentinel calf program), then both tests (bvdv-1 and bvdv-2) should be requested, as a low value to 1 type might show a 10-fold higher unequivocal titer to the other type. in this instance, the higher titer value would be indicative of an infection with that type of virus. neosporosis neosporosis in cattle is usually manifested in 2 patterns that may overlap. in the endemic pattern, there is an increased abortion rate in the herd, generally > 5% per year. this pattern persists year after year over a long period. in the epidemic pattern, > 30% pregnant cows may abort with a few months. midterm abortion is the characteristic of neosporosis in cattle with no other clinical signs. not all seropositive cows abort. congenital neosporosis occurs in 80 90% of calves born to seropositive cows. in many cases, congenital infection results in the birth of persistently infected calves that have precolostral antibodies against neospora. these calves can transmit the infection to their offspring when they become pregnant and perpetuate the infection in the herd. less often, congenitally infected calves have neurological deficits at birth ranging from mild proprioceptive deficits to severe paralysis, resulting from inflammatory lesions in the brain and spinal cord. currently, the main diagnostic test to identify neospora in tissues from aborted fetuses consists of detection of n. caninum dna by rtpcr. fetal tissues most often used for rtpcr are brain, heart, lung, liver, and fetal membranes. demonstration of microscopic lesions in h&e-stained sections of brain, heart, liver, skeletal muscle and fetal membrane, and demonstration of the organism in tissues by immunohistochemistry are recommended as complementary tests. neospora antibodies in fetal serum or fluids can be demonstrated in some aborted fetuses by indirect immunofluorescent antibody test. presence of neospora antibodies in maternal serum can be assessed by indirect immunofluorescent antibody test or by elisa. these tests are not always standardized across various diagnostic laboratories and appropriate cut-off values should be established using standardized sera. neospora serologic tests in maternal serum should be interpreted with caution and are useful for determination of herd exposure but not to identify neospora as the cause of abortion in individual cases. antibody titers in maternal serum may fluctuate throughout pregnancy and in some cases, titers may be below the cut off value at the time of abortion. regardless of aborting cows’ titers below the cut clinical theriogenology 2021; 13: 217 off value, it is appropriate to compare the presence of neospora antibody titers between groups of cows that aborted to groups of cows that did not. the percentage of abortion in seropositive pregnant cows is 3 times higher than in seronegative cows and proportion of repetitive abortion in subsequent pregnancies was up to 5 times higher in seropositive cows than in seronegative cows.68 in goats, there was a significant association between increased maternal igg titers in serum in the second half of pregnancy and congenital transmission of n. caninum.26,69,70 conclusion there are few perfect diagnostic tests, as it is usually possible to have false-positives and false-negative test results. therefore, it is impossible to prove that a herd/flock is free from a disease even with large sample size, as there is a chance that an infected animal may have been missed or that the test result is wrong.71 for accurate determination of the cause of abortion in ruminants it is important to take a multidimensional approach and use a battery of diagnostic tests done in parallel, including histopathology, rtpcr, bacterial culture, virus isolation, fetal antibodies in serum or fluid from thorax or abdomen, and maternal serology.72 ultimately, linking molecular, microbiological and serological findings to grossly and/or microscopically visible lesions is always recommended to mitigate against false-positive and false-negative results.4 conflict of interest authors have no affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. references 1. de la concha-bermejillo a, romano j: pregnancy loss in cattle. clinical theriogenology 2021;3:167-180. 2. de la concha-bermejillo a, romano j: pregnancy loss in small ruminants. clinical theriogenology 2021;3:194-205. 3. de la concha-bermejillo a, romano j: pregnancy loss in ruminants. clinical theriogenology 2021;3:181-193. 4. borel n, frey cf, gottstein b, et al: laboratory diagnosis of ruminant abortion in europe. vet j 2014;200:218-229. https://doi.org/10.1016/j. tvjl.2014.03.015. 5. anderson m: disorders of cattle. in: njaa bl: editor. kirkbride’s diagnosis of abortion and neonatal loss in animals. 4th edition, oxford; wileyblackwell: 2012. p. 13-48. https://doi.org/10.1002/9781119949053.ch3. 6. moeller rb: disorders of sheep and goats. in: njaa bl, editor. kirkbrides diagn. abort. neonatal loss anim., oxford, uk: wileyblackwell; 2012, p. 49-87. https://doi.org/10.1002/9781119949053.ch3. 7. 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agerholm js, hewicker-trautwein m, peperkamp k, et al: virusinduced congenital malformations in cattle. acta vet scand 2015;57:54. https://doi.org/10.1186/s13028-015-0145-8. 32. schlafer d, foster r: female genital system. in: maxie m, editor. jubb kennedy palmer’s pathol. domest. anim. female genit. syst., vol. 3. 6th ed., elsevier; 2016, p. 358-464. 33. de la concha-bermejillo a: cache valley virus is a cause of fetal malformation and pregnancy loss in sheep. small rumin res 2003;49:1-9. https://doi.org/10.1016/s0921-4488(03)00050-6. 34. de la concha-bermejillo a: cache valley virus. in: chase c, lutz k, mckenzie e, editors. blackwell’s five-minute veterinary consult ruminant. 2nd edition, hoboken, nj: john wiley & sons: 2017. p. 140-141. 35. collisson ew, edwards jf, de la concha bermejillo a, et al: ovine fetal malformations induced by in utero inoculation with main drain, san angelo, and lacrosse viruses. am j trop med hyg 1997;56:171-176. https://doi.org/10.4269/ajtmh.1997.56.171. 36. de regge n, van den berg t, georges l, et al: diagnosis of schmallenberg virus infection in malformed lambs and calves and first indications for virus clearance in the fetus. vet microbiol 2013;162:595-600. https:// doi.org/10.1016/j.vetmic.2012.11.029. 37. oberst rd: viruses as teratogens. vet clin north am food anim pract 1993;9:23-31. https://doi.org/10.1016/s0749-0720(15)30668-x. 38. maclachlan n, osburn b: induced brain lesions in calves infected with bluetongue virus. vet rec 2008;162:490-491. https://doi.org/10.1136/ vr.162.15.490-b. 39. wouda w, peperkamp n, rumen m, et al: epizootic congenital hydranencephaly and abortion in cattle due to bluetongue virus serotype 8 in the netherlands. tijdschr diergeneeskd 2009;134:422-427. 40. evans t: reproductive toxicity and endocrine disruption. in: gupta r, editor. veterinary toxicology, 3rd edition, st. louis, mo; elsevier: 2007, p. 206-244. 41. james l, shupe j, binns w, et al: abortive and teratogenic effects of locoweed on sheep and cattle. am j vet res 1967;28:1379-1388. 42. navarro m, cristofol c, carretero a, et al: anthelmintic induced congenital malformations in sheep embryos using netobimin. vet rec 1998;142:86-90. https://doi.org/10.1136/vr.142.4.86. 43. whitlock bk, kaiser l, maxwell hs: heritable bovine fetal abnormalities. theriogenology 2008;70:535-549. https://doi.org/10.1016/j. theriogenology.2008.04.016. 44. lefebvre r: fetal mummification in the major domestic species: current perspectives on causes and management. vet med (auckl) 2015:6:233-244. https://doi.org/10.2147/vmrr.s59520. 45. lefebvre rc, saint-hilaire é, morin i, et al: retrospective case study of fetal mummification in cows that did not respond to prostaglandin f2α treatment. can vet j 2009;50:71-76. 46. ghanem me, suzuki t, akita m, et al: neospora caninum and complex vertebral malformation as possible causes of bovine fetal mummification. can vet j 2009;50:389-392. 47. woolums a: chlamydiosis. in: chase c, lutz k, mckenzie e: editors. blackwell’s five-minute veterinary consult ruminant. 2nd edition, hoboken, nj; john wiley & sons: 2017. p. 219-220. 48. wilson k, livingstone m, longbottom d: comparative evaluation of eight serological assays for diagnosing chlamydophila abortus infection in sheep. vet microbiol 2009;135:38-45. https://doi.org/10.1016/j. vetmic.2008.09.043. 49. de la concha-bermejillo a: q fever: an overview. 105th u. s. anim. health assoc., 2002, p. 391-314. 50. cilia g, bertelloni f, fratini f: leptospira infections in domestic and wild animals. pathogens 2020;9:573. https://doi.org/10.3390/ pathogens9070573. 51. van de weyer lm, hendrick s, rosengren l, et al: leptospirosis in beef herds from western canada: serum antibody titers and vaccination practices. can vet j 2011;52:619-626. 52. grooms dl, bolin ca: diagnosis of fetal loss caused by bovine viral diarrhea virus and leptospira spp. vet clin north am food anim pract 2005;21:463-472. https://doi.org/10.1016/j.cvfa.2005.02.010. 53. smith c, ketterer p, mcgowan m, et al: a review of laboratory techniques and their use in the diagnosis of leptospira interrogans serovar hardjo infection in cattle. aust vet j 1994;71:290-294. https:// doi.org/10.1111/j.1751-0813.1994.tb03447.x. 54, grégoire f, bakinahe r, petitjean t, et al: laboratory diagnosis of bovine abortions caused by non-maintenance pathogenic leptospira spp.: necropsy, serology and molecular study out of a belgian experience. pathogens 2020;9:413. https://doi.org/10.3390/pathogens9060413. 55. wynwood sj, burns ma, graham gc, et al: serological diagnosis of leptospirosis in bovine serum samples using a microsphere immunoassay. vet rec open 2016;3:e000148. https://doi.org/10.1136/ vetreco-2015-000148. 56. rodrigues hoffmann a, welsh cj, varner pw, et al: identification of the target cells and sequence of infection during experimental infection of ovine fetuses with cache valley virus. j virol 2012;86:4793-800. https://doi.org/10.1128/jvi.06858-11. 57. chung s, livingston cj, edwards j, et al: congenital malformations in sheep resulting from in utero inoculation of cache valley virus. abstract europe pmc. am j vet res 1990;51:1645-1648. 58. rodrigues hoffmann a, dorniak p, filant j, et al: ovine fetal immune esponse to cache valley virus infection. j virol 2013;87:5586–5592. https://doi.org/10.1128/jvi.01821-12. 59. wang h, nattanmai s, kramer ld, et al: a duplex real-time reverse transcriptase polymerase chain reaction assay for the detection of california serogroup and cache valley viruses. diagn microbiol infect dis 2009;65:150-157. https://doi.org/10.1016/j.diagmicrobio.2009.07.001. clinical theriogenology 2021; 13: 219 60. desmecht d, vanden bergh r, sartelet a, et al: evidence for transplacental transmission of the current wild-type strain of bluetongue virus serotype 8 in cattle. vet rec 2008;163:50-52. https://doi. org/10.1136/vr.163.2.50. 61. waldvogel as, anderson ga, phillips dl, et al: infection of bovine fetuses at 120 days’ gestation with virulent and avirulent strains of bluetongue virus serotype 11. comp immunol microbiol infect dis 1992;15:53-63. https://doi.org/10.1016/0147-9571(92)90102-w. 62. de la concha-bermejillo a: bluetongue virus. in: chase c, lutz k, mckenzie e: editors. blackwell’s five-minute veterinary consult ruminant. 2nd edition, hoboken, nj; john wiley & sons: 2017. p. 91-93. 63. smith db, meyers g, bukh j, et al: proposed revision to the taxonomy of the genus pestivirus, family flaviviridae. j gen virol 2017;98:21062112. https://doi.org/10.1099/jgv.0.000873. 64. walz ph, chamorro mf, falkenberg s, et al: bovine viral diarrhea virus: an updated american college of veterinary internal medicine consensus statement with focus on virus biology, hosts, immunosuppression, and vaccination. j vet intern med 2020;34:1690-1706. https:// doi.org/10.1111/jvim.15816. 65. maya l, macías-rioseco m, silveira c, et al: an extensive field study reveals the circulation of new genetic variants of subtype 1a of bovine viral diarrhea virus in uruguay. arch virol 2020;165:145-156. https:// doi.org/10.1007/s00705-019-04446-z. 66. braun u, hilbe m, peterhans e, et al: border disease in cattle. vet j 2019;246:12-20. https://doi.org/10.1016/j.tvjl.2019.01.006. 67. corbett em, grooms dl, bolin sr, et al: use of sentinel serology in a bovine viral diarrhea virus eradication program. j vet diagn invest 2011;23:511-515. https://doi.org/10.1177/1040638711403412. 68. mazuz ml, fish l, reznikov d, et al: neosporosis in naturally infected pregnant dairy cattle. vet parasitol 2014;205:85-91. https:// doi.org/10.1016/j.vetpar.2014.06.009. 69. mesquita lp, nogueira ci, costa rc, et al: antibody kinetics in goats and conceptuses naturally infected with neospora caninum. vet parasitol 2013;196:327-333. https://doi.org/10.1016/j.vetpar.2013.03.002. 70. dubey jp, schares g, ortega-mora lm: epidemiology and control of neosporosis and neospora caninum. clin microbiol rev 2007;20:323367. https://doi.org/10.1128/cmr.00031-06. 71. michael e, smith me, katabarwa mn, et al: substantiating freedom from parasitic infection by combining transmission model predictions with disease surveys. nat commun 2018;9:4324. https://doi.org/10.1038/ s41467-018-06657-5. 72. vidal s, kegler k, greub g, et al: neglected zoonotic agents in cattle abortion: tackling the difficult to grow bacteria. bmc vet res 2017;13:373. https://doi.org/10.1186/s12917-017-1294-y. clinical theriogenology 2021; 13: 220 laboratory use in pregnancy loss diagnosis 2018: nerve dysfunction in the penis of an angus bull nerve dysfunction in the penis of an angus bull college of veterinary medicine, auburn university, auburn, al a 4-year-old angus bull was presented for spiral deviation of the penis and inability to impregnate cows. at the end of the previous breeding season, the bull was removed from the breeding herd for seven months of sexual rest with no health concerns. a routine breeding soundness examination performed by the referring veterinarian revealed a corkscrew deviation of the penis during electroejaculation. the owner noticed during natural service attempts that the penis spiraled and coitus could not be achieved. the theriogenology service performed a breeding soundness examination. during electroejaculation, spiraling was observed and mild blushing was seen on the free portion of the penis just proximal to the glans penis. a test mating was observed and upon mounting the corkscrew deviation was noted. a cavernosagram was performed to evaluate blood flow within the penis. the results did not definitively indicate the presence of a shunt. a nerve stimulation test to evaluate nerve function in the penis determined no sensation to be present in the glans penis. even though the bull was able to achieve an erection, lack of sensation in the glans penis would not allow the bull to achieve intromission. the stimuli in the glans penis are conducted over the dorsal nerves of the penis to the pudendal nerves, which connect to the spinal cord and transfer stimulation to the brain. if 75% of the sensory fibers in the dorsal nerves are damaged, the bull usually cannot ejaculate.1 the best way to evaluate function of the dorsal nerve is to observe the bull successfully achieve intermission and ejaculation.1 intromission was not achieved by this bull and did not evoke action potentials nor sensory nerve or motor nerve conduction velocities as determined by the nerve stimulation test. this test provides consistent and highly reproducible measurement of sensory-system function of the penis.1 reference 1. wolfe df, moll hd: examination and special diagnostic procedures of the penis and prepuce. in: wolfe df, moll hd, editors. large animal urogenital surgery. baltimore: williams and wilkins; 1998. p. 221-231. clinical theriogenology • volume 10, number 3 • september 2018353 m. pelletier, m. edmondson, j. gard brix refractometer scoring of immunoglobulin g in postpartum mares™ colostrum   brix refractometer scoring of immunoglobulin g in postpartum mares’ colostrum julie storme,a patrick mccue,a christian bisiau,a ann hessb adepartment of clinical sciences and bdepartment of statistics colorado state university, fort collins, co abstract newborn foals are dependent on ingestion of high-quality colostrum for their immune protection. colostrum quality (immunuoglobulin g [igg]) can be determined by the single radial immunodiffusion (srid) test or estimated by brix refractometer. goal was to compare srid test findings (igg concentrations) and brix refractometer readings (igg percentage) in colostrum samples collected from mares during the first 12 18 hours postpartum. a total of 56 colostrum samples were collected from 10 postpartum mares. postfoaling, colostrum igg concentrations were 21,785 ± 5,592 mg/dl and igg percentage was 27.9 ± 3.7%. at 12 hours postfoaling, igg concentrations decreased to 1,713.9 ± 1,380.4 mg/dl and its percentage to 11.4 ± 1.6%. there was a high correlation (r = 0.9661) between srid test results and brix refractometry scores. in summary, brix refractometry can be used to monitor colostrum quality. keywords: mare, colostrum, immunoglobulin g, single radial immunodiffusion test, refractometer introduction passive transfer of immunoglobulins through ingestion of colostrum is critical to the survival of equine neonates.1,2 the epitheliochorial placenta of the equine does not allow for any substantial transplacental transfer of immunoglobulins during pregnancy.3,4 intestinal absorption of antibodies from colostrum is highest within the first 12 hours of life and very little absorption occurs after 24 hours.5-7 evaluation of colostrum quality immediately after foaling can be beneficial in predicting the efficacy of passive transfer of immunoglobulins.8,9 foals born to mares with good quantity and quality colostrum are more likely to have adequate passive antibody transfer. clinical situations associated with a limited amount or poor quality colostrum include premature leakage of colostrum, inadequate mammary development in maiden mares and fescue toxicity.10-12 early intervention of at-risk foals by oral treatment of supplemental colostrum from a colostrum bank is an easier and less expensive alternative than intravenous plasma treatment.12,13 the gold standard for measurement of immunoglobulin g (igg) concentrations in plasma or colostrum is the single radial immunodiffusion (srid) test.14 unfortunately, srid test requires ~ 18 24 hours to perform,9 making it unsuitable when rapid clinical decisions have to be made. the colostrometer, a second method of colostrum evaluation, measures specific gravity or density of colostrum.15 although specific gravity measurements of colostrum are correlated with igg concentrations, the colostrometer is cumbersome, the procedure is time consuming, and prone to errors due to the requirement to measure an exact volume of colostrum.8 brix or sugar refractometer has been described as a rapid, inexpensive and repeatable technique to estimate the quality of equine colostrum.8,16,17 quality of equine colostrum as estimated by a brix refractometer is highly correlated with the igg content of colostrum as determined by immunoradiodiffusion,8 or immunoturbidimetric assay.17 in addition, the brix refractometer score of mare colostrum collected immediately after foaling has been reported to be correlated with igg in foal plasma collected 12 48 hours after birth.17 other reasons for evaluation of mare colostrum besides prevention of failure of passive transfer include informed management decisions on harvesting good quality colostrum from individual mares for a frozen colostrum bank and monitoring depletion of antibodies in colostrum of mares seropositive for anti-red blood cell (rbc) antigens for the prevention of neonatal isoerythrolysis in at-risk foals.17 it is common practice to muzzle foals born from mares that are seropositive for antirbc antibodies for 24 hours after birth, provide an alternative source of colostrum and repeatedly strip out and discard the potentially harmful colostrum from the dam to prevent clinical disease.18 clinical theriogenology • volume 12 number 4 • december 2020486   objectives were to monitor the decreases in igg content of colostrum during the first 12 18 hours in normal postpartum mares using srid and brix refractometry tests and to determine the correlation between these 2 techniques. the clinical rational was to assess whether brix refractometry could be used in the preventive management of neonatal isoerythrolysis and in the selection of colostrum to be harvested and stored in a colostrum bank. materials and methods all procedures were approved by institutional animal care and use committee. a total of 10 late-term pregnant mares were used. all mares foaled under veterinary supervision and without assistance. an assessment of the health status of each foal was made in the early postpartum period. foals were subsequently monitored for the time interval from birth to when the foal first stood and the time to when the foal first nursed. continued observations were made throughout the first day to confirm that all foals were active and had nursed appropriately. a small volume (< 1 ml) of colostrum was collected from each mare immediately after foaling and subsequently at ~ 2 hour intervals for a period of 12 18 hours. one drop from each fluid sample was applied to a brix refractometer (animal reproduction systems, chino, ca) and the brix score was recorded as a percentage.8,16,19 the remaining colostrum samples were subsequently submitted to the colorado veterinary diagnostic laboratory for measurement of equine igg concentrations (mg/dl) using a single radial immunodiffusion (srid) test (equine igg test kit, cat. # 828411, triple j farms, bellingham, wa). data analyses statistical analysis was performed using r 3.6.1 statistical software. nonlinear regression was used to fit a 4-parameter logistic curve to the data. the correlation coefficient (r value) between srid values and brix score was also determined. all data are presented as the mean ± standard deviation. results average intervals from birth to first standing and birth to first nursing for 10 foals were 47.9 ± 45.8 and 155.6 ± 68.2 minutes, respectively. a total of 56 colostrum samples were collected from 10 postpartum mares. average srid igg concentrations of colostrum immediately after foaling was 21,785 ± 5,592 mg/dl. a steady decline in igg was noted over time as foals started to nurse and continued nursing (figures 1 and 2). individually, the igg concentrations in the colostrum of all 10 mares decreased to < 5,000 mg/dl by 9.1 ± 2.8 hours postfoaling (figure 1). overall, the igg concentration decreased to 1,713.9 ± 1,380.4 mg/dl at 12 hours after foaling (figure 2). figure 1 concentrations of igg (mg/dl) in equine colostrum as determined by single radial immunodiffusion test from immediately after birth to 12 18 hours postpartum in 10 mares; note: a nonlinear logistic regression curve was fit to data. clinical theriogenology • volume 12 number 4 • december 2020 487   figure 2. igg concentrations in colostrum (mg/dl) for 10 individual postpartum mares as determined by srid test average brix refractometry score of colostrum samples collected immediately after foaling was 27.9 ± 3.7%. refractometry score declined rapidly over time as foals were nursed (figures 3 and 4). the brix percentage decreased to < 15% by 9.3 ± 3.0 hours postfoaling and was 11.4 ± 1.6% at 12 hours postfoaling. figure 3. brix refractometry score (%) for equine colostrum from immediately after birth to 12 18 hours in 10 mares; note: a nonlinear logistic regression curve was fit to data. clinical theriogenology • volume 12 number 4 • december 2020488   figure 4. brix refractometry score (percentage) for colostrum from 10 individual postpartum mares the correlation coefficient (r) between srid values and brix scores for all 56 mammary fluid samples was 0.9661 (figure 5). figure 5. relationship between igg content measured by srid test (mg/dl) and brix refractometry score (percentage) 0 5,000 10,000 15,000 20,000 25,000 30,000 35,000 5 10 15 20 25 30 35 ig g s r id m g/ dl brix score % igg srid versus brix % clinical theriogenology • volume 12 number 4 • december 2020 489   discussion mares begin to produce colostrum during last 2 weeks of pregnancy by sequestering antibodies from their blood into fluid produced within alveoli of the mammary gland.20 production of new colostrum decreases or ceases at the time of foaling or shortly thereafter.21 the high antibody content of colostrum is gradually depleted as the foal nurses and the fluid volume is replaced by production of milk. the transition of fluid within the mammary gland from colostrum to milk occurs over the first 24 hours in a normal postpartum mare. results confirmed that brix refractometry scores are highly correlated to a quantitative measurement of igg in colostrum as determined by srid test. brix refractometry had the advantages of being ultra-rapid, simple to perform, low cost, and easy to interpret. primary clinical indication for routine use of the brix refractometer to evaluate mare colostrum is in the prevention and management of passive transfer of immunoglobulins.12,16 early identification of poor-quality colostrum in a postpartum mare allows for early intervention through oral supplementation with frozen-thawed colostrum from an on-site colostrum bank within the first few hours of life. routine evaluation of colostrum quality and therapeutic intervention has largely eliminated the need for intravenous plasma transfusions on well managed equine breeding farms. observations regarding the health and development of the neonatal foal are also critical in the prevention of failure of passive transfer. foals generally stand within 1 hour after birth and begin to nurse within 2 hours.22,23 another clinical indication for use of the brix refractometer for immediate evaluation of colostrum in a postpartum mare is the determination of quality of the colostrum for banking.12,24,25 in general, it is recommended that colostrum be harvested for frozen storage if the brix score is ≥ 20 to 23%.8,12,17 colostrum with a brix score of < 20% is of poorer quality and should not be banked for future use. our study results suggest that it is best to harvest colostrum for frozen storage very early in the postpartum period and not to wait even for a few hours since suckling significantly reduced igg content. furthermore, igg content in mammary fluid of mares being suckled by healthy foals are low by 9 12 hours postpartum and are essentially just milk and no longer considered to be colostrum. finally, monitoring colostrum quality using a brix refractometer can be used as 1 component of a management plan for the prevention of clinical cases of neonatal isoerythrolysis or ‘jaundice foal syndrome’. clinical neonatal isoerythrolysis may occur if a late-term pregnant mare has antirbc antibodies directed against her foal’s red blood cell antigens (i.e. aa, qa, etc.) and the newborn foal ingests a substantial quantity of high-titer antirbc colostrum.26,27 serotesting late-term pregnant mares during last 2 weeks prior to their due date can be used to determine if there are antirbc antibodies present and if the foal is at risk of clinical disease.27 seronegative pregnant mares are at no risk of having a foal develop clinical ni. in contrast, a foal born to a seropositive mare may be at risk, depending on the red blood cell antigen type of the foal and the type and titer of antirbc antibodies in the mare. laboratory tests used to identify foals born from seropositive mares and therefore at risk of developing ni include hemolytic and agglutination cross-matching assays.28 the jaundice foal agglutination (jfa) test is performed by mixing red blood cells from the foal into serial dilutions of the mare’s colostrum followed by centrifugation. agglutination of rbcs in the bottom of the centrifuge tube at a dilution of 1:16 or higher is considered significant. if a jfa test is not available, prevention of clinical disease in at-risk foals is based on prevention of foal from nursing for 24 36 hours and harvesting and discarding the potentially harmful colostrum from the mare.18 the former is usually accomplished by muzzling the foal, administration of supplemental colostrum and subsequently feeding a commercial equine milk substitute. the latter is accomplished by repeatedly stripping and discarding colostrum from the mare’s udder every 2 hours for 24 hours. this timeframe is based on the physiological assumption that the foal cannot absorb any significant quantity of antibodies after 24 hours post birth. conclusion brix refractometry score for equine colostrum was highly correlated with the igg concentration as determined by the single radial immunodiffusion assay. evaluation of colostrum quality in the clinical theriogenology • volume 12 number 4 • december 2020490   immediate postpartum period is valuable in determination of which colostrum to harvest for banking and which newborn foals are at potential risk of failure of passive transfer. monitoring colostrum quality over time may also be valuable in the management of foals at risk of neonatal isoerythrolysis. additional laboratory and field studies are needed to confirm the relationship between the brix refractometer score and specific antirbc antibody concentration in mare colostrum. conflict of interest none to declare. references 1. jeffcott lb: the transfer of passive immunity to the foal and its relation to immune status after birth. j reprod fertil 1975;suppl 23:727-733. 2. mcguire tc, crawford tb, hallowell al, et al: failure of colostral immunoglobulin transfer as an explanation for most infections and deaths of neonatal foals. j am vet med assoc 1977;170:1302-1304. 3. chucri tm, monteiro jm, lima ar, et al: a review of immune transfer by the placenta. j reprod immunol 2010;87:14-20. 4. borghesi j, mario lc, rodrigues mn, et al: immunoglobulin transport during gestation in domestic animals and humans a review. open j anim sci 2014;4:323-336. 5. jeffcott lb: studies on passive immunity in the foal: ii. the absorption of 125i-labelled pvp (polyvinyl pyrrolidone) by the neonatal intestine. j comp path 1974;84:279-289. 6. raidal sl, mctaggart c, penhale j: effect of withholding macromolecules on the duration of intestinal permeability to colostral igg in foals. aust vet j 2005;83:78-81. 7. vivrette sl: assessment and modification of passive transfer. in: equine reproduction. 2nd edition. mckinnon ao, squires el, vaala we, et al: editors. ames; wiley-blackwell: 2011. p. 346-352. 8. chavatte p, clément f, cash r, et al: field determination of colostrum quality by using a novel, practical method. proc am assoc equine pract 1998; p. 206-209. 9. davis r, giguère s: evaluation of five commercially available assays and measurement of serum total protein concentration via refractometry for the diagnosis of failure of passive transfer of immunity in foals. j am vet med assoc 2005;227:1640-1645. 10. clabough dl, levine jf, grant gl, et al: factors associated with failure of passive transfer of colostral antibodies in standardbred foals. j vet int med 1991;5:335-340. 11. leblanc mm, tran t, baldwin jl, et al: factors that influence passive transfer of immunoglobulins in foals. j am vet med assoc 1992;200:179-183. 12. nath lc, anderson ga, savage cj, et al: use of stored equine colostrum for the treatment of foals perceived to be at risk for failure of transfer of passive immunity. j am vet med assoc 2010;236:1085-1090. 13. massey re, leblanc mm, klapstein ef: colostrum feeding of foals and colostrum banking. proc am assoc equine pract 1991;37:1-8. 14. young km, lunn dp: immunodiagnostic testing in horses. vet clin n am-equine 2000;16:79-103. 15. leblanc mm, mclaurin bi, boswell r: relationships among serum immunoglobulin concentration in foals, colostral specific gravity, and colostral immunoglobulin concentration. j am vet med assoc1986;189:57-60. 16. cash rs: colostral quality determination by simple refractometry, a preliminary investigation. centaur 1995;11:56-59. 17. cash rs: colostral quality determined by refractometry. equine vet educ 1999;11:36-38. 18. mccue pm, ra ferris, ea swain, et al: foal formulary and field protocol guide. fort collins, colorado state university: 2017. p. 66-68. 19. mccue pm: evaluation of colostrum quality: brix refractometry. in: dascanio j, mccue p: editors. equine reproductive procedures. ames, wiley-blackwell: 2014. p. 297-298. 20. knottenbelt dc, holdstock n, madigan je: the role of colostrum in immunity. in: knottenbelt dc, holdstock n, madigan je, editors. equine neonatology, medicine and surgery, philadelphia, wb saunders: 2004. p.15-18. 21. delouis c: physiology of colostrum production. ann rech vét 1978;9:193-203. 22. magdesian kg: neonatology. in: orsini ja, tj divers, editors. equine emergencies. 3rd edition, st. louis; saunderselsevier: 2014. p. 486-521. 23. mccue pm, ferris ra: parturition, dystocia and foal survival: a retrospective study of 1047 births. equine vet j 2012;44:22-25. 24. lester gd: colostrum: assessment of quality and artificial supplementation. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction, 2nd edition, ames; wiley-blackwell: 2011. p. 99-110. 25. mccue pm: colostrum banking. in: dascanio j, mccue p, editors. equine reproductive procedures. ames; wileyblackwell: 2014. p. 299-301. 26. boyle ag, magdesian kg, ruby re: neonatal isoerythrolysis in horse foals and a mule foal: 18 cases (1988–2003). j am vet med assoc 2005;227:1276-1283. clinical theriogenology • volume 12 number 4 • december 2020 491   27. johnson jr: neonatal isoerythrolysis. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction, 2nd edition; ames, wiley-blackwell: 2011. p. 353-360. 28. whiting jl: neonatal isoerythrolysis. in: robinson ne, editor. current therapy in equine medicine, 5th edition, philadelphia; saunders: 2003. p. 636-640.   clinical theriogenology • volume 12 number 4 • december 2020492 2018: embryo transfer as a reproductive management tool embryo transfer as a reproductive management tool peter j. hansen department of animal sciences, university of florida, gainesville fl introduction – embryo transfer is more than just a tool for genetic selection regardless of whether generated by superovulation (otherwise called multiple ovulation – embryo transfer; moet) or by in vitro production (ivp), the most common purpose of transfer of cattle embryos today is for genetic improvement. the advent of genomics means that genetically-superior females can be identified with acceptable accuracy and these females can be used to produce up to 100 or more embryos from a cow or heifer each year. embryo transfer can also be used to rapidly establish a herd of high-producing animals, as exemplified by the large numbers of embryos exported from north america to other countries to establish high-yielding dairy herds. production of embryos in vitro is also an effective means of utilizing sexed semen because one straw of sexed semen can be used to produce multiple embryos in vitro. the pregnancy rates following transfer into recipients is similar between cows receiving embryos produced sexed semen and those receiving embryos produced with conventional semen (xu et al., 2006; rasmussen et al., 2012). a less well appreciated use of the bovine embryo is as a tool for improving pregnancy rates. in general, the percent of cows pregnant after receiving an embryo is not appreciably higher than pregnancy success following artificial insemination (ai). however, embryo transfer can improve fertility of cow populations when reproduction is suboptimal because of heat stress. there is also some evidence in dairy cattle that embryo transfer can improve pregnancy rates of lactating cows classified as repeat breeders. the physiological basis for improvement in fertility using embryo transfer pregnancy rates can be improved in infertile cows by embryo transfer because the procedure bypasses certain causes of pregnancy failure that occur before the time when an embryo is typically transferred into cows (day 7-8 after ovulation). among the reasons why a cow fails to become pregnant after ai are anovulation, fertilization failure and early embryonic death. for an embryo transfer recipient, these causes are reduced because most embryos used for transfer are the product of a successful fertilization (with the exception of a few parthenotes) and the embryo has been capable of developing to the morula stage or blastocyst stage when it is typically transferred. embryo transfer during heat stress the best documented example of the use of embryo transfer to improve fertility is for the heatstressed cow. much of the negative effect of heat stress on pregnancy establishment involves negative effects on the oocyte or the developing embryo. the oocyte can be damaged by heat stress as early as 105 days before ovulation (torres-júnior et al., 2008) and remains sensitive to heat stress through oocyte maturation on the day of ovulation (putney et al., 1989b). development of the early embryo is also inhibited by heat stress but it becomes resistant to elevated temperature by about the 8-cell stage of development (hansen, 2013). experimentally, heat stress at day 1 after estrus reduced embryonic development but heat stress at days 3, 5, and 7 had no effect (ealy et al., 1993). what this means is that the morula and blastocyst stage embryos that are typically transferred into recipients are largely resistant to damage by maternal hyperthermia and subsequent development is unlikely to be compromised by heat stress. the effectiveness of embryo transfer for improving pregnancy rate during heat stress is shown by representative experiments depicted in figure a. the improvement in fertility with et as compared to ai has been seen when embryos were produced by moet (putney et al., 1989a; drost et al., 1999; rodrigues et al., 2004; vasconcelos et al., 2011) or by ivp (ambrose et al., 1999; al-katanani et al., 2002; block et al., 2010; stewart et al., 2011). the poor cryosurvival of ivp embryos means, however, that pregnancy rate was improved during heat stress when ivp embryos were transferred fresh but not when transferred after conventional freezing or vitrification (see data from drost et al., 1999; block et al., 2010 and stewart et al., 2011 presented in figure a). clinical theriogenology • volume 10, number 3 • september 2018229 using embryo transfer, the summer decline in fertility of lactating cows can be largely eliminated. this idea is shown graphically for an experiment in brazil in figure 1b (rodriques et al. (2004) and there are other examples in the literature (putney et al., 1988; chebel et al., 2008; vasconcelos et al., 2011; ferraz et al., 2016). one important consideration when carrying out embryo transfer during heat stress is that estrous behavior is reduced in cows exposed to heat stress. accordingly, the technique is usually performed using fixed-time embryo transfer based on one or more ovulation synchronization protocols such as ovsynch. in such systems, the day of ovulation (i.e., day of insemination for timed ai protocols) is considered day 0 when calculating synchrony with the embryo. not every cow responds successfully to ovulation synchronization protocols so an embryo is transferred only into those recipients with a functional corpus luteum as determined by rectal palpation or ultrasonic examination. repeat breeder cows another possible use for embryo transfer is to improve fertility of the repeat-breeder cow. these animals are defined as those that have not become pregnant after multiple inseminations (usually three). there are three experiments supporting the idea that embryo transfer can increase fertility for these type of animals as compared to fertility after ai (table). each of these studies involved small numbers of animals and further work is warranted. moreover, it is unlikely that embryo transfer will prevent all sources of infertility associated with the repeat-breeding condition. ribeiro et al. (2016) observed that negative effects of disease incidence (uterine or non-uterine) before breeding on fertility was apparent in cows subjected to either ai or embryo transfer. can embryo transfer be used to improve pregnancy rates in the absence of infertility? based on what was stated at the beginning of this paper, one would expect that pregnancy rate following embryo transfer would usually be higher than pregnancy after ai because failure of achieve pregnancy because of oocyte defects, anovulation, misdiagnosis of estrus, errors in insemination, poor semen quality, fertilization failure, and disruptions in early embryonic development would be prevented. surprisingly, however, there is not usually a fertility advantage for embryo transfer as compared to ai unless there is some underlying cause of infertility such as heat stress or the repeat-breeder condition. this phenomenon is illustrated in the experiment of rodrigues et al. (2004) depicted in figure b. although pregnancy rates were much higher for embryo transfer than ai in the warm months, pregnancy rates were similar for embryo transfer and ai in cool months of the year. similar results were seen in studies with lactating cows in wisconsin (sartori et al., 2006) and florida including one performed in cool months (rasmussen et al., 2012) and one using data collected year-round (ribeiro et al., 2016). the reason why et does not meet the promise of improving fertility in most situations is not known. it may be that technical limitations in embryo production or transfer limit the expected improvement in pregnancy rate. clinical theriogenology • volume 10, number 3 • september 2018 230 figure. enhancement of pregnancy rates during heat stress using embryo transfer. data in panel a represent results from various experiments in the summer in florida. abbreviations are as follows: ai: artificial insemination; eg, frozen in ethylene glycol; f, fresh; gly, frozen in glycerol; ivfet, embryo transfer with an in vitro produced embryo; moet, multiple ovulation embryo transfer; tai, timed artificial insemination; tet-ivf, timed embryo transfer with an in vitro produced embryo; vit, vitrified. the numbers in the graph represent the day of gestation at which pregnancy diagnosis was carried out. panel b represents data from a commercial dairy in brazil in which cows were either inseminated or received an embryo produced by superovulation (rodriques et al., 2004). asterisks represent months in which pregnancy rate was different between ai and et. the figure is reproduced from hansen (2013). clinical theriogenology • volume 10, number 3 • september 2018231 table. improvement in fertility in repeat-breeder dairy cows by embryo transfer (et) as compared to artificial insemination (ai). study location embryo type definition of repeat-breeder treatment number of animals pregnancy rate, percent tanabe et al., 1985 usapennsylvania moet >3 infertile services ai at estrus 22 50 et at estrus 23 70 son et al., 2007 south korea moet 3 or more infertile services ai at estrus 27 18.5 timed ai 13 7.7 timed et 13 53.8 block et al., 2010 usa – florida ivp > 3 infertile services timed ai 33 21.1 timed etfresh 25 60.0 timed etvitrified 31 29.0 conclusion based on the experiments conducted to date, there is a high degree of confidence that embryo transfer can improve pregnancy rate when heat stress is a factor. although the evidence is less clear, there may also be an improvement in fertility using embryo transfer for repeat-breeder cows. the decision to use embryo transfer for fertility enhancement depends on an individual cow’s fertility, the expected improvement in pregnancy rate caused by using embryo transfer, the cost of the embryo available for transfer, and the economic value of getting a cow pregnant. for a discussion of the economic value of embryo transfer, see papers by ribeiro et al. (2012) and kaniyamattam et al. (2018). references al-katanani ym, drost m, monson rl, et al: pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions. theriogenology 2002; 58:171-182. ambrose jd, drost m, monson rl, et al: efficacy of timed embryo transfer with fresh and frozen in vitro produced embryos to increase pregnancy rates in heat-stressed dairy cattle. j dairy sci 1999; 82:2369-2376. block j, bonilla l, hansen pj: efficacy of in vitro embryo transfer in lactating dairy cows using fresh or vitrified embryos produced in a novel embryo culture medium. j dairy sci 2010; 93:5234-5242. chebel rc, demétrio dg, metzger j: factors affecting success of embryo collection and transfer in large dairy herds. theriogenology 2008; 69:98-106. drost, m, ambrose jd, thatcher m-j, et al: conception rates after artificial insemination or embryo transfer in lactating dairy cows during summer in florida. theriogenology 1999; 52:1161-1167. ealy ad, drost m, hansen pj: developmental changes in embryonic resistance to adverse effects of maternal heat stress in cows. j. dairy sci 1993; 76:2899-2905. ferraz pa, burnley c, karanja j, et al: factors affecting the success of a large embryo transfer program in holstein cattle in a commercial herd in the southeast region of the united states. theriogenology 2016; 86:1834-1841. hansen pj: cellular and molecular basis of therapies to ameliorate effects of heat stress on embryonic development in cattle. anim reprod 2013;10:322-333. kaniyamattam k, block j, hansen pj, et al: economic and genetic performance of various combinations of in vitro-produced embryo transfers and artificial insemination in a dairy herd. j dairy sci 2018;101:1540-1553. putney dj, drost m, thatcher ww: influence of summer heat stress on pregnancy rates of lactating dairy cattle following embryo transfer or artificial insemination. theriogenology 1989a;31:765-778 putney dj, mullins s, thatcher ww, et al: embryonic development in superovulated dairy cattle exposed to elevated ambient temperatures between the onset of estrus and insemination. anim reprod sci 1989b;19:37-51. clinical theriogenology • volume 10, number 3 • september 2018 232 putney dj, thatcher ww, drost m, et al: influence of environmental temperature on reproductive performance of bovine embryo donors and recipients in the southwest region of the united states. theriogenology 1988; 30:905-922. rasmussen s, block j, seidel ge, et al: pregnancy rates of lactating cows after transfer of in vitro produced embryos using xsorted sperm. theriogenology 2012;79:453-461. ribeiro es, galvão kn, thatcher ww, et al: economic aspects of applying reproductive technologies to dairy herds. anim reprod 2012;9:370-387. ribeiro es, gomes g, greco lf, et al: carryover effect of postpartum inflammatory diseases on developmental biology and fertility in lactating dairy cows. j dairy sci 2016;99:2201-2220. rodrigues ca, ayres h, reis el, et al: artificial insemination and embryo transfer pregnancy rates in high production holstein breedings under tropical conditions. proc 15th int congr anim reprod 204;2:396 (abstr). sartori r, gümen a, guenther jn, et al: comparison of artificial insemination versus embryo transfer in lactating dairy cows. theriogenology 2006;65:1311-1321. son ds, choe cy, cho sr, et al: a cidr-based timed embryo transfer protocol increases the pregnancy rate of lactating repeat breeder dairy cows. j reprod dev 2007; 53:1313-1318. stewart bm, block j, morelli p, et al: efficacy of embryo transfer in lactating dairy cows during summer using fresh or vitrified embryos produced in vitro with sex-sorted semen. j dairy sci 2011; 94:3437-3445. tanabe ty, hawk hw, hasler jf: comparative fertility of normal and repeat-breeding cows as embryo recipients. theriogenology 1985;23:687-696. torres-júnior jr de s, pires m de fa pires, de sá wf, et al: effect of maternal heat-stress on follicular growth and oocyte competence in bos indicus cattle. theriogenology 2008; 69:155-166. vasconcelos jl, jardina dt, sá filho og, et al: comparison of progesterone-based protocols with gonadotropin-releasing hormone or estradiol benzoate for timed artificial insemination or embryo transfer in lactating dairy cows. theriogenology 2011; 75:1153-1160. xu j, guo z, su l, et al: developmental potential of vitrified holstein cattle embryos fertilized in vitro with sex-sorted sperm. j dairy sci 2006; 89:2510-2518. clinical theriogenology • volume 10, number 3 • september 2018233 clinical theriogenology • volume 10, number 3 • september 2018 234 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice bull reproductive development and sperm production enhancement bull reproductive development and sperm production enhancement ahmed tibary,a muhammad waqas,b michela ciccarelli,a cristian patinoa adepartment of veterinary clinical sciences and center for reproductive biology washington state university, pullman, wa bdepartment of theriogenology, university of agriculture faisalabad, pakistan abstract the onset of puberty and daily sperm production of bulls are fundamental aspects that impact natural breeding and semen production centers. critical molecular and endocrine aspects of testicular development, spermatogenesis and functions are reviewed. methods to enhance sperm production (gonadotropin supplementation, immunization against inhibin and induction of temporary hypothyroidism) and findings are discussed. keywords: spermatogenesis, sertoli cell, endocrinology, puberty, hypothyroidism introduction important aspects of bull testicular development and sperm production have been reviewed.1-3 breeding soundness examinations of thousands of bulls are performed every year before they are used for natural breeding or enter artificial insemination centers. however, on their first examination, approximately 20 25% of yearling beef bulls fail to meet minimum standards for testicular size and sperm morphology.4,5 for high genetic value bulls entering artificial insemination centers for semen collection and cryopreservation, maximum sperm production is required at each collection. development of reproductive system and in particular testicular function, starts during fetal life and continues through postnatal period until puberty (table). these developmental periods are primarily affected by genetics and environmental factors. testicular function and spermatogenic efficiency are mainly influenced by factors during prenatal period, prepubertal growth phase and post-pubertal maturation phase. understanding bull gonadal development facilitates optimal management of seedstock dams and young bulls. objectives are to review bull developmental biology, factors affecting spermatogenesis and potential ways to improve sperm production. development of reproductive system in the fetus understanding testicular differentiation and development has facilitated substantial improvement in recent years using molecular tools and advancements in genomics. differences in gene expression can be detected as early as blastocyst stage.6,7 differentiation of inner cell mass into somatic germ layers (ectoderm, endoderm, and mesoderm) follows hatching. hypothalamus, pituitary gland, and penis derive from ectoderm, whereas gonads, epididymides, ductus deferens and urinary system derive from mesoderm. somatic germ cells are pluripotent except some yolk sac inner lining cells that differentiate into primordial germ cells (pgcs). undifferentiated gonad forms after passive or amoeboid migration of pgcs into genital/gonadal ridge. this phenomenon is likely controlled by several molecular signals that are not yet completely understood. high mitotic index is recognized in pgcs and they rapidly populate genital ridge; 1000 2000 pgcs are already present in genital ridge by 25 days and gonads can be identified at 28 29 days of pregnancy. several key player genes (wilms tumor-1 factor, lim1 transcription factor, and steroid factor) are identified in gonadal development.8,9 wilms tumor-1 factor and zinc finger like transcription factor (produced by sertoli cells) have important roles in testicular cord assembly and regulating development of fetal leydig cells and peritubular myoid cells.10 differentiation of fetal testes is determined by sry gene (y chromosome sex determining region). expression of sry begins on day 37 and peaks on day 39.11 this gene encodes for a protein of high mobility group and regulates several other genes involved in gonadal differentiation into testis. one of these genes, sox9, is essential in differentiation of fetal sertoli cells. furthermore, gata4 (required for expression of sry gene and formation of genital ridge) and dmrt are also implicated. clinical theriogenology • volume 12 number 3 • september 2020 245 table. chronology of major events during fetal and postnatal development of bull reproductive system period major events embryonic and fetal development blastocyst differences between male and female embryos in gene expression hatching primordial germ cells derived from inner lining of yolk sac day 25 1000 2000 primordial germ cells migrate to genital ridge days 28 29 presence of undifferentiated gonad days 37 39 sry expression days 41 42 differentiation of male gonad, testicular chords, fetal leydig cells and sertoli cells day 47 masculinization of external genitalia due to effects of testosterone and androstenedione from fetal leydig cells day 50 start of paramesonephric ducts regression days 56 58 appearance of seminal vesicles and prostate day 60 differentiation of scrotum day 70 branching of seminal vesicles, differentiation of epididymis, stabilization of mesonephric ducts day 80 complete regression of paramesonephric duct; testes begin transabdominal phase of descent day 90 presence of major components of male reproductive system day 110 formation of epididymis, testis begin inguinoscrotal phase of descent day 120 testicular descent into scrotum postnatal development month 1 degeneration of fetal leydig cells, increased adult leydig cells and undifferentiated sertoli cells; proliferation of prespermatogonia month 5 appearance of primary spermatocytes; seminiferous tubule lumens begin to form months 5 8 presence of primary and secondary spermatocytes in seminiferous tubules months 7 9 complete detachment of penis from prepuce months 8 10 presence of mature sperm in seminiferous tubule lumens months 8 12 puberty: ejaculate with > 50 million sperm and > 10% progressive motility months 12 16 maturation differentiation of bovine testis starts at 41 42 days. at this stage, testicular cords lack a lumen and consist of undifferentiated sertoli cells. by 60 70 days, rete testis appears at end of testicular cords and become connected to each other. concurrently, first generation of fetal leydig cells (from mesonephros) appear in mesenchyme. after birth, fetal leydig cells degenerate and are replaced by adult leydig cells. external genitalia become masculinized by day 47 under effects of testosterone and androstenedione produced by leydig cells. by 60 days, scrotum is well developed. paramesonephric ducts start regressing by day 50 and are completely regressed by day 80. seminal vesicles and prostate appear by 56 58 days.12 by end of first trimester, all components of reproductive system are present except for epididymis (begins to form at 110 days.13) internal and external genitalia complete development occurs after 100 days. descent of testes within scrotum occurs at 100 120 days of pregnancy. testicular descent is mediated by insulin-like peptide 3 (from fetal leydig cells) that initiates transabdominal phase around 80 90 days of pregnancy. inguinoscrotal phase of testicular descent is mediated by androgens. this early maturation of hypothalamo-pituitary gonadal axis in bovine has been confirmed. concentrations of insulin-like peptide 3 and testosterone in maternal plasma are significantly higher at 4 and 8 months of pregnancy in cows carrying a male versus female fetus.14 prepubertal development onset of puberty in bulls is highly dependent on nutrition and body growth. slow growth is correlated to slower testicular development and delayed puberty. in some breeds, scrotal circumference is highly correlated to body weight.15 at birth, testes are composed of solid (no lumen) chords comprised of primordial germ cells, fetal leydig cells and undifferentiated sertoli cells (figure 1).16 accessory sex glands are not functional and penis is adherent to prepuce. fetal leydig cells degenerate clinical theriogenology • volume 12 number 3 • september 2020246 in first postnatal month and are replaced by adult leydig cells. gnrh receptors in anterior pituitary increase significantly from 6 10 weeks.17 increased frequency of gnrh pulses and corresponding increases in lh concentrations occur during cell differentiation. number of undifferentiated sertoli cells increases rapidly.16,18 during prepubertal period, serum fsh and inhibin concentrations are high, but decline with onset of rapid testicular growth.19 fsh has an important role in prepubertal proliferation of sertoli cells,20 as it increases at 4 25 weeks.21 figure 1: histological appearance of bull testis at 1 month (a, b), 5 months (c, d), 8 months (e, f), 10 months (g, h); note: development of a lumen at 5 months, initiation of spermatogenesis at 8 months, appearance of sperm in lumen by 10 months. (left column x 400, right column x 1000) proliferation of prospermatogonia began at 1 month, and primary spermatocytes appeared ~ 5 months.22 age of puberty was negatively correlated with magnitude of lh secretion in first 5 months of life,23 a highly heritable trait.24 detachment of the peno-preputial adhesions occurs ~ 6 weeks prior to puberty. as puberty approaches, testicular and epididymal growth accelerate and become almost linear. low-frequency lh pulses increase serum testosterone concentrations produced by a rapidly increasing number of adult leydig cells, promoting establishment of spermatogenesis.16 primary and subsequently secondary clinical theriogenology • volume 12 number 3 • september 2020 247 spermatocytes are detected 5 8 months (figure 1). mature sperm are present in seminiferous tubules from 8 10 months. puberty (ejaculate with at least 50 million sperm and at least 10% progressive motility) occurs when scrotal circumference reaches 28 30 cm in bos taurus2 and 27 28 cm in bos indicus.25 following puberty, bulls enter a phase of maturation characterized by increases in accessory sex gland weights, sperm production and proportion of morphologically normal sperm. this phase of maturation varies considerably amongst bulls, lasting 3 4 months in bos taurus.26 effect of nutrition on early development nutrition of the dam profoundly affects testicular development.27 heifers fed 2.4 x recommended energy and protein requirement in the first 6 months of pregnancy produced calves that had smaller testes and lower serum testosterone concentrations compared to calves from heifers fed 1.9 x or 0.7 x recommended ration.27 santa gertrudis bull calves from dams fed low-protein diet had delays in onset of puberty and sexual maturity than those from dams fed high-protein diet.28 however mechanism of this action remains unclear. bull calves fed diets to achieve 1.4 1.5 kg/day average daily gain reach puberty earlier with greater paired testes weight and daily sperm output.2,29,30 in general, with an average daily gain of 1 kg/day, puberty occurs at 8 12 months.31 preweaning early development of bulls was largely dependent on maternal milk production. bull calves born to heifers and aged dams (> 9 years) had smaller scrotal circumference due to lower hypothalamo-pitutary activity.2 however, effects of diet on onset of puberty are confounded by genetics and greater feed intake does not consistently result in earlier puberty.32 it is important to note that a high-energy diet, such as often fed in bull testing stations, may have adverse effects on testicular development and spermatogenesis. young bulls fed high energy diets have better average daily gain and thicker backfat, but their scrotal circumference may not be altered. 2,3,34 however, these bulls have more morphologically abnormal sperm due to poor testicular thermoregulation attributed to increased scrotal fat.2,33,34 enhancement of sperm production enhancement of sperm production in valuable bulls is important to satisfy the demand for more doses of semen (particularly sexed semen) for artificial insemination. in addition to care and nutrition of the pregnant dam, several other postnatal strategies were investigated to increase bull sperm production. unilateral castration increased the contralateral testicular size but failed to increase sperm production. in recent years, our understanding of the role of sertoli cell as support cell for spermatogenesis, provides new approaches to enhance sperm production. the role of sertoli cells in spermatogenesis was studied in a variety of species.20,35 strong positive correlation between sertoli cell number and sperm production capacity in bulls was established.20 sertoli cell number correlated with number of spermatogonial stem cell niches in mice, critical for spermatogenic activity and sperm production.36,37 postnatal expansion of sertoli cell population was described in several mammalian species as a 2 wave linear increase in sertoli cell population.37 termination of sertoli cell proliferation appears to be an extremely important biological point for manipulating spermatogenesis.37 increases in testicular estrogen concentrations arrest sertoli cell expansion in swine but apparently not in bulls.35 in the latter, based on early cytological studies, sertoli cell number plateaus at 6 7 months.22 however, based on cell proliferation marker ki67 and expression of sertoli cell marker sox9, sertoli cell proliferation ceases at 4.5 5 months (figure 2).38 during prepubertal development, sertoli cell proliferation is stimulated by fsh but inhibited by thyroid hormone concentrations. this relationship between sertoli cell proliferation and thyroid hormone concentrations was demonstrated in boars39 and rams.40 in bulls, thyroxine concentrations were negatively correlated with testicular size at puberty.41 these observations led to development of hormonal methods during early development to enhance sperm production. these hormonal methods clinical theriogenology • volume 12 number 3 • september 2020248 include immunization against inhibin, gnrh injections, fsh injections, and establishment of a transient hypothyroidism. immunization against inhibin in early postnatal period increases serum fsh concentrations, testicular size, and daily sperm production in bulls.42 however, it was transient and required multiple vaccinations. there was also a great variability of the response among individuals. administration of gnrh to prepubertal bulls (120 µg/kg) bid from 4 8 weeks hastened puberty by 6 weeks compared to untreated bulls.43 figure 2. cross-sectional view of seminiferous epithelium.38 green cells are sox9 positive sertoli cells, red cells are ki67 positive dividing cells. white arrow points to a dividing sertoli cell stained with both sox9 and ki67 at 4.5 months. pattern filled arrow indicates a ki67 positive dividing germ cell at 5 months; note: at 5 months none of the sox9 positive sertoli cells express ki67. bull calves given exogenous fsh every 2 days for 4 8 weeks had more sertoli cells number at 56 weeks and attained puberty 5 weeks earlier than nontreated calves.44 in a more recent study, administration of porcine fsh (30 mg, folltropin-v in 2% hyaluronic acid) from 35 91 days increased endogenous fsh at 70 days and sertoli cell numbers per seminiferous tubule at 93 days,45 with increase in endogenous fsh due to a positive feedback loop through activin a produced by sertoli cells. fsh-treated bulls had on an average 4 more sertoli cells per seminiferous tubule section than untreated bulls. 46 however, there was no difference in spermatogonial cell number.46 potential for enhancement of sperm production after induction of transient hypothyroidism in bulls (based on negative relationship between sertoli cell proliferation and circulating thyroid hormones concentrations) was investigated.38 to induce hypothyroidism, bull calves received methimazole (2 clinical theriogenology • volume 12 number 3 • september 2020 249 mg/kg bw, bid) from 4 6 months. age of puberty was delayed with increased sperm production. bulls subjected to transient hypothyroidism had 30 180% more sperm per ejaculate than control bulls, with no effect on sperm motility, morphology, post-thaw survivability or rates of ivf cleavage and blastocyst development. treated bulls had a 22% higher mean paired testes weight and 50% higher epididymal weight compared to untreated bulls. finally, treated bulls had a 2.3-fold increase in number of sertoli cells per cross section of seminiferous tubule than control bulls. these promising results are impetus for further studies. conclusion in the current era of genomic testing, sire selection can be performed very early in life and bull development and sperm production are increasingly important for efficient use of elite sires. in this context, studies are needed to dissect sertoli cell-specific role in sperm production and methods to enhance it. 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between early and late maturing bulls, and changes in semen characteristics at puberty. theriogenology 1995;43:569-578. 24. corbet n, burns b, johnston d, et al: male traits and herd reproductive capability in tropical beef cattle. 2. genetic parameters of bull traits. anim prod sci 2013;53:101-111. 25. chase c, chenoweth p, larsen r, et al: growth, puberty and carcass characteristics of brahman-, senepol-, and tulisired f1 angus bulls. j anim sci 2001;79:2006-2015. 26. lunstra d, echternkamp s: puberty in beef bulls: acrosome morphology and semen quality in bulls of different b reeds. j anim sci 1982;52:638-648. 27. sullivan t, micke g, greer r, et al: dietary manipulation of bos indicus x heifers during gestation affects the prepubertal reproductive development of their bull calves. anim reprod sci 2010;118:163-170. 28. copping kj, ruiz-diaz md, rutland cs, et al: peri-conception and first trimester diet modifies reproductive development in bulls. reprod fert develop 2018;30:703-720. 29. brito l, barth a, rawlings n, et al: effect of nutrition during calfhood and peripubertal period on serum metabolic hormones, gonadotropins and testosterone concentrations, and on sexual development in bulls. domest anim endocrinol 2007;33:1-18. 30. dance a, thundathil j, blondin p, et al: enhanced early-life nutrition of holstein bulls increases sperm production potential without decreasing postpubertal semen quality. theriogenology 2016;86:687-694. 31. barth ad, brito lfc, kastelic jp: the effect of nutrition on sexual development of bulls. theriogenology 2008;70:485-494. 32. harstine b, maquivar m, helser l, et al: effects of dietary energy on sexual maturation and sperm production in holstein bulls. j anim sci 2015;93:2759-2766. 33. bollwein h, janett f, kaske m: effects of nutrition on sexual development of bulls. anim reprod (belo horizonte) 2017;14:607-613. 34. bourgon sl, amorim mdd, chenier t, et al: relationships of nutritional plane and feed efficiency with sexual development and fertility related measures in young beef bulls. anim reprod sci 2018;198:99-111. 35. berger t: testicular estradiol and the pattern of sertoli cell proliferation in prepuberal bulls. theriogenology 2019;136:60-65. 36. oatley mj, racicot ke, oatley jm: sertoli cells dictate spermatogonial stem cell niches in the mouse testis. biol reprod 2011;84:639-645. 37. oatley mj, kaucher av, yang q, et al: conditions for long-term culture of cattle undifferentiated s permatogonia. biol reprod 2016;95:1-10. 38. waqas ms, ciccarelli m, oatley mj, et al: enhanced sperm production in bulls following transient induction of hypothyroidism during prepubertal development. j anim sci 2019;97:1468-1477. 39. sun y, yang w, luo h, et al: thyroid hormone inhibits the proliferation of piglet sertoli cell via pi3k signaling pathway. theriogenology 2015;83:86-94. 40. oluwole oa, bartlewski pm, hahnel a: relationships of serum thyroid hormones and follicle-stimulating hormone concentrations to sertoli cell differentiation during the first wave of spermatogenesis in euthyroid ram lambs. reprod biol 2013;13:150-160. 41. cestnik v: higher thyroid hormone levels in neonatal life result in reduced testis volume in postpubertal bulls. int j androl 1998; 21:352-357. 42. bame jh, dalton jc, degelos sd, et al: effect of long-term immunization against inhibin on sperm output in bulls. biol of reprod 1999;60:1360-1366. 43. madgwick s, bagu et, duggavathi r, et al: effects of treatment with gnrh from 4 to 8 weeks of age on the attainment of sexual maturity in bull calves. anim reprod sci 2008;104:177-188. 44. bagu et, madgwick s, duggavathi r, et al: effects of treatments with lh or fsh from 4 to 8 weeks of age on the attainment of puberty in bull calves. theriogenology 2004;62:861-873. 45. harstine br, cruppe lh, abreu fm, et al: impact of a timed-release fsh treatment from 2 to 6 months of age in bulls i: endocrine and testicular development of beef bulls. theriogenology 2018;105:142-149. 46. harstine br, cruppe lh, abreu fm, et al: impact of a timed-release follicle-stimulating hormone treatment from one to three months of age on endocrine and testicular development of prepubertal bulls. j anim sci 2017;95:16691679. clinical theriogenology • volume 12 number 3 • september 2020 251 016_ms-016 tibary -1 color all figures ultrasonography of the bull genital system harry momont school of veterinary medicine, university of wisconsin-madison, madison, wi abstract ultrasonography has become a mainstay of reproductive medicine. it is used less frequently for examination of male cattle than females. this paper describes the technical aspects of diagnostic ultrasonography of the bull genital system, provides examples of normal findings, and discusses commonly encountered abnormalities. keywords: cattle, bull, ultrasonography, reproductive organs, testis introduction ultrasound refers to any sound waves with frequencies above the threshold of human hearing (> 20 khz). medical applications of ultrasound can be either diagnostic or therapeutic. the author is not aware of any reports on the use of therapeutic ultrasonography for the bull reproductive system, so the focus will be on diagnostic applications. diagnostic ultrasonography produces images of internal tissues and relies on ultrasound frequencies, typically, in 1 15 mhz range. higher frequencies are correlated with shorter wavelengths that allows for finer image detail. lower frequency ultrasound has longer wavelengths that suffer less from attenuation as they pass through tissue, allowing for imaging of deeper structures. best image is generally obtained by using the highest frequency transducer that will penetrate to the depth of the structure that one wishes to examine. the viewed image is the result of coherent echoes returned to the transducer by reflection that occurs when the transmitted ultrasound beam encounters tissues with differing acoustic impedance properties. major advantages of diagnostic ultrasonography are, it is noninvasive, produces images in real time, and can be packaged in a portable system that allows for point-of-care use in large animal facilities. additionally, unlike radiography, it does not use ionizing radiation and is generally regarded as safe. the production of image artifacts is a significant issue when interpreting ultrasonographic studies and they are a part of almost every imaging session. common artifacts (e.g., reverberation, distal enhancement, attenuation, speed propagation error, reflection, refraction, and beam width artifact) can be attributed to equipment or operator limitations (e.g., selection of an inappropriate transducer frequency, incorrect instrument settings, patient movement, and poor patient preparation) or tissue interactions with the sound beam that cannot be anticipated or properly compensated for by the imaging software. a basic understanding of ultrasonographic principles and common artifacts is crucial to interpreting images. there are texts1-3 available that cover these basics and it is highly recommended to have 1 of them as a reference material. a detailed knowledge of normal anatomy is crucial for the correct interpretation of ultrasound studies. an excellent bovine reproductive anatomy guide can be obtained from the national association of animal breeders.4 recommended reading materials include general reviews of male genital pathology5,6 and bull genital ultrasonography.7,8 a mental and digital library of normal sonograms can be obtained by repeatedly examining the reproductive tracts of normal bulls. b-mode ultrasonography brightness or b-mode is the system that produces the classic gray-scale images in real time that most of us associate with ultrasonography. first report on b-mode ultrasonography for examination of the bull scrotum was published in 1987.9 a number of subsequent studies failed to confirm additional substantial benefit when ultrasonography was used as an adjunct to the standard bull breeding soundness examination (bse).10-12 consequently, ultrasonography is not a basic component of a standard bull bse as recommended by the society for theriogenology.13,14 the major advantage of ultrasonography is its ability to confirm, localize, and monitor the progression of gross morphological lesions of the reproductive tract. it is recommended for prepurchase examinations of older bulls, for all cases of infertility or subfertility in valuable animals, and for bulls with palpable or visible scrotal lesions. additionally, abnormal seminal parameters (e.g., low semen volume, pyospermia, teratospermia, hemospermia, azoospermia, and oligospermia) call for an ultrasound examination. safety of everyone involved in the examination should be a priority when working with bulls. mature bulls can be unpredictably aggressive, and their large size increases the danger. whereas younger bulls are generally less aggressive, in author’s experience they are more prone to kicking than mature bulls. a good stock or chute with a squeeze and well-trained handlers make the job much easier. sedation is generally not required; however, it can be used for fractious animals. acquiring an image requires that the transducer be coupled to smooth scrotal skin. this will require stretching the scrotal skin by pulling down on the testis, along with the application of a coupling agent. author prefers to cover the transducer for all examinations by inserting a small amount of coupling gel in a disposable sleeve or glove. scrotal contents (spermatic cord, vascular cone, epididymis, and testis) are examined in sagittal and transverse planes. representative images should be labeled, measured, and preserved. the probe may be applied to the caudal, lateral, or cranial surface of the scrotum to obtain the best image of the target tissues. author prefers a linear transducer that will cover most of the testis of a mature bull in the sagittal view. the linear transrectal probe used for routine examination of female cattle is nearly ideal for this purpose. a transducer frequency of 5 8 mhz provides a good image with adequate depth. a higher-frequency transducer may be required for detailed imaging of epididymis; however, the cost of an additional ultrasound probe can be substantial. one should purchase a system that gives the best image after testing image quality and durability under field conditions. normal testis (figure 1) has a relatively homogeneous parenchyma with a stippled, medium echogenicity. the parenchyma surrounds a centrally located and more hyperechoic (whiter) mediastinum testis. the width of the mediastinum testis is about 5 10 mm and increases with age. a second age-related ultrasonographic correlation is the gradual increase in echogenicity that accompanies the onset of puberty.11,15 when examining peripubertal bulls the author has commonly observed an aberrant feature (figure 2). small, scattered hyperechoic foci may be randomly distributed in the parenchyma or concentrated in 1 area of the testis and can vary widely in number. less commonly they encircle the mediastinum testis, suggesting involvement of the straight tubules. similar lesions appeared in human males16 and are classified as microlithiasis, small foci of mineralization of unknown etiology and significance. histological basis for this lesion in the bull has not been established and its impact on fertility is likewise unknown; however, it does not appear to be a substantial factor in bull infertility. figure 1. ultrasonogram of a normal testis of a yearling holstein. note mediastinum testis (arrows) appearing as a linear structure in sagittal view on the left and appearing in cross section in transverse view on the right. mediastinum testis is more hyperechoic than surrounding parenchyma. images were obtained with a 5 mhz linear transducer placed on the caudal aspect of scrotum using alcohol as a coupling agent. scale for the left image is cm. figure 2. sagittal view of a testis of a mature bull with scattered hyperechoic foci distributed throughout the parenchyma. note normal mediastinum testis (arrow). there is another common abnormality (figure 3) encountered when examining bovine testis. these larger hyperechoic foci have been identified as fibrotic in nature17 and may progress in time to mineralization. unlike smaller lesions noted above, they are often accompanied by acoustic shadowing. lesions may be more focal in nature or radiate outward from the mediastinum testis in a pattern, suggesting involvement of individual seminiferous tubules.18 these lesions may represent figure 3. sagittal (left) and transverse (right) views of a yearling dairy bull testis with mild testicular fibrosis. normal mediastinum (short white arrows) is in longitudinal section (left) and in cross-section in transverse view (right). fibrosis (longer arrows on right) appears to radiate from the mediastinum suggesting involvement of discrete seminiferous tubules. note acoustic shadowing is below the fibrotic tissue on the extreme right of the image. figure 4. sagittal view of a yearling bull testis with a history of oligospermia and low semen volume. note the dilated, hypoechoic rete testis in longitudinal view between 2 arrows. a discrete local degeneration of the germinal tubules; however, care should be exercised in attaching significance to them as they are often noticed in bulls with normal spermiograms.17 a reduction in sperm production capacity is expected in bulls with large amounts of fibrotic tissue in the parenchyma. whereas the precise pathogenesis of these fibrotic lesions is not clearly defined, some suggested etiologies include infectious or inflammatory conditions, developmental defects of the seminiferous tubules or their connecting ducts, autoimmunity, obesity, and aging.18 when trueperella pyogenes was injected into the testis of rams, the initial hypoechoic response attributable to inflammation and edema was followed by the development of hyperechoic lesions that were confirmed to be fibrotic tissue.19 furthermore, an outbreak of bovine respiratory syncytial virus in a group of bulls was associated with an increased prevalence of fibrotic lesions; however, a cause-andeffect relationship could not be confirmed.17 in theory, the pathological mechanism of obesity is a disruption of scrotal thermoregulation. however, scrotal insulation that resulted in a dramatic decrease in semen quality had no fibrotic lesions of the testis in a follow up (4 6 months) examination.20,21 trauma and age are also considered as factors for progressive fibrosis of the ventral testicular parenchyma; however, it has also been described as a normal feature of aging.22 hypoechoic lesions are less commonly encountered. they may represent delayed sexual development, degeneration, or tumors. lesions with mixed echogenicity are also less common and the author has observed them with tumors, inflammation, or abscesses. a specific hypoechoic lesion of the bovine testis is seen in figure 4. first reported in 1991,23 fluid distention of the rete testis secondary to outflow obstruction results in the hypoechoic, dilated rete testis. younger bulls with a history of persistent low semen volume and oligospermia should be monitored for development of this ultrasound finding. a congenital aplasia somewhere along the sperm outflow tract is usually observed in postmortem or after gonadectomy. the lesion may be acquired in older bulls. a complete scrotal examination should include an assessment of the spermatic cord, vascular cone, scrotal wall, and epididymis. the vascular elements can be assessed by both b-mode and doppler ultrasonography. doppler ultrasonography will be discussed later. lesions associated with the epididymis include cysts, sperm granulomas, and congenital aplasia. cystic structures in the region of the head of the epididymis may represent proximal mesonephric duct remnants and are generally benign.5 other scrotal pathologies encountered in this area include inguinal and scrotal hernias, scrotal hydrocoele (figure 5), and vascular abnormalities. other genital tissues ultrasonography of the pelvic accessory sex organs, prepuce, and penis may be accomplished using a the transrectal or transdermal approach. a recent review is available.8 alcohol alone may provide adequate coupling to produce a transdermal image; however, dense, long, or matted hair will likely require shaving in addition to using a coupling agent. special care should be taken when examining the distal penis or the prepuce. bulls are more likely to object to this manipulation and sedation may be required. figure 5. sagittal view of a mature beef bull testis diagnosed with scrotal hydrocoele. note the accumulation of hypoechoic fluid in the vaginal process (arrow) contrasts with the more echoic normal testis parenchyma below the fluid and the caudal scrotal wall above it. small dark areas in the testis periphery are branches of intratesticular artery. doppler ultrasonography a thorough coverage of doppler ultrasonography will not be attempted here. more detailed information is readily available in recent reviews on the subject.3,24 basic function of doppler ultrasonography is to detect movement within the image. in veterinary medicine, this usually means detecting red blood cells (rbcs) movement through a vessel. doppler ultrasonography can be used to detect and evaluate blood flow in the scrotum and testis. color doppler is readily available on most mid-range portable systems and not typically in basic machines used for on-farm purposes. in color doppler mode, the transducer functions in both b-mode and doppler mode to detect blood flow in tissue and depicts movement as color on the gray scale b-mode image in real time. the color (usually blue or red) indicates movement away from or toward the transducer. pulsed wave is another doppler imaging option routinely available with more expensive portable systems. in this mode, the velocity of the moving tissue (rbcs) is displayed as a spectral graph from which peak systolic and end diastolic velocity can be determined and used to calculate various indices of arterial blood flow (e.g., mean velocity, resistive index and pulsatility index). doppler ultrasonography has proven useful in human medicine for evaluating circulatory disruption associated with testis torsion, varicosities, and tumors.25 the resistive index has some correlation with disrupted testes function in bulls.26 presently, however, the greatest utility of doppler ultrasound appears to be the ability to confirm blood flow in tissue using the color doppler function. its use as a screening tool for bull infertility requires more detailed study. future directions b-mode ultrasonography will remain the standard for examination of the bull, enhanced by continued improvements in image quality and affordability. doppler studies will gain acceptance as veterinarians in large animal practice acquire systems with these capabilities. doppler, contrast-enhanced ultrasonography, and tissue elastography are examples of imaging modalities that are now being used in human medicine.25 conclusion ultrasonography is a safe, noninvasive method for obtaining diagnostic and prognostic information about infertile bulls. competency with both b-mode and doppler techniques can be readily achieved by repeated imaging the genital tract of normal, fertile bulls. the most frequently encountered lesions are easily detected by instruments with average or better image quality. author performs ultrasound studies for all cases of infertility involving bulls at collection centers and for valuable natural service sires. author does not use it for routine screening of young sires in the absence of grossly detectable lesions. it is important to consider that the most frequently detected ultrasonic changes in young bulls are of questionable value to predict future fertility. conflict of interest none to declare. references 1. ginther oj: ultrasonic imaging and animal reproduction. fundamentals. cross plains, wi; equiservices publishing: 1995. 2. blond l buczinski s: basis of ultrasound imaging and the main artifacts in bovine medicine. vet clin north am food anim pract 2009; 25:553-565. 3. mattoon j, nyland t: fundamentals of diagnostic ultrasound. in: mattoon j, nyland t: editors. small animal diagnostic ultrasound. 3rd edition, st. louis; elsevier saunders: 2015. p. 1-49. 4. mullins k, saacke r: illustrated anatomy of the bovine male and female reproductive tracts. from gross to microscopic. blacksburg, va: germinal dimensions (available from naab-css.org), 2003. 5. mcentee k: reproductive pathology of domestic animals. san diego; academic press: 1990. p. 224-383. 6. foster r: male genital system. in: maxie g: editor. jubb, kennedy, and palmer’s pathology of domestic animals. volume 3. 6th edition, st. louis: elsevier: 2016. p. 465-510. 7. gnemmi g, lefebvre rc: ultrasound imaging of the bull reproductive tract: an important field of expertise for veterinarians. vet clin north am food anim pract 2009; 25:767-779. 8. momont h, keeler m, nicholson m: ultrasound examination of the reproductive tract. in: hopper, r: editor. bovine reproduction. 2nd edition, hoboken; wiley-blackwell: 2021. p. 120-129. 9. pechman r, eilts b: b-mode ultrasonography of the bull testicle. theriogenology 1987;27:431-441. 10. eilts be, pechman rd: b-mode ultrasound observations of bull testes during breeding soundness examinations. theriogenology 1988;30:1169-1175. 11. brito l, barth a, wilde r, et al: testicular ultrasonogram pixel intensity during sexual development and its relationship with semen quality, sperm production, and quantitative testicular histology in beef bulls. theriogenology 2012;78:69-76. 12. tomlinson m, jennings a, macrae a, et al: the value of transscrotal ultrasonography at bull breeding soundness evaluation (bse): the relationship between testicular parenchymal pixel intensity and semen quality. theriogenology 2017;89:169-177. 13. chenowith p, hopkins f, spitzer j, et al: guidelines for using the bull breeding soundness examination form. clinical theriogenology 2010;2:43-50. 14. koziol jh, armstrong cl: society for theriogenology manual for breeding soundness examination of bulls. 2nd edition, society for theriogenology (therio.org): 2018. p. 123-126. 15. brito l, silva a, unanian m, et al: sexual development in early and late maturing bos indicus a bos indicus × bos taurus crossbred bulls in brazil. theriogenology 2004;16:1198-1217. 16. balawender k, orkisz s, wisz p: testicular microlithiasis: what urologists should know. a review of the current literature. cent european j urol 2018;71:310-314. 17. barth a, alisio l, aviles a, et al: fibrotic lesions in the testis of bulls and relationship to semen quality. anim reprod sci 2008;106:274288. 18. barth a, kastelic j: testicular degeneration. in: hopper, r: editor. bovine reproduction. 2nd edition, hoboken; wiley-blackwell: 2021. p. 144-150. 19. gouletsou p, fthenakis g, cripps p, et al: experimentally induced orchitis associated with arcanobacter pyogenes: clinical, ultrasonographic, seminological and pathological features. theriogenology 2004;62:1307-1328. 20. sidibe m, franco l, fredriksson g, et al: effects on testosterone, lh and cortisol concentrations, and on ultrasonographic appearance of induced testicular degeneration in bulls. acta vet scand 1992;33:191196. 21. arteaga a, barth a, brito l: relationship between semen quality and pixel-intensity of testicular ultrasonograms after scrotal insulation in beef bulls. theriogenology 2005;64:408-415. 22. humphrey j, ladds p: a quantitative histological study of changes in the bovine testis and epididymis associated with age. res vet sci 1975;19:135-141. 23. andersson m, alanko m: ultrasonography revealing the accumulation of rete testis fluid in bull testicles. andrologia 1991;23:75-78. 24. ginther oj: ultrasonic imaging and animal reproduction. colordoppler ultrasonography. cross plains, wi; equiservices publishing: 2007. 25. huang d, sidhu p: focal testicular lesions: colour doppler ultrasound, contrast-enhanced ultrasound and tissue elastography as adjuvants to diagnosis. br j radiol 2012;85(spec iss 1):s41-53. 26. gloria a, carluccio a, wegher l, et al: pulse wave doppler ultrasound of testicular arteries and their relationship with semen characteristics in healthy bulls. j anim sci biotechnol 2018;9:14-20. ltrasonog 2018: accuracy of whelping date calculator based on fetalultrasonographic measurements accuracy of whelping date calculator based on fetal ultrasonographic measurements gilad fefer, assaf lerer, erika first, gilad fefer, amy yeager, mariana diel de amorim, soon hon cheong department of clinical sciences, college of veterinary medicine, cornell university, ithaca, ny gestation length in the bitch is 65 (+/1) days from luteinizing hormone (lh) peak. prediction of whelping date can be fairly accurate within a 3-day window when proper progesterone monitoring is performed. an accurate due date is desirable as some breeds are known to have higher rate of dystocia and elective cesarean section needs to be planned. in cases where progesterone monitoring has not been performed and an lh peak date could not be estimated, ultrasonographic measurements of the fetus and gestational sac have demonstrated high accuracy to predict the gestational age.1 these formulas require the sonographer to manually calculate and average the results in order to provide useful information. it is therefore desirable to have a computerized online whelping calculator application that a practitioner can input fetal measurements and immediately obtain a predicted whelping date. our objective was to develop and validate a whelping calculator application that will generate a predicted whelping date based on fetal ultrasound measurements. the purpose of our study was to test our hypothesis that the current formula used in the website application will be able to predict the whelping date to within a 3-day window based on ultrasound measurements. methods a webpage (https://vetapp-sb.vet.cornell.edu/whelping_calc) was created by the cornell veterinary medicine information technology based on the mathematical formulas previously published by yeager et al.1 to test the performance of the website, a retrospective study design was used. data of fetal ultrasonographic measurements were obtained from medical records of patients (n=14) with known whelping date; or known date of emergency or elective cesarean section (progesterone <2ng/ml) to test the calculator. fetal measurements used for the calculators were: crown-rump length (n=11); biparietal diameter (n=3); fetal trunk/abdomen diameter (n=11), inner-chorionic cavity (n=7) and outer-chorionic cavity (n=10). results the average gestation from the progesterone based presumptive lh peak was 65.7 ± 2.0 days, and 65.1 ± 1.5 days based on ultrasonographic predicted lh peak. there was a significant linear relationship between ultrasonocraphic predicted gestational age and progesterone based lh peak gestational age (0.557±0.224, p=0.037 jmp pro 13). in this preliminary study population, the accuracy to predict whelping date within the 3-day due date window was 40% for progesterone based due date and 64.3% based on ultrasonographic based due date. if the window of accuracy were extended to predict whelping within +/2 days from the predicted due date, the accuracy was 70% for progesterone based due date and 85.7% for ultrasonographic based due date. all bitches whelped within +/3 days of either predicted due dates. discussion/conclusions ultrasonographic measurement is significantly correlated with progesterone based due dates and can be a very useful tool to predict whelping dates especially in cases where progesterone monitoring was not performed. further development and validation of this free web application is underway with more retrospective cases being collected and additional prospective studies are being planned. keywords: canine, ultrasound, fetus, lh whelping calculator reference 1. yeager ae, mohammed ho, meyers-wallen v, et al: ultrasonographic appearance of the uterus, placenta, fetus, and fetal membranes throughout accurately timed pregnancy in beagles. am j vet res 1992;53:342-351. clinical theriogenology • volume 10, number 3 • september 2018 312 lactational anestrus in caribbean donkeys   lactational anestrus in caribbean donkeys erik w. peterson, brandi n. roberts, ninian j. cameron-blake, robert o. gilbert, hilari m. french ross university school of veterinary medicine, st kitts and nevis, west indies summary lactational anestrus is common in many species, but not in equids. this case report documents an unusual finding of eight donkeys (from a group of 25) on the caribbean island of st. kitts that remained anestrus after foaling during the physiological breeding season. twenty-five feral female donkeys in advanced pregnancy were acquired. no breeding history was known and all pregnancies were monitored via weekly transabdominal ultrasonography. all jennies foaled without complications and weekly transrectal ultrasonographic examinations were conducted to monitor uterine involution and cyclicity. four foals died from septicemia within the first week and all of their dams soon re-established ovarian cyclicity. thirteen jennies had a foal heat with overt signs of estrus within 2 weeks after foaling, then returned to normal cyclicity. however, the remaining eight jennies did not ovulate or display signs of estrus until their foals were weaned, ~4 months later. following weaning, all eight jennies promptly returned to normal cyclicity. that these eight jennies returned to normal cyclicity following weaning supported our diagnosis of lactational anestrus during the physiological breeding season, a phenomenon apparently never reported in donkeys. keywords: donkey, jenny, lactation, anestrus, caribbean case presentation history twenty-five feral female donkeys were obtained from the island of nevis and transported to the sister island of st. kitts for a research study, approved by the ross university school of veterinary medicine animal care and use committee. jennies were estimated to be 3 to 13 years of age, with body condition scores of 4 to 6.1 all jennies were confirmed to be in late gestation via transabdominal ultrasonography,2 and all subsequently foaled without complications between march and july (spring/summer) 2016. jennies and foals were monitored daily after foaling for nursing and general health. four foals died from septicemia within their first week, although their dams had no apparent health issues. beginning 1 week after foaling, transrectal ultrasonography was done once weekly. uterine horn diameter and endometrial edema, scored on a scale of 0 to 3, were evaluated. ovaries were monitored for ovarian follicular and corpora lutea development. jennies were teased twice weekly using jacks to detect estrus behavior. estrus behavior was graded as follows: 0 rejects the presence of the jack; 1 indifferent to the jack’s presence; 2 performs mouthing behavior only; 3 performs mouthing behavior, backs up to the jack, squatting, urination and clitoral winking. once weekly for 20 weeks, starting 5 weeks before weaning, blood samples were collected from each jenny by jugular venipuncture. serum was separated within 3 hours after collection and stored at -80oc. within 1 month after foaling, 17 jennies had displayed behavioral signs of estrus and had ultrasonographic evidence of ovarian follicular development, uterine horn and endometrial edema, ovulation and cl formation. however, the remaining eight jennies (32%), all with foals, had no behavioral signs of estrus, and based on transrectal ultrasonographic examinations, smaller uterine horns, no evidence of ovulation and no cl. serum samples were thawed and analyzed with a commercial enzyme immunoassay for quantitative determination of progesterone (arbor assay, k025-h5; ann arbor, mi, usa). validation was done to assess possible matrix effects when this assay was used for donkey serum. pools of serum were prepared from blood samples collected from jennies during estrus, diestrus and early pregnancy. each pool was subsequently diluted in a serial manner (1:1, 1:2, 1:4, 1:8, 1:16, and 1:32) to determine parallelism with the reference standard curve. four of six dilutions were within the linear range of the 453 clinical theriogenology • volume 10, number 4 • december 2018   standard curve, with a cv of 26%, indicative of parallelism.11 thereafter, the assay was conducted in accordance with the manufacture’s protocol, using 50 µl of sample diluted 1:16 (estrous cycle samples) or 1:32 (pregnancy samples) with assay buffer. samples with progesterone concentrations outside the range of the reference standard were further diluted and re-analyzed as necessary. within-assay cv and sensitivity were 17% and 0.05 ng/ml, respectively. in all eight jennies with lactational anestrus, serum progesterone concentrations were < 4.0 ng/ml from 5 weeks before weaning to 1 week after weaning (figure 1), in stark contrast to the remaining jennies with physiologic postpartum estrous cycles (figure 2). however, beginning 1 week after weaning, progesterone concentrations in subsequent estrous cycles began to fluctuate, similar to jennies with regular postpartum estrous cycles (figure 3). serum progesterone concentrations were consistent with behaviors in response to teasing and ovarian follicular development, as monitored ultrasonographically. outcome all eight jennies (100%) affected by lactational anestrus ovulated 1 to 2 weeks after their foals were weaned and thereafter continued to cycle similar to their herd mates. discussion the influence of housing and nutrition on this condition was questionable. all jennies were housed together in an outdoor pasture, received the same diet of pasture supplemented with sugar cane grass twice daily and ad libitum access to a fresh clean water source. all were in similar body condition (body condition scores of 4 to 6), with no apparent loss of body condition after foaling. in contrast, three jennies increased at least by one body condition score grade during the time of observation. therefore, it is unlikely that the lack of estrus in these eight jennies was related to nutrition, as widely documented in many species.3-6 st. kitts is on the 17th parallel north, where daylight only varies by approximately 2 hours throughout the year. daylight affects the reproductive cycle in mares, although donkeys cycle throughout the year, even in more northern latitudes.7-10 weekly monitoring of estrus behavior as well as uterine and ovarian structures throughout the winter demonstrated no change in average interovulatory interval in herd mates. therefore, shortened exposure to daylight was an unlikely cause of anestrus. in contrast to the mare, relatively higher serum progesterone concentrations observed at all stages of the cycle agreed with earlier reports.11,12 as noticed, progesterone concentrations can be suprabasal at ovulation.11 although postpartum mares can have ovarian inactivity for known and unknown reasons,13 in the current study, the most likely cause was lactation, as suggested for mares.11 although horses are seasonally polyestrous, the majority of donkeys ovulate year-round, even at temperate latitudes (43°n), although the duration of estrus and interovulatory interval fluctuate due to seasonality.13 conversely, horse mares are sensitive to seasonal changes in daylength, even in temperate climates such as southern mexico (latitude 15-22°n).14 almost all horse mares ovulate soon after foaling, with a very small minority entering an anovulatory period, most often associated with foaling in the nonovulatory season.15 although the failure to return to normal estrus following parturition has been attributed to factors such as shortened daylight hours, disease or nutritional deficits in equids, all seem unlikely in our population. based on daily monitoring plus weekly transrectal ultrasound examinations, there were no indications that ovarian abnormalities or disease caused anestrus. additionally, all jennies received the same nutrition and were in a similar body condition, making a nutritional disorder doubtful. lactating mares with a foal at foot commonly do not demonstrate estrus behaviors due to the presence of the foal.15 however, in this case, weekly transrectal ultrasonography detected no ovarian activity, making it unlikely that these jennies experienced silent estrus. that each jenny began cycling normally 1 to 2 weeks after weaning supported our diagnosis that eight of the 25 donkeys entered a true lactational anestrus during the physiological breeding season. 454clinical theriogenology • volume 10, number 4 • december 2018 learning points  eight of 25 donkeys in the caribbean experienced lactational anestrus  cyclicity did not return until foals were weaned  lactational anestrus is not common in equids references 1. pearson ra, ouassat m: a guide to live weight estimation and body condition scoring of donkeys. centre for tropical veterinary medicine, university of edinburgh 2000;1-21. 2. crisci a, rota a, panzani d, et al: clinical, ultrasonographic, and endocrinological studies on donkey pregnancy. theriogenology 2014;81:275-283. 3. loudon as, mcneilly as, milne ja: nutrition and lactational control of fertility in red deer. nature 1983;302:145147. 4. peters ar, lamming ge: lactational anoestrus in farm animals. oxf rev reprod biol 1990;12:245-288. 5. dunn tg, kaltenbach cc: nutrition and the postpartum interval of the ewe, sow and cow. j anim sci 1980;51:29-39. 6. richards mw, wettemann rp, schoenemann hm: nutritional anestrus in beef cows: body weight change, body condition, luteinizing hormone in serum and ovarian activity. j anim sci 1989;67:1520-1526. 7. palmer e, driancourt ma: some interactions of season of foaling, photoperiod and ovarian activity in the equine. livest sci 1983;10:197-210. 8. ginther oj: reproductive biology of the mare: basic and applied aspects. 2nd edition, cross plains: equiservices; 1992. p. 124 9. ginther oj, scraba st, bergfelt dr: reproductive seasonality of the jenney. theriogenology 1987;27:587-592. 10. saltiel a, calderon a, garcia n, et al: ovarian activity in the mare between latitude 15 degrees and 22 degrees n. j reprod fert supplement 1982;32:261-267. 11. díaz-duran m, zarco l, boeta am: ovarian dynamics and estrous cycle length in the donkey (equus asinus). theriogenology 2017;103:1-8. 12. hoffmann b, bernhardt aw, failing k, et al: profiles of estrone, estrone sulfate and progesterone in donkey (equus asinus) mares during pregnancy. tierärztliche praxis großtiere 2014;42:32-39. 13. nagy p, guillaume d, daels p: seasonality in mares. anim reprod sci 2000;60:245-262. 14. boeta m, porras a, zarco la, et al: ovarian activity of the mare during winter and spring at a latitude of 19° 21′ north. j equine vet science 2006;26:55-58. 15. ginther oj, whitmore hl, squires el: characteristics of estrus, diestrus, and ovulation in mares and effects of season and nursing. am j vet res 1972;33:1935-1939. 455 clinical theriogenology • volume 10, number 4 • december 2018   figure 1. weekly serum progesterone concentrations in eight jennies with lactational anestrus. figure 2. weekly serum progesterone concentrations in 13 normally cycling jennies. figure 3. average serum progesterone concentrations of lactational anestrus and normally cycling jennies. 0 10 20 30 40 50 60 ‐5 ‐4 ‐3 ‐2 ‐1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 p ro ge st er o n e  (n g/ m l) weeks from weaning  0 5 10 15 20 25 30 35 40 45 ‐5 ‐4 ‐3 ‐2 ‐1 0 1 2 3 4 5 6 7 8 9 10 p ro ge st er o n e  (n g/ m l) weeks from weaning lactational anestrus normally cycling 456clinical theriogenology • volume 10, number 4 • december 2018 000_frontal ms_1 forematter for december 2018 ms_2 letter1 for december 2018 ms_2 letter2 for december 2018 ms_2 letter3 for december 2018 ms_3 table of contents ms_4 therio news for december 2018_bw 2019 therio preview therio recap with pics steiner-new officers 2018 therio sponsors 2018 exhibitors-sft board noms 2018 auction donations 2019 bartlett nomination act call for noms call for abstracts 2019 for ctj the society for theriogenology and american college of theriogenologists issue a call for research and case abstracts to be presented at the annual therio conference july 24-27, 2019 in savannah, georgia. 1. competitive category: 2. non‐competitive category: 3. case abstract category: 4. poster category: general: guidelines: what to send and how to send it abstracts not adhering to these guidelines will not be considered for presentation. deadline: abstracts must be received no later than february 15, 2019 for consideration. call for 2019 student case presentations.pdf call for student case presentations-2019 annual therio conference student case presentations call for student case presentations also available at www.therio.org guidelines for application submission society for theriogenology student case presentation competition annual therio conference, savannah, georgia, july 24-27, 2019 ms_5 editorial 001a_ms_6 roberts color pictures 002_ms_7 root kustritz 003_ms_8 ferrer 004_ms_9 wiley 005_ms_10 wallace 006_ms_11 peterson 007_ms_12 nobre 008_ms_13 sidelinger 009_ms_14 mirando how to perform neurological examination in a neonatal foal monica aleman department of medicine and epidemiology school of veterinary medicine, university of california, davis, ca abstract neurological examination on a neonatal foal is performed concurrently with the physical examination. first step in examination is observation. neurological examination consists of evaluation of behavior, state of consciousness, cranial nerve function, posture (head, neck, trunk, limbs, and tail) and postural reactions (limbs), segmental (spinal) reflexes, palpation, and gait evaluation. to assess most segmental reflexes, the foal must be in lateral recumbency and relaxed. key points include safety, observation and palpation, knowing normal, including development of important milestones, and (if possible) conducting serial examinations over time. keywords: foal, neonate, neurology, neurodevelopment, reflexes introduction neonatal foals, as prey animals, in order to survive, must develop major milestones relatively quickly after birth.1 this evolutionary adaptation provides specific neurologic development, maturation, and function. for an equine clinician, it is essential to become familiar with normal neurologic status during neonatal period.1 a complete clinical history of mare and foal should be obtained, including pregnancy and periparturient events. a thorough physical examination of the foal is important to determine the overall health status.1 common neonatal disorders manifest with similar clinical signs to foals with neurologic disease and might include weakness, reduced muscle tone, recumbency, reduced or absent suckle reflex, and dysphagia.1 therefore, ruling out common neonatal disorders is paramount to direct proper therapy and supportive care. alertness, response to environment, and movement are different in utero, during birth, and in extrauterine life. in the birth canal, foals appear in a drowsy/sleepy state, become minimally responsive, and movement is depressed. however, as precocious species, neonatal foals must rapidly transition to extrauterine life.1 neonatal foals are able to stand and nurse in a relatively short interval after birth compared to predator species. foals are fully visual and have auditory function comparable to adult horse.1 nervous system continues to mature after birth. for example, although cerebellar layers in neonatal foals are already distinct histologically at birth, cell differentiation and migration into various layers continue after birth.1 cerebellar development and myelination in various parts of nervous system might explain the ‘bouncy’ gait in neonatal foals.1 neurologic examination in a neonatal foal is performed concurrently with general physical examination. neurological examination consists of assessing behavior, state of consciousness, cranial nerves, posture and postural reactions, segmental (spinal) reflexes, palpation, and gait evaluation.1-3 key points include observation and palpation, knowing normal, tailoring the examination to the individual (safety, domestication, and cooperation), and, if possible, the importance of conducting serial examinations over time.1 furthermore, it is critical to recognize autonomic nervous system (parasympathetic, sympathetic, and intrinsic enteric) as integral part of the nervous system.1 neurologic examination in neonatal foals neurologic status of the normal neonatal foal goes through a transition from in utero to extrauterine life. the apgar score was adopted from human medicine to assess neonates in the postfoaling period and consists of evaluating the following variables: 1. heart rate (normal: regular, 60 beats per minute [abnormal if undetectable, irregular or < 60 bpm]) 2. respiratory rate (normal: regular, 60 breaths per minute [abnormal if undetectable, irregular or < 60 bpm]) 3. mucous membranes (normal: pink) 4. muscle tone (normal: strong enough to remain in sternal recumbency) 5. responsiveness a. nasal stimulation (expected response: strong grimace, sneeze) b. ear tickle (expected response: head shake) c. back scratch (expected response: attempts to stand) this evaluation is performed shortly after birth and repeated within 15 minutes postfoaling to determine if veterinary intervention is needed. important postbirth milestones include, time to sternal recumbency 1 2 minutes, alert and responsive to external (tactile, visual, and auditory) stimuli within 5 minutes, suckle reflex within first 20 minutes, vocalizing in response to dam’s nickering within 30 minutes, time to stand within 60 minutes (> 2 hours is considered abnormal), and time to nurse 2 hours (> 3 hours is abnormal). neurologic examination must cover all areas cited for adult horses. determine mentation, observe behavior and posture as history is recorded and foal is examined. states of consciousness include bright alert and responsive, obtunded, stuporous, and comatose (figure 1).1-3 1. obtunded: quiet, a neurologic status and not due to systemic disease (response is lethargy). in this state, the foal remains responsive to stimuli (visual, tactile, and sound) and reacts to environment. there could be degrees of obtundation (e.g., mild, moderate, and severe) on which the stimuli might have to be stronger for a response; however, the key to recognize this state is the ability to always respond to nonpainful stimuli. in this state, foals could be standing or recumbent. foals with moderate and certainly with severe obtundation are recumbent. 2. stuporous: severely altered mental status, unresponsive to minimal to moderate stimuli. profound painful stimulus (pinching skin or foot with hemostats) generates a response from the foal (‘waking up’); however, the response dissipates (goes back to stuporous) as soon as stimulus stops. in this state, foals are always recumbent. 3. comatose: the foal is unresponsive to any kind of stimuli including profound painful stimuli. profound stimuli might result in reflexive movement but not voluntary movement and the foal will be unresponsive. needless to say, foals in this state are always recumbent. figure 1. states of consciousness in neonatal foals, mild (left) and moderate to severe (middle) obtundation; note foal with mild obtundation is standing and appearing ‘sleepy’. foal with a more severe degree of obtundation is recumbent and responsive to nonpainful stimuli. stuporous foal (right) is recumbent and requires painful stimuli to respond transiently. normal neonatal foal is bright alert, curious and responds to environment, bonds with mare, draws closer to mare, seeks udder, nurses, and sleeps. it is important to note that neonatal foals have their entire sleeping in recumbency, different to older foals (> 2 months of age) and certainly adults. sleeping while standing in neonatal foals is abnormal (figure 2). figure 2. sleep in neonatal foals. left: normal sleep, right: abnormal sleep characterized by sleeping while standing. cranial nerve deficits might be apparent during the initial observation before approaching the patient. cranial nerves are evaluated in order (from i to xii) or they can be evaluated by their functional regions.1-3 author prefers functional regions starting with sense of smell (subjective); all eye functions (menace, palpebral fissure size, palpebral reflex, corneal reflex, dazzle reflex, pupillary light reflex, adaptation to light and darkness, eye globe position and retraction, physiologic nystagmus, and tear production), jaw/facial motor, sensation, and symmetry, and nursing (suction, tongue tone and movement, and gag reflex).1-3 do not alter your method of evaluation and always be consistent to avoid overlooking abnormalities. menace response is a learned response (i.e., not a reflex) that develops within 7 10 days in neonatal foals. foals are completely visual and brainstem reflexes are present at birth (e.g., pupillary light reflex, corneal reflex, and palpebral reflex). dazzle reflex is a subcortical reflex and is evaluated using a bright light close to the eye. normal reflex consists of fast sharp blink that is present since birth. also, auditory function is present at birth and comparable to adults based on brainstem auditory evoked response (brainstem auditory evoked responses or hearing test).1 neonatal foals have physiologic nystagmus at birth. this is the normal conjugate eye movement (both eyes moving in the same direction) when the head is moved from side to side. absence of physiological nystagmus indicates bilateral vestibular disease. head posture in neonatal foals has a ‘flexed’ appearance at the atlanto-occipital joint compared to adults and their stance is wide-based that becomes narrower within days. head posture also changes as foal matures. observation of posture during urination and defecation is also important. palpation is essential to detect areas of apparent pain, local temperature, muscle tone and symmetry, joint extension and flexion, and tail tone among others. tactile stimuli result in brisk exaggerated responses and reactions in normal foals compared to older animals. segmental (spinal) reflexes that can be evaluated in foals include cervicofacial, cutaneous trunci, biceps, triceps, patellar, gastrocnemius, flexor (withdrawal), anal, and perianal. the cross-extensor reflex may or may not be present in the neonatal period. if present, it is not considered abnormal and will subside within a few days. extensor thrust reflex can also be observed in normal neonatal foals.1 as a reminder, in order to assess most segmental reflexes (e.g., biceps, triceps patellar, gastrocnemius, and flexor), the foal has to be in lateral recumbency and relaxed. increased muscle tone will result in difficulty assessing and interpreting these segmental reflexes.1 neonatal foals have a hypermetric gait that becomes less ‘bouncy’ within a few days. effects of systemic disease, orthopedic disease, congenital anomalies, motor deficits (from initiation of movement by the forebrain all the way to the nerves, neuromuscular junction, and muscle as the executers), and weakness can result in recumbency.1 cutaneous sensation is evaluated to investigate presence or absence of sensory function. nociception (conscious perception of pain) is only evaluated, if voluntary motor function is absent or difficult to interpret. neuroanatomical localization main divisions of the nervous system are brain, spinal cord, and peripheral.1-3 brain has 3 functional anatomical areas: cerebrothalamus, brainstem, and cerebellum. note that although thalamus is anatomically part of brainstem, functionally it forms part of forebrain (cerebrothalamus). spinal cord consists of spinal cord segments: c1 c5/6, c6 t2, t3 l3, l4 s2, s-caudal.1-3 peripheral system consists of nerve rootlets, roots, ganglia, nerve, and neuromuscular junction. neuromuscular system neuromuscular system has central (lower motor neurons [lmn]) and peripheral (nerve rootlets, roots, ganglia [sensory], nerves, and neuromuscular junction) components.1 neuromuscular disorders can be diffuse or can involve only a single nerve. diffuse neuromuscular disease induces generalized weakness, difficulty supporting weight, base-narrow stance, paresis or paralysis, muscle fasciculations, and tendency to become recumbent. segmental reflexes can be decreased or absent in neuromuscular disease. the most common diffuse neuromuscular disease of foals is botulism. focal lmn disease or neuropathy leads to specific signs pertaining to the region affected (e.g., specific gait deficits and focal muscle atrophy). conflict of interest none to declare. references 1. aleman m: neurological examination of horses. proc am assoc equine pract 2015;61:181-190. 2. adams r, mayhew ig: neurological examination of newborn foals. equine vet j 1984;16:306-312. 3. mackay rj: neurologic disorders in neonatal foals. vet clin north am equine pract 2005;21:387-406. how to perform neurological 2017 squamous cell carcinoma and papilloma virus in the prepuce of a boer buck � � squamous cell carcinoma and papilloma virus in the prepuce of a boer buck e.d. grimmer,a i.f. canisso,a s.h. hsiao,b e.f. garretta adepartment of veterinary clinical medicine, college of veterinary medicine, university of illinois urbana-champaign, urbana il; bveterinary diagnostic laboratory, college of veterinary medicine, university of illinois urbana-champaign, urbana il abstract case description: a 4.5-year-old boer buck was presented for chronic posthitis and reluctance to breed. clinical findings: gross examination of the buck’s prepuce revealed numerous small ulcerated masses. histopathology and immunohistochemistry of the biopsied preputial tissue identified squamous cell carcinoma with cytoplasmic immunoreactivity for papillomavirus antigens present in the neoplastic epithelial cells. clinical relevance: squamous cell carcinoma (scc) occurs sporadically worldwide in a variety of anatomical sites in small ruminants. papillomavirus has been occasionally reported in goats and other wild caprine species as a suggested cause of neoplasia. this report describes a unique case of posthitis associated with scc and papillomavirus in a breeding buck. keywords: genital neoplasia, goat, squamous cell carcinoma, papilloma virus background squamous cell carcinoma is a primary malignant neoplasm of the squamous epithelium that has been reported in a variety of domestic animals. the reported predilection sites of scc in small ruminants are ears, eye and eyelids, base of the horns, on the shoulder, back, and flank regions, lateral to the umbilicus, udder, hind limbs, anus, perineum, vulva, and on the inner aspect of the tail.1 squamous cell carcinoma is not uncommon in small ruminants and occurs sporadically worldwide.2 the suggested predisposing factors for scc include ultraviolet exposure of non-pigmented epithelium, genetic predisposition, and viral involvement. papillomaviruses (pv) are dna viruses that typically cause hyperproliferative lesions of the mucosal and cutaneous epithelia.3 in addition to the typical papilloma lesions, pv infection is also associated with tumor development in several species.2-6 to the best of our knowledge, preputial scc in a goat with strong, diffuse, cytoplasmic immunoreactivity for papillomaviral antigen has not been described. case presentation a boer buck, 4.5-years-old, weighing 86 kg with a body condition score of 3 (range 1 minimal and 5 maximum), was presented to the university of illinois veterinary teaching hospital with a four month history of swelling and purulent discharge from the prepuce and a reluctance or inability to breed. similar signs were first noticed by farm personnel nine months prior to admission but the clinical signs subsided with unknown topical treatments. however, the clinical signs recurred and increased in severity about four months prior to admission. the buck was treated once with ceftiofur crystalline free acid and oxytetracycline (doses unknown) and the prepuce was flushed daily with water during the month prior to presentation. the buck was able to urinate and defecate normally and demonstrated a good appetite. upon physical examination, there was alopecia and crusting of the skin of the flanks, scrotum, dorsal neck, and ventral abdomen, and the patient was pruritic (fig. 1a). based on a skin scraping, an acetate tape test, and a trichogram, the dermatologic condition was diagnosed as chorioptic mange with secondary pyoderma and malassezia spp infection. the internal lamina of the prepuce was partially prolapsed, slightly swollen, ulcerated and partially covered with gray exudate. a corynebacterium renale infection was the primary differential for the ulcerated and inflamed prepuce, as it is a common finding with small ruminant posthitis cases. the right testicle was firmer than the left testicle. the scrotal circumference of 28.5 cm was smaller than the 32-40 cm normal circumference range expected for a prospective breeding buck. ultrasound examination 595 clinical theriogenology • volume 9 number 4 • december 2017 � � of the buck’s testes revealed numerous hyperechoic areas present in the parenchyma of both testes. this finding was suggestive of testicular degeneration and dystrophic testicular mineralization. a complete blood count and biochemistry panel, along with a breeding soundness examination were offered but declined by the owner. the rest of the examination was unremarkable. treatment to treat the chorioptic mange and dermatitis, the buck was treated with ivermectin 1% at 200 mcg/kg subcutaneously once every two weeks for a total of three treatments. the animal was also bathed with gentle shampoo and lime sulfur dip once while the buck was hospitalized. to treat the suspected corynebacterium renale induced posthitis, the buck was initially treated for five days with meloxicam (0.5 mg/kg, po, q 24 hours), florfenicol (20 mg/kg, im, q 48 hours), daily flushing of the prepuce with 500 mls of a 1:40 dilution of chlorohexidine solution every 12 hours, and a topical ointment consisting of lanolin, scarlet oil, and oxytetracycline that was applied to the interior of the preputial cavity. the buck appeared to improve during initial treatments as indicated by decreased preputial swelling and discomfort; however, four days after stopping treatment, the preputial swelling returned. preputial and penile examination since the presumed bacterial posthitis did not resolve with treatment after nine days in the hospital, further investigation was deemed necessary. the buck was sedated with an intravenous combination of ketamine (0.1 mg/kg), butorphanol (0.025 mg/kg) and xylazine (0.05 mg/kg). the buck was positioned in lateral recumbency for examination of the penis and prepuce. gray, inspissated material was present in the preputial cavity. with difficulty, the penis was exteriorized and numerous small, raised, round, coalescing masses, many of which were ulcerated, were observed covering most of the prepuce (fig. 1 b-d). adhesions between the prepuce and the penis were also present. three 4 mm punch biopsies were taken from representative affected areas of the prepuce. a biopsy sample was submitted for histopathology. a sample of the inspissated exudate and two punch biopsy specimens were submitted for bacterial culture and antibiotic sensitivity. the buck was treated with ceftiofur hydrochloride (2 mg/kg intramuscularly) once daily for seven days to help control the secondary bacterial infection of the prepuce. meloxicam (0.5 mg/kg orally) was continued once daily and morphine (0.2 mg/kg intramuscularly), as needed for pain relief. histopathology and microbiology histopathologic evaluation revealed that the specimen comprised polymorphic, infiltrative islands of non-keratinizing, squamous, epithelial cells with dyskeratosis and central cystic degeneration supported by inflamed fibrous stroma. the epithelial cells were polygonal with indistinct cellular borders and a small to moderate amount of amphophilic, slightly vacuolated cytoplasm. nuclei were abnormally large, round to oval, central and hypochromatic with vesicular chromatin and prominent nucleoli. there was marked anisocytosis, marked anisokaryosis, frequent apoptosis, and 30 mitotic figures per ten 400x fields. the overlying epithelium was ulcerated. the microscopic diagnosis was scc. an immunohistochemical stain using a broad spectrum antibody cocktail against bpv-1/1h8 and camvir1 papillomaviral antigens (biocare medical, llc, concord, ca) was applied to the specimen. there was diffuse, cytoplasmic immunoreactivity for papillomaviral antigen in all the neoplastic epithelial cells (fig. 2). aerobic culture of the biopsy specimen resulted in a mix culture of moderate amounts of proteus, alpha streptococcus ovis, staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa. antimicrobial susceptibility results indicated that the staphylococcus aureus and pseudomonas aeruginosa were susceptible to several antibiotics, including ceftiofur, enrofloxacin, gentamicin, and florfenicol. 596clinical theriogenology • volume 9 number 4 • december 2017 � � outcome after the definitive diagnosis of scc of the prepuce was made, euthanasia was recommended because the buck could not function as a breeding animal, progression of local disease with possible metastasis would occur, and treatment options were limited. the owners elected to take the buck back to the farm and the buck was discharged from the hospital with meloxicam (0.5 mg/kg po every 24 hours). it was recommended to keep the buck isolated from the other goats and not use him for live-cover breeding. according to the owners, the buck appeared to do well after returning to the farm. he had a good appetite and gained weight. however, because he was unable to perform as a breeding buck, the owners elected to euthanize him five months after being discharged from the hospital. a necropsy was not performed. discussion the prepuce is not a predilection site for scc in goats and this is the first report of this type of tumor involving the prepuce in goats. in addition, this is the first description of papilloma virus infection being associated with scc in goats. infection of humans with certain types of pv is associated with oropharyngeal, penile, and cervical scc.4,7 infection with equine papilloma virus is associated with scc of the penis in horses.6 papilloma virus infection is usually species-specific; however, infection of horses with bovine papilloma viruses types 1 and 2 is associated with development of sarcoids.3 it is unknown if the papillomavirus in the present animal induced scc, but it is possible that the papilloma viral infection identified in the preputial epithelium preceded the scc and could have been responsible for the neoplastic changes in the preputial epithelium. options for treatment of scc in the prepuce of domestic mammals are surgical excision, cryotherapy, hyperthermia, radiation, chemotherapy, and intralesional chemotherapy.8-11 the biologic behavior of cutaneous scc is locally invasive and not often metastatic. based on this, the treatment of choice for tumors in amenable locations is wide surgical excision.12 a combination of two or more methods of treatment is recommended for complete resolution.13 intralesional chemotherapy has been used in horses with scc in various locations and with varying results.14,15 in the present case, intralesional chemotherapy could not be pursued due to the fact that chemotherapeutic agents are illegal in food animals in the united states. surgical excision was not possible because such a large excision would not permit normal penile function during mating. penile amputation or a perineal urethrostomy were possible, but not acceptable for a breeding buck. cryotherapy or hyperthermia are appropriate for small, localized lesions but were not deemed viable options because of the large area affected. the general health of the buck appeared to be compromised based on the chronic dermatologic conditions and abnormalities of the prepuce. however, the clinical history precludes us from being completely certain of the chronological order of the lesions and any potential immunosuppression playing a role in the development of the skin condition and the preputial masses. it is also unknown what role the farm treatments may have played in the occurrence of scc and pv infection. in conclusion, we describe the first case of papillomavirus associated with scc in the prepuce of a goat. while papillomavirus infection is associated with development of scc in some species, the broader implications for pv infection and risk of scc in goats is not clear at this time. future reports of scc in goats should be screened for the presence of pv to determine if there is an association between pv infection and risk for this type of tumor. finally, this case serves as a reminder to thoroughly evaluate the prepuce in all cases of preputial abnormalities. learning points � the prepuce is not a predilection site for scc in goats and this is the first report of this type of tumor involving the prepuce in goats. � this is the first description of papilloma virus infection being associated with scc in goats. � there is strong evidence in the literature concerning other species to indicate that papillomaviruses are responsible for inducing scc, as may be the case in this buck. 597 clinical theriogenology • volume 9 number 4 • december 2017 � � acknowledgement photographs of the goat’s penis were courtesy of dr. jamie stewart. conflict of interest no financial support was received for this project and none of the authors have any conflict of interest or bias for the publication of this report references 1. ahmed af, hassanein kma: ovine and caprine cutaneous and ocular neoplasms. small rumin res 2012;106:189200. 2. borzacchiello g, roperto f: bovine papillomaviruses, papillomas and cancer in cattle. vet res 2008;39:1-19. 3. bogaert l, van poucke m, de baere c, et al: bovine papillomavirus load and mrna expression, cell proliferation and p53 expression in four clinical types of equine sarcoid. j gen virol 2007;88:2155-2161. 4. boscolo-rizzo p, schroeder l, romeo s, et al: the prevalence of human papillomavirus in squamous cell carcinoma of unknown primary site metastatic to neck lymph nodes: a systematic review. clin exp metastasis 2015;32:835-845. 5. nasir l, campo ms: bovine papillomaviruses: their role in the aetiology of cutaneous tumours of bovids and equids. vet dermatol 2008;19:243-254. 6. knight cg, munday js, peters j, et al: equine penile squamous cell carcinomas are associated with the presence of equine papillomavirus type 2 dna sequences. vet pathol 2011;48:1190-1194. 7. shabbir m, kayes o, minhas s: challenges and controversies in the management of penile cancer. nat rev urol 2014;11;702-711. 8. ruslander d, kaser-hotz b, sardinas, jc: cutaneous squamous cell carcinoma in cats. compend contin educ pract vet 1997;19:1119-1129. 9. lana se, ogilvie gk, withrow sj, et al: feline cutaneous squamous cell carcinoma of the nasal planum and the pinnae: 61 cases. j am anim hosp assoc 1997;33:329-332. 10. gallegos j, schmiedt cw, mcanulty jf: cosmetic rostral nasal reconstruction after nasal planum and premaxilla resection: technique and results in two dogs. vet surg 2007;36:669-674. 11. webb jl, burns re, brown hm, et al: squamous cell carcinoma. compend contin educ vet 2009:3:133-142 12. gibbons pm, lamb l, mansell j: presentation, treatment, and outcome of squamous cell carcinoma in the perineal region of 9 goats. can vet j 2015;56:1043-1047. 13. taylor s, haldorson g: a review of equine mucocutaneous squamous cell carcinoma. equine vet j 2013;25:374-378. 14. theon ap, pascoe jr, meagher dm: perioperative intratumoral administration of cisplatin for treatment of cutaneous tumors in equidae. j am vet med assoc 1994;205:1170-1176. 15. theon ap, pascoe jr, galuppo ld, et al: comparison of perioperative versus postoperative intratumoral administration of cisplatin for treatment of cutaneous sarcoids and squamous cell carcinomas in horses. j am vet med assoc 1999;215:1655-1660. � 598clinical theriogenology • volume 9 number 4 • december 2017 � � figure 1. photographs of a 4.5-year-old boer buck presented with preputial squamous cell carcinoma. a.) cranial view of the scrotum showing crusting dermatitis. b.) cranial view of the partially prolapsed, erythematous prepuce. c.) lateral view of the partially exteriorized prepuce with gray, inspissated material present in the preputial cavity. d.) lateral view of the exteriorized penis with numerous squamous cell carcinoma lesions. figure 2. histopathology of preputial masses taken from a 4.5-year-old boer buck with squamous cell carcinoma. nuclei are abnormally large, hypochromatic with vesicular chromatin, contain prominent nucleoli and frequent mitotic figures. h&e stain. bar = 50μm. insert: there is diffuse, cytoplasmic immunoreactivity in all the neoplastic epithelial cells by an immunohistochemical stain using cocktail broad spectrum antibodies against papillomaviral antigens. (editor’s note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) 599 clinical theriogenology • volume 9 number 4 • december 2017 vaginopexy: a new look at an old technique vaginopexy: a new look at an old technique darcie r. sidelinger, f. kevin walters, richard m. hopper, e. heath king department of pathobiology and population medicine, college of veterinary medicine mississippi state university, mississippi state, ms abstract vaginal and cervicovaginal prolapse are common in cattle. once the prolapse has been reduced, several retention methods can be employed. each method has benefits, as well as limitations. this article introduces a novel modification to an established technique and discusses advantages and disadvantages compared to other options. keywords: minchev, prolapse, vaginopexy, bovine, bühner introduction vaginal prolapse is a common reproductive malady of cattle. although overall occurrence of vaginal prolapse is low, with reports of less than 2% among several populations,1,2 breeds predisposed to vaginal prolapse include herefords, charolais, limousin, and shorthorns, as well as bos indicus breeds3 and composites. nutritional issues can predispose to vaginal prolapse, including diets deficient in calcium or too high in estrogenic compounds, as well as consumption of low-quality roughages.3,4 other conditions thought to contribute include previous injury to vaginal or cervical tissues or increased abdominal pressure in late gestation due to a cow being over-conditioned, tenesmus, or chronic coughing.3,4 because of the likely genetic component to the condition and the high probability of recurrence, culling should be recommended for affected cattle.3 for economic reasons, this is most commonly done after weaning their calf. an iatrogenic etiology for vaginal prolapse occurs due to hormonal manipulation performed during the superovulation of embryo donor cows.3 just as there are many etiologies and much variation in both the presentation and severity of these cases, there are also many treatment options. replacement of prolapsed tissues is straightforward, but varies in difficulty depending on severity, duration of the prolapse, and whether the bladder is also prolapsed. management of an acute vaginal prolapse allows a plethora of temporary retention techniques, as these are typically easily reduced and tissue damage is minimal. however, management of chronic vaginal prolapse is often challenging and requires more rigid fixation and stabilizing of the vaginal vault.4 a grading system for vaginal prolapse was established to aid decision-making regarding reduction. it is based on a scale of i-iv, as described by wolfe, et al.5 a discontinuous prolapse of vaginal tissue with increased abdominal pressure (i.e. lying down) would be considered grade i. grade ii describes a persistence of vaginal prolapse that may or may not include the bladder. grade iii includes the bladder and cervix in addition to the vaginal mucosa. a grade iv prolapse classifies a grade ii or iii prolapse that is further affected by another process (infection, trauma, necrosis).5 replacement is usually facilitated by epidural anesthesia. a pudendal nerve block or sacral paravertebral block can be utilized for more challenging cases. following replacement, retention is most often accomplished using a method of external fixation, which involves minimizing the vulvar opening to prevent prolapse of vaginal tissues but allowing for urination. the most common retention method, the bühner stitch, is a purse-string suture placed with the use of a specialized needle (bühner needle). alternate techniques can be utilized that allow parturition while maintaining fixation. these are the winkler (cervicopexy) technique, the minchev (vaginopexy) technique, or one of several modifications of the minchev, including those that utilize specialized pins. vaginopexy, utilizing one of the variations of the minchev technique, fits the need for temporary or “semi-permanent” retention, while allowing parturition followed by culling at a more economically opportune time. further simplification of this procedure with the use of a gerlach needle, as will be described, should increase utilization of this procedure. 463 clinical theriogenology • volume 10, number 4 • december 2018 modification of the minchev technique using a gerlach needle the suggested adaptation is a modification of the traditional minchev technique6 utilizing a gerlach needle (figure 1; displayed with a bühner needle for context) to pass either a length of #8 polyamide thread suture or umbilical tape (1/4 or 3/8 inch wide) externally from within the vagina. in order to accomplish this, the cow is first prepared by cleaning the perineum and the prolapse. after cleaning, a regional anesthetic is used, most often a caudal epidural. the prolapsed tissue is then reduced. next, the gerlach needle is introduced into the vagina while using the thumb and forefinger to carefully guard the vaginal mucosa from the point (tip) of the needle (figure 2). next, the cranial aspect of the vaginal wall (near the caudal cervix, at about the 10 or 2 o’clock position) is engaged and then manipulated in such a way as to retract the tissue caudally for subsequent entry through the sacrosciatic ligament (located 4-5 cm lateral from midline and immediately caudal to the shaft of the ilium) to the exterior. as the sacrosciatic ligament forms the majority of the lateral pelvic wall, it will be engaged during this procedure. care should be taken to avoid the rectum, sciatic nerve, and pudendal artery. once the cranial vaginal wall is engaged and the point of exit identified, the needle is passed, only once, from inside the vagina to the exterior of the cow. a length of suture is prepared and one free end is passed through the eye of the gerlach needle to half the length of the suture. with the two free ends maintained outside the cow, the gerlach needle is then pulled back through into the pelvis and out through the vulva. the loop of suture is then cut to make two free ends (figure 3). the ends are passed through each of two holes that have been previously prepared in a syringe case top (figures 4a & 4b) and tied in order to secure the syringe case top. the authors prefer syringe case tops from monojecttm 60 cc syringes; however, any smooth plastic disc could be functional in this use (i.e. bottom of a 500 ml fluid bottle or a sample cup lid), as long as care is taken to remove sharp edges. the syringe case tops are prepared by filing the lip down to about ¼ to ½” thickness and two holes are drilled into the top using the gerlach needle. the slack in the suture material is then drawn taut from the exterior and tied over a gauze roll located caudal to the cow’s ilium. the procedure is then repeated on the other side. discussion vaginal prolapse, a reproductive malady of the cow, can occur for many reasons. the most important issue to consider is the suggested hereditary etiology and the high likelihood of recurrence. replacement followed by culling is the recommended management. replacement can be accomplished in a variety of ways. the focus of this paper is a modification of the minchev technique. an advantage of the method, as described, over other minchev variations, is that the gerlach needle is easier to pass than a large s-shaped needle and less traumatic than the trocar as described in another modification.6 likewise, it seems to be less traumatic and subjectively less painful for the cow than methods that utilize pins and plastic sleeves. the described procedure can also be performed as quickly as those which utilize the commercially available pins. furthermore, the syringe cap replaces the need for rolled 4x4 gauze in the vagina of the cow, allowing for further ease during calving. the procedure commonly referred to as the bühner technique is easy to perform, rarely fails, and has few complications. however, it must be removed prior to parturition, which dictates very close observation of the cow by the owner/herdsman, and it is not an appropriate technique in the rare case when permanent retention is indicated. additionally, it must sometimes be removed prematurely when tissue damage results due to the constrictive nature of what is essentially a “purse-string” suture placed in an area subject to continual fecal contamination. winkler’s cervicopexy is the most technically difficult technique and thus, is reserved for very specific situations. as this is a permanent retention method it should only be utilized when the etiology is believed to be iatrogenic: specifically, an embryo donor cow who has had numerous superovulation treatments. in this case, the hormonal influence is implicated in contributing to the prolapse and therefore, it is not considered hereditary. a permanent fixation is then an option for treatment in order to maintain these cows as donors. 464clinical theriogenology • volume 10, number 4 • december 2018 summary the minchev technique and its modifications have few complications. it allows for calving without the necessity of removing the stitch. lastly, the modification of the minchev technique discussed here utilizes very inexpensive and readily available supplies with the only necessary purchase being the relatively inexpensive gerlach needle. references 1. patterson dj, bellows ra, burfening pj: effects of caesarean section, retained placenta and vaginal or uterine prolapse on subsequent fertility in beef cattle. j anim sci 1981;53:916-921. 2. woodward rr, quesenberry jr: a study of vaginal and uterine prolapse in hereford cattle. j anim sci 1956;15:119124. 3. peter at: vaginal, cervical, and uterine prolapse. in: hopper rm, editor. bovine reproduction. ames: wileyblackwell; 2015. p. 383-395. 4. miesner m, anderson d: management of uterine and vaginal prolapse in the bovine. vet clin north am food anim pract 2008;24:409-419. 5. wolfe df, carson rl, powe ta, pugh dg: surgery of the vestibule, vagina, and cervix. large animal urogenital surgery. baltimore: williams and wilkins; 1999. p. 397-412. 6. habel re: a modified operation for vaginal prolapse. j am vet med assoc 1957;130:344-345. figure 1. gerlach needle (left) & buhner needle (right). figure 2. operator’s hand holding gerlach needle in a guarded position for introduction into the vaginal cavity. 465 clinical theriogenology • volume 10, number 4 • december 2018 figure 3. suture in gerlach needle being cut by the operator. figure 4a. “syringe case tops” prepared for use. 466clinical theriogenology • volume 10, number 4 • december 2018 figure 4b. suture threaded through the holes in the “syringe case top” and tied in a knot. figure 5. external view of bilateral completed modified minchev technique; stay sutures tied over gauze externally. (editor’s note: online edition of the manuscript has color photographs) 467 clinical theriogenology • volume 10, number 4 • december 2018 000_frontal ms_1 forematter for december 2018 ms_2 letter1 for december 2018 ms_2 letter2 for december 2018 ms_2 letter3 for december 2018 ms_3 table of contents ms_4 therio news for december 2018_bw 2019 therio preview therio recap with pics steiner-new officers 2018 therio sponsors 2018 exhibitors-sft board noms 2018 auction donations 2019 bartlett nomination act call for noms call for abstracts 2019 for ctj the society for theriogenology and american college of theriogenologists issue a call for research and case abstracts to be presented at the annual therio conference july 24-27, 2019 in savannah, georgia. 1. competitive category: 2. non‐competitive category: 3. case abstract category: 4. poster category: general: guidelines: what to send and how to send it abstracts not adhering to these guidelines will not be considered for presentation. deadline: abstracts must be received no later than february 15, 2019 for consideration. call for 2019 student case presentations.pdf call for student case presentations-2019 annual therio conference student case presentations call for student case presentations also available at www.therio.org guidelines for application submission society for theriogenology student case presentation competition annual therio conference, savannah, georgia, july 24-27, 2019 ms_5 editorial 001a_ms_6 roberts color pictures 002_ms_7 root kustritz 003_ms_8 ferrer 004_ms_9 wiley 005_ms_10 wallace 006_ms_11 peterson 007_ms_12 nobre 008_ms_13 sidelinger 009_ms_14 mirando 2018: equipment for your andrology laboratory equipment for your andrology laboratory natalie s. fraser the university of queensland, gatton, qld, australia introduction andrology is the field of medicine that deals with physiology and disease of the male reproductive system. for any practitioner offering theriogenology services to clients, this potentially represents half of your caseload! an andrology laboratory that is adequately equipped and well maintained can improve productivity and quality of the services provided. sufficient equipment must be available for the number of staff working in the laboratory and for the laboratory to function efficiently at maximum caseload; this will be dependent on the individual practices’ volume. consideration should also be given to having backup equipment where appropriate to ensure that the andrology laboratory can continue to function in the event of equipment failure or repairs. practitioners should also be aware of the need for quality control measures and equipment maintenance to ensure that results obtained are accurate, as it has been demonstrated in other species that adherence to strict quality control guidelines are necessary for consistent results.1-3 basic laboratory equipment basic laboratory equipment is necessary for macroscopic parameter assessment, including color, volume, and ph assessment. supplies and consumables that may be of use include semen collection cones/receptacle, microcentrifuge tubes, glass microscope slides and coverslips, micropipettes with different ranges and corresponding tips, stage or slide warmer, beakers, graduated cylinders, 15ml conical tubes with caps, disposable 3ml graduated pipettes, an analytical balance, centrifuge, and water bath. semen handling supplies can be stored in a laboratory incubator set to 37°c. if frozen semen is to be stored on premises, a liquid nitrogen dewar will be needed for storage. stains for semen analysis buffered formol saline can be helpful for preparation and storage of semen samples for phasecontrast or differential interference contrast microscopy. approximately 1ml of buffered formol saline can be added to a microcentrifuge tube, with a few drops of raw ejaculate added until the solution appears cloudy. these samples can be labelled and stored in the refrigerator after morphological assessment for future review if required. alternately, eosin-nigrosin (hancock’s stain) can be used for routine morphology as well as assessment of live vs. dead sperm. dead sperm cells, with a damaged plasma membrane, are colored by eosin; living cells do not absorb the stain. the background is stained dark from nigrosin. other stain options, such as romanowski stains (diff-quick®) or spermac may also be helpful depending on the results of clinical examination or morphologic assessment. foreign cells (erythrocytes, leukocytes, round cells) are readily identified on air dried smears after staining with romanowski stains, and spermac allows clear visualization of the head, acrosome, equatorial region, centrepiece, and tail of the sperm. microscopy the microscope is one of the most used pieces of equipment in the andrology laboratory, and both bright field and contrast microscopy have their advantages in the laboratory for different applications. a bright field microscope will be ideal for examining stained preparations, such as for morphology, and can be used for evaluating motility if the field diaphragm is closed to enhance contrast and improve sperm visualization. phase microscopy is an important technology that allows one to view live organisms and internal structures without staining. a microscope with good quality phase contrast optics is extremely helpful in the andrology laboratory. ideal specifications of a microscope configured for andrology include 10x, 20x, 40x objectives (all positive low phase contrast), 100x oil immersion objective, phase telescope for proper alignment of phase rings, wide-field eyepieces (12.5x preferred), an extra eyepiece of built-in grid reticle, clinical theriogenology • volume 10, number 3 • september 2018197 and heated stage.4 systems with video capability will also need intermediate magnification capability and camera oculars/camera adapters (c-mount for video camera). differential interference contrast (dic) produces an image with more uniform greys, as opposed to the flatter image with darker cellular features seen with phase contrast.5 with dic, wallaston prisms are utilized that result in very sharp, 3-d like images, making it very useful for studying sperm. in bulls, morphologic assessment with eosin-nigrosin stained slides using bright field microscopy was deemed adequate when compared to formal saline wet mounds examined with dic, although dic was preferred as it was more effective in visualizing major defects.6 tools for determining sperm concentration sperm concentration can be determined by manual or automated methods. the hemocytometer is considered the gold standard for determining concentration of canine sperm, as well as other species.7,8 it consists of a specialized microscope slide with a grid system; sperm are counted using one of several methods within the grid.9 with this counting method, semen samples must be diluted prior to use to allow identification of individual cells within the grid. once the hemocytometer is loaded and allowed to equilibrate, sperm on both sides of the counting grid should be assessed to ensure that both sides are within 10% of each other. if greater than 10% discrepancy between the two sides is noted, the hemocytometer should be reloaded and the counting process repeated. automated devices include the nucleocounter®, photometric devices (ie spermacue® or densimeter®), and computer-assisted sperm analysis (casa) systems. the nucleocounter® has been assessed in canine sperm and provides assessment of not only sperm concentration, but also viability with reasonable repeatability.10 this device provides automated cell counting with a built-in fluorescence microscope using propridium iodide, and provides a hardware-based image analysis. photometric devices, on the other hand, determine concentration of sperm by determining the percentage of light transmission. poor sample handling (ie, fingerprints on cuvettes) or non-sperm material such as other cell types or debris will result in inaccurate results as these artefacts will also reduce light transmission through the sample. semen samples with concentrations at either extreme (highly concentrated or dilute) will yield significantly less accurate results. computer-assisted sperm analysis (casa) manual semen analysis does not provide any information on the kinematics of sperm motion. however, casa systems should not be viewed as a panacea for poor semen analysis techniques, as accurate assessment is dependent on the specific computer algorithm and the initial setup and validation process. manual assessment is much more accurate in discerning amoung debris or immotile/dead sperm heads.11 dependent on the specific casa system purchased, program functions may include assessment of motility, sperm morphology, concentration, dna fragmentation, acrosome status, and vitality. computer-assisted sperm analysis systems typically consist of a phase-contrast microscope, a camera, stage warmer, an image digitizer, and a computer to save/analyze data.12 this allows the system to analyze cell motion from successive image frames. most systems have a playback feature to allow the user to validate whether cells were properly identified by the software. computer-assisted sperm analysis systems have the significant advantage of providing accurate, rapid, and simultaneous analysis of multiple sperm parameters such as concentration, motility/velocity assessment, linearity of sperm movement, beat cross frequency, and amplitude of the lateral head displacement.13,14 the main disadvantage to these systems is the need for standardization and validation of the system; the choice of internal image setting such as minimum cell size, frame rate, and analysis time clearly influences the results obtained.12,15 thus, these systems are only as good as the user defined settings. dilution of the semen sample, the type of diluent used, temperature, and the type of counting chamber may also result in variability of results.13,14 cryopreservation units semen cryopreservation can be performed using incredibly low-tech equipment. a polystyrene box can be utilized to hold liquid nitrogen (ln2). a floating rack can be purchased commercially or clinical theriogenology • volume 10, number 3 • september 2018 198 made from polystyrene box lids or metal test tube racks. with a little imagination, many items readily available around the clinic can be re-purposed for semen freezing. controlled rate freezing units (biological freezers) may improve post-thaw motility in some dogs.16 freezing of canine semen in a polystyrene box has been compared to a protocol using an ultralow temperature freezer (152°c).17 after freezing and thawing, there were no significant differences in sperm motility or the percentage of live or abnormal spermatozoa between the two techniques. this led the authors to conclude that ultra-freezers could be used as potential alternative to liquid nitrogen for storage. references 1. filimberti e, degl'innocenti s, borsotti m, et al: high variability in results of semen analysis in andrology laboratories in tuscany (italy): the experience of an external quality control (eqc) programme. andrology 2013;1:401-407. 2. punjabi u, wyns c, mahmoud a, et al: fifteen years of belgian experience with external quality assessment of semen analysis. andrology 2016;4:1084-1093. 3. čipak a, stanić p, đurić k, et al: sperm morphology assessment according to who and strict criteria: method comparison and intra-laboratory variability. biochem med (zagreb) 2009;19:87-94. 4. björndahl l, mortimer d, barratt clr, et al: a practical guide to basic laboratory andrology. cambridge: cambridge university press; 2010. 5. dascanio jj: wet mount evaluation of sperm. in: dascanio j, mccue p, editors. equine reproductive procedures. hoboken (nj): wiley-blackwell; 2014. p. 389-391. 6. freneau ge, chenoweth pj, ellis r, et al: sperm morphology of beef bulls evaluated by two different methods. anim reprod sci 2010;118:176-181. 7. douglas-hamilton dh, smith ng, kuster ce, et al: capillary-loaded particle fluid dynamics: effect on estimation of sperm concentration. j androl 2005;26:115-122. 8. kuster c: sperm concentration determination between hemacytometric and casa systems: why they can be different. theriogenology 2005;64:614-617. 9. lorton sp: evaluation of semen in the andrology laboratory. in: chenoweth p, lorton s, editors. animal andrology: theories and applications wallingford (uk): cab international; 2014. p. 100-143. 10. daub l, geyer a, reese s, et al: sperm membrane integrity in fresh and frozen-thawed canine semen samples: a comparison of vital stains with the nucleocounter sp-100. theriogenology 2016;86:651-656. 11. vasan ss: semen analysis and sperm function tests: how much to test? indian j urol 2011;27:41-48. 12. rijsselaere t, van soom a, maes d, et al: w. computer-assisted sperm analysis in dogs and cats: an update after 20 years. reprod domest anim 2012;47 suppl 6:204-207. 13. rijsselaere t, van soom a, tanghe s, et al: new techniques for the assessment of canine semen quality: a review. theriogenology 2005;64:706-719. 14. verstegen j, iguer-ouada m, onclin k: computer assisted semen analyzers in andrology research and veterinary practice. theriogenology 2002;57:149-179. 15. rijsselaere t, van soom a, maes d, et al: effect of technical settings on canine semen motility parameters measured by the hamilton-thorne analyzer. theriogenology 2003;60:1553-1568. 16. rota a, rota a, martini m, et al: evaluation of dog semen quality after slow (biological freezer) or rapid (nitrogen vapours) freezing. reprod nutr dev 2005;45:29-37. 17. batista m, alamo d, gonzalez f, et al: influence of the freezing technique (nitrogen liquid vs ultrafreezer of -152 degrees c) and male-to-male variation over the semen quality in canarian mastiff breed dogs. reprod domest anim 2006;41:423-428. clinical theriogenology • volume 10, number 3 • september 2018199 clinical theriogenology • volume 10, number 3 • september 2018 200 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype true /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice semen extender type and cold storage of tissue affect cryosurvival of alpaca epididymal sperm semen extender type and cold storage of tissue affect cryosurvival of alpaca epididymal sperm maria s. ferrer,a lisa williamson,a roberto a. palomares,b david j. hurley,b alejandro hoyosjaramillo,b jose urdanetab adepartment of large animal medicine and bpopulation health, college of veterinary medicine university of georgia, athens, ga abstract the objectives of this study were to determine the ability of liposomeand dmfa-containing semen extenders to support cryosurvival of alpaca epididymal sperm, and to evaluate the effect of epididymal cold storage on post-thaw viability. it was hypothesized that both semen extenders provide appropriate cryoprotection, and post-thaw sperm viability does not differ when alpaca epididymal sperm are cryopreserved immediately after castration or after 24 h of cold storage. sperm were recovered from one epididymis from each male (n = 10) on the day of castration, and from the other one after 24 h of tissue storage at 4c. on each day, each sample was divided into two aliquots, which were cryopreserved in semen extender optixcell or botucrio. pre-freezing sperm parameters did not differ between samples obtained on the day of castration or 24 h later. motility of sperm cryopreserved in botucrio on the day of castration did not differ from pre-freezing values. use of optixcell or delaying sperm cryopreservation decreased total (p = 0.002) and progressive sperm motility (p = 0.041) compared with pre-freezing values seen in sperm recovered on the day of castration. membrane integrity was lowest when sperm were cryopreserved in optixcell after cold storage. treatment had no effect on dna integrity. it was concluded that the dmfa-containing semen extender botucrio was superior to the liposome-containing semen extender optixcell at preserving post-thaw viability of alpaca epididymal sperm. cold storage of epididymides for 24 h negatively affected post-thaw motility and membrane integrity of sperm. therefore, cryopreservation with botucrio immediately after castration is recommended for best preservation of post-thaw viability of alpaca epididymal sperm. keywords: camelid, male, diluent, freezing, post-thaw introduction harvest and cryopreservation of epididymal sperm enables preservation of genetic material in the event of unexpected death of an animal. while reports of this technique exist in camelids, an efficient protocol for epididymal sperm cryopreservation has not been developed. initial reports have shown that lactose-based semen extenders provided better post-thaw motility than citrateor tris-based extenders.1 using lactose-based extenders, it was then shown that final concentrations of 3 to 4% glycerol and 1% equex stm paste provided ideal cryoprotection.2 however, mean post-thaw motility was low, ranging from 14.4 to 27%.1,2 later work testing alternative cryprotectants demonstrated that post-thaw motility was improved when dimethylsulfoxide (dmso) or diacetylamide (dma) were used instead of ethylene glycol or glycerol.3,4 with addition of these cryoprotectants, mean post-thaw motility ranged from 31 to 34%.3,4 amides, like dma and dimethylformamide (dmfa) are small hydrophilic molecules with better cell penetration and less toxicity than glycerol.4,5 adding dmfa to the semen extender improved cryosurvival of stallion epididymal sperm.6 in bulls, addition of liposomes improved post-thaw semen quality.7,8 liposomes can incorporate into the plasma membrane and change the lipid phase transition, decreasing the sensitivity of sperm to low temperatures.7 the use of liposome and dmfa containing commercial semen extenders for cryopreservation of alpaca sperm has not been yet tested. in addition to semen extenders, holding time could also have an impact on post-thaw semen quality. the ability to hold epididymides for a prolonged time allows practitioners to transport tissue to the laboratory for further processing and cryopreservation. this practice is routinely performed in stallions since cold storage for up to 96 h does not affect quality or fertility of cryopreserved equine sperm.9,10 in spite of this apparent advantage, there have been no direct comparisons on post-thaw motility of alpaca epididymal sperm cryopreserved immediately after castration or after 24 h of cold storage. 419 clinical theriogenology • volume 10, number 4 • december 2018 the objectives of this study were to determine the ability of commercial semen extenders containing liposomes and dmfa to support cryosurvival of alpaca epididymal sperm, and to evaluate the effect of cold storage on post-thaw sperm parameters. it was hypothesized that both semen extenders provide appropriate cryoprotection, and that post-thaw sperm parameters do not differ when alpaca epididymal sperm are cryopreserved immediately after castration or after 24 h of cold storage. materials and methods animals and castrations twenty testes were collected from 10 male alpacas. the alpacas were of huacaya and suri breeds, and 1 to 7 y old. they were presented to the veterinary medical center of the university of georgia for elective castration. the reproductive history of these males was not known. the alpacas were anesthetized with xylazine (0.4 mg/kg), ketamine (4 mg/kg) and butorphanol (0.04 mg/kg), all of them given intramuscularly. the testes and epididymides were removed using a standard closed castration technique. prior to castration, the deferent ducts were ligated with 2 pds ii. each pair of testes with epididymides was placed in a clean whirlpack bag without any addition of media, and was placed at 4°c in the truck’s refrigerator until processing. experimental design and sperm collection   consequent to arrival to the laboratory, one epididymis from each male was immediately processed, while the other testis remained at 4c until processing the following day. tissue was processed as previously described, with some modifications.1,2 the parietal vaginal tunic was opened, and the testes and epididymides were exposed and rinsed with 0.9% sodium chloride solution. a ligature (2-0 pds ii) was placed at the junction between the corpus and cauda epididymides. cauda epididymides, including at least 1 cm of the proximal ductus deferens, were separated and transferred to a petri dish. connective tissue containing superficial blood vessels around the cauda epididymis was removed by dissection with scissors. the tissue was then covered with fractionated milk-based semen extender (inra96, imv technologies, maple grove, mn, usa), and minced into small fragments with a scalpel. while there is no information in alpacas, the use of inra96 resulted in better preservation of membrane integrity and fertility of liquid-stored camel semen compared with other commercial extenders.11 sperm were allowed to swim up into the medium by incubation for 15 min at 38°c. after incubation, the tissue fragments were removed with forceps. the medium containing sperm was filtered through a stallion semen gel filter (max-flow semen gel filters, nasco, ft. atkinson, wi, us) into a 15-ml tube to remove small tissue fragments and debris. a complete semen evaluation was performed as described below, including evaluation of volume, sperm concentration, morphology and motility. then, each sample was divided into two aliquots. both aliquots were centrifuged at 900 x g for 10 min. this centrifugation force has no negative effects on sperm motility or viability, and minimizes sperm losses in the supernatant.12 after discarding the supernatant, one aliquot was resuspended with the liposome-containing semen extender optixcell (imv technologies, maple grove, mn, usa) to dilute sperm to a final concentration of 25 x 106/ml. there are no reports in the literature on the ideal sperm concentration for alpaca semen cryopreservation. the concentration used was selected to enable the division of the samples into the number of aliquots needed. sperm diluted in optixcell were placed in the refrigerator at 4°c for 5 h for equilibration. after equilibration, pre-cooled 0.5 ml french straws were loaded. the other centrifuged aliquot was diluted with the dmfa-containing semen extender botucrio (botupharma usa, phoenix, az, usa) until a final sperm concentration of 25 x 106/ml was reached. sperm diluted in botucrio were loaded into 0.5-ml french straws at room temperature, and equilibrated at 4°c for 15 min. next, all straws were placed horizontally on a rack, 3 cm above liquid nitrogen for 15 min. the straws were plunged in liquid nitrogen, loaded into 10-mm goblets, and transferred to the storage tank. all straws were stored at 196c until evaluation. the cryopreservation protocols used were based on instructions from the manufacturer of each semen extender. the other epididymis from each male was maintained in the plastic 420clinical theriogenology • volume 10, number 4 • december 2018 bag at 4°c for 24 h. after 24 h of cold storage, the cauda epididymides were processed as described for the contralateral testis. each sample was also divided into two aliquots, diluted with optixcell or botucrio, and cryopreserved following the same protocols as described above. two weeks after freezing, one straw from each aliquot was thawed in a water bath at 38c for 30 s, and sperm motility and membrane integrity were evaluated. another straw was thawed for evaluation of dna integrity. semen evaluation immediately after recovery of sperm, sample volume was determined using a graduated 15-ml centrifuge tube. sperm concentration and motility were evaluated with a computer assisted semen analyzer (casa; spermvision professional, minitube of america, verona, wi, usa). the settings of the instrument were: field depth of view 20 m, pixel to m ratio 130 to 100, cell area 18 to 80 m, frames acquired 30, frame rate 60 hz, aoc cut off static cells 5 and dsl cut off 4.5 µm/s (progressive motility). semen was placed in a 20-µl sperm analysis chamber over the heated specimen stage at 38c. mean percentage of total (tmot) and progressively (pmot) motile sperm was assessed from all cells present in seven fields with a x 20 phase-contrast objective. phase contrast microscopy was used for assessment of sperm morphology. sperm were diluted 1:10 (v:v) with formalin buffered solution, and a 5l drop was placed on a microscope slide. evaluation was done at 100x magnification under oil immersion, and 100 sperm were classified based on their morphological characteristics. two weeks after freezing, one straw from each aliquot was thawed in a water bath at 38c for 30 s, and sperm motility and membrane integrity were evaluated using casa. in addition to tmot and pmot, other motion parameters analyzed post-thaw were: average path velocity (vap), straight-line velocity (vsl), curvilinear velocity (vcl), amplitude of lateral head displacement (alh), beat cross frequency (bcf), straightness (str), linearity (lin), wobble (wob), curvilinear distance (dcl), straight line distance (dsl), and distance of average path (dap). membrane integrity was evaluated using fluorescence microscopy under a 40x objective. sperm were incubated for 10 min at 38c with the vital stains sybr14 and propidium iodide (live/dead kit, minitube of america) following instructions from the manufacturer. all sperm presented in 10 fields were classified as membrane-intact (green fluorescence) or membrane-damaged (red fluorescence) by a built-in software within the casa system (spermvision professionaltm, minitube of america), and mean percentages were reported. sperm chromatin structure was evaluated on frozen-thawed semen using acridine orange and flow cytometry (accuri c6 plus, bd biosciences, san jose ca, usa). frozen-thawed sperm were washed in phosphate buffer solution by centrifugation at 600 x g for 5 min. after removing the supernatant, the pellet was re-suspended to 0.5 ml in tne buffer (0.186 g disodium etda, 0.79 g tris-hcl, 4.380 g nacl in 500 ml deionized water, ph 7.4, 4°c). then, 15 l of the resuspended sample was diluted with 185 l of tne buffer. this dilution provided a final sperm concentration of 2 x 106/ml. semen was incubated for 30 s after addition of 400 l of acid-detergent solution (2.19 g nacl, 1 ml 2n hcl, 0.25 ml triton x, q.s. 250 ml deionized water, ph 1.2, 4°c). finally, 1.2 ml of a 2 g/ml working solution of acridine orange (invitrogen, waltham, ma, usa, cat. no. a3568) in buffer (3.8869 g citric acid monohydrate, 8.9429 g na2hpo4, 4.3850 g nacl, 0.1700 g disodium edta, q.s. 500 ml water, ph 6) was added and semen was incubated for 3 min at room temperature. the sample flow was initiated for 1 min to allow for hydrodynamic equilibration, after which data collection was initiated. the flow rate was adjusted to around 200 cells/sec. from each sample, 20 000 events in the forward and 90º light scatter population representing whole sperm were analyzed using 0.2-m filtered 18 mega-ohm ultra-pure water as the sheath fluid. a gate containing sperm was selected based on dot plot distribution of forward (size) versus side scatter (complexity parameter) to eliminate debris and somatic cells from the analysis. the green and red signals were detected using a 5mwatt blue argon laser (488 nm) and emission filters (535 ± 30 nm for green and 585 ± 30 nm for red). baseline background fluorescence signal was initially evaluated in unstained samples. the control area was marked on unstained samples with < 1 % of cells registering as positive for both signals. the main population represented sperm that emitted more green than red fluorescence (native double-stranded dna). cells located to the right and below the main 421 clinical theriogenology • volume 10, number 4 • december 2018 population represented sperm with increased red fluorescence and decreased green fluorescence (denatured single-stranded dna). a modified dna fragmentation index (mdfi) was calculated as the percentage of sperm with denatured dna (red) divided by the percentage of total sperm with fluorescence emission (green + red). statistical analysis statistical analysis was performed using sas package (sas institute, cary, nc, usa). distribution of the data were tested for normality using a shapiro wilk test. variables that did not follow a normal distribution underwent logarithmic transformation. means were compared between treatments using anova, including the fixed effect of treatment. paired comparisons were made using a tukey’s test. data were expressed as mean  se and differences were considered significant if p < 0.05. results sperm were obtained from 70% (7/10) of the epididymides processed on the day of castration, and 60% (6/10) of those processed after 24 h of cold storage. pre-freezing sperm parameters did not differ between the samples obtained on the day of castration or 24 h later (p > 0.05; table 1). the proportion of pre-freezing total and progressive motility retained (% of initial motility) after 24 h of cold storage was 65.1±10.9% and 49.9±16.1%, respectively. motility of sperm cryopreserved in botucrio on the day of castration did not differ from prefreezing values. however, total (p = 0.002) and progressive (p = 0.041) sperm motility were lower when optixcell was used or cryopreservation was delayed for 24 h (figure). post-thaw membrane integrity was also affected by treatment and was lowest when sperm were cryopreserved in optixcell after cold storage (p = 0.039; figure). no other sperm motion parameters differed with treatment (table 2). similarly, post-thaw mdfi did not differ with treatment. mean mdfi was 2.5  1.8% and 4.3  3.1% for sperm cryopreserved immediately in botucrio or optixcell, respectively; and 3.3  1.8% and 4.1  3.1% for sperm cryopreserved after cold storage in botucrio or optixcell, respectively. discussion to the authors’ knowledge, this is the first study directly comparing post-thaw quality of alpaca epididymal sperm recovered on the day of castration and after 24 h of cold storage. without direct comparisons between holding times, recommendations for immediate or delayed processing of alpaca epididymides could not be made. while mean sperm motility in this study was not statistically different between storage times, storage of testes with epididymides at 4c for 24 h resulted in loss of 35% and 51% of total and progressively motile sperm, respectively. moreover, post-thaw motility was significantly decreased by cold storage compared with pre-freezing values of sperm recovered on the day of castration. while the ability to preserve sperm viability for a prolonged time would allow practitioners to ship the epididymides of deceased males to a centralized facility for sperm recovery and cryopreservation, based on the results of this study, this practice is not recommended in alpacas. this is in contrast with findings in other species. in stallions and bucks, sperm viability and fertility were maintained when epididymides were cooled for 48 to 96 h.9,10,13 differences in diluent, cryoprotectant or sperm membrane composition may account for the discrepancies among species. the osmolality of the semen extender was shown to affect survival of cold-stored ram epididymal sperm.14 it was proposed that since the osmolality of the epididymal medium increases with post-mortem time, epididymal sperm might adapt to an increasingly hypertonic environment, requiring the use of semen extenders with higher osmolality for best response to cryopreservation.14 the effect of storage time on osmolality of epididymal contents recovered postcastration, and the effect of osmolality of semen extender on post-thaw viability of alpaca sperm require investigation. this study was also first to evaluate the ability of commercial semen extenders containing dmfa or liposomes to support cryosurvival of alpaca sperm. the use of a diluent containing dmfa provided the best preservation of post-thaw motility. the liposome-containing semen extender was less suitable for 422clinical theriogenology • volume 10, number 4 • december 2018 cryopreservation of alpaca epididymal sperm, resulting in decreased post-thaw motility and membrane integrity at both processing times. adding dmfa to freezing extenders also had a beneficial effect on post-thaw motility of stallion epididymal sperm.7 this is thought to be because dmfa is a small hydrophilic molecule with good cell penetration and low cytotoxicity.4,5 in this study, sperm were recovered from 60 to 70% of the epididymides. this number was lower than previously reported (90-100%). previous studies have included animals of 2 to 4 y of age and testicular size was a criterion for inclusion in the study. males in this study were between 1 and 7 y old. no attempt was made to identify the donor of each tissue or measure testicular size. it is possible that the epididymides that yielded no sperm belonged to young peri-pubertal animals. on the other hand, the number of sperm recovered from each male that did yield a sample was higher than previously reported. reported mean recovery ranged from 75.3 to 143.1 x106 sperm/male.1,2 in this study, a mean of 560 x106 sperm/male (range 203 to 898.5 x106 sperm/male) was obtained. pre-freezing sperm motility was comparable to previous reports.1,2,15 the mean percentage of morphologically normal sperm was 29%. the main abnormality present was cytoplasmic droplets (45%). cytoplasmic droplets can be present in more than 60% of alpaca sperm in the terminal segment of the cauda epididymis, and are lost when sperm reach the ductus deferens.16 while these findings represent physiological changes that occur during epididymal transit, the effect of epididymal sperm morphology on alpaca fertility is not known. in conclusion, a dmfa-containing commercial semen extender (botucrio) was superior to a liposome-containing semen extender (optixcell) at preserving post-thaw motility and membrane integrity of cryopreserved alpaca epididymal sperm. cold storage of epididymides for 24 h negatively affected post-thaw sperm motility and membrane integrity. therefore, cryopreservation with botucrio immediately after castration is recommended for best preservation of post-thaw viability of alpaca epididymal sperm. references 1. morton km, bathgate r, evans g, et al: cryopreservation of epididymal alpaca (vicugna pacos) sperm: a comparison of citrate-, trisand lactose-based diluents and pellets and straws. reproduction, fertility and development 2007;19:792-796. 2. morton km, evans g, maxwell wmc: effect of glycerol concentration, equex stm® supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen-thawed epididymal alpaca (vicugna pacos) sperm. theriogenology 2010;74:311-316. 3. terreros mc, huanca wl, arriaga ic, et al: efecto de tres crioprotectores en la criopreservacion de espermatozoides epididimarios de alpaca. rev inv vet peru 2015;26:420-426. 4. canorio pariona n, paredes arnedo f, valdivia cuya m: agentes crioprotectores alternativos para el congelamiento lento de espermatozoides epididimarios de alpaca (vicugna pacos). rev inv vet peru 2015;26:434-443. 5. morillo rodriguez a, balao da silva c, macıas-garcıa b, et al: dimethylformamide improves the in vitro characteristics of thawed stallion spermatozoa reducing sublethal damage. reprod dom anim 2012;47:995-1002. 6. álvarez c, gil l, gonzález n, et al: equine sperm post-thaw evaluation after the addition of different cryoprotectants added to inra 96 extender. cryobiology 2014;69:144-148. 7. ansari ms, rakha ba, akhter s, et al.: optixcell improves the postthaw quality and fertility of buffalo bull sperm. theriogenology 2016;85:528-532. 8. miguel-jiménez s, mogas t, peña ai, et al: post-thaw changes in sperm membrane and ros following cryopreservation of dairy bull semen using four different commercial extenders. anim reprod 2016;13:573. 9. james an, green h, hoffman s, et al: preservation of equine sperm stored at 4 c for 24, 48, 72 and 96 hours. theriogenology 2002;58:401-404. 10. monteiro ga, papa fo, zhan fs, et al: cryopreservation and fertility of ejaculated and epididymal stallion sperm. anim reprod sci 2011;127:197-201. 11. skidmore ja, morton km, billah m: artificial insemination in dromedary camels. anim reprod sci 2013;136:178186. 12. ferrer ms, lyle sk, eilts be, et al: factors affecting sperm recovery rates and survival after centrifugation of equine semen. theriogenology 2012;78:1814-1823. 13. turri f, madeddu m, gliozzi tm, et al: effect of testicle postmortem storage on goat frozen-thawed epididymal sperm quality as a tool to improve genebanking in local breeds. animal 2014;8:440-447. 14. tamayo-canul j, alvarez m, mata-campuzano m, et al: effect of storage method and extender osmolality in the quality of cryopreserved epididymal ram spermatozoa. anim reprod sci 2011;129:188-199. 423 clinical theriogenology • volume 10, number 4 • december 2018 15. santiani a, ugarelli a, evangelista-vargas s: characterization of functional variables in epididymal alpaca (vicugna pacos) sperm using imaging flow cytometry. anim reprod sci 2016;173:49-55. 16. delhon g, von lawzewitsch i: ductus epididymidis compartments and morphology of epididymal spermatozoa in llamas. anat histol embryol 1994;23:217-225. table 1. pre-freezing parameters of epididymal sperm collected on the day of castration or after 24 h of cold storage. data are expressed as mean ± sem. parameter day of castration 24 h after castration total sperm motility (%) 50.16.1 34.216.1 progressive sperm motility (%) 33.75.7 19.56.2 normal sperm (%) 29.15 29.86 abnormal acrosomes (%) 0.80.5 0.30.3 abnormal heads (%) 6.13.1 72.2 abnormal midpieces (%) 6.72.1 3.51.1 kinked tails (%) 7.82.3 8.13 proximal droplets (%) 32.56.8 38.34 distal droplets (%) 10.82.2 9.72.3 coiled tails (%) 4.42 2.31.2 detached heads (%) 1.41.1 0.80.8 sperm concentration (x106/ml) 53.610.9 71.412.5 sample volume (ml) 6.30.3 3.70.1 total sperm (x106) 326.955.9 271.954.2 total progressively motile sperm (x106) 121.428.8 64.923.9 total normal motile sperm (x106) 28.85.8 14.14.8 table 2. motion parameters of epididymal alpaca sperm after cryopreservation in botucrio or optixcell on the day of castration (0) or after 24 h of cold storage (24). dcl=curvilinear distance, dap=distance of average path, dsl=straight line distance, vcl=curvilinear velocity, vap=average path velocity, vsl=straight line velocity, lin=linearity, str=straightness, wob=wobble, bcf= beat cross frequency, alh= amplitude of lateral head displacement. data are expressed as mean ± sem. variable botucrio0 optixcell0 botucrio24 optixcell24 dcl (m) 34.52 35.84.5 34.52.4 32.85.2 dap (m) 17.90.9 16.70.9 16.90.5 15.82.1 dsl (m) 10.50.4 10.20.4 100.4 10.40.9 vcl (m/sec) 75.54.5 79.28.6 75.64.6 70.511 vap (m/sec) 39.72 38.21.7 37.41.2 34.64.7 vsl (m/sec) 23.51.2 23.91.2 22.51.1 22.82.2 lin 0.310.01 0.30.04 0.30.03 0.340.03 str 0.590.02 0.620.02 0.60.03 0.670.02 wob 0.520.01 0.50.04 0.50.03 0.510.04 bcf (hertz) 14.70.6 14.91.6 14.70.5 12.81.8 alh (m) 3.80.3 3.90.1 3.50.09 3.10.4 424clinical theriogenology • volume 10, number 4 • december 2018 figure. motility of alpaca epididymal sperm prior to freezing (prefreeze), and after cryopreservation in botucrio or optixcell on the day of castration (0) or after 24 h of cold storage (24). membrane integrity was only evaluated after cryopreservation. data are expressed as mean ± sem. a,bindicate differences among treatments (p < 0.05). 425 clinical theriogenology • volume 10, number 4 • december 2018 000_frontal ms_1 forematter for december 2018 ms_2 letter1 for december 2018 ms_2 letter2 for december 2018 ms_2 letter3 for december 2018 ms_3 table of contents ms_4 therio news for december 2018_bw 2019 therio preview therio recap with pics steiner-new officers 2018 therio sponsors 2018 exhibitors-sft board noms 2018 auction donations 2019 bartlett nomination act call for noms call for abstracts 2019 for ctj the society for theriogenology and american college of theriogenologists issue a call for research and case abstracts to be presented at the annual therio conference july 24-27, 2019 in savannah, georgia. 1. competitive category: 2. non‐competitive category: 3. case abstract category: 4. poster category: general: guidelines: what to send and how to send it abstracts not adhering to these guidelines will not be considered for presentation. deadline: abstracts must be received no later than february 15, 2019 for consideration. call for 2019 student case presentations.pdf call for student case presentations-2019 annual therio conference student case presentations call for student case presentations also available at www.therio.org guidelines for application submission society for theriogenology student case presentation competition annual therio conference, savannah, georgia, july 24-27, 2019 ms_5 editorial 001a_ms_6 roberts color pictures 002_ms_7 root kustritz 003_ms_8 ferrer 004_ms_9 wiley 005_ms_10 wallace 006_ms_11 peterson 007_ms_12 nobre 008_ms_13 sidelinger 009_ms_14 mirando 2013: effect of increasing student engagement on multiple-choice test scores in the theriogenology diagnostics course effect of increasing student engagement on multiple-choice test scores in the theriogenology diagnostics course margaret v. root kustritz university of minnesota college of veterinary medicine,, st. paul, mn abstract third year veterinary students in two consecutive years completed a multiple-choice examination as a pre-test before exposure to course content. students in group i completed a take-home assignment and then “graded” an assignment provided by the instructor. students in group ii completed only the take-home assignment. students in both groups completed an on-line multiple-choice examination as a post-test two weeks after completion of assignments. students in both groups showed significant improvement from the pre-test to the post-test. mean pre-test scores did not differ significantly between the two groups (p = 0.57). mean post-test scores did vary significantly (p = 0.02) with group i scores higher than those for group ii, demonstrating value in increasing student engagement in the material. keywords: theriogenology education, active learning, introduction active learning is use of educational methodologies that provide students with opportunities to work with information presented to them, by reflection, evaluation, analysis, synthesis, and communication.1,2 active learning often is equated with group work, which serves to help students not only to master content but also to learn skills in the discipline of interest including teamwork and conflict resolution.3 other types of active learning involve individual students working either inside or outside of class and may include asking students to process or translate course information into unusual expressive modes, for example poems or songs; requiring students to argue on behalf of unfamiliar positions; or asking the student to articulate the clearest or muddiest point in a presentation.4-6 another example of an active learning instructional strategy is asking students to find or identify mistakes or to critique another student’s work.4.5.7 this forces the student to evaluate their grasp of the material, which often is strengthened as they struggle to defend why they think another student is in error, and promotes critical thinking.5 this is an authentic teaching tool as it mirrors what many people are asked to do in their daily work.4 peer-instruction can vary from the “convince your neighbor” scenario espoused by eric mazur, which requires students to discuss concepts within class and to reclassify that material within their own thinking so as to be able to explain it to others, to formalized systems where students with higher levels of demonstrated competency provide instruction and feedback to students earlier in a given program of study.8,9 peer instruction also can include assessment of other students’ work and providing of constructive feedback. this study evaluated differing levels of student engagement in a short, required course in theriogenology to determine if increased student engagement was associated with increased test scores on a multiple-choice assessment. materials and methods theriogenology diagnostics is a required course for third year veterinary students. it is a multispecies survey course and is the only required clinical theriogenology course in the core curriculum; other material is provided in species-specific courses available elsewhere in the tracked curriculum. students participated in this study in two consecutive years. in both years, students were not given advance notice of the study to prevent their studying for the pre-test portion. in the first year (group i), students completed a six-question multiple-choice assessment as a pre-test and then were presented with course information about canine reproduction for two hours, supported by notes available electronically or as hard copy. students then had two weeks to complete a take-home assignment; students could work on the assignment together and could use any resources but each student had to hand in a completed assignment 63 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 individually. the assignment was graded by the instructor. the instructor then compiled an example of that completed take-home assignment with some correct and some incorrect responses, with the incorrect responses those most commonly seen on student assignments, and all students were required to “grade” that second assignment, with the instructor assessing their effort as pass or fail. two weeks after completing that second assignment, students logged onto the course website and took the same sixquestion multiple-choice assessment, this time as a post-test. in the subsequent year (group ii), students took the pre-test, were presented with information, and completed the take-home assignment as with the previous group, but did not receive or “grade” the second assignment. they took the post-test on-line two weeks after handing in the first assignment. grades on the pre-test and post-test were compared within classes using the paired t-test. grades were compared between groups i and ii for the pre-test and post-test using the unpaired (students) t-test. significance was set at p < 0.05. results there were 96 participants in group i and 82 participants in group ii. participation was not 100% in either group because some students did not arrive in class until after presentation had begun, precluding their ability to take the pre-test as a true measure of their prior knowledge. these students completed the preand post-tests for their own learning but were not included in the study. the two classes were similar in composition. participants in group i were from a class of 97 students, 20 men and 77 women. mean age at admission was 25.4 years and mean grade point average (gpa) on required courses at admission was 3.5. participants in group ii were from a class of 100 students, 25 men and 75 women. mean age at admission was 24.4 years and mean gpa on required courses at admission was 3.6. data for preand post-testing scores in both groups were normally distributed by visual assessment. mean (± standard deviation) pre-test score for students in group i was 1.59 ± 0.95. mean post-test score was 3.78 ± 1.05. this difference was significant, at p < 0.0001. mean pre-test score for students in group ii was 1.51 ± 0.95. mean post-test score was 3.41 ± 1.04. this difference was significant, at p < 0.0001. mean pre-test scores did not differ significantly between the two groups (p = 0.57). mean posttest scores did vary significantly (p = 0.02) with group i scores higher than those for group ii. discussion both groups demonstrated significant gains in knowledge as assessed by multiple-choice testing. between the groups, increasing student engagement was associated with a greater gain in knowledge as assessed by multiple-choice testing. this is supported by a body of literature showing increased performance on assessments from students participating in active learning compared to passive learners.6,7,10-14 other courses completed in that same semester were the same between the two groups. the author was the single faculty member responsible for small animal theriogenology training in both years. the groups were similar in gender distribution, mean values for academic achievement, and mean age. splitting of a class in one year and having half of that class complete the work of group i and the other half complete the work of group ii may have decreased unexplained variation between the groups. the author chose not to do this because of the automatic decrease in sample size with splitting of a class of pre-defined total size of 100, because there was greater likelihood of student anxiety altering scores, especially in group i, whose members were required to do more work than those in group ii, and because it is the author’s experience that when classes are split, students will “try out” the other alternative. while the author has no problem with the increased learning that occurs during this experimentation, it does negate clear-cut differences between treatments in experimental groups. this has clearly occurred to this author with splitting of a class at her home institution for another study.15 one could argue that because the study was implemented over two successive years, the students in group ii heard about it from those in group i, somehow giving them an advantage. this was disproven by the students in group i performing better on the post-test than those in group ii. 64clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 the study design did not permit identification of which aspects of the assignments were most beneficial in increasing student test scores. many other tools for assessment could have been employed, including other types of assignments or a closed-book examination. this study does not attempt to prove that the chosen method is the best method for student instruction in theriogenology, or to demonstrate that retention was improved with one method compared to another. conclusion students do not always enjoy being forced to evaluate each other’s work; a study evaluating use of an interactive web-based training tool for ethics instruction showed that students were less satisfied with the training technique but also were better able to demonstrate achievement of learning objectives.10 use of an asynchronous instructional strategy that required peers to evaluate each other’s work provided students with the benefits of active learning and increased their understanding of the material. references 1. fink ld: creating significant learning experiences: an integrated approach to designing college courses. san francisco: jossey-bass; 2003. 2. michael j: where’s the evidence that active learning works? advan physiol educ 2006;30:159-167. 3. machemer p, crawford p: student perceptions of active learning in a large cross-disciplinary classroom. active learn higher educ 2007;8:9-30. 4. lang jm: the benefits of making it harder to learn. chron higher educ 2012;58:a33-a34. 5. faust jl, paulson dr: active learning in the college classroom. j exc coll tchg 1998;9:3-24. 6. prince m: does active learning work? a review of the research. j engin educ 2004;93:223-231. 7. reese-durham n: peer evaluation as an active learning technique. j coll sci teach 2011;40:338-343. 8. mazur e: peer instruction. upper saddle river (nj): prentice hall; 1997. 9. baillie s, shore h, gill d, et al: introducing peer-assisted learning into a veterinary curriculum: a trial with a simulator. j vet med educ 2009;36:174-179. 10. root kustritz mv, copes l: use of a civil discourse website for ethics training. j vet med educ 2013 (in press). 11. sharkey l, overmann j, flash p: evolution of a course in veterinary clinical pathology: the application of case-based writing assignments to focus on skill development and facilitation of learning. j vet med educ 2007;34:423-430. 12. levy dp, merenstein b: working with stories: an active learning approach to theories of deviance. teach sociol 2005;33:66-73. 13. yoder jd, hochevar cm: encouraging active learning can improve students’ performance on examinations. j instr physchol 2005;32:91-95. 14. mullen et: teaching an engaged analysis class through active learning. prob res issues math undergrad studies 2012;22:186-200. 15. root kustritz mv: use of stories as a way to increase retention of clinical small animal theriogenology information. clin therio 2009;1:347-348. 65 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 2018: sperm and seminal plasma micrornas in the context ofbull sperm fertility sperm and seminal plasma micrornas in the context of bull sperm fertility vanmathy kasimanickam, ramanathan kasimanickam department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa abstract spermatogenesis consists of mitotic proliferation of spermatogonia, meiosis of spermatocytes, haploid differentiation of spermatids, and genesis of male gametes. male germ cells have a complex transcriptome. phase-specific gene expression patterns are seen by the active transcription of the genome of male germ cells, however new rnas are not transcribed in haploid sperm. the rnas originated from the diploid germ stem cells are carried to the oocyte upon fertilization. sperm have protein-coding messenger rnas, and many noncoding rnas, including micrornas (mirnas). the mirnas regulate gene expression, mainly by repressing post-transcription or by inhibiting translation of their target messenger rnas. clinical studies have exploited the well-defined profiles of mirnas in sperm and seminal plasma, and human sperm or seminal plasma mirnas have been proposed as potential biomarkers for male factor infertility. we have explored the expression of mirnas in bull sperm or seminal plasma and correlated with their field fertility. remarkably, a few studies explored mirnome profiling in bull sperm and seminal plasma using deep sequencing and mapping technologies. in future studies, whole-mirnome profiling using highly sensitive and specific deep sequencing and mapping technologies should be used to examine bull sperm and seminal plasma. once novel and known mirnas are screened by deep sequencing technologies, functionality should be validated using realtime pcr technology. most of all, standardization of the nomenclature and abundancy specific to cattle is needed to include mirnome profile in bull fertility testing. contents micrornas (mirnas), a recently discovered class of non-coding rnas, function as epigenetic regulators of gene expression, either by degrading target mrna or inhibiting its translation. consequently, mirnas have critical roles in regulating development and function. in addition, expression of mirnas in tissues and biofluids are perturbed in developmental and functional aberrations, making them as promising molecular biomarkers for various cell types, tissues and biofluids. sperm have thousands of rnas, including messenger rna (mrna), micro rna (mirna), interference rna (rnai) and antisense rna (asrna). since sperm chromatin is condensed and compacted until fertilization, these sperm rnas are not transcribed from sperm nuclear dna, but they originate from diploid cells (spermatogonia and primary spermatocytes) and subsequently contribute to fertilization and embryo development.1-3 sperm-borne mirna-34c has been identified to be required for the first cell division in mouse zygote.4 the expression levels of mir-21, mir-34c, mir-140 and mir-375 were positively correlated with number of blastomeres and embryo quality.5 based on expression levels of the mir-34 family in bovine sperm, oocytes and embryos, these mirnas were implicated in transfer of epigenetic messages from gametes to offspring.6 in addition, mir-30d-5p, mir-320b, mir-10b-3p, mir1291, and mir-720 were predominant in follicular fluid in women undergoing in vitro fertilization, and expression levels of mir-202-5p, mir-206, mir-16-1-3p, and mir-1244 in follicular fluid varied significantly between follicles containing a fertile versus non-fertile oocyte.7 epigenetic signatures including mirnas in germ cells are vulnerable to the alterations in parental environment, and these epigenetic signatures can influence the embryo development and can mediate transgenerational programming.8,9 rna molecules even in transcriptionally inert mature sperm are of primary interest, as they play important roles in transgenerational programing and these rna population may be altered by environmental change, even via intercellular communication of epididymosomes. in dairy and beef cattle production, bull fertility is an important economic trait, although cow fertility is more often considered in genetic selection. male and female fertility have low heritability values; however, genetic and epigenetic factors from both gametes influence fertilization and embryo development. with increased use of artificial insemination and other assisted reproductive technologies in cattle breeding, semen quality and sire fertility are scrutinized continuously. decision to use or discard the clinical theriogenology • volume 10, number 3 • september 2018223 semen is often based on semen quality or field fertility. no single sperm bioassay is presently available to accurately predict bull fertility, however, many sperm assays have shown to be highly correlated with in vivo fertility. although various powerful tests including computer-assisted sperm analysis and various flow cytometric analyses have been developed, their accuracy and repeatability are still uncertain. sperm rna profiling is a potential tool to assess sperm fertility, as they regulate sperm functions, including motility in the female reproductive tract, capacitation, acrosome reaction, fertilization and postfertilization events. therefore, sperm-borne mirnas could serve as epigenetic regulators of genes required for fertilization and post-fertilization events. these sperm-specific mirnas can alter gene expression in a heritable manner, without transgenerational alterations of dna sequences, in addition to histone modification, dna methylation and acetylation.10 bull sperm have many mirnas; expression level varied between bulls differing in non-return rates, implying they may be a marker of bull fertility.11 several mirnas were differentially expressed between highand low-fertility holstein bulls; targeted gene expression analyses confirmed involvement of these differentially expressed mirnas in spermatogenesis and early embryonic development.12 in frozen-thawed sperm, 178 mirnas were investigated, of which seven (mir-502-5p, mir-1249, mir-320a, mir-34c-3p, mir-19b-3p, mir-27a-5p and mir-148b-3p) were differentially expressed between highand moderate-fertility holstein bulls.13 seminal plasma mirnas were stable, with 692 mirnas identified in human seminal plasma by solexa sequencing. in addition, 19 were altered in count between azoospermia and asthenozoospermia patients and a control group.14 two selected seminal plasma mirnas, mir-19b and let-7a, differed in expression level between idiopathic infertile males and fertile controls.15 thirty-seven mirnas were differentially expressed in seminal plasma of cancer patients compared to control, with upregulated mir142 and downregulated mir-34b validated using rt-qpcr.16 five mirnas (hsa-mir-34b, hsa-mir-34b, hsa-mir-34c-5p, hsa-mir-429, and hsa-mir-122) were variously expressed in seminal plasma of infertile patients and proposed as seminal plasma markers for infertile humans. we profiled 84 highly prioritized bovine mirnas in seminal plasma of highand low-fertility bulls and identified 56 mirnas differentially expressed (table; kasimanickam & kasimanickam, unpublished data). furthermore, in boar semen, there was an association of normalized threshold cycle values of sperm and seminal plasma micrornas (figure 1; kasimanickam & kasimanickam, unpublished data). summary in summary, numerous micrornas are present in sperm in humans and animals. these mirnas may originate from germ stem cells, are relatively inert in sperm, but carry messages to oocytes and can influence fertilization and postfertilization events without altering dna sequences. additionally, mirnas are abundant in seminal plasma. the mirnas in seminal plasma are stable and easily quantified. seminal plasma mirnas may originate from sperm or they may shuttle between sperm and seminal plasma to facilitate cell-to-cell communication. sperm and seminal plasma mirnas are differentially detected during developmental progression, whereas expression levels are altered in pathologic conditions. in most situations, expression levels of mirnas and their target genes are indirectly proportional. high‐throughput sequencing and mapping technology (figure 2) continue to identify known and novel mirnas in sperm and seminal plasma, although these technologies are mostly adopted for humans. prediction and pathway analysis identify target genes and clarify epigenetic function. notably, a few studies explored mirnome profiling in bull sperm and seminal plasma using deep sequencing and mapping technologies. in future studies, whole-mirnome profiling using highly sensitive and specific deep sequencing and mapping technologies should be used to examine bull sperm and seminal plasma. once novel and known mirnas are screened by deep sequencing technologies, functionality should be validated using realtime pcr technology. most of all, standardization of the nomenclature and abundancy specific to cattle is needed to include mirnome profile in bull fertility testing. to further explore their role in spermatogenesis, temporal profiling of mirnas in various cell types, spermatogonia, primary spermatocytes, secondary spermatocytes, and spermatids are essential. clinical theriogenology • volume 10, number 3 • september 2018 224 references 1. boerke a, dieleman sj, gadella bm: a possible role for sperm rna in early embryo development. theriogenology 2007;68:s147-155. 2. bourchis d, voinnet o: a small-rna perspective on gametogenesis, fertilization, and early zygotic development. science 2010;330:617-622. 3. dadoune jp: spermatozoal rnas: what about their functions? microsc res tech 2009;72:536-551. 4. liu wm, pang rt, chiu pc, et al: sperm-borne microrna-34c is required for the first cleavage division in mouse. proc natl acad sci usa 2012;109:490-494. 5. jiang w, liu n: correlation between the levels of mir-21, mir-34c, mir-140 and mir-375 in the sperm from in vitro fertilization patients and the embryo quality. zhong nan da xue xue bao yi xue ban 2015;40:864-871. 6. tscherner a, gilchrist g, smith n, et al: microrna-34 family expression in bovine gametes and preimplantation embryos. reprod biol endocrinol 2014;12:85. 7. machtinger r, rodosthenous rs, adir m: extracellular micrornas in follicular fluid and their potential association with oocyte fertilization and embryo quality: an exploratory study. j assist reprod genet 2017;34:525-533. 8. rodgers ab, morgan cp, bronson sl, et al: paternal stress exposure alters sperm microrna content and reprograms offspring hpa stress axis regulation. j neurosci 2013;33:9003-9012. 9. rodgers ab, morgan cp, leu na, et al: transgenerational epigenetic programming via sperm microrna recapitulates effects of paternal stress. proc natl acad sci usa 2015;112:13699-13704. 10. al-gazi mk, carroll m: sperm-specific micrornas their role and function. j hum genet clin embryol 2015;1:003. 11. robertson lr, feugang jm, rodriguez-osorio n, et al: microrna sequences of bull spermatozoa. reprod fertil dev 2008;21;147. 12. govindaraju a, uzun a, robertson l, et al: dynamics of micrornas in bull spermatozoa. reprod biol endocrinol 2012;10:82. 13. fagerlind m, stalhammar h, olsson b, et al: expression of mirnas in bull spermatozoa correlates with fertility rates. reprod domest anim 2015;50:587-594. 14. wang c, yang c, chen x, et al: altered profile of seminal plasma micrornas in the molecular diagnosis of male infertility. clin chem 2011;57:1722-1731. 15. wu w, hu z, qin y, et al: seminal plasma micrornas: potential biomarkers for spermatogenesis status. mhr basic sci reprod med 2012;18:489-497. 16. pelloni m, coltrinari g, paoli d, et al: differential expression of mirnas in the seminal plasma and serum of testicular cancer patients. endocrine 2017;57:518-527. table: relative expression of seminal plasma mirnas between highand low-fertility holstein bulls. layout 01 02 03 04 -05 06 07 08 09 10 11 12 a bta-let-7f -4.05 bta-mir101 -10.99 bta-mir103 -1.20 bta-mir125a -2.38 bta-mir125b -2.23 bta-mir126-3p 1.12 bta-mir128 -5.75 bta-mir145 5.07 bta-mir148a -5.65 bta-mir151-3p 1.09 bta-mir151-5p 5.91 bta-mir16b -32.60 b bta-mir181a 1.41 bta-mir18 -1.46 bta-mir18b 2.02 bta-mir199a-5p 20.11 bta-mir205 -1.81 bta-mir20a -1.09 bta-mir21-5p 3.84 bta-mir221 1.66 bta-mir222 1.06 bta-mir26a -3.71 bta-mir26b -3.21 bta-mir27a-3p -1.76 c bta-mir27b -1.91 bta-mir29a -9.26 bta-mir30b-5p -1.50 bta-mir30d 2.35 bta-mir31 8.37 bta-mir320a 5.09 bta-mir34b -8.60 bta-mir484 7.70 bta-mir499 1.14 bta-mir99a-5p -7.97 bta-mir5p -1.55 bta-let7d -1.37 d bta-let7g -1.87 bta-let-7i 1.08 bta-mir17-5p -1.01 bta-mir107 7.57 bta-mir10a -4.41 bta-mir10b -3.66 bta-mir122 1.26 bta-mir124b 2.41 bta-mir127 3.32 bta-mir132 2.65 bta-mir138 6.64 bta-mir139 6.55 e bta-mir140 3.33 bta-mir142-3p 3.15 bta-mir142-5p 13.45 bta-mir148b -1.07 bta-mir150 7.14 bta-mir15b -6.51 bta-mir17-3p 4.01 bta-mir17-5p 8.61 bta-mir181b 4.80 bta-mir181c 1.46 bta-mir186 -8.50 bta-mir191 -5.34 f bta-mir192 1.66 bta-mir193a-3p 12.83 bta-mir193a-5p 6.93 bta-mir1991-3p 9.03 bta-mir199b 6.80 bta-mir200a -11.03 bta-mir200b -2.59 bta-mir200c -1.57 bta-mir20b 17.78 bta-mir210 4.40 bta-mir21.3p 13.48 bta-mir214 23.68 g bta-mir215 7.20 bta-mir218 2.52 bta-mir22.5p 1.16 bta-mir23a -1.39 bta-mir23b-3p -1.48 bta-mir24-3p 2.35 bta-mir25 -2.15 bta-mir29b -2.66 bta-mir29c -14.58 bta-mir30a-5p 1.50 bta-mir30c -1.97 bta-mir30e-5p -4.09 positive numbers indicate upregulated mirnas and negative numbers indicate down regulated mirnas in high fertile bulls (scr +7; n=3) compare to low fertile bulls and high (scr -4; n=3) clinical theriogenology • volume 10, number 3 • september 2018225 figure 1. normalized threshold cycle values for sperm and seminal plasma mirnas for three high-fertile landrace boars. within a boar, normalized cycle values had similar trends for sperm and seminal plasma mirnas clinical theriogenology • volume 10, number 3 • september 2018 226 figure 2. deep sequencing and mapping analyses of rna from sperm or seminal plasma. mirna library prep. of sperm or seminal plasma high-throughput sequencer sequencing file: highly sensitive and specific mirna sequences image analysis & base calling 3' adaptor trimming & normalization map mrna unclassified mirdeep novel mirna map repeats unclassified mirdeep novel mirna map rfam unclassified mirdeep novel mirna map genome unclassified mirdeep 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice editorial editorial dear theriogenologists, you might have noticed that you have a new editor for our professional journal. four decades of learning and growing experiences in theriogenology have prepared me for this important assignment. have i arrived or perfected to know everything in theriogenology? no, and no one can; i am still learning. i am very thankful to the task force and the executive board of sft to have chosen me as the editor of our professional journal. i am equally thankful to dr. charles franz for his able leadership in the process of selecting a successor to the founding editor of clinical theriogenology, dr. bob youngquist. bob was a strong supporter during my purdue academic career. furthermore, i am grateful that i met all the seven ideal qualities that he wished to see in his successor. it is a privilege to follow his footsteps in editing. i am sure that we will recognize bob at an appropriate time for serving all of us very well. i am aware of the responsibilities of this position and looking forward to serve with assistance from the talented members of the editorial board, copy editor dr. john kastelic (who has been a source of tremendous wisdom and strength, right from my graduate program/residency days at uw madison), leadership and membership of sft/act, franz management, and omnipress. moving forward, at the ripe time we plan to resubmit a medline application for indexing. this task should only be attempted if our journal can address six important deficiencies noted by medline. the first five issues are major; they pertain to the quality of our journal and depth of scholarship. in this regard, we have decided to approach colleagues to create reviews in their areas of expertise, keeping a focus on applied aspects of theriogenology, the reason why this journal was created. please write to me (petera@purdue.edu), if you wish to contribute. we really need your support as we continue to build our journal! the final issue on the list is to have a few international members serving on the editorial board. i am reaching out to our international theriogenologists who have a track-record in scholarship. it is a threeyear term and the major responsibility is to review 2 or 3 papers a year. please let me know (petera@purdue.edu) if you have a desire to serve. thanks. augustine t. peter 395 clinical theriogenology • volume 10, number 4 • december 2018 introduction pregnancy loss in sheep and goats results in reproductive wastage, and thereby making it economically relevant. a wide range of causes, either infectious or noninfectious, resulted in pregnancy loss in small ruminants.1,2 some infectious causes of pregnancy loss in small ruminants are zoonotic diseases and may cause serious clinical disease and abortion in humans.3 important causes of infectious abortion in small ruminants include brucella melitensis, campylobacter spp., chlamydia abortus, coxiella burnetii, leptospira spp., listeria monocytogenes, salmonella spp., yersinia pseudotuberculosis, aspergillus spp., bluetongue virus (btv), border disease virus (bdv), cache valley virus (cvv), caprine herpes virus-1 (cphv-1), neospora caninum, and toxoplasma gondii.1,2 multiple pathogen infections can occur in some outbreaks of abortion in small ruminants.4 some infectious causes of abortion such as brucella melitensis, akabane disease virus, and rift valley fever virus; wesselsbron disease virus and schmallenberg virus are exotic and reportable in us. noninfectious causes of abortion in small ruminants are diagnosed less frequently and include toxic plants, toxins, nutritional deficiencies, and environmental factors.2,5,6 goats are more susceptible to pregnancy loss than sheep because goats require a functional corpus luteum throughout pregnancy. any cause, infectious or noninfectious that results in luteolysis in goats will terminate pregnancy. in sheep, the placenta is responsible for progesterone production after day pregnancy loss in small ruminants andrés de la concha-bermejillo,a juan romanob atexas a&m veterinary medical diagnostic laboratory blarge animal clinical sciences, college of veterinary medicine & biomedical sciences texas a&m university, college station, tx abstract pregnancy loss in sheep and goats is an important cause of economic loss to livestock producers. causes of pregnancy loss are noninfectious and infectious. noninfectious causes include toxic plants and toxins, nutritional deficiencies, medications, and environmental factors. infectious causes are more commonly diagnosed than noninfectious causes and include bacterial, mycotic, viral, and parasitic infections. several of the infectious causes of pregnancy loss in small ruminants are transmissible to humans, either directly or indirectly, and pose substantial threats to public health. identification of the cause of pregnancy loss is of prime importance to implement control programs to reduce economic impact and the risk of human infection. veterinary diagnostic laboratories help in identifying, preventing, and controlling pregnancy loss. additionally, laboratory testing is an important part of any surveillance program, in identifying transboundary animal diseases and in testing livestock and their specimens for export purposes. keywords: cattle, abortion diseases, pregnancy loss, embryonic mortality 55 of pregnancy and ewes can maintain pregnancy after this period in the absence of a functional corpus luteum.7 although sporadic abortions may occur in any flock or herd, often epizootic outbreaks occur with > 50% pregnancy loss. when several abortions occur in close succession, further investigations must be carried out to determine the cause so that control measures can be implemented. diagnosis of abortion in small ruminants is difficult, because it is common for fetuses to die in utero and to be expelled after several days. these fetuses exhibit advanced autolysis that hampers the interpretation of postmortem findings. fetal membranes are often soiled with dirt and hay and contaminated with saprophytic bacteria that can interfere in culturing pathogens responsible for abortion.2 the objective is to review common causes of abortion in small ruminants, with an emphasis on infectious causes and human health implications, and to outline an approach for investigation of abortion outbreaks, describing available tests for diagnosis. further information on abortion causes in ruminants is reviewed in the proceedings.8,9 prevention of human infection several infectious causes of abortion in small ruminants are zoonotic diseases. prevention of human infection should be an important goal in the control of these infections. zoonotic clinical theriogenology 2021; 13: 194 diseases are transmitted to humans through direct contact with infected animals or fomites, by inhalation of airborne microorganisms, and by ingestion of farm animal products.3 most common route of transmission from small ruminants to humans is via consumption of raw milk or fresh cheese from sheep and goats. only pasteurized milk should be consumed by humans. all milk sold across state lines or internationally must be pasteurized and meet the standards of us pasteurized milk ordinance.10 owners, farm workers, and veterinarians are often exposed to sheep and goat zoonotic diseases during lambing, kidding or by handling aborted fetuses and fetal membranes of infected animals. general precautions that should be taken during lambing/kidding or when handling aborted fetuses include the use of personal protective equipment (ppe). basic ppe comprises disposable rubber gloves, safety googles, coveralls and rubber boots. although the use of n95 masks is highly desirable, it is not always possible. to be effective, ppe should have the level of protection needed for the infectious organism involved and should be adequately fitted for the person using it.11 all dirty clothes should be left at the farm for disinfection and washing or placed in biohazard bags and washed separately. all contaminated trash and rubber boots should be disinfected and trash disposed of properly. samples sent to the laboratory should be packed and labelled appropriately.8 pregnant women, small children, and immunosuppressed individuals should refrain from handling infected animals or assisting in lambing and kidding.12 infectious causes of pregnancy loss in small ruminants brucellosis brucella melitensis is the main cause of abortion in goats and sheep in many parts of the world where it is also an important cause of human disease. b. melitensis infection has been eradicated from the us and currently is a reportable disease.13 infection with b. abortus occurs occasionally in small ruminants. brucella ovis is a less pathogenic species of brucella and more commonly associated with epididymitis and infertility in rams, but occasionally it causes abortion, stillbirth, and increased perinatal mortality.14 in contrast to other brucella species, b. ovis lacks zoonotic potential.15 brucella melitensis is the most virulent member of the brucella species and accounts for most cases of human brucellosis. transmission of b. melitensis infection from animal to human may occur through direct contact, inhalation of airborne microorganisms, and ingestion of farm animal products. consumption of raw milk or fresh cheese made with unpasteurized milk from an infected sheep or goat is the main way of transmission from animals to humans.16 human brucellosis (‘undulant fever’) is characterized by a nonspecific clinical syndrome with relapsing fever and arthritis and in some cases hepatomegaly, splenomegaly, endocarditis, and abortion.17 abortion due to brucella melitensis in small ruminants commonly occurs during the last 2 months of pregnancy. the organism has high affinity for trophoblast cells and causes placentitis.18 diagnosis of brucellosis is the center of any control program and is based on bacterial culture and serological testing. the polymerase chain reaction (pcr) methods provide additional means of detection.19 campylobacteriosis campylobacter spp. are important animal pathogens and opportunistic human pathogens. several species and subspecies of campylobacter cause pregnancy loss and infertility in ruminants. the mammal-associated campylobacter (c.) fetus is comprised of 2 subspecies: c. fetus subsp. venerealis and c. fetus subsp. fetus, with both being well-known causes of reproductive failures in ruminants.20 c. fetus subsp. venerealis causes bovine genital campylobacteriosis, characterized by infertility and abortion. c. fetus subsp. fetus is most frequently isolated from abortions in sheep. since the 1990s, c. jejuni has been associated with ovine abortion in the us.21 in sheep, the reproductive disease associated with c. fetus subsp. fetus and c. jejuni is commonly known as epizootic abortion.22 these bacteria are transmitted orally via fecal contamination or from infected fetal membranes or fluids. abortion generally occurs in the last trimester and abortion storms occur in naïve flocks.23 cases of campylobacteriosis and abortion occur in pregnant women during the second and third trimesters of pregnancy.24 in pregnant ewes, infection results in placentitis and fetal infection; abortion typically occurs in the last trimester of pregnancy or there is birth of weak lambs. fetal membranes lesions may be subtle. in some cases, fetal membranes appear edematous, thickened, and leathery. cotyledons may be covered with brown to red exudate. the fetus is often autolyzed and has red-tinged fluid in body cavities. in some aborted fetuses, peritonitis is present, characterized by a thin layer of fibrin covering liver capsule with strings of fibrin in the peritoneum. in ~ 25% of aborted fetuses, there are white, often circular areas of necrosis in the liver ranging from 1 to 5 cm. inflammation of fetal membranes, pneumonia, serositis, hepatitis, and encephalitis are common microscopic lesions. culture of the organism from fetal membranes and fetal stomach content is the preferred diagnostic method.18 in the us, a vaccine from inactivated cultures of campylobacter fetus and campylobacter jejuni is available for sheep that requires a first dose before breeding and a booster in 60 to 90 days. annual revaccination is recommended. antibiotic treatment is used to clear the infection in carrier animals.13,23 chlamydiosis chlamydia abortus (ch. abortus), formerly known as chlamydophila abortus or chlamydia psittaci serovar 1 is an obligate intracellular organism that causes abortions in sheep and goats, and clinical theriogenology 2021; 13: 195 occasionally in deer, cattle and llamas. in sheep, the disease is often referred as enzootic abortion of ewes. it is the most commonly diagnosed infectious cause of lamb loss in many countries world-wide. ch abortus can be a cause of abortion in humans.25 transmission to naïve ewes and goats and to susceptible humans is via contact with tissues and fluids of aborted fetuses, vaginal discharges, and fetal membranes of aborted ewes.26 between > 30 60% of pregnant susceptible animals may abort when the organism is first introduced to a naïve flock or herd. once the infection becomes endemic, the abortion rate decreases and animals that abort are generally newly introduced naïve and young animals. when infection occurs early in pregnancy, sheep and goats may abort late in that pregnancy, but when infection occurs late in pregnancy, infected animals usually abort in the following pregnancy. lambs and kids infected congenitally may abort during their first pregnancy.27 infected ewes and does may give birth to weak lambs or kids that fail to survive beyond 24 hours. some clinically healthy lambs and kids born from infected ewes/does can be a source of infection for naïve animals and represent a substantial zoonotic risk. the risk of transmission of infection from vaginal discharges of infected ewes/does is substantially reduced or virtually eliminated after several days, once vaginal discharges have ceased.26 in advanced stages of ch. abortus fetal infection, there is severe diffuse inflammation of fetal membranes, characterized by cotyledons that are dark red to brown and intercotyledonary areas that are thickened with a leathery appearance. microscopically, there was loss of trophoblast and suppurative inflammation throughout the hilar zone of placentomes, and vasculitis.4 basophilic microorganisms that stain magenta with gimenez stain are present in trophoblast cells. focal areas of coagulative necrosis can be observed in the liver and spleen of some aborted fetuses.7,28 subclinical infection with ch. abortus may substantially affect bovine herd health and production.29 in some countries, a commercial live, attenuated ch. abortus 1b vaccine strain is used for the prevention of chlamydiosis in sheep. recently, it became apparent that the unattenuated vaccine strain caused placentitis indistinguishable from placentitis caused by ch. abortus wild-type.30 if chlamydia is suspected to be present in a flock/herd, treatment with a long-acting oxytetracycline preparation (20 mg/ kg) will reduce the severity of infection and losses resulting from abortion. the treatment has to start after 95 100 day of pregnancy, the point at which pathologic changes start to occur. further doses can be subsequently given at 2-week intervals until the time of lambing. although such treatment reduces losses and limits the shedding of infectious organisms, it does not eliminate the infection nor reverse any pathologic damage already done to the placenta, thus abortions or the delivery of stillborn or weakly lambs can still occur, and the shed organisms are a source of infection for other naıve animals.31 moreover, antibiotic treatment has not been critically evaluated and this approach has the potential to create microbial resistance. serological tests including enzyme–linked immunosorbent assay (elisa) and complement fixation to detect the presence of anti-ch. abortus antibodies are able to actually prove the presence of an immunological response and thus an infection. complement fixation titers of 1:16 1:32 are considered positive for most laboratories. the titer may increase to > 1:80 in 2 3 weeks after abortion. currently, the best diagnosis of test in aborted fetuses is real time polymerase chain reaction (rtpcr) of fetal membranes and fetal tissues.25 in the us, an inactivated vaccine of ch. abortus, abortigenic serovar is available for sheep that required the first dose 60 days prior to breeding and a booster 30 days later. annual revaccination is recommended. in europe, some live attenuated vaccines were associated with abortion in sheep (type 1b). use of live attenuated vaccines presents some drawbacks: require adequate handling, possible transmission to humans, cannot be given to pregnant females and to animals receiving antibiotics and vaccinated animals cannot be commingled with unvaccinated animals.30 coxiellosis coxiellosis, also known as q (query) fever, is a highly infectious zoonotic disease caused by the intracellular bacterium coxiella burnetii that primarily affects goats, sheep, and less frequently cattle. other species less commonly affected include dogs, cats, rabbits, a variety of wild and domestic mammals, and birds. in the us, c. burnetii is classified as a category b potential aerosolized biological weapon.12 transmission is mainly by ingestion or inhalation of contaminated fetal membranes or uterine discharges, or by inhalation of contaminated dust from barns.32,33 ewes and does can shed the bacteria in milk. c. burnetii infections in sheep and goats are characterized by late term abortions, stillbirths, and weak neonates that often die during the first few days after birth. in naïve herds, > 50 70% of susceptible ewes or goats may abort. in subsequent years, once infection becomes endemic in the herd, the abortion rate may be < 5%. abortions tend to occur primarily in younger naive animals. other than losing pregnancy, ewes and goats that abort do not have any clinical signs. the most common lesion is inflammation and thickening of fetal membranes and leathery appearance with large quantities of white–yellow, creamy or reddish-brown exudate at the edges of the cotyledons and in the intercotyledonary areas. histologically, large amounts of necrotic debris are present on the chorionic surface, with severe necrosis of the chorionic epithelium. large numbers of microorganisms may be present in the cytoplasm of chorionic epithelial cells, but these microorganisms need to be differentiated from other microorganism that cause clinical theriogenology 2021; 13: 196 placentitis in small ruminants, including brucella and campylobacter species. the stroma of the chorioallantois is infiltrated with large numbers of mononuclear leukocytes and some neutrophils.12,33 bacterial identity in fetal membranes can be confirmed by immunohistochemistry. isolation of the microorganism in cell culture, embryonated chicken eggs and laboratory animals is considered dangerous and is rarely used. currently, the preferred diagnostic method is amplification of c. burnetii dna by rtpcr. several serologic tests including indirect immunofluorescence, elisa and complement fixation test are used for the detection of c. burnetii antibodies in maternal serum. c. burnetii igm and igg phase ii specific antibodies can be detected 2 weeks postinfection and remain increased for up to 13 weeks. antibodies directed against c. burnetii phase i increase generally 4 weeks later compared to phase ii antibodies. serum antibodies in infected animals can be detected for months to years.12,33 leptospirosis leptospirosis is a worldwide zoonosis caused by pathogenic spirochetes that belong to the genus leptospira. a large amount of scientific information exists on the disease in cattle, but less information exists about leptospirosis in sheep and even less in goats.34 relative to cattle, sheep and goats have been considered resistant to leptospiral infection.35 animal infection most frequently occurs through direct contact with leptospira-contaminated urine or indirectly from interaction with contaminated water and/or soil.36 leptospiral infection in sheep and goats occurs in many countries. affected sheep and goats may act as carriers of the organism. leptospira infection in small ruminants is often subclinical; however, younger animals may have fever, dyspnea, depression, anorexia, jaundice, and may eventually die. in a study in spain to determine causes of pregnancy loss in small ruminants, 1.7 and 2.6% of abortions were caused by leptospira spp. in sheep and goats, respectively, with the majority of these abortions caused by serovar pomona.37 laboratory tests are essential to achieve an accurate diagnosis of the infection. microscopic agglutination test is used to determine the presence of antibodies in serum of infected animals and determine flock/herd status. in aborted fetuses, rtpcr is the recommended test for confirmation of leptospira spp. as cause of abortion.38 listeriosis listeria monocytogenes, serovars 4b and 5 (l. ivanovii), is the etiological agent of listeriosis in small ruminants. l. monocytogenes is a ubiquitous facultative saprotroph present in soil, plants, ground water, and vegetation.39 silage is a common source of infection for ruminants. the organism is shed in the feces of healthy carriers and sick animals. listeria monocytogenes is a severe human foodborne infection characterized by gastroenteritis, meningitis, encephalitis, abortions, and perinatal infections.40 l. monocytogenes infections in small ruminants can result in 3 separate clinical syndromes, including encephalitis in adult animals, abortions, and neonatal septicemia. abortions and neonatal septicemia can occur concurrently with the encephalitic syndrome in the same flock or herd, but more commonly, they occur as separate entities. abortions in cattle, sheep and goats usually occur in the last trimester of pregnancy and up to 50% of the susceptible pregnant animals may abort in an outbreak.18,41 aborted fetuses are retained in utero for several days before being expelled and may have evidence of autolysis. retention of the fetal membranes and their inflammation are common. dystocia, metritis and neonatal septicemia occurs in some cases. grossly aborted fetuses may have multifocal areas of necrosis ranging from 1 to 3 mm in the liver and less often in the lungs, heart, kidney, spleen, and brain. fetal membranes exhibit extensive thickening and yellow discoloration of intercotyledonary areas and cotyledons.18 bacterial isolation from fetal membranes, fetal stomach content or fetal tissues is the diagnostic method of choice. better results are obtained with the cold enrichment procedure.42 detection and genetic characterization of l. monocytogenes dna is by rtpcr of fetal brain and other fetal tissues.43 salmonellosis salmonellosis is an uncommon disease in sheep, but severe outbreaks may result in heavy animal losses. sheep can be infected with several species of salmonella of the family enterobacteriaceae. ovine salmonellosis may be an important zoonotic reservoir for human infection.44 profuse diarrhea is common and pregnant ewes may abort. salmonella abortus-ovis is a bacterial pathogen that can cause abortions and stillbirths in pregnant ewes, and mortality in neonates.45 abortion storms occur in naïve flocks when this organism is first introduced, but once the organism becomes endemic in the flock, ewes generally develop protective immunity, and abortions are sporadic or limited to younger or newly introduced animals. stress including transportation, comingling, drastic changes in weather, and other factors often precipitate abortion outbreaks in infected flocks. some ewes that abort may not have other clinical signs of disease apart from abortion; however, some may have fever, anorexia, diarrhea, and may die. other salmonella serovars including s. dublin, s. typhimurium, and s. montevideo also can cause abortion in pregnant ewes, with or without systemic illness in the ewe. clinical theriogenology 2021; 13: 197 retention of fetal membranes is common. inflammation of fetal membranes (grossly indistinguishable from inflammation caused by other bacteria) can be apparent. the skin/ wool of aborted fetuses is often stained with meconium. microscopically, evidence of meconium aspiration in the lung is noticed. salmonella spp. can be isolated from the liver, spleen or the gastrointestinal tract of aborted fetuses, fetal membranes, and from vaginal secretions of affected ewes.18 the pcr allows the detection of s. abortusovis dna up to 3 months after infection in samples that were negative by culture.46 yersiniosis yersinia pseudotuberculosis, the cause of yersiniosis is a gram-negative aerobic or facultative anaerobic rod-shaped bacterium of the family enterobacteriaceae that infects small ruminants, cattle, humans, and several animal species.47 clinical disease in 1to 6-month-old goat kids can be manifested as sudden death or diarrhea. the disease occurs more frequently in winter and spring and appears to be predisposed by stressful situations including drastic changes in weather, transportation, comingling, and starvation. in pregnant goats, infection might result in abortion and the birth of weak kids. the infection has also been associated with mastitis in goats and cows. transmission occurs mainly through the fecal-oral route, after ingestion of contaminated feed or water.48 fetal infection is characterized by abortion. fetal membranes are thickened and appear white with opaque white foci on cotyledons. histologically, suppurative inflammation of fetal membranes and suppurative pneumonia of fetuses. the organism can be isolated from fetal membranes, stomach content, liver and lung. in case of an abortion storm, treatment of goats with tetracycline has been useful. other broad-spectrum antibiotics may also be useful. animals receiving antibiotic treatment must undergo the determined milk and meat withdrawal period prior to milk and meat entering the human food supply.49 bluetongue bluetongue (bt) is an insect-transmitted, noncontagious disease of ruminants caused by bluetongue virus (btv), the prototype virus of the genus orbivirus in the family reoviridae. btv exists throughout much of the world between latitudes of ~ 40 50 degrees n and 35 degrees s.50-52 bt clinical disease in north america occurs most commonly in sheep and deer, and sporadically in cattle and goats. it has been described in south american camelids.53 abortion has been reported in dogs vaccinated with a commercial multivalent modified live vaccine against canine distemper virus, and adenovirus and parvovirus contaminated with btv-11.54 currently, there are 28 btv serotypes recognized worldwide. in the us, serotypes 10, 11, 13, and 17 are endemic. btv-2 is isolated sporadically in the us. nonendemic serotypes ((1, 3, 5, 6, 9, 12, 14, 19, 22, and 24) normally present in mexico and central america may sporadically appear in the us.53 insects of the genus culicoides (biting midges) serve as true biologic vectors for the transmission of btv infection between susceptible ruminants. in the us, the main vector of btv is culicoides sonorensis (previously known as c. varipennis). after infection, sheep can remain viremic for up to 54 days and cattle for 60 100 days. long term infection with btv-25 has been reported in healthy goats in switzerland.55 transmission can occur transplacentally or by semen.52 some of the new btv serotypes (btv-25, btv-26, btv-27) are thought to also be transmitted horizontally without vector involvement.56 from 5 to 50 -75% of susceptible animals may have clinical signs when btv is first introduced into a naïve population; however, once the infection becomes endemic, the morbidity and mortality may be as low as 1 2%. clinical disease in sheep is characterized by fever, depression, salivation, facial swelling, panting, nasal discharge, hyperemia of the muzzle, lips, ears, oral ulcerations, and coronitis.53 btv vertical transmission from dam to fetus in enzootic areas is considered negligible. in these areas, btv-induced pregnancy loss and congenital malformations have been associated with btv strains from modified live virus vaccine strains. infection of cattle, sheep, and goats with the european btv-8 wild-type strain demonstrated a high incidence of transplacental transmission and pregnancy loss in natural circumstances.57,58 transplacental transmission of wild-type btv-8 is approximately 10 41.7% in cows and up to 69% in sheep.59 infection of pregnant cows with wild-type btv-8 can cause abortion, stillbirth, and fetal malformations (hydranencephaly).60 goats are less susceptible to btv-8 infection than sheep and cattle.61 modified live virus (mlv) vaccines are used in california to protect sheep against infection with btv serotypes 10, 11, and 17 and an mlv vaccine to btv serotype 10 is licensed for use in sheep throughout the us. adverse effects of btv mlv vaccines are fetal malformations and recombination of btv vaccine strains with field strains giving rise to new strains of virus.53 serologic diagnosis of btv relies upon the detection of btv antibodies by competitive elisa, virus neutralization, agar gel immunodiffusion, and complement fixation tests. btv can be isolated from heparinized blood, semen or tissues (spleen, lymph nodes, and lung) of infected animals by inoculation of susceptible sheep or embryonated chicken eggs. subsequent adaptation to cell culture and serotyping of the virus may be necessary. assays based on rtpcr are used to detect btv rna in clinical samples (e.g. blood or spleen).62 in the us, a nationally licensed commercial modified live vaccine is commercially available for sheep and goats to prevent type 10 bluetongue virus infection. lambs are vaccinated > 3 months of age and ewes are vaccinated 3 weeks prior to breeding or after lambing. additional vaccines in ca to types 10, 11, and clinical theriogenology 2021; 13: 198 17 for sheep and types 10,11, 13, and 17 for wildlife are available through a state license. other states use inactivated btv provisionally to control outbreaks. border disease border disease is caused by bdv that belongs to the genus pestivirus in the family flaviviridae, together with bovine viral diarrhea virus types1 (bvdv-1) and 2 (bvdv-2) and classical swine fever virus (csfv).63 it is known that many pestiviruses exhibit a broad species tropism.64 small ruminants can abort with bovine viral diarrhea virus (bvdv).65 sheep persistently infected with bdv readily transmit the virus to calves seronegative to bvd virus.66 several subgenotypes of bdv have been described to date. bdv infections occur mainly in sheep and goats but also in cattle, pigs, and wild even-toed ungulates.63 infection of pregnant ewes and goats with bdv during the first trimester of pregnancy often results in fetal death and resorption. infection during the second and last trimester of pregnancy causes fetal malformation, fetal mummification, abortion, stillbirth, and birth of weak or normal lambs.18 some of the lambs infected congenitally had low birth weight, abnormalities in wool pigmentation and severe neurological signs including ataxia or rhythmic contractions of the hind limbs, ears, tail, and head (hairy shakers).2 examination of the brain reveals cerebellar hypoplasia, porencephaly, hydranencephaly, cerebellar dysplasia, nonsuppurative meningoencephalomyelitis with hypomyelinogenesis, and arthrogryposis.67,68 diagnosis of pregnancy loss by bdv in sheep and goats is complex. virus isolation, detection of bdv antibodies in fetal serum or fluids from cavities and amplification of bdv nucleic acid by rtpcr should be performed concurrently for accurate diagnosis.18 cache valley virus cache valley is an arthropod-borne viral infection, afflicting a variety of domestic and wild ruminants including sheep, goats, deer, cattle, caribou and also pigs, horses, raccoons, woodchuck, turtles, rabbits, foxes, and humans, caused by cvv, a bunyavirus in the family bunyaviridae, bunyamwera group.69,70 other north american bunyaviruses including lacrosse virus, san angelo virus, and main drain virus and other exotic bunyaviruses such as akabane, schmallenberg, and rift valley fever viruses cause similar congenital malformations in sheep and goats.2,71 cvv is endemic throughout the us, canada, mexico and some countries of central america.69 transmission of cvv to ruminants occurs through bites of infected mosquitos, including several species of aedes, psorophora, anopheles, coquillettidia, culex, and from culiseta inornata. majority of infections in adult ruminants are subclinical, but cvv infection in pregnant ewes and goats and less often in other ruminants may result in embryonic mortality, fetal mummification, stillbirth, and various degrees of musculoskeletal or central nervous system abnormalities including arthrogryposis, hydranencephaly, hydrocephalous, microcephalous, porencephalia, cerebellar hypoplasia, scoliosis, torticollis, and lordosis.70 cvv-experimentally induced fetal malformation in sheep occurs when the virus infects the fetus between days 27 and 54 of pregnancy. after the second month of pregnancy, the ovine fetus becomes immunocompetent and develops a cellular and humoral immune response that controls and clears cvv without apparent consequences to the fetus.72 dystocia may be a sequela in ewes that deliver full-term malformed offspring. ewes that are cvv seropositive at breeding are resistant to reinfection with cvv, but not against other related bunyaviruses.72,73 because fetuses infected in second and third trimesters develop an immune response that clears cvv, demonstration of cvv-neutralizing antibodies in the serum or thoracic or abdominal fluid of malformed fetuses is the best method to establish diagnosis. virus isolation from full-term aborted fetuses with cvv-induced malformations is unsuccessful. presence of cvv antibodies in maternal serum is an indication of exposure but not a proof that fetal malformation was caused by cvv. lack of antibodies in maternal serum rules out cvv as the cause of fetal malformation. presence of serum cvv antibodies before breeding in ewes and goats is protective of transplacental fetal infection with cvv, but not against other related bunyaviruses.69,70 some diagnostic laboratories offer a gel-based pcr for the demonstration of cvv-rna in tissues of aborted fetuses. cross contamination in gel-based pcr platforms is common and its use is discouraged. an rtpcr assay has been developed for the detection of california (cal) serogroup viruses and cvv, for use in human surveillance, but is not routinely used for cvv in small ruminants.74 until, rtpcr methodology is validated in small ruminants, the detection of cvv-rna in late term malformed ruminant fetuses, the results of gel-based pcr are difficult to interpret. breeding ewes outside of the mosquito season may help reduce cvv fetal infections. however, short-term changes in weather patterns during a particular season may result in renewed vector activity and increased risk of fetal infection.69 caprine herpesvirus-1 caprine herpesvirus 1 (cphv-1) belongs to the subfamily of alphaherpesviruses that contains seven genetically-related viruses. cphv-1 is closely related to bovine herpesvirus 1 (bohv-1), responsible for infectious bovine rhinotracheitis (ibr).75 cphv-1 infection of goats can cause vulvovaginitis and neonatal systemic infection. cphv-1infection of pregnant goats may result in abortion without other clinical signs of infection.76 clinical theriogenology 2021; 13: 199 microscopic lesions in aborted fetuses consists of multifocal areas of necrosis with minimal cellular infiltration in liver, necrotic bronchiolo-alveolitis and necrosis in other organs including thymus, spleen, kidneys, intestine, and adrenal gland. characteristic herpesvirus eosinophilic intranuclear inclusions are difficult to find, but can be observed more frequently in cells surrounding the areas of necrosis especially in adrenal glands. necrotizing vasculitis may be present in fetal membranes’ villi.18,77,78 diagnosis can be established by observation of characteristic microscopic lesions in conjunction with virus isolation and amplification of cphv-1dna by rtpcr.77 neosporosis neosporosis is caused by neospora caninum, an apiclomplexa parasite closely related to t. gondii. neospora was initially misidentified as toxoplasma, but was subsequently differentiated based on host preferences, etiology, morphological, and genetic differences.79 dogs and other canids (coyotes, wolves, other) are definitive host.80 cattle, sheep, goats, white-tailed deer, other ruminants, and horses are intermediate hosts.81 neosporosis is a major cause of abortion in cattle, sheep and goats. infection in small ruminants occurs either by ingesting sporulated oocysts shed by dogs and other canids in feces that contaminate food and water, or vertically from infected ewes and goats to the fetus after recrudescence of a chronic infection during pregnancy. transplacental infection may result in abortion, fetal mummification, birth of a congenitally-infected offspring with clinical neuromuscular signs, or a live and healthy progeny. fetal membranes may have areas of necrosis and mineralization, but the intercotyledonary areas are unremarkable. multifocal areas of necrosis may be apparent in the brain and liver of few of the n. caninum aborted fetuses.18,82 the clinical outcome of n. caninum in pregnant sheep and goats is dependent to some extent on the stage of pregnancy when animals are infected.83 diagnosis in aborted fetuses is established by demonstration of characteristic microscopic lesions in the tissues of aborted fetuses, together with demonstration of the organism by immunohistochemistry or by amplification of n. caninum dna by rtpcr in brain, spinal cord, or other tissue of aborted fetuses.84 detection of antibodies to n. caninum is by elisa or immunofluorescent antibody tests.85 toxoplasmosis toxoplasmosis is caused by toxoplasma gondii, a coccidia – intracellular protozoan parasite of the phylum apicomplexa. the parasite is present virtually in all warm-blooded animals including sheep, goats, and in nearly one-third of humans. goats are very susceptible to infection. cats (felidae) are the only known definitive hosts and thus, the main reservoirs of infection and main source of infection for small ruminants. unsporulated oocysts are shed in the cat’s feces. small ruminants are intermediate hosts and contract the infection by eating contaminated grass and/or animal feed with feces of infected felines. small ruminants do not shed oocysts in feces. ewes and does that abort cannot infect ‘clean ewes’ or does in the flock or herd, unless naïve pregnant ewes and does ingest fetal membranes and fetal fluids of infected animals. cats become infected after consuming intermediate hosts harboring tissue cysts. cats may also become infected directly by ingestion of sporulated oocysts.86 in humans, t. gondii is most frequently transmitted horizontally following ingestion of environmentally resistant oocysts excreted by cats, or via ingestion of undercooked meat containing persistent asexual stages (bradyzoites) residing in intermediate hosts’ tissues.87 t. gondii infection in pregnant sheep and goats may cause early embryonic death, fetal mummification, abortions, stillbirth, and the birth of weak kids or lambs.88 t. gondii-induced abortion can occur in ewes and goats of all ages, although maiden ewes and goats are most affected. most ewes and does are asymptomatic before and after abortion. when a flock or herd of naïve pregnant ewes or does are first infected with t. gondii, the infection results in an epidemic outbreak with high abortion rate. the pregnancy loss is less drastic in subsequent lambing/kidding seasons. sheep and goats that abort as a result of t. gondii infection develop immunity and are more resistant to abort a second time, but subsequent pregnancies may result in the birth of weak offspring and shedding of the organism in fetal membranes, fetal fluids, and milk.82,88 histologic examination of fetal tissues and fetal membranes, detection of t. gondii in tissues by immunohistochemistry, amplification of t. gondii dna by rtpcr, and serological examination of the fetus and the dam for the presence of antibodies are all useful in diagnosis of toxoplasmosis. t. gondii is ubiquitous in the environment. presence of antibodies in maternal serum is an indication of exposure and not a confirmation of the cause of abortion.88,89 prevention of toxoplasmosis involves keeping cats away from feed, feed troughs, and pens with pregnant ewes or goats. maintaining clean bedding and clean pen areas is important. grain and feed should be stored in covered containers. barn cats may play an important role in rodent control, but the population, particularly of kittens must be kept under control. kittens younger than 6 months are more likely to be carriers of t. gondii than adult cats. delivery/nursing of kittens by queens must not be permitted in pens with pregnant ewes/goats and feed barns. all adult cats should be neutered or spayed. cats should not be allowed to consume fetal membranes or fetuses and should not be fed raw meat.90 there is no approved treatment for toxoplasmosis in small ruminants; however, feeding the coccidiostat decoquinate during pregnancy may reduce lamb losses.91 a live vaccine for the protection of sheep against abortion due to t. gondii is used in some countries, but the vaccine is not licensed in the us.92 the vaccine available commercially contains live attenuated clinical theriogenology 2021; 13: 200 toxoplasma (strain s48) for the prevention of abortion in sheep. the vaccine provided life-long protection; however, its downsides are potential risk to people that handle the vaccine, expensive, short shelf-life and could revert to a pathogenic strain.92 noninfectious causes of pregnancy loss veratrum californicum veratrum californicum is a plant that grows primarily in the high mountain ranges of the western us. veratrum californicum fed to ewes ~ at days 12 14 of pregnancy can cause congenital cyclopia and other defects of the cranium and brain in lambs, in addition to prolonged pregnancy. ingestion of the plant between days 19 21 of pregnancy causes embryonic death. maternal exposure between days 24 30 results in fetal cleft palate. metacarpal, metatarsal, and tracheal cartilage defects result from maternal exposure during days 27 36 of pregnancy.93 astragalus pubentissimus sheep and cattle grazed on astragalus pubentissimus (locoweed) plant early in pregnancy may abort or give birth to congenitally deformed offspring including arthrogryposis, aplasia of the lower jaw, hypermobility of the hock and stifle joints, and contracted tendons, especially the ankles.94 dexamethasone ewes experimentally treated with 0.25 mg/kg body weight on days (1, 3, and 5) during first trimester, days (51, 53, and 55) during second trimester, and days (101, 103, and 105) during the third trimester had decreased plasma progesterone concentrations and aborted. abortion appeared to be a consequence of the adverse effects of decreased placental progesterone concentrations.95 prostaglandin f2α goats depend on progesterone from the cl throughout pregnancy. exogenous treatment or endogenous release of pgf2α at any time during pregnancy will result in pregnancy loss in goats. two doses of 5 mg pgf2α given intramuscularly 24 hours apart at ~ 3 months of pregnancy in does results in abortion and 70% of does retained fetal membranes.96 pregnancies in ewes carrying a single embryo can be terminated by 1 dose of 100 µg of cloprostenol given ~ day 21 of pregnancy, but 2 injections given about 7 days apart are necessary when multiple embryos are present. however, the ewe placenta takes over progesterone production at ~ day 55 of pregnancy and ewes will not abort when exogenous pgf2α is given or endogenous release after 60 70 days of pregnancy.97 benzimidazoles benzimidazole compounds have teratogenic effects in domestic and experimental animals. giving ewes netobimin, a benzimidazole compound, on day 17 of pregnancy resulted in fetal skeletal and congenital renal malformations.98 albendazole, a broad-spectrum anthelmintic, should not be given to female cattle during first 45 days of pregnancy or to ewes or does during the first 30 days of pregnancy. vitamin e and selenium deficiency congenital nutritional muscular dystrophy caused by vitamin e and selenium deficiency is uncommon but has been reported in sheep and cattle.99 affected lambs may have bilateral symmetrical chalky-white streaks in skeletal muscles and in the heart. histologically, necrosis, degeneration, and calcification of myofibers are observed. congenital goiter calves, lambs, and kids with goiter are born partially or completely hairless and are either born dead or die soon after birth.100-102 congenital hypothyroidism can be the result of iodine deficiency or excess, due to goitrogenic compounds that interfere with thyroid hormone synthesis, or due to hereditary defects in thyroid hormone synthesis. common goitrogenic compounds include thiouracil, sulfonamide, and plants of the brassica group. affected and aborted full-term fetuses/newborns exhibit severe, bilateral thyroid enlargements, alopecia, myxedema, swelling of the tongue and laryngeal edema, the latter causing asphyxia.18 body condition score reducing body condition (bcs) score in the last 45 60 days prior to lambing or kidding increase the risk of pregnancy loss and/or pregnancy toxemia. the most efficient and accurate method to assess energy adequacy in ewes and goats is to perform and record the bcs by using an objective 15 scoring system, with 1 being extremely thin and 5 being extremely obese.103 the bcs is determined by palpating the amount of fat and muscle covering on the spinous processes and transverse processes in the lumbar region. most healthy productive ewes and goats will have a score of 2.5 3.5. ewes or does with a score of 1 2 should be examined and fed to attain a higher score, whereas those with a score > 3.5 should be fed less. dietary changes should be carried out slowly and abrupt reduction in total energy intake should always be avoided. it is convenient to assess the bcs at prebreeding, at breeding, and 3 months postbreeding. at prebreeding, animals < 2.5 are fed more, whereas those > 3.5 are fed less to arrive a score of 2.5 3 at breeding. changes in feed management are avoided during the first month of pregnancy. at 3 months of pregnancy, evaluation is required to arrive at an ideal score of 3.5 prior lambing/kidding. the primary predisposing cause of pregnancy toxemia is inadequate nutrition during late pregnancy. animals may not be fed according to the number of fetuses or because of insufficient energy density of the ration and decreased rumen-reticular capacity due to clinical theriogenology 2021; 13: 201 fetal growth. fetal growth is 70% in the last 6 weeks prior to delivery; therefore, the requirements need to be satisfied accordingly. moreover, the increase in the number of fetuses will affect the energy needs of the dam. females with a poor bcs (≤ 2) or that are over conditioned (≥ 4) and carrying more than 1 fetus are at highest risk of developing pregnancy loss and toxemia, although the condition can occur even in ideally conditioned ewes on an adequate ration. gastrointestinal parasites many species of nematodes and cestodes cause parasitic gastritis and enteritis in sheep and goats. the most important of these are haemonchus contortus, teladorsagia (ostertagia) circumcincta, trichostrongylus axei, intestinal species of trichostrongylus, nematodirus spp, bunostomum trigonocephalum, and oesophagostomum columbianum. cooperia curticei, strongyloides papillosus, trichuris ovis, and chabertia ovina also may be pathogenic in sheep. even subclinical infections depresses appetite, impairs gastric digestion, and reduces use of metabolizable energy and protein with the consequent loss in bcs. clinical signs associated with gastrointestinal parasitism are shared by many diseases and conditions; however, a presumptive diagnosis based on signs, grazing history, and season is often justified. infection usually can be confirmed by demonstrating nematode eggs or tapeworm segments in fecal examination. in small ruminants during late pregnancy and early lactation, the presence of gastrointestinal parasites appears to be more deleterious due to the higher energy requirements with reduced capacity of the digestive system. presence of parasites with inadequate feeding program in pregnant females create ideal conditions to begin an outbreak of pregnancy loss. ‘diagnostic attitude’ for a pregnancy loss case an adequate history is paramount for the diagnostic investigation of pregnancy loss, specifically the following information: the total population at risk, number of females that have pregnancy loss, category of females, age, stage of pregnancy, fresh or decomposed fetus, movements of animals (male and female) within the property or outside the property and time, purchase of new animals, change in source of water, food (quality and quantity), vaccination (type and time), deworming (product and time), and medications. to optimize reaching a diagnosis, correct sampling from more than 1 abortion is required. fresh material is crucial and the following material is required: fetus, fetal membranes, and maternal blood. contaminants, such as feces, urine, or dirt material should be removed gently from the specimens under biosecurity protocols without washing the sample with water or with antiseptics. to avoid mistakes, contact diagnostic laboratory about submitting samples, samples required and preservation, and shipping method. managing the pregnancy loss in the flock/herd females with pregnancy loss are kept away from healthy pregnant females until a diagnosis is determined. reduce or avoid any movement of animals. measure of biosecurity and biocontainment need to be in place immediately. pregnancy loss is considered infectious until it is proven to be noninfectious. therefore, a good clinical history, assessment, and submission of adequate samples to the laboratory are paramount. every person involved with animals needs to wear gloves, boots, protecting clothing, and masks. it is important to have the order of work from healthy to sick, from young to old, and from clean to dirty for cleaning or feed management purposes. no child, pregnant women, elderly, or immunocompromised person should assist. problem females should not go for auction or to another flock/herd as breeding animal until the cause is determined. conclusion it is obvious that pregnancy loss in small ruminants is an important cause of economic losses for producers. additionally, some infectious causes of pregnancy loss in small ruminants are transmitted to humans and pose an important human health risk. rapid and accurate identification of the cause of pregnancy loss is of prime importance. the diagnosis of small ruminant pregnancy loss involves an investigative effort among the owner, the veterinary practitioner, and the veterinary diagnostic laboratory. for an accurate diagnosis, a battery of diagnostic tests is conducted in parallel 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embryos on luteolysis and termination of early pregnancy in sheep with cloprostenol. biol reprod 1982;26:263-269. https://doi.org/10.1095/biolreprod26.2.263. 98. navarro m, cristofol c, carretero a, et al: anthelmintic induced congenital malformations in sheep embryos using netobimin. vet rec 1998;142:86-90. https://doi.org/10.1136/vr.142.4.86. 99. nisbet di, renwick cc: congenital myopathy in lambs. j comp pathol therapeut 1961;71:177-in15. https://doi.org/10.1016/ s0368-1742(61)80022-2. 100. falconer i: studies of the congenitally goitrous sheep. the iodinated compounds of serum, and circulating thyroid-stimulating hormone. biochem j 1966;100:190-196. https://doi.org/10.1042/bj1000190. 101. cheema a, shakoor a, shahzad a: congenital goitre in goats. pakistan vet j 2010;30:58-60. 102. singh ras: diseases of thyroid in animals and their management. in: payan carreira r: editor. insights from veterinary medicine, intech; 2013. https://doi.org/10.5772/55377. 103. kenyon pr, maloney sk, blache d: review of sheep body condition score in relation to production characteristics, new zeal j agric res 2014;57:38-64, doi: 10.1080/00288233.2013.857698 clinical theriogenology 2021; 13: 205 pregnancy loss in small ruminants 2018: effects of coenzyme q10 (coq10) on equine semen quality after cryopreservation effects of coenzyme q10 (coq10) on equine semen quality after cryopreservation a. ruiz, s. waqas, w. bayly, a. tibary comparative theriogenology, department of veterinary clinical sciences, college of veterinary medicine, center for reproductive biology, washington state university, pullman, wa mammalian spermatozoa are highly susceptible to free radicals because of a high concentration of polyunsaturated fatty acids in their membranes and a lack of cytoplasmic antioxidants. coenzyme q10 (coq10) is a powerful antioxidant when reduced (c0q10-ubiquinol), preventing lipid peroxidation in sperm membranes. coq10 concentrates in sperm mitochondria and has been shown to improve sperm motility and fertility after oral supplementation in infertile men. addition of coq10 to semen extender prior to cooling and cryopreservation has also been shown to improve equine sperm motility parameters. the aim of the present work was to determine if post-thaw incubation with coq10 has any effect on equine sperm quality. frozen semen from 6 stallions of various breeds and fertility was used. semen was cryopreserved using a standard protocol: semen was collected, extended to 50 million/ml spermatozoa in inra 96®, centrifuged (600 g for 10 minutes) and the supernatant discarded. sperm aliquots were suspended to 200 x106 sperm/ml in e-z freezing “le”® extender, loaded into 0.5 ml straws and cryopreserved 4 cm above liquid nitrogen for 20 minutes before being plunged into liquid nitrogen. the experiment was conducted in 5 replicates with two straws per stallion per replicate. semen was thawed at 37°c for 30 seconds, split into 3 aliquots and incubated at 37°c with no coq10 addition (control group), with extender containing coq10 (sigma-aldrich company, st. louis, mo) at 40 µg/ml (treatment 1) or coq10 at 80 µg/ml (treatment 2). post-thaw sperm total motility (tm) and progressive motility (pm) were assessed using casa (spermvision®, mofa, verona, wi), viability (v) was assessed using sybr-pi stain and plasma membrane functionality (pmf) using host, at 0, 1 h, and 2 h following incubation. data were analyzed by rm anova with coq10 treatment as main factor using sas. significance was set at p<0.05. results are expressed as average for all stallions. sperm quality decreased significantly during incubation in all samples. there was no significant effect of treatment on any of the sperm parameters studied (table). in conclusion, post-thaw addition of coq10 to the extender did not have any effect on motility, viability and plasma membrane integrity of equine sperm. it is possible that coq10 was not incorporated in sperm in this form. future studies will focus on the nutritional coq10 supplementation and its effect on semen quality parameters. table: effects of treatment and time on semen quality parameters a,b,c different superscript letters within the same column indicate significant differences, p< 0.05. keywords: equine sperm, frozen semen, antioxidants, coenzyme q10 time treatment tm pm v pmf 0h control 32.26 +/1.25a 20.45 +/0.85a 29.64 +/1.55a 16.93 +/1.09abc treatment 1 31.49 +/1.70ab 19.50 +/1.10a 27.21 +/1.22ab 15.86 +/1.04abc treatment 2 30.89 +/1.95ab 19.14 +/1.09ab 27.44 +/1.23ab 17.96 +/1.18ab 1h control 28.37 +/1.45abc 15.97 +/1.19bc 27.05 +/1.66ab 17.40 +/0.63a treatment 1 27.18 +/1.59bcd 15.32 +/1.17cd 26.11 +/1.21ab 16.16 +/0.91abc treatment 2 28.83 +/1.86abcd 15.48 +/1.07c 26.18 +/1.32ab 16.26 +/0.94abc 2h control 25.06 +/1.41cde 13.23 +/1.04cde 25.74 +/1.22ab 15 +/0.93bc treatment 1 24 +/1.45de 12.30 +/0.96de 25.46 +/1.53ab 14.53 +/0.96c treatment 2 22.52 +/1.30e 11.47 +/0.78e 25.21 +/1.39b 14.66 +/1.14bc clinical theriogenology • volume 10, number 3 • september 2018311 cervix and myometrium: role in preterm and normal term birth in mare cervix and myometrium: role in preterm and normal term birth in mare hossam el-sheikh ali and barry ball gluck equine research center, department of veterinary science university of kentucky, lexington, ky abstract myometrial activation and cervical remodeling during term and preterm labor involve complex molecular mechanisms. progress in elucidating these mechanisms has been slow and based on classical molecular techniques such as realtime quantitative reverse transcription polymerase chain reaction. recently, using ribonucleic acid sequencing, we have elucidated key regulators and molecular mechanisms triggering these events. objective is to summarize changes in myometrial and cervical transcriptome during placentitis and normal prepartum period in comparison to normal pregnancy. keywords: mare, cervix, myometrium, pregnancy, progestins, parturition, placentitis introduction placentitis induced preterm labor and term labor share a common pathway that includes 3 major events: myometrial activation, cervical remodeling and chorioallantois activation (separation and rupture).1-4 understanding molecular mechanisms of these 3 events holds potential for development of new diagnostic tools and therapies to forestall placentitis induced preterm labor. recently, using a transcriptomic approach (i.e. ribonucleic acid sequencing), we elucidated key regulators and molecular mechanisms, triggering these events in an experimental model of acute ascending placentitis. objective is to summarize our recent findings, with a focus on mechanisms underlying myometrial activation and cervical remodeling during equine placentitis and normal prepartum period in comparison to normal pregnancy. myometrial activation key event in placentitis induced preterm labor and term labor is myometrial activation with subsequent initiation of labor.1,5 myometrial activation involves complex myriad of coordinated changes involving immune and hormonal factors, upregulation of several contraction-associated proteins (caps), and arrangement of cytoskeletal machinery that provides uterus capacity to generate force and contract1,5 mechanisms underlying myometrial activation during equine placentitis and normal prepartum period are summarized (figures 1 and 2). progestins and progesterone receptor signaling (ligand receptor signaling) in mammals studied,6-8 progestins, acting through its nuclear receptor (pr, also known as pgr), plays a central role in maintaining myometrial quiescence during pregnancy through blockage of inflammatory cascade and suppression of caps. recently, we reported that myometrial tissue concentrations of 5α dihydroprogesterone (5α dhp, also known as dhp), allopregnanolone (3α dhp) and 20α hydroxy 5α dihydroprogesterone (20α dhp) were lower (progestin withdrawal) in mares with experimentally induced acute placentitis compared to ageand pregnancy-matched controls.1 this local reduction in 5α dhp and its downstream metabolites is attributed to a decline in enzymes responsible for synthesis of these progestins, such as 5α reductase type 1 (5α r1 also known as srd5a1) and aldo-keto reductase family 1 member c23 (akr1c23, also known as akr1c1).1 moreover, expression of nuclear pr is downregulated (functional progestin withdrawal) in equine myometrium during placentitis. a closer look at pr-isoforms proteins (i.e. pr-a and pr-b) revealed a decrease in pr-b to pr-a ratio.1 it is worth noting that in primates, progesterone (p4) promotes myometrial quiescence through pr-b-mediated antiinflammatory actions.9 at labor, pr-a becomes more predominant and inhibits antiinflammatory actions of pr-b and stimulates proinflammatory gene expression.9 downregulation in progestin-pr signaling is in turn associated with activation of nf-κb pathway and upregulation of proinflammatory cytokines (e.g. il1β), as well as upregulation clinical theriogenology • volume 12 number 3 • september 2020340 of transcripts coding for caps (e.g. ptgs2 and gja4).1 together, these findings suggest that placentitis induces localized progestin withdrawal and progestin functional withdrawal in myometrium that lead to myometrial activation through activation of inflammatory cascade and upregulation of caps. in prepartum myometrial transcriptome, although we did not identify any significant changes in geneses coding for srd5a1, akr1c1 and pr, we identified upregulation of aldehyde dehydrogenases (aldh1a1, aldh1a2, and aldh1a3). it is worth noting that aldh1 family is involved in conversion of retinaldehyde to retinoic acid, which in turn decreases pr transcription.10 therefore, upregulation of aldhs during prepartum period might contribute to myometrial preparation for labor. figure 1. progestin-progesterone receptor signaling during equine placentitis. a) pr-b/ pr-a ratio in myometrium during pregnancy and placentitis. b) molecular mechanisms underlying progestin withdrawal and progestin functional withdrawal in equine myometrium during placentitis. activation of inflammatory cascade in myometrium during inflammatory reaction in pathogenic infection, inflammatory cascade is initiated by immune recognition of pathogen mediated through toll-like receptors (tlrs), which are primary and earliest detection mechanisms for pathogens.11 among known tlrs, tlr2 and tlr4 are responsible for recognition of gram positive and negative bacteria, respectively.12 recently, we identified streptococcus induced placentitis to be associated with upregulation of tlr2 in myometrial samples retrieved from placentitis group in comparison to control group.2 moreover, this upregulation is associated with upregulation of a wide array of tlr2-dependent downstream molecules in inflammatory cascade.2 these findings highlight central role of tlr2 in triggering inflammatory signals in myometrium during streptococcus induced placentitis. therefore, targeting tlr2 through therapeutic inhibition (antagonism) might be beneficial for prevention and/or treatment of streptococcus induced placentitis. this notion is supported by reports in primates that treatment of amniotic infection using tlr antagonists (tlra) resulted in a downregulation of proinflammatory cytokines with subsequent delay or prevention of preterm birth.13 in prepartum myometrium, we identified upregulation of several inflammation related genes (e.g. ↓srd5a1 ↓akr1c23 ↓pr ↓3α-dhp ↓20α-dhp ↓5α-dhpp4 progestins withdrawal progestin functional withdrawal downregulation in progestin signaling myometrial activation myometrium (acute placentitis) ↑ pro-inflammatory cytokines↑ contraction-associated proteins placentitispregnancy myometrial activation myometrial quiescence ↑pr-b/pr-a ↓pr-b/pr-a a) b) clinical theriogenology • volume 12 number 3 • september 2020 341 tnfaip6, icam1, socs3, cxcr4).2 upregulation of these genes might reflect presence of sterile inflammatory signaling in myometrium during prepartum stage in mare. myometrial infiltration with leukocytes in women, myometrial infiltration with leukocytes is hallmark of switching myometrium from a quiescent to a contractile state during term and preterm labor.14-16 similarly, we reported that equine placentitis is associated with increased myometrial infiltration with leukocytes.1,2 moreover, we elucidated chemokine signaling mechanisms implicated in upregulation of several chemotactic factors including; c-c motif chemokine ligand (ccl2, 4, and 8), c-x-c motif chemokine ligand (cxcl1, 2, 3, 6, 8, and 9) and calgranulins (s100a8 and s100a9).1,2 in equine prepartum myometrium (330 days ga), although we identified upregulation of ccl2, cxcl1, cxcl3, and cxcl6, we did not observe marked leukocytic infiltration in myometrium.2 this might reflect an early chemotaxis event taking place in prepartum myometrium in preparation for labor. myometrial apoptosis during placentitis, equine myometrium is associated with a significant upregulation of apoptosis related transcripts, including: caspases (casp3, casp4, casp7), activating transcription factor 3 (atf3), fas cell surface death receptor (fas), fos proto-oncogene subunit (fos), activator protein 1 (ap-1), and baculoviral iap repeat containing 3 (birc3).2 myometrial apoptosis occurred during chorioamnionitis in women17,18 and infection-induced labor in mice.19 additionally, apoptosis by itself might be a key event in switching myometrial cells from quiescent to contractile status.17,18,20 uterine contraction associated genes placentitis induced myometrial inflammation is associated with upregulation of several contraction-associated transcripts, including prostaglandin endoperoxide synthase 2 (ptgs2, also cyclooxygenase 2; cox2), prostaglandin e receptor 3 (ptger3), gap junction alpha 4 (gja4, also known as connexin-37; cxn37 or cx37), matrix metallopeptidases (mmp1 and mmp8) plus downregulation relaxin (rln).2 role of these genes in myometrial activation during term and preterm labor is well established in women and mice.21 for example, ptgs2 is essential for synthesis of prostaglandin f2α (pgf2α), which is a potent uterotonic (ecbolic).21 contrarily, gjas are believed to play a critical role in preterm and term labor by forming gap junctions in myometrium, which increase myometrial cell coupling with subsequent generation of synchronous myometrial contractions.21-24 cervical remodeling cervical remodeling is transformation of cervix from a rigid, tightly closed structure into a flaccid and open one to permit fetal delivery during term and preterm labor.25-27 cervical remodeling consists of 4 overlapping phases (i.e. softening, ripening, dilation, and postpartum repair).25-27 cervical remodeling requires decreases in cervical collagen concentrations (i.e. extracellular matrix (ecm) degradation) and wide dispersing of collagen fibers through increasing cervical water content (i.e. cervical hydration), with subsequent decrease of cervical tensile strength to allow cervical dilation.28 mechanisms underlying cervical remodeling events during equine placentitis and normal prepartum period are summarized below. extracellular matrix (ecm) degradation during placentitis, we reported upregulation of several proteases, including mmps (e.g. mmp1, 8, 13 and 14).29 these mmps are believed implicated in cervical collagen degradation during equine placentitis.29 contrarily, cervix from prepartum mares (330 d ga) was not associated with significant change in mmps expression. clinical theriogenology • volume 12 number 3 • september 2020342 figure 2. inflammatory events and associated molecular mechanisms in equine myometrium during placentitis (a) and prepatum period (b). cervical hydration along with ecm degradation, cervical hydration is another important component in cervical remodeling in which water content increases in cervical tissue, reducing collagen density.29 cervical hydration could be achieved through various mechanisms, e.g. upregulation of aquaporins (aqps) water channels and hydrophilic proteoglycans, as well as increased vascular permeability.25-27,29 for instance, cervical remodeling during placentitis is associated with upregulation of aqp9 (water transporter), aggrecan (acan; a hydrophilic proteoglycan), plus vascular permeability-related genes such as rac family small gtpase (rac) and nitric oxide synthase (enos, also known as nos3).29 altogether, these findings highlight possible molecules implicated in cervical ecm degradation and cervical hydration during placentitis. conclusion this review provides a brief overview of key regulators and molecular mechanisms underlying myometrial activation and cervical remodeling during placentitis and prepartum period. strategies to block identified key regulators and associated pathways (e.g. using tlras) hold potential for therapies to forestall placentitis-induced preterm birth. acknowledgement supported by albert g. clay endowment of university of kentucky. ascending placentitis (streptococcus equi subspecies  zooepidemicus) ↑ chemokines (↑ccl2, ↑cxcl2, ↑cxcl6,  ↑s100a8, ↑s100a9,…) ↑ leukocytes infiltration & inflammatory cascade ↑ cytokines & inflammatory nediators (↑il1β, ↑il1α, ↑il8,  ↑tnfaib6, ↑icam1) toll-like receptors activation ↑tlr2 prostaglandin synthase and receptors (↑ptgs2, ↑ptger3) prepartum period ↑ chemokines (↑ccl2, ↑cxcl1, ↑cxcl3,  ↑cxcl6) ↑ chemoattractants for leukocytes infiltration & inflammation ↑ inflammatory mediators (↑tnfaib6, ↑icam1) transcription factors ↑stat3, ↑egr1, ↑f2r oxidative stress (↑dusp1, ↑dusp5, ↑dusp6,  ↑myc) a) b) transcription factors ↑stat1, ↑nfkb1a, ↑junb,  ↑e2f1, ↑foxm1, ↑hif1a  myometrial activation preparation for myometrial activation ? clinical theriogenology • volume 12 number 3 • september 2020 343 conflict of interest none to report. abbreviations 20αdhp; 20α-hydroxy-5α dihydroprogesterone 3αdhp; allopregnanolone 5α-dhp; 5α-dihydroprogesterone akr1c23; aldo-keto reductase family 1 member c23 aldh1; aldehyde dehydrogenases ap-1; activator protein 1 aqps; aquaporins atf3; activating transcription factor 3 birc3; baculoviral iap repeat containing 3 caps; contractions-associated proteins casp; caspase ccl; c-c motif chemokine ligand cxcl; c-x-c motif chemokine ligand cxcr4; c-x-c motif chemokine receptor 4 dusp; dual specificity phosphatase ecm; extracellular matrix enos/nos3; nitric oxide synthase fas; fas cell surface death receptor gja4/cxn37; gap junction alpha 4/connexin-37 icam1; intercellular adhesion molecule 1 il; interleukin mmp; matrix metallopeptidases myc; myc proto-oncogene, bhlh transcription factor nf-κb; nuclear factor-κb p4; progesterone pgf2α; prostaglandin f2α pr/pgr; progesterone receptor ptger3; prostaglandin e receptor 3 ptgs2/cox2; prostaglandin-endoperoxide synthase 2/cyclooxygenase 2 rac; rac family small gtpase rln; relaxin s100a; calgranulins srd5a1; 5α reductase type 1 tlr; toll-like receptors tlra; tlr antagonists tlrs; toll-like receptors tnfaip6; tnf alpha induced protein 6 references 1. el-sheikh ali h, legacki el, loux sc, et al: equine placentitis is associated with a downregulation in myometrial progestin signaling. biol reprod 2019;101:162-176. 2. el-sheikh ali h, legacki el, loux sc, et al: transcriptomic analysis reveals the key regulators and molecular mechanisms underlying myometrial activation during equine placentitis. biol reprod 2020; in press. 3. el-sheikh ali h, legacki el, scoggin ke, et al: steroid synthesis and metabolism in the equine placenta during placentitis. reproduction 2020;159:289-302. 4. fernandes cb, ball ba, loux sc, et al: uterine cervix as a fundamental part of the pathogenesis of pregnancy loss associated with ascending placentitis in mares. theriogenology 2019;45:167-175. 5. lyle sk: immunology of infective preterm delivery in the mare. equine vet j 2014;46:661-668. 6. wu s-p, demayo fj: progesterone receptor signaling in uterine myometrial physiology and preterm birth. in: forrest d, tsai s: editors. current topics in developmental biology. cambridge; academic press, 2017: p. 171-190. 7. merlino aa, welsh tn, tan h, et al: nuclear progesterone receptors in the human pregnancy myometrium: evidence that parturition involves functional progesterone withdrawal mediated by increased expression of progesterone receptor-a. j clin endocrinol metab 2007;92:1927-1933. 8. mesiano s, wang y, norwitz er: progesterone receptors in the human pregnancy uterus: do they hold the key to birth timing? reprod sci 2011;18:6-19. clinical theriogenology • volume 12 number 3 • september 2020344 9. tan h, yi l, rote ns, et al: progesterone receptor-a and-b have opposite effects on proinflammatory gene expression in human myometrial cells: implications for progesterone actions in human pregnancy and parturition. j clin endocrinol metab 2012;97:e719-e730. 10. chwalisz k, fahrenholz f, hackenberg m, et al: the progesterone antagonist onapristone increases the effectiveness of oxytocin to produce delivery without changing the myometrial oxytocin receptor concentrations. am j obstet gynecol.1991;165:1760-1770. 11. medzhitov r: toll-like receptors and innate immunity. nat rev immunol 2001;1:135-145. 12. akira s, takeda k: toll-like receptor signalling. nat rev immunol 2004;4:499. 13. adams waldorf km, persing d, novy mj, et al: pretreatment with toll-like receptor 4 antagonist inhibits lipopolysaccharide-induced preterm uterine contractility, cytokines, and prostaglandins in rhesus monkeys. reprod sci 2008;15:121-127. 14. thomson aj, telfer jf, young a, et al: leukocytes infiltrate the myometrium during human parturition: further evidence that labour is an inflammatory process. hum reprod 1999;14:229-236. 15. shynlova o, nedd-roderique t, li y, et al: myometrial immune cells contribute to term parturition, preterm labour and post-partum involution in mice. j cell mol med 2013;17:90-102. 16. mackler am, green lm, mcmillan pj, et al: distribution and activation of uterine mononuclear phagocytes in peripartum endometrium and myometrium of the mouse. biol reprod 2000;62:1193-1200. 17. lirussi f, rakotoniaina z, madani s, et al: adrb3 adrenergic receptor is a key regulator of human myometrial apoptosis and inflammation during chorioamnionitis. biol reprod 2008;78:497-505. 18. leroy m-j, dallot e, czerkiewicz i, et al: inflammation of choriodecidua induces tumor necrosis factor alpha-mediated apoptosis of human myometrial cells1. biol reprod 2007;76:769-776. 19. muhle ra, pavlidis p, grundy wn, et al: a high-throughput study of gene expression in preterm labor with a subtractive microarray approach. am j obstet gynecol 2001;185:716-724. 20. shynlova o, oldenhof a, dorogin a, et al: myometrial apoptosis: activation of the caspase cascade in the pregnant rat myometrium at midgestation. biol reprod 2006;74:839-849. 21. arthur p, taggart mj, zielnik b, et al: relationship between gene expression and function of uterotonic systems in the rat during gestation, uterine activation and both term and preterm labour. j physiol 2008;586:6063-6076. 22. sakai n, tabb t, garfield re: modulation of cell-to-cell coupling between myometrial cells of the human uterus during pregnancy. am j obstet gynec 1992;167:472-480. 23. balducci j, risek b, gilula nb, et al: gap junction formation in human myometrium: a key to preterm labor? am j obstet gynec 1993;168:1609-1615. 24. cook jl, zaragoza db, sung dh, et al: expression of myometrial activation and stimulation genes in a mouse model of preterm labor: myometrial activation, stimulation, and preterm labor. endocrinology 2000;141:1718-1728. 25. mahendroo m: cervical remodeling in term and preterm birth: insights from an animal model. reproduction 2012;143:429-438. 26. liggins g: the cervix in pregnancy and labor, clinical and biochemical investigation. edinburgh. cuurchill livingrtone.1981:1-9. 27. leppert pc: anatomy and physiology of cervical ripening. clin obstet gynecol 1995;38:267-279. 28. read cp, word ra, ruscheinsky ma, et al: cervical remodeling during pregnancy and parturition: molecular characterization of the softening phase in mice. reproduction 2007;134:327-340. 29. el-sheikh ali h, legacki el, loux sc, et al: transcriptome analysis reveals the key regulators and molecular mechanisms underlying equine cervix remodeling during placentitis and prepartum period. proc annu conf plant anim genome 2019; po0311. clinical theriogenology • volume 12 number 3 • september 2020 345 024_ms-024 el-sheikh 2018: diagnostic approaches to pregnancy loss diagnostic approaches to pregnancy loss frances o. smith smith veterinary hospital, inc., burnsville, mn abstract pregnancy loss can occur from events closely associated with conception up to just prior to parturition. death of the embryo or fetus can be the result of hormonal, toxic, environmental and/or bacterial or viral agents. in many species of animals embryonic losses are higher than perinatal losses. in the dog, pregnancy diagnosis occurs midway through the gestation period further complicating the diagnosis of embryonic losses. the bitch is luteal dependent throughout pregnancy so luteolytic events can results in fetal loss or abortion. death of the fetus can result in mummification, maceration or abortion. viruses are a common cause of mummification in dogs and cats. aborted fetuses often are autolyzed due to death 24-28 hours prior to delivery. history including housing, nutrition, supplements, vaccination status, exercise, illness and medications including topical and parasite control products should be obtained. keywords: pregnancy loss, infectious agents, progesterone, environment bacterial agents brucellosis brucella canis is a small, gram-negative intracellular coccobacilli. it is the most significant cause of pregnancy loss and infertility in the canine species and is an under diagnosed and under reported cause of pregnancy failure in the canine. clients perceive that a canine cannot have brucella canis if it sexually naïve. brucella infection is spread both orally and venereally through mucosal contact with infected material.1 vaginal discharge, semen and fluid and tissues from abortion contain the highest number of organism but urine, blood, milk, saliva and feces also contain organisms. the bacteria can survive in both chilled and frozen semen. infection can cause infertility, early embryonic death, fetal resorption and late term abortion. infected bitches often do not display any signs of illness. abortion often occurs after day to day 50 and is accompanied by voluminous vaginal discharge. brucella canis has zoonotic potential and has been diagnosed in laboratory workers and a young child exposed to an infected puppy. all breeding animals should be screened for brucella canis prior to breeding. screening tests include the tube agglutination test, indirect fluorescent antibody, rapid slide immunodiffusion, and agar gel immunodiffusion using cell wall antigen (agidcwa). definite diagnosis involves blood culture, agar gel immunodiffusion using cytoplasmic protein antigens (agidcpa).vaginal swab polymerase chain reaction (pcr) is a good choice for brucellosis diagnosis in bitches suspected to be infected.2 incidence of brucella canis has increased in the united states as a result of imported dogs and the united states interstate dog trade. salmonella and escherichia coli salmonella is a gram negative bacillus that is transmitted through the gastrointestinal tract via contaminated feed or water. in the united states salmonella has been identified as a pathogen in dog kennels feeding a raw diet based on chicken. salmonella has zoonotic potential particularly in the very young, the very old and the immunocompromised and can result in severed clinical disease in humans. infection with salmonella can results in abortion, stillbirth, and poor puppy survival. definite diagnosis of salmonella associated abortion requires culture of fetal tissues and membranes. several cases have been associated with the feeding of raw diets including a case of feline stillbirth with salmonella thyphimurium. escherichia coli while commonly identified in case of metritis, mastitis and pyometra has been identified in cases of abortion. escherichia coli is the most common bacteria isolated from the canine vagina. escherichia coli produces an endotoxin that has been shown to be an abortifacient in other species. clinical theriogenology • volume 10, number 3 • september 2018185 campylobacter species campylobacter are gram negative curved rods. while they have been isolated from the feces of normal dogs they have been associated with abortion in dogs. campylobacter may require special identification techniques including culture of the organism from fetal or neonatal tissues including stomach or stomach contents as well as from the placenta and vaginal fluid from the bitch. special incubation techniques are necessary to culture this organism so refer to laboratory guidelines for proper sample collection and sample handling. campylobacter infection can be zoonotic. infections can be animals to human or vice versa. other bacteria associated with canine abortion beta hemolytic streptococci have been isolated from a group of bitches with a history of abortion, infertility and neonatal death. this bacterium has also been isolated from the vagina of normal bitches and queens. group g streptococcus canis is a zoonotic pathogen that has been reported to cause reproductive disease and abortion in dogs as well as toxic shock and neonatal sepsis in cats. leptospirosis associated infertility and abortion in dogs is most commonly associated with serovar bratislava. dogs are considered a maintenance host for serovars icterohaemorrhagiae, canicola and grippothyphosa. dogs are incidental hosts for infection with autumnalis, australis, tarassovi, ballum, bataviae and bratislava. infection in an incidental host is typified by low susceptibility, severe pathogenic effects and poor transmission. leptospires are often transmitted through urine or contaminated water. infection occurs when the organism penetrates mucous membranes or abraded skin. mycoplasma and ureaplasma species are often implicated by breeders as cause of infertility resorption, stillborn puppies and neonatal death. these gram negative cell wall free bacteria are among the many bacteria part of normal vaginal flora in the bitch and have been cultured from the vagina from over 60% of bitches. breeders often request enrofloxacin as a preemptory attempt to solve their perceived mycoplasma induced reproductive issues. arbitrary use of antibiotics is not warranted and contributes to multi-drug resistance. protozoa there is experimental evidence that toxoplasma gondii can infect puppies in utero. there have not been reports of naturally occurring toxoplasma infections causing abortion in the dog. neosporum caninum is a widespread protozoan parasite that has been shown to be transplacentally transmitted. seroprevalence of neosporum caninum in dogs appears to be relatively high but it is unknown if the organism is an important cause of canine abortion. raw diets can increase the risk of infection. viral causes of pregnancy loss in the canine canine minute virus also known as canine parvovirus 1 (cpv1) has been proposed as a cause of early pregnancy loss.3 the more commonly seen canine parvovirus (cpv2) has not been associated with reproductive issues in the bitch. canine distemper virus and canine adenovirus can both cause spontaneous abortion in the bitch. appropriate vaccination can prevent infection with either of the viruses. herpes virus of canines is a controversial cause of reproductive failure in the dog. infection in healthy adult dogs is often unrecognized. infection of a naïve bitch during pregnancy or a resurgence of the virus in a bitch may be associated with early embryonic loss, resorption, abortion, stillbirth or most importantly infection of the neonates during passage through the vagina. puppies infected at birth often succumb to fulminant infections. the herpes virus is heat labile so clinical infections in the neonate may be lessened by providing a well heated environment as the virus cannot replicate at normal body temperature. canine herpes virus has been demonstrated in populations of dogs throughout the world. serologic testing has limited value as antibody to the virus is only detectable for about 100 days. a canine herpes virus vaccine is available in some countries but is not presently available in the united states. clinical theriogenology • volume 10, number 3 • september 2018 186 luteal insufficiency the bitch is dependent upon the production of progesterone in the corpus luteum throughout pregnancy. plasma progesterone levels of 2-3ng/ml are required to maintain pregnancy. progesterone levels below 2-3ng/ ml for 2448 hours routinely result in loss of pregnancy. it is difficult to determine if the progesterone levels drop due to loss of the pregnancy itself due to other causes or if the loss of pregnancy is due to the low progesterone. definite proof that luteal insufficiency is a cause of pregnancy loss has not been defined. there are limited data that prove that declining progesterone levels over multiple days is the cause of repeated pregnancy loss. in two bitches examined at this author’s clinic there were two cycles each where the bitch suffered apparent luteal in sufficiency that were successfully supported by supplementation with altrenogest at 0.088mg/kg beginning when progesterone levels decreased to approximately 5ng/ml and discontinued several days prior to expected whelping date. other authors have recommended the use of progesterone in oil or micronized progesterone orally. use of these medications can result in masculinization of female fetuses and an increase in cryptorchid male puppies. if the hypoluteoidism is associated with shortened interestrous intervals, a genetic basis for the issue may be suspected. daughters of bitches with luteal insufficiency have been observed to also exhibit luteal insufficiency. the placental hormone relaxin rapidly decreases after death of all the fetuses in a litter so monitoring relaxin levels can aid in monitoring placental health.4 uterine disease there are numerous publications implicating uterine pathology as a contributing factor to infertility and total or partial pregnancy loss in the bitch. in a study by fernando et al a group of seven bitches with historic pregnancy loss had uterine biopsies by laporatomy.5 all seven bitches had intraluminal uterine dilation; three bitches had ovarian cysts. histopathologic changes were found in six of the seven bitches and included endometritis (three of seven), pseudoplacentational endometrial hyperplasia (two of six), pyometra (one of six), and cystic endometrial hyperplasia together with mucometra (one of six). the uterine contents revealed high leukocyte counts in all of these bitches. no bacteria or viruses were isolated from the uterine lumen. other causes of pregnancy loss there is a report in the literature (bartos) inferring that there is a sociobiological origin of pregnancy failure in domestic dogs associated with return of a mated bitch to her home where there is a male dog present who is not the sire of the litter. bitches mated elsewhere but returned home to be housed in a home pack were four times more likely to remain pregnant than a bitch housed individually after returning home. the highest proportion of successful pregnancies occurred in bitches mated at home and staying home. hypothyroidism has never been proven to be a cause of ppregnancy loss in the canine. likewise, diabetes mellitus, hypoadrenocorticism or hyperadrenocorticism are more likely to interfere with becoming pregnant than maintaining pregnancy. body condition is important for pregnancy maintenance and neither under or over conditioning is desirable. if energy and nutrients are at suboptimal levels pregnancy can be compromised. the pregnant bitch should be fed an all life stages food or one labeled for pregnancy and lactation once pregnancy is confirmed by ultrasonography. a food labeled for adult maintenance is not intended for pregnancy and can result in inadequate nutrition. a bitch with an average litter size for her breed should be expected to gain approximately one-third of her pre-pregnancy weight during her pregnancy. supplements, vitamins, nutriceuticals and topical preparation should only be used in a pregnant bitch if the label indicates that it is safe. androgens, estrogens, glucocorticoids, antifungals or antineoplastic agents should not be used in pregnant bitches. parasiticides should only be used if the label indicates the product has been tested as safe for pregnant and breeding animals. topical ophthalmic and otic preparations often contain very potent corticosteroid products and have been proven to have systemic effects including suppression of the hypothalamic-pituitary axis. certain plastics such as bisphenol can serve as endocrine disruptors and as an estrogen source. investigation of pregnancy failures clinical theriogenology • volume 10, number 3 • september 2018187 in a kennel situation should warrant a physical inspection of the housing and management practices in the kennel. summary the causes of pregnancy failure in the bitch are many. the value of history, physical examination of the bitch and the environment are vital to obtaining a diagnosis and a treatment plan designed to minimize the recurrence of such loss in a bitch references 1. givens md, marley msd: infectious causes of embryonic and fetal mortality. theriogenology 2008; 70:281-282. 2. reid lb, soares rm, vasconcellos sa, et al :a polymerase chain reaction for detection of brucella canis in vaginal swabs of naturally infected bitches. theriogenology 2007;68:1260-1270. 3. verstegen j,dhaliwal g,verstegen-onclin k:canine and feline pregnancy loss due to viral and non-infectious causes: a review. theriogenology 2008;70:304-319. 4. johnson ca:highrisk pregnancy and hypoluteoidism in the bitch. theriogenology 2008;70:1423-1430. 5. bartos l, bartosova j, chaloupkova h, et al: a sociobiological origin of pregnancy failure in domestic dogs. sci rep 2016;26;6:22188. clinical theriogenology • volume 10, number 3 • september 2018 188 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice clinical theriogenology 2022; 14: 22 congenital hypospadias and vertebral malformation in a barbados blackbelly neonate brittany diehl,a federico grosso,b marley iredale,c michael dark,d catalina cabreraa adepartment of large animal clinical sciences, bdepartment of small animal clinical sciences cdepartment of comparative, diagnostic and population medicine, college of veterinary medicine, university of university of florida, gainesville, fl, dveterinary diagnostic laboratories, college of veterinary medicine and emerging pathogens institute, university of florida, gainesville, fl abstract a male purebred barbados blackbelly, twin to another male, was born with obvious external congenital abnormalities. male twin was normal. abnormal lamb had a short torso and spinal column. at 1 week, the lamb was presented with dyspnea and lethargy, and euthanasia was elected. lamb was submitted for full necropsy and for additional diagnostics. after postmortem, in addition to histopathology, radiographs and computed tomographs were obtained. lamb had congenital vertebral and occipital malformations, meningitis, hypospadias, and renal malformation. to authors’ knowledge, hypospadias and vertebral malformation in the barbados blackbelly breed has not been reported. apparently, barbados blackbelly breed harbors closely related genetics, thus vigilant monitoring for genetic malformations is suggested. keywords: barbados blackbelly, twin lamb, congenital, hypospadias, vertebral malformation case presentation a congenitally abnormal, purebred barbados blackbelly male lamb was born as a twin to a normal, healthy male lamb. they were born in central florida to a dam and sire with a history of healthy lamb offspring in previous years; however, this was the first time that they were bred to each other. owner observed that the lamb had excessively long limbs, a short torso and vertebral column, and external genital malformations (figure 1). owner described urination to be occurring from the perineal region. initially, the lamb was unable to stand and was rejected by the ewe. with some assistance, the lamb was able to stand and nurse from a bottle within the first few hours. lamb was alert, consuming milk replacer from a bottle, and was able to urinate and defecate. at 1 week, the lamb was presented with lethargy and dyspnea. due to welfare concerns, euthanasia was recommended and elected. owner submitted the lamb for a full necropsy and further diagnostics. radiology and computed tomography immediately after postmortem, orthogonal radiographs of the skull, vertebral column, thorax and abdomen of the lamb were performed with a digital radiography plate (cxdi-50g digital plate, canon, lake success, ny). additionally, single mediolateral radiographs of right thoracic and pelvic limbs were obtained. lamb had normal vertebral formula with 7 cervical, 13 thoracic, 7 lumbar, 4 sacral, and 5 coccygeal vertebrae. occipital condyles were subjectively thickened and elongated. remainder of the skull appeared within normal limits. cervical vertebrae were substantially larger than the thoracic and lumbar vertebrae with various sized and shaped vertebral endplates and vertebral bodies. figure 1. external visualization of the lamb. note short torso and long appendages. there was severe narrowing of c2 c3 and c4 c5 intervertebral disc spaces with widening of c3 c4, and especially, c5 c6 intervertebral disc spaces (figure 2). multiple shortened, misshapen and fused thoracic, lumbar, sacral, and coccygeal vertebrae with associated moderate thoracolumbar kyphosis and scoliosis were observed. thoracic and lumbar vertebrae were markedly widened. spinous processes of multiple thoracic and lumbar vertebrae were fused and misshapen. severe mid to caudal thoracic rib crowding secondary to the vertebral malformations (figure 3) was observed. sternum was composed clinical theriogenology 2022; 14: 23 of 7 radiographically normal sternebrae with s5 and s6 fusion. diffuse increases in pulmonary opacity with decreased visualization of pulmonary vessels were noted, likely, a postmortem finding attributed to pulmonary atelectasis and pulmonary congestion. otherwise, the thorax appeared normal. in the abdomen, a diffuse loss of peritoneal serosal detail was observed, probably associated with lamb’s young age. gastrointestinal tract contained a mild to moderate amount of gas. skin margins were irregular in the penile region. no osseous abnormalities were observed in right thoracic and pelvic limbs. to further assess radiographic changes, a whole-body postmortem computed tomography (ct) was performed with a 160-slice (aquilion prime) multidetector row, helical ct unit (toshiba medical systems, tustin, ca). scan was performed in helical acquisition mode with a scan thickness of 0.5 mm, a pitch of 0.8, 100 ma, 120 kvp, tube rotation time of 0.5 seconds, and a 512 x 512 matrix. radiographic and ct findings were similar: multiple vertebral malformations (figure 4), abnormally elongated occipital condyles (figure 5), a normal sternum and a normal appendicular skeleton. necropsy and histopathology there was ~ an 8 cm, shallow, linear defect in the integument and urethra between rectum and prepuce base (figure 6) that divided the scrotum with 1 testis on either side of the defect (hypospadias). testes, pelvic urethra, bladder and ureters were grossly and microscopically unremarkable. right kidney was ~ twice the size of the left kidney and was irregularly shaped. histopathology revealed no prominent lesions within either kidney. following removal of head and dissection of masticatory muscles, substantial malformation of the occipital condyles was noticed that caused deformation of foramen deformation and magnum and cerebellum herniation. on dissection of the epaxial and hypaxial muscles, severe irregularity of lumbar vertebrae shape was observed that caused moderate scoliosis and kyphosis. figure 2. postmortem lateral radiograph of skull and cervical vertebral column (a), ventrodorsal radiograph of cervical vertebral column (b) and sagittal multiplanar ct reconstruction in a bone window of lamb (c). note severe narrowing of c2 c3 and c4 -c5 intervertebral disc spaces (arrows) with widening of c3 c4 and c5 c6 intervertebral disc spaces (arrowheads). figure 3. postmortem lateral radiograph (a), and lateral three-dimensional ct reconstruction (b) of the thoracolumbar vertebral column of the lamb. note multiple shortened, misshapen and fused thoracic and lumbar vertebrae (braces) with mid to caudal thoracic rib crowding (wide arrow) and kyphosis (asterisks) secondary to the vertebral malformations, and fused and misshapen spinous processes of multiple thoracic and lumbar vertebrae (braces). figure 4. postmortem ventrodorsal radiograph (a), and dorsal (b) and ventral (c) three-dimensional ct reconstructions of thoracolumbar vertebral column of the lamb. note multiple shortened, misshapen and fused vertebrae (braces) with mid to caudal thoracic rib crowding and throacolumbar scoliosis (asterisks) secondary to vertebral malformations, and markedly widened multiple vertebrae (wide arrows). clinical theriogenology 2022; 14: 24 figure 5. caudal three-dimensional ct reconstructions of lamb’s skull. note thickened occipital and elongated condyles (arrows). figure 6. shallow, linear defect in the integument between rectum and prepuce base, consistent with hypospadias sepsis was evident, characterized by meningitis and valvular endocarditis. definitive cause of sepsis was not histologically identifiable. cerebellum herniation and sepsis were likely the cause of lethargy. genetic analysis lamb’s hair roots were submitted to the university of california-davis veterinary genetics laboratory for dna analysis to test for ovine hereditary chondrodysplasia (spider lamb syndrome). assay used is an allele specific pcr of the c.1719t>a variant. results revealed n/n, meaning that the lamb was homozygous for tyrosine in the fgfr3 gene, indicating 2 normal copies. no sequencing was performed and thus flanking sequence was not evaluated. discussion ovine hereditary chondrodysplasia (spider lamb syndrome) is a recessive, semilethal inherited disorder in young lambs resulting in skeletal deformities.1 this syndrome is a result of a mutation within the gene for fibroblast growth factor receptor 3 (fgfr3). frequently, affected lambs have spinal deformities, long bone defects, and underdeveloped musculature.1 although the findings in this lamb were compatible with multiple congenital vertebral malformations and caudal occipital malformation, hereditary chondrodysplasia was considered a less likely differential diagnosis in the absence of proximal ulnar, glenoid, sternebrae and facial abnormalities.10 negative genetic testing suggested that this lamb did not have the well-characterized single-nucleotide polymorphism that causes spider lamb syndrome; however, this does not rule out other, spontaneous mutations in fgfr3. hypospadias can be defined as an imperfect closure of the external urethra in a male9 and is commonly classified into 4 types: palanetic, penile, scrotal, and perineal.8 this lamb demonstrated a case of penile, scrotal, and perineal hypospadias. hypospadias has been described in multiple livestock species, and in humans. it is among the most common congenital malformation in human male neonates, whereas these defects apparently occur as a result of a multifactorial disorder that has been associated with genetic and environmental factors.6 however, broadly this process of morphogenesis appears to be incredibly complex and is largely not understood. in a survey of the occurrence of congenital defects in sheep, the urogenital system was involved in 22.9% of the cases, and the most common male urinary defects were hypospadias and renal agenesis.3 reported concurrent developmental abnormalities in other animals with hypospadias include other urogenital abnormalities, such as penile aplasia, an incomplete ventral sheath, cryptorchidism, and atresia ani.5,7 none of these concurrent genitoanal abnormalities were present in this lamb. however, this lamb did present with marked, lumbar vertebral and occipital malformation, and moderate cerebellar herniation. limbs and the external genitalia may originate from nearby cell lines during embryogenesis.4 together with this information, these findings suggest that these congenital malformations may have occurred early in the development (differentiation of the embryo mesoderm). although hypospadias and associated congenital abnormalities etiology remain unknown, lack of abnormalities in the twin suggests that an infectious or environmental process had no effect; however, it cannot be ruled out. therefore, it is more likely that this abnormality was initiated within the developmental process of that individual embryo, although it remains unclear if it is product of inheritable genetic mutations. clinical theriogenology 2022; 14: 25 barbados blackbelly sheep breed originated in barbados, easternmost island of caribbean chain and west africa. they are known for their ability to withstand harsh tropical climates and heavy parasite burdens.2 currently, to our present knowledge, there are no specific perpetuating congenital abnormalities harboring within the purebred of barbados blackbelly lineage. however, the closeness of genetics within the breed makes a case for close monitoring of congenital defects that may be the result of recessive congenital genes. likewise, due to increasing implications of viruses, plants, and medicinal or chemical products, it is important to continue to be vigilant in our efforts to identify environmental teratogens that may be responsible for congenital defects.3 producers of sire and dam of this lamb, were informed, to follow up for potential future congenital defects associated with the lines of sire and dam to avoid perpetuating a flaw within the breed. this was the first occurrence in either of their flocks. to our knowledge, a case of hypospadias and vertebral malformation in the barbados blackbelly breed has not been reported. learning points • hypospadias and concurrent congenital malformations can occur in several mammalian species, often without a definitive explanation. • occurrence of congenital malformations within specific breeds is important to monitor, specifically in those with closely related genetics, • complications in embryogenesis should be considered when malformations of the external genitalia and appendages are present, conflict of interest authors disclose no conflicts of interest. acknowledgement authors thank the producer for willingness to facilitate further evaluation of the lamb. references 1. beever je, smit ma, meyers sn, et al: a single-base change in the tyrosine kinase ii domain of ovine fgfr3 causes hereditary chondrodysplasia in sheep. anim genet 2006;37:66-71. 2. de almeida am: barbados blackbelly: the caribbean ovine genetic resource. trop amin health prod 2018;50:239-250. 3. dennis sm: congenital defects of sheep. vet clin north am food anim pract 1993;9:203 217. 4. herrera am, cohn, mj: embryonic origin and compartmental organization of the external genitalia. sci ref 2014;4:6896. doi: 10.1038/srep06896. 5. jeong w, seong y, kim i, et al: radiographic diagnosis of hypospadias in three korean native calves. j vet clin 2008;25:557-559. 6. joodi m, amerizadeh f, hassanian sm, et al: the genetic factors contributing to hypospadias and their clinical utility in its diagnosis. j cell physiol 2019;234:5519-5523. 7. kumi-diaka j, osori di: perineal hypospadias in two related bull calves, a case report. theriogenology 1979;11:63-164. 8. ladds pw: congenital abnormalities of the genetalia of cattle, sheep, goats, and pigs. vet clin north am food anim pract 1993;9:127-144. 9. radostitis, om, gay cc, hinchcliff kw, et al: veterinary medicine. 10th edition, london; saunders co: 2007. p. 573. 10. vanek ja, walter pa, alstad ad: radiographic diagnosis of hereditary chondrodysplasia in newborn lambs. j am vet med assoc 1989;194:244-248. 2011: effective treatment of uterine disease in certified organic dairy herds effective treatment of uterine disease in certified organic dairy herds gustavo m. schuenemann,a troy a. brick,b santiago basa adepartment of veterinary preventive medicine and bdepartment of veterinary clinical sciences, college of veterinary medicine, the ohio state university, columbus, oh abstract postpartum metritis and clinical endometritis are common uterine disorders in lactating dairy cows that negatively impact reproductive performance. for certified organic herds, implementation of preventive management practices such as proper nutrition and calving management are critical to prevent uterine diseases from occurring. several alternative therapies such as garlic tincture, aloe vera, vitamins, and vegetable oils have been used by organic dairy producers. despite the use of these alternative therapies to treat postpartum metritis, there is no scientific evidence available in the literature that can promote or discourage these practices. for organic (and conventional) herds, the implementation of proactive management strategies that control the risk factors for uterine diseases should be a top priority. keywords: certified organic, dairy cattle, uterine diseases introduction organic food in the us, primarily produce and dairy products, reached retail sales over $21.1 billion in 2008 and for several years, organic farmers struggled to produce enough supply to meet the growing domestic demand.1 the organic dairy industry has been growing steadily since 1990 not only in the us, but also in europe. the dairy sector has been one of the fastest growing segments of the organic commodities, almost doubling the number of certified milk cows every year between 1997 and 2005 in the us.1 additionally, the retail demand for organic milk doubled and the retail milk prices were roughly double the conventional prices between 2004 and 2006.1 the 2005 us dairy survey showed that the size (small dairy operations) and location of the farm (northeast and upper midwest) increased the likelihood of organic practices as an alternative to improve farm income.2,3 environmental stewardship and milk price along with long term production contracts between the processors and producers have been the top reasons for converting to organic or adding a new organic dairy operation. management practices for certified organic dairies organic dairy production has gained increasing attention because of consumer concerns about food safety, animal welfare, and environmental impacts of intensive livestock operations.4 the national organic program (nop) standards for dairy and livestock require that cattle are actively grazing and at least 30% of the total dry matter intake (on a daily basis) be provided from grazing pasture for a minimum of 120 days during the growing season (nop access to pasture [livestock], february 12, 2010). although well managed pastures provide plentiful high quality forage, harvesting through grazing and balancing dairy rations present challenges. additionally, the us standard prohibits the use of antibiotic drugs and synthetic hormones in certified organic dairies. if a lactating dairy cow is treated with prohibited substances (e.g., antibiotics), the organic status of the animal is lost. it is important to note that from an animal welfare point of view, no animal must suffer and veterinary care should not be withheld to preserve the organic status of an animal. the whole certified organic process relies on a number of preventive management practices such as nutrition management that ultimately assures cows’ health. postpartum uterine diseases (e.g., retained fetal membranes, metritis), respiratory diseases, and mastitis require special attention. an observational field study in wisconsin revealed that the overall prevalence of mastitis, metritis, foot infections, and respiratory diseases was greater for conventional than organic dairy herds.5,6 risk factors for metritis and endometritis postpartum metritis and clinical endometritis (ce) are common uterine disorders in lactating dairy cows that negatively impact reproductive performance; thus, diminishing profitability and clinical theriogenology • volume 3 number 4 • december 2011555 sustainability of dairy operations. metritis (puerperal; usually within 14 days postpartum) is defined as the inflammation of all layers of the uterus and characterized by fetid uterine discharge with fever, anorexia, and/or decreased milk production (systemically ill).7,8 clinical endometritis is defined as the inflammation of the endometrial lining of the uterus characterized within 21-40 days in milk (dim) by mucopurulent or more significant vaginal discharge (clinical diagnosis) or the presence of polymorphonuclear cells (≥5%, cytology diagnosis); usually without systemic signs of illness. lactating dairy cows subjected to poor nutrition management (body condition score at calving of <2.75; scale 1-5) increased the risk for uterine diseases with increased levels of non-esterified fatty acids and betahydroxybutyrate in early postpartum cows.12 furthermore, retained fetal membranes, stillbirths, abortions, dystocic births and twins12 as well as hygiene of the perineum region at calving (presence of manure and dirt around the birth canal) increased the likelihood of postpartum metritis and clinical endometrtis.9 management and treatment options for clinical endometritis endometritis is an inflammation of the endometrial lining of the uterus without systemic signs of illness.7 clinical endometritis negatively impacts reproductive performance (delays time to conception and increases the risk for culling after calving) in dairy cattle; thus, diminishing profitability and sustainability.7,10 a recent study in three herds showed that 18% of lactating dairy cows suffer from ce at 35 dim.12 proactive management that targets the risk factors should be a top priority to prevent both puerperal metritis and ce. for instance, cow comfort (e.g., 30 inches of feed bunk space and 80% stocking density in relation to stalls) during the preand postpartum period reduced the risk for uterine diseases such as metritis and ce. for clinical cases, administration of prostaglandin f2α analog (pg)12-14 and antimicrobial agents such as ceftiofur hydrochloride and penicillin7,15,16 are frequently used to treat these cows in conventional herds. antimicrobial therapy for cows with clinical signs of uterine disease is recommended in certified organic herds, but the organic status is lost and the animal must exit the herd. therefore, the use of alternative therapies such as garlic tincture, aloe vera, vitamins, and vegetable oils have been used by organic dairy producers.5,17 despite the use of these alternative therapies to treat uterine diseases (e.g., metritis), there is no scientific evidence available in the literature that can promote or discourage these practices. recently, a field trial showed that homeopathic remedies (lachesis compositum [lachesis], carduus compositum [carduus], and traumeel lt [traumeel]) were not effective to prevent ce (based on clinical and fertility outcomes).18 the use of lytic enzyme preparations from bacillus subtilis (lysosubtilin) was assessed as alternative treatment for cows with postpartum endometritis compared to neofur or uterosan.19 lysosubtilin (2 x 106 iu) was dissolved in 100 ml of distilled water and administered intrauterinarily twice per week until recovery in cows with ce as opposed to cows treated with neofur or uterosan. this field trail showed that cows treated with a lytic enzyme preparation (lysosubtilin) had improved therapeutic outcomes (clinical cure), reduced calving-to-conception interval, and improved conception risk compared to cows treated with neufur or uterosan. the use of intra-abdominal hypertonic glucose (20%) was previously reported to control acute peritonitis in rabbits.20 furthermore, an in vitro study has shown that mannose (a sugar monomer) inhibits the adhesion of bacteria to the epithelial cells of the equine endometrium.21 this suggests that the use of 50% dextrose in water (a hypertonic solution), that targets the uterine environment rather than the bacterial pathogen itself, may be a viable and effective strategy for organic lactating dairy cows diagnosed with ce. recently, we investigated the use of intrauterine dextrose (200 ml; 50% dextrose in water) in a randomized clinical trail under field conditions.22 lactating dairy cows (n=760) from two conventional herds were screened using vaginoscopy for ce at 26±3 dim and scored using a 0-3 scale.8,23 cows scored as 2 or 3 were stratified by parity and randomly allocated into one of three treatment groups: 1) control (n=83), 2) subcutaneous administration of ceftiofur crystalline free acid (ccfa, n=75; 6.6 mg/kg; excede®; pfizer animal health, new york, ny), or 3) intrauterine infusion (equine infusion pipettes) of 200 ml 50% dextrose (n=79; vedco, saint joseph, mo). in this study, all cows (with or without ce) were presynchronized with two injections of pg given 14 days apart (starting at 26±3 dim) followed by clinical theriogenology • volume 3 number 4 • december 2011 556 ovsynch (ov; gonadotropin releasing hormone [gnrh]-7 d-pg-56 h-gnrh 16 h-timed-artificial insemination [ai]) 12 days later. this study showed that cows that received intrauterine dextrose had improved clinical cure compared to control groups. also, pregnancy per ai (pai) in dextrose treated cows (29.8%) tended to differ (p=0.1) from cows in the control (21.1%) or ccfa treated groups (19.7%); whereas pai in dextrose cows was not different from cows without ce (39.1%).22 it is important to note that intrauterine infusion is a procedure that requires proper training and strict aseptic precautions to prevent further uterine damage. final remarks postpartum metritis and ce are common uterine disorders in lactating dairy cows that negatively impact reproductive performance; thus, diminishing profitability and sustainability of dairy operations. for certified organic herds (as well as conventional herds), implementation of preventive management practices such as proper nutrition and calving management are key to prevent uterine diseases from occurring. for clinical cases, the use of intrauterine dextrose showed promising clinical and fertility results. however, further investigation is needed with certified organic herds to confirm and determine the underlying mechanisms for these findings. acknowledgements the authors wish to thank the collaborating dairy farms for assisting and providing the animals used in the endometritis study. also appreciation is extended to dr. j. daniels and dr. c. pinto (department of veterinary clinical sciences) for their input and bacteriology analysis. the effectiveness of the intrauterine dextrose administration on cows diagnosed with clinical endometritis was supported in part by the usda-nifa animal health (formula fund). references 1. dimitri c, oberholtzer l: marketing u.s. organic foods: recent trends from farms to consumers. 2009 eib-58. economic research service/usda. (http://www.ers.usda.gov/publications/eib58/eib58.pdf; accessed on january 25, 2010). 2. mcbride w, greene c: a comparison of conventional and organic milk production systems in the us. selected paper at the american agricultural economics association annual meeting, july 29-august 1, 2007, portland, or. (http://ageconsearch.umn.edu/handle/9680/; accessed on january 25, 2010). 3. usda-nass 2008: farms, land in farms, and livestock operations 2007 summary. (http://usda.mannlib.cornell.edu/mannusda/viewdocumentinfo.do?documentid=1259; accessed on february 15, 2010). 4. sundrum a: organic livestock farming, a critical review. livest prod sci 2001;67:207-215. 5. pol m, ruegg pl: treatment practices and quantification of antimicrobial drug usage in conventional and organic dairy farms in wisconsin. j dairy sci 2007;90:249-261. 6. ruegg pl: management of mastitis on organic and conventional dairy farms. j anim sci 2009;87:43-55. 7. leblanc sj, duffield tf, leslie ke, et al: defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows. j dairy sci 2002;85:2223-2236. 8. sheldon im, lewis gs, leblanc s, et al: defining postpartum uterine disease in cattle. theriogenology 2006;65:15161530. 9. schuenemann gm, nieto i, bas s, et al: dairy calving management: effect of perineal hygiene scores on metritis [abstract]. j dairy sci 2011. 10. gilbert ro, shin st, guard cl, et al: prevalence of endometritis and its effects on reproductive performance of dairy cows. theriogenology 2005;64:1879-1888. 11. heuwieser w, tenhagen ba, tischer m, et al: effect of three programmes for the treatment of endometritis on the reproductive performance of a dairy herd. vet rec 2000;146:338-341. 12. dubuc j, duffield tf, leslie ke, et al: risk factors for postpartum uterine diseases in dairy cows. j dairy sci 2010;93:5764-5771. 13. kasimanickam r, duffield tf, foster ra, et al: the effect of a single administration of cephapirin or cloprostenol on the reproductive performance of dairy cows with subclinical endometritis. theriogenology 2005;63:818-830. 14. galvão kn, frajblat m, brittin sb, et al: effect of prostaglandin f2 alpha on subclinical endometritis and fertility in dairy cows. j dairy sci 2009;92:4906-4913. 15. mcdougall s: effect of intrauterine antibiotic treatment on reproductive performance of dairy cows following periparturient disease. n z vet j 2001;49:150-158. clinical theriogenology • volume 3 number 4 • december 2011557 16. okker h, schmitt ej, vos pl, et al: pharmacokinetics of ceftiofur in plasma and uterine secretions and tissues after subcutaneous postpartum administration in lactating dairy cows. j vet pharmacol ther 2002;25:33-38. 17. nodpa 2009: northeast organic dairy producers alliance. nodpa news. volume 9, issue 1. p 1-39 (http://www.nodpa.com/january_2009_final_low-res.pdf; accessed on february 10, 2010). 18. arlt s, padberg w, drillich m, et al: efficacy of homeopathic remedies as prophylaxis of bovine endometritis. j dairy sci 2009; 92:4945-4953. 19. biziulevichius ga, lukauskas k: in vivo studies on lysosubtilin: 2. efficacy for treatment of post-partum endometritis in cows. vet res 1998;29:47-58. 20. narat jk: experimental study upon the use of intra-abdominal injections of hypertonic glucose solution in the treatment of peritonitis. ann surg 1923;78:357-363. 21. king ss, young da, nequin lg, et al: use of specific sugars to inhibit bacterial adherence to equine endometrium in vitro. am j vet res 2000;61:446-449. 22. brick ta, bas s, daniels jb, et al: impact of intrauterine dextrose therapy on conception of lactating dairy cows with clinical endometritis [abstract]. j dairy sci 2011. 23. williams ej, fischer dp, pfeiffer du, et al: clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle. theriogenology 2005;63:102-117. clinical theriogenology • volume 3 number 4 • december 2011 558 2018: mixed germ cell tumor in the placenta of an arabian mare mixed germ cell tumor in the placenta of an arabian mare college of veterinary medicine, auburn university, auburn, al a 14 year old arabian mare was confirmed pregnant at 14 days via transrectal ultrasonography. the fetal heartbeat was present at 45 days and again at 5 months gestation. at 9 months gestation, the mare was presented for thick, clear vaginal discharge. the mare had travelled as a show mare and was current on vaccinations. she previously delivered multiple foals with no complications. upon physical examination, the mare was clinically normal. temperature, heart rate, and respiratory rate were within normal limits. the fetus could not be palpated or detected by ultrasonography transrectally. the fetal heartbeat was visualized on transabdominal ultrasonography but was not recorded. the allantoic fluid contained hyperechoic flecks. treatment for placentitis was instituted and included trimethoprim sulfamethoxazole tablets (9,600/1,920 mg orally twice daily), altrenogest (22 mg orally twice daily), and flunixin meglumine (400 mg iv twice daily).2 the vaginal discharge remained unchanged despite treatment. five days after the initial examination, the fetus was aborted. the fetus appeared grossly normal for his gestational age. the mare retained the placenta overnight. the following morning, gentle traction was required to remove the placenta as a large mass was holding it within the cervix. after removal, multiple masses were noted on the placenta. the fetus and placenta were submitted for necropsy. the placental masses and additional lesions found in the fetus’ liver were confirmed to be a mixed germ cell tumor. placental germ cell tumors rarely metastasize to the foal. metastasis occurs by the placental germ cell tumor sending cells through the umbilical vein to the fetal liver.1 the mare was treated for two days after the abortion with uterine lavages and infusion. she was re-bred to a different stallion two months later and delivered a normal foal. this case represents a rare neoplasia as a cause for vaginal discharge and abortion. keywords: equine, placenta, tumor, fetus references 1. bockenstedt mm, fales-williams a, haynes js: equine placental mixed germ cell tumor with metastasis to the foal. vet pathol 2015;52:360-363. 2. bailey cs, macpherson ml, pozor ma, et al: treatment efficacy of trimethoprim sulfamethoxazole pentoxifylline and altrenogest in experimentally induced equine placentitis. theriogenology 2010;74:402-412. clinical theriogenology • volume 10, number 3 • september 2018 358 c. ikard, a.k. johnson, k. varga 2018: processing techniques for canine semen processing techniques for canine semen natalie s. fraser the university of queensland, gatton, qld, australia introduction the use of artificial insemination with either cooled or frozen canine semen continues to gain in popularity world-wide. processing and extension of canine semen is typically straightforward; however, sperm selection methods more commonly used in other species can be adapted for use with canine sperm. processing after collection can be used to remove seminal plasma and prostatic fluid, to increase the concentration of the ejaculate, and to preferentially select normal sperm. supplies and materials that may be helpful include a variable speed centrifuge, preferably with hanging buckets, plastic or glass 15ml conical tubes, disposable pipettes, flat-bevel needles, and larger-volume (1 ml to 5 ml) micropipettors and disposable tips. extension a variety of commercially prepared semen extenders are available for use specifically with canine semen. cooling and freezing media can also be prepared within the laboratory, but this requires the practitioner to assume all responsibility for quality control of raw materials and final product composition (ph, osmolality, bacterial contamination, etc). given the ease of procurement, high quality, and low costs of commercially prepared extenders, this is likely a more practical option for practitioners. semen may be extended for the purposes of cooled storage/transport or cryopreservation. this process involves dilution of semen, and then cooling and storage at appropriate temperatures until insemination. cooled semen has several advantages over frozen semen, namely its procedural ease, no need for specialized equipment for preparation or use,1 and possibly fewer restrictions on transport/importation.2 extenders for cooling typically contain fructose or glucose, and fructosecontaining extenders may result in longer sperm longevity.2 extended semen should be stored at 4-12°c; temperatures below 4°c may result in reduced sperm longevity. when stored at an appropriate temperature, sperm longevity up to 23 days has been reported,2 but prolonged storage is typically impractical and unnecessary and results in decreased physical and functional characteristics. extension for cryopreservation can be performed with single-step or two-step protocols.3 ideally, the sperm rich fraction should be isolated with minimal collection of prostatic fluid. centrifugation is performed to concentrate the sperm and remove excess prostatic fluid; sperm washing can be performed as an initial step, based on clinician preference and the specific protocol used. specific canine freezing media can be purchased, or human sperm cryopreservation media can be used. the most common cryoprotectant in canine freezing media is glycerol, and glycerol appears to outperform amide cryoprotectants for canine sperm.4,5 although most freezing media used for canine sperm contains egg yolk, soy bean based extender has been evaluated for use in this species.6 although not in common use, the soy based extenders have a distinct advantage of being free from animal proteins which may be beneficial when frozen semen is subjected to international transport. sperm selection methods a wide variety of sperm selection methods are currently available, including sperm washing,7,8 swim up,9,10 density-gradient centrifugation,11-13 single-layer centrifugation,8,14-16 and glass wool filtration.11 these methods vary greatly in the recovery rate and sperm quality assessment parameters such as motility, morphology, chromatin integrity, viability and acrosome integrity.17 sperm washing washing of sperm can be achieved by diluting the ejaculate with extender, and centrifuging the sample in order to separate the seminal plasma from sperm. the supernatant can be manually removed with a disposable 3ml pipette or needle and syringe. selection of centrifugation speed needs to balance reduction in sperm loss while mitigating damage to sperm. higher centrifugation speeds improve sperm clinical theriogenology • volume 10, number 3 • september 2018201 recovery and produce a more tightly packed pellet. however, higher centrifugation speeds are also associated with increased dead/moribund sperm.7 the pellet produced contains dead, moribund, and abnormal spermatozoa as well as viable spermatozoa.8 recommended centrifugation speeds range from 700-900g for 5-10 minutes. setting the centrifuge deceleration speed to the lowest setting will result in less turbulence to disrupt the sperm pellet during braking. swim up swim-up is a method of purification most commonly used for selection of sperm for procedures such as in-vitro fertilization, and is considered a basic manipulation for this procedure. in a standard swim up procedure, the sperm are washed, and the resultant pellet is over layered with medium and incubated at 37°c in 5% co2.18 after incubation, the supernatant containing actively mobile sperm is removed. modification of this technique may reduce sperm lost during the centrifugation phase.8 briefly, a small volume of ejaculate is placed under a layer of medium; semen samples are then incubated in a vertical position, and the top layer of medium is removed and re-suspended, allowing collection of sperm that have “swam up”.8 most viable sperm are recovered after an in vitro incubation step of 15 minutes; as the time of incubation increases, so does the number of degenerated or damaged cells.9 density-gradient centrifugation density-gradient centrifugation separates normal from abnormal spermatozoa based on isopyknic point; sperm are centrifuged through layers of colloid. after removal of the supernatant containing the colloid layers and discarded sperm, the subsequent pellet is re-suspended in washing medium and centrifuged again to remove any residual colloid prior to insemination or freezing. this technique results in selection of a sperm population that demonstrates improved quality (ie, higher motility, viability, and acromsome integrity).12 density-gradient centrifugation has also been used to successfully separate erythrocytes from sperm.13 the main drawbacks of the technique are related to technical competency; formation of the colloid layers is integral to successful separation, which requires some practice on behalf of the user to master. preparation of the conical tubes can be time consuming, particularly if large volumes need to be prepared. centrifugation speeds are typically slower with an increased centrifugation time, for example, 20 min at 300 x g. single-layer centrifugation this technique is similar to density-gradient centrifugation, but is simplified in that it only uses one layer of colloid. from a practical point of view, this results saving time and effort in preparation of the sample whilst still selecting a normal highly motile sperm population.16 current research regarding the separation of other cell types from single-layer centrifugation is lacking. glass wool filtration glass wool filtration is easily performed in practice, with no specialized equipment needed. glass wool is packed to a depth of 6mm in a 1-ml disposable syringe barrel (ie, tuberculin syringe). the column is rinsed 2-3 times with 1ml of medium to remove loose glass wool fibers and other particles prior to use. the end of the syringe barrel is placed in a 1.5ml eppendorf tube, perforated at the lid to accommodate the tip of the syringe.11 additional holes are punched in the tube to equilibrate pressure, and the tube and column can then be placed in a rack in the incubator. glass wool filtration can be used to remove cryo-damaged sperm after freezing-thawing,11 and this technique is thought to trap mechanically-damaged spermatozoa. sex sorting of canine semen sex-sorted semen has become common in cattle reproduction, but is not commonly practiced or commercially available for canine sperm at this time. however, this technology has been evaluated under field conditions for the purposes of research.19 conception rates in both the xand ysorted sperm clinical theriogenology • volume 10, number 3 • september 2018 202 groups were lower than unsorted control groups (25% and 20% vs. 55%, respectively), but both sorted groups produced approximately 86% of the puppies being born the desired sex. conclusions a variety of processing techniques are available for canine semen; these techniques are commonly used in other species and can be adopted in the laboratory with minimal investment in additional equipment. although these techniques can be successfully used to select normal sperm from abnormal, removal of other cell types (i.e., red blood cells or leukoycytes) presents a significant clinical advantage for processing canine semen. further research and improvements on these techniques may improve success rates with assisted reproductive technologies in canids. references 1. goericke-pesch s, klaus d, failing k, et al: longevity of chilled canine semen comparing different extenders. anim reprod sci 2012;135:97-105. 2. ponglowhapan s, essen-gustavsson b, linde forsberg c: influence of glucose and fructose in the extender during long-term storage of chilled canine semen. theriogenology 2004;62:14981517. 3. brito mm, lucio cf, angrimani ds, et al: comparison of cryopreservation protocols (single and two-steps) and thawing (fast and slow) for canine sperm. anim biotechnol 2017;28:67-73. 4. futino do, mendes mc, matos wn, et al: glycerol, methyl-formamide and dimethyl-formamide in canine semen cryopreservation. reprod domest anim 2010;45:214-220. 5. lopes kr, costa ll, lima gl, et al: dimethylformamide is no better than glycerol for cryopreservation of canine semen. theriogenology 2009;72:650-654. 6. sanchez-calabuig mj, maillo v, beltran-brena p, et al: cryopreservation of canine sperm using egg yolk and soy bean based extenders. reprod biol 2017;17:233-238. 7. rijsselaere t, van soom a, maes d, et al: effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa. theriogenology 2002;57:1669-1681. 8. dorado j, galvez mj, demyda-peyras s, et al: differences in preservation of canine chilled semen using simple sperm washing, single-layer centrifugation and modified swim-up preparation techniques. reprod fertil devel 2015;28:1545-1552. 9. bukowska d, kempisty b, sikora j, et al: the effect of swim-up purification and incubation of cells on sperm viability in dogs of different ages. vet med (praha) 2011;56:248-254. 10. sanchez r, risopatron j, schulz m, et al: canine sperm vitrification with sucrose: effect on sperm function. andrologia 2011;43:233-241. 11. kim sh, yu dh, kim yj: apoptosis-like change, ros, and dna status in cryopreserved canine sperm recovered by glass wool filtration and percoll gradient centrifugation techniques. anim reprod sci 2010;119:106-114. 12. dorado j, alcaraz l, duarte n, et al: centrifugation on puresperm® density-gradient improved quality of spermatozoa from frozen-thawed dog semen. theriogenology 2011;76:381-385. 13. phillips tc, dhaliwal gk, verstegen-onclin km, et al: efficacy of four density gradient separation media to remove erythrocytes and nonviable sperm from canine semen. theriogenology 2012;77:39-45. 14. morrell j, rodriguez-martinez h, linde-forsberg c: single layer centrifugation on a colloid selects motile and morphologically normal spermatozoa from dog semen: preliminary results. reprod domest anim 2008;43:61-68. 15. dorado j, galvez mj, morrell jm, et al: use of single-layer centrifugation with androcoll-c to enhance sperm quality in frozen-thawed dog semen. theriogenology 2013;80:955-962. 16. dorado j, alcaraz l, galvez mj, et al: single-layer centrifugation through puresperm(r) 80 selects improved quality spermatozoa from frozen-thawed dog semen. anim reprod sci 2013;140:232-240. clinical theriogenology • volume 10, number 3 • september 2018203 17. morrell j, rodriguez-martinez h: biomimetic techniques for improving sperm quality in animal breeding: a review. open androl j 2009;1:9. 18. jameel t: sperm swim-up: a simple and effective technique of semen processing for intrauterine insemination. j pak med assoc 2008;58:71-74. 19. wei yf, chen fl, tang ss, et al: birth of puppies of predetermined sex after artificial insemination with a low number of sex-sorted, frozen-thawed spermatozoa in field conditions. anim sci j 2017;88:1232-1238. clinical theriogenology • volume 10, number 3 • september 2018 204 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2017: fetal mummification of a 270 day fetus in a mare   fetal mummification of a 270 day fetus in a mare morgan e. agnew, patricia l. sertich department of clinical studies, new bolton center, school of veterinary medicine, university of pennsylvania, kennett square, pa summary a 12 year old, selle francais mare was presented for pregnancy evaluation. a transabdominal ultrasound examination by the referring veterinarian had revealed a lack of fetal fluids and no visible fetal heartbeat. on presentation, the mare was bright and alert. transabdominal and transrectal ultrasound examinations were performed; both confirmed the referring veterinarian’s findings. on transrectal palpation, the cervix was tightly closed and the uterus was partially involuted. bony protuberances of the fetus were palpable transrectally. a vaginal examination was performed. with manipulation, the cervix did not fully dilate; however, one hand could be passed into the uterus which was contracted around bony portions of a large fetus. the client elected a cesarean section to eliminate the risk of uterine or cervical trauma. a partially mummified 17 kg fetus, with a crown rump length of 86 cm was removed from the uterus. the mare recovered uneventfully after surgery. background this case describes the second report in the literature of a fetus that developed past 200 d of gestation prior to mummification, without the mare displaying signs of systemic illness. it also describes successful removal of the large fetus via cesarean section. case presentation a 12 year old, primiparous, selle francais mare was presented to the university of pennsylvania new bolton center for pregnancy evaluation at approximately 350 d of gestation. the mare was initially examined 33 days prior to admission by the referring veterinarian at approximately 317 d gestation, when premature mammary gland development was noted by the owner. palpation and ultrasonography per rectum at that time revealed a thickened combined thickness of the uterus and placenta, the viability of the fetus was not assessed transabdominally at this time. trimethoprim sulfa and altrenogest administration for suspected placentitis were initiated. the mammary gland development regressed and no signs of premature labor were observed. eleven days after the mare’s expected due date, at approximately 350 d gestation, the referring veterinarian performed a second transrectal and transabdominal ultrasound examination. at that time, no fetal fluids were visible, the fetus had no heartbeat and there was no fetal movement. the mare was referred to new bolton center that same day for further evaluation. on presentation, the mare was bright and alert with all vital parameters within normal limits. the mare had no vaginal discharge, a slightly developed mammary gland and milk could be expressed from both teats. bloodwork was unremarkable with a packed cell volume of 35%, total protein of 7.0 gm/dl, white blood cell count of 6.78x103/µl and a creatinine of 1.2 mg/dl. transabdominal ultrasonography was performed and the uterus was seen to be partially involuted and no fetal fluids were visible. hyperechoic structures consistent with fetal bones were visualized in the right caudo-dorsal inguinal area; no fetal heartbeat was present. palpation and ultrasonography per rectum were performed. bony protuberances of the fetus were easily palpable through the wall of the uterus, and were visible on ultrasound as hyperechoic structures in the uterine lumen. treatment transvaginal manual delivery of the fetus was attempted. the cervix was initially closed, but with gentle manipulation one hand was passed through the cervix to evaluate fetal size and position. the fetus was relatively large with leathery contracted tissue, making it difficult to assess. the suspected presentation, position and posture were anterior longitudinal, dorsoiliac with the head and neck flexed. a sterile nasogastric tube was passed through the cervix and a solution of methylcellulose lubricant and clinical theriogenology • volume 9, number 2 • june 2017213   water was pumped into the uterus around the fetus. the cervix failed to fully dilate, the uterus was inelastic and the fetus was rigid making manipulations to correct the position difficult. since the owner’s primary concern was preserving fertility of the mare, a cesarean section was recommended at this time to prevent uterine or cervical trauma. the mare was pre-medicated with 0.5 mg/kg xylazine intravenously (iv) and induced with 2.2 mg/kg ketamine iv and 0.05 mg/kg midazolam iv. anesthesia was maintained with isoflurane. the fetus was removed through a midline laparotomy incision. the fetus was female and weighed 17 kg with a crown rump length of 86 cm, suggesting that the fetus died around 270 days gestation. there was no malodorous exudate or fetal maceration suggestive of active infection. both the limbs and nose of the fetus were contorted. the presentation position and posture was confirmed as per vaginal assessment. additionally, the right front limb was flexed at the carpus and the left front limb was flexed at the elbow. the hind limbs were bilaterally flexed at the hock. the skin was diffusely brown black, leathery and easily sloughed. viscera were in varying degrees of dehydration. the fetal membranes were friable brown and leathery, and were removed in entirety with the fetus. (see figure) the mare recovered from general anesthesia uneventfully. after surgery, the mare was administered 22,000 iu/kg of potassium penicillin iv qid, 6.6 mg/kg of gentamicin iv sid and 1.1 mg/kg of flunixin meglumine iv bid for 72 hours. the mare was administered maintenance, 60 ml/kg/day, iv fluids at 1-2 l lactated ringer’s solution/hour for the first 24 hours after surgery. the uterus was lavaged with 5 l sterile saline 24 hours following surgery. the effluent was slightly blood tinged with a few blood clots and all the fluid was recovered. following the lavage, a single dose of 20 iu oxytocin was administered iv to evacuate the uterus. outcome palpation and ultrasonography per rectum 48 hours after surgery revealed that the left ovary contained a corpus luteum and multiple small follicles < 20 mm in diameter. the right ovary contained multiple small follicles <15 mm in diameter. the uterus had excellent tone and no intraluminal fluid. the cervix was closed and no fluid was viaualized in the vagina. the mare was discharged with instructions to continue oral administration of a 1.1 mg/kg dose of flunixin meglumine once daily for three days. it was recommended the mare be kept on stall rest for one month, then turned out in a small paddock for one month before being turned out with a group of brood mares. discussion fetal mummification of a singleton fetus and fetal maceration are rarely reported in the mare. the feto-placental unit becomes the dominant source for progestogens once endometrial cups regress between 90-120 d of gestation.1 without a functioning feto-placental unit a fetus is generally aborted shortly after death, before mummification occurs.1 fetal mummification was first reported in 1984 by ginther and pierson in describing the ultrasonographic anatomy and pathology of the equine uterus.2 on transrectal ultrasound of 300 ponies and 110 horses several mummified fetuses were identified.2 one mare reportedly had the mummy removed and became pregnant when bred that same season, no subsequent pregnancy data were given on the other cases.2 there are limited other case reports available with several causes proposed for the mummification. in mares carrying a singleton fetal mummy altrenogest administration is thought to cause retention of the dead fetus. there are several case reports in the literature that seem to support this hypothesis, however many pregnant mares administered altrenogest abort shortly after the fetus dies.3,4 in both reports mares were administered altrenogest through 150 d of pregnancy or later.3,4 fetuses were between 140 and 200 d of gestation at death based on crown rump length or last recorded palpation with fetal movement.3,4 in one case the mare expelled the fetus without assistance,4 while the fetus was manually delivered transvaginally from the other mare.3 both mares were discharged without any systemic complications.3,4 in one mare, histological evaluation of an endometrial biopsy sample the following year was classified as category iii due to chronic inflammation and moderate fibrosis.3 clinical theriogenology • volume 9, number 2 • june 2017 214   a second group of case reports involve early fetal death. in these cases, the cause of fetal death was unknown, but authors hypothesized that retention of the fetus occurred because retained endometrial cups continued to produce equine chorionic gonadotropin and stimulate development of secondary corpora lutea.5,6 none of the mares were administered altrenogest.5,6 in one case the mare was evaluated by palpation and ultrasonography per rectum at nine months of gestation and found to have a retained fetal mummy.5 the fetal mummy was manually removed transvaginally and based on crown rump length the age at death was estimated at 50 d.5 this mare was rebred and produced a foal the following year.5 in a second case a fetal mummy was manually removed transvaginally from a mare evaluated at 10 months of gestation.6 the fetus had brachygnathia and gastroschisis.6 the crown rump length was 18 cm, suggesting fetal death occurred around 95 d.6 the mare was rebred and diagnosed pregnant at 14 d, but aborted several months later.6 there is one case report that attributes fetal mummification to a congenitally short uterine body in a mare administered altrenogest.7 the mare had a history of consecutive abortions.7 a mummified fetus that appeared to be 80-90 d of age based on crown rump length was aborted at 273 d gestation.7 fetal mummification of a late gestation fetus has been previously described in a single case report. a 15 year old mare was evaluated at 375 days gestation for vaginal discharge and a history of abdominal discomfort 17 days prior to admission.8 the mare had retained a dead fetus that could not be removed transvaginally due to size of fetus and inelasticity of uterine wall.8 a term, partially mummified fetus was removed via cesarean section; the fetus was in transverse position and had never engaged in the cervix.8 the mare recovered uneventfully from surgery.8 this case describes a retained mummified fetus that had developed after 200 d of gestation with no systemic signs other than slight mammary gland development. like the gilbert report, the fetus was too large to deliver transvaginally through a partially closed cervix with a noncompliant uterus and therefore a cesarean section was performed. learning points  late term fetal death and mummification is possible in the mare without any systemic signs of illness.  cesarean section is a viable option to deliver a large mummified fetus when the cervix is not fully dilated and the uterus is noncompliant. references 1. löfstedt rm: abnormalities of pregnancy. in: equine reproduction. 2nd ed. chichester (uk: wiley-blackwell; 2011. p. 2445-2446. 2. ginther o, pierson r: ultrasonic anatomy and pathology of the equine uterus. theriogenology 1984;21:505-516. 3. mccue p, vanderwall d, squires e: fetal mummification in a mare. j equine vet sci 1997;17:267-269. 4. barber j, troedsson m: mummified fetus in a mare. j am vet med assoc 1996;208:1438-1440. 5. threlfall w: singleton mummified fetus in a standardbred mare. equine vet educ 2010;17:235-239. 6. newcombe j: mummified singleton foetus in a mare. aust equine vet 1998;16:33-34. 7. meyers p, varner d: abortion of a mummified fetus associated with short uterine body in a mare. j am vet med assoc 1991;198:1768-1770. 8. gilbert r, bosu w, levine s, et al: intrauterine death and onset of mummification of a single equine foetus. equine vet j 1989;21:301-302. clinical theriogenology • volume 9, number 2 • june 2017215   figure. photograph of the mummified fetus showing the posture within the uterus. (editor’s note: the photograph in this manuscript is available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 9, number 2 • june 2017 216 1 contact allan gunn allangunn@bigpond.com © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 9972, http://dx.doi.org/10.58292/ct.v16.9972 research report effect of incorrect storage of bull semen samples on sperm morphology assessment allan gunn,a mark bakerb aschool of agricultural, environmental and veterinary sciences, faculty of science and health, and the gulbali institute, charles sturt university, wagga wagga, australia bpriority research centre, school of environmental and life sciences, university of newcastle, callaghan, australia abstract thirteen pairs of bull semen samples were assessed. one of each pair of samples was inadvertently stored in phosphate buffered saline (pbs) that was used for analysis of motility immediately after collection. other sample of the pair that was correctly stored in buffered formol saline (bfs) was assessed later. proportion of morphologically normal sperm was different (p = 0.0021) between the 2 storage methods. most of the abnormalities in the pbs samples were loose and detached heads. closer examination of these sperm illustrated changes in the composition of the tail, with an apparent loss of the plasma membrane. this serendipitous error allowed documentation of the importance of correct storage of semen samples for morphological assessment of sperm, and the tertiary defects detected with the incorrect storage of semen samples in semen extender. tertiary defects should always be considered a possibility during morphological assessment of sperm via spermiogram. keywords: bull sperm, tertiary morphological abnormalities, detached heads introduction fertility has been defined as the ability to produce young, that for a male means there is a requirement to produce viable sperm. assessment of the quality of the ejaculate from a male animal requires examination of a semen sample via spermiogram. color of the ejaculate, and sperm motility and morphology are assessed to identify the likelihood of bulls that are infertile or subfertile. sperm morphology is arguably the most important criterion for assessing fertility, with teratozoospermia an often under-rated aspect of subfertility or compromised fertility.1 features determining semen quality are interrelated to at least some degree.2 sperm morphology are classified in several ways: 1. primary, secondary or tertiary classification based on the origin of the lesion (testis, epididymis or handling/processing respectively), 2. major or minor abnormalities based on the actual or perceived detrimental effects on fertility, 3. anatomical location of the lesion on the sperm (head, midpiece, tail), and 4. compensable or noncompensable defects, depending on the effect of the relative number of sperm required in the breeding dose.1,2 we report observing teratozoospermia because of inadvertent incorrect storage (phosphate buffered saline [pbs]) of semen samples. morphological defects were classified anatomically, with most samples initially submitted having a large proportion of morphologically abnormal sperm. aim of this report is to illustrate the differences, and to highlight the importance of, correct storage (in 10% buffered formal saline [bfs]). materials and methods semen was collected from clinical cases (animal care and ethics committee approval was not required) via electroejaculation, and samples (for sperm morphological assessment) were submitted in 1.5 ml snap top eppendorf tubes containing 1.0 ml diluent. samples initially assessed were submitted in diluent consisting of isotonic pbs that was used to assess bull-side motility. these samples were prepared 10 days after collection, and microscopically assessed 11 and 12 days after collection. the correct samples, consisting of 200 µl of raw semen collected onsite and placed in 1 ml 10% bfs, were submitted and assessed 2 months after collection. at the laboratory, the contents of the eppendorf tubes were gently agitated, and 20 µl of the eppendorf tube contents were placed on a 25 x 75 mm microscope slide (westlab 25 x 75 x1  mm; www.westlab.com.au). a 22 x 40 mm cover slip mailto:allangunn@bigpond.com http://dx.doi.org/10.58292/ct.v16.9972 http://www.westlab.com.au 2 citation line: clinical theriogenology 2024, 16, 9972, http://dx.doi.org/10.58292/ct.v16.9972 (menzel-glasser; deckglasser 22 x 40 # 1) was placed over the drop and left overnight to settle. sperm on the slide were evaluated morphologically using oil immersion 1000 x differential interference microscopy (nikon eclipse 80i). sperm morphology was assessed, and the anatomical abnormalities were recorded. one hundred sperm were counted and classified per slide. data were analysed by comparing the median percent normal morphology in each group using paired wilcoxon test and p value was set at ≤ 0.05 for significance. results thirteen pairs of samples were submitted and analysed after the initial submission error. comparison of the 13 paired pbs and bfs samples revealed substantial differences in sperm morphology between the storage methods. for good fertility there should be a minimum of 70% morphologically normal sperm in an ejaculate, with not > 20% having nuclear abnormalities or proximal droplets, and not > 25% acrosomal or tail abnormalities.3 bfs samples had 12/13 with ≥ 68% morphologically normal sperm and 1/13 of the pbs samples had ≥ 68% normal sperm with 5/13 having < 20% normal sperm. there were 2 samples in the bfs cohort that had ≥ 19% loose or detached heads compared to 12 in the pbs cohort that had ≥ 35% (7 of them had ≥ 55% loose or detached heads [table 1]). most abnormalities recorded in the pbs samples were a combination of loose and detached heads. there were differences (p = 0.0021) in the percentage of morphologically normal sperm between (figure 1) pbs and bfs groups. most of the abnormal sperm were classified as either loose (figure 2 a-b; b.) or detached heads (figure 2 a-b; a.). a substantial proportion of remaining tail portions (figure 2 a-b; c.) were abnormal. they were not recorded as such, as they had already been recorded as detached heads. discussion bull breeding soundness examination has evolved from the origins of what is now the society for theriogenology, with the intention of determining the likelihood of infertile and subfertile bulls. male fertility is determined by spermiogram assessment, with sperm morphology as the prime indicator of male fertility4 and is correlated to sperm dna damage.5 unacceptable sperm morphology has been the most common reason cited for not having bulls classified as satisfactory breeding animals.6 table 1. proportion of normal and abnormal (tail, midpiece, and head abnormalities) sperm in the samples of ejaculates stored in phosphate buffered saline (pbs) or buffered formol saline (bfs) sample number percent normal percent abnormal tail percent abnormal midpiece percent abnormal head percent loose/ detached head 1 pbs 21 6 1 0 72 1 bfs 82 0 11 1 6 2 pbs 58 1 2 0 39 2 bfs 82 0 7 0 11 3 pbs 63 2 0 0 35 3 bfs 75 0 2 0 23 4 pbs 2 32 1 0 65 4 bfs 81 2 6 1 10 5 pbs 69 0 3 0 28 5 bfs 68 0 13 0 19 6 pbs 39 2 3 0 56 6 bfs 86 0 3 0 11 7 pbs 19 4 2 0 75 7 bfs 83 0 8 0 9 8 pbs 61 2 2 0 35 8 bfs 85 0 2 1 12 9 pbs 49 4 5 0 42 9 bfs 89 0 7 1 3 10 pbs 9 4 1 0 86 10 bfs 76 0 9 1 14 11 pbs 0 0 0 0 100 11 bfs 84 0 2 1 13 12 pbs 62 0 0 0 38 12 bfs 89 0 5 3 3 13 pbs 0 7 1 0 92 13 bfs 41 2 37 13 7 http://dx.doi.org/10.58292/ct.v16.9972 citation line: clinical theriogenology 2024, 16, 9972, http://dx.doi.org/10.58292/ct.v16.9972 3 there were a substantial number of detached heads in most of the original (pbs) samples that were assessed from this cohort of bull ejaculates (table 1). reasons for detached heads (a small proportion [5.1 ± 0.74%] is acceptable) include: bulls with testicular hypoplasia (implicated as a hereditary trait in at least some animals), transient phenomenon in some bulls with testicular degeneration or other inflammatory conditions, and conditions associated with testicular hyperthermia.2 a relatively commonly reported reason for detached heads is senescence as a result of duct stasis.2 other reported defects manifesting as detached heads include the stump tail sperm defect that on closer examination include a rudimentary tail or short stump as a tail. decapitated sperm defect that typically has > 80% of the sperm affected with most part of tails motile and have a characteristic loop in the midpiece.7 in the context of this report (figure 2 a-b; b, c) the short tail sperm defect that has been reported in pigs and anecdotally in cattle7 could be considered a possibility. tails in these samples were not short and the condition is unlikely to occur in such a high proportion of submitted samples. sperm tails were often not critically assessed in the initial examination in these samples as a result of them being detached or loose and were documented as such in the morphological assessment. it illustrates that close examination of the tail, and their attachments to the head were abnormal, with the plasmalemma of the tail being obviously abnormal, probably as a result of bacterial consumption of the phospholipids.8 during sperm morphological assessment, some aberrant movement of the material on the pbs slides was detected and noted. retrospectively, this is abnormal in bfs preserved samples, and is likely because of the presence of motile bacteria or other microorganisms. agglutination of sperm preserved in bfs can be ameliorated by the use of formol citrate.3 tertiary defects are often referred to in texts associated with assessing a spermiogram. they are however, unlikely to have any effect on the fertility of the bull, resulting in diagnostic errors and incorrect decisions on the outcome of the animals concerned. in particular, poor handling and the use of inappropriate extender or in this case preservative, can result in the incorrect diagnosis due to these tertiary defects (table 2).8 conclusion inadvertent error of assessing incorrectly processed semen samples has allowed for a serendipitous opportunity to report abnormalities that are most likely due to incorrect storage of samples submitted for morphological assessment of an ejaculate. it is clear from these observations that appropriate collection and storage of samples for morphological assessment is required when assessing sperm morphology. incorrect sample preparation and storage should be considered as a reason for an abnormal spermiogram, especially when a large proportion of detached heads, with or without tail and midpiece plasma membrane abnormalities/deficits, are detected in a semen sample.8 figure 1. box and whisker plot of the proportion (%) of normal sperm for each of the treatment groups: buffered formal saline (bfs) and extender (pbs); dark line is the median, upper and lower box limits represent the upper and lower quartiles; whisker lines represent the lower and upper data extremes and circles represent individual outlier data points. figure 2. a-b. photomicrographs (differential interference contrast microscopy x 1000) of sperm from semen samples submitted in semen extender (pbs); a. detached heads, b. loose head, and c. detached tails without plasma membranes. http://dx.doi.org/10.58292/ct.v16.9972 4 citation line: clinical theriogenology 2024, 16, 9972, http://dx.doi.org/10.58292/ct.v16.9972 acknowledgement authors thank dr. victoria brookes for data analysis. conflict of interest none to report and no funding is associated. author contributions authors conceived the idea, ag wrote substantial part of the manuscript, mb reviewed it, and both authors approved submission. references 1. kaya a, birler s, enwall l, et al: determinants of sperm morphology. in: chenoweth pj, lorton sp: editors. animal andrology theories and applications. wallingford, oxfordhshire, uk; cabi: 2014. p. 34–56. 2. barth ad, oko rj: abnormal morphology of bovine spermatozoa. ames, iowa; iowa state university press: 1989. 3. barth ad. bull breeding soundness. 3rd edition, sakatoon, sakatchewan, canada; western canadian association of bovine practitioners: 2013. 4. ostermeier gc, sargeant ga, yandell bs, et al: relationship of bull fertility to sperm nuclear shape. j androl 2001;22:595–603. doi: 10.1002/j.1939-4640.2001.tb02219.x 5. enciso m, cisale h, johnston sd, et al: major morphological sperm abnormalities in the bull are related to sperm dna damage.  theriogenology 2011;76:23–32. doi: 10.1016/j. theriogenology.2010.12.034 6. carson rl, wenzel jg: observations using the new bull-breeding soundness evaluation forms in adult and young bulls. vet clin north am food anim pract 1997;13:305–311. doi: 10.1016/ s0749-0720(15)30343-1 7. brito lfc. applied andrology in cattle (bos taurus). 1st edition, wallingford, oxfordshire, england, uk; cabi: 2014. 8. beggs ds: veterinary bull breeding soundness. eight mile plains, qld, australia; australian cattle veterinarians; australian veterinary association ltd: 2013. table 2. abnormal spermiogram findings (tertiary defects) because of sample preparation error is illustrated (reproduced8 with permission) 8.8.11 defects caused by sample preparation several sperm problems, including those related to movement and morphology, can be caused by environmental factors which occur after the sample is collected that can lead to errors in diagnosis and prognosis. common problems listed in this category are listed below: sample preparation error indicators in sperm cold shock • observation of particular sperm movements, such as moving backwards, circling and ‘shimmering’ in place. • suspiciously large difference between motility and live/dead assessments (especially if sperm morphology is otherwise good). • increased distal midpiece reflexes without accompanying retained droplets poor handling (temperature, contamination, rough handling, inappropriate extender) • decreased percent intact acrosomes • increased loose/degenerating acrosomes • presence of evident numerous bacteria • excessive clumping • increased bent and coiled tails • increased loose/detached sperm heads • evidence of crystal formation nonisotonic media • increased distal midpiece reflexes and tail abnormalities poor preparation/staining/microscopy • sperm stained too dark, too light • sperm too concentrated or too sparse on the slide • ‘halo’ effect and ‘cracks’ in the stain monolayer • undetected subtle sperm defects (especially if consistent) • curiously increased numbers of narrow heads excessive motion or torsion (coverslip) • increased loose/detached sperm heads • evidence of disruption (broken midpieces and tails, sperm debris) of the sperm preparation p. 92 examination of sperm bull breeding soundness evaluation. http://dx.doi.org/10.58292/ct.v16.9972 https://doi.org/10.1002/j.1939-4640.2001.tb02219.x https://doi.org/10.1016/j.theriogenology.2010.12.034 https://doi.org/10.1016/j.theriogenology.2010.12.034 https://doi.org/10.1016/s0749-0720(15)30343-1 https://doi.org/10.1016/s0749-0720(15)30343-1 _hlk149118499 _hlk153015088 2018: evolution of cervine, caprine and ovine sex-sorted semen processing evolution of cervine, caprine and ovine sex-sorted semen processing clara gonzález-marín stgenetics, navasota, tx introduction it has been three decades since the first publication showing that flow cytometry was a reliable method to separate x and y chromosome bearing sperm based on their difference in dna content (johnson et al., 1989). ten years after that publication, the first commercial straw containing 2.1 x 106 frozen sex-sorted bovine sperm was released to the market for standard artificial insemination (garner and seidel, 2008). several improvements have happened in the flow cytometry technology since that first commercial straw was released, such as the introduction of orienting nozzles, digital processing, multiple headed sorters, and automation, in a new generation of faster and more efficient sperm sorters known as genesis (sharpe and evans, 2009; evans, 2010). significant enhancements in sperm handling, preparation for sorting and media composition have also allowed for sperm quality and conception rates of sex-sorted sperm to reach levels that are comparable with non-sorted (conventional) semen (vishwanath et al., 2014; vishwanath, 2014; gonzález-marín et al., 2018; de graaf et al., 2014). the result is a complete overhaul of the conditions under which sperm is processed and sorted, known commercially as sexedultra™. today, beef and dairy cattle sperm sorting laboratories are operating commercially in more than 25 locations, in 14 countries, with an annual production of more than eight million straws. in the past few years, small ruminant industries have been progressively testing and implementing the sperm sex-sorting technology for application in their specific environments. the demand for sheep and goat products has increased considerably worldwide since these small ruminants are easily managed, require a relatively small initial investment and their short generation interval lends itself to a fast return on investment for farmers. also, dairy goat production keeps drawing the attention of producers due to the health benefits of milk and the popular cheeses and dips made from it. on the other hand, cervine industries have been slowly integrating sex-sorted sperm into their artificial insemination practices for antler trophy hunting and genetic improvement of the herds. sex-sorted sperm will allow these growing industries to produce optimal proportions of males and females, improve herd management and increase the rate of genetic progress. keywords: sex-sorted sperm, fertility, small ruminants cervine sex-sorted sperm – the bucks win the captive deer breeding industry has experienced an important period of growth worldwide in recent years (garde et al., 2006; gao et al., 2011). there has been an increased trend toward deer gaming farms where the main financial profits rely on antler trophies. given that males have the highest economic value, sex-sorted sperm represents significant management cost-savings. in vitro sperm quality studies comparing post-thaw motility and dna fragmentation kinetics of sex-sorted and conventional sperm of red and white-tailed deer have shown equal or better semen characteristics for the sex-sorted samples (kjelland et al., 2011). commercial production of cervine sex-sorted sperm started in 2009 in the headquarters for stgenetics in navasota, tx. since then, there has been a steady market for both fresh and frozen sexsorted sperm in white-tailed deer in the united states, amounting to many thousands of straws produced every year. in vitro quality analysis of white-tailed deer sperm including total visual motile sperm, acrosome integrity, computer assisted sperm analysis (casa) for total and progressive motile and gender purity is part of the commercial production routine, and demonstrates equal or better semen characteristics for the sex-sorted compared to conventional samples (table 1). clinical theriogenology • volume 10, number 3 • september 2018205 table 1. number of fresh and frozen straws produced for white-tailed deer in navasota, tx in the past four years. percent visual motile, intact acrosomes, casa motile and progressive, and gender purity were measured 20 minutes after thawing the straws for frozen semen and at day 1 after sorting for fresh semen. straws produced % visual motile % intact acrosomes % casa motile % casa prog. % gender purity 2014 conventional frozen 2588 55.1 70.5 55.6 46.4 sex-sorted frozen 7537 59.9 73.8 71.2 65.8 91.5 sex-sorted fresh 439 61.6 78.2 70.5 52.5 91.5 2015 conventional frozen 2197 56.1 70.4 65.2 37.5 sex-sorted frozen 8676 59.0 72.3 66.5 38.8 92.1 sex-sorted fresh 826 63.5 79.1 77.0 55.3 90.3 2016 conventional frozen 1466 58.8 70.5 63.4 44.5 sex-sorted frozen 9347 60.7 77.1 68.4 42.7 91.3 sex-sorted fresh 699 74.9 87.3 88.1 63.5 90.7 2017 conventional frozen 1455 57.5 71.0 62.7 43.2 sex-sorted frozen 7354 62.5 79.3 71.1 43.4 90.9 sex-sorted fresh 837 74.1 86.6 87.5 62.7 90.6 total conventional frozen 7706 56.9 70.6 61.7 42.9 sex-sorted frozen 32914 60.5 75.6 69.3 47.7 91.5 sex-sorted fresh 2801 68.5 82.8 80.8 58.5 90.8 information on fertility of sex-sorted deer sperm is very vague. some reports point towards the resilience of deer sperm to withstand the sorting process and maintain good fertility. fertility trials with red deer using 3 x 106 sex-sorted sperm on two separate ranches showed that pregnancy results were similar using conventional and sex-sorted sperm (brigans et al., 2010). other studies show a slightly lower fertility of y sorted sperm when using iberian red deer (anel-lopez et al., 2017), although pregnancy rates were significantly higher when hinds were inseminated closer to ovulation induction, so lower fertilities could be attributed to the need of devising synchronization protocols for this specific species when using sex-sorted sperm samples (anel-lopez et al., 2018). there are no published reports on the application of sexedultra™ in deer semen, but personal communication from the sorting laboratory confirms that, when the proper breeding management and synchronization protocols are used, sex-sorted frozen cervine sperm presents pregnancy rates of ~93-95% to those of conventional (70% vs 74%), and fresh sex-sorted sperm is achieving average conception rates 5-8% better than those of conventional. gender purity in the field is 92-95% (personal communication. jared templeton, global production manager. stgenetics). caprine sex-sorted sperm – the kids matter goat sperm sex-sorting has become a recent interest, especially as the dairy goat industry continues to expand. in 2013, one of the few publications regarding sex-sorted goat semen reported successful sorting and birth of kids after laparoscopic intrauterine artificial insemination (lai) with about 32 x 106 sperm per insemination of either sex-sorted or conventional sperm. in this report, fertility was lower for sex-sorted sperm, but the success of the technique was demonstrated (bathgate et al., 2013). clinical theriogenology • volume 10, number 3 • september 2018 206 the magnitude of the dna content between the sex-determining gametes varies among species. the average difference between x and y sperm dna content in bovine sperm is 3.8% (garner, 2006) while, for caprine, this difference is closer to 4.3%, so the separation of x and y chromosome bearing sperm using flow cytometry is not a problem when using ram sperm. also, the implementation of sexedultra™ procedures and straw freezing, have resulted in a successful commercialization of caprine sex-sorted sperm that started at the end of 2015. since then, over 11,000 sex-sorted straws have been produced for lai purposes with an average post-thaw visual motile sperm of 60%, a casa total and progressive motile of 67% and 59% respectively, intact acrosomes of 74% and a gender purity of 93%. in small scale field trials in waco, tx, 75 does were divided into two groups and inseminated with sex-sorted fresh (2 x 106 sperm per insemination) or sex-sorted frozen sperm (4 x 106 sperm per insemination). pregnancy rates were 57% for fresh and 49% for frozen sperm. these results are comparable to conventional semen used in the same farm (personal communication. earl peacock, owner. premiere semen). further trials in camperdown (australia) with 150 does split into three groups and inseminated using lai with conventional frozen semen at 20 x 106 sperm per insemination and sex-sorted frozen sperm at 4 and 2 x 106 sperm per insemination demonstrated no difference in pregnancy rates between conventional and sex-sorted sperm, and between the two types of sex-sorted sperm doses (personal communication. ponneelan ganesan, laboratory manager. sexing technologies australia). most caprine inseminations occur trans-cervically, so the current challenge is to be able to deliver a good fertile dose of sex-sorted semen that can be used for this purpose. therefore, researchers are investigating the optimal dose for trans-cervical insemination in sheep. contemporaneous ejaculates from four high genetic value bucks were processed as conventional semen (208 straws) and sex-sorted sperm at concentrations of 4 x 106 (160 straws) and 8 x 106 (287 straws). sex-sorted sperm presented an average post-thaw visual motile sperm of 62%, intact acrosomes of 77% and a gender purity of 93%, and has now been released to the field for trans-cervical insemination. ovine sex-sorted sperm the exception to the rule the use of sex-sorted sperm has genetic, management and financial benefits for dairy, wool and/or meat sheep production. catt et al. (1996) presented the first report using ram sex-sorted sperm, where 85 conventional, 92 female-sorted and 74 male-sorted ram sperm were injected into in vitro matured sheep oocytes and placed into the oviducts of 28 estrous sheep. one pregnancy was diagnosed by ultrasonography after 55 days from an oocyte injected with 'male-sorted' sperm. besides this initial study, the use of ivf and icsi is not commercially relevant for the ovine industry, so research has been focused on sex-sorted, fresh and frozen sperm to be used in lai. the first pregnancies after lai with sex-sorted frozen-thawed sperm were achieved using low numbers of sperm per dose (2-4 x 106). the overall pregnancy rate for ewes inseminated with sex-sorted sperm was half that of conventional controls (140 x 106 sperm; hollinshead et al., 2002). further testing of in vitro quality parameters of sex-sorted ram spermatozoa showed a reduced total and progressive motility and a tendency towards premature capacitation in sex-sorted sperm compared to conventional (hollinshead et al. 2003). combining these findings suggested that sex-sorted ram spermatozoa had a reduced fertilizing lifespan, which would explain the decrease in fertility. in subsequent field experiments, the fertility problems were shown to be partly improved by increasing the number of sexsorted sperm per insemination (hollinshead et al. 2003), but this was not a viable solution considering the commercial imperative to minimize the number of sex-sorted sperm per lai dose. however, de graaf et al. (2006) later reported that sex-sorted ram spermatozoa presented higher motility, viability, acrosome integrity and mitochondrial activity than non-sorted controls. in vivo studies supported these in vitro results, demonstrating that sex-sorted ram sperm result in similar or superior fertilization/lambing percentages than conventional controls (de graaf et al., 2007; beilby et al., 2009). it appeared that the sex-sorting process could select a functionally superior population of sperm in terms of both in vitro and in vivo function from the ejaculate, resulting in sex-sorted ram sperm with a superior fertilizing lifespan inside of the female reproductive tract compared with conventional sperm from the clinical theriogenology • volume 10, number 3 • september 2018207 same ejaculate. since that moment, ram sex-sorted sperm was considered an exception to the long-held rule that sex-sorting negatively impacted sperm function to an extent where fertility was compromised. for the past three years, researchers at stgenetics have been working to make the sex preselection technology a commercially viable and effective reproductive management option for the sheep industry. the research performed has been focused on adapting sexedultra™ bovine sperm sorting procedures for ovine semen, and to replace the pellet freezing method (evans and maxwell, 1987) that was used in all other previous experiments using sex-sorted ovine sperm, since this method is not a commercially viable option. in the first field trial (new zealand, 2017), ejaculates from two rams were split and processed in one of two methods: conventional or sex-sorted. conventional (conv) semen was processed at 60 x 106 per dose and sex-sorted sperm was processed at a gender purity of 92% as fresh semen at 1 x 106 cells per dose (fresh1m), fresh semen at 2 x 106 cells per dose (fresh2m), cryopreserved in pellets at 3 x 106 cells per dose (cryo3m) and cryopreserved in straws at 6 x 106 cells per dose (cryo6m). percent visual sperm motilities were analyzed after final dilution (0 h) and after 24 h of incubation at 18°c for sex-sorted fresh semen, and after thawing (0 h) and after 3 h incubation at 37°c for sex sorted cryopreserved semen. no differences (p < 0.05) were found in percent total motile between fresh1m and fresh2m at 0 h (73.0% vs 73.0%) or after 24 h (71.0% vs 71.0%). however, sperm motility was greater in fresh semen compared to cryo3m at 0 h (73.0% vs 69.0%) and after incubation (71.0% vs 63.0%). percent total motile sperm was the lowest in cryo6m at 0 h (44.0%) and 3 h after incubation (43.0%). no statistical differences (p < 0.05) were found in the percentage of ewes lambing after insemination between treatments (table 2). this study confirmed that sex-sorted ram sperm are equally fertile to conventional. in fact, 6 x 106 cryopreserved sex-sorted sperm and 2 x 106 sex-sorted fresh sperm presented numerically superior fertility when used in lai than conventional sperm inseminated at higher concentrations. additionally, no difference was observed between fresh and cryopreserved sex-sorted sperm, which would allow more flexibility once the product is commercialized. finally, sorted sperm cryopreserved at 6 x 106 per straw presented numerically higher conception rates than sorted sperm cryopreserved at 3 x 106 pellet, which would ensure ease of use in the sorting laboratories and in the field. table 2. pregnancy rate and lambing information after insemination of synchronized ewes with 60 x 106 total frozen-thawed conventional, 3 or 6 x 106 total frozen-thawed sex-sorted, and 1 or 2 x 106 total fresh sex-sorted ram sperm. type/dose of semen ewes inseminated lambed (%) born/ewes lambing conventional frozen-thawed 60x106 60 41.8 1.6 sex-sorted fresh 1x106 57 35.8 1.4 sex-sorted fresh 2x106 56 43.6 1.4 sex-sorted frozen 3x106 (pellet) 62 32.8 1.4 sex-sorted frozen 6x106 (straw) 51 56.1 1.3 it is likely that the promising results in sheep with sex-sorted sperm could in part be due to lai procedures, where the sperm are placed at the tips of the uterine horns. more trials are needed at this time to determine optimal synchronization protocols and the minimum sex sorted sperm per dose that would allow a corresponding decrease in the associated cost per dose, but there is no question that this product could become an important breeding option at the elite stud level as well as the commercial farm level in the next couple of years. the future of small ruminant sperm sorting in the past decade, sheep and goat production has increased by about one-third due to their economic value as efficient converters of low-quality forages into quality meat, milk, and wool. the deer gaming farms have also experienced an important period of growth worldwide. clinical theriogenology • volume 10, number 3 • september 2018 208 for these growing industries, it is imperative to use all available pregnancies to modify the offspring sex-ratio in order to generate productive animals (females or males), allowing for faster genetic progress and increased production while reducing wastage. sperm sex-sorting by flow cytometry is the only reliable technology to separate x and y chromosome bearing sperm based on their difference in dna content. the technology has now been validated for all three species on the basis of laboratory analysis and live births, and incorporates modified flow cytometric sorting instrumentation and sexedultra™. acknowledgments the talented r&d and production teams at sexing technologies and stgenetics, and all the field trial participants in the united states and overseas. references anel-lópez l, garcía-álvarez o, parrilla i, et al: effect of sex-sorting and cryopreservation on the post thaw sperm quality of iberian red deer spermatozoa. theriogenology 2017; 89:206-213. anel-lópez l, garcia-álvarez o, tarantini t, et al: influence of insemination time on the fertility of sex sorted frozen-thawed ysperm in red deer. theriogenology 2018; 113:171-175. bathgate r, mace n, heasman k, evans g, et al: birth of kids after artificial insemination with sex-sorted, frozen-thawed goat spermatozoa. reprod domest anim 2013; 48:893-898. beilby kh, grupen cg, thomson pc, et al: the effect of insemination time and sperm dose on pregnancy rate using sex-sorted ram sperm. theriogenology 2009; 71:829-835. bringans m, kjelland m, lenz, rw, et al: artificial insemination of red deer using sex-sorted sperm. proc 5th world deer congress 2010; 21-23. catt sl, catt jw, gomez mc, et al: birth of a male lamb derived from an in vitro matured oocyte fertilised by intracytoplasmic injection of a single presumptive male sperm. vet rec 1996; 139:494-495. de graaf sp, evans g, maxwell wmc, et al: in vitro characteristics of fresh and frozen-thawed ram spermatozoa after sexsorting and re-freezing. reprod fertil dev 2006; 18:867-874. de graaf sp, evans g, maxwell wmc, et al: successful low dose insemination of flow cytometrically sorted ram spermatozoa in sheep. reprod domest anim 2007; 42,648-653. de graaf sp, leahy t, vishwanath r: biological and practical lessons associated with the use of sexed semen. reprod domes rumin 2014;viii:507-522. evans g, maxwell wmc: salamon’s artificial insemination of sheep and goats. sydney: butterworths.; 1987. p. 127-130. evans km: interpretation of sex-sorting process and new developments. proc 25th tech conf ai reprod 2010; 93-98. gao qh, wang he, zeng wb, et al: embryo transfer and sex determination following superovulated hinds inseminated with frozen–thawed sex-sorted y sperm or unsorted semen in wapiti (cervus elaphus songaricus). anim reprod sci 2011; 126:245250. garde jj, martínez-pastor f, et al: the application of reproductive technologies to natural populations of red deer. reprod domest anim 2006; 41:93-102. garner dl, seidel jr ge: history of commercializing sexed semen for cattle. theriogenology 2008;69:886-895. gonzález-marín c, góngora ce, gilligan tb, et al: in vitro sperm quality and dna integrity of sexedultra™ sex-sorted sperm compared to non-sorted bovine sperm. theriogenology 2018;114:40-45. hollinshead fk, o'brien jk, maxwell wm, et al: production of lambs of predetermined sex after the insemination of ewes with low numbers of frozen-thawed sorted xor y-chromosome-bearing spermatozoa. reprod fertil dev 2002; 14:503-508. hollinshead fk, gillan l, o’brien jk, et al: in vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing. reprod fertil dev 2003; 15:351-359. johnson la, flook jp, hawk hw: sex preselection in rabbits: live births from x and y sperm separated by dna and cell sorting. biol reprod 1989;41:199-203. kjelland me, gonzález-marín c, gosálvez j, et al: dna fragmentation kinetics and postthaw motility of flow cytometric-sorted white-tailed deer sperm. j anim sci 2011;89:3996-4006. sharpe jc, evans km: advances in flow cytometry for sperm sexing. theriogenology 2009;71,4-10. vishwanath r, sedoglavich n, evans km, et al: industrial use of sorting flow cytometry in gender preselection: lessons learned. xxix cong int soc adv cytometry 2014; abstr no 147. vishwanath r: sexedultra – raising the fertility bar of sexed sorted semen. proc 25th tech conf ai reprod 2014:57-61. clinical theriogenology • volume 10, number 3 • september 2018209 clinical theriogenology • volume 10, number 3 • september 2018 210 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2018: planning and management of mares and recipients for healthy pregnancies and foals planning and management of mares and recipients for healthy pregnancies and foals claire card department of large animal clinical sciences, western college of veterinary medicine, university of saskatchewan, saskatoon, sk, canada developmental origins of health and disease the developmental origins of health and disease (dohad) in horses is a growing area of investigation that concerns the way the maternal environment influences the foal’s future health. maternal environmental clues program important physiologic adaptations in the foal such as: conformation, energy homeostasis, osteochondral health, and thyroid function.1 this topic was also investigated and described as ‘fetal programming’ where placental, fetal and postnatal development were studied.2,3 maternal nutrition during pregnancy, stress and exposure to harmful substances during critical time periods are known to have an effect on the overall health of her offspring.1-3 the foundational research on the dohad involves studies on epigenetic mechanisms. epigenetic mechanisms ‘epigenetic’ is a term applied to the myriad complex interactions between the genome and environmental factors during development and differentiation. epigenetic mechanisms are induced by modifications of the cellular environment. epigenetic mechanisms include dna methylation and posttranscriptional modification.4 the methylation of dna can block binding of transcription factors, this is a mechanism where expression of unwanted or surplus genes may be suppressed. methylation of dna is referred to as an ‘epigenetic mark’. as a cell divides these epigenetic marks are propagated, which becomes a non-genetic means of altering physiologic functions. the epigenetic marks may result in physiologic effects a very long time after the event that resulted in their formation, occurred.1 for example, in pregnant rats a low protein diet has deleterious effects on the health of her offspring, where the rat mother’s low protein diet results in the birth of pups with altered mrna expression attributed to epigenetic processes.5 periconceptional period what has recently been described is how the periods just before and just after fertilization, in what is called the pre and periconceptional period, are important in the dohad.1,4 the periconceptional period spans the time from fertilization to blastocyst development. patterns of gene expression in the embryo are modulated by events such as dna methylation and histone modification, in a process termed ‘epigenetic regulation’. the dynamics of the methylation pattern is also affected by processes during development, and are influenced by maternal age, parity and gender. ‘deep epigenetic modifications’ occur during the development of the gametes.1,4 the mother’s and father’s nutritional status at the time of gamete formation is therefore important. research has shown that gamete development is influenced by the nutritional status of the dam and sire during their production, after fertilization and during the periconceptual period.4 assisted reproductive procedures may impact health and alter subsequent metabolism, cardiovascular function and body weight of the individual.1,6 the milieu during pregnancy and neonatal life, has been reported to have long-term effects. there is however some plasticity in individuals, because epigenetic modification occurs throughout the life of the individual.1 in females, oocytes are formed in fetal life and then decrease over the life of the individual, in males, testicular germinal epithelial cells begin active division at puberty and constantly produce sperm through the process of spermatogenesis. therefore, epigenetic process that involved gametes may produce different effects due to the functional and structural differences in male and female gametes. effect on assisted reproductive technologies the conditions for successful embryo technologies include optimized culture media for gametes and embryos.5 problems with in vitro fertilized embryos, such as large offspring syndrome, are believed to be a result of epigenetic processes.4,5,7 in vivo and in vitro derived equine embryos show different patterns clinical theriogenology • volume 10, number 3 • september 2018259 of gene expression.6 the difficulties with somatic cell clone nuclear transfer are also attributed to altered gene regulation by epigenetic processes during embryonic genome activation, which leads to developmental defects in embryos.7 in utero development many factors that influence a mare’s health also play a role in the health of the mare’s foals. the health of the brood mare in terms of her metabolism, nutrition, and whether assisted reproductive technologies, such as embryo transfer or intracytoplasmic sperm injection, were utilized to establish the pregnancy, have been shown to affect: feto-placental development, foal birth weight, and foal growth.8 the effects of breed using cross over studies of larger and small breed embryo donors and embryo recipients has been described in horses.1,8 walton and hammond (1938) using artificial insemination crossed shetland pony mares and shire draft horses to illustrate these differences, and they showed that the mare carrying the pregnancy regulated fetal size, and post-natal growth rate of the foal.1,8 as expected of these crosses the shire offspring of shetland pony mares remained smaller as adults and shetland offspring of shire mares remained larger as adults.8 other researchers studied osteochondral health of foals born following a pregnancy that involved embryo transfer and a size mismatch. this research illustrated why matching donor and recipient mare size is advantageous in reducing osteochondrosis in foals.9 nutrition and feeding practices in the feral state mares would typically foal in spring near the time of lush pasture availability, and then encounter less abundant feed in autumn and even less in winter. the feral mare supports the final growth of the foal often using her energy reserves.1 foaling would coincide around the time the resurgent growth of pasture occurs. the national research council (nrc) however recommends feeding mares a maintenance diet through the fifth month of pregnancy and then increasing the mare’s plane of nutrition to account for fetal growth.10 while this feeding recommendation makes sense to prevent weight loss during pregnancy, there is a mismatch between the natural ebb and flow of energy that a feral horse would encounter and the subsequent body weight changes a feral mare would experience induced by those energy flows, and the currently recommended nrc feeding practices. how our feeding practices influences epigenetic events in horses is currently an area requiring further investigation. half of the cost of keeping horses is feeding them.11 an evidence-based feeding program is best to optimize horse health. some very interesting epidemiologic studies showed that mares fed concentrate during pregnancy had foals of similar birth weight compared to mares that were fed only forage. the foals of belgian ardennes mares fed the concentrate however had a prevalence of osteochrondrosis of 33-38% while those mares fed forage had an eight-fold reduction in ocd in their foals compared to the grain fed mares, that is a 4% prevalence of ocd.12 the foals born to the grain-fed mares, however had better regulation of glucose, but delayed puberty, and thinner cannon bones. the effects of grain feeding the mare on the foal’s subsequent glucose homeostasis, likely occurs through epigenetic mechanisms and the subsequent changes in the processes and hormones that regulate metabolism and maturation.1,12 the pregnant mare is often overfed during pregnancy (eating for two) and underfed during lactation. the reality is that many mares enter the breeding season already overweight. a report on body condition of horses in saskatoon, saskatchewan, canada revealed that 20% of horses are over-conditioned and 8% are obese.13 it is common for many brood mares to have a bcs of 7/9. in europe it is estimated that mares in late pregnancy are fed from 10-40% more than their nutritional needs.1 practitioners should encourage mare owners not to normalize excess body condition in mares. the trends in body condition scores or mare weights should be recorded as part of herd management. mares should enter into pregnancy with a healthy body condition. ideally mares should have a bcs of 6/9 during pregnancy. adult horses with free access to pasture may spend up to 90% of their 24-hour time budget eating. mares with free access to forage may consume 3% of their body weight in kilograms when they only require 1.5-2%, for maintenance and early-mid pregnancy.10,11 mare’s access to pasture may need to be limited, as pasture provides 24-7 availability of feed, and unlike the feral horse a subsequent decrease in feed availability is not encountered. pasture grasses are high in vitamins and beta carotene, they are also clinical theriogenology • volume 10, number 3 • september 2018 260 full of highly digestible carbohydrates.15 lush grass may be problem for horses with heavier body condition, and for mares that have endocrine or metabolic problems. these endocrine (for examine pituitary pars intermedia dysfunction; ppid) and equine metabolic syndrome mares are predisposed to develop laminitis. laminitis in horses is now reported to be a syndrome, rather than just an acute event, and it is reported that endocrine factors are involved in many horses.14 supplementation of most mares with concentrate or richer forage such as alfalfa, does not need to occur before the last third of pregnancy. it is common although unwise, for mare owners not to have a feed analysis performed on their hay. an analysis of the hay assists with the choice of a ration balancer and or mineral mixture. as the mare nears foaling the fetus will gain about 0.5 kg/day. the mare’s nutritional needs increase so she needs 20% more digestible energy to maintain body weight. this may often be accomplished by feeding more hay or a higher quality hay. hay nets with small diamonds provide a more controlled access to the feed in a manner that more closely mimics natural grazing.10,11 vitamins and minerals the relationship between micronutrients and health was recognized in north america in the 1930’s when iodine deficiency and thyroid disease, which characteristically resulted in goiter formation, was identified in both people and horses. iodine deficiency was linked to the low iodine content of the soil and diet, which resulted in goiter formation, and hypothyroidism.11,15 other micronutrient deficiencies, such as selenium, were identified and associated with white muscle disease in horses.16 copper deficiency in horses was associated with poor hair coat, anemia and orthopedic disease. zinc deficiency was associated with poor hair coat, lower fertility and impaired immune function, copper deficiency results in poor hair coats, anemia and orthopedic disease.10,11 micronutrients are an essential part of any feeding program. micronutrients include trace minerals important for horses such as the aforementioned: iodine, copper, selenium, and zinc but also includes some vitamins such as a and d.10,11 these nutrients are key to equine health as they are required for: metabolic function, growth, milk production, and reproduction. requirements for vitamins and minerals are higher during pregnancy. an ongoing concern is that because horse owners do not routinely analyze their forages, they guess at the level of mineral supplementation required for their mares, and may use only mineral blocks for supplementation. this commonly results in horses having an inadequate nutritional intake of micronutrients, such as iodine, selenium, zinc and copper in their diet.10,11 in addition, consumption of minerals by horses is uneven, when minerals are provided ad libitum in salt-based blocks, or as a loose mixture. most horses do not consume enough of the block minerals to meet their requirements. in spite of changes in climate and agricultural practices there has been little monitoring of changes in the important micronutrients in the equine population. a 2014 study by the saskatchewan forage council of trace minerals in pastures showed that native and cultivated pasture grasses had insufficient copper and zinc levels to meet the needs of beef cattle.15 as horses graze these same areas, similar results should be expected. other trace minerals were shown to vary by plant species, season and soil zone, indicating that mineral supplementation programs should be based on measuring actual values. selenium deficiency may result in lower glutathione peroxidase and vitamin e levels.17 waldner and coworkers in reported in a comprehensive series of studies of beef cattle herds in western canada that: forages were often deficient in zinc and copper, 43% of cows were copper deficient, vitamin e deficiencies were present in abortuses/stillbirths, and selenium deficiency was present in some neonatal calves.18 forages for horses are grown in the same regions and soils as cattle, and contain the same plant species, hence it is likely that they have similar nutritional characteristics, including micronutrient deficiencies. authors report micronutrients may be monitored through laboratory analysis of mane hair or serum analysis19,20 micronutrient levels have been reported in mare and foal serum and mane hair.19,20 feeding practices have been reported to affect mineral levels in mane hair.20 fetal foal hair forms from about seven months of pregnancy to term, hence the trace minerals in foal mane hair at birth represent a snapshot of the foal’s in utero exposure. the analysis of the trace minerals in a foal’s mane hair may be a useful tool to clinical theriogenology • volume 10, number 3 • september 2018261 analyze the nutrition program of the dam. a periodic analysis of mare serum and newborn foal hair for trace mineral levels would provide valuable information on the feeding program of the farm. the thyroid health of the mother has been identified as a key determinant of her foal’s future health and disease. dietary factors such as iodine deficiency or excess in mares, are known to cause congenital goiter in foals. changes in thyroid hormone levels are related to iodine in the diet in cases of deficiency and excess. exposure of pregnant mares to excess nitrate, plants that disrupt iodine utilization, and trace mineral deficiencies have been found to be risk factors for a disease of foals called congenital hypothyroidism dysmaturity syndrome (chds). the consequences of chds are dramatic and include: thyroid hyperplasia, limb contracture/tendon ruptures, mandibular prognathism, umbilical problems, and severe carpal and tarsal boney dysgenesis. due to abnormal musculoskeletal development, chds foals are either euthanized, or experience high rates of developmental orthopedic disease.21,22 horses may be exposed to goitrogenic plants and seeds (brassica family) due to cultivation and spread of oilseed plants (mustard, canola) and through consumption of pasture weeds. goitrogenic plants contain glucosinolates that when hydrolyzed, are converted into compounds that are either thyrotoxic or that interfere with the uptake or organification of iodine.23 even if iodine intake is adequate these plants may result in iodine depletion over time. the dams of hypothyroid foals also have impaired thyroid function.22 other plants such as fescue grass infected with the fungal endophyte, neotyphodium coenophialum, also pose a risk to pregnant mares. ingestion of endophyte infected fescue may lead to a series of clinical problems such as: prolonged gestation, dystocia, agalactia (ergopeptides mimic dopamine and act to suppress prolactin secretion), failure of passive transfer, and suppressed fetal thyroid function. mares need to be off fescue pasture for a month before foaling, and/or administered domperidone orally daily for 10 days prior to parturition to help prevent premature separation of the placenta (red bag delivery) and agalactia.24,25 nutrition summary appropriate feeding of pregnant mares and stallions is important for the long term health of the foal. epigenetic processes begin during gametogenesis of the parents and continue in the periconceptual period. fetal development and postnatal growth of the foal is affected by the conditions of pregnancy, such as uterine size, and feeding of the dam. recipients of embryos should be similar in size and breed to the donor mares. minimal grain feeding to pregnant mares should be considered to prevent metabolic alterations that may lead to osteochondrosis in her foal. mineral salt block access alone is unlikely to supply sufficient trace minerals for the requirements of pregnancy. plants such brassica family mustard plants and endophyte infected fescue may result in disease and suppress thyroid function. serum or milk trace mineral analysis is a direct way to detect mineral depletion, while function markers such as thyroxine, vitamin e, or glutathione peroxidase are more helpful in identifying diseases caused by an underlying deficiency, or glandular dysfunction.23 embryo transfer recipient selection the recent research points to the need for excellent multi-generational management of horses. embryo transfer recipient mares should preferably be similar in size and breed to donor mares to minimize epigenetic problems. mares under the age of 12 are generally preferred, as this corresponds to the most fertile years of a mare’s life. preferably recipient mares are tractable and have had a successful foaling. the recipients should have no history of reproductive problems, and they should be in good body condition at breeding. they should have normal estrous cycles. excellent perineal conformation is desirable.26 synchronization of recipients hormonal treatment may be utilized with anestrus, transitional and cycling, recipient mares to simulate the conditions of a mare that has been in estrus, or align the donor and recipient mare’s ovulation. the ovulation of donor and recipient mares should be closely aligned. generally, it is desirable for the recipient mare to ovulate from one day before the donor’s ovulation and up to three days after the donor’s ovulation.26 clinical theriogenology • volume 10, number 3 • september 2018 262 seasonal asynchrony between donors and recipients if the donor mare is cycling because she was treated with artificial light, and the available recipient mares are in deep anestrus or transition, anestrus and transitional mares may be treated to prepare them for an embryo transfer. the protocol for the prospective recipient is started on the day the donor ovulates. the recipient is administered 6.6 mg estradiol 17β im for two consecutive days followed by 5 to 7 days of a short-acting progesterone preparation (200 mg, im, q 24h). she is then ready for a transfer. supplemental progesterone must then be provided using daily administration of altrenogest (0.44 mg/kg) or treatment using long-acting progesterone formulations (1,500 mg, progesterone im), or long acting altrenogest (150 mg) im once weekly, and continued if the pregnancy has been established for 100 days.27 there is a microparticle compounded formulation of altrenogest that has activity for 30 days. an alternative protocol involves on the day of the donor ovulation beginning recipient treatment with estradiol benzoate for 3 days once daily im using 5mg, then 3 mg, and followed by 2mg of estradiol benzoate and 400 mg of long-acting progesterone weekly until 120 days.27 cycling but asynchronous donors and recipients there are newer hormonal strategies to try for aligning donor mares and recipients. recipient mares in early diestrus (days 1-5) may be administered two doses of prostaglandin f2alpha (pgf) 24 hours apart as the early corpus luteum is more resistant to prostaglandin, and mares from day 5-16 may be administered one 5 mg sq dose of pgf. the follicular structures on the ovary at the time of pgf treatment in cycling mares determines the time to estrus. follicles typically grow at 3 mm per day. mares typically start to exhibit signs of estrus when a dominant follicle reaches 30 mm. the timing of the pgf treatment therefore needs to include consideration for the follicle size of the donor and recipient to align their estrus. ovulation induction agents are then utilized to further tighten the synchrony.26 to align diestrus mares with estrus donor mares, diestrus recipient mares may be administered pgf and 10 mg estradiol-17β im on the day of or the day after the donor mare’s ovulation (day -4). the administration of estradiol-17β was repeated on day -3 (10 mg estradiol-17β im), day -2 (20 mg estradiol17β im), and day -1 (10 mg estradiol-17β im). on day 0 (named to mimic the ovulation day), mares received long-acting altrenogest (300 mg im). recipient mares treated in this fashion, appear to be able to receive embryos from day +3 to day +8 after long-acting altrenogest (300 mg im). prior to the transfer of the embryo the recipient mare received a second treatment with altrenogest and had uterine features assessed. in these recipient mares on days +10 to +12 a second treatment with long acting progesterone (1500 mg) was administered.28 if the transferred embryo is present in the recipient mare, then long acting progesterone is administered once a week until 100 days or until supplementary corpora lutea form. altrenogest has also been administered.28 management of pregnant mares mare group size it is strongly recommended to keep pregnant mare groups stable, with small groups sized (fewer than 20 mares), with mares of similar ages, after the fifth month of pregnancy.29 pregnant mares should not have fenceline contact with horses that come and go from the farm, or new arrivals. large groups of mares in enclosed areas may create stress as horses have a strong social hierarchy. horses when confined constantly vie for preferred access to food, water, and shelter and have a social ordering that controls access.28 in feral conditions, bands of horses are often small and are comprised of around three mares and a stallion. disruption in the social order may results in the shedding of equine herpesvirus 1. mares become susceptible to ehv-1 abortion after the fifth month of pregnancy.30 dental care mares with poor body conditions should have a thorough dental examination performed and their teeth floated if needed. generally, it is preferabe to perform the necessary dental work under sedation. if clinical theriogenology • volume 10, number 3 • september 2018263 heavy sedation is required and there is concern expressed by the owner regarding the stimulatory actions of the alpha agonists on the myometrium, the alpha agonist sedatives may be reversed.31 vaccination the veterinarian plays a major advisory role concerning vaccination along with local disease pressures, the risk of exposure, the risk and consequences of the disease, vaccine effectiveness, potential for vaccine related adverse events and cost of the vaccine. biosecurity and biosecurity practices of the facility should be considered. recommended information about vaccines includes: vaccines are 100% effective in preventing disease, pathogen strains may change over time, horse age and vaccine history influences response, booster vaccinations are generally required for good immunity, and vaccinations are complementary to good management. a good starting point is the information on core and risk-based vaccines for mares (table) published by the american association of equine practitioners.30 equine herpesvirus (ehv-1) abortion is the cause of viral abortion pregnancy. key points to remember is that colostral antibodies do not prevent infection with ehv-1 in foals, horses are infected with ehv-1 as foals, and a high percentage of infected horses develop latency. no ehv-1 vaccine that can eliminate the ehv-1 carrier state. the available vaccines do not protect against infection, reinfection, or coinfection with similar strains of ehv-1, hence the goal with vaccination is to modulate the immune response to reduce shedding, viremia and clinical disease. veterinarians often recommend vaccination of mares against ehv-1 (viral abortion) using a killed virus vaccine* at 5, 7 and 9 months of pregnancy, but there is no substitution for good management in the prevention of herpesviral (ehv-1) abortion. in addition, core vaccinations are usually administered at 4-6 weeks before a mare’s due date, to increase the levels of specific antibodies present in her colostrum.30,32 parasites and deworming tick burdens vary by location and weather factors. there are no tick products approved for use in horses. ticks are usually manually removed. ticks may carry certain infectious diseases (lyme disease), borrelia burgdorferi, and piroplasmosis (theileria equi), and babesia caballi.30 rather than blanket deworming, fecal egg counts are commonly performed. low burdens are generally considered to be less than 200 eggs per gram (epg) of feces, and these horses are not treated. higher burdens of nematodes such as strongyles should be treated using a product labeled for use in pregnant mares, such as ivermectin. because coprophagy is a normal behavior of foals, the dam’s manure is a source of parasites. good stall, paddock and pasture hygiene decreases the risk of high worm burdens.33 hoof care it is important for mares to have their hooves trimmed on a regular basis every 6-8 weeks. this is particularly important for mares that retired from competition due to injury.31 breeding management managed breedings allow the practitioner to precisely know the ovulation date. mares should have an ultrasonographic examination at 15 and 25 days after ovulation to determine if twins are present. mares that have a poor perineal conformation should have caslick’s surgery performed. prefoaling management prior to foaling a mare wellness examination is often planned. at this time the mare may be administered: a vitamin e selenium injection, booster vaccinations, and dewormed if her fecal egg count is high. a rectal and ultrasonographic examination should be performed to determine the presentation of the foal. in the majority of cases the head of the foal and orbit maybe easily identified confirming the foal is in anterior presentation. if the head cannot be identified a transabdominal ultrasonographic examination may be performed to visualize the foal’s chest and the location of foal’s heart within the chest. the foal’s heart will be located closer to the mare’s pelvis in an anterior presentation, rather than the posterior presentation where the fetal stomach is closer to the mare’s pelvis than the heart. mares with a posterior presentation are clinical theriogenology • volume 10, number 3 • september 2018 264 candidates for induction of parturition. the mare’s vulva should be checked for the presence of caslick’s surgery and if present, the caslick’s surgery should be opened. there are increasingly convenient methods of predicting the time of parturition using calcium/magnesium or ph indicator strips. a rising prefoaling mammary secretion calcium and magnesium level and a decrease in ph from above ph of 7.0 to below 6.7 indicates impending foaling and the mare should be observed carefully for signs of foaling.34 *pneumobort-k; zoetis references 1. chavatte-palmer p, peugnet p, robles m: developmental programming in equine species: relevance for the horse industry. anim frontiers 2017;7:48-54. 2. allen wr, wilsher s, turnbull c, et al: influence of maternal size on placental, fetal and postnatal growth in the horse. i. development in utero. reproduction 2002;123:445-453. 3. allen, wr, wilsher s, tiplady c et al: the influence of maternal size on preand postnatal growth in the horse: iii. postnatal growth. reproduction 2002;123:445-453. 4. chavatte-palmer p, robles m, tarrade a, et al: gametes, embryos, and their epigenome: considerations for equine embryo technologies. j equine vet sci 2016;41:13-21. 5. bertoldo, m, locatelli y, o'neill c, et al: impacts and interactions between environmental stress and epigenetic programming during early embryo development. reprod fertil dev 2015;27:1125-1136. 6. smits k, goossens k, van soom a, et al: in vivo-derived horse blastocysts show transcriptional upregulation of developmentally important genes compared with in vitro-produced horse blastocysts. reprod fertil dev 2011;23:364375. 7. xin l, yizhi w, yuanpeng g, et al: h3k9 demethylase kdm4e is an epigenetic regulator for bovine embryonic development and a defective factor for nuclear reprogramming. development 2018;145:158. 8. walton a, hammond j: the maternal effects on growth and conformation in shire horse-shetland pony crosses. proc r soc lond series b, biol sci 1938;125:311-335. 9. van der heyden l, lejeune jp, caudron i, et al: association of breeding conditions with prevalence of osteochondrosis in foals. vet rec 2013;172:68. 10 national research council: nutrient requirements of horses. 6th edition. washington dc: national academies press; 2007. 11. cymbaluk n: feeding horses. regina: government of saskatchewan; 2015. p 1-56. https://www.saskhorse.ca/download/healthwelfare/feeding-horses.pdf 12. peugnet p, robbles m, mendoza l, et al: effects of moderate amounts of barley in late pregnancy on growth, glucose metabolism, and osteoarticular status of pre-weaning horses. plos one 2015;11(12);e0122596 doi:10:1031/journal.pone.01225 13. kosolofski h, gow c, robinson ka: prevalence of obesity in equine population of saskatoon and surrounding area. can vet j 2017;58:968-970. 14. patterson-kane jc, karikoski np, mcgowan cm: paradigm shifts in understanding equine laminitis. vet j 2018;231:3340. 15. saskatchewan forage council report 2014. http://www.saskforage.ca/images/pdfs/projects/adf%20trace%20mineral%20status%20of%20sk%20pastures_web.p df 16. muirhead t, wichtel j, stryhn h, et al: the selenium and vitamin e status of horses in prince edward island. can vet j 2010;51:979-985. 17. karren bj, thorson jf, cavinder ca, et al: effect of selenium supplementation and plane of nutrition on mares and their foals: selenium concentrations and glutathione peroxidase. j anim sci 2010;88:991-997. 18. waldner c, blakely b: evaluating micronutrient concentrations in liver samples from abortions, stillbirths, and neonatal and postnatal losses in beef calves. j vet diag invest 2014;26:376-389. 19. ghorbani a, mohit a, darmani kuhi h: effects of dietary mineral intake on hair and serum mineral contents of horses. j equine vet sci 2015;35: 295-300. 20. palm f, aurich c, steininger b, et al: analysis of selenium, zinc and copper in hair of horse mares and their neonates. tierärzliche umschau 2012; 67:512-519. 21. koikkalainen k, knuuttila a, karikoski k, et al: congenital hypothyroidism and dysmaturity syndrome in foals: first reported cases in europe. equine vet educ 2014;26:181-189. 22. diel de amorim m, messer n, card c: primary hypothyroidism in mares from a herd affected with congenital hypothyroidism. proc am assoc equine pract 2015;61:49. 23. tripathi mk, mishra as: glucosinolates in animal nutrition: a review. anim feed sci tech 2007;132:1-27. 24. cross d, reinemeyer c, prado j, et al: efficacy of domperidone gel in an induced model of fescue toxicosis in periparturient mares. theriogenology 2012;78:1361-1370. clinical theriogenology • volume 10, number 3 • september 2018265 25. garrett l, heimann e, wilson l et al: reproductive problems of pregnant mares grazing fescue pastures. j anim sci 1980;51(suppl1):237. 26. mccue p: equine embryo transfer: clinical perspectives. clin therio 2017;9:369-375. 27. kaercher f, kozicki le, camargo ce et al: embryo transfer in anovulatory recipient mares treated with estradiol benzoate and long-acting progesterone. j equine vet sci 2013;33:205-209. 28. oliveira neto v, canisso i, segabinazzi l: synchronization of cyclic and acyclic embryo recipient mares with donor mares. anim reprod sci 2018;190:1-9. 29. fureix c, bourjade m, henry s: exploring aggression regulation in managed groups of horses (equus caballus). applied anim behav sci 2012;138):216-228. 30. vaccination guidelines aaep http://www.aaep.org/info/vaccination-guidelines-265 31. chopin j: preventive medicine for brood mare farms. in: mckinnon ao, squires el, vaala w, et al, editors. equine reproduction. 2nd edition. boston: wiley blackwell; 2011. p2747-2752. 32. pusterla n, wilson wd, madigan j, et al: equine herpesvirus-1 myeloencephalopathy; a review of recent developments. vet rec 2009;180:279-289. 33. scoles g, ueti m: vector ecology of equine piroplasmosis. annu rev entomol 2015;60:561-580. 34. korosue k, murase h, sata f, et al: comparison of ph and refractometry index with calcium concentrations in preparturient mammary gland secretions of mares. j am vet med assoc 2013;242:242-248. table: core and risk-based vaccines for use in preventive medicine programs in pregnant mares and embryo transfer recipient mares core vaccines tetanus eastern western venezuelan encephalitis west nile virus rabies risk based vaccines ehv -1 ehv-4 eva influenza anthrax botulism strangles leptospirosis clinical theriogenology • volume 10, number 3 • september 2018 266 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice case report: bilateral indirect inguinal herniation in a male alpaca case report: bilateral indirect inguinal herniation in a male alpaca humberto nobre, jessica rush, rochelle jensen, jenna stockler, misty edmondson, julie gard department of veterinary clinical sciences, college of veterinary medicine auburn university, auburn, al summary inguinal hernias are extremely rare in male south american camelids (sacs). it is important to differentiate and classify herniation, since treatment depends on the diagnosis. hernias can be either congenital or acquired. congenital hernias are due to defects during embryogenesis, whereas acquired hernias occur postnatally due to trauma, dilation and/or weakness of soft tissue. hernias are further classified as direct or indirect. direct herniation occurs when herniated contents go through a tear or rent in tissue. direct herniation is usually an emergency, due to strangulation of hernial content, warranting immediate surgical correction. indirect herniation occurs when the herniated content goes through a normal anatomical structure. although indirect herniation is usually not considered an emergency, they still warrant surgical correction. males have a predisposition to indirect inguinal herniation; although this condition is not documented in females, they can have direct inguinal herniation. a sexually intact male alpaca was presented with a history of depression, decreased body condition score, diarrhea, and intermittent rear leg lameness. bilateral scrotal enlargement was observed, with palpable content in the inguinal region. the condition was classified as an acquired, indirect inguinal hernia. after reducing the hernia by a prescrotal approach a bilateral orchiectomy was performed. keywords: alpaca, inguinal hernia, chronic weight loss case presentation history an 8-year-old male alpaca was presented to the auburn university college of veterinary medicine small ruminant service with a 6-week history of depression, decreased body condition score (thinner than herd mates), diarrhea, and intermittent left rear lameness. on physical examination, the alpaca was quiet, alert, and responsive with no lameness. vital signs were: rectal temperature 37.5°c (99.5°f), heart rate 52 beats/min, and respiratory rate 20 breaths/min. the alpaca had pink mucous membranes and a capillary refill time < 2 seconds. a fecal sample was retrieved and on a mcmaster's fecal egg count (fec), there were 8,200 eggs/gram. this animal and all herd mates were given moxidectin 0.1% oral drench (1ml per 22 pounds of body weight). the following week, the animal was presented and bilateral inguinal hernias were apparent within the inguinal canals and scrotum, with loops of small bowels palpable within hernial sacs. however, testes and external genitalia had no abnormalities. body weight was 50 kg (110 pounds) with body condition score 2/9, rectal temperature 38.4°c (101.3°f), heart rate 52 beats/min and respiratory rate 20 breaths/min. the alpaca had pink mucous membranes and a capillary refill time < 2 seconds. normal urination and normal defection were observed. the alpaca was bright and alert with signs of mild abdominal discomfort. another fec was performed again and there were only 50 eggs per gram, indicating that moxidectin had been effective. there was mild hind limb ataxia or potential weakness. blood was submitted for chemistry and hematology and results were: aspartate amino transferase 1,664 u/l (reference interval: 127 – 420 u/l), creatinine 1.3 mg/dl (1.4 – 3.2 mg/dl), erythrocytes (rbc) 5.95 x 106/µl (10.5 – 17.2 x 106/ul), hemoglobin 6.4 g/dl (11.9 – 19.4 g/dl), mean corpuscular volume 33.2 fl (22.2 – 29.9 fl), mean corpuscular hemoglobin concentration 32.3 g/dl (39.3 – 46.8 g/dl), and white blood cells 5.27 x 103/µl (reference interval: 8 – 21 x 103/µl). the interpretation was a regenerative anemia; although no obvious etiology was apparent, regeneration suggested hemorrhage or hemolysis, perhaps with early iron deficiency. meningeal worm (paralaphostrongylus tenuis) can be a cause of ataxia in sacs and had occurred on this farm. therefore, a cerebrospinal (csf) tap was performed at the lumbosacral junction, but no abnormalities were detected. 457 clinical theriogenology • volume 10, number 4 • december 2018 bilateral inguinal hernias were apparent in each inguinal canal and a small loop of bowel could be palpated (figure 1). however, other bowels were not in the scrotum as previously observed, indicating intermittent inguinal herniation. ultrasonographic examination of the scrotum and inguinal canals revealed the presence of loops of bowel within protruding inguinal sacs. attempts were made to manually replace the herniated bowel into the abdomen, it immediately returned. therefore, surgery to correct hernias and castrate the animal was planned. feed and water were withheld for 48 and 8 hours, respectively, prior to surgery. after being sedated with xylazine (0.25 mg/kg) and induced with ketamine (5 mg/kg) via a jugular catheter, the alpaca was intubated and maintained under general anesthesia with isoflurane in oxygen. once in an appropriate anesthetic plane, the alpaca was placed in trendelenburg position and manual reduction of the herniated contents attempted, but were unsuccessful. the ventral abdomen was prepared for sterile surgery in a routine fashion. a 7-cm right paramedian skin incision was made 1.5-2 cm lateral to the distal sheath, the skin undermined and the sheath reflected off midline. an additional 7-cm incision was made through the linea alba (figure 2). intra-abdominal retraction of small intestine and omentum from the scrotum returned herniated contents to the abdomen. the retracted intestine was grossly normal with no adhesions. the linea alba was closed with 2-0 polydioxanone in a simple continuous suture pattern. subcutaneous tissue was closed using 2-0 polydioxanone suture (simple continuous suture pattern) and skin closed using 2-0 poliglecaprone 25 (simple interrupted), an open castration was performed using a prescrotal approach. the right testis was pushed cranially and a 3-cm pre-scrotal incision was made with a number 10 scalpel blade. the skin and subcutaneous tissue were incised to expose the parietal vaginal tunic. the spermatic fascia was stripped and the cauda epididymis bluntly dissected. the vaginal process was incised longitudinally to expose the internal structures of the spermatic cord. the testicular artery and vein, and ductus deferens were ligated using one circumferential and an additional distal transfixing ligature using 2-0 polydioxanone suture. the testicular artery and vein and ductus deferens were crushed with a hemostat and transected distal to the ligatures. the cremaster muscle and vaginal process were ligated together in the same manner as the testicular vasculature and ductus deferens, crushed with a hemostat and then transected distal to the ligatures. the same technique was performed to the left testis (figure 3). it was the surgeon’s preference to use a prescrotal versus inguinal approach, due to the size of hernia sacs present in the inguinal areas. inguinal canals were obliterated to prevent reoccurrence of inguinal hernias. the right inguinal canal was located by palpation and a 1-cm skin incision made medial to the caudal border of the external inguinal ring and extended cranially, resulting in a 5-cm incision. the incision was carefully continued through all subcutaneous layers until the peritoneum was identified. the external inguinal ring was closed in a two layers simple continuous pattern. the internal abdominal oblique muscle was closed in a single layer using 0 non-absorbable polyamide suture (simple continuous), whereas fascia and external abdominal oblique muscle were closed in a single layer using 0 non-absorbable polyamide (simple interrupted). non-absorbable material was used to achieve permanent closure of external inguinal rings. the subcutaneous layer was closed using 2-0 polydioxanone suture (simple continuous) and skin was closed using 2-0 poliglecaprone 25 suture (simple interrupted). the left inguinal canal was similarly closed. the alpaca recovered uneventfully, with no evidence of pain upon recovery or thereafter, and only one dose 2.5 mg (0.25 ml) of butorphanol was given intramuscularly after surgery. ceftiofur sodium 140 mg (2.8 mg/kg) of was given intravenously once daily for 5 days. twelve days after surgery, the alpaca was sedated with 0.2 mg/kg butorphanol and 2mg/kg xylazine added to 20 mg/kg of ketamine, administered intravenously. he was placed in dorsal recumbency and the midline and inguinal sutures removed without incident. there was no evidence of infection, inflammation, edema or pain. the alpaca was bright, alert and responsive with a normal appetite. he was able to defecate and urinate normally. additionally, there was no lameness or ataxia after surgery, only a slight wide-based stance. 458clinical theriogenology • volume 10, number 4 • december 2018 outcome this male alpaca had bilateral acquired, indirect inguinal hernias with heavy internal parasitism, likely haemonchus contortus. the hernia was reduced by a prescrotal approach and a bilateral orchiectomy performed. discussion physical anomalies in sacs in the usa are attributed to inbreeding and reduced genetic diversity.1 congenital defects have potential to cause economic losses; those that are genetic in origin and not recognized may perpetuate within the narrow gene pool in north america, leading to even greater losses.1 inguinal hernias are extremely rare in male sacs. they are a genetic recessive disorder and therefore, should only be repaired after castration. the left side is more commonly affected. strangulation of the intestine within the hernia, will cause abdominal pain. however, due to the stoical nature of sacs, affected animals are often found dead.2 inguinal hernia repair in a llama (lama guanicoe) has been reported.3 however, bilateral inguinal hernias are not common in any species. indirect hernias in ruminants occur on the left side ~90% of the time, due to rumen pressure and cattle commonly being in sternal recumbency with their left rear leg pulled caudally. many affected bulls were markedly over-conditioned, followed by rapid weight loss.4 this scenario can predispose other male species to inguinal herniation. although sacs do not have a rumen, in recumbent animals, the third compartment of the stomach can increase abdominal pressure towards the left inguinal area. consequently, a higher prevalence of left side inguinal herniation in sacs has been reported. inguinal hernia results in herniation of abdominal contents (usually omentum or jejunum) through the internal inguinal ring. if this continues, inguinal hernia results, as abdominal contents move through the external inguinal ring into the scrotum, enlarging the neck of the scrotum or causing obvious enlargement of the scrotum.5 acquired inguinal hernia occurs in adult bulls following trauma, or due to age-related relaxation and stretching of the abdominal musculature.5 however, indirect herniation through the inguinal ring is usually not a dire emergency, as loops of bowel are not strangulated. inguinal herniation occurs most often in bulls and rarely in cows. inguinal hernias occur in camelids, more often in male llamas than male alpacas, but more commonly than in female camels.6 correction is performed in cattle via either a standing flank laparotomy or inguinal approach and simple interrupted or continuous suture patterns used.4 due to the age and physical presentation of this alpaca’s hernias, the diagnosis was acquired indirect inguinal hernias. depletion of the inguinal fat pad occurs with weight loss, enabling structures (e.g. intestines) to herniate through the inguinal ring. this alpaca's acquired indirect inguinal hernias were likely secondary to weight loss caused by heavy parasitism. haemonchus contortus is a hematophagous gastrointestinal parasite of small ruminants and camelids. heavy burdens of this parasite can cause anemia, hypoproteinemia, weight loss, and even mortality. haemonchus contortus is also becoming a major health concern in new world camelids.7 severity of parasite infection is determined by clinical signs, including pale mucous membranes, poor body condition, diarrheaand fec (modified mcmasters).7 it is imperative that camelids be monitored for parasites, particularly animals with poor body condition, signs of weakness, pale mucous membranes, or dependent edema. in this case, the initial weakness was attributed to anemia and muscle atrophy secondary to heavy parasitism. learning points  intact male animals (sacs, small ruminants and ruminants) with chronic weight loss and low body condition score are considered at risk for development of inguinal hernias.  inguinal hernia is a differential diagnosis when a male animal is presented with hind limb lameness and/or ataxia in combination with soft tissue swelling in the inguinal regions and/or a swollen scrotum.  parasite prevention and control must be part of routine herd work. 459 clinical theriogenology • volume 10, number 4 • december 2018 acknowledgments the authors thank clinicians of the department of small ruminants and food animal at auburn university college of veterinary medicine for their expertise and support. conflict of interest no conflict of interest to declare. references 1. leipold hw, hiraga th, johnson lw: congenital defects in the llama. vet clin north am food anim pract 1994;10:401-420. 2. duncanson gr: sedation, anesthesia, surgical conditions and euthanasia. in: veterinary treatment of llamas and alpacas. london, uk: cabi; 2012. p. 75. 3. weaver, a.d: inguinal hernial repair in a llama (lama guanicoe). vet rec 1991;128:356-357. 4. riddell m: developmental anomalies of the scrotum and testes. in: wolfe d, moll h, editors. large animal urogenital surgery. 2nd edition, baltimore: williams and wilkins; 1998. p. 283-294. 5. st jean g: male reproductive surgery. vet clin north am food anim pract 1995;11:55-94. 6. ramadan ro, abdin-bey mr: abdominal and inguinal hernias in camel (camelus dromedarius) in saudia arabia (field cases). emir j food agric 2001;13:57-61. 7. edwards ee, garner bc, williamson lh, et al: pathology of haemonchus contortus in new world camelids in the southeastern united states: a retrospective review. j vet diagn invest 2016;28:105-109. 460clinical theriogenology • volume 10, number 4 • december 2018 figure 1. bilateral inguinal hernias with a loop of small bowel within the inguinal canals. arrows point to the most cranial border of the hernias (alpaca’s head on the top of the picture). figure 2. a right paramedian skin incision lateral to the distal sheath with an additional incision through the linea alba (alpaca’s head on the right side of the picture). 461 clinical theriogenology • volume 10, number 4 • december 2018 figure 3. an open castration technique via a prescrotal approach (alpaca’s head on the left side of the picture). (editor’s note: online edition of the manuscript has color photographs) 462clinical theriogenology • volume 10, number 4 • december 2018 000_frontal ms_1 forematter for december 2018 ms_2 letter1 for december 2018 ms_2 letter2 for december 2018 ms_2 letter3 for december 2018 ms_3 table of contents ms_4 therio news for december 2018_bw 2019 therio preview therio recap with pics steiner-new officers 2018 therio sponsors 2018 exhibitors-sft board noms 2018 auction donations 2019 bartlett nomination act call for noms call for abstracts 2019 for ctj the society for theriogenology and american college of theriogenologists issue a call for research and case abstracts to be presented at the annual therio conference july 24-27, 2019 in savannah, georgia. 1. competitive category: 2. non‐competitive category: 3. case abstract category: 4. poster category: general: guidelines: what to send and how to send it abstracts not adhering to these guidelines will not be considered for presentation. deadline: abstracts must be received no later than february 15, 2019 for consideration. call for 2019 student case presentations.pdf call for student case presentations-2019 annual therio conference student case presentations call for student case presentations also available at www.therio.org guidelines for application submission society for theriogenology student case presentation competition annual therio conference, savannah, georgia, july 24-27, 2019 ms_5 editorial 001a_ms_6 roberts color pictures 002_ms_7 root kustritz 003_ms_8 ferrer 004_ms_9 wiley 005_ms_10 wallace 006_ms_11 peterson 007_ms_12 nobre 008_ms_13 sidelinger 009_ms_14 mirando economic impact of reproductive performance in dairy herds and approaches for program selection economic impact of reproductive performance in dairy herds and approaches for program selection julio giordano dairy cattle biology and management laboratory department of animal science, cornell university, ithaca, ny abstract implementation of an effective reproductive management program is critical to success of dairy operations, as herd reproductive performance is inextricably linked to farm profitability. therefore, understanding the multiple sources of variation in herd cash flow affected by reproductive management is fundamental to support informed decisions when defining reproductive management or level of desired herd performance. ideally, selecting a reproductive management program or level of desired reproductive performance is based on an objective measure of expected profitability. approaches available for determination of the economic impact of reproductive management programs include: (1) decision support tools that simulate dairy herd dynamics and economics; (2) extrapolation of expected trends in profitability gain for changes in reproductive metrics (e.g. 21 day pregnancy rate or days open); and (3) extrapolation of results from research experiments conducted through simulation or on commercial dairy farms. this review includes a summary of the main factors associated with reproductive management that contribute to variation in herd profitability and a brief description of approaches available to support selection of reproductive management programs based on their expected economics. keywords: profitability, pregnancy, dairy cow, dairy heifer introduction effects of reproductive performance of dairy cattle on profitability of dairy operations have long been recognized. total milk sales, number of cows replaced and number of replacement heifers born are among the major sources of income affected by reproductive performance of lactating dairy cows. rearing costs and opportunity cost of delayed lactation are 2 major factors driving economics for dairy heifers. costs of feed, labor and veterinary inputs are also influenced by reproductive programs, as lactation length, dry period length, days in rearing and number of services to conceive depend primarily on reproductive performance. therefore, when selecting a reproductive management strategy, all factors that contribute to variations in reproductive and economic performance need to be considered. it remains a major challenge for dairy producers, veterinarians, and consultants to define the reproductive management strategy that maximizes profitability for a farm operation. numerous management strategies and reproductive technologies can be implemented in dairy farms to optimize reproductive efficiency; however, it is usually challenging to determine their economic benefits or drawbacks in the field. this is primarily due to the multiple factors affected by changes in reproductive management and the time required to quantify productive and economic impact of changes in reproductive performance. consequently, practitioners must be aware of biological, management and economic factors that drive herd profitability and various approaches available to identify reproductive management strategies that optimize profitability for specific dairy herds. these include: general herd profitability trends expected in response to changes in reproductive performance; use of software decision making tools based on models that simulate herd dynamics and economics; and data from research studies conducted with simulation tools or field experiments. reproductive management and performance factors that affect herd profitability due to multiple inputs and outputs affected by reproductive management programs and the reproductive performance of dairy herds, several economic parameters are affected by changes in reproductive management and respective herd performance. among the most relevant are effects of timing of pregnancy on milk production efficiency and replacement dynamics (cows sold and calves born) and cost of reproductive program implementation. reproductive performance can also have direct 329 clinical theriogenology • volume 11, number 3 • september 2019 implications on herd health and milk production; however, these are more complex and difficult to quantify. milk production efficiency number of lactations and productive efficiency per lactation in the lifetime of cows is defined by the occurrence and timing of pregnancy; therefore, cows with frequent calvings will have more lactation peaks and a greater lifetime total yield compared to cows with less frequent calvings. in addition, feed intake and milk yield during lactation are not completely synchronized, thereby cows convert feed into milk with different efficiencies according to stage of lactation. although feed intake has a somewhat similar pattern of change than milk yield during lactation, rate and timing of increase and decrease of feed intake and milk yield are uncoupled in early and late lactation. whereas maximum dry matter intake usually occurs several weeks after cows reach peak milk yield, rate of intake decline after peak is more gradual than milk yield. therefore, the ratio of milk value produced to feed costs, usually known as income over feed cost (iofc), is high early in lactation but decreases as lactation progresses. therefore, timing of pregnancy during each lactation cycle becomes a critical factor for cow profitability, as it determines the amount of time that cows spend in the most efficient part of their lactation curve throughout their lifetime (highest iofc), which in turn, affects overall profitability of the dairy operation. replacement dynamics dynamics of cows leaving the herd due to reproductive failure or other reasons and number of replacements born can be dramatically affected by timing of pregnancy.1 to maintain herd size constant, cows that leave the herd due to sale or death are replaced immediately by a recently calved heifer. for cows sold, the farm incurs a cash cost of replacement. this is the difference between the market value (if the replacement is bought) or the cost of rearing until calving (if the replacement heifer is raised by the operation) and salvage value. value of the calf born from the replacement animal contributes to offset the cash cost of culling. as failure to become pregnant during lactation and delayed pregnancy result in a greater proportion of cows culled and the cash cost of replacing cows is usually negative, reduced reproductive performance can lead to substantial economic losses for dairy herds. of note, the economic detriment of an excessive replacement rate due to poor reproductive performance is exacerbated under market conditions characterized by elevated replacement heifer costs, unfavorable prices for cull cows and poor value of calves born. reproductive program cost regardless of the type of method to submit cows for insemination, all reproductive programs incur a cost of implementation. calculation of this cost should include hormonal treatments (if used), ai, detection of estrus during lactation, pregnancy testing and labor associated with each activity. although research studies have suggested that cost of reproductive programs is not the main contributor to cash flow differences between reproductive management programs, reproductive cost can generate noticeable profitability differences when input costs are substantial or many ai services per cow are required to achieve the desired level of reproductive performance. general trends for economics of reproductive performance although trends and associations between metrics of reproductive performance and herd profitability hold true for certain scenarios and conditions, in general it is not possible to generalize results from deterministic or even stochastic estimations of herd performance and profitability to all herds and management conditions. thus, focus should be on extrapolating trends and the expected magnitude of rate of gain in profitability, rather than extrapolating absolute values for differences in profitability associated with certain changes in metrics of reproductive performance. for example, it is well accepted that increasing the 21 day pregnancy rate (21dpr) follows the law of diminishing variable returns whereby, the same level of gain in 21dpr results in greater profitability gains at low versus high levels of reproductive performance. nevertheless, the magnitude of 330clinical theriogenology • volume 11, number 3 • september 2019 the change in profitability for the same gain of 21dpr may vary dramatically from herd to herd and the value of multiple economic and performance inputs used for calculation. similarly, number of days from calving to conception, also known as days open (do), has been extensively used to quantify economic impact of reproductive performance.2-4 the basis of such approach has been that maximum profitability of a dairy herd is observed when cows have a specific number of do that results in an ideal calving interval. cows that conceive after the ideal number of do incur economic losses. then, the economic value of a program or specific strategy under consideration is determined by comparing its economic outcome to that of a hypothetical scenario in which all cows have an ideal number of do. numerous epidemiological studies provided insights regarding implications of reproductive performance for dairy herds based on the calculation of the cost of do. however, a major caveat of this approach is that the difference in do generated by various reproductive programs is unknown, unless herd dynamics are compared in the field or simulated and the cost of do usually changes dramatically based on specific herd and economic conditions. thus, practitioners should understand limitations of direct extrapolation of economic estimations, due to changes in reproductive management across herds and within a herd over time, given multiple interactions among inputs and outputs and their expected absolute values. decision making tools the myriad of factors that affect farm costs and revenues, and the lag time between changes in reproductive performance and realization of benefits or losses, creates challenges for economic analyses of reproductive management programs under field conditions. therefore, simulation models have been created to incorporate both performance and economic field data in combination with herd dynamics simulation to predict potential impact of various reproductive management strategies on profitability of dairy farms.3-7 some simulation models used in research settings7 have also been used to develop decisionsupport tools and are readily available for use by stakeholders. for example, university of wisconsin cornell university (uwcu)-dairyrepro$ tool available at: university of wisconsin: dairymgt.info: tools: reproduction or cornell university: http://www.ansci.cornell.edu/dm/resources.html. the uwcu-dairyrepro calculates and compares economic value of dairy reproductive programs, including fixed time artificial insemination (ftai), heat detection (hd), and combinations of ftai and hd programs. this tool also enables inclusion of automated estrus detection systems (e.g. activity monitors) for detection of estrus in lactating dairy cows. the model behind the tool applies probabilistic reproduction survival curves with expected monetary values to determine the difference in net present value (npv) of a current (i.e. program currently used by farm of interest) versus an alternative (i.e. program under consideration for implementation) reproductive program for a specific dairy farm. thus, this tool facilities economic-based decision making, as users can compare, between programs, expected direction and magnitude of economic differences. research on the impact of management interventions on the profitability of dairy cattle examples of research studies conducted with simulation or collection of data from field experiments for lactating dairy cows are provided to exemplify impacts of reproductive management on dairy herd profitability. more details about these studies are described in the literature.7-8 combination of artificial insemination at detected estrus and fixed time artificial insemination or all fixed time artificial insemination the dairy industry has widely adopted the use of hormonal protocols for synchronization of estrus and ovulation. although some dairies rely solely on synchronization of ovulation and perform ftai for all services, the majority implement programs combining ftai and artificial insemination at detected estrus (aie). in farms that use combined programs, it is frequent to observe different levels of performance for cows receive aie and cows that receive ftai. thus, determination of the impact of 331 clinical theriogenology • volume 11, number 3 • september 2019 different proportions of cows aie and ftai and the respective success of each type of ai services can be useful to aid producers and consultants in their decision-making process. in this regard, giordano et al.7 simulated a set of plausible scenarios of reproductive management and performance observed in dairy herds. a reproductive program that consisted of all ftai (aftai) was used as baseline for comparison to multiple combined programs with varying proportions of cows aie (30 70%) and varying levels of fertility (low = 25%, medium = 30%, and high = 35%; table 1). this broad range of performance represented a wide range of herd reproductive performance usually observed in dairy farms in the us. the net value (nv) differences between the 16 programs combining aie and ftai (2 to16) with the aftai are presented in figure 1. results indicated that fertility of cows aie and the consequent change in the population of cows reaching ftai are critical factors that affect profitability in herds combining ftai and aie. when compared to aftai, programs with 25% conception rate (cr) to aie services had a lower nv at all levels of ed, suggesting that allowing more cows to receive ftai is more economical. this is because cows that would have a greater chance of becoming pregnant after synchronization are prevented from getting a ftai and therefore subject to a longer time interval until they are re-inseminated and have another chance to conceive. the detriment of receiving aie was greater as the percentage of cows receiving aie increased from 30 70% as demonstrated by the continued decrease in nv. however, as cr to aie increased from 25 30%, a major nv positive change was observed for all programs. thus, when 30 70% of cows receive aie and cr is 30%, neither major gains nor losses should be expected if cows receive aie or ftai. lastly, when compared to aftai programs combining aie and ftai with 35% cr had substantially greater nv at all levels of ed. a practical implication of these results is that when 30 to 70% of cows receive aie and the cr is 25%, profitability will be reduced when compared to a program like aftai. thus, allowing cows to receive ftai would be a preferred strategy. conversely, when cr to aie is 30 or 35% there would always be an economic advantage, albeit of different magnitude, when cows are allowed to be aie. although minor differences were observed when cr after ed inseminations is 30%, there is a clear benefit of inseminating cows in estrus when cr is 35%. economics of manipulating the duration of the voluntary waiting period insemination and conception risks after the end of the voluntary waiting period (vwp) are the 2 major determinants of time to pregnancy during lactation. nonetheless, duration of the vwp can also influence timing of pregnancy, because it determines when cows become eligible for insemination. through its impact on timing of pregnancy, vwp duration has a direct effect on profitability of dairy cows. traditionally, dairy farms in the us began inseminating cows at ~ 40 50 dim because suboptimal estrus detection and fertility to ai required that cows received multiple services to conceive. in recent years, however, better reproductive management programs that ensure inseminating cows by a set dim, led to increases in reproductive and productive performance of well-managed dairy herds. improved detection of estrus and fertility reduces the number of inseminations needed to conceive and variation in the interval for cows to become pregnant. thus, there is an opportunity to better control timing of pregnancy during lactation and thereby maximize profitability. in this regard, manipulating vwp duration may be a simple and inexpensive change, with potential to impact profitability of dairy herds. in a recent experiment, we evaluated reproductive performance, herd exit dynamics and economics of dairy cows managed with variable duration of the vwp. we hypothesized that extending vwp duration from 60 88 dim impacts reproductive performance and profitability of lactating holstein cows. cows from 3 commercial farms in ny state received ftai after synchronization with the doubleovsynch protocol at 60 ± 3 or 88 ± 3 dim. reproductive performance and cash flow for up to 18 months after calving in the experimental lactation were compared. results of this experiment are presented in detail in the literature.1,8 for primiparous cows, cash flow per slot per 18 months or per day was similar for the vwp60 and vwp88 treatment, despite a $68 numerical difference in favor of the vwp88 group (table 2). interestingly, most of the difference between treatments was due to greater replacement cost for the 332clinical theriogenology • volume 11, number 3 • september 2019 table 1. expected reproductive performance of programs used for simulation in the case study. first ai second and subsequent ai program number program aiea before 1st ftaib cr aiec before 1st ftai cr ftai aie before ftai cr aie before ftai cr ftai 1 aftaid 42 30 2 ftai+aie 2e 30 25 40 30 25 30 3 ftai+aie 3 40 25 38 40 25 30 4 ftai+aie 4 50 25 36 50 25 30 5 ftai+aie 5 60 25 34 60 25 28 6 ftai+aie 6 70 25 32 70 25 28 7 ftai+aie 7 80 25 30 80 25 28 8 ftai+aie 8 30 30 40 30 30 30 9 ftai+aie 9 40 30 38 40 30 30 10 ftai+aie 10 50 30 36 50 30 30 11 ftai+aie 11 60 30 34 60 30 28 12 ftai+aie 12 70 30 32 70 30 28 13 ftai+aie 13 80 30 30 80 30 28 14 ftai+aie 14 30 35 40 30 35 30 15 ftai+aie 15 40 35 38 40 35 30 16 ftai+aie 16 50 35 36 50 35 30 17 ftai+aie 17 60 35 34 60 35 28 18 ftai+aie 18 70 35 32 70 35 28 19 ftai+aie 19 80 35 30 80 35 28 apercentage of cows ai after estrus detection (aie) before first time ai. bftai = fixed time artificial insemination. cconception rate (cr) of cows ai after detection of estrus. dprogram all fixed time artificial insemination (aftai) relied on 100% ftai for all services after synchronization of ovulation for first postpartum ai with the presynch-ovsynch protocol whereas the ovsynch protocol was used for second and subsequent ai services. the voluntary waiting period was 72 d and the interval between 2 successive inseminations was 42 days. eprograms combining ftai with estrus detection (ftai + aie 2 to 19), used the same synchronization of ovulation protocols than ftai except that ai after aie was added between the end of the vwp set at 50 dim and the first ftai at 72 dim and in between ftai services (aie was discontinued at the time of the first gnrh of the ovsynch protocol for all ai services) vwp60 treatment, because the small differences observed for the rest of the parameters offset each other. the difference in replacement cost was primarily due to greater cost in the subsequent lactation because a slightly greater percentage of second-lactation cows left the herd before the end of the 18 month period. 333 clinical theriogenology • volume 11, number 3 • september 2019 thus, when attempting to extrapolate results of our experiment to other farms, it is important to recognize the dominance of replacement cost over total profitability, as a different replacement cost dynamics (i.e. different culling pressure and different cash cost of culling) may be observed across herds and changing market conditions. indeed, when the potential effect of changes in economic conditions was simulated, replacement costs accounted for up to ~ 80% of total variation in cash flow. figure 1. difference in net value (nv; $/cow per year) for 15 programs combining estrus detection and timed ai with an all fixed time artificial insemination program (aftai). interestingly, for multiparous cows, results were opposite to those of primiparous cows. although a statistically significant difference between treatments was not observed, cows in the vwp60 were more profitable by $85 per slot per 18 months than cows in the vwp88 treatment (table 2). in this case, however, replacement cost was greater for the vwp88 reflecting increased culling pressure in nonpregnant cows in the vwp88 treatment in later lactation. such contrast in results for overall cash flow likely reflected differences in milk production persistency (i.e. lactation curves are less persistent for multiparous than primiparous) and the interaction between parity and risk of leaving the herd as lactation progressed. although to a lesser extent than for primiparous cows, replacement cost explained a substantial proportion of numerical economic differences for results with fixed economics values or when we simulated varying economic conditions. in summary, economic outcomes for our experiment suggest that extending the vwp from 60 to 88 dim when using aftai to submit cows for first service may result in greater (numerical) profitability for primiparous cows, primarily through a reduction in replacement costs. conversely, the same extension of the vwp duration for multiparous cows may lead to economic losses (numerical), primarily due to greater replacement cost and reduced iofc that cannot be offset by reduced reproductive program costs. results from our experiment should be interpreted with caution because in spite of the large number of cows included, we did not detect statistically significant differences for overall cash flow, all cows received rbst and the particular replacement dynamics of the herds involved in our research may have been affected by individual farm management decisions and economic conditions during the trial. 334clinical theriogenology • volume 11, number 3 • september 2019 table 2. effect of extending duration of the voluntary waiting period from 60 to 88 dim on revenues and expenses during 18 months after calving in the experimental lactation. primiparous multiparous vwp60a (n = 480) vwp88b (n = 471) differ encec p valued vwp60 (n = 785) vwp88 (n = 789) differe nce p value $/slot4 $/slot milk iofc 3,806 3,803 -3 0.95 4,363 4,324 -39 0.38 calf value 100.5 102.9 2.4 0.59 80.78 77.98 -2.80 0.43 replacement cost 327.3 259.0 -68.3 0.07 624.9 673.6 48.7 0.16 reproductive cost 97.58 91.28 -6.30 < 0.01 104.14 93.63 -10.5 < 0.01 bst cost 215.9 221.6 5.7 < 0.01 219.4 224.9 5.5 < 0.01 operating expenses 1,512 1,512 1,512 1,512 cash flow per 18 mo 1,756 1,824 68 0.32 2,006 1,921 -85 0.19 cash flow per day 3.25 3.37 0.12 0.32 3.71 3.56 -0.15 0.19 avwp60 = first service timed ai at 60 ± 3 dim after the double-ovsynch protocol. bvwp88 = first service timed ai at 88 ± 3 dim after the double-ovsynch protocol. cdiff = difference between vwp88 and vwp60 (diff = vwp88-vwp60). dslot is the unit of space occupied by each cow enrolled in the experiment for an 18 month period after calving in the experimental lactation. conclusion the relationship between herd reproductive performance and profitability is complex due to multiple factors, inputs and outputs affected by reproductive performance. availability of reproductive decision support tools provides practitioners with new opportunities to help their clientele. decision support tools can be used to simulate impacts of reproductive performance and herd dynamics on profitability of a specific dairy herd. thus, practitioners can help dairy farm managers make informed decisions regarding choice of reproductive management programs or strategies to achieve a desired level of performance. otherwise, extrapolation of expected economic responses to changes in reproductive performance or results from research studies can be used to guide on-farm decisions. conflict of interest there are no conflicts of interest to declare. references 1. stangaferro ml, wijma r, masello m, et al: extending the duration of the voluntary waiting period from 60 to 88 days in cows that received timed artificial insemination after the double-ovsynch protocol affected the reproductive performance, herd exit dynamics, and lactation performance of dairy cows. j dairy sci 2017;101:717-735. 2. louca a and legates je: production losses in dairy cattle due to days open. j dairy sci 1968;51:573-583. 3. groenendaal h, galligan dt, mulder ha: an economic spreadsheet model to determine optimal breeding and replacement decisions for dairy cattle. j dairy sci 2004;87:2146-2157. 4. meadows c, rajala-schultz pj, frazer gs: a spreadsheet-based model demonstrating the nonuniform economic effects of varying reproductive performance in ohio dairy herds. j dairy sci 2005;88:1244-1254. 5. de vries, a: economics of delayed replacement when cow performance is seasonal. j dairy sci 2004;87:2947-2958. 6. giordano jo, fricke pm, wiltbank mc, et al: an economic decision-making support system for selection of reproductive management programs on dairy farms. j dairy sci 2011;94:6216-6232. 7. giordano jo, kalantari as, fricke pm, et al: a daily herd markov-chain model to study the reproductive and economic impact of reproductive programs combining timed artificial insemination and estrus detection. j dairy sci 2012;95:5442-5460. 8. stangaferro ml, wijma r, masello m, et al: economic performance of lactating dairy cows submitted for first service timed artificial insemination after a voluntary waiting period of 60 or 88 days. j dairy sci 2018;101:7500-7516. 335 clinical theriogenology • volume 11, number 3 • september 2019 336clinical theriogenology • volume 11, number 3 • september 2019 omniblank: << 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/useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice contact peyton draheim; sophie boorman pfdraheim@gmail.com; sophieboorman@me.com © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9647, http://dx.doi.org/10.58292/ct.v15.9647 case report paraphimosis of 7 years’ duration in a percheron stallion peyton draheima, sophie boormanb, reid hansonc, candace lymanc ahagyard equine medical institute, lexington, ky, usa bvirginia-maryland regional college of veterinary medicine, blacksburg, va, usa ccollege of veterinary medicine, auburn university, auburn, al, usa abstract a 15-year-old percheron stallion was presented to auburn university veterinary teaching hospital for evaluation of paraphimosis of 7 years’ duration. available history indicated that the paraphimosis was subsequent to a kick injury that occurred 7 years prior. vital parameters were within normal limits. the penis and prepuce were markedly fibrotic and swollen, extending to the level of the distal tarsus, and evidence of chronic penile exteriorization was present. due to apparent lack of penile sensation and the chronicity of the lesion, phallectomy and castration were recommended. distal phallectomy via the williams’ technique was performed under general anesthesia, concurrently with closed castration. no excessive intraoperative hemorrhage was noted. within 30 min postoperatively, the patient experienced mild hemorrhage from the stoma. around 12 h post-surgery, the gelding exhibited dull mentation, pale mucous membranes, tachycardia, and tachypnea. the gelding was administered infusions of aminocaproic acid, 0.05% formalin, and a whole blood transfusion. by the following afternoon, packed cell volume had increased from 18 to 22% and total solids increased from 5.0 to 6.8 g/dl; vital parameters were within normal limits. the gelding was discharged 5 days postoperatively with normal vital parameters. medications included phenylbutazone, trimethoprim sulfamethoxazole, and red cell®. follow up telephone consultation with the owner several months postoperatively indicated the gelding had made a full recovery. this case describes resolution of a severely protracted paraphimosis and successful management of common, albeit concerning, postoperative complications. keywords: paraphimosis, stallion, phallectomy, castration, penile paralysis, hemorrhage introduction paraphimosis is the inability of the animal to retract the penis into its normal position within the prepuce. known causes of paraphimosis in the horse include trauma during breeding, debilitation and malnourishment, equine herpesvirus-1 infection, unresolved priapism, rabies, and administration of phenothiazine-derivative tranquilizers, most commonly acepromazine.1 regardless of the initial cause, the ultimate consequence of paraphimosis is accumulation of edema that is then exacerbated by gravity. the increased weight of the prolapsed and edematous penis and internal lamina of the prepuce can lead to chronic stretching of the internal pudendal nerves, resulting in permanent penile paralysis. in order to resolve paraphimosis and prevent penile paralysis, prompt treatment is warranted. while some cases may be resolved with medical management, more protracted and severe cases generally require surgical intervention. this paper describes the treatment and management of postoperative complications of chronic paraphimosis in a stallion. history and physical examination a 15-year-old percheron stallion was presented to auburn university veterinary teaching hospital (auvth) for evaluation of paraphimosis of 7 years’ duration. available history indicated that the paraphimosis was subsequent to a kick injury that occurred 7 years prior. due to a recent change in ownership, no further history was available other than the horse’s current vaccination status. vital parameters and mentation were within normal limits. the stallion had a body condition score of 3 on a 9-point scale. a complete blood count, chemistry panel, and fibrinogen were performed and were within normal limits. the preoperative packed cell volume (pcv) was 32% with a total solids (ts) of 8.7 g/dl. the penis and prepuce were markedly fibrotic and edematous, extending to the level of the distal tarsus, and the penile epithelium was thickened, friable, and cool to the touch (figure 1). there was a lack of response by the stallion when a firm pinch was applied to the penile epithelium, suggesting nerve damage. due to the apparent lack of sensation and the extreme mailto:pfdraheim@gmail.com mailto:sophieboorman@me.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9647 citation line: clinical theriogenology 2023, 15, 9647, http://dx.doi.org/10.58292/ct.v15.9647 53 protracted nature of the paraphimosis in this case, partial phallectomy and castration were recommended. treatment the stallion received preoperative antibiotics and anti-inflammatories (6.6 mg/kg gentamicin iv; 22,000 iu/kg procaine penicillin im; 1.1 mg/kg flunixin meglumine iv). following sedation with xylazine (1 mg/kg iv) and induction of general anesthesia with propofol (0.35 mg/kg iv) and ketamine (2.2 mg/kg iv), the patient was intubated and maintenance of anesthesia was accomplished with isoflurane in oxygen. the scrotum and penis were aseptically prepared and a stallion urinary catheter was placed (figure 2). the use of electrocautery and an esmarch tourniquet at the base of the penis provided hemorrhage control intraoperatively. a urethrostomy was created by making three separate 3cm-long incisions, forming a triangular-shaped wedge on the ventrum of the penis with the apex of the triangle directed proximally (figure 3). the urethral catheter was removed and a stoma was created by apposing the urethral mucosa to the preputial epithelium with simple interrupted sutures (2-0 poliglecaprone 25) (figure 4). the penis was then amputated approximately 5 mm distal to the urethrostomy site (figure 5). a simple interrupted suture pattern provided compression and closure of the corpus cavernosum around the urethral lumen. prior to final closure, the tourniquet was removed to observe for and repair focal areas of hemorrhage. finally, a closed castration was performed routinely with serra emasculators (figure 6). no excessive intraoperative hemorrhage was noted. following an uncomplicated recovery from anesthesia, the gelding experienced mild postoperative hemorrhage from the  penile stoma shortly after standing and swelling of the penile stump was noted (figure 7). vital parameters at this time were unremarkable. around 12 h postoperatively, the patient was observed to have dull mentation, pale mucous membranes, tachycardia (72 beats per minute), and tachypnea (32 breaths per minute), though no active blood loss from the penile stump was noted during this time. the pcv and ts at this time were 18% and 5.0 g/dl, respectively. the gelding was administered intravenous infusions of aminocaproic acid (3.5 mg/kg/min for 15 min, then 0.25 mg/kg/ min as a continuous rate infusion), 0.05% formalin (50 ml 10% neutral buffered formalin diluted in 1 l of isotonic fluids),2 and received an unmatched whole blood transfusion (7 l). within 12 h of initiating treatment, physical parameters stabilized. there was not a significant amount of blood within the gelding’s stall; total estimated blood loss from the penile stump was 0.5 l. transabdominal ultrasonographic examination did not reveal the presence of increased free fluid within the abdomen. by the following afternoon, the pcv had increased from 18 to 22% and the ts had increased from 5 to 6.8 g/dl; vital parameters remained within normal limits. the gelding was discharged 5 days post-surgery with normal postoperative physical exam findings and a pcv of 16%. medications dispensed for continued administration at home included phenylbutazone (1.5 g po q12 h), trimethoprim sulfamethoxazole (tms; 30 mg/kg poq12 h), and an oral figure 1. color photograph of a 15-year-old percheron stallion with chronic paraphimosis, with the distal end of the fibrosed penis extending to the level of the distal tarsus. figure 2. color photograph of the surgical field during sterile preparation for distal phallectomy and castration. roll gauze has been used to secure the distal penis to a hoist to provide traction throughout the procedure. http://dx.doi.org/10.58292/ct.v15.9647 54 citation line: clinical theriogenology 2023, 15, 9647, http://dx.doi.org/10.58292/ct.v15.9647 vitamin-iron-mineral supplement (red cell®; 30 ml po q12 h). recommendations provided to the owners included stall rest, daily hydrotherapy of the scrotum, and brief hand walks twice daily to manage postoperative scrotal edema. four days after discharge, the owners returned the gelding for assessment citing concerns of dull mentation; vital parameters were within normal limits except for a mild tachycardia (56 beats per minute). the urethral stoma appeared to be healing appropriately. the pcv was 16% and ts was 7.4 g/dl at presentation. the gelding remained stable throughout observation and was discharged 2 days later. recommendations included continued stall rest and hydrotherapy in addition to application of femycin ointment (lanolin, dexamethasone, and oxytetracycline) to the prepuce twice daily. an update provided by the owners several months after surgery indicated that the gelding had fully recovered and was being used under saddle (figure 8). discussion this case report describes the successful treatment of chronic paraphimosis in an aged stallion. the size of the penis, the poor condition of the horse, and the chronicity of the condition likely contributed to the postoperative anemia. this case highlights the need for judicious postoperative monitoring following phallectomy; it may be worth considering blood transfusions if the penis to be removed is particularly large. the horse in this case report was diagnosed with chronic paraphimosis, in which an animal is unable to retract the flaccid penis into the prepuce. initially, the penis of the affected horse may appear slightly rigid rather than flaccid, which may lead to the premature and erroneous diagnosis of priapism rather than paraphimosis. this rigidity is due to the rapid pooling and clotting of blood within the corpus cavernosum penis (ccp), which occurs within 2–5 h of the inciting event.3 priapism, which differs from paraphimosis in that it is a persistent erection, is primarily due to stasis of blood within the ccp, caused either by an excessive volume of arterial inflow or impeded venous outflow. a case of priapism may progress to paraphimosis if left untreated, as the protruded penis becomes edematous and the combination of muscle fatigue and stretching of the pudendal nerves leads to an inability of horse to retract the penis. priapism may be managed with injection of diluted phenylephrine into the ccp, irrigation of the ccp with heparinized saline, or the creation of a shunt from the ccp to the corpus spongiosum penis (csp).1 paraphimosis may be successfully managed medically if a diagnosis is made and treatment is initiated promptly. medical treatment of paraphimosis differs from that of priapism, highlighting the importance of an accurate diagnosis in cases of chronic penile protrusion. managing paraphimosis should focus on resolving the inciting cause, mitigating preputial edema, and preventing further trauma of exposed tissues.4 in this case, medical treatment was not attempted due to the severe chronicity of the disease. figure 3. color photograph of the urethrostomy site. the incision extends to the level of the stallion urinary catheter (indicated by the asterisk). the distal end of the penis is at the top of the image. figure 4. color photograph of the urethrostomy site after the urethral mucosa was sutured to the penile epithelium with absorbable simple interrupted sutures. the distal end of the penis is at the top of the image. http://dx.doi.org/10.58292/ct.v15.9647 citation line: clinical theriogenology 2023, 15, 9647, http://dx.doi.org/10.58292/ct.v15.9647 55 the stallion in this report presented with extensive damage to the body of the penis. paraphimosis results in prolonged exposure of the penis and prepuce which exacerbates edema and promotes muscle fatigue and pudendal nerve damage; the exposed epithelium becomes inflamed and friable, predisposing the underlying loose connective tissues to bacterial invasion. infection of the underlying tissues leads to fibrosis, which permanently inhibits the normal telescoping action of the prepuce.5 replacement of the penis into the preputial cavity is essential in resolution of the condition. a common method of retaining the penis within the prepuce involves the use of a probang device, which has been previously described.6 the use of a purse string suture or towel clamps to narrow the preputial orifice has also been described, though these methods tend to cause more inflammation to the already vulnerable prepuce.4 the degree of inflammation and edema dictates whether or not penile repulsion will be initially possible or if reduction of edema must be accomplished before the penis can be replaced within the prepuce. if the penis cannot be replaced, methods of reducing edema include compressive elastic bandaging (i.e., esmarch), manual massage under sedation or anesthesia, topical application of a hyperosmolar agent, and systemic administration of non-steroidal anti-inflammatories. another method has been described whereby a 5-liter pressure infusion bag was applied over the prolapsed penis and prepuce in order to provide constant pressure. the amount of pressure may be adjusted as edema wanes without necessitating removal of the device.7 the use of an abdominal sling or bandage to maintain the penis and prepuce in a less dependent position is also useful in preventing the accumulation of further edema. the relatively inelastic preputial ring may act as a constrictor band and prevent replacement of the penis into the preputial cavity. in these cases a preputiotomy may be performed, whereby the preputial ring is incised longitudinally to relieve this constricting effect. the incision is left open to heal by second intention.8 if penile paralysis develops subsequent to paraphimosis, erectile function is likely permanently lost; however, affected stallions should retain the ability to ejaculate and may be trained to ejaculate with the aid of manual stimulation.9 successful chemical ejaculation of a stallion with penile paralysis has also been reported.10 in the described case, the severe and protracted nature of the paraphimosis negated the consideration of any medical treatment options. many options have been reported for surgical management of paraphimosis. the williams’ technique of partial phallectomy under general anesthesia was performed in this case. other surgical methods of distal phallectomy include the vinsot and scott’s.11,12 the vinsot technique is largely similar to the described william’s technique, though contrary to the william’s technique, the apex of the triangular urethrostomy is directed distally rather than proximally. a modified vinsot technique of partial phallectomy has also been described that can be performed in the standing sedated horse, negating the need for general anesthesia in debilitated horses or in cases where client finances are a constraint.13 the standing method was not selected in this case due to the size of the penis and figure 5. color photograph of the urethrostomy site post-transection of the distal penis and prior to compression of the ccp. the esmarch tourniquet (asterisk) has not yet been removed. figure 6. color photograph of the surgical sites post-phallectomy and post-castration. the penile stump has been returned to its normal placement within the prepuce and sheath. http://dx.doi.org/10.58292/ct.v15.9647 56 citation line: clinical theriogenology 2023, 15, 9647, http://dx.doi.org/10.58292/ct.v15.9647 the risk of hemorrhage. finally, scott’s technique of partial phallectomy involves the creation of a circumferential incision at the intended site of transection. dissection is continued to the level of the urethra. a segment of the urethra, approximately 4–5 cm in length, is dissected away from the distal end of the penis and is ultimately stretched over the end of the penis and sutured to the penile or preputial epithelium.12 the reason that the william’s technique was selected in this case was to minimize the chances of postoperative formation of a urethral stricture at the urethrostomy site, as the other described techniques pose a higher risk of stricture formation. the horse in this case was castrated under the same anesthetic episode as the distal phallectomy. when castration is indicated in a case of paraphimosis, it is typically performed several weeks prior to phallectomy to minimize the chances of the horse obtaining an erection post-phallectomy, which increases the chances of significant hemorrhage from the surgical site. however, in the described case, the client was reluctant to have the horse undergo two separate episodes of general anesthesia for financial reasons. further, the horse’s size and age gave the surgical team reason to reduce the number of anesthetic episodes. while castration can also be performed in the standing horse,14 the age of this stallion and size of the testicles necessitated meticulous ligation of the spermatic cord vasculature, which is more efficiently accomplished with the horse in dorsal recumbency under general anesthesia. postoperatively, this horse presumably experienced significant hemorrhage from the surgical site(s), as evidenced by the altered physical examination findings noted at 12 h postoperatively. however, there was not an abnormal amount of blood found in the stall, nor was any free fluid noted on transabdominal ultrasound. these findings make the question of where exactly the hemorrhage originated from, or where the resultant frank blood went, a perplexing one. it has thus been postulated that rather than an overt hemorrhagic event, the gelding’s pcv decreased so dramatically as a result of the acute removal of a significant pool of blood within the fibrosed penis. alternatively, it is possible that the horse was actually dehydrated on presentation with a pcv of 32% and a ts of 8.7 g/dl, as this ts value is slightly above the published reference range for draft horses (6.4–8 g/dl) but the pcv is within the reference range (24–42%).15 intravenous fluids during and after surgery could have thus led to hemodilution and signs of anemia. when the intravenous administration of aminocaproic acid failed to alleviate clinical signs, intravenous formalin was administered, and ultimately a 7  l whole blood transfusion was performed. perhaps prophylactic intraoperative administration of whole blood, rather than postoperative once the horse was showing signs of shock, would have prevented the occurrence of such complications. however, blood transfusions are rarely necessary in these cases, and as the patient was clinically stable with an adequate pcv prior to surgery (32%), there was no indication for such measures. conclusion this report describes the successful surgical treatment of an extremely protracted case of paraphimosis in a mature stallion, as well as the management of significant postoperative complications. when treated appropriately and in a timely manner, paraphimosis can be resolved and the horse can return to normal function. however, if allowed to progress, penile paralysis may result and may require surgical management. due to the high degree of vascularity of relevant anatomy, there is a risk of hemorrhage associated with partial phallectomy as well as with routine castration. this case demonstrates that these complications can be managed figure 7. color photograph of the postoperative swelling of the penile stump. figure 8. color photograph taken several months postoperatively. the gelding had improved in body condition since hospitalization. http://dx.doi.org/10.58292/ct.v15.9647 citation line: clinical theriogenology 2023, 15, 9647, http://dx.doi.org/10.58292/ct.v15.9647 57 successfully and the horse may return to normal function with appropriate postoperative care. references 1. sprayberry ka, lu kg: managing reproduction emergencies in the field: part 1: injuries in stallions; injury of the external portion of the reproductive tract and gestational conditions in the mare. vet clin equine 2021;37:339–366. doi: 10.1016/j. cveq.2021.04.007 2. moreno cr, delph km, beard wl: intravenous formalin for treatment of haemorrhage in horses. equine vet educ 2021;33:460–465. doi: 10.1111/eve.13451 3. mcdonnell sm: managing the paralyzed penis, priapism or paraphimosis in the horse. equine vet educ 2005;17:310–311. doi: 10.1111/j.2042-3292.2005.tb00398.x 4. schumacher j, varner dd: abnormalities of the penis and prepuce. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 1130–1144. 5. simmons ha, cox je, edwards gb, neal pa, urquhart ka: paraphimosis in seven debilitated horses. vet rec 1985;116:126–127. doi: 10.1136/vr.116.5.126 6. koch c, o’brien t, livesey ma: how to construct and apply a penile repulsion device (probang) to manage paraphimosis. proceedings of american association of equine practitioners, las vegas, 2009 december; p. 338–341. 7. brouwer ed, ribera t, climent f, et al.: alternative method to facilitate resolution of paraphimosis after penile trauma in the horse. equine vet educ 2017;29:655–658. doi: 10.1111/ eve.12556 8. walker df: surgery of the penis. in: walker df, vaughan jt, editors. bovine and equine urogenital surgery, philadelphia, pa; lea & febiger: 1980. p. 10. 9. love cc, mcdonnell sm, kenney rm: manually assisted ejaculation in a stallion with erectile dysfunction subsequent to paraphimosis. j am vet med assoc 1992;200:1357–1359. 10. feary dj, moffett pd, bruemmer je, et al.: chemical ejaculation and cryopreservation of semen from a breeding stallion with paraphimosis secondary to priapism and haemorrhagic colitis. equine vet educ 2005;17:299–304. doi: 10.1111/j.20423292.2005.tb00396.x 11. schumacher j, varner dd: surgery of the penis and prepuce. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 1130–1144. 12. schumacher, j: penis and prepuce. in: auer ja, stick ja: editors. equine surgery. 4th edition, st. louis; elsevier/saunders: 2012. p. 840–866. 13. arnold ce, brinsko sp, love cc, et al.: use of a modified vinsot technique for partial phallectomy in 11 standing horses. j am vet med assoc 2010;237:82–86. doi: 10.2460/javma.237.1.82 14. kilcoyne i, watson jl, kass ph, et al: incidence, management, and outcome of complications of castration in equids: 324 cases (1998–2008). j am vet med assoc 2013;242:820–825. doi: 10.2460/javma.242.6.820 15. gelser dr, goble do, held jp: normal hematology and serology of the clydesdale draft horse. equine prac 1984;6:7–11. http://dx.doi.org/10.58292/ct.v15.9647 https://doi.org/10.1016/j.cveq.2021.04.007 https://doi.org/10.1016/j.cveq.2021.04.007 https://doi.org/10.1111/eve.13451 https://doi.org/10.1111/j.2042-3292.2005.tb00398.x https://doi.org/10.1136/vr.116.5.126 https://doi.org/10.1111/eve.12556 https://doi.org/10.1111/eve.12556 https://doi.org/10.1111/j.2042-3292.2005.tb00396.x https://doi.org/10.1111/j.2042-3292.2005.tb00396.x https://doi.org/10.2460/javma.237.1.82 https://doi.org/10.2460/javma.242.6.820 2018: intrauterine therapies in mares ? a clinically applicable review intrauterine therapies in mares – a clinically applicable review s.h. cheong department of clinical sciences, college of veterinary medicine, cornell university, ithaca ny introduction intrauterine infusions in mares are commonly used by veterinarians to treat a variety of conditions. the intrauterine route offers some advantages over systemic treatments such as the ability to achieve higher concentration of drugs or compounds than is possible through systemic administration, the ability to use potentially toxic substances such as disinfectants, and allow direct contact of the compound to the endometrium. conversely, the disadvantages of intrauterine infusions include the risk of irritation, contamination, poor retention, and deactivation by uterine content. the choice to include intrauterine infusions can be complicated by the increasing number of compounds and products that have been proposed. this paper summarizes some of the established groups of compounds used as intrauterine treatments and discusses emerging compounds that have been proposed but not yet used in routine practice. antibiotics bacterial endometritis is the most common reason for intrauterine treatment and it is not surprising that antibiotics are the most established group of compounds used as intrauterine infusion in mares. antibiotic resistance is a major concern in veterinary and human medicine and judicious use of antibiotics is essential. it is recommended that antibiotic use be based on bacterial culture and sensitivity results or based on morphological appearance of the bacteria on cytological examination. further, intrauterine antibiotic treatment should be avoided during diestrus as it may promote fungal endometritis. the main concern for intrauterine infusion in mares is the potential irritation to the endometrium from antibiotics. recommendations are available for the buffering or dilution of some antibiotics to minimize endometrial irritation. biological material in the intrauterine fluid may reduce efficacy of antibiotics and thus uterine lavage to remove excess biological material in the uterus is recommended prior to intrauterine infusion of antibiotics. some bacteria may form biofilm that protects the bacteria against antibiotics. thus, treating mares with suspected biofilm producing bacteria with biofilm reducing agents prior to intrauterine antibiotic infusion will improve efficacy of the antibiotics. aminoglycoside amikacin sulfate. amiglyde-v®a is licensed for intrauterine use in mares at the dose of 2g (8ml) daily for three consecutive days diluted in 200 ml sterile saline for treatment of endometritis, metritis, and pyometra in mares. alternatively, amikacin can be buffered by adding at least equal volumes of 8.4% sodium bicarbonateb to reduce endometrial irritation. it should not be used on in horses intended for human consumption. amikacin has been demonstrated to be effective clinically in mares against escherichia coli, klebsiella sp., and pseudomonas sp. in addition, a large number of pathogenic bacteria especially gram-negative bacteria have been shown to be susceptible against amikacin in vitro but its effectiveness may be poorer for gram-positive bacteria. it is not recommended to infuse amikacin on the day of breeding if possible due to the potential detrimental effect on sperm. gentamycin sulfate. sparhawk laboratories inc., obtained fda approval for generic gentamycin sulfate solution use for intrauterine infusion for the treatment of bacterial metritis and improving fertility of mares. it is not to be used in horses intended for human consumption. the recommended dose for intrauterine infusion is 2.0 – 2.5g (20 to 25ml) diluted with 200 – 500ml of sterile saline daily for theee to five days during estrus. the large volume recommended is not easy to retain in the uterus and an alternative method is to buffer gentamycin with a minimum of equal volume of 8.4% sodium bicarbonateb. spectrum of efficacy is better for gram-negative bacteria and it is not recommended to infuse gentamycin on the day of breeding for the potential impairment of sperm. clinical theriogenology • volume 10, number 3 • september 2018267 cephalosporin ceftiofur sodium. naxcel®c is not labeled for intrauterine use in mares. several papers have recommended the dose of 1 g diluted in 100 ml sterile saline.1,2 spectrum of efficacy includes grampositive and gram-negative bacteria. fluoroquinolone enrofloxacin. is not labeled for intrauterine use in mares. the injectable preparation of enrofloxacin results in severe endometrial inflammation when infused into the uterus at 2.5mg/kg on three consecutive days and is not advisable for intrauterine treatment in mares.3 more recently, a compounded water-based preparation of enrofloxacind has been evaluated for intrauterine use at a dose of 50ml of 2.5% suspension and has been shown to be safe and did not induce worsening endometrial biopsy score.4 spectrum of efficacy includes gram-positive and gram-negative bacteria. ciprofloxacin. is not labeled for intrauterine use in mares. the use of the injectable preparation of ciprofloxacine for intrauterine treatment 60ml of 10mg/ml preparation in mares resulted in seven of ten mares with normal uterine cytology after 24 hours while the remaining two mares had mild inflammation and one mare had moderate inflammation on cytology.5 while the endometrial response to ciprofloxacin is milder than the injectable form of enrofloxacin, it is not yet determined if the long-term detrimental effects seen for enrofloxacin will occur in mares treated with intrauterine ciprofloxacin. spectrum of efficacy includes gram-positive and gram-negative bacteria. penicillin ampicillin. is not labeled for intrauterine use in mares. ampicillinf 2g diluted in at least 60ml of sterile saline has been proposed but a lower concentration (i.e., more dilute preparation) is recommended as higher concentrations can be irritating to the endometrium.6 spectrum of efficacy includes grampositive bacteria and susceptible escherichia coli. ticarcillin with or without clavulanate potassium. the commercial ticarcillin disodium with clavulanate potassium (timentin®)g has been voluntarily withdrawn by the manufacturer and is no longer available. recommendation of use was 3 to 6g reconstituted in a larger volume of at least 150 to 200ml of sterile saline and is effective against gram-positive bacteria and pseudomonas. potassium penicillin. pfizerpen®h is not labeled for intrauterine use in mares. the dose of 5 million iu in 100ml of sterile saline has been shown to reduce growth of streptococcus equi subspecies zooepidemicus7 and the spectrum of efficacy includes gram-positive bacteria. procaine penicillin. is not labeled for intrauterine use in mares. the spectrum of efficacy includes gram-positive bacteria. procaine penicillin is substantially cheaper than potassium penicillin. polymyxin polymyxin b. polymixin b sulfate is not approved for intrauterine infusion in mares. the spectrum of activity includes gram-negative organisms including pseudomonas. the recommended intrauterine dose of polymixin b is 1 million iu diluted in at least 60ml sterile saline. antifungal fungal endometritis is commonly treated with intrauterine medications. many practitioners do not submit fungal cultures and sensitivity as there are only a few laboratories that offer antifungal susceptibility testing and the turnaround time can be lengthy. there are no approved antifungal drugs for intrauterine infusion in mares. most yeast isolates have good susceptibility to antifungal drugs but mold isolates are much more likely to be resistant to antifungal drugs.8 therefore a multimodal therapy using antifungal drugs and other treatments is indicated especially for fungal endometritis with mold isolates. the most common antifungal drugs used for intrauterine infusion are: clotrimazole, nystatin, miconazole, fluconazole and amphotericin b in that order.6 the course of treatment is recommended to be for five to seven days. clinical theriogenology • volume 10, number 3 • september 2018 268 azole – imidazole clotrimazole. the recommended dose is 500 to 700mg of clotrimazole diluted in 50ml of sterile saline. clotrimazole tablets 100mg can be crushed and resuspended in saline and there are compounded preparations available. miconazole. the recommended dose of miconazole for intrauterine treatment is 1,200mg ovules insert of monistat®i. azole – triazole fluconazole. fluconazole tablets does not dissolve easily in water and requires a vehicle usually dmso to dissolve. add at least 1ml of dmso for each 200mg fluconazole tablet and dilute in sterile water to at least 60ml. compounded fluconazole preparations for intrauterine infusions are also available. polyenes amphotericin b. the recommended dose for intrauterine treatment with amphotericin b is 100 to 200mg of fungizonej 50mg per vial. dilute two to four vials of amphotericin b in sterile saline to at least 100ml. nystatin. the recommended dose of nystatin for intrauterine treatment is 0.5 million to 2.5 million iu (5g) suspended in 10ml of sterile saline. non-specific antifungal lufenuron. the recommended dose for lufenuron treatment is two doses of 266mg program®k infused intrauterine and an additional 266mg dose to be applied to the vaginal vault and clitoral area. acetic acid. distilled vinegar at a concentration of 2% of the commercially available product is a popular treatment for fungal endometritis. the actual concentration of acetic acid in household distilled vinegar varies from high 20’s to high 30’s % thus the actual concentration of acetic acid used for uterine lavage is much lower than 2%. preparation of vinegar lavage typically is done by adding 20ml of distilled vinegar to 1l of sterile saline. it is unclear if adding distilled vinegar to lactated ringer’s solution which contains buffers will reduce efficacy and potentially reduce endometrial irritation from acidic ph. antiseptic povidone iodine. povidone iodine is more commonly used during the postpartum period but also when treating fungal endometritis. betadine®l solution is available in 5% and 10% strength preparations thus it is important to know which preparation is being used to calculate the dilution. in a 10% solution, there is only 1% active iodine thus to prepare a 0.05% povidone iodine solution, add 5ml of 10% solution to 995ml sterile saline.9 higher concentrations of povidone iodine may cause irritation to the endometrium and lower doses are recommended. hydrogen peroxide. traditionally recommended to be used at 1% strength or by diluting 20ml of 3% hydrogen peroxide to 60ml with sterile saline. this concentration of hydrogen peroxide has been shown to be able to reduce biofilm biomass and reduce escherichia coli and klebsielle pneumoniae colony forming units.10 the same group has recently recommended infusion of 60 to 120ml of 3% hydrogen peroxide for treatment of fungal endometritis.11 dimethyl sulfoxide. dimethyl sulfoxide (dmso) is indicated for the reduction of uterine inflammation and to reduce biofilm. dilute 50 to 200ml of dmso in 1l sterile saline followed by uterine lavage with sterile saline or lactated ringer’s solution without dmso. tricide® and tris edta. chelators has been shown to enhance antibiotic efficacy and proposed to disrupt biofilm. the recommendation of use is to infuse 250 to 500ml of tricide®m into the uterus and lavage the uterus with lactated ringer’s solution to remove the debris. n-acetylcysteine. n-acetylcysteine is a known mucolytic and is effective to treat endometritis from biofilm producing organisms.10 dilute 30ml of acetylcysteine (20% or 200mg/ml) into 150ml of clinical theriogenology • volume 10, number 3 • september 2018269 sterile saline and infuse into the uterus. the uterus should be lavaged to recover the uterine content 12 to 24 hours after infusion. the efflux fluid content should be examined for the presence of evidence of mucus and biofilm and repeat treatment as necessary. ceragenins. ceragyn™n products contains purifect™ technology molecules that disrupt bacterial cell membrane. there are two ceragyn products the ceragyn lavage and ceragyn infusion. ceragyn lavage is diluted in sterile lactated ringer’s solution and can be used to lavage mares 24 hours before and 12 hours after insemination. kerosene. kerosene is a very controversial compound used for intrauterine infusion. the common indication for use is typically a barren mare that has poor uterine biopsy grade even grade iii. the subsequent fertility results of previously barren mares are surprisingly good.1,12 some care should be observed when performing kerosene intrauterine infusion. first, the equipment used should not contain rubber or weak plastics. second, the mare should be kept outdoors where the kerosene that may be discharged from the vagina will not cause fires. the infusion volume is typically between 90ml and 500ml and the kerosene should be removed by uterine lavage the following day. mycobacterium cell wall immunostimulant. the indication of using settle™o mycobacterium cell wall fraction immunostimulant is to enhance immune function of the mare to overcome endometritis caused by streptococcus equi subspecies zooepidemicus.13 intrauterine infusion dose is 1.5ml of settle™ diluted in sterile lactated ringer’s solution to a volume of 25 to 50ml. bactivate. latent bacterial infections evade detection and action of the immune system and antibiotics by remaining dormant. bactivatep is a compound that reactivates these dormant bacteria making them more susceptible to treatment.14 the indication of bactivate is to treat mares suspected to have latent bacterial infection. it is recommended to collect a pre-infusion sample for culture by biopsy or low-volume lavage in early estrus. infuse 10ml of bactivate and collect a repeat culture sample 24 hours after infusion and treat for bacterial endometritis. regenerative medicine currently, intrauterine use of autologous or allogenic biologics in equine practice is not commonplace. several groups have begun testing these methods and the results are promising. most of these products are theorized to regulate the immune response of the uterus to infection and contamination. autologous plasma autologous plasma is the use of plasma collected from the mare, processed, and then infused into the uterus to moderate the local immune response. intrauterine infusion with autologous plasma reduced the number of neutrophils after streptococcus equip subspecies zooepidemicus inoculation but did not reduce the growth of the bacteria.7 autologous plasma intrauterine infusion after breeding was also beneficial for pregnancy rates.15 overall the results are encouraging but much more work has to be done to convince the industry to adopt this treatment. allogeneic plasma allogenic plasma is the use of frozen plasma usually meant for foals with failure of passive transfer to be infused into the uterus. this reduces the work required to produce the plasma for infusion and is likely more appealing to the practitioner. platelet-rich plasma platelet-rich plasma has been used clinically to modulate inflammatory response in mares for orthopedic conditions but the use in reproduction is still not well established. there are several reports of the beneficial effects of platelet-rich plasma16-19 but the production of platelet-rich plasma is somewhat difficult in the field and a little costly to use the established systems. the increasing use of platelet-rich plasma in equine orthopedics will likely help the development of this method as more locations are able to produce the product by the sports medicine group in-house and use it in the reproductive unit. clinical theriogenology • volume 10, number 3 • september 2018 270 mesenchymal stem cells mesenchymal stem cell isolation technology is constantly being improved and many biotechnology companies are focusing on improving yield and application of bone-marrow derived, adipose derived and umbilical cord blood derived mesenchymal stem cells from horses. the use of allogenic bone-marrow derived mesenchymal stem cells to modulate the immune response to dead-sperm cells20 and adipose derived mesenchymal stem cells were successfully observed in both uterine horns and body in transplanted mares.21,22 references 1. scoggin cf: endometritis: nontraditional therapies. vet clin north am equine practice. 2016;32:499–511. 2. leblanc mm: advances in the diagnosis and treatment of chronic infectious and post–mating-induced endometritis in the mare. reprod domest anim 2010;45(s2):21-27. 3. odriguez js, han s, nielsen s, et al: consequences of intrauterine enrofloxacin infusion on mare endometrium. j equine vet sci 2012;32:106-111. 4. schnobrich mr, pearson lk, barber bk, et al: effects of intrauterine infusion of a water-based suspension of enrofloxacin on mare endometrium. j equine vet sci 2015;35:662-667. 5. trundell da, ferris ra, hennet mr, et al: pharmacokinetics of intrauterine ciprofloxacin in the mare and establishment of minimum inhibitory concentrations for equine uterine bacterial isolates. j equine vet sci 2017;54:5459. 6. dascanio jj: how and when to treat endometritis with systemic or local antibiotics. proc annu conv am assoc equine pract; 2011. p. 24-31. 7. troedsson mh, scott ma, liu ik: comparative treatment of mares susceptible to chronic uterine infection. am j vet res 1995;56:468-472. 8. beltaire ka, cheong sh, coutinho da silva ma: retrospective study on equine uterine fungal isolates and antifungal susceptibility patterns (1999–2011). equine vet j 2012;44(s43):84-87. 9. perkins nr: equine reproductive pharmacology. vet clin north am equine practice 1999;15:687-704. 10. ferris ra, mccue pm, borlee gi, et al: in vitro efficacy of nonantibiotic treatments on biofilm disruption of gramnegative pathogens and an in vivo model of infectious endometritis utilizing isolates from the equine uterus. j clin microbiol 2016;54:631-639. 11. ferris ra: therapeutics for infectious endometritis: a clinical perspective. rev bras reprod anim 2017;41:175-179. 12. bracher v, neuschaefer a, allen wr: the effect of intra-uterine infusion of kerosene on the endometrium of mares. j reprod fertil suppl 1991;44:706-707. 13. rogan d, fumuso e, rodriguez e, et al: use of a mycobacterial cell wall extract (mcwe) in susceptible mares to clear experimentally induced endometritis with streptococcus zooepidemicus. j equine vet sci 2007;27:112-117. 14. petersen mr, skive b, christoffersen m, et al: activation of persistent streptococcus equi subspecies zooepidemicus in mares with subclinical endometritis. vet microbiol 2015;179:119-125. 15. pascoe dr: effect of adding autologous plasma to an intrauterine antibiotic therapy after breeding on pregnancy rates in mares. biol reprod monogr ser 1 1995;539-543. 16. metcalf es: the effect of platelet-rich plasma (prp) on intraluminal fluid and pregnancy rates in mares susceptible to persistent mating-induced endometritis (pmie). j equine vet sci 2014;34:128. 17. metcalf es, scoggin k, troedsson mht: the effect of platelet-rich plasma on endometrial pro-inflammatory cytokines in susceptible mares following semen deposition. j equine vet sci 2012;32:498. 18. reghini mfs, bussiere mcc, neto cr, et al: effect of use of platelet rich plasma on post-breeding uterine inflammatory response of mares. j equine vet sci 2014;34:127. 19. segabinazzi lg, friso am, correal sb, et al: uterine clinical findings, fertility rate, leucocyte migration, and cox-2 protein levels in the endometrial tissue of susceptible mares treated with platelet-rich plasma before and after ai. theriogenology 2017;104:120-126. 20. ferris ra, frisbie dd, mccue pm: use of mesenchymal stem cells or autologous conditioned serum to modulate the inflammatory response to spermatozoa in mares. theriogenology 2014;82:36-42. 21. mambelli li, winter ghz, kerkis a, et al: a novel strategy of mesenchymal stem cells delivery in the uterus of mares with endometrosis. theriogenology 2013;79:744-750. 22. mambelli li, mattos rc, winter gh, et al: changes in expression pattern of selected endometrial proteins following mesenchymal stem cells infusion in mares with endometrosis. plosone 2014;9:e97889. footnotes a amiglyde-v®, zoetis, kalamazoo, mi b sodium bicarbonate injection 8.4%, hospira inc., lake forest, il c naxcel®, zoetis, kalamazoo, mi d enrofloxacin uterine flush, rood & riddle veterinary pharmacy, lexington, ky e ciprofloxacin, hospira inc., lake forest, il clinical theriogenology • volume 10, number 3 • september 2018271 f polyflex®, bohringer ingleheim vetmedica, duluth, ga g timentin®, galaxosmithkline, durham, nc h pfizerpen®, roerig, new york, ny i monistat®, insight pharmaceuticals, tarrytown, ny j fungizone, x-gen pharmaceuticals inc., horseheads, ny k program®, novartis animal health, surrey, uk l betadine® purdue pharma l.p., stamford, ct m tricide®, medical molecular therapeutics, athens, ga n ceragyn™, pureshield life sciences llc, walnut creek, ca o settle™, novavive, athens, ga p bactivate, bojesen and petersen biotech aps, copenhagen, denmark clinical theriogenology • volume 10, number 3 • september 2018 272 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2018: pharmacokinetics of intra-rectal altrenogest administration in horses pharmacokinetics of intra-rectal altrenogest administration in horses k.e. ellis,a k.a.von dollen,a k. mirandi,a t.m. beachler,a c.s. bailey,a j.l. davis,b s.k. lylea adepartment of clinical sciences, college of veterinary medicine, north carolina state university, raleigh, nc bdepartment of biomedical sciences and pathobiology, virginia-maryland college of veterinary medicine, blacksburg, va hospitalized high-risk pregnant mares being held nil per os (npo) due to medical or surgical events present a dilemma for multi-modal therapy aimed at pregnancy maintenance, which commonly includes oral altrenogest as the supplemented progestin. some long-acting intramuscular preparations do not reach peak concentrations until 24 h after administration and repeated im injections can cause severe complications, such as abscess formation and clostridial myonecrosis. rectal administration of medications is a recognized route for achieving systemic concentrations, but there are currently no data available on the pharmacokinetics of rectal altrenogest administration in horses. the purpose of this study was to determine the pharmacokinetics of altrenogest following per os (po) or per rectum (pr) administration in mares. using a randomized two-way crossover study design, six horses received altrenogest (0.088 mg/kg; po or pr q 24 h for 5 d), with a 7day washout period. plasma samples were collected prior to administration (0 h) and at 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 12, 24, 48, 72, 96.0, 96.25, 96.5, 97.0, 97.5, 98.0, 98.5, 99.0, 100, 102, 104, 108, 112, 120, 144, 168, and 192 h after administration. the concentrations of altrenogest were determined by ultra-high performance liquid chromatography with tandem mass spectrometry. assay validation and quality assurance were performed according to published guidelines. administration pr produced therapeutic plasma levels in all mares following initial dosing (cmax 2.54 + 1.18 ng/ml) with a markedly decreased time to maximum concentration (tmax 0.83 + 0.26 h ) when compared to po administration (tmax 4.25 + 4.83 h). plasma concentrations only persisted above presumed therapeutic concentrations (0.5 ng/ml) for a mean of 5.5 h (range 3-8 h) following pr administration, while serum concentrations remained > 0.5 ng/ml for at least 24 h in all horses following po administration. the calculated half-life (t½) of pr administration (2.82 + 1.07 h) was correspondingly decreased when compared to po administration (7.01 + 3.13 h). relative bioavailability of altrenogest following pr administration was only 5.47%. in conclusion, altrenogest is rapidly absorbed following pr administration in the horse and reaches therapeutic concentrations, making this a viable method of treatment in npo mares. however, the decreased bioavailability and shorter detection time would require a shorter dosing interval. results of this study suggest 0.088 mg/kg pr q 4-8 h would be necessary to maintain therapeutic concentrations over a 24 h period. keywords: horse, altrenogest, pharmacokinetics, intra-rectal administration clinical theriogenology • volume 10, number 3 • september 2018313 introduction over, 11,300 species of reptiles have evolved on every continent except antarctica. at this moment in history, we are observing the biological annihilation of the natural world. anthropogenic induced environmental alterations such as habitat loss and degradation, invasive species introduction, environmental pollution, infectious disease, unsustainable harvesting, illegal trade, and global climate changes are all substantial threats to current reptile populations worldwide. one in 5 reptile species is threatened with extinction. this is the harsh reality of our lifetime. zoological institutions utilizing both in-situ and ex-situ conservation strategies have contributed to reptile conservation; however, assisted reproductive technologies may be 1 of the last hopes to preserve and conserve reptilian biodiversity for some species. we briefly review what has been completed to date in order to help develop assisted reproductive technologies (art) for reptiles and discuss where art is applicable to reptiles. gamete collection techniques males postmortem collection of samples can be used for endangered species that die in zoological settings and from animals subject to mass-mortality events to preserve genetic diversity.1 during the reproductive season, epididymis and ductus deferens distend with sperm because these are the sites where sperm undergo in vivo maturation and storage prior to ejaculation. therefore, postmortem sperm recovery should focus on sperm present in the epididymis and ductus deferens. once the male reproductive developing assisted reproduction for reptiles, what’s next? sean perry mississippi aquarium, gulfport, ms abstract assisted reproductive technologies (art) are often used as management tools to protect endangered vertebrate taxa; however, progress in developing art for reptiles has been at a tortoise’s pace! as proven useful for other species, the creation of functional and sustainable art in reptiles can strengthen our conservation abilities by allowing zoos and aquariums to capture genetic material from select important individuals. this is a review of reptile research in art dating back to the 1970’s, highlighting important areas that still need investigation. topics include artificial insemination, gamete collection, gamete storage, and genome resource banking. additionally, potential application of in-vitro/ex-ovo fertilization, intracytoplasmic sperm injection (icsi), cloning (somatic cell nuclear transfer), and genetic editing to reptiles, and the hurdles that need to be overcome, are included. for all reptile taxa, utilizing short-term gamete storage and genome resource banking, in conjunction with timed artificial insemination or ex-ovo incubation, could lead to profound advances in reptilian art, with the hope of mitigating the loss of reptile biodiversity. keywords: reptiles, assisted reproduction, conservation, gamete tract is collected, sperm can be harvested by expressing the ductus deferens and/or macerating the epididymis under a dissection scope. samples should not be collected directly from the testes, as testicular sperm have poor motility (1%); even after being exposed to a phosphodiesterase inhibitor (caffeine), motility only increased to 7%.2 successful ai has been performed using sperm recovered in crocodilians at postmortem.3,4 currently, postmortem collection is the best method to conduct experiments to develop art for reptiles, as it provides large sample volumes from model reptile species that are necessary to further develop art. antemortem semen collection was performed in a wide range of reptile species via various methodologies, including coelomic massage, digital phallic massage, a vibrational method, and electroejaculation. coelomic massage was used successfully to collect semen from snakes and small lizards. in snakes, semen was collected by gently massaging the ventral caudal third of the coelomic cavity using light digital pressure in a caudoventral direction toward the cloaca.5-13 similarly, digital ventral coelomic massage was used in small geckos and skinks to collect semen samples.14,15 in crocodilians, digital phallic massage was used to successfully collect semen under mild sedation and manual restraint. success with this strategy was reported in american alligators (alligator mississipiensis), broad-nosed caiman (caiman latirostris), saltwater crocodiles (crocodylus porosus), and philippine crocodiles (crocodylus mindorensis). to collect semen, the crocodilian is placed in ventral recumbency and straddled across 2 supporting structures; the phallus extruded clinical theriogenology 2021; 13: 383 from the proctodeum using a gloved finger; and the phallus base at the level of the urodeum massaged until a sample is produced within the sulcus spermaticus.3,4,16-21 no reports exist of using this method in other species, although investigation may be warranted, especially in chelonians. one report described the use of a human vibrational device to collect semen from chelonians. male turtles were held upright and an appropriately sized vibrator set to its highest frequency and placed against the carapace and moved along the carapace in linear and circular motions. if no erection was noted after carapacial stimulation, then the vibrator was placed on the plastron for 7 22 minutes. semen collection success was species-specific, with good success in blanding’s turtles (emydoidea blandingii) (12/12), but poor success in painted turtles (chrysemys picta) (4/30) and wood turtles (glyptemys insculpta) (2/17).22 the most widely used method to collect semen antemortem from reptiles is electroejaculation (eej), used to successfully and safely collect ejaculates from all reptile taxa other than the tuatara (sphenodon punctatus). this is the preferred antemortem method to collect semen in larger lizards and chelonians because their anatomy limits the use of some of the other methodologies. semen collection by eej in lizards and chelonians is typically performed under anesthesia or heavy sedation; however, some studies did not sedate animals for this procedure. various electroejaculation units and protocols were reported to be successful for collecting semen from reptiles. most had a variable voltage/ amperage power source with a plastic or metallic probe. much of the variation in eej techniques is due to the variation in the size of animals, unique species anatomy, and variable/limited availability of appropriately sized probes. in lizards, eej was first performed in green iguanas.23 since then, multiple studies were conducted to evaluate the safety and efficacy of eej in lizards, including veiled chameleons (chameleo calyptratus), spiny lava lizards (tropidurus spinulosis), spiny lizards (sceloporus torquatus), and grand cayman blue iguana hybrids (cyclura lewisi x nubila).23-30 safety of repeated eej in anesthetized veiled chameleons demonstrated that repeated anesthesia and eej can be performed safely and successfully at least once weekly in this species.24 overall animal health was assessed using physical examination, complete blood counts, biochemistry values, cloacal external morphology, and cloacal mucosal inflammation with endoscopy. transient cloacal inflammation was observed after eej, but resolved spontaneously.24 this study demonstrated that repeated semen collection can be done safely using eej in reptiles without impacting the long-term health of the reptiles. to date, eej effects on peripheral corticosterone (stress-related) concentrations in reptiles was not evaluated. in chelonians, eej is the most common antemortem method used to collect semen. eej was used to collect semen from leopard tortoises (stigmochelys pardalis), green sea turtles, red-eared slider turtles, a galapagos tortoise (chelonoidis nigra), a ploughshare tortoise (astrochelys yniphora), olive ridley turtles, hawksbill turtles, and the black marsh turtle (siebenrockiella crassicollis).31-38 eej was performed with and without anesthesia in chelonians with no associated discomfort or trauma; however, no studies specifically evaluated those effects.31,38 in reptiles anesthesia and sedation techniques were improved in the past 20 years. sedation or anesthesia is recommended for eej to limit discomfort. an aversion to performing eej in chelonians exists because a single report of a death that occurred in a ploughshare tortoise 30 days after semen collection due to suspected renal disease.31,33 however, no reports exist in the literature associating renal failure and eej in any species. in snakes, eej was successful in checkered garter snakes (thamnophis marcianus) as an alternative to coelomic massage. eej in snakes pose a special problem because of the distance from the cloaca to the gonads. for the garter snakes, a special elongated eej probe was used due to the cranial position of the reproductive tract. in larger snakes, eej may be the only method that can be used to collect semen because coelomic massage can be challenging.31,39 in crocodilians, eej was used successfully to collect semen from american alligators and saltwater crocodiles.18,20 females there are no reports of antemortem or postmortem collection with in vitro oocyte maintenance and maturation for reptiles. oocyte recovery in live reptiles is complicated. numerous factors contribute to this, including reproductive season/timing, ovary type/structure, ovulation timing, or deposition of albumen or egg shell. we do not have well established antemortem collection maintenance methods. viviparous reptile species should be the first investigated to develop antemortem collection methods, as these species are reproductively more similar to mammals, and techniques are likely more transferable. semen handling and genome resource banking a fully functional genome resource bank for reptiles is necessary for continued conservation efforts. a complete genome resource bank could help maximize our ability to store reproductive cells and tissues. the technology and techniques required to develop, maintain, and grow a genome resource bank include our ability to routinely collect sperm, oocytes, embryos, and somatic cells. once these samples are collected, indefinite storage is required, which could be achieved using cryopreservation. semen handling and extension semen handling and extension methods were investigated in at least 1 species from each reptile taxon, except the tuatara. standard semen handling methods should be adhered to when handling reptile sperm. a general trend is that reptile sperm can be successfully stored at low temperatures for shorter durations. species evaluated include the american alligator (alligator mississippiensis), saltwater crocodile (crocodylus porosus), broad snouted caiman (caiman latirostris), green iguana (iguana iguana), green anole (anolis carolinensis), mccann’s skink (oligosoma maccanni), hybrid grand cayman blue iguanas (cyclura lewisi x nubila), eastern water skink (eulamprus quoyii), corn snake (pantherophis guttatus), banded watersnake (nerodia fasciata), clinical theriogenology 2021; 13: 384 red eared slider (trachemys scripta elegans), leopard tortoise (stigmochelys pardalis), olive ridley(lepidochelys olivacea), and hawskbill sea turtle (eretmochelys imbricata).10,15,19,20,21,23,30,34,35,38,40-42 semen extenders are summarized (table 1). semen cryopreservation/vitrification in reptiles, only a few reports exist describing experiments evaluating sperm cryopreservation and none exist exploring vitrification. species that were studied to date include the saltwater crocodile, american alligator, argentine black and white tegu (tupinambis (salvator) merianae), eastern water skink (eulamprus quoyii), argus monitor (varanus panoptes), red diamond rattlesnake (crotalus ruber), burmese python (python bivittatus), and the red eared slider turtle.3,4,16,17,25,41-49 the most extensive work regarding reptilian cryopreservation taxon species media temperature (°c) total motility (%) duration crocodilians american alligator (alligator mississippiensis) best with egg yolk and/ or milk 5 25 50 2 5 days saltwater crocodile (crocodylus porosus) pbs 30 < 30 120 minutes broad snouted caiman (caiman latirostris) best with egg yolk 5 > 60 5 days lacertilians green iguana (iguana iguana) modified ham’s f-10 + test yolk buffer 5 (equitainer) 60 24 hours green anole (anolis carolinensis) ham’s f-10 inra 96 sperm wash media 4 25 12 hours mccann’s skink (oligosoma maccanni) ham-f 10 4 70 5 days hybrid grand cayman blue iguanas (cyclura lewisi x nubila) inra 96 test yolk buffer 4 4 41 24 hours eastern water skink (eulamprus quoyii) pbs tlhepes ham’s f-10 20 70 16 hours serpentes corn snake (pantherophis guttatus) modified ham’s f-10 + test yolk buffer 5 (equitainer) > 50 48 hours banded watersnake (nerodia fasciata) inra 96 test yolk buffer sperm wash media 4 > 60 72 hours chelonians red eared slider (trachemys scripta elegans) same as n. fasciata 4 > 60 96 hours leopard tortoise (stigmochelys pardalis) modified ham’s f-10 + test yolk buffer 4 0 24 hours olive ridley sea turtle (lepidochelys olivacea) test yolk buffer tyrode medium with albumin, lactate, pyruvate beltsville poultry semen extender 3% sodium citrate buffer phosphate buffered saline eel 1% bovine serum albumin ham f-10 4 0 5 6 hours hawskbill (eretmochelys imbricata) same as for l. olivacea 4 < 5 6 hours table 1. semen extenders that have been evaluated for short-term storage of reptile sperm clinical theriogenology 2021; 13: 385 was performed in saltwater crocodiles (table 2). as with other taxa, methods to cryopreserve reptile sperm were not consistent across species, although, major themes emerged when reptile sperm are cryopreserved. first, although the effects of cold shock on sperm was only evaluated in saltwater crocodiles and argus monitors, both appeared to be tolerant of cold shock.45,48 second, cryoprotectant type and concentration affected prefreeze motility. cryoprotectant cytotoxicity and the protective effects against cryodamage is a complex multifactor interaction in reptiles.50 third, dmso and glycerol were used across species and have promise. finally, freeze periods vary taxon species media cryoprotectant concentration storage container dreeze rate (°c/minute) crocodilians saltwater crocodile johnston, et al: 2014b, johnston, et al: 2017 dulbecco’s pbs w/o ca2+ and mg2+ dmso dimethylacetamide (dma) glycerol 0.68 m [5%] 1.35 m [10%] 2.7 m [20%] 250 µl french straws 6 21 dulbecco’s pbs w/o ca2+ and mg2+ trehalose raffinose sucrose 0.3 m 250 µl french straws 6 21 dulbecco’s pbs w/o ca2+ and mg2+ glycerol +sucrose 0.68 m [5%] + 0.2 m sucrose 0.68 m [5%] + 0.3 m sucrose 1.35 m [10%] + 0.2 m sucrose 1.35 m [10%] + 0.3 m sucrose 250 µl french straws 6 21 american alligator dmso glycerol 5% 10% 20% 30% lacertilians argentine black and white tegu (tubinambis merianae) young, et al: 2017 test-yolkm199 with hepes dmso glycerol 8% 12% 16% cryovials 0.3 1 6.3 eastern water skink (eulamprus quoyii) (hobbs, et al: 2018) pbs w/o ca2+ and mg2+ tris-yolk buffer beltsville poultry semen extender dmso 1.35 m 200 µl french straws 6 argus monitor (varanus panoptes) pbs w/o ca2+ and mg2+ dmso glycerol 5% 10% 15% 250 µl french straws 32.1 serpentes red diamond rattlesnake (crotalus ruber) (zacariotti, et al: 2011) lake’s extender test-yolk buffer dmso glycerol 2% 4% 4% 8% 10% cryovials see abstract for protocols burmese python (python bivittatus) (young, et al: 2017) test-yolk buffer dmso glycerol dmso + glycerol 8 % 12% 16% 4:4% 6:6% 8:8% cryovials 0.3 table 2. published semen cryopreservation methods that have been applied to reptile species (please directly refer to the source for methodologies) clinical theriogenology 2021; 13: 386 among studies but it appears that faster freezes may preserve a higher proportion of cells, although this remains species specific. sperm integrity may be more important than motility when measuring success, but this remains to be determined. studies using vitrification as a cryopreservation method have not been performed in reptiles. artificial insemination artificial insemination (ai) with successful fertilization, oviposition/ parturition, and production of offspring has only been reported in 3 snake species and 1 crocodilian. ai was successful with freshly collected or short-term extended semen. currently, no guidelines exist for recommendations regarding insemination volume, sperm concentration, and semen deposition location. use of cryopreserved semen needs to be investigated further. american alligator was the first reptile to be successfully artificially inseminated with fresh sperm and produce live offspring.16,18,19 currently, ai has limited value for the american alligator industry but should be pursued for threatened and endangered crocodilians. successful ai was reported in 3 species of snakes: corn snakes on 2 separate occasions, a garter snake (thamnophis marcianus), and an amazon tree boa (corallus hortulanus).12,39,51 corn snakes were the first snake species demonstrating offspring could be produced using fresh and cooled semen following posthibernation insemination. in this case, no follicular monitoring was performed, and all animals were inseminated at the same time. sperm was deposited into the oviduct using a metallic ball tip syringe. insemination was performed in 10 females, but only 3 females produced eggs. two of these clutches produced offspring with 33% (5/15) and 38% (5/13) hatch rates. genetic testing was performed on the offspring from both clutches to confirm paternity with microsatellites.12 other reports of successful ai in snakes include 3 clinical cases, 2 corn snakes and an amazon tree boa where the female snakes were monitored for follicular progression prior to insemination. ai was performed using endoscopic assisted oviductal catheterization and a transcervical insemination catheter. the corn snakes used in this study had successfully produced eggs in prior years from natural breeding, whereas the amazon tree boa was a wild-caught adult with an unknown reproductive history. paternity was never confirmed for either species. in the corn snakes, it was reported that from the 2 clutches all eggs were fertile and hatched. the authors reported that the amazon tree boa underwent parturition 4 months after insemination and successfully produced 2/7 live young. paternity testing was not performed in these cases. in lizards, a single report of ai exists. a micropipettor was used to inseminate a mccann’s skink at the lateral side of the cloaca. no offspring were produced from the procedure, but environmental issues and a disease outbreak may have contributed to this outcome.15 reports of ai in chelonians are limited. based on the literature, ai was only attempted in the ploughshare tortoise and the yangtze giant softshell turtle (rafetus swinhoei). in 1982, ai was attempted in a ploughshare tortoise following semen sample collection via eej. the female laid a clutch of 7 eggs 12 days after the ai procedure. a single dead embryo was reported in this clutch. it was concluded that this clutch was not secondary to the ai due to quick oviposition. additional clutches were laid with dead embryos and one live hatching in december 1982 and february 1983. these latter events could have been related to the ai or sperm storage from natural copulation in 1982. this was never further investigated with paternity testing.33 in the case of the yangtze giant softshell turtle, ai was performed thrice since 2015 in the last known female of the species, and she successfully laid eggs following insemination, but none have been fertile. when performing ai in reptiles confirmation of paternity is recommended. multiple paternity was documented in all major groups of reptiles and is suggestive of high levels of female promiscuity.52 multiple paternity arises in reptiles via 2 routes: 1) either mating occurs with more than 1 male during the same reproductive cycle, or 2) mating occurs with 1 or more males during each reproductive cycle and sperm storage occurs.52 tests for multiple paternity in animals with sperm storage is needed because of this reproductive strategy. additionally, alternative mating strategies such as parthenogenesis occur in reptiles complicating paternity confirmation. in order to confirm success with ai, testing is needed for each offspring to confirm paternal lineage and rule out parthenogenesis. in-vitro/ex-ovo fertilization/intracytoplasmic sperm injection in-vitro/ex-ovo fertilization and icsi have not been described in any reptile to date. this likely stems from our incomplete knowledge of ovulation timing. additionally, a major hurdle to overcome is developing a method to harvest the ovum following ovulation but before fertilization in a minimally invasive fashion. currently, successful ovum collection would require surgical or terminal procedures following ovulation due to their large size, as is performed in birds.53 reptiles, like birds, undergo polyspermic fertilization; thus in-vitro/ex-ovo fertilization would likely be the preferred technique to allow for successful fertilization and developmental progression. if icsi is attempted in reptiles, coinjection of sperm extract would likely enhance development. in avian species this is required, as injection with a single sperm is not successful.54 coinjection is needed for icsi-generated zygotes for avian species.53 we should look at birds as the model to enhance these technologies in reptiles. cloning/somatic cell nuclear transfer no reports exist in the literature using cloning or somatic cell nuclear transfer in reptiles. genetic editing in reptiles genetic editing in reptiles is in its infancy. transgenic leopard geckos and ball pythons (python regius) have been successfully developed inserting genes with lentiviral vectors to induce fluorescent proteins.55,56 the first successful report of genetic editing using the crispr/cas9 system was reported in the brown clinical theriogenology 2021; 13: 387 anole (anolis sagrei). surgical ovarian microinjection introduced a cas9 protein coupled to a mixture of 3 distinct synthetic tyr guide rnas into immature oocytes to terminate the tyrosinase gene that resulted in phenotypic albino brown anoles.57 conclusion since the1970s, great advances that laid the groundwork to apply art in reptiles. so, what is next? where should our limited resources be focused in developing art for reptiles? currently, biodiversity loss is outpacing our ability to innovate new art technologies for reptiles. additionally, we are limited in our ability to successfully reproduce some of these threatened and endangered species in captivity. efforts should be focused, and we should preserve what species we have that involves developing a robust genome resource bank exclusively for reptiles and preserving the extant genetic biodiversity. success with artificial insemination will progress with further refinement of techniques as we better understand the female reptile reproductive anatomy and physiology. these are technologies that are successful and being utilized in other species. with time, research and success, more individuals will realize art’s role in reptile conservation. conflict of interest there are no conflicts of interest to declare. references 1. clulow j, clulow s: cryopreservation and other assisted reproductive technologies for the conservation of threatened amphibians and reptiles: bringing the arts up to speed. reprod fert develop 2016;28:1116-1132. 2. depeiges a, dacheux jl: acquisition of sperm motility and its maintenance during storage in the lizard, lacerta vivipara. reproduction 1985;74:23-27. 3. larsen re, cardeilhac p, godwin f: artificial insemination in the american alligator. proc society for theriogenology 1988. p. 285-292. 4. larsen re, cardeilhac pt, lane t: semen extenders for artificial insemination in the american alligator. aquaculture 1984;42:141-149. 5. watson pf: ai and the preservation of semen. in: lamming ge: editor. marshall’s physiology of reproduction, vol 2, male reproduction. london; churchill livingstone: 1990. p. 747-869. 6. fitch hs: criteria for determining sex and breeding maturity in snakes. herpetologica 1960;16:49-51. 7. millar j, watson p: cryopreservation of gametes and embryos in reptiles and 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turtles. herpetol rev 2013;44:595-600. 23. zimmerman dm, mitchell ma, perry bh: collection and characterization of semen from green iguanas (iguana iguana). am j vet res 2013;74;1536-1541. 24. perry sm, konsker i, lierz m, et al: determining the safety of repeated electroejaculations in veiled chameleons. j. zoo wildl med 2019;50:557-569. 25. perry sm: can assisted reproductive technologies help conserve 300 million years of evolution? a first attempt at developing these technologies for male reptiles. lsu doctoral dissertations. 5184. 2019. https://digitalcommons.lsu.edu/gradschool_dissertations/5184 26. mitchell ma, funcke s, bublat a, et al: determining the value of electroejaculation as a method of semen collection in lizards and chelonians. proc 2nd international con on avian, herpetological and exotic mammal medicine 2015. 27. lópez jg, chiaraviglio m, cardozo g: electrostimulation is an effective and safe method for semen collection in medium-sized lizards. clinical 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chelonia mydas. br j herpetol 1982;6:200-202. 33. juvik jo, meier de, mckeowen s: captive husbandry and conservation of the madagascar ploughshare tortoise, geochelone yniphora. proc 1st international symposium on turtles & tortoises: conserv and captive husbandry 1991. p. 127-137. 34. tanasanti m, sujaritthanyatrakul c, dhanarun k, et al: electroejaculation and semen evaluation in olive ridley turtle (lepidochelys olivacea) and hawksbill turtle (eretmochelys imbricata) in thailand. proc 4th international symposium on seastar2000 and asian bio-logging science (8th seastar2000 workshop) 2009. p. 29-32. 35. sirinarumitr k, patthong y, jaimjaturong p, et al: extender for sperm dilution in olive ridley turtle (lepidochelys olivacea) and hawksbill turtle (eretmochelys imbricata) semen. proc 5th international symposium on seastar2000 and asian bio-logging science (9th seastar2000 workshop) 2010. p. 7-10. 36. kimskulvech s, suttiyotin p: stimulation patterns for erection and ejaculation using electroejaculator in black marsh turtles. thai j vet med 2012;42:325-31. 37. kawazu i, maeda k, koyago m, et al: semen evaluation of captive hawksbill turtles. chelonian conserv biol 2014; 13:271-278. 38. zimmerman dm, mitchell ma: semen collection and ejaculate characteristics of the leopard tortoise (stigmochelys pardalis). conserv physiol 2017;5. 39. quinn h, blasedel t, platz jr cc: successful artificial insemination in the checkered garter snake thamnophis marcianus. int zoo yearb 1989;28:177-183. 40. perry sm, mitchell ma: evaluation of commercial semen extenders and crystalloids for short term cooled extension of epididymal spermatozoa in squamates and chelonians. proc society for theriogenology 2019. 41. perry sm, mitchell ma: a standardized approach to developing cryopreservation protocols for reptile semen, the red eared slider, a model. proc exotics con 2019. p. 656. 42. hobbs r, keogh l, james k, et al: sperm cryopreservation in eulamprus quoyii (eastern water skink). reprod fertil and dev 2018;31:180-181. 43. young c, ravida n, curtis m, et al: development of a sperm cryopreservation protocol for the argentine black and white tegu (tupinambis merianae). theriogenology 2017:87:55-63. 44. young c, ravida n, rochford m, et al: sperm cryopreservation in the burmese python (python bivittatus) as a model for endangered snakes. reprod fert dev 2017;30:185-186. 45. campbell l, cafe sl, upton r, et al: a model protocol for the cryopreservation and recovery of motile lizard sperm using the phosphodiesterase inhibitor caffeine. conserv physiol 2020:8:9. 46. zacariotti r, guimarães m, jensen t, et al: cryopreservation of snake semen: are we frozen in time? reprod fert dev 2011;24:171-171. 47. johnston sd, lever j, mcleod r, et al: extension, osmotic tolerance and cryopreservation of saltwater crocodile (crocodylus porosus) spermatozoa. aquaculture 2014;426-427:213-221. 48. johnston sd, qualischefski e, cooper j, et al: cryopreservation of saltwater crocodile (crocodylus porosus) spermatozoa. reprod fertil dev 2017;29:2235. 49. mattson kj, devries at, krebs, j, et al: cryopreservation of corn snake, elaphae gutatta, semen. reprod fert develop 2008;21:179-180. 50. mazur p, leibo sp, chu eh: a two-factor hypothesis of freezing injury. evidence from chinese hamster tissue-culture cells. exp cell res 1972;71:345-355. 51. oliveri m, bartoskova a, spadola f, et al: method of semen collection and artificial insemination in snakes. j exot pet med 2018;27:75-80. 52. uller t, olsson m: multiple paternity in reptiles: patterns and processes. mol ecol 2008:17;2566-2580. 53. mizushima s: fertilization 2: polyspermic fertilization. in: sasanami t: editor. avian reproduction: from behavior to molecules. singapore; springer: 2017. p. 105-123. 54. mizushima s, hiyama g, shiba k, et al: the birth of quail chicks after intracytoplasmic sperm injection. development. 2014;141:3799. 55. hull k, hodgson d, clark b, et al: lentiviral transgenesis of the leopard gecko, eublepharis macularius. afr j microbiol res 2014;8:1070-1079. 56. mozdziak pe, petitte jn: transgenic snakes and methods of making. us patent 7,663,019. 2010. washington dc. 57. rasys am, park s, ball re, et al: crispr-cas9 gene editing in lizards through microinjection of unfertilized oocytes. genetics 2019;28:2288-2292. clinical theriogenology 2021; 13: 389 diagnosis and medical treatment of pyometra in the queen aime johnson auburn university college of veterinary medicine auburn, al abstract although the incidence of feline pyometra is lower than canine, it can have devastating effects in breeding animals. owners are requesting medical management of pyometra for valuable breeding animals more often and the practitioner must be able to provide recommendations and guidance. pyometra is most often diagnosed in older animals, but there is now a disease process observed in animals as young as 9 10 months. pathogenesis, clinical signs, diagnosis, and medical treatment are discussed. keywords: pyometra, queen, cystic endometrial hyperplasia, endometritis introduction accumulation of purulent material within the uterus is referred to as pyometra. this occurs during the luteal phase when a corpus luteum (cl) is present. because there is no pregnancy, this luteal phase is often called a ‘pseudopregnancy’ because it lasts 40 days rather than the full 63 days of pregnancy. incidence of pyometra in the cat is much lower than the bitch where 20% of females will develop pyometra by age 10.1 pyometra developed in 2.2% european cats by the age of 13 years.2 lower incidence of pyometra in the cat is likely due to less overall prolonged progesterone exposure since they are induced ovulators, have a shorter nonpregnancy luteal phase (40 days) and are seasonal. pyometra occurs in middle age or older queens, 3 4 weeks after ovulation (spontaneous, induced, or mated), and can be life threatening. pathogenesis most studies evaluated the pathogenesis of pyometra in dogs; sadly, it is inferred that development in cats is similar. cystic endometrial hyperplasia (ceh) is an underlying uterine pathology that has been implicated as a precursor to the development of pyometra. when uterus is subjected to prolonged periods of progesterone exposure without pregnancy, progesterone induces changes in the endometrium. progesterone decreases uterine contractions, causes thickening of the endometrium, proliferation of the endometrial glands, and increases glandular secretions.3 progesterone also decreases the ability of uterus to respond to bacterial assault by decreasing the inflammatory response. changes within the uterus with ceh predispose the animal to development of pyometra. once ceh has damaged the uterus and progesterone decreases uterine overall ability to eliminate infection, bacteria entering through the vaginal area and cervix are able to colonize and replicate. the most common bacteria isolated in queens is escherichia coli (e. coli), followed by streptococcus spp and staphylococcus spp.4 ‘atypical’ pyometra? although in past literature, ceh-pyometra complex has been long discussed as a single disease, there is increasing support that ceh and pyometra are actually 2 separate disease processes and although they often occur together, they can also occur separately. this is likely the case in young cats that develop pyometra. the author has observed this clinical presentation in her feline research colony. these queens are between 10 months and 2 years and some have not been bred by a male. pyometra has been historically described as the sequalae to uterine changes following prolonged exposure to progesterone. therefore, spontaneous ovulation must be considered in unmated queens presenting with pyometra. spontaneous ovulation (ovulation without coitus) may occur in ~ one third of cats.5 incidence of spontaneous ovulation increased as body weight increased.5 although factors affecting spontaneous ovulation are not clearly known, they are observed more frequently in cats housed within the visual, olfactory, and auditory presence of an intact male. in grouphoused queens, the incidence of spontaneous ovulation prior to addition of a male was 0 22% compared to 33 57% after a male was housed within olfactory, visual, and auditory presence in the room.6 even with multiple spontaneous ovulations, uterine pathology does not seem to be the underlaying predisposition in this group of queens. many queens are < 1 year and may have only been cyclic a few months. their successful response to medical management and return to fertility indicates that the underlying uterus is not damaged. it is suspected that endometritis, inflammation within the endometrium, is occurring in the absence of permanent pathological changes. endometritis caused by the entry of ascending bacteria can be undetected until the queen is mated. once the queen ovulates, progesterone closes the cervix, decreases the myometrial contractions, increases the glandular fluid, and reduces the inflammatory response within the uterus all allowing for prime bacterial growth. the typical presentation of this group are young queens, either not yet bred (but in the same room as a male) or placed with a male for the first time. these queens remain bright and alert, systemically healthy, but may have a slight fever. purulent vaginal discharge in their cage where they sit, or on their tail/vulva is the most consistent clinical sign observed. these queens remain stable, continue to eat and drink and do not require supportive care. ultrasonography of the uterus confirms pyometra. uterine loops distended with flocculent material can be observed in both uterine horns. vaginal cytology will identify degenerative neutrophils in high numbers and progesterone concentrations > 2 ng/ml, indicating the presence of a cl. these queens respond well to medical treatment (described in the next section) and often only require a few days of prostaglandin therapy to clear the uterus. these queens are allowed to rest for 1 month and then exposed to a male. approximately 80 85% of cats will successfully produce a litter following treatment. anecdotally, these queens are predisposed to another episode of pyometra later in their breeding career; however, this has not been confirmed by research. the author has had 2 valuable queens that were treated medically for pyometra twice in their breeding career and produce litters following both episodes. one queen was spayed following a litter after her second treatment and the other was spayed after producing 2 more litters and then being diagnosed with a third pyometra. both queens were first diagnosed with pyometra at 11 12 months of age and neither was assessed for underlaying chronic endometritis at any time. further research is necessary with these young queens to determine the cause of pyometra in relation to uterine pathology. however, if this is observed commonly in a colony, subclinical endometritis should be suspected. diagnosis pyometra can be suspected based on history (placed with a male 3 4 weeks ago), signalment (older intact queen), and/ or clinical signs. clinical signs include any of the following and depend on severity of the disease: purulent vaginal discharge, pyrexia, anorexia, lethargy, and abdominal distension. abdominal radiographs may reveal soft tissue mass opacity; however, radiographs alone are not diagnostic. ultrasonography is the most ideal diagnostic tool for identifying pyometra. by scanning the uterus starting near the bladder and extending cranially, fluid-filled loops of uterus become visible. pyometra causes segmental enlargement of uterus in cat, unlike in bitch where the uterus is uniformly enlarged. pyometra must be differentiated from pregnancy. uterine distension caused by pyometra is often not uniform and there will be areas with various fluid diameters within the uterus. in pregnancy, all fluid-filled structures should measure similarly. echogenicity of the fluid may also give an indication; inflammatory debris within the uterus with pyometra will appear flocculent. pockets of uterine fluid should be measured along each uterine horn to assess uterine distension. those same pockets should be remeasured daily to assess response to treatment. vaginal cytology should be performed to confirm the presence of inflammatory cells within the discharge. huge numbers of degenerative neutrophils will be present along with a few parabasal or small intermediate cells in cases of pyometra. additional testing prior to and throughout treatment may include complete blood count and serum chemistry to assess the severity of systemic disease and response to treatment. acute phase proteins (e.g., serum amyloid a, serum hp, and serum albumin) were higher in cats affected with pyometra when compared to normal cats. following surgical correction (ovariohysterectomy), the acute phase proteins returned to normal concentrations within 2 weeks. this suggested that measurement of acute phase proteins may be used as a biomarker for response to treatment.7 determination of serum progesterone concentrations is necessary; however, it can wait a few days into treatment. documentation that progesterone concentrations are baseline following initiation of treatment is more important than knowing the initial value. medical treatment when considering medical treatment of pyometra, the overall health status of the queen must be evaluated. if there is comorbidity including peritonitis or sepsis, medical therapy is not recommended. medical treatment should only be attempted when the queen is not systemically compromised, a valuable breeding animal, has not exhibited signs of infertility with past mating attempts, and preferably young (< 4 years of age). supportive care and stabilization are essential when treating a pyometra in any species. queen tends not to be as systemically ill as the bitch, but the severity of clinical signs in the queen is directly correlated to the amount of uterine distension.8 medical treatment begins with antibiotic therapy. the most common bacteria isolated in cases of feline pyometra is e. coli, followed by streptococcus spp., staphylococcus aureus, klebsiella spp., and other fecal bacteria. antibiotics should be chosen based on vaginal culture and susceptibility results. since these results are not available immediately, broad spectrum antibiotics that especially target e. coli should be initiated immediately. amoxicillin/clavulanic acid (15 25 mg/kg orally twice a day or intravenously 3 times a day) is an option as the initial treatment of choice. this author prefers initiating treatment with enrofloxacin (5 mg/kg orally or subcutaneously every 24 hours) to target the uterus directly. since enrofloxacin caused retinal degeneration in cats at higher doses,9 other fluroquinolones (e.g., pradofloxacin) may be substituted. antibiotic therapy should be adjusted once final culture and susceptibility patterns are known. therapy should be continued for 10 14 days after uterine distension has resolved. prostaglandin f2α (pgf2α) therapy is essential by lysing the cl, opening the cervix, and inducing uterine contractions. two forms of pgf2α are available; native prostaglandin (dinoprost tromethamine) that is marked through zoetis as lutalyse, and cloprostenol, a synthetic prostaglandin marketed by merck as estrumate. both are considered off-label in companion animals. side effects are largely dose dependent and include vocalization, salivation, panting, restlessness, vomiting, diarrhea, excessive grooming, and tenesmus. signs begin shortly after treatment and continue for no more than 1 hour.10 cats should be hospitalized and monitored continuously when first treated with prostaglandins and with each increasing dose to observe an individual’s side effects. if extensive side effects are observed, decreasing the dose or switching to the other form of pgf2α may be helpful. lutalyse treatment should start at 10 -15 µg/kg subcutaneously 3 4 times a day for the first day, followed by 25 µg/kg subcutaneously 3 4 times a day on day 2, and increasing to 50 µg/kg subcutaneously 2 4 times a day on day 3 until uterine clearance and luteolysis is complete. cloprostenol given subcutaneously at 5 µg/kg once a day for 3 days was effective in treating open pyometra.11 cloprostenol has a longer half-life and may have a longer duration of efficacy making dosing frequency less. although cloprostenol may cause fewer side effects, lutalyse is preferred by this author because it causes stronger uterine contractions and faster emptying of the uterus. starting with a low dose and increasing both dose and frequency over time minimizes side effects. with the described dosage, transient hypersalivation, vomiting, and diarrhea are the most common side effects observed. a third type of treatment is a dopamine agonist. cabergoline and bromocriptine are prolactin antagonists and therefore have an antiluteotropic effect on the cl. this therapy should be used in combination with pgf2α to remove the cl and open the cervix. cabergoline is preferred because side effects are minimal. cabergoline can be obtained from most human pharmacies and prescribed at a dose of 5 µg/kg orally once daily. bromocriptine is more cost effective but requires more frequent dosing and has higher incidence of side effects such as vomiting, lethargy, and diarrhea. the dose for bromocriptine is 10 25 µg/kg orally every 8 hours.12 treatment with either medication should be continued for 5 7 days and can be discontinued once progesterone concentrations remain baseline. nonsurgical uterine lavage (transcervical) has been described in large nondomestic feline species as a treatment for pyometra..13 this technique is gaining popularity in the dog; however, it is still not practical in domestic cat due to the small size of the vaginal canal. monitoring response to treatment aggressive therapy is important if return to fertility is desired. monitoring medical management should be an intense process with daily examinations, ultrasonography, and assessments of the queen overall. even if medical management is initiated, the queen may not respond and still require ovariohysterectomy. daily physical examination is important to assess overall wellbeing of the queen. some can be very stoic and subtle signs indicating systemic compromise may be missed. once medical management is initiated, the cat should be held in the hospital for about 2 3 hours to assess reaction to medications. if the queen is systemically stable, hydrated, and eating and drinking normally, the queen can return home for outpatient treatment. if the queen is compromised, dehydrated, or has moderate to severe clinical signs associated with pgf2α treatment, hospitalization is necessary. daily ultrasonography of the uterus should be performed for contents and measurement of uterine diameter. with treatment, a 50% decrease in uterine fluid and an increase in the amount of vaginal discharge is expected within 2 days. if uterine diameter is not decreasing despite treatment, the frequency of lutalyse can be increased up to 4 times daily. serum progesterone concentrations should be determined 2 3 days after initial treatment. progesterone concentrations should return to baseline (< 2 ng/ml). feline cl is more refractory to prostaglandins than canine cl and may require longer treatment to fully lyse the cl. once baseline progesterone concentrations are reached (and the uterus is free of fluid), prostaglandin therapy should be continued for another 24 hours. serum progesterone concentrations can be determined again (2 4 days later) to make sure that the concentrations are still baseline and the cl has not rebounded. medical treatment can be considered effective if uterus is reducing in diameter, free fluid is resolving, vaginal discharge that initially increased is reducing, and the inflammatory leukogram is improving. once these are occurring, prostaglandin and cabergoline therapy can be discontinued. antibiotic therapy should be continued for 10 14 days after resolution of clinical signs. final ultrasonography of the uterus to assess the presence of intraluminal fluid is necessary at the end of antibiotic therapy. medical treatment is considered a failure if there is minimal or no decrease in uterine diameter within 4 5 days after starting treatment, the queen becomes systemically compromised, or uterine pathology is observed over the course of treatment. in these cases, ovariohysterectomy should be performed. prognosis for future fertility decreases the longer the treatment is required. prognosis if queen responds well to medical treatment, return to fertility will depend on underlaying uterine pathology. younger queens are more likely to return to a successful breeding career than older queens because underlaying ceh is more prevalent as queens age. regardless, queens should be bred ~ 4 weeks after resolution of clinical signs. they should be kept away from intact males until ready for breeding to decrease the incidence of spontaneous ovulation. if queen fails to become pregnant with mating, a relapse of pyometra is more likely. if endometritis is suspected in a queen, treatment with antibiotics around mating may reduce the chance of development of pyometra. this should be instituted only on a case-by-case basis to prevent unnecessary and overuse of antibiotics. after mating, the queen should be monitored closely via abdominal ultrasonography starting 20 30 days to assess for either pregnancy or pyometra. once the queen is no longer needed for breeding, ovariohysterectomy should be performed. uterus must be removed (versus ovariectomy) to prevent any possibility of recurrence should the queen be exposed to hormones later. conclusion pyometra is less common in queen than in bitch. however, medical management is similar. with aggressive therapy, prognosis is positive and a return to fertility is possible in the right case. conflict of interest none to declare. references 1. jitpean s, hagman r, strom holst b, et al: breed variations in the incidence of pyometra and mammary tumours in swedish dogs. reprod domest anim 2012;47 suppl 6:347-350. 2. hagman r, strom holst b, moller l, et al: incidence of pyometra in swedish insured cats. theriogenology 2014;82:114-120. 3. cox je: progestagens in bitches: a review. j small anim pract 1970;11:759-778. 4. hagman r: pyometra in small animals. vet clin north am small anim pract 2018;48:639-661. 5. binder c, aurich c, reifinger m, et al: spontaneous ovulation in cats-uterine findings and correlations with animal weight and age. anim reprod sci 2019;209:106167. 6. gudermuth df, newton l, daels p, et al: incidence of spontaneous ovulation in young, group-housed cats based on serum and faecal concentrations of progesterone. j reprod fertil suppl 1997;51:177184. 7. vilhena h, figueiredo m, ceron jj, et al: acute phase proteins and antioxidant responses in queens with pyometra. theriogenology 2018;115:30-37. 8. blanco pg, rube a, lopez merlo m, et al: uterine two-dimensional and doppler ultrasonographic evaluation of feline pyometra. reprod domest anim 2018;53 suppl 3:70-73. 9. wiebe v, hamilton p: fluoroquinolone-induced retinal degeneration in cats. j am vet med assoc 2002;221:1568-1571. 10. davidson ap, feldman ec, nelson rw: treatment of pyometra in cats, using prostaglandin f2 alpha: 21 cases (1982-1990). j am vet med assoc 1992;200:825-828. 11. garcia mitacek mc, stornelli mc, tittarelli cm, et al: cloprostenol treatment of feline open-cervix pyometra. j feline med surg 2014;16:177-179. 12. hollinshead f, krekeler n: pyometra in the queen: to spay or not to spay? j feline med surg 2016;18:21-33. 13. hildebrandt tb, goritz f, boardman w, et al: a non-surgical uterine lavage technique in large cats intended for treatment of uterine infection-induced infertility. theriogenology 2006;66:1783-1786. diagnosis and 2018: body wall tear in a standardbred mare with live foal outcome body wall tear in a standardbred mare with live foal outcome college of veterinary medicine, cornell university, ithaca, ny body wall tears, such as prepubic tendon rupture in late term pregnant mares, are life threatening conditions; however some cases have been successfully managed until foaling. a ten-year-old multiparous standardbred mare at 336 days in gestation was presented with a chief complaint of colic and elevated serum amyloid a (saa). the mare was tachycardic, tachypnic, and had marked ventral edema that extended caudally. she had elevated creatinine kinase, aspartate aminotransferase, and saa, and a mild neutrophilia with left shift. reproductive examination revealed an active fetus with normal heart rate, combined thickness of the uterus and placenta of 13.4 mm and abnormal echogenicity of allantoic fluid which may be caused by placentitis. gastrointestinal colic and uterine torsions were ruled out. treatment with systemic broad-spectrum antimicrobials, anti-inflammatory drugs and pain management was instituted. the following day, ultrasonographic examination of the ventral body wall revealed the caudal portion of the rectus abdominal muscle to have a superficial partial tear bilaterally. medical management of abdominal wall disease was elected in attempt to save both foal and mare. an abdominal support bandage was applied and the mare was monitored hourly. the mare developed bloody mammary secretions on day 4 and foaled on day 5 after presentation. the foal was in dorsosacral position which was corrected and a large filly was delivered alive per vagina. the filly became septic but was successfully treated and then grafted onto a nurse mare before being discharged. ultrasonography after foaling showed the mare to have a complete bilateral prepubic tendon rupture. a year following discharge, both the filly and mare are alive. the mare is being used as an embryo transfer donor. clinical theriogenology • volume 10, number 3 • september 2018 360 o.f. cox, s.h. cheong, m. diel de amorim immune system™s role in placental detachment immune system’s role in placental detachment cristina rosales,a brett tennent-brown,a david hanlon,b christina marth,a mark stevenson,a natali krekelera amelbourne veterinary school, faculty of veterinary and agricultural sciences university of melbourne, australia bmatamata veterinary services, matamata, new zealand retained fetal membranes (rfm) can have serious implications for mare health and reproductive potential. however, mechanisms leading to physiological detachment of membranes in an appropriate time frame remain poorly understood. based on previous studies in horses and other species, we hypothesised that an inflammatory process including leukocyte migration and increased cytokine activity is necessary for membrane detachment. objectives were to investigate the role of leukocyte numbers and the expression of proinflammatory cytokines interleukin (il)1ß, il6, and il8 in this process. thirty three spontaneously foaling mares on a thoroughbred stud farm in new zealand and 4 healthy full term mares without signs of impending labour sourced from a local abattoir were studied. blood was collected from the umbilical artery and vein while the umbilicus was still attached and biopsies were collected from fetal membranes and the endometrium immediately after membranes were released. leukocyte counts were obtained from the umbilical blood samples using the unopette system (beckton, dickinson and company, auckland, new zealand) and compared using two sided student’s t test. total rna was extracted from biopsy samples and reverse transcribed. messenger rna levels of il1ß, il6 and il8 mrna were investigated using a quantitative real-time polymerase chain reaction. all gene expression data were log transformed and analysed using anova and tukey’s post hoc test. neutrophil, lymphocyte and overall leukocyte numbers were higher (p < 0.001) in the umbilical artery compared to the umbilical vein. messenger rna levels of il1ß, il6, and il8 were 15, 22, and 839 times higher (p < 0.001) in endometrial samples from foaling versus control mares. in fetal membranes, il8 expression was 14 times higher (p < 0.001) in foaling mares, whereas il6 expression was nearly 3 times greater in control mares. levels of il1ß mrna were not significantly different between the 2 groups. based on presence of fetal leukocytes within fetal membranes and increased expression of pro inflammatory cytokine genes by the endometrium, we inferred that both maternal and fetal immune systems contributed to an inflammatory reaction at the site of placental detachment during parturition. future studies are needed to confirm the presence of the corresponding proteins in the tissues and to determine whether this recruitment is decreased in mares developing rfm. keywords: mares, retained fetal membranes, neutrophils, proinflammatory cytokines 407 clinical theriogenology • volume 11, number 3 • september 2019 408clinical theriogenology • volume 11, number 3 • september 2019 omniblank: clinical theriogenology 2022; 14: 82 immunoglobulin g, white blood cell, and fibrinogen concentrations among dairy cows with and without endometritis during transition period ali bazzazan,a nelson cardenas,a dominic dolbec,b mariela segura,b réjean lefebvreab adepartment of clinical sciences, bgremip, faculty of veterinary medicine, university of montreal saint-hyacinthe, quebec, canada abstract postpartum reproductive performance closely depends on uterine health. under normal circumstances, almost 100% of cows have uterine contamination within first 2 weeks after calving. whereas the innate immune and reproductive systems usually eliminate most offending microbes, persistent infection still reportedly occurs in ~ 20% of postpartum cows. our purpose was to estimate concentrations of systemic immune indicators (igg, white blood cells, and fibrinogen) during the transition period in dairy cows with and without endometritis. fifty-nine multiparous cows were systematically and consecutively enrolled during the dry period and examined 6 times from 40 days before to 40 days after calving. cows in the diseased group (n = 11) were identified based on 4 criteria (presence of trueperella pyogenes grade > 2 in the uterus, clinical endometritis, subclinical endometritis, and cervicitis). cows in the control group (n = 11) were negative for all 4 criteria. prevalence of trueperella pyogenes, clinical endometritis, subclinical endometritis, and cervicitis was 25, 16, 23, and 31%, respectively. concentrations of igg, white blood cells, and fibrinogen did not change over the period or vary among control and diseased cows. in conclusion, systemic indicators of inflammation were not good markers for diagnosing or monitoring endometritis in postpartum dairy cows. keywords: diary cattle, inflammatory markers, uterine disease introduction reproductive health of cows is the foundation of productivity in the dairy industry. although entire transition period is critical for dairy cows, postpartum reproductive performance is closely dependent on uterine health. for cattle, the transition period is essentially a period of stress management that requires metabolic adjustments in the face of a substantial negative energy balance and immune system modulations.1 these adjustments enable a smooth transition from pregnancy state to restoration of uterine condition, ovarian cyclicity, and establishment of subsequent pregnancy.2-4 under normal circumstances, almost 100% of cows have uterine contamination within the first 2 weeks after calving.5,6 although the innate immune system usually eliminates all offending microbes, persistent infection or inflammation reportedly occurred in 15 40% of postpartum cows.6 postpartum uterine disease is commonly associated with escherichia coli (e. coli), trueperella pyogenes (t. pyogenes), fusobacterium necrophorum (f. necrophorum), and prevotella species.4 numerically, the most common pathogens were e. coli (37% of pathogenic bacteria isolated) and t. pyogenes (49%).7 when contamination of the uterus persists beyond 4 weeks postpartum, the uterine infection is labelled as endometritis. clinical and subclinical endometritis are prevalent conditions in postpartum dairy cows, causing economic losses due to decreases in both milk production and fertility.8 clinical endometritis is defined as inflammation of the endometrium with purulent vaginal discharge (pvd) and in the absence of systemic clinical disease at > 21 days postpartum,4 whereas subclinical endometritis is defined as the presence of > 18% polymorphonuclear neutrophils (pmns) in uterine cytosmears (21 33 days postpartum) or > 10% pmns (34 47 days postpartum).9 cellular and humoral mechanisms of nonspecific and specific immunity have important roles in the resolution of postpartum endometritis. first and most substantial cell type recruited during uterine inflammation is the pmn.10 these cells, along with monocytes, are recruited from the blood circulation into uterine lumen to eliminate bacteria.11 like pmns and macrophages, endometrial cells also have toll-like receptors (tlrs) that recognize pathogens present in the uterus. for example, tlr4 recognizes lipopolysaccharides in the cell wall of gram-negative bacterial species, initiating an innate immune response that triggers the release of antibacterial substances from responding cells.12 this inflammatory response results in other inflammatory cells attracting pmns, amplifying an inflammatory loop.13 endometrial cells also secrete diverse molecules, including cytokines, whose presence in uterine secretions reflected the intensity of endometrial inflammation.14 fibrinogen response is a useful indicator of the presence of inflammation, bacterial infection, and surgical trauma in catclinical theriogenology 2022; 14: 83 tle. fibrinogen binds specifically to cd11/cd18 integrins on the cell surface of migrated phagocytes, triggering a cascade of intracellular signals that leads to degranulation, phagocytosis, cellular cytotoxicity, and delayed apoptosis. integrins, a group of adhesion molecules that are expressed on leucocytes, have an important role in immune function.15 plasma fibrinogen concentrations were higher in cows with subclinical and clinical mastitis than in control cows.14 immunoglobulins (ig) also have a protective role against pathogens in the bovine uterus.16 however, the specific functions of the isotopes iga, igg, and igm in the genital tract are still not clear. innate and adaptive immunity, both local and systemic, have an essential role in defending the uterus against postpartum infection. we hypothesized that clinical endometritis is associated with an increase in systemic indicators of inflammation in dairy cows. the hypothesis was tested by comparing concentrations of igg, white blood cells (wbc), and fibrinogen during the transition period among dairy cows with and without endometritis. materials and methods animals procedures were carried out in compliance with canadian council on animal care guidelines, and the animal care committee of the université de montréal approved the experimental protocol. multiparous cows (n = 59) were selected based on sequential date of calving (june 2016 february 2017) from 3 commercial dairy herds in quebec (canada). herd size was from 70 130 lactating cows. reproductive and health data were compiled in a databank using health record management software (dsahr, saint-hyacinthe, québec, canada). rolling herd average milk production was ~ 9000 kg. cows were housed in tie stall barns and milked twice daily. cows were fed a total mixed ration consisting of mainly corn silage or hay silage formulated to meet the dietary requirements of lactating dairy cows at each stage of production.17 farms were visited weekly by the same veterinarians. cows were vaccinated intramuscularly against e. coli on day 40 and on day 26 before calving (2 ml, j-vac, merial inc., athens, ga), and against types 1 and 2 bvd, ibr, pi-3, and brsv on day 15 40 after calving (2 ml, bovi-shield gold® fptm 5 l5, zoetis, parsippany, nj). cows were injected with 5 ml of selenium on day 60 after calving (mu-se, intervet canada corp., a subsidiary of merck and co. inc, kirkland, qc, canada). experimental design this was an observational prospective cohort study in which cows (n = 59) were systematically and consecutively enrolled during the dry period and then examined 6 times from 40 days before to 40 days after calving. six examinations (exam 1 6) were performed: on days 40 ± 4, 26 ± 4 and 12 ± 4 before calving (dbc), and on days 7 ± 4, 21 ± 4 and 35 ± 4 after calving (dac). examinations included assessment for lameness, cyclicity, and body condition. milk, blood, and endometrial samples (via cytobrush) were collected, and transrectal and vaginal examinations were performed. before calving, each cow had a transrectal examination to confirm pregnancy. after calving, the reproductive tract was examined transrectally and ultrasonographically to determine cervical and uterine horns’ diameters, assess fluid in uterus, and characterize ovarian structures (corpus luteum, dominant follicle, and follicular cyst). additionally, a uterine endometrial cytobrush examination (described below) and vaginoscopy were performed. cows were enrolled in a systematic synchronization protocol with 2 injections of prostaglandins given at a 14-day interval starting at the end of the voluntary waiting period (60 dim) with estrus observation. reproductive data were collected until at least 300 days in milk (dim). throughout the sampling period, none of the 22 cows in the control and diseased groups received any antibiotics. diseased cows (n = 11) were identified based on 4 criteria. in decreasing order of importance, these were: 1) cows with t. pyogenes grade ≥ +2 in the uterus (culture-based method only on exam 5); 2) cows with pvd of grade ≥ 2;18 3) cows with > 18% pmns on endometrial cytology (number of pmns/number of total cells);19 and 4) cows with grade 2 cervicitis.20 control cows (n = 11) were negative for all 4 criteria (t. pyogenes grade < 2, pvd < 2, pmns < 5%, and cervicitis grade < 2). vaginal and transrectal examinations cervicitis was assessed20 and vaginal discharge was examined.18 following transrectal palpation, vulva was cleaned of feces with a wet paper towel and then wiped with gauze soaked in isopropyl alcohol. for vaginoscopy, a multiple-use vaginal speculum (50 cm long) was inserted through the vulva and into vagina up to the outer cervical os. no lubricant was used. with a light source, the vaginal cavity and cervical os were examined visually for discharge and categorized using a 4-point classification system.18 vaginal content was classified as follows: 0 = no discharge, 1 = clear and translucent mucus, 2 = cloudy mucus with or without flecks of pus (< 50% pus), 3 = mucopurulent discharge (> 50% pus), and 4 = purulent discharge with fetid smell.7 transrectal examination was performed first for the sake of convenience. however, detection of vaginal discharge by vaginoscopy was not enhanced by a preceding transrectal palpation of the uterus.9 cervix was assessed and classified20 as follows: c0 = cervix without abnormality; c1 = second cervical fold swollen with no redness, and c2 = second cervical fold swollen with redness. blood sampling at each examination, blood samples were collected from the coccygeal vein using 10-ml vacutainer k2 edta blood collection tubes for hematology analysis, and without an anticoagulant for biochemistry analysis (becton, dickinson and company, franklin lakes, nj). collected samples were immediately placed on ice for further processing at the laboratory within < 3 hours. once clotting was complete in the red top tube, the serum was separated by centrifugation (3,000 revolutions per minute for 10 minutes). beta-hydroxybutyrate concentration was then measured using a freestyle precision neo kit (abbott laboratories ltd, abbott park, il). finally, 3.0-ml aliquots of serum were labelled and placed at – 80°c for long-term storage and further analysis. after 30 minutes at room temperature and gentle agitation, the anticoagulant-containing tube was subject clinical theriogenology 2022; 14: 84 to a complete hematology analysis (vetscan hm5 hematology analyzer, abaxis global diagnostics, union city, ca). quantification of serum igg and fibrinogen concentrations bovine igg concentrations were measured21 by elisa using a commercial bovine igg elisa quantitation set (e10-118, bethyl laboratories, montgomery, tx). endometrial cytology sampling uterine cytobrush samples (n = 59) were collected for routine endometrial cytology and bacteriology assessment. briefly, a sterile cytobrush (cytosoft, camarillo, ca) was screwed onto a stainless-steel rod (65 cm x 4 mm) and was then protected within a sterile stainless-steel tube. the rod-guard apparatus was inserted into a hard protective plastic sheath (imv technologies, l’aigle, france) before being inserted into a second protective sheath (30-cm long sani-shield rod protector, agtech, manhattan, ny) to reduce the risk of vaginal and cervical contamination. instrument was pushed through the cervix and the sterile cytobrush gently rotated clockwise (360 degrees) on the endometrium to collect cellular material. cytobrush was then retracted into the stainless-steel tube and hard plastic protective sheath, and withdrawn. the bristles of the brush were rolled over a sterile microscope slide (fischer scientific, toronto, on, canada), and then the brush tip was cut off and packaged for bacterial culturing. slide preparation and staining the smear on the slide was allowed to dry at room temperature for 10 -15 minutes. cytology smears were stained with modified wright giemsa stain using a hematek automated slide stainer (miles scientific, napierville, il). slides were examined under the microscope at 250 and 400 x magnifications and various inflammatory cell types (pmns, lymphocytes, and monocytes), and endometrial epithelial cells were counted. for each slide, a total of 300 cells were counted by a single observer. cows were classified as endometritis positive if > 18% pmns was present at 5 weeks after calving. uterine bacterial culturing and identification uterine cytobrush samples for bacteriology testing were collected only at 21 days after calving (exam 5) for routine bacterial culturing (aerobic and anaerobic) using standard methods for bacteriological testing (api system, biomérieux, marcy l’étoile, france). cytobrush samples were stored in a culture tube (starplex scientific inc., etobicoke, on, canada) and transported at room temperature to the faculty of veterinary medicine’s diagnostic laboratory within 3 hours. for microbiological analysis, the brushes were scraped onto sheep blood agar (soy agar with 5% sheep blood; becton, dickinson and company, sparks, md). plates were incubated for 48 hours at 35°c under aerobic conditions and then examined. when growth was observed, colony types were identified based on morphology, pigmentation, and hemolytic patterns. tiny beta-hemolytic, catalase-negative colonies demonstrating coliform gram-positive rods were identified as t. pyogenes, f. necrophorum and p. melaninogenicus. these were then isolated using the standard procedure at the faculty of veterinary medicine’s diagnostic laboratory (ponbac-019). for other bacterial species, cytobrush samples were scraped directly onto brucella agar containing neomycin (100 g/ml) and incubated anaerobically at 35°c for 5 days. when gram-negative rods were observed, colonies were examined using the api 20 a gallery system for identifying f. necrophorum and p. melaninogenicus. for isolation of e. coli, cytobrush samples were scraped directly on blood agar and macconkey agar (oxoid inc., ottawa, on, canada) at 37°c. at the oie reference laboratory for escherichia coli (ecl; faculty of veterinary medicine, university of montreal), 5 typical lactose-positive e. coli colonies from the macconkey agar plates were streaked with blood agar for isolation and further identification. isolates were submitted to 3 biochemical tests (indole spot, simmon’s citrate, and motility) for confirmation of e. coli. isolates of e. coli were stored in tryptic soy broth containing 30% glycerol at – 80°c (becton, dickinson and company, sparks, md). data analyses reproductive data were obtained from the health records databank. we calculated descriptive statistics and then analyzed the data using sas v. 9.2. the model used herd as the random effect. statistical power and sample size calculations were performed before analysis. based on reports,18,21 the following values were deemed indicative of endometritis: 18% pmns on endometrial cytology, > grade 2 pvd, cervicitis score > 2, and 15 35 mg/ml of igg in the serum. we determined that 11 cows per group were required based on a 10% difference between the control and disease groups, and using a one-sided test with 95% confidence interval and 80% power (g*power, version 3.1.9.2, germany).22 data were not normally distributed and so were transformed using logarithm base 10 to normalize the distribution. a linear mixed model was used, with farm and cow nested within farm as random effects, and health status (control and diseased animals) and period (1 6) as fixed effects. means for each health status at each time were compared. alpha level was adjusted downward using the benjamin-hochberg procedure. for ordinal scores, the cochran-mantel-haenszel test was used to examine potential differences in the distribution of score values between control and diseased cows. significance was set at p ≤ 0.05. results descriptive statistics for the study population initial potential sample size was 250 multiparous (2nd 5th lactation) cows. from the original group of cows (n = 83), individuals were eliminated from the dataset because of culling (n = 5), use of uterine antibiotics (n = 4), missing data (n = 10), and metabolic diseases (n = 5). mean day of sampling for exams 1 6 were: 40 (45 37 dbc), 28 (31 24 dbc), 13 (17 10 dbc), 4 (0 7 dim), 17 (14 21 dim), and 31 (28 35 dim), respectively. the remaining 59 cows had the following prevalence: clinical endometritis (exam 6) 23%; cytological endometritis (exam 6) 16%; t. pyogenes positive on culture (exam 5) 25%; and cervicitis (exam 6) 31%. there were no differences (p > 0.5) among 6 sampling times in blood concentrations of wbcs (figure 1) and fibrinogen (figure 2). clinical theriogenology 2022; 14: 85 descriptive statistics for the study groups (control versus diseased cows) body condition score, lameness score, and cyclicity status were not different (p > 0.05) between control (n = 11) and diseased groups (n = 11). control cows were free of t. pyogenes (score ≤ 1) at exam 5; clinical figure 1. wbc (c/ml) in cows (n = 59) overtime of examination; note the lack of difference at examinations. figure 2. serum fibrinogen (g/dl) in cows (n = 59) over time of examination; note the lack of difference at examinations. -6 wkbc -4 wkbc -2 wkbc 0 +1 wkbc +3 wkbc +5 wkbc weeks before and after calving w bc c /m l ca lv in g -6 wkbc -4 wkbc -2 wkbc 0 +1 +3 wkbc +5 wkbc weeks before and after calving se ru m fi br in og en (g /d l) -6 wkbc -4 wkbc -2 wkbc 0 +1 wkbc +3 wkbc +5 wkbc weeks before and after calving se ru m fi br in og en (g /d l) ca lv in g figure 1. wbc (c/ml) in cows (n = 59) overtime of examination; note the lack of difference at examinations. figure 1. wbc (c/ml) in cows (n = 59) overtime of examination; note the lack of difference at examinations. figure 2. serum fibrinogen (g/dl) in cows (n = 59) over time of examination; note the lack of difference at examinations. -6 wkbc -4 wkbc -2 wkbc 0 +1 wkbc +3 wkbc +5 wkbc weeks before and after calving w bc c /m l ca lv in g -6 wkbc -4 wkbc -2 wkbc 0 +1 +3 wkbc +5 wkbc weeks before and after calving se ru m fi br in og en (g /d l) -6 wkbc -4 wkbc -2 wkbc 0 +1 wkbc +3 wkbc +5 wkbc weeks before and after calving se ru m fi br in og en (g /d l) ca lv in g figure 2. serum fibrinogen (g/dl) in cows (n = 59) over time of examination; note the lack of difference at examinations. endometritis (score ≤ 1); subclinical endometritis (< 5% pmns on endometrial cytology) at exam 5; and cervicitis (score ≤ 1) at exam 6. in diseased cows, 100% (n = 11) had t. pyogenes (score ≥ 2) on exam 5, whereas 65% (n = 7) had clinical endometritis (score ≥ 2 of pvd), 36% (n = 4) had subclinical endometritis (≥ 18% pmns on endometrial cytology), and 55% (n = 6) had cervicitis (score of 2) at exam 6. diseased cows met at least 3 of 4 criteria. prevalence of t. pyogenes at exam 5 (n = 15) was 25%. in control cows, the distribution of pvd scores shifted to lower values (p = 0.04) from exam 4 to exam 6. however, this effect was not observed in diseased cows. by contrast, there was more vaginal discharge in diseased cows at exam 5 and exam 6 compared to exam 4 (p < 0.04 and p < 0.002, respectively). at exam 6, diseased cows had more (p = 0.008) pvd than the control cows. mean pmn counts on endometrial cytology were not different (p > 0.05) from exam 4 to exam 6 in control cows. in diseased cows, mean pmn counts on the endometrial cytology at exam 4 were lower (p < 0.0005) than at exams 5 and 6. although mean pmn count at exam 4 was clinical theriogenology 2022; 14: 86 different (p > 0.05) between control and diseased cows, it was higher at exam 5 and exam 6 for diseased cows compared to the control group (p < 0.0001 and p < 0.02, respectively). cows with pvd score ≥ 2 had a higher (p = 0.0001) mean pmn count on endometrial cytology than those with lower scores. cows with t. pyogenes had a higher mean pmn count on endometrial cytology at both exams 5 and 6 compared to cows without this bacterial species (p = 0.0001 and p = 0.02, respectively). similarly, cows with t. pyogenes were more likely to have clinical endometritis (n = 11; 73%) and subclinical endometritis (n = 10; 67%) at exam 5 (table). cervicitis scores were higher in diseased group compared to control group at exams 5 and 6 (p = 0.04 and p = 0.002, respectively). of the 22 diseased and control cows, 8 of 11 control cows and 4 of 11 diseased cows were pregnant after the first artificial insemination. concentrations of igg, wbc components (pmns, monocytes, lymphocytes, and eosinophils), and fibrinogen did not change (p > 0.05) over period or vary among control and diseased cows at any examination (figures 3a and 3b, figures 4a and 4b, and figures 5a and 5b). farm did not explain any variation (p = 0.23) in the data. a lack of variation (p > 0.05) over period (transition) for diseased and control groups and between groups at various examinations for all 3 inflammation indicators was also observed when the postpartum diseases (clinical and subclinical endometritis, presence of t. pyogenes, and cervicitis) were analyzed individually. discussion postpartum endometritis may reflect a generalized systemic inflammatory environment that can be attributed to a systemic metabolic condition that is common in the transition period.23 alternatively, it may be the direct result of impaired immune defences. to distinguish between these 2 possibilities and cast more light on the pathogenesis of postpartum endometritis, we sampled at 6 time points during the transition period. this study was also unique in defining the diseased animal based on several conditions. these are, in decreasing order of importance: 1) t. pyogenes (≥ 2 grade); 2) clinical endometritis (≥ 2 purulent vaginal discharge); 3) subclinical endometritis (≥ 18% pmns), endometritis (≥ 18% pmns), and 4) cervicitis (score of 2). t. pyogenes is associated with inflammation and infection of the endometrium in vivo and in vitro. it is the most prevalent uterine bacterial species at 3 weeks postpartum and is associated with a substantial reduction in pregnancy rate.24 disease definition used in our study was based on combining various chronic postpartum diseases, assuming that 2 conditions (clinical and table. temporal changes in polymorphonuclear cells (pmn), purulent vaginal discharge (pvd), and cervicitis in control and diseased cows exam 4 exam 5 exam 6 pmn ≥ 18% control 11 (24%) 14 (30%) 6 (13%) pmn ≥ 18% diseased 2 (13%) 10 (67%) 4 (27%) pvd ≥ 2 control 20 (43%) 21 (46%) 7 (15%) pvd ≥ 2 diseased 4 (27%) 11 (73%) 7 (47%) cervicitis (score = 2) control 14 (30%) 14 (30%) 12 (26%) cervicitis (score = 2) diseased 8 (53%) 11 (73%) 7 (47%) table. temporal changes in polymorphonuclear cells (pmn), purulent vaginal discharge (pvd), and cervicitis in control and diseased cows figure 3a. blood igg concentrations in control (n = 11) cows; note the lack of difference at examinations exam 4 exam 5 exam 6 pmn ≥ 18% control 11 (24%) 14 (30%) 6 (13%) pmn ≥ 18% diseased 2 (13%) 10 (67%) 4 (27%) pvd ≥ 2 control 20 (43%) 21 (46%) 7 (15%) pvd ≥ 2 diseased 4 (27%) 11 (73%) 7 (47%) cervicitis (score = 2) control 14 (30%) 14 (30%) 12 (26%) cervicitis (score = 2) diseased 8 (53%) 11 (73%) 7 (47%) -6wkbc -4wkbc -2wkbc 0 +1wkac +3wkac +5wkac weeks before and after calving ca lv in g bl oo d ig g c on ce nt ra tio n (m g/ m l) figure 3a. blood igg concentrations in control (n = 11) cows; note the lack of difference at examinations clinical theriogenology 2022; 14: 87 figure 3b. blood igg concentrations in diseased cows (n = 11); note the lack of difference at examinations figure 4a. wbc in control (n =11) cows; note the lack of difference at examinations -6wkbc -4wkbc -2wkbc 0 +1wkac +3wkac +5wkac bl oo d ig g c on ce nt ra tio n (m g/ m l) weeks before and after calving -6wkbc -4wkbc -2wkbc +1wkac +3wkac +5wkac weeks before and after calving w bc c /m l ca lv in g ca lv in g figure 3b. blood igg concentrations in diseased cows (n = 11); note the lack of difference at examinations figure 3b. blood igg concentrations in diseased cows (n = 11); note the lack of difference at examinations figure 4a. wbc in control (n =11) cows; note the lack of difference at examinations -6wkbc -4wkbc -2wkbc 0 +1wkac +3wkac +5wkac bl oo d ig g c on ce nt ra tio n (m g/ m l) weeks before and after calving -6wkbc -4wkbc -2wkbc +1wkac +3wkac +5wkac weeks before and after calving w bc c /m l ca lv in g ca lv in g figure 4a. wbc in control (n =11) cows; note the lack of difference at examinations weeks before and after calving figure 4b. wbc in diseased cows (n = 11); note the lack of difference at examinations figure 5a. fibrinogen concentration in control cows (n = 11); note the lack of difference at examinations w bc c /m l -4wkbc -2wkbc +1wkac +3wkac +5wkac -6wkbc ca lv in g -6wkbc -4wkbc -2wkbc +1wkac +3wkac +5wkac weeks before and after calving s er um fi br in og en (g /d l) ca lv in g figure 4b. wbc in diseased cows (n = 11); note the lack of difference at examinations clinical theriogenology 2022; 14: 88 weeks before and after calving figure 4b. wbc in diseased cows (n = 11); note the lack of difference at examinations figure 5a. fibrinogen concentration in control cows (n = 11); note the lack of difference at examinations w bc c /m l -4wkbc -2wkbc +1wkac +3wkac +5wkac -6wkbc ca lv in g -6wkbc -4wkbc -2wkbc +1wkac +3wkac +5wkac weeks before and after calving s er um fi br in og en (g /d l) ca lv in g figure 5a. fibrinogen concentration in control cows (n = 11); note the lack of difference at examinations figure 5b. fibrinogen concentration in diseased cows (n = 11); note the lack of difference at examinations -6wkbc -4wkbc -2wkbc +1wkac +3wkac +5wkac weeks before and after calving ca lv in g se ru m fi br in og en (g /d l) figure 5b. fibrinogen concentration in diseased cows (n = 11); note the lack of difference at examinations subclinical endometritis) are more detrimental to fertility than is a single disease.25 proportion of pmns at 5 and 7 weeks postpartum has been associated with concomitant bacterial infection24 and with impaired reproductive performance.18,27 since the most suitable threshold for the diagnosis of subclinical endometritis is still being debated, we used a threshold of 18% pmns on endometrial cytology28 for samples obtained between 20 and 33 dim. definition of disease chosen was meant to increase the likelihood of finding differences between control and diseased groups. sample size calculation and using a systematic and consecutive enrollment design strengthened the experimental design. as less is known about the pathogenesis of postpartum endometritis, especially the role of systemic inflammatory markers, our study provided new insights into the systemic immune response of dairy cows to postpartum endometritis throughout the entire transition period. prevalence of clinical and subclinical endometritis and of cervicitis at exam 5 (n = 59) were similar to reported values for a similar time point during the postpartum period.7,18,20 we observed a decrease in prevalence from exam 5 to exam 6, similar to the prevalence of clinical and subclinical endometritis that declined from 25.4% at 3 weeks to 14.7% at 8 weeks postpartum.29 similarly, prevalence of cervicitis throughout the early part of the postpartum period decreased. like endometritis, this was most likely due to spontaneous cure that is expected to decrease with number of days postpartum. contrary to previous studies, the prevalence of t. pyogenes at exam 5 was 4 times greater than the prevalence reported at 28 clinical theriogenology 2022; 14: 89 dim (6.3%),30 and 1.2 times less than the prevalence reported at 21 days postpartum (30.2%).31 however, the sample size in our study limited this comparison. on day 21 postpartum, < 40% of cows had t. pyogenes in uterine samples obtained using low-volume uterine lavage.31 as prevalence of t. pyogenes was not greater than studies that had only 1 sampling, multiple sampling over the whole transition period probably did not affect results in our study. definitively associated with endometrial lesions and fertility, t. pyogenes is the most recognized etiological agent of postpartum endometritis.32 however, prevalence of t. pyogenes varies with herd, type of facility, management, and location. cows with t. pyogenes (score ≥ 2) exhibited a greater probability of having clinical and/or subclinical endometritis (p = 0.008 and p = 0.02, respectively), but not cervicitis (p > 0.05). we are the first group to assess systemic immune indicators of inflammation over the entire transition period (day – 40 to day + 35) in dairy cows with and without endometritis. there was no difference in igg concentrations between exam 1 and exam 6, and among diseased and control cows at any examination throughout the entire transition period. immunoglobulins have an important role in immune defence by serving as opsonin that enhance phagocytosis, and by stimulating the classical complement pathway.14 as igg is produced in part from the endometrium (mainly igg1), with the balance of igg1 derived from peripheral circulation and all igg2 derived from peripheral circulation,33,34 one would expect variations in igg concentrations in the blood stream of cows during the transition period.35 decreased igg concentrations in the peripheral circulation around calving could be explained by the immunosuppressant status of pregnancy-associated physiological phenomena, the effect of cortisol at calving, and the stress associated with high-producing cows in the postpartum period.36 some researchers have suggested that postpartum uterine diseases are related to an immunosuppressant status during the postpartum period, but they have provided no details about what specific features of this status could be involved. in our study, control and diseased cows did not have any locomotor, mammary gland or metabolic diseases that could have been potential sources of inflammation causing the changes in inflammatory indicators. therefore, our chances of observing differences among control and diseased cows due to uterine anomalies alone was improved. based on the results, and assuming that a large portion of total igg in the uterine lumen is synthesized systemically, estimation of igg serum concentrations is not a good inflammatory indicator to predict or monitor postpartum endometritis in dairy cows.37 in addition to measuring variation in igg concentrations, several researchers also reported a decrease in peripheral blood lymphocyte counts.21,38 by contrast, as was the case for igg, we did not observe variation in wbc numbers. peripheral blood concentrations of total leukocytes or individual types (pmns, monocytes, lymphocytes, and eosinophils) in the 2 groups of cows did not vary throughout the transition period, or differ between control and diseased cows at examinations. in normal cows, the prepartum period is usually accompanied by peripheral leukocytosis,39 followed by leucopenia during the first week after calving.40 cows with subclinical endometritis had higher blood leukocyte counts and elevated pmn concentrations compared to control postpartum cows between days 45 and 55.41 fibrinogen concentrations were not different between control and diseased cows at examinations during the transition period. fibrinogen is a secreted plasma protein that is the precursor of fibrin and is used to form secondary hemostatic plugs at sites of vascular injury, in several inflammatory and traumatic conditions in cattle.42 analyses of metabolic blood parameters, serum inflammatory mediators, antibodies, and the cellular composition of cow blood for concentrations of elements like beta-hydroxybutyrate, haptoglobin and sialic acid around calving have been proposed, but none was satisfactory for disease prediction.43 increased fibrinogen concentrations (hyperfibrinogenemia) may be associated with inflammation or with dehydration. although the ratio of plasma proteins/ fibrinogen may help to distinguish dehydration from inflammation, dehydration was not evident in our study. conclusion concentrations of systemic inflammatory indicators (igg, white blood cells, and fibrinogen) did not change over time nor did they differ between control and diseased postpartum cows at examinations. therefore, we suggest that systemic inflammatory markers are not good indicators for diagnosing or monitoring endometritis in postpartum dairy cows. conflict of interest none to declare. references 1. goff jp, horst rl: physiological changes at parturition and their relationship to metabolic disorders. j dairy sci 1997;80:1260-1268. 2. drillich m: an update on uterine infections in dairy cattle. slov vet res 2006;43:11-15. 3. overton tr, yasui t: practical applications of trace minerals for dairy cattle. j anim sci 2014;92:416-426. 4. sheldon im, lewis gs, leblanc sj: defining postpartum uterine disease in cattle. theriogenology 2006;65:1516-1530. 5. leblanc sj: postpartum uterine disease and dairy herd reproductive performance: a review. vet j 2008;176:102-114. 6. sheldon im, williams ej, miller an, et al: uterine diseases in cattle after parturition. vet j 2008;176:115-121. 7. williams ej, fischer dp, pfeiffer du, et al: clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle. theriogenology 2005;63:102-117. 8. sheldon im, dobson h: postpartum uterine health in cattle. anim reprod sci 2004;82:295-306. 9. kasimanickam r, duffield t, foster r, et al: a comparison of the cytobrush and uterine lavage techniques to evaluate endometrial cytology in clinically normal postpartum dairy cows. can vet j 2005;46:255-259. 10. herath s, dobson h, sheldon im: use of the cow as a large animal model of uterine infection and immunity. j reprod immunol 2006;69:13-22. 11. galvão kn, flaminio mjbf, brittin sb, et al: association between uterine disease and indicators of neutrophil and systemic energy status in lactating holstein cows. j dairy sci 2010;93:2926-2937. clinical theriogenology 2022; 14: 90 12. herath s, lilly st, santos nr, et al: expression of genes associated with immunity in the endometrium of cattle with disparate postpartum uterine disease and fertility. reprod biol endocrinol 2009;7:55, https://doi.org/10.1186/1477-7827-7-55. 13. sheldon im, cronin j, schuberth hj: defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle. biol reprod 2009;81:1025-1032. 14. singh j, murray rd, mshelia g, et al: the immune status of the bovine uterus during theperipartum period. vet j 2008;175:301-309. 15. alberts b, johnson a, lewis j, et al. molecular biology of the cell. 4th edition. new york: garland science; 2002. 16. dhaliwal gs, murray rd, woldehiwet z: some aspects of immunology of the bovine uterus related to treatments for endometritis. anim reprod sci 2001;67:135-152. 17. national academies of sciences, engineering, and medicine. 0. nutrient requirements of dairy cattle: eighth revised edition. washington, dc: the national academies press. 18. tison n, bouchard e, descoteaux l, et al: effectiveness of intrauterine treatment withcephapirin in dairy cows with purulent vaginal discharge. theriogenology 2017;89:305-317. 19. gilbert ro: management of reproductive disease in dairy cows. vet clin north am food anim pract 2016;32:387-410 20. hartmann d, rohkohl j, merbach s, heilkenbrinker, et al: prevalence of cervicitis in dairy cows and its effect on reproduction. theriogenology 2016;85:247-253. 21. herr m, bostedt h, failing k: igg and igm levels in dairy cows during the periparturient period. theriogenology 2011;75:377-385. 22. faul f, erdfelder e, buchner a: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. behav res methods 2007;39:175-191. 23. sordillo lm, contreras ga, aitken sl: metabolic factors affecting the inflammatory response of periparturient dairy cows. anim health res rev 2009;10:53-63. 24. foldi j, kulcsar m, pecsi a, et al: bacterial complications of postpartum uterine involution in cattle. anim reprod sci 2006;96:265-281. 25. dubuc j, duffield tf, walton js: risk factors for postpartum uterine diseases in dairy cows. j dairy sci 2010;93:5764-5771. 26. gilbert ro, shin st, guard cl, et al: prevalence of endometritis and its effects on reproductive performance of dairy cows. theriogenology 2005;64:1879-1888. 27. islam r, kumar h, nandi s: determination of anti-inflammatory cytokine in periparturient cows for prediction of postpartum reproductive diseases. theriogenology 2013;79:974-979. 28. kasimanickam r, duffield tf, foster ra, et al: endometrial cytology and ultrasonography for the detection of subclinical endometritis in postpartum dairy cows. theriogenology 2004;62:9-23. 29. gautam g, nakao t, koike k, et al: spontaneous recovery or persistence of postpartumendometritis and risk factors for its persistence in holstein cows. theriogenology 2010;73:168-179. 30. leblanc sj, duffield tf, leslie ke, et al: the effect of treatment of clinical endometritis on reproductive performance in dairy cows. j dairy sci 2002;85:2237-2249. 31. prunner i, pothmann h, wagener k, et al: dynamics of bacteriologic and cytologic changes in the uterus of postpartum dairy cows. theriogenology 2014;82:1316-1322. 32. barlund cs, carruthers td, palmer cw: a comparison of diagnostic techniques for postpartum endometritis in dairy cattle. theriogenology 2008;69:714-723. 33. corbeil lb, schurig gd, duncan jr, et al: immunoglobulin classes and biological functions of campylobacter (vibrio) fetus antibodies in serum and cervicovaginal mucus. infect immun 1974;10:422-429. 34. beutler b, ulevitch rj: how we detect microbes and respond to them: the toll-like receptors and their transducers. j leukoc biol 2003;74:479-485. 35. fischer c, drillich m, heuwieser w, et al: selected proinflammatory factor transcripts in bovine endometrial epithelial cells are regulated during the oestrous cycle and elevated in case of subclinical or clinical endometritis. reprod fertil dev 2010;22:818829. 36. lucy mc: stress, strain, and pregnancy outcome in postpartum cows. anim reprod 2019;16:455-464. published online 2019 oct 23. doi:10.21451/1984-3143-ar2019-0063. 37. hazlett l, wu m: defensins in innate immunity. cell tissue res 2011;343:175-188. 38. kim ih, na kj, yang mp: immune responses during the peripartum period in dairy cows with postpartum endometritis. j reprod dev 2005;51:757-764. 39. mateus l, da costa ll, bernardo f, et al: influence of puerperal uterine infection on uterine involution and postpartum ovarian activity in dairy cows. reprod domest anim 2002;37:31-35. 40. guidry aj, paape mj, pearson re: effects of parturition and lactation on blood and milk cell concentrations, corticosteroids, and neutrophil phagocytosis in the cow. am j vet res 1976;37:1195-1200. 41. raboisson d, mounié m, maigné e: diseases, reproductive performance, and changes in milk production associated with subclinical ketosis in dairy cows: a meta-analysis and review. j dairy sci 2014;97:7547-7563. 42. hussain am, daniel rcw: phagocytosis by uterine fluid and blood neutrophils and hematological-changes in postpartum cows following normal and abnormal parturition. theriogenology 1992;37:1253-1267. 43. kasimanickam r, kasimanickam v, olsen j, et al: associations among serum proand anti inflammatory cytokines, metabolic mediators, body condition, and uterine disease in postpartum dairy cows. reprod biol endocrinol 2013;11:103. comparison of short-term complications following cesarean section or cesarean section with concurrent ovariohysterectomy in bitches and queens: a pilot study   comparison of short-term complications following cesarean section or cesarean section with concurrent ovariohysterectomy in bitches and queens: a pilot study margaret v. root kustritz college of veterinary medicine, university of minnesota, st. paul, mn abstract cesarean section is a common treatment for canine and feline dystocia. ovariohysterectomy may or may not be recommended concurrently. there are apparently no data in the literature providing evidence to guide decision-making regarding the advantages and disadvantages of performing these procedures concurrently. animal signalment, history upon presentation, medical and anesthetic management of dystocia, and incidence of short-term complications were compared between bitches and queens undergoing cesarean section alone and those undergoing concurrent cesarean section and ovariohysterectomy. short-term complications were defined as those happening after recovery from anesthesia and extubation and within several days after patient discharge. complete data were available for two queens undergoing cesarean section alone and two queens undergoing concurrent cesarean section and ovariohysterectomy, and for 20 bitches undergoing cesarean section alone and 22 bitches undergoing concurrent cesarean section and ovariohysterectomy. no short-term complications were reported in any of these patients. an important limitation was small sample size; therefore, further investigations, perhaps by pooling data from several institutions, would be beneficial. keywords: cesarean section, dystocia, ovariohysterectomy, spay, bitch, queen introduction reported incidence of dystocia is 2.0-5.0% and 3.3-5.8% in dogs and cats, respectively, with a predisposition in some breeds.1-9 cesarean section (c-section) is performed in 54-66% of dogs and in 7479% of cats with dystocia.1,4-6,10 reported complications of c-section include hemorrhage, infection or dehiscence of the incision, and peritonitis.2,11,12 complications may occur due to underlying disease in the animal or surgeon error.12 mortality rate for the bitch or queen during or immediately after c-section is 02%.11,13 it is not uncommon for veterinarians to recommend or for clients to request ovariohysterectomy (ohe) at the time of c-section. reasons to conduct the two procedures concurrently include: the bitch is spared a subsequent anesthetic episode (assuming she will be spayed later); bitches that require c-section should not be rebred, which may or may not be true depending on the cause of the dystocia and the reason for the c-section; uterine pathology, e.g. uterine rupture or infection; and this is an opportunity to perform ohe for population control.10,14 some argue that risks of concurrent c-section and ohe are excessive. it is not difficult to expect increased morbidity and potentially increased mortality in bitches and queens undergoing two invasive surgical procedures concurrently. blood pressure rises when the linea alba is incised and as ovaries are manipulated in healthy bitches undergoing ohe.15-17 commonly reported complications of ohe include: hemorrhage at the ovarian pedicle or from other intra-abdominal vessel; inadvertent ligation of the distal ureter; trauma to other intra-abdominal organs; persistent bloody vulvar discharge; and infection or dehiscence of the incision.17-28 reported concerns specific to concurrent csection and ohe are: lack of milk production due to dehydration, anemia, or uncontrolled pain postsurgically; hemorrhage; and decreased mothering responses.29 other possible concerns with concurrent ohe and c-section are physiologic responses in the dam, including: changes in blood pressure or hematologic parameters secondary to removal of the blood-filled uterus and associated placental tissues; increased duration of anesthesia and surgery if procedures are performed concurrently, with associated complications such as hypothermia and tissue damage; and ethical concerns for the bitch. veterinarians, breed clubs, and others interested in ensuring optimal health of breeding bitches rely on scientific literature to help them make decisions. in one retrospective study, number of days hospitalized and complications were reportedly higher in 10 bitches that had undergone both c-section and ohe than in those that had undergone c-section alone.1 complications were not described in that 409 clinical theriogenology • volume 10, number 4 • december 2018   study. other studies described overall complications of populations of animals undergoing either csection or ohe, some of which likely underwent both procedures, but again without comparing complications between c-section or ohe alone and the two surgeries performed concurrently.4,6,10,19 the author is unaware of any published studies specifically evaluating short-term complications of c-section versus c-section with concurrent ohe. the goals of this pilot study were to identify reported short-term complications associated with csection alone versus c-section with concurrent ohe and to identify trends in patient signalment or medical and anesthetic management of these cases that may have contributed to development of complications. materials and methods the electronic medical record was searched for the procedure code for c-section. complete records were either available electronically or hard copies were retrieved to ensure that the author had all available data. data recorded included signalment, history upon presentation, and medical and anesthetic management. breed but not weight was recorded, as weight varies in late gestation (due to litter size). similarly, body condition score was not reported because it can be difficult to assess in late-gestation bitches. short-term complications were defined as those happening after recovery from anesthesia and extubation and within several days after patient discharge. results the electronic medical record was searched from 2001 through 2017. fifty-four medical records were identified. five records were incomplete. three bitches were presented for hysterotomy due to pregnancy loss of undefined cause (n = 1) or for removal of a non-viable fetus and fetal membranes (n = 2). of the remaining cases, four were queens, two of which underwent c-section alone and two that underwent c-section with concurrent ohe, and 42 were bitches, 20 of which underwent c-section alone and 22 that underwent c-section with concurrent ohe. history and management of the queens are shown (table 1). anesthetic protocol was only reported in detail for the two queens that underwent c-section with concurrent ohe; one queen received midazolam and butorphanol and was maintained on sevoflurane, whereas the other was induced with propofol and maintained on sevoflurane. reason for the concurrent ohe was not reported in either queen. all kittens were born alive and discharged with the dam. no short-term complications were reported for any queens. history and management of the 20 bitches that underwent c-section alone are shown (table 2). mean + sd age at the time of presentation was 4.2 + 1.8 years. breed was variable, with no breed overrepresented. three bitches were of toy breeds and one was of a giant breed. anesthetic protocol was reported in detail for 10. two were maintained on isoflurane and also received propofol (n = 1) and alfaxalone (n = 1). eight were maintained on sevoflurane and also received hydromorphone and propofol (n = 4); hydromorphone, diazepam, and propofol (n = 2); fentanyl and propofol (n = 1), or diazepam and propofol (n = 1). two bitches suffered transient hypotension under anesthesia that responded to medical therapy. all pups were born alive and sent home with the dam for 13 bitches; all pups were stillborn, could not be resuscitated, or had congenital defects incompatible with life at the time of c-section for four bitches; and there was a combination of live and stillborn pups for three bitches. no short-term complications were reported for any bitches that underwent c-section alone. one bitch on a raw, homemade diet had a poor appetite and was not attentive to the pups the first day after surgery; however, that resolved after she was fed a commercial diet. history and management for the 22 bitches that underwent c-section and concurrent ohe are shown (table 3). mean age at the time of presentation was 4.2 + 2.0 years. breed was variable, with no breed over-represented. five bitches were of toy breeds and one was of a giant breed. reason for the concurrent ohe was unreported in 21 bitches, whereas a uterine tear was present in one bitch. anesthetic protocol was reported in detail for 13. seven were maintained on isoflurane and also received alfaxalone (n = 2); propofol (n = 2); atropine, midazolam, hydromorphone, and propofol (n = 1); diazepam, 410clinical theriogenology • volume 10, number 4 • december 2018   hydromorphone, and propofol (n = 1); hydromorphone and propofol (n = 1). six were maintained on sevoflurane and also received hydromorphone and propofol (n = 2); atropine, hydromorphone, and propofol (n = 2); diazepam, hydromorphone, and propofol (n = 1); and fentanyl and propofol (n = 1). one bitch with aspiration pneumonia in late gestation had a prolonged capillary refill time and a decline in pcv from 35 to 19% intra-operatively; she responded to transfusion with packed rbcs. one bitch with pregnancy toxemia had an unreported decline in pcv intra-operatively; she responded to transfusion with blood products. one bitch with respiratory distress had low oxygen saturation and could only maintain normal oxygen saturation after intubation and oxygen therapy; however, this resolved after surgery, and she had a prolonged but uneventful recovery. all pups were born alive and sent home with the dam for 13 bitches; all pups were stillborn, could not be resuscitated, or had congenital defects incompatible with life at the time of c-section for three bitches; and there was a combination of live and stillborn pups for six bitches. no short-term complications were reported for any bitch that underwent c-section with concurrent ohe. discussion in this study, no short-term complications were noted either for c-section alone or for c-section with concurrent ohe. all cases were managed in a referral hospital where the receiving clinician had immediate access to a board-certified theriogenologist and all procedures were completed by boardcertified anesthesiologists and surgeons. this, coupled with availability of pharmaceuticals and monitoring equipment that may not be available at all facilities, may have decreased the likelihood of complications. one limitation of this study was the limited number of cases. this is a referral hospital in a large metropolitan area with many clinics that offer high-quality small animal reproductive services, which limits such cases presenting to the author’s institution. another limitation was a lack of complete followup on all cases, as some undoubtedly were presented solely because emergency service was available and they subsequently completed follow-up with their regular veterinarian. for many cases, complete analysis of records permitted the author to access information from telephone conversations with clients in the days following surgery. a final limitation, common in retrospective studies, was the lack of consistent data in medical records. for example, surgical reports often did not clarify if ohe had been performed post-hysterotomy or if an en bloc ohe had been performed. this could have affected incidence of shortterm complications. in one study describing en bloc ohe as a treatment for dystocia, examples of postsurgical complications included: anemia requiring blood transfusion in three queens; disseminated intravascular coagulation (dic) in a queen; bloody vulvar discharge in a bitch; uroperitoneum in a bitch; and death of a queen.30 as another example, use of preor intra-operative antibiotics was variably recorded. for many cases, antibiotic use was identified by the author reviewing the anesthesia record, the surgical record, the pharmacy request screen, or the final bill. consequently, there was limited ability to determine when the antibiotic had been administered. presumably some bitches received antibiotics and it either was not recorded or was not retrieved. consequently, use of preand intra-operative antibiotics could not reliably be reported. data regarding dispensed antibiotics were consistent in the record, as described above. use of antibiotics generally is not required unless there is systemic infectious disease, ongoing uterine pathology in bitches or queens left intact, or contamination of the peritoneal cavity with uterine contents during either c-section or ohe.11 the rationale for using or withholding antibiotics was not consistently apparent. two studies evaluating post-surgical complications in dogs and cats based on retrieval of hard copy or electronic records demonstrated under-reporting in computer records.19,23 both hard copies and electronic records had some deficiencies in this study, more likely due to lack of training and tracking of record completion, rather than variation in the medium used. most studies in dogs and cats identify uterine inertia as the most common cause of dystocia.1,4,10,30 in a study describing 510 c-section procedures in 453 bitches, lack of progression of labor was the 411 clinical theriogenology • volume 10, number 4 • december 2018   reported cause of dystocia in 73.1% of cases.2 however, in the present study, specific causes of dystocia were difficult to identify from the medical record. variability of breed and range of age of bitches and queens in this case series matched those reported in the literature.2,13 increased body weight was associated with increased incidence of complications of ohe in several studies.17,20 that was not supported by data in this study, although weight of the bitches and queens in this case series was not retrieved from the medical record, due to concerns regarding inability to differentiate lean body weight from pregnancy weight, with varying litter size. various anesthetic protocols were used, consistent with the literature.2,13 the lack of short-term complications precluded making associations with medical or anesthetic management. however, the variability of medical and anesthetic management protocols suggests that such associations would be unlikely unless sample size or the effect were large. the author looks forward to other publications with larger sample sizes, perhaps as a collaborative effort from several institutions, to provide more definitive evidence. references 1. gaudet da: retrospective study of 128 cases of canine dystocia. j am anim hosp assoc 1985;21:813-818. 2. ayres se, thomas pga: population characteristics of 453 bitches undergoing 510 cesarean section procedures between 1999 and 2009 – a retrospective study. clin therio 2012;2:451-465. 3. evans km, adams vj: proportion of litters of purebred dogs born by caesarean section. j small anim pract 2010;51:113-118. 4. ekstrand c, linde-forsberg c: dystocia in the cat: a retrospective study of 155 cases. j small anim pract 1994;35:459-464. 5. gunn-moore da, thrusfield mv: feline dystocia: prevalence, and association with cranial conformation and breed. vet rec 1995;136:350-353. 6. munnich a, kuchenmeister u: dystocia in numbers – evidence-based parameters for intervention in the dog: causes for dystocia and treatment recommendations. reprod domest anim 2009;44 (suppl 2):141-147. 7. bergstrom a, nodtvedt a, lagerstedt a-s, et al: incidence and breed predilection for dystocia and risk factors for cesarean section in a swedish population of insured dogs. vet surg 2006;35:786-791. 8. humphreys j: dystocia in cats. vet rec 1974;95:353. 9. linde-forbserg c, eneroth a: abnormalities in pregnancy, parturition, and the periparturient period. in: ettinger sj, feldman ec, editors, textbook of veterinary internal medicine, 5th edition, philadelphia; wb saunders; 2000. p. 15271538. 10. walett darvelid a, linde-forsberg c: dystocia in the bitch: a retrospective study of 182 cases. j small anim pract 1994;35:402-407. 11. sammarco j, kahn a: cesarean section in dogs: indications, techniques. available at: http://veterinarynews.dvm360.com/cesarean-section-dogs-indications-techniques?id=&sk=&date=&pageid=3. accessed 04-04-18. 12. baltodano la, memon ma, king c: incidence of post-surgical complications of ovariohysterectomy and orchiectomy in field conditions. clin therio 2013;5:37-43. 13. moon pf, erb hn, ludders jw, et al: perioperative management and mortality rates of dogs undergoing cesarean section in the united states and canada. j am vet med assoc 1998;213:365-369. 14. gilroy ba, deyoung dj: cesarean section. anesthetic management and surgical technique. vet clin north am 1986;16:483-494. 15. hoglund ov, olsson k, hagman r, et al: comparison of haemodynamic changes during two surgical methods for neutering female dogs. res vet sci 2011;91:159-163. 16. tallant a, ambros b, freire c, et al: comparison of intraoperative and postoperative pain during canine ovariohysterectomy and ovariectomy. can vet j 2016;57:741-746. 17. berzon jl: complications of elective ovariohysterectomies in the dog and cat at a teaching institution: clinical review of 853 cases. vet surg 1979;8:89-91. 18. burrow r, batchelor d, cripps p: complications observed during and after ovariohysterectomy of 142 bitches at a veterinary teaching hospital. vet rec 2005;157:829-833. 19. pollari fl, bonnett bn, bamsey sc, et al: postoperative complications of electives surgeries in dogs and cats determined by examining electronic and paper medical records. j am vet med assoc 1996;208:1882-1886. 20. muraro l, white rs: complications of ovariohysterectomy procedures performed in 1880 dogs. tierarztliche praxis 2014;42:297-302. 21. adin ca: complications of ovariohysterectomy and orchiectomy in companion animals. vet clin north am 2011;41:1023-1039. 412clinical theriogenology • volume 10, number 4 • december 2018   22. roberts ml, beatty ja, dhand nk, et al: effect of age and surgical approach on perioperative wound complication following ovariohysterectomy in shelter-housed cats in australia. j feline med surg open reports 2015; 1. doi:10.1177/2055116915613358. 23. pollari fl, bonnett bn: evaluation of postoperative complications following elective surgeries of dogs and cats at private practices using computer records. can vet j 1996;37:672-678. 24. pearson h: the complications of ovariohysterectomy in the bitch. j small anim pract 1973;14:257-266. 25. okkens ac, van de gaag i, biewenga wj, et al: urological complications following ovariohysterectomy in dogs. tijdschr diergeneeskd 1981;106:1189-1198. 26. harris kp, adams vj, fordyce p, et al: comparison of surgical duration of canine ovariectomy and ovariohysterectomy in a veterinary teaching hospital. j small anim pract 2013;54:579-583. 27. kennedy kc, tamburello kr, hardie rj: peri-operative morbidity associated with ovariohysterectomy performed as part of a third-year veterinary surgical-training program. j vet med educ 2011;38:408-413. 28. bowlt blacklock kl, langer p, halfacree z, et al: canine ovariohysterectomy: a survey of surgeon concerns and surgical complications encountered by newly graduated veterinarians. j vet med educ 2016;43:184-189. 29. traas am: surgical management of canine and feline dystocia. theriogenology 2008;70:337-342. 30. robbins ma, mullen hs: en bloc ovariohysterectomy as a treatment for dystocia in dogs and cats. vet surg 1994;23:48-52. 413 clinical theriogenology • volume 10, number 4 • december 2018   ta bl e 1. s ig na lm en t a nd c as e m an ag em en t d at a fo r q ue en s u nd er go in g ce sa re an se ct io n al on e or c es ar ea n se ct io n w ith c on cu rre nt o va rio hy ste re ct om y. pr o ce d u re br ee d / a g e pr es en ti n g h is to ry ra d io g ra ph s / u lt ra so u n d o x y to ci n th er a py ? pr ea n es th et ic bl o o d w o r k a n ti bi o ti cs d is pe n se d a t d is ch a rg e cse ct io n on ly a by ss in ia n / 4 y ea rs le th ar gy , va ria bl e an em ia ov er th e la st w ee k ra di og ra ph s a nd ul tra so un d n o pc v 2 3% n o cse ct io n on ly d sh / 1 ye ar pr ol on ge d la bo r, on e sti llb or n ki tte n bo rn a t ho m e ra di og ra ph s y es – tw o liv e ki tte ns b or n pc v 3 8% n o cse ct io n w ith co nc ur re nt o h e si am es e / 3 ye ar s pr ol on ge d la bo r ra di og ra ph s a nd ul tra so un d n o pc v 3 9% , h g b 13 .3 g m /d l n o cse ct io n w ith co nc ur re nt o h e d sh / 4 ye ar s pr ol on ge d la bo r, hi sto ry o f p el vi c fra ct ur e ra di og ra ph s n o pc v 2 9% , pl at el et n um be r no rm al (e sti m at e) n o 414clinical theriogenology • volume 10, number 4 • december 2018   ta bl e 2. s ig na lm en t a nd c as e m an ag em en t d at a fo r b itc he s u nd er go in g cse ct io n al on e. br ee d / a g e pr es en ti n g h is to ry ra d io g ra ph s / u lt ra so u n d o x y to ci n th er a py ? pr ea n es th et ic bl o o d w o r k a n ti bi o ti cs d is pe n se d a t d is ch a rg e en gl ish b ul ld og / 2 y ea rs pr ol on ge d la bo r, gr ee n vu lv ar di sc ha rg e u ltr as ou nd n o pc v 4 4% , h g b 15 .2 g m /d l n o en gl ish b ul ld og / 2 y ea rs g re en v ul va r d isc ha rg e 4 da ys pr io r t o pl an ne d cse ct io n ra di og ra ph s n o pc v 4 1% , h g b 13 .9 g m /d l y es st an da rd po od le / 2 ye ar s pr ol on ge d la bo r n on e re po rte d y es – n o ef fe ct pc v 3 6% , h g b 12 .4 g m /d l, pl at el et n um be r 38 9, 00 0 n o a m er ic an bu lld og / 2 ye ar s pr ol on ge d la bo r n on e re po rte d y es – n o ef fe ct n on e re po rte d n o be rn es e m ou nt ai n do g / 3 ye ar s pl an ne d cse ct io n as b itc h en te re d la bo r ra di og ra ph s n o pc v 5 2% , h g b 17 .8 g m /d l, pl at el et n um be r 32 6, 00 0 n o en gl ish c oc ke r sp an ie l / 3 ye ar s pr ol on ge d la bo r ra di og ra ph s y es – n o ef fe ct pc v 2 9% , h g b 9. 9 gm /d l y es la br ad or re tri ev er / 3 ye ar s st ill bo rn th e da y be fo re , n o co nt ra ct io ns si nc e. ra di og ra ph s a nd ul tra so un d y es – 6 p up s bo rn li ve pc v 4 2% , h g b 14 .3 g m /d l n o la br ad or re tri ev er / 3 ye ar s pr ol on ge d la bo r, re fe rri ng d v m g av e ox yt oc in to n o ef fe ct ra di og ra ph s y es – 2 p up s bo rn li ve pc v 3 2% , h g b 10 .9 g m /d l n o be rn es e m ou nt ai n do g / 4 ye ar s fi ve p up s b or n liv e at h om e, pr ol on ge d la bo r f or fi na l p up ra di og ra ph s n o pc v 3 8% , h g b 12 .9 g m /d l n o si be ria n hu sk y / 4 y ea rs pr ol on ge d la bo r ra di og ra ph s n o pc v 3 7% , h g b 12 .9 g m /d l, pl at el et n um be r 53 1, 00 0 n o 415 clinical theriogenology • volume 10, number 4 • december 2018   ch ih ua hu a / 4 ye ar s pr ol on ge d la bo r, tw o pu ps b or n liv e at h om e th at la te r d ie d ra di og ra ph s y es – n o ef fe ct n on e re po rte d n o pu g / 4 y ea rs pr ol on ge d la bo r ra di og ra ph s a nd ul tra so un d n o n on e re po rte d n o ita lia n gr ey ho un d / 4 ye ar s pr ol on ge d la bo r, ha d re ce iv ed de xa m et ha so ne e ar lie r i n pr eg na nc y fo r m is m at in g ra di og ra ph s n o pc v 3 4% , h g b 11 .7 g m /d l y es si be ria n hu sk y / 5 y ea rs el ec tiv e cse ct io n n on e re po rte d n o n on e re po rte d n o d ac hs hu nd / 5 ye ar s pr ol on ge d la bo r ra di og ra ph s y es – 1 p up bo rn li ve n on e re po rte d n o si be ria n hu sk y / 6 y ea rs el ec tiv e cse ct io n fo r s in gl e, la rg e pu pp y ra di og ra ph s n o n on e re po rte d n o co lli e / 6 y ea rs pr ol on ge d la bo r ra di og ra ph s y es – 2 p up s sti llb or n pc v 3 9% n o iri sh w ol fh ou nd / 7 ye ar s pr ol on ge d la bo r, bi tc h in a tri al fib ril la tio n an d he ar tw or m po sit iv e n on e re po rte d n o pc v 3 4% , h g b 12 .2 g m /d l n o sa m oy ed / 7 ye ar s pr ol on ge d la bo r, on e liv e pu p an d on e sti llb or n at h om e n on e re po rte d n o n on e re po rte d y es a fg ha n ho un d / 8 ye ar s pr ol on ge d la bo r ra di og ra ph s y es – n o ef fe ct pc v 4 5% , h g b 15 .2 g m /d l y es 416clinical theriogenology • volume 10, number 4 • december 2018   ta bl e 3. s ig na lm en t a nd c as e m an ag em en t d at a fo r b itc he s u nd er go in g co nc ur re nt c -s ec tio n an d o h e. br ee d / a g e pr es en ti n g h is to ry ra d io g ra ph s / u lt ra so u n d o x y to ci n th er a py ? pr ea n es th et ic bl o o d w o r k a n ti bi o ti cs d is pe n se d a t d is ch a rg e pu g / 1 y ea r pr ol on ge d la bo r n on e re po rte d n o n on e re po rte d y es ch ih ua hu a / 1 ye a r pr ol on ge d la bo r ra di og ra ph s n o pc v 4 2% , h g b 14 .3 g m /d l n o y or ks hi re te rri er / 2 ye ar s pr ol on ge d la bo r, on e liv e an d on e sti llb or n pu p at h om e u ltr as ou nd n o pc v 3 7% , h g b 12 .6 g m /d l n o g ol de n re tri ev er / 2 ye ar s re sp ira to ry d ist re ss in la te ge sta tio n / d ia gn os ed w ith as pi ra tio n pn eu m on ia ra di og ra ph s a nd ul tra so un d n o pc v 3 5% , h g b 11 .9 g m /d l, pr ol on ge d cr t y es ro ttw ei le r / 2 ye ar s pr ol on ge d la bo r ra di og ra ph s n o pc v 3 9% , h g b 14 .0 g m /d l n o po m er an ia n / 3 ye ar s pr ol on ge d la bo r, pr io r h ist or y of p el vi c in ju ry ra di og ra ph s n o pc v 3 4% , pl at el et n um be r in cr ea se d (e sti m at e) y es be rn es e m ou nt ai n do g / 3 ye ar s pr ol on ge d la bo r u ltr as ou nd n o pc v 3 9% n o d ac hs hu nd / 3 ye ar s ta rry st oo ls, le th ar gy / di ag no se d w ith p re gn an cy to xe m ia ra di og ra ph s n o pc v 2 5% n o a m er ic an st af fo rd sh ire te rri er / 3 ye ar s pr ol on ge d la bo r, tw o liv e pu ps bo rn a t h om e an d th en la bo r sto pp ed ra di og ra ph s a nd ul tra so un d n o pc v 3 4% , h g b 11 .6 g m /d l n o en gl ish b ul ld og / 4 y ea rs re sp ira to ry d ist re ss in la te ge sta tio n n on e re po rte d n o pc v 3 5% , h g b 12 .0 g m /d l n o la br ad or re tri ev er / 4 ye ar s pr ol on ge d la bo r, co nc ur re nt sta ge ii v ag in al h yp er pl as ia . fi ve p up s b or n liv e at h om e n on e re po rte d n o n on e re po rte d n o g ol de n re tri ev er / 4 ye ar s pr ol on ge d la bo r ra di og ra ph s y es – n o ef fe ct pc v 4 1% , h g b 14 .4 g m /d l, y es 417 clinical theriogenology • volume 10, number 4 • december 2018   pl at el et n um be r no rm al pu g / 5 y ea rs n on e re po rte d ra di og ra ph s n o n on e re po rte d y es a m er ic an st af fo rd sh ire te rri er / 5 ye ar s pr ol on ge d la bo r, gr ee n vu lv ar di sc ha rg e ra di og ra ph s n o pc v 3 9% , h g b 13 .9 g m /d l n o m in ia tu re pi ns ch er / 5 ye ar s o ve rd ue b as ed o n br ee di ng da te s, te m pe ra tu re d ro pp ed to 99 f fo ur d ay s a go , i nt er m itt en t co nt ra ct io ns ra di og ra ph s n o pc v 4 1% , h g b 14 .5 g m /d l n o ca va lie r k in g ch ar le s s pa ni el / 5 y ea rs pr ol on ge d la bo r ra di og ra ph s y es – 2 p up s bo rn li ve pc v 4 5% , h g b 15 .3 g m /d l n o en gl ish se tte r / 6 ye ar s pr ol on ge d la bo r, gr ee n vu lv ar di sc ha rg e n on e re po rte d n o pc v 3 9% , h g b 13 .3 g m /d l n o be ar de d co lli e / 6 ye ar s pr ol on ge d la bo r ra di og ra ph s n o pc v 4 1% n o iri sh w ol fh ou nd / 6 y ea rs pr ol on ge d la bo r, 2 pu ps b or n liv e at h om e ra di og ra ph s n o pc v 3 7% , h g b 16 g m /d l, no rm al p la te le t nu m be r (e sti m at e) n o w hi pp et / 7 ye ar s pr ol on ge d la bo r, gr ee n vu lv ar di sc ha rg e ra di og ra ph s n o pc v 4 1% y es bi ch on fr ie s / 7 ye ar s pr ol on ge d la bo r, tw o sti llb or n at h om e ra di og ra ph s n o pc v 3 4% , h g b 11 .6 g m /d l n o sa m oy ed / 9 ye ar s pr ol on ge d la bo r, ga ve b irt h to 9 pu ps a t h om e ra di og ra ph s y es , b y ow ne r pc v 3 2% n o 418clinical theriogenology • volume 10, number 4 • december 2018 000_frontal ms_1 forematter for december 2018 ms_2 letter1 for december 2018 ms_2 letter2 for december 2018 ms_2 letter3 for december 2018 ms_3 table of contents ms_4 therio news for december 2018_bw 2019 therio preview therio recap with pics steiner-new officers 2018 therio sponsors 2018 exhibitors-sft board noms 2018 auction donations 2019 bartlett nomination act call for noms call for abstracts 2019 for ctj the society for theriogenology and american college of theriogenologists issue a call for research and case abstracts to be presented at the annual therio conference july 24-27, 2019 in savannah, georgia. 1. competitive category: 2. non‐competitive category: 3. case abstract category: 4. poster category: general: guidelines: what to send and how to send it abstracts not adhering to these guidelines will not be considered for presentation. deadline: abstracts must be received no later than february 15, 2019 for consideration. call for 2019 student case presentations.pdf call for student case presentations-2019 annual therio conference student case presentations call for student case presentations also available at www.therio.org guidelines for application submission society for theriogenology student case presentation competition annual therio conference, savannah, georgia, july 24-27, 2019 ms_5 editorial 001a_ms_6 roberts color pictures 002_ms_7 root kustritz 003_ms_8 ferrer 004_ms_9 wiley 005_ms_10 wallace 006_ms_11 peterson 007_ms_12 nobre 008_ms_13 sidelinger 009_ms_14 mirando 1 contact augustine t. peter petera@purdue.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9973, http://dx.doi.org/10.58292/ct.v15.9973 editorial editor’s note balogh, roberto palomares, robyn ellerbrock, jamie stewart, and karen wolfsdorf), and to drs. jimmy alexander and david christiansen (student abstracts) for their dedication and due diligence. special thanks to ms. beth gibson of our management office for collecting documents and being a go-to-person for this important endeavour. we express our profound appreciation to copy editor, dr. john kastelic and production editor, ms. emma csemiczky. at the conference, it was good for me to visit with a few guest editors of upcoming special issues of the journal, plus editorial board members. sharing the growth of the journal with the society for theriogenology executive board in person has always been an important remit for me. i do enjoy attending christian veterinary mission breakfast during the conference. i am thankful to my wife of 46 years, damayanthy, for not only helping me to pursue my daily 2-hour commitment to our journal but also accompanying me to the conference. if you have any suggestions to improve our journal quality, please feel welcome to write to me (petera@purdue.edu) or contact any of the editorial board members of clinical theriogenology. i am very thankful for their support. regards, augustine dear theriogenology friends, a strategic change has been made for the conference issue of clinical theriogenology. as of this year, not every podium presentation will be published; only those selected by the editorial board will be published after peer review. it is our pleasure to publish the bartlett address and abstracts presented at the 2023 conference in this conference issue. dr. stuart meyers gave the bartlett address as the recipient of  2023 bartlett award for lifetime achievement in theriogenology, presented annually to a distinguished individual who has made important contributions to theriogenology. this award intended to cement the efforts of the society for  theriogenology, american college of theriogenologists (act), and theriogenology foundation towards common goals in animal reproduction, to reward and inspire excellence, improve the visibility of theriogenology, and to annually recognize the efforts of the founders: particularly honors dr. david e. bartlett, the act’s first president. among his many accomplishments, dr. bartlett was responsible for deriving the terms ‘theriogenology’ and ‘theriogenologists’ and was instrumental in gaining avma recognition for the act in 1971. dr. meyers has promised to submit a review to our journal and we look forward to publishing it after peer review. we express our gratitude to the act scientific information/ abstract committee (drs. misty edmondson [chair], orsolya mailto:petera@purdue.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9973 mailto:petera@purdue.edu comparison of intramuscular vehicles, with or without progesterone, on serum progesterone concentrations in noncycling mares comparison of intramuscular vehicles, with or without progesterone, on serum progesterone concentrations in noncycling mares jamie kaczor, charles scoggin, cassandra cromer, holly hersey, ryan ferris,a etta bradecamp, maria schnobrich rood and riddle equine hospital, lexington, ky asummit equine, newberg, or progesterone (p4) supplementation, daily or weekly, is common in clinical broodmare practice. the objective was to evaluate serum concentrations of progesterone following a single intramuscular injection of 3 vehicles (biorelease, bet labs, lexington, ky; and cotton seed and sesame seed oils, rood and riddle veterinary pharmacy, lexington, ky), with or without p4. anestrus light horse mares (n = 48) were used; they had no luteal tissue, ovarian follicles d 20 mm and serum p4 concentrations < 0.5 ng/ml over a 2 week interval. mares were randomly allocated into 6 treatment groups: 1500 mg p4 in 10 ml biorelease (br + p4; n = 12); 1500 mg p4 in 10 ml sesame seed oil (sso + p4; n = 12); 1500 mg p4 in 15 ml cotton seed oil (cso + p4; n = 11); 10 ml biorelease without p4 (br only; n = 5); 10 ml sesame seed oil without p4 (sso only; n = 4); and 15 ml cotton seed oil without p4 (cso only; n = 4). day 0 was designated as the day on which mares were given p4. mares were monitored by transrectal ultrasonography every 3 days and serum collected on days 1 14, 17, and 20. serum p4 concentrations were determined using the clinical enzyme immunoassay analyzer, tosoh a1a 900. one-way anova was used to compare differences in mean p4 concentrations among treatment groups, with significance set at p < 0.05. serum p4 concentrations were higher (p < 0.003) by day 7 in the br + p4 group compared to all other groups (table). by day 14 no difference (p = 0.127) in serum p4 was detected among treatment groups. serum p4 concentrations were > 1.0 ng/ml in non-cycling mares for at least 7 days after a single intramuscular injection of 1500 mg p4 when delivered in biorelease, sesame seed oil or cotton seed oil. table. mean + sem serum p4 concentrations (ng/ml) in mares given three vehicles, with or without p4. group day 0 day 1 day 4 day 7 day 10 day 14 day 21 br + p4 0.13±0.04a 16.33±1.99a 8.04±0.54a 8.10±1.06a 2.73±0.60a 0.71±0.28a 0.15±0.04a sso + p4 0.10±0.06a 23.73±2.99a,b 7.00±1.09a 2.79±0.52b 1.02±0.22b 0.29±0.07a 0.08±0.03a csso +p4 0.07±0.02a 23.63±2.04b 6.7±0.93a 3.33±0.84b 1.20±0.27b 0.38±0.13a 0.05±0.02a br only 0.06±0.04a 0.04±0.02c 0.04±0.02b 0.06±0.04c 0.02±0.02c 0.06±0.04a 0.06±0.04a sso only 0.00±0.00a 0.00±0.00c 0.00±0.00b 0.03±0.03c 0.20±0.20b,c 0.0±0.0.0a 0.00±0.00a cso only 0.03±0.03a 0.00±0.00c 0.03±0.03b 0.03±0.03c 0.00±0.00c 0.05±0.03a 0.05±0.05a a-cwithin a column, means without a common superscript differed (p < 0.05). keywords: equine, progesterone, blood, progesterone supplementation 411 clinical theriogenology • volume 11, number 3 • september 2019 412clinical theriogenology • volume 11, number 3 • september 2019 omniblank: a review of pyometra in small animal medicine: incidence, pathophysiology, clinical diagnosis, and medical management   a review of pyometra in small animal medicine: incidence, pathophysiology, clinical diagnosis, and medical management grayson b. wallace,a margret l. casalb anorth oatlands animal hospital & reproduction center, leesburg, va; bschool of veterinary medicine, university of pennsylvania, pa abstract pyometra is a common reproductive disorder involving an acute or chronic bacterial infection of the uterus, with accumulation of purulent exudate in the lumen. this disorder is hormonally driven, primarily by uterine exposure to progesterone and estrogen priming during repeated estrous cycles. traditionally, pyometra is thought to occur in conjunction with cystic endometrial hyperplasia as “cystic endometrial hyperplasia-pyometra complex”. cystic endometrial hyperplasia commonly, but not necessarily, precedes development of pyometra. pyometra is more widely recognized as a reproductive condition of bitches, but can also occur in queens. pyometra is classified as being either openor closedcervix, based on the presence or absence of vaginal discharge. diagnosis of pyometra is based on reproductive history, clinical signs, blood work, and imaging. depending on extent of endotoxemia and cervical patency, patient presentation is variable, ranging from mild clinical signs to decompensated shock. regardless, pyometra should be considered a potentially life-threatening medical emergency, independent of apparent stability, since sepsis, peritonitis, and uterine rupture are possible sequelae. in all cases, initial stabilization and institution of appropriate antibiotic therapy are warranted. ovariohysterectomy is the traditional treatment of choice for pyometra. recently, medical management has become a viable alternative to ovariohysterectomy, enabling preservation of future fertility of bitches and queens. the most effective medical protocols involve low-dose prostaglandin treatment in conjunction with antiprogestins or dopamine agonists. although medical therapy can be effective in stable cases, recurrence during future estrous cycles is common. keywords: pyometra, bitch, queen, medical management, cystic endometrial hyperplasia incidence and signalment affecting nearly 25% of intact bitches by 10 years of age, pyometra is a reproductive disorder primarily occurring in mature intact bitches and queens during diestrus.1 pyometra is essentially a disease caused by bacterial infection of a uterus primed with progesterone (p4) and its risk of development increases with age and repetitive estrous cycles. in bitches, the mean age of pyometra diagnosis is reported to be 7.25 years (range, 4 months to 16 years).2 the incidence of pyometra increases with maturity and may be >65% in intact bitches >9 years old.2 incidence increases in bitches >4 years and nulliparous bitches.3 prior hormonal treatment with progestins or estrogens can greatly increase the risk of pyometra development.4-6 even low doses of estradiol benzoate can increase (by a multiple of 6.5) the incidence of pyometra within 4 months after treatment.5 prior exposure to reproductive hormonal therapy and congenital abnormalities of the genital tract, e.g. vaginal strictures, can predispose to development of pyometra. most bitches and queens present with pyometra during diestrus while their uterus is under progesterone dominance. the majority of bitches develop pyometra within 3 months (mean, 5 to 6 weeks) after the onset of their last proestrus.7 however, bitches may present with pyometra at any stage of their estrous cycle. pregnancy may confer a protective benefit, and there are reports of cases with a normal pregnancy in one horn and concurrent pyometra in the contralateral horn.2,8 pseudocyesis (aka pseudopregnancy) is not associated with pyometra.8 in up to 30% of cases of canine pyometra that present during anestrus p4 <1.0 ng/ml), it is believed the actual onset was during p4 dominance. these may be 435 clinical theriogenology • volume 10, number 4 • december 2018   chronic insidious cases in which signs were not recognized until the end of a normal luteal phase.9 lifetime risk of pyometra development is variable (15 to 24%), depending on characteristics of the female population (age, breed, and geographic location).1,10 in recent studies, incidence varies dramatically among breeds, suggesting breed predisposition. 1,11-14 furthermore, the strength of factors that confer protection against development of pyometra is variable depending on breed.11 in a 2001 study of swedish insured bitches, overall risk of developing pyometra was ~24% by 10 years; however, there was considerable variability among breeds (10 to 54%).1 in a similar 2012 study, proportion of bitches affected by pyometra was highest in the following breeds: bernese mountain dog (66%), great dane (62%), leonberger (61%), rottweiler (58%), irish wolfhound (58%), staffordshire bullterrier (54%), bullterrier (52%), newfoundland (50%), collie (44%), and old english sheepdog (42%).12 other at-risk breeds identified include: saint bernard, chow chow, miniature schnauzer, irish terrier, airedale terrier, rough collies, cavalier king charles spaniels, golden retrievers, bullmastiffs, dogue de bordeaux, and english cocker spaniels. 1,13,14 there was a significant effect of breed on mean age of presentation, with some breeds being significantly younger, e.g. dogue de bordeaux (3.3 years) and bullmastiff (5.4 years), whereas others presented significantly older, e.g. yorkshire terriers (9.4 years) and border collies (10.3 years).13 breeds identified at decreased risk for development of pyometra include: drevers, german shepherd dogs, miniature dachshunds, dachshunds, and swedish hounds.1 breed popularity was accounted for in these studies. pyometra is less common in queens, likely because cats, as induced ovulators, have less cumulative uterine exposure to p4. regardless, pyometra is still one of the most common reproductive disorders in intact queens. the overall prevalence of pyometra in the queen has not been as well documented, but the incidence in swedish insured cats was one cat per 588 cat years at risk, with overall prevalence of 2.2% by 13 years of age.15 dow reported clinically diagnosing pyometra in about one cat per three dogs diagnosed.16 as in bitches, incidence in queens increases with maturity, with an average age reported 4 and 7.2 years.15,17 affected queens’ age at presentation can range from 1 to 20 years.2 the majority of queens present for pyometra during diestrus, within 2 to 5 weeks after induced ovulation.17 therefore, use of progestins that simulate diestrus (for long-term suppression of estrus) in queens increases the risk of developing cystic endometrial hyperplasia (ceh) and pyometra.18,19 recently, significant breed predispositions for pyometra development have been reported in exotic and oriental cat breeds, including the siberian cat, ocicat, korat, siamese, ragdoll, maine coon, bengal and sphynx, with the latter breed being at greatest risk.15 as in dogs, differences in incidence among cat breeds suggest a genetic component to development of pyometra. pathophysiology pyometra is a reproductive endocrine disorder typically associated with diestrus or the luteal phase, when the uterus is under the influence of p4. as a bacterial infection of the uterus, pyometra causes intraluminal accumulation of purulent exudate and inflammatory infiltrates and often initiates systemic inflammation and sepsis, which can be life threatening. the complete etiology of pyometra has not yet been fully elucidated. however, pyometra is generally considered an abnormal uterine response to estrogen and p4 affecting the endometrium that enhances bacterial colonization of the uterus.20 estrogen priming of the uterus enhances growth and vascularization of the endometrium and potentiates the stimulatory effect of p4 on the uterus through sensitization of uterine p4 receptors, enhancing their binding.21 physiologic formation of a corpus luteum after every ovulatory cycle leads to relatively prolonged uterine exposure to elevated serum p4 concentrations. this prolonged p4 dominance occurs with every estrous cycle in the bitch and each ovulatory cycle in the queen. p4 increases endometrial proliferation and glandular secretions, decreases myometrial contractions and causes cervical closure.21 additionally, p4 has an inhibitory effect on uterine cellular immunity by decreasing neutrophil chemotaxis 436clinical theriogenology • volume 10, number 4 • december 2018   and phagocytic ability and facilitating bacterial adherence to the endometrium.22,23 effects of p4 on the uterus promote accumulation of glandular secretions within the closed lumen; coupled with an inhibitory effect p4 on leukocytes, this creates an ideal environment for pregnancy or bacterial growth by ascending infection in cases of pyometra. uropathogenic strains of escherichia coli are the most common bacteria isolated in bitches and queens with pyometra.24, 25 pyometra is strongly associated with ceh, the most common uterine disorder in older intact bitches and queens. ceh is characterized by abnormal endometrial gland proliferation and hypersecretion leading to cyst formation, endometrial thickening, and accumulation of viscous intraluminal glandular fluid.7 the cumulative effect of repeated p4 exposure during diestrus leads to development of ceh.26 although ceh is a subclinical non-inflammatory disorder of the aging uterus, not associated with any signs other than infertility,27 it can be associated with accumulation of sterile intraluminal uterine fluid, resulting in development of mucometra (viscous fluid), hydrometra (watery fluid), or hematometra (bloody fluid). due to their distinct clinical and histopathologic presentations, pyometra and ceh are generally considered separate disease entities. historically, ceh was considered a prerequisite for pyometra.26,28 although ceh precedes pyometra-endometritis in the majority of cases, there is evidence that these conditions can develop independently.29 whereas all intact bitches will eventually develop ceh with age, not all will develop pyometra.9 in rare cases, pyometra can develop in the absence of ceh.21 it was recently postulated that pyometra could develop as the result of local uterine irritation.30 subclinical bacterial infections have been postulated as causing excessive endometrial proliferation (aka a trophoblastic reaction, similar to implantation), leading to increased glandular secretions and bacterial proliferation and ultimately pyometra.30-36 this was validated by evidence that ceh can develop from a trophoblastic reaction to any endometrial irritation in a p4-primed uterus; consequently, uterine biopsies should not be collected during diestrus. 37, 38 uterine reaction to bacterial invasion could explain concurrent presence of ceh in pyometra cases. in both theories, p4 clearly has a critical role in development of pyometra, either by initiating ceh through chronic exposure, or as an essential component for induction of a trophoblastic reaction. this may explain development of pyometra in young bitches without pre-existing ceh.9 although the full etiology of pyometra has not been completely elucidated, it is generally regarded as multifactorial. innate immune response and bacterial virulence undoubtedly are important in development of pyometra. high serum p4 concentrations during the luteal phase suppress local cellular immunity in the uterus.9 it was recently reported that bitches could have subclinical endometritis without developing overt pyometra;39 perhaps mild, chronic bacterial contamination of the uterus occurs in younger bitches prior to escalating into clinical pyometra.39 ascension of vaginal bacterial flora through the relaxed cervix to infect a p4-primed uterus presumably results in pyometra. the pathogen predominantly associated with pyometra is escherichia coli (70% of cases), but other vaginal commensal bacteria, e.g. staphylococcus aureus, enterobacter, pseudomonas, klebsiella, proteus, and streptococcus spp., have less commonly been recovered.24, 40 in most cases of pyometra, uropathogenic strains of e. coli are isolated with specific virulence factors enhancing adherence. these uropathogenic virulence factors, in conjunction with the diestral uterine environment, facilitate infection of the urogenital tract and development of pyometra.41,42 concurrent urinary tract infections are common (20 to 70%) in bitches with pyometra.43 uterine infections with e.coli frequently release lipopolysaccharide (lps), a bacterial endotoxin, which can lead to development of serious systemic disease and inflammation. for example, lps produced by some pathogenic strains of e. coli can cause antidiuretic hormone insensitivity in the distal convoluted 437 clinical theriogenology • volume 10, number 4 • december 2018   tubules and collecting ducts, reducing renal concentrating ability. this endotoxin-induced transient glomerular and tubular kidney dysfunction leads to clinical signs of polyuria/polydipsia frequently associated with pyometra in bitches.44 elevated serum endotoxin concentrations in dogs with pyometra have been associated with a higher risk of death.45 a clinically applicable method to measure endotoxin or an indicator of its release, such as prostaglandin f2α (pgf2α) metabolite, would be very valuable in rapid evaluation of pyometra.46 furthermore, in uterine tissues, pyometra significantly uterine up-regulates genes associated with chemokines, cytokines, proteases, inflammatory cell extravasation, antibacterial action, the complement system, and innate immunity.47 endotoxemia associated with pyometra may progress to sepsis, systemic inflammatory response (sir), or disseminated intravascular coagulation. systemic inflammatory response has been reported in up to 50% of bitches with pyometra and has been associated with a poor prognosis and longer hospitalization.46,48 in cases of pyometra-induced sepsis/sir, serum concentrations of inflammatory markers such as c-reactive protein (crp), serum amyloid a (saa) and pgf2α metabolite significantly increased.46,49,50 appropriate surgical treatment of pyometra should abrogate the marked inflammatory response. therefore, monitoring serum concentrations of acute phase proteins, e.g. saa, crp, and haptoglobin, may be useful to determine necessity for surgical intervention, post-operative detection of complications (ongoing infection), and resolution.51 additional complications of pyometra-induced sepsis include bacteremia or thomboemboli to other organs (e.g. brain, heart, or eye), leading to multi-organ dysfunction.47 clinical presentation the majority of patients with pyometra present with a history of recent estrus. in bitches, the classic time frame is 1 to 4 months after estrus,14 whereas queens typically present within 4 weeks.17 in any ill intact female patient, pyometra should be considered as a differential and a thorough reproductive history taken (i.e. parity, last estrus and prior hormonal therapy). affected bitches typically present with lethargy, anorexia, vomiting, polyuria/polydipsia, and/or vaginal discharge (depending on cervical patency). presentation can vary widely from gait abnormalities and hind limb lameness (more common with closed cervix pyometra), abdominal distention alone, nonspecific symptoms, or decompensated septic shock.52 no relation has been demonstrated between nature of clinical signs and severity of uterine lesions.9 classic presenting signs of pyometra in queens (figure 1) include purulent vaginal discharge, anorexia, lethargy, emesis, unkempt appearance, and a palpable uterine enlargement/abdominal distention.17,53 in contrast to the bitch, clinical signs in queens are typically fewer, milder, or more nebulous, with polyuria/polydipsia rarely present.54 cervical patency determines the presence or absence of vaginal discharge and is an important consideration in case management. open-cervix pyometra typically results in milder systemic signs; commonly a hemorrhagic purulent discharge may be the only clinical abnormality. in contrast, closedcervix pyometra cases typically present without overt vaginal discharge, but marked systemic signs of illness are often apparent due to resorption of endotoxins into circulation leading to endotoxemia and/or bacteremia. severe cases may present in septic shock. classic clinical findings of pyometra include vaginal discharge (80%), pyrexia (47%), polyuria/polydipsia (<50%), and emesis.9 unspecific findings include lethargy and anorexia and in more advanced cases, signs of shock may include tachycardia, tachypnea, hypotension and pale or injected mucous membranes. abdominal palpation may reveal large tubular organ/uterine enlargement55,56 (more common in queens) and may elicit pain. when pyometra is suspected, abdominal palpation should be performed very carefully, as excessive pressure can rupture a friable uterus. volume of vaginal discharges 438clinical theriogenology • volume 10, number 4 • december 2018   are related to cervical patency and character is purulent to hemorrhagic to mucoid. fastidious grooming may mask a vaginal discharge. abdominal imaging ultrasonography of the urogenital tract is the most important diagnostic tool in a pyometra workup. typical findings include uteromegaly, thickened uterine walls, proliferative endometrial changes, fluid-distended convoluted tubular horns and anechoic to hyperechoic luminal fluid.57,58 uterine fluid in pyometra is usually homogenous, but can be flocculent and there may be slow swirling.59 ultrasonography is an important tool to differentiate pyometra from other soft tissue causes of uterine distention such as early pregnancy (>24 days gestation), uterine torsion, mucometra, hydrometra and hematometra. furthermore, if uterine torsion or rupture is suspected, ultrasonography can be used to detect abdominal free fluid, which is suggestive of peritonitis and has a poor prognosis. mucometra and hematometra commonly appear ultrasonographically as thin-walled uterine distention with homogenous echodense to hypoechoic luminal fluid, whereas luminal contents are typically anechoic in hydrometra.54 ultrasonography is also critical for detection of ceh, endometrial integrity, and uterine wall thickening (figures 2 and 3). ceh is reliably diagnosed ultrasonographically once it has progressed to a moderate to advanced state, characterized by 1 to 4 mm anechoic cysts within a thickened endometrium.60 furthermore, ultrasonographically apparent (i.e. severe) ceh was associated with a poorer response to medical therapy (antibiotics and pgf2α).60 the ultrasonographic appearance of uterine luminal fluid and endometrium in conjunction with cytology and bloodwork can help differentiate sterile uterine disorders from pyometra, although definitive diagnosis is ultimately based on uterine culture and uterine histopathology after ovariohysterectomy (ovh). abdominal imaging by radiography is not as sensitive or specific as ultrasonography. in dogs with pyometra, the uterus appears as a sausage-like fluid filled tubular organ between the descending colon and the urinary bladder (figure 4). however, conditions causing soft tissue/fluid uterine distention, including early pregnancy (<45 days gestation, prior to fetal calcification), sterile uterine luminal fluid disorders, uterine cancer and uterine torsion can yield similar images. additionally, abdominal radiographs may be non-diagnostic in open-cervix pyometra with minimal uterine distension. vaginal diagnostics if pyometra is suspected, vaginal discharge cytology, culture, and vaginal mucosal cytology should be performed. if the cervix is at least slightly patent, the discharge present in the pericervical cranial vagina may be representative of uterine contents; a sample should be collected with a guarded swab and speculum, subjected to cytology and also subjected to aerobic culture and sensitivity, especially if medical management is planned. in most pyometra cases, a large number of degenerate neutrophils and intracellular or extracellular bacteria are apparent. cytology of the cranial vaginal discharge can be useful to differentiate pyometra from the following sterile uterine disorders producing luminal fluid and potentially associated with ceh: mucometra (low neutrophil count, endometrial red blood cells (rbcs) and proteinaceous debris), hydrometra (few rbcs and white blood cells (wbcs) with abundant mucus), and hematometra (abundant rbcs with minimal wbcs and mucus).56 vaginal mucosal cytology should also be performed to rule out other causes of discharge, e.g. estrus or vaginitis. if purulent vaginal discharge is present but uteromegaly is not apparent on ultrasonography, endoscopic vaginoscopy can be pursued. due to the friable nature of the vaginal tract during diestrus, vaginoscopy should be performed with caution and may require sedation. the purpose of endoscopic vaginoscopy would be to evaluate cervical patency and to evaluate the vaginal tract for alternate sources of vaginal discharge such as a vaginal mass or polyp, foreign body, lower urinary tract infection (uti), or idiopathic vaginitis. 439 clinical theriogenology • volume 10, number 4 • december 2018   laboratory findings complete blood count pyometra classically presents with a marked leukocytosis characterized by neutrophilia with a left shift, toxic neutrophilic changes, and monocytosis. the leukocytosis is typically more severe in closedcervix pyometra cases. percentage of band neutrophils is the most sensitive laboratory parameter for differentiating pyometra from sterile uterine luminal fluid disorders: >19.9% band neutrophils is 94.2% sensitive and 70% specific for pyometra.40 mild to moderate normocytic, normochromic, nonregenerative anemia of chronic disease may be present, though evaluation of anemia can be complicated by concurrent dehydration/hemoconcentration. up to 25% of dogs with pyometra have a normal complete blood count (cbc), especially if pyometra is incidentally detected early in its course by ultrasonography.9 blood serum chemistry patients with pyometra have variable changes in serum chemistry values. the most common abnormalities include: azotemia [increased blood urea nitrogen (bun) and creatinine], increased liver enzymes (alkaline phosphatase and alanine transaminase), hyperbilirubinemia, hypercholesterolemia, hyperglobulinemia, hypoalbuminemia, and hyperproteinemia.40 azotemia may be variably present (12 to 37%) in affected bitches, and may be due to pre-renal dehydration and/or transient, endotoxin-induced renal tubular damage.43 these renal changes are typically transient and reversible once appropriate treatment is instituted. however, bun >60 mg/dl has been associated with acute renal failure and a poor prognosis. some strains of e. coli produce cytotoxic necrotizing factors, which in conjunction with dehydration, can cause reversible hepatocellular damage or hypoxia.23 serum concentrations of inflammatory markers, c-reactive protein (crp) and pgf2α metabolite, are typically elevated in serum of animals with pyometra and can help differentiate pyometra from sterile cystic endometrial hyperplasia disorders.61,62 lactate concentrations are variable and not considered diagnostic.63,64 urinalysis in patients with pyometra, urinalysis may reveal dilute urine (isothenuria to hyposthenuria), bacteriuria (concurrent uti), glucosuria, and/or proteinuria. free-catch urine collection is acceptable since the main purpose of the urinalysis in cases of pyometra is to evaluate specific gravity, renal function, and presence of co-morbidities. ultrasound-guided cystocentesis is not recommended due to the potential puncture of a friable, distended uterus. hormone assays serum p4 concentrations should be evaluated to determine stage of the estrus cycle. in the majority of cases of pyometra, p4 will be significantly elevated, indicative of diestrus. however, in a minority of cases, p4 may be baseline (<1 ng/ml) indicating anestrus. in medical management of pyometra, baseline p4 is necessary to determine the degree of luteolytic drugs needed; serial p4 measurements are done until luteolysis is achieved (p4 <1.0 ng/ml). therapy stabilization due to the insidious onset and nonspecific signs of pyometra, many patients present in advanced stages and may require stabilization prior to initiating diagnostics, surgical or medical therapy. initial examination should include immediate evaluation of metabolic, hemodynamic, hydration, and 440clinical theriogenology • volume 10, number 4 • december 2018   hematologic status. these parameters should be monitored closely throughout diagnostics, treatment, and post-therapy. once pyometra is diagnosed, all patients should be started on broad-spectrum bactericidal antibiotics. prior to initiating therapy, especially surgery, correction of electrolyte, hydration, and acidbase imbalances should be instituted. for patients presenting hypovolemic and dehydrated, appropriate intravenous (iv) fluid therapy with crystalloids should be immediately initiated. azotemic patients with persistent hypotension may require constant rate intravenous infusion of low-dose dopamine or dobutamine, in addition to aggressive fluid resuscitation. therapy for other concurrent life-threatening abnormalities (e.g. renal failure, pulmonary dysfunction, cardiac arrhythmias, and hypoglycemia) should be instituted before surgery. in patients presenting in decompensatory septic/endotoxic shock, more aggressive treatment with oxygen therapy and iv hypertonic saline and colloids should be instituted.65 deteriorating cases with endotoxemia may benefit from corticosteroids (prednisolone sodium succinate 30-60 mg/kg iv). 66 endotoxemia due to systemic lps released from bacteria may reach severe to lethal levels in pyometra cases.67 initiation of antibiotic therapy can worsen endotoxemia by increasing systemic lps concentrations (up to 2000-fold). therefore, it was proposed that endotoxemic bitches be given anti-lps (e.g., polyvalent equine anti-endotoxin hyperimmmune plasma) at a dose of 0.5 mg/kg subcutaneously (sc) in cases of delayed surgery or administered iv after dilution in 100-300 ml lactated ringer’s solution (lrs) if surgery is to be performed immediately. no side effects were reported, but overall efficacy is unknown.67 treatment to promote stabilization should be immediately instituted in all patients presenting with pyometra. in very critical cases, surgery may be required after only a few hours of emergency medical stabilization. however, if the patient is more stable, surgical and anesthetic risks may be reduced and stability improved by initiating medical therapy to lyse the corpora lutea, dilate the cervix, and promote evacuation of purulent uterine fluid.66 antibiotic therapy broad-spectrum antibiotic therapy should be started at presentation in all pyometra cases. since e. coli is by far the most common pathogen isolated in pyometra, empiric antibiotic selection should target gram-negative bacteria. e. coli isolates in pyometra are typically identical to those in the patient’s urine or blood48 and in some cases (10%) may be multidrug resistant.68 acutely, a combination of a fluoroquinolone and an extended spectrum penicillin is preferred. fluoroquinolones have superior penetration in the genitourinary tract.69 other appropriate antibiotic choices for certain cases of pyometra include third-generation cephalosporins and aminoglycosides; however due to risk of renal damage, the latter should not be used in patients with renal dysfunction. 70 long-term antibiotic therapy should be based on results of a bacterial culture and sensitivity profile of purulent exudate and continued for at least 2 weeks post-surgery or 4 to 6 weeks with medical management. surgical versus medical management after diagnosis, stabilization, and initiating antibiotic treatment, the underlying cause of pyometra should be addressed. the severity of clinical presentation and breeding potential of the animal informs the decision to pursue medical or surgical management. historically, surgical ohe was the only treatment for pyometra. ohe is still the treatment of choice in the majority of pyometra cases, especially in nonbreeding animals, because it directly removes the source of the purulent exudate and endotoxin release. however, surgery and anesthesia, especially in critical patients, can be risky, and ohe sterilizes the animal. over the last 20 years, new, successful medical approaches have been proposed, and medical management of pyometra is now considered a viable alternative to surgery in stable cases where there is a desire to preserve fertility. medical management of pyometra may be pursued in the following cases: 441 clinical theriogenology • volume 10, number 4 • december 2018   valuable breeding bitches/queens (< 6 years), animals with increased anesthetic risks (normothermic critical cases or geriatric cases with significant co-morbidities), cases with monetary restrictions, or to improve patient status prior to surgery.55 medical therapy is contraindicated if peritonitis is suspected. if the patient’s condition deteriorates or if clinical improvement is not apparent within 2 days of initiating medical therapy, reconsideration of surgical management is warranted. surgical management ovariohysterectomy is generally the preferred treatment for pyometra. once the patient has been stabilized, antibiotic therapy instituted, and surgical risk minimized, surgery can be performed, under general anesthesia and careful monitoring. the uterus can be grossly enlarged and quite friable, especially with closed-cervix pyometra. therefore, careful handling of the uterus with minimal pressure is essential to minimize risk of uterine rupture and/or purulent material leaking from the uterine tubes into the peritoneal cavity.66 a ventral abdominal approach, with an incision from xyphoid to pubis, is recommended to minimize risk of uterine rupture. the abdominal cavity should be closely examined for the presence of purulent material or evidence of peritonitis, which has a poor prognosis. a traditional three-clamp technique is sufficient for ligation of ovarian pedicles, although large vessels in the broad ligament should be individually ligated. it is essential to avoid lacerating the distended uterine body. the uterus should be carefully isolated from the abdominal cavity with saline-soaked laparotomy sponges before transection just anterior to the cervix. clamping the uterine body is not recommended, as it may rupture. the uterine body should be transfixed prior to ligation. after removal of the uterine stump, the residual uterine lumen should be lavaged with warm isotonic saline and omentalized. after complete removal of the uterus, cervix, and both ovaries, the peritoneal cavity should be copiously lavaged. a sample of uterine fluid should be obtained directly from the lumen of the uterus after removal and submitted for aerobic and anaerobic culture and sensitivity. in ~10% of cases, peritonitis may be present.50 in these cases, a culture of the peritoneal cavity should be obtained prior to lavage and a closed active suction drainage system placed before abdominal closure.71 otherwise, the laparotomy incision should be closed with the same routine triple layer closure used in routine ohe. in the case of uterine stump pyometra, surgical management should be performed as outlined above, to remove the uterine stump, with exploration to identify and remove residual hormone-producing ovarian tissue. all surgically managed pyometra cases should be intensively monitored for 24-48 hours postoperatively. signs of sepsis, hypovolemia, hypotension, azotemia, anemia, hypoglycemia, hyperproteinemia, liver or renal dysfunction, and acid-base derangement should be immediately addressed. supportive treatments and intravenous fluids should be continued for at least 24 hours, or until the patient is eating and drinking on their own. serum biochemistry and complete blood cell counts which may dramatically increase after ohe should be monitored post-operatively until abnormalities resolve (typically within 5 days). peritonitis is the most common complication, followed by septicemia/endotoxemia, anorexia, pyrexia, vomiting, renal insufficiency, and hepatic disease. most complications resolve within 2 weeks.66 the survival rate after surgery is 92%, thus the prognosis is generally very good.43 renal status preand post-operatively is the best prognostic indicator. for patients that have renal dysfunction at initial presentation, follow-up measurement of persistent proteinuria (urine protein creatinine ratio) is recommended. other factors that affect prognosis for pyometra include cervical patency, dehydration status, uterine rupture (septic abdomen/peritonitis) and important co-morbidities. medical management medical management of pyometra is a viable treatment option in cases of pyometra with stable presentation and there is a desire to preserve breeding potential (table). it also can be used to further stabilize cases prior to surgery by converting a closed-cervix pyometra into an open-cervix pyometra. 442clinical theriogenology • volume 10, number 4 • december 2018   medical treatment can be successful, but owners should be warned of possible future recurrence and potential reduction in fertility. case selection for medical therapy is important to optimize outcome. ultimately, multiple factors contribute to efficacy of medical treatment, including cervical patency, degree of systemic involvement, age, follicular cysts, or presence or absence of ceh.55 medical treatment involves pgf2α (natural or synthetic agonist), dopamine agonists, and p4 receptor antagonists (antiprogestins), either alone or in combination. there are many protocols for medical management of pyometra, but the aims are similar. the first goal is to remove effects of p4, either by luteolysis or blocking p4-receptor binding. removing the p4 influence facilitates cervical relaxation, permits myometrial contractions, and improves local uterine immunity, enabling natural expulsion of purulent uterine discharge through an open cervix (enhanced with ecbolics). this goal can be accomplished using prostaglandins (alone or in combination with dopamine agonists) and/or p4-receptor antagonists. uterine contractions that facilitate uterine drainage are induced directly by pgf2α or indirectly by p4 receptor antagonists by removing the effect of p4 on the uterus. the second goal of medical management is prevention of bacterial proliferation through appropriate antibiotic therapy. prostaglandin f2α and agonists success in treatment of pyometra has been reported with the use of pgf2α therapy alone or in combination. repeated microdosing of pgf2α acts directly on corpora lutea to induce luteolysis or block p4 synthesis, decreasing p4 concentrations, resulting in cervical opening and decreased glandular secretions.72 furthermore, prostaglandins directly promote uterine contractions, which with a patent cervix, permit expulsion of uterine discharge. pgf2α treatment is therefore contraindicated in closedcervix pyometra, especially if uterine walls are thin, due to the increased risk of uterine rupture and ensuing peritonitis. therapeutic pgf2α is available in a natural form (e.g., dinoprost tromethamine, lutalyse®, zoetis, parsippany-troy hills, nj) and in a synthetic form (e.g., cloprostenol sodium, estrumate®, merck animal health, madison, nj). synthetic prostaglandins (pgf2α analogs) can be used in lieu of natural pgf2α, and have been associated with prolonged activity, enhanced specificity for uterine smooth muscle, and less side effects (especially emesis). however, pgf2α analogs have reduced ability to stimulate uterine contractions.9 although pgf2α is standard of practice for medical management of open pyometra in the usa, these compounds are off-label in dogs and cats, so it is imperative to obtain written consent before starting treatment. treatment with pgf2α can have important, dose-dependent side effects, especially at higher doses. these side-effects include panting, emesis, hypersalivation, hypothermia, diarrhea, urination, anxiety/shivering, ataxia, and abdominal contractions. additional side effects present only in queens include vocalization, grooming, kneading, mydriasis, and lordosis.53 prostaglandin f2α has a narrow therapeutic index, and in rare cases (especially with higher doses and a closed cervix) may cause shock and death. therefore, frequent administration of low doses through subcutaneous (sc) or intramuscular (im) injections is recommended. adverse effects typically develop within 5-30 minutes and should abate within another 30 minutes. tolerance develops quickly, and reduction of side effects generally occurs with each subsequent dose. hospitalization (at least at the onset of pgf2α therapy) is recommended, due to frequency of side effects and dosing, as well as potential toxicity. reduction of side effects is accomplished with low-dose protocols (administered frequently), fasting 1 to 2 hours before treatment, walking the patient 15-30 minutes after administration, using pgf2α analogs, and pre-treating with atropine 0.025 mg/kg sc 15 minute prior to injection, to minimize parasympathetic effects.55,73 additionally, combination protocols of prostaglandins with antiprogestins or dopamine agonists are reported to minimize side effects and may improve overall therapeutic success.74,75 443 clinical theriogenology • volume 10, number 4 • december 2018   multiple protocols for pgf2α therapy have been proposed. cloprostenol, a synthetic pgf2α agonist, can be dosed at 1-5 µg/kg sc every 24 hours until resolution.2,76 for dinoprost tromethamine, low-dose, high-frequency dosing protocols have been most successful, including substantial reductions in side effects. protocols progressively increasing doses have also been successful and minimize adverse reactions. most authors now recommend natural pgf2α dosing at 100-250 µg/kg/day divided into small frequent doses (three to six times daily), and in some cases, the overall dosage is gradually increased over a few days. in dogs, a successful well-tolerated low-dose protocol for dinoprost tromethamine is as follows: 10 µg/kg sc, given in three to five doses on day 1; 25 µg/kg sc in three to five doses on day 2; and 50 µg/kg sc in three to five doses on days 3 to 10, or until resolution of vaginal discharge.9 in cats, the preferred protocol for dinoprost is similar: 10-15 µg/kg sc every 8 hours on day 1; 25 µg/kg sc every 8 hours on day 2; and 50 µg/kg sc every 8 hours for days 3 to 10, or until complete uterine evacuation.66 anecdotally, intravaginal administration of dinoprost tromethamine has been described in the dog (150 µg/kg inserted vaginally at 0.3 ml/kg every 12 hours until resolution) with reported success.77 pgf2α treatment may need to be continued for 7 to 10 days, or until complete uterine emptying occurs.48 clinical improvement is expected within 48 hours of initiating prostaglandin treatment. copious vaginal discharge is typically observed for >48 hours, then gradually diminishes. a rapid reduction in uterine diameter (determined with ultrasonography) should be evident within 24-48 hours.55 ultrasonographic monitoring of uterine evacuation is important to determine efficacy and duration of therapy. the uterus should be re-assessed frequently enough to assess uterine emptying to determine duration of therapy, and a follow-up uterine ultrasonographic examination is recommended after conclusion of therapy. resolution of pyometra involves complete uterine emptying confirmed ultrasonographically as lack of intraluminal fluid, cessation of vaginal discharge, baseline p4 (<1 ng/ml), and return to normal appetite and activity. pyometra resolution is reported with pgf2α therapy in 75 to 100% of cases, depending on the study and protocol.23 medical prostaglandin treatment in queens is recommended only for open-cervix pyometra, as success with closed-cervix pyometra is poor.78 future fertility after prostaglandin therapy is 75 to 87% in bitches.21 recurrence of pyometra after pgf2α treatment is variable, with 5% recurrence for bitches that became pregnant on their next estrus to as high as 70% recurrence in all treated bitches followed for 27 months.21,79 dopamine agonists dopamine agonists, such as cabergoline or bromocriptine, can be used (off-label) in combination with pgf2α to treat pyometra. prolactin is a major luteotroph that supports long-term maintenance of a functional corpus luteum in the bitch. dopamine agonists have antiprolactin activity and therefore decrease p4 synthesis by the corpus luteum by decreasing lueteotrophic prolactin concentrations as early as 25 days after the luteinizing hormone peak. when used synergistically in conjunction with pgf2α, dopamine agonists can potentiate the rapid p4 decrease which leads to cervical patency typically within 24-48 hours.75 bromocriptine is less popular, as it must be given more frequently (tid) with food and is more likely to cause emesis. the dosing protocol is similar in both bitches and queens. the typical dosage regimen of bromocriptine is 10 to 20 µg/kg po every 8 hours. cabergoline is traditionally dosed at 5 µg/kg po every 24 hours, but the dose can be divided and given every 12 hours to reduce gastrointestinal side effects. both are given until p4 concentrations return to baseline (<1 ng/ml) when used in combination with pgf2α. progesterone receptor antagonists (antiprogestins) antiprogestins such as aglepristone can be successfully used alone or in combination with pgf2α for medical management of pyometra. unfortunately, aglepristone is not currently available in north 444clinical theriogenology • volume 10, number 4 • december 2018   america, but is marketed in europe and australia for veterinary use (ru 534, alizin® virbac animal health, france). as a p4 receptor antagonist, aglepristone is a competitive inhibitor for intrauterine p4 receptors (binds receptors at three times the fixation rate of p4); consequently, it dramatically inhibits the action of p4 on the uterus and decreases intraluminal p4 concentration.80 very few adverse effects have been reported with the use of aglepristone. however, it should not be used in patients with compromised hepatic or renal function. aglepristone can be used relatively safely in both closedand open-cervix pyometras, since it has negligible uterotonic effects. when used to convert a closed-cervix pyometra into an open-cervix case, aglepristone (administration on days 1 and 2) safely perpetuates cervical opening with minimal uterine contractions by 25 hours (range 4 to 48 hours) after the first injection.74 cervical opening is associated clinically with voiding of copious amounts of purulent discharge and significant improvement and increased appetite. in bitches with baseline p4 (<1.0 ng/ml) there is a positive effect of aglepristone alone.80,81 recurrence of pyometra on subsequent estrous cycles after treatment with aglepristone is generally thought to be lower, from no occurrence within 1 year to ~18%.55,80,82 multiple protocols have been developed for the use of aglepristone. to optimize the efficacy of aglepristone the following protocol was developed: 10 mg/kg sc once on days 1, 2, 8, +/days 15, and 30 (after day 8, treatment efficacy is determined weekly prior to additional injections).55 pyometra is considered completely resolved once all vaginal discharge ceases and the uterine lumen has returned to its baseline diameter. resolution rates of 45 to 60% have been reported with this method.74 the use of aglepristone in combination with prostaglandin therapy results in better recovery rates for pyometra, due to their synergistic actions. in the treatment of closed-cervix pyometra, pgf2α therapy should start 24-48 hours after initial aglepristone injection, once the cervix opens; this allows cervical opening by aglepristone prior to pgf2α inducing uterine contractions, thereby optimizing uterine evacuation. combining aglepristone treatment with pgf2α for 5 to 10 days increases reported success rates to 72 to 100% in bitches and 90% in queens.10,80,83 follow up care and monitoring with medical management medical therapy for pyometra should be accompanied by careful monitoring. a dramatic increase in vaginal discharge should be apparent within 24-36 hours and persist for 7-10 days. weekly cbc’s are recommended; the left shift should resolve within the first week, with neutrophilia resolved within 10 to 15 days after starting medical management.66 any biochemistry abnormalities on presentation should also be followed until resolution. serial uterine ultrasonographic examinations are recommended to assess response to treatment, with a visible reduction in luminal diameter in 5 to 7 days. the patient should be re-evaluated 10 to 20 days after recovery to assess the need for additional treatment, including antibiotics.81 with medical management, the rate of pyometra recurrence within 2 years is 20 to 70% in bitches and 14% in queens.53,75,84 after medical management for pyometra, 40 to 70% of bitches whelp normally,2 with fertility generally better in younger animals. since pregnancy is considered protective, it is recommended the bitch or queen be bred on her next cycle. references 1. egenvall a, hagman r, bonnett bn, et al: breed risk of pyometra in insured dogs in sweden. j vet intern med 2001;15:530-538. 2. johnson sd, kustritz mvr, olson pns: disorders of the canine uterus and uterine tubes 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secondary to pyometra in dogs. j am vet med assoc 1999;215:1283-1287. 66. krekler n, hollinshead f: pyometra. in: monnet e, editor. small animal soft tissue surgery. 1stedtion, hoboken: wiley-blackwell; 2013. p. 625-634. 447 clinical theriogenology • volume 10, number 4 • december 2018   67. wessels bc, wells mt: antiendotoxin immunotherapy for canine pyometra endotoxemia. j am anim hosp assoc 1989;25:455-460. 68. hagman r, greko c: antimicrobial resistance in escherichia coli isolated from bitches with pyometra and from urine samples from other dogs. vet rec 2005;157:193-196. 69. brown sa: fluoroquinolones in animal health. j vet pharmacol ther 1996;19:1-14. 70. lee s, cho j, shin n, et al: identification and antimicrobial susceptibility of bacteria from the uterus of bitches with pyometra. korean j vet res 2000;40:763-767. 71. halfacree z: optimising the treatment of the pyometra patient. british small animal veterinary conference; birmingham uk; 2014. p. 124-126. 72. renton jp, boyd js, harvey mj: observations on the treatment and diagnosis of open pyometra in the bitch (canis familiaris). j reprod fertil suppl 1993;47:465-469. 73. hubler m, arnold s, casal m, et al: use of a low dose prostaglandin f2 alpha in bitches. schweiz arch tierheilkd 1991;133:323-329. 74. fieni f: clinical evaluation of the use of aglepristone, with or without cloprostenol, to treat cystic endometrial hyperplasia-pyometra complex in bitches. theriogenology 2006;66:1550-1556. 75. corrada y, arias d, rodriguez r, et al: combination dopamine agonist and prostaglandin agonist treatment of cystic endometrial hyperplasia-pyometra complex in the bitch. theriogenology 2006;66:1557-1559. 76. romagnoli s: canine pyometra: pathogenesis, therapy, and clinical cases. 27th world small animal vet assoc congress; granada, spain, 2002. 77. gabor g, siver l, szenci o: intravaginal prostaglandin f2 alpha for the treatment of metritis and pyometra in the bitch. acta vet hung 1999;47:103-108. 78. barsanti ja: genitourinary infections. in: green ce, editor. infectious diseases of the dog and cat. 3rd edition, st louis: elsevier; 2006. p. 958. 79. memon ma, mickelsen wd: diagnosis and treatment of closed-cervix pyometra in a bitch. j am vet med assoc 1993;203:509-512. 80. gobello c, castex g, klima l, et al: a study of two protocols combining aglepristone and cloprostenol to treat open cervix pyometra in the bitch. theriogenology 2003;60:901-908. 81. threlfall wr: diagnosis and medical management of pyometra. semin vet med surg (small anim) 1995;10:21-29. 82. jurka p, max a, hawrynska k, snochowski m: age-related pregnancy results and further examination of bitches after aglepristone treatment of pyometra. reprod domest anim 2010;45:525-529. 83. nak d, nak y, tuna b: follow-up examinations after medical treatment of pyometra in cats with the progesteroneantagonist aglepristone. j feline med surg 2009;11:499-502. 84. meyers-wallen vn, goldschmidt mh, flickinger gl: prostaglandin f2 alpha treatment of canine pyometra. j am vet med assoc 1986;189:1557-1561. table. medical management protocols for the treatment of pyometra. protocols drug(s) and dosing side effects and indications references low dose pgf2α lutalyse® (low dose) 20-50 mcg/kg sc every 2-4 hours until complete uterine evacuation may require hospitalization due to high dosing frequency. romagnoli 200276 titrating dose pgf2α lutalyse® (titrating dose) 10 mcg/kg sc for 3-5 doses on day 1; 25 mcg/kg sc for 3-5 doses on day 2; 50 mcg/kg sc for 3-5 doses on days 3-10 (or until resolution of vaginal discharge) this dosing regimen minimizes side effects of pgf2α verstegen et al. 20089 high dose pgf2α (>0.2 mg/kg every 24) lutalyse® (high dose) 250 500 mcg/kg sc every 24 hours for 3 days worse pgf2α side effects increased risk of meyers-wallen et al. 198684 448clinical theriogenology • volume 10, number 4 • december 2018   no longer recommended uterine rupture contraindicated in closed-cervix pyometra (risk of peritonitis/rupture) intravaginal infusion of pgf2α lutalyse® (intravaginally) 150 mcg/kg topically (as a vaginal infusion at 0.3 ml/kg) every 12 hours for 3-12 days (until resolution) treatment of opencervix pyometra. intravaginal administration helps minimize systemic side effects of pgf2α gabor et al. 199977 pgf2α in queens* lutalyse® (medium dose) 100 mcg/kg sc every 12-24 hours for 3-5 days or lutalyse® (titrating dose) 10-15 mcg/kg sc every 8 hours on day 1; 25 mcg/kg sc every 8 hours on day 2; 50 mg/kg sc every 8 hours for day 3-10 (or until complete uterine emptying) significant side effects of pgf2α dosing (especially with first doses) decreased side effects of pgf2α with this dosing regimen davidson 199253 krekler and hollinshead 201366 pgf2α analog** cloprostenol (synthetic pgf2α agonist) 1-5 µg/kg sc every 24 hours until resolution treatment may need to be continued for 7-10 days or until complete uterine emptying is observed32,79,34 johnston, kustritz and olson 20012 romagnoli 200276 pgf2α analog + dopamine agonist cloprostenol (pgf2α analog) 1-2 µg/kg sc every 24 hours x 7 days with cabergoline (dopamine agonist) 5 mcg/kg po every 24 hours for up to 14 days or bromocriptine (dopamine agonist) 10-20 µg/kg po every 8 hours for up to 14 days dosing protocol is similar in both bitches and queens dopamine agonist should be given for at least 7 days or until resolution when used in combination with pgf2α bromocriptine is less popular since it must be given every 8 hours with food and is associated with more emesis corrada et al. 2006 75 krekler and hollinshead 201366 antiprogestin aglepristone not currently fieni 200674 449 clinical theriogenology • volume 10, number 4 • december 2018 10 mg/kg sc once for only on days 1, 2, 8, 15*, and 30* *(after dosing on day 8, evaluate treatment efficacy weekly prior to additional doses)* available in us very few adverse effects, but aglepristone is contraindicated with compromised hepatic or renal function negligible uterotonic effects, so can be used with open or closed cervix pyometras. (reported resolution rates of 45% by 28 days to 60% by 90 days) fieni, topie and gogny 201455 antiprogestin + pgf2α analog aglepristone (antiprogestin) 10 mg/kg sc once on days 1, 3, 8, and 15* cloprostenol (pgf2α analog) 1 mcg/kg sc once on days 3 and 8 (or once on days 3, 5, 8, 10, 12, and 15) after dosing on day 15, then weekly doses until complete resolution on u/s 100% resolution rates reported when protocol used in open-cervix pyometra with closed-cervix pyometra, pgf2α therapy is commenced 24-48 hours after initial aglepristone dose (allows cervical opening by aglepristone)74 gobello et al. 200380 *in queens, the early cl is refractory to pgf2α-induced luteolysis, therefore a higher daily dosing of pgf2α may be required for a longer period in early diestrus.66 use of ecbolics is contraindicated in both dogs or cats unless patency of the cervix can be induced or develops naturally. medical prostaglandin treatment in queens is recommended only in the treatment of open-cervix pyometra, as it has had poor success in inducing cervical patency in close-cervix pyometra cases in cats.78 **synthetic pgf2α analogs have been associated with prolonged activity, enhanced specificity for uterine smooth muscle, and reduced observable side effects (especially emesis). however, they have comparatively reduced ecbolic activity therefore are not as effective as natural pgf2α for uterine emptying.9 450clinical theriogenology • volume 10, number 4 • december 2018 figure 1. pyometra in a two-year-old intact female cat. the uterine horns are moderately enlarged and mildly segmented. after the uterus was opened, purulent material containing neutrophils and bacteria was discovered. figure 2. ultrasonographic appearance (longitudinal view, right horn) of early cystic endometrial hyperplasia in a five-year-old intact female cocker spaniel. the uterus is empty with normal wall stratification. cursors indicate uterine thickness. there are few small cysts (arrows) throughout the endometrium up to three mm in diameter. 451 clinical theriogenology • volume 10, number 4 • december 2018 figure 3. ultrasonographic appearance (transverse view, left horn) of a pyometra in a two-year-old intact female pit bull terrier. the uterine horn is markedly distended with echogenic fluid (36.5 mm in diameter). the uterine wall is thickened (7.57 mm) with irregular and heterogeneous endometrium with numerous cystic lesions representing cystic endometrial hyperplasia. figure 4. radiological appearance (ll view) of a pyometra in an eight-year-old intact female pit bull terrier. the abdomen is distended and there is a tortuous, large tubular, well-defined soft tissue opacity present in the caudal abdomen. the structure displaces the descending colon dorsally, the urinary bladder ventrally, and the small intestine cranially. differential diagnoses for this radiographic appearance include hydro-, muco-, hemo-, pyometra, and pregnancy before fetal ossification. (editor’s note: online edition of the manuscript has color photographs) 452clinical theriogenology • volume 10, number 4 • december 2018 000_frontal ms_1 forematter for december 2018 ms_2 letter1 for december 2018 ms_2 letter2 for december 2018 ms_2 letter3 for december 2018 ms_3 table of contents ms_4 therio news for december 2018_bw 2019 therio preview therio recap with pics steiner-new officers 2018 therio sponsors 2018 exhibitors-sft board noms 2018 auction donations 2019 bartlett nomination act call for noms call for abstracts 2019 for ctj the society for theriogenology and american college of theriogenologists issue a call for research and case abstracts to be presented at the annual therio conference july 24-27, 2019 in savannah, georgia. 1. competitive category: 2. non‐competitive category: 3. case abstract category: 4. poster category: general: guidelines: what to send and how to send it abstracts not adhering to these guidelines will not be considered for presentation. deadline: abstracts must be received no later than february 15, 2019 for consideration. call for 2019 student case presentations.pdf call for student case presentations-2019 annual therio conference student case presentations call for student case presentations also available at www.therio.org guidelines for application submission society for theriogenology student case presentation competition annual therio conference, savannah, georgia, july 24-27, 2019 ms_5 editorial 001a_ms_6 roberts color pictures 002_ms_7 root kustritz 003_ms_8 ferrer 004_ms_9 wiley 005_ms_10 wallace 006_ms_11 peterson 007_ms_12 nobre 008_ms_13 sidelinger 009_ms_14 mirando 2017 news from the society for theriogenology and the american college of theriogenologists from the president of the society for theriogenology fall has arrived in the rocky mountains. the golden aspen leaves that surround utah valley are a sign of the changing weather. the majestic towering peaks surrounding springville, utah are covered in snow. the small ruminant and cervid breeding seasons are winding down, and i find my schedule finally subsiding. i hope that the changing season finds each of you well and that 2017 has been a year filled with success and much happiness. the 2017 conference, held in fort collins, co, was a tremendous success. the scientific program was outstanding! the session chairs did an excellent job of selecting speakers whose expertise and lectures were appreciated by all who attended. an equine embryo transfer symposium and wet lab were held, with help from the colorado state university equine reproduction center, and provided participants with hands-on experience with embryo collection techniques, evaluation of embryos, cooled-transport of embryos, non-surgical transfer of embryos and vitrification. a student wet-lab was also held, which provided students with a variety of experiences in advanced equine reproductive technologies. over the past several years, the board of directors of the sft have worked tirelessly to improve the society and to promote theriogenology. we have placed significant attention on recruiting and retaining student members. i am happy to report that student involvement in the annual conference has increased exponentially since the initiation of these efforts. the merck student quiz bowl, student chapter of the year contest and t-shirt design contest all encourage student involvement and have grown each year in participation and popularity. i strongly believe that these student members will be the future of the society, and that investment in them will yield a tangible and successful legacy and will ensure perpetual continuity for the sft. the theriogenology foundation continues to do great things in promotion of the sft. the working dog project is a collaborative effort of the broad institute of mit and harvard and the theriogenology foundation. its goal is to use genetics to guide the selection of dogs for specific training and career paths. this project will apply cutting-edge genomics to investigate how genetic variation influences two key working dog behaviors: scenting and retrieving. this initiative will promote and enable, large-scale, collaborative research into the genetics of dog behavior, with the goal of providing the working dog community with the new tools they need to accelerate the breeding and training of successful working dogs. it is an exciting time for the society for theriogenology. the future is bright. i am humbled to gaze at the statue of nandi each morning as i enter the clinic. i am honored to have this opportunity to serve you. onward and upward we must continue! please feel free to contact me with ideas and suggestions on how we can better serve you. i can be reached at isaac@docbott.com or at (801)489-9676. dr. isaac bott president, society for theriogenology 517 clinical theriogenology • volume 9 number 4 • december 2017 from the president of the american college of theriogenologists greetings to my fellow act diplomates! i am so very grateful to be a member of the act and absolutely thrilled to serve as president! i am a canadian, born on a dairy farm northwest of edmonton, alberta, canada. i graduated from the u. of saskatchewan in 1982, spent 2 years in private practice, then 1 year in a clinical residency at my alma mater. in 1985, i moved to the u. of wisconsin-madison, where i studied under the tutelage of dr. o.j. ginther. most of my thesis research involved using ultrasonography to examine the heifer reproductive tract. what an incredible opportunity! after completion of my phd (1990), i spent 22 years as a research scientist at the agriculture and agri-food canada lethbridge research centre, conducting research in bovine reproduction (cows and bulls). in 2012, i joined the university of calgary, faculty of veterinary medicine, my first full-time appointment in academia, where i am privileged to be able to spend most of my time helping others to build their careers, improve their lives and indeed, the world around them. my ‘dream job’ was to serve as co-editor-in-chief of theriogenology for more than a decade and i continue to spend considerable time editing papers and giving workshops and presentations on scientific writing. in the near future, i plan be more involved with our clinical theriogenology journal. the 2017 meeting in ft. collins in early august was a great success, with just under 500 participants. there were 80 abstracts, including 35 competitive abstracts, 24 non-competitive abstracts, 7 case reports and 14 posters submitted. in addition, the act qualifying examination was delivered electronically, with 22 candidates sitting the examination. of those, 12 (54.5%) passed, including 11 of 18 standard route candidates and 1 of 4 alternate route candidates. the examination is our way of adding to our ranks and on behalf of the membership, i extend my sincere appreciation to the exam committee for their efforts to prepare and deliver the examination. the theriogenology foundation (tf) had a very successful fundraiser at ft. collins (>$76k raised). the tf is supporting the working dog project (in collaboration with the broad institute of mit and harvard). the objective is to identify genetic loci associated with key behavioral traits of military and service dogs. this project has incredible potential to really improve lives! it is highly ambitious and also very expensive. in addition, the tf also provides financial support for theriogenology residencies, travel and research. please visit their website (www.theriofoundation.org/) for more details, including purchasing sympathy cards. also, please encourage your clients to support this most worthy cause. the executive board and all of the act committees have been busy. the training and credentialing committee is refining the general information guide (gig) and developing the maintenance of certification (moc), as this will begin in 2020. we are mandated to implement this process and there is no doubt that it will take considerable time, effort and money to bring this to fruition. a sincere thank you to those who recently completed terms in various aspects of the act. our organization depends on the dedicated efforts of many volunteers. every year, we need persons to step forward to replace those who have completed their terms. a call for nominations will come out in 2018. ideally, we should have multiple candidates for each position, in order to maintain a diversity of experience, expertise and opinion. please identify individuals whom you think would be willing and able participants, and encourage them to go forward. the act currently has solid finances, but we also have some large expenses on the horizon, especially as we prepare for maintenance of certification. please promote the financial viability of the 518clinical theriogenology • volume 9 number 4 • december 2017 act by paying your membership dues. prompt payment is encouraged, either by mail or on the website (www.theriogenology.org). save the date for next year's conference august 1-4, 2018 to be held in milwaukee, wisconsin! dr. peter hansen will deliver a talk on epigenetics. there is a strong food-animal focus to this meeting, including the expected launch of a revised bull breeding soundness evaluation manual (the current version is more than 3 decades old)! in addition, there will be many interesting presentations for those who work with horses, dogs, cats and other species. the act is truly an amazing group! our strength is in our diversity. furthermore, our success and our future depend on the efforts of many diplomates to take an active role in our organization, at all levels, formally and informally, including making our clients, colleagues and administrators aware of what we do and why it is important. we all serve as ambassadors for our organization and our discipline; that is a huge responsibility, that we need to take very seriously. if you have a need, an idea, or a complaint, please do not hesitate to contact me and communicate your message. i can be reached at 403-210-8660 or by email (jpkastel@ucalgary.ca). i look forward to hearing from you and i hope to see many of you in milwaukee next fall. sincerely, john kastelic act president, 2017-8 519 clinical theriogenology • volume 9 number 4 • december 2017 future annual theriogenology conference dates sponsored by the society for theriogenology and the american college of theriogenologists the conference planning committee is developing outstanding programs! make plans to join us at the following locations: 2018 august 1-4 milwaukee, wisconsin 2019 july 24-27 savannah, georgia 2020 july 20-25 pittsburgh, pennsylvania 520clinical theriogenology • volume 9 number 4 • december 2017 2018 therio conference and bovine symposium preview the 2018 therio conference will be held at the beautiful hilton milwaukee city center august 1-4. milwaukee offers natural beauty bordering lake michigan and offers plenty in exceptional outdoor adventures. the 2018 therio conference will focus on the production animal program, along with small animal and equine tracks. the conference will begin wednesday with a limited number of veterinarians being given the opportunity to participate in the bovine symposium. the opening session abstracts will be presented wednesday evening followed by the opening session abstract reception. thursday morning begins at 7:30 am with the presentations of the bartlett award and address and the theriogenologist of the year award and address. the dr. jerry rains memorial abstract competition and student case presentations will follow along with the society for theriogenology and american college of theriogenologists business meetings. the opening reception in the exhibit hall will be thursday evening with the opportunity to visit with our vendors, relax with a cocktail, and enjoy light hors d’oeuvres. the theriogenology foundation silent auction will open for browsing and bidding during the reception. make plans to join us for an thrilling thursday evening fun night event off-site at an exclusive venue to be announced very soon. the annual merck student quiz bowl will also be held at the fun night event this year. the plenary session will begin friday’s events and will feature dr. peter hansen speaking on epigenetics of cow's reproduction and dr. mark mirando’s presentation on how the usda-nifa funding and program promote bovine reproduction, roadmap for future directions, and how we can be successful in obtaining grants from usda-nifa. continuing education sessions will begin following the opening session and tracks in small animal, production animal and equine will be offered. the unveiling of the revised bull breeding soundness evaluation manual is also expected to launch during the meeting. friday evening will feature the annual awards dinner reception and awards dinner, followed by the theriogenology foundation’s live auction. saturday will begin with a presentation by current act diplomates for those interested in learning more about becoming a certified veterinarian in theriogenology. a full day of ce will also be offered saturday with tracks in small animal, production animal and equine. as soon as the educational tracks wrap up, we will head to miller park for the group outing to watch the milwaukee brewers take on the colorado rockies. group rates are available at the conference hotel until july 3, 2018 or the room block is sold out. reservations can be made through the link on the conference website or by contacting the hotel and asking for the society for theriogenology group rate. we hope that you will make plans to join us in the beautiful city of milwaukee. more information will follow in the near future regarding a full agenda, symposium details, and conference registration. 521 clinical theriogenology • volume 9 number 4 • december 2017 2017 therio conference recap beautiful fort collins, colorado provided the scenery for the 2017 annual therio conference. a total of 497 attendees gathered for the conference and symposium august 2-5 at the hilton fort collins. the week began with the much anticipated act exam and concluded with a splash down the poudre river at the therio group white water rafting adventure. the equine embryo transfer and embryo vitrification educational course for veterinarians kicked off the meeting wednesday. the daylong course led by drs. pat mccue and ryan ferris provided half a day of lecture and half a day of lab. the event was at full capacity and allowed participants the opportunity for a practical laboratory experience from some of the world leaders in equine embryo transfer. the board meetings for act, sft, and tf were held throughout the day. wednesday evening was packed with fun-filled activities including: the opening session abstracts, opening session abstract reception, a craft beer tasting to benefit the foundation, and the student quiz bowl, sponsored by merck animal health and hosted by the infamous ron mcdaniel. first place in the quiz bowl went to oklahoma state university with lincoln memorial university following in second place. thursday morning began by honoring dr. pat mccue with the theriogenologist of the year award, sponsored by merck animal health, and the david e. bartlett award honoring dr. cliff shipley. eight presenters competed during the dr. jerry rains memorial abstract competition, sponsored by merck animal health. the sft and act business meetings were held after the abstract competition prior to the student/ mentor reception and student case presentations. the act business meeting recognized the 12 candidates that successfully passed this year’s act exam. veterinary students were given the opportunity to attend a student equine wet lab at colorado state university’s equine reproduction laboratory. students were given a hands-on opportunity led by drs. ferris and mccue to learn …...continued on the following page. first place quiz bowl winners: oklahoma state university pictured (left to right): jo eike, michelle shaw, and nikki collins second place quiz bowl winners: lincoln memorial university pictured (left to right): julia antczak, jaclyn adkins, and sarah grigoleit 522clinical theriogenology • volume 9 number 4 • december 2017 about common reproductive procedures performed in equine clinical practice. the fun night event at the fort collins museum of discovery, sponsored by merck animal health and zoetis, was held thursday evening. laughter was provided by dr. kevin fitzgerald and an interactive tour of the museum closed out the evening’s events. plenary speakers, dr. cheryl asa, sponsored by the act, and dr. kevin fitzgerald started friday’s sessions with “nonsurgical methods for reproductive management of captive and freeranging wildlife populations” and “denver zoo, mongolia project: effects of progress upon endangered species”. the act educator’s forum, sponsored by the theriogenology foundation, focused on theriogenology curriculum for veterinary students. small animal, production animal, equine, and large animal abstracts were held during the morning sessions followed by small animal, production animal, and equine tracks in the afternoon. the awards dinner reception was held in the atrium of the hotel followed by the awards dinner, sponsored by merck animal health and aurora pharmaceutical, in the ballroom. prior to dinner, dr. anita migday, president of the theriogenology foundation, welcomed the audience. dr. elinor karlsson with the broad institute of mit and harvard introduced “the working dog project” with a captivating video. dr. jimmy alexander recognized the student case presentation winners: chun kuen mak, national taiwan university (first place); kaitlyn mccombs, auburn university (second place); brittany shumack, auburn university (third place); jenna ward, texas dr. patrick mccue with his wife diane dr. clifford shipley with his wife victoria congratulations to the 2017 act diplomates! pictured left to right (front): dr. morgan agnewatglen, pa; dr. kelli beavers-baton rouge, la; dr. jenna dockweiler-wheat ridge, co; dr. jamie stewart-urbana, il; dr. stephanie walbornn-bryan, tx left to right (back): dr. yehonatan berkowicdavis, ca; dr. jenny katrine boye-davis, ca; dr. jasmin hyatt-matamata, nz; dr. b. matt dredge-rexburg, id; dr. gabriel davolli-baton rouge, la not pictured: dr. dinesh dadarwal-saskatoon,sk 523 clinical theriogenology • volume 9 number 4 • december 2017 a&m university (fourth place); sarah jacobs, university of california-davis (fifth place); and christina thompson, university of california-davis (sixth place). winners of the tee shirt design contest, sponsored by bovine services, were auburn university (first place) and washington state university (second place). the award for student chapter of the year resulted in university of california-davis placing first, with auburn university in second place. dr. pete sheerin, president of sft, presented the student chapter of university of california-davis with the scoty award. dr. carlos pinto, abstract committee chair for 2018, presented the dr. jerry rains memorial abstract competition awards, sponsored by merck animal health. this year’s winners were: dr. tessa fiamengo (first place), dr. jamie stewart (second place), brian blawut(third place) and dr. raphael malbrue (fourth place). dr. mike thompson presented the dr. john steiner award for practitioner excellence to dr. anita migday. unanimously approved by the executive board, dr. migday epitomizes the objectives of the society in her leadership and promotion of theriogenology. ron mcdaniel and dr. carol mcleod led the live auction which generated a grand total of $60,000.00 for the foundation. the evening concluded by drawing the winning raffle ticket for the 2018 conference package, which includes free conference registration, 3 nights lodging at the hilton city center milwaukee, an awards dinner ticket and a travel stipend. the winner was dr. andrew lovelady. the winner of the first annual 50/50 raffle, which received half of the total ticket sales, was dr. gary warner. the conference wrapped up saturday with a roundtable discussion focusing on “careers in theriogenology” led by drs. pete sheerin, jill colloton, marty greer, wendy vaala, and richard hopper and “so you want to become a diplomate?” hosted by several current act members. the equine track, sponsored by rood and riddle equine hospital and veterinary pharmacy, small animal track, sponsored by idexx, and the production animal track, sponsored by lane manufacturing inc., continued throughout the day with informative ce sessions. while some headed home after saturday’s sessions, others stayed an extra day to enjoy the therio group rafting trip sunday to venture down the cache le poudre. the 2017 annual therio conference in fort collins held fun for all and included great networking, sight-seeing, outdoor adventures and breathtaking scenery. make plans to join us in the beautiful city of milwaukee, wisconsin, august 1-4, 2018. dr. john steiner award for excellence in practice dr. anita migday abstract winners (left to right): bryan blawut (third place), dr. jamie stewart (second place), and dr. tessa fiamengo (first place) not pictured: dr. raphael malbrue (fourth place) student case presentation winners (left to right): christina thompson (sixth place), sarah jacobs (fifth place), jenna ward (fourth place), brittany shumack (third place), kaitlyn mccombs (second place), and chun kuen mak (first place) 524clinical theriogenology • volume 9 number 4 • december 2017 dr. john steiner award for practitioner excellence new sft/act of�icers installed for 2017-2018 new sft officers were installed thursday, august 3 at the sft business meeting. officers installed dr. isaac bott, springville, utah, president; dr. robyn wilborn, auburn university, alabama, president elect; dr. colin palmer, saskatoon, saskatchewan, vice president; dr. peter sheerin, new freedom, pennsylvania, immediate past president; and dr. jill colloton, edgar, wisconsin, secretary/treasurer. new elected members of the board of directors are drs. kristina baltutis, burlington, north carolina; misty edmondson, auburn, alabama; and dan tracy, fort collins, colorado. new act officers were installed thursday, august 3 at the act business meeting. officers installed were dr. john kastelic, calgary, alberta, president; dr. reed holyoak, stillwater, oklahoma, president elect; dr. richard hopper, mississippi state, mississippi, vice president; dr. ram kasimanickam, pullman, washington, immediate past president; dr. jack d. smith, mississippi state, mississippi, secretary; and dr. leonardo brito, madison, wisconsin, treasurer. dr. herris maxwell, auburn, alabama, was elected to the board of directors. dr. peter sheerin, sft immediate past president (left) is presented the nandi by incoming sft president dr. isaac bott. new act president dr. john kastelic thanks immediate past president dr. ram kasimanickam for his service. dr. anita migday dr. mike thompson presented the dr. john steiner award for practitioner excellence to dr. anita migday during the 2017 therio conference in fort collins, colorado. unanimously approved by the executive board, dr. migday epitomizes the objectives of the society with her leadership and promotion of theriogenology and her dedication to education. as president of the theriogenology foundation since 2012, she has worked tirelessly in support and promotion of the discipline and has developed the foundation into an organization that furthers the missions of all three theriogenology organizations. the award is intended to recognize veterinarians who epitomize the objectives of the society which are to establish and promote standards of excellence, to disseminate emerging information to veterinarians and students, to serve as a global resource for scientific exchange, to foster client education and awareness, and to encourage a network of collegial exchange. dr. migday earned both bachelor and master of science degrees from the college of agriculture in 1978, and then graduated from the ohio state university college of veterinary medicine in 1980. she purchased slade veterinary hospital in 1982 from dr. edward slade, who established the animal hospital in 1946. dr. migday’s efforts have resulted in a modern facility and progressive practice, which have been recognized for high standards of excellence through accreditation by the american animal hospital association since 1985. dr. migday also spent six years in local broadcast (wvbf 105.7 fmboston, wkox 1200 amframingham, and wcvb ch. 5boston) giving tips as a pet health expert, and hosted the national pbs series, “cats & dogs” in the mid1980s. the show offered a video encyclopedia of pet care, beginning in the examination room and continuing across the nation in exploration of the human/companion animal bond. in 1989, dr. migday was appointed by governor michael dukakis to the board of registration in veterinary medicine for the commonwealth of massachusetts and sat as chairman from 1993 to 1994. she and her husband raise, train, and exhibit bullmastiff dogs and have consistently produced breed champions and obedience titlists since 1992. in her spare time, you may find dr. migday at the dojo, on the tennis court, or on a yoga mat. 525 clinical theriogenology • volume 9 number 4 • december 2017 thank you to all of our 2017 therio conference sponsors and exhibitors 2017 conference sponsors pla�num plus sponsor merck animal health pla�num sponsors lane manufacturing, inc. rood and riddle equine hospital and veterinary pharmacy gold sponsors aurora pharmaceu�cal sonosite, a fujifilm company idexx laboratories silver sponsors american college of theriogenologists professional embryo transfer supply, inc. theriogenology founda�on zoe�s bronze sponsors biomerieux bovine services, llc completevites, llc mars veterinary michelson found animals founda�on mul�min usa, inc. parnell tosoh bioscience, inc. 2017 conference exhibitors animal reproduc�on systems, inc. aurora pharmaceu�cal bcf technology biomerieux breeder's choice, inc. chemometec a/s chris�an veterinary mission e.i. medical imaging fer�lity technology resources, inc. hagyard pharmacy hamilton biovet llc idexx imv technologies usa jorgensen laboratories karl storz veterinary endoscopy of america lane manufacturing, inc. merck animal health minitube usa mofa global mul�min usa, inc. nestle purina pla�num performance professional embryo transfer supply, inc. rood and riddle equine hospital and veterinary pharmacy sonosite, a fujifilm company spectrum labs success concepts llc tosoh bioscience, inc. universal imaging, inc. viasensor veterinary perinatal special�es vetlab supply vetoquinol usa zoe�s do you know someone who would make a great director of the sft board? in accordance with policy, the society of theriogenology requests your suggestions for the board of directors. names received from the general membership will be forwarded to the nominating committee for consideration. directors serve a term of three years, beginning at the annual therio conference. if you would like to suggest an active society member for service as a director on the board, please submit the following information to the executive of�ice no later than march 21, 2018. please ensure the individual’s willingness to serve on the board (if elected) prior to submission. please direct any questions to dr. peter sheerin, chair of the nominating committee at petesheerin@nandivet.com. when suggestions are submitted we will need the following contact information on both the person submitting the suggestion and also the suggested individual: name, address, phone, email, and practice type. mail, email, or fax information to: charles f. franz, dvm executive director society for theriogenology 761 tiger oak drive pike road, al 36064 fax: 334-270-3399 email: charles@franzmgt.com you may submit nominations online at http://therio.org/ boardnominationform you must be logged in to the sft website to access the board nomination form located under board and of�icers. 526clinical theriogenology • volume 9 number 4 • december 2017 thank you to all of the individuals, companies, and organizations that supported the theriogenology foundation by donating to the 2017 live and silent auctions individuals dr. leaa arnold dr. jane barber dr. gail colbern nita franz dr. o.j. ginther alan kalter dr. ram kasimanickam john lichty dr. victor medina dr. anita migday dr. jay randall dr. joann randall dr. tom riddle dr. peter ryan dr. will schultz dr. phil senger dr. mike thompson dr. gary warner dr. dwight wolfe a huge thank you to all the following individuals for all their efforts in planning the 2017 therio conference. annual conference program chair dr. isaac bo� equine track program chairs drs. ryan ferris and patrick mccue small animal track program chairs drs. milan hess and kara kolster produc�on animal track program chair dr. ed gilsleider educator’s forum program chair dr. hilari french live auc�on chair dr. carol mcleod silent auc�on chair dr. joann randall equine symposium and student wet lab drs. ryan ferris and patrick mccue 2017 funds raised cra� beer tas�ng $5,360.00 2018 raffle tickets $4,300.00 50/50 raffle tickets $1,375.50 live auc�on $60,000.00 silent auc�on $5,097.00 total $76, 132.50 companies and organizations auburn university student chapter auburn university theriogenology department hagyard equine medical ins�tute lane manufacturing inc. mofa global ohio state university theriogenology department purdue college of veterinary medicine rood & riddle equine hospital 527 clinical theriogenology • volume 9 number 4 • december 2017 theriogenology foundation 2017 in review research grants the theriogenology founda�on offers research grants up to $5,000 for proposals that focus on all aspects of theriogenology including (infer�lity, endocrinology, neonatology, obstetrics, etc.). in excep�onal circumstances, grants up to $10,000 may be considered. ac�ve diplomates, honorary members of the american college of theriogenologists, veterinarians who are society for theriogenology members in private prac�ce, industry or on faculty, or residents enrolled in act approved residency programs (including the alternate route) and have a minimum of one year remaining in their training program are eligible to apply for these grants. �� one bovine research grant was awarded in 2017: dr. jennifer koziol, purdue university “descrip�on of the prepu�al microbiota of bulls using 16s r-dna profiling” �� one small animal research grant was awarded in 2017: dr. meghann haase-berglund, red dog veterinary relief services, llc “histologic evalua�on of parovarian nodules in the cat” �� one equine research grant was awarded in 2017: dr. natalie fraser, university of queensland “evalua�on of cytokine and chemokine profile of equine seminal plasma” the working dog project the goal of the working dog project is to iden�fy gene�c explana�ons for why some dogs succeed in working dog programs while others do not, and to help be�er target individual dogs to different programs, such as assistance versus law enforcement work. this new project is based at the broad ins�tute of mit and harvard, in cambridge, massachuse�s, and is generously funded by the theriogenology founda�on and akc reunite. the working dog project has received one grant in the amount of $200,000. act student outreach the act seeks to promote awareness of the college and to encourage veterinary students to aspire to diplomate status a�er gradua�on. the purpose of this fund is to facilitate travel and related expenses for non-local act diplomates invited by student groups to visit and teach at veterinary schools. act/tf hopes that these visits will s�mulate student enthusiasm and interest in the college by broadening students’ awareness of the many opportuni�es open to our diplomates. this opportunity is open to all student groups. it is not required that the school has an ac�ve student chapter of the sft. �� one grant in 2017 has been awarded to: purdue university to host dr. ahmed tibary to speak on south american camelid produc�on travel grants in 2017 the tf also awarded $ 15,200 in student travel grants to assist veterinary students who were travelling to a�end the therio conference in fort collins, co. these students were presen�ng abstracts, posters, student case presenta�ons or taking part as a quiz bowl team member at the conference. $550.00 was awarded in a resident/intern travel grant this year and $750 was awarded in new faculty travel grants. akc, akc-chf, tf residency travel grants all of the residencies are made possible by generous grants from the american kennel club and the akc canine health founda�on. each resident was awarded $750 towards their travel to the annual therio conference. student externship grants student externship grants are grants intended to support experien�al educa�onal advancement of students interested in theriogenology. so far in 2017, $2,900 in externship grants have been awarded. student chapter grants the tf receives and considers requests for funding of student chapter grants intended to provide educa�onal advancement of students interested in theriogenology. furthermore, primary considera�on is given to funding requests which provide an educa�onal experience in theriogenology that is beyond the scope of the basic curriculum. the tf may consider all or par�al funding of a request based on the annual budget and the merit of the educa�onal experience to students. funding to cover a speaker’s travel expense is also available. �� one student chapter grant in the amount of $700 has been awarded in 2017: university of california-davis zemjanis outreach fund this grant is intended to aid veterinarians traveling from developing countries to the united states for educa�onal purposes or for sending a trainer to a developing country to conduct training onsite. one request has been awarded for 2017 in the amount of $500. 2017 tf grants awarded as of 11/1/17 student travel 26 grants $15,200 resident/intern travel 1 grant $550 new faculty travel 2 grants $750 act student outreach 1 grant $1,000 student externship 4 grants $2,900 student chapter 1 grant $700 research-bovine 1 grant $5,000 small animal 1 grant $2,057 equine 1 grant $5,000 akc, akc-chf, tf travel 4 grants $3,000 zemjanis outreach 1 grant $500 educator’s forum 1 grant $2,500 working dog project 1 grant $200,000 528clinical theriogenology • volume 9 number 4 • december 2017 nominations are now being sought for the 201 david e. bartlett award for lifetime achievement in theriogenology, presented annually to a distinguished individual who has made important contributions to the field of theriogenology. the award intended to cement the efforts of the society and college toward common goals in animal reproduction, to reward and inspire excellence, improve the visibility of theriogenology, and to annually recognize the efforts of the founders particularly honors dr. david e. bartlett, first president of the college. among his many accomplishments, dr. bartlett was responsible for deriving the terms “theriogenology” and “theriogenologists,” and was instrumental in gaining hard-won avma recognition for the american college of theriogenologists in 1971. the 201 david e. bartlett award for lifetime achievement in theriogenology will be presented during the annual therio conference in fort collins, colorado. the recipient will be notified in advance, allowing family and friends to be on hand. the honoree will address those in attendance and will have the option of submitting a contribution to the conference proceedings. the honoree will receive $1,000 cash, an engraved statue of nandi, and complimentary registration, hotel accommodations and transportation to the 201 conference. please submit the accompanying preliminary entry form and nominate an individual to add to the list of preeminent recipients. entry forms must be received no later than january 4, 201 . nominations may also be made online at http://www.therio.org/bartlettawardform. you must be signed in as a sft member to access the form. it is located under the 201 therio conference and also under the bartlett award recipient list on the sft website. ���8���� � �� ���������� ���������� 2018 dr. david e. bartlett award for lifetime achievement in theriogenology preliminary entry form sponsored by the theriogenology foundation in cooperation with the society for theriogenology & american college of theriogenologists purpose: to reward a distinguished theriogenologist who has made important contributions to the field of theriogenology. nominee: full address: current employment and position: home phone: business phone: fax: email: provide a short description of nominee’s qualifications for this award. use a separate sheet of paper containing a maximum 350 word narrative bio, highlighting nominee’s key accomplishments. nominated by: full address: home phone: business phone: fax: email: nominations must be received by january 4, 201 to: theriogenology foundation, bartlett award 761 tiger oak drive pike road, al 36064-3063 529 clinical theriogenology • volume 9 number 4 • december 2017 support your foundation we hope that you will visit our website (www. theriofoundation.org) which provides information on all aspects of the theriogenology foundation including grant opportunities, fundraising activities and updated success stories of the work being done through the theriogenology foundation. in addition to the giving options we currently offer, we now have a planned giving option available through the website for those of you who would like to be creative in your support of the foundation. there are several options that you can choose from such as adding the tf into your will or naming tf as a beneficiary in your retirement fund. you may consider annual giving or a gift to honor someone who is no longer with us. please visit the tf website and look under “giving” to find out more about these opportunities. this is the time of year when funds are raised for tf as members of sft and act are paying their member dues. please do not forget to make a donation when you renew your dues. you may pay online, by phone (334.395.4666) or by check. receiving these donations is important to the future of tf and they help us support educational and research projects throughout the year. your clients may also contribute to tf through the website. donations may be made in honor of you, their veterinarian, or they may wish to contribute their stud fees rather than submit them to the owner, and these donations would be tax deductible. the tf welcomes all donations as we work toward funding the future of theriogenology. �� ��������������� ����� to board the nominating committee of the act and the executive board are calling for nominations for the following positions: with the position of vice president, moving to president elect, president and then immediate past president. active diplomates may be considered for any of these positions. travel is required to board meetings in january and august. travel costs for the january board meeting are reimbursed by the college. please forward nominations to dr. no later than march 1, 201 biographical sketches and information along with ballots will be included in the spring act newsletter. the following information should be included with submissions for nominations: 1. name, school, year of graduation and position running for, 2. year achieving diplomate status and route (residency vs practitioner), 3. species emphasis and main focus of work, 4. qualifications specific for the post you are seekinginclude education, personal experience, vision etc. please restrict the above comments to 350 words or less. send submission by march 1, 201 to: email: 201� annual sft �����������!��� � it is that time of year again! make sure you do not miss out on any of the great benefits that the society has to offer: for search on web site business tool for referrals therio conference society website, including past proceedings and newsletters library electronic articles free of charge annual therio conference online quarterly publication of the society for theriogenology large animal listservs for case discussion with specialists with varied discipline interests journal for your convenience you can renew your dues at the sft web site, www.therio.org! 530clinical theriogenology • volume 9 number 4 • december 2017 ��������� �� � ���� ��� � � � ���������� ��������� �������� � �� � �� � ���� ��� � � � ����� *call for student case presentations also available at www.therio.org attention to all sft student members! the sft program committee is pleased to announce the call for submission of student theriogenology case presentations at the annual therio conference in milwaukee, wisconsin. the competition will be held during the annual conference, august 1-4, 2018. the purpose of this competition is to promote student interest in sft, to encourage investigative and communication skills and allow students greater participation in our annual meeting. all sft student members are qualified to enter the competition. the student theriogenology case presentations will be held in milwaukee, wisconsin. it will give the sft membership a look at future leaders and participants of the sft. six student cases will be selected for presentation at the annual conference competition. do you have a clinical case presentation you have given or that you are preparing? you can submit it, as long as it is on theriogenology. a copy of the guidelines and submission instructions can be obtained from the sft website, your sft faculty liaison, or dr. alexander. please refer to the “guidelines for application submission” for preparation of your manuscript. abstracts must be submitted by march 10, 2018. please submit them in microsoft word format to tara beasley at the society office, therioconference@franzmgt.com, with a copy to your advisor. include the words "student case presentation" in subject line. an acknowledgement of your submission should be received within 24 hours. if you do not receive acknowledgement, please contact the office at 334.395.4666. a panel of practitioners and academicians will select six manuscripts for presentation in milwaukee and notify the students by april 11, 2018. the students will be responsible for making their own travel arrangements and accommodations. conference hotel information will be available on the sft website. please be aware that this is a competition. the contestants will be judged on both the quality of their investigation and the quality of the presentation. therefore, you must attend the meeting in milwaukee to be eligible for the awards. the sft will award six cash prizes: first place $ 650 fourth place $ 375 second place $ 525 fifth place $ 300 third place $ 450 sixth place $ 200 more than one student may be involved with the presentation of a case or research project, however only one prize will be awarded per presentation. all students (presentations) who are accepted to present abstracts may be awarded a travel stipend. grant information is available on the theriogenology foundation website, www.theriofoundation.org. travel grants must be applied for via the website. please contact your sft faculty liaison for more information and for assistance in preparing your manuscript. you may also contact dr. james h. alexander at the email address below. we hope you will participate in this event. we look forward to hearing from you. sincerely, james h. alexander, dvm jalexanderdvm@gmail.com 531 clinical theriogenology • volume 9 number 4 • december 2017 ���� ��� ������������� ���� ���������� ��� �������� ���! ����!�� �� � ���� ��� � � � �������� � ���� ��������� �������� � �� "�#��$��% "�&��������"���!�� ���'"������ all applicants must be student members of the society for theriogenology. this includes student members graduating in the spring of 2018. an abstract of the student case report must be received by march 10, 2018. please include the following with your submission: 1. name, home address and school address 2. telephone number 3. email address 4. name, title, address, email address and phone number of the faculty member working with you. 5. abstract of your case (300 words or less in the body of the abstract). a) title and authors b) the importance of the case to theriogenology c) a discussion of the diagnostic approach and treatment options considered. d) results/discussion e) references 6. please copy your submission email to your advisor. you will receive an email acknowledgement of your submission within 24 hours (during business hours). if you do not receive an acknowledgment, please call the office at 334.395.4666. we will not need any photos, graphs, or diagrams at this time. prior to the conference you will be required to submit a copy of your power point presentation to the sft office to the attention of tara beasley at the society office, therioconference@franzmgt.com, no later than june 25, 2018. you will also be required to bring a copy with you to the conference. you will be allowed to update your presentation after june 25, up until 4 hours before your presentation. case presentations must be submitted by march 10, 2018. please submit your presentations in microsoft word format to tara beasley at the sft office, therioconference@franzmgt.com. include "student case presentation" in the subject line. the abstract should be in normal style, times new roman font, 12-font size, single spacing, and 1” margins. six presentations will be selected to be presented orally. you will be notified no later than april 11, 2018, whether your presentation has been selected. further instructions will follow notification. each presentation will be 10 minutes in length. time has been allotted for questions after each presentation. those not selected for oral presentation may be selected for a poster presentation. a panel of academicians and private practitioners will review manuscripts. please be aware that this is a competition. the contestants will be judged on both the quality of their investigation and the quality of the presentation. therefore, you must attend the meeting in milwaukee to be eligible for the awards. the sft will award six cash prizes: first place $ 650 fourth place $ 375 second place $ 525 fifth place $ 300 third place $ 450 sixth place $ 200 more than one student may be involved with the presentation of a case, however only one prize will be awarded per presentation. questions can be submitted to: james h. alexander, dvm at jalexanderdvm@gmail.com 532clinical theriogenology • volume 9 number 4 • december 2017 call for abstracts-2018 ________________________________________________________________________________ the society for theriogenology and american college of theriogenologists issue a call for research and case abstracts to be presented at the annual therio conference august 1-4, 2018 in milwaukee, wisconsin. applica�on deadline is february 15, 2018. abstracts will be considered in four categories: _________________________________________________________________________________________ 1. compe��ve category: abstracts submi�ed for this category must have a graduate student, resident, veterinary student, or undergraduate student as the first author (only one submission per first author will be accepted in this category). abstracts on material that has already been published or has been accepted for publica�on at the �me of submission will not be considered. these abstracts will be judged on the basis of scien�fic merit (wri�en). the eight abstracts with the highest wri�en scores will be selected for presenta�on during the abstract compe��on. first authors chosen to present in the abstract compe��on will receive one complimentary registra�on for the mee�ng. the abstracts will be presented during a plenary session and judged for presenta�on quality (oral and understanding of scien�fic basis). financial awards will be given to the four presenters with the highest total score (wri�en plus oral). the first author (graduate student, resident, veterinary student, or undergraduate student) will be required to present the abstract during the abstract compe��on at the therio conference. oral presenta�ons will be 10 minutes in length; an addi�onal two to three minutes will be allowed for ques�ons only. time limits will be strictly enforced for the abstract compe��on; exceeding the 10-minute �me limit will result in disqualifica�on from the compe��on. other high-scoring abstracts not selected for the abstract compe��on will be chosen for presenta�on during a noncompe��ve session, and one of the authors will be required to present the abstract at the therio conference during a general session or educa�onal session. presenters of these abstracts will not receive complimentary registra�on for the conference. if the appropriate author is not available to present the abstract at the therio conference, you may pe��on the chair of the scien�fic abstract commi�ee to request permission for an alterna�ve author to present the informa�on. in the case of a compe��ve abstract for which the submi�ng graduate student/resident is not available to present the abstract but for which a suitable subs�tute presenter can be found, the abstract may s�ll be included for presenta�on during the compe��ve session but it will not be scored and will not be eligible for awards. 2. non-compe��ve category: all individuals, including prac��oners, faculty, graduate students, residents and veterinary students are encouraged to submit abstracts to be considered for presenta�on during a general session or an educa�onal session (there may be more than one submission per first author in this category). abstracts on material that has already been published or has been accepted for publica�on at the �me of submission will not be considered. presenters in this category will not receive a complimentary registra�on to the conference. oral presenta�ons will be 10 minutes in length; an addi�onal two to three minutes will be allowed for ques�ons only. 3. case abstract category: all individuals, including prac��oners, faculty, graduate students, residents, are encouraged to submit note-worthy case reports for considera�on for presenta�on during a general or educa�onal session. abstracts on material that has already been published or has been accepted for publica�on at the �me of submission will not be considered. case reports will not be considered for inclusion in the compe��ve category. there may be more than one submission per first author in this category. presenters in this category will not receive a complimentary registra�on to the conference. 4. poster category: all individuals, including prac��oners, faculty, graduate students, residents, are encouraged to submit abstracts/case reports to be presented as posters. abstracts on material that has already been published or has been accepted for publica�on at the �me of submission will not be considered. there may be more than one submission per first author in this category. presenters in this category will not receive a complimentary registra�on to the conference. posters are normally displayed in the exhibit hall. you are encouraged to be present as o�en as possible during exhibit hall breaks to answer ques�ons about your poster. however, all poster presenters are expected to be present during the friday lunch hour when the conference will adver�se your presence. abstracts and case abstracts not selected for oral presenta�on may be given the op�on to be presented as posters. general: presenters of research abstracts, posters, and case abstracts will not be reimbursed for travel or other expenses incurred in presen�ng an abstract, nor will they receive an honorarium. all accepted abstracts will be printed in the annual therio conference proceedings, clinical theriogenology. 533 clinical theriogenology • volume 9 number 4 • december 2017 submission of an abstract for considera�on in either the compe��ve or any noncompe��ve sessions is considered a commitment that, should the abstract be accepted in any of the categories, the appropriate author (as described in the “call for abstracts”) will a�end the therio conference to present the abstract in a formal oral or poster session. in the event that no suitable person is available to present an accepted abstract or poster at the therio conference, the submi�ng/presen�ng author will be barred from submission of scien�fic abstracts for considera�on for presenta�on at the therio conference and will not be allowed to speak at the therio conference for a period of five (5) years. all co-authors will be barred from submission of scien�fic abstracts for considera�on for presenta�on at the therio conference and will not be allowed to speak at the therio conference for one (1) year. penalized authors have the right to pe��on the execu�ve board (eb) of the american college of theriogenologists for a reduc�on or waiver of the penalty. a wri�en pe��on that includes the reason for the absence from the mee�ng should be submi�ed to the chair of the scien�fic abstract commi�ee no later than one week before the therio conference. the chair and other members of the abstract commi�ee will review the pe��on and present the case to the eb at the august board mee�ng. authors will be no�fied of the decision of the board by email a�er the conclusion of the therio conference. ___________________________________________________________________________________________________________ guidelines: what to send and how to send it electronically: submit via email (address on top of submission form) or on a cd (compa�ble with microso� word) general: authors should read the guide for authors for the journal clinical theriogenology. specific instruc�ons for submission of abstracts can be found on the act website (www.theriogenology.org) under “call for abstracts”. font: single spaced, 12 point times new roman length: no longer than one page paper: 8.5” x 11” margins: 1.5” top and bo�om – 1” le� and right header: the �tle, authors, and their affilia�ons must be centered at the top of the page. content: abstracts should state clearly the hypothesis and aims of the project, describe the methods used, and summarize the findings. keywords (up to five) should be listed at the bo�om of the page. authors are encouraged to consider the criteria for judging provided on the website. form: you must complete and submit the submission form for your abstract. abstracts not adhering to these guidelines will not be considered for presenta�on. deadline: abstracts must be received no later than february 15, 2018 for considera�on. if you have not received an acknowledgement that we have received your abstract submission by february 15, 2018, please call our office at 334.395.4666 the act/sft abstract submission author agreement form must be signed by all authors. a copy of the completed form must be submi�ed along with the abstract. the form is available on both the act and sft websites. access the required call for abstracts applica�on form on either the act website at www.theriogenology.org or the sft website at www.therio.org under the 2018 therio conference heading, listed under educa�on and events on both sites. those submi�ng abstracts for considera�on will be no�fied by march 30, 2018 if their abstract(s) were accepted or declined. 534clinical theriogenology • volume 9 number 4 • december 2017 about milwaukee milwaukee’s nicknamed cream city because of its distinctive cream colored brick and brew city because of its rich brewing history. milwaukee borders the 22,300 square miles of lake michigan. the lake actually touches 14,200 square miles of wisconsin. the milwaukee riverwalk spans nearly 3 miles along the milwaukee river, taking you through downtown milwaukee. downtown milwaukee boasts over 150 restaurants and 17 museums. heralded as the "city of festivals," milwaukee showcases its rich heritage with a procession of ethnic and cultural celebrations throughout the summer. the henry w. maier festival park along the shores of lake michigan becomes a portal to the world as guests are invited to explore the arts, cuisine, history and entertainment of a new culture nearly every weekend. arts and theater lovers will find so much to love in milwaukee. the theater scene boasts opera companies, a world-class symphony, a nationally acclaimed ballet company and an array of professional dance and theater companies performing at venues all across the metro area. enjoy some of the nation’s best youth performers, the biggest broadway hits on tour and experience a special night (and day) when the arts community opens its doors for art lovers of all kinds. biking, hiking, paddling and more – it's easy to find fun and adventure in milwaukee's great "urban outdoors." there are 15 golf courses, over 150 state county parks, and over 130 miles of bike trails. downtown arenas are home to professional and college basketball, soccer, ice hockey, and roller derby, while miller park is home to the milwaukee brewers. with all the many options for entertainment throughout the city, the entire family is sure to enjoy their visit to milwaukee. photos credits: visitmilwaukee.org for more information about milwaukee, go to www.visitmilwaukee.org to view upcoming events, restaurant and museum guides, and more! 535 clinical theriogenology • volume 9 number 4 • december 2017 safety and efficacy of intrauterine kerosene infusions to enhance endometrial cup regression in mares safety and efficacy of intrauterine kerosene infusions to enhance endometrial cup regression in mares giorgia podico,a igor canisso,a claire kaplan,a robyn ellerbrock,b vitor mercadante,c nicholas dias,c scott austin,a patrick roady,a robert douglas,d fabio limaa adepartment of veterinary clinical medicine, college of veterinary medicine university of illinois urbana-champaign, urbana, il; bdepartment of large animal medicine college of veterinary medicine, university of georgia, athens, ga cdepartment of animal and poultry sciences virginia polytechnic institute and state university, blacksburg, vi; dbet laboratory, lexington, ky endometrial cup retention after early abortion can result in immediate or long term reproductive failure. mares with active endometrial cups have abnormal estrous cycles and persistent mineralized structures have been occasionally reported one or two seasons after initial abortion. although intrauterine infusions with kerosene were proposed as a method to enhance removal of endometrial cups, no controlled studies have evaluated kerosene efficacy. objectives were to evaluate side effects and efficacy of intrauterine kerosene infusions to enhance regression of endometrial cups. we hypothesized that: i) intrauterine kerosene infusions hasten regression of endometrial cups without detrimental effects on the endometrium; and ii) mares have negligible systemic side effects after intrauterine infusions. nine light horse breed mares were enrolled in the study after an artificially induced abortion (n = 12) at 58 62 days of pregnancy. mares were randomly allocated an intrauterine infusion with 500 ml kerosene (ker; n = 6) or 500 ml saline (cont; n = 6) on days 21 and 35 after pregnancy termination. uterine biopsies were collected upon completion of abortion at day 7, 21, 35, and 49, routinely processed and stained with h & e, and graded by a pathologist. physical examinations, a complete hemogram, and serum biochemistry were performed before intrauterine infusion and daily for 2 days after the procedure. uterine lavage was performed 24 hours after each infusion. serum samples were collected right before the abortion induction and at 7, 21, 28, 35, 42, and 49 days after pregnancy termination for assessment of serum ecg concentrations. continuous data were analyzed with mixed procedure with repeated measures in sas and categorical data with logistic procedure in sas. significance was set as p < 0.05. treatment had no effect on complete blood cell count or serum chemistry values or on objective or subjective parameters of physical examinations. mean ± sem ecg concentrations decreased over time, with no difference between treatment groups (day 0 cont 88.7 ± 8.5, ker 87.3 ± 8.5; day 7 cont 86.0 ± 8.5, ker 82.00 ± 8.5; day 21 cont 79.3 ± 8.5, ker 74.3 ± 8.5; day 28 cont 73.0 ± 8.5, ker 61.5 ± 8.5; day 35 cont 59.1 ± 8.5, ker 45.2 ± 8.5; day 42 cont 58.5 ± 8.5, ker 34.0 ± 8.5; day 49 cont 45.2 ± 8.5, ker 25.3 ± 8.5). histologically, endometrium had no signs of increased fibrosis or degeneration related to treatment group. in conclusion, although kerosene infusions did not appear to have detrimental effects on mare health, there were no indications that kerosene infusions enhanced regression of endometrial cups. keywords: endometrial cups, kerosene, mares, ecg 425 clinical theriogenology • volume 11, number 3 • september 2019 426clinical theriogenology • volume 11, number 3 • september 2019 omniblank: a delayed timed artificial insemination 78 hours after cidr removal does not affect pregnancy outcomes in dairy heifers treated with a 4 day cosynch + cidr protocol a delayed fixed time artificial insemination 78 hours after cidr removal does not affect pregnancy outcomes in dairy heifers treated with a 4 day cosynch + cidr protocol roberto palomares,ab maria ferrer,b joão bittar,a alejandro hoyos-jaramillo,a juan gutierrez-añez,a heidi fishman-holland,b adriana camacho,a justin graham,c renato rizzoc departments of apopulation health and blarge animal medicine college of veterinary medicine, university of georgia, athens, ga cbrooksco dairy, quitman ga use of 4 day cosynch + cidr + ftai (fixed time artificial insemination) at 72 hour after cidr removal in dairy heifers has resulted in adequate pregnancy per ftai (55.0%), not significantly different from that in the 5 day cosynch + cidr (63.3%). a subsequent study demonstrated that heifers in the 4 day group had smaller follicular diameter after cidr removal and before ovulation, and longer interval from cidr removal to ovulation (90 96 hours) compared to heifers in the 5 day group (84 90 hours), which might affect pregnancy outcomes and influence the optimal timing for ai. based on these results, we hypothesized that prolonging the interval from cidr removal to ftai by 6 hours (ftai at 78 hour after cidr removal) in heifers treated with the 4 day cosynch + cidr protocol does not affect ftai and pregnancy loss (pl) compared to control heifers subjected to the modified 5 day cosynch + cidr + ftai at 72 hour after cidr removal. objective was to compare p/tai and pl in dairy heifers treated with day cosynch + cidr + ftai at 78 hours post cidr removal versus 5 day cosynch + cidr + ftai at 72 hours after cidr removal. twelve-month old holstein heifers (n = 796) were randomly assigned to 1 of 2 groups. heifers received an intravaginal cidr insert containing 1.38 g of progesterone for 4 days (4 day cosynch + cidr; n = 401) or 5 days (5 day cosynch + cidr; n = 396). at the time of cidr removal, 25 mg of pgf2α was injected intramuscularly. finally, heifers were artificially inseminated and received 100 μg of gnrh intramuscularly at either 78 or 72 hours after cidr removal, for 4 and 5 day groups, respectively. artificial insemination was performed by an experienced technician, using commercial frozen thawed semen from a single sire. pregnancy diagnosis was performed by transrectal ultrasonography 32 days after ftai. in addition, pregnancy was reconfirmed 60 days after ftai to calculate pl. categorical data (ftai and pl) were analyzed using proc logistic and chi square tests, using statistical analysis system (sas®). heifers in the 4 day cosynch + cidr group (monday friday protocol) had an adequate pregnancy per ftai (55.4%; 222/401), which was not different (p = 0.4) from that in the 5 day cosynch + cidr group (59.5%; 235/395). pregnancy loss rate was not different between groups and was within reference ranges for dairy heifers (6.6 and 4.4% for 4 and 5 day groups, respectively). in conclusion, 4 day cosynch + cidr protocol with ftai at 78 hours after cidr withdrawal resulted in adequate pregnancy per ftai and pl in dairy heifers. pregnancy rate was similar to that with 5 day cosynch + cidr with ftai at 72 hours after cidr removal. these results supported our hypothesis that ftai at 78 hours after cidr removal within the 4 day cosynch + cidr protocol does not affect pregnancy per ftai and recapitulated previous findings, suggesting that this protocol may represent a promising tool for ovulation synchronization of dairy heifers and would facilitate breeding programs by making it suitable for a monday friday schedule. keywords: 4 day cosynch, controlled internal drug release, dairy heifer, timed artificial insemination 439 clinical theriogenology • volume 11, number 3 • september 2019 440clinical theriogenology • volume 11, number 3 • september 2019 omniblank: anti müllerian hormone concentrations and antral follicle number in dairy and beef heifers subjected to a modified 5 day cosynch + cidr protocol anti müllerian hormone concentrations and antral follicle number in dairy and beef heifers subjected to a modified 5 day cosynch + cidr protocol sarah stanley,a heidi fishman-holland,b joao bittar,a alejandro hoyos-jaramillo,a maria ferrer,b natalie norton,a juan gutierrez-añez,a adriana camacho,a roy berghaus,a justin graham,c renato rizzo,c roberto palomaresa departments of apopulation health and blarge animal medicine, college of veterinary medicine university of georgia, athens, ga, cbrooksco dairy, quitman, ga anti müllerian hormone (amh) concentration is associated with ovarian antral follicle count (afc) and ovulatory response and is a potential biomarker for ovarian status of replacement heifers.1,2 however, only a few studies have compared amh concentrations and afc between beef and dairy heifers, under commercial management conditions. the objective was to compare amh concentration and afc between dairy and beef heifers subjected to 5 day cosynch + cidr protocol and to evaluate the possible association between amh and afc with pregnancy rate to fixed time artificial insemination (ftat). we hypothesized that beef and dairy yearling heifers have comparable amh concentrations and afc at the time of enrolment in the breeding program and that there is no association between amh concentration and afc at the time of cidr insertion and ftat. a total of 80 holstein and 94 angus 1 year old heifers received an intravaginal cidr containing 1.38 g of progesterone for 5 days. at cidr removal, 25 mg of pgf2± was given im and 72 hours after cidr removal, 100 μg of gnrh was given im and heifers were artificially inseminated with frozen-thawed semen. immediately before cidr insertion, heifers were evaluated by transrectal ultrasonography (trus) to determine reproductive tract score and afc (> 3 mm). blood samples were concurrently collected for determination of amh concentration using a commercial elisa (anshlabs®, webster, tx). pregnancy diagnosis was performed by trus 32 days after ftat. data were analyzed using stata® (statacorp llc, version 15.1). concentrations of amh and afc were compared between groups using a two-sample student’s t test. logistic regression and chi-square were used to determine if there was an association between amh concentration, afc and ftai. beef heifers had higher (p < 0.0001) amh concentrations than dairy heifers (387.44 ± 41.5 versus 178.20 ± 22.0 pg/ml, respectively). however, afc was not different between beef and dairy heifers (5.02 ± 0.25 versus 5.81 ± 0.3 follicles). based on logistic regression analysis, amh concentrations and afc at initiation of the protocol did not influence ftat either in dairy (52.5%) or in beef (39.3%) heifers (p > 0.05). in conclusion, beef heifers had significantly higher amh concentrations than dairy heifers, but afc did not differ. further studies are necessary to evaluate ahm concentration as a potential biomarker to assess heifer reproductive status before initiating a synchronization protocol. keywords: anti müllerian hormone, antral follicle number, heifers, beef, dairy references 1. ireland jj, zielak-steciwko ae, jimenez-krassel f, et al: bio repro 2009;80:954-964. 2. rico c, fabre s, médigue c, et al: bio reprod 2009;80:50-59. 419 clinical theriogenology • volume 11, number 3 • september 2019 420clinical theriogenology • volume 11, number 3 • september 2019 keywords: anti müllerian hormone, antral follicle number, heifers, beef, dairy references 1. ireland jj, zielak-steciwko ae, jimenez-krassel f, et al: bio repro 2009;80:954-964. 2. rico c, fabre s, médigue c, et al: bio reprod 2009;80:50-59. omniblank: anatomical location of bovine embryos following superstimulation in beef heifers anatomical location of bovine embryos following superstimulation in beef heifers caitlin wiley,a marianna jahnke,a patrick j. gunn,b tyler dohlmana aveterinary diagnostic and production animal medicine, college of veterinary medicine, iowa state university, ames, ia; bdepartment of animal science, iowa state university, ames, ia abstract since the advent of non-surgical embryo recoveries in cattle, there has always been uncertainty regarding the location of embryos at the time of recovery. despite thousands of embryo recoveries performed annually, there is limited research describing embryo location 6-7 d after ovulation in cattle subjected to a superstimulation protocol. the objective was to determine location of embryos within the reproductive tract of superstimulated heifers. non-pregnant, yearling beef heifers (n = 12; 577 ± 31 kg) were allocated into three replicates and began a timed, 17-d, superovulation controlled internal drug release-based protocol, with artificial insemination done twice. at 6.5 d after first artificial insemination, heifers were slaughtered at a commercial abattoir. immediately after exsanguination, the reproductive tract was excised and each side sectioned into 4 segments (section 1 for the uterine tube and sections 2 to 4 for the uterine horn, with section 4 including the uterine body). each section was isolated, flushed, and flush media filtered and embryos identified and enumerated. corpora lutea were also enumerated. there were differences among uterine sections in number of embryos recovered (p < 0.0001), with embryos recovered from all sections. section 2 (tip of horn) contained the greatest number and percentage of embryos, followed by sections 3, then 4 and finally 1 (53.5, 33.9, 6.8 and 5.8%, respectively). in summary, embryos were located throughout the tract, with the majority located in the tip and middle third of the uterine horn. keywords: embryo, superovulation, migration, uterine horn introduction since the 1970s, the embryo transfer (et) industry has been striving to improve embryo recovery rates in superovulated donors. evidence-based improvements have been made in superovulation protocols and recovery techniques. initiating ovarian stimulation concurrent with follicular wave emergence drastically improved embryo yield.1-3 with regard to recovery technique, growing interest to transition recovery and transfer techniques from surgical to non-surgical occurred in the late 1970s. as non-surgical embryo recoveries were becoming more feasible, newcomb et al. used a surgical approach to determine embryo migration in the uterus at d 3-7 after insemination. they reported that more embryos were present in the tip of uterine horn compared to the base of the uterus.4 therefore, it was suggested that non-surgical recoveries should emphasize flushing the tips of the horns. additionally, adoption of non-surgical embryo recovery enabled et to be performed at a producer’s operation, which led to substantial growth in the industry.5 currently with conventional et, non-surgical embryo recoveries are done ~6-7 d after ovulation. following fertilization in the uterine tube, embryonic development from zygote to 16-cell embryos occurs over ~4 d in the uterine tube.6,7 migration into the uterus occurs at ~day 5, when the embryo has further developed and undergone compaction to form a morula.8 with this time interval to recovery, most embryos are at a compact and mature developmental stage.9, 10 there are 2 general approaches for catheter placement within the uterus for non-surgical embryo recovery. one approach involves placing the catheter just anterior to the internal cervical os and flushing both horns and body simultaneously, known as a “body flush”.11 as a large volume of flushing medium is required for adequate recovery, this approach is best suited to donors with smaller uteri (e.g. heifers). the other approach, “horn flushing,” involves placing the catheter in each horn (separately) and advancing it cranially, at least to the external bifurcation or as far cranially as possible.12 although the catheter must be placed more cranially then for a body flush, a smaller volume of flush medium can be used. however, without knowledge of embryo location at this stage of development, there is the potential for embryos to be left in the uterus if catheter placement is too far cranial. 427 clinical theriogenology • volume 10, number 4 • december 2018 the objective of this study was to determine the location of embryos within the uterus of superovulated heifers at the time of embryo recovery. it was hypothesized that the majority of embryos are located towards the tips of the uterine horns. materials and methods general all protocols and procedures used were approved by the iowa state university institutional animal care and use committee. the project was conducted at the iowa state university zumwalt station research center in ames, ia, in january 2016 – february 2016, with heifer slaughter and recovery of reproductive tracts occurring at amend packing company, des moines, ia. single-sourced heifers from the iowa state university mcnay beef research farm were used. animals and methods non-pregnant, purebred angus yearling beef heifers (n = 12; 577 ± 31 kg) were used. prior to treatment, all heifers were subjected to a transrectal reproductive ultrasound examination to confirm that there were no apparent reproductive abnormalities and that the heifers were cycling. heifers were assigned to 1 of 3 replicates based on weight and were penned in these replicate groups for the duration of the study. initiation of superovulation protocols for each replicate were started at 1-wk intervals. all heifers were subjected to superstimulation by utilizing a timed, 17-d, controlled internal drug relase (cidr; eazi-breed™, zoetis inc., kalamazoo, mi)-based protocol with gonadotropin releasing hormone (gnrh; cystorelin®, merial llc, duluth, ga) and prostaglandin f2α (pgf2α; lutalyse®, kalamazoo, mi) with a decreasing total dosage of 240 mg follicle stimulating hormone (fsh; folltropinv®, vetoquinol, lavaltrie, québec, canada) administered twice daily for 4 d (table 1). heifers were artificially inseminated (ai) following a timed-ai schedule in conjunction with observed signs of standing estrus, with a second ai done 12 h later by the same technician. at each ai, 1 unit of frozen semen from the same ejaculate of 1 bull was used, with gnrh (150 µg) given im at first insemination. at 6 d after the initial ai, transrectal ultrasonography was done to estimate the number of cls on each ovary. approximately 12 h later, heifers were taken to a commercial abattoir where they were stunned via captive bolt and exanguinated. reproductive tracts were excised within 15 min after slaughter. initial processing was performed at the abattoir. within the next 30 min, the length of each tract was measured in its natural state and each horn was sectioned into even thirds, with the uterine body split between the caudal third of each horn (figure). zip ties were placed at the edges of these sections and the uterus was cut distal to the zip tie while being held over open ziplock bags, for a total of 4 sections per side (total of 8 sections per heifer). once in individual ziplock bags and labeled accordingly, commercial flush medium (vigro™ complete flush; bioniche, pullman, wa) was added to preserve embryos and tissue for transportation to the laborabory. ovaries were excised and placed in individual labelled bags. samples were transported in a cooler at room temperature (approximately 60-min drive). embryo recovery at the laboratory, each section of uterus was opened with mayo scissors in its own bag and with a catheter tip syringe, approximately 60 ml flushing medium was used to lavage the tissue, scissors, and bag that held the tissues. uterine tubes were flushed, starting at the infundibulum, with 5 ml of flushing medium, followed by air, into a bag. all media from each section was then filtered through emcon™ filters (mai animal health). each filter from each section was thoroughly rinsed into labeled squaregridded search dishes and searched for embryos using a stereomicroscope. all recovered embryos were evaluated according to international embryo technology society standards by 2 persons (both were certified by the american embryo transfer association). ovaries were evaluated for number of (corpora lutea) cls and anovulatory follicles to determine recovery rate. to improve accuracy, structures were marked with pins, and then dissected with a scalpel for final enumeration. 428clinical theriogenology • volume 10, number 4 • december 2018 statistical analysis data were analyzed using sas 9.4 (sas institute inc., cary, nc) using the mixed procedure, with heifer serving as the experimental unit and section as the fixed main effect. animal within replicate (group) served as the random effect. both section length and total number of cls were initially included as covariates, but section length was subsequently removed due to a lack of significance (p = 0.70). statistical significance was designated as p ≤ 0.05. results in total, embryos were present in all sections, including the uterine tubes. for combined total recovery from all sections, there was a difference among sections in location of embryos (p < 0.001; table 2). in terms of total proportion of embryos recovered, there was a difference between section 2 (tip of uterine horn) and section 3 (middle third of uterus; p = 0.05; figure). however, both of these sections had more embryos than either section 1 or section 4 (uterine tube and caudal uterine horn and body, resepctively; p < 0.05). there was no difference between the percentage of embryos in section 1 versus section 4 (p = 0.90). there was a difference between embryo counts from section 1 compared to section 2, and section 1 compared to section 3 (p = 0.0005, p = 0.006; respectively). furthermore, sections 2 and 4 and sections 3 and 4 also differed (p = 0.002, p = 0.017; respectively). when total number of embryos recovered from each of the sections were compared, there were no differences between sections 2 and 3 (p= 0.37), nor between sections 1 and 4 (p = 0.65). however, there was a difference between the combined totals from sections 2 and 3 relative to that from sections 1 and 4 (p < 0.05). as total number of cls increased, so did number of embryos recovered (p < 0.01). based on cl counts and total embryos recovered per uterus, the average recovery rate was 49% (range, 0 to 92%). discussion general the objective was to determine location of embryos in the reproductive tract of superovulated heifers at the usual time of recovery. although research has focused on the environment of the uterine tube and uterus during embryo formation and development,13-15 little is known about embryo location after entering the uterus relative to time of embryo recovery for multiple ovulation embryo transfer. therefore, heifers were slaughtered when non-surgical recovery would have been done (day 6.5 following insemination). based on previous work using slaughtered cows, the reproductive tract was excised as soon as possible after exsanguination, to mitigate negative effects of declining ph following death.16 after the tract was excised, measurements and sectioning were immediately performed to decrease potential post-mortem movement of embryos. although the recovery rate was not 100 percent even with ex vivo recovery, it was assumed that not all ova were captured in the infundibulum, due to the degree of ovarian stimulation. these results are relevant for embryo transfer operators conducting nonsurgical embryo recoveries. embryo quality stages and grades of embryos were not described, due to poor embryo quality. despite prebreeding semen evaluation, observation of standing estrus, and an experienced ai technician, resulting embryos were poor. poor embryo quality of abattoir-collected embryos has been reported16 and attributed to a drastic decrease in intrauterine ph after slaughter due to a change from aerobic to anaerobic metabolism. however, in another study involving embryo recovery from slaughtered cows on day 4 after estrus and subsequently placed in culture medium for further growth in vitro, there was no reference made to ph affecting embryo quality.17 in the current study, efforts were made to process the tissues and recover embryos as soon as possible, to minimize potential post-mortem effects. 429 clinical theriogenology • volume 10, number 4 • december 2018 embryo location as the et industry transitioned from surgical to non-surgical embryo recoveries, there were questions raised about the efficiency of embryo recovery.4,11,12 in an experiment4, embryos were recovered between days 3 and 7 after estrus via a surgical approach, with flushing of uterine tubes conducted separate from uterine flushing. as the day of embryo collection following estrus increased, so did number of developmental stage 4-7 embryos, as well as number of degenerate embryos present in the uterus. interestingly, in a previous study,4 there were several embryos in the uterine tube on day 6, compared to days 7-8. similarly, in the present study, 5.8% of embryos were in the uterine tube on day 6.5 after insemination and likely would not have been recovered by conventional flushing. if the interval from ovulation to recovery were prolonged, there is the potential for an increased proportion of embryos being recovered from the uterus. however, these embryos would likely be more mature, limiting their suitability for cryopreservation. further investigation into the percentage of quality embryos still in the uterine tube at time of recovery may be of merit. in previous work,4 ligation of the uterus into 2 sections revealed a greater proportion of embryos in the cranial half of the uterine horn compared to the uterine tubes and base of the uterus. although most embryos were in the uterine tip section, there were embryos in the caudal half of the uterus (uterine base section). it should be noted this previous experiment was all performed using a surgical flushing technique in vivo.4 in the current study, the uterus was sectioned into thirds, plus the uterine tube, for a total of 4 sections per horn, 8 sections per tract, enabling more precise localization of embryos. accounting for individual animal variation, the total length of each uterus was taken into consideration and each uterus was evenly sectioned into thirds from the utero-tubal junction to the internal cervical os. these variations in section lengths among heifers were evaluated, but did not impact migration of embryos at the time of recovery. embryos were in the uterine tube and all 3 sections of the uterus, with the largest percentages in the tip and middle third (53.5 and 33.9%, respectively). furthermore, embryos were also present in the base of the horns. based on these results, placement of the catheter close to the tip of the horn would likely fail to recover embryos that have migrated closer to the uterine body. therefore, if a horn flush is to be conducted, placing the catheter at the level of the uterine bifurcation should enable recovery of most embryos, although there is the potential for some to be in the uterine body. whereas this study utilized nulliparous, yearling heifers, a similar study using mature, multiparous cows would be of value to compare embryo migration between the two reproductive statuses. an ultrasonographic examination of the ovaries was done ante mortem to predict stimulatory response. these results were not included, because the ovaries were examined <12 h before slaughter. there can be a wide variation in accuracy of evaluating ovaries and counting number of cls, especially with a donor who has a high response to stimulation, based on the ultrasonographer’s skill and experience. ovarian overstimulation may have contributed to discrepancies between cl counts and embryo recovery. especially in heifers, there can be physical limitations on ovaries with regard to the number of developing follicles that they can support and subsequently ovulate.18 repeating this study with a larger sample size, may yield more conclusive results. additionally, even though cls were accounted for, progesterone concentrations were not evaluated. progesterone is known to cause relaxation of the smooth muscle of the uterine tube, which leads to a decreased rate of transport through the uterine tube.19 however, with superovulated cows, whether or not progesterone concentrations impact migration of embryos through the uterine tube or uterus remains unknown. conclusion at 6.5 d after insemination in superovulated heifers, embryos were located throughout the reproductive tract, although the majority localized towards the tip and middle third of the uterine horn. with regard to catheter placement for recovering embryos, the closer the catheter is towards the tip of the uterine horn, the greater the risk of missing embryos. whether the catheter is placed within the uterine body or each horn, the focus of the recovery should be to distend the tip and middle third of the horn to improve recovery rates while potentially incorporating the body of the uterus to maximize recovery. 430clinical theriogenology • volume 10, number 4 • december 2018 further research is warranted to determine if the developmental stage and/or quality of the embryo impacts its migration and whether other factors such as the age of donor or perhaps progesterone concentrations during superstimulation influence embryo location at the time of recovery. acknowledgements the authors thank claire andresen and allie lundberg for their help in processing samples and assisting in the laboratory. conflict of interest none of the authors have any conflicts of interest to disclose. references 1. nasser lf, adams gp, bo ga, et al: ovarian superstimulatory response relative to follicular wave emergence in heifers. theriogenology 1993;40:713-724. 2. baracaldo mi, martinez mf, adams gp, et al: superovulatory response following transvaginal follicle ablation in cattle. theriogenology 2000;53:1239-1250. 3. bo ga, adams gp, pierson ra, et al: effect of progestogen plus estradiol-17 beta treatment on superovulatory response in beef cattle. theriogenology 1996;45:897-910. 4. newcomb r, rowson lea, trounson ao: the entry of superovulated eggs into the uterus. luxembourg, commission of the european communities, 1976:1-13. 5. hasler jf: current status and potential of embryo transfer and reproductive technology in dairy cattle. j dairy sci 1992;75:2857-2879. 6. barnes fl, eyestone wh: early cleavage and the maternal zygotic transition in bovine embryos. theriogenology 1990;33:141-152. 7. betteridge kj, flechon je: the anatomy and physiology of pre-attachment bovine embryos. theriogenology 1988;29:155-187. 8. dorniak p, bazer fw, spencer te: physiology and endocrinology symposium: biological role of interferon tau in endometrial function and conceptus elongation. j anim sci 2013;91:1627-1638. 9. lawson ras, rowson lea, moor rm, et al: experiments on egg transfer in the cow and ewe: dependence of conception rate on the transfer procedure and stage of the oestrous cycle. j reprod fertil 1975;45:101-107. 10. trounson ao, willadsen sm, rowson lea: storage of cow eggs at room temperature and low temperatures. j reprod fertil 1976;46:173-178. 11. hasler jf: forty years of embryo transfer in cattle: a review focusing on the journal theriogenology, the growth of the industry in north america, and personal reminisces. theriogenology 2014;81:152-169. 12. rowe rf, delcampo mr, critser jk, et al: embryo transfer in cattle non surgical transfer. am j vet res 1980;41:1024-1028. 13. sirard ma: resumption of meiosis: mechanism involved in meiotic progression and its relation with developmental competence. theriogenology 2001;55:1241-1254. 14. rizos d, ward f, duffy p, et al: consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. mol reprod dev 2002;61:234-248. 15. hyttel p, grondahl c, madison v, et al: cell biological aspects of bovine oocyte maturation, fertilization and initial embryonic-development in vivo and in vitro. arch tierz 1990;33:517-529. 16. deleeuw am: number and viability of embryos collected in vivo or from the excised uteri of slaughtered donor cows. theriogenology 1992;37:907-913. 17. dias fcf, costa e, adams gp, et al: effect of duration of the growing phase of ovulatory follicles on oocyte competence in superstimulated cattle. reprod fertil dev 2013;25:523-530. 18. lerner sp, thayne wv, baker rd, et al: age, dose of fsh and other factors affecting superovulation in holstein cows. j anim sci 1986;63:176-183. 19. bennett wa, watts tl, blair wd, et al: patterns of oviductal motility in the cow during the estrous cycle. j reprod fertil 1988;83:537-543. 431 clinical theriogenology • volume 10, number 4 • december 2018 table 1. experimental timeline of protocol for superovulation of heifers. day time activities 0 am1 cidr™-insertion + 25 mg pgf2α 4 am 150 µg gnrh 5 pm 40 mg fsh 6 am/pm 40 mg fsh / 30 mg fsh 7 am/pm 30 mg fsh / 30 mg fsh 8 am/pm 30 mg fsh / 20 mg fsh + 25 mg pgf2α 9 am 20 mg fsh + 25 mg pgf2α + cidr™-removal 10 pm ai + 150 µg gnrh 11 am ai 16 pm ultrasound ovaries, document cls 17 am slaughter, excise and process uterus, locate embryos 1 am to pm was ~ a 12-h interval 432clinical theriogenology • volume 10, number 4 • december 2018 table 2. embryos recovered from each section. section1 1 2 3 4 sem2 p value left recovery, % 9.5 bc 59.1a 25.8 b 5.6 c 7.0 <0.001 right recovery, % 0 b 52.1a 41.7 a 6.3 b 11.9 0.01 total recovery, % 3 5.8 c 53.5a 33.9 b 6.8 c 9.6 <0.001 left recovered, n 0.33 b 2.08a 1.08 b 0.25 b 0.30 <0.001 right recovered, n 0 b 1.08ab 1.42 a 0.42 ab 0.41 0.08 total recovered, n 3 0.33 b 3.17a 2.50 a 0.67 b 0.52 <0.001 a-c within each row lsmeans that do not share a common superscript differed (p ≤ 0.05). 1 section: 1=uterine tube; 2=tip of horn; 3=middle third of horn; 4=base of horn (refer figure) 2 sem: n=12 3combined left and right sides for each section. 433 clinical theriogenology • volume 10, number 4 • december 2018 key1: lo: left ovary ro: right ovary l1: left uterine tube r1: right uterine tube l2-l4: sections of left uterine horn r2-r4: sections of right uterine horn     figure. designation of sections (uterine tubes and uterus). each side was divided into 4 sections, the uterine tube (l1/r1), and 3 equal lengths of uterine horn (l2-4/r2-4) with the uterine body bisected in half longitudinally.   434clinical theriogenology • volume 10, number 4 • december 2018 000_frontal ms_1 forematter for december 2018 ms_2 letter1 for december 2018 ms_2 letter2 for december 2018 ms_2 letter3 for december 2018 ms_3 table of contents ms_4 therio news for december 2018_bw 2019 therio preview therio recap with pics steiner-new officers 2018 therio sponsors 2018 exhibitors-sft board noms 2018 auction donations 2019 bartlett nomination act call for noms call for abstracts 2019 for ctj the society for theriogenology and american college of theriogenologists issue a call for research and case abstracts to be presented at the annual therio conference july 24-27, 2019 in savannah, georgia. 1. competitive category: 2. non‐competitive category: 3. case abstract category: 4. poster category: general: guidelines: what to send and how to send it abstracts not adhering to these guidelines will not be considered for presentation. deadline: abstracts must be received no later than february 15, 2019 for consideration. call for 2019 student case presentations.pdf call for student case presentations-2019 annual therio conference student case presentations call for student case presentations also available at www.therio.org guidelines for application submission society for theriogenology student case presentation competition annual therio conference, savannah, georgia, july 24-27, 2019 ms_5 editorial 001a_ms_6 roberts color pictures 002_ms_7 root kustritz 003_ms_8 ferrer 004_ms_9 wiley 005_ms_10 wallace 006_ms_11 peterson 007_ms_12 nobre 008_ms_13 sidelinger 009_ms_14 mirando background equine cervix is an important structure that acts as a defense mechanism for the uterus.1-3 cervical impairment in the mare is challenging and moderately common in the middle-aged maiden mare. failure of the cervix to fully dilate in estrus typically results in fluid accumulation in the uterus, especially postbreeding, that may lead to uterine infections and subsequent subfertility. due to the muscular nature of the cervix, manual dilation can be performed to treat this condition.4,5 adjunct treatments include local topical pharmacologic agents, most notably, misoprostol (pge1) or butylscopolamine bromide (buscopan®, boehringer ingelheim, vetmedica inc., st joesph, mo). misoprostol aids in cervical ripening in women and many domestic species, and is commonly used in mares.5 butylscopolamine bromide causes smooth muscle relaxation and is labeled for parenteral use in spasmodic colic. topical application for cervical relaxation remains anecdotal. we successfully used butylscopolamine topically for cervical relaxation in the breeding management of a mare that apparently had cervical impairment. case presentation history a 10-year-old quarter horse/paint horse mare was presented for breeding management. the mare was recently retired from an athletic career due to substantial coffin joint osteoarthritis. managing cervical impairment in the mare: a case study hannah smith, margo macpherson college of veterinary medicine, university of florida, gainesville, fl abstract a 10-year-old quarter horse mare was presented for breeding management. mare had no previous reproductive system examination. no apparent problems were noticed during initial reproductive system examination at presentation. mare was artificially inseminated with cooled transported semen. minimal uterine fluid accumulation was evident postbreeding and the cervix appeared closed; uterine lavage was performed. fourteen days postovulation, mare was examined for pregnancy. large volume of cellular fluid was present in the mare’s uterus with no evidence of pregnancy. endometrial culture and cytology results suggested bacterial endometritis. therapy included daily large volume uterine lavage, systemic and intrauterine antimicrobials, ecbolic agents, and topical application of butylscopolamine bromide over the cervix. on a subsequent posttherapy estrus, uterus was lavaged before and after insemination and butylscopolamine bromide was applied topically over the cervix to facilitate cervical relaxation and expulsion of uterine fluid. two bilateral embryonic vesicles were observed during pregnancy diagnosis and 1 was reduced successfully. mare carried a healthy singleton fetus to term and foaled a healthy colt. keywords: mare, cervix, uterus, bacterial endometritis, uterine lavage mare had not been bred previously. to hasten ovulation, mare was maintained under artificial lighting for a period of 3 months prior to presentation. clinical examination initial evaluation of the mare revealed an overall good body condition score (6/9). mammary glands were small and consistent with a nulliparous mare. perineal conformation was good and the mare had excellent apposition of vulvar lips with no vulvar discharge. mare’s reproductive tract was evaluated via transrectal palpation and ultrasonography. cervix had moderate tone and uterus had mild tone with a trace of edema of the uterus without intraluminal fluid. both ovaries had small follicles and a corpus luteum was visualized in the left ovary. it was determined that the mare was in diestrus. mare was given 250 µg of cloprostenol sodium (estrumate®, merck animal health, madison, nj) intramuscularly to hasten return to estrus. because the mare was a maiden, there was no indication to perform culture and cytology, as no potential fomites had passed the cervix. mare’s reproductive tract was examined every day via transrectal palpation and ultrasonography. six days after cloprostenol treatment, a 40 x 35 mm follicle was identified in the left ovary. edema was noted in the uterus with 5 mm of anechoic intraluminal fluid and the cervix appeared mildly dilated. mare was given intramuscularly 1.8 mg of deslorelin clinical theriogenology 2021; 13: 370 acetate (sucromate®, thorne bioscience llc, louisville, ky) to induce ovulation and 20 iu of oxytocin intramuscularly 3 times daily, to aid in uterine clearance. mare was inseminated with 40 ml of cooled transported semen 24 hours after deslorelin treatment. the insemination dose contained 1.3 x 109 progressively motile, morphologically normal sperm. oxytocin treatment was continued every 8 hours for 24 hours after insemination for uterine clearance. mare’s reproductive tract was evaluated 24 hours after insemination via transrectal palpation and ultrasonography. a corpus luteum was identified in the left ovary indicating recent ovulation. a moderate amount of cellular fluid was visualized in the uterus and uterine body (figures 1). mare’s uterus was lavaged using 5 liters of sterile saline (0.9% nacl). the initial fluid return was mildly cloudy and was recovered with intravenous oxytocin treatment. it was presumed that the intraluminal fluid accumulation in the uterus was due to lack of cervical dilation and the problem was resolved with uterine lavage. mare was discharged with plans to return 14 days after ovulation for pregnancy diagnosis. mare’s reproductive tract was examined via transrectal palpation and ultrasonography at 14 days postovulation for evidence of pregnancy. multiple small to medium follicles were noted on both ovaries and a corpus luteum was identified in the left ovary. cervix felt toned and elongated. mare’s uterus was palpably toned and mild edema was identified. substantial amount (up to 50 ml) of moderately echogenic fluid was detected in the uterine lumen, predominantly in the right uterine horn. fluid was also noted in the vagina. an embryonic vesicle was not detected. uterine cytology had marked neutrophilic inflammation with intracellular bacteria. streptococcus equi spp zooepidemicus was isolated from a bacterial culture of uterine fluid. fungal elements were not noted from either uterine cytology or culture. mare was diagnosed with bacterial endometritis. treatment plan for endometritis treatment was multi-faceted; included daily uterine lavage for 5 days, ecbolic agents, and antimicrobial treatment based on culture and sensitivity. initial uterine lavage, using 5 liters of sterile 0.9% nacl solution, was performed on the first day. fluid was infused into the mare’s uterus, 1 liter at a time, and effluent recovered. the effluent was initially mucoid and opaque in nature but became watery and more translucent with repeated lavage. fluid return was adequate during the first 2 days, but became more difficult to recover by the third lavage. immediately after uterine lavage each day, 5 million iu k+ penicillin reconstituted in 50 ml sterile water was infused into the mare’s uterine lumen. additionally, due to bacterial sensitivity to ceftiofur, the mare was intramuscularly treated with 3.2 gm ceftiofur crystalline free acid (excede®, zoetis, kalamazoo, mi). despite appropriate lavage therapy and use of ecbolic agents, intraluminal fluid retention persisted on day 3 of treatment, largely due to poor cervical dilation. digital evaluation of the cervix during uterine lavage on day 3 revealed a poorly relaxed cervix that was tortuous. on day 4 of treatment, to aid in cervical muscular relaxation, 60 mg (3 ml) of butylscopolamine bromide (buscopan®) was mixed with 1 ml sterile lubricant and infused into the cervical lumen via a sterile artificial insemination pipette. the mixture was massaged into the cervix, and cervical relaxation was digitally induced immediately after application. butylscopolamine bromide is a smooth muscle relaxant labeled for use in spasmodic colic episodes, and also has anecdotal evidence of cervical relaxation. this treatment was used with the 2 subsequent uterine lavages. in addition to cervical massage and relaxation, fluid recovery was managed by manipulating the catheter into the base of the horn by deflating the cuff and by using digital guidance in the cervix. fluid was transrectally massaged to the catheter figure 1. transrectal ultrasonogram of uterus; note substantial fluid accumulation in the uterine body (right) clinical theriogenology 2021; 13: 371 tip. the process was repeated on day 5 of treatment and fluid recovery was confirmed by transrectal ultrasonography. mare was intramuscularly treated with 20 iu of oxytocin 3 times daily for all 5 days of treatment, to aid in uterine evacuation. ecbolic agents were given 4 hours after antimicrobial infusion. mare received 250 µg of cloprostenol on day 1 of treatment to hasten return to estrus and on day 4 of treatment for of its ecbolic potency. mare was evaluated 15 days after treatment to determine if the bacterial endometritis had been resolved and for possible breeding management. mare’s reproductive tract was evaluated via transrectal palpation and ultrasonography. both ovaries were active, and the cervix was toned. uterus had soft tone palpably, mild edema, and was free of intraluminal fluid. endometrial cytology had rare degenerate neutrophils but no evidence of bacteria. endometrial culture had no bacterial growth. bacterial endometritis that was noted had resolved. mare was given 250 µg of cloprostenol to hasten return to estrus. on day 3 after treatment, the mare began to exhibit signs of estrus when exposed to a teaser stallion, as evidenced by clitoral eversion, urination, and posturing. daily evaluation of the mare’s reproductive tract revealed mild uterine edema and trace amounts of anechoic intraluminal fluid. intramuscular 3 times daily oxytocin (20 iu) treatment was initiated. on day 7 after cloprostenol treatment, 1.8 mg of deslorelin acetate was given to induce ovulation. butylscopolamine bromide (60 mg in 3 ml) was mixed with 1 ml sterile lubricant and infused into the cervical lumen via a sterile artificial insemination pipette, and uterine lavages with 5 liters of 0.9% nacl began 24 hours before insemination. mare was inseminated with 39 ml of cooled transported semen 24 hours after deslorelin treatment. the insemination dose contained 1.5 x 109 progressively motile morphologically normal sperm. six hours after insemination, butylscopolamine bromide was again applied to the cervix, and a postbreeding uterine lavage was performed. a single dose of cloprostenol was given after lavage, as ovulation had not occurred, to aid in uterine clearance. morning after insemination, double ovulation was documented on the left ovary. for 3 days following insemination, uterine lavage with 5 liters 0.9% nacl was performed, with the aid of butylscopolamine bromide applied topically to the cervix. oxytocin treatment continued 3 times daily throughout the mare’s time in hospital. the mare was discharged with an appointment to evaluate for twin pregnancy. outcome mare’s reproductive tract was examined on day 14 postovulation for evidence of twin pregnancy. multiple small to medium follicles were noted on both ovaries and 2 corpora lutea were identified on the left ovary. cervix was toned and elongated and uterus was palpably toned, and no uterine edema or intraluminal fluid was identified. two embryonic vesicles were located (figure 2). a 16 mm vesicle was identified in the left uterine horn and a 14 mm vesicle was identified in the right uterine horn. mare was given intravenously 500 mg flunixin meglumine, 60 mg butylscopolamine bromide, twice 150 mg of xylazine, and orally 20 ml altrenogest (regumate®, merck animal health, madison, nj). after multiple unsuccessful attempts to manually crush an embryonic vesicle, the procedure was discontinued. mare was hospitalized overnight and a transrectal ultrasonography was performed the following morning. mare had a 21 mm embryonic vesicle at the base of left uterine horn and an 18 mm embryonic vesicle at the base of right uterine horn. using the ultrasound transducer, pressure was placed on the right embryonic vesicle until it was visibly flattened and a rapid downward motion applied with a successful embryo crush performed. free fluid was noted in the base of the right uterine horn. left uterine horn vesicle remained intact. mare was given intravenously 500 mg flunixin meglumine and orally 20 ml altrenogest daily for 3 days after embryonic reduction. mare figure 2. transrectal ultrasonograms of uterine horns with embryonic vesicles in uterine horns clinical theriogenology 2021; 13: 372 was discharged with directions to continue 10 ml of once daily oral altrenogest at a dose of 10 ml until the day of next pregnancy diagnosis. mare returned 29 days postovulation for pregnancy diagnosis after twin reduction. transrectal palpation and ultrasonography revealed a good toned uterus and a palpable bulge was noted at the base of left uterine horn. cervix was closed and a small amount of uterine edema was noted with no intraluminal fluid. both ovaries had multiple small to medium sized follicles. an embryo, consistent with 29 days of pregnancy, was noted at the base of the left horn. an active embryonic heartbeat was detected. mare was discharged with the recommendation to continue daily altrenogest treatment until her next pregnancy evaluation. mare returned on day 61 postovulation for pregnancy evaluation. transrectal palpation and ultrasonography evaluation revealed a distinct bulge at the base of the left horn, consistent with 60 days pregnancy; the rest of the uterus, including the left uterine horn and cervix, had a good tone, and there was no evidence of uterine edema. a viable fetus with a heartbeat was visualized. the size and the position of the fetus were as expected for this stage of the pregnancy. both ovaries were visualized and had small follicles. altrenogest treatment continued through 120 days of pregnancy. mare foaled a healthy colt in the spring of 2021. discussion streptococcus equi spp zooepidemicus (s. zooepidemicus) is a beta-hemolytic gram-positive cocci, and the pathogen most commonly isolated from the uterus of mares.6 s. zooepidemicus is an opportunistic pathogen and part of the resident flora in the caudal reproductive tract, and endometritis is frequently the result of an ascending infection. poor anatomic conformation is generally associated with infection, as the bacteria must pass through the 3 anatomical barriers of the mare’s reproductive tract, namely, the vulva, the vestibulovaginal sphincter, and the cervix. furthermore, semen is not sterile, and although most commercial semen extenders contain antibiotics, bacterial contamination during artificial insemination is possible. any intrauterine procedure may introduce bacterial pathogens into the uterus, despite efforts to maintain sterility. treatment of bacterial endometritis consists of appropriate antimicrobial therapy, along with uterine lavages and ecbolic agents. oxytocin and cloprostenol treatment aid in uterine evacuation by stimulating uterine contractions. the effect is more pronounced and longer lasting with cloprostenol, and effects lasting up to 5 hours. timing of treatment in relation to ovulation is important, as postovulation cloprostenol treatment was associated with decreased corpus luteum function.8 embryo enters the uterus from the uterine tube between 5 and 6 days postovulation; therefore, the uterine environment must be conducive to maintaining a pregnancy by that point. intrauterine antibiotic infusions are performed daily for 3 7 days, depending on the severity of infection. uterine lavage is typically performed for at least during the first 3 days to aid in clearing inflammatory products. although these procedures are best performed during estrus, they may be continued beyond ovulation if needed, for up to 3 days. beyond this time uterine defense mechanisms decreases substantially under the influence of progesterone. it is noteworthy that treatment beyond 2 3 days postovulation has been associated with decreased pregnancy rates by altering steroid hormone receptors.8 equine cervix is an important structure that acts as a defense mechanism for the uterus. mare’s cervix is a simple cylindrical structure with no transverse rings. it has a normal length of 5 7 cm and a diameter of 2 5 cm.1,3 cervix has longitudinal mucosal or cervical folds that are continuous with endometrial folds of the uterine body and horns.2 mare’s cervical canal is lined with simple columnar epithelial cells that consist of alternating nonciliated goblet-like cells that produce mucin, and kinociliated cells that aid in the caudal flow of mucus. cervix is highly vascularized that ensures a rapid immunological reaction to bacterial invasion. hormonally influenced changes in cervical tone and secretions during the estrous cycle are important physical and physiological barriers to protect the uterus and facilitate uterine clearance, fertilization, pregnancy maintenance, and parturition.3 cervical changes during the estrous cycle in mares are primarily regulated by steroid hormones. progesterone tightens and tones the cervix whereas estrogen dilates and relaxes the cervix. cervix can be best assessed by vaginal speculum examination or by manual digital palpation.3 during estrus, the cervix should be dilated and loose, allowing uterine clearance. however, in some aged maiden mares, physiologic malfunction or cervical fibrosis may prevent proper dilation. cervical incompetence typically results in fluid accumulation in the uterus, especially postbreeding that may lead to uterine infections and subsequent subfertility. failure of cervical dilation may be congenital or acquired, and mares with this condition frequently accumulate fluid in the uterus. fluid can become a nidus for infection, and create a hostile environment for conceptus. cervical adhesions are acquired during parturition. although suspected age-related fibrosis of the cervix is most commonly encountered in older maiden mares, occasionally, young maiden mares may exhibit inadequate cervical relaxation and patency during estrus.4 due to the muscular nature of the cervix, manual dilation can be achieved. however, the dilation may not be long lasting. adjunct treatments include local topical application of pharmacologic agents,5 most notably, prostaglandin e1 (misoprostol) or butylscopolamine bromide (buscopan®). misoprostol aids in cervical ripening in women and many domestic species, and is commonly used in mares; however, efficacy data are inconsistent. misoprostol (200 µg) applied intracervically 2 hours before breeding resulted in 6 8 hours of cervical dilation5 and may be effective until 24 hours after treatment.6 labelled intravenous butylscopolamine bromide treatment results in smooth muscle relaxation. although topical use butylscopolamine bromide for cervical relaxation remains anecdotal, it is possible to have a rapid response. clinical theriogenology 2021; 13: 373 diligent breeding management is imperative when dealing with subfertile or problem mares. anatomical issues should be addressed, and endometritis, whether bacterial or sterile breeding induced, must be recognized rapidly and treated appropriately. approach to management should be multi-modal. uterine lavage to evacuate fluid and debris from the uterus should be performed. ecbolic agents should be used to aid in continued uterine clearance. antimicrobial(s) therapy should be initiated as needed, based on endometrial culture and cytology. identifying and addressing cervical impairment, treating bacterial endometritis, and clearing fluid accumulation in the uterus during breeding management enabled this mare to have a healthy pregnancy. learning points • equine cervix is a muscular organ and provides important defense mechanism for the uterus. • changes in cervical tone are regulated by steroid hormones of the estrous cycle. • failure of the cervix to dilate properly can contribute to poor uterine clearance and subsequent endometritis. • whereas manual dilation of the equine cervix is possible, topical application of pharmacological aid in the process. • although bacterial endometritis has a good prognosis for future fertility when diagnosed rapidly and managed appropriately, susceptible mares are more prone to recurrence of the condition in subsequent estrous cycles. conflict of interest none to report. references 1. tibary a: failure to dilate. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 2724-2730. 2. sertich p: cervix adhesions. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p 2721-2723. 3. bergfelt dr: anatomy and physiology of the mare [internet]. veteriankey. com. [cited 2021 may 12]. available from: https://veteriankey.com/ anatomy-and-physiology-of-the-mare/. 4. leblanc mm, mckinnon ao: breeding the problem mare. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 2733-2736. 5. nie gj, barnes aj: use of prostaglandin e1 to induce cervical relaxation in a maiden mare with post breeding endometritis. equine vet educ 2003;15:172-174. 6. mcnaughten j, pozor m, macpherson m, et al: effects of topical application of misoprostol on cervical relaxation in mares. reprod domest anim 2014;49:1057-1062. 7. rasmussen cd, haugaard mm, petersen mr: streptococcus equi subsp. zooepidemicus isolates from equine infectious endometritis belong to a distinct genetic group. vet res 2013;44:26. 8. card c: hormone therapy in the mare. in: samper j: editor. equine breeding management and artificial insemination. 2nd edition; elsevier health sciences: 2009. p. 89-97. clinical theriogenology 2021; 13: 374 anesthesia for small animal cesarean surgery and neonatal resuscitation sandra allweiler angell animal medical center 350 s huntington ave, boston, ma abstract anesthesia is a crucial part of cesarean surgery in small animals. approximately 16% of bitches have dystocia during whelping and > 60% of bitches with dystocia have a cesarean surgery. performing an emergency cesarean surgery is a routine procedure of paramount importance in small animal obstetrics.1 anesthetic management must be tailored to the mother and neonates. current anesthesia practice assesses each patient to arrive at the best protocol aimed to reduce fetal distress and maximizing neonatal outcomes. keywords: cesarean surgery, neonatal resuscitation, pregnancy introduction pregnancy is an adaptive multisystem maternal response that allows fetal growth to occur in the best possible environment. to better understand the challenges of providing anesthesia for pregnant animals, we need to comprehend the physiology of pregnancy. the emergency nature of many cesarean surgeries often results in the limited availability of trained and knowledgeable staff. the anesthesia provider must prepare for neonatal resuscitation and postdelivery care when planning for cesarean surgery. puppy mortality is ~ two-thirds greater during emergency circumstances compared to scheduled cesareans.2 in addition, maternal mortality during emergency delivery is increased.3 pregnancy creates physiologic and anatomic adaptations associated with an increased metabolic demand imposed by growing fetal and uterine mass. this adaptation returns to prepartum values within 4 12 weeks postpartum in women.4 cardiovascular system oxygen consumption increases to meet the metabolic demand associated with the growing fetus and reaches its peak during the last trimester. cardiac output increases by 30 40% and gravid uterine blood flow are 20 40 times higher than nonpregnant flow. blood flow to the skin and mammary glands will also increase. the increased cardiac output during pregnancy is associated with a decrease in vascular tone and arterial blood pressure (10%) and an increase in heart rate (55%) and stroke volume. in beagles, blood volume was increased by 23%.5 because cardiac work is increased during pregnancy and parturition, the cardiac reserve is decreased. patients with previously well-compensated heart disease may suffer pulmonary congestion and heart failure caused by additional cardiac workload. plasma estrogens decreased peripheral vascular resistance, increasing cardiac output, whereas systolic and diastolic blood pressures remain unchanged.6 during labor and in the immediate postpartum period, cardiac output increased an additional 10 25% due to blood being extruded from the contracting uterus.7 uterine and placental blood flow are not autoregulated, and factors that affect the flow of blood (hypotension, anesthetic drugs causing vasoconstriction and/or pain) will result in decreased fetal oxygen and nutrition. due to the downregulation of α and β adrenergic receptors, and increased vasodilation from prostaglandin drugs, increased dosing of vasopressors and chronotropic drugs may be required. ephedrine improves blood pressure in pregnant ewes without decreasing uterine blood flow, whereas dopamine effectively improved maternal blood pressure but decreased uterine blood flow and increased uterine vascular resistance at high doses. dobutamine treatment increased heart rate markedly and decreased uterine blood flow without changes to map and uterine tone.8 uterine blood flow decrease is dose-dependent and greater with dopamine than with dobutamine. recommendations to support blood pressure in pregnant dogs and cats after fluid resuscitation are with a bolus of intravenous ephedrine (0.03 0.1 mg/kg) and dobutamine (2 4 mg/ kg/min). plasma volume increases relatively more than red cell mass, resulting in pregnancy-related anemia. the magnitude of this correlates with fetal numbers. blood loss during normal parturition is buffered by the increased blood volume and red cell mass. volume resuscitation reflexes that increase heart rate, vasopressin, and cortisol concentrations are reduced in pregnant dogs and this reduction may contribute to the inability of pregnant animals to achieve normal cardiovascular homeostasis during hemorrhage.9 based on human literature, conventional guidelines for the treatment of hypotension secondary to hemorrhage during cesarean surgery recommend a rapid infusion of a crystalloid solution to restore normal blood pressure as quickly as possible. this premise is based, in part, on clinical studies, and substantial laboratory data; hemorrhagic shock in animals produced with a controlled hemorrhage model was reversible when shed blood was replaced with 2 3 times that volume of crystalloid solution. however, resuscitation with crystalloid fluids means that large amounts are needed that may induce acidosis and coagulopathy, interstitial edema formation, and microcirculation impairment. furthermore, rapid fluid resuscitation is not without consequences and can worsen the outcome by disruption of early soft thrombus, coagulopathy, hemodilution, and rebleeding. restrictive or permissive resuscitation has recently been advocated as an alternative to the current standard care. in animal studies, military settings, and studies of nonpregnant trauma patients, controlled hypotensive resuscitation has been investigated. aggressive fluid resuscitation to restore near-normal mean arterial pressure of 80 mm hg during uncontrolled hemorrhage induced massive blood loss and excessive hemodilution.10 controlled fluid resuscitation to maintain a mean arterial pressure of 40 mm hg in the presurgical treatment of severe and uncontrolled hemorrhagic shock decreased further blood loss, prevented excessive hemodilution and coagulopathy, improved the early survival rate, and reduced apoptosis of the visceral organs. studies evaluating controlled fluid resuscitation in pregnant small animals are needed. respiratory system tidal volume increases by 70%, despite the enlarged abdomen and anterior movement of the diaphragm due to an increase in respiration rate and minute ventilation. oxygen consumption increases by 20% owing to the developing fetus, placenta, uterine muscle, and mammary tissue. arterial oxygen tension remains unchanged. pregnancy also affects the mechanism of ventilation. airway conductance is increased by progesterone-induced relaxation of bronchial smooth muscles. lung compliance is unaffected. the functional residual capacity is reduced by the anterior movement of the diaphragm and leads to decreased oxygen reserves within the lungs. hypoventilation and apnea can more readily result in hypoxemia and hemoglobin desaturation. hypoxemia is exacerbated by increased oxygen consumption during labor. it is advised to preoxygenate every patient before induction of anesthesia and monitor for signs of respiratory depression and hemoglobin desaturation. gastrointestinal system gastrointestinal tract is affected by 2 main changes during pregnancy; smooth muscle relaxation from progesterone and relaxin effects, and expansion of the growing uterus. as a consequence, there is prolonged gastrointestinal transit time and slower gastric emptying leading to constipation. concentrations of peptide hormone and gastrin increase in pregnant dogs, causing an increase in the production of stomach acid. rapid intubation with a cuffed endotracheal tube will help protect the airway and prevent aspiration. there may be a benefit of h2 antagonists and proton pump inhibitors; however, further research is warranted. food should be withheld before planned cesarean surgery due to the increased risk of regurgitation and decreased lower esophageal sphincter tone. liver maintains the normal size and blood flow; however, albumin production is decreased, increasing plasma volume and perhaps changing drug pharmacokinetics and dynamics. central nervous system minimum alveolar concentration is decreased by 16 40% due to increased endorphin production. weight gain metabolic rate during pregnancy increases ~ 15% (humans). during the last 3 4 weeks of pregnancy, a bitch’s body weight will increase by 15 25%. in queens, the bodyweight increases in linear growth, with a total weight gain of up to 38% of prepregnant body weight at the end of pregnancy.11 epidural and spinal compartment regional anesthesia (epidural or subarachnoid) has the advantages of technique simplicity, minimal exposure to the fetus to drugs, less intraoperative bleeding, muscle relaxation, and analgesia. epidural veins are thin-walled and valveless. the volume of the epidural and the subarachnoid space is reduced as a result of increased size in epidural veins. epidural pressure is unchanged in pregnancy but increases during contraction. there is an increased sensitivity and distribution to local anesthetic agents during pregnancy and parturition. due to venous enlargement, only two-thirds of the nonpregnant epidural dose is required for regional analgesia. spinal cord terminates at the level of the sixth lumbar vertebra in dogs and between l7 and mid-sacrum in cats. the advantage of epidurals and spinal analgesia is that there are minimal effects on the fetus. local anesthetics given into the epidural space may result in systemic hypotension due to vasodilation. animals that have received spinal or epidural analgesia should be monitored until their motor function has returned. drugs, placenta, and fetus all drugs used in medicine have the potential to produce both beneficial and harmful effects, particularly in pregnancy where these effects must be considered by 2 or more patients, rather than 1 patient. lipophilic molecules (up to a molecular weight of 1000) diffuse simply across the placenta. hydrophilic molecules diffuse slower. less is known regarding the extent of drug passage across the placenta in dogs and cats. placenta has some ability to metabolize drugs and some carrier ability via proteins on the maternal and fetal sides. developing fetuses are most vulnerable to teratogenic drug effects during the first trimester (the first 20 days of pregnancy in dogs and cats). most anesthetic drugs have molecular weights < 300 daltons and are lipid-soluble. the large surface of the placenta allows anesthetic drugs to readily cross. in sheep, medetomidine, propofol, ketamine, and etomidate rapidly cross the placenta to develop high fetal concentrations. exceptions are glycopyrrolate, a large polar molecule, and neuromuscular blocking agents. inhalation anesthetics are highly lipid-soluble and cross the placenta. isoflurane, sevoflurane, and desflurane have low blood:gas coefficients. if the neonate breathes at delivery, these agents are rapidly cleared. in healthy beagles, sevoflurane induction resulted in a briefer loss of palpebral reflex, negative tail clamp response, time to tracheal intubation, and better quality of induction compared to isoflurane. both anesthetics were associated with a rapid and smooth recovery.12 effects of opioids and nsaids maternal treatment of opioids will result in a placental transfer of the opioids to the fetus. the extent of this transfer differs among opioids and solubility has been postulated as the cause.13 placental transfer of buprenorphine tends to be low (< 10% of placental buprenorphine reaches the fetus), whereas highly lipid-soluble opioids such as fentanyl, reach higher concentrations and persist in the fetus long after clearing the maternal circulation.14-16 respiratory depression is the most notable difference in neonates as small changes in tidal volume or respiratory rate can result in life-threatening hypoxia. after a cesarean surgery, a newborn is dependent on its respiratory system for gas exchange, and opioid-induced respiratory depression can lead to increased neonatal mortality. in addition, an apparent lack of milk production with buprenorphine in rats decreased viability and lactation indices. nonsteroidal antiinflammatory drugs nonsteroidal antiinflammatory drugs (nsaids) inhibit inflammation and produce analgesia by decreasing prostaglandin synthesis. prostaglandins cause vasodilation, increase vascular permeability, and sensitize pain receptors to noxious stimuli. nsaids have minimal effects on normal cardiovascular and respiratory systems and have been studied in humans during pregnancy and lactation. in the third trimester, nsaids and aspirin are usually avoided because of substantial fetal risks such as renal injury, oligohydramnios, constriction of the ductus arteriosus (with potential for persistent pulmonary hypertension in the newborn), necrotizing enterocolitis, and intracranial hemorrhage. maternal parenteral treatment or ingestion of most nsaids results in low infant exposure via breastmilk, such that both cyclooxygenase-1 and cyclooxygenase-2 inhibitors are generally considered safe, and preferable to aspirin when breastfeeding. excretion in breast milk is minimal.17 patient management preanesthetic evaluation should include a thorough physical examination and evaluation of blood work, emphasizing packed cell volume, total proteins, glucose, and calcium concentrations for animals in active labor. an intravenous catheter should be placed to give intravenous fluids and drugs. preoxygenation is critical because oxygen delivery to the placenta is an important factor when it comes to fetal mortality. delivering oxygen by face mask for 3 minutes prolongs the onset of desaturation in healthy dogs induced with propofol.18 drug choices propofol or alfaxalone are the drugs of choice for anesthetic induction in cesarean surgery. they both have a short half-life, rapid hepatic and extrahepatic clearance, and manageable respiratory and cardiovascular side effects. compared to propofol, alfaxalone induction resulted in improved puppy viability and may provide a slightly better quality of anesthesia for bitches.19 although there was no significant difference in puppy mortality, the alfaxalone group had a higher apgar score compared to the propofol group in the first 60 minutes of life with no impact on long-term viability.20 apgar score provides a simple and useful tool to evaluate canine newborns.21 this may be of clinical relevance when performing a cesarean surgery after-hours with limited support to observe puppy recovery. giant-size purebred pups had a higher apgar score with alfaxalone compared to propofol.21 data comparing alfaxalone and propofol in cats are sparse. alfaxalone had a less adverse influence on respiration compared to propofol in cats premedicated with medetomidine and may be a better choice when mechanical ventilation was unavailable.23 ketamine has been associated with decreased puppy vigor. the use of inhalation anesthetics for maintenance has the benefit that inhalant anesthetics are mostly eliminated through ventilation; however, inhalants depress ventilation, therefore it is recommended to maintain a light plane of anesthesia to minimize inhalant delivery across the placenta. remifentanil is an ultrashort-acting opioid that is metabolized by esterase throughout the body. remifentanil crossed the human placenta rapidly, metabolized, and redistributed, without changing the ph value of neonatal umbilical arterial blood. remifentanil did not change apgar scores and was without neonatal adverse effects.24 the author routinely uses remifentanil as continuous rate infusions (0.1 0.2 µg/kg/ minute) in cesarean surgeries to reduce inhalant concentrations and minimize systemic effects of inhalants. fetal physiology and neonatal resuscitation placenta acts as a barrier similar to the blood-brain barrier in the sense that drugs that cross the blood-brain barrier will also cross the placenta to the fetus. the fetal environment is more acidic compared to the maternal side, and more basic drugs (e.g., lidocaine) can become trapped in the fetus. the first breath after birth is stimulated by fetal hypoxia, hypercarbia, a lower body temperature, and increased sensory input from passing through the birth canal. neonatal resuscitation involves supplying tactile stimulation by vigorously drying it with an absorbent towel, emphasizing the perianal and abdominal areas. the oral cavity should be cleared of fluid by using a suction bulb; however, strong suction should be avoided to reduce damage to the delicate neonatal mucosa. bradycardia is a sign of hypoxemia and if spontaneous breathing is not obvious, vigorous rubbing should be continued and supplemental oxygen should be given via an endotracheal tube. a short-bladed laryngoscope can help visualize epiglottis and a flexible 14or 16-gauge intravenous catheter can be modified into an endotracheal tube. acupuncture point gv 26 has been successfully used for stimulating respiration in cpr and neonatal resuscitation. gv 26 is located in the midline of the philtrum of dogs and cats, level with the lower edge of the nostrils. gv 26 can resuscitate newborn animals that have a heartbeat but that are delayed in taking their first breath. neonatal heart rate is best counted by palpating a precordial pulse. normal heart rate should be > 200 beats per minute.25 using atropine to stimulate heart rate proves ineffective because bradycardia is not parasympathetically mediated at that age, but rather caused by severe hypoxia. if opioids were given, naloxone should be used sublingually or through the umbilical vein to reverse opioid-based respiratory depression. if drugs are given through the umbilical vein, a thin-walled structure within the umbilical stump, they should be diluted to 0.5 ml volume to facilitate injection and absorption. there is no evidence in the literature to suggest that doxapram stimulates ventilation in the newborn. conflict of interest none to declare. acknowledgment author thanks b.c ebert for his continuous support. references 1. bergstrom a, nodtvedt a, lagerstedt as, et al: incidence and breed predilection for dystocia and risk factors for cesarean section in a swedish population of insured dogs. vet surg 2006;35:786-791. 2. moon pf, erb hn, ludders jw, et al: perioperative risk factors for puppies delivered by cesarean section in the united states and canada. j am anim hosp assoc 2000;36:359-368. 3. moon pf, erb hn, ludders jw, et al: perioperative management and mortality rates of dogs undergoing cesarean section in the united states and canada. j am vet med assoc 1998;21:3:365-369. 4. chang j, streitman d: physiologic adaptations to pregnancy. neurol clin 2012;30:781-789. 5. kaneko m, nakayama h, igarashi n, et al: relationship between the number of fetuses and the blood constituents of beagles in late pregnancy. j vet med sci 1993:55:681-682. 6. ueland k, parer jt: effects of estrogens on the cardiovascular system of the ewe. am j obstet gynecol 1966;96:400-406. 7. ueland k, hansen jm: maternal cardiovascular dynamics. ii. posture and uterine contractions. am j obstet gynecol 1969;103:1-7. 8. fishburne ji, meis pj, urban rb et al: vascular and uterine responses to dobutamine and dopamine in the gravid ewe. am j obstet gynecol 1980;137:944-952. 9. brooks v, keil l: hemorrhage decreases arterial pressure sooner in pregnant compared with nonpregnant dogs: role of baroreflex. am j physiol 1994;266:h1610-1619. 10. lu yq, cai xj, gu lh, et al: experimental study of controlled fluid resuscitation in the treatment of severe and uncontrolled hemorrhagic shock. j trauma 2007;63:798-804. 11. root kustritz mv: clinical management of pregnancy in cats. theriogenology 2006;66:145-150. 12. johnson ra, striler e, sawyer dc, et al: comparison of isoflurane with sevoflurane for anesthesia induction and recovery in adult dogs. am j vet res 1998:59:478-481. 13. bragg p, zwass ms, lau m, et al: opioid pharmacodynamics in neonatal dogs: differences between morphine and fentanyl. j appl physiol 1995;79:1519-1524. 14. matthews ka: pain management for the pregnant, lactating, and neonatal to pediatric cat and dog. vet clin north am small anim pract 2008;38:1291-308. 15. nanovskaya t, deshmukh s, brooms m, et al: transplacental transfer and metabolism of buprenorphine. j pharmacol exp ther 2002;300:26-33. 16. cooper j, jauniaux e, gulbis b, et al: placental transfer of fentanyl in early human pregnancy and its detection in fetal brain. br j anaesth 1999;82:929-931. 17. bloor m, paech m: nonsteroidal anti-inflammatory drugs during pregnancy and the initiation of lactation anesth analg 2013;116:10631075. 18. mcnally em, robertson sa, pablo ls: comparison of time to desaturation between preoxygenated and non-preoxygenated dogs following sedation with acepromazine maleate and morphine and induction of anesthesia with propofol. am j vet res 2009;70:13331338. 19. metcalfe s, hulands‐nave a, bell m, et al: multicentre, randomised clinical trial evaluating the efficacy and safety of alfaxalone administered to bitches for induction of anaesthesia prior to caesarean section. aust vet j 2014;92:333-338. 20. doebeli a, michel e, bettschart r, et al: apgar score after induction of anesthesia for canine cesarean section with alfaxalone versus propofol. theriogenology 2013;80:850-854. 21. veronesi mc, panzani s: an apgar scoring system for routine assessment of newborn puppy viability and short-term survival prognosis. theriogenology 2009;72:401-407. 22. melandri m, salvatore a, et al: effects of alfaxalone or propofol on giant-breed dog neonates viability during elective caesarean sections. animals (basel) 2019;nov 9:96. 23. campagna i, schwarz a, et al: comparison of the effects of propofol or alfaxalone for anaesthesia induction and maintenance on respiration in cats. vet anaesth analg 2015;42:484-492. 24. kan re, hughes sc, rosen ma, et al: intravenous remifentanil: placental transfer, maternal and neonatal effects. anesthesiology; 1998;88:1467-1474. 25. root-kustritz: history and physical examination of the neonate. small animal pediatrics: first 12 months of life. st louis; elsevier saunders: 2011. thesi 2017: the effect of early postpartum intervention on the reproductive performance of anovulatory anestrus new zealand dairy cows the effect of early postpartum intervention on the reproductive performance of anovulatory anestrus new zealand dairy cows jasmin hyatt, greg jarratt, fiona hollinshead, david hanlon matamata veterinary services, matamata, nz* abstract the objective of the current study was to determine the reproductive performance of anovulatory anestrus (aa) cows treated with a routine protocol at 14 to 35 d postpartum (pp). a prospective cohort study was performed over two seasons involving 9,630 cows in 24 herds. cows not showing signs of estrus and without a corpus luteum (cl) identified on transrectal ultrasound were classified as aa (n=507). in the first season all cows were treated with an intravaginal progesterone device (cue mate 1.56 g w/w progesterone [p4]). the device was removed six days later and equine chorionic gonadotropin (ecg; 400 iu) administered im. two days later, gonadotropin releasing hormone (gnrh; 100 ug) was administered im and fixed-time ai (ftai) performed 16 to 20 h later. in the first season, all cows determined to be aa were allocated to the treatment group. in the second season, cows were randomly assigned to the treatment group or untreated control group. multivariable logistic regression analysis was used to determine the effect of treatment, cow age, parity, days pp, uterine horn diameter, ovarian diameter, ovarian follicle diameter, metricheck™ score and body condition score on reproductive performance. treatment of aa cows was associated with significant reductions in both the interval from presentation date to conception date and the interval from individual calving date to conception date by an average of 7 d. additionally, pregnancy rate to ftai was improved in treated cows when compared to control cows (25% (95ci: 21-29%) vs. 14% (95ci: 9-19%) respectively; p = 0.01). keywords: bovine, anestrus, intravaginal progesterone, uterine involution introduction the pasture-based dairy industry of new zealand relies on a concentrated seasonal calving pattern to align peak pasture growth with the time of greatest energy demand. this system requires all cows within a herd, regardless of their individual calving date, to commence breeding on the same calendar date (planned start of mating; psm). cows which have not resumed cyclical activity pp by this date are termed aa. anovulatory anestrus cows are a significant cause of reproductive wastage as a direct result of a decreased number of cows inseminated and reduced conception and pregnancy rates.1,2 in the majority of well-managed herds, all cows failing to display signs of estrus by psm are commonly assessed by a veterinarian via transrectal palpation. cows without a cl are classified as being aa. the prevalence of aa cows within a herd is influenced by calving date, breed, nutrition and peri-partum disease incidence and is documented to range from 15 to 27% in new zealand dairy herds.1,3-5 uterine involution and the resumption of normal ovarian cyclicity is reported to occur on average within 35 d pp in pasture-fed new zealand dairy cows.6 the process of involution involves uterine contractions, cellular shrinkage and necrosis, sloughing of the caruncles and regeneration of the endometrium.7,8 transrectal ultrasonography of the uterus enables an accurate measurement of uterine diameter and the associated degree of involution, as well as visualization of the presence of any free intrauterine fluid.8 given the accepted average time for uterine involution to occur and the associated return to cycling and accompanying fertility, the current “industry standard” is to wait at least 28 to 35 d pp to examine and treat aa cows.6 this protocol therefore dictates that all cows in the herd that calve in the five weeks leading up to psm are not eligible for aa treatment. pharmacologic programs for aa cows are initiated with the aim of inducing estrus and ovulation in these animals to improve the overall reproductive efficiency of the herd.1,5,6 standard aa treatment protocols initiated at least 28 d pp involve continuous p4 exposure via a controlled intravaginal releasing device for a period of six to eight days to suppress luteinizing hormone (lh) *findings from the 2014 season within this study were published and presented as an abstract at the 2016 conference of the society for theriogenology. clinical theriogenology • volume 9, number 2 • june 2017137 and follicle stimulating hormone (fsh) pulses and prime the hypothalamus for an lh surge. at the initiation of p4 treatment, administration of gnrh is commonly performed to synchronize follicular waves.9 following removal of the intravaginal p4 device, a second dose of gnrh is administered 48 h later to induce ovulation.9 often the administration of ecg at the time of device removal is performed as it has been shown to increase pregnancy rates and decrease median days to conception in aa cows.10,11 the routine induction of calving in cows with a calving date late in the season using longacting corticosteroids is a management technique that has previously been commonplace in the seasonal new zealand dairy industry.12 premature induction of calving enables artificial tightening of the calving spread by advancing the calving date of cows otherwise due to calve late in the season. this procedure was proscribed in new zealand in 2014 on animal welfare grounds, resulting in an increased number of cows calving later in the season and an associated broadening of the calving period. late calving cows are a significant source of economic loss as they are less likely to conceive by the end of mating, are more likely to conceive later in the mating period, and are less likely to remain in the herd for more than two consecutive seasons.1 in seasonally calving herds, late calving cows produce less milk in the subsequent season because all cows are ‘dried off’ on the same calendar date, regardless of individual calving dates. therefore, late calving cows have reduced lactation length and fewer days in milk (dim), reducing the income from that season. therefore recent focus has been on additional tools to bring forward potential conception dates within herds, particularly focusing on late-calving cows. a potential method of advancing conception dates within a herd is to reduce the wait period pp before aa cows are eligible for treatment. therefore the objective of the current study was to determine the reproductive performance of aa cows treated with intravaginal p4, ecg and gnrh as early as 14 to 35 d pp. additionally, clinical and historical parameters were assessed pre-treatment to determine their influence on reproductive performance. clinical examination included an assessment of body condition score (bcs), endometritis using the metricheck™ (simcro ltd, hamilton, new zealand) tool and transrectal ultrasound to identify uterine diameter, ovarian diameter and ovarian structures present. historical parameters analyzed included cow age, parity, herd, breed and season. a prospective cohort study was performed involving 9,630 cows in 24 herds. we hypothesized that the early intervention and treatment of aa cows would improve their reproductive performance by increasing the number of cows available for breeding at psm. additionally, the examination of measurable clinical and historical parameters may be useful to predict the success of treatment, enabling immediate on-farm recommendations to be made. materials and methods animals all cows from 24 seasonally-calving, predominantly pasture-based dairy herds that were 14 to 35 d pp and not showing visible signs of estrus were presented for veterinary examination at psm (n = 534, mean age: 5.1 years). pre-mating estrus detection was carried out with the aid of tail paint in all herds prior to presentation.13 all cows presented had no history of dystocia or pp disease during the current season. all herds were located in the waikato region of new zealand and were examined during the 2014 and 2015 spring breeding seasons. cows diagnosed with uterine pathology or the presence of a cl were excluded from the trial. of the 534 cows examined meeting the initial criteria, 507 were confirmed via ultrasound examination as being truly aa. breed representation was; friesian (n = 149), jersey (n = 93) and friesian-jersey cross (n = 265). a pilot study was performed during the first study season. due to all herds involved being commercial operations and the unknown potential outcomes of treatment or non-treatment, all cows in the pilot study were allocated into the treatment group (treated; n=231). after attaining results of the pilot study, cows during the subsequent and second season were randomly assigned to the treatment group (treated; n=142) or left untreated (control; n=134). clinical examination on initial examination, each cow was body condition scored on a scale of 1 (emaciated) to 10 (obese)14 and examined for the presence of vaginal exudate as an indication of endometritis using a clinical theriogenology • volume 9, number 2 • june 2017 138 metricheck™ device as previously described.15 briefly, the intravaginal device consists of a steel rod attached to a concave silicon hemisphere. advancement of the hemisphere to the cranial extent of the vaginal fornix followed by caudal retraction enables visual assessment of any material gathered in the silicon surface. material obtained by the metricheck™ device was scored as follows: 0 = no discharge, 1 = clear mucus, 2 = flecks of purulent material within otherwise clear mucus, 3 = mucopurulent but <50% purulent material, 4 = mucopurulent with >50% purulent material and 5 = mucopurulent with >50% purulent material and an odor.15 in each season, all examinations were performed by the same veterinarian (2014 = jh, 2015 = gj). ultrasound examination a transrectal ultrasound (easi-scan; bcf ultrasound australasia; auckland, new zealand; broadband straight linear rectal probe; 5mhz) examination of the uterus and ovaries was performed. combined uterine horn diameter was determined by the addition of the largest, cross-sectional diameter of each horn 4 cm cranial to the bifurcation of the uterine horns.8 pressure with the ultrasound transducer was minimized in order to obtain a circular cross section. the entire uterus was visualized to assess for the presence of intrauterine fluid. if present, fluid was measured at its largest cross-sectional diameter to the nearest mm. visualization of each ovary enabled calculation of ovarian diameter by averaging two perpendicular measurements of cross-sectional diameter. ovarian activity was described as follows: msf = multiple small follicles (≤ 5 mm), mmf = multiple medium follicles (> 5 mm), cl = corpus luteum, or the diameter of the largest follicle/s if there were ≤ 2 large follicles present. treatment protocol an intravaginal controlled release progesterone device (cue mate 1.56 g w/w p4, bioniche animal health [a/asia] pty. ltd., armidale, nsw, australia) was inserted per vagina into all treatment group animals on day 0. on day 6, the intravaginal device was removed and cows administered 400 iu ecg im (pregnecol, bayer new zealand ltd., auckland, new zealand). on day 8, 100 ug gnrh (ovurelin, bayer new zealand ltd., auckland, new zealand) was administered im. fixed-time artificial insemination with frozen semen was performed 16 to 20 h following gnrh administration. all inseminations were performed by experienced artificial insemination (ai) technicians. detection of estrus and ai continued for four to six weeks following ftai. thereafter, herd sires were used for natural service for three to five weeks. all cows were examined for pregnancy by transrectal sector ultrasonography at 42 d and 100 d after ftai. fetal age assessment of trunk diameter at both 42 d and 100 was performed to enable accurate calculation of conception dates.16 additional data additional data collected for each cow enrolled in the study included herd, breed, calving date and age at the time of treatment. data were retrieved from veterinary practice records, the herd owner and the record-keeping database infovet (infovet, zoetis new zealand ltd, auckland, new zealand). statistical analysis multivariable logistic regression analysis was used to assess the effect of cow age, breed, bcs, metricheck™ score, herd, days pp, season, combined uterine horn diameter, ovarian diameter, ovarian follicular diameter and treatment on each of the following reproductive performance parameters: pregnancy rate to ftai (pr to ftai; proportion of cows conceiving within 2 d of the initial ftai/number of enrolled cows), 21 d in-calf rate (21 d icr; proportion of cows conceiving within 21 d of ftai/ number of enrolled cows), and end of season in-calf rate (spr; proportion of cows pregnant at 100 d after ftai/ number of enrolled cows). all independent variables (including interactions) were initially included (full model) and then a backward, step-wise model building process was used where non-significant (wald’s test, p > 0.05) interactions were removed sequentially, followed by non-significant main effects. after each variable was dropped from the model, a likelihood ratio test was performed to compare the new, reduced model to the previous model in order to determine significance of the reduced model. survival analysis was used to analyze the time-to-event outcomes of days to conception after the psm and days to conception after gnrh treatment. all independent variables (including interactions) were entered into a cox proportional clinical theriogenology • volume 9, number 2 • june 2017139 hazards regression model. a backward, step-wise model building process was then used where nonsignificant (wald’s test, p > 0.05) interactions were removed sequentially, followed by nonsignificant main effects. all analyses were performed using the software package r version 3.1.3.17 results effect of treatment on reproductive performance treated cows had a significantly higher pr to ftai when compared to control cows (25% (95ci: 21-29%) vs. 14% [95ci: 9-19%], respectively; p = 0.01, table 1). there was no difference between the treatment and control groups in terms of 21 d icr (39% [95ci: 34-45%] vs. 33% [95ci: 25-41%], respectively; p = 0.2, table 1) or spr (72% [95ci: 68-77%] vs. 73% [95ci: 65-80%], respectively; p = 0.8, table 1). the mean interval from the administration of gnrh to conception date was significantly shorter in treated cows when compared to control cows (24  20 d vs. 31  22 d, respectively; p = 0.007, table 2). similarly, the interval from calving to conception was significantly reduced in treated cows (60  21 d vs. 67  23 d; p = 0.005, table 2). effect of days postpartum on reproductive performance the number of days pp was categorized into three groups; ≤ 24 d, 25 to 29 d and ≥ 30 d. there was no difference in pr to ftai (23% [95ci: 16-32%] vs. 21% [95ci: 15-27%] vs. 22% [95ci: 17-28%], respectively; p = 0.8, table 3), 21 d icr (42% [95ci: 33-52]%) vs. 33% [95ci: 2640%] vs. 39% [95ci: 33-46%], respectively; p = 0.2, table 3) or spr (69% [95ci: 60-78%] vs. 73% [95ci: 66-80%] vs. 73% [95ci: 67-79%], respectively; p = 0.8, table 3) amongst the three groups. effect of uterine horn diameter on reproductive performance combined uterine horn diameter was categorized into < 4.5 cm and ≥ 4.5 cm. overall the combined horn diameter did not affect pr to ftai (21.6% [95ci: 10-29%] vs. 22.6% [95ci: 9-29%], respectively; p = 0.8), 21 d icr (35% [95ci: 29-47%] vs. 43% [95ci: 32-54%], respectively; p = 0.6) or spr (72% [95ci: 68-78%] vs. 73% [95ci: 69-78%], respectively; p = 0.9). effect of clinical parameters on reproductive performance when controlled for treatment and days pp, there was no significant effect of herd, cow age, breed, bcs, metricheck™ score, ovarian diameter or ovarian follicular diameter on any of the reproductive performance outcomes. discussion the current study aimed to determine the reproductive performance of aa cows identified and treated between 14 and 35 d pp with intravaginal p4, ecg and gnrh. assessment of clinical and historical parameters pre-treatment was also performed with the objective of determining any possible effect on reproductive performance to aid in predicting the success of treatment. this is the first study in new zealand to assess reproductive performance of treated aa cows compared to non-treated controls in the very early pp period. a significant increase in pregnancy rate to ftai was demonstrated in treated cows when compared to non-treated controls (25% vs 14%, respectively). this is most likely a direct result of p4 ‘priming’ of the reproductive tract during the period of intravaginal device insertion, with increased p4 concentrations in the preceding luteal phase increasing the likelihood of conception.5,18-20 however, the overall pr to ftai of treated cows in this study was reduced compared to previous studies that demonstrated pr to ftai following aa treatment to be 48.9%,21 47.1%,22 38.8%.5 this is likely a result of those studies using a treatment protocol involving intravaginal p4 and estradiol benzoate (eb) which is no longer commercially available in new zealand.23 alternatively, this result may have been influenced by the reduced pp wait period in the current study. numerous studies have examined the impact of aa cows in both new zealand and overseas.1,5,21,22,24-28 a delay in the resumption of normal ovulatory estrus cycles has been associated with environmental factors including decreased bcs, peri-partum disease, reduced energy intake, negative energy balance and suckling.1,29 the current study has demonstrated that the innovative clinical theriogenology • volume 9, number 2 • june 2017 140 early treatment of aa cows can lead to conception occurring on average by 60 d pp, significantly less than non-treated controls. in a seasonal herd, this advancement of conception date has considerable production and economic benefits. in this study, treated cows demonstrated a 7 d reduction in the interval from calving and time of treatment to conception when compared to non-treated controls. the most significant advantage of earlier conception in seasonal herds is an extra 7 dim per cow, increasing the income from that season. in seasonally calving dairy herds, all cows are dried off on the same date, regardless of their calving date. with the current milk price of $6/kg milk solids, seven days of extra production provides an additional $84 per cow per lactation assuming production of 2kgms/day. in addition, the cumulative positive effects of earlier conception continue into future seasons. firstly, it brings the calving date for the following season forward, aligning the current season’s late calving cows with the rest of the herd. with the banning of pharmaceutical calving induction in 2014, this presents a potential alternative management tool to advance conception dates and bring about the consolidation of the calving pattern.1 furthermore, an earlier calving date next season lengthens the exposure of individuals to the following breeding season, increasing the likelihood of early conception and reducing the risk of not being pregnant at the end of the season. this effect is cumulative, with each season bringing forward the potential calving date and subsequent conception dates.1 the current study demonstrated no significant effect of days pp at the time of first presentation on the reproductive performance of both control and treated cows. this is very positive, indicating a significant economic benefit can be gained from treating aa cows sooner pp than the current industry standard of 35 d. this advantage is in agreement with a previous study in which no significant difference was noted in mean calving to conception interval when comparing the reproductive performance of cows treated 14 to 21 d pp with those 22 to 42 d pp and those greater than 42 d.30 whilst this aforementioned study also demonstrated a significant reduction in season pregnancy rate and submission rate in cows less than 21 d pp when compared to those cows greater than 21 d pp, control animals were not included in the trial and therefore conclusions are better drawn from the current study which did include control animals. one of the objectives of the current study was to analyze the effect of specific clinical and historical parameters measured at the time of treatment on reproductive performance. this would allow for practical recommendations to be made on-farm at the time of treatment based on clinical findings to aid in predicting the success of treatment. in our study there was no effect of bcs at the time of presentation on the outcome variables. whilst this is in agreement with a previous study,24 an improvement in reproductive performance in cows with higher body condition scores has also been reported.21 the narrow range of condition scores in the current study may account for the lack of effect of bcs in our study. whilst there was no significant effect of uterine diameter on reproductive performance when results from both seasons were combined, results from the 2014 season had indicated that cows with a combined uterine horn diameter of <4.5cm had significantly improved pr to ftai than cows >4.5cm. previous studies have identified that increased uterine volume and delayed involution can be associated with decreased fertility in multiparous, lactating dairy cattle, specifically a reduction in pr to ftai.21 it is possible in the current study that the discrepancy in uterine diameter results between seasons may have been an operator effect, with two different operators each performing measurements for a single season. whilst this may indicate that ultrasound examination is a less useful aid in a practical setting, we have only examined the use of two individuals. these potential discrepancies may have been overcome by increasing the number of operators involved or further standardising the methodology of measurements taken transrectally. however, the 2014 results alone suggest that if consistent measurements are taken it may be possible for the measurement of uterine diameter to become a valuable predictive tool in the success of early aa treatment. additional studies are necessary to further analyze the predictability of uterine horn diameter on reproductive performance. the current study did not demonstrate an association between metricheck™ score and reproductive performance. this is in contrast to previous studies that found a significant reduction in conception, pregnancy and non-return rates in metricheck™ positive animals.15,31,32 however, a previous new zealand study described the reduced specificity of this diagnostic tool for the detection clinical theriogenology • volume 9, number 2 • june 2017141 of endometritis, with only 37% of cows metricheck™ positive (scores 2 and greater) showing evidence of endometrial inflammation on cytology.33 the short interval from parturition to diagnosis in the current study may further increase over-diagnosis of endometritis using this technique, which may explain the lack of correlation between metricheck™ score and reproductive performance.31-34 conclusion the present study demonstrated a significant improvement in reproductive performance when aa cows were treated as early as 14 d pp. the potential advancement of conception date by 7 d provides economic benefits by increasing the subsequent lactation length and dim per treated cow. additionally, the resulting synchronization of ovarian cyclicity of late calving cows with the rest of the herd at psm is likely to have significant positive economic and production impacts in a seasonally calving industry during subsequent seasons. acknowledgements the support and co-operation of the participating herd owners and their staff is gratefully acknowledged. cue-mates, gnrh and ecg were kindly donated by bioniche animal health australasia. references 1. rhodes fm, mcdougall s, burke cr, et al: invited review: treatment of cows with an extended postpartum anestrous interval. j dairy sci 2003;86:1876-1894. 2. xu zz, burton lj: reproductive efficiency in lactating dairy cows. proc nz soc an prod 1996;56:34-37. 3. mcdougall s, burke cr, williamson nb, et al: the effect of stocking rate and breed on the period of postpartum anoestrum in grazing dairy cattle. proc nz soc anim prod 1995;55:236-238. 4. rhodes fm, clark ba, nation dp, et al: factors influencing the prevalence of postpartum anoestrus in new zealand dairy cows. proc nz soc anim prod 1998;58:79-81. 5. mcdougall s, compton cwr, hanlon dw, et al: reproductive performance in anestrous dairy cows following treatment with two protocols and two doses of progesterone. theriogenology 2005;63:1529-1548. 6. xu zz, burton lj, macmillan kl: reproductive performance of lactating dairy cows following oestrus synchronisation with progesterone, oestradiol and prostaglandin. nz vet j 1996;44:99-104. 7. senger p: the puerperium and lactation. pathways to pregnancy and parturition. 3rd ed. redmon(or): current conceptions inc; 2012. p. 316-23. 8. silvestre ft, bartolome ja, kamimura s, et al: postpartum suppression of ovarian activity with a deslorelin implant enhanced uterine involution in lactating dairy cows. anim reprod sci 2009;110:79-95. 9. kasimanickam r: pharmacological intervention of estrous cycles. in: hopper r, editor. bovine reproduction. ames(ia):wiley; 2014. p. 304-313. 10. xu zz, burton lj, macmillan kl: treatment of post-partum anoestrous dairy cows with progesterone, oestradiol and equine chorionic gonadotrophin. nz vet j 1997;45:205-207. 11. bryan ma, bo g, mapletoft rj, et al: the use of equine chorionic gonadotropin in the treatment of anestrous dairy cows in gonadotropin-releasing hormone/progesterone protocols of 6 or 7 days. j dairy sci 2013;96:122-131. 12. welch ras: the use of corticosteroids to induce parturition. nz vet j 1977;25:224-225. 13. macmillan kl, curnow rj: the application of oestrus synchronization in new zealand dairy herds. proc nz soc anim prod 1976;36:50-57. 14. dairynz: dairynz body condition scoring hamilton, new zealand: dairynz ltd.; [updated february 2012. available from: https://www.dairynz.co.nz/media/443179/bcs_reference_guide.pdf. 15. mcdougall s, macaulay r, compton c: association between endometritis diagnosis using a novel intravaginal device and reproductive performance in dairy cattle. anim reprod sci 2007;99:9-23. 16. kahn w: sonographic fetometry in the bovine. theriogenology 1989;31:1105-1121. 17. r development core team (2008). r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. isbn 3-900051-07-0, url http://www.r-project.org<http://www.rproject.org/>. 18. rosenberg m, kaim m, herz z, et al: comparison of methods for the synchronization of estrous cycles in dairycows. 1. effects on plasma progesterone and manifestation of estrus. j dairy sci 1990;73:2807-2816. 19. folman y, kaim m, herz z, et al: comparison of methods for the synchronization of estrous cycles in dairy-cows. 2. effects of progesterone and parity on conception. j dairy sci 1990;73:2817-25. 20. holness dh, sprowson gw, sheward c, et al: studies on plasma progesterone concentrations and fertility in friesland dairy-cows during the postpartum period. j agric sci 1981;97:649-654. 21. hanlon dw, wichtel jj, xu zz, et al: the reproductive performance of anoestrous dairy cows following treatment with progesterone and oestradiol prior to the start of mating. nz vet j 2000;48:136-143. 22. xu zz, burton lj, mcdougall s, et al: treatment of noncyclic lactating dairy cows with progesterone and estradiol or with progesterone, gnrh, prostaglandin f-2 alpha, and estradiol. j dairy sci 2000;83:464-470. 23. kasimanickam r: pharmacological intervention of estrous cycles. in: hopper rm, editor. bovine reproduction. ames(ia): wiley; 2015. p. 304-313. clinical theriogenology • volume 9, number 2 • june 2017 142 24. hanlon dw, davidson pj, hittmann ar, et al: supplementing previously treated anestrous dairy cows with progesterone does not increase first-service conception rate. theriogenology 2005;63:239-45. 25. mcdougall s, compton cwr, anniss fm: effect of exogenous progesterone and oestradiol on plasma progesterone concentrations and follicle wave dynamics in anovulatory anoestrous post-partum dairy cattle. anim reprod sci 2004;84:303-314. 26. rhodes fm, clark ba, mcdougall s, et al: insemination at the second of two induced oestrous periods in anoestrous dairy cows increases conception rates to first service. nz vet j 1999;47:39-43. 27. segwagwe bve, malmo j, macmillan kl, et al: the reproductive performance of dairy cows with anovulatory anoestrus that were injected with either gonadotrophin-releasing hormone or oestradiol benzoate as part of a retreatment process after insemination. j s afr vet assoc 2007;78:6-11. 28. saxena ms, gupta sk, sanwal pc, et al: plasma progesterone profile during anestrus and anovulatory estrus in crossbred cattle. j vet physiol allied sci 1990;9:27-30. 29. crowe ma: resumption of ovarian cyclicity in post-partum beef and dairy cows. reprod domest anim 2008;43:20-28. 30. nation dp, macmillan kl, taufa vk: efficacy of an anoestrous treatment for dairy cows is reduced if administered within three weeks of calving. proc nz soc anim prod 1997;57:228-230. 31. lambertz c, volker d, janowitz u, et al: evaluation of vaginal discharge with the metricheck device and the relationship to reproductive performance in postpartum dairy cows. anim sci j 2014;85:848-852. 32. leblanc sj, duffield tf, leslie ke, et al: defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows. j dairy sci 2002;85:2223-2236. 33. peter at, jarratt gm, hanlon dw: accuracy of diagnosis of clinical endometritis with metricheck tm in postpartum dairy cows. clinl therio 2011;3:461-465. 34. krueger l, koerte j, tsousis g, et al: transrectal doppler sonography of uterine blood flow during the first 12 weeks after parturition in healthy dairy cows. anim reprod sci 2009;114:23-31. table 1: reproductive performance of treated and control cows group n pr to ftai (%) 95% ci 21 d icr (%) 95% ci spr (%) 95%ci treatme nt 373 25 (21-29) 39 (34-45) 72 (68-77) control 134 14 (9-19) 33 (25-41) 73 (65-80) p-value 0.01 0.2 0.8 table 2: effect on conception date of treated and control cows group n mean gnrh to conception interval (days) mean calving to conception interval (days) treatme nt 373 24  20 60 21 control 134 31 22 67 23 p-value 0.007 0.005 table 3: reproductive performance of days postpartum days postpartum n pr to ftai (%) 95%ci 21 d icr (%) 95%ci spr (%) 95%ci ≤ 24 111 23 16-32 42 33-52 69 60-78 25-29 188 21 15-27 33 26-40 73 66-80 ≥ 30 208 22 17-28 39 33-46 73 67-79 total 507 22 38 73 p-value 0.8 0.2 0.8 clinical theriogenology • volume 9, number 2 • june 2017143 clinical theriogenology • volume 9, number 2 • june 2017 144 omniblank: 2019 bartlett address: comparative theriogenology: international perspectives the 2019 bartlett address comparative theriogenology: international perspectives ahmed tibary comparative theriogenology section, department of veterinary clinical sciences college of veterinary medicine, washington state university, pullman, wa introduction imagine my great surprise when, instead of telling me about his latest cases or adventures in reindeer theriogenology, dr. isaac bott unexpectedly informed me that i was selected for the bartlett award. i was speechless. it is beyond my wildest dreams to be selected for such an honor. i am grateful and humbled knowing there are a great number of deserving people within our society and college. as is customary, i am asked to stand before my peers and friends and deliver the bartlett address. i would like to share with you my experiences over the years and the impact this specialty has had on my professional and personal life. beginnings my family has a long agricultural tradition. our farms included not only crops and tomatoes but also a little bit of every traditional production animal species (cattle, sheep, and goat). my main interest was horses. my father was a member of our hometown racing commission (one of only 3 race tracks in the country at that time). therefore, i grew up involved with thoroughbreds and anglo-arab breeding and racing. i spend all my free time sailing, riding and going to the track weekly during the racing season. i have to admit that i was – and still am very fond of cats. i owned a breeding colony of siamese. it was no surprise to anyone that i ended up selecting veterinary studies when i graduated from high school. i obtained my “doctorate en medecine vétérinaire” in 1980, from the only veterinary school in morocco, the “institut agronomique et vétérinaire hassan ii". the 6 year education program paralleled that of european schools and in fact many of my professors were from french, belgian or german veterinary schools. the veterinary curriculum was developed with novel ideas and included amongst other subjects a requirement in rural sociology, economics, and statistics, for which i remain very grateful because of how much this background helped me later. i developed an interest in reproductive medicine in my 4th year of veterinary medicine, guided by my first mentors, german veterinarians dr. gero hanschke and professor dr. peter glatzel with whom i had my first publications. dr. peter glatzel was my main advisor and mentor for my dvm thesis which allowed me to introduce a structured breeding soundness examination for stallions at the national stud farms and particularly for the barb and arab-barb horses for the first time. i also became involved in donkey semen cryopreservation from two endangered species the catalan and poitou which were heavily used for mule production. this experience was the final impetus to become a theriogenologist. thanks to the support of my advisor, i was hired as an instructor at the veterinary college, where i practiced as a clinician in the area of reproduction which included all aspects of reproductive medicine and surgery, neonatology, and bovine mastitis. during these years and under the leadership of professor lahlou-kassi, i had the opportunity to meet many reproductive specialists from around the world who were interested in moroccan prolific sheep and horses. my professional development did not become a reality until i obtained a scholarship to go to the university of minnesota for advanced training through a us-aid program. minnesota experience what a wonderful experience it was! i arrived in minnesota in july 1982, and after a short intensive english program i started my graduate studies in the theriogenology section. there i found the 175 clinical theriogenology • volume 11, number 3 • september 2019 perfect union between a great theriogenology faculty (drs. ray zemjanis, brad seguin, shirley johnston, norm williamson, and mel fahning) and a great reproductive physiology faculty in animal science (drs. ed graham, bo crabo, and al hunter). all these people have greatly and positively impacted my learning, professional and personal growth. i was most fortunate to get involved with faculty from other units notably dr. ashley robinson and dr. don johnson who mentored me in epidemiology and international work, and dr. al weber who introduced me to the fascinating world of cytogenetics. i was surrounded by a wonderful group of residents and graduate students many of whom have become successful theriogenologists and leaders in the field (drs. harry momont, farhat vahdat, hisham fahmi, fran smith, doug freeman, carlos gradil, miguel fortin, rebecca davies, juan samper, peggy root, stefano romagnoli, juan romano, jane barber, gary nie, atushi tajima, and sue swanson). it is within this group that i learned the science of gamete physiology and cryopreservation, fundamentals of clinical theriogenology and was initiated in advanced techniques in theriogenology which i put into practice as soon as i got back to morocco upon finishing my ms degree and completing my prelim to continue my phd research in morocco. back to morocco education that i received in minnesota opened many doors to me and within a few months i was in charge of the cryopreservation lab and equine reproduction at my school. i was very fortunate to have the confidence of several large equine (royal stud farms, national stud farms, equestrian federation) and bovine (santa gertrudis ranches and dairies) operations. i met other mentors who helped me along the way, in particular dr. abdelhafid yacoubi who included me in the reproductive program of a large herd of santa gertrudis (ranch adarouch). from 1985 1989, i participated in training of veterinarians and providing clinical services to large and small animal clients. however, i believe that my major contribution was to serve as a mentor for 2 women; drs. samira manar and nadia lotfi who wanted to work with large animals and who are now leaders in their respective fields (small ruminant reproduction and bovine reproduction). i was fortunate that the field was wide open to engage in many activities in research and development. i got involved in dairy cattle management, helping to introduce dairychamp® (which i translated to french), extension activities (ram breeding soundness examination) and even served as the director of the first veterinary program in peace corps. i was fortunate to be requested to help with reproductive issues on wildlife at the national zoological park working on large cats (particularly the barbary lions), gazelles, buffaloes, and other creatures, thanks to my friend and colleague dr. brahim haddane. i left morocco in the winter of 1989, with the promise to then general director of the iavhassan ii, dr. m’hamed sedrati who was a great mentor and supporter, to continue serving the institution whenever needed. did i say that in addition to wanting to discover new horizons in my discipline, i have also left something very dear in minnesota? my soon to be wife brigitte. back to minnesota i was welcomed again into the theriogenology team at minnesota where i worked as a clinical assistant professor and helped with dr. graham’s research and teaching as he was in fading health. the support i received in those uncertain years will remain forever engraved in my memory. it is during this second “tour” that i had the great chance to meet and learn from dr. patty olson and dr. jerry olson who mentored me along with all the other faculty for the theriogenology boards. i kept my contacts with my alma mater and continued to provide lectures and even initiated a series of books on equine reproduction in french. 176clinical theriogenology • volume 11, number 3 • september 2019 abu dhabi years in the fall of 1991, i was asked to travel to abu dhabi for a consultation on a project on camel embryo transfer that my colleague, dr. abdelhaq anouassi, had initiated with the veterinary technician i trained (mr. mustapha adnani). i had never worked on camels before, except to help colleagues with ultrasonography for their research. dr. anouassi had done tremendous work at inra-france on camel reproductive endocrinology and i was bringing a clinical approach to this lab. together, we spent long days working on development of reproductive evaluation techniques in camels which allowed us to publish a book on camelid theriogenology after 7 years. we expanded the labs to include equine with the collaboration of friends (drs. ahmed abdelhamid, kamal pasha, andrew daglish, and peter davis). my only regret during those years is not to have published more on our clinical observations on camels and equine, notably outbreaks of habronemiasis in stallions and the effect of high heat and humidity on reproductive function in horses. i was lucky that my contract was only for the breeding season which left me 4 months to travel back to morocco and the usa to work on my publications and to teach. throughout that time, i wanted to return to my passion: veterinary education and research, and was looking for a position in theriogenology. a break came finally in 1998 where i interviewed both at the university of massachusetts equine reproduction (dr. doug freeman, chair) and at wsu for a dairy cattle position (dr. shirley johnston, chair). i was offered both but opted to go to wsu because it is was tenure-track and was a veterinary program which would allow me to continue my involvement with clinical training and research. wsu: a new beginning i arrived pullman in the summer of 1998, already 20 years ago! dr. mushtaq memon, was mainly doing small animal theriogenology and dr. duane michelson was in charge of bovine reproduction. i thank them both for their encouragement and for introducing me to their clients. my position was in field disease investigation unit (fdiu) as a reproduction specialist. having had most of my work experience outside of the usa university system, i accepted (not without a challenge to my pride, i have to admit) to start from the beginning and go through all the painful processes of pretenure, tenure, promotion etc. i was very lucky to have started with fdiu under the leadership of dr. clive gay. it was a wonderful opportunity for professional growth and i am very grateful for his guidance as well as that of all the members of the team (drs. larry fox, john gay, and dale hancock). i was able to participate in his research on lupine toxicosis. the work in the fdiu was exciting, but my heart was in developing a clinical theriogenology program. i was allowed under the leadership of dr. warwick bayly to move into the clinic and develop the comparative theriogenology service. obviously, no service can be developed without the support of other critical clinical specialties and i am indebted to my colleagues in equine and agricultural animal medicine and surgery for their support. in particular, dr. claude ragle was very supportive and became a family friend, kidnaping me once to go on one of his mule rides in the wilderness. during my tenure at wsu, i was able to make progress toward tenure due to the support from colleagues from the college and from animal science, in particular drs. jerry reeves and phil senger. it is the support of these fine colleagues and that of the students who honored me with so many teaching awards including the norden distinguished teacher awards in 2001 and 2005 that allowed me to “survive’ the pretenure part of my career. the comparative theriogenology program began to strengthen in 2007 when ms. shirley sandoval joined me as the lead veterinary instruction technologist. her practical experience and her passion for her work allowed us to provide several services. we initially started capitalizing on the lack of services for camelids. i had never touched an alpaca prior to my arrival to wsu and soon we were receiving referrals from several states. from there we went on to develop an equine theriogenology program, then a small ruminants service. the program continued to strengthen thanks to wonderful 177 clinical theriogenology • volume 11, number 3 • september 2019 residents and graduate students (drs. cheryl fite, jacobo rodriguez, lisa pearson, yessinia picha, aymen elzawam, hamid alkar, alexis campbell, salman waqas, michella ciccarelli, agustin ruiz, and cristian patino). all had an impact on the program that went beyond my expectation. i pause here to thank dr. harmon rogers who involved them all in small animal theriogenology. in research, i continued working with my colleagues in abu dhabi on camel projects, but on campus i was fortunate to be included in research programs of dr. subramaniam srikumaran on big horn sheep and dr. jon oatley on spermatogonia stem cells transfer. my return to the us allowed me to finally be able to actively participate in the society for theriogenology and the american college of theriogenologists, and later the theriogenology foundation. it was a great honor for me to serve in the leadership of these organizations. this allowed me to get to know other clinicians and researchers from across north america and beyond, with similar interest. i felt and still feel welcomed by all. on international work being able to participate in research and continuing education abroad is certainly one of the highlights of my life and professional career. as a young veterinarian in morocco, i was on the receiving end of teaching from visiting professors and scientists. it is only natural that i would want to return the favor. i was given this opportunity by several people as i continued to learn. first and foremost, i never lost contact with my alma mater which continues to nourish my professional and personal life and where i continue to collaborate in teaching, research and continuing education with colleagues and former students. it is in the spirit of international work and thanks to my continuous involvement with veterinary education in north africa that i was able to associate some colleagues from wsu and other theriogenologists such as dr. carla carleton to my work there. one of the activities i am most proud of is that i was able to use my experience and all i have learned on camels in the middle east to train wonderful veterinarians and technicians from the 91st and 96th us civil affair battalions, who in turn could help many people who depend on these animals for their livelihood. i thank professors jorg and christina aurich for involving me in their equine course and resident seminars in vienna. professors luis losino and marcelo miragaya have been wonderful friends and made me discover many aspects of equine and camelid science in argentina and chile. professors julio sumar and edwin mellisho provided opportunities for involvement in peru. dr. marco alvarenga gave me the opportunity to discover brazilian equine industry. these international events allowed me to share experiences with several great theriogenologists. i have often wondered why i got all these invitations. i always came to the same conclusion, there are aspects of these international interactions that go beyond technical or scientific expertise. people like to share their experiences in a respectful and fun atmosphere. this is why i always try to involve my previous residents in international work. i am very grateful to dr. jacobo rodriguez and dr. alexis campbell who help me with camel projects in qatar. international involvement includes also the ability to receive overseas students. unfortunately, such experiences are slowly disappearing particularly for students from developing countries. at wsu, and under the leadership of dr. erik stauber, i was fortunate to work with international veterinary students who later become theriogenologists, drs. natali krekler, maria ferrer, gislaine dujovne. i know that this an effort of the theriogenology foundation through the zemajin funds which has benefited one of my graduate students (dr. y. picha). concluding remarks and pet peeves i cannot end without sharing some pet-peeves that i have developed over the years. the list is long but i limited to top 4. 1) do not use the verb “utilize” when “use” is sufficient, 2) we do not do 178clinical theriogenology • volume 11, number 3 • september 2019 “preg check” nor do we “sleeve’ or “arm”, we do pregnancy diagnosis and reproductive evaluations, 3) we do not do rectal palpation but “transrectal palpation”, 4) last, it may not be true for all but when you are so invested in your specialty, everything that touches it is personal. i have to conclude with some sort of words of wisdom. for me it is simple: what goes around comes around, so i tried to offer young aspiring theriogenologists what others had offered to me throughout my career. i have been asked frequently in recent months about when i will be retiring. my answer is, why retire from such an exciting profession and specialty? many of the concepts of what theriogenology is, laid out by my predecessors, still apply. i urge all theriogenologists to think big, think international and may you all continue to enjoy our specialty in health and happiness! 179 clinical theriogenology • volume 11, number 3 • september 2019 180clinical theriogenology • volume 11, number 3 • september 2019 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /pagebypage /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile 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/metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename (srgb iec61966-2.1) /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [612.000 792.000] >> setpagedevice 2013: fundamentals of bull selection fundamentals of bull selection ramanathan kasimanickam,a ahmed tibary,a john kastelicb adepartment of veterinary clinical sciences, washington state university, pullman, wa; bproduction animal health, university of calgary, calgary, ab, canada abstract bull selection is a critically important decision, as bulls contribute to genetics of the farm well beyond their lifetime. irrespective of the method of breeding (natural service or artificial insemination), a sire will produce more calves in his lifetime compared to a cow. in addition, the genetic make-up of each calf is 50% from the calf’s sire, and of the 50% from the dam, half of that is from her sire and one quarter from her grandsire, etc. thus, a calf's genetics is derived from several generations of bulls. this paper discusses important criteria and provides guidelines for successful bull selection. keywords: bull selection, genetic improvement, estimated progeny differences, breeding management introduction genetic make-up of all bulls is not equal – the range is very wide! to achieve economic success, it is important to select bulls with genetics that sire offspring with desirable traits.1 for breeding purposes, bulls are grouped into three major categories: 1) maternal bulls used for breeding heifers (to produce calves with low birthweight, but good weaning weight and perhaps future cows); 2) maternal bulls used for breeding cows (to produce future cows); and 3) terminal bulls used for breeding cows (to produce market cattle). one, two, or all three categories can be useful in a breeding program, depending on the farm’s goals and resources (e.g. nutrition and environment). in that regard, veterinarians should assist producers in selecting appropriate bulls. there are several factors and steps involved in the selection process. farm goals farm goals are the basis for bull selection; they should guide selection of bulls with traits of economic importance.2,3 in addition, it is essential that the farm environment and the producer’s production and marketing strategies are also considered. key questions include:  how will this sire be used in the overall breeding program?  will the bull be used to breed heifers, mature cows, or both?  will replacement females be retained?  how will the calf crop be marketed?  what are the nutritional resources?  what are the labor and management resources?  what are the environmental conditions? farm strengths and weaknesses good records are essential to recognize overall strengths and weaknesses of a cattle operation. knowing basic production and reproduction parameters (e.g. length of breeding and calving seasons, calving and weaning percentage, weaning weights, average daily gain, carcass data, etc.), is necessary to assess overall performance of the farm, areas of strength and those needing improvement; this will indicate what trait(s) should be given highest priority during bull selection.2 selection priorities emphasize factors expected to have the largest impact on profitability that can be impacted by bull selection. income is derived from weaning percentage, weight at sale, value per pound, carcass merit, etc. although costs are related to a combination of several production and reproduction traits, bull selection should focus on a few priority traits (weighted for economic importance) rather than attempting clinical theriogenology • volume 5 number 2 • june 201397 to change many traits simultaneously. because performance is a function of both genetics and environment/management, the potential economic advantage of superior genetics can be annulled by poor management. therefore, veterinarians should assist producers in evaluating the impact of management (nutrition and animal health), as well as the impact of genetics, based on the priorities of that farm.3 utilization of selection tools once selection priorities have been established (through close examination of farm goals and current status), there are several useful tools to assist in making genetic improvement. for a commercial cattle operation, there are two basic ways to provide genetic improvement: crossbreeding and selection of parent animals. genetic improvement using these pathways can relate to farm goals. for example, if the emphasis is to improve reproductive parameter (fertility) and longevity, crossbreeding should be emphasized and selection of parent animals given a lower priority. in contrast, if the focus is to improve carcass traits (increased marbling or decreased back-fat) selection of parent animals should be a much higher priority than crossbreeding. crossbreeding crossbreeding, namely mating sires and dams of different breeds, usually results in calves with beneficial characteristics from both breeds. crossbreeding is an important production practice, as it offers advantages from both heterosis and complementary.4 utilization of different breeds in a complementary fashion through a structured crossbreeding strategy provides opportunities for improvement in multiple traits.5 cattle producers can gain hybrid vigor and complementarity simply by crossing appropriate breeds. however, sustaining acceptable levels of hybrid vigor and breed complementarity over the long term requires a well-planned crossbreeding system. useful criteria for designing crossbreeding systems6,7 include:  advantages of component breeds – the breed included in a crossbreeding system must be well chosen and must bring favorable characteristics to the offspring  hybrid vigor – in crossbreeding, the offspring are often bred to have better vigor than either parent, along with a specific number of desired traits. most crossbreeding systems do not achieve maximal hybrid vigor; backcrossing should be limited to maintain acceptable levels of hybrid vigor.  breed complementarity – refers to production of a more desirable offspring by crossing breeds that are genetically different from each other, but have complementary attributes. a good example would be mating a charolais bull (growth and retail yield) to a gelbviehangus cross cow. the result – the cow has good milk production and fertility, and the calf has more growth/retail yield. the characteristics gained from this mating complement each other.  strength of performance – an ideal crossbreeding system should produce a consistent product. it is much easier to market a uniform group of animals than a varied one. from the management aspect, it is also much easier to manage a female population that is essentially one type, rather than one made up of many types, each with its own requirements. design a crossbreeding system with less variation to offer increased consistency.  replacement consideration – in terms of hybrid vigor, the eventual female is an f1. although many commercial producers would like to have entire herds of f1 females, it is difficult to produce a continuous supply of f1s without a compromise. this can be addressed by either maintaining purebred parent populations to cross to produce f1s or purchasing all the replacements needed. in practice, neither of these methods is optimal for most producers. a number of crossbreeding systems manage to overcome the replacement female quandary by allowing breeders to produce replacement heifers from their own hybrid populations. however, this expediency results in loss of hybrid vigor, breed complementarity and simplicity. clinical theriogenology • volume 5 number 2 • june 2013 98  ease of application – the breeding program should be relatively simple. expensive and/or complex systems requiring high levels of management are not ideal, as complex breeding systems often conflict with vital management practices unrelated to breeding. therefore, crossbreeding systems should fit with other aspects of cattle production.  precision of genetic prediction – the higher the accuracy of genetic prediction, the more predictable the offspring and lower selection risk. because relatively little performance information on commercial animals is recorded and even less is analyzed and reported, the accuracy of prediction in a commercial operation is limited to the accuracy of prediction for seed stock inputs to the crossbreeding system – typically sires. in many cases, accurate estimated progeny differences (epds; for at least some traits) are available for purebred sires, and crossbreeding systems using purebred sires benefit as a result. although the effect of breed difference is powerful (table 1), the choice of individuals from within a breed is also very important. poor choices of breeds and animals from within a breed will have a lasting impact on the success of any crossbreeding program. crossbreeding systems are in two general categories: 1) production of replacement females as well as market cattle (rotational and composite systems); and 2) production of market cattle (terminal cross).9 in rotational (or composite) systems, bulls must be selected with maternal traits in mind as well as growth and carcass traits, since replacement heifers are retained from within the herd. terminal systems allow for greater emphasis on selection for growth and carcass traits in bulls, since female replacements are supplied from outside the herd. the genetic merit of a terminal bull for maternal traits is of no consequence, since his female progeny will not be bred. calving ease must be considered (particularly for heifer dams), regardless of the type of crossing system. briefly, different traits respond to heterosis to varying extents (reproduction and longevity are strongly influenced, growth is moderately influenced, carcass traits are minimally influenced, etc.). parent animals in any breeding program, there are four basic pathways of genetic improvement, corresponding to the total of four sources of parental genes of male and female progeny.10,11 these four pathways are: • male parents of male progeny (sires of males, sm) • female parents of male progeny (dams of males, dm) • male parents of female progeny (sires of females, sf) • female parents of female progeny (dams of females, df) sires of males since typically only limited numbers of young bulls are subjected to performance and progeny testing, and every sire can produce tens of thousands of doses of semen, we need only a few sires to produce these young bulls each generation. thus we need to select only the top one or two percent of tested bulls as sires of sons. these sires have high accuracy of genetic evaluation, since progeny tests generally give high accuracy. the generation interval will, however, be at least six years, due to the interval from birth of the young bull, through the birth of his first crop of test daughters, through their first calving. sires of females since there are many more cows than bulls used, many more bulls are required to produce the necessary amount of semen each generation. in an efficient scheme, the top 10-15% of young bulls can be selected, giving lower selection intensity than for sires of sons. accuracy of selection is the same as for sires of sons, because they are chosen on the basis of the same information. the generation interval is, however, approximately one year longer, since it takes time to breed a large population of cows and better bulls will be retained and used longer than those of lesser merit. clinical theriogenology • volume 5 number 2 • june 201399 dams of males since there are numerous cows, but only limited numbers of sons tested, dams of sons can be selected very intensely; perhaps only the best 0.1 to 0.5% are needed. however, evaluation is based on the dam’s performance, which has lower accuracy than a progeny test. these cows could be bred in their second lactation (based on their first lactation performance and part of their second lactation performance), so that they would be approximately 4.5 to five years old at the birth of their sons. dams of females dairy cows have a very low reproductive rate, producing less than one live calf per year (based on average calving intervals and mortality of fetuses and calves). allowing for disease and other losses of growing heifers and that only half the calves are females, only approximately one in three calvings result in a potential replacement heifer for the dairy herd. since average life in the herd in many western countries is often not much over three lactations, the average cow barely has sufficient time to produce a replacement before she leaves the herd. consequently, there is limited opportunity for selection of dams of cows, with perhaps 90% of all cows required for breeding. accuracy of selection would be very similar to that for dams of sires. however, generation interval is generally increased by one to two years since the average cow requires close to three calvings to produce a replacement. accounting for young bulls in the sire to female path, only the use of progeny-tested sires, to breed cows to produce herd replacements is taken into account. however, young bulls also contribute to the next generation of females.12 semen from young bulls can represent as much as 20% of all inseminations in a practical breeding program and should be considered (figure). in that regard, genetic superiority and generation interval for sires of females must be computed as a weighted average. figure. pathway following incorporation of young bulls in a breeding program. utilizing genomic selection the advent of dna sequencing and high-throughput genomic technologies has elucidated numerous single nucleotide polymorphisms (snp) in cattle. genomic selection refers to selection preferences based on genomic estimated breeding values (gebv).12-14 these gebv are calculated by estimating snp effects from prediction equations, which are derived from a reference population (a subset of animals) that have snp genotypes and phenotypes for traits of interest. the correctness of gebv depends on the heritability of the trait, and the degree of associations between selection candidates and the reference population, and the size of the reference population used to obtain prediction equations. clinical theriogenology • volume 5 number 2 • june 2013 100 the advantage of genomic selection is it allows breeders to identify genetically superior animals at an earlier age, even before they reach sexual maturity. genomic selection involves identifying which snps are linked to important functional traits and selecting bulls with dna patterns to produce superior priority traits.15,16 recent advances in dna/genomic technology and decision support tools will enhance selection accuracy.17 these approaches not only provide information for selection of bulls with the genetic make up for superior priority traits, but concurrently provide information to eliminate bulls with undesirable genetic traits.18 however, there are still improvements to be made, as some bulls with a very promising genomic assessment have poor actual performance or progeny. using only genomic selection this approach is short-sighted; the use of all available tools, including phenotype, genetic information, information from close relatives and individual performance, is strongly recommended. ongoing assessment of daughter phenotypes is essential because prediction equations for calculating gebv should be restructured periodically to increase the size of the reference population, account for changes over time in herd management practices or the genetic background of mates, and maintain high levels of linkage disequilibrium (the non-random association of alleles at two or more loci) between the reference population and the selection candidates. this genomic information has many advantages, including:  potential to speed genetic improvement  enables bull selection from a much wider genetic pool  provides earlier information regarding genetic differences between siblings  predicts genetic merit of young animals with more accuracy  improves the reliability of current progeny testing for low-heritability traits this technology provides artificial insemination (ai) companies with the ability to carry out accurate selection of bulls at a young age. hence this technology allows ai companies to purchase fewer bulls, knowing that only males with the highest genetic merit will be marketable. this could reduce sire expansion costs by up to 92%, due to the substantial costs associated with housing and feeding 19 genomic testing many progressive breeders are using genomic testing for the majority of their cows and heifers to identify females that received the most favorable combination of genes from their parents. the use of young genomic bulls by breeders as sires of replacement females (sire female (sf) pathway) continues to increase in popularity. among dairy producers, there has been a major shift toward the use of genomic bulls in the sf pathway. the total number of units of semen sold from young dairy sires increased by 13%, between 2006 and 2010,20 and the increased acceptance of genomics among dairy cattle producers has allowed extensive marketing of genomic bulls. at present, a low-density (ld) chip with 6,909 snp21 and a medium-density (50k) chip with 54,609 snp22 are the products used by breeders, and gebv for production, health, and conformation traits can be computed using genotypes from either chip. recently, the ld chip replaced the 3k chip with 2,900 snp23 because of greater gains in reliability and improved readability among snp genotypes. genomic testing services are currently offered by breed associations, ai stud services, and some private companies. the ld chip costs $43 to $55 per animal and reliabilities of the resulting gebv for production traits in holsteins are approximately 60 to 65%. the 50k chip is more costly ($125 to $135 per animal), but reliabilities for production traits in holsteins are roughly 70%, whereas they are up to 95% for angus. the tests that are currently being included in the us beef industry are comprised of either 384 snp or 50k snp, although the research community is commonly using 50k or 770k genomic tests for discovery of new traits (e.g. feed efficiency and disease susceptibility). cost undoubtedly hinders use of this technology; however, recent advances show promise in dramatically decreasing the cost of genotyping.24 within-breed genomic predictions based on medium density (i.e. 50k) genotypes add accuracy, particularly to young animals, for several traits (table 2). the test also includes the following:  arthrogryposis multiplex clinical theriogenology • volume 5 number 2 • june 2013101  coat color  multi-sire parentage  horned/polled  bvd-pi diagnostic test methodology related to the use of this technology in crossbred or composite animals is critically needed to fully benefit the commercial livestock sector. the importance of adoption will be getting commercial producers to realize the value of, and thus pay for, increased gebv accuracy. there is a still a need to collect and routinely record phenotypic information by seed-stock producers and commercial producers need to realize that gebv, and economic index values, are critically important for optimizing selection. in that regard, although genomic technology makes these tools stronger, it does not replace them. establishing benchmarks estimated progeny difference values for sires can be utilized to establish benchmarks.18 estimated progeny differences are a useful selection tool for many of the traits (table 3). it is noteworthy that epds predict the expected performance of calves sired by a bull, compared to the expected performance of calves sired by another bull. they are based on:  performance records of an individual, records of relatives and progeny  adjustments for differences in the genetic merit of mates  genetic correlations (genetic relationship between traits) to improve estimates  accounting for genetic changes over time and genetic relationships among individuals  increased validity of some estimates, due to associations  total herd reporting, so records are provided on more individuals  adjustments for differences among contemporary groups in environment and management (e.g. climate and nutrition)  current epds are directly comparable within a breed for all individuals (males and females) in all locations and management systems across all years. many breed associations have epds on individual animals that can be obtained from published sire summaries and searchable internet databases. breed associations also publish epd data to indicate where individual animals rank within the breed for specific traits. selection for improved efficiency should account for production, reproduction, longevity, and feed costs.26 the use of epds is a tool to aid purebred and commercial producers in selecting potential breeding stock. cattlemen must clearly define their breeding objectives and determine what sort of bull is needed to meet these goals and then carefully consider the epds of the bull (as well as his relatives), if possible. the basic steps to choosing a herd sire are: retaining ownership birth weight and carcass traits (weight, yield grade, quality grade) are of primary importance. maternal traits and mature weight must be considered if daughters are being retained for breeding. selling calves as yearlings birth weight and yearling weight are of primary importance. maternal traits, such as milk, stayability and mature size are important if replacement heifers are retained. stayability is the probability of daughters remaining in the herd for at least six years and have the opportunity to become part of the breeding herd. clinical theriogenology • volume 5 number 2 • june 2013 102 cow/calf operation if calves are sold at weaning, birth weight and weaning weight are the primary considerations. maternal traits (milk, stayability) and mature size must also be considered if replacement heifers are being selected. bull selection becomes more complex as the number of breeding objectives increases. for example, rapid growth and superior maternal traits are not usually found in the same genetic package. producers selecting their own replacements heifers while retaining ownership beyond weaning must make a decision. they can either find a sire that does all jobs acceptably well (a balance of maternal and growth traits), or split the cow herd, breeding some cows to a sire with strong maternal traits (calving ease, milk, stayability) to produce replacement heifers, and breeding the rest to a terminal sire (growth, carcass traits) to produce feeder or slaughter cattle. the table below provides some general guidelines for using epds in commercial scenarios (table 4). expected progeny differences are easily interpreted. they are expressed in various units, depending on the specific trait. to demonstrate how epds work, consider the following examples. example a bull milk epd 1 +20 2 +5 difference 10 lb interpretation: due to high milking ability, daughters of bull 1 wean claves 15 pounds heavier than daughters of bull 2. example b interpretation: 4% fewer calves sired by bull 1 need assistance at delivery compared to calves sired by bull 2. preparation for selection there are many sources of bulls that warrant consideration, including production sales, test stations and private sales. of critical importance is that the bull be from a trustworthy source which will provide assurance of satisfaction and good service. it may be necessary to consider several sources in order to find an appropriate bull. examination and evaluation of the bull's performance record to determine those that meet the epd and other specifications that have been established. follow rigorously the selection criteria and qualifications/specifications that have been established. all of these criteria should be set before selection. select those which meet the specifications and eliminate those that do not meet the specifications. once selection has been narrowed to only bulls which meet the criteria, further refining of the evaluation and selection may be used. having a list of suitable bulls prior to arrival at the auction or farm not only saves time, but also assists in ensuring that an appropriate bull is purchased. upon narrowing the list of potential bulls, they can be evaluated for suitability of phenotypic traits and the potential candidate list shortened. not all relevant traits have epd (e.g., disposition, structural and foot soundness, fleshing ability, etc.) and, therefore, must be evaluated visually (up to 3% of young bulls are eliminated after visual inspection).10 bull calving ease epd 1 + 7 2 + 3 difference 4% clinical theriogenology • volume 5 number 2 • june 2013103 how to use epd percentile tables most of the major breed associations publish epd percentile tables, which indicate where a bull ranks within a breed for each of the traits of interest. some breeds even produce multiple tables-current (active) sires, current (active) dams, non-progeny sires, etc. for most producers, a comparison against the active sires would be appropriate. for example, assume we are interested in an angus bull with a calving ease epd (ce) of 5.5, weaning weight epd (wwt) of 38, milk epd (mlk) of 5, and yearling weight epd (ywt) of 62. using the table from the american angus association, this bull is in the top x1% of the breed for calving ease, the top x2% for weaning weight growth, the top x3% for milking ability, and the top x4% for yearling weight growth. consequently, we expect this bull will be relatively easy calving, high growth, and produce daughters with about average (for angus) milking ability. evaluation of bull breeding potential before purchase and before each breeding season, bulls should be evaluated to ensure that they meet the society for theriogenology breeding soundness evaluation criteria for structural and physical soundness, sperm motility and morphology, and be free of venereal diseases.27-29 reasons that bulls are classified as unsatisfactory potential breeders include: 25% due to poor sperm parameters (usually morphology), seven percent due to inadequate scrotal circumference, and three percent have poor structural conformation.27 test for genetic defects genetic defects occur due to random mutations in the genome; they are more common in purebred cattle, although crossbreds that contain a breed from an at-risk population are also at risk. several genetic defects are autosomal recessive, whereas environmental factors can produce phenotypes similar to genetic defects. there are dna tests for several genetic defects, which can confirm whether the defect is due to genetic or environmental causes. with advancements in molecular technology, genetic defects can be effectively managed in cattle populations. instead of abolishing entire lines of cattle, carrier animals can be identified and either culled or used in certain circumstances with confidence. numerous genetic defects are currently being monitored by us breed associations and others likely will be discovered in the future. managing newly purchased bulls purchasing a new bull is a relatively infrequent occurrence. this further emphasizes the importance of selecting an appropriate bull, particularly in single-sire farms. the value of an appropriate bull should not be underestimated. investments in good genetics will pay dividends, both shortand longterm, through the influence the bull has on each calf crop, as well as his daughters that are retained for breeding. care and management of the newly acquired bull is essential. genetic progress and increased profit can be achieved only if the bull has satisfactory reproductive performance. with most new sires purchased as yearling bulls, management prior to, during and after the first breeding season is particularly important. plan ahead by acquiring a new yearling bull at least 60 to 90 days prior to the breeding season, so ample time is available to allow for adjustment to a new environment, commingling with other bulls with known temperament and disease status, and getting the bull in proper breeding body condition. bulls must be properly acclimated to the environment and management conditions present on the farm; lack of adaptation is likely to lead to poor reproductive performance. it is important to maintain performance record for bulls to monitor their economically important productive and reproductive performance parameter and to determine progress. in multi-sire breeding schemes, some producers may increase the intensity of their management by testing all or a sub-set of calves for parentage to determine the number of calves actually sired by each bull and how each bull’s progeny performed. some herds may elect to determine parentage on only the best performing calves and the poorest performing calves as an aid in bull culling decisions. clinical theriogenology • volume 5 number 2 • june 2013 104 conclusions bull selection is one of the most important producer decisions, as it offers an opportunity to enhance the genetic merit and profitability of the farm. to effectively select sires, producers must use selection tools such as epd indices and understand both withinand between-breed differences of these indices. in addition, producers must also accurately and objectively assess their current genetics, nutritional resources, and management. this will help producers with decision making. the selection and addition of bulls must not only meet revenue-improving priority traits, but also compliment other important production traits. recent advances in dna/genomic technology and decision support tools will enhance selection accuracy. producers who utilize these advances in beef cattle genetics in the selection process should not only increase profit from improved revenue and reduced production costs, but best match genetics to their operation’s production demands. also it is important to use all available tools such as phenotypic, genetic information, information from close relatives, and individual performance. references 1. xu s, martin tg, muir wm: multistage selection for maximum economic return with an application to beef cattle breeding. j anim sci 1995;73:699-710. 2. pruzzo l, cantet rj, fioretti cc: risk-adjusted expected return for selection decisions. j anim sci 2003;81:29842988. 3. nkrumah jd, basarab ja, wang z, et al: genetic and phenotypic relationships of feed intake and measures of efficiency with growth and carcass merit of beef cattle. j anim sci 2007;85:2711-2720. 4. fitzhugh ha jr, long cr, cartwright tc: systems analysis of sources of genetic and environmental variation in efficiency of beef production: heterosis and complementarity. j anim sci 1975;40:421-432. 5. wolfová m, wolf j, kvapilík j, et al: selection for profit in cattle: ii. economic weights for dairy and beef sires in crossbreeding systems. j dairy sci 2007;90:2456-2467. 6. mcallister aj: is crossbreeding the answer to questions of dairy breed utilization? j dairy sci 2002;85:2352-2357. 7. vanraden pm, sanders ah: economic merit of crossbred and purebred us dairy cattle. j dairy sci 2003;86:10361044. 8. franke de, derouen sm, williams ar, et al: direct and maternal breed additive and heterosis genetic effects for reproductive, preweaning, and carcass traits. proc symp tropically adapted breeds, regional project s-1013, am soc anim sci, southern section meeting; 2005. p 204-209. 9. cundiff lv, gregory ke: what is systematic crossbreeding? proc cattlemen’s college; cattle industry annu meet and trade show, natl cattlemen’s beef assoc; 1999. 10. van raden pm, freeman ae: potential genetic gains from producing bulls with only sires as parents. j dairy sci 1985;68:1425-1431. 11. de roos ap, schrooten c, veerkamp rf, et al: effects of genomic selection on genetic improvement, inbreeding, and merit of young versus proven bulls. j dairy sci 2011;94:1559-1567. 12. meuwissen the, hayes bj, goddard me: prediction of total genetic value using genome-wide dense marker maps. genetics 2001;157:1819-1829. 13. dassonneville r, brøndum rf, druet t, et al: effect of imputing markers from a low-density chip on the reliability of genomic breeding values in holstein populations. j dairy sci 2011:94:3679-3686. 14. hayes bj, bowman pj, chamberlain aj, et al: invited review: genomic selection in dairy cattle: progress and challenges. j dairy sci 2009;92:433-443. 15. van eenennaam al, van der werf jh, goddard me: the value of using dna markers for beef bull selection in the seedstock sector. j anim sci 2011;89:307-320. 16. de roos ap, schrooten c, veerkamp rf, et al: effects of genomic selection on genetic improvement, inbreeding, and merit of young versus proven bulls. j dairy sci 2011;94:1559-1567. 17. sherman el, nkrumah jd, li c, et al: fine mapping quantitative trait loci for feed intake and feed efficiency in beef cattle. j anim sci 2009;87:37-45. 18. fouilloux mn, renand g, gaillard j, et al: genetic correlation estimations between artificial insemination sire performances and their progeny beef traits both measured in test stations. genet sel evol 2000;32:483-499. 19. schaeffer lr: strategy for applying genome-wide selection in dairy cattle. j anim breed genet 2006;123:218-223. 20. olson km, hutchison jl, vanraden pm, et al: change in the use of young bulls. am dairy sci assoc/am soc anim sci joint annu meet;2011 (poster). 21. illumina. bovineld genotyping beadchip. accessed jan. 16, 2013. http://www.illumina.com/documents/products/datasheets/datasheet_bovineld.pdf. 22. illumina. bovinesnp50 genotyping beadchip. accessed jan. 16, 2013. http://www.illumina.com/documents/products/datasheets/datasheet_bovine_snp5o.pdf. clinical theriogenology • volume 5 number 2 • june 2013105 23. illumina. goldengate bovine3k genotyping beadchip. accessed jan. 16, 2013. http://www.illumina.com/documents/products/datasheets/datasheet_bovine3k. pdf. 24. thallman rm, hanford kj, quaas rl, et al: estimation of the proportion of genetic variation accounted for by dna tests. proc 41st annu res symp and annu meet, beef improvement federation;2009. p. 184-209. 25. wiggans gr,vanraden pm, cooperta: the genomic evaluation system in the united states: past, present, future. j dairy sci 2011;94:3202-3211. 26. golden bl, garrick dj, newman s, et al: economically relevant traits a framework for the next generation of epd. proc beef improv fed 2000;32:2-13. 27. kennedy sp, spitzer jc, hopkins fm, et al: breeding soundness evaluations of 3,648 yearling beef bulls using the 1993 society for theriogenology guidelines. theriogenology2002;58:947-961. 28. ball l, dargatz da, cheney jm, et al:. control of venereal disease in infected herds. vet clin north am food anim pract 1987;3:561-574. 29. farin pw, chenoweth pj, tomky df, et al: breeding soundness, libido and performance of beef bulls mated to estrus synchronized females. theriogenology 1989;32:717-725. table 1. units and percentage of heterosis by trait8 trait bos taurus crossbred dams bos taurus crossbred calves bos taurus by bos indicus crossbred dams bos taurus by bos indicus crossbred calves units % units % units % units calving rate 3.5 3.7 3.2 4.4 15.4 4.3 calf survival 0.8 1.5 1.4 1.9 1.6 1.8 -1.4 birth weight 1.6 1.8 1.7 2.4 1.36 16.2 11.4 weaning weight 18.0 3.9 16.3 3.9 0.97 17.0 78.5 table 2. production and reproduction traits§ currently tested using genomic techniques. angus* holstein25 jersey25 brown swiss25 calving ease direct birth weight weaning weight yearling weight dry matter intake yearling height yearling scrotal (cm) docility milk mature weight mature height carcass marbling carcass rib-eye area carcass fat carcass weight milk (kg) fat (kg) protein (kg) fat (%) protein (%) productive life (mo) scs daughter pregnancy rate sire calving ease daughter calving ease sire stillbirth daughter stillbirth milk (kg) fat (kg) protein (kg) fat (%) protein (%) productive life (mo) scs daughter pregnancy rate milk (kg) fat (kg) protein (kg) fat (%) protein (%) productive life (mo) scs daughter pregnancy rate *american angus association currently utilizes genetic correlations between traits and their genetic indicators offered by ingenity (384 snp) and pfizer (50k) for the following traits. §it should be noted that the genetic markers available will vary by breed. clinical theriogenology • volume 5 number 2 • june 2013 106 table 3. list of production and reproduction traits with expected progeny differences growth trait epds carcass trait epds maternal/paternal trait epds birth weight weaning weight yearling weight mature weight mature height average daily gain feed efficiency feed intake (dry matter and residual) carcass weight marbling rib-eye area fat thickness tenderness quality grade (percent choice) milk protein fat daughter pregnancy rate docility calving ease (maternal and direct) stayability (longevity) scrotal circumference heifer pregnancy rate table 4. recommendations for epds for various commercial scenarios use of individual breed birth weight weaning weight yearling weight milk terminal sire on mature cows large carcass not excessive high high not relevant bull to use with heifers small to medium size low moderate moderate consider if keeping heifers sire replacement heifers medium size maternal low to moderate moderate to high moderate to high varies clinical theriogenology • volume 5 number 2 • june 2013107 2018: reproductive challenges in small mammals reproductive challenges in small mammals kurt k. sladky department of surgical sciences, school of veterinary medicine, university of wisconsin, madison, wi the objective of this presentation is to briefly describe normal reproductive structures and function in small mammal species, and primarily focus on clinical diagnosis and management of common reproductive disorders with an emphasis on pet species of rodents (rats, guinea pigs, gerbils, chinchillas, hamsters, degus), rabbits, hedgehogs, sugar gliders, and ferrets. rabbits there are several unique features of rabbit reproductive anatomy and physiology worth addressing. in the female, the paired ovaries are connected to paired oviducts, which connect to the long, well-vascularized, and slightly convoluted uterine horns.1,2 the uterine horns each have their own cervix, which separates the uterus from the vagina. the rabbit vaginal vestibule is relatively long, wide and flaccid compared to other mammals.1 these unique features are important to understand, particularly when performing an ovariohysterectomy. in the male, the paired, scrotal testicles are elongated and more flaccid compared to many other mammals. the skin of the scrotum is relatively thin and wrinkled. the inguinal canals remain open after the testicles descend, whereas most male mammal inguinal canals constrict after testicular descension.1,2 after castration, herniation of abdominal organs can occur through these open inguinal canals, so appropriate closure of underlying tissues should be a concern. rabbits are induced ovulators, and ovulation occurs approximately 10 hours after coitus. wild rabbits have a seasonal pattern to reproduction, but pet rabbits will breed year-round if allowed.1,2 sex determination can be somewhat challenging in rabbits. one needs to press down on either side of the external genital region in order to express the penis or vulva. the expressed penis is cylindrical in shape. the vulva also protrudes from the perineum and can easily be mistaken for a penis. look for the vertical vulvar slit and two distinctive blood vessels visible running along each side of the vulvar protuberance into the vagina.1 uterine neoplasia early elective ovariohysterectomy in female rabbits is relatively common, making uterine neoplasia much less common than 10 years ago. uterine tumors are the most common reproductive tract neoplasms of older, intact female rabbits and the incidence increases dramatically with age.1,2 depending on the publication, greater than 75% of intact female rabbits will develop uterine tumors by 6-7 years of age.1 uterine adenocarcinoma is the most common of the uterine neoplasms. these tumors are slow to metastasize, but metastatic disease occurs, and can affect the lungs, liver, brain, and other organ systems. other uterine tumors reported include adenoma, leiomyoma and leiomyosarcoma.1,2 diagnosis includes abdominal palpation to confirm suspicion of a mass affect, and imaging modalities (ultrasonography, ct or contrast ct scan) with biopsy and histology or fine needle aspiration and cytology. a contrast ct scan may also help identify metastatic disease, which will provide prognostic information, with metastatic disease being a poor to grave prognosis. treatment of any uterine tumor relies on removing the affected uterus and ovaries via ovariohysterectomy. pyometra pyometra is uncommon in pet female rabbits, but occurs more commonly in females maintained for breeding.1 typical clinical signs include a rabbit with a depressed attitude, a mucopurulent vulvar discharge, and a palpably enlarged uterus. sepsis can occur with chronicity. imaging (ultrasonography or ct scan), in addition to a vulvar swab with cytology, will help with diagnosis. treatment includes ovariohysterectomy, bacterial culture and sensitivity of the intrauterine material, and broad-spectrum antibiotics. clinical theriogenology • volume 10, number 3 • september 2018167 dystocia dystocia is uncommon in rabbits, but is reported in the literature. gestation ranges from 28-36 days in length.1,2 dystocia may be more common in rabbits that are obese, produce larger fetuses, have a narrower pelvis, or experience uterine inertia.1,2 pregnant rabbits in dystocia may be presented with a history of straining, depressed attitude, vaginal discharge, and passage of some fetuses. imaging (ultrasonography and radiographs) will help to characterize the presence of remaining fetal skeletons. medical treatment includes fluid therapy, sedation, analgesics, oxytocin (1-3 iu/rabbit, subcutaneously), and gentle removal of the fetuses and fetal membranes.1 however, cesarian section is typically indicated, and may include ovariohysterectomy after removal of any viable fetuses. cryptorchidism cryptorchidism refers to the failure of one or both testicles to fully descend into the scrotum. testicular descent occurs by approximately 12 weeks of age in male rabbits.1 with cryptorchidism, the testicle or testicles may remain in the caudal abdomen or within the proximal inguinal canal. a word of caution; during normal handling and physical examination, males with descended testicles, commonly retract their testicles temporarily into the inguinal canal making it appear that the testicles are not descended. sedation will relax the rabbit and allow proper assessment of testicular descent. ultrasonography may help identify whether the testicles are intra-abdominal versus within the proximal inguinal canal. as with other mammals, cryptorchidism may lead to an increased incidence of testicular neoplasia. castration via the inguinal canal or via an intra-abdominal approach is indicated in these cases. testicular tumors testicular tumors tend to occur in older, intact males and are not commonly observed in pet rabbits, as many are neutered earlier in life. sertoli cell tumors, seminomas, interstitial cell tumors and gonadoblastoma are reported.1,2 surgical removal of the affected testicle is the treatment of choice, and removal of the unaffected testicle at the same time is recommended. treponematosis treponema paraluiscuniculi (“rabbit syphilis”) is a spirochete that can affect both male and female rabbits, and is most common in intact rabbits as the organism is commonly transmitted through sexual contact.1,2 vertical transmission of treponema from dam to the fetus can occur during vaginal passage during parturition.1 clinical signs include ulcerative, crusty, or vesicular lesions associated with the mucocutaneous junctions of the mouth, eyes, nose, and genitals. diagnosis is based on appearance of lesions, and silver staining of skin scrapes or biopsy specimens of the lesions.1 serology is also available at some commercial laboratories with submission of plasma samples. penicillin procaine with benzathine is most commonly used for treating treponematosis, but any penicillin must be administered subcutaneously and not orally in rabbits. hypersexuality and hyperaggression hypersexuality and hyperaggression occasionally occurs in male and female rabbits, even after castration/ovariohysterectomy. this behavior may be associated with a hormone secreting tumor, or without an obvious cause.1 behaviors include excessive mounting, biting, fighting, and urinating. imaging (ct scan or ultrasonograpgy) is recommended in order to rule out endocrine or other organ system tumors. a contrast ct scan may be helpful in ruling out pituitary tumors. surgery should be considered if a discrete tumor can be identified and removed. gonadotropin releasing hormone (gnrh) agonists, such as deslorelin implants, may be implanted to try to decrease the behavior by reducing reproductive hormones.3,4 euthanasia is not uncommon with hyperaggressive rabbits, as owners are not able to deal with, or provide appropriate care for these rabbits. clinical theriogenology • volume 10, number 3 • september 2018 168 rodents for the purpose of these proceedings, “rodent” refers to the following: rats, guinea pigs, chinchillas, gerbils, hamsters and degus. reproductive disorders of rodents are common and are predominantly neoplastic diseases. in all species listed, females have bilateral ovaries and a bicornuate uterus, consisting of two uterine horns, a uterine body, a cervix and a vagina, except for the rat, in which the two uterine horns join together and open into the vagina.5-8 males have two scrotal testicles, and open inguinal canals, much like rabbits. all have a penis and a prepuce, and rats have an os penis. mammary tissues are extensive in many rodents of both sexes, particularly in rats and mice, which is important in some disease processes. determining sex is relatively straightforward in most rodent species, with anogenital distance larger in males than in females. as with rabbits, sex determination in young animals can be more challenging. ovarian cysts ovarian cysts, or cystic rete ovarii, are common in guinea pigs, gerbils, and some hamster species, however, the etiology is unknown. prevalence in guinea pigs increases with age and ranges from 30-90% depending on which publication one reads. middle-aged (2to 4-year-old) sows are most commonly affected.6-8 definitive underlying mechanisms associated with reproductive history and cyst development have not been determined. concurrent diseases associated with ovarian cysts include leiomyomas, granulosa cell tumors, cystic endometrial hyperplasia, and endometritis.6-8 clinical signs may include abdominal distension, nonpruritic bilateral alopecia especially of the flanks (functional follicular cysts), lethargy, and anorexia. frequently, the cystic structures are palpable during a physical examination, but overzealous palpation can cause rupture of the cyst. diagnostics include imaging (e.g., ultrasonography) to visualize the cysts; pancytopenia may be observed secondary to hyperestrogenism on a complete blood count, but cystic ovaries do not always produce excessive estrogens. the current treatment of choice is surgical ovariohysterectomy. there is interest in gnrh agonists, but a single published study found no effect on cystic ovaries in guinea pigs administered deslorelin implants.8 future research with gnrh agonists will be beneficial.9 female reproductive neoplasia uterine tumors. uterine tumors are common in female rodents with malignancy diagnosed most frequently in hamsters and gerbils, and benign uterine tumors more common in rats.5,6 benign endometrial stromal tumors are most common in older female rats, with sarcomas most common in mice, and adenocarcinomas in hamsters and gerbils. in guinea pigs, uterine tumors include leiomyomas and leiomyosarcomas. approximately 50% of these guinea pig uterine tumors are malignant, but metastasis is not common.7,8 clinical signs in all rodents include hemorrhagic vaginal discharge, abdominal distention, and abdominal pain.5,6 imaging (ct scan and ultrasonography) may help with diagnosis and for evaluating the animal for metastatic disease. fine needle aspirate of uterine masses may provide a cytologic diagnosis. the treatment of choice is ovariohysterectomy with histopathologic evaluation. prevention of all reproductive neoplasms is best accomplished by ovariohysterectomy of young animals. ovarian tumors. ovarian tumors are most common in rats, and most of these are diagnosed as tubular ovarian adenomas.5,6 other ovarian tumors diagnosed in rodents include granulosa cell tumors, thecal cell tumors, malignant granulosa cell tumors, ovarian mesotheliomas, tubular adenocarcinomas, and papillary cystadenomas/cystadenocarcinomas.5-8 imaging (ct scan and ultrasonography) may help with diagnosis and for evaluating the animal for metastatic disease. ovariohysterectomy is the treatment of choice, but the prognosis will be poor if the tumor is malignant or if metastatic disease is present. mammary gland neoplasia. mammary gland neoplasia is particularly common in rats and mice. malignant mammary adenocarcinoma and fibrosarcomas are most common in mice, while benign mammary fibroadenomas are very common in rats and guinea pigs of both sexes; adenocarcinomas also occur in rats and guinea pigs.5-8 mammary tumors are not common in most hamster species, but djungarian hamsters have a higher prevalence of mammary tumors than other hamster species.6 in rats, mammary tumors can occur almost anywhere on the body, as mammary tissue is diffuse and extensive. in clinical theriogenology • volume 10, number 3 • september 2018169 female rats, there is evidence that ovariohysterectomy early in life (between 3-6 months of age) will protect the rat from developing mammary tumors as the rat ages.5,6 there is anecdotal evidence that ovariohysterectomy at the time of first mammary tumor surgical removal, may reduce or delay recurrence of the mammary tumor, but there are no systematically derived data supporting this hypothesis. fine needle aspirate of mammary masses may provide a cytologic diagnosis. surgical excision is the treatment of choice for mammary tumors in any species, but recurrence is common, and in those rodent species in which malignant tumors are excised, the prognosis is poor. surgical excision can be very difficult depending on location of the tumor(s); inguinal and perineal masses can be particularly problematic due to close proximity to other vital structures. many benign fibroadenomas are well-vascularized, so care should be taken during surgery to minimize hemorrhage. dehiscence of the surgical site is common in guinea pigs, partially due to the fact that their immune system reacts dramatically to foreign suture material. more recently, subcutaneous deslorelin implants have been administered in rats after surgical excision of mammary tumors, with the hope of delaying recurrence without putting the rat through an invasive ovariohysterectomy. our service is monitoring efficacy of this approach, but we have not collected enough data at this time. pregnancy toxemia pregnancy toxemia is most commonly observed in guinea pigs either just before or just after parturition.7,8 there are two forms of pregnancy toxemia. the first is metabolic toxemia with ketosis occurs late in pregnancy and early postpartum, which may be associated with stress and starvation (negative energy balance). predisposing factors include obesity, lack of exercise, large fetal loads, change in diet and/or environment, heat stress, and primiparity.7,8 clinical signs include anorexia, dehydration, and with chronicity, ataxia, dyspnea and death. breath can smell like ketones with ketonemia. diagnostic testing includes a blood sample (hypoglycemia), urinalysis (ketonuria), and imaging (ct scan and/or ultrasonography).7,8 treatment will include force-feeding, and fluids with dextrose, but the prognosis is poor. the second form of pregnancy toxemia is vascular compromise associated with the uterus compressing its own vascular supply or that of the kidneys or gastrointestinal tract, leading to tissue ischemia and hypertension.7,8 this is equivalent to preeclampsia in women and occurs in late pregnancy. diagnosis includes indirect blood pressure to confirm hypertension, and ultrasonography with doppler to evaluate blood flow. treatment includes fluid therapy, analgesics and cesarian section, with a poor prognosis. with a poor prognosis for both forms or pregnancy toxemia, prevention is critical and includes decreased stress and good nutrition during pregnancy, and encouraging exercise. dystocia dystocia is more common in guinea pigs than other rodents or rabbits, and it has been attributed to large pups, narrow pelvic canals, or fusion of the pubic symphysis.7,8 the pubic symphysis is a fibrocartilaginous bridge in female guinea pigs, which calcifies and is permanently fused if the female has not been bred by 5-6 months of age. other contributing factors include obesity, hypovitaminosis c, and uterine inertia.7,8 sows at risk should be monitored and should undergo a cesarian section at the time of parturition. male reproductive neoplasia testicular tumors are reported in rodents. in rats, testicular interstitial cell tumors (leydig cell tumors) are most common.5,6 other tumors documented in mice, guinea pigs, hamsters, gerbils and chinchillas include seminomas, epididymal adenoma/adenocarcinoma, amongst others.5-8 surgical orchiectomy is the treatment of choice for testicular tumors in any rodent species. fur ring and paraphimosis fur ring and paraphimosis are most common in male chinchillas.9 fur ring is characterized by fur encircling the glans penis. smegma can also accumulate in this same penile region causing adhesion of the prepuce to the glans penis.9 clinical signs may include excessive grooming of the prepuce, straining to clinical theriogenology • volume 10, number 3 • september 2018 170 urinate or chronic exposure of the glans penis (paraphimosis). balanoposthitis, or infection of the prepuce and glans penis can occur, causing the same clinical signs. in severe cases, ability to urinate is compromised and the condition becomes emergent. treatment includes removing any fur or smegma from the prepuce and penis using cotton tipped applicators and an antimicrobial solution (dilute chlorhexidine).9 topical antimicrobial ointments can be applied to the affected area locally, and systemic antibiotics and non-steroidal anti-inflammatories can be administered. ferrets the female reproductive tract is similar to the cat, with paired ovaries, uterine horns, a short uterine body and a single cervix.11 females are induced ovulators, and the vulva swells during estrus. males have a small prepuce and a palpable os penis. once descended, the testicles are contained in a scrotum. many diseases of the reproductive tract that occur in other small mammals, do not occur in most pet ferrets, since early ovariohysterectomy and castration (≤ 6 weeks of age) is extremely common prior to sale in the united states. in european countries, however, intact ferrets are more common and early spay and neutering is much less common. deslorelin implants can be an effective alternative to surgical castration in intact ferrets.12 some common neoplastic diseases of the endocrine system produce reproductive hormones, which cause prostatomegaly, aggressive and reproductive behavior in males and vulvar enlargement in females, but these are considered independent of true reproductive diseases. because most pet ferrets in the united states are spayed and neutered early in life, most commonly, neoplastic diseases are diagnosed. female reproductive diseases female reproductive diseases most commonly include neoplastic diseases.11,13 in intact females, ovarian tumors are most common, but uterine tumors also occur. metastatic disease associated with ovarian or uterine tumors is not reported in the literature, so ovariohysterectomy is the treatment of choice with any female reproductive tract neoplastic disease.11,13 mammary tumors in female ferrets are rare and have been reported as benign and malignant.11,13 surgical excision, much like dogs and cats, to include the affected mammary chain, is recommended. male reproductive diseases male reproductive diseases most commonly include preputial tumors, but intact males may develop testicular tumors, cryptorchidism, and primary prostatic diseases.11,13 preputial adenomas and adenocarcinomas (apocrine gland tumors) have been diagnosed and can occur in castrated males as well as intact males. most preputial gland tumors are malignant and infiltrate local tissues, as well as metastasize to lymph nodes and lungs.11 clinical signs include raised masses of varying color associated with the prepuce. overgrooming by the male may cause dermatitis or ulcerative lesions. imaging (contrast ct scan) will help diagnose metastatic lesions providing prognostic information. surgical removal is the treatment of choice, but this can be a complicated procedure due to the aggressive infiltration of the tumor into adjacent tissues. sugar gliders sugar gliders have become popular pets during the past ten years. they are nocturnal marsupials native to forests of australia and new guinea.14 the “glider” name derives from the patagium used for gliding from tree branch to tree branch in the forest, that stretches from hind limbs to forelimbs. sugar gliders have a cloaca where the gastrointestinal, urinary and reproductive tracts meet and exit.14 male sugar gliders can be distinguished from females by the presence of an oval-shaped scent gland on the top of the head and a ventral midline scent gland located near the thoracic inlet.14 females have a pouch in which young are raised, much like other marsupial species. male anatomy is unique as the males have a bifurcated penis and two testicles contained in a pendulous scrotum located cranial to the cloaca on ventral midline. males urinate from the base of the bifurcated penis, not the distal tip.14 the female sugar clinical theriogenology • volume 10, number 3 • september 2018171 glider has two uterine horns, and two vaginas that end in a septate, sac-like structure. reproduction is polygamous and seasonally polyestrus.14 elective ovariohysterectomy is uncommonly performed in female sugar gliders due to the risk of hemorrhage in such small individuals, but castration of the males is common and an extremely simple procedure with the proper instruments. reproductive diseases mutilation of the penis, scrotum, cloaca and tail in males is the most common disease process of the sugar glider reproductive tract, and a condition that is poorly understood.14,15 hypotheses associated with the cause of this behavior include single housing or improper socialization, lack of access to females, and poor husbandry (inappropriate nutrition, not maintaining nocturnal environment). however, the cause remains unknown. males will attack their organs, chewing them, and even eviscerating themselves. this is an emergency situation, and surgical repair or euthanasia are the options. if the injuries can be repaired and the glider survives, it is imperative that the animal does not have access to the same tissues once it recovers from anesthesia (use of e-collars and body coverings are necessary). if the tips of the penis are affected, the tissue can be surgically removed without consequences to urination, as the urethra exists at the penile base. with severe damage, the prognosis is grave and the euthanasia should be considered. there is some anecdotal information that castration may be preventative. orchidectomy orchidectomy is relatively simple using a co2 laser or a ligasure® system. this will minimize post-surgical self-trauma compared to using scalpel and sutures.15 african hedgehogs african pygmy hedgehogs are very popular pets. they are not rodents, but members of the insectivore family, erinaceidae.16 african hedgehogs are nocturnal and native to the savannah regions of east and central africa. they are smaller than european hedgehogs, and their integument is covered in spines, which are a deterrent to predators. if you are able to prevent a conscious hedgehog from rolling into a ball, sex determination is relatively easy. the male has a prominent prepuce and the penis is large and palpable. two testicles are present in a para-anal sac, without a true scrotum.16 the female has an obvious external vulva, a single vagina with two prominent uterine horns ending in a cervix without a uterine body. african hedgehogs are polyestrous and breed throughout the year in captivity.16 although not confirmed, african hedgehog females are thought to be induced ovulators. elective ovariohysterectomy and castration are less commonly performed in hedgehogs compared to other small mammals, but the prevalence of reproductive tract neoplasia makes therapeutic ovariohysterectomy common in older females. the most common reproductive diseases of african hedgehogs are neoplastic. uterine neoplasia uterine neoplasia is very common in in middle-age to older female hedgehogs, and malignancy is common.16,17 in a survey of uterine tumors, most were reported in females 3-5-yeasr of age and included adenocarcinomas, endometrial stromal sarcomas, endometrial polyps, and adenoleiomyoma/adenoleiomyosarcoma. 17 all hedgehogs with uterine disease are presented with similar clinical signs; hematuria and weight loss. imaging (ct scan and/or ultrasonography) is important to rule out reproductive versus urinary diseases, and to try to determine whether metastatic disease is present. ovariohysterectomy is the treatment of choice for any female hedgehogs with uterine disease. mammary neoplasia mammary neoplasia is also common in female hedgehogs, and adenocarcinomas are most common.16 these tumors can be invasive and grow large enough that many females are presented with ulcerative and necrotic lesions of the skin overlying the masses. diagnosis includes fine needle aspirates clinical theriogenology • volume 10, number 3 • september 2018 172 and cytology or biopsy and histopathology. imaging may be useful for prognosis since metastatic lesions in lymph nodes and lungs are common. these tumors are well-vascularized, so hemostasis is critical when surgically excising the mass. preventing access to the surgical wound is important in hedgehogs because many will obsessively lick and chew the wound until it dehisces. references 1. harcourt-brown fm: disorders of the reproductive tract of rabbits. vet clin north am exot anim pract 2017;20:555587. 2. klaphake e, paul-murphy j: disorders of the reproductive and urinary systems: in: quesenberry k, carpenter j, editors. ferrets, rabbits and rodents, clinical medicine and surgery. 3rd edition. st. louis: elsevier; 2011. p. 217-231. 3. risi e: control of reproduction in ferrets, rabbits and rodents. reprod domest anim 2014;49:81-86. 4. schoemaker nj: gonadotrophin-releasing hormone agonists and other contraceptive medications in exotic companion animals. vet clin north am exot anim pract 2018;21:443-464. 5. martorell j: reproductive disorders in pet rodents. vet clin north am exot anim pract 2017;20:589-608. 6. brown c, donnelly tm: disease problems of small rodents: in: quesenberry k, carpenter j, editors. ferrets, rabbits and rodents, clinical medicine and surgery. 3rd edition. st. louis: elsevier; 2011. p. 354-372. 7. kondert l, mayer j: reproductive medicine in guinea pigs, chinchillas and degus. vet clin north am exot anim pract 2017;20:609-628. 8. hawkins mg, bishop cr: disease problems of guinea pigs: in: quesenberry k, carpenter j, editors. ferrets, rabbits and rodents, clinical medicine and surgery. 3rd edition. st. louis: elsevier; 2011. p. 295-310. 9. schuetzenhofer g, goericke-pesch s, wehrend a: effects of deslorelin implants on ovarian cysts in guinea pigs. schweiz arch tierheilk 2011;153:416-417. 10. mans c, donnelly tm: disease problems of chinchillas: in: quesenberry k, carpenter j, editors. ferrets, rabbits and rodents, clinical medicine and surgery. 3rd edition. st. louis: elsevier; 2011. p. 311-325. 11. jekl v, hauptman k: reproductive medicine in ferrets. vet clin north am exot anim pract 2017;20:629-663. 12. schoemacker nj, van deijk r, muijlaert b, et al: use of a gonadotropin releasing hormone agonist implant as an alternative for surgical castration in male ferrets (mustela putorius furo). theriogenology 2008;70:161-167. 13. pollock c: disorders of the reproductive and urinary systems: in: k quesenberry k, carpenter j, editors. ferrets, rabbits and rodents, clinical medicine and surgery. 3rd edition. st. louis (mo): elsevier; 2011. p. 46-61. 14. ness r, johnson-delaney ca: sugar gliders: in: quesenberry k, carpenter j, editors. ferrets, rabbits and rodents, clinical medicine and surgery. 3rd edition. st. louis: elsevier; 2011. p. 393-410. 15. miwa y, sladky kk: small mammals: common surgical procedures of rodents, ferrets, hedgehogs, and sugar gliders. vet clin north am exot anim pract 2016; 19:205-244. 16. ivey e, carpenter jw. african hedgehogs: in: quesenberry k, carpenter j, editors. ferrets, rabbits and rodents, clinical medicine and surgery. 3rd edition. st. louis: elsevier; 2011. p. 411-427. 17. mikaelian i, reavill dr, practice a: spontaneous proliferative lesions and tumors of the uterus of captive african hedgehogs (atelerix albiventris). j zoo wildl med 2004;35:216-220. clinical theriogenology • volume 10, number 3 • september 2018173 clinical theriogenology • volume 10, number 3 • september 2018 174 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2018: atrophy of a bull testis resulting from hypertrophy of a scrotallymph node atrophy of a bull testis resulting from hypertrophy of a scrotal lymph node college of veterinary medicine, university of tennessee, knoxville, tn importance hyperplasia of a scrotal lymph node causing atrophy and degeneration of a testis of bulls or other domestic animals has not been previously reported. hyperplasia of a scrotal lymph node may impart an hourglass appearance to the scrotum of a bull. diagnostic approach and treatment a 2 year-old angus bull was presented because of abnormal scrotal shape first noted 4 months previously. exudate in the semen and decreased seminal quality coincided with the change in scrotal appearance. the bull's scrotum had an hourglass shape, the left testis was small, and the right testis was substantially smaller than the left. ultrasonographically, both testes appeared heterogenous and hyperechogenic. an approximately 9.5-cm x 4.5-cm x 4.5-cm mass, hyperechogenic at its center and hypoechogenic at its periphery, causing the hourglass appearance, was found in the neck of the scrotum. evaluation of semen showed degenerate and non-degenerate neutrophils, some containing bacteria, and only 5 sperm per high-powered field; sperm motility was 60 percent. every sperm examined had an abnormality of the head or midpiece. trueperella pyogenes, a bacteria known to cause mastitis and metritis in cows, was cultured from the semen.1 the bull anesthetized and the right testis and mass were excised. results and discussion histological examination revealed the mass to be a non-infected, hyperplastic scrotal lymph node.2 this enlarged node, sometimes called the superficial inguinal lymph node3 caused the bull's scrotum to assume an hourglass shape, a shape most commonly indicative of inguinal herniation. the right testis and its epididymis histologically appeared to suffer from chronic atrophy and degeneration. these changes likely resulted from compromise of blood flow caused by impingement on the spermatic cord by the enlarged lymph node. the left testis was expected to hypertrophy and increase production of sperm after excising the right testis and mass. keywords: bull, testis, lymph node references 1. ribeiro mg, risseti rm, bolaños cad, et al: trueperella pyogenes multispecies infections in domestic animals: a retrospective study of 144 cases (2002 to 2012). vet q 2015;35:82-87. 2. jubb kvf, kennedy pc, palmer nc: in: maxie mg, editor. jubb, kennedy, and palmer’s pathology of domestic animals: vol. 3, 6th ed., st. louis:elsevier; 2016. p. 498. 3. corner la, alton gg, iyer h. distribution of brucella abortus in infected cattle. aust vet j 1987;64:241-244. clinical theriogenology • volume 10, number 3 • september 2018355 o. j. schumacher, j.l. klabnik-bradford, p.y. mulon, t.m. prado 2018: programming of development during embryonic and fetal life incattle and its consequences for subsequent production programming of development during embryonic and fetal life in cattle and its consequences for subsequent production peter j. hansen department of animal sciences, university of florida, gainesville fl introduction – capacity for health and productive function begins to form in utero at an early stage in their education, animal scientists are taught that an animal’s phenotype depends on a combination of genetic and environmental effects. indeed, efforts to increase the productive capacity of livestock have depended largely on two approaches – selection of allelic variants to genes that confer superior production and manipulating the environment to enhance the ability of animals to realize the potential of those genes. from a genetics point of view, environment includes everything that is not genetic including, as examples, nutrition, meteorological conditions, exposure to pathogens, and human interventions such as treatment with growth regulators or reproductive hormones, vaccinations and other veterinary procedures. while the importance of the environment for optimal health and production is well recognized, it is less widely understood that the function of an adult organism depends not only on environmental determinants after birth but also on environmental conditions it was exposed to before birth during embryonic and fetal development. this concept has been variously termed developmental programming, fetal programming or developmental origins of health and disease. for mammals, the prenatal environment of a developing embryo or fetus is established by its mother and changes in maternal environment can alter postnatal phenotype of the developing organism. we are only beginning to understand the degree to which developmental programming affects health and productive function of cattle. experiments performed to date are indicative that developmental programming can occur throughout gestation, from the early stages of development of the preimplantation embryo to the final weeks of gestation. moreover, developmental programming can shape many functions of adult cattle and in a way that influences economically-important traits. the purpose of this review is to provide some examples of developmental programming in cattle to illustrate the wide range of influences the prenatal environment can have on postnatal function. emphasis will not be on mechanisms although these are likely to include epigenetic modifications in the developing organism as well as in patterns of development of specific cell lineages that give rise to adult tissues. by providing examples of developmental programming, we can appreciate the potential for manipulating the embryo and fetus to positively or negatively affect health and production of the postnatal animal. the take-home message is that there is opportunity for animal managers and veterinarians to use information about developmental programming to increase the efficiency of milk and meat production. programming during the preimplantation period as revealed by the in vitro produced embryo one of the most extreme examples of an abnormal environment during the preimplantation period is the in vitro produced (ivp) embryo. such embryos are exposed to concentrations of nutrients, salts and growth factors that differ from those present in vivo and the substratum for culture (usually plastic) is different from that established by the oviductal and endometrial epithelium. not surprisingly, then, the ivp embryo differs from the embryo produced in vivo in terms of morphology, gene expression, cryotolerance and competence to establish pregnancy (see review by hansen, 2015 and recent data on competence to establish pregnancy by ferraz et al., 2016). alterations in the characteristics of the embryo caused by ivp can result in offspring with altered characteristics in the postnatal period. this concept was first recognized by observations that calves born following ivp sometimes exhibit a range of developmental disorders termed the large offspring syndrome that is characterized most prominently by excessive body growth (farin et al., 2006). recently, it was reported that alterations in physiological function extend to adult life, at least when ivp embryos are produced using fertilization with reverse-sorted sperm (siqueira et al., 2017). in this study, characteristics of females born from embryo transfer using embryos produced by ivp with conventional semen (ivpclinical theriogenology • volume 10, number 3 • september 2018163 conv; 345 heifer calves born alive and 218 with lactation records), ivp with reverse-sorted semen (ivprs; 685 heifers born alive and 430 with lactation records) and multiple-ovulation embryo transfer (moet; 249 heifer calves born alive and 183 with lactation records) were compared to females born as a result of artificial insemination (ai; 3,465 ai females born alive and 2,037 with production records for first lactation). lactational performance of the offspring was adjusted by using genomic estimates for milk, fat or protein as a covariate. the most striking difference between groups was in lactational performance during first lactation (table 1). in particular, ivp-rs females produced less milk, fat and protein than cows of other groups. the difference in average projected actual milk yield between cows produced using ivp-sexed and cows produced by ai was 321 kg. table 1. effects of technique used to produce a pregnancy on first-lactation milk yield of the resultant offspring after adjusting for genomic predicted transmitting ability for yield.1 ai ivp-conv ivp-rs moet p-value projected actual milk yield, 305 d (kg) 11038±31a 10946±100ab 10717±76b 10891±149ab 0.0014 projected actual fat yield, 305 d (kg) 388.3±1.2a 385.6±3.9ab 377.1±3.0b 384.7±5.8ab 0.0072 projected actual protein yield, 305 d (kg) 334.6±1.0a 336.5±3.3a 327.1±2.5b 331.2±4.8ab 0.0318 1 the p-values are for the main effect of reproductive technique. within a row, means without a common superscript differ at p < 0.05. it should be noted that these results do not come from a controlled experiment and there could be unknown biases that affect the results including the fact that neither dams nor sires of the animals studied were assigned randomly to treatment. in mice, the specific culture system used to produce embryos can affect the nature of developmental programming (fernández-gonzalez et al., 2004; donjacour et al. 2014; rexhaj et al., 2015) and effects observed by siqueira et al. (2017) might not be observed with other ivp systems. the fact that ivp was only associated with lower milk yield when reverse sorting was used for fertilization is suggestive that damage of the sperm during sorting may have been responsible for the observed programming. there are two other examples where the specific culture conditions in which ivp embryos developed changed the characteristics of the resultant offspring. addition of the cytokine colonystimulating factor 2 (csf2) to culture medium on day 5 of culture (when embryos are typically at the morula stage) resulted in changes in growth profiles of the resultant calves produced after embryo transfer (kannampuzha-francis et al., 2015). there were no differences in birth weight or withers height between heifers derived from ivp with or without csf2. subsequently, however, body growth (weight and height) through 13 mo of age was greater for the csf2 group. in another experiment, addition of the wnt antagonist dickopf-related protein 1 (dkk1) to culture medium at day 5 of culture resulted in calves of lower birth weight than calves derived from embryos cultured without dkk1 (tribulo et al., 2017). these results suggest that, for embryos produced in vivo, changes in maternal environment could conceivably result in changes in secretion of regulatory molecules like csf2 or dkk1 from the maternal reproductive tract that alter the developmental program of the embryo. studies to examine this hypothesis are warranted. effects of maternal protein nutrition on postnatal growth and reproduction experiments in australia with pregnant beef heifers indicate that protein nutrition during pregnancy can affect prenatal and postnatal growth of male and female offspring (micke et al., 2010a, 2010b, 2011). in these experiments, pregnant beef heifers were fed diets that were either high (240% of recommended intake) or low in crude protein (70% of recommended intake) during the first or second trimesters of gestation. there was no effect of maternal diet in the first trimester on calf birth weight but postnatal growth was affected in a sex-specific manner. for male offspring, those that were derived from clinical theriogenology • volume 10, number 3 • september 2018 164 dams receiving a low protein diet were heavier than those from dams fed a high protein diet. the opposite trend was apparent for female offspring. cross-sectional area of the semitendinosus and longissimus dorsi muscles followed a similar pattern: low protein nutrition of the dam was associated with larger muscle size for males but not for females. developmental programming caused by maternal protein nutrition during the second trimester was different than for programming during the first trimester. calves from mothers fed a low protein in the second trimester had lower birth weights than calves fed a high protein diet. after 29 days of age, there was no effect of treatment on body weight or, in general, on muscle growth. cross-sectional area of longissimus dorsi at slaughter was increased for male and female progeny from low protein dams although this effect was removed when area was adjusted for carcass weight. there is a report by martin et al. (2007) that fertility of female offspring can also be affected by maternal protein nutrition during the last trimester of gestation. heifers born from dams that received a protein supplement three times per week were more fertile in their first breeding season than heifers from dams that were not supplemented. in particular, maternal diet did not affect birth weight or age at puberty but more heifers from supplemented dams became pregnant after a 45 day breeding season (93%) than heifers from non-supplemented dams (80%). differences were particularly pronounced when examining the percent of heifers that became pregnant early in the breeding season. the percent of heifers that calved in the first 21 days of the breeding season was 77% for heifers from supplemented dams vs 49% for heifers from control dams. antral follicle count of heifers can be influenced by maternal energy intake of their dams an important determinant of fertility in dairy cattle is the number of ovarian follicles recruited into the growing pool during a follicular wave. the size of the follicular pool is usually estimated as the number of follicles > 3 mm in diameter (antral follicle count; afc) or by measuring concentrations of antimullerian hormone (amh) in the blood. several studies in dairy cattle found that the size of the afc was associated with fertility and productive herd life with a low or high afc or amh concentration being deleterious and with an intermediate afc or amh concentration being desirable (mossa et al., 2012; jimenez-krassel et al., 2015, 2017). one determinant of afc could be the nutritional environment the embryo or fetus is exposed to during earlyand mid-gestation. mossa et al. (2013) individually fed beef heifers a diet that provided either 1.2 or 0.6 times the energy requirements for maintenance from 11 days before insemination until day 110 of gestation. as illustrated in table 2, heifers born from undernourished cows had a reduction in afc from 7-86 weeks of age and in the number of visible antral follicles when the reproductive tract was collected at slaughter at 95 wk of age. in addition, circulating amh concentrations from 7-86 wk of age were lower in heifers from undernourished cows. table 2. characteristics of heifers as affected by energy intake of their dams from 11 days before insemination to 110 days of gestation (mossa et al., 2013). trait control dams underfed dams birth weight (kg) 40.5 + 1.0 39.5 + 2.0 placental weight at birth (kg) 5.6 + 0.6 5.3 + 0.5 age at puberty (weeks) 95 + 0.2 95 + 0.2 mean antral follicle > 3 mm count, 86 wk of age 23.6 + 19.9 15.8 + 1.8* visible antral follicles at slaughter, 95 wk of age 47 + 5 32 + 6* * treatment effect, p<0.05 conclusions and implications this paper represents a sampling of experiments showing that events in the prenatal life of a developing bovine can have effects that alter the function of the animal after birth. clearly, more work is needed to identify aspects of physiology of the pregnant female that cause long-term changes in its offspring and to identify mechanisms responsible for developmental programming. doing so will clinical theriogenology • volume 10, number 3 • september 2018165 generate new opportunities to maximize an animal’s genetic potential for production by allowing us to shape postnatal function through manipulation of maternal environment during gestation. references donjacour a, liu x, lin w, et al: in vitro fertilization affects growth and glucose metabolism in a sex-specific manner in an outbred mouse model. biol reprod 2014; 90:80. farin pw, piedrahita ja, farin ce: errors in development of fetuses and placentas from in vitro-produced bovine embryos. theriogenology 2006; 65:178-191. fernández-gonzalez r, moreira p, bilbao a, et al: long-term effect of in vitro culture of mouse embryos with serum on mrna expression of imprinting genes, development, and behavior. proc natl acad sci usa 2004; 101:5880-5885. ferraz pa, burnley c, karanja j, et al: factors affecting the success of a large embryo transfer program in holstein cattle in a commercial herd in the southeast region of the united states. theriogenology 2016; 86:1834-1841. hansen pj: developmental programming in the preimplantation period: can it be exploited to enhance postnatal function in cattle? anim reprod 2015;12:428-436. jimenez-krassel f, scheetz dm, neuder lm, et al: concentration of anti-müllerian hormone in dairy heifers is positively associated with productive herd life. j dairy sci 2015;98:3036-3045. jimenez-krassel f, scheetz dm, neuder lm, et al: a single ultrasound determination of follicles ≥3 mm in diameter in dairy heifers is predictive of a reduced productive herd life. j dairy sci 2017;100:5019-5027. kannampuzha-francis j, denicol ac, loureiro b, et al: exposure to colony stimulating factor 2 during preimplantation development increases postnatal growth in cattle. mol reprod dev 2015;82:892-897. martin jl, vonnahme ka, adams dc, et al: effects of dam nutrition on growth and reproductive performance of heifer calves. j anim sci 2007;85:841-847. micke gc, sullivan tm, soares magalhaes rj, et al: heifer nutrition during earlyand mid-pregnancy alters fetal growth trajectory and birth weight. anim reprod sci 2010a;117:1-10. micke gc, sullivan tm, gatford kl, et al: nutrient intake in the bovine during early and mid-gestation causes sex-specific changes in progeny plasma igf-i, liveweight, height and carcass traits. anim reprod sci 2010b;121:208-217. micke gc, sullivan tm, mcmillen ic, et al: protein intake during gestation affects postnatal bovine skeletal muscle growth and relative expression of igf1, igf1r, igf2 and igf2r. mol cell endocrinol 2011;332:234-241. mossa f, walsh sw, butler st, et al: low numbers of ovarian follicles ≥3 mm in diameter are associated with low fertility in dairy cows. j dairy sci 2012; 95:2355-2361. mossa f, carter f, walsh sw, et al: maternal undernutrition in cows impairs ovarian and cardiovascular systems in their offspring. biol reprod 2013;88:92. rexhaj e, pireva a, paoloni-giacobino a, et al: prevention of vascular dysfunction and arterial hypertension in mice generated by assisted reproductive technologies by addition of melatonin to culture media. am j physiol heart circ physiol 2015; 309:h1151-1156. siqueira lgb, dikmen s, ortega ms, et al: postnatal phenotype of dairy cows is altered by embryo production in vitro using reverse x-sorted semen. j. dairy sci. 2017; 100:5899-5908. tríbulo p, bernal ballesteros bh, ruiz a, et al: consequences of exposure of embryos produced in vitro in a serum-containing medium to dickkopf-related protein 1 and colony stimulating factor 2 on blastocyst yield, 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2011: error in pregnancy diagnosis by per-rectal palpation in beef cows error in pregnancy diagnosis by per-rectal palpation in beef cows r. kasimanickam,a w.d. whittier,b a. tibary,a b. inmanb adepartment of veterinary clinical sciences, washington state university, pullman, wa; bdepartment of large animal clinical sciences, virginia polytechnic institute and state university, blacksburg, va abstract the objective of this study was to determine the error rate of pregnancy diagnosis while differentiating artificial insemination (ai) pregnancy from bull-bred pregnancy in a synchronized breeding program. angus-cross beef cows (n=1941) were artificially inseminated using ovsynchcidr or co-synch-cidr synchronization protocols. bulls were introduced two weeks after ai to breed cows that were not pregnant from ai. the pregnancy status was diagnosed and days in gestation of each cow were determined by per-rectal palpation 70 d after ai. projected gestation length was determined based on days of gestation at pregnancy diagnosis and projected calving date. the gestation length (281.2 ± 4.71 d; mean ± sd) for the study population was retrospectively calculated from breeding and calving dates. these projected and actual gestation lengths were matched to determine the error in diagnosing pregnancies resulting from ai or by natural service. the error in pregnancy diagnosis was classified as error due to over-estimation (diagnosed pregnant to ai but pregnant to bulls) or under-estimation (diagnosed pregnant to bulls but pregnant to ai) using mean ± 2 sd as cut-off. the error rate for pregnancy diagnosis was estimated as 7.0 % (3.4% due to under-estimation and 3.6% due to over-estimation) in differentiating ai pregnancy from bull-breeding pregnancy for the gestation length of 281 days. similarly, the error rate for gestation lengths of 279 and 283 days were estimated as 5.8% and 7.1%, respectively. the error rate varied from 4% to 13% among clinicians. in conclusion, estimation of pregnancy rate from ai breeding programs should account for the error in pregnancy diagnosis accuracy. keywords: beef cows, breeding program, per-rectal palpation, pregnancy diagnosis, error rate introduction pregnancy is a measure of success in breeding programs. in clinical practice various diagnostic techniques have been adopted for diagnosing pregnancy. the most commonly used technique for diagnosing pregnancy is per-rectal palpation of the uterus and its contents.1-3 other methods in use are transrectal ultrasonography,4,5 and measurement of progesterone,5-7 pregnancy specific protein5,8 and early conception factors9 in serum. in beef synchronization programs, resynchronization of non-pregnant cows during the breeding season is not commonly utilized by producers due to repeated handling and difficulties in managing the cows. the most common practice is introduction of bulls two weeks after fixed-time ai (ftai) to impregnate cows that are not pregnant from ftai.11 this practice maximizes number of cows pregnant at the end of the breeding season while minimizing management. in order to measure the success of ai breeding programs it is necessary to distinguish ftai pregnancy from bull-breeding pregnancy. it is a challenge to differentiate ftai pregnancy from bull-bred pregnancy accurately by per-rectal palpation of the gravid uterus and its contents when the gestational difference is minimal. the objective of this study was to determine the error rate in determining cows pregnant to ai from cows pregnant to bulls by per-rectal palpation in a beef synchronization program. materials and methods cattle and synchronization protocols crossbred angus beef cows (n=1941) were artificially inseminated using ovsynch-cidr or co-synch-cidr synchronization programs. briefly, cows were treated with 100 g gonadotropinreleasing hormone (gnrh; cystorelin, merial, athens, ga) and a controlled internal drug release device (cidr; eazi-breed cidr, pfizer animal health, new york, ny) on day 0, 25 mg dinoprost tromethamine (pgf; lutalyse, pfizer animal health, new york, ny) and cidrs were removed on day 7 and either 100 g gnrh 48 h after pgf on day 9 (ovsynch-cidr), and ftai 16 43 h after gnrh on day 10 or 100 g gnrh on day 10 (co-synch-cidr) at the time of ftai. angus bulls were penned with cows two weeks after ftai (1:40 bull:cow ratio) to breed cows that were not pregnant for 45-50 days. pregnancy diagnosis pregnancy status was diagnosed for each cow by per-rectal palpation 70 d after ftai. once pregnancy was confirmed, the per-rectal palpation criteria1 used to differentiate ftai pregnancy from bull-bred pregnancy (70 and < 55 d pregnancy, respectively) were: the size of the gravid horn (7.0 to 10.0 cm vs. 5.0 to 6.5 cm), presence or absence of fluid in the non-gravid horn, size of the amniotic vesicle (5.5 to 6.5 cm [extra-large hen egg size] vs. 3.5 to 4.5 cm [small hen egg size]). all criteria were used to confirm the gestational age. six clinicians performed pregnancy diagnosis and their experience varied from one to 25 years. data management and analysis only cows inseminated with the semen from angus sires were used in the analysis. the mean ± sd gestation length was 281.2 ± 4.71 d when cows that calved between 260 and 300 d after ftai were considered. in order to prevent gestation length overlap between ftai and natural service pregnancies, the pregnancy error study population was restricted. the mean ± sd time between ftai and parturition in cows that conceived to bull breeding was 302 ± 4.71 d, only cows with gestation lengths of ≥ 271 d and ≤ 291 d were included in the analysis. projected calving dates for a gestation length of 281 d were calculated based on estimated days of gestation at pregnancy diagnosis. these dates were compared to actual calf birthdates to determine error in pregnancy diagnosis. error was classified as error due to over-estimation (diagnosed pregnant to ai but pregnant to bulls) or underestimation (diagnosed pregnant to bulls but pregnant to ai) using the mean ± 2 sd as the cut-off. similarly the error rate for gestation lengths of 279 and 283 d were also estimated. influence of parity was examined using chi-square analysis. data were analyzed with a statistical software program (sas version 9.1 for windows, sas institute, cary, nc). descriptive analysis was performed to estimate mean gestation length. arcsine transformation was applied to perform the analysis. the non-parametric one-way procedure of the sas system was used to perform the kruskal-wallis test to compare the difference among clinicians for total error rate and error rate due to over-estimation and under-estimation. influence of parity (2, 3, 4, and >4) was examined using chi-square analysis results the error rate, based on mean ± sd, in differentiating ftai pregnancy from bull-bred pregnancy is given in table 1. the error rate for pregnancy diagnosis was estimated to be 7.0% (3.4% due to under-estimation and 3.6% due to over-estimation) in differentiating ftai pregnancy from bull-bred pregnancy for the gestation length of 281 days. similarly the error rate for gestation lengths of 279 and 283 days were estimated to be 5.8% (1.4% due to under-estimation and 4.4% due to over-estimation) and 7.1% (3.7% due to under-estimation and 3.4% due to over-estimation), respectively. the error rate varied from 4% to 13% among clinicians. the year of graduation, number of animals examined and total error rate for clinicians are shown in table 2. frequency distribution histogram for gestation length based on actual calving date is shown in the figure. there was no influence of parity on the error of pregnancy diagnosis. table 1. error rate* in differentiating ai pregnancy from bull pregnancy (n=1941) gestation length (d) number underestimated pregnancies error rate due to under-estimation number overestimated pregnancies error rate due to over-estimation total number of errors total error rate 279 28 0.0144 85 0.0438 113 0.0582 281 66 0.0340 70 0.0361 136 0.0701 283 72 0.0371 65 0.0335 137 0.0706 * mean gestation length ± 2 sd 44 table 2. the year of graduation, number of animals examined and total error rate for clinicians who participated in the study clinician year of graduation number of animals total error rate 279 281 283 1 1979 679 0.040 0.048 0.046 2 1993 369 0.043 0.055 0.060 3 1991 330 0.068 0.072 0.071 4 1984 233 0.076 0.077 0.080 5 2004 194 0.087 0.130 0.130 6 2004 136 0.095 0.120 0.120 291288285282279276273270 120 100 80 60 40 20 0 gestation fr eq ue nc y histogram of gestation figure. distribution histogram for number of cows (n=1510) that calved after various gestation lengths. mean 281.0 days standard deviation = 4.2 days discussion in this study, errors in assessing gestation length by per-rectal palpation to determine whether the cow was pregnant to ftai or natural service were estimated. the error rate for pregnancy diagnosis was estimated to be 7.0 % for gestation lengths of 281 d. the mean (± sd) gestation length of 281.0 d (± 4.2) in the present study is consistent with that previously reported.12 in this study, the error due to over-estimation was similar to the error due to under-estimation (3.6% and 3.4%, respectively). if all cows were successfully synchronized the difference in gestation length would be approximately 21 days between cows that conceived to ftai and cows that conceived to the first service by bulls. however some cows may not have been synchronized and it is possible that the difference between an ftai pregnancy and a bull-bred pregnancy may be as short as 14 days (bulls introduced 14 days after ftai). experience of the clinician performing the examination may account for some of the errors in estimating gestation length. in the present study the error rate decreased with increased years of experience. other possible factors might be rumen fill, position of the uterus, ability of the examiner to retract the uterus and its contents, variation in the size of the gravid horn 45 and size of the amniotic vesicle. wisnicky and cassida described per-rectal palpation of the amniotic vesicle as an aid in determining pregnancy status in cattle.13 other investigators used crown-rump length14 and head and trunk diamters15 to determine gestational age using transrectal ultrasonography. these characteristics cannot be accurately employed in per-rectal palpation because vigorous and extended manipulation of uterus and fetus are required. in addition, these features are difficult to determine during 55 to 70 days of pregnancy due to the rigidity of amniotic vesicle. the first report of pregnancy diagnosis in cattle by per-rectal palpation dated from the early 1800’s.16 currently per-rectal palpation is the most common method used for pregnancy diagnosis in cattle. in a beef cattle operation early detection of non-pregnant cows is the main benefit of pregnancy diagnosis.17 it allows producers to make critical management decisions several months prior to calving, including marketing decisions based on pregnancy status. it also allows determination of the success of ai programs. veterinary schools across the united states and in other countries continue to train their students in the art of per-rectal palpation for diagnosis of pregnancy in dairy cattle. because of its widespread use and the number of bovine practitioners trained to perform the procedure, per-rectal palpation will likely remain a mainstay for pregnancy diagnosis in cattle. application of ultrasonography can be beneficial in determining gestation length. even though ultrasonographic measurement of crown-rump length and head and trunk diameters is correlated with gestational age, measuring these parameters may be time-consuming. features such as fetal and placentome size determined by ultrasonography may be useful to differentiate between gestational ages of 55 and 70 days. estimations of error rate by transrectal ultrasonography and cost-benefit analysis are needed to determine the advantage of ultrasonography over per-rectal palpation for differentiating between a pregnancy due to ai or to natural service at a subsequent estrus. the error in pregnancy diagnosis found in this study shows that it may influence the estimation of ai and synchronization program success. in conclusion, estimation of pregnancy rate in a beef ai program should account for the error in pregnancy diagnosis. acknowledgements the authors thank the clinicians who participated in the study. the authors also extend their gratitude to the staff of virginia department of corrections for their support with data collection. references 1. roberts sj: veterinary obstetrics and genital diseases. 2nd ed. ithaca, ny: published by the author; 1971. p. 14-35. 2. momont h: rectal palpation: safety issues. bovine pract 1990;25:122-123. 3. youngquist rs: pregnancy diagnosis. in: youngquist rs, editor. current therapy in large animal theriogenology. philadelphia: wb saunders co., 1997. p. 295-303. 4. ball pj, logue dd: ultrasound diagnosis of pregnancy in cattle. vet rec 1994;134:532. 5. sasser rg, ruder ca: detection of early pregnancy in domestic ruminants-review. j reprod fertil suppl 1987;34:261-271. 6. shemesh m, ayalon n, shalev e, et al: milk progesterone measurement in dairy cows: correlation with estrus and pregnancy determination. theriogenology 1978;9:343-353. 7. pennington ja, spahr sl, lodge jr: factors affecting progesterone in milk for pregnancy diagnosis in dairy cattle. br vet j 1976;132:487-495. 8. maurer rr, ruder ca, sasser rg: effectiveness of the protein b radioimmunoassay to diagnose pregnancy in beef cattle. j anim sci suppl 1 1985;61:390 [abstract]. 9. nancarrow cd, wallace al, grewal as: the early pregnancy factor of sheep and cattle. j reprod fertil suppl 1981;30:191-199. 10. cordoba mc, sartori r, fricke pm: assessment of a commercially available early conception factor (ecf) test for determining pregnancy status of dairy cattle. j dairy sci 2001;84:1884-1889. 11. barber ka: maximizing the impact of dairy and beef bulls through breeding technology. j dairy sci 1983;66:2661-2671. 12. lamb gc, larson je, geary tw, et al: synchronization of estrus and artificial insemination in replacement beef heifers using gonadotropin-releasing hormone, prostaglandin f2alpha, and progesterone. j anim sci 2006;84:3000-3009. 13. wisnicky w, cassida le: a manual method for diagnosis of pregnancy in cattle. j am vet med assoc 1948;113:451. 14. white ir, russel aj, wright ia, et al: real-time ultrasonic scanning in the diagnosis of pregnancy and the estimation of gestational age in cattle. vet rec 1985;117:5-8. 15. white ir, mckelvey wa, busby s, et al: diagnosis of pregnancy and prediction of fetal age in red deer by realtime ultrasonic scanning. vet rec 1989;124:395-397. 46 16. cowie ta: pregnancy diagnosis tests: a review. wallingford, uk: commonwealth agricultural bureaux; 1948. joint publication no. 13. p. 11-17. 17. zemjanis r: diagnosis and therapeutic technique in animal reproduction. 2nd ed. baltimore: the williams and wilkins co; 1971. 47 48 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 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[612.000 792.000] >> setpagedevice genomics, transcriptomics, and proteomics of normal and abnormal equine placenta: better understanding of late pregnancy function and dysfunction genomics, transcriptomics, and proteomics of normal and abnormal equine placenta: better understanding of late pregnancy function and dysfunction barry ball, shavahn loux, pouya dini, harutaka murase, hossam ali el-sheikh gluck equine research center, department of veterinary science university of kentucky, lexington, ky abstract high-throughput methods to assess genomics, gene messenger rna expression, and protein composition of tissues and body fluids have led to rapid advancement in understanding of normal and abnormal function of many body systems across a wide variety of animal (mammalian and nonmammalian) species. over several years, these techniques were applied to study normal physiology and disease of pregnant mare, focusing on placental and fetal fluids. although our understanding of endocrine aspects of pregnancy in mares is reasonably advanced, much of our understanding related to placental function and dysfunction remains limited. this review covers studies that detailed normal pregnancy changes in fetal and maternal placenta, along with changes in gene expression in a number of late-pregnancy diseases. keywords: equine, placenta, transcriptome, placentitis, pregnancy introduction advent of high-dimensional biological techniques, including genomics, transcriptomics, proteomics, lipidomics, and metabolomics and increasing application of these techniques marks a landmark in research to understand normal physiologic function and pathophysiology of disease in animals. in particular, high-throughput rna sequencing (rna-seq) allows quantitative evaluation of gene expression in a tissue or cell and investigation of different isoforms of transcript present. these approaches generate massive datasets that require high-capacity computing platforms for bioinformatics analysis. fortunately, equine genome sequencing1 and subsequent updates to the equine genome (equcab3.0)2 provided detailed information on equine genome. this information, in combination with information overload regarding gene structure and function, cellular, biological, and disease pathways from biomedical research in humans and laboratory species, heralded a new era in veterinary medical research. objective is to summarize recent studies in authors’ laboratory using these approaches to better understand normal physiology and pregnancy disease in mare. transcriptomics of normal fetal and maternal placenta across pregnancy much research on chorioallantois (ca) and endometrium (en) as fetal and maternal portions of placenta in domestic animals has focused on early pregnancy (maternal recognition of pregnancy) or very late pre-term changes. we examined changes in messenger rna (mrna) transcripts in both ca and en across pregnancy (1.5, 4, 6, 10, and 11 months) in mare.3 large number of differentially expressed genes (dge) were identified, with 5,932 and 3,667 deg in ca and en, respectively. greatest difference in expression occurred at 4 or 11 months of pregnancy. unsurprisingly, most highly expressed genes in ca and en were related to either endocrine or immune function. highly expressed genes included endocrine related transcripts (rln, cyp19a1, hsd3b2, spp1, pla2g10, inhba), immune-related transcripts (cst3, ctsl, serpina3, serpina6, serpina14, spink7, spink9, ltf, s100a6, slpi), iron binding proteins (acp5, fth1, hba2, lcn2, serpina14), and serine protease inhibitors (serpina3, serpina6, serpina14, spink7, spink9). others included extracellular matrix proteins (ecm1, sparc, mmp26), transport proteins (acp5, gm2a, hba2, lcn2), and antioxidants (prdx1, sod3). evaluation of gene expression networks provides ability to examine cellular or biological pathways associated with changes in transcript abundance. in fetal and maternal placenta, many pathways associated with cell growth, mitosis, metabolism, oxidative stress, angiogenesis, and steroidogenesis were upregulated, whereas immune-related pathways, including b cell activation, leukocyte and lymphocyte activation and immune response, were downregulated. these findings are consistent with fetal placenta clinical theriogenology • volume 12 number 3 • september 2020 333 needs for continued growth, steroid synthesis, and transport of materials to fetus, along with protecting allogeneic fetus from maternal immune response. analysis of placental transcriptome across pregnancy has also been useful in better understanding endocrine function of both en and ca during equine pregnancy.4 in particular, evaluation of various isoforms of steroidogenic enzymes in ca and en across pregnancy reveals close coordination between these 2 tissues for pregnane and estrogen synthesis regulation during pregnancy. similarly, changes in steroid receptors during pregnancy imply differences in relative importance of receptor types during pregnancy. nuclear progesterone receptor has lower and relatively constant expression in ca and en for most of pregnancy; however, membrane associated progesterone receptor (pgrmc1) is highly expressed in en and ca, which suggest that placental effects of pregnanes in mares may be mediated by these receptors in mares.4 microrna in chorioallantois, endometrium and circulation in normal and abnormal equine pregnancy in addition to protein coding messenger rna (mrna), there are a variety of small noncoding rna (ncrna) that are detected with rna-seq. these ncrna appear to regulate expression of mrna and provide additional information about gene function. of ncrna, microrna (mirna) are small (20 22 nucleotides) rna that regulate protein coding genes. expression of mirna clusters have been described in human placenta, including human chromosome 14 (c14mc) that appears to be highly conserved across eutherian mammals and seems to have an important role in placental development.5 in horse, orthologous mirna cluster is located on eca24 (c24mc).6 expression of mirna in c24mc in equine placenta was higher in earlier pregnancy, but declined with advancing pregnancy.6 target mrna of mirna in c24mc cluster had a reciprocal expression pattern (increased with pregnancy), and many transcripts regulated by these mirnas were related to angiogenesis and vascularization of placenta with advancing pregnancy.6 unlike mrna, mirna have a relatively long half-life in circulation, and changes in circulating mirna have been identified in normal and abnormal pregnancy in animals and in humans, with potential application as diagnostic biomarkers.7-10 in an initial, pcr-based study, we identified 1 mirna that was differentially expressed in late pregnancy (mir-374b) and 4 mirna that were differentially expressed during pregnancy in mares (mir-454, mir-133b, mir-486-5p, and mir-204b).11 these pregnancyspecific mirna targeted pathways related to placentation, angiogenesis, and endocrine function during pregnancy.11 members of c24mc cluster of mirna were also detected in circulation of pregnant mares.12 microrna from c24mc cluster were more highly expressed in circulation during early pregnancy, consistent with their expression pattern in placenta. serum enrichment with mir-1247-3p, mir-134-5p, mir-382-5p, and mir-433-3p at day 25 pregnancy and mir-1247-3p, mir-134-5p, mir409-3p, and mir-379-5p at day 45 pregnancy suggest that these mirnas are involved in early pregnancy events.12 one goal of examining mirna expression in serum during pregnancy was to evaluate use of mirna in blood as potential biomarkers during abnormal equine pregnancy. for this study, rna-seq was used to screen ncrna expression in ca, en and blood of mares with experimentally induced placentitis at ~ 280 days of pregnancy.13 tissues collected between 3 5 days after inoculation in treated mares and uninoculated mares of comparable pregnancy used as controls.13 analysis of ncrna expression in blood, ca and en revealed 658 and 507 mirna for tissue and blood, respectively. principal component analysis of these data revealed distinct clustering of samples based upon tissue of origin and disease state. a total of 50 ncrna were differentially expressed between control and placentitis tissue samples. differentially expressed mirna included 26 in ca, 20 in en and 9 in serum. of 9 mirna that changed in serum, 6 also exhibited parallel changes in either ca or en.13 many mirna that were upregulated in equine placentitis were also upregulated in women with chorioamnionitis, suggesting that aspects of these disease processes are conserved. many mirna that were dysregulated in equine placentitis were associated with altered immune function, in particular, regulation of inflammation mediating cytokines il6 and il8, as well as activation of macrophages and clinical theriogenology • volume 12 number 3 • september 2020334 lymphocytes. whether or not changes identified in circulating mirna in mares with experimental placentitis will have utility in diagnosis of spontaneous equine placentitis remains to be determined. although different changes detected between controls and treated mares, magnitude of changes may not lend themselves to good diagnostic tests, and it is likely that a panel of mirna will need to be evaluated as possible biomarkers. changes in transcriptome during abnormal equine pregnancy nocardioform placentitis nocardioform placentitis (np) remains a poorly understood disease of placenta in mares. it is characterized by late-term abortions and fetal growth retardation associated with a distinct placental lesion located typically at ventral aspect of placenta, distinct from cervical star. np is associated with gram-positive, branching actinomycetes including amycolatopsis spp., crossiela equi along with more recently characterized isolates of streptomyces atiruber and streptomyces silaceus.14-16 during 2017 foaling season, we collected placenta from mares suspect for nocardioform placentitis (n = 4) and 4 normal placentas as controls. rna isolated from these tissues was analyzed by rna-seq.17a total of over 3,000 genes were differentially expressed in placenta from mares with np. signaling pathways related to inflammation (cytokines and chemokines), pattern recognition receptors (toll-like receptors), apoptosis (caspases), hypoxia, angiogenesis and antimicrobial peptides upregulated in placenta from mares with np were compared to normal term placenta. ascending bacterial placentitis in addition to nocardioform placentitis, ascending bacterial placentitis remains an important cause of late-term pregnancy loss in mares. we examined changes in transcriptome of both ca and en recovered from mares with experimentally induced placentitis (streptococcus equi spp. zooepidemicus) at 8 days after initial transcervical inoculation and ~ 290 days of pregnancy. uninoculated mares served as controls; ~ 3000 genes were differentially expressed in ca and ~ 1000 deg were detected in en from mares with ascending placentitis. upregulated pathways in ca included inflammation, interleukin and integrin signaling, angiogenesis, apoptosis, toll-receptor signaling, and b cell and t cell activation (elsheikh and ball, unpublished). upregulated pathways in en included inflammation and integrin signaling, and toll receptor signaling. these changes, in turn, were associated with dysregulation of placental steroidogenesis, angiogenesis, nutrient transport, and hypoxia. a number of matrix metalloproteases were also upregulated in ca that may be associated with degradation of extracellular matrix and resultant placental separation. related pathways and mechanisms associated with this dataset are illustrated (figure 1). premature placental separation premature placental separation (pps; red bag placenta) is a common and poorly understood problem in foaling mare. premature separation of ca from endometrium without rupture of ca at cervical star during late first stage and second stage labor may result in significant fetal hypoxia if not identified quickly and corrected by opening chorioallantois to assist foal delivery. incidence of pps varies with study, but cited as 1.6% of 1,047 foaling with a mortality rate of 17.6%,18 0.9% of abortions presented in normandy france19 or 4.7% of reproductive losses in central kentucky.20 during late abortions associated with mare reproductive loss syndrome (mrls), incidence of pps was reported as 28% of cases.21 these data likely underestimate pps frequency, because many such cases are likely not presented to diagnostic laboratories if neonate is not overtly affected during delivery. clinically, pps has been variably associated with problems such as endophyte-infected fescue,22 placental inflammation associated with viral (ehv 1) placentitis,23 and ascending bacterial placentitis.24 although clinical presentation of equine pps is well known, underlying pathophysiology of problem in mare is poorly understood. therefore, we examined holistic changes in gene expression in ca of mares with premature placental separation using next generation sequencing technology. we performed rna-seq on ca from clinical theriogenology • volume 12 number 3 • september 2020 335 figure 1. schematic illustration of possible mechanisms associated with ascending placentitis in mare. mares with pps (n = 33) and mares with normal parturition as controls (n = 4). resulting rna-seq data were subjected to standard analysis pipeline to examine differentially expressed genes and associated pathways and upstream regulators (murase, el sheikh ali and ball, unpublished data). a large number of differentially expressed genes (dge) were identified with 5,932 and 3,667 deg in ca and en, respectively. a number of genes associated with extracellular matrix, including collagens, proteoglycans, and metalloproteinase inhibitors were upregulated in mares with pps compared to control ca. key upstream regulators identified include transcripts associated with hypoxia, inflammation, extracellular matrix, and cell adhesion (figure 2). hydrops allantois hydrops conditions are rare in mare and there is little information about underlying pathogenesis of these diseases in any species. we evaluated ca from formalin-fixed paraffin embedded (ffpe) tissues collected from archival materials of cases of hydrops allantois submitted to university of kentucky veterinary diagnostic laboratory (n = 10) compared to ffpe of pregnancy-matched normal control mares. rna was isolated from ffpe tissues from both groups for assessment of expression of genes related to angiogenesis and steroidogenesis.25 capillary density was reduced and expression of angiogenic genes was lower in ca from hydrops allantois cases, while transcripts related to hypoxia increased compared to controls (figure 3).26 interestingly, expression of genes associated with estrogen synthesis and estrogen receptors were also downregulated, which suggests a possible role of estrogen in dysregulation of placental angiogenesis in these cases. clinical theriogenology • volume 12 number 3 • september 2020336 figure 2. schematic diagram of changes associated with premature placental separation in mare. proteomic analysis of equine fetal fluids in normal and abnormal pregnancy analysis of biological materials by mass spectrometry can also be used to determine protein composition of body fluids, including amniotic and allantoic fluids.27,28 proteome of fetal fluids from control mares and from mares with experimentally induced placentitis was determined by ltq orbitrap mass spectrometry.27,28 overall, a total of 130 proteins were characterized in amniotic and/or allantoic figure 3. schematic illustration of changes associated with hydrops allantois in mare. clinical theriogenology • volume 12 number 3 • september 2020 337 fluid, with a total of 18 proteins upregulated in amniotic fluid from mares with placentitis. three proteins (haptoglobin, plasminogen isoforms) were present only in amniotic fluid in placentitis. an additional 15 proteins were upregulated in amniotic fluid, including proteins in serpin superfamily, immunoglobulins, apolipoproteins, transferrin, thyroxine binding globulin and serum albumin.28 interestingly, both, positive acute phase proteins (haptoglobin, alpha-1antiproteinase, and alpha-2-macroglobulin) and negative acute phase proteins (transferrin albumin) were increased in amniotic fluid from mares with inflamed placenta. a number of these proteins are regulated by inflammatory modulating cytokine (il6) and change during placental inflammation in women.28 allantoic fluid had relatively fewer proteins change in placentitis presence and most of these proteins were in common with those of amniotic fluid (alpha-1-antiproteinase, serotransferrin, and transferrin). similar results were obtained in a second study (increases in transferrin, lactoferrin and alpha-1-antiproteinase in allantoic fluid of mares with placentitis).27 alpha-1antiproteinase is an anti-inflammatory protein and modulates tissue-damaging effects of neutrophil enzymatic proteins. serum concentrations of this protein are used as an acute-phase protein to detect inflammation in humans.27 conclusion application of high-dimensional biology to normal and abnormal equine pregnancy is essential. data from our studies provide researchers a valuable resource to address specific research questions and to formulate new research hypotheses concerning pregnancy in mare. acknowledgement supported by albert g. clay endowment of university of kentucky. conflict of interest none to report. references 1. wade cm, giulotto e, sigurdsson s, et al: genome sequence, comparative analysis, and population genetics of the domestic horse. science 2009;326:865-867. 2. kalbfleisch ts, rice es, depriest ms, jr., et al: improved reference genome for the domestic horse increases assembly contiguity and composition. commun biol 2018;1:197-197. 3. loux sc, dini p, el-sheikh ali h, et al: characterization of the placental transcriptome through mid to late gestation in the mare. plos one.2019;14:e0224497-e0224497. 4. loux s, conley aj, el-sheikh ali h, et al: new insights in equine steroidogenesis: an in-depth look at steroid signaling in the placenta. reproduction 2020;160:65-82. 5. morales-prieto d, ospina-prieto s, schmidt a, et al: elsevier trophoblast research award lecture: origin, evolution and future of placenta mirnas. placenta 2014;35:s39-s45. 6. dini p, daels p, loux sc, et al: kinetics of the chromosome 14 microrna cluster ortholog and its potential role during placental development in the pregnant mare. bmc genomics 2018;19:954-954. 7. kotlabova k, doucha j, hromadnikova i: placental-specific microrna in maternal circulation--identification of appropriate pregnancy-associated micrornas with diagnostic potential. j reprod immunol 2011;89:185-191. 8. miura k, miura s, yamasaki k, et al: identification of pregnancy-associated micrornas in maternal plasma. clin chem 2010;56:1767-1771. 9. ura b, feriotto g, monasta l, et al: potential role of circulating micrornas as early markers of preeclampsia. taiwan j obstet gynecol 2014;53:232-234. 10. ngo tt, moufarrej mn, rasmussen m-lh, et al: noninvasive blood tests for fetal development predict gestational age and preterm delivery. science 2018;360:1133-1136. 11. loux sc, scoggin ke, bruemmer je, et al: evaluation of circulating mirnas during late pregnancy in the mare. plos one 2017;12:e0175045. 12. dini p, el-sheikh ali h, carossino m, et al: expression profile of the chromosome 14 microrna cluster (c14mc) ortholog in equine maternal circulation throughout pregnancy and its potential implications. int j mol sci 2019;20:6285. 13. loux sc, fernandes cb, dini p, et al: small rna (srna) expression in the chorioallantois, endometrium and serum of mares following experimental induction of placentitis. reprod fert develop 2019:10.1071/rd18400. 14. donahue jm, williams nm, sells sf, et al: crossiella equi sp nov., isolated from equine placentas. int j syst evol micr 2002;52:2169-2173. clinical theriogenology • volume 12 number 3 • september 2020338 15. labeda dp, donahue jm, williams nm, et al: amycolatopsis kentuckyensis sp. nov., amycolatopsis lexingtonensis sp. nov. and amycolatopsis pretoriensis sp. nov., isolated from equine placentas. int j syst evol micr 2003; 53:1601-1605. 16. labeda dp, price np, kroppenstedt rm, et al: streptomyces atriruber sp. nov. and streptomyces silaceus sp. nov., two novel species of equine origin. int j syst evol micro 2009;59:2899-2903. 17. ball b, loux s, kennedy l, et al: transcriptomic analysis of the chorioallantois from mares with nocardioform placentitis. j equine vet sci 2018;66. 18. mccue p, ferris r: parturition, dystocia and foal survival: a retrospective study of 1047 births. equine vet j 2012;44:22-25. 19. laugier c, foucher n, sevin c, et al: a 24-year retrospective study of equine abortion in normandy (france). j equine vet sci 2011;31:116-123. 20. hong cb, donahue jm, giles rc, jr., et al: equine abortion and stillbirth in central kentucky during 1988 and 1989 foaling seasons. j vet diagn invest1993;5:560-566. 21. cohen nd, carey vj, donahue jg, et al: descriptive epidemiology of late-term abortions associated with the mare reproductive loss syndrome in central kentucky. j vet diagn invest 2003;15:295-297. 22. putnam mr, bransby di, schumacher j, et al: effects of the fungal endophyte acremonium coenophialum in fescue on pregnant mares and foal viability. am j vet res 1991;52:2071-2074. 23. smith k, whitwell k, blunden a, et al: equine herpesvirus‐1 abortion: atypical cases with lesions largely or wholly restricted to the placenta. equine vet j 2004;36:79-82. 24. canisso i, ball ba, erol e, et al: editors. comprehensive review on equine placentitis. proc am assoc eq pract 2015;61:490-509. 25. dini p, esteller‐vico a, scoggin ke, et al: extraction of rna from formalin‐fixed, paraffin‐embedded equine placenta. reprod domest anim 2019;54:627-634. 26. dini p, carossino m, loynacha a, et al: equine hydrallantois is associated with impaired angiogenesis in the placenta. placenta 2020;93:101-112. 27. canisso if, loux s, scoggin ke, et al: fetal-fluid proteome analyses in late-term healthy pregnant mares and in mares with experimentally induced ascending placentitis. reprod fert develop 2019;31:1486-1496. 28. loux s, ball b: the proteome of fetal fluids in mares with experimentally-induced placentitis. placenta 2018;64:71-78. clinical theriogenology • volume 12 number 3 • september 2020 339 023_ms-023 ball 2018: association between inter puppy interval at whelping andstillbirth risk association between inter puppy interval at whelping and stillbirth risk a.j. cornelius, s.h. cheong college of veterinary medicine, cornell university, ithaca, ny prolonged interval between puppy births increases the risk of the subsequent puppy being stillborn. there are general guidelines of what constitutes a normal inter-puppy interval (ipi) but there are no studies quantifying the change of stillbirth risk at these time points. our objectives were to: 1) identify the optimal threshold for ipi associated with stillbirth and report the performance of the commonly used threshold of 2-4 hours; and 2) determine if covariates such as birth order and sex of puppy are associated with stillbirth risk. we hypothesize that 1) the optimal threshold for ipi is between the 1-4 hours with a tradeoff between over diagnosis of puppies at increased stillbirth risk and missing puppies that will be stillborn as the ipi threshold increases; and 2) covariates such as birth order affects stillbirth rates. study design retrospective study was performed using whelping data from a guide dog colony. birth outcome (stillborn or born alive) was used as the dependent variable. covariates evaluated included: sex of the puppy, birth order, if the puppy was the last pup in the litter, litter size, breed and total whelp time. the ipi cannot be determined for the first puppy born in a litter and these were excluded from the analyses. association between stillbirth and ipi was evaluated using logistic regression (jmp pro ver 13) and the optimal threshold for ipi was determined by receiver operator characteristics (roc) curve. the effect of birth order on stillbirth risk was evaluated using mixed-model anova (proc glimmix of sas ver 9.4). covariates were evaluated individually and offered to the final model if p < 0.10 and retained if p < 0.05. results a total of 3,594 puppies from 493 litters were evaluated and the stillbirth rate was 6.6%. puppies that were born live had lower median and mean ipi (32 and 47 minutes) compared with stillborn puppies (65 and 102 minutes). roc analysis showed the optimal ipi threshold to identify stillborn puppy was 62 minutes (area under the curve=61%) with a positive predictive value (ppv)=14.5% and the negative predictive value (npv)=95.9%. puppies with an inter pup interval of >60 minutes were 3.7 times more likely to be stillborn with ppv=13.5% and npv=95.9% at this threshold. the odds ratio for being stillborn was 5.9, 4.7 and 9.3 respectively for puppies with an ipi of >120 minutes (ppv=28.4%, npv=93.7%), >180 minutes (ppv=31.8%, npv=94.5%), and >240 minutes (ppv=36.5%, npv=94.2%). the final model for stillbirth retained ipi, birth order, being the last puppy in the litter, and sex of the puppy. male puppies are 34% more likely to be stillborn compared with female pups; birth order was significantly associated with stillbirth; and surprisingly the last puppy in the litter were 2.4 times more likely to be stillborn even after controlling for birth order. discussion/conclusion the optimal threshold for ipi in our dataset was 62 minutes which is lower than the 2-4 hours typically accepted as normal. when making decision to intervene during whelpings, the ppv is more useful than npv as the ppv informs us the likelihood that the puppy with ipi >62 minutes has a probability of 14.5% to be stillborn and increases to 36.5% when ipi >240 minutes. the high npv just mean the puppy born within 62 minutes from the previous puppy has a 95.9% probability to be alive but even for the 4.1% stillborn puppy with the low ipi, there is no action to be taken. the proportion of female puppies that were stillborn was 5.8% compared with 7.3% in male puppies but the last puppy in the litter, regardless of litter size has a stillbirth rate of 18.7% compared with non-last puppies at 4.6%. male pups and more importantly, last pups in the litter were at higher risk of stillbirth. keywords: canine, stillbirths, inter puppy interval, birth order clinical theriogenology • volume 10, number 3 • september 2018 310 clinical theriogenology 2022; 14: 339 l-carnitine and acetyl-l-carnitine enhance stallion sperm quality during semen storage at 5°c fernando lisboa,a lorenzo segabinazzi,b felipe hartwig,a camila freitas-dell`aqua,a frederico papa,a josé dell`aqua jr,a adepartment of veterinary surgery and animal reproduction, sao paulo state university (unesp) school of veterinary medicine and animal science, botucatu, brazil bross university school of veterinary medicine, basseterre, st. kitts, west indies abstract carnitine, a powerful antioxidant, has an essential role in sperm energy metabolism. among carnitines, only l-carnitine’s effect on stallion semen has been tested and not acetyl-l-carnitine. therefore, we aimed to determine the ideal concentrations of l-carnitine (lc) and acetyl-l-carnitine (ac) and their effects on stallion semen cooled at 5℃ for up to 48 hours. semen was extended to 50 x 106 sperm/ml in commercial extender (control), and concentrations of 5, 10, and 15 mmol/l of lc and ac were evaluated in experiment 1. sperm motility and plasma membrane integrity were assessed by casa and epifluorescence microscopy, respectively. in experiment 2, the combination of the intermediate doses of lc (10 mmol/l) and ac (10 mmol/l) was tested. sperm parameters were evaluated as in experiment 1 and in addition, dna fragmentation index (dfi), production of reactive oxygen species (ros), and lipid peroxidation (perox) were evaluated by flow cytometry. all analyses were performed at 0, 24, and 48 hours after semen collection, processing, and cooled-stored at 5°c. in experiments 1 and 2, the groups supplemented with lc and ac or lc+ac had higher plasma membrane integrity and motility parameters compared to control group (p < 0.05). the lc and ac combination did not change sperm parameters compared to lc or ac alone (p > 0.05). no differences (p > 0.05) were observed for dfi, ros, and perox. in conclusion, lc and ac’s addition, alone or in combination, enhanced sperm motility and plasma membrane integrity of stallion sperm after cooled-storage at 5℃ for up to 48 hours. keywords: stallion, carnitine, sperm metabolism, cooled semen, sperm viability introduction l-carnitine use as a nutraceutical drug is consolidated in equine reproduction since various reports have demonstrated positive results after oral supplementation.1-3 however, only a few reports have documented the effects of carnitines inculsion in stallion semen.4-6 carnitines modulate several metabolic functions in sperm (e.g., beta-oxidation of fatty acids, acetyl-coa, and free coa ratio, using pyruvate and lactate as energetic substrates to produce adenosine triphosphate).7 only the l-isomer of carnitines is biologically active and is concentrated in tissues that require high amounts of energy, such as skeletal, cardiac muscles, and specialized organs of the reproductive tract (e.g., epididymis).8 l-carnitine is carried from blood to epididymis through active transport and is accumulated into sperm through passive transport.7 these interactions are androgen dependent.1,9 for this reason, concentrations of carnitine in the epidydimal fluid are 2,000 fold higher than in the blood, suggesting that this molecule is highly related to fertility. 4 l-carnitine is a carrier of fatty acids in energy metabolism since it can cross the internal mitochondrial membrane it has an essential role in the oxidative pathways and atp synthesis.10,11 l-carnitine needs to be acetylated through the carnitine acetyltransferase enzyme to cross the mitochondrial membrane, forming acetyl-l-carnitine. after crossing the mitochondrial membrane, acetyl-l-carnitine is dissociated into acetyl-coa to synthesize atp.7 in summary, the acetylated form of carnitine is responsible for providing acetyl groups for sperm motility.12,13 the entry of l-carnitine and its conversion into acetyl-l-carnitine in sperm is evidence of good epididymal function.1 furthermore, there is a correlation between l-carnitine/acetyl-l-carnitine ratio in semen and sperm progressive motility, suggesting that carnitines might contribute to sperm viability during the cooling process.14 additionally, carnitines have a powerful antioxidant action by reducing the availability of phospholipids for lipid peroxidation and increasing concentrations and activity of antioxidant enzymes (e.g., superoxide dismutase and glutathione peroxidase).15 we hypothesized that adding l-carnitine and acetyl-l-carnitine could improve equine sperm quality during cooled storage, either by carrying acetyl groups available in the extender to the inside mitochondria or by providing them directy by its acetylated form. we aimed to evaluate the isolated and assoclinical theriogenology 2022; 14: 340 ciated inclusion of l-carnitine and acetyl-l-carnitine in semen extender on sperm motility and viability parameters of cooled equine semen stored at 5℃ for up to 48 hours. materials and methods reagents used were purchased from sigma-aldrich (st. louis, mo). experimental protocols were approved by the ethics committee for animal use (são paulo state university [unesp] protocol #74/2012). this study was carried out from march to may of 2013. animals twelve stallions (6 of each quarter horse and mangalarga marchador breed), aged between 5 and 15 years, were enrolled. animals were kept in stalls, fed 3 kg of balanced grain and with free access to hay, water, and trace minerals. stallions were in reproductive management and had been collected 3 times a week. therefore, no washout semen collections were needed before the study. experimental design semen collections were performed using a botucatu artificial vagina (botupharma, botucatu, são paulo, brazil) at 72 hour intervals off a dummy mount in the presence of a teaser mare. after semen collection, each ejaculate was filtered, and the sperm concentration was determined with a hemocytometer chamber. thereafter, semen samples were equally split into groups and extended to 50 x 106 sperm/ml using a commercially available kenney-type (skim milk-based, skm) extender (botu-semen, botupharma), containing 20 g/l of skm, glucose, bicarbonate buffer, 1 g/l of gentamicin sulfate, and 1 g/l of sodium penicillin supplemented or not with l-carnitine or acetyl-l-carnitine. all extenders had ph fixed between 6.8 7.0 and osmolarity at 340 360 mosm. all semen samples were extended at a minimum dilution of 2:1 (v:v; extender:semen).16 after extension, samples were stabilized for 15 minutes at room temperature (22°c) and then stored in a passive semen cooling container (botuflex, botupharma) at 5°c for 24 hours. container was maintained in the laboratory at room temperature to avoid any additional interference. after this period, samples were transferred to a temperature-controlled refrigerator (minitube do brasil®, porto alegre, rio grande do sul, brazil) at 5°c out to 48 hours postcooling. experiment 1 inclusion of various concentrations of l-carnitine and acetyl-l-carnitine into semen extender on sperm parameters of cooled sperm two ejaculates of each stallion (n = 24) were divided into: control group, semen was extended in botu-semen; lc1, botu-semen supplemented with 5 mmol/l of l-carnitine; lc2, botu-semen supplemented with 10 mmol/l of l-carnitine; lc3, botu-semen supplemented with 15 mmol/l of l-carnitine; ac1, botu-semen supplemented with 5 mmol/l of acetyl-l-carnitine; ac2, botu-semen supplemented with 10 mmol/l of acetyl-l-carnitine; ac3, botu-semen supplemented with 15 mmol/l of acetyl-l-carnitine. sperm motility parameters and pmi were evaluated immediately before cooling (0 hour), 24, and 48 hours. experiment 2 effects of the combination of l-carnitine and acetyl-l-carnitine on sperm parameters in cooled semen as there were no differences among treated groups in experiment 1, intermediate doses of l-carnitine and acetyl-l-carnitine were chosen to test the combination of these molecules on sperm parameters. two ejaculates (n = 24) of each stallion were divided into: control, semen extended in botu-semen; lc, botu-semen supplemented with 10 mmol/l of l-carnitine; ac, botu-semen supplemented with 10 mmol/l of acetyl-l-carnitine; lc/ac, botu-semen supplemented with 10 mmol/l of l-carnitine and 10 mmol/l of acetyl-l-carnitine. sperm motility parameters, pmi, dna fragmentation (dna), lipid peroxidation (perox), and reactive oxygen species (ros) were evaluated immediately before cooling before cooling (0 hour), 24, and 48 hours. sperm analyses sperm kinetics sperm motility parameters were evaluated using computer-assisted sperm analysis (ivos 12, hamilton thorne inc., beverly, ma) using customized settings for equine sperm.17 for each sample, the percentages of total motility (tm), progressive motility (pm), average path velocity (vap, µm/s), straight-line velocity (vsl, µm/s), curvilinear velocity (vcl, µm/s), and rapid sperm (rap) were evaluated. each sample was incubated in a dry bath at 37°c for 10 minutes before each evaluation. for each sample, 5 random fields were assessed. plasma membrane integrity plasma membrane integrity was performed by epifluorescence microscopy (leica microsystem-dmlb, germany) based on the association of the fluorescent probes propidium iodide and 6-carboxyfluorescein diacetate.18 carboxifluorescein diacetate–positive cells were considered sperm with intact plasma membrane and pi-positive sperm with damaged plasma membrane. flow cytometry analyses flow cytometric analyses were carried out with fortessa lsr equipment (becton dickinson, mountain view, ca) equipped with blue (488-nm, 100 mw), red (640-nm, 40 mw), and violet (405-µm, 100 mw) lasers that were quality-controlled daily using cs&t beads and facs diva software (bd biosciences). the filter configurations for the pmts (photomultiplier tubes) measuring fluorescence emission of the applied fluorochromes were 450/50 nm (h342), 530/30 nm (fitc); 660/20 nm (apc); and 694/50 nm (pi). auto-fluorescence and single-color controls were acquired to perform spectral overlap compensation using the automated compensation matrix feature in facs diva software. fluorescence minus the clinical theriogenology 2022; 14: 341 controls was used to identify staining regions. flow cytometry data were plotted using bi-exponential plots with axes < 0 to ensure that all data were visible and properly compensated. thereafter, histograms were generated for analysis. auto-fluorescence and controls of each fluorochrome were acquired to adjust wave overlap and compensation using the matrix compensation of the manufacturer’s software. the percentage of positive sperm was considered for each assay. for each assay, at least 10,000 cells per sample were analyzed, and the data were extracted using the manufacturer’s software (bd facsdiva™ v6.1). all samples were extended in modified talp-pva19 at 5 x 106 sperm/ml concentration. composition of the talp-pva medium was: 100 mm nacl, 3.1 mm kcl, 25.0 mm nahco3, 0.3 mm nah2po4, 21.6 mm dl 60% sodium lactate, 2.0 mm cacl2, 0.4 mm mgcl2, 10.0 mm hepesfree acid, 1.0 mm sodium pyruvate, 1.0 mg/ml polyvinyl alcohol-pva, and 25 µg/ml gentamicin. dna fragmentation index this assay was performed as described.20,21 first, semen was diluted in a buffer solution (0.186 g of disodium edta, 0.790 g of tris-hcl, 4.380 g nacl in 500 ml deionized water, ph 7.4) to 1 x 106 sperm/ml. then, 400 μl of acid detergent solution (2.19 g naci, hci 1.0 ml of 2n solution, 0.25 ml triton-x, in quality standards program 250 ml of deionized water was added, ph 1.8) was added, incubated for 30 seconds and then added 1 ml of acridine orange dye solution (ao; 3.8869 g of citric acid monohydrate, 8.9429 g na2hpo4, 4.3850 g naci, 0.1700 g of disodium edta, 4 μg/ml of acridine orange solution 1 mg/ml, in quality standards program 500 ml of water, ph 6.0). samples were then analyzed by flow cytometry in 5 minutes, and the data generated were analyzed using the winlist 6.0 software (verify software house). the dna fragmentation index (dfi, %) was generated from the analysis of 10,000 cells marked with acridine orange. the idf is the proportion, expressed as a percentage of sperm dna and fragmented dna (red fluorescence) divided by total fluorescence. ros production intracellular hydrogen peroxide production was assessed using dihydrorhodamine 123 (dhr, d23806, life technologies, são paulo, são paulo, brazil). after reacting with ros such as hydrogen peroxide or peroxide nitrite, dihydrorhodamine 123dhr is oxidized into a fluorescent compound (rhodamine 123) that is retained within the cell. each 500 μl aliquot of extended semen in talp-pva had 1.5 μm propidium iodide (diluted in talp-pva), and 1 μm dhr (diluted in dimethyl sulfoxide) added before being incubated at 37°c for 20 minutes under the light. lipid peroxidation lipid peroxidation assessments were carried out with the fluorescent probe c11-bodypy (d-3861; molecular probes, carlsbad, ca), a lipophilic fluorophore sensitive that reacts with oxygen and peroxynitrite and, once oxidized, converts its fluorescence from red to green. lipid peroxidation was accessed in the total sperm population of each sample. each aliquot of semen (2 x 106 sperm/ml talp-pva extended in 489.5 μl) was added c11bodipy581/591 (0.5 μl, solution 1 mg/ml), propidium iodide (5 μl, solution 50 μg/ml), hoescht 33,342 (5 μl, solution 100 μg/ml) and then the mixture was incubated for at 37°c for 30 minutes. after incubation, the samples were washed twice at 300 x g for 5 minutes, and the pellet was resuspended in 500 μl of talp-pva and then analyzed by flow cytometry.22 data analyses graphpad prism 8.0.1. (graphpad software, san diego, ca) was used. the gaussian distribution of the semen parameters was evaluated using the shapiro-wilk normality test. semen parameters were assessed with a one-way anova and tukey’s as a posthoc test. stallion was accounted as a random effect. ejaculate order, time of storage, and the treatment groups as fixed effects. data are presented as mean ± sd. results there were no effects (p > 0.05) of ejaculate order and stallions displayed minimal variation (p > 0.05), in raw semen parameters. the overall gel-free volume, sperm concentration/ ml, and total sperm ejaculated were 36 ± 16 ml, 264 ± 164 x 106, and 8 ± 3.9 × 109, respectively. experiment 1 motility parameters were enhanced in fresh semen in lc1 group compared to control group (p < 0.05; figure 1a,d,g). progressive motility of fresh semen was higher (p > 0.05) in all groups supplemented with l-carnitine (lc1, lc2, and lc3) compared to control group (figure 1d). inclusion of ac in semen did not change (p > 0.05) sperm motility in fresh semen samples (figure 1a,d,g). plasma membrane integrity was higher (p > 0.05) in fresh semen samples in lc2 and l3 groups than control (figure 1j). semen in the other groups had intermediate pmi (p > 0.05, figure 1j). sperm motility parameters and pmi decreased (p > 0.05) over time in all groups (figure 1). after 24 hours of storage at 5°c, higher (p > 0.05) tm was observed in all groups supplemented with lc and ac (figure 1b). semen in all lc groups and ac3 had higher (p > 0.05) pm and rap than the control group, but not different (p > 0.05), than ac1 and ac3 (figure 1e). at 48 hours, tm, pm, and rap were higher (p > 0.05) in all groups supplemented with lc or ac compared to the control group (figure 1f). however, pm was higher (p > 0.05) in the lc2 group compared to the others, except lc3 (figure 1f). higher (p > 0.05) pmi was observed in all lc groups at 24 and 48 hours after cooling compared to control (figure 1k,l). the ac groups had intermediate (p > 0.05) pmi values at the same time points (figure 1k,l). values for vap, vcl, and vsl are highlighted (table 1). sperm velocity parameters were reduced (p > 0.05) over time in all groups, and the addition of l-carnitine change vap and vsl in fresh semen samples compared to control. clinical theriogenology 2022; 14: 342 figure 1. mean ± sd of sperm motility parameters and plasma membrane integrity (pmi) of stallion semen (n = 24 ejaculates) in vitro supplemented with various concentrations of l-carnitine (lc) or acetyl-l-carnitine (ac) at 0 (a,d,g,j), 24 (b,e,h,k), and 48 hours (c,f,i,l). ct, semen extended in milk-based extender (botu-semen) without supplementation; lc1, botu-semen supplemented with 5 mmol/l of l-carnitine; lc2, botu-semen supplemented with 10 mmol/l of l-carnitine; lc3, botu-semen supplemented with 15 mmol/l of l-carnitine; ac1, botu-semen supplemented with 5 mmol/l of acetyl-l-carnitine; ac2, botu-semen supplemented with 10 mmol/l of acetyl-l-carnitine; ac3, botu-semen supplemented with 15 mmol/l of acetyl-l-carnitine. different superscripts denote effects of time (a,b,c) and differences (p > 0.05) among groups within each time point (a,b,c). clinical theriogenology 2022; 14: 343 table 1. mean ± sd of sperm kinetic parameters evaluated by casa of cooled equine semen (n = 24 ejaculates) supplemented or not with various concentrations of l-carnitine (lc) or acetyl-l-carnitine (ac) at moments 0, 24, and 48 hours (n = 24). time (hour) group vap (µm/s) vsl (µm/s) vcl (µm/s) t0 ct 123 ± 14ac 94 ± 12ab 229 ± 23aa lc1 135 ± 9aa 103 ± 7aa 239 ± 47aa lc2 134 ± 9aab 103 ± 7aa 237 ± 49aa lc3 131 ± 12ab 102 ± 10aa 240 ± 22aa ac1 133 ± 9aab 99 ± 6aab 246 ± 21aa ac2 132 ± 10aab 100 ± 7aab 245 ± 23aa ac3 129 ± 10aab 98 ± 7aab 238 ± 22aa t24 ct 111 ± 13ba 87 ± 11aa 211 ± 22aba lc1 115 ± 15ba 91 ± 12ba 207 ± 46ba lc2 116 ± 14ba 90 ± 20ba 207 ± 46ba lc3 113 ± 16ba 91 ± 12ba 210 ± 31ba ac1 115 ± 15ba 91 ± 11ba 208 ± 46ba ac2 114 ± 13ba 91 ± 10ba 214 ± 25ba ac3 112 ± 13ba 90 ± 11ba 209 ± 23ba t48 ct 103 ± 19ba 75 ± 13ba 205 ± 33ba lc1 108 ± 15ba 83 ± 12cb 212 ± 29ba lc2 108 ± 18ba 84 ± 13bb 207 ± 33ba lc3 103 ± 17ca 81 ± 13cab 199 ± 29ba ac1 105 ± 16ca 81 ± 11ca 206 ± 29ba ac2 104 ± 17ca 80 ± 12ca 204 ± 31ba ac3 102 ± 17ca 80 ± 12ca 200 ± 30ba vap, average path velocity; vsl, straight line velocity; vcl, curvilinear velocity. group ct (botu-semen®); lc1 group (botu-semen® + 5 mmol/l of lc); lc2 group (botu-semen® + 10 mmol/l of lc); lc3 group (botu-semen® + 15 mmol/l of lc); ac1 group (botu-semen® + 5 mmol/l of ac); group ac2 (botu-semen® + 10 mmol/l of ac); group ac3 (botu-semen® + 15 mmol/l of ac). t0 (15 minutes of incubation), t24 (24 hours of refrigeration) and t48 (48 hours of refrigeration). different superscripts denotes effect of time in the same group (a,b,c) and among groups at the same moment (a,b) (p < 0.05). experiment 2 supplementation did not change (p > 0.05) tm and rap in fresh semen samples (figure 2a,g). however, semen supplemented with lc had higher (p > 0.05) pm at 0 hour than semen in control group, whereas ac and lc/ac had intermediate values not different (p > 0.05) than others (figure 2d). sperm motility parameters and pmi decreased (p > 0.05) over time in all groups (figure 2). after cooled storage for 24 and 48 hours, tm, pm, and rap were higher (p > 0.05) in all treated groups compared to control (figure 2). velocity parameters (vap, vcl, and vsl) are highlighted in table 2. sperm velocity parameters were reduced (p > 0.05), overtime in all groups. additionally, canitines enhanced (p > 0.05) pmi at all time points (0, 24, and 48 hours) in compared to control (figure 2j,k,l). however, carnitines did not change (p > 0.05) the sperm dfi, production of ros, or perox over cooling storage compared to unsupplemented samples (figure 3). discussion we determined whether the inclusion of l-carnitine and acetyl-l-carnitine, alone or in combination, in a milk-based extender, improved stallion semen quality after storage at 5°c for up to 48 hours. although the benefits of l-carnitine as a nutraceutical have been suggested,1-3 only a few studies have reported the effects of the inclusion of l-carnitine into semen extender on stallion sperm parameters.4-6 furthermore, to the best of the authors’ knowledge, there is no study evaluating the inclusion of acetyl-l-carnitine into equine semen extender. sperm motility parameters improved by the inclusion of l-carnitine (5, 10, and 15 mmol/l) in fresh semen. it suggested a rapid effect of l-carnitine on sperm metabolism that increased the availability of atp for sperm motility.4 furthermore, sperm parameters of oligospermic stallions improved by including carnitines in semen.1 additionally, inclusion of l-carnitine in chemically-defined medium for stallion semen kept at room temperature resulted in better tm and pm, and reduced oxidative dna damage.4 it is known that l-carnitine carries fatty acids through the internal mitochondrial membrane that clinical theriogenology 2022; 14: 344 improved the β-oxidation of fatty acids and consequently increased the production of energy by the cell,11 improving sperm kinetics.4,6 interestingly, a positive correlation between plasma concentrations of l-carnitine and sperm quality was reported in stallions6,14 and humans.7 furthermore, nutraceutical supplementation of l-carnitine improved sperm parameters in both species.1,3,23 higher availability of l-carnitine might be associated with improved energy metabolism in sperm. oral supplementation of l-carnitine has been suggested to improve spermatogenesis by reducing the percentage of abnormal sperm, such as sperm with detached heads and tails.1 additionally, oral supplementation with 40 g/day of l-carnitine for over 6 weeks improved conception rates in standardbred stallions.24 however, in another report, oral supplementation with l-carnitine (10 mg/day) did not affect sperm parameters of an arabian stallion.25 besides different doses in each study, the authors hypothesized that the different results may be associated with the individual seminal plasma concentrations of carnitines, which makes some stallions benefit more from l-carnitine supplementation. figure 2. mean ± sd of sperm motility parameters and plasma membrane integrity (pmi) of stallion semen (n = 24 ejaculates) in vitro supplemented with l-carnitine (lc) or acetyl-l-carnitine (ac) at moments 0 hour (a,d,g,j), 24 hours (b,e,h,k), and 48 hours (c,f,i,l). ct, semen extended in botu-semen; lc, botu-semen supplemented with 10 mmol/l of l-carnitine; ac, botu-semen supplemented with 10 mmol/l of acetyl-l-carnitine; lc/ac, botu-semen supplemented with 10 mmol/l of l-carnitine and 10 mmol/l of acetyl-l-carnitine. different superscripts denote effects of time (a,b,c) and differences (p > 0.05) among groups within each time point ( a,b,c). clinical theriogenology 2022; 14: 345 table 2. mean ± sd of sperm kinetics parameters evaluated by casa of cooled equine semen (n = 24 ejaculates) supplemented or not with l-carnitine (lc), acetyl-l-carnitine (ac), and their combination (lc/ac) at moments 0, 24, and 48 hours (n = 24). time (hour) group vap (µm/s) vsl (µm/s) vcl (µm/s) t0 c 123 ± 15aa 94 ± 12aa 228 ± 23aa lc 133 ± 12aa 103 ± 7ab 247 ± 18aa ac 132 ± 10aa 100 ± 7ab 244 ± 23aa lc/ac 130 ± 12aa 100 ± 8ab 241 ± 22aa t24 c 111 ± 13ba 88 ± 10aa 210 ± 22aba lc 118 ± 17ba 94 ± 12bb 216 ± 25ba ac 114 ± 13ba 91 ± 10bab 214 ± 24ba lc/ac 113 ± 13ba 92 ± 10bab 208 ± 25ba t48 c 103 ± 19ba 75 ± 13ba 205 ± 33ba lc 107 ± 18ca 84 ± 13cb 207 ± 33ba ac 104 ± 17ba 80 ± 12cb 200 ± 41ba lc/ac 105 ± 15ba 83 ± 12cb 205 ± 26ba vap, average path velocity; vsl, straight line velocity; vcl, curvilinear velocity. group ct (botu-semen®); lc1 group (botu-semen® + 5 mmol/l of lc); lc2 group (botu-semen® + 10 mmol/l of lc); lc3 group (botu-semen® + 15 mmol/l of lc); ac1 group (botu-semen® + 5 mmol/l of ac); group ac2 (botu-semen® + 10 mmol/l of ac); group ac3 (botu-semen® + 15 mmol/l of ac). t0 (15 minutes of incubation), t24 (24 hours of refrigeration) and t48 (48 hours of refrigeration). different superscripts denotes effect of time in the same group (a,b,c) and among groups at the same moment (a,b) (p < 0.05). figure 3. mean ± sd of sperm with dna fragmentation index (dfi), production of reactive oxygen species (ros), and lipid peroxidation (perox) of stallion semen (n = 24 ejaculates) in vitro supplemented with l-carnitine (lc) or acetyl-l-carnitine (ac) at moments 0 (a,d,g), 24 (b,e,h), and 48 hours (c,f,i). ct, semen extended in botu-semen; lc, botu-semen supplemented with 10 mmol/l of l-carnitine; ac, botu-semen supplemented with 10 mmol/l of acetyl-l-carnitine; lc/ac, botu-semen supplemented with 10 mmol/l of l-carnitine and 10 mmol/l of acetyl-l-carnitine. clinical theriogenology 2022; 14: 346 besides improving the energy metabolism of sperm, carnitines have an antioxidant role.15 carnitines reduce the availability of free lipids, as carnitines transport fatty acids through the mitochondrial membrane for β-oxidation and the consequent production of atp that may reduce lipid peroxidation and oxidative stress.7 furthermore, this substance increased the activity of antioxidant enzymes (e.g., superoxide desmutase and glutathione peroxidase) that reduced lipid peroxidation.15 interesingly, the inclusion of l-carnitine in semen extenders has been suggested to reduce oxidative dna damage of equine sperm.4 however, in the present study, the inclusion of either l-carnitine and acetyl-l-carnitine, or their combination, did not change sperm dna fragmentation, ros, or perox. these results, in part, may be associated with the group of stallions used in this study, as they were not selected based on semen cooling ability or fertility rates, or with the semen storage method. also, reults might have been different if semen was stored at room temperature with no seminal plasma.4 however, cooling semen at 5°c reduced sperm metabolism,26 and ros generation was enhanced when the metabolism was accelerated27 also in abnormal sperm.28 furthermore, seminal plasma posses antioxidants mechanism that scavenge ros to prevent cellular damage15,27 that may have influenced the oxidative stress in the present study. it is also important to note that the inclusion of acetyl-l-carnitine alone or in combination with l-carnitine resulted in greater sperm motility parameters and pmi than the control group. a correlation between the acetyl-l-carnitine/l-carnitine ratio and progressive motility postcooling has already been reported.4,6,14 however, an imbalance in the acetyl-l-carnitine/l-carnitine ratio can cause an absence of sperm motility.29 in fact, the carnitine acetylated form releases acetyl-coa to the krebs cycle for energy production; however, when in excess, it blocks the pyruvate dehydrogenase activity, interrupting energy production through glycolysis.30 in conclusion, inclusion of carnitines improved sperm parameters of stallion semen extended in a milk-based extender and stored at 5°c for up to 48 hours. the combination of l-carnitine and acetyl-l-carnitine did not prove to be more efficient than including 1 of them alone in equine semen extender. further studies are needed to evaluate the effect of these substances in semen of subfertile or stallions with poor semen cooling ability. funding são paulo research foundation (fapesp, grant 2012/11658-4). conflict of interest none to declare. references 1. stradaioli g, sylla l, zelli r, et al: effect of l-carnitine administration on the seminal characteristics of oligoasthenospermic stallions. theriogenology 2004;62:761-777. 2. de arruda rp, da silva df, alonso ma, et al: nutracêuticos na reprodução de tourosegaranhões. revista brasileira de zootecnia 2010;39:393-400. 3. bazzano m, laus f, spatern, et al: use of nutraceuticals in the stallion: effects onsemen quality and preservation. reprod domest anim 1-7 (2021) doi:10.1111/rda.13934 4. gibb z, lambourne sr, quadrelli j, et al: l-carnitine and pyruvate are prosurvivalfactors during the storage of stallion spermatozoa at room temperature. biol reprod 2015;93:1-9. 5. potter ds, webb gw, onyango b, et al: l-carnitine and pyruvate inclusion indiluents for cold-stored stallion spermatozoa. j equine vet sci 2015;35:440. 6. nery, ihav et al: effects of l-carnitine on equine semen quality during liquid storage. biopreserve biobank 2020;18:403-408. 7. jeulin c, lewin lm: role of free l-carnitine and acetyl-l-carnitine in post-gonadal maturation of mammalian spermatozoa. hum reprod update1996;2:87-102. 8. chiu mn, blackman mr, wang c, et al: the role of carnitine in the male reproductive system. ann n y acad sci 2004:1033; 177-188. 9. enomoto a, wemp mf, tsuchida h, et al: molecular identification of a novel carnitine transporter specific to human testis: insights into the mechanism of carnitine recognition. j biol chem 2002;277:3626236271. 10. fritrz ib, yue kt: long-chain carnitine ccyltransferase and the role of acylcarnitine. j lipid res 1963;4:279-288. 11. hoppel c: the role of carnitine in normal and altered fatty acid metabolism. am j kidney dis 2003;41:4-12. 12. bruns k, casillas er: partial purification and characterization of an acetylcarnitine hydrolase from bovine epididymal spermatozoa. arch biochem biophys 1990;277:1-7. 13. casillas er, erickson bj: the role of carnitine in spermatozoan metabolism: substrate induced elevations in the acetylation state of carnitine and coenzyme a in bovine and monkey spermatozoa. biol reprod 1975;12:275-283. 14. stradaioli, g, sylla l, zelli r, et al. seminal carnitine and acetylcarnitine content and carnitine acetyltransferase activity in young maremmano stallions. anim reprod sci 2000;64:233 245. 15. neuman sl, lin tl, heste py: the effect of dietary l-carnitine on semen traits of white leghorns. poult sci 2002;86:2228-2235. 16. kenney rm, bergman rv, cooper wl, et al: minimal contamination techniques for breeding mares: techniques and preliminary findings. proc am assoc equine pract 1975;21:327 336. 17. carneiro jam, canisso if, bandeira rs, et al: effects of coenzyme q10 on semen cryopreservation of stallions classified as having good or bad semen freezing ability. anim reprod sci 2018;192:107-118. 18. harrison rap, vickers, se: use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. j reprod fertil 1990;88:343-352. 19. parrish jj, susko-parrish j, winer ma: capacitation of bovine sperm by heparin. biol reprod 1988;38:1171-1180. 20. evenson dp, thompson l, jost l: flow cytometric evaluation of boar semen by the sperm. theriogenology 1994;41:637-651 21. evenson d, jost l: sperm chromatin structure assay is useful for fertility. methods cell sci 2000;22:169-189. 22. guasti pn, freitas-dell’aqua cp, maziero rrd, et al: validation of flow cytometry for assessment of membrane lipid peroxidation of equine spermatozoa. int sym anim bio reprod 2012;929. clinical theriogenology 2022; 14: 347 23. garolla a, maiorino m, roverato a, et al: oral carnitine supplementation increases sperm motility in asthenozoospermic men with normal sperm phospholipid hydroperoxide glutathione peroxidase levels. fertil steril 2005;83:355-361. 24. morris, l, & gibb z. oral supplementation with l-carnitine improves stallion fertility. j equine vet sci 43, s82. 25. rosa fac, spers a, rodrigues phm: efeito da l-carnitina na alimentação de garanhões puro sangue árabe na congelação do sêmen. in congresso de integração em biologia da reprodução 2001;174. 26. christine a: recent advances in cooled-semen technology. anim reprodsci 2008:107;268-275. 27. baumber j, ball ba, gravance cg, et al: the effect of reactive oxygen species on equine sperm. j androl 2000;21:895-902. 28. iwasaki a, gagnon c: formation of reactive oxygen species in spermatozoa of infertile patients. fertil steril 1992:57;409-416. 29. golan rr, weissenberg r llm: carnitine and acetylcarnitine in motile and immotile human spermatozoa. 1984;7:484-494. 30. beattie ds: bioenergetics, mitochondria, and oxidative metabolism. in: devlin tm: editor. textbook of biochemistry with clinical correlations. 4th edition, new york, john wiley & sons: 1997.p.542-589. 2017 optimization of a protocol for cryopreservation of electroejaculated beef bull semen under ambulatory conditions � optimization of a protocol for cryopreservation of electroejaculated beef bull semen under ambulatory conditions maria s. ferrer,a david j. hurley,b roberto a. palomares,b anne-claire bullington,a joão henrique jabur bittarb adepartment of large animal medicine and bpopulation health, college of veterinary medicine, university of georgia, athens, ga abstract the overall goal was to optimize and simplify a cryopreservation protocol under ambulatory conditions. it was hypothesized that removing seminal plasma by centrifugation would improve postthaw semen quality, and that increasing equilibration to 24 h and freezing in liquid nitrogen vapor within the neck of the tank would not affect post-thaw quality of electroejaculated bull semen. one ejaculate was collected from nine beef bulls using electroejaculation, and was divided into four aliquots: nc5: noncentrifuged, equilibrated for 5 h; nc24: non-centrifuged, equilibrated for 24 h; ce5: centrifuged, equilibrated for 5 h; ce24: centrifuged, equilibrated for 24 h. straws were held horizontally on a rack 3cm above liquid nitrogen for 10min before plunging in liquid nitrogen. in addition, some straws equilibrated for 5h were held vertically 2cm over liquid nitrogen within the neck of the tank (ncneck, ceneck). post-thaw sperm motility (casa), membrane integrity (sybr14/pi), acrosome integrity (fitc-pna) and apoptosis (anv) were evaluated and compared using anova. sperm motility, membrane integrity or apoptosis did not differ among treatments. however, centrifuged semen equilibrated for 24h had significantly lower dsl, vsl, lin, str and wob than non-centrifuged semen equilibrated for 5h. centrifugation increased the percentage of acrosome-damaged spermatozoa (nc5 5.8±3, ce5 42.2±10.7, nc24 4.8±2.5, ce24 32.5±8.9, ncneck 17.9±7.2, ceneck 42.3±12.5%; p=0.002). extending the equilibration time had no effect on the parameters evaluated in non-centrifuged semen. removal of seminal plasma by centrifugation is not recommended since it had a negative effect on acrosome integrity. keywords: bull, cryopreservation, centrifugation, equilibration, liquid nitrogen vapor introduction use of frozen semen has become the standard for artificial insemination in cattle. current management of bulls for semen freezing involves housing bulls at specialized centers. the bulls are trained to ejaculate into an artificial vagina, and semen is collected and frozen on a regular basis until the desired number of insemination doses is obtained. such practice involves a high cost to bull owners related to animal transportation, quarantine and housing, in addition to the cost of semen freezing itself. small producers, especially of rare breeds, often want to cryopreserve semen from bulls that have genetic value for their programs, but cannot afford the cost of semen freezing at a specialized facility. the optimization of a method to collect and freeze semen at the farm would reduce the cost of customfreezing bull semen and favor a more widespread use of this technology. most of the reports in the literature describe and address cryopreservation of dairy bull semen in semen freezing centers, collected with an artificial vagina, and processed under controlled laboratory conditions. such bulls are intensively selected based on their production of excellent quality semen with good freezing ability.1 some of the protocols used and reported may not be suitable for client-owned beef bulls selected for the genetic value to the producer’s breeding program and not for their freezing ability. cryopreservation under ambulatory conditions requires making adjustments to protocols to overcome specific challenges. these challenges are presented by the working conditions in ambulatory practice and critically involve the lack of facilities and personnel trained for semen collection with an artificial vagina, long working hours, time constraints due to other routine duties, uncontrolled environment with exposure to unfavorable ambient conditions, and limited availability of equipment like automated semen freezers. therefore, the overall goal of this study was to advance the art of on-farm semen freezing by the 563 clinical theriogenology • volume 9 number 4 • december 2017 � optimization and simplification of a method to freeze electroejaculated beef bull semen under ambulatory conditions. collecting and processing semen for cryopreservation on-farm requires the use of electroejaculation, which yields samples with variable concentrations of seminal plasma, and which can be contaminated with urine. while the removal of seminal plasma is not routine practice in the cattle industry, seminal plasma is routinely removed before freezing equine and canine semen. it has been demonstrated that this practice improves survival and membrane stability of spermatozoa during cryopreservation.2 therefore, removal of seminal plasma and other contaminating fluids may improve the post-thaw quality of frozen bovine semen obtained by electroejaculation. however, reports provide conflicting data of the costs and benefits of centrifugation of bovine semen. in some studies, sperm velocity, mitochondrial potential, oxidative stress and dna integrity were improved by centrifugation and removal of seminal plasma prior to cryopreservation.3,4 in contrast, sperm motility and membrane integrity did not change, and acrosome integrity and in vitro fertility decreased with centrifugation in one of these studies.4 because of the variation and discrepancy in findings, the effect of centrifugation on post-thaw bovine semen quality is still not clear and needs to be further evaluated. furthermore, no studies have evaluated the effect of centrifugation in bos taurus beef bulls collected with electroejaculation. it is unknown if breed differences may exist in seminal plasma composition that may result in differential effects on beef bull semen quality when it is removed. optimization of time management is another critical factor for a busy ambulatory practice. most current protocols involve an equilibration time of at least 2h,5 time during which a clinician is prevented from providing other services while waiting at the farm. the ability to equilibrate semen overnight would allow transport of semen to the laboratory during equilibration. there the freezing process could be completed under controlled conditions on the following day. this would reduce the number of work hours required for freezing of semen, or allow practitioners to peruse other activities while the semen equilibrates. previous studies have demonstrated that equilibration of semen overnight may actually be beneficial for post-thaw quality of semen collected with an artificial vagina in a controlled environment from dairy bulls.1,6 however, transient exposure of semen to varying environmental and handling conditions at the farm could result in sublethal damage that may affect sperm survival during prolonged times at refrigeration temperature. furthermore, the interaction between removal of seminal plasma by centrifugation and equilibration time has not been previously evaluated in cattle. finally, if the semen freezing process is completed on the farm, an automated semen freezer is rarely available and a manual method is most often required. the straws are loaded under refrigeration conditions and frozen in liquid nitrogen vapor phase. this is performed by holding straws horizontally on a rack, 3 to 6cm above liquid nitrogen.5,7 therefore, a container capable of safely holding liquid nitrogen and fitting the rack needs to be available. after rack freezing, the straws are plunged in the liquid nitrogen and placed into goblets, which are transferred to the tank for storage. the freezing process could be simplified and the amount of materials needed could be minimized if the straws could be held vertically in the liquid nitrogen vapor within the neck of the storage tank. the objectives of this study were to evaluate the effect of removal of seminal plasma by centrifugation, prolongation of the equilibration time and freezing in the liquid nitrogen vapor within the neck of the tank on post-thaw semen quality. it was hypothesized that removing seminal plasma by centrifugation would improve post-thaw semen quality, and that increasing the equilibration time to 24 h and freezing in the liquid nitrogen vapor within the neck of the tank would not affect post-thaw quality of electroejaculated beef bull semen. materials and methods experimental animals nine 17-month old angus bulls were used in the study. no attempt was made to select bulls based on freezing ability to replicate commercial conditions of the local industry. initial total and progressive sperm motility was 65.2 ± 4.3% and 62.7 ± 4.4%, respectively (mean ± sem). mean percentage of morphologically normal spermatozoa was 71.4 ± 3.6%. three bulls had less than 70% 564clinical theriogenology • volume 9 number 4 • december 2017 � normal spermatozoa (46, 64 and 65% normal spermatozoa). the main finding in these three bulls was presence of distal droplets. while spermatozoa with distal droplets were not counted as morphologically normal here, presence of distal droplets does not affect fertility in bulls and is not considered a morphological defect by many clinicians. in addition, these bulls had sperm post-thaw motility comparable with the other bulls. therefore, they were included in the study. mean sperm concentration, volume and total sperm count was 492.2 ± 109.9 x 106 spermatozoa/ml, 4.5 ± 0.3 ml and 2.1 ± 0.5 x 109 spermatozoa, respectively. scrotal circumference ranged between 34.5 and 38 cm. no abnormalities were noted in the prepuce, penis, scrotum or accessory sex glands of any of the animals. bulls were group-housed in one pasture at the university of georgia’s research farm, and were fed water, hay and mineral salt ad libitum. the nine bulls were included in the first experiment. three of the bulls were then turned out with the university’s cow herd for breeding and were unavailable for the second study. therefore, six bulls were included in the second experiment. the study was performed following guidelines from the institutional animal care and use committee. experiment 1: effect of centrifugation, equilibration time and vapor freezing method on post-thaw semen quality a cleanout semen collection was performed one week prior to the study. on the day of the study, one ejaculate was obtained from each of nine bulls. the cloudy sperm-rich fraction was collected using the automatic setting on an electroejaculator (pulsator iv, lane manufacturing inc., denver, co) during penis protrusion or erection. sperm concentration was evaluated using a densimeter. immediately after collection, 200�l of an antibiotic cocktail (css antibiotic mix, imv technologies, maple grove, mn) was added per ml of raw semen. each 200�l of antibiotic cocktail contained 100�g of tylosin, 500�g of gentamycin and 300/600�g of linco-spectin. semen was then placed in a water bath at 34�c for 5min. pre-warmed (34�c; optixcell�, imv technologies) was added to dilute the semen to make a final concentration of 50 x 106 spermatozoa/ml. each ejaculate was then divided into four aliquots (table): nc5: non-centrifuged, equilibrated for 5h; nc24: non-centrifuged, equilibrated for 24h; ce5: centrifuged, equilibrated for 5h; ce24: centrifuged, equilibrated for 24 h. non-centrifuged aliquots (nc5 and nc24) were refrigerated immediately after dilution. centrifuged aliquots (ce5 and ce24) were centrifuged at 800 x g for 10min, the supernatant was removed and the pellet was resuspended to 50 x 106 spermatozoa/ml in optixcell�. semen collection, dilution and centrifugation were performed bull-side at the farm. the extended semen was initially placed within the truck’s refrigerator at the farm. transportation time from the farm to the laboratory was approximately 30min. upon arrival at the laboratory, semen was moved to the laboratory’s refrigerator within a pre-cooled closed-cell extruded polystyrene box to complete equilibration at 5�c for 5h (nc5 and ce5) or 24h (nc24 and ce24). cooling rate was not calculated in this study, but bovine spermatozoa tolerate variable cooling rates between 50 and 100�c/min.5 after equilibration, pre-cooled 0.5ml straws were loaded. next, the straws were placed horizontally on a rack 3cm above liquid nitrogen for 10min within a closed-cell extruded polystyrene box. the straws were then plunged in liquid nitrogen, loaded into 13-mm goblets submerged in the liquid nitrogen, and transferred to the storage tank. in addition, half of the straws equilibrated for 5h, both non-centrifuged and centrifuged (ncneck and ceneck), were exposed to liquid nitrogen vapor within the neck of the storage tank (table). the straws were loaded into pre-cooled 13-mm goblets within the refrigerator. only the bottom goblet of each cane was filled with straws. the canes were then held vertically within the neck of the tank so that the bottom of the goblet was 2cm above the liquid nitrogen. the distance from liquid nitrogen was modified to ensure all semen and extender mixture within the straw theoretically reached a temperature of -40�c at the warmest point before plunging into liquid nitrogen. this is the temperature at which 95% of water in the extended semen is frozen.7 while temperature was not measured in this study, it was expected to be -160�c at the bottom of the straw (2 cm above liquid nitrogen) and -40�c at the top of the straw (15 cm above liquid nitrogen).7 after 10min, the canes were lowered to rapidly submerge the straws into the liquid nitrogen. all straws were stored at 196�c until evaluation. two weeks after freezing, one straw from each aliquot was thawed in a water 565 clinical theriogenology • volume 9 number 4 • december 2017 � bath at 38�c for 30s, and sperm motility, membrane integrity, acrosome integrity and apoptosis were evaluated (see evaluation of sperm parameters). statistical analysis was performed using statistical analysis software (sas, sas institute, cary, nc). normally distributed data were expressed as mean ± sem. mean motion parameters, percentage of membrane intact, acrosome intact and apoptotic spermatozoa were compared among the six treatments (nc5, ncneck, nc24, ce5, ceneck and ce24) using anova, including the fixed effect of treatment, and the interaction between centrifugation and equilibration time. paired comparisons were made using a tukey’s test. differences were considered significant if p < 0.05. table. summary of treatments and conditions treatment nc5 ncneck nc24 ce5 ceneck ce24 centrifugation no no no yes yes yes equilibration time (h) 5 5 24 5 5 24 vapor freezing horizontal vertical horizontal horizontal vertical horizontal experiment 2: effect of centrifugation on sperm motion and acrosome integrity in fresh semen a negative effect of centrifugation on acrosome integrity of frozen-thawed semen was noted in the first experiment. experiment 2 was performed to determine if this effect was caused by mechanical damage during centrifugation, or by cryodamage resulting from removal of seminal plasma. one week after the first experiment, another ejaculate was collected from each of six bulls using electroejaculation. pre-warmed (34 �c) optixcell� was added to make a final concentration of 50 x 106 sperm/ml. each ejaculate was then divided into two aliquots. one aliquot served as the non-centrifuged (nc) control treatment while the other aliquot was centrifuged at 800 x g for 10min (ce). the supernatant was removed and the pellet was resuspended to a final concentration of 50 x 106 sperm/ml in optixcell�. evaluation of sperm motility, membrane integrity and acrosome integrity was then performed in fresh semen (see evaluation of sperm parameters). semen was not frozen in this experiment. mean motion parameters, percentage of membrane intact and acrosome intact spermatozoa were compared between centrifuged and non-centrifuged aliquots using a t test. evaluation of sperm parameters phase contrast microscopy was used for assessment of sperm morphology immediately after semen collection. spermatozoa were diluted 1:10 in formalin buffered solution (animal reproduction systems, chino, ca). a wet mount was prepared and 100 spermatozoa were classified based on their morphological characteristics under oil immersion at x100 magnification. sperm motility was evaluated with a computer assisted semen analyzer (casa; spermvision professional, minitube of america, verona, wi). the settings of the instrument were: field depth of view 20�m, pixel to �m ratio 130 to 100, cell area 18 to 80�m, frames acquired 30, frame rate 60hz, aoc cut off static cells 5 and dsl cut off 4.5μm/s (progressive motility). mean percentage of total (tmot) and progressively (pmot) motile spermatozoa was assessed from all cells present in seven fields with a x 20 phase-contrast objective. other motion parameters analyzed were: average path velocity (vap), straight-line velocity (vsl), curvilinear velocity (vcl), amplitude of lateral head displacement (alh), beat cross frequency (bcf), straightness (str), linearity (lin), wobble (wob), curvilinear distance (dcl), straight line distance (dsl), and distance of average path (dap). for evaluation of acrosome and plasma membrane integrity, 1μl of a 1μg/ml solution of fluorescein isothiocyanate-labeled peanut agglutinin (fitc-pna; cat. no. f-2301-1, ey laboratories, san mateo, ca) and 1μl of a 6�m solution of propidium iodide (pi; live/dead kit, molecular probes) were added to a 1ml suspension containing 5 x 106 spermatozoa. semen was incubated at 38�c in 5% co2 in air for 10min. the percentage of membrane-intact (pi negative) and live acrosome-damaged (pi negative and pna positive) spermatozoa was evaluated by flow cytometry. 566clinical theriogenology • volume 9 number 4 • december 2017 � sperm apoptosis was evaluated using fitc-labelled annexin v stain (anv, cat. no. a13199, molecular probes, eugene, or). spermatozoa were washed once by centrifugation at 600 x g for 5min and were resuspended to 4 x 105 /ml in binding buffer (0.1 m hepes, 1.4 m sodium chloride, 25mm calcium chloride in distilled water, ph 7.4). then, 10μl of fitc-annexin v was added to 1ml of sperm suspension, and semen was incubated for 15min at room temperature in the dark. semen was washed by centrifugation once to remove unbound stain, and was resuspended to 0.5ml in binding buffer. then 0.5μl of a 6�m solution of pi was added. cells were evaluated by flow cytometry and were classified as membrane-intact (anv negative/pi negative), early apoptotic (anv positive/pi negative), late apoptotic (anv positive/pi positive), or necrotic (anv negative/pi positive). a fluorescence flow cytometry analyzer (accuri� c6 generation 1, bd biosciences, san jose, ca) was used in this study. from each sample, 10,000 events in the forward and 90º light scatter population representing whole sperm were analyzed using 0.2-�m filtered 18 mega-ohm ultra pure water as the sheath fluid. a gate containing spermatozoa was selected based on dot plot distribution of forward (size) versus side scatter (complexity parameter) to eliminate debris and epithelial cells from the analysis. the fitc (green) and pi (red) signals were detected using a 5mwatt blue argon laser (488 nm) and emission filters (535 ± 30 nm for fitc and 585 ± 30 nm for pi). baseline background fluorescence signal was initially evaluated in unstained samples. compensation for fitc emission into the pi detector or vice versa was done by adjusting digital compensation to remove the green signal from the red detector and the red signal from the green detector with samples containing only fitc or pi. the areas of single and dual staining were established by setting quadrants on spermatozoa labeled only with pi or fitc. fluorescence emission data were collected using a 7.2 log logarithmic digital signal collection system for green fluorescence (fitc) in the fl1 detector and red fluorescence (pi) in the fl3 detector. the control quadrant (lower left) was marked on unstained samples with < 1% of cells registering as positive in the upper left, upper right and lower right quadrants. results experiment 1: effect of centrifugation, equilibration time and vapor freezing method on post-thaw semen quality the percentage of total and progressively motile spermatozoa was not affected by treatment. however, there was an effect of treatment on dsl (p=0.038), vsl (p=0.045), lin (p=0.008), str (p=0.007) and wob (p=0.034; fig. 1). there was also an interaction between centrifugation and equilibration time on lin (p=0.007), str (p=0.005) and wob (p=0.028). centrifuged semen equilibrated for 24 h had lower dsl, vsl, lin, str and wob than non-centrifuged semen equilibrated for 5h frozen traditionally or within the neck of the tank (fig. 1). no other motion parameters differed. 567 clinical theriogenology • volume 9 number 4 • december 2017 � figure 1. sperm motion parameters in centrifuged (ce) and non-centrifuged (nc) frozen-thawed bovine semen equilibrated for 5 h(5) or 24 h(24), and exposed to liquid nitrogen vapor within the neck of the tank (neck). a,bp<0.05. tmot=total motility (%), pmot=progressive motility (%), dcl=curvilinear distance (�m), dap=distance of average path (�m), dsl=straight line distance (�m), vcl=curvilinear velocity (�m/sec), vap=average path velocity (�m/sec), vsl=straight line velocity ((�m/sec), lin=linearity, str=straightness, wob=wobble, bcf= beat cross frequency (hertz), alh= amplitude of lateral head displacement (�m). the percentage of membrane-intact, necrotic, early apoptotic and late apoptotic cells did not differ significantly with treatment (fig. 2). the percentage of live spermatozoa with damaged acrosomes was higher in centrifuged than non-centrifuged samples (p=0.002; fig. 2). when semen was frozen at the neck of the tank, acrosomal damage did not differ with centrifugation but was higher than in noncentrifuged samples frozen traditionally. there was also an interaction between centrifugation and equilibration time on late apoptotic cells (p=0.014) and live acrosome damaged cells (p=0.001). 568clinical theriogenology • volume 9 number 4 • december 2017 � figure 2. percentage of necrotic, apoptotic, membrane-intact and live acrosome-damaged spermatozoa in centrifuged (ce) and non-centrifuged (nc) frozen-thawed bovine semen equilibrated for 5 h (5) or 24 h (24), and exposed to liquid nitrogen vapor within the neck of the tank (neck). a,bp=0.002. experiment 2: effect of centrifugation on sperm motion and acrosome integrity in fresh semen sperm motion parameters, plasma membrane integrity and acrosome integrity did not differ between centrifuged and non-centrifuged fresh semen (p>0.05; fig. 3). figure 3. sperm motion parameters, plasma membrane integrity and acrosome integrity in centrifuged (ce) and non-centrifuged (nc) fresh bovine semen. tmot=total motility (%), pmot=progressive motility (%), dcl=curvilinear distance (�m), dap=distance of average path (�m), dsl=straight line distance (�m), vcl=curvilinear velocity (�m/sec), vap=average path velocity (�m/sec), vsl=straight line velocity ((�m/sec), lin=linearity, str=straightness, wob=wobble, bcf= beat cross frequency (hertz), alh= amplitude of lateral head displacement (�m), mi=membrane integrity (% spermatozoa), ad=damaged acrosomes (% spermatozoa). 569 clinical theriogenology • volume 9 number 4 • december 2017 � discussion this study evaluated ways to optimize and simplify the protocol for cryopreservation of electroejaculated bovine semen under ambulatory conditions. centrifugation was performed to remove seminal plasma and other contaminating fluids from electroejaculated samples, to minimize the interaction of the sperm plasma membrane with seminal plasma proteins and these contaminating fluids. however, centrifugation had no beneficial effect on sperm motility, membrane integrity or apoptosis. furthermore, there was an interaction between centrifugation and equilibration time that resulted in a negative effect of removing seminal plasma on some motion parameters when semen was equilibrated for 24h. a negative effect of bovine seminal plasma on post-thaw sperm motility and viability was reported when semen was frozen in egg yolk-based diluents.3,8-11 in this study, a liposome containing diluent was used. it is thought that liposomes modify sperm membranes by exchanging lipids and cholesterol, lowering the lipid phase transition temperature and improving cryotolerance.6 how liposomes interact with seminal plasma components to alter plasma membrane stability is not known. the seminal plasma components that increase sperm sensitivity to cold shock originate from the seminal vesicles.10 because electroejaculation results in a higher contribution of seminal vesicle secretions than collection with an artificial vagina,12 it seemed plausible that removal of seminal plasma would improve post-thaw motility of electroejaculated spermatozoa. however, this hypothesis was rejected in studies in which egg yolk-free diluents were used, including the present one.4 as mentioned, there was an interaction between centrifugation and equilibration time on some sperm motion parameters. the lowest values for dsl, vsl, wob, lin and str were observed in centrifuged samples equilibrated for 24h. this is contrary to a previous report in which some of these parameters increased after removal of seminal plasma.4 however, seminal plasma was removed by filtration and equilibration was limited to 4h. it is possible that centrifugation caused sublethal mechanical damage to spermatozoa. while the damage was not immediately evident, extending the equilibration time could have resulted in further sperm damage and decreased cryotolerance. because vsl, str and lin are positively correlated with bull fertility,13 the centrifugation protocol used in this study is not recommended if the equilibration time will be extended to 24h. previous studies also found an improvement in post-thaw semen quality after equilibration for 24h in non-centrifuged semen.1,4 no beneficial effect of a prolonged equilibration was observed in this study. differences in breed, semen collection methods and semen freezing protocols may account for the discrepancy. moreover, transient exposure of semen to less controlled environmental and handling conditions at the farm could result in sublethal damage that may affect sperm survival during prolonged exposure to refrigeration temperatures. this may counteract the beneficial effect of prolonged equilibration. despite the lack of a benefit on semen quality, extending the equilibration time did not have a negative effect in non-centrifuged semen and may therefore be beneficial relative to time management in a busy ambulatory practice. this would also allow for semen to be shipped to a semen processing center after on-farm collection for further processing and freezing. membrane integrity and sperm apoptosis were not affected by centrifugation or equilibration time. however, acrosome integrity was negatively affected by removal of seminal plasma by centrifugation. similar findings were reported when seminal plasma was removed by centrifugation in bulls.4 it is possible that centrifugation caused mechanical damage to the sperm plasma and acrosome membranes.4 however, acrosomal damage was also reported when seminal plasma was removed by filtration.4 seminal plasma contains proteins that stabilize the sperm plasma membrane and prevent premature capacitation. cryopreservation induces capacitation-like changes in bovine spermatozoa14 and removal of seminal plasma could render spermatozoa more susceptible to cryocapacitation. this could lead to premature and spontaneous acrosome reaction. because acrosome response to centrifugation was not evaluated in fresh semen in the first experiment, a second experiment was performed to determine if the acrosomal changes seen were observed immediately after centrifugation and prior to freezing. acrosome integrity did not differ with centrifugation in fresh semen, possibly indicating that the 570clinical theriogenology • volume 9 number 4 • december 2017 � differences seen in frozen semen were due to a higher susceptibility to cryocapacitation rather than direct damage caused by centrifugation. lastly, freezing semen in the neck of the tank yielded a post-thaw motility, membrane integrity, apoptosis and acrosome integrity similar to traditional vapor freezing. however, acrosomal damage was higher than in non-centrifuged samples frozen traditionally. holding straws vertically may have delayed the freezing curve of spermatozoa on the top of the straws, which were in a warmer region of the liquid nitrogen vapor, allowing for acrosomal damage or cryocapacitation to occur. post-thaw motility in this study was lower than previously reported. total and progressive postthaw motility of electroejaculated spermatozoa frozen in botubov was 54% and 41%, respectively.4 semen diluted in optixcell� had a post-thaw motility between 56% and 66%.6,15 the difference could be due to the use of beef bulls that were not selected for their freezing ability or optimal semen quality in this study. however, since semen was collected at the farm, it was exposed to uncontrolled ambient conditions like temperature, humidity, sunlight and dust. how these conditions affected post-thaw semen quality is unknown. an attempt to control ambient conditions was made by maintaining semen warm within a water bath, working under the shade, in the cleanest possible place, and in the most time-efficient manner. another difference between this study and previous ones was the use of a manual freezing method instead of a programmable freezer. although manual and automated methods were not compared here, it can be speculated that the uncontrolled freezing curve may have also decreased post-thaw semen quality. based on the findings of this study, a recommended protocol for semen freezing under ambulatory conditions would involve collecting semen using electroejaculation, diluting the semen with the extender immediately after collection, and initiating refrigeration of the diluted semen at the farm. extending the equilibration time had no effect on the parameters evaluated as long as centrifugation was not performed. therefore, equilibration of non-centrifuged semen could safely take place while semen is being transported to the laboratory for up to 24h, where the freezing process could be completed using a programmable freezer. removal of seminal plasma by centrifugation had a negative effect on acrosome integrity. therefore, centrifugation of electroejaculated bovine semen extended in optixcell� at 800 x g for 10 min is not recommended for cryopreservation. references 1. anzar m, kroetsch t, boswall l: cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes. anim reprod sci 2011;126:23-31. 2. brinsko sp, crockett ec, squires el: effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. theriogenology 2000;54:129-136. 3. lymberopoulos ag, khalifa tta: sperm chromatin stability during in vitro manipulation of beef bull semen. reprod domest anim 2010;45:307-314. 4. peres campanholi s, monteiro fm, ribeiro dias ea, et al: effect of seminal plasma removal before cryopreservation of bovine semen obtained by electroejaculation on semen quality and in vitro fertility. theriogenology 2017;89:114121. 5. sathe s, shipley cf: cryopreservation of semen. in: hopper rm, editor. bovine reproduction. ames(ia): wiley blackwell; 2015. p. 662-670. 6. fleisch a, malama e, witschi u, et al: effects of an extension of the equilibration period up to 96 hours on the characteristics of cryopreserved bull semen. theriogenology 2017;89:255-262. 7. santos mv, sansinena m, zaritzky n, et al: mathematical prediction of freezing times of bovine semen in straws placed in static vapor over liquid nitrogen. cryobiology 2013;30-37. 8. martinus rd, molan pc, shannon p: deleterious effect of seminal plasma in the cryo-preservation of bovine spermatozoa. n z j agr res 1991;34:281-285. 9. moustafa ar, meszaros i: interrelationship between the total protein content of bovine seminal plasma and behaviour of the spermatozoa after freezing and thawing. acta vet acad sci hung 1980;28:403-408. 10. fulka j, surcova m, valenta h: protein polymorphism of the seminal vesicles of bulls and the sensitivity of bull spermatozoa to cold shock. in:proc 9th eur anim blood group conf; 1965. p. 381-386. 11. baas jw, molan pc, shannon p: factors in seminal plasma of bulls that affect the viability and motility of spermatozoa. j reprod fertil 1983;68:275-280. 571 clinical theriogenology • volume 9 number 4 • december 2017 � 12. rego jpa, moura aa, nouwens as, et al: seminal plasma protein profiles of ejaculates obtained by internal artificial vagina and electroejaculation in brahman bulls. anim reprod sci 2015;160:126-137. 13. farrell pb, presicce ga, brockett cc, et al: quantification of bull sperm characteristics measured by computerassisted sperm analysis (casa) and the relationship to fertility. theriogenology 1998;49:871-879. 14. comier n, sirard ma, bailey jl: premature capacitation of bovine spermatozoa is initiated by cryopreservation. j androl 1997;18:461-468. 15. miguel-jiménez s, mogas t, peña ai, et al. post-thaw changes in sperm membrane and ros following cryopreservation of dairy bull semen using four different commercial extenders. anim reprod 2016;13:573. 572clinical theriogenology • volume 9 number 4 • december 2017 reproductive management in dairy herds reproductive management in dairy herds james ferguson department of clinical studies, school of veterinary medicine university of pennsylvania, new bolton center, pa abstract reproduction influences farm profitability. cows should become pregnant between 55 and 115 days postcalving to optimize milk production, calves born per year and minimize culls for reproductive failure. pregnancy rate, heat (estrus) detection rate times conception, is the critical driver of reproductive efficiency. economic returns associated with reproduction are optimal when pregnancy rate is > 25%. to achieve a pregnancy rate > 25%, conception rate needs to be > 33% and first insemination heat detection needs to be > 70%. herd managers should select a voluntary wait period between 50 70 days that optimizes conception rate and use a management program to control first insemination intensity. pregnancy examination should be scheduled to control days between inseminations. keywords: cattle, reproductive management, pregnancy rate introduction milk sales account for 80 90% of income on dairy farms and sale of calves and cull cows account for 10 20% of income.1 milk sales are a function of the number of cows milking, parity, average days in milk, management, and genetic merit. however, milk production is dependent on the successful birth of a calf, that makes reproduction an essential component of a successful dairy operation. due to the shape of the lactation curve, peak production is reached by 30 50 days postcalving and then declines at a consistent rate; therefore, cows produce milk most efficiently when they calve every 11 13 months. since pregnancy gestation averages ~ 280 days, the goal of reproductive programs on dairy farms should be for pregnancy to occur between 55 115 days postcalving. since uterine involution and resumption of ovarian cycling are not complete until 30 50 days postcalving, management programs should begin insemination 50 70 days postcalving to achieve optimal intervals between successive calving. to become pregnant, cows must be inseminated in estrus, that occurs every 21 days in a cow (range; 18 24 day).2 standing estrus is relatively short, lasting 5.1 10.6 hours.3 depending on the number of cows around estrus, the number of mounts per estrus period may range from 6.2 12.8 mounts per cow.4 a mount may last for < 20 seconds, so observation to detect a cow in estrus needs to be committed for at least 15 minutes at a time and occur 3 4 times in a day. various factors influence the intensity of expressed estrus by a cow such as number of cows around estrus, flooring, and level of milk production.2,3 having more than 2 cows in estrus increases activity and the probability of observing estrus. cows with greater milk production have lower serum estrogen, shorter periods of estrus expression, and fewer estrus mounts.4,5,6 a major challenge in reproductive management is observing cows in estrus. average heat (estrus) detection rate for 9,480 herds in the dairy records management service (drms, raleigh, nc) in the ne is 45.1%. multiple factors influence efficiency of estrus detection. housing, flooring, cattle grouping, number of cows in and around estrus, level of milk production, parity, and frequency and intensity of observation all contribute to the probability of observing cows in estrus.6 however, the major factor in low estrus detection is failure to commit sufficient time throughout the day to detect cows in estrus. particularly as herds have increased in size and cows are spread across multiple groups, committing labor to just watch for estrus is a low priority on many farms. many approaches may be employed to improve estrus detection on dairy farms. there is visual observation, heat detection aids, radio telemetry, activity monitors, progesterone biosensors, and estrus synchronization programs.8-11 evaluating the efficiency of estrus detection is important. estrus detection needs to be quantitated to evaluate the efficiency of a reproductive program. days open and calving interval are outcomes of management and are not critical control points for reproductive control. heat detection is the dominant management control point of reproduction and it must be quantitated to assess a program on a dairy farm. various methods have been used to estimate heat detection rates (hdr) from herd records.12 a simple clinical theriogenology • volume 12 number 3 • september 2020 309 method is to distribute first inseminations from the vwp by 21-day periods and calculate the number of animals inseminated within each 21-day period by the number of available animals to inseminate. this will give a value for first insemination heat detection rate (fsthdr). distributing days between sequential inseminations by periods < 10, 10 17, 18 24, 25 35, 36 48 and > 48 days gives an estimate of estrus detection for repeat inseminations (18 24 and 36 48 days), heat detection errors (< 10 and 10 17 days), early pregnancy losses (25 35 days), and gross inefficiency in repeat insemination efficiency (rpthdr) (> 48 days). management has direct control of detecting cows in estrus for insemination. since pregnancy depends on insemination, cows have to be observed in estrus to initiate reproductive outcomes. conception, establishing a pregnancy, is the second critical component of a herd program. conception rate (cr) to first service for 9,480 herds in the drms in the ne is 42%. with an hdr of 45% and a cr of 42%, pregnancy rate would be 18.9%. the question is how well does this approach a reproductive optimum? ultimately, herd reproductive performance is a function of heat detection rate (hdr) and cr. since estrus is a periodic event, occurring every 21 days, the combination of hdr and cr define the pregnancy rate (pr), that is the proportion of open cows that become pregnant every 21 days.13,14 the reported pr from the 9,480 herds in the drms records is 19.4%. this is slightly higher than the prior estimate of 18.9%, as culling cows with reproductive problems would result in a slightly higher pr than hdr x cr. pregnancy rate determines the economic value of reproduction.1,14,15 the proportion of cows pregnant every 21 days following the voluntary wait period (vwp) determines the average milk produced per day (due to the shape of the lactation curve and period of recalving), the number of calves born per year, and the number of animals culled for reproductive failure. these contribute to the value of getting a cow pregnant within each 21-day period from the vwp. the proportion of open cows pregnant with each 21-day window sums to determine the overall herd value of reproduction. figures 1a and 1b display income over feed cost for pr from 7.5 100% for five milk production classes (305 day production): 21,000, 23,000, 26,000, 28,500, and 33,000 lb. milk price was $20/cwt, calf value was $175/head; heifer rearing cost was $1500/head and cull cow value was $750/head. lactating cow feed cost was $0.12/lb of dry matter and dry cow feed cost was $0.06/lb of dry matter. income is higher with greater milk production and pregnancy rate (figure 1a). marginal value decreases more steeply when pr is < 25%, whereas the change in value above 25% is less (figure 1c). therefore, economically, the goal for herd management is to achieve a 25% pr or greater. furthermore, with a pr of 19.4%, the average drms herd is losing $165 a year due to low reproductive efficiency. figure 1a. income over feed cost for five levels of milk production for pregnancy rates varying from 7.5 to 100%. milk price is $20/cwt, calf value is $175/head; heifer rear cost is $1500/head and cull cow value is $750/head. lactating feed cost is $0.12/lb of dry matter and dry cow feed cost is $0.06/lb of dry matter. milk production m305 is 21,000, 23,000, 26,000, 28,500, or 33,000 lb per 305 days of production. 0 1000 2000 3000 4000 5000 0% 5% 10%15%20%25%30%35%40%45%50%55%60%65%70%75%80%85%90%95%100% of c: m ilk ($ 20 ), ca lv es ($ 15 0) , re pl ac em en t ( $7 50 ) pregnancy rate income vs pr clinical theriogenology • volume 12 number 3 • september 2020310 figure 1b. marginal change in income over feed cost as a function of pregnancy rate (same assumptions as figure 1a). figure1c. marginal change in value as pregnancy rate declines from 100%. losses below a pregnancy rate of 25% are six-fold the losses above a pregnancy rate of 25%. factors influencing pregnancy pregnancy rate is a function of hdr and cr.13,14 heat detection rate has different attributes, depending whether detecting estrus in cows for first insemination versus detecting cows in estrus postinsemination. cows for first insemination are not pregnant and therefore may be induced into estrus using prostaglandin or combinations of prostaglandin (pgf2α) and gonadotrophin releasing hormone (gnrh). therefore, first inseminations may be clustered in groups of cows within a 21-day window from the vwp. however, once inseminated, a cow cannot be induced into estrus until the farm manager is certain the cow is not pregnant. return to estrus at 21 days postinsemination must be based on visual observation or an analysis of hormonal indicators of nonpregnancy status, such as declines in progesterone or bovine pregnancy glycoproteins. most pregnancy tests are not accurate until 28 35 days postinsemination. therefore, clustering of open cows for reinsemination cannot be done until they are confirmed not pregnant. first insemination hdr (fsthdr) can be clustered in a 21-day window from -450 -400 -350 -300 -250 -200 -150 -100 -50 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 ch an ge in a nn ua l i nc om e/ co w , $ pregnancy rate marginal change in value, $ clinical theriogenology • volume 12 number 3 • september 2020 311 the vwp; repeat hdr (rpthdr) for reinsemination cannot be clustered in a 21-day period, but at best at a 42 48 day period. in addition 100% of cows are available for first insemination whereas only (1fstcr)*100% of cows are available for second insemination, with a declining proportion at each sequential service. figures 2a, b, c present pregnancy rate as a function of conception rate across all services (2a), of first insemination heat detection rate (2b), and of repeat heat detection rate (2c) for 534 observations from 325 herds, largely in pennsylvania. to achieve a pr of 25% or greater, cr needs to be > 33 35%. conception may be greater than this and not achieve a 25% pr, due to low rates of heat detection. to achieve a pr of 25%, fsthdr needs to be > 70% (figure 2b) and a higher proportion of herds achieve a pr of 25% when fsthdr is 100%; that occurs when a synchronized first insemination program is employed. to achieve a pr of 25%, rpthdr needs to be > 50% (figure 2c); however, rpthdr has a lower association with pr than fsthdr and cr. figure 2a. pregnancy rate for conception rate across all services for 539 herds. for a pregnancy rate of 25%, conception rate needs to be at least 33% or more. however, conception rates greater than 35% may not achieve 25% pregnancy rates due to low heat detection rates. figure 2b. pregnancy rate for first service heat detection for 539 herds. first insemination heat detection rate has to be above 70% to achieve a 25% pregnancy rate. clinical theriogenology • volume 12 number 3 • september 2020312 figure 2c. pregnancy rate for repeat heat detection rate for services after first service for 539 herds. repeat heat detection rate needs to be greater than 50% to have a greater probability of achieving a pregnancy rate of 25%. mean values for herd observations with pr > 25% and for herd observations with pr < 25% are shown (table 1). except for mean days to first insemination, the proportion of interbreeding intervals less than 10 days and between 25 to 35 days and the percent of annual culls, all reproductive indices are superior for herds with pr > 25%. herds with a pregnancy rate of > 25% realized $333/cow more income if milk price was $20/cwt, calves had a value of $150/head, replacement cost minus cull cow value was $750/cow, lactation feed cost was $0.12/lb of dry matter and dry cow feed cost was $0.08/lb of dry matter. although the lower pregnancy rate group produced more total milk, it occurred over a longer period of time, reducing the efficiency of milk produced per day, reducing calves born annually, and increasing replacement costs due to fewer pregnancies over a 126-day breeding period. table 1. mean values for 325 herds and 534 observations for mean reproductive indices for herds with pregnancy rates equal to and > 25% and herds with < 25% pregnancy rate ---------------------------------------------------------------------------------------------------------------------------------------------------- item pr < 0.25 pr > 0.25 mean + sem mean + sem p value --------------------------------------------------------------------------------------------------------------------------------------------------- number observations 359 175 <.0001 voluntary wait period, d 52.38 0.68 61.53 0.82 <.0001 days to first insemination, d 78.97 0.70 77.56 0.83 0.0718 proportion first insemination rate 0.638 0.011 0.825 0.014 <.0001 proportion conception rate all services 0.351 0.005 0.436 0.005 <.0001 proportion repeat insemination rate 0.479 0.009 0.560 0.011 <.0001 pregnancy rate 0.185 0.003 0.305 0.004 <.0001 interval between inseminations 43.76 0.56 37.86 0.63 <.0001 proportion of inseminations < 10 days 0.026 0.003 0.034 0.003 0.0152 proportion of inseminations 10 17 days 0.038 0.002 0.036 0.002 0.4491 proportion of inseminations 18 24 days 0.237 0.007 0.256 0.008 0.04 proportion of inseminations 25 35 days 0.128 0.004 0.120 0.005 0.1875 proportion of inseminations 36 48 days 0.280 0.012 0.353 0.015 <.0001 proportion of inseminations > 49 days 0.294 0.009 0.218 0.011 <.0001 pregnant after 126 days breeding period 0.693 0.004 0.880 0.005 <.0001 calving interval, days 457.0 1.4 415.2 1.5 <.0001 mean lactation age of herd, years 2.37 0.02 2.48 0.020 <.0001 proportion of active cows culled 0.229 0.010 0.243 0.012 0.2952 total milk produced/cow, kg/cow 12,490 14.9 12,044 15.0 <.0001 milk produced per day, kg/cow 27.40 0.05 29.01 0.06 <.0001 annual value of income /cow, $ 1,833.0 9.2 2,166.0 10.5 <.0001 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 pr rpthdr rpthdr and pr clinical theriogenology • volume 12 number 3 • september 2020 313 good reproductive performance is possible using visual heat detection. figure 3 presents data from a farm for first insemination and repeat insemination using visual observation to manage insemination. heat detection rates are >70% and pr is >25% on this farm. good reproductive management may be achieved with visual observation. it will be reflected in first insemination and repeat insemination frequencies.first insemination frequencies will be spread somewhat uniformly over 21 day periods. frequency distribution of first insemination by days in milk from the voluntary wait period of 48 days is shown in figure 3 with corresponding tables. . distribution by 21-day periods of first inseminations from the voluntary wait period: heat detection cows bred: 838 total cows: 1101 all lactations wait period 1-48 49-69 70-90 91-111 112-132 > 133 total number of cows: 41 584 153 37 16 7 838 cumulative %: 4.9% 74.6% 92.8% 97.3% 99.2% 100.0% heat detection: 4.9% 73.3% 71.8% 61.7% 69.6% 100.0% mean hde: 72.4% 95% ci: 3.1% range: 69.3% <----> 75.5% distribution of days between inseminations by category intervals: interval analysis: distribution of days between breeding services sum of 1 2, 2 3 and 3 4 all lactations interval < 10 10 17 18 24 25 35 36 48 > 49 n # cows: 37 55 476 75 102 72 % cows: 4.5% 6.7% 58.3% 9.2% 12.5% 8.8% 817 long: 8.8% abnormal: 17.4% ratio: 4.7 posthdr: 70.8 figure 3. the graph is the frequency distribution of first insemination by days in milk from the voluntary wait period of 48 days. grouping by 21-day periods from the vwp allows calculation of first insemination rates. the first insemination intensity (first insemination heat detection rate, fsthdr) was 72.4%. pregnancy rate in this herd was 31.3%. conception rate to all services was 44.3%. next is the frequency distribution of days between inseminations grouped by <10, 10 17, 18 24, 25 35 and > 48 days. 0 5 10 15 20 25 30 35 40 45 c ow s days distribution days to 1st breeding clinical theriogenology • volume 12 number 3 • september 2020314 figures 4a, b, c presents the same graphics for pregnancy rate but from a group of herds with superior reproductive performance (149 herds). a subset of these herds was reported.15 conception rate is strongly associated with pr, and almost all these herds have a cr > 35%. pregnancy rate is also strongly associated with fsthdr. repeat heat detection rate (rpthdr) is not strongly associated with pr, but all these herds have rpthdr greater than 50%. these herds averaged a pr of 36.0% (sd 6.0%), cr of 48.0% (sd 7.5%), fsthdr of 91.4% (sd 9.8%), and a rpthdr of 64.8% (sd 9.3%). these herds were large (mean size, 1530 cows) but ranged in size from 81 to 21,000 cows. herd size does not inhibit good reproductive performance. conception rate in these superior herds is not significantly better than cr in drms records. what makes these herds superior in reproductive performance is high rates of first insemination efficiency figure 4a. pregnancy rate and conception rate to all services for 149 herds with excellent reproductive performance from across the us. if conception rate is greater than 35%, no herd has a pr less than 25%. figure 4b. pregnancy rate for first insemination rates for 149 herds with excellent reproductive performance across the us. only one herd with a first insemination rate greater than 75% has a pregnancy rate less than 25%. y = 0.6884x + 0.0345 r² = 0.734 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% 55% 20% 25% 30% 35% 40% 45% 50% 55% 60% 65% 70% pr eg na nc y r at e conception rate pr superior herds y = 0.2751x + 0.1132 r² = 0.1974 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% 55% 50% 55% 60% 65% 70% 75% 80% 85% 90% 95% 100% pr eg na nc y r at e, % first insemination rate prall clinical theriogenology • volume 12 number 3 • september 2020 315 figure 4c. pregnancy rate for repeat insemination for 149 herds with excellent reproductive performance from across the us. there is no strong relationship between repeat insemination rates and pregnancy rate, but all most all these herds have repeat insemination rates > 50%. based on the observations in figures 2, 3 and 4, cr is the main driver of pr. herd management must achieve a cr > 35% to achieve a pr of 25%. secondly, management must control fsthdr and achieve rates greater than 70%. repeat insemination heat detection rate is less critical, but still should be managed to control days between breeding. the average days between inseminations for herds represented in figure 4 was 32.9 days (+/4.4 days). these herds employed excellent management control of reproduction. important control points two most important control points to achieve a pr of 25% are herd conception rate and first insemination rate.1,8 regressing pr on cr yields an r2 of 0.70; regressing pr on fsthdr yields an r2 of 0.40; regressing pr on rpthdr yields an r2 of 0.09. therefore, priorities are conception, first insemination intensity, then repeat insemination intensity. herd conception rate is a function of health postcalving. cows with any postpartum problem have an odds ratio (or) of first service conception rate relative to healthy cows of 0.54. conditions postpartum associated with reduced cr include ovulatory dysfunction, body condition loss of more than ¾ of a unit, and infectious and metabolic diseases. cows that have more than one problem postpartum have a further reduction in first service cr with an or of 0.36. approximately, 20 30% of problem cows have more than one condition. the average herd experiences about 50% of cows with some health issue postcalving, whereas well-managed herds can reduce this to 30%. it is difficult to eliminate all health issues postcalving. good nutrition and management practices are necessary to reduce risk of health problems postcalving. the fertility in healthy cows will determine the cr in a herd, as they will be the most fertile group in the herd. if 50% of cows have no health problems postcalving and cr in these cows is 45%, then herd cr will be 36% and a pr of 25% is achievable. if healthy cows are 50% of animals calving but have a cr of 40%, then herd cr will be 32%, and a pr of 25% is achievable, but is less likely. if cr in healthy cows is 35%, then more than 80% of cows need to have no health problem to achieve a pr of 25%. the take home point is that farm managers need to do all they can to ensure high rates of conception in healthy cows and reduce the proportion of cows with problems postcalving. in addition to cow health, high rates of conception are also dependent on proper time of insemination relative to estrus, good semen handling and placement, and using bulls with good fertility. control of first insemination reproductive management programs can be structured as follows: chose a vwp that fits the biology of the herd and maximizes cr. typically 50 70 days provides a reasonable range for y = 0.0084x + 0.3593 r² = 0.0002 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% 55% 40% 45% 50% 55% 60% 65% 70% 75% 80% 85% 90% 95% 100% pr eg na nc y r at e repeat insemination rate prall clinical theriogenology • volume 12 number 3 • september 2020316 establishing a vwp that should be associated with good fertility. secondly, chose a program to control first insemination intensity. lastly coordinate veterinary visits and days postinsemination for pregnancy exam to reduce the proportion of cows that are inseminated greater than 48 days from the previous insemination. management has some influence on cr. however, management has total control over first insemination intensity and this is the most important management control point for herd reproductive efficiency.8,13-15 first insemination intensity (fsthdr), is the proportion of cows first inseminated in 21 days from the vwp. this is totally under the control of management. to achieve a pr of 25%, fsthdr needs to be greater than 70%. figure 3 has the frequency distribution of first inseminations by days in milk for a herd with a pr of 31.3%. this herd has a fsthdr of 73.1%, mainly performed using visual observation, apparent by the uniform frequency distribution across the 21-day periods. the vwp was 48 days. conception rate across all inseminations is 44.4%. the combination of intense first service insemination and high cr results in the high pr. repeat inseminations are important, but do not have the great impact on pr as cr and fsthdr. presented in figure 3 is the distribution of days between inseminations in categories by day: < 10, 10 17, 18 24, 25 35 and > 48 days. this herd inseminated 58.3% of repeat inseminations between 18 to 24 days following a previous insemination; that represents a regular inter-estrus interval. this is more than double the proportion observed for mean values in table 1 for herds with pr < 25% and herds with pr > 25%. the most critical proportion in this table is the frequency of cows with interval between inseminations > 48 days. this herd is only 8.8%, whereas herds with pr less than 25%, this proportion is 29.4%. a significant delay in reinsemination and timely pregnancy examination can reduce this proportion significantly. there are many ways to control first insemination.8 the optimal program for a herd will depend on farm management. milk progesterone, radio telemetry, and activity monitors require the investment in equipment to improve hdr. synchronization programs depend on injection of hormones at appropriate timing of the estrus cycle. visual heat detection requires ancillary aids such as tail paint or pressure sensitive tail patches and 3 4 observation periods per day to increase detection to rates greater than 70%. thus, all programs have some additional cost to control insemination intensity. what is best for any given farm depends on the management structure of the herd. synchronization programs prostaglandin was first proposed as a program to manage insemination by clustering estruses in cows at specific times.13-14 however, depending on day of estrous cycle at the time of prostaglandin injection, cows may not respond to the injection, and time to estrus could vary from 1 to 7 days in cows that seemed to respond to the injection. the variation in time to estrus depended on the stage or follicular development on the ovary, and the day of the estrus cycle of the cow. sequential injections of prostaglandin could synchronize groups of cows in estrus within weekly periods, but could not synchronize follicular waves, so time to estrus following injection was variable. therefore, although estrus detection could be focused on specific weekly periods, cows had to be inseminated on observed estrus to have good rates of conception. cows could be inseminated on a schedule following two injections 11 14 days apart, but conception was only optimal if inseminated twice, on days 3 and 4 post injection. additionally, prostaglandin injections had no benefit to induce estrus in anovulatory cows, which could be 10 20% of cows following a vwp of 50 days. a method using gnrh combined with a prostaglandin injection that synchronized luteal regression and ovulation so timed insemination could be managed on a specific appointment (ovsynch) was proposed.16 the program consisted of an injection of gnrh to induce ovulation and initiate a new follicular wave. seven days later, a corpus luteum should be responsive to prostaglandin with an emergent dominant follicle. following the 7-day prostaglandin injection, 2 days later, a gnrh injection should induce ovulation of a new dominant follicle, that 16 hours later should be fertilizable.16,17 thus, timed artificial insemination (tai) could be scheduled following the ovsynch program. initial application in a commercial herd demonstrated the benefit of using this program compared to typical herd management.18 clinical theriogenology • volume 12 number 3 • september 2020 317 the ovsynch program induced ovulation in anovular cows. however, fertility in these cows was typically poor. conception rates are typically 20% in cows entering an ovsynch program that have not previously ovulated. since anovulatory cows have low serum progesterone, although they may have ovulatory follicles on the ovary, follicles induced to ovulate tend to be more mature and produce lower concentrations of progesterone.17,19 both poorer quality follicles and lower progesterone production reduce fertility in anovular cows.19 alternative methods to improve cr in anovular cows have been proposed. furthermore, as ovsynch programs were evaluated, it was observed that only 65 to 70% of cows were synchronized, due to several problems with initiating the program in a random group of cows. 20 the first gnrh injection of the ovsynch protocol may not induce ovulation due to a regressing or immature follicle on the ovary. when these cows received prostaglandin and gnrh for ovulation and insemination, they tended to have a larger and less fertile follicle than cows that ovulated to the first gnrh injection.21 in addition 5 15% of cows had only partial luteolysis after the prostaglandin injection, disrupting sperm transport due to elevations in progesterone. other problems include smaller follicles in a subgroup of cows with insufficient progesterone output postinsemination, in addition to the problem with cows with larger, over-mature follicles. various additional methods were proposed to improve stage of follicular development and follicular quality prior to the ovsynch schedule. these recommendations included using progesterone intravaginal devices in cows with no cl at the gnrh injection,22 presynchronization with sequential prostaglandin injections prior to ovsynch to increase optimal stage of estrus cycle for synchronization,23,24 sequential use of gnrh prior to initiation of ovsynch,25 and combinations of estrus detection following prostaglandin prior to ovsynch.26 various programs used to control first insemination are shown (table 2) and various programs used on a group of farms to achieve excellent reproductive performance are detailed. 15 there was no difference in outcome for the various approaches used to control fsthdr. table 2. programs to control first insemination ----------------------------------------------------------------------------------------------------------------------------------------------- 1. pgf2α pgf22α pgf22α watch estrus 7 days 14 days, watch estrus 7 days 2. ovsynch gnrh pgf22α gnrh tai day 0 7 days 2 days 12 to 16 hours 3. presynch-ovsynch pgf2α pgf22α ovsynch day 0 14 14 5. selectsynch gnrh pgf22α watch estrus gnrh/tai day 0 7 72 hours 84 hours after pgf2α 6. g6g pgf2α gnrh ovsynch day 0 2 8 7. cidr7 cidr pull cidr/pgf22α watch estrus day 0 7 8 11 8. cidr6 cidr pgf22α pull cidr watch estrus day 0 6 7 8 11 9 anovulatory cows gnrh pgf22α gnrh ovsynch day 0 7 10 17 10 no cl at gnrh gnrh/2cidr pull2cidr/pgf22α pgf22α gnrh/tai 0 of ovsynch 5 6 8 ---------------------------------------------------------------------------------------------------------------------------------------------- figure 5 presents distribution of first inseminations associated with various programs. figure 5a presents first service distribution for a herd using weekly tai with a presynch ovsynch program. figure 5b presents first service distribution for a herd using a tai program every 14 days. figure 5c presents a herd using estrus detection following prostaglandin in the presynch portion of the program than tai is cows not inseminated in estrus. distribution of first service reveals the pattern of fsthdr control employed within a herd. first insemination needs to be greater than 70% to achieve a pr of 25% or better. conception rate needs to be > 35% to achieve pr of 25% or better. clinical theriogenology • volume 12 number 3 • september 2020318 after first insemination, cows not pregnant need to be reinseminated in a timely fashion. cows returning in one estrus interval and observed in estrus will be inseminated between 18 to 24 days following a prior insemination. if that estrus is missed, the next opportunity will be 36 to 48 days. with scheduled regular pregnancy diagnosis, nonpregnant cows should be detected between 30 to 40 days postinsemination. cows diagnosed open could be scheduled into a resynchronization program. effect of gnrh injections at 19, 26, and 33 days were compared following insemination with pregnancy examination at 26 days using ultrasonography. 27 open cows then were assigned to a prostaglandin and gnrh tai program beginning at 26, 33 and 39 days following the initial gnrh.27 the greatest pregnancy at tai was for cows begun on postsynchronization at 33 days postinsemination. therefore, resynchronization can be used in cows not re-inseminated by 24 days postinsemination. double ovsynch improved synchronization for repeat inseminations compared to 1 ovsynch synchronization.28 figure 6 presents distribution of days to second insemination for herds using various approaches to control reinsemination. figure 6a presents the frequency distribution for a herd using visual observation to detect estrus. distribution is spread over many days, since first insemination is also spread over many days (figure 5a). figure 6b is the frequency distribution of days in milk for second insemination in a herd using primarily resynchronization. cows are clustered from 36 to 48 days for rebreeding. figure 6c presents distribution of days to second insemination for a herd using visual figure 5a. distribution of first inseminations in a herd using 100% presynch ovsynch every 7 days figure 5b. distribution of first inseminations in a herd using an ovsynch program every 14 days clinical theriogenology • volume 12 number 3 • september 2020 319 figure 5c. herd using estrus detection following prostaglandin presynch and then applying ovsynch in cows not inseminated in estrus observation and then using resynchronization for cows not reinseminated by 30 days postbreeding. most critical is the herd in figure 5a has only 8.8% of repeat inseminations over 48 days from the previous service, the herd in figure 5b has only 4.4% of days between breeding over 48 days, and the herd in figure 5c has only 7.2% of days between breeding over 48 days. the critical control point for repeat inseminations is a pregnancy diagnosis program to reduce days between breeding over 48 days to less than 10% of inseminations. figure 6a. herd using visual observation figure 6b. herd using resynchronization primarily (herd in figure 3 for firs insemination) figure 6c. herd using heat detection and then re-synchronization figure 6. frequency distribution of days to second insemination for different management programs clinical theriogenology • volume 12 number 3 • september 2020320 conclusion control of first insemination is the critical control point in managing herd reproduction. monitoring fertility in healthy cows establishes baseline fertility in the herd and provides an estimate of the wellbeing of transition cows and insemination protocols. healthy cows should have fstcr greater than 50%. first service intensity needs to be greater than 70% and pregnancy performed on a timely basis before 40 days postinsemination. many opportunities exist to control first insemination. the best strategy will depend on herd management. automatic activity monitoring versus synchronized breeding programs could not be generalized, but depended on management within each herd.29 conflict of interest there are no conflicts of interest to declare. references 1. cabera ve: economics of fertility in high-producing dairy cows on confined tmr systems. animal 2014;8:s1: 211-221. 2. forde n, beltman me, lonergan, p, et al: oestrous cycles in bos taurus cattle. anim reprod sci 2011;124:163-169 3. dransfield mbg, nebel rl, pearson re, et al: timing of insemination for dairy cows identified in estrus by a radiotelemetric estrus detection system. j. dairy sci 1998;81:1874-1882. 4. sveberg g, refsdal ao, ehard hw, et al: sexually active groups in cattle a novel estrus sign. j dairy sci 2013;96:4375-4386. 5. lopez h, satter ld, wiltbank mc: relationship between level of milk production and estrous behavior of lactating dairy cows. anim reprod sci 2004;81:209-233. 6. diskin mg, sreenan jm: expression and detection of oestrus in cattle. reprod nutr dev 2000;40:481-491. 7. roelofs j, lόpez-gatius f, hunter rhf, et al: when is a cow in estrus? clinical and practical aspects. theriogenology 2010; 74:327-344. 8. nebel rl, jobst sm: evaluation of systematic breeding programs for lactating dairy cows: a review. j dairy sci 1998;81:1169-1174. 9. aungier spm, roche jf, sheehy m, et al: effects of management and health on the use of activity monitoring for estrus detection in dairy cows. j dairy sci 2012;95:2452-2466. 10. nebel rl, dransfeld mg, jobst sm, et al: automated electronic systems for the detection of oestrus and timing of ai in cattle. anim reprod sci 2000;60-61:713-723. 11. delwiche m, tang x, bondurant r, et al: estrus detection with a progesterone biosensor. transactions of the asae 2001;44:2003-2008. 12. heersche g, nebel rl: measuring efficiency and accuracy of detection of estrus. j dairy sci 1994;77:2754-2761. 13. ferguson jd, galligan dt: prostaglandin synchronization programs in dairy herds part 1. compend cont ed north am 1993;15:646-654. 14. ferguson jd, galligan dt: prostaglandin synchronization programs in dairy herds part ii. compend cont ed north am 1993;15:1127-1130. 15. ferguson jd, skidmore a: reproductive performance in a select sample of dairy herds. j dairy sci 2013;96:12691289. 16. pursley jr, mee mo, wiltbank mc: synchronization of ovulation in dairy cows using pgf22α and gnrh. theriogenology 1995;44:915-923. 17. wiltbank mc, sartori r, herlihy mm, et al: managing the dominant follicle in lactating dairy cows. theriogenology 2011;76:1568-1582. 18. pursley jr, kosorok mr, wiltbank mc: reproductive management of lactating dairy cows using synchronization of ovulation. j dairy sci 1997;80:301-306. 19. wiltbank mc, baez gfm, garcia-guerra a, et al: pivotal periods for pregnancy loss during the first trimester of gestation in lactating dairy cows. theriogenology 2016;86:239-253. 20. bello nm, steibel jp, pursley jr: optimizing ovulation to first gnrh improved outcomes to each hormonal injection of ovsynch in lactating dairy cows. j dairy sci 2006;89:3413-3424. 21. colazo mg, behrouzi a, ambrose dj, et al: diameter of the ovulatory follicle at time artificial insemination as a predictor of pregnancy status in lactating dairy cows subjected to gnrh-based protocols. theriogenology 2015;84:377383. 22. bisinotto rs, ribeiro es, lima fs, et al: targeted progesterone supplementation improves fertility in lactating dairy cows without a corpus luteum at the initiation of the timed artificial insemination protocol. j dairy sci 2013;96:22142225. 23. navanukraw c, redmer da, reynoolds lp, et al: a modified presynchronization protocol improves fertility to timed artificial insemination in lactating dairy cows. j dairy sci 2004;87:1551-1557. clinical theriogenology • volume 12 number 3 • september 2020 321 24. silva e, sterry ra, kolb d, et al: effect of pretreatment with prostaglandin f22α before resynchronization of ovulation on fertility of lactating dairy cows. j dairy sci 2007;90:5509-5517. 25. carvalho pd, guenther jn, fuenzalida mj, et al: presynchronization using a modified ovsynch protocol or a single gonadotropin-releasing hormone injection 7 d before an ovsynch-56 protocol for submission of lactating dairy cows to first timed artificial insemination. j dairy sci 2014;97:6305-6315. 26. gumen a, keskin a, yilmazbas-mecitoglu g, et al: effect of presynchronization strategy before ovsynch on fertility at first service in lactating dairy cows. theriogenology 2012;78:830-838. 27. fricke pm, caraviello dz, weigel ka, et al: fertility of dairy cows after resynchronization of ovulation at three intervals following first timed insemination. j dairy sci 2003;86:3941-3950. 28. giordano jd, wiltbank mc, guenther jn, et al: increased fertility in lactating dairy cows resynchronized with doubleovsynch compared with ovsynch initiated 32 d after timed artificial insemination. j dairy sci 2012;95:639-653. 29. neves rc, leslie ke, walton js, et al: reproductive performance with an automatic activity monitoring system versus a synchronized breeding program. j dairy sci 2012;95:5683-5693. clinical theriogenology • volume 12 number 3 • september 2020322 021_ms-021 ferguson 2 intracytoplasmic sperm injectionproduced equine embryos: transport, thawing, and transfer rob foss equine medical services, inc., columbia, mo abstract intracytoplasmic sperm injection (icsi)-produced equine embryos are transferred at the early blastocyst stage, similar to a day 6.5 in vivo-produced embryo and will benefit from a recipient mare that ovulated 3 5 days before transfer. frozen-thawed and vitrified-thawed embryos may have an even narrower window of synchrony with recipients for best results. day of blastocyst formation is a useful indicator of embryo quality and potential for establishing a successful pregnancy; early embryonic loss is higher for blastocysts that formed on days 9 and 10 after icsi than those formed earlier. keywords: intracytoplasmic sperm injection, equine embryo, embryo transfer introduction production of equine embryos via intracytoplasmic sperm injection (icsi) and their subsequent transfer is becoming more important for commercial horse production. many of these embryos are potentially valuable and contain genetics prized by their owners, so it is in the best interest of veterinarians and clients to handle and transfer these embryos as optimally as possible. intracytoplasmic sperm injection-produced embryos (hereafter referred to as icsi embryos) differ from in vivo-produced embryos. although they can be handled similarly, some adjustments are necessary. careful selection of appropriate recipient mares, embryo handling, and timing of transfer are necessary to optimize results. comparison of in vivo and intracytoplasmic sperm injection-produced embryos equine icsi embryos differ from their in vivo counterparts morphologically and metabolically.1 in vitro culture conditions can be successful for viable embryo production; however, conditions are not similar to those that exist in utero (uterus or oviduct), leading to many differences. one of the more prominent morphological differences of icsi embryos is the lack of development of an embryonic capsule. the capsule, a mucin-like glycoprotein, is normally produced from secretions from the trophoblast cells, and forms between them and the zona pellucida. a very strong, elastic structure, it presumably provides protection for the developing blastocyst after shedding zona pellucida. a capsule does not form on icsi embryos in vitro; however, it develops after embryos are transferred into a recipient uterus. due to the injection-induced breach in the zona, there is a short interval after transfer when icsi embryos may be directly exposed to the uterine immune system and therefore inflammation. the discrete inner cell mass that develops in the early blastocyst in vivo is not observed in icsi embryos, another result of in vitro culture. the delay in the aggregation of inner cell mass cells may contribute to the higher rate of monozygotic twinning after transfer of equine icsi embryos. zona pellucida appears to be somewhat hardened by in vitro culture. this has a role when the early blastocyst starts expanding and is limited in its expansion by the zona. during in vivo development, the zona thins as the blastocyst expands and then, after the capsule forms, thins enough until it flakes away. during in vitro, the zona does thin; however frequently the trophoblast layer will herniate through the icsi-generated hole in the zona. this, when the embryo is transferred, would further expose the otherwise unprotected blastocyst to the uterine environment. herniation of trophoblast and blastocoele might also predispose to monozygotic twinning, forming a separate blastocyst outside the zona pellucida. an icsi embryo, growing in culture instead in an oviduct, is subjected to physiologic stress throughout the culture period. without the conditions provided by oviductal epithelium, the various cytokines and other ‘embryo-friendly’ compounds, embryos do grow; however, they are probably compromised by the stress of adapting to in vitro culture conditions. there are differences in gene activation of the icsi embryo compared to in vivo embryos.2 problems with chromosomal segregation are more common in vitro leading to embryos with ploidy issues, i.e., too many or too few chromosomes in daughter cells.3 all of these factors contribute to the fact that at our current state of knowledge, icsi embryos, on average, are less likely to establish a pregnancy and then maintain a viable pregnancy to term than their in vivo counterparts. author’s practice advises clients to expect a 10% lower pregnancy rate with icsi embryos versus in vivo embryos, and approximately twice the rate of early embryonic loss. transfer of icsi embryos transfer of icsi embryos is usually done at the early blastocyst stage as it is starting to expand but before much expansion occurs. this permits transfer of a uterine stage embryo that has not had trophoblast cells herniate through the breach in the zona. currently, developmental status of the icsi embryo is roughly equivalent to that of a day 6.5 in vivo embryo, although the embryo may be anywhere from 6 to 10 days after fertilization by icsi. synchrony selection of a recipient mare to receive an icsi embryo should consider the developmental stage of the embryo. preferred synchrony for in vivo days 7 and 8 embryos is usually considered ideal at -1 or -2 (a recipient that is on day 5 or 6 after ovulation), but with a window of +1 to -3 (recipient days 8 4). this suggests that the preferred recipient for an icsi early blastocyst would be a recipient that was days 4 or 5 postovulation. reports on the success of icsi embryo transfer have suggested that the window of synchrony for transfer is narrower for icsi embryos than their in vivo counterparts, although these reports utilized frozen-thawed embryos.4,5 author’s clinical practice has not identified a narrower window of acceptable synchrony for embryos that have not been frozen. data from 926 transfers (table 1) suggested a trend toward higher pregnancy rates with day 3, 4, and 5 recipient mares and no statistical difference with days 2 through 7 recipients. table 1. pregnancy by day after recipient ovulation recipient day pregnant/transferred pregnant (%)* noncyclic 21/25 84.0 a 7 45/58 77.6 a 6 146/181 80.6 a 5 225/274 82.1 a 4 229/278 82.3 a 3 86/105 81.9 a 2 3/5 60.0 a total 755/926 81.5 *within a column, percentages without a common superscript differed (p < 0.05) day of development day of development, representing the number of days following icsi that blastocyst formation occurs, has a distinct effect on results of embryo transfer. as expected, on average, the faster an embryo reaches the early blastocyst stage the healthier it is and the more likely to produce a viable pregnancy. data (table 2) from the same group of 926 transfers were compared by day of blastocyst development and pregnancy rate. a fairly consistent trend of decreasing pregnancy rate with increasing days of development was evident; however, the difference was not significant until days 9 and 10. table 2. pregnancy by day of blastocyst development day pregnant/transferred pregnant (%)* 6 36/39 92.3 a 7 288/348 82.7 a 8 232/278 83.5 a 9 147/185 79.5 a,b 10 52/76 68.4 b total 755/926 81.5 a,bwithin a column, percentages without a common superscript differed (p < 0.05) early embryonic loss early embryonic loss is more common with icsi embryos than with in vivo embryos; author’s practice informs the clients to expect ~ double the rate of embryonic loss. most of these losses occur before day 30 of pregnancy and are usually preceded by a period of slower than expected embryonic growth or they appear as an empty trophoblastic vesicle. there is a smaller group that appear normal at the heartbeat stage that are lost by 45 days, and an even smaller group that is lost by 60 days. early embryonic losses for the group of 926 transfers (discussed previously) by recipient mare day postovulation are summarized (table 3). the overall total loss rate was 17.7% with no significant differences related to synchrony. table 4 has the same group classified by day of blastocyst development. a consistent trend in increased loss with increased day of development was noticed, becoming significant by days 9 and 10 post-icsi. table 3. early embryonic loss by day of recipient cycle recipient day pregnant/transferred %* losses %* noncyclic 21/25 84.0a 2/21 9.5a 7 45/58 77.6a 10/45 22.2a 6 146/181 80.6a 24/146 16.4a 5 225/274 82.1a 46/225 20.4a 4 229/278 82.3a 35/229 15.2a 3 85/105 81.9a 14/86 16.2a 2 3/5 60.0a 3/3 100a total 755/926 81.5 134/755 17.7 *within a column, percentages without a common superscript differed (p < 0.05) table 4. early embryonic loss by day of blastocyst development day pregnant/transferred % loss % 6 36/39 92.3a 4/36 11.1a 7 288/348 82.7a 34/288 11.8a 8 232/278 83.5a 35/232 15.1a 9 147/185 79.5a,b 41/147 27.8b 10 52/76 68.4b 20/52 38.5b,c total 755/926 81.5 134/755 17.7 a-cwithin a column, percentages without a common superscript differed (p < 0.05) grading icsi embryos traditionally embryos have been graded by evaluating morphological characteristics at a single point in time with the goal of providing useful information concerning the estimated ability of a given embryo transfer to result in an ongoing pregnancy. similar morphologic evaluation of icsi embryos generally does not give the same ability to estimate the pregnancy potential of a given embryo. icsi embryos are transferred at an early blastocyst stage that will appear remarkably similar for vigorous healthy embryos and those that are not so vigorous and healthy. day of blastocyst formation/transfer has a higher correlation to pregnancy than morphology. tables 4 and 5 suggest that early embryonic loss and pregnancy rate decreased with increasing day of blastocyst formation. since owners and veterinarians alike want to know the grade of an individual embryo, author’s practice grades embryos based on day of blastocyst formation, adjusted for morphologic abnormalities. typically, days 6 and 7 blastocysts are graded as grade 1, day 8 blastocysts are graded as grade 1.5, grade 2 for day 9, and grade 2.5 for day 10. history noted during the culture period may also be used to adjust grades. blastocyst formation normally occurs 2 days after morula compaction and if this period is longer, the embryo will be graded lower. table 5. pregnancy for vitrified/thawed embryos by day of recipient cycle recipient day pregnant/transferred pregnant (%) noncyclic 11/15 73.3 a 7 4/6 66.6 a 6 5/12 41.7 a 5 62/81 76.5 a 4 115/157 73.2 a 3 23/28 82.1 a total 220/299 73.5 *within a column, percentages without a common superscript differed (p < 0.05) monozygotic twins occurrence of monozygotic twins is more likely following transfer of icsi embryos than in vivo embryos with an incidence of ~ 1.5%.6 these twin pregnancies occur following the transfer of a single embryo and are typically monochorionic, so that only a single vesicle is seen on initial pregnancy examinations. a diagnosis for twin pregnancy is not performed until more than 1 embryo-proper is visible in the vesicle. twin reduction of monozygotic twins by cranio-cervical dislocation is possible but generally not successful,7 so it is often prudent to consider this a pregnancy loss and abort the pregnancy. it is interesting to note that monozygotic twinning in human ivf embryos occurs ~ 1.7 2.5%,8 the lower rate from transfers on day 2 or 3 with the higher rate from transfers on day 5 or 6. this is an indication that it is the culture environment that leads over time to an increased incidence of twinning. this may be from ineffective migration of inner cell mass cells leading to the formation of two inner cell masses. twin production via herniation of trophoblast with some inner cell mass cells through the piezo hole in the zona is a less likely explanation as 2 embryonic vesicles should be formed in this manner leading to a dichorionic twin pregnancy. shipping of icsi embryos since icsi laboratories are often geographically distant from recipient mares, shipping becomes necessary. the icsi embryo, being produced in only an imitation of its natural environment, may be considered stressed, so additional stress should be avoided. stresses to consider are temperature, duration of transport, and selection of culture/holding medium providing ph maintenance and embryo nutrition. temperature or osmotic stress are also a possibility, if the embryo is moved to a different medium (after shipping) before transfer. icsi embryos are generally transported in passive heating/ cooling devices, such as the equocyte (hamilton biovet, ipswich, ma), or portable incubators capable of active heating and cooling (e.g., micro q [micro q technologies, scottsdale, az]). equine icsi embryos can tolerate several hours at room temperature, but pregnancy rates decreased as the interval at room temperature increased.7 addition of nutritional support in the form of 10 mm glucose can increased the survival time, up to 50% pregnancy rate at 48 hours.7 currently, author’s practice utilizes a modified culture medium for shipping in an incubator (micro q) at 38°c. the shipping medium can be used for transfer of the embryo so that temperature and osmotic changes are avoided, and an attempt is made to minimize transport time as much as possible. thawing and transfer of vitrified embryos vitrified icsi embryos can be thawed and transferred in most situations and practices that can transfer fresh embryos. due to the number of embryos produced in the nonbreeding season and surplus embryos produced during the breeding season, many are vitrified. vitrification gives an opportunity not only for storage but also for shipping and transfer into a specific recipient or even back into the donor. author’s practice has utilized thawing into a 0.3 m sucrose solution with timed stepwise reduction in sucrose concentration for several years, but have utilized a single step procedure for the last 2 years.9 this procedure consists of plunging the device containing the embryo (e.g., cryolock [bio tech inc., alphareta, ga]) or open pulled straw, directly from liquid nitrogen into a petri dish containing the thawing solution warmed to 38°c. this thawing solution is gmops plus (vitrolife, goteborg, sweden) in the author’s practice but commercial embryo holding medium can also be used. embryo is held in the thawing solution on a warm stage for 5 minutes and then either prepared for transfer or placed in culture. if a specific recipient mare is to be used, directly transferring the embryo may be preferable, but if multiple recipients are available, culture for a short interval may be preferable as it allows evaluation of embryo re-expansion. embryo re-expansion will take place rather quickly, 2 3 hours, in most instances but other embryos that may not have tolerated the freezing as well may take a day or even 2. this can drastically affect embryo-recipient synchrony, so culturing improves recipient selection in those instances. there is also a difference in timing between embryo re-expansion and resumption of growth. embryonic vesicles detectable via ultrasonography on the first day of pregnancy are generally smaller or even a day later than expected from transfer of fresh icsi embryos. this delay in resumption of growth may explain the narrower window of synchrony reported for transfer of frozen-thawed icsi embryos.4,5 results (table 5) from transfer of vitrified-thawed embryos in the author’s practice broken down by recipient mare’s day of cycle. there is a trend toward higher pregnancy rates from days 3, 4, and 5, but it does not reach the level of significance. this may be since the majority of these embryos were cultured until re-expansion versus directly transferred into recipients. conflict of interest none to declare. references 1. tremolada j, stout t, lagutina i, et al: effects of in vitro production on horse embryo cytoskeletal charactereistics and blastocyst capsule formation. biol reprod 2003;69:1895-1906. 2. hisey e, ross p, meyers s: a review of oct4 functions and applications to equine embryos. j equine vet sci 2021;99:103394. 3. ducheyne k, rizzo m, cuervo-arango j, et al: in vitro production of horse embryos predisposes to micronucleus formation, wheras time to blastocyst formation affects likelihood of pregnancy. reprod fert dev 2019;31:1830-1839. 4. cuervo-arango j, claes a, stout t: in vitro-produced horse embryos exhibit a very narrow window of acceptable recipient mare uterine synchrony compared with in vivo-derived embryos. reprod fertil dev 2019;31:1904-1911. 5. cuervo-arango j, claes a, stout t: the recipient’s day after ovulation and the number of corpora lutea influence the likelihood of pregnancy in mares following transfer of icsi frozen embryos. theriogenology 2019;135:181-188. 6. djikstra a, cuervo-arango j, stout t, et al: monozygotic multiple pregnancies after transfer of single in vitro produced equine embryos. eq vet j 2020;52:258-261. 7. foss r: unpublished data. 8. kanter j, boulet s, kawwass j, et al: trends and correlates of monozygotic twinning after single embryo transfer. obstet gynecol 2015;125:111-117. 9. canesin h, ortiz i, filho a, et al: effect of warming method on embryo quality in a simplified equine embryo vitrification system. theriogenology 2020;151:151-158. ytoplasmic clinical theriogenology 2022; 14: 209 meeting the demand for laparoscopic artificial insemination in sheep and goats rachael gately department of environmental and population health, cummings school of veterinary medicine tufts university, north grafton, ma abstract demand for laparoscopic artificial insemination in small ruminants is growing exponentially. use of a well-designed estrus synchronization protocol allows does and ewes to be inseminated in a timely manner with good conception rates. females are sedated with an intramuscular cocktail to facilitate inversion and laparoscopic access to their uterus for deposition of fresh or frozen semen directly into the lumen of both uterine horns. keywords: laparoscopy, artificial insemination, small ruminants introduction use of assisted reproductive technologies (art) is becoming more popular within small ruminant industries. requests for laparoscopic artificial insemination (lai) services increase each year as producers look for ways to advance genetics in a quick and cost-effective manner. availability of global importation of semen from bucks and rams to the united states has further driven the demand for this surgical procedure. laparoscopic artificial insemination allows semen to be deposited intrauterine in these 2 species with difficult to pass or impassable cervixes. the thought of performing laparoscopic surgery in small ruminants can be daunting to the veterinarian. with a basic understanding of synchronizing estrus and a review of laparoscopic equipment and procedure, a general practitioner should be able to competently add laparoscopic artificial insemination to their list of service offerings. estrus synchronization a standard small ruminant controlled internal drug release (cidr) synchronization protocol is utilized. does and ewes have a cidr placed on day 0. a dose of prostaglandin is given on day 11. the cidr is removed on day 12 and a dose of pg600® (intervet/merck animal health, madison, nj) is given intramuscularly. food and water are removed mid-day on day 13 and a teaser ram or buck is turned in with females. laparoscopic artificial insemination is performed on day 14 at 48 54 hours after cidr removal. sedation does and ewes are sedated prior to lai using a combination of ketamine, xylazine, and butorphanol. ketamine (1 mg/kg) is combined with 20 mg/ml xylazine (0.05 mg/kg) and butorphanol (0.025 mg/kg) to make a stock solution for the lai procedure. ewes are given intramuscularly 0.02 ml/kg whereas does respond variably to an intramuscular dose of 0.01 0.02 ml/kg. after 10 minutes, the sedated females are ready to be stood alongside an artificial insemination table and rolled into dorsal recumbency. patient preparation once in dorsal recumbency on the artificial insemination table, the front limbs of the female are hooked under the corresponding leg brackets at pastern joint level. hind limbs are secured to their corresponding leg brackets using the attached rope and ratcheted down. female’s eyes are covered using a towel and halter combination or a custom-designed blindfold. a 30 x 30 cm area cranial to the udder is clipped using commercial sheep shears followed by a surgical blade. the area subsequently undergoes a surgical scrub using alternating chlorhexidine scrub-soaked gauze and sterile saline-soaked gauze. artificial insemination procedure artificial insemination table is inverted. two stab incisions (3 5 cm) are made through the skin ~ 12 cm cranial to the udder on either side of midline. a teat cannula or veress needle is inserted bluntly through 1 stab incision, puncturing into the abdominal cavity. tubing leading from a carbon dioxide insufflator is connected and the abdomen is insufflated at 5 7 liters per minute. teat cannula or veress needle is then removed and immediately replaced with a trocar. a laparoscope is introduced via the trocar and the uterus is visualized. semen is requested to be prepared while the second trocar is placed through the second skin stab incision. the prepared artificial insemination gun is inserted through the second trocar by an assistant and handed off to the technician performing the insemination. needle of the artificial insemination sheath is clinical theriogenology 2022; 14: 210 aligned perpendicular to the base of 1 uterine horn. a quick stab is made, presumably penetrating the uterine lumen. assistant plunges half of the semen dose. gun is then removed from that uterine horn and moved to the opposite horn. similar stab technique is used to insert the needle into the second uterine horn, and the remaining semen in the straw is deposited. insemination gun is removed, trocar stopcocks are opened, and excess carbon dioxide is drained from the abdomen. then trocars are removed and 1 staple is placed in the skin over each incision. blindfold is removed, and the female is rolled off the table. ewes readily walk away whereas goats may lie sedated for up to 45 minutes. conflict of interest none to declare. 2017 mares behaving badly: a review of methods for estrus suppression in the mare � mares behaving badly: a review of methods for estrus suppression in the mare* kathleen hornberger, candace c. lyman, elizabeth coffman, g. reed holyoak center for veterinary health sciences, oklahoma state university, stillwater, ok abstract mares are seasonally polyestrus and based on the presence or absence of reproductive hormones, specifically estrogen and progesterone, their behavior can vary throughout the year. concerns pertaining to mare behavior are often attributed to their estrous cycle, however undesirable mare behaviors need to be differentiated from other non-reproductive causes of undesirable behavior. while behaviors consistent with estrus are described as increased frequency of approaching the stallion, leaning hindquarters toward the stallion, relaxed facial muscles, slightly lowered head, ears turned toward the side, posturing, clitoral eversion, and passive urination, common client complaints such as kicking, tail swishing, or poor attitude may be related to the diestrus phase of the estrous cycle. display of these behaviors alone may not directly relate to the issues with the estrous cycle and, in particular, if not displayed under controlled teasing conditions with a stallion. multiple treatments have been utilized by veterinarians for estrus suppression and manipulation, however some of them are poorly effective while others may actually promote estrous behavior. treatments can be categorized as progestogen supplementation, extension of the luteal phase, suppression of ovarian follicular activity, ovariectomy, and non-traditional or alternative therapies. this review details the individual therapies available for the potential treatment of estrous behavior and each therapy’s associated efficacy. keywords: estrus behavior, mare, estrus suppression, review introduction mares are frequently presented by owners and trainers for undesirable behavior perceived to correlate with cyclicity. however, a variety of other issues may result in declining mare performance, including musculoskeletal, gastrointestinal, behavioral, and urinary tract disorders. thus, it is important to combine a baseline knowledge of the normal mare estrous cycle and associated reproductive behaviors with a complete physical examination and potential ancillary tests (endoscopy, lameness examination, etc.) before attributing the behaviors to stages of the reproductive cycle. we will therefore briefly review normal estrous cycle and associated behaviors in the mare. following this, various treatment options will be discussed while focusing on their individual level of empirical evidence of success, advantages, and disadvantages. the equine estrous cycle mares are long-day, seasonally polyestrous breeders. the mare’s reproductive seasonality is regulated by photoperiod and can be broken down into four phases throughout the year: anestrus, spring transitional period, the ovulatory season (further divided into estrus and diestrus), and autumn transitional period. however, a small percentage of mares will ovulate year round and never enter the remaining three phases.1,2 the beginning of the ovulatory season is marked by the first ovulation which may be detected by regular examinations by ultrasound per rectum or serial determination of serum progesterone concentrations. as stated above, each cycle thereafter is divided into two stages: estrus and diestrus. estrus averages 6.5 days in length and is characterized by sexual receptivity, dominant follicle emergence, basal concentration of plasma progesterone (<2 ng/ml), and ovulation. estrogen begins to rise 6 to 8 days prior to ovulation and its presence is due to increased follicular growth. high concentrations of estrogen with baseline progesterone concentrations are positively correlated to expression of estrus behavior. the length of this estrus period becomes abbreviated as day length increases and summer solstice approaches. ovulation marks the end of estrus (day 0), however, mares ������������������������������������������������������������ *�presented in part at the oklahoma veterinary medical summer seminar, june 2016� 583 clinical theriogenology • volume 9 number 4 • december 2017 � can still exhibit estrus behavior up to 24-48 hours following ovulation. diestrus averages 15-16 days, is initiated after ovulation, and extends through luteolysis. this stage is characterized by the presence of a corpus luteum (cl) in the ovary, high concentrations of plasma progesterone, and non-receptive behavior toward the stallion. estrogen is not the main dictator for reproductive behavior but rather behavior is regulated by the concentration of progesterone; basal concentrations of progesterone will allow for varying degrees of behavior consistent with estrus. it should be noted that the ovulatory season is the only period of the year during which there is endogenous progesterone by the cl a structure present in the ovary as a result of ovulation of one or more follicles. this concept needs to be understood in order to be able to determine whether or not a behavior is associated with a cyclical pattern and, if it is, what the dominant hormone is at the time the behavior is expressed. moreover, in most mares, these phases are differentiated by the distinct behaviors when mares are teased under controlled conditions to a stallion. it is only when using a stallion to tease a mare that one can attribute a particular behavior to a particular stage of the estrous cycle. in addition, findings from reproductive ultrasound examination and serum progesterone concentrations can aid in determining in which phase of the estrous cycle is the mare. anestrus occurs during the time of year when shortened day length increases pineal melatonin production and a subsequent decrease in hypothalamic gonadotropin releasing hormone (gnrh) production. during this time of low gnrh production, most mares are anovulatory and generally reproductively quiescent. although ovulation does not occur during this phase, some follicular growth is sustained. there is no luteal source of progesterone during this phase and as a result mares may exhibit varying degrees of estrus behavior and stallion receptivity. spring transition occurs during the period of increasing daylight, whereas autumn transition occurs during the period of decreasing daylight. both periods are characterized by estrus without ovulation. it is during these timeframes that owners will become frustrated with unpredictable periods of estrus behavior since mares will produce follicular waves without ovulation which may extend for many days to weeks. during the spring transition there is a progressive increase in follicular activity with development of up to three anovulatory follicular waves prior to the ovulatory wave.3 it has been hypothesized that failure of ovulation during spring transition is due to insufficient stores of pituitary luteinizing hormone (lh) or lack of steroidogenic competence of ovarian follicles.4,5 during the autumn transition there is a progressive decline in luteal and follicular activity marked by a decrease in steroidogenesis. thus in this regard, the autumn transition can be characterized by a long anovulatory follicular phase or a shortened luteal phase. lastly, anovulatory hemorrhagic follicles are more common in autumn transition. these occur when a dominant follicle fails to ovulate and subsequently, the follicular lumen fills with blood. some anovulatory hemorrhagic follicles appear to luteinize and synthesize progesterone, however do not appear to be responsive to prostaglandin f2-alpha as the average interovulatory interval is 38.5±15 days.6 in summary, autumn transition is accompanied by erratic estrous cycles characterized by long follicular phase/short luteal phase or anovulatory hemorrhagic cycles. this transition can take up to 60 days or more. estrus behavior it is important to understand the range of behavior in normal mares during estrus in order to assess whether or not a behavior is attributable to ovarian hormone production (or lack thereof). these behaviors can only be objectively evaluated in the presence of a stallion under controlled teasing conditions. such estrous behaviors include increased frequency of approaching the stallion, leaning hindquarters toward the stallion, relaxed facial muscles, slightly lowered head, ears turned toward the side, posturing, clitoral eversion, bent forelimb, vulvar winking, and urination. posturing is the most significant sign of receptivity and is characterized by a full body response: arched tail, flexed stifles and hocks, tipped pelvis, and weight distribution to the front limbs; this posture allows the mare to support the weight of a stallion during breeding.1 in the absence of a stallion, estrus behavior may be directed towards inanimate objects or other animals including geldings and mares, and even cattle, however one cannot truly conclude the mare is in heat based on her behavior in the absence of a stallion. in contrast, 584clinical theriogenology • volume 9 number 4 • december 2017 � during diestrus and thus under the influence of progesterone, mares are non-receptive to stallion advances and may squeal, bite or kick. other non-receptive behaviors include pinned ears and tail swishing. given that the absence of progesterone is a pre-requisite for displaying estrus behavior in mares, behaviors consistent with estrus are observed in anestrus mares and even ovariectomized mares. in fact, the adrenal gland serves as a small source of sex steroid production (estradiol, progesterone, and testosterone) and it is hypothesized by some to play a role in facilitating paradoxical estrus behavior.7 paradoxical estrus behavior is unique to the mare and is thought to be important for maintaining a bond between a stallion and his harem of mares in the wild, as they band year-round. this phenomenon is of vital importance clinically because bilateral ovariectomy may not improve estrus behavior, actually the objectionable behavior may be exacerbated. in fact, bilateral ovariectomy may be performed to make a “jump” or “teaser” mare for use in situations of semen collection from stallions. while some “jump” mares are useful just after ovariectomy and require no supplemental estrogen to display estrus behavior, some mares may require estrogen therapy. behavior problems in the performance mare: etiology and clinical evaluation often mares are presented to veterinarians for complaints of estrus behavior affecting performance. common complaints from owners that are not associated with estrus behavior include kicking, biting, squealing, stomping in the trailer, tail swishing, and poor attitude. not all owners can rightfully discriminate actual estrus behavior from other behavioral issues associated with poor discipline or medical problems. described behaviors by some owners will be the result of inadequate training e.g. the inability of an owner to catch a mare in a pasture is likely not estrus related. however, some mares do fail to perform at their best due to strong estrus behavior and may become difficult to manage, perform erratically, or appear lame.8 estrogen has also been associated with laxity in ligaments so lameness may be exacerbated during estrus.8 common complaints from owners associated with estrus include elevating tail or winking during performance, leaning on fences toward other horses or stimuli, slowing or stopping at other horses while riding, wet stall/legs/tail due to excessive urination, posturing in the arena, or intermittent colic. intermittent colic can occur due to ‘mittelschmerz’, which is pain associated with ovulation. whereas estrus behavior can interfere with performance or be an inconvenience, it is important to exclude other causes of poor performance and undesirable behavior before assigning a diagnosis. it is important to obtain a thorough history of the types of behaviors observed, when these behaviors are occurring, and whether they are cyclic in nature. some mares may possess a desirable demeanor during estrus and exhibit undesirable behaviors during diestrus. a thorough physical examination along with reproductive and lameness examinations should be performed to diagnose whether the mare’s behavior/demeanor is truly reproductive in origin. a complete reproductive examination should include trans-rectal palpation, trans-rectal ultrasound, external examination, vaginal speculum examination, serum progesterone concentration, and teasing behavior to a stallion. granulosa-theca cell tumors can be characterized by variable unwanted behavior depending on the predominant hormone. undesirable behavior and back soreness can also occur with ‘mittelschmerz’. mares that exhibit pain at the time of ovulation can be painful on palpation of the ovary, painful when touching their flanks, or painful while riding. these mares may show resistance if saddling occurs near the time of ovulation. mares may require serial reproductive examinations to determine the frequency of the behavior and the relationship to the estrous cycle. if the behavior is deemed not related to the estrous cycle, it is important to systematically evaluate other organ systems to determine other medical problems that may be occurring. musculoskeletal conditions such as joint and/or back pain should be ruled out as a primary cause of poor performance. gastrointestinal abnormalities such as enteroliths or gastric ulcers must be considered as a cause for intermittent pain. while urogenital conditions such as uroliths, vaginitis, and pneumovagina can be confused as causes of estrus associated behaviors due to increased frequency of urination or vulvar winking, these conditions can be ruled out during a thorough urogenital examination. treatment for estrus suppression can be pursued once other causes of poor performance and undesirable behavior are ruled out and it is determined the mare is free from ovarian pathology. it is 585 clinical theriogenology • volume 9 number 4 • december 2017 � important to emphasize to owners that estrus behavior is a normal behavior, some mares exhibit it more intensely than others and that there are several treatment options available to suppress estrus behavior with varying efficacy. depending on the owner’s goals mares with overt estrus behavior may need to be treated either for a prolonged period of time, or only short-term (i.e. to get through a show). some treatment options are superior to others, with each having associated advantages and disadvantages. not all treatments may be suitable for all mares given that behavioral problems may occur at different stages of the estrous cycle. progestogen therapy progesterone has a strong negative feedback effect on lh secretion by the adenohypophysis9,10 and can be implemented throughout the year as a therapy for estrus suppression. exogenous progestogens can be administered to bind to and activate progesterone receptors thereby suppressing estrus behavior. plasma progesterone concentrations greater than 1 ng/ml need to be maintained to block estrus behavior.11 progesterone has a stronger negative impact on lh secretion than it does on follicle stimulating hormone (fsh) secretion, therefore, follicular activity is present due to incomplete fsh suppression, and ovulations are still possible.12 mares that demonstrate misbehavior associated with ‘mittelschmerz’ pain may not benefit from exogenous progestogens as ovulation, and thus ovulatory pain, is still possible. in humans, progesterone has a tranquilizing effect on the central nervous system,13 thus it can be argued that progestogen administration may not be ideal for high performance athlete mares. natural progesterone natural progesterone can be compounded in an intramuscular injectable oil-based form. one hundred milligrams of progesterone per day is sufficient to block estrus behavior in mares.14 anecdotally, this is the quickest acting therapy available and achieves adequate suppression within 1-2 days of administration. a long-acting intramuscular formulation is available that is given every 7-10 days for suppression of estrus behavior by maintaining concentration of serum progesterone greater than 2ng/ml.15 advantages of natural progesterone include its high efficacy at suppression of estrus behavior, its rapid onset, and its capacity to be administered year-round for estrus suppression. disadvantages of natural progesterone include associated injection site swelling and pain which is not ideal for performance mares, the need for multiple injections, and the associated human safety issues due to liposolubility and ease of trans-dermal absorption. altrenogest altrenogest (regumate®; merck animal health; summit, nj) is a progestin considered to be the ‘gold-standard’ therapy for the suppression of unwanted estrus behavior. it has been fda approved for the use in horses and is administered orally once daily. estrus suppression is accomplished within 3 days of treatment initiation. because of its high efficacy, administration of altrenogest and observation for behavioral improvement may be used as a diagnostic trial when it is unclear whether a particular behavior is due to reproductive cyclicity. altrenogest has been recently compounded into an intramuscular injectable.16 there are two formulations available: a 12 day formulation (altrenogest biorelease la150,betpharm, lexington, ky ) and a 30 day formulation (mp500, betpharm, lexington ky). however, these injectable forms are not fda approved and the studies available have only been completed by the compounding drug company.16 mp500 contains 500mg altrenogest encapsulated in lactide-glycolide microparticles and is designed to release approximately 16.6mg altrenogest/day over a 30 day period.16 a similar amount of altrenogest (450mg) is formulated as a long release la150 form which inhibited the onset of estrus for approximately 15 days, indicating a greater availability and more rapid release in the la150 formulation when compared to the mp500 formulation.16 thus, mp500 would be more useful for longer-term suppression of estrus in performance mares, whereas la150 would be suited for shorter suppression (approximately 2 weeks duration).16 586clinical theriogenology • volume 9 number 4 • december 2017 � advantages of oral use of regumate® include its high efficacy, ability to be implemented as therapy year-round, absence of reported long-term adverse consequences on mare fertility and body condition, and oral administration.17 disadvantages of regumate® include the associated human safety issues with ease of trans-dermal absorption, the daily dosing regimen, and the cost of the product. the advantages of formulated, intramuscular altrenogest include its reduced frequency of administration when compared to its oral formulation, its progestogenic effect to successfully block return to estrus, and its ability to be implemented year-round. disadvantages of injectable altrenogest include injection site soreness, cost, and lack of long-term fertility studies. synthetic progestin implants various hormone implants are available for use in other species and have been unsuccessfully used in mares for estrus suppression.18,19 synovex®s (zoetis inc.; kalamazoo, mi) is a bovine growth promotor implant. this implant was developed for use in steers and heifers to promote weight gain and feed efficiency. one subcutaneous implant consists of 8 pellets containing 200mg progesterone and 20mg estradiol, which are slowly absorbed over a period of 100-150 days. assuming a constant rate of degradation, these implants would release 1.3-2.0 mg progesterone daily.20 implanting 1, 4, and 10 devices subcutaneously in mares resulted in return to estrus within the expected timeframe as determined by behavior when teased to a stallion; accordingly, serum progesterone concentrations returned to <0.5ng/ml following cessation of the natural luteal phase.18 in conclusion, the synovex®s implant does not provide the minimum amount of progesterone required to prevent estrus behavior in the mare and therefore it is not an efficacious therapy for estrus suppression in mares. other injectable progestins numerous injectable progestins, including synthetic progestins, formulated for human use have been administered off-label to mares in an attempt to suppress estrus behavior. the perceived advantage of these preparations is that they may be administered periodically instead of daily. however, use of these products does not ensure that the compound will bind the mare’s progesterone receptors and result in suppression of estrus behavior. the most common progestins available are the human contraceptive medroxyprogesterone acetate (depo-provera® pfizer injectables; new york, ny), hydroxyprogesterone caproate (makena®, amag pharmaceuticals, inc; waltham, ma), and megestrol acetate (mga 200 premix®, zoetis; parsippany, nj). all three of these progestins have been examined with regard to their ability to maintain pregnancy in the mare as an indicator of maintaining adequate blood progesterone concentrations.21 all three failed to maintain early pregnancy when administered daily beginning on day 16 of gestation following prostaglandin administration on day 18.21 medroxyprogesterone acetate is the only one that has been studied in regards to its ability to suppress estrus behavior in mares and failed to do so.22 the above studies support the notion that none of these progestins can suppress estrus behavior in mares, likely due to their inability to bind to the equine progesterone receptors.21 however, many owners and veterinarians continue to use these products with the perception that they are effective. this further indicates that many perceived misbehaviors are likely unrelated to reproductive hormones or cyclicity. currently, the only effective synthetic progestogen in mares is altrenogest. prolongation of the luteal phase extending the function of the cl will prolong the luteal phase (diestrus) and the production of endogenous progesterone. because estrus suppression methods based on this mechanism of action relay on ovulation and subsequent cl formation, they can only be used in cycling mares. pregnancy and pseudopregnancy prior to pharmacological methods of estrus suppression becoming available, pregnancy was used as a method for suppressing estrus behavior. the gestation length in a mare is quite variable and can range from 320-380 days in length. during the early period, the primary cl is responsible for endogenous progesterone production and subsequent pregnancy maintenance and suppression of estrus 587 clinical theriogenology • volume 9 number 4 • december 2017 � behavior. by day 35, the endometrial cups begin forming and produce equine chorionic gonadotrophin (ecg) which is responsible for luteinization of ovarian follicles. equine chorionic gonadotropin is continually secreted and progesterone reaches maximal concentrations at 90-120 days gestation, when the placenta begins to maintain pregnancy via placental progestogen production. pregnancy is currently not recommended as a method of estrus suppression. in addition, an important disadvantage to pregnancy is the regulations by differing sports governing bodies regarding the stage of pregnancy during which a mare can compete. pseudopregnancy, or false pregnancy, is accomplished when an embryo is reduced after the period of maternal recognition of pregnancy. embryos can be reduced via the trans-rectal pinching technique after day 16. manual reduction of embryos between day 16-22 resulted in prolonged interestrus interval (mean 82 days) in ten of eleven mares.23 this method is effective at suppressing estrus behavior due to maintenance of endogenous progesterone production for at least 60 days.23 however, this method may present ethical issues by termination of a viable pregnancy and cost accompanying breeding and cycle management. similar to the above recommendations pertaining to pregnancy and estrus suppression, this is also not a currently recommended method to suppress estrus. intrauterine marble the intrauterine marble is a well-known method for attempting estrus suppression among clients and veterinarians. however, its efficacy is questionable. the use of an intrauterine marble for the suppression of estrus was first reported in the netherlands with a success rate of 75%,24 and its presence was thought to mimic a conceptus or induce a low-grade endometrial inflammation, resulting in prostaglandin depletion and subsequent prevention of luteolysis.24 a 25or 35-mm marble was placed in the uterine lumen during early diestrus (within 24 hours of ovulation) and resulted in prolonged luteal function up to 90 days; however, the reported success rate of 75% did not account for the 6 of 24 mares that expelled the marble and included mares who did not respond for multiple cycles. if the 24 mares that started the study are included, only 4/24 (16%) responded on their first cycle. this is very similar to the reported incidence of spontaneous persistent cl (13%) formation from that same study.24 however, intrauterine placement of a 20mm water filled polypropylene ball inserted 2 to 4 days after ovulation did not produce evidence of continuous prostaglandin release.25 t hey reported that 9 of 12 mares (75%) had a prolonged luteal phase (44-75 days) which is a different efficacy than that which had been previously reported. the advantages to the intrauterine marble technique are its ease of administration and relatively low cost, leading to its widespread use despite varying degrees of efficacy. the biggest disadvantage to using the intrauterine marble technique is that there is a high probability of failure with the potential for marble expulsion in a majority of mares. in addition, there are well-documented cases of diffuse endometrial damage induced from glass shards embedded into the endometrium, in addition to cases of pyometra.26 therefore intrauterine marbles have been shown to be unreliable by a number of studies, with numerous significant complications reported. there are other superior methods available to suppress estrus. exogenous oxytocin serial oxytocin administration is a practical and efficacious method of prolonging cl function in the mare. administration of 60 units of oxytocin in the muscle twice daily on days 7 to 14 prolonged cl function in 6 out of 6 mares through day 30.27 it was later reported that 60 units of oxytocin in the muscle once daily on days 7-14 was equally effective.28 a lower dose of 10 units of oxytocin in the muscle or intravenously once daily on days 7 to 14 was unsuccessful.29 a later study found the effectiveness of suppressing estrus behavior following administration of 60 units of oxytocin in the muscle once daily on day 7 to 14 of the mare’s cycle.30 this study concluded that cl function was prolonged for about 2 months in 2 of 3 treated mares with weak expression of estrus behavior noted.30 administration of 60 units of oxytocin in the muscle once daily is effective at prolonging cl function in 60-70% of treated mares when administered on day 7-14. this is the most common protocol implemented clinically.28 the advantages associated with this method of estrus suppression include its 588clinical theriogenology • volume 9 number 4 • december 2017 � efficacy, low cost, and reversibility with administration of exogenous prostaglandin. the disadvantages of this method include the requirement of reproductive cyclicity, multiple injections, and cycle management to accurately identify the day of ovulation. if the owner wants to forgo cycle management costs, one option is to initiate treatment with 60 units of oxytocin in the muscle once daily for 29 consecutive days.31 this option resulted in prolonged cl function in over 70% of treated mares, and cost to the owner is minimal.31 with once daily or twice daily treatments of 60 units of oxytocin, there were no adverse effects or reactions observed in any of the mares.31 diestrual ovulation in contrast to other species, mares can ovulate during diestrus in the presence of high circulating concentrations of progesterone. the frequency of diestrus ovulations varies according to breed, with thoroughbreds and quarter horses having as many as 20% of ovulations occurring during diestrus, while the frequency is very low in ponies32,33 diestrual ovulations that occur 1-4 days prior to luteolysis may cause a prolonged luteal phase, as the immature cl does not undergo luteolysis in the face of endogenous endometrial prostaglandin production. induction of a diestrual ovulation using 3000 iu of human chorionic gonadotropin (hcg) intravenously successfully resulted in a prolonged luteal phase (2-3 months) in 4 of 5 treated mares that developed a follicle >30mm in size.34 however, not all mares developed a large enough follicle to induce ovulation on their first cycle; 2 of five mares developed inducible follicles on their first cycle (both responded to hcg), one mare developed an inducible diestrual follicle on her second cycle (responded to hcg), one mare developed one inducible diestrual follicle on her third cycle (did not respond to hcg), and one mare never developed an inducible diestrual follicle.34 in that same study, 1 of 4 control, saline-treated mares had a spontaneous diestrual ovulation and developed a prolonged luteal phase.34 advantages of this option for treatment of estrus suppression include its effectiveness if conditions are met (30mm late diestrual follicle that responds to hcg treatment). however, a major disadvantage is that only a small percentage of mares will be presented with a diestrual follicle large enough to respond to treatment and they may or may not respond to hcg treatment. another limitation that hinders its use in practice includes the necessity of multiple reproductive tract ultrasonograms to identify an appropriately-sized diestrual follicle capable of responding to an ovulation induction agent. intrauterine plant oil in 2011 it was reported that the intrauterine infusion of 1ml of either coconut or peanut oil on day 10 after ovulation prolonged the luteal phase in 11 of 12 treated mares as assessed by concentrations of progesterone above baseline for 30 days.35 estrus behavior was not documented. a follow-up study found that when 1ml fractionated coconut oil was infused into the uterus on day 10 in 12 mares, all 12 mares returned to estrus as expected with a normal decline in progesterone at the time of luteolysis.36 based on the conflicting data, more work is needed to determine whether intrauterine plant oil administration is an effective method for estrus suppression in mares and whether there are any associated side effects with its administration. if effective, it would require some cycle management to identify the day of ovulation with resultant prolongation of endogenous progesterone production. as with other methods of prolongation of luteal function, reproductive cyclicity must be present. suppression of ovarian follicular activity suppressing ovarian function is done by targeting and subduing the hypothalamicadenohypophyseal-ovarian axis. by down-regulating the hypothalamic production of gnrh, there is a subsequent reduction in pituitary gonadotropin production and a resultant suppression of ovarian activity. by employing these methods the reduction in follicular growth and subsequent lack of ovulation result in a decrease in endogenous estrogen production and no endogenous progesterone production. typically mares that undergo these therapies exhibit behavior similar to their anestrus behavior; therefore, it may not suppress or solve behavior problems if mares are reported to exhibit estrus behavior in the winter 589 clinical theriogenology • volume 9 number 4 • december 2017 � months. the therapies listed below may be a useful test of the likelihood of the efficacy of ovariectomy for behavioral modification. gnrh agonist when a gnrh agonist is administered, it is postulated that the adenohypophysis becomes hyperstimulated and eventually desensitized.37 with desensitization, there is a reduction in gonadotropin release and a subsequent lack of ovulation (due to reduced lh) and reduction in follicular growth (due to reduced fsh). ovuplant™ (peptech animal health pty limited; north ryde nsw 2113, australia) is a deslorelin-containing, slow-release implant that functions to hasten ovulation in mares. field reports indicate that ovuplant™ is associated with a delayed return to estrus in mares following implantation,38 with a prolonged interovulatory interval when compared to induction of ovulation with intravenous hcg.39 in addition, administration of prostaglandin to induce luteolysis following ovuplant™ resulted in a prolonged interovulatory period compared to mares without the implant by an average of 6.2 days.38,40,41 as expected, plasma concentration of lh and fsh were lower in treated compared to control mares.38 however, removal of the deslorelin implant within 48 hours after administration resulted in normal interovulatory intervals and fsh secretion.40 when ovuplant™ is used with the goal of estrus suppression, the implant is left in place in order to maintain a decrease in gonadotropin production with a resultant suppression of ovarian activity. although this option offers ease of administration, the duration of estrus suppression is short-lived (until follicular growth resumes) and dose dependent. this method does hold an increased cost associated with cycle monitoring, unpredictable return to estrus, and problems with availability of the implant (not available in the usa). gnrh antagonist gonadotropin releasing hormone antagonists act by inhibiting the action of gnrh and thereby inhibiting gonadotrophin secretion and subsequent ovarian activity. the advantage of a gnrh antagonist over gnrh agonists in that they have a more rapid onset.42 for this reason, administration of gnrh antagonists is more frequent that their agonist counterparts.42 antarelix™ ([n-ac-d-nal(2)‘, d-pclphe*, d-pa1(3)3, d-(hci)6, lys(ipr)*, d-ala”] gnrh; 100 pg/kg in 10 ml of 5% mannitol, europeptides, argenteuil, france) is a gnrh antagonist that has been shown to postpone ovulation in mares, thereby increasing the interovulatory interval.43 when a single subcutaneous dose of antarelix™ (100ug/kg) was administered to mares on day 8 following ovulation, the estrous cycles of treated mares were significantly longer than control mares (33.5±3.8 days vs. 24.0±1.1 days).44 the prolongation was attributed to a delay in the development of a large preovulatory follicle due to a reduction in fsh and lh concentrations.43 although ovarian activity can be suppressed, behavioral estrus can be inconsistent with this treatment; in addition, frequent administration, short-term effect, and highly variable responses among mares also reduce the indications for clinical application of this treatment option. gnrh vaccine vaccination with a gnrh carrier protein and adjuvant can induce an immune response to a gnrh protein, resulting in neutralization of the deca-peptide via production of anti-gnrh antibodies. by neutralizing gnrh, there is a decrease in gonadotropin production and a subsequent reduction in ovarian activity. mare response, as indicated by antibody titers to gnrh immunization, is variable and estrusrelated behavior may correlate with the achieved level of ovarian suppression.44 there are several gnrh vaccines available on the market. for instance, equity® (zoetis australia pty ltd.; rhodes nsw, australia) is perhaps the most widely applied. it requires two doses administered four weeks apart and is labeled for use in australia for estrus suppression in mares and fillies not intended for breeding. in a study done by pfizer animal health, all treated mares exhibited increased gnrh antibody titers, reduction in ovarian size, decreased folliculogenesis, and anestrus-like behavior (average of 3 months duration) within two weeks of the second immunization.45 the length of ovarian shutdown following the second immunization varied from 4-23 weeks in 10 of 16 vaccinated mares whereas the remaining 6 vaccinated mares did not return to 590clinical theriogenology • volume 9 number 4 • december 2017 � cyclicity for the remainder of the trial which was 14-18 weeks from the onset of initial injection.45 a second retrospective study identified abnormal prolonged acyclicity for 1-3 stud seasons in retired thoroughbred racing mares that all had an associated history of equity® administration.46 improvac® (pfizer laboratories ltd; sandton, south africa) is another gnrh vaccine available and is labeled for immunocastration to control boar taint in meat. improvac® may be used off-label to suppress cyclicity in mares with resumption of ovarian function and expression of estrus behavior being highly variable.47 all mares in this study had suppressed cyclicity within 8 weeks of the first vaccination and cyclicity remained suppressed for a minimum of 23 weeks.47 improvac® is not labeled for use in animals intended for breeding due to the potential for extended ovarian suppression. vaccination against gnrh is successful and effective at suppressing ovarian function and may or may not be effective at estrus behavior suppression. the duration of ovarian suppression and potential estrus behavior suppression is variable and unpredictable. thus, due to the potential of ovarian suppression of unknown length it is not recommended for use in potential breeding mares. injection site reactions have been reported with all gnrh vaccines with variably severity from slightly raised flat swelling at the site of injection to systemic pyrexia the day after injection.44,45,47 gonadotropin releasing hormone vaccines are not currently available in the united states. porcine zona pellucida vaccine the porcine zona pellucida vaccine (pzp) vaccine is derived from porcine zona pellucida protein 3 (pzp3). this binding site on the zona pellucida is similar across domestic species. the pzp3 binding site also happens to be the same binding site for a spermatozoon, therefore, this vaccine was developed as a contraceptive to prevent sperm binding and subsequent acrosome reaction and fertilization. recently, the effect of the vaccine on ovarian function and estrous cyclicity was discovered.48 five out of 6 immunized mares showed intermittent to sustained evidence of ovarian suppression for greater than 5 weeks following the second immunization. ovarian volume, follicle counts, maximum follicle diameter, and pregnancy rate in treated mares were significantly lower than control mares.48 this study highlights the suppressive effect of the pzp vaccine on a majority of mares is consistent with previous studies,49 however, behavior was not evaluated. although this may be a potential therapy for estrus suppression via ovarian suppression, future research is required to investigate resulting behaviors and potential adverse side effects of vaccination.49 bilateral ovariectomy bilateral ovariectomy is the most effective therapy for behavioral modification in mares who exhibit adverse behavior during diestrus or mares that exhibit adverse behavior due to ‘mittelschmerz’ as bilateral ovariectomy will result in abolishment of luteal potential and abolishment of ovulation and its associated discomfort. bilateral ovariectomy may also be effective in mares that show estrus behavior problems only during the ovulatory season and do not exhibit estrus behavior problems during anestrus. however, bilateral ovariectomy may not be effective in those mares that exhibit overt estrus behavior during the ovulatory season and also overt estrus behavior during anestrus as bilateral ovariectomy will result in the same endogenous hormonal environment as is present during anestrus or even times of ovarian suppression. therefore, when considering ovariectomy as a treatment option for overt estrus behavior, it would be useful to observe response to therapy aimed at suppression of ovarian activity (see above) or anestrus behavior. in general, client satisfaction with bilateral ovariectomy is relatively high with 78% (18/23) of owners being satisfied or very satisfied with the procedure.50 veterinarians must keep in mind that an ovariectomy may be classified as successful by an owner even if a behavior was not reproductively related. the advantage of this treatment option is that potential long-term success. however, due to mare variability, bilateral ovariectomy may not be effective in eliminating unwanted behavior. additionally, ovariectomy poses surgical risk as well as irreversible loss of breeding potential. case selection is therefore extremely important in considering this option. 591 clinical theriogenology • volume 9 number 4 • december 2017 � alternative therapies auricular acupuncture acupuncture is a form of alternative medicine that is becoming more commonplace in the equine industry. anecdotal reports suggest application of an ear staple as a method of suppressing estrus. the premise behind the ear staple is that it stimulates the auricular vagal branches which results in an increase in parasympathetic nervous system tone.51 this results in pineal melatonin release and consequent decreased production of gnrh from the hypothalamus. the hypothalamic-adenohypophyseal-ovarian axis becomes quiescent, similar to anestrus. however, there is no empirical evidence supporting this theory; thus, it is not recommended as a valid treatment option for estrus suppression in the mare. herbal remedies mare magic (mare magic; shelton, wa) is an herbal supplement marketed “to help influence a quiet disposition in mares and geldings”. its active ingredient is dried raspberry leaf which has been implemented in treating morning sickness, preventing miscarriage, and shortening and easing labor in women.52 raspberry leaf tablets are touted as being safe for pregnant women and may be beneficial for women during parturition by causing spontaneous rupture of the membranes and shortening labor duration.53 however, its effect in horses regarding pregnancy, parturition, or estrus suppression etc. has not been empirically assessed. there is little to no evidence supporting its application. conclusions several therapies for estrus suppression are available with variable efficacy and advantages and disadvantages. any time a mare is presented for undesirable behavior related to the estrous cycle or reproductive cyclicity, it is important to ensure that the behavior problem(s) are not related to medical or training issues. once a diagnosis is made, treatment selection should be based on the time of year the behavior occurs, specific behaviors observed, the duration of suppression needed, and the cost associated with each treatment implementation. by far, the most reliable way to suppress estrus behavior any time of the year is administration of progestogens—compounded natural progesterone or altrenogest. however, oxytocin injections during the ovulatory season are found to be effective, low cost, and an excellent alternative to administration of progestogens. references 1. ginther oj. reproductive biology of the mare: basic and applied aspects. 2nd ed. cross plains: equiservices; 1992. p. 107-108. 2. king ss, nequin lg, drake s, et al: progesterone levels correlate with impending anestrus in the mare. j equine vet sci 1988;8:109-111. 3. ginther oj: folliculogenesis during the transitional period and early ovulatory season in mares. j reprod fertil 1990;90:311-320. 4. schauer sn, briant c, ottogalli m, et al: supplementation of equine early spring transitional follicles with luteinizing hormone stimulates follicle growth but does not restore steroidogenic activity. theriogenology 2011;75:1076-1084. 5. hart pj, squires el, imel kj, et al: seasonal-variation in hypothalamic content of gonadotropin-releasing hormone (gnrh), pituitary receptors for gnrh, and pituitary content of luteinizing-hormone and follicle-stimulating-hormone in the mare. biol reprod 1984;30:1055-1062. 6. mccue pm, squires el: persistent anovulatory follicles in the mare. theriogenology 2002;58:541-543. 7. asa cs, goldfoot da, garcia mc, et al: dexamethasone suppression of sexual-behavior in the ovariectomized mare. horm behav 1980;14:55-64. 8. hinrichs k: irregularities of the estrous cycle and ovulation in mares (including seasonal transition). in: youngquist rs, editor. current therapy in large animal theriogenology. philadelphia: saunders; 1997. p. 153. 9. ginther oj, utt md, bergfelt dr,et al: controlling interrelationships of progesterone/lh and estradiol/lh in mares. anim reprod sci 2006;95:144-150. 10. gastal el, bergfelt dr, nogueira gp, et al: role of luteinizing hormone in follicle deviation based on manipulating progesterone concentrations in mares. biol reprod 1999;61:1492-1498. 11. hawkins dl, neely dp, stabenfeldt gh: plasma progesterone concentrations derived from the administration of exogenous progesterone to ovariectomized mares. j reprod fertil suppl 1979:211-216. 12. daels pf, mccue pm, demoraes mj, et al: persistence of the luteal phase following ovulation during altrenogest treatment in mares. theriogenology 1996;46:799-811. 592clinical theriogenology • volume 9 number 4 • december 2017 � 13. schumacher m, hussain r, gago n, et al: progesterone synthesis in the nervous system: implications for myelination and myelin repair. front neurosci-switz 2012;6:1-22 14. loy rg, swan sm: effects of exogenous progestogens on reproductive phenomena in mares. j anim sci 1966;25:821826 15. vanderwall dk, marquardt jl, woods gl: use of a compounded long-acting progesterone formulation for equine pregnancy maintenance. j equine vet sci 2007;27:62-66. 16. storer wa, thompson dl, gilley rm, et al: evaluation of injectable sustained release progestin formulations for suppression of estrus and ovulation in mares. j equine vet sci 2009;29:33-36. 17. hodgson d, howe s, jeffcott l, et al: effect of prolonged use of altrenogest on behaviour in mares. vet j 2005;169:321-325. 18. mccue pm, lemons ss, squires el, et al: efficacy of synovex-s(r) implants in suppression of estrus in the mare. j equine vet sci 1997;17:327-329. 19. dujovne ga, johnson ak, wilborn rr, et al: use of etonogestrel implants to suppress estrous behavior in mares. clin therio 2015;7:35-44. 20. mccue pm: estrus suppression in performance horses. j equine vet sci 2003;23:342-344. 21. mckinnon ao, lescun tb, walker jh, et al: the inability of some synthetic progestagens to maintain pregnancy in the mare. equine vet j 2000;32:83-85. 22. gee ek, deluca c, stylski jl, et al: efficacy of medroxyprogesterone acetate in suppression of estrus in cycling mares. j equine vet sci 2009;29:140-145. 23. lefranc ac, allen wr: nonpharmacological suppression of oestrus in the mare. equine vet j 2004;36:183-185. 24. nie gj, johnson ke, braden td, et al: use of an intra-uterine glass ball protocol to extend luteal function in mares. j equine vet sci 2003;23:266-273. 25. del alamo mmr, reilas t, kindahl h, et al: mechanisms behind intrauterine device-induced luteal persistence in mares. anim reprod sci 2008;107:94-106. 26. turner rm, vanderwall dk, stawicki r: complications associated with the presence of two intrauterine glass balls used for oestrus suppression in a mare. equine vet educ 2015;27:340-343. 27. vanderwall dk, rasmussen dm, woods gl: effect of repeated administration of oxytocin during diestrus on duration of function of corpora lutea in mares. j am vet med assoc 2007;231:1864-1867. 28. vanderwall dk, rasmussen dm, carnahan kg, et al: effect of administration of oxytocin during diestrus on corpus luteum function and endometrial oxytocin receptor concentration in cycling mares. j equine vet sci 2012;32:536-541. 29. gee ek, gillespie l, bolwell cf: effect of oxytocin on suppression of oestrus in mares exhibiting normal oestrous cycles. nz vet j 2012;60:189-193. 30. vanderwall dk, agnew me, schnobrich mr et al: effect of administration of oxytocin during diestrus on the duration of corpus luteum function and estrous behavior in cycling mares. clin therio 2012;4:400. 31. vanderwall dk, parkinson kc, rigas j: how to use oxytocin treatment to prolong corpus luteum function for suppressing estrus in mares. j equine vet sci 2016;36:1-4. 32. hughes jp, couto ma, stabenfeldt gh: luteal phase ovulations: what are the options? proc annu meet soc therio 1985; 123-125. 33. wesson ja, ginther oj: influence of season and age on reproductive activity in pony mares on the basis of a slaughterhouse survey. j anim sci 1981;52:119-129. 34. hedberg y, dalin am, santesson m, et al: a preliminary study on the induction of dioestrous ovulation in the mare--a possible method for inducing prolonged luteal phase. acta vet scand 2006;48:12. 35. wilsher s, allen wr: intrauterine administration of plant oils inhibits luteolysis in the mare. equine vet j 2011;43:99105. 36. de amorim md, nielsen k, cruz rks, et al: progesterone levels and days to luteolysis in mares treated with intrauterine fractionated coconut oil. theriogenology 2016;86:545-550. 37. reissmann t, felberbaum r, diedrich k et al: development and applications of luteinizing hormone-releasing hormone antagonists in the treatment of infertility-an overview. hum reprod 1995; 10; 1974-1981 38. johnson ca, thompson dl, kulinski km, et al: prolonged interovulatory interval and hormonal changes in mares following the use of ovuplant (tm) to hasten ovulation. j equine vet sci 2000;20:331-366. 39. morehead ja, blanchard tl: clinical experience with deslorelin (ovuplant (tm)) in a kentucky thoroughbred broodmare practice (1999). j equine vet sci 2000;20:358-362. 40. farquhar vj, mccue pm, carnevale em, et al: deslorelin acetate (ovuplant) therapy in cycling mares: effect of implant removal on fsh secretion and ovarian function. equine vet j 2002;34:417-420. 41. mccue pm, farquhar vj, carnevale em, et al: removal of deslorelin (ovuplant) implant 48 h after administration results in normal interovulatory intervals in mares. theriogenology 2002;58:865-870. 42. stout tae, colenbrander b: suppressing reproductive activity in horses using gnrh vaccines, antagonists or agonists. anim reprod sci 2004;82-3:633-643. 43. watson ed, pedersen hg, thomson srm, et al: control of follicular development and luteal function in the mare: effects of a gnrh antagonist. theriogenology 2000;54:599-609. 44. dalin am, andresen o, malmgren l: immunization against gnrh in mature mares: antibody titres, ovarian function, hormonal levels and oestrous behaviour. j vet med a 2002;49:125-131. 593 clinical theriogenology • volume 9 number 4 • december 2017 � 45. elhay m, newbold a, britton a, et al: suppression of behavioural and physiological oestrus in the mare by vaccination against gnrh. aust vet j 2007;85:39-45. 46. robinson sj, mckinnon ao. prolonged ovarian inactivity in broodmares temporally associated with administration of equity. aust eq vet 2006; 25: 85-87 47 imboden i, janett f, burger d, et al: influence of immunization against gnrh on reproductive cyclicity and estrous behavior in the mare. theriogenology 2006;66:1866-1875. 48. tarr cj, bertschinger hj, fosgate gt et al: ovarian function in pony mares undergoing porcine zona pellucide immunocontraception. clin therio 2015;7:308. 49. bechert u, bartell j, kutzler m, et al: effects of two porcine zona pellucida immunocontraceptive vaccines on ovarian activity in horses. j wildlife manage 2013;77:1386-1400. 50. kamm jl, hendrickson da: clients' perspectives on the effects of laparoscopic ovariectomy on equine behavior and medical problems. j equine vet sci 2007;27:435-438. 51. rong pj, zhao jj, li yq, et al: auricular acupuncture and biomedical research-a promising sino-austrian research cooperation. chin j integr med 2015;21:887-894. 52. mcfarlin bl, gibson mh, o'rear j, et al: a national survey of herbal preparation use by nurse-midwives for labor stimulation. review of the literature and recommendations for practice. j nurse midwifery 1999;44 :205-216. 53. simpson m, parsons m, greenwood j, et al: raspberry leaf in pregnancy: its safety and efficacy in labor. j midwifery womens health 2001;46:51-59. 594clinical theriogenology • volume 9 number 4 • december 2017 2011: effect of presynchronization with gnrh 7 days prior to resynchronization with co-synch on pregnancy wastage in lactating dairy cows effect of presynchronization with gnrh 7 days prior to resynchronization with co-synch on pregnancy wastage in lactating dairy cows a. alkar,a a. tibary,a j.r. wenz,a r. nebel,b r. kasimanickama adepartment of veterinary clinical sciences, washington state university, pullman, wa; bselect sires inc., plain city, oh abstract increased secretion of prostaglandin f2α (pgf) between d 30 and 36 of pregnancy causes luteal regression and embryonic loss. in addition, administration of gonadotropin releasing hormone (gnrh) or human chorionic gonadotropin (hcg) has been held responsible for early embryonic death when administered at pregnancy diagnosis between 29 and 42 days after insemination. the objective of this study was to determine the effect of gnrh administration at 28±3 d after artificial insemination (ai), 7 d prior to the initiation of resynchronization with co-synch, on pregnancy wastage rate (late embryonic and early fetal loss rate; pwr) for the previous insemination. all parity (2.38±1.45; range 1-10) lactating holstein cows (n=1417) from a dairy farm in washington state were enrolled. cows that were not detected in estrus by 28±3 d (day -7) after ai were assigned to receive either gnrh (100 μg, im; n=693) or no gnrh (control; n=724). cows not detected in estrus during 7 d following gnrh administration were presented for pregnancy diagnosis (35±3 d after ai, day 0) and non-pregnant cows were submitted for resynchronization with co-synch protocol for subsequent timed ai. the cows that were not pregnant to resynchronization were resubmitted for co-synch resynchronization with presynch-gnrh for a second time or to other breeding protocols. cows were examined for pregnancy at 35 d after ai and pregnant cows were submitted to determine pregnancy status for a second time approximately at 60 d after ai. the overall pwr was 13.9% (142/1044). the pwr for gnrh and control groups was 14.8% (75/506) and 12.5% (67/538), respectively (p=0.27). in conclusion, the gnrh treatment 7 d prior to initiation of resynchronization with co-synch, when administered at 28±3 d after a previous breeding, did not increase pregnancy wastage compared to controls. keywords: dairy cows, gnrh, presynchronization, resynchronization, pregnancy wastage introduction spontaneous abortion of dairy cows is an increasingly important problem that contributes substantially to low herd viability and production inefficiency. embryonic mortality for cows confirmed pregnant between 35 to 45 days of gestation typically has ranged from 8 to 10%, with pregnancy loss often exceeding 14% in some herds.1,2 sreenan et al calculated that pregnancy rates of 90% and calving rates of 55% are normal for heifers and moderate yielding dairy cows, indicating an overall embryonic and fetal mortality rate of about 40%.3 these authors concluded that few embryos are lost in the days immediately after fertilization and up to day 8 after ovulation and that about 70 to 80% of the total embryonic loss is sustained between days 8 and 16 after insemination, a further 10% between days 16 and 42 and a further 5 to 8% between day 42 and term. it is suggested that the rate of late embryonic loss, after the fourth week of gestation, may be higher in high-producing dairy cows than in either moderateyielding cows or in heifers. a study by vasconcelos et al indicated that the pattern of loss might be different in high-yielding dairy cows with such cows showing a higher increment of late embryonic loss.4,5 vasconcelos et al reported that 20.2% of the embryos were lost between days 28 and 98 after ai in intensively managed dairy cows yielding between 11,000 and 12,000 kg of milk per lactation.4,5 while infectious diseases are a primary focus of pregnancy wastage prevention, infectious agents probably cause less than half of the pregnancy wastages.6 schallenberger et al reported that a series of increases in secretion of pgf between d 30 and 36 of pregnancy caused luteal regression and embryonic loss.7 in addition, among many non-infectious causes, administration of gnrh or hcg after ai has been held responsible for early embryonic death when administered at pregnancy diagnosis between 29 and 42 clinical theriogenology • volume 3 number 4 • december 2011467 days after insemination.8 the objective of this study was to determine the effect of gnrh administration at 28±3 d after ai, 7 d prior to the initiation of resynchronization with co-synch, on pwr for the previous insemination. materials and methods animal enrollment and data collection all parity (2.38±1.45; range 1-10) lactating holstein cows (n=1417) from a dairy farm in washington were enrolled from december 2009 to july, 2010. cows were housed in free-stall barns; primiparous and multiparous cows were housed separately and milked thrice daily at 8 h intervals. cows were fed a total mixed ration, twice daily, to meet or exceed the dietary requirements for lactating holstein cows weighing 1200 to 1400 lb and producing 60 to 80 lbs of 3.5% fat corrected milk. lists of all eligible cows including injection schedules, reproductive events, pregnancy examinations, health events, and milk yield data on the test date closest to the date of subsequent insemination were generated, tracked, and recorded using a commercial on-farm computer software programs (dairycomp 305, valley agricultural software, tulare, ca or dhi plus, dhi computing service, provo, ut). treatment cows that were not detected in estrus by 28±3 d (day -7) after ai were assigned to receive either gnrh (100 μg, im; n=693 or no gnrh (control; n=724). cows not detected in estrus during 7 d following gnrh administration were presented for pregnancy diagnosis (35±3 d after ai, day 0) and non-pregnant cows were submitted for resynchronization with co-synch protocol for subsequent breeding. briefly, cows received 100 μg gnrh, im, on day 0 (35±3), 25 mg pgf on day 7 (42±3), and 100 μg gnrh, im, and insemination 72 h later on day 10 (45±3). the protocol was repeated for the cows that were not pregnant to resynchronization. it should be noted that some non-pregnant cows were submitted to other breeding protocols. pregnant cows were submitted for pregnancy diagnosis for a second time at approximately 60 d after ai. the pregnancy wastage was determined by cows that were diagnosed as pregnant at 35 days after insemination and subsequently observed to be non-pregnant at 60 d after ai. the pwr was calculated as number of cows pregnant 35 days after insemination and not pregnant at 60 days after insemination divided by total number of cows pregnant at 35 days after insemination. statistical analysis multivariate logistic model (proc logistic, sas version 9.1 for windows, sas institute, cary, nc) was used to examine the effect of treatments on pwr. the variables included in the model were treatment (gnrh vs. no gnrh), parity (1, 2 and 2+), and appropriate interaction. the ‘p’ value at 0.05 was considered significant. results accounting for parity (p<0.001), the pwr between gnrh and control groups was not different (p>0.1). the pregnancy/ai (p/ai) at 35 days after insemination was 66.0% (935/1417). the pwr at 60 days after insemination was 13.8% (129/935) for the previous breeding. the pwr was not different between gnrh and control groups (gnrh: 15.2% [69/453] vs. control: 12.4% [60/482]; p=0.36). there were 482 cows (gnrh: n=217; control: n=265) that were submitted for a second breeding. out of those, 293 cows were eligible for resynchronized timed ai. the p/ai for resynchronized timed artificial insemination was 37.2% (109/293). the pwr was 11.9% (13/109) for resynchronized timed artificial insemination. the pwr for resynchronized breeding was not different between gnrh and control groups (gnrh: 11.3% [6/53] vs. control: 12.5% [7/56]; p=0.29). the overall pwr was 13.9% (142/1044). the pwr for gnrh and control groups was 14.8% (75/506) and 12.5% (67/538), respectively (p=0.27). no parity by treatment interaction (p>0.1) was recorded. clinical theriogenology • volume 3 number 4 • december 2011 468 discussion the reason for the administration of gnrh prior to the initiation of resynchronization is to improve the synchrony of follicular wave emergence and thereby conception rates. in addition, it may reduce pregnancy wastage by inducing accessory corpora lutea and increasing progesterone concentrations in pregnant cows. however, administration of gnrh has been held responsible for early embryonic death when administered after insemination. chebel et al demonstrated that initiation of the ovsynch/tai protocol with the administration of 100 µg of gnrh prior to pregnancy diagnosis did not affect pre-enrollment pregnancy rates and pregnancy loss during the first 42 days of gestation.9 stevenson et al administered hcg, gnrh or saline at pregnancy diagnosis between 29 and 42 days after insemination and observed the pregnancy status for four weeks.8 they observed 15.4% total pregnancy losses. the pregnancy loss varied from 11.3% in controls to 22.3% in hcg-treated females. however, no significant treatment effect on pregnancy loss was detected. moreira et al studied the effect of bovine somatotropin and resynchronization with gnrh on pregnancy rates of lactating dairy cows.10 the authors suggested that resynchronization of cows with initiation of the ovsynch protocol on day 20 after the initial ai decreases embryo survival to the prior ai. treatment with gnrh is followed by a rapid secretion of luteinizing hormone, and is also associated with a transitory increase in plasma estradiol.11 these events may stimulate the secretion of pgf, which could result in luteolysis and terminate pregnancy. thus it appeared reasonable to test the effect of gnrh on pregnancies from previous insemination when administered as a strategy for resynchronization. in this study, administration of gnrh at 28±3 d after ai did not increase pregnancy wastage in lactating dairy cows compared to cows that were not treated. treatment with gnrh during the early embryonic period,12-15 and at pregnancy diagnosis8,16 clearly increases the number of additional corpora lutea. however it has failed to reduce fetal loss in any of the studies performed. similarly, using gnrh on day 21 or 23 of gestation as a resynchronization strategy, was found to have no effect on subsequent pregnancy losses in cows that were pregnant at the time of treatment.17,18 it seems there are inconsistencies in the effect of gnrh administration and embryonic survival.19,20 even though the gnrh administration is able to induce accessory corpora lutea, progesterone concentrations remained similar in cows that developed accessory corpora lutea compared to cows that did not. the reason might possibly be the higher rate of liver catabolism in high-producing animals. thus, gnrh administration is not beneficial in reducing early embryonic loss in those animals. el-zarkouny and stevenson also showed similar results with progesterone-releasing vaginal inserts.21 dairy cows of unknown pregnancy status received a progesterone-releasing insert or no insert from d 13 to d 21 after ai to resynchronize estrus in non-pregnant cows. this treatment had no effect on established pregnancies. in this study, parity affected the pregnancy wastage but there was no treatment by parity interaction. the overall pregnancy wastage rates have been found to be similar in heifers, beef cows and lowto moderate-producing dairy cows; however, the embryo survival rate is lower in high-producing dairy cows. it is unlikely that this is an ageor parity-related phenomenon but it may be due to the direct and indirect effects of milk production. there are several non-infectious factors that may affect early embryonic survival such as genetic causes, parity, plasma concentrations of progesterone after ai, energy balance after calving and at the time of insemination, dry-matter and protein intake, milk production, and twinning.22,23 in order for progesterone supplementation to effectively increase embryo survival, treatment would have to be targeted rather than treatment of all cows.17 the targets should be high-producing cows, cows bred during peak milk production, and in herds with the history of high early embryonic death due to noninfectious causes, and cows with loss of body condition.20,21,24-28 in conclusion, the gnrh treatment 7 d prior to initiation of resynchronization with co-synch, when administered at 28±3 d after a previous breeding, did not increase pwr compared to controls. clinical theriogenology • volume 3 number 4 • december 2011469 acknowledgements the authors thank select sires inc., plain city, oh for partial financial support for this study and extend their gratitude to the participating producer. references 1. forar al, gay jm, hancock dd: the frequency of endemic fetal loss in dairy cattle: a review. theriogenology 1995;43:989-1000. 2. thurmond mc, picanso jp, jameson cm: considerations for use of descriptive epidemiology to investigate fetal loss in dairy cows. j am vet med assoc 1990;197:1305-1312. 3. sreenan jm, diskin mg, morris, dg: embryo survival in cattle, a major limitation to the achievement of high fertility. bsas occasional publication 2001;26:93-104. 4. vasconcelos jlm, silcox rw, lacerda ja, et al: pregnancy rate, pregnancy loss, and response to heat stress after ai at 2 different times from ovulation in dairy cows. biol reprod 1997;56(suppl 1):140 (abstract). 5. vasconcelos jlm, sangsritavong s, tsai sj, et al: acute reduction in serum progesterone concentrations after feed intake in dairy cows. theriogenology 2003;60:795-807. 6. barr bc, anderson ml: infectious diseases causing bovine abortion and fetal loss. vet clin north am food anim pract 1993;9:343-368. 7. schallenberger e, schams d, meyer hhd: sequences of pituitary, ovarian and uterine hormone secretion during the first 5 weeks of pregnancy in dairy cattle. j reprod fertil 1989;37(suppl):269-276. 8. stevenson js, tiffany sm, inskeep ek: maintenance of pregnancy in dairy cattle after treatment with human chorionic gonadotropin or gonadotropin-releasing hormone. j dairy sci 2008;91:3092-3101. 9. chebel rc, santos jep, cerri rla, et al: effect of resynchronization with gnrh on day 21 after artificial insemination on pregnancy rate and pregnancy loss in lactating dairy cows. theriogenology 2003;60:1389-1399. 10. moreira f, risco ca, pires mfa, et al: use of bovine somatotropin in lactating dairy cows receiving timed artificial insemination. j dairy sci 2000;83:1237-1247. 11. stevenson js, phatak ap, rettmer i, et al: postinsemination administration of receptal: follicular dynamics, duration of cycle, hormonal responses, and pregnancy rates. j dairy sci 1993;76:2536-2547. 12. lopez-gatius f, santolaria p, martino a, et al: the effects of gnrh treatment at the time of ai and 12 days later on reproductive performance of high producing dairy cows during the warm season in northeastern spain. theriogenology 2006;65:820-830. 13. sterry ra, welle ml, fricke pm: treatment with gonadotropin-releasing hormone after first timed artificial insemination improves fertility in noncycling lactating dairy cows. j dairy sci 2006;89:4237-4245. 14. stevenson js, portaluppi ma, tenhouse de, et al: interventions after artificial insemination: conception rates, pregnancy survival, and ovarian responses to gonadotropin-releasing hormone, human chorionic gonadotropin, and progesterone. j dairy sci 2007;90:331-340. 15. wallace ld, breiner ca, breiner ra, et al: administration of human chorionic gonadotropin at embryo transfer induced ovulation of a first wave dominant follicle, and increased progesterone and transfer pregnancy rates. theriogenology 2011;75:1506-1515. 16. bartolome ja, kamimura s, silvestre f, et al: the use of a deslorelin implant (gnrh agonist) during the late embryonic period to reduce pregnancy loss. theriogenology 2006;65:1443-1453. 17. bartolome ja, sozzi a, mchale j, et al: resynchronization of ovulation and timed insemination in lactating dairy cows iii. administration of gnrh 23 days post ai and ultrasonography for non-pregnancy diagnosis on day 30. theriogenology 2005;63:1643-1658. 18. mann ge, lamming ge: the influence of progesterone during early pregnancy in cattle. reprod domest anim 1999;34:269-274. 19. starbuck mj, dailey ra, inskeep ek: factors affecting retention of early pregnancy in dairy cattle. anim reprod sci 2004;84:27-39. 20. stronge ajh, sreenan jm, diskin mg, et al: post-insemination milk progesterone concentration and embryo survival in dairy cows. theriogenology 2005;64:1212-1224. 21. el-zarkouny sz, stevenson js: resynchronizing estrus with progesterone or progesterone plus estrogen in cows of unknown pregnancy status. j dairy sci 2004;87:3306-3321. 22. grimard b, freret s, chevallier a, et al: genetic and environmental factors influencing first service conception rate and late embryonic/foetal mortality in low fertility dairy herds. anim reprod sci 2006;91:31-44. 23. lucy mc: reproductive loss in high-producing dairy cattle: where will it end? j dairy sci 2001;84:1277-1293. 24. rabiee ar, macmillan kl, schwarzenberger f: the effect of level of feed intake on progesterone clearance rate by measuring faecal progesterone metabolites in grazing dairy cows. anim reprod sci 2001;67:205-214. 25. silke v, diskin mg, kenny da, et al: extent, pattern and factors associated with late embryonic loss in dairy cows. anim reprod sci 2002;71:1-12. clinical theriogenology • volume 3 number 4 • december 2011 470 26. wiltbank m, lopez h, sartori r, et al: changes in reproductive physiology of lactating dairy cows due to elevated steroid metabolism. theriogenology 2006;65:17-29. 27. bech-sabat g, lopez-gatius f, yaniz jl, et al: factors affecting plasma progesterone in the early fetal period in high producing dairy cows. theriogenology 2008;69:426-432. 28. rhinehart jd, starbuck-clemmer mj, flores ja, et al: low peripheral progesterone and late embryonic ⁄ early fetal loss in suckled beef and lactating dairy cows. theriogenology 2009;71:480-490. clinical theriogenology • volume 3 number 4 • december 2011471 clinical theriogenology • volume 3 number 4 • december 2011 472 omniblank: 2018: association between subclinical hypocalcemia and postpartumhealth disorders in dairy cattle association between subclinical hypocalcemia and postpartum health disorders in dairy cattle carlos a. risco center for veterinary health sciences, oklahoma state university, stillwater, ok introduction in dairy cattle, parturition and the onset of lactation lowers blood calcium concentrations (hypocalcemia) that can result in a clinical (milk fever) or subclinical condition. cows with milk fever have paresis and require prompt calcium treatment. in contrast, in the sub-clinical form of hypocalcemia (sch) the lower blood calcium concentration does result in paresis but does affect smooth muscle function (huber et al., 1981) and immune function affecting health (risco et al., 1984, 1994; martinezpatino et al., 2012). many dairy producers have adopted the practice of feeding acidogenic diets prepartum that has contributed to a notable reduction in the incidence of milk fever with an average reported of 2.4 % (reinhardt et al., 2011). however, despite the application of these diets, the prevalence of subclinical hypocalcemia (sch) remains at 25 and 47% in primiparous and multiparous cows, respectively. because of the higher prevalence and impact on health, there has been a resurgence in the interest to develop sch prevention and therapeutic strategies. this paper discusses the association between hypocalcemia, postpartum health disorders and risk for metritis related to a reduction in immune function in dairy cattle. in addition, therapeutic considerations for sch are discussed. association of calcium status to postpartum health disorders parturition and the onset of lactation predisposes cows to hypocalcemia characterized by a plasma calcium concentration < 8.5 mg/dl (martinez et al., 2012; chapinal et al., 2011). in cows with milk fever, decreased blood calcium concentration is accentuated and results in flaccid paralysis and eventually coma, if the animal is not treated. there is evidence that cows with milk fever, despite calcium treatment develop postpartum disorders. the association between milk fever and postpartum health disorders was evaluated in 33 holstein dairy herds that involved 2,190 cows (curtis et al, 1983). there were highly significant associations between milk fever and dystocia, retained fetal membranes (rfm), ketosis, and mastitis. the odds ratios for these associations were 6.5, 3.2, 8.9, and 8.1, respectively. grohn et al. (1990), after evaluating the lactational and health records of over 61,000 dairy cows in finland, found that milk fever was a significant risk factor for dystocia, rfm and clinical ketosis. the latter was associated with silent heats, cystic ovaries and infertility. the effect of hypocalcemia on cow health goes beyond the clinical symptom of paresis. in a study involving sheep, huber et al. (1980) demonstrated a true cause and effect relationship between hypocalcemia and normal smooth muscle contractility in the ruminant stomach. the major conclusions of this study were that: 1) ruminal contractions ceased long before signs of hypocalcemia were observed, 2) ruminal dysfunction may occur substantially before the clinical signs of hypocalcemia. in a study that evaluated the association between hypocalcemia and uterine prolapse, hypocalcemia without paresis was more common in cows affected with uterine prolapse when compared to cows without a prolapsed uterus (risco et al., 1984) and cows with prolapsed uterus remained hypocalcemic during the first 7 days postpartum (risco et al., 1994). subclinical hypocalcemia has also been associated with digestive disorders, a higher risk of culling and lower fertility. dairy cows that had abomasal displacement in an iowa study had low blood calcium content preceding displacement (hull et al., 1973). similarly, cows with hypocalcemia without paresis where 4.8 times more likely to develop left displacement of the abomasum (massey et al., 1993). low serum calcium, <2.2 to 2.4 mmol/l from wk 1 through wk 3 postpartum were associated with reduced pregnancy at first ai (chapinal et al, 2012). association between hypocalcemia and immune function hypocalcemia is a stressor and cortisol is a major contributor to immune suppression. typically, cows have a 3-4 fold increase in plasma cortisol as a component of the parturition process. however, sch cows can have a 5-7 fold increase in plasma cortisol on the day of parturition and cows with milk fever may exhibit plasma cortisol concentration that are 10-15 fold higher than pre-calving plasma cortisol concentration (horst et al., 1982). because immune suppression has been reported to begin 1-2 weeks prepartum (kehrli et al., 1998[a,b]); clinical theriogenology • volume 10, number 3 • september 2018219 horst et al., 1982) and the surge of cortisol is confined to the day of parturition, cortisol probably plays more of a contributing than a causal role in immune suppression. the response of immune cells is complex. generally, a compound such as a cytokine will bind to receptors on the immune cell surface, which then initiates an increase in intracellular ionized calcium (ica) concentrations which act as a second messenger to alter intracellular metabolism, initiating the phagocytic and killing response of the cell. the source of calcium for this response is from the endoplasmic reticulum and mitochondria of the cell (bréchard and tschirhart, 2008). immune cells of hypocalcemic cows have less cytosolic ica available for activation and consequently, function is suppressed. as hypocalcemia develops in the extra cellular fluid, there is also a concomitant reduction of ica2+ in the endoplasmic reticulum of the immune cells. because of the insufficient stores of calcium in the endoplasmic reticulum, the response of immune cells to activating stimuli is blunted in cows with hypocalcemia (kimura et al., 2006). a series of experiments at the university of florida (martinez et al., 2012; 2014) were conducted to establish the associations between suboptimal blood calcium concentrations, immune function, physiological responses and uterine infections. martinez-patino et al (2012) evaluated the peripartal calcium concentration in cows at low or high risk (dystocia, retained placenta) of developing metritis during the first 12 days postpartum. on the basis of receiver operator characteristic curves, sch was defined as a serum calcium concentration ≤ 8.59 mg/dl (< 2.124 mm) in at least 1 sample in the first 3 days postpartum. also, cows with sch (< 8.59 mg/dl) had elevated concentrations of nefa and bhba in serum during the first 12 days in milk, reduced neutrophil function and increased risk of developing metritis, compared to normocalcemic cows. this increased incidence of metritis in sch cows was observed regardless of the risk group (high vs. low) for metritis at calving. interestingly, the ability to maintain calcium concentration in blood during the first three days after calving was more important than the absolute calcium concentration; the greater the drop in calcium concentration in the first three days postpartum, the greater the probability of developing metritis later postpartum. martinez-patino et al (2014) induced sch (ica < 1.00 mm) in healthy nonlactating dairy cows, simulating the loss of calcium of an early postpartum dairy cow. cows with sch had reduced rumen contractions, dry matter intake, elevated nefa and glucose concentrations and decreased concentrations of insulin in plasma. the low calcium concentration likely compromised pancreatic release of insulin (littledike et al., 1968) which may explain the increased concentrations of blood glucose and lipid mobilization based on elevation in plasma nefa concentrations. cows induced to develop sch had a faster decline of cytosolic ica in neutrophils after ionophore stimulation, which likely explains the compromised phagocytic and oxidative burst activities of these cells against pathogenic bacteria. results from this study suggest that increased risk of diseases observed in cows that develop sch in early lactation is, in part, caused by suppressed function of immune cells mediated by reduced cytosolic ica concentrations. calcium therapeutic considerations calcium therapy by intravenous (iv) or oral route, is directed at maintaining normal plasma calcium concentrations. in cases of milk fever immediate parenteral iv calcium therapy (8.5 to 11.5 g calcium) is warranted. however, oral calcium supplementation is recommended for cows with undetected sch or those in stage 1 milk fever (oetzel, 2011). total serum calcium dynamics were evaluated after prophylactic treatment of sch after parturition in 33 multiparous jersey x holstein crossbreed cows (blanc et al 2014). compared to oral calcium treatment, iv calcium administration resulted in total serum calcium concentrations that reached hypercalcemic levels (11.4 mg/dl) that peaked 1 hour after treatment and declined to hypocalcemic levels by 24 hours after treatment. the short duration of hypercalcemia observed after iv ca supplementation may have impaired the ability of the animal to maintain calcium homeostasis. according to goff (1999), hypercalcemia causes a decrease in blood parathyroid hormone levels and an increase in thyrocalcitonin release, which decreases renal and bone ca reabsorption, decreases calcitrol conversion to 1, 25-dihydroxyvitamin d, increases aciduria, and depletes blood of cations. whether or not the hypocalcemia that resulted from iv ca treatment in the blanc et al. (2014) study affected health and productivity was not reported. oral supplementation of soluble calcium salts are commercially available that are suitable to treat sch cases and prevent relapses in milk fever cases after iv calcium treatment. oral calcium compounds have been clinical theriogenology • volume 10, number 3 • september 2018 220 developed to take advantage of passive diffusion of ionized calcium across cellular tight junctions in the rumen and intestines (goff & horst, 1993, 1994). the effect of oral calcium supplementation in early postpartum dairy cows has been studied but results have been inconsistent. a field study evaluated the effects of oral drenching with additional energy or energy plus calcium on blood parameters and performance of postpartum dairy cows (stokes et al., 2001). treatments were 9.5l water (control), 9.5l water plus 300ml (310g) propylene glycol (pg), or 9.5l water plus 0.68kg calcium propionate (cp). cows received the assigned drench within 4 h of calving and again 24 h after calving. drenching with pg or cp had no effect on plasma concentrations of calcium and glucose, or milk yield compared with control cows. however, cows receiving either pg or cp at calving had a significantly lower incidence of metritis compared with control animals. averaged across all trial periods, animals receiving pg had 3.1 kg/d greater milk production than those receiving the control. another study performed by melendez et al. (2003) that evaluated the effect of no oral calcium treatment, 60g of calcium as cacl2 , 110g of calcium as calcium propionate in combination with 400g of propylene glycol on calving-related disorders, fertility and milk yield. the results showed no effect of treatment on incidence of diseases, milk yield or reproductive performance. in contrast, a study performed by oetzel (1996) evaluated the effects of supplementation with oral ca on incidence of hypocalcemia and early postpartum diseases. the treatments consisted of no oral calcium, or 54g of oral calcium as cacl2 gel containing 1.5g of mg and 8.2g of p to cows 12h before expected calving, immediately after calving, at 12 and at 24h after calving. treatment with 54g of oral calcium increased serum calcium concentrations by 0.72 mg/dl during the first 2 days postpartum, and reduced the incidence of clinical and subclinical hypocalcemia, but no effect of treatment was found for retained fetal membranes but it did reduce the incidence of displaced abomasum. a study by oetzel and miller (2012) evaluated the effect of oral administration of two doses of 43g of ca as cacl2 and caso4, after calving and the second one 8 to 35 h later, on health and milk yield. the authors observed no differences in serum calcium concentrations, but a reduced incidence of health events in the first 30 days postpartum in cows considered lame. in addition, cows with a previous lactation mature equivalent greater than 105% of herd average produced 2.9 kg more milk in the first test postpartum when treated with calcium compared with untreated controls in the same milk category. studies have been conducted to evaluate the benefits of different doses and duration of calcium supplementation on cohort of dairy cows with different susceptibility to hypocalcemia. oral calcium supplementation with 43g or 86g calcium as cacl2 and caso4 (bovikalc bolus, boehringer ingelheim, st. joseph, mo) increased ica concentrations in blood, but the increase in ica and total calcium lasted fewer than 2 hours with 43g and fewer than 8 hours with 86g (martinez-patino et al., 2016[a]). supplementing oral calcium at these doses reduced the incidence and prevalence of sch and these reductions were greater when supplementation was extended to 4 days in milk. interestingly, despite the reduction in the incidence of sch, oral calcium supplementation increased the incidence of metritis in primiparous cows considered to be at low risk of metritis. conversely, multiparous cows supplemented with oral calcium had reduced incidence of diseases other than metritis and ketosis. another study that involved 450 holstein cows, evaluated the effects of oral calcium supplementation on milk yield, body condition, pregnancy per ai, and days to pregnancy in holstein cows considered to be of low or high risk of developing metritis (martinez-patino et al., 2016 [b]). cows were randomly assigned to control (no calcium supplementation), 86g of calcium ((bovikalc bolus, boehringer ingelheim, st. joseph, mo) at calving and 1 day postpartum, or 86g of ca at calving and 1 day postpartum followed by 43g/d on d 2 to 4 postpartum. supplementation with oral ca had no effect on the change of body condition in the first 32 days postpartum. interactions with risk of metritis and production potential in multiparous cows were observed in the first 30 days of lactation. within multiparous cows, those with greater potential for production benefited from supplemental calcium, whereas cows of less production potential had depressed milk yield when they received oral calcium. supplementing primiparous cows with oral calcium lowered pregnancy per ai and rate of pregnancy. in contrast, the same strategy to multiparous cows improved pregnancy per ai and increased pregnancy rate. based on these results, the authors suggest that oral calcium supplementation at calving should be avoided in primiparous cows and target only populations at high risk of developing hypocalcemia 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acid-base status, energy metabolites, and health of postpartum dairy cows. j dairy sci 2016a:99:8397-8416. martinez n, sinedino ldp, bisinotto rs, et al: effects of oral calcium supplementation on productive and reproductive performance in holstein cows. j dairy sci 2016b:99:8417-8430. massey cd, wang c, donovan ga: hypocalcemia at parturition as a risk factor for left displacement of the abomasum in dairy cows. j am vet med assoc 1993;203:852-853. melendez p, donovan ga, risco ca, et al: effect of calcium-energy supplements on calving-related disorders, fertility and milk yield during the transition period in cows fed anionic diets. theriogenology2003;60:843-854. oetzel gr: non-infectious diseases: milk fever. in: fuquay jw, mcsweeney pl, editors. encyclopedia of dairy sciences, vol. 2. san diego: academic press; 2011. p. 239-245. oetzel gr: effect of calcium chloride gel treatment in dairy cows on incidence of periparturient diseases. j am vet med assoc 1996;209:958-961. oetzel gr, miller be: effect of oral calcium bolus supplementation on early-lactation health and milk yield in commercial dairy herds. j dairy sci 2012;95:7051-7065. reinhardt ta, lippolis jd, mccluskey bj, et al: prevalence of subclinical hypocalcemia in dairy herds. vet j 2011:188:122-124. risco ca, reynolds jp, hird d: uterine prolapse and hypocalcemia in dairy cows. j am vet med assoc 1984;185:1517-1521. risco ca, drost m, thatcher ww: effects of retained fetal membranes, milk fever, uterine prolapse or pyometra on postpartum uterine and ovarian activity in dairy cows. theriogenology1994;42:183-190. stokes sr, goff jp: evaluation of calcium propionate and propylene glycol administered into the esophagus of dairy cattle at calving. prof sci 2001;17:115-122. clinical theriogenology • volume 10, number 3 • september 2018 222 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2017 calving date prediction based on transrectal ultrasonography determination of gestational age in beef cattle � calving date prediction based on transrectal ultrasonography determination of gestational age in beef cattle r.k. kasimanickam,a rabie l. abdel aziz,a,c v.r. kasimanickam,a,b c. formend adepartment of veterinary clinical sciences, bschool of molecular biosciences, college of veterinary medicine, washington state university, pullman, wa; cdepartment of theriogenology, faculty of veterinary medicine, beni-suef university, beni-suef 62512, egypt; dtrinity farm, ellensburg, wa abstract in cattle, accurate gestation length determination during pregnancy diagnosis helps to determine calving date. this aids beef cattle producers to estimate length of calving season, labor availability and labor cost and to make nutritional management decisions. the objective of the study was to determine the error rates for projected calving date based on ultrasound determined gestational age in comparison to actual gestational age (derived as the number of days from artificial insemination [ai] date). reproductive tract ultrasound examinations were performed in 8887 angus cross beef females that were artificially inseminated in 12 spring calving herds between 2011 and 2016 to determine gestational age of the embryo or fetus. only pregnancies estimated to be between 30 and 120 days (n = 6355 heifers and n = 2532 cows) were used in this study. the projected calving dates for actual and ultrasonography based gestational ages were determined using gestational length for beef cattle breed. actual calving date, number of calves delivered at birth, gender of calf born, calf weight, and incidence of assisted birth were recorded. in heifers, the error rates for calving date prediction for actual and ultrasonography gestational ages were 22.1 and 9.9% respectively (p<0.0001); whereas in cows the error rates for actual and ultrasonography gestational ages were 13.5 and 9.1% respectively (p<0.01). the results of the multivariate analysis revealed dam’s age and sire’s calving ease estimated progeny difference (epd) score influenced the accuracy of calving date prediction based on ultrasound determined gestational age (p<0.01). in conclusion, ultrasound determination of calving prediction was more accurate than traditional breeding date based calving prediction. keywords: beef female, pregnancy diagnosis, ultrasonography, calving, error rate introduction in bovine practice, various techniques have been successfully adopted for the diagnosis of pregnancy. the most commonly used techniques are per-rectal palpation of the reproductive tract and transrectal ultrasonography of the reproductive tract and its contents.1-5 these two methods are direct, reliable and fairly quick methods for diagnosis of pregnancy. other methods in use are estimation of progesterone,5-7, pregnancy specific protein5.8 and early conception factors9 concentrations in serum. under most on-farm conditions, pregnancy can be rapidly and accurately diagnosed using ultrasound as early as 26 days after breeding.10 sensitivity and specificity of pregnancy diagnosis in lactating dairy cows based on ultrasonographic detection of uterine fluid as well as embryonic membranes from 28 to 35 days after ai was 96% and 97%, respectively.11 use of ultrasound to diagnose pregnancy offers several advantages over rectal palpation such as accurate earlier pregnancy diagnosis, establishing fetal age, determination of fetal gender, and identifying abnormalities in embryonic or fetal development. sizes of maternal, placental and fetal components evaluated during ultrasonography examination have been used to explain reliable parameters to predict gestational age in cattle.9,12-14 the manifestation of a heartbeat, crown rump length, occipitonasal length, orbital diameter, abdominal diameter, umbilical cord diameter, amniotic sac diameter, thoracic diameter, head circumference, and chest depth of the fetus and placentome size are all proposed approaches to estimate gestational age.9,12-18 studies have shown that several factors contribute to the growth rate of developing embryo and fetus and that growth rate is not always uniform. both maternal and embryonic or fetal factors, as well as farm management factors, can contribute to less than perfect prediction. in addition, in utero development may be linked to increased risk of smaller sized calves12,13 or associated with larger birth size. twin calves, calves born to primiparous cows and heifer calves tend to be lighter at birth compared 573 clinical theriogenology • volume 9 number 4 • december 2017 � to singletons, calves born to multiparous cows, or bull calves respectively.15-17 poor maternal nutrition or reduced uterine capacity can limit conceptus growth.19.20 in addition fetuess in larger offspring syndrome, an overgrowth disorder caused by assisted reproductive technologies in ruminants, featured with excessive birth weight. pregnancy is a measure of success for any breeding programs of beef cattle. non-pregnant cows need to be identified as early as possible after the breeding season in order to determine reproductive efficiency, pregnancy rate and for culling decisions. further, accurate gestation length determination helps to determine calving date which helps producer to estimate length of calving season, labor availability and labor cost. this also assists producers to make nutritional management preferences for the pregnant females. the objective of the study was to determine the error rates for projected calving date for ultrasound determined gestational age in comparison to actual gestational age (derived as the number of days from the most recent ai date). materials and methods: pregnancy diagnosis reproductive tract ultrasound examinations were performed in 8887 beef females (6355 heifers and 2532 cows) that were artificially inseminated in 12 spring calving herds between 2011 and 2016. transrectal ultrasonography was performed by one clinician using a real-time, b-mode ultrasound scanner with a 5-mhz transducer (aloka 500, sysmed lab inc., chicago, il or sonoscape s8, universal diagnostic solutions, oceanside, ca). only pregnancies between 30 and 120 days were included in this study. the following embryo or fetal parameters unique to specific stages of development were used to estimate the gestational age an embryo (1 cm wide) with heartbeat by day 30 of gestation; differentiation of the head and abdominal regions by day 35 of gestation; budding of the limbs by day 45 of gestation; crown to rump length, abdominal, thoracic, head (width and/or occipitonasal length), placentome and umbilical cord sizes; amniotic sac and orbital diameters. data management only cows bred to angus sires and cows that calved between 260 and 300 d after fixed time ai (ftai) were used in the analysis. gestational age difference was calculated as actual (i.e., days since most recent breeding) minus estimated (i.e., based on estimate from ultrasonography) age of the conceptus. therefore, an underestimation (positive gestational age difference) means the ultrasound estimated age of the conceptus was younger than the actual gestational age and an overestimation (negative gestational age difference value) means the ultrasound-estimated age of the conceptus was older than the actual gestational age. projected calving dates for a gestation length of 283 d were calculated for ultrasonography gestational age and actual breeding dates. these dates were compared to actual calf birthdates to determine error in pregnancy diagnosis. the error rate was defined as number of calving over/underestimated (occurred before 278 and after 287 days after ftai) divided by total number of calving. sensitivity analysis for the gestation lengths 281 and 285 d were also calculated. data including age of the dam, body condition of the dam (1 to 9; 1, emaciated; 9, obese), sire of the calf, sire’s calving ease estimated progeny difference (epd) score, date of insemination, date of ultrasound pregnancy diagnosis, date of calving, number of calves recorded at birth, gender of calf born, calf weight, dystocia in the subsequent calving, and stillbirths were recorded. age records were categorized as 1, 2, 3 to 6, 6 to 10 and >10. body condition were categorized as <5, 5 to 7 and >7. statistical analysis data were analyzed with a statistical software program (sas version 9.4 for windows, sas institute, cary, nc). differences in the mean gestation length were analyzed using anova (proc glm of sas). the bartlett test was used to assess homogeneity of variance. the data for heifer and cow groups were analyzed separately. wherever variances for the mean gestation length were heterogeneous, 574clinical theriogenology • volume 9 number 4 • december 2017 � a log10 transformation was performed. all values are presented with non-transformed values. all pairwise differences between factor level means were analyzed by tukey’s method. factors associated to differences in birthweight of calf were determined by ordinal regression analysis. variables included in the model were calf’s gender, dystocia, calving ease estimated progeny difference and age of the dam. factors associated gestational age differences were determined using a mixed model. fixed variables included in the model were calf gender, calf body weight, sires calving ease epd and age of the dam and dam’s body condition score. location clustered with in the year (year [location]) was included as random variables in the model. factors associated with prediction accuracy of calving date by ultrasound based gestational age were determined using a mixed model. fixed variables included in the model were calf gender, calf body weight, sires calving ease epd score and dams’ age. year (location) offered as random effects. the error rates on the accuracy of calving date prediction for ultrasound and actual gestational ages in heifers and cows were calculated. the error rate was defined as number of calving that occurred outside the gestation length window divided by total number of calving (for gestation length 283 ± 7 days [mean ± 2 standard deviation {sd}], any calving that occurred < 276 or > 290 days was considered as error in calving prediction for both methods). results the mean (± sd) gestation length for the study population is 283.68 ± 3.52 days. the frequency histogram for gestation length based on breeding date is given in the figure. least square mean (± sem) gestational length for heifers and cows are given in table 1. the gestation length was different for gender of calf, calves that experienced difficult birth or not, sire’s calving ease epd and parity of cow. calf’s gender (p<0.0001), incidence of dystocia (p<0.01), sire’s calving ease epd (p<0.0001) and age of the dam (p<0.0001) influenced the mean birth weight of calves (table 2). the mean (± sem) birth weight of male and female calves were 82 ± 0.22 and 78 ± 0.36, respectively. the mean (± sem) birth weight of calves that experienced dystocia or not were 86 ± 0.32 and 79.0 ± 0.28, respectively. the result of multivariate analysis for the effect of calf gender, dystocia, calf birth weight, and dam age on the gestation length is given in table 3. the calf gender, dystocia, calf birth weight, and dam age affected the gestation length (p<0.01). the result of multivariate analysis for the effect of calf gender, dystocia, calf birth weight, and dam age on the accuracy of calving date prediction based on ultrasound pregnancy diagnosis is given in table 4. age of dam and sire’s calving ease epd score influenced the accuracy of calving prediction (p<0.01). the differences in the accuracy of calving prediction for both methods is given in table 5. for 283 ± 7 days gestation length in heifers, the error rate for calving date prediction accuracy by ultrasonography and calving calendar was 22.1 and 9.9% respectively; whereas for similar gestation length in cows, the error rate for calving date prediction accuracy by ultrasonography and calving calendar was 13.5 and 9.1%, respectively. similarly for 285 ± 11 days gestation length in heifers, the error rate for calving date prediction accuracy by ultrasonography and calving calendar was 18.3 and 8.4%, respectively; whereas for similar gestation length in cows, the error rate for calving date prediction accuracy by ultrasonography and calving calendar was 9.1 and 4.7%, respectively. in addition, the differences on the accuracy of calving date prediction based on ultrasound pregnancy diagnosis and breeding date for different gestational ages are given in table 6. discussion in this study, errors rates for calving date prediction were evaluated to determine whether producers can rely on breeding date or ultrasonography pregnancy diagnosis. the error rates for calving date prediction was lower for ultrasonography compared to breeding date validated the importance of ultrasonography pregnancy diagnosis. in this study, gender of the calf, age of dam, calf sire epd and dystocia influenced the gestational length. the effects of gender of the calf were significant for birth weight, (heifer calf 76.3 lbs vs. bull calf 81.2 lbs). birth weight of calves that were born to dams in 2, 3 to 6 and >6 years age groups were 575 clinical theriogenology • volume 9 number 4 • december 2017 � significantly different, 72.3, 2 80.6 and 82.4 lbs, respectively. calves born to sires with greater calving ease epd had lower birth weight. birth weight for calves that were delivered with assistance was greater (82.3 lbs) compared to calves that were born normally (79.0 lbs). it is plausible that the traits that contributed to increased birth weight may have led to prolonged gestation length. in this study, the gestational age using ultrasonography was determined using different fetal and maternal parameters. pregnancies were examined with at least two measurable characteristics defined in the material and methods. embryo size, fetal size (crown-rump length), placentome sizes, size of the head (width and/or occipitonasal length) of the fetus, and diameters of the abdomen and thorax were visualized more frequently, and the diameter of umbilicus less frequently. studies that investigated the associations of gestational age and fetal parameters concluded that fetal size provided the most precise estimate of gestational age. hannum et al showed residual sd + 4.5 days for fetal size, ± 6.9 to 8.7 days for head length and the diameters of trunk, head and nose and ± 12.6 days for uterine diameter.14 in the current study the residual sd was +3.5 days. the placenta plays a crucial role in the development of the fetus. placental characteristics such as the weight and volume of the placentomes, sizes including length, height and change in the placentomes density was used to estimate gestational age. during transrectal ultrasonography pregnancy diagnosis, placentomes were first visible around day 35 of gestation. these can be viewed as flattened, semicircular elevations on the surface of the uterine lumen. results from studies that evaluated the usefulness of placentome parameters to determine gestational ages, revealed that placentomes sizes are not significantly associated with gestational age during both transrecatal21 and transabdominal9 untrasonography. blankenvoorde claimed that there was no significant effect of the breed and age of the dam or uterine horn (gravid vs. non-gravid) on placentome size (p > 0.05) but observed significant association between gestational age and placentome size (p< 0.001).22 collectively, significant variations in size of placentomes impedes their use as a criteria for fetal ageing. however, a recent study claimed that the measurement of several placentomes sizes could be used to determine fetal age in late gestation.23 others have noted that there is a significant increase in the average placentome length with increasing gestational age should be taken into account while determining fetal age.23 there was a strong positive correlation between estimated gestational age from ultrasonography and actual gestational age based on the breeding date consistent with similar studies that used ultrasonography to estimate gestational age in cows.3,24 overestimation of the gestational age by ultrasonography was observed in the current study. fitzgerald et al observed overestimation of the embryonic age and underestimation of the fetal age and suggested that alterations in the conceptus-touterine lumen volume ratio may have contributed to this inconsistency.24 it is interesting to note that the error rate for ultrasonography in heifers was less for 60 to 90 days and higher for 30 to 60 and 90 to 120 days of gestation; whereas the error rate in cows was higher for 60 to 90 days gestational age and less for 30 to 60 and 90 to 120 days of gestation in the current study. it is possible that a combination of distinct fetal differentiation in older-stage pregnancies along with greater uterine capacity in multiparous cows may interfere with the estimation of gestational age using ultrasonography and may account for some of this prediction error. it should be noted that conceptus-to-uterine lumen volume ratio was not studied in the current study. however when fetal, abdominal and thorax sizes were considered to estimate fetal ages, differences in the uterine volume in relation to position of the fetus may have contributed to the error. the error rate for calving date prediction for ultrasonography was minimal when pregnancy diagnosis occurred between 60 and 90 days gestational age compared to 30 and 60 or 90 to 120 days gestational ages. normal intrauterine growth takes place in phases–an embryonic and a fetal phase.25 the embryonic phase consists of proliferation, organization and differentiation of the embryo, whereas the fetal phase consists of continuing growth and functional maturation of the various tissues and organs. the fetal phase of intrauterine development depends on genetic, placental, and maternal factors. these factors plausibly contribute to the asymmetric fetal growth which occurs in late gestation. it is possible that variation in proliferation, organization and differentiation of the embryo during embryonic phase (<60 days gestational age) and asymmetry growth during later fetal phase (>90 days gestational age) 576clinical theriogenology • volume 9 number 4 • december 2017 � could be contributed to higher error rate compared to transition phase from embryo to fetus ( between 60 and 90 days gestational age) with minimal error rate. in heifers, the error rates were higher than for cows. kramer et al. concluded that first parity influenced the calving date prediction.26 crews jr. suggested a linear effect of age of dam on gestation in which increasing age of dam was associated with longer gestation27 consistent with the current study. it should be noted that the size (length and width) of the uterus increase progressively with increasing parity, and therefore, gestational age may be overestimated because of the increased conceptus-to uterine lumen ratio in heifers. tactical use of reproductive ultrasound can help enhance the overall productivity of the herd. given the seasonal nature of the beef farming, the use of ultrasound for reproductive management tools tends to be concentrated during various stages of the production cycle. pregnancy diagnosis at an early stage of pregnancy provides a tool for many producers, both seedstock and commercial, to identify aiimpregnated versus cleanup bull-sired calves.28 in addition, earlier detection of non-pregnancy diagnosis assists with management decisions to cull open cows. further identification of cows that were to become pregnant very late in the breeding season also assists with management decisions to sell them as pregnant animals in order to maintain a short calving season. this study was conducted to determine the error rates for projected calving date based on ultrasound determined gestational age in comparison to actual gestational age. the results from the study revealed that the ultrasound determination of calving prediction was more accurate than traditional breeding date based calving prediction. acknowledgements the authors thank all beef cattle producers who participated in this study for their support. dr. rabie abdel aziz, beni-suef 306 university, beni-suef, egypt was financially supported by egyptian government research 307 scholarship (sab 2086), egypt cultural and educational bureau, the arab republic of egypt. references 1. roberts sj: veterinary obstetrics and genital diseases. 2nd ed. ithaca(ny): published by the author; 1971. p. 14-35. 2. momont h: rectal palpation: safety issues. bovine pract 1990;25:122-123. 3. youngquist rs: pregnancy diagnosis. in: youngquist rs, editor. current therapy in large animal theriogenology. philadelphia: wb saunders co., 1997. p. 295-303. 4. ball pj, logue dd: ultrasound diagnosis of pregnancy in cattle. vet rec 1994;134:532. 5. sasser rg, ruder ca: detection of early pregnancy in domestic ruminants-review. j reprod fertil suppl 1987;34:261271. 6. shemesh m, ayalon n, shalev e, et al: milk progesterone measurement in dairy cows: correlation with estrus and pregnancy determination. theriogenology 1978;9:343-353. 7. pennington ja, spahr sl, lodge jr: factors affecting progesterone in milk for pregnancy diagnosis in dairy cattle. br vet j 1976;132:487-495. 8. maurer rr, ruder ca, sasser rg: effectiveness of the protein b radioimmunoassay to diagnose pregnancy in beef cattle. j anim sci suppl 1 1985;61:390 [abstract]. 9. white ir, russel aj, wright ia, et al: real-time ultrasonic scanning in the diagnosis of pregnancy and the estimation of gestational age in cattle. vet rec 1985;117:5-8. 10. filteau v, descôteaux l. predictive values of early pregnancy diagnosis by ultrasonography in dairy cattle. proc am assoc bovine pract annu mtg 1998; 31:170-171. 11. nation dp, malmo j, davis gm, et al: accuracy of bovine pregnancy detection using transrectal ultrasonography at 28 to 35 days after insemination. aust vet j 2003; 81:63-65. 12. beal we, perry rc, corah lr. the use of ultrasound in monitoring reproductive physiology of beef cattle. j anim sci 1992;70:924-929. 13. bergamaschi macm, vicente wrr, barbosa rt, et al: ultrasound assessment of fetal development in nelore cows. arch zootec 2004;53:371-374. 14. hunnam jc, parkinson tj, lopez-villalobos n, et al: association between gestational age and bovine fetal characteristics measured by transcutaneous ultrasound over the right flank of the dairy cows. aust vet j 2009;87:379383. 577 clinical theriogenology • volume 9 number 4 • december 2017 � 15. singh ns, gawande oh, mishra op, et al: accuracy of ultrasonography in early pregnancy diagnosis in doe. asianaustralas j anim sci 2004;17:760-768. 16. nwaogou ic, anya ko, agada pc: estimation of feetal age using ultrasonic measurements of different foetal parameters in red sokoto goats (capra hircus). veterinarski arhiv 2010;80:225-233. 17. abdelghafar rm, ahmed bh, ibrahim mt, et al: prediction of gestational age by transabdominal real-time ultrasonographic measurements in saanen goats (capra hircus). glob veterinaria 2011;6:346-351. 18. harris rm, snyder bg, meyer r, et al: the relationship of bovine crown-rump measurement to fetal age. agripracitce. 1983;4:16-22. 19. long nm, vonnahme ka, hess bw, et al: effects of early gestational undernutrition on fetal growth, organ development, and placentomal composition in the bovine. j anim sci 2009;87:1950-1959. 20. fowden al, forhead aj. endocrine mechanisms of intrauterine programming. reproduction 2004;127:515-526. 21. adeyinka fd1, laven ra, lawrence ke, et al: association between placentome size, measured using transrectal ultrasonography, and gestational age in cattle. n z vet j. 2014;62:51-56. 22. blankenvoorde g. determination of gestational age in dairy cattle using transrectal ultrasound measurements of placentome size [thesis]. palmerston north (nz); masey university; 2011. 23. lawrence ke, adeyinka fd1, laven ra, et al:�assessment of the accuracy of estimation of gestational age in cattle from placentome size using inverse regression. n z vet j. 2016;64:248-252. 24. fitzgerald am, ryan dp, berry dp. factors associated with the differential in actual gestational age and gestational age predicted from transrectal ultrasonography in pregnant dairy cows. theriogenology 2015;84:358-364. 25. kasimanickam rk, kasimanickam vr, kastelic jp. intrauterine fetal growth restriction, adaptation and programming– a review. clin therio 2012;4;133-148. 26. kramer rw, smith dr, rupp gr, et al: estimation of calving date in beef cattle with real-time ultrasound. prof anim sci 2016;32:322-327. 27. crews jr dh: age of dam and sex of calf adjustments and genetic parameters for gestation length in charolais cattle. j anim sci 2006;84:25-31 28. kasimanickam r, whittier wd, tibary a, et al: error in pregnancy diagnosis by per-rectal palpation in beef cows. clin therio 2011;3:43-47. 578clinical theriogenology • volume 9 number 4 • december 2017 � table 1. gestation length least squares mean ± sem for calf gender, sires’ calving ease epd, and dam age and breed age group effect n gestation length heifer gender bull 3396 281.11 ± 0.66a heifer 2959 283.63 ± 0.67b sire calving ease epd � 5 1067 282.91 ± 0.52a 6 to 10 3056 281.22 ± 0.43ab >10 2232 280.90 ± 0.31b dystocia yes 181 284.12 ± 0.16a no 6174 281.19 ± 0.73b cow gender bull 2367 283.23 ± 0.52a heifer 1981 285.41 ± 0.56a sire calving ease epd � 5 563 281.92 ± 0.63a 6 to 10 1668 283.67 ± 0.34b >10 2117 285.23 ± 0.54c age 2 832 281.25 ± 0.32a 3 to 6 1519 282.19 ± 0.29ab 7 to 10 1144 283.92 ± 0.32b >10 853 285.55 ± 0.39c dystocia yes 219 286.12 ± 0.22a no 4129 283.94 ± 0.85b table 2. effect of calf’s gender, dystocia, calving ease estimated progeny difference (epd) score, and dam’s age on birth weight of the calf predictor coef se coef z p odds ratio lower 95% ci upper 95% ci constant 7.97635 1.00703 7.92 0 calf’s gender -0.86312 0.120359 -7.17 0.0001 0.42 0.33 0.53 dystocia 1.03038 0.215744 2.48 0.013 1.36 1.16 1.81 calving ease epd score -0.05432 0.010617 -5.12 0.000 0.56 0.33 0.68 age of dam (yrs) -0.21567 0.024057 -8.96 0.000 0.81 0.77 0.84 table 3. multivariate analysis for the effect of gender of calf, dystocia, calving ease epd, birth weight of calf, and age of the dam on gestation length. predictor coef se coef z p odds ratio lower 95% ci upper 95% ci constant 8.73983 1.09137 8.01 0 gender of calf -0.0486 0.116411 -0.42 0.0167 1.95 1.76 2.20 dystocia 0.372988 0.14422 3.66 0.0142 1.58 1.31 2.96 calving ease epd -0.02125 0.005781 -3.68 0.0001 0.88 0.87 0.89 age of dam 0.0304 0.022754 3.34 0.0116 1.17 1.11 1.23 calf birth weight 0.414523 0.161287 4.12 0.0018 1.40 1.22 1.51 579 clinical theriogenology • volume 9 number 4 • december 2017 � table 4. multivariate analysis for the effect of gender of calf, dystocia, sire calving ease epd, birth weight of calf, age of the dam on the accuracy of calving date prediction based on ultrasound pregnancy diagnosis. predictor coef se coef z p odds ratio lower 95% ci upper 95% ci constant 3.27163 0.389028 8.41 0 calf gender -0.1326 0.207646 -0.64 0.0523 0.88 0.58 1.32 dystocia 0.298869 0.659154 0.45 0.65 1.35 0.37 4.91 age of dam (yrs) 0.390264 0.150928 2.59 0.01 1.48 1.1 1.99 sire calving ease epd 0.219147 0.05572 3.93 0 1.25 1.12 1.39 calf birth weight 0.004658 0.040815 0.11 0.909 1.42 0.75 4.87 580clinical theriogenology • volume 9 number 4 • december 2017 �t ab le 4 . e rr o r ra te f o r ca lv in g d at e p re d ic ti o n f o r u lt ra so n o g ra p h y a n d a ct u al g es ta ti o n al a g es p ar am et er h ei fe r (n = 6 3 5 5 ) c o w s (n = 4 3 4 8 ) b as ed o n a i d at e an d ac tu al c al v in g b as ed o n u s g es ta ti o n al ag e an d a ct u al c al v in g b as ed o n a i d at e an d a ct u al c al v in g b as ed o n u s g es ta ti o n al ag e an d a ct u al c al v in g # o f ca lv es b o rn o u ts id e o f 2 8 1 ± 7 ( % ) 1 2 4 8 ( 1 9 .6 )* * * 5 8 8 ( 9 .3 )* * * 4 1 3 ( 9 .5 )* 2 7 8 ( 6 .4 )* # o f ca lv es b o rn o u ts id e o f 2 8 3 ± 7 ( % ) 1 4 0 4 ( 2 2 .1 )* * * 6 2 9 ( 9 .9 )* * * 5 8 5 ( 1 3 .5 )* 3 9 5 ( 9 .1 )* # o f ca lv es b o rn o u ts id e o f 2 8 5 ± 7 ( % ) 1 1 6 3 ( 1 8 .3 )* * 5 3 4 ( 8 .4 )* * 3 9 5 ( 9 .1 )* 2 0 7 ( 4 .7 )* w it h in h ei fe rs a n d w it h in r o w s: * = < 0 .0 5 ; * * = < 0 .0 1 ; w it h in c o w s an d w it h in r o w s: * * = < 0 .0 1 ; * * * = < 0 .0 0 1 ; 581 clinical theriogenology • volume 9 number 4 • december 2017 � table 5. error rate for calving date prediction for ultrasonography and actual gestational ages at different stages (gestation length 283±7). group embryo or fetal age @ us n error rate (%) based on us determination of gestational age and actual calving (n) error rate (%) based on breeding date and actual calving (n) heifer 30 to 45 393 10.9 (43) 24.4 (96) 46 to 60 1006 10.5 (106) 24.1 (243) 61 to 75 1914 9.2 (176) 21.3 (407) 76 to 90 1756 9.7 (170) 21.2 (372) 91 to 105 874 10.5 (92) 22.4 (196) 106 to 120 413 10.2 (42) 21.8 (90) total 6355 9.9 (629) 22.1 (1404) cow 30 to 45 222 7.2 (16) 15.3 (34) 46 to 60 721 9.6 (69) 11.8 (85) 61 to 75 1314 9.7 (128) 14.0 (184) 76 to 90 1222 9.2 (112) 13.5 (165) 91 to 105 556 7.7 (43) 13.1 (73) 106 to 120 313 8.9 (28) 14.7 (46) total 4348 9.1 (396) 13.5 (587) table 6. error rate (%) due to over or under estimation in gestational ages and calving prediction. gestation length estimation heifer cow breeding date (n=1404) ultrasound (n=629) breeding date (n=585) ultrasound (n=395) 281 under 70.8 (994) 15.7 (99) 66.3 (388) 29.9 (118) over 29.2 (410) 84.3 (530) 33.7 (197) 70.1 (277) 283 under 67.2 (943) 13.8 (87) 61.4 (359) 28.1 (111) over 32.8 (461) 86.2 (542) 38.6 (226) 71.9 (284) 285 under 69.2 (972) 16.9 (106) 64.8 (256) 26.6 (105) over 30.8 (432) 83.1 (523) 35.2 (139) 73.4 (290) figure. histogram of gestation length frequency;mean ± sd: 283.68 ± 3.52 0 100 200 300 400 500 600 700 800 2 6 0 2 6 5 2 6 9 2 7 1 2 7 3 2 7 5 2 7 7 2 7 9 2 8 1 2 8 3 2 8 5 2 8 7 2 8 9 2 9 1 2 9 3 2 9 5 2 9 6 2 9 8 3 0 0 n u m b er o f fe m al es gestation length 582clinical theriogenology • volume 9 number 4 • december 2017 2018: management of the high risk mare management of the high risk mare claire card department of large animal clinical sciences, western college of veterinary medicine, university of saskatchewan, saskatoon, sk, canada context for many mare owners late pregnancy is a time of nervous anticipation and expectation. while greater than 90% pregnancies are healthy and result in uncomplicated deliveries, there are however mares whose pregnancy poses a health risk to them.1,2 there are also mares that develop life-threatening medical and surgical problems during their pregnancy, and problems that put the mare’s pregnancy at risk.2 mares that are presented with a history of pregnancy or fetal loss, or that have serious medical conditions, which may compromise the desired outcomes of pregnancy, such as: birth of a healthy, term foal, and a healthy postpartum mare are considered ‘high risk’.3 commonly 5% of pregnancies are lost after 100 days of gestation.3 the foals arising from troubled pregnancies may result in the birth of foals that are non-viable, or that require intensive care.3,4 life threatening complications may also arise as a consequence of a mare’s foaling, and these injuries may endanger a mare in future pregnancies.5 the developmental consequences of high risk pregnancy for the fetal foal are often severe.4,6 the recognition, intervention, and prevention strategies for high risk pregnancies in mares will be discussed. conditions associated with ‘high risk’ mares or pregnancies ‘high risk’ mares include those with a previous and often repeat history of delivering a compromised foal. compromised foals include: stillborn foals, premature/dysmature/prolonged gestation foals, septic foals, foals with neonatal isoerythrolysis, ischemic encephalopathy, twin foals, and neonatal maladjustment foals.3,4 the ‘high risk’ category also includes mares that have birth related problems such as: premature placental separation (red bag), dystocia, placentitis, protracted labor, and uterine artery hemorrhage/hematoma.4 some mares may develop physical abnormalities during pregnancy including: laminitis, hydrops, abdominal wall disease, or have a narrow or fractured pelvis.1,2 mares carrying foals with genetic problems (example hydrocephalus) are also at risk. mares with advanced age (>20 years) during pregnancy are also at higher risk of complications. placentitis is one of the main causes of ‘high risk’ pregnancy in mares.4,6 other ‘high risk’ mares include those that may develop complicated medical or surgical problems. mares may become ‘high risk’ from environmental exposures such as: grazing pastures or forages containing fungal endophytes, such as fescue infected with neotyphodium coenophialum; consumption of toxic plants (locoweed astragalus spp), goitrogenic plant species (brassica species i.e mustard foals), or other toxins (for example, heavy metals). history the mare’s health history and past problems during pregnancy should be discussed. it is important to determine the mare’s breeding date(s) or exposure to a stallion. it is important to establish if the mare was evaluated for a twin pregnancy, and when this examination(s) occurred in the pregnancy. determine how long the clinical signs, such as colic-like signs, or precocious udder development have been present, and what other clinical signs have been seen, such as vaginal discharge. determine if the mare been dripping or streaming milk, or has material on her legs compatible with leaking mammary secretion. take note of the tilt, shape, and size of vulva and integrity of the vulvar seal and if the mare has had caslick’s surgery. mares that have a history of problems during pregnancy or that are at risk of problems should be monitored carefully, generally with serial ultrasound examination of the reproductive tract, fetus, blood work, and milk electrolytes. for mares that are high risk a practitioner should consider a referral to a practice or location with facilities readily and able to: manage obstetrical emergencies, perform cesarian sections, or that provide neonatal intensive care services. the birth of a foal at a location where there is an intensive care unit facilitates rapid intervention during and after birth. careful monitoring of the ‘high risk’ mare and clinical theriogenology • volume 10, number 3 • september 2018283 the pregnancy using serial ultrasonographic examinations, and milk electrolytes, is useful. having knowledgeable foaling attendants present at parturition also may help to optimize the outcome. high risk mares-examination assessment of a ‘high risk’ mare should be systematic and thorough. a complete physical examination should be performed, including examination of the mare’s digital pulses and mucus membrane color. the tools used to recognize a ‘high risk’ mare include: history, vaginal examination, transrectal palpation and ultrasound, abdominal wall examination, transabdominal placental and fetal monitoring, serial examination of mammary secretion electrolytes, serum chemistry/cbc and biomarker analysis.3 vaginal examination a vaginal examination is used to evaluate the status of the cervix and to investigate mares with vaginal discharge. a healthy pregnant mare with good perineal conformation should have a pale, dry vagina, and a tight cervix with no discharge until near term. the mucus plug of the cervix is tacky and light tan in color. this plug generally is passed within 12 hours of parturition at term. the vaginal examination of a mare must be performed using strict aseptic technique. the mare’s perineum is prepared as for artificial insemination. a sterile speculum may be used, and/or a careful manual, quick, vaginal examination performed. a careful, efficient, gentle examination may be performed with a surgically gloved hand of the external portion of the cervix to determine if it is softened, open or closed. avoid excessive handling of the cervix. if there is a vaginal discharge a few slides should be prepared using a wright giema stain to examine the cells present (there should be no neutrophils or red blood cells and no bacteria/fungi). to differentiate the cytology of a liquefying cervical plug from a purulent discharge, the plug cytology will have very few cells in it. if inflammatory cells are present on the sample it should be submitted for culture and sensitivity. the presence of red blood cells or neutrophils in a vaginal discharge is abnormal. if neutrophils are present examine them for intracellular bacteria (determine if cocci or rods) and gram stain a slide to determine the class of bacteria. if bacteria are present the determine of the bacterial morphology and gram classification should inform the choice of an antibiotic. it also serves as a starting point if the foal is born septic and needs treatment. the most common bacterial agents involved in ascending placentitis include 𝛽hemolytic streptococcus such as streptococcus equi zooepidemicus, and e. coli. in spite of having bacterial placentitis, infected mares seldom develop fevers, but when infection occurs through the ascending route they may have vaginal discharge. vaginal discharge is not always however a constant finding.4 transrectal palpation and ultrasonographic examination rectal examination is used to determine: cervical tone, the fill of the uterus, fetal orientation and assessment of the fetal compartment (combined thickness of the uterus and placenta [ctup], fetal fluid depth and character).7 the fetal parts that are commonly assessed include the fetal cranium, orbit and limbs (carpi). the goal is to measure the orbital diameter (length and width in mm) the sum of which is correlated with gestational age (to a month). location of the cranium of the fetus informs the examiner that the fetus is in anterior presentation. the foal may be assessed in terms of the skeletal development by evaluating the shape of the growth plates. the uteroplacental unit is a fluid filled organ, and the ventral surface of the uteroplacental unit is evaluated, and the location where the combined thickness of the uterus and placenta (ctup) is measured is usually adjacent to the vaginal branch of the uterine artery (figure 1). the areas adjacent to the cervix is examined for the presence of an exudate, placental separation and edema (figure 2). rough guidelines for ctup are: <8 mm 271-300 days, <10mm 301330, <12 mm after 330 days. thumb rule is that after the eighth month of pregnancy the ctup should match the month of gestation.7 thicker ctup measurements may signal trouble, such as ascending placentitis. it has been our experience that once a placenta has an increased ctup in a case of placentitis it may or may not completely return to within normal limits after therapy.3 clinical theriogenology • volume 10, number 3 • september 2018 284 abdominal wall examination in healthy pregnant mares, typical measurements of the thickness of the muscles in millimeters of the ventral abdomen include (mean + standard deviation; sd): rectus abdominus 16.5+6, mm pectoralis ascendens 10.5+3 mm, and cutaneous trunci 9+1.5 mm and only slight edema is usually present in pregnant mares. older mares are predisposed to ventral abdominal wall disease because studies show that the abdominal wall muscles weaken with age.8 an examiner needs to be aware that there may be fat interspersed between the muscle layers. there was little variation from left to right side of the abdomen in muscle measurements and surprisingly similar results with large breed and light horse mares, and before and after parturition. when abdominal wall disease is suspected an examination should be concerned about underlying causes such as: placentitis, dropsical conditions, and twins. excessive edema, thickening and muscle fiber separation are all signs of abdominal wall muscle compromise. the excessive weight of the fetus and fetal fluid poses a risk of ventrolateral abdominal wall disease.9 ventrolateral abdominal wall disease may also arise idiopathologically. clinical signs include reluctance to move or a change in gait. mares will develop hot painful ventral edema, and as the condition deteriorates, discoloration of the abdominal wall, and serum weeping through the skin. this is evidence of severe myonecrosis. in advanced cases the abdominal wall may fail at the aponeurosis of the internal oblique muscle in the lower flank area(s), which leads to a unilateral and bilateral lower flank hernia. the myonecrosis may dissect across the ventral abdomen involving the prepubic tendon, or arise on either side of the pelvis. there usually is bloody mammary secretion, which is pathognomonic for abdominal wall disease. the prepubic tendon is complicated mixture of insertion fibers. when the prepubic tendon area ruptures the mare’s pelvis tilts, the abdomen drops, and the result is displacement of the udder cranially, ahead of the stifle (figure 3). they may develop a fatal ileus as the bowel may be located adjacent to the skin. in very serve cases the skin may also rupture leading to eventration. the problem may be unilateral or bilateral.9 transabdominal ultrasonographic examination transabdominal ultrasonographic examination is performed by systematically evaluating the abdominal wall integrity and the quadrants of the abdomen. a 3.5 or 5 mhz probe is used. the abdomen is scanned from the udder to the sternum to locate the uterus. the examination is accomplished by wetting the hair of the mare’s abdomen using alcohol and mineral oil or ultrasongraphic couplant. the fetal allantoic and amniotic fluid compartments may be visualized and the fetal fluids evaluated in terms of quantity and character. some floating particulate material is considered normal (vernix, protein), excessive matter usually indicates fetal asphyxia/fetal diarrhea, or bacterial growth in the fetal fluids. hippomanes may be identified in the allantoic fluid. the ultrasonographic scan is usually most productive in the caudal quadrant of the abdomen near the udder. the ventral abdomen of the mare from the xiphoid to the udder on both sides should be examined. the orientation of the foal can be confirmed. the ctup is measured in a number of locations (normal <2.2cm). the fetal orientation is ascertained, (in anterior presentation the fetal heart is located in the anterior of its thorax close to the mare’s pelvis) and then the thorax is located in order to observe the fetal heart rate. the fetal and non-fetal uterine horns are identified (or not if there are twins).7,11,12 the fetal aorta, and distance between the ribs may be measured to assist in determining fetal age, and growth over time.7 fetal assessment a fetal assessment, also called a biophysical profile, should include an evaluation of the ctup of the placenta in various locations. a determination of fetal fluid depth/character, fetal tone, aortic diameter, and fetal heart rate. fetal body parts may be measured to confirm fetal age fetal ribs shadows are used to help locate the fetal heart. fetal heart rate and character may be evaluated (figure 4). a fetal and maternal ekg may be obtained. cardiac function in regards to fetal movement should be determined, as fetal movement in the absence of heart rate accelerations indicates fetal hypoxia. the mean fetal heart rated (fhr) varies from 75-85 bpm in a non-stressed fetus. prolonged fhr <40 or >110 bpm indicates clinical theriogenology • volume 10, number 3 • september 2018285 significant distress. note that the fhr normally should vary and fetal activity is normally associated with transient increases in fhr. if the fhr does not increase with fetal movement this is also a sign of hypoxia or distress.12 clinical chemistry and cbc the mare’s cbc and chemistry do not change significantly during a healthy pregnancy.7 in abdominal wall disease the mare’s creatine phosphokinase (ck), alkaline phosphatase, aspartate amino transferase enzymes (ast) and gamma glutamyl transferase (ggt) will be elevated, however these changes may not reflect the severity of the abdominal wall compromise. a cbc is useful to determine if there is elevated fibrinogen suggesting long standing chronic inflammation. a stress leukogram may be noted. only in the rare really ill systemically sick mares that are endotoxemic will there be a left shift with toxic change noted.11 biomarkers biomarkers of placentitis include steroids, elevations in acute phase protein serum amyloid a, relaxin, alpha fetoprotein (which may not be consistently elevated) and microrna’s. in placentitis serial progestogen levels may be increased or decreased, serum amyloid a may be increased (>7 mg/l), and serum fibrinogen may be elevated. 3,13,14 fetal adrenal activation may result in rising progesterone, while placental failure may be associated with falling progesterone.6 mammary secretion testing prefoaling mammary secretion should be evaluated in mares with premature lactation. in mares approaching their due date the use of ph strips to determine milk ph, and water hardness test strips may be used to measure changes in calcium and magnesium that are indicate of readiness to foal. the ph of the mammary secretion typically decreases to ph<6.5 near delivery, and the calcium and magnesium levels (measured as water hardness or calcium carbonate equivalents) in samples diluted 1 part milk to 5 parts water (1:6 dilution), to measure changes in calcium magnesium levels (>200 mmol calcium carbonate equivalents, or 40 mg/dl indicates readiness to foal), or a calcium titration method to determine calcium levels may be used (>200 mmol, 200 ppm).15 hormonal analysis serial hormonal analysis of serum progestagen and estrogen values may help predict if the placenta is healthy and is maintaining endocrine functions. progestagens are monitored as an indicator of placental function and fetal stress. during pregnancy excessively high or low progestagen levels indicate a problem. in the experimental infection models of streptococcus equi zooepeidemicus placentitis, mares that had acute reactions and aborted early had high levels of progestagens while more chronically infected mares had lower levels of progestogen.4,6 fetal stress may cause premature adrenal activation and the production of increased amounts of precursors for the progestagens. progestagen values measured as progesterone in the coat-a-count® ria typically range from 2-6 ng/ml from 180-310 days of gestation and may increase or decrease + 2 ng/ml on a daily basis, hence the recommendation to monitor the levels changing over time. progestagen levels (cross reactive in progesterone assays) over 10 ng/ml are considered elevated.4,6 a sudden drop in progestagen is often associated with impending abortion. synthetic progestagens such as altrenogest (regumate®) do not interfere or cross-react in the progesterone ria. fetal death is associated with a decrease in maternal estrogen levels. a sudden drop in progesterone is often associated with impending abortion. interventions for specific conditions of ‘high risk’ mares the specific therapy for a number of conditions is discussed below. therapy for placentitis if a cause can be determined the therapy is aimed at minimizing inflammation, improving fetal viability using medications such as antibiotics, tocolytics, steroids, anti-endotoxin or anti-inflammatory clinical theriogenology • volume 10, number 3 • september 2018 286 agents, and hormones. for twin pregnancy prevention is the key! in mares with placentitis mares need to be monitored for preterm delivery and managers need to be prepared for birth of a neonate that may need intensive care. neonatal intensive care tools (heat lamps, plasma transfusion, antibiotics, 24 hour supportive care) need to be readily available.4,6 the goals of therapy are to fight or eliminate infection, decrease inflammation and cytokine formation, and decrease myometrial activity. therapy will often delay preterm delivery due to placentitis, but seldom is sufficient to allow a term delivery.4 the initial choice of antibiotic should be based on cytology, gram stain, and culture. a culture and sensitivity are used to confirm the organism and to choose the appropriate antibiotic. the most commonly used antibiotic is trimethoprim sulfa. studies using ceftiofur showed that this antibiotic does not readily cross the equine placenta. to decrease inflammation anti-inflammatories such as flunixin meglumine (utilized when endotoxemia is present) or phenylbutazone are often administered. inflammatory products may lead to increased uterine contractions. pentoxyphylline is used to decrease cytokine production. the direct evidence of the benefit of pentoxyphylline still requires further investigation. dexamethasone may be used to suppress the proinflammatory cytokines, and has been used sparingly in dosages from 25-50 mg sid once or for a few days. altrenogest (double dose 0.088 mg/kg po) has been shown to be effective in stopping cloprostenol induced abortion experimentally.4 in one report mares with streptococcus induced placentitis had gestational lengths that were longer than untreated mares. recently the use of estradiol cypionate has been examined as a means of improving fetal outcomes.16 to decrease myometrial contractility tocolytics such as clenbuterol (beta agonists) need to be given 2-3 times a day to decrease uterine contractions.17 generally clenbuterol administration is associated with uterine relaxation and improved fetal perfusion. clenbuterol is no longer available in injectable form, but only as a syrup. this medication will not stop abortion or parturition, but may modulate uterine contractions.18 altrenogest may also decrease uterine contractions and may improve fetal outcomes. the cox-1 cox-2 inhibitors such as firocoxib may decrease endometrial prostaglandin section, however there is little information on their use during pregnancy. the non-steroidal antiinflammatory (nsaid) medications, such as flunixin meglumine, are commonly administered to mares with placentitis because of their anti-endotoxic effects. the minimum duration of treatment for mares is usually 2 weeks after which a mare should be reassessed. in some cases mares may need to be maintained on antibiotic and nsaid therapy until delivery. the table lists common medications utilized to treat placentitis. consequences of placentitis. veterinary bills are large. the foal is often born septic or endotoxemic. the foal may have neonatal maladjustment syndrome (nms). foals may never reach their full size, they may develop debilitating developmental orthopedic disease. neonatal intensive care unit graduates often are suboptimal athletic performers. preterm delivery is also associated with full or partial retention of the placenta, which may trigger its own set of problems in the mare. the mare has impaired reproductive performance including: uterine sub-involution and difficulty conceiving that breeding season. laminitis many cases of laminitis during pregnancy are idiopathic, but others may be related to excessive body condition. ideal weight during pregnancy should result in a body condition score of 6/9. mares that gain weight easily which can lead to mechanical overloading of the hooves and laminitis. in one study 42% of draft horses had a bcs of >7.19 mares may be grazing on pasture where they may spend 90% of the time consuming grass. the highly digestible carbohydrates in the grass may be a problem. draft mares and ponies may have experienced previous episodes of laminitis, which puts them at risk of a reoccurrence. their hoof care may also be neglected which may make the mechanical forces on their hooves worse.14 owners may report a reluctance to move, laying down alot, or are stiff in the shoulders. a physical examination should include evaluation of the mare’s stance. a typical laminitic mare will have a clinical theriogenology • volume 10, number 3 • september 2018287 ‘sawhorse’ stance if the forefeet are involved and a shifting leg stance if all 4 hooves are involved. reluctance to lift the feet is present. the hooves may be warm. palpation of the digital pulses shows prominent, bounding pulses. positive hoof testing is usually present along the toe. in severe cases sinking may be noted near the coronet band. walking and turning the mare may also reveal the gait problem. the heart rate usually rises dramatically when a mare with laminitis is walked. the heel of the hoof usually strikes the ground first in laminitis. palmer digital nerve blocks will improve the lameness. lateral radiographs of the coffin bone (p3) assist in determining if rotation or sinking of the hooves is present. treatment includes removal of any source of endotoxins, oral phenylbutazone, icing the hooves, trimming the hooves if needed, and application of a support device such as “soft-rides” or homemade versions of these on the bottom of the hoof. don’t forget to get them off the grass! limiting the hours grazing or grazing muzzles may be needed to keep mares from getting too fat. hydropic conditions mares with excessive girth may have hydropic conditions, may be found to be carrying twins or are excessively conditioned. (body condition score of >8/9). hydrops is a rare medical problem in mares that arises from a placental or occasionally fetal dysfunction. most cases are however idiopathic and result in a progressive and life threatening accumulation of fetal fluid. the excess fetal fluid accumulation may result in the mare’s abdominal circumference increasing at a rate of more than 4 cm per day. 9 there may be colic signs, respiratory distress and inappetence in mares that relates to their hemodynamic compromise. most commonly the fluid accumulates in the allantoic cavity, causing hydrallantois, but occasionally the amniotic fluid is involved and causes hydramnios, or when both compartments are involved, it is called polyhydramnios. the excess weight of the fetal fluid leads to cardiovascular compromise of the mare, and abdominal wall stress. in hydrallantois the fetus may not be palpated per rectum, and in hydramnios there is excessive amniotic fluid. these mares may experience abdominal wall rupture.7,11 termination of the pregnancy is almost always needed. currently preloading the mare with intravenous fluid, then slow drainage of the fetal fluid (over 2-4 hours) with concurrent aggressive fluid replacement, is recommended to save the life of the mare.11 twins genetically thoroughbred, warmblood and draft mares are more likely to have twins. prevention and early intervention are the key. extensive management of some mares is also a factor where mares may not be evaluated during early pregnancy using ultrasonography. statistics show in general very poor survival of twins, with only 1 in 300,000 having both twins survive the neonatal period. twins are usually aborted or born about a month premature. they may not reach their full mature size and are prone to developmental orthopaedic disease.3 ventrolateral abdominal wall disease history includes a large amount of ventral edema, a reluctance to walk, development of an abnormal gait, or a history of “falling.” this is a disease that accompanies obesity, age, and may arise as a complication associated with hydrops or twins. in most cases it is idiopathic. excessive weight on the mare’s abdomen results in a pathologic hot, painful, pitting, ventro-lateral edema that indicates underlying muscle damage. the ventral flank is often afflicted first (at the aponeurosis of the internal abdominal oblique) followed by extension to the ventral abdomen and rectus abdominus. in most advanced cases the internal abdominal oblique muscle and rectus abdominus muscle ruptures (aka prepubic tendon rupture), and the pelvis tips. the udder develops a bloody secretion and becomes displaced so the teat is visible ahead of the stifle. the mare may develop a fatal ileus, or eventration.7 fetal abnormalities many breeds are known for specific heritable conditions and have genetic tests available for them. a breed with a long list of fetal abnormalities is the friesian. this breed has a high inbreeding coefficient. there is genetic testing available for “water head”, (hydrocephalus) from a defect in the clinical theriogenology • volume 10, number 3 • september 2018 288 b3galnt2 gene, and dwarfism caused by a mutation in the b4galt7 gene (fenway foundation http://fenwayfoundation.com/forms/). both have a simple recessive mode of inheritance so there is a 1:4 chance if two carriers are mated of having a foal with the abnormality. junctional epidermolysis bullosa testing is also available for belgians, but this disease does not cause antepartum problems. testing is available at the university of kentucky (http://getgluck.ca.uky.edu/content/submission-forms).20 decision to terminate a pregnancy and/or induce parturition it is difficult to determine if the medical problems of the mare are severe enough to warrant the termination of pregnancy. under most circumstances elective induction of parturition should only be performed in mares when medically necessary. the mare should be greater than 330 days of gestation, have colostrum in her udder, a drop in mammary secretion ph below 6.5 and an elevation in milk calcium/magnesium (>200 mmol / l). her cervix should be relaxed. it is best to seek advice in these cases as many of these mares may require intensive care or hospitalization. the most common method to induce parturition is the use of oxytocin. although prostaglandin f2-alpha has been used. fetal maturation may be hastened by the administration of dexamethasone at 100mg/day. induction of parturition in mares at around 320 days of pregnancy with dexamethasone involves 4 consecutive days of administration at this dose with parturition occurring approximately four days later. there are a variety of doses utilized to induce parturition including a low dose method of 3.5-5 iu of oxytocin (given once daily until parturition occurs) and 20 iu may be utilized to induce foaling within one hour.21 the low dose method appears to work more reliably if the mare in not moved from her home farm to a new location. if the cervix is very tight a pretreatment with prostaglandin e1 may be utilized intra-cervically 4 hours before oxytocin induction.22 attendants should be prepared to open a prematurely separated chorioallantoic membrane (redbag) and assist delivery of a foal and potentially using the exit procedure developed by at the university of pennsylvania. prevention strategies in advance of breeding have genetic testing performed of mares and stallions. ensure the mares have regular hoof care, vaccination and deworming. have an ultrasonographic examination for pregnancy status performed are least twice during early pregnancy from 15-35 days to detect twins. perform a caslick’s vulvoplasty on mares that have sloping or tipped in perineal conformation to help prevent ascending bacterial infections. retire mares >20 years of age from breeding, or perform embryo transfer. avoid excessive weight gain during pregnancy. provide a balanced diet to pregnant mares with adequate amounts of mineral. if pregnancies are lost perform necropsies to determine the cause.23 monitor mares closely during pregnancy and keep them in stable isolated groups, preferably with mares close to the same age, and away from other horses. references 1. tibary a, pearson lk: mare problems in the last month of pregnancy. proc am assoc equine pract 2012;58:350-358. 2. frazer gs, embertson r, perkins nr: complications of late gestation in the mare. equine vet educ 1997;9:306-311. 3. bucca s: diagnosis of the compromised equine pregnancy. vet clin north am equine pract 2006;22:749-761. 4. lyle s, paccamonti d: high risk pregnancy in the mare-practical implications for the practitioner. pferdeheilkunde 2010;26:29-35. 5. frazer gs: postpartum complications in the mare. part 2: fetal membrane retention and conditions of the gastrointestinal tract, bladder and vagina. equine vet educ 2003;15:91-100. 6. leblanc m, mcpherson m, sherrin p: ascending placentitis what we know about pathophysiology, diagnosis and treatment. proc am assoc equine pract 2004;50:127-143. 7. bucca s: assessment of feto-placental well-being in the mare from mid-gestation to term: transrectal and transabdominal ultrasonographic features. theriogenology 2005;64:542-557. 8. anderson s, bracamonte j, carmalt j, et al: ex vivo comparison of bursting strength of ventral median and right ventral paramedian celiotomies in horses. vet surg 2013;42:468-472. 9. wolfsdorf, k: ventral abdominal hernia and prepubic tendon rupture. in robinson ne, editor. current therapy in equine medicine. philadelphia: saunders; 2003. p. 310-311. 10. hanson r, todhunter r: herniation of the abdominal wall in pregnant mares. j am vet med assoc 1986;189:790-793. clinical theriogenology • volume 10, number 3 • september 2018289 11. diel de amorim m, chenier t, card c et al: treatment of hydropsical conditions using transcervical gradual fetal fluid drainage in mares with or without concurrent abdominal wall disease. j equine vet sci 2018;64:81-88. 12. reef v, vaala we, worth lt et al: ultrasonographic evaluation of the fetus and intrauterine environment in healthy mares during late gestation. vet radiol ultrasound 1995;36:533-541. 13. troedsson mht, miller l: equine placentitis. pferdeheilkunde 2016;32: 49-53. 14. canisso i, ball b, scoggin k et al: alpha-fetoprotein is present in the fetal fluids and is increased in plasma or mares with experimentally induced ascending placentitis. anim reprod sci 2015;154:48-55. 15. korosue k: testing mammary gland secretions to help predict when a mare will foal. vet rec 2013;173:216-217. 16. curcio b, canisso i, pazinato f et al: estradiol cypionate aided treatment for experimentally induced ascending placentitis in mares. therio 2017; 102:98-107 17. robinson ne: clenbuterol and the horse. proc am assoc equine pract 2000;46:229-233. 18. palmer e, chavatte-palmer p, duchamp g et al: lack of effect of clenbuterol for delaying parturition in late pregnant mares. theriogenology 2002;58:797-799. 19. kosolofski h, gow c, robinson k: prevalence of obesity in equine population of saskatoon and surrounding area. can vet j 2017;58:968-970. 20. sabbagh m: genetic and environmental analysis of dystocia and stillbirths in draft horses. animal 2014;8:184-191. 21. witkowski m, pawlowski k: clinical observations of the course of oxytocinor prostaglandin e2/oxytocin-induced parturition in mares. polish j vet sci 2014;17:347-351. 22. villani m, romano g: induction or parturition with daily low-dose oxytocin injections in pregnant mares at term: clinical applications and limitations. reprod domest anim 2008;43:481-483. 23. schlafer d: postmortem examination of the equine placenta, fetus, and neonate: methods and interpretation of findings. proc am assoc equine pract 2004;50:144-157. clinical theriogenology • volume 10, number 3 • september 2018 290 figure 1: a. transrectal image of the uterus and placenta. b. the combined thickness of the uterus and placenta (ctup) is shown by the connected markers. figure 2: transrectal ultrasonographic image of the pericervical area including the cervix (dashed arrow) and uteroplacental tissue (solid line) in a pregnant mare. amnion ctup ctup vaginal branch uterine artery a b cervix ctup clinical theriogenology • volume 10, number 3 • september 2018291 figure 3: a. mare in late pregnancy with a ruptured prepubic tendon. b. transabdominal image of the abdominal muscles showing excess edema in the cutaneous trunci (solid arrow), disorganized muscle fibers and hemorrhage of the rectus abdominus (dashed line arrows). c. transabdominal image of the abdominal muscles of a periparturient mare showing mild edema of the cutaneous trunci (solid arrow), and intact rectus abdominus (dashed line arrows). a b a c rectus abdominus cutaneous trunci cutaneous trunci rectus abdominus clinical theriogenology • volume 10, number 3 • september 2018 292 figure 4: transrectal ultrasonographic image of the a. fetal orbit, b fetal lens, and c. transabdominal image of the fetal thorax and aorta. (editor’s note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) a b aorta c clinical theriogenology • volume 10, number 3 • september 2018293 table: medications administered to high risk mares with placentitis antimicrobials trimethoprim sulpha 30 mg/kg bid po gentamicin 6.6 mg/kg sid iv or im potassium penicillin g 22,000 iu / kg iv qid procaine penicillin g 22,000 iu/kg bid anti-inflammatories pentoxyfylline 8.5 mg/kg po bid flunixin meglumine 1.1 mg/kg bid iv phenylbutazone 4 mg/kg bid po meclofenamic acid 2.2 mg/kg sid po tocolytics clenbuterol 0.6 μg/kg iv drip then tid po progesterone 0.6 mg mg/sid im hormones estradiol cypionate 10 mg im / mare / every 3 days altrenogest 0.088 mg/kg sid po (double dose) clinical theriogenology • volume 10, number 3 • september 2018 294 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice urogenital surgery in camelids urogenital surgery in camelids ahmed tibary,a alexis campbell,a jacobo rodriguez,c cristian patinoa adepartment of veterinary clinical science college of veterinary medicine and center for reproductive biology cvista equine colorado, fort collins, co abstract surgical interventions on the urogenital system are some of the most common procedures performed in veterinary practice. the present paper describes indications, techniques, possible complications and postoperative care of urogenital surgeries in camelids. potential complications are discussed, based on retrospective analysis of clinical cases seen by the authors over the past 28 years of theriogenology practice. although not part of the urogenital system, excision of the soft palate and rectal prolapse in camels are also discussed, as these disorders are related to reproductive activity in this species. keywords: cesarean section, ovariectomy, laparoscopy, cryptorchydectomy, prolapse introduction urogenital surgeries are undoubtedly among the most commonly performed procedures in veterinary practice. they can be divided into 3 general categories: elective surgeries, surgeries to reestablish reproductive function, and emergency surgeries. elective surgeries are primarily used to eliminate reproductive function (castration, ovariectomy), to prepare teaser animals (vasectomy), or to perform advanced reproductive techniques. surgeries to restore or preserve normal reproductive function include, unilateral gonadectomy, and repair of the reproductive tract anatomy. emergency surgeries in the female include cesarean section, laparotomy, and vaginal and uterine prolapse replacement. emergency surgeries in the male, include primarily unilateral castration of an injured testis, preputial disorders, and urolithiasis. excision of the soft palate is a frequent procedure in the dromedary camel. the present paper describes the indications, postoperative care and possible complications associated with reproductive surgeries based on the authors’ experience. some of the surgical techniques have been described elsewhere and are not detailed in this paper. general consideration the primary consideration prior to surgery is the approach to analgesia and anesthesia. these protocols are generally dictated by the type of surgery, species and the overall health of the patient. in the field, most elective surgeries are performed using a combination of chemical and physical restraint (table 1). deep sedation and field anesthesia may be obtained with higher doses of the same drugs. heavier sedation and regional or local anesthesia is excellent for more involved surgeries, such as cryptorchidectomy and flank cesarean section. general anesthesia is mostly indicated in advanced techniques such as midline cesarean section, cystotomy, and laparoscopy. in the field, general anesthesia may be obtained with injectable anesthetics. in a hospital setting, general anesthesia is preferably induced with injectable anesthetics and maintained with gas (e.g. isoflurane in oxygen). details are summarized in table 1. in nonemergency surgeries, feed should be withheld for 24 48 hours prior and water for 12 24 hours prior, depending on the size of the animal. broad spectrum antibiotics and tetanus toxoid should be administered prior to surgery. postoperative pain management may include butorphanol tartrate (0.05 0.1 mg/kg im), flunixin meglumine (2.2 mg/kg), morphine (0.10.25 mg/kg im, every 24 hours) or meloxicam (1 mg/kg, every 48 72 hours in alpacas and llamas, 0.5 mg/kg, orally every 48 72 hours in camels). it is extremely important to consider the degree of hydration and any potential compromise of kidney function, especially when using drugs known to be nephrotoxic. clinical theriogenology • volume 12 number 3 • september 2020 271 table 1. common drug protocols used by the authors for sedation and anesthesia in camelids (doses are in mg/kg bodyweight, unless otherwise stated) drug alpacas and llamas camels remark sedation acepromazine 0.15 im/sq 0.03 – 0.1 im 0.01 0.02 iv urolithiasis butorphanol 0.05 0.2 im 0.03 0.05 iv or im standing castration detomidine 0.04 0.06 im 0.02 0.05 im restraint diazepam 0.5 0.2 im 0.2 0.3 im light to moderate sedation xylazine 0.1 0.5 iv 0.2 0.6 im 0.1 0.25 iv 0.3 0.4 im recumbent sedation at high doses anesthesia (recumbency) diazepam + ketamine d: 0.2 03 im + k: 5 8 im d: 0.2 0.3 + k: 5 8 im d: 0.1 0.2 + k: 3 -5 iv xylazine + ketamine x: 0.22 0.44 iv + k: 2.2 2.5 iv x: 0.22 0.44 im + k: 10 -15 im 10 minutes later x: 0.35 + k: 5 8 im x: 0.25 + k: 3 5 iv anesthesia 30 60 minutes butorphanol + ketamine + xylazine alpacas: b: 0.046 + k: 4.6 + x: 0.46 im llamas: b: 0.037 + k: 3.7 + x: 0.37 im b: 0.3 + k: 3-4 + x: 0.2-0.3 im anesthesia 30 40 minutes triple drip: xylazine (1 mg/ml), ketamine (1 2 mg/ml) in 5% guaifenesin induction: 0.6 1.1 ml/kg maintenance: 2.2 mg/kg/hour induction: 1.1 ml/kg maintenance: 2 ml/kg/hour maintenance cri to effect propofol for induction 2 3.5 iv can be used for maintenance, but expensive urogenital surgeries in the female camelid cesarean section (hysterotomy) cesarean section in llamas and alpacas may be performed using a left flank or ventral midline approach.1-3 the flank approach is the best choice under field conditions and for severely compromised patients. in camels, although a left ventrolateral approach has been described,4 the authors recommend only using a higher left flank approach because of risks of herniation using a ventrolateral approach.2,5 for a flank approach, most females tolerate the surgery using local anesthesia with lidocaine administered in a line or inverted “l” block anesthesia (lidocaine diluted to 1% with isotonic bicarbonate or saline, with the total dose not to exceed 4.4 mg/kg) following sedation, caudal epidural and physical restraint in the sitting sternal (“cush”) position. the surgical procedure has been described by the authors and is similar to that performed in ruminants, except that the skin incision is oblique, extending from the angle formed by tuber coxae to the base (ventral aspect) of the last rib.1-3 the incision line should be parallel to the direction of the quadriceps when the animal is sitting in the sternal position (figure 1). the subcutaneous muscle and fascia and the external oblique muscle are incised, whereas the internal oblique and transverse abdominal muscle may be gridded along the muscle fibers. the gravid uterine horn (always the left) is grasped around a fetal limb and gently exteriorized from the abdominal incision (figure 2). a uterine incision is made over the limb at the greater curvature and the fetus is exteriorized (figure 3). if the placenta is still attached, it should be left in place but peeled away from the uterus 2 4 cm along the entire uterine incision to avoid incorporating it into the uterine closure, resulting in subsequent dehiscence. because of the type of placentation (epitheliochorial and microcotyledonary), mural bleeding is common and hemostasis is provided by over-sewing (“whipstitching”) the margins of clinical theriogenology • volume 12 number 3 • september 2020272 figure 3. flank approach for cesarean section in camelids. uterine incision and delivery of the fetus. figure 4. flank approach for cesarean section in camelids. uterine suture using an inverting utrecht pattern. figure 6. midline approach for cesarean section in an alpaca with a 360° uterine torsion figure 1. flank approach for cesarean section in camelids with the patient restrained in sternal position. note the oblique direction of the incision figure 2. flank approach for cesarean section in camelids. exteriorization and incision of the uterus. figure 5. flank approach for cesarean section in a llama. skin suture clinical theriogenology • volume 12 number 3 • september 2020 273 the uterine incision in a continuous interlocking pattern using absorbable suture material. the uterine wall is closed in a cushing, utrecht, or lambert pattern using absorbable suture material (figure 4). if the uterus appears healthy, a single layer closure is sufficient; however, if there is compromise, a double layer closure is recommended. the uterine wall and abdominal cavity may be lavaged with a warm sterile saline solution containing antibiotics (penicillin g potassium 22,000 units/kg, ampicillin sodium 20 mg/kg, or ceftiofur sodium 1 mg/kg), and heparin (20 40 units/kg). carboxymethyl cellulose (14 ml/kg, intraperitoneally) may be used to prevent postoperative adhesions. the abdominal cavity and skin are closed in the same manner as for ruminant cesarean section (figure 5). the midline celiotomy approach is performed in alpacas and llamas in dorsal recumbency under general anesthesia; this is the preferred approach if the uterus is compromised or has undergone torsion (figure 6). a midline celiotomy incision (25 30 cm in alpaca and 35 40 cm in llamas) is made through the skin, subcutaneous fat, cutaneous trunci muscle and linea alba from ~ 4 cm cranial to the border of the mammary gland extending towards the umbilical scar. the uterus is identified by direct palpation and exteriorized. an incision is made through the uterine wall along the greater curvature. the fetus is removed and the umbilicus clamped and transected. uterine closure is as described above. this technique can be used to resolve preterm uterine torsion without delivery of the fetus if it is still alive.6 if the fetus is dead or at term (dystocia due to uterine torsion), the fetus is delivered, then the torsion corrected. the linea alba is closed using absorbable suture material (no. 2 polyglycolic acid or no. 1 polydioxanone or polyglactin 910) in an appositional interrupted (horizontal mattress, cruciate) or continuous suture pattern. closure of the skin may be achieved with staples, horizontal mattress suture pattern, ford interlocking suture pattern or preferably where possible, a subcuticular suture pattern (no. 2-0 polyglactin 910 or polyglecaprone). postoperative care includes pain management for 3 5 days. antimicrobial prophylaxis should be continued for 5 7 days, depending on the condition of the uterus and fetus at the time of surgery. fluid therapy may be indicated in some cases. the dam should be monitored for clinical signs of postpartum metritis and toxemia. the placenta is generally expelled within a few hours of surgery if the cervix is open or 2 4 days if it was closed at the time of surgery. oxytocin may be administered (20 iu im in camels and 5 10 iu im in south american camelids) every 4 hours during the first 24 hours postsurgery, if the cervix is open. administration of cloprostenol im (125 250 µg in alpacas, 250 µg in llamas, and 500 750 µg in camels) is recommended if the surgery was performed to remove a dead fetus due to uterine torsion. complications of cesarean section in camelids include retained fetal membranes, incisional infection, herniation, peritonitis, intestinal adhesions, and infertility (table 2). however, these complications are minimal when the surgery is performed early in dystocia.7-9 the most common complications seen in camel cesarean sections, performed by the authors in the field, were incisional infections (myasis) and herniation. the rebreeding success rate is excellent (> 70%) following cesarean section, and most females will rebreed successfully 3 4 months postsurgery.6-9 the earliest successful rebreeding post cesarean section seen by the author is 45 days. the authors generally recommend at least 45 60 days of sexual rest, with a thorough prebreeding examination prior to any breeding following a cesarean section. ovariectomy/ovariohysterectomy ovariectomy is usually performed to prevent sexual activity and eliminate pregnancy risk or to remove a diseased organ (e.g. ovarian masses, ovariobursal adhesions).10 ovariohysterectomy is rarely performed in camelids, but may be considered in cases of uterine or cervical masses and mucometra.11 ovariectomy or ovariohysterectomy should ideally be performed during the luteal phase of the cycle, so that the uterus is relaxed and complications associated with hemorrhage are reduced. alternately, females should be given progesterone (progesterone in oil im or intravaginal cidr) for 7 10 days prior to surgery. clinical theriogenology • volume 12 number 3 • september 2020274 table 2. indication and outcome of cesarean sections in camelids reference campbell et al: 2013* miller et al: 2013* tibary et al: 2015** no. females 24 31 76 primarous (%) 45.8 multiparous (%) 54.2 76 uterine torsion (%) 61 38 0.5 fetal maldisposition (%) 21.7 failure of cervical dilation (%) 8.7 17.5 81.65 other (%) 8.6 22 dam survival (%) 91.2 86 92.1 neonate survival (%) 46 59 81.6 complications retained fetal membranes (%) 8.3 88 15.7 metritis (%) 2.9 other complications (%) 20 postsurgical fertility (%) 70 90.5 70.8 *llamas and alpacas, **dromedary camel recipients in an embryo transfer center in alpacas and llamas, ovariectomy may be performed using a parainguinal, ventral midline or flank approach. laparoscopic and laparoscopic-assisted techniques are described in subsequent sections of the present paper. the choice of a particular technique depends on the age of the animal, the side of the ovary concerned (unilateral or bilateral), and the status of the ovary (normal versus abnormal). the flank approach is considered the best for camels.2,10 the technique is similar to the approach described for cesarean section, except that the incision is vertical and gridding is possible (figure 7). for a ventral midline approach, the anesthetized animal is placed in dorsal recumbency. a small (6 8 cm) incision is made on the ventral midline just cranial to the udder and continued into the abdominal cavity as described for cesarean section. the surgeon introduces 2 fingers into the abdominal cavity. the urinary bladder is identified, and the uterus is recognized in its dorsal aspect by following 1 of the horns to the uterine bifurcation. vaginal manipulation with a sterile tube speculum or blunt ended rod by an assistant may help the surgeon locate the uterus. one uterine horn is grasped between the fingers and pulled towards the surgical incision. both horns are exteriorized by gentle traction, followed by exteriorization of the ovaries. for ovariectomy, the vascular pedicle of the ovary is isolated by passing forceps through the mesovarium, making sure to incorporate the ovarian artery and vein. a size 0 absorbable suture material is used to transfix the ovarian pedicle before transection. large ovarian masses (e.g. granulosa theca cell tumor, teratoma) may require placement of overlapping transfixing sutures on the pedicle to prevent hemorrhage (figure 7). for hysterectomy or ovariohysterectomy, the mesometrium and broad ligament of each uterine horn are transected after ligation of blood vessels. transfixation and circumferential ligatures, using absorbable suture, are placed proximal to the cervix. the surgeon should make sure to include the large uterine vessels located on each side. the uterus is transected at the level of the body between 2 hemostatic forceps. if the remaining portion of the uterine body is large, it should be closed with an inverting suture pattern before replacing it in the abdomen. removal of 1 horn or portion of a uterine horn (a partial hysterectomy) is sometimes used in research settings or for treatment of pathology confined to one side of the abdomen. the technique is similar to a total hysterectomy, although a flank approach would be more possible. the vasculature supplying the ovary and ipsilateral horn is ligated and transected. pregnancy is only possible if the left uterine horn with the ipsilateral ovary are normal and maintained. however, embryos may still be collected from females with right uterine horn and normal ipsilateral ovary. clinical theriogenology • volume 12 number 3 • september 2020 275 figure 7. paralumbar flank approach to ovariectomy in camels; a b) removal of an ovary encapsulated with ovariobursal adhesions, c) removal of an ovarian teratoma postoperative care should include antimicrobial therapy and pain management. there are very limited risks of complication with ovariectomy. however, ovariohysterectomy may have a higher risk, due to intraoperative hemorrhage, adhesion formation and stump complications. laparoscopic techniques laparoscopy is widely used in reproductive research and for exploration of the abdomen and urogenital system abnormalities.12-14 the technique requires the use of a rigid laparoscope with a diameter of 6 10 mm and various lens angles, depending on the indication for the procedure. for most reproductive techniques in alpacas, we use a 6 mm diameter laparoscope with a 0 or 30° angle. this allows for minimal incisional size for portal placements. standing laparoscopy may be used in llamas and camels using a 10 mm diameter laparoscope. for alpacas, laparoscopy is usually performed on the sedated or anesthetized animal in dorsal recumbency on a surgical table that can be tilted into trendelenburg position. animals should be fasted for at least 24 hours prior to the procedure to reduce forestomach compartment (c-1) fill and reduce risk of regurgitation. standing laparoscopy standing laparoscopy is performed mainly in llamas and camels for in situ observation of the genital organs or ovariectomy (figure 8). the female is restrained in stocks and sedated with butorphanol tartrate. the left paralumbar fossa is prepared for aseptic surgery and 2% lidocaine is infused into the subcutaneous and muscular tissues in an inverted “l” pattern to desensitize the region (figure 8).15,16 a small (10 15 mm) skin incision is made in the craniodorsal portion of the paralumbar fossa caudal to the12th rib (4.5 cm in llamas, 8 12 cm in camels) and ventral to the transverse processes of the lumbar vertebrae (8 cm in llamas and 12 15 cm in camels). this provides a portal for a 30° laparoscope, which is placed after penetrating the abdominal cavity through the abdominal musculature and peritoneum using an appropriate trocar. the abdomen is insufflated with co2 to a pressure of 10 mm hg. clinical theriogenology • volume 12 number 3 • september 2020276 figure 8. standing laparoscopy in llamas and camels. sites for laparoscope (1) and instrument portals (2 & 3) figure 9. ventral midline laparoscopy technique. placement of the endoscope portals (1) and manipulation forceps portal (2). figure 10. examples of abnormalities diagnosed by laparoscopic examination of the reproductive tract in alpacas. a) ovarian hypoplasia/dysgenesis, b) ovarian neoplasia, c) uterus unicornis, d) ovarian neoplasia, e) hydrosalpinx, f) adhesions figure 11. laparoscopic-assisted ovariectomy in an alpaca. ovary is dislodged from its bursa (a b) then held with forceps from the pedicle and elevated to body wall (c d). an allis forceps is introduced through a midline incision to hold the ovary and exteriorize it (e f). figure 12. laparoscopic-assisted ovariectomy in an alpaca. pedicle of exteriorized ovary (a) is a transfixing ligature placed before excision (b). clinical theriogenology • volume 12 number 3 • september 2020 277 each ovary is identified by following the respective uterine horn from the uterine bifurcation to the tip of the uterine horn. this manipulation may be difficult if the urinary bladder is distended. catheterization of the bladder with a foley catheter prior to the procedure is helpful. once the genital tract has been located and inspected, a second incision is made in the skin of the caudodorsal portion of the left paralumbar fossa caudal to the 12th rib (10 cm in llamas and 15 18 cm in camels) and ventral to the transverse process of the lumbar vertebrae (10 cm in llamas and 15 20 cm in camels). this incision will provide the portal for a manipulation instrument (grasping forceps) to be inserted through a cannula fitted with a sharp trocar. a small incision is then made in the skin of the caudoventral region of the left paralumbar fossa caudal to the 12th rib and ventral to the transverse processes of the lumbar vertebra to provide a portal for the introduction of a ligature guide holding a loop of size 0 polydioxanone. the ovary is grasped with the forceps through the suture loop. the ligature is pushed to the level of the ovarian pedicle and tightened around it. the ligature guide is removed, and scissors are introduced to cut the end of the ligature and transect the ovarian pedicle at the base of the ovary. the ovary is removed from the abdomen with the grasping forceps. all instruments are removed. it is important to ensure that excess gas is expelled prior to removal of the cannulas to avoid postoperative discomfort. to close portal incisions, use a single cruciate suture with 3-0 polyglyconate for the external oblique muscle and a single cruciate suture of 3-0 monofilament nylon for the skin. ventral abdominal approach the anesthetized patient is placed in dorsal recumbency. the ventral abdomen is clipped and surgically prepared from the cranial edge of the mammary gland to the xiphoid. the laparoscope portal is made over the umbilical scar or a few centimeters caudal to it (figure 9). a 10 15 mm skin incision (size depending on the diameter of the laparoscope) is made on the midline at the level of the umbilicus or 3 5 cm caudal to it, depending on the technique and length of the endoscope.1, 17-20 a controlled 2 3 mm stab incision is made on the linea alba and a teat canula or a verses needle is used to penetrate through the linea alba and peritoneum into the abdomen. adequate penetration of the abdomen can be verified by injecting sterile saline through the canula. the abdominal cavity is insufflated with co2 to a partial pressure of 10 -15 mm of hg. a cannula fitted with a pyramidal trocar is inserted at this level through the linea alba and peritoneum into the abdomen. the trocar is removed and replaced with a laparoscope. the pelvic inlet is identified by tilting the surgical table to 40 degrees to elevate the hind legs and displace the abdominal viscera cranially (trendelenburg position). for in situ examination of the reproductive tract, instruments portals are made lateral (left and right) to the midline midway between the scope portal and the caudal aspect of the fold of the flank. the uterus is located under the urinary bladder and each uterine horn is followed to its tip and elevated to allow visualization of the ipsilateral ovary. atraumatic grasping forceps are used to manipulate the uterus and bladder for location and visualization of the ovaries (figure 10). for ovariectomy, the ovary is dislodged from the ovarian bursa by gentle manipulation until the mesovarium and proper ligament of the ovary are isolated (figure 11). hemostasis may be provided by two hulka clips across the mesovarium or proper ovarian ligament, or a suture loop may be placed around these structures. the proper ligament is incised with scissors proximal to the clips or suture.17,18 alternately, the ovary is grasped at the level of its pedicle after exteriorization from its bursa and elevated to the abdominal wall (figure 11). an incision is made through the skin and linea alba as far caudal as possible, to avoid excessive tension on the ovary. a pair of allis forceps is introduced through the incision into the abdomen and the ovary is grasped exteriorized (figure 12). a transfixing suture is placed on the ovarian pedicle, using pds-0, prior to excision of the ovary with scissors (figure 12). the procedure is repeated on the other ovary.19 closure of the external abdominal musculature (portal locations) and the linea alba is achieved using 2 polyglycolic acid in a cruciate and simple continuous pattern, respectively. the skin is closed with 2-0 polypropylene using a simple cruciate suture. for laparoscopic ovariohysterectomy, the female is prepared in the same manner as for ventral midline laparoscopic ovariectomy. however, this technique requires more expertise in the handling of surgical instruments through laparoscopy. the procedure starts with ligation of the ovarian vessels. the clinical theriogenology • volume 12 number 3 • september 2020278 mesovarium is exposed and incised, after hemostasis has been established using a series of clips (ligaclip®) inserted into the left portal. the broad ligament is transected close to the uterine horn caudal to the uterine body, avoiding the uterine artery. the same procedure is repeated on the other side. once both uterine horns are freed, a ligature loop is introduced and passed around the uterine horns and ovaries all the way to the level of the uterine body, where it is tightened. a second ligature loop is placed in the same manner around the uterine body to provide adequate hemostasis. the uterus is transected, after placing a third loop along the portion of the uterus to be removed (to prevent loss of uterine content into the abdomen). the uterus and ovaries are removed from the abdominal cavity from 1 of the instrument portals after increasing its size.20 laparoscopic assisted ovum pickup in preliminary trials by the authors, laparoscopic ovum pickup can be an alternative to laparotomy for alpacas.21 the technique is similar to that describe above for laparoscopic-assisted ovariectomy. once the ovary is exteriorized from its bursa, an 18 gauge, 6 cm needle attached to an aspiration pump is introduced through the abdominal wall and follicles are aspirated. the recovery rate obtained (56.1%) was lower than the laparotomy technique. rectovaginal tear perineal lacerations are classified using the same system described for mares. the majority of cases observed by authors are third-degree and occasionally second-degree perineal lacerations. the occurrence of third-degree lacerations is common after severe dystocia, due to the small perineal body in these species (figure 13). superficial lacerations can be repaired quickly under local anesthesia using a caslick’s procedure if the tear has already epithelialized. second-degree lacerations require reconstruction of the perineal body, generally after sedation and epidural anesthesia. repair of the third-degree perineal laceration and rectovaginal fistulas should be delayed until the tissue has granulated and epithelialized. although some practitioners may perform this operation as early as 2 weeks after injury, it is the authors’ preference to wait 4 8 weeks postpartum. repair of third-degree laceration can be performed standing in camels. in llamas and alpacas, we prefer to have the patient heavily sedated or under general anesthesia in a sternal position (figure 14). in sedated animals, epidural anesthesia is helpful, as rectal and vulvar tissues need to be retracted using suture or retractors. repair techniques are similar to those described for the mare and consist of dissection of the rectal and vaginal walls, with the goal of creating a new separate rectovaginal shelf. the rectal and vaginal layers are closed with monofilament absorbable suture (2-0 or 3-0 pds in llamas and alpacas, 0 or 1 pds in camels). the perineal body is sutured separately, followed by skin suture.22 rectovaginal fistula in the authors’ experience, rectovaginal fistulas in camelids involve primarily the vestibular area (rectovestibular fistulas). they are better corrected by converting them to a complete rectovaginal tear prior to correction. chronic vaginal prolapse vaginal prolapse is relatively common in camelids, and particularly in heavy breeds of camels, in the last 2 months of pregnancy.23 predisposing factors include age (older females), parity, and body condition (obese or very thin females).3 in late-term animals, vaginal prolapse may be the first sign of a uterine torsion. prolapse of the entire vagina and exteriorization of the cervix is rare. prolonged periods of prolapse lead to increased inflammation, resulting in severe necrosis of the vaginal mucosa. in some cases, ascending infectious placentitis is possible. in camels, myiasis of the prolapsed tissue is not uncommon (figure 15). chronic cases are at risk of abortion and/or rectal prolapse due to persistent tenesmus. rectal and vaginal prolapse may be the only sign of uterine torsion, dystocia or abortion. clinical theriogenology • volume 12 number 3 • september 2020 279 tenesmus figure 13. third-degree perineal laceration following dystocia and fetotomy in a llama figure 14. third-degree rectovaginal laceration repair in an alpaca. a b) incision and dissection to separate the rectum from vaginal vestibule, c d) apposition of the flaps of tissue with sutures, ensuring that the rectal mucosa is apposed and the vestibular mucosa is everted into the vestibule. e f) the perineal body is sutured using a simple interrupted pattern figure 15: myasis complication of a chronic vaginal prolapse in a dromedary camel figure 16. cercalge suture technique in an alpaca with recurrent vaginal prolapse due to rupture of the vestibule-vaginal sphincter. left to right, the vaginal tissue is cleaned and replaced, a purse string suture is placed around the vestibular suture and a caslick suture is placed to reduce the side of the vulva. figure 17. uterine prolapse in a dromedary camel (left) and an alpaca (right) figure 18. episioplasty to repair a narrow vulva opening (incomplete atresia vulvi) clinical theriogenology • volume 12 number 3 • september 2020280 the prognosis for the life of the fetus and dam is relatively good if the condition is treated early. prolapsed vaginal tissue is cleaned and replaced after sedation and epidural analgesia. in camels, the vaginal tissue is maintained in place by a bühner suture around the vulva.23 in alpacas and llamas, the vulva can be sutured with light umbilical tape, using a long postmortem needle in purse string or a shoelace suture pattern.24 sheep vaginal prolapse retainers have been used successfully to manage vaginal prolapse in alpacas.25 recurrent vaginal prolapse in postpartum alpacas and camels has been observed by the authors. in these cases, the vestibulovaginal sphincter may be compromised and the bladder may be entrapped in the prolapsed tissue. after replacement, the vestibular sphincter is held in place with an encircling suture using heavy resorbable suture material (figure 16). although the general recommendation is not to rebreed females with vaginal prolapse during pregnancy, there is no evidence for a hereditary nature of vaginal prolapse. uterine prolapse partial or total uterine prolapse can occur secondary to dystocia, abortion, manual removal of a retained placenta or excessive use of oxytocin (high dosage and frequency). uterine prolapse is more common in camels than in llamas and alpacas, and is often associated with hypocalcemia, selenium deficiency and retained fetal membranes, particularly in dairy camels (figure 17).2,26-28 uterine prolapse generally occurs immediately (the first 60 minutes) after parturition or abortion. techniques for replacement are similar to those reported in cattle under sedation and epidural analgesia. the placenta is often easily peeled off and should be removed, if possible, prior to replacement of the uterus. the female is positioned in sternal recumbency, with the hind quarters slightly elevated. the uterus should be inspected for any lacerations or hemorrhage. the area of major risk for hemorrhage is located near the cervix where the uterine artery may be exposed. the uterus is cleaned with warm dilute povidone iodine solution before replacement. the vulva is sutured with a bühner suture in camels or a shoelace pattern in alpacas and llamas. uterine prolapse tend to recur if the uterine horns are not fully extended. hysterectomy may be considered if the uterus has sustained severe damage; however the prognosis for survival in these cases is poor.27,29 vulvoplasty/episioplasty episioplasty is often considered in females with atresia vulvi or a recessed vulva (figure 18). the surgery is straight forward and aims at extending the commissures of the vulva. however, owners should be warned about the possible hereditary nature of atresia vulvi and breeding these females should be discouraged.30-32 vaginal adhesions vaginal adhesions are common complications of overt obstetrical manipulations or fetotomy in camelids.3 laser surgery may be considered in some cases;33 however adhesions often reform and in some cases, the surgery cannot be performed without risk of affecting the urethra (figure 19). figure 19. vaginal adhesions in alpaca following fetotomy clinical theriogenology • volume 12 number 3 • september 2020 281 urogenital surgeries in the male camelid castration castration at an early age delays closure of the long-bone physes, resulting in tall, post-legged geldings and a predisposition to early onset of degenerative osteoarthritis or patellar luxation. the authors’ recommended age for elective castration is 15 months for alpacas, 18 months for llamas and 2 years for camels. precastration considerations include examination of the scrotum for testicular descent, lesions or adhesions. presurgical administration of tetanus toxoid vaccination and antimicrobial therapy is recommended (procaine penicillin g is generally given at 22,000 u/kg sc or im before castration and daily for 3 days, or long acting ceftiofur 6.6 mg/kg sc once). although the prescrotal technique has been described most practitioners use the scrotal approach. the scrotal technique can be performed on the standing animal in llamas and camels, although lateral recumbency is recommend for alpacas.1,34 standing castration is performed after sedation with butorphanol alone or combined with xylazine, followed by local scrotal and testicular infiltration of lidocaine. if castration is to be performed in lateral recumbency, a combination of xylazine, ketamine, and butorphanol is indicated. the scrotum is prepared for aseptic surgery and an incision is made along the most ventral aspect of the scrotum, with the scrotal skin taught by holding the testis firmly into the scrotum. the testis is exteriorized and held with a towel clamp while the testicular cord is gently stripped from fat and connective tissue, using a piece of gauze (figure 20). the testis is removed using an emasculator (adult llamas) or after transfixation ligation of the spermatic cord with no. 0 (llamas, camels) or no. 2-0 (young llamas and alpacas) absorbable suture material. unilateral castration is indicated in breeding males with a unilateral testicular disorder such as orchitis, trauma, or neoplasia (figure 21).29 the procedure is similar to conventional castration, except that a primary closure is recommended to promote fast healing and reduce the effects on the remaining testis. the vaginal tunic is closed with a simple interrupted pattern using absorbable suture material. excess scrotal skin is removed and the subcutaneous tissue is apposed and closed with a simple continuous pattern. the skin is closed with a simple interrupted pattern using nonabsorbable suture material. postoperative care for castration includes antimicrobial therapy and confinement in a small pen for 24 hours. topical antiseptic and fly spray are indicated under farm conditions. the animal should be observed for excessive bleeding or swelling, exudative discharge due to infection, and difficulty urinating. however, postcastration complications are rare (table 3), and excessive bleeding can be managed by scrotal packing with gauze. hemostatic agents are very helpful in controlling the hemorrhage in some cases. under field conditions, the most common complication in camels is local wound infection and scirrhous cord development (figure 22). it is important to inform the client that some males may continue displaying copulatory activity even after castration. cryptorchidectomy cryptorchidism is more frequent in alpaca and llamas than in camels.35,36 in 1 study in alpacas, there was 3% unilateral cryptorchidism in 792 animals. the left testis seems to be slightly more affected than the right testis (58.3 versus 41.7%).37 table 3. complications following castration to remove normal descended testes in camelids complications llamas and alpacas* camels* no. % no. % no complications 168 92.3 47 73.4 anesthesia complication 1 0.5 slight hemorrhage 4 2.2 severe hemorrhage 1 0.5 severe scrotal edema/preputial edema 8 4.4 15 23.4 scirrhous cord 2 3.1 total 182 64 *all of these castrations were performed by veterinary student clinical theriogenology • volume 12 number 3 • september 2020282 figure 20. standing castration in dromedary camels figure 21. unilateral castration with primary closure in a dromedary with a severe testicular hemorrhage and hematoma due to a biting injury figure 22. scirrhous cord in a 12 year-old male dromedary figure 22. scirrhous cord in a 12 year old male dromedary following castration figure 23. serum testosterone concentrations in intact male alpacas (n = 8) and cryptorchid unilaterally castrated alpacas (n = 3) before and after hcg challenge figure 24. ultrasonogram of a retained testis in a bactrian camel (percutaneous inguinal, a b) and alpaca (transrectal) (c d, ub: urinary bladder clinical theriogenology • volume 12 number 3 • september 2020 283 surgical approaches for cryptorchidectomy include flank, inguinal and parainguinal laparotomy40 or laparoscopic assisted techniques.36 for camels, the parainguinal approach is favored by the authors in camels.35 the retained testis is often in proximity to the vaginal ring and may be retrieved using a spay hook. in some cases, direct exploration of the abdominal cavity is necessary. for the parainguinal approach, the inguinal canal is located by palpation and a 1 cm skin incision is made medial to the caudal border of the external inguinal ring and extended cranially 3 4 cm. the incision is continued carefully through all tissue layers into the peritoneum. the abdominal cavity is entered using 2 fingers and the retained testis is identified by palpation of the area around the incision. the cryptorchid testis usually lies just lateral to the vaginal ring. once identified, the testis is grasped and brought up to the incision site (figure 25). the spermatic cord is ligated with 2-0 polyglactin 910 and resected. the internal abdominal oblique muscle is closed with 0 polyglactin 910 in a continuous pattern. the fascia of the external abdominal oblique muscle is closed with 1 polyglactin 910 in a simple interrupted or simple continuous pattern. to close the skin, a subcuticular closure in a simple continuous pattern is sufficient. postoperative care should include nonsteroidal antiinflammatories and antimicrobials for 3 and 5 days, respectively. laparoscopy-assisted cryptorchidectomy offers the advantages of speed and reduced manipulation of the abdominal contents. the animal is placed in dorsal recumbency under general anesthesia and the ventral abdomen is prepared for surgery. the laparoscope portal may be placed over the umbilical scar. the intraabdominal testis is located and the second portal is placed laterally to the prepuce and used to introduce grasping forceps. the testis is grasped and elevated toward the inguinal area where a small parainguinal incision is made to exteriorize the testis (figure 26).36 a complete laparoscopic technique for cryptorchidectomy has been described and requires the use of 3 ports. the laparoscope portal is placed as described above and 2 instrument portals are placed on each side, ~ 5 cm from the prepuce. one portal is used to grasp the testis while the other is used to introduce laparoscopic, bipolar, electrocautery forceps. the spermatic cord is cauterized and transected and the testis is removed through the grasping instrument portal (figure 27). the portal may need to be enlarged at this point. portal sites are closed with a simple interrupted suture pattern. the skin is closed in cruciate pattern.36 vasectomy vasectomy is performed primarily for preparation of teaser males and for experimental studies where natural induction of ovulation (presence of ß subunit of nerve growth factor in seminal plasma) without fertilization is desired. in alpacas and llamas, vasectomy may be performed via laparoscopy or a prescrotal technique under general anesthesia (figure 28, 29).36,41 the inguinal region is clipped and surgically prepared. the spermatic cord is palpated lateral to the penis, caudal to the inguinal ring. a 2 cm skin incision is made over the spermatic cord and the vaginal tunic opened with a stab incision. the ductus deferens is isolated with hemostats. a 2 cm segment is isolated by blunt dissection and 2 ligatures of 2-0 polydioxanone are placed on each side of the segment prior to incision. the tunic is left open and the skin closed using 2-0 polydioxanone in a subcuticular pattern.42,43 epididymectomy is an alternative to vasectomy, but is not favored by the authors (figure 30).29 vasectomized males are sterile by 3 weeks after surgery. preputial prolapse, paraphimosis preputial lacerations are usually a consequence of masturbation behavior (breeding the ground or objects) or complications from foreign bodies within the prepuce. hair rings around the penis are common in llamas and suri alpacas. presenting complaints may be similar to those for urolithiasis. in some cases, the only sign is preputial bloody or purulent discharge. complications resulting in local adhesions and prevention of urination are possible.2,44 the penis and prepuce are evaluated under heavy sedation or general anesthesia. early management of preputial and penile injuries should center on providing adequate protection of the traumatized tissue (and prevention of infection and complication with urine scalding ointment containing lanoline, castor oil and antibiotics).45 clinical theriogenology • volume 12 number 3 • september 2020284 figure 30. cauda epididymectomy in camel figure 25. parainguinal approach to cryptorchidectomy in a bactrian camel figure 26. laparoscopic-assisted cryptorchidectomy in a male alpaca figure 27. laparoscopic cryptorchidectomy in a male llama figure 28. laparoscopic vasectomy in a male alpaca. a) identification of the vaginal ring, b d) transection of the vas deferens figure 29. inguinal vasectomy technique in camelids clinical theriogenology • volume 12 number 3 • september 2020 285 replacement of the healthy prolapsed preputial mucosa and its retention with a purse string suture is helpful in early cases (figure 31). daily cleaning of the sheath with saline and application of local antiinflammatory and antimicrobial ointment for 3 5 days will reduce the chance of further complications. sutures may be removed after 7 10 days. excessive preputial prolapse with slight necrosis requires circumferential resection and anastomosis of the prepuce. long standing lesions have a poor prognosis, particularly if there is overt cellulitis and tissue necrosis (figure 32). paraphimosis is a common complication (figure 33). tissue necrosis is a common problem with these cases and may include the penis due to pressure ischemia. surgical debridement and/or phallectomy may be required in these cases. in valuable racing camels, sand masturbation, the leading cause of preputial problems, is prevented by application of preputial rings (figure 34). soft palpate (dulla excision) the soft palate (dulla) is exteriorized frequently during the rutting season in the dromedary.46 impaction of this diverticulum with food or foreign bodies results in entrapment of the tissue under the molars and traumatization during mastication (figure 35). severe edema and abscess formation are common complications, resulting in a permanent exteriorization and dysphagia.47,48 in rare cases, the soft palate is swollen but not exteriorized and blocks respiration, which may lead to asphyxiation.49 surgical management (i.e. palatectomy) is the best course of action. excision of the prolapsed “dulla” is performed under heavy sedation or general anesthesia. the prolapsed tissue is excised after careful ligation of large vessels.49-52 postoperative care includes nsaids, antimicrobials and tetanus prophylaxis. animals should be on soft food for 3 to 4 days after surgery.29,51 palatectomy is often performed at a young age in racing camels to improve airflow during the breeding season. rectal prolapse rectal prolapse is common in obese male camels following excessive breeding, but can also be the results of other factors (e.g. diarrhea, excessive tenesmus, neoplasia, urolithiasis).53,54 it is classified in 3 degrees or types: type 1 involves the rectal mucosa and submucosa, type 2 involves the full thickness of the rectum and type 3 may include intussusception of some small colon (figure 36).55 types 1 and 2 are the most commonly encountered in breeding males and require sexual rest and sometimes surgical intervention. the rectal mucosa is replaced manually and retained with a purse string suture around the anal sphincter using a heavy nonabsorbable suture. the suture is left in place for 5 10 days. mucosal resection and anastomosis are indicated if the mucosa is damaged. amputation of the rectum is the last resort and carries a poor prognosis.53,54,56 cystotomy urolithiasis is relatively common in camelids. the etiology of urinary calculi in these species is not well understood but is suggested to be similar to that in other domestic ruminants.57,58 clinical signs include persistent straining, odontoprisis, anorexia and ileus, anorexia, dribbling blood-tinged urine, and signs of abdominal discomfort.58,59 more severe clinical signs ensue in cases of complete blockage and rupture of the urethra or bladder. fluid from abdominocentesis or the preputial swelling has increased creatinine concentration.59 increased serum urea nitrogen and creatinine concentrations suggest presence of uroperitoneum. transcutaneous ultrasonography of the ventral abdomen may show subcutaneous free fluid and tissue edema in the case of urethral rupture and large volumes of free fluid in the abdominal cavity in the case of urinary bladder rupture. in the latter case, the urinary bladder may not be possible to image. transrectal ultrasonography may reveal dilation of the pelvic urethra if the bladder is intact. prognosis is grave in the presence of hydroureter and hydronephrosis. uroliths are often located in the distal penile urethra, ~ 7 12 cm from the penile orifice but may occasionally be found immediately proximal to the sigmoid flexure. muscle relaxation and sedation may be obtained with acepromazine and diazepam (0.1 mg/kg slow iv). the urethral recess (diverticulum) at the ischial arch makes catheterization and retropulsion of uroliths impossible. the penis should be exteriorized and the tip of the glans penis (urethra) examined for presence of calculi (figure 37). clinical theriogenology • volume 12 number 3 • september 2020286 figure 31. preputial mucosa prolapse and replacement in an alpaca figure 32. preputial necrosis in an alpaca figure 33. paraphimosis in a camel. a) before treatment, b c) treatment with bandaging and emollient creams then suture in place, d) penis 7 months after treatment figure 34. preputial ring for prevention of masturbation in dromedary camels figure 35. traumatized permanently exteriorized soft palate in a dromedary (courtesy of dr. t. mahendra 51) clinical theriogenology • volume 12 number 3 • september 2020 287 figure 36. rectal prolapse in a dromedary camel caused by excessive mating figure 37. exteriorization of the penis and attempt to flush uroliths following catheterization of the urethra several surgical approaches have been attempted in alpacas and llamas, including urethrotomy, bladder marsupialization, tube cystotomy, and penile reefing.58 tube cystotomy technique is similar to that used in small ruminants on the anesthetized patient in dorsal recumbency using a paramedian approach. after exposure of the bladder, an incision is made into the bladder wall and the uroliths are removed followed by flushing of the urethra. a foley catheter (12 18 fr) is placed through a stab incision in the body wall of the inguinal region, inserted into the bladder and sutured in placed using a purse string suture (figure 38). the cystotomy incision is closed using an inverting suture pattern. in camels, urethrotomy is the most common salvage procedure.60 however, a case of successful management with tube cystotomy was recently described in a bactrian camel.61 overall, these surgeries carry a poor prognosis, particularly for reproduction. figure 38. tube cystotomy in a llama clinical theriogenology • volume 12 number 3 • september 2020288 conclusions surgical interventions on the urogenital systems are common in camelid practice. veterinarians providing theriogenology services should be trained for a wide range of reproductive surgeries. most of the surgical techniques are similar to those practiced in other large domestic animal species. however, case management should take into account anesthetic management and species anatomical differences. as data continue to accumulate on postoperative care and possible complications, veterinarians can provide better assessments when communicating with clients. conflict of interest there are no conflicts of interest to declare. references 1. tibary a: surgery of the reproductive tract in lamoids. in: youngquist r, threfall w: editors. current therapy in large animal theriogenology. 2nd edition, philadelphia; saunderselsevier: 2007. p. 905-918 2. tibary a, anouassi a: surgery of the reproductive tract in the female. in: tibary a, anouassi a: editors. theriogenology in camelidae: anatomy, physiology, bse, pathology and artificial breeding. abu dhabi, uae: actes editions, institut agronomique et veterinaire hassan ii, rabat morocco: 1997. p. 375-390. 3. tibary a, rodriguez js, anouassi a, et al: management of dystocia in camelids. stillwater: proc am assoc of bovine pract 2008; p.166-176. 4. elias e: left ventrolateral cesarean section in three dromedary camels (camelus dromedarius). vet surg 1991;20:323325. 5. lopes maf: flank caesarean section in a bactrian ceml (camelus bactrianus) restrained ins ternal recumbency and post-operative treatment with enteral fluid therapy. j camelid sci 2013;6:122-131. 6. pearson lk, rodriguez js, tibary: uterine torsion in late gestation alpacas and llamas: 60 cases (2000-2009). small ruminant res 2012;105:268-272. 7. campbell a, pearson l, tibary a: cesarean section in alpacas and llamas at a referral center technique, survival, and postoperative fertility: 24 cases (2000-2012). clinical theriogenology 2013;5:360. 8. miller ba, brounts sh, anderson de, et al: cesarean section in alpacas and llamas: 34 cases (1997-2010). j am vet med a 2013;242:670-674. 9. tibary a, pearson lk, anouassi a: cesarean section in camels (camelus dromedarius). complications and postsurgical fertility. clinical theriogenology 2015;7:329. 10. tibary a, anouassi a: retrospective study on an unusual form of ovario-bursal pathology in the camel (camelus dromedarius). theriogenology 2001;56:415-424. 11. egloff c, gerspach c, rütten m, et al: pyometra and persistent hymen in an alpaca. tierärztliche praxis ausgabe g, grosstiere/nutztiere 2013;41:185-189. 12. bravo pw, sumar j: laparoscopic examination of the ovarian activity in alpacas. anim reprod sci 1989;21: 271-281. 13. campbell a, tibary a: infertility of female and male camelids. clinical theriogenology 2017;9:311-319. 14. tibary a, ruiz a: laparoscopia y cirurgica laparoscpica. in: e mellisho ed. tecnica de investigacion reproductiva en camelidos. lima, peru: asociacion peruana de reproduccion animal: 2018. p. 33-44. 15. king mr, hendrickson da, southwood ll, et al. laparoscopic ovariectomy in two standing llamas. j am vet med a 1998;213:523-525. 16. yarbrough tb, snyder jr, harmon fa: laparoscopic anatomy of the llama abdomen. vet surg 1995;24:244-249. 17. morrow ck, barrington gm, johnson lw, et al: how to perform laparoscopic ovariectomy in llamas and alpacas. vet med 1998;93:295-296. 18. rodgerson dh, baird an, lin hc, et al: ventral abdominal approach for laparoscopic ovariectomy in llamas. vet surg 1998;27:331-336. 19. pearson l, campbell a, tibary a: laparoscopic-assisted ovariectomy in alpacas (vicugn pacos). clinical theriogenology 2013;5:372. 20. carpenter em, hendrickson da, anderson de: laparoscopic ovariectomy and ovariohysterectomy in llamas and alpacas. vet clin n am-equine 2000;16:363-375. 21. ruiz a, fumoso f, patino c, et al: laparoscopic follicular aspiration and oocyte recovery in alpacas (vicugna pacos). proceeding 5th conferences of the international society of camelid research and development 2018; p. 360-365. 22. anwar s, purohit gn: occurrence and surgical repair of third degree perineal lacerations in adult female camels (camelus dromedarius) by one-stage (goetz) technique. open vet j 2013;3:75-79. 23. tibary a, anouassi a: reproductive disorders of the female camelidae. in: tibary a: editor. theriogenology in camelidae: anatomy, physiology, bse, pathology and artificial breeding. abu dhabi, uae: actes editions, institut agronomique et veterinaire hassan ii : 1997: p. 317-368. clinical theriogenology • volume 12 number 3 • september 2020 289 24. pearson lk, rodriguez js, tibary a: disorders and diseases of pregnancy. in: cebra c, anderson de, tibary a, et al: editors. llama and alpaca care. st. louis; w.b. saunders: 2014. p. 256-273. 25. mcnaughten jw, pozor ma, mallicote mf, et al: non-surgical management of vaginal prolapse in a late gestation alpaca (lama pacos). clinical theriogenology 2014;6:489-493. 26. gutierrez c, corbera ja, morales i, et al : uterine prolapse in 2 dromedary camels. can vet j 2001;42:803-804. 27. van straten m: periparturient conditions affecting camels (camelus dromedarius) in israel and their treatments. revue d'élevage et de médecine vétérinaire des pays tropicaux 2000;53:101-104. 28. agab h: diseases and causes of mortality in a camel (camelus dromedarius) dairy farm in saudi arabia. j camel pract res 2006; 13:165-169. 29. tibary a, anouassi a: pathology and surgery of the reproductive tract and associated organs in the male camelidae. in: tibary a, anouassi a: edotors. theriogenology in camelidae: anatomy, physiology, bse, pathology and artificial breeding. abu dhabi, uae: actes editions, institut agronomique et veterinaire hassan ii: 1997. p. 115132. 30. hallowell gd, potter tj, mills nj: labial fusion causing urinary tract obstruction in an alpaca cria. veterinary record 2007;161:862. 31. tibary a, anouassi a, memon ma: an approach to the diagnosis of infertility in camelids: retrospective study in alpaca, llamas and camels. j camel pract res 2001;8:167-179. 32. wilkins pa, southwood ll, bedenice d: congenital vulvar deformity in 6 alpacas. j amer vet med a 2006;229:263265. 33. bennett j, kennel a, stanhope cr: surgical correction of an acquired vaginal stricture in a llama, using a carbondioxide laser. j amer vet med a1998;212:1436-1437. 34. tibary a, anouassi a. standing castration in camels. j camel pract res 2004;11:125-127. 35. garcia pereira fl, allen a, anouassi a, et al : parainguinal cryptorchidectomy under general anaesthesia in a bactrian camel (camelus bactrianus). j camel pract res 2004;11:103-107. 36. tibary a, ruiz a, campbell a: laparoscopic-assisted cryptorchidectomy in alpacas (vicugna pacos). proceedings 5th conferences od the international society of camelid research and development 2018; p.361-363. 37. sumar j: studies on reproductive pathology in alpacas. ms thesis dept obstet and gynaec, college of vet med, swedish,univ of agric sci, uppsala. 1983. 38. fite c: endocrine and clinical effects of hcg in lhrh immunized and non-immunized male alpacas (vicugna pacos). pullman, wa: departmen of veterinary clinical sciences college of veterinary medicine, washington state university; 2006. 39. ciccarelli m, tibary a, campbell aj, et al: effect of age and castration on serum anti-müllerian hormone concentration in male alpacas. theriogenology 2018;105:174-177. 40. parker je, semevolos sa. use of a parainguinal approach for cryptorchidectomy in alpacas. j am vet med a 2002;220:1835-1836. 41. bravo pw, sumar j: evaluation of intra-abdominal vasectomy in llamas and alpacas. j amer vet med a 1991;199:1164-1169. 42. pearson l, sandoval s, tibary a: pre-scrotal vasectomy in alpacas (vicugna pacos).technique and complication rate. clinical theriogenology 2012;4:424. 43. pearson lk, campbell aj, sandoval s, et al: effects of vasectomy on seminal plasma alkaline phosphatase in male alpacas (vicugna pacos). reprod domest anim 2013;48:995-1000. 44. tibary a, pearson lk. reproductive emergencies in camelids. clinical theriogenology 2014;6:579-592. 45. koziol jh, edmondson ma, wolfe df, et al: successful resolution of a preputial prolapse in an alpaca using medical therapy. canadian vet j 2015;56:753-755. 46. tibary a, pearson lk, anouassi a: applied andrology in camelids. in: chenoweth pj, lorton s: editors. animal andrology: theory and applications. wallingford, cabi: 2014. p. 418-449. 47. reece jf, chawla sk: prolapse of the soft palate in a male arabian camel (camelus dromidarius). vet rec 2001;149:656-657. 48. gahlot tk, jhirwal sk, bishnoi p, et al: facial paralysis, glossoplegia and injured soft palate in a camel. j camel pract res 2004;11:157-158. 49. m'zah a, ben said ms, matoussi a, et al: excision of the soft palate in a male dromedary. revue de médecine vétérinaire 1993;144:885-890. 50. biswadeep j, abhishek s: resection of entrapped, over-distended soft palate in a male dromedary camel (camelus dromedarius). international j livestock res 2014;4:54-57. 51. mahendra t, bishnoi p, palecha s, et al: surgical soft palate excision a clinical study of 8 camels. intas polivet 2016;17:567-568. 52. al-sobayil fa, ahmed af: surgery of the injured dulla in dromedary camels (camelus dromedarius). iran j vet surg 2011;6:17-22. 53. madeh s: rectal prolapse in camels (camelus dromedarius): clinical findings and treatment outcomes. j camel pract res 2019;26:99-103. 54. sickinger m, brachthäuser l, köhler k, et al: rectal prolapse in a male llama: a case report. wiener tierärztliche monatsschrift. 2012;99:47-51. clinical theriogenology • volume 12 number 3 • september 2020290 55. ramadan ro: surgery and radiology of the dromedary camel. al ahsa, 31982: college of veterinary medicine, king faisal university, box 1757;1994. 56. zabady mk, fouda ta, abdin-bey mr, et al: rectal prolapse in dromedary camels. j camel pract and res 2011;18:337-344. 57. gutierrez c, corbera ja, faye b: obstructive phosphate urolithiasis in a dromedary camel: a case report. j camel pract res 2008;15:77-79. 58. duesterdieck-zellmer kf, metre dcv, cardenas a, et al: acquired urethral obstruction in new world camelids: 34 cases (1995-2008). aust v j 2014;92:313-319. 59. tibary a, rodriguez j, sandoval s. reproductive emergencies in camelids. theriogenology 2008;70:515-534. 60. kaswan bl, mahendra t, purohit nr, et al: surgical management of urinary retention in three camels (camelus dromedarius). j camel pract res 2015;22:141-144. 61. rosser jm, jacob si, brounts sh: use of tube cystostomy in the surgical management of obstructive urolithiasis in a bactrian camel. j am vet med a 2019;254:868-873. clinical theriogenology • volume 12 number 3 • september 2020 291 019_ms-019 tibary 2 antisperm antibodies and immune mediated infertility. are we missing something? antisperm antibodies and immune mediated infertility. are we missing something? maria ferrer department of large animal medicine, college of veterinary medicine university of georgia, athens, ga abstract immune mediated infertility was first reported in stallions in the 1990s. dysregulation of the tolerogenic testicular environment leads to development of autoimmune orchitis (ao), defined as an autoimmune reaction to germ cell and sperm antigens. ao is characterized by testicular interstitial infiltration with leukocytes, immunoglobulin binding to germ cells in seminiferous tubules, disruption of spermatogenesis, production of antisperm antibodies (asas) and infertility. clinical diagnosis of ao is based on detection of asas. furthermore, asas can cause infertility by preventing sperm binding to oviductal cells, undergoing capacitation and binding to the zona pellucida. however, icsi circumvents these effects and enables pregnancies to be obtained from males with asas. other reported treatments include glucocorticoid treatment for immunosuppression, or laboratory techniques to select asa-free sperm. although ao is poorly understood in stallions, presence of sperm-directed igg and iga has been described in stallions with poor semen quality and infertility. immune mediated infertility may represent an overlooked cause of reduced reproductive efficiency in stallions. keywords: autoimmune orchitis, equine, stallion, semen, sperm directed antibodies first reports of antisperm antibodies in stallions the presence of antisperm antibodies (asas) in stallions was first reported in the 1990s. two case reports simultaneously linked the presence of sperm directed igg in serum or seminal plasma to infertility.1,2 both infertile stallions had a history of scrotal trauma 6 months to 2 years prior to diagnosis of asas. on presentation, these stallions were oligospermic and asthenospermic. however, 1 week after treatment with glucocorticoids, sperm motility and fertility temporarily recovered. concurrently, asa titers decreased, providing evidence for an effect of asas on stallion fertility.1,2 autoantigenicity of stallion sperm was confirmed in 1994 when two stallions were immunized with autologous sperm.3 immunization induced an increase in serum igg and seminal plasma iga titers that peaked in 2 4 weeks, and returned to prevaccination levels by 20 weeks. concomitant with increased asa titers, both stallions developed oligospermia and teratospermia.3 testicular immunology and the origin of asas initiation of spermatogenesis leads to expression of new proteins by developing germ cells that are recognized as foreign by the immune system (neoantigens) since immune self-tolerance is established in utero. however, these neoantigens are normally tolerated without inducing immune responses. testicular immune privileged status is maintained by tissue physical structures, local immunosuppressive milieu and systemic immune tolerance working cooperatively. the blood testes barrier (btb) is formed during puberty by specialized junctions between adjacent sertoli cells and is supported by the basement membrane of seminiferous tubules and peritubular myoid cells.4,5 the btb physically divides seminiferous tubules into basal and adluminal compartments. spermatogenesis is initiated within the basal compartment, where type a spermatogonia divide and start their differentiation. type b spermatogonia progress to preleptotene spermatocytes, which then translocate to the adluminal compartment. the progression of meiosis, spermiogenesis and spermiation occurs within the adluminal compartment, where contact between neoantigens and the immune system is prevented by the btb.4 however, it must be noted that preleptotene spermatocytes, type b and type a spermatogonia within the basal compartment also express neoantigens capable of inducing an autoimmune response. permeability of the btb is regulated by fsh and testosterone.6 in seasonal breeding animals, the absence of fsh results in increased permeability of the btb during the nonbreeding season.6 in addition, sperm can interact with the immune system at the rete testis and throughout 361 clinical theriogenology • volume 11, number 3 • september 2019 the excurrent duct system, as a blood tissue barrier is not present at these sites. other factors are therefore also involved in preventing an autoimmune response against germ cell antigens. sertoli cells are key players in testicular immune tolerance. apoptotic germ cells and their antigens are removed from the seminiferous tubules by phagocytosis by sertoli cells. prior to spermiation, redundant cytoplasm and plasma membrane of elongated spermatids is packaged into a large cytoplast, the residual body, retained in the seminiferous tubule and a small cytoplast, the cytoplasmic droplet, attached to the sperm. residual bodies contain germ cell antigens that are captured by sertoli cells in the adluminal compartment and are released basally and into the interstitial space through sertoli cell basal projections, bypassing the btb by intracellular transport.7 the release occurs at spermiation and egressed neoantigens are crucial for maintenance of systemic tolerance mediated by regulatory t (treg) cells.7,8 sertoli cells function as nonprofessional tolerogenic antigen presenting cells by inducing enrichment of treg.9 the testis and draining lymph nodes are enriched for sperm specific tregs that normally suppress autoreactive t cells.10 within the interstitial space, resident macrophages and dendritic cells also help maintain the tolerogenic environment. resident macrophages express the m2 phenotype, characterized by production of antiinflammatory cytokines.11 testicular dendritic cells also have an immature and tolerogenic phenotype. resident dendritic cells capture neoantigens and maintain immunologic tolerance by routinely migrating to draining lymph nodes and presenting self-antigens to lymphocytes in a tolerogenic manner.12 paracrine factors involved in testicular immune regulation include testosterone from leydig cells, tgf β1 produced by leydig and sertoli cells, activin a expressed by most testicular cells and il10 from resident macrophages.13 dysregulation of the tolerogenic environment leads to development of autoimmune orchitis (ao), which can be primary or secondary to inflammation, infection, trauma or toxins, among others. ao is defined as an autoimmune reaction to testicular antigens characterized clinically by presence of asas and infertility.14 several components of the innate immune system are present in the male genital tract. in the testis, toll like receptors (tlrs) are expressed in all immune, somatic and germ cells.15 in particular, activation of tlr 2 and 4 is critical for development of ao in mice models.16 microbial components and damaged germ cells induce pro-inflammatory cytokine production by sertoli cells through tlr activation.15 heat shock proteins (hsp60 and hsp70), disulfide isomerase er 60 and outer dense fiber protein 2 have been proposed as damage associated molecular patterns (damps) released by germ cells in a rodent model of experimental auto immune orchitis (eao).17 in addition, activation of tlrs in sertoli cells disassembles junctional complexes involved in the btb and sertoli cell germ cell interaction, resulting in sloughing of germ cells.15 furthermore, activation of tlrs in leydig cells suppresses steroidogenesis, altering testicular function through decreased intratesticular testosterone concentration. activation of tlrs in germ cells directly induces apoptosis.15 these effects are mediated by inflammatory cytokines, which also induce recruitment of macrophages, neutrophils, mast cells and lymphocytes to the testis. recruited leukocytes further contribute to production of proinflammatory cytokines and mast cells contribute to tissue fibrosis and thickening of the basement membrane. under inflammatory conditions, immature testicular dendritic cells overcome immune tolerance. proinflammatory cytokines induce maturation of testicular dendritic cells (dc) that capture germ cell antigens released to the interstitium during the course of the disease.18,19 these inflammatory dc migrate to the draining lymph nodes and activate t lymphocytes. sensitized lymphocytes in turn migrate to the testes where progressive amplification of the autoimmune response leads to chronic ao with continuous antigen presentation of dc to lymphocytes in lymph nodes and testis.18,19 although pathophysiology of ao in stallions is not known, eao has been induced by immunization with autologous sperm.20 stallions had increased serum igg and seminal plasma iga titers 1 week after immunization, concurrent with decreases in sperm numbers and morphologically normal sperm. chronic inflammatory changes around the rete testis, hypospermatogenesis and presence of immature germ cells and low sperm numbers in the epididymis was described on histopathology.20 362clinical theriogenology • volume 11, number 3 • september 2019 diagnosis of autoimmune orchitis clinical diagnosis of ao is based on detection of asas. the current consensus in human andrology is that only sperm-bound igg and iga are relevant to fertility.14 therefore, assays considered most relevant are those that detect igg or iga directly bound to sperm (direct assays). indirect techniques detect asas in serum or seminal plasma. serum or seminal plasma from the infertile animal are incubated with sperm from a “normal,” asa-free donor. binding of serumor seminal plasma derived antibodies to donor’s sperm was detected in stallions using a sperm agglutination or immobilization test, indirect immunofluorescence or enzyme linked immunosorbent assay.1-3,20,21 other indirect tests used in human medicine include immunocytochemistry, indirect mixed antiglobulin reaction and indirect immunobead binding test.14 direct techniques detect asas already bound to the infertile male’s sperm at ejaculation. the direct mixed antiglobulin reaction and immunobead binding tests (dibt) are most commonly used in human andrology.14 in 2010, ferguson et al. reported cross reaction between human and equine asas and used a dibt to demonstrate sperm-bound asas in a stallion with unexplained infertility.22 although the dibt has promise for clinical applications in the field, it has not been further validated in stallions. in addition, the dibt is based on counting motile sperm bound to antibody-coated beads. therefore, the estimation is subjective and requires good sperm motility in samples from infertile patients.14,22 instead, flow cytometry allows objective and quantitative estimation of asas on the surface of living or dead sperm and is a sensitive, specific and repeatable test. flow cytometry also allows identification of antibody class, isotype and load.23 a direct diagnostic test was standardized in veterinary medicine to detect sperm-bound asas using flow cytometry and was used to characterize igg and iga binding to bovine and equine sperm.24-26 lastly, testicular biopsies in men with testicular dysfunction of unknown origin often reveal inflammatory changes characterized by peritubular infiltration with lymphocytes, nonresident macrophages and mast cells, translocation of macrophages and mast cells from the interstitium to the seminiferous tubules, tubular atrophy, hyalinization of basement membrane with deposition of immunoglobulins and complement and interstitial fibrosis, representing varying stages of ao.8 clinical manifestations and indications for testing immune mediated infertility does not have a pathognomonic clinical presentation. in men, testing for asas is recommended if sperm agglutination occurs in the absence of clinical infection, sperm motility is < 30%, there is poor mucus penetration, or unexplained infertility.14 in stallions, asas have been identified in the center of sperm clumps, indicating that they are involved in agglutination (ferrer, unpublished); presence of this phenomenon warrants investigation of asas. low sperm motility was also identified in iga and igg positive stallions and bulls,1,2,25,26 and therefore asa investigation may be indicated in cases of asthenospermia. in addition, presence of iga was associated with poor sperm morphology in stallions and bulls.25,26 testing teratospermic animals may also be indicated. role of asas in unexplained infertility in normospermic stallions is less clear. in one study, 30% of unsatisfactory breeder stallions had significant iga binding, whereas no satisfactory breeder stallions were iga positive.25 therefore, iga binding is less likely to be associated with idiopathic infertility. however, igg binding was equally prevalent (30%) among unsatisfactory and satisfactory breeders.25 a similar prevalence of igg positive sera (34%) was described among subfertile, unsatisfactory breeder stallions, whereas only 5% satisfactory breeders were igg positive.20 some of the igg positive satisfactory breeder stallions developed low efficiency of spermatogenesis with time, suggesting they were in the early stages of ao.20 conversely, some of these stallions could have been in the recovery phase of testicular pathology. persistence of asas has been implicated in long-term effects of genital infection on human fertility. even when semen quality returns to normal values, asas are estimated to persist for an average of 5 years.27 persistence of asas for 1.5 2 years was described in stallions and bulls after traumatic or infectious orchitis.1,28 therefore, testing normospermic stallions with unexplained infertility for sperm-directed igg is recommended.25 363 clinical theriogenology • volume 11, number 3 • september 2019 additionally, presence of sperm bound iga and igg was associated with poor motility and membrane integrity of cooled stallion sperm.29 testing stallions with poor tolerance to cooling for asas may be recommended. effects on fertility initial reports linked presence of asas in serum or seminal plasma to infertility in stallions.1,2,20,21 however, mechanisms by which asas affect equine fertility have not been described. using a model of eao in bulls, sperm bound asas reduced ability of bovine sperm to bind to oviductal explants in vitro.30 this effect was mediated primarily by igg. since bi is correlated with non-return rates, it was proposed that sperm bound igg may affect bovine fertility by reducing ability of sperm to form an oviductal reservoir.30 similarly, antiserum raised against proteins from the periacrosomal membrane reduced ability of equine sperm to attach to oviductal epithelial cells in vitro.31 the antiserum recognized integral membrane components present on epididymal sperm and spermatids, as well as adsorbed seminal plasma proteins present in ejaculated sperm.31 experimentally induced asas prevent sperm capacitation in vitro in bulls.32 membrane fluidity changes associated with sperm capacitation were prevented by sperm-bound iga. similarly, asas blocked sperm capacitation in humans by preventing removal of cholesterol from the plasma membrane and inhibiting membrane fluidity changes.33,34 furthermore, sperm-bound iga reduced ability of bovine sperm to bind to the zona pellucida in vitro.32 in men, asas block sperm zona binding by inhibiting capacitation-associated increases in surface expression of mannose receptors.34 binding to the zona pellucida or exposure to soluble factors in the vicinity of the cumulus oocyte complex stimulates the acrosome reaction, for which capacitation and loss of cholesterol is also a prerequisite. however, bovine and human asas did not affect the ability of sperm to undergo the acrosome reaction in response to calcium ionophore a23187.32,34 stimulation of the acrosome reaction with calcium ionophore a23187 is not physiological and bypasses the activation of calcium channels that occur during sperm capacitation. when a more physiologic stimulus was used (i.e. zona pellucida proteins), the acrosome reaction was inhibited by human asas.34 lastly, experimentally induced asas can affect fertilization rates in vitro and in vivo.35-37 bovine asas in sera of immunized bulls and heifers had a negative effect on in vitro fertilization (ivf).35,36 mating of female wallabies to males with sperm-bound asas resulted in 20% in vivo fertilization rate compared to 95% in females mated to control, nonimmune males.37 mechanisms proposed for these effects were alterations in sperm motility, or inhibition of sperm capacitation, zona pellucida binding and oocyte penetration.35,37 immunolocalization of experimentally induced asas was evaluated in bulls using immunofluorescence. both igg and iga bound mainly to the acrosomal, equatorial and post-acrosomal areas, and to cytoplasmic droplets.24 however, antigen specificity is not known. in stallions, 62 and 70 kd proteins were identified as autoantigens.3 in humans, antibodies against fertilization antigen 1 (fa 1) inhibited sperm capacitation, acrosome reaction and zona pellucida binding. addition of anti fa 1 antibodies to the ivf medium also decreased fertilization rate in cattle.38 ylp12 is another protein involved in binding to the zona pellucida protein zp3. anti ylp12 antibodies inhibited sperm zona binding in humans and mice.39 antibodies against other antigens, e.g. epithelial cadherin, crisp 1, zonadhesin, p34h and saga 1, blocked sperm–zona binding in humans and rodents.40 most of these antigens are located on the sperm membrane around the acrosomal area. treatment to date, there are no critically tested treatments for male immune mediated infertility. immunosuppressive compounds have been used to decrease asa production. treatment of stallions with dexamethasone (20 mg iv sid) or prednisolone (0.5 g iv bid) for 1 3 weeks decreased asa titers, improved semen quality and increased pregnancy rates.1,2 the effect was transient and semen quality deteriorated after treatment was discontinued.1 whereas glucocorticoids have been used in humans, fertility was transiently increased in only 20% of men during prednisolone treatment.41 other studies have 364clinical theriogenology • volume 11, number 3 • september 2019 failed to identify an increase in pregnancy rate or semen quality, in spite of decreased igg binding.42 benefits of glucocorticoid treatment are controversial and their side effects include potential endocrine disruption with a detrimental effect on semen quality.43 laboratory techniques aim at removing asa bound sperm from the ejaculate, or removing asas from the sperm surface. in stallions, washing by centrifugation did not remove asas. however, single layer centrifugation enriched a population of asa-free sperm and improved semen quality and tolerance to cooling (ferrer, unpublished). similar findings were reported in men.44,45 immunomagnetic separation was also able to reduce asa bound sperm in humans, although with a low efficiency.46 proteases have been used to destroy asas on the sperm surface. although these treatments were successful at reducing asa binding, they also caused damage to the sperm.45 lastly, ivf circumvents the problem of the reduced sperm reservoir. in consequence, ivf has helped achieve pregnancies in some, but not all, reports in human medicine.45 controversial results with ivf are likely due to the heterogeneity in antibody class and antigen specificity. because intracytoplasmic sperm injection (icsi) circumvents most of the known effects of asas on sperm function, pregnancy rates are similar between asa positive and asa negative men after icsi. therefore, icsi is currently the most commonly used treatment for asa mediated male infertility in humans.45 antisperm antibodies in mares females can also mount an immune response against sperm. indeed, due to the lack of tolerogenic sperm antigen presentation, females mount a stronger immunological response to sperm antigens than males.7 when mares were immunized with sonicated sperm, serum igg was detected using elisa from 1 to 5 weeks after immunization, with a detectable serum iga response present on week 5.47 immunized mares were then inseminated over 5 consecutive estrous cycles the following breeding season. while asa titers had returned to prevaccination levels, serum igg rapidly increased by 1 week postovulation in the first insemination cycle and remained elevated throughout the study. similarly, uterine iga increased after the first insemination in 1 mare and after the fourth insemination in the other mare.48 serum iga and uterine igg titers did not differ from levels observed in control mares. in spite of increased asa titers, pregnancy rates were not different between immunized and control mares.48 however, definitive conclusions on the effect of mare asas on fertility cannot be made due to limited numbers of mares in each group (n = 2). conclusion presence of asas represent a relevant entity in male infertility. advances have been made in diagnostic methods and understanding how asas affect sperm function. however, pathophysiology of ao is poorly understood in domestic animals. without this knowledge, designing effective treatments will remain challenging. nevertheless, recommendations in this review regarding indications for testing and semen processing techniques may improve sample quality. conflict of interest there are no conflicts of interest to declare. references 1. papa fo, alvarenga ma, lopes md, et al: infertility of autoimmune origin in a stallion. equine vet j 1990; 22:145-146. 2. zhang j, ricketts sw, tanner sj: antisperm antibodies in the semen of a stallion following testicular trauma. equine vet j 1990;22:138-141. 3. teuscher c, kenney rm, cummings mr, et al: identification of 2 stallion sperm-specific proteins and their autoantibody response. equine vet j 1994;26:148-151. 4. cheng cy, mruk dd: the blood-testis barrier and its implications for male contraception. pharmacol rev 2012; 64:16-64. 5. rode k, sieme h, richterich p, et al: characterization of the equine blood-testis barrier during tubular development in normal and cryptorchid stallions. theriogenology 2015;84:763-772. 6. stanton pg: regulation of the blood-testis barrier. seminars in cell & dev boil 2016;59:166-173. 365 clinical theriogenology • volume 11, number 3 • september 2019 7. tung ks, harakal j, qiao h, et al: egress of sperm autoantigen from seminiferous tubules maintains systemic tolerance. j clin invest. 2017;127:1046-1060. 8. fijak m, pilatz a, hedger mp, et al: infectious, inflammatory and ‘autoimmune’ male factor infertility: how do rodent models inform clinical practice? human reprod update, 2018;24:416-441. 9. secco vd, riccioli a, padula f, et al: mouse sertoli cells display phenotypical and functional traits of antigenpresenting cells in response to interferon gamma. biol reprod 2008;78:234–242. 10. jacobo p: the role of regulatory t cells in autoimmune orchitis. andrologia 2018;50,e13092. https://doi.org/10.1111/and.13092. 11. bhushan s, meinhardt a: the macrophages in testis function. j reprod immunol 2017;119:107-112. 12. rival c, guazzone va, von wulffen w, et al: expression of co-stimulatory molecules, chemokine receptors and proinflammatory cytokines in dendritic cells from normal and chronically inflamed rat testis. mol human reprod 2007;13:853-861. 13. chen q, deng t, han d: testicular immunoregulation and spermatogenesis. semin cell dev biol 2016;59:157-165. 14. silva ca, cocuzza m, carvalho jf, et al: diagnosis and classification of autoimmune orchitis. autoimmunity reviews 2014;13:431-434. 15. hedger mp: toll-like receptors and signalling in spermatogenesis and testicular responses to inflammation—a perspective. j reprod immunol 2011;88:130-141. 16. liu z, zhao s, chen q, et al: roles of toll-like receptors 2 and 4 in mediating experimental autoimmune orchitis induction in mice. biol reprod 2015;63:1-11. 17. fijak m, iosub r, schneider e, et al: identification of immunodominant autoantigens in rat autoimmune orchitis. j pathol 2005;207:127-138. 18. guazzone va, hollwegs s, mardirosian m, et al: characterization of dendritic cells in testicular draining lymph nodes in a rat model of experimental autoimmune orchitis. int j androl 2010;34:276-289. 19. rival c, guazzone va, von wulffen w. exppression of co-stimulatory molecules, chemokine receptors and proinflammatory cytokines in dendritic cells from normal and chronically inflamed rat testis. mol human reprod 2007;13:853-861. 20. kenney rm, cummings mr, teuscher c, et al: possible role of autoimmunity to spermatozoa in idiopathic infertility of stallions. j reprod fertil suppl 1998;56:23-30. 21. day mj: detection of equine antisperm antibodies by indirect immunofluorescence and the tube-slide agglutination test. equine vet j 1996;28:494-495. 22. ferguson ac, morrison lc, rodger ja et al: detection of antisperm antibodies in equine semen using the immunobead test. anim reprod sci 2010;121s:s151-s152. 23. räsänen ml, hovatta ol, penttilä im, et al: detection and quantitation of sperm-bound antibodies by flow cytometry of human semen. j androl 1992;13:55-64. 24. sardoy mc, anderson de, george a, et al: standardization of a method to detect bovine sperm-bound antisperm antibodies by flow cytometry. theriogenology 2012;78:1570-1577. 25. ferrer ms, miller lmj: equine sperm-bound antisperm antibodies are associated with poor semen quality. theriogenology 2018;118:212-218. 26. ferrer ms, laflin s, anderson de, et al: prevalence of bovine sperm-bound antisperm antibodies and their association with semen quality. theriogenology 2015;84:94-100. 27. hinting a, soebaldi dm, santoso ri: evaluation of the immunological cause of male infertility. andrologia 1996;28:123-126. 28. vlok i, ferrer m, sardoy c, et al: serum anti-sperm antibodies associated with orchitis in a bull. clin theriogenology 2009;1:125. 29. ferrer ms, miller lmj, george a, et al: sperm-bound antisperm antibodies affect motility of cooled stallion spermatozoa. j equine vet sci 2016;43:s64-s65. 30. ferrer ms, anderson de, miller lmj, et al: effect of bovine sperm-bound antisperm antibodies on oviductal binding index. reprod dom anim 2016;51:287-293. 31. thomas pga, ball ba, ignotz gg, et al: antibody directed against plasma membrane components of equine spermatozoa inhibits adhesion of spermatozoa to oviduct epithelial cells in vitro. biol reprod 1997;56:720-730. 32. ferrer ms, klabnik-bradford j, anderson de, et al: sperm-bound antisperm antibodies prevent capacitation of bovine spermatozoa. theriogenology 2017;89:58-67. 33. nakagawa k, yamano s, kamada m, et al: sperm-immobilizing antibodies suppress an increase in the plasma membrane fluidity of human spermatozoa. fertil steril 2004;83:1054-1058. 34. benoff s, cooper gw, hurley i, et al: antisperm antibody binding to human sperm inhibits capacitation induced changes in the levels of plasma membrane sterols. am j reprod immunol 1993;30:113-130. 35. kim ca, parrish jj, momont hw, et al: effects of experimentally generated bull antisperm antibodies on in vitro fertilization. biol reprod 1999;60:1285-1291. 36. menge ac, stone wh, tyler wj, et al: immunological studies on fertility and sterility. iv. fertility of cattle and rabbits inseminated with semen treated with antibodies produced against semen, spermatozoa and erythrocytes. j reprod fertil 1962;3:331-41. 366clinical theriogenology • volume 11, number 3 • september 2019 https://doi.org/10.1111/and.13092 37. asquith kl, kitchener al, kay dj: immunization of the male tammar wallaby (macropus eugenii) with spermatozoa elicits epididymal antigen-specific antibody secretion and compromised fertilization rate. j reprod immunol 2006;69:127-147. 38. conrood sa, westhusin me, naz rk: monoclonal antibody to human fertilization antigen-1 (fa-1) inhibits bovine fertilization in vitro: application in immunocontraception. biol reprod 1994;51:14-23. 39. naz rk, chauhan sc: human sperm-specific peptide vaccine that causes long-term reversible contraception. biol reprod 2002;67:674-680. 40. vazquez-levin mh, marin-briggiler ci, veaute c: antisperm antibodies: invaluable tools toward the identification of sperm proteins involved in fertilization. am j reprod immunol 2014;72:206-218 41. hendry wf, hughes l, scammell g et al: comparison of prednisolone and placebo in subfertile men with antibodies to spermatozoa. lancet 1990; i:85-88. 42. haas gg jr, manganiello p: a double-blind, placebo controlled study of the use of methylprednisolone in infertile men with sperm-associated immunoglobulins. fertil steril 1997; 47:295-301. 43. barth ad, bowman pa: the sequential appearance of sperm abnormalities after scrotal insulation or dexamethasone treatment in bulls. can vet j 1994;35:93-102. 44. almagor m, margalioth ej, yaffe h: density differences between spermatozoa with antisperm autoantibodies and spermatozoa covered with antisperm antibodies from serum. human reprod 1992;7:959-961. 45. shibahara h, shiraishi y, suzuki m. diagnosis and treatment of immunologically infertile males with antisperm antibodies. reprod med biol 2005;4:133-141. 46. ryan m, drudy l, cottell e, et al: preparation of antibody free spermatozoa by in vitro immunodepletion using immunobeads. andrologia 1994;26:247-250. 47. lee c, nie gj, joo hs, et al: an enzyme-linked immunosorbent assay (elisa) for the detection of antisperm antibodies in horse serum. theriogenology 1993;40:1117-1126. 48. nie gj, lee c, momont hw, et al: equine antisperm antibodies (easa): preliminary study of the clinical response following breeding in immunized mares. theriogenology 1993;40:1107-1116. 367 clinical theriogenology • volume 11, number 3 • september 2019 368clinical theriogenology • volume 11, number 3 • september 2019 omniblank: evaluation of angiogenesis in equine hydrops evaluation of angiogenesis in equine hydrops pouya dini,a,b peter daels,a mariano carossino,d alan loynachan,c karen wolfsdorf,e el-sheikh ali,b kirsten scoggin,b barry ballb afaculty of veterinary medicine, ghent university, merelbeke, belgium bgluck equine research center and cveterinary diagnostic laboratory department of veterinary science, university of kentucky, lexington, ky dlouisiana animal disease diagnostic laboratory and department of pathobiological sciences school of veterinary medicine, louisiana state university, baton rouge, la ehagyard equine medical institute, lexington, ky hydrops conditions refer to excessive accumulation of fluid in the amniotic (hydramnios) or allantoic (hydrallantois) cavities during the last trimester of pregnancy. although this condition is uncommon in mares, consequences of untreated hydrops allantois/amnion can be, very negative, including abdominal wall hernias, prepubic tendon rupture, cardiovascular shock associated with unattended abortion or foaling and dystocia. it has been postulated that hydrallantois is associated with structural and/or functional changes in the chorioallantoic membrane such as dysfunction of chorionic ion pumps. macroscopic examination of affected placentas suggests areas with small, sparse villi or avillous areas on the chorionic surface. however, it remains undetermined whether this is due to villi atrophy or hypoplasia. in humans and mice, disruption in expression of the imprinted rtl1 gene (retro transposon gag like) is associated with hydrops. in previous studies, we evaluated expression of the rtl1 locus in the equine placenta throughout gestation and localized this protein in the endothelial cells within microvilli capillaries. in the present study, we hypothesized that the expression of angiogenic genes and the number of chorioallantoic (ca) capillaries are altered in hydrops placentae. therefore, we evaluated angiogenesis in the hydrops placenta. expression of 14 angiogenic genes was compared between equine hydrops (n = 10) and normal, 10 month pregnant ca samples (n = 4). protein expression of rtl1 was evaluated by immunohistochemistry and vascular density was evaluated by immunostaining with von willebrand factor. anova analyses revealed 4 differentially expressed transcripts between normal and hydrops samples. dvl1 (dishevelled segment polarity protein 1) had lower expression, whereas ednra (endothelin receptor type a), nrp1 (neuropilin), and tyro3 (tyro3 protein tyrosine kinase) had higher expression in hydrops samples (p < 0.05). protein expression of rtl1 and the number of vessels were lower in hydrops samples. in agreement with our hypothesis, there was an alteration in expression of select angiogenic genes and disruption in capillary vessel formation. interestingly, in human placentae, altered expression of dvl1 significantly reduced placental estradiol production through modification of cytochrome p450 family 19 subfamily a member 1 (cyp19a1) promoters. furthermore, there are lower plasma estradiol concentrations in ewes with hydrallantois. however, a direct link between reduced dvl1 expression and estradiol production in equine hydrops needs to be elucidated. in conclusion, equine hydrops was associated with an alteration in placental angiogenic features. keywords: hydrops, hydrallantois, hydramnion, placenta, equine, angiogenesis 405 clinical theriogenology • volume 11, number 3 • september 2019 406clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2011: association of carcass quality traits, genetic and management factors on scrotal circumference development in yearling angus bulls association of carcass quality traits, genetic and management factors on scrotal circumference development in yearling angus bulls r. kasimanickam,a s.p. greiner,b a. tibary,a b. inman,c w.d. whittierc adepartment of veterinary clinical sciences, washington state university, pullman, wa; bdepartment of animal and poultry sciences and cdepartment of large animal clinical sciences, virginia polytechnic institute and state university, blacksburg, va abstract the objective of this study was to estimate the association among various carcass traits, genetic and management factors on scrotal circumference development in yearling angus bulls. data obtained from angus bulls (n=1374) were retrospectively evaluated. pearson correlation coefficient analysis was performed to estimate the linear relationship between the variables. the mean ( sem) scrotal circumference (sc), body weight, and carcass traits were different among age groups, locations, years and expected progeny difference (epd) groups (p<0.05). for yearling bulls, positive correlations were observed between the following: body weight and sc (r=0.11; p=0.0001), age (d) and sc (r=0.22; p=0.0001), sc-epd and adjusted yearling sc (r=0.50; p=0.0001), 240 d sc and yearling sc (r=0.42; p=0.0001), sc and adjusted yearling rump fat (r=0.07; p=0.008), sc and adjusted yearling rib fat (r=0.12; p=0.0001), sc and adjusted yearling rib eye area (r=0.13; p=0.0001), sc and adjusted yearling percent intramuscular fat (r=0.06; p=0.05). bull average daily weight gain during the trial period was positively correlated with growth differences in sc during the same period (r=0.13; p=0.0001). the sc growth per day was higher for bulls completing the trial period at < 1 year of age compared to those > 1 yr, and higher sc epd values were associated with more sc growth during the trial period. in summary, the current study shows significant but moderate to poor positive associations among yearling bull sc and various growth and carcass quality traits, so we conclude that the best method to increase sc in angus bulls would be by independent bull selection based on the sire sc-epd trait. keywords: sire, progeny, scrotal circumference, carcass trait, epd introduction proper bull selection is the most rapid way to change the genetic component of a cattle herd. selecting bulls that are genetically superior through national cattle evaluation programs can double the genetic change compared to within herd selection.1 however, reproductive traits also must be considered in genetic improvement even though their heritability is low.2-4 the most important single determining factor for bulls attaining satisfactory potential breeder status is larger sc, accounting for 66 to 72% of failures to achieve satisfactory breeding soundness status.4 scrotal circumference has been found to be positively correlated with growth and reproductive traits. several studies have found moderate to high genetic correlations between sc and growth traits.5-7 bourdon and brinks suggested that positive genetic correlations between scrotal circumference and growth traits also inherently affects fertility in females.6 thus, the selection of bulls with larger sc and higher growth rates may improve reproductive efficiency in female offspring. beef seedstock breeders have available epds for carcass traits including rib eye muscle area and subcutaneous fat thickness at the rib and rump for use as selection criteria. these data are collected from real-time ultrasound scanning of yearling bulls and heifers in seedstock herds. producers also use sc-epd as a selection criterion. determination of the effectiveness of using different traits for selection is a complex problem. factors affecting decisions about selection criteria include use for sire, dam, or individual selection, potential number of animals measured and the intensity of selection possible, relationship with other desirable effects and age at measurement. the producers’ perception is that selection for carcass traits, especially less external fat and bigger rib eye area results in smaller sc. the objective of this study was to estimate the association among various carcass traits, genetic and management factors on sc development in yearling angus bulls. 49 materials and methods multiple farm source angus bulls (n = 1374) were brought to virginia beef cattle improvement association-sponsored bull test stations at two locations (location 1 and location 2) in virginia, for a four month evaluation period. data from bulls entered in the test stations from 2002 to 2006 were retrospectively evaluated. bull management bulls entering the test stations were managed under the auspices of the virginia beef cattle improvement association. weaning age and age at arrival at the test station varied among farms. bulls born august through november were designated as senior bulls (9.0 to 11 months of age), whereas bulls born january through march were designated as junior bulls (6.5 to 8.5 months of age). at location 1, senior bulls arrived in july, and junior bulls in november. at location 2, both senior and junior bulls arrived in october. at arrival, senior and junior bulls were divided into two pens of 50 to 70 bulls based on age, weight, and breed at both locations. each bull had negative brucellosis and tuberculosis tests or was from a certified and accredited herd, and negative anaplasmosis and persistently infected bovine viral diarrhea virus tests. on arrival, bulls were weighed, vaccinated for infectious bovine rhinotracheitis, parainfluenza type 3 virus, bovine viral diarrhea virus, bovine respiratory syncytial virus, seven species of clostridia, and pasteurella and were dewormed. all bulls were subjected to examination of the scrotum and its contents and measurement of sc. bulls with significant abnormalities were rejected from the test. bulls were fed a transition ration for a period of 14 d, at which time they received at ration formulated to achieve daily weight gain of 3.75 pounds (72% total digestible nutrients, 13.5% crude protein) when offered ad libitum. at location 1, the ration was composed of primarily corn silage, dry corn, and soybean meal. similar feedstuffs were offered at location 2 from 2002 through 2004. in 2005, a commercial dry feed consisting of corn gluten feed, wheat midds, cottonseed hull pellets, peanut hulls, soy hulls, dry distillers’ grains was utilized. all diets included an ionophore. bulls were maintained at the bull test stations for a test period of 112 to 133 days. at the end of the test period, bulls were weighed and subjected to reproductive examination procedures including sc measurement and extension of the penis either manually or by stimulation with an electroejaculator and transrectal examination to assess the internal reproductive organs. pre-slaughter carcass characteristics were measured by a beef improvement federation certified technician with an ultrasound scanner using a 3.5 mhz, 17.2 cm linear array transducer (aloka 500v; corometrics medical systems, wallingford, ct). measurements were taken when the average age of the bull group was approximately one year and measurements were adjusted to yearling values. bulls were measured for rib fat thickness between the twelfth and thirteenth ribs over the longissimus muscle, for rib eye area over longissimus muscle area between the twelfth and thirteenth rib and for the rump fat thickness at the p8 site (p8 site is located over the gluteus muscle on the rump, at the intersection of a line through the pin bone parallel to the chine and its perpendicular through the third sacral crest). predictions of percent intramuscular fat were made on images collected from a longitudinal scan of the longissimus muscle made longitudinally over eleventh to the thirteenth rib and analyzed with software developed by iowa state university.8 expected progeny differences for individual bulls tested and their sires were obtained from the american angus association (http://www.angus.org/registeredangus/index.html). the variables of interest were bull’s age, weight, average daily weight gain, sc, adjusted 240 day sc, adjusted yearling sc, bull and sire sc epd, adjusted yearling ultrasound rib fat, adjusted yearling ultrasound rib eye area, adjusted yearling ultrasound percent intramuscular fat. 50 statistical analyses data were analyzed with a statistical software program (sas version 9.1 for windows, sas institute, cary, nc). pearson correlation coefficient analysis and univariate regression analysis was performed to estimate the linear relationship between the sc and carcass quality variables. mean (±sem) daily sc and carcass traits among age groups (junior vs. senior bulls), test stations (1 vs. 2), years (2002-03, 2003-04, 2004-05, 2005-06) and epd groups were determined and the differences in the mean were analyzed using general linear model. results mean ( sem) sc, body weight, and carcass traits in yearling angus bulls are shown in the table. the yearling sc and daily sc growth were different among age group, years and sc-epd (p<0.05), but not different between the test stations (p>0.1). average daily gain was different among test stations, years, age groups and average daily gain (adg)-epd (p<0.05). the carcass traits were significantly different with the exception that the yearling rump fat thickness was not significantly different among stations and age groups. for yearling bulls, positive correlations were observed between the following: body weight and sc (r=0.11; p=0.0001), age (d) and sc (r=0.22; p=0.0001), sc-epd and adjusted yearling sc (r2=0.25; p=0.0001), 240 d sc and yearling sc (r=0.42; p=0.0001), sc and adjusted yearling rump fat (r=0.07; p=0.008), sc and adjusted yearling rib fat (r=0.12; p=0.0001), sc and adjusted yearling rib eye area (r=0.13; p=0.0001), sc and adjusted yearling percent intramuscular fat (r=0.06; p=0.05). bull adg during the trial period was positively correlated with growth differences in sc during the same period (r=0.13; p=0.0001). discussion in the current study associations among carcass quality traits, genetic, and management factors on sc development in yearling angus bulls were studied. in general, positive associations among the variables of interest were observed. scrotal circumference was positively correlated to age and body weight and has been shown to be associated with height,6 weight,9 age10 and age of the dam.6,11 selection for any growth traits may increase sc. however in young bulls all these factors are confounded with weight. reported heritability estimates for yearling sc are moderate to high.5-7,12-15 in the current study, the sc growth during the trial period was higher in junior bulls than in senior bulls (table 1). the sc growth also increased with an increase in the sc-epd scores. it seems logical that sires’ and progeny’s sc-epd would be correlated since the sires' sc-epd is included in the calculation of the sons' sc-epd. in fact, since the progeny sc-epd often did not have their own sc measurement nor any progeny data included in the calculation, most of the data contributing to calculation of the progeny (yearling bulls in this study) scepd are from pedigree sources. however, dam effects are also included in this calculation so that this analysis allows an assessment of the mating decisions made by the breeders of these bulls. this positive correlation suggests that breeders are not simply cancelling a negative sc-epd in the dams' pedigrees with positive sc-epd from sires. rather, this finding documents that continued progress in selection for larger sc is probably occurring. in the present study, factors such as age groups, and year of the trial affected sc growth during the trial period. results indicated that the junior bulls had higher sc growth and average daily gain than senior bulls during the trial period. differences in adg were observed between the test stations. however, there were no differences in sc growth between test stations. the trial during 2002-2003 resulted in higher sc growth but lower adg compared to subsequent years. correlations among sc and adjusted yearling rump fat (r2=0.02; p=0.0001), sc and adjusted yearling rib fat (r2=0.02; p=0.0001), sc and adjusted yearling rib eye area (r2=0.02; p=0.0001), sc and adjusted yearling percent intramuscular fat (r2=0.003; p=0.05) were low. it is possible that these factors are confounded with growth traits. 51 these data would suggest that the selection for growth and carcass traits that is currently being used in angus breeding decisions does not counter the selection for increased sc. it should be noted that correlations between yearling sc and other growth and carcass traits were positive indicating that selection for yearling sc is not adversely affecting carcass traits in the angus breed. the main objective of the cow-calf segment of the beef industry is production of calves. changes in body composition potentially affect the production of calves by decreased calf survival and increased pubertal age and postpartum interval. leaner breeds tend to be associated with these factors.16,17 it seems possible to change age at puberty either by direct selection or by selection based on sc in bulls.18 selection for direct and maternal calving ease or smaller birth weights likely decreases calving difficulty.19 bennett and gregory concluded that larger sc was correlated with decreased maternal calving difficulty and increased 200 d weight.20 therefore, cows of leaner genotypes may need to be selected for improved reproductive ability. selection for reproduction could be achieved alternatively by terminal crossing to partially disassociate the slaughter animals from the cow’s genotype.17 the positive associations between growth and carcass characteristics and sc described in this study suggest that successful selection for all of these traits is occurring. determination of the effectiveness of using different traits for selection is a complex problem. factors affecting decisions about selection criteria include use for sire, dam, or individual selection, potential number of animals measured and the intensity of selection possible, relationship with other desirable effects and age at measurement. in summary, the current study shows significantly positive but moderate to poor strength of associations among yearling bull sc and various growth and carcass quality traits. this study suggests that the best method to increase sc in angus bulls would be by independent bull selection based on the sire sc-epd trait. acknowledgement the authors thank all owners who participated in the study and test station staff of the virginia beef cattle improvement association. references 1. hough jd, benyshek ll, mabry jw: direct and correlated response to yearling weight selection in hereford cattle using nationally evaluated sires. j anim sci 1985;61:1135-1344. 2. kriese la, bertrand jk, benyshek ll: age adjustment factors, heritabilities and genetic correlations for scrotal circumference and related growth traits in herford and brangus bulls. j anim sci 1991;69:478489. 3. coulter gh, foote rh: bovine testicular measurements as indicators of reproductive performance and their relationship to productive traits in cattle: a review. theriogenology 1979;11:297-311. 4. chenoweth pj, chase cc, thatcher mj, et al: breed and other effects on reproductive traits and breeding soundness categorization in young beef bulls in florida. theriogenology 1996;46:1159-1170. 5. knights sa, baker rl, gianola d, et al: estimates of heritabilities and of genetic and phenotypic correlations among growth and reproductive traits in yearling angus bulls. j anim sci 1984;58:887-893. 6. bourdon rm, brinks js: scrotal circumference in yearling hereford bulls: adjustment factors, heritabilities and genetic, environmental and phenotypic relationships with growth traits. j anim sci 1986;62:958-967. 7. nelson ah, benyshek ll, johnson mh, et al: the effect of changing of dam correction factors on beef national genetic evaluation programs. j anim sci 1989;67(suppl 2):6(abstract). 8. wilson de, zhang h, rouse gh, et al: using real-time ultrasound to predict intramuscular fat in the longissimus dorsi of live beef animals. beef research report, iowa state university, ames. a.s. leaflet r 1017, 1993; p. 29-31. 9. coulter gh, foote rh: relationship of body weight to testicular size and consistency in growing holstein bulls. j anim sci 1977;44:1076-1079. 10. coulter gh, larson ll, foote rh: effect of age on testicular growth and consistency of holstein and angus bulls. j anim sci 1975;41:1383-1389. 11. lunstra dd, gregory ke, cundiff lv: heritability estimates and adjustment factors for the effects of bull age and age of dam on yearling testicular size in breeds of bulls. theriogenology 1988;30:127-136. 12. coulter gh, rounsaville tr, foote rh: heritability of testicular size and consistency in holstein bulls. j anim sci 1976;43:9-12. 13. latimer fg, wilson ll, cain mf, et al: scrotal measurements in beef bulls: heritability estimate, breed and test station effects. j anim sci 1982;54:473-479. 52 14. neely jd, johnson bh, dillard eu, et al: genetic parameters for testis size and sperm number in hereford bulls. j anim sci 1982;55:1033-1040. 15. gipson ta, vogt dw, massey jw, et al: associations of scrotal circumference with semen traits in young beef bulls. theriogenology 1985;24:217-225. 16. cundiff lv, gregory ke, koch rm, et al: genetic diversity among cattle breeds and use to increase beef production efficiency in a temperate environment. proc 3rd world cong genet appl livest prod ix. lincoln, ne 1986;271-282. 17. bennett gl, williams cb: implications of genetic changes in body composition on beef production systems. j anim sci 1994;72:2756-2763. 18. morris ca, bennett gl, johnson dl: selecting on pubertal traits to increase beef cow reproduction. proc nz soc anim prod 1993;53:427-432. 19. meijering a, postma, a: responses to sire selection for dystocia. livest prod sci 1985;13:251-266. 20. bennett gl, gregory ke: genetic (co)variances for calving difficulty score in composite and parental populations of beef cattle: ii. reproductive, skeletal, and carcass traits. j anim sci 2001;79:52-59. 53 t ab le . m ea n ( s e m ) sc ro ta l c ir cu m fe re nc e, b od y w ei gh t, an d ca rc as s tr ai ts in y ea rl in g a ng us b ul ls r ai se d in te st s ta ti on s be tw ee n 6. 5 an d 8. 5 (j un io r bu ll s; n = 8 32 ) or 9 to 1 1 m on th s of a ge ( se ni or b ul ls ; n = 5 42 ). y ea rl in g s c ( cm ) d ai ly s c g ro w th (c m /d )* y ea rl in g w ei gh t (k g) a d g ( kg /d )* y ea rl in g % i m f y ea rl in g ri b ey e ar ea y ea rl in g ru m p fa t t hi ck ne ss l oc at io n s ta ti on 1 34 .8 0 .9 a 0. 07 0 0. 00 1a 12 08 .3 9 .7 a 3. 68 0 .0 92 a 3. 28 0 .0 6a 12 .2 7 0. 83 a 0. 32 0 .0 8a s ta ti on 2 34 .6 0 .7 a 0. 07 3 0. 00 3a 12 95 .8 1 2. 5b 3. 52 0 .1 08 b 3. 34 0 .0 9a 12 .7 6 0. 79 b 0. 32 0 .0 1a y ea r 20 02 -0 3 35 .1 0 .7 c 0. 04 0 0. 00 7c 11 80 .9 7 .8 c 3. 10 0 .0 84 c 2. 95 0 .1 0c 11 .7 8 0. 92 c 0. 28 0 .0 4c 20 03 -0 4 34 .3 0 .4 d 0. 03 5 0. 01 1d 12 45 .3 8 .3 d 3. 72 0 .0 97 d 3. 30 0 .0 9d 12 .3 3 0. 67 d 0. 31 0 .0 6cd 20 04 -0 5 34 .3 0 .5 d 0. 03 6 0. 01 0d 12 84 .9 6 .5 e 4. 00 0 .1 14 e 3. 50 0 .0 5d 13 .1 4 0. 93 e 0. 34 0 .0 7de 20 05 -0 6 35 .0 0 .9 c 0. 03 4 0. 01 2d 12 97 .0 1 1. 2f 3. 57 0 .1 02 df 3. 49 0 .0 3d 12 .8 2 0. 89 e 0. 35 0 .0 2e a ge ju ni or 30 .2 0 .6 a 0. 08 1 0. 00 4a 10 44 .4 1 2. 5a 3. 81 0 .1 07 a 3. 02 0 .0 6a 11 .0 6 0. 78 a 0. 28 0 .0 6a s en io r 31 .5 0 .6 b 0. 05 0 0. 00 7b 11 81 .5 1 5. 6b 3. 19 0 .1 16 b 2. 86 0 .0 4b 11 .1 9 0. 59 a 0. 29 0 .0 6a s ir e s c -e p d < 0. 5 33 .6 0 .8 g 0. 04 9 0. 00 9g 99 8. 2 9. 1g 2. 78 0 .0 98 g 3. 05 0 .0 5g 11 .9 2 0. 70 g 0. 27 0 .0 6g  -0 .5 to 0 33 .9 0 .4 g 0. 05 4 0. 00 6g 11 44 .5 8 .9 h 3. 01 0 .0 89 h 2. 98 0 .0 3g 12 .1 4 0. 56 g 0. 28 0 .0 4gh  0 to 0 .5 34 .6 0 .9 h 0. 06 7 0. 00 8h 11 60 .4 1 0. 4i 3. 30 0 .0 84 h 3. 68 0 .0 7h 13 .3 9 0. 81 h 0. 31 0 .0 1hi  0 .5 to 1 34 .8 0 .7 h 0. 08 8 0. 00 8h 11 97 .4 8 .2 j 3. 67 0 .1 04 i 4. 87 0 .0 6i 13 .6 8 0. 66 h 0. 33 0 .0 8i  1 35 .8 0 .5 i 0. 10 6 0. 01 1i 12 47 .4 7 .6 k 3. 86 0 .1 34 i 6. 26 0 .0 4j 14 .2 4 0. 86 h 0. 34 0 .0 8i *d ur in g te st p er io d of 1 12 to 1 33 d ay s. a, b l in es w ith d if fe re nt s up er sc ri pt s di ff er ( p < 0 .0 5) ; l oc at io n ef fe ct a nd / o r ag e ef fe ct . c, d, e, f l in es w ith d if fe re nt s up er sc ri pt s di ff er ( p < 0 .0 5) ; y ea r ef fe ct . g, h, i,j ,k l in es w it h di ff er en t s up er sc ri pt s di ff er ( p < 0 .0 5) ; s ir e s c -e pd e ff ec t 54 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags 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false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2017 a case of uterine inertia in an unobstructed birth canal of normal litter size in a flemish giant rabbit (oryctolagus cuniculus) corrected by repeated oxytocin treatment and supportive care a case of uterine inertia in an unobstructed birth canal of normal litter size in a flemish giant rabbit (oryctolagus cuniculus) corrected by repeated oxytocin treatment and supportive care s. arizmendi, a.t. peter, s.b. thompson department of veterinary clinical sciences, college of veterinary medicine, purdue university, west lafayette, in summary a one-year-old female flemish giant rabbit that never kindled before was presented forty-eight hours after delivery of three kits. vaginal examination revealed the presence of a fetus in anterior presentation of normal size in an unobstructed birth canal. radiographs identified the presence of two normal sized apparently dead fetuses, which was confirmed by transabdominal ultrasound examination. repeated oxytocin treatment and supportive care enabled the doe to deliver two dead fetuses. keywords: flemish giant rabbit, dystocia, uterine inertia, reproduction background although dystocia is rare in rabbits,1, it can occur secondary to factors influenced by the doe or the fetus. maternal factors include anatomical abnormalities, variations in pelvic sizes and uterine inertia. fetal factors include malposition, malpresentation, fetal monsters, and fetal death. uterine inertia represents the main cause of dystocia in rabbits.2 primary uterine inertia is multi-factorial with genetic, mechanical, hormonal, and physical components. causes of primary uterine inertia include hypocalcemia, obesity, and overstretching of the myometrium. secondary uterine inertia, commonly referred to as uterine fatigue, occurs due to exhaustion from having previously prolonged contractions. large litter size can be a reason for uterine inertia.3 the present case describes a dystocia in a flemish giant rabbit due to uterine inertia that was resolved by repeated oxytocin treatment and supportive care. case presentation a one year-old female flemish giant rabbit was presented for dystocia to the veterinary teaching hospital. forty-eight hours prior to presentation, the doe had three viable kits at home and the owners did not notice the expulsion of fetal membranes. the information on the time taken to deliver the first three kits was not recorded. two hours prior to admission to the hospital, the doe started having uterine contractions and seemed restless. the doe was bred 31 days before she had the first kit and this was her first litter. on physical examination, the doe was found to be bright, alert, responsive, and there was evidence of straining. she was in desirable body condition (13kgs; bcs 4/5). an edematous, swollen vulva with a hemorrhagic vaginal discharge was noticed. the perivulvar area and hind limbs were mildly stained with the hemorrhagic vaginal discharge. strong and synchronous femoral pulses were palpated. respiratory rate and heart rate were within normal limits. a lower than normal body temperature was observed based on the ear temperature (100°f) recording. her mucous membranes were pink and moist with a capillary refill time of <2 seconds. mammary glands felt normal on palpation. on abdominal palpation, a pendulous abdomen was felt although no obvious structure was identified. on vaginal examination, a round firm structure was palpated within two centimeters from the vulva. abdominal radiographs were obtained in order to identify the structure within the vaginal canal and to evaluate the presence or absence of a fetus. ventrodorsal and left lateral images were obtained (figs. 1 and 2). the ventral abdominal wall was moderately pendulous. two large soft tissue structures extending from the caudal abdomen that contained two full term fetuses were identified. multifocal gas opacities overlying the fetus on the left side of the abdomen were noted. the skull of the fetus positioned on the cranial pelvic inlet (fig. 1) appeared to be smaller in size than that of the pelvic canal. presence of gas within the region of the heart, aorta, and liver was noted on the fetus located on the right side of the abdomen (fig. 2). a transabdominal ultrasound examination with doppler revealed the presence of two fetuses that lacked heartbeats. 601 clinical theriogenology • volume 9 number 4 • december 2017 figure 1: ventrodorsal (vd) radiograph, note the sizes of fetal skull and pelvic inlet figure 2: left lateral radiograph, note the presence of gas in the fetus on the right side. treatment future breeding was discouraged and an ovariohysterectomy was recommended but declined by the owner. at this time, the owners elected medical management. the doe was given butorphanol 602clinical theriogenology • volume 9 number 4 • december 2017 (0.1mg/kg, s/c) and two units of oxytocin (i/m), and placed in a quiet, dark room. thirty minutes later, the doe was examined and on palpation, no fetus could be detected in the birth canal; however, contractions were strong. three more units of oxytocin were then administered intramuscularly. one hour later, the doe was inspected for any evidence of progression toward delivery. the doe was palpated and while being examined, she urinated crystalized urine. she was then administered 100 ml of subcutaneous fluid (normosol-r) to maintain moisture within the mucosa. the doe was given five more units of oxytocin and within thirty minutes of administration, a dead fetus was expelled. it appeared fullterm; similar in size to the three live ones and no congenital abnormalities were evident based on external observation. the patient was discharged with one dead fetus remaining in the uterus. overnight hospitalization was discussed which was declined by the owner. prior to discharge, the doe was administered another dose of butorphanol subcutaneously. the doe was sent home with meloxicam (0.23 mg/kg q 12 hrs po) and antibiotics (trimethoprim sulfa 15 mg/kg q 12 hrs po) for a week. clear instructions were given to the owners at the time of discharge of the patient and regular communications were maintained to determine whether further treatment was required. the client was advised to watch for the last dead fetus to be expelled. according to the owner, the second dead fetus that was expelled at home the next morning was also of normal size with no obvious evidence of decomposition; the doe appeared normal and was nursing the three kits. outcome ten hours after being discharged from the hospital, the doe delivered the second dead fetus. a total of three live and two dead kits were delivered. the doe was treated with antibiotics and pain medications for a week after delivery. the doe and kits continued to thrive and no further medical intervention was warranted. discussion the time of kindling in this case, 31 days from breeding, was normal. normal gestation of a doe is approximately 31 to 32 days with a range from 28 to 36 days.1,4 factors affecting gestation length include the age of the doe, weight, genetics, litter size, nutritional status, husbandry practice, and environmental hygiene. depending on the breed of rabbit, litter size ranges from four to 12 kits.1 parturition in rabbits is a relatively quick process and is usually completed in thirty minutes following the initial onset. it is rare for the young to be delivered several hours apart. hence, in this case stage ii and iii extended beyond their normal duration. similar to other multitoccus species, the first and second stages of labor in rabbits tend to occur simultaneously.5 it is not known whether the shedding of fetal membranes of the first three kits occurred. the clients failed to observe shedding while at home nor was there evidence in the hospital during the time the doe was under medical management for dystocia. placental delivery most likely occurred without notice and the doe might have swiftly consumed them, a maternal behavior known among breeders. furthermore, the doe appeared healthy at the time of presentation and during its stay in the hospital with no clinical signs of retention of fetal membranes. published literature does not provide information on retained fetal membranes in rabbits. certain husbandry and nutrition practices have been associated with decreased fertility of does, stillbirths, and decreased survival of kits. obesity and nutritional deficiencies are amongst the common causes. the level of feed intake during the last week of gestation has been shown to affect kit’s survival and birth weight.6,7 this doe’s feed intake remained consistent throughout the entire pregnancy and the clients have adopted standard nutritional practice. use of oxytocin, an ecbolic agent, in this patient was justified given the unobstructed birth canal with normal presentation, position, and posture of the proximal dead fetus as diagnosed by vaginal, radiography, and transabdominal ultrasound examination. although fetal measurements were not obtained, it is advisable to determine parameters such as the biparietal diameter. the dose of oxytocin chosen was based on prior reports,1,8 wherein one to three units of oxytocin have been administered intramuscularly to assist in uterine contraction. failure of response within 30 minutes necessitated an additional treatment with a higher dose. as reported for dogs,9 calcium treatment might have helped in 603 clinical theriogenology • volume 9 number 4 • december 2017 this case. since the contractions was strong after the initial treatment calcium administration was considered but not administered. measurement of peripheral calcium would have been useful to determine whether calcium administration was necessary but the client declined the test. butorphanol was used to treat this doe to avoid excessive anxiety10 and its influence or adverse effects on uterine contraction in rabbit are not known. a study in human obstetrics suggested that it had no adverse effects on uterine activity.11 prolonged stage ii led to the death of these fetuses.8 as reported in a previous case,8 the prognosis would have been better for these two kits, if the patient were admitted after the first evidence of dystocia. although the present case differs from the one reported previously8 in terms of breed, age, animal husbandry, and the condition of the dead fetuses delivered, both does responded to oxytocin treatment and supportive care. the reason for the uterine inertia remains unknown in this case given the normal litter and fetal sizes. immaturity of the doe and obesity might have contributed to uterine inertia. learning points � uterine inertia represents the main cause of dystocia in rabbits � abdominal radiographic information is useful in diagnosing and correcting dystocia � butorphanol treatment is useful in obstetrical cases � repeated oxytocin treatment resolves uterine inertia in an unobstructed birth canal acknowledgements the authors thank drs. hock gan heng and chee kin lim of the diagnostic imaging service for their assistance. references 1. quesenberry ke, carpenter jw: ferrets, rabbits, and rodents: clinical medicine and surgery. st. louis: saunders; 2004. p.57. 2. harkness j, wagner j: the biology and medicine of rabbits and rodents. baltimore: williams and wilkins; 1995. p. 3344. 3. ozimba ce, lukefar sd: comparison of rabbit breed types for postweaning, litter growth, feed efficiency, and survival performance traits. j anim sci 1991;69:349-350. 4. paul-murphy j: critical care of the rabbit. vet clin north am exotic anim pract 2007;10:437-461. 5. bishop cr: reproductive medicine of rabbits and rodents. vet clin north am exotic anim pract 2002;5:507-535. 6. rommers j, meijerhof r, noordhuizen j, et al: the effect of level of feeding in early gestation on reproductive success in young rabbit does. anim reprod sci 2004;81:151-158. 7. easson w: a review of rabbit and rodent production medicine. semin avian exot pet 2001;10:131-139. 8. dickie e: dystocia in a rabbit (oryctolagus cuniculus). can vet j 2011;52:80-83. 9. darvelid aw, linde-forsberg c: dystocia in the bitch: a retrospective study of 182 cases. j small anim pract 1994;35:402-407. 10. portnoy lg, hustead dr: pharmacokinetics of butorphanol tartrate in rabbits. am j vet res 1992:53:541-543. 11. abboud tk, afrasiabi a, zhu j, et al: bupivacaine/butorphanol/epinephrine for epidural anesthesia in obstetrics: maternal and neonatal effects. reg anesth 1989:14:219-224. 604clinical theriogenology • volume 9 number 4 • december 2017 granulosa cell tumor in a double yellow-headed amazon parrot (amazona oratrix) granulosa cell tumor in a double yellow-headed amazon parrot (amazona oratrix) julia bumberry,a reed holyoak,a lauren schmidt,a ian kanda,a joão brandão,a acenter for veterinary health sciences, oklahoma state university, stillwater, ok a 25 year old, intact female, double yellow-headed amazon parrot (amazona oratrix) presented with acute diarrhea, coelomic distention, respiratory distress and reluctance to perch. ultrasound-guided coelomocentesis was performed. cytology of coelomic fluid indicated exudative effusion containing macrophages and heterophils, but no infectious organisms. a coelomic computed tomography scan revealed coelomic fluid, compressed caudal air sacs and a coelomic mass, presumptively oviductal or ovarian in origin. a 4.7 mg subcutaneous deslorelin implant was placed interscapular. coelomic exploratory surgery was performed, with an intention to remove the mass via salpingohysterectomy or ovariosalpingohysterectomy. during surgery, a cystic structure that filled majority of caudal coelom was drained of thick, amber fluid. deep to this structure, a mass associated with left oviduct was identified, along with a second suspected mass in the right coelom. due to severity of adhesions throughout coelom, association of masses to ureters and large vessels and high surgical risk, owner elected to discontinue surgery and pursue palliative treatment. patient was maintained on deslorelin implants and regular therapeutic coelomocentesis as needed. three months after initial presentation, the patient died. at necropsy, there was a malignant granulosa cell tumor that originated from the ovary and metastasized into lymphatics and surrounding tissues. this presentation of an ovarian granulosa cell tumor was unusual in psittacines. surgical excision of neoplastic tissue is challenging, but medical management may provide conservative treatment for reproductive neoplasia and secondary coelomic ascites in psittacines. keywords: psittacine, parrot, ovarian, neoplasia, coelomocentesis, deslorelin 415 clinical theriogenology • volume 11, number 3 • september 2019 416clinical theriogenology • volume 11, number 3 • september 2019 omniblank: introduction acupuncture and herbals are 2 common components of traditional chinese veterinary medicine (tcvm) and are becoming popular in equine reproduction. potential physiological mechanisms of acupuncture applied to reproduction include endorphin-mediated changes of gonadotropin concentrations, and direct effects on gonadal paracrine and autocrine control of steroidogenesis.1 acupuncture is practiced with dry needles, electroacupuncture, moxibustion, or aqua-acupuncture with vitamin b-12 utilizing a selection of 361 points located along energetic channels (meridians) that connect these points. cun (anatomical chinese inch) is a unit of measurement relative to the scale of the body used to locate acupuncture points. for instance, on the back, the width of the last rib is equal to 1 cun.2 conditions that theriogenologists most commonly seek assistance from tcvm practitioners in our practice are in 3 general categories: prebreeding or postbreeding intrauterine fluid accumulation, urine pooling/vesicovaginal reflux, and pregnancy maintenance. although practitioners have described protocols based on western diagnoses for these conditions, integrating tcvm theory in western treatments can greatly increase success.3 expanded reviews on acupuncture use in equine reproduction are available.1,4 this review focusses on integrating tcvm theory in western-based reproduction management. traditional chinese veterinary medicine theory and bian zheng treatment in tcvm is enhanced by incorporating its diagnosis or bian zheng with western medicine. for example, for several of the tcvm conditions described below, findings from transrectal acupuncture and herbals in equine reproduction kristina lu, lauren javernick hagyard equine medical institute,. lexington, ky abstract combining traditional chinese and western veterinary medicines can improve the outcome in refractory equine reproduction cases. basic understanding of diagnostic and treatment principles can help a practitioner to prescribe acupuncture and herbals or to coordinate and communicate with a veterinarian trained in traditional chinese veterinary medicine. theories, diagnoses, potential acupuncture points, and herbals for common reproductive cases are discussed. keywords: equine reproduction, acupuncture, traditional chinese veterinary medicine palpation and ultrasonography of reproductive tract may be similar; however, favorable response to treatment is only possible if points are chosen that best fit the tcvm diagnosis. additionally, the bian zheng is imperative in choosing herbals. theory of tcvm incorporates yin and yang, 5 element theory, and 8 principles theory with the goal of achieving and maintaining energy balance. yin and yang balance each other. examples of yin include solid organs, female, moon, dark, and cold. examples of yang include hollow organs, male, sun, light, and heat. each yin organ is paired with a yang organ or organ activity (table). each organ is associated with an energy meridian. in addition to meridians associated with organs, there are 8 extraordinary channels. commonly discussed 2 channels are governing (aka du mai) and conception (aka ren mai) vessels. a third channel is the penetrating vessel (chong mai), important because it helps to regulate cyclicity, menstruation in women, conception, and pregnancy. conception and penetrating vessels originate from uterus. conception vessel makes meridian connections with governing vessel and stomach meridians, whereas penetrating vessel makes a meridian connection with kidney meridian. five element theory describes the relationship among elements: wood, fire, earth, metal, and water. elements and their corresponding meridians feed into each other and balance 1 another (table). individual constitutions and personalities fall within these elements with a perfectly balanced individual having aspects of all 5 elements. individual’s personality can help identifying the element that they are most associated with, clinical theriogenology 2021; 13: 255 and thus the organ system and existing disharmony that might be most important to rebalance. eight principles theory aids in identifying bian zheng include yin and yang, interior and exterior, hot and cold, and excess or deficiency. examination in tcvm varies with practitioner and often includes signalment, main complaint, evaluation of tongue for color, size and coating, evaluation of pulse (comparing pulses in the left and right carotid arteries in the horse), assessment of horse’s overall condition, assessment of hair and skin quality, evaluation of temperature fluctuations over the body, and assessment of reactivity and recesses at various acupuncture points over the body. effective implementation of tcvm is by combining acupuncture with herbals. particularly, herbals should be included in chronic cases of subfertility to achieve the tcvm balance. herbal therapies improve underlying deficiencies and excesses commonly observed in subfertility whereas acupuncture helps with uterine clearance and tone, nerve damage, and musculoskeletal pain. a trained tcvm practitioner should prescribe herbal formulas and evaluate animals’ response. acupuncture is relatively safe and if used inappropriately will likely have no response. inappropriate use of herbal formulas can result in worsening patient’s condition. unlike western medications, herbals can take several months to change the animal’s pattern and clinical signs. subtle changes should be noticed within 2 3 weeks of starting herbals; however, to correct severe deficiencies, months of treatment may be necessary. during this period, patient should be monitored periodically to ensure that the pattern has not changed warranting a different herbal formulation or discontinuation. herbals utilized for excess conditions are more potent, therefore duration between evaluations should be shorter (1 per week) whereas duration between evaluations of a chronic deficient condition can be longer (every 4 8 weeks). common patterns observed in equine subfertility there is a lack of scientific data on specific acupuncture protocols that fit all horses based on western conditions, requiring practitioners to rely on classic chinese pattern differentiation to choose points. core points for reproduction, the locations are detailed below, include bai hui, yan-chi, and bl-23.5 kidney qi, yang and jing deficiency in tcvm, the kidney is the organ meridian directly associated with reproduction. kidney is the yin organ associated with water element, whereas bladder is the yang element. deficiencies in kidney qi (energy), yang (appears as qi deficiency with coldness), and jing (qi that is born with or prenatal qi) have similar treatment approaches in horses. examples of clinical manifestations of kidney qi, yang or jing deficiency include anestrus, lack of follicular development, anovulatory follicles, prolonged diestrus, and failure to display estrus signs. this group of patterns can also be present in stallions and can present as oligospermia, decreased sperm quality, or decreased libido. physically, the patient may display a decreased body condition, or lack of strength and/or poor performance. upon closer inspection, tongue appears pale, pulse is weak and deep, and the back and extremities may be cold. potential acupuncture points to incorporate in treatment include:2 cv-4/6 (to tonify qi, dry needle) – on ventral midline, 3 cun and 1.5 cun caudal to umbilicus respectively kid-3 (to tonify kidney yin and jing) – between medial malleolus and calcaneal tuberosity table. five elements of traditional chinese veterinary medicine, yin and yang organs, and meridians tcvm element yin organ/meridian (# of points) yang organ/meridian (# of points) wood liver (14) gallbladder (44) fire heart (9) and pericardium (9) samll intestine (19) and triple heater (23) earth spleen (21) stomach (45) metal lung (11) large insttine (20) metal kidney (27) bladder (67) conception vessel (24) governing vessel (28) clinical theriogenology 2021; 13: 256 kid-7 (to tonify qi) – 2 cun directly proximal to kid-3 at cranial border of the tendon bl-26 (gates of yuan source qi) – 3 cun lateral to lumbosacral space gv-1 (to warm yang) – in the depression between anus and tail gv-3 (for kidney yang/gi deficiency) – in the depression between l4 and l5 gv-4 (for yang deficiency) – in the depression between l2 and l3 bai-hui (for yang deficiency) – on dorsal midline at the lumbosacral space yan-chi (important point for infertility) – midway between tuber coxae and shen-peng shen shu (for yang or qi deficiency) – 2 cun lateral to bai-hui shen peng (for yang or qi deficiency) – 2 cun cranial to shen-shu shen jiao (for yang or qi deficiency) – 2 cun caudal to shen-shu epimedium is a useful herbal formula derived from the classical formula sheng jing san. its principles of treatment include tonification of kidney yang and nourishment of yin and jing. it should be used with caution in patients with inflammation or infection (excess heat).6 liver-kidney yin deficiency itcvm theory describes the elements of feeding into 1 after the other, similar to seasons. the order of elements is wood to fire to earth to metal to water and back to wood. kidney is associated with water. liver is associated with wood. hence, kidney yin deficiency and liver yin deficiency can travel together. clinical signs include anestrus, irregular estrous cycles, or repeated failure to achieve pregnancy. a small amount of thick, mucoid to mucopurulent vulvar discharge may be present. haircoat and hooves may be dry. eyelids may be swollen, and the mare may demonstrate blepharospasm or epiphora when exposed to light. on examination, tongue is red and dry, and pulse is fast, weak, and thin. potential acupuncture points to incorporate in treatment include:2 kid-3 (to tonify kidney yin and jing) – between medial malleolus and calcaneal tuberosity kid-6 (to tonify kidney yin) – between calcaneal tuber and talus kid-7 (to tonify qi) – 2 cun directly proximal to kid-3 at the cranial border of the tendon sp-6 (master point of the caudal abdomen and urogenital tract, to tonify yin and qi) – 3 cun proximal to medial malleolus and just caudal to tibial border bl-18 (back shu association point for liver meridian) – a pair of points at the 13th and 14th intercostal spaces, 3 cun lateral from midline bl-23 (back shu association point for kidney meridian) – 3 cun lateral to l2-l3 vertebral space bl-52 (paired with bl-23) – 3 cun lateral to bl-23 yan-chi (important point for infertility) – midway between tuber coxae and shen-peng shen shu (to tonify kidney qi) – 2 cun lateral to bai-hui shen peng (to tonify kidney qi) -2 cun cranial to shen-shu zuo gui wan can be a useful herbal formula for liver-kidney yin deficiency. its treatment principle is to nourish yin and tonify kidney qi and jing. several herbs in this formula also nourish liver yin.6 ophiopogon formula is an excellent kidney yin tonic useful for horses with insulin dysregulation and/or pituitary pars intermedia dysfunction that have a bian zheng consistent with yin deficiency. in addition to nourishing yin, it clears heat and promotes body fluids. its classical antecedent is mai men dong san.6 liver-kidney yin deficiency mares with liver qi and blood stagnation may have ovaries that contain several medium to large follicles without regularly growing to a dominant follicle or ovulation. additional clinical signs include anestrus, irregular estrous cycles, and a thick vulvar or vaginal discharge or coating during estrus. mare may exhibit abdominal discomfort. qi-blood stagnation is also observed in stallions associated with musculoskeletal pain inhibiting breeding behavior or ejaculation. this is an excess pattern. on examination, the tongue appears purple with a thin coating and a wiry pulse. acupuncture points can include:2 bl-18 (back shu association point for liver meridian) – a pair of points at the 13th and 14th intercostal spaces, 3 cun lateral from midline bl-21 (back shu association point for stomach, to move qi) – caudal to last rib, 3 cun lateral from midline bl-23 (back shu association point for kidney meridian) – 3 clinical theriogenology 2021; 13: 257 cun lateral to l2-l3 vertebral space bl-26 (gates of yuan source qi) – 3 cun lateral to lumbosacral space bl-52 (paired with bl-23) – 3 cun lateral to bl-23 liv-1 (for liver qi stagnation) – craniomedial hind limb coronary band liv-3 (for liver qi stagnation) – on craniomedial hind cannon bone, 1/3 of distance from tarsus to fetlock gb-34 (to soothe liver qi) – cranial and distal to the head of fibula in the interosseus space between tibia and fibula, between long and lateral digital extensor tendons gb-44 (gallbladder ting point) – craniolateral hind limb coronary band li-4 (to move qi and clear heat) – between second and third metacarpal 1/3 of distance from carpus to fetlock st-40 (to move qi) – 8 cun proximal and 0.5 cun lateral to lateral malleolus a useful herbal formulation is lotus formula, a formula based on the classical formula huo zue yu zi tang. its principles of treatment are to nourish blood, clear heat and resolve blood stasis. it is contraindicated during pregnancy and should be discontinued at breeding.6 phlegm damp and spleen-kidney qi deficiency clinical signs include chronic endometritis or chronic vulvar discharge. additional signs include loss of appetite, diarrhea, weakness or fatigue, and ventral or distal edema. the tongue is pale and wet and greasy or with a white coating. pulse is deep and weak or choppy. acupuncture points could include:2 bl-20 (back shu association point for spleen meridian) – 3 cun lateral to midline at17th intercostal space bl-23 (back shu association point for kidney meridian) – 3 cun lateral to l2-l3 vertebral space bl-26 (gates of yuan source qi) – 3 cun lateral to lumbosacral space st-36 (potent qi tonic point) – 0.5 cun lateral to tibial crest st-40 (to move qi and clear damp) – 8 cun proximal and 0.5 cun lateral to lateral malleolus sp-6 (master point of the caudal abdomen and urogenital tract, to tonify yin and qi) – 3 cun proximal to medial malleolus and just caudal to tibial border sp-9 (helps clear damp and transform phlegm) – at the level of patellar ligaments, 0.5 cun cranial to saphenous vein qi-hai-shu (for qi deficiency) – at the 16th intercostal between longissimus dorsi and iliocostalis muscles gv-4 (for yang deficiency) – in a depression between l2 and l3 cv-1 (local point for the uterus) – halfway between anus and vulva bai-hui (for yang deficiency) – on dorsal midline at lumbosacral space shen shu (to tonify kidney qi) – 2 cun lateral to bai-hui include heat-clearing points for uterine damp heat, such as: li-4 (to move qi and clear heat) – between second and third metacarpal 1/3 distance from carpus to fetlock gv-14 (to clear heat) – on dorsal midline in a depression in front of t1 spinous process wei-jian (to clear heat) – at the tail tip useful herbal formulations are wan dai fang for spleen qi deficiency with damp without pathogens, or phellodendron and plantago combination for uterine damp heat with pathogens. wan dai fang’s principle of treatment is to strengthen spleen qi, eliminate damp and resolve vaginal discharge. phellodendron and plantago is based on the classical formula bai che san. its treatment principle is to clear damp heat and resolve vaginal discharge. phellodendron and plantago are not contraindicated during pregnancy.6 pregnancy following points are commonly used for equine pregnancy maintenance that a practitioner can modify based on pattern assessment.2 first trimester pregnancy loss is often associated with blood or jing (essence, prenatal qi) deficiency and kidney deficiency, whereas pregnancy loss beyond first trimester is often associated with liver-blood stasis or sinking/collapsing spleen qi.7 bai-hui (calms mind to reduce fetal hyperactivity) bl-18&19 (harmonizes liver, association points of liver and gallbladder) – bl-19 is at the15th intercostal space, 3 cun lateral to dorsal midline clinical theriogenology 2021; 13: 258 bl-20&21 (tonifies spleen qi, association points of spleen and stomach) qi-hai-shu (tonifies spleen qi) – at the 16th intercostal space between longissimus dorsi and iliocostalis muscles da-feng-men, tian-men, an-shen (calms mind, used for fetal restlessness) – da-feng men is a triangle of points with apex at rostral forelock and legs of 0.5 cun; tian-men is at the atlanto-occipital joint; an-shen is in a depression behind the ears several points listed in veterinary literature are contraindicated in pregnant animals, including points around abdomen and lumbosacral region:2 du/cv-1 through 6, yan-chi, bl-23 through 28, bl-52, li-4, sp-6, bl-60, bl-40, st-36, and bl-67. a useful herbal for pregnancy maintenance is pregnancy smoother or yun bao modified from bai zhu san. it strengthens spleen qi and nourishes liver blood to maintain pregnancy.6 bai zhu san is originally mentioned in the yuan heng’s classical collection on treatment of equine diseases (yuan heng liao ma ji) written in the 1600s. this is a classical equine specific herbal formula and includes herbs that maintain pregnancy by strengthening spleen qi, tonifying blood, and smoothing liver qi. conclusion acupuncture and herbals for equine reproduction cases are most effective for chronic subfertility when used in conjunction with western management. although protocols are available based on western diagnoses, treatment is substantially more successful when combined with acupuncture point choices and herbals based on the animal’s bian zheng. herbals in particular should only be prescribed by a veterinarian trained in tradition chinese veterinary medicine. conflict of interest none to declare references 1. schofield wa: use of acupuncture in equine reproduction. theriogenology 2008;70:430-434. 2. xie h, preast v: introduction to acupuncture points. in: xie h, preast v: editors. xie’s veterinary acupuncture, ames; blackwell publishing: 2007. p. 13-26. 3. lu kg, javernick l: acupuncture in mare reproduction. in: dascanio jj, mccue p: editors. equine reproductive procedures, 2nd edition, ames; wiley blackwell: 2021. p. 143-147. 4. rathgeber ra: use of acupuncture in equine reproduction. proc am assoc equine pract 2011;138-140. 5. schmalberg j, xie h: the clinical application of equine acupuncture. j equine vet sci 2009;29:753-760. 6. ma, a: commonly used formulas. in: ma, a: editor. clinical manual of chinese veterinary herbal medicine, gainesville; ancient art press: 2016. p. 51-306. 7. maciocia g: diagnosis. in: maciocia g: editor. the foundations of chinese medicine, new york; churchill livingstone: 1989. p. 143-174. clinical theriogenology 2021; 13: 259 acupuncture and herbals in equinereproduction 2018: management of dystocia and fetotomy in a miniature horse mare management of dystocia and fetotomy in a miniature horse mare college of veterinary medicine, auburn university, auburn, al a five year old miniature horse mare was presented for signs of colic lasting 3 hours. no breeding date was known and pregnancy was not confirmed, but owners suspected late term pregnancy. physical examination was unremarkable other than elevated pulse (88 bpm) and a full udder. transabdominal ultrasonography revealed a fetus with no heartbeat and minimal placental fluids. no fetal movement was elicited on transrectal palpation. on transvaginal palpation, the cervix was open, but no fetal extremities were palpable. the mare was determined to have a dystocia and the fetus was presumed deceased. the mare was anesthetized to better assess fetal position. fetal position was cranial longitudinal, dorsal sacral, with bilateral shoulder and ventral neck flexion. due to position of the foal, prolonged dystocia leading to death of the foal, and small size of the mare, fetotomy was chosen. when the fetus is dead, fetotomy is often preferred over cesarean section because the mare’s recovery is shorter with less after care.1 two different studies showed a mare survival rate of 95.8% after fetotomy2 compared to 85% after cesarean section.3 a single cut was made at the base of the fetal neck using a fetotome, and the head and neck were detached. the head and neck slipped cranially, so the bilateral shoulder flexion was corrected and the body was removed from the uterus utilizing chains on the front limbs. the head and neck were then removed. the uterus was examined and lavaged with isotonic distilled water. no lacerations or rupture were identified. the placenta was retained for 12 hours and standard treatment was initiated. the placenta was passed uneventfully and the mare was discharged after 3 days. this case demonstrates a mare that presented for colic but was experiencing a dystocia. it also indicates when a fetotomy is chosen over cesarean section. keywords: dystocia, fetotomy, equine references 1. brinkso sp, blanchard tl, varner dd, et al: dystocia and postparturient disease. in: manual of equine reproduction. 3rd ed. st. louis: mosby/elsevier; 2011. p. 131-142. 2. carluccio a, contri a, tosi u, et al: survival rate and short term fertility rate associated with the use of fetotomy for resolution of dystocia in mares: 72 cases (1991-2005). j am vet med assoc 2007; 230:1502-1505. 3. freeman de, hungerford ll, schaeffer d, et al: caesarean section and other methods for assisted delivery: comparison of effects on mare mortality and complications. eq vet j 1999; 31:203-207. 4. frazer gs: dystocia and fetotomy. in: samper jc, pycock jf, mckinnon ao, editors. current therapy in equine reproduction, st. louis: saunders elsevier; 2007. p. 417-433. clinical theriogenology • volume 10, number 3 • september 2018351 r. thomas, a. johnson, f. pinaffi 2018: dystocia because of uterine torsion complicated by perosomus elumbisin a stillborn angus calf resulting from a consanguineous mating dystocia because of uterine torsion complicated by perosomus elumbis in a stillborn angus calf resulting from a consanguineous mating riley e. thompson,a sam lawton,a jessica l. petersen,c alyssa b. helms,a allison watson,b mee-ja sula,b david steffen,c brian k. whitlocka adepartment of large animal clinical sciences, and bdepartment of biomedical and diagnostic sciences, college of veterinary medicine, university of tennessee, knoxville, tn; cuniversity of nebraskalincoln, lincoln, ne a six-year-old female multiparous angus cow (~ 700 kg bw; five parities) was presented on emergency for dystocia. the cow had begun stage two labor earlier that morning, but failed to progress at which time the owner contacted the university of tennessee veterinary medical center (utvmc). the attending veterinarian performed vaginal and rectal examinations on the cow following administration of a caudal epidural anesthetic (5 ml lidocaine; 20 mg/ml) and diagnosed the etiology of the dystocia as uterine torsion. the uterus was rotated toward the left (counterclockwise) with a rotation of approximately 360. the cow was sedated with xylazine (40 mg iv) and acepromazine (20 mg iv), cast using the double half hitch method, and placed in left lateral recumbency. the uterine torsion was partially corrected by rolling the cow with the aid of a plank (three episodes) and ultimately completely corrected (final 90°) by manual detorsion via the vagina. a vaginal examination of the cow revealed a partially dilated cervix (8 to 10 cm), a moderately contracted uterus, and a fetus in cranial longitudinal presentation and dorsosacral position, with the head, neck, and forelimbs extended. epinephrine was administered (10 mg iv) to the cow as a tocolytic to better allow manipulation of the fetus, and placement of obstetric chains and snare to the forelimbs and head, respectively. manual traction, for a duration of approximately 30 minutes by two individuals, completely dilated the cervix and ultimately delivered a stillborn male fetus presumptively diagnosed as a perosomus elumbis (pe). a vaginal examination revealed no additional fetuses and minimal partial thickness uterine and vaginal tears. ceftiofur crystalline free acid (4.5 g sq) and flunixin meglumine (1.5 g iv) were administered to the cow. upon receiving the full patient history, it was determined that the calf was the result of a consanguineous (mother to son) mating. tissue samples from the affected fetus and blood samples from the dam, sire, and ten half-siblings were collected for parentage verification and genetic testing. the stillborn calf was submitted for necropsy to the utvmc. the calf weighed 19.2 kg, had a skull that appeared short and rounded with maxillary brachygnathism, and bilateral exophthalmos. bilaterally symmetric arthrogryposis and partial lumbar, and complete sacral and coccygeal vertebral aplasia (consistent with pe) were observed. an 18-cm long abdominal midline defect was observed with abdominal organ herniation and atresia ani, otherwise, all abdominal organs were present. radiographic and computed tomography examinations were consistent with and supported the diagnosis of pe. perosomus elumbis is a congenital abnormality of unknown origin that is characterized by aplasia of the lumbar vertebrae and spinal cord. perosomus elumbis has been reported in several domestic species, including cattle. while this condition has been reported in holsteins and herefords, it has not yet been reported in angus cattle. one case of pe was associated with a holstein calf infected with bovine viral diarrhea virus. this is the first report of pe associated with a consanguineous mating. keywords: perosomus elumbis, angus, calf, congenital abnormality, consanguineous mating clinical theriogenology • volume 10, number 3 • september 2018331 2018: equine placental microbiome equine placental microbiome jenny l. sones, babiche a. heil louisiana state university school of veterinary medicine, baton rouge, la introduction the mammalian placenta has long been regarded as a sterile organ that nourishes and protects the growing fetus during gestation. there is recent evidence in mares that the placenta harbors a unique microbiome.1 while this work is in its infancy in the horse, there is a growing amount of data in human literature to support the role of the placental microbiome in pregnancy success and adverse outcomes. the following paper will review and highlight this up and coming area of interest in equine reproduction. background the placenta is a vital, but transient organ of pregnancy that ensures fetal growth and survival until the time of delivery. it is composed of fetal membranes in intimate association with the mother’s uterus. placental development and fetal survival relies on tolerance by the maternal environment. the placenta is responsible for maternal-fetal exchanges of nutrients and waste as well as tolerance of the semi-allogeneic fetus that is composed of maternal and paternal antigens.2 the placenta also provides pregnancy maintenance by producing a number of pro-gestational hormones. thus, it’s immunologic and endocrinologic functions have been well characterized and studied. in both women and mares, the placenta has been thought of as a sterile organ during pregnancy. however, it was recently elucidated in women that the placenta harbors a unique microbiome and this was discovered using molecular biology, specifically metagenetic, techniques.3 as a result, a potential new role of the placenta during pregnancy has been proposed. the taxonomic profile of the human placental microbiome was discovered to be most similar to the oral microbiome followed closely by the vaginal microbiome and further from the fecal microbiome.3 this finding was found to be consistent in horses and the results are described below.1 there is strong rationale to support challenging the dogma of a sterile placenta in horses. distinct uterine microbiomes have been reported between diestrual mares (day 7 after ovulation) carrying an embryo and those that are open using metagenetics. proteobacteria and bacteroidetes were associated with culture positive samples at ovulation. sphingobium (proteobacteria) and sphingobacteriales (bacteroidetes) are associated with mares carrying embryos at day 7 after ovulation and rhodocyclaceae and enterobacteriaceae (proteobacteria) are associated with mares not carrying embryos.4 also, amniotic fluid taken at delivery from healthy equine pregnancies has yielded bacterial growth.5 additionally, foal meconium on postnatal day 1, prior to suckling, is dominated by the firmicutes phyla consisting primarily of the genera enterococcus, bacillus and lactococcus. there were no differences between mare milk and foal fecal bacterial diversity in meconium or feces from postnatal day 2 and 7. recent evidence suggests there are associations between placental microbiome communities and adverse pregnancy outcomes in women, including preterm birth and preeclampsia.3,6 indeed, preterm birth in people has been associated with intrauterine infections ascending from the lower genital tract as well as intra-amniotic and extra-uterine infections.7 while the vaginal lactic acid bacteria have been evaluated in the mare by traditional culture methods, metagenomic sequencing of the vaginal microbiome in the context of healthy pregnancies and adverse pregnancy outcomes has not yet been reported in the mare. novel metagenetic analyses of the equine placenta, similar to that done in humans, in relation to other extra-placental body sites in healthy mares, will provide the foundation for a better understanding of the relationship between the equine placenta and resident bacterial populations during uncomplicated healthy pregnancies. clinical theriogenology • volume 10, number 3 • september 2018279 figure. the equine fetoplacental unit in relation to profiles of extra-placental bacterial populations associated with equine placentitis (oral, fecal, vaginal). methods to characterize the equine placental microbiome the microbiome consists of the microorganisms and their genomes within a specific region of the body. it is important to know that while traditional identification of bacteria utilizes cultivating methods (i.e. aerobic and anaerobic culture), metagenetic techniques utilize non-cultivating methods. these involve isolation of 16s ribosomal dna and sequencing of the amplified bacterial dna. therefore, metagenetic techniques yield live and dead bacteria populations. metagenetic analysis of the placenta in horses should therefore be superior to cultivating methods that may fail to identify dormant bacteria that may reside at the maternal-fetal interface in pregnant mares. however, until these new methodologies are robustly tested and validated, the clinician should perform cultivating until non-cultivating methods are routinely available and used. to characterize the first equine placental microbiome, university owned mares were used for sampling prior to and at the time of foaling (n=15). collection of samples within thirty days of foaling, swabs from the buccal mucosa of the oral cavity, the cranial vaginal using sterile instruments and speculum, and a fresh fecal sample were collected and stored at -80°c. at the time delivery, placenta and specific regions of fetal membranes (gravid and non-gravid chorioallantois) were collected, dissected and stored at -80°c. dna isolation and amplification of 16s rrna frozen samples were thawed and individually processed for dna extraction. samples were homogenized, centrifuged and resuspended in 400 µl of nuclease-free water. four hundred µg of lysozyme was added and incubated for 12 hrs at 56°c to maximize bacterial dna extraction. isolation of dna was performed using a commercial dna isolation kit powersoil® (mo-bio laboratories inc) according to the manufacturer’s instructions. dna concentration was determined using the qubit® 2.0 flurometer (life technologies, grand island, ny) and dna integrity assessed by electrophoresis. the v3 and v4 domain of bacterial 16s rrna8 was amplified by pcr. primer sequence for 16s amplicon pcr was the 341f and 785r primers as previously described9 and optimized for the illumina miseq platform. the earth microbiome project (http://www.earthmicrobiome.org)10 was used to select 150 different 12 bp clinical theriogenology • volume 10, number 3 • september 2018 280 error-correcting golay barcodes for 16s rrna as previously described.9 the pcr reaction was performed in triplicate containing: microbial genomic dna (25ng), 1x gotaq green master mix (promega, madison, wi), 1 mm magnesium chloride, and 10 µm of each primer. the pcr conditions for 16s rrna consisted of an initial denaturation step of: 3 min at 94°c; followed by 35 cycles of 94°c for 45 secs, 50°c for 1 min, and 72°c for 90 secs; and the final elongation step of 72°c for 10 min. no template controls and two positive controls (mock microbial communities) were added to each pcr plate. replicate amplicons were pooled and purified with a qiaquick pcr purification kit (qiagen, valencia, ca) and visualized by electrophoresis. amplicons were quantified using qubit® 2.0 flurometer then the amplicon aliquots were standardized to the same concentration. miseq sequencing final equimolar libraries were sequenced using the miseq reagent kit v3 (600 cycles) using the miseq platform (illumina inc., san diego, ca). sequence reads were analyzed using the quantitative insights into microbial ecology (qiime) program and assigned to operational taxonomic units (otus). these otus identify the genera of bacteria present in the sample. discriminant analysis (jmp pro 12) was used to evaluate the correlation between bacterial taxa and the prevalence of each type of bacteria in the placental samples with the potential source of bacteria from extra-placental body sites. different prevalence of bacteria in each sample was used as a covariate in a stepwise discriminant analysis model. variables were removed in a stepwise manner until the only variable with a p< 0.005 were retained in the final model. results relative abundance within the chorioallantois demonstrated three main phyla represented in the gravid horn (firmicutes, proteobacteria, bacteroidetes) and those same three phyla plus actinobacteria in the non-gravid horn. the most abundant phyla within the oral, fecal, and vaginal samples (firmicutes and proteobacteria) were also detected in the chorioallantois. the gravid horn and feces of the mare have different populations of bacteria. bacillus, mycoplasma, and gemella are all higher in relative abundance in the fecal samples of the mare (p<0.0001). conversely, clostridium and moraxella are higher in the gravid horn samples (p<0.0001). the most abundant bacterial phyla in gravid and nongravid chorioallantois share significant overlap, suggesting similar, but not identical, environments within different compartments of the chorioallantois. studies are still in progress to evaluate the cervical star region of the chorioallantois, which is a key area of the equine placenta where ascension of bacteria from the lower reproductive tract would occur. discussion placentitis is the most common cause of equine preterm birth in the united states and accounts for one-third of all late-term abortions/fetal loss of horses in this country.11 the most common cause of placentitis is bacteria ascending from the lower reproductive tract initiating an infectious inflammatory response in the placenta.12 oral and fecal pathogens (ie streptococcus equi subspecies zooepidemicus, esherichia coli) are often cultured in cases of equine placentitis, but it is not clear why some mares are susceptible to these organisms and others are not. furthermore, diffuse bacterial placentitis cases, such as nocardioform, do not follow the same pathogenesis of ascending placentitis cases. therefore, the mechanism needs further elucidation. future direction would include evaluation of the vaginal microbiome in mares. it has been shown by cultivating methods that the vagina of the mare is rich in lactobacillus and enterococcus species.13 this is of particular interest as lactobacillus cispatus has been used with a success rate of 69-90% when used to colonize vaginas of sexually active healthy women seeking assisted reproductive techniques. colonizing the transfer-catheter tip with lactobacillus crispatus at the time of embryo transfer may increase the rates of implantation and live birth rate while decreasing the rate of infection.14 phyla of relatively high abundance in oral and vaginal samples correspond to those found in the chorioallantois, indicating possible associations between placental and extra-placental microbiota, yet clinical theriogenology • volume 10, number 3 • september 2018281 there are significant differences between the gravid horn and the fecal samples. to the authors’ knowledge, this is the first report to characterize the equine placental microbiome by metagenetics. further studies include pyrosequencing of equine placentae from mares with adverse pregnancy outcomes, i.e. placentitis and fetal growth restriction, to determine if the equine placenta has a unique microbiome in health and disease. several maternal risk factors are known or hypothesized to impact the human placental microbiome, including obesity/excessive gestational weight gain, diabetes, and even periodontal disease.15 as this area of research grows, it will be important to analyze equine placental microbiomes in the context of overall mare health status before and during pregnancy. this knowledge will also help guide the way we diagnose and treat dysbiosis of the mare’s reproductive tract in an effort to prevent adverse pregnancy outcomes in the horse. this author advises caution and judicious usage of interventions to alter the microbiome of the mare’s reproductive tract until an exhaustive microbiota characterization has been performed. significance diagnosing placental infection during equine pregnancy is extremely difficult since outward clinical signs are often not present. understanding the relationship between the equine placenta and resident bacterial populations during healthy and diseased pregnancies could provide the opportunity to use extra-placental sources (oral, fecal and/or vaginal) as “proxies” for predicting placentitis and associated adverse outcomes. further investigations are required to evaluate clinical cases of equine placentitis as well as the mare’s uterine microbiome before pregnancy. identification of bacterial targets in extra-placental body sites as causative in adverse pregnancy outcomes would revolutionize the way we manage pregnancy in the mare. references 1. xia yx, cornelius aj, donnelly cg, et al: metagenomic analysis of the equine placental microbiome. clin therio 2017;9:452. 2. moffett a, loke c: immunology of placentation in eutherian mammals. nat rev immunol 2006; 6:584-594. 3. aagaard k, ma j, antony km, et al.: the placenta harbors a unique microbiome. sci transl med 2014; 6:237. 4. sathe s, leiken a, plummer p: metagenomic sequencing of the uterine microbial environment during estrus and early pregnancy in mares. clin therio 2017;9:453. 5. hemberg e, einarsson s, kútvölgyi g, et al: occurrence of bacteria and polymorphonuclear leukocytes in fetal compartments at parturition; relationships with foal and mare health in the peripartum period. theriogenology 2015;84:163-169. 6. amarasekara r, jayasekara rw, senanayake h, et al: microbiome of the placenta in pre-eclampsia supports the role of bacteria in the multifactorial cause of pre-eclampsia. j obstet gynaecol res 2015;41:662-669. 7. cao b, stout mj, lee i, et al: placental microbiome and its role in preterm birth. neoreviews 2014;15:537-545. 8. hanafy ka, krumenacker js, murad f: no, nitrotyrosine, and cyclic gmp in signal transduction [review] med sci monit 2001;7:801-819. 9. hannken t, schroeder r, stahl ra, wolf g: angiotensin ii-mediated expression of p27kip1 and induction of cellular hypertrophy in renal tubular cells depend on the generation of oxygen radicals. kidney int 1998;54:1923-1933. 10. harada k, komuro i, shiojima i, et al: pressure overload induces cardiac hypertrophy in angiotensin ii type 1a receptor knockout mice. circulation 1998;97:1952-1959. 11. leblanc m m: ascending placentitis in the mare: an update. reprod domest anim 2010; 45: 28-34. 12. macpherson m l: diagnosis and treatment of equine placentitis. vet clin north am equine pract 2006; 22:763-776. 13. fraga m, perelmuter k, delucchi l, et al: vaginal lactic acid bacteria in the mare: evaluation of the probiotic potential of native lacrobacillus spp. and enterococcus spp. strains. antoine van leeuwenhoek 2007; 93:71-78. 14. sirota i, zarek sm, segars jh: potential influence of the microbiome on infertility and assisted reproductive technology. semin reprod med 2014;32:35-42. 15. pelzer e, gomez-arango lf, barrett hl, et al: review: maternal health and the placental microbiome. placenta 2017;54:30-37. clinical theriogenology • volume 10, number 3 • september 2018 282 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype true /parseiccprofilesincomments true /embedjoboptions true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2018: effect of feeding glucosinolates to mares on triiodothyronine levels effect of feeding glucosinolates to mares on triiodothyronine levels m.f. lopez-rodriguez,a n. cymbaluk,a b. laarveld,b e.c. serrano-recalde,a c.e. carda adepartment of large animal clinical sciences, western college of veterinary medicine and bdepartment of animal and poultry science, university of saskatchewan, saskatoon, sk, canada brassicaceae family plants contain glucosinolates (gsl), which when ingested form metabolites that suppress thyroid function by inhibiting iodide uptake and organification. experimental data on the tolerance of horses to gsl are lacking, however consumption of gsl during pregnancy has been linked to hypothyroidism in dams and their foals. our null hypothesis was that chronic feeding of gsl would result in normal thyroid function in non-pregnant mares. a 3-month feeding trial was performed using isonitrogenous and isocaloric diets, with oriental mustard powder included as a source of gsl. the effect of gsl on triiodothyronine (t3), the biologically active form of thyroid hormone, was studied. mares were assigned to four diet groups: control (n = 6), propylthiouracil (ptu; n = 6; 4 mg/kg po sid), low gsl (n = 7; 20 mmol gsl / day), and high gsl (n = 6; 35 mmol gsl / day). thyroid releasing hormone (trh) stimulation tests (1 mg iv, sigma chemical company) were performed at 0, 6 and 12 weeks with blood obtained at 0 and 2 hours after trh administration. serum t3 and thyroxine (t4) levels were measured using siemen’s immunlite method (prairie diagnostic services, saskatoon, sk). triiodothyronine parameters measured were: basal (pre-), post-trh (2 hr), fold change, post-pre trh, and t3/t4 ratio. data were analyzed at (p<0.05; spss version 24) by shapiro-wilk, and median tests with pairwise bonferroni corrected post-hoc comparisons. there was a significant effect of week among groups on median t3: fold change (p=0.008), where week 0 was greater than week 12 (p=0.003) and week 6 was greater than week 12 (p=0.003); post-pre data (p=0.039) with week 6 values greater than week 12 (p=0.020); t3/t4 ratio (p<0.001) with week 0 greater than week 6 and 12. there was a significant effect of group on t3 median: pre values (p<0.001), with ptu group less than control (p<0.001), low gsl (p<0.001) and high gsl (p=0.002); post values (p=0.002), with ptu group less than control (p=0.002), low gsl (p=0.02) and high gsl (p=0.024), and control values were greater than (p=0.046) the high gsl group; post-pre median values (p=0.001), with control greater than ptu (p=0.002), low gsl (p=0.039) and high gsl groups (p=0.046); and t3/t4 ratio (p<0.001) with control greater than the ptu (p=0.031) group. in conclusion, ptu and gsl groups resulted in lower t3 values over time, with the ptu group more dramatically affected compared to the gsl group, with control group having greater median t3 post values than high gsl mares, as well as greater t3 post-pre levels than high and low gsl mares. in addition, dietary tolerance of mares to gsl is less than 20 to 35 mmol per day, as there is evidence of thyroid suppression at this level, and should be avoided. keywords: thyroid, glucosinolates, triiodothyronine, propylthiouracil clinical theriogenology • volume 10, number 3 • september 2018 314 2018: enrofloxacin administration to late pregnant mares does not cause articular lesions or decrease tendon strength in the resulting foals enrofloxacin administration to late pregnant mares does not cause articular lesions or decrease tendon strength in the resulting foals r.e. ellerbrock,a i.f. canisso,a p.j. roady,a g. podico,a b. curcio,a f.s. lima,a z. lib adepartment of veterinary clinical medicine, broy carver biotechnology center, university of illinois urbana-champaign, urbana, il enrofloxacin would be an excellent antimicrobial to treat bacterial infections in pregnant mares if proven safe. previous work demonstrated that enrofloxacin crosses the equine placenta and reaches therapeutic concentrations in the fetal fluids without apparent affects on the fetus, and that ciprofloxacin, an active metabolite of enrofloxacin, accumulates in the allantoic fluid over time (i.e., up to 1,000 ng/ml). however, it is unknown if in utero exposure to enrofloxacin and ciprofloxacin affect cartilage development in the weight-bearing foal. we hypothesized that enrofloxacin administration to late-term pregnant mares would not produce cartilaginous lesions or tendonopathies in the resulting foal. the objectives of this study were to determine: (i) if in utero exposure to enrofloxacin and ciprofloxacin resulted in clinical lameness or lesions in articular cartilage or tendons of the foal, and (ii) if in utero exposure resulted in decreased tensile strength or cartilage thickness in the resulting foals. clinically healthy mares (7-23 years old) were enrolled in the study at 280 days gestation, and were assigned to: 1) control (n=5), 2) therapeutic dose of enrofloxacin (n=6, 7.5mg/kg po), or 3) double therapeutic dose enrofloxacin (n=6, 15 mg/kg po). enrofloxacin was administrated once a day for 14 days, starting 280 days gestation. maternal plasma samples were collected daily and plasma preserved at -80oc. transabdominal ultrasound guided fetal fluid sampling was performed in a subset of mares (n=4) at 330335d gestation, and fetal fluids were collected for enrofloxacin and ciprofloxacin concentrations. mares were allowed to carry the foals to term. after foaling, mares and foals were maintained on pasture for 30d, until foal euthanasia. proximal articular surfaces of foal long bones and fetal membranes were examined macroscopically and stained with he for histopathological evaluations. tissues were graded for structural, cellular, and extracellular changes. tensile testing was conducted on superficial flexor tendons by loading until failure (instron 8511, norwood, ma). histomorphometric and extracellular matrix analyses were evaluated using a non-parametric kruskal-wallis one-way analysis of variance. enrofloxacin and ciprofloxacin concentrations were measured by lc-ms/ms. tensile data were analyzed using anova with repeated measures and tukey post-hoc test in r version 3.2.2 (https://www.rproject.org/). significance was set at p<0.05. peak plasma concentrations were 1040 ± 228 ng/ml (enrofloxacin) and 210 ± 25 ng/ml (ciprofloxacin) for the 7.5 mg/kg group and 1638 ± 246 ng/ml (enrofloxacin) and 327 ± 21 ng/ml (ciprofloxacin) for the 15 mg/kg group on day 1. both enrofloxacin and ciprofloxacin were still detectable in the amniotic (7.4 ±4.7, 15.3 ±12.4 ng/ml) and allantoic (11.2 ±1.0, 148.3 ± 61.1 ng/ml) fluids thirty to forty days after administration of the last dose. no differences were seen in macroscopic, cytological, or extracellular matrix lesions in the cartilage of foals from control or treated groups. no differences in tendon yield stress were detected between the control (7.6± 1.0 mpa), 7.5mg/kg (8.6 ± 1.5 mpa) and 15 mg/kg (9.2 ± 1.9 mpa) groups. these findings suggest that enrofloxacin administration to the late pregnant mare reaches therapeutic drug concentrations in the fetus and fetal fluids without detrimental effects on the cartilage and tendons of the resulting foal. additionally, ciprofloxacin concentrations in the allantoic fluid may remain elevated for many days after administration of last enrofloxacin dose. this suggests that enrofloxacin may be a useful antibiotic option for late pregnant mares suffering severe bacterial infections. keywords: fluoroquinolone, equine, toxicity, foal, pregnancy clinical theriogenology • volume 10, number 3 • september 2018309 2018: impact of age and castration on canine prostate collagen organization impact of age and castration on canine prostate collagen organization h. ruetten,a k. wegner,b s. colopy,a h. zhang,d s. sandhu,a m. romero,e,f c. vezinaa,b,g aschool of veterinary medicine, bmolecular and environmental toxicology center, dcollege of agriculture & life sciences, gcarbone cancer center, university of wisconsin-madison, madison wi; ephysiology and biomedical engineering, fnephrology and hypertension, mayo clinic college of medicine and science, rochester, mn prostate-related urinary signs such as incomplete bladder emptying and straining are common in older intact male dogs and men. a longstanding paradigm is these symptoms derive from an aging-related increase in prostate volume but new evidence suggests additional mechanisms. for example, human prostatic collagen content was recently associated with tissue stiffness and urinary retention. the implications of which are huge for the veterinary community because treatment of fibrosis would not impact fertility like current alpha reductase inhibitors. the present study tested whether canine prostatic collagen increases with advancing age and whether castration reduces prostatic collagen content. we also used mice as a tractable model to test whether castration changes baseline urination and whether testosterone supplements reverse these changes. the canine study involved prostates obtained at necropsy divided into four groups: young (≤3 years) intact, young neutered, old (≥5 years) intact, and old neutered (n=9, 4, 4, and 14 respectively). formalin fixed paraffin embedded prostate sections were stained with picrosirius red (psr), imaged with florescent microscopy, and quantitatively analyzed for collagen fiber thickness, length, and density using novel computer software (ct fire, developed at lociuniversity of wisconsin-madison). the c57bl/6j mouse study design involved three groups: sham castrated, castrated + placebo implant, castrated + slow release testosterone implant (n=8). urinary function was evaluated by anesthetized cystometry. transverse canine prostate sections (figure) were characterized by four histologically distinct regions: urethral, periurethral, peripheral, and capsule. the prostatic urethra harbored a thick band of densely packed collagen. collagen bundles radiated from the urethra and into the periurethral region where they branched before extending into the peripheral region. collagen fibers in the peripheral region changed orientation before networking with the circumferential fibers of the capsule. collagen fibers were denser in periurethral and peripheral regions of neutered male prostates compared to intact. prostatic peripheral region collagen fibers were denser and the capsular collagen fibers thicker in old intact male prostates compared to compared to young intact males (p = 0.0251 and 0.0470, respectively). in mice, castration reduced void duration and time between voids, and testosterone supplementation returned void duration to that of intact but did not alter the reduced time between voids. consistent with our hypothesis, this study revealed aging related increases in collagen density in intact males, and surprisingly castration was protective against these increases. contrary to our hypothesis, castration resulted in increased glandular collagen density. these studies are the first to characterize regional collagen organization in the canine prostate and demonstrate the impact of castration on mouse voiding behavior. supported in part by nih u54 dk104310 & dk100227. keywords: benign prostatic hyperplasia, collagen, fibrosis, cystometry, prostate figure. psr stained prostate section imaged with fluorescent microscopy clinical theriogenology • volume 10, number 3 • september 2018319 2017 ultrasound anatomy of the penis in normal bos taurus bulls � ultrasound anatomy of the penis in normal bos taurus bulls amy e. schkeeper, robert c. cole, william r. brawner, julie a. gard, herris s. maxwell, misty a. edmondson department of clinical sciences, college of veterinary medicine, auburn university, auburn, al abstract the bull penis is subject to various medical conditions that have the potential to cause permanent damage to the penile tract, and thus devastating financial loss to the owner. such conditions include: trauma with hematoma formation, abscess formation, vascular shunting, urethral obstruction, and fistula formation. the purpose of this study is to examine and document the sonographic appearance of the penis in a subset of normal bos taurus bulls extending from the distal bend of the sigmoid flexure through the glans. a second purpose is to provide an anatomical reference correlating ultrasound, computed tomography, magnetic resonance imaging, and gross images. four locations in the bull penis were chosen: the distal bend of the sigmoid flexure (s), the glans (g), and two equidistant locations in between (a and b). bulls presenting for breeding soundness examination were recruited and were divided into three age groups: 15-18 months, 22-24 months, and 36 months and older. the hypothesis was that no significant differences (p < 0.5; with a confidence interval of 95%) in the penile measurements of bulls in and between the different age groups, including the measurements between locations a, b, and s, would be found. following numerous measurements of the bull penis from bulls in three different age groups and at three different locations a, b, and s no significant differences in measurements were found supporting our hypothesis. the data were analyzed using the general linear model (glm) for analysis of variance (anova) and scheffe’s test for multiple comparisons. a p value of < 0.05 is considered significant. keywords: fibroelastic, urethra, comparison, correlative, ct, mri introduction a bull’s usefulness is primarily related to the viability of his reproductive tract. swelling of the preputial region is a common presenting complaint that, when associated with penile injury and dysfunction, can lead to devastating economic and genetic losses to the cattle producer.1-3 causes of penile dysfunction and/or preputial swelling include penile hematomas, abscesses, urinary obstruction or fistula formation due to urethral calculi, hair rings, papillomas and vascular shunting. hematomas can form in bulls from various causes including premature separation of the interdigitating attachment (joins the epithelium of the free portion of the penis with the integumentary epithelium of the penile portion of the internal lamina of the prepuce), rupture of the tunica albuginea (“broken” or “fractured” penis) and less commonly, rupture of superficial vessels of the prepuce.1,2 hematomas often resolve on their own without notice, but have the potential to serve as a nidus for infection, resulting in an abscess; which can lead to phimosis.2 abscesses may also occur secondary to lacerations or foreign body penetration.2 accurate and rapid diagnosis is crucial since the course of treatment of these conditions can be vastly different. while diagnosis can be suggested via location and symmetry of the swelling, the convenience, portability, and availability of base line descriptions of cross-sectional imaging via ultrasound has potential for aiding in the diagnosis of penile abnormalities. ultrasound is a developing, minimally invasive imaging modality, that practitioners are finding invaluable for the examination of food animals, particularly for reproductive assessments.4 ultrasound of the testes and epididymis as well as the accessory sex glands including the prostate, the bulbourethral gland, the ampullae, and vesicular glands, has been described in the literature and is typically done with little or no sedation.5-11 the only reports of ultrasonic evaluation of the bull penis include two single transverse images of a normal penis, two penile abscesses, two penile hematomas, and one method for injecting contrast medium into the crus penis.7,8,12,13 although, the penis has been mentioned as part of an ultrasound examination of the reproductive tract, the sonographic appearance of the penis in the normal bull has not yet been thoroughly documented. once normal findings are completely documented, the use 545 clinical theriogenology • volume 9 number 4 • december 2017 � of ultrasound for diagnosing bovine penile problems can progress beyond detection of large abscesses and hematomas. in human medicine, ultrasound is commonly used in diagnosis of causes of impotence including vascular shunting.14 these sometimes subtle changes could not be accurately identified if the normal ultrasonographic appearance was not established first. bulls have a fibroelastic penis which is firm at all times and forms a sigmoidal flexure in the nonaroused state (the distal curve of the “s” shape opens cranially).15 the penis has a cylindrical shape with a urethral groove. the penis consists of a root, body and glans penis. the root consists of the origin of the erectile tissue (corpus cavernosum and corpus spongiosum) and the muscles of the penis. the paired corpus cavernosa (crura) make up the majority of the penis and arise alongside the ischiatic arch, medial to the ischiatic tuberosity, and course ventromedially to form the body of the penis.16 the corpus spongiosum (bulb of the penis) surrounds the urethra, originating along the midline of the ischiatic arch, between the crura.16 the thick tunica albuginea surrounds the erectile tissues and has an outer layer of longitudinal fibers and an inner circular layer. the inner layer is filled with a dense system of trabeculae that form a central axial column of fibrous tissue and radiate towards the tunica.15 between the fibrous tissues are small cavernous blood spaces along the length of the penis, representing the corpus cavernosum penis.15 the paired retractor penis muscles originate from the first and second coccygeal vertebra and pass along the ventral caudal surface of the penis to attach to the ventrolateral surface of the penis at the distal bend of the sigmoid flexure – these retract the penis and prepuce into the sheath after erection.17 the free part of the penis has a counterclockwise twist when viewed from behind, making the urethral orifice lie along the right side of the penis.15 the glans is not well developed and is made up of loose connective tissue encompassing a thin layer of plexiform veins.15 the objectives of this study were to determine the normal ultrasonographic appearance of the penis in normal bulls and to provide a cross-sectional atlas of the penis correlating gross anatomy with ultrasound, computed tomography (ct), and magnetic resonance imaging (mri) and to determine if variations occur in penile measurements at various sites of the penis and if the differences are determined to be significantly between the age groups sampled. methods animals bos taurus bulls presenting for breeding soundness examination (bse) between november 2015 through february 2016 were recruited for this study on a voluntary basis with informed client consent. exclusion criteria included any history of reproductive or urinary tract injuries or medical problems related to the penis or urethra (including penile trauma, penile hematomas, retropreputial abscesses, penile vascular shunting, urethral calculi, stranguria, or hematuria). this study was approved by and in accordance with requirements of our institutional animal care and use committee. animals were grouped based on age: group 1 (15-18 months), group 2 (20-24 months), and group 3 (36 months and older). ultrasound examination ultrasound examination was performed (a.e.s) after a bse was performed with the bull restrained in a squeeze chute with a mechanical headgate. a wooden bar was placed behind the pelvic limbs so as to confine the bull to the front portion of the chute, limit back and forth motion, and prevent or slow kicking. semen was collected from all bulls for the bses using electroejaculation. the results of the bses were not included in this study. no sedation was given to any bull prior to or during the ultrasound examination. if a bull was not cooperative for the ultrasound examination, the examination was terminated and bull was excluded from this study. an 8l (4-12mhz) wide band linear probe with a portable ultrasound machine (ge logiq e, carlsbad, ca) was used to obtain transverse images of the penis from the level of the distal bend of the sigmoid flexure and extending distally. images at the distal bend of the sigmoid flexure were obtained caudal to the scrotum from behind the patient. these images were both obtained from above and below the crossbar in the chute, depending on ease of examination and positioning of the patient. the remainder of the images were obtained cranial to the scrotum alongside the patient (either the left or right of the patient). isopropyl alcohol was applied generously to 546clinical theriogenology • volume 9 number 4 • december 2017 � the skin of the sheath as the coupling agent. the hair was not clipped in any patient. the majority of the images were scanned at 10 mhz. subjective assessment of ease of examination and cooperation of the bull was recorded as good (bull remained stationary or displayed minimal swaying or shifting feet) or fair (bull swayed left and right, moved forwards and backwards, and lifted back feet). images were recorded both with and without pressure specifically to assess the corpus spongiosum and urethra. pressure involved the amount of contact force required with the probe. this could not be objectively measured, but degree of pressure used was considered adequate when the entire corpus cavernosum could be visualized. the images were labeled at four locations during the examination: s – the distal bend, g – the glans, and at two equidistant locations between the distal bend and the glans – a and b with a being more proximal. image analysis and review were not performed (a.e.s) until after the examination to limit the time required for the bull to be restrained. images were oriented with the left side of the patient being on the left side of the screen. measurements (table and appendix) image analysis and review was performed using either the built in software (ge, carlsbad, ca) on the ultrasound machine for the ultrasound images or a dicom viewing software (osirix, switzerland) for the ct and mr images. various measurements of the penile structures were made at the four previously described locations: skin thickness (a,b,g); both layers of the ventral and dorsal aspect of the tunica albuginea individually and as a whole (s,a,b,g); the height, width, circumference, and area of the corpus cavernosum (s,a,b,g); the height of both parts of the corpus spongiosum together and separately, in both the ventral and dorsal aspects, with and without pressure (s,a,b,g); the height, circumference, and area of the corpus spongiosum as a whole, with and without pressure (s,a,b,g); the height of the urethra with and without pressure (s,a,b,g); the height of one of the ventral vascular channels of the penis (s,a,b); the height of the largest preputial vessel in the field of view (s,a,b); the height of the ventral aspect of the fibrofatty tissue in the glans (g); the height and width of the fibrofatty tissue of the glans (g); and the height of the retractor penis muscles (s). note, the tunica was measured at the level of one of the ventral vascular channels. pressure was not applied at location g as the penis is very movable at this location. the number of preputial vessels in the field of view was recorded (s,a,b,g). subjective assessment of the quality of images was recorded as excellent (visualization of the penile structures from the ventral skin through the dorsal tunica with clear distinction of the dorsal margin of the dorsal tunica), good (visualization through the corpus cavernosum with clear distinction of the dorsal aspect of the corpus cavernosum), fair (visualization through the mid corpus cavernosum with difficult distinction of the dorsal aspect of the corpus cavernosum), and poor (visualization through the ventral aspect of the corpus cavernosum with difficult distinction of the dorsal aspect of the corpus spongiosum) (s,a,b,g). the echogenicity and echotexture of all measured structures were observed and recorded. gross, computed tomography, and magnetic resonance imaging the penile tract of a 2y old 727kg angus bull was removed from a patient euthanized for reasons unrelated to the reproductive or urinary tract. this bull had no history of reproductive or urinary tract trauma or other reproductive/urinary medical problems. the penile tract was removed proximal to the proximal bend of the sigmoid flexure. ultrasound of this penile tract was not performed. the cross sectional imaging was obtained for representative purposes to compare against live animal imaging. helical transverse ct images of the removed penile tract were obtained with 5.0 mm thickness slices and reconstructed into 0.6 mm thickness slices with a 64 slice ct scanner (100 kvp, 80 ma, 1s tube rotation, 0.984:1 pitch, fov 24 x 24 cm, matrix 512 x 512; ge light-speed; ge medical systems, milwaukee, wi). t1-weighed transverse mr images (tr 400.0 ms, te 11.5 ms, 5.0 mm, fov 25 x 25 cm) were also obtained using a 1.5 t scanner and a spine coil (infinion, philips medical systems, andover, ma). the penile tract was positioned to be in a retracted state for both the mr and ct scans. the same measurements and observations were obtained at the previously described four locations, excluding measurements of the skin and assessment of the preputial vessels. in addition, hounsfield units (hu) 547 clinical theriogenology • volume 9 number 4 • december 2017 � were measured at each location and averaged. cuts through the penile tract at the four measured locations were made using a 10 scalpel blade. statistics data were analyzed using the general linear model (glm) for analysis of variance (anova) and scheffe’s test for multiple comparisons. a p value of < 0.05 was considered significant. results examination thirty-four bulls were recruited: 15 in group 1 (15-16 months of age), 4 in group 2 (22-24 months of age), and 15 in group 3 (36-75 months of age). bos taurus bulls of the representative breeds; angus, hereford, simmental, simmental-angus, and red angus, were utilized in this study in all three age groups. the bulls were in good body condition having a score between 6-7 on a 1-9 point scale. additionally, all bulls included in the study were determined to have passed the society for theriogenology guidelines for breeding soundness examination. withers height and weight were not included as parameters in this study since, aside from appropriate body condition score, are not included in breeding soundness evaluation. all bulls displayed good cooperation for the ultrasound examination cranial to the scrotum. approximately half of the bulls in each group held their penis in a mildly retracted state with the glans located in the distal third between the termination of the sheath and the scrotum, while the remainder of the bulls held their penis in a very retracted state with the glans located in the proximal third of the sheath. with the bulls in the more retracted state, only locations g and b were able to be imaged cranial to the scrotum while locations a and s were both imaged caudal to the scrotum. approximately, a third of the bulls in each group displayed fair cooperation for the examination caudal to the scrotum. all images were considered diagnostic with the majority being classified as good to excellent quality in each group at locations a, b, and g, and considered fair in the majority of bulls in each group at location s. sonographic appearance locations a and b (fig. 1a) locations a and b were indistinguishable sonographically. the corpus cavernosum (ccp) was bilobed and had a fine, mildly hyperechoic texture when enough pressure was applied and when the ultrasound probe was held perpendicular to the axis of the penis. if the pressure was lessened or if the probe was not perpendicular, the central portion of each crus became hypoechoic with distal acoustic shadowing and the remainder of the ccp became mildly heterogeneous. a ventral vascular channel in the most ventral portion of each crus was anechoic with a hyperechoic dorsal and ventral rim and with variable distal acoustic enhancement. this channel occasionally narrowed or collapsed with pressure. the corpus spongiosum (csp) and urethra varied in appearance depending on the amount of pressure. without pressure, the csp was egg-shaped and had a large coarse hypoechoic outer layer with a smaller inner layer that was mildly hypoechoic with a fine echotexture. with pressure, the outer layer of the csp decreased in size, became circular to ovoid in shape, and became hyperechoic with a fine echotexture. this layer of the csp was not always distinguishable from the surrounding tunica without manipulation of pressure. the inner layer of the csp became somewhat smaller in size with pressure, and maintained a hypoechoic fine texture. the urethra was anechoic and collapsed in the majority of patients with or without pressure, though the location was always visible. the tunica that surrounds the ccp and csp could be easily distinguished into its longitudinal and circumferential parts. the circumferential fibers of the tunica that surrounds the ccp was homogenously hypoechoic, and not always visible along the dorsal aspect. this hypoechoic part of the tunica maintained the same width surrounding the ccp though became slightly thicker at the ventral junction of the crus. the longitudinal fibers of the tunica that surround both the ccp and csp were hyperechoic with a fine echotexture. the skin surrounding the penis was hypoechoic, thin, and maintained the same width around the penis. in some bulls, air (hyperechoic reverberating shadows) was seen between the penile skin and the prepuce, which, if in enough quantity, would hinder visualization of the penis. the air dissipated enough to adequately assess 548clinical theriogenology • volume 9 number 4 • december 2017 � the penis within a few minutes. the number of preputial vessels seen on the images obtained varied from 1 unilaterally to 6 bilaterally, and varied vastly in size. all vessels were anechoic with a hyperechoic ventral and dorsal rim and had variable distal acoustic enhancement. sonographic appearance location g (fig. 2a) at location g, the penis had already begun its counterclockwise rotation (as seen from behind), with the csp and urethra noted on the left of the patient. the urethra was no longer visible at this location. the two layers of the csp became indistinguishable and the csp appeared as an ill-defined, slightly heterogeneous hyperechoic circular structure. the csp was often only visible if followed from proximal to the glans. the ccp extended distally as a small process being homogenously mildly hyperechoic with a circular cross-section. ventral vascular channels were no longer visible. the tunica could still be differentiated into the longitudinal and circular layers with the entire tunica being easily visualized. the fibrofatty portion of the glans surrounded the tunica and was primarily hypoechoic with radiating hypo and hyperechoic bands coursing towards the axis of the penis. this fibrofatty tissue had a slightly hyperechoic rim, which was adjacent to the penile skin. sonographic appearance location s (fig. 3a) location s was the most difficult location to image primarily due to the thickness of the tissues caudal to the penis resulting in additional pressure needed to visualize the entire ccp. the ccp and csp were similar in appearance to locations a and b though the center of each crus of the ccp was almost always hypoechoic with distal acoustic shadowing. the urethra dilated as it coursed proximal to the distal bend of the sigmoid flexure and the ccp and csp became somewhat distorted due to the angle of the probe with respect to the cranial course/bend of the penis. the appearance to the ventral vascular channels and preputial vessels was similar as locations a and b. computed tomography (fig. 1b,2b,3b) locations a, b, and s had a similar appearance. when describing ct images, terms including hyper, hypo and isoattenuating are used. similar to radiography, ct is a measure of the attenuation of xrays. structures that appear white are hyperattenuating, while structures that appear black are hypoattenuating. the ccp was again noted to be bilobed. the core of the crus of the ccp were slightly hyperattenuating with an average hu of 115. the outer portion of the ccp was slightly hypoattenuating with an average hu of 75. the ventral vascular channels were easily seen as symmetric hypoattenuating circular tubes coursing through the ventral aspect of the ccp. the csp was ovoid and homogenously hypoattenuating with an average hu of 55. the urethra was only seen as a small amount of gas in the central portion of the csp in the proximal bend of the sigmoid flexure. the tunica appeared homogenously hyperattenuating with an average hu of 100. the distinction between longitudinal and circumferential fibers was not evident. the skin could not be distinguished from the tunica. the retractor penis muscles were seen as symmetric crescent hypoattenuating shapes along the ventral aspect of the penis with an average hu of 42. at location g, the ccp was seen as a small, homogenous, mildly hypoattenuating cylindrical extension with an average hu of 75. the csp and urethra could not be identified. the tunica was seen as a hyperechoic rim surrounding the ccp with an average hu of 100. the fibrofatty tissue surrounding the tunica was symmetric and mildly hypoattenuating with an average hu of 24. the skin was seen as a hyperechoic rim surrounding the fibrofatty tissue with an average hu of 80. measurements of the visualized structures were obtained and were within the ranges of the respective ultrasonographic measurements. magnetic resonance imaging (fig. 1c,2c,3c) locations a, b, and s had a similar appearance. the mr image is based on signal intensity. when describing mr images, the terms hyper, hypo, and isointense are utilized. structures that appear bright (white) on mr are said to be hyperintense while structures that are dark (black) are said to be hypointense. the ccp was again noted to be bilobed. the core of the crus of the ccp was hypointense 549 clinical theriogenology • volume 9 number 4 • december 2017 � with the outer portion being well-defined and hyperintense. the ventral vascular channels were seen as small symmetric ovoid regions of hyperintensity – these were hyperintense with respect to the outer portion of the ccp. the csp was homogeneously hyperintense, being isointense to the ventral vascular channels. the urethra was only seen as a small amount of gas (signal void) in the central portion of the csp in the proximal bend of the sigmoid flexure. the distinction between longitudinal and circumferential fibers was easily seen with the circumferential fibers being hypointense (isointense to the core of the ccp), and with the longitudinal fibers being mildly hypointense (hypointense with respect to the outer portion of the ccp). the skin was seen as a thin hypointense band encircling the tunica. the retractor penis muscles were seen as symmetric crescent hyperintense shapes along the ventral aspect of the penis – these were hyperintense with respect to the csp. at location g, the ccp was seen as a very small, hypointense center with a hyperintense rim. the hypointense longitudinal fibers of the tunica surrounded the ccp, though the circumferential fibers could not be distinguished. the fibrofatty tissue was very hyperintense surrounding the tunica and was mildly asymmetric with the portion incorporating the csp being thicker. the csp could not be distinguished from the fibrofatty tissue. the skin was not visible. measurements of the visualized structures were obtained and were within the ranges of the respective ultrasonographic measurements. gross anatomy (fig. 1d,2d,3d) the architecture of the penis was similar at locations a, b, and s. the ccp was seen to be made up of radiating fibers directed towards the central portion of each crus. the core of each crus was dense and very firm with the outer portion being less dense and thus, less firm. the ventral vascular channels were easily seen and gas filled. the csp was palpably soft with a denser central portion and a less dense, more cavitary outer portion. the circumferential fibers of the tunica were palpably very firm while the longitudinal fibers were less firm. the skin was seen as a very thin layer surrounding the tunica. the retractor penis muscles were both soft. at location g, the ccp was somewhat soft with a firm tunica surrounding it. the csp was also soft. the fibrofatty tissue in the glans was palpably spongy. measurements a detailed chart of all measurements is below (table and appendix.). there were no significant differences found between any recorded measurements within the different age groups (p < 0.5). hence, these findings support our hypothesis that no statistical differences would be found when comparing measurements taken at specific locations on the penis; a, b, and s in the groups evaluated. the thickness of the penile skin averaged 0.1 cm for locations a, b, and s, and 0.05 cm for location g, with overlap of the ranges of each group. the dorsal aspect of the tunica was only visible in approximately half of the patients at locations a and b, and in only one patient at location s. the height of the circumferential fibers of the tunica was similar between all locations, ventrally and dorsally with an average of 0.1 cm. the height of the longitudinal fibers and the height of the tunica as a whole had similar averages between the dorsal and ventral aspects at locations a, b, and s with an average of 0.27 cm for the longitudinal fibers and 0.36 cm for the whole tunica. although the high end of the range for the ventral aspect of both the longitudinal fibers and whole tunica exceeded that of the dorsal aspect. the longitudinal fibers and the whole tunica were smaller at location g compared to the other three locations with an average of 0.15 cm and 0.19 cm, respectively. the measurements for the ccp tended to increase from the youngest to oldest age groups at locations a and b, and tended to increase from location b to a to s. there was overlap in the ranges and no significant difference in the averages (p<0.05). the average height of the ccp at locations a, b, and s was 1.26 cm and at location g was 0.25 cm. the csp height as a whole was similar between locations a, b, and s, and increased when pressure was released with an average of 0.40 cm with pressure and 0.61 cm without pressure. the height of the csp at location g averaged 0.24 cm. the average urethral height was similar amongst locations a, b, and s at 0.02 cm, and was not visualized at location g. the height of the ventral vascular channel with pressure was similar between locations a, b, and s with an average of 0.1 cm. the average height increased without pressure at locations a and b (0.17 cm), and there was overlap of the ranges. the average height of the ventral vascular channel did not 550clinical theriogenology • volume 9 number 4 • december 2017 � differ at location s with or without pressure. the diameter of the largest preputial vessel was similar between locations a, b, and s with pressure, but increased to a greater extent at locations a and s without pressure. the largest diameter in any location was 0.37 cm. the average height of the retractor penis muscle was 0.40 cm. conclusions ultrasound is a fast and non-invasive method of evaluating the bull penis. all bulls in this study were cooperative for the scans of the distal penis cranial to the scrotum, though scanning the distal bend of the sigmoid flexure caudal to the scrotum proved more difficult, yet feasible. the images obtained for this study were all deemed diagnostic with good to excellent quality in the portion of the penis cranial to the scrotum, and fair caudal to the scrotum. the hair did not need to be clipped in any patient, thus decreasing the time needed to remain in the chute. in our experience, a bull can be scanned when confined between two swing gates. however, the use of squeeze chute and head catch with a wooden bar behind the pelvic limbs limited the back and forth motion of the bull thus facilitating the exam. this study provides an anatomical reference for the bull penis from the level of the distal bend to the glans using ultrasound, ct, mri, and gross images. although ct and mri are invaluable cross sectional imaging modalities in people and small animals, due to the size of the bovine patient, it is not possible to use these modalities to evaluate the penis unless it is for gross specimens. this emphasizes the need to understand the ultrasonographic appearance of the penis as this is the only imaging modality capable of providing cross sectional images in a live patient. this information can then be used as the basis for further studies of the bovine penis. with the normal values and appearance of the penis established, alterations in the tunica albuginea and ccp, including vascular shunting in cases of penile trauma or congenital shunting and occlusion of the vascular channels can be studied. the information provided herein will aid in future diagnoses of urinary obstruction, vascular shunting, tearing of the tunica, hematoma, and abscess formation. it may limit the need for more challenging imaging procedures such as cavernosography and urethrography. limitations of this study are that all bulls had a breeding soundness examination utilizing electroejaculation performed immediately prior to scanning, though as the bull penis is fibroelastic, this is not expected to alter the dimension of the penis. other potential limitations of the study include the challenges of imaging the penis at the level of the sigmoid flexure. this is an important area to image as it is the most common site of injury with a fractured penis. this was more difficult than areas distal to the scrotum, but with experience and practice, we found that diagnostic images could be obtained in this region. although not part of this study, light sedation could be given that may potentially make imaging that region easier. ultrasound examination requires the use of variable pressure with the probe no matter what organ is being imaged. some patients require more probe pressure than others and certain locations require more pressure than other locations (sigmoid vs the distal penis). as a result, there is no practical way to standardize the amount of pressure required. with practice the clinician will come to understand how pressure (or lack thereof) can affect the image. this was shown when evaluating the ccp and ventral vascular channels, as well as the csp and urethra. for example, it is possible that if too much pressure is applied, a dilated obstructed urethra could be artifactually collapsed causing the clinician to misdiagnose a urethral obstruction. furthermore, with experience, we believe the clinician will be able to gauge how much probe pressure is required and will learn to vary the probe pressure when needed as is routinely done with ultrasoungraphy of the ovary when determining follicular diameter. this is a clear limitation of ultrasound as operator variability will have an effect not only on measurement values, but image quality. despite this limitation, the values that are reported here will serve as a baseline and could be revised as more studies are performed. only bos taurus bulls of the most common breeds (angus, simmental, and hereford) were utilized in the study to represent the most common subset of the bull population that is seen within the united states. bos indicus breeds were not included in this study due to the possibility of adding confounding variables to the measurements taken. a similar study, assessing bos indicus bulls needs to be preformed which would allow for statistical comparisons within bos indicus bull populations and for valid comparisons to the measurements taken from bulls of bos taurus lineage. 551 clinical theriogenology • volume 9 number 4 • december 2017 � acknowledgments the authors thank sound eklin (ge logiq e – carlsbad, ca) for providing an ultrasound machine for this project. we also thank the faculty, staff, and students at auburn university who helped in the recruitment and handling of the patients. lastly, we thank dr. james wright for his assistance with the statistics. this work was supported by the department of clinical sciences at auburn university, college of veterinary medicine references 1. barth a: testicular degeneration in: hopper rm, editor. bovine reproduction. ames(ia): wiley-blackwell; 2015. p. 103-108. 2. maxwell h: inability to breed due to injury or abnormality of the external genitalia of bulls. in: hopper rm, editor. bovine reproduction. ames(ia): wiley-blackwell; 2015. p. 113-130. 3. simpson km, streeter rn: bovine urolithiasis in: hopper rm, editor. bovine reproduction. ames(ia): wileyblackwell; 2015. p. 172-180. 4. ginther oj: how ultrasound technologies have expanded and revolutionized research in reproduction in large animals. theriogenology 2014;81:112-125. 5. abou-el-roos, mea: evaluation of buffalo-bulls by using modified serving capacity test and diagnostic ultrasound. j egypt vet med associ 2004;64:17. 6. abu-seida, am: ultrasonographic diagnosis of some scrotal swellings in bulls. pak vet j 2012;32:378-381. 7. gnemmi g, lefebvre rc: ultrasound imaging of the bull reproductive tract: an important field of expertise for veterinarians. vet clin north am food anim pract 2009;25:767-779. 8. gnemmi g, lefebvre rc: bull anatomy and ultrasonography of the reproductive tract in: descôteaux l, colloton j, gnemmi g, editors. practical atlas of ruminant and camelid reproductive ultrasonography. ames(ia): wileyblackwell; 2010, p. 143-162. 9. kastelic jp, brito lfc: ultrasonography for monitoring reproductive function in the bull. reprod domest anim suppl 3 2012;47:45-51. 10. momont h, checura c: ultrasound evaluation of the reproduction tract of the bull in: hopper rm, editor. bovine reproduction. ames(ia): wiley-blackwell; 2015. p. 79-91. 11. streeter rn, step dl: diagnostic ultrasonography in ruminants. vet clin north am food anim pract 2007;23:541574. 12. anderson de, st-jean g, desrochers a, et al: use of doppler ultrasonography and positive-contrast corpus cavernosography to evaluate a persistent penile hematoma in a bull. j am vet med assoc 1996;209:1611-1614. 13. nöthling jo, irons pc, gerber d: ultrasound-guided injection of contrast medium into the crus penis for diagnosis of erection failure in bulls. theriogenology 2002;57:1199-1205. 14. bacar mm, batislam e, altinok d, et al: sildenafil citrate for penile hemodynamic determination: an alternative to intracavernosal agents in doppler ultrasound evaluation of erectile dysfunction. urology 2001;57:623-626. 15. nickel r, schummer a, seiferle e: male genital organs of the ruminants in: schummer a, nickel r, sack o, editors. the viscera of the domestic mammals. 2nd ed. new york: springer-verlag; 1979. p. 333-339. 16. nabors b, linford r: anatomy of the reproduction system of the bull in: hopper rm, editor. bovine reproduction. ames(ia): wiley-blackwell; 2015. p. 5-10. 17. beckett sd, wolfe df. anatomy of the penis, prepuce, and sheath: cattle, sheep, and goats in: wolfe df, moll d, editors. large animal urogenital surgery. baltimore: williams-wilkins; 1998. p. 201-209. 552clinical theriogenology • volume 9 number 4 • december 2017 � table. measurements obtained with ultrasound measurements in cm or cm2 location a location b location g location s tunica dorsal aspect of the tunica .34 (0.18-0.45) n16 0.25 (0.21-0.34) n16 0.34 (0.34-0.34) n1 ventral aspect of the tunica .39 (0.27-0.62) 0.42 (0.27-0.69) 0.19 (0.12-0.28) 0.42 (0.15-0.61) ccp height 1.28 (0.81-1.77) 1.17 (0.72-1.52) 0.25 (0.14-0.43) 1.33 (0.85-1.74) width 2.45 (1.81-1.77) 2.42 (2.00 -3.07) 2.48 (2.07-3.05) circumference 6.93 (4.98-9.24) 6.63 (5.34-8.29) 0.99 (0.52-1.54) 7.16 (6.04-9.00) csp with pressure height .42 (0.19-0.68) 0.35 (0.22-0.58) na 0.44 (0.21-0.74) circumference 1.57 (0.89-2.12) 0.56 (0.43-1.75) na 1.62 (0.89-2.9) csp without pressure height .62 (0.41-0.87) 0.56 (0.43-1.00) 0.24 (0.11-0.42) 0.65 (0.30-0.92) circumference 2.09 (1.61-2.47) 1.83 (1.46-2.72) 0.70 (0.37-1.35) 2.16 (1.22-2.90) urethra height with pressure 0.04 (0.00-0.09) 0.02 (0.00-0.15) not visible 0.01 (0.00-0.11) height without pressure 0.17(0.10-0.29) 0.02 (0.00-0.19) not visible 0.16 (0.00-0.56) ventral vascular channel height with pressure 0.09 (0.01-0.18) 0.10 (0.05-0.21) na 0.10 (0.03-0.21) height without pressure 0.17 (0.10-0.29) 0.16 (0.10-0.24) na 0.11 (0.04-0.15) n = number of bulls included in the calculation. if no n value is recorded, the structure was measured in all 34 bulls. 553 clinical theriogenology • volume 9 number 4 • december 2017 � figure 1. transverse image through the penis at location a with ultrasound (a), ct (b), mri (c), and gross (d) images. ventral is on the top of the image and left is on the left of the image. 1 (ccp), 2 (ventral vascular channels), 3 (csp dense inner portion), 4 (csp less dense outer portion), 5 (urethra), 6 (circumferential fibers of the tunica), 7 (longitudinal fibers of the tunica), 8 (skin), 9 (preputial vessel). 554clinical theriogenology • volume 9 number 4 • december 2017 � figure 2. transverse image through the penis at location g with ultrasound (a), ct (b), mri (c), and gross (d) images. ventral is on the top of the image and left is on the left of the image. 1 (ccp), 4 (csp less dense outer portion), 6 (circumferential fibers of the tunica), 7 (longitudinal fibers of the tunica), 8 (skin), 11 (fibrofatty tissue). 555 clinical theriogenology • volume 9 number 4 • december 2017 � figure 3. transverse image through the penis at location s with ultrasound (a), ct (b), mri (c), and gross (d) images. ventral is on the top of the image and left is on the left of the image. 1 (ccp), 2 (ventral vascular channels), 3 (csp dense inner portion), 4 (csp less dense outer portion), 5 (urethra), 6 (circumferential fibers of the tunica), 7 (longitudinal fibers of the tunica), 8 (skin), 9 (preputial vessel), 10 (retractor penis muscle). (editor’s note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) 556clinical theriogenology • volume 9 number 4 • december 2017 � a p p en d ix m ea su re m en ts in c m o r c m 2 g ro up �1 �a vg �� g ro up �2 �a vg �� g ro up �3 �a vg �� al l�g ro up s�a vg )� lo ca tio n a sk in � ve nt ra l�h ei gh t� 0. 09 �(0 .0 6� � 0. 13 )� 0. 1� (0 .0 9� �0 .1 1) � 0. 1� (0 .0 4� �0 .1 6) � 0. 1� (0 .0 4� �0 .1 6) � do rs al �a sp ec t�o f�t he �tu ni ca � w ho le �tu ni ca �h ei gh t� 0. 26 �(0 .1 8� � 0. 41 )� 0. 42 �(0 .4 2� �0 .4 2) � 0. 33 �(0 .2 7� �0 .4 5) � 0. 34 �(0 .1 8� �0 .4 5) � lo ng itu di na l�f ib er s� he ig ht � 0. 16 �(0 .0 9� 0. 33 )� 0. 3� (0 .3 0� �0 .3 0) � 0. 23 �(0 .1 6� �0 .3 9) � 0. 23 �(0 .0 9� �0 .3 9) � ci rc um fe re nt ia l�f ib er s� he ig ht � 0. 1� (0 .0 7� � 0. 12 )� 0. 15 �(0 .1 5� �0 .1 5) � 0. 1� (0 .0 6� �0 .1 4) � 0. 12 �(0 .0 6� �0 .1 5) � � ve nt ra l�a sp ec t�o f�t he �tu ni ca � w ho le �tu ni ca �h ei gh t� 0. 35 �(0 .2 7� 0. 43 )� 0. 4� (0 .3 4� �0 .4 8) � 0. 41 �(0 .2 9� �0 .6 2) � 0. 39 �(0 .2 7� �0 .6 2) � lo ng itu di na l�f ib er s� he ig ht � 0. 26 �(0 .1 9� � 0. 33 )� 0. 3� (0 .2 5� �0 .3 9) � 0. 33 �(0 .2 4� �0 .4 8) � 0. 3� (0 .1 9� �0 .4 8) � ci rc um fe re nt ia l�f ib er s� he ig ht � 0. 09 �(0 .0 5� 0. 13 )� 0. 11 �(0 .0 9� �0 .1 7) � 0. 1� (0 .0 6� �0 .1 8) � 0. 1� (0 .0 5� �0 .1 8) � � cc p� he ig ht � 1. 12 �(0 .8 1� � 1. 51 )� 1. 35 �(1 .0 5� �1 .5 4) � 1. 36 �(0 .8 9� �1 .7 7) � 1. 28 �(0 .8 1� �1 .7 7) � w id th � 2. 35 �(2 .0 9� 2. 59 )� 2. 39 �(1 .8 1� �2 .6 9) � 2. 63 �(1 .9 3� �3 .1 6) � 2. 45 �(1 .8 1� �3 .1 6) � ci rc um fe re nc e� 6. 48 �(5 .2 6� � 7. 43 )� 6. 97 �(5 .1 � � 8. 13 )� 7. 33 �(4 .9 8� �9 .2 4) � 6. 93 �(4 .9 8� �9 .2 4) � ar ea � 2. 35 �(1 .4 � 3. 29 )� 2. 88 �(1 .5 7� �3 .6 0) � 3. 22 �(1 .5 8� �4 .7 0) � 2. 82 �(1 .4 0� �4 .7 0) � � cs p� w ith �p re ss ur e� de ns er ,�i nn er �p or tio n� ve nt ra l�h ei gh t� 0. 04 �(0 .0 3� � 0. 07 )� 0. 06 �(0 .0 6� �0 .0 6) � 0. 06 �(0 .0 3� �0 .1 2) � 0. 06 �(0 .0 3� �0 .1 2) � le ss �d en se ,�o ut er � po rt io n� do rs al �h ei gh t� 0. 11 �(0 .0 6� � 0. 15 )� 0. 13 �(0 .0 2� �0 .2 6) � 0. 13 �(0 .0 7� �0 .2 1) � 0. 12 �(0 .0 2� �0 .2 6) � le ss �d en se ,�o ut er � po rt io n� ve nt ra l�h ei gh t� 0. 19 �(0 .1 2� � 0. 30 )� 0. 3� (0 .1 1� �0 .4 0) � 0. 26 �(0 .1 1� �0 .4 3) � 0. 25 �(0 .1 1� �0 .4 3) � w ho le �c sp �in cl ud in g� ur et hr a� 0. 32 �(0 .2 3� � 0. 44 )� 0. 51 �(0 .3 3� �0 .6 8) � 0. 42 �(0 .1 9� �0 .6 0) � 0. 42 �(0 .1 9� �0 .6 8) � ci rc um fe re nc e� 1. 2� (0 .8 9� � 1. 62 )� 1. 93 �(1 .7 1� �2 .0 8) � 1. 59 �(0 .9 8� �2 .1 2) � 1. 57 �(0 .8 9� �2 .1 2) � ar ea � 0. 11 �(0 .0 5� � 0. 21 )� 0. 28 �(0 .2 3� �0 .3 2) � 0. 2� (0 .0 7� �0 .3 4) � 0. 2� (0 .0 5� �0 .3 4) � � cs p� w ith ou t�p re ss ur e� de ns er ,�i nn er �p or tio n� ve nt ra l�h ei gh t� 0. 09 �(0 .0 6� � 0. 16 )� 0. 12 �(0 .0 8� �0 .1 5) � 0. 11 �(0 .0 5� �0 .1 7) � 0. 11 �(0 .0 5� �0 .1 7) � le ss �d en se ,�o ut er � po rt io n� do rs al �h ei gh t� 0. 2� (0 .1 3� � 0. 29 )� 0. 27 �(0 .2 1� �0 .3 2) � 0. 23 �(0 .1 � � 0. 38 )� 0. 23 �(0 .1 � � 0. 38 )� le ss �d en se ,�o ut er � po rt io n� ve nt ra l�h ei gh t� 0. 38 �(0 .2 9� � 0. 50 )� 0. 28 �(0 .1 9� �0 .3 7) � 0. 38 �(0 .2 7� �0 .5 0) � 0. 35 �(0 .1 9� �0 .5 0) � w ho le �c sp �in cl ud in g� ur et hr a� 0. 61 �(0 .4 5� � 0. 76 )� 0. 60 �(0 .4 1� �0 .7 8) � 0. 69 �(0 .5 8� �0 .8 7) � 0. 62 �(0 .4 1� �0 .8 7) � ci rc um fe re nc e� 2. 00 �(1 .6 1� � 2. 41 )� 2. 06 �(1 .7 1� �2 .4 1) � 2. 20 �(1 .7 6� �2 .4 7) � 2. 09 �(1 .6 1� �2 .4 7) � ar ea � 0. 32 �(0 .2 0� � 0. 46 )� 0. 33 �(0 .2 3� �0 .4 3) � 0. 37 �(0 .2 3� �0 .4 7) � 0. 34 �(0 .2 0� �0 .4 7) � 557 clinical theriogenology • volume 9 number 4 • december 2017 � � u re th ra � he ig ht �w ith �p re ss ur e� 0. 01 �(0 .0 0� � 0. 09 )� 0. 09 �(0 .0 0� �0 .0 3) � 0. 01 �(0 .0 0� �0 .0 8) � 0. 04 �(0 .0 0� �0 .0 9) � he ig ht �w ith ou t�p re ss ur e� 0. 02 �(0 .0 0� 0. 07 )� 0. 08 �(0 .0 0� �0 .1 5) � 0. 05 �(0 .0 0� �0 .1 9) � 0. 05 �(0 .0 0� �0 .1 9) � � ve nt ra l�v as cu la r�c ha nn el � he ig ht �w ith �p re ss ur e� 0. 08 �(0 .0 4� � 0. 18 )� 0. 07 �(0 .0 1� �0 .1 3) � 0. 11 �(0 .0 6� �0 .1 7) � 0. 09 �(0 .0 1� �0 .1 8) � he ig ht �w ith ou t�p re ss ur e� 0. 15 �(0 .1 1� 0. 22 )� 0. 19 �(0 .1 9� �0 .1 9) � 0. 18 �(0 .1 0� �0 .2 9) � 0. 17 �(0 .1 0� �0 .2 9) � � pr ep ut ia l�v es se l� la rg es t�d ia m et er �w ith � pr es su re � 0. 21 �(0 .1 0� � 0. 35 )� 0. 14 �(0 .0 6� �0 .2 3) � 0. 20 �(0 .0 9� �0 .3 7) � 0. 18 �(0 .0 6� �0 .1 0) � la rg es t�d ia m et er � w ith ou t�p re ss ur e� 0. 26 �(0 .1 5� 0. 33 )� 0. 26 �(0 .2 3� �0 .2 9) � 0. 26 �(0 .1 3� �0 .3 7) � 0. 26 � � 0. 13 � � 0. 37 )� lo ca tio n b sk in � ve nt ra l�h ei gh t� 0. 09 �(0 .0 6� � 0. 12 )� 0. 10 �(0 .0 6� �0 .1 5) � 0. 93 �(0 .0 4� �0 .1 4) � 0. 10 �(0 .0 4� �0 .1 5) � do rs al �a sp ec t�o f�t he �tu ni ca � w ho le �tu ni ca �h ei gh t� 0. 25 �(0 .2 3� � 0. 34 )� *� 0. 25 �(0 .2 1� �0 .2 8) � 0. 25 �(0 .2 1� �0 .3 4) � lo ng itu di na l�f ib er s� he ig ht � 0. 19 �(0 .1 4� 0. 29 )� *� 0. 18 �(0 .1 3� �0 .2 3) � 0. 19 �(0 .1 3� �0 .2 9) � ci rc um fe re nt ia l�f ib er s� he ig ht � 0. 09 �(0 .0 6� � 0. 11 )� 0. 18 �(0 .1 8� �0 .1 8) � 0. 11 �(0 .0 9� �0 .1 3) � 0. 12 �(0 .0 6� �0 .1 8) � � ve nt ra l�a sp ec t�o f�t he �tu ni ca � w ho le �tu ni ca �h ei gh t� 0. 34 �(0 .2 7� 0. 43 )� 0. 48 �(0 .3 5� �0 .6 9) � 0. 45 �(0 .3 1� �0 .5 0) � 0. 42 �(0 .2 7� �0 .6 9) � lo ng itu di na l�f ib er s� he ig ht � 0. 26 �(0 .1 9� � 0. 33 )� 0. 39 �(0 .2 5� �0 .6 0) � 0. 35 �(0 .2 4� �0 .4 1) � 0. 33 �(0 .1 9� �0 .6 0) � ci rc um fe re nt ia l�f ib er s� he ig ht � 0. 09 �(0 .0 6� 0. 12 )� 0. 10 �(0 .0 8� �0 .1 2) � 0. 11 �(0 .0 7� �0 .1 6) � 0. 10 �(0 .0 6� �0 .1 6) � � cc p� he ig ht � 1. 04 �(0 .7 2� � 1. 27 )� 1. 18 �(0 .8 5� �1 .4 9) � 1. 28 �(1 .0 4� �1 .5 2) � 1. 17 �(0 .7 2� �1 .5 2) � w id th � 2. 34 �(2 .0 0� 2. 76 )� 2. 39 �(2 .0 0� �2 .6 0) � 2. 53 �(2 .1 2� �3 .0 7) � 2. 42 �(2 .0 0� �3 .0 7) � ci rc um fe re nc e� 6. 25 �(5 .3 4� � 7. 58 )� 6. 69 �(5 .4 0� �7 .5 8) � 6. 95 �(5 .4 9� �8 .2 9) � 6. 63 �(5 .3 4� �8 .2 9) � ar ea � 2. 09 �(1 .4 0� 3. 19 )� 2. 55 �(1 .7 7� �3 .3 6) � 2. 83 �(1 .8 2� �3 .9 5) � 2. 49 �(1 .4 0� �3 .9 5) � � cs p� w ith �p re ss ur e� de ns er ,�i nn er �p or tio n� ve nt ra l�h ei gh t� 0. 04 �(0 .0 1� � 0. 07 )� 0. 07 �(0 .0 4� �0 .1 0) � 0. 05 �(0 .0 3� �0 .1 4) � 0. 06 �(0 .0 1� �0 .1 4) � le ss �d en se ,�o ut er � po rt io n� do rs al �h ei gh t� 0. 10 �(0 .0 6� 0. 18 )� 0. 14 �(0 .0 7� �0 .2 2) � 0. 13 �(0 .0 8� �0 .2 2) � 0. 12 �(0 .0 6� �0 .2 2) � le ss �d en se ,�o ut er � po rt io n� ve nt ra l�h ei gh t� 0. 21 �(0 .1 1� � 0. 34 )� 0. 36 �(0 .2 3� �0 .4 2) � 0. 23 �(0 .1 0� �0 .4 3) � 0. 27 �(0 .1 0� �0 .4 3) � w ho le �c sp �in cl ud in g� ur et hr a� 0. 33 �(0 .2 2� � 0. 54 )� 0. 28 �(0 .2 8� �0 .2 8) � 0. 41 �(0 .3 1� �0 .5 8) � 0. 35 �(0 .2 2� �0 .5 8) � ci rc um fe re nc e� 1. 29 �(0 .9 1� � 1. 75 )� 0. 43 �(0 .4 3� �0 .4 3) � 0. 69 �(0 .4 6� �1 .0 0) � 0. 56 �(0 .4 3� �1 .7 5) � ar ea � .0 13 �(0 .0 6� � 0. 22 )� 0. 26 �(0 .1 4� �0 .3 4) � 0. 17 �(0 .0 5� �0 .3 7) � 0. 19 �(0 .0 5� �0 .3 7) � � cs p� w ith ou t�p re ss ur e� de ns er ,�i nn er �p or tio n� 0. 10 �(0 .0 6� � 0. 04 �(0 .0 4� �0 .0 4) � 0. 09 �(0 .0 5� �0 .1 4) � 0. 08 �(0 .0 4� �0 .1 7) � 558clinical theriogenology • volume 9 number 4 • december 2017 � ve nt ra l�h ei gh t� 0. 17 )� le ss �d en se ,�o ut er � po rt io n� do rs al �h ei gh t� 0. 20 �(0 .1 6� � 0. 27 )� 0. 14 �(0 .1 4� �0 .1 4) � 0. 23 �(0 .1 2� �0 .4 2) � 0. 19 �(0 .1 2� �0 .4 2) � le ss �d en se ,�o ut er � po rt io n� ve nt ra l�h ei gh t� 0. 35 �(0 .2 4� 0. 47 )� 0. 28 �(0 .2 8� �0 .2 8) � 0. 41 �(0 .3 1� �0 .5 8) � 0. 35 �(0 .2 4� �0 .5 8) � w ho le �c sp �in cl ud in g� ur et hr a� 0. 56 �(0 .4 7� � 0. 65 )� 0. 43 �(0 .4 3� �0 .4 3) � 0. 69 �(0 .4 6� �1 .0 0) � 0. 56 �(0 .4 3� �1 .0 0) � ci rc um fe re nc e� 1. 91 �(1 .6 � 2. 12 )� 1. 46 �(1 .4 6� �1 .4 6) � 2. 11 �(1 .8 4� �2 .7 2) � 1. 83 �(1 .4 6� �2 .7 2) � ar ea � 0. 29 �(0 .2 0� � 0. 35 )� 0. 17 �(0 .1 7� �0 .1 7) � 0. 35 �(0 .2 6� �0 .5 6) � 0. 27 �(0 .1 7� �0 .5 6) � � u re th ra � he ig ht �w ith �p re ss ur e� 0. 00 �(0 .0 0 0. 05 )� 0. 04 �(0 .0 0� �0 .1 2) � 0. 02 �(0 .0 0� �0 .1 5) � 0. 02 �(0 .0 0� �0 .1 5) � he ig ht �w ith ou t�p re ss ur e� 0. 01 �(0 .0 0� � 0. 03 )� 0. 00 �(0 .0 0� �0 .0 0) � 0. 48 �(0 .0 0� �0 .1 9) � 0. 02 �(0 .0 0� �0 .1 9) � � ve nt ra l�v as cu la r�c ha nn el � he ig ht �w ith �p re ss ur e� 0. 09 �(0 .0 5� 0. 15 )� 0. 09 �(0 .0 6 �0 .1 3) � 0. 11 �(0 .0 5� �0 .2 1) � 0. 10 �(0 .0 5� �0 .2 1) � he ig ht �w ith ou t�p re ss ur e� 0. 17 �(0 .1 2� � 0. 24 )� *� 0. 15 �(0 .1 0� �0 .1 9) � 0. 16 �(0 .1 0� �0 .2 4) � � pr ep ut ia l�v es se l� la rg es t�d ia m et er �w ith � pr es su re � 0. 18 �(0 .0 8� 0. 30 )� 0. 17 �(0 .1 3� �0 .2 0) � 0. 20 �(0 .0 8� �0 .3 1) � 0. 18 �(0 .0 8� �0 .3 1) � la rg es t�d ia m et er � w ith ou t�p re ss ur e� 0. 21 �(0 .1 5� � 0. 29 )� *� 0. 2� (0 .2 � � 0. 2) � 0. 21 �(0 .1 5� �0 .2 9) � lo ca tio n g sk in � ve nt ra l�h ei gh t� 0. 05 �(0 .0 3� � 0. 08 )� 0. 06 �(0 .0 4� �0 .0 8) � 0. 05 �(0 .0 4� �0 .0 8) � 0. 05 �(0 .0 3� �0 .0 8) � fi br of at ty �ti ss ue � ve nt ra l�h ei gh t� 0. 20 �(0 .0 7� 0. 29 )� 0. 26 �(0 .2 0� �0 .3 5) � 0. 20 �(0 .1 1� �0 .3 4) � 0. 22 �(0 .0 7� �0 .3 5) � en itr e� w id th � 1. 92 �(1 .4 9� � 2. 1) � 1. 78 �(1 .5 3� �2 .0 6) � 1. 91 �(1 .3 8� �2 .2 4) � 1. 87 �(1 .3 8� �2 .2 4) � en tir e� he ig ht � 0. 94 �(0 .2 9� 1. 61 )� 1. 08 �(0 .9 7� �1 .1 6) � 1. 01 �(0 .7 4� �1 .2 8) � 1. 01 �(0 .2 9� �1 .6 1) � ci rc um fe re nc e� 4. 94 �(3 .9 5� � 5. 36 )� 4. 61 �(4 .1 8� �4 .9 4) � 4. 75 �(3 .8 7� �5 .2 0) � 4. 77 �(3 .8 7� �5 .3 6) � ar ea � 1. 61 �(1 .1 4� 1. 97 )� 1. 53 �(1 .2 7� �1 .7 5) � 1. 51 �(0 .9 9� �1 .7 5) � 1. 55 �(0 .9 9� �1 .9 7) � � ve nt ra l�a sp ec t�o f�t he �tu ni ca � w ho le �tu ni ca �h ei gh t� 0. 19 �(0 .1 3� � 0. 28 )� 0. 17 �(0 .1 2� �0 .2 6) � 0. 19 �(0 .1 3� �0 .2 4) � 0. 19 �(0 .1 2� �0 .2 8) � lo ng itu di na l�f ib er s� he ig ht � 0. 14 �(0 .1 0� 0. 19 )� 0. 15 �(0 .1 1� �0 .2 3) � 0. 14 �(0 .0 9� �0 .2 0) � 0. 15 �(0 .0 9� �0 .2 3) � ci rc um fe re nt ia l�f ib er s� he ig ht � 0. 06 �(0 .0 4� � 0. 12 )� 0. 34 �(0 .0 3� �0 .0 5) � 0. 05 �(0 .0 3� �0 .0 8) � 0. 05 �(0 .0 3� �0 .1 2) � � cc p� he ig ht � 0. 25 �(0 .1 4� � 0. 40 )� 0. 27 �(0 .1 9� �0 .3 5) � 0. 25 �(0 .1 6� �0 .4 3) � 0. 25 �(0 .1 4� �0 .4 3) � ci rc um fe re nc e� 0. 97 �(0 .5 2� � 1. 35 )� 1. 01 �(0 .8 0� �1 .2 4) � 0. 98 �(0 .6 9� �1 .5 4) � 0. 99 �(0 .5 2� �1 .5 4) � ar ea � 0. 08 �(0 .0 2� � 0. 14 )� 0. 08 �(0 .0 5� �0 .1 2) � 0. 08 �(0 .0 4� �0 .1 8) � 0. 08 �(0 .0 2� �0 .1 8) � 559 clinical theriogenology • volume 9 number 4 • december 2017 � � cs p� w ho le �h ei gh t� 0. 23 �(0 .1 1� � 0. 42 )� 0. 25 �(0 .1 5� �0 .3 4) � 0. 24 �(0 .1 6� �0 .3 4) � 0. 24 �(0 .1 1� �0 .4 2) � ve nt ra l�h ei gh t� 0. 16 �(0 .1 0� 0. 33 )� 0. 17 �(0 .0 9� �0 .2 2) � 0. 17 �(0 .1 3� �0 .2 5) � 0. 17 �(0 .0 9� �0 .3 3) � ci rc um fe re nc e� 0. 68 �(0 .3 7� � 1. 35 )� 0. 70 �(0 .4 1� �0 .8 9) � 0. 71 �(0 .5 7� �0 .9 4) � 0. 70 �(0 .3 7� �1 .3 5) � ar ea � 0. 04 �(0 .0 1� 0. 14 )� 0. 04 �(0 .0 1� �0 .0 6) � 0. 04 �(0 .0 2� �0 .0 7) � 0. 04 �(0 .0 1� �0 .1 4) � lo ca tio n s sk in � ve nt ra l�h ei gh t� 0. 07 �(0 .0 3� � 0. 16 )� 0. 12 �(0 .1 0� �0 .1 5) � 0. 11 �(0 .0 6� �0 .2 6) � 0. 10 �(0 .0 3� �0 .2 6) � do rs al �a sp ec t�o f�t he �tu ni ca � w ho le �tu ni ca �h ei gh t� *� *� 0. 34 �(0 .3 4� �0 .3 4) � 0. 34 �(0 .3 4� �0 .3 4) � lo ng itu di na l�f ib er s� he ig ht � *� *� 0. 27 �(0 .2 7� �0 .2 7� 0. 27 �(0 .2 7� �0 .2 7� ci rc um fe re nt ia l�f ib er s� he ig ht � *� *� 0. 09 �(0 .0 9� �0 .0 9) � 0. 09 �(0 .0 9� �0 .0 9) � � ve nt ra l�a sp ec t�o f�t he �tu ni ca � w ho le �tu ni ca �h ei gh t� 0. 42 �(0 .2 8� 0. 61 )� 0. 42 �(0 .3 8� �0 .4 6) � 0. 42 �(0 .1 5� �0 .5 1) � 0. 42 �(0 .1 5� �0 .6 1) � lo ng itu di na l�f ib er s� he ig ht � 0. 31 �(0 .0 7� 0. 15 )� 0. 31 �(0 .2 7� �0 .3 5) � 0. 31 �(0 .0 8� �0 .4 0) � 0. 31 �(0 .0 7� �0 .4 0) � ci rc um fe re nt ia l�f ib er s� he ig ht � 0. 11 �(0 .0 7� � 0. 15 )� 0. 12 �(0 .0 9� �0 .1 4) � 0. 12 �(0 .0 8� �0 .1 8) � 0. 12 �(0 .0 7� �0 .1 8) � � cc p� he ig ht � 1. 21 �(1 .0 0� 1. 50 )� 1. 33 �(0 .8 5� �1 .7 1) � 1. 45 �(1 .1 9� �1 .7 4) � 1. 33 �(0 .8 5� �1 .7 4) � w id th � 2. 26 �(2 .0 7� 2. 57 )� 2. 53 �(2 .1 3� �3 .0 5) � 2. 67 �(2 .3 1� �3 .0 5) � 2. 48 �(2 .0 7� �3 .0 5) � ci rc um fe re nc e� 6. 57 �(6 .0 4� 7. 45 )� 7. 22 �(6 .2 3� �8 .0 0) � 7. 69 �(6 .6 3� �9 .0 0) � 7. 16 �(6 .0 4� �9 .0 0) � ar ea � 2. 54 �(2 .0 0� � 3. 10 )� 3. 06 �(2 .3 0� �3 .9 3) � 3. 44 �(2 .3 3� �4 .6 0) � 3. 01 �(2 .0 0� �4 .6 0) � � cs p� w ith �p re ss ur e� de ns er ,�i nn er �p or tio n� ve nt ra l�h ei gh t� 0. 05 �(0 .0 3� 0. 13 )� 0. 06 �(0 .0 5� �0 .0 7) � 0. 06 �(0 .0 2� �0 .1 4) � 0. 05 �(0 .0 2� �0 .1 4) � le ss �d en se ,�o ut er � po rt io n� do rs al �h ei gh t� 0. 13 �(0 .0 7� � 0. 18 )� 0. 17 �(0 .1 4� �0 .2 0) � 0. 16 �(0 .0 8� �0 .2 7) � 0. 15 �(0 .0 7� �0 .2 7) � le ss �d en se ,�o ut er � po rt io n� ve nt ra l�h ei gh t� 0. 21 �(0 .1 0� 0. 39 )� 0. 33 �(0 .2 5� �0 .4 7) � 0. 26 �(0 .1 1� �0 .4 1) � 0. 26 �(0 .1 0� �0 .4 7) � w ho le �c sp �in cl ud in g� ur et hr a� 0. 36 �(0 .2 1 � 0. 56 )� 0. 52 �(0 .4 2� �0 .6 5) � 0. 44 �(0 .2 2� �0 .7 4) � 0. 44 �(0 .2 1� �0 .7 4) � ci rc um fe re nc e� 1. 38 �(0 .8 9� 1. 92 )� 1. 85 �(1 .3 3� �2 .3 2) � 1. 65 �(1 .8 8� �2 .9 )� 1. 62 �(0 .8 9� �2 .9 )� ar ea � 0. 15 �(0 .0 6� � 0. 28 )� 0. 27 �(0 .1 3� �0 .4 2) � 0. 21 �(0 .0 7� �0 .4 5) � 0. 21 �(0 .0 6� �0 .4 5) � � cs p� w ith ou t�p re ss ur e� de ns er ,�i nn er �p or tio n� ve nt ra l�h ei gh t� 0. 06 �(0 .0 0� � 0. 22 )� 0. 05 �(0 .0 0� �0 .0 1) � 0. 07 �(0 .0 0� �0 .1 8) � 0. 06 �(0 .0 0� �0 .2 2) � le ss �d en se ,�o ut er � po rt io n� do rs al �h ei gh t� 0. 16 �(0 .1 0� � 0. 23 )� 0. 18 �(0 .1 7� �0 .1 9) � 0. 21 �(0 .1 6� �0 .4 1) � 0. 19 �(0 .1 0� �0 .4 1) � le ss �d en se ,�o ut er � po rt io n� ve nt ra l�h ei gh t� 0. 28 �(0 .1 5� � 0. 59 )� 0. 30 �(0 .1 8� �0 .4 2) � 0. 31 �(0 .1 0� �0 .5 5) � 0. 30 �(0 .1 0� �0 .5 9) � 560clinical theriogenology • volume 9 number 4 • december 2017 ���� ��� ��* n ot �v isi bl e w ho le �c sp �in cl ud in g� ur et hr a� 0. 60 �(0 .3 0� � 0. 88 )� 0. 65 �(0 .6 2� �0 .6 7) � 0. 71 �(0 .5 6� �0 .9 2) � 0. 65 �(0 .3 0� �0 .9 2) � ci rc um fe re nc e� 2. 12 �(1 .2 2� 2. 80 )� 2. 06 �(2 .0 2� �2 .1 0) � 2. 30 �(1 .8 8� �2 .9 0) � 2. 16 �(1 .2 2� �2 .9 0) � ar ea � 0. 36 �(0 .1 2� � 0. 59 )� 0. 32 �(0 .3 2� �0 .3 2) � 0. 41 �(0 .2 7� �0 .6 4) � 0. 37 �(0 .1 2� �0 .6 4) � � u re th ra � he ig ht �w ith �p re ss ur e� 0. 02 �(0 .0 0� 0. 11 )� 0. 02 �(0 .0 0� �0 .0 7) � 0. 01 �(0 .0 0� �0 .1 0) � 0. 01 �(0 .0 0� �0 .1 1) � he ig ht �w ith ou t�p re ss ur e� 0. 16 �(0 .0 0� � 0. 46 )� 0. 14 �(0 .0 0� �0 .2 8) � 0. 18 �(0 .0 0� �0 .5 6) � 0. 16 �(0 .0 0� �0 .5 6) � � ve nt ra l�v as cu la r�c ha nn el � he ig ht �w ith �p re ss ur e� 0. 07 �(0 .0 3� 0. 12 )� 0. 10 �(0 .0 7� �0 .1 3) � 0. 13 �(0 .0 5� �0 .2 1) � 0. 10 �(0 .0 3� �0 .2 1) � he ig ht �w ith ou t�p re ss ur e� 0. 09 �(0 .0 4� � 0. 15 )� 0. 10 �(0 .1 0� �0 .1 0) � 0. 14 �(0 .1 4� �0 .1 4) � 0. 11 �(0 .0 4� �0 .1 5) � � pr ep ut ia l�v es se l� la rg es t�d ia m et er �w ith � pr es su re � 0. 18 �(0 .1 4� 0. 30 )� 0. 13 �(0 .0 9� �0 .1 7) � 0. 19 �(0 .0 8� �0 .3 5) � 0. 17 �(0 .0 8� �0 .3 5) � la rg es t�d ia m et er � w ith ou t�p re ss ur e� *� *� 0. 29 �(0 .2 9� �0 .2 9) � 0. 29 �(0 .2 9� �0 .2 9) � � re tr ac to r�p en is� m us cl e� he ig ht � 0. 37 �(0 .3 0� 0. 48 )� 0. 39 �(0 .3 5� �0 .4 5) � 0. 44 �(0 .3 0� �0 .5 5) � 0. 40 �(0 .3 0� �0 .5 5) � 561 clinical theriogenology • volume 9 number 4 • december 2017 2015: retraction-necrosis of the spermatic cord, epididymis, and testis of a friesian stallion caused by salmonella abortus-equi. clin therio 2014;6:377. retraction—necrosis of the spermatic cord, epididymis, and testis of a friesian stallion caused by salmonella abortus-equi bouchard ap, prado tm, schumacher j, donnell rl: necrosis of the spermatic cord, epididymis, and testis of a friesian stallion caused bysalmonella abortus-equi [abstract]. clin therio 2014;6:377. an issue raised by this report concerns the histological identification of s. abortus equi as the cause of suppurative orchitis and epididymitis in a stallion. this histological diagnosis was not supported by the results of the bacteriologic culture of tissue, which established that the organism cultured was s. hartford,which belongs to the salmonella c1 serogroup. s. abortus equi, on the other hand, is classified in the b serogroup. accordingly, our published report stating that infection was caused by s. abortus equi is in error. clinical theriogenology • volume 7, number 1 • march 201575 2018: tauroursodeoxycholic acid supplementation improves bovine in vitro fertilization rates tauroursodeoxycholic acid supplementation improves bovine in vitro fertilization rates y.l. lee,a e.k. kennedy,a y.j. chung,a j.e. graham,a m.s. roberson,b s.h. cheonga adepartment of clinical sciences and bdepartment of biomedical sciences, college of veterinary medicine, cornell university, ithaca, ny bile acids are traditionally known for their role in lipid digestion and absorption but recently have been shown to be endocrine signaling molecules. in an observational study, women with high levels of ursodeoxycholic acid in follicular fluid had oocytes with better developmental potential. another study showed that supplementation of the taurine conjugated ursodeoxycholic acid (tudca) to bovine embryos during the culture phase improves resistance to endoplasmic reticulum stress and increases development rates. the effects of supplementation during the maturation phase, where the oocytes are in contact with follicular fluid have not been studied; therefore, the aim of our study was to determine the effects of tudca supplementation during the maturation phase or culture phase on embryo development rates. we hypothesized that tudca supplementation during maturation phase improves embryo development. study design bovine oocytes were collected from slaughterhouse ovaries and assigned to one of 4 treatment groups: control group with no tudca exposure, in vitro maturation (ivm) group supplemented with 200 µm of tudca during the oocyte maturation phase only, in vitro culture (ivc) group supplemented with 200 µm tudca during the embryo culture phase only, and ivm/ivc group which were supplemented with 200 µm tudca during the maturation and culture phases of embryo production. embryos were evaluated on days 3, 7 and 10 after fertilization to determine cleavage, blastocyst, and hatched blastocyst rates, respectively. the effects of treatment on cleavage, blastocysts and hatched blastocyst rates were determined using a mixed-model anova (proc glimmix) of sas version 9.4. treatment was the fixed effect and the effect of week was included as a random variable to control for replicate variability. post-hoc tukey test was used to determine differences between treatment groups. results a total of 896 embryos were included in the study. there were no differences in cleavage rates between treatment groups (p=0.44). blastocyst rates were higher in the ivm group (60% of cleaved embryos) compared with the control group (42% of cleaved embryos; p=0.0004) and the ivc group (40% of cleaved embryos; p=0.006); and tended to be higher than the ivm/ivc group (47% of cleaved embryos; p=0.07). the hatch blastocyst rates were higher in the ivm group (p=0.02) compared with the ivc group and tended to be higher compared with the control group (p=0.08). discussion/conclusion we found that tudca supplementation during the ivm stage improved blastocyst rates and tended to improve hatched blastocyst rates compared with the control group. we did not observe an improvement of blastocyst rates in the ivc group compared with the control group in our study. tudca may have application as a supplement for bovine in vitro fertilization to improve embryo development rates. acknowledgment this work was supported by the usda national institute of food and agriculture, research animal health project nycv-480867. keywords: tauroursodeoxycholic acid, in vitro fertilization, bovine clinical theriogenology • volume 10, number 3 • september 2018303 2018: understanding and managing age-related subfertility in the stallion understanding and managing age-related subfertility in the stallion regina m. turner new bolton center, school of veterinary medicine, university of pennsylvania, kennett square, pa abstract age-related or idiopathic testicular degeneration is a common cause of subfertility and infertility, particularly in middle aged and older stallions. this manuscript describes the problem of idiopathic testicular degeneration in the equine breeding industry and summarizes what is known about the pathophysiology of the disease. additionally, the clinical signs of idiopathic testicular degeneration are reviewed so that the clinician can more quickly and accurately arrive at a diagnosis. the practitioner is provided with practical information on how to more effectively manage affected stallions and what, if anything, can be done to improve reproductive performance of these animals both in the field and in a referral setting. finally, the findings of current research on the disease are presented with a discussion of potential future therapies. keywords: stallion, subfertility, aging, testicular degeneration introduction aging is evident in most tissue and organ systems, and the testis is not immune. the mechanisms of aging are difficult to identify and poorly understood. idiopathic testicular degeneration (itd) refers to the adverse effects of aging on the testis and is a common cause of acquired and often progressive infertility in stallions.1,2 idiopathic testicular degeneration most often affects middle aged or older animals, but can be seen in much younger animals as well.3 regardless of the age of onset, itd is typically progressive and results in a steady decline in fertility, sometimes ending in sterility. economic losses resulting from this disease in the equine breeding industry are substantial and stem from losses of breeding fees, increased management costs and loss of valuable male genetics. in the past, the pathophysiology of the disease was poorly understood and even today, itd is clinically characterized largely by the appearance of a common set of progressive clinical signs including, but not limited to, an increase in palpable softness and a decrease in size of the testicular parenchyma and a decline in semen quality. this manuscript will review the clinical approach to itd and our current understanding of the disease. finally, new research that sheds light on the pathophysiology of itd and potential therapeutic approaches will be presented. clinical approach history a complete history is necessary before one can make an accurate diagnosis of itd since both clinically and histologically, itd may be indistinguishable from testicular hypoplasia. since testicular degeneration is an acquired condition, while testicular hypoplasia is congenital, a firm diagnosis of itd can be made only if the stallion has a history of declining reproductive efficiency, decreasing testicular size, decreasing semen quality, or some combination of these things. as such, information on the stallion’s past book sizes, seasonal pregnancy rates, average numbers of heat cycles per pregnancy, testicular measurements and past semen analyses all can be very helpful. it should be kept in mind that many animals with testicular hypoplasia often are affected by degeneration as well.2 not all cases of testicular degeneration are idiopathic. testicular trauma, recent fever, administration of anabolic steroids or other potentially damaging substances all can cause testicular degeneration. as such, when obtaining a history, one should also attempt to determine if the animal has any history of, for example, testicular trauma, recent illness associated with fever, or drug administration. in cases of traumatic or thermal injury to the testes, the onset of infertility is generally sudden and closely associated with the inciting incident. if steroids or other harmful agents are involved, the progression of the problem may be acute or more gradual. in cases in which a history reveals a likely inciting cause, clinical theriogenology • volume 10, number 3 • september 2018295 removal of the cause can allow for restoration of full testicular function. thus, unless the spermatogonial stem cells have been permanently damaged, the prognosis for future fertility is much better than for cases of itd. if a stallion is presented with a history of declining fertility over time with no apparent inciting incident then itd should be suspected, particularly in older animals. although itd is considered to be a slowly progressive problem, some cases may present for a perceived acute onset of infertility or subfertility, particularly if semen quality and testicular parameters were not being routinely monitored. clinical signs and diagnosis cases of itd can present with a range of clinical signs. mild cases of itd may not be associated with any noticeable change in testicular character. specifically, studies on germ cell loss rates in stallions indicate that itd can be present before any clinically significant decrease in testicular size can be appreciated.4 as such, early signs of itd may only be noticed if semen quality is being frequently and carefully monitored. a gradual decline in overall semen quality (including a decline in total sperm numbers and/or declines in the percentages of motile and morphologically normal sperm) may be the only clinical signs early in the disease. as the disease progresses, clinical signs become more apparent and include decreasing testicular size (most often affecting both testes similarly, but infrequently affecting one more than the other), palpable softening of the testicular parenchyma, decreasing sperm numbers, low daily sperm output (dso) per ml of testis, the appearance of increasing numbers of immature round spermatogenic cells and/or multinucleate giant cells in the ejaculate and an overall decline in semen quality.2,5-7 in advanced cases, stallions may become azoospermic. because the size of the epididymis usually does not change in cases of itd, the epididymis may seem to be disproportionately large with respect to testicular size.1 if a stallion’s fertility is not regularly monitored, some cases of itd present for what is perceived to be an acute onset of subfertility or infertility. in fact, in many of these cases, the problem was more likely progressive over time but went unnoticed until it had become a severe problem. in severe, end-stage itd, the testicles may become overly firm.8 monitoring and diagnostic techniques in stallions that can be followed over time, it is recommended that testicular measures be obtained at least annually. in general, the more frequently the measurements can be obtained, the better as this allows more precise identification of any trends towards a decrease in testicular size. at some top farms, the testes of valuable stallions are measured monthly. measurements can be obtained either with calipers or ultrasonographically and ideally should be performed by the same individual to minimize variation. use care not to distort the shape of the testes by overly aggressive manipulation during measurement. although not absolutely necessary, sedation of the stallion can help facilitate testicular measurement by allowing the stallion to relax and the testicles to descend passively, thus minimizing the need to pull the testicles down into the scrotum and potentially distorting measurements. measurements should include total scrotal width, as well as length, width and height of each testis individually. length, width and height measures then should be used to calculate testicular volume and to determine if the stallion is producing appropriate sperm numbers for its testicular size. the volume of a single testis can be calculated using the following formula: 4/3  x (length of testis (cm))/2 x width of testis (cm))/2 x height of testis (cm))/2 and total testicular volume equals: volume of the left testis + volume of the right testis.9 additionally, dso per ml of testis can be calculated by dividing the total number of sperm in the ejaculate at dso by the total testicular volume. low dso/ml of testis, together with a low percentage of clinical theriogenology • volume 10, number 3 • september 2018 296 morphologically normal sperm in the ejaculate have been recommended as good indicators of the possible presence of itd.9 frequent examination and measurement of the testes facilitate early identification of trends suggestive of itd (e.g., decreasing testicular size/volume). semen analysis (preferably with the stallion at dso) also should be performed at least annually and, when possible, much more frequently (i.e., for stallions breeding by artificial insemination, each ejaculate should be analyzed). however, this may not be practical for thoroughbred stallions breeding exclusively by natural cover. stallions at sexual rest typically have large epididymal stores of sperm. therefore, until this sperm reserve is essentially exhausted or stabilized, total sperm numbers at sexual rest can be highly variable. therefore, gradual downward trends in total sperm numbers and semen quality will be more difficult to identify if ejaculates are only examined when the stallion is at sexual rest. a better option is to examine the stallion’s semen quality after sperm reserves are depleted (i.e., when the stallion has reached dso). this requires serial semen collections (2 to 3 ejaculations/day for 3 to 5 days). once dso is reached, ejaculated sperm numbers become more consistent, thus making subtle changes in sperm numbers and semen quality more apparent. idiopathic testicular degeneration might be suspected if a stallion’s total sperm numbers or semen quality are declining progressively over time or if a stallion at dso is producing low sperm numbers for his testicular volume. another hallmark of itd is the appearance of immature spermatogenic cells (round cells) in the ejaculate. in an unstained semen sample, these cells can sometimes be confused with white blood cells. however, because different stages of spermatogenic cells typically appear in a single ejaculate, spermatogenic cells usually vary in size while white blood cells are more homogeneous. analysis of a mmooddiiffiieedd rroommaannoowwsskkyy (diff-quik®) stained semen sample can facilitate identification of neutrophils and lymphocytes and so, by process of elimination, can aid in the identification of spermatogenic cells. multinucleated giant cells also may be present.2,5 keep in mind that low numbers of immature spermatogenic cells may be found in the ejaculates of normal stallions.10 because of the variation in plasma hormone levels seen in normal and subfertile stallions, circulating hormone levels may not be a good predictor of mild to moderate itd.4 in severe cases, elevated follicle stimulating hormone (fsh) and luteinizing hormone (lh) as well as low plasma estradiol are consistent with a diagnosis of itd. however, by the time these hormonal changes become consistent and apparent, the disease is typically advanced and can be diagnosed based on testicular size and character as well as semen quality and sperm numbers. ultrasonographic evaluation of testes affected by itd is often unrewarding since the ultrasonographic appearance usually is not remarkable. nonetheless, ultrasonographic evaluation of the testes is recommended both to obtain accurate testicular measurements and to rule out possible inciting causes of itd or other testicular pathologies such as testicular neoplasia. histopathology of affected testes reveals a common group of spermatogenic abnormalities including cytoplasmic vacuolization and a loss of the normal architecture of the seminiferous epithelium.5 the diameter of the seminiferous tubules may be decreased and immature spermatogenic cells may be shed into the lumen of the seminiferous tubule. in more severe cases, these immature (or ‘round’) spermatogenic cells may appear in the ejaculate in increasing numbers, as described above. as itd progresses, there is an increased loss of germ cells from the seminiferous tubule. in the most extreme cases, fibrous tissue may be present and tubules can become almost devoid of spermatogenic cells and may be left with only sertoli cells and few spermatogonia. fibrosis and calcification of the testicular parenchyma also may be seen. (11) keep in mind that even normal testes can have some focal areas of abnormal spermatogenesis. thus, the percentage of the testicular parenchyma that is affected as well as the severity of the histological lesions should be taken into account before a diagnosis of td is made. because histopathologic findings can help to define itd (and testicular hypoplasia), evaluation of a testicular biopsy sample does provide definitive evidence of these conditions. however, in practice, testicular biopsy is rarely indicated. once the clinician has obtained an adequate history and has performed a complete physical and reproductive examination, a diagnosis of itd can usually be made with some confidence and a biopsy sample is not necessary. additionally, there is some concern that a clinical theriogenology • volume 10, number 3 • september 2018297 single biopsy sample may not be representative of the condition of the entire testis and thus may not be of significant prognostic value. if a biopsy sample is to be taken, the testes should be examined ultrasonographically prior to obtaining the biopsy.12 the ultrasonographic appearance of the parenchyma can help the clinician to choose a representative site for sampling. several reports have indicated that obtaining testicular biopsy samples in the stallion can be done safely and with minimal permanent damage to the remaining testicular parenchyma.13,14 however, many of these studies were performed on normal stallions and thus the risk to an already compromised testicle (e.g. a degenerating testicle) is more difficult to ascertain. clinicians must carefully weigh the diagnostic benefits of obtaining a biopsy sample against the risk of damaging some portion of an already marginally functional testicular parenchyma. treatment if there is a known or suspected cause of the degeneration (e.g., fever, toxin), successful treatment or removal of the inciting cause should at least prevent further progression of the disease and may allow for complete recovery. in cases of unilateral testicular damage or degeneration, some have recommended removal of the affected testis. the reasoning behind this recommendation is that the damaged testicular tissue could result in the production of anti-sperm antibodies that might adversely affect sperm produced by the normal testis.15 additionally, removal of one testis often results in hypertrophy of the remaining testis and a resultant increase in sperm numbers. the practice of unilateral castration is debatable, however, as there are reports of acceptable fertility in stallions with unilateral itd in which the affected testis was not removed.1 as would be expected for a tissue autologous disease, clinical treatments designed to drive testicular function have proven ineffective. although there are some reports of the successful use of gonadotropin releasing hormone (gnrh) therapy as a treatment for infertility in stallions16,17 these successes have not been duplicated in controlled studies.18-20 gonadotropin releasing hormone therapy has been highly successful in treating men with hypogonadotropic-hypogonadism, but this condition has not been clearly documented in stallions and our studies strongly suggest that a lack of gonadotropins is not the underlying cause of severe itd in the horse. in addition, our studies on xenografts of equine testes severely affected by itd have identified no improvement in the condition of the testes following treatment of host mice with exogenous gonadotropins or provision of the mice with a source of endogenous hormones from normal, functional testis xenografts.21 if all of this information is taken together, the use of gnrh implants or pulsatile administration of gnrh as a treatment for stallion infertility in general or itd specifically becomes highly questionable. if this therapy is to be attempted, it has been suggested that treatment must start early, before the testis has reached a severe state of degeneration.22 there is support in the literature for beneficial effects of fat-soluble antioxidants (e.g., docosahexaenoic acid (dha)) on semen quality in stallions.23 however, proven benefits so far have been limited to improved longevity of sperm motility in cooled, stored semen samples. whether or not this improvement translates into increased pregnancy rates, particularly in subfertile stallions, has not been tested. we have used our xenografting model to study the histological effects of dha supplementation on degenerate stallion testes and have identified no benefit.21 nonetheless, it is a common practice to supplement subfertile stallions with neutriceuticals containing dha or similar substances (e.g., prosperm , minitube of america, verona, wi). management in breeds allowing artificial insemination, semen cryopreservation is recommended for any genetically valuable stallion when the animal is at the peak of its fertility. this provides a genetic ‘insurance policy’ against reproductive loss should a stallion’s fertility be compromised by itd as it ages. since there is no proven treatment for itd, the basis of dealing with this problem centers around stallion and mare management. the veterinarian first should determine the number of progressively clinical theriogenology • volume 10, number 3 • september 2018 298 motile, morphologically normal sperm that the stallion is capable of producing while on a schedule set up to mimic what would be required during the breeding season. the stallion’s mare book then should be adjusted accordingly to insure that the stallion is not overused. if a stallion with low or marginal sperm numbers is required to serve too frequently, it is not uncommon for the animal’s sperm numbers to drop below what would be required for a minimum insemination dose. limiting the animal’s book so that it has one or more days of sexual rest between each ejaculate often can help boost sperm numbers and improve pregnancy rates in mares. if possible, the semen quality of each ejaculate should be monitored to be certain that each mare is receiving a minimum insemination dose. addition of an extender to the ejaculate may help improve longevity of sperm motility in some cases. for stallions breeding by natural cover, reinforcement breeding is highly recommended to maximize the number of sperm delivered to the mare.24 the use of deep horn reinforcement breeding may further increase the chances of pregnancy when dealing with small numbers of sperm. semen from stallions with itd should be handled with particular care. mares should be inseminated as quickly as possible after semen collection. semen should be carefully evaluated as to its suitability for cooled transport. however, in many cases of moderate to severe itd, sperm longevity of motility is poorly maintained and pregnancy rates may be significantly reduced in mares bred with cooled semen. if this is the case, it may be prudent to discontinue the use of shipped semen and only breed mares on site with fresh, extended semen or by natural cover. semen processing techniques can be used in an attempt to boost semen quality. for stallions with poor quality seminal plasma, centrifugation of semen with removal of seminal plasma and subsequent resuspension in semen extender can increase sperm longevity. in cases where a high percentage of sperm morphologic defects are present, gradient separation of sperm can result in a higher quality insemination dose. however, sperm numbers are typically greatly reduced. one or more laboratory trials of gradient separation are recommended before semen is needed to breed mares. these laboratory trials allow one to tailor the technique to each stallion and to determine in advance if the improvement in semen quality is likely to outweigh the loss of sperm numbers. more intensive mare management also can be used to improve pregnancy rates. with judicious use of ovulation induction agents, mares can reliably be bred very close to the time of ovulation, and in extreme cases within six hours after ovulation, thus minimizing the requirement for sperm longevity. the routine use of deep horn, low volume insemination can increase pregnancy rates when sperm numbers are limited. a final option for management is the use of assisted reproductive techniques such as intracytoplasmic sperm injection (icsi). intracytoplasmic sperm injection allows for the production of offspring even from severely azoospermic stallions. however, the expense is significant and not all breed registries approve of this technique. pathophysiology and future avenues for treatment in both moderate and severe cases of itd, the primary defect resides within the testis itself, and not with the hypothalamus, the pituitary or the extratesticular environment.25 earlier endocrinologic work26 suggested that the primary defect in equine itd resides within the sertoli cell. however, more recent work in mice suggests a somewhat more complicated basis for age-related declines in male fertility. while there is now evidence for some direct effects of aging on the spermatogonial stem cell (scc) itself,27 a highly significant contributor to infertility in old male mice was shown to result from failure of the spermatogonial stem cell (ssc) niche, a specific testicular microenvironment comprised of a variety of cellular and molecular components. in vivo cell retransplantation experiments demonstrated that ssc self-renewal could continue normally for several years past the normal life span of the donor animal if the sscs were maintained by a young ‘niche’.28 in other words, if sscs can be maintained in a ‘young’ somatic niche environment, their function (including self-renewal and potentially differentiation through spermatogenesis) could be maintained well beyond what would be defined as old age in the donor. in rodents, there is evidence that this niche is based within the interstitial compartment, in proximity to interstitial blood vessels and involving input from the leydig cells.29 sertoli cell function also is likely to be part of the niche, but the role of sertoli cells in the niche may be influenced by input clinical theriogenology • volume 10, number 3 • september 2018299 from the interstitial compartment. if these observations also hold true for the stallion, then declines in sertoli cell function remain a central factor responsible for age-related declines in fertility. however, the full picture likely involves much more than sertoli cell function alone, and probably is heavily influenced by input from the interstitial compartment. taking all this together, it appears that numerous cell types and cell interactions are required to create a ‘young’ testicular environment. in support of this hypothesis, we have shown that exposing aged spermatogonia to young testicular somatic cells results in improved survival of the spermatogonia compared to aged spermatogonia exposed only to aged testicular somatic cells.30 given the evidence that aging of somatic cells and aging of the ssc niche play large parts in compromising the function of aging sscs, studies have been undertaken to determine what cellular functions are altered in aging testicular somatic cells. in brown norway rats, one of the best laboratory models for testicular aging, it has been determined that leydig cells from aged rats produce less testosterone than do leydig cells from young rats.31-33 since then, researchers have identified several differences in aged leydig cells that could adversely affect the function of the steroidogenic pathway (differences in lh-receptor number, camp production, pk-a activity, cholesterol transport, activities of steroidogenic enzymes, etc.).33-38 additionally, a factor originating from both leydig and peritubular myoid cells (colony stimulating factor 1 (csf1)) has been implicated as a stimulator of spermatogonial stem cell self-renewal in mice.29 thus, changes in function of even peritubular myoid cells could adversely affect the ssc niche and be involved in age-related declines in spermatogenesis. finally, an inability of the sertoli cells to respond to signals (e.g. changes in fsh responsiveness) or to produce factors required to regulate ssc self-renewal and differentiation (e.g. glial cell-derived neurotrophic factor; gdnf) has been implicated as part of the aging process.29 thus, the functions of numerous cell types are affected by aging and the list of cell functions that are altered is long and growing. although until recently none of these studies have focused on the horse, the work that has been done in other species can be used as a ‘short cut’ to identifying age-related problems in the stallion testis. we have used a xenografting model to show that testicular function in tissue from stallions affected with itd is not rescued by grafting the tissue into a young extratesticular environment.25 these findings confirm that, in cases of itd, a primary testicular defect is present. however, if healthy, prepubertal tissue is co-grafted in physical contact with degenerate testicular tissue, we observe an improvement in the degenerate grafts.39 these findings suggest that there is a beneficial paracrine effect of the young tissue on the old tissue. in other words, as might be expected from a tissue autologous disease, improvement in the disease is seen only when the ‘treatment’ originates from within a normal testis. this finding suggests that cell-based therapies (e.g. forms of stem cell therapy) may be the best approach to arriving at a treatment for the adverse affects of aging in the equine testis. cell-based therapies can provide transforming results for some disease processes. however, these therapies are fraught with difficulties including isolation and storage of therapeutic cells, complicated and sometimes invasive cell delivery techniques, and maintenance of the therapeutic cell population in the recipient (immunologic rejection, cell death, etc.). if the specific proteins that are responsible for the beneficial effect of the young cell population on the old cells could be identified and isolated, it may be possible to move towards a more standard method of treatment potentially involving, for example, periodic systemic protein or hormone injections. in addition to leading to potential therapeutic approaches to itd, these studies also lead us to a better understanding of the pathophysiology of itd and thus provide means of identifying the disease in its early stages and/or developing methods to prevent its occurrence and progression. we have recently completed experiments designed to identify whole-transcriptome differences in gene expression between cell populations from adult, fertile testes vs. gene expression in cell populations from age-matched, degenerate testes. we now are in the process of mining a large dataset comprised of thousands of genetic sequences in order to identify those genes whose expression are significantly altered between the two tissue types. within this dataset, we have identified numerous genes and signaling pathways that are affected by aging and that are likely to influence testicular function in the horse. by developing therapies targeted to affect the function of these pathways, we may be able to develop a clinical theriogenology • volume 10, number 3 • september 2018 300 simpler approach to the treatment of itd. in addition to potential therapeutic approaches to itd, these studies also lead us to a better understanding of the pathophysiology of itd and thus provide means of identifying the disease in its early stages and/or developing methods to prevent its occurrence and progression. acknowledgment portions of this work were supported by a grant from the grayson jockey club research foundation, inc. references 1. blanchard t, varner d: testicular degeneration. in: mckinnon ao, voss jl, editors. equine reproduction. philadelphia: lea and febiger; 1993. p. 855-860. 2. watson ed, clarke cj, et al: testicular degeneration in 3 stallions. equine vet j 1994;26:507-510. 3. gehlen h, bartmann cp, et al: azoospermia due to testicular degeneration in a breeding stallion. j equine vet sci 2001;21:137-139. 4. blanchard tl, johnson l: increased germ cell degeneration and reduced germ cell:sertoli cell ratio in stallions with low sperm production. theriogenology 1997;47:655-677. 5. mcentee k: reproductive pathology of domestic animals. mcentee k, editor. san diego: academic press, inc.;1990. 6. blanchard t, johnson l, et al: increased germ cell loss rates and poor semen quality in stallions with idiopathic testicular degeneration. j equine vet sci 2000;20:263-265. 7. blanchard tl, johnson l, et al: low daily sperm output per ml of testis as a diagnostic criteria for testicular degeneration in stallions. j equine vet sci 2001;21:11-35. 8. varner d, schumacher j, et al: diseases and management of breeding stallions. goleta(ca): american veterinary publications; 1991. 9. love cc, garcia mc, et al: evaluation of measures taken by ultrasonography and caliper to estimate testicular volume and predict daily sperm output in the stallion. j reprod fert suppl 1991;44:99-105. 10. swerczek tw: immature germ cells in the semen of thoroughbred stallions. j reprod fertil suppl 1975;23:135-137. 11. humphrey jd, ladds pw: a quantitative histological study of changes in the bovine testis and epididymis associated with age. res vet sci 1975;19:135-141. 12. turner rm: ultrasonography of the genital tract of the stallion. in: reef vb, editor. equine diagnostic ultrasound. philadelphia: w.b. saunders company; 1998. p. 446-479. 13. delvento vr, amann rp, et al: ultrasonographic and quantitative histologic assessment of sequelae to testicular biopsy in stallions. am j vet res 1992;53:2094-2101. 14. faber nf, roser jf: testicular biopsy in stallions: diagnostic potential and effects on prospective fertility. j reprod fertil suppl 2000;56:31-42. 15. zhang j, ricketts sw, et al: antisperm antibodies in the semen of a stallion following testicular trauma. equine vet j 1990;22:138-141. 16. evans jw, finely m: gnrh therapy in a stallion of low fertility. j equine vet sci 1990;10:182. 17. shiner ka, pickett bw, et al: clinical approaches to diagnosis and treatment of subfertile stallions. proc annu conv am assoc equine pract 1993. 18. douglas rh, umphenour n: endocrine abnormalities and hormonal therapy. vet clin north am equine pract 1992;8:237-249. 19. blue bj, pickett bw, et al: effect of pulsatile or continuous administration of gnrh on reproductive function of stallions. j reprod fertil suppl 1991;44:145-154. 20. roser jf, hughes jp: use of gnrh in stallions with poor fertility: a review. proc annu conv am assoc of equine pract; 1994. 21. turner rm, rathi r, et al: xenografting to study testis function in stallions. anim reprod sci 2006;94:161-164. 22. brinsko sp: gnrh therapy for subfertile stallions. vet clin north am equine pract 1996;12:149-160. 23. brinsko sp, varner dd, et al: effect of feeding a dha-enriched nutriceutical on the quality of fresh, cooled and frozen stallion semen. theriogenology 2005;63:1519-1527. 24. blanchard tl, love cc, et al: role of reinforcement breeding in a natural service mating program. proc annu meet am assoc equine pract 2006. 25. turner rm, rathi r, et al: xenografting restores spermatogenesis to cryptorchid testicular tissue but does not rescue the phenotype of idiopathic testicular degeneration in the horse (equus caballus). reprod fertil dev 2010;22:673-683. 26. stewart bl, roser jf: effects of age, season, and fertility status on plasma and intratesticular immunoreactive (ir) inhibin concentrations in stallions. domest anim endocrinol 1998;15:129-139. 27. schmidt ja, abramowitz lk, et al: in vivo and in vitro aging is detrimental to mouse spermatogonial stem cell function. biol reprod 2011;84:698-706. 28. ryu by, orwig ke, et al: effects of aging and niche microenvironment on spermatogonial stem cell self-renewal. stem cells 2006;24:1505-1511. clinical theriogenology • volume 10, number 3 • september 2018301 29. oatley jm, oatley mj, et al: colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal. development 2009;136:1191-1199. 30. turner rm, zeng w, et al: paracrine factors from normal equine testicular tissue improve the condition of xenografts of degenerate testicular tissue. anim reprod sci 2010;121s:s198-s9. 31. chen h, hardy mp, et al: age-related decreased leydig cell testosterone production in the brown norway rat. j androl 1994;15:551-557. 32. zirkin br, santulli r, et al. testicular steroidogenesis in the aging brown norway rat. j androl 1993;14:118-123. 33. liao c, reaven e, et al: age-related decline in the steroidogenic capacity of isolated rat leydig cells: a defect in cholesterol mobilization and processing. j steroid biochem mol biol 1993;46:39-47. 34. chen h, hardy mp, et al. age-related decreases in leydig cell testosterone production are not restored by exposure to lh in vitro. endocrinology 2002;143:1637-1642. 35. lin t, vinson ne, et al: the aging leydig cell. viii. protein kinase activity. j androl 1983;4:324-330. 36. culty m, luo l, et al: cholesterol transport, peripheral benzodiazepine receptor, and steroidogenesis in aging leydig cells. j androl 2002;23:439-447. 37. luo l, chen h, et al: temporal relationships among testosterone production, steroidogenic acute regulatory protein (star), and p450 side-chain cleavage enzyme (p450scc) during leydig cell aging. j androl 2005;26:25-31. 38. luo l, chen h, et al: are leydig cell steroidogenic enzymes differentially regulated with aging? j androl. 1996;17:509-515. 39. turner rm, zeng w, et al: paracrine factors from normal testicular tissue improve the condition of xenografts of degenerate testicular tissue. anim reprod sci 2010;121s:s198-s199. clinical theriogenology • volume 10, number 3 • september 2018 302 << /ascii85encodepages false 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice clinical theriogenology 2022; 14: 348 development and validation of an ovine cesarean surgery model and rubric philippa gibbons,a elizabeth devine,b david dutton,a tiffany pulliam,b stacy anderson,b julie huntb atexas tech school of veterinary medicine, amarillo, tx blincoln memorial university college of veterinary medicine, harrogate, tn abstract clinical skills are learned through deliberate practice with specific feedback. however, it is not possible to provide such experience to each student in food animal reproductive procedures (e.g., cesarean surgery). aims of this study were to create an ovine cesarean surgery model to facilitate skill acquisition, to create a scoring rubric to assess performance, and to gather validation evidence for the model and rubric. a commercially available ovine manikin was modified using poured silicone to create suturable skin, muscle, and uterus containing a commercially available fetal lamb manikin that could be manipulated and delivered. a scoring rubric consisting of a 16-item checklist and 5 global rating scores (grs) was created. model was tested by veterinarians (n = 14, experts) and veterinary students (n = 16, novices) in a validation study that evaluated evidence in content, internal structure, and relationship with other variables (level of training). most experts (93%) felt that the model would be helpful to teach procedural steps; also experts (100%) agreed that the model would improve students’ skill in performing cesarean surgery. novices scored lower than experts on the checklist and total grs, and novices’ surgical times were longer than experts’ times. content evidence and relationship with other variables evidence supported validation of the model and rubric. checklist reliability (alpha = 0.58) could be improved by increasing the number of items and further refining them. keywords: simulation, model, ovine, cesarean surgery introduction cesarean surgery is performed in ruminants when maternal/ fetal characteristics make vaginal delivery difficult/impossible, or on an elective basis due to the value of the fetus or due to conditions other than dystocia (e.g., pregnancy toxemia). cesarean surgery is frequently performed in an emergency and must be completed quickly and accurately to save lives of dam and fetus. although small ruminant practitioners have not expressed competencies expected of new veterinary graduates, bovine practitioners expect new graduates to be able to perform bovine cesarean surgery independently with little supervision.1 performing a cesarean surgery requires accurate identification of tissue layers, fetal manipulation and delivery, and proficiency in suturing individual layers upon closure.2 complications from cesarean surgery can occur due to errors in surgical technique and may include trauma to the uterus, gastrointestinal organs, and abdominal wall; peritoneal cavity contamination; and inadequate uterine closure.2 surgical training has slowly transitioned from the ‘see one, do one, teach one’ paradigm (a level of competence could be reached from observation of surgery)3,4 to deliberate practice (skill is gained through repetitive practice with specific feedback).5-7 surgical practice can be acquired using live animals, cadavers, or models. however, the availability of live animals requiring cesarean surgery is limited and typically seasonal, making it challenging to provide every veterinary student with adequate educational opportunities utilizing live animals. likewise, it is unusual to find cadavers with a full-term fetus to practice cesarean surgery, and if such cadavers are available, the typical drawbacks of cadavers still exist, including lack of bleeding, onset of rigor, and postmortem tissue changes.8 model-based training permits students to repetitively practice their skills until reaching competence without any risk to animal welfare, and models have proven valuable in teaching other surgical procedures including canine castration,9,10 canine ovariohysterectomy,11,12 and bovine castration.13 studies have confirmed the superiority of veterinary surgical models compared to learning surgical skills using laboratory manuals and/ or videos,11,14 cadavers,15 and live animals.16 a meta-analysis of human medical education studies also demonstrated that for an array of medical and surgical skills, skills taught on models were learned more effectively than skills taught using traditional methods (e.g., observation, lectures, and other hands-off learning techniques).17 a recent review of veterinary surgical models identified a scarcity of large animal surgical models and recommended further development in this area.18 authors are unaware of a commercially available model for teaching ruminant cesarean surgery nor any privately constructed model that has been previously published. first aim of this study was to create a cost-efficient ovine cesarean surgery model that would serve as a model for ruminant cesarean surgery to clinical theriogenology 2022; 14: 349 improve veterinary students’ proficiency with this procedure. second aim was to create a rubric to score performance on the cesarean surgery procedure. final aim was to validate the model and scoring rubric using a framework of evidence in content, relationship with other variables (level of training), and internal structure.19-21 the validation framework stated that if the model and rubric are valid for use in teaching veterinary students to perform ovine cesarean surgery, then: 1) experienced veterinarians will rate the model as easy to use, reasonably realistic, and suitable for use in student training (content evidence), 2) experienced veterinarians will achieve higher scores and greater surgical efficiency while performing ovine cesarean surgery on the model than students (evidence of relationship with other variables – level of training), and 3) veterinarians’ and students’ checklist scores will demonstrate adequate reliability (internal structure evidence). we hypothesized that an ovine cesarean surgery model could be created that had adequate features to be acceptable to experienced veterinarians and to differentiate the performances of students from those of veterinarians. further, we hypothesized that checklist scores would attain at least an acceptable measure of reliability. materials and methods model development and content evaluation study was approved (# 969 v.0) by lincoln memorial university (lmu) institutional review board. large animal faculty at lmu college of veterinary medicine worked with college’s model builder to create and test model prototypes in an iterative process. final model is described here. for ovine body, a commercially available life size standing ovine artificial insemination model (anatomoulds, pretoria, south africa) was used. a stiff copper cable was bolted to the metal frame to simulate spine, and a mock last rib of copper was added. fabric over the left paralumbar fossa was cut to make a flap (~ 20 x 30 cm). hook and loop tape was stitched to the inside of the fabric body wall. the body wall where the left paralumbar fossa incision would be performed was created by pouring 4 layers of different colored soft silicone rubber (smooth-on, easton, pa) to represent skin, external abdominal oblique, internal abdominal oblique, and transverse muscle layers. perforated clear plastic food wrap was used between the silicone layers so that they could be differentiated and undermined. hook and loop tape was glued around the edge of the suture pad to attach it to the ovine mannikin (figure 1). figure 1. ovine cesarean surgery model: a) ovine mannikin with silicone suture pad, b) clear plastic food wrap separates the skin layer from the subcutaneous layer below it uterus was made by pouring several layers of soft silicone rubber over a 27-cm diameter bowl. a strip of the uterus was designated as the greater curvature; this region had additional layers of poured silicone and a layer of 4-way stretch power mesh material to allow the uterus to be thick enough (3 4 mm) for partial thickness suturing. placenta was made by pouring a thin layer of silicone over the same mold as the uterus, with thicker discs of silicone to imitate ovine cotyledonary placentation. two fabric ties were included in the uterus to attach it to the metal frame dorsally so that it hung in the abdomen. for clinical theriogenology 2022; 14: 350 lambs, commercially available soft toys (viahart toy company, wills point, tx) were used. for leg bones, 1.27 cm diameter polyvinyl chloride pipes were inserted into the lamb mannikin. limbs were articulated using rivets so that the fore and hindlimb joints bent in the correct anatomical directions (figure 2). foam and poly-fill padding was placed into the abdominal cavity to represent other abdominal organs. figure 2. ovine cesarean surgery model: a) the ovine mannikin with uterus installed in the abdomen, b) the lamb inside the uterus, c) the lamb mannikin with its spine and articulated limbs after model’s completion, a convenience sample of 14 veterinarians who were experienced in performing ruminant cesarean surgery tested the model and provided survey feedback about its features. these veterinarians were employed by 2 veterinary schools in the region; 6 were board-certified in a specialty (2 in theriogenology, 2 in surgery, and 2 in internal medicine), and 8 were general practice veterinarians. data were collected using a series of 5-point likert scales ranging from ‘strongly disagree’ to ‘strongly agree’ about ease of use, fidelity, and perceived ability to enhance student learning. veterinarians were invited to provide written comments about the model (appendix 1). novice to expert comparison a convenience sample of third year veterinary students (n = 16) who had never performed a ruminant cesarean surgery was recruited from students enrolled in a food animal elective course (n = 31). prior to using the model, students had a 50-minute lecture regarding ruminant cesarean surgery and read an article describing the procedure.2 all students were enrolled in the lmu surgical skills training program, and their prior instruction included repetitive practice of surgical skills on the canine castration model, canine ovariohysterectomy model, and several suturing and ligation task trainers. students were not exposed to the ovine cesarean surgery model prior to the start of the study. students, along with 14 veterinarians who had provided survey feedback, were recorded using wide angle action video cameras (gopro hero 6, gopro, san mateo, ca) while preparing for and performing cesarean surgery on the ovine model. preparation included identifying landmarks and choosing where to make the incision. although the model could simulate an inverted l-line block for anesthesia if desired, this feature was not used. veterinarians performed the surgical procedure according to the technique were comfortable in using and not necessarily the standardized technique taught to the students. ovine model was positioned standing on top of a low table to simulate an acceptable position for performing ovine cesarean surgery.22 for the first 2 participants, veterinarians, cameras were worn on a head mount. after researchers observed that there was too much motion in these videos, cameras were subsequently attached to a nearby rod using a flexible arm. cameras were oriented to exclude participant’s identifying features whenever possible. recorded surgeries were scored and timed by 1 investigator who was experienced in performing ruminant cesarean surgery and in teaching students to perform the procedure. rater was not involved in development of the model or rubric and was blinded to the identity and group of the person performing the procedure. surgical time was recorded from the start of the incision to end of the final stitch. the video recordings were viewed on a computer monitor, and the rater could pause or replay the recordings as needed. a rubric to score the video recordings was developed by faculty members who were experienced at performing, teaching, and assessing ruminant cesarean surgery and general surgical skills. rubric consisted of 16 checklist items and 5 global rating scales. the 16 checklist items represented the steps for performing the procedure on a live animal and were each scored with 0 point awarded for unsatisfactory performance for that step and 1 point awarded for satisfactory performance of that step. maximum checklist score was 16 points. five global rating scores (grs) were awarded independently from the checklist score and were awarded for tissue handling, instrument handling, efficiency of time and motion, suturing, and overall grs. clinical theriogenology 2022; 14: 351 each grs was scored on a 1 6 scale with 1 point awarded for a very poor performance, 2 for poor, 3 for borderline unsatisfactory, 4 for borderline satisfactory, 5 for good, and 6 for excellent (appendix 2). rubric was not shared with study participants. student surveys after performing simulated cesarean surgery, students completed a survey evaluating the model’s features and ease of use on a 5-point likert scale. students were invited to write comments for its continued improvement (appendix 3). data analyses data were analyzed using spss version 28 (ibm). categorical and ordinal data (survey data, grs, and scores from individual steps on the checklist) were compared using mann-whitney u-tests. a shapiro-wilk normality test was performed on continuous data (surgical time, checklist score, and total grs); checklist score percentage and total grs were normally distributed but not time. performance scores were compared using student’s t-tests, and surgical time was compared using a mann-whitney u-test. for variables analyzed with a t-test, levene’s test was used to confirm homogeneity of variance. hedge’s g was used to evaluate effect sizes because the group sizes were unequal. benchmarks suggested by cohen were used for interpreting effect sizes; 0.2 was considered small, 0.5 medium, and 0.8 large.23 cronbach’s alpha was used to assess internal consistency of checklist scores. results model development to produce a model deemed acceptable by the teaching faculty, model developers went through 2 revisions of the lamb, 4 revisions of the abdominal closure pad, and 2 revisions of the uterus to get the correct thickness and texture. entire manikin, including lamb, cost $732 to construct. replacement parts for each use, including closure pad, uterus, and placenta, were $38 per use. if not ripped, uterus could be patched with silicone and reused for a second surgery for a $10 cost savings. content evaluation fourteen veterinarians provided survey feedback after using the model, providing content evidence for the model’s validation. all veterinarians agreed to strongly agreed that the model was easy to use, and the majority of veterinarians (13/14, 93%) agreed that adequate landmarks were present and that the model felt realistic. eleven veterinarians (79%) felt that the model’s materials looked realistic. nine veterinarians (64%) felt that the model adequately replicated the actual tactile experience; 1 veterinarian (7%) disagreed with this statement whereas remaining 4 veterinarians (29%) were neutral. thirteen veterinarians (93%) felt that the model was able to teach preparation and skills required to perform the skill, and that the model would improve animal welfare by allowing students to first perform the skill on the model; 1 veterinarian (7%) was neutral on these statements. all veterinarians felt that the model would increase students’ learning of the skill. thirteen veterinarians (93%) felt that the model was adequate to prepare students for performing a live animal cesarean surgery; 1 veterinarian (7%) was neutral on this statement. one veterinarian (7%) felt a concern that the model could teach students poor technique; that veterinarian also left a comment explaining that the model’s skin was not a good representation of the live animal, and that this could result in poor suturing technique. survey item results are summarized (table 1). thirteen veterinarians left a total of 25 written comments about the model (table 2). table 1. veterinarians’ survey responses after using the model question strongly disagree n (%) disagree n (%) neutral n (%) agree n (%) between agree and strongly agree* n (%) strongly agree n (%) model was easy to use* 5 (36%) 1 (7%) 8 (57%) adequate landmarks were present 1 (7%) 10 (71%) 3 (21%) materials looked realistic* 1 (7%) 2 (14%) 7 (50%) 1 (7%) 3 (21%) materials felt realistic* 1 (7%) 11 (79%) 1 (7%) 1 (7%) i feel that students can safely utilize this model 3 (21%) 11 (79%) model was able to teach the preparation and steps required to perform this skill 1 (7%) 7 (50%) 6 (43%) model adequately replicates the actual tactile experience when performing this skill 1 (7%) 4 (29%) 8 (57%) 1 (7%) model will improve animal welfare by allowing students to first perform the skill on the model 1 (7%) 1 (7%) 12 (86%) model will increase students’ learning ability by first performing the skill on the model 1 (7%) 13 (93%) model is adequate to prepare students for performing a live animal cesarean surgery 1 (7%) 4 (29%) 11 (79%) model could teach students poor technique 3 (21%) 10 71%) 1 (7%) *one veterinarian marked in between agree and strongly agree clinical theriogenology 2022; 14: 352 table 2. veterinarians’ comments about the model, paraphrased. numbers in parentheses indicate how many veterinarians made that comment. add fluid in the uterus (6) very good model/very useful (4) skin should be tougher/suture pulls through (3) muscle layers should be tougher/puncture resistant (2) add peritoneal fluid (2) silicone material doesn’t mimic live tissue very well; had more memory than typical small ruminant skin (2) add a peritoneum (2) add more landmarks – e.g., transverse vertebral wings, pin bone, etc. (2) tissue layers didn’t open up like they would in real life (2) add a rumen good layers for suturing skin feel is excellent surprisingly life like shows all steps necessary sterility reminders will need to be provided by supervisor materials a bit sticky uterus should have same thickness throughout uterus should be thicker so students can do utrecht as a partial thickness pattern fetus was outside of the uterus when i entered the abdomen. skin layer released from the model during my approach. uterus was a bit hard to identify. i thought it was possibly intestine or other tissue. ideally should bleed novice to expert comparison two expert videos were excluded because they were recorded using a head mount, and there was too much motion in the video recordings for accurate scoring. six student videos were excluded from analysis because the video files were lost or incomplete. this left 12 expert videos and 10 student videos for scoring. on average, experts received a checklist percentage score of 73.4% (sd = 12.5%); this was higher (p = 0.042) than the average checklist score achieved by students (mean = 62.4%, sd = 15.2%, g = 0.88). on average, although experts tended to receive an overall higher grs of 3.67 (sd = 1.30) it was not different (p = 0.058) from the overall grs awarded to students (mean 2.90, sd 0.88, g = 0.59). on average, the sum of experts’ 4 grs was 16.25 (sd = 3.67); this was higher (p = 0.043) than the sum of students’ 4 grs (mean = 13.50, sd = 3.44, g = 0.75). experts spent a median of 42 minutes (iqr = 8.75) performing the surgery; this was less than (p = 0.03) the time spent by novices (median 69.00, iqr = 30.75). although experts may have varied slightly in how they performed the procedure, the low iqr (8.75 minutes) suggested that these small variations did not have much impact on procedural time. alpha for the 13-item checklist was 0.58. student surveys fifteen of the 16 students (94%) agreed or strongly agreed that the model was easy to use and had adequate landmark structures. ten students (63%) agreed or strongly agreed that the materials looked and felt realistic; although students had not previously performed ovine cesarean surgery and could not rate tissue realism specific to that surgery, they could rate tissue realism generally due to their experience performing small animal ovariohysterectomies and orchidectomies. all students agreed or strongly agreed that they could safely utilize the model. student survey responses are summarized (table 3). one student left a comment regarding the difficulty to suture deeper layer; it was unclear if this referred to the uterus or the abdominal transversus muscle layer. no other student comments were received. clinical theriogenology 2022; 14: 353 table 3. students’ survey responses after using the model question strongly disagree n (%) disagree n (%) neutral n (%) agree n (%) strongly agree n (%) model was easy to use 1 (6%) 6 (38%) 9 (56%) adequate landmarks were present 1 (6%) 7 (44%) 8 (50%) the materials looked and felt realistic 1 (6%) 5 (31%) 7 (44%) 1 (6%) i feel that i can safely utilize this model 1 (6%) 15 (94%) discussion “days of ‘see one, do one, teach one’ are long gone, and we are now in the era of evidence and outcome medicine; that is, ‘see many, learn from the outcome; do many with supervision and learn from the outcome; and finally, teach many with supervision and learn from the outcome’”.24 learning to perform a surgical procedure takes practice and repetition,5 and a well-designed model gives veterinary students the opportunity for repetitive practice and frees them from the necessity of animal or cadaver availability. in this study, the ovine cesarean surgery model and rubric were assessed using a validation framework of content evidence, relationship with other variables (e.g., level of training) evidence, and internal structure evidence. experts rated the model’s features and value for training students highly, offering content evidence in support of validation. experts’ comments indicated their desire for a rumen to be added to the model, and lubrication or fluid to simulate the slippery uterine environment. after completion of the study, the model was modified to include a rumen, simulated by a mostly deflated free-hanging rubber ball that must be pushed out of the way to access uterus. lubrication or fluid in the uterine environment poses more of a logistical challenge and is being considered for future iterations of the model. novices felt that the model was easy to use and possessed adequate landmark structures to orient them to the task. experts scored significantly higher than novices on the checklist and total grs, which indicates that the model had adequate features to differentiate novice from expert performance. experts also performed the procedure faster than novices, though the time to perform the procedure varied widely among novices. other studies in veterinary education have also demonstrated that novices require longer time to perform simulated procedures.25-28 students performing ovine cesarean surgery on the model may have benefitted from the presence of a second surgeon or technician to assist during surgery. however, the design of the study required that the surgeon perform the procedure alone to limit the influence of the second person’s skills, knowledge, and abilities. when included in the veterinary curriculum, the mock cesarean surgery could be performed by 2 students, allowing the primary surgeon to have the benefit of the assistant and granting students more training time with the model—once as assistant surgeon and once as primary surgeon. scores produced by the 16-item checklist had poor to questionable internal consistency (α = 0.58), which indicates that performance among the various items was not closely correlated. this suggests that the steps performed during ovine cesarean surgery require a diversity of skills that are not necessarily closely related. these skills include knowledge-based items (e.g., leaving the feal membranes in the uterus rather than peeling it away and selecting appropriate suture patterns for closure). these skills also include technical items such as making a smooth incision and isolating muscle layers for incision. modifying the checklist to contain more items would increase the internal consistency, as would including items that are more correlated with one another. also, some of the individual skills contained within the cesarean surgery procedure may be able to be taught separately using task trainers (e.g., suture trainers for large animal skin and hollow organs). our study had some limitations. first, surgical performance was evaluated using video recordings rather than being observed in person. although this allowed a single blinded scorer to score all performances, evaluating a 3-dimensional task using a 2-dimensional recording can potentially impact the accuracy of performance ratings.29 previous research has indicated that assessing surgical skills using video recordings is a useful way of improving study flexibility and allowing blinding.30 second, some participants’ videos were either lost, incomplete, or unsuitable for scoring because of using a head mounted camera that impaired the rater’s ability to evaluate performance. other studies in veterinary education have also excluded some participants’ videos from analysis due to technological malfunctions with cameras and video recordings.9,29,31 statistical significance was observed despite lower number of participants. third, surgical performance was evaluated by a single rater; this did not allow for the calculation of inter-rater reliability. inter-rater reliability could be assessed in a subsequent study using the model and rubric. fourth, the model was placed in standing rather than lateral recumbency that is more common for ovine cesarean surgery. model accommodates either positioning. finally, the study was performed with a relatively small cohort of veterinarians and veterinary students. additional studies involving students from multiple institutions could generate further evidence for generalizability to other settings. research comparing students’ surgical performance on this model to their performance on the live animal procedure would provide further evidence in support of validation. conclusion a cost-effective ovine cesarean surgery model has been created that met the content evidence and relationship with other variables evidence components of the validation framework. reliability of scores produced by the checklist in this study could be further increased by adding more items and including items clinical theriogenology 2022; 14: 354 that are more correlated with one another. model allowed students to practice cesarean surgery in a safe, standardized, lowstress environment. teaching students to perform cesarean surgery on a model will allow them to perform the procedure repetitively to hone their skills while receiving instructor feedback, before performing the procedure on a live patient. acknowledgement authors acknowledge bill collingsworth for his contributions in creating and producing the model used in this study. funding southeast veterinary educational consortium (a group of veterinary colleges in southeastern us). funder had no role in study design; collection, analyses, or interpretation of data; in writing the report; or in the decision to submit the article for publication. conflict of interest authors have no conflict of interest to report. author contributions philippa gibbons: conceptualization, methodology, investigation, resources, writing – review & editing, supervision elizabeth devine: conceptualization, methodology, investigation, writing – review & editing dave dutton: formal analysis, writing – review & editing tiffany pulliam: conceptualization, methodology, investigation, writing – review & editing stacy anderson: conceptualization, methodology, resources, writing – review & editing julie hunt: conceptualization, methodology, formal analysis, investigation, resources, data curation, writing – original draft, supervision data availability statement data that support the findings of this study are available from the corresponding author (julie hunt) upon reasonable request. references 1. morin de, constable pd, troutt hf, et al: surgery, anesthesia, and restraint skills expected of entry-level veterinarians in bovine practice. j am vet med assoc 2002;221:969-974. 2. newman k, anderson d: caesarean section in cows. vet clin north am food anim 2005;21:73-100. 3. halsted ws: the training of the surgeon. bull johns hop hosp 1904. 4. rodriguez-paz jm, kennedy m, salas e, et al: beyond “see one, do one, teach one”: toward a different training paradigm. postgrad med j 2009:244-249. 5. ericsson ka: deliberate practice and the acquisition and maintenance of expert performance in medicine and related domains. acad med 2004;79:s70-81. 6. ericsson ak, krampe rt, tesch-römer c: development of elite performance and deliberate practice. psychol rev 1993;100:363-406. 7. duvivier rj, van dalen j, muijtjens am, et al: the role of deliberate practice in the acquisition of clinical skills. bmc med educ 2011;11:1-7. 8. carpenter l, piermattei d, salman m, et al: a comparison of surgical training with live anesthetized dogs and cadavers. vet surg 1991;20:373378. 9. hunt ja, heydenburg m, kelly ck, et al: development and validation of a canine castration model and rubric. j vet med educ 2020;47:78-90. 10. motta t, carter b, sweazy e, et al: development and validation of a low-fidelity, low-cost surgical simulation model to teach canine orchiectomy. clinical theriogenology 2018;10:125-139. 11. read e, vallevand a, farrell r: evaluation of veterinary student surgical skills preparation for ovariohysterectomy using simulators: a pilot study. j vet med educ 2016;43:190-213. 12. annandale a, scheepers e, fosgate gt: the effect of an ovariohysterectomy model practice on surgical times for final-year veterinary students’ first live-animal ovariohysterectomies. j vet med educ 2020;47:44-55. 13. anderson s, miller l, gibbons p, et al: development and validation of a bovine castration model and rubric. j vet med educ 2021;48:96104. 14. baillie s, christopher r, catterall a, et al: comparison of a silicon skin pad and a tea towel as models for learning a simple interrupted suture. j vet med educ 2019;47:516-522. 15. griffon dj, cronin p, kirby b, et al: evaluation of a hemostasis model for teaching ovariohysterectomy in veterinary surgery. vet surg 2000;29:309-316. 16. olsen d, bauer m, seim h, et al: evaluation of a hemostasis model for teaching basic surgical skills. vet surg 1996;25:49-58. 17. mcgaghie wc, issenberg sb, cohen er, et al: does simulationbased medical education with deliberate practice yield better results than traditional clinical education? a meta-analytic comparative review of the evidence. acad med 2011;86:706-711. 18. hunt j, simons m, anderson s: if you build it, they will learn: a review of models in veterinary surgical education. vet surg 2022;51:5261. 19. kane m: an argument-based approach to validity. psychol bull 1992;112:527. 20. american association of educational research, american psychological association, national council on measurement in education: standards for educational and psychological testing. american educational research association 1999. 21. cook d, beckman t: current concepts in validity and reliability for psychometric instruments: theory and application. am j med 2006;119:166-e7. 22. tibary a, pearson l, van metre d, et al: surgery of the sheep and goat reproductive system and urinary tract. in: fubini s, ducharme n, editors. farm animal surgery. 2nd edition. philadelphia, pennsylvania: wb saunders: 2017. p. 571-595. 23. cohen j: statistical power analysis for the behavioral sciences, 2nd edition. hillsdale, nj: lawrence erlbaum associates: 1988. 24. rohrich r: “see one, do one, teach one”: an old adage with a new twist. plast reconstr surg 2006;118:257-258. clinical theriogenology 2022; 14: 355 25. uson-gargallo j, uson-casaus j, perez-merino e, et al: validation of a realistic simulator for veterinary gastrointestinal endoscopy training. j vet med educ 2014;41:209-217. 26. banse h, mcmillan c, warren a, et al: development of and validity evidence for a canine ocular model for training novice veterinary students to perform a fundic examination. j vet med educ 2021;48:620628. 27. tapia-araya a, uson-gargallo j, enciso s, et al: assessment of laparoscopic skills in veterinarians using a canine laparoscopic simulator. j vet med educ 2016;43:71-79. 28. fransson b, chen c, noyes j, et al: instrument motion metrics for laparoscopic skills assessment in virtual reality and augmented reality. vet surg 2016;45:5-13. 29. tan j-y, ma iwy, hunt ja, et al: video recording in veterinary medicine osces: feasibility and inter-rater agreement between live performance examiners and video recording reviewing examiners. j vet med educ 2021;48:485-491. 30. williamson ja, farrell r, skowron c, et al: evaluation of a method to assess digitally recorded surgical skills of novice veterinary students. vet surg 2018;47:378-384. 31. williamson ja, brisson ba, anderson sl, et al: comparison of 2 canine celiotomy closure models for training novice veterinary students. vet surg 2019;48:966-974. appendix 1. model evaluation survey* completed by veterinarians please rate the statements below using a scale of strongly disagree, disagree, neutral, agree, and strongly agree. adequate landmarks were present materials looked realistic materials felt realistic model was easy to use model was able to teach the preparation and steps required to perform the skill model will improve animal welfare by allowing students to first perform the skill on this model model will improve students’ learning ability by first performing the skill on this model model is adequate to prepare students for performing a live animal caesarean surgery students can safely utilize this model model could teach students poor technique ________ *adapted from: williamson ja: construct validation of a small-animal thoracocentesis simulator. j vet med educ 2014;41:384-389. appendix 2. rubric used to score expert and novice performances on the model checklist (yes/no) 1. palpates landmarks for appropriate placement of incision 2. makes incision in the correct location on the paralumbar fossa 3. creates skin incision of appropriate length (10 15 cm) 4. makes smooth incision through skin 5. isolates external abdominal oblique and either incises sharply or lifts and uses scissors 6. isolates internal abdominal oblique and either incises sharply or lifts and uses scissors 7. isolates transversus and lifts and uses scissors to incise 8. makes similar length incisions through skin and muscle layers 9. palpates and correctly identifies uterus 10. brings fetus’s hind limb to the body wall incision 11. makes uterine incision approximately 10 cm in length (equal to fetal foot to hock length) 12. leaves placenta in the uterus (unless it comes away with the lamb) 13. uses inverting suture pattern to close uterus (utrecht preferred; cushing accepted if performed well) 14. closes muscle layer using simple continuous pattern (either individual layers or all in one) 15. closes skin using ford interlocking pattern 16. ends skin closure with 1 2 simple interrupted at ventral end of incision global rating scales (very poor, poor, borderline unsatisfactory, borderline satisfactory, good, excellent) 1. tissue handling 2. instrument handling 3. efficiency of time and motion 4. quality of suturing 5. overall global rating score appendix 3. model evaluation survey* completed by students please rate the statements below using a scale of strongly disagree, disagree, neutral, agree, and strongly agree. adequate landmarks were present materials looked/felt realistic model was easy to use model was suitable to teach the preparation and steps required to perform the skill model was suitable to give a general idea of the actual tactile experience when performing the skill i feel the model will increase student learning ability by first performing the skill on this model i feel students can safely utilize this model ________ *adapted from: williamson ja: construct validation of a small-animal thoracocentesis simulator. j vet med educ 2014;41:384-389. 2018: effect of omega 3, 6 and 9 fatty acids and vitamin e on canine semen motility, morphology and cryopreservation effect of omega 3, 6 and 9 fatty acids and vitamin e on canine semen motility, morphology and cryopreservation c. barstow,a r. wilborn,a a. johnson,a j. brendemuehl,b y. zhao,c k. willianc acollege of veterinary medicine, auburn university, auburn al; b college of agriculture, auburn university, auburn al; c college of science and mathematics, auburn university, auburn, al there are currently a number of supplements commercially available that claim improvement of male fertility. there are few documented studies in stud dogs investigating the efficacy of these supplements, most notably those containing fatty acids. it has been found that supplementation with vitamin e, which has a similar action as fatty acids, did show some success in dogs. additionally in horses, supplementation with dha, an omega 3 fatty acid, did improve poor semen quality. the objective of this study was to investigate the effects of a commercial omega fatty acid supplement (om3 gold 1000; spectrum veterinary; phoenix, az), on canine semen quality parameters and survival through the cryopreservation process. each capsule contained 144 mg of linoleic acid, 500 mg of linolenic acid, 203 mg of oleic acid and 20 iu of vitamin e. twelve dogs were assigned to either the treatment (n=8) or control group (n=4), with owners and kennel managers blinded to the treatment. the eight treated dogs were further broken down into below average (n=4) and average (n=4) with regard to initial post thaw motility of 19% and 52%, respectively. all treatment dogs received one capsule of om3 gold 1000 by mouth twice daily for a period of 8 weeks. ejaculates were collected prior to initiation of treatment (time zero) on all dogs and then every two weeks for 3 months. motility, morphology and total sperm number were evaluated on each collection. ejaculates were cryopreserved at time zero and again at 8 weeks, with post-thaw motility as the measured observation. after 8 weeks, total sperm number did not differ between the treatment (546 million to 708 million) (p=0.063) and control groups (625 million to 701 million). treated dogs in the below average group had an increased post thaw motility from 19% to 51% (p=0.043), whereas control dogs went from 52% to 57% after 8 weeks. normal morphology in the below average treated group started at 65% and increased to 74% (p=0.058) after 8 weeks, however the control dogs went from 57% to 53%. fatty acid analysis via gas chromatography was performed on the serum and seminal plasma at time zero and 8 weeks after treatment. in all treatment dogs, there was a decrease in concentration of omega 6 linoleic acid (p=0.027) in the serum, whereas there was a decrease in concentration of omega 6 arachidonic acid (p=0.023) in the seminal plasma. it appears that supplementation with om3 gold 1000 may help improve post-thaw motility and decreasing the concentration of pro-inflammatory omega 6 fatty acids. keywords: canine, sperm, fatty acid, supplementation clinical theriogenology • volume 10, number 3 • september 2018 324 2018: endometrial cyst ablation using uterine alligator forceps in three broodmares endometrial cyst ablation using uterine alligator forceps in three broodmares b.d. radny, j.l. klabnik-bradford, t.m. prado college of veterinary medicine, university of tennessee, knoxville, tn introduction uterine or endometrial cysts vary in shape and size and can be seen as single or multiple cysts on ultrasound. the presence of endometrial cysts are important because they can be mistaken for a pregnancy by resembling an embryo on ultrasound or palpation, interfere with embryo mobility and induce pregnancy loss. the risk of these consequences are greater when the collective volume of the cyst(s) is higher.1,2 diagnostic approach and treatment three mares, varying in breed and ages, between 6 to 22 years, underwent a breeding soundness examination at the university of tennessee during the 2017 breeding season. all three mares had a single endometrial cyst at the uterine lumen that ranged from 25 to 55 mm in diameter. mares were artificially inseminated during the previous estrous cycle, but did not become pregnant. cyst ablation was recommended to allow establishment of pregnancy. different techniques were offered, including co2:yag laser ablation technique,1,2 however the clients elected a new technique–partial removal of the cyst wall via uterine alligator forceps. theoretically, this would prevent the cyst from refilling with minimal scaring. results and discussion in order to ablate each cyst with the uterine biopsy tool, the cervix was dilated. next, the tool, driven by the clinician’s hand, was introduced vaginally and continued into the uterine lumen. the cyst was held between the index and middle finger as the instrument was held against the cyst. a piece of cyst wall was cut, as is performed for a uterine biopsy. transrectal ultrasonography confirmed complete ablation of the cyst. following cyst ablation, two of the mares bred on the next estrous cycle, became pregnant, and are expected to foal in the spring of 2018. the third mare came back into estrus late in the season; the owner decided to wait until spring 2018 to breed her. keywords: mare, endometrium, cyst, pregnancy, biopsy forceps references 1. pycock jf: breeding management of the problem mare. in: samper jc, editor. equine breeding management and artificial insemination. 2nd edition. st. louis: saunders, elsevier; 2009. p. 141-142. 2. leblanc m, lopate c, knottenbelt d, et al: the mare. in: knottenbelt d, leblanc m, lopate c, et al, editors. equine stud farm medicine and surgery. london: saunders, elsevier; 2003. p. 203-304. clinical theriogenology • volume 10, number 3 • september 2018 330 postpartum factors and fertility in dairy cows postpartum factors and fertility in dairy cows james ferguson department of clinical studies, school of veterinary medicine university of pennsylvania, new bolton center, pa abstract normal reproductive function should return by 50 days postcalving and depends on complete uterine involution, clearance of uterine pathogens, and resumption of ovarian cycling. delays in resumption of ovarian cycling beyond 35 days postcalving, failure to clear uterine pathogens, excessive adipose tissue mobilization and body condition loss, increased blood ketones, and decreased serum glucose are associated with decreased pregnancy to first insemination and increased pregnancy loss in cows that do conceive. overall, health issues and delays in ovulation have an odds ratio of conception to first insemination of 0.54 0.39 relative to cows with early ovulation and no infectious or metabolic health problems. embryonic losses are approximately 16.5% in cows with no health problems and 27% in cows with postcalving health problems. significant proportions of cows with health problems challenge the ability of farm managers to achieve pregnancy rates that result in economic optimal reproductive performance. keywords: postpartum cows, fertility, postpartum health reproductive goals birth of a calf initiates lactation. ideally, dairy cows would begin to produce milk at 22 24 months of age and calve every 11 13 months until replaced by a genetically superior female.1 however, removal may occur for nongenetic purposes, such as for health problems, reproductive failure, or injury. typically, cows remain in a herd for 2.5 3 lactations2 until replaced by a younger female, but health and reproductive failure may influence the age at which a cow leaves the herd. herd profitability is influenced by the dynamics of age at first calving, age of cow at replacement, annual rate of replacement, genetic intensity of replacement, and the annual frequency of animals recalving.1 a successfully managed reproductive program controls the age at first calving (22 24 months), the period of annual recalving (calving interval, 11 13 months), the reason for herd removal (primarily low production), and the sale of female and male animals. annual recalving should occur every 11 13 months, with a rest period of 40 60 days between sequential lactations.1 since pregnancy is ~280 days in bos taurus cows (range; 270 295 days), to maintain 11 13 month calving intervals requires that cows become pregnant 55 115 days postcalving. since annual replacement (culling) rates are typically about 25 35% in a dairy herd, 65 70% of cows which calve should become pregnant within this interval, with new replacements maintaining the dairy population. management practices that control insemination have a major influence on achieving this goal.1-3 however, cow physiology also has a role determining if pregnancy will be achieved within economically optimal intervals. the economic success of herd reproductive programs depends on successful integration of management practices and cow physiology.1,4-5 pregnancy rate (pr) is a metric that captures management (insemination rate) and cow fertility (conception rate) in one variable.1-3 it represents the proportion of nonpregnant (open) cows that become pregnant every 21 days.2,3 the average estrous cycle of the cow, 21 days (range; 18 24 days), delineates the time period for determining rates of insemination and conception. for this paper, conception will be defined as cows diagnosed as pregnant between 30 45 days postinsemination, relative to a particular time period of insemination. true, physiologic conception (fertilization of an ovulated oocyte) are close to 85 90% in dairy cows but are not observable on dairy farms.5 embryonic loss of fertilized oocytes prior to 35 days are high (40 60% of fertilized oocytes) and still significant up to 45 days postinsemination (10 15% of pregnancies from 28 to 45 days).5 pregnancy rate depends on insemination rate (ir) and conception rate (cr). insemination rate is calculated as nonpregnant (open) cows inseminated within a 21-day period, divided by the proportion of clinical theriogenology • volume 12 number 3 • september 2020292 nonpregnant cows available to inseminate. conception rate is calculated as the proportion of cows diagnosed as pregnant, divided by the total cows inseminated within the concurrent 21-day interval. every 21 days following the voluntary waiting period (vwp), the proportion of open cows that become pregnant are determined by the pr. pregnancy rate is the combination of ir (heat detection rate) times cr. survival curves of pregnancy with days postcalving describe overall pr for a herd. for optimal economic returns, pr needs to be > 25% (figure 1). pregnancy rates < 25% have 6fold economic loss compared to pr > 25% (figure 1). to achieve a pr > 25%, insemination rates need to be > 70%, and cr needs to be at least > 35%. management controls ir. cow fertility is the major controller of cr. this paper will examine cow factors influencing pr. a companion paper will examine management factors influencing insemination rates. figure 1. pregnancy rate (pr) on the x-axis and change in economic value (losses) relative to a pregnancy rate of 1.00 on the left y-axis, with potential reproductive culls on the right y-axis. cows that become pregnant in the first 21 days after the voluntary waiting period have a pregnancy rate of 1.00. with each successive 21 days, pr declines proportionally. the distribution of cows within each 21-day window determines the herd overall pr. cows not pregnant by 150 days, typical for most herds, are at risk for culling, the proportion on the right y-axis. reproductive losses are much greater when the mean herd pr is below 25% and potential reproductive culls increase. parturient cow ideally, by 50 days postpartum, a cow is fully capable of becoming pregnant and carrying a pregnancy to term.6 this requires a fully regenerated and repaired uterus and resumption of estrous cycles prior to 50 days postpartum.6-8 during this period, milk production increases to peak yield, prioritizing nutrient demands for glucose, amino acids, and minerals.9 peak milk production occurs by 30 50 days postcalving, yet feed intake will not peak until 100 120 days postcalving. this creates negative balances in energy, protein, and minerals, especially in the first 2 weeks postcalving.10-17 positive balances will not be realized until 8 weeks postcalving, as feed intake increases and milk production declines from peak.16 at calving, homeorhetic signals coordinate nutrient flows to support lactation through alterations in tissue utilization of glucose, increased gluconeogenesis in the liver, mobilization of fatty acids from adipose tissue, and mineral mobilization from bone.4,9,15 central control centers in the arcuate nucleus of the hypothalamus sense the degree of disparity in nutrient balance and turn on, dampen, or shut down hypothalamic centers that control gnrh release, impacting the initiation of ovarian function.14,18 endocrine and metabolic signals influence central control centers that determine when reproductive function will commence. endocrine and metabolic factors in the week prior to calving and in the first several weeks postcalving influence resumption of ovarian cycling and fertility at first insemination.15,19 clinical theriogenology • volume 12 number 3 • september 2020 293 during the first weeks postcalving, homeostatic processes may be disrupted, resulting in ill health in the postpartum period (milk fever, ketosis, fatty liver, displaced abomasum); furthermore, infectious diseases (metritis, mastitis) further impact the reproductive axis and influence ovulation and future fertility (table 1).19-21 in addition, elevations in nonesterified fatty acids (nefa), serum beta hydroxy butyric acid (bhba), and reductions in calcium have been associated with increased days to first ovulation, lower conception at first insemination, and reduced pregnancy rates.22-26 energy balance, metabolic stress, and infectious disease combine to reduce fertility in the postpartum cow.19 table 1. postpartum conditions and fertility in dairy cows measured as the odds of conceiving at first insemination (or, odds ratio)1. ______________________________________________________________________________________________ item4 signalment action level (%)2 or fstcr3 -------------------------------------------------------------------------------------------------------------------------------------------- dystocia calving difficulty > 10 0.80,0.75 twins > 5 die death losses < 30 days > 5 culling first 60 days > 10 milk fever hypocalcemia > 2 0.85 retained placenta > 24 hours > 8 0.68 puerperal metritis fever > 39.7oc, off feed > 10 0.56,0.64,0.43,0.37 chronic metritis purulent discharge > 30d > 20 0.57, 0.68, 0.34, 0.24 0.381, 0.71, 0.70, 0.67 subclinical endometritis proportion of neutrophils in uterine cytology > 20 <1.00 ketosis b-oh butyrate > 1 mmol/l > 10 0.69, 0.50, 0.79 displaced abomasum > 3 anestrus 0.47, 0.38, 0.42 ovulatory dysfunction > 40 dim first ovulation > 20 body condition loss 30 dim > ¾ score loss > 15 0.545,0.689 body condition postpartum score < 2.5 at 30 dim > 15 0.650 one clinical disease in first 30 dim > 30 0.52, 0.79 two or more clinical disease in first 30 dim > 10 0.57 ------------------------------------------------------------------------------------------------------------------------------------------------ 1or = odds ratio, 2action level = proportion of animals with the condition divided by the number calving 3fstcr = first service conception rate 4 dystocia = based on calving difficulty score of 2 or greater 4 dim = days in milk 4 body condition based on a 5 point scale, 1 being emaciated, 2 thin, 3 adequate, 4 fat, 5 obese fertility is the outcome of multiple systems acting in concert in the postpartum dairy cow.18,19 the immune, metabolic, endocrine, and gastrointestinal systems undergo adaptations following calving to coordinate lactation, uterine repair, feed intake and resumption of reproductive cycling.15,19 excessive inflammation and infection, increased metabolic disparity between nutrient intake and output in milk, and gastrointestinal upset can disrupt the coordination of nutrient flows and lead to impairment of health and fertility.20,21,27 homeorhetic controls are achieved through changes in somatomedins postcalving.2,4,28,29 serum insulin and insulin growth factor 1 (igf1) decrease, whereas serum growth hormone, prolactin, parathyroid hormone, and glucocorticoids increase.4,15,18,28,29 immediately postcalving, serum glucose declines and serum nefa increases, released from adipose tissue.4,15,30 amino acids are released from skeletal muscle. plasma calcium declines precipitously on the day of calving and returns to homeostatic concentrations over next 4 days. liver gluconeogenesis increases to meet the demand for glucose by the mammary gland using propionate absorbed from the rumen, and lactate and amino acids released from skeletal muscle.17 serum bhba increases due to incomplete beta oxidation of nefa in the liver and peripheral tissues. liver triglycerides increase due to incomplete incorporation of nefa into triglycerides for export from the liver as low-density lipoproteins. reduced serum insulin and increased insulin resistance in clinical theriogenology • volume 12 number 3 • september 2020294 insulin-sensitive tissues direct glucose to the mammary gland and reduce glucose availability to muscle and adipose tissue. increases in inflammatory cytokines from uterine endothelial cells, macrophages, and neutrophils in response to uterine infection stimulate the liver to produce acute phase proteins, such as haptoglobin, serum amyloid a, and ceruloplasmin, and decrease synthesis of negative acute phase proteins, such as albumin, and vitamin and hormone carriers.20,21,27 reactive oxidation compounds increase, increasing the demand for antioxidant protection.31 decreases in plasma insulin, igf1, calcium and glucose combined with increases in nefa, bhba, and inflammatory compounds influence the hypothalamic-pituitary-ovarian axis and may disrupt ovarian follicular growth, steroidogenesis, and ovulation.4,18,29 in addition, failure to clear pathogenic bacteria from the uterus further impacts the ability of the cow to establish and maintain pregnancy.7,8,32,33 physical conditions at calving, such as dystocia, twin births, and uterine prolapse create risk factors for reduced fertility.34-38 disease in the postpartum period, the first 30 days in milk, such as metritis, ketosis, milk fever, displaced abomasum, mastitis, and lameness, increase the risk for low fertility.34-42 increased negative energy balance, calving problems, uterine disease and metabolic disease disrupt uterine involution, ovarian activity, and pregnancy maintenance. cow factors influencing fertility postpartum cows experience multiple challenges postcalving. parturition is an inflammatory process and inflammatory cytokines increase, influencing hepatic function and exerting central influences on feed intake and reproductive control.20,21,27 uterine bacterial contamination is present in over 90% of cows in the first 3 weeks postcalving, further contributing to inflammatory challenges.7,8 parturition is associated with depression in innate immunity, increasing the risk of bacterial contamination becoming a significant infection.7,8 specific bacteria have a greater risk of causing uterine pathology.7,8 uterine infection may present as an acute puerperal metritis observed in the first 2 weeks postcalving. puerperal metritis is characterized by a fetid, watery vaginal discharge, fever > 39.5oc, and a depressed, anorexic cow. failure to clear pathogenic bacteria by 3 4 weeks postcalving can result in a chronic endometritis or subclinical endometritis associated with purulent to mucopurulent vaginal discharge or an increased presence of polymorphonuclear cells in uterine lavages.7,8,32,33 superimposed on inflammatory and infectious challenges are metabolic challenges associated with the negative energy balance postcalving.4 in the first 3 weeks postcalving, significant elevations in serum nefa (> 0.70 meq/l), and bhba (> 10 mg/dl), and reduced serum concentrations of glucose are associated with reductions in conception at first ai.22-26 serum glucose is important in coordinating insulin and igf1 in the early postpartum period and low serum glucose in the first week postcalving is associated with lower fstcr.4 in addition elevations of nefa in the week prior to calving are associated with reduction in fstcr. elevations in serum nefa, bhba and decreases in serum calcium and glucose have been associated with reduction in fstcr in cows with no apparent clinical conditions postcalving.22-26,30,40 metabolic stresses late in pregnancy and early postcalving influence fstcr. metabolic influences in early postpartum could alter follicular and ovum development which would reduce fertility at first insemination. 43 changes in body condition (bc) postpartum seem to support this hypothesis, as cows with more extreme condition loss have lower fertility.44-46 similarly, cows with more body weight loss have fewer fertilized oocytes and transferable embryos.47 cows that experience more negative energy balance, more condition and body weight loss, and have greater serum nefa in the first week postcalving have lower fertility at first insemination. health problems postpartum exacerbate energy balance, as feed intake is often depressed, which may compound problems with fertility at first insemination. conception at first insemination postcalving for cows with various reproductive disorders is summarized (table 2). postpartum disorders have an odds ratio (or) < 1, indicating a reduction in cr to first insemination. cows with no health problems postcalving and that ovulate prior to first insemination have an odds ratio of first service of 1; they are the sentinel fertility group in a herd. clinical theriogenology • volume 12 number 3 • september 2020 295 table 2. odds ratio of pregnancy at first insemination from various references ------------------------------------------------------------------------------------------------------------------ author year herds cows group condition or sem ----------------------------------------------------------------------------------------------------------------- fourichon et al: 1999 meta-analysis studies used varied with condition physical dystocia 0.802 0.025 physical still birth 0.739 0.081 metabolic milk fever 0.852 0.483 metabolic retained placenta 0.681 0.014 infectious metritis 0.563 0.036 metabolic cystic ovaries 0.786 0.058 metabolic anestrus 0.471 0.022 metabolic ketosis 0.754 0.256 metabolic displaced abomasum 1.000 physical lameness 0.852 0.123 ribeiro et al: 2013 2 957 physical dystocia 0.460 0.128 infectious metritis 0.370 0.122 infectious endometritis 0.680 0.138 infectious mastitis 0.700 0.140 infectious pneumonia 0.490 0.255 metabolic digestive problem 0.220 0.122 physical lameness 0510 0.235 metabolic 1 subclinical condition 0.820 0.158 metabolic >1 subclinical condition 0.520 0.105 metabolic nefa > 0.70 meq/l 0.420 0.084 metabolic bhba> 10 mg/dl 0.690 0.112 metabolic ca < 2.14 mmol/l 0.890 0.153 general 1 postpartum disease 0.640 0.110 general > 1 postpartum disease 0.340 0.089 santos et al: 2009 8 5,719 physical dystocia 0.750 0.064 infectious metritis 0.660 0.056 infectious endometritis 0.620 0.056 infectious fever >39.5oc 0.600 0.043 infectious mastitis 0.840 0.117 infectious pneumonia 0.630 0.242 metabolic ketosis 0.500 0.082 metabolic digestive problem 0.780 0.224 physical lameness 0.570 0.094 general 1 postpartum disease 0.790 0.056 general > 1 postpartum disease 0.570 0.054 toni et al: 2015 3 1498 infectious metritis 0.640 0.071 infectious endometritis 0.340 0.056 metabolic cystic ovaries 0.260 0.037 metabolic anestrus 0.380 0.089 physical lameness 0.540 0.041 harman et al: 1996 6,227 44,450 infectious endometritis 0.235 0.036 infectious other disease 0.653 0.128 infectious other infertility disease 0.342 0.044 infectious mastitis 0.786 0.058 metabolic anestrus 0.418 0.026 metabolic cystic ovaries 0.439 0.021 metabolic ketosis 0.786 0.041 physical lameness 0.515 0.172 francos and mayer 1988 nr 14,573 infectious metritis 0.303 0.022 clinical theriogenology • volume 12 number 3 • september 2020296 infectious endometritis 0.381 0.012 metabolic retained placenta 0.363 0.012 metabolic anestrus 0.431 0.008 lee et al: 1989 5 1,059 infectious metritis 0.538 0.177 infectious endometritis 0.709 0.103 metabolic retained placenta 0.493 0.122 metabolic cystic ovaries 0.538 0.137 physical lameness 0.527 0.165 leblanc et al. 2002 27 1,865 infectious endometritis 0.700 0.083 ferguson et al. 1995 4 566 infectiousfever > 39.5oc 0.350 0.075 ospina et al: 2010 91 2,770 metabolic nefa > 0.27 1 week prior 0.681 0.095 metabolic nefa > 0.72 1 week after 0.730 0.119 metabolic bhba > 10 mg/dl 1 week 0.873 0.176 bcs bcs > 3.75 1.041 0.239 chapinal et al: 2012 55 1,919 bcs bcs > 4 versus < 1.00 bcs bcs 3.25-3.75 vs > 4.0 0.710 0.089 bcs bcs< 3.0 versus > 4.0 0.540 0.084 metabolic ca < 2.3 mmol/l week-1 0.670 0.105 metabolic ca < 2.2 mmol/l week +1 0.770 0.096 kim and jeong 2019 2 790 general one disease postpartum 0.550 0.089 bcs bcs at ai <3 versus >3 0.640 0.119 bcs bcs at 30 day < 2.75 versus >2.75 0.792 0.155 loeffler et al: 43 9,369 infectiousmetritis 0.740 0.099 infectiousendometritis 0.670 0.128 infectiousmastitis 0.530 0.110 metabolic cystic ovarian disease 0.530 0.115 bcs bcs < 2.5 versus 2.75-3.25 0.650 0.117 bcs bcs > 3.5 versus 2.73-3.25 0.740 0.161 bruinje et al: 2017 2 748 metabolic no luteal phase versus 1 normal 0.260 0.059 metabolic one abnormal luteal phase versus none 0.746 0.119 metabolic no luteal phase versus 2 normal 0.381 0.066 metabolic ovulation > 63 days versus earlier 0.461 0.115 barletta et al. 2017 1 232 bcs bcs loss vs bcs gain 0.198 0.068 bcs bcs maintain versus bcs gain 0.326 0.130 santos et al: 2009 4 6396 anestrus no ovulation 65 d versus ovn 0.584 0.038 bcs calving bcs < 3 versus bcs 3 3.5 0.848 0.060 bcs calving bcs > 3.75 versus bcs 3 3.5 1.058 0.072 bcs at ai bcs < 3 versus 3 3.5 0.727 0.403 bcs at ai bcs > 3.75 versus 3 3.5 1.236 0.119 ------------------------------------------------------------------------------------------------------------------- 1papers reporting hazard of pregnancy or relative risk converted to odds ratio using the formula or = (rr rr*cr reference group)/ (1 rr*cr reference group); cr = conception rate; rr = relative risk cows that had dystocia, 1 postpartum disease, multiple postpartum diseases, metabolic stress, lameness, and ovulatory dysfunction have reduced cr at first insemination. body condition at calving, bc at insemination, and bc loss have variable effects on cr to first insemination. in general, bc < 2.5 and bc loss > 1 unit have lower odds of conceiving at first insemination. cows with bc > 3.75 at calving and that have maintained bc from calving to first insemination have cr that are better or not different from cows with no health problems postcalving. clinical theriogenology • volume 12 number 3 • september 2020 297 milk production (figure 2) for cows with no postpartum problem compared to cows that had a health issue or delivered twin calves (897 total calving, milk production was from 841 cows distributed as follows; normal (452) metritis (131), retained placenta (106), dystocia (168), milk fever (15), ketosis (57), displaced abomasum (85), twins (47)). milk production was reduced in cows with problems in the first 10 weeks postcalving, reducing total lactation yield in 44 weeks by 439 kg of milk. figure 2. milk production by week postcalving for cows with no postpartum disease and cows with a problems postcalving (metritis, retained placenta, dystocia, ketosis, displaced abomasum, and twins). data from university of pennsylvania marshak dairy, 900 cows. total difference in milk -439 kg over 44 weeks postcalving. a random effects model was used to calculate an overall or of cr for cows with postpartum problems and bc change. general groupings were constructed for the data (table 3). table 3. random effects model across studies for the mean odds ratio (or) for postpartum conditions. ------------------------------------------------------------------------------------------------------------------------------ condition postpartum or se 95% confidence range ---------------------------------------------------------------------------------------------------------------------------------------- anestrus, cod, nonovulatory 0.466 0.048 0.373 0.559 bc change <-1 unit from calving 0.440 0.263 -0.075 0.955 bc change > -1 unit from calving 0.502 0.089 0.327 0.677 bc at ai 2.75 3.25 0.581 0.665 -0.722 1.884 bc at calving 2.75 3.5 0.648 0.197 0.263 1.033 bc at calving > 3.75 0.936 0.083 0.774 1.098 bc at calving < 3 0.616 0.117 0.387 0.846 bc at ai > 3.75 0.939 0.185 0.576 1.301 bc at ai < 3 0.671 0.129 0.418 0.924 one postpartum disease 0.617 0.104 0.412 0.821 more than one postpartum disease 0.390 0.113 0.169 0.612 metritis, mastitis postpartum 0.478 0.043 0.394 0.561 postpartum ketosis, elevated nefa 0.499 0.044 0.413 0.585 two metabolic conditions 0.502 0.216 0.080 0.925 dystocia, lameness, still birth 0.610 0.063 0.487 0.732 overall mean 0.542 0.020 0.502 0.581 ------------------------------------------------------------------------------------------------------------------------------------- cod = cystic ovarian disorder nonovulatory = no ovulation prior to first insemination bc = body condition, scale 1 to 5, with 1 emaciated, 2 thin, 3 average, 4 fat, and 5 obese ai = artificial insemination overall, having a postpartum problem, significant bc loss, or having thin bc at insemination had an or of 0.542 (se 0.020) on insemination to first insemination. if cr in normal cows is 50%, the cr in -10 0 10 20 30 40 50 0 10 20 30 40 50 m ilk p ro du ct io n, k g/ da y week postcalving milk prodution and postpartum health healthy unhealthy difference clinical theriogenology • volume 12 number 3 • september 2020298 problem cows would be 35.1%. if heat detection (insemination rates) were similar in both groups of cows, which is unlikely as cows with reproductive problems often have delayed first ovulation, normal cows would achieve a pr of 25.0% if cr was 33.3% and insemination rates 75%. cows with reproductive problems would have a cr of 21.3%, which would result in a pr of 16.0% if insemination rates were 75%. this would correspond to a hazard rate of pregnancy of 0.76 compared to normal cows. effects on survival curves for normal cows and reproductive problem cows are shown (figure 3). the lower pr reduces milk produced per day due to longer calving intervals, fewer calves born per year, and a higher risk of culling due to more open cows at or beyond 230 days postcalving. in addition, cows with reproductive problems due to health issues produce about 439 kg less milk over a 305 days lactation, primarily due to lower yields in the first 10 weeks postcalving. the result would be a reduction in income of $404.00 per cow per year (milk income, $0.17/lb; calf value, $150/head; replacement costs, $750/cull cow). figure 3. failure time (pregnancy rate) curves for cows with normal postpartum conditions () versus cows with reproductive disease or late first ovulation (). normal cows have a pregnancy rate of 25.0% (insemination rates of 75% and cr of 33.3%), whereas the reproductive disease cows have a pregnancy rate of 18.4% (insemination rate of 75% and cr of 24.5%, based on a mean or of 0.65 compared to normal cows for reproductive disease). voluntary wait period is 50 days. the hazard rate of pregnancy for the reproductive disease cows is 0.828 relative to the normal cows. initiation of ovulation reinitiation of ovarian cycling early postpartum is critical for full restoration of fertility.6,10-14,48 ovulation of a healthy ovum capable of fertilization and normal embryonic development is essential for maintenance of pregnancy.47 follicular development affects the quality of the ovum and of the corpus luteum that develops following ovulation, influencing embryonic development, progesterone secretion, and the maintenance of pregnancy.47 typically, it has been observed that several estrous cycles prior to the breeding period are necessary to ensure a healthy ovum and sufficient progesterone secretion to support pregnancy.6 to that end, earlier first ovulation and normal luteal phases prior to first insemination are associated with higher pregnancy rates.49 the degree of energy balance nadir,16 the days to energy balance nadir,13 and uterine bacterial contamination can influence to day to first ovulation50 and the quality of ovum and progesterone produced by the subsequent corpus luteum.8 negative energy balance, the difference in energy output in milk minus energy intake from feed, is a factor influencing resumption of luteal activity postpartum and subsequent fertility.13 majority of cows experience negative energy balance postcalving, with the nadir typically occurring within the second week (figure 4). majority of cows return to positive energy balance by week 8, with a range from 4 12 weeks (figure 4). cows tolerate moderate degrees of negative energy balance in the early 0 0.2 0.4 0.6 0.8 1 1.2 0 50 100 150 200 250 300 pr op or tio n co w s o pe n days post calving proportion open with dim normal rep. disease clinical theriogenology • volume 12 number 3 • september 2020 299 postpartum period, but there seems to be a threshold that impairs ovarian function and fertility.16 days to first ovulation were negatively correlated with the nadir of negative energy balance, rather than the time.16 the greater the negative energy balance, the longer the days to first ovulation. the greater the degree of negative energy balance postpartum, the greater the serum concentrations of nefa and bhba, and the lower the concentration of glucose, insulin, and igf-1.10-12,15,19 negative energy balance is greater in cows with greater bc loss over 30 days postcalving. cows with nefa > 0.7 meq/l, bhba >10 mg/dl had lower serum concentrations of insulin, igf-1, and glucose in the first 1 3 weeks postcalving. figure 4. daily energy balance (energy intake in feed energy output in milk) by week postpartum in 40 lactating holstein dairy cows (upper panel). energy output in milk and energy intake from feed (mcal/day) to energy balance (eb) and body condition change from calving body condition score (bschng) on a 5-point scale, using quarter-point increments, by week postcalving (lower panel). it is not possible to measure energy balance on a farm, but body condition score (bcs) is a good estimator of body fat,51 and change in bcs is a good estimate of fat mobilization and cumulative negative energy balance.52,53 one unit of bc loss was associated with 42 or 54 kg of fat mobilized to support milk production, corresponding to 420 and 564 mcal of net energy of lactation used for milk production from body fat.52,53 these estimates of tissue loss and energy depend upon how fat the cow is; a cow with a bcs of 4 will lose more fat with a higher energy value when losing 1 unit of condition than a cow with a bcs clinical theriogenology • volume 12 number 3 • september 2020300 of 3.54 in addition, cows fat at calving will lose more bc than thinner cows. generally, 1 unit of bc loss corresponds to -300 to -500 mcal of cumulative negative energy balance for cows with a bcs of 2.5 4.0. body condition loss is greatest between calving and 30 days in milk, then tends to flatten over the next 4 8 weeks before increasing, as cows accumulate positive energy balance (figure 4). cows seem to tolerate up to ¾ of bcs loss before a reduction in first service conception rate is observed. when bc loss is > ¾ of bcs, the odds ratio of first service conception rate (fstcr) is 0.34 0.50 relative to no change in bc in the first month postcalving. meta-analysis (15 studies) of bc and bc loss observed heterogeneity of bc loss and fstcr, with significant fertility reduction in cows with low bc at calving or at first ai (< 2.5) and in cows with bc loss > 1unit of bcs. 44 this would tend to support the observation that cows tolerate ¾ of bc loss. thinner cows, particularly < 2.5 bc, had lower fertility than cows with greater bc.44,45 cows in thinner (< 3.0) or moderate bcs (3.35 3.75) at calving had a lower odds of pregnancy at first service (or = 0.54, or 0.71, respectively) compared to fat cows (> 4.0 bcs) at calving. 23 body condition loss and bc at first insemination create the heterogeneity in associating condition score with conception rates. the influence of energy balance on fstcr may operate through delays in first ovulation. cows that ovulate after 35 days postpartum have lower first service conception and lower pregnancy rates.48 longer days to first ovulation is associated with lower energy balance, later days to energy balance nadir, lower body condition score at 30 days postpartum, metritis, abnormal calving, ketosis, and increased serum nefa in the first week postpartum.10-14,16,40,41,46,49,50,56 longer days to first ovulation postcalving is consistently associated with lower fstcr. ovulation of the first dominant follicle occurred at 21 days postcalving in 30 50% of cows10-14,16,40,41,46,49,50,56 (figure 5). at 50 days postcalving, 20% of cows had not ovulated (figure 5) resulting in lower first service conception rates. cows that were in synchronized ovulation and timed artificial insemination program had a fstcr of ~ 20% lower than cycling contemporary cows. metritis, figure 5. cumulative proportion of cows with first ovulation by days postcalving across studies.10-14,16,40,41,46,49,50,56 ketosis, digestive upsets, and calving difficulties increased the proportion of anovulatory cows at 50 days postcalving to 30% and further reduction in fstcr occurred in cows that had greater risk of endometritis. embryonic mortality postpartum conditions reduced conception at first insemination with higher pregnancy loss after insemination.5,47,57 losses in the first week were due to fertilization failure, from days 8 27 due to failure of maternal recognition of pregnancy, from 28 60 days due to improper placental development, and from 60 90 days, due to placental growth.47 on dairy farms, fertilization failure and y = -0.0155x2 + 2.6065x 10.915 r² = 0.8555 0 10 20 30 40 50 60 70 80 90 100 110 0 10 20 30 40 50 60 70 80 90 100 cu m ul at iv e pr op or tio n fi rs t ov ul at io n, % days to first ovulation proportion days to first ovulation 10 studies clinical theriogenology • volume 12 number 3 • september 2020 301 embryo losses are not apparent, except as return to estrus within 24 days from insemination. losses from 8 27 days may only be apparent as returns to estrus between 25 35 days after insemination, but inseminations in this time period may be confounded with prior inseminations of cows that were not truly in estrus. losses from 28 60 days and 60 90 days would be observable as cows checked pregnant at an examination from 28 42 days postinsemination then either returning to estrus after examination or not pregnant at a subsequent examination. these losses were characterized as late embryonic losses based on the classification; early embryonic loss (15 17 days postinsemination), late embryonic loss (17 42 days postinsemination), and fetal loss (after 50 days postinsemination).57 due to pregnancy losses and fertilization failure following insemination, it is estimated that only about 30 35% of inseminations result in a live calf.5,47 with this degree of loss, it would require 5 or more inseminations to achieve 85% probability of inseminated cows recalving. on most farms, with routine veterinary reproductive programs, pregnancy loss will be most apparent from 30 60 days after pregnancy examination. typically, cows are first examined for pregnancy ~ 28 42 days postinsemination. pregnancy loss was ~ 12% at 60 days postinsemination after a pregnancy examination at 30 days postinsemination.47 however, this may be influenced by heat stress and prior postpartum conditions. postpartum conditions will not only lower conception rates, they will also increase late embryonic loss. conditions (figure 6) that result in pregnancy loss were classified.45,49,59-84 classifications were organized into two groupings: "no major problem:" (bcsmain = maintenance of body condition from calving; norm = cows with no health problems postcalving and ovulatory before insemination; bcsgain = cows gaining body condition from calving to insemination; bcsloss = cows with body condition loss from calving with no other identified health problems; tai = ovulatory cows inseminated on timed insemination; ov = cows that had ovulated prior to first insemination); "problem:" (dis = cows with 1 postpartum disease; met = cows with subclinical metabolic disease; phy = cows with a physical problem such as lameness or dystocia; inf = cows with metritis, endometritis, mastitis, pneumonia; multdis = cows with more than one condition post calving; abn = general classification for other abnormal conditions postcalving). classes were examined using a random effects model in sas model with study as random effect. a total of 94 observations were analyzed from 28 studies. data from cows classified as "no major problem" had a pregnancy loss of 16.5% (sem + 2.3%) similar to 12% reported.47 however, cows with problems postcalving had a loss of 27.2% (sem + 3.1%). cows that had not ovulated by the voluntary wait period had a pregnancy loss of 20.3%, intermediate to cows without a problem and cows with problems postcalving. this analysis should be viewed cautiously, as more descriptive analysis is needed. however, it points that the loss of pregnancy in cows with no problems are significant and losses in cows with problems are about double the loss in cows with no problems. proportion of pregnancy loss 30 days from first insemination (figure 7) suggested wide variation, with higher losses in cows that had a lower cr at 30 days postinsemination. overall impact on herd performance most dairy farms calve cows year-round, have cows of multiple parities, and have a proportion of cows that experienced health problems postcalving. in addition, cows with no apparent health issues may clinical theriogenology • volume 12 number 3 • september 2020302 figure 6. random effects model of the proportion of pregnancies lost from first insemination after a "30" day initial pregnancy examination for categories of conditions: bcsmain = maintenance of body condition from calving; norm=cows with no health problems postcalving and ovulatory before insemination; bcsgain= cows gaining body condition from calving to insemination; bcsloss = cows with body condition loss from calving with no other identified health problems; tai = ovulatory cows inseminated on timed insemination; ov = cows that had ovulated prior to first insemination; dis = cows with one postpartum disease; met = cows with subclinical metabolic disease; phy = cows with a physical problem such as lameness or dystocia; inf = cows with metritis, endometritis, mastitis, pneumonia; multdis = cows with more than one condition postcalving; abn = general classification for other abnormal conditions postcalving. the lines represent a mean loss of 16.5% (se 2.3%) and 27.2% (se 3.1%), with error bars representing the 95% confidence range for the two estimates. figure 7. proportion of pregnancies lost against the conception rate at 30 days after insemination. not ovulate by 50 days postcalving. healthy cows that ovulates prior to 50 days postcalving had the best fertility in the herd and the lowest pregnancy loss. given the pattern of first ovulation (figure 5), 81% of healthy cows would ovulate prior to 50 days postpartum and 19% of cows would not. for cows with health problems, 67% would ovulate by 50 days and 33% would not. cows with delayed ovulation and health issues will have an or of 0.54 for first service cr. cows with more than one health problem or a health problem combined with delayed ovulation will have an or of fstcr of 0.29 (table 3). the impact on fstcr would be as follows: if healthy cows have a fstcr of 50%, then healthy cows with ovulation later than 50 days would have a fstcr of 35%, as would cows with one health problem that ovulated prior to 50 days. cows with multiple health problems and with late first ovulation would have a fstcr of 22%. pregnancy losses will also be influenced by these conditions. cows with no health problems that ovulated prior to 50 days would have a pregnancy loss of 16.5%, which would reduce 0% 5% 10% 15% 20% 25% 30% 35% 40% pr eg na nc y l os s, % pregnancy loss after 30 day examination y = -0.152ln(x) + 0.7772 r² = 0.1121 0% 20% 40% 60% 80% 100% 0 10 20 30 40 50 60 70 80 pr op or tio n lo ss a t 4 5 to 6 0 da ys a fte r i ns em in at io n conception rate to first insemination at 30 post insemination pregnancy loss versus conception rate at 30 days clinical theriogenology • volume 12 number 3 • september 2020 303 table 4. impact of heat detection rate (insemination rate), conception rate based on a pregnancy exam at 30 days postinsemination, or of cr of 0.54 in cows with delayed ovulation after 50 days postpartum and with one health problem, and an or of 0.28 in cows with two or more health problems or health problem and delayed ovulation. pregnancy loss after 30 days was 16.5% for cows with no health problems and 27.2% for cows with delayed ovulation or one or more health problems. break even dollars were calculated relative to a pr of 25%. ------------------------------------------------------------------------------------------------------------------- hdr healthy cows cr needed to break even % % of cows calving % of insemination pregnant at 30 days _____________________________________________________________________________ 50 50 76 50 60 73 50 70 69 50 80 66 60 50 70 60 60 67 60 70 63 60 80 60 70 50 64 70 60 61 70 70 57 70 80 54 80 50 58 80 60 55 80 70 51 80 80 48 ------------------------------------------------------------------------------------------------------------- pregnancy following first insemination from 50 to 42%. healthy cows that ovulated after 50 days appear to have a pregnancy loss by 60 days of 20%, resulting in a pregnancy outcome at 60 days of 28%. cows with health problems have a pregnancy loss of 28%; pregnancy at 60 days would be 25%. pregnancy at 60 days in cows with multiple problems would be 20%. herd reproductive performance would be a composite of the proportion of healthy cows and cows with significant body condition loss and severe negative energy balance, infectious disease (metritis, endometritis, mastitis), metabolic disease (ketosis, displaced abomasum, lameness), subclinical metabolic conditions (elevated nefa and bhba, and hypocalcemia). the standard of fertility in the herd would be healthy cows with first ovulation prior to 50 days postcalving. all other groups of cows would have lower reproductive performance, both in terms of pregnancy at 30 days postinsemination and maintenance of pregnancy to 60 days postinsemination. depending on the fstcr in healthy cows and the proportion of healthy cows, the ability to achieve an optimal economic return on reproduction will be limited. impact of heat detection rate (hdr, insemination rate) on cr in healthy cows at 30 days postinsemination to achieve a pr of 25% is summarized (table 4). it requires a herd with more than 50% of cows calving with no health problem and an insemination rate of 70% or better to have a cr in healthy cows below 55% to achieve a herd pr of 25%. this is a challenge on many dairy farms. typically, on most farms, only 50% of cows have no problems postcalving. average hdr are below 50% according to dhia summary statistics. therefore, reproductive performance is below a pregnancy rate of 25% on most farms. this represents a significant loss of income. there are major opportunities to improve reproduction by improving health of cows in transition from the dry period to lactation and employing programs to ensure high rates of insemination. clinical theriogenology • volume 12 number 3 • september 2020304 conclusion to prevent health problems postcalving in less than 50% of cows calving requires a sound management program prior to calving and in the first month postcalving. housing, feed bunk access, and adequately formulated rations are a must for decreasing health conditions postcalving. it is beyond the scope of this paper to identify adequate rations for late pregnancy, but those are described elsewhere.85-87 conflict of interest there are no conflicts of interest to declare. references 1. cabera ve: economics of fertility in high-producing dairy cows on confined tmr systems. 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follicular development and fertility. proc annu am conv assoc bovine pract 1992;24:39-43. 44. lόpez-gatius f, yániz j, madriles-helm d: effects of body condition score and score change on the reproductive performance of dairy cows: a meta-analysis. theriogenology 2003;59:801-812. 45. santos jep, juchem so, cerri rla, et al: effect of bst and reproductive management on reproductive performance of holstein dairy cows. j dairy sci 2004;87:868-881. 46. shrestha hk, nakaob t, suzukic t, et al: relationships between body condition score, body weight, and some nutritional parameters in plasma and resumption of ovarian cyclicity postpartum during pre-service period in highproducing dairy cows in a subtropical region in japan. theriogenology 2005;64:855-866. 47. wiltbank mc, baez gfm, garcia-guerra a, et al: pivotal periods for pregnancy loss during the first trimester of gestation in lactating dairy cows. theriogenology 2016;86:239-253. 48. gautam g, nakao t, yamada k, et al: defining delayed 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(7th rev. ed.).2001; national academy of sciences, washington, dc 55. barletta rv, maturana filho m, carvalho pd, et al: association of changes among body condition score during the transition period with nefa and bhba concentrations, milk production, fertility, and health of holstein cows. theriogenology 2017;104:30-36. 56. opsomer g, gröhn yt, hertl j, et al: risk factors for post partum ovarian dysfunction in dairy cows in belgium: a field study. theriogenology 2000;53:841-857. 57. santos jep, rutigliano hm, sa filho, mf: risk factors for resumption of postpartum estrous cycles and embryonic survival in lactating dairy cows. anim reprod sci 2009;110:207-221. 58. wathes dc: mechanisms linking metabolic status and disease with reproductive outcome in the dairy cow. reprod domest anim 47(suppl 4) 2012;304-312. 59. barletta rv, marturana filho m, carvalho pd, et al: association of changes among body condition score during the transition period with nefa and bhba concentrations, milk production, fertility, and health of holstein cows. theriogenology 2017;104:30-36. 60. borchardt s, haimeri p, heuwieser w: effect of insemination after estrous detection on pregnancy per artificial insemination and pregnancy loss in a presynch-ovsynch protocol: a meta-analysis. j dairy sci 2016;9:2248-2256. 61. bruinje tc, colazo mg, ribeiro es, et al: using in-line milk progesterone data to characterize parameters of luteal activity and their association with fertility in holstein cows. j dairy sci 2018:102:780-798. 62. cartmill ja, el-zarkouny sz, hensley ba, et al: an alternative ai breeding protocol for dairy cows exposed to elevated ambient termperature before or after calving. j dairy sci 2001;84:799-806. 63. cartmill ja, el-zarkouny sz, hensley ba, et al: stage of cycle, incidence and timing of ovulation, and pregnancy rates in dairy cattle after three timed breeding protocols. j dairy sci 2001;84:1051-1059. 64. cerri rla, santos jep, juchem so, et al: timed artificial insemination with estradiol cypionate or insemination at estrus in high-producing dairy cows. j dairy sci 2004; 87:3704-3715. 65. chebel rc, santos jep, cerri rla, et al: effect of synchronization with gnrh on day 21 after artificial insemination on pregnancy rate and pregnancy loss in lactating dairy cows. theriogenology 2003;60:1389-1399. 66. chebel rc, santos jep, reynolds jp, et al: factors affecting conception rate after artificial insemination and pregnancy loss in lactating dairy cows. anim reprod sci 2004;84:239-255. 67. chebel rc, santos jep, cerri rla, et al: reproduction in dairy cows following progesterone insert presynhronization and resynchronization protocols. j dairy sci 2006;89:4205-4219. 68. chebel rc, santos jep: effect of inseminating cows in estrus following a presynchronization protocol on reproductive and lactation performance. j dairy sci 2010;93:4632-4643. 69. fricke pm, guenther jn, wiltbank mc: efficacy of decreasing the dose of gnrh in a protocol for synchronization of ovulation and timed ai in lactating dairy cows. theriogeology 1998;50:1275-1284. 70. fricke pm, giordano jo, valenza a, et al: reproductive performance of lactating dairy cows managed for first service using timed artificial insemination with or without detection of estrus using an activity-monitoring system. j. dairy sci 2014;97:2771-2781. 71. galvāo kh, sá filho mf, santos jep: reducing the interval from presynchronization to initiation of timed artificial insemination improves fertility in dairy cows. j dairy sci 2007;90:4212-4218. 72. gümen a, guenther jn, wiltbank mc: follicular size and response to ovsynch versus detection of estrus in anovular and ovular lactating dairy cows. j dairy sci 2003;86:3184-3194. 73. labèrnia j, lόpez-gatius f, santolaria p, et al: influence of management factors on pregnancy attrition in dairy cattle. theriogenology 1996;45:1247-1253. 74. lopez h, kanitz fd, moreira vr, et al: reproductive performance of dairy cows fed two concentrations of phosphorus. j dairy sci 2004;87:146-157. 75. lόpez-gatius f, santolaria p, yániz j, et al: factors affecting pregnancy loss from gestation day 38 to 90 in lactating dairy cows from a single herd. theriogenology 2002;57:1251-1261. 76. lόpez-gatius f, yániz j, madriles-helm d: effects of body condition score and score change on the reproductive performance of dairy cows: a meta-analysis. theriogenology 2013;59:801-812. 77. moreira f, risco ca, pires mfa et al: use of bovine somatotropin in lactating dairy cows receiving timed artificial insemination. j dairy sci 2000;83:1237-1247. 78. pereira rv, caixeta ls, giordano jo, et al: reproductive performance of dairy cows resynchronized after pregnancy diagnosis at 31 (+/3 days) after artificial insemination (ai) compared with resynchronization at 31 (+/3 days) after ai with pregnancy diagnosis at 38 (+/3 days) after ai. j dairy sci 2013;96:7630-7639. 79. pereira rv, wiltbank mc, vaconcelos jlm: expression of estrus improves fertility and decreases pregnancy losses in lactating dairy cows that receive artificial insemination or embryo transfer. j dairy sci 2016;99:2237-2247. 80. santos jep, thatcher ww, chebel rc, et al: the effect of embryonic death rates in cattle on the efficacy of estrus synchronization programs. anim reprod sci 2004;82-83:513-535. clinical theriogenology • volume 12 number 3 • september 2020 307 81. szenci o, beckers jf, humblot p, et al: comparison of ultrasonography, bovine pregnancy-specific protein b, and bovine pregnancy-associated glycoprotein 1 tests for pregnancy detection in dairy cows. theriogenology 1998:50:77-88. 82. shrestha hk, nakao t, suzuki t, et al: relationship between body condition score, body weight, and some nutritional parameters in plasma and resumption of ovarian cyclicity postpartum during pre-service period in high-producing dairy cows in a subtropical region in japan. theriogenology 2005;64:855-856. 83. toledo mz, baez gm, garcia-guerra a, et al: effect of feeding rumen-protected methionine on productive and reproductive performance of dairy cows. plos one 2017;12:e0189117. 84. vasconcelos jlm, silcox rw, rosa gjm, et al: synchronization rate, size of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows. theriogenology 1999;52:1067-1078. 85. santos, jep, bisnotto rs, ribeiro es, et al: applying nutrition and physiology to improve reproduction in dairy cattle. reprod fertil suppl 2010;67:387-403. 86. drackley jk, dann hm, douglas, gn, et al: physiological and pathological adaptations in dairy cows that may increase susceptibility to periparturient diseases and disorders. ital j anim sci 2005,4:323-344. 87. overton tr, waldron mr: nutritional management of transition dairy cows: strategies to optimize metabolic health. j dairy sci 2004;87(e suppl):e105-119. clinical theriogenology • volume 12 number 3 • september 2020308 020_ms-020 ferguson 1 introduction semen collection and cryopreservation provide an excellent opportunity for small ruminant producers via artificial insemination with frozen semen. laparoscopic artificial insemination (lai) with fresh semen yields the best results with a high-quality frozen semen. there are multiple variables surrounding the collection that can affect the quantity and quality of semen doses. furthermore, extending and freezing semen can affect the postthaw quality of semen. not all frozen semen is created equally. using high quality semen for lai is critical for obtaining acceptable or better conception rates. semen collection semen is collected from bucks and rams either via an artificial vagina (av) during their mount on a female in behavioral estrus or via electroejaculation (ee). although semen collected using an av is preferred, semen collected via ee provides acceptable postthaw semen quality. generally, ee collection provides a higher volume of semen; however, it has lower concentration of sperm because of additional seminal plasma in the ejaculate. additional seminal plasma reduces resistance to cold shock and decreases postthaw sperm survival. furthermore, ee collection has a risk of urine contamination. additionally, ee also increases the chance of having cellular debris and dirt from the prepuce in the final extended ejaculate. impact of season generally, semen collection during breeding season produces the highest volume of quality semen for either fresh lai or cryopreservation for later use. on average, depending on season, a buck or ram produces an ejaculate ranging from 0.5 to 1.5 ml, semen that is collected during the breeding seacryopreservation of small ruminant semen made simple rachael gately department of environmental and population health, cummings school of veterinary medicine tufts university, north grafton, ma abstract successful cryopreservation of small ruminant semen is critical to prosperous artificial insemination programs. semen collection with the intent to cryopreserve is achieved by either via the use of an artificial vagina or an electroejaculator. semen collected during the natural breeding season most often has the best prefreezing and postthawing viability. ejaculates from rams and bucks can be preserved using commercially available optixcell ii extender and freezing via ‘floatation over liquid nitrogen technique‘ for a short interval. keywords: semen, cryopreservation, small ruminant, artificial insemination son will provide a higher volume of slightly less concentrated semen. it is not uncommon, in breeding season, to obtain 0.8 2.0 ml of semen from a mature male small ruminant with a concentration of 2 6 x 109 sperm/ml. semen collected outside of breeding season typically yields 0.4 0.6 ml with a concentration of 5 9 x 109/ml. furthermore, collection during summer months has heat stress effect on male fertility. collection during winter months adds a substantial degree of difficulty to ensure that cold shock is not encountered. semen contact with temperatures lower than 37°c can substantially lower fertility. evaluation of semen quality there is no single measure that can effectively evaluate buck or ram semen quality. considering a combination of measures provides the best assessment of semen fertility. semen volume, concentration, progressive motility, and morphology are all values that can help determine a buck or ram’s fertility after a fresh collection. furthermore, these affect the quantity of straws that can be frozen and have a direct effect on postthaw quality. it is imperative to thaw a straw of semen after the freezing process to evaluate at least the survivability (+/motility) and concentration of the semen in that straw. teaching clients to request this information when purchasing frozen semen can be helpful. immediately after collection, the ejaculate is placed into a digital dry block heater at 34°c. a 50 µl aliquot of semen is diluted with 450 µl of optixcell ii (imv technologies usa, maple grove, mn) extender at 34°c to a 1:10 dilution factor. a 7.5 µl drop of diluted semen is placed onto the disposable base sampling chip of the isperm semen analyzer (aidmics biotechnology co., ltd, taipei city 10647, taiwan) with a pipette. a cover chip is placed over the base chip before the sample collector is attached to the optical lens module on the ipad. initial sperm total motility percentage and concentration (109 sperm per ml) of the sample is obtained with a 4-view analysis on the isperm device. extension and freezing a liposome-based extender optixcell ii (imv technologies, maple grove, minnesota, usa) is preferred. extenders are freshly prepared on each collection day. the stock solution of extender is stored in a 4°c refrigerator. optixcell ii is prepared by diluting 15 ml of optixcell ii extender with 30 ml of art distilled water for reproductive techniques. the prepared extenders are left to equilibrate in a 34°c digital dry block warmer for 20 minutes before use. the amount of extender required is calculated to reach a final dilution of 200 x 106 sperm/ml, ~ 100 x 106 sperm per 0.5 ml straw. optixcell ii, held at 34°c in a digital dry block warmer, is slowly incorporated into the ejaculate in a dropwise manner in order to avoid osmotic shocks. the conical tube with extended semen is gently rocked until thoroughly mixed. conical tubes of extended semen are taken out of the 34°c digital dry block warmer and kept for 20 minutes at ambient room temperature. during this equilibration, the semen is loaded into 0.5 ml french straws at ambient temperature. straws are sealed using a commercial ultrasonic sealer or with a pvc powder and water combination. the straws are then placed onto a metal semen straw rack in a 4°c refrigerator to equilibrate. after 4 hours has elapsed, the straws are frozen. the freezing process involves floating the samples on a metal and styrofoam rack 7 cm above the surface of liquid nitrogen in the vapors for 10 minutes before plunging into the liquid nitrogen for storage. straws are then placed into 10 or 20 straw canes under liquid nitrogen before transferred to a cannister in a liquid nitrogen tank for storage. postthaw evaluation semen straws are removed from liquid nitrogen storage tank and placed in a 37°c water bath for 1 minute and then dried with a paper towel. straws are cut with scissors and samples are emptied in glass vials kept on 37°c digital dry block warmer and swirled for 1 minute. a 7.5 µl sample is loaded with a pipette to the isperm base chip and analyzed for total motility and concentration. conflict of interest none to declare. meeting the demand for laparoscopic artificial insemination in sheep and goats rachael gately department of environmental and population health, cummings school of veterinary medicine tufts university, north grafton, ma abstract demand for laparoscopic artificial insemination in small ruminants is growing exponentially. use of a well-designed estrus synchronization protocol allows does and ewes to be inseminated in a timely manner with good conception rates. females are sedated with an intramuscular cocktail to facilitate inversion and laparoscopic access to their uterus for deposition of fresh or frozen semen directly into the lumen of both uterine horns. keywords: laparoscopy, artificial insemination, small ruminants introduction use of assisted reproductive technologies (art) is becoming more popular within small ruminant industries. requests for laparoscopic artificial insemination (lai) services increase each year as producers look for ways to advance genetics in a quick and cost-effective manner. availability of global importation of semen from bucks and rams to the united states has further driven the demand for this surgical procedure. laparoscopic artificial insemination allows semen to be deposited intrauterine in these 2 species with difficult to pass or impassable cervixes. the thought of performing laparoscopic surgery in small ruminants can be daunting to the veterinarian. with a basic understanding of synchronizing estrus and a review of laparoscopic equipment and procedure, a general practitioner should be able to competently add laparoscopic artificial insemination to their list of service offerings. estrus synchronization a standard small ruminant controlled internal drug release (cidr) synchronization protocol is utilized. does and ewes have a cidr placed on day 0. a dose of prostaglandin is given on day 11. the cidr is removed on day 12 and a dose of pg600® (intervet/merck animal health, madison, nj) is given intramuscularly. food and water are removed mid-day on day 13 and a teaser ram or buck is turned in with females. laparoscopic artificial insemination is performed on day 14 at 48 54 hours after cidr removal. sedation does and ewes are sedated prior to lai using a combination of ketamine, xylazine, and butorphanol. ketamine (1 mg/kg) is combined with 20 mg/ml xylazine (0.05 mg/kg) and butorphanol (0.025 mg/kg) to make a stock solution for the lai procedure. ewes are given intramuscularly 0.02 ml/kg whereas does respond variably to an intramuscular dose of 0.01 0.02 ml/kg. after 10 minutes, the sedated females are ready to be stood alongside an artificial insemination table and rolled into dorsal recumbency. patient preparation once in dorsal recumbency on the artificial insemination table, the front limbs of the female are hooked under the corresponding leg brackets at pastern joint level. hind limbs are secured to their corresponding leg brackets using the attached rope and ratcheted down. female’s eyes are covered using a towel and halter combination or a custom-designed blindfold. a 30 x 30 cm area cranial to the udder is clipped using commercial sheep shears followed by a surgical blade. the area subsequently undergoes a surgical scrub using alternating chlorhexidine scrub-soaked gauze and sterile saline-soaked gauze. artificial insemination procedure artificial insemination table is inverted. two stab incisions (3 5 cm) are made through the skin ~ 12 cm cranial to the udder on either side of midline. a teat cannula or veress needle is inserted bluntly through 1 stab incision, puncturing into the abdominal cavity. tubing leading from a carbon dioxide insufflator is connected and the abdomen is insufflated at 5 7 liters per minute. teat cannula or veress needle is then removed and immediately replaced with a trocar. a laparoscope is introduced via the trocar and the uterus is visualized. semen is requested to be prepared while the second trocar is placed through the second skin stab incision. the prepared artificial insemination gun is inserted through the second trocar by an assistant and handed off to the technician performing the insemination. needle of the artificial insemination sheath is aligned perpendicular to the base of 1 uterine horn. a quick stab is made, presumably penetrating the uterine lumen. assistant plunges half of the semen dose. gun is then removed from that uterine horn and moved to the opposite horn. similar stab technique is used to insert the needle into the second uterine horn, and the remaining semen in the straw is deposited. insemination gun is removed, trocar stopcocks are opened, and excess carbon dioxide is drained from the abdomen. then trocars are removed and 1 staple is placed in the skin over each incision. blindfold is removed, and the female is rolled off the table. ewes readily walk away whereas goats may lie sedated for up to 45 minutes. conflict of interest none to declare. preservat diagnosis and management of cystic endometrial hyperplasia in a potbellied pig diagnosis and management of cystic endometrial hyperplasia in a potbellied pig julie strachota,a carrie jacobs,a dalen agnew,b jennifer robertsa adepartment of large animal clinical sciences bdepartment of pathobiology and diagnostic investigation college of veterinary medicine, michigan state university, lansing, mi cystic endometrial hyperplasia (ceh) has been reported in many species, including humans. endometrial cysts arise from the glandular epithelium. as the condition progresses, endometrial polyps develop from larger cysts. depending on the species, ceh leads to infertility and predisposes females to a variety of uterine conditions including neoplasia and pyometra. in september 2018, an 8 year old intact, nulliparous female potbellied pig was presented for evaluation of tissue prolapsing from her vulva. the owner noticed prolapsed tissue 30 hours prior to her presentation and reported the pig was in estrus. an irregularly shaped, dark red tissue (~ 8 cm long) with a black perimeter protruded from her vulva. under general anesthesia, digital palpation determined that the tissue was prolapsed through her cervix. the tissue was ligated, amputated, and submitted for histopathology. results confirmed that the tissue was uterine in origin and supported the diagnosis of ceh, although neoplasia could not be ruled out. ovariohysterectomy was recommended. in october 2018, a routine ovariohysterectomy was performed under general anesthesia. the uterine horns were significantly enlarged with engorged vasculature. a 6 cm stalk of tissue was noted on the serosa of the lateral aspect of the left uterine horn. transection and hemostasis of the broad ligament was achieved using a vessel sealing and tissue dividing device (ligasuretm). the patient recovered uneventfully and was hospitalized for observation overnight. histopathological examination of the tissues revealed chronic, diffuse cystic endometrial hyperplasia with no evidence of neoplasia. numerous edematous and necrotic polyps were noted projecting from endometrial cysts. the serosal polyp identified at surgery contained endometrium, indicating that adenomyosis had also occurred. protruding tissue excised during initial presentation was a large necrotic polyp originating from the cystic endometrium. prevalence of ceh in aged potbellied pigs has been reported to be as high as 75%, with incidence of neoplastic lesions as high as 60%. the incidence of uterine pathology in potbellied pigs is high because these animals are long lived and often nulliparous pets. therefore, to decrease the risk of developing uterine disease such as ceh and uterine neoplasia in pet pigs, ovariohysterectomy should be performed as early as possible. keywords: cystic endometrial hyperplasia, potbellied pig, reproduction, uterus 487 clinical theriogenology • volume 11, number 3 • september 2019 488clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2018: comparison between the 5-day cosynch and 7-day estradiol-based protocols for synchronization of ovulation and timed artificial insemination in beef suckled cows comparison between the 5-day cosynch and 7-day estradiol-based protocols for synchronization of ovulation and timed artificial insemination in beef suckled cows j.a. bartolome,a l.o. zapata,a h. romero harry,b m.g. bilbao,a s. perez wallace,c m.f. farcey,a l. gelid,b m.s. ferrerd afacultad de ciencias veterinarias, universidad nacional de la pampa, argentina, beea inta anguil, anguil, la pampa, argentina, czoetis srl, buenos aires, argentina, dcollege of veterinary medicine, university of georgia, athens, ga the objective was to compare pregnancy per artificial insemination (ai) and follicular dynamics in beef suckled cows using either 7-day estradiol-based protocol or the 5-day cosynch for timed artificial insemination (tai) during four breeding seasons. we hypothesized that pregnancy per ai would be higher with the 5-day cosynch protocol and, that estrous cycle status, days postpartum (dpp), fat depth and plasma progesterone would modify this effect. every year since 2014, cows with more than 30 dpp were randomly assigned into two treatment groups. cows in the 7-day estradiol-based group (n = 428) received 2.5 mg of estradiol benzoate and a 0.5 g progesterone intravaginal device (pid) on day -9, pid removal, 0.125 mg of cloprostenol and 0.5 mg of estradiol cipionate on day -2 and tai 48 h later (day 0). cows in the 5-day cosynch group (n = 428) received 100 μg of gnrh and a 0.5 g pid on day 8, pid removal and 0.125 mg of cloprostenol on day -3, a second dose of cloprostenol on day -2, and 100 μg of gnrh and tai on day 0. on day -9, estrous cycle status (cyclic or anestrus) based on clinical signs at per rectum palpation and ultrasonography of the genital tract was recorded. in a subset of cows (n = 79) the size of dominant follicle was determined between day 2 and day 0 using transrectal ultrasonography of the ovaries. in a subset of cows (n = 340), dpp, fat depth (mm) and plasma progesterone concentration (ng/ml) were obtained on day – 9. pregnancy per ai was determined by per rectum ultrasonography of the uterus on day 30 after tai. pregnancy per ai was higher for cows in the 5-day group than for cows in the 7-day group (50.9 % vs. 41.3 %, p = 0.01) and was also higher for cyclic cows than for cows in anestrus (54.3 % vs. 33.2 %, p < 0.0001). there was also a significant effect of breeding season (p = 0.0002), sire (p = 0.03) and an interaction between treatment group by breeding season (p = 0.03). the dominant follicle was larger for the cows in the 5-day group (10.1 ± 0.19 mm) than in the 7-day group (8.9 ± 0.18 mm, p = 0.01), and there was also an effect of day (p < 0.0001). in the subset of cows were dpp, fat depth and plasma progesterone concentration were obtained on day – 9, pregnancy per ai was higher for cows in the 5-day group than in the 7-day group (44.0 % vs. 30.8 %, p = 0.006), higher for cows with ≥ 55 dpp (47.0 % vs. 29.6 %, p = 0.001), higher for cows with fat depth ≥ 0.50 mm (44.7 % vs. 29.7 %) and higher for cows with high (≥ 1 ng/ml) plasma progesterone concentration (47.2 % vs. 28.7 %, p = 0.01). there was also an interaction between breeding season and treatment group (p = 0.002) and a tendency for interaction between treatment group and plasma progesterone concentration (p = 0.07). in cows with high plasma progesterone concentration, pregnancy per ai was higher for cows in the 5-day group (60.5 %) compared to cows in the 7-day group (34.9 %), but there was no difference in cows with low plasma progesterone. in conclusion, pregnancy per ai was higher for cows in the 5-day group. the increase in pregnancy per ai with the 5-day protocol was greater when plasma progesterone concentration at initiation of the treatment was higher than 1 ng/ml. keywords: beef suckled cows, artificial insemination, synchronization, ovulation, pregnancy clinical theriogenology • volume 10, number 3 • september 2018 308 2018: new and recurrent hemorrhagic anovulatory follicles in tropical jennies new and recurrent hemorrhagic anovulatory follicles in tropical jennies e. hughes, r. ambrosia, m. landers, r.o. gilbert, h. french ross university school of veterinary medicine, st. kitts, west indies a group of eight mature, cycling jennies were examined regularly via transrectal ultrasonography during various trials characterizing reproductive behavior in donkeys in a tropical environment. this communication describes hemorrhagic anovulatory follicles (haf) in two jennies. in april, the first of these donkeys developed a 42.75-mm diameter presumptive preovulatory follicle, which developed an internal structure typical of a haf. this ovarian structure remained ultrasonographically evident for 37 days and grew to a diameter of 68.6-mm before beginning its regression. the interovulatory period of the cycle characterized by the haf (19 days) was shorter than the preceding cycle (25 days) and two subsequent cycles (23 days each) that followed during this study. this jenny continued to return to normal behavioral estrus each cycle. the progesterone concentrations measured on mondays, wednesdays, and fridays during the cycle dominated by the haf were not different from those of the presumably normal ovulatory cycles of this jenny. six months later, this same donkey again produced a presumptive haf of 37.6-mm in diameter. on this occasion during which the haf persisted, the interovulatory interval was lengthened to 42 days. a progesterone profile is not available for this cycle. another jenny with no previous history of haf development, despite monitoring of many cycles, developed a presumptive haf in february on day 22 after her previous ovulation. this structure reached a diameter of 46.4-mm. this jenny had received 75μg of cloprostenol (0.3 ml estrumate™) on day 7 of the cycle. the haf persisted until the time of writing on day 29 after ovulation. the average interovulatory interval for preceding cycles in this donkey was 24 days. again, no comparative progesterone profile is available for this case. these observations confirm that hafs may occur and recur in donkeys.this may be a spontaneous occurrence without any treatment intervention, as in the first jenny described, or may follow administration of prostaglandin, which has previously been described as a risk factor in mares. including these and other donkeys monitored for several different experiments, this group has followed approximately 210 cycles in 25 donkeys over a period of 2 years without encountering other cases, suggesting that occurrence of hafs in tropical jennies is rare. keywords: anovulation, follicle, prostaglandin, donkeys clinical theriogenology • volume 10, number 3 • september 2018345 2018: 64,xy (sry-negative) phenotypic mare 64,xy (sry-negative) phenotypic mare p.m. mccue, r.a. ferris, j.a. storme department of clinical sciences, colorado state university, fort collins, co a 7-year-old american quarter horse mare was presented for reproductive evaluation. the owner’s goal was to have the mare carry her own foal to term, but the mare had never been observed to show behavioral estrus and had not received any medications. physical examination revealed a phenotypically normal mare with a normal vulva and clitoris and a small mammary gland consistent with that of a maiden mare. transrectal ultrasonographic examination showed very small gonads adjacent to the tip of each uterine horn. the gonadal structures were 1.5 cm in length and devoid of any visible follicles. speculum examination revealed a normal vaginal vault and a normal external cervical os. videoendoscopic examination revealed a normal uterine body, two uterine horns and a utero-tubular junction at the tip of each horn. hormone analysis indicated low concentrations of anti-müllerian hormone (amh) (0.01 ng/ml; normal range 0.1 to 3.8 ng/ml), inhibin (0.21 ng/ml; normal range 0.1 to 0.7 ng/ml), testosterone (27.6 pg/ml; normal range 20 to 45 pg/ml) and progesterone (0.12 ng/ml). a blood sample was subsequently submitted for chromosomal analysis, which indicated that the mare had a 64, xy karyotype. further analysis of the y chromosome revealed that the sex-determining region of the y chromosome (sry) was absent. specifically, pcr test results indicated that the mare was negative for all male-specific y chromosome markers. fluorescent in situ hybridization (fish) test results confirmed that the small chromosome fragment was a portion of the heterochromatic part of the y chromosome. the entire male specific region of y (msy) and the pseudoautosomal region on the y were absent. the gonads were subsequently removed by standing laparoscopic surgery. histologically the gonads were devoid of any normal ovarian or testicular structures. development of a male or female reproductive tract is normally dictated by the genetic sex of the spermatozoon that fertilizes the oocyte. if the sperm carries a normal y chromosome, the horse should have a 64, xy karyotype. the presence of an sry region on the y chromosome should drive differentiation of the gonads toward testicular development. production of amh by sertoli cells and testosterone by leydig cells of a normal testis would suppress development of the müllerian duct and stimulate development of the wolffian duct, respectively. conversion of testosterone to dihydrotestosterone (dht) by the enzyme 5α-reductase in peripheral tissues should lead to formation of a penis and scrotum. however, in the present case, absence of an sry region on the y chromosome lead to failure of gonadal development and subsequent low concentrations of the hormones amh, testosterone and dht led to formation of female internal and external genitalia. a chromosomal abnormality should be suspected in adult mares with bilaterally small, inactive ovaries during the physiologic breeding season that have not been treated with exogenous hormones or vaccinated against gonadotropin releasing hormone. keywords: equine, genetic infertility, sex-reversal syndrome, chromosomal abnormality clinical theriogenology • volume 10, number 3 • september 2018305 2018: occurrence of proximal droplets in performance-tested beef bulls occurrence of proximal droplets in performance-tested beef bulls z.j. bartenslager, l. strickland, f.d. kirkpatrick, l. schneider, a.m. saxton, j.l. edwards, k.g. pohler, j.k. smith, f.n. schrick department of animal science, university of tennessee, knoxville, tn the presence of spermatozoa proximal droplets in performance-tested bulls during breeding soundness examinations causes concern regarding future fertility. a retrospective analysis was performed to evaluate various parameters related to breeding soundness examination (bse) classification and occurrence of proximal droplets in bulls. results of bse from 755 bulls (12 – 16 months of age) that represented 8 breeds collected over a 4-year period at two 84-day performance testing facilities were included in the following analysis regarding proximal droplets and fertility. other facility-specific criteria for eligibility included average daily gain, adjusted 365-day weight, frame score (≥ 5.0), and minimum scrotal circumference (≥ 31 cm). bulls were then either classified as satisfactory (successfully passed all aspects of the bse as set forth by the society for theriogenology) or unsatisfactory (unsuccessfully completed the semen portion of the bse). data observations included body weight and scrotal circumference measurements collected at onand off-test dates, along with evaluations of spermatozoa motility and morphology conducted during the first bse. descriptive statistics were performed using the frequency procedure of sas 9.4 and pivot tables in microsoft excel. separate linear mixed models were used to determine if morphological defect-deferral and proximal droplet deferral were associated with scrotal circumference on-test and off-test, age, on-test and off-test weight, average daily gain and scrotal circumference growth. logistic regression models did not identify an impact of location, breeds, or year. the incidence of proximal droplets accounted for 80.95 % (170/210) of primary defects. on-test scrotal circumference was greater in bulls that passed than those that were deferred for overall sperm morphology (32.94  0.20 cm vs. 31.98  0.32 cm, respectively; p = 0.01) and the incidence of proximal droplets (32.9 ± 0.18 cm vs. 31.4 ± 0.44 cm, respectively; p = 0.001). however, off-test scrotal circumference did not differ between bulls that passed or were deferred (37.3 ± 0.12 cm vs. 37.3 ± 0.22 cm, respectively), and was not related to sperm morphology (p = 0.89) or the incidence of proximal droplets (p = 0.31). scrotal circumference growth was greater for bulls that were unsatisfactory when compared to bulls that were satisfactory (0.070 ± 0.003 vs. 0.060 ± 0.002 cm/day, respectively; morphology (p = 0.0009), proximal droplets (p = 0.002)). furthermore, age was inversely related to deferrals on morphology (p = 0.0005) and proximal droplets (p < 0.0001), with younger bulls exhibiting increased morphological issues. on-test body weight was increased in bulls with morphological failure (p = 0.04), but had no difference in relation to proximal droplet failure (p = 0.35). off-test weight was not affected by classification status on either sperm morphology (p = 0.10) or occurrence of proximal droplets (p = 0.54). average daily gain of the bulls during the 84-day test had no impact on bse classification related to sperm morphology and occurrence of proximal droplets. in summary, bull age and scrotal circumference growth during the test period were important factors associated with unsatisfactory classification due to sperm morphology and occurrence of proximal droplets. occurrence of proximal droplets has been previously associated with bull maturity; however, the cause of its relationship to rapid scrotal circumference growth in performance-tested bulls remains unclear. keywords: proximal droplet, bulls, fertility, scrotal circumference, breeding soundness examination clinical theriogenology • volume 10, number 3 • september 2018 344 2018: pregnancy loss following twin ablation on a thoroughbred breeding farm in beijing pregnancy loss following twin ablation on a thoroughbred breeding farm in beijing allan gunn,ab elizabeth jones,ab victoria brookesabc acharles sturt university, school of animal and veterinary sciences, nsw, australia; bthe graham centre for agricultural innovation, pugsley place, nsw, australia; csydney school of veterinary science, the university of sydney, camden, nsw, australia introduction the occurrence of twins is recognised as one of the most important and preventable causes of reproductive wastage in thoroughbred horses. early manual reduction (ablation) to a single vesicle under ultrasonographic guidance is the accepted method to prevent the occurrence of twin fetuses in equines. evidence to support peri-ablation treatment of mares with nsaids and progestagens is lacking. the objective of this study was to assess the pregnancy outcome of thoroughbred mares with twin conceptuses following manual reduction without any peri-ablation pharmaceuticals. we hypothesised that a higher rate of pregnancy loss would occur in this group of mares. materials and methods the study took place in beijing, china with the first day of the northern hemisphere breeding season designated as 14 february 2001. mares were bred according to accepted thoroughbred breeding practices, with per rectum ultrasonographic pregnancy diagnosis carried out at 14—19 (epd), 21—30 (mpd), and 40—65 (fpd) days after service. all mares that were pregnant at epd and had completed pregnancy diagnoses at mpd and fpd (as required) prior to 03 july 2001 were included in the study. twin pregnancies were detected at epd, and typically the largest of the two vesicles was manually ablated. no pharmacological treatment was available for administration to any mare. a univariable logistic regression model in which the outcome was maintenance or loss of pregnancy following epd and the determinant was ablation of a twin conceptus. results the total number of mares eligible for the study was 361 mares. only 4 mares were pregnant more than once (twice each); therefore, clustering was not accounted for in the analysis. the proportion of mares with twin conceptuses was 12.1 %. of the 44 mares that underwent twin ablation, 4 lost pregnancies post-epd (6.8 %). of the 321 mares with single conceptuses diagnosed at epd, 31 lost pregnancies post-epd (9.7 %). there was no significant difference (or =1.46, 95 % ci 0.48— 6.42, p = 0.55) between these two groups. discussion the twinning rate in this population is consistent with that reported in other studies, as are the rates of pregnancy losses post-epd to fpd with and without ablation of a conceptus despite the lack of peri-ablation pharmaceutical administration. conclusion this study does not support the hypothesis that the manual ablation of a twin conceptus in mares without nsaid and progestagen treatment increases pregnancy loss between epd and fpd. keywords: mare, twin ablation, pregnancy loss, progestagens clinical theriogenology • volume 10, number 3 • september 2018335 2018: the effects of estrous cycle and pregnancy on echocardiographic variables in healthy bitches the effects of estrous cycle and pregnancy on echocardiographic variables in healthy bitches swanand r. sathe, jessica l. ward department of veterinary clinical sciences, college of veterinary medicine, iowa state university, ames, ia pregnancy causes alterations in maternal hemodynamics, including increased stroke volume, cardiac output, and heart rate. associated echocardiographic changes are well-described in women, but have been less consistently documented in bitches. furthermore, it remains unknown whether the estrous cycle itself affects echocardiographic measurements in bitches, and whether hemodynamic changes in canine pregnancy vary with litter size. the objectives of this study were (1) to characterize changes in maternal echocardiographic variables throughout the reproductive cycle in bitches; and (2) to determine whether magnitude of changes in pregnancy are related to fetal number. nine healthy client-owned breeding bitches were enrolled prospectively and followed longitudinally throughout a single reproductive cycle. physical examination, echocardiography (including left ventricular strain and 3-dimensional imaging), blood pressure, and plasma volume calculations were performed at 4 time points as determined by serum progesterone estimation and breeding dates: (1) proestrus (day 3-5 of bleeding), (2) early pregnancy (day 25-30 after breeding), (3) late pregnancy (day 45-60 after breeding), and (4) anestrus (30 days after weaning). fetal echocardiography was also performed during late pregnancy visits. data were compared across time points using paired t-tests. mean estimated plasma volume increased by 36% in early pregnancy (p = 0.009) and 62% in late pregnancy (p = 0.0009). no significant changes were observed at any time point in heart rate, blood pressure, echocardiographic measurements of left ventricular size or wall thickness, or echocardiographic calculations of stroke volume or cardiac output. fetal echocardiography was feasible in a subset of fetuses for each bitch. there was a significant correlation between estimated total fetal cardiac output and maternal cardiac output in late pregnancy (r = 0.86). the incidence of physiologic heart murmurs ranged from 5/9 bitches in proestrus to 1/9 in late pregnancy, attributed to variations in aortic outflow velocity. this study demonstrated that hemodynamic alterations in pregnant bitches do not result in consistently detectable echocardiographic changes, suggesting that cardiac screening could be diagnostic at any time during a bitch’s reproductive cycle. larger litters may be associated with higher maternal hemodynamic demand during pregnancy. physiologic heart murmurs were common in this study population but were not obviously associated with reproductive cycle. keywords: echocardiography, cardiac output, heart murmur, fetus, cardiac output clinical theriogenology • volume 10, number 3 • september 2018 322 2011: impacts of metabolism and nutrition during the transition period on fertility of dairy cows impacts of metabolism and nutrition during the transition period on fertility of dairy cows j.e.p. santos, r.s. bisinotto, e.s. ribeiro, f.s. lima, w.w. thatcher department of animal sciences; university of florida, gainesville, fl abstract in early postpartum, high-producing dairy cows undergo a period of extensive tissue catabolism because of negative nutrient balance. metabolic imbalances can lead to diseases, which are known to depress fertility in dairy cows. negative nutrient balance has been associated with compromised immune and reproductive functions in dairy cows, and a compromised immune system leads to greater risk of diseases. low circulating concentrations of glucose and insulin associated with elevated concentrations of non-esterified fatty acids and ketone bodies postpartum have detrimental effects on the oocyte, granulosa and immune cells, disrupting their metabolism and reducing viability. therefore, minimizing the extent and duration of negative nutrient balance in early lactation is expected to improve fertility. reductions in circulating concentrations of ca around parturition are also linked with impaired immune competence and have recently been linked with uterine diseases in dairy cows. manipulation of the diet to influence metabolic health might benefit fertility. dietary additives that influence rumen or intermediary metabolism to favor postpartum health, and supplementation with specific fatty acids during early lactation and the breeding period are potential alternatives to offset dietary insufficiencies and reestablish metabolic health in early lactation. keywords: dairy cow, nutrition, infertility, transition management introduction dairy cows must become pregnant and deliver a calf in order undergo proper mammary development and lactogenesis. reproduction is also critical to production because it determines when primiparous cows become multiparous leading to increments in milk yield, alters the average milk yield per day of calving, affects the number of replacement animals available and the risk of culling, and influences the rate of genetic progress. it is well described that poor health during the transition period suppresses fertility in dairy cows. perhaps the most debated aspect is the association between negative nutrient balance in early lactation and subsequent resumption of ovulatory cycles, and establishment and maintenance of pregnancy in high-producing dairy cows. during periods of negative energy balance (neb), extensive lipolysis and products from fat metabolism seem to influence oocyte competence and subsequent embryo development. in addition, impaired metabolic health often leads to immunosuppression and the occurrence of diseases that further reduce fertility. managing cows in the transition period in a proactive manner to minimize nutrient imbalances is mandatory for proper health, but it often benefits reproduction. clinical and subclinical diseases reduce fertility of dairy cows the transition from the nonlactating pregnant state to nonpregnant lactating requires the highproducing dairy cow to drastically adjust its metabolism so that nutrients can be partitioned to support milk synthesis, a process referred to as homeorrhesis. a sharp increase in nutrient requirements generally occurs when feed intake still is depressed in early lactation, which causes extensive mobilization of body tissues, particularly body fat, but also amino acids, minerals and vitamins. despite tight homeostatic controls and homeorrethic adjustments to cope with the changes in metabolism caused by milk production, 45 to 60% of dairy cows across different levels of milk production, breeds and management systems, develop metabolic and infectious diseases in the first months of lactation.1,2 calving related disorders and diseases that affect the reproductive tract are major contributors to depression of fertility. dystocia, metritis, and clinical endometritis were observed in 14.6, 16.1, and 20.8%, respectively, of postpartum dairy cows in large us confinement herds.2 cows that presented at least one of the aforementioned disorders were 50 to 63% less likely to resume ovarian cyclicity by the end of the voluntary waiting period, and 25 to 38% less likely to become pregnant following the first clinical theriogenology • volume 3 number 4 • december 2011579 artificial insemination (ai) postpartum compared with healthy cows. moreover, cows with dystocia and those diagnosed with clinical endometritis were 67 and 55% more likely to lose their pregnancies during the first 60 days of gestation than healthy cows. the negative effects of reproductive disorders on subsequent fertility are also observed in dairy cows kept under grazing systems.1 even though the prevalence of dystocia, metritis, and clinical endometritis are numerically less in grazing-based herds (8.2, 5.7, and 14.7%, respectively), cows with metritis had 2.7-fold increased odds of being anovular at 50 days postpartum compared with unaffected herdmates. cows affected with uterine diseases had marked depression in pregnancy per ai at the first postpartum ai and increased risk of pregnancy loss. in fact, when diseases were classified as clinical (calving problem, metritis, clinical endometritis, mastitis, pneumonia, digestive problems, lameness), subclinical or both (subclinical hypocalcemia, subclinical ketosis, and severe neb based on excessive plasma nonesterified fatty acids [nefa]), or both, affected cows had increased anovulation, reduced pregnancy per ai, and increased pregnancy loss (table 1).1 these data strongly suggest that diseases in early lactation have a profound impact on fertility of dairy cows and maintaining metabolic health to minimize the risk of clinical and subclinical health problems are expected to benefit fertility of dairy cows. nutrient balance and reproduction in dairy cows energy balance in early lactation has been positively associated with reproductive performance of dairy cows.3 the severity and length of neb can be estimated through changes in body condition score (bcs). cows that lost more body condition during the first 65 days postpartum were more likely to be anovular at the end of the voluntary waiting period, had decreased pregnancy per ai, and increased risk of pregnancy loss after the first ai postpartum.4 using circulating concentration of nefa as an indicator of the energetic status of grazing dairy cows in the first two weeks postpartum, ribeiro et al showed that cows under neb (nefa ≥ 0.7 mm) were less likely to resume ovarian cyclicity before 50 days postpartum and to become pregnant to the first ai of the breeding season.1 others have reported similar results in dairy herds managed under confinement. the rate of pregnancy in the first 70 days of breeding was 16% less for cows with blood nefa ≥ 0.7 mm than for those with concentrations below this threshold in early lactation.5 ketosis resulting from extensive fat mobilization has also been associated with compromised fertility. both the relative circulating concentration of β-oh-butyrate (bhba) and the duration of elevated bhba concentrations were negatively associated with the probability of pregnancy following the first postpartum ai.6 in fact, for every 100 µm increase in bhba concentration on weeks one and three after calving, the proportion of pregnant cows was reduced by 2 and 3%, respectively. furthermore, the rate of pregnancy within 70 days after the end of the voluntary waiting period was 13% smaller among cows with blood bhba ≥ 100 µm compared with herdmates with concentrations below 100 µm.5 therefore, circulating concentrations of these metabolites can be used as indicators of excessive lipid mobilization that interfere with fertility. furthermore, as the prevalence of cows with elevated concentrations of blood nefa or bhba increases, reproductive performance declines.7 in the latter study, the 21-day cycle pregnancy rate was reduced by 0.9 percentage units in herds on which more than 15% of the sampled cows had nefa ≥ 0.7 mm, and by 0.8 percentage units if more than 15% of the sampled cows had bhba ≥ 115 µm. the reduction in fertility associated with low nutrient intake and neb is, at least in part, mediated by the damaging effects on immunity and postpartum health. exposing immune cells in vitro to nefa at concentrations compatible with those observed in high-producing postpartum dairy cows (0.12 to 1 mm) has been shown to reduce function and viability. increasing the concentration of nefa in the culture media abridged the synthesis of interferon-γ and igm by peripheral blood mononuclear cells.8 furthermore, nefa reduced phagocytosis-dependent oxidative burst in polymorphonuclear leucocytes.9 when concentrations of nefa in the culture medium were further increased to 2 mm, polymorphonuclear oxidative burst was not altered, but more leukocytes underwent necrosis, thereby impairing function. not only nefa, but also bhba has been implicated with immunosuppression in postpartum dairy cows. incubation of bovine neutrophils with increasing concentrations of bhba reduced phagocytosis, extracellular trap formation, and killing.10 in vivo observations support the clinical theriogenology • volume 3 number 4 • december 2011 580 immunosuppressive effects of neb. cows under severe neb had increased nefa and bhba, which was associated with decreased leukocyte numbers.11 it is likely that cows that are unable to recover feed consumption after parturition and, therefore, remain in more severe neb, are more susceptible to diseases. it is known that reduced nutrient intake and neb even before calving are associated with poor uterine recovery from parturition and the occurrence of uterine diseases.12 these observations seem to be linked with changes in patterns of endometrium gene expression mediated by the energetic status of the cows. wathes et al evaluated global gene expression of the endometrium of cows at two weeks postpartum.11 they observed that several probes linked with inflammation and active immune response were still upregulated in cows undergoing severe neb compared with those exhibiting a more modest caloric deficit, suggesting a delay in uterine involution. in addition, cows that developed uterine diseases in early postpartum had greater concentrations of nefa and bhba around calving than healthy cows.12,13 it is important do highlight that the occurrence of diseases early postpartum can further accentuate the adverse effects of neb, as sick cows have reduced appetite and oftentimes lose more body weight than healthy cows. energy balance, ovarian function and oocyte competence energy balance to an extent determines when normal reproduction resumes after parturition in most mammals.14 in cattle, undernutrition has been linked with reduced frequency luteinizing hormone (lh) pulses by the pituitary gland,14 which is known to support follicle growth, maturation and ovulation. restricted supply of oxidizable fuels during neb limits the ability of hypothalamic neurons to sustain the gonadotropin releasing hormone (gnrh) pulse generator.15 this is thought to be mediated by glucose, which is a preferred substrate for neuron energy metabolism.15 under a favorable nutritional status, the hormonal milieu to which the hypothalamus and pituitary gland are exposed favors the release of gnrh and gonadotropins. for instance, in addition to oxidizable substrates, metabolic hormones such as insulin, insulin-like growth hormone-1 (igf-1), and leptin have increased concentrations when nutrient intake is adequate, and all of them play a role in potentiating the secretion of gnrh and gonadotropins.15 cows under neb have limited hepatic expression of growth hormone (gh) receptor 1a triggered by low circulating concentrations of insulin.16,17 this phenomenon uncouples the gh/igf-1 axis which reduces the synthesis of igf-1 by the liver. reduced concentrations of igf-1 have been associated with diminished follicle sensitivity to lh, growth and steroidogenesis.17,18 conversely, the increase in circulating concentrations of insulin as energy balance improves seems to be one of the signals to reestablish the gh receptor expression in the liver and restore igf-1 synthesis in dairy cows.16 restricting follicular growth and synthesis of estradiol delay resumption of ovulation postpartum and might compromise oocyte quality, which likely hampers estrous detection and pregnancy in dairy cows. in addition to extensive nutrient shortage, high producing dairy cows also undergo extensive ovarian steroid catabolism. this is thought to be mediated by the high dry matter intake and consequent increased splanchnic blood flow.19 hepatic blood flow doubles in the first three months postpartum averaging 1,147 l/h on the week preceding parturition and 2,437 l/h on the third month postpartum.20 the increased clearance of ovarian steroids can have important implications to the reproductive biology of dairy cows and indirectly influence follicle development,21 which can have implications to oocyte quality and subsequent embryo development. reduced circulating concentrations of estradiol because of hepatic catabolism in cows with high dry matter intake can result in shorter and less intense estrus period.22 in addition, estradiol catabolism requires follicles to grow for longer periods of time to be able to trigger estrus and ovulation.21,23 longer periods of follicular dominance reduce embryo quality24 and pregnancy per ai in cows inseminated on estrus25 or following timed ai.26 a commonly discussed hypothesis put forth by jack britt suggested that developmental competence of the oocyte and the steroidogenic capacity of the follicle in dairy cows are influenced by the environment in which the follicles developed from the primary follicle to the pre-ovulatory state. follicles from cows undergoing extensive neb would suffer metabolic and biochemical changes that would influence the microenvironment to which they are exposed to and, consequently, the competence clinical theriogenology • volume 3 number 4 • december 2011581 of their oocytes. this hypothesis has never been fully tested, although it is clear that the catabolic state in early lactation influenced follicle growth and oocyte quality. although the follicle is capable of controlling fluctuations in glucose availability, which generally results in concentrations in the follicular fluid greater than those observed in blood, intra-follicular glucose concentrations also decline around parturition.27 it has been shown that glucose is critical for adequate oocyte maturation, affecting cumulus expansion, nuclear maturation, cleavage, and subsequent blastocyst development. in fact, glucose concentrations compatible with those observed in cows suffering from clinical ketosis (1.4 mm) were shown to reduce cleavage and the proportion of embryos developing to blastocysts.26 although the oocyte does not directly use glucose as an energy source, it is has to be readily available for cumulus cells for glycolysis to provide pyruvate and lactate, oocyte’s preferred substrates for atp production.29 therefore, it is possible that hypoglycemia in early lactation might compromise oocyte competence in dairy cows. extensive fat mobilization and the release of large amounts of nefa into the bloodstream have been shown to exert a direct effect on fertility of postpartum dairy cows. concentrations of nefa in the follicular fluid parallel those of serum, and they increase around parturition.30 maturation of oocytes in vitro in the presence of saturated fatty acids reduced oocyte competence and compromised the initial development of embryos. specifically, the addition of palmitic and stearic acids to the maturation medium induced apoptosis and necrosis of cumulus cells, which was associated with impaired fertilization, cleavage, and development to the blastocyst stage.30 changes in circulating concentrations of bhba are promptly reflected in the follicular fluid.27 however, in vitro models developed to study the effects of subclinical ketosis on fertility of dairy cows have failed to demonstrate a direct effect of bhba on oocyte competence, which seems only to aggravate the responses to low concentrations of glucose during oocyte maturation.28 calcium homeostasis and uterine health during early postpartum in early lactation, synthesis and secretion of colostrum depress systemic concentration of ca, which often results in reduced availability of ionized ca (ca2+) for cellular metabolism. surveys in the us indicate that 25% of the primiparous and more than 41% of the multiparous cows are subclinically hypocalcemic (ca < 8.0 mg/dl) in the first 48 hours after calving.31 mild depressions in serum ca concentrations postpartum have been implicated with uterine health. martinez et al32 observed that cows with serum ca < 8.59 mg/dl in at least one of the first three days postpartum had reduced neutrophil phagocytic and killing activities in vitro, increased odds of developing fever (adjusted or = 3.5; 95% ci = 1.1-11.6) and metritis (adjusted or = 4.5; 95% ci = 1.3-14.9) and these associations were observed for both, cows considered to be of high or low risk of developing metritis based on calving problems. the authors concluded that the attributable risk for a cow to develop metritis because of low serum ca was 75.3%. ionized ca is an important second messenger in cellular signal transduction, and fluctuations in intracellular ca2+ concentrations are critical to activate immune cells.33 cows with milk fever have reduced intracellular stores of ca2+,34 which might explain the increased risk of infectious diseases in these cows.1 collectively, these data suggest that ca status is linked with immune cell function and play a role on the risk of uterine diseases of dairy cows. cows suffering from uterine diseases have delayed postpartum ovulation, reduced pregnancy per ai, and increased pregnancy loss.2 improving periparturient metabolism through management and nutrition cow movement and dry period length regrouping of cows induces social tensions that disturb feeding and resting patterns, and often causes a temporary depression in dry matter intake.35 the changes in behavior with movement of cows between groups have lead veterinarians and researchers to advise producers against regrouping at the imminence of calving. it is thought that regrouping would further suppress intake and increase the risk of ketosis and fatty liver. however, it is unknown if regrouping when cows are moved to the close up pen is detrimental to health and production in the subsequent lactation. researchers at the university of clinical theriogenology • volume 3 number 4 • december 2011 582 wisconsin have attempted to address this question and their findings refuted the concept that weekly addition of cows to the close up group is detrimental to postpartum metabolism and production.36 it seems that when appropriate feedbunk space and number of stalls are available, transition cows can adapt to the weekly regrouping. a strategy to improve postpartum intermediary metabolism is to manipulate the length of the dry period. reducing the dry period from 55 to 34 days increased bcs between weeks two and eight postpartum and reduced the concentrations of plasma nefa at week three postpartum,37 suggesting improved energy status postpartum. when energy balance was measured, cows subjected to a 28-day dry period experienced a less severe neb postpartum, which resulted in reduced bcs and body weight losses compared with cows having the traditional 56 days dry.38 some of the benefit to energy balance is the result of less milk production, particularly in cows starting their second lactation.37,39 the improved energy balance with short dry period likely explains the earlier first postpartum ovulation and reduction in anovular cows.40,41 despite changes in energy status and an earlier resumption of estrous cyclicity, cows with a dry period of 28 to 35 days had similar reproductive performance to those with a standard eightweek dry period.40-42 nevertheless, in observational studies, extending the exposure of cows to the prepartum diet was associated with reduced days open and increased proportion of pregnant cows at weeks six and 21 after the initiation of the breeding season.43 manipulating prepartum nutrient intake by diet formulation altering caloric intake prepartum influences postpartum metabolism in dairy cows. ad libitum nutrient intake during the entire dry period tends to increase body weight and bcs prepartum and predispose cows to increased lipid mobilization in early lactation.44 several studies have evaluated the impact of manipulating the energy density of the prepartum diet on postpartum performance. in some cases, nutrient intake was restricted not by altering the diet formulation, but by limiting the amount of feed offered. a summary of studies in which caloric intake was restricted in late gestation is depicted in table 2. on average, cows consumed 14.6 and 19.8 mcal/day for the low and high caloric intake, respectively. restricting caloric intake prepartum, although suggested to improve metabolism,44 reduced production of fat-corrected milk. the reduction in production averaged 2 kg/day. in the same data set, concentrations of bhba in early lactation were mostly unaffected and averaged 6.8 mg/dl for both, low and high caloric intake cows. nevertheless, concentrations of bhba were greater (p < 0.05) for high than low caloric intake cows in two studies,44,45 but the opposite was observed in another study in which low prepartum caloric intake resulted in greater concentrations of bhba postpartum.46 therefore, restricting caloric intake prepartum can be used to minimize lipid mobilization and triacylglycerol accumulation in the liver, but at the expense of milk production. altering the protein content of prepartum diet has little impact on performance of postpartum multiparous cows; however, increasing prepartum dietary protein from 12.7% to 14.7% of the diet dry matter with a high rumen undegradable protein source enhanced milk production in primiparous cows.46 nonetheless, protein had negligible impacts on measures of reproduction. time to resumption of ovulation postpartum, days open and pregnancy per ai were all not affected by prepartum dietary protein concentration. similarly, the incidence of diseases postpartum was not affected by prepartum dietary protein. therefore, diets for cows in the last weeks of gestation should contain between 12% (multiparous cows) and 15% (primigravid cows) crude protein to result in an estimated 1 kg/day of metabolizable protein intake.48 feeding postpartum diets to increase blood insulin a number of studies have demonstrated the importance of insulin as a signal mediating the effects of acute changes in nutrient intake on reproductive parameters in dairy cattle. feeding more dietary starch or enhancing the ruminal fermentability of starch in the diet usually results in increased plasma insulin concentrations. insulin mediates recoupling of the gh/igf-1 axis,16 which is important for follicle development and ovulation. gong et al fed cows of lowand high-genetic merit isocaloric diets that differed in the ability to induce high or low insulin concentrations in plasma.49 feeding the highclinical theriogenology • volume 3 number 4 • december 2011583 starch diet reduced the interval to first postpartum ovulation and resulted in a greater proportion of estrous cyclic cows within the first 50 days postpartum. nevertheless, this response has not been consistent.50 it is important to remember that although diets high in starch favor increases in plasma insulin, excessive amounts of readily fermentable starch have the potential to suppress dry matter intake and offset any potential benefits of dietary manipulation on ovarian function. altering metabolism by supplementing ionophores to periparturient dairy cows another method to increase blood insulin is to supplement the diets of dairy cattle with ionophores. monensin is commonly used in cattle diets to selectively increase the microbial population in the rumen that favor propionate production and n conservation. feeding monensin typically increases blood glucose and insulin and reduces nefa and bhba.51 therefore, feeding monensin improves metabolic health, and these responses have resulted in reduced incidence of ketosis, displaced abomasum, and mastitis.52 when monensin was supplemented as a controlled-release capsule, it reduced the incidence of metritis.52 surprisingly, feeding monensin to dairy cows during the transition period has not been shown to hasten resumption of ovulation postpartum, to reduce days to pregnancy, or to increase the rate of pregnancy in spite of consistent improvements in metabolic health.52,53 diet manipulation to improve hepatic lipid metabolism during periods of extensive fat mobilization, fat accumulates in the hepatic tissue. in early lactation cows with relatively low plasma nefa concentrations (0.36 mm), the liver extracted 724 g of nefa from blood over a 24 hour period.20 thus, in cows with concentrations of nefa above 1 mm, as those with extensive lipid mobilization immediately after calving, the liver might remove as much as 2 kg of nefa per day, the equivalent of 20% of its weight. most of these nefa reaching the liver are oxidized for energy production or converted into bhba, with a smaller contribution for synthesis of very low-density lipoprotein (vldl). the bovine liver has limited capacity to synthesize and secrete vldl, thereby compromising export of triacylglycerols during periods of extensive hepatic nefa uptake. the resulting hepatic lipidosis has been associated with retained placenta, ketosis, displaced abomasum, and impaired immune function and reproduction.54,55 thus, reducing the risk of lipid-related disorders might improve reproduction of dairy cows. supplementation of periparturient dairy cows with rumen-protected choline has been used as a strategy to improve lipid metabolism and alleviate hepatic lipidosis. when feed intake was restricted to 30% of the maintenance to simulate a period of neb and induce hepatic lipidosis, the supplementation of rumen-protected choline reduced triacylglycerol accumulation in the liver.56 furthermore, the inclusion of supplemental choline in the diet from approximately 25 days before to 80 days after calving reduced loss of body condition postpartum and concentrations of bhba, which resulted in less incidence of clinical and subclinical ketosis despite the increase in fat-corrected milk.57 although feeding rumen-protected choline reduced morbidity, and improved metabolic health, no benefits were observed for reproduction. supplemental rumen-protected choline did not affect the resumption of postpartum estrous cyclicity, pregnancy per ai at the first and second inseminations, or maintenance of pregnancy in the first 60 days of gestation. improving ca homeostasis postpartum the most common method to improve postpartum ca status is the manipulation of the dietary cation-anion difference (dcad) prepartum.58,59 reducing the dcad by feeding salts with strong anions decreases blood ph and enhances the affinity of the parathyroid hormone (pth) to the pth receptor present on cells in the bones, intestine, and kidneys.58 although feeding strong anions reduces feed intake during supplementation, the improved postpartum ca metabolism often results in greater postpartum feed intake.60 feeding acidogenic diets prepartum did not reduce the incidences of retained placenta, lameness and subclinical ketosis.58 however, supplementing cows with calcium chloride in a gel formulation 12 hours before the expected calving and at 0, 12, and 24 hours after calving reduced the incidence of clinical and subclinical hypocalcemia, and displacement of abomasums.61 despite the benefits of feeding acidogenic diets on ca homeostasis and the link between serum ca and uterine diseases and reproduction clinical theriogenology • volume 3 number 4 • december 2011 584 in dairy cows,32 interval to first insemination and pregnancy were not affected by feeding a low dcad diet prepartum.59 additional research is needed with properly powered experiments to critically evaluate the impact of reducing subclinical hypocalcemia by manipulating the dcad of prepartum diets or supplementing ca postpartum on reproduction of dairy cows. fatty acid supplementation and postpartum health lipids are important molecules that serve as a source of energy and are critical components of the physical and functional structure of cells. they play important regulatory roles in cell metabolism and serve as molecules affecting transduction pathways that control cell activity and proliferation. some fatty acids are considered essential as they cannot be synthesized by mammalian cells and have to be consumed in the diet. using growing rats, burr and burr demonstrated that diets low in fat interfered with growth, health and ovulation, events then reversed after feeding the polyunsaturated fa c18:2 n-6 (linoleic acid) and c18:3 n-3 (α-linolenic acid).62 the prostaglandin (pg) f2α synthesized by the endometrium plays an important role in reproduction of dairy cows. during parturition, eicosanoids are produced in copious amounts and play an important role in regulating uterine contractions and expulsion of the placenta and uterine contents. prostaglandin f2α is an important eicosanoid that regulates corpus luteum lifespan and might influence uterine defense mechanisms. feeding diets that differ in fatty acid profile influences the composition of the uterine tissues, which in turn affects the secretion of pgf2α in the early puerperium.63 santos et al reviewed studies in which transition cows were fed diets differing in fatty acid profile.63 prepartum supplementation with ca salts of long chain fa rich in n-6 fa reduced the incidence of retained placenta, metritis and mastitis compared with cows not fed fat prepartum.63 similarly, supplementing prepartum diets with 2% ca salts of either palm oil or a blend of c18:2 n-6 and transoctadecenoic fa reduced the severity of uterine disease postpartum.64 it is known that polyunsaturated fa are capable of modulating immune response, and n-6 fa usually potentiate the inflammatory response, whereas n3 fa tend to depress this response. recent work by our group evaluated cellular responses and innate immunity of dairy cows fed ca salts containing mostly saturated/monounsatured fa (palm oil), n-6 fa (safflower oil), or n-3 fa from fish oil.65 cows fed ca salts high in linoleic acid (n-6 fa) tended to have (p < 0.10) more linoleic acid in the caruncular tissue than cows fed palm oil. the n-6:n-3 ratio of fa was greater (p < 0.05) in caruncular tissue of cows fed ca salts containing safflower compared with that of cows fed ca salts of palm oil. neutrophils from cows fed ca salts of safflower oil rich in n-6 fa had increased ability to kill bacteria in vitro. similarly, feeding more n-6 fa during the transition period increased the acute phase response and induced neutrophils to secrete an increased amount of pro-inflammatory cytokines.65 on the other hand, when cows in the breeding period received either ca salts of palm oil (saturated or monounsatured fa) or of fish oil (n-3 fa), the latter induced a period of suppressed inflammatory responses. these data suggest that it is possible to manipulate the innate immunity by altering the fatty acid makeup of the diet, although additional studies are needed to determine the exact combination of fa required to optimizing postpartum health. nevertheless, the combination of dietary fa that enhanced immune response around calving and suppressed during breeding resulted in the highest proportion of pregnant cows following the first two postpartum inseminations.66 conclusions dairy cows during the periparturient period undergo metabolic distress because of the initiation of lactation. in many cows, homeorrhetic controls are not able to sustain homeostasis and disease eventually develops. in fact, 30 to 50% of the postpartum dairy cows are diagnosed with a disease event in the first two to three months postpartum. such problems are negatively associated with pregnancy. in fact, diseases extend the period of anovulation, reduce pregnancy per ai, and increase pregnancy loss. it is expected that implementation of nutritional and health programs that address the needs of dairy cows to minimize drastic changes in intermediary and mineral metabolism tend to favor metabolic health and subsequent reproduction. in many cases, these responses have not been demonstrated in controlled clinical theriogenology • volume 3 number 4 • december 2011585 research. nevertheless, common sense dictates that cows that undergo transition without health problems are more fertile. references 1. ribeiro es, lima fs, ayres h, et al: effect of postpartum diseases on reproduction of grazing dairy cows. j dairy sci 2011;94(e-suppl 1):63 (abstract). 2. santos jep, bisinotto rs, ribeiro es, et al: applying nutrition and physiology to improve reproduction in dairy cattle. in: lucy mc, pate jl, smith mf, et al, editors. reproduction in domestic ruminants vii. nottingham: nottingham university press; 2011. p. 387-404. 3. butler wr: energy balance relationships with follicular development, ovulation and fertility in postpartum dairy cows. livest prod sci 2003;83:211-218. 4. santos jep, rutigliano hm, sá filho mf: risk factors for resumption of postpartum cyclicity and embryonic survival in lactating dairy cows. anim reprod sci 2009;110:207-221. 5. ospina pa, nydam dv, stokol t, et al: associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern united states. j dairy sci 2010;93:1596-1603. 6. walsh rb, walton js, kelton df, et al: the effect of subclinical ketosis in early lactation on reproductive performance of postpartum dairy cows. j dairy sci 2007;90:2788-2796. 7. ospina pa, nydam dv, stokol t, et al: association between the proportion of sampled transition cows with increased nonesterified fatty acids and β-hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level. j dairy sci 2010;93:3595-3601. 8. lacetera n, scalia d, franci o, et al: short communication: effects of non-esterified fatty acids on lymphocyte function in dairy heifers. j dairy sci 2004;87:1012-1014. 9. 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jlmr, vanholder t, mateusen b, et al: non-esterified fatty acids in follicular fluid of dairy cows and their effect on developmental capacity of bovine oocytes in vitro. reproduction 2005;130:485-495. 31. reinhardt ta, lippolis jd, mccluskey bj, et al: prevalence of subclinical hypocalcemia in dairy herds. vet j 2011;188:122-124. 32. martinez n, risco ca, maunsell f, et al: evaluation of peripartal calcium status and neutrophil function of dairy cows of low or high risk of developing uterine diseases. proc annu conf am assoc bovine pract; 2011(abstract). 33. lewis rs: calcium signaling mechanisms in t lymphocytes. annu rev immunol 2001;19:497-521. 34. kimura k, reinhardt ta, goff jp: parturition and hypocalcemia blunts calcium signals in immune cells of dairy cattle. j dairy sci 2006;89:2588-2595. 35. von keyserlingk ma, olenick d, weary dm: acute behavioral effects of regrouping dairy cows. j dairy sci 2008:91:1011-1016. 36. coonen jm, maroney mj, crump pm, et al: short communication: 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dry period length on reproduction during the subsequent lactation. j dairy sci 2009;92:3081-3090. 42. santschi de, lefebvre dm, cue ri, et al: incidence of metabolic disorders and reproductive performance following a short (35-d) or conventional (60-d) dry period management in commercial holstein herds. j dairy sci 2011;94:33223330. 43. degaris pj, lean ij, rabiee ar, et al: effects of increasing days of exposure to prepartum transition diets in reproduction and health in dairy cows. aust vet j 2010;88:84-92. erratum in aust vet j 2010;88:189. 44. douglas gn, overton tr, bateman hg ii, et al: prepartal plane of nutrition, regardless of dietary energy source, affects periparturient metabolism and dry matter intake in holstein cows. j dairy sci 2006;89:2141-2157. 45. janovick na, boisclair yr, drackley jk: prepartum dietary energy intake affects metabolism and health during the preparturient period in primiparous and multiparous holstein cows. j dairy sci 2011;94:1385-1400. 46. santos je, depeters ej, jardon pw, et al: effect of prepartum dietary protein level on performance of primigravid and multiparous holstein dairy cows. j dairy sci 2001;84:213-224. 47. kanjanapruthipong j, homwong n, buatong n: effects of prepartum roughage neutral detergent fiber levels on periparturient dry matter intake, metabolism, and lactation in heat-stressed dairy cows. j dairy sci 2010;93:2589-2597. 48. national research council: nutrient requirements of dairy cattle. 7th ed. washington (dc): national academy press; 2001. 49. gong jg, lee wj, garnsworthy pc, et al: effect of dietary-induced increases in circulating insulin concentrations during the early postpartum period on reproductive function in dairy cows. reproduction 2002;123:419-427. 50. garnsworthy pc, fouladi-nashta aa, mann ge, et al: effect of dietary-induced changes in plasma insulin concentrations during the early post partum period on pregnancy rate in dairy cows. reproduction 2009;137:759-768. 51. duffield tf, rabiee ar, lean ij:. a meta-analysis of the impact of monensin in lactating dairy cattle. part 1. metabolic effects. j dairy sci 2008;91:1334-1346. 52. duffield tf, rabiee ar, lean ij: a meta-analysis of the impact of monensin in lactating dairy cattle. part 2. health and reproduction. j dairy sci 2008;91:2328-2341. 53. abe n, lean ij, rabiee a, et al: effects of sodium monensin on reproductive performance of dairy cattle. ii. effects on metabolites in plasma, resumption of ovarian cyclicity and oestrus in lactating cows. aust vet j 1994;71:277-282. 54. jorritsma r, jorritsma h, schukken yh, et al: relationships between fatty liver and fertility and some periparturient diseases in commercial dutch dairy herds. theriogenology 2000;54:1065-1074. 55. bobe g, young jw, beitz dc: invited review: pathology, etiology, prevention, and treatment of fatty liver in dairy cows. j dairy sci 2004;87:3105-3124. 56. cooke rf, silva del río n, caraviello dz, et al: supplemental choline for prevention and alleviation of fatty liver in dairy cattle. j dairy sci 2007;90:2413-2418. 57. lima fs, sá filho mf, greco lf, et al: effects of feeding rumen-protected choline on incidence of diseases and reproduction of dairy cows. vet j 2011. in press. clinical theriogenology • volume 3 number 4 • december 2011587 58. goff jp: macromineral disorders of the transition cow. vet. clin north am food anim pract 2004;20:471-494. 59. seifi ha, mohri m, farzaneh n, et al: effects of anionic salts supplementation on blood ph and mineral status, energy metabolism, reproduction and production in transition dairy cows. res vet sci 2010;89;72-77. 60. degroot ma, block e, french pd: effect of prepartum anionic supplementation on periparturient feed intake, health, and milk production. j dairy sci 2010;93:5268-5279. 61. oetzel gr: effect of calcium chloride gel treatment in dairy cows on incidence of periparturient diseases. j am vet med assoc 1996;209:958-961. 62. burr go, burr mm: the nature and role of the fatty acids essential in nutrition. j biol chem 1930;86:587-621. 63. cullens fm, staples cr, bilby tr, et al: effect of timing of initiation of fat supplementation on milk production, plasma hormones and metabolites, and conception rates of holstein cows in summer. j dairy sci 2004;86 (suppl 1):308 (abstract). 64. santos jep, bilby tr, thatcher ww, et al: long chain fatty acids of diet as factors influencing reproduction in cattle. reprod domest anim 2008;43:23-30. 65. silvestre ft, carvalho tsm, crawford pc, et al: effects of differential supplementation of fatty acids during the peripartum and breeding periods of holstein cows: ii. neutrophil fatty acids and function, and acute phase proteins. j dairy sci 2011;94:2285-2301. 66. silvestre ft, carvalho tsm, francisco n, et al: effects of differential supplementation of fatty acids during the peripartum and breeding periods of holstein cows: i. uterine and metabolic responses, reproduction and lactation. j dairy sci 2011;94:189-204. 67. douglas gn, rehage j, beaulieu ad, et al: prepartum nutrition alters fatty acid composition in plasma, adipose tissue, and liver lipids of periparturient dairy cows. j dairy sci 2007;90:2941-2959. 68. rabelo e, rezende rl, bertics sj, et al: effects of preand postfresh transition diets varying in dietary energy density on metabolic status of periparturient dairy cows. j dairy sci 2005;88:4375-4383. 69. rabelo e, rezende rl, bertics sj, et al: effects of transition diets varying in dietary energy density on lactation performance and ruminal parameters of dairy cows. j dairy sci 2003;86:916-925. 70. doepel l, lapierre h, kennelly jj: peripartum performance and metabolism of dairy cows in response to prepartum energy and protein intake. j dairy sci 2002;85:2315-2334. 71. hayirli a, keisler dh, doepel l, et al: peripartum responses of dairy cows to prepartal feeding level and dietary fatty acid source. j dairy sci 2011;94:917-930. clinical theriogenology • volume 3 number 4 • december 2011 588 table 1. association among clinical and subclinical diseases and fertility responses in dairy cows health problem estrous cyclic * aor (ci) † p healthy 95.6 a 1.00 -- subclinical disease only 88.9 b,c 0.35 (0.16-0.76) <0.01 clinical disease only 93.0 a,b 0.63 (0.23-1.75) 0.37 subclinical and clinical disease 83.5 c 0.23 (0.10-0.50) <0.01 health problem pregnant d 30 *,¶ aor (ci) p healthy 73.5 a 1.00 -- subclinical disease only 63.1 b 0.67 (0.44-0.99) 0.05 clinical disease only 54.8 b,c 0.44 (0.26-0.75) <0.01 subclinical and clinical disease 50.0 c 0.39 (0.24-0.61) <0.01 health problem pregnant d 65 *,¶ aor (ci) p healthy 66.2 a 1.00 -- subclinical disease only 57.1 a,b 0.72 (0.49-1.05) 0.09 clinical disease only 46.3 b,c 0.45 (0.26-0.76) <0.01 subclinical and clinical disease 42.1 c 0.39 (0.25-0.61) <0.01 data from ribeiro et al.1 numbers for estrous cyclic, and pregnancy on days 30 and 65 after ai represent the proportion of cows affected from a population of 957 lactating cows in two dairy farms. †aor = adjusted odds ratio; ci = confidence interval. a,b,c superscripts within a day of pregnancy differ (p < 0.07). contrasts: *effect of uterine disease (healthy vs. all others) p < 0.05; ¶ additive effect of metritis and clinical endometritis (clinical endometritis only + metritis only vs. metritis and clinical endometritis) p < 0.05. table 2. effect of prepartum caloric intake on fat-corrected milk (kg/d) prepartum intake‡ reference low caloric intake high caloric intake p§ douglas et al67 35.6 37.9 ns douglas et al44 40.8 39.8 ns rabelo et al68,69 38.5 40.4 0.59 doepel et al70 39.1 40.3 ns hayirli et al71 33.7 35.2 0.27 janovick et al45 40.5 46.1 0.09 kanjanapruthipong et al47 26.1 28.4 0.04 average 36.3 38.3 ‡prepartum caloric intake (net energy for lactation) averaged 14.6 and 19.8 mcal/cow/day for the low and the high caloric intake, respectively. §ns = not significant (p > 0.10). clinical theriogenology • volume 3 number 4 • december 2011589 clinical theriogenology • volume 3 number 4 • december 2011 590 omniblank: 2018: determining the lowest dose of prostaglandin needed for luteolysisof a mature corpus luteum in cycling donkeys in the caribbean determining the lowest dose of prostaglandin needed for luteolysis of a mature corpus luteum in cycling donkeys in the caribbean m. landers, a. kent, e. peterson, r. gilbert, h. french ross university school of veterinary medicine, st. kitts, west indies by gaining a better understanding of the jenny’s reproductive characteristics efforts can be made to improve breeding management for population control, or in the case of endangered species, better reproductive efficiency. although donkeys are often treated similarly to horses there is evidence that they metabolize agents very differently. dinoprost tromethamine is a luteolytic agent used in cycling mares to control time of estrus. the labeled dose is known to cause adverse effects mimicking colic behavior but studies have shown that one-eighth of the labeled dose is effective and does not cause unwanted side effects. the aim of this study was to determine the lowest effective dose of dinoprost tromethamine in cycling jennies in the caribbean. the study also characterized adverse side effects at each tested dose of dinoprost tromethamine. seven reproductively sound, non-pregnant caribbean jennies between 3 and 12 years of age were monitored via transrectal ultrasonography daily to detect follicular and corpus luteum (cl) formation. seven days after the formation of a cl one of four dinoprost tromethamine treatment doses (full, half, quarter, lowest) was administered to donkeys arranged in a cross-over design. they were monitored for 30 minutes following treatment and pulse, respiration and behavior recorded every 5 minutes. after treatment, the measurement of the two largest follicles as well as the cl were recorded daily until a new ovulation occurred. seven days later, donkeys received a different assigned treatment. they continued in this cycle until all four treatments were concluded. estrous cycle lengths were extracted from this and compared by treatments. cycle length was analyzed by mixed effects multiple linear regression using jenny as a random variable and treatment dose as fixed effect. overall respiration rate and heart rate were similar throughout all treatment groups but behavior was notably different. for the adverse effect of cramping the full dose treatment group had 7/7 adverse effects, the half dose had 3/7, the quarter dose had 1/7 and the lowest dose had 0/7 effects. all treatment doses of dinoprost tromethamine were effective at reducing the estrous cycle length compared to the jennies’ normal (untreated) cycle (p < 0.0001). estrous cycles were reduced by an average of 6.6 days for the full dose, 5.7 days for the half dose, 6.6 days for the quarter dose and 4.4 days for the lowest dose. the lowest dose showed the most variable response (p = 0.001), while still reducing cycle length overall. the results of these experiments correlate to those found in mares and show that lower doses of dinoprost tromethamine effectively shorten estrous cycles in the jenny. future research will test the efficacy of a different synthetic prostaglandin, cloprostenol used at lower than conventional doses. keywords: donkey, luteolysis, corpus luteum, dinoprost tromethamine clinical theriogenology • volume 10, number 3 • september 2018347 when and how to perform a transvaginal ovarian biopsy in a mare when and how to perform a transvaginal ovarian biopsy in a mare claire card western college of veterinary medicine, university of saskatchewan, sk, canada abstract transvaginal ovarian biopsy (tvob) is a clinical tool to confirm the presence of an ovarian abnormality and additionally may be used in research to obtain serial ovarian samples to study physiologic functions without negative effects on mare health and fertility. circumstances where the tvob is useful as a clinical diagnostic or research tool include: discordant ovarian tumor biomarker results and clinical findings; biomarkers are elevated but it is unclear which ovary is abnormal or both ovaries are abnormal; or serial biopsies are desired. details regarding how to perform the tvob procedure are described and case examples included to illustrate its clinical application. keywords: transvaginal ovary biopsy, mare, ovarian tumor introduction investigators have developed a variety of transvaginal ultrasound guided clinical techniques that have been applied to the equine ovary, e.g. follicle aspiration for oocyte collection (tuga), transfollicular oocyte transfer, luteal biopsy (tvlb) and ovarian biopsy (tvob).1-3 transvaginal ultrasound guided aspiration (tuga) of follicles in the mare was reported in 1988.1 the tuga procedure is repeatedly performed in mares to recover oocytes; ovaries of mares that had undergone many tuga had fibrosis in the ovarian stroma, but there were no reported effects of repeated tuga on fertility.4 luteal biopsies in the mare and cow were described by slough et al. and aerts et al.5,6 health and fertility effects of transvaginal ultrasound guided luteal biopsy (tvlb) along with transvaginal ultrasound guided ovarian biopsy (tvob) in mares was reported, with fertility unaffected.3 in previous studies, experiments were done on abbatoir-derived equine ovaries. tvlb /tvob allows serial sampling of the ovary which has the positive benefit of reducing the number of horses required for research, while enabling study of various physiologic functions. the tvob was developed to assist clinical decision making by obtaining biopsy information from abnormal areas of an ovary or ovaries.3 review of ovarian abnormalities in most clinical settings, an abnormal ovary is identified though transrectal palpation of the ovary followed by a transrectal ultrasonographic examination. a mare’s history may include concern with the mare’s behavior such as: anestrus, stallion-like behavior or frequent estrus. abnormal transrectal findings may be present and include: abnormal ovarian texture, abnormal ovarian size, inability to discern the ovulation fossa, abnormal mobility of the ovary, adhesions to the ovary, and presence of static structures on the ovary. these findings may be associated with neoplastic and nonneoplastic processes.7 neoplastic processes include tumors, e.g. granulosa theca cell tumor (gtct), the most common ovarian tumor in the mare and arises from gonadostromal cells.7 other gonadostromal tumors (thecoma), germ cell tumors (teratoma, dysgerminoma), epithelial tumors (cystadenoma), mesenchymal tumors (e.g. leiomyoma), and metastatic tumors have been reported.3 the gtct has been reported in mares with a wide age range, from fetuses to aged mares, and may be unilateral or bilateral. the classic presentation of a gtct is an enlarged ovary with a “cluster of grapes” appearance, along with a small, inactive contralateral ovary. mares with gtct may exhibit anestrus, stallion-like behavior, persistent estrus or short interestrus intervals. the gtct are associated with unusual echotextural features; however, small tumor masses maybe be present in ovaries that appear to have a normal echotextural appearance.7 a few mares with gtct continue to have estrous cycles, may become pregnant and foal. reproductive activity may continue for a number of years, but in most cases, there is negative feedback on the pituitary from tumor products from the abnormal ovary, resulting in a small, static contralateral ovary. epithelial tumors and gtct in mares may present with a relatively normal echotextural appearance.8 387 clinical theriogenology • volume 11, number 3 • september 2019 as not all ovarian abnormalities arise from neoplastic tissue, a clinician should be aware that other abnormalities have been reported, including ovarian abscesses, foreign bodies, torsions, large follicular cystic structures, cystic embryonic remnants and other physiologic structures that may or may not enlarge the ovary, including persistent anovulatory follicles, epidermal inclusion cysts, supplementary corporal lutea and hematomas.9 ultrasonography of the ovary may identify both normal tissue such as stromal, luteal, and follicular tissues, as well as abnormal tissue, including tumors. other imaging modalities to visualize the equine ovary, as magnetic resonance imaging (mri), computerized tomography (ct), and diffraction enhanced radiography, are not performed due to the large size of mares, deep abdominal position of their ovaries, cost and availability. in some cases, ancillary tests such as a cbc, serum chemistry, serum amyloid a, and abdominocentesis may be helpful to identify ovarian inflammation, e.g. when a foreign body or abscess is present in the ovary.9 when an ovarian abnormality, such as a tumor is suspected, usually a serum sample is obtained for ovarian tumor biomarker analysis, including inhibin, testosterone and anti müllerian hormone (amh).10,11 analysis of serum concentrations of amh has been shown to have a high sensitivity and specificity for detection of gtct in mares, when compared to measurement of inhibin or testosterone. androgen concentrations may be elevated in mares with ovarian cystadenomas, although most other ovarian tumors do not produce reliable biomarkers.10,11 it is noteworthy that biomarker analysis for ovarian neoplasia indicates that certain types of tumor tissue are present, but the analysis does not confirm if one or both ovaries are affected, nor does it detect all types of ovarian neoplasia. prior to development of the tvob, a laparoscopic approach was used to examine the gross appearance of ovaries suspected to be neoplastic.3 ovariectomy was then performed to provide a definitive diagnosis and allow a return to reproductive function. a less expensive option than laparoscopy is a targeted tvob to obtain ovarian tissue for histopathology, which may be utilized in advance of surgery to identify the ovary or ovaries with neoplasia.7 when to perform a tvob the tvob is predominately utilized in a clinical setting when there is a need to confirm the presence of an ovarian tumor and when biomarker analysis (inhibin, testosterone and amh) are discordant with clinical findings. criteria for the use of a tvob include: abnormal ovarian texture or consistency, inconclusive hormonal findings, questionable or abnormal ultrasonographic echotexture of the ovary, undiagnosed behavioral or estrous cycle irregularities, difficulty accessing biomarker laboratories, need to rule out ovarian neoplasia as a cause of behavioral problems, and investigation of possible bilateral tumor involvement. health and fertility of mares undergoing tvlb or tvob of neoplastic ovarian tissue have been described, with fertility not affected by tvob.3 how to perform a tvob transrectal ultrasonography is performed using a linear-array transrectal transducer to confirm the ovary to be biopsied and area(s) on each ovary targeted for biopsy. a tuga transducer (7.5 10 mhz) (minitube®, ingersoll, ontario, canada) is utilized, or a vaginal transducer housing is disinfected with cold sterilant, rinsed with distilled water and a microconvex 7.5 10 mhz ultrasound transducer disinfected with 70% alcohol, is secured into the probe housing. a sterile insemination pipette or similar sterile plastic tube may be inserted into the needle track and secured in place, if desired. the tvob device is a modified, 14-gauge, 20 cm, single-action semiautomatic spring-loaded biopsy needle (supercore®, argon medical, frisco, tx), modified to be 65 cm long, with the tip beveled to a taper point. the tvob device may be sterilized with either gas or ultraviolet light, or the handle and needle portion wiped with alcohol or disinfectant wipes and the distal portion of the needle immersed in a cold sterilization media (cidex®) for at least 10 minutes. two tvob devices should be prepared for each procedure, so a different device may be used per ovary if both ovaries are biopsied, and in order to have a spare device if a malfunction occurs. if cold sterilization media is used, it should be rinsed off the end of 388clinical theriogenology • volume 11, number 3 • september 2019 the biopsy device with sterile water or saline, with the notch in the biopsy needle exposed. the instrument is then placed on a sterile field. experienced individuals require < 10 minutes after sedation in most mares to perform tvob. depending on mare temperament, a combination of sedatives and analgesics are given, such as: acepromazine (atravet® boehringer ingleheim, ridgefield, ct) 0.04 mg/kg 0.01 0.02 mg/kg iv; butorphanol tartrate (torbugesic®, zoetis, kalamazoo, mi), 0.01 0.02 mg/kg iv; detomidine hydrochloride (dormosedan®, zoetis) 0.04 0.08 mg/ kg iv; or xylazine (rompun®, bayer, mississauga, ontario, canada) 0.2 0.5 mg/kg iv. the mare’s head should be positioned to prevent airway obstruction as her head drops due to sedation. hyoscine butylbromide (buscopan®, boehringer ingelheim) 0.1 mg/ kg iv is then administered to relax the rectum. the perineum is cleansed as for artificial insemination. the vaginal probe is covered, lubricated, and inserted into the vagina using a sterile gloved hand and arm and positioned lateral to the cervix, ipsilateral to the ovary to be biopsied. operator then removes their hand from the vagina, inserts their arm in rectum, removes any manure and manipulates the target ovary cranial into the abdomen and then up and over the broad ligament so the ovary is positioned adjacent to the transducer. the entry point to the needle channel on the vaginal transducer should be kept covered either by a sterile plastic cover (minitube®, delavan, wi), or sterile towel during this manipulation to prevent contamination of the biopsy needle channel port. the ovary is carefully examined using transvaginal ultrasonography. transducer and ovary are manipulated so the region of the ovary to biopsied is easily visualized and there is no bowel or large blood vessel visible between the ovary and transducer. biopsy instrument should be handled using sterile gloves and is cocked by pulling back on the tip of the handle, resulting in an audible click. biopsy needle tip and side of the needle are then lightly lubricated using a small quantity of a sterile lubricant (minitube®). the biopsy needle is inserted carefully into the needle channel of the transducer and advanced until it meets resistance in the front of the vagina. at this time, the veterinarian supporting the ovary through the rectum against the vaginal wall must hold the ovary steady and firmly against the vaginal transducer. an on-screen biopsy needle track (guide) may or may not be used. a tvob needle positioned for obtaining a biopsy within an ovary is shown (figure 1). if a needle track guide is used, the ovary must be positioned so the region to be biopsied is accessible, with the track bisecting the area of interest. the depth needed to reach the target area to be biopsied in the ovary is assessed by evaluating centimeter marks on the monitor. the biopsy needle is grasped at the desired distance to be advanced into the ovary and the ovary entered using a controlled stabbing motion under real-time visualization. the entrance of the biopsy device into the ovary of the mare may elicit some motion in some mares. if the mare moves excessively during the procedure, additional sedation should be administered. for safety reasons, a biopsy sample should not be captured when the mare is moving. the authors recommend that only the side of the needle should be used for advancing the needle for positioning, as manipulation of the device’s handle may result in accidentally firing the instrument. an unplanned firing of the biopsy instrument may puncture the mare’s rectum or the veterinarian’s hand in the rectum. the biopsy needle is guided into or near the target area by visualizing the bright echo produced by reflections generated from the diamond scoring on the needle tip. solid areas of suspect ovarian tissue should be targeted for biopsy, as better samples are obtained compared to cystic areas. a 2 step biopsy method involves the veterinarian visualizing the area of interest in the ovary and advancing the biopsy needle tip under direct visualization to just behind the biopsy location. the needle tip is then advanced to the first stop into the exact location to be biopsied, by applying gentle pressure on the tip of handle and then the needle is moved slightly to fill the notch on the biopsy needle. the biopsy instrument is then fired by firmly pushing on the end of the instrument to reach the second stop and an audible click will be heard. using this 2 step method, the biopsy needle tip does not advance when firing, rather the cutting sheath moves and closes over the notched portion of the needle to capture the sample. alternatively, the other choice of technique is called the 1 step procedure. for this, the biopsy needle tip is advanced to the perimeter of the tissue of interest, accounting for fact the needle will advance 2 cm during firing. the biopsy needle should not be held during firing with either method and only the handle 389 clinical theriogenology • volume 11, number 3 • september 2019 of the device should be manipulated during firing, as holding the needle prevents the cutting sheath from advancing and may cause the device to malfunction. figure 1. transvaginal ultrasound image of a biopsy needle in position to capture a sample using transvaginal ovarian biopsy technique. needle is visible as a white line casting an acoustic shadow. white arrow indicates where the needle enters the ovary, and horizontal arrow indicates needle tip. the biopsy needle is then removed from the biopsy channel and placed flat on the sterile field. the captured sample is accessed by pulling back on the tip of the handle to re-cock the instrument (an audible click is heard), then advancing the needle to the first stop by gentle pressure on the end of the instrument to expose the captured biopsy core sample (figure 2). needles (22 gauge) are often used to remove the sample from the needle notch. care should be taken when removing biopsies, as they are thread like and easily stretched. biopsy cores are placed in a small amount of fixative (10% buffered formalin). the biopsy needle may be rinsed with sterile saline or wiped with sterile gauze and the procedure repeated to capture at least three core samples in the area(s) of interest. captured ovarian fluid may be dropped onto sterile glass slides, dried and processed for cytology. following completion of the sample collections, the vaginal probe is removed. a small amount of blood may be present on the tip of the probe from the biopsy needle puncturing the cranial vaginal wall. fixed tissues for histological examination are processed in a routine manner and stained using hematoxylin and eosin (figure 2). it may also be possible to perform a tvob procedure through an ultrasound-guided flank approach with a nonmodified device without the extension of the biopsy needle, by pushing the ovary against the body wall. however, heavy, very enlarged ovaries are somewhat cumbersome to hold in position with either technique. figure 2. image of an ovarian biopsy sample (left panel) in the notch of 14 gauge needle of transvaginal ovarian biopsy device (black arrows) and histology slide (right panel) containing ovarian samples obtained during transvaginal ovarian biopsy. 390clinical theriogenology • volume 11, number 3 • september 2019 undesired outcomes and complications mares with a solitary large cystic structure or multi-cystic ovarian tissue may be problematic to biopsy, as there is little tissue to capture. in these cases, more solid areas of the mass should also be targeted for biopsy. occasionally non-diagnostic samples are obtained that have been obtained off target. an instrument may malfunction. gastrointestinal or rectal puncture may occur during the procedure due to: bowel present between the transducer and ovary; mare movement; over zealous insertion of the biopsy needle; failure to identify the location of the biopsy needle before firing; positioning errors in areas difficult to biopsy; operator error, especially when learning the technique; or inadvertently firing the biopsy device. the veterinarian’s hand may also be penetrated by the biopsy needle, in which case the biopsy needle will have to be retracted through the rectum and ovary. the mare’s rectum may be injured during manipulation. the possibility of biopsy needle puncture through the rectum should be disclosed to the client. during the tvob’s we have performed, a few of these rectal and hand punctures have occurred; mares were treated with nsaids, antibiotics and rectal temperatures were measured for 3 days, but no complications occurred in the mares. first aid should be available for the operator. ovarian abscess or infection may occur if the rectum is punctured or if inadvertent needle contamination occurs during the procedure. a biopsy instrument that punctures the rectum should be considered contaminated and should be either cold sterilized or gas sterilized before it is used again. ideally, ovarian biopsy instruments are single use. a rectal injury, such as minor bleeding from the rectum or a small laceration of the mucosa, may occur with this type of ovarian manipulation, and should be investigated when discovered and treated accordingly. we have not detected neoplastic tissue seeding other locations in the abdomen as a result of the tvob procedure for gtct. aftercare aftercare includes monitoring the mare’s physical examination parameters (temperature, pulse, respiration), appetite and demeanor for 3 days after tvob. generally, mares are not treated with nsaids or antibiotics before or after the tvob procedure, except when rectal bleeding is detected and appropriate measures are taken, or an inadvertent rectal puncture has occurred, then antibiotics and nsaids should be administered. case 1 history august 2018. initial examination by rdvm included the following history: mare was reported to have aggressive, out-of-character behavior since spring of 2018. there was a thick-walled structure with a hyperechoic center and appearance of a corpus hemorrhagicum on the right ovary. there were multiple small follicles on the left ovary and grade 1 uterine edema. the mare was given pgf2α 14 days before referral. biomarker analysis progesterone was 2.6 ng/ml, consistent with active luteal tissue; inhibin (32.3 pg/ml) was normal (reference range: 92 100 ng/ml); amh (12.9 ng/ml) was elevated (reference range: 0.1 6.9 ng/ml); and testosterone (45.8 pg/ml) marginally elevated (reference range: 12 45 pg/ml for a non-pregnant mare). ovariectomy was recommended based on biomarkers. in november 2018, mare (figure 3) was presented for ovariectomy. ovaries were normal (figure 3). hormonal analysis was repeated, with testosterone < 0.2 pg/ml and amh within reference ranges. a tvob was performed and histology of the ovarian tissue had normal stromal and luteal tissue (figure 4). the owner elected to take the mare home and no surgery was performed. 391 clinical theriogenology • volume 11, number 3 • september 2019 figure 3. case 1 picture of a quarter horse mare (left) panel, and ultrasound images of the mare’s left ovary (center panel) and right ovary (right panel) in november 2018. figure 4. case 1 histologic image of stromal (left panel) and luteal (right panel) tissue obtained using a transvaginal ovarian biopsy from the mare’s ovaries. diagnosis transiently elevated biomarkers, ovarian tissue within reference range. case 2 history an aqha 10 year old broodmare was presented with a history of signs of aggression and persistently enlarged ovaries. mare was owned by a veterinarian. owner reported that the mare had unexplained infertility with no inflammation on endometrial cytology and a grade iib endometrial biopsy. upon referral, based on transrectal palpation and ultrasonography, ovaries were within normal limits in consistency, size and echotextural features. biomarker analysis testosterone and inhibin concentrations were within normal ranges, and due to cost, amh was not analyzed. karyotype analysis was wnl. owner requested a biopsy of both ovaries. diagnosis histology of biopsies obtained using tvob indicated healthy ovarian tissue. owner reported the mare was inseminated twice in 2017 with cooled semen, but failed to become pregnant. in 2018, she was inseminated with cooled semen and became pregnant, but experienced early embryonic loss by 30 days. 392clinical theriogenology • volume 11, number 3 • september 2019 case 3 history a 12 year old warmblood mare was presented with a history of repeated estrus. in july 2018, she had an enlarged right ovary containing luteal tissue and a small left ovary. at a repeat evaluation in february 2019, there was an enlarged (> 10 cm) firm ovary and a very small left ovary. uterus and cervix were moderately soft and there was grade 2 -3 uterine edema and scant grade 0 intrauterine fluid. the mare was considered a valuable brood mare and the owners did not want to go ahead with ovariectomy until confirmation of ovarian neoplasia was obtained. biomarkers at the july examination, concentrations of all ovarian tumor biomarkers were within normal limits. a follow up transrectal and ultrasonographic examination confirmed the large right ovary and small left ovary. a tvob was performed in february, but did not yield a diagnostic sample. biomarker analysis and repeat tvob were scheduled (figure 6). ultrasonographic appearance of a gtct may be quite variable. three ultrasonograms that illustrate the variable appearance of gtct are shown (figure 5). a tvob is useful in many cases to determine if both ovaries contain gtct; note the gtct tissue obtained from a tvob (figure 6) and compared it to tissue in figure 4. figure 5. ultrasound appearance of three ovaries that each contain a granulosa theca cell tumor (gtct). the enlarged ovary in left panel has a classic appearing (gtct), center panel contains an ovary with a gtct that appears within normal limits, and right panel contains a gtct that appears as a large cystic structure. figure 6. histologic appearance of granulosa theca cell tumor tissue obtained using a transvaginal ovarian biopsy. 393 clinical theriogenology • volume 11, number 3 • september 2019 discussion while there have been some extensive studies regarding serum concentrations of amh and inhibin in mares with gtct, reference ranges provided by laboratories should still be considered a guide to be interpreted and applied in the context of other diagnostic tests. the clinician should integrate results of transrectal palpation and ultrasonography, ovarian tumor biomarker analysis and ovarian biopsy in cases where the diagnosis is not straight forward. case 1 illustrates a mare where 2 ovarian tumor biomarkers were initially increased and then returned to within normal limits. in our equine practice, we have identified occasional mares with this same amh profile; that is, elevated amh concentrations that then decrease to the reference range. case 2 is an example of a mare where it was unlikely that a gtct was present, however not all tumors produce elevated ovarian tumor biomarkers, or are visible using ultrasonography. the owner wanted to rule out neoplasia as a factor in the mare’s poor fertility using tvob as a modality. case 3 represents a mare that is presented with an abnormal ovary detected with ultrasonography, without an elevation in ovarian tumor biomarkers. this may arise as a result of the tumor being predominately comprised of thecal tissue, i.e. a thecoma, another type of tumor (teratoma, cystadenoma, secondary metastasis) or it might accompany an error in sample handling or laboratory analysis. the history of the contralateral small ovary and persistently enlarged right ovary were highly suggestive of a gtct tumor; however other ovarian abnormalities are also associated with 1 small ovary.12 evaluation of the amh reference ranges for gtct versus other ovarian abnormalities may result in changes in the reported reference range.12 in conclusion, tvob has specific applications for cases where the nature of an ovarian abnormality is not diagnosed, or to determine if 1 or both ovaries are affected by neoplasia. the tvob procedure has not been associated with impairment of fertility and may provide useful diagnostic information. acknowledgement funding for this research was provided from the equine health research fund at the university of saskatchewan, western college of veterinary medicine and the national science and engineering research council of canada. we thank clients of the large animal clinic at the western college of veterinary medicine for their cooperation with the tvob research. thanks to the researchers including: drs. diel de amorim, dawn nairn, ilse dedden, ellie ripley, lyndsay rogers, kayla nielsen, stephen manning and maria lopez rodriguez who assisted with research and development of tvlb and tvob. conflict of interest author claims no direct or indirect affiliation with any of the manufacture’s listed in the text. information regarding the equipment and various manufacturers is solely based on experience and is for readers’ reference only. references 1. cook nl, squires el, ray bs, et al: transvaginal ultrasonically guided follicular aspiration of equine oocytes: preliminary results. j equine vet science 1992;12:204-207. 2. hinrichs k, digiorgio lm: embryonic development after intrafollicular transfer in mares. j repro fertil suppl 1991;44:369-374. 3. diel de amorim m, nairn ld, manning s, et al: evaluation of diagnostic utility, safety considerations, and effect on fertility of transvaginal ultrasound-guided ovarian biopsy in mares. theriogenology 2016;5:1030-1036. 4. velez ic, arnold , jacobson cc, et al: effects of repeated transvaginal aspiration of immature follicles on mare health and ovarian status. equine vet j 2012;44:78-83. 5. slough tl, rispoli la, carnevale em, et al: temporal gene expression in equine corpora lutea based on serial biopsies in vivo. j anim sci 2011;89:389-396. 6. aerts jmj, oste m, bols pej: development and practical applications of a method for repeated transvaginal, ultrasound-guided biopsy collection of the bovine ovary. theriogenology 2005;64:947-957. 7 card ce. ovarian neoplasia. in mckinnon ao, squire el, vaala we et al: editors. equine reproduction. 2nd edition, ames: wiley-blackwell, 2011. p. 2707-2716. 8. hinrichs k, frazer gs, degannes rvg, et al: serous cystadenoma in a high plasma testosterone values. j am vet med assoc 1989;194:381-382. 394clinical theriogenology • volume 11, number 3 • september 2019 9. randleff-rasmussen, gray as. metallic foreign body in the ovary of a broodmare. eq vet educ 2018;30:237-240. https://doi.org/10.1111/eve.12618 10. ball ba, almeida j, conley aj. determination of serum anti-mullerian hormone concentrations for the diagnosis of granulosa-cell tumours in mares. equine vet j 2013;45:199-203. 11. ball ba, conley aj, almeida j, et al: a retrospective analysis of 2,253 cases submitted for endocrine diagnosis of possible granulosa cell tumors in mares. j equine vet sci 2014;34:307-313. 12. murase h, ball b, tanbyuenyong s, et al: serum anti-müllerian hormone concentrations in mares with granulosa cell tumors versus other ovarian abnormalities. j equine vet sci 2018;60:6-10. 395 clinical theriogenology • volume 11, number 3 • september 2019 https://doi.org/10.1111/eve.12618 396clinical theriogenology • volume 11, number 3 • september 2019 omniblank: genetic predisposition for developing equine arteritis virus carrier state: adopting proper biosecurity measures when breeding mares to a persistently infected stallion genetic predisposition for developing equine arteritis virus carrier state: adopting proper biosecurity measures when breeding mares to a persistently infected stallion udeni balasuriya, mariano carossino, zelalem mekuria, reed holyoaka louisiana animal disease diagnostic laboratory and department of pathobiological sciences school of veterinary medicine, louisiana state university, baton rouge, la aveterinary clinical sciences, veterinary medical teaching hospital oklahoma state university, stillwater, ok abstract outbreaks of equine viral arteritis (eva) have occurred around the world and there is evidence of an increase in global incidence of the disease in past years. these outbreaks have had important economic consequences in terms of direct financial losses to the equine industry. following natural infection, 10 70% of stallions can become persistently infected carriers of equine arteritis virus (eav) and continuously shed virus in their semen. carrier stallions have a central role in maintenance and perpetuation of the virus and are responsible for initiating major outbreaks of eva. furthermore, occurrence and spread of eav has been facilitated by increasing horse movement around the world for competition and breeding and by trade of fresh or frozen semen and embryos. control and preventive measures include prophylactic vaccination and control of movement of stallions used for natural or artificial breeding by serological testing and subsequent virologic assessment of their semen before initiation of each breeding season, as well as during pre-export and import quarantines. here, we review the latest findings regarding genetic predisposition of stallions to become long-term eav carriers, current laboratory tests for diagnosis of eav in semen, as well as biosecurity measures that should be implemented when breeding mares to persistently infected stallions. keywords: equine arteritis virus, eav, equine viral arteritis, eva, persistent infection, biosecurity introduction and background equine viral arteritis (eva) is a reproductive and respiratory disease that affects members of the family equidae, which includes horses, mules, donkeys and zebras.1-10 eva is caused by equine arteritis virus (eav), a single-stranded, positive sense rna virus that belongs to the family arteriviridae, genus alphaartevirus in the order nidovirales.1,11,12 the molecular biology of eav, and clinical features and pathogenesis of eva have been recently reviewed in several publications.6,13-18 the virus is distributed throughout the world; most eav infections are subclinical in nature and may go undiagnosed. however, some virulent strains periodically cause marked outbreaks of disease and they could be associated with abortion, neonatal mortality and establishment of persistent infection in stallions.2,8,19-27 the incubation period varies from 2 -14 days (usually 6 8 days after venereal exposure) and the disease is clinically characterized by fever (> 41°c), depression, leukopenia, excessive lacrimation, anorexia, conjunctivitis, rhinitis and nasal discharge, urticaria of the head, neck, trunk, and hind limbs, and edema, which is most pronounced over the eyes (supraorbital), the abdomen, prepuce, scrotum, mammary glands, and hind limbs.3,4,9,27-40 abortion is a frequent outcome in naïve pregnant mares and generally occurs 10 30 days after exposure to eav and at any time between 3 and 10 months of gestation.2,23,27,35,37,41,42 congenital infection in neonatal foals is characterized by severe fulminating bronchointerstitial pneumonia. foals that are 1 to 3 months old can develop a progressive “pneumoenteric” syndrome, leading to death frequently associated with secondary bacterial infections.24,25,43,44 most importantly, a variable proportion of acutely infected stallions (10 70 %) may become persistently infected.22,45 the virus only persists in the stallion’s reproductive tract and is continuously and exclusively shed in semen after clinical recovery from the acute phase of the infection (carrier state).29,34, 45 the carrier state is testosterone-dependent and there is no evidence of persistent infection in mares, geldings or foals.27,46-48 persistently infected stallions shed eav in their semen for either a short interval (ranging from several weeks to < 1 year post infection; defined as short term shedders or short term carriers) or a long interval (> 1 year post infection to lifelong; defined as long term persistently 369 clinical theriogenology • volume 11, number 3 • september 2019 infected shedders or carriers) without adverse effects on semen quality or reproductive capacity.22,34,49-51 in these stallions, eav is only detectable in the reproductive tract and viral persistence occurs, despite the presence of high serum neutralizing and mucosal antibodies.22,34,50,52 viral shedding in semen frequently begins at 5 days post infection; viral titers in seminal plasma are variable but frequently high, ranging from 101 to > 107 pfu/ml.22,29,34 analysis of multiple tissues from the reproductive tract of eav carrier stallions unequivocally confirmed the ampullae as the primary site of eav persistence. dual immunohistochemistry staining confirmed eav localizes in vimentin positive fibrocytes and mononuclear cells (t and b lymphocytes and macrophages) but not in the glandular epithelium.34 persistently infected stallions have a central role in maintenance, perpetuation and evolution of the virus in the horse population. the occurrence and spread of eav has been facilitated by increasing horse movement around the world for competition and breeding purposes and by trade of fresh or frozen semen and embryos.26,53-58 development of the carrier state in stallions directly reduces commercial value of these animals, with higher costs for breeding and barriers in national and international movement of persistently infected stallions and commercialization of their semen and embryos. genetic predisposition of stallions for developing eav carrier state recently, it has been demonstrated that establishment of eav long-term persistent infection (ltpi) correlates with in vitro susceptibility of a subpopulation of cd3+ t lymphocytes to eav infection;33,59 consequently, stallions with the cd3+ t lymphocyte susceptibility phenotype are at higher risk of becoming long-term, persistently infected carriers compared to those that lack this phenotype. a genome wide association study (gwas) demonstrated that these phenotypes are associated with the cxcl16 gene located in equine chromosome 11 (eca11).60 subsequently, studies identified 2 allelic variants of cxcl16 (namely cxcl16s and cxcl16r) that differ by 4 nonsynonymous nucleotide substitutions in exon 1 and have a very strong association with 2 cd3+ t lymphocyte phenotypes and with either establishment of long-term persistence (cxcl16s) or the early viral clearance in stallions (cxcl16r).61 of the 2 encoded proteins, the cxcl16s isoform has eav receptor activity and is associated with the cd3+ t lymphocyte susceptible phenotype and establishment of ltpi in stallions, whereas the cxcl16r isoform lacks receptor activity and results in a cd3+ t lymphocyte resistant phenotype and early viral clearance in stallions (short term carriers).61,62 moreover, the secretory from of the cxcl16 protein is the ligand for the chemokine receptor cxcr6 that is predicted to be expressed on cd4+ and cd8+ t cells, nkt cells and nk cells. thus, it has been suggested that equine cxcl16 and cxcr6 may be 2 major cellular proteins associated with the eav carrier state in stallions. specific mechanisms mediating modulation of the cxcl16/cxcr6 axis and viral immune evasion in the male reproductive tract are currently under investigation in our laboratory. since genetic studies clearly established a very strong correlation between stallion genotype and likelihood of establishment of long-term persistence following infection, we recently developed an allelic discrimination quantitative pcr (qpcr) for cxcl16 genotyping (balasuriya and graves, 2018, unpublished). this assay detects presence of the susceptibility allele (cxcl16s), allowing identification of stallions more prone to become long-term carriers if exposed to eav. therefore, it is recommended that sexually mature colts and stallions with the susceptibility allele should be vaccinated yearly against eav to prevent establishment of the carrier state following natural infection. local inflammatory and mucosal antibody responses to eav during long-term persistent infection interestingly, the primary site of eav ltpi is in the ampulla of the reproductive tract rather than immunologically privileged tissues (i.e. testis). recent studies characterized viral tropism as well as local inflammatory and mucosal antibody responses to eav infection during the ltpi.34,50,63 using immunohistochemistry (ihc) and dual immunofluorescence, it has been demonstrated that eav has a specific tropism for vimentin-positive stromal cells (e.g. fibrocytes and tissue macrophages) and cd8+ t and cd21+ b lymphocytes, but not for cytokeratin-positive glandular epithelial cells in the male reproductive tract, with the highest number of virus infected cells within the ampullae of the ductus deferens. viral persistence is also associated with a moderate to severe, multifocal lymphoplasmacytic 370clinical theriogenology • volume 11, number 3 • september 2019 ampullitis with extensive involvement of t lymphocytes (cd3+, cd4+, cd8+ and cd25+), clusters of cd21+ b lymphocytes, diverse ig secreting plasma cells, macrophages (iba1+, cd163+ and cd204+) and dendritic cells (cd83+).34,50 most importantly, infiltrating lymphocytes have enhanced expression of cxcl16 and cxcr6.49,63 moreover, transcriptome analysis of the ampulla of eav ltpi stallions demonstrated that the local cd8+ t lymphocyte response is predominantly orchestrated by a specific subset of transcription factors (mainly eomes, prdm1 [blimp-1], nfatc2, tbx21 [t bet]), which are associated with presence of the cxcl16s allele and likely related to a local th1 response.63 also, there was upregulation of t cell exhaustion related markers and homing chemokines/chemokine receptors (including cxcl16/cxcr6). extensive characterization of the antieav mucosal antibody response in long-term carrier stallions and comparative analysis with the serological response identified major differences in immunoglobulin isotypes in seminal plasma.50 the serological response is primarily mediated by virusspecific igm and igg1, although virus-specific serum iga, igg3/5, igg4/7, and igg6 isotype responses are not detected. in contrast, eav specific immunoglobulin isotypes in seminal plasma included iga, igg1, igg3/5, and igg4/7. interestingly, seminal plasma igg1 and igg4/7 possess virus neutralizing activity similar to that of serum igg1, whereas seminal plasma iga and igg3/5 do not. however, virus neutralizing igg1 and igg4/7 in seminal plasma are not effective in preventing viral infectivity and mechanism(s) enabling eav to evade neutralizing mucosal antibody response in semen is unknown. role of microrna in eav long term persistent infection in stallions micrornas are noncoding rna molecules ~ 22 24 nucleotides in length that participate in posttranscriptional regulation of gene expression through specific degradation of mrna targets or blockade of their translation.64 recently, we investigated the role of equine seminal exosome-associated mirnas and their potential role during eav ltpi in the reproductive tract.49 seminal exosomes participate in cell to cell communication by specific cargo delivery, which may include proteins (e.g. cytokines, growth factors), specific lipids, and coding and small noncoding rnas with potential regulatory functions including micrornas (mirnas).65,66 our studies demonstrated that eav ltpi is associated with specific downregulation of seminal exosome associated mirna eca-mir-128, along with enhanced expression of cxcl16 in the male reproductive tract. interestingly, target evaluation and in silico analysis identified cxcl16 mrna as a putative target of eca-mir-128; furthermore, we determined that expression of these (cxcl16 and seminal exosome-associated eca-mir-128) in the male reproductive tract are inversely correlated. based on these findings, we inferred that seminal exosome-associated ecamir-128 is implicated in regulation of the cxcl16/cxcr6 axis in the reproductive tract of persistently infected stallion. however, further investigation is warranted to identify its specific mechanism in modulating the cxcl16/cxcr6 axis in the reproductive tract of the eav long-term carrier stallion. how other eca-mir-128 regulated pathways contribute to modulating the cxcl16/cxcr6 axis favoring long-term eav persistent infection in the stallion reproductive tract is not well known. in summary, recent studies indicate that pathogenesis of eav persistence in the male reproductive tract is complex and involves multiple host factors. evolution of eav during persistent infection despite the presence of neutralizing antibodies in serum and seminal plasma, eav persists in the reproductive tract of carrier stallions and is transmitted via semen. eav evolves during persistent infection in the reproductive tract, leading to emergence of genetic variants with distinct neutralization phenotypes due to amino acid substitutions in major viral neutralization sites located in the gp5 major envelope glycoprotein, conferring the ability to successfully escape humoral immunity.20,21,26,58,67,68 however, these investigations did not provide a complete picture of eav evolution during long-term persistent infection, because they were confined either to a few selected viral open reading frames (orf5 or orfs 2 7) or based on consensus genomic sequences. availability of next generation sequencing technology has overcome these deficiencies and allowed identification of rapidly evolving regions of the viral genome likely to contribute to survival of eav in the reproductive tract of 371 clinical theriogenology • volume 11, number 3 • september 2019 experimentally and naturally infected stallions. recently, sequential viruses isolated from nasal secretions, buffy coat cells and semen of experimentally infected and naturally infected eav carrier stallions (followed for 726 days and 7 and 10 years post infection, respectively) have been deep sequenced to elucidate the intra-host micro evolutionary process after a single transmission event.69 analysis of variants from nasal secretions and buffy coat cells during acute infection indicated a lack of extensive positive selection; however, characteristics of the mutant spectra were different in the two sample types. in contrast, the virus population in the semen during acute infection has undergone a selective bottleneck, as reflected by a reduction in population size and multiple sites in the virus genome that were under diversifying selection. during persistent infection, there is extensive genome-wide purifying selection which shaped the variant diversity in stallion reproductive tract. furthermore, during persistent infection, extensive genome wide purifying selection shaped variant diversity in the stallion reproductive tract. overall, the nonstochastic nature of eav evolution during persistent infection was driven by active intra-host selection pressure. among the open reading frames within the viral genome, orf3, orf5 and the nsp2 encoding region of orf1a accumulated the majority of nucleotide substitutions during persistence, with orf3 and orf5 having the highest intra-host evolutionary rates. eav evolved at a rate of 4 x 10-4 nucleotide substitutions/site/year during natural infection in the reproductive tract. these findings provide a novel insight into the evolutionary mechanisms of eav, identifying critical regions of the viral genome likely associated with establishment and maintenance of persistent infection in the stallion reproductive tract. identification of eav carrier stallions current eva control and prevention strategies partly rely on identification of eav carrier stallions. the current approach for initial identification of eav carrier stallions is dependent on demonstration of anti-virus neutralizing antibodies in serum. if the stallion is determined to be seropositive (neutralizing antibody titer e 1:4) and has no certified history of vaccination against eva or confirmation of seronegative status prior to initial vaccination, virologic assessment of their semen either by virus isolation in cell culture or demonstration of viral nucleic acids by molecular diagnostic testing is required to determine their infection status and potential carrier state.52,70-72 an alternative method for identification of eav carrier stallions or infective semen is to perform test breeding of the stallion (or semen derived from the stallions) to 2 naïve mares. briefly, this method consists of test breeding 2 seronegative (naïve) mares twice, each on 2 consecutive days (total of 4 covers). the mares should be kept in quarantine and tested for the presence of neutralizing antibodies to eav after 14 and 28 days post breeding. if the test is performed properly and strict quarantine is maintained, seroconversion in both mares bred to the stallion is evidence of eav infection and thus, indicates that the stallion (or the semen) is persistently infected. there could be 2 outcomes: (i) if both mares seroconvert, the stallion is a carrier of eav and should be reported to the state veterinarian (please refer below for guidelines to breed eav carrier stallions to a seropositive mare); or (ii) if both mares remain seronegative, identify the stallion as a “seropositive nonshedding stallion” or “seropositive noncarrier”. stallion is qualified for breeding but needs to be vaccinated annually. however, this method of screening stallions for eav carrier state is expensive, time consuming and not practical for routine identification of carrier stallions. for serological testing, whole blood should be collected into vacutainer® tubes without anticoagulant (for separation of serum). semen samples submitted for virologic assessment should contain the sperm-rich fraction of the ejaculate.19,45 preejaculatory fluids are inappropriate, since presence of virus in this fluid is inconsistent or variable. frozen semen straws from eav seropositive stallions should be tested for presence of virus by virus isolation or viral nucleic acids by molecular testing. currently, virus isolation and virus neutralization test (vnt) are the world organization for animal health (oie) prescribed tests for international trade (oie manual of diagnostic tests and vaccines for terrestrial animals).70 a competitive enzyme linked immunosorbent assay (celisa) was recently developed and validated for detection of eav-specific antibodies and could be considered as an alternative assay for serological diagnosis of eva.73-76 even though virus isolation is the gold standard for detection of eav in semen and the oie prescribed test for international trade, performance of an rt-qpcr and a reverse 372clinical theriogenology • volume 11, number 3 • september 2019 transcription insulated isothermal pcr (rt-iipcr) described in literature have equal or higher sensitivity compared to virus isolation in semen samples.77-89 therefore, molecular assays could be used as an alternative method for eav diagnosis.77-79,88 biosecurity measures when breeding mares to a persistently infected stallion eva control and prevention measures are primarily based on identification of eav carrier stallions and vaccination of susceptible horses, including stallions. commercially available vaccines (modified live virus vaccine [mlv; arvac®zoetis, kalamazoo, mi] and inactivated vaccine [artervac®], zoetis) are protective and can prevent establishment of the carrier state, although arvac® (zoetis) is better characterized than the inactivated product. it is extremely important to prevent sexually mature colts and stallions from being exposed to eav during outbreaks of eva. spread of eav could be prevented by implementing proper biosecurity control programs on breeding farms, racetracks, horse shows and veterinary clinics and hospitals. it is imperative to identify and isolate the index case and horses that come in contact, thereby minimizing or eliminating direct or indirect contact of susceptible horses with the secretions and excretions of eav-infected horses. if an outbreak of eva on a farm is suspected based on clinical signs and history, state veterinarian should be notified, affected and in-contact horses should be isolated, movement of horses on and off the farm should be discontinued (quarantine), at-risk horses should be vaccinated and breeding activity should be stopped to prevent further spread of virus. stalls and equipment on the affected premises should be decontaminated with disinfectants (phenolic, chlorine, iodine or quaternary ammonium compounds). quarantine is discontinued when no additional clinical cases of eva or serologic evidence of infection are observed for 3 4 weeks. stallions in exposed premises should be tested for shedding of the virus as described above and follow the guidelines described below during breeding of seropositive stallions to naïve mares.15 eav carrier stallions have pivotal roles in transmission and maintenance of eav infection in horse populations.27,45 therefore, outbreaks of eva can be prevented by identification of persistently infected stallions and institution of management practices to prevent introduction of eav-infected horses. the us department of agriculture (usda) animal and plant health inspection service (aphis), equine viral arteritis: uniform methods and rules, describes minimum standards for detecting, controlling and preventing eva, as well as minimum eva requirements for interstate and intrastate movement of horses.72 guidelines for prevention and control of eav in breeding stallions 1. all stallions should be tested for neutralizing antibodies to eav before they are vaccinated with mlv or inactivated eav vaccines. testing should be done at least 60 days prior to breeding. 2. a neutralization antibody titer of 1:4 or greater (vnt titer ≥ 1:4) is regarded as positive. owners of stallions that are seropositive should provide a valid vaccination certificate (stallion should have been confirmed seronegative before first vaccination against eav). 3. nonvaccinated seropositive stallions (no vaccination history) for eav (vnt titer ≥ 1:4) should be tested for carrier state by test breeding or testing semen samples for presence of eav by virus isolation/molecular testing of 2 separate semen samples derived from 2 separate collections or by test breeding, as indicated above. if semen samples are negative for eav by virus isolation/molecular testing, identify stallions as eav noncarriers. 4. management of eav carrier stallions: carrier stallions must be housed, handled and bred in a facility isolated from noncarrier stallions and mares. carrier stallions should be approved by the state veterinarian for breeding. carrier stallions should be bred only to mares that are seropositive either by natural exposure or by vaccination with neutralizing antibody titers ≥ 1:64 determined at least 30 days prior to breeding. 373 clinical theriogenology • volume 11, number 3 • september 2019 5. if seronegative (titer < 1:4), stallions should be vaccinated with the mlv vaccine after collecting a serum sample as proof. the vaccination needs to be officially recorded. 6. if stallions were to be vaccinated for the first time, they should be isolated for 28 days post vaccination before the breeding season or semen collection for artificial breeding purposes can begin. vaccinated stallions should receive annual boosters. guidelines for breeding a mare to an eav shedding stallion or insemination with eav contaminated semen: 1. stallions that are confirmed semen shedders and carriers of eav can be used for breeding purposes, provided stringent requirements are met.27,45,54,55,90 2. carrier stallions should be kept physically isolated and bred only to mares that are seropositive from either previous natural exposure or vaccination (not < 3 weeks previously). it is also critical that carrier stallions are kept isolated and collected separately to prevent contamination of collection equipment, teasers, and premises with ejaculate, because eav can be transmitted to susceptible horses by indirect aerosol contact. when embryo transfer is used for breeding, it is highly recommended that both donor and recipient mares are vaccinated against eva if the former are to be bred with eav-infective semen.56 3. mares to be bred to eav carrier stallions should be tested for neutralizing antibodies at least 30 days prior to breeding. 4. neutralizing antibody titer of ≥ 1:64 is regarded as protective against eav; therefore, these mares can be bred to an eav shedding carrier stallion (natural breeding) or insemination with eav infective semen (artificial breeding) without being vaccinated. 5. mares that are seronegative to eav should be vaccinated with mlv vaccine and the vaccination recorded. vaccinated mares should be isolated for 21 days and should not be bred during this period. after 21 days, breed the shedding stallion (natural breeding) or inseminate with eav infective semen (artificial breeding). 6. mares should be kept isolated from other nonvaccinated or seronegative horses for 3 weeks (21 days) after being bred to a shedding stallion or after insemination with infective semen. 7. management of mares after annual booster vaccination: annual booster vaccination 21 days prior to breeding is required and no isolation necessary following booster vaccination. there is no need for isolation of mares after breeding to a carrier stallion for the second time. general guidelines for preventing stallions from becoming carriers of eav 1. standard biosecurity measures for the prevention of infectious diseases should be implemented on breeding farms e.g. horses that travel for competitions or comingle with horses coming from outside the farm should be vaccinated. 2. stallions that are shuttled between northern and southern hemispheres should be tested for eav (neutralizing antibodies and carrier state) by an accredited laboratory. 3. prepubertal colts should be genetically tested for presence of the cxcl16 genotype (cxcl16ss or cxcl16sr) using an allelic discrimination real time pcr. colts that carry the susceptible genotype (cxcl16s) should be vaccinated after 6 months of age. if no genetic testing is to be performed, all colts should be vaccinated between 6 and 12 months of age. 4. more widespread screening of stallion populations for eav, as well as harmonization of various diagnostic tests (e.g. virus isolation, real time pcr, vnt and elisa) as well as utilization of molecular approaches as prescribed tests for international trade by the oie should be promoted and would be highly beneficial to satisfy current demands of the equine industry. 374clinical theriogenology • volume 11, number 3 • september 2019 conclusion equine arteritis virus continues to be an important infectious disease of horses; its occurrence can be associated with substantial economic loss to the equine industry due to abortions, neonatal mortality and establishment of the carrier state. there is sufficient evidence that increase in the incidence of eva in past years is associated with movement of horses for breeding purposes and commercialization of semen. eva prevention and control strategies are primarily focused on identification of carrier stallions; therefore, substantail efforts are being undertaken in this regard, as well as in trying to understand mechanisms of pathogenesis of eav in the stallion reproductive tract. it is anticipated that these will identify host factors associated with persistent infection and may, in the future, enable strategies to induce viral clearance in carrier stallions other than surgical castration. future research focused on improvement of diagnostic assays and vaccines is also of high priority. implementation of strict eva surveillance programs capable of identifying carrier stallions may lead to eav eradication. however, considering large value of international trade of horses, semen, and embryos, enactment of eva eradication programs will require joint international efforts towards implementation of a universal code of practice. acknowledgement studies were supported by the agriculture and food research initiative competitive grant number 2013-68004-20360 from the usda national institute of food and agriculture (usda-nifa). current work on eav is supported by louisiana state university, school of veterinary medicine startup funds (pg 002165) to 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theriogenology • volume 11, number 3 • september 2019 20. balasuriya ub, hedges jf, nadler sa, et al: genetic stability of equine arteritis virus during horizontal and vertical transmission in an outbreak of equine viral arteritis. j gen virol 1999;80:1949-1958. 21. hedges jf, balasuriya ub, timoney pj, et al: genetic divergence with emergence of novel phenotypic variants of equine arteritis virus during persistent infection of stallions. j virol 1999;73:3672-3681. 22. timoney pj, mccollum wh: equine viral arteritis: further characterization of the carrier state in stallions. j reprod fertil suppl 2000;56:3-11. 23. bryans jt, crowe me, doll er, et al: the blood picture and thermal reaction in experimental viral arteritis of horses. cornell vet 1957;47:42-52. 24. del piero f, wilkins pa, lopez jw, et al: equine viral arteritis in newborn foals: clinical, pathological, serological, microbiological and immunohistochemical observations. equine vet j 1997;29:178-185. 25. vaala we, hamir an, dubovi ej, et al: fatal, congenitally acquired infection with equine arteritis virus in a neonatal thoroughbred. equine vet j 1992;24:155-158. 26. zhang j, timoney pj, shuck km, et al: molecular epidemiology and genetic characterization of equine arteritis virus isolates associated with the 2006-2007 multi-state disease occurrence in the usa. j gen virol 2010;91:2286-2301. 27. timoney pj, mccollum wh: equine viral arteritis. vet clin north am equine pract 1993;9:295-309. 28. mccollum wh, timoney pj, wayne r, et al: editors. response of vaccinated and non-vaccinated mares to artificial insemination with semen from stallions persistently infected with equine arteritis virus. fifth international conference of equine infectious diseases; 1988; lexington, ky. 29. campos jr, breheny p, araujo rr, et al: semen quality of stallions challenged with the kentucky 84 strain of equine arteritis virus. theriogenology 2014;82:1068-1079. 30. maclachlan nj, balasuriya ub, rossitto pv, et al: fatal experimental equine arteritis virus infection of a pregnant mare: immunohistochemical staining of viral antigens. j vet diagn invest 1996;8:367-374. 31. zhang j, go yy, huang cm, et al: development and characterization of an infectious cdna clone of the modified live virus vaccine strain of equine arteritis virus. clin vaccine immunol 2012;19:1312-1321. 32. balasuriya ub, snijder ej, heidner hw, et al. development and characterization of an infectious cdna clone of the virulent bucyrus strain of equine arteritis virus. j gen virol 2007;88:918-924. 33. go yy, cook rf, fulgencio jq, et al: assessment of correlation between in vitro cd3+ t cell susceptibility to eav infection and clinical outcome following experimental infection. vet microbiol 2012;157:220-225. 34. carossino m, loynachan at, canisso if, et al: equine arteritis virus has specific tropism for stromal cells and cd8+ t and cd21+ b lymphocytes but not glandular epithelium at the primary site of persistent infection in the stallion reproductive tract. j virol 2017;91:pii: e00418-00417. 35. bryans jt, doll er, knappenberger re: an outbreak of abortion caused by the equine arteritis virus. cornell vet 1957;47:69-75. 36. mccollum wh, timoney pj, tengelsen la: clinical, virological and serological responses of donkeys to intranasal i noculation with the ky-84 strain of equine arteritis virus. j comp pathol 1995;112:207-211. 37. mccollum wh, timoney pj: editors. the pathogenic qualities of the 1984 strain of equine arteritis virus. grayson foundation international conference of thoroughbred breeders organizations; 1984; lexington, ky. 38. balasuriya ub, heidner hw, davis nl, et al: alphavirus replicon particles expressing the two major envelope proteins of equine arteritis virus induce high level protection against challenge with virulent virus in vaccinated horses. vaccine 2002;20:1609-1617. 39. balasuriya ub, snijder ej, van dinten lc, et al: equine arteritis virus derived from an infectious cdna clone is a ttenuated and genetically stable in infected stallions. virology 1999;260:201-208. 40. timoney pj, editor clinical, virological, and epidemiological features of the 1984 outbreak of equine viral arteritis in the thoroughbred population in kentucky, usa. grayson foundation international conference of thoroughbred breeders organizations; 1984; ireland. 41. clayton h: 1986 outbreak of eav in alberta, canada. j equine vet sci 1987;7:101. 42. coignoul fl, cheville nf: pathology of maternal genital tract, placenta, and fetus in equine viral arteritis. vet pathol 1984;21:333-340. 43. mccollum wh, timoney pj, lee jr jw, et al: editors. features of an outbreak of equine viral arteritis on a breeding farm associated with abortion and fatal interstitial pneumonia in neonatal foals. eighth international conference on equine infectious diseases; 1998; dubai, uae. 44. golnik w, michalska z, michalak t: natural equine viral arteritis in foals. schweiz arch tierheilkd 1981; 123:523-533. 45. timoney pj, mccollum wh, murphy tw, et al: the carrier state in equine arteritis virus infection in the stallion with specific emphasis on the venereal mode of virus transmission. j reprod fertil suppl 1987;35:95-102. 46. little tv, holyoak gr, mccollum wh, et al: editors. output of equine arteritis virus from persistently infected stallions is testosterone-dependent. sixth international conference on equine infectious diseases; 1991; cambridge, uk. 47. mccollum wh, little tv, timoney pj, et al: resistance of castrated male horses to attempted establishment of the carrier state with equine arteritis virus. j comp pathol 1994;111:383-388. 48. holyoak gr, little tv, mccollum wh, et al: relationship between onset of puberty and establishment of persistent i nfection with equine arteritis virus in the experimentally infected colt. j comp pathol 1993;109:29-46. 376clinical theriogenology • volume 11, number 3 • september 2019 49. carossino m, dini p, kalbfleisch ts, et al: downregulation of microrna eca-mir-128 in seminal exosomes and enhanced expression of cxcl16 in the stallion reproductive tract are associated with long-term persistence of equine arteritis virus. j virol 2018. 50. carossino m, wagner b, loynachan at, et al: equine arteritis virus elicits a mucosal antibody response in the reproductive tract of persistently infected stallions. clin vaccine immunol 2017;24. 51. campos jr: effects on semen quality and on establishment of persistent equine arteritis virus (eav) infection in stallions following experimental challenge with the kentcky 84 (ky84) strain. lexington, ky: university of kentucky; 2012. 52. timoney pj, mccollum wh, roberts aw: editors. detection of carrier state in stallions persistently infected with equine arteritis virus. american association for equine practitioners (aaep); 1987. 53. olguin perglione c, cordoba m, echeverria mg, et al. equine viral arteritis outbreak in argentina 114 anual meeting of the united states animal health association (usaha); minneapolis, mn2010. 54. timoney pj. factors influencing the international spread of equine diseases. vet clin north am equine pract 2000;16:537-551. 55. timoney pj: the increasing significance of international trade in equids and its influence on the spread of infectious diseases. ann n y acad sci 2000;916:55-60. 56. broaddus cc, balasuriya ub, timoney pj, et al: infection of embryos following insemination of donor mares with equine arteritis virus infective semen. theriogenology 2011;76:47-60. 57. balasuriya ub, evermann jf, hedges jf, et al: serologic and molecular characterization of an abortigenic strain of equine arteritis virus isolated from infective frozen semen and an aborted equine fetus. j am vet med assoc 1998;213:1586-1589. 58. miszczak f, legrand l, balasuriya ub, et al: emergence of novel equine arteritis virus (eav) variants during persistent infection in the stallion: origin of the 2007 french eav outbreak was linked to an eav strain present in the semen of a persistently infected carrier stallion. virology 2012;423:165-174. 59. go yy, zhang j, timoney pj, et al: complex interactions between the major and minor envelope proteins of equine arteritis virus determine its tropism for equine cd3+ t lymphocytes and cd14+ monocytes. j virol 2010;84: 4898-4911. 60. go yy, bailey e, cook dg, et al: genome-wide association study among four horse breeds identifies a common haplotype associated with in vitro cd3+ t cell susceptibility/resistance to equine arteritis virus infection. j virol 2011;85:13174-13184. 61. sarkar s, bailey e, go yy, et al: allelic variation in cxcl16 determines cd3+ t lymphocyte susceptibility to equine arteritis virus infection and establishment of long-term carrier state in the stallion. plos genet 2016;12:e1006467. 62. sarkar s, chelvarajan l, go yy, et al: equine arteritis virus uses equine cxcl16 as an entry receptor. j virol 2016;90:3366-3384. 63. carossino m, dini p, kalbfleisch t, et al: long-term persistent infection with equine arteritis virus is associated with the upregulation of specific cd8+ t lymphocyte transcription factors, inhibitory receptors, and the cxcl16/cxcr6 axis in the ampullae of the stallion reproductive tract. plos pathog 2019;(in revision). 64. bartel dp: micrornas: genomics, biogenesis, mechanism, and function. cell 2004;116:281-297. 65. vojtech l, woo s, hughes s, et al: exosomes in human semen carry a distinctive repertoire of small non-coding rnas with potential regulatory functions. nucleic acids res 2014;42:7290-7304. 66. valadi h, ekstrom k, bossios a, et al: exosome-mediated transfer of mrnas and micrornas is a novel mechanism o f genetic exchange between cells. nat cell biol 2007;9:654-659. 67. balasuriya ub, hedges jf, smalley vl, et al: genetic characterization of equine arteritis virus during persistent infection of stallions. j gen virol 2004;85:379-390. 68. balasuriya ub, hedges jf, maclachlan nj: molecular epidemiology and evolution of equine arteritis virus. adv exp med biol 2001;494:19-24. 69. nam b, mekuria z, carossino m, et al: intra-host selection pressure drives equine arteritis virus evolution during persistent infection in the stallion reproductive tract. j virol 2019. 70. world organisation for animal health (oie). equine viral arteritis. in: oie biological standards commission, manual of diagnostic tests and vaccines for terrestrial animals. 2. 7th edition, paris, france: oie; 2016. 71. world organisation for animal health (oie). infection with equine arteritis virus. in: oie, editor. terrestrial animal health code. 24th edition, paris, france2016. 72. united states department of agriculture animal and plant health inspection service (usda-aphis). equine viral arteritis: uniform methods and rules. in: united states department of agriculture animal and plant health inspection service (usda-aphis). 2004. 73. chung c, wilson c, timoney p, et al: validation of an improved competitive enzyme-linked immunosorbent assay to detect equine arteritis virus antibody. j vet diagn invest 2013;25:727-735. 74. pfahl k, chung c, singleton md, et al: further evaluation and validation of a commercially available competitive elisa (celisa) for the detection of antibodies specific to equine arteritis virus (eav). vet rec 2016;178:95. 377 clinical theriogenology • volume 11, number 3 • september 2019 75. chung cj, grimm al, wilson cl, et al: enhanced sensitivity of an antibody competitive blocking enzyme-linked immunosorbent assay using equine arteritis virus purified by anion-exchange membrane chromatography. j vet diagn invest 2015;27:728-738. 76. chung c, wilson c, timoney p, et al: comparison of an improved competitive enzyme-linked immunosorbent assay with the world organization for animal health-prescribed serum neutralization assay for detection of antibody to equine arteritis virus. j vet diagn invest 2013;25:182-188. 77. balasuriya ub, leutenegger cm, topol jb, et al: detection of equine arteritis virus by real-time taqman reverse t ranscription-pcr assay. j virol methods 2002;101:21-28. 78. miszczak f, shuck km, lu z, et al: evaluation of two magnetic-bead-based viral nucleic acid purification kits and three real-time reverse transcription-pcr reagent systems in two taqman assays for equine arteritis virus detection in semen. j clin microbiol 2011;49:3694-3696. 79. lu z, branscum aj, shuck km, et al: comparison of two real-time reverse transcription polymerase chain reaction assays for the detection of equine arteritis virus nucleic acid in equine semen and tissue culture fluid. j vet diagn invest 2008;20:147-155. 80. fukunaga y, wada r, sugita s, et al: in vitro detection of equine arteritis virus from seminal plasma for identification of carrier stallions. j vet med sci 2000;62:643-646. 81. gilbert sa, timoney pj, mccollum wh, et al. detection of equine arteritis virus in the semen of carrier stallions by using a sensitive nested pcr assay. j clin microbiol 1997;35:2181-2183. 82. mankoc s, hostnik p, grom j, et al: comparison of different molecular methods for assessment of equine arteritis virus (eav) infection: a novel one-step mgb real-time rt-pcr assay, pcr-elisa and classical rt-pcr for detection of highly diverse sequences of slovenian eav variants. j virol methods 2007;146:341-354. 83. ramina a, dalla valle l, de mas s, et al: detection of equine arteritis virus in semen by reverse transcriptase p olymerase chain reaction-elisa. comp immunol microbiol infect dis 1999;22:187-197. 84. st-laurent g, morin g, archambault d. detection of equine arteritis virus following amplification of structural and n onstructural viral genes by reverse transcription-pcr. j clin microbiol 1994;32:658-665. 85. starick e: rapid and sensitive detection of equine arteritis virus in semen and tissue samples by reverse transcriptionpolymerase chain reaction, dot blot hybridisation and nested polymerase chain reaction. acta virol 1998;42:333-339. 86. westcott dg, king dp, drew tw, et al: use of an internal standard in a closed one-tube rt-pcr for the detection of equine arteritis virus rna with fluorescent probes. vet res 2003;34:165-176. 87. hans a, gaudaire d, manuguerra jc, et al: combination of an unbiased amplification method and a resequencing microarray for detecting and genotyping equine arteritis virus. j clin microbiol 2015;53:287-291. 88. carossino m, lee pa, nam b, et al: development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (rt-iipcr) assay for detection of equine arteritis virus in equine semen and tissue samples using the pockit system. j virol methods 2016;234:7-15. 89. carossino m, loynachan at, james maclachlan n, et al: detection of equine arteritis virus by two chromogenic rna in situ hybridization assays (conventional and rnascope(r)) and assessment of their performance in tissues from aborted equine fetuses. arch virol 2016;161:3125-3136. 90. timoney pj, mccollum wh, roberts aw, et al: status of equine viral arteritis in kentucky for 1986. vet rec 1987;120:282. 378clinical theriogenology • volume 11, number 3 • september 2019 2018: the 2018 bartlett address: ruminations from the back 40 the 2018 bartlett address ruminations from the back 40 walter johnson professor (retired), department of population medicine, ontario veterinary college, university of guelph, guelph, on, canada it is a wonderful and great honor to be the recipient of the 2018 bartlett award. previous bartlett award winners comprise a ‘who’s-who’ list of distinguished theriogenologists, and to be included in this esteemed group is a humbling experience. it is gratifying to be recognized in this manner by colleagues. it is an interesting exercise to reflect upon one’s professional life and career. i grew up on a mixed farm in the peace river district of northern alberta. my father was born on that farm in a pioneer environment and spent his life growing his farm into a very successful enterprise. my mother was validictorian at her graduation from the school of nursing at the vancouver general hospital. initially their farm was a traditional mixed enterprise with work and saddle horses, beef and milk cows, pigs, chickens, dogs and cats, transitioning from the horse era to the machinery era. as a result i gained experience with a wide variety of farm animals, an excellent background for a budding veterinarian. there are many people who had an influence on my development as a person and my professional career. of course the first were my parents. they encouraged their children to work hard, participate in all sports and community activities, and get a good education. more currently, my present family, wife joyce, daughter christine and two lovely grandaughters sadie and eliza, have a huge influence on my wellbeing. following graduation from the western college of veterinary medicine (wcvm) in 1972, i worked in a mixed practice in fairview, alberta with dr. jim henderson. it was a new practice expanding to two veterinarians, and in 1974 it expanded again to three veterinarians with the addition of dr. richard mcwatt. it was basically a beef cattle practice with a few dairies, the occasional swine and small ruminant case, and a significant small animal component. a mixed practice such as this is great training for a young, novice veterinarian as it requires a broad base of talents in a variety of disciplines and people skills. the experience provides a solid foundation for young veterinarians who may later transition to professional specialization. in 1978 i returned to wcvm to do graduate studies, obtaining a masters of veterinary science (mvsc) in 1980. the mvsc is a combination clinical and research program. my research component investigated factors governing maintenance of pregnancy in cows. it was a huge learning experience on how to develop, perform and publish a research project. a large portion of the clinical experience included the development of non-surgical bovine embryo transfer techniques. the mvsc program is multi-spieces, including considerable equine and canine reproduction. the broadly based program provided a solid foundation for future professional aspirations. for the next several years i worked for the wcvm reproductive research trust providing onfarm embryo transfer service to producers in western canada. it was a rewarding experience to work with some of the best cattle, on the best farms and ranches, with the best producers in western canada. it was an exhilerating experience to participate in the production of ‘ litters’ of calves from top quality cows. in 1983 i joined the theriogenology group at the ontario veterinary college (ovc). at that time the ovc was in an expansion phase with an increase in faculty and new physical facilities. both of the incumbent theriogenology faculty were leaving, dr. barker retiring and dr. bosu moving to wisconsin. the new theriogenology group consisted of four rookies, brian buckrell, wayne etherington, neil savage and myself, necessitating in a dramatic and steep learning curve for each of us for survival. my position at the ovc included teaching undergraduate and graduate students, providing clinical service in the hospital, managing a research program and administrative responsiblities. in 1986, i became a diplomate, american college of theriogenologists. my tenure at ovc was an enormously rewarding career. veterinary students are the cream-ofthe-crop, highly selected, smart, motivated, intelligent young people and are a pleasure to be associated clinical theriogenology • volume 10, number 3 • september 2018159 with. i remained at ovc, progressing through the various academic rankings, until retirement as a professor in 2008. the past 40 years has brought many prominent advancements to the veterinary profession. specifically influencing the theriogenology discipline, some of the more notable were the introduction of prostaglandin and gonadotropin releasing hormone (gnrh), the development of ultrasonography, the advancement of in vitro fertilization (ivf), and the discovery of cloning. recent graduates accept these as routine procedures. what changes might occur in the next half century? my community committment included serving on the board of the college of veterinarians of ontario (cvo), the licensing body for veterinarians in ontario, becoming president in 1999. during this time i served on the national examining board committee. one of the responsibilities of this committee was participation in the avma veterinary college accreditation program. it was a great learning experience to observe the strengths and weaknesses of other veterinary colleges. i also served on the board of the canadian embryo transfer association (ceta) for many years after being part of the team responsible for the inception of ceta, concluding with a lifetime membership. it is always a satisfying experience to participate in the administration of the various organizations with which you are involved and which impact your life. there are many people to recognize for their contribution to my career journey. a few of these colleagues follow but the list is long and i apologize to those i’ve missed. dr. reuben mapletoft. when i returned to graduate school in 1978, dr. mapletoft had recently joined the faculty at the wcvm bringing his interest in bovine embryo transfer. it was an exciting time as the embryo transfer industry was changing from the clinical, surgical procedures to the more practical onfarm, non-surgical techniques. i had the great pleasure of working very closely with reuben for several years and we remain friends and colleagues. drs. bill cates and frank bristol were the theriogenolgy faculty at wcvm during my undergraduate and graduate programs and were very instrumental in my basic theriogenology training. dr. bill adams was primary advisor for my graduate program, providing wonderful guidance and friendship. drs. brian buckrell, cathy gartley and tracey chenier were colleagues and friends with whom i had the pleasure of working in the theriogenology group at ovc. drs. keith betteridge, stanley leibo and nada loskutoff were faculty in the ovc embryo research laboratory, with whom i worked on many projects. dr. wayne martin, an epidemiologist, was the initial chair of the department of population medicine, and taught experimental design and statistical analysis to faculty and graduate students, always encouraging us to plan quality research projects. one of the most enjoyable parts of my career was involvement with graduate students, whether as the primary advisory, committee member or supportive faculty. we were blessed with many wonderful graduate students, each bringing their own interests and expertise to our program. james thompson is on faculty at the college of veterinary medicine, texas a & m, cathy gartley provides small animal theriogenology service at ovc, tracey chenier provides equine theriogenolgy service at ovc, claudia jimenez is at facultad de medicina veterinaria y de zootecnia,universidad nacional de colombia, bogata, columbia, alejandro estrada is at facultad de medicine veterinaria y zootechnia, universidad veracruzana, veracruz, mexico, ramanathan kasimanickam is on faculty at the college of veterinary medicine, washington state university, cyril stephen is on faculty at the school of animal and veterinary science, charles sturt university, waga waga, nsw, australia, ken christy provides bovine embryo transfer service in ontario, lee morris worked with dr.t.allen in cambridge, england and presently provides assisted reproductive technologies in equine practice in new zealand, pedro jou provided a speciality reproductive equine practice in ontario until his early death, rasa levstein is in small animal practice in ontario, scott madill is in veterinary practice in australia, andria acs works with norvartis health in lausanne, switzerland, and hernan ortegon is with the alberta department of agiculture. the doctor of veterinary science (dvsc) program includes a research component including writing a thesis, and a multi-species clinical component. many of our graduate students have successfully clinical theriogenology • volume 10, number 3 • september 2018 160 completed the act board examination, becoming diplomates of the american college of theriogenologists. i am a strong advocate of multi-species undergraduate and graduate student programs as i believe the comparative medicine aspect of veterinary medicine provides a very solid career foundation. it is my understanding that the theriogenology programs at many colleges are being downsized. this is certainly the case at ovc where there are now two theriogenology faculty, down from four faculty several years ago. it is well documented that efficient, economic survival of any animal production operation begins with a successful reproductive program. our college of theriogenologists must continue to lobby deans of veterinary colleges in the importance of quality theriogenology teaching programs in their schools. in conclusion, i consider myself one of the lucky people who enjoyed going to work every day. the veterinary profession is a wonderful profession, and teaching veterinary students is challenging and extremely rewarding. recognition by colleagues as the recipient of the 2018 bartlett award is a wonderful conclusion. thank you. clinical theriogenology • volume 10, number 3 • september 2018161 clinical theriogenology • volume 10, number 3 • september 2018 162 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2011: the value of pregnancy diagnosis a revisit to an old art the value of pregnancy diagnosis – a revisit to an old art james d. ferguson, david t. galligan university of pennsylvania, school of veterinary medicine, kennett square, pa abstract rectal palpation (rp) for pregnancy diagnosis between 35 to 50 days post-insemination has been a fixture of veterinary practice since the 1950’s. in the last decade ultrasound (us) has replaced palpation for pregnancy diagnosis as it facilitates early days to diagnosis, typically 26 to 28 days post-insemination. more recently, blood tests for bovine pregnancy proteins (bp) have become available. these tests typically can be used by 30 days post-insemination in cows more than 90 days post-calving. in general all three tests have similar sensitivity and specificity for pregnancy diagnosis. attributes differ slightly in cost, but differ significantly in veterinary involvement in performing the test. blood pregnancy testing may be performed by cattle managers with no veterinary intervention, which has significant implications for veterinary practice. no matter the test, the value of pregnancy diagnosis is in identifying non-pregnant cows earlier post-insemination and returning them to a re-insemination program. heat detection rate (hdr) and conception rate (cr) influence the optimal days post-insemination for pregnancy diagnosis. as hdr and cr increase, the value of pregnancy diagnosis must be performed earlier post-insemination. however, earlier pregnancy diagnosis post-insemination detects more pregnancies which will be lost due to early embryonic mortality, dictating re-testing of pregnant animals in a more timely fashion. herd programs structured on weekly and biweekly testing are more valuable than those scheduled monthly. the optimal pregnancy test post-insemination is influenced by eed, hdr and cr, number of cows tested within each period and cost of herd visit and test. programs should be structured on a herd-to-herd basis based on utility to the producer. keywords: pregnancy diagnosis, dairy cattle, rectal palpation, ultrasound, biopryn introduction the value of a pregnant cows decreases with increasing days in milk at pregnancy (days open, figure 1). the value of a pregnant cow is influenced by days open, the value of milk, the value of a calf, the cost of replacement for reproductive failure, and the age and producing value of the cow.1 as seen in figure 1, with a milk value of $0.10/lb the value of a day open is -$2.18/day; if milk is valued at $0.17/lb then the value of a day open is -$3.48 (calves are valued at $100/head, replacement heifers at $1200/head and cull reproductive cows at $500/head for the analysis). the essence of a reproductive program is “to get cows pregnant fast” after calving. driving this system are insemination efficiency (hdr) and cr (defined as cows determined pregnant after insemination by a diagnostic test). the combination of these events is the pregnancy rate, the proportion of cows pregnant every 21 days from the voluntary waiting period (vwp). heat detection is nuanced, as management of first insemination is different than management of repeat insemination due to the phenomenon of early embryonic death (eed) and the need to ensure cows are not pregnant prior to applying technologies to control estrus. hence the value of routine pregnancy examination; non-pregnant cows (open cows) may be identified sooner post-insemination than management may observe and reassigned to an insemination protocol, reducing days between inseminations and days open. rectal palpation for pregnancy examination became a staple of veterinary practice due to the convergence of several changes in the dairy industry: artificial insemination (ai), increasing herd size, and the recognition of the economic value of reduced days open. however, ai necessitated accurate estrus detection, which became more difficult with larger cattle groups and more confinement on concrete floors. with these developments in the 1950’s and 1960’s, the veterinary profession was able to promote the economic benefit of routine reproductive examinations as an aid in the control of reproductive performance. reproductive programs were marketed under the concept of “herd health” as they benefited the economic performance of the farm by improving the performance of aggregate groups of cows. it was clinical theriogenology • volume 3 number 4 • december 2011559 clinical theriogenology • volume 3 number 4 • december 2011559 a win-win situation for the dairy producer and the veterinarian. in many regions, veterinary services became structured around the herd check, a regularly scheduled visit of the veterinarian to the farm to examine cows for reproductive status. diagnosis of open and pregnant cows has been based on manual examination of the reproductive tract per rectum between 35 to 50 days post-insemination.2-4 positive diagnosis of pregnancy was defined by the palpable presence of an embryonic vesicle, fetal membrane slip, or a fetus. absence of these structures indicated an “open cow”, identified for return to estrus. rectal palpation was viewed as safe for the fetus and reliable as a diagnostic tool, although pregnancy losses associated with rp were variable between palpators. 3,5,6 abbitt et al observed that palpation for fluctuation of fluid alone was accurate and safe for pregnancy diagnosis, as membrane slip increased fetal loss. with advancement of skill, practitioners found they could reduce the days post-insemination for examination, and many veteran practitioners could successfully palpate cows from 30 days post-breeding. a diagnosis of “open” in a cow, to be of value, must reduce the days to next insemination compared with days if the diagnosis had not been made.7,8 thus, reproductive examinations should be linked with management interventions that make use of the information to reduce the days to next insemination.8-10 prostaglandin f2α (pgf) treatment in open cows with a mature corpus luteum makes it possible to shorten the time to next estrus and focus attention on estrus detection in treated cows. it is critical to accurately identify an open cow so pgf will not abort a pregnant cow. in addition the diagnostic test should not cause injury of the embryo which can result in abortion. incorporation of gonadotropin releasing hormone (gnrh) injections in sequence with pgf treatment has made it possible to schedule ai at specific times after a diagnosis of not pregnant, enhancing the likelihood that a cow is re-inseminated in a timely fashion.11,12 gonadotropin releasing hormone in combination with pgf can be used for timed ai from about 26 days post-insemination in open cows.11 the association between a veterinary “open” diagnosis with application of a managed breeding program has greatly increased reproductive efficiency in dairy herds. currently, us and bp have begun to replace the time honored art of rp for pregnancy diagnosis13-15 ultrasound may reduce the days post-insemination for a diagnosis of pregnancy to 26 to 28 days post-insemination for a skilled operator.15-17 the bp test is diagnostic from 30 days postinsemination as long as the cow is at least 90 days post-calving.14,17 both these tests offer producers options for employing methods to manage repeat insemination in open dairy cows. veterinary reproductive diagnostic programs interface with management in controlling days between sequential inseminations. the value in reduction in days to pregnancy is a function of the value of a day open.9 the relative value of the diagnostic test is determined by the reduction in days relative to not having performed the test multiplied by the proportion of cows influenced by the test. therefore the value of pregnancy diagnosis will be influenced by the time post-insemination the test may be performed, the proportion of cows available to be tested, and the errors associated with the test. costs associated with testing include the test itself, factors associated with employing the test (for the veterinarian: the trip fee to the farm; for the producer: the time spent to perform the herd check or bleeding cows, for the blood test, the cost of mailing and time waiting for information), and possible losses associated with misdiagnosis or time delays due to a questionable diagnosis which requires re-testing at a later period. the distribution of days between inseminations is presented in table 1 for several scenarios. the days are divided into categories as follows: <10 days (heat detection errors); 10 to 17 days (heat detection errors and short intervals); 18 to 24 days (normal estrous interval); 25 to 35 days (heat detection errors, extended interval due to eed); 36 to 48 days (a long interval due to a missed earlier estrus or a long interval due to eed); >= 49 days (long intervals). the proportion of cows that have a period over 48 days between inseminations ranges from 5.6% to 51.9% (table 1). the proportion over 48 days is related to the hdr and the reproductive management program employed by the farm management. since most pregnancy tests are only viable at the earliest 26 to 28 days post-insemination and at best only 60% of cows can only be induced into estrus for re-insemination by 3 to 5 days after a diagnosis of not pregnant with a pgf injection, routine pregnancy testing can only significantly reduce the proportion of cows that have more than 48 days between inseminations. clinical theriogenology • volume 3 number 4 • december 2011 560clinical theriogenology • volume 3 number 4 • december 2011 560 the proportion of pregnancies lost from the days post-insemination of a first pregnancy diagnosis to a second confirming diagnosis of pregnancy at 65 to 70 days post-insemination are presented in figure 2 (early embryonic [and fetal] death). first diagnosis of pregnancy was made by various methods depending on the day of examination and the figure is a compilation across multiple studies (references available on request) in dairy cows. the proportion of pregnancy loss is high at 21 to 24 days postinsemination. from 5 to 25% of pregnancies were lost by 65 days post-insemination in studies where first examination was made at 26 to 32 days post-insemination (figure 2). losses were 5 to 19% when first examination was between 37 to 45 days post-insemination and only 2% when first examination was at 50 days post-insemination (figure 2). embryonic and fetal loss is variable between studies and therefore it would be expected to be variable across herds. it may be expected that eed would influence the value of early pregnancy diagnosis by increasing the apparent “false pregnancy” diagnosis via higher pregnancy loss post-diagnosis. this could extend days between insemination in a proportion of cows by withholding them from re-breeding for a period of time. slightly later pregnancy testing will be associated with less eed, reducing “false pregnancy” diagnosis, but increasing days between insemination for cows not pregnant at diagnosis. early pregnancy diagnosis (28 to 32 days) has value in identifying open cows sooner, but has a negative value in determining pregnancy in a proportion of cows that will be open in the next seven to 14 days, which may delay repeat insemination in this group of animals if not seen in estrus and re-examination does not occur until 65 days post-insemination. thus, early open diagnosis must be weighed against increased eed in pregnant cows when examined early post-insemination. materials and methods several models were constructed based on a decision tree for flow of pregnant and open cows presented in figure 3. pregnancy was modeled based on the structure of pregnancy losses in figure 2 as follows: cr (defined as pregnant cows by days post-insemination) = 0.37 + 107.1*exp(-0.2845*d); where d = days post-insemination. the intercept, 0.37, was then varied to examine cr from 0.22 to 0.54 with a similar form of eed. embryonic and fetal losses by 65 days post-insemination as a function of days post-insemination was modeled from figure 2 as follows: eed = -0.02 – 50*exp(-0.23*d); where d= days post-insemination. all models were constructed to control number of days to next insemination in open cows and cows with eed following a prior first service. days to next insemination were either determined by hdr or were based on assigning cows to a re-synchronization program at time of pregnancy diagnosis. mean days to next insemination for an underlying hdr was estimated as 21*1/hdr. days to next insemination were valued based on the cost of a day open, which ranged from $2.00 to $5.00. diagnostic tests for pregnancy were valued as the reduction in days to next insemination possible through routine herd visits compared to a management program with no pregnancy testing and days to next insemination was managed based on hdr. the first model examined the influence of hdr and cr on the value of time of pregnancy testing from 21 to 60 days post-insemination. heat detection rate and cr could vary; eed was set to zero. this model was used to examine the general value of time of examination post-insemination with varying hdr and cr when sensitivity and specificity were 100%. cost of examination was $3.00 and value of a day open was $2.25. secondly, a model was constructed to compare three time periods post-insemination for pregnancy diagnosis: 28 days, 32 days and 39 days. this model was used to examination the interactions of sensitivity and specificity of a test on the value of the test. sensitivity and specificity were varied from 70% to 100% and eed was varied by time post-insemination by weighting the coefficients in the model clinical theriogenology • volume 3 number 4 • december 2011561 clinical theriogenology • volume 3 number 4 • december 2011561 described above. pregnancy losses varied at 28 days post-insemination from 5.6 to 19.5%, at 32 days from 2.3 to 12.4%, and at 39 days post-insemination from 0.3 to 3.3%. cows which lost a pregnancy could first be seen in estrus by one week after pregnancy loss and then 21 days later if not inseminated. if not seen in estrus, they were examined at 65 days, along with all previously pregnant cows, and if open, assigned to a re-synchronization program. the value of a day open was $2.25 and the cost of a test was $3.00. a third model was constructed to evaluate three pregnancy test programs: us begun at 26 days post-insemination; rp begun at 35 days post-insemination; and bp begun at 32 days post-insemination. these programs were then modeled for a herd visit weekly, biweekly, triweekly (data not shown), or monthly. cost of tests varied between $1.00 to $9.00 for us, $0.50 to $5.00 for rp, and $2.00 to $6.00 for bp. trip fee for us and rp examinations varied from $9.00 to $100, and cows examined per test period ranged from 1 to 100 animals. the basis of each test was the identification of true pregnancies (sensitivity of the test) and true open cows (specificity). sensitivity and specificity for each test were varied from 0.8 to 1.0. a decision tree was constructed based on possible diagnostic findings at pregnancy examination: a cow may be found open (not pregnant), pregnant, or be classified for re-examination if diagnosis was not clear (questionable diagnosis). data for bp suggest that 10% to 15% of samples may have questionable diagnosis. these cows were retested at the next scheduled herd test for model three, which was weekly, biweekly, triweekly, or monthly. pregnant cows could lose the pregnancy as a function of a model fit to the loss data in figure 2 and as described above. pregnancy losses varied at 28 days post-insemination from 5.6 to 19.5%, at 32 days from 2.3 to 12.4%, and at 39 days post-insemination from 0.3 to 3.3%. all pregnant cows at the first examination were re-examined at 65 days post-insemination. all eed cows could be observed in estrus beginning one week following the eed and then were assumed to have normal estrous cycles and were re-inseminated based on hdr. for all programs and diagnostic tests, cows classified as open at examination were assigned to a re-synchronization protocol.11,12 re-synchronization resulted in re-insemination of open cows ten days after the days at pregnancy test. false positive cows, cows diagnosed as pregnant that were open, lost days to next insemination due to the false information and could be re-inseminated based on the hdr. false open cows, cows truly pregnant diagnosed as open, were aborted as they were placed into a resynchronization program. this resulted in a days to next insemination of ten days from the time of examination. the cost of abortion of pregnant cows was assigned as marginal loss of -$46. the additional loss assigned to an aborted cow was not the full value of the pregnancy, as these cows were reinseminated within ten days from the time of examination. superimposed on the value of test information for reproductive status is the frequency of herd visit, typically weekly, bi-weekly, or monthly. statistical models for models two and three combined categorical and continuous variables in regression models using proc glm in sas statistical software. statistical models examined value (value of reduced days open – cost of test and trip fee) as a function of hdr, cr, exam, eed, sensitivity, and specificity. models were as follows: model two: value = u + examk + hdri + crj + eed + sens + spec + eijkl where u = overall mean, examk = the kth examination time post-insemination, 28, 32, 29, hdri = ith hdr, 03, 0.4, 0.5, 0.6., 0.7, 0.8, crj = jth cr, 0.30, 0.40, eed = as continuous variable from 1 to 6% of pregnancy, sens = sensitivity as a continuous variable from 0.7 to 1.0, spec = specificity as a continuous variable from 0.7 to 1.0, eijkl = residual error model three regressed value of program (us examination beginning at 26 days post-insemination, rp beginning at 35 days post-insemination, and bp beginning at 32 days post-insemination) for herd visits clinical theriogenology • volume 3 number 4 • december 2011 562clinical theriogenology • volume 3 number 4 • december 2011 562 performed weekly, bi-weekly or monthly on the inverse of hdr, the inverse of cr, the percent eed based on time of examination, the number of cows tested at each frequency of test, the cost of herd visit, the cost of the test, the value of an open day, and the sensitivity and specificity of each test. in addition, the bp test had questionable test results. up to three levels of interaction between variables of inverse hdr, cr, sensitivity and specificity were included in the model. backward elimination was performed when three way interactions were not significant. proc glm in sas statistical software was used with the solution option for the regression analysis. results and discussion a total of 882 and 2005 simulations were done for model two and model three, respectively. model one was used to examine the influence of hdr and cr on losses based on days to next insemination in a deterministic fashion. in general, pregnancy testing prior to 42 days (36 to 48 days) post-insemination is more valuable than after 42 days, as this corresponds to open cows passing through a second opportunity for estrus detection and re-insemination, reducing the proportion of open cows the test may benefit (figure 4). as hdr increased the value of pregnancy testing decreased. as hdr increases to 70%, pregnancy testing becomes less valuable, and may be negative in value even prior to 36 (42) days because more cows are observed in estrus and re-inseminated. an increase in cr has a similar influence on decreasing value of examination, but the influence has less magnitude than for hdr (figure 5). after a pregnancy test, the probability of re-insemination has to be increased by 50% or more for the pregnancy test to be of value.7 not shown is the influence of the boost in hdr needed for the examination to be profitable. model one assumed a100% efficiency in boost in re-insemination; if the boost in re-insemination efficiency declined below 60%, the value of a pregnancy test prior to 42 days was lost. this is slightly higher than that observed by oltenacu et al,7 but the relative value will be influenced by the value of a day open and cost of the test. oltenacu et al found that risk of aborting a cow had to be less than 5% for rp to be profitable.7 in model two, the risk of aborting a pregnant cow was related to sensitivity of the test, as cows diagnosed as open were assigned to a re-synchronization program. in figures 6 and 7 and table 2 the influence of sensitivity and specificity of a pregnancy test on value of the test is presented. the influence of sensitivity is almost four-fold the effect of specificity (table 2) and the relative value of pregnancy testing becomes negative with decreasing sensitivity below 90% with varying hdr and cr (figure 6). specificity can decrease to 70% and still have a positive value with varying cr and hdr (figure 7). reducing sensitivity decreased the value of pregnancy diagnosis due to losses associated with aborting a cow through missed diagnosis as open. these cows were assigned to a re-synchronization program, forcing re-insemination in ten days. this reduced the losses of aborting a pregnant cow compared with losses due to damage to the embryo, which may delay diagnosis to time of re-examination and prolong time to re-insemination, increasing possibility of culling due to long days open. it is likely that rp would have the greatest probability of causing abortion compared to us, and the bp test would have little chance of inducing abortion. decreasing specificity in model two was associated with decreasing test value, as cows falsely diagnosed as pregnant were not assigned to re-synchronization, and these cows may not be found open until re-examination at 65 days. increasing hdr decreased these losses, as open cows would have a higher probability of being seen in estrus at an earlier time than 65 days at re-examination. if specificity of a test is low, it may be more profitable to re-examine pregnant cows sooner than 65 days, particularly if scheduled visits are done frequently, but the test would need to have a high sensitivity. in general rp, us, and bp tests increase in sensitivity with time post-insemination and false diagnosis should decline with time post-insemination.13,15-17,19 pregnancy losses after re-examination increase the risk of culling, as these cows will not be re-examined, and therefore become more costly. this was not modeled in this paper. in model two, questionable diagnosis was assessed based on 0 to 15% of examinations. cows with questionable diagnosis were re-examined at the next scheduled herd visit, weekly, bi-weekly, or monthly. later examination improved sensitivity and specificity of all tests. questionable diagnosis was clinical theriogenology • volume 3 number 4 • december 2011563 clinical theriogenology • volume 3 number 4 • december 2011563 not as negative as decreasing sensitivity or specificity, particularly on a one week program, as cows were more likely to have a correct diagnosis and, if open, be re-assigned to a re-insemination program sooner. cost of examination increased slightly, but if a herd visit was scheduled, or if blood samples scheduled for collection, then the cost was small compared to the error of a false diagnosis. the bp test is more likely to have a questionable diagnosis than rp or us. questionable diagnosis with re-examination was preferable to false diagnosis. a problem with rp and us is that operators tend to view their diagnosis as 100% accurate, when they are not. thus, questionable diagnosis may be overlooked as an option in fewer examinations than should be. factors identified as significant in model one and two which influence the value of a pregnancy test include underlying hdr and cr, time post-insemination the test is done, the sensitivity and specificity of the test, and abortion caused by the test. this is consistent with galligan et al who found hdr and sensitivity of a test the two major factors influencing value.9 in general, the test should be as early as possible post-insemination, it should be combined with a re-synchronization program, and it should have a high sensitivity. model three compared three programs: us examination at 26 days, bp test at 32 days, and rp at 35 days post-insemination, scheduled at varying frequencies (tables 3, 4 and 5). for weekly herd visits, the us model examined cows from 26 to 32 days post-insemination; for the bp test cows were tested from 32 to 38 days post-insemination; for rp cows were tested from 35 to 41 days post-insemination. the days post-insemination were increased for the bi-weekly and monthly herd visits based on the time interval of visits. for model three, sensitivity and specificity varied from 80 to 100%, the baseline level of 80% determined from model two. these ranges included reported or calculated sensitivity and specificity of us, rp, and the blood pregnancy test.13,14,16,20,21 overall, all three tests had similar sensitivity and specificity, but optimal time of examination differ. two thousand five simulations were run to examine the value of the three methods and programs. cows available for examination for each test period ranged from 1 to 100 cows, with a mean of 15.9 (sd 12.0) cows, which would correspond to a herd size of approximately 500 cows having a weekly herd check. the mean trip fee was $50.00 (sd $25.10) and ranged from $9 to $100 per visit. mean value of days open was $2.80 (sd $ 0.70) and ranged from $2.00 to $5.00. mean value for hdr was 0.477 (sd 0.21, range 0.1 to 0.9) and mean value for cr was 0.38 (sd 0.06; range 0.222 to 0.517). embryonic mortality was modeled based on figure 2 and was higher for earlier examinations. questionable diagnoses were re-examined at the next scheduled herd test. mean values for benefit of the three programs by hdr were similar (table 3) when sensitivity and specificity and costs of the tests were similar and the average trip fee was $50.00 and 15 cows were examined at each period. weekly and bi-weekly programs were not greatly different in value, although biweekly programs tended to have slightly lower value relative to weekly visits (table 3; figure 8 presents the difference in biweekly and monthly programs relative to a weekly herd program). weekly and bi-weekly programs were similar as most cows were examined prior to 42 days post-insemination. monthly (and tri-weekly, data not shown) programs had a lower value as more cows were examined after 42 days post-insemination, giving a second opportunity for estrus detection, reducing open cows that could be managed by the test. it was anticipated that eed would influence the time of the test, but the effect was not significant even with rates varying from 4 to 20%, particularly for us at 26 days. in general, values for all three weekly programs were very similar as a function of hdr (figure 9). each program became negative in value when hdr exceeded 60%. low hdr herds obtain the greatest benefit from a consistent program. possibly depending on labor costs for hdr on a dairy, herd managers should consider using routine herd programs to control post-insemination and eliminate hdr. this would depend on relative costs of herd testing versus labor for heat detection. table 4 presents regression models for the weekly herd visits for the three pregnancy tests. heat detection rate and cr had significant interaction influencing value of testing in all programs, which is not surprising given the data in model one. interestingly, hdr interacted with sensitivity and specificity as did sensitivity and specificity with each other. this interaction represents changing predictive value positive and negative with changes in underlying prevalence of open and pregnant cows at examination. clinical theriogenology • volume 3 number 4 • december 2011 564clinical theriogenology • volume 3 number 4 • december 2011 564 heat detection rate (and to a lesser extent cr) strongly influence open cows at time of examination, altering prevalence, changing predictive values for the tests. these interactions capture this effect. surprisingly, cost of an us test, varying between $1 to $9, did not significantly influence value of examination. cost of rp and to a lesser extent, cost of the bp test influenced value. possibly this was related to the earlier time of examination in the us program versus the bp and rp programs. trip fee (for rp and us) and number of cows tested at examination did influence value of a test and the program. as cost of herd visit increased, value of us and rp declined. this did not influence bp, as no herd visit is associated with the test. in general, if fewer than ten cows were tested then bp test was more valuable than us or rp (figure 10, table 5). however, this benefit may change with differences in sensitivity and specificity and cost of test (table 5). if the sensitivity or specificity of the bp test decreased then us became more valuable a test at similar costs. rectal palpation in general was less valuable, as it was performed later days post-insemination. however, if rp at 39 days were combined with routine gnrh at 32 days in all cows in a re-synchronization program, then rp may be a more valuable option. this was not modeled in this paper and would depend on relative costs of gnrh and rp. based on published literature, sensitivity of us and rp have a value of 0.97 and a specificity of 0.87.13,15,16 the bp test has a sensitivity of 0.90 to 0.95 and a specificity of 0.85 to 0.90.13,14,20 if costs of the tests are $2.75 to $3.00 and a trip fee is $50 and at least ten cows are examined each week, then relative values of the programs would be: us, $37.00; rp, $35.10; bp, $37.80 to $34.96, with lower or higher specificity. these values are similar and suggest there is no strong economic difference among the tests. herd managers should select a program which integrates with their management system and affords them the greatest flexibility in managing their time for pregnancy testing. the effects of management were not included in this model. conclusion at the sensitivity and specificity (80 to 100%), questionable diagnosis (10 to 15%, for the bp test) and cost ($2.00 to $5.00 per test) examined in this study, us, rp, and bp provide similar options for producers for pregnancy testing, if incorporated with a herd re-insemination program. the value of all three tests was influenced by underlying heat detection and cr. heat detection had the greatest influence on the value of the tests, value decreasing with increasing hdr. weekly or biweekly test intervals are more valuable than monthly or tri-weekly intervals (not shown), even when only one to two cows are tested, particularly for the bp test when there are fewer than ten cows to examine. producers and veterinarians need to design herd programs which integrate pregnancy testing with re-insemination programs which have the greatest utility for the producer. questionable diagnosis was not as negative, as cows were re-examined one week later, increasing cost of examinations to a small extent, but not aborting cows by assigning them to re-synchronization programs and more quickly assigning open cows to re-insemination. after first insemination, identifying pregnant and non-pregnant (open) cows as early as feasible has economic value to the producer if open cows can be re-inseminated more quickly.7,8 resynchronization protocols11,12 have been effective when gnrh injections have been initiated between 25 to 33 days post-insemination. this requires an accurate pregnancy diagnosis prior to an injection of pgf one week following the gnrh injection. pregnancy diagnosis may be made using the blood bp test, us, or by rp.12 what has become apparent with earlier pregnancy diagnosis is the high rate of eed which occurs between 28 to 42 days post-insemination and continues up to 90 days post-insemination. embryonic loss rates from 28 to 42 days have typically been in the range of 10 to 20% and have averaged about 14%.22,23 fetal loss from 42 days to 90 days has been lower, but still has been in the range of 5%.22,23 in fact, using serum progesterone concentrations at 20 to 23 days post-insemination, 60% to 70% of cows have elevated progesterone,24 suggesting they are not returning to estrus, but only approximately half of these cows are pregnant by 30 to 40 days post-insemination. reproductive efficiency is greatly reduced by the high loss of embryos from 21 to 42 days post-insemination. twin pregnancies, low body clinical theriogenology • volume 3 number 4 • december 2011565 clinical theriogenology • volume 3 number 4 • december 2011565 condition, uterine infection, and heat stress have been implicated as causes23 but many losses occur with no apparent cause. references 1. devries a: economic value of pregnancy in dairy cattle. j dairy sci 2006;89:3876-3885. 2. wisnicky w, casidale: a manual method for the diagnosis of pregnancy in cattle at an early stage of gestation. j am vet med assoc 1948;113:451-452. 3. abbitt b, ball l, kitto gp, et al: effect of three methods of palpation for pregnancy diagnosis per rectum on embryonic and fetal attrition. j am vet med assoc 1978;173:973-977. 4. thompson ja, marsh we, calvin ja, et al: pregnancy attrition associated with pregnancy testing by rectal palpation. j dairy sci 1994;77:3382-3387. 5. gowan ew, etches rj, bryden c, et al: factors affecting the accuracy of pregnancy diagnosis in cattle. j dairy sci 1982;65:1294-1302. 6. thurman mc, picanso jp: fetal loss associated with palpation per rectum to diagnose pregnancy in cows. j am vet med assoc 1993;203:432-435. 7. oltenacu pa, ferguson jd, lednor aj: economic evaluation of pregnancy diagnosis in dairy cattle: a decision analysis approach. j dairy sci 1990;73:2826-2831. 8. pitcher p, galligan dt: decision analysis and economic evaluation of rapid milk progesterone assays. j am vet med assoc 1990;197:1586-1590. 9. galligan dt, ferguson j, munson r, et al: economic concepts regarding early pregnancy testing. proc am assoc bovine pract 2009. p 48-53. 10. mee jf: the role of the veterinarian in bovine fertility management on modern dairy farms. theriogenology 2007;68(suppl 1):s257-s265. 11. fricke pm, caraviello dz, weigel ka, et al: fertility of dairy cows after resynchronization of ovulation at three intervals following first timed insemination. j dairy sci 2003;86:3941-3950. 12. silva e, sterry ra, kolb d, et al: effect of interval to resynchronization of ovulation on fertility of lactating holstein cows when using transrectal ultrasonography or a pregnancy-associated glycoprotein enzyme-linked immunosorbent assay to diagnose pregnancy status. j dairy sci 2009;92:3643-3650. 13. szenci o, beckers jf, humbolt p, et al: comparison of ultrasonography, bovine pregnancy-specific protein b and bovine pregnancy-associated glycoprotein 1 tests for pregnancy detection in dairy cows. theriogenology 1998;50:7788. 14. sasser rg, ruder ca, ivani ka, et al: detection of pregnancy by radioimmunassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation. biol reprod 1986;35:936-942. 15. pieterse mc, szenci o, willemse ah, et al: early pregnancy diagnosis in cattle by means of linear-array real-time ultrasound scanning of the uterus and a qualitative and quantitative milk progesterone test. theriogenology 1990; 33:697-707. 16. nation dp, malmo j, davis gm, et al: accuracy of bovine pregnancy detection using transrectal ultrasonography at 28 to 35 days after insemination. aust vet j 2003;81:63-65. 17. romano j, thompson ja, forrest dw, et al: early pregnancy diagnosis by transrectal ultrasonography in dairy cattle. theriogenology 2006;66:1034-1041. 18. humblot f, camous s, martal j, et al: diagnosis of pregnancy by radioimmunoassay of a pregnancy-specific protein in the plasma of dairy cows. theriogenology 1988;30:257-267. 19. alexander bm, johnsonms, guardia ro, et al: embryonic loss from 30 to 60 days post breeding and the effect of palpation per rectum on pregnancy. theriogenology 1995;43:551-556. 20. garmo rt, refsdal ao, karlberg k, et al: pregnancy incidence in norwegian red cows using nonreturn to estrus, rectal palpation, pregnancy-associated glycoproteins, and progesterone. j dairy sci 2007;91:3025-3033. 21. breed mw, guard cl, white me, et al: comparision of pregnancy diagnosis in dairy cattle by use of a commercial elisa and palpation per rectum. j am vet med assoc 2009;235:292-298. 22. lopez-gatius f, santolaria p, yaniz j, et al: factors affecting pregnancy loss from gestation day 38 to 90 in lactating dairy cows from a single herd. theriogenology 2002;57:1251-1261. 23. lopez-gatius f, santolaria p, yaniz jl, et al: timing of early foetal loss for single and twin pregnancy in dairy cows. reprod domest anim 2004;39:429-433. 24. cox nm, thompson fn, culver dh: milk progesterone to predict reproductive status in a commercial dairy herd. j dairy sci 1978;61:1616-1621. clinical theriogenology • volume 3 number 4 • december 2011 566clinical theriogenology • volume 3 number 4 • december 2011 566 table 1. distribution of days between inseminations (%) of inseminations and mean interval between first and second service. herd num. <10 10-17 18-24 25-35 36-48 >=49 mean, days -------------------------------------------------------------------------------------------------------------------- hdr40 563 0.9 4.4 15.1 12.4 15.3 51.9 60.1 hdr70 1596 6.9 3.3 47.9 18.6 11.9 11.4 32.8 resynch-32d 1636 0.6 1.1 6.1 3.6 78.0 10.7 44.2 ussynch-26d 843 4.4 4.5 21.9 46.5 17.1 5.6 31.2 hdrresynch-39d 509 5.3 4.9 44.0 13.9 10.4 21.41 31.1 biopryn 625 1.1 2.7 15.2 5.9 62.2 12.8 69.8 hdr40: herd using heat detection to manage reproduction with a 40% efficiency of detection hdr70: herd using heat detection to manage reproduction with a 70% efficiency of detection resynch-32d: herd using a pre-synch first insemination program and a re-synch program in open cows beginning at 32 days post-insemination with rectal palpation at 39 days and non-pregnant cows injected im with pgf and given gnrh two days later and re-inseminated 16 hours later. ussynch-26d: herd using ultrasound examination to diagnose open and pregnant cows at 26-32 days post-insemination and assigning cows to a re-synchronization program based on ultrasound scan of ovarian structures on a weekly schedule hdresynch-39: a herd using heat detection for post-insemination management and palpation of cows not re-inseminated by 39 days post-insemination and assigning non-pregnant cows to a resynchronization program biopryn: herd using re-synchronization program based on blood pregnancy examination at 32 days postinsemination with low intensity estrus detection. clinical theriogenology • volume 3 number 4 • december 2011567 clinical theriogenology • volume 3 number 4 • december 2011567 table 2. regression output for model two examining value of pregnancy testing for varying heat detection rate (hdr) conception rate (cr), early embryonic death (eed), examination day (exam), sensitivity and specificity of the test. number of observations: 882. item beta sem f-test p< -------------------------------------------------------------------------------------------------------------------- exam 28 20.319 1.404 14.47 <.0001 exam 32 20.555 1.401 14.67 <.0001 exam 39 18.326 1.402 13.07 <.0001 cr 0.3 3.947 0.178 22.17 <.0001 cr 0.4 0 . . . hdr 0.3 26.036 0.318 81.98 <.0001 hdr 0.4 18.508 0.308 60.01 <.0001 hdr 0.5 11.997 0.318 37.77 <.0001 hdr 0.6 7.068 0.319 22.14 <.0001 hdr 0.7 3.133 0.304 10.31 <.0001 hdr 0.8 0 . . . eed -81.857 1.100 74.39 <.0001 sensitivity 46.733 0.919 50.87 <.0001 specificity 13.408 0.960 13.97 <.0001 r2 = 0.958 -------------------------------------------------------------------------------------------------------------------- value is calculated as the difference in reduction in mean days to re-insemination for hdr detection alone (21*1/hdr) compared with identification of nonpregnant cows at three days post-insemination (28, 32, or 39 days) and assigning open cows to a post-synchronization program on day of examination. the difference in days is multiplied by the value of a day open, $2.25, to assign a monetary value. cost of test, $3, and trip fee per cow examined, $2, are subtracted from the value difference. early embryonic death (eed) is indexed from 0.8 to 1.0 to effect pregnancy loss rates of 1% to 6% at 32 days post-insemination. range in sensitivity and specificity: 0.7 to 1.0. clinical theriogenology • volume 3 number 4 • december 2011 568clinical theriogenology • volume 3 number 4 • december 2011 568 table 3. least square means for three pregnancy tests as a function of heat detection rate and schedule of herd visits. hdr us_26 sem rp_35 sem bp_32 sem -------------------------------------------------------------------------------------------------------------------- weekly visit 0.1 -197.46 1.52 -196.62 1.55 -201.48 1.46 0.2 -98.69 1.08 -96.23 1.09 -98.63 1.03 0.3 -43.11 0.91 -39.62 0.92 -42.53 0.87 0.4 -20.03 0.97 -17.14 0.99 -19.68 0.93 0.5 -7.19 0.94 -4.45 0.96 -7.59 0.90 0.6 0.48 0.96 2.91 0.98 -0.09 0.92 0.7 4.79 0.93 6.54 0.95 3.22 0.90 0.8 6.61 1.20 8.50 1.22 4.77 1.15 0.9 1.77 2.70 3.49 2.74 0.82 2.59 biweekly visits 0.1 -200.05 1.51 -188.98 1.46 -189.53 1.36 0.2 -98.24 1.06 -88.45 1.03 -88.08 0.96 0.3 -40.08 0.89 -32.74 0.86 -34.32 0.81 0.4 -17.14 0.96 -11.70 0.93 -13.50 0.87 0.5 -4.33 0.93 -0.53 0.90 -3.65 0.84 0.6 2.87 0.95 5.25 0.92 2.18 0.86 0.7 6.83 0.92 8.31 0.89 4.61 0.83 0.8 8.12 1.19 9.22 1.15 5.28 1.07 0.9 -0.09 2.67 1.95 2.58 0.11 2.41 monthly visits 0.1 -182.19 1.43 -175.31 1.38 -170.70 1.25 0.2 -82.04 1.00 -76.74 0.97 -72.45 0.88 0.3 -27.28 0.85 -23.90 0.82 -22.89 0.74 0.4 -7.80 0.91 -5.37 0.88 -5.51 0.80 0.5 2.03 0.88 3.69 0.85 1.56 0.77 0.6 6.66 0.90 7.68 0.87 5.06 0.79 0.7 8.82 0.87 9.50 0.84 5.93 0.77 0.8 9.07 1.12 9.68 1.09 5.70 0.99 0.9 1.47 2.52 2.91 2.45 1.30 2.22 us_26 = ultrasound examination for pregnancy performed at a minimum of 26 days postinsemination rp_35 = rectal palpation for pregnancy examination performed at a minimum of 35 days postinsemination bp_32 = blood pregnancy protein test performed at a minimum of 32 days post-insemination senstivity and specificity of tests ranged from 80% to 100%; all open cows re-assigned to a postsynchronization program and re-inseminated within 10 days of the test; mean cr was 0.36 and mean trip fee was $50 and cost of mean cost of tests $3.00. negative values indicate more value relative to the underlying heat detection rate in the herd. positive values indicate the test had less value than the herd program clinical theriogenology • volume 3 number 4 • december 2011569 clinical theriogenology • volume 3 number 4 • december 2011569 t ab le 4 . v al ue o f th re e w ee kl y re pr od uc ti ve te st p ro gr am s, u lt ra so un d ex am in at io n at 2 6 to 3 2 da ys , r ec ta l p al pa ti on a t 3 5 to 4 1 da ys , a nd b lo od pr eg na nc y te st 3 2 to 3 8 da ys p os tin se m in at io n as a f un ct io n of h ea t d et ec ti on r at e, c on ce pt io n ra te , c os t o f tr ip to f ar m , n um be r of c ow s te st ed w ee kl y, v al ue o f a da y op en , e ar ly e m br yo ni c de at h (e e d ) fo r ea ch te st a t 2 8, 3 5 an d 42 d ay s po st -i ns em in at io n, c os t o f te st , a nd s en si ti vi ty a nd sp ec if ic it y of e ac h te st . --------------------------------------------------------------------------------- u ltr as ou nd w ee kl y r 2 0. 94 9 r ec ta l p al pa ti on w ee kl y r 2 0. 94 6 b lo od te st w ee kl y r 2 0. 95 0 be ta se m t_ te st p < be ta se m t_ te st p < be ta se m t_ te st p < in te rc ep t -5 86 .3 00 10 7. 36 3 -5 .4 6 < .0 00 1 -5 06 .1 46 10 7. 31 9 -4 .7 2 < .0 00 1 -6 23 .5 76 10 5. 13 0 -5 .9 3 < .0 00 1 in ve rs e h d r 20 2. 68 5 29 .8 99 6. 78 < .0 00 1 17 0. 25 9 29 .2 25 5. 83 < .0 00 1 19 1. 77 8 28 .9 22 6. 63 < .0 00 1 in ve rs e c r -4 .8 16 1. 18 4 -4 .0 7 < .0 00 1 2. 37 8 1. 22 9 1. 94 0. 05 31 1. 50 3 1. 17 0 1. 29 0. 19 89 in ve rs eh d r *i nv er se c r 1. 94 1 0. 34 6 5. 6 < .0 00 1 -4 .2 32 0. 35 9 -1 1. 8 < .0 00 1 -3 .9 56 0. 34 2 -1 1. 58 < .0 00 1 t ri p f ee -0 .1 13 0. 01 2 -9 .4 9 < .0 00 1 -0 .1 07 0. 01 2 -8 .6 5 < .0 00 1 n um be r c ow s t es te d -0 .2 11 0. 02 5 -8 .4 8 < .0 00 1 -0 .2 01 0. 02 6 -7 .7 7 < .0 00 1 0. 05 8 0. 02 3 2. 52 0. 01 18 v al ue o f d ay so pe n 11 .5 41 0. 42 1 27 .4 3 < .0 00 1 11 .2 57 0. 43 7 25 .7 7 < .0 00 1 -2 1. 02 6 16 .8 44 -1 .2 5 0. 21 21 e e d , 2 8, 4 2, 3 5 4. 73 3 4. 77 5 0. 99 0. 32 17 -1 77 .6 75 64 .6 47 -2 .7 5 0. 00 6 10 .4 09 0. 41 1 25 .3 < .0 00 1 c os t o f t es t -0 .2 92 0. 42 3 -0 .6 9 0. 49 01 -1 .2 91 0. 48 5 -2 .6 6 0. 00 79 -0 .8 27 0. 48 0 -1 .7 2 0. 08 49 s en si tiv ity o f t es t 72 4. 96 8 11 8. 99 5 6. 09 < .0 00 1 60 4. 40 7 11 8. 87 2 5. 08 < .0 00 1 73 3. 16 5 11 6. 56 2 6. 29 < .0 00 1 in ve rs eh d r *s en si tiv ity o f te st -2 33 .1 60 33 .2 42 -7 .0 1 < .0 00 1 -1 78 .8 74 32 .2 44 -5 .5 5 < .0 00 1 -2 01 .8 16 32 .1 06 -6 .2 9 < .0 00 1 s pe ci fi ci ty o f te st 78 1. 74 7 11 8. 51 5 6. 6 < .0 00 1 64 5. 65 3 11 8. 84 6 5. 43 < .0 00 1 79 5. 03 5 11 6. 20 2 6. 84 < .0 00 1 in ve rs eh d r *s pe ci fi ci ty o f te st -2 62 .2 74 33 .0 15 -7 .9 4 < .0 00 1 -2 00 .2 69 32 .5 57 -6 .1 5 < .0 00 1 -2 32 .7 44 31 .9 61 -7 .2 8 < .0 00 1 s en si tiv ity *s pe ci fi ci ty o f te st -8 39 .6 28 13 1. 09 8 -6 .4 < .0 00 1 -6 84 .1 52 13 1. 39 0 -5 .2 1 < .0 00 1 -8 44 .7 25 12 8. 50 9 -6 .5 7 < .0 00 1 in ve rs eh d r *s en si tiv ity *s pe ci f ic it y of te st 25 9. 64 7 36 .6 85 7. 08 < .0 00 1 19 0. 56 8 35 .9 53 5. 3 < .0 00 1 22 4. 77 2 35 .4 61 6. 34 < .0 00 1 in ve rs e h d r = 1 / h d r , r an ge h d r 0 .3 to 0 .9 , m ea n h d r 0 .4 77 s d 0. 21 0 in ve rs e c r = 1 /c r , r an ge c r 0 .2 2 to 0 .5 1, m ea n c r 0 .3 85 s d 0. 05 9 t ri p fe e, c os t o f w ee kl y he rd v is it , r an ge $ 9. 00 to $ 10 0, m ea n $5 0. 7 sd 2 5. 0 n um be r of c ow s te st ed e ac h w ee k: r an ge 1 to 1 00 , m ea n 15 .9 s d 12 .0 v al ue o f da ys o pe n, r an ge $ 2 to $ 5, m ea n $2 .8 1 sd 0 .7 0 e e d 28 : r an ge 4 .2 % to 3 0. 9% , m ea n 11 .5 % s d 5. 85 e e d 35 : r an ge 2 .3 % to 1 0% , m ea n 4. 1% , s d 1. 6% e e d 4 2 ra ng e 2% to 4 .2 % m ea n 2. 5% s d 0. 4% s en si ti vi ty o f te st s: r an ge 0 .8 to 1 .0 0, m ea n 89 .8 s d 4. 9 s pe ci fi ci ty o f te st s: r an ge 0 .8 to 1 .0 , m ea n 90 .1 s d 4. 8 c os t o f te st s: u lt ra so un d ra ng e: $1 .0 0 to $ 9. 1, m ea n $3 .0 8 sd 0 .6 6; r ec ta l p al pa tio n ra ng e: $ 0. 50 to 5 .0 00 , m ea n $2 .9 7 sd 0 .6 0 b lo od t es t r an ge : $2. 00 to $ 6. 00 , m ea n $3 .0 5 sd 0 .5 7 clinical theriogenology • volume 3 number 4 • december 2011 570 table 5. relative value for ultrasound examination at 26 days for pregnancy (us_26), rectal palpation at 35 days for pregnancy (rp), and blood pregnancy test at 32 days (bp_32) for varying costs, number of cows examined and sensitivity of tests. input items sensitivity/specificity test value,$-------------------------------------- trip,$ num us rp blood us_26 rp bp_32 -------------------------------------------------------------------------------------------------------------------- cost per cow, $ 3.00 3.00 2.75 50 20 95/95 95/95 95/95 21.90 19.61 20.06 50 20 85/95 85/95 85/95 19.04 16.91 17.47 50 20 85/85 85/85 85/85 15.42 13.52 14.23 50 10 95/95 95/95 95/95 19.40 17.11 20.06 50 12 95/95 95/95 95/95 20.23 17.94 20.06 cost per cow, $ 2.13 0.00 2.75 50 10 95 95 95 20.06 19.36 20.06 cost per cow, $ 3.00 3.00 5.00 50 10 95 95 95 19.40 17.11 18.28 50 10 85 85 85 16.54 14.41 15.89 cost per cow, $ 3.00 0.00 2.75 50 12 95 95 95 20.23 20.20 20.06 cost per cow, $ 3.00 3.00 2.75 100 30 95 95 95 21.07 18.78 20.06 100 30 85 85 85 18.21 16.08 17.66 100 20 95 95 95 19.40 17.11 20.06 ------------------------------------------------------------------------------------------------------------------- clinical theriogenology • volume 3 number 4 • december 2011571 clinical theriogenology • volume 3 number 4 • december 2011571 figure 1. value of a pregnant cow at two different values of milk, $0.10/lb and $0.17/lb. 1b. data presented as a value of a day open, with milk at $0.10/lb value is $2.18/day, with milk at $0.17/lb value is $3.46/day.value declines as days in milk increases. y = ‐2.1797x + 525 r² = 0.9921 y = ‐3.4647x + 895.68 r² = 0.994 v al u e  o f  p re gn an t  c o w days in milk clinical theriogenology • volume 3 number 4 • december 2011 572clinical theriogenology • volume 3 number 4 • december 2011 572 figure 2. proportion of pregnacies lost by 65 days post-insemination from time of first insemination. data collected from multiple diagnostic tests used for first diagnosis of pregancy based on either progesterone concentration (first examination 21 days), ultrasound, rectal palpation, and ruminant blood pregnancy protein – b. re-examination for pregnancy at 65 – 70 days was by rectal palpation. 10 20 30 40 50 60 days post-insemination first pregnancy diagnosis -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 p er ce nt p re gn an cy l os s by 6 5 da ys pregnancy losses clinical theriogenology • volume 3 number 4 • december 2011573 clinical theriogenology • volume 3 number 4 • december 2011573 insemination pregnantd days post‐insemination pregnant = 0.37 + 107.1exp(‐.2845*day) preg. loss to 65 days =  ‐0.02 – 50exp(‐0.23*day) opend = 1 ‐ pregnantd days post‐insemination hdr 1 ‐ hdr re – ai21 eedd opend open pregnantd 21 26 ‐42 42 65 re – ai36‐48 eedd hdr*(1‐hdr) hdr opend re – ai25‐35 hdr (1‐hdr)*hdr (1 – hdr)2 pregnantd opend (1 – hdr)3 (1‐hdr) (1‐hdr)2 (1 – hdr)3 (1‐hdr) re – ai36‐64 figure 3. schematic of pregnant and open cows by time post-insemination. hdr = heat detection rate. eed = early embryonic or fetal loss. pregnancy is a function of days post-insemination: cr = 0.37 + 107.1*exp(-0.2845*day post-insemination); eed loss to 65 days postbreeding by day post-insemination = -0.02 -50*exp(-0.23*day post-insemination). cows either open or pregnant at day of examination were functions of three rates: hdr, cr, and eed. hdr was varied from 0.10 to 0.90; cr varied from 0.22 to 0.52 by varying the intercept of the model; eed varied by day post-insemination at ranged from a proportion of 0.309 to 0.042 of pregnancies between 26 days to 32 days post-insemination; eed after this time ranged as a proportion of pregnancies from 0.10 to 0.02. clinical theriogenology • volume 3 number 4 • december 2011 574clinical theriogenology • volume 3 number 4 • december 2011 574 figure 4. influence of heat detection rate (hdr, %) on value of a diagnostic test to determine pregnancy and open status from 21 to 60 days post-insemination when non-pregnant cows are assigned for rebreeding. sensitivity and specificity were set to 100% for the test. embryonic mortality set to zero. value of a day open was set to $2.25 and cost of palpation to $3.00 per cow. steps are created by cows having an opportunity for a second and third estrus with time post-insemination, reducing the proportion of open cows at examination and reducing the possible reduction in days open from the baseline associated with hdr rates. conception rate was 30%. figure 5. influence of conception rate (cr) and heat detection rate (hdr) on value of pregnancy/nonpregnant diagnosis by days post-insemination. sensitivity and specificity were set to 100% for the test. embryonic mortality set to zero. value of a day open was set to $2.25 and cost of pregnancy test was $3.00. as cr increases the value of the test decreases at a given hdr, represented here as 0.40. -100 -80 -60 -40 -20 0 20 40 60 80 21 24 27 30 33 36 39 42 45 48 51 54 57 60 days post-insemination v al u e, $ hdr 30 hdr 40 hdr 50 hdr 60 hdr 70 -100 -80 -60 -40 -20 0 20 40 21 24 27 30 33 36 39 42 45 48 51 54 57 60 days post-insemination v al u e, $ hdr 0.4 cr 0.3 hdr 0.4 cr 0.4 hdr 0.4 cr 0.5 clinical theriogenology • volume 3 number 4 • december 2011575 clinical theriogenology • volume 3 number 4 • december 2011575 figure 6. influence of decreasing sensitivity and increasing false open diagnosis on value of examination at 32 days post-insemination with cr = 30% and hdr of 40% (1), 50% (2), 60% (3) and 70% (4) for controlling days to next insemination. figure 7. influence of decreasing specificity (false pregnant) on value of examination at 32 days postinsemination with cr = 30% and hdr of 40% (1), 50% (2), 60% (3) and 70% (4) for controlling days to next insemination. 1 2 3 4 hdr 40% (1) to 70% (4) and cr 30% -15 -10 -5 0 5 10 15 20 v al ue , $ sens 95% sens. 90% sens 80% influence of false open 1 2 3 4 hdr 40% (1) to 70% (4) and cr 30% 0 5 10 15 20 v al ue , $ spec. 90% spec. 80% spec. 70% influence of false pregnant clinical theriogenology • volume 3 number 4 • december 2011 576clinical theriogenology • volume 3 number 4 • december 2011 576 figure 8. the relative value of biweekly and monthly herd visits compared to weekly visits in dollars at different heat detection rates. figure 9. relative values of ultrasound examination at 26 days (us_26), rectal examination at 35 days, and blood pregnancy test at 32 days (blood_32) post-insemination as a function of heat detection rate. ‐20 ‐18 ‐16 ‐14 ‐12 ‐10 ‐8 ‐6 ‐4 ‐2 0 0 0.2 0.4 0.6 0.8 1 v al u e  r e la ti ve  t o  a  w e e kl y  h e rd  v is it , $ heat detection rate biweekly visit monthly visit ‐20 0 20 40 60 80 100 120 0 0.2 0.4 0.6 0.8 1 v al u e  o f  w e e kl y  p re gn an cy  e xa m , $ heat detection rate us_26 rec_35 blood_32 clinical theriogenology • volume 3 number 4 • december 2011577 clinical theriogenology • volume 3 number 4 • december 2011577 figure 10. relative value of ultrasound examination versus blood pregnancy test as a function of number of cows tested weekly. cost of tests $3.00, ultrasound test includes trip fee. sensitivity and specificity of tests equal. ‐10 ‐5 0 5 10 15 20 25 30 35 0 10 20 30 40 r e la ti ve  v al u e  b lo o d  t e st  ‐ u lt ra so u n d   e xa m , $ number of cows tested weekly series1 clinical theriogenology • volume 3 number 4 • december 2011 578clinical theriogenology • volume 3 number 4 • december 2011 578 identifying etiologies of subfertility in stud dogs kara kolster staff veterinarian, springfield veterinary center, glen allen, va introduction subfertility can be defined as sporadic success in a male’s ability to sire litters or semen parameters that are substantially below expected normal. subfertility can be congenital or acquired. dogs with acquired conditions have a history of previous normal fertility. however, if breeding is not attempted until later in life, it may not be possible to determine if fertility problems are congenital or acquired. if a cause of subfertility is identified, appropriate management and intervention may help to improve fertility in a particular stud dog. unfortunately, in more than 50% of cases, a definitive cause may not be determined.1 in such cases, empirical treatment (e.g., dietary supplements and hormonal treatment) may be beneficial. laboratory evaluation of semen accurate semen analysis for diagnosis of subfertility requires knowledge of the appropriate tools and tests to use, and evaluation of as many parameters as possible. semen evaluations have traditionally been performed manually; however, more recently computer-assisted sperm analysis (casa) systems have become popular.2 advantages of casa systems are their ability to rapidly and objectively analyze semen parameters without the influence of human variability.3 a major disadvantage is their inability to accurately detect sperm morphological abnormalities. motility evaluation manual semen motility evaluation is performed by placing a drop of raw semen on a warmed slide with a coverslip and estimating the percent motile sperm as viewed under 100 400 x bright field microscopy. if semen concentration is high, dilution with physiologic saline allows evaluation of individual sperm movement and provides a more accurate estimate of progressive motility. casa systems assess total and progressive sperm motility by obtaining multiple digital images of a field of sperm in rapid succession, identifying individual sperm, and tracking those sperm across frames.3 anecdotally, manual evaluation results in higher estimation of percent motile sperm compared to casa evaluation. subfertile dogs frequently have lower total and progressive motility compared to dogs exhibiting normal fertility.1 viability evaluation some newer casa systems can evaluate sperm viability, defined as membrane integrity, by fluorescent labeling. this is an important advancement as viability may correlate better than progressive motility to fertility.4 live:dead ratio of a sample stained with eosin-nigrosin and identification of membrane intact sperm using a hypoosmotic swelling test are manual methods of evaluating viability that do not require special equipment. the nucleocounter sp-100 (chemometec, allerod, denmark) has lower variability in evaluating sperm membrane integrity compared to human-analyzed tests.5 total sperm count manual evaluation of an ejaculate concentration is performed using a neubauer hemocytometer and a standard dilution, thereby allowing calculation of total sperm count. the reader is referred to literature6,7 for details. casa systems also analyze semen samples at a standard dilution, allowing the system to calculate concentration of a raw sample. other semen analyzers are commercially available that are not part of a casa system. these can be photometers or densimeters that measure concentration of a semen sample based on transmittance of light through the sample as compared to a standard buffer. all photometers and densimeters are calibrated to a certain concentration range; therefore, samples outside this range must be diluted for accurate measurement. extraneous particles in the sample (e.g., white blood cells or undissolved components of semen extender) may falsely elevate the measured sample concentration. additionally, the parameters by which a casa system identifies and analyzes sperm vary by species, instrument, and settings. differences in the technical settings of these instruments can influence the results.8 this is an important consideration when comparing results among laboratories and in fresh versus chilled or cryopreserved semen containing egg yolk extender. correct dilution in any methodology is vital to obtain an accurate result. when comparing multiple samples to each other, it is valuable to have analyses performed by the same person to reduce the risk of human variability. casa systems have lower variability than human-derived (e.g., hemocytometer), measurements of semen concentration.9,10 the nucleocounter sp-100 has been more accurate than traditional casa systems or densimeters because it excludes background debris from measurement.3 for this reason, it is also useful for measuring sperm concentration in semen extended with egg yolk. morphology evaluation sperm morphology is perhaps one of the most important albeit most commonly misused tests in semen analysis. morphology is correlated with fertility in multiple species, including dogs, bulls, and humans.1,11-14 accurate evaluation requires appropriate stains, quality bright field, phase-contrast, or differential interference contrast microscopy, and knowledge and experience in identifying sperm abnormalities. canine semen morphology is commonly evaluated with bright field microscopy using an eosin-nigrosin stain or a modified giemsa stain.15 phase-contrast microscopy uses light to produce a high contrast image of transparent sperm, eliminating the need for stain. a minimum of 200 sperm should be counted, and percentage of normal and abnormal sperm calculated.7 some casa systems have the ability to evaluate morphology; however, this is generally limited to crude analysis of the sperm head and tail coiling, and accuracy is highly dependent on the software settings of a particular instrument.3 results of casa morphology evaluations are not comparable to evaluation by a trained human.3,16,17 there are several systems for categorizing sperm abnormalities. in the more common classification system, primary abnormalities are considered those that develop during spermatogenesis or spermiogenesis and are caused by pathologic processes in the seminal epithelium, whereas secondary abnormalities are considered those that originate during transport through or storage in the epididymis, and tertiary abnormalities are those caused after ejaculation by rough handling or environmental conditions.12 another system classifies sperm with major abnormalities as those with severe aberrations that are generally believed to be incapable of fertilization, and minor abnormalities as those that are less likely to cause changes in fertility.11,12 this creates some confusion as there is often not detailed knowledge of the effect on fertility of specific morphologic abnormalities in the dog. a system of classifying morphologic abnormalities as compensable or noncompensable, depending on whether increasing sperm dosage has an impact on fertility or not, could be useful for determining insemination doses.12 however, similar to major and minor classification, there is limited information on which abnormalities are compensable versus noncompensable in the dog. a newer morphological classification system groups defects based on their location on the sperm cell; head, midpiece, or tail.12 this has also not been correlated to fertility; however, it may be beneficial in tracking a dog’s morphological changes over time as the categories are more descriptive. determining etiology of subfertility many insults to the spermatogenic cycle result in oligo-astheno-teratospermia. specific deviations from a normal spermiogram, combined with a complete history of breeding and husbandry, can help narrow down possible etiologies and guide toward appropriate further diagnostic tests. timing and variety of morphologic abnormalities observed in an ejaculate may help to determine the time and type of insult to spermatogenesis. for example, increased scrotal temperature in bulls caused sperm mitochondrial defects ~ 14 days after insult, and nuclear vacuoles ~ 24 days after insult.12 spermatocytes undergoing meiosis and cells undergoing spermiogenesis are most susceptible to damage.11 determination of serum hormone concentrations may be useful to further characterize infertility. male dogs with at least 12 months duration of subfertility had lower basal testosterone concentrations than dogs of normal fertility.18 increased follicle stimulating hormone (fsh) can be a marker of primary testicular failure.6,19 this occurs due to decreased negative feedback from inhibin that is normally produced by sertoli cells.6 increased fsh in the face of declining semen quality or azoospermia carries a poor prognosis because testicular changes are likely irreversible.20 these tests should be performed by a laboratory using assays validated for dogs. determination of alkaline phosphatase concentrations in semen can be used to diagnose ductus deferens patency. nearly all alkaline phosphatase present in a normal ejaculate is contributed by the second (sperm rich) fraction.21 the cutoff of 5,000 u/l is generally used to indicate a complete ejaculate. low alkaline phosphatase in an azoospermic sample can indicate bilateral epididymal obstruction or incomplete ejaculation.21 high alkaline phosphatase concentrations in an azoospermic ejaculate indicate failure of spermatogenesis and carries a poor prognosis for future fertility. anti-müllerian hormone (amh) is produced in the male exclusively by sertoli cells. determination of amh concentrations in dogs can be used for determining gonadectomy status,22 and diagnosing testicular atrophy and sertoli cell tumor.23-25 ultrasonography is an important tool in evaluation of stud dog fertility. it has been used for some time in evaluation of prostatic disease and testicular tumors. more recently, b-mode and doppler ultrasonography findings have been correlated to current fertility in the dog.18,25 increased echogenicity of testis was associated with fewer morphologically normal sperm.25 conversely, hypoechoic testis was associated with poor morphology.27 it is possible that both increased and decreased echogenicity may represent changes in the testicular architecture that lead to decreased semen quality. rate of blood flow through the testicular artery is positively correlated to current semen quality, likely because it represents a marker for the rate of spermatogenesis.18,28 testicular size and total sperm output are positively correlated with body weight in dog; however, among dogs of comparable body weight, testicular volume, as measured by ultrasonography, has not been a reliable indicator of fertility.6,18 conclusion tests discussed should provide a reasonably complete picture of the nature of subfertility. is it primarily due to oligospermia, teratozoospermia, or to prostatic or testicular abnormalities? understanding the anatomic and physiologic processes involved in sperm production can help pinpoint the stage at which it went wrong. furthermore, breeding history can help narrow down the timeframe at which subfertility may have started, allowing the clinician and owner to investigate potential insults to spermatogenesis that occurred at that time. hormonal tests (e.g., fsh and amh) can help determine the prognosis for return to fertility. a logical approach to diagnose and determine the etiology of subfertility, starting with an accurate semen evaluation, provides the best chance to correct or manage a subfertile stud dog. conflict of interest none to declare. references 1. domoslawska a, zdunczyk s: clinical and spermatological findings in male dogs with acquired infertility: a retrospective analysis. andrologia 2020;52:1-7. 2. amann rp, waberski d: computer-assisted sperm analysis (casa): capabilities and potential developments. theriogenology 2014;81:517. 3. amann rp, katz df: reflections on casa after 25 years. j androl 2004;25:317-325. 4. fraser l, gorszczaruk k, strzezek j: relationship between motility and membrane integrity of boar spermatozoa in media varying in osmolality. reprod domest anim 2001;36:325-329. 5. daub l, geyer a, braun j, et al: sperm membrane integrity in fresh and frozen-thawed canine semen samples: a comparison of vital stains with the nucleocounter sp-100. theriogenology 2016;86:651656. 6. johnston sd, root kustritz mv, olson pns: canine and feline theriogenology. philadelphia: w.b. saunders; 2001. 7. root kustritz mv: the value of canine semen evaluation for practitioners. theriogenology 2007;68:329-337. 8. rijsselaere t, van soom a, maes d, et al: computer-assisted sperm analysis in dogs and cats: an update after 20 years. reprod domest anim 2012;47(suppl 6):204-207. 9. scofield db, baumber-skaife j, loomis pr: improvements in equine semen processing techniques that aid optimal fertility. clinical theriogenology 2016;8:465-469. 10. hansen c, vermeiden t, vermeiden jpw, et al: comparison of facscount af system, improved neubauer hemocytometer, corning 254 photometer, spermvision, ultimate and nucleocounter sp100 for determination of sperm concentration of boar semen. theriogenology 2006;66:2188-2194. 11. barth ad, oko rj: abnormal morphology of bovine spermatozoa. ames: iowa state university press; 1989. 12. koziol jh, armstrong cl: manual for breeding soundness evaluation of bulls, 2nd edition. matthew, al. society for theriogenology 2018. 13. zinaman mj, brown cc, selevan sg, et al: semen quality and human fertility: a prospective study with healthy couples. j androl 2000;21:145-153. 14. ombelet w, bosmans e, janssen m, et al: semen parameters in a fertile versus subfertile population: a need for change in the interpretation of semen testing. human reprod 1997;12:987-993. 15. kruger tf, ackerman sb, simmons kf, et al: a quick, reliable staining technique for human sperm morphology. arch androl 1987;18:275-277. 16. lu jc, huang yf, lu nq: computer-aided sperm analysis: past, present and future. andrologia 2014;46:329-338. 17. steigerwald p, krause w: estimation of sperm morphology using a new casa system. andrologia 1998;30:23-27. 18. de souza mb, england gb, mota filho ac, et al: semen quality, testicular b-mode and doppler ultrasound, and serum testosterone concentrations in dogs with established infertility. theriogenology 2015;84:805-810. 19. douglas rh, umphenour n: endocrine abnormalities and hormonal therapy. vet clin north am equine pract 1992;8:237-249. 20. kelsey tw, mcconville l, edgar ab, et al: follicle stimulating hormone is an accurate predictor of azoospermia in childhood cancer survivors. schlatt s. ed. plos one 2017;12(7):e0181377. doi:10.1371/ journal.pone.0181377. 21. tornelli a, arauz m, baschard h, et al: unilateral and bilateral vasectomy in the dog: alkaline phosphatase as an indicator of tubular patency. reprod domest anim 2003;38:1-4. 22. themmon apn, kalra b, visser ja, et al: the use of antimullerian hormone as diagnostic for gonadectomy status in dogs. theriogenology 2016;86:1467-1474. 23. giudice c, banco b, veronesi mc, et al: immunohistochemical expression of markers of immaturity in sertoli and seminal cells in canine testicular atrophy. j comp pathol 2014;150:208-215. 24. banco b, veronesi mc, giudice c, et al: immunohistochemical evaluation of the expression of anti-mullerian hormone in mature, immature and neoplastic canine sertoli cells. j comp pathol 2012;146:18-23. 25. holst bs, dreimanis u: anti-mullerian hormone: a potentially useful biomarker for the diagnosis of canine sertoli cell tumours. bmc vet res 2015;11:166. 26. moxon r, bright l, pritchard b, et al: digital image analysis of testicular and prostatic ultrasonographic echogenicity and heterogeneity in dogs and the relation to semen quality. anim reprod sci 2015;160:112-119. 27. arteaga aa, barth ad, brito lfc: relationship between semen quality and pixel-intensity of testicular ultrasonograms after scrotal insulation in beef bulls. theriogenology 2005;64:408-415. 28. england g, bright l, pritchard b, et al: canine reproductive ultrasound examination for predicting future sperm quality. reprod domest anim 2017;52(suppl 2):202-207. identifying etiologies 2018: causes of newborn mortality in devon rex kittens causes of newborn mortality in devon rex kittens holly omoto, michelle anne kutzler department of animal and rangeland sciences, college of agricultural sciences, oregon state university, corvallis, or introduction even in well-managed catteries, up to 16% of kittens may be stillborn and up to 20% of kittens may die before weaning.1 the objective of this study was to identify gross pathology lesions in newborn purebred kittens who died spontaneously. we hypothesized that lesions consistent with congenital causes of mortality would be more common than infectious causes. methods post-mortem examinations were performed on 17 newborn (up to 3 days old) devon rex kittens. the palate, heart, lungs, liver, spleen, kidneys, bladder, umbilical stump, and urogenital area were examined for abnormalities. a sample of lung tissue was floated in water to evaluate atelectasis, and urine was aspirated from the bladder to evaluate hematuria. the age at death, the dam’s and sire’s blood types, and clinical signs present before death were also recorded. results no gross pathological abnormalities were found in 17.6% (3/17) of the kittens. of these kittens with no abnormalities, signs before death were hypothermia (n=1), dystocia (n=1), and dyspnea (n=2). there was one kitten with two signs before death, dystocia and dyspnea. a cleft palate was found in one kitten (5.8%), and no other congenital defects were found in the remaining kittens. no kittens died from neonatal isoerythrolysis. hematuria (a sign associated with neonatal isoerythrolysis, ni) was present in 17.6% (3/17) of the kittens, and icterus (another sign associated with ni) was identified in one kitten. however, all four of these kittens were produced by blood type b sires, indicating that ni was not the cause.2 pyothorax was present in 35.3% (6/17) of the kittens, and dyspnea was reported in 4/6 of those kittens prior to death. one kitten with pyothorax had atelectasis. discussion about 13% of devon rex litters have at least one congenital defect, which is consistent our findings.3 however, our findings revealed that 52.9% (9/17) of the kittens up to 3 days of age died from infectious causes (pyothorax and hematuria not associated with ni). additional diagnostic testing is planned to determine the etiologic agents involved with the infectious causes of feline newborn mortality. keywords: feline, mortality, necropsy, neonatal, perinatal references 1. malandain é, little s, casseleux g, et al: diseases of the neonate. in: malandain é, editor. practical guide to cat breeding. aimargues(france): aniwa sas on behalf of royal canin; 2006. p. 164-179. 2. silvestre-ferreira ac, pastor j: feline neonatal isoerythrolysis and the importance of feline blood types. vet med int 2010;2010:1-8. 3. little s: congenital defects of kittens. in: tufts’ canine and feline breeding and genetics conference; 200 clinical theriogenology • volume 10, number 3 • september 2018325 clinical theriogenology 2022; 14: 32 avian reproductive tract diseases and surgical resolutions scott echols,a,b,c brian speerb aechols veterinary services, salt lake city, ut bmedical center for birds, oakley, ca cparrish creek veterinary hospital and diagnostic center, centerville, ut abstract reproductive tract disease is a commonplace in avian practice, particularly among female birds. unique anatomy and physiology of the avian reproductive tract results in a highly productive but poorly accessible organ system. affected hens are commonly presented with nonspecific lethargy, coelomic swelling and sometimes laying abnormal eggs. reproductive diseases are less common in male birds; however, they are well described. although many reproductive tract diseases can be managed medically, there is a definite need for surgical intervention in some cases. because of internal location, various disease processes and even normal physiologic processes can dramatically alter patient presentations. for this reason, a clear understanding of avian female anatomy and male reproductive tracts is vital to diagnose and treat disorders. this manuscript focuses on recognition and treatment (emphasizing surgical management) of avian reproductive tract diseases. because of the unique avian reproductive anatomy and frequent need for surgical correction of disorders, the organization of the manuscript starts with descriptions of coelom and basic surgical principles. keywords: birds, reproductive anatomy, ovarian and oviductal problems, testicular disease, surgical and medical treatments introduction primarily due to high egg producing hens, reproductive tract diseases are common in pet and even commercial avian practice.1 with advances in diagnostics and therapeutics available to birds, many reproductive tract diseases can be medically managed. however, several diseases can only be treated surgically. because of past literature describing procedures such as salpingohysterectomy as ‘difficult and risky’, many practitioners are rightfully cautious about performing avian reproductive tract surgery.1 as with diagnostics and therapeutics, anesthetic techniques, instrumentation, pain management, a better understanding of respiratory physiology and surgical techniques have all advanced in avian medicine. due to medical and surgical progress and public awareness, avian practitioners are being called upon to manage more complex behavioral, medical, and surgical related problems in birds with greater success.1 surgeon’s experience and proper instrumentation are vital to minimize tissue trauma, bleeding, and anesthetic time and to improve outcomes. analgesics should be considered in all surgical cases. description of anesthetic management, postsurgical care, and analgesics are beyond the scope of this manuscript and are adequately described. coelomic anatomy of birds because of avian reproductive tracts’ location, a basic understanding of the coelom is beneficial.2,3 coelom refers to the primordial body cavity lined by mesoderm and formed by fusion of lateral body folds early in embryonic development. within the coelomic cavity are individual and unique compartments, each lined by mesothelium and capable of producing serosal fluid. ‘lower’ reptile species have 2 cavities (pericardial and pleuroperitoneal) and more highly developed reptiles have 3 (pericardial, pleural, and peritoneal), whereas birds have 8 distinct coelomic cavities. interrelationship of these cavities with the respiratory tract is complex. eight coelomic cavities of birds include, 1 pericardial and 1 intestinal peritoneal cavity (ipc), 2 pleural cavities, and 4 hepatic peritoneal cavities. all visceral organs except right and left liver lobes are contained within ipc. pericardial cavity is situated ventrally on the midline, just dorsal to sternum and is restrained by fibrous attachments to surrounding structures, most notably sternum. apex of the sac is intimately associated with and positioned between 2 liver lobes. parietal pleura surrounding each lung and mesothelial coating of visceral pleura applied to the lung itself form a small, clinical theriogenology 2022; 14: 33 potential pleural cavity due to fixed dimensions of avian lung in most species. parietal and visceral pleura develop fibrous cross connections starting along lung’s ventromedial surface.2 birds have 5 distinct peritoneal cavities. four hepatic peritoneal cavities (hpc) surround right and left liver lobes. a single, midline ipc contains most of the gastrointestinal tract, gall bladder (if present), reproductive tract, and spleen. hepatic peritoneal cavities are composed of 2 paired structures, larger left and right ventral hepatic peritoneal cavities, and much smaller right and left dorsal hepatic peritoneal cavities. hpcs are divided on the midline into right and left entities by the ventral mesentery. in chicken, left dorsal hepatic peritoneal cavity communicates with intestinal peritoneal cavity via a small opening that is covered on the ipc side only by abdominal air sacs. a single ipc is centrally located in mid to caudal coelom and contains proventriculus, ventriculus, gall bladder (if present), spleen, intestines, and male or female reproductive tracts. the only coelomic cavity containing air sacs (left and right abdominal) is ipc. a ventral midline incision made caudal to the ventriculus leads directly and specifically into ipc, not into ‘coelom’ or ‘coelomic cavity’, and not into respiratory tract. right and left subpulmonary cavities (air sacs) are structures unique to birds and strictly speaking, are not true coelomic cavities as they are not lined by mesothelium. their intimate relationships with coelomic cavities, however, require understanding for the purposes of surgical intervention. subpulmonary cavities predominately include air sac system of birds; cervicocephalic, clavicular, cranial thoracic, and caudal thoracic air sacs. surgical principles and patient preparation for coeliotomy, the patient is usually placed in lateral or dorsal recumbency. birds have a very thin epidermis that is diffusely supplied by capillaries in the dermis, unlike what is observed in mammals. there is less subcutaneous tissue, and dermis is firmly attached to the underlying muscle fascia. compared to mammals, most blood vessels are comparatively less protected by surrounding tissues, leaving more potential for hemorrhage. body contour feathers are easily plucked in the direction of their growth once the patient is anesthetized. some species of bird may have particularly thin skin, predisposing them to iatrogenic tears when feathers are plucked. water soluble gel or masking tape may be used to keep down and contour feathers under control. surgical preparation of the skin is performed with betadine, chlorhexidine, and/or isopropyl alcohol. patient preparation with excessive amounts of saline or alcohol can predispose the patient to hypothermia. draping the patient as quickly as possible with clear plastic drapes helps to retain heat better than using cloth drapes. clear, transparent plastic drapes also allow respiration to be monitored during the procedure. supplemental heat (warm water bottles, circulating warm water blankets, heat lamps, and forced warm air) should be provided to the patient. surgical instruments magnification is a common and important component of many coelomic procedures, viewing the frequent small patient size and resultant needs for fine instrumentation and microsurgical skills. magnification makes hand tremors more apparent, and necessitates slow, deliberate motions for greatest efficiency. this in turn can result in a slower surgical pace for the procedure(s) that may require increased detail to anesthesia protocols and patient support methods used during the procedure. although magnification greatly adds to visualization of the direct surgical field, it limits surgeon’s ability to visualize peripheral fields. this makes an assistant necessary when utilizing the operating microscope, and helpful when utilizing magnifying loupes. fine and delicate instruments are essential for avian surgery. gauze pads (2 x 2) can be cut from standard 4 x 4 sponges or can be specifically purchased. a basic surgery pack should contain a long jeweler’s forceps with fine delicate tip but flat handles, round handled microsurgical forceps, a castroviejo pen needle holder of at least 5.25 inch length, with fine jaws and rounded handles, and a spring-loaded microsurgical scissors of at least 7 inches with rounded handles. sterile cotton tipped applicators should also be available for point hemostasis and blunt tissue retraction. hemostatic clips are also useful and come in a variety of sizes. right angled appliers are also available for special applications in deep cavities. heiss blunt retractors, alm retractors and ring retractors are suitable for use in medium to smaller species.4 halsted’s principles of surgery should be followed when performing surgery in any species. these principles are itemized below: gentle handling of tissue meticulous hemostasis preservation of blood supply strict aseptic technique minimum tension on tissues accurate tissue apposition obliteration of dead space surgical approaches to coelomic cavities approaches commonly made include a ventral midline approach, a midline-l approach, a midline-t approach, and left and right lateral approaches that may or may not include l flaps. modifications of these basic approaches also can be used, depending on the patient and desired exposure of surgical site. these surgical approaches generally require the bird to be in dorsal or lateral recumbency. if ascites or substantial organomegaly is present, elevating the proximal half of the body may help reduce pressure on heart, lungs, and more cranial air sacs, improving ventilation. to specifically enter the ipc directly from the body wall without penetrating an air sac, use 1 of 2 approaches. a midline, paramedian or transverse incision in the ventral body wall will enter the ipc when made caudal to posthepatic septum clinical theriogenology 2022; 14: 34 end and medial to distal limits of caudal thoracic and abdominal air sacs. the other site for direct entry from the body wall to ipc is the left or right postischial approach. a small skin incision dorsal to pubis and just caudal to ischium allows a blunt puncture through the thin body wall dorsolateral to coprodeum.2,3 female reproductive tract diseases understanding the avian female reproductive tract anatomy, especially, its blood supply and drainage, is vital to proper diagnosis and treatment (especially surgery). during sexual and egg laying activity, blood supply to ovary and oviduct can be substantial. due to potential vascularity, being mindful of local blood supply and minimizing bleeding can often determine the outcome. focus will be given to basic structure and vasculature of ovary and oviduct. if possible, the following surgeries should be performed when the female is reproductively inactive as blood supply is often reduced. ovarian anatomy a right and left ovary and oviduct are present in the embryologic stages of all chicks, but the right half regresses due to müllerian inhibiting substance action prior to hatch. although a persistent right oviduct with or without a functional right ovary is present in some birds, most birds only have a left female reproductive system. brown kiwi is an exception and normally has a functional left and right ovary. interestingly, in chicken about 480,000 oocytes develop by hatching. of these, ~ 2,000 are visible as a mass of small ova and only 250 500 reach maturity and ovulate within the lifespan of domestic species and even fewer mature in wild species.1 ovarian follicles are arranged hierarchically. largest follicle (f1) will ovulate on the next day, second largest (f2) on the following day, and so on. ovary is attached to the cranial renal division and dorsal body wall by the mesovarian ligament and receives its blood supply from ovarian artery that originates off the left cranial renal artery or directly off the aorta. accessory ovarian arteries may also arise from other adjacent arteries. ovarian artery further divides into many branches with the greatest blood flow directed to any large preovulatory follicles present. ovarian veins unite into main anterior and posterior veins that drain into the overlying vena cava. multiple left ovarian veins may exist and drain into the cranial oviductal vein that enter the common iliac vein and finally into the caudal vena cava.5 in first author’s experience, the cranial oviductal vein is too short or poorly developed to recognize grossly. instead, multiple short veins seem to enter the common iliac vein over the length of its contact with the dorsum (base) of the ovary. ovarian diseases cystic ovarian disease although the cause is often unknown, cystic ovarian disease has been reported in several bird species. cystic ovaries are sometimes secondary to neoplasia. depending on their size ovarian cysts can be observed incidentally; small ovarian cysts can cause none to mild coelomic distension whereas large and/or numerous cysts can be associated with readily identifiable ascites. many cysts are large and can often be diagnosed noninvasively using ultrasonography, ct, and/or mri. cysts can be treated by ultrasonography guided transabdominal aspiration or more directly via celiotomy or endoscopy. if collected, evaluate the fluid for evidence of infection or other abnormalities. severe cystic disease may require partial or complete ovariectomy and should include biopsy for histopathological evaluation. leuprolide acetate has also been suggested to reduce or resolve ovarian cysts in birds and may offer a noninvasive treatment option.1 use of desorelin (a gnrh agonist) has also been recommended anecdotally.6 it is important to note that no peer reviewed publications have documented the benefit of using either leuprolide acetate or deslorelin to treat ovarian cysts in birds. in authors’ experience, aspiration or physical removal is the only means to remove ovarian cysts, if clinically indicated. oophoritis ovarian infections can be life-threatening and are often associated with septicemia. salmonella pullorum is the etiologic agent of pullorum disease of poultry and most frequently affects ovary.1 clinically affected birds usually have more severe, albeit general signs of illness and if not treated quickly, peritonitis and death may result. irregular follicular shapes and sizes noted on ultrasonography, mri or ct may be related to infectious oophoritis or neoplastic disease. abnormally shaped, colored or partially ruptured follicles identified during celiotomy or endoscopy should be carefully aspirated for cytological and microbiological analysis and patients treated with broad-spectrum antibiotics, initiated pending culture results. storz injection needle with teflon guide (karl storz veterinary endoscopy america, goleta, ca) is particularly useful as an endoscopic means to aspirate ovarian follicles. if possible, completely drain the abscessed follicle(s), being careful not to contaminate coelom. partial or complete ovariectomy may be required for chronically infected and caseated follicles. neoplasia of ovary and oviduct ovarian neoplasia is reported with some frequency in birds and can be associated with egg retention, ascites, cystic ovarian disease, medullary hyperostosis, coelomic hernias, oviductal impaction, and general malaise. thirty eight percent of usda inspection service mature fowl condemnation is the result of neoplastic disease, with most from the genital tract; ‘there is indeed a unique propensity for hens (poultry) to develop cancer of the reproductive system in the almost total absence of tumors at other sites’.7 granulosa cell tumors and ovarian adenocarcinomas are most frequently reported but carcinomas, leiomyosarcomas/leiomyomas, adenomas, teratomas, dysgerminomas, fibrosarcomas, lipomas, and lymphomatosis have all been identified in bird ovaries. oviductal tumors are less common than ovarian neoplasia and include adenocarcinomas/adenomas, adenomatous hyperplasia, carcinoma, and carcinomatosis.1 granulosa cell tumors and possibly other reproductive tract neoplasms may be functional and cause increased plasma hormone concentrations. granulosa cell tumors are common in older female budgerigars (melopsittacus undulatus) and may clinical theriogenology 2022; 14: 35 be functional resulting in hyperestrogenism.8 polyostotic (medullary) hyperostosis may also result as a paraneoplastic syndrome with functional ovarian and oviductal neoplasms. interestingly, hyperestrogenism did not cause polyostotic hyperostosis in several species of birds with various neoplastic and nonneoplastic reproductive tract diseases.9 clinical signs vary and are nonspecific for most reproductive tract diseases and include coelomic swelling, dyspnea, ascites, poor or altered reproductive performance and lethargy. if the mass compresses the overlying lumbar or sacral nerve plexuses, lameness (usually left sided) may be observed. diagnosis can be further supported using radiography, ultrasonography, mri, ct, exploratory celiotomy, endoscopy, and biopsy. once a definitive diagnosis is made, options for therapy include chemotherapy, radiation therapy, and partial or complete ovariectomy, but all carry a guarded prognosis if the neoplastic tissue cannot be completely removed. ovarian surgery partial and ‘complete’ ovariectomy ovariectomy in hens is a challenging and oftentimes high-risk procedure. ovariectomy has been used in many poultry studies and this procedure is mentioned throughout literature. unfortunately, most papers poorly describe specific details of ovariectomy or its complications. to cite a few examples; it was stated that ovariectomized birds ‘lost considerably more blood than sham-operated hens’.10 incompletely ovariectomized turkeys (number not provided) were excluded.11 ovariectomy has also been described by ‘destroying ovarian tissue by local application of small pieces of dry ice.’12 although it has been stated that the short stalk of cranial renal artery is what makes ovariectomy difficult, first author suggested that the intimate and lengthy attachment to the overlying common iliac vein is what makes this procedure risky.1 as discussed, multiple small veins often connect directly into the common iliac vein. it is often venous, and not arterial, bleeding from a lacerated common iliac vein that usually causes life-threatening hemorrhage during ovariectomy. as with oviduct, ovary can dramatically change in size and vascularity with sexual and egg laying activity. as a result, every attempt should be made to medically or behaviorally downregulate bird’s reproductive activity to reduce vascularity to ovary. oftentimes, diseases requiring ovariectomy do not allow attending clinician’s time to ‘condition’ the avian patient prior to surgery. ovariectomy should be reserved for ovarian diseases such as cancer, chronic recurring cysts, persistent follicular activity, oophoritis and other diseases that cannot be managed medically and are life-threatening without further treatment. whereas some birds such as cockatiels appear to stop ovulating after the salpingohysterectomy, other species such as ducks may continue chronic and possibly life-threatening ovulation necessitating hormonal therapy or ovariectomy.13 behavioral and/or medical management, discussed elsewhere in literature, is necessary after a partial ovariectomy, to prevent internal or ectopic ovulation. a cranial left lateral celiotomy often provides the best left ovary exposure. clean the surgical field of fluid and debris to best visualize the ovary and its vasculature. surrounding organs may need to be gently retracted using moistened cotton-tip applicators or other nontraumatic instruments. a ventral midline, singly or combined with a transverse approach, can also be used to reach the ovary from a caudal approach. first step to ovariectomy is to debulk its mass, regardless of size, in order to visualize the ovarian attachment to the overlying common iliac vein and any other vessels. if the ovary is inactive or juvenile, little debulking is needed. if present, remove large preovulatory follicles by twirling as discussed below. aspirate and drain any cystic follicles present, being careful not to spill contents into the coelomic cavity, especially if there is concern of oophoritis. when aspirating follicles, guide a small gauge (23 25) butterfly catheter needle into the most visibly avascular portion to reduce hemorrhage and have an assistant provide distant suction. using this aspiration technique, a substantial amount of an active and/or cystic ovary can be debulked. blood filled follicles may represent previously ruptured blood vessels from an invasive mass and warrants caution when attempting debulking. alternatively, large ovarian follicles can be twirled using cotton tip applicators. use the cotton tip applicators to rotate the follicle in 1 direction continuously until it separates from its pedicle. this may require 15 30 full rotations until the follicle is free. once free, simply remove the follicle. once the fluid component is minimized, progressively clamp or hemostatically clip the ovarian mass closer to its base. when used properly, angled debakey neonatal vascular clamps are atraumatic, will rest in the surgical site without obstructing view and seem to provide some hemostasis to the ovarian mass. once a section of the mass is hemostatically clipped or clamped, surgically excise or cauterize and remove the ventral-most ovarian segment. reassess the mass and move the clamp (or place new hemostatic clips) closer to the base and repeat the excision process. this process is repeated until the overlying vasculature is clearly identified, and the course of the common iliac vein is observed. once the mass has been debulked, several options exist for complete or partial ovariectomy. an electrocautery ball electrode was used effectively to coagulate ovarian follicles in immature females.14 similar procedure resulted in ovarian regeneration and subsequent ovulatory activity in mature hens. some juvenile bird ovaries were gently ‘peeled’ in toto from caudal to cranial off its dorsal attachments with no or minimal bleeding.6 in these cases, ovarian caudal end is grasped with angled hemostats and pulled in a cranial direction with a clear separation, and minimal effort, from the dorsally located common iliac vein. if attempting this procedure, stop if any resistance is noted to prevent tearing the overlying vein. another technique with juvenile or sufficiently debulked ovaries is to place hemoclips in the potential space between the ovarian base and the common iliac vein. gently lift the ovarian caudal pole and place a small to medium hemostatic clip from caudal to cranial across the ovarian vascular supply. although difficult without good exposure, a last hemostatic clip clinical theriogenology 2022; 14: 36 can be placed from cranial to caudal in the same manner in an attempt to ligate the more cranially located ovarian artery. with the blood supply adequately clamped, the ovary can be gently shaved off with precise radiosurgery using an ellman b ‘loop’ series or blade electrode (ellman international, inc., new york, ny) or left to die without a blood supply. first author has successfully performed ovariectomies in adult hens using this technique. obvious complications include hemorrhage when trying to remove the hemostatically clipped ovary and inadequate, blind, placement of the hemostatic clips. second author has used another approach when the ovarian attachment to the overlying common iliac vein is too long to hemoclip or there is erosion into the overlying vessel and the entire ovary must be removed for the bird’s survival (as with otherwise untreatable cancer).6 debulk the ovarian mass as described above. once clearly identified, hemostatically clip the common iliac vein just caudal to the ovary and cranial to its junction with caudal renal vein. next, hemostatically clip the common iliac vein just cranial to the ovary and caudal to its junction with caudal vena cava. if performed properly, the ovarian artery and common iliac veins are effectively clamped, allowing one to carefully dissect the entire ovary from the overlying vessel(s). if needed, the ventral wall of the common iliac vessel can be safely removed. there is the real potential of damaging left adrenal gland, substantially altering blood flow through the renal portal system and the cranial renal division and causing physical damage to overlying kidney and lumbar and/or sacral nerve plexus(es). none of the above described ovariectomy procedures have been satisfactorily studied in pet bird species and each carries a marked risk to patient. with each procedure, closure is routine. more often, a partial ovariectomy is performed and only the abnormal ovarian tissue is removed. oviductal anatomy oviduct, or salpinx, develops from the left müllerian duct and can be divided into 5 regions.15 cranial most region is the infundibulum (site of fertilization) that engulfs the ovulated ovum. next, ovum moves into the largest region, the magnum. magnum produces albumin that surrounds the developing egg. as the ovum progresses caudally it enters the isthmus where the inner and outer shell membranes are formed. egg is ‘plumped’ with water and solutes, calcified to form a shell and pigments are deposited during its prolonged stay in the shell gland or ‘uterus’. shell gland transfers the complete egg through the uterovaginal sphincter and into vagina. uterovaginal area contains sperm storage tubules allowing many species to store viable sperm for prolonged periods (> 21 days in turkey hens).15 vagina terminates at cloaca and coordinates with shell gland to ultimately expel egg. oviduct is suspended within the coelomic cavity via a dorsal and ventral ligament.15,16 cranial, middle, and caudal oviductal arteries running in the dorsal mesentery, supply blood to oviduct. origins of each vessel vary between species, but some generalization can be made.16 cranial oviductal artery arises from the left cranial renal artery, aorta, or external iliac artery. middle oviductal artery arises from the left ischiatic artery or its branch, the medial renal artery. finally, the caudal oviductal artery arises from the left internal iliac artery or the pudendal artery. veins draining the cranial oviduct empty into caudal vena cava (via common iliac vein), whereas those draining the caudal oviduct enter the renal portal or hepatic systems. oviductal diseases congenital defects congenital defects have been identified in birds and include large cysts on a rudimentary oviduct of a budgerigar and discontinuous or atretic oviducts in domestic fowl. persistence of right ovary and/or oviduct has been reported in numerous species.1 first author has observed an association between the presence of right oviducts and nonspecific reproductive tract problems in hens (e.g. cystic ovarian follicles, and excessive egg laying). cystic dilatation is common with persistent right oviducts in poultry species. each defect should be evaluated on a case-by-case basis and may require surgical modification or removal based on associated clinical signs. ectopic ovulation, ectopic eggs, and egg yolk peritonitis ectopic ovulation occurs when infundibulum fails to engulf an ovum or fails to retain ovum because of oviductal rupture or reverse peristalsis. ectopic ovum is not well developed. potential causes include infundibulum failure from oviductal fat, trauma or disease, exuberant reverse peristalsis, and oviductal disease. ectopic ovulation occurs frequently and has been reported in 28.6% of necropsied birds from 9 orders.17 authors have also observed ectopic ovulation associated with a persistent right oviduct in several avian species. ectopic ovulation usually results in mild, self-resolving, sterile yolk coelomitis and usually requires no or minimal supportive therapy (fluid therapy, anti-inflammatories, and others). partially and completely shelled ectopic eggs result when a developing egg enters the ceolomic cavity through an oviductal rupture or via reverse peristalsis from oviductal or even cloacal disease. anything affecting the oviduct function such as cloacal or oviductal masses (including egg binding, impactions, and neoplasia), salpingitis, cystic hyperplasia, and oddly shaped or large eggs can result in ectopic eggs. a large ectopic egg can cause a penguin-like stance and is often associated with ascites and varying degrees of depression in many birds. diagnosis can often be suspected using radiography, ultrasonography, mri, ct, and sometimes endoscopy (depending on how much debris is in the coelom); however, celiotomy is often required for definitive diagnosis. ectopic eggs should always be considered when conservative therapy for egg binding fails. partially and fully formed ectopic eggs should be surgically removed after stabilizing the patient and determining underlying cause(s). severe sterile and life-threatening septic egg yolk peritonitis may also result from ectopic ovulation or eggs. acute egg yolk peritonitis may result in marked depression, anorexia, and ascites and rarely, respiratory distress and death. secondary diseases resulting from sterile and septic yolk peritonitis include pancreatitis, splenitis, yolk-thromboembolic’ disease, hepatitis, nephritis, and coelomic adhesions. septic yolk peritonitis may obviously result in or from septicemia and has been most noted with coliforms such as escherichia coli, yersinia pseudoclinical theriogenology 2022; 14: 37 tuberculosis and staphylococcus spp.18 coelomocentesis and cytologic fluid analysis and culture are used to definitively diagnose yolk peritonitis. if needed, celiotomy and endoscopy can be used to assess the associated internal pathology. severe egg yolk peritonitis, especially when associated with bacteria, includes aggressive supportive care, antimicrobials, identifying and resolving causative factors if possible and occasionally may require surgical removal, irrigation of the caudal coelomic cavity and placing a drain tube. if irrigation is used, be careful to prevent fluid from entering the respiratory system. egg binding and dystocia egg binding, dystocia and other reproductive tract diseases (e.g. egg yolk peritonitis and cystic ovary) are common problems in pet bird medicine. oviposition is the expulsion of egg from the oviduct and is conducted by vigorous contraction of the uterine muscles and peristalsis of the vagina. egg binding is simply defined as prolonged oviposition (egg is arrested in oviduct longer than normal for the given species) whereas dystocia implies the developing egg is within the distal oviduct either obstructing the cloaca or prolapsed through the oviduct-cloacal opening. dystocia is often more advanced than egg binding alone, has many potential causes and is commonly associated with functional (malformed eggs, cloacal masses, and obesity), metabolic (calcium imbalance and nutritional deficiencies), environmental (temperature changes, lack of exercise and other stressors) and hereditary diseases. captive conditions (readily available food, water, light, appropriate temperature, and frequently a constant mate) tend to promote reproductive activity in pet birds. as a result, excessive egg laying, prolonged broody behavior and reproductive tract disease are common, especially, in smaller birds such as cockatiels and budgerigars. secondary complications of excessive egg laying include egg binding, dystocia, egg yolk peritonitis, acquired metabolic bone disease, pathologic fractures, prolapsed cloaca and oviduct, and salpingitis. additionally, some of these ‘over productive’ hens are on a poor diet, are nutritionally depleted and have other underlying diseases complicating their reproductive problem. several options are available for treatment of eggbound birds. most birds can produce a shelled egg from a follicle in < 24 hours. although knowledge of individual species is important, most birds that have an egg present for > 24 hours are considered ‘egg bound’. eggs place pressure on surrounding organs, especially the kidneys, and can cause progressive problems the longer egg binding continues. smaller the bird, the more serious egg binding becomes. stable birds can often be managed conservatively (pain management, fluid therapy, supplemental heat, nutritional and hormonal support, and others) to help the patient deliver the bound egg. if needed, the egg can be manually manipulated or crushed to facilitate removal. following techniques should only be considered if more conservative therapy fails, or pressure must be relieved immediately. due to the potential for substantial tissue damage, antibiotics are often indicated. one can gently push the egg toward the cloaca and (hopefully) out. prelubricate the cloaca with a water-soluble lubricant (h-r lubricant, carter products, new york, ny) to facilitate the egg’s removal. another option is to aspirate the egg’s contents (pass an 18 20 gauge needle through the cloaca if the egg is visualized, and aspirate) and then crush the egg with digital pressure. if no adhesions or oviductal torsion, masses or tear(s) are present, the bird will oftentimes pass the crushed egg within 24 hours. aspirating the egg through the coelomic wall results in minimal to severe (laceration) trauma to the oviduct and is not recommended. salpingohysterectomy is needed in otherwise nonresponsive cases. once egg binding has been resolved, every attempt should be made to downregulate the bird’s reproductive system. simple methods include removing the mate, nest box(es) or favorite toy(s) when present, changing the diet (usually improving quality), decreasing the photoperiod to a maximum of 10 12 hours a day, modifying the cage setup and toys and in general, altering the bird’s normal daily activities. gnrh agonists (leuprolide acetate and deslorelin) may also be beneficial. the intent herein is to limit environmental influences on reproductive activity and stop the bird’s desire to breed. as supported in domestic fowl, once incubation behavior is established, the ovarian hormones are not required for maintenance or the readiness to brood day-old chicks.19 this implies that drugs intended to decrease production of ovarian hormones, such as leuprolide acetate, alone may not be sufficient to stop a bird’s reproductive behaviors. this further supports the need for environmental, dietary, and other behavioral modifications with leuprolide acetate use to decrease reproductive activity in birds. oviductal cystic hyperplasia cystic oviductal hyperplasia or dilatation has been reported in budgerigars, other psittacines and poultry.1 although little etiologic information is forwarded, cysts may occur secondary to improper formation of the oviduct. affected oviducts are often thickened with white to beige masses and distended with brown or white mucoid fluid. affected birds may show no signs (and the oviductal hyperplasia is discovered incidentally) or signs typical of reproductive tract disease. antimicrobials may be tried if organisms are recovered from aspirated samples, otherwise salpingohysterectomy is indicated. oviductal impaction an impacted oviduct is usually distended and simply contains caseated material and misshapen, ruptured, soft-shelled, partially, or fully formed eggs. potential causes include excess mucin and albumin secretion secondary to inspissated egg material and cystic hyperplasia. salpingitis is often found concomitantly, especially in older birds. metritis, salpingitis, egg binding, dystocia and neoplasia commonly precede oviductal impactions. typical of most reproductive tract diseases, vague clinical signs with or without coelomic swelling and ascites are common with oviductal impaction. some birds may show persistent ‘broodiness’ with recent cessation of egg laying. oviductal impactions have been reported in budgerigars, cockatiels, canaries, african grey parrots (psittacus erithacus) and poultry.1 definitive diagnosis is made at celiotomy or sometimes via clinical theriogenology 2022; 14: 38 ultrasonography, mri, ct, and endoscopy with aspiration of the oviductal contents. chronic oviductal impactions may be observed incidentally during exploratory celiotomy and are often associated with a history of sudden cessation of egg laying several months or years prior to presentation. acute impactions may be treated by salpingotomy, culture and appropriate antibiotic use and oviductal flushing, whereas severe or chronic disease are best treated with salpingohysterectomy. oviductal prolapse powerful coelomic contractions combined with the process of oviposition can result in oviductal prolapse which is often secondary to dystocia. predisposing factors may include large or abnormally shaped eggs, malnutrition, general debilitation and systemic illness, disease of the oviduct and sometimes, normal egg laying. in turkeys selected for high meat yield, decreased vaginal collagen has been associated with uterine prolapse.1 uterus is most commonly prolapsed; however, vagina and other portions of oviduct may also prolapse. cloacal prolapsed tissue, if present, should be distinguished from the oviductal prolapse. because the exposed tissue can rapidly become devitalized and infected, aggressive treatment with warm saline flushes, antibiotics and replacement of the prolapsed oviduct is warranted. if the prolapsed oviduct is edematous, topical dextrose, dimethyl sulfoxide (dmso) and/or steroids may be needed to reduce the swelling. if an egg is present in the prolapsed or coelomic oviductal tissue, ovocentesis and digital crushing is often needed to reduce associated pressure and aid in egg-shell removal. after stabilizing the bird, remove the egg medically if possible and replace the prolapsed tissue. two transcloacal sutures may be required to prevent the immediate recurrence of prolapsed tissue. surgical removal is indicated when the oviduct is necrotic and/or the egg (or its fragmented shell) cannot be removed medically or pass on its own. if an oviductal torsion is present caudal to egg (within the oviduct), attempting to force deliver the egg will often result in further damage. oviductal torsion, neoplasia, adhesions, and other anatomic disorders should be considered if a bound egg cannot be delivered without forceful techniques and surgical options should be pursued. oviductal torsion oviductal torsion has been reported infrequently as a cause of egg binding. oviductal torsion may occur after a tear of the dorsal, and possibly ventral, oviductal ligament(s). in 4 reported cases, all birds presented with signs of egg binding and/or general lethargy and had a history of previously laying ‘many eggs’ prior to the oviductal torsion.20 a cockatiel presented thin with lethargy, depression and coelomic distension, and died despite emergency therapy. of the 3 birds presented live, 2 cockatiels were treated with salpingohysterectomy and 1 eclectus parrot (eclectus roratus vosmaeri) was treated with a hysterotomy (salpingotomy), egg removal, torsion correction, and subsequent closure of the oviductal ligament tear. all birds recovered uneventfully from surgery. eclectus parrot successfully laid normal clutches after surgery. salpingitis and metritis salpingitis, inflammation of the oviduct or salpinx, is common in birds. in poultry, salpingitis has been listed as the most prevalent form of reproductive tract disease. e. coli infections are common in poultry and can cause salpingitis, but streptococcus spp., mycoplasma gallisepticum, acinetobacter spp., corynebacterium spp., salmonella spp., and pasteurella multocida have all been implicated from various species.1 some ground-nesting species, such as anseriformes and emus, may develop nonlactose fermenting, gram negative (pseudomonas aeruginosa, proteus mirabilis, p. vulgaris) salpingitis. noninfectious salpingitis can also occur, especially with chronic, sterile oviductal impactions. metritis is inflammation within the uterine portion of the oviduct and may result from or cause egg binding, chronic oviductal impaction and rupture, coelomitis, and septicemia. prosthogonimus ovatus and other related trematodes (flukes) can inhabit the oviduct of anseriformes and galliformes and result in salpingitis with heavy infestations. other infectious agents ascending from the vagina or cloaca or descending from air sacculitis, pneumonia and septicemia can also cause salpingitis. specifically, in poultry, vent cannibalism has been implicated as a precursor to salpingitis. birds with nonseptic salpingitis or metritis often have vague signs of illness, whereas septic birds are usually clinically very ill. a distinctive feature is that egg-shell deformities and embryonic and neonatal infections are often secondary to metritis. presumptive diagnosis may be made with ultrasonography, mri, ct, and occasionally with endoscopy. definitive diagnosis is made during celiotomy or endoscopy with aspiration of oviductal fluid for cytologic and microbiologic analysis or if the oviduct has no liquid contents, biopsy with culture. base antibiotic use on culture and sensitivity results. if trying to spare the oviduct, repeated endoscopic evaluation, direct and indirect oviductal flushing, and long-term antimicrobials are recommended. salpingohysterectomy is indicated for severe cases or those nonresponsive to appropriate antimicrobials. oviductal surgery salpingohysterectomy salpingohysterectomy, is the surgical removal of oviduct, from infundibulum to uterus, and is indicated for chronic egg laying and any oviduct disease that cannot be medically managed and is reported as the ‘therapy of choice for overproduction of eggs.’1 every attempt should be made to understand the bird’s overall health status prior to surgery, as the patient should ideally be stable. birds with septic yolk-peritonitis generally carry a poor prognosis. patients with underlying health problems such as various lung, liver, and kidney diseases, can also complicate surgery. otherwise, healthy salpingohysterectomy candidates typically do well and surgery is often straightforward especially when the oviduct is small and inactive. oviductal hypertrophy occurs secondary to elevated estrogen concentrations during sexual activity and can take up most of the left side of the ipc. this oviductal hypertrophy includes increased vascularity and risk of bleeding during surgery. if the patient is stable, time permits, and increased reproductive tract vascularity is suspected, the authors will ‘condition’ the bird prior to surgery. ‘conditioning’ includes improving nutritional status (if necessary) and attempting to downregulate the bird’s sexclinical theriogenology 2022; 14: 39 ual cycle as discussed under ‘egg binding and dystocia’. this process may take weeks to months and often results in decreased vascularity and lower patient morbidity. in first author’s experience, a left lateral approach offers the best exposure to the left ovary and oviduct, however a ventral midline approach can also be used and is best for overall reproductive tract evaluation. perform a left lateral celiotomy. after incising through the left abdominal air sac, ovary and oviduct are readily visible. gently retract the cranial oviduct (infundibulum area) out the incision and hemostatic clip or cauterize suspensory ligament vessels as needed. closer the bird is to laying, the larger the vessels present. depending on the size, the cranial, middle and/or caudal oviductal artery(ies) may need to be hemoclipped or cauterized. once visualized, hemoclip the base of the oviduct just proximal to its junction with the cloaca. excise the oviduct. well-developed preovulatory follicles (f1 and f2 +/f3, and f4) may pose a risk for intra-coelomic ovulation and can usually be easily removed as described for ‘ovarian surgery.’ if the follicle has a well-developed vascular pedicle, use hemoclips and then excise the follicle or twirl the follicle. domestic chickens have a pause in laying that increased with the number of follicles removed compared to sham operated hens.21 cystic follicles should either be aspirated (drained) or removed. whereas normal follicles have a yellow appearance and are filled with thick yolk, cystic follicles are pale to translucent and generally contain clear watery fluid. if the follicle is accidentally incised, yolk will leak into the coelom. simply ‘mop up’ excess yolk and other fluid if present. collect culture and samples for histopathologic evaluation as needed. an endoscopic approach to salpingohysterectomy of juvenile cockatiels has been described.22 a left lateral coelomic endoscopic approach (left leg pulled caudally) was performed on juvenile (3 11 months) cockatiels. once visualized, the supporting ligament of the infundibulum was carefully pulled laterally toward the coelomic entry site using flexible endoscopic grasping forceps (karl storz veterinary endoscopy, inc., goleta, ca). this action broke down the supporting structures (ventral and dorsal suspensory ligaments of the cranial oviduct and uterus) and separated the salpinx (oviduct) from the overlying kidney, caudal vena cava and left ureter. next, a cotton-tipped applicator was placed in the cloaca and was used to better visualize the uterus-cloacal junction and ensure the salpinx was ‘peeled’ from the surrounding tissues. salpinx was exteriorized and then crushed and cut with microsurgical forceps and scissors, respectively, at the point of exit from the coelomic cavity, just cranial to the uterovaginal sphincter. endoscope was replaced to check for hemorrhage and closure was routine. endoscopic salpingohysterectomy has several distinct benefits and limitations. as indicated,22 this procedure was acceptable in juvenile birds due to a poorly developed blood supply of the oviduct and that if attempted in mature, egg-producing cockatiels, may result in fatal hemorrhage. additionally, this procedure required an endoscope and 2 surgeons. although the endoscopic surgery was limited to young birds, there was minimal hemorrhage and could be performed safely and quickly (estimated to take < 10 minutes with experience) and offers an option for juvenile salpingohysterectomy.22 caesarian section and reproductive tract sparing caesarian section is indicated when the bird’s reproductive capabilities need to be spared and is typically limited to egg binding with an otherwise normal, or minimally diseased oviduct. depending on the location of the egg, a caudal left lateral or ventral midline approach is used. oviduct should be incised directly over the bound egg and away from prominent blood vessels. after removing the egg, inspect the oviduct for other abnormalities and collect biopsies and cultures as needed. close the oviduct in a single simple interrupted or continuous layer using fine (4 0 or smaller) absorbable suture material. coelomic closure is standard. authors recommend resting the hen from reproductive stimuli for 2 4 weeks or longer as dictated by culture and/or histopathologic results. male reproductive tract diseases compared to females, male reproductive diseases are less frequently reported; however, they are well described. a clear understanding of the anatomy is essential to make a diagnosis and to properly treat conditions identified. medical and surgical management of male reproductive tract diseases, caponization, and vasectomy are described. testicular anatomy reproductive anatomy of most male birds consists of 3 main gross structures: testes, epididymis, and ductus deferens. some birds also possess a phallus. paired testes are located ventral to their respective left or right cranial renal division. mesorchium connects the testes to the dorsal body wall. left testis is typically larger than the right in most young birds, but this relationship can change as the bird ages. in seasonal breeders, such as some passerines, testes can increase 300 500 times in size and should not be interpreted as neoplasia. large active testes can also be readily evident radiographically. in addition to size, the color of the testes can also change with fluctuating hormone concentrations ranging from black in the sexually immature or inactive cockatoos to white or yellow in the chicken.1 epididymis is located at the testicular hilus, or dorsomedial aspect of testis. ductus deferens continue from epididymis as highly convoluted tubes running lateral to and alongside the ureters and then terminate at the urodeum as a papillae ventral to ureteral ostium. testicular artery arises from the cranial renal artery and provides most of the arterial blood supply to testis. an accessory testicular artery may arise directly from the aorta. venous drainage is returned either directly to caudal vena cava or forms a common stem with adrenal veins. in pekin drakes (anas platyrhynchos), 2 testicular veins empty directly into caudal vena cava.23 given the diversity within the class aves, it is likely that multiple variations of the testicular vasculature exist. clinical theriogenology 2022; 14: 40 although most birds lack a copulatory organ, some birds possess a nonprotrusible (galliformes) or protrusible (ratites and anseriformes) phallus. domestic chickens and turkeys have a nonintromittent phallus, consisting of a median and 2 lateral phallic bodies, on the floor of the lip of the vent. lymphatic flow through the phallic bodies and their laterally associated lymphatic folds result in tumescence. because the lymphatic folds and lateral phallic bodies accumulate more fluid than the median body, phallus everts during tumescence producing a groove for semen to travel. semen is deposited when phallus contacts the hen’s everted oviductal opening. male avian reproductive system diseases orchitis inflammation of the testis, or orchitis, is usually due to bacterial infections and may originate from septicemia, renal obstruction, cloacitis or even prolapsed or ulcerated phalli. affected birds may show signs of septicemia. however, first author has seen cases of focal orchitis with no associated clinical signs or reduced fertility only. orchitis may be diagnosed via cytology and/or microbiologic analysis via aspiration through endoscopy or celiotomy when whole testis appears abnormal or, biopsy when focal lesions are seen. ultrasonography and contrast ct or mri may help isolate lesions within the testis. initial treatment for bacterial orchitis should include antibiotics based on culture and sensitivity results. if a focal granulomatous lesion is observed and appropriate antimicrobials have proven ineffective, the testis can be partially ablated. clamp with hemostats or surgically clip the testicular tissue dorsal (towards the blood supply) to the lesion(s) and remove using cold excision, laser, or radiosurgery. avian testicular tissue has great regenerative capabilities and may redevelop following partial ablation. enbloc surgical removal of the affected testicle is indicated for diffuse, nonmedically responsive orchitis. testicular neoplasia avian testicular neoplasia most commonly includes sertoli and interstitial cell tumors, seminomas, teratomas, and lymphoproliferative diseases. sertoli cell tumors appear to be more prevalent testicular neoplasm in birds. reported neoplasms of epididymis and ductus deferens include leiomyosarcoma and carcinoma. chronic weight loss, coelomic swelling and unilateral paresis are most commonly associated with testicular cancer. radiographs, ct, mri, ultrasonography may all be used to help make a diagnosis. surgical removal of the affected testis is the treatment of choice and carries a good prognosis if metastasis is not present. as noted in literature and in authors’ experience, many testicular tumors are cystic. cystic testicular masses can be aspirated and drained during surgery to reduce their mass and facilitate removal. some testicular tumors may metastasize as has been reported in a guinea fowl (numida meleagris) with malignant seminoma.24 cystic testicular disease nonneoplastic cystic testicular disease is very infrequently reported, and its importance is unknown.1 cystic dilatation of the seminiferous tubules (and testes) has been produced in fowl fed a diet high in sodium. cystic testes have also been noted in chickens fed egg albumen as a source of protein. dilatation of seminiferous tubules, but not gross cystic testicular change, has been noted in roosters affected with epididymal cysts and stones of unknown origin. as mentioned above, consider cancer first when cystic testis is diagnosed. cystic testis should be drained, biopsied and ideally removed. disorders of phallus male waterfowl have a protrusible phallus, which is highly variable and particularly long and corkscrewed in the muscovy duck (cairina moschata).6 partial and complete phallic prolapses are possible in waterfowl with large phallus and are usually secondary to trauma, local infection, and masturbation. over exuberant vent sexing and mating, neisseria spp. (suspected to be sexually transmitted in geese and the cause of ‘goose gonorrhea’) and contamination have all been implicated causes of phallic infections.1 a prolapsed phallus may become enlarged, ulcerated and/or necrotic compounding the problem. frostbite and resultant necrotizing dermatitis of a prolapsed phallus has been noted in ostriches. birds with severe prolapse and infection may be substantially depressed and often lose interest in copulation. clean exposed phallus and carefully debride abnormal tissue prior to replacement. topical antibiotic creams, dmso and systemic antibiotics may be beneficial, and their use is based on clinical findings. cloaca may need partial closure (via transcloacal sutures) to prevent recurring prolapses. if the prolapse is prolonged and will not stay when replaced, use 4-0’ monofilament absorbable suture to gently tack the phallus to its resting position within the cloacal mucosa. severely necrotic phallus often needs surgical debridement. using absorbable suture in an encircling pattern, ligate the phallus proximal to necrotic tissue. it is best if there is a clear demarcation from healthy tissue. amputate the tissue distal to the ligature ensuring that all necrotic tissue is removed. male reproductive system surgery castration clinical castration is infrequently discussed, especially in comparison to salpingohysterectomy, suggesting that male reproductive tract diseases are relatively uncommon. although caponization is common in the poultry industry (performed between 1 2 weeks or up to 6 weeks of age depending on the breed), routine castration is rare in pet birds. as a result, there is little information regarding the behavior and physiologic altering effects of castration in pet birds. caponized chickens (capons) have increased coelomic fat weight, total hepatic lipid content and saturated fatty acid percentage compared to intact birds. medical consequences, if any, of this body change are not known. castrated gambel’s (callipepla gambelii) and scaled (c. squamata) quail have reduced or eliminated courtship behaviors and lower rates of male-male threats.25 however, the castrates maintained ornate plumage, exhibited overt aggression, and clinical theriogenology 2022; 14: 41 frequently won contests when actually engaged. yearling european starlings (sturnus vulgaris) castrated when not actively reproductive were more aggressive compared to uncastrated controls.26 it was suggested that ‘nonreproductive aggression in yearling male starlings is independent of gonadal sex steroids and it even increased after castration.’26 these limited results suggest that persistent ‘male’ behaviors are either already learned at castration, result from hormones other than testosterone or another source of testicular hormones is still present postcastration. it is known that some species have an appendix epididymis extending from epididymis into adrenal gland that may secrete androgens after castration. authors have performed castration in roosters in effort to stop crowing. castrated roosters did exhibit reduced crowing behavior; however, it did not stop. authors concluded that castration was not appropriate or effective to eliminate crowing behavior in roosters. until further studies are available, castration should be used judiciously to alter avian behaviors, especially in adult birds and should always be considered secondary to more conservative methods of behavior management. however, castration has real benefit with testicular cancer, abscesses/granulomas, cysts, and other conditions that may not respond to medical management alone. several methods of castration have been advocated and include simple extraction (caponization), laser ablation, intracapsular suction, enbloc surgical excision and endoscopic orchidectomy. even with early age caponization, testis regrowth is well documented.6 this supports the need for complete removal of testis, the reason why the authors prefer enbloc surgical excision. caponization is typically performed in young male chickens to create meat that is apparently more tender, juicier, and tastier than in the intact rooster. heavy chicken breeds are caponized at 2 4 weeks, whereas some slow-growing meat-type birds after 6 weeks.6 as the bird ages, tunica albuginea of the testis becomes hard making caponization more difficult and time consuming. the procedure is typically performed without anesthesia with the bird held or strapped to a table. a sterile preparation is given to the appropriate side and an incision is made between the last 2 ribs through the lateral body wall that is then spread with a ‘spreader’. performed correctly, specialized caponizing forceps are used to enter the incision, delicately hold entire testis, and pull with a twisting motion until testis is free and removed. wound is disinfected and left to close by second intention. incompletely caponized birds may regrow testis and the birds tend to develop secondary sex characteristics unlike true capons. a similar technique using standard curved forceps is described in 9 10 weeks japanese quail (coturnix coturnix japonica).27 use a cranial left lateral approach or ventral midline incision with transverse flap to evaluate testis. due to cranial location, the lateral celiotomy is often extended cranially by cutting the last 2 ribs to improve testicular exposure. with a left lateral approach, puncture through the caudal thoracic and/or the abdominal air sac(s) to expose left testis. right testis may be exposed through the same incision by cutting through the midline junction of the corresponding air sacs or the process may be repeated with a right lateral celiotomy. with gentle traction, pull the testis ventrally and surgically clip the dorsal blood supply. use of a right-angle surgical clip often makes the approach easier. if 2 clips can be placed, then incise between the surgical clips and remove the testis. otherwise, use radiosurgery to carefully free testis from the surgical clip and vascular cord. cautery should destroy any remaining testicular cells attached to the surgical clip. be careful to not damage the overlying blood vessels, kidney, or adrenal gland. alternatively, if testicular blood supply is small, a hemostat can be temporarily used in the place of a surgical clip and testis is pulled free. leave the hemostat on the vascular stump for 1 2 minutes prior to release. use direct pressure hemostasis as needed. diode laser excision can also be used through this approach and may be performed without the need for direct hemostasis. closure is routine. multiport endoscopic orchidectomy has been described in carneau pigeons.28 whereas the details of the procedure are described,28 endoscopic orchidectomy produced good results in 10 of 11 pigeons with a mean surgical time of 39 minutes. mild hemorrhage and partial necrosis of the cranial renal pole was noted in 27% of tested birds and represented the most common complication of surgery. surgical failure (regrowth of testis) in 1 bird was considered due to surgeon’s inexperience. when performed using appropriate equipment and techniques, endoscopic orchidectomy was successful and safe in pigeons. this technique could potentially be used in poultry and waterfowl. as expected, large testes are more difficult to remove endoscopically.28 in authors’ experiences, orchidectomy is more often needed for clinically abnormal (cancer, cysts, and others) and often large testes requiring celiotomy. vasectomy vasectomy is a useful technique to produce ‘teaser males’ and aid in population control and has been described in small passerines and budgerigars.29 in anesthetized budgerigars, a 3 mm incision, 7 mm lateral to the cloacal sphincter (vent), was used for the initial approach. careful dissection was made through the coelomic musculature and fat. an operating microscope was used to find and aid in the removal of a 5 mm section of vas deferens. only skin incision was closed. it was recommended performing left and right vasectomy 2 weeks apart.29 two of 12 birds died postoperatively and 1 had preexisting disease. other complications were postoperative tenesmus for 2 days and accumulation of droppings around the vent in 3 of the remaining 10 birds. this procedure was successful (no semen upon collection attempts) in 9 of the 10 surviving birds.30 anesthetized bengalese (lonchura striata) and zebra (taeniopygia guttata) finches have been vasectomized similarly to the procedure described above.29 in anesthetized finches, a 3 mm incision 5 mm lateral to the cloaca was made using an operating microscope. muscle and fat were incised to locate seminal glomera (glomus). it was noted that the seminal glomera of bengalese finch were ‘obvious and highly accessible,’ and that of the zebra finch was ‘less obvious and in some cases difficult to locate.’ vas deferens was carefully separated from the ureter clinical theriogenology 2022; 14: 42 and ‘1 or more pieces’ were removed with no ligature and the skin was closed. a single (14 days apart) and bilateral vasectomies were performed successfully, and was successful in 12 of 12 bengalese and 14 of 15 zebra finches.29 in larger species, vas deferens zig zags lateral to the ureter and can be transected endoscopically or via celiotomy. either a left, and sometimes right, lateral coelomic approaches are used. ureter is avoided to prevent damage. in vasectomized (just distal to epididymis) roosters, spermatogenesis ceased within 5 7 days.31 authors prefer endoscopic vasectomy in large birds, most commonly, as a means of population control in gallinaceous birds. ductus deferens is identified endoscopically (as if evaluating kidney) and grasped just distal to the epididymis and ~ 5 8 mm is removed with simple traction. at this location, ductus deferens is usually not closely associated with ureter.32 depending on the species, both ductus deferens may be approached through 1 endoscopic portal. alternatively, 2 endoscopic entry points (left and right) can be used. it should be recognized that vasectomy does not stop courtship and copulation. conflict of interest there are no conflicts of interest to declare. references 1. echols ms: surgery of the avian reproductive tract. seminars in avian and exotic pet med 2002;11:177-195. 2. taylor wm: pleura, pericardium, and peritoneum: the coelomic cavities of birds and their relationship to the lung-air sac system. in: speer: editor. current veterinary therapy in avian medicine and surgery. st louis; elsevier. 2016. p. 345-362. 3. mison m, mehler s, echols s, et al: approaches to the coelom and selected procedures. in: speer: editor. current veterinary therapy in avian medicine and surgery. st louis; elsevier: 2016; p. 638-657. 4. rubin ja, runge jj: principles of microsurgery. in: speer: editor. current veterinary therapy in avian medicine and surgery. st loui; elsevier: 2016; p. 631-638. 5. baumel jl: systema cardiovasculare. in: baumel jl, kings as, breazile je, editors. handbook of avian anatomy: nomina anatomica avium. 2nd edition, cambridge; nuttall ornithological club: 1993. p. 407-475. 6. echols ms: soft tissue surgery. in: greenacre c, morishita t: editors. backyard poultry medicine and surgery: a guide for veterinary practitioners. indianapolis; wiley: 2021. p. 381-433. 7. fredrickson tn: ovarian tumors of the hen. environ health perspect 1987;73:35-51. 8. bauck l: neoplasms. in: rosskopf wj, woerpel rw: editors. diseases of cage and aviary birds. baltimore; williams and wilkins: 1996. p. 480-489. 9. baumgartner r, hatt-j-m, dobeli m, et al: endocrinologic and pathologic findings in birds with polyostotic hyperostosis. j avian med surg 1995;9:251-254. 10. zadworny d, etches rj: effects of ovariectomy or force feeding on the plasma concentrations of prolactin and luteinizing hormone in incubating turkey hens. biol repro 1987;36:81-88. 11. proudman ja, opel h: daily changes in plasma prolactin, corticosterone, and luteinizing hormone in the unrestrained, ovariectomized hen. poultry sci 1989;68:177-184. 12. terada o, shimada k, saito n: effect of oestradiol replacement in ovariectomized chickens on pituitary lh concentrations and concentrations of mrnas encoding lh β and α subunits. j repro fertil 1997;111:59-64. 13. hudelson ks, hudelson p: a brief review of the female avian reproductive cycle with special emphasis on the role of prostaglandins and clinical applications. j avian med surg 1996;10:67-74. 14. altman rb: soft tissue surgical procedures. in: altman rb, clubb sl, dorrestein gm, et al: editors. avian medicine and surgery. philadelphia; wb saunders: 1997. p. 704-732. 15. johnson al: reproduction in the female. in: whittow gc, editor. sturkie’s avian physiology. 5th edition, san diego; academic press: 2000. p. 569-596. 16. female reproductive system. in: king as, mclelland j, editors. birds their structure and function. 2nd edition, philadelphia; bailliere tindall: 1984. p. 145-165. 17. keymer if: disorders of the avian female reproductive system. avian pathol 1980;9:405-419. 18. romagnano a: avian obstetrics. sem in avian exotic pet med 1996;5:180-188. 19. lea rw, richard-yris ma, sharp pj. the effect of ovariectomy on concentrations of plasma prolactin and lh and parental behavior in the domestic fowl. gen comp endocrinol 1996;101:115-121. 20. harcourt-brown nh: torsion and displacement of the oviduct as a cause of egg-binding in four psittacine birds. j avian med surg 1996;10:262-267. 21. johnson pa, brooks c, wang sy: plasma concentrations of immunoreactive inhibin and gonadotropins following removal of ovarian follicles in the domestic hen. biol repro 1993;49:1026-1031. 22. pye gw, bennett ra, plunske r: endoscopic salpingohysterectomy of juvenile cockatiels (nymphicus hollandicus). j avian med surg 2001;15:90-94. 23. kremer a, budras kd: the blood supply of the testis in pekin drakes (anas platyryhnchos l.). macroscopic, light microscopic, and scanning electron microscopic studies. anat anz 1990;171:73-87. 24. golbar hm, izawa t, kuwamura m, et al: malignant seminoma with multiple visceral metastasis in a guinea fowl (numida mealagris) kept in a zoo. avian dis 2009;53:143-145. 25. hagelin jc: castration in gambel’s and scaled quail: ornate plumage and dominance persist, but courtship and threat behaviors do not. horm behav 2001;39:1-10. 26. pinxten r, de ridder e, de cock m, et al: castration does not decrease nonreproductive aggression in yearling male european starlings (sturnis vulgaris). horm behav 2003;43:394-401. 27. busso jm, satterlee dg, roberts ml, et al: testosterone manipulation postcastration does not alter cloacal gland growth differences in male quail selected for divergent plasma corticosterone stress response. poult sci 2010;89:2691-2698. clinical theriogenology 2022; 14: 43 28. hernandez-divers sj, stahl sj, wilson gh, et al: endoscopic orchidectomy and salpingohysterectomy of pigeons (columba livia): an avian model for minimally invasive endosurgery. j avian med surg 2007;21:22-37. 29. birkhead tr, pellatt je: vasectomy in small passerine birds. vet rec. 1989;125:646. 30. samour jh, markham ja: vasectomy in budgerigars (melopsittacus undulatus). vet rec 1987;120:115. 31. janssen sj, kirby jd, hess ra: identification of epididymal stones in diverse rooster populations. poult sci 2000;79:569-574. 32. samour j: vasectomy in birds: a review. j avian med surg 2010;24:169-173. clinical theriogenology 2022; 14: 333 editor’s note dear theriogenology friends, paradigm shift i am so thankful to the ad hoc committee (drs. riley thompson [committee chair], divakar ambrose, and brian whitlock) for their hard work and approval of our leadership to move our journal to its next stage of its growth (open access) with the ultimate goal of getting recognized (pubmed indexing) in the scientific world. yes, our journal will become ‘open access’, incidentally, this will be the last hard copy issue! my profound thanks to omnipress staff and project managers (kristi connelly, jessica fogarty, and lisa poster) who were very helpful during the last 4 years. process a link will be in place in appropriate portal(s) to submit manuscripts to our journal. we will inform (via our list servers) once the platform becomes available (november). manuscripts accepted by the editor for publication shall be published online either continuously as they are accepted and edited, and/or collated into online issues. manuscript that are processed currently, including 4 canine symposium papers, and those that will be submitted to the journal (editor) will be processed for open access via open academia publishing services. we will stive to maintain our journal’s current typographic design and layout (including citations and references) initiated by dr. ahmed tibary. it took a while by the founding editor, dr. bob youngquist (10 years) and me (4 years) along with the copy editor (dr. john kastelic) to arrive at the current clinical theriogenology style and policy. thank you, bob, john, and ahmed. special issues we will collate manuscripts that are prepared for those 15 special issues and publish them online. i will continue to be in touch with the guest editors and bring special issues to fruition. conference issue conference issue will be eliminated. instead, conference proceedings (manuscripts, presentation notes, and abstracts) will be compiled for the online conference website and not on the open access website. however, excellent quality manuscripts from the proceedings will be chosen by the editorial board for the peer-review process to be included in the open access journal. this will encourage speakers to accept presentations for the conference without the requirement of submitting a full proceedings article, though a shortened document of presentation notes will still be required. welcome to our new executive director many colleagues may be aware that dr. charles franz has passed on the rein of executive directorship to ms. betsy griener. watching betsy’s involvement with our journal activities (via recent zoom meetings), i do firmly believe that our journal will continue to grow under betsy’s leadership. charles has done a fine job and personally, it was a wonderful experience for me to serve along with charles on the act board and with our journal for the last 4 years. thank you, charles, for your 2 decades of friendship and tremendous support, we will be in touch. please feel free to write to me (petera@purdue.edu), if you have questions or concerns, thank you all. wish you all a wonderful new year! regards, augustine 2018: the use of isperm technology for on-farm measurement of equine sperm motility and concentration the use of isperm technology for on-farm measurement of equine sperm motility and concentration christa r. moraes, bryan blawut, marco a. coutinho da silva department of veterinary clinical sciences, college of veterinary medicine, the ohio state university, columbus, oh the isperm® is a newly released semen analysis tool from aidmics biotechnology co. ltd, which allows an ordinary ipad mini to be transformed into a hand-held microscope with objective semen analysis software for equine available through the apple store (version 4.5.2). the aim of this study was to compare isperm® values for sperm motility and sperm concentration to current acceptable methods for semen analysis and to determine the agreement with these methods using statistical methods. two ejaculates from each of five standardbred stallions were used in this study to compare sperm motility and concentration. for motility analysis, spermatozoa were diluted to approximately 30 x106/ml in inra 96. an aliquot was then snap-frozen in liquid nitrogen and used to prepare samples with varying motilities by systematically adding a percentage of dead spermatozoa to the fresh sample (live:dead; 100:0, 75:25, 50:50, 25:75). each sample was measured in triplicate by casa (ceros, hamilton thorne, inc.) and isperm®. endpoints included total motility (tm), progressive motility (pm) and average path velocity (vap). for concentration determination, samples were centrifuged at 500 × g for 10 minutes and resuspended to approximately 480 x106/ml. a serial dilution was then prepared to achieve concentrations of 240, 120, 60 and 30 x106/ml, which spans the reported accuracy interval for isperm® (20 to 500 mil/ml). samples were evaluated in triplicates with both the isperm® and nucleocounter sp-100 (chemometec a/s). data were analyzed by first testing for the differences between the means of each method using a linear mixed model in r statistical software. the model accounted for the effect of stallion, ejaculate within stallion and the method of measurement. the ‘lme4’ and ‘lmertest’ r-packages were used for model fitting and hypothesis testing. the agreement between the two continuous measurements for each method was then investigated by computing lin’s concordance correlation coefficient in the ‘epir’ r-package. this measurement combines measures of both precision and accuracy to determine how close the data are to the line of perfect concordance with values ranging from 0 (no agreement) to 1 (perfect agreement). results are reported as the lin’s coefficient with the associated 95% confidence interval in parentheses. means for both tm and vap were different between casa and isperm® readings (p<0.001). however, pm means were not different between casa and isperm® values (p = 0.852). for concentration, means were not different between nucleocounter and isperm® values for the five concentrations analyzed (p = 0.578). the lin’s concordance correlation coefficient for tm was 0.831 (0.774, 0.875). for pm, the correlation coefficient was 0.883 (0.836, 0.917). average path velocity had a much lower correlation coefficient of 0.072 (0.043, 0.101). finally, the correlation coefficient for concentration compared by isperm® and nucleocounter was 0.953 (0.936, 0.966). with values close to the line of perfect concordance for tm, pm and concentration it is concluded that there is good repeatability between the two sets of data for each method used. since the software is still undergoing development, it is recommend that users maintain the software updated with the latest developments. the isperm® will introduce a low cost and affordable method for on farm semen analysis for breeders and veterinarians. as a result, more farms will have access to accurate sperm analysis tools which will help to standardize semen processing procedures leading to better overall quality of semen used for artificial insemination. keywords: spermatozoa, motility, equine, isperm®, casa clinical theriogenology • volume 10, number 3 • september 2018341 2018: unilateral segmental aplasia of the uterine horn in a labradorretriever unilateral segmental aplasia of the uterine horn in a labrador retriever marthina greer veterinary village and international canine semen bank wi/il, lomira, wi a 4 year old intact female labrador retriever was presented pregnant with one fetus. she was scheduled for an elective cesarean section because she was pregnant with a single fetus. her pre-operative bloodwork showed leukocytosis with a left shift. at surgery, she had one normal uterine horn with a normal, full-term, viable fetus that was successfully delivered. the opposite horn was incomplete, ending in a blind pouch approximately 10 cm proximal to the cervix containing purulent material suggestive of pyometra. this blind pouch/segmental aplastic horn, was removed, leaving the normal horn intact and in situ. she went on to be bred another time and produced a litter of four pups. one bitch puppy from this litter was retained by the owner for his breeding program. on her first litter, she was found to have two normal uterine horns and produced a litter of ten pups. clinical theriogenology • volume 10, number 3 • september 2018323 2018: application of gene-editing in livestock ? updates and prospects application of gene-editing in livestock – updates and prospects kiho lee, kyungjun uh, junghyun ryu department of animal and poultry sciences, virginia tech, blacksburg, va introduction genetic improvements are essential for sustainable production in livestock. traditionally, genetic selection has been based on phenotypes related to efficient production or economically valuable traits; livestock has been bred for these traits to enhance profit. for instance, cattle have been selectively bred to produce more meat or more milk, and pigs have been crossed to improve productivity, reproductive traits, and resistance to diseases. these breeding programs have utilized the natural genetic variation present in an animal population. on the other hand, genetic engineering technology can precisely modify a genome to improve genetic value of livestock.1 the approach can selectively edit genetic traits without segregating other economically valuable traits seen in breeding-based genetic improvements; therefore, the approach can dramatically reduce the genetic value of livestock with a minimum number of breedings. success of genetic engineering in livestock has been historically low due to technical challenges. however, the recent development of gene-editing technology allows these modifications to be highly efficient.2 historical approach of genetic engineering the first report of genetic engineering in mammals was in 1976 by introducing exogenous moloney leukemia virus (m-mulv) into a mouse genome and transmitting it to its offspring.3 engineering of these viral vector systems allowed us to deliver exogenous dna into livestock genomes as a form of transgenesis including pigs4 and cattle.5 although successful, application of the approach has been limited because of difficulties in constructing effective viral vectors and integration of viral genes into host genomes. pronuclear injection, on the other hand, is technically more convenient, and unlike virus-mediated transgenesis, there is no limit to the size of exogenous dna that can be integrated into the genome. in 1980, gordon et al.6 successfully introduced exogenous dna into the mouse genome by pronuclear microinjection. the pronuclear injection approach was also applied to develop numerous transgenic pig lines7-9 and cattle.10 however, this technique has limitations because the exogenous dna integrates to a random location of genome11 therefore, no targeted disruption is possible and the copy number of integrated exogenous dna cannot be controlled. in mice, use of embryonic stem (es) cell based genetic engineering allow us to overcome these issues. homologous recombination-based gene targeting approach permits us to modify genome in es cells, then es cells can be used to generate a chimeric mouse carrying the genetic modifications.12 breeding the chimeric mice establishes genetically engineered mouse models carrying desired genetic modifications. this technique has been the main approach to generate knockout mouse models, which are valuable animal models in biological sciences.13-15 however, the lack of true es cells in livestock has prohibited this approach in producing genetically engineered livestock. alternatively, application of somatic cell nuclear transfer (scnt), i.e. cloning, allows us to bypass the need of es cells in production of genetically engineered livestock. early experiments used embryonic cells from cleavage stage embryos or blastocysts as donor nuclei: cattle16,17 and pigs.18 later, it was demonstrated that somatic cells, such as fetal derived fibroblasts19 or adult derived cells,20 could have full term development through scnt when used as donor cells. this was a groundbreaking report in cellular reprogramming, but also indicated that genetic engineering of donor cells followed by scnt could be an ideal system to produce genetically engineered livestock. the strategy was quickly adopted and resulted in the production of genetically engineered livestock21-24 for various applications. however, efficiency of gene targeting through conventional hr is extremely poor thus only limited reports of genetically engineered livestock have been made. recent developments in gene-editing technology allow us to introduce site-specific genetic modifications in somatic cells at high efficiency. specifically, the use of engineered endonucleases such as zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens), and clustered regularly interspaced short palindromic repeat (crispr)/crispr-associated 9 (cas9) system can introduce homozygous or biallelic clinical theriogenology • volume 10, number 3 • september 2018239 modifications in somatic cells in various livestock species.25-27 in addition, the systems are powerful enough to introduce genetic modifications during embryogenesis, thus bypassing the need of scnt in generating genetically engineered livestock.28 these gene-editing systems furnish unprecedented opportunities to enhance the genetic value of livestock species. mechanistic action of gene-editing system: engineered endonucleases the gene-editing systems are labeled as engineered endonucleases because they can bind to a specific region of genome and induce a double-strand break (dsb) in the chromosome, allowing introduction of targeted modification into the locus (reviewed in2,29). endogenous dna repair pathways, non-homologous end joining (nhej) or homologous recombination (hr), are vigorously activated by response to the dsb. the repair through nhej does not rely on template dna, thus it may result in the formation of small insertion or deletion (indels). these small indels often cause frameshifts on the coding region of the target gene, resulting in a targeted disruption. the repair through hr is accurate at the nucleotide level, as it utilizes the dna template, in nature sister chromatid, to correct damages caused by the dsb. introducing donor dna with engineered endonucleases can induce targeted insertion of specific dna sequences or exogenous genes into the dsb locus through homology-directed repair (hdr) pathway. the modifications through hdr is specific at the nucleotide level. genetically engineered livestock by zfns zinc-finger nucleases (zfns) were the first developed engineered endonucleases, consisting of zinc-finger proteins, which recognize specific dna sequences, and foki enzyme, a non-specific endonuclease. each zinc-finger protein motif, originally identified from xenopus oocytes,30 can recognize and bind to three nucleotides. the zinc-finger proteins can be engineered to recognize any three nucleotides in the genome, and these proteins can be connected to recognize a longer dna sequence. these zinc-finger proteins are fused with a restriction endonuclease foki to induce a site-specific dsb in the genome. the use of zfns dramatically improved the efficiency of gene targeting. the first publication using human cells demonstrated that the system can induce targeted modifications at 1,000fold higher, compared to the conventional gene targeting approach.31 the zfns were first applied in livestock to generate biomedical pig models.32,33 for agricultural application, zfns were first used to generate genetically engineered cattle with a different milk composition. genetically engineered cattle carrying modified beta-lactoglobulin (blg) gene, known as one of the major milk allergens, was produced by utilizing zfns.34 in this study, first zfns were used to alter blg gene in bovine cell lines through nhej repair pathway. then, two cell lines containing biallelic mutation in the blg locus were used as nucleus donors for scnt. a total of eight live animals were born through the scnt, but only one cow survived over a month. the report did not comment on the quality of milk from the cow, probably due to extended time required to breed and obtain milk from the cow. another study applied zinc-finger nickases (zfnickases) to produce dairy cattle which can be resistant to staphylococcus aureus (s. aureus)-induced mastitis. use of zfnikase instead of zfns allowed them to utilize hdr pathway instead of nhej because zfnikases trigger site-specific singlestrand break (ssb) without causing dsb. in this study, through the zfnickases-mediated hdr, the lysostaphin gene was inserted into the endogenous β-casein locus (csn2) in bovine fetal fibroblasts.35 lysostaphin, which is naturally produced in staphylococcus simulans (s. simulans), is known to be effective in treating staphylococcus aureus (s. aureus)-induced mastitis, the most consequential disease in dairy cattle. the lysostaphin knock-in fibroblast cells were used for scnt and the gene-targeted calves were born. analysis of milk from the cows indicated that the lysostaphin was present in the milk and in vitro assay demonstrated the milk’s ability to kill s. aureus. the zfns were also applied to generate genetic engineered pigs for agricultural improvements. myostatin (mstn) gene plays a negative regulatory role in muscle development and naturally occurring mutation in mstn has been identified in multiple species such as sheep36 and cattle,37 famously known in the belgian blue breed of cattle. these animals present better feed efficiency, growth rate, and higher muscle mass. this naturally occurring mutation was introduced into pigs by disrupting the mstn in clinical theriogenology • volume 10, number 3 • september 2018 240 somatic cells using zfns followed by scnt.38 the mstn null pigs displayed higher growth rate and increased muscle mass compared to wild-type control, indicating these line of pigs can increase productivity, if incorporated into production system. as shown above, the use of zfns significantly increased the efficiency of generating genetically engineered livestock and provided new opportunities to design animals with higher genetic values. however, assembly of zfns can be technically challenging, and effectiveness of zfns was limited to gc-rich regions of the genome.39 genetically engineered livestock by talens transcription activator-like effector nucleases (talens) are designed by fusing a tal effector dna binding domain to foki enzyme, which introduces a dsb. the tal effector was originally isolated from plant pathogenic bacteria, xanthomonas.40,41 the binding specificity of talen is determined by a central domain of tandem, 33–35 amino acid repeats, and one talen motif can bind to a single base pair.42,43 in contrast to the zfns, talens have a greater design flexibility with the ability of single base recognition of tal effector. the first application of talens on genetically engineered livestock production was on the pig and cattle. use of talens effectively introduced targeted modifications in somatic cells, as well as in embryos.27 the paper produced a pig model for human metabolic syndromes as a proof of concept. the same group reported the possibility of introducing the hornless phenotype, exists in breeds such as angus,44 into dairy cattle by introgression of a candidate polled allele into the genome of somatic cells isolated from dairy cattle. in a follow-up study, these cell lines were used for scnt to produce five hornless calves, which, as expected, presented the hornless phenotype.45 physical dehorning of cattle is typically carried out in the dairy industry to protect animals and producers from accidental injury. however, the process is costly and painful for the animals. this talen-mediated introgression of a specific allele into a breed demonstrates that gene-editing technology can be used to improve economic value to producers and contribute to animal welfare as well. the talens were also used to generate pigs carrying targeted modifications without having to apply scnt.28 genetically engineered pigs potentially resistant to african swine fever virus were produced by direct injection of talen mrnas targeting rela gene. in this study, zfns were also able to introduce a targeted modification in the rela region, but only one allele was modified through zfns, whereas talens were able to disrupt both alleles during embryogenesis. this was the first report of producing genetically engineered livestock with site specific modifications without having to apply scnt. similar approaches were used to generate mstn-mutant cattle and sheep.46 in this study, oocytes were fertilized in vitro, and then talen mrna targeting mstn was injected into the zygotes. the cultured blastocysts were transferred to surrogates and live mstn-mutant animals were born. not all animals showed increased muscle phenotype because the modifications introduced by talens did not cause a frame shift of coding sequences in many of the gene-edited animals. these studies demonstrated that zygote injection of talen mrna can be an alternative to the laborious and technically difficult scnt technique for genetically engineering livestock. use of talen also allowed for generation of mstn-mutant goats by a targeted disruption of mstn in somatic cells followed by scnt.47 similar to the application of zfnickase, transcription activator-like effector nickase (tale nickase) was also applied to introduce a specific gene into a specific location of the bovine genome. in 2015, tale nickase system was used to produce cattle resistant to tuberculosis, a chronic infectious disease, by introducing the sp110 gene under the control of a macrophage specific promoter, macrophage scavenger receptor 1 (msr1) promoter.48 expression of the sp110 gene, obtained from tuberculosisresistant mouse strain, allowed the genetically engineered cows to manage the growth of mycobacterium bovis (m. bovis) in their system and efficiently be resistant to the low dose of m. bovis. application of talens expanded the production of gene-edited animals and demonstrated that introducing specific mutations during embryogenesis was possible. however, although simper than zfn assembly, constructing effective talen sets has been the main limitation on the wide use of the technology. clinical theriogenology • volume 10, number 3 • september 2018241 genetically engineered livestock by crispr/cas9 system clustered regularly interspaced palindromic repeats (crispr) is an adaptive immune mechanism presented in bacteria cells against exogenous dna from virus or plasmid.49,50 the guide sequences within the crispr system, which corresponds to the viral dna, can be easily replaced by a target of interest. this engineered single guide rna (sgrna) combined with tracr-rna can bind to a target dna sequence, and recruit cas9 endonuclease protein to induce dsb on the target site.51,52 both zfn and talen systems require a series of assembly to generate a construct; however, design of 20 bp grna for the target sequence is only needed to establish a crispr/cas9 system for gene-editing. due to the simplicity, crispr/cas9 system has become the leading gene-editing system to develop gene-edited livestock. the first application of crispr/cas9 system in livestock was to produce a pig model for von willebrand disease in humans.53 for agricultural purposes, the first report was to produce pigs, resistant to porcine reproductive and respiratory syndrome (prrs) virus.54,55 although prrs virus causes substantial economic losses in north america, europe, and asia, there is no effective vaccine against the virus. piglets lacking functional siglec1 (cd169), a candidate primary viral receptor for prrs virus infection, were generated by the conventional approach without the use of gene-editing system.56 establishing the pigs required two breeding cycles because only one siglec1 allele could be modified through the conventional gene targeting approach. unfortunately, these pigs were not resistant to the prrs virus. when crispr/cas9 was used to disrupt cd163, another putative receptor for prrs virus, cd163 null pigs were established without incorporating any breeding.55 some of these pigs were generated by injecting crispr/cas9 system into fertilized embryos. challenging these pigs with prrs virus demonstrated that, indeed, the cd163 null piglets were resistant to prrs,54 indicating that incorporation of these pigs into production can dramatically reduce economic losses associated with the prrs virus. the first cd163 knockout paper also proved that direct injection of crispr/cas9 system could be highly effective in generating knockout pigs; efficiencies were 100% in the study. using a similar approach, we generated rag2/il2rg double knockout pigs without breeding or scnt.57 all seventeen pigs produced through the approaches carried modification on both rag2 and il2rg. they also presented expected phenotype, primary immunodeficiency. to the best of our knowledge, this was the first report of pigs lacking all major lymphocytes: b, t, and nk cells. the direct injection of crispr/cas9 system indicates that scnt may not always be necessary to generate genetically engineered livestock carrying multiple gene-edits, thus avoiding developmental abnormalities associated with scnt. the study utilized nhej to introduce site-specific modifications; however, we also showed that exogenous dna fragment could be integrated into a specific locus through hdr during embryogenesis in pigs.58 crispr/cas9 system has also been applied to generate genetically engineered cattle to prevent diseases. the bovine prnp gene, responsible for mad cow disease, was successfully modified using crispr/cas9 system,59 similar to a previous study in which prnp gene was disrupted by using talen system.60 although prnp-mutant cattle have not been generated, this study suggested the possibility that cattle with a resistance to mad cow disease can be created through crispr/cas9 system. more importantly, the efficiency of targeted disruptions in these studies was much more efficient compared to publications related to the original production of prnp knockout cattle.24, 61 in addition to disrupting endogenous genes to improve disease resistance, crispr/cas9 system targeting an african swine fever virus gene, cp204l, was incorporated into the pig genome to effectively prevent the viral infection. porcine cell lines carrying the crispr/cas9 system was resistant to the african swine fever virus,62 indicating the approach can be used to provide an enhanced immunity to pigs. above examples focused on generating gene-edited livestock for disease prevention. the crispr/cas9 system was also used to improve genetic value of livestock by introducing or replacing specific dna sequences. the japanese black cattle breed is known for producing high quality meat. however, the breed is susceptible to isoleucyl-trna synthetase (iars) syndrome, a recessive disease in japanese black cattle caused by a single nucleotide substitution.63 the recessive genetic trait was successfully replaced/repaired by utilizing crispr/cas9 system.64 the study demonstrates that harmful clinical theriogenology • volume 10, number 3 • september 2018 242 genetic traits can be precisely replaced, while keeping valuable genetic information unmodified. similarly, gene-edited pigs that have improved thermogenesis capability were produced by replacing endogenous uncoupling protein 1 (ucp1) gene, known to be a key element for brown adipose tissue-mediated thermogenesis and regulating energy homeostasis, by utilizing crispr/cas9 system.65 pigs have lost functional ucp1 during evolution, thus showing susceptibility to cold, which leads to high neonatal mortality and decreased production efficiency. in this study, first adiponectin promoter driven mouse ucp1 gene was inserted into the endogenous porcine ucp1 locus through crispr/cas9-mediated gene insertion in porcine fetal fibroblast cells. then, murine ucp1 overexpressing pigs were produced by scnt using the cells. the gene-edited pigs showed improved ability to maintain body temperature, decreased fat deposition, and increased carcass lean percentage. this report indicates that gene-editing technology can improve genetic value of pigs, production efficiency, and animal welfare. conclusion the use of gene-editing systems has provided new opportunities to improve the genetic value of livestock. due to available funding opportunities, most gene-edited livestock is generated for biomedical purposes.26,57,66-69 however, examples introduced here certainly illustrate the potential impact of geneedited livestock in the agriculture industry and an upcoming line of animals that may be introduced into the food production system. many of the gene-edited animals only rely on endogenous nhej pathway to introduce targeted modification, thus no foot-print remained in their genome. because there is no integration of exogenous dna sequence, the word ‘transgenic’ does not apply to these animals. the fda approval of transgenic salmon also suggests that more genetically engineered or gene-edited animal products may be introduced into the food production system.70 the development of gene-editing technology will certainly lower the existing barriers in producing genetically engineered livestock, thus widening the use of genetically engineered livestock in agriculture. references 1. lotti sn, 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opin microbiol 2009;12:37-43. 42. deng d, yan c, pan x, et al: structural basis for sequence-specific recognition of dna by tal effectors. science 2012;335:720-723. 43. moscou mj, bogdanove aj: a simple cipher governs dna recognition by tal effectors. science 2009;326:1501. 44. long cr, gregory ke: inheritance of the horned, scurred, and polled condition in cattle. j hered 1978;69:395-400. 45. carlson df, lancto ca, zang b, et al: production of hornless dairy cattle from genome-edited cell lines. nat biotechnol 2016;34:479. 46. proudfoot c, carlson df, huddart r, et al: genome edited sheep and cattle. transgenic res 2015;24:147-153. 47. yu b, lu r, yuan y, et al: efficient talen-mediated myostatin gene editing in goats. bmc dev biol 2016;16:26. 48. wu h, wang y, zhang y, et al: tale nickase-mediated sp110 knockin endows cattle with increased resistance to tuberculosis. proc nat acad sci 2015;112:e1530-e1539. 49. horvath p, barrangou r: crispr/cas, the immune system of bacteria and archaea. science 2010;327:167-170. 50. wiedenheft b, sternberg sh, doudna ja: rna-guided genetic silencing systems in bacteria and archaea. nature 2012;482:331. 51. jinek m, chylinski k, fonfara i, et al: a programmable dual-rna–guided dna endonuclease in adaptive bacterial immunity. science 2012:337:816-821. clinical theriogenology • volume 10, number 3 • september 2018 244 52. cong l, ran fa, cox d, et al: multiplex genome engineering using crispr/cas systems. science 2013;339:819-823. 53. hai t, teng f, guo r, et al: one-step generation of knockout pigs by zygote injection of crispr/cas system. cell res 2014;24:372-375. 54. whitworth km, rowland rr, ewen cl, et al: gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. nat biotechnol 2015;34:20. 55. whitworth km, lee k, benne ja, et al: use of the crispr/cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. biol reprod 2014;91:78. 56. prather rs, rowland rr, ewen c, et al: an intact sialoadhesin (sn/siglec1/cd169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus. j virol 2013;87:9538-9546. 57. lei s, ryu j, wen k, et al: increased and prolonged human norovirus infection in rag2/il2rg deficient gnotobiotic pigs with severe combined immunodeficiency. sci rep 2016;6:25222. 58. ryu j, prather rs, lee k: use of gene-editing technology to introduce targeted modifications in pigs. j anim sci biotechnol 2018;9:5. 59. bevacqua r, fernandez-martín r, savy v, et al: efficient edition of the bovine prnp prion gene in somatic cells and ivf embryos using the crispr/cas9 system. theriogenology 2016;86:1886-1896. 60. choi w, kim e, yum s-y, et al: efficient prnp deletion in bovine genome using gene-editing technologies in bovine cells. prion 2015;9:278-291. 61. kuroiwa y, kasinathan p, matsushita h, et al: sequential targeting of the genes encoding immunoglobulin-mu and prion protein in cattle. nat genet 2004;36:775-780. 62. hübner a, petersen b, keil gm, et al: efficient inhibition of african swine fever virus replication by crispr/cas9 targeting of the viral p30 gene (cp204l). sci rep 2018;8:1449. 63. hirano t, kobayashi n, matsuhashi t, et al: mapping and exome sequencing identifies a mutation in the iars gene as the cause of hereditary perinatal weak calf syndrome. plos one 2013;8:e64036. 64. ikeda m, matsuyama s, akagi s, et al: correction of a disease mutation using crispr/cas9-assisted genome editing in japanese black cattle. sci rep 2017;7:17827. 65. zheng q, lin j, huang j, et al: reconstitution of ucp1 using crispr/cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity. proc nat acad sci 2017;114:e9474-e9482. 66. lee k, kwon dn, ezashi t, et al: engraftment of human ips cells and allogeneic porcine cells into pigs with inactivated rag2 and accompanying severe combined immunodeficiency. proc nat acad sci 2014;111:7260-7265. 67. kang jt, cho b, ryu j, et al: biallelic modification of il2rg leads to severe combined immunodeficiency in pigs. reprod biol endocrinol 2016;14:74. 68. klymiuk n, blutke a, graf a, et al: dystrophin-deficient pigs provide new insights into the hierarchy of physiological derangements of dystrophic muscle. hum mol genet 2013;22:4368-4382. 69. li p, estrada jl, burlak c, et al: efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide rna and carbohydrate selection. xenotransplantation. 2015;22:20-31. 70. ledford h: salmon approval heralds rethink of transgenic animals. nature 2015;527:417-418. clinical theriogenology • volume 10, number 3 • september 2018245 clinical theriogenology • volume 10, number 3 • september 2018 246 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2018: assessment of fertility in male cats through cytologic evaluation of testicular aspirates assessment of fertility in male cats through cytologic evaluation of testicular aspirates camille cordero aponte,a stephen horvath,b david pontius,b rebecca garabed,c christopher premanandana adepartment of veterinary biosciences, bdepartment of veterinary clinical sciences, and cdepartment of veterinary preventive medicine, college of veterinary medicine, the ohio state university, columbus, oh assessment of male feline fertility plays an important role in breeding management and biomedical research. common methods of assessing fertility in tomcats include analysis of semen samples collected through an artificial vagina and electroejaculation. samples are usually evaluated for characteristics such as concentration, progressive motility and morphology. nevertheless, obtaining semen samples can be a difficult task and are usually low in volume. in addition, testicular biopsies can be utilized to assess testicular function, but these may result in post-biopsy complications that can affect fertility. it has been proven that testicular fine needle aspiration (fna) serve as a tool for evaluation of fertility in reproductive settings.1 compared to testicular biopsies, fna has been proven to be a less invasive, quicker, and easier approach to obtaining valuable information regarding fertility in multiple species, including dogs, bulls and stallions.1-3 in dogs, in particular, the use of testicular fine needle aspiration and cytology has a role in analyzing canine fertility.1 in this study, testicles were collected after orchiectomy from fifty cats ranging from six months to six years of age. each testicle was aspirated for cytology and subsequently sectioned for impressions smears and histological evaluation. two hundred cells were counted per slide and identified appropriately as spermatogonia, spermatocytes, spermatids, spermatozoa and sertoli cells. the quantified cell populations were used to establish a sertoli cell index (sei) and sperm cell index (si) for each testicle. the testicular cytology counts were subsequently grouped into five groups according to age ranges. the groups were divided as follows: one year of age (group 1, n=15), greater than one year (group 2, n=7), two years (group 3, n=13), greater than 2 years to three years (group 4, n=5), greater than three years (group 5, n=4). our results showed slight variations in numbers between aspirates and impressions for all postpubescent testes, but particularly in cats of one year of age. cats in the one year group had aspirate sei and si (±standard deviation) of 22.7±10.9 and 36.6± 7.7, respectively. the sei and si of impressions at one year were 18.3± 8.5 and 33.9± 8.6, respectively. only sertoli cells were observed in pre-pubertal samples (si and sei were not calculated). the groups consisting of testes from older felines displayed a more consistent range of indices. the sei and si for aspirates from group 2 were 23.9±8.5 and 38.4±12.6, group 3, 14.9±4.6 and 35.7±10.4, group 4, 12.3±2.3 and 37.5±11.2, and group 5, 15.4±4.6 and 32.5±10.6, respectively. the (sei) and (si) for impressions from group 2 were 15.9±6.1 and 34.0±6.7, group 3, 13.8±4.7 and 37.9±4.7, group 4, 15.4± 5.5 and 40.3± 5.9, and group 5, 9.5±2.0 and 42.7±4.7, respectively. the sei may be a more valuable index for providing information about potential problems affecting spermatogenesis due to less variation between cats presenting normal testicular pathology. the sei was found to be much higher in cats with testicular degeneration compared to cats with normal testicular pathology. in conclusion, testicular fna can be an effective adjunct method of assessing fertility in male cats. keywords: spermatogenesis, feline, aspirate, testicular, cytology references 1. santos m, marcos r, caniatti m. cytologic study of normal canine testis. theriogenology. 2010; 73:208-214. 2. chapwanya a, callanan j, larkin h, keenan l, et al:breeding soundness evaluation by semen analysis, testicular fine needle aspiration cytology and trans-scrotal ultrasonography. irish vet j. 2008; 61:315-318. 3. leme dp, papa fo. cytological identification and quantification of testicular cell types using fine needle aspiration in horses. equine vet. j. 2010; 32:444-446. clinical theriogenology • volume 10, number 3 • september 2018 320 2013: top ten foal medicine papers published within the last year top ten foal medicine papers published within the last year l. chris sanchez department of large animal clinical sciences, university of florida, gainesville, fl abstract this talk will cover what i consider to be 10 of the best papers published in the last year regarding neonatal foals. many of these come from a supplement to the equine veterinary journal focused on perinatology. they are presented in random order – not in countdown fashion – and will be briefly summarized in the proceedings. some will be used as examples of areas in which significant new information was gained in 2012. i have left a few slots open to discuss articles published between the time proceedings were submitted until the meeting – that way i can cover some late-breaking reports. keywords: foal, medicine, perinatology 1. corley ktt, corley mmb: hospital treatment as a foal does not adversely affect future sales performance in thoroughbred horses. equine vet j 2012;44:87-90. this paper tackled an age old question: is it financially worthwhile to hospitalize neonatal foals? they retrospectively evaluated thoroughbred neonatal hospital admissions from 2007 and 2008 which were subsequently sold at public auction. they used horses sold before and after as controls. they had 63 foals in the study (19 presented to be sold as foals, 39 as yearlings, 5 as 2-year olds). the bottom line from their study was that if foals made it to the sales, they did not have a significant difference in buyback rate or sales price, relative to controls. 2. furr m, cohen nd, axon je, sanchez lc, pantaleon l, haggett e, campbell r, tennent-brown b: treatment with histamine-type 2 receptor antagonists and omeprazole increase the risk of diarrhoea in neonatal foals treated in intensive care units. equine vet j 2012;44:80-86 this paper tackled another old adage – you might as well treat neonatal foals with ulcer prophylaxis as it won’t hurt. while that’s a very tough thing to prove one way or another, this paper took a good first step. it was a multi-center retrospective study evaluating the records of 1710 foals from six referral hospitals (private and academic). the use of ulcer prophylaxis (histamine-2 receptor antagonists, omeprazole, or sucralfate) varied substantially among hospitals (6 to 97%), as did the occurrence of diarrhea (7.5 to 35.9%); survival was similar among hospitals, ranging from 74.5 to 87.2%. the use of anti-ulcer drugs was associated with an increased chance of foals developing in-hospital diarrhea. thus, while other factors may certainly have played a role in this, the statement that such drugs are completely innocuous in this population merits further investigation. 3 and 4. venner m, rodiger a, laemmer m, et al: failure of antimicrobial therapy to accelerate spontaneous healing of subclinical pulmonary abscesses on a farm with endemic infections caused by rhodococcus equi. vet j 2012;192:293-298. venner m, astheimer k, lämmer m, giguère s: efficacy of mass antimicrobial treatment of foals with subclinical pulmonary abscesses associated with rhodococcus equi. j vet intern med. 2013;27:171-176. these papers evaluated the authors’ hypothesis that antimicrobial treatment of foals with ultrasonographic evidence of pulmonary abscesses but not clinical disease is unnecessary. both studies followed foals on a farm with endemic r. equi pneumonia in randomized, double-blinded, placebocontrolled prospective trials. the first study enrolled 128 foals with ultrasonographic evidence of pulmonary abscesses ≥1.0 cm in diameter and had four treatment groups (tulathromycin im, azithromycin po, azithromycin plus rifampin po, and glucose po as a placebo). the second study had 108 foals, used a larger abscess cutoff (pulmonary abscesses 5-10 cm in diameter) and had five treatment groups (tulathromycin im, doxycycline po, doxycycline with rifampin po, azithromycin with rifampin po, or saline im as a placebo). in the first study, 14/32 foals treated with a placebo recovered without therapy, 545 clinical theriogenology • volume 5, number 4 • december 2013 and none of the measured parameters (response to initial therapy, duration of therapy, removal from the study, days to removal from the study) differed among groups. in the second, most (22/25) foals in the placebo group recovered without treatment, and the percentage of foals with disease progression did not differ significantly among groups. interestingly, a smaller proportion of foals needed additional therapy or were removed from the second study. the authors’ hypothesis was that farm personnel thought that foals treated with a placebo would not recover, thus were quicker to pull them from the first study, but for the second study, they were more comfortable giving the foals time to recover. 5. himler m, hurcombe sda, griffin a, barsnick rj, rathgeber ra, macgillivray kc, toribio re: presumptive nonthyriodal illness syndrome in critically ill foals. equine vet j 2012; 44 (suppl 41):43-47. this is one of several papers of late to shed further light on the hypothalamic-pituitary-adrenal (hpa) axis and its adaptations in the young foal. they evaluated total and free t4, total, free and reverse t3 in healthy, sick non-septic and septic foals. all thyroid hormones except rt3 were decreased in both sick foal groups, and non-surviving septic foals had lower tt4, ft4, tt3, and ft3 concentration than their surviving counterparts 6. jellyman jk, allen vl, forhead aj, holdstock nb, and fowden al: hypothalamic–pituitary– adrenal axis function in pony foals after neonatal acth-induced glucocorticoid overexposure. equine vet j 2012;44 (suppl. 41):38-42. following the hpa axis theme, this study evaluated hpa programming in foals receiving either saline or long-acting acth for the first five days of life. they found that basal cortisol concentrations were higher in acth-treated foals at three but not 13 weeks, and the cortisol response to short-acting acth did not differ between groups. thus, it does not appear that rises in plasma cortisol concentration during the neonatal period (such as in response to stressful events) re-program the hpa axis in the relative short term. 546clinical theriogenology • volume 5, number 4 • december 2013 prostaglandin facilitated mare foal bonding in a pregnant nurse mare prostaglandin facilitated mare foal bonding in a pregnant nurse mare justin mcnaughten rhinebeck equine, rhinebeck, ny nurse mares provide an invaluable service for orphaned foals; however, there is often negative public perception surrounding the nurse mare industry. although pharmacologically induced lactation can reduce the number of nurse mare foals, some nurse mare owners stipulate that a nurse mare must be rebred and returned in foal. catastrophic injury and illness affecting the broodmare after foaling is often unpredictable and may occur anytime during a foal’s first few months of life. unpredictability and length of physiologic breeding season may contribute to an excessive number of foals that need nurse mares. although the need for nurse mares may not be eliminated, the following case report demonstrates that the number of nurse mares could be reduced. a 9 year old multiparous paint mare was presented as a nurse mare for a 48 day old orphan foal whose dam was euthanized on humane grounds. mare foal bonding was achieved following prostaglandin administration. while on farm, this nurse mare was bred via live cover and became pregnant. a 40 day old foal was orphaned when its dam was euthanized during colic surgery. due to lack of nurse mare availability, the original orphan foal was weaned at 147 days in an attempt to graft the new orphan to this mare. prior to the second mare foal bonding session, transrectal ultrasonography was performed to confirm fetal viability. plasma progesterone concentrations were 8.42 ng/ml. the mare was administered altrenogest (regumate,® 0.044 mg/kg, po) followed by a dose of prostaglandin (lutalyse,® 10 mg, im) to facilitate mare foal bonding. nurse mare bonding was successful and this mare remained on altrenogest for 30 days. transrectal ultrasonography and blood sampling were repeated on days 5 and 22 following prostaglandin treatment and circulating progesterone concentrations were 0.79 and 5.18 ng/ml, respectively. with appropriate progestin supplementation, prostaglandins may still be utilized in mare foal bonding in a pregnant nurse mare, despite luteolytic effects. keywords: equine, nurse mare bonding, prostaglandin, pregnancy maintenance 489 clinical theriogenology • volume 11, number 3 • september 2019 490clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2011: impacts of nutrition on reproduction of dairy cattle impacts of nutrition on reproduction of dairy cattle j.e.p. santos department of animal sciences, university of florida, gainesville, fl abstract nutrition has many important impacts on the reproductive performance of cattle. inadequate caloric intake in adult cattle has a detrimental impact on reproductive activity of the female bovine. cows under negative energy balance have extended periods of anovulation. postpartum anestrus, as well as infertility, is magnified by losses of body condition during the early postpartum period. resumption of ovulatory cycles is associated with energy balance, and the underlying mechanisms seem to be associated with metabolic signals and regulatory hormones primarily insulin and insulin-like growth factor (igf) i, which link nutritional status with gonadotropin secretion, re-coupling of the growth hormone-igf system, and follicle maturation and ovulation. feeding diets that promote increases in plasma glucose and insulin may improve the metabolic and endocrine status of cows in early lactation. nevertheless, feeding excess of starch to promote increases in insulin and glucose might suppress intake of early lactating dairy cows, thereby precluding benefits to cyclicity. feeding behavior of dairy cows during the transition period, particularly a decline in feed intake before calving, is associated with risk of postpartum uterine disease. because metritis and more chronic forms of uterine diseases have profound negative effects on pregnancy in dairy cows, providing adequate bunk space and an environment to maximize feed intake might potentially improve fertility of dairy cows. specific nutrients and dietary ingredients have been implicated in reproduction in cattle. excess of dietary protein has been suggested as detrimental to fertility, although the evidence is weak. addition of moderate amounts of supplemental fat to the diet improves caloric intake, modulates prostaglandinf2 (pgf) secretion by the uterus, affects ovarian dynamics, enhances luteal function and embryo quality, and has moderate positive effects on fertility. more specifically, some fatty acids (fa) might impact fertilization rate and embryo quality in dairy cows. on the contrary, some dietary ingredients, such as gossypol, when ingested in large quantities decrease fertility of dairy cows because of negative effects on embryo quality and pregnancy maintenance. keywords: nutrition, dairy cow, periparturient disease, infertility, gossypol introduction selection of dairy cattle for milk yield has linked the endocrine and metabolic controls of nutrient balance and reproductive events so that reproduction in dairy cattle is compromised during periods of nutrient shortage, such as in early lactation. the energy costs to synthesize and secrete hormones, ovulate a follicle, and sustain an early developing embryo are probably minimal compared to the caloric needs for maintenance and lactation. however, the metabolic and endocrine cues associated with negative energy balance (neb) impair resumption of ovulatory cycles, oocyte and embryo quality, and establishment and maintenance of pregnancy in dairy cattle. as the demands for milk synthesis increase, reproductive functions may be depressed when no compensatory intake of nutrients is achieved. numerous recent studies have found that reproductive performance is compromised by the nutrient demands associated with high levels of production. milk yield increases at a faster rate in the first four to six weeks after parturition than caloric intake, consequently high yielding cows will experience some degree of negative balance of nutrients during the early postpartum period. when cows experience a period of neb, the blood concentrations of nonesterified fatty acids (nefa) increase, at the same time that igf-i, glucose, and insulin are low. these shifts in blood metabolites and hormones might compromise ovarian function and fertility. it has also been reported that energy balance and dry matter intake (dmi) might affect plasma concentrations of progesterone, which may interfere with follicle development and maintenance of pregnancy.1,2 during the last decades, genetic selection and improved management of herds have dramatically increased milk production of dairy cows, at the same time that fertility has decreased.3 selection for higher milk production in dairy cattle has changed endocrine profiles of cows so that blood concentrations clinical theriogenology • volume 3 number 4 • december 2011591 of bovine somatotropin and prolactin have increased; whereas insulin has decreased.4 these hormonal changes and the increased nutrient demands for production might negatively impact reproduction of dairy cows. however, adequate nutrition and sound management have been shown to offset depression of fertility in herds with average milk production exceeding 12,000 kg/cow/yr.5,6 several nutritional strategies have been proposed to improve reproduction of dairy cattle with no detrimental effect on lactational performance. maximizing dmi during the transition period, minimizing the incidence of periparturient problems, feeding of diets that promote increased concentrations of insulin in early lactation, adding supplemental fat to diets, and manipulating the fa content of fat sources are expected to benefit reproduction in dairy cattle. however, factors such as high incidence of metabolic diseases early postpartum, poor body condition score (bcs) at first insemination, and excessive gossypol concentrations in plasma are detrimental to fertility of dairy cattle. nutrition and postpartum uterine health and fertility epidemiological studies have clearly demonstrated strong relationships between postparturient diseases and subsequent reproductive performance in dairy cattle. cows diagnosed with clinical hypocalcemia were 3.2 times more likely to experience retained placenta (rp) than cows that did not have clinical hypocalcemia.7 whiteford and sheldon also found that hypocalcemia was associated with occurrence of uterine disease in lactating dairy cows.8 markusfeld reported that 80% of cows with ketonuria developed metritis.9 a major risk factor for uterine disease is rp. generally, cows with rp have increased risk of developing metritis compared with cows not experiencing rp. both metritis and rp double the risk of cows remaining with uterine inflammation at the time of first postpartum insemination.10 in the us, a recent usda study indicated that the incidence of rp in dairy cows was 7.8 ± 0.2%.11 a 2006 study on five dairy farms in israel observed that rp was diagnosed in 13.1% (9.4 to 18.1%) and 9.2% (3.6 to 13.8%) of multiparous and primiparous cows, respectively.12 in the same study, metritis affected 18.6% (15.2 to 23.5%) and 30% (19.4 to 42.3%) of the multiparous and primiparous cows, respectively. both rp and metritis can have devastating effects on reproductive efficiency in lactating dairy cows, with reduced conception rates and extended intervals to pregnancy.12 in fact, not only does the clinical disease negatively affect fertility of dairy cows; but subclinical endometritis, a disease characterized by increased proportion of neutrophils in uterine cytology without the presence of clinical signs of inflammation of the uterus, has major deleterious effects on conception rates of lactating dairy cows at first postpartum insemination. a rising story suggests that feed intake and feeding behavior around parturition might mediate some of the increased risk for uterine diseases in dairy cattle.13-15 hammon et al observed that cows developing uterine disease postpartum experienced reduced dmi beginning one week before calving.13 similarly, cows diagnosed with severe metritis after calving were already consuming less dry matter two weeks prior to calving.14 in the same study, even cows that subsequently developed mild metritis had reduced dmi one week before calving compared with cows with healthy uteri. the same group observed that cows subsequently developing metritis spent significantly less time eating before and after calving than cows that did not develop metritis.15 these data indicate that suppressed intake of nutrients or alterations in feeding behavior prior to calving are major risk factors for development of metritis postpartum. a potential link between nutrient intake and development of uterine diseases may be the immune status of the cow. kimura et al evaluated neutrophil function in 142 periparturient dairy cows from two herds by evaluating chemotaxic and killing activity of those cells.16 the authors observed that 14.1% of the cows developed rp. neutrophils isolated from blood of cows with rp had reduced ability to migrate to placental tissue and reduced myeloperoxidase activity, a marker for oxidative burst and killing activity of neutrophils. interestingly, the reduced neutrophil function was observed between one and two weeks prior to calving, which suggests that the reduced innate immune function may be part of the cause of rp rather than a consequence of the disease. in fact, cows that developed uterine disease, either clinical metritis or subclinical endometritis, experienced reduced dmi and neutrophil function prior to calving.13 clinical theriogenology • volume 3 number 4 • december 2011 592 these data strongly suggest that inadequate nutrient intake before calving might predispose cows to impaired immune function; and, subsequently, increased risk for uterine diseases that negatively affect reproduction. because intake of nutrients seems to influence energy status and immune function of dairy cows, both of which seem to be related to risk of uterine diseases; it is prudent to suggest that nutritional and management strategies that optimize nutrient intake around parturition should improve uterine health and subsequent fertility of dairy cows. perhaps, of equal or greater importance than the diet composition is the environment to which the preparturient cow is subjected. inadequate cow comfort, competition for space, and hierarchical status can influence the ability of the cow to consume nutrients; which can consequently predispose her to uterine disease.13-15 resumption of postpartum estrous cycles the onset of lactation creates an enormous drain of nutrients in high producing dairy cows; which, in many cases, antagonizes the resumption of ovulatory cycles. during early postpartum, reproduction is deferred in favor of individual survival. therefore, in the case of the dairy cow, lactation becomes a priority to the detriment of reproductive functions. during periods of energy restriction, oxidizable fuels consumed in the diet are prioritized toward essential processes such as cell maintenance, circulation, and neural activity. homeorhetic controls in early lactation assure that body tissue, primarily adipose stores, will be mobilized in support of milk production. therefore, the early lactation dairy cow that is unable to consume enough energy-yielding nutrients to meet the needs of production and maintenance, will sustain high yields of milk and milk components at the expense of body tissues. this poses a problem to reproduction, as delayed ovulation has been linked repeatedly with energy status.3 caloric deprivation reduces the frequency of pulses of luteinizing hormone (lh); thereby impairing follicle maturation and ovulation. furthermore, undernutrition inhibits estrous behavior by reducing responsiveness of the central nervous system to estradiol by reducing the estrogen receptor  content in the brain.18 generally, the first postpartum ovulation in dairy cattle occurs 10 to 14 days after the nadir of neb.3 severe weight and bcs losses caused by inadequate feeding or illnesses are associated with anovulation and anestrus in dairy cattle. in fact, cows with low bcs at 65 days postpartum are more likely to be anovular, which compromises reproductive performance at first postpartum insemination.19 prolonged postpartum anovulation or anestrus extends the period from calving to first artificial insemination (ai) and reduces fertility during the first postpartum service.19 in fact, anovular cows not only have reduced estrous detection and conception rates, but also have compromised embryo survival.20 on the other hand, an early return to cyclicity is important in regard to early conception. the timing of the first postpartum ovulation determines and limits the number of estrous cycles occurring prior to the beginning of the insemination period. typically, in most dairy herds, fewer than 20% of cows should be anovulatory by 60 days postpartum.19 estrous expression, conception rate, and embryo survival improved when cows were cycling prior to an estrous synchronization program for first postpartum insemination.20,21 resumption of ovarian activity in high producing dairy cows is determined by energy status of the animal. therefore, feeding management that minimizes loss of body condition during the early postpartum period and incidence of metabolic disorders during early lactation should increase the number of cows experiencing a first ovulation during the first four to six weeks postpartum. protein and reproduction lactating dairy cattle require large quantities of metabolizable amino acids for synthesis of milk protein. it is typical for lactating rations to contain crude protein between 16 and 18% of total dry matter. diets with limited crude protein can compromise microbial growth and rumen fermentation, which often reflects in declines in feed intake and milk production. on the other hand, feeding protein in excess to what is needed by the cows has been implicated in increased in ammonia and urea concentrations in blood and milk, which have been used as markers for reduced fertility.22 the suggested decline in fertility of clinical theriogenology • volume 3 number 4 • december 2011593 cattle fed excess protein is caused by alterations in uterine physiology with a decline in uterine ph during the early luteal phase of the estrous cycle.22 a more acidic uterine environment is less conducive with maintenance of pregnancy in cattle.23 this effect seems to be restricted to the early stages of embryo development.24 because high-producing lactating dairy cows are more efficient in utilizing protein sources when diets are moderate in crude protein and are balanced for the supplies of metabolizable protein and limiting amino acids, it is not justified to feed diets with protein concentrations that will increase urea n and harm fertility.25 caloric intake and reproduction caloric intake appears to have the greatest impact on energy status of lactating dairy cows. santos et al reported that most of the variation in energy balance in early postpartum high-producing holstein cows is determined by caloric intake and little by the amount of calories secreted in milk.26 therefore, selecting for less milk yield with no changes in nutrient intake is unlikely to be a productive method to improve energy status of early lactation dairy cows. therefore, differences among cows in the severity of neb are more related with how much energy they consume than with how much milk they produce. during periods of neb, blood concentrations of glucose, insulin, and igf-i are low; as well as the pulse frequency of gonadotroin releasing hormone (gnrh) and lh. plasma progesterone concentrations are also affected by the energy balance of dairy cows. these metabolites and hormones have been shown to affect folliculogenesis, ovulation, and steroid production in vitro and in vivo. the exact mechanism by which energy status affects secretion of releasing hormones and gonadotropins is not well defined; but it is clear that lower levels of blood glucose, igf-i, and insulin may mediate this process. it has been suggested that neb influences reproduction of dairy cows by impacting the quality and viability of the oocyte of the ovulatory follicle and the corpus luteum (cl) resultant of the ovulation of that follicle. because there is substantial evidence that metabolic factors can influence early follicular development, it is conceivable that changes in metabolism during periods of neb could influence preantral follicles destined to ovulate weeks later during the breeding period. to test this hypothesis, kendrick et al randomly assigned 20 dairy cows to one of two treatments formulated so that cows consumed either 3.6% (high energy) or 3.2% (low energy) of their body weight.27 follicles were transvaginally aspirated twice weekly and oocytes were graded based upon cumulus density and ooplasm homogeneity. cows in better energy balance (high energy) had greater intrafollicular igf-i and plasma progesterone levels and tended to produce more oocytes graded as good. therefore, neb not only delays resumption of ovulatory cycles, but it might also influence the quality of occytes once cows are inseminated. nutritional manipulation to increase nutrient intake nutritional efforts to minimize the extent and duration of neb may improve reproductive performance. the first and most important factor that affects caloric intake in dairy cows is feed availability.28 therefore, dairy cows should have continual access to a high quality, palatable diet to assure maximum dmi. however, dmi is limited during late gestation and early lactation, which can compromise total caloric intake and reproductive performance. several nutritional management strategies have been proposed to increase caloric intake during early lactation. feeding high quality forages, increasing the concentrate:forage ratio, or adding supplemental fat to diets are some of the most common ways to improve caloric intake in cows. a number of studies have demonstrated the importance of insulin as a signal mediating the effects of acute changes in nutrient intake on reproductive parameters in dairy cattle. in early postpartum dairy cattle under neb, reduced expression of hepatic growth hormone receptor 1a (ghr-1a) is thought to be responsible for the lower concentrations of igf-i in plasma of cows.29 because igf-i is an important hormonal signal that influences reproductive events such as stimulation of cell mitogenesis, hormonal production, and embryo development, among other functions; increasing concentrations of igf-i early clinical theriogenology • volume 3 number 4 • december 2011 594 postpartum are important for early resumption of cyclicity and establishment of pregnancy. it is interesting to note that insulin mediates the expression of ghr-1a in dairy cows, which results in increased concentrations of igf-i in plasma.30,31 because igf-i and insulin are important for reproduction in cattle, feeding diets that promote greater insulin concentrations should benefit fertility. gong et al fed cows of low and high genetic merit isocaloric diets, that differed in the ability to induce high or low insulin concentrations in plasma.31 the diets that induced high insulin reduced the interval to first postpartum ovulation and increased the proportion of cows ovulating in the first 50 d postpartum. feeding fat and fatty acids to influence reproduction feeding fat to dairy cattle usually improved the risk for pregnancy, although responses have not been consistent.33 when fat feeding improved production and increased body weight loss, primiparous cows experienced reduced pregnancy risk at first ai;34 although pregnancy to ai was extremely high in the unsuplemented cows. however, ferguson et al observed a 2.2 fold increased risk of pregnancy at first ai and all ai in lactating cows fed 0.5 kg/d of fat, which tended (p = 0.08) to enhance the proportion of pregnant cows at the end of the study (93 vs. 86.2%).35 in grazing cows, supplementation with 0.35 kg of fa improved the risk of pregnancy after the first postpartum ai; although a similar proportion of cows were pregnant at the end of the study.36 feeding calcium salts of long chain fatty acids (ca-lcfa) of palm oil improved pregnancy of dairy cows,37 although the authors did not report statistical significance. on the other hand, others did not observe improvements in fertility of dairy cows supplemented with ca-lcfa38,39 or oilseeds;40 which might be attributed to increased milk yield and body weight losses.34,39 because the benefits of feeding fat may originate from specific fa,41,42 others have evaluated whether feeding fa differing in the degree of saturation might influence fertility of cows. the essential fa of the n-6 and n-3 families are available in much smaller supply to ruminants than nonruminants because of microbial biohydrogenation of fa in the rumen,43 suggesting that their supplementation may benefit reproduction.33,42 three recent studies explored the role of n-6 and n-3 fa supplementation to lactating dairy cows on risk of pregnancy after the first postpartum ai.44-46 when cows were fed 0.75 kg of fat from flaxseed, a source rich in c18:3 n-3, or sunflower seed, a source rich in c18:2 n-6; pregnancy tended (p = 0.07) to be greater for cows fed n-3 fa. however, a similar response was not observed by others when cows were fed flaxseed as the source of n-3 fa.45,46 when cows received a sequence of fa feeding of ca salts rich in n-6 during the transition period and n-3 during the breeding period, the cumulative first and second ai pregnancy was improved primarily because cows fed n-3 fa during the breeding period had less pregnancy loss.47 juchem et al evaluated the effect of feeding cows preand postpartum ca-lcfa of either mostly saturated and monounsaturated fa or a blend of c18:2 n-6 and trans-octadecenoic fa.48 they observed that cows fed unsaturated fa had 1.5 times greater risk of pregnancy either at 27 or 41 days after ai compared with cows fed mostly saturated fa. improvements in pregnancy risk when cows were fed c18:2 n-6 and trans-octadecenoic fa were supported by improved fertilization and embryo quality in non-superovulated lactating dairy cows.49 because n-3 fa can suppress uterine secretion of pgf2α,50-52 it is thought that they have the potential to improve embryonic survival in cattle.53 in three of five experiments, feeding n-3 fa either as flaxseed rich in c18:3 n-344,46 or fish oil rich in eicosapentanoic acid (epa) and docosahexanoic acid (dha)47 reduced pregnancy losses in lactating dairy cows after the first postpartum ai. on the other hand, when n-6 fa were fed as ca-lcfa, pregnancy losses were similar to those observed for cows fed ca-lcfa of palm oil.47,48 collectively, these data suggest that feeding fat to dairy cows generally improves fertility and responses are observed when the caloric density of the ration increased with fat feeding. also, these data suggest that fertility responses to fat feeding is altered according to the type of fa supplemented in the diet. feeding n-3 fa from oilseeds has improved pregnancy risk in some, but not all studies; however feeding n-3 fa as ca-lcfa containing fish oils does not seem to influence risk of pregnancy. on the clinical theriogenology • volume 3 number 4 • december 2011595 other hand, feeding ca-lcfa rich in n-6 and trans-octadecenoic fa improved pregnancy in lactating dairy cows. although feeding n-3 fa has not consistently improved pregnancy risk, it has reduced pregnancy losses in dairy cows. source of selenium and reproduction during the immediate postpartum period, the cow’s immune system is challenged severely,54 and the innate and humoral defense systems are reduced. the incidence of diseases and disorders can be high during this time period and have a negative impact on reproductive performance. for example the risk of pregnancy (odds ratio) was reduced if cows had rp or lost one bcs unit.12,19 reduction in adaptive and innate immunity at parturition increases the risk of health disorders such as rp, metritis, and mastitis. selenium has long been associated with immunity. cattle supplemented with se-yeast had an 18% increase of se in plasma in comparison to sodium selenite in some studies.55 some regions of the us are deficient in se, particularly the southeast; whereas other states, such as california, are mostly adequate in se. we have conducted an experiment to evaluate a supplemental source of organic selenium on reproductive and immune responses by dairy cows in florida and california.10,56,57 objectives were to evaluate effects of organic se on health and reproductive performance of dairy cows. cows were assigned prepartum at approximately 25 days prior to expected day of calving to one of two sources of se, organic se (se-yeast [sy]; sel-plex®, alltech, nicholasville, ky) or inorganic sodium se (sodium selenite, ss) fed at 0.3 ppm (dm basis) until 80 days posptartum. in both sites, cows followed the same study protocol and health was monitored daily throughout the study. rectal temperature was recorded each morning for 10 days postpartum. in florida, vaginoscopic evaluation of the reproductive tract was performed at five and 10 days postpartum. cows were evaluated for incidence of rp, metritis, puerperal metritis, subclinical endometritis by uterine cytology, ketosis, displacement of abomasum, and mastitis. cows had their ovulation synchronized for first postpartum ai. plasma se concentrations increased with days postpartum, but source of se did not influence se concentrations in cows in california. however, in florida, feeding sy improved plasma se concentrations (0.087 vs. 0.069 ± 0.004 μg/ml; p < 0.01). incidence of postpartum diseases did not differ between treatments in both sites, but cows fed sy had smaller incidence of purulent vaginal discharge than those fed ss in florida. diet altered frequency of multiparous cows detected with > 1 event of fever (rectal temperature > 39.5ºc; sy, 13.3% [25/188] vs ss,25.5% [46/181]; p < 0.05); but the sy effect was not observed in primiparous cows, which had a much higher frequency of fever (40.5%). vaginoscopy discharge scores at five and 10 days postpartum were better for the sy group; namely, 47.1 (217/460) vs 35.0% (153/437) clear, 43.4 [200/460] vs 47.8% [209/437]) mucopurulent, and 9.3 (43/460) vs 17.1% (75/437) purulent for sy and ss groups, respectively (p < 0.05). feeding organic se (sy) improved uterine health and second service pr during summer. diet failed to alter first service pregnancy rates in california and florida, and second service pregnancy rate in california. however, second service pregnancy rate in florida was greater for cows fed sy than ss [sy, 17% (34/199) vs ss, 11.3% (24/211); p < 0.05]. the benefit of sy on second service pregnancy rate is intriguing. we hypothesize that cows of the sy group were better able to re-establish an embryo-trophic environment at second service following either early or late embryonic losses. measures of innate and humoral immune responses were unaltered by source of se in california, but cows fed sy in florida had improved neutrophil function and serum titers against ovalbumin. our findings indicated that feeding sy improved measures of humoral and cellular immunity, uterine health, and second service pregnancy rate in cows in florida which is known as a se deficient state. however, in california source of se had no impact on health, measures of immune response, or reproductive performance. consumption of gossypol from cotton byproducts and reproduction gossypol was first discovered by chinese scientists after noticing that no children were born for more than a decade in a village where people cooked food with cottonseed oil. since then, innumerable clinical theriogenology • volume 3 number 4 • december 2011 596 reports in the literature have confirmed the anti-fertility effect of gossypol in mammals. gossypol disrupts cell membrane metabolism, affects glycolysis, influences mitochondrial and energy metabolism in the cell, and increases fragility of cell membranes, such as in red blood cells. in fact, erythrocyte fragility has been one of the indicators of potential gossypol toxicosis. risco et al were among the first to show that gossypol can be toxic and even kill growing cattle.58 they fed rations with 200, 400 or 800 mg/kg of free gossypol (fg) to bull calves for 120 days. the diets with 400 and 800 mg/kg of fg were considered to be toxic and could potentially cause the death of growing ruminants. baby calves have little ability to detoxify gossypol; therefore toxicity can be easily induced by feeding cotton products. the negative effects of gossypol on fertility of ruminants are clear in males. studies at university of florida and kansas state university have shown that as little as 8 g/d of fg fed to young bulls reduced sperm quality and sexual activity.59,60 however, the female ruminant seems to be relatively insensitive to the anti-fertility effect of gossypol because of rumen detoxification; but in vitro data indicate some inhibition of embryonic development and ovarian steroidogenesis.61 more recently, a series of experiments by our group demonstrated that consumption of up to 40 mg of fg/kg of bodyweight did not influence follicle and luteal development in dairy heifers, but feeding a diet with 40 mg of fg/kg of body weight reduced embryo quality and development in vivo and in vitro.62-64 these effects likely explain the reduced risk of pregnancy in dairy cows with high plasma gossypol concentrations,65 and compromised embryo survival after transfer.66 therefore, it is prudent to feed lactating dairy cows amounts of cottonseed that result in low plasma gossypol concentrations. conclusions inadequate intake of nutrients and inadequate body reserves during early lactation are the major factors affecting reproductive performance of dairy cows. improving energy balance by increasing caloric intake through additional non-fiber carbohydrates or supplemental fat in the diet reduces days to first ovulation and improves conception postpartum. strong evidence suggests that management of cows during the prepartum period affects uterine health. inadequate intake of nutrients prepartum and altered feeding behavior increases the risk of metritis in dairy cows. supplementation with unsaturated fa of the n-3 and n-6 families usually improves fertility, as long as it does not interfere with rumen microbial metabolism. it is critical that improved methods to protect these unsaturated fa are required if precise calculations of the supply of unsaturated lipids are to be utilized in dairy cattle ration formulation to improve fertility. source of se might influence health and reproduction of dairy cows, but response seems to be dependent upon the background se concentrations in dietary ingredients. lastly, although lactating dairy cows can consume substantial amounts of gossypol with no detrimental effects on health and lactation, when plasma gossypol concentrations exceed 5 g/ml, embryo development and establishment and maintenance of pregnancy are compromised. references 1. vasconcelos jlm, sangsritavong s, tsai sj, et al: acute reduction in serum progesterone concentrations after feed intake in dairy cows. theriogenology 2003;60:795-807. 2. villa-godoy a, hughes tl. emery rs, et al: association between energy balance and luteal function in lactating dairy cows. j dairy sci 1988;71:1063-1072. 3. butler wr: energy balance relationships with follicular development, ovulation and fertility in postpartum dairy cows. livest prod sci 2003;83:211-218. 4. bonczeck rr, young cw, wheaton je, et al: responses of somatotropin, insulin, prolactin, and thyroxine to selection for milk yield in holsteins. j dairy sci 1988;71:2470-2478. 5. nebel rl, mcgilliard ml: interactions of high milk yield and reproductive performance in dairy cows. j dairy sci 1993;76:3257-3268. 6. jordan er, fourdraine rh: characterization of the management practices of the top milk producing herds in the country. j dairy sci 1993;76:3247-3256. 7. curtis cr, erb hn, sniffen cj, et al: association of parturient hypocalcemia with eight periparturient disorders in holstein cows. j am vet med assoc 1983;183:559-561. 8. whiteford lc, sheldon im: association between clinical hypocalcaemia and postpartum endometritis. vet rec 2005;157:202-203. clinical theriogenology • volume 3 number 4 • december 2011597 9. markusfeld o: relationship between overfeeding, metritis and ketosis in high yielding dairy cows. vet rec 1985;116:489-491. 10 rutigliano hm, lima fs, cerri rla, et al: effects of method of presynchronization and source of selenium on uterine health and reproduction in dairy cows. j dairy sci 2008;91:3323–3336. 11. nahms: dairy 1996 part iii: reference of 1996 dairy health and health management. online. available: http://nahms.aphis.usda.gov/dairy/dairy96/dr96pt3.pdf. 12. goshen t, shpigel ny: evaluation of intrauterine antibiotic treatment of clinical metritis and retained fetal membranes in dairy cows. theriogenology 2006;66:2210-2218. 13. hammon ds, evjen im, dhiman tr, et al: neutrophil function and energy status in holstein cows with uterine health disorders. vet immun immunopathol 2006;113: 21-29. 14. huzzey jm, veira dm, weary dm, et al: prepartum behavior and dry matter intake identify dairy cows at risk for metritis. j dairy sci 2007;90:3220-3233. 15. urton g, von keyserlingk ma, weary dm: feeding behavior identifies dairy cows at risk for metritis. j dairy sci 2005;88:2843-2849. 16. kimura k, goff jp, kehrli me jr, et al: decreased neutrophil function as a cause of retained placenta in dairy cattle. j dairy sci 2002;85:544-550. 17. wade gn, jones jj: neuroendocrinology of nutritional infertility. am j regul integr comp physiol 2004;287:12771296. 18. hileman sm, lubbers ls, jansen ht, et al: changes in hypothalamic estrogen receptor-containing cell numbers in response to feed restriction in the female lamb. neuroendocrinology 1999;69:430-437. 19. santos jep, bilby tr, thatcher ww, et al: long chain fatty acids of diet as factors influencing reproduction in cattle. reprod domest anim 2008;43(suppl 2):23-30. 20. santos jep, thatcher ww, chebel rc, et al: the effect of embryonic death rates in cattle on the efficacy of estrous synchronization programs. anim reprod sci 2004;82-83:513-535. 21. santos jep, juchem so, cerri rla, et al: effect of bst and reproductive management on reproductive and lactational performance of holstein dairy cows. j dairy sci 2004;87:68-881. 22. butler wr: review: effect of protein nutrition on ovarian and uterine physiology in dairy cattle. j dairy sci 1998;81:2533-2539. 23. ocon om, hansen pj: disruption of bovine oocytes and preimplantation embryos by urea and acidic ph. j dairy sci 2003;86:1194-1200. 24. rhoads ml, rhoads rp, gilbert ro, et al: detrimental effects of high plasma urea nitrogen levels on viability of embryos from lactating dairy cows. anim reprod sci 2006;91:1-10. 25. noftsger s, st-pierre nr: supplementation of methionine and selection of highly digestible rumen undegradable protein to improve nitrogen efficiency for milk production. j dairy sci 2003;86:958-969. 26. santos jep, bisinotto rs, ribeiro es, et al: applying nutrition and physiology to improve reproduction in dairy cattle. in: lucy mc, pate jl, smith mf, et al, editors. reproduction in domestic ruminants vii. nottingham: nottingham university press; 2011. p. 387-404. 27. kendrick kw, bailey tl, garst as, et al: effects of energy balance on hormones, ovarian activity, and recovered oocytes in lactating holstein cows using transvaginal follicular aspiration. j dairy sci 1999;82:1731-1740. 28. grant rj, albright jl: feeding behavior and management factors during the transition period in dairy cattle. j anim sci 1995;73:2791-2803. 29. radcliff rp, mccormack bl, crooker ba, et al: plasma hormones and expression of growth hormone receptor and insulin-like growth factor-i mrna in hepatic tissue of periparturient dairy cows. j dairy sci 2003;86: 920-3926. 30. rhoads rp, kim jw, leury bj, et al: insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows. j nutr 2004;134:1020-1027. 31. butler st, marr al, pelton sh, et al: insulin restores gh responsiveness during lactation-induced negative energy balance in dairy cattle: effects on expression of igf-i and gh receptor 1a. j endocrinol 2003;176:205-217. 32. gong jg, lee wj, garnsworthy pc, et al: effect of dietary-induced increases in circulating insulin concentrations during the early postpartum period on reproductive function in dairy cows. reproduction 2003;123:419-427. 33. santos, j.e.p., h.m. rutigliano, and m.f. sá filho: risk factors for resumption of postpartum cyclicity and embryonic survival in lactating dairy cows. anim reprod sci 2009;110:207-221. 34. sklan d, kaim m, moallem u, et al: effect of dietary calcium soaps on milk yield, body weight, reproductive hormones, and fertility in first parity and older cows. j dairy sci 1994;77:1652-1660. 35. ferguson jd, sklan d, chalupa wv, et al: effects of hard fats on in vitro and in vivo rumen fermentation, milk production, and reproduction in dairy cows. j dairy sci 1990;73:2864-2879. 36. mcnamara s, butler t, ryan dp, et al: effect of offering rumen-protected fat supplements on fertility and performance in spring-calving holstein–friesian cows. anim reprod sci 2003;79:45-56. 37. schneider p, sklan d, chalupa w, et al: feeding calcium salts of fatty acids to lactating cows. j dairy sci1988;71:2143-2150. 38. scott ta, shaver rd,. zepeda l, et al: effects of rumen-inert fat on lactation, reproduction, and health of high producing holstein herds. j dairy sci 1995;78:2435-2451. clinical theriogenology • volume 3 number 4 • december 2011 598 39. sklan d, moallem u, folman y: effect of feeding calcium soaps of fatty acids on production and reproductive responses in high producing lactating cows. j dairy sci 1991;74:510-517. 40. schingoethe dj, casper dp: total lactational response to added fat during early lactation. j dairy sci 1991;74:26172622. 41. staples cr, burke jm, thatcher ww: influence of supplemental fats on reproductive tissues and performance of lactating cows. j dairy sci 1998;81:856-871. 42. staples cr, thatcher ww: effects of fatty acids on reproduction of dairy cows. in: garnsworthy pc, wiseman j, editors. recent advances in animal nutrition. nottingham: nottingham university press; 2005. p. 229-256. 43. juchem so: lipid digestion and metabolism in dairy cows: effects on production, reproduction and health [dissertation]. davis(ca): university of california; 2007. 44. ambrose dk, kastelic jp, corbett r, et al: lower pregnancy losses in lactating dairy cows fed a diet enriched in αlinolenic acid. j dairy sci 2006;89:3066-3074. 45. fuentes mc, calsamiglia ss, sánchez c, et al: effect of extruded linseed on productive and reproductive performance of lactating dairy cows. livest sci 2008;113:144-154. 46. petit hv, twagiramungu h: conception rate and reproductive function of dairy cows fed different fat sources. theriogenology 2006;66:1316-1324. 47. silvestre ft, carvalho ts, francisco n, et al: effects of differential supplementation of fatty acids during the peripartum and breeding periods of holstein cows: i. uterine and metabolic responses, reproduction, and lactation. j dairy sci 2011;94:189-204. 48. juchem so, cerri rla, villaseñor m, et al: supplementation with calcium salts of linoleic and trans-octadecenoic acids improves fertility of lactating dairy cows. reprod domest anim 2010;45:55-62. 49. cerri rla, juchem so, chebel rc, et al: effect of fat source differing in fatty acid profile on metabolic parameters, fertilization and embryo quality in high-producing dairy cows. j dairy sci 2009;92:1520-1531. 50. mattos r, staples cr, williams j, et al: uterine, ovarian, and production responses of lactating dairy cows to increasing dietary concentrations of menhaden fish meal. j dairy sci 2002; 85:755-764. 51. mattos r, guzeloglu a, badinga l, et al: polyunsaturated fatty acids and bovine interferon- modify phorbol esterinduced secretion of prostaglandinf2α and expression of prostaglandin endoperoxide synthase-2 and phospholipase-a2 in bovine endometrial cells. biol reprod 2003;69:780-787. 52. mattos r, staples cr, arteche a, et al: the effects of feeding fish oil on uterine secretion of pgf2α, milk composition, and metabolic status of periparturient holstein cows. j dairy sci 2004;87:921-932. 53. mattos r, staples cr, thatcher ww: effects of dietary fatty acids on reproduction in ruminants. rev reprod 2000;5:38-45. 54. goff jp: transition cow nutrition: effects on immune function and postpartum health. proc annu meet conv, dairy cattle reproduction council; 2006. p. 1-8. 55. weiss wp: selenium nutrition of dairy cows: comparing responses to organic and inorganic selenium forms. in: lyons tp, jacques ka, editors. alltech’s nineteenth annual symposium, nutritional biotechnology in the feed and food industries. nottingham: nottingham university press; 2003. p. 333-343. 56. silvestre ft, silvestre dt, crawford c, et al: effect of selenium (se) source on innate and adaptive immunity of periparturient dairy cows. biol reprod annual meeting; 2006. special issue. p. 132. 57. silvestre ft, silvestre dt, santos jep, et al: effects of selenium (se) sources on dairy cows. j anim sci 2006;89(suppl 1):52. 58. risco ca, holmberg ca, kutches a: effect of graded concentrations of gossypol on calf performance: toxicological and pathological considerations. j dairy sci 1992;75:2787-2798. 59. chenoweth pj, chase cc jr, risco ca, et al: characterization of gossypol-induced sperm abnormalities in bulls. theriogenology 2000;53:1193-1203. 60. velasquez-pereira j, chenoweth pj, mcdowell lr, et al: reproductive effects of feeding gossypol and vitamin e to bulls. j anim sci 1998;76:2894-2904. 61. randel rd, chase cc jr, wyse sj: effects of gossypol and cottonseed products on reproduction of mammals. j anim sci 1992;70:1628-1638. 62. coscioni ac,villaseňor m, galvão kn, et al: effect of gossypol intake and plasma gossypol concentrations on follicle development and luteal function in dairy heifers. j dairy sci 2003;86(suppl 1):240. 63. coscioni ac, villaseňor m, galvão kn, et al: effect of gossypol intake on plasma and uterine gossypol concentrations and on embryo quality and development in superovulated holstein dairy heifers. j dairy sci 2003; 86(suppl 1):240. 64. villaseñor m, coscioni ac, galvão kn, et al: gossypol disrupts embryo development in heifers. j dairy sci 2008;91: 3015-3024. 65. santos jep, villaseňor m, depeters ej, et al: type of cottonseed and gossypol in diets of lactating dairy cows: plasma gossypol, reproduction, and health. j dairy sci 2003;86:892-905. 66. galvão kn, santos jep, coscioni ac, et al: embryo survival from gossypol-fed heifers after transfer to lactating cows treated with human chorionic gonadotropin. j dairy sci 2006;89: 2056-2064. clinical theriogenology • volume 3 number 4 • december 2011599 clinical theriogenology • volume 3 number 4 • december 2011 600 omniblank: introduction findings of recent studies regarding potential side effects of surgical castration on health or behavior in males and females have placed european veterinarians in a dilemma. it has become difficult for them to give accurate advice to dog owners. practioners prefer to avoid harming and endangering the future health of animals. they are also aware that sterilization is important to prevent uncontrolled reproduction and control the risk of abandoned puppies or adoption by irresponsible owners. this short review is not intended to provide answers, but to raise questions about the future of surgical sterilization. gonadectomy and cancers a review that analyzed data on the role of neutering in cancer development had inconclusive findings.1 bitch owners in france consider ovariectomy from the perspective of preventing mammary tumors. common concern is, does early neutering, substantially reduce the risk of mammary tumors in the bitch? this belief is based on a study published in 1969 that has not since been repeated.2 however, a meta-analysis of 13 selected reports in english language peer-reviewed journals, had to exclude 10 manuscripts because of their high bias.3 of the 3 remaining, 1 study detected an association between neutering and a reduced risk of mammary tumors. there were no evidence of association in 2 studies. authors concluded that the evidence that neutering reduces the risk of mammary neoplasia, and the evidence that age of neutering has an effect, are judged to be is there a future for surgical contraception in the dog? alain fontbonne, cindy maenhoudt, bruna mendes ecole nationale vétérinaire d’alfort, centre d’etude en reproduction des carnivores maisons-alfort (paris), france abstract many scientific studies have focused on the potential detrimental effects of surgical castration. some studies observed an increase in the risk of developing certain types of cancers in neutered animals, males and females. however, there often appears to have been a breed predisposition. there are contradictory data about neutering effect on lifespan, probably due to the fact that sterilized and intact dogs do not face similar risks in their daily life. castration often reduces unwanted behaviors, but may increase certain types of aggression towards other dogs or even humans, depending on the age when neutered. other potentially increased risks reported in castrated dogs and bitches are bone and joint problems, including predisposition to hip dysplasia in some breeds, urinary incontinence in bitches, development of a ‘puppy coat’ or even loss of cognitive functions. vasectomy or hysterectomy without ovariectomy may be surgical alternatives, but the best approach is probably to refer to guidelines for a rational choice to satisfy owners and optimize dogs’ health. keywords: neuter, dogs, cancers, lifespan, behavior, bones, joints, cognitive functions, alternatives weak and are not a sound basis for firm recommendations. even if this belief appears to be probably true for most veterinary practitioners following bitches throughout their lives (as very few bitches spayed at an early age develop mammary tumors), it is not appropriate to present this as a fact. hemangiosarcomas are frequent cancers in the dog, especially in some breeds such as the golden retriever. some studies4 have pointed out the susceptibility of this specific breed, especially in spayed bitches, but age may have a larger effect on cancer-related mortality than reproductive status. however, the risk of hemangiosarcomas in golden retrievers is 4 times greater in females ovariectomized after 1 year of age compared to intact females or females neutered earlier in life.5 regarding lymphomas and lymphosarcomas, a retrospective study published in 2018 on 6,201dogs6 suggested an increased risk for neutered animals to develop this disease, both in males (or = 2.8; 95%ci) and females (or = 4.4; 95%ci). however, breed and sex are also important factors, independent of whether the animals are sterilized or not. osteosarcomas are the third cause of mortality in golden retrievers and have even a higher prevalence in giant breeds (e.g. rottweilers). neutered golden retrievers animals (male and female) had substantailly higher risk for osteosarcoma.4 a study in rotweilers7 suggested an increased risk in gonadectomized clinical theriogenology 2021; 13: 161 animals (3 times in females and 4 times in males) to develop osteosarcoma, specifically, if the animals are spayed before 1 year of age. although the prevalence of prostatic carcinomas is lower (0.6%) in the general population there is an increased risk in castrated male dogs.8 some breeds (e.g. bouvier des flandres) appear to have more cancer in females than in males. according to some authors,9 castrated animals from both sexes may have an increased risk. transitional cell carcinomas of the prostate are also more likely to occur in castrated males.10 mast cell tumors are rare in the general canine population but some breeds are believed to be predisposed such as golden retrievers (incidence: 2.4%), boxers, boston terriers, or labradors. in males, vizslas11 was identified to have increased risk of cancer in castrated dogs after 1 year compared to dogs castrated earlier or in intact dogs. in females, there was an increased risk for cancer after ovariectomy.12 reasons explaing potential predisposition to develop some types of cancers in gondectomized animal is not clear. after removal of gonads, lh is continously elevated at supraphysiologic concentrations.13 as there are lh receptors in many neoplastic tissues (e.g. lymphomas, hemangiosarcomas, mastocytomas, transitional cell carninomas, and osteosarcomas), it may be assumed that lh receptor activation in these various tissues may have a detrimental role in castrated animals. however, this remains to be confirmed. gonadectomy and lifespan several studies in the us based on a large number of animals, males or females suggested that castrated animals live longer than intact ones. for example, 1 study14 based on 40,000 sterilized and intact domestic dogs reported that sterilization was strongly associated with an increased lifespan. also, data presented in 2013, based on 2.2 m dogs had similar findings.15 spayed dogs lived an average of 11.6 years, 23% longer than intact female dogs, whereas neutered dogs lived an average of 11.1 years, 18% longer than intact male dogs.15 however, there may be some bias in these comparisons, as intact animals, especially male dogs, may live a more ‘at-risk’ life, perhaps due to the lifestyle of the owners or the medical care of pets that may differ between castrated and intact dogs, or because the hormonal drive of dogs that may lead them to face more dangerous situations. for example, intact dogs are more than twice as likely to be hit by a car or bitten by another animal as neutered dogs. sterilization decreased the risk of infections,14 perhaps due to the withdrawal of progesterone and testosterone, which have an immunosuppressive effect. also, intact male dogs were more likely than sterilized dogs to roam and to fight with other dogs, and intact bitches may have more dominance aggression than spayed females. such behaviors may increase the risks of infectious or traumatic, direct or indirect causes of death. some factors linked with castration are to be taken into account when studying lifespan and gonadectomy. for example, median lifespan was shorter in overweight animals compared to normal weight dogs, with breed specificities.6 however, these differences were highest in yorkshire terriers and least in german shepherd. according to these authors, it is important to promote healthy body condition in castrated animals. more than 10 years data on rottweilers suggested that ovariectomy at a young age decreased the risk of mammary tumors.17 according to these authors, it is important to consider issues more broadly and not only in terms of risk for certain diseases, but in terms of the effect on the whole body. in contrast to what was believed and taught for decades (‘ovariectomy is good for health’), several studies indicate that the presence of ovaries is part of an overall system that promotes longevity. certainly, bitches that are not spayed have a higher risk of developing mammary tumors and pyometra. however, these authors studied whether bitches that developed mammary tumors or pyometra during their lifetime had a clearly shortned lifespan or not. nearly 50% of mammary tumors in dogs are benign and also due to good medical care of pets in western countries (currently, diagnosis and surgical treatment of mammary tumors and pyometra are usually performed early). therefore, the lifespan of the majority of affected bitches is not compromised. on a population level, mammary tumors or pyometra did not affect their longevity.17 in contrast, bitches < 4.3 years had increased (p = 0.002) longevity of 33% compared to females spayed early, an average increase in lifespan of 17 months. in fact, other diseases that are clearly more serious for longevity and linked to sterilization have to be considered: lymphomas and osteosarcomas in particular, not to mention other diseases (e.g. obesity and diabetes) directly or indirectly linked to hormonal changes following ovariectomy. in other words, ovario-hysterectomy performed in young bitches would substantially reduce life expectancy. studies are to be continued in other breeds and on a larger number of animals before drawing firm conclusions. gonadectomy and behavior male dogs it is important to differentiate between undesirable behaviors potentially linked to testosterone and behavioral disorders. undesirable behaviors (urinary marking, roaming, mounting people’s legs or inter-male aggression) are often considered by the owners as normal phenomenon. two-thirds of dogs encountered at least 1 of 4 undesirable behaviors as per a survey (virbac, unpublished data). very often, owners are bothered by them (for example, 31% of the owners were bothered by inter-male aggression and 51% by urinary marking), but in the majority of cases (nearly 75%), they do not discuss it with their veterinarian or do not even consider mentioning, because they feel that these normal behaviors in male dogs for which nothing can be done to decrease the inconvenience it causes. what is the effect of gonadectomy on these undesirable behavioral situations? some studies considered this ; for example, there were clinical theriogenology 2021; 13: 162 modifications of behavior in 122 dogs after surgical castration.18 urinary marking inside the house decreased by 69% and outside decreased by 23% but was not completely stopped. roaming, when it was induced by bitches in estrus, decreased by 64%, but roaming due to other causes (e.g. hunting dogs) decreased only by 16%. mounting on people’s legs decreased by 57%, mounting on other males decreased by 43%, and mounting on bitches decreased by 41%.18 however, mounting inanimate objects (e.g. cushions) decreased only by 26% and was even increased by 5% in some. regarding inter-male aggression, 23 dogs had this unwanted behavior, 13 dogs improved, but 1 dog even became more agressive after castration. most other studies had similar numbers with slight differences. for example, 48 of 35 (73%) did not have any modifications on urinary marking outside and only 9 dogs (19%) had a decreased frequency of urinary marking, and 4 dogs (8%) even began to sit down to urinate as prepubertal animals.19 we can therefore roughly estimate that, after castration, undesirable behaviors decrease, but are not completely eliminated in ~ 60% of cases. the relative failure of gonadectomy toward inter-male dominance or other unwanted behaviors is not surprising. a study20 conducted in free-roaming dogs in chile reported that castrated dogs had no reduction in sexual activity or fighting behavior with other males. in other words, these undesirable behaviours are only partly linked to testosterone. furthermore, aggresion towards humans was another problem that was addressed in most studies. after castration, aggression towards stragers decreased in 22%, but increased in 4% of animals.18 in the same study, aggression towards familiar people also increased by 4%. in other words, after castration, some male dogs may become more agressive towards other dogs and also towards people. a retrospective study21 on 13,498 male dogs agression to humans and 13,237 dogs agression to other dogs used the c-barq scale (canine behavioral assessment & research questionnaire), a standardized, behavioral evaluation tool. it appeared that some dogs became more agressive with strangers, especially if castrated between 7 and 12 months of age, a common practice in europe. these authors therefore concluded that their data did not support the view that gonadectomy will predictably reduce aggressive behavior. veterinarians need to consider these facts when counseling and informing owners about the advantages and disadvantages of castration in order to avoid potential owner complaints or even legal actions against the veterinarian if the dog bites someone (e.g. a child). what about medical castration? in 2007, subcutaneous implants containing the gnrh super-agonist deslorelin (suprelorin®, virbac) was released on the european market. surgically castrated male dogs and dogs implanted with suprelorin® 4.7 mg were compared.22 effects on mounting, urine marking, roaming and inter-male dominance were identical. in other words, when a veterinarian hesitates to surgically castrate a male with an undesirable behavior without knowing whether it will improve the dog’s behavior or not, using an implant may be a good alternative to test the effect of castration. if it works, then the dog may be surgically castrated later, being more confident about the success of the procedure. behavioural pathology is an even more complicated matter when we consider gonadectomy. among 1,644 dogs, significantly more neutered male dogs were admitted to a teaching hospital for aggression and phobias compared to the percentage of neutered dogs admitted to other departments of the same hospital.23 the authors concluded that ‘these findings deserve attention from the veterinary community and suggest to reexamine the recommendations of gonadectomy for dogs’. in other words, gonadectomy may be useful in case of slightly unwanted behaviors, but may become contraindicated in case of real behavioral disorders. among 2,505 castrated male dogs from the vizsla breed had 1.3 more risk to develop a behavioral disorder, and even more if castrated before 6 months of age (1.8 times more risk).11 therefore, if a dog is presented with a potential behavioral disorder, it is wise to seek advice from a behavioral specialist or at least to begin by a behavioral treatment before ‘jumping’ to have surgical gonadectomy. a lakeland terrier that had anxiety separation was castrated by a veterinarian (owners were not told that castration could increase the problem).24 after castration, it started to howl at night and the author felt that it would have been wise to postpone castration while waiting for the advice of a behaviorist. the potential negative effect of gonadectomy on behavioral disorders is especially interesting to bear in mind, when we know that thousands of dogs are abandoned in europe, many for unwanted behavior. different behavioral disorders may be associated with an insufficient duratoin of exposure to gonadal hormones before castration.25 the authors wrote that ‘shelters are inundated by dogs desplaying undesirable behaviours.’ it remains to be known as to how many inappropriate castrations result in behavioral issues and subsequent abandoment. female dogs this issue is not so clear in females, although some publications suggest a potential increase of agressions in spayed bitches.26,27 significantly higher ‘dominance aggression’ towards family members was observed more in spayed bitches.28 in labrador retrievers, owners of spayed bitches observed more frequent or more intense fear reaction in their animals in response to loud noises, unfamiliar objects approaching on or near the sidewalk, or if they were approached by unknown dogs barking, growling or jumping.29 in contrast to popular belief, gonadectomy did not inevitably result in a behaviorally more stable dog.29 as in male dogs, caution should be taken by veterinarians when owners are requesting ovariectomy in a bitch for behavioral disorders. other problems potentially linked with gonadectomy bone and joint problems among 1,170 intact german shepherd male dogs, 7% were diagnosed with at least 1 of 3 diseases (hip dysplasia, elbow dysplasia, and clinical theriogenology 2021; 13: 163 cranial cruciate ligament rupture) and in neutered male dogs (neutered before 1 year of age), this incidence was significantly higher (21%).30 similarly, females spayed before 1 year of age had a significantly higher risk (17%) to develop at least 1 of these 3 orthopedic disorders compared to intact females (5%). in contrast, another study31 reported that neutering was not associated with these musculoskeletal diseases (including hip dysplasia). regarding hip dysplasia, in neutered male dogs of various breeds and sizes were significantly more likely to have clinical signs of hip dysplasia than dogs in the other study groups (intact males, intact females, and spayed females) (or = 1.21; 95% ci = [1.18-1.24]).32 however, breed is another important risk factor to consider. in a study5 of 759 golden retrievers aged 1 8 years, the incidence of hip dysplasia in male dogs neutered before 12 months of age was twice that in the group of male dogs neutered after 12 months of age ((10.3 versus 5.1%, respectively, and significantly different). in adult dogs, sex steroid hormones have major rolse in maintenance of bone tissue and its remodeling. they have an inhibitory effect on bone resorption, enabling a balance between resorption and osteosynthesis.33 in women with bilateral ovariectomy, an increase in bone turnover and excessive bone resorption were observed.33 however, regulation of this turnover can then be controlled by treatment with sex hormones. these same physiological hypotheses are put forward in dogs. in addition to their actions on bone tissue, these hormones are involved in other parts of the musculoskeletal system. in women, for example, it has been reported that estrogens act on joint cartilage, subchondral bone, muscles, ligaments and synovial membranes.34 concerning the potential increased risk of early spaying, it is now well known that castrating male or female dogs before puberty delays closure of growth cartilage in long bones (femur and humerus). the time of closure of the growth centers was significantly delayed in dogs neutered at 7 weeks of age, compared to thsoe neutered at 7 months of age or older.35 it may predispose to ligament problems (including hip dysplasia) or even epiphyseal fractures, in association with the weight gain and obesity often encountered after gonadectomy. early neutering (before 5.5 months of age) was significantly associated with an increase in hip dyspasia (or = 1.70; 95% ci = [1.04-2.78]).36 gonadectomized animals may be also predisposed to other ligamentary diseases such as rupture of the crucial ligament or elbow dysplasia. in labradors, the incidence of elbow dysplasia was significantly higher in dogs neutered at 6 months of age (4.2%) and between 2 and 8 years of age (2.2%) compared to the whole dog group (0.6%).37 urinary incontinence in bitches the association between spaying and the subsequent development of urinary incontinence in bitches is well known, although not fully confirmed scientifically. there appears to be an increased risk of urinary incontinence after spay in certain large breeds, e.g. boxer, rottweiler, doberman, irish setter, weimaraner, springer spaniel, bobtail, and giant schnauzer, wheras dogs of other breeds with a similar body weight like the german shepherd and the labrador were rarely affected.38 the surgical method of spaying, ovariectomy versus ovariohysterectomy, appears to have no effect on the risk for urinary incontinance, whereas the risk factor ‘age at neutering’ is still controversially discussed. the timing of gonadectomy relative to the onset of puberty may have an influence on the degree of incontinence. according to studies performed in switzerland,39 60% of early spayed bitches had an uncontrolled loss of urine not only while sleeping but also when awake, during resting, and occasionally in a sitting position or on a walk. in contrast, bitches spayed after puberty are mainly incontinent during sleep, and even though more dogs spayed after the first estrus become incontinent, the frequency of incontinent episodes is considerably lower. a systematic review40 of 7 selected peer-reviewed original english analytic journal articles that considered the effect of neutering or age at neutering on the risk of urinary incontinence concluded 4 articles to be at high risk of bias. of the remaining 3 studies that were at moderate risk of bias, there was some weak evidence that neutering, particularly before 3 months of age, increases the risk of urinary incontinence. for these authors, the evidence overall was neither consistent nor strong enough to allow for strict recommendations on the effect of neutering or age at neutering on the risk of urinary incontinence. dematological problems certain breeds, such as the irish setter, cocker spaniel, longhaired dachshund, eurasier, chow-chow, golden retriever, newfoundland and landseer, longhaired belgian shepherd, afghan hound, etc, have a predisposition for coat changes after spaying. affected dogs have substantial increase of wool hair and decrease in hair color intensity. the coat resembles that of young dogs and is called ‘puppy coat.’41 these may be so severe that breeders advise against spaying. however, it is unknown to what extent reproductive hormones influence hair growth. from our own experience, this may happen in male dogs and in bitches after surgical gonadectomy. gnrh agonists subcutaneous implants may help to restore – at least partially – the initial quality of the coat.41 loss of cognitive functions some authors hypothesize that cognitive decline in neutered senior dogs appears to progress more rapidly than in intact dogs.42 their results suggest that the presence of circulating testosterone in aging sexually intact male dogs may slow the progression of cognitive impairment, at least among dogs that already have signs of mild impairment. estrogens would be expected to have a similar protective role in sexually intact female dogs; unfortunately, too few sexually intact female dogs were available for inclusion in this study to test this hypothesis. androgen deprivation results in decreased synaptic connections in the hippocampus of rodents and nonhuman primates, associated with an increased amyloid deposition in the brain.43 however, another study44 in beagles (10 years old) based on brain histological analyses, refuted this conclusion. clinical theriogenology 2021; 13: 164 other surgical approaches although most studies on surgical castration are focused on gonadectomy, there are other forms of desexing that enables a dog to keep their gonads intact while preventing reproduction.13,21,45 in male dogs, vasectomy is a surgical alternative method for desexing. this procedure consists of surgical transection of the vas deferens, leading to azoospermia and consequently, infertility, without apparently affecting testicular structures or testosterone concentrations.45,46 even if vasectomy may be a suitable option for male contraception and useful in free-roaming and/or feral dogs as a potentially effective means of population control,45 it has not been widely adopted in practice. it appears that it does not result in changes in libido nor other male-specific behaviors in dogs,45 and specific complications appear to be rare and include spermatocele,47 sperm granuloma,47,48 and testicular degeneration.49 nevertheless, there is a need to further explore the long-term consequences of vasectomy. in beagles, vasectomy led to irreversible damage to testes structure and other health problems.50act on joint cartilage, subchondral bone, muscles, ligaments and synovial membranes.34 in female dogs, other surgical approaches (other then ovariohysterectomy and ovariectomy) are not widely used in practice. however, surgical transection of the uterine tubes (ovary-sparing hysterectomy) is more frequently performed.45 their medical consequences are not well studied and like vasectomy for male dogs, these alternative desexing methods do not change the hormonal status of the bitch.45 therefore, the expected various health consequences are similar to those in intact individuals, inasmuch as the susceptible organs remain in the body. more research is needed on the medical complications of these alternative surgical aproaches. how to make a rational choice, satisfy the owners and optimize the health of the dog in small dog breeds, the occurrences of cancers and joint disorders were low or even close to zero in both intact and neutered dogs, with exceptions of boston terrier and shih tzu, where there was a significant increase in cancers after neutering.51 these authors proposed and published interesting guidelines for the best age for neutering in 35 breeds, to avoid increasing long-term health risks of neutering. same authors52 suggested the risks of 1 or more joint disorders following early neutering can be predicted on the basis of the dog’s body weight. those dogs that are expected to reach at least 20 kg as adults had a significantly increased risk of having 1 or more joint disorders, that is up to 20% above the level of intact dogs. conflict of interest none to report. references 1.smith an: the role of neutering in cancer development. vet clin north am small anim pract 2014;44:965-975. 2. schneider r, dorn cr, taylor do: factors influencing canine mammary cancer development and postsurgical survival. j natl cancer inst 1969;43:1249-1261. 3. beauvais w, cardwell jm. brodbelt dc: the effect of neutering on the risk of mammary tumours in dogs-a systematic review. j small anim pract 2012;53:314-322. 4. kent ms, burton jh , dank g, et al: association of cancer-related mortality, age and gonadectomy in golden retriever dogs at a veterinary academic center (1989-2016). plos one. 2018;13:1-12. 5. torres de la riva g, hart bl, farver tb, et al: neutering dogs: effects on joint disorders and cancers in golden retrievers. plos one. 2013;8:1-7. 6. bennett pf, taylor r, williamson p: demographic risk factors for lymphoma in australian dogs: 6201 cases. j vet intern med 2018;32:2054-2060. 7. cooley dm, beranek bc, schlitter dl, et al: endogenous gonadal hormone exposure and bone sarcoma risk. cancer epidemiol biomarkers prev 2002;11:1434-1440. 8. teske e, naan ec, van dijk em, et al: canine prostate carcinoma: epidemiological evidence of an increased risk in castrated dogs. mol cell endocrinol 2002;197:251-255. 9. mutsauers aj, widmer wr, knapp dw: canine transitional cell carcinoma. j vet intern med 2003;17:136-144. 10. bryan jn, keeler mr, henry cj, et al: a population study of neutering status as a risk factor for canine prostate cancer. prostate 2007;67:1174-1181. 11. zink mc, farhoody p, elser se, et al: evaluation of the risk and age of onset of cancer and behavioral disorders in gonadectomized vizslas. j am vet med assoc 2014;244:309-319. 12. white cr, hohenhaus ae, kelsey j, et al: cutaneous mcts: association with spay/neuter status, breed, body size, and phylogenetic cluster. j am anim. hosp assoc 2011;47:210-216. 13. kutzler ma. possible relationship between long-term adverse health effects of gonad-removing surgical sterilization and luteinizing hormone in dogs. animals 2020;10:599:1-11. 14. hoffman jm, creevy ke, promislow de: reproductive capability is associated with lifespan and cause of death in companion dogs. plosone 2013;8:1-7. 15 . h t t p s : / / f r. s c r i b d . c o m / d o c u m e n t / 1 4 01 9 61 71 / banfield-pet-hospital-state-of-pet-health-2013-report 16. salt c, morris pj, wilson d, et al. association between life span and body condition in neutered client-owned dogs. j vet intern med 2019;33:89-99. 17. waters dj, kengeri ss, maras ah, et al: life course analysis of the impact of mammary cancer and pyometra on age-anchored life expectancy in female rottweilers: implications for envisioning ovary conservation as a strategy to promote healthy longevity in pet dogs. vet j 2017;224:25-37. 18. maarschalkerweerd rj, endenburg n, kirpensteijn j, et al: influence of orchiectomy on canine behaviour. vet rec 1997;140:617-619. 19. andersson a, linde-forsberg c: castration and progestagen treatment of male dogs, part 2. eur j companion anim pract 2002;12:178-185. 20. garde e, pérez ge, vanderstichel r, et al: effects of surgical and chemical sterilization on the behavior of free-roaming male dogs in puerto natales, chile. prev vet med 2016;123:106-120. 21. farhoody p, mallawaarachchi i, tarwater pm, et al: aggression toward familiar people, strangers, and conspecifics in gonadectomized and intact dogs. front vet sci 2018;5:18. clinical theriogenology 2021; 13: 165 22. de gier j and vinke cm. use of deslorelin to control hypersexuality in male dogs. proc 7th evssar congress 2010:9-11. 23. bamberger m, houpt ka. signalment factors, comorbidity, and trends in behavior diagnoses in dogs: 1,644 cases (1991-2001). j am vet med assoc 2006;229:1591-1601. 24. what is your client thinking. will neutering change his behaviour ? vet rec12 2019:70. 25. mcgreevy pd, wilson b, starling mj, et al: behavioural risks in male dogs with minimal lifetime exposure to gonadal hormones may complicate population-control benefits of desexing. plos one. 2018;13:1-14. 26. podberscek al, serpell ja: aggressive behaviour in english cocker spaniels and the personality of their owners. vet rec 1997;14:73-76. 27. reisner ir, houpt ka, shofer fs: national survey of owner-directed aggression in english springer spaniels. j am vet med assoc 2005;227:1594-1603. 28. o’farrell v, peachey e: behavioural effects of ovario-hysterectomy on bitches. j small anim pract 1990: 595-598. 29. balogh o, borruat n, andrea meier a, et al: the influence of spaying and its timing relative to the onset of puberty on urinary and general behaviour in labrador retrievers. reprod domest anim 2018;53:1184-1190. 30. hart bl, hart la, thigpen ap, et al: neutering of german shepherd dogs: associated joint disorders, cancers and urinary incontinence. vet med sci 2016;2:191-199. 31. howe lm, slater mr, boothe hw, et al: long-term outcome of gonadectomy performed at an early age or traditional age in dogs. j am vet med assoc 2001;218:217-221. 32. witsberger th, villamil ja, schultz lg, et al: prevalence of and risk factors for hip dysplasia and cranial cruciate ligament deficiency in dogs. j am vet med assoc 2008;232:1818-1824. 33. almeida m, laurent mr, dubois v, et al: estrogens and androgens in skeletal physiology and pathophysiology. physiol rev 2017;97:135-187. 34. miyatake k, muneta t, ojima m, et al: coordinate and synergistic effects of extensive treadmill exercise and ovariectomy on articular cartilage degeneration. bmc musculoskelet disord 2016;17:238 :1-11. 35. salmeri k, bloomberg m, scruggs s, et al: gonadectomy in immature dogs: effects on skeletal, physical and behavioral development. j am vet med assoc 1991;198:1193-1203. 36. spain cv, scarlett jm, houpt ka: long-term risks and benefits of earlyage gonadectomy in dogs. j am vet med assoc 2004;224:380-387. 37. hart bl, hart la, thigpen ap, et al: long-term health effects of neutering dogs: comparison of labrador retrievers with golden retrievers. plos one 2014;9:1-10. 38. arnold s, arnold p, hubler m, et al: urinary incontinence in spayed female dogs: frequency and breed disposition. schweizer archiv für tierheilkunde 1989;131:259-263. 39. stöcklin-gautschi nm, hässig m, reichler im, et al: the relationship of urinary incontinence to early spaying in bitches. j reprod fertil (suppl 57) 2001;57:233-236. 40. beauvais w, cardwell jm, brodbelt dc: the effect of neutering on the risk of urinary incontinence in bitches a systematic review. j small anim pract 2012;53:198-204. 41. reichler im, welle m, eckrich c, et al: spaying-induced coat changes: the role of gonadotropins, gnrh and gnrh treatment on the hair cycle of female dogs. vet dermatol 2008;19:77-87. 42. hart bl: effect of gonadectomy on subsequent development of age-related cognitive impairment in dogs. j am vet med assoc 2001;219:51-56. 43. janowsky js: the role of androgens in cognition and brain aging in men. neuroscience 2006: 138;1015-1020. 44. waters dj, shen s, glickman lt: life expectancy, antagonistic pleiotropy, and the testis of dogs and men. prostate 2000;43:272-277. 45. urfer sr, kaeberlein m: desexing dogs: a review of the current literature. animals 2019;9:1086:1-28. 46. whyte j, sarrat r, torres a, et al: experimental vasectomy: comparison of the testicular structure with various surgical techniques. actas urol esp 1998;22:178-183. 47. pérez-marín cc, lopez r, dominguez jm, et al: clinical and pathological findings in testis, epididymis, deferens duct and prostate following vasectomy in a dog. reprod domest anim 2006;41:169-174. 48. mayenco aguirre am, garcia fernandez p, sanchez muela m: sperm granuloma in the dog: complication of vasectomy. j small anim pract 1996;37:392-393. 49. whyte j, sarrat r, torres a, et al: effects of vasectomy on the testicular structure of the dog. actas urol esp 1997;21:446-452. 50. zhang y, wang x, chen z, et al: long-term reproductive consequences of no-scalpel vasectomy in beagles. j huazhong univ sci technolog med sci 2012;32:899-905. 51. hart bl, hart la, thigpen ap, et al: assisting decision-making on age of neutering for 35 breeds of dogs: associated joint disorders, cancers, and urinary incontinence. front vet sci 2020;7:388:1-14. 52. hart bl, hart la, thigpen ap, et al: assisting decision-making on age of neutering for mixed breed dogs of five weight categories: associated joint disorders and cancers. front vet sci 2020;7:472 :1-12. clinical theriogenology 2021; 13: 166 is there a future for surgical contraception in thedog? 2018: acupuncture increases matrix metalloproteinase activity in bovine caruncles after calving acupuncture increases matrix metalloproteinase activity in bovine caruncles after calving katrina hiebel,a alfred menino,b michelle kutzlerb adepartment of integrated science and bdepartment of animal and rangeland sciences, oregon state university, corvallis, or introduction placental release is a complex physiological process involving matrix metalloproteinase (mmp) lysis to separate cotyledon from caruncle. if mmp activity is absent or diminished placental retention can result due to lack of collagenase activity.1 acupuncture has been used as a treatment for other reproductive disorders in dairy cattle including delayed uterine involution, repeat breeding, and uterine infections.2 the objectives of this study were to determine the effects of acupuncture in dairy cows on caruncular mmp activity at 0, 2, and 4 hours after calving. we hypothesized that acupuncture would increase caruncular mmp activity. methods immediately after natural calving, cows were restrained in a stanchion. the perineum was aseptically prepared and a caruncle was obtained from the body of the uterus, and flash frozen. caruncle collection was repeated at 2 and 4 hours after calving. acupuncture was administered to cows (n=6) following each caruncle collection (at 0 and 2 hours after calving). untreated controls (n=9) were kept in a stanchion for 15 minutes without any stimulation. acupuncture was applied to points reported to relax the cervix and stimulate uterine contractions: bladder 31, 32, and 34, baihui, guanyuanshu, and governing vessel-1. frozen caruncles (0.5 g) were homogenized in solubilization buffer and centrifuged. the supernatant was collected and the protein concentration was determined. zymography was performed by electrophoresis on 8% polyacrylamide and 0.6% gelatin. gels were incubated in a 2.5% triton x-100 renaturing buffer and stained with coomassie blue r-250. gels were immersed in destaining solution to develop lytic zones (bands) indicating enzymatic activity. enzymatic activity was quantified using a digital camera and true 16-bit data capture system (gel logic 2200 pro, bio-techniques). average band intensities (indicating enzyme activity) for each molecular mass category between treatment groups and time points were compared using student’s t-test. significance was defined as p<0.05. results five distinct calcium-dependent gelatinases (234, 143, 117, 59, and 49 kda) were found in the bovine caruncles at 0, 2, and 4 hours after calving. there was a trend for acupuncture to decrease the activity of the 234 kd gelatinase at 4 hours after calving compared to controls (p=0.08). the average enzyme activities did not differ between acupuncture and controls for the 143, 117, and 59 kda gelatinases. enzyme activity of the 49 kda gelatinase significantly increased from 2 to 4 hours after calving in acupuncture-treated cows and significantly decreased from 0 to 2 hours after calving in control cows. discussion the lowest molecular mass gelatinase (49 kda) corresponds to the active form of mmp type 2. these results are supported by previous research in our laboratory that showed that acupuncture increased tissue concentrations of mmp type 2 from 2 to 4 hours after calving.3 keywords: caruncle, cow, matrix metalloproteinase, placentome, retained placenta acknowledgment this project was funded by the united states department of agriculture-national institute of food and agriculture (animal health project #ore00141). references 1. mcnaughton ap, murray rd: structure and function of the bovine fetomaternal unit in relation to the causes of retained fetal membranes. vet rec 2009;165:615-622. 2. habacher g, pittler mh, ernst e. effectiveness of acupuncture in veterinary medicine: systematic review. j vet intern med 2006;20:480-488. 3. hiebel k, gentle l, kutzler m: acupuncture increases matrix metalloproteinase type-2 immunoexpression and tissue concentration in bovine caruncles after calving clin therio 2017;9:469. clinical theriogenology • volume 10, number 3 • september 2018 316 2018: a comparison of two protocols for pregnancy termination in mares a comparison of two protocols for pregnancy termination in mares giorgia podico, robyn e. ellerbrock, claire kaplan, fabio lima, igor f. canisso department of veterinary clinical medicine, college of veterinary medicine, university of illinois urbana-champaign, urbana il in horses, pregnancy termination may be performed to eliminate unwanted or twin pregnancies, after a diagnosis of embryonic/fetal or maternal abnormalities, to eliminate a conceptus of an undesired sex, or after legal disputes between parties. while a single systemic injection of prostaglandin f2α (pgf2α) is effective to terminate equine pregnancies during the first 4 wks of gestation, mares are more resistant to the abortifacient effects of pgf2α from 5-12 wks of gestation, and thus require multiple systemic injections (3-5 injections of sodium cloprostenol, 250 µg/mare q 24 h) to be effective. recently, investigators reported that intracervical administration of 500 mcg sodium cloprostenol effectively terminated 85-100% of 60-d pregnancies in 24 h. while this approach could potentially result in uterine infection, it has the advantage of requiring only a single administration. we hypothesized that systemic administration of sodium cloprostenol is equally effective to the intracervical administration. thus, the objective of this study was to compare two protocols of prenancy termination in mares by assessing maternal progesterone concentrations, fetal heart rate, and time from treatment to loss of fetal heartbeat. light breed pregnant mares (60 d of gestation) were randomly assigned to receive cloprostenol intramuscularly (500 µg, im, q 12 h, n=4) or intracervically (500 µg extended in 10 ml of saline solution, q 48 h, n=5).1 immediately after intracervical cloprostenol infusion, the cervix was manually dilated as previously described. immediately before cloprostenol administration, and then at 6-hour intervals, mares were assessed for pregnancy fluids appearance and fetal heart rates using a sectorial convex rectal transducer (5.5 mhz ivo ibex). plasma samples were collected immediately (0h) before and after (1 h, 6 h, 12 h, 24 h, and 48 h) cloprostenol administration for progesterone determination with an immunoassay. statistical analyses were performed with anova (one-way and repeated measures), significance was set as p<0.05. there were no differences in time from administration of cloprostenol to loss of fetal heartbeat (intramuscular 31.5 ± 6.1 h, range 18-48.5 h; intrauterine 40.2 ± 4.3 h, range 23.5-48 h), in fetal heart rate preceding death, or in maternal plasma progesterone concentrations (p>0.05). progesterone concentrations decreased rapidly after either route of cloprostenol administration (p<0.05). by 48 h after treatment, only two mares in each group had progesterone concentrations below 1 ng/ml. cloprostenol is known to present fewer side effects (e.g., colic-like signs) than dinoprost, and negligible side effects (sweating, discomfort, and loose manure) were observed after either route of drug administration in this study. the mean time to abortion with intrauterine administration of cloprostenol observed here was longer than the seminal report1 but similar to a recent study by the same authors.2 it is unknown whether this difference would be observed in a larger number of treated mares. regardless, our results suggest that both intrauterine and systemic methods of cloprostenol administration are equally effective to terminate 60-day equine pregnancies. keywords: abortion, early fetal loss, prostaglandin f2α, horses references 1. aguilar j, luzuriaga i, casale p, et al: transcervical administration of pgf2α analog to interrupt gestation in mares. reprod domest anim 2012;47(suppl 4):416-613. 2. cuervo-arango j, aguilar jj, vettorazzi ml, et al: ecg concentrations, luteal structures, return to cyclicity, and postabortion fertility in embryo transfer recipient mares. theriogenology 2015;84:1003-1013. clinical theriogenology • volume 10, number 3 • september 2018 342 funding opportunities at the national institute of food and agriculture: program priorities and tips for successful grant writing funding opportunities at the national institute of food and agriculture: program priorities and tips for successful grant writing mark a. mirando, adele m. turzillo u.s. department of agriculture, national institute of food and agriculture, institute of food production and sustainability, division of animal systems, washington, d. c. abstract the national institute of food and agriculture is the agency within the united states department of agriculture that provides extramural funding for competitive grants in research, education and extension. several grant programs exist in this agency that support both basic and applied research in bovine reproduction, the most notable of which is the animal reproduction program area priority within the agriculture and food research initiative. other programs within this initiative, while not dedicated solely to work in reproductive biology, also include opportunities for work on reproduction. the animal health and disease program area of this initiative provides funding for research on infectious diseases of the reproductive tract. the critical agriculture research and extension and the exploratory research program area priorities are two unique opportunities that offer funding for work in all areas in the food and agricultural sciences, including reproduction in cattle. opportunities for education support at the agency include the predoctoral fellowships, postdoctoral fellowships, and research and extension experiences for undergraduates program, as well as the national needs graduate and postgraduate fellowship grants, higher education challenge grants and higher education multicultural scholars programs. to stimulate technological innovations in the private sector, small business innovation research program competitively awards grants to qualified small businesses to address important problems in agriculture. to address veterinary shortage situations, especially those in food animal medicine, the veterinary medicine loan repayment program helps qualified veterinarians offset a significant portion of debt incurred in pursuit of their veterinary degrees in return for service in designated high-priority veterinary shortage areas. the veterinary services grant program provides funding for education, extension, and training in veterinary medicine of agricultural animals, as well as grants to enhance rural veterinary practices. most federal grant programs are highly competitive, so skill and experience in applying for grants is needed to achieve success in securing grant funding. recommendations for preparation of a meritorious grant proposal include careful reading of the program announcement, solicitation or request for applications to fully understand the scope, eligibility and requirements of the program; writing the grant proposal well in advance of the submission deadline to allow time for thoughtful preparation, critique by an experienced colleague, revision, and proof reading; and understanding the review process and criteria by which the proposal will be evaluated. keywords: beef, dairy, cattle, reproduction, federal funding, competitive grant, veterinary introduction the food, conservation, and energy act of 2008 (public law 110-246; i.e., 2008 farm bill) authorized the creation of the national institute of food and agriculture (nifa) from its predecessor agency (the cooperative state research, education and extension service) within the u.s. department of agriculture. nifa is authorized by the u.s. congress to provide extramural competitive and capacity grant funding for research, education and/or extension projects in the food and agricultural sciences. more than 30 different competitive grant funding opportunities are offered by nifa annually. several of these programs support work in bovine reproduction; however, each program has specific programmatic priorities, eligibility restrictions, and other requirements that may be statutory and/or specifically designed by nifa to target national needs or prevent duplication. this paper briefly reviews the priorities, eligibility restrictions and other requirements for these opportunities. some important strategies for writing grant proposals to enhance success in obtaining competitive grants from nifa and other funding agencies are also discussed. clinical theriogenology • volume 10, number 4 • december 2018469 https://www.gpo.gov/fdsys/pkg/plaw-110publ246/pdf/plaw-110publ246.pdf https://nifa.usda.gov/ as do most federal grant funding agencies, nifa uses "application" as the official term for grant proposal, and these two terms will be used interchangeably during the subsequent discussion. nifa and most other federal funding agencies require submission of grant applications electronically through www.grants.gov. submission of a grant application requires several additional steps beyond downloading the funding opportunity announcement (foa) or request for applications (rfa, sometimes referred to as request for proposals or rfp) that solicits grant applications. these steps include completing the official application package specifically associated with the foa; obtaining a data universal numbering system (duns) number from dun and bradstreet; and registering via the system for award management to conduct business with the federal government. all of these steps must be completed prior to electronic submission of a grant application to www.grants.gov. the agriculture and food research initiative (afri) competitive grant program the afri program was initially authorized in the 2008 farm bill to replace the national research initiative, which was in existence since 1991. the 2014 farm bill (agricultural act of 2014, public law 113-79) subsequently reauthorized the afri program to competitively award research, education and extension grants in six broad priority areas of agricultural and food science. these priority areas are: a) plant health and production and plant products; b) animal health and production and animal products; c) food safety, nutrition, and health; d) bioenergy, natural resources, and environment; e) agriculture systems and technology; and f) agriculture economics and rural communities. in 2018, nifa reorganized the afri program into three rfa (see https://www.nifa.usda.gov/afri-request-applications) that address the six farm bill priority areas. these rfa are the foundational and applied science rfa, the sustainable agricultural systems rfa, and the education and workforce development rfa. each of these rfa provides opportunities for research, education and extension projects in bovine reproduction, as discussed subsequently. afri animal reproduction program area priority included within the animal health and production and animal products priority area of the afri foundational and applied science rfa is the animal reproduction program area priority. this program area priority has been in existence in various forms since before the beginning of the national research initiative and has undergone substantial modification over that time.1 it currently solicits basic and applied research projects on molecular, cellular, genomic, genetic and whole animal aspects of reproductive biology to improve reproductive efficiency or enhance reproductive management of agricultural animals, including cattle. projects are solicited in four broad priority areas: a) gonadal function, including production, function, and preservation of gametes; b) hypothalamic-pituitary axis; c) embryonic and fetal development, including interaction between the conceptus and its uterine environment; and d) microbiome of the reproductive tract. these priorities were developed based, in part, on input from stakeholders.2 collectively, these three priorities are intended to span the entire spectrum of reproductive biology in farm animals. grant types solicited by the animal reproduction program area priority are standard grants, conference grants, and food and agricultural science enhancement (fase) grants (i.e., strengthening standard grants, new investigator grants, strengthening conference grants, seed grants, equipment grants, and sabbatical grants). whereas eligibility for most afri grants is very broad and includes almost all entities within the u.s., the intent of the fase program is to strengthen and build capacity. therefore, institutional and individual eligibility for fase grants is subject to specific restrictions. a detailed description of all afri grant types, including eligibility requirements, is available at https://nifa.usda.gov/resource/afri-request-applications-resources. description of the fase program can be accessed at https://nifa.usda.gov/afri-fase-epscor-program. other afri program area priorities within the foundational and applied science rfa although not dedicated solely to work in reproductive biology, other programs within afri also include opportunities for work on reproduction. for example, the afri animal health and disease program area priority supports projects focused on diseases of livestock and aquaculture species, clinical theriogenology • volume 10, number 4 • december 2018 470 http://www.grants.gov/ http://www.dnb.com/ https://sam.gov/portal/sam/#1 https://sam.gov/portal/sam/#1 http://www.grants.gov/ https://nifa.usda.gov/program/agriculture-and-food-research-initiative-afri https://www.gpo.gov/fdsys/pkg/plaw-113publ79/pdf/plaw-113publ79.pdf https://www.nifa.usda.gov/afri-request-applications https://nifa.usda.gov/funding-opportunity/agriculture-and-food-research-initiative-foundational-applied-science-program https://nifa.usda.gov/funding-opportunity/agriculture-and-food-research-initiative-foundational-applied-science-program https://nifa.usda.gov/resource/afri-request-applications-resources https://nifa.usda.gov/afri-fase-epscor-program including those affecting the reproductive tract with impacts on fertility. for cattle, these include metritis, brucellosis, leptospirosis, infectious bovine rhinotracheitis, bovine viral diarrhea, vibriosis and trichomoniasis. the critical agriculture and research and extension (care) program area priority solicits projects that integrate late-stage applied research with extension to quickly yield solutions to local, regional, or national problems that can be rapidly implemented by agricultural producers. the exploratory research program area seeks innovative, proof-of-concept projects in areas not previously addressed or where a novel approach could result in high impact breakthroughs. finally, the program titled "dual purpose with dual benefit: research in biomedicine and agriculture using agriculturally important domestic animal species" is an interagency program offered jointly by nifa and the eunice kennedy shriver national institute of child health and human development. the foa for this program is posted on the website of the national institutes of health, and nifa's support for this program is derived from the foundational and applied science program. the scientific priorities for the dual purpose with dual benefit program include assisted reproductive technologies, developmental programming, stem cell biology, metabolism related to obesity and adiposity, and infectious reproductive diseases. the program is entering its ninth and final year of existence, and the deadline for submission of applications was september 27, 2018. afri sustainable agriculture systems rfa the sustainable agriculture systems rfa is new in afri in 2018. it solicits large, systems-level coordinated agricultural projects (cap) with budgets up to $10 million for 5 years in duration. priorities for this program include increasing agricultural productivity; optimizing water and nitrogen use efficiency; protecting yield losses from stresses, diseases, and pests; reducing food-borne diseases; and advancing development of biobased fuels, chemicals, and coproducts. thus, a project including bovine reproduction, within the broader context of dairy or beef production systems, could fit the priorities of sustainable agriculture systems program. grant applications submitted to the sustainable agriculture systems program area must be fully integrated. the nifa definition of an integrated project is one that includes and integrates in a meaningful way at least two of the three components of the agricultural knowledge system (i.e., research, education and extension).3 for cap grant applications submitted to the sustainable agriculture systems program area, all three components of the agricultural knowledge system are required. a more detailed description of the cap grant and integrated project types can be found at https://nifa.usda.gov/resource/afri-request-applications-resources. afri education and workforce development rfa several opportunities exist within the afri education and workforce development rfa for educational projects on bovine reproduction. preand postdoctoral fellowships are offered to support scholarly training within all six of the afri farm bill priority areas, including animal health and production and animal products.4 applications are submitted by the individual preor postdoctoral scholar, typically through the student's institution. up to two years of funding can be requested to support student projects that are research, education or extension focused. projects may also be integrated. eligibility for afri fellowships is restricted to u.s. citizens, nationals and permanent residents. additional eligibility restrictions are based on progress toward completion of the doctoral degree for predoctoral fellowships and time after completion of the doctoral degree for postdoctoral fellowships; further details are clearly described in the rfa. the education and workforce development rfa also provides opportunities for undergraduate student fellowships in the research and extension experiences for undergraduates (reeu) program area. the reeu program area solicits applications from institutions of higher education to provide substantial experiential learning opportunities in research and extension for undergraduate students. as with the other program areas in this rfa, the reeu program area is not restricted to any one discipline within the food and agricultural sciences but broadly covers all disciplines; however, grant applications can emphasize specific disciplines and sub-disciplines, such as bovine reproduction. clinical theriogenology • volume 10, number 4 • december 2018471 https://grants.nih.gov/grants/guide/pa-files/par-16-366.html https://grants.nih.gov/grants/guide/pa-files/par-16-366.html https://nifa.usda.gov/funding-opportunity/afri-sustainable-agricultural-systems-competitive-grants-program https://nifa.usda.gov/resource/afri-request-applications-resources https://nifa.usda.gov/funding-opportunity/agriculture-and-food-research-initiative-education-workforce-development other higher education competitive grant programs nifa offers several funding opportunities that support education in the food and agricultural sciences, including education focused on animal production and health. each of these programs can support educational programs on cattle reproduction. the national needs graduate and postgraduate fellowships grants program provides grants to support a group of graduate and postgraduate fellowships, typically at a single institution.4 a major difference between this program and afri predoctoral fellowships is that the application is submitted by a team of faculty mentors instead of the individual predoctoral scholar. a second major difference is that a national needs graduate and postgraduate fellowships grant can support several fellowships on a single grant, whereas each afri predoctoral fellowship supports the scholarly activity of only one individual student. the higher education challenge grants program focuses on improving formal baccalaureate, master's, or doctor of veterinary medicine (dvm) degree programs and curricula in the food and agricultural sciences. projects supported by this program will: 1) address a state, regional, national, or international educational need; 2) involve a creative or non-traditional approach toward addressing that need that can serve as a model to others; 3) encourage and facilitate better working relationships in the university science and education community, as well as between universities and the private sector, to enhance program quality and supplement available resources; and 4) result in benefits that will likely transcend the project duration and usda support. the purpose of the multicultural scholars program is to increase multicultural diversity of the professional and scientific workforce in the food and agricultural sciences. this program funds competitive grants to colleges and universities to provide scholarships to support recruitment, retention, mentoring, and training of multicultural scholars in higher education programs leading to either a baccalaureate degree within food and agricultural science disciplines or to the dvm degree. these include animal science, dairy science, and veterinary degree programs. small business innovation research (sbir) program the sbir program provides grants to qualified small businesses to support research related to important scientific problems and opportunities in agriculture that could lead to significant public benefits. a major goal of this program is to increase private sector commercialization of innovations derived from usda-supported research and development efforts. the sbir program offers both phase i and phase ii grants. funding for phase i projects supports research to prove the scientific or technical feasibility of an approach or concept. successful phase i awardees may apply for phase ii funding to support continued research that may lead to the development of a new product based upon results obtained during phase i. topic areas in the phase i solicitation include animal production and protection, with emphasis on three priorities: 1) improving production efficiency (including improved fertility of livestock); 2) improving the safety and/or quality of end products derived from animals; and 3) improving animal health and well-being. thus, the sbir program has supported and will continue to support projects focused on bovine reproduction. veterinary medicine loan repayment and veterinary services grant programs the veterinary medicine loan repayment program (vmlrp), authorized by the national veterinary medical services act (public law 108-161), aims to help qualified veterinarians offset a significant portion of the debt incurred in pursuit of their dvm degree. in return for loan repayment assistance of up to $25,000 per year, awardees agree to provide veterinary services in certain highpriority, nifa-designated veterinary shortage situations (https://nifa.usda.gov/vmlrp-map) for three years. vmlrp awards are made competitively, and priority is given to agreements with veterinarians for the practice of food animal medicine. the veterinary services grant program was authorized by the 2014 farm bill as a companion program to the vmlrp to incentivize service in veterinarian shortage situations. this program expands on the educational loan repayments offered under the vmlrp by offering competitive grant funding for clinical theriogenology • volume 10, number 4 • december 2018 472 https://nifa.usda.gov/program/national-needs-graduate-and-postgraduate-fellowship-grants-program-funding-opportunity-nnf https://nifa.usda.gov/funding-opportunity/higher-education-challenge-hec-grants-program https://nifa.usda.gov/funding-opportunity/higher-education-multicultural-scholars-program-msp https://nifa.usda.gov/program/small-business-innovation-research-program-sbir https://nifa.usda.gov/funding-opportunity/small-business-innovation-research-program-phase-i https://nifa.usda.gov/funding-opportunity/small-business-innovation-research-program-phase-ii https://nifa.usda.gov/program/veterinary-medicine-loan-repayment-program https://www.congress.gov/108/plaws/publ161/plaw-108publ161.pdf https://nifa.usda.gov/vmlrp-map https://nifa.usda.gov/program/veterinary-services-grant-program education, extension and training programs with an emphasis on food animal medicine and food safety. projects may include veterinary telemedicine and other distance-based education, and should focus on providing veterinary students, veterinarians, veterinary technicians, and/or other health professionals the skills needed to relieve veterinarian shortage situations or support veterinary practices public health. grants are also available to support establishment or expansion of veterinary services to relieve veterinarian shortage situations in rural areas across the u.s. tips for successful grant writing obtaining a grant from a funding agency is a highly competitive process. available funding is limited and grant application submission rates are high such that most federal funding agencies are generally only able to fund 10 to 20% of the applications submitted. a very common situation is one in which the amount of funding needed to support all of the high quality proposals submitted to a program far exceeds the amount of funding available. whereas poor ideas will not be funded regardless of how well they are presented in a grant application, many excellent ideas go unfunded because they are not well packaged and presented.5,6 thus, improving the packaging and presentation of the good ideas in a grant application can increase its likelihood for success, a process often referred to as grantsmanship.5,6 given the highly competitive nature of the granting arena, developing skill and gaining experience in grant proposal writing is paramount to achieving success. this paper will highlight a few of the many strategies and recommendations for enhancing success at writing grant proposals, regardless if the application is submitted to nifa or another funding agency. the first strategy is to carefully read the foa, rfa, rfp, program announcement or whatever the solicitation may be called. these foa are a treasure trove of valuable information such as the program or agency's funding priorities, eligibility restrictions, proposal preparation guidelines, submission deadlines, funding restrictions, review criteria, and contact information of program staff. all of this information is critical to proposal preparation. for example, a clear understanding of the program's funding priorities allows the applicant to determine if his/her project is highly relevant (and thus more likely to form the basis for a successful proposal) or lies on the fringes with less relevance, thereby allowing the applicant to avoid the scenario of “trying to force a square peg into a round hole”. knowing the eligibility restrictions can prevent futile submission to a program for which one is ineligible. other guidelines in the solicitation will facilitate better planning and preparation of an application, including instructions for developing an appropriate budget and organizing the application according to the recommended or required format. a second important recommendation is to start preparing the grant application early and well ahead of the submission deadline.5,6 this includes organizing the project team, developing a timeline for proposal preparation, obtaining critical feedback on a penultimate draft of the proposal from knowledgeable colleagues, and allowing sufficient time to complete administrative procedures at your institution to permit submission of a mistake-free application by the submission deadline. for new programs that are in their first year of solicitation, information describing program priorities may be unavailable or insufficient to begin proposal preparation ahead of rfa release. however, for many other programs the priorities are fairly consistent from year to year or change only slightly. experienced and successful proposal writers recognize this and begin early! work on applications to these programs can and should be initiated well before the anticipated release of the rfa. in contrast, waiting until just a month or two before the submission deadline limits the time available for serious polishing and may preclude the opportunity to obtain critical feedback from experienced and successful colleagues. some of the most frequent criticisms from reviewers of unsuccessful proposals are that the application appeared to be prepared in a rush, was not thoughtfully assembled, and was not carefully proofread. starting the writing process early also facilitates on-time submission of the proposal, ideally well ahead of the submission deadline. at most academic institutions, the sponsored projects office requests receipt of the final proposals from the applicants up to two weeks before the submission deadline. this time is required to add additional information to the proposal and carefully submit multiple applications from their institution to meet the same submission deadline. electronic submission of applications during clinical theriogenology • volume 10, number 4 • december 2018473 the last few hours or minutes before the deadline increases the likelihood that mistakes will be made during the submission process that lead to an application being rejected. early submission well ahead of the deadline is also preferred because if the applicant finds important technical or scientific errors in the proposal, they can request withdrawal of the initial application followed by resubmission (ahead of the deadline) of a revised application. it was suggested6 that proposals should not be submitted too early or too late relative to the deadline because proposals are often reviewed in the order submitted, and being reviewed in the middle of a review panel meeting provides a more favorable outcome than review towards the beginning or end. however, our experience indicates that order of review does not influence final ranking of a proposal (r = 0.168 0.100, mean = 0.038 for 4 panels each reviewing 75 to 119 proposals). thus, the hypothesis that it is better to submit an application so that it arrives in the middle of the pack is not tenable and that suggestion should be rejected. the best advice is to start preparing the grant application early and then submit it well before the deadline! two other recommendations that go hand-in-hand are to understand the review criteria and learn about the review process. most funding agencies publish the criteria by which applications will be evaluated, either in the solicitation or on their website. at nifa, all rfa include the specific review criteria for each program. understanding the review criteria provides valuable insight into the required components of the proposal; how to best organize ideas, objectives, and expected outcomes; and components or aspects of the proposal that will be weighted more heavily than others by the reviewers. learning the review process requires greater effort than understanding the review criteria but also can be extraordinarily enlightening. the review process used by nifa has been described previously7 and is available on the nifa website (https://nifa.usda.gov/resource/nifa-peer-review-process-competitivegrant-applications). moreover, funding agencies are continuously looking for expert peer reviewers to provide thoughtful, detailed and critical evaluations of proposals upon which to base funding decisions. serving as either an ad hoc reviewer of a few proposals or as a review panel member to review a larger cohort of proposals can provide an opportunity to gain significant insight into the review process while contributing an important service to the greater scientific community. whereas service as an ad hoc reviewer may require several hours to review each proposal and provide detailed written reviews (which are typically submitted electronically), service on a review panel involves substantially more time and effort. after reviewing each assigned proposal individually and submitting written reviews, panelists typically devote several days to discussing proposals as a group, either virtually or in person at the funding agency's location. each review panelist may be asked to evaluate as many as 10 to 20 applications. this provides an invaluable learning opportunity because there is typically a wide range in the quality of proposals submitted to any program. a first-time panelist quickly observes what to do and what to avoid when preparing a proposal, and it becomes readily apparent how poor packaging of an otherwise good idea can reduce its chance of garnering funding. most agencies maintain an extensive database of expert reviewers, derived in part from previous applicants to the agency's funding opportunities. for experts who wish to volunteer their service as a reviewer but have no previous history with a specific agency, addition to a reviewer database may be possible by contacting the program staff who manage programs for which they have appropriate expertise. a brief email highlighting how the individual's expertise aligns with the program's priorities along with a detailed curriculum vitae will assist the program staff in adding you to the reviewer database. program staff usually value this information and appreciate the volunteering of service as a reviewer. first-time reviewers should keep in mind, however, that program staff will often evaluate a new reviewer's ability to provide critical and thoughtful reviews by requesting an ad hoc review or two before inviting them to serve on a review panel. most agencies also exclude applicants to the program from reviewing proposals during that funding cycle in order to avoid conflicts of interest, either actual or perceived. the strategies and recommendations discussed herein are just a few of those that can be considered to improve the quality of a grant application; many other important suggestions are excluded from this discussion due to space limitations. recommended additional resources on grantsmanship are available on the nifa website (https://nifa.usda.gov/resource/nifa-peer-review-process-competitiveclinical theriogenology • volume 10, number 4 • december 2018 474 https://nifa.usda.gov/resource/nifa-peer-review-process-competitive-grant-applications https://nifa.usda.gov/resource/nifa-peer-review-process-competitive-grant-applications https://nifa.usda.gov/resource/nifa-peer-review-process-competitive-grant-applications grant-applications) and elsewhere.5,6 collectively, these resources provide a myriad of suggestions which, when utilized carefully and effectively, can improve the quality of grant applications and enhance the likelihood of success. summary and conclusions nifa is the agency within usda that provides extramural funding for competitive grants in the food and agricultural sciences. several nifa competitive grant programs support work on various aspects of reproduction in beef and dairy cattle. as with most federal grant programs, nifa grant programs are highly competitive; thus, skill and experience in grant writing are essential to secure grant funding. whereas poor ideas are rarely funded, many excellent ideas are not funded because they are poorly packaged into a weak grant proposal. improving the quality of the proposal through superior grantsmanship can greatly enhance the likelihood of garnering funding. acknowledgments the findings and conclusions in this publication have not been formally disseminated by the u.s. department of agriculture and should not be construed to represent any agency determination or policy. references 1. mirando ma: the national research initiative competitive grants program in animal reproduction: changes in priorities and scope relevant to u.s. animal agriculture. j anim sci 2007;85 (e. suppl.):e11-e15. doi:10.2527/jas.2006-506. 2. mirando ma, hamernik dl: funding priorities in animal reproduction at the united states department of agriculture's cooperative state research, education, and extension service. biol reprod 2006;74:459-462. doi:10.1095/biolreprod.105.048686 3. mirando ma, bewley jm, blue j, amaral-phillips dm, corriher va, whittet km, arthur n, patterson dj: reinventing extension as a resource what does the future hold? j anim sci 2012;90:3677-3692. doi:10.2527/jas.20115074. 4. mirando ma, turzillo am, ali r: funding opportunities for phd programs in animal, dairy and poultry science at the usda national institute of food and agriculture. j anim sci 2015;93 (suppl. s3):260. 5. ogden te: towards more effective grantsmanship. in: introduction to molecular and cellular research. washington (dc), the endocrine society; 1992. p. 133-134. 6. mackenzie dr, angle js: principles of grantsmanship: a manual on organizing a competitive grant proposal. college of agriculture and natural resources, university of maryland, college park. 1995. 7. jacobs-young c., mirando ma, palmisano a:usda-csrees national research initiative support for agricultural research. in: loebenstein g, thottappilly g, editors. agricultural research management. new york: springer publishing; 2007. p. 259-270. clinical theriogenology • volume 10, number 4 • december 2018475 https://nifa.usda.gov/resource/nifa-peer-review-process-competitive-grant-applications clinical theriogenology 2022; 14: 334 ovulation induction in caribbean jennies in the tropics hilari french, lorenzo segabinazzi, ava kent, shelby nester, erik peterson, robert gilbert ross university school of veterinary medicine, basseterre, st. kitts, west indies abstract the population of some endangered donkey breeds in tropical regions has declined recently. therefore, there is an increasing interest to improve the reproductive management of these animals. ovulation induction agents are often used in equine practice to optimize reproduction techniques. however, limited information exists to support its use in donkeys. therefore, we compared 2 ovulation-inducing agents (commonly used in equine practice) to hasten ovulation in caribbean jennies in the tropics. five cycles of 8 caribbean jennies were used. jennies were monitored via transrectal ultrasonography and in 4 estrous cycles they had ovulation induced with either 2,500 iu of hcg or 1.8 mg of deslorelin acetate when a follicle measuring between 27 30 mm or > 30 mm in diameter was diagnosed. in the control-assigned cycle, jennies were not treated with hormones and had spontaneous ovulation. follicle diameters affected (p < 0.05) the interval to ovulation. when given with a follicle > 30 mm, either treatment reduced (p < 0.05) the interval to ovulation. a higher percentage of jennies ovulated within 48 hours after hcg or deslorelin acetate with > 30 mm follicle (p < 0.05), and with deslorelin acetate with 27 30 mm follicle (p < 0.05) than in the control-assigned cycle. in conclusion, caribbean jennies in tropics with > 30 mm follicle responded to ovulation induction with hcg or deslorelin acetate, with ovulation occurring within 48 hours. keywords: donkey, breeding, mule, hcg, gnrh analog introduction interest in donkey production has increased worldwide. in the americas, donkeys have been used for agricultural work and for producing top mules whereas in the orient, donkeys are used to produce milk for cosmetic manufacturing and for meat.1,2 in addition, many donkey breeds have been threatened with extinction.3-5 therefore, there is an increasing interest to improve the reproductive management in donkey species. guidelines developed for horses have been used in donkeys over the years for reproductive management. one of the most common techniques applied in horse breeding programs is ovulation induction to manipulate the reproductive cycle and optimize breeding management. this technique improves artificial insemination efficiency, particularly in advanced assisted reproduction techniques, as it predicts ovulation timing. although this procedure is routinely used in horses, limited information exists to support its use in donkeys. recently, some studies have reported the efficacy of gnrh analogs (e.g., deslorelin acetate, buserelin, and histrelin acetate) and human chorionic gonadotropin (hcg) to hasten ovulation in jennies.6-9 although gnrh analogs and hcg have similar efficacy to hasten ovulation in jennies,6,9 these hormones act differently on the ovaries. human chorionic gonadotropin binds to lh receptors in granulosa cells directly to induce ovulation10 whereas gnrh analogs induce endogenous release of lh by the adenohypophysis.11,12 in mares, hcg has been reported to have a shorter interval to induce ovulation than gnrh analogs;13 however, it has not been observed in brazilian northwest9 or martina franca6 jennies in the subtropical and temperate environment, respectively. to the best of the authors’ knowledge, there has been no study evaluating ovulation-inducing agents in jennies in tropical regions. although there are only a few studies on follicular dynamics of jennies, they differ in results that may be attributed to the differences in breeds and location of the donkeys.14-16 these differences conceivably could affect response to ovulation inducing agents. additionally, the population of donkeys in some countries in the tropical zone (e.g., brazil, ecuador, and colombia) has declined that may lead these animals to be threatened with extinction.3 therefore, we compared deslorelin acetate, a gnrh analog, and hcg to hasten ovulation in caribbean jennies in the tropical zone. materials and methods study was approved by the institutional animal care and use committee (protocol 15.12.032). it was conducted from sepclinical theriogenology 2022; 14: 335 tember 2018 to april 2019 at ross university school of veterinary medicine, basseterre, st. kitts, west indies (17°18′n 62°44′w), close to the equator (tropical zone). five estrous cycles of 8 reproductively sound, nonpregnant caribbean jennies, ages 3 12 (± 5) years were enrolled. jennies were housed in outdoor grass paddocks under natural light, fed freshly-cut new guinea grass (megathyrsus maximus) with free access to water. jennies were examined via transrectal ultrasonography (5 mhz linear transducer, sonosite, bothell, wa) to monitor ovarian activity and follicular growth. five days after detection of a corpus luteum in 1 ovary, jennies received intramuscular prostaglandin f2α (pgf2α; 0.25 mg cloprostenol sodium, estrumate®, merck animal health, madison, nj) for induction of estrus. four days after pgf2α treatment, jennies started to be monitored daily. five successive estrous cycles of each jenny was studied and randomly assigned to each of 5 treatment groups in a crossover design. in the control-assigned cycle, jennies were not treated with hormones and had spontaneous ovulation, whereas the other 4 estrous cycles were used to evaluate the ovulation-inducing treatment at various follicular sizes. jennies had ovulation induced with either intravenous 2,500 iu of hcg (chorulon, msd animal health, summit, nj) or 1.8 mg of intramuscular deslorelin acetate (sucromate®, thorn bioscience llc, louisville, ky) as follows: hcg was given when a follicle measuring between 27 and 30 mm (g1) or > 30 mm (g2) in diameter was diagnosed; deslorelin acetate was given when a follicle measuring between 27 and 30 mm (g3) or > 30 mm (g4) in diameter was diagnosed. starting 24 hours posttreatment, jennies were monitored every 6 hours via transrectal ultrasonography until ovulation. number of jennies ovulating within 48 hours, the interval between ovulation-inducing agent treatment and ovulation, preovulatory follicular size, length of estrous cycle, and endometrial edema (0: no edema, 1: mild edema, 2: moderate edema, 3: evident edema, 4: exacerbated edema)17 were recorded. jennies did not receive an injection of pgf2α to bring them back to estrus between treatments. all jennies were submitted to every treatment. data analyses graphpad prism 8.0.1. (graphpad software, san diego, ca) was used. data are summarized as mean ± sd. the gaussian distribution of the data was evaluated using the kolmogorov-smirnov normality test. mixed model and tukey’s posthoc test were used to compare the interval between treatment of an ovulation-inducing agent, follicular diameters, number of follicles > 27 mm, and days of the cycle. edema scores were considered categorical variables and analyzed with nonparametric tests (kruskal-wallis test followed by dunn’s test). chi-square test was used to compare the success of the ovulation-inducing treatments (the percentage of jennies ovulating within 48 hours after treatment or > 48 hours after treatment, indicating unsuccessful induction) and between intervals until ovulation in control and treated cycles. multiple linear regression model was used to evaluate the relationship between ovulation and endometrial edema score. ovulation was evaluated by a logistic regression model, where ovulation was the dependent variable and edema score the explanatory variable. correlations between ovulation and edema score were categorized as high (r ≥ 0.7), moderate (0.5 ≤ r < 0.7), and poor (r < 0.5). significance was set at p ≤ 0.05 for all tests and p < 0.10 and > 0.05 as a statistical trend. results follicular diameter at ovulation induction and the length of the estrous cycle, were similar (p > 0.05) among groups. however, jennies in g3 tended to have smaller (p = 0.068) follicular diameter 12 hours before ovulation was detected compared to control cycle whereas this parameter was similar (p > 0.1) to control cycle in other groups. more jennies in the g3 group (gnrh, 27 30 mm) ovulated (p < 0.05) within 48 hours compared to control-matched group, but not different (p > 0.05) than g1 (hcg, 27 30 mm; table). jennies in g1 group tended to have more ovulations (p = 0.056) within 48 hours after treatment than control group. a higher percentage of jennies had ovulation diagnosed (p = 0.015) within 48 hours in both g2 and g4 groups (hcg and gnrh, > 30 mm, respectively) compared to control-assigned cycle. however, there was no difference (p > 0.05) among treated groups in the percentage of jennies ovulating within 48 hours (table). the mean interval between the detection of a predefined follicular size and ovulation was longer (p < 0.001) in jennies with 27 30 mm follicles with the respective matched cycle (table). also, in g2 and g4 groups, this interval was shorter (p < 0.05) by treatments compared to control-matched cycle (table). additionally, there were no differences in the number of follicles > 27 mm, the number of ovulations per cycle, or the endometrial edema score among assigned cycles (table). all jennies had single ovulation throughout the experimental cycles, and no jennies failed to ovulate. edema score during the predefined follicular sizes and ovulation were not associated events in these jennies (r = 0.0186). discussion we evaluated hcg and deslorelin acetate use to induce ovulation in caribbean jennies in the tropics. although ovulation-inducing agents (e.g., hcg and gnrh analogs) have been described to induce ovulation in jennies,6-9,18 there was no study assessing the efficacy of these hormones to hasten ovulation in jennies in a tropical environment. recently, some countries in the tropical zone (e.g., brazil, ecuador, and colombia) have reported a decrease in herd number of donkeys, and therefore, there has been an increase in interest to improve the breeding management of this species to prevent threatened extinction.3 it is noteworthy that none of the jennies failed to ovulate during the study that was conducted in the tropics at ~ 17° north during the winter period (september 2018 april 2019). although a case of hemorrhagic anovulatory follicle has already been reported in caribbean jennies,30 donkeys’ ovulation seem not to be affected by seasons since this species display less seasonality than horses in subtropical and temperate zones.1,15 however, the follicular dynamics and reproductive management of this species are still not well described.14,15,19 in a study by our group,20 the follicular dynamics of caribbean jennies in tropics were described which contradicted the existing information regarding the estrous cycle in this species in temperate and subtropical zones.14,15,19 one follicular wave clinical theriogenology 2022; 14: 336 per cycle was described in egyptian jennies,14 2 or 3 waves in jennies in ethiopia,19 and we characterized 2 waves in caribbean jennies.20 we speculated that these differences are associated with the breed, origin, and location of the animals in each study. in the first study of our group, the maximum follicular diameter before ovulation in caribbean jennies was 34.64 ± 2.91 mm,20 prompting us to test both intervals (27 30 and > 30 mm follicle) for ovulation induction. although the diameter of preovulatory follicles have a large variation based on donkey’s breed (30 45 mm),6,7,9,18,21-23 in a brazilian study assessing the efficacy of histrelin acetate to hasten ovulation in northwest jennies with various follicle diameters, follicles 29 32 mm had the most satisfactory response.7 in another study, the same authors reported that hcg and histrelin acetate had similar efficacy to induce ovulation in jennies with 29 32 mm follicles, with 90 and 100% of ovulation occurring between 36 48 hours after treatment, respectively.9 similar to other authors description, in the present study, the interval between detection of > 30 mm follicle and ovulation was reduced by treatments (hcg or deslorelin acetate). however, when jennies had ovulation induction treatment with 27 30 mm follicle, there was no change in this interval compared to the control-matched cycle. some jennies with 27 30 mm follicles had a potential to ovulate, but others were not able to respond to exogenous lh stimulus. it may be associated with the follicular maturation at 27 30 mm, as fewer jennies responded to the treatment (hcg, 37.5%; gnrh, 50%) with ovulation within 48 hours. immature follicles fail to respond to exogenous gonadotropins due to lack of granulosa cells’ lh receptors.24 in both hcg and deslorelin acetate groups, a higher proportion of jennies ovulated within 48 hours after ovulation induction, implying that some jennies with follicles under 30 mm may have ovulation potential.7 however, ovulation induction using hcg or deslorelin acetate was most efficient in caribbean jennies with > 30 mm follicles. deslorelin acetate induced ovulation within 48 hours more consistently than hcg at a follicular diameter between 27 and 30 mm in jennies in the present study. this lack in response to hcg is interesting in caribbean jennies. the hcg is a glycoprotein with lh-like biological activity that binds directly to lh receptors in granulosa cells to induce ovulation,10 whereas gnrh analogs act at the anterior pituitary to induce the secretion of endogenous lh.11,12 therefore, hcg might have a faster response and might be more efficient than gnrh analog, as hcg does table. variables measured at several predefined follicular sizes in jennies with spontaneous ovulation (control) and in jennies that had the ovulation induced with hcg or deslorelin acetate (gnrh) variables estrous cycle assigned follicle diameter 27 30 mm > 30 mm mean number of follicles > 27 mm control hcg gnrh 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.3 ± 0.4 1.3 ± 0.4 mean number of ovulations per cycle control hcg gnrh 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 mean diameter of follicles with predefined follicular size (mm) control hcg gnrh 28.9 ± 0.9 28.4 ± 1.1 28.1 ± 0.9 32.1 ± 1.6 32.2 ± 1.3 31.7 ± 1.5 mean diameter of preovulatory follicle 12 hours before ovulation (mm) control hcg gnrh 37.2 ± 2.3* 34.9 ± 2.4 32.7 ± 1.9* 37.2 ± 2.3 37.0 ± 3.3 34.4 ± 1.9 mean interval from detection of predefined follicular size to ovulation (hours) control hcg gnrh 121 ± 31.1.0x 92.3 ± 40.4x 79.5 ± 37.8x 86.8 ± 28.8ya 50.3 ± 22.4yb 42.7 ± 8.7yb mean length of estrous cycle control hcg gnrh 23.8 ± 2.8 23.9 ± 3.9 22.9 ± 2.2 23.8 ± 2.8 22.5 ± 2.1 21.6 ± 2.1 median/mode of edema score at each specific time lap control hcg gnrh 0.5/0 0.5/0 0.5/0 1.5/1 1.0/1 1.0/1 percentage of jennies responding with ovulation within 48 hours control hcg gnrh 0%b* 37.5%ab* 50.0%a 12.5%b 62.5%a 75.0%a percentage of jennies with unsuccessful induction of ovulation control hcg gnrh 100% 62.5% 50.0% 87.5% 37.5% 25.5% edema score: 0, no edema, and 4, exacerbated edema. unsuccessful induction of ovulation: ovulation detected > 48 hours after administartion of induction agent. lines with different superscript capital letters (x,y) and columns with different superscript lowercase letters (a,b) represent differences (p < 0.05). columns with superscript (*) trend to be different (p < 0.10 and > 0.05). clinical theriogenology 2022; 14: 337 not need endogenous lh to induce ovulation.13 however, this was not observed in this study. it is also important to note that repeated doses of hcg may induce antibody development in mares and may reduce its effectiveness.10,25,26 although hcg antibody development in jennies is still unknown, it is an important information to take into account in advanced breeding programs, especially whenever a female jenny or mare will be bred many times in a season. jennies used in the present study were feral and had not been manipulated previously. although hcg antibody was not assessed in this study, the authors assumed that our results were not affected by hcg antibodies. one jenny in each treated cycle (hcg and gnrh) ovulated before 36 hours after treatment. we speculated that it may be associated with an early endogenous hormonal stimulus in these jennies. also, the number of follicles ≥ 27 mm was similar among groups and all jennies had single ovulation in all cycles. of interest was the observation by other authors who reported that the percentage of jennies with multiple ovulations varied between 5 to 32% depending on the breed and location;7,27-29 however, all caribbean jennies in the present study had single ovulation in all estrous cycles. it may be a specific characteristic of this donkey breed; however, it must be evaluated in a larger number of animals. it was also noted in the present study that caribbean jennies did not have an endometrial edema pattern associated with the phase of estrus that has been described by other authors.7 in mares, a pronounced endometrial edema pattern was closely related to the estrous cycle and higher secretion of estradiol by the preovulatory follicle that decreased spontaneously before ovulation.24,31 therefore, endometrial edema cannot be used to predict the best time to induce ovulation in jennies or the time close to ovulation. it is important to recognize that the present study was conducted with a limited number of jennies and estrous cycles. it is possible that if a larger number of jennies was used, different results could have been obtained. however, we feel that the results provided a good representation of the caribbean donkeys’ response in tropics to ovulation-inducing agents. we concluded that ovulation induction using hcg or deslorelin acetate in caribbean jennies in tropics is most suitable with a follicle > 30 mm. gnrh induced ovulation within 48 hours more consistently than hcg; however, at a follicular diameter of 27 or 30 mm, the effect was not as predictable as it is in mares induced at a follicular diameter of 35 mm. further studies are needed to improve the breeding management of this species in various environments. funding intramural grant by the center for intregrative mammalian research of ross university school of veterinary medicine. conflict of interest none to declare. references 1. canisso if, panzani d, miró j, et al: key aspects of donkey and mule reproduction. vet clin north am equine pract 2019;35:607642. 2. canisso if, davies morel mcg, mcdonnell s: strategies for the management of donkey jacks in intensive breeding systems. equine vet educ 2009;21:652-659. 3. carneiro gf, cavalcante lucena je, de oliveira barros l: the current situation and trend of the donkey industry in south america. j equine vet sci 2018;65:106-110. 4. cecchi f, ciampolini r, ciani e, et al: demographic genetics of the endangered amiata donkey breed. ital j anim sci 2006;5:387-391. 5. save foundation. donkey breeds in europe: inventory, description, need for action, conservation report 2007/2008. monitoring institute for rare breeds and seeds in europe. accessed in 2021. http://www.agrobio diversity.net/topic_network/donkey/ donkey/report2007_2008.pdf http://www.agrobio diversity.net/ topic_network/don 6. carluccio a, panzani s, tosi u, et al: efficacy of hcg and gnrh for inducing ovulation in the jenny. theriogenology 2007;68:914-919. 7. oliveira sn, canuto l, segabinazzi lgtm, et al: histrelin acetateinduced ovulation in brazilian northeastern jennies (equus asinus) with different follicle diameters. theriogenology 2019;136:95-100. 8. camillo f, vannozzi i, tesi m, et al: induction of ovulation with buserelin in jennies: in search of the minimum effective dose. anim reprod sci 2014;151:56-60. 9. oliveira sn, segabinazzi lgtm, canuto l, et al: comparative efficacy of histrelin acetate and hcg for inducing ovulation in brazilian northeastern jennies (equus africanus asinus). j equine vet sci 2020;92:103146. 10. wilson cg, downie cr, hughes jp, et al: effects of repeated hcg injections on reproductive efficiency in mares. j equine vet sci 1990;10:301-308. 11. meinert c, silva j, kroetz i, et al: advancing the time of ovulation in the mare with a short-term implant releasing the gnrh analogue deslorelin. equine vet j 1993;25:65-68. 12. mumford el, squires e, jochle e, et al: use of deslorelin shortterm implants to induce ovulation in cycling mares during three consecutive estrous cycles. anim reprod sci 1995;39:129-140. 13. boakari yl, ferreira jc, canesin hs, et al: influence of two ovulation-inducing agents on the pituitary response and follicle blood flow in mares. theriogenology 2017;100:95-99. 14. derar ri, hussein ha: ovarian follicular dynamics during the estrous cycle in jennies in upper egypt. vet med int 2011;860511. 15. contri a, robbe d, gloria a, et al: effect of the season on some aspects of the estrous cycle in martina franca donkey. theriogenology 2014;81:657-661. 16. miragaya mh, neild dm & alonso ae: a review of reproductive biology and biotechnologies in donkeys. j equine vet sci 2018;65:5561. clinical theriogenology 2022; 14: 338 17. mccue p, scoggin c, lindholm a: estrus. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 1716-1727. 18. oliveira jv, oliveira pvlf, melo e oña cm, et al: strategies to improve the fertility of fresh and frozen donkey semen. theriogenology 2016;85:1267-1273. 19. kebede h, lemma a, negussie h: ultrasonographic studies on ovarian dynamics and associated estrus manifestations of jennies under controlled management, ethiopia. trop anim health prod 2012;44:1965-1970. 20. ambrosia r, gilbert ro, bergfelt d, et al: follicular and luteal dynamics of the estrous cycle under tropical conditions in caribbeanbased donkeys. clinical theriogenology 2018;10:329. 21. rota a, panzani d, sabatini c, et al: donkey jack (equus asinus) semen cryopreservation: studies of seminal parameters, post breeding inflammatory response, and fertility in donkey jennies. theriogenology 2012;78:1846-1854. 22. pérez-marín cc, vizuete g & galisteo jj: embryo recovery results in hispano-arabe horse and spanish donkey breeds. livest sci 2017;206:76-81. 23. quaresma m & payan-carreira r: characterization of the estrous cycle of asinina de miranda jennies (equus asinus). theriogenology 2015;83:616-624. 24. samper jc: induction of estrus and ovulation: why some mares respond and others do not. theriogenology 2008;70:445-447. 25. siddiqui mar, gastal el, gastal mo, et al: effect of hcg in the presence of hcg antibodies on the follicle, hormone concentrations, and oocyte in mares. reprod domest anim 2009;44:474-479. 26. roser jf, kiefer bl, evans jw, et al: the development of antibodies to human chorionic gonadotrophin following its repeated injection in the cyclic mare. j reprod fertil 1979;27:173-179. 27. meira c, ferreira jcp, papa fo, et al: ovarian activity and plasma concentrations of progesterone and estradiol during pregnancy in jennies. theriogenology 1998;49:1465-1473. 28. blanchard tl, taylor ts, love cl: estrous cycle characteristics and response to estrus synchronization in mammoth asses (equus asinus americanus). theriogenology 1999;52:827-834. 29. perez-marin cc, galisteo i, perez-rico a, et al: effects of breed, age, season, and multiple ovulations on cyclic, pgf2α-induced, and postpartum estrus characteristics in spanish jennies. theriogenology 2016;85:1045-1052. 30. hughes e, ambrosia r, gilbert ro, et al: hemorrhagic anovulatory follicle in a tropical jenny. clinical theriogenology 2018;10:119-124. 31. samper jc: a review of a practitioner’s perspective on endometrial edema. pferdeheilkunde 2010;26:14-18. 5 contact jessica cowley j.cowley@auburn.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9646, http://dx.doi.org/10.58292/ct.v15.9646 review report management of urolithiasis in breeding bulls jessica cowley and richard hopper department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa abstract urolithiasis is commonly encountered and frequently a fatal disease in the ruminant species. clinical signs of urolithiasis are related to both its severity and chronicity. after complete obstruction of 24–48 h, bladder or urethral obstruction can occur. the varying urinary environments affect the type of stone development: phosphatic stones occur when cattle consume high phosphate diets; silica calculi form when grazing native grasses in the western us; and calcium stones form when cattle consume high amounts of lush clover grass or alfalfa hay. medical management is rarely successful as lone treatment but is used in conjunction with a surgical procedure. for a bull to return to reproductive function, the surgical procedure chosen must leave the urethra intact. the two procedures discussed presently are the ischial urethrostomy and tube cystostomy. keywords: bovine, urinary stones, ischial urethrostomy, tube cystostomy introduction urolithiasis is a condition affecting most veterinary species and is a significant economic disease in ruminants. fatalities from urolithiasis have been reported to range from 3 to 20%.1,2 urolithiasis is commonly associated with small ruminants or castrated males of all ruminants, but stones form in several urinary environments and breeding bulls can develop this disease as well. this article will focus on diagnosis, pathophysiology, and treatment of urolithiasis for bulls intended to return to reproductive function. clinical signs each case of urolithiasis can present with different clinical signs based on the chronicity and severity of the obstruction (figure 1). hemorrhage and necrosis of the urethral mucosa are evident at the site of obstruction, but the bladder will also show signs of mucosal trauma (figure 2). in cattle, the most common site of obstruction is the distal portion of the sigmoid flexure.3 cattle can present with acute colic-type symptoms including bruxism, kicking of the abdomen, and a wide base stance. an enlarged urinary bladder may be present and upon rectal palpation urethral pulsations can be observed. urethral pulsations can also be palpated just below the anus on midline. the chronicity of the obstruction will determine secondary effects of urolithiasis, such as urethral rupture or bladder rupture. urethral rupture occurs at the site of the obstruction, predisposed by mucosal hemorrhage and necrosis caused by the stone. urine subsequently accumulates in the subcutaneous tissues, commonly in the preputial region (figure 3). in cases of bladder rupture, the abdomen can be enlarged and filled with large amounts of blood-tinged fluid, creating a fluid wave. in cases of chronic urethral obstruction, hydronephrosis, hydroureter, and bladder wall hypertrophy are often present. pathophysiology multiple factors are involved in urolith formation; however, a requirement is high concentrations of crystalloids that aggregate to form crystals.4 urine contains substances that act as inhibitors of crystallization (mucopolysaccharides, ions, and organic acids), but when urine becomes supersaturated with crystalloids, these inhibitors become exceeded.4 the crystalloids are no longer soluble in the urine and form precipitates (i.e., supersaturation). these precipitates enlarge and form calculi as more minerals become insoluble. supersaturation of the urine is not solely responsible for stone formation. all stones increase in occurrence during winter months presumptively due to decreased water consumption.4 dehydration, or rather water deprivation, with resultant concentration of urinary minerals, appears to be a potential contributing factor in mailto:j.cowley@auburn.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9646 6 citation line: clinical theriogenology 2023, 15, 9646, http://dx.doi.org/10.58292/ct.v15.9646 the development of all types of uroliths.4,5 other less common factors that have been reported to increase urolith formation include hypovitaminosis a, hypervitaminosis d, and estrogenic intake.2,6,7 exact etiologies of stone formation vary with the type of urolith formed and specific predisposing factors are listed below. types of stones phosphatic calculi ruminants consuming diets with high levels of phosphorus (almost all grain-based diets) are prone to developing struvite (magnesium ammonium phosphate hexahydrate) or apatite (calcium phosphate) calculi. it is unknown exactly what role phosphorus plays in urolith formation, but rations where the calcium to phosphorus ratio is less than 2:1 are particularly prone to cause outbreaks of urolithiasis.4 struvite stones appear to be affected by magnesium and other minerals present in the diet as well. phosphatic calculi are typically white or gray, smooth, radiopaque, and easily broken.4 silica calculi these uroliths are primarily a problem of ruminants grazing native rangeland in western north america.6 unpolymerized silicic acid is dissolved in ruminal fluid of the grazing animal, absorbed, and excreted in urine. cattle on rangeland typically have intermittent access to water, resulting in periods of water deprivation, leading to formation of highly concentrated urine at times. 4 calculi develop as silicic acid concentrates and polymerizes to polysilicic acid.4 urine ph does not alter silica urolith formation. silica uroliths are hard, smooth, white to brown, and radiopaque.4 calcium-based calculi the two primary types of calcium-based calculi observed are calcium carbonate and calcium oxalate. calcium carbonate uroliths form in small ruminants grazing lush, clover pastures or fed high amounts of alfalfa hay. these stones are round, smooth, and copper colored.4 calcium oxalate crystals are often present in normal urine, but form in low calcium diets and in periods of decreased water intake. oxalate stones are dense, hard, white to yellow, and smooth or jagged.4 calcium based calculi are less commonly observed in cattle compared to small ruminants.1,7 stone analysis the type of urolith is presumptively identified based on outward physical characters such as shape and color, as described above.7 since there is overlap in the gross appearance of a urolith, and most stones are of mixed composition, it is helpful for chemical analysis to be performed on collected stones. determination of the composition of uroliths collected at time of surgery is used for herd management and prevention of future cases.4 uroliths may be submitted to a number of diagnostic labs for analysis (minnesota urolith center, university of minnesota college of veterinary medicine or g.v. ling urinary stone analysis laboratory, uc davis school of veterinary medicine). figure 1. urethral calculi visible on preputial hairs. figure 2. bladder mucosal trauma. picture courtesy of department of pathobiology, auburn university veterinary teaching hospital. figure 3. subcutaneous swelling due to urine accumulation in the prescrotal region. http://dx.doi.org/10.58292/ct.v15.9646 citation line: clinical theriogenology 2023, 15, 9646, http://dx.doi.org/10.58292/ct.v15.9646 7 treatment most cases of urethral obstruction will need to be corrected by surgical intervention.5 patients often develop dehydration and severe electrolyte abnormalities, especially if bladder rupture has occurred, indicating the need for medical therapy no matter the treatment option chosen.7,8 a combination of surgical and medical management is often needed to ensure a good prognosis and return to breeding capacity.5 medical management very few cases of urethral obstruction can be corrected by medical management alone. medical management is aimed at relief of the obstruction and correction of electrolyte abnormalities. certain phenothiazine tranquilizers can have antispasmodic effects on the tunica albuginea and straightening of the sigmoid flexure aiding in passage of urethral stones.3,9 acepromazine (acepromazine injection, vetone, mwi, boise, id, 0.05–0.1 milligram/kilogram (mg/kg) intravenous (iv) or intramuscular (im)) has been used for this purpose, but its efficacy has not been proven.10,11 alpha-2 adrenergic agonists (anased® 20, xylazine injection, akorn animal health, lake forest, il) should not be used due to their diuretic effect.12 non-steroidal anti-inflammatories are beneficial for patient recovery and irritated urinary tract mucosa.11 patients with uroperitoneum often require preoperative stabilization of hypovolemia and correction of electrolyte abnormalities. initial boluses of hypertonic saline followed by physiologic saline can be used to correct volume deficits and hyponatremia. obstructed patients should not be supplemented with potassium since many patients are already suffering from hyperkalemia.11 hyperkalemia can induce bradycardia and fatal cardiac arrthymias.4 severe elevations of potassium can be corrected by administration of dextrose 50% (dextrose 50% injection, vetone, mwi, boise, id).4 slow drainage of fluid from the abdomen reduces pressure on the diaphragm and slows progression of metabolic derangements caused by uroperitoneum. acute renal failure is an occasional sequela to urethral obstruction and should be taken into consideration during physical exam and bloodwork evaluation. depending on the composition of the urinary stone present, acidification of the urine can result in breakdown of the stones. phosphatic calculi form in alkaline urine and may be broken down by acidifying the urine. silica and calcium-based calculi form in a range of urine ph levels  and  will not be broken down by acidification of the urine.4 ammonium chloride (ammonium chloride af animal feed grade, basf, ludwigahafan, germany) at an initial dose of 200 mg/kg orally once daily will acidify the urine, increasing by 50 mg/kg to keep the urine ph between 6 and 6.5.5 surgical correction the method of surgical correction chosen is highly dependent on the proposed outcome of the animal. for castrated animals that are intended for human consumption, economic considerations may be the most important deciding factor. in the circumstance of breeding bulls, the most important factor is preserving their breeding capacity by maintaining a patent urethra. ischial urethrostomy sometimes referred to as a “high perineal urethrostomy,” the approach for an ischial urethrostomy is 5 centimeter (cm) below the rectum within the ischial arch (figure 3).13 the patient is restrained in a standing position and a caudal epidural is performed. an alternative local block is the pudendal nerve block. the surgical site is clipped and surgically prepped. an incision is made beginning 5 cm below the rectum and continued for 10 cm, deep enough to incise through the dense layer of fascia overlying the retractor penis muscles. blunt dissection is used between the retractor penis muscles to visualize the bulbospongiosus muscle (figure 5). care should be figure 4. ischial urethrostomy incision site. figure 5. ischial urethrostomy incision anatomy.13 http://dx.doi.org/10.58292/ct.v15.9646 8 citation line: clinical theriogenology 2023, 15, 9646, http://dx.doi.org/10.58292/ct.v15.9646 taken to incise directly on midline through the bulbospongiosus muscle into the urethra (figure 6). hemorrhage from the bulbospongiosus muscle is common and a fair amount of hemorrhage from the corpus spongiosum may occur (figure 7). inexperienced surgeons can have difficulty reaching the urethra due to the depth of the bulbospongiosus muscle and urethra. once the urethra is opened, a catheter can be passed into the bladder. a 20–28 french foley catheter with a balloon can be used to maintain urine evacuation from the bladder (silicone foley catheters 23–34 inches long. agtech inc, manhattan, ks) (figure 8).5 normograde flushing of the distal urethra can be attempted by passing a catheter distally from the incision site. the authors have infused a mixture containing 5 milliliters (mls) lidocaine (lidocaine hcl 2% injection), 5 ml dexamethasone (dexamethasone injection 2 mg/ml) and 10–20 ml saline into the distal urethra to provide the potential benefit of decreasing urethral inflammation as well as decreasing discomfort from urethral spasm. the advantage of an ischial urethrostomy instead of a perineal urethrostomy is that the breeding potential of the bull can be salvaged. the urethra at the site of an ischial urethrostomy has a larger diameter, resulting in less concern of stricture formation. tube cystostomy originally described in 1965 for steers, a tube cystostomy is the procedure of choice when urinary bladder or urethral rupture has occurred.14 the authors believe a tube cystostomy is the best option, even when the bladder is intact, for a bull in which future use as a breeder is desired. tube cystostomy provides diversion of urine from the bladder for relief of urethral spasm or swelling and allows for stone passage. a tube cystostomy procedure can be performed standing or via a ventral midline incision. for a standing procedure a left flank approach is preferred. the bull is administered caudal figure 7. ischial urethrostomy incision. figure 6. midline palpation of the urethra at the ischial urethrostomy incision site. figure 8. using hemostats to direct a catheter into the urethra during an ischial urethrostomy procedure.13 http://dx.doi.org/10.58292/ct.v15.9646 citation line: clinical theriogenology 2023, 15, 9646, http://dx.doi.org/10.58292/ct.v15.9646 9 epidural anesthesia and the left flank is clipped and surgically prepped. paralumbar anesthesia is achieved by surgeon’s preference, commonly via an inverted l block. the urinary bladder is visualized and exteriorized as much as possible. infusion of lidocaine into the bladder via an extension set and small gauge needle can be helpful for bladder relaxation. a stab incision through the abdominal wall is made 8–10 cm caudo-lateral from the surgical approach incision for insertion of a 20–30 french foley.5 if a ventral midline incision is performed, the stab incision for insertion of the foley is made lateral to the initial incision at least 3 cm from the prepuce.5 for both approaches, the foley catheter is passed into the abdomen by aid of a stylet to insert the foley into the cranioventral aspect of the bladder near the apex. the balloon is distended, and a purse-string suture is placed in the seromuscular layer of the bladder around the foley with care taken to not crimp the catheter. tension is placed on the catheter to move the bladder close to the abdominal wall. a chinese finger trap suture is placed around the exterior portion of the foley to the exterior abdominal wall (figure 9). a one-way valve is placed on the end of the foley using the finger of an exam glove and the initial abdominal incision is closed in a routine three layer closure (figure 10). administration of ammonium chloride acidifies the urine causing breakdown of the stone(s).4,5 urine ph is monitored to ensure adequate doses are given as described above. the foley catheter is left in place for a minimum of 7 days or until urine flow through the urethra has been re-established.5 the catheter is occluded and observation of normograde voiding of urine indicates that the offending urinary calculi have passed. the foley is typically kept in place for 2–3 days past observation of normal urination to ensure re-obstruction has not occurred. surgical considerations the surgical procedure chosen is highly case specific with important consideration given to the systemic health of the patient. the success of a tube cystostomy procedure relies on establishing normal urine flow, which is only accomplished after passing of the obstructive stones. oral urinary acidifying agents are used to break down phosphatic stones. if silicaor calcium-based calculi are suspected, other surgical techniques besides a tube cystostomy, for example, an ischial urethrostomy, should be considered. post-operative management after surgery, all blood work abnormalities should be reassessed and corrected. continued assessment of hydration status is necessary and mild diuresis is helpful to correct azotemia and prevent blood clot formation. antimicrobial therapy is warranted to prevent urinary tract infections when an indwelling tube is present. beta-lactams (penicillins and cephalosporins) are commonly chosen due to their good spectrum of activity and urinary excretion.15 it is recommended to submit urine cultures for antimicrobial decisions since the most common bacteria present after foley catheter placement are aerococcus and enterococcus and 42% of cultures were resistant to two or more antibiotics.16 currently, there are no approved antibiotics labeled for post-operative surgical procedures or for treatment of urinary tract infections in ruminants and regulations for extra-label drug use must be followed. prevention it may not always be easy to pinpoint the causes of urolith formation. the most important preventative measure is always ready access to water. maintenance of appropriate hydration will result in less concentrated urine, preventing supersaturation and crystallization. increasing salt intake may increase water intake, producing more dilute urine. provision of loose salt, rather than salt blocks, increases cattle intake. analysis of the diet, including minerals, may be useful to identify predisposing factors. a general guideline for prevention of phosphatic calculi is to maintain the calcium-to-phosphorus ratio at a level greater than 2:1.4 this calcium-to-phosphorus figure 9. diagram of a tube cystostomy placement in a bull. figure 10. placement of a foley catheter after a tube cystostomy procedure in a bull. http://dx.doi.org/10.58292/ct.v15.9646 10 citation line: clinical theriogenology 2023, 15, 9646, http://dx.doi.org/10.58292/ct.v15.9646 ratio may be facilitated by decreasing the amount of grain fed. prevention of silicate uroliths is difficult when pastures contain high levels of unpolymerized silicic acid. management is dependent on adequate water intake. prevention of calcium carbonate uroliths can potentially be accomplished by substituting grass hay for alfalfa. conclusion when treating bulls for urolithiasis, an important consideration is maintenance of their breeding capacity by ensuring integrity of the urethra. surgical and medical management are often combined to ensure the best prognosis and return to breeding service. prevention of urolithiasis is based on free choice water availability, but other production techniques can be implemented depending on the type of offending stones. familiarizing oneself with the diagnosis, pathophysiology, treatment, and prevention of urinary stones in breeding bulls is helpful for any practitioner. acknowledgments the authors would like to thank jennie c. hill for her creation of the drawings used in the publication. conflict of interest none to declare. references 1. hawkins ww, jr: experimental production and control of urolithiasis. j am vet med assoc 1965;147:1321–1323. 2. udall rh, jensen r: studies on urolithiasis. ii. the occurrence in feedlot lambs following implantations of diethylstilbestrol. j am vet med assoc 1958;133:514–516. 3. oehme fw, tillmann h: diagnosis and treatment of ruminant urolithiasis. j am vet med assoc 1965;147:1331–1339. 4. byers sr: urolithiasis. in: smith bp, van metre dc, pusterla n: editors. large animal internal medicine. 6th edition, st. louis; mosby: 2020. p. 956–1005. 5. grissett g: management of urolithiasis. in: hopper rm: editor. bovine reproduction. 2nd edition, hoboken; wiley-blackwell: 2021. p. 230–241. 6. swingle kf, marsh h: vitamin a deficiency and urolithiasis in range cattle. am j vet res 1956;17:415–424. 7. larson b: identifying, treating, and preventing bovine urolithiasis. vet med 1996;91:366–377. 8. sockett d, knight a: metabolic changes associated with obstructive urolithiasis in cattle. compend contin educ vet 1984;6: s311–s315. 9. eh s: urinary calculi in feedlot cattle. report on treatment with amino promazine. j am vet med assoc 1960;137:665–667. 10. murray m: urolithiasis in a ram. compend contin educ vet 1985;7:s269–s273. 11. van metre dc, house jk, smith bp, et al: obstructive urolithiasis in ruminants: medical treatment and urethral surgery. compend contin educ vet 1996;18:317–327. 12. newcomer b: anesthetic management of specific procedures. in: lin h, passler t, clark-price s: editors. farm animal anesthesia. 2nd edition, hoboken; wiley-blackwell: 2022. p. 189–206. 13. wolfe df: urolithiasis. in: wolfe df, moll hd: editors. large animal urogential surgery. 2nd edition, baltimore; williams & wilkins: 1999. p. 349–359. 14. hastings dh: retention catheters for treatment of steers with ruptured bladders. j am vet med assoc 1965;147:1329–1330. 15. ewoldt j, jones m, miesner m: surgery of obstructive urolithiasis in ruminants. vet clin north am food anim pract 2008;24: 455–465. doi: 10.1016/j.cvfa.2008.06.003 16. chigerwe m, mavangira v, byrne ba, et al: antibiotic resistance patterns of bacteria isolated from indwelling foley catheters following tube cystostomy in goats with obstructive urolithiasis. j vet diagn invest 2017;29:316–320. doi: 10.1177/ 1040638717695607 http://dx.doi.org/10.58292/ct.v15.9646 https://doi.org/10.1016/j.cvfa.2008.06.003 https://doi.org/10.1177/104063871769​5607 2015: comparison of teaching paradigms in theriogenology among schools of veterinary medicine by use of a uniform assessment tool comparison of teaching paradigms in theriogenology among schools of veterinary medicine by use of a uniform assessment tool margaret v. root kustritz,a c. scott baileyb acollege of veterinary medicine, university of minnesota, st. paul, mn; bdepartment of clinical science, college of veterinary medicine, north carolina state university, raleigh, nc abstract information regarding specific teaching methodologies in theriogenology was provided by 18 north american and caribbean schools. at least one student each from 12 of those schools completed an on-line, multiple-choice assessment tool and provided demographic information; 45 students were included in a regression analysis comparing score on the completed assessment with various parameters. none of the specific parameters evaluated had a statistically significant association with score, including presence or absence of tracking in the curriculum; course structure; contact hours in lecture or laboratory; or availability of active learning, hands-on laboratories, or training using models. primary source of variance was the individual student. keywords: education, theriogenology introduction the field of veterinary medicine is undergoing significant change as sophistication and cost of medical care rise and number of patient visits declines, leading to decreased income and reduced salaries even as changes in state funding of veterinary colleges drive rising tuition and greater student debt.1,2 many veterinary colleges have undertaken curriculum review and revision with an eye toward greater efficiency of teaching to minimize rising costs of education and control increasing tuition costs for students. this has led to changing paradigms in teaching modalities used in various disciplines and, some suggest, may be associated with changing faculty population, as maintaining a cadre of highly specialized faculty members may not be as cost-effective for colleges as employing faculty members who are willing to provide entry-level training both within and outside of their field of specialty. there also is a greater trend toward student-centered learning, with recognition that true learning is the student’s responsibility and is facilitated by good instruction. principles of good instruction include encouraging contact between students (peer learning and group work) and between students and faculty, giving prompt feedback, recognizing and promoting different ways of learning, and engaging students in discipline-specific critical thinking.3,4 students must be comfortable with general knowledge and skills before they are expected to demonstrate knowledge and competence in discipline-specific skills.5 attention must be paid to students’ need to learn both information (declarative knowledge) and skills (procedural knowledge). similarly, attention must be paid to cognitive, emotional or attitudinal, and physical needs of the learner. one author suggests that brick-and-mortar universities may be replaced by on-line learning unless we can create value for learners in face-to-face interactions, by focusing less on reciting content to students and concentrating more on application of content and higher level cognitive processing, and development of intellectual curiosity and other professional attributes of learners.6 if we are helping them grow into professionals in our field, we have to help students recognize that their knowledge and skills are bounded by the arts of problem solving, implementation, and improvisation, and to permit them to practice manipulation of knowledge and skills to develop that artistry of practice.7 in theriogenology training, lectures and live-animal laboratories have been the norm for decades. lectures are an efficient way of explaining complex material to a large group of students at one time. concerns about lectures are that students are passive, not active learners, and that the lecture is teachercentered, not learner-centered.8 there are many ways to make these large-group teaching sessions more active for the learners, better engaging their attention and enhancing their learning and retention.9-13 however, lectures are best used for helping students achieve cognitive and perhaps attitudinal objectives, not for acquisition of psychomotor skills. clinical theriogenology • volume 7, number 1 • march 201559 laboratories with live animals or use of samples directly collected from those animals are excellent for helping students gain competence in psychomotor skills but are less good for helping students achieve cognitive or attitudinal competencies.8 hands-on work should promote development of understanding of information presented prior to the laboratory and acquisition of procedural skills.14 learners of all ages, but particularly adult learners, value authentic experiences and live-animal laboratory training is the ultimate authentic pre-clinical experience available in veterinary training.15 individualization of instruction and distribution of practice with student reflection and feedback from instructors over time should enhance ability of students to achieve competence.14,16 laboratories permit students the opportunity to practice skills in a safe environment by working with teaching animals instead of client-owned animals, while still working within the confines of safe and humane animal handling guidelines. however, maintaining teaching animals is expensive and teaching of laboratories is laborintensive for faculty. determination of what is core content is part of what guides course and curriculum development. the american college of theriogenologists (act) established a core curriculum for all species (appendix 1). a survey of practicing veterinarians from across north america verified which components of this core curriculum were absolutely key for general practice by species and better defined which content and opportunities for practice were best made available to students while still in veterinary school.17 procedures defined as most valuable in large animal practice were transrectal palpation and dystocia management, and in small animal species were vaginal cytology assessment and dystocia management. the group of practitioners surveyed felt that they had been presented with the necessary content as students but not to the extent required for competence prior to graduation, especially for large animal skills. other concerns brought forward were lack of exposure to small animal theriogenology training in general at many colleges and lack of follow-through on the effect of tracking during veterinary school on subsequent performance of graduate veterinarians. there is ongoing concern regarding what is the best overall teaching paradigm for theriogenology instruction as veterinary schools make economic decisions that may impact student learning and competence in this discipline as graduate veterinarians. there also is growing pressure from nonveterinarians, who provide aspects of theriogenology service for animals that may be permitted by the veterinary practice act in some states because there is a perceived or documented lack of availability of suitably skilled veterinarians to perform the work.1 in this study, student achievement on a set of multiple-choice questions covering all species and topics in theriogenology as defined in the core curriculum was used to evaluate teaching paradigms at participating veterinary colleges. hypotheses are the following: (1) students from institutions with theriogenology programs mandating focus on at least two species will have higher scores than those with species-focused programs, (2) students from institutions with dedicated boarded faculty members and residency or graduate student training will have higher scores than those with limited theriogenology programs, (3) number of required contact hours will be positively correlated with student scores, (4) students who take required didactic courses in all species will have higher scores than students who take required species-specific courses or only have elective didactic multi-species courses available to them, (5) students from institutions that teach theriogenology as a stand-alone discipline will have higher scores than those with integration of theriogenology material into medicine or surgery courses, (6) students who are exposed to active learning methodologies will have higher scores than those in traditional lectures, (7) students with practical experience in the form of hands-on laboratories with a given species will have higher scores for questions in that species than students without hands-on experience, (8) students trained using alternate models (simulations) will have higher scores by species than those without models, but will have lower scores than those with live-animal laboratory experience in that species, and (9) students at schools that do not track will have higher scores than students from schools that track. materials and methods this study protocol was evaluated and approved by an institutional review board. a teaching survey was generated to address hypotheses as described above and to repeat collection of information clinical theriogenology • volume 7, number 1 • march 2015 60 gleaned from the survey previously described (appendix 2).18 the survey was sent to faculty liaisons of the society for theriogenology (sft) at all north american and caribbean veterinary schools (n=35). multiple-choice questions were solicited from members of the teaching educators in the sft and act. questions were edited for grammar and formatted for uniformity. one hundred questions were chosen to represent the species and topic distribution of the core curriculum (appendix 1). answers were written for all questions explaining the correct and incorrect foils (figure). the questions were posted on a moodle site hosted at the university of minnesota and student access was set up as anonymous guest accounts; investigators had access to basic demographic data and an email address for each participant but no personal information was collected. fourth-year students from all schools that completed the teaching survey were invited to participate through their faculty liaisons, and reminders sent to those faculty liaisons to pass along to their students every two weeks. the site was made available in early september, when students traditionally begin study for the north american veterinary licensing examination (navle) and remained open through the first navle testing window, closing in late december. students were incentivized to participate by a drawing for an ipad mini™ from email addresses of all students who attempted all 100 questions and got at least 70% correct. students could come and go from the testing instrument as they chose and were not given guidance regarding whether or not they could or should use outside resources. as students completed answering a given question, the correct response would be displayed to give them instant feedback and enhance their learning. to assess relationships between each variable and student score, mixed models were used, with a random effect for school, using the score as a response and each variable as a predictor. one score was a significant low outlier (4.34 standard deviations below the mean). because all student entries were anonymous, the investigators could not contact the student to determine if this was a true score, reflecting lack of ability despite significant effort by the student in completing the assessment, or a false score, reflecting lack of student effort. the investigators chose not to include this outlier in the statistical analysis so as not to generate spurious results. significance was set at p < 0.05. results theriogenology teaching complete surveys were received from 18 schools (51.4% response rate). participating schools were atlantic (prince edward island), auburn, colorado, florida, georgia, guelph (ontario), louisiana, minnesota, north carolina, ohio, oregon, pennsylvania, purdue, ross, tuskegee, washington state, western (saskatchewan), and wisconsin. total enrollment at participating schools ranged from 224 to 900, with a mean enrollment of 413 +/164 students (mean +/sd; n=14). six schools (33%) did not allow tracking at any point in the curriculum. of the other 12 schools, one offered tracking beginning in the first year, eight in the third year, and three in the fourth year of the curriculum. tracks available included small animal, equine, bovine or food animal, mixed, and other (exotics, wildlife, public/corporate, research/public health); not all tracks were available at all schools. in general, the small animal track was most subscribed, with the highest percentage of students in this track at eight schools and an overall mean percentage of 44 +/20%. percentages for the other tracks were 10 ± 7% for equine, 9 ± 5% for bovine/food animal, 22 ± 15% for mixed track, and 10 ± 15% for other tracks. number of faculty members board-certified in the act or equivalent foreign specialty college averaged 4 ± 2, with a range from 1 to 10. number of residents in programs approved by the act averaged 1 ± 1, with a range from 0 to 3. seven of the responding schools had no resident in training at the time of this survey. nine of the schools with residents reported that their residents were in a combined residency/graduate degree program; only one resident from these schools was not in such a program. the other two schools with residents reported that those residents were completing residency training only. number of dedicated theriogenology graduate students averaged 1 ± 1, with a range from 0 to 4. thirteen of the responding schools had no theriogenology graduate students. schools were asked to self-report areas of emphasis, with six options to choose from (dairy, beef, swine, small ruminant, equine, small animal). only two schools reported emphasis on all six options. clinical theriogenology • volume 7, number 1 • march 201561 two schools reported emphasis on all options but swine. two schools reported emphasis on all but swine and small ruminants. one school reported emphasis on all but swine and beef cattle. seven schools reported emphasis on three options; all reported emphasis on dairy cattle and horses. two schools reported emphasis on only two options; horses were emphasized at both schools. two schools did not report areas of emphasis. four schools reported that all information related to theriogenology, including reproductive biology, clinical theriogenology didactic and laboratory teaching, and clinical rotations, was taught by individuals board-certified in theriogenology. eleven schools reported that reproductive physiology was taught by faculty members outside of theriogenology. other areas in the curriculum taught by nontheriogenology faculty included food animal laboratories (5), food animal rotations (4), small ruminant rotations (4), surgery (2), small ruminant laboratories (2), exotics clinical rotations (2), exotics laboratories (1), small animal clinical rotations (1), and equine rotations (1). there was great variability in number or required and elective lecture hours by discipline or species (table 1). reproductive biology and physiology was taught exclusively in first year at seven schools, exclusively in second year in six schools, and exclusively in third year in one school. reproductive physiology training was spread across several years in four schools. the remaining schools did not report when in the curriculum this content was presented. species-based theriogenology lectures were taught exclusively in second year in two schools and exclusively in third year in nine schools, and were spread across several years in six schools. one school did not report when in the curriculum this content was presented. all responding schools stated that theriogenology was taught as a stand-alone discipline, with five schools stating that some theriogenology content also was presented in medicine, surgery, and production courses. for required lecture courses, 14 schools presented theriogenology content in a comparative course and two schools covered theriogenology content scattered through systems courses. for elective lecture courses, 14 schools presented theriogenology content by species and one school provided comparative content. fourteen schools provided content specific to herd health, with number of hours of content ranging from 3 to 50. several schools noted that this was hard to quantify as material was presented across many courses in the curriculum. all schools reported time spent on small animal genitourinary disease in the curriculum with 11 schools reporting discussion of this content in theriogenology and medicine courses. fourteen schools reported that no theriogenology text was required. of those schools requiring a text, it was reported that few students actually purchased that text. materials were made available for later review by students at 15 schools; specific materials provided included taped lectures, narrated powerpoint presentations, and podcasts. most schools reported that they also provide notes as hard or electronic copies. active learning, defined as provision of course materials to encourage student engagement as a means of enabling them to understand and encode new knowledge, was provided at 11 schools with specific examples including use of cases (individual or group work) and use of audience response systems. active learning generally was reported to be very instructor-dependent and number of hours of time spent in active learning was not well reported by the schools. no school reported use of the qualifying examination available through the national board of veterinary medical examiners as a tool to determine student readiness for pre-clinical training. there was great variability in number or required and elective laboratory hours by species (table 2). laboratories were offered exclusively in second year by one school, exclusively in third year by eight schools, and across several years by six schools. the other schools did not report in which years of the curriculum laboratories were offered. live animal laboratories were available for students at all schools except one. of 14 schools reporting which species of animals were available for live-animal laboratories, all 14 reported availability of horses, 13 of cattle, 9 of dogs, 7 of small ruminants/camelids, and 3 of pigs. thirteen schools (72%) reported that all students had an opportunity to learn transrectal palpation on either cows or horses, although it was not required at all schools. hours available in the curriculum for practice to refine transrectal palpation skills was variably reported, with some schools providing contact hours and others providing number of laboratories or clinical rotations. seventeen schools (94%) reported that all students completed a canine or feline ovariohysterectomy in the first three years of the clinical theriogenology • volume 7, number 1 • march 2015 62 curriculum. one school reported that 90% of students completed an ovariohysterectomy, and one school reported that less than 10% of students completed this in the first three years but that all students at that school performed ovariohysterectomy as a requirement during the fourth year. schools generally reported that no students or very few students had an opportunity to participate in a cesarean section in any species in the first three years of the curriculum. eleven of 18 schools (61%) reported using models or simulators in laboratories for theriogenology training. models described included excised reproductive tracts (fresh or frozen/thawed) (6), dead calves (2), and use of the breed n betsy model (2). clinical rotations were required for all students or required for students within specific tracks at 12 schools. a comparative rotation was required at nine schools, with some schools also requiring species-specific rotations, depending on student track. species-specific rotations were the sole requirement at two schools. elective rotations were available at 11 schools. fourteen schools (78%) offered a comparative rotation, 12 schools offered a food animal rotation, 11 schools offered an equine rotation, and 10 schools offered a small animal rotation. other services on which students may see theriogenology cases included large animal ambulatory, small animal medicine, large animal medicine, shelter medicine, small animal surgery, large animal surgery, food animal/ruminant rotation, general or community practice, emergency, swine rotation, poultry rotation, and production medicine. a theriogenologist was routinely called to consult on medical or surgical cases involving the reproductive tract that were not transferred to/maintained by the theriogenology service at 17 schools; one school does not maintain a teaching hospital or clinical outreach services. reproductive emergencies (dystocia, postpartum disease, testicular/scrotal disease, pyometra/metritis) were seen by the theriogenology service at those same 17 schools, with 16 schools reporting seeing emergencies in horses, 14 in cattle, 8 in small ruminants and camelids, and 7 in small animals. fifteen schools reported on the percentage of students that completed an ovariohysterectomy in a dog or cat during their fourth year. mean percentage was 94% (± 10%) with a range from 70-100%. percentage of students that participated in a cesarean section during their fourth year was extremely variable, with most schools reporting that 10% or less of students would participate in a cesarean section in any species. students were most likely to participate in a cesarean section in a cow. schools were asked to describe student participation in extracurricular activities. fifteen schools (83%) hosted a student chapter of the society for theriogenology. other student clubs in which students might gain theriogenology experience included agricultural animal club, bovine club, canine club, diagnostic imaging club, equine club, farm animal club, food animal club, orphan kitten project, palpation team, production animal medicine club, student initiative for reservation veterinary services, and the student chapters of the american animal hospital association, american association of bovine practitioners, american association of equine practitioners, american association of feline practitioners, american association of small ruminant practitioners, and american veterinary medical association. average number and range of lectures and laboratories available by species was variable. mean number of lectures and laboratories offered annually in cattle was 9 ± 8, in horses was 3 ± 2, in small animals was 2 ± 1, and in exotics was 0.5 ± 0.5. responses from assessment one hundred seventy five students signed into the assessment tool. sixty-nine students completed at least one question. forty-five students completed all 100 questions and the associated demographic data to permit the investigators to link their score with their school. fourteen students were from pennsylvania; 8 were from minnesota; 6 were from wisconsin; 3 each were from guelph and north carolina; 2 each were from auburn, florida, ohio, and ross; and 1 each was from louisiana, oregon, and washington state. at least one student completed the assessment for each of 12 schools. only data from those schools was included in the following statistical analysis. total score averaged 65 (± 8) with a range of 48 to 77. scores split out by species were for bovine 17 ± 2 with a range of 13 to 22 (24 questions, mean score = 71%), for equine 12 ± 3 with a range of 4 to 18 (21 questions, mean score = 57%), for small animal 14 ± 2 with a range of 10 to 20 (22 clinical theriogenology • volume 7, number 1 • march 201563 questions, mean score = 64%), for small ruminant 6 ± 1 with a range of 1 to 7 (7 questions, mean score = 86%), for other species 9 ± 2 with a range of 3 to 13 (14 questions, mean score = 64%), and for nonspecies questions 8 ± 2 with a range of 4 to 11 (12 questions, mean score = 67%). none of the specific parameters evaluated had a statistically significant association with score, including presence or absence of tracking in the curriculum; course structure; contact hours in lecture or laboratory; or availability of active learning, hands-on laboratories, or training using models. primary source of variance was the individual student. ninety-two percent of the variability was associated with the students, with estimated school to school standard deviation of 2.2 points and estimated student within school standard deviation of 7.7 points. ninety-five percent confidence intervals for the difference due to each variable were also calculated; the bounds for three of the seven were less than 10 points different, and the largest was a 16 point difference. statistical analysis was repeated looking at individual sets of questions by species or discipline (bovine, equine, small ruminant, small animal, other species, basic science). again, no variance was shown due to school or specific parameters and the primary source of variance was the individual student. due to insufficient data, hypotheses concerning variation in scores associated with depth of program as evidenced by number of faculty, residents, and graduate students; comparative versus species-specific required course content; and provision of theriogenology as a standalone discipline versus a component of surgery and medicine courses could not be evaluated. discussion there is little research specifically evaluating effect of lecture and laboratory work, either alone or in combination, on acquisition of specific skills in veterinary medicine. this study showed no difference in scores on a uniform assessment tool relative to a variety of parameters that may have impacted student learning. this agrees with studies comparing student learning in physical science, natural science, general biology, home economics, and computer training between lecture-demonstration, laboratory, and individual student work including workbooks and term papers, which showed no differences between groups in knowledge or practical application of that information.19-23 this may be due to individual student abilities; in one study comparing lecture training to laboratory training in an integrated science course, high achieving students were equally successful with either method while low achieving students were more successful with laboratory training alone than with lecture training alone.24 this was supported by demonstration in the current study of the preponderance of variation in scores being due to the individual student. in a study comparing lecture only to lecture plus laboratory training in earth science, the latter was superior, with a 33% increase in student mastery of concepts as evaluated by multiple-choice assessment.25 learning in lecture with accompanying laboratories is enhanced if strict attention is paid to alignment of content to ensure integration of concepts and appropriate sequencing of material presented.26 it is perhaps not surprising that no difference was shown in this study, as there were so many more variables than in those studies that did show a difference, including variations in prerequisite coursework and life experience in students, sequencing of content within the curriculum, and presence or absence of significant extracurricular activities that would reinforce coursework. recent work suggests that extracurricular activities, if entered into by students with a goal of enhancing their learning, are associated with increased grade point average, making it clear that such parameters should be addressed when comparing learning at various institutions.27 this study suffered from insufficient data despite concerted efforts by the authors to gather information about the schools and to encourage students to complete the assessment. future studies may be more successful if they focus on change within a given school’s curriculum as this may give more opportunity to harvest data and will decrease some of the variability inherent in a large, multi-institutional study. this study also suffered from self-selection by students in accessing and completing these questions. it may be that students who chose to participate were only those who had a particular interest in theriogenology or only those who were particularly concerned about theriogenology as they prepared for the board examination. this would have skewed the results. information about theriogenology teaching is valuable for comparison as schools consider changing their curriculum. it is difficult to compare information from this study with previous work, clinical theriogenology • volume 7, number 1 • march 2015 64 which contained information from 31 north american and caribbean veterinary schools. in that study, didactic training in at least one species was required at all participating schools. laboratory training in at least one species was required at 75.0% and clinical experience was required at 72.7%.18 the current study showed great variability in required versus elective course offerings either as lectures or as laboratories. some of this variability is likely driven by interests and skills of the faculty members at that institution. it has been demonstrated that student interests are driven by their classmates and faculty mentors and it may well be that some schools, with very engaging faculty members and peer support for learning, provide a rich educational experience even with limited contact time and other resources.28,29 conclusion this study used a uniform assessment tool to gauge the amount of variance in student scores due to differing teaching methodologies between institutions. sample size was small despite concerted efforts to increase engagement by schools and students. regression analysis identified the individual student as the primary source of variance, with no parameters relative to differing teaching paradigms a significant source of variance. smaller, more focused studies, may be required to identify subtle effects of teaching methodology on student performance. identification of a within-school effect of student of 7.7 points can be used to calculate appropriate sample size for future studies. acknowledgements the authors wish to thank dr. aaron rendahl and xiao zhong for performing the statistical analysis. references 1. felsted ke: economic trends and issues in veterinary medicine. available at: https://www.avma.org/about/governance/documents/felsted_economic_trends_and_issues.pdf. accessed october 13, 2013. 2. shepherd aj, pikel l: employment, starting salaries, and educational indebtedness of year-2013 graduates of us veterinary medical colleges. j am vet med assoc 2013;243:983-987. 3. chickering aw, gamson zf: seven principles for good practice in undergraduate education. in: american association for higher education bulletin. available at: http://wwwtemp.lonestar.edu/multimedia/sevenprinciples.pdf. accessed october, 14, 2013. 4. bain k: how do they conduct classes? in: what the best college teachers do. cambridge (ma): harvard university press; 2004. p. 98-134. 5. schunk dh: content-area learning. in: learning theories: an educational perspective. upper saddle river (nj): pearson prentice hall; 2004. p. 389-433. 6. bowen ja: teaching naked: how moving technology out of your college classroom will improve student learning. san francisco: jossey-bass; 2012. p. 232-233. 7. schon da: preparing professionals for the demands of practice. in: educating the reflective practitioner. san francisco: jossey-bass; 1987. p. 3-21. 8. thomas pa: step 4: educational strategies. in: kern de, thomas pa, hughes mt, editors. curriculum development for medical education: a six-step approach. baltimore: the johns hopkins university press; 2009. p. 57-83. 9. doyle t: from lecturer to facilitator. in: learner-centered teaching: putting the research on learning into practice. sterling (va): stylus; 2011. p. 51-61. 10. filene p: lecturing. in: the joy of teaching: a practical guide for new college instructors. chapel hill (nc): university of north carolina press; 2005. p. 47-55. 11. meyers c, jones tb: the case for active learning. in: promoting active learning: strategies for the college classroom. san francisco: jossey-bass; 1993. p. 3-18. 12. sachdeva ak: large group teaching. in: distlehorst lh, dunningham gl, folse jr, editors. teaching and learning in medical surgical education: lessons learned for the 21st century. new york: psychology press; 2000. p. 85-98. 13. cantillon p: teaching large groups. bmj 2003;326:437-440. 14. mckeachie wj: laboratory teaching. in: teaching tips: strategies, research and theory for college and university teachers. lexington (ma): dc heath and co.; 1994. p. 135-137. 15. zemelman s, daniels h, hyde a: the seven structures of best practice teaching. in: best practice: today’s standards for teaching and learning in america’s schools. portsmouth (ny): heinemann; 2005. p. 227-265. 16. moulton c-ae, dubrowski a, macrae h, et al: teaching surgical skills: what kind of practice makes perfect? annals surg 2006;244:400-409. clinical theriogenology • volume 7, number 1 • march 201565 17. root kustritz mv, chenoweth pj, tibary a: efficacy of training in theriogenology as determined by a survey of veterinarians. j am vet med assoc 2006;229:514-521. 18. root kustritz mv, tibary a, chenoweth pj: availability of theriogenology training at north american and caribbean veterinary colleges. j vet med educ 2006;33:140-144. 19. balcziak lw: the role of the laboratory and demonstration in college physical science in achieving the objectives of general education [dissertation]. st. paul: university of minnesota; 1954. 20. dearden dm: an evaluation of the laboratory in a college general biology course. j experiment educ 1960;26:241247. 21. trotter vy: a comparison of the laboratory and lecture-demonstration methods of teaching survey of food preparation for freshman home economics at the university of vermont. ohio state university, 1960 (unpublished). 22. bradley rl: lecture demonstration versus individual laboratory work in a natural science course at michigan state university [dissertation]. east lansing: michigan state university; 1963. 23. romeu jl, alemzadeh j: a statistical assessment of an experiment to compare traditional versus laboratory approach in teaching introductory computer programming. j educ tech systems 1999;27:319-324. 24. odubunmi o, baogun ta: the effect of laboratory and lecture teaching methods on cognitive achievement in integrated science. j res sci teach 1991;28:213-224. 25. forcino fl: the importance of a laboratory section on student learning outcomes in a university introductory earth science course. j geosci educ 2013;61:213-221. 26. dibiase wj, wagner ep: aligning general chemistry laboratory with lecture at a large university. school sci math 2002;102:158-171. 27. jones ml, rush br, elmore rg, et al: level of and motivation for extracurricular activity are associated with academic performance in the veterinary curriculum. j vet med educ 2014;41:275-283. 28. gwinner kp, prince jb, andrus dm: attracting students into careers in food supply veterinary medicine. j am vet med assoc 2006;228:1693-1704. 29. university of missouri college of veterinary medicine agscholars program: available at www.cvm.missouri.edu/prevet_ag_mats/agscholars.pdf. accessed october 9, 2014. you are vaccinating an 8 month old golden retriever and notice that the dog has only one testicle in his scrotum. they are hoping to use the dog for breeding. which of the following is your best advice for the owners about this dog? a. he will be sterile and he will not develop the normal secondary sex characteristics. b. he’s too young for you to be able tell them if this will be a problem. c. he was probably born with only one testicle (monorchid), and he should still be fertile. d. he should be castrated because this is most likely a heritable condition. cryptorchidism is failure of one or both testes to descend into the scrotum. the process by which the gubernaculums assist in decent of the testes into the scrotum should be complete by 8 weeks of age. the retained testicle is not able to produce spermatozoa and tends to be smaller than the scrotal testicle because of the higher temperature inside the body; descended testes are normal. monorchidism is extremely rare in dogs. since cryptorchidism is inherited, it is important not to breed this male as he can pass it on to his offspring. figure. example question clinical theriogenology • volume 7, number 1 • march 2015 66 table 1. required and elective lecture hours discipline/species mean +/sd required lecture hours range required lecture hours mean +/sd elective lecture hours range elective lecture hours reproductive biology / physiology 19 +/17 5-57 2 +/6 0-26 equine 9 +/5 0-20 7 +/9 0-28 food animal 11 +/7 0-31 9 +/11 0-41 small animal 7 +/4 0-15 5 +/6 0-14 small ruminant 3 +/3 0-10 2 +/6 0-8 other* 3 +/2 0-8 1 +/1 0-4 total 52 +/23 19-132 24 +/25 0-76 * swine, camelid, exotics, biotechnology table 2. required and elective laboratory hours species mean +/sd required laboratory hours range required laboratory hours mean +/sd elective laboratory hours range elective laboratory hours equine 4 +/4 0-13 10 +/11 0-30 food animal 5 +/6 0-16 9 +/10 0-28 small animal 1 +/8 0-12 1 +/1 0-4 small ruminant 0 +/1 0-4 1 +/2 0-8 other* 2 +/4 0-15 1 +/2 0-9 total 12 +/13 0-40 22 +/22 0-58 * histology, semen evaluation, pathology, swine, obstetrics, fetotomy clinical theriogenology • volume 7, number 1 • march 201567 appendix 1. core curriculum theriogenology core curriculum suggested prerequisites to veterinary school medical terminology endocrinology genetics topics in the veterinary curriculum 1) basic sciences a) anatomy i) male and female, representative large and small animal species b) histology i) gonads ii) uterus iii) uterine tubes iv) mammary tissue c) endocrinology i) hypothalamus ii) pituitary iii) gonads iv) uterus v) placenta vi) thyroid vii) adrenal d) embryology and placentation e) reproductive biology i) sex determination and differentiation ii) estrous cycle, representative species iii) folliculogenesis / oogenesis / ovulation / luteinization iv) seasonal, environmental, nutritional effects v) puberty vi) pharmacologic manipulation of estrus vii) intro to advanced reproductive technologies – semen cryopreservation, cloning, embryo manipulation, etc. viii) spermatogenesis / sperm maturation ix) emission / ejaculation x) fertilization / embryo movement / maternal recognition of pregnancy xi) pregnancy / parturition xii) artificial insemination xiii) contraception – surgical, pharmacologic, immunologic xiv) mammary gland development / lactation / milk biology f) pharmacology i) use of reproductive hormones and analogues ii) use of antibiotics / withdrawal times g) behavior 2) clinical sciences a) bovine i) female (1) estrous cycle / breeding management (2) pharmacologic manipulation of estrus (3) anestrus (4) cyclical aberrations (5) pregnancy diagnosis / pregnancy management (6) dystocia management / obstetrics, fetotomy (7) abortion (8) periparturient disorders – hydrops, vaginal / uterine eversion, metritis, retained fetal membranes, endometritis (9) infectious diseases / disorders of the reproductive tract (10) mastitis ii) male (1) infertility, male (2) infectious diseases / disorders of the reproductive tract iii) general (1) herd health / reproductive performance goals / economics / record keeping (2) artificial insemination / embryo transfer clinical theriogenology • volume 7, number 1 • march 2015 68 (3) testing for heritable disorders / creation of breeding plans to minimize genetic disorders (4) neoplasia of the reproductive tract iv) techniques (1) history taking / physical examination / creation of a diagnostic and treatment scheme (2) transrectal reproductive examination (3) ultrasound (4) breeding soundness examination / semen collection and evaluation (5) passage of pipette / biopsy instrument / insemination pipette into uterus (6) neonatal resuscitation (7) anesthesia (8) surgery (a) cesarean section (b) teat surgery (c) caslick’s (d) ovariectomy (e) castration b) equine i) female (1) estrous cycle / breeding management (2) pharmacologic manipulation of estrus (3) anestrus (4) cyclical aberrations (5) pregnancy diagnosis / pregnancy management (6) dystocia management / obstetrics (7) abortion (8) periparturient disorders – metritis, retained fetal membranes (9) endometritis (10) infectious diseases / disorders of the reproductive tract (11) mastitis ii) male (1) infertility, male (2) infectious diseases / disorders of the reproductive tract iii) general (1) herd health / reproductive performance goals / economics / record keeping (2) artificial insemination / embryo transfer (3) testing for heritable disorders / creation of breeding plans to minimize genetic disorders (4) neoplasia of the reproductive tract iv) techniques (1) history taking / physical examination / creation of a diagnostic and treatment scheme (2) transrectal reproductive examination (3) ultrasound (4) breeding soundness examination / semen collection and evaluation (5) passage of pipette / biopsy instrument / insemination pipette into uterus (6) neonatal resuscitation (7) anesthesia (8) surgery (a) cesarean section (b) perineal reconstruction (c) caslick’s (d) ovariectomy (e) castration (descended and cryptorchid) c) small animal i) female (1) estrous cycle / breeding management (2) pregnancy diagnosis / pregnancy management (3) dystocia management / obstetrics (4) abortion (5) periparturient disorders – metritis, sips, eclampsia (6) infectious diseases / disorders of the reproductive tract (7) mastitis (8) mammary neoplasia ii) male clinical theriogenology • volume 7, number 1 • march 201569 (1) infertility, male (2) infectious diseases / disorders of the reproductive tract iii) general (1) artificial insemination (2) contraception (3) testing for heritable disorders / creation of breeding plans to minimize genetic disorders (4) neoplasia of the reproductive tract iv) techniques (1) history taking / physical examination / creation of a diagnostic and treatment scheme (2) breeding soundness examination / semen collection and evaluation (3) neonatal resuscitation (4) anesthesia (5) surgery (a) cesarean section (b) ovariectomy / ovariohysterectomy (c) castration (descended and cryptorchid) d) porcine i) estrous cycle / breeding management ii) parturition / obstetrics iii) infectious causes of reproductive loss iv) breeding soundness examination, male v) artificial insemination e) ovine / caprine i) estrous cycle / breeding management ii) pregnancy diagnosis / pregnancy management iii) abortion iv) parturition / obstetrics v) breeding soundness examination, male clinical theriogenology • volume 7, number 1 • march 2015 70 appendix 2. teaching survey theriogenology teaching survey 2013 1) faculty and training number of boarded or board-eligible faculty members: number of residents currently in training: number of residents concurrently completing a graduate degree: number of graduate students (no concurrent residency): 2) total student enrollment (all years of the curriculum): 3) is tracking permitted? if no, go to question 3. if tracking is permitted, which year in the curriculum is the first year where it is permitted? [ ] 1st year [ ] 2nd year [ ] 3rd year [ ] 4th year what is the rough percentage of students in each track in your current clinical year? please designate by putting an x in the box appropriate for each species. track rough percentage in each track < 5 5-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 >90 small animal equine bovine mixed exotic / wildlife practice other which of the following do you believe is a focus of theriogenology training at your institution, based on emphasis in the curriculum and number of faculty involved in training/research? please check all that apply. [ ] dairy cattle (including cow/calf and herd management) [ ] beef cattle (including herd management) [ ] swine [ ] sheep / goat / camelid [ ] equine [ ] small animal 4) didactic coursework – please complete the table below. please total the lecture hours by species, required versus elective, and year in curriculum; we are not concerned in this table if they are taught over multiple courses course required hours elective hours year in curriculum taught by diplomate?* lectures (this does not include rotations – see question 4) reproductive biology / physiology clinical equine clinical food animal clinical small animal clinical small ruminant clinical theriogenology • volume 7, number 1 • march 201571 clinical other laboratory hours equine food animal small animal small ruminant other * act, ecar, australian fellow for required courses, does your institution have stand-alone courses by species or one or more courses covering all species of interest? for elective courses, does your institution have stand-alone courses by species or one or more courses covering all species of interest? is theriogenology primarily taught as a discipline or is theriogenology material covered in other courses (e.g. medicine or surgery)? in lectures: how many hours in the curriculum are dedicated to herd health/ herd reproductive management vs. individual management/disease management? are canine urogenital diseases discussed? is this a component of a medicine or theriogenology class? is a theriogenology textbook required? please list required texts. are lectures available for review outside of class? are “alternative teaching tools” applied to teach theriogenology (case-based learning/ problem-based learning / small group learning / flipped classroom)? if yes, please describe briefly: how many hours of the theriogenology curriculum are devoted to alternative teaching methods? in laboratories: do all students have an opportunity to learn transrectal palpation on either cows or horses? how many hours are available in the curriculum to practice transrectal palpation skills? what percentage of students completes a canine or feline ovariohysterectomy as a component of a teaching laboratory in the first three years of the curriculum? what percentage of students participates in a c-section in the first three years of the curriculum (species?)? are live animal theriogenology labs available for horses, cows, pigs, avian, canine? are these labs required or elective? are artificial teaching tools (palpation models) used instead of live animals for all students? available for some students? do students at your institution take the qualifying examination or another capstone assessment to assess basic science knowledge before beginning didactic work in clinical disciplines? 5) clinical coursework – please complete the table below. rotation required or elective? taught by diplomate?* comments general therio small animal therio equine therio food animal therio other * act, ecar, australian fellow if theriogenology is covered on other clinical services, what are they (e.g. general practice, medicine)? is a theriogenologist routinely called to consult on medical or surgical cases involving the reproductive tract that are not transferred to/maintained by the theriogenology service? are reproductive emergencies (dystocia, postpartum disease, testicular/scrotal disease, pyometra/metritis) seen by the theriogenology service? if so, in what species? what percentage of students completes a canine or feline ovariohysterectomy in the fourth year of the curriculum? what percentage of students participates in a c-section in the fourth year of the curriculum (species?)? 6) extracurricular training opportunities for dvm students does your institution have a student chapter of the society for theriogenology? does your institution have other student clubs that have lectures or wet labs with a focus on theriogenology (palpation team, etc.)? clinical theriogenology • volume 7, number 1 • march 2015 72 please list the approximate number of lectures/labs available per year through these activities for each species group: sa: equine: food animal: exotic: thank you for taking the time to complete this survey clinical theriogenology • volume 7, number 1 • march 201573 2018: the early conceptus and circulating blood progesteroneconcentrations in caribbean jennies the early conceptus and circulating blood progesterone concentrations in caribbean jennies b.n. roberts, r.o. gilbert, d.r. bergfelt, e.w. peterson, j.c. samper, h.m. french ross university school of veterinary medicine, st kitts, west indies donkeys are essential in many parts of the world. few studies have explored the early pregnancy in jennies. the aim of this study was to describe the development of the early conceptus in jennies: day of first detection of the embryonic vesicle (ev), day of fixation, morphological changes of ev, detection of the embryo proper and heart beat, and day of emergence of the allantoic sac. additionally, this study compared circulating blood progesterone during non-pregnant and pregnant cycles in relation to corpus luteum (cl) volume and ev diameter. eight caribbean jennies were monitored through four non pregnant estrous cycles via daily transrectal ultrasonography and every other day blood collection for determination of serum progesterone concentration. at estrus, the same jennies were naturally bred to a jack of proven fertility daily until ovulation (day 0). thereafter, daily ultrasound examinations and blood collection continued. the diameter of the ev was measured to evaluate growth rate. day of fixation was defined as the day the embryo ceased movement and remained in the same location during sequential 10minute examinations for 2 hours. subsequent to fixation, daily ultrasonography was used to monitor morphological shape changes, detection of the embryo proper, fetal heart beat, and emergence of allantoic sac. monitoring of the conceptus, cl volume and progesterone concentration ceased on day 30 of pregnancy. six of the ev were identified on day 9 and the remaining two on day 10, with the mean diameter 2.4 mm ( 0.77 sem). during the mobility phase, growth rate of the embryo showed linear progression at 3.75 mm/d (±0.19 se). from day 18 to 28, growth rate plateaued and the slope was not significantly different from zero. linear growth resumed on day 28. fixation occurred between day 13 and 16 with the mean diameter of the vesicle 23.9 ( 0.47 se). in 7/8 jennies, the vesicle size one day prior to fixation was less than 22 mm. once the ev became greater than 22 mm in size, fixation occurred. this suggests that size of the vesicle is more important to fixation than day of pregnancy. morphological changes, day of first detection of the embryo proper (mean 20.8±0.36 se), embryonic heart beat (mean 22±0.26 se) and emergence of the allantoic sac (mean 24.4±0.32 se) were consistent with past studies in mares and jennies. for the first 10 days after ovulation, progesterone concentrations were significantly higher in pregnant donkeys compared to their non-pregnant cycles (p<0.001). from day 10 to 16, the diameter of the ev was significantly positively correlated to cumulative progesterone exposure (p<0.0001). cumulative progesterone exposure up to day 9 was higher for those jennies in which the conceptus was detected on day 9 than in those for whom first detection was on day 10. these results suggest that the effects of early pregnancy on luteal function occur sooner than previously thought. this is the first report of different progesterone profiles during the early pregnancy of donkeys and the first correlation between progesterone and embryo growth. keywords: donkey, early pregnancy, progesterone, corpus luteum clinical theriogenology • volume 10, number 3 • september 2018 340 2018: primary hypothyroidism in a miniature jenny associated with neonatal congenital hypothyroidism dysmaturity syndrome primary hypothyroidism in a miniature jenny associated with neonatal congenital hypothyroidism dysmaturity syndrome m.f. lopez-rodriguez,a m. rhodes,c m. diel de amorim,a g. starrak,b c. carda adepartment of large animal clinical sciences; bdepartment of small animal clinical sciences, western college of veterinary medicine, university of saskatchewan, saskatoon, sk; and cnanton veterinary clinic, nanton, ab, canada a 7-year-old miniature jenny with a history of stillborn foals in 2012 and 2013 was maintained on pasture from spring through fall, fed grass hay and oats in winter, and had access to blue and brown salt blocks. she was bred at pasture and was reported to be a few weeks overdue at foaling. her udder development and milk production were normal, and her placenta was expelled within 4 h. a miniature jack foal was born in an uncomplicated foaling. he had an abnormal appearance and crooked front legs but was able to stand and nurse. the foal was examined shortly after birth, and administered selenium, tetanus toxoid, and an enema. the significant findings included: prolonged gestation, mandibular prognathism, front limb angular limb deformities, carpal and tarsal bone dysgenesis, and subnormal body temperature, which were signs consistent with congenital hypothyroidism dysmaturity syndrome (chds). six weeks later the foal developed respiratory signs and was euthanized. due to the history of stillborn/chds foals, a series of tests were performed on the miniature jenny to determine if she was hypothyroid and if dietary risk factors for chds were present (i.e. low trace mineral status or exposure to nitrates or glucosinolates [gsl]). serum samples were submitted to prairie diagnostic services, saskatoon, sk for evaluation of concentration of vitamin e, trace minerals, tri-iodothyronine (t3) and total thyroxine (t4) (immulite assay). mane hair was submitted for determination of selenium. a thyrotropin releasing hormone (trh) response test was performed, by drawing blood (pre), administering 0.5 mg of trh iv, and taking a post-stimulation sample 4 h later. an adequate response to trh is a doubling or more of pre-thyroid hormones by 4 h. the pasture contained gsl plants. serum cobalt at 0.928 ppm and serum selenium at 0.108 ppm, were considered marginal for equids, and hair selenium at 0.42 ppm, was deficient. the t3 pre-trh level was 1.83 nmol/l and post-trh levels was 4.05 nmol/l (fold change 2.2). the pre-trh and the post-trh t4 level were both 10 nmol/l. as donkey t4 levels are higher than horses, we interpreted the resting t4 value as deficient. the lack of a post-trh t4 response was compatible with thyroid dysfunction. the chds has been reported in donkey neonates, where clinical signs included hypothermia. the failure of t4 to increase following trh administration indicated the miniature jenny had primary hypothyroidism, which may be related to consumption of gsl through pasture and/or hay. the gsl interfere with iodide uptake and organification and have been reported to cause materno-fetal thyroid dysfunction. the low serum and hair selenium, and low cobalt level indicated inadequate dietary mineral intake by the miniature jenny, which is a risk factor for chds, and which may have contributed to the jennie’s thyroid dysfunction. keywords: hypothyroidism, jenny, hypothermia, thyroxine, trh clinical theriogenology • volume 10, number 3 • september 2018337 evaluation of bull breeding soundness examination evaluation of bull breeding soundness examination jennifer n. roberts, daniel l. grooms, eileen r. thompson, andrew g. huff department of large animal clinical sciences, college of veterinary medicine michigan state university, east lansing, mi abstract records from 2,887 bull breeding soundness examination conducted at michigan state university from 2007-2017 were analyzed to validate, verify, and evaluate the process. all bulls were examined using society for theriogenology bull breeding soundness guidelines. with a conservative α of .001, nominal logistic regression was used to identify influential factors and potential sources of bias, and determine predictive accuracy of the breeding soundness examination itself in classifying reproductive potential. of 2,887 records evaluated, 82% of bulls were classified as satisfactory potential breeders, 3% unsatisfactory, and 15% deferred. factors identified as influential in final classification of bulls included season, veterinarian bias, presence of white blood cells, and percent morphologically normal sperm. season of year and presence of white blood cells in ejaculate significantly affected the outcome. routine and consistent evaluation of bulls is important for economic success of beef cow-calf operations. further studies are needed to determine if the seasonal effect is related to ambient temperature or underlying physiology of spermatogenesis in the bull. some components of the breeding soundness examination may be unnecessary. we concluded that veterinarians and farmers could save time and money by reducing the number of data collection points while increasing reproductive potential of the herd by using only influential factors in the process. breeding soundness examination has been useful in evaluation of reproductive potential in bulls; however, alternate formulas of reproductive potential may have higher predictive accuracy and validity and may be more cost effective. keywords: breeding soundness examination, validity, process improvement, resource allocation introduction breeding soundness examination (bse) of beef bulls is critical to reproductive success of cowcalf operations. infertile or sub-fertile bulls can negatively impact reproductive performance of the cow herd, resulting in poor conception rates, delayed conception, decreased calf crop, and reduced weaning weights which cause economic losses. previous studies examining benefits of performing a bse concluded that bulls with >70% morphologically normal sperm produce more calves than bulls with <50% normal sperm and that quality of sperm in herd sires is directly related to the size of the calf crop.1 additionally, bulls not subjected to bse prior to the breeding season had 6% lower conception rates than those selected for use, based on the outcome of a bse.2 the breeding potential of a bull is often assessed prior to sale or at the beginning of each breeding season. fertility and physical soundness can change over time and may be affected by various factors, including genetics, environment, stress, body condition, trauma and age.3 additionally, there is variation among veterinarians conducting bses, particularly when measuring scrotal circumference and percent morphologically normal sperm, which may result in some satisfactory bulls failing the bse.4 however, an annual bse is an adequate screening tool and aids in identification of bulls with sub-optimal fertility so they can be culled from the herd to improve farm production efficiency and profitability. the society for theriogenology (sft) established guidelines for conducting a bull bse that evaluates breeding potential of a bull, based on standards for scrotal circumference and sperm motility and morphology. 5 these criteria are intended to identify bulls with potential to attain pregnancy in ≥25 healthy, cycling cows during a 65-70 day breeding period.6 to be classified as a satisfactory potential breeder, bulls are evaluated in each of these categories and must meet a minimum recommended scrotal circumference based on age, >70% morphologically normal sperm and >30% motility. in addition, the bull must also be physically sound and free from reproductive or genetic defects. in addition to evaluation of sperm motility and morphology, cytologic examination of the ejaculate is also performed during a bse. cytologic examination can be beneficial for identification of 397 clinical theriogenology • volume 10, number 4 • december 2018   white blood cells (wbc) or other round cells such as epithelial cells or immature spermatids in the ejaculate, a procedure not performed routinely. during epididymal transit, abnormal sperm are eliminated via phagocytosis by macrophages; therefore, up to 85% of bulls may have wbc present in the ejaculate, regardless of fertility.7 in human semen, leukocytes induce peroxidative damage to sperm and reduced fertilizing potential.8 although the impact of leukocytes in bull semen is unclear and no formal threshold has been outlined for classification of a satisfactory bull based on wbc, the presence of >5 wbc per high power field (hpf) at 1000x magnification has been suggested as a cut off and may be useful during clinical examination of bulls.9 the objective was to evaluate records from bses conducted by veterinarians at the michigan state university (msu) veterinary medical center and at remote sites during the msu extension spring bull test program from 2007-2017. specific goals were to identify trends in age, breed, reproductive soundness and other factors that influenced outcomes of bses in michigan beef bulls and to evaluate effectiveness of the bse. several hypotheses motivated the investigation: (h1) season is associated with gross motility and morphology; (h2) season is associated with bse outcome; (h3) individual veterinarians systematically perform the bse differently from each other, potentially introducing bias into this study sample of bse results; and, (h4) that the bse is a valid predictor of reproductive potential. materials and methods data used in this study were collected during routine clinical evaluation of bulls at the msu veterinary medical center or at remote sites for the msu extension spring bull test program from 2007-2017. records were included in the analysis if all data points were present on the paper bse form: age, breed, date of exam, gross motility, morphology, scrotal circumference, wbc count as measured per high power field, and final classification. each bse record included the name of the veterinarian conducting the bse. a total of 2,887 records were analyzed and 435 were excluded due to incomplete or inaccurate information in the bse record. all examinations followed the 1993 sft bull bse guidelines.5 bulls were examined visually for physical defects prior to reproductive examination. scrotal circumference measurements were obtained for each bull and transrectal palpation was done to assess internal reproductive structures. semen was collected via electroejaculation into a plastic collection cone maintained in a sleeve filled with warm water to minimize effects of cold temperatures on sperm. after collection, a drop of semen was pipetted onto a warmed slide to assess gross motility at 40x magnification. eosin nigrosin stain was mixed with the ejaculate on a slide, smeared and allowed to dry prior to observation under oil immersion at 1000x magnification on a bright field microscope for evaluation of sperm morphology. finally, a cytologic preparation of one drop of semen spread onto a slide and stained with dip quick stain (jorgensen laboratories, inc., loveland, co) was used to quantify wbcs present in the ejaculate. each bull was classified as satisfactory, unsatisfactory, or deferred based on minimum standards of the sft guidelines. a power calculation was conducted prior to analysis, with an α of 0.05, 5 degrees of freedom, an effect size of 0.10, and a sample size of 2,887 yielded a power of 0.99. data were analyzed with jmp® pro version 13.0.0 (sas institute inc., cary, north carolina, usa). descriptive statistics were calculated. all statistical tests were performed with a highly conservative α of 0.001, and a conservative statistical significance value of 0.001 was used as a cutoff value.10 a variable named season was created by grouping bses conducted during fall/winter months (october – march) or spring/summer months (april – september) to measure effects of ambient temperature on bse outcomes. nominal motility descriptions (i.e., poor, fair, good, very good) were transformed into ordinal values (i.e., 1-4) to yield more information in the analyses. bivariate tests were used to determine if there were associations between all combinations of: age, breed, season, gross motility, morphology, scrotal circumference, veterinarian conducting the exam, wbc count, and bse classification results. after bivariate tests were performed, nominal logistic regression was performed, controlling for age, breed, season, gross motility, morphology, scrotal circumference, veterinarian conducting the exam, and wbc count, to identify predictors of bse outcomes and to determine what elements of the bse are most useful. after model construction, multiple combinations of variables used in the bse method were 398clinical theriogenology • volume 10, number 4 • december 2018   used to construct different bse models, and these new bse models were compared using bayesian information criterion (bic). this approach was used to determine if the bse exam itself could be improved by comparing the goodness of fit of the new bse models versus the bse method used in michigan from 2007-2017. false discovery rates (fdr) were calculated for each variable to determine the importance of each measurement within the bse method itself and to reduce the likelihood of false positives during hypothesis testing. lastly, effect likelihood ratio tests were used to conservatively test for associations between factors and bse outcomes. results in this study, 38.6% of bulls presented for bse in michigan were between 10 and 18 months of age, resulting in a right-skewed histogram of age at bse (figure 1). of the bulls examined in michigan the median age was 24 months, and the mean age was 30 months (figure 1). angus bulls represented 50% of all bulls examined, whereas red angus, beef crossbred, polled hereford, simmental, and maineanjou bulls made up an additional 40% of bulls examined (data not shown). there were no significant associations between breed and final classification of the bull. of the bulls presented for bse, 82%, or approximately 4 in 5 bulls, were classified as satisfactory potential breeders. the remainder of bulls were classified as deferred or unsatisfactory, with 4.7 times the number of failing bulls placed into the deferred category. (h1) season is associated with gross motility and morphology: with an alpha of .001, the initial exploratory regression indicated that time of year, or season, was associated with motility and morphology (p < 0.01 for both measures). (h2) season is associated with bse outcome: with an alpha of .001, the effect likelihood ratio test (table 4) indicated that time of year had a χ2 of 20.951 (p < 0.002). initial exploratory regression values indicated an association (p < 0.01) between time of year and bse outcome (table 1). the fdr had a log worth of 4.55 (p < 0.001) for time of year (table 3). when analyzing for the effect of season via nominal regression, season had an estimate of 0.847 with a standard error of 0.251 (p < 0.001). (h3) individual veterinarians systematically performed the bse differently from each other; thus, veterinarians potentially introduced bias into this study’s sample of bse results: veterinarians that conducted the bse where analyzed as a group and individually. with an alpha of .001, during the initial exploratory regression (table 1), veterinarians were analyzed together as a group, and all variables in the model were associated with veterinarians, except for motility (p = 0.08) and wbc (p = 0.47). in contrast, when veterinarians were analyzed individually in the nominal logistic regression (table 2), there was no association between veterinarians and bse outcomes. when veterinarians were combined, the false discovery rate (table 3) yielded a log worth of 1.56 for veterinarians in bse outcomes (p = 0.02). the effect likelihood ratio test indicated a weak association between veterinarians (combined) and bse outcomes (p = 0.027). (h4) bse is a valid predictor of reproductive potential: comparing bse results to predicted bse values using nominal logistical and contingency analysis demonstrated that bse is capable of accurately predicting a satisfactory exam result (figure 2). however, this comparison also indicated that bse does not predict deferred or unsatisfactory results with a high degree of accuracy (figure 3). based on the receiver operating characteristic curve, bse misclassifications were more likely for deferred and unsatisfactory results (figure 3). a comparison of models using bayesian information criteria indicated that the bse would have a higher predictive validity if the number of variables in the bse exam were reduced to the most influential variables in the bse (tables 3-5). 399 clinical theriogenology • volume 10, number 4 • december 2018   bulls with higher wbc count and poor morphology were more likely to be classified as unsatisfactory (p < 0.001). season also influenced the outcome of the bses analyzed in this study. the % normal morphology, high wbc count (1-2), time of year, age in months, and scrotal circumference were highly predictive of bse outcome (table 3). the false discovery rate also indicated that there was a low probability of false positives in bse’s measurements of % normal morphology, wbc count, time of year, age in months, and scrotal circumference when compared against bse outcomes in michigan from 2007-2017. the fdr also indicated that % normal morphology (196.94) and wbc (24.44) were the most influential measurements in the bse (table 3). these results indicated that the bse method could be improved by reducing the number of data points collected in the bse. discussion of the 2,883 bull records examined from the last 10 years, 82% of bulls were classified as satisfactory potential breeders. this was consistent with previous surveys indicating rates of 65-85% for satisfactory classification of bulls presented for bse.11 however, this also means that 1 in 5 bulls will not be classified as a satisfactory potential breeder during a bse, further emphasizing the importance of this examination and its implications for herd reproductive performance and production efficiency. over time, young bulls may be added to the herd as herd sires as the older bulls are culled for various reasons, including declines in fertility or physical abnormalities.4 as indicated in figure 1, 38.6% of bulls presented for bse in michigan are 10-18 months of age, whereas very few bulls >60 months of age were presented for evaluation. this could be because some producers may elect to only test young bulls and not examine older bulls every year. however, due to the above-mentioned factors impacting fertility, testing each bull on an annual basis is important to ensure optimal reproductive performance of the herd. additionally, older bulls are more likely than younger bulls to develop degenerative conditions that can impact physical soundness and fertility. one study identified a significant effect of breed on scrotal circumference of yearling bulls, with breed accounting for 12% of variation in scrotal circumference measurements.12 in the present study, angus bulls represented 50% of bulls examined and no significant associations were identified between breed and any variable used in the bse. therefore, there was no relationship between breed and breeding soundness as measured by the bse. the distribution of breeds in michigan is like other studies conducted in the southern united states; however, previous studies of bulls evaluated by the bse in canada indicated a more even breed distribution, with a smaller proportion of angus bulls.12, 13 with such a large percentage of angus bulls in this study’s sample, there could be an expectation that angus bulls would have associations with the bse outcomes, although none were identified. this further indicates that breed may have no relationship with bse outcomes. whereas several factors influenced the outcome of the bses conducted through the msu bse program from 2007 to 2017, including percent morphologically normal sperm, presence of wbc in the ejaculate, age of bull, scrotal circumference, motility, season, and veterinarian performing the examination, % normal morphology was the most influential factor in the final classification of the bull. these findings were consistent with a previous study in which 83% of bulls classified solely based on morphology were satisfactory breeders and the remaining 17% were unsatisfactory, whereas breed, age, and sc did not significantly affect the percent of morphologically normal or abnormal sperm.14 white blood cell count was identified as a significant factor in final classification; bulls with higher wbc counts were more likely to be classified as unsatisfactory (p < 0.001). white blood cells could indicate infection or inflammation within the reproductive tract of the bull, leading to production of abnormal sperm. additionally, macrophages in the epididymal ducts are responsible for eliminating abnormal sperm and may be present in higher numbers in the ejaculate of bulls with a high percentage of morphologically abnormal sperm.7 although there are no current recommendations for a minimum number of wbc in the ejaculate, a cutoff of >5 wbc per hpf has been suggested and cytologic evaluation of the ejaculate should be considered an important component of a bse.9 season of the year when the examination was performed also influenced parameters evaluated, including sperm motility and morphology (table 2). this was consistent with previous findings that bulls 400clinical theriogenology • volume 10, number 4 • december 2018   subjected to bse during winter months had lower semen quality and were more likely to be classified as unsatisfactory or deferred compared to bulls examined in the summer.3 bses are often performed in field conditions that are not ideal for survival of sperm and cold shock can decrease motility and increase abnormal morphology. despite the use of slide warmers, block heaters and warm water baths around the collection cone to reduce cold shock to sperm, the value of field assessment of motility is variable. furthermore, it has been suggested that other seasonal factors may be related to changes in bull fertility, depending on the time of year, including changes in photoperiod, feed and pasture availability, or cold stress. although this study only examined the factors that influence the outcome of the bse on the day of examination, effects of season could also be related to ambient temperatures in the 2 months before the bse as adverse temperatures, either high or low, may negatively impact spermatogenesis leading to changes in motility and morphology. further studies are required to determine how much these factors contribute to seasonal variations in bull semen quality and reproductive performance, or if the effects on bse outcome in colder months are only because of the effects of lower ambient temperatures on the day of evaluation. in the past, motility has been considered an important measure of bull fertility during a bse, and the impact of environmental conditions during motility assessment may have a significant impact on final classification of the bull.15 the value of motility assessment during a bse could be debated, as most studies evaluating the impact of sperm motility on bull fertility have been conducted using post-thaw motility measurements of frozen-thawed bull semen.16-19 effects of ambient temperature and the subjective nature of motility assessment in field bses is well known and reflected in the low minimum threshold for motility outlined in the sft bull bse guidelines.5 in this study, motility was not an important predictor of bse outcomes and therefore could be eliminated from the bse. further investigation into the value of motility in the bse is warranted. based on this study’s results, differences between individual veterinarians conducting bses exist and are a source of bias within the overall population of bse records examined. to correct this source of bias, veterinarians could receive more structured and systematic training on bses, or better and more precise tools to measure the variables in the bse could be used (e.g., computer-assisted semen analysis [casa]). when conducting field bses, however, use of casa may be limited or impractical due to expense and availability. based on nominal regression results (table 2), veterinarians performed bses differently from each other, introducing a source of bias into this study sample of bse results. this factor should be considered when interpreting the results of the bse, especially when making comparisons of individual bulls when two veterinarians completed the bse for an individual bull. the fourth hypothesis, that the bse is a valid predictor of the bull’s reproductive potential, was partially supported; however, the bse method can likely be improved by reducing the number of bse variables in the formula to classify the bull as unsatisfactory, deferred, or satisfactory potential breeder. this statement was supported by all results from this study. focusing on analyses described in tables 3, 4, and 5, scrotal circumference, age in months, time of year, wbc count, and % normal morphology were the most influential factors in the bse method used in michigan from 2007-2017. other factors were of little or no value to in predicting bse outcomes (i.e., % primary, veterinarians, motility scale, breed). after identifying these surprising results, various combinations of the bse’s variables were compared against bse outcomes and the bic results clearly indicated that the number of variables in the bse method could be reduced. this finding may be controversial; however, a follow-up study comparing at least two methods of bse against fertility success is warranted to validate this study’s findings. some of these results were not completely surprising as the population of animals selected for this study was not a truly random sample. this study sample excluded bulls not selected for bse based on prior selection criteria implemented by the farmer such as genetic merit or desired phenotype. furthermore, survival of the bulls evaluated in this study bse were selected using the bse itself, and this partially violates the assumption of independence. considering that producers use the bse to select the best bulls, finding an untested bull population for comparison would be difficult and likely costly. this study used highly conservative assumptions to help correct for these methodological issues. 401 clinical theriogenology • volume 10, number 4 • december 2018   our results indicated that the bse accurately predicts a satisfactory classification, but does not accurately predict unsatisfactory or deferred classifications. misclassifications are more likely to occur for bulls in deferred or unsatisfactory classifications; this is problematic for producers, as culling a bull with false positive unsatisfactory classification likely results in unwarranted culling (figures 2&3). this finding could be particularly important for young bulls that may require more than one bse to achieve a satisfactory classification. in a study evaluating spermiograms of pubertal bulls between 11 and 15 months of age, only 42% of bulls within this age range had a mature spermiogram defined as >60% motility and >70% normal morphology.20 based on previous evaluations of bse results over time, young bulls and older bulls usually accounted for most of the deferred and unsatisfactory classifications, with most deferrals of young bulls due to immaturity.13 misclassification of a bull as deferred or unsatisfactory may result in culling from the herd, loss of genetic potential, and increased cost to the producer to replace the bull. measurement of scrotal circumference (sc) was not as influential in the final classification of the bull as wbc count and % morphologically normal sperm. scrotal circumference has been included as a standard in the bse, due to its correlation with total testis weight, sperm production capacity, fertility of offspring, and earlier pubertal development of female offspring.21-23 furthermore, bulls with sc ≤34 cm are more likely to have a higher percentage of morphologically abnormal sperm compared to bulls with sc >34 cm and sc is highly correlated to % morphologically normal sperm in bulls between 11 and 15 months of age.20 although it may be important to identify young bulls that do not meet the minimum criteria for sc by age, sc may not be an important measurement in older bulls during routine bse, as the correlation between sc and sperm production capacity decreases as bulls age.21 future research to improve the predictive value of a bse should be directed to determine how bulls in different locations and time periods may differ. while this study yielded important results regarding the importance of season and wbc, future studies should seek to include counterfactual data as a basis of comparison. future studies should include the pregnancy outcomes of cows bred to bulls classified as satisfactory using the modified methodology proposed by the findings of this study. such findings may allow for modifications to the bse procedure that more accurately predict reproductive potential of bulls, while potentially reducing the number of analyses that need to be performed in the bse itself. this could save veterinarians time and resources when conducting bses. routine and consistent evaluation of bulls is important for economic success of beef cow-calf operations. understanding the physiology of spermatogenesis and factors impacting the outcome of the bse can aid veterinarians in providing valuable information during a bse. that the wbc count in the ejaculate was an influential factor in bull classification during bse indicated this should not be overlooked. accurate and complete examination records allow for periodic analysis of data to provide evidence-based recommendations to producers seeking to select highly fertile breeding bulls as herd sires. overall, the bse must be critically analyzed to determine which of the current measurements are necessary and most predictive of reproductive potential of herd sires to improve process efficiency for veterinarians and producers. acknowledgements the authors thank ms. katherine koebel for her assistance with data entry prior to analysis. 402clinical theriogenology • volume 10, number 4 • december 2018   references 1. fitzpatrick la, fordyce g, mcgowan mr, et al: bull selection and use in northern australia: part 2. semen traits. anim reprod sci 2002;71:39-49. 2. wiltbank jn, parish nr: pregnancy rate in cows and heifers bred to bulls selected for semen quality. theriogenology. 1986;25:779-783. 3. barth ad, waldner cl: factors affecting breeding soundness classification of beef bulls examined at the western college of veterinary medicine. can vet j 2002;43:274-284. 4. waldner cl, kennedy ri, palmer cw: a description of the findings from bull breeding soundness evaluations and their association with pregnancy outcomes in a study of western canadian beef herds. theriogenology 2010;74:871883. 5. chenoweth pj, spitzer jc, hopkins, fm: a new bull breeding soundness evaluation form. proc annual meeting of the society for theriogenology 1992; p. 63-70. 6. kastelic jp, thundathil jc: breeding soundness evaluation and semen analysis for predicting bull fertility. reprod dom anim 2008;43:368-373. 7. zart al, jurgielewicz vcl, fernandes ce: seminal leucocytary profile in beef bulls. reprod dom anim. 2014; 49:719-724. 8. johanisson e, campana a, luthi r, et al: evaluation of ‘round cells’ in semen analysis: a comparative study. hum reprod update. 2000;6:404-412. 9. sprecher dj, coe ph, walker rd: relationships among seminal culture, seminal white blood cells, and the percentage of primary sperm abnormalities in bulls evaluated prior to the breeding season. theriogenology 1999;51:1197-1206. 10. colquhoun d: an investigation of the false discovery rate and the misinterpretation of p-values. r soc open sci. 2014;1:140216. 11. chenoweth pj, mcpherson fj: bull breeding soundness, semen evaluation and cattle productivity. anim reprod sci. 2016;169:32-36. 12. guerra ág, hendrick s, barth ad: increase in average testis size of canadian beef bulls. can vet j 2013;54:485-490. 13. carson r, koziol j, wenzel j, et al: twenty year trends of bull breeding soundness examinations at a teaching hospital. clinical theriogenology 2014; 6:495-501. 14. menon ag, barkema hw, wilde r, et al: associations between sperm abnormalities, breed, age, and scrotal circumference in beef bulls. can j vet res 2011;75:241-247. 15. chenoweth pj: bull breeding soundness exams and beyond. proc applied reproductive strategies in beef cattle workshop 2002; p. 174-180. 16. stalhammer em, janson l, philipsson j: the impact of sperm motility on non-return rate in preselected dairy bulls. reprod nutr dev 1994;34:37-45 17. farrell pb, presicce ga, brockett cc, et al: quantification of bull sperm characteristics measured by computerassisted sperm analysis (casa) and the relationship to fertility. theriogenology 1998;49:871-879. 18. kasimanickam r, nebel rl, peeler id, et al: breed differences in competitive indices of holstein and jersey bulls and their association with sperm dna fragmentation index and plasma membrane integrity. theriogenology 2006;66:1307-1315. 19. sellem e, broekhuijse mlwj, chevier l, et al: use of combinations of in vitro quality assessments to predict fertility of bovine semen. theriogenology 2015;84:1447-1454. 20. arteaga a, baracaldo m, barth ad: the proportion of beef bulls in western canada with mature spermiograms at 11 to 15 months of age. can vet j 2001;42:783-787. 21. willett el, ohms ji: measurement of testicular size and its relation to production of spermatozoa by bulls. j dairy sci 1957;40:1559-1569. 22. smith ba, brinks js, richardson gv: relationships of sire scrotal circumference to offspring production and growth. j anim sci 1989;67:2881-2885. 23. coulter gh, foote rh: bovine testicular measurements as indicators of reproductive performance and their relationship to productive traits in cattle: a review. theriogenology1979;11:297-311. 403 clinical theriogenology • volume 10, number 4 • december 2018   table 1. summary of initial exploratory regression values with an alpha of .001. tim e of y ear v eterinarian breed a ge in m onths scrotal circum ference m otility scale % n orm al m orphology % prim ary % secondary w hite blood cell count breeding exam status time of year x veterinarian <0.01 x breed <0.01 <0.01 x age in months <0.01 <0.01 <0.01 x scrotal circumference <0.01 <0.01 <0.01 <0.01 x motility scale <0.01 0.08 <0.01 <0.01 <0.01 x % normal morphology <0.01 <0.01 <0.01 0.02 <0.01 <0.01 x % primary <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 x % secondary <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.01 0.01 x white blood cell count 0.99 0.47 1.00 0.39 0.04 <0.01 <0.01 <0.01 <0.01 x breeding exam status <0.01 <0.01 <0.01 0.10 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 x 404clinical theriogenology • volume 10, number 4 • december 2018   table 2. nominal regression results for the bse measurements controlling for potential sources of bias against bse outcomes with an alpha of .001. term estimate std error χ 2 prob > χ 2 lower 99.9% upper 99.9% intercept -1.975 7,534.421 0.00 0.999 -24,794.192 24,790.240 time of year [summer] -0.847 0.251 11.34 < 0.001 -1.763 -0.078 veterinarian [1] -1.274 4,319.803 0.00 0.999 -14,215.705 14,213.155 veterinarian [2] 14.962 21,599.018 0.00 0.999 -71,057.185 71,087.110 veterinarian [3] -3.268 4,319.803 0.00 0.999 -14,217.699 14,211.161 veterinarian [4] -4.206 4,319.803 0.00 0.999 -14,218.636 14,210.223 veterinarian [5] -2.812 4,319.803 0.00 0.999 -14,217.242 14,211.616 age in months -0.032 0.006 22.09 < 0.001 -0.053 -0.009 scrotal circumference 0.096 0.044 4.61 0.031 -0.0513 0.243 motility scale 0.250 0.200 1.56 0.211 -0.408 0.909 % normal morphology 0.224 0.015 223.30 < 0.001 0.174 0.273 % primary -0.0209 0.015 1.88 0.170 -0.071 0.028 % secondary 0 0 . . . . white blood cell count [1-2] -6.704 1.028 42.52 < 0.001 -10.908 -3.450 white blood cell count [2-3] -20.617 10,818.086 0.00 0.998 -35617.818 3,5576.583 white blood cell count [3-4] 18.176 9,1967.897 0.00 0.999 -302604.65 302,641.001 table 3. false discovery rates for each measurement within the breeding soundness exam data with an alpha of .001. source log worth p value % normal morphology 196.94 < 0.001 white blood cell count 24.44 < 0.001 time of year 4.55 < 0.001 age in months 4.36 < 0.001 scrotal circumference 3.06 < 0.001 % primary 1.87 0.01 veterinarian 1.56 0.02 motility scale 1.48 0.03 breed 0.00 0.99 % secondary 405 clinical theriogenology • volume 10, number 4 • december 2018   table 4. effect likelihood ratio tests with an alpha of .001. source df χ 2 p value time of year 2 20.951 < 0.001 veterinarian 10 20.231 0.027 breed 100 51.224 1.000 age in months 2 20.120 < 0.001 scrotal circumference 2 14.105 < 0.001 motility scale 2 6.841 0.032 % normal morphology 2 906.972 < 0.001 % primary 2 8.610 0.013 % secondary 0 0 . white blood cell count 6 127.864 < 0.001 table 5. comparison of model fit using bayesian information criterion, controlling for veterinarians, seasonality, and bull age. model bic current method of bse including all variables: scrotal circumference, motility, % normal, % primary, % secondary, wbc 2,258.97 new bse with 5 measures: scrotal circumference, motility, % normal, % primary, wbc 2,243.21 new bse with 4 measures: scrotal circumference, % normal, % primary, wbc 2,241.88 new bse with 3 measures: scrotal circumference, % normal, wbc 2,235.08 new bse with 2 measures: % normal, wbc 2,251.35 new bse with 1 measure: % normal 2,369.94 406clinical theriogenology • volume 10, number 4 • december 2018   figure 1. histogram of age in months of bulls presented for bses in michigan (n=2,887) from 2007-2017. the median age of bulls examined was 24 months. figure 2. a mosaic plot of a contingency analysis of most likely breeding exam status comparing bse results to nominal logistical regression predicted bse values. the bar on the right of the mosaic plot depicts the actual results of the bse in michigan from 2007-2017. the bars to the left indicate that the bse accurately predicted satisfactory exam results, but the bse did not accurately predict deferred or unsatisfactory results. 407 clinical theriogenology • volume 10, number 4 • december 2018   figure 3. a receiver operating characteristic (roc) graph of the bse’s outcomes predicted outcomes compared against actual outcomes (blue = deferred, red = satisfactory, green = unsatisfactory). the roc curves indicate that the bse can be improved by reducing the amount of false positive test results, especially in bse misclassifications of deferred and unsatisfactory. these false positives often lead to reexaminations or culling, and likely result in lost time, money, and other resources for veterinarians and producers. 408clinical theriogenology • volume 10, number 4 • december 2018 000_frontal ms_1 forematter for december 2018 ms_2 letter1 for december 2018 ms_2 letter2 for december 2018 ms_2 letter3 for december 2018 ms_3 table of contents ms_4 therio news for december 2018_bw 2019 therio preview therio recap with pics steiner-new officers 2018 therio sponsors 2018 exhibitors-sft board noms 2018 auction donations 2019 bartlett nomination act call for noms call for abstracts 2019 for ctj the society for theriogenology and american college of theriogenologists issue a call for research and case abstracts to be presented at the annual therio conference july 24-27, 2019 in savannah, georgia. 1. competitive category: 2. non‐competitive category: 3. case abstract category: 4. poster category: general: guidelines: what to send and how to send it abstracts not adhering to these guidelines will not be considered for presentation. deadline: abstracts must be received no later than february 15, 2019 for consideration. call for 2019 student case presentations.pdf call for student case presentations-2019 annual therio conference student case presentations call for student case presentations also available at www.therio.org guidelines for application submission society for theriogenology student case presentation competition annual therio conference, savannah, georgia, july 24-27, 2019 ms_5 editorial 001a_ms_6 roberts color pictures 002_ms_7 root kustritz 003_ms_8 ferrer 004_ms_9 wiley 005_ms_10 wallace 006_ms_11 peterson 007_ms_12 nobre 008_ms_13 sidelinger 009_ms_14 mirando 2018: testicular size as an indicator of poor sperm output in white-tailed (odocoileus virginianus) and mule (odocoileus hemionus) deer bucks testicular size as an indicator of poor sperm output in white-tailed (odocoileus virginianus) and mule (odocoileus hemionus) deer bucks with rising popularity of cervid hunting, there is an increased demand for epididymal harvesting as a final option to preserve genetics of valuable bucks. while seasonality plays a crucial role in reproductive characteristic fluctuations in bucks, little is known about minimal testicular volume and sperm output.1 here we describe morphological and histological characteristics of abnormal testes from captive white-tailed deer (wtd) and mule deer (md) bucks and a normal white-tailed deer (cont) in rut. all three testicle pairs were obtained postmortem examination during peak rut (oct to dec) and shipped overnight on ice for routine epididymal sperm harvest. cauda epididymis and vas deferens were removed, dissected, and flushed with optixcell extender (imv technologies). the cont flush yielded a total output of 2.5 billion sperm with 56% motility. in comparison, the wtd flush yielded dilute semen (<10 million sperm/ml) with 50% motility. the md flush revealed no mature spermatozoa, but numerous spheroids. testes measurements were used to estimate testicular volume, as shown in the table. the cont testes exceeded the minimum recommended volume of 25 cm3, whereas the wtd and md testes were subpar. testes were fixed in 10% formalin, paraffin-embedded, and stained with hematoxylin and eosin and masson’s trichrome. the wtd testes had marked collagen and connective tissue present within interstitial spaces. additionally, there was reduced spermatogenesis within seminiferous tubules compared to cont. the md testes showed no evidence of spermiogenesis within the seminiferous tubules and increased collagen deposition and disorganization within the interstitial space. lack of spermiogenesis in the md suggests potential hereditary hypoplasia, whereas the wtd may have been undergoing testicular atrophy.2 though different etiologies, both findings indicate that testicular size can be useful as an indicator of sperm output and a predictor of subor infertility in wtd and md. establishment of minimal testicular volume necessary for a successful epididymal flush would prevent unnecessary shipment of unusable testes. table. testicular volume estimation (tv=0.5236×l×w2; l=length, w=width)1 keywords: cervidae, epididymal sperm harvest, hypogonadism, semen quality references 1. stewart j, schmidt l, ellerbrock re, et al: effects of seasonality on male white-tailed deer (odocoileus virginianus) reproductive characteristics. am assoc small rumin pract research summary, omaha, ne. 2017. 2. marburger rg, robinson r, thomas jw: genital hypoplasia of white-tailed deer. j mammal 1967;48:674-676. cont wtd wtd md left right left right left right length (cm) 7.8 7.5 3.9 4.5 4.4 3.8 width (cm 3.7 4.5 2.5 3.2 2.0 2.0 volume (cm3) 55.91 79.52 12.76 24.13 9.21 7.98 clinical theriogenology • volume 10, number 3 • september 2018359 n. sugaia, c.f. shipleya, r.e. ellerbrockb, j.l. stewarta acollege of veterinary medicine, university of illinois urbana-champaign, urbana, il bcollege of veterinary medicine, university of georgia, athens, ga 2018: development of a subjective scoring system for evaluating corpus luteum function in beef cattle   development of a subjective scoring system for evaluating corpus luteum function in beef cattle bethany j. funnell,a austin egloff,b emily g.taylor,b kara stewartb aveterinary clinical sciences, college of veterinary medicine, purdue university, west lafayette, in; bdepartment of animal sciences, college of agriculture, purdue university, west lafayette, in abstract the objective of this project was to develop a scoring system to evaluate blood flow through the corpora lutea and circulating progesterone concentrations in heifers and cows through the progression of a normal, nonmanipulated estrous cycle. sixteen, non-lactating, beef females were subjected to reproductive tract ultrasound scans every other day for the duration of an entire normal bovine estrous cycle (22 days). ovarian structures (follicles and corpora lutea) were identified and measured. color doppler mode was used to visualize blood perfusion through luteal tissue and was given a subjective score based on percent of the corpus luteum that showed color (0, no color; 1, small color at periphery; 2, up to 80% color; 3, >80% color). venous blood samples were collected at the time of ultrasound examination, and serum progesterone values were used to confirm corpus luteum function. the area of the corpus luteum was correlated with serum progesterone concentrations (p <0.0001, r=0.45). doppler score was strongly correlated with serum progesterone concentrations (p < 0.0001, r=0.62) and area of the corpus luteum (adjusted for lacunas) (p <0.0001, r=0.48). peak serum progesterone concentrations were not correlated with age (p=0.74), weight (p=0.80), or bcs (p=0.13). there was a tendency for peak progesterone concentration to be correlated with the area of the corpus luteum (p=0.07, r=0.46). doppler score was not correlated (p = 0.59) with ultrasonographer. in summary, doppler scores can be utilized as an estimation of corpus luteum function, irrespective of scanning technician, and may be a more reliable measure than by corpus luteum area determination, alone. keywords: bovine, ultrasound, progesterone, doppler score, corpus luteum, perfusion introduction food animal veterinary medicine has embraced ultrasound technology, and the use of this diagnostic tool is continuing to rapidly expand in both private practice, and well as in research applications. many of the more advanced capabilities of ultrasound were initially cost prohibitive, such that the addition of these capabilities were restricted by economics to research or referral institutions. as with most technological advancements, costs of adding additional capabilities to ultrasound units have decreased to approach potential value as an added diagnostic tool or veterinary practice service, if a routine application would exist. doppler ultrasonography has been used extensively in cardiac evaluation for many years, but more recently, blood flow and perfusion of tissues of the reproductive tract has gained the attention of some researchers. more and more research trials are published, in which doppler (color flow and power) is used to attempt to further elucidate normal processes, as well as potentially illustrate physiologic responses to treatments. capturing doppler images on portable ultrasound units is a very quick process that requires very little additional training for the skilled ultrasonographer. the practical implementation and interpretation of these images remains a challenge. progesterone and corpus luteum size there is no doubt that adequate circulating progesterone (p4) is an integral component to the reproductive success of the bovine. higher progesterone in circulation has positive influences on the developmental competence of the oocyte, enhances conceptus development, and is critical for the establishment and maintenance of pregnancy.1 pregnancy in the cow has many direct and indirect economic benefits,2 and attempts to maximize the opportunities to establish pregnancies are incessant. many studies have shown a positive correlation between corpus luteum (cl) size and circulating progesterone concentrations.3-11 as the cl is developing (days 0-7 of the estrous cycle), progesterone concentration increases proportionally to cl growth, nearly doubling between day 4 and day 7 of the cycle.4 however, the cl will increase in size during the static phase (day 7 to day 14) by only clinical theriogenology • volume 10 number 1 • march 201835   approximately 25 percent, but progesterone concentrations will double, again, during this period of time.12 this is believed to be due to the angiogenic potential of the cl and the development of capillaries within the luteal tissue which permit greater delivery of blood (and signaling molecules) to the luteal tissue, as well as increased transportation of progesterone (and other signaling molecules) from the cl.13 after exposure to prostaglandin f2α (pgf), both progesterone and cl size decrease, but not in a proportional manner. within 48 hrs of pgf administration, circulating progesterone levels decreased dramatically (80% decline in 48 hrs).4,14 in contrast, the cl size decreased more gradually, with negative growth pattern (regression phase) similar in change and duration as the positive growth pattern (growth phase), rather than exhibit a dramatic decrease in size, as progesterone levels would suggest.4,15 this finding supports the theory that cl perfusion is impacted in a much more immediate manner, by the decrease in blood volume circulating through the luteal tissue, as well as an increase in the resistance to blood perfusion through the luteal tissue. using doppler as an estimate of cl function researchers in a previously mentioned study evaluated luteal blood flow in conjunction with cl size and progesterone concentrations.4 blood flow was estimated using image capture and pixelflux software (version 1.0, chameleon software, leipzig, germany). not surprisingly, luteal blood flow changes mirrored that of progesterone, but not cl size. indeed, all three measures (cl size, luteal blood flow, and progesterone concentration) all increased in a linear fashion, nearly doubling from day 4 to day 7 of the cycle. as cl growth slowed during the static phase, luteal blood flow continued to increase comparable to progesterone (doubling, again, from day 7 to day 14).4 this finding suggests that there may be opportunities to utilize doppler technology to more accurately classify a cl as functional or nonfunctional, rather than using cl size to estimate function. it has been suggested that a luteal blood flow measure of >0.6cm2 could be a reliable indicator for cl function.4 in contrast to this suggested standard, findings of a study by scully, et al,9 did not observe as dramatic and divergent blood flow change between pregnant and nonpregnant beef heifers on day 18 after insemination, but rather concluded that the cl blood flow ratio (percent of the cl area with color pixilation) was increased in pregnant heifers on day 18, and could be more diagnostic of cl function. current quantification systems are not yet available on smaller, more portable cow-side ultrasound units, which have greatly limited the applicability of this tool in an on-farm setting. as acceptance of doppler technology for cl assessment becomes a staple of the bovine reproductive examination, if can be assumed that the manufacturers of the ultrasound units would add such applications as standard features to their machines. until quantitative evaluation systems are available as software applications on the smaller, more widely used ultrasound machines, an alternative would be to use a subjective score to classify blood flow through the cl.3,17 a functional cl is critical to the success of the transfer of embryos, and a subjective visual assessment system may identify some recipients that would not be suitable candidates for transfer, if doppler scoring indicates that cl perfusion is suboptimal. in addition, some researchers are attempting to utilize cl blood flow as a very early indicator of pregnancy status (day 16-20 after estrus),3,7-9,17 which is especially valuable in the identification of pregnant and non-pregnant dairy cows, so that appropriate interventions can be implemented in a timely manner (prior to the onset of the next standing estrus).3,17-19 likewise, the greatest expense in an embryo transfer program is recipient costs, and the early identification of pregnant and non-pregnant recipients can aid in the economic management of the herd.8 some researchers have shown reliability and repeatability in open versus not open diagnoses.3,7,8 in one study, the positive predictive value of pregnancy was 65.1%, and the negative predictive value was excellent at 98.5%, with accuracy of 74.8%.7 these results were assessed very near the time of onset of the next estrus (day 20), and utilized subjective evaluations of cl perfusion. the objective of this project to correlate serum p4 concentrations with a subjective visual doppler score, to be used as a chute-side assessment of cl function. such a scoring system has potential utility in many bovine reproductive capacities. clinical theriogenology • volume 10 number 1 • march 2018 36   materials and methods all procedures and sample collections were performed in accordance with the guidelines of the purdue animal care and use committee. seven non-pregnant nulliparous, five non-pregnant primiparous, and four non-pregnant multiparous bos taurus (angus x simmental) females (all nonlactating) were housed in a dry-lot and were fed to maintenance, according to nrc recommendations. body weight and body condition score (bcs) were recorded for all cows at the start of the study. all females were subjected to reproductive ultrasound examination (micromax, fujifilm sonosite inc, bothell, wa) at a frequency of 7.5 mhz by one of three technicians every other day for the duration of one complete bovine estrous cycle. ovarian structures (follicles, corpora lutea) were mapped and measured. if a corpus luteum (cl) was present, a color-flow doppler sonography subjective score was recorded by the technician on a scale from 0-3 based on the following criteria: 0) no doppler color; 1) small doppler color on periphery of cl; 2) up to 80% of cl having doppler color; and 3) greater than 80% of cl having doppler color (fig. 1). blood samples were collected by jugular venipuncture in 10 ml evacuated tubes (vacutainer, becton, dickinson and company, franklin lakes, nj) on the day of ultrasound examination, and were placed on ice immediately after collection. samples were cooled for 12-24 hours, and were centrifuged at 2800 x g for 20 minutes at 4°c, and serum was removed and stored at -20oc until assayed. plasma progesterone concentration was determined using an in vitro diagnostic kit; siemens 06603261 immulite® progesterone kit (siemens medical solutions diagnostics, los angeles, ca). to validate the progesterone assay for bovine plasma, parallelism, recovery and repeatability were checked. dilution curves of plasma samples and pure progesterone standards were parallel. when known amounts of progesterone were added to bovine plasma, recovery rates ranged from 89.5% to 104%. eight plasma samples were evaluated 10 times each and the intra-assay cv was 5.8%, indicating good repeatability. the inter-assay cvs for a known range of 0.52-1.26 mg/dl was 12.81, 5.99-11.1 mg/dl was 5.33 17.130.5 mg/dl was 7.79. correlation analyses were conducted using the spearman correlation coefficients in proc corr of sas version 9.3 (sas inst. inc., cary, nc). variables analyzed were doppler score (dop), progesterone serum concentrations (prog), area of the cl (area), and day of ultrasound (day). additional correlations between peak serum progesterone (the highest recorded progesterone concentration during the cycle) concentration and age (heifers, two year-olds and 3+ years), weight, bcs and area of the cl were performed. correlation between doppler score and ultrasound technician were evaluated. any pvalue less than 0.05 was considered significant. p-values >0.05 and ≤0.10 were considered a tendency. results the area of the cl was correlated with serum progesterone concentrations (p <0.0001, r=0.45). peak serum progesterone concentrations were not correlated with age (p=0.74), weight (p=0.80), or bcs (p=0.13). there was a tendency for peak progesterone concentration to be correlated with the area of the cl (p=0.07, r=0.46). doppler score was strongly correlated with serum progesterone concentrations (p < 0.0001, r=0.62). doppler score was also significantly correlated with area of the cl (adjusted for lacunas) (p <0.0001, r=0.48). ultrasonographer was not correlated with doppler score (p = 0.59). discussion data from this project support the historical standards of circulating progesterone when determining cyclicity status,20-22 and this information is validated by the visible presence of luteal tissue as well as the presence of adequate cl blood perfusion. circulating progesterone levels were well correlated with blood flow through the corpus luteum based on ultrasonographic evaluation of the cl utilizing color doppler as a means of subjectively evaluating blood flow. scores assigned to cl blood flow were repeatable between scanning individuals, and repeatability implies a degree of reliability, or consistency across individuals in the evaluation of cl function. this technique can therefore be utilized to assess the function (progesterone production) of a clinical theriogenology • volume 10 number 1 • march 201837   corpus luteum. assessment, or scoring, of cl function may serve a practical purpose, by providing an additional piece of information in a fertility examination, and presenting a clearer picture to the clinician of a given animals cyclicity status. a corpus luteum can persist in the ovary of a cow for an extended period of time, physical destruction of the luteal tissue is a function of the immune system, and is a process that occurs over time.13,23,24 however, decrease in progesterone concentrations subsequent to pgf exposure occurs at a much more accelerated rate, thereby indicating that the drop in progesterone is not simply due to luteal tissue destruction and removal. power doppler evaluation of the dynamics of blood flow through the corpus luteum before and immediately after pgf administration has shown that after an initial increase in cl blood flow (30 min to 2 hr after pgf administration), there was a significant decrease in vessel diameter and increased resistance to blood flow through the cl, effecting a dramatic decrease in the concentration of circulating progesterone in a very short period of time.14 in contrast, in a study by herzog et al,4 the findings did not detect an increase in blood flow in response to pgf exposure. however, due to the short duration of the blood flow increase (30-120 minutes) it is possible to have missed this event. from a practical standpoint, if this blood flow increase is present during a single doppler evaluation, it may be impossible to correctly differentiate between a functioning cl and one that is in the early stages of luteolysis. conclusion utilization of doppler ultrasonography presents a very real opportunity for practitioners to make rapid, cow-side judgments regarding the function of the cl in the bovine female. however, the routine, practical use of this technology requires further exploration. from a purely observational standpoint, this study is in agreement with others, illustrating that cl function can be assessed by visualization of blood perfusion, and that these subjective assessments are repeatable between ultrasonographers. ultrasound examination, utilizing b mode (presence or absence of a cl) and doppler (color flow or power) using the proposed scoring system, with refinement and further large-scale studies, may provide a more rapid and less invasive means to categorize cows as having >1ng/ml of circulating progesterone (cycling) or <1 ng/ml of circulating progesterone (not cycling), than by venipuncture and serum progesterone assays. the doppler scoring system may aid in identification of “open” versus “not open” cows prior to early pregnancy diagnosis. alternatively, and perhaps just as importantly, this scoring system may provide a means to estimate circulating progesterone concentrations at the initiation of an estrus and ovulation synchronization program, in an effort to maximize the developmental competence of the oocyte to be ovulated from a manipulated ovulation. each pregnancy in a bovine animal is an investment, and the value of a potential pregnancy is not one that a client is willing to put at risk without very significant evidence supporting an open diagnosis. shortcomings still exist in our ability to differentiate between a functioning cl and one that is undergoing the very early stages of luteolysis. given the conflicting reports regarding the immediate changes to cl blood flow, more work needs to be done to identify more objective means of quantifying blood flow and perfusion (cl function), as well as appropriate timing of evaluation of function, if we are to identify and institute early interventions (16-20 days after estrus) in the presumably open cow. some of the larger, more costly, and less portable ultrasound machines have the capability to provide real-time quantifications of blood perfusion based on doppler color. however, these machines are too valuable and cumbersome to be brought to the chute or the headlocks for use in routine bovine reproductive practice. another significant challenge of quantifying cl blood flow is that often ovarian vessels not associated with the active cl may show color in the scan box or area, and may therefore give falsely elevated perfusion estimates. many of the more recent publications site the use of image capture and importation of images to image analysis software to report quantification of blood flow through luteal tissues.1,4,6,9 this software allows for estimation perfusion of only the area and structure of interest (cl) by allowing the user to establish a perimeter within which pixilation would be assessed. although effective, this process does not allow the practitioner an immediate quantitative result on which treatment and management decisions can be based. the ultimate goal will be to develop quantitative values as a reliable clinical theriogenology • volume 10 number 1 • march 2018 38   indication of the continuation or termination of the lifespan of a cl in the case of pregnancy or lack of pregnancy, respectively, within a timeframe (day 18-20) that would be convenient for incorporation of an additional ultrasound scan in the normal, routine reproductive management of cattle (once weekly examinations). with the increased durability and portability of other computational tools (i.e. ipads, tablets, laptop computers, etc), there may be an immediate opportunity to bring image analysis software to the barns where the cows are evaluated, if the ultrasound unit itself does not have image analysis software installed. our group is in the process of developing more objective and quantitative parameters for the assessment of the function of ovarian structures in order to produce an immediate cowside result by utilizing a combination of the aforementioned technologies. conflict of interest the authors declare no conflicts of interest. references 1. lonergan p: influence of progesterone on oocyte quality and embryo development in cows. theriogenology 2011;76:1594-1601. 2. devries a.: economic value of pregnancy in dairy cattle. j dairy sci 2006;89:3876-3885. 3. utt md, johnson iii gl, beal we: the evaluation of corpus luteum blood flow using color-flow doppler ultrasound for early pregnancy diagnosis in bovine embryo recipients. theriogenology 2009;71:707-715. 4. herzog k, brockhan-ludemann m, kaske m, et al: luteal blood flow is a more appropriate indicator for luteal function during the bovine estrous cycle than luteal size. theriogenology 2010; 73:691-697. 5. luttgenau j, beindorff n, ulbrich se, et al: low plasma progesterone concentrations are accompanied by reduced luteal blood flow and increased size of the dominant follicle in dairy cows. theriogenology 2011; 76:12-22. 6. bollwein h, luttgenau j, herzog k: bovine luteal blood flow: mechanism and clinical relevance. reprod fertil develop 2013; 25:71-79. 7. pugliesi g, miagawa bt, paiva yn, et al: conceptus-induced changes in the gene expression of blood immune cells and the ultrasounds-accessed luteal function in beef cattle: how early can we detect pregnancy? biol reprod 2014;91:95,1-12. 8. guimaraes crb, oliveira me, rossi jr, et al: corpus luteum blood flow evaluation on day 21 to improve the management of embryo recipient herds. theriogenology 2015;84:537-241. 9. scully s, evans aco, carter f, et al: ultrasound monitoring of blood flow and echotexture of the corpus luteum and uterus during early pregnancy of beef heifers. theriogenology 2015;83:449-458. 10. brogan pt, henning h, stout tae, et al: relationship between colour flow doppler sonographic assessment of corpus luteum activity and progesterone concentrations in mares after embryo transfer. anim reprod sci 2016;166:22-27. 11. kaya s, kacar c, polat b, et al: association of luteal blood flow with follicular size, serum estrogen and progesterone concentrations, and the inducibility of luteolysis by pgf2α in dairy cows. theriogenology 2017;87:467-172. 12. mann ge: corpus luteum size and plasma progesterone concentration in cows. anim reprod sci 2009;115:296-299. 13. shirasuna k, nitta a, sineenard j, et al: vascular and immune regulation of corpus luteum development, maintenance, and regression in the cow. domest anim endocrinol 2012;43:198-211. 14. ginther oj, silva la, arauja rr, et al: temporal associations among pulses of 13, 14-dihydro-15 keto-pgf2α, luteal blood flow, and luteolysis in cattle. biol reprod 2007;76:506-513. 15. luttgenau j, bollwein h: evaluation of luteal blood flow by using color doppler ultrsonography. reprod biol 2014;14:103-109. 16. miyamoto a, kobayashi s, arata s, et al: prostaglandin f2α promotes the inhibitory action of endothelin-1 on the bovine luteal function in vitro. j endocrinol 1997;152:r7-11. 17. siqueira lgb, areas vs, ghetti am, et al: color doppler flow imaging for the early detection of nonpregnant cattle at 20 days after timed artificial insemination. j dairy sci 2013;96:6461-6472. 18. kelley de, galvao kn, mortensen cj, et al: using doppler ultrasonography on day 34 of pregnancy to predict pregnancy loss in lactating dairy cattle. j dairy sci 2017;100:3266-3271. 19. matsui m, miyamoto a: evaluation of ovarian blood flow by colour doppler ultrasound: practical use for reproductive management. vet j 2009;181:232-240. 20. swanson lv, hafs hd, morrow da: ovarian characteristics and serum lh, prolactin, progesterone and glucocorticoid from first estrus to breeding size in holstein heifers. j anim sci 1972;34:284-293. 21. martin o, friggens nc, dupont j, et al: data-derived reference profiles with corepresentation of progesterone, estradiol, lh, and fsh dynamics during the bovine estrus cycle. theriogenology 2013;79:331-343. 22. broes a, leblanc sj: comparison of commercial progesterone assays for evaluation of luteal status in dairy cows. can vet j 2014;55:582-584. 23. miyamoto a, shirasuna k, wijayagunawardane mpb, et al: blood flow: a key regulatory component of corpus luteum function in the cow. domest anim endocrinol 2005;29:329-339. clinical theriogenology • volume 10 number 1 • march 201839   24. smith gw, meidan r: ever-changing cell interactions during the life span if the corpus luteum: relevance to luteal regression. reprod biol 2014;14:75-92. doppler score: 0 doppler score: 1 doppler score: 2 doppler score: 3 fig. 1. visual scoring of the level of perfusion of the cl (0: no perfusion, 1: low, 2: medium, 3: high) (editor’s note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 10 number 1 • march 2018 40 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy 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/preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] /pagelayout /singlepage >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice genomic testing does not correlate with performance trait phenotype in crossbred calves genomic testing does not correlate with performance trait phenotype in crossbred calves mackenzie roberts, michelle kutzler department of animal and rangeland sciences, oregon state university, corvallis, or interest in genetic improvement of performance traits in beef cattle using genomic testing is increasing. genomic testing is especially appealing for young animals who have no progeny of their own on which to judge their genetic merit. in crossbred production systems, medium density genomic panels measuring 50,000 single nucleotide polymorphisms are compared to values from purebred individuals. however, research in crossbred cattle validating accuracy of genomic testing results when compared to actual traits expressed by individual animals (phenotype) is lacking. the objective was to compare actual performance traits in weaned calves to results provided by 2 independent genomic testing companies (neogen, lansing, mi and zoetis, parsippany, nj). it was hypothesized that genomic testing results for performance traits are highly correlated with phenotype. crossbred (predominately angus) calves (n = 94) pastured at oregon state university's soap creek ranch were included in this study. birth and weaning weights were recorded for each calf. on the first day after weaning, calves were restrained in a squeeze chute and 10 ml of blood was collected from the tail vein into vacutainer® tubes with edta. as calves were released from the chute, docility was scored using a 1 6 scale.1 in accordance with instructions provided by each genomics testing provider, 1 or 2 drops of blood from each calf were deposited onto each manufacturers’ blood cards and they were shipped overnight to each manufacturer for testing. on the second day after weaning, calves were restrained in a squeeze chute for ultrasonographic evaluation of two carcass traits using a 5 mhz, linear array transducer. briefly, hair was shaved over the area of interest and 75% ethanol, followed by ultrasound coupling gel, was applied to the skin. ribeye area (in2) and backfat thickness (in) were measured by the same technician at a point three fourths the distance from the medial end of the longissimus dorsi muscle (12th 13th rib interface).2 averages of 3 ultrasound measurements for both carcass traits from each calf were used for comparison with genomic results. docility scoring was repeated as calves were released from the chute and average docility score for each calf was compared to genomic testing. comparisons between actual phenotype traits and genomic results were made using simple linear regression. correlations between zoetis’s genomic results and actual phenotype were very weak (birth weight (r2 = 0.0493), weaning weight (r2 = 0.0020), docility (r2 = 0.0006), back fat (r2 = 0.0040), and rib eye area (r2 = 0.0039) and not significant (p > 0.05). correlations between neogen’s genomic results and actual phenotype were also very weak (birth weight (r2 = 0.1165), weaning weight (r2 = 0.00159), docility (r2 = 0.0188), back fat (r2 = 0.0039) and rib eye area (r2 = 0.0007) and not significant (p > 0.05). we concluded that there was little cost benefit to producers of crossbred cattle to use genomic testing for prediction of performance traits. keywords: backfat thickness, birth weight, docility score, ribeye area, weaning weight references 1. beef improvement federation. guidelines for uniform beef improvement programs (8th edition). university of georgia: athens, ga. 2002; p. 26-27. 2. houghton pl, turlington lm: application of ultrasound for feeding and finishing animals: a review. j anim sci 1992;70:930-941. 427 clinical theriogenology • volume 11, number 3 • september 2019 428clinical theriogenology • volume 11, number 3 • september 2019 omniblank: virulence factors and antimicrobial resistance of escherichia coli isolated from canine pyometra and its relationship to biofilm formation virulence factors and antimicrobial resistance of escherichia coli isolated from canine pyometra and its relationship to biofilm formation tessa fiamengo,a erin runcan,a dubraska diaz-campos,a christopher premanandan,b marco coutinho da silvaa departments of aveterinary clinical sciences and bveterinary biosciences college of veterinary medicine, the ohio state university, columbus, oh our laboratory recently demonstrated that a majority of escherichia. coli (e. coli) isolates associated with canine pyometra produce biofilm both in vivo and in vitro. biofilm, an extracellular matrix of polysaccharides, protects a microorganism from the host immune system and antimicrobial agents. moreover, multiple virulence factors and antimicrobial resistance genes have been identified in this microorganism which play a role in its pathogenesis. we hypothesized that e. coli isolates capable of biofilm production display increased resistance to antimicrobials and carry more resistance genes and virulence factors. the objectives were to compare virulence factors and susceptibility patterns to antimicrobials between strains of e. coli that are biofilm producers versus non producers. fourteen strains of e. coli isolated from clinical cases of canine pyometra were utilized. of the 14 strains, 11 strains were biofilm forming and 3 strains were non biofilm forming. bacteria were cultured on macconkey and blood agar for 24 hours. antimicrobial susceptibility testing to commonly used antibiotics and in vitro biofilm production testing were performed at the ohio state university veterinary medical center microbiology laboratory. whole genome sequencing of isolates was performed at the ohio department of agriculture, animal disease and diagnostic laboratory to determine presence of known virulence factors and antimicrobial resistance genes. in vitro antimicrobial sensitivity, presence of antimicrobial resistance genes, and virulence factors were compared between groups by fisher’s exact test, using statplus software. significance was set at p < 0.05. biofilm producers were resistant to more antimicrobials (9/19) than nonbiofilm producers (2/19). all e. coli samples (14/14) were resistant to amoxicillin/clavulanic acid and ampicillin, common antibiotics used for treatment of pyometra. all strains (14/14) contained at least 1 antimicrobial resistance gene and 50% (7/14) of strains contained multiple resistance genes. there was no relationship between ability of biofilm formation and presence of multiple drug resistance genes. in total, 18 virulence factors were identified. multiple virulence factors were present in all strains (14/14), including factors associated with uropathogenic e. coli in humans. there were no differences between the number or type of virulence factors present and the organism’s ability to form biofilm. in conclusion, biofilm producing e. coli were more likely to be resistant to more antimicrobials than nonbiofilm producing bacteria. however, there were no relationships between the organism’s ability to produce biofilm and presence of individual virulence factors or antimicrobial resistance genes. surprisingly, all strains of e. coli evaluated were resistant to antibiotics commonly used in veterinary practice. this underlies the importance of antimicrobial stewardship and judicious antimicrobial use. keywords: escherichia coli, biofilm, canine, pyometra, antimicrobial resistance 403 clinical theriogenology • volume 11, number 3 • september 2019 404clinical theriogenology • volume 11, number 3 • september 2019 omniblank: effects of granulocyte colony stimulating factor on the peripheral leukocyte function in postpartum dairy cows effects of granulocyte colony stimulating factor on the peripheral leukocyte function in postpartum dairy cows dinesh dadarwal,a kira crooks,a khawaja ahmed,b ryan dickinson,b colin palmera alarge animal clinical sciences, bveterinary pathology, western college of veterinary medicine university of saskatchewan, saskatoon, canada recombinant bovine granulocyte colony stimulation factor (gcsf) causes migration of myeloid cells, mostly neutrophils, into peripheral circulation. it is unknown whether this systemic neutrophilia is also accompanied by an increase in peripheral neutrophil function, namely phagocytic capability. we hypothesized that gcsf treatment enhances phagocytic capabilities of peripheral neutrophils in postpartum cows. holstein cows were injected with either saline (n = 7) or gcsf (n = 8) at ~ 21 (+/3) days postpartum. blood samples were collected on the day of injection and on 3, 6, 10, and 21 days. blood samples were incubated with fluorescein isothiocyanate (fitc) labeled staphylococcus aureus, neutrophils were stained with primary then secondary antibodies and analyzed by flow cytometry. normally distributed data were compared using student’s t test and nonnormally distributed data were compared using the wilcoxon rank sum test (stata 13.1 software). phagocytosis capability was compared between the 2 groups using flowjo software (tree star, usa). on the recruitment day, cows from control and treatment groups did not differ with regards to median body condition score, lactation number, body weight and leukocyte count. control and treatment group cows had similar (p > 0.41) total and differential leukocyte counts on day 0. however, cows treated with gcsf had increased (p < 0.01) total peripheral leukocyte and neutrophil counts on days 3, 6, and 10 compared to control group cows. treatment also increased (p < 0.03) phagocytosis of fitc labelled staphylococcus aureus on day 6 after treatment. in conclusion, gcsf increased phagocytic activity of blood neutrophils in treated cows. keywords: pegbovigrastim, immunomodulation, neutrophil function, postpartum cattle acknowledgement funded by adf summer research fund and faculty startup fund, university of saskatchewan 409 clinical theriogenology • volume 11, number 3 • september 2019 410clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2015: in memorium: dr. stanley mark dennis in memorium: dr. stanley mark dennis carla l. carleton department of large animal clinical sciences, college of veterinary medicine, michigan state university, east lansing, mi dr. stanley m. dennis, at 90 years of age, our colleague, friend, educator, theriogenologist and reproductive pathologist passed away on wednesday, april 1, 2015, in topeka, ks. he is survived by his wife, barbara; immediate and extended family of two daughters, two sons, one sister, 14 grandchildren, and eight great-grandchildren. he was predeceased by his first wife, naldi, and their eldest daughter and son. stanley was born in australia. at 17, he enlisted in the australian army during world war ii and was stationed in new guinea. after discharge, he obtained his veterinary degree (bvsc) and phd from the university of sydney. he worked for seven years in dairy practice and three years as senior veterinary pathologist in the animal health and diagnostic laboratory in western australia. dr. dennis earned fellowships in the royal college of veterinary surgeons (frcvs), the royal college of pathologists (frcpath) in england, and from the australian college of veterinary scientists (facvsc). he is a diplomate of the american college of theriogenologists (act). in addition, he served as act president from 1990 to 1991 and was a member and chairman of the act certifying examination committee. dr. dennis was professor and head of the department of veterinary pathology at kansas state university. during that time, he helped establish and administer the faculty of veterinary medicine (cvm) at ahmadu bello university in zaria, nigeria. after 26 years at kansas state, dr. dennis served as founding dean of the school of veterinary medicine, faculty of medical sciences, at the university of the west indies in trinidad (st. augustine campus, trinidad and tobago), and as dean at ross university school of veterinary medicine in st. kitts, british west indies. he left retirement for the third time when he became dean at the college of veterinary medicine and biomedical sciences at the american university of antigua (aua) for two years. in 2001, dr. dennis was awarded a member of the british empire (mbe) by queen elizabeth ii for meritorious contributions to international veterinary medicine for his service as dean of the school of veterinary medicine at ross university. as an internationally-recognized researcher in reproductive pathology and pregnancy wastage in domestic animals, he published more than 242 scientific papers and oversaw the academic programs of 34 doctoral and three master’s degree candidates. as one of my professors during my veterinary studies, dr. dennis was extraordinary for his mentorship, enthusiasm for all things theriogenology and pathology, and for creating passion in us to learn and enjoy being challenged. i was in an accident during veterinary school and missed one of dr. dennis’ examinations and had to make it up. i went to his office and he said, “you’ll take the same exam as your classmates.” he was always fair, but that seemed unexpected. then i took the exam, orally, question by question, sitting across from dr. dennis. “number 1, the stem is ‘___.’ now, foil a – why or why not?, foil b – why or why not?,” etc. i had to answer/explain every single multiple choice option to his satisfaction. it was very fortunate that i shared his enthusiasm for pathology or my studies might have been abbreviated. i was fortunate to enroll in his graduate class (offered summer term for at least a decade at ksu cvm), pregnancy wastage in domestic animals, prior to sitting for the act certifying examination. what wonderful preparation. many aspiring act diplomates benefited from his vast knowledge and interest in abortion diseases of animals. one clear memory of mine, to which i still refer in my teaching, is the time of death classification. the system originator must have had a sense of humor, as tod is german for death. his sense of humor could be quite dry. years ago, the act examination was given in two increments. the written at a location administered by one’s mentor, an act diplomate, and immediately before the fall conference, an oral examination by committee members. i had passed the written portion of the act certifying examination and was in nashville for the oral examination. i remember some tense clinical theriogenology • volume 7, number 4 • december 2015405 moments during the two groups of questioners. all candidates had to wait until day’s end to be informed of the outcome. as many early in their careers, i couldn’t afford the headquarters hotel and stayed down the road at some ‘cheaper venue’. i returned to the opryland hotel and was told to find dr. dennis who was to inform me of my outcome. the hotel had a grand staircase built in two sweeping curves. i was at the bottom, saw dr. dennis to the left at the top and all he did was glare and indicate with one finger that i was to come to him. such a long staircase – he didn’t once flinch or change his expression. i made it to the top, stood in front of him – cringing. he stuck out his hand, “congratulations, dr. carleton!” good grief – he was laughing. i remember being so relieved. i didn’t want to disappoint him. i last saw stan at an sft conference two years ago. it was my delight to chat with stan and barbara, and then introduce him to dr. jennifer roberts, my colleague at michigan state. dr. dennis was unchanged – always interested in the next generation – the next theriogenologist to carry on his life’s work. it was wonderful to take time for lunch with dr. dennis, barbara and jennifer – to meet and talk with an individual who meant so much to me and many others. he was one of the best and shall sorely be missed. though dr. dennis had a diverse and remarkable life in veterinary medicine spanning six decades in many countries, he was a simple man who loved helping others. his supportive influence inspired many young veterinarians to pursue careers both in theriogenology and pathology. i invite those of you who knew him, or who appreciated his many contributions, to consider making a memorial gift to kansas state university, college of veterinary medicine (101 trotter hall, manhattan, ks 66506; vetmed@kstate.edu), christ the king catholic church (5973 25th street, topeka, ks 66614), or the theriogenology foundation which dr. dennis supported (http://www.theriofoundation.org). clinical theriogenology • volume 7, number 4 • december 2015 406 reproductive hormones of pregnant mares in early aluteal cycles and reproductive parameters of cycles following treatment reproductive hormones of pregnant mares in early aluteal cycles and reproductive parameters of cycles following treatment chun mak,a erin oberhaus,b victor medina,a carlos pintoa adepartment of veterinary clinical sciences, school of veterinary medicine, louisiana state university baton rouge, la; bschool of animal sciences, louisiana agricultural experiment station louisiana state university agricultural center, baton rouge, la an aluteal cycle, a progesterone-deprived environment, can be induced in mares by serial administration of prostaglandin f2α (pgf2α) after ovulation. we determined reproductive hormone profiles of pregnant mares in first 7 days of aluteal cycles and reproductive parameters of cycles after treatment. we hypothesized that: 1) pregnant mares sustain concentrations of plasma luteinizing hormone (lh) in early aluteal cycles; 2) occurrence of multiple ovulations are greater in post treatment cycles of aluteal cycle mares compared to control cycle mares; 3) interovulatory interval (ioi) of aluteal cycle mares is shorter than the ioi of control cycle mares. after ovulation, 8 light breed cycling mares were randomly assigned to 1 of the following 3 treatment cycles; control: serial treatment of saline solution; aluteal: serial treatment of pgf2α; aluteal supplemented: serial treatment of pgf2α + a single injection of long-acting altrenogest. each mare was subjected to all 3 treatment cycles in a randomized order. embryos were collected on day 7 via uterine lavage to confirm pregnancy. the dosage of serial treatment of pgf2α has been described.1 a single long acting injection of 225 mg of altrenogest (biorelease altrenogest la 150, bet pharm, lexington, ky) was given im to mares on day 0 (i.e. day of ovulation) in aluteal-supplemented cycles. all mares received 10 mg of pgf2α im immediately after embryo collection. daily blood samples from all mares were collected from days 0 to 7. plasma concentrations of progesterone, lh, and follicle stimulating hormone (fsh) were measured by radioimmunoassays in samples collected daily from days 0 to 7. hormone concentrations, number of multiple ovulations, and ioi were analyzed using anova or chi square, as appropriate. the overall p value was set to d 0.05 for statistical significance. the ioi is reported as mean ± sem. mean concentrations of plasma progesterone remained below 1.0 ng/ml for the first 7 days in aluteal cycles and aluteal supplemented cycles; both were lower than control cycles starting from days 3 to 7. mean concentrations of plasma lh were greater in aluteal cycles than the other 2 cycles from days 1 to 7, but plasma fsh concentrations were not significantly different among treatment cycles. multiple ovulations were more common in aluteal cycles (63%; 10/16) compared to aluteal supplemented cycles (13%, 2/15), but not control cycles (34%, 14/41). mean iois after aluteal cycles (12.7 ± 0.7 days) were shorter than control (17.0 ± 0.4 days) and aluteal supplemented cycles (15.9 ± 0.6 days). in conclusion, mares with aluteal cycles had a characteristic hormonal profile of low plasma progesterone and persistently elevated plasma lh concentrations. occurrence of multiple ovulations in aluteal cycle mares was greater than aluteal supplemented cycle mares and the following ioi in aluteal cycle mares was shortened. these results may lead to novel strategies to manipulate the reproductive cycle of mares. keywords: aluteal, equine, lh, pgf2α, progesterone reference 1. coffman ea, pinto crf, snyder hk, et al: antiluteogenic effects of serial prostaglandin f2α administration in cycling mares. theriogenology 2014:82:1241-1245. 423 clinical theriogenology • volume 11, number 3 • september 2019 424clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2018: next generation dna sequencing, culture and cytology results in29 mares with suspected endometritis next generation dna sequencing, culture and cytology results in 29 mares with suspected endometritis m.r. schnobrich, e.a. bradecamp, c.f.scoggin rood and riddle equine hospital, lexington, ky endometritis is a common cause of infertility in the mare, caused most frequently by microbial (bacteria and fungi) colonization of the endometrium. traditional methods to diagnose infectious endometritis in the mare include aerobic culture and cytologic evaluation of fluid, swabs, or tissue obtained from the uterine lumen or endometrium. next generation dna sequencing (ngs) is a method of microbial diagnostics using 16s sequencing that is used in human medicine to identify microorganisms not easily identified by traditional culture techniques. the objective of this study was to compare ngs (pathogenius laboratory, lubbock, tx) results to traditional methods of diagnosing infectious endometritis. a group of 29 light-horse mares evaluated in clinical practice for reproductive management in central kentucky were used as subjects. the subjects were 500 -700 kg, mean age 9.4 years and included 25 barren, 1 maiden, and 2 foaling mares. transrectal palpation and ultrasonography was performed and samples were obtained from subjects with: one ≥ 25mm follicle present on an ovary, moderate uterine edema, and a relaxed cervix. a uterine lavage was performed with 250ml-1l of fluid and the efflux split. one sample was submitted for aerobic culture and cytologic evaluation, the other sample was submitted for ngs. processing of samples for cytologic evaluation included allowing the fluid to settle for 1h prior to removing 50ml of fluid and centrifuging for 10 minutes at 800g.the pellet was smeared onto a slide and evaluated at 100x for the presence of leukocytes as previously described.1 quantitative pcr was performed prior to ngs to determine microbial load. the ngs sequencing was reported as: negative = no microbial dna isolated, low = less than 105 bacteria/fungi/ml, medium =105-107 bacteria/fungi/ml or high = greater than 107 bacteria/fungi/ml. the microbial dna isolated was listed by genus, species, and the percentage of microbial dna present. aerobic culture of fluids recovered growth on 100% (29/29) samples, 17.2% (5/29) had more than one bacteria isolated, and the most common isolates were: e. coli 44.8% (13/29), beta-hemolytic streptococcus 41.4% (12/29), pseudomonas sp. 10.3% (3/29), and klebsiella sp. 6.7% (2/29). of the samples that had growth following culture, 17.2% (5/29) of these were negative with ngs, 17.2% (5/29) were low, 37.9% (11/29) 2/9) were medium, and 27.6% (8/29) were high. the most common microbe identified on ngs was e.coli 37.9% (11/29) with 72.7% (8/11) identified on culture. psuedomonas sp and klebsiella sp were identified in 17.2% (5/29) samples, but only 60% (3/5) and 20% (1/5), respectively, isolated these bacteria with culture. interestingly only 6.8% (2/9) of ngs samples identified beta-hemolytic streptococcus, and of these only 50% (1/2) were also isolated with culture. cytologic analysis of 28.5% (8/28) of samples had > 3 leukocytes per hpf, 28.6% (8/28) had 1-2 leukocytes per hpf, and 42.8% (12/28) had no leukocytes observed. this study describes the ngs results compared to traditional methods for identifying bacterial endometritis in a population of mare’s which were identified as having microbial growth isolated from endometrial samples. further work is required to determine the utility of this diagnostic tool in identifying clinical and subclinical cases of infectious endometritis. keywords: endometritis, equine, next generation sequencing reference 1. riddle wt, leblanc mm, stromberg aj: relationships between uterine culture, cytology and pregnancy rates in a thoroughbred practice. theriogenology 2007;68:395-402. clinical theriogenology • volume 10, number 3 • september 2018 338 2018: transcutaneous ultrasound-guided twin reduction in a quarter horse mare transcutaneous ultrasound-guided twin reduction in a quarter horse mare college of veterinary medicine, texas a&m university, college station, tx twin pregnancies maintained through late gestation are undesirable due to complications such as abortion, dystocia and may prove fatal to the mare,1 as well as complicate the resumption of a normal estrous cycle and rebreeding of the mare.2 current options for treatment of a twin pregnancy include manual reduction per rectum, cranio-cervical dislocation, transvaginal ultrasound-guided, or transcutaneous ultrasound-guided twin reduction.2 a healthy, 9-year-old quarter horse mare was referred to the texas college of veterinary medicine for transcutaneous ultrasound-guided twin reduction. abdominal ultrasonography identified two fetuses in the right ventral abdomen, both with normal tone and heart rate. the larger fetus (thoracic diameter 7-8 cm) was present in the right cranioventral abdomen, while the smaller fetus (thoracic diameter 67 cm) was present in the right inguinal region. the mare was sedated and the right caudoventral abdomen was clipped, aseptically prepared, and a local anesthetic (carbocaine) was applied. using ultrasound guidance, an 18g 3.5” spinal needle was inserted into the heart of the smaller fetus and 6 million units (15-20 mls) of procaine penicillin g (ppg) was injected to induce cardiac arrest. the larger fetus maintained a normal heart rate (93 bpm), tone, and rhythm throughout the procedure. postoperatively the mare was treated with flunixin meglamine, ppg, and altrenogest and recovered normally. abdominal ultrasonography later verified the presence of the larger fetus with a normal heart rate (131 bpm) and tone, and the smaller fetus, which had no heartbeat and decreased tone. the mare delivered a viable-term foal in addition to the mummified remains of the non-viable fetus without complications. this case highlights a common reproductive problem in the mare and is an example of the optimum outcome of a transcutaneous, ultrasound-guided twin reduction procedure. keywords: mare, twin, pregnancy, reduction, transcutaneous, ultrasound-guided references 1. miller a, woods gl: diagnosis and correction of twin pregnancy in the mare. vet clin north am equine pract 1988;4:215-220. 2. tan dks, krekeler n: success rates of various techniques for reduction of twin pregnancy in mares. j am vet med assoc 2014;245:70-78. clinical theriogenology • volume 10, number 3 • september 2018 350 s.r. grissom, c. navas de solis, a.m. ripley, c.c. love 2017: pilot study: serum concentration of insulin-like growth factor 1 in intact and spayed dogs pilot study: serum concentration of insulin-like growth factor 1 in intact and spayed dogs margaret v. root kustritz department of veterinary clinical sciences, college of veterinary medicine, university of minnesota, st. paul, mn abstract insulin-like growth factor 1 (igf-1) has been positively associated with body size and negatively associated with lifespan in dogs. gonadectomy has been associated with increased lifespan in dogs. insulin-like growth factor 1 was assayed in serum harvested from 50 intact and 50 spayed female dogs as they were surrendered to a humane organization. serum igf-1 concentrations were significantly lower in spayed female dogs than in intact bitches. overlap in serum igf-1 concentrations in the groups in this study was too great to permit a single sample to be used to differentiate spayed from intact dogs. keywords: insulin-like growth factor 1, gonadectomy, ovariohysterectomy, ovariectomy, lifespan introduction insulin-like growth factor 1 is a protein similar in appearance to pro-insulin at the molecular level that binds to insulin receptors with low affinity.1 the insulin/igf-1 signaling pathway mediates growth by stimulating increase in cell size and number through the action of insulin-like peptides.2 the growth hormone (gh)/igf-1 signaling pathway also stimulates growth. growth hormone stimulates release of igf-1 which in turn stimulates anabolic and mitogenic activity of gh.1,3 insulin-like growth factor 1 receptors are present in almost all tissues in humans during embryogenesis and igf-1 has been demonstrated as an essential component of embryogenesis of the gonads in mice.4 in adult humans, igf1 is produced primarily in the liver but also is produced in other tissues and may act synergistically with gh at distant sites or locally.1 variation in concentration of igf-1 has been associated with physical characteristics of dogs and with various disorders (table).5-22 a positive correlation of igf-1 concentrations with height, weight, and general body size of dogs has been reported.5,10,23 genetic studies have identified igf-1 as a primary determinant of variant body size within dog breeds.24-26 many studies have documented increased lifespan as a benefit of gonadectomy.6,18-22 exact mechanism of this association has not been defined although some authors cite as possible reasons increased owner investment in animals they have paid to have altered and decrease in risky behaviors by the dogs after gonadectomy, such as roaming.18-20 cause of death varies between populations of intact and gonadectomized dogs, with intact dogs more likely to die of infectious disease or trauma and gonadectomized dogs more likely to die of cancer.22 decreased concentrations of igf-1 have been associated with increased longevity in many species.15,16,27-32 a negative correlation of concentrations of igf-1 with age has been reported in dogs.5 when comparing small breed to large breed dogs, the increased lifespan of smaller dogs appears to be due to slower aging in those breeds.33 it has been suggested that lower concentrations of igf-1 may delay aging and extend lifespan, perhaps through association with greater resistance to oxidative stress.32,34-36 data from one study suggested that baseline concentrations of igf-1 were lower in gonadectomized dogs than in intact dogs.5 this suggests that igf-1 may be associated with the increased longevity noted after gonadectomy. if decreased concentrations of igf-1 can be demonstrated in ovariectomized versus intact bitches, it would lend support to the hypothesis that decrease in igf-1 is the cause for this effect. if values are well enough differentiated between intact and ovariectomized dogs, this could be another tool to help humane organizations and practicing veterinarians determine intact status in female dogs. while non-invasive tests for luteinizing hormone (lh) and anti-mullerian hormone (amh) to determine intact status have been described, many people rely on palpation of a spay scar, which is not a specific measure of intact status, or waiting for the dog to show signs of heat, which increases risk of unintentional breeding and hormone exposure associated with mammary neoplasia and pyometra.37,38 clinical theriogenology • volume 9 number 1 • march 201747 skilled practitioners with access to high quality equipment also may be able to identify whether or not a dog is intact using ultrasonography; negative findings may be equivocal. hypotheses evaluated in this study were the following: hypothesis #1: serum igf-1 concentrations will be significantly lower in spayed female dogs than in intact bitches. hypothesis #2: the difference in concentration between spayed and intact dogs will be great enough to permit measurement of igf-1 concentration in serum to be used as a sensitive and specific measure of intact status in female dogs. materials and methods blood was drawn from 50 spayed and 50 intact female dogs as they entered a humane organization.* animals were relinquished by appointment or were transferred from another humane facility. animals were enrolled as they were relinquished or transferred; no specific exclusion criteria were required. breed and age of dog were identified by the owner of relinquished animals; for all other dogs, breed and age were estimated by trained intake staff. bitches were identified as spayed by a spay scar (24 dogs) or a tattoo (26 dogs). duration from spay to relinquishment was not recorded. bitches admitted as intact underwent ovariohysterectomy within days of entry, before being placed for adoption. blood was collected at the time of admission, spun down, and the serum removed. serum was frozen and shipped as directed by the commercial laboratory which performed assay for igf-1.†b statistical analysis included descriptive statistics, pearson’s correlation coefficient, and the unpaired t-test. significance was set at p < 0.05. results breeds of dogs in the intact group were american blue heeler cross (1), australian cattle dog cross (1), basset hound cross (2), beagle (1), beagle cross (1), boxer (1), catahoula leopard dog cross (1), chihuahua (12), collie cross (1), dachshund cross (1), german shepherd (1), german shepherd cross (4), golden retriever cross (1), hound cross (5), jack russell terrier cross (1), labrador retriever cross (5), miniature pinscher cross (1), pit bull (1), pit bull cross (1), retriever cross (1), shih tzu (2), and terrier cross (5). breeds of dogs in the spayed group were american staffordshire terrier (1), australian cattle dog (1), beagle cross (1), basenji cross (1), border terrier cross (1), chihuahua (27), chow chow cross (1), coonhound cross (1) hound cross (2), labrador retriever (1), labrador retriever cross (1), manchester terrier (1), retriever cross (3), rottweiler (1), st bernard (1), shepherd cross (3), shih tzu cross (1), and terrier cross (2). overall mean weight was 26.3±18.5 lbs. mean weight of the intact dogs was 28.1±16.7 lbs. mean weight of the spayed dogs was 24.5±20.0 lbs. difference in weight between the groups was not significant. overall mean age was 19.2±9.3 months. age was not correlated with igf-1 concentration (r= 0.17). mean age of the intact dogs was 21.9±9.7 months. mean age of the spayed dogs was 16.5±8.1 months. difference in age between the groups was significant (p=0.003). overall mean igf-1 concentration was 37.3±25.0 nmol/l. overall correlation between weight and igf-1 concentration was moderately positive (r=0.67). mean igf-1 concentration in intact dogs was 42.1±27.6 nmol/l and was positively correlated with weight (r=0.62). mean igf-1 concentration in spayed dogs was 32.5±21.3 nmol/l and was strongly correlated with weight (r=0.74). difference in igf-1 concentration between the groups was significant (p=0.05). * animal humane society, golden valley, mn † michigan state university diagnostic center for population and animal health, lansing mi clinical theriogenology • volume 9 number 1 • march 2017 48 discussion the primary concern about this study is the lack of information about the dogs due to sampling from a humane society population. it is clear that all dogs were correctly identified as intact, as they were spayed within days of entering the facility. however, it is possible that some dogs identified as having been spayed were actually intact, and did not show signs of heat while in the facility prior to adoption. however, this would have artificially skewed the mean concentration for igf-1 in the population of gonadectomized dogs higher than was reported, which simply strengthens the findings of the study. we also do not know if dogs identified as spayed had undergone ovariectomy or ovariohysterectomy. it is possible, although very unlikely in the region from which this sample was drawn, that some dogs identified as spayed had undergone hysterectomy. inability to define exact means of surgical sterilization in all dogs unavoidably introduced variation. no information was gathered about body condition score of dogs from which samples were drawn. obesity is common in gonadectomized dogs.39-44 obesity is associated with increased concentrations of igf-1 in humans.11-13 if the same is true in dogs and if the gonadectomized dogs in this study were more obese than the intact dogs in the study, again this would have skewed the true concentration of igf-1 higher in the gonadectomized dogs, strengthening the findings of the study. future studies would benefit from normalization of igf-1 concentrations with body condition scoring. this study does not address the actual mechanisms by which either gonadectomy or decrease in concentrations of igf-1 increase lifespan. the majority of studies to date have demonstrated that gonadectomy increases lifespan but in one population of exceptionally long-lived rottweilers, remaining intact (ovarian exposure) was associated with increased lifespan, suggesting that some factor released from the ovary promoted longevity in that group of dogs.45 this increase in lifespan was not associated with intensity or tempo of reproductive effort.46 canine ovarian follicles contain igf-1 but concentrations are well correlated with plasma concentrations of igf-1, suggesting a lack of direct connection between presence or absence of ovaries and secretion of igf-1.23 because duration from spaying to relinquishment was not recorded and may well not have been known for many of the dogs, it is possible that the variation noted in this study was altered by variability in time from gonadectomy to a rise in igf-1 concentrations in serum. similarly, stage of the estrous cycle was not identified in those bitches who underwent ovariohysterectomy at the humane society. it has been demonstrated that concentrations of gh and other growth factors, including igf-1, are higher during the luteal phase of the estrous cycle in bitches.47-49 considerations of cost and logistics of the surgery service at the humane organization supporting this study prevented use of serum progesterone concentration measurement or histopathologic assessment of excised ovaries as a means of determining stage of the estrous cycle of bitches in the intact group. hypothesis #1 was supported in this study as spayed dogs had significantly lower serum concentrations of igf-1 than intact dogs in this sample. although there was a positive correlation of weight with igf-1 concentration, there was no significant difference in weight between the groups, removing that variable as a cause for the difference in this sample. there was no correlation of age with igf-1 concentration, so although there was a significant difference in age between the groups, this variable cannot be a cause for the difference in this sample. hypothesis #2 was not supported in this study. overlap in serum igf-1 concentrations in the groups in this study was too great to permit a single sample to be used to differentiate spayed from intact dogs. conclusion there is a growing body of evidence documenting benefits and detriments of gonadectomy in dogs.50-54 in general, benefits of gonadectomy of female dogs are a reduction in incidence of mammary neoplasia and pyometra when aged. reported detriments associated with gonadectomy include neoplasia, including osteosarcoma, hemangiosarcoma, transitional cell carcinoma, lymphosarcoma, and mast cell tumor; orthopedic disease, including anterior cruciate ligament injury and hip dysplasia; obesity; and urinary incontinence. better understanding of the mechanisms underlying physiologic changes induced clinical theriogenology • volume 9 number 1 • march 201749 by gonadectomy, including effects of a decrease in concentrations of igf-1 and other hormones, will permit veterinarians to make the best decisions regarding recommendations for gonadectomy of individuals or populations of dogs to balance pet overpopulation with individual animal health. acknowledements the author thanks dr. jonna swanson and the veterinarians and staff at the animal humane society for their assistance in collecting samples. funding for this study was provided by the american kennel club canine health foundation (chf grant 02136-a) references 1. laron z: insulin-like growth factor 1 (igf-1): agrowth hormone. j clin pathol mol pathol 2001;54:311-16. 2. gokhale rh, shingleton aw: size control: the developmental physiology of body and organ size regulation. wires dev biol 2015;4:335-356. 3. david a, hwa v, metherell la, et al: evidence for a continuum of genetic, phenotypic, and biochemical abnormalities in children with growth hormone insensitivity. endocrinol rev 2011;32:472-497. 4. pitetti j-l, calvel p, romero y, et al: insulin and igf1 receptors are essential for xx and xy gonadal differentiation and adrenal development in mice. plos genetics 2013;9:e1003160:1-17. 5. greer ka, hughes lm, masternak mm: connecting serum igf-1, body size, and age in the domestic dog. age 2011;33:475-483. 6. greer ka, canterberry sc, murphy ke: statistical analysis regarding the effects of height and weight on life span of the domestic dog. res vet sci 2007;82:208-214. 7. tvarijonaviciute a, martinez-subiela s, carrillo-sanchez jd, et al: effects of orchidectomy in selective biochemical analytes in beagle dogs. reprod domest anim 2011;46:957-963. 8. dabrowski r, szczubial m, kostro k, et al: serum insulin-like growth factor-1 and c-reactive protein concentrations before and after ovariohysterectomy in bitches with pyometra. theriogenology 2015;83:474-477. 9. jitpean s, strom holst b, hoglund ov, et al: serum insulin-like growth factor-1, iron, c-reactive protein, and serum amyloid a for prediction of outcome in dogs with pyometra. theriogenology 2014;82:43-48. 10. jaillardon l, martin l, nguyen p, et al: insulin-like growth factor type i concentrations in healthy dogs and dogs with spontaneous primary hypothyroidism. vet j 2011;190:e95-e99. 11. frystyk j, vestbo e, skjaerbaek c, et al: free insulin-like growth factors in human obesity. metabolism 1995;44:37-44. 12. nam sy, lee ej, kim kr, et al: effect of obesity on total and free insulin-like growth factor (igf)-1, and their relationship to igf-binding protein (bp)-1, igfbp-2, igfbp-3, insulin, and growth hormone. intl j obes relat metab disord 1997;21:355-359. 13. louveau i, gondret f: regulation of development and metabolism of adipose tissue by growth hormone and the insulin-like growth factor system. domest anim endocrinol 2004;27:241-255. 14. strage em, lewitt ms, hanson jm, et al: relationship among insulin resistance, growth hormone, and insulin-like growth factor 1 concentrations in diestrous swedish elkhounds. j vet intern med 2014;28:419-428. 15. pedersen hd, falk t, haffstrom j, et al: circulating concentrations of insulin-like growth factor-1 in dogs with naturally occurring mitral regurgitation. j vet intern med 2005;19:528-532. 16. gayet c, bailhache e, dumon h, et al: insulin resistance and changes in plasma concentration of tnfalpha, igf1, and nefa in dogs during weight gain and obesity. j anim physiol a anim nutr 2004;88:157-165. 17. spichiger ac, allenspach k, zbinden y, et al: plasma insulin-like growth factor-1 concentration in dogs with chronic enteropathies. vet med (praha) 2006;51:35-43. 18. bronson rt: variation in age at death of dogs of different sexes and breeds. am j vet res 1982;43:2057-2059. 19. michell ar: longevity of british breeds of dogs and its relationship with sex, size, cardiovascular variables and disease. vet rec 1999;145:625-629. 20. moore ge, burkman kd, carter mn, et al: causes of death or reasons for euthanasia in military working dogs: 927 cases (1993-1996). j am vet med assoc 2001;219:209-214. 21. kraft w: geriatrics in canine and feline internal medicine. eur j med res 1998;3:31-41. 22. hoffman jm, creevy ke, promislow de: reproductive capability is associated with lifespan and cause of death in companion dogs. plos one 2013;8:e61082. doi:10.1371/journal.pone.0061082. 23. reynaud k, chatant-maillard s, batard s, et al: igf system and ovarian folliculogenesis in dog breeds of various sizes: is there a link? j endocrinol 2010;26:85-92. 24. rimbault m, beale hc, schoenebeck jj, et al: derived variants at six genes explain nearly half of size reduction in dog breeds. genome res 2013;23:1985-1995. 25. sutter nb, bustamante cd, chase k, et al: a single igf1 allele is a major determinant of small size in dogs. science 2007;316:112-115. 26. hoopes bc, rimbault m, liebers d, et al: the insulin-like growth factor 1 receptor (igf-1r) contributes to reduced size in dogs. mamm genome 2012;23:780-790. clinical theriogenology • volume 9 number 1 • march 2017 50 27. chase k, lawler df, mcgill dl, et al: age relationships of postmortem observations in portuguese water dogs. age 2011;33:461-473. 28. querioga f, perez-alenza md, silvan g, et al: crosstalk between gh/igf-1 axis and steroid hormones (progesterone, 17-beta-estradiol) in canine mammary tumours. j ster biochem molec biol 2008;110:76-82. 29. antebi a: genetics of aging in caenorhabditis elegans. plos genetics 2007;3:1565-1571. 30. hsieh cc, deford jh, flurkey k, et al: implications for the insulin signaling pathway in snell dwarf mouse longevity: a similarity with the c. elegans longevity paradigm. mech ageing dev 2002;123:1229-1244. 31. al-regaiey ka, masternak mm, bonkowski m, et al: long-lived growth hormone receptor knockout mice: interaction of reduced insulin-like growth factor 1/insulin signaling and caloric restriction. endocrinology 2005;146:851-860. 32. tatar m, bartke a, antebi a: the endocrine regulation of aging by insulin-like signals. science 2003;299:1346-1351. 33. kraus c, pavard s, promislow del: the size-life span trade-off decomposed: why large dogs die young. am nat 2013;181:492-505. 34. kenyon c: the plasticity of aging: insights from long-lived mutants. cell 2005;120:449-460. 35. berryman de, christiansen js, johannsson g, et al: role of the gh/igf-1 axis in lifespan and health span: lessons from animal models. growth horm igf res 2008;18:455-471. 36. jimenez ag: physiological underpinnings in life-history trade-offs in man’s most popular selection experiment: the dog. j comp physiol b 2016. doi:10.1007/500360-016-1002-4. 37. lofstedt rm, vanleeuwen ja: evaluation of a commercially available luteinizing hormone test for its ability to distinguish between ovarectomized and sexually intact bitches. j am vet med assoc 2002;220:1331-1335. 38. themmen ap, kalra b, visser ja, et al: the use of anti-mullerian hormone as diagnostic for gonadectomy status in dogs. theriogenology 2016;86:1467-1474. 39. mason e: obesity in pet dogs. vet rec 1970;86:612-616. 40. david g, rajendran ei: the aftereffects of spaying in bitches and cats. cheiron 1980;9:193-195. 41. colliard l, ancel j, benet jj, et al: risk factors for obesity in dogs in france. j nutr 2006;136:1951s-1954s. 42. crane sw: occurrence and management of obesity in companion animals. j small anim prac 1991;32:275-282. 43. edney atb, smith pm: study of obesity in dogs visiting veterinary practices in the united kingdom. vet rec 1986;118:391-396. 44. sloth c: practical management of obesity in dogs and cats. j small anim prac 1992;33:178-182. 45. waters dj, kengeri ss, clever b, et al: exploring mechanisms of sex differences in longevity: lifetime ovary exposure and exceptional longevity in dogs. aging cell 2009;8:752-755. 46. kengeri ss, maras ah, suckow cl, et al: exceptional longevity in female rottweiler dogs is not encumbered by investment in reproduction. age 2013;35:2503-2513. 47. lee wm, kooistra hs, mol ja, et al: ovariectomy during the luteal phase influences secretion of prolactin, growth hormone, and insulin-like growth factor-1 in the bitch. theriogenology 2006;66:484-490. 48. kooistra hs, okkens ac: secretion of prolactin and growth hormone in relation to ovarian activity in the dog. reprod domest anim 2001;36:115-119. 49. mol ja, selman pj, sprang epm, et al: the role of progestins, insulin-like growth factor (igf) and igf-binding proteins in the normal and neoplastic mammary gland of the bitch: a review. j reprod fertil 1997;suppl 51:339-344. 50. reichler im: gonadectomy in cats and dogs: a review of risks and benefits. reprod domest anim 2009;44(suppl 2):29-35. 51. romagnoli s: surgical gonadectomy in the bitch and queen: should it be done and at what age? proc southern european vet conf congreso nacional avepa, barcelona spain, 2008. 52. root kustritz mv: determining the optimal age for gonadectomy of dogs and cats. j am vet med assoc 2007;231:1665-1675. 53. root kustritz mv: optimal age for gonadectomy in dogs and cats. clin therio 2010;2:177-181. 54. root kustritz mv: effects of surgical sterilization on canine and feline health and on society. reprod domest anim 2012;47 (suppl 4):214-222. table. causes of variation in blood concentrations of igf-1 and in lifespan of dogs associated with decreased igf-1 associated with increased igf-1 gonadectomy increasing age small body size acute inflammation including post-surgical inflammation, disease conditions chronic inflammation large body size chronic disorders including obesity associated with decreased lifespan associated with increased lifespan large body size obesity gonadectomy clinical theriogenology • volume 9 number 1 • march 201751 clinical theriogenology • volume 9 number 1 • march 2017 52 omniblank: equine intracytoplasmic sperm injection laboratory: an overview of management challenges rob foss equine medical services, inc., columbia, mo abstract process of equine in vitro embryo production requires consistent monitoring, controlling, and adjustment of several factors in the laboratory. this is a brief introduction to some of the more important factors. keywords: intracytoplasmic sperm injection, equine embryo, laboratory introduction in vitro embryo production via intracytoplasmic sperm injection (icsi) is a laboratory-intensive process that involves much more than the procedure of sperm injection. establishing and maintaining a functional productive environment that is suitable for embryo production and growth has many facets. in utero, oocytes and embryos are in an extremely protected and nourishing environment; however, in vitro they are potentially subject to, and sensitive to, numerous environmental challenges. although the act of sperm injection is important to be performed well, the real challenge is in the creation and maintenance of a homeostatic ‘embryo-friendly’ laboratory. the realities of the equine reproduction and embryo transfer industry offer further challenges with planning and communication with multiple parties. air quality air, of course, permeates every corner of the laboratory, and although seemingly innocuous, air quality can make or break in vitro embryo production. laboratory air is evaluated for particulates, toxic gases, and volatile organic compounds (vocs). particulates can be a source for contamination of any exposed culture medium but can generally be controlled by hepa air filtration. the target level for particulates in an in vitro fertilization (ivf) laboratory is < 10,000 particles from 0.5 to 10 microns.1 minimizing the number of people in the laboratory and meticulous cleaning can also help keep airborne particulates low. regardless of the quality of room air and media, culture dishes should be prepared under laminar flow of hepa filtered air in a laminar flow cabinet (i.e., tissue culture hood). although other toxic gases are uncommon in the laboratory, vocs are a substantial concern, potentially crippling production of viable embryos.2,3 volatile organic compounds arise from several sources including paints, adhesives, upholstery, dry wall, wood composites, plastics, cleaning agents, disinfectants, vehicles, petroleum products, aerosol solvents, and cosmetic products, especially, those with fragrances. the target level for total voc concentration in an ivf laboratory is < 500 µg per cubic meter (400 800 parts per billion, depending on the molecular size) and it is < 5 µg per cubic meter for aldehydes (embryos are particularly sensitive).1 precise measurement of airborne vocs, especially of specific chemicals, is best performed with gas chromatography; however, a single sample may not give a reliable picture of the daily fluctuations produced by human activity and the environment. more commonly, laboratory air can be monitored with handheld photoionization detectors or gas sensitive semiconductors, but these sensors vary in their ability to detect various types of vocs.4 these units are also difficult to calibrate for the lower concentrations that are necessary for ivf laboratories. monitoring is important to identify the source of and amelioration for any surges in vocs. heating, ventilation, and air conditioning (hvac) system of the laboratory can be a substantial contributor to overall air quality. laboratory should maintain positive pressure to prevent incursions of contaminants from other rooms in the building. positive pressure is maintained by increasing the inflow of air from the air handler into the room or limiting the outflow. this may require additional outside air being brought into the system that will generally also decrease voc concentration. however, at times, external air may be a source of toxicity and has to be completely shut off, such as in periods of high ozone concentrations or vocs from road work, painting, roofing, or other activities. active filtration and removal of vocs may be necessary in several laboratory locations. this can be provided by filtration through activated charcoal and potassium permanganate filters that are in standalone filtration units or in the hvac system. filters in the hvac system should be installed on the conditioned side of the air handling unit.1 since vocs become more volatile at higher temperatures, activated charcoal is less effective and could even release vocs previously absorbed if exposed to increased temperature. toxicity toxins present in the laboratory system can harm or stop embryo production and there are many potential sources. plasticware (petri dishes, centrifuge tubes, culture tubes) and mineral oil, used to cover incubating oocytes and embryos, can be sources of toxins. plasticware and mineral oil produced specifically for in vitro fertilization use should be screened with mouse embryo assay (mea) in which mouse zygotes are cultured to the blastocyst stage.6 plastics not screened are a risk, as plastics are a common source of vocs. a good generalization is that harder plastics, such as polystyrene, release less vocs than softer plastics such as polypropylene. all plasticware should have its packaging wrap opened for 2 4 weeks to allow vocs to ‘off-gas’ before use. mineral oil, a petroleum distillate, is used to overlay micro-drops of culture medium during incubation. mineral oil provides a barrier to contamination and evaporation. mineral oil is prone to peroxidation, especially when exposed to light. being hydrocarbon petroleum distillates, mineral oil or paraffin oil has to be extremely well-refined, screened via mea, and washed with a fluid containing protein to remove lipophilic and hydrophilic toxins to be considered safe for embryology. incubator the incubator is the heart of an embryo culture system, a complex system that provides appropriate temperature, humidity, and atmosphere. embryo culture media use a bicarbonate-based buffer system that balances bicarbonate and co2 to arrive at the target ph. oxygen is reduced to 5% to mimic oxygen tension in the reproductive tract and minimize oxidative damage. to produce this environment, carbon dioxide and oxygen sensors control and balance carbon dioxide and nitrogen gas supplies. these sensors can be calibrated to standard gases; however, drift over time will require periodic monitoring and adjustment, and oxygen sensors are particularly prone to failure without warning, making monitoring even more imperative. the incubator can also be a source of vocs and new incubators must be ‘burned in’ through a high temperature disinfection cycle and run for several weeks to ‘off-gas’ harmful vocs. temperature maintenance is another function of the incubator. digital thermometry has very small drift, so once calibrated it should maintain accuracy. equine embryos can handle a range of temperatures,6 but blastocyst production should be the best in the center of that range. since most incubators have multiple shelves there is an overall temperature difference of up to 0.1 0.2°c between the top and bottom shelves. this difference can affect blastocyst production7 and the overall incubator temperature should be adjusted so that all shelves are as close to the ideal production temperature as possible. culture medium culture medium provides the developing embryo, electrolytes, carbohydrates (pyruvate, lactate, and glucose for energy), amino acids, vitamins, nucleic acid precursors, chelators, antioxidants, antibiotics, proteins, and other growth factors. culture media for equine embryos have not received the research and development as several other species. however, generally they are developed based on information derived from those species. the first reliable medium for in vitro equine blastocyst production was a complex tissue culture medium, dmem/ f12 that demonstrated the early equine embryo’s tolerance and the early blastocyst’s affinity for a high glucose concentration (17 mm). although a commercial equine medium is now available, most laboratories still prepare their own. some use sequential media, starting with a relatively low glucose medium the first 4 5 days of culture and then move to a higher glucose, usually more complex medium. nonsequential media are used by some to avoid the stress of change. in vitro culture of most mammalian embryos has moved away from using serum in the culture medium, largely because it is somewhat unpredictable in its composition from batch-tobatch, and so also its function in the medium. addition of another protein source (e.g., bovine serum albumin, and several cytokines) has generally replaced the functions of serum and frequently improved results in other species. however, this has not been the case with equine culture media. most equine labs still use fetal bovine serum in equine embryo culture, if not during the first days, at least starting at day 5. despite the inconsistency in batches, it still provides useful growth factors and cytokines. early embryos have limited ability to control their internal ph, so maintaining a desirable ph of the culture medium is essential. equine icsi embryos tolerate a fairly wide range of ph; however, maximum embryo production generally occurred optimally within a ph range between 7.25 and 7.35.6 this is maintained by a balance between the bicarbonate in the medium and the concentration of co2 in the incubator. most media are designed to maintain an appropriate ph at 5 or 6% co2, although a higher co2 concentration is required as height above sea level increases. the ph should be monitored in medium equilibrated in the incubator at regular intervals and with any change in culture medium lot or formulation. the ph is then adjusted by either regulating the co2 concentration or by the addition of acid or base to the medium. light light exposure had deleterious effects on embryos, especially certain wavelengths.8,9 farther towards the ultraviolet spectrum the more harmful light is to embryos. mouse zygotes were exposed to 15 minutes of cool white light, 15 minutes of warm white light, or 1 minute of sunlight; of these, 73% of controls, 58% of those exposed to warm white fluorescent light, 44% of those exposed to cool fluorescent light, and 25% of those exposed to sunlight developed to term.9 it is imperative that laboratory light should be in the ‘warm’ spectrum, kept at low levels, and oocyte and embryo exposure to light should be minimal. communication the equine icsi laboratory has unique communication challenges. there are usually many parties involved in the entire process that all must be either kept informed of progress and/ or consulted. first there is the mare owner who not only wants to be updated at each stage, and also has to plan ahead, knowing which semen to use. laboratory has to know from the owner where to send the embryos produced, how many to send, etc. veterinarian or facility collecting the oocytes has to know the progress, as this might affect planning for the next cycle of the mare. stallion/semen owner has to be contacted to give permission to use stored semen or to supply semen. stallion owner will also like to know the outcome. the facility receiving embryos has to know the progress periodically so that they can prepare suitable recipient mares. conversely, not every curious person that might call wanting to know what is going on with a mare’s oocytes has the right to know, so dispensing information properly can be challenging. to handle all of these needs, a laboratory should have a defined communication plan or protocol. conflict of interest none to declare. references 1. mortimer d, cohen j, mortimer s, et al: cairo consensus on the ivf laboratory environment and air quality: report of an expert meeting. reprod biomed online 2018. 2. johnson j, boone w, bernard r: the effects of volatile compounds (vc) on the outcome of in vitro mouse embryo culture. fertil steril 1993;(suppl 1):s98-99. 3. agarwal n, chattopadhyay r, ghosh s, et al: volatile organic compounds and good laboratory practices in the in vitro fertilization laboratory: the important paramaters for successful outcome in extended culture. j assis reprod genet 2017;34:999-1006. 4. spinelle l, gerboles m, kok g, et al: review of portrable and lowcost sensors for the ambient air monitoring of benzene and other volatile organic compounds. sensors 2017;7:1520. 5. van den abbeel e, vitrier e, lebrun f, et al: optimized bioassay for the detection of embryology contaminants. hum reprod 1999:114. 6. brom-de-luna j, salgado r, canesin h, et al: equine blastocyst production under different co2 concentrations during early cleavage. reprod fert dev 2019;31:1823-1829. 7. foss r: unpublished data. 8. ottosen l, hindkjar j, ingelsled j: light exposure of the ovum and preimplantation embryo during art procedures. j asist reprod genet 2007;24:99-103. 9. korhonen k, sjovall s, viitanen et al: viability of bovine embryos following exposure to green filtered or wider bandwidth light during in vitro embryo production. hum reprod 2009;24:308-314. equine intracytoplasmic 2018: effect of liposome-containing diluent and centrifugation on motion parameters and membrane integrity of electroejaculated cooled bovine spermatozoa   effect of liposome-containing diluent and centrifugation on motion parameters and membrane integrity of electroejaculated cooled bovine spermatozoa maria s. ferrer,a roberto a. palomares,b anna-claire bullington,a joão henrique jabur bittarb adepartments of large animal medicine and bpopulation health, college of veterinary medicine, university of georgia, athens, ga abstract the objectives were to evaluate the effect of optixcell diluent and centrifugation on cooled bovine sperm viability. it was hypothesized that motility and membrane integrity of cooled spermatozoa diluted in optixcell would be higher than in frozen semen for at least 48 h, and removing seminal plasma by centrifugation would improve cooled sperm viability. first, semen was collected from nine bulls and extended in optixcell. an aliquot was frozen, while the remaining semen was refrigerated for 96 h. motility and membrane integrity were higher in spermatozoa cooled for up to 48 h than in frozen semen. sperm motility, vsl, dsl, str and bcf did not differ between 0 and 72 h of cooling, but decreased at 96 h. next, semen was collected from 11 bulls and refrigerated for 72 h with (control) or without (centrifuged) seminal plasma. motion parameters did not differ with treatment. in non-centrifuged samples, motility, vap, dsl and bcf decreased at 72 h. in centrifuged samples, motility decreased at 72 h and velocity at 48 h. viability of cooled bovine semen diluted in optixcell was superior than in frozen semen for 48 h. removal of seminal plasma by centrifugation did not improve preservation of cooled sperm viability. keywords: cryopreservation, bovine, centrifugation, motility, chilled semen introduction the use of cooled shipped semen for artificial insemination is a routine procedure in the equine and canine industries.1,2 maintaining semen at refrigeration temperature lowers the metabolic rate of spermatozoa and decreases overgrowth of bacteria, extending the lifespan of spermatozoa.3 in general, pregnancy rates achieved with cooled semen are higher than with frozen semen.4,5 this is because freezing and thawing induce sperm damage associated with plasma membrane reorganization, accumulation of intracellular ions, osmotic stress, cryoprotectant toxicity and formation of intracellular ice crystals, altering sperm viability and function.6,7 these changes in the sperm plasma membrane decrease the ability of spermatozoa to attach to oviductal epithelial cells and form an oviductal reservoir, decreasing their survival time within the female’s reproductive tract.8 therefore, to achieve acceptable pregnancy rates with frozen semen, insemination of an appropriate number of spermatozoa and accurate timing of insemination close to ovulation are of utmost importance.5 in cattle, frozen semen is almost exclusively used for artificial insemination in most countries, except new zealand where cooled semen is used in 95% of artificial inseminations.3 efficient use of frozen semen requires estrus synchronization and timed artificial insemination (tai) or accurate estrus detection since timing of insemination with respect to ovulation is critical for fertilization. unfortunately, ovulatory response to estrus synchronization protocols is variable in terms of percentage of cows ovulating and timing of ovulation.9 this can have a negative impact on pregnancy rates after tai with frozen semen. the higher survival of cooled spermatozoa in the cow’s tract could allow for higher flexibility in the timing of insemination, improving pregnancy rates after tai. the improved fertility would also allow for a reduction in clinical theriogenology • volume 10 number 1 • march 201825   the number of spermatozoa per insemination dose, maximizing the number of doses obtained per ejaculate and optimizing the use of genetically superior bulls.3 in addition, while a complete semen evaluation is still needed to ensure proper semen handling and packaging, the cost and complexity of semen processing for cooling is substantially reduced compared to freezing. semen can be collected and processed for shipment at the farm, decreasing the costs associated with transporting, testing, and housing bulls in semen freezing centers. furthermore, semen cooling could be an alternative for bulls with poor post-thaw sperm survival. on the other hand, bulls in a cooled semen program may need to have semen collected multiple times within a breeding season to meet the needs of producers requesting the shipments. this may increase labor at the farm during the breeding season. in spite of the potential advantages of this technology, few studies have evaluated the use of cooled semen in cattle. a decrease in sperm motility, membrane integrity, acrosome integrity and pregnancy rate was observed when semen was cooled for 48 h in some studies.10,11 however, sperm motility and in vitro fertilizing ability were preserved for 6 days in another study.3 differences in breed, semen extender, cryoprotectant, semen collection method and semen processing protocol may account for the variation in reported results. a noteworthy protocol difference in the later study was the removal of seminal plasma by centrifugation prior to cooling. this practice was shown to improve survival and membrane stability of stallion spermatozoa during cooling.12,13 furthermore, semen collection on-farm requires the use of electroejaculation since the facilities and personnel required for semen collection using an artificial vagina are rarely available. electroejaculation yields samples with variable concentration of seminal plasma, and sometimes contaminated with urine. therefore, centrifugation and removal of seminal plasma and contaminating fluids could improve quality of bovine cooled semen obtained by electroejaculation, or could prolong storage times. however, to date the effect of centrifugation on survival of cooled spermatozoa has not been critically evaluated in cattle. furthermore, the ability of liposome-containing diluents to support viability of cooled bovine spermatozoa has not been tested. liposome-containing diluents, such as optixcell®, have been shown to provide improved cryoprotection during freezing and increased pregnancy rates compared with other semen diluents like tryladil, bioxcell or tris-egg yolk.14,15 liposomes added to the semen extender can incorporate into the plasma membrane and change the lipid phase transition.14 this decreases the sensitivity of spermatozoa to cooling and improves their survival.14 in addition, optixcell® is a chemically defined diluent, free of animal proteins. the diluent is clear and free of particles, allowing accurate evaluation of sperm function using modern technologies, such as computer-assisted semen analysis or flow cytometry. the objectives of this study were to evaluate the ability of the liposome-containing diluent optixcell to support viability of cooled bovine spermatozoa during prolonged storage, to compare viability of cooled and frozen spermatozoa diluted in optixcell, and to evaluate the effect of centrifugation on cooled sperm viability. it was hypothesized that motility and membrane integrity of cooled spermatozoa diluted in optixcell would be higher than in frozen semen for at least 48 h, and that removing seminal plasma by centrifugation would improve sperm motility and membrane integrity during cold storage of electroejaculated bovine semen. clinical theriogenology • volume 10 number 1 • march 2018 26   materials and methods experiment 1: motility and membrane integrity of cooled spermatozoa diluted in optixcell compared with frozen semen nine 17-month old angus bulls were included in this study. a cleanout semen collection was performed one week prior to the study. on the day of the study, one ejaculate was obtained from each bull. the cloudy sperm-rich fraction was collected using electroejaculation (pulsator iv, lane manufacturing inc., denver, co) during penis protrusion or erection. sperm concentration was evaluated using a densimeter and sperm morphology was evaluated using phase contrast. the mean percentage of morphologically normal spermatozoa was 71.4±3.8%. immediately after collection, 200 l of an antibiotic cocktail (css antibiotic mix, imv technologies, maple grove, mn) was added per ml of raw semen. each 200 l of antibiotic cocktail contained 100 g of tylosin, 500 g of gentamycin and 300/600 g of linco-spectin. semen was then placed in a water bath at 34c for 5 min. pre-warmed (34c) optixcell (imv technologies) was added to a final concentration of 50 x106 spermatozoa/ml. the extended semen was placed in a refrigerator at 5c for 5 h. after 5 h, pre-cooled 0.5 ml straws were loaded. the straws were held horizontally in a rack 3 cm above liquid nitrogen for 10 min. the straws were then plunged in the liquid nitrogen and stored at -196c until evaluation. the remaining semen was maintained in the refrigerator at 5c for 96 h. at 0, 24, 48, 72 and 96 h of cold storage, an aliquot of each sample was warmed to 38c for 10 min for evaluation of sperm motility. in addition, membrane integrity was evaluated at 24 and 48 h. two weeks after freezing, one straw from each sample was thawed in a water bath at 38c for 30 s, and sperm motility and membrane integrity were evaluated after allowing the semen to warm at 38ºc for 10 min. statistical analysis was performed using sas package (sas institute, cary, nc). distribution of the data was tested for normality using a shapiro wilk test. normally distributed data were expressed as mean ± sem. sperm motion parameters and membrane integrity were compared among treatments using anova for repeated measurements. paired comparisons were performed using a tukey’s test. differences were considered significant if p < 0.05. experiment 2: effect of centrifugation on motility and membrane integrity of cooled spermatozoa eleven angus bulls (17 m to 3 y old) were included in the study. a cleanout semen collection was performed one week prior to the study. on the day of the study, one ejaculate was obtained from each bull. the cloudy sperm-rich fraction was collected using electroejaculation. sperm concentration was evaluated using a densimeter, and sperm morphology was evaluated using phase contrast. the mean percentage of morphologically normal spermatozoa was 72.8±3.7%. an antibiotic cocktail (css antibiotic mix, imv technologies; 200 l/ml of semen) was immediately added to the semen, which was placed in a water bath at 34c for 5 min. prewarmed (34c) optixcell was added to a final concentration of 50 x106 spermatozoa/ml. each ejaculate was divided into two aliquots. one aliquot was refrigerated without centrifugation (nc treatment). sperm concentration in undiluted semen ranged from 156 to 998 x106 spermatozoa/ml (492.2±109.9 x106 spermatozoa/ml, mean±sd). the dilution ratio (semen:extender) in nc semen ranged from 1:2 to 1:19, with an average ratio of 1:8. the other aliquot was centrifuged at 800 x g for 10 min. the supernatant was removed and the pellet was resuspended in optixcell to a final concentration of 50 x106 spermatozoa/ml (ce treatment). clinical theriogenology • volume 10 number 1 • march 201827   both aliquots were placed in a refrigerator at 5c for 72 h. at 0, 24, 48 and 72 h of cold storage, an aliquot of each sample was warmed to 38c for 10 min for evaluation of sperm motility. in addition, membrane integrity was evaluated at 24 and 48 h. sperm motion parameters and membrane integrity were compared among storage times within treatment using anova for repeated measurements. paired comparisons were performed using a tukey’s test. comparisons between treatments within storage time were done using a paired t test. evaluation of sperm parameters sperm motility was evaluated with a computer assisted semen analyzer (spermvision professional, minitube of america, verona, wi). the settings of the instrument were: field depth of view 20 m, pixel to m ratio 130 to 100, cell area 18 to 80 m, frames acquired 30, frame rate 60 hz, aoc cut off static cells 5 and dsl cut off 4.5 µm/s (progressive motility). mean percentage of total (tmot) and progressively (pmot) motile spermatozoa was assessed from all spermatozoa present in 7 fields with a 20x phase-contrast objective. other motion parameters analyzed were average path velocity (vap), straight-line velocity (vsl), curvilinear velocity (vcl), amplitude of lateral head displacement (alh), beat cross frequency (bcf), straightness (str), linearity (lin), wobble (wob), curvilinear distance (dcl), straight line distance (dsl), and distance of average path (dap). membrane integrity was evaluated using fluorescence microscopy under a 40x objective. spermatozoa were incubated for 10 min at 38c with the vital stains sybr14 and propidium iodide (pi; live/dead kit, minitube of america) following instructions from the manufacturer. all spermatozoa present in 10 fields were classified as membrane-intact (green fluorescence) or membrane-damaged (red fluorescence) by a built-in software within the casa system (spermvision professional, minitube of america), and mean percentages were reported. phase contrast microscopy was used for assessment of sperm morphology. spermatozoa were diluted 1:10 in formalin buffered solution (animal reproduction systems, chino, ca). a wet mount was prepared and 100 spermatozoa were classified based on their morphological characteristics under oil immersion at 100x magnification. results experiment 1: motility and membrane integrity of cooled spermatozoa diluted in optixcell compared with frozen semen total motility (p<0.0001), progressive motility (p=0.0001), dsl (p=0.005), vsl (p=0.001), str (p=0.009) and bcf (p=0.007) differed significantly among treatments (fig. 1). total and progressive sperm motility did not differ for 72 h, but were lower at 96 h than 0, 24 and 48 h. motility was higher in spermatozoa cooled for up to 48 h than in frozen semen. however, sperm motility did not differ between semen cooled for 72 or 96 h, and frozen. dsl, vsl, str and bcf were lowest in spermatozoa cooled for 96 h. the percentage of spermatozoa with intact membranes did not differ among cooling times but was higher in semen cooled for 24 h (88.5 ± 3.3%) or 48 h (82.5 ± 3.9%) than frozen semen (42±7.6%) (p<0.0001). no other parameters differed with treatment (p>0.05). experiment 2: effect of centrifugation on motility and membrane integrity of cooled spermatozoa there was no difference in any of the parameters between centrifuged and noncentrifuged samples at any time (p>0.05). among non-centrifuged samples, total motility clinical theriogenology • volume 10 number 1 • march 2018 28   (p=0.0065), progressive motility (p=0.006), dsl (p=0.0068), vap (p=0.005), vsl (p=0.003) and bcf (p=0.03) differed with time (fig. 2). total motility, progressive motility, vap and bcf did not change for the first 48 h. however, these parameters decreased at 72 h. dsl was not different between 0 and 24 h, but decreased at 48 h. vsl decreased in all cooled samples compared with the initial value. the percentage of membrane-intact spermatozoa was not different between 24 h (88.5±3.3%) and 48 h (82.5±3.9%) of cooling in the presence of seminal plasma. among centrifuged samples, total motility (p=0.013), progressive motility (p=0.007), dcl (p<0.0001), dap (p<0.0001), dsl (p=0.005), vcl (p<0.0001), vap (p=0.0001), vsl (p=0.026), wob (p=0.026) and alh (p=0.029) differed with time (fig. 3). sperm motility did not differ between 0 and 48 h, but was lower at 72 h. dcl and vcl were higher at 24 h than the rest of the treatments. dap and vap decreased at 48 h. wob was not different among cooled samples, but was lower at 24 h than 0 h. the percentage of membrane-intact spermatozoa was not different between 24 h (81.6±5.3%) and 48 h (77.5±4.7%) of cooling in the absence of seminal plasma. discussion use of frozen semen has become the standard for artificial insemination in cattle. however, the freezing process induces changes in sperm structure and function that alter their viability and longevity in the female reproductive tract. because cooled semen does not undergo freezing and thawing, sperm damage is minimized resulting in higher viability and fertilizing ability.3,16 this allows for a reduction in the insemination dose, optimizing the use of genetically superior bulls. it also increases pregnancy rates compared with frozen semen, improving the efficiency of tai. however, the benefit of storing cooled bovine semen is limited to 24 h.10,16,17 extending the storage period would simplify the management of semen shipments and inseminations. in an attempt to prolong storage time, egg yolk, lecithin or glycerol were added to the semen extender.11,17 however, a significant decline in sperm motility and membrane integrity was still observed at 48 h. pregnancy rates were lower after tai with semen cooled for 48 h than frozen semen.11 oxidative stress and accumulation of reactive oxygen species during liquid preservation produced irreversible sperm damage, with a decrease in sperm motility and fertility.11 in this study, a liposome-containing diluent (optixcell) provided appropriate cryoprotection to preserve sperm motility during cooling for a longer period than previously reported.10,11,16,17 sperm motion parameters and membrane integrity were better preserved in semen cooled for up to 48 h than in frozen semen. while a decline was observed after 72 or 96 h, motion parameters were still comparable with frozen semen. optixcell® is a chemically defined commercial diluent that contains liposomes and is free of animal proteins. the diluent is clear and free of particles, allowing accurate evaluation of sperm function using modern technologies, such as casa or flow cytometry. in previous studies, this extender provided improved cryoprotection during freezing and increased pregnancy rates compared with tryladil, bioxcell or tris-egg yolk.14,15 liposomes added to the semen extender can incorporate into the plasma membrane and change the lipid phase transition.14 this decreases the sensitivity of spermatozoa to cooling and improves their survival.14 prolonged storage of cooled semen for 6 days was reported using a catalase containing semen extender.3 while the longer preservation of sperm function could be attributed to semen clinical theriogenology • volume 10 number 1 • march 201829   extender composition, semen was also centrifuged and seminal plasma was completely removed prior to cooling.3 high concentrations of seminal plasma in cooled stallion semen can be detrimental to sperm quality and fertility.12,13,18-20 centrifugation and removal of seminal plasma improved equine sperm motility after storage times of more than 24 h,12 and decreased production of reactive oxygen species and degradation of dna.19,21 in bulls, a high molecular weight fraction of seminal plasma has been shown to reduce sperm motility and viability.22 therefore, seminal plasma was removed in the second part of this study in an attempt to improve sperm motility and membrane integrity during cooling. however, removal of seminal plasma did not improve semen quality during cold storage under the conditions of this study. sperm velocity (dcl, vcl, dap, vap) in centrifuged samples was generally higher at 24 h of cooling than at any other time points, including initial values. in a previous study, addition of glycerol to the medium increased sperm velocity during cooling.17 it was thought that glycerol induced cellular dehydration, making spermatozoa lighter and increasing velocity.17 it could be speculated that incubation of spermatozoa for 24 h in optixcell, which contains glycerol, allowed for a more prolonged equilibration time with subsequently more dehydration and lighter spermatozoa.23 also speculative, the prolonged equilibration could have allowed for more incorporation of glycerol into the cell, which could then be metabolized acting as an additional energy source.24,25 more prolonged cooling may result in cell damage, possibly explaining why the same increase in velocity was not observed at 48 or 72 h. this initial increase in velocity was not observed when semen was cooled in presence of seminal plasma. it is possible that seminal plasma proteins adsorbed to the plasma membrane stabilized the membrane and decreased its permeability to water or glycerol. this was the first study evaluating use of optixcell for bull semen cooling, and the effect of removing seminal plasma on motility of cooled spermatozoa. cooled bovine semen diluted in optixcell remained suitable for artificial insemination for up to 48 h, as assessed by sperm motion parameters and membrane integrity. during this period, sperm motility and membrane integrity were superior in cooled semen than frozen semen. removal of seminal plasma by centrifugation did not improve preservation of sperm motion or membrane integrity in cooled semen, and therefore is not necessary. the effect of these findings on pregnancy rate, and the performance of semen processed using the protocol tested here in a tai program need to be evaluated. references 1. aurich c: recent advances in cooled-semen technology. anim reprod sci 2008;107:268-275. 2. peña f, núñez-martínez i, morán jm: semen technologies in dog breeding: an update. reprod domest anim 2006;41(s2):21-29. 3. verberckmoes s, van soom a, dewulf j, et al: comparison of three diluents for the storage of fresh bovine semen. theriogenology 2005;63:912-922. 4. jasko dj, moran dm, farlin me, et al: pregnancy rates utilizing fresh, cooled and frozen-thawed stallion semen. procannu conv am assoc equine pract 1992;38:649-660. 5. miller cd: optimizing the use of frozen–thawed equine semen. theriogenology 2008;70:463-468. 6. amann rp, picket bw: principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. j equine vet sci 1987;7:145-173. 7. sieme h, harrison rap, petrunkina am: cryobiological determinants of frozen semen quality, with special reference to stallion. anim reprod sci 2008;107:276-292. 8. watson pf: the causes of reduced fertility with frozen semen. proc 14th intl cong anim reprod, stockholm, sweden, 481-492, 2000. 9. roelofsa jb, bouwmana eg, dielemanb sj, et al.: influence of repeated rectal ultrasound examinations on hormone profiles and behaviour around oestrus and ovulation in dairy cattle. theriogenology 2004;62:1337-1352. 10. crespilho am, papa fo, santos mp, et al: use of cooled bull semen as a strategy to increase the pregnancy rate in fixed-time artificial insemination programs-case report. am j anim vet sci 2012;7:175-179. clinical theriogenology • volume 10 number 1 • march 2018 30   11. crespilho am, nichi m, guasti pn, et al: sperm fertility and viability following 48 h of refrigeration: evaluation of different extenders for the preservation of bull semen in liquid state. anim reprod sci 2014;146:126-133. 12. brinsko sp, crockett ec, squires el: effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. theriogenology 2000;54:129-136. 13. barrier-battut i, bonnet c, giraudo a, et al: removal of seminal plasma enhances membrane stability on fresh and cooled stallion spermatozoa. reprod domest anim 2013;48:64-71. 14. ansari ms, rakha ba, akhter s, et al: optixcell improves the postthaw quality and fertility of buffalo bull sperm. theriogenology 2016;85:528-532. 15. miguel-jiménez s, mogas t, peña ai, et al: post-thaw changes in sperm membrane and ros following cryopreservation of dairy bull semen using four different commercial extenders. anim reprod 2016;13:573. 16. borges-silva jc, silva mr, marinho db, et al: cooled semen for fixed-time artificial insemination in beef cattle. reprod fertil dev 2016;28:1004-1008. 17. papa pm, maziero rd, guasti pn, et al: effect of glycerol on the viability and fertility of cooled bovine semen. theriogenology 2015;83:107-113. 18. jasko dj, hathaway ja, schaltenbrand vl, et al: effect of seminal plasma and egg yolk on motion characteristics of cooled stallion spermatozoa. theriogenology 1992;37:1241-1252. 19. kareskoski m, sankari s, johannisson a, et al: the association of the presence of seminal plasma and its components with sperm longevity in fractionated stallion ejaculates. reprod domest anim 2011;46:1073-1081. 20. pickett bw, sullivan jj, byers ww, et al: effect of centrifugation and seminal plasma on motility and fertility of stallion and bull spermatozoa. fertil steril 1975;26:167-174. 21. morrell jm, georgakas a, lundeheim n, et al: effect of heterologous and homologous seminal plasma on stallion sperm quality. theriogenology 2014;82:176-183. 22. baas jw, molan pc, shannon p: factors in seminal plasma of bulls that affect the viability and motility of spermatozoa. j reprod fertil 1983;68:275-280. 23. mazur p: freezing of living cells: mechanisms and implications. am j physiol 1984;247:125-142. 24. mann t, white ig: metabolism of glycerol, sorbitol and related compounds by spermatozoa. nature 1956;178:142143. 25. mohri h, masaki j: glycerokinase and its possible role in glycerol metabolism of bull spermatozoa. j reprod fertil 1967;14:179-194. clinical theriogenology • volume 10 number 1 • march 201831   fig. 1. motion parameters of bovine spermatozoa cooled for 96 h or frozen (n=9). a,b,cp<0.05. tmot=total motility (%), pmot=progressive motility (%), dcl=curvilinear distance (m), dap=distance of average path (m), dsl=straight line distance (m), vcl=curvilinear velocity (m/sec), vap=average path velocity (m/sec), vsl=straight line velocity ((m/sec), lin=linearity, str=straightness, wob=wobble, bcf= beat cross frequency (hertz), alh= amplitude of lateral head displacement (m). clinical theriogenology • volume 10 number 1 • march 2018 32   fig. 2. motion parameters of bovine spermatozoa cooled for 72 h without centrifugation (n=11). a,bp<0.05. tmot=total motility (%), pmot=progressive motility (%), dcl=curvilinear distance (m), dap=distance of average path (m), dsl=straight line distance (m), vcl=curvilinear velocity (m/sec), vap=average path velocity (m/sec), vsl=straight line velocity ((m/sec), lin=linearity, str=straightness, wob=wobble, bcf= beat cross frequency (hertz), alh= amplitude of lateral head displacement (m). clinical theriogenology • volume 10 number 1 • march 201833   fig. 3. motion parameters of centrifuged bovine spermatozoa cooled for 72 h (n=11). a,bp<0.05. tmot=total motility (%), pmot=progressive motility (%), dcl=curvilinear distance (m), dap=distance of average path (m), dsl=straight line distance (m), vcl=curvilinear velocity (m/sec), vap=average path velocity (m/sec), vsl=straight line velocity ((m/sec), lin=linearity, str=straightness, wob=wobble, bcf= beat cross frequency (hertz), alh= amplitude of lateral head 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] /pagelayout /singlepage >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2018: luteinizing hormone receptor expression in canine t-lymphomacell lines luteinizing hormone receptor expression in canine t-lymphoma cell lines sabrina gust,a alyssa ettinger,a takuya mizuno,b michelle kutzlera adepartment of animal and rangeland sciences, oregon state university, corvallis, or; blaboratory of veterinary internal medicine, the united graduate school of veterinary medicine, yamaguchi university, 1677 – 1 yoshida, yamaguchi 753 – 8515, japan introduction canine lymphoma is a common malignant tumor in dogs. luteinizing hormone receptors (lhr) have been found in normal and neoplastic canine lymphatic tissue.1 the lhrs have also been found in human leukemia cell lines.2 therefore, we hypothesized that lhr would also be present in canine lymphoma cell lines. the objectives of the study were to determine if lhr is expressed in cultured canine t-lymphoma cells and to quantify the level of cellular expression of lhr in 3 different cell lines. methods t-lymphoma cell lines (clc, ema, clk) established from primary canine lymphomas were generously donated from yamaguchi university (yamaguchi, japan). following receipt, cells were thawed and cultured in r10 complete medium (rpmi1640; vwr life science, visalia, ca) supplemented with 10% fetal bovine serum (#10803-034; vwr) and 100 u/ml penicillin with 100 μg/ml streptomycin (#sv30082.01; hyclone, south logan, ut) at 37ºc at a humidified 5% co2 incubator. cells were cultured in fresh r10 complete media every 2-3 days at a ratio of 1:4 (ema, clk) or 1:9 (clc). at 75-85% confluence, cells were washed to remove culture media and transferred to eppendorf tubes. nonspecific binding was blocked with mouse seroblock fcr (1:10 dilution; bio-rad antibodies, richmond, ca). cells were incubated with goat polyclonal lhr (1:50 dilution; sc-26341, santa cruz biotechnology, dallas, tx) for 20 minutes at room temperature. cells were washed and then incubated with goat f(ab’)2 igg negative control:rpe (1:10 dilution; bio-rad antibodies, richmond, ca). the primary antibody was omitted in the negative controls. the cells were analyzed on a cytoflex flow cytometer (beckman coulter, indianapolis, in) at the oregon state university core facility. results the clc, ema, and clk cell lines expressed lhr in 45%, 10%, and 35% of cells. in all cell lines, the cell population that expressed lhr was smaller in size (forward scatter) and more granular (side scatter). there was no positive staining evident in any of the negative control samples. discussion this is the first study to show lhr expression in canine lymphoma cell lines. our laboratory is currently investigating the pro-neoplastic effects of lhr activation (e.g. cell proliferation) using these cell lines. keywords: cell culture, dog, flow cytometry, luteinizing hormone, lymphoma acknowledgment research funded by oregon state university incentives program references 1. ettinger a, zwida k, kutzler m: normal and neoplastic canine lymphocytes express luteinizing hormone receptors. clin therio 2017;9:428. 2. abdelbaset-ismail a, borkowska s, janowska-wieczorek a, et al: novel evidence that pituitary gonadotropins directly stimulate human leukemic cells-studies of myeloid cell lines and primary patient aml and cml cells. oncotarget 2016;7:3033-3046. clinical theriogenology • volume 10, number 3 • september 2018321 uterine flush fluid proteome of mares in estrus and diestrus: qualitative differences and functional classification uterine flush fluid proteome of mares in estrus and diestrus: qualitative differences and functional classification firdous khan,a,b tracey cheniera adepartment of population medicine, ontario veterinary college university of guelph, guelph, on, canada bdepartment of large animal medicine and surgery school of veterinary medicine, st. george’s university, true blue, grenada analysis of the uterine flush fluid proteome is a minimally invasive approach for evaluating changes in uterine luminal composition across stages of the estrous cycle in mares. objectives were to evaluate qualitative differences in the uterine flush fluid proteome between mares in estrus and mares in diestrus and to perform a functional classification of proteins either unique to each stage or common between the 2 stages. uterine flush fluid samples were collected from 8 light breed mares in either estrus (n = 5) or diestrus (n = 3). mares were 3 8 years of age and reproductively healthy, based on transrectal examination, gross appearance of uterine flush fluid and endometrial cytology, culture and biopsy. proteomic analysis of uterine flush fluid was conducted using label-free liquid chromatography tandem mass spectrometry. proteins exclusively detected in estrus or diestrus and those common to both stages were identified using scaffold software (version scaffold_4.4.8, proteome software inc., portland, or). functional classification of identified proteins into gene ontology (go) categories [cellular component (cc), molecular function (mf) and biological process (bp)] was performed using protein analysis through evolutionary relationships (www.pantherdb.org) classification system version 11.0.1 of 172 proteins identified, 51 and 28 were exclusively detected in mares in estrus and diestrus, respectively, with 93 proteins common to both stages. most represented terms in various go categories were similar among the 3 subsets of proteins. most represented cc terms were cell (go:0005623) and extracellular region (go:0005623), most represented mf terms were binding (go:0005488) and catalytic activity (go:0003824) and most represented bp terms were cellular process (go:0009987) and metabolic process (go:0008152). however, in relatively less represented terms, there were some differences between protein subsets unique to estrus or diestrus. for instance, immune system process (go:0002376) was represented by proteins unique to estrus, whereas developmental process (go:0032502) was represented by proteins unique to diestrus. in conclusion, proteomic analysis of the uterine flush fluid enabled identification of subsets of proteins unique to estrus or diestrus, or common to both stages. based on functional classifications, a majority of the proteins were involved in basic functions such as maintenance of cellular and metabolic processes. results of this study can serve as a baseline for future research focused on finding stage-specific protein markers or evaluating differences in the uterine flush fluid proteome between normal mares and those with uterine disease. keywords: equine, uterus, proteome, estrus, diestrus reference 1. mi h, huang x, muruganujan a, tang h, et al: panther version 11: expanded annotation data from gene ontology and reactome pathways, and data analysis tool enhancements. nucleic acids res 2017;45:d183-189. 529 clinical theriogenology • volume 11, number 3 • september 2019 530clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2017: a single post-mating uterine infusion of penicillin does not improve pregnancy rates in maiden thoroughbred broodmares a single post-mating uterine infusion of penicillin does not improve pregnancy rates in maiden thoroughbred broodmares c.f. scoggin,a c.a. burden,b m.r. schnobrich,a e.a. bradecampa aleblanc reproduction center, rood and riddle equine hospital, lexington, ky; bgoulburn valley equine hospital, congupna, victoria, australia abstract past studies have found improved pregnancy rates in mares treated with post-mating intrauterine (iu) therapies and/or oxytocin. these treatments are thus commonplace in broodmare practice. in some instances, these treatments are given regardless of a mare’s history, current status and examination findings. the purpose of this study was to evaluate the effect of a single iu infusion of saline or procaine penicillin g on pregnancy and live-foal rate in maiden mares. thirty-eight maiden thoroughbred broodmares were evaluated over at total of 50 estrous cycles. all mares had a negative uterine culture prior to each mating. during each breeding cycle, all mares received 10 iu oxytocin (im) 4-6 h after mating and either an infusion of physiologic saline (control; n=24 cycles) or penicillin g (ppg; n=26 cycles) 16-24 h post-breeding. if a mare failed to become pregnant on her first cycle, another treatment was performed without consideration to the treatment given on the previous cycle. day 0 was defined as the day of ovulation. pregnancy status was determined by transrectal ultrasonography. pregnancy rate per cycle was 72.00% (36/50), and day 45 pregnancy and live-foal rates were 100% (36/36) and 90.6% (29/32), respectively. first-cycle pregnancy rates did not differ (p=0.278) between control (83.3%; 15/18) and ppg cycles (65.0%; 13/20). no difference (p=1.0) was found in live-foal rates between control (87.5%; 14/16) and ppg mares (93.8%; 15/16). these results indicate a single post-mating infusion does not appear to affect pregnancy and live-foal rates in maiden thoroughbred mares. keywords: broodmare, antibiotics, penicillin, ecbolics, intrauterine treatments, endometritis introduction post-mating treatment with iu infusion of antibiotics and ecbolics is common in many large broodmare practices, especially in regions that utilize live-cover breeding programs. justifications for use are: reducing the incidence of infectious endometritis and assisting with fluid drainage.1 in the authors’ practice area (central ky), it is not unusual for mares to receive a single intrauterine (iu) infusion of antibiotics 24-48 hours after breeding. in many cases, these treatments are performed irrespective of the mares’ status, history or findings from the post-mating examination. most commonly used agents for iu infusion are those with antibacterial activity, such as ticarcillin (+/clavulonic acid) and ceftiofur sodium.2 pycock et al. reported improved pregnancy rates in mares receiving iu infusion of antibiotics within 72 hours of mating compared to untreated controls.3 these researchers also demonstrated that a combination therapy of intravenous oxytocin with iu infusion of antibiotics provided superior pregnancy rates compared to either treatment alone and untreated controls. unfortunately, this study did not control for age or the reproductive status (e.g., maiden, barren and foaling) of the mares. because both are influential predictors of fertility (for a review see4), results may have been confounded if one or more of the treatment groups contained, for example, an increased proportion of young mares. despite the apparent usefulness of iu antibacterial infusions, their routine use is superfluous and potentially counterproductive by selecting for resistant organisms.5 frequent and unscrupulous use of antibiotics can lead to microbial resistance, which have important public health concerns.6 in broodmare practice, iu antibiotic infusions has been associated with an increased susceptibility to fungal endometritis.1,7 finally, cumulative treatments can be costly and affect the breeding farms’ profitability. it thus seems prudent for both clinicians and farm managers to consider a more judicious approach to the use of prophylactic antibiotics. breeding-induced endometritis is a normal physiologic response following coitus, and the majority of mares are capable of resolving the inflammation without any intervention.8 studies evaluating risk factors for bacterial infections can be useful in guiding future treatment strategies. the clinical theriogenology • volume 9 number 1 • march 201753 use of iu antibiotic infusions is one area where antimicrobial use can potentially be stemmed, especially in certain subsets of mares where the risk of developing a clinical case of infectious endometritis is low. the purpose of this study was to evaluate the effect a single infusion of either saline or penicillin g had on pregnancy and live-foal rates in maiden thoroughbred mares. materials and methods this study involved 38 maiden thoroughbred mares, 3-7 years of age (4.46±0.72 years) over 50 estrous cycles. it took place during the 2015 northern hemisphere breeding season in bourbon county, kentucky (38.2098° n, 84.2530° w). mares were housed at night in two separate barns beginning december first of the previous year or upon arrival to the farm. supplemental lighting of ~10 ft-candles per stall was provided from 4 to 11 pm each night. during the day, mares were turned out in two separate fields (>50 acres/field) unless they were under exercise restriction due to an injury. mares were fed two quarts of 12% protein concentrate twice daily and free-choice grass hay while stalled. fresh water was provided ad libitum. the first mares were bred february 16th and the last mare bred may 25th. mares were teased daily and examined when displaying signs of estrus or a change in behavior. reproductive evaluations consisted of transrectal palpation and ultrasonography and a vaginal speculum examination. a uterine culture was obtained on each mare prior breeding and submitted to a clinical veterinary microbiologic laboratory.* samples were considered negative when no growth was observed after 48 hours of incubation. mares were deemed suitable for mating when they had one or more large (≥35 mm) preovulatory follicles. the day prior to breeding, mares were administered 2500 iu human chorionic gonadotropin (hcg) intravenously to induce ovulation on the first cycle. if mares failed to become pregnant on the first cycle, then 1.8 mg deslorelin acetate (sucromatetm, thorn bioscience, louisville, ky) was administered intramuscularly on subsequent cycles to indue ovulation. mares were bred by live-cover mating to commercial thoroughbred stallions of acceptable fertility. four to six hours after each cover, all mares received a single dose of 10 iu oxytocin im. sixteen to twenty-four hours after mating, a separate reproductive examination was performed to detect ovulation (defined as day 0) and identify any unusual findings, such as the presence and amount of intraluminal fluid, excessive edema, and pneumovagina. regardless of the findings, a single iu infusion was performed of either 35 ml physiologic saline (control) or 5 million units procaine penicillin g (ppg) qs to 35 ml in physiologic saline. treatments were chosen by a blinded technician by selecting from one of two infusions. if a mare failed to become pregnant on the first cycle, another treatment was performed without consideration to the treatment given on the previous cycle. once a mare had ovulated, the only other treatment she received was a caslick’s procedure (either complete or a repair); no additional oxytocin injections or iu infusions were performed. the first pregnancy examination was performed on day 14 and subsequent examinations were performed on days 16, 23, 35, 45, and 60, as well as during mid-gestation. live-foal rates were determined by evaluating produce records reported to the north american jockey club. pregnancy and live-foal rates were compared between groups by two-tailed fishers exact tests for small counts. significance was defined as p<0.05. results this study involved 38 mares, and foaling data were available for 32 of these mares. live foals were reported from 29 of these mares; one mare died prior to foaling; one mare aborted at mid-gestation; and the third mare delivered a dead foal. foaling data were not available for six mares: three mares were removed from the study after day 45 of gestation due to illness, and no reports were filed for three mares. conveniently but not intentionally, these exclusions yielded the same sample sizes (16 mares/group) between the two treatment groups when foaling data were compared between these two groups. first-cycle, per cycle, day 45 and live-foal rates are provided in the table. firstand per-cycle pregnancy rates are represented graphically in the figure. thirty-eight mares were included in the * rood and riddle veterinary laboratory, lexington, ky clinical theriogenology • volume 9 number 1 • march 2017 54 pregnancy data. foaling data were available for only 32 mares (32/38; 84.21%) for reasons described above. table. pregnancy and live-foal data obtained from the current study. no differences (p>0.05) were observed in any of the measured parameters. treatment group no. mares no. cycles per cycle pregnancy rate (%) first-cycle pregnancy rate (%) day 45 pregnancy rate (%) livefoal rate* control 18 24 87.50a 83.33a 79.17a 87.50 (14/16)a ppg 20 26 65.38a 65.00a 65.38a 93.75 (15/16)a total 38 50 76.00 73.68 100.00 90.63 (29/32) *only 32 mares were included in the live-foal data; the ratios are provided in parentheses. avalues in columns with similar letters are similar (p>0.05) overall pregnancy rate per cycle was 76.00% (38/50), while first-cycle, day 45 and live-foal rates were 73.68% (28/38), 100.0% (38/38) and 90.63% (29/32), respectively. no difference (p = 0.1) was observed in pregnancy rate per cycle between control and ppg mares. similarly, no significant interaction was seen between the two treatments groups with respect to first-cycle (p = 0.278) and day 45 (p = 0.352) pregnancy rates, as well as live-foal (p = 1.0) rates. discussion results from this study indicate that a single post-mating iu infusion of penicillin the day after live-cover mating did not significantly affect pregnancy or live-foal rates in maiden thoroughbred mares. no significant interactions were observed between mares receiving a single iu infusion of saline or penicillin when evaluating first-cycle and seasonal pregnancy rates, as well as live-foal rates. furthermore, data from this study suggest that uterine infusions did not negatively impact fertility, but that cannot be stated with absolute certainty due to a lack of negative controls (e.g., mares receiving no iu treatments) in this study. antibiotic residues been found in the milk of dairy cows receiving iu infusions of procaine penicillin g,9 and there is conjecture that a prevailing residue within the endometrium may adversely affect embryonic or fetal development. no data were collected to determine if such a residue occurs; however, if a residue does form after a single infusion of procaine penicillin g, results from this study indicate that pregnancy and live-foal rates were not adversely affected. finally, data contained herein provide further evidence of what has been known for some time in clinical practice: young maiden mares appear to be at very low-risk of clinical infectious endometritis and thus require minimal postmating management. the researchers chose to study this topic because uterine infusions are a fairly common practice in areas of high equine breeding activity, particular live-cover operations. in some instances, it is used as blanket or stopgap treatment on large stud farms because of pressure to achieve high pregnancy rates. however, commercial operations are also businesses and must be treated as such to remain profitable. because there does not appear to be a clinical benefit for a single iu infusion in young reproductively normal mares, this procedure could be abandoned in this subset of mares without sacrificing fertility yet improving profitability. in the usa, a single uterine infusion can range in cost from tens to hundreds of dollars, depending on the product. assuming a median cost of $50 per infusion and using the mare numbers from this study, reduction in farm expenses would be ~$2,500 (e.g., 50 cycles x $50 = $2,500). findings from this study may also have pertinence with respect to public health. antimicrobial resistance is of immediate concern to health professions and the global community, and strategies to curb emergence are in high demand.10 overuse is one of the main reasons attributed to the development of clinical theriogenology • volume 9 number 1 • march 201755 resistance, and livestock programs are considered purveyors of resistance due to the shear quantities of antimicrobials used in agricultural production systems.11,12 while the amount of antimicrobials used for iu infusions in mares is relatively low compared to volumes used in food-producing species, antimicrobial stewardship should apply to all clinicians. veterinarians should assume some of the onus in promoting judicious use of antimicrobials, regardless of how much or little they use. pregnancy and live-foal rates reported in this study were consistent with—if not slightly higher— than previously published reports.13-15 this is most likely due to the present study including only young and healthy maiden mares. past studies have evaluated the efficacy of iu antibiotic infusion3 or uterine irrigation in conjunction with a single iu antibiotic infusion,16 but they either did not control for age or involved only barren mares. because age and status can affect mare fertility, direct comparison of fertility data in the present study with that of the past should be interpreted with caution. it should also be mentioned that in these previous studies oxytocin was administered six hours after breeding. oxytocin was used in a similar manner in the current study, and its clinical benefit should not be overlooked. it is a potent ecbolic that is highly useful in assisting both normal and subfertile mares with uterine fluid clearance.17 additionally, treatment with oxytocin alone has been shown improve pregnancy rates in mares receiving 25 iu (iv) within 48 hours of mating compared to untreated controls.3 the use of a single iu infusion was similar to what was performed in previously cited studies. while a clinical benefit of a single iu infusion has been noted in previously cited studies, a direct association with improved pregnancy rates and its effect in combating infectious endometritis remains in question. for example, published systemic doses for procaine penicillin in adult horses are 22,00044,0000 unit/kg twice daily or 12 million units per dosage. the dosage used in the present study (5 million units) was previously reported in the literature as adequate for treating susceptible infections.18 however, it is less than half that and given only as a sole infusion. whether or not therapeutic concentrations were created and maintained with this treatment is unknown but warrants further investigation. along these same lines, the choice of penicillin g, and specifically procaine penicillin g, could have influenced pregnancy rates. the drug used in this study was compounded from the commercially available intramuscular injection by dissolving 5 million units of penicillin g (17 ml of a 300,000 units/ml) in approximately 18 ml of physiologic saline. even though this formulation is relatively uncommon from a clinical standpoint,2 it should have good activity against streptococcus equine ssp zooepidemicus (beta-strep),19 which is the most common bacteria isolated from mares with infectious endometritis.20 furthermore, the author has used it with good clinical success in confirmed cases of betastrep endometritis, and procaine penicillin g was a component of an antibiotic used in a past study evaluating the efficacy of iu infusions.3 there were a few drawbacks to the current study design. first, as previously mentioned, there were no negative controls. second, this study involved only a small number of test subjects (n=38) and reproductive cycles (n=50) and certain mares had to be excluded after day 45 due to extenuating circumstances. thus, the statistical power of this study was relatively low. also, endometrial cytology data were not available for these mares; all mares had only a “clean” uterine culture. the combination of a culture and cytology increases the sensitivity of diagnosing clinical endometritis,21 so there is a possibility that a culture alone failed to identify mares with low-grade endometritis, which could adversely affect pregnancy rates. finally, there were three foaling reports that had yet to be filed at the time of this writing, and all three mares were part of the control group. if all three report a dead foal, then the live-foal percentage would drop to ~74% and approach statistical significance. in conclusion, results from this study indicate that a single post-mating iu infusion of penicillin has no effect on pregnancy or live-foal rates in young maiden mares. per cycle pregnancy rates for control mares verged on a trend (p=0.1) of being higher than ppg mares. unfortunately, the limitations of this study do not allow for the claim that iu infusions are contraindicated, but data presented herein could suggest that such treatment is unnecessary to achieve adequate fertility in young maiden mares. other factors, such as past reproductive history, proper breeding management, and stallion selection appear to be much more important factors dictating pregnancy and live-foal rates in horses. clinical theriogenology • volume 9 number 1 • march 2017 56 references 1. liu ikm, troedsson mht: the diagnosis and treatment of endometritis in the mare: yesterday and today. theriogenology 2008;70:415-420. 2. dascanio j: how and when to treat endometritis with systemic or local antibiotics. proc annu conv am assoc equine pract; 2011. p. 24-31. 3. pycock jf, newcombe jr: assessment of the effect of three treatments to remove intrauterine fluid on pregnancy rate in the mare. vet rec 1996;138:320-323. 4. scoggin cf: not just a number: effect of age on fertility, pregnancy and offspring vigour in thoroughbred brood-mares. reprod fertil dev 2015;27:872-879. 5. cooke cd: prophylactic intra-uterine antibacterial therapy. equine vet educ 2015;27:554-555. 6. sabtu n, enoch da, brown nm: antibiotic resistance: what, why, where, when and how? br med bull 2015;116:105113. 7. stout tae: fungal endometritis in the mare. pferdeheilkunde 2008;1:83-87. 8. woodward em, troedsson mht: inflammatory mechanisms of endometritis. equine vet j 2015;47:383-389. 9. black wd, mackay al, doig pa, et al: a study of drug residues in milk following intrauterine infusion of antibacterial drugs in lactating cows. can vet j 1979;20:354-357. 10. world health organization. antimicrobial resistance in: factsheets: world health organization, media centre, 2016, url: http://www.who.int/mediacentre/factsheets/fs194/en/. 11. gilchrist mj, greko c, wallinga db, et al: the potential roled of concentrated animal feeding operations in infectious disease epidemics and antibiotic resistance. environ health perspect 2007;115:313-316. 12. levy sb, marshall b: antibacterial resistance worldwide: causes, challenges and responses. nat med 2004;10:s122s129. 13. allen wr, brown l, wright m, et al: reproductive efficiency of flatrace and national hunt thoroughbred mares and stallions in england. equine vet j 2007;39:438-445. 14. bosh ka, powell d, shelton b, et al: reproductive performance measures among thoroughbred mares in central kentucky, during the 2004 mating season. equine vet j 2009;41:883-888. 15. nath lc, anderson ga, mckinnon ao: reproductive efficiency of thoroughbred and standardbred horses in northeast victoria. aust vet j 2010;88:169-175. 16. knutti b, pycock jf, van der weijden gc, et al: the influence of early post-breeding uterine lavage on pregnancy rate in mares with intrauterine fluid accumuluations after breeding. equine vet educ 2000;12:267-270. 17. leblanc mm, neuwirth l, mauragis d, et al: oxytocin enhances clearance of radiocolloid from the uterine lumen of reproductive normal mares and mares susceptible to endometritis. equine vet j 1994;26:279-282. 18. leblanc mm, mckinnon ao: breeding the problem mare in: mckinnon a, squires e, vaala w, et al., eds. equine reproduction. ames (ia): wiley-blackwell; 2011. p. 2620-2642. 19. singh br: antimicrobial sensitivity assay and antimicrobial chemotherapy in animals: a practical approach in: singh br,somvanshi r, eds. disease of animals: diagnosis and management. izatnagar-243: indian veterinary research institute, 2013. p. 7-31. 20. jackson ps, allen wr, ricketts sw, et al: the irritacy of chlorhexidine gluconate in the genital tract of the mare. vet rec 1979;105:122-124. 21. riddle wr, leblanc mm, stromberg aj: relationships between uterine culture, cytology, and pregnancy rates in a thoroughbred practice. theriogenology 2007;68:395-402. clinical theriogenology • volume 9 number 1 • march 201757 figure. day 15 and 45 pregnancy rates of control and ppg mares 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 90.00% 100.00% p re gn an cy r at es ( % ) intrauterine treatment comparison of pregnancy rates between two different iu treatments procaine penicillin g control a a a a day 15 day 45 clinical theriogenology • volume 9 number 1 • march 2017 58 2018: reproductive challenges in pet birds and reptiles reproductive challenges in pet birds and reptiles kurt k. sladky department of surgical sciences, school of veterinary medicine, university of wisconsin, madison, wi the objective of this presentation is to describe normal reproductive structures and function in reptile and avian species, and primarily focus on clinical diagnosis and management of common reproductive disorders in captive birds and reptiles. reptiles our understanding of normal reptile reproductive strategies and, conversely, reproductive disorders is complicated by the fact that there is an enormous diversity of species across the class reptilia, one of the most phylogenetically diverse animal classes with four main orders: chelonia (turtles, terrapins, tortoises), squamata (lizards), serpentes (snakes), and crocodilia (alligators, crocodiles, caiman, gharial). reproductive activity tends to be seasonal in most reptile species, but may exists year around, particularly in tropical species. while most reptile species require sexual intercourse to produce offspring, some species are parthenogenic (e.g., komodo dragons, whiptail lizards, boa constrictors, etc.); that is, the occurrence of unisexual lines whereby reproduction occurs without any involvement of males or their sperm. in addition, females can lay infertile eggs when a male is not present.1 most reptile species are oviparous (i.e., egg layers), but a few species give birth to live young (i.e., ovoviviparous and viviparous; for example, skinks, horned lizards, boas, adders, and some chameleons to name a few).1 sex differentiation is commonly temperature dependent and establishing sex through sexually dimorphic phenotypes ranges from relatively simple (e.g. many chelonians and lizards) to difficult (some snake species, skinks, monitor lizards, etc.). determining sex of a reptile can be particularly challenging in younger animals. methods include phenotypic characteristics (e.g., concave plastron in chelonians, femoral pore size in some lizards, longer forelimb nails in aquatic turtles, longer tails in aquatic turtles, cloacal spurs in boid snakes, eversion of hemipenes in lizards and snakes), use of snake probes, imaging modalities (e.g., ultrasonography, computed tomography[ct] scan), or use of endoscopy (cloacoscopy versus coelioscopy). anatomically, female reptiles have two ovaries, two oviducts, and the reproductive tract exists into the cloaca; in some snakes, the left oviduct is vestigial and nonfunctional. females of some reptile species can store semen from a male for more than one reproductive season. males have two testicles and either a phallus (chelonians and crocodilians) or hemipenes symmetrically positioned on each side of the cloaca (snakes and lizards). the phallus and hemipenes are intromittent organs used exclusively for reproduction, and there is no direct connection to the urinary system.1 common reproductive disorders: diagnosis and management males hemipenile plugs are very common in some captive male lizard and snake species.2-4 hemipenile plugs are slightly elongated structures protruding from the tip of the hemipene. the exposed portion tends to be dry, while the remainder within the hemipenile orifice tends to be moist with a caseous consistency.3 these plugs are generally composed of hard waxy plugs of seminal fluid and cellular debris that build up in the inverted hemipenes.3 chronic build-up and retention of these plugs appear to cause discomfort, and many clinicians will mistake these plugs for necrotic prolapsed hemipenes. fortunately, hemipenile plugs are relatively easy to remove by grasping the material with a forceps and gently pulling the plug straight out of the orifice. rarely do retained hemipenile plugs require surgical intervention, which may include resection of the hemipene or hemipenes. recurrence may occur in the future. prolapse of the phallus or hemipenes is a common male reptile condition, which may have no significant consequences with natural resolution, or may become a medical problem if prolonged. prolapse may occur under normal conditions in which males become sexually aroused and develop erect phalluses or hemipenes, or under disease conditions, such as the presence of uroliths, neurologic clinical theriogenology • volume 10, number 3 • september 2018175 dysfunction, chronic constipation amongst others.1-6 under normal conditions, the erectile tissues will regress and the prolapsed organ(s) will retract into normal position. under abnormal conditions, the organs may remain prolapsed and the tissues become edematous and necrotic due to vascular compromise and excoriation. in cases in which the tissues remain viable, the phallus or hemipene may be moistened and the erectile tissues reduced back into normal anatomical position. medical management may also include a mu-opioid analgesic (e.g., morphine or hydromorphone) and anti-inflammatory drugs such as meloxicam or carprofen.7 in chronic cases in which tissues are no longer viable, a phallectomy (amputation of the phallus or hemipene) may be necessary. it is important to remember that the reproductive organs of male reptiles are not connected to the urinary tract, so that amputation will not affect ability to pass urine; it will only affect reproductive ability. if only one hemipene needs amputation in snakes or lizards, the remaining hemipene can function as a viable reproductive organ. phallectomy in chelonians or crocodilians will permanently affect the ability to reproduce. orchidectomy. elective castration of male reptiles is uncommon, primarily due to the fact that testicles are intracoelomic and the procedure is invasive. surgical removal of a testicle or testicles may be necessary in cases of testicular tumors, or in a last-ditch effort to alter aggressive behavior or hypersexual behavior in pet male reptiles.1,2,5 more recently there is interest, but no published evidence, of administering gonadotropin releasing hormone (gnrh) agonists (e.g., deslorelin implants or leuprolide acetate depot) to hyper-aggressive or hypersexual reptiles in order to attempt to change these behaviors.8 females preovulatory follicular stasis is a common reproductive disorder in female reptiles. typically, it is associated with non-ovulation of preovulatory follicles or absence of regression of these follicles, which causes follicles to continue to enlarge or remain static.2-4 it commonly occurs under conditions in which social or environmental cues are not present, husbandry is inappropriate, or the female has other health problems. it is clinically manifested as hyporexia or anorexia and lethargy, and requires medical or surgical intervention. with chronicity, manipulation and palpation of the coelomic cavity can contribute to rupture of the large follicles causing yolk coelomitis. imaging modalities (e.g., ct scan and/or ultrasonography) are necessary to make a diagnosis of follicular stasis, and serial imaging over a period of time may help with the diagnosis and surgical planning. medical management is generally considered futile. surgical management of this condition is common and includes ovariectomy and/or ovariosalpingectomy, with bilateral ovariectomy being more straightforward than ovariosalpingectomy. these procedures are described in the published literature.1-6 dystocia can occur in female reptiles under a variety of conditions. it is, typically, defined as retention of some or all eggs or fetuses during parturition. dystocia is typically categorized as either nonobstructive or obstructive.3,6 nonobstructive dystocia is most commonly associated with poor husbandry (e.g., inappropriate temperature, humidity or substrate, lack of ultraviolet light source, starvation or obesity, etc.), or infection/inflammation of the oviduct (salpingitis). obstructive dystocia is associated with the presence of abnormally large eggs or fetuses, old injuries to the bones of the pelvic canal, uroliths, or other pathologic conditions. imaging modalities (e.g., ct scan, radiographs, and/or ultrasonography) are necessary to evaluate number and sizes of eggs and fetuses, as well as some pathologic conditions. it is important to remember that ectopic eggs can occur, especially in chelonians, and may be localized within the urinary bladder or the coelomic cavity, which may require endoscopy, cloacoscopy and/or surgical intervention.3,6 medical therapy can be instituted in cases of nonobstructive dystocia, and includes supportive care (fluids, calcium gluconate, anti-inflammatories), and oxytocin, which stimulates oviductal contraction. the administration of calcium gluconate to stimulate the oxytocin receptors is recommended before starting the hormone treatment.3,6 the efficacy of oxytocin for facilitating parturition is variable depending on the species, and its use can lead to oviductal rupture and may increase the possibility of any retained eggs being expressed into the urinary bladder, if present. more invasive therapeutic approaches include percutaneous ovocentesis of larger eggs, and surgical intervention and removal of retained eggs or fetuses (salpingotomy, ovariosalpingectomy). clinical theriogenology • volume 10, number 3 • september 2018 176 salpingitis is inflammation or infection of the salpinx (oviduct), which can interfere with reproduction. diagnostic tests include imaging (ultrasonography, ct scan), biopsy with histopathology, cytology, microbial culture and antimicrobial sensitivity. surgical intervention may be necessary and would include salpingectomy and ovariosalpingectomy. yolk coelomitis occurs when follicles or eggs rupture outside of the reproductive tract, causing yolk to be present in the coelomic cavity.1-6 imaging (ct scan, ultrasonography) may be useful in making the diagnosis of coelomitis, and celiocentesis with cytology may provide supporting evidence of the presence of yolk in the coelom. chronic yolk coelomitis can cause significant inflammation, adhesions and fibrosis, and surgical treatment is complicated and, generally unrewarding. prognosis of yolk coelomitis in any female reptile should be considered poor. neoplasia of the reproductive tract is not common in male or female reptiles.9 testicular tumors (seminoma, sertoli and leydig cell tumors) are most commonly presented in the published literature. ovarian tumors include teratomas, dysgerminomas, granulosa cell tumors, amongst others. leiomyomas and leiomyosarcomas of the oviduct have been reported in female reptiles. ovariectomy and ovariosalpingectomy. elective ovariectomy of female reptiles is uncommon due to invasiveness of the procedure. ovariectomy and/or ovariosalpingectomy may be necessary in cases of follicular stasis, dystocia, and reproductive neoplasia. surgical removal of the ovaries may also occur in an effort to prevent future egg laying. more recently, there is interest, but little published evidence, of administering gnrh agonists (e.g., deslorelin implants or leuprolide acetate depot) to dampen reproductive hormones through negative feedback mechanisms and prevent egg production.8 one publication indicated that reproductive activity was suppressed in a female green iguana after administration of a deslorelin implant, and hyper-aggressive behavior diminished in a bearded dragon after administration of deslorelin.8 birds most detailed information on reproduction in birds exists for few species. anatomically, males have bilateral testes and females of most bird species have a functional left ovary and oviduct, with regression of the right side of the reproductive tract. some species of raptors and ratites have functional right and left reproductive tracts. while most male bird species do not have a phallus, many anseriforms exhibit quite large phalluses. in most birds, copulation occurs with mutual cloacal eversion (“cloacal kiss”) and transfer of semen from the male ejaculatory papilla to the female cloaca. bird species with a phallus will intromit the phallus into the female cloaca. female birds can store sperm for up to several months. reproduction may be seasonal in some species, such that reproductive tissues of both males and females are stimulated by appropriate hormones, become active (follicles develop in the ovary and testicles grow), and sexual behavior commences. seasonally dependent reproduction is associated with a variety of environmental cues, such as changing photoperiod and the presence of other birds. females will lay infertile eggs without a male present. common reproductive disorders: diagnosis and management males and females cloacal prolapse and flaccid cloaca are among the most common clinical conditions in pet birds, particularly cockatoo species due to excessive reproductive behavior. other causes of cloacal prolapse include: dystocia, coelomitis or coelomic mass, or inflammation/infection of the reproductive tract. many of these cases are associated with chronic sexual behavior, during which the bird’s focus of sexual attention is commonly a bonded human. this is considered a “rule out” diagnosis in which all potential medical causes are ruled out leaving sexual behavior as the primary focus. for non-behavioral causes, the underlying problem must be diagnosed and appropriately treated. if the tissues prolapsed appear viable, the prolapsed tissue can be cleaned and replaced and two sutures placed for a period of 7-10 days. if the tissue is necrotic, identification of the organ is imperative, as surgical resection will be necessary. for sexual behavioral causes, medical and surgical management are combined to try to control this condition, but eliminating the problem is almost impossible. typically, the first step is subcutaneous administration clinical theriogenology • volume 10, number 3 • september 2018177 of a gnrh agonist (e.g., deslorelin implant or leuprolide acetate depot) in order to try to dampen the behavior, with the hope that the cloacal prolapse will resolve.8,10,11 in cases in which the prolapse does not resolve or the cloaca is chronically flaccid, non-inflammatory metallic sutures (stainless steel or niobium wire) must be placed for months at a time and then replaced every 6-12 months as needed. niobium is used in human jewelry and is considered hypoallergenic. chronic and inappropriate reproductive behavior may lead to cloacal prolapse or flaccid cloaca, but is commonly associated with feather destructive behavior (i.e., feather-picking) and self-mutilation. in these cases, behavior modification is recommended using positive reinforcement paradigms, but is extremely difficult and time consuming, requiring constant and long-term commitment by the caregiver. several husbandry-related changes are suggested, including diet changes to reduce fat content and changing the way the caregiver handles/pets the bird to avoid sexual stimulation. gonadotropin releasing hormone agonists are used to try to alter or prevent the behaviors, which may have a seasonal component.8,10-12 antianxiety and antidepressant drugs have been tried, but published documentation is sparse and, generally, anecdotal. males neoplasia (testicular tumors) is more common in some species (e.g., budgies) and have been identified as seminomas, leydig and sertoli cell tumors, adenocarcinomas, leiomyosarcomas and lymphosarcoma.13,14 clinical signs may include coelomic distension and a variety of ill bird signs (fluffed feathers, anorexia, depressed behavior, etc.). diagnosis may be made using imaging modalities with ultrasound-guided aspiration and cytology. in male budgies with hormone-secreting testicular tumors, the normally blue cere may turn brown and a unilateral hind limb lameness may be observed. treatment for most avian neoplastic diseases is considered difficult and unrewarding. surgical excision of a reproductive tumor is theoretically possible, but with an overall poor prognosis, surgical intervention may be futile. medical management through chemotherapy is not well studied and would carry a guarded to poor prognosis. our clinical service has used a gnrh agonist (deslorelin implant, 4.7 mg) in a male budgie with a testicular tumor and unilateral hind limb lameness to attempt to shrink the tumor, which reestablished limb function. regular replacement of the implant provided an added two years to the budgie’s life. phallus prolapse (waterfowl) is not uncommon in waterfowl species, and may be associated with hypersexual behavior, trauma, or phallus infection. continued trauma to the prolapsed organ may result in tissue necrosis. medical management may include cleaning, lubrication, and reinsertion of the phallus with or without cloacal sutures, antimicrobials, and nonsteroidal anti-inflammatories (e.g., meloxicam).13,14 with chronic prolapse and tissue necrosis, phallectomy may be necessary, which will eliminate reproduction, but not interfere with urination. females chronic egg laying is a common problem in companion birds, such as psittacines, backyard chickens and waterfowl, with caregivers opposed to this behavior in one group (psittacines) and taking advantage of it in the other (chickens, ducks).13,14 most commonly chronic egg laying refers to a nonseasonal pattern in which there are regular, year-round, clutches. chronic egg laying can cause diseaserelated issues, such as hypocalcemia and other nutritional deficiencies, weight loss, pathologic bone fractures, and predisposition to dystocia and ectopic egg formation. it can also be psychologically disturbing to the caregiver. husbandry recommendations may include changing the diet by reducing fat and carbohydrate intake, decreasing the photoperiod, and avoiding sexually stimulating the bird through touching. since surgical ovariectomy is risky in adult females, administration of gnrh agonists (deslorelin implant or leuprolide acetate depot) to diminish reproductive hormones and reproductive behavior is a commonly used medical approach.8,10-12 gonadotropin releasing hormone agonists must be regularly administered, and there is anecdotal evidence in some species that the duration of effect may decrease after each successive administration. clinical theriogenology • volume 10, number 3 • september 2018 178 dystocia/egg-binding can occur in female birds at almost any time in life and does not appear to be clearly associated with primiparity or multiparity, although there may be a predisposition in older primiparous birds. clinically, birds in dystocia become hyporexic, fluffed, regularly strain, and lose interest in perching. with chronicity, the bird becomes exhausted, obtunded and can pass away. the physical examination may provide evidence of a palpable egg, but sometimes the egg remains in the oviduct more cranially and imaging is necessary to demonstrate the presence of an egg. differentiating an ectopic egg from an intra-oviductal egg can be challenging even with imaging. ultrasonography and/or ct scan may help identify if the egg is within the oviduct or vagina. in cases in which the female bird remains active, alert and responsive, initial medical therapy may be may help with oviposition, unless the egg appears massive. medical therapy includes subcutaneous fluids, calcium gluconate, oxytocin and placing the bird in a warm incubator (85-90˚f). if this does not have any effect within several hours with repeated oxytocin administration, or if the bird is presented in a depressed, weakened state, the bird should be sedated or anesthetized and the egg either extracted in toto using caudal external pressure and lubrication, or broken down through transcloaca ovocentesis. percutaneous ovocentesis is only an option when the egg cannot be visualized per cloaca and in emergent situations. surgical intervention is rarely necessary when an experienced clinician is involved in the case. surgical intervention includes salpingotomy, extraction of the egg through the surgical site and surgical closure. ectopic eggs can occur when the calcified or partially calcified egg is expressed directly into the coelomic cavity. making the diagnosis can be difficult and multiple imaging modalities may be necessary (radiographs, ultrasonnography and ct scan).15 an ectopic egg requires surgical exploration of the coelomic cavity and extraction of the egg. the coelomic cavity may need flushing with warm sterile saline after the egg is removed. yolk coelomitis occurs when an ovum is ovulated or ruptured into the coelomic cavity. yolk adheres to internal organs and causes inflammation and adhesions. with chronicity, ascites is a common sequela. ultrasonography can be useful in diagnosing coelomitis and celiocentesis with cytology may provide supporting evidence of the presence of yolk in the coelom. treatment is generally unrewarding and the prognosis is poor. administration of gnrh agonists may help stop follicular and egg development, which may help the bird feel better and provide extra months of life. nonsteroidal antiinflammatories may help with discomfort. salpingitis refers to inflammation or infection of the oviduct, and most commonly occurs as an ascending bacterial infection from the cloaca. clinical signs will be nonspecific for oviduct inflammation/infection, so ultrasonography and/or ct scan may help define the problem. a cbc may be indicative of inflammation through the presence of a leukocytosis, which is also nonspecific to organ system. with a definitive diagnosis, antimicrobial and anti-inflammatory medications will likely be necessary. neoplasia of the reproductive tract is not uncommon in female birds.13,14 a variety of tumor types have been documented in the literature, including: adenomas and adenocarcinomas, leiomyomas and leiomyosarcomas, granulosa cell tumors, and lymphosarcoma.13,14 clinical signs may include coelomic distension and a variety of ill bird signs (fluffed feathers, anorexia, depressed behavior, etc.). diagnosis may be made using imaging modalities with ultrasound-guided aspiration and cytology. as with males, surgical excision of a reproductive tumor is theoretically possible, but with an overall poor prognosis, surgical intervention may be futile. medical management through chemotherapy is not well studied and would carry a guarded to poor prognosis. references 1. denardo d: reproductive biology: in: mader d, editor. reptile medicine and surgery. 2nd ed. st. louis: elsevier; 2006. p. 376-390. 2. hedley j: reproductive diseases of reptiles. in practice 2016;38:457-462. 3. knotek z, cermakova e, oliveri m: reproductive medicine in lizards. vet clin north am exot anim pract 2017;20:411-438. 4. di girolamo n, selleri p: reproductive disorders in snakes. vet clin north am exot anim pract 2017;391-409. clinical theriogenology • volume 10, number 3 • september 2018179 5. sykes jm: updates and practical approaches to reproductive disorders in reptiles. vet clin north am exot anim pract 2010;13:349-373. 6. innis cj, boyer th: chelonian reproductive disorders. vet clin north am exot anim pract 2002;5:555-578. 7. sladky kk: analgesia. in: mader dr, divers s, editors. current therapy in reptile medicine and surgery. 3rd ed. st. louis: elsevier, 2014, p. 217-228. 8. schoemaker nj: gonadotrophin-releasing hormone agonists and other contraceptive medications in exotic companion animals. vet clin north am exot anim pract 2018;21:443-464. 9. garner mm, hernandez-divers sj, raymond jt: reptile neoplasia: a retrospective study of case submissions to a specialty diagnostic service. vet clin north am exot anim pract 2004;7:653-671. 10. mans c, pilny a: use of gnrh agonists for medical management of reproductive disorders in birds. vet clin north am exot anim pract 2014;17:23-33. 11. petritz oa, lierz m, samour j: advancements in methods for decreasing reproductive success. in: speer bl, editor. current therapy in avian medicine and surgery. st. louis: elsevier; 2016. p. 446-460. 12. molter c, fontenot d, terrell s: use of deslorelin acetate implants to mitigate aggression in two adult male domestic turkeys (meleagris gallopavo) and correlating plasma testosterone concentrations. j avian med surg 2015;29:224-230. 13. carrasco dc, gonzalez ms: reproductive disorders in commonly kept fowl. vet clin north am exot anim pract 2017;20:509-538. 14. scagnelli am, tully tn: reproductive disorders in parrots. vet clin north am exot anim pract 2017;20:485-507. 15. mans c, sladky kk: clinical management of ectopic egg retention in a timneh african grey parrot (psittacus erithacus timneh). j am vet med assoc 2013;242:963-968. clinical theriogenology • volume 10, number 3 • september 2018 180 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice effects of nerve growth factor ? added to extenders for cryopreservation of electro ejaculated and epididymal harvested bull semen effects of nerve growth factor β added to extenders for cryopreservation of electro ejaculated and epididymal harvested bull semen jamie stewart,ab igor canisso,a giorgia podico,a edgar garrett,a fabio limaa a department of veterinary clinical medicine college of veterinary medicine, university of illinois, urbana, il bdepartment of large animal clinical science, virginia-maryland college of veterinary medicine virginia polytechnic institute and state university, blacksburg, vi nerve growth factor β (ngf) is a protein in seminal plasma of mammalian species. seminal plasma ngf concentrations and sperm bound ngf expression have both been positively associated with sire conception rates in bulls. since bovine sperm express ngf in the head and tail and its receptor (trka) in the acrosomal cap, nucleus, and tail regions, it is likely that ngf influences sperm function. seminal plasma ngf mrna expression was positively associated with maintenance of post thaw functional membrane integrity in bull sperm, suggesting a role in sperm cryotolerance. based on the aforementioned findings, we hypothesized that ngf supplementation enhances quality of frozen thawed bull sperm. objectives were to compare postthaw semen quality from ejaculated and epididymal harvested bull sperm incubated with purified ngf prior to cryopreservation. semen was obtained from angus x simmental crossbred bulls (n = 10) collected by electroejaculation, followed by castration and epididymal sperm harvest 3 days later. semen samples from each bull were preextended with a commercial extender (optixcell imv, maple grove, mn) and divided into one of 4 treatments to achieve a final sperm concentration of 4 x 106 spermatozoa/ml and a final ngf concentration of 0, 0.5, 5 and 50 ng/ml (cont, low, med and high, respectively). immediately after final dilution, samples were manually loaded into 0.5 ml semen straws, sealed ultrasonically, and incubated in a cold room at 5°c for 3 5 hours per manufacturer’s recommendation. straws were then placed 4 cm above liquid nitrogen for 15 minutes and then submerged and stored in liquid nitrogen until post-thaw analysis. prefreeze sperm motility was assessed in each sample prior to treatment using computer-aided sperm analysis. frozen straws were thawed and incubated at 37°c for postthaw motility assessment in 30 minute intervals for 4 hours. samples were stained with fluorescent probes for evaluation of sperm viability (sybr 14/pi), acrosomal integrity (fitc pna/pi), and chromatin stability (acridine orange) using flow cytometry. kruskal wallis rank sum test and anova were used for statistical analyses, with significance set at p < 0.05. postthaw sperm motility and velocity parameters were decreased, whereas linearity (lin) was increased in high versus cont ejaculated samples (p < 0.01), but no differences were observed in epididymal samples (p = 0.22). high ejaculated samples had a lower amplitude of lateral head displacement (alh) at 2.5 and 3 hours postthaw (p < 0.01). postthaw viability, acrosome integrity, and dna fragmentation index were not affected by ngf treatment in either ejaculated or epididymal sperm (p ≥ 0.15). treatment with ngf did not significantly improve cryotolerance of sperm collected by electroejaculation or epididymal harvest in bulls. supplementation of extender with high concentrations of ngf decreased postthaw curvilinear velocity and alh and increased lin, suggesting a potential role in preventing premature sperm hyperactivation and capacitation. although the current study did not support a role of ngf in improving sperm cryotolerance, further studies should address potential effects on sperm fertility within the female reproductive tract. keywords: bovine, motility, nerve growth factor β, spermatozoa 441 clinical theriogenology • volume 11, number 3 • september 2019 442clinical theriogenology • volume 11, number 3 • september 2019 untitled omniblank: reproductive effects of equine endocrine disease reproductive effects of equine endocrine disease teresa burns college of veterinary medicine, the ohio state university, columbus, oh abstract endocrine disease is very common in domestic horses and ponies and has become even more so as members of these populations are currently living relatively longer. older animals are now often successfully fulfilling their intended use (competition, pleasure riding and reproduction) well into their third decade. therefore, managing influence of endocrine diseases (many of which increase in frequency with age) on fertility and reproductive outcomes will likely be more important for veterinary practitioners in the future. relatively little is known about specific effects of equine metabolic syndrome and pituitary pars intermedia dysfunction on reproductive function in horses, although preliminary information describing some effects on fertility, ovulation efficiency, implantation, duration of pregnancy and lactation have been published. further work characterizing these influences will ideally lead to establishment of concrete guidelines and best practices to optimally manage older breeding animals affected by endocrine disease. keywords: equine metabolic syndrome, pituitary pars intermedia dysfunction, insulin dysregulation, pregnancy hormonal effects of obesity and insulin dysregulation on equine reproductive function horses with equine metabolic syndrome (ems) by definition have insulin dysregulation (id) and are frequently persistently hyperinsulinemic.1 mares with ems are suspected to have altered reproductive physiology, with some investigators correlating elevated serum insulin concentrations and poor fertility. body condition score and percentage body fat were reported to be inversely correlated to systemic insulin sensitivity and directly (positively) correlated to blood expression levels of il1 and tnfα in mares;2 these changes were more prominent in older animals, suggesting a relationship between age-related systemic insulin resistance (ir) and age-related decline in fertility in mares. when systemic ir was experimentally induced in mares with a heparin and lipid infusion, an increased interovulatory period and higher peak luteal progesterone concentrations resulted.3 insulin has also been evaluated as a potential candidate for the signal for equine maternal recognition of pregnancy (mrp); however, after observing that 1) insulin administration did not affect luteal size, diestrus length, interovulatory interval, or circulating lh concentrations and 2) insulin was not detected in yolk sac fluid of 10 14 day equine conceptuses, investigators concluded that insulin is unlikely to be the signal of mrp in equids.4 the role(s) of insulin (and hyperinsulinemia associated with endocrine disease) in reproductive dysfunction associated with ems is not well described, with further work needed. serum concentrations of leptin, an adipokine produced primarily by white adipose tissue, have been positively correlated with body condition score (bcs) and degree of adiposity in many species, including humans, rodents and horses. serum leptin concentration is considered to principally reflect body fat composition and to be a circulating ‘marker’ of adiposity (e.g. leptin concentration is directly related to body weight in lusitano mares5). however, hyperleptinemia is also associated with systemic ir in horses (even when controlling for bcs), suggesting a more important role for leptin in pathophysiology of systemic id in horses. overweight or obese mares have been reported to display continuous ovarian cyclicity (i.e. these mares often fail to enter seasonal anestrus) at a higher rate than lean mares, suggesting links among nutrition, glucose and lipid metabolism and reproductive function. serum leptin concentrations correlated well with fat mass (which generally changes slowly), but they also may be altered more acutely by nutrition and medications. hyperleptinemia remained stable when mares were exposed to varied management schemes (pasture versus hay in dry lots versus grain meal feeding), but variability in serum leptin concentrations was minimized when mares were fed hay (which also minimized variability in insulin and glucose concentrations).6 feed restriction for as little as 24 hours significantly lowered serum leptin concentrations in horses, which, due to the time course involved, was clearly not a result of significant reduction in body fat mass.7 leptin does not appear to be secreted in a pulsatile fashion in horses, but a circadian rhythm in its concentration has been reported;8 furthermore, it has also been reported that leptin concentrations are lower in fasted versus fed states, but unaffected by ovariectomy or melatonin implants (making a direct link between leptin and the hypothalamo397 clinical theriogenology • volume 11, number 3 • september 2019 pituitarygonadal axis less clear). leptin concentrations can also be manipulated pharmacologically; dexamethasone or propylthiouracil increase serum leptin concentrations in mares and geldings after a relatively short interval.9,10 treatment with clenbuterol for 6 months altered body fat and leptin concentrations in light breed mares in another study, with no effect on seasonal anestrus or cyclicity.11 seasonality (especially occurrence and duration of seasonal anestrus) has been shown to be controlled to some extent by metabolic hormones and correlated with body condition. older mares were more likely to cycle continuously and to enter seasonal anestrus later than younger mares in 1 study; the investigators reported that propensity for continuous cycling was associated with bcs, body fat percentage, and serum leptin concentration (all of which were highest in summer months).12 welsh pony mares (a breed likely more relevant to a discussion of id and ems than many light breeds, given recent descriptions of their genetic predispositions) fed to achieve low bcs always displayed seasonal anestrus, which persisted longer than in mares that were ‘well fed’ (only 40% of these mares displayed seasonal anestrus and it was shorter than in other groups).13 there were large differences in growth hormone, igf1, and leptin concentrations between well fed and restricted groups, and glucose, insulin, growth hormone and leptin concentrations were highly correlated with duration of ovulatory activity. however, in a population of mares not predisposed to id/ems, ‘flushing’ (i.e. acute increase in plane of nutrition, body condition score, and body fat percentage ~ 3 weeks prior to the breeding season) stimulated ovarian activity in stressed maiden standardbred mares (and presumably improved their reproductive efficiency);14 hormone concentrations were not evaluated, only first seasonal ovulation (which occurred earlier in mares with higher bcs). little information exists regarding the role of plane of nutrition and endocrine disease status on reproductive performance during pregnancy and lactation in horses. feeding mares to obesity during gestation had no detectable effect on postpartum reproductive performance (interval to first and second ovulation after foaling, conception rates and early pregnancy loss rates were similar to mares maintained at a moderate bcs).15 even a high bcs produced by overfeeding during gestation did not seem to adversely affect postpartum reproductive performance in multiparous mares. based on clinical experience, foals may have difficulty nursing (at least initially) from overconditioned mares that accumulate a large amount of adipose tissue within or near their mammary glands, which can efface the teats and make them less prominent and readily available to the foal. additionally, mares with historical or current endocrinopathic laminitis may have increased orthopedic pain that is difficult to control as they gain weight during the third trimester. even if reproductive efficiency itself does not suffer directly due to overconditioning, these welfare concerns for mare and foal are good reasons to maintain ideal bcs in broodmares during and after/before pregnancy. finally, in other species, robust evidence in multiple species supports a role for maternal nutrition and metabolism in ‘programming’ of future metabolic pathways of offspring while still in utero. although fetal macrosomia is a common complication in children born to women with ir and diabetes mellitus (gestational or otherwise), children born prematurely to affected mothers, while initially often reported to have intrauterine growth restriction and low birth weight, later have significantly increased risk of obesity, ir, and type ii diabetes mellitus in adulthood. evidence for gestational programming in horses is very limited, but a few studies have investigated links between maternal and fetal/neonatal metabolism in equids. leptin concentrations in nursing mares were shown not to affect leptin, triglyceride, or free fatty acid concentrations in their foals,16 but maternal nutritional restriction during mid-gestation was associated with altered pancreatic responsiveness following birth in their (otherwise normal) foals.17 in this study, mid gestational maternal nutritional restriction was associated with an increased acute insulin response to glucose (enhanced pancreatic ß cell responsiveness) in neonates, although no differences in response to acth stimulation or exogenous insulin administration was observed between foals from high-condition mares compared to those in moderate condition. the long-term significance of these findings remains to be determined, but this preliminary evidence for gestational metabolic programming in horses may represent another excellent reason for maintaining endocrine health of broodmares. medical management of broodmares with equine metabolic syndrome/insulin dysregulation optimization of nutrition and exercise management, as well as management of endocrinopathic laminitis if present, are mainstays of treatment for horses and ponies with ems; strategies recommended for the general equine population can also be used for managing affected broodmares, particularly in the first and second 398clinical theriogenology • volume 11, number 3 • september 2019 trimesters of gestation (when metabolic demands and weight gain are not yet significantly different from the nonpregnant state). indeed, these strategies may be appropriate for most broodmares, as ir is physiologic and progressive during normal pregnancy (this would be expected to be enhanced and more likely pathologic in animals that have been diagnosed with ems before becoming pregnant). provision of a diet primarily composed of grass hay (ideally containing < 10% nonstructural carbohydrate on a dry matter basis) and some sort of ration balancer to ensure adequate trace mineral and vitamin intake are central to dietary management of ems cases; nutritional goals include minimizing dietary nonstructural carbohydrate (sugars, starches and fructans) and encouraging safe, gradual weight loss if needed. although timing and rate of weight loss that is appropriate and safe for broodmares is currently unclear, prevention of excessive weight gain during gestation would seem to be a reasonable goal in this population. continuous consumption of forage is considered ideal for horses and ponies in general, but timing of feeding was recently shown to affect fertility;18 mares with continuous access to forage had higher rates of fertility and fewer estrus abnormalities than mares offered forage only at night. continuous forage access may be important for maximizing fertility in broodmares generally, but particularly those who may have dietary restriction as a part of a treatment plan for ems/ir. during the last third trimester and onset of lactation, most broodmares require supplemental calories in the form of concentrate feed to maintain body weight and support newly increased metabolic demands; composition of that diet can affect insulin sensitivity of the mare, even in the absence of a significant change in body weight or composition. in a study comparing effects of dietary composition on insulin sensitivity in broodmares, provision of a diet rich in sugar and starch enhanced systemic ir when compared to forage only or diet rich in fat and fiber; this effect was enhanced in mares with higher bcs.19 the authors recommended using fat and fiber rich diets to provide supplemental calories in broodmares, in addition to avoiding overweight or obese body condition in this population; this recommendation seems particularly important when considering what is now known in horses and other species about gestational metabolic programming.20-22 effects of exercise and medications on management of ems and ir in broodmares have not been investigated, but these strategies might be useful as well. exercise in particular may prove highly valuable, as it is likely safe (at least in the first and second trimesters, and as long as the animal is sound and comfortable), inexpensive, and highly effective in improving insulin sensitivity both acutely following an exercise bout and chronically in conjunction with weight loss and improvement in body composition. as little as 30 minutes of trot exercise daily is thought to be useful, as this amount of exercise elicits improvement in insulin sensitivity in the absence of any change in bcs or body weight in both lean and obese (though nonpregnant) mares;23 this benefit was observed almost immediately following institution of the exercise program, but it did not persist long past discontinuation of the protocol. consistency, therefore, appears to be important to maximize benefits from exercise. regarding pharmaceutical use in the management of broodmares with ems, there are no studies of safety or efficacy of medications such as levothyroxine or metformin in pregnant horses and ponies. based on known risks to the fetus associated with maternal hyperthyroidism in humans,24,25 levothyroxine use during equine pregnancy should not be assumed to be empirically safe. the efficacy of metformin in improving ir in horses remains to be characterized more fully, but the drug appears to be reasonably safe in horses.26 metformin has not been evaluated for safety in pregnant mares; however, this drug is frequently used in diabetic pregnant women with an excellent safety profile.27 if diet and exercise fail to achieve therapeutic goals in an at risk pregnant mare, metformin treatment may be helpful. effects of pituitary pars intermedia dysfunction on equine reproductive function as equine populations age and as horse owners maintain health of their animals well into their third (and fourth) decades, the number of middle aged and geriatric horses presented for reproductive management will undoubtedly increase. mares can conceive and carry foals well into their third decade, and this will certainly occur more commonly as management and medical progress increases the number of geriatric animals cared for by equine veterinary practices. aged mares are more likely to require veterinary assistance for fertility issues. even in the absence of a direct adverse effect of pars intermedia dysfunction (ppid) on reproductive performance, affected broodmares are likely to require veterinary management for an expected age-related decline in fertility; veterinarians will likely encounter these mares with increasing frequency in the future, 399 clinical theriogenology • volume 11, number 3 • september 2019 making knowledge of ppid testing and treatment that much more relevant and important. although effects of ppid on equine reproductive performance have not been evaluated in great depth, it seems reasonable to assume that there should be some influence, given current understanding of the pathophysiology. the pars intermedia is involved in regulation of certain seasonal functions in equids (e.g. growing and shedding hair), and the seasonality of equine reproduction has also long been recognized; initial evidence was presented in 1979 that the pineal-hypothalamo-pituitary axis influences annual seasonal patterns of reproductive activity in horses.28 effects of cortisol and related metabolites on reproductive hormone secretion have long been held to negatively affect fertility; however, horses with ppid have inconsistently elevated serum cortisol concentrations and results of at least 1 study have suggested that even when cortisol secretion is high, effects on subsequent fertility are minimal (although mares diagnosed with ppid were not specifically evaluated).29 dopaminergic neurodegeneration in certain subsets of hypothalamic neurons has been reported to be directly involved in pathogenesis of ppid, and pergolide (a dopaminergic agonist), is currently the recommended treatment for the disease. the role of dopamine in control of reproductive cyclicity in mares has been evaluated (albeit in otherwise normal mares, not those with ppid),30 and it has been suggested that dopamine may facilitate control of reproductive seasonality in mares and exert tonic inhibition on reproductive activity during the anovulatory season. therefore, whereas mares with ppid may cycle regularly (indeed, they may be more likely to cycle for a greater part of the year than normal mares; pharmacologic inhibition of dopaminergic neurotransmission resulted in increased follicular development and plasma estrogen and prolactin concentrations in mares),31 effective treatment for ppid in the form of dopaminergic agonists may adversely affect cyclicity. moreover, this treatment is highly likely to suppress prolactin secretion (in fact, protocols for induction of lactation in mares have been described using dopaminergic antagonists),32,33 suggesting that hypogalactia and agalactia are more likely to be problematic in lactating mares receiving pergolide for treatment of ppid.31 medical management of broodmares with pars intermedia dysfunction treatment of horses with ppid involves administration of medication to suppress secretory activity of pars intermedia (current fda approved drug of choice is pergolide, a dopaminergic agonist) and management changes.34 in pregnant and lactating animals, pergolide administration may increase the risk of prolonged gestation, premature placental separation and hypogalactia or agalactia. it may be appropriate in this case to manage ppid affected mares according to established recommendations for mares grazing endophyte-infested fescue pasture during gestation (also a condition caused by dopamine agonism);35-37 strategies such as temporary discontinuation of pergolide therapy 30 days prior to the expected foaling date and judicious use of dopaminergic antagonists such as domperidone or sulpiride may be useful to prevent prolonged gestation and to encourage normal lactation. mares should be monitored after foaling to ensure that they have adequate milk production; poor foal growth and incessant or continuous nursing behavior suggest that milk production may be inadequate and supplementation with milk replacer, goat’s milk, and or a milk-based creep feed may be helpful. pergolide therapy is often reinstituted at approximately 30 days after foaling; however, if the mare’s ppid is poorly controlled without medication, she should receive treatment sooner, and alternative sources of nutrition for the foal should be investigated (including a healthy, lactating nurse mare). alternatively, affected mares that don’t tolerate withdrawal of pergolide therapy may be used most effectively as embryo donors if their genetics are sufficiently valuable. however, pergolide treatment might also be expected to affect oocyte and or embryo harvest from these mares. further work is needed to optimize these protocols for mares with ppid to help guide best practice. although not all horses with ppid have systemic ir, this is not an uncommon finding in these animals.38 provision of sufficient calories to support lactation in ir horses (particularly those that have recently suffered a bout of laminitis) requires careful attention to composition of the diet to avoid inciting complications associated with hyperinsulinemia. feedstuffs with calories primarily coming from fat and fermentable fiber should be offered, and sugars, starches, and fructans (non-structural carbohydrates) in the diet should be minimized. body condition of affected mares should be carefully maintained within a normal range to ensure sufficient reserves to support lactation while avoiding over conditioning (excessive body weight would be expected to complicate 400clinical theriogenology • volume 11, number 3 • september 2019 clinical laminitis if it should occur, particularly in late gestation when body weight is already substantially increased). conclusion ems and ppid are very common conditions in horses and ponies (including those that are used for reproduction), and a concerted and extensive research effort over the past 20 years has yielded effective therapeutic and management strategies to minimize effects of these diseases on both length and quality of life and (preliminarily) reproductive efficiency in affected animals. future work will improve upon these initial efforts and serve to generate best practices for optimizing reproductive efficiency for equids affected by endocrine disease. conflict of interest the author claims no direct or indirect affiliation with any of the product manufacturers referenced in the body of this document and has no other conflicts of interest to declare. references 1. frank n, geor rj, bailey sr, et al: american college of veterinary internal medicine. equine metabolic syndrome. j vet intern med 2010;24:467-475. 2. vick mm, adams aa, murphy ba, et al: relationships among inflammatory cytokines, obesity, and insulin sensitivity in the horse. j anim sci 2007;85:1144-1155. 3. sessions dr, reedy se, vick mm, et al: development of a model for inducing transient insulin resistance in the mare: preliminary implications regarding the estrous cycle. j anim sci 2004;82:2321-2328. 4. rambags bp, van rossem aw, blok ee, et al: effects of exogenous insulin on luteolysis and reproductive cyclicity in the mare. reprod domest anim 2008;43:422-428. 5. ferreira-dias g, claudino f, carvalho h, et al: seasonal reproduction in the mare: possible role of plasma leptin, body weight and immune status. domest anim endocrinol 2005;29:203-213. 6. storer wa, thompson dl,jr, waller ca, et al: hormonal patterns in normal and hyperleptinemic mares in response to three common feeding-housing regimens. j anim sci 2007;85:2873-2881. 7. mcmanus cj, fitzgerald bp: effects of a single day of feed restriction on changes in serum leptin, gonadotropins, prolactin, and metabolites in aged and young mares. domest anim endocrinol 2000;19:1-13. 8. buff pr, morrison cd, ganjam vk, et al: effects of short-term feed deprivation and melatonin implants on circadian patterns of leptin in the horse. j anim sci 2005;83:1023-1032. 9. cartmill ja, thompson dl,jr, gentry lr, et al: effects of dexamethasone, glucose infusion, adrenocorticotropin, and propylthiouracil on plasma leptin concentrations in horses. domest anim endocrinol 2003;24:1-14. 10. cartmill ja, thompson dl, jr, storer wa, et al: endocrine responses in mares and geldings with high body condition scores grouped by high vs. low resting leptin concentrations. j anim sci 2003;81:2311-2321. 11. fitzgerald bp, reedy se, sessions dr, et al: potential signals mediating the maintenance of reproductive activity during the non-breeding season of the mare. reprod suppl 2002;59:115-129. 12. fitzgerald bp, mcmanus cj: photoperiodic versus metabolic signals as determinants of seasonal anestrus in the mare. biol reprod 2000;63:335-340. 13. salazar-ortiz j, camous s, briant c, et al: effects of nutritional cues on the duration of the winter anovulatory phase and on associated hormone levels in adult female welsh pony horses (equus caballus). reprod biol endocrinol 2011;9:130. 14. vecchi i, sabbioni a, bigliardi e, et al: relationship between body fat and body condition score and their effects on estrous cycles of the standardbred maiden mare. vet res commun suppl 2010;34:41-45. 15. kubiak jr, evans jw, potter gd, et al: postpartum reproductive performance in the multiparous mare fed to obesity. theriogenology 1989;32:27-36. 16. kedzierski w, kusy r, kowalik s: plasma leptin level in hyperlipidemic mares and their newborn foals. reprod domest anim 2011;46:275-280. 17. ousey jc, fowden al, wilsher s, et al: the effects of maternal health and body condition on the endocrine responses of neonatal foals. equine vet j 2008;40:673-679. 18. benhajali h, ezzaouia m, lunel c, et al: temporal feeding pattern may influence reproduction efficiency, the example of breeding mares. plos one 2013;8:e73858. 19. hoffman rm, kronfeld ds, cooper wl, et al: glucose clearance in grazing mares is affected by diet, pregnancy, and lactation. j anim sci 2003;81:1764-1771. 20. ojha s, fainberg hp, sebert s, et al: maternal health and eating habits: metabolic consequences and impact on child health. trends mol med 2015;21:126-133. 21. rodriguez l, panadero mi, roglans n, et al: fructose only in pregnancy provokes hyperinsulinemia, hypoadiponectinemia, and impaired insulin signaling in adult male, but not female, progeny. eur j nutr 2016;55:665-674. 401 clinical theriogenology • volume 11, number 3 • september 2019 22. smith cj, ryckman kk: epigenetic and developmental influences on the risk of obesity, diabetes, and metabolic syndrome. diabetes metab syndr obes 2015;8:295-302 doi: 10 2147/dmso s61296 ecollection 2015;8:295-302. 23. powell dm, reedy se, sessions dr, et al: effect of short-term exercise training on insulin sensitivity in obese and lean mares. equine vet j suppl 2002;34:81-84. 24. sheehan pm, nankervis a, araujo junior e: maternal thyroid disease and preterm birth: systematic review and meta-analysis. j clin endocrinol metab 2015;100:4325-4331. 25. van der kaay dc, wasserman jd, palmert mr: management of neonates born to mothers with graves' disease. pediatrics. 2016://schemas.openxmlformats. 26. durham ae, rendle di, newton je: the effect of metformin on measurements of insulin sensitivity and beta cell response in 18 horses and ponies with insulin resistance. equine vet j 2008;40:493-500. 27. rowan ja, hague wm, gao w, et al: metformin versus insulin for the treatment of gestational diabetes. n engl j med 2008;358:2003-2015. 28. sharp dc, vernon mw, zavy mt: alteration of seasonal reproductive patterns in mares following superior cervical ganglionectomy. j reprod fertil suppl 1979;27:87-93. 29. berghold p, mostl e, aurich c: effects of reproductive status and management on cortisol secretion and fertility of oestrous horse mares. anim reprod sci 2007;102:276-285. 30. besognet b, hansen bs, daels pf: induction of reproductive function in anestrous mares using a dopamine antagonist. theriogenology 1997;47:467-480. 31. brendemuehl jp, cross dl: influence of the dopamine antagonist domperidone on the vernal transition in seasonally anoestrous mares. j reprod fertil suppl 2000:185-193. 32. guillaume d, chavatte-palmer p, combarnous y, et al: induced lactation with a dopamine antagonist in mares: different responses between ovariectomized and intact mares. reprod domest anim. 2003;38:394-400. 33. chavatte-palmer p, arnaud g, duvaux-ponter c, et al: quantitative and qualitative assessment of milk production after pharmaceutical induction of lactation in the mare. j vet intern med 2002;16:472-477. 34. mcfarlane d: equine pituitary pars intermedia dysfunction. vet clin north am equine pract 2011;27:93-113. 35. blodgett dj: fescue toxicosis. vet clin north am equine pract 2001;17:567-577. 36. evans tj: endocrine alterations associated with ergopeptine alkaloid exposure during equine pregnancy. vet clin north am equine pract 2002;18:371-378, viii. 37. mccann js, caudle ab, thompson fn, et al: influence of endophyte-infected tall fescue on serum prolactin and progesterone in gravid mares. j anim sci 1992;70:217-223. 38. klinkhamer k, menheere pp, van der kolk jh: basal glucose metabolism and peripheral insulin sensitivity in equine pituitary pars intermedia dysfunction. vet q 2011;31:19-28. 402clinical theriogenology • volume 11, number 3 • september 2019 2018: management of abortion and mummification due to t-cell lymphomain a labrador retriever management of abortion and mummification due to t-cell lymphoma in a labrador retriever college of veterinary medicine, auburn university, auburn, al a pregnant 1.5-year old labrador retriever was presented at 45 days gestation for brown vaginal discharge. a physical examination was normal except for the presence of mucoid, non-odorous, brown vaginal discharge. ultrasonography revealed a small fetus near the cervix that lacked a heartbeat. all other fetuses were normal. a vaginal culture was obtained and treatment with amoxicillin trihydrate/clavulanate potassium was initiated.1,2 the vaginal discharge lessened initially, but increased again by 49 days gestation. mammary development was also noted at this time. culture revealed a heavy growth of proteus mirabilis and antibiotic therapy was changed to cefpodoxime. at 52 days gestation, the vaginal discharge decreased, but a second deceased fetus was noted on ultrasonography and radiography. tests for canine herpes virus and brucella canis were negative. despite these results, acyclovir was initiated and cefpodoxime was continued. the dam remained clinically healthy and afebrile. at 60 days gestation, decreased fetal heart rates indicated fetal stress. fetal kidney definition and intestinal peristalsis were present on ultrasonography. six viable puppies and one fetal mummy were delivered via cesarean section. one month later, the dam was presented for vomiting and distended abdomen. chemistry profile, complete blood count, abdominal ultrasonography, radiographs, and abdominocentesis suggested lymphoma. severe adhesions throughout the abdomen were found on exploratory laparotomy, and the patient was euthanized without recovery. necropsy results confirmed a final diagnosis of t-cell high grade lymphoma with metastasis to most abdominal organs. there is evidence in rodents and horses that estrogen and progesterone may influence the growth and development of lymphoid malignancies. the frequency and pathology of neoplasia causing canine abortions and fetal mummification is not often discussed.1,2 this case represents the importance of management of undiagnosed abortion and fetal demise, with an outcome resulting in healthy offspring, but a life-threatening condition for the dam. keywords: lymphoma, pregnancy, abortion references 1. root kustritz m: pregnancy diagnosis and abnormalities of pregnancy in the dog. theriogenology 2005;64:755-765. 2. verstegen j, dhaliwal g, verstegen-onclin k: canine and feline pregnancy loss due to viral and non-infectious causes: a review. theriogenology 2008;70:304-319. 3. henson k, alleman ar, kelley lc, et al: immunohistochemical characterization of estrogen and progesterone receptors in lymphoma of horses. vet clin pathol 2000;29:40-46. clinical theriogenology • volume 10, number 3 • september 2018361 b.e. shumack, r.r. wilborn, a.k. johnson, c.l. barstow  2017: perineal laceration following obstructive dystocia in a bitch   perineal laceration following obstructive dystocia in a bitch jenna c. dockweiler, soon hon cheong, robert o. gilbert* department of clinical sciences, college of veterinary medicine, cornell university, ithaca, ny summary a three-year-old longhaired whippet was presented after whelping for evaluation of vaginal hemorrhage. her owners reported an obstructive dystocia which had spontaneously resolved. on examination, a second-degree perineal laceration was discovered. surgical repair was performed five weeks after the initial injury and was successful in closing the wound. to the authors’ knowledge, this is the first report of perineal laceration and the first description of its repair in a bitch. keywords: perineal laceration, bitch, dystocia background dystocia in the bitch is not uncommon, affecting approximately 16% of canine pregnancies and accounting for 56% of reproductive emergencies.1,2 causes for dystocia include maternal causes such as primary uterine inertia or secondary uterine inertia and fetal causes such as oversized fetuses causing physical obstruction of the birth canal.3 a retrospective study evaluated maternal vs. fetal causes of dystocia and found fetal causes represent only 24.7% of dystocia causes.4 the most common fetal cause of dystocia in this study was malpresentation.4 of all dystocia cases, 60-80% are ultimately treated by cesarean section.5 the present case describes a bitch with obstructive dystocia that spontaneously resolved, but resulted in a perineal laceration. to the authors’ knowledge, this condition has not been reported in the bitch. case presentation a three-year-old primiparous longhaired whippet bitch was presented to the cornell university hospital for animals for evaluation of postpartum vaginal and vulvar bleeding. her owners reported the first puppy was malpostured, and initially only a single limb was visible outside the vulva. after several minutes of active contractions that failed to advance the obstructed fetus, a second fetus was delivered simultaneously alongside the malpostured fetus and the bitch began to bleed from her caudal reproductive tract. she whelped seven additional puppies without difficulty before she was presented for evaluation. all puppies survived and were apparently healthy. she was bred by natural mating on days 3 and 5 after the lh peak based on progesterone timing. no abnormalities of the reproductive tract were noted on digital vaginal examination or vaginal speculum examination prior to breeding. on presentation, the bitch was bright, alert, and responsive. her vital signs and general physical condition were within normal limits. on examination of the vulva, a tear extending straight dorsally approximately 2.5 cm from the dorsal commissure was noted. the tear extended to the vestibulovaginal junction. digital vaginal examination revealed a full-thickness laceration of the dorsal commissure of the vulva and caudal vagina with involvement of the perineal body (second-degree perineal laceration). no communication with the rectum was noted on vaginal or rectal examination. antibiotics (cephalexin 25 mg/kg) and analgesic medications (tramadol 3 mg/kg) were administered for two weeks in preparation for surgical repair. treatment repair of the laceration was delayed until five weeks postpartum to ensure the tissue was viable. appearance of the laceration at the time of surgical repair is depicted in figure 1. the patient was anesthetized using propofol to effect for induction and was maintained on isoflurane inhalant. she was                                                         * current address: ross university school of veterinary medicine, st. kitts, west indies  clinical theriogenology • volume 9 number 1 • march 201777   placed in sternal recumbency and the surgical area was clipped and aseptically prepared. the laceration was located and the edges of the wound were freshened using metzenbaum scissors (figure 2). the muscles and mucosa were closed in a simple continuous pattern using poliglecaprone suture (monocryl®, ethicon, somerville, nj). the skin was closed using non-absorbable suture (ethilon®, ethicon, somerville, nj) in a simple interrupted pattern (figure 3). recovery from anesthesia was uneventful. outcome the bitch removed the skin sutures eight days after surgery, but the underlying suture layer remained intact. the skin was allowed to heal by second intention. the wound healed without complication. based on digital vaginal examination after repair, the authors feel this bitch could be bred naturally and whelp without complication. the owner does not plan to breed her a second time. discussion perineal lacerations are classified as first-degree, second-degree, or third-degree depending on which tissues are involved. a first-degree laceration is characterized by involvement of the vulvar mucosa only.6 a second-degree laceration is characterized by involvement of the vulvar mucosa and the perineal body.6 a third-degree laceration involves tearing of the vulvar mucosa, perineal body, rectum, and anal sphincter.6 of the major domestic species, the mare is most likely to suffer a perineal laceration following delivery of a foal. heavily muscled or primiparous mares are more often affected.6 perineal laceration may result during unassisted delivery of a large foal, with overzealous assistance at delivery, or as a sequela to dystocia.6,7 third-degree perineal lacerations may result from the fetal hoof catching on the dorsal aspect of the vagina at the vestibulovaginal junction and penetrating through the vagina and rectum.7 in most cases, repair is attempted at six to eight weeks after delivery to allow for the resolution of swelling and granulation of the wound.6,7 although these injuries are not life-threatening, future fertility is affected if the wound is not appropriately repaired.6 reported complications of dystocia in the bitch include metritis, uterine rupture with secondary septic peritonitis, and fetal or maternal death.5,8,9 to the authors’ knowledge there are no reports of perineal laceration in the bitch, although perineal hernia with retroflexion of the urinary bladder is a reported sequela of dystocia.10 episiotomy is described as a treatment for obstructive dystocia, but it is not commonly utilized.5 in this case, it is possible the perineal trauma in the bitch would have been less severe if an episiotomy were performed or if obstetrical manipulations had corrected the malposture of the first fetus. the present case describes a second degree perineal laceration resulting from an obstructive dystocia in a bitch. this condition and its repair has not previously been reported in this species. learning points  perineal laceration is a possible sequela to dystocia in the bitch.  surgical repair of the laceration is relatively simple, although no information on future fertility of bitches with perineal lacerations exists in the literature at this time.  owners or others who attend whelpings should be counseled on proper fetal presentation, position, and posture, and given instructions on how to manipulate a fetus and when to seek veterinary attention. conflict of interest the authors have no conflicts of interest or sources of funding to disclose. references 1. bergström a, nødtvedt a, lagerstedt as, et al: incidence and breed predilection for dystocia and risk factors for cesarean section in a swedish population of insured dogs. vet surg 2006;35:786-791. clinical theriogenology • volume 9 number 1 • march 2017 78   2. martins-bessa a, cardoso l, costa t, et al: reproductive emergencies in the bitch: a retrospective study. j hellenic vet med soc 2015;66:231-240. 3. münnich a, küchenmeister u: dystocia in numbers – evidence-based parameters for intervention in the dog: causes for dystocia and treatment recommendations. reprod domest anim 2009;44:141-147. 4. darvelid aw, linde-forsberg c: dystocia in the bitch: a retrospective study of 182 cases. j small anim pract 1994;35:402-407. 5. traas am: surgical management of canine and feline dystocia. theriogenology 2008;70:337-342. 6. leblanc mm: common peripartum problems in the mare. j equine vet sci 2008;28;709-715. 7. frazer gs: post partum complications in the mare. part 2: fetal membrane retention and conditions of the gastrointestinal tract, bladder and vagina. equine vet educ 2003;15:91-100. 8. fresno l, marco a, calvo ma, et al: fibrino-purulent necrotizing metritis as a post-surgical complication after a prolonged delivery in a bitch: a case report. int j vet med: res reports 2014;1-8. 9. humm kr, adamantos se, benigni l, et al: uterine rupture and septic peritonitis following dystocia and assisted delivery in a great dane bitch. j am anim hosp assoc 2010;46:353-357. 10. sontas bh, apaydin so, toydemir ts, et al: perineal hernia because of retroflexion of the urinary bladder in a rottweiler bitch during pregnancy. j small anim pract 2008;49:421-425. figure 1. perineal laceration five weeks after whelping. the dorsal aspect of the vulvar lips have healed together, while the remainder of the laceration is complete. clinical theriogenology • volume 9 number 1 • march 201779   figure 2. intra-operative view of the laceration after freshening the edges of the wound. figure 3. view of the laceration following repair. (editor’s note: the photographs in this manuscript are available in color in the online edition of clinicl theriogenology.) clinical theriogenology • volume 9 number 1 • march 2017 80 2013: embryo transfer and pregnancy diagnosis embryo transfer and pregnancy diagnosis misty a. edmondson department of clinical sciences, college of veterinary medicine, auburn university, auburn, al abstract embryo transfer (et) is a very effective technique for quickly improving the genetics. while the application of this technology is extensive in the cattle industry, it is a growing technology for small ruminants with considerable progress being made with regard to estrus synchronization and superovulation. early pregnancy diagnosis and determination of fetal number are of significant value in goat reproductive herd health management. the value of pregnancy determination is in the identification of non-productive females and does bearing multiple fetuses. keywords: embryo transfer, pregnancy diagnosis, gender determination, doe introduction embryo transfer is a very effective technique for quickly improving the genetics. while the application of this technology is extensive in the cattle industry, it is a growing technology for small ruminants. rapid genetic improvement is accomplished by shortening the generational interval and can potentially double the genetic gain in a herd. the main limitations for wider use in goats are the naturally occurring anestrus period, the inconsistency of response to superovulatory protocols, fertilization failure, and the need for surgical collection and transfer of embryos. however, considerable progress has been made with regard to estrus synchronization and superovulation. early pregnancy diagnosis and determination of fetal number are of significant value in goat reproductive herd health management. frequently, clinical signs such as failure to return to estrus after breeding, enlarging abdomen, and developing mammary glands to make a presumptive diagnosis of pregnancy. however, there are many limitations to using these clinical signs to diagnose pregnancy. the value of pregnancy determination is in the identification of non-productive females and does bearing multiple fetuses. embryo transfer breeding programs based on multiple ovulations and embryo transfer use genetically superior females to increase the genetic diversity and progress in the herd. a successful et program requires advanced planning and attention to detail in donor and recipient selection, synchronization of donor and recipient, and successful recovery and transfer of high quality embryos.1 embryo transfer can be performed in or out of the breeding season. however, the best response is obtained during the breeding season when donors and recipients are cycling normally. donor and recipient management donor and recipient selection and management are critical for the success of an et program. recipient and donor does must be synchronized to cycle together. donors respond most successfully to estrus synchronization and superovulation when they are young, healthy, and cycling normally with does between two and five years of age responding best to synchronization and superovulation protocols.2 donors should be in good body condition but not over-conditioned. they should also be current on vaccinations for any infectious diseases that are locally prevalent. donors should be acclimated to new pastures or pens, feeding, and handling two to four months prior to the beginning of the et program to help minimize stress.3 stress appears to be cause premature luteal regression in some breeds (e.g., boer). recipients should also be healthy animals with proven reproductive ability that are in good body condition (bcs 3 to 3.5) and cycling normally.2 does that are two to four years of age with a history of good maternal characteristics and adequate potential for milk production are preferred. in addition, recipients should also be current on vaccinations. 483 clinical theriogenology • volume 5, number 4 • december 2013 synchronization most et programs rely on the use of exogenous hormones to induce and synchronize estrus in donors and recipients. synchronization protocols commonly use progestin sponges or controlled internal drug release devices (cidrs). the accurate detection of estrus is critical to the success of et programs. teaser bucks are often used to provide more accurate detection of estrus. the same method of estrus detection should be used for both donor and recipients; however, superior results have been obtained if progestin sources are removed from recipients 12 hours before they are removed from donors.1 superovulation superovulation of donor animals is achieved by injecting equine chorionic gonadotropin (ecg) or pituitary extracts of follicle stimulating hormone (fsh). the ecg is administered in a single dose 48 hours prior to removal of the progesterone source. equine cg has a longer half-life and is associated with overstimulation of the ovaries leading to the release of large numbers of oocytes, an increased proportion of unfertilized embryos and poorer quality embryos. in addition, antibody formation is a common side effect after treatment with ecg, even at small doses, which could have a negative impact on future fertility of the donor.4 the donor may also be superovulated with an fsh product either alone or in combination with ecg two days before the end of the artificially created luteal phase.5 the fsh preparations, usually of porcine or ovine origin, have a half-life of six hours and are administered twice a day beginning 48 hours before removal of the progestin source. the fsh preparations are superior to ecg in ovulation rates, fertilization rates and percentage of good-quality embryos. does typically exhibit estrus between 24 and 36 hours after progestin removal. gonadotropin releasing hormone (gnrh) has been administered at 30 to 36 hours after removal of the progestin to induce a strong pre-ovulatory luteiniing hormone (lh) surge which is required to maximize the probability of ovulation. careful and frequent observation of does for estrus with the aid of a teaser animal will ensure accurate detection and recording of the time of estrus. donors may be hand-mated 12 to 24 hours after estrus detection. laparoscopic deposition of frozen-thawed semen into the uterine horns 24 hours after the animal is first seen in estrus produces optimum results. if donor animals are to be bred naturally, one buck should be housed with one or two super-ovulated does.6 embryo recovery and handling embryos are usually harvested from the donor’s uterus on day 5 to 7 after breeding. in most cases, surgical collection of embryos is performed. however, laparoscopic and non-surgical embryo collections have also been developed. there are numerous methods to recover embryos including surgical techniques, laparoscopic techniques and non-surgical techniques. surgical embryo collection involves fasting the animals prior to the procedure in preparation for anesthesia. once under general anesthesia, the animal is prepared for surgery. during the surgery, the uterus and ovaries are exteriorized and examined. the uterus is then flushed with harvest medium to harvest embryos by catheterizing each uterine horn. the abdominal incision is then closed in routine fashion. prostaglandin f2α (pgfα) should then be administered to the donor to ensure lysis of luteal tissue. if embryos are to be collected prior to the fourth day following breeding, oviductal flushing is necessary. laparoscopic collection of embryos can also be performed be exteriorizing the tip of the uterine horn and flushing in the same manner described for surgical embryo collection. laparoscopic collection reduces the severity of adhesions that result from the handling required in the surgical approach. laparoscopic collection of embryos requires considerable skill and is not practical as a routine field procedure. this method allows the operator to visualize the ovary and corpus luteum (cl) and more easily exteriorize the uterine horn. advantages of laparoscopic collection of embryos include reduced surgical time and a small abdominal incision. non-surgical or transcervical embryo collection methods avoid the risk of postsurgical adhesions and maintains the value of genetically superior donors after embryo collections. several reports of successful non-surgical embryo collections in goats have been published.7-9 embryo recovery rates 484clinical theriogenology • volume 5, number 4 • december 2013 appear to be similar to those attained with surgical embryo collections. however, surgical recovery is still the most common method used for commercial recovery of embryos. the flush medium that is recovered during flushing is examined with a dissecting microscope. the embryos are retrieved and placed into a holding dish after washing. before transfer into recipients or freezing, the embryos are carefully evaluated for stage of development and quality. embryos in the morula or blastocyst stages are expected when embryos are collected at day 5 or 6. the international embryo transfer society (iets) has defined handling procedures to reduce the risk of disease transmission during et.1 the embryos are pulled into a small-bore intravenous catheter attached to a 1 ml syringe for immediate transfer into recipients. embryos may also be frozen if immediate transfer is not available or desired. \ transfer of embryos recipients are chosen for transfer of embryos based on the greatest synchrony of estrus to the donor doe which is one of the most important factors affecting success of et. most transfers are performed surgically. the recipient is anesthetized and prepared for surgery. during surgery, the ovaries are examined for the presence of a cl. once a cl is identified, the uterine horn on the ipsilateral side is exteriorized. embryos harvested prior to day 4 are then transferred into the oviducts through the fimbriae using a catheter or pipette. older embryos (embryos collected after day 4) are transferred into the uterine horns. before closing the abdominal incision, one should examine the catheter or pipette to ensure that no embryos have been retained. two embryos are most commonly transferred per recipient.1 laparoscopic transfer of embryos is performed by placing the animal under general anesthesia and preparing the recipient for surgery. the embryos are loaded into a 0.5 ml straw which is placed into a cassou insemination gun with an injection tip. the cassou gun is inserted into the abdomen through a cannula, the needle inserted into an avascular area of the tip of the uterine horn and the plunger of the gun depressed to express the embryos into the uterine horn.1 an average of eight to ten0 transferable embryos can be expected per flush with acceptable pregnancy rates of 60% to 80% for the transfer of two fresh embryos per recipient.10 pregnancy rates from the transfer of frozen embryos are much lower. there are many factors that can affect the success of an et program. pregnancy diagnosis early pregnancy diagnosis and determination of fetal number are very important for goat reproductive herd health management. often, signs such as failure to return to estrus after breeding, enlarging abdomen, and developing mammary glands are used to make a presumptive diagnosis of pregnancy. however, there are many limitations to using these clinical signs to diagnose pregnancy. pathologic conditions of the uterus and ovaries, physiologic anestrus late in the breeding season, and outof-season breeding also may cause post-breeding anestrus in non-pregnant does. in addition, some does exhibit estrus behavior during pregnancy. ultrasonography and hormonal assays are the most useful methods for pregnancy diagnosis. abdominal palpation, ballotment and radiography have limited usefulness. the value of pregnancy determination is in the identification of non-productive females and does bearing multiple fetuses. early identification of multiple fetuses allows for the implementation appropriate nutritional and management programs. this includes dividing the herd into groups based on pregnancy status to improve the health care of these animals as well as decreasing production costs. imaging ultrasonography. ultrasonography for pregnancy determination has included amplitude modulation (a-mode or amplitude modality), doppler, and real-time (b-mode or brightness modality) imaging. a-mode imaging has been used to detect pregnancy between 70 and 100 days of gestation where fluid density is interpreted as pregnancy. a-mode is considered accurate (90-95%) but the 485 clinical theriogenology • volume 5, number 4 • december 2013 accuracy falls significantly after 145 days of gestation. however, a-mode is considered unreliable because it may yield false positive results in cases of hydrometra or a large bladder. doppler ultrasonography can be used to detect movement (fetal movements, fetal heart beat, and blood flow through the middle uterine artery) which may indicate pregnancy. the external doppler technique has an accuracy of 100% during the second half of gestation but is not as effective at 50 to 75 days or earlier. transrectal doppler ultrasonography may be attempted at 25 to 30 days after breeding; however, waiting until 35 to 40 days produces better results. false negative and false positive results are common and determining fetal number is quite difficult.1 pregnancy diagnosis via ultrasonography in the doe is now performed almost entirely with realtime (b-mode) ultrasonography. linear array real-time ultrasound transducers can be used transrectally to diagnose pregnancy between 18 and 60 days of gestation. a plastic extension can be made from polyvinyl chloide pipe to allow easy introduction of the transducer into the rectum. a 5or a 7.5 mhz transducer is recommended for rectal scans. after 60 days of gestation, the gravid or pregnant uterus is pulled over the brim of the pelvis and down into the abdomen which can make transrectal ultrasonography difficult.1 transabdominal ultrasonography with a 3.5or 5-mhz linear or sector scanner is used 28 to 30 days of gestation. the transducer is placed on the hairless or clipped area of the abdomen high in the inguinal area, preferably in the right flank. a fluid such as ultrasound gel, vegetable oil, or methylcellulose should be used to couple the ultrasound transducer to the skin. the transducer beam is aimed toward the pelvis, and the abdomen is scanned by slowing sweeping the transducer cranially. identification of the triangular-shaped bladder provides an excellent landmark. the uterus is typically located dorsal or cranial to the bladder. pregnancy may be diagnosed by finding a fetus and numerous fluid-filled uterine luminal sections (less reliable). the fetal bones form shadows, and the fetal ribs produce a characteristic striated appearance. the fetal heartbeat can be first detected by 26 days and easily recognized as early as 30 days to 35 days after breeding. twin pregnancies are best determined between 45 and 90 days of gestation.1 radiography. the use of radiography is used more commonly in small ruminants than in domesticated livestock species but is still rarely used even in these species. radiographs are only useful after ossification of the fetal bones. radiography is highly accurate and may also be used to accurately assess fetal number. although, the use of radiography is limited due to the cost and late stage of fetal bone ossification. abdominal palpation and ballottement during the last half of gestation, the gravid uterus or fetus may be palpated through the abdominal wall.1 ballottement may also be used to diagnose pregnancy in late gestation with a large fetus. ballottement is performed by gently pushing a fisted hand into the caudoventral abdomen of the female. in a pregnant female in late gestation, the fetus should be pushed away from the fist and then return in a fluid wave to “bump” the fist. ballottement is unable to assess the viability of the fetus and may only be accurately performed later in gestation. hormone assays the measurement of hormone assays in blood, milk, or urine provides an alternative method of pregnancy when ultrasonography may not be available. estrone sulfate. estrone sulfate is the major estrogen produced by the fetoplacental unit during pregnancy. measurement of estrone sulfate has been used to diagnose pregnancy in cattle, goats, and sheep. estrone sulfate can be detected in the urine, serum or milk after day 50 of gestation and are nearly 100% accurate. a positive test indicates a viable fetus. however, false positive results are possible with hemolyzed serum samples. in addition, false negatives may occur if samples are collected before 50 days of gestation.1 due to the limited number of laboratories performing this assay and the expense of the test, very few people use this assay for pregnancy diagnosis. pregnancy-specific protein b. pregnancy-specific protein b (pspb) is produced by the binucleate giant cells of the placenta throughout gestation. this hormone assay can be used to detect 486clinical theriogenology • volume 5, number 4 • december 2013 pregnancy any time after 30 days of gestation in goats. both false positive and false negative results are possible and few laboratories offer this test. multiple fetuses result in higher levels of pspb.11 progesterone. the progesterone assay is not a test for pregnancy but more accurately detects non-pregnant animals. goats require progesterone from the cl throughout gestation to maintain pregnancy. plasma or serum progesterone concentrations below 1 to 2 ng/ml at 21 days in the doe indicate that the doe is not pregnant due to the absence of a cl.1 an elevated progesterone concentration may indicate pregnancy, hydrometra, pyometra, early embryonic death, fetal mummification, or irregular estrous cycle. the accuracy of blood progesterone levels is 80% to 100% for non-pregnancy and 67% to 100% for pregnancy.1 fetal age determination the size of the fetus as determined by ultrasonography can be used to estimate gestational age. the crown-to-rump length of the fetus in relation to gestational age has been estimated for saanen and alpine goat breeds. a crown-to-rump length of 40 mm is equivalent to 45 days of gestation, 100 mm to 60 days, and 250 mm to 90 days. fetal age has also been estimated for dairy and pygmy goats by using biparietal diameter with ultrasonography.12,13 fetal sex determination fetal sex determination can be made using real-time ultrasonography by visualization of the genital tubercle of the fetus between 55 and 75 days of gestation.1 the genital tubercle begins its development between the hind limbs and moves back towards the tail in females and forwards towards the umbilicus in males. in addition, females should have two teats visible between the hind limbs with the absence of the triangular-shapes scortum.1 the penis and prepuce are located just caudal to the umbilical cord.14 fetal sexing is more difficult with twin or triplet pregnancies. conclusion advanced reproductive technologies have helped advance the goat industry. embryo transfer has become a very effective technique for quickly improving genetics within a herd. while the application of this technology has been used extensive in the cattle industry, it is a growing technology for small ruminants. rapid genetic improvement is accomplished by shortening the generational interval and can potentially double the genetic gain in a herd. embryo transfer is becoming more common as progress has been made with regard to estrus synchronization and superovulation. in addition, early pregnancy diagnosis and determination of fetal number have become important aspects in goat reproductive herd health management. imaging, palpation or ballotment, and hormone assays are the most common methods of pregnancy diagnosis. early pregnancy determination allows the producer to identify nonproductive females and does bearing multiple fetuses which might need additional nurtition. references 1. edmondson ma, roberts jf, baird an, et al: theriogenology of sheep and goats. in: pugh dg, baird an, editors. sheep and goat medicine. 2nd edition. st. louis: elsevier saunders; 2012. p. 150-230. 2. wallace jm, milne, aitken rp: effect of weight and adiposity at conception and wide variations in gestational dietary intake on pregnancy outcome and early postnatal performance in young adolescent sheep. biol reprod 2010;82:320330. 3. ishwa ak, memon ma: embryo transfer in sheep and goats: a review. small rumin res 1996;19:35-43. 4. bodin l, drion pv, remy b, et al: anti-pmsg antibody levels in sheep subjected to oestrus synchronization. reprod nutr dev 1997;37:651-660. 5. husein mq, bailey mt, ababneh mm, et al: effect of ecg on the pregnancy rate of ewes transcervically inseminated with frozen-thawed semen outside the breeding season. theriogenololgy 1998;49:997-1005. 6. hill j: maximizing the results of goat embryo transfer programs. proc am assoc small rumin pract res symp health disease; 1996. p. 120. 7. pereira rj, sohnery b, holtz w: nonsurgical embryo collection in goats treated with prostaglandin f2α and oxytocin. j anim sci 1998;76:360-363. 8. melican d, gavin w: repeat superovulation, non-surgical embryo recovery transfer in transgenic dairy goats. theriogenology 2008;69:197-203. 487 clinical theriogenology • volume 5, number 4 • december 2013 9. agrawal kp, goel ak, tyagi s: successful non-surgical embryo recovery from a goat. indian j exp biol 1991;29:1144. 10. rowe jd: reproductive management in sheep and goats. proc am assoc small rumin pract res symp health disease; 1996. p. 39. 11. rowe jd, east ne: reproductive management – part i: estrous cycles, synchronization, aritificial insemination, pregnancy diagnosis. small rumin mixed anim pract, western vet conf; 1998. p. 37. 12. haibel gk: real-time ultrasonic fetal head measurement and gestational age in dairy goats. theriogenology 1988;30:1053-1057. 13. haibel gk, perkins nr, lidl gm: breed differences in biparietal diameters of second trimester toggenburg, nubain, and angora goat fetuses. theriogenology 1989;32:827-834. 14. santos mhb, moraes ep, bezerra fq, et al: early fetal sexing of saanen goats by use of transrectal ultrasonography to identify the genital tubercle and external genitalia. am j vet res 2007;68:561-564. 488clinical theriogenology • volume 5, number 4 • december 2013 2018: evaluating the efficacy of a silicone y-design intrauterine device as a horse contraceptive in a captive breeding trial evaluating the efficacy of a silicone y-design intrauterine device as a horse contraceptive in a captive breeding trial c.c. lyman,a j.m. baldrighi,a m.yanos,a s.s. germaine,b a. kane,b g.r. holyoaka acenter for veterinary health sciences, oklahoma state university, stillwater, ok; bu.s. department of interior, bureau of land management, lakewood, co due to continued on-range population growth there are upwards to 70,000 feral horses against a blm range ecologist established goal of 27,000. therefore, the need to identify an effective means of contraception for feral horses on the western range remains a blm priority. as reported at the 2017 therio conference we experienced unexpectedly high loss rates over the first two months with a previously published silastic o-ring intrauterine device (iud) and also with other iud size and durometer (hardness) variations.1 we also observed that a majority of the iuds were expelled within 14 days; we considered that this may be a result of uterine expulsatory forces or by short cycling the mares and inducing estrus with subsequent frequent breeding behavior after placement of the iud. therefore in the next phase of this study we hypothesized that co-administration of a long-acting progesterone at the time of iud insertion would increase iud retention by preventing these aftereffects. furthermore, in human iuds a fundus seeking design ameliorates normal uterine expulsion forces, therefore, we extrapolated from those data to formulate an iud ‘y’ design (of 50 and 60 durometers) to test for a >75% retention rate. two acclimated breeding pods, with one stallion per group, were used in this study. within each group ten mares randomly received either a 50 or 60 durometer y-design iud. additionally, every other mare received an intramuscular injection of either saline, or long-acting progesterone. this process allowed for an equally randomized, split plot design of equal numbers of mares (ten mares) in each treatment for iud hardness, and co-administration of progesterone. of the mares receiving a 50 durometer iud, eight of ten retained their iud over the first two months (20% loss rate, 80% retention). of the mares receiving a 60 durometer iud, nine of ten retained their iud over the first two months (10% loss rate; 90% retention). co-administration of long-acting progesterone aided in retention over the initial cycle’s expulsive forces as ten of the ten treated mares retained their iud over the first two months (100%). at this phase of the study these data indicate that the 60 durometer y-design iuds are superior to 50 durometer y iuds: 90% vs. 80% retention over 60+ days. also, that co-administration of progesterone helps overcome the initial 14-day loss rate: none of the mares that received progesterone at the time of iud placement lost their iud, whether she received a 50 or 60 durometer iud. keywords: intrauterine device, feral horse, y-design, o-ring, progesterone reference 1. baldrighi jm, lyman cc, hornberger k, et al: evaluating the efficacy and safety of silicone o-ring intrauterine devices as a horse contraceptive through a captive breeding trial. clin therio 2017;9:417. clinical theriogenology • volume 10, number 3 • september 2018 336 analytic approach for endometrial polymorphonuclear cells cytology threshold for the definition of cytological endometritis in primiparous and multiparous dairy cows analytic approach for endometrial polymorphonuclear cells cytology threshold for the definition of cytological endometritis in primiparous and multiparous dairy cows shaked drukera,b, ron sicsica, michael van stratena,b, tamir goshena,c, tal raza akoret school of veterinary medicine, robert h. smith faculty of agriculture food and environment, the hebrew university of jerusalem, rehovot, israel bhachaklait, mutual society for veterinary services, caesarea, israel cthe veterinary services, ministry of agriculture and rural development rishon lezion, israel cytological endometritis (cem) is usually diagnosed based on proportion of polymorphonuclear cells (pmn) in endometrial cytology. incidence of cem varies greatly among studies, ranging from 12 45%, presumably reflecting diversity among herds, but potentially due to diagnostic criteria, including parity, threshold of pmn proportion, as well as interval from calving to cytology sample collection. our objective was to examine whether primiparous and multiparous cows should be diagnosed for cem by different criteria and timing, using a combination of selected reproductive performance outcomes [interval to first service and to pregnancy, pregnancy rate at 180 days-in-milk (dim), first service conception rate, and number of ais (artificial inseminations) to pregnancy]. two endometrial cytobrush cytology samples were collected from holstein dairy cows (n = 213; 133 multiparous, 80 primiparous), at 30 40 and at 60 70 dim; slides prepared and stained with diff-quick and pmn proportions evaluated blindly. pmn proportion thresholds were set at ≥ 1 to ≥ 10, ≥ 15, and ≥ 20%, for diagnosis of cem. data were analyzed by cox’s proportional hazard model for time to first service and time to pregnancy (from calving and from first service), controlling for the effect of farm. cytological endometritis in primiparous cows were best identified at 30 40 dim, whereas in multiparous cows, cem was best diagnosed at 60 70 dim. in primiparous cows, a threshold of ≥ 7% at 30 40 dim was associated with significant reductions in the majority of reproductive performance parameters analyzed (time to first service and to pregnancy, pregnancy rate at 180 dim, first service conception rate, and number of ais to pregnancy), with specificity, sensitivity and positive predictive value of 85.2, 41.2, and 59.2%, respectively. however, at 60 70 dim, none of the pmn thresholds were associated with reduced reproductive performance in primiparous cows with cem. in contrast, in multiparous cows, a threshold of ≥ 3% at 60 70 dim was associated with significant reductions in the majority of the reproductive performance parameters analyzed, with specificity, sensitivity and positive predictive value of 83.1, 39.1, and 62%, respectively. however, at 30 40 dim, none of the pmn thresholds were associated with reduced reproductive performance in multiparous cows with cem. differences between primiparous and multiparous cows could be related to metabolism, immune function, and uterine involution. in conclusion, diagnosis of cem in primiparous and multiparous cows should be made using different pmn threshold criteria and at different intervals after calving. keywords: dairy cows, endometritis, cytobrush, pmn threshold 429 clinical theriogenology • volume 11, number 3 • september 2019 430clinical theriogenology • volume 11, number 3 • september 2019 omniblank: nerve growth factor ? increases small luteal cell number and enhances steroidogenic activity in bovine corpus luteum nerve growth factor β increases small luteal cell number and enhances steroidogenic activity in bovine corpus luteum jamie stewart,a,b vitor mercadante,c nicholas dias,c stephanie stella,a laís cunha,a igor canisso,a fabio limaa acollege of veterinary medicine, university of illinois, urbana, il bvirginia-maryland college of veterinary medicine, and ccollege of agricultural and life sciences virginia polytechnic institute and state university blacksburg, va nerve growth factor β (ngf) is a seminal plasma protein that stimulates bovine theca cell proliferation and steroidogenesis in vitro and is luteotrophic in vivo, yet there is limited information on how it alters complex dynamics associated with ovulation and corpus luteum (cl) formation in cattle. objectives were to evaluate the effects of systemically administered purified bovine ngf on vascularity and steroidogenic function of pre ovulatory follicles and subsequent cl in cattle. our hypothesis was that systemic administration of ngf to heifers with a pre-ovulatory follicle increases vascularity and steroidogenic function of follicle, decreases the time to ovulation, and improves subsequent luteotrophic function through increased vascularity and small luteal cell number in the cl. holstein heifers (n = 12) were synchronized using a gnrh prostaglandin and intravaginal progesterone protocol (5 day cidrsynch) and randomly assigned to 1 of 2 treatment groups: 12 ml phosphate-buffered saline (cont) or 250 µg purified ngf in 12 ml phosphate-buffered saline (ngf) given im at time 0 (presence of preovulatory follicle). using a crossover design, a second replicate was performed at ~ 1 month after completion of first replicate. transrectal ultrasonography with doppler and blood sampling were performed every 4 hours from 0 32 hours to evaluate follicle size and vascularity, time to ovulation, and serum estradiol concentrations. transrectal ultrasonography was performed daily to assess cl size and vascularity, and blood was obtained every 2 days to measure serum progesterone concentrations. on days 9 and 14, cl biopsy was performed on a subset of heifers (n = 6 heifers/treatment/time) to assess mrna expression of steroidogenic enzymes and lh receptor by qpcr and to determine the histological ratio of small to large luteal cells. statistical analyses were performed using a general linear mixed model with repeated measures, using heifer id as a random variable (r version 3.4.3). there was a main effect of ngf treatment on follicle diameter (p = 0.02), but no effect on serum estradiol concentrations (p = 0.95) or time to ovulation (p = 0.42). treatment with ngf tended to increase cl diameter (p = 0.10) and increased serum progesterone concentrations (p = 0.04) from days 10 12. there was a higher (p < 0.01) percentage of small luteal cells and a tendency for increased lh receptor gene expression (p = 0.09) in the cl of ngf treated heifers. consistently, there were increased expressions (p ≤ 0.05) of genes for steroidogenic acute regulatory protein and 3β hydroxysteroid dehydrogenase in the cl of ngf treated heifers. treatment with ngf did not alter vascularity (p ≥ 0.16) of the follicle or cl. we concluded that systemic administration of purified ngf to heifers interacted with pre ovulatory follicles to alter downstream cl formation and function. luteotrophic effect of ngf was attributed to increased small luteal cell number and enhanced steroidogenic enzyme activity within the cl. keywords: bovine, luteotrophic, nerve growth factor β, ovulation, progesterone acknowledgement this project was funded by the united states department of agriculture national institute of food and agriculture (hatch funds # 1014712). 445 clinical theriogenology • volume 11, number 3 • september 2019 446clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2013: rabbit reproduction rabbit reproduction swanand r. sathe,a clifford f. shipleyb adepartment of veterinary clinical sciences, college of veterinary medicine, iowa state university, ames, ia; bagricultural animal care and use program, college of veterinary medicine, university of illinois, urbana, il introduction rabbit production (also referred to as cuniculture) is the agricultural practice of breeding and raising domestic rabbits, usually for meat, fur, research, show and as pets. the domestication of and rearing of rabbits for meat and fur production began in the 16th century, and has evolved from a simple backyard production of hutch raised rabbits to extensive operations capable of producing thousands of animals. the european union continues to be the world’s largest producer of rabbit meat accounting for about 55% of the global production, followed closely by china. in the united states the rabbit industry is relatively small and no reliable statistics exist describing the meat industry in the terms of number of producers and tonnage of meat produced. in fact in the us, rabbits are not recognized as an agricultural commodity by the department of agriculture and are instead regulated by the food and drug administration. hence they are not regarded as a separate species in the agricultural census. the american rabbit breeders association inc. (www.arba.net) is the single largest and oldest organization in the us dedicated to the development, promotion and improvement of the rabbit industry. rabbit meat has started gaining popularity due to several advantages over red meat. it is low in fat, cholesterol and sodium, high in protein and has a pleasant taste similar to chicken meat. owing to their small body size, short generation interval and high reproductive capacity, ability to grow rapidly on feeds not directly consumed by humans (grain free) and the potential for genetic improvement, rabbit meat is an attractive primary source of meat in many regions of europe and africa. the two most popular breeds of rabbits raised for meat production are the new zealand white and the californian owing to their high meat to bone ratio characteristics and white fur. fur producers prefer the rex and the american chinchilla breeds. rabbits are a valuable animal model for biomedical research, including studies in cardiovascular medicine, neurophysiology, ophthalmology, immunology, asthma research, arthritis and toxicology. with respect to embryological research, the rabbit has several advantages over other animal models as its embryonic morphology is archetypical for mammals during many phases of uterine development (e.g., gastrulation, neurulation, gonad development) and its late implantation makes it relatively accessible at these early stages of development.1 due to the phenomenon of induced ovulation in rabbits after coitus (or artificially by hormonal treatments), it is easier to time pregnancies with an accuracy unsurpassed in other common laboratory animals. this greatly facilitates the analysis of the highly dynamic developmental processes over a broad interval of developmental stages, from blastocyst formation to neurulation at four to nine days after coitus. moreover, the high cell numbers and yield in rabbit blastocysts (the largest spherical blastocyst in mammals), relatively late implantation at a time when gastrulation is already proceeding, detailed morphologic and molecular knowledge on gastrulation stages, and a hemochorial placenta structure similar to the human placenta make the rabbit an ideal reproductive animal model for studying human health.2 in the research industry new zealand whites are the preferred breed as they are less aggressive in nature and have fewer health problems compared to other breeds. keywords: rabbit, reproductive management, reproductive disease reproductive anatomy and physiology male the reproductive system of the male rabbit or buck is comprised of the external genitalia (penis, prepuce and scrotal sac), internal genitalia (testis and spermatic cords) and a full set of accessory sex glands (vesicular, prostate and bulbourethral glands). the testes descend at about 12 weeks after birth 465 clinical theriogenology • volume 5, number 4 • december 2013 and lie in two separate hemiscrotal sacs cranial to the penis. the inguinal canal does not close in rabbits and hence they are termed “functional cryptorchids” as the position of the testes depends on many factors including body position, body temperature, breeding activity, gastrointestinal tract filling, and the amount of abdominal fat. they are also able to voluntarily pull up the testes when they are frightened or during fighting. the short back-slanted penis lies caudal to the testes and points forward when erect. the glans penis is not well developed, is tapered, and covered by the prepuce. these anatomical peculiarities hold special consideration when attempting to perform orchiectomies in bucks. it is highly recommended to perform surgical closure of the open inguinal canal to prevent postsurgical hemiscrotal herniation of the abdominal viscera. likewise due to the caudal position of the penis, it is possible to adopt a pre-scrotal approach with a single incision on the midline during orchiectomies3 as an alternative to a scrotal approach. depending on the breed and level of nutrition bucks can reach sexual maturity between four and eight months of age. new zealand white bucks have been known to have an increased daily sperm output from 20 weeks of age to a mature level at 31 weeks of age. ejaculate volume for bucks can range between 0.5 to 1.5 milliliters per ejaculate with a concentration ranging from 10 to 300 million spermatozoa per milliliter. this varies among bucks and depends on the breed, frequency of collection and level of stimulation. rabbits can experience testicular recrudescence in the wild with a reduction in the testicular size in autumn and a peak increase in late spring. female in the female rabbit or doe, the ovaries, oviducts and the uterus are paired organs similar to other species of mammals. the ovaries are not enclosed in an ovarian bursa as is found in carnivores and rodents but instead are surrounded by fat which also envelopes the mesovarium and mesosalpinx. female rabbits have a bicornuate duplex uterus lacking a uterine body and having two separate cervixes that open directly and separately into an elongated vaginal canal which is about six to ten cm long. the urethra opens midway along the ventral aspect of the vaginal body at the vaginal vestibule. the external genitalia of the doe include a urogenital sinus, which is continuous with the vagina and whose external lips form the vulva. the color and moistness of the vulva can be used as an indicator of sexual receptivity. a doe with a moist red or pink vulva is more likely to be receptive to the buck that a one with a pale and dry one. puberty in does is dependent on the breed and plane of nutrition with some does showing receptivity as early as three and one-half months of age and capable of conception at four to four and onehalf months. it is however advisable to delay mating in does until they have achieved at least 80 percent of their mature body weight. rabbits do not have a defined estrous cycle, but are induced ovulators like cats and ferrets. ovulation occurs about ten to11 hours after coitus or can be induced by injecting luteinizing hormone (lh). though rabbits do not have an estrous cycle, they are not in constant heat as sometimes thought. follicular development occurs in waves as in other species and these follicles persist for around 12 to 14 days coincident with the doe’s receptivity. in absence of coitus the follicles become atretic and a new wave starts after about four days. does are less receptive to the male during these four days. thus a doe has a cycle of about 16 to 18 days, with about 12 to 14 days of receptivity and four days of quiescence. determining the sex of a newborn (kit) may be done accurately a day or two after birth. at this age the external genitalia of both sexes may be similar in appearance and hence needs to be carefully examined. the newborn may be restrained on their back in the palm of the hand with the head extended towards the heel of the hand. after pressing the tail back and down, gentle pressure may be applied to the external genitalia to expose the reddish mucous membrane. in the case of a doe a slit is visible with slight depression next to the anus, while in the case of a buck a small circular opening might be visible. as bucks get older it is possible to extrude and expose the penis. bucks will also have a greater anogenital distance than does. alternatively, young rabbits may also be sexed at weaning or by around three weeks of age when the external genitalia will have a more defined appearance. 466clinical theriogenology • volume 5, number 4 • december 2013 breeding management as mentioned above, the ideal age for breeding is primarily breed dependent along with a proper plane of nutrition. smaller breeds (polish) can be bred as early as four months of age, medium sized breeds (new zealand whites and californians) at six months, while it is best to wait until at least nine to 12 months to breed the larger breeds (flemish giants) of rabbits. bucks should be put to service typically a month later than does, as they take a longer time to mature sexually. does that are sexually receptive regardless of age usually have a moist, swollen and reddened vulva, whereas the vulva may be small and whitish in instances where the female is not receptive. although this is a reliable indicator of ‘when to breed’, it is advisable to maintain age, breeding records and dates before deciding to include a doe in the breeding program. receptive does are usually restless and rub their chins on the cage, water and feed troughs. does can be aggressive towards other individuals placed in their cage, hence they should always be taken to the buck’s cage for mating. this also reduces the chances of territorial fights in case where the buck is presented to the doe in her cage. if the doe is truly receptive mating occurs immediately and when it is complete the buck usually falls to his side. sometimes male rabbits emit a cry towards the end of mating before falling and this often coincides with ejaculation. does can be bred two or three times to the buck after they are placed in the cage as this increases the chances of conception. sometimes breeders may rebreed the doe to a different buck. if mating does not take place the doe can be presented to another buck. however, if she is not receptive even then, she should be put away and tried again after a few days. bucks and does should not be left unattended for longer than a few minutes as there is a risk of the buck getting injured. if mating does not occur in a few minutes, it is advisable to remove the doe to another buck’s cage. occasionally, it may be necessary to physically restrain a female for mating, especially in case of shy does. pregnancy and gestation the gestation period in rabbits ranges from 29 to 32 days. since rabbits are induced ovulators, a short mating bout including ejaculation is sufficient to induce a rapid release of lh from the anterior pituitary. plasma levels of lh have been shown to rise within three minutes after copulation and ejaculation and reach a plateau within 15 minutes.4,5 rabbits ovulate ten to11 hours after coitus and only one ejaculation is sufficient to induce ovulation in a majority of cases unlike cats which require multiple intromissions and ejaculations in order to ovulate.5-7 fertilization occurs at around ten hours after mating and the rabbit blastocyst attaches to uterine epithelium late in embryogenesis as compared to other mammals.2 implantation starts at day 6 plus 18 h; while definitive mesometrial chorioallantoid placentation occurs at day 8.8 the rabbit placenta is discoid and hemodichorial (two cellular layers of chorion between the maternal and the fetal blood) in structure and similar to primate placentas in terms of growth and fetomaternal exchanges during pregnancy.9 gestation length may sometimes be prolonged especially if the doe is carrying small litters and has been observed to extend up to 35 days. pregnancy diagnosis pregnancy status in does may be determined by test-mating does by returning them to the buck’s cage. this is not a reliable method as some pregnant does may accept service, while some non-pregnant ones may decline. also, noting an increased abdominal girth or gain in flesh, though indicative, is not always reliable. palpation of the caudal abdomen to feel the developing fetuses is a faster and more reliable method of pregnancy diagnosis in rabbits. most breeders prefer palpating at about two weeks after mating to feel the marble shaped fetuses developing in the uterus. however with experience and care it is possible to feel the growing embryos as early as ten days without causing them injury. if no developing embryos are felt by ten to 12 days, the doe can be returned to the buck’s cage for mating. however many does may not breed at ten to 12 days due to psuedopregnancy and these animals should be checked again at 18 days to determine receptivity. palpating earlier than day ten is generally not recommended due to the fear of injuring the developing embryos and also because it is easy to confuse them with fecal pellets in the caudal abdomen. with practice it is also possible to become proficient in identifying retained fetuses or abnormalities of the reproductive tract such as abscesses, cysts or tumors. 467 clinical theriogenology • volume 5, number 4 • december 2013 does may sometimes experience fetal loss prior to day 19 of gestation and may resorb the fetuses. hence breeders prefer re-palpating by 28 to 29 days before putting the doe into a nest box, to ascertain that she is indeed pregnant. real-time abdominal ultrasonography, though not widely used as the preferred method, may also be useful to detect presence of embryonic vesicles in the uterine lumen reliably as early as the ninth day of gestation. again with experience some practitioners are able to diagnose pregnancy as early as the seventh or eighth day when the diameter of the embryonic vesicle is in the range of 8 to 9.5 mm.10 parturition and maternal care parturition or kindling in rabbits usually occurs by day 30 to 32 after breeding. a few days before the anticipated date, a nest box containing bedding material such as straw or shavings should be provided to the doe. does will also pull fur from their dewlap and mammary gland area to complete the nest. parturition usually occurs in the early morning and lasts for about 30 minutes, with the kits born every one to five minutes. kits are hairless, blind and deaf at birth. occasionally in cases of large offspring kindling can be delayed for a day or two. it is important to check the doe to make sure that there is no dystocia. the number of kits in the litter varies among breeds and can range from fewer than four (small breeds) to eight to ten in larger breeds like the new zealand whites and flemish giant. does usually eat the placenta upon completion of parturition and may occasionally resort to cannibalism if they are disturbed or nervous. those that have not kindled by day 32 to 34 of gestation may be induced by injecting oxytocin (1 to 3 units per animal). does usually nurse the newborn immediately after birth and later only once a day for a short period of time. colostrum is produced for the first two to three days and thereafter regular lactation begins. does bearing large litters are often in a negative energy balance owing to the greater demands placed on lactation and also have reduced appetite prior to kindling. it is desirable to gradually increase the amount of feed in the first week after kindling to meet these demands until full feed intake is reached. in intensive breeding systems does may be re-bred within 24 hours after kindling. however, this type of breeding management also increases the percentage of culled does per year. several breed-back schedules (e.g., at 7, 14, 21, 28, 35 and 42 days) are followed by breeders based on their level of experience and facilities available. however, an ideal re-breeding schedule usually depends on the condition of the doe after the previous parturition. does with large litters and lactating heavily should not be bred until the young are weaned. litters are usually weaned by 28 days of age, as milk production is declining and the young have started consuming solid feed. common reproductive disorders psuedopregnancy psuedopregnancy can occur in does due to a sterile mating, in does mounting each other, or sometimes when does mount the young in their own litter. it may also be seen in pet rabbit does housed singly. the condition can last for 15-17 days and does may not be receptive to the buck during this period. matings occurring during psuedopregnancy usually are not fertile. towards the end of pseudopregnancy does may display nesting behavior and pull out fur. mammary development is most pronounced in the first ten days of false pregnancy, after which involution typically follows. psuedopregnancy resolves spontaneously but may recur or may result in hydrometra or pyometra.11 ovariectomy is the treatment of choice and may be performed after the mammary glands undergo involution. pregnancy toxemia pregnancy toxemia is an often fatal metabolic disorder which occurs in rabbits during the last week of gestation. though the underlying mechanism is unknown, it is more common in obese does and in cases of inadequate caloric intake. in addition environmental changes and stress can predispose a pregnant doe to this condition. reduced feed intake may also be caused due to hairball formation occurring when does pull their fur for nest building. clinical signs include weakness, depression, incoordination, anorexia, abortion, convulsions, and coma and can progress over one to five days, or death may occur acutely. supportive therapy with fluids to correct metabolic derangements, combined 468clinical theriogenology • volume 5, number 4 • december 2013 with calcium gluconate and corticosteroids are the mainstay of the treatment plan. pregnancy toxemia carries a very grave prognosis, and treatment is usually unrewarding. the best approach is prevention by avoiding fasting or undernutrition during late pregnancy and by preventing obesity and sudden stress at all times. dystocia and retained fetuses dystocia is not common in rabbits since normal delivery is usually complete within 30 minutes and rarely are the kits delivered several hours apart. the normal presentations in does are anterior and breech and it may be essential to palpate does 24 hours after delivery to determine if any fetuses have been retained. does may be predisposed to dystocia if they are obese, have a narrow pelvic canal, are carrying large fetuses or experiencing uterine inertia. does may have persistent contractions and may continue straining. occasionally a greenish-brown vaginal discharge may be observed. radiographs may provide a definitive diagnosis and reveal abnormalities of the fetus or a narrow pelvic canal. oxytocin (13 units im per animal) may aid uterine contractions, while in cases of uterine inertia 5 to 10 ml of 10% calcium gluconate orally 30 minutes before administering the oxytocin may prove useful. does are kept in a quiet dark room after administration. if the medical therapy does not work, a cesarean section is indicated although prognosis can be guarded. abortion and fetal resorption abortion can occur in does after day 19 of gestation, whereas resorption usually occurs in the event of embryonic or fetal death before day 19. as with other species, obtaining a thorough clinical and breeding history of the individual and the herd is of utmost importance. aborting animals should always be checked for presence of retained fetuses. all aborted material should be submitted for bacterial culture and histopathology. there are numerous causes for abortion and fetal resorption in rabbits including infections, stress, trauma, neoplasia, inappropriate drug use and dietary imbalances such as deficiencies of vitamins a and e and protein. listeriosis should be considered in does experiencing late term abortions.12 uterine adenocarcinoma uterine adenocarcinomas are the most common tumors of the genital tract in rabbits. their occurrence is independent of breeding history but is largely affected by age. certain breeds such as tan, french silver, havana, and dutch, older than four years of age have an incidence of 50 to 80 percent.13,14 uterine adenocarcinomas are slow growing tumors, with local invasion to adjacent peritoneal structures and sometimes hematogenous spread to the lungs, liver and brain.13 early clinical signs, such as decreased fertility, small litter size, and an increased incidence of fetal retention or resorption and stillbirths may be recognized in a breeding doe. cystic mammary glands can develop concurrently with uterine hyperplasia or adenocarcinoma. in late stages, clinical signs may range from depression, anorexia and ascites to dyspnea if pulmonary metastases are present. diagnosis can be made based on clinical signs, abdominal palpation, radiology and abdominal ultrasound. ascites may be present. palpation of the caudal abdomen may reveal an enlarged uterus or nodular masses (1 to 5 cm in diameter), or both. chemotherapy is not often attempted; but ovariohysterectomy may be curative if the tumor is confined to the reproductive tract. animals should be reexamined every few months for several of years to monitor for any signs related to metastasis. uterine adenocarcinomas can be best prevented with an ovariohysterectomy in pet rabbits before they reach two years of age. pet owners may be advised to conduct an annual or semi-annual examination of intact animals three years of age or older. cannibalism though cannibalism is more of behavioral issue and not a primary reproductive disorder, it is nevertheless an important concern to breeders as it can significantly affect the production efficiency of a breeding unit. it is common for does to eat their placenta and dead kits after kindling. they may also devour the entire litter in some instances. various theories to explain this behavior have been proposed including the doe’s level of feeding and low energy status, environmental stress such as loud noises, 469 clinical theriogenology • volume 5, number 4 • december 2013 handling by strangers and mixing with strange animals. certain lines or families of rabbits are more prone to cannibalize their young than others and this could be attributed to their nervous nature. breeders usually prefer culling does that cannibalize two litters in a row in absence of management errors. venereal spirochetosis or rabbit syphilis rabbit syphilis or vent disease is caused by a spirochete treponema paraluiscuniculi and can be spread by direct contact as well as by the venereal route. it is a disease of major concern in rabbitries as it can result in a decreased rate of conception and increased incidence of metritis, placenta retention, and neonatal deaths.15 lesions first appear on the skin of the perineum and genitalia and begin as areas of redness that progress to edema, vesicle formation, ulcerations, and scabs in both does and bucks. the lesions are painful and can impair breeding activity. syphilis is easily treatable with long-acting penicillin and tetracyclines in water. the disease can be introduced into the herd after introducing a new animal (buck or doe) or after loaning a buck to service someone else’s does. infected animals may be identified by serologic surveys with a microhemagglutination test or the rapid plasma reagin (rpr) card test which are very specific. risk of t. paraluiscuniculi infection increases with parity and does that have had six or more litters are often found seropositive as are bucks that have been in a breeding program for more than six to 12 months.16 metritis, pyometra and orchitis does may occasionally suffer from metritis and pyometra, with venereal transmission being the most common route of infection. the most common organisms isolated are pasteurella multocida and staphylococcus aureus. clinical signs include anorexia, lethargy, weakness, enlarged abdomen and vaginal discharge. history of the doe may indicate a recent parturition, psuedopregnancy or an inability to rebreed. in mild cases, it may be possible for the doe to kindle successfully or she may suffer from abortions and fetal resorption. nulliparous does are at an equal risk of developing metritis and pyometra. diagnosis may be reached based on clinical signs, abdominal palpation, ultrasonography and radiography. though antibiotic, fluid and supportive therapy may be attempted, it is extremely difficult to achieve effective drainage of the tenacious uterine exudate in affected does. ovariohysterectomy remains the treatment of choice mainly due to the high incidence of p. multocida infections. bucks on rare occasions may suffer from orchitis and epididymitis due to venereal transmission or due to bite injuries on the scrotum caused during fighting. clinical signs may include swelling of the testes, micro or macro abscesses, fever, anorexia, weight loss and low conception rates. p. multocida is the organism most often isolated from clinical cases. treatment of choice is castration and antibiotic therapy. bucks should be housed separately to prevent fighting and subsequent injuries. similarly, they should not be left unsupervised with a female in a cage during mating as females can be very aggressive. reduced fertility fertility issues are usually multifactorial in rabbits and may include malnutrition (e.g., excess vitamin a, deficiencies of vitamins a, d, or e), heat stress, systemic illness, nitrate contamination of food or water, environmental disturbances, a decrease in daylight, endometrial carcinoma, metritis, or pyometra. old age and reproductive senescence or breeding at too young an age are additional causes of infertility. vitamin e deficiencies are known cause myodystrophy leading to abortions, stillbirths and neonatal mortality. hyperand hypo-vitaminosis a are also major nutritional causes of reproductive failure and infertility. breeders and owners should be advised regarding proper nutrition of their animals using the national research council dietary guidelines. references 1. püschel b, daniel n, bitzer e, et al: the rabbit (oryctolagus cuniculus): a model for mammalian reproduction and early embryology. cold spring harb protoc 2010;doi:10.110/pdb.emo139. 2. fischer b, chavatte-palmer p, viebahn c, et al: rabbit as a reproductive model for human health. reproduction 2012;144:1-10. 3. capello v: surgical techniques for orchiectomy of the pet rabbit. exot dvm 2005;7:23-32. 470clinical theriogenology • volume 5, number 4 • december 2013 4. jones ef, bain jb, odell wd: postcoital luteinizing hormone release in male and female rabbits as determined by radioimmunoassay. fertil steril 1976;27:848-852. 5. bakker j, baum mj: neuroendocrine regulation of gnrh release in induced ovulators. front neuroendocrinol 2000;21:220-262. 6. johnson lm, gay vl: luteinizing hormone in the cat. ii. mating-induced secretion. endocrinology 1981;109:247-252. 7. wildt de, seager swj, chakraborty pk: effect of copulatory stimuli on incidence of ovulation and on serum luteinizing hormone in the cat. endocrinology 1980; 107:1212-1217. 8. denker hw: implantation. the role of proteinases, and blockage of implantation by proteinase inhibitors. adv anat embryol cell biol 1977;53:3-123. 9. mcardle am, denton km, maduwegedera d et al: ontogeny of placental structural development and expression of the renin–angiotensin system and 11b-hsd2 genes in the rabbit. placenta 2009;30:590-598. 10. ypsilantis p, saratsis p: early pregnancy diagnosis in the rabbit by real time ultrasonography. world rabbit sci 1999;7:95-99. 11. patton nm: colony husbandry. in: manning pj, ringler dh, newcomer ce, editors. the biology of the laboratory rabbit. new york: academic press; 1994. p. 27-45. 12. watson gl, evans mg: listeriosis in a rabbit. vet pathol 1985;22:191-193. 13. baba n, von haam e: animal model for human disease: spontaneous adenocarcinoma in aged rabbits. am j pathol 1972;68:653-656. 14. weisbroth sh: neoplastic diseases. in: manning pj, ringler dh, newcomer ce, editors. the biology of the laboratory rabbit. new york: academic press; 1994. p. 259-292. 15. paré ja, paul-murphy j: disorders of the reproductive and urinary systems. in: quesenberry k, carpenter jw, editors. ferrets, rabbits, and rodents clinical medicine and surgery. 3rd ed. st louis: elsevier; 2012. p. 183-193. 16. digiacomo rf, talburt cd, lukehart sa, et al: treponema paraluiscuniculi infection in a commercial rabbitry: epidemiology and serodiagnosis. lab anim sci 1983;33:562-566. 471 clinical theriogenology • volume 5, number 4 • december 2013 472clinical theriogenology • volume 5, number 4 • december 2013 omniblank: 2018: fetal mummification due to neospora caninum in an angus cow fetal mummification due to neospora caninum in an angus cow college of veterinary medicine, purdue university, west lafayette, in a mature angus cow was presented to the purdue university veterinary teaching hospital for a suspected abortion. the owner noticed that the cow had placenta hanging from her vulva but was showing no signs of active labor. the cow was palpated per rectum by the owner and a hard structure was palpated in the vaginal vault. the cow was from a 35 head beef herd that has had five abortions in the past two years. in the present year, the herd has experienced three abortions in addition to this case. upon presentation and physical examination, the cow was systemically healthy. approximately 20 cm of fetal membranes was protruding from the vulva. rectal and vaginal palpation revealed a 4cm x 3cm x 16cm hard, discoid structure within the vagina vault with a palpably empty orbit. the cow was administered a caudal epidural and a fetal mummy and associated placenta was extracted from the vagina. the fetus and placenta were submitted for necropsy and histopathology. the fetus was found to be positive for neospora caninum by pcr testing with no other bacteria or viral organisms isolated. the dam was also found to be positive for neospora caninum by elisa. neospora caninum is a major cause of abortion in both dairy and beef cattle. cattle become infected following ingestion of the oocyst. cows may abort from three months gestation to term with most abortions occurring at five to six months of gestation. fetuses may also be stillborn, born alive with clinical signs or born clinically normal but persistently infected. 1,2 mummified fetuses are not a common presentation associated with neospora infections. this case demonstrates that in cases of mummified fetuses, neospora caninum should be considered as a differential diagnosis. neospora is an economically important disease world-wide affecting both the dairy and beef industries. keywords: neospora caninum, bovine, mummification references 1. dubey jp, schares g: neosporosis in animals--the last five years. vet parasitol 2011;180:90-108. 2. dubey jp: neosporosis in cattle. vet clin north am food anim pract 2005;21:473-483. clinical theriogenology • volume 10, number 3 • september 2018 352 c.a. hegg, j.h. koziol, a. powers, s. tinkler 2011: international teaching and research opportunities sponsored by fulbright scholar program international teaching and research opportunities sponsored by fulbright scholar program mushtaq a. memon fulbright ambassador, department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa the fulbright program was established by the us congress to ‘‘enable the government of the united states to increase mutual understanding between the people of the united states and the people of other countries.’’ the core fulbright scholar program sends more than 800 us faculty and administrators to 125 countries to lecture or conduct research around the world each year. unfortunately, only 28 faculty members from the us veterinary colleges have used fulbright scholar opportunities in the last 20 years (1989–2009). considering recent worldwide events, such as the global dispersion of the asian strain of highly pathogenic avian influenza and pandemic h1n1 affecting human and animal species, the importance of awareness and education of veterinarians to such global issues is obviously urgent. therefore, fulbright scholarships represent an important opportunity to gain experience and bring this time-critical information back to fellow veterinarians and veterinary students. veterinarians who wish to contribute to internationalization of the curricula and their campuses should consider applying for fulbright scholar support to launch their career in this pivotal direction. for details about the fulbright scholar program, eligibility, and application procedures, please see the following reference for the web link. keywords: fulbright scholar, international, teaching, research reference us scholars – fulbright scholar program http://www.cies.org/us_scholars/ . clinical theriogenology • volume 3 number 4 • december 2011501 clinical theriogenology • volume 3 number 4 • december 2011 502 omniblank: a molar pregnancy in a hereford donor cow a molar pregnancy in a hereford donor cow lauren merritt, james wenzel, jessica rush, lawrence cofield, salman waqas, jenna stockler, julie gard college of veterinary medicine, auburn university, auburn, al a 6 year old hereford donor cow was referred for a mass in the tip of her left uterine horn. cow had undergone several embryo collections that had yielded unfertilized, degenerate ova. cow underwent transrectal palpation and ultrasonography. a multi locular mass enveloped by two separate compartments similar to amnionic and allantoic cavities, and bilateral cystic ovaries were visualized on ultrasonography. vaginoscopy revealed a closed cervix with no evidence of urine pooling or any abnormalities. a complete blood count, serum chemistries, and an abortion profile were submitted. cystic ovaries were treated by administering 2,500 iu of hcg iv and transvaginal aspiration of cystic follicles. cow was put on a cidr synch program to induce estrus for possible evacuation of mass from uterus. cow was given 20 iu of oxytocin im while in estrus. additionally, misoprostol paste was smeared on her cervical os and placed within her cervix via a mare infusion pipette to assist in dilating cervix for hysteroscopy. however, cervix did not dilate enough to allow a hysteroscope to pass through cervix. so, a foley catheter was placed and the uterus was massaged and flushed many times with sterile saline to dislodge the mass. pieces of tissue were collected and submitted for histopathology. tissue was determined to be placenta, confirming the diagnosis of a molar pregnancy. molar pregnancies are rare and characterized by abnormal growth of trophoblastic cells leading to formation of intrauterine cystic masses. this case emphasizes the need of a follow up reproductive tract ultrasonography 30 days following a nonsurgical collection to detect any abnormalities such as pregnancies and cystic conditions. this case also demonstrates the importance of keeping all diagnostic possibilities in mind, regardless of perceived prevalence. keywords: cystic ovaries, molar pregnancy, ultrasonography 533 clinical theriogenology • volume 11, number 3 • september 2019 534clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2011: collecting and processing semen from cervids, sheep and   collecting and processing semen from cervids, sheep and goats manoel tamassia new jersey department of agriculture, trenton, nj abstract the ability to freeze semen from domestic species has provided the farming community with an inexpensive way to preserve and propagate superior genetics. during cryopreservation sperm will endure severe physical and chemical challenges. there is significant difference in sperm from different species. the sperm shape, size and lipid composition will affect its ability to survive the freezing and thawing process. an understanding of the freezing requirements for the different species is necessary to increase sperm survival. the purpose of this review is to present to the practitioner the basic information necessary for the successful cryopreservation of sperm in small ruminants and deer. keywords: sperm, cryopreservation, reproduction, small ruminants introduction the purpose of this article is to provide important background information for understanding the process of cryopreservation of semen in small ruminants and deer. differences in semen and cryopreservation techniques exist among domestic farmed animals and several reviews with more detailed information are available.1,2 semen collection the use of an artificial vagina (av) is the preferred method of semen collection since the technique is successful in a high percentage of bucks and rams and a representative ejaculate is obtained. semen collection with an av should be made during attempts to breed a mount, ideally an estrual female. many males, especially if used to hand breeding, will mount a nonestrual female if she is sufficiently restrained to prevent rearing and limit lateral movement. semen samples can also be collected using electroejaculation. several types of electroejaculators and probes are available commercially. independent of the model used, the rectum of the animal should be emptied prior to collection. a ram probe can be used to collect bucks. the desired response is penile extension followed by ejaculation. one main advantage of electroejaculation is that it can be used in the absence of a mount female or when the male is unable or unwilling to mount. semen from farmed deer is obtained by using either an av or electroejaculation3. most stags are not tame or trained to be collected using an av. electroejaculation must be used when the objective is to repeatedly recover samples from selected males. electroejaculation must be performed in combination with effective and secure anesthesia (http://www.ivis.org/special_books/heard/caulkett2/ivis.pdf). electroejaculated semen can have a different composition when compared with semen collected by more natural methods (e.g., av). this difference is due to differential stimulation of seminal glands by the electroejaculation probe resulting in high variability between ejaculates.4 alternatively, semen can be collected post-mortem by recovery of epididymal spermatozoa, from hunt harvested animals.5-9 these techniques have been tested not only in deer, but also in many other wild ruminant species.4 semen evaluation the principal objective is to assess the number of motile, morphologically normal spermatozoa in the ejaculate prior to freezing. volume of semen, sperm concentration, proportion of motile sperm and spermatozoa morphology must be measured. values must be determined with precision to provide useful information to be used in the dilution and freezing process. of these variables sperm motility is the most difficult and inaccurate measurement. motility should be estimated on a warm slide (35° c) using a good microscope, preferably equipped with phase-contrast illumination for an accurate evaluation. stained slides of semen can be prepared for morphological evaluation of sperm. a common stain is eosinclinical theriogenology • volume 3 number 4 • december 2011507   nigrosin as it also permits the assessment of viable (unstained) sperm. semen should have at least 70% normal sperm cells with good motility and concentration. normal sperm concentration varies from animal to animal with a normal ejaculate containing at least a billion sperm cells in sheep and goats. the estimated semen output from red deer collected by electroejaculation is more than three billion sperm/ejaculate. in the same study, 2.5 billion/sperm were collected post mortem.10 semen concentration will vary with the age of the animal, method of collection and health status.10 in some deer semen collection, using electroejaculation, one may observe a thick gel fraction. this should be collected separately from the sperm-rich portion as sperm will stay trapped in the gel.11,12 sperm concentration can be determined accurately with the use of a hemocytometer. other methods of counting cells are also available and will not be discussed here. the total number of sperm in the ejaculate can be estimated by multiplying the number of sperm/ml (concentration) by the volume of the ejaculate. this product multiplied by the percentage of normal sperm gives the number of normal sperm in the ejaculate. seminal plasma for years, the only available model used for successful cryopreservation of small ruminant semen was the technique used for cattle. however, this technique did not work well with goat semen. the success was limited due to the deleterious interaction between the bulbourethral gland secretion and egg yolk used on the semen extender. this was known to be caused by an egg yolk coagulating enzyme.13 this enzyme was shown to hydrolyze egg yolk phospholipids into lysophospholipids such as lysolecithins, which are toxic to spermatozoa.14 this problem is not seen with bull, boar, rabbit and ram semen. to successfully freeze goat semen it is recommended to remove the seminal plasma if egg yolk or milk extender is being used. however, seminal plasma secretions appear to be beneficial to preservation of sperm integrity during freezing in the absence of egg yolk. the removal of the seminal plasma is carried out by dilution of ejaculated sperm in an isotonic buffer followed by centrifugation (washing method). washing increases the storage duration of chilled goat sperm and also improves the survival of spermatozoa after freezing and thawing. sperm dilution or concentration semen samples from farm animals have been diluted prior to freezing based solely on volume (v/v) or to a specific final sperm concentration. the second method being preferred as it provides uniformity to the samples necessary for commercialization of straws. an insemination dose should have forty to one-hundred million viable sperm. this concentration has been reported to achieve acceptable pregnancy rates in small ruminants. the number of sperm/insemination dose can be reduced drastically if the semen is to be deposited deep into the uterus. sperm concentration can be determined accurately with the use of a hemocytometer. the hemocytometer is a device used to count red and white blood cells but it can also be used to count sperm. sperm must be diluted with water or buffered formalin. the objective is to immobilize the sperm cells and to dilute them sufficiently so that you will count 100 to 400 sperm cells in the five squares. the central counting area of 25 large squares has an area of 1 square mm, and the coverglass rests 0.1 mm above the floor of the chamber. thus, the volume over the central counting area is 0.1 microliter. in other words, to calculate the number of sperm/ml of original sample one must calculate the mean number of sperm counted for each chamber (i.e. for each of the central counting areas 25 squares). then multiply the mean obtained in the two chambers by 10,000 to obtain the number of cells/ml of diluted sample. finally, multiply the count obtained in by the dilution factor. once the concentration of sperm in the ejaculate has been determined, the sample will need to be diluted with extender or concentrated (through centrifugation) to adjust the insemination dose to fit into one straw or cryovial. the most common diluents used to extend sperm contain either non-fat dried skin milk or egg yolk. clinical theriogenology • volume 3 number 4 • december 2011 508   cryopreservation cryopreservation of sperm is a simple process that involves the complex balancing of many reagents with the objective of obtaining satisfactory results (viable sperm that can fertilize the oocyte and result in a normal pregnancy). the process can be intuitive but knowledge of sperm physiology for each species is important for success. semen extenders are a complex mixture of reagents. the use of diluents in cryopreservation of sperm is necessary as they provide a source of energy, protect the cells from temperature related damage and maintain an environment where the sperm can survive. egg yolk and skim milk in semen extenders exert a protective effect against cold shock upon chilling or freezing of spermatozoa. egg yolk is one of the most commonly used components of cryoprotectants used in sperm cryopreservation. in general a cryoprotectant medium contains a penetrating cryoprotectant (glycerol, ethylene glycol or dimethyl sulfoxide), a non-penetrating cryoprotectant (egg yolk or milk), a buffer (tris, hepes), one or more sugars (glucose, lactose, fructose, saccharose or trehalose), salts (sodium citrate, citric acid), and antibiotics (penicillin and streptomycin). proteins present in the extender (from milk, egg yolk or other protein sources present in the synthetic extenders) help to stabilize the cell membranes and act as a non-penetrating osmotic factor protecting sperm from cold damage during freezing. the beneficial effect of egg yolk can be attributed to the presence of phospholipids, cholesterol and low density lipoproteins.15 salts and sugars are added to maintain an isotonic solution. sugars provide energy for the sperm cells and larger sugar molecules serve as osmotic regulators helping to dehydrate the sperm during the freezing process. buffers are added to maintain a physiological ph level and should work over a wide temperature range. antioxidants added or present in the egg yolk slow or prevent oxidation avoiding cell deterioration and death. other antioxidants are added in some commercial semen extender formulas. antibiotics are added to control bacterial growth from contamination that occurs during collection and through processing. the freezing process is quite simple, i.e. extended sperm is exposed to cold and then plunged into liquid nitrogen. however the process is more involved than this simple description. the concentration and morphology of the ejaculate is determined and the final dilution is made (e.g. 200 million/sperm/ml). addition of glycerol to the extender may be made in one step or in multiple steps (e.g. one step diluents and two step diluents). the diluted sperm sample is cooled to 5° c for at least 90 minutes and then frozen in pellets or straws. freezing in pellets is accomplished with the aid of a block of dry ice (solid co2 with the surface temperature of -79° c) in which cavities have been carved. these holes are filled with 0.1 to 0.3 ml of extended semen and left to freeze for 2 to 4 minutes. pellets are then transferred to labeled cryovials containing the expected volume necessary for an artificial insemination dose. an alternative to freezing semen in pellets is freezing it in straws. straws are easily labeled and can be easily and efficiently stored in semen tanks. straws (0.5 or 0.25 ml) are loaded with semen after dilution and equilibration at 5° c for a period of time (varies according to the diluent being used) and placed on a rack over liquid nitrogen (3-4 cm) for 7-8 minutes (goat semen) and then plunged into liquid nitrogen. the semen concentration is adjusted prior to freezing to contain one insemination dose per straw. programmable freezers can also be used to freeze semen in straws. they are convenient to freeze large numbers of straws and allow precise controlling of the freezing curve. these machines can be programmed with multiple freezing steps for better results. this makes comparison of results during freezing very difficult. some programmable embryo freezers may be used for this purpose. there are several “recipes” for semen extenders listed in the scientific literature. for the practitioner we recommend the use of commercially available semen extenders. these formulas are available for a reasonable cost and eliminate the need to mix and keep reagents. they also avoid the need of having scientific equipment to mix, adjust ph, and have a reduced risk of contamination among other challenges. most commercially available freezing solutions will require the addition of egg yolk and all handling should be done on a clean environment. some commercial semen extenders have shown to keep sperm at room temperature for extended periods of times (several days). refrigeration of extended semen at 5º c for several days is also possible. this allows semen to be collected on a farm and moved to the clinical theriogenology • volume 3 number 4 • december 2011509   clinic or laboratory for evaluation, extension and freezing. it also avoids the need to freeze semen increasing the number of breeding doses as it prevents sperm loss during the freezing and thawing process. some commonly used commercial extender names are: andromed ®, bioxcell ®, triladyl ®, biladyl ®, bovipro cryoguard ®. thawing quality of the frozen semen can be dramatically affected by the thawing method used. some of these effects are due to the interaction of the sperm with the components of the semen extender and others are entirely related to the thawing process. the use of epididymal vs. ejaculated sperm also affects sperm survival after thawing. thawed frozen sperm should be evaluated for motility and longevity post-thaw. the best evaluation of success remains the pregnancy or offspring rate. differences in post-thaw motility, viability and fertility may be attributed to the extender used, the cooling rate, the thawing method and the method of freezing (straw vs. pellets) among other variables. thawing sperm is determined by the method used to freeze the sperm. sperm pellets should be thawed in a dry test tube submerged in a 37º c waterbath for 15-30 seconds. several temperatures have been used to successfully thaw semen frozen in straws. straws were successfully thawed in 37º c water for 30 seconds to 2 minutes, or 40º c for 20 seconds or even the scalding temperature of 70º c for 7 seconds. attention to temperature and timing becomes much more important as temperatures above 37º c are used. to avoid problems with fertility, one should always follow the thawing recommendations directed by the freezing institution. semen should be thawed immediately before insemination. thawed semen should be used in its entirety once thawed. one should not try to refreeze the thawed semen as it is very likely to yield disappointing results. split semen straws, i.e. using one straw to inseminate several females, is not recommended. however it has been done successfully. the rate of success for split straws will vary and is directly related to the quality of the thawed semen, the concentration, the site of semen deposition and method of insemination. references 1. amoah ea, gelaye s: biotechnological advances in goat reproduction. j anim sci 1997;75:578-585. 2. chemineau p, baril g, leboeuf b, et al: implications of recent advances in reproductive physiology for reproductive management of goats. j reprod fertil suppl 1999;54:129-142. 3. martinez-pastor f, martinez f, alvarez m, et al: cryopreservation of iberian red deer (cervus elaphus hispanicus) spermatozoa obtained by electroejaculation. theriogenology 2009;71:628-638. 4. asher gw, berg dk, evans g: storage of semen and artificial insemination in deer. anim reprod sci 2000;62:195211. 5. blash s, melican d, gavin w: cryopreservation of epididymal sperm obtained at necropsy from goats. theriogenology 2000;54:899-905. 6. hishinuma m, suzuki k, sekine j: recovery and cryopreservation of sika deer (cervus nippon) spermatozoa from epididymides stored at 4 degrees c. theriogenology 2003;59:813-820. 7. ikeda h, kikuchi k, noguchi j, et al: effect of preincubation of cryopreserved porcine epididymal sperm. theriogenology 2002;57:1309-1318. 8. kundu cn, chakrabarty j, dutta p, et al: effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium. reproduction 2002;123:907-913. 9. santiago-moreno j, toledano-diaz a, pulido-pastor a, et al: effect of egg yolk concentration on cryopreserving spanish ibex (capra pyrenaica) epididymal spermatozoa. theriogenology 2006;66:1219-1226. 10. martinez af, martinez-pastor f, alvarez m, et al: sperm parameters on iberian red deer: electroejaculation and postmortem collection. theriogenology 2008;70:216-226. 11. gizejewski z: effect of season on characteristics of red deer (cervus elaphus l.) semen collected using modified artificial vagina. reprod biol 2004;4:51-66. 12. gizejewski z, snochowski m, mayntz m: fractions of the semen of red deer (cervus elaphus)--their occurrence and characteristics in different periods of season. pol j vet sci 2003;6:219-223. 13. roy a. egg yolk-coagulating enzyme in the semen and cowper's gland of the goat. nature 1957;179:318-319. 14. pellicer-rubio mt, magallon t, combarnous y: deterioration of goat sperm viability in milk extenders is due to a bulbourethral 60-kilodalton glycoprotein with triglyceride lipase activity. biol reprod 1997;57:1023-1031. 15. pace mm, graham ef: components in egg yolk which protect bovine spermatozoa during freezing. j anim sci 1974;39:1144-1149. clinical theriogenology • volume 3 number 4 • december 2011 510 2017 bovine reproductive ultrasound for practitioners bovine reproductive ultrasound for practitioners jill colloton bovine services, llc, edgar, wi abstract the use of ultrasound has become common for examination of the female bovine reproductive tract. early pregnancy and twin diagnosis, confirmation of embryonic and fetal viability, fetal gender determination, identification of fetal anomalies, and accurate assessment of ovarian structures and uterine health are benefits of ultrasound. keywords: ultrasound, pregnancy, embryonic and fetal loss, fetal anomaly, genital tubercle, corpus luteum, follicular cyst, uterine pathology the embryo and fetus under ideal conditions the bovine embryo can be visualized ultrasonographically as early as 21 days after insemination.1 under field conditions it is more practical and accurate to wait until at least 27 days in adult cows.2 heifers can be scanned for pregnancy with excellent accuracy a day or two earlier. the embryonic period lasts from one to 42 days of gestation. the risk of pregnancy loss is highest during this period because the placenta has not fully developed. signs of embryonic death at this point include lack of heartbeat, cloudy amniotic or chorioallantoic fluid, and separation of the chorioallantoic membrane. twins can be detected by presence of a shared chorioallantoic membrane (twin line), two embryos, and usually two corpora lutea. over 50% of pregnant cows with two corpora lutea will have twins,3 unless they have been resynchronized with gonadotropin releasing hormone (gnrh) prior to pregnancy examination. resynchronized cows will have an even higher percentage of multiple corpora lutea without a concurrent increase in twins. during the fetal period from 42 days to parturition the differentiation of organs progresses. at approximately 45 days testicular development begins. this is of particular interest in the case of twin pregnancies. if a male fetus dies after this time, but the female twin remains, the female may be a freemartin. by 50 days fetal movement can easily be detected to verify viability. at 55 days the external genitalia are positioned in their final locations, allowing gender determination until the fetus is beyond reach of the ultrasonographer, sometimes as late as 130 days. the optimal time for many ultrasonographers is 60 to 80 days, when nearly 100% accuracy can be achieved.4 at this time the genital tubercle is very visible and completely migrated to its final position. also, the fetus is still within reach and small enough to orient under the transducer. the genital tubercle will become the penis in the male and the clitoris in the female. hence, it is located behind the umbilicus and cranial to the thighs in the male and under the tail head and caudal to the thighs in the female. in both genders it usually appears as a hyperechoic bi-lobed structure, although it sometimes appears to have one or three lobes. teats and scrotum may also be visualized, but it is inadvisable to use these soft tissue structures alone for the diagnosis of fetal gender. specular reflection artifacts in the umbilicus can look similar to the male genital tubercle. embryonic and fetal anomalies abembryonic vesicle an amniotic vesicle is seen either without any visible embryo, or with the embryo outside the vessicles. such embryos rarely survive even if they have a heartbeat on the day of first examination. recheck examination usually reveals an empty amniotic sac or complete lack of uterine contents. schistosomus reflexus the fluid-filled fetal stomach can be visualized outside the body cavity. in most cases the spinal column is severely bent, sometimes into a v shape. 615 clinical theriogenology • volume 9 number 4 • december 2017 fetal ascites in a normal fetus the abdominal organs completely fill the cavity with no visible peritoneal fluid. anechoic areas around the internal organs indicate fetal ascites. in some cases there is also fluid around the heart or lungs. careful assessment should be made to differentiate fetal ascites from a dead fetus with fluid accumulation. fetal anasarca fluid is accumulated under the skin rather than in the peritoneal space. follow-up sometimes reveals that the fluid is no longer visibly present. in other cases there is no change or fetal death occurs. umbilical hernia an abnormally large umbilicus may indicate an umbilical hernia. if the defect is very large the umbilicus may contain tissues of various densities representing abdominal organs. amorphous globosus or acardiac twin these anomlies are differentiated from fetal monsters by lack of a heartbeat and the presence on a normal fetus. these anomalies usually do not grow large so are rarely a problem at parturition. they have no gender so are not of concern for freemartinism. conjoined twins conjoined twins are very rare and must be differentiated from normal twins lying close together. normal vs abnormal ovarian structures with a good field ultrasound unit the presence of a corpus luteum (cl) can be identified as early as two days after ovulation, and even earlier with a research quality machine. there is a wide variation in the ultrasonographic appearance of normal corpora lutea. metestrus cls are more heterogeneous and smaller than diestrus cls or those of pregnant cows. up to 80% of cls have a fluid cavity at some time in their development, usually early. some cavitary cls have an echogenic center, probably clotted blood remaining from ovulation. the differentiation of cavitary cls and cystic cls is perhaps a matter of academic semantics. this author prefers not to use the term cystic cl unless the structure is proven by repeated examinations to be persistent beyond the normal life of a cl follicular cysts have been defined as thin-walled fluid-filled follicular structures greater than 22mm in diameter. a follicular cyst is differentiated from a cavitary cl, luteal cyst, or luteinizing follicular cyst by the absence of a visible luteal rim. follicular cysts are benign in most cases, with luteal structures and follicular waves developing normally on the same or contralateral ovary. benign cysts can persist throughout several cycles and into pregnancy. thorough examination of both ovaries is essential to properly classify follicular cysts. if luteal tissue is detected the cyst is benign. if no luteal tissue is seen a second examination must be performed in about five to fourteen days to determine if a cl has developed. if not, the cyst is likely preventing normal cyclicity. ovulation scars are seen as small hyperechoic foci within the ovarian stroma. they tend to be more visible in older cows and embryo donors. these are normal and do not block the entire ultrasound beam as calcifications do. granulosa cell tumors are very heterogenic masses usually occupying the entire ovary. they are composed of highly vascularized dense tumor tissue. the acoustic mismatch between the dense neoplastic tissue and the fluid in the blood vessels creates specular reflections throughout the tumor. ovarian follicular dysplasia (ofd) has been described.5 this syndrome begins with bilateral development of clustered solid follicles that eventually progress to a granulosa theca cell tumor. early cases have the same ultrasonographic appearance as truly anovular or pre-pubertal ovaries. later in development the ovaries increase in size, showing increased hyperechogenicity, decreased fluid in the follicles, and hyperechogenic shadows due to mineralization of dysplastic follicles. 616clinical theriogenology • volume 9 number 4 • december 2017 uterine and vaginal pathology in most cases of metritis/endometritis echogenic purulent material can be seen in the uterine lumen. this fluid may be any shade of light gray to very bright white depending on its density. intraluminal fluid in cases of pyometra is usually voluminous and of medium echogenicity. cases of chronic subclinical metritis/endometritis usually have little intraluminal fluid of higher echogenicity. occasionally hyperechoic foci are seen within the endometrium itself, with no intraluminal fluid visible. these foci may be due to bacterial invasion of the tissue from prior pyometra. the ultrasonographer should be careful not to confuse small volumes of echogenic intraluminal fluid with specular reflections due to estrus. mucometra is an accumulation of sterile anechoic or slightly echoic fluid in the uterine lumen not associated with pregnancy. specular reflections may or may not be present. the endometrium is not thickened as it is in estrus. mucometra does not appear to be related to ovarian structures. it is critical to confirm the absence of an embryo or fetus in cases of mucometra. when in doubt the ultrasonographer should perform a second examination a few days later to confirm the diagnosis. mucometra in heifers may be related to the presence of a complete hymen or other congenital blockage of the tubal portions of the reproductive tract. the cause in adult cows is unknown. uterine lymphosarcoma is identified by very firm thickening of the uterine wall that may be localized or throughout the entire uterus. the affected uterine tissue is homogeneous and dense enough to cause partial shadow artifacts. uterine, cervical or vaginal abscesses can be distinguished from metritis, cervicitis or vaginitis by the presence of a capsule around the purulent material. uterine abscesses have a poor prognosis for future fertility. cervical and vaginal abscesses may or may not be of concern depending on size and location. oviducts are normally difficult to visualize with field ultrasound units. visible fluid accumulation occurs with salpingitis or blockage. the normal vagina presents as a thin white line representing the confluence of opposite sides of the vaginal wall. in cases of vaginitis echogenic material is visible in the vaginal vault. unless echogenic material is also present in the uterus prognosis for fertility is fair to good if a protective sheath is used on the insemination gun at breeding. vaginitis may be primary or caused by urine pooling. pneumovagina presents as a reverberation artifact. staging the reproductive cycle in proestrus the uterus becomes more heterogeneous as blood flow increases. anechoic mucous begins to accumulate in the lumen about three days before ovulation. one, or sometimes more, large (16-20mm) follicles will be present. the cl is usually still visible, but is becoming smaller and more heterogeneous. during estrus the uterus is most hetergenous. varying amounts of anechoic mucous are present in the uterine lumen. the large difference in acoustic impedance between the dense endometrium and the fluid mucous often creates specular reflection artifacts on the inner wall of the endometrium. the endometrium thickens to about 2.5 times its normal volume under the influence of estrogen.6 a mature follicle is present until ovulation at approximately 12 hours after the end of standing heat. sometimes a portion of the follicular fluid is not expelled during ovulation. in these cases it is difficult to determine if ovulation has occurred. a small, heterogeneous regressed cl may still be seen throughout proestrus and estrus. after ovulation metestrus begins with the development of a new wave of small follicles (less than 8mm). in the first day or two of the cycle these small follicles are all that is visible on the ovaries. by day two or three a small, heterogenic cl becomes visible, often with an anechoic fluid filled cavity. occasionally the cavity is filled with very hyperechoic material, probably clotted blood. during diestrus the uterus is homogeneous and a medium shade of gray. there should be little or no fluid in the lumen. the cl will also be homogeneous and a medium shade of gray. the cl may or may not have a fluid cavity. about 80% of cls have at a cavity some stage in development, usually early. the cavity may be filled with clear fluid, probably due to incomplete expulsion of follicular fluid 617 clinical theriogenology • volume 9 number 4 • december 2017 during ovulation. dense material in the cavity may be clotted blood remaining after ovulation. the fluid cavity tends to fill in over time, but may persist into pregnancy. progesterone production is dependent on the volume of luteal tissue: the size of a cavity is irrelevant. follicles will be present in diestrus ranging in size from 2mm to 20mm depending on the stage of follicular wave cycles. embryo transfer donors should be assessed for uterine health and ovarian function before beginning superovulation protocol. if the cow is examined during follicular recruitment it may be possible to predict the response to future superovulation. cows that recruit large numbers of follicles at the beginning of each follicular wave can be expected to produce more follicles after follicle stimulating hormone treatment. it is usually not necessary to examine the donor cow on the day of insemination unless very rare or expensive semen is to be used. in those cases it is helpful to know if enough ovulatory size (8mm or more) follicles are present to warrant insemination. care must be taken to scan gently over the ovaries without manipulation or pressure to avoid disrupting the bursa. on collection day ultrasound is much more useful than manual palpation for counting the number of cls. small cls, cavitary cls and cls imbedded deep in the ovary can be seen. some cavitary cls will have very thin walls and may be difficult to differentiate from unovulated follicles. when these fluid-filled structures are seen the cow should still be flushed. it is possible that more embryos than expected will be retrieved. recipients should be evaluated for uterine health and ovarian function prior to beginning estrus synchronization. this is more important for adult cows than for heifers due to increased risk of uterine or ovarian pathology in parous animals. it is critical that ultrasound is used to identify cls before embryo transfer. because ultrasound is much more accurate than manual palpation for identifying small or fluid filled cls more animals will be used. almost 100% of cls in recipients are diagnosed with ultrasound, but only 80% with manual palpation. synchronization protocols with ultrasound earlier pregnancy diagnosis with ultrasound plus higher service rate with synchronization can dramatically increase pregnancy rate. in addition, accurate assessment of ovarian structures improves success of synchronization and reduces unnecessary use of progesterone supplementation. it has been shown that the presence of a cl and the availability of an ovulatory size (8mm or greater) follicle at g1 improve conception rates for presynch/ovsynch.8 presence of a functional (producing blood progesterone levels of at least 1ng/ml) cl is more important and easier to evaluate than presence of a potentially ovulatory follicle. the ultrasonographer should keep in mind that cls may be visible in proestrus and metestrus, but are not producing appreciable amounts of progesterone at those times. careful evaluation of follicular structures and the uterus as described in the section on staging the cycle can assist in determining which animals are at an appropriate stage to begin a protocol. ultrasound is also helpful for troubleshooting presynch failures. when cattle are presynchronized with prostaglandin (presynch) or double ovsynch before beginning ovsynch all normal, cycling animals should have a cl when the first gnrh injection (g1) is scheduled. an examination of ovaries at this time will reveal if presynchronization was successful. if many cows do not have cls at this time the protocol has failed. poor compliance, poor cow health, low body condition scores, nutrition problems, etc., are possible reasons for failure. if pregnancy examination is done at the appropriate time ovarian structures in open cows can help troubleshoot ovsynch failures. normal, cycling open cows bred 28-35 days before examination should have a good cl. if not, there is a problem with management, cow health, compliance, cystic follicles, or embryonic loss. if pregnancy examinations are done prior to 28 days it is more difficult to evaluate the absence of a cl in open cows. they may be cycling normally and are in early metestrus, or they may be anovular. 618clinical theriogenology • volume 9 number 4 • december 2017 resynchronization prior to pregnancy examination has become a popular way to expedite reinsemination of open cows. the most common method is to give gnrh approximately seven days prior to pregnancy examination. open cows then should be ready for the prostaglandin injection of the protocol, saving seven days. this type of resynchronization at 19 days has been shown to be less effective than at 26 or 32 days,8 probably because the cl does not have time to mature adequately to become prostaglandin responsive. adding a second dose of prostaglandin approximately 24 hours after the first may improve regression of the cl in these animals.9 artifacts beam width artifact when one ultrasound beam passes through two tissues of different densities at the same depth a beam width artifact is produced. two echoes reach the transducer at the same time, but only one is read. these artifacts are most common on the lateral walls of fluid filled structures and cause the sides of the structure to appear more echogenic than the contents. crystal defects crystal defects are seen as black vertical lines extending vertically from the transducer face. they can be differentiated from shadow artifacts or reflection/refraction artifacts because they always stay in the same location on the monitor and always begin at the face of the transducer. they may be caused by broken crystals in the transducer or by broken wires connecting the crystals to the power source in the unit. enhancement artifacts enhancement artifacts are a function of machine settings. because ultrasound beams are attenuated over time and distance it is necessary to increase the gain (volume) at deeper levels so that similar tissues at various depths from the transducer appear the same. however, when less echogenic material such as fluid is near the face of the transducer the beam is not attenuated and structures below the fluid appear brighter than they should. mirror image artifacts mirror image artifacts are a type of reverberation artifact. they are most common when the pelvic bone lies below the structure being examined. understanding mirror image artifacts helps avoid mistaking one structure for two. reverberation artifacts repeated echoing of ultrasound waves between two strong interfaces creates reverberation artifacts. one interface is usually the rectal wall/transducer. the second interface is usually gas or bone. each time the wave is echoed it is reduced by attenuation. hence, each reverberated echo is weaker than the previous one. also, the time delay between echoes results in each later echo being seen farther down on the ultrasound screen. the resulting image on the screen consists of equidistant, increasingly smaller, parallel artifacts. shadowing material dense enough to block or attenuate the ultrasound beam causes shadowing. most obvious are shadows caused by bone. less dramatic shadows can also be caused by manure between the transducer and rectal wall leading to a poor quality dark image. reflection and refraction shadows fluid reflects and refracts ultrasound waves just as the surface of a lake reflects and refracts light waves. when the ultrasound beam is either refracted at an angle into the fluid or reflected away from the fluid it is not echoed back to the transducer. hence, no image will be seen below the point of 619 clinical theriogenology • volume 9 number 4 • december 2017 reflection/refraction. these reflection and refraction shadows are commonly seen at the lateral edges of fluid filled structures. specular reflection specular reflection artifacts occur when two interfaces of very different densities are aligned horizontally to each other and perpendicular to the ultrasound beam. they are most often seen when a fluid filled structure is adjacent to denser tissue. they appear as thin bright horizontal lines of varying lengths. this artifact is very common in estrus. references 1. curran s, pierson r, ginther o: ultrasonographic appearance of the bovine conceptus from days 10 through 20. j am vet med assoc 1986;189:1289-1302. 2. romano j: early pregnancy diagnosis by transrectal ultrasonography in dairy cattle. theriogenology 2006;66:10341041. 3. lópez-gatius f, garcía-ispierto i, hunter rhf: factors affecting spontaneous reduction of corpora lutea and twin embryos during the late embryonic/early fetal period in multiple-ovulating dairy cows. theriogenology 2010;73:293299. 4. curran s, kastelic j, ginther o: determining the sex of the bovine fetus by ultrasonic assessment of the relative location of the genital tubercle. anim reprod sci 1989;19:217-227. 5. roberts j, steel s, gard j, et al: morphological characteristics of ovarian follicular dysplasia (ofd) observed by ultrasound in four florida beef herds. clin therio 2016;8:325. 6. souza a, silva e, cunha a, et al: ultrasonographic evaluation of endometrial thickness near timed ai as a predictor of fertility in high-producing dairy cows. theriogenology 2011;75:722-733. 7. bicalho rc, galvao kn, guard cl, et al: optimizing the accuracy of detecting a functional corpus luteum in dairy cows. theriogenology 2008;70:199-207. 8. fricke pm, caraviello dz, weigel ka, et al: fertility of dairy cows after resynchronization of ovulation at three intervals following first timed insemination. j dairy sci 2003;86:3941-3950. 9. wiltbank cm, baez gm, cochrane f, et al: effect of a second treatment with prostaglandin f2� during the ovsynch protocol on luteolysis and pregnancy in dairy cows. j dairy sci 2015;98:8644-8654.� 620clinical theriogenology • volume 9 number 4 • december 2017 2013: fetal loss in goats fetal loss in goats d.g. pugh college of veterinary medicine, auburn university, auburn, al loss of the embryo, fetus, or conceptus during gestation is referred to as fetal loss or wastage or abortion (note: these terms will be used interchangeably during this discussion). loss rates of five percent are commonly encountered in goat flocks. infectious agents, stress, some drugs, malnutrition, and toxic materials (plants, chemicals, mycotoxins, etc) may all be causes of this condition. keywords: non-infectious abortion, infectious abortion, zoonotic diseases non-infectious fetal wastage stress (malnutrition, predator attack, weather changes, etc) can be a common cause of pregnancy loss in the doe due to her dependency on progesterone from the corpus luteum. angora goats may be more prone to stress related abortions than other breeds. periods of excessive heat can also cause pregnancy wastage, particularly during the early embryonic period. malnutrition (energy/protein/vitamin/mineral deficiencies, excesses, or inappropriate nutrient ratios) can in some instances result in fetal wastage. maintaining an optimal body condition score for the doe(s) while insuring adequate dietary intake will help maximize normal pregnancy, fetal development, and parturition. some drugs have been associated with fetal losses (phenothiazine, levamisole, xylazine, acepromazine, corticosteriods, estrogenic compounds, prostaglandins, etc.) and thus should be either avoided or used in a very judicious fashion during gestation. plants that accumulate nitrate (jimsonweed, johnson grass, lamb’s quarter, oat hay, pigweed, sorghum, sweet clover, sunflower, and many others) and can cause nitrate-nitrite toxicosis resulting in fetal losses. some common causes of infectious fetal loss chlamydiosis (chlamydophila abortus) chlamydophila abortus (a gram-negative intra-cellular organism) is one of the most common causes of infectious fetal loss in goats in north america. when introduced into naive goat flocks, fetal loss rates as high as 25% to 60% may be encountered. chlamydial organisms can also cause epididymitis, keratoconjunctivitis, pneumonia, and polyarthritis. most commonly the transmission of c. abortus is via oro-nasal contact of aborted tissues, vaginal discharges or contaminated neonates. aborting does shed the organism in the uterine discharge, fetus, and placenta, particularly during the first three weeks after abortion. however, some birds (pigeons, sparrows) serve as reservoir hosts, while ticks and other arthropods may be vectors for disease transmission. signs in the infected doe may include: late term abortions (100d to near term), anorexia, fever, bloody vaginal discharge, delivery of a fresh, autolyzed fetus, and/or weak kids, and possibly retained fetal membranes. the placenta should be examined for thickened, white, gray, yellow, or red cotyledons. cytologic or histologic evaluation of cotyledonary impression smears (gimenez or modified ziehl-neelsen stain) for elementary bodies, or necrotizing vasculitis of placental vessels will aid in the diagnosis. aborted kids may have white spots on the liver, grossly. a definitive diagnosis can be made by culturing the organism or with polymerase chain reaction (pcr) from fresh placenta, stomach content or fetal tissue. serological testing may also be of value, particularly if using paired serum samples of aborting does (two to three weeks apart), or by finding antibodies in blood/serum of the aborted kid. the prophylactic use of tetracycline in all pregnant does may be of value in controlling, preventing, or arresting the continued spread of abortions in a herd. sheep vaccine may be used (extra label) in goats, but the efficacy is not completely known. quarantine of aborting does and proper disposal of aborted fetuses and tissues is critical in order to minimize the spread. 489 clinical theriogenology • volume 5, number 4 • december 2013 toxoplasmosis toxoplasma gondii can cause abortion, fetal mummification, stillbirth, and the birth of weak lambs and kids. domestic cats develop a transplacental infection after ingesting infected rodents or birds. kittens that become infected in utero, can shed t. gondii oocytes in feces. infected kittens/young/immunosuppressed cats may bury feces in hay and feed bins. does become infected when ingesting oocyst contaminated feedstuffs. toxoplasma can invade and multiply in the doe’s placenta then infect the fetus, causing necrosis of the placenta, particularly the cotyledons, with resultant abortion, stillbirth, or the birth of weak kids. does infected prior to breeding usually do not abort, while those infected between one and three months of gestation undergo fetal wastage or mummification. does which are infected from three to five months of gestation abort. most nonimmunosuppressed does show no overt signs (other than occasional increased rectal temperature) at the time of abortion. does with concurrent immunosuppression can develop neurologic disease. on gross examination of the placenta, the cotyledons are grey-white to yellow, with focal areas of calcification and necrosis. t gondii antibodies in aborted fetal fluids or presuckling blood indicates transplacental infection, and can confirm the diagnosis. the absence of t. gondii antibodies by one week after abortion can usually rule out toxoplasma as the cause. high antibody titers in a doe is not diagnostic of recent infection, but the absence of antibodies can usually rule out toxoplasmosis. a positive diagnosis of toxoplasmosis requires isolation/culture of the t gondii from the placenta or fetal tissues. all samples for culture should be transported the laboratory on ice. toxoplasmosis is best controlled by preventing pregnant doe exposure to infective oocytes. management protocols useful for prevention should include: (1) fetal membranes and aborted materials not used in diagnostics should be incinerated; (2) kittens and pregnant queens should be kept from pasture and feedstuffs used for pregnant does; (3) spayed queens kept in barns may prevent feral pregnant queens from nesting; (4) keep feedstuffs in areas or containers that minimize cat defecation in the feed, feeders, and/or feed handling equipment. the inclusion decoquinate, monensin, and lasalocid may be useful in toxoplasmosis control, but the clinician should always be mindful to avoid the use of pharmaceuticals in an extra-label fashion. the clinician should also be mindful of the zoonotic potential for toxoplasmosis. people should only consume pasteurized goat milk. q (query or queensland) fever q fever is caused by coxiella burnetii (an intracellular rickettsial microorganism), and can be a cause abortion in goats throughout much of the world. c. burnetti can be carried and shed in the placenta, uterine fluids, colostrum, and milk of many ruminant animals. these infected animals can serve as sources for infection, as can grazing contaminated pastures, and vector tick bites. pregnant does that become infected will occasionally develop placentitis, anorexia and depression, and have late term abortions and stillbirths. does that have aborted develop immunity. isolation of c. burnetii from the placenta or aborted tissues can confirm a diagnosis of q fever. identification of the organism via ziehl-neelsen staining of cotyledonary or fetal abomasal tissue is also diagnostic. fluorescent antibody testing of frozen placenta can also be rewarding. antibody titers >1:20 suggest c. burnetii exposure, but are not confirmative of the cause of fetal wastage; however a four fold increase in the aborted doe’s acute and convalescent serum titers indicates a recent infection. the infected doe can carry and shed c. burnetii indefinitely. as with other causes of abortion, all placentas and aborted tissues not used for diagnostic purposes should be incinerated. the use of some antibiotics (chlortetracycline) in susceptible does may reduce the incidence of abortions. cats, cattle, and sheep can all serve as a source of infection, thus their monitoring and control may be of benefit in prevention. because of the zoonotic potential q fever, all goat milk intended for human consumption should be pasteurized, and infected does identified and culled. listeriosis 490clinical theriogenology • volume 5, number 4 • december 2013 listeria monocytogenes (gram-positive non–acid fast facultative microaerophilic organism) can cause meningoencephalitis, abortion, and septicemia in goats. the causal organism can be found in feces, plant material, silage, soil and water. ingestion by the doe of contaminated feedstuffs in early pregnancy may result in abortion and stillbirth or weak neonates when ingested later in gestation. both abortion and neurologic conditions may occur simultaneously in goat herds. fetal wastage will usually occur in late gestation. the abortion may be associated with fever, inappetence, septicemia, and the expulsion of an autolyzed fetus. a confirmative diagnosis can be made by culturing l monocytogenes from the placenta, fetal tissues, or uterine discharge. the disease may be minimized or prevented by: (1) avoiding grazing by susceptible animals on contaminated pastures or feeding spoiled silage; (2) inclusion of chlortetracycline in feed supplements; (3) administration of long-acting oxytetracycline preparations during abortions storms; (4) use of a vaccine where available. as listeriosis may be zoonotic, care should be taken to minimize transmission to people by exposure to aborted tissues. leptospirosis goats are more resistant to leptospira interrogans infections and fetal wastage than are other domestic species, but are occasionally infected if exposed to infected/contaminated urine. of the seven or eight serovars commonly encountered in most domestic animals, only icterohaemorrhagiae, grippotyphosa, and pomona have been associated with fetal wastage in goats. infected does may present with anemia, inappetence, fever, hemoglobinuria, icterus, neurologic disease, late term abortion, and death. the diagnosis can be confirmed by isolation of the causal organism (which is uncommon), and by using darkfield microscopy, immunofluorescence testing, and silver stains on the placenta and fetal tissues. paired sera from aborted does showing a four fold increase in serum titer in aborted and convalescent does is very suggestive of leptospirosis. vaccination in endemic areas may have some value, however the clinician should avoid the extralabel use of vaccines and pharmaceuticals. avoiding contaminated urine (rodent control, clean water, etc.) and the addition of chlortetracycline to feed supplements may all be useful in prevention. leptospirosis is potentially a zoonotic disease, thus care should be exercised when handling materials suspected of contamination. mycoplasmosis mycoplasma organisms can cause arthritis, keratoconjunctivitis, mastitis, and vulvovaginitis and fetal wastage. fetal wastage occurs in the final trimester of gestation, and aborting does will shed the organism in amniotic fluid, milk and placenta. confirmative diagnosis can be made by culture and serotyping of the organism from placenta or fetal tissues. some antibiotics (tetracyclines and tylosin) may be of benefit to prevent the continued spread if used during abortion outbreaks. salmonellosis salmonella abortus-ovis infection in goats can produce septicemia, metritis and fetal wastage. stressful conditions (overcrowding, malnutrition, parasitism, antibiotic use, etc.) may predispose or exacerbate the incidence of this condition. birds and other wildlife and domestic ruminants may serve as carriers and sources of contamination. in herd outbreaks up to 70% of the does may have fetal wastage, with retained fetal membranes, metritis and signs of septicemia; all occurring in the final month of gestation. a confirmative diagnosis can be obtained by culturing salmonella from aborted tissues. the use of antimicrobials, based on culture and sensitivity patterns, and/or autogenous vaccines may be of some benefit in both prevention and control. salmonella organisms which are associated with abortions should be considered to be zoonotic. campylobacterosis (vibrosis) campylobacter fetus and/or c jejuni (gram-negative microaerophilic rods) are rarely documented in cases of fetal loss of goats in north america. infection may occur when pregnant 491 clinical theriogenology • volume 5, number 4 • december 2013 does ingest water or feedstuffs contaminated by the organism; usually after it has been shed from the gastrointestinal tract of sheep, dogs and some birds. fetal loss is characterized by stillbirths, weak kids, late gestational expulsion of fresh fetuses and maternal diarrhea. findings of placental edema with necrotic and/or swollen cotyledons, fetal subcutaneous edema, pleuritis, hepatic disease (gray targets on liver surface) and peritonitis would be indicative of campylobacter. isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharge will provide a definitive diagnosis. antibiotic therapy (penicillin, tetracycline, streptomycin) during late gestation, the early kidding season, or during an outbreak may decrease the incidence of fetal loss. a vaccine is available for sheep, and may be of use (in an extra-label fashion) for goats. on endemic premises an autogenous bacterin may be of value. c. jejuni is associated with mild gastroenteritis in people. selected references mobini s, heath am, pugh dg: theriogenology. in: pugh dg, editor. sheep and goat medicine. maryland heights (mo): saunders; 2002. p 129-186. mobini sc, wolfe d, pugh dg: flock health. in: pugh dg, editor. sheep and goat medicine. maryland heights (mo): saunders; 2002. p. 421-434. edmondson m, roberts j, baird am, et al: theriogenology of sheep and goats. in: pugh dg, baird an, editors. sheep and goat medicine, 2nd ed. maryland heights (mo): elsevier saunders; 2012. p.150-230. scharko p, johnson j, mobini s, et al: flock amd herd health. in: pugh dg, baird an, editors. sheep and goat medicine, 2nd ed. maryland heights (mo): elsevier saunders; 2012. p. 539-556. 492clinical theriogenology • volume 5, number 4 • december 2013 1 contact eduardo prado eprado@utk.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10269, http://dx.doi.org/10.58292/ct.v16.10269 case report vaginal lipoma with cervicovaginal and rectal prolapse in a cow eduardo prado, tulio prado, elizabeth whitt, alison bradley, pierre-yves mulon department of large animal clinical sciences, college of veterinary medicine, university of tennessee, knoxville, tn, usa abstract a 5-year, mixed breed cow, was presented with cervicovaginal and rectal prolapses. cow had a vaginal mass on the left caudal aspect of cervicovaginal prolapse. rectal prolapse was resolved; however, vaginal mass prevented reduction of vaginal prolapse. next day, vaginal mass was resected that made vaginal prolapse reduction and vulval closure (bühner stich) possible. histopathology identified the mass as infected vaginal lipoma. keywords: cow, prolapse, lipoma, vagina background lipoma is a benign tumor that occurs as a result of fat accumulation in various parts of body affecting normal function of affected organs.1 however, in cows, vaginal lipomas have not been reported as a common pathology. vaginal lipoma, reported in a cow, was suggested as a possible cause of dystocia.2 vaginal eversion (commonly known as vaginal prolapse) is associated with several factors (breed, estrous cycle stage, diet, perivaginal fat accumulation, and others). vaginal or cervicovaginal prolapse is more common in pregnant cows, months or weeks before parturition.3 vaginal prolapses are classified into 4 grades, depending on the severity, chronicity, and inclusion of cervix.3 as a consequence of chronic straining rectal prolapse ensues. rectal prolapse is classified into 4 grades, depending on the anatomical/structural involvement.4 in the present case, pedunculated tumor in the vagina resulted in vaginal and rectal prolapses and prevented reduction of vaginal prolapse. case presentation a 5-year, mixed-breed cow, was presented with cervicovaginal and rectal prolapse. cow was bright, alert, and responsive; vital parameters (prolapse prevented obtaining rectal temperature) were within normal limits. cow had a body condition score of 8 out of 9 and weighed 400 kg. a month before, owner noticed a mass in the caudal vaginal aspect. cow had 2 calves and the last calving was 6 months ago. as per owner, cow was not bred after last calving, was not current on vaccines, and was dewormed (pour on dewormer) 7 months ago. cow was with 4 other cows and a bull was in pasture. cow was restrained in a chute, both prolapses (figure 1) were washed with water and dilute iodine solution. rectal prolapse (grade ii) was first resolved. cervicovaginal prolapse (grade iii, protrusion of vagina and cervix3) was properly visualized to identify the vaginal mass. rectal and reproductive tract tissue were mildly affected but still viable. cow was able to urinate and defecate. treatment high epidural (between last sacral and first coccygeal vertebrae) was performed with 5 ml of lidocaine before resolving (via manual reduction) rectal prolapse. once rectum was returned to its normal position, using umbilical tape, pursestring suture (figure 2) was placed on the skin around anal opening. despite several attempts, resolving cervicovaginal prolapse was unsuccessful because of vaginal mass. cow received antibiotics (6.6 mg/kg crystalline ceftiofur) subcutaneously (base of ear; based on proven efficacy for reproductive tract via this route5). intravenous flunixin meglumine (1.1 mg/kg) was given. next day, a 12 x 8 cm mass was removed surgically. cow was held in headlock, regional anesthesia was applied, and sedation was performed via caudal tail vein with 1 ml (10 mg/ml) acepromazine. cow also received 7 ml of mepivacaine mailto:eprado@utk.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10269 2 citation line: clinical theriogenology 2024, 16, 10269, http://dx.doi.org/10.58292/ct.v16.10269 hydrochloride via high epidural. surgical drape was placed over hindquarters and a window was created in the drape to expose the cervicovaginal prolapse. prolapsed mass was thoroughly cleaned with iodine scrub. foley urinary catheter was placed and the balloon was inflated. line block was performed with lidocaine around the mass and in surrounding healthy tissue. mass was dissected using electrocautery and removed by blunt dissection with doyen scissors. remaining abnormal tissue was removed with a scalpel. most likely differentials for this mass were various neoplasia, lipoma, or polyp. during surgery, bleeding was controlled by simple circumferential ligatures using 2-0 tapered absorbable monocryl and electrocautery. after vaginal mass removal, area was flushed with sterile water. margins of the incision was closed (interrupted horizontal mattress pattern followed by several cruciate and simple interrupted patterns with 2-0 tapered absorbable monocryl). because of immediate cervicovaginal prolapse reduction was not possible, a compressive bandage of vetwrap and elastikon was placed overnight around the cervicovaginal prolapse. cow received another dose of intravenous flunixin meglumine (1.1 mg/kg). next day, substantial reduction in inflammation of the prolapse was noticeable. manual reduction was performed under low epidural block (5 ml of mepivacaine hydrochloride). bühner stich (figure 3) was applied with umbilical tape to prevent recurrence of prolapse. outcome next day, cow was discharged with oral meloxicam (1 mg/ kg, once a day for 1 week). histopathological diagnosis of the mass was vaginal lipoma with secondary infection. owner was reached after 5 days; owner mentioned that the cow was grazing in the field with others in the herd. it was recommended to watch for any vulvar discharges, behavior change, inappetence, lethargy, straining, or any sign of discomfort with the possibility of suture removal in 2 weeks. discussion vaginal lipomas in cattle have been reported once in a 5-year, friesian-holstein cow.2 although tumors of cows’ genital organs have low prevalence, it does occur and most common vaginal tumor being fibropapilloma.6,7 other tumors diagnosed were leiomyomas, leiomyosarcomas, fibromas, fibrosarcomas, fibroleiomyosarcomas, and squamous cell carcinomas.6 early diagnosis and treatment provide better prognosis. surgical resection is recommended for vaginal tumors.7 even though a vaginal lipoma may or may not interfere directly with fertility, it could be a cause of dystocia or vaginal/cervicovaginal and rectal prolapses. learning points • complete examination of the reproductive tract should be performed in prolapse cases • vaginal tumors require surgical resection and histopathology analysis is necessary for final diagnosis figure 1. rectal and cervicovaginal prolapses. figure 2. resolved rectal prolapse. http://dx.doi.org/10.58292/ct.v16.10269 citation line: clinical theriogenology 2024, 16, 10269, http://dx.doi.org/10.58292/ct.v16.10269 3 • depending on the size and location of vaginal lipoma(s), resulting abnormal pressure to reproductive tract and constant straining might result in vaginal and rectal prolapses figure 3. resolved cervicovaginal prolapse. conflict of interest none to report. references 1. hendrick mj: mesenchymal tumor of the skin and soft tissues. in: meuten dj: editor. tumors in domestic animals. hoboken; john wiley & sons: 2020. p. 158-159. proquest ebook central. available from: https://ebookcentral.proquest.com/lib/utk/detail.action?docid=4737344 [cited 30 april 2024]. 2. el-maghraby hm: a retrospective study on some surgical affections of the perineum and vagina in farm animals. zag vet j 2002;30:84-100. 3. peter at, king eh: management of vaginal, cervico-vaginal, and uterine prolapse. in: hopper rm: editor. bovine reproduction, 2nd edition, hoboken; john wiley & sons: 2021. p. 563-566. companion website. available from: www.wiley.com/go/hopper/ bovine [cited 30 april 2024]. 4. anderson de: rectal prolapse. in: food animal practice, 5th edition, amsterdam; elsevier: 2009. p. 125-128. 5. risco ca, youngquist rs, shore md: postpartum uterine infections. in: youngquist rs, threlfall wr: editors. current therapy in large animal theriogenology, 2nd edition, amsterdam; elsevier: 2007. p. 341-342. 6. martz p, oezcan-martz a, bittner l, et al: case reports of genital tract tumours in cows. vet med-czech 2020;65:401-408. doi: 10.17221/124/2020-vetmed 7. musal b, ulutas p, aydogan a: vaginal fibrosarcoma in a cow. ir vet j 2007;60:424-425. doi: 10.1186/2046-0481-60-7-424. pmid: 21851697; pmcid: pmc3113825. http://dx.doi.org/10.58292/ct.v16.10269 https://ebookcentral.proquest.com/lib/utk/detail.action?docid=4737344 https://ebookcentral.proquest.com/lib/utk/detail.action?docid=4737344 http://www.wiley.com/go/hopper/bovine http://www.wiley.com/go/hopper/bovine https://doi.org/10.17221/124/2020-vetmed 2015: diagnosis of canine and feline neonatal death: a retrospective study of 107 cases (2000-2010) diagnosis of canine and feline neonatal death: a retrospective study of 107 cases (2000-2010) chaley n. young,a gary haldorson,b mushtaq a. memona adepartment of veterinary clinical sciences and bdepartment of veterinary microbiology and pathology, college of veterinary medicine, washington state university, pullman, wa abstract the objective of the study was to characterize common causes of canine neonate death by evaluating diagnostic records and thus provide information to practitioners regarding potential preventative options. a total of 107 canine and feline neonates ranging from 0 to 7 days of age submitted to washington state animal disease diagnostic laboratory (waddl) during the years 2000-2010 were evaluated. gross examination of all tissues and body cavities; as well as collection of representative samples for bacteriology, virology and histopathology was performed. a diagnosis was made 72% of the time when a dead neonate was submitted to waddl. an infectious cause was responsible in 41% of submissions, and 37% had a non-infectious cause of death. the most common cause of death of the cases submitted to waddl over the ten year period was septicemia accounting for 15% of the diagnoses. this was closely followed by aspiration pneumonia, which accounted for 14% of the diagnoses. the information gained from the study gives insight on the most common causes of neonatal deaths and facilitates the understanding of how to prevent neonatal deaths in small animal populations. keywords: neonate, septicemia, aspiration pneumonia, diagnosis introduction puppies and kittens at birth are blind, poikilothermic, and have limited mobility. due to their immature status, neonates have complete reliance on their mother for life outside the womb and are highly susceptible to the environment. a recent comprehensive retrospective study1 of 10,810 litters of 224 breeds registered in the norwegian kennel club in 2006 and 2007 shows perinatal mortality in 24.6% of the litters. eight percent of the puppies died before eight days after birth, with 4.3% as stillbirth and 3.7% as early neonatal mortality. prematurity of the neonate is a significant cause of death in the young. there are many vital anatomical and physiological changes that take place during late gestation and after birth of a puppy or kitten. the first respiration is stimulated by hypoxia, hypercapnia, and cooling of the skin immediately after delivery and it must be taken within the first eight to ten minutes of life to prevent permanent brain damage or death.2 the secretion of surfactant in the lungs is the last thing to develop in the gestation of the neonate and with prematurity, surfactant production is deficient and pulmonary function is hindered. low birth weight is often accompanied by immature physiologic processes and inadequate production and release of lung surfactant and has been correlated with increased neonatal deaths.3 prematurity also results in an overall weak puppy or kitten and predisposes them to aspiration pneumonia, which leads to death in most cases.1 neonates are unable to regulate their own body temperature so it is important that they be in a warm environment. the normal body temperature of a neonate is above 36.1°c. hypothermia is reported to cause bradycardia, cardiovascular failure, neuronal injury, and ileus.2 when the body temperature drops below 35°c, the neonate has trouble with oxygenation, which leads to hypoxia and stasis of the gastrointestinal tract. this stasis further exacerbates the risk for aspiration pneumonia, which often results in death of the neonate. there are several infectious agents that are known risks to the lives of neonatal puppies and kittens. these include feline panleukopenia virus, canine parvovirus, canine and feline herpes virus, and a number of bacterial organisms. fading puppy and fading kitten syndrome was not considered a cause of death in this study. fading puppy or kitten syndrome is a name given to describe a combination of several clinical signs, which can lead to neonatal deaths but is not specific for one disease entity.4 in this study, necropsy records of canine and feline neonates age 0 to 7 days submitted to waddl over a ten year period between 2000 and 2010 were reviewed. the information gained from the postmortem examination of these neonates was analyzed to establish the most common causes of neonatal clinical theriogenology • volume 7, number 1 • march 201553 deaths in order to better understand how to prevent and control the rate of neonatal deaths in small animal populations. materials and methods whole carcasses of the deceased neonates were submitted to waddl for necropsy from pet owners and private veterinary practitioners. upon receipt of the carcasses at waddl, a thorough necropsy was performed following typical postmortem examination protocol.5 gross examination was performed on all tissues and body cavities including the brain and representative samples of tissues were collected for histopathology. the gastrointestinal tract was evaluated in its entirety for amount and character of ingesta. at every necropsy, sterile samples of lung, liver, kidney, and spleen were collected for microbiologic testing. if abnormalities were found in other tissues on gross or histopathologic examination, those tissues were also collected for microbiologic testing. diagnostic laboratory records on all canine and feline neonates between the ages of zero and seven days submitted to waddl from the years 2000 to 2010 were evaluated. a total of 107 cases were collected. the information was analyzed and put into a spreadsheet for organization and analysis. information was categorized regarding the answers to the following questions: was an infectious agent responsible for the death? could a cause of death be determined? from those not determined, was a complete analysis completed including bacteriology and virology? from this information a table was formulated of the most common diagnoses as well as the most common reasons for failure to obtain a diagnosis. results from the data collected, there is a 72% (77/107) chance of obtaining a definitive or strongly suspected diagnosis regarding cause of death when a neonatal cadaver is submitted to waddl for diagnosis. the most common cause of death in this study was septicemia and accounted for 15.8% (17/107) of the deaths. of the neonates who died of septicemia, six originated from omphalitis and one from esophageal perforation. the source of septicemia was not identified in the remaining cases. aspiration pneumonia accounted for 15% (16/107) of deaths in this study and was the second most common cause. six neonates in this study (5% or 6/107) died of congenital abnormalities including ventricular septal defect, cleft palate, spina bifida, other congenital heart defects and atresia jejuni (table). the other known causes of death were less frequent and a complete list can be found in the table. in 30/107 cases (28%), a definitive cause of death was not established. some main reasons for not obtaining a diagnosis include the absence of an identifiable lesion, poor tissue quality, and the need for further workup. possible factors which were not confirmed, but that may have contributed to the death of the neonates include hypoglycemia, hypothermia, environmental causes, metabolic derangement, in-utero stress, premature or complications during cesarean section anesthesia, suffocation, freezing/thawing tissue artifact and congenital cardiac arrhythmias. other causes related to poor tissue samples include autolysis, excessive postmortem bacterial growth and method of euthanasia. in some cases, bacterial and/or viral cultures were not performed based upon client interest, and thus the presence or absence of organisms that could have been responsible for the death of the neonate was not confirmed. discussion in this study, an infectious cause of death was considered anything that had a bacterial or viral component that was directly responsible for the neonate death. aspiration pneumonia was considered an infectious cause because, in most cases, the aspiration of materials which contain bacteria were inhaled into the lungs and caused an infection within the lungs and sometimes septicemia leading to the death of the neonate. fading puppy and fading kitten syndrome was not considered a cause of death in this study. fading puppy or kitten syndrome is a name given to describe a combination of several clinical signs which can lead to neonatal deaths but is not specific for a single disease entity.4 hypothermia, clinical theriogenology • volume 7, number 1 • march 2015 54 hypoglycemia, anorexia, dehydration, etc. are all signs which would be included in fading puppy/kitten syndrome but could be caused by environmental, genetic, or infectious causes. there is no definitive mechanism described by the use of the term fading puppy/kitten syndrome and therefore it was not used as a cause of death in this study. in this study, septicemia was found to be the number one identified cause of death among neonates. there are several routes of infection in the neonate which lead to sepsis. the most common causes of neonatal sepsis are tail docking, umbilical cord infections, respiratory infections, gastrointestinal infections, urinary tract infections, and skin infections.4,6 in this study, 35% (6/17) of the deaths caused by sepsis were due to infection originating from the umbilicus. there was also one puppy that died of septicemia in this study from an esophageal perforation causing pleuritis, most likely secondary to complications with tube feeding. in one study it was found that e. coli, k. pneumoniae, and b-hemolytic streptoccous sp. caused septicemia in suckling puppies from mothers with mastitis caused by the same bacteria.7 the most common bacteria isolated from septic neonates are staphylococcus, streptococcus, e. coli, klebsiella, enterobacter, clostridium, and salmonella.4 predisposing conditions that lead to sepsis in neonates include inadequate colostrum, hypothermia and hypoglycemia, poor nutrition, viral infections, endoparasitism, or a dam with metritis or mastitis.4 sepsis in the neonate is difficult to detect antemortem because clinical signs may be subtle. some signs include crying and reluctance to nurse, as well as decreased urine output and cold extremities.6 treatment of sepsis includes aggressive fluid therapy and fresh or fresh-frozen plasma from a wellvaccinated dog or cat. this will help to boost the immune status to that of a regularly suckling kitten or puppy.6 electrolyte and blood glucose measurements are also important as well as adequate nutrition and environmental temperature. antibiotics therapy should be based on culture and sensitivity.6 antibiotics commonly used in the neonate include cephalosporins, penicillins, clavulanic acid, macrolides, and trimethoprim-sulfonamides.8 prevention of sepsis in the neonate is the key to decrease neonatal mortality. it is clear that the use of proper aseptic techniques is important in neonates in order to prevent infection and subsequent sepsis. umbilical cords should be kept clean, properly ligated and dipped in an antimicrobial such as iodine to prevent umbilical infections. owners should be advised of the importance of cleaning and properly handling the umbilical cords of the puppies or kittens when delivered at home. the importance of good hygiene and health of the dam should also be stressed. many gastrointestinal and respiratory infections can be prevented by keeping neonates in a clean environment at the correct temperature and by keeping the dam well-vaccinated and healthy. milk production should be closely monitored as well as color and character of the milk. the teats of the dam should be examined daily for signs of mastitis including heat, firmness, redness, or pain. by taking preventative measures and doing pre-whelping and post-whelping examinations on the bitch or queen, many of these causes of neonatal septicemia can be prevented. aspiration pneumonia was the second most common cause of neonatal death found in this study. neonatal puppies and kittens have immature organ systems including their lungs and immune system. this puts them at high risk for developing infections, especially within the lungs. in several instances aspiration pneumonia was diagnosed based on the presence of protein globules with or without the presence of bacteria within the airways and tube feeding of puppies was suspected to be responsible for aspiration pneumonia. tube feeding is needed when the neonate is too weak to nurse or the mother is unable to care for her litter. it is unclear in these cases whether the aspiration and death was due to the tube feeding itself or the condition predisposing the neonate to need to be tube fed. it is important that clients are properly informed and instructed on how to tube feed and the complications that may arise if it is done improperly. clients should be taught what to look for if the puppies should develop aspiration pneumonia so they can begin treatment as soon as possible. with proper instruction and practice with the veterinarian, some causes of aspiration pneumonia can be avoided. signs of aspiration include milk replacer flowing out of the nostrils, difficulty breathing, coughing, and fever.9 the neonate will be weak and lethargic and may be found separate from others in the litter. bacterial pneumonia in the neonate should be treated with antibiotics. a transtracheal wash clinical theriogenology • volume 7, number 1 • march 201555 can be performed and a culture and sensitivity obtained on the contents in order to select the most specific and effective antibiotic therapy. it is most beneficial to give antibiotics iv or intraosseous in the neonate.4. a number of reasons were found for inability to obtain definitive diagnoses. maternal care that is degraded by poor health, condition, and behavior of the dam is one important factor that cannot easily be evaluated upon necropsy of the puppy or kitten. for example, if the dam suffers from agalactia and is not producing enough milk, several deaths can occur due to malnutrition of the neonates. on necropsy, there will be a lack of ingesta in the gastrointestinal tract suggesting that the neonate died of anorexia and hypoglycemia. however, there are several other reasons why a neonate may lack ingesta (including other illness in the neonate) and a pathologist would not be able to make a definitive diagnosis based on this finding alone. if this finding is consistent throughout an entire litter of neonates on necropsy, it can be more suggestive of a diagnosis of agalactia. the dam can also have other conditions such as mastitis or there may simply be some mismothering, which hinders the neonates’ ability to thrive. all of these potential complications with the dam would make diagnosis based solely upon necropsy of the neonate difficult. hypoglycemia and hypothermia are significant causes of death. in general, they cannot be definitively diagnosed as a cause of death by necropsy. it is not possible to determine the antemortem temperature or blood glucose level of a neonate after it is deceased, and therefore a definitive diagnosis of hypothermia or hypoglycemia cannot be made. diagnoses were also not made because of severe artifacts within the tissues caused by freezing and thawing of the submitted specimens and by the method of euthanasia. the damage caused by these processes can mask a true cause of disruption to the tissues and prevent a diagnosis from being made. the amount of time that is allowed to elapse between the time of death and the time of tissue sampling is also very critical. the amount of autolysis that occurs is directly related to this time interval and the ability to make a diagnosis is closely related to the amount of autolysis present. the best way to maximize the chance of getting a diagnosis is to minimize the time between the death of the neonate and the time of tissue sampling. there were three instances within this study where in utero fetal stress was suspected. when a neonate endures in utero stress, it is born weak and has a higher possibility of complications and failure to thrive. there may be no associated gross changes evident on necropsy but they will cause neonatal death within the first days of life. there are also causes such as suffocation, congenital cardiac arrhythmias, and complications with anesthesia during a cesarean section which may cause death but do not have specific lesions to allow for a definitive diagnosis. in this study, 19 of the 31 cases in which the cause of death was unknown bacteriology or virology was not performed due to the request of the submitter. thus 61% of the cases in which the cause of death was unknown did not have a complete examinaion. if the additional testing would have been permitted by the submitter, there is a greater chance that a diagnosis may have been made. many factors inhibited the ability to establish a definitive diagnosis, many of which may have been prevented. there are several things a veterinarian or animal owner can do to increase the chances of obtaining a diagnosis when submitting a sample for necropsy. autolysis is one of the major factors that occurs postmortem that can hinder the ability to obtain a definitive diagnosis. narrowing the time interval between the time of neonatal death and the time the tissue samples can be collected is crucial to improving the diagnostic quality of the tissues. the neonate should be refrigerated immediately after death, packaged properly, and sent to the diagnostic laboratory as soon as possible. freezing should be avoided unless there will be an extended interval between death and necropsy in order to avoid the artifacts and damages that freezing has on the tissues. also, the method of euthanasia should be taken into account if it is a necessary step. the best way to euthanize a neonate without disruption of tissues is iv injection of euthanasia solution. a complete history should be submitted with every necropsy submission. information about the dam, the whelping of the litter, the environment, and the health of the littermates are all important when establishing a diagnosis. it is important to know if the dam is vaccinated and if she has had any previous clinical theriogenology • volume 7, number 1 • march 2015 56 health problems. information about whether this is her first litter or if she has had previous litters and if there were any health concerns in previous litters are all important. also, it is important to know if she had a natural delivery or if a cesarean section was required. the number of puppies or kittens that were in the litter and information on the status of the littermates are also valuable, as well as if there was neonatal mortality in previous litters. it is helpful to know about the neonates’ environment. was the area kept clean and isolated or were other animals in contact with the litter? was the litter kept at an adequate temperature and was there any mismothering? it is also desirable to have complete history about the neonate itself. information about its normality at birth and a specific timeline of events that occurred prior to its death is important in differentiating specific diagnoses. all of this information enhances the pathologists’ ability to put together the pieces of the puzzle and determine a specific cause of death for the neonate. there are several factors that need to be taken into consideration when evaluating the results of this study. the environmental factors of the pacific northwest vary significantly from other areas of the country including parasite load and prevalence of specific infectious diseases. results from studies in other parts of the country may vary significantly. also, the neonates submitted to waddl were mostly purebred and most likely were submitted by breeders. this may skew the results from those found in the general population as many neonatal cadavers are not submitted to diagnostic laboratories. references 1. tønnessen r, borge ks, nødtvedt a, et al: canine perinatal mortality: a cohort study of 224 breeds. theriogenology. 2012;77:1788-801. 2. grundy s: clinically relevant physiology of the neonate. vet clin north am small anim prac 2006;36:443-459. 3. lawler df: neonatal and pediatric care of the puppy and kitten. theriogenology 2008;70:384-392. 4. fortney w: managing the sick neonate. proc west vet conf;2007. 5. lamm cg, njaa bl: clinical approach to abortion, stillbirth, and neonatal death in dogs and cats. vet clin north am small anim pract. 2012;42:501-513. 6. mcmicheal m: pediatric emergencies. vet clin north am small anim pract 2005;35:421-434. 7. schafer-somi s, spergser j, breitenfellner j, et al: bacteriologic status of canine milk and septicaemia in neonatal puppies-a retrospective study. j vet med 2003;50:343-346. 8. johnston sd, root kustritz mv, olson pns: the neonate-from birth to weaning. in: canine and feline theriogenology. philadelphia: wb saunders; 2001. p. 200-224. 9. cohn l: successful treatment of bacterial pneumonia. proc west vet conf; 2010. clinical theriogenology • volume 7, number 1 • march 201557 table : causes of 107 kitten and puppy neonates death diagnosed at washington state animal disease diagnostic laboratory during 2000-2010 diagnosis frequency percent (frequency/107) infectious septicemia 17 15.8 yes aspiration pneumonia 15 14.0 yes infectious pneumonia 6 5.6 yes prematurity 6 5.6 no anorexia 6 5.6 no congenital defect 6 5.6 no non-infectious pneumonia 4 3.7 no trauma 4 3.7 no canine herpesvirus 1 infection 2 2.8 yes meningitis 2 2.8 yes panleukopenia virus infection 1 0.9 yes parvovirus infection 1 0.9 yes endotoxemia 1 0.9 yes clostridial myonecrosis 1 0.9 yes cerebral hemorrhage 1 0.9 no infant respiratory distress syndrome 1 0.9 no infectious enteritis 1 0.9 yes bacterial peritonitis 1 0.9 yes intestinal intussusception 1 0.9 no unknown 30 28 clinical theriogenology • volume 7, number 1 • march 2015 58 2017: clinical relevance of follicular dynamics for optimal reproductive management of cycling mares   clinical relevance of follicular dynamics for optimal reproductive management of cycling mares b. matthew dredge,a dirk k. vanderwallb adepartment of animal and food science, brigham young university–idaho, rexburg, id and bdepartment of animal, dairy and veterinary sciences, school of veterinary medicine, utah state university, logan, ut abstract reproductive hormones, such as prostaglandin f2 alpha, are widely utilized in equine reproductive management. the use of transrectal palpation and ultrasonography, along with an understanding of the physiology of follicular dynamics, and appropriate use of those reproductive hormones, help the equine practitioner better manage single mares for breeding or even groups of mares requiring estrus and ovulation synchronization for breeding or advanced reproductive techniques. much research has illuminated critical events that occur during the diestrus period in the mare that assist the practitioner in making the most appropriate treatment decisions. this paper reviews the information gained from that research allowing practitioners to incorporate the knowledge into everyday reproductive practice. keywords: follicular dynamics, prostaglandin f2alpha, cycling mare, estrus synchronization, ovulation synchronization introduction with the discovery that prostaglandin f2 alpha (pgf2α) is the endogenous luteolytic hormone in sheep,1 and later in the horse in the early 1970’s,2,3 a powerful new dimension of reproductive management was added to the toolbox of the equine practitioner (i.e. the ability to “short-cycle” a mare by abbreviating the luteal phase). however, this discovery was before ultrasonographic examination of the reproductive tract of the mare was possible and the physiology of follicular dynamics (i.e. primary and secondary follicular waves) were fully understood, which contributed to the occurrence of frustrating inconsistencies in mares following treatment with pgf2α. the use of transrectal palpation and ultrasonographic examinations of the reproductive tract of the mare has partially eliminated these apparent frustrating inconsistencies by allowing the practitioner to better know the ovarian/follicular status of the mare. for example, the mare that is presented with a history of showing recent estrous behavior and ultrasonographic evidence of a corpus luteum (cl) without a secondary dominant follicle is generally not challenging to the practitioner. in contrast, the mare that is presented with a history of exhibiting recent estrous behavior, has ultrasonographic evidence of a cl, and also has a large (>30 mm) secondary dominant follicle (i.e. a “diestrus follicle”) can be challenging to manage. therefore, this review will focus on how to optimally manage the cycling diestrus mare, based on the current understanding of follicular dynamics and its impact on how an individual mare responds to exogenous pgf2α. physiology of primary and secondary follicular waves mares are considered monovular, since they predominantly develop one ovulatory follicle during estrus.4 terminology referring to the events associated with follicular dynamics throughout the estrous cycle are listed in the table.5 all cycling mares have a primary major follicular wave that begins in late diestrus resulting in the selection of the dominant (ovulatory) follicle. that follicle is associated with the return to estrus after luteolysis causes regression of the cl that originated during the previous ovulatory estrus (figure16). it is important to remember the progression of an ovarian follicle within a follicular wave during the ovulatory season as depicted in figure 2.6 some mares only develop the ovulatory primary follicular wave (top panel figure 1) as can be seen ultrasonographically with a single dominant follicle and edema of the endometrial folds where the dominant follicle will most often proceed to ovulation (figure 3).7 other mares will develop an earlier (in diestrus) major secondary follicular wave that is associated with selection of a dominant (≥25mm) follicle in mid-diestrus that, without clinical theriogenology • volume 9, number 2 • june 2017145   pharmacological intervention, will have one of three outcomes: 1) regression (atresia), 2) ”silent” ovulation (incidence ranges from rarely in ponies,8 4% of cycles in quarter horses and appaloosas,9 to 25% of cycles in thoroughbreds10 and standardbreds11), or 3) development into a hemorrhagic anovulatory follicle (haf)7 (incidence range from 2.5%12 35.8%13) as shown in the middle panel of figure 1. these mares have a functional cl and a secondary dominant follicle, but are not physiologically or behaviorally in estrus due to progesterone being the dominant hormone when both progesterone and estrogen are being secreted (figure 4).14,15 a third group of mares will develop a secondary minor wave that is not associated with selection of a dominant follicle (the largest follicle remains <28mm), as shown in the bottom panel of figure 1. the varying degrees of follicular development among each “type” of mare dramatically influences their response to a luteolytic dose of pgf2α (i.e., how soon they come into behavioral estrus and ovulate). therefore, it is essential to know the follicular status of an individual mare at the time of pgf2α administration for optimal reproductive management. the importance of knowing the ovarian follicular status of mares when administering pgf2α given the variability of follicular wave patterns among mares, it is important to know the status of the ovaries in mares at the time of pgf2α treatment. this will enable the practitioner to more accurately predict the mare’s response following administration of pgf2α (i.e. approximately how soon she will come into estrus and ovulate).12,16 the challenge is to predict (to the degree possible) the expected interval from pgf2α treatment to ovulation. when treating with pgf2α, the interval from treatment to ovulation is related to the mare’s follicular wave status (i.e. size of ovarian follicles at the time of treatment as shown in fig. 1).17-19 burden et al. recently confirmed the findings of early studies, showing an inverse relationship between the size of follicle and the interval of treatment to ovulation (i.e. the larger the size of the diestrus follicle at the time of pgf2α treatment, the shorter the interval to estrus/ovulation).12 as noted above, the potential outcomes of large secondary diestrus follicles are regression, ovulation, or formation of haf. therefore, treating a mare with pgf2α without knowledge of the ovarian status could result in unexpected outcomes based on the presence of a large secondary diestrus follicle. the potential outcomes of pgf2α treatment in a diestrus mare knowledge of the ovarian status of mares under consideration for pgf2α treatment allows the practitioner to make informed management decisions. research has shown that some variation exists as to when the luteal cells of the equine cl will be responsive to single dose of pgf2α.2,3,20,21 it is currently common practice to treat recently ovulated mares no sooner than day five of the cycle (ovulation equals day 0).17,19,22 this point supports the importance of using transrectal palpation and ultrasonography to determine the ovarian status of the mare. given the wide variation in length of estrus in mares (three to seven days, or longer) and the fact that signs of estrus continue for 24 to 48 hours after ovulation,23 it becomes difficult to precisely determine when the cl is “mature” and sensitive to a single dose of pgf2α without knowing the day of ovulation. when emergence of a secondary follicular wave does not occur in early diestrus, there will not be a large secondary dominant follicle present, so mares will have minimal follicular activity at the time of pgf2α administration (figure 5). once the cl is mature, five days after ovulation, treatment with one single administration of pgf2α should reliably result in luteolysis, causing the mare return to estrus in three to five days.2 based on the experience of the authors, the interval from treatment to ovulation in mares with this follicular status can be longer than ten days, which is consistent with data from burden et al., which showed that when all secondary follicles were <10mm in diameter, the average interval to ovulation after treatment with pgf2α was 11.8 days.12 this situation occurs when emergence of the next primary follicular wave has not yet occurred, meaning no follicles >10 mm are present at the time of treatment (figure 5). in contrast, mares that have a secondary follicular wave in early diestrus and develop a large secondary dominant follicle will generally have a shorter interval from pgf2α treatment to ovulation. clinical theriogenology • volume 9, number 2 • june 2017 146   burden et al. determined that the interval to ovulation after treatment with pgf2α for secondary follicles <35 mm in diameter was inversely proportional to the diameter of the largest follicle present at the time of treatment.12 secondary follicles 20-24 mm and 30-34 mm in diameter had an average interval to ovulation after treatment of 9.1 days and 7.8 days, respectively.12 therefore, a mare with a 34 mm follicle on the left ovary, a cl on the right ovary, and lack of edema of the endometrial folds (figure 6) would have an expected interval from treatment to ovulation of approximately 7.8 days. secondary follicles that are large (≥35 mm) can have a treatment to ovulation interval of 48 hours.12 based on the research, a mare with a ≥35 mm follicle on the right ovary, smaller follicles and cl on the left ovary, and lack of edema of the endometrial folds could have an interval from treatment to ovulation within 48 hours (figure 4). some large follicles may even ovulate within 24 hours after treatment without the mare displaying behavioral estrus.12,17,24,25 some mares may have large follicles (≥35 mm) at the time of treatment which regress and become replaced by a dominant follicle of the next primary follicular wave.24 this regression can be followed by serial transrectal palpation and ultrasonographic examinations (figures 4 and 7) some studies have indicated that pregnancy rates were decreased in pgf2α induced estrous cycles when compared to pregnancy rates from spontaneous estrous cycles.13,18,26-28 lindeberg et al. indicated that pgf2α treatment may exert a negative influence on large diestrus follicles, however, it was also indicated that pregnancies doubled in pgf2α induced cycles when the ovaries contained small follicles (<30mm) at the time of treatment.18 nielsen et al. indicated that only the mares in their project showing poor estrous signs were treated with pgf2α indicating these mares may have had ongoing reproductive irregularities.26 cuervo-arango et al. found pregnancy rates and embryo recovery rates strongly correlated with an increased interval of treatment with pgf2α to ovulation29 while agnew et al. found administration of pgf2α to mares with large diestrus follicles appeared to have lower pregnancy rates when compared to mares bred on spontaneous estrus cycles.28 in contrast to these studies, metcalf and thompson found no difference in pregnancy rates when comparing pgf2α induced estrous cycles and spontaneous estrous cycles.30 clinically, it can be challenging to know if a diestrus follicle ≥35mm is viable. in addition to the studies mentioned previously, studies by samper et al.24 and burden et al.12 demonstrate that mares with larger diestrus follicles can develop uterine edema within 24 hours, and the development of uterine edema is a strong indicator of follicle viability. ultimately, more research is needed in this area to determine the fertility of large (>35 mm), diestrus follicles. the ability to more accurately manage mares with diestrus follicles will increase breeding season efficiency. estrus/ovulation synchronization programs the purpose of estrus/ovulation synchronization can vary from timed insemination to nuclear transfer. regardless of the purpose of the synchronization, the goal is to align the ovarian activity of a group of mares in order to synchronize both estrus and ovulation. older protocols, such as the use of pgf2α and progesterone, can be effective at synchronizing estrus, but fail to synchronize ovulation, leaving a variable time interval from the end of treatment to ovulation of the mares in the synchronization protocol, while newer protocols, such as progesterone, estrogen, pgf2α, and an ovulation induction agents in combination, are effective in synchronizing both estrus and ovulation. it is important to consider what is occurring in the ovary with each of the techniques/protocols used in order to better understand the expected outcome. the following paragraphs will describe some of the older and newer protocols used for estrus/ovulation synchronization. prostaglandin f2α can be and is used in mare estrus synchronization programs although it is rarely used as the sole method of synchronization.31 in the instance of a single timed insemination or the single client desiring to shorten the diestrus period, the practitioner can attempt to inform the client of the expected interval of treatment to ovulation without knowing the follicular status of the ovaries. however, if it is not known whether the mare has only one primary follicular wave, or multiple follicular waves (see figure 1), giving the client the typical time period of three to five days for return to estrus after treatment could potentially cause frustration. based on the information discussed in the previous section, a mare clinical theriogenology • volume 9, number 2 • june 2017147   can fall into any of the described scenarios and have a wide range of interval from treatment with pgf2α to ovulation. knowing the follicular status of the ovaries via transrectal palpation and ultrasonography can assist in giving the client a more informed estimate of when the mare should return to estrus and proceed to ovulation. for example, when presented with a mare that has a 34mm diestrus follicle and a mature cl (at least 5 days post-ovulation), as depicted in figure 6, treatment with pgf2α on the same day of the ultrasonographic examination would likely result in a shorter interval from treatment to return to estrus (average interval to ovulation 7.8 days12), whereas if a mare is presented with a diestrus follicle(s) ≤10mm and a mature cl as depicted in figure 5, one could expect an average of 11.8 days12 from the time of treatment to ovulation. thus treating single mares with pgf2α alone, where tight synchrony is not the goal of treatment, usually does not lead to frustration. however, synchronization of multiple mares with pgf2α only can lead to a group of mares that are not closely synchronized. most synchronization programs utilize some form of exogenous progesterone to provide negative feedback on the adenohypophysis, suppressing luteinizing hormone (lh) secretion. after the prescribed period of time administering progesterone, pgf2α is administered to lyse any residual luteal tissue that may be present and the mares are checked in three to five days for return to estrus.32,33 however, the primary limitation of this estrus synchronization program is that progesterone alone only suppresses lh secretion by the adenohypophysis, not follicle stimulating hormone (fsh) secretion.33 therefore, maturation of the follicle is halted and ovulation can be inhibited; however, without suppression of fsh, follicular waves can and do develop, as shown in the middle and bottom panels of figure 1 when mares are under the influence of elevated levels of endogenous progesterone. the progesterone eliminates behavioral estrus even in the face of endogenous estrogen as discussed previously; however, ovarian activity and follicular waves are not suppressed, which results in greater variability in return to estrus and ovulation after treatment. several products are available for progesterone treatment with typical treatment regimens being daily dosing for 10 days with oral altrenogest at 0.044 mg/kg or intramuscular injection of progesterone in oil at a dose of 150 mg/day. another injectable progesterone product is available in a slow-release formulation that maintains elevated progesterone for approximately seven days.34 altrenogest has also become available in an injectable slow-release formulation. prostaglandinf2α is typically given on day 10 of the daily oral or injectable treatment protocols or ten days after injection with the slow-release progesterone or altrenogest products with expected return to estrus in three to four days and ovulation in seven to ten days following pgf2α treatment. the appropriate use of ovulation inducing agents, described elsewhere32, most commonly human chorionic gonadotropin (hcg) or gonadotropin releasing hormone (gnrh) analogs, can help tighten ovulation synchrony when used in conjunction with progesterone treatments. estrus synchronization programs that utilize a combination of progesterone and estradiol-17β obtain suppression of lh secretion, as described above, as well as suppression of fsh secretion from the adenohypophysis and therefore, suppression of ovarian follicular development.35 suppression of the ovary in this manner provides much tighter synchrony than can be achieved by progesterone alone because recruitment of follicles for follicular waves does not occur in the absence of fsh.32 traditionally, progesterone and estradiol-17β were mixed in vegetable oil at concentrations of 50 mg/ml and 3.3 mg/ml, respectively, and administered intramuscularly at a daily dose of 150 mg progesterone and 10 mg estradiol-17β for ten days with administration of pgf2α on the last day of treatment.35 in the early 1990’s, research into sustained release of these hormones to alleviate the need for daily injections was conducted.36,37 although no food and drug administration (fda) approved products are currently available, compounded products are available with sustained release of progesterone and estradiol-17β. one injection of this long-acting/slow-release progesterone and estrogen product results in ovarian suppression occurring for approximately ten days. prostaglandinf2α is used at the end of the synchronization treatment in order to eliminate any luteal tissue which may still be present in the ovary. the mares are then checked three to five days after the last day of treatment to assess ovarian follicular status. the appropriate use of ovulation inducing agents, as mentioned above, will further synchronize ovulation, tightening overall synchrony of mares in the synchronization program. clinical theriogenology • volume 9, number 2 • june 2017 148   summary in summary, treating mares with pgf2α during the breeding season is a common practice and having a basic understanding of primary and secondary follicular waves helps the practitioner when treatment decisions must be made. the use of transrectal palpation and ultrasonography to assess ovarian follicular status can greatly augment the effectiveness and efficiency with which pgf2α treatment can be utilized by practitioners, since the diameter of secondary follicles is inversely proportional to the time of treatment with pgf2α to ovulation. synchronization programs based on pgf2α treatment alone do not provide the tight synchrony seen with other programs due to the variation of primary and secondary follicular waves experienced in the ovaries of mares. the use of progesterone in combination with estrogen, followed by treatment with pgf2α at the end of the treatment period, and the appropriate use of ovulation inducing agents provides the tightest synchrony available. in conclusion, the optimal use of pgf2α in mares requires an understanding of the underlying physiological events associated with follicular dynamics throughout the estrous cycle. references 1. mccracken ja, carlson jc, glew mc, et al: prostaglandin f2a identified as a luteolytic hormone in sheep. nature 1972;238:129-134. 2. douglas rh, ginther oj: effect of prostaglandin f2alpha on length of diestrus in mares. prostaglandins 1972;2:265268. 3. allen wr, rowson le: control of the mare's oestrous cycle by prostaglandins. j reprod fertil 1973;33:539-543. 4. ginther oj, beg ma, bergfelt dr, et al: follicle selection in monovular species. biol reprod 2001;65:638-647. 5. beg ma, bergfelt d: folliculogenesis. in: mckinnon, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. ames: wiley-blackwell; 2011. p. 2009-2019. 6. bergfelt d: anatomy and physiology of the mare. in: samper jc, editor. equine breeding management and artificial insemination. 2nd ed. st. louis: saunders/elsevier; 2009.p. 113-131. 7. ginther oj: major and minor follicular waves during the equine estrous cycle. j equine vet sci 1993;13:18-25. 8. ginther oj, whitmore hl, squires el: characteristics of estrus, diestrus, and ovulation in mares and effects of season and nursing. am j vet res 1972;33:1935-1939. 9. ginther oj, pierson ra: regular and irregular characteristics of ovulation and the interovulatory interval in mares. j equine vet sci 1989;9:4-12. 10. hughes jp, stabenfeldt gh, evans jw: clinical and endocrine aspects of the estrous cycle of the mare. proc annu conv am assoc equine pract 1972; p.119-151. 11. sirois j, ball ba, fortune je: patterns of growth and regression of ovarian follicles during the oestrous cycle and after hemiovariectomy in mares. equine vet j 1989;21:43-48. 12. burden ca, mccue pm, ferris ra: effect of cloprostenol administration on interval to subsequent ovulation and anovulatory follicle formation in quarter horse mares. j equine vet sci 2015;35:531-535. 13. cuervo-arango j, newcombe jr: the effect of cloprostenol on the incidence of multiple ovulation and anovulatory hemorrhagic follicles in two mares: a case report. j equine vet sci 2009;29:533-539. 14. asa cs, goldfoot da, garcia mc, et al: the effect of estradiol and progesterone on the sexual behavior of ovariectomized mares. physiol behav 1984;33:681-686. 15. hayes ke, ginther oj: role of progesterone and estrogen in development of uterine tone in mares. theriogenology 1986;25:581-590. 16. samper jc, geertsema bs, hearn p: rate of luteolysis, folliculogenesis and interval to ovulation in mares treated with a prostaglandin analogue on day 6 or 10 of the estrous cycle. proc annu conv am assoc equine pract 1993; 169-170. 17. newcombe jr, jöchle w, cuervo-arango j. effect of dose of cloprostenol on the interval to ovulation in the diestrous mare: a retrospective study. j equine vet sci 2008;28:532-539. 18. lindeburg h, koskinen e, huhtinen m, et al: influence of pg administration and follicle status on the number of conceptuses. theriogenology 2002;58:571-574. 19. card c: hormone therapy in the mare. in: samper jc, editor. equine breeding management and artificial insemination. 2nd ed. st. louis: saunders/elsevier; 2009.p. 89-97. 20. bergfelt dr, pierson ra, ginther oj: regression and resurgence of the cl following pgf2α treatment 3 days after ovulation in mares. theriogenology 2006;65:1605-1619. 21. brendemuehl j: effect of oxytocin and cloprostenol on luteal formation, function and pregnancy rates in mares. theriogenology 2002;58:623-626. 22. hughes jp, loy rg: variations in ovulatory response associated with the use of prostaglandins to manipulate the lifespan of the normal diestrous corpus luteum or the prolonged corpus luteum of the mare. proc annu conv am assoc equine pract 1978;173. 23. day ft: sterility in the mare associated with irregularities of the oestrous cycle. vet rec 1939;51:1113. clinical theriogenology • volume 9, number 2 • june 2017149   24. samper jc: induction of estrus and ovulation: why some mares respond and others do not. theriogenology 2008;70:445-447. 25. marinone ai, losinno l, fumuso e, et al: the effect of mare's age on multiple ovulation rate, embryo recovery, posttransfer pregnancy rate, and interovulatory interval in a commercial embryo transfer program in argentina. anim reprod sci 2015;158:53-59. 26. nielsen jm, kofoed bock ts, ersbøll ak: factors associated with fertility in horses in a danish equine practice after artificial insemination with frozen–thawed semen. anim reprod sci 2008;107:336-337. 27. cuervo-arango j, newcombe jr: risk factors for the development of haemorrhagic anovulatory follicles in the mare. reprod domest anim 2010;45:473-480. 28. agnew me, schnobrich mr, stromberg a, et al: pregnancy outcomes in thoroughbred mares administered different doses of cloprostenol. clin therio 2015;7:335. 29. cuervo-arango j, mateu-sánchez s, aguilar jj, et al: the effect of the interval from pgf treatment to ovulation on embryo recovery and pregnancy rate in the mare. theriogenology 2015;83:1272-1278. 30. metcalf es, thompson mm: the effect of pgf2α-induction of estrus on pregnancy rates in mares. j equine vet sci 2010;30:196-199. 31. squires el: hormonal manipulation of the mare: a review. j equine vet sci 2008;28:627-634. 32. bradecamp ea: synchronization of ovulation. in: mckinnon, squires e, vaala w, et al, editors. equine reproduction.2nd ed. ames: wiley-blackwell; 2011.p. 1870-1878. 33. pinto cr, meyers pj: control and synchronization of the estrous cycle and ovulation. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: saunders/elsevier; 2007. p. 91-98. 34. burns pj, morrow c, abraham j: evaluation of biorelease p4 la 300 in the mare. proc int symp on equine embryo transfer 2008: p. 82-83. 35. loy rg, pemstein r, o'canna d, et al: control of ovulation in cycling mares with ovarian steroids and prostaglandin. theriogenology 1981;15:191-200. 36. burns pj, ball ba, tice tr, et al: a preliminary report on the efficacy of biodegradable microspheres for the controlled release of progesterone and estradiol for synchronization of ovulation in mares. theriogenology 1990;33:202. 37. blanchard tl, varner dd, burns pj, et al: regulation of estrus and ovulation in mares with progesterone or progesterone and estradiol biodegradable microspheres with or without pgf2α. theriogenology 1992;38:1091-1106. (editor’s note: images in this manuscript appear in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 9, number 2 • june 2017 150   table. terminology commonly used in association with follicular dynamics in mares. reprinted with permission from john wiley and sons.5 characteristic definition follicular wave a cohort or group of antral follicles (mean 7 to 11) that are first detected (≥ 6 mm in diameter) within 1 to 2 days of one another and increase in diameter similarly until follicle deviation. follicle or wave emergence the earliest ultrasonic detection of follicles (≥ 6 mm) of a wave compatible with tracking individual growing and regressing follicles in a reliable and consistent manner. emergence of a wave is a useful reference point for aligning the emergence of waves that occur at different times among mares. common growth phase the period from emergence of a major follicular wave to beginning of deviation (6 to 7 days). all follicles of the wave grow at a similar rate (about 3 mm/day) during the common growth phase. follicle deviation the dissociation in diameter between the largest follicle and next largest follicles of a major wave that begins, on average, when the largest follicle reaches 22.5 mm. the largest follicle continues to grow whereas the next largest follicles cease growth and regress after the beginning of deviation. the beginning of deviation is calculated to occur at the ultrasound examination preceding the first examination with an apparent change in diameter difference between two largest follicles. the beginning of deviation is a useful reference point for aligning the growth profiles of follicles of a wave for comparing structural and functional characteristics within and among mares. dominant follicle the largest follicle of a major wave that continues growth after deviation, reaches ≥ 28 mm and becomes ovulatory or anovulatory. subordinate follicles the next largest follicles of a major wave relative to a dominant follicle, that cease growth after deviation, reach < 28 mm and eventually regress. dominance phase the period from the beginning of deviation until the beginning of the pre-ovulatory phase (approximately 7 to 8 days). growth of all subordinate follicles of the wave ceases during this period and typically no new follicular waves emerge. pre-ovulatory phase the period beginning when the dominant follicle of the major primary wave attains ≥ 30 mm and until ovulation or formation of a hemorrhagic follicle. the pre-ovulatory follicle attains the developmental capacity to respond to an endogenous or exogenous ovulatory stimulus. major follicular wave a follicular wave that develops at least one dominant follicle (≥ 28 mm) and corresponding subordinate follicles (< 28 mm) during the transitional period, estrous cycle, and early pregnancy. minor follicular wave a follicular wave that does not develop a dominant follicle. instead, the largest follicle of the wave reaches < 28 mm during the transitional period, estrous cycle, and early pregnancy. primary follicular wave a major follicular wave that emerges during late diestrus where the dominant follicle becomes pre-ovulatory and results in the primary ovulation at the end of estrus. secondary follicular wave a major wave that emerges during late estrus of the previous estrous cycle or early diestrus where the dominant follicle either is anovulatory and regresses or forms an anovulatory hemorrhagic follicle or results in secondary ovulation during middiestrus. clinical theriogenology • volume 9, number 2 • june 2017151   figure 1. various profiles of follicular wave patterns during the equine estrous cycle depicting ovulation (ov) of the dominant follicle (≥30 mm) of a primary major wave at the end of estrus and ov or anovulation and formation of a hemorrhagic follicle (ahf) or re regression of the dominant follicle of a secondary major wave during diestrus. alternatively, either minimal follicular growth (<15 mm) or a minor follicular wave (largest follicle, <30 mm) precedes the primary major wave. reprinted with permission from elsevier.6 clinical theriogenology • volume 9, number 2 • june 2017 152   figure 2. the morphological characteristics of the adult equine ovary (a) during the ovulatory season. note the sequential transformation of a primordial follicle to a dominant follicle and corpus luteum following ovulation. reprinted with permission from elsevier.6 clinical theriogenology • volume 9, number 2 • june 2017153   figure 3. top ultrasound image of an ovary 16 days after ovulation with a dominant follicle. bottom an ultrasound image from the same mare demonstrating her uterus with edema of the endometrial folds. tic marks on the right side of the images equal 10 mm. clinical theriogenology • volume 9, number 2 • june 2017 154   figure 4. the top image displays a cl and small follicle on the left ovary and a large secondary follicle on the right ovary 7 days after ovulation. the bottom image is the uterus from the same mare, displaying no edema within the endometrial folds. tic marks on the right side of the image equal 10 mm. clinical theriogenology • volume 9, number 2 • june 2017155       figure 5. the top-left image shows multiple small follicles, all roughly 10 mm in diameter in the left ovary with a cl in the right ovary (top-right) 3 days after ovulation. the bottom image shows the uterus without edema of the endometrial folds. tic marks on the right side of the images equal 10 mm. clinical theriogenology • volume 9, number 2 • june 2017 156     figure 6. the top-left image shows the left ovary with a roughly 34mm follicle. the top-right image shows the right ovary with a corpus luteum. the bottom image is of the uterine horns from this mare showing no edema of the endometrial folds. this mare is 9 days after ovulation. tic marks on the right side of the images equal 10 mm. clinical theriogenology • volume 9, number 2 • june 2017157   figure 7. these images are from the same mare as figure 4, 6 days after figure 4 was obtained (13 days after ovulation). the cl in the left ovary has regressed, the follicle in the right ovary has regressed, and no edema of the endometrial folds is present. tic marks on the right side of the images equal 10 mm. clinical theriogenology • volume 9, number 2 • june 2017 158 2011: neutrophil prevalence in dismount semen samples of thoroughbred stallions   neutrophil prevalence in dismount semen samples of thoroughbred stallions* allan j gunn, victoria j. brookes barn veterinary services, muswellbrook, nsw, australia introduction semen dismount samples from thoroughbred stallions are routinely monitored after natural mating.1,2 due to the relative ease of obtaining the sample, it is tempting to use features such as neutrophil presence to predict fertility.3-6 there are reports that neutrophils in a dismount sample from a stallion may indicate an inflammatory or infectious process in the reproductive tract of that stallion, or that they may be from the mare with an inflamed, and possibly infected reproductive tract with which the stallion has mated.2 the presence of neutrophils in dismount samples has been reported to decrease the odds of pregnancy.7,8 however, neutrophil prevalence in dismount samples of stallions with no reported fertility problems is unknown. the aim of this study was to investigate the prevalence of neutrophils in stallion dismount samples from stallions with no perceived fertility problems. the study group consisted of commercial thoroughbred stallions on a breeding farm in eastern australia. keywords: stallion, dismount semen sample, neutrophils materials and methods study design the seven thoroughbred stallions chosen for the study were all used commercially for the full length of the 2009 southern hemisphere breeding season. no abnormalities of the reproductive tracts of any of the stallions were detected. the stallions all resided on the same farm. the sampling strategy was convenience sampling. dismount semen samples from the distal penis and urethra were collected after natural mating from as many matings as possible, monday through friday from 10 september to 7 december 2009 (figure 1). the date, the time of collection and the stallion were recorded for each sample. sample processing and assessment the samples were examined within 24 hours of collection. after agitating the sample, a 25 microliter drop was smeared across a micrscope slide, air dried and stained with modified wright giemsa stain according to the procedure described by schumacher and moll9 (figures 2 and 3). the length of the slide was scanned initially at 100x magnification, then more critically evaluated at 1000x magnification with an oil immersion objective. numbers of neutrophils were counted in each field and the sample was categorized according to table 1. the number of slides falling into each category was counted. category number of neutrophils per high power field (hpf) 0 0 1 <1 per 5 hpf 2 1 per 5 hpf <1 per 3 hpf 3 >1 per 3 hpf<1 per hpf 4 1-2 per hpf 5 >2 per hpf table 1. sample category according to number of neutrophils in stallion semen dismount sample.                                                              *data presented at australian college of veterinary scientists science week, 1-3 july 2010 and association of applied animal andrology 7th biennial meeting, sydney, australia, 28-29 august 2010.   clinical theriogenology • volume 3 number 4 • december 2011477   figure 1. collection of a dismount sample from the penis of a stallion. analysis the prevalence of neutrophils in each category was calculated, with a 95% confidence interval. the number of samples collected from each stallion was compared to expected numbers (chi-square test), to determine that a particular stallion was not underor over-represented by the sampling method. the number of samples containing neutrophils for each stallion was also compared to expected numbers (chi-square test), to investigate whether samples with neutrophils were more likely to come from one stallion than another. data on the number of services for each stallion per week were not available. however, the number of samples collected each week and the number of samples with neutrophils each week was examined, to ascertain that the samples had been taken over the entire season, and to investigate whether neutrophil presence could be associated with a particular time of season. clinical theriogenology • volume 3 number 4 • december 2011 478     figure 2. neutrophils, stratified squamous cells and a spermatozoan.   figure 3. spermatozoa and ciliated epithelial cell. anucleate stratified  squamous epithelial  cells.  active neutrophils  aged/degenerating  neutrophils spermatozoan ciliated epithelial  cell spermatozoa clinical theriogenology • volume 3 number 4 • december 2011479   results four hundred eighteen samples were collected (table 2). stallion total services (n) samples (n) samples with neutrophils (n) 1 257 91 27 2 167 63 8 3 96 38 9 4 210 93 20 5 115 42 8 6 115 40 6 7 148 51 6 total 1108 418 84 p value of x2 statistic p=0.71 p=0.20 table 2. total number of services, number of samples collected and number of samples containing neutrophils, by stallion. the samples were categorized according to neutrophil presence, and the prevalence of neutrophils calculated for categories 1 to 5 (table 3). category number of samples percent of total prevalence of neutrophils (95% confidence interval) 0 334 80.0 1 42 10.0 0.10 (0.16, 0.24) 2 11 2.6 0.03 (0.01, 0.04) 3 9 2.1 0.02 (0.01, 0.04) 4 12 2.9 0.03 (0.01, 0.04) 5 10 2.4 0.02 (0.01, 0.04) total 418 100 0.20 (0.16, 0.24) table 3. number and percent of stallion semen dismount samples in each neutrophil score category. a graph was plotted of the number of samples collected weekly and the number of samples with neutrophils (categories 1-5) over time (figure 4). figure 4. graph showing the weekly number of stallion semen dismount samples collected and the number of samples with neutrophils. 0 10 20 30 40 50 60 1 2 3 4 5 6 7 8 9 10 11 12 13 week (10.09.09-07.12.09) n um be r o f s am pl es samples collected samples with neutrophils clinical theriogenology • volume 3 number 4 • december 2011 480   discussion the results of this study show that neutrophils are present in 20% (95% confidence interval, 16%-24%) of dismount semen samples from this population of thoroughbred stallions with no detected fertility problems from a commercial breeding operation in australia. the sampling strategy (convenience) did not significantly overor under-represent any of the stallions (p>0.05). the number of samples containing neutrophils was not significantly different between each stallion (p>0.05). the sample collection appears to be spread over the entire breeding season. samples with neutrophils are also evenly distributed over time, and appear to be of similar proportion to number of samples collected each week (figure 1), indicating that neutrophil presence in stallion dismount samples is a consistent feature. the origin of the neutrophils was not determined in this study, and further work is required to establish whether they are from the mare or the stallion, or both. further work is also required to determine the outcome of individual matings where neutrophils are present compared to those without neutrophils. conclusion in this study, approximately 20% of dismount samples from thoroughbred stallions contained neutrophils. in the authors’ opinion, neutrophil presence in a dismount semen sample should not be used as an indicator of stallion fertility. acknowledgements the authors thank john chopin for advice in preparing the manuscript. references 1. umphenour nw, sprinkle ta, murphy hq: natural service. in: mckinnon ao, voss jl, editors. equine reproduction. philadelphia: lea and febiger; 1992. p. 805-807. 2. conboy hs: significance of bacteria affecting the stallion’s reproductive system. in: samper jc, pycock jf, mckinnon ao, editors. current therapy in equine reproduction. st. louis: w.b. saunders; 2007. p. 234. 3. haag fm: evaluation of ‘dismount’ semen in thoroughbred horse breeding. j am vet med assoc 1959;134:312314. 4. gravance cg, champion z, liu ikm, et al: sperm head morphometry analysis of ejaculate and dismount semen samples. anim reprod sci 1997;47:149-155. 5. love cc, varner dd, thompson ja: intraand inter-stallion variation in sperm morphology and their relationship with fertility. j reprod fertil suppl 2000;56:93-100. 6. koyago m, nakada k, tsunoda n, et al: change in morphology of spermatozoa from dismount semen during the breeding season in thoroughbred stallions in japan. j equine sci 2009;20:1-5. 7. blanchard tl: the subfertile stallion. in: samper jc, pycock jf, mckinnon ao, editors. current therapy in equine reproduction. st. louis: w.b. saunders; 2007. p. 240-241. 8. blanchard tl, thompson ja, brinsko sp, et al: sources of variation in fertility of thoroughbred stallions. anim reprod sci 2010;121 suppl:28-29. 9. schumacher j, moll h.d: handling of fluid specimens. in: schumacher j, moll hd, editors. a manual of equine diagnostic procedures. jackson (wy): teton newmedia. internet publisher: international veterinary information service, last updated: 31-may-2010. available at: http://sss.ivis.ort/advances/schumacher/toc.asp clinical theriogenology • volume 3 number 4 • december 2011481 clinical theriogenology • volume 3 number 4 • december 2011 482 omniblank: rethinking sperm: lessons from mitochondrial function rethinking sperm: lessons from mitochondrial function evelyn bulkeley, azarene foutouhi, stuart meyers department of anatomy, physiology, and cell biology school of veterinary medicine, university of california, davis, ca abstract biological nature of age-related declining fertility in males of any species, including stallions, has been elusive. in horses, economic costs to the breeding industry are frequently considered extensive. mitochondrial function in ejaculated sperm, essential for sperm motility, is reflected by the dynamic processes of adenosine triphosphate production, mitochondrial oxidative efficiency and production of reactive oxygen species; this balance may become compromised in aging stallions and during cryopreservation process. this presentation will focus on mitochondrial integrity and function as an avenue for understanding the pathophysiology of sperm undergoing cryopreservation and male aging. keywords: sperm, mitochondrial function, oxidative phosphorylation, reactive oxygen species introduction conventional thought in sperm biology holds that the sperm flagellum is powered by adenosine triphosphate (atp) produced by a complex of mitochondrial enzyme systems including the electron transport chain (etc). as such, the powerhouse of the cell, the mitochondrion, is likely the foundation of sperm pathophysiology and structure.1-3 there are species differences in energy requirements for mitochondria and as optimal conditions for sperm function are becoming evident, individually tailored sperm handling and storage techniques4-6 are likely to become clinical realities. media modifications for sperm processing and storage may become necessary to provide appropriate energy substrates to minimize sperm damage and improve sperm longevity after cryopreservation.5 damage to sperm mitochondria is one of the most marked organelle changes during cryopreservation that is likely responsible for the majority of loss in motility and fertility after a freeze-thaw cycle.7,8 an increased understanding of sperm mitochondrial bioenergetics provides strong rationale to make considerable improvements in preservation and assisted reproduction techniques. mitochondria also have their own genome; therefore, investigations of mitochondrial dna may be useful to identify currently unknown causes of sperm dysfunction.9.10 the purpose of this review is to discuss current knowledge of sperm mitochondrial biology, focusing on appropriate sperm mitochondrial energy sources, mitochondrial production of reactive oxygen species and determination of future research directions that may improve sperm function, morphology and fertility. anatomy and physiology of sperm mitochondria equine sperm contain 40 50 helical mitochondrial gyres (turns) per cell in the midpiece composed of single mitochondria that line up end to end during spermiogenesis.11-13 functions of mitochondria in sperm include atp production for motility, reactive oxygen species (ros) production and redox signaling, and apoptosis.12,14 there is also growing evidence that sperm mitochondria may also be involved in intracellular calcium signaling and homeostasis, well-established functions of somatic mitochondria. equine sperm possess machinery for cellular processes of glycolysis, citric acid cycle, and oxidative phosphorylation (oxphos) to meet large energy requirements for motility and capacitationrelated events within the female reproductive tract, including hyperactivated motility, required for fertilization. further, sperm mitochondria are also known to be involved in lipid biogenesis, dna replication of its own genome and protein and hormone production. during spermatogenesis, sperm undergo a reduction in cytoplasm and re-organization of cellular organelles (i.e. spermiogenesis). mitochondria become organized end-to-end in a spiral formation around the sperm’s midpiece flagellar machinery in a fashion that appears to prevent the dynamic functions of mitochondrial fusion and fission. these functions are inherent in somatic cell mitochondria in which this amazing organelle may adapt to 353 clinical theriogenology • volume 11, number 3 • september 2019 physiological changes and are thus capable of self-replication, reduction and consolidation. the so-called mitochondrial sheath is tethered and overlain by the sperm’s fibrous sheath. mitochondria are comprised of 4 sub compartments: outer mitochondrial membrane (omm), intermembrane space (ims), inner mitochondrial membrane (imm) and mitochondrial matrix.15 the ims separates the omm and imm and is the site of proton accumulation from etc activity, which ultimately generates the electrochemical gradient and transmembrane potential (mitochondrial membrane potential). the imm, which is largely impermeable, houses the enzyme complexes of the electron transport chain (etc) and is thus the site of atp production by oxidative phosphorylation.16 the etc is a series of favorable sequential redox reactions, beginning with electron donors of low reduction potential. enzyme complexes 1, 3, and 4 pump protons across their gradient to acidify the mitochondrial intermembrane space, establishing a proton motive force that powers the fifth complex, atp synthase, via chemiosmosis to generate atp. each etc enzyme is itself composed of multiple subunits arranged dynamically and tethered through the mitochondrial membrane to the sperm’s fibrous sheath. the omm has nonspecific pores called porins, protein complexes that allow for passage of ions and most metabolites < 10 kda into the mitochondria.16,17 the imm encloses the matrix, with many cristae or invaginations increasing the surface area available for energy production. the inner mitochondrial membrane must be intact and impermeable for sperm mitochondria to be fully functional.18,19 the mitochondrial membrane potential describes a proton gradient formed by passage of electrons along the electron transport chain which is then used by atp synthase to make atp.20 decreased membrane potential, which occurs by membrane depolarization, indicates mitochondrial damage and the cell’s subsequent inability to meet energy demands. hyperpolarization, by contrast, can lead to increased cellular damage through reactive oxygen species production and lipid peroxidation.21 function of sperm mitochondria it was recently reported that production of atp by equine sperm for motility is primarily driven by oxphos rather than glycolysis.5,22 this is different than metabolism in many other species, e.g. humans and nonhuman primates, which still have essential sperm functions such as flagellar motility when oxphos is inhibited.9,23 it is reasonable to consider that there may be a metabolic shift to glycolysis with aging and semen cryopreservation when mitochondria become less efficient, due to an altered physiological state of the sperm. since glycolysis is less efficient than oxphos (only producing 2 atp per molecule of glucose, whereas oxphos produces 30) this could at least partially explain decreased motility in both cryopreservation and aging. in the aging testis, there is decreased antioxidant buffering capacity, which includes decreased superoxide dismutase enzyme activity as well as less active intramitochondrial catalase and peroxidase.24 mature sperm are particularly susceptible to oxidative damage for several reasons, including: (i) diminished cytoplasm, which houses the majority of cellular antioxidant enzymes; (ii) membranes with a high content of polyunsaturated fatty acids (pufas), which are particularly susceptible to peroxidative damage; and (iii) lack of transcription and translation machinery leaves mature sperm without typical dna repair mechanisms used by somatic cells to combat against oxidative damage. ultimately, this leaves sperm more susceptible to oxidative cell and dna damage which could lead to decreased oxphos. cryopreservation significantly damages sperm after thawing, but exact causes and targets of cryoinjury have not been determined. possible causes include ros production and apoptosis, whereas potential targets include plasma and mitochondrial membranes, known to have high pufa content. postthaw sperm motility is typically 50% of initial motility; this percentage may be lower and more variable for stallions than for other species.14 mitochondria are responsible for producing the majority of ros that can cause oxidative cellular damage during cryopreservation.14,22,25 the daily yield of ros can reach 107 molecules of superoxide anion per mitochondrion and negative effects on sperm include decreased fertility, motility, morphology, viability, altered membrane potential and mtdna damage.14,26 the high variation among males, especially stallions, is an industry problem and may originate in sperm mitochondria. 354clinical theriogenology • volume 11, number 3 • september 2019 assessing sperm mitochondria although one of the most popular methods of assessing mitochondrial function has been chemical probe-based measurements of mitochondrial membrane potential (mmp), conclusions have been complicated by issues of reliability and a lack of appropriate controls. the mmp refers to the electrochemical gradient across the inner mitochondrial membrane which is necessary to enable mitochondria to generate atp by oxphos. probing the magnitude of mmp in motile and nonmotile sperm can provide insights into the extent of that specie’s reliance on oxphos, as reported for dog and stallion sperm. volpe and coworkers reported on the relationship between mmp and sperm motility in dogs of known fertility, ranging from 16 months to 10 years of age.27 using fluorescent markers for viability and the fluorophore jc1 for mmp along with computer assisted sperm motility analysis, they reported that sperm of low motility were correlated with a high inner mitochondrial membrane potential, suggesting atp production via oxidative phosphorylation is not essential for fertility in male dogs. while these results provide exciting prospects for development of extenders of ideal substrate composition for long term preservation of sperm from valuable working dogs and stallions, these and the results of other such studies are recently being questioned. mmp probes are cationic and their rate of accumulation within the mitochondria is inversely proportional to inner mitochondrial membrane potential. the popular mmp probe jc1 allows dual color high/low assessment of inner mitochondrial membrane potential. however, like the other mmp probes used in sperm, jc1, which is sensitive to factors of intracellular stress such as ros, is not reliable as a quantitative measure of mmp without appropriate controls. ideally, high/low mmp controls would include using an inhibitor of atp synthase (e.g. oligomycin) and an uncoupler of mmp, such as trifluorormethoxy carbonylcyanide phenylhydrazone (fccp) or 2,4 dinitrophenol (dnp); these are ionophores that dissipate the proton motive force by transporting hydrogen ions across the mitochondrial membrane. davilla and coworkers28 challenged the previous understanding that oxidative phosphorylation is the dominant metabolic pathway for sperm motility in the stallion. similar to volpe and coworkers,27 davilla and coworkers28 used the mmp probe jc 1 and computer assisted sperm motility analysis, but also included dnp and oligomycin as controls in their analysis of ejaculates from spanish stallions. these authors used 60 µm oligomycin, whereas 1 µm is the commonly used concentration in studies of human, boar and stallion sperm.28 consistent with other studies, inhibition of mitochondrial respiration by mitochondrial uncouplers dramatically reduces mmp and sperm motility. to explore whether glycolysis alone can support sperm motility, davila and coworkers28 aimed to inhibit glycolysis by incubating stallion sperm in the presence of 2 deoxy d glucose (2 dg) rather than glucose. although 2 dg is a weak competitive inhibitor of glycolysis through its inhibition of glucose 6 phosphate production, its metabolism to 2 deoxyglucose 6 phosphate results in accumulation of phosphate binding products that cannot undergo further substrate level phosphorylation. consequently, most of the phosphate in the sperm is bound, making it unavailable for atp synthesis in oxidative phosphorylation. in the presence of 2 dg application, sperm have increased mmp and decreased atp production via oxidative phosphorylation as well as any atp lost due to the inhibition of glucose 6 phosphate production in glycolysis. davila et al28 reported that inhibition of glycolysis resulted in decreased sperm motility. however, their results described a reduction in total motility, but no change in progressively motile sperm. this distinction is important, as the significance of motility measures of sperm vary widely. progressive motility describes an efficient consistent forward movement, whereas nonprogressive motility refers to sperm making tight circles or are moving but not making forward progression. although the glycolytic pathway likely provides support for other sperm functions (e.g. capacitation), its role in the ability of stallion sperm to reach the egg is unlikely. that glycolysis can provide the energy necessary for motility of stallion sperm has been disproved by other more recently developed methods. the use of mmp as a method of defining reliance on oxidative phosphorylation, though valuable in enabling comparison with previous studies, can be unreliable. in aerobic organisms, oxygen serves as the terminal electron acceptor; its reduction to water results in depletion of oxygen. measuring this oxygen consumption provides an indirect measure of atp produced by oxidative phosphorylation in sperm and reliably indicates mitochondrial function when employed alongside adequate mitochondrial effector drug treatments. to facilitate measurement of 355 clinical theriogenology • volume 11, number 3 • september 2019 oxygen consumption in a variety of cell types, an oxygen biosensor system has been developed using a microplate system with an oxygen impermeable matrix and oxygen-sensitive, ruthenium-based fluorophore. as oxygen is consumed from the media by the cells, the ruthenium-based probe fluoresces in proportion to oxygen depletion. in contrast to the approach used by davila,28 this method was used by darr et al.5 to determine major substrates for motility and mitochondrial function in stallion sperm. production of low levels of ros is a normal byproduct of mitochondrial function, resulting from low levels of electron leakage during normal etc activity. these free radicals are highly reactive and can quickly oxidize lipids, cause dna mutations and lead to apoptosis. to minimize damage caused by free radicals such as peroxides and superoxide anions, sperm produce antioxidant substances to scavenge free radicals. reducing agents such as glutathione peroxidase and superoxide dismutase, which are produced during normal metabolism, exert antioxidant effects by converting free radicals into nonradical forms. although antioxidants are an efficient protective mechanism, excessive reactive oxygen species generation can overwhelm protective effects provided by these reducing agents. in a system primarily relying on glycolysis for progressive motility in sperm, supplementation with substrates supportive of oxidative phosphorylation would result in increased ros production. as a result, inadequate substrate composition of storage media can lead to mitochondrial dysfunction and oxidative damage; therefore, it is crucial to provide optimum storage conditions. since oxidative phosphorylation provides the energetic support for stallion sperm motility, the oxygen biosensor assay was utilized to determine the preferred substrate for sperm storage.3,5 consumption of oxygen was highest in groups provided with the substrates lactate or pyruvate; additional testing indicated a dose-dependent response of increasing mitochondrial activity with increasing substrate concentrations. because glucose can only be utilized through glycolysis, it was expected that lactate and pyruvate would better support sperm motility and mitochondrial function. progressive motility was highest in lactateand pyruvate-supplemented groups. understanding the preferred metabolic pathways of sperm is important, as being able to control metabolism is the basis of developing extenders and media. in sperm, slowing metabolism during preservation and increasing metabolism just before insemination is the ultimate goal. measurement of oxygen consumption is a reliable, high-throughput method of investigating dependency on oxidative phosphorylation for sperm motility, despite limitations to the oxygen biosensor assay. there are several methods to analyze preferred metabolic pathways underlying motility of sperm from various species. however, many of these methods suffer from a variety of pitfalls that limit interpretability of the resultant data. although the underlying metabolic preference for sperm motility has been well established in some species, there is an ongoing quest to identify ideal substrates for long term storage and artificial insemination calcium and sperm motility our recent studies demonstrated that stallion sperm motility is fueled primarily by mitochondrialproduced atp and that both mitochondrial oxidative function and motility decrease significantly with cryopreservation.5,30 additionally, cryopreservation alters stallion sperm ca2+ i signaling and homeostasis,31 known to have important roles in sperm motility.32,33 because mature sperm lack an intact er, the predominant intracellular ca2+ store in most cell types, it was previously believed that influx of extracellular ca2+ is the sole ca2+ source and regulator of ca2+ i homeostasis.34 however, there is now evidence that intracellular ca2+ stores are present in sperm of several species, including humans, mice and cattle,35,36 but ca2+ i stores have not yet been identified or characterized in stallion sperm. in other cell types, mitochondria act as ca2+ i stores, in addition to the er, with an important role in maintenance of ca2+ i homeostasis.37 specifically, increased oxphos activity likely triggers mitochondrial ca2+ uptake through the mitochondrial ca2+ uniporter, resulting in increased ca2+ mt stores, which increase mitochondrial oxidative function and atp production.38 investigation of ca2+ mt uptake in sperm are limited, but considerable evidence suggests maintenance of ca2+ mt homeostasis is essential for motility regulation in human39 and bovine sperm.40 we demonstrated that mitochondrial oxygen consumption (mitox) is an indicator of mitochondrial oxidative balance and is likely involved mechanistically in calcium function. a new understanding of ca2+ homeostasis in context with sperm oxidative function will 356clinical theriogenology • volume 11, number 3 • september 2019 support development of rational semen and stallion therapeutic intervention and is needed in the ai industry. cryopreservation-induced alterations in ca2+ i dynamics may significantly contribute to reductions in mitochondrial oxidative function and motility that we previously reported for cryopreserved stallion sperm. however, the role of sperm mitochondria in ca2+ i regulation and its subsequent impact on atp production and motility have never been investigated in stallions. sperm morphology and reactive oxygen species despite being widely accepted as a negative indicator of sperm health in most species, implications of increased ros in stallion sperm have been highly debated.41 stallion sperm fuel motility almost exclusively via mitochondrial-produced atp, in contrast to most species, which use primarily glycolytic42,43 pathways to produce atp. increased mitochondrial function and etc electron flow results in increased passive electron leakage, increases in ros production and oxidative stress accompany increases in mitochondrial activity. it has been proposed that the sperm in an ejaculate that generate high ros concentrations are not defective, but actually may be the highly motile, more robust sperm and that biomarkers of oxidative stress are a positive indicator of stallion fertility.44 although not validated, this concept has been challenged by studies that have correlated increased ros with decreased motility, viability and mitochondrial function.3,5 ros evaluation is a promising prospective addition to diagnostic semen evaluation for stallion infertility if it can be definitively established as a positive or negative biomarker of stallion sperm function. in stallions, a positive correlation between pregnancy rates and percentage of morphologically normal sperm and a negative correlation between pregnancy rates and midpiece or tail abnormalities has been reported.45 ros production in human sperm has been positively correlated with abnormal sperm morphologies including abnormal heads, cytoplasmic droplets, midpiece, and tail abnormalities.46,47 if morphologic abnormalities are correlated with elevated ros concentrations in stallion sperm, this will strongly implicate ros as a negative biomarker of sperm function and fertility in stallions. recent technological advances in imaging flow cytometry demonstrates strong promise for evaluation of stallion semen.48 the imagestream® instrument has been successfully used for evaluation of human49 and bull50 semen. imagestream® technology is unique in that it enables generation of high throughput flow cytometry data with parallel fluorescence and brightfield microscopy, which has tremendous potential for uses in both clinical and research settings. within a research setting, imagestream® technology can be used to generate morphologic and fluorescence data with sample sizes large enough for statistical significance. further, the ability of the imagestream® to analyze fixed or live cells allows a wider range of applications in semen assessment than fluorescence microscopy alone, which requires nonmotile cells for accurate morphologic assessment. although the cost and size of the imagestream® system limits clinical applications, it is possible that fixable assays for negative biomarkers can be performed by clinicians and sent to a laboratory for further analysis. conclusion this review has focused on the function and structure of sperm mitochondria and their role in sperm motility and morphology, primarily in stallions and other livestock. greater understanding of negative and positive influences on sperm motility and abnormal morphology, which are highly variable in stallions, would have great benefit to the breeding industry, particularly for stallion management. improved precision and accuracy of sperm measurements may be gained without additional samples taken during a breeding soundness exam or when shipping chilled or frozen semen. in this review, we also highlighted the translational nature of basic cell biology and its application to sperm and ultimately to various breeding industries. conflict of interest authors claim no direct or indirect affiliation with any manufacturer listed in text. any information regarding equipment and various manufacturers is for readers’ reference only. 357 clinical theriogenology • volume 11, number 3 • september 2019 acknowledgement authors thank uc davis center for equine health for generous research and animal support for some of the studies cited in this manuscript. references 1. losano j, padin j, mendez-lopez i, et al: the stimulated glycolytic pathway is able to maintain atp levels and kinetic patterns of bovine epididymal sperm subjected to mitochondrial uncoupling. ox med cell longev id 2017: 1682393;1-8. 2. hu c, zhuang x, wei y, et al: comparison of mitochondrial function in boar and bull spermatozoa throughout cryopreservation based on jc-1 staining. cryo letters 2017;38;75-79. 3. darr c, cortopassi g, datta s, et al: mitochondrial oxygen consumption is a unique indicator of stallion sperm spermatozoal health and varies with cryopreservation media. theriogenology 2016:86;1382-1392. 4. davila m, munoz p, tapia j, et al: inhibition of mitochondrial complex i leads to decreased motility and membrane integrity related to increased hydrogen peroxide and reduced atp production, while the inhibition of glycolysis has less impact on sperm motility. 2015:plos one 10:e0138777. 5. darr c, varner d, teague s, et al: lactate and pyruvate are major sources of energy for stallion sperm with dose effects on mitochondrial function, motility, and ros production. biol reprod 2016:95;1-11. 6. gibb z, lambourne s, quadrelli j, et al: l-carnitine and pyruvate are prosurvival factors during the storage of stallion spermatozoa at room temperature. biol reprod 2015:93;1-9. 7. ball b: oxidative stress, osmotic stress and apoptosis: impacts on sperm function and preservation in the horse. anim reprod sci 2008:107;257-267. 8. gonzalez-fernandez l, morrell j, pena f, et al: osmotic shock induces structural damage on equine spermatozoa plasmalemma and mitochondria. theriogenology 2012:78:415-422. 9. may-panloup p, chrétien mf, savagner f, et al: increased sperm mitochondrial dna content in male infertility. hum reprod 2003:18:550-556. 10. malik a, czajka a: is mitochondrial dna content a potential biomarker of mitochondrial dysfunction? mitochondrion 2013:13:481-492. 11. ho h, wey s: three dimensional rendering of the mitochondrial sheath morphogenesis during mouse spermiogenesis. microsc res tech 2007:70:719-723. 12. ramalho-santos j, amaral s: mitochondria and mammalian reproduction. mol cell endocrinol 2013:379:74-84. 13. varner d, johnson l: from a sperm’s eye view: revisiting our perception of this intriguing cell. in: mckinnon ao, vaala we, varner d: editors. equine reproduction. 2nd edition, ames: wiley-blackwell; 2011. p. 1491-1497. 14. peña fj, rodríguez martínez h, tapia ja, et al: mitochondria in mammalian sperm physiology and pathology. reprod domest anim 2009:44:345-349. 15. mannella c: structure and dynamics of the mitochondrial inner membrane cristae. bbamol cell res 2006: 1763; 542-548. 16. nicholls d, ferguson s. cell biology of the mitochondrion. chapter 10. in: bioenergetics. 4th edition. san diego: academic press; 2013. p. 303-325. 17. ortega ferrusola c, gonzález fernández l, salazar sandoval c, et al: inhibition of the mitochondrial permeability transition pore reduces “apoptosis like” changes during cryopreservation of stallion spermatozoa. theriogenology 2010:74;458-465. 18. paoli d, gallo m, rizzo f, et al: mitochondrial membrane potential profile and its correlation with increasing sperm motility. fertil steril 2011:95;2315-2319. 19. piomboni p, focarelli r, stendardi a, et al: the role of mitochondria in energy production for human sperm motility. int j androl 2011:35;109-124. 20. saraste m: oxidative phosphorylation at the fin de siècle. science 1999:283;1488-1493. 21. wolken g, arriaga e: simultaneous measurement of individual mitochondrial membrane potential and electrophoretic mobility by capillary electrophoresis. anal chem 2014:86;4217-4226. 22. gibb z, lambourne s, aitken: the paradoxical relationship between stallion fertility and oxidative stress. biol reprod 2014:91;77. 23. hung p, miller m, meyers s, et al: sperm mitochondrial integrity is not required for hyperactivated motility, zona binding, or acrosome reaction in the rhesus macaque. biol reprod 2008:79;367-375. 24. meyers sa: cryostorage and oxidative stress in mammalian spermatozoa. in: agarwal a, aitken j, alvarez j: editors. studies on men’s health and fertility, oxidative stress in applied basic research and clinical practice. 1st edition, new york: springer; 2012. p. 41-56. 25. aitken r, lambourne s, gibb z: the john hughes memorial lecture: aspects of sperm physiology—oxidative stress and the functionality of stallion spermatozoa. j eq vet sci 2014:34:17-27. 26. wei y: mitochondrial dna alterations as ageing-associated molecular events. mutat res 1992:275;145-155. 27. volpe s, leoci r, aiudi g, et al: relationship between motility and mitochondrial functional status in canine spermatozoa. reprod domest anim 2009:2;275-278. 358clinical theriogenology • volume 11, number 3 • september 2019 28. davila m, muñoz p, gallardo bolaños j, et al: mitochondrial atp is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation, reproduction 2016:152;683-694. 29. mookerjee s, brand m: measurement and analysis of extracellular acid production to determine glycolytic rate. journal of visualized experiments/: jove 2015:106;53464. pmc 30. darr c, moraes l, scanlan t, et al: sperm mitochondrial function is affected by stallion age and predicts post-thaw motility. j eq vet sci 2017:50;52-56, 31. albrizio m, moramarco a, nicassio m, et al: localization and functional modification of l-type voltage-gated calcium channels in equine spermatozoa from fresh and frozen semen. theriogenology 2015:83;421-429. 32. publicover s, harper c, barratt, c: [ca2+]i signaling in sperm--making the most of what you’ve got. nat cell biol 2007:9;235-242. 33. miller, m, mansell s, meyers s, et al: flagellar ion channels of sperm: similarities and differences between species. cell calcium 2015:58;105-113. 34. lishko p, mannowetz n: catsper: a unique calcium channel of the sperm flagellum. curr opin physiol 2018:2;109-113. 35. correia j, michelangeli f, publicover s: regulation and roles of ca2+ stores in human sperm. reproduction 2015:150;r56-r76. 36. lawson c, dorval v, goupil s, et al: identification and localisation of serca 2 isoforms in mammalian sperm. mol hum reprod 2018:13;307-316. 37. rizzuto r, de stefani d, raffaello a, et al: mitochondria as sensors and regulators of calcium signalling. nat rev mol cell biol 2012:13;566-578. 38. bravo-sagua r, parra v, lópez-crisosto c, et al: calcium transport and signaling in mitochondria. comp physiol 2017:7623-634. 39. bravo a, treulen f, uribe p, et al: effect of mitochondrial calcium uniporter blocking on human spermatozoa. andrologia 2015:47;662-668. 40. rodriguez p, satorre m, beconi m. 2012. effect of two intracellular calcium modulators on sperm motility and heparininduced capacitation in cryopreserved bovine spermatozoa. anim reprod sci 2012;131:135-142. 41. gibb z, lambourne s, curry b, et al: aldehyde dehydrogenase plays a pivotal role in the maintenance of stallion sperm motility. biol reprod 2016:94;133. 42. darr c, martorana k, scanlan t, et al: the effect of low oxygen during the early phases of sperm freezing in stallions with low progressive motility: can we improve post-thaw motility of stallion sperm? j eq vet sci 2016:42;44-51. 43. ford, w: glycolysis and sperm motility: does a spoonful of sugar help the flagellum go round? hum reprod update 2006:12;269-274. 44. lei xg, zhu jh, cheng wh, et al: paradoxical roles of antioxidant enzymes: basic mechanisms and health implications. physiol rev 2016:96:307-364. 45. saacke, r: sperm morphology: its relevance to compensable and uncompensable traits in semen. theriogenology 2008:70;473-478. 46. aziz n, saleh ra, sharma r, et al: novel association between sperm reactive oxygen species production, sperm morphological defects, and the sperm deformity index. fertil steril 2004:81;349-354. 47. said tm, aziz n, sharma r, et al: novel association between sperm deformity index and oxidative stress-induced dna damage in infertile male patients. asian j androl 2005:7;121-126. 48. bulkeley e, meyers s: novel use of imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species (ros) in stallion sperm. j eq vet sci 2018:66:45. 48. buckman c, george t, friend s, et al: high throughput, parallel imaging and biomarker quantification of human spermatozoa by imagestream flow cytometry. syst biol reprod med 2009:55:244-251. 49. kennedy c, krieger k, sutovsky m, et al: protein expression pattern of pawp in bull spermatozoa is associated with sperm quality and fertility following artificial insemination. mol reprod dev 2014:81:436-449. 359 clinical theriogenology • volume 11, number 3 • september 2019 360clinical theriogenology • volume 11, number 3 • september 2019 16. nicholls d, ferguson s. cell biology of the mitochondrion. chapter 10. in: bioenergetics. 4th edition. san diego: academic press; 2013. p. 303-325. 20. saraste m: oxidative phosphorylation at the fin de siècle. science 1999:283;1488-1493. omniblank: 2018: attaining reproductive solutions through activity monitoring attaining reproductive solutions through activity monitoring r. l. nebel select sires inc., plain city, oh abstract today’s activity monitoring systems do much more than just identify cow/heifer for insemination. the addition of rumination monitoring allows the identification of health events along with reproductive events. performance of automated activity monitoring (aam) systems in combination with timed artificial insemination (tai) compared to tai exclusively has been widely researched and adopted. detecting a high percentage of cows in estrus is essential to maintain reproductive performance in dairy herds using ai. the dairy records management systems reported a mean yearly estrus detection rate on 8,749 us holstein herds (including all reproductive management strategies) as 46.5% in january of 2018.1 automated activity monitoring technologies are an available alternative to supplement or replace visual estrus detection. parameters with potential for aam include mounting events, activity level, lying time, rumination and eating times, feeding events, progesterone levels, body temperature, and more. the automatic measurement of chewing and ruminating activity can enable the early detection of feeding deficiencies and assist in ration adjustments. feeding and ruminating behaviors have traditionally been monitored through visual observation or video recording methods, but these methods are time consuming and only practically used in research settings. commercially marketed technologies recording feeding behavior, rumination, and lying behavior performed well when compared with visual observation. rumination behavior was most variable from visual observation. similar performance to visual observations and other validated technologies indicates dairy cattle behavioral monitors to provide accurate information for use in dairy cow management. introduction activity monitoring systems are now the backbone of many herd reproductive management systems. this technology is helping herds achieve reproductive results that seemed impossible only a decade ago. the growth in usage and performance of aam technology is one of the reasons we are now seeing many herds achieving a pregnancy rate of thirty percent or higher. activity monitoring systems do much more than just identify cow/heifer for insemination they provide new insight into individual animal characteristics. the addition of rumination monitoring allows the identification of health events along with reproductive events. the milk a cow produces today is dependent on what she consumed yesterday thus it allows for an advanced signal prior to any clinical signs. it must be remembered that an increase in activity is a secondary sign of estrus and systems that minimize false positive alerts (cow with an estrus alert that are not in estrus), and maximize true or positive alerts will give optimum results. every aam system has three basic components: sensor, receiver of data from the sensor to the software, and software. neck activity, ear activity, leg activity, step count, lying bouts, lying time, rumination, feeding time, and reticulorumen temperature are useful as predictors of estrus. rumination time is positively associated with milk yield in early-lactation across all parities. algorithms combining decreases in rumination and increasing lying or resting minutes are used to declared health alerts. performance of aam monitoring systems in combination with tai compared to tai have been reported. monitoring of activity for detection of estrus detection of estrus was the initial function of an amm systems.2,3 despite the widespread adoption of hormonal synchronization protocols that allow for tai plays an important role in the reproductive management of most dairies. increase physical activity is a secondary expression of estrus in cattle, and a new generation of aam systems that continuously monitor physical activity to predict timing of insemination are now available. seventy to eighty percent of eligible lactating cows and seventy to ninety percent of eligible dairy heifers monitored using an amm system are usually detected in estrus. there are many issues that influence the percentage of animals detected by a monitoring system. these clinical theriogenology • volume 10, number 3 • september 2018247 issues always relate back to the nutrition, cow comfort and general health. extended the length and higher the peak in activity, the more likely a cow will be detected accurately by the system. recent work done at the university of british columbia4 reported factors related to peak activity levels and duration when monitoring cows with commercially available activity systems. each cow was fitted with an activity collar and a leg-mounted pedometer. the neck collar and leg-mounted pedometer had an estrus detection rate of 89.6 percent and 85.5 percent, respectively, during the trial. this research showed that a low body condition score was associated with reduced peak and duration. they also reported more pregnancies per insemination from cows that had a higher peak activity level. lactation and secondary signs of heat were also related to estrus. this recent canadian study,4 reproductive performance between two approaches to management of first insemination on commercial farms, one based on the use of an amm system combined with tai if cows were not identified in estrus by 25 days after the voluntary waiting period and the other based exclusively on tai. heavy reliance on an aam system was compared with a double ovsynch protocol, which has been reported to result in high pregnancies per insemination, particularly in primiparous cows, in intensively managed confined dairy herds 5,6 the study design provided for all cows in each treatment to be inseminated by 88 days in milk. the design also allowed a proportion of cows in the aam + ovsynch treatment to be inseminated twice by the time cows in the double ovsynch treatment received their first insemination, which explains the difference between the proportions of cows pregnant in the aam + ovsynch treatment at first ai and at 88 days in milk. the proportion of cows pregnant at 88 days in milk was not a biological or specific economic endpoint but, given the design, was used to describe reproductive performance with the two reproductive management practices. one of the potential advantages of using an aam system is to have the opportunity to re-inseminate cows at approximately three-week intervals. overall, the treatments offered similar performance, as shown in previous studies 7,8 yet, as in other trials with multiple herds, relative performance of amm systems compared with tai differed between herds.7,9,10 one important result from these studies is the effect of treatments differed between the two herds. cows in the double ovsynch treatment in one herd were more likely to become pregnant at first ai than cows in the aam + ovsynch treatment. on the contrary, in the other herd, cows in the aam + ovsynch treatment were more likely to be pregnant by 88 days in milk than cows in the double ovsynch treatment. the interaction of treatment with herd remained for the time to pregnancy up to 200 days in milk. these interactions point to differences among herds in the relative performance of tai and incorporation of an aam system. this variability among herds has been shown previously for amm systems,7,9 but has not been reported for double ovsynch (i.e., no farm by treatment interactions; 5,6 these studies were not designed to explain the variability in response to treatment between herds, but it can be hypothesized that differences in farm-specific characteristics and management, as well as the use of different amm systems, might have played a role, as may be expected, aam systems performed better in the herd in which the insemination rate was greater. although the authors could not formally explain the reasons behind the differences between herds, they noted that the success of estrus detection was likely an important determinant of the results that were obtained. in the aam + ovsynch treatment, the insemination rate was 57% greater in herd 1 than in herd 2 from 50 to 88 days in milk (1.52 vs. 0.97 inseminations per 21 cow-days). the probability of pregnancy at first ai and by 88 days in milk was also greater in herd 1 than in herd 2 for aam + ovsynch treatment. this difference is also reflected in the proportion of cows not identified in estrus by the aam before 75 days in milk. the proportion of cows (7% in herd 1 and 36% in herd 2) not detected in estrus between 50 and 75 days in milk is in the expected range, but this represents the ends of the distribution.8,9 their results with aam + ovsynch are consistent with controlled trials11 and observational studies12,13 that show that a greater insemination rate is a key determinant of good performance when employing estrus detection as part of a reproductive management program. barring a substantial reduction in pregnancies per insemination, more inseminations will result in more pregnancies per unit of time, in this case contributing to the difference between treatments in the proportion of cows pregnant by 88 days in milk in herd 1. factors such as the prevalence of lameness, stocking density in the breeding pens, and the sensitivity of the aam system are likely involved in the probability of identifying cows in estrus by an aam system, but were not assessed in these studies. clinical theriogenology • volume 10, number 3 • september 2018 248 timing of insemination several research studies over the last five years in lactating dairy cows and heifers have indicated that insemination closer to the time of ovulation with gender sorted semen may improve conception rates.14,15 a previous study indicated that insemination between 8 to 16 hours after activity thresholds16 was ideal for use with conventional semen in lactating holstein cows. bombardelli, and co-workers14 reported the optimal timing of insemination using heatime aam system (scr engineers ltd., netanya, israel) occurred somewhere between 23 to 41 h after the reaching the activity threshold activity level in lactating jersey cows. inseminating 16 plus hours after the onset of activity defined by cowmanager aam technology (agis, harmelen, the netherlands) resulted in in a greater conception rate compared to breeding early (0 to 8 hours) in lactating jersey cows and heifers. although there were no significant differences in conception rate between 8 and 16 and 16 plus hours, the heifer and cow data along with data from other studies suggests breeding 16 to 24 h after the onset of activity should maximize conception rates when using gender sorted semen. rumination monitoring the automatic measurement of chewing and ruminating activity can enable the early detection of feeding deficiencies and assist in ration adjustments.17 feeding and ruminating behaviors have traditionally been monitored through visual observation or video recording methods,18 but these methods are time consuming and only practically used in research settings. additionally, monitoring animal behaviors using visual observation is subjective and open to observer interpretation.19 monitoring rumination and feeding behaviors with precision aam technologies could replace subjective visual observations while providing useful and continuous measures of these behaviors. technologies recording feeding and rumination behaviors have traditionally quantified these behaviors using chewing activity monitors (pressure and strain recorders;17,20,21 chewing activity (pressure and strain) and feeding behavior monitors are primarily used in research settings, but commercially available rumination and feeding behavior quantification methods have recently been developed and evaluated. bikker et al.22 evaluated a technology (cowmanager sensoor, agis, harmelen, the netherlands) monitoring rumination and feeding behavior through head movement and found these behaviors to be closely related to visual observations. the rumination time, chewing cycles, as well as rumination bouts detected by a different aam system (smartbow gmbh, jutogasse 3, 4675 weibern, austria) were highly associated (r > 0.99) with the analyses of video recordings.23 algorithm testing revealed in an underestimation of the average ± standard deviation rumination time per 1-h period by this aam system of 17.0 ± 35.3 s (i.e., −1.2%), compared with visual observations. the average number ± standard deviation of chewing cycles and rumination bouts was overestimated by 59.8 ± 79.6 (i.e., 3.7%) and by 0.5 ± 0.9 (i.e., 1.6%), respectively, compared with the video analyses. in summary, the agreement between the smartbow system with video analyses was excellent. from a practical and clinical point of view, the detected differences were negligible. however, further research is necessary on testing the system under various field conditions and on evaluating the benefit of implementing rumination data into herd management decisions. similarly, schirmann et al.18 evaluated a technology (hr tag, scr engineers ltd., netanya, israel) quantifying rumination sounds through a microphone and microprocessor and found a strong correlation between visual observations and this technology. another method to quantify feeding behaviors is through technologies describing when cows approach feeding areas. many of these types of technologies have been evaluated and found to be highly correlated with visual methods.24,25 these findings indicate the potential for technology performance to vary among aam systems for the same parameters through different methods. health alerts lying behavior is a parameter frequently quantified by aam.26-28 time spent lying can indicate cow comfort, welfare, and health changes.29 proudfoot et al.30 found sick or ill cattle spent more time lying apart from the herd. compared with other parameters measured by precision dairy-monitoring clinical theriogenology • volume 10, number 3 • september 2018249 technologies (e.g., feeding behavior, rumination, and activity), standing and lying events are easily visually monitored but the process remains time-consuming. previous studies evaluating lying behavior have reported strong correlations between technologies and visual or video monitoring. lying behaviors measured by the hobo data logger (hobo pendant g acceleration data logger, onset computer corporation, pocasset, ma) have strongly matched video monitoring observations.28 similarly, the afiact pedometer plus (afimilk, s.a.e. afikim, kibbutz afikim, israel) and icetag (a version of the iceqube intended for research; icerobotics ltd., edinburgh, scotland) monitored dairy cow lying behavior and observations were closely related to video monitoring observations.31,32 conclusion activity monitoring provides adequate opportunity to monitor cow health, comfort, and welfare but human factors related to successful adoption cannot be overlooked nevertheless activity information must be interpret and appropriate action taken. these systems change the way producers manage their animals. the primary successful application has been detection of estrus; however, rumination monitoring potential return on investment is greater by early detection of health events, decreasing death lost, and days in the hospital pen as well as early treatment to minimize antibiotic treatment is now being realized. references 1. drms: dairymetrics. dairy records management systems, raleigh, nc. 2018. 2. kiddy ca: variation in physical activity as an indication of estrus in dairy cows. j dairy sci 1977;60:235-243. 3 dransfield mbg, nebel rl, pearson re, et al: timing of insemination for dairy cows identified in estrus by a radiotelemetric estrus detection system. j dairy sci 1998;81:1874-1882. 4. denis-robichaud j, cerri rla, jones-bitton a, et al: performance of automated activity monitoring systems used in combination with timed artificial insemination compared to timed artificial insemination only in early lactation in dairy cows. j dairy sci 2018;101:624-636. 5. souza ah, ayres h, ferreira rm, et al: a new presynchronization system (double-ovsynch) increases fertility at first postpartum timed ai in lactating dairy cows. theriogenology 2008;70:208-215. 6. herlihy mm, crowe ma, diskin mg, et al: effects of synchronization treatments on ovarian follicular dynamics, corpus luteum growth, and circulating steroid hormone concentrations in lactating dairy cows. j dairy sci 2012;95:743-754. 7. neves rc, leslie ke, walton js, et al: reproductive performance with an automated activity monitoring system versus a synchronized breeding program. j dairy sci 2013;95:5683-5693 8. fricke pm, giordano jo, valenza a, et al: reproductive performance of lactating dairy cows managed for first service using timed artificial insemination with or without detection of estrus using an activity monitoring system. j dairy sci 2014;97:2771-2781. 9. dolecheck ka, heersche jr g, bewley jm: retention payoff–based cost per day open regression equations: application in a user-friendly decision support tool for investment analysis of automated estrus detection technologies. j dairy sci 2016;99:10182-10364. 10. burnett ta, madureira aml, silper bf, et al: integrating an automated activity monitor into an artificial insemination program and the associated risk factors affecting reproductive performance of dairy cows. j dairy sci 2017;100:50055018. 11. tenhagen b-a, drillich m, surholt r, et al: comparison of timed ai after synchronized ovulation to ai at estrus: reproductive and economic considerations. j dairy sci 2004;87:85-94. 12. ferguson jd, skidmore a: reproductive performance in a select sample of dairy herds. j dairy sci 2013;96:12691289. 13. denis-robichaud j, cerri rla, jones-bitton a, et al: performance of automated activity monitoring systems used in combination with timed artificial insemination compared to timed artificial insemination only in early lactation in dairy cows. j dairy sci 2017;101:624-636. 14. bombardelli gd, soares hf, chebelrc: time of insemination relative to reaching activity threshold is associated with pregnancy risk when using sex-sorted semen for lactating jersey cows. theriogenology 2015;85:533-539. 15. nebel rl: time of insemination relative to onset of activity threshold of cowmanager® is associated with pregnancy risk when using gender selected™ semen for jersey cattle. dairy vet sci j 2018;5:1-3. 16. stevenson js, hill sl, nebel rl, et al: ovulation timing and conception risk after automated activity monitoring in lactating dairy cows. j dairy sci 2014;97:1-13. 17. zehner n, niederhauser jj, nydegger f, et al: validation of a new health monitoring system (rumiwatch) for combined automatic measurement of rumination, feed intake, water intake and locomotion in dairy cows. proc int conf agric eng cigr-ageng, 2012:c-438. clinical theriogenology • volume 10, number 3 • september 2018 250 18. schirmann k, von keyserlingk mag, weary dm, et al: technical note: validation of a system for monitoring rumination in dairy cows. j dairy sci 2009;92:6052-6055. 19. weary dm, huzzey j, von keyserlingk m: board-invited review: using behavior to predict and identify ill health in animals. j anim sci 2009;87:770-777. 20. beauchemin ka, zelin s, genner d, et al: an automatic system for quantification of eating and ruminating activities of dairy cattle housed in stalls. j dairy sci 1989;72:2746-2758. 21. kononoff pj, lehman ha, heinrichs aj: technical note: a comparison of methods used to measure eating and ruminating activity in confined dairy cattle. j dairy sci 2002;85:1801-1803. 22. bikker jp, van laar h, rump p, et al: technical note: evaluation of an ear-attached movement sensor to record cow feeding behavior and activity. j dairy sci 2014;97:2974-2979. 23. devries tj, von keyserlingk mag, weary dm, et al: technical note: validation of a system for monitoring feeding behavior of dairy cows. j dairy sci 2003;86:3571-3574. 24. chapinal n, veira dm, weary dm, et al: technical note: validation of a system for monitoring individual feeding and drinking behavior and intake in group-housed cattle. j dairy sci 2007;90:5732-5736. 25. mcgowan j, burke c, jago j: validation of a technology for objectively measuring behavior in dairy cows and its application for estrous detection. proc n z soc anim prod 2007; p. 136. 26. o’driscoll k, boyle l, french p, et al: the effect of out-wintering pad design on hoof health and locomotion score of dairy cows. j dairy sci 2008; 91:544-553. 27. ledgerwood dn, winckler c, tucker cb. evaluation of data loggers, sampling intervals, and editing techniques for measuring the lying behavior of dairy cattle. j dairy sci 2010;93:5129-5139. 28. haley db, rushen j, de passille am: behavioral indicators of cow comfort: activity and resting behavior of dairy cows in two types of housing. can j anim sci 2000;80:257-263. 29. proudfoot kl, jensen mb, weary dm, et al: dairy cows seek isolation at calving and when ill. j dairy sci 2014;97:2731-2739. 30. mattachini g, antler a, riva e, et al: automated measurement of lying behavior for monitoring the comfort and welfare of lactating dairy cows. livest sci 2013;158:145-150. 31. mattachini g, riva e, bisaglia c, et al: methodology for quantifying the behavioral activity of dairy cows in freestall barns. j anim. sci 2013;91:4899-4907. clinical theriogenology • volume 10, number 3 • september 2018251 clinical theriogenology • volume 10, number 3 • september 2018 252 omniblank: << /ascii85encodepages false 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1 contact lorenzo segabinazzi lgseg@hotmail.com © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10243, http://dx.doi.org/10.58292/ct.v16.10243 technique report modified diff-quick staining for canine sperm morphology mariana mazzuchini,a guilherme novello,a lorenzo segabinazzib adepartment of veterinary surgery and animal reproduction, school of veterinary medicine and animal science, são paulo state university, botucatu, brazil bross university school of veterinary medicine, basseterre, st. kitts, west indies abstract sperm morphology assessment requires specialized microscopes or stains. diff-quick (dq) is considered a universal stain that is cost-effective; however, morphological evaluation of sperm using dq staining is poor and not encouraging. therefore, this study investigated modifications to the dq protocol to improve identification of morphological defects of dog sperm and compared the modified dq techniques with eosin-nigrosin, karras, and differential interference contrast microscopy [dic]). one ejaculate from each of 9 dogs was used. to perform the proposed modified dq techniques (dq1 and dq2), dried semen smears were fixed by immersing for 10 seconds in solution 1 of dq and 5 minutes each in solutions ii and iii of the kit. after the third stain solution, slides in dq1 were rinsed in water whereas slides in dq2 were not rinsed but were vertically supported to facilitate stain drainage. results suggested that the standard dq protocol overestimated normal sperm and detached heads whereas underestimated abnormal heads and total defects compared to dic, karras, eosin-nigrosin, and dq2. acrosome abnormalities were only detectable with karras, dic, and dq2. in conclusion, prolonging exposure to dq staining solutions enhanced sensitivity in sperm morphological evaluation, and avoiding rinse as a final step in the dq protocol improved visualization of certain acrosome defects in dog sperm. therefore, modified dq techniques can serve as a viable alternative for dog sperm morphology evaluation in clinical practice. keywords: canine, semen, dip-quick, breeding soundness examination, male dog introduction canine breeding market development is ongoing. primary purpose of dog breeders is to select animals with desired breed standards; therefore, the lack of selection for fertility has become a problem in companion animals, as many purebred dogs are subfertile. additionally, inbreeding, a common practice in the dog industry, has decreased reproductive capacity, with low semen quality in male dogs.1,2 therefore, breeding soundness examination is essential to determine male dog reproductive ability for breeders. andrological examination include physical and genital examinations, and semen collection and analysis.3 physical examination is essential to determine congenital abnormalities, mating capability, and dog’s general health. in contrast, specific genital examination can identify problems of prostate, testes, and penis.3,4 more specifically, semen evaluation is essential to predict fertilizing capacity via determining sperm concentration, total and progressive motility, sperm plasma membrane integrity, and sperm morphology.5 sperm are translucent and visible under light microscopy for motility assessment and for sperm concentration via neubauer chamber. however, sperm morphology evaluation requires special microscopes, such as differential interference contrast microscopy (dic) or staining techniques to highlight sperm structures.1,6 several stains have been used in clinical practice to identify various cell types. among staining techniques, the diff-quick stain is considered as a universal cytological stain due to its low cost and availability. however, poor results of sperm smears stained with diff-quick discouraged its use for sperm morphology evaluation.7,8 although increasing the duration of slides exposure to diff-quick stains improved the sensitivity of this assay for dog9 and stallion10 sperm analysis, the sensitivity to detect certain major defects (e.g. head, acrosome, and midpiece) is still low.11 in addition, although studies12,13 have used diff-quick staining to assess dog sperm, to the best of our knowledge, there are only a few studies5,13,14 that compared various staining methods and their capacity to highlight sperm morphology disorders. therefore, we compared 2 commonly used staining techniques (karras and eosin-nigrosin) for sperm morphology evaluation in several species, specialized dic, and 3 diff-quick staining protocols to mailto:lgseg@hotmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10243 2 citation line: clinical theriogenology 2024, 16, 10243, http://dx.doi.org/10.58292/ct.v16.10243 evaluate their efficacy in identifying sperm morphological disorders in dogs. materials and methods study protocol was approved by the animal care and use committee of são paulo state university. study was conducted at the department of veterinary surgery and animal reproduction of são paulo state university, from march to july 2020. sampling strategy and slides staining semen samples collected from 9 dogs during students’ practical laboratories were used for this study. dogs belonged to and were housed at the department of veterinary surgery and animal reproduction of são paulo state university and had semen collected by digital stimulation technique. from each semen aliquot, 5 semen smears were prepared and an aliquot was diluted with 10% formol saline for evaluation using dic microscopy (leica microsystems, wetzlar, germany). each smear was stained and evaluated using the techniques described (table 1). for modified karras, dried semen smears were fixed with methanol for 10 seconds and dried on a slide warming table. afterward, the slides were immersed sequentially in rose bengal, tannin, and victoria blue dye for 90, 60, and 20 seconds, respectively, lightly rinsed with water after each stain, and dried at the end.15 eosin-nigrosin staining was performed by mixing 1 drop of semen and 1 drop of stain on a slide, then spreading the mixed semen-stain sample similar to blood smear and air-dried.16 to perform modified diff-quick techniques (dq1 and dq2), dried semen smears were fixed by immersing for 10 seconds in solution 1 (fixative-methanol) of diff-quick kit (instant prov; newprov). afterward, the slide was immersed for 5 minutes in each of the solutions ii (buffered solution of eosin y) and iii (buffered solution of thiazine dyes consisting of methylene blue and azure a) of the kit. following each step, the excess stain was removed from slides by tapping the slides vertically on a paper towel to facilitate stain drainage. after third stain solution, slides in the dq1 were rinsed in water and then airdried. in contrast, slides in dq2 were not rinsed after the final solution but were vertically supported to facilitate stain drainage and air drying. slides evaluation all slides were assessed by 2 experienced and blinded evaluators. stained semen smears were evaluated under an optical microscope (jenamed 2 zeiss: carl zeiss) whereas dic analysis was carried out with a phase contrast microscope using a wet-mount preparation. analyses were performed under 1000 x magnification with immersion oil. two hundred sperm from each sample were evaluated (100 sperm by each evaluator). abnormalities were classified as head abnormalities, midpiece defects, tail defects, proximal cytoplasmic droplets, distal cytoplasmic droplets, detached heads, and acrosome disorders. additionally, acrosome anomalies were counted separately, determining the number of sperm with acrosome defects in each 100 sperm. data analyses data were analyzed with graphpad prism 9.0.1. (graphpad software, san diego, ca, usa). gaussian distribution was evaluated by the kolmogorov-smirnov normality test. sperm morphology defects were tested using kruskal-wallis and dunn’s tests. dogs were accounted as a random effect whereas the staining groups as fixed effects. significance was set at p < 0.05 for all tests and significant tendency was determined with 0.05 < p < 0.1. data are presented as mean ± sem. degrees of linear correlations between methodologies were tested using spearman correlation test. high correlation was considered when r ≥ 0.7, moderate correlation when r ≥ 0.5 but < 0.7, and poor correlation when r < 0.5. results standard diff-quick overestimated the identification of normal sperm and detached heads (p < 0.05) and underestimated the identification of abnormal heads and total defects (p < 0.05) compared to dic, karras, eosin-nigrosin, and modified dq2 but was similar compared to modified dq1 (p > 0.05). modified dq1 presented intermediate results not different from other techniques (p > 0.05). all tests had the same efficacy for tail defects, detached head, and proximal and distal droplets (p > 0.05). modified dq1, standard diff-quick, and eosin-nigrosin techniques were not able to highlight acrosome abnormalities (detached acrosome and absence of acrosome) that were observed using karras, dic, and dq2 (p < 0.05, table 2). however, it is worth noting that the modified dq2 was enabled detection of detaching acrosome but no absent or swollen acrosome that were observed in the karras and dic staining techniques (p < 0.05). representative images of wet-mounted sperm slides evaluated by dic and sperm smears stained with classical sperm staining techniques karras and eosin-nigrosin are presented (figure 1). although there were differences in the number of sperm defects observed between staining techniques as described above, a moderate (r > 0.5) to high correlation (r ≥ 0.7) for the number of normal sperm was observed among all techniques table 1. staining techniques and their descriptions staining technique description karras modified karras staining.15 eosin-nigrosin manufacturer instruction (botuvital, botupharma, brazil).16 diff-quick (dq) manufacturer instructions (instant prov, newprov, brazil). modified dq1 (dq1) each smear was immersed for 5 minutes in solutions 2 and 3 of the dq-staining and rinsed with distilled water at the end.9 modified dq2 (dq2) each smear was immersed for 5 minutes in solutions 2 and 3 of the dq-staining and air-dried.8 http://dx.doi.org/10.58292/ct.v16.10243 citation line: clinical theriogenology 2024, 16, 10243, http://dx.doi.org/10.58292/ct.v16.10243 3 (table 3). however, standard diff-quick had poor correlation compared to dic and karras, respectively, for proximal droplets (r = -0.018 and r = 0.395) and tailless heads (r = 0.271 and r = 0.331). in addition, dic, karras, eosin-nigrosin, and dq2 had moderate to high correlations between each other to identify all sperm abnormalities (r > 0.05, table 3). representative images of sperm smears stained with 3 diffquick techniques are presented (figure 2). discussion our objective was to evaluate the ability of various diff-quick staining techniques to highlight morphological abnormalities in dog sperm. diff-quick is the most commonly available in human and veterinary medicine as a universal cytological stain8 and has been reported as a quick and reliable technique for human17,18 cat,19 and bull20 sperm morphology evaluation. however, in contrast, the poor delineation and staining of the cranial pole of the head and the contours of the sperm midpiece affect the sensitivity of diff-quick to identify major defects, such as head, acrosome, and midpiece defects and proximal droplets in stallion7 and dog sperm,11 even though basic alterations in sperm morphology (e.g. oversized or undersized heads, bent tails, and tailless heads) and the presence of immature germ or rounded cells can be detected using this technique.11 the reason for the poor staining of stallion and dog sperm compared to other species (e.g. human17,18 cat, clique ou toque aqui para inserir o texto.,19 and bull20) is not well known, but it has been hypothesized that structural changes in contents of the acrosome among species may be responsible for those differences.21 in addition, authors suggested that the overestimation of detached heads in diff-quick stained slides is associated with poor delineation and staining of the cranial pole of the head11 that caused the identification of these sperm as ‘normal’ detached heads. similar results have also been reported in stallion sperm stained with diff-quick.8 studies suggested that the increased exposure of the smears to diff-quick staining solutions could improve the sensitivity of this assay in dog9 and stallion10 semen smears analysis. it is worth noting that even though the increased time of smears in solutions ii and iii of diff-quick staining improved the identification of more specific abnormalities in dog sperm smears, the deletion of the rinsing step at the end of the staining protocol produced more reliable results for sperm morphological analysis. similar results have also been observed using this technique for staining stallion sperm table 2. mean and standard error of the mean of sperm morphological defects according to the technique performed morphology dic karras eosin-nigrosin modified dq1 modified dq2 diffquick normal 75.4 ± 2a 76.1 ± 1.7ab 76.4 ± 1.5ab 81.3 ± 1.9bc 77.8 ± 2.1ab 84.3 ± 2.4c abnormal heads 3.2 ± 0.6a 2.2 ± 0.4ab 2.5 ± 0.4ab 1.4 ± 0.3bc 2.3 ± 0.6ab 0.4 ± 0.2c abnormal midpiece 5.8 ± 0.9ab 7.4 ± 1.2a 6.4 ± 1.1ab 4.3 ± 0.8bc 6.6 ± 1.0ab 2.4 ± 1.3c pathologic tails 7.0 ± 1.4 8.1 ± 1.2 7.6 ± 1.1 7.0 ± 0.9 6.3 ± 1.2 5.8 ± 0.9 proximal droplet 1.1 ± 0.5 0.5 ± 0.1 0.6 ± 0.2 0.2 ± 0.1 0.2 ± 0.1 0.1 ± 0.1 distal droplet 4.4 ± 1.9 3.4 ± 1.1 3.1 ± 1.1 3.1 ± 1.6 3.3 ± 1.5 2.2 ± 2.0 detached head 1.7 ± 0.2a 2.1 ± 0.5a 2.2 ± 0.3a 2.8 ± 0.6ab 2.2 ± 0.5a 4.8 ± 0.5b all defects 24.6 ± 2.1a 23.9 ± 1.7ab 23.6 ± 1.5 18.7 ± 1.9bc 22.2 ± 2.1ab 15.7 ± 2.4c acrosome 1.8 ± 0.4a 1.4 ± 0.3a 0b 0b 1.3 ± 0.3a 0b a-cwithin a row, means without a common superscript differ (p < 0.05) modified diff-quick (dq) 1 and dq 2 consisted of slide fixation for 10 seconds in solution 1 (fixative-methanol) of diff-quick stain (instant prov; newprov, brazil). afterward, the slide was immersed for 5 minutes in each of solutions ii (buffered solution of eosin y) and iii (buffered solution of thiazine dyes consisting of methylene blue and azure a of the diff-quick kit. following each step, excess stain was removed from slides by tapping the slides vertically on a paper towel to facilitate stain drainage. after the third stain solution, slides in modified dq 1 were rinsed in water and then air-dried. in contrast, slides in modified dq 2 were not rinsed after the final solution but were vertically supported to facilitate stain drainage and air-dried. figure 1. canine morphological sperm analysis under a. differential interference contrast microscopy, b. karras, and c. eosin-nigrosin staining techniques. black arrow indicates sperm with a damaged acrosome and white arrow indicates sperm with an underdeveloped head and damaged midpiece. http://dx.doi.org/10.58292/ct.v16.10243 4 citation line: clinical theriogenology 2024, 16, 10243, http://dx.doi.org/10.58292/ct.v16.10243 table 3. correlation of dog sperm morphology assessed with various staining techniques technique dic karras eosin-nigrosin modified dq1 modified dq2 normal dic 1.000 karras 0.887 1.000 eosin-nigrosin 0.846 0.898 1.000 modified dq1 0.798 0.657 0.708 1.000 modified dq2 0.891 0.812 0.734 0.742 1.000 diff-quick 0.882 0.782 0.796 0.909 0.800 abnormal heads dic 1.000 karras 0.811 1.000 eosin-nigrosin 0.901 0.868 1.000 modified dq1 0.656 0.317 0.546 1.000 modified dq2 0.821 0.864 0.909 0.404 1.000 diff-quick 0.743 0.540 0.636 0.859 0.476 abnormal midpieces dic 1.000 karras 0.892 1.000 eosin-nigrosin 0.909 0.937 1.000 modified dq1 0.717 0.834 0.809 1.000 modified dq2 0.808 0.923 0.798 0.690 1.000 diff-quick 0.536 0.783 0.621 0.692 0.759 tail defects dic 1.000 karras 0.926 1.000 eosin-nigrosin 0.940 0.967 1.000 modified dq1 0.886 0.779 0.821 1.000 modified dq2 0.904 0.855 0.894 0.763 1.000 diff-quick 0.913 0.792 0.852 0.859 0.872 proximal droplets dic 1.000 karras 0.986 1.000 eosin-nigrosin 0.926 0.926 1.000 modified dq1 0.396 0.445 0.496 1.000 modified dq2 0.973 0.955 0.945 0.496 1.000 diff-quick -0.101 -0.080 -0.227 -0.158 -0.172 distal droplets dic 1.000 karras 0.974 1.000 eosin-nigrosin 0.977 0.982 1.000 modified dq1 0.975 0.987 0.969 1.000 modified dq2 0.979 0.985 0.966 0.994 1.000 diff-quick 0.959 0.972 0.981 0.941 0.939 (continued) http://dx.doi.org/10.58292/ct.v16.10243 citation line: clinical theriogenology 2024, 16, 10243, http://dx.doi.org/10.58292/ct.v16.10243 5 human sperm analysis with the ability to highlight acrosome abnormalities,18 in domestic animals, this staining technique has been less effective in identifying acrosome defects compared to other staining methods.8,14 however, it is also worth noting that even though the modified technique proposed herein highlighted sperm with detaching acrosome, besides dic and karras, no other method was able to identify sperm with an absent or swollen acrosome in the present study. an increment in time in each stain (> 5 minutes) associated with not rinsing the slides might result in a better outcome for viewing acrosome abnormalities in dogs. however, future studies are needed to address the effectiveness of diff-quick table 3. (continued) technique dic karras eosin-nigrosin modified dq1 modified dq2 tailless heads dic 1.000 karras 0.822 1.000 eosin-nigrosin 0.636 0.634 1.000 modified dq1 0.741 0.793 0.521 1.000 modified dq2 0.711 0.523 0.304 0.545 1.000 diff-quick 0.168 0.074 0.139 0.408 0.294 total defects dic 1.000 karras 0.862 1.000 eosin-nigrosin 0.904 0.933 1.000 modified dq1 0.798 0.718 0.769 1.000 modified dq2 0.748 0.709 0.751 0.657 1.000 diff-quick 0.890 0.803 0.867 0.897 0.761 acrosome defects dic 1.000 . . . karras 0.834 1.000 . . . modified dq1 0.746 0.643 . . 1.000 modified diff-quick (dq) 1 and dq 2 consisted of 10 seconds of fixation in solution 1 (fixative-methanol) of diff-quick stain (instant prov; newprov, brazil). afterward, the slide was immersed for 5 minutes in each of solutions ii (buffered solution of eosin y) and iii (buffered solution of thiazine dyes consisting of methylene blue and azure a) of the diff-quick kit. following each step, excess stain was removed from slides by tapping the slides vertically on a paper towel to facilitate stain drainage. after the third stain solution, slides in the modified dq 1 were rinsed in water and then air-dried. in contrast, slides in modified dq 2 were not rinsed after the final solution but were vertically supported to facilitate stain drainage and air-dried. figure 2. canine sperm smears stained with a. standard diff-quick, b. modified diff-quick 1, and c. modified diff-quick 2. slides were assessed using an optical microscope. black arrow indicates sperm with a detached acrosome. smears when compared to the standard diff-quick technique.8 under-stained sperm have also been associated if a slide with air-dried semen smear is dipped several times in each of 3 solutions, as recommended for blood smear staining10 that may corroborate with the findings of the present study in which sperm undergo a different staining process compared to cytological smears. another interesting finding of this study was that the proposed modified diff-quick technique (dq2) enabled identification of sperm with detaching acrosome. although diff-quick has been suggested as an easy and effective alternative for http://dx.doi.org/10.58292/ct.v16.10243 6 citation line: clinical theriogenology 2024, 16, 10243, http://dx.doi.org/10.58292/ct.v16.10243 stain to highlight the cranial region of sperm head or possible acrosome abnormalities if slides were left longer in the stains. conclusion diff-quick staining sensitivity for sperm morphological evaluation can be improved by prolonged exposure to the staining solutions. this technique has proven to be an alternative for assessing dog sperm morphology and can easily be introduced in clinical practice, although some care should be taken for acrosome evaluation. although no rinsing of slides at the end of staining protocol (modified dq 2) improved the visualization of some major defects (e.g. detaching acrosome) compared to the technique suggested,9 the current technique was unable to highlight acrosome defects in the same way as other standard methods for sperm evaluation, such as dic and karras. conflict of interest none to report. references 1. dai c, zhang z, shan g, et al: advances in sperm analysis: techniques, discoveries and applications. nat rev urol 2021;18: 447–467. doi: 10.1038/s41585-021-00472-2 2. marelli sp, beccaglia m, bagnato a, et al: canine fertility: the consequences of selection for special traits. reprod domest anim 2020;55:4–9. doi: 10.1111/rda.13586 3. lopate c: applied animal andrology: dog. in: chenoweth pj, lorton s: editors. manual of animal andrology. wallingford, uk: cabi international; 2022. p. 160–186. 4. soderberg sf: canine breeding management. vet clin north am small anim pract 1986; 16:419–433. doi: 10.1016/s0195 5616(86)50051-6 5. kolster ka: evaluation of canine sperm and management of semen disorders. vet clin north am small anim pract 2018;48:533–545. doi: 10.1016/j.cvsm.2018.02.003 6. arruda r, silva d, affonso f, et al: methods for assessment of sperm morphology and function: actual moment and future challenges. rev bras reprod anim 2011;35:145–151. 7. brito lfc, greene lm, kelleman a, et al: effect of method and clinician on stallion sperm morphology evaluation. theriogenology 2011;76:745–750. doi: 10.1016/j.theriogenology.2011. 04.007 8. segabinazzi lg, mercês chaves lf, araujo ea, et al: dip quick staining modified for morphological evaluation to equine spermatozoa. j equine vet sci 2017;55:71–75. doi: 10.1016/j. jevs.2017.02.015 9. kustritz m: determining the optimal age for gonadectomy of dogs and cats. j am vet med assoc 2007;231:1665–1675. doi: 10.2460/ javma.231.11.1665 10. pozor ma, zambrano gl, runcan e, macpherson m. usefulness of dip quick stain in evaluating sperm morphology in stallions. proc am ass equine practnrs 2012;58:506–510. 11. randall cj, negri ap, quigley km, et al: sexual production of corals for reef restoration in the anthropocene. mar ecol prog ser 2020;635:203–232. doi: 10.3354/meps13206 12. soler c, alambiaga a, martí ma, et al. dog sperm head morphometry: its diversity and evolution. asian j androl 2017;19: 149–153. doi: 10.4103/1008-682x.189207 13. wysokin’ ska a, wójcik e, chłopik a: evaluation of the morphometry of sperm from the epididymides of dogs using different staining methods. animals 2021;11:1–11. doi: 10.3390/ ani11010227 14. surmacz p, niwinska a, kautz e, et al: comparison of two staining techniques on the manual and automated canine sperm morphology analysis. reprod domest anim 2022;57:678–684. doi: 10.1111/rda.14100 15. papa fo, alvarenga ma, carvalho im, et al: karras spermatic coloration modified by the use of barbatimão (stryphnodendrum barbatiman). arq bras med vet zootec 1988;40:115–123. 16. hancock jl: a staining technique for the study of temperature-shock in semen. nature 1951:166: 323–324. doi: 10.1038/167323b0 17. oehninger s, kruger tf: sperm morphology and its disorders in the context of infertility. fertil steril 2020;2:75–92. doi: 10.1016/j. xfnr.2020.09.002 18. kruger tf, ackerman sb, simmons kf, et al: a quick, reliable staining technique for human sperm morphology. syst biol reprod med 1987;18:275–277. doi: 10.3109/01485018708988493 19. mota pc, ramalho-santos j: comparison between different markers for sperm quality in the cat: diff-quick as a simple optical technique to assess changes in the dna of feline epididymal sperm. theriogenology 2006;65:1360–1375. doi: 10.1016/j.theriogenology. 2005.08.016 20. bastos yhgb, da silva cf, gomes gm, et al: evaluation of different coloration techniques for smears obtained by aspiration biopsy puncture of bull testis. rev saúde 2015;6:05. doi: 10.21727/ rs.v6i2.961 21. foster ja, gerton gl: the acrosomal matrix. adv anat embryol cell biol 2016;220:15-33. doi: 10.1007/978-3-319-30567-7_2 http://dx.doi.org/10.58292/ct.v16.10243 https://doi.org/10.1038/s41585-021-00472-2 https://doi.org/10.1111/rda.13586 https://doi.org/10.1016/s0195-5616(86)50051-6 https://doi.org/10.1016/s0195-5616(86)50051-6 https://doi.org/10.1016/j.cvsm.2018.02.003 https://doi.org/10.1016/j.theriogenology.2011.04.007 https://doi.org/10.1016/j.jevs.2017.02.015 https://doi.org/10.1016/j.jevs.2017.02.015 https://doi.org/10.2460/javma.231.11.1665 https://doi.org/10.2460/javma.231.11.1665 https://doi.org/10.3354/meps13206 https://doi.org/10.4103/1008-682x.189207 https://doi.org/10.3390/ani11010227 https://doi.org/10.3390/ani11010227 https://doi.org/10.1111/rda.14100 https://doi.org/10.1038/167323b0 https://doi.org/10.1016/j.xfnr.2020.09.002 https://doi.org/10.1016/j.xfnr.2020.09.002 https://doi.org/10.3109/01485018708988493 https://doi.org/10.1016/j.theriogenology.2005.08.016 https://doi.org/10.1016/j.theriogenology.2005.08.016 https://doi.org/10.21727/rs.v6i2.961 https://doi.org/10.21727/rs.v6i2.961 https://doi.org/10.1007/978-3-319-30567-7_2 2018: effect of inflammatory response related to mastitis on dairy cattle reproduction effect of inflammatory response related to mastitis on dairy cattle reproduction carlos a. risco,a mohammad o. dahlb acenter for veterinary health sciences, oklahoma state university, stillwater, ok; bcollege of veterinary medicine, university of florida, gainesville, fl introduction mastitis is the inflammation of the mammary gland and is a common disease that affects dairy cattle health and wellbeing. economic losses attributed to mastitis include lower milk production, increase in the amount of discarded milk, labor and medical costs, and premature culling (fetrow, 2000). in addition to these costs, over the past two decades there has been mounting evidence that both clinical and subclinical mastitis reduce reproductive efficiency in dairy cattle (kumar et al., 2017; dahl et al., 2017). this paper discusses the effect of mastitis on dairy cattle reproduction from an inflammatory response basis of the mammary gland to infection. inflammatory response due to mastitis inflammation due to mastitis occurs when bacteria enter the mammary gland, multiply and produce toxins, enzymes, and cell-wall components, which stimulate production of various inflammatory mediators. the main impact of gram-negative bacteria is through lipopolysaccharides (lps), a component of the cell wall (sordillo and babiuk, 1991). the phagocytizing activity of neutrophils and macrophages destroys the bacteria and releases lps stimulating release of inflammatory mediators, particularly prostaglandin f2α (pgf2α). in addition, lps and lipopolysaccharide binding protein binds to granulocytes and macrophages stimulating synthesis of pro-inflammatory cytokines, particularly tumor necrosis factor-α (tnf-α) and interleukins (il-1α, il-1β, il-6, and il-8); tnf-α and il-1 are responsible for pyrogenic response to endotoxins (danek and zurek, 2014). experimentally induced coliform mastitis through intramammary inoculation of klebsiella pneumonia caused an increase of pgf2α in both milk and plasma (cullor, 1990). intravenous infusion of e. coli lps increased pgf2α and decreased progesterone concentrations in plasma (cullor, 1990). intramammary infusion of e. coli lps or streptococcus uberis, elevated somatic cell count (scc) in milk, and increased the activity of the inflammatory cytokines, tnf-α, il-1β, and il-8 in the milk, lymph, and circulation (rainard and paape, 1997; hockett et al., 2000; hoeben et al., 2000; paape et al., 2002; persson waller et al., 2003; rambeaud et al., 2003). inflammatory response due to mastitis can also be related to elevated nitrous oxide (no). hansen et al. (2004) reported that intramammary infusion of lps and streptococcus uberis caused an increase in no concentrations in blood or milk. gram-positive bacteria can also produce toxins, extracellular enzymes, or enterotoxins that can cause a host inflammatory response including release of pgf2α and pro-inflammatory cytokines that result in pyrexia (barker et al., 1998). clinical mastitis induced via intramammary infusion of streptococcus uberis suspension increased pgf2α concentration following oxytocin administration (hockett et al., 2000). effect of mastitis on dairy cattle reproduction fertility poor fertility from the inflammatory and systemic response to clinical mastitis is related to an effect on the estrous cycle, impairment of oocyte maturation and development before ovulation, and delayed ovulation or anovulation. a study by cullor (1990) indicated that mastitic cows have an altered interestrus interval (less than 18 days or more than 24 days), related to luteolysis as a function of pgf2α and/or pro-inflammatory cytokines. that is, pgf2α affects corpus luteum blood flow (miyamoto and shirasuna, 2009), and cytokines have extreme cytotoxic activity on the corpus luteum (schams and berisha, 2004). hansen et al. (2004) proposed a potential pathway by which infection of the mammary gland can reduce embryonic survival by modulating the hypothalamic–pituitary axis, ovary, oocyte and even the embryo. endotoxins from gram-negative bacteria can disrupt luteinizing hormone (lh) secretion due to clinical theriogenology • volume 10, number 3 • september 2018253 induction of secretion of cytokines such as ifn-a as well as cortisol. the loss of lh support can potentially lead to anovulation, delayed ovulation or formation of follicular cysts, due to inhibition of the lh surge. reduced lh secretion may also reduce progesterone secretion from the corpus luteum and thereby alter uterine function. in addition to apoptosis, tnf-a, disrupts the process of oocyte maturation. cytokines can also cause increased secretion of other molecules that are disruptive to oocytes and embryos. in response to tnfα and il-1b, pgf2α is produced by several tissues including endometrium that can cause luteolysis and interfere with oocyte maturation. elevated body temperature can also inhibit oocyte development before ovulation, processes in the oocyte occurring around the time of maturation (hansen et al 2004). pregnancy survival early embryonic stages are sensitive to inflammatory responses. in rats, blastocysts have a receptor for tnf-α (pampfer, 2001) which can cause apoptosis to embryo blastomeres leading to reduce inner cell mass number and affect embryo development (hansen et al., 2004). further, an in-vitro study reported that increased local concentrations of lps, pgf2α, has a deleterious effect on oocyte function and no on embryonic development (soto et al., 2003). another possible pathway for pregnancy loss is that pregnancy survival is compromised by the effect of pgf2α on the corpus luteum (cl) causing luteolysis and termination of pregnancy (pate, 1994). this effect could be greater during late embryonic and early fetal development stages, as there is evidence that cl tissues from the luteal phase is different from that for the pregnant phase in the protein expression level (chung et al., 2012). in a previous observational study, risco el al. (1999) showed that cows affected with clinical mastitis during the first 45 d of gestation were 2.7 times as likely to experience pregnancy loss within the following 90d after diagnosis of mastitis, compared to cows not affected with clinical mastitis. similarly, a recent matched case control study indicated that the odds of pregnancy loss in primiparous holstein cows were 2.2 times higher in cows affected with clinical mastitis during early gestation, compared to cows without mastitis (dahl et al., 2018, unpublished). there is evidence that mastitis before or after breeding negatively impacts embryo survival. circulating inflammatory mediators may reach the ovaries and affect follicular growth, and subsequently compromise oocyte and embryo development. ribeiro et al. (2016) reported that the quality of embryos and the number of live embryos 1 to 5 or 6 d after breeding was reduced in cows exposed to clinical mastitis and other diseases before breeding. in addition, the uterine concentration of interferon tau (ifnτ), was reduced in cows with clinical mastitis before breeding (ribeiro et al., 2016). interferon tau is involved in the pregnancy recognition process by stimulating gene expression in the endometritum necessary for embryo elongation and implantation (brooks et al., 2014). in a recent matched case control study limited to multiparous holstein cows with clinical mastitis within 42 days before breeding in parities four or later, were more likely to experience pregnancy loss, compared to cows without mastitis in parities two or three (or = 3.25, or 2.69, respectively; dahl et al., 2018 unpublished). impact of mastitis and parity on embryo development and pregnancy may be exacerbated in older cows because of agerelated changes in gene characteristic of bovine endometrial cells. endometrial cells obtained from aged cows exhibited spontaneously higher levels of inflammatory signaling and dysfunction of cell division associated with chronic inflammation, that may lead to lower ifn-τ in older (> 150 months) cows compared to younger (28 to 68 months) cows (tanikawa et al., 2017). in another study, (hernandez et al., 2012), clinical mastitis during early lactation in combination with low body condition (bcs ≤ 2.75) at 70 dim increased the risk of pregnancy loss in dairy cows. in that study, it was suggested that events of clinical mastitis during the first 10 to 50 days in milk can induce a prolonged negative energy balance in cows leading to losses of body weight and body condition days before or after insemination, and low body condition is a known predisposing factor for embryonic loss. reproductive performance adequate reproductive performance of the lactating herd is paramount to increase and sustain profitability in a dairy herd. improved reproductive performance reduces the time from calving to clinical theriogenology • volume 10, number 3 • september 2018 254 conception; thereby increasing the amount of milk produced per day of herd lifetime, reduces the number of cows culled for reproductive failure, and accelerates the rate of genetic progress, which collectively increase herd income (risco et al, 1998). observational studies indicate that clinical mastitis lowers reproductive performance from an increase in the number of days to first service, days non-pregnant, and services per conception (moore et al., 1991; barker et al., 1998). these changes can be related to the effect of clinical mastitis on the estrous cycle, cl function, oocyte maturation, delayed ovulation or anovulation, and embryonic survival. influence of pathogen causing mastitis the type of pathogen causing mastitis has a similar effect on fertility in dairy cattle. barker et al. (1998) reported that services per conception did not differ among cows with clinical mastitis caused by gram positive or negative pathogens. in agreement with this finding, other studies have shown that gram positive or negative bacteria had a similar effect on conception (schrick et al., 2001; santos et al., 2004). similarly, risco et al. (1999) reported no difference in abortion among bacteria types. the implication is that the inflammatory response elicited by gram-positive bacteria is similar to that of gram-negative bacteria. however, huszenicza et al. (1998) reported significantly higher rate of premature luteolysis in mastitis induced by gram negative compared to gram positive bacteria. however, the occurrence of mastitis and type of pathogen in relation to time of breeding affects conception rate. hertl et al. (2010) found that the occurrence of clinical mastitis between 14 days before artificial insemination (ai) or 7 days after ai, resulted in a significant effect on probability of conception and was affected by pathogen type. mastitis due to gram-negative bacteria occurring between 8 and 14 days before ai was associated with a 32% reduction in conception rate, while mastitis caused by grampositive and gram-negative bacteria from 1 to 7 days before ai was associated with a 50% reduction in probability of conception. similarly, all types of clinical mastitis occurring from 0 to 7 days after ai were associated with reduced conception rates. gram positive bacteria reduced the probability of conception by 47%, while gram negative reduced it up to 80%. effect of subclinical versus clinical mastitis from the studies mentioned above, the systemic inflammatory response due to clinical mastitis is well known. in contrast, there has been a paucity of research on the systemic response from inflammation in cows with subclinical mastitis. this lack of information, may lead to the belief that the subclinical form of mastitis may not adversely affect fertility. however, various studies have indicated that subclinical mastitis does have an impact on fertility. subclinical mastitis, defined as the presence of the same pathogen in at least two consecutive milk samples, with an elevated somatic cell count (scc), resulted in lower reproductive performance of lactating cows, and was comparable to cows with clinical mastitis (schrick et al, 2001). furthermore, a negative effect of a linear scc ≥ 4.5 within 30 days before ai on embryo survival was reported (moore et al, 2005). pinedo et al. (2009) evaluated the effect of subclinical mastitis (lnscc ≥4.5) during early lactation on reproductive performance, and estimated their association with the risk of abortion in a population of central-southern chilean dairy cattle. in that study, time to first breeding was 21.8 days longer in cows with at least one high lnscc before the first breeding compared to controls. cows with at least one high lnscc before the fertile breeding had an increment in time to conception of 48.7 days and required on average 0.49 more services to conceive. the odds of conception at first service in cows with a high lnscc within 30 d before or after breeding were 0.85 (0.81 to 0.89; 95% confidence interval) [0.82 (0.78 to 0.87; 95% confidence interval)] times the odds of conception for cows without a high lnscc during that period. cows registering a high lnscc during the first 90 d of gestation had an increased risk of abortion. in another study, lavon et al. (2010) reported that that subclinical, long-term mastitis and short-term clinical events induced delayed ovulation in about 30% of the cows. this finding was associated with low estradiol concentrations that subsequently induced low or delayed or no lh surge, without any change in pulsatile lh secretion, suggesting that mastitis directly affected follicular functioning. clinical theriogenology • volume 10, number 3 • september 2018255 conclusion inflammation associated with clinical mastitis negatively affects reproductive performance in dairy cattle. in particular, lps and pgf2α can have deleterious consequences on oocyte function, inflammatory cytokines like tnf-α and no can disrupt embryonic development. although, subclinical mastitis also impairs reproductive performance, its deleterious effect on fertility is yet to be determined. consequently, veterinarians engaged in reproductive management of dairy cattle, should ensure that mastitis control practices are optimal in order to 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2001:84:1407-1412. soto p, natzke rp, hansen pj: identification of possible mediators of embryonic mortality caused by mastitis: actions of lipopolysaccharide, prostaglandin f2alpha, and the nitric oxide generator, sodium nitroprusside dihydrate, on oocyte maturation . am j reprod immunol 2003;50:263-272. sordillo lm, babiuk la: controlling acute escherichia coli mastitis during the periparturient period with recombinant bovine interferon-gamma. vet microbiol 1991;28:189-198. tanikawa n, ohtsu a, kawahara-miki r, et al: age-associated mrna expression changes in bovine endometrial cells in vitro. reprod biol endocrinol 2017;15:63. clinical theriogenology • volume 10, number 3 • september 2018257 clinical theriogenology • volume 10, number 3 • september 2018 258 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2011: determination of core content throughout the curriculum determination of core content throughout the curriculum jared danielson college of veterinary medicine, iowa state university, ames, ia this contribution is written in response to a request to assist the academic theriogenology community in their efforts to: 1. define a theriogenology core for veterinary schools and 2. determine whether or not students are actually learning what has been identified as core. this is part of a process that is often referred to as a needs assessment or front-end analysis. i will describe the aspects of a needs assessment that are most relevant to these two questions, discuss what members of the theriogenology community have already done to answer these questions, and then suggest some additional steps that you might consider taking, both as a community and as individuals. determining the core curriculum with discrepancy analysis determining the core is another way of saying “deciding what should be taught to everybody” in a particular area of study. there a number of ways to approach this decision. one common approach is to use a discrepancy analysis. as the term implies, a discrepancy analysis involves determining the discrepancy, or gap, between what students should know, and what they currently know. therefore, at the heart of this particular curricular problem is the ability to measure first what veterinary graduates of avma-accredited colleges need to know about theriogenology upon graduation, and second, what veterinary graduates of avma-accredited colleges actually do know about theriogenology upon graduation. the discussion in this paper assumes that the reader is primarily interested in preparing veterinary students to become general practitioners. a similar approach could be employed for defining a core for preparing specialists, researchers, and so forth. what students should know a common approach to determining what students need to know (or be able to do) is often referred to as task analysis. a task analysis involves examining the knowledge and skills of a competent professional in the area for which the students are preparing. since this discussion targets a general veterinary practitioner, in our context this approach requires two assumptions. first, we assume that there is a definable set of important theriogenology knowledge and skills relevant to the general practitioner, and not specific to a specialty. second, we assume that most experienced general practitioners possess these knowledge and skills. these may not always be completely safe assumptions. for instance, if theriogenology knowledge and skills vary greatly among common species, there may be some skills that are, by their nature, specialized in some species but core in others. second, it is possible for experienced general practitioners to lack knowledge or skills in a specific area, such as an area of emerging emphasis or transitioning technology. in such cases, it may not be safe to rely entirely on a task analysis with experienced practitioners to establish the gold standard of what graduates should know. nonetheless, generally speaking, answering those two questions should provide the information needed to determine the required theriogenology content in the veterinary curriculum. identifying the problem a discrepancy analysis defines the gap between what a practitioner knows and what the recent graduate knows. however, it can also be important to determine the practical implications of that discrepancy. for example, are there some common skills that are learned quickly and easily in the field, but are not practical to teach in an academic setting because of cost or other factors? do some knowledge and skills have much greater implications for patient outcome, cost, client satisfaction, and so forth, than others? being clear about the real or potential problems associated with specific knowledge gaps can be very useful for determining what constitutes the core. clinical theriogenology • volume 3 number 4 • december 2011493 what you already know two recent papers represent a very good start at needs assessment for the theriogenology education community. in one, published in javma, root kustritz, chenoweth, and tibary surveyed practicing veterinarians to determine which of a number of theriogenology procedures had the most value in practice and were performed most competently by new veterinary graduates.1 in the other, published in jvme, root kustritz, tibary and chenoweth surveyed north american and caribbean colleges of veterinary medicine to determine how much emphasis was given to theriogenology instruction given a variety of factors, such as species area and instruction type (lecture, lab, clinical).2 since they share the same authors, i will refer to these studies as “the javma study” and “the jvme study” respectively in this paper. based on these two studies you now know some interesting things that support a number of conclusions with implications for action or further study: 1. as a community, you have a reasonable list of theriogenology procedures that matter to practicing veterinarians, and graduates generally perform these procedures well. the procedures included in the javma study were those most commonly taught in veterinary school and requested for continuing education. respondents were given the opportunity to suggest other procedures that might be important, but it does not appear that such a list emerged. furthermore, graduates’ competence at any given skill was highly correlated with that particular skill’s perceived value in practice.1 2. as a community, you have a reasonably good idea of what matters to practitioners. some skills, such as diagnosis and management of dystocia, seem to be important for all respondents and across all species. other procedures, such as artificial insemination, are less important to practitioners.1 this sort of information provides the framework for a “recommended curriculum”, defined as the knowledge and skills needed to perform the procedures identified by practicing veterinarians as at least having some importance (perhaps a “2.5” or “3” on the scale used in the study.) 3. as a community, you have a reasonably good idea of where improvement is needed. the javma study identified some clear species-specific needs, such as improvement of instruction/learning with vaginal cytologic interpretation, brucellosis testing, and performance of cesarean sections in dogs, dystocia management in cats, and more instruction generally with llamas, alpacas, and exotic species.1 these might easily be identified as global deficiencies, deserving of attention from veterinary schools in general. 4. required instruction in theriogenology varies remarkably across veterinary schools. in the jvme study, total required lecture hours ranged from 4 to 60 and total required laboratory hours ranged from 3 to 42. required clinical exposure to theriogenology likewise varied considerably. when required hours were broken down by species, the standard deviation of hours across schools was often nearly as large as the mean.2 this suggests that as veterinary educators, we have very little idea of how many hours of theriogenology instruction might be “optimal” or even minimally required in veterinary medical education. clearly, some graduates are becoming licensed with minimal formal exposure to theriogenology, at least as identified by the respondents to the jvme survey. 5. while the global trend of actual competence matched value in practice well, there may be important missing curriculum-specific information. respondents to the javma study were evenly divided between those who said they had a good veterinary education in theriogenology, and those who said they did not. standard deviations for competency at graduation are relatively large, given the scale (about 20% of the full scale value), though no larger than the standard deviations for the rated value in practice of a given procedure.1 these results, while beneficial, beg the question of whether or not students feeling unprepared can be grouped systematically in some way. this idea will be discussed further in the next section. things you can do as a community the studies cited above are a strong start to defining a theriogenology core. in fact, one defensible approach for moving forward might be to make sure that all programs are covering the most common procedures identified in the javma study, pat yourselves on the back, and focus your attention elsewhere. however, if the goal is to establish and support a coherent and defensible core across colleges, clinical theriogenology • volume 3 number 4 • december 2011 494 there is more information that would prove helpful. the following are four suggestions regarding directions your community might consider in conducting future research and evaluation: identify how the general trends are represented at the local level, if at all. this discussion is an extension of the issues raised in points 4 and 5 of the prior section, in which it was observed that the amount of time dedicated to theriogenology instruction varies greatly across institutions, and that while about half of surveyed veterinarians in the javma study felt their theriogenology training was adequate, half felt that it was not.1 an important question for those wanting to define a theriogenology core is: how are these halves divided? do most graduates of the college that requires 60 lecture hours of theriogenology feel well-prepared (and perform well) while most graduates of the college that requires 4 hours feel unprepared? there are other potential/reasonable break-downs. perhaps many graduates with a large or mixed animal focus feel well prepared while most graduates with a small animal focus feel less well prepared. if your community can determine how perceived/measured skills/knowledge relate to specific curricular practices, you will have a stronger case to make when recommending a theriogenology core to colleges and other stakeholders. the kind of data produced by the two studies discussed above, if combined, could produce very insightful implications for curricular practice. consider, for instance, figure 1, which represents hypothetical data charting the relationship between credit hours dedicated to theriogenology instruction (lab and lecture combined) and competence (1-5; 1=completely lacking competence, 3 = minimally competent, and 5 = extremely competent). if this were a real dataset, it would provide a compelling case that if schools want their graduates to feel competent in their theriogenology ability, they need to dedicate in the neighborhood of 40+ hours to formal teaching. furthermore, 60-75 hours would seem to be the sweet spot, being associated with good competence overall (about 4 on the 5 point scale), with additional time (beyond about 75 hours) not being associated with significantly greater levels of competence. you might also encounter implications for exceptional curricular practice, such as the hypothetical institution that achieved the third highest competence ratings with its graduates, while investing slightly less than 40 hours of formal teaching. such a school might merit a visit and potential emulation from schools that were trying to improve either effectiveness or efficiency. of course, this hypothetical dataset generally envisions a strong linear relationship between credit hours and competence. this represents a reasonable hypothesis, but it is by no means the only possible relationship between credit hours and competence. it might be that other factors (such as clinical exposure, related case load, species-specific exposure in medicine courses, and so forth) have a more powerful impact on competence than simple hours in classroom and laboratory. a study such as the theriogenology javma study1 combined with the theriogenology jvme study2 could provide the information needed to answer these questions. the sampling procedure would need to ensure that veterinary respondents could be associated with a specific college and curriculum. because the complexity of establishing this sort of sampling procedure and ensuring that it were followed adequately might render such an approach impractical, there is another approach that could serve the 0 20 40 60 80 100 120 1 2 3 4 5 c re d it  h o u rs  d e d ic at e d  t o  t h e ri o competence figure 1 ‐ hypothetical case ‐ credit hours vs. competence clinical theriogenology • volume 3 number 4 • december 2011495 same purpose. that second approach might be to share measures across veterinary schools, measuring students’ perceived and actual competence during their clinical training, where reasonable response rates can still be ensured. this approach, “shared benchmarks” is discussed next. shared benchmarks in the business world companies benefit by benchmarking their performance against competitors’ performance. in very simple terms, companies compare results from measures that they share in common, and then attempt to emulate other companies’ successes. entertainment companies compare ratings or dollars at the box office, car companies compare horsepower, gas mileage, percent of cars still on the road after ten years, and so forth. universities use benchmarks such as research dollars and numbers of publications cited. unfortunately, in veterinary medical education, we have few shared benchmarks of educational success that are of any value, and certainly none that are specific enough to inform meaningful changes in theriogenology curricula. however, that does not mean that the theriogenology community could not create such measures and share data. a test could be designed and administered to theriogenology students at multiple schools at similar points of the curriculum, or identical clinical assessments could be implemented in clinical rotations. such tests or assignments could be designed to emphasize knowledge and skills that are considered fundamental by veterinary practitioners. with adequate involvement from colleges, curricular differences could be associated with learning outcomes. this approach would avoid the challenges of sampling error inherent in survey research with employers or alumni, and would eliminate uncontrolled factors such as employers’ potentially faulty memory of competence, and skills/abilities learned on the job after graduation. explicitly identify employer expectations at a skill-specific level in addition to associating global trends with curricular attributes as discussed in the suggestions above, more might also be learned from practicing veterinarians about their expected level of competence by procedure. the javma study showed that graduates tend to feel most competent with theriogenology procedures that are considered more valuable in practice, and less competent with skills that are considered less valuable. this is a desirable trend, and is valuable information. in addition to this information, it may be worth asking employers what level of competence they expect from new graduates for each skill. for instance hubbell, saville and moore asked veterinarians to identify whether the proficiency expected for a number of clinical equine skills were “able to perform unsupervised,” “able to perform with little supervision”, “able to perform with much supervision,” and “no proficiency expected.”3 this approach could provide additional information regarding which skills are “day 1” and which are “year 1”, and might also provide more species-specific trends. identify any recent change the two studies cited in the “what you know” section are now five years old. that is not long in the curricular life-cycle, so the information contained in them is likely still to be current. however, the last six years have seen two important changes that might have affected veterinary curricula for avmaaccredited schools. first, many schools are continuing to implement new outcomes assessment efforts, and to receive accreditation visits for the first time under the new outcomes assessment requirements. second, many colleges are experiencing unprecedented budget cuts. either of the factors could substantially affect curricula, either for better or worse. therefore, follow-up studies to both the javma and jvme studies seem warranted. of the four suggestions above, it is my opinion that the theriogenology community in veterinary medical education would derive the most benefit from pursuing the first two. these would provide information that would be both specific and generalizable (e.g., representative of many institutions rather than just a few.) however, solutions such as those require active participation from many partnering colleges and/or departments; sometimes such participation can be challenging to obtain. therefore, i also offer some suggestions that could be implemented on a smaller scale, and could benefit the individual clinical theriogenology • volume 3 number 4 • december 2011 496 instructor seeking to better identify his or her own “core.” the “community” and “individual” recommendations are by no means mutually exclusive. things that the individual instructor/small groups of instructors can do determine what students already know the discrepancy analysis approach as it was presented in the first section of this paper focuses entirely on the actual outcome of participating in a curriculum when compared with a desired outcome. another important and often overlooked aspect of needs assessment is determining a number of student characteristics, including what they already know when they enter a given course or curriculum. this is not likely to be a course of action that can be taken by the entire theriogenology community and prove to be generalizable, since results are likely to vary considerably among schools. nonetheless, sometimes the results of a pre-test can be surprising and enlightening strictly at the local level. for example, one colleague decided to implement a pre-test in his core required medicine course. to his surprise, on average students scored 50% on the test, indicating significantly greater knowledge than he had assumed. similarly, another colleague implemented an examination intended to measure clinical competence to a variety of students throughout the curriculum. to his surprise, a few students knew enough to pass the test during their first semester of veterinary school. this point reflects the fact that we do not know what our students already know unless we test them. at least one respondent replied to the root kustritz et al javma study that “my undergrad courses were nearly as good as my veterinary school training in regard to [reproductive] physiology.”1 if this comment happened to be representative of students in your courses at your institution, it might suggest that some material currently taught by clinical faculty in veterinary school might be shifted to earlier courses in the professional curriculum or may have already been treated adequately in prerequisite courses, allowing more to be covered in clinical theriogenology courses. learning task analysis there are many ways to conduct a task analysis, (e.g., analyze what experts know that learners need to learn).4 the root kustritz et al javma study1 represents one approach to broadly identifying procedures that are important to practitioners. this can be considered a task analysis, as it explores the tasks that are considered of greatest importance to practitioners. another and complementary approach is referred to as a learning task analysis.5 this approach also can be done in a number of ways, but in all cases involves breaking a task into its component parts and determining how those parts are related, usually in terms of prerequisite relationships. this is an exercise that most instructors engage in at least informally, when first designing a course. instructors might often think of this as “deciding what to teach first.” detailed learning task analyses are often time-consuming and tedious to construct, so they are generally used only when they are considered necessary – for instance when designing stand-alone instructional software tutorials. nonetheless, learning task analyses can also be a useful tool when limited time and/or resources or inadequate performance create the need for a better than average understanding of what is being taught in a curriculum. figure 2 represents a hierarchical learning task analysis for learning neurolocalization of spinal cord lesions. note that the ultimate targeted task for this analysis is the top one in the figure: “given a case with a spinal cord lesion, describe and justify the process that would be required to localize a lesion within one of the following segments (c1-c5, c6-t2, t3-l3, l4s2).” directly prerequisite to that task are four others: “describe/explain the principle of central recognition of pain”, “describe/explain the principle and testing of proprioception”, and so forth. each of those knowledge/skills also has prerequisite knowledge and skills, and so forth, all the way clinical theriogenology • volume 3 number 4 • december 2011497     figure 2 ‐ hierarchical learning task analysis for neurolocalization of spinal cord lesions. courtesy of k. bolser  describe/explain the principle of local  spinal reflexes. note: withdrawal and  patellar tendon tap are examples.  (principle)  define and describe  synapse, neuron, signal  transduction  (declarative knowledge)  given a case with a spinal cord lesion, describe and justify the  process that would be required to localize a lesion within one of  the following segments (c1‐c5, c6‐t2, t3‐l3, l4‐s2).  correctly classify and describe the role  of each type of neuron (concept) distinguish between primary  sensory and projection  neurons (concept)  distinguish between upper  motor and lower motor  neurons (concept)  describe the  characteristics and  roles of motor  neurons (concept)  describe the  characteristics and roles  of sensory neurons  (concept)  diagram a typical  thoracic spinal  nerve (declarative  knowledge)  list the peripheral nerves  that supply the thoracic  limb, and those that supply  the pelvic limb.  include the  spinal cord segments from  which they arise.  (declarative knowledge)  describe/explain  the principle of  voluntary motor  activity. (principle)  describe/explain the  principle and testing  of proprioception  (principle)  describe/explain  the principle of  central  recognition of  pain (principle)  clinical theriogenology • volume 3 number 4 • december 2011 498 down to defining and describing “synapse, neuron. . .” etc. in the case of figure 2, a line is drawn horizontally across the figure above the bottom three boxes, indicating that the knowledge represented by those boxes has already been learned, and is not intended to be included in the instruction for which this analysis was conducted. analyses such as the one demonstrated in figure 2 have several practical purposes for those who wish to define an instructional core. first, they help to ensure that essential knowledge/skills are not forgotten, or that, if forgotten, they are identified if their absence proves to be problematic. this is particularly important when the core is primarily being established by experts in a field. while experts are very good at knowing what experts need to know, they are often not very good at remembering the learning path that got them to their expert level, or how the knowledge pieces all fit together. they have often either done something so many times that they have automated it, forgetting, essentially, how all the pieces fit together, or they have learned the knowledge and skills without ever formally articulating them.6 it is in this way that we become proficient speakers of a native language without defining parts of speech or diagramming sentences. learning task analyses, then, are a tool for experts to use in identifying pesky omissions in content areas that consistently give students trouble. the second benefit of a learning task analysis, is that it can help the instructor to identify key knowledge/skills upon which many other knowledge or skills are dependent. consider, for example, figure 2. note that there is one skill, “correctly classify and describe the role of each type of neuron,” that serves as the pathway between all of the second-level skills, and nearly all of the fourth, fifth, and sixth level skills. assuming that this learning task analysis is correct, this skill is certainly one that a wise instructor would wish to pay attention to when teaching, reviewing, or testing students’ readiness to learn the applied task of neurolocalization. less is more an analogy can be drawn between well-meaning instructors who design curricula and wellmeaning parents who pack for a child’s overnight hiking trip. some parents might pack in an effort to ensure that their child is prepared for every eventuality, including things such as a portable cot and inflatable sleeping pad for comfort, several changes of clothing in case they get wet, an extra blanket for warmth, extra snacks to share with friends, etc. all of these items are helpful, and their inclusion is reasonable. however, this “just-in-case” mentality can produce a backpack so heavy that the child can barely get it from the porch to the car, much less through an all-day hike. as a result, things are removed from the backpack for the purpose of lightening it. however, the decision regarding what stays and what goes is made by the child several hundred yards into the hike, and not by the parent who took such care in packing. similarly, some instructors see it as their duty to force into the curriculum every valuable detail that they have acquired over years of practice or study. they feel that the greatest disservice they can do to their students is to omit some someday-useful fact. however, research suggests that “just-in-case” curricular packing can lead to learning of unrelated facts without the ability to make sense of those facts.7 note that this is not an argument to water-down the curriculum or to omit factual learning. it is simply a reminder that wise instructors know that students will not learn everything, and that it is the responsibility of the instructor to carefully pack the proverbial curricular backpack with a load that can be carried. if we, as teachers, don’t choose what not to include in the curriculum, our students certainly will. conclusion available evidence suggests that, in general, the theriogenology community in veterinary medical education is providing adequate knowledge and skills to graduating veterinarians. however, several important questions remain unanswered, specifically, how are general trends represented at the local level, how do employers specifically identify the importance of given procedures, and have trends changed substantially in the past 5-6 years since the available work was done? the theriogenology community could benefit greatly from benchmarking using shared measures with advanced students across multiple programs. individual programs could also benefit by determining what students already clinical theriogenology • volume 3 number 4 • december 2011499 know when they enter courses that emphasize theriogenology, conducting learning task analyses, and remembering that “less is more” when adding content to the curriculum. references 1. root kustritz mv, chenoweth pj, tibary a: efficacy of training in theriogenology as determined by a survey of veterinarians. j am vet med assoc 2006;229:514-521. 2. kustritz mv, tibary a, chenoweth pj: availability of theriogenology training at north american and caribbean veterinary colleges. j vet med educ 2006;33:140-144. 3. hubbell ja, saville wj, moore rm: frequency of activities and procedures performed in private equine practice and proficiency expected of new veterinary school graduates. j am vet med assoc 2008;232:42-46. 4. jonassen d, hannum wh, tessmer m: handbook of task analysis procedures. new york: praeger publishers; 1989. 5. smith pl, ragan tj: instructional analysis: analyzing the learning task. in: instructional design. 3rd ed. hoboken: john wiley & sons; 2005. 6. bereiter c, scardamalia m: surpassing ourselves: an inquiry into the nature and implications of expertise. chicago: open court publishing co; 1993. 7. bransford jd, brown al, cocking rr: the design of learning environments.in: how people learn: brain, mind, experience, and school. washington: national academy press; 2000. clinical theriogenology • volume 3 number 4 • december 2011 500 2011: colic associated with bilateral seminoma in a cryptorchid american miniature horse stallion colic associated with bilateral seminoma in a cryptorchid american miniature horse stallion r.a. smith, l.k. pearson, l.n. wise, a. tibary department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa equine testicular neoplasms are rare, likely due to the practice of early castration. testicular tumors originate from germ cells, sex-cord stroma, or other cells. the most commonly reported testicular tumor in older (11 to 22 years, mean 16.5 years) cryptorchid stallions is seminoma.1 most reported cryptorchid seminomas are incidental findings after cryptorchidectomy. reports of testicular tumors as a differential for a colic syndrome, such as in this report, are few. a 13-year-old bilaterally cryptorchid american miniature horse stallion was referred to the washington state university-veterinary teaching hospital for colic of 12 hours duration. initial evaluation demonstrated normal heart rate, decreased gastrointestinal borborygmi, and a small volume of nasogastric reflux. transabdominal ultrasonography revealed moderate small intestinal distention, thickened intestinal walls, and associated ileus. two large masses of heterogenous echotexture were presumed to be the cryptorchid testes due to their location. abdominocentesis, complete blood count, and serum biochemistry panel were consistent with chronic inflammation. despite medical therapy with intravenous fluids and non-steroidal anti-inflammatories, the horse remained depressed and mildly painful with ongoing nasogastric reflux. exploratory laparoscopy was performed and demonstrated a nonstrangulating compression of the jejunum by the right testis, which accounted for the clinical signs. bilateral cryptorchidectomy was performed. the horse recovered uneventfully from surgery. histopathology confirmed malignant seminoma in both testes. further diagnostics to identify metastases were declined by the owner. often in clinical practice, differential diagnoses for colic overlook reproductive etiologies. in this case, the history of cryptorchidism assisted clinicians in making the decision to pursue surgical therapy for colic. this case presented several unique features not commonly reported: bilateral cryptorchidism in an older stallion, neoplasia of both retained testes, and presentation to the veterinarian for colic signs. this case also raised issues of welfare in cryptorchid stallions and reinforces the importance of client education to seek removal of cryptorchid testes. keywords: equine; testes; neoplasia; pathology; laparoscopy; ultrasonography reference 1. valentine ba: equine testicular tumours. equine vet educ 2009;21:177-178. clinical theriogenology • volume 3 number 4 • december 2011601 clinical theriogenology • volume 3 number 4 • december 2011 602 omniblank: telemetric monitoring of calving using a novel calf alert device telemetric monitoring of calving using a novel calf alert device cyril stephen,ab don menzies,c david swain,c kym patison,c nicholas corbet,c scott normanab aschool of animal and veterinary sciences, charles sturt university, wagga wagga, nsw, australia bgraham centre for agricultural innovation, wagga wagga, nsw, australia ccentral queensland university, rockhampton, qld, australia in extensive grazing systems, lack of methods to monitor calving has been an impediment to characterizing and managing calf losses. a device that could telemetrically detect time and location of a portion of parturition events would allow timely recording of calving-related data, increase opportunities to conduct necropsies and be useful to quantify and derive scientifically based conclusions for neonatal calf mortality and morbidity. objective was to build and evaluate a telemetric calf alert device suitable for identification of the time and location of calving events in extensive beef production herds in northern australia. a prototype device was selected from several preliminary designs, based on cow safety and 100% vaginal retention as tested in a 28 day field trial in 5 nonpregnant cows. the device was further developed incorporating a taggle™ pcb (printed circuit board) and a battery (life of 2 years) within a cargo pod. taggle™ location system consists of 3 components a transmitter that outputs a low-power radio signal; a sensitive terrestrial receiver that detects the taggle™ ping and its time of arrival; and a method of transmitting the time of ping arrival at each receiver to a central location server. when taggle™ ping is detected by 3 or more receivers, the time difference of arrival of these signals is used to calculate the location of the transmitter. device transmits signals every 15 minutes and can transmit to receivers up to 7 km away. device was active from the time of intravaginal insertion, sending out attenuated signals detectable by receivers. at device expulsion during parturition, improved reception of the unattenuated signal allowed the timing and location of calving event to be transmitted. a field trial was conducted where devices were inserted into the cranial vagina of 20 early (2 3 months) pregnant cows, with 20 contemporary controls. cows were monitored monthly until calving, using an intravaginal endoscope and complete blood counts, plus assessment of acute phase serum proteins. with modifications following the initial field trial, the device was further tested in 80 mid to late gestation cows. devices were monitored over 150 days for reception and location data utilizing taggle™ terrestrial receivers mounted at heights of 12 15 metres. parturition events and locations were confirmed by daily visual surveillance of calving paddocks. there was an 85% retention rate of the modified calf alert devices in pregnant cows, without any adverse effects on cow or calf. modified devices functioned without interruption throughout the trial period and 83% of inserted devices provided locations throughout the data collection period. modified devices detected calving date in 66% of cows, with calving location derived for 64% of cows. precision of calving location was within 100 metres in a 150 hectare network, and 200 metres in a 1050 hectare network. we successfully developed and deployed a calf alert device that can be placed within the vagina of pregnant beef cattle for at least 6 months to provide calving time and location alerts by sms or computer messaging. ability to detect calving within 24 hours of actual calving date would enable neonatal calves to be monitored, thereby increasing the opportunity to identify, investigate, and address any calf losses. keywords: calf alert device, telemetry, calving, receiver, location 449 clinical theriogenology • volume 11, number 3 • september 2019 450clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2018: canine pyometra: a retrospective study of 21 cases canine pyometra: a retrospective study of 21 cases daesik kim, hoyoung kang, cheolkyu han, hyunsuk so, donghee park, youngung kim, namsoo kim department of veterinary surgery, college of veterinary medicine, chonbuk national university, iksan, korea purpose pyometra is a common life-threatening disease of intact bitches caused by the hormonal effects on the uterus, with bacterial infections. this should be differentiated with hydrometra, hematometra and mucometra. pyometra can be treated with medical therapy or surgery. from 2015 to 2016, 21 cases diagnosed with pyometra were treated with ovariohysterectomy at chonbuk national university animal hospital. materials and methods the bitches selected were diagnosed with pyometra and were treated with ovariohysterectomy. criteria evaluated included the percent ratio of ovaries and uterus to body weight, antibiotic susceptibility, open or closed type, age, and type of fluid discharged. the antibiotic susceptibility test was categorized as ampicillin (am10), amikacin (an30), cefazolin (cz30), enrofloxacin (enr5), amoxicillin/clavulanic acid (amc30), sulfamethoxazole/trimethoprim (sxt), clindamycin(cc2) and metronidazole (mtz5). results the average weight of the ovaries and uterus was 6.5% (2.2 ~ 10%) of the body weight. when antibiotic susceptibility was established, the effective ratio of the corresponding antibiotic was confirmed. the results were as follows: ampicillin (am10) 0%, amikacin (an30) 52%, cefazolin (cz30), enrofloxacin (enr5) 29%, amoxicillin/clavulanic acid (amc30) 38%, sulfamethoxazole/trimethoprim (sxt) 43%, clindamycin 10% (cc2), and metronidazole (mtz5) 10%. the average age was 7.5 (2 to 15) years old. twenty three percent of bitches showed closed type. the types of fluids discharged were pyometra 88%, hematometra 4%, mucometra 8% and hydrometra 0%. conclusion we checked statistics of various features of pyometra, such as percent ratio of ovaries and uterus to body weight, antibiotic susceptibility, open or closed type, age and type of fluid discharged. through accumulating more data, it is anticipated that more effective treatment will be possible for the pyometra. keywords: pyometra, antibiotic susceptibility, bitch clinical theriogenology • volume 10, number 3 • september 2018 346 introduction equine metabolic syndrome (ems) has been associated with infertility in the mare. the mechanisms by which this condition occurs has not been completely elucidated. in preparation for winter, during autumn months horses begin to accumulate energy in the form of body fat. this process is mainly controlled by the hypothalamic-pituitary axis and the secretion of peptides (adrenocorticotropic hormone [acth], alpha melanocyte stimulating hormone [αmsh], corticotropin-like intermediate peptide [clip], β-endorphins) from the pituitary pars intermedia that stimulate an increase in appetite, adipogenesis, and the development of a long, thick haircoat.1 in ems, excess body fat occurs because similar level of feeding is maintained during winter that is perceived by the body as an ‘excess,’ especially carbohydrates, because physiologically it should be a period of feed shortage.2 ems is suspected in obese or normal mares that have ectopic fat deposits, abnormal estrous cycles, anovulatory follicles, estrous cycles during winter, and laminitis. effect of endocrinopathies on fertility in the mare part 1: equine metabolic syndrome maria cadario,a louis archbaldb aequine reproduction specialty practice, ocala, fl bdepartment of large animal clinical sciences college of veterinary medicine, university of florida, gainesville, fl abstract obesity and insulin resistance (ir) have been associated with subfertility in the mare. there has been a substantial increase in the prevalence of overweight and obese horses in recent years (45 50% from 4.5% in 1998). some of these mares, with a history of good sport performance, enter breeding programs with high expectations of embryo production or pregnancy. without losing perspective of the reproductive problems associated with ageing and obesity, it is very important to consider metabolic problems (e.g., equine metabolic syndrome [ems] and pituitary pars intermedia dysfunction [ppid; i.e., equine cushing’s disease]) that may directly impact fertility. however, ems and ppid differ widely in their pathophysiology; ppid is neurological in origin, whereas ems is the result of overfeeding horses with high metabolic efficiency, especially during winter. generally, ems and ppid are associated (and often confused with each another) because they share several clinical manifestations, especially, when or if ppid has concurrent insulin dysregulation manifestations (abnormal and excessive distribution of adipose tissue, ir, laminitis, and subfertility). a large proportion of obese and ir mares continue to cycle during winter, have longer inter-ovulatory intervals, and a higher incidence of anovulatory follicles. diagnosis of ems requires a detailed history and physical evaluation combined with appropriate laboratory tests. once identified, ems should be primarily managed with a low-carbohydrate diet and a good exercise program. if necessary, medication (e.g., levothyroxine or metformin) can be used. however, there are no critical studies regarding safety or efficiency of these drugs. keywords: obesity, mare, fertility, insulin resistance, anovulatory follicles equine metabolic syndrome equine metabolic syndrome is defined as a ‘collection of risk factors for endocrinopathic laminitis’.3 these risk factors are insulin dysregulation (id) and/or ir, obesity, adipose dysregulation, and less frequently, cardiovascular changes; regardless, the key factor is id. adipose dysregulation also has an important role by increasing blood concentrations of leptin and proinflammatory cytokines, and by decreasing adiponectin. leptin is responsible for maintaining body condition by suppressing appetite and increasing energy outflow.3 persistent high leptin concentrations are associated with hyperinsulinemia in nonpregnant ponies.4 adiponectin increases insulin sensitivity and reduces inflammation. lower than normal concentrations of adiponectin have been associated with an increase in serum amyloid-a, a marker for inflammation, and with id.3 horses with ems have a history of or are predisposed to laminitis, and frequently exhibit abnormal estrous cycles.5 obesity is manifested by regional, localized, or ectopic deposition of subcutaneous adipose tissue in the nuchal ligament (i.e., ‘crested’ or bulging neck), tail base, shoulders, mammary gland and prepuce, especially in geldings. these horses appear to be genetically predisposed to need fewer calories to maintain adequate body weight (i.e., ‘easy keepers’). some breeds (e.g., ponies, quarter horses, morgans, arabians, and saddlebreds) are predisposed to have this condition.6 pathophysiology amount of insulin needed for glucose uptake into a cell is highly regulated and is called ‘insulin sensitivity.’ when a horse develops ir, there is a decrease in insulin-dependent glucose uptake in skeletal and adipose tissue, creating an increase in plasma concentrations of glucose.2,6 this in turn generates hyperinsulinemia in an effort by the pancreas to compensate for cellular insensitivity that perpetuates this condition. information on predisposing factors to id is limited; however, obesity appears to be a recurring factor. obesity is not observed as the sole cause of ems, since this syndrome also diagnosed in lean horses; however, when present, it exacerbates id.3 in horses with ems, the fat deposits are composed of enlarged adipocytes and infiltrated macrophages that behave like endocrine tissue. excess fat deposits will release cortisol, adipokine peptides (e.g., leptin, adiponectin), and proinflammatory cytokines (e.g., tumor necrosis factor-α, (tnf-α), interleukin (il)-1 and il-8. these hormones, proteins and factors may adversely affect glucose absorption by cells, producing inflammation and possibly vascular endothelial damage.2,7 elevated tnf-α concentrations would inhibit normal function of cellular insulin receptor favoring ir. they also reduce secretion of adiponectin (anti-inflammatory peptide and insulin sensitivity promoter) by adipocytes creating a proinflammatory state and increasing ir. conversely, secretion of leptin is increased with obesity, insulin, and tnf-α; however, as with insulin, these mares appear to develop ‘leptin resistance,’ failing to regulate appetite or energy balance.8 obesity in horses, especially ponies, is a risk factor for development of laminitis. the simplest explanation is excessive weight on the hoof and inter-laminar surface.2,4,6,9 mechanisms by which obesity and id causes laminitis have not been completely elucidated. current theories include changes in intracellular insulin signaling resulting in endothelial dysfunction and mechanisms involving insulin-like growth factor 1 (igf-1).3,10,11 obesity and ir appear to be responsible for mares continuing to cycle during winter, having longer inter-ovulatory intervals, prolonged luteal phases, and a higher incidence of anovulatory follicles. possible systemic and local mechanisms of action of insulin on reproduction in the mare a few studies linked ems, ir and/or hyperinsulinemia with subfertility. it was proposed that signals sent to the brain regarding metabolic status may somehow control seasonality, especially the occurrence and length of seasonal anestrus.8 aggressive feed restriction led to cessation of reproductive activity during winter whereas obese mares displayed continuous estrous cycles during winter.12 similarly, a large proportion of obese mares continue to cycle during winter compared to proper weight or slender mares.13 after several investigations it was concluded that the cause for the loss of the seasonal anestrus was not plasma leptin concentrations but the excess energy balance.12 in mares, physiological recognition of the availability of metabolic substrate for cyclicity would be more important than the perception of a change in the photoperiod. subsequent studies reported aberrations in estrous cycle length and progesterone concentrations in obese/ir mares compared to normal mares.14-16 transient induction of hyperinsulinemia in mares using exogenous lipid-heparin infusion lengthened the inter-ovulatory interval with a marginal increase of the luteal phase, and higher peaks of progesterone concentrations; however, with no effect on lh concentrations compared to control mares.14 insulin in horses, like in women, may act directly at the level of ovary by stimulating steroidogenesis since progesterone concentrations were higher in mares with ir without an increase in lh concentrations.14 obese mares had longer inter-ovulatory intervals, prolonged luteal phases or elevated progesterone concentrations, and a higher incidence of anovulatory follicles compared to obese mares with moderate feed restriction.15 progesterone concentrations remained elevated between 37 and 78 days in 83% of obese mares whereas in mares subjected to feed restriction, increased progesterone concentrations did not exceed 22 days.15 it was speculated that the persistence of elevated progesterone concentrations in obese mares was due to persistent corpus luteum (cl) or luteinization of anovulatory follicles.15,16 transrectal ultrasonographic evaluation in obese mares demonstrated the presence of persistent anovulatory follicles during elevated progesterone concentrations, suggesting that 1 or more mechanisms for triggering ovulation is affected.15,17 some preliminary data indicated that transient ir did not lead to differences in lh or fsh concentrations compared to normal mares and suggested that elevated insulin concentrations may directly affect the ovary rather than the hypothalamic-pituitary axis.14 with respect to the link between insulin and maternal recognition of pregnancy, exogenous insulin treatment during days 7 17 of diestrus had no effect on luteal size, diestrus length, inter-ovulatory interval, or circulating lh concentrations.18 similarly, obesity produced by overfeeding in peripartum did not adversely affect subsequent estrus duration, time to first and second postfoaling ovulations, and rates of pregnancy or early embryonic death.19 possible mechanisms that associate hyperinsulinemia/id with ovarian dysfunction and anovulatory follicles in mares polycystic ovary syndrome (pcos) in women has been used as a model to understand effects of hyperinsulinemia on fertility in mares. equine metabolic syndrome and pcos have similar clinical signs (e.g., ovulatory dysfunction, obesity, and hyperinsulinemia/ir). in women, chronic hyperinsulinemia/ir has been associated with an increase in the duration of follicular phase due to a high incidence of ovarian (anovulatory) follicles.20 supposedly, high insulin concentrations stimulated the selective release of gnrh/lh and increased ovarian steroid secretion.20-22 increased pulses of gnrh selectively stimulate the secretion of lh, but not fsh. lh stimulates the expression of cytochrome p450c17 in follicular cells that increases the production of 17α-hydroxyprogesterone and testosterone. increases in testosterone concentrations adversely affected the secretion of fsh. hormonal environment of increased lh and decreased fsh concentrations reduced or inhibited follicular maturation, decreased the number of granulosa cells, decreased aromatization and estrogen production, and inhibited ovulation.20 metformin treatment to women with metabolic syndrome and pcos decreased plasma concentrations of insulin, lh, and androgens, thus supporting the leading role of hyperinsulinemia in this endocrine alteration.20,23 however, there are 2 fundamental differences between the effect of hyperinsulinemia on the reproductive system of mares compared to women. mares with ir have a longer estrous cycle due to a longer diestrus period, and not a longer follicular phase. more importantly, higher concentrations of insulin did not affect the hypothalamic pituitary axis in mares since lh and fsh concentrations are similar for mares with or without ir.14 therefore, in mares, the mechanism of action appears to be at the ovarian level. higher concentrations of insulin may act as an lh-like molecule, bind to insulin or the igf-1 receptors at the theca cells, and stimulate production of 17α-hydroxyprogesterone and testosterone. this synergic action with endogenous lh will have a negative impact on follicular development, maturation, and ovulation. in ponies that developed hemorrhagic anovulatory follicles (haf) repeatedly, higher estradiol concentrations occurred 3 days before the formation of haf.24 in these ponies, follicular diameter, lh, fsh, and progesterone concentrations were similar on day 0 (ovulation) and day -1 (previous) compared to follicles that ovulated, suggesting that the focus (formation of haf) should not be strictly on hypothalamic-pituitary-gonadal axis. interestingly, the body condition of mares used for this experiment (4 16 years old) was higher throughout the experiment (body score > 7) and 3 of 4 ‘haf repeaters’ mares did not enter the anovulatory season later in the year. although the metabolic status of these mares is not known, obesity and/or hyperinsulinemia might have affected ovarian steroidogenesis. furthermore, substantial correlation of insulin, leptin, adiponectin, and cytokines concentrations with serum and follicular fluid suggested the role of hyperinsulinemia.25 granulosa cells expressed receptors for insulin, leptin, adiponectin, tnf-α, il-6 and il-1b indicating that they have the potential to respond to these hormones and factors that have a local effect on steroidogenesis, granulosa cell proliferation, oocyte maturation and early stages of embryo development.25 additionally, gene expression of tissue inhibitor of metalloproteinase -2 (timp2) in granulosa cells was increased, essential for activation of matrix metalloproteinase-2 (mmp-2). inadequate timp2 expression will inhibit activation of mmp-2 and disrupt tissue remodeling necessary for ovulation. this could contribute to obesity-related formation of persistent anovulatory follicles.26 disruption of normal follicular concentrations by these factors may affect follicular development and maturation, ovulation, pregnancy, and even the metabolic future of the offspring. impacts of insulin resistance on follicular growth, size at ovulation and response to human chorionic gonadotrophin (hcg) were studied.27 dominant follicle (f1) had similar size at ovulation in ir mares compared to unaffected mares. mares with ir had more subordinate follicles, and their second largest follicle (f2) was larger in diameter than in normal mares, almost establishing codominance with f1. the impact of these findings on fertility was not studied. although it was not significant, because of the number of mares per group (n = 4), hcg treatment induced ovulation before 48 hours in 2 of 4 mares with ir inducing ovulation in all (n = 4) normal mares.27 as practitioners, we consider the lack of response to ovulatory drugs as 1 of the first signs of an anovulatory follicle. in summary, most studies point towards an alteration in steroidogenesis at the ovary in mares with ems. insulin has gonadotrophic and steroidogenic effects in other species. in mare, the excess insulin may have an lh-like effect on theca cells, producing an increase in androgen/estrogen concentrations affecting normal follicular development and ovulation.20,24 it may also affect steroidogenesis once a cl is formed, or in anovulatory follicles by increasing progesterone production, as shown in early studies.14,15 equine metabolic syndrome and pregnancy normal pregnant mares have slower glucose clearance and greater insulin secretion than nonpregnant mares. it appears to be a kind of ‘physiologic ir’ created to have high glucose concentrations, since uptake by the feto-placental unit depends on the existing concentration gradient. pregnant mares with ems have an exacerbated insulin and glucose response to high carbohydrate diets during pregnancy.28-30 diagnosis common clinical features in horses with ems are obesity, and the characteristic regional distribution of body fat (neck, shoulders, base of the tail). these horses also need less feed to maintain body weight (i.e., ‘easy keepers’), or they may have difficulty losing weight. in addition, these horses have a predisposition, or history of previous bouts of laminitis and abnormal estrous cycles. there are static and dynamic tests to identify ir/id. static tests insulin and glucose concentrations: this test is ideal for the identification of horses with moderate to severe insulin dysregulation. baseline insulin values > 20 μu/ml are indicative of id and values > 30 μu/ml are considered diagnostic.6, 8,11 adipokines: blood concentrations of adiponectin is associated with obesity and id, and low blood concentrations are considered a risk factor for laminitis.3,31-33 dynamic tests oral sugar test: this is easily performed and is very sensitive to identify mild to moderate cases of id.3,5 combined glucose-insulin test: evaluates tissue insulin sensitivity and beta cell responsiveness.3,5,34 treatment diet and exercise: treatment is aimed at decreasing excess weight gain and, if necessary, gradual weight loss. the goal is also to keep insulin concentrations to < 30 μu/ml. affected horses should have a diet composed of grass hay; low (i.e., < 10%) nonstructural carbohydrates (nscsugar, starches and fructanes); and a ration balancer to meet mineral and vitamin requirements. exercise, if possible, is recommended to burn calories and to stimulate glucose absorption. mares should have continuous access to forage, especially if they have dietary restrictions due to ems, since it is proven to enhance fertility.8,35 grain and concentrates should be given in small amounts or eliminated from the diet, and time on pasture should be limited.36 pregnant mares with ems should be fed in the same way as nonpregnant ems mares for the first 2 trimesters. the necessary increase in calories needed during the last trimester and lactation should be coming from fat and fiber.28-30 exercise has a proven beneficial effect on insulin sensitivity in nonpregnant mares. it is recommended to do no more than 30 minutes of trot exercise/day in the third trimester.8 pharmaceuticals: they are supposed to stimulate weight loss and insulin sensitivity. however, there are no studies regarding safety or efficacy of these drugs. supplementation with oral 24 96 mg/day of levothyroxine (thyro l®, lloyd inc, shenandoah, ia) is recommended if the mare loses weight very slowly, or cannot reach the desired weight, cannot exercise due to laminitis or, her hyperinsulinemia persists even after reaching the desired weight.6,37 this approach is not recommended in pregnant mares since it may produce congenital goiter in the foal, as reported in humans.38 in humans, metabolic syndrome is successfully treated with biguanide (metformin) that is also safe to use in pregnant women. however, this drug is not well absorbed in horses, and that there is low bioavailability and minimal systemic effects on id.39 the exact mechanism of action in horse is not known, but it may decrease the absorption of glucose at the level of enterocytes. recommended dose is oral 15 30 mg/kg (twice or thrice a day) 30 minutes before each meal. conclusion pituitary pars intermedia dysfunction and ems have been associated with subfertility in the mare, probably through their effects on the estrous cycle and ovarian follicular dynamics. however, confounding factors are age and insulin dysregulation. it is important to make an accurate diagnosis since treatment on ‘a trial-and-error basis’ is not without adverse consequences. use of appropriate laboratory tests in conjunction with clinical signs is paramount in the diagnosis and management of this condition. conflict of interest none to declare. references 1. donaldson mt, mcdonell sm, schanbaucher bj, et al: variation in plasma adrenocorticotropic hormone concentration and dexamethasone suppression tests results with season, age, and sex in healthy ponies and horses. j vet intern med 2005;19:217-222. 2. johnson pj, ganjam sk, turk jr, et al: obesity paradigm: an introduction to the emerging discipline of adipobiology. proc am assoc equine pract 2006; p. 41-50. 3. durham ae, frank n, mcgowan c, et al: eceim consensus statement on equine metabolic syndrome. j vet intern med 2019;33:335-349. 4. morgan ra, mcgowan tw, mcgowan cm: prevalence and risk factors for hyperinsulinemia in ponies in queensland, australia. aust vet j 2014;92:101-106. 5. johnson pj: equine ms. j eq vet sci 2003;23:373-374. 6. frank n: managing equine ms. compend contin educ vet 2008;3:348-355. 7. poretsky l, cataldo na, rosenwaks z, et al: the insulin-related regulatory system in health and disease. endocr rev 1999;20:535-582. 8. burns t: effects of common equine endocrine diseases on reproduction. vet clin north am equine pract 2016;32:435-449. 9. treiber kh, kronfeld ds, hess tm, et al: evaluation of genetic and metabolic predisposition and nutritional risk factors for pasture associated laminitis in ponies. j am vet med assoc 2006;228:15381545. 10. geor r, frank n: metabolic syndromefrom human organ disease to laminar failure in equids. vet immunol immunopathol 2009;129:151-154. 11. frank n: insulin resistance in the horse. proc am assoc equine pract 2006; p. 51-54. 12. gentry lr, thompson dl, gentry gt, et al: the relationship between body condition, leptin, and reproductive and hormonal characteristics of mares during the seasonal anovulatory period. j anim sci 2002;80:2695-2703. 13. fitzgerald bp, reedy se, sessions dr, et al: potential signals mediating the maintenance of reproductive activity during the nonbreeding season of the mare. reprod suppl 2002;63:335-340. 14. sessions dr, reedy, vick mm, et al: development of a model for inducing transient insulin resistance in the mare: preliminary implications regarding the estrous cycle. j anim sci 2004;82:23212328. 15. vick mm, sessions dr, murphy ba, et al: obesity is associated with altered metabolic and reproductive activity in the mare: effect of metformin on insulin sensitivity and reproductive cyclicity. reprod fertil dev 2006;18:609-617. 16. fitzgerald bp, sessions dr, vick mm, et al: obesity and reproduction in the mare. in: the chronically infertile mare workshop. proc of the havemeyer foundation 2008; p. 23. 17. mccue pm, squires el: persistent anovulatory follicles in the mare. theriogenology 2002;58:541-543. 18. rambags bpb, van rossem aw, block ee, et al: effect of exogenous insulin on luteolysis and reproductive cyclicity in the mare. reprod domest anim 2008;43:422-428. 19. kubiak jr, evans jw, potter gd, et al: postpartum reproductive performance in the multiparous mare fed to obesity. theriogenology 1989;32:27-36. 20. castro acuña v, martinez-martinez l, cravioto mc: insulin resistance in polycystic ovaries syndrome. revista de investigacion clinica 2004;56:763-772. 21. diamanti-kandarakis e, bergiele a: the influence of obesity on hyperadrenogenism and infertility in the female. obese rev 2001;2:231-238. 22. nestler je: obesity, insulin, sex steroids and ovulation. int j obes relat metab disord 2000;24:s71-s73. 23. sattar n, hopkinson ze, greer ia: insulin sensitizing agents in polycystic ovary syndrome. lancet 1998;351:305-306. 24. ginther oj, gastal el, gastal mo, et al: conversion of a viable preovulatory follicle into a hemorrhagic anovulatory follicle in mares. anim reprod 2006;3:29-40. 25. sessions-bresnahan dr, carnevale em: the effect of equine metabolic syndrome on the ovarian follicular environment. j anim sci 2014;92:1485-1494. 26. sessions dr, vick mm, fitzgerald bp: expression of mmp-2 and timp in equine granulosa cells and the impact of elevated insulin in vitro. biol reprod 2007;77:156. 27. prado, julio cesar, “follicular dynamics in insulin resistant mares “ master’s thesis, university of tennessee, 2016. https://trace. tennessee.edu/utk_gradthes/4303 28. fowden a, comline rs, silver m; insulin secretion and carbohydrates metabolism during pregnancy in the mare. equine vet j 1984;16:239-246. 29. george la, staniar wb, cubbit ta, et al: evaluation of the effect of pregnancy on insulin insensitivity, insulin secretion, and glucose dynamics in thoroughbred mares. am j vet res 2011;72:666-674. 30. hoffman rm, kronfeld ds, cooper wl, et al: glucose clearance in grazing mares is affected by diet, pregnancy, and lactation. j anim sci 2003;81:1764-1771. 31. menzies-gow nj, harris pa, elliott j: prospective cohort study evaluating risk factors for the development of pasture-associated laminitis in the united kingdom. equine vet j 2017;49:300-306. 32. menzies-gow nj, knowles ej, rogers i, et al: validity and application of immunoturbidimetric and enzyme-linked immunosorbent assays for the measurement of adiponectin concentration in ponies. equine vet j 2018;51:33-37. 33. carter ra, treiber kh, geor rj, et al: prediction of incipient pasture-associated laminitis from hyperinsulinemia, hyperleptinemia and generalized and localized obesity in a cohort of ponies. equine vet j 2009;41:171-178. 34. morgan ra, keen ja, mcgowan cm: treatment of equine metabolic syndrome: a clinical case series. equine vet j 2016;48:422426. 35. benhajali h, ezzaouia m, lunel c, et al: temporal feeding pattern may influence reproduction efficiency, the example of breeding mares. plos one 2013;8:e73858. 36. chameroy ka, frank n, elliot sb, et al: effect of supplement containing chromium and magnesium on morphometric measurements, resting glucose insulin concentrations and insulin sensitivity in laminitic, obese horses. eq vet j 2011;43:494-499. 37. douglas rh: endocrine assessment and management of insulin resistance and ppid. proc primer congreso argentino de reproducción equina 2009; p. 81-83. 38. sheehan pm, nankervis a, araujo junior e, et al: maternal thyroid disease and preterm birth: systematic review and meta-analysis. j clin endocrinol metab 2015;100:4325-431. 39. hustance jl, firshman am, mata je: pharmacokinetics and bioavailability of metformin in horses. am j vet res 2009;70:665-668. effect of endocrinopathies 2013: goat herd health; reproductive management, feeding the doe, care of the kid goat herd health; reproductive management, feeding the doe, care of the kid d.g. pugh southern traxx vet services, waverly, al reproductive management practices regardless of the species of small ruminant the clinician is treating, there are several herd/flock health practices that are useful in enhancing productivity and improving reproductive health. included in these practices are: (1) perform pregnancy diagnosis on all bred females 45 to 60 days after breeding (note: a linear array 3.5-5 mhz transducer can be used in most small ruminant females). proper restraint, use of a coupling agent, and knowledge of the reproductive tract of each species lends itself to excellent results. (2) maintain a separate area for confirmed pregnant females and do not mix any new animals with pregnant small ruminants. (3) if the herd has a history of disease-associated pregnancy wastage, and efficacious vaccines are available, then the breeding females should be vaccinated for those specific abortion causing diseases one month before the start of breeding. in herds with a history of infectious abortion, consider adding ionophores and/or tetracycline to feeds or mineral mixtures. (note: the clinician should always be mindful of united states department of agriculture regulations concerning feed additives). (4) monitor and record all herd members’ body condition score. prevent overconditioning and provide plenty of exercise for parturient does. scores of 2.5 to 3.5 (on a 5-point system) for goats are desirable. periodically check pregnant does for ketones with urine sticks in herds with a history of pregnancy toxemia. if animals examined have higher than normal ketone values, take steps to correct the problem (e.g., feed niacin and/or ionophores, feed more grain, force more exercise). (5) trim feet as needed, and maintain good hoof care. avoid hoof disease late in gestation. (6) provide a clean, dry, draft-free area for maternity pens, or a well-drained, clean pasture with shelter. (7) give yearly vaccination boosters of toxoids for clostridium perfringens type c and d and c. tetani and other required vaccinations for that herd 30 days before kidding. (8) insure a well-designed parasite control program is instituted. preference should be given to programs that minimize the need to deworm, and enhance refugia by selective or targeted deworming (famacha©). life cycle feeding goats have greater nutritional requirements on a body-weight basis than do cows. good-quality grass forage or browse with free access to fresh water and a trace mineral/salt supplement is will meet the demands of most goats. range or pastured goats, does in late gestation or early lactation, and bucks used for breeding may require supplemental concentrates. a complete, loose, trace-mineralized salt containing selenium (in deficient areas) should be offered free choice year-round to all goats. salt and/or mineral blocks designed for cattle are commonly used may not be suitable for goats and may be associated with broken teeth or injured mouths. the caregiver should avoid over-supplementation of concentrate. when feeding does of poor to moderate body condition (<3) increasing their energy intake three to four weeks prior to breeding increases the ovulation rate. this increased feeding, or flushing, can be done by feeding good quality forages free choice, allowing creep grazing, feeding concentrates, or increasing the amount of concentrate the animals are receiving. pregnant does should receive supplemental feed four weeks before and after kidding; however over-conditioning should be avoided. obesity is associated with dystocia, pregnancy toxemia, and raises production cost. bucks can be fed a ration used as maintenance for does. the clinician should be cognizant of the potential for urinary calculi and feed males rations to minimize the incidence of this disease. a creep fed starter ration and good quality hay should be provided to kids from the first week of life feeding to minimize the incidence of pregnancy toxemia pregnancy toxemia is a rarely diagnosed condition of late term gestation in goats carrying multiple fetuses that are unable to consume energy adequate to meet metabolic demands. environmental conditions that decrease energy intake yet increase energy demands (e.g., cold, wet, windy conditions) predispose pregnant does to this condition. extremes in body condition (e.g., obesity, emaciation) also 493 clinical theriogenology • volume 5, number 4 • december 2013 predispose does to pregnancy toxemia. does carrying twins or triplets require much more energy than those carrying a single fetus. some does are not capable of consuming an adequate amount of feed to meet demands of late gestation particularly if the feed is of poor quality. chronic illnesses (e.g., parasitism, foot rot, foot scald, caprine arthritis-encephalitis, etc.) that cause weight loss and depressed appetite can lead to this condition. if energy supply does not keep pace with demands, the liver may become overwhelmed with free fatty acids and may release toxic levels of ketone bodies. the signs of pregnancy toxemia are depression, incoordination, circling, teeth grinding, tremors, and stargazing. the diagnosis can be confirmed by documenting an increase in urine and blood ketone concentrations. treatment should include oral propylene glycol, sodium propionate, or intravenous glucose solutions and should be initiated immediately. dietary strategies useful in prevention of pregnancy toxemia include: (1) feeding to meet the energy, protein, mineral and vitamin requirements of late gestation; (2) decreasing stress in late gestation (e.g., parasite and predator control), (3) dietary supplementation of niacin and/or ionophores during late gestation; (4) identifying the does carrying multiple fetuses, separating them from those pregnant with one fetus, and feeding them accordingly; (5) providing adequate bunk space to insure adequate intake by all does; (6) monitoring body condition score throughout gestation of all pregnant does; (7) keep reserve feed for emergencies; and (8) cull all poor-doing non-pregnant does and does with chronic disease. feeding the kid all newborn kids should receive 8% to 10% of their body weight in colostrum within three to 12 hours after birth. the dam’s colostrum is best but if it is not available from the dam frozen colostrum can be thawed and used. all colostrum stored frozen and fed to should be free from diseases. if possible, colostrum should be fed for two to three days. if the kid has been orphaned and is to be hand-fed then feeding 10% to 20% of their body weight of good quality species specific milk is preferred. hand-fed kids, lambs, or crias can be switched to a 2-3x daily feeding regimen by the third week of life. if a dry milk replacer powder is used, it should be thoroughly mixed with water and heated to ≈103°f before feeding. all feeding equipment should be cleaned and dried after use. kids can consume small amounts of dry feed during the first two to three weeks of life but may not be able efficiently utilize forage nutrients until two to three months of age. creep feeding can help reduce the stress associated with weaning, increase nutrient intake, and hasten the time to weaning and reduce labor associated with handfeeding. when weaning, it should be the only stressful event (weather changes, new hay, new feed, farm management changes, etc.). weaned animals should have access to good quality, highly digestible forages (14-16% crude protein). other reading pugh dg, baird an: sheep and goat medicine, 2nd ed. philadelphia: elsevier; 2011. smith mc, sherman dm: goat medicine, 2nd ed. ames (ia): wiley-blackwell; 2009 committee on the nutrient requirements of small ruminants: nutrient requirements of small ruminants: sheep, goats, cervids, and new world camelids: animal nutrition series. washington dc: the national academies press; 2007. 494clinical theriogenology • volume 5, number 4 • december 2013 idiopathic infertility in a friesian stallion idiopathic infertility in a friesian stallion lindsay hilburger, lacey rosenberg, anna mitchell, dickson varner,a soon cheong, caitlin castaneda, fahad alshanbari,a mariana amorim college of veterinary medicine, cornell university, ithaca, ny acollege of veterinary medicine, texas a&m university, college station, tx a 7 year old friesian stallion was presented for breeding soundness examination after failing to establish pregnancy in multiple warmblood mares. his breeding history included multiple unsuccessful breeding attempts over 2 consecutive breeding seasons using fresh, cooled, and frozen thawed semen with appropriate management. physical examination findings were unremarkable. a breeding soundness examination was performed. in first collection, semen volume was 105 ml with a concentration of 73 million/ml. progressive motility was 70% with 5.4 x 109 progressively motile sperm (pms). in second collection, ejaculate volume was 75 ml with 50% progressive motility, a concentration of 44 million/ml and a total of 1.6 x 109 pms. predicted daily sperm output was 3.9 x 109. a semen sample was sent to texas a&m for a sperm chromatin structure assay (scsa) and acrosome reaction assay. total and progressive motility after a 24 hour cool test were 33 and 47% respectively, after centrifugation and morphology consisted of 54% normal sperm. scsa results revealed no increase in sperm dna fragmentation, but sperm had substantially decreased acrosomal reaction activation. karyotyping and genotyping were performed and revealed a normal sry gene and a nonsusceptibility genotype at fkbp6 locus. owners elected for castration. histology of both testes demonstrated mild inflammation and degenerative changes, but these changes were not severe enough to explain infertility. presumptive reason for reduced fertility was impaired acrosomal reaction (iar), a well established cause of male subfertility in humans and animals. a recent study identified fkbp6 as a candidate susceptibility gene for iar1 leaving the mechanism of impaired acrosomal reaction unknown. friesian breed has a high inbreeding coefficient and is genetically predisposed to multiple conditions such as dwarfism, immune mediated disorders, megaesophagus, aortic rupture and orthopedic disorders.2,3 this case has potential implications for another genetic cause for infertility, such as locus other than fkbp6. keywords: acrosomal reaction, friesian, genetic, infertility, stallion references 1. raudsepp t, mccue me, das pj, et al: genome-wide association study implicates testis-sperm specific fkbp6 as a susceptibility locus for impaired acrosome reaction in stallions. plos genet 2012;8:12 2. ploeg m, saey v, de bruijn cm, et al: aortic rupture and aorto-pulmonary fistulation in the friesian horse: characterisation of the clinical and gross post mortem findings in 24 cases. equine vet j 2013;45:101-106 3. boerma s, back w, sloet van oldruitenborgh-oosterbaan mm: the friesian horse breed: a clinical challenge to the equine veterinarian? equine vet educ 2012;24:66-71. 501 clinical theriogenology • volume 11, number 3 • september 2019 502clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2015: laparoscopic artificial insemination with frozen-thawed semen in a bitch laparoscopic artificial insemination with frozen-thawed semen in a bitch mushtaq a. memon, boel a. fransson, harmon a. rogers department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa summary laparoscopic artificial insemination (l-ai) in a bitch is described. breeding management of a six-year-old rottweiler bitch was accomplished by blood progesterone and luteinizing hormonal assays. for l-ai, the bitch was anesthetized, and the surgical site prepared under aseptic techniques. two incisions about 1.5 cm in length were made, one for the laparoscope and the other for the instrument portal. the uterus was elevated to the ventral abdomen where a 20g indwelling catheter was threaded into the body of the uterus and fed into each horn individually delivering 2.5ml of frozen-thawed semen. the incisions were closed, and the bitch discharged two hours after l-ai. the bitch’s pregnancy was confirmed by transabdominal ultrasonography. on day 60 after insemination the bitch vaginally delivered two stillborn puppies. because of abdominal contractions without delivery of remaining fetuses, the bitch was taken to a veterinary clinic where a cesarean section was performed. five live and three dead puppies were delivered. laparoscopic artificial insemination provides another option for depositing frozen-thawed semen in the canine uterus. the main advantage of l-ai compared to traditional surgical ai is the quick recovery. keywords: laparoscope, artificial insemination, frozen-thawed semen, bitch case presentation a six year-old female rottweiler was presented for pre-breeding examination and artificial insemination using frozen thawed semen. three years earlier the bitch was inseminated by transcervical insemination (tci) but pregnancy did not occur. two years earlier the bitch was bred by natural service resulting in four puppies delivered via cesarean section these breedings were with different sires. the bitch was current on vaccination and was negative for brucella canis. the bitch was not on any medication and did not have any previous medical conditions. on physical examination, the bitch was bright, alert and responsive. she was in desirable body condition (47 kg; 3.5/5 bcs), her hair coat was soft and shiny, no musculoskeletal conformation abnormalities were noticed. ears, eyes, nose and mouth were clean, clear and free of any discharge or debris. no lymph nodes were palpably abnormal. her rectal temperature, respiratory rate and heart rate were within normal limits. her mucous membranes were pink and moist with a capillary refill time of <2 seconds. mammary chains were normal. a mild clear, viscous discharge from vulva was noted. the vulva was swollen and felt warm to the touch. vaginal examination revealed a moderate stricture at the vestibule vaginal junction approximately 3 cm. from the vulvar opening. the stricture was not considered to be sufficient to prevent normal whelping. no pain was elicited with flexion and extension of all joints. abdominal palpation was within normal limits. neurologic examination of the cranial nerves and proprioception were within normal limits. breeding management blood samples were collected for progesterone (p4) and luteinizing hormone (lh) assays. progesterone was assayed by radioimmunoassay, whereas the lh was detected by enzymeimmunoassay(witness lh test kits, synbiotics, kansas city, mo). cotton-tipped swabs were used to collect vaginal cells which were smeared on to microscope slides, stained with a commercial romanowsky stain variant (diff quik, jorgensen labs, loveland, co) and evaluated microscopically. transcervical insemination was attempted using an endoscope (karl storz, tuttingen, germany). many vaginal folds were visualized, further characterizing estrus activity. several attempts were made over a 15 minute period to pass a catheter through the cervix into the uterus but were unsuccessful. clinical theriogenology • volume 7, number 4 • december 2015431 laparoscopic artificial insemination the bitch was premedicated with hydromorphone (0.1 mg/kg) and dexmedetomidine (2 mcg/kg) as a mixed intramuscular injection. anesthesia was induced with intravenous injections of midazolam (0.2 mg/kg), ketamine (3.6 mg/kg), and dexmedetomidine (1 mcg/kg), and maintained by isoflurane. the surgical site, including the ventral abdomen from the xyphoid to the pubis was prepared using routine aseptic techniques and draped. two ventral midline incisions were made, one for the laparoscope and one for an instrument portal, both approximately 1.5 cm in length. the visual portal was located immediately caudal to the umbilicus and the caudal instrument portal 3 cm cranial to the pubic rim. the uterus was grasped with an atraumatic tissue forceps, elevated to the ventral abdominal wall where a 5f central silicon catheter (v-cath picc, neomedical, mila international inc, erlanger, ky) was placed transabdominally, utilizing the 18 g over-the-needle peel-away introducer of the catheter kit. the introducer entered the body of the uterus in an oblique angle, aiding cranial advancement of the catheter. the catheter was advanced 10-15 centimeters, and 2.5ml of properly thawed and prepared semen delivered into each horn. the abdomen and its organs were free of any adhesions or obvious lesions. the incisions were closed in three layers including the abdominal wall, subcutis and intradermal. the bitch recovered uneventfully, and was free of any outward signs of pain including posturing, vocalizing and general attitude. she was discharged two hours postoperatively. no further pain medication was deemed necessary. outcome on day 60 after insemination the bitch vaginally delivered two stillborn puppies. because of abdominal contractions without delivery of remaining fetuses, the bitch was taken to a veterinary clinic where a cesarean section was performed. five live and three dead puppies were delivered. discussion to our knowledge, this is the first report on successful outcome of a laparoscopic ai (l-ai) with frozen-thawed semen in the bitch. laparoscopic ai with fresh canine semen was reported by silva, et al.1 in dogs, laparoscopic surgery has been associated with less pain and faster recovery, compared to open procedures.2-4 in addition, intraoperative oxidative stress,5 c-reactive protein levels,6 and hemodynamic changes7 are minimized with laparoscopy. therefore, the minimally invasive nature of the surgery in this case may have contributed to the successful outcome. however, laparoscopic surgery entails limitations including the requirement for specialized equipment and skills. the unsuccessful attempts to catheterize the cervix in our case may have been due to nonpatency of the cervix as a result of a previous cesarian section. in addition to challenging canine breeding management, ai requires additional efforts, especially ai with frozen-thawed semen. frozen-thawed sperm is viable for a short time, therefore intrauterine insemination is recommended. pregnancy rates of 10-60% are reported with vaginal insemination with frozen-thawed semen.8-11 semen deposition into the uterus via the vagina is difficult due to the unique cervical anatomy which is unfavorable for catheterization without visualization of the cervical os.1,8,9,12,13 the long canine vagina, which can be up to 29cm long in large breeds of dogs13 is another difficulty in reaching the cervix through the vagina. due to these reasons, endoscopic catheterization or tci is commonly used for canine artificial insemination with frozen-thawed semen. numerous tci techniques are reported.15-18 however, tci requires practice and sometimes can be challenging.14,19 surgical insemination in the uterus is another technique to deposit semen directly into the uterus.20 the main disadvantage of surgical insemination is the time required for surgical preparation and post-surgical recovery of the patient. laparoscopic artificial insemination offers quick recovery. in the case reported, the bitch was released from the hospital two hours after l-ai. learning points • laparoscopic artificial insemination provides one more technique for depositing frozen-thawed semen into the canine uterus. clinical theriogenology • volume 7, number 4 • december 2015 432 • the main advantage of l-ai compared to traditional surgical ai is rapid recovery. acknowledgements the authors wish to thank alpine animal clinic, helena, mt for referring the case to the veterinary teaching hospital at washington state university, and to dakota woodward for attending the case. references 1. silva ldm, onclin k, snaps f, et al: laparoscopic intrauterine insemination in the bitch. theriogenology 1995;43:615-623. 2. hancock rb, lanz oi, waldron dr, et al: comparison of postoperative pain after ovariohysterectomy by harmonic scalpel-assisted laparoscopy compared with median celiotomy and ligation in dogs. vet surg 2005;34:273-282. 3. davidson eb, moll hd, payton me: comparison of laparoscopic ovariohysterectomy and ovariohysterectomy in dogs. vet surg 2004;33:62-69. 4. culp wt, mayhew pd, brown dc: the effect of laparoscopic versus open ovariectomy on postsurgical activity in small dogs. vet surg2009;38:811-817. 5. lee jy, kim mc: comparison of oxidative stress status in dogs undergoing laparoscopic and open ovariectomy. j vet med sci 2014;76:273-276. 6. kjelgaard-hansen m, strom h, mikkelsen lf, et al: c-reactive protein as a quantitative marker of the inflammatory stimulus of aseptic elective soft tissue surgery. vet clin pathol 2013;42:342-345. 7. hoglund ov, olsson k, hagman r, et al: comparison of haemodynamic changes during two surgical methods for neutering female dogs. res vet sci 2011;91:159-163. 8. silva ldm, onclin k, snaps f, et al: comparison of intravaginal and intra-uterine insemination of bitches with fresh or frozen semen. vet rec 1996;17:154-157. 9. linde-forsberg c, strom hb, govette g: comparison of data from vaginal vs. uterine insemination of frozen thawed dog semen. a retrospective study. theriogenology 1999;52:11-13. 10. rota a, iguer-ouada m, verstegen j, et al: fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without equex stm paste. theriogenology 1999;52:1045-1058. 11. thomassen r, farstad w, krogenaes a, et al: artificial insemination with frozen semen in dogs: a retrospective study. j reprod fertil suppl 2001;57:341-346. 12. wilson ms: non-surgical intrauterine artificial insemination in bitches using frozen semen. j reprod fertil suppl 1993;47:307-311. 13. linde-forsberg c: intrauterine insemination in the dog using the scandinavian transcerival catheter and a comparison with other methods. in: concannon pw, england g, verstegan j, et al, editors. recent advances in small animal reproduction. itaca (ny): international veterinary information services; 2001. http://www.ivis.org/ 14. wilson ms: endoscopic transcervical insemination in the bitch. in: concannon pw, england g, verstegan j, et al, editors. recent advances in small animal reproduction. ithaca (ny): international veterinary information services; 2003 http://www.ivis.org/ 15. thomassen r, sanson g, krogenaes a, et al: artificial insemination in dogs: a retrospective study of 10 years using a non-surgical approach. theriogenology 2006;66:1645-1650. 16. hayashi k, morita r, takuya a, et al: evaluation of transcervical insemination using frozen semen by flexible endoscope in dogs. j vet med sci 2013;75:315-318. 17. kustritz mvr, rice r: retrospective evaluation of breeding management data and breeding type and correlation with successful breeding of bitches. clin therio 2013;5:213-219. 18. mason sj, rous nr: comparison of endoscopic-assisted transcervical and laparotomy insemination with frozenthawed dog semen: a retrospective clinical study. theriogenology 2014;82:844-850. 19. pretzer sd, lillich rk, althouse gc: single, transcervical insemination using frozen-thawed semen in the greyhound: a case series study. theriogenology 2006;65:1029-1036. 20. brittain d, concannon pw, flanders ja, et al: use of surgical intrauterine insemination to manage infertility in a colony of research german shepherd dogs. lab anim sci 1995;45:404-407. clinical theriogenology • volume 7, number 4 • december 2015433 clinical theriogenology • volume 7, number 4 • december 2015 434 omniblank: 1 contact joaquin paredes bparedes@uoguelph.ca © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10273, http://dx.doi.org/10.58292/ct.v16.10273 case report disorders of sexual development in small ruminants joaquin paredes,a luzasz czochara,b,c daniel villagomez,b,c,d allan kingb,c adepartment of population medicine bdepartment of biomedical sciences, ontario veterinary college, university of guelph, guelph, on, canada ckaryotekk inc, guelph, on, canada ddepartamento de producción animal, universidad de guadalajara, zapopan, mexico abstract intersex conditions, also known as disorders of sexual development (dsd), are uncommon in small ruminants. this report describes in detail, 2 cases of dsd in small ruminants, including clinical presentation, histopathological and cytogenetic analysis that led to the final diagnoses. case 1, a mixed-breed ewe, was assessed due to a reported ram-like behavior at ~ 6 months of age. physical examination revealed abnormal external genitalia with enlarged clitoris and bilateral inguinal gonads, histologically confirmed as testicular tissue. karyotyping revealed sex chromosome blood chimerism (54xx, 54xy), implicating freemartinism as the cause of dsd. case 2 was a 1-year nigerian dwarf cryptorchid male goat presented for castration. during surgery, bilateral ovoid gonads attached to a bicornuate uterus were identified. histology revealed testicular tissue and uterus alongside vas deferens were consistent with persistent müllerian duct syndrome. this report describes presentations, findings, and features of dsd, a rare occurrence in small ruminants. keywords: intersex, small ruminant, gonadal differentiation, freemartinism, karyotyping background intersex conditions are rare pathologies with severe impacts on reproductive function, often rendering affected animals infertile.1–3 these conditions originate due to abnormalities in the chromosomal, gonadal or phenotypic sex of affected individuals.4 most disorders of sexual development (dsd) present challenging phenotypes, often prompting practitioners to consult theriogenologists regarding their cause and prognosis. thus, knowing the ‘common features of uncommon conditions’ holds value for the accurate diagnostic workup in these clinical cases. freemartinism is the most common dsd reported in cattle and affects > 90% of females from a heterosexual twin pregnancy.5 conversely, this condition is less common in sheep, where it affects ~ 1-5% of females from a male-bearing multiple pregnancy.6 however, freemartin ewes have a much wider range of phenotypes compared to cattle.7 in this context, we present a case of freemartinism in a sheep (case 1) with extreme masculinization features. the presented case can assist readers to understand differences between sheep and cattle in the presentation of freemartins.3 in contrast to freemartinism, other dsds such as persistent müllerian duct syndrome (pmds) are exceedingly rare in ruminants. typical features of this condition have been mainly described in dogs,8 highlighting the relevance of a comparative approach for dsd diagnosis. here we present a case of pmds in a goat, with phenotypic, histologic, and cytogenetic features like those described in dogs. case 1: intersex sheep case presentation a 6-month, rideau arcott mixed-bred ewe, was evaluated ~ 2 months after purchase due to male-like behavior. external reproductive examination revealed small vulvar opening and enlarged clitoris (figure 1, a). in the inguinal region, there were paired gonads near the abdominal wall, with the long axis of the gonad in horizontal position. no defined scrotal sac was detected (figure 1, b). gonads were symmetrical, ~ 5 cm in diameter and 9-10 cm long, with elastic consistency and freely movable in the inguinal region. epididymis, spermatic cord, and testicular vascular cone were readily palpable. no other abnormalities were detected. mailto:bparedes@uoguelph.ca http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10273 2 citation line: clinical theriogenology 2024, 16, 10273, http://dx.doi.org/10.58292/ct.v16.10273 outcome approximately 2 weeks after examination, a routine open castration was performed by the referring veterinarian. inguinal gonads were removed; examination revealed tunica albuginea, epididymis, and testicular vascular cone. excised tissues were fixed in 10% buffered formalin and submitted for histopathological examination. after surgery, blood and skin samples were submitted to our laboratory for cytogenetic analysis. animal recovered uneventfully from surgery and no complications were reported by the owner in the following 2 weeks. histologic evaluation excised tissues were processed, sectioned, routinely stained with hematoxylin and eosin (h&e) and evaluated under light microscopy. gonadal parenchyma had hypoplastic seminiferous tubules, with sertoli cells in the basal compartment and leydig cells in the interstitial compartment with no spermatogonia (figure 2, a). these findings supported testicular tissue but without evidence of spermatogenesis. furthermore, there was no evidence of ovarian tissue. epididymis tail was well-developed, with a pseudostratified ciliated columnar lining epithelium and a defined muscular layer but no sperm were visible in the lumen (figure 2, b). cytogenetic evaluation standard cytogenetic techniques were applied to obtain metaphase spreads from blood leukocytes and skin fibroblasts, as described.9 karyotype composition and sex chromosome identification was performed either in leishman and giemsa stained or g-banded spreads (n = 50 cells) under light microscopy. karyotyping results from blood leukocytes revealed 84% of cells with (2n = 54xx) karyotype (42/50 cells) and 16% of spreads with (2n = 54, xy) (8/50 cells), demonstrating the presence of blood chimerism (2n = 54xx/54xy) in this case (figure 3, a-b). karyotyping from skin fibroblast revealed a normal female karyotype (2n = 54xx) in all cells evaluated (50/50). pcr results blood samples were collected in edta-coated blood collection tubes (bd – canada, oakville, on, canada) and dna figure 1. external genitalia of case 1: a. abnormal vulva and enlarged clitoris; b. inguinal bilateral gonads. figure 2. histologic sections of gonadal tissue (h&e) from case 1: a. seminiferous tubules without spermatogenesis (400 x magnification); b. epididymal tail had no extragonadal sperm reserves (100 x magnification). http://dx.doi.org/10.58292/ct.v16.10273 citation line: clinical theriogenology 2024, 16, 10273, http://dx.doi.org/10.58292/ct.v16.10273 3 was extracted from blood leukocytes, as described.9 after incubation with red cell lysis buffer (140 mm ammonium chloride and 17 mm tris ph 7.65), leukocytes were pelleted by centrifugation (10 minutes x 700 g), washed with 0.9% nacl and resuspended in a solution containing 0.1 m tris:40 mm edta. cell lysis was performed in a lysis buffer (0.1 tris, 40 mm edta, 0.5 mm nacl, 0.2% sds). following tissue digestion and cell lysis, dna was extracted using  standard phenol-chloroform with phase separation technique. extracted dna was used for polymerase chain reaction (pcr)  for the sex determining region (sry) gene, using primers  designed for cattle, sheep, and goats, as described by  our group  (f: 5’-ccaattaagccggtcacagt-3’ r: 5’-gcacaagaaagtccaggctc-3’).9 the reaction yielded a 162 bp amplicon run through an electrophoresis gel; the pcr was positive for the sry gene, confirming the presence of blood chimerism (54,xx/54,xy-sry positive (figure 8). case 2: intersex goat case presentation a 1-year, nigerian dwarf male cryptorchid goat was referred to the ontario veterinary college – teaching veterinary hospital for castration. external examination of the genitalia revealed normal prepuce and penis, and small scrotum with no evidence of testis in either in scrotum or inguinal area, leading to a presumptive diagnosis of abdominal cryptorchidism. animal’s head had 2 small horns (~ 1.5 2 cm long), ruling out polled intersex syndrome (pis). outcome approximately 36 hours after admission, cryptorchidectomy was performed under general anesthesia. surgery via inguinal approach revealed 2 round gonads ~ 2 cm in diameter in the inguinal canal, close to the internal inguinal ring. on exteriorization, well-developed smooth and bicornuate tubular tract attached to the gonads (figure 4, a) was noticed. gonads and tubular tract were surgically removed (figure 4, b) and fixed in 10% formalin for histological examination. blood (cytogenetic analysis) and samples were sent to our laboratory. animal recovered uneventfully and was discharged from the hospital 3 days later. histologic evaluation histologic evaluation of the gonadal tissue revealed seminiferous tubules lined by sertoli cells in the basal compartment and occasionally scattered spermatogonia. findings were compatible with testicular tissue (figure 5, a-b). however, there was no evidence of spermatogenesis nor ovarian tissue detected. the tubular bicornuate tissue had a well-develop endometrium, myometrium, and serosa, plus a vas deferens with a well-defined lumen, muscular and serosa layer (figure 6, a-b). based on these findings, a diagnosis of pmds was made. cytogenetic evaluation g-banded karyotype from case 2 revealed a normal male goat karyotype (2n = 60, xy) in all cells evaluated (n = 50 cells) (figure 7). figure 4. cryptochidectomy: a. gonad and attached bicornuate duct; b. excised ovoid gonads with extended bicornuate ducts. figure 3. g-banding of leukocytes metaphase spreads from case 1: a. 2n = 54xy; b. 2n = 54xx. http://dx.doi.org/10.58292/ct.v16.10273 4 citation line: clinical theriogenology 2024, 16, 10273, http://dx.doi.org/10.58292/ct.v16.10273 pcr results case 2 pcr was positive for the sry gene (figure 8) and thus confirmed a normal male karyotype (2n = 60xy – sry positive). discussion freemartinism has been reported far less frequently in sheep than in cattle. case 1 was an ewe with extreme masculinization features including ambiguous genitalia and inguinal testes, whereas the blood chimerism (xx, xy) is a hallmark for freemartinism in all domesticated ruminant species. in our case, cell chimerism (2n = 54xx, 54xy) was only present in blood leukocytes but not in skin fibroblasts (2n = 54xx), leading to the final diagnosis of freemartinism. despite no confirmation that the animal was born from a male-bearing twin or figure 6. histologic section of the bicornuate ducts (h&e) from case 2: a. uterus alongside a muscular vas deferens (right side of the image) (25 x magnification); b. endometrial tissue of the same specimen (200 x magnification). figure 5. histological examination of gonadal tissue (h&e) from case 2: a. seminiferous tubules (200 x magnification); b. seminiferous tubules containing sertoli cells and scattered spermatogonia without evidence of active spermatogenesis (400 x magnification). figure 7. g-banding of leukocytes metaphase spreads from case 2 (2n = 60, xy). figure 8. pcr results for sry gene: lane 1. ladder (100 bp); lane 2. h2o control; lane 3. female control (bovine, blood); lane 4. male control (bovine, blood); lane c1. case 1; lane c2. case 2. http://dx.doi.org/10.58292/ct.v16.10273 citation line: clinical theriogenology 2024, 16, 10273, http://dx.doi.org/10.58292/ct.v16.10273 5 multiple pregnancy, all clinical, histologic and cytogenetic analyses were consistent with previous reports of freemartinism in sheep.10 in cattle, freemartinism affects > 90% of heifers born from a heterosexual twin pregnancy3 whereas in sheep, the incidence is as low as 1-5% in male-bearing multiple pregnancies.6,10 the more common development of vascular placental anastomosis between fetuses is responsible for the high incidence of freemartin in cattle11 but this phenomenon is less frequent in small ruminants.2,3 it has been suggested that in highly prolific sheep breeds, male-bearing pregnancies with 4 or 5 lambs might present an increased risk for freemartinism, potentially due to increased placental contact between fetuses.10 the presented case (case 1) involved a mixed-rideau arcott sheep, a breed with an average of 2.6 lambs per birth.12 however, as information on the litter was not available, it is not known whether this factor had a role in this case. some reports have suggested that certain rams have higher incidence of freemartin lambs than others.9 however, no information regarding the sire and half-siblings was available; therefore, it was not possible to determine what other predisposing factor might have had a role in the presented case. descended testes is a phenotypic feature rarely reported in freemartin cattle.13 approximately 31% of freemartin sheep have descended testes-like gonads with epididymis and testicular vascular cone,10 and constitute the most extreme forms of masculinization of this condition. perhaps the notable testicular gonadal differentiation observed in freemartin sheep was linked to development of placental vascular anastomosis between fetuses at an earlier stage. dsd such as pmds are exceedingly rare in ruminants. the presented case (case 2) was a goat, and the main features included cryptorchidism, uterine development, and normal male goat karyotype (2n = 60xy). interestingly, similar features have been described in other species (e.g. dogs) with pmds. affected individuals are xy, with male-like behavior, a well-developed penis and prepuce, bilateral cryptorchids and a concomitant development of the oviducts, uterine tubes, cervix and vagina connected to the prostate.14 in this species, ~ 50% of affected individuals have cryptorchidism,14 and it is common for these cryptorchid dogs to develop testicular neoplasms later in life. consequently, the condition may remain unnoticed until signs of testicular tumors and estrogen exposure are present.8,15 apart from testicular tumors, further complications of pmds include hydrometra,16 pyometra,17 and uterine leiomyoma18 in dogs. this condition has been primarily described in miniature schnauzers and has a known genetic component.19 a mutation in the antimüllerian hormone receptor 2 (amhr2) is responsible for this condition and has an autosomal recessive mode of inheritance.19,20 there are few reports of this condition in ruminants and affected individuals present with male phenotype and bilateral cryptorchidism.21 the genetic basis and mode of inheritance has not been yet defined for goats and there is no known breed predisposition. cryptorchidism, testicular tissue, and vas deferens along well-developed uterus are characteristic features of pmds in goats21 and dogs.19 other potential dsd in goats include the well-known pis, considered a recessive inherited sex-reversal syndrome linked to the homozygous polledness trait. affected animals have normal female karyotype (60, xx) with variable degrees of masculinization including external male genitalia and testicular gonadal development. this condition arises in homozygous females with a mutation in the foxl2 gene that is responsible for the polledness trait and also responsible for ovarian development.22,23 in case 2, the presence of small horns and xy karyotype ruled out pis. these cases demonstrated that dsd can have a wide range of phenotypes (e.g. freemartinism in small ruminants), whereas in contrast, other conditions such as pmds may have typical features across species. understanding the range of clinical presentations and key features of various dsds is not only relevant for diagnostic workup, but also essential for understanding mechanisms driving reproductive development in mammals. learning points • freemartins are characterized by (xx/xy) blood chimerism in ruminants. although less common in sheep, it can present with marked masculinization features compared to cattle. • the most extreme form of freemartinism in sheep can include well-developed descended testes and spermatic cord with no evidence of spermatogenesis. • pmds is an exceedingly rare dsd in ruminants. key features reported in other species, including cryptorchidism, well-developed uterus, and vas deferens, plus normal male karyotype are also features present in goats with pmds. conflict of interest none to declare. acknowledgement authors thank dr jeff rau and dr camila cantarelli for clinical management of these cases and elizabeth st john for pcr preparations. references 1. christensen bw: disorders of sexual development in dogs and cats. vet clin north am small anim pract 2012;42:515–526. doi: 10.1016/j.cvsm.2012.01.008 2. albarella s, d’anza e, galdiero g, et al: cytogenetic analyses in ewes with congenital abnormalities of the genital apparatus. animals 2019;9:1–10. doi: 10.3390/ani9100776 3. peretti v, ciotola f, albarella s, et al: xx/xy chimerism in cattle: clinical and cytogenetic studies. sex dev 2008;2:24–30. doi: 10.1159/000117716 4. villagómez daf, parma p, radi o, et al: classical and 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https://doi.org/10.1111/age.12918 1 contact dale kelley dale.kelley@okstate.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10071, http://dx.doi.org/10.58292/ct.v16.10071 research report stallion sperm concentration measurements: experience and equipment dale kelley, reed holyoak department of veterinary clinical sciences, college of veterinary medicine oklahoma state university, stillwater, ok, usa abstract accuracy is paramount in evaluating sperm concentration and can be a challenge to those with minimal laboratory experience. purpose of the study was to determine the effect of operator experience on sperm concentrations using 4 methods: makler® counting chamber, equine densimeter, isperm, and nucleocounter®. there was no difference (p = 0.64) between experienced and novice processors for makler® counting chamber; was difference (p = 0.005) for equine densimeter; was no difference (p = 0.35) for isperm; and was tending toward difference (p = 0.068) for nucleocounter®. correlation between bias and magnitude for makler® counting chamber was –0.74 (p = 0.003); for equine densimeter was –0.44 (p = 0.11); for isperm was 0.06 (p = 0.83), and nucleocounter® was –0.52 (p = 0.06). makler® counting chamber produced a mean sperm concentration similar to an experienced processor but had significant variation within novice processors. equine densimeter significantly overestimated sperm concentrations with novice processors but had the least variation. the isperm performed poorly for both experienced and novices and produced significantly different concentrations than other methods. thus, isperm cannot be recommended to accurately measure sperm concentration. lastly, nucleocounter had no difference in mean sperm concentrations or variation and was the best system for a novice processor to gain accurate and repeatable sperm concentration measurements. keywords: stallion, sperm concentration, laboratory experience introduction laboratory work can be challenging for veterinarians new to processing semen, since a few receive formal training. accurately determining sperm concentration sperm in an ejaculate is an essential step in analyzing and processing semen1 and depends on proper dilution technique, laboratory skills, and equipment knowledge. several types of equipment exist to determine sperm concentration with varying working principles. the simplest method relies on a microscope and visual differentiation and counting of sperm on a slide with a known volume (hemocytometer or makler counting chamber). a second, common, type of equipment is a spectrophotometer. this measures light impedance through the ejaculate to determine sperm concentration using a standard curve. a third type of equipment coined computer-assisted sperm analysis (casa) uses a computer system coupled to a microscope to identify and count sperm and can perform sperm motion analysis. finally, another common system to determine sperm concentration uses fluorescent dyes to stain nuclei to count sperm via either flow cytometry or a cell counter.1 there were  differences2,3 among photometric, direct counting and fluorescent cell counting methods in evaluating stallion sperm concentration. each of these methods of determining sperm concentration has their advantages and disadvantages in terms of accuracy, time required to perform, and operational skills. purpose of this study was to determine the effect of operator experience level on determining sperm concentrations using each of these 4 distinct methods. materials and methods animal use was approved by the institutional animal care and use committee. three ejaculates from 2 stallions were utilized. briefly, stallions were collected using a missouri artificial vagina, phantom, and tease mare. after semen was collected the volume of semen was determined and semen was gently mixed and evenly divided among an experienced and 6 novice semen processors. experienced processor was a diplomate of the american college of theriogenologists with multiple years of experience in equine theriogenology. novice mailto:dale.kelley@okstate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10071 2 citation line: clinical theriogenology 2024, 16, 10071, http://dx.doi.org/10.58292/ct.v16.10071 processors were 4th year veterinary students with little to no previous experience in semen processing. four methods were selected to determine sperm concentration based on different principles of measurement. they were: makler® counting chamber (sefi-medical instruments, ltd., santa ana, ca, usa) to represent manual analysis; equine densimeter (model 591b, animal reproduction systems, chino, ca, usa) to represent a spectrophotometer-based system; isperm (mcasa; aidmics biotechnology co, ltd, taipei city, taiwan) to represent a casa based system; and nucleocounter® (sp-100; chemometec a/s, allerod, denmark) to represent a fluorescent-dye based system. prior to study initiation, novice processors (n = 6) were given a training session specific to each equipment. throughout the study, processors had access to written instruction on equipment operation. instructions for makler® counting chamber were as follows. semen was diluted 1:1 with 10% buffered formal saline4 and placed on a slide. after semen mixture was well mixed and a 5 μl drop was placed on the center of the disc area using a micropipette. a coverslip was then placed on the 4 pins and gently pressed down. sperm heads were counted within each grid and those that touched the top or left lines, whereas those touching the bottom or right lines were not counted. a line of 10 squares were counted and multiplied by 2 (to account for dilution), this represented the sperm concentration (106/ml). a second strip of 10 squares was counted and the sperm concentration determined as before; average of 2 counts was used to determine sperm concentration. to represent the spectrophotometer-based systems, equine densimeter was used. novice processors were trained to operate by following directions on the screen. briefly, 3.42 ml of formalin 10 (animal reproduction systems) was added to a cuvette and system was zeroed. next, the cuvette was removed and 180 μl of raw semen was added to formalin 10 solution in the cuvette, mixed and placed back in the machine to determine sperm concentration. prior to use, isperm was set up according to manufacturing instructions. novice processors were instructed to use isperm according to manufacturer directions. initially, semen was diluted 1:1 with semen extender (inra96, imv technologies, osseo, mn, usa) and if needed, further dilutions were made with semen extender. a base chip was then mounted on the sample collector. one of 3 sampling loading methods were taught, and the method used was at operator discretion. method 1 added 7.5 μl of extended semen sample on a vertical base chip, method 2 used a dropper to add the extended semen sample into the cover chip, and method 3 dipped the base chip into the extended semen sample. after the application of the extended semen sample, base chip was pressed vertically into the cover chip until a ‘click’ was heard and continued to be pressed for an additional 1–2 seconds. once this was completed the sample collector/base chip/cover chip combination was attached to the ipad and sperm concentration was determined. sample collector was rotated to perform a 4-view analysis. if any errors occurred, analysis was repeated. for nucleocounter® novice processors were instructed on operation and semenview software including how to select the species for analysis and change the dilution factor to correspond to the ideal reading range for sperm concentration. novice processors were free to set the dilution factor (df), select the appropriate amount of reagent (s100; chemometec) and sample volume based on the df utilized. briefly, depending on the df used a volume of reagent was added to a clean tube. next, the desired volume of sample was added to the reagent. these volumes (reagent and sample) were based on manufacturer tables. next the sample was gently mixed, and a cassette tip was placed in the solution, sample aspirated, and the cassette placed in the machine and sample was analyzed. after the training sessions novice processors determined sperm concentration without supervision. data analyses mean sperm concentrations were analyzed using sas (9.4) general linear model procedure by operator experience (novice vs. experienced), method of semen concentration analysis (makler counting chamber, equine densimeter, isperm, and nucleocounter®) and interaction between experience and method. means statement with least squares difference was used to determine mean sperm concentration by method and make pairwise comparisons. the sgplot procedure was used to generate bland–altman plots (difference and mean concentration between experienced and novice processors). t-test was used to compare experience level for each method (test for zero bias). independence of bias was tested using the corr procedure using the bias (difference between experienced and novice) and magnitude (average of experienced and novice). data were represented as mean ± standard deviation. results mean sperm concentration for makler® counting chamber, equine densimeter, isperm and nucleocounter® for experienced operator was 107.1 ± 13.7, 97.6 ± 13.1, 148.6 ± 63.4, and 109.3 ± 16.4 (106/ml), respectively (table 1). mean sperm concentrations for the makler® counting chamber, equine densimeter, isperm, and nucleocounter® for novice operator were table 1. mean sperm concentration (106/ml) and standard deviation (std) for each method by operator experience. analysis method experienced operator novice operator p value mean std mean std makler counting chamber 107.1 13.7 109.9 29.2 0.64 densimeter 97.6 13.1 109.2 19.1 0.005 isperm 148.6 63.4 161.7 61.3 0.35 nucleocounter 109.3 16.5 121.9 27.7 0.068 http://dx.doi.org/10.58292/ct.v16.10071 citation line: clinical theriogenology 2024, 16, 10071, http://dx.doi.org/10.58292/ct.v16.10071 3 109.9 ± 29.2, 109.2 ± 19.1, 161.7 ± 61.3, and 121.9 ± 22.7 (106/ml), respectively (table 1). there was no significant effect of experience level (p = 0.72), there was a significant effect of method (p = 0.18), and no significant interaction between experience and method (p = 0.99). there was no difference in mean sperm concentration values between makler® counting chamber, equine densimeter, and nucleocounter®; however, isperm had significantly higher sperm concentration values (p < 0.05) than other methods. bland–altman plots for makler counting chamber, equine densimeter, isperm, and nucleocounter are provided (figure 1). mean difference in sperm concentration assessments between experienced and novice operator for makler® counting chamber was -2.8 ± 24.7; for equine densimeter was -11.6 ± 14.7; for the isperm was -13.1 ± 56.5; and nucleocounter® was -12.6 ± 26.6 (106/ml). t-test result between experienced and novice processors for makler® counting chamber) was not different (p = 0.64); for equine densimeter was different (p = 0.005); for isperm was not different (p = 0.35); and for nucleocounter® there was a tendency toward difference (p = 0.068). correlation between the bias and magnitude for makler® counting chamber was -0.74 (p = 0.003); for equine densimeter was -0.44 (p = 0.11); for isperm was 0.06 (p = 0.83); and nucleocounter® was -0.52 (p = 0.06). discussion differences between and within human5,6 and bovine7 andrology laboratories were observed and thus it is not surprising in our study to find differences in agreement  among different equipment and operator experience level. makler® counting chamber had similar mean sperm concentration values between experienced and novice processors. the correlation was significant, and the negative value indicated significantly more variation in the sperm concentration values with novice processors. the variation in sperm concentration assessments with novice processors can be due to several factors such as accuracy of dilutions, uniformly mixing semen prior to performing dilutions, mixing prior to pipetting samples, chamber filling, how the sperm were counted under the microscope, sample variation and mathematical errors. the accuracy of dilutions can be impacted by both the precision of pipetting and type of pipet used. two types of pipets were available, air displacement and positive displacement. positive displacement pipets were more accurate than air displacement pipets.3 additionally, with air figure 1. bland-altman plot difference (experienced minus novice operator sperm concentration) to mean ([experience plus novice operator sperm concentration]/2) for a. makler counting chamber; b. densimeter; c. isperm; and d. nucleocounter. for each figure (a–d) blue line represents a difference = 0. dashed red line represents 2 standard deviations (stds) and green dashed line represents 3 stds. http://dx.doi.org/10.58292/ct.v16.10071 4 citation line: clinical theriogenology 2024, 16, 10071, http://dx.doi.org/10.58292/ct.v16.10071 displacement pipets novice users may inadvertently aspirate too much fluid by depressing the plunger too far or not aspirate enough fluid all of which can impact accuracy. another source of variation can be how the sperm were counted under the microscope. in general, sperm should be counted when the head is either in the grid or lying on 2 adjacent lines to prevent double counting with adjacent squares; whether this was carried out correctly each time is unknown but is a potential source of error. lastly, since a 1:1 dilution was made the count needed to be doubled to determine the concentration. in this case it is unlikely mathematical errors contributed to the variation but this is more likely with hemocytometer as not only the dilution factor has to be accounted for but also the volume of sample analyzed.8 although the mean sperm concentrations were similar between experience level, the amount of variation within the novice group makes it difficult to recommend this as an accurate method. with equine densimeter novice processors obtained significantly higher mean sperm concentration values than the experienced operator; however, the variation between experience levels was not different. the higher mean sperm concentration obtained by novices could be due to pipetting error either with the densimeter media or semen sample or mixing of sample. interestingly, there was no difference in variation among groups. this could be due to the fact equine densimeter has direction for operation on the led as the sample run that may result is less variation in the procedure. this suggested that novices using an equine densimeter will generally overestimate sperm concentrations but with less variation. the isperm assessed significantly higher concentrations than other methods but there was no difference related to experience on the concentration and variation. this system is subjected to the same problems as the makler® counting chamber, such as mixing, dilution, and chamber filling but is also subjected to software misidentification.9 for boar sperm, casa system had higher agreement with the hemocytometer with dilute samples but this was lost in more concentrated semen samples.10 casa-based systems (e.g. isperm) is considered to be inaccurate and only give a rough estimate of sperm numbers10 and are not recommended by who11 or the national association of animal breeders.12 with nucleocounter® there was no significant difference in sperm concentration values or variation between experience levels. studies have used nucleocounter as a gold standard for determining sperm concentration13,14 but this system is subjected to errors with dilution and dilution factor settings. using a dilution rate outside optimal limits will decrease accuracy.1 additionally, excessive time to read could impact accuracy since a fluorescent dye is used.1 this appears to be the best method for novices to evaluate sperm concentration since the mean and variation in sperm concentration were not significantly different. each system that measured sperm concentration had their advantages and disadvantages in how they performed with novice processors. makler® counting chamber produced a mean sperm concentration similar to an experienced operator but had significant variation. equine densimeter significantly overestimated sperm concentration with novice processors but had the least variation. the isperm performed poorly for both experienced and novices and produced significantly different concentration than other methods. thus, isperm cannot be recommended to accurately measure sperm concentration. lastly, nucleocounter had no difference in mean sperm concentrations or variation and was the best system for a novice processor to gain accurate and repeatable sperm concentration measurements. regardless of the system employed to evaluate sperm concentration, it is important to take time to become familiar and comfortable with the laboratory techniques and operation of the equipment to ensure accurate results. conflict of interest none to report. references 1. brito lfc, althouse gc, aurich c, et al: andrology laboratory review: evaluation of sperm concentration. theriogenology 2016;85:1507–1527. doi: 10.1016/j.theriogenology. 2016.01.002 2. rigby sl, varner dd, thompson ja, et al: measurement of sperm concentration in stallion ejaculates using photometric or direct sperm enumeration techniques. proc ann conv am assoc equine pract 2001;47:236–238. 3. comerford kl: validation of a commercially available fluorescence-based instrument to evaluate stallion spermatozoal concentration and comparison to photometric systems. ms thesis, texas a & m university. oak trust library; 2010. 4. makler a: the improved ten-micrometer chamber for rapid sperm count and motility evaluation. fertil steril 1980;33:337–338. doi: 10.1016/s0015-0282(16)44606-6 5. lemmens l, van den hoven l, van vrouwerff nj, et al: external quality control and training of semen analysis in the netherlands: starting point for further reduction of outcome variability. asian j androl 2022;24:15. doi: 10.4103/aja.aja_46_21 6. jørgensen n, auger j, giwercman a, et al: semen analysis performed by different laboratory teams: an intervariation study. int  j androl 1997;20:201–208. doi: 10.1046/j.1365-2605.1997. 00052.x 7. brito lf: a multilaboratory study on the variability of bovine semen analysis. theriogenology 2016;85:254–266. doi: 10.1016/j. theriogenology.2015.05.027 8. absher m: chapter 1 – hemocytometer counting. in: kruse pf, patterson mk, editors, tissue culture. academic press; 1973, p. 395–397. doi: 10.1016/b978-0-12-427150-0.50098-x 9. zinaman mj, uhler ml, vertuno e, et al: evaluation of computerassisted semen analysis (casa) with ident stain to determine sperm concentration. j androl 1996;17:288–292. doi: 10.1002/ j.1939-4640.1996.tb01784.x 10. hansen c, vermeiden t, vermeiden jpw, et al: comparison of facscount af system, improved neubauer hemocytometer, corning 254 photometer, spermvision, ultimate and nucleocounter sp-100 for determination of sperm concentration of boar semen. theriogenology 2006;66:2188–2194. doi: 10.1016/j.theriogenology.2006.05.020 11. world health organization: who laboratory manual for the examination and processing of human semen. geneva, switzerland; who press, world health organization: 2010. http://dx.doi.org/10.58292/ct.v16.10071 https://doi.org/10.1016/j.theriogenology.2016.01.002 https://doi.org/10.1016/s0015-0282(16)44606-6 https://doi.org/10.4103/aja.aja_46_21 https://doi.org/10.1046/j.1365-2605.1997.00052.x https://doi.org/10.1046/j.1365-2605.1997.00052.x https://doi.org/10.1016/j.theriogenology.2015.05.027 https://doi.org/10.1016/j.theriogenology.2015.05.027 https://doi.org/10.1016/b978-0-12-427150-0.50098-x https://doi.org/10.1002/j.1939-4640.1996.tb01784.x https://doi.org/10.1002/j.1939-4640.1996.tb01784.x https://doi.org/10.1016/j.theriogenology.2006.05.020 citation line: clinical theriogenology 2024, 16, 10071, http://dx.doi.org/10.58292/ct.v16.10071 5 12. brito l, beckman b, cardwell b, et al: naab-css semen quality control program minimum guidelines. proceedings of the 24th technical conference on artificial insemination and reproduction. 2012, p. 37–41. 13. hoogewijs mk, de vliegher sp, govaere jl: influence of counting chamber type on casa outcomes of equine semen analysis. equine vet j 2012;44:542–549. doi: 10.1111/ j.20423306. 2011.00523.x 14. anzar m, kroetsch t, buhr mm. comparison of different methods for assessment of sperm concentration and membrane integrity with bull semen. j androl 2009;30:661–668. doi: 10.2164/jandrol.108.007500 http://dx.doi.org/10.58292/ct.v16.10071 https://doi.org/10.1111/j.2042-3306.2011.00523.x https://doi.org/10.1111/j.2042-3306.2011.00523.x https://doi.org/10.2164/jandrol.108.007500 31 contact eduardo arroyo eduardo.arroyo@wsu.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 review report cryptorchidism in stallions eduardo arroyo,a ahmed tibarya,b acomparative theriogenology, department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa, usa bcenter for reproductive biology, washington state university, pullman, wa, usa abstract cryptorchidism is the most common disorder of sexual development in the male. the etiopathogenesis of cryptorchidism remains poorly understood and may involve genetic and environmental factors. the condition is obvious in horses with a good history, but diagnosis of cryptorchidism can be challenging in an apparently gelded horse presenting with stallion-like behavior and hemicastrated horses. determination of the location of the retained testis is important for the selection of the surgical approach for cryptorchidectomy. the objective of this paper is to review current knowledge regarding pathogenesis of cryptorchidism in the equine, and methods for diagnosis including ultrasonographic determination of the location of the retained testis. a discussion of cryptorchidectomy approaches and possible complications is presented. keywords: equine, testis, sexual disorders, cryptorchid, male introduction cryptorchidism, the failure of one or both testes to complete testicular descent into the scrotum, is the most common disorder of sexual development in males.1 in the equine, the reported prevalence of cryptorchidism ranges from 2 to 12%.2–4 in the equine industry, cryptorchid horses may be referred to as rigs, ridglings, or high flankers by some horse owners. geldings with stallion-like behavior sometimes are referred to as false rigs.5 a multicenter study on 5009 cryptorchid horses showed that  some breeds (american quarter horses, american saddlebreds, and percherons) are overrepresented while others (thoroughbred, standardbred, morgan, tennessee walking horse and arabian) are less represented.2 this breed predisposition was confirmed by other authors.3,6,7 a high incidence of cryptorchidism was reported in friesian,8 swedish icelandic,9 and mangalarga horses.6 this suggests a genetic component in the etiology of the defect. the authors have seen a high incidence in some akhal teke colts (5/16) and paso fino colts (7 /18), the latter being sired by a cryptorchid stallion. a review of the records of 76 colts born at washington state university’s arabian herd, showed a cryptorchidism incidence of 2.6%. cryptorchidism may be suspected upon examination of yearlings, but most cases are diagnosed at 2 years of age or older as most male horses not intended for breeding are castrated at this age.10 although uncommon, cryptorchidism may be associated with other more severe urinary or urogenital malformations such as renal dysplasia, ectopic ureters, uretectasia,11 umbilical or inguinal hernias, and hypoplastic testis.2 this emphasizes the importance of thorough physical examination prior to castration to avoid surgical complications. in horses with no clear castration history, it can be clinically challenging to distinguish between geldings with stallion-like behavior and bilateral or unilaterally castrated (hemicastrated) cryptorchid horses. hemicastrated cryptorchid horses may have been sold as geldings and started to display stallion-like behavior later in life. the reported prevalence of hemicastration among stallions undergoing castration is around 4.5%, while the prevalence of hemicastration in cryptorchid horses can be as high as 41%.11–13 in one study, the prevalence of hemicastration in suspected unilateral cryptorchid horses aged 4 years, 5 years and > 6 years or more was 57%, 73%, and 84%.10,13 hemicastration (removal of the descended testicle) may be intentional, due to the inability to complete the surgery when attempting a cryptorchidectomy, or due to surgical error in which a portion of the epididymis is misidentified as a testicle and removed during an attempted castration.10 mailto:eduardo.arroyo@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9648 32 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 several ethical issues are raised when dealing with cryptorchidism. in some countries and for some breed registries, cryptorchid stallions disqualify them from being approved for breeding. breeding of unilateral cryptorchid stallion is discouraged. hemicastration of unilateral cryptorchid horses is strongly discouraged when they are intended to be sold as gelding. some breed associations require reporting hemicastration prior to registering the horse. other associations require that stallion owners disclose hemicastration, because of cryptorchidism, in their breeding contracts. veterinarians are often asked for treatment options that will promote or enhance testicular descent. human chorionic gonadotropin (hcg) has been used in humans to promote testicular descent and has been tested in horses as well. in a small trial, inguinal retained testes descended into the scrotum in 4/8 colts after treatment with 2500 iu of hcg twice weekly for 4 weeks.14 acupuncture has also been reported anecdotally to promote testicular descent.15 the aim of this paper is to review the etiopathogenesis, diagnosis, and surgical management of cryptorchidism in horses. a special emphasis is given to clinical procedures for the determination of the location of the retained testes. pathophysiology of cryptorchidism in the normal horse, testicular descent is completed between 30 days prepartum and 10 days after birth. testicular descent in mammals was reviewed thoroughly in a landmark paper by amann and veeramacheneni.1 the vaginal process, the gubernaculum, and the inguinal canal are the anatomic structures involved in the process of testicular descent. at mid-pregnancy (~5 months), the equine fetal gonad is large enough to restrict the passage of the fetal testes through the inguinal canal1 (figure 1). in mid-pregnancy, the length of the caudal gonadal ligament, size of the testis, small diameter of the inguinal ring, and the lack of tension on the gubernaculum prevent descent of the testes through the inguinal canal. after the 8th month of gestation, the gubernaculum proprium begins to decrease in size and only the fibrous connection within the inguinal canal holds the vaginal process outside the inguinal ring. in late pregnancy or early in the neonatal period, the testes have descended into the scrotum and the vaginal ring constricts to about 1 cm in diameter preventing their return to an abdominal position.16–18 testicular descent into the scrotum occurs in 2 phases: the transabdominal phase and the inguinoscrotal phase. the anatomical changes leading to testicular descent are controlled by hormonal signals such as insulin-like peptide 3, it’s receptor that belongs to the relaxin family peptide 2 (rxfp2), and testosterone.1,19,20 insulin-like peptide 3 (insl3) is a member of the insulin hormone superfamily expressed in the developing testes and is involved in the transabdominal phase of testicular descent through a proper development of the gubernaculum.20 insl3, rxfp2, nuclear receptor 5 orphenus 1, and hoxa10 are all implicated with the estrogen receptor 1 controlled growth of the gubernaculum. additionally, androgen receptor (ar) is involved in the regression of the cranial suspensory ligament of the testis. testicular descent is also regulated by calcitonin/calcitonin-related polypeptide alpha gene (calca) acting on the genitofemoral nerve.21 polymorphism in the ar, tgfbr3, and hoxa11 genes and polymorphism in genetic loci coding for cytoskeleton-associated proteins are also considered to be contributing factors for cryptorchidism.22 cryptorchidism has been attributed to 4 primary mechanisms: failure of the gubernaculum to enlarge the inguinal ring, failure of regression of the gubernaculum, primary testicular abnormalities (cystic rete testis or neoplasia) and persistence of the suspensory ligament of the testis.1,3,23,24 it has been shown that equine cryptorchid testes have a decreasein immunoreactive insl-3 in the testicular interstitial cells.25 also, serum insl3 was found to be lower in bilaterally cryptorchid stallions and unilaterally cryptorchid stallions following removal of the scrotal testis compared to intact and unilateral cryptorchid stallions with 1 scrotal testis.20 these observations strongly suggest the potential role of the insl-3 gene in the pathogenesis of cryptorchidism. other genes have been associated with cryptorchidism in various species.26 role of genetics in cryptorchidism the role of genetics and the heritability of cryptorchidism have long been suspected. in the 1940s, hamori suggested a hereditary nature of cryptorchidism based on observation of the defect in 11 of 17 related horses.27 in the early 1950s, a unilateral cryptorchid anglo-arab stallion in the trakehner stud book sired 8 cryptorchid colts out of 24 (2 bilateral and 6 unilateral cryptorchids).28 recently, the heritability of cryptorchidism was estimated to be between 0.12 and 0.32 in swedish-born icelandic horses.9 in friesian horses, a prevalence of cryptorchidism of 14.2% and a heritability estimate of 0.13 were reported in one study.29 investigation of 7 candidate genes potentially involved in abnormal testicular descent (androgen receptor, insulin-like peptide 3, relaxin/insulin like family peptide receptor 2, nuclear receptor 5 orpheus 1 gene, and the hoxa 10) failed to demonstrate an association.21 genomic studies showed that 9% of cryptorchid horses share the same 200-kb deletion around akr1c genes in eca29. the region is considered as a putative risk factor for cryptorchidism. tspy and etsty2 showed significant copy number variations between cryptorchid and normal males.30 no genetic tests are currently available for heritable genetic defects associated with cryptorchidism.31 cryptorchidism may be associated with other disorders of sexual differentiation32,33 (figures 2, 3). several cases of 64 xx, figure 1. large fetal testes (arrow) in abdominal location in a 7-month-old fetus. http://dx.doi.org/10.58292/ct.v15.9648 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 33 sry negative disorder of sexual development with ambiguous external genitalia, stallion-like behavior have been described in various breeds.34–39 in one case, cryptorchidism was associated with an autosomal 27 trisomy.40 in these intersex animals, the abdominal gonad can be testes,38,39,41 bilateral ovotestis,42,43 a single ovotestis, or an ovary and a testis.44,45 all these observations point to a multifactorial etiopathogenesis of cryptorchidism involving possibly genetics, and other external factor such as fetal exposure to endocrine disruptors. location of the retained testis a retained testis may be located anywhere along the course of normal testicular descent. for classification, 3 locations are described: complete abdominal cryptorchidism when the testis and epididymis are located entirely within the abdomen (figure 4); partial abdominal cryptorchidism when the testis is in the abdomen and the epididymis in the inguinal canal (figure 5); inguinal cryptorchidism when the testis and epididymis are located within the inguinal canal or adjacent to the external inguinal ring within the inguinal fascia (figure 6). cryptorchidism is predominantly unilateral, occurring with equal frequency on the left and right testis; only 10 to 15% of cases are bilateral (table).29,46–50 early studies showed an equal frequency of left and right side cryptorchidism in older ponies and other breeds of horses but a higher incidence of right side cryptorchidism was reported in younger ponies.12 in a recent study on friesian horses, the right testis was retained significantly more often than the left (64.5% vs. 35.5%, n = 188).29 according to many publications, the left testicle is more likely to be retained abdominally than the right.6,7,13 however, in a study on 94 cases, the abdominally retained testes were rightsided and left-sided in 48.9% and 51.1% of the cases respectively.8 in bilateral cryptorchid cases, both testes are generally found in the abdominal position and only a small proportion are bilaterally inguinal or a combination of an inguinal and an abdominal testis.6,8,47 effect of cryptorchidism on fertility the role of testicular thermoregulation on establishment and maintenance of normal spermatogenesis is well established.51 transcriptomic analysis showed downregulation of 11 genes associated with spermatogenesis in cryptorchid compared to normal testes.22 bilaterally cryptorchid horses are sterile while unilaterally affected horses have varying degrees of fertility. in one case report, a unilaterally cryptorchid stallion achieved a 90% live foal rate with an average of 1.4 cycles per pregnancy despite a high percentage of abnormal spermatozoa.52 in another case, a unilaterally cryptorchid stallion achieved a foaling rate of 17.7% on 259 mares compared to 42.5% on control mares (n = 2457) bred to a non-cryptorchid stallion.28 fertility of cryptorchid horses may decrease over the years as illustrated by a case of a unilaterally cryptorchid stallion that achieved pregnancy rates of 81%, 90%, 73%, and 48% in the first, second, third, and 4th breeding seasons, respectively.53 histopathology of retained testis because of the lack of appropriate testicular thermoregulation, the undescended testis undergoes several histological and endocrine disturbances.54,55 a 45% and 31% reduction in seminiferous tubules density was observed in abdominal and inguinal testes respectively, compared to a normal descended testicle.56 on histopathology seminiferous tubules have a wider lumen with vacuolation of the cells within the figure 2. abnormal vulvar conformation and large clitoris (left), normal mammary gland (right), in a 4-year-old xx-sex reverse horse with bilateral abdominal testes. figure 3. same horse in figure 2 after teasing a mare in heat (maybe not). http://dx.doi.org/10.58292/ct.v15.9648 34 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 seminiferous epithelium (figure 7). seminiferous tubules may be poorly differentiated and appear as solid cords with sertoli cell cytoplasm filling the tubules (figure 8). some tubules are lined by only 1 or 2 layers of spermatogonia. in inguinal retained testes, germ cells can be found at different stages of maturation but not beyond the stage of secondary spermatocytes, whereas in abdominal testes spermatogenesis is arrested at type a or b spermatogonia.55,57,58 a few spermatogonia and occasional degenerating primary spermatocytes may be present in some cases.40 atypical germ cells may be seen in the basal area of the seminiferous tubules.53 the population of leydig cells observed in cryptorchid testes is similar to that of the scrotal testes.56 with advancing age, the testicular parenchyma of the retained gonad undergoes further degenerative changes and fibrosis with increase thickening of the albuginea. these changes are more pronounced in abdominal than in inguinal testes.56,59,60 severe testicular compromise following torsion of the spermatic cord may lead ischemia,59,61,62 tissue necrosis and atrophy, which may sometimes be confused with true monorchidism, a much less common condition.63–67 histochemical studies of cryptorchid testes revealed a disruption of the morphology and histochemistry of both leydig and sertoli cells. leydig cells are poorly differentiated and have a deviation in α-actin expression.59 sertoli cells of cryptorchid testes show degenerative changes and alteration of the distribution of vimentin, a component of the intermediate figure 5. laparoscopic view of a partial abdominal testis. figure 6. laparoscopic view of an inguinal testis. figure 4. laparoscopic view of a complete abdominal testis. the spermatic cord of the retained testis is indicated by the arrow. http://dx.doi.org/10.58292/ct.v15.9648 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 35 filaments postulated to play an important role in regulation of spermatogenesis.59,68,69 abdominally retained testes sometimes become enlarged due to neoplastic changes. the retained testis can become extremely large (up to 60 cm) in some cases.70 it is not clear if these changes are a cause or a result of cryptorchidism.24,54,58,71 the most common neoplasms described in undescended testes include teratomas and seminomas.32 seminomas can be malignant and metastasize to peritoneal cavity and other organs, resulting in progressive debilitation and various clinical signs such as colic, fever, weight loss, tachycardia and dyspnea59,70,72,73 (figures 9, 10). development of seminoma may be due to over expression of aromatase. the excess estrogens may lead to a suppression of insl-3 responsible for testicular descent and is associated with reproductive disturbances including testicular neoplasia in humans.74,75 several cases of teratomas of the retained testes have been described in the literature76–81 (figure 11). the morphological, gross appearance, and ultrasonographic features of teratomas vary depending on the tissue present. in some cases, large cystic formations may be detected through ultrasonography along with tissue of various echogenicity82,83 (figure 12). other neoplasms reported in undescended testes including carcinomas,53 interstitial cell tumors,71,84 malignant sertoli cells tumors,85 and leiomyosarcoma.86 horses with abdominal retention of a testis may sometimes present with an acute abdomen (colic) due to torsion of the spermatic cord61 or jejunal entrapment and strangulation.73,87 diagnostic approach diagnosis of cryptorchidism is usually evident when animals are presented with a single scrotal testis and no history of attempted castration. in cases of ‘geldings’ with no scrotal testes but presenting with stallion-like behavior, diagnosis becomes more challenging, especially when the history of castration is lacking or uncertain. in the latter case, clinical and endocrinal evaluations are necessary to establish the diagnosis.88 clinical evaluation of all suspect cryptorchid horses is important prior to surgery to locate the retained testis, and determine its size and morphological features.88,89 figure 7. photomicrograph of a section of an abdominal testis: st = seminiferous tubules showing vacuolization, arrow indicates leydig cells (h& e stain; bar = 100µm). figure 8. photomicrograph of a section of an abdominal testis. the seminiferous tubule is filled with the cytoplasm of sertoli cells (h& e stain; bar = 20µm). table. prevalence of cryptorchidism based on side and location of the retained testis authors number of cases unilateral (%) bilateral (%) position of the retained testis abdominal (%) inguinal (%) incomplete abdominal (%) bartman 2001 67 86.6 13.4 56.7 43.3 n/a cattelan et al. 2004 42 95.2 4.8 64 34 n/a cox et al. 1979 100 81 19 33 67 n/a hartman et al. 2015 601 86.5 13.3 49.9 40.1 8.7 hughes 2006 100 90 10 47 53 n/a huppes et al. 2017 280 85.7 14.3 65 46 n/a leipold et al. 1986 100 93 7 60 39 n/a roderson & hansen, 1997 100 86 14 59 41 n/a stratico et al. 2020 70 92.8 7.2 54.3 24.3 21.4 http://dx.doi.org/10.58292/ct.v15.9648 36 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 endocrine diagnostic tests endocrine testing is helpful to differentiate cryptorchid horses with no scrotal testis from geldings with stallion like behavior unrelated to the presence of gonadal tissues. mature equine testes, whether descended or undescended, produce testosterone, estrogens, and anti-müllerian hormone (amh). detection and quantification of these hormones are the basis for endocrine diagnostics.90 androgens basal serum testosterone concentrations in normal and cryptorchid stallions are generally higher (> 100pg/ml) than in geldings (< 40pg/ml). contrary to common belief, the epididymis and spermatic cord of the horse do not produce testosterone.91 unfortunately, relying on a single measure of serum testosterone may be associated with inconclusive results due to diurnal fluctuations, seasonal effects (lower out of the breeding season) and age-related variations (lower in horses < 2 years or > 9 years of age) variations.90,92–95 serial determination of serum testosterone concentration before and after stimulation with hcg or gnrh improves diagnostic accuracy,96 as testosterone concentration is expected to rise within 25 to 35 minutes following administration treatment.91 the most commonly protocol used, is to determine serum testosterone concentration on blood samples taken, before and 2 hours after intravenous administration of 6000 to 12,000 iu hcg.95,97 a marked rise of testosterone level (>  100 pg/ml), consistent with presence of testicular tissue, is expected in cryptorchid stallions. even with this protocol 6.7% of results may be inconclusive.97 because a larger increase in serum testosterone concentration occurs between 24 and 76 hours after hcg administration.98 an additional sample collected 24 to 48 hours after challenge is often recommended to improve diagnostic sensitivity. testosterone sensitivity is 85% and specificity is 91%. an amplified enzyme immunoassay for serum androstenedione has been shown to have a sensitivity and specificity of 92% and 93% respectively when used to detect the presence of testicular tissue but is not commercially available for routine use. estrogens because estrogen biosynthesis occurs in both descended and cryptorchid testis in mature stallions,98 determination of estrone sulphate concentration in serum has been proposed for diagnosis of cryptorchidism. this test may not be consistently accurate in horses less than 3 years of age.98 reference ranges depend on the time of the year the sample was taken and vary from 10 to figure 9. gross (top) and ultrasonographic (bottom) of an abdominal testis with seminoma in a bilateral cryptorchid miniature horse. http://dx.doi.org/10.58292/ct.v15.9648 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 37 figure 10. photomicrographs of histological section of the testis shown in figure 9: metastatic seminoma with several abnormal mitotic figure (a, c), giant neoplastic cells (b) and raft of neoplastic cells within a blood vessel (d). figure 11. gross appearance of a testicular teratoma from an abdominal cryptorchid stallion. http://dx.doi.org/10.58292/ct.v15.9648 38 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 50 ng/ml for normal horses and from 0.1 to 10 ng/ml for cryptorchid horses. diagnosis of cryptorchidism could be achieved via measurement of the concentration of unconjugated estrogens in feces, but this assay is not commonly used.90 estrone sulphate sensitivity is 88% and specificity is 84%.99 anti-müllerian hormone (amh) amh is produced by sertoli cells and has proven to be an excellent biomarker for presence of testicular tissue. serum amh is higher in cryptorchid stallions (32.4±5.0 ng/ml) than in either intact stallions (14.7±2.4ng/ml) or geldings (0.07±0.01 ng/ ml).100,101 serum amh concentrations are higher during the breeding season in stallions.100 a high specificity for diagnosis of cryptorchidism has been shown for all ages.20,94,101 false negative results are possible if the testes have undergone necrosis or were not sufficiently differentiated (i.e., low number of sertoli cells).102 clinical evaluation and location of the retained testicle determination of the location of the retained testis is important when deciding on the appropriate surgical approach.8,88,89,103 ultrasonography is an important aspect of the examination of suspected cryptorchid horses, not only for location of the testis but also to determine the size and if potential pathology is present.71,85 for a complete examination, the horse should be sedated and placed in stocks. the authors prefer to sedate the horse with detomidine (0.01–0.02 mg/k, iv) and butorphanol (0.01–0.02 mg/kg, iv). the general clinical approach for determination of the location of the testis includes external inspection and palpation, followed by percutaneous ultrasonography (inguinal and transabdominal), and transrectal palpation and transrectal ultrasonography if the previous examinations are inconclusive. ultrasonographic examination of the lateral aspect of the flank has been described. it is performed by the authors only when transrectal examination is not possible (small breeds or very young horses)104 (figure 13). inspection and external palpation the inguinal area is first inspected visually, and each external inguinal ring is palpated from the ipsilateral side. the external inguinal ring varies in length from 1 to 4 centimeters.89,105 inguinal testes may be recognized by palpation of the epididymal tail. inguinal palpation allows identification of 60% of the superficially retained testes and 7% of the deep inguinal testes with a specify of 100%. identification of the retained testes by inguinal palpation can be difficult, particularly in younger horses. palpation of fatty tissue or the ligament of the cauda epididymis may be confused with a testis especially in young colts.106 complete abdominal testes cannot be palpated in the inguinal region. percutaneous ultrasonography although a linear transducer may be used for percutaneous ultrasonography, the authors prefer to use a convex 3.5 to 5 mhz transducer. higher frequencies (7.5 to 10 mhz) for linear transducers may be helpful to have a better definition of superficial testes. the inguinal area is cleaned, sprayed with 70% isopropyl alcohol, or coupling gel to improve the image figure 12. transrectal ultrasonogram (top) and gross (bottom) appearance of cystic testicular teratoma from an abdominal cryptorchid horse. figure 13. region of the flank to scan ultrasonographically. note that this scanning is often not necessary compared to inguinal and lower abdominal scanning. http://dx.doi.org/10.58292/ct.v15.9648 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 39 quality. the transducer is placed over the external inguinal ring oriented in a dorsolateral fashion, following the course of the inguinal canal107 (figure 14). if the testis cannot be visualized, the examination continues by placing the transducer more caudal over the midline, aiming at the urinary bladder. the examination proceeds by moving the transducer cranially from midline to the fold of the flank104 (figure 15). the retained testis can be recognized by the characteristic hyperechoic appearance of the tunica albuginea and the presence of a central vein104,107 (figures 16). an inguinal retained testis is often misshapen and has a less echogenic parenchyma than normal scrotal testis. the superficial inguinal and deep inguinal cryptorchid testis can be accurately localized in 100% and 93% of the cases respectively.108,109 in one report that included 127 horses with 141 cryptorchid testes, the sensitivity and specificity of percutaneous inguinal ultrasonography to determine the location of the testis were 98% and 97%, respectively.110 abdominal testes are often visualized on the ventral abdominal wall within a few centimeters of or adjacent to the urinary bladder (figures 17, 18) and lying between the intestinal loops or colon haustra. left abdominal testis are more difficult to visualize. they may be found between the left ventral colon and the spleen. other factors that may hinder visualization of the abdominal testes include higher location due to short testicular cord, gas, or food-filled large colon. examination of the horse after a period of 24 to 36 hours fasting may improve ultrasonographic localization of the abdominal testis.104 the combination of inguinal and abdominal ultrasonographic examination allow location of the testes with very figure 14. illustration of the area for percutaneous of inguinal ultrasonographic evaluation of a suspected cryptorchid horse. figure 15. illustration of the area for per cutaneous low abdominal ultrasonographic evaluation of a suspected cryptorchid horse. http://dx.doi.org/10.58292/ct.v15.9648 40 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 high sensitivity (97.6%) and specificity (100%).104,111 however, some authors reported only a 50% localization of incomplete abdominal testis with a specificity of 75%.109 other authors reported localization of the abdominal testis in 72.7% of the cases.108 this difference of results may be due to a difference in experience and/or type of horses examined, gas or content in the large colon. transrectal palpation and ultrasonography transrectal palpation and ultrasonography should be performed on cryptorchid horses whenever possible if the percutaneous ultrasonographic exam is inconclusive. in addition to heavy sedation, administration of a spasmolytic (n-butyl scopolamine bromide, 0.3 mg/kg slow iv) helps to reduce intestinal peristalsis and relaxes the rectum to allow a more thorough examination. the objective of transrectal palpation is not to find the testes, but rather to determine if the vas deferens is entering the internal inguinal ring. the inguinal ring is palpated just cranial and ventrolateral to the brim of the pelvis as a slit-like opening accommodating one or 2 fingers (figure 19). palpation of the inguinal ring may be facilitated by the aid of a second examiner introducing two fingers in the external inguinal ring. the vas deferens can be felt entering the inguinal ring in cases of incomplete abdominal or inguinal cryptorchidism. the location of the retained tests could be determined in 67% to 94% of the cases. experienced clinicians are significantly more likely to correctly determine the location of the testis.47 an incorrect diagnosis is more likely to occur in patients who have undergone a prior attempt at castration. transrectal ultrasonography should be performed in a systematic manner scanning the entire internal genitalia. after fecal evacuation and palpation of the inguinal ring, a 5 to 10 mhz frequency linear transducer is introduced into the rectum. the accessory sex glands are often larger and active in a cryptorchid horse compared to a gelding88 (figure 20). the technique used by the authors is similar the one described by pozor et al. and consists of following the ampullae along their course.107 the ampulla of the vas deferens is identified on each side just cranial to the pelvic urethra (figures 20, 21). in inguinal and incomplete abdominal cryptorchids, the ampulla and vas deferens gradually curve laterally and ventrally toward the inguinal ring. in the case of abdominally retained testis, the ampulla runs craniolaterally or craniodorsally without bending. a sweeping motion from midline to the lateral abdominal wall while moving the hand cranially usually allows the identification of the testis. the testis may be recognized first as one approaches the tail of the epididymis or the pampiniform plexus and testicular artery (figures 21–23). using this approach of examination including transrectal palpation and ultrasonography, the authors were able to locate 100% of the testes in unilaterally cryptorchid horses (n = 43) and 9 out of 10 bilaterally retained testes.88 the sensitivity and specificity of ultrasonographic examination of suspected cryptorchid horses including transrectal evaluation is 91 and 97% for abdominal testis and 88 and 99% for inguinal testes.8,46 a 100% agreement between testicular location determined by ultrasonography and surgery has been reported in some studies.112 although rare, failure to locate the testicle in a cryptorchid stallion may be due to true monorchidism,113–115 or ischemic necrosis of the testis following spermatic cord torsion.116 cryptorchidectomy surgical approaches for equine cryptorchidectomy can be grouped into 2 large categories: traditional surgical techniques, and minimally invasive techniques (laparoscopic techniques). traditional surgical techniques include inguinal, para-inguinal, suprapubic, and flank approaches. the choice of a surgical technique is primarily dictated by the location and size of the retained testis. with exception for the flank and laparoscopic techniques, which are performed in the standing horses, all approaches are performed under general anesthesia in the recumbent horse. figure 16. ultrasonogram of an inguinal testis (t). note the hyperechoic albuginea (arrow). http://dx.doi.org/10.58292/ct.v15.9648 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 41 patient preparation and preoperative considerations cryptorchidectomy is an elective procedure and should be performed on otherwise healthy horses. as for any surgery, a thorough physical examination and pre-operative blood work should be conducted prior to surgery. food is withheld for 12 to 24 hours for conventional surgeries. longer fasting (24 to 36 hours) is recommended for laparoscopic approaches to reduce the risk of perforation of viscera during introduction of the trocar and allows a better exploration of the abdominal cavity.8,117,118 preoperative medication consists of tetanus prophylaxis and administration of a nonsteroidal anti-inflammatories (phenylbutazone 4.4 mg/kg iv or per os; or flunixin meglumine 1.1 mg/kg iv, or meloxicam 0.6mg/kg iv). flunixin meglumine is the preferred option for all horses undergoing laparoscopic cryptorchidectomy to minimize the abdominal discomfort associated with carbon dioxide insufflation. antibiotics are administered preoperatively.119–121 sedation for standing cryptorchidectomy is achieved using xylazine (0.5 mg/kg iv) or detomidine (0.01–0.02 mg/kg iv) in combination with butorphanol (0.01 mg/kg iv). intravenous constant rate infusion of detomidine (20 mg/l) and butorphanol (10 mg/l) is given to effect following sedation.120,122,123 some authors recommend a caudal epidural with detomidine (0.03–0.06 mg/kg) diluted to 10 ml with 0.9% saline. however, this treatment has been associated with collapse.118,124,125 for flank and laparoscopic techniques, the paralumbar fossa region is desensitized with local injection of lidocaine in an inverted ‘l’ pattern.113,126 general anesthesia is required for inguinal, para-inguinal, and suprapubic surgeries and for laparoscopic approach in dorsal recumbency. the most common anesthesia protocol used for field surgery is the triple drip (guaifenesin 5% to 10%, xylazine 10% in combination with ketamine).57,127–129 however, injectable anesthesia protocols usually limit time available for surgery, and inhalational anesthesia is preferred when surgical time is anticipated to exceed 60 minutes. inguinal approach the inguinal approach to cryptorchidectomy has been described by several authors.105,128,130,131 the anesthetized horse is placed in figure 17. ultrasonogram of abdominal testis testis (t) showing clearly an anechoic area corresponding to the central vein (top arrow), and a teratoma. http://dx.doi.org/10.58292/ct.v15.9648 42 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 dorsal recumbency, and the entire inguinal area is sterilely prepared for surgery and draped. an 8 to 12 cm incision is made through the skin over the superficial (external) inguinal ring or directly in the scrotum. the inguinal fascia is dissected bluntly to expose the superficial inguinal ring. if the retained testis is inguinal, it should be easily exposed and excised. it is important to open the common vaginal tunic to expose the testes and the epididymis prior to ligation or emasculation. in case of partial abdominal testis, the vaginal process containing the tail of the epididymis is identified, grasped with sponge forceps, and incised with scissors. the testicle is exteriorized by gentle traction on the tail of the epididymis and the proper ligament of the testis. the spermatic cord and cremaster may be emasculated together or separately. some authors prefer to place a single transfixing suture proximal to the site of emasculation. the incision may be left to close by second intention. primary closure is advocated by some practitioners and consists of suturing the deep fascia using a simple continuous pattern and finishing with a subcuticular pattern to oppose the skin edges. in abdominal cryptorchidism, the vaginal process remains inverted into the abdomen and can be everted into the inguinal canal by identifying the gubernaculum (narrow fibrous band of tissue). it is important to avoid confusing the gubernaculum and the genital femoral nerve. once everted the vaginal process is opened with scissors and the testis is exposed by traction on the epididymis after digital dilation of the internal inguinal ring. sometimes, the inguinal extension of the gubernaculum is deep and cannot be palpated readily. a long (25 cm) foerster sponge forceps is introduced into the inguinal canal along the fingers of the surgeon and the apex of the vaginal process is grasped at the level of the inguinal ring and everted (figure 24). a deeper exploration of the abdominal cavity using two fingers through blunt penetration through the inguinal ring is necessary to identify the vas deferens, epididymis, or testis. an even deeper manual exploration may be needed if these structures cannot be identified. following identification, exteriorization, and excision of the gonad, the superficial inguinal ring is closed with heavy suture to prevent evisceration, and the skin is closed routinely with a subcuticular suture pattern. para-inguinal approach the para-inguinal approach (also known as the danish method) to cryptorchidectomy was developed in the late 1800s.132 the technique underwent several modifications, the most notable one was proposed by wilson and reinertson in 1987.4,105,132–134 this approach should be considered if the testis is determined to be abdominal. the horse is prepared as described above. a 4 to 6 cm incision is made through the skin and the aponeurosis of the external abdominal oblique muscle 2 cm medial, parallel to the superficial inguinal ring (figure 25). the internal abdominal oblique muscle is opened along its muscle fibers and the peritoneum is entered bluntly with the fingers. using one or two fingers placed into the abdomen, the ductus deferens, epididymis or gubernaculum can be identified coursing in the direction of the inguinal ring just caudal and lateral to the incision and then followed toward the tail of the epididymis and proper ligament of the testis (figure 26). the testicle is exteriorized by traction on the proper ligament and emasculated. the incision may be extended to accommodate the entire hand for better exploration if the gubernaculum and epididymis cannot be identified. the aponeurosis of the external abdominal oblique muscle is closed in a continuous pattern using heavy absorbable material. the subcutaneous tissues are opposed. flank approach cryptorchidectomy using the flank approach is used for abdominally retained testes.105,135 the major disadvantage of the technique is the long healing period.10 the horse is prepared for standing surgery under heavy sedation. the flank ipsilateral to the affected side is clipped, desensitized, and scrubbed for surgery. a 10to 15-cm incision is made through the skin and subcutaneous tissue in the paralumbar fossa. the external abdominal oblique muscle is transected in the direction of the skin incision. the internal abdominal oblique and the transverse abdominal muscles are split in direction of the muscle fibers to expose the peritoneum. the retroperitoneal fat and peritoneum are penetrated bluntly using the fingers. the area of the inguinal ring is explored digitally or manually to locate the vas deferens, tail of epididymis, or testis. the testis can be exteriorized by gentle traction and the spermatic cord emasculated or transected after placement of single transfixing suture136 (figure 27). if the testis cannot be exteriorized, an écraseur is used to transect the testicular vasculature.130 once the abdominal testis is removed, each layer is closed separately with continuous or interrupted pattern. figure 18. examples of ultrasonograms of abdominal testes visualized by abdominal scanning (t = testis, ub = urinary bladder, arrow indicates intestinal loops). http://dx.doi.org/10.58292/ct.v15.9648 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 43 some authors forego suture of the peritoneum and the transverse abdominal mucle.105 suprapubic paramedian approach the suprapubic paramedian approach is used in cases of bilateral cryptorchidism or unknown castration history.130,137 a longitudinal skin incision is made 5 to 10 cm lateral to the ventral midline beginning at the level of the preputial orifice and extending caudally. the abdominal tunic and the ventral sheath of the rectus abdominis muscle are incised. the rectus abdominis muscle is separated by blunt dissection along the muscle fibers which are in the same direction of the incision. the abdominal cavity is penetrated by bluntly perforating the dorsal rectus sheath, retroperitoneal fat, and peritoneum with the fingers. the testis and associated structure are figure 19. illustration of transrectal palpation of the vaginal ring and the vas deferens (arrow) entering it. figure 20. transrectal ultrasonography of a cryptorchid stallion: arrows indicate bulbourethral gland (a), protate (b and c), ampulla of the vas deferens (d) (ub: urinary bladder). figure 21. transrectal ultrasonography of a cryptorchid stallion: ub: urinary bladder, t = testis arrow indicate vas deferens. http://dx.doi.org/10.58292/ct.v15.9648 44 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 generally found near the inguinal ring. the testis can also be located by following the vas deferens found in the genital fold of the bladder. in bilateral cryptorchid horse, both testes can be removed from the same incision although exteriorization of the contralateral testis is difficult and may require the use of an écraseur. the ventral rectus sheath and subcutaneous cutaneous tissues are closed with absorbable suture material in a continuous patter or interrupted pattern. the skin is sutured separately with interrupted suture. alternately, a subcuticular pattern with absorbable suture can be used to close the skin. laparoscopic cryptorchidectomy laparoscopic cryptorchidectomy has become the most used approach in horses.120 techniques vary slightly depending on the location of the testis and preference of the surgeon.127,138,139 laparoscopic cryptorchidectomy is preferably performed in the standing horse121,138 under heavy sedation and local analgesia. however, laparoscopic cryptorchidectomy in dorsal recumbency under general anesthesia has also been described.127,139 here we briefly described the standing approach. figure 22. transrectal ultrasonograms in a horse with inguinal cryptorchidism. note the curving of the ampulla of the vas deference (top) and misshapen testis (t, bottom image). figure 23. transrectal ultrasonograms in a horse with inguinal cryptorchidism: note the abnormal testis. http://dx.doi.org/10.58292/ct.v15.9648 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 45 the horse is heavily sedated and placed in stocks. the tail is wrapped and secured to the side. a urinary catheter is placed. the left and right paralumbar fossa are clipped from the tuber coxae to the 15th intercostal space and from the epaxial musculature to the level of the stifle and scrubbed. the sites of the 3 portals are desensitized by lidocaine 2% infiltration (figure 28). the first portal is located 5 cm below the dorsal margin of the internal abdominal oblique muscle, between the last rib and the tuber coxae. the second portal is located in the 17th intercostal space, below an imagined line drawn horizontally through the ventral border of the tuber coxae. the third portal is located 5 to 10 cm ventral to the initial portal, following the caudal aspect of the rib, in the middle of the flank. a final sterile scrub is performed, and the patient is sterilely draped. a 2 to 3 cm vertical incision is made through the skin in the  17th intercostal space. a 12-mm laparoscopic cannula with a blunt-tip obturator is introduced into the skin incision and  passed through the intercostal musculature and the figure 24. cryptorchidectomy: inguinal approach, exteriorization of the testicle. figure 25. parainguinal approach to cryptorchidectomy. illustration of the superficial inguinal ring (a) and the incision site (b) in unilateral cryptorchid stallion in dorsal recumbency. figure 26. cryptorchidectomy: parainguinal approach, exteriorization of the testicle. http://dx.doi.org/10.58292/ct.v15.9648 46 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 peritoneum punctured to enter the abdominal cavity. this step is considered the most dangerous due to the risk of perforation of the gastrointestinal structures. insufflation of the abdominal cavity with co2 using a veress needle or a trocar catheter helps avoid damaging viscera during insertion of the trocar.136 the risk of perforation of abdominal viscera can also be reduced using a threaded endotip cannula. a 10 mm, 30-degree laparoscope is introduced into the abdomen to allow visualization of abdominal viscera. a second portal site is then made under intra-abdominal laparoscopic guidance in the paralumbar fossa, midway between the last rib and the tuber coxae and approximately 3 cm ventral to the first portal.138,140 in cases of bilateral cryptorchidism, the contralateral testis can be visualized by manipulating the endoscope under the small colon or by creating a defect in the mesocolon. in some case, it is preferable to approach each testis from its ipsilateral flank particularly if the gubernaculum is short or testicular abnormalities are found.119,136 once the testis has been clearly isolated, a 32 cm, 18g tip laparoscopic injection needle is used to infiltrate the testicular pedicle with approximately 15 ml of 2% lidocaine. several techniques have been described to ensure hemostasis and remove the retained testis. emasculation can be performed extracorporeally after exteriorization through a low flank incision. however, the most common approach is to perform an intraabdominal ligation and transection of the spermatic cord using intrabdominal loop of polydioxanone suture (endoloop ligature, ethicon endosurgery cincinnati, oh).136 recently, bipolar electrosurgery has become more available and is used to ensure hemostasis prior to transection of the mesorchium and vas deferens. some authors prefer the use of bipolar vessel sealing devices using high current and low voltage (ligasure®, covidien, dublin, ireland).111,113,114,122,136,141,142 the abdominal testis and associated structures can be grasped with claw forceps and removed through the caudal ventral instrument portal after ventral extension of the incision. the use of a morcellator to divide the tissues of the retained testis into smaller segments has been described to facilitate extraction through a 12 to 20 mm incision.121 laparoscopic castration of cryptorchid horses using spermatic cord ligation without transection has been described but the technique fails to induce proper hemostasis in 3.4 to 5.6% of the cases.83,117,143,144 plasma testosterone concentrations should be determined 7 days after surgery to verify success of the procedure.8 following cryptorchidectomy, all trocars are removed after deflation and the skin incisions are closed in using skin staples. postoperative care and complications following cryptorchidectomy postoperative care following cryptorchidectomy depends on the approach used. in open surgeries, stall rest for 24 to 36 hours is recommended. horses that have undergone a procedure using the inguinal approach can return to normal levels of exercise within 7 to 10 days of surgery. for horses that have undergone more invasive techniques, hand walking for 2 to 3 weeks is recommended prior to gradual return to normal activity. incisions must be protected from flies and wounds cleaned routinely.130 for non-invasive procedures, post operative antimicrobial treatment is not necessary if appropriate antibiotic therapy was administered perioperatively and there were no perceived breaks in sterility. anti-inflammatory drugs are continued for 3 to 5 days. horses that have undergone standing laparoscopic cryptorchidectomy are usually discharged 1 to 3 days after the procedure with instruction to hand-walk for 5 to 7 days.121 reported complication rates of conventional open cryptorchidectomy range from 7.6 to 12.3%.58,132 the most catastrophic complications are hemorrhage, evisceration, septic peritonitis, and clostridial infections. 147 in one report, 2 horses figure 28. laparoscopic cryptorchidectomy portal sites. figure 27. cryptorchidectomy: flank approach. http://dx.doi.org/10.58292/ct.v15.9648 citation line: clinical theriogenology 2023, 15, 9648, http://dx.doi.org/10.58292/ct.v15.9648 47 undergoing conventional cryptorchidectomy developed diarrhea and colic which required euthanasia.145 other complications include fever, hydrocele, septic funiculitis, pyrexia, lameness, and penile damage.45,149 complication rate following laparoscopic cryptorchidectomy ranges from 0% to 33%. this large variation depends on how complications are defined. intraoperative complications are primarily related to lack of identification of the testis or presence of abnormally developed testis,114,148 bowel perforation,8,146 failure to pull an inguinal testis into the abdomen, poor visual field due to small intestinal distension, and inappropriately placed portals.145 standing laparoscopic castration is generally safe and rapid (30 to 40 minutes) when performed under optimal conditions but requires practice.142 laparoscopic cryptorchidectomy under general anesthesia is associated with increased surgical time and increased risk for diarrhea and colic.145 complications associated with a large incision in the paralumbar fossa for testis extraction are substantially reduced using a morcellator.121 conclusion cryptorchidism is a frequent complaint in equine reproduction. progress has been made in the study of testicular descent and factors controlling it. the hereditary nature of cryptorchidism is still debated and raises questions about the appropriateness of using unilaterally cryptorchid for breeding. theriogenologists should be trained in the diagnosis and surgical treatment of the condition. endocrine diagnosis of presence of testicular tissue can be achieved reliably with a single serum level of amh. in a few exceptions, where tissue is abnormal or has undergone severe degeneration, this test may not be diagnostic. clinical evaluation and determination of the location of the retained testicle are important steps in decision making for selection of the appropriate surgical approach. location of the testicle can be achieved with high sensitivity and specify with a combination of the transcutaneous and transrectal ultrasonography. special attention should be given to the size of the testis and any abnormalities of testicular parenchyma. several surgicalapproaches have been described for cryptorchidectomy. standing laparoscopic approaches are becoming the preferred method for partial and complete abdominal cryptorchidism, as they are minimally invasive with a fewer 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fetal membranes in an african elephant (loxodanta africana) jack kottwitz,a carolina lopes da cruz torres alpino,a john f. roberts,b amelia s. munstermanc aveterinary department, the montgomery zoo, montgomery, al; bthompson, bishop, sparks state veterinary medical diagnostic laboratory, auburn, al; cdepartment of clinical sciences, college of veterinary medicine, auburn university, auburn, al* summary a 30 year old, nulliparous female african elephant gave birth to a stillborn, full term male calf. the cow had developed an elevated white blood cell count five weeks prior to parturition, however labor and parturition were without complications for the cow. the calf was stillborn with thoracic limb arthrogryposis, renal agenesis, and evidence of fetal stress prior to parturition. fetal membranes were observed protruding from the vulva within 24 hours of birth, but that tissue did not pass spontaneously. gentle manual extraction six days after parturition removed the protruding material which was determined to be amnion and a portion of the umbilical cord. the cow developed severe ventral abdominal edema and a significant leukocytosis, but remained afebrile with a normal appetite. signs of labor were noted 86 days after the stillbirth, with the remaining chorioallantois and portion of the umbilical cord being passed spontaneously overnight. ninety days postpartum, a large mucoid mass was passed. at that point the cow’s ventral edema resolved completely, the white blood cell count returned to within normal limits. hematology values remained within normal limits over the subsequent six months. keywords: african elephant, dystocia, loxodanta africana, retained fetal membranes, stillbirth case presentation a 30 year old female african elephant (loxodanta africana) gave birth to a full term (> 610 days of gestation), stillborn, 120.5 kg male calf after showing intermittent signs of labor for 24 hr. an elevated white blood cell count (16.7 x 103 cells/μl; reference range 10.32 ± 2.837 x 103 cells/μl1) with neutrophilia (10.6 x 103 cells/μl; reference range 3.455 ± 1.737 x 103 cells/μl) was first noted 41 days prior to parturition. the cow demonstrated no observable signs of illness and maintained a normal appetite and attitude until parturition. the cow had an apparently normal parturition 36 hours after serum progesterone levels returned to baseline, with the hardest labor lasting approximately 2.5 hours prior to the delivery of the stillborn calf. the calf had arthrogryposis of the thoracic limbs with both carpi fixed at 90 degrees flexion (figure 1). both eyes had pronounced hemorrhagic conjunctiva (figure 2). additional signs of fetal stress including petechial hemorrhage on the endocardial and epicardial surfaces were noted during gross necropsy (figure 3). a significant portion of the lungs sank when placed in formalin confirming failure to initiate normal respiration. the primary bronchi contained green mucoid material consistent with aspirated meconium. the stomach also contained similar thick mucoid green material. the cecum was hyperemic with the colon distended with soft green meconium. there was no right kidney. fetal membranes protruded from the cow’s vulva within 24 hours of birth, but that material did not pass spontaneously. based on physical appearance of the protruding tissue, it was initially thought that the bulk of the fetal membranes were in the vaginal vestibule. the temperament of the cow did not permit extensive palpation of the perineal region or an attempt at ultrasonographic examination of the reproductive tract. a wet towel was tied to the protruding fetal membranes to attempt to utilize gravity to assist in the evacuation of retained tissues. an incomplete portion of the fetal membranes composed of amnion and a portion of the umbilical cord was passed six days later. no chorioallantois was present. * present addresses: department of anatomy, physiology and pharmacology, college of veterinary medicine, auburn university, auburn, al (kottwitz); college of veterinary medicine, centro universitario serra dos orgaos, teresopolis, rio de janerio, brazil (alpino); the center for animal care sciences, smithsonian conservation biology institute, national zoological park, washington, dc (roberts). 459 clinical theriogenology • volume 5, number 4 • december 2013 histopathologic examination of the umbilical cord revealed numerous hairs incorporated into the outer tunic, suggesting the umbilical cord was compressed tightly against the fetus during parturition. the cow’s attitude and appetite remained normal after parturition, but she developed ventral abdominal edema approximately 36 hours after parturition that increased over the next two weeks. a hemogram performed the day after parturition revealed a mild leukocytosis (14.6 x 103 cells/μl) with moderate numbers of degenerate neutrophils. vulvar discharge consisting of large drops of thick, dark red, mucoid material was noted after the allantois was passed. ciprofloxacin (pack pharmaceuticals, llc, buffalo grove, il; 2.5 mg/kg po twice per day for two weeks) was initiated. direct bacterial culture of the vaginal canal or vaginal vestibule was not possible due to lack of cooperation by the cow. culture of the vulvar discharge revealed heavy growth of e. coli and beta-hemolytic streptococcus. both bacteria were susceptible to ciprofloxacin. repeat complete blood counts (cbc) at nine and 16 days after parturition showed little change in the white blood cell (wbc) count, although degenerate neutrophils were no longer observed. a cbc performed 25 days postpartum revealed significant leukocytosis (25.8 x 103 cells/μl), neutrophilia (19.3 x 103 cells/μl) and a left shift. the wbc count remained elevated through the next month, yet the cow continued to have normal attitude, appetite and activity levels with an intermittent vulvar discharge. a repeated culture of the discharge showed light growth of beta-hemolytic streptococcus with no e. coli growth. based on the continued elevated wbc count and little apparent response to ciprofloxacin, the decision was made to change antibiotics. oral ampicillin (dava pharmaceuticals, fort lee, nj; 8 mg/kg po twice per day) was chosen based on cost of treatment and antibiotic susceptibility. two days after discontinuing the ciprofloxacin and starting oral ampicillin the cow became anorexic. because of concern about possible gastrointestinal effects from the oral ampicillin, the antibiotic was not administered on the third day. that evening there was a significantly increased volume of thick, red, mucoid material from the vulva. fetal membranes were passed overnight, 86 days after parturition (figure 4). blood drawn that morning revealed continued leukocytosis (23.5 x 103 cells/μl) with neutrophilia (17.6 x 103 cells/μl). ampicillin was continued. the cow’s appetite continued to be decreased for the next four days, at which time a large amount of dark reddish, mucoid material was passed. after that material passed, the cow’s appetite and activity level quickly returned to normal over the next 24 hours. the remaining ventral abdominal edema resolved over the next week. the cbc performed 93 days after parturition revealed decreased but still elevated wbc count (18.4 x 103 cells/μl). hematology performed the next week revealed a normal wbc count (13.0 x 103 cells/μl). ampicillin was continued for an additional two weeks, then discontinued as the wbc count remained within normal limits. the leukogram remained within normal limits and there was no additional vulvar discharge or other abnormalities noted in biweekly examinations for the next six months. discussion there are few reports of retained fetal membranes in elephants, with this case representing the first report in an african elephant. expulsion of the fetal membranes in elephants usually occurs within ten hours of birth.2 retained fetal membranes in domestic animals can be defined as membranes that have not passed within 24 hours of parturition.3 risk factors associated with retained fetal membranes in domestic cattle and horses include twins, dystocia, fetal death, stillbirth, induced parturition, abortion, milk fever and geriatric dams.3,4 dystocia and stillbirth are considered common in captive asian and african elephants in north america.5 age of the mother may play a significant role in the incidence of dystocia and stillbirth. pregnancies in captive nulliparous african cows over the age of 24 years have only resulted in dystocia or stillbirth in north america.5,6 in addition, there has been only a small number of calves born to multiparous cows over the age of 24 years.5 age of the cow must be considered in this case of retained fetal membranes and stillbirth there has been one confirmed case of arthrogryposis of an african elephant calf which caused angular limb deformity that was considered to be the likely cause of dystocia.7 etiologies of arthrogryposis in domestic species include genetic predisposition, exposure to akbane virus or bluetongue virus (btv) or ingestion the teratogens anagyrine or piperidine found in plants in the 460clinical theriogenology • volume 5, number 4 • december 2013 fabaceae family which includes lupine.7 the cow had no known exposure to toxic plants known to cause arthrogryposis. bluetongue virus exposure is a possibility, as the virus is considered endemic in the southeastern united states.8 however, btv was not confirmed in this case. salmonella spp. has also been implicated as a potential cause of abortion and stillbirth in african elephants.9 in this case, the cow showed no clinical signs of salmonellosis and cultures performed during necropsy of the calf, the maternal feces, and the fetal membranes proved to be negative for this etiological agent. both the dam and the calf were also negative for elephant endotheliotropic herpesviruses. first calving, maternal age, and arthrogryposis most likely contributed to difficult passage of the fetus and stress during parturition ultimately resulted in fetal death. the elevation in the wbc count six weeks prior to parturition is suggestive of placentitis of unknown etiology. retention of the fetal membranes prevented complete and thorough examination of fresh tissues to determine possible etiologies. retention of the fetal membranes can have a wide variety of effects on the dam in domestic animals, ranging from fatal laminitis and death in domestic horses to spontaneous passage of the retained tissue and return to normal breeding soundness in domestic cattle.3,4,10 fetal retention has occurred in african elephants for time periods ranging from three months to over seven years without death of the mother, suggesting a unique ability of elephants to sequester the uterus and prevent systemic infection.2,6,11,12 the elevated wbc count of the cow with the subsequent decline after membrane expulsion indicates an immune response that continued as long as the fetal membranes were retained. this cow had a history of irregular estrous cycles for the three years prior to becoming pregnant with absent or incomplete records before that time. the estrous cycle during which conception occurred was the only normal cycle for which records were available. a large, polypoid urogenital mass was also passed prior to this normal cycle. a complete reproductive tract evaluation, including transrectal ultrasonography, performed four years prior to this birth indicated no apparent abnormalities at that time. it was not possible to fully evaluate the reproductive tract for the presence of other abnormalities or masses after that evaluation was performed. the cow’s irregular reproductive cycle history and passage of the urogenital mass caused concern for the presence of unidentified uterine abnormalities. the potential for unknown uterine abnormalities combined with the apparent ability of elephants to retain fetal tissues without subsequent maternal death and uterine ruptures associated with the administration of oxytocin in elephants precluded attempts to administer ecbolic drugs. the duration of retained fetal membranes increases the likelihood of significant fibrosis within this cow’s uterus. direct evaluation of the endometrium by means of a biopsy is not possible due to the anatomy and size of an african elephant. therefore while it is unlikely that the uterus is capable of supporting another pregnancy, maternal age and the potential for a negative outcome suggests that future breeding is ill-advised. learning points  african elephants appear capable of retaining fetal membranes for several months without outward detrimental effects. references 1. international species information system: physiological data references values for loxodanta africana. apple valley (mn): international species information system; 2002. 2. schmitt dl: reproductive system. in: fowler me, mikota sk, editors. biology, medicine, and surgery of elephants. ames (ia): blackwell publishing; 2006. p. 347-355. 3. leblanc sj: postpartum uterine disease and dairy herd reproductive performance: a review. vet j 2008;176:103-114. 4. leblanc mm: common peripartum problems in the mare. j equine vet sci 2008;28:709-715. 5. gage lj: neonatal elephant mortality. in: fowler me, miller re, editors. zoo and wild animal medicine: current therapy. 6th ed. philadelphia: wb saunders; 2008. p. 365-368. 6. hermes r, saragusty j, schaftenaar w, et al: obstetrics in elephants. theriogenology 2008;70:131-144. 7. gage lj, schmitt dl: dystocia in an african elephant (loxodonta africana). proc am assoc zoo vet; 2003. p 88. 8. maclachlan nj: bluetongue: history, global epidemiology, and pathogenesis. prev vet med. 2011;102:107-111. 461 clinical theriogenology • volume 5, number 4 • december 2013 9. emanuelson ka, kinzley ce: salmonellosis and subsequent abortion in two african elephants. proc am assoc zoo vet; 2000. p. 269-274. 10. ishii m, kobayashi s, acosta tj, et al: effective oxytocin treatment on placental expulsion after foaling in heavy draft mares. j vet med sci 2009;71:293-297. 11. chatchote t, pongsopawijit p, thongtip n, et al: dystocia following prolonged retention of a dead fetus in an asian elephant (elephas maximus). theriogenology 2006;66:1284-1291. 12. foerner jj: dystocia in the elephant. in: fowler me, miller re, editors. zoo and wild animal medicine: current therapy. 4th ed. philadelphia: wb saunders; 1999. p. 522-525. figure 1: stillborn, full term, 120.5 kg male african elephant calf born to 30 year old primiparous cow. note the thoracic limb arthrogryposis with the legs fixed at 90 degrees flexion at the carpi and severe hyperemia of the right eye, suggestive of fetal stress during parturition. the etiological agent that caused the arthrogryposis may have also contributed to the retained fetal membranes of this case report. figure 2: close-up of the eye of stillborn, full term 120.5 kg male african elephant calf showing significant hemorrhage of the conjunctival tissues, thought to be indicative of fetal stress during parturition. 462clinical theriogenology • volume 5, number 4 • december 2013 figure 3: cardiac petechiation, thought to be an indicator of hypoxia and fetal stress during parturition. the cause of fetal stress likely contributed to retention of the fetal membranes. figure 4: choriollantois and retained umbilical cord fragment delivered 86 days after parturition from a 30 year old african elephant cow. unfortunately due to the prolonged retention time of the fetal membranes, autolysis prevented complete diagnostic evaluation. (editor’s note: the photographs in this paper appear in color in the online version of clinical theriogenology.) 463 clinical theriogenology • volume 5, number 4 • december 2013 2018: congenital peritoneopericardial diaphragmatic hernia in a family of persian cats congenital peritoneopericardial diaphragmatic hernia in a family of persian cats carol a. margolis,a maja zakosek pipan,b margret l. casala asection of medical genetics, department of clinical sciences and advanced medicine, mj ryan veterinary hospital of the university of pennsylvania, philadelphia, pa;bclinic for reproduction and large animals, veterinary faculty, university of ljubljana, ljubljana, slovenia a 16-month-old primiparous persian queen was presented 7 weeks after breeding for pregnancy confirmation and radiographic fetal count. four fetal skeletons were identified. incidentally, an enlarged cardiac silhouette with superimposed gas filled opacities was discovered, suggestive of a peritoneopericardial diaphragmatic hernia (ppdh). therefore, the patient was referred for a cesarean section and concurrent ppdh repair. one male kitten had an omphalocele and was humanely euthanized. the three remaining male kittens appeared normal on physical examination. however, two kittens were having difficulty nursing, were dyspneic and failed to thrive during the first week of life. one of the dyspneic kittens died at 10 days of age, upon which radiographs were obtained from the second dyspneic kitten and images were highly suggestive of ppdh. humane euthanasia was elected due to poor prognosis, and necropsy findings confirmed the presence of ppdh in both kittens. the tom of this litter was also bred to the queen’s sister producing one normal female kitten and one stillborn male with an omphalocele. survey radiographs were obtained from both the tom and the second queen, and no abnormal anatomic structures were seen. a review of the 3-generation pedigree revealed common ancestry; the tom’s sire was a littermate to both queens (minimum coefficient of inbreeding 12.5%). omphalocele and ppdh have both been described as congenital midline defects in cats and dogs. unfortunately, neither of the kittens with omphalocele were available for necropsy. however, a published report of 58 cases of ppdh revealed that about half of affected dogs and about a quarter of affected cats also had umbilical hernias, abdominal wall hernias, or sternal defects, suggesting that omphalocele is associated with ppdh. other reports described weimaraner dogs and longhaired cat breeds as being overrepresented suggesting an inherited trait and over 63% of affected cats were males. in our study, the majority of cats affectced with midline defects (ppdh or omphalocele) were males, and they were directly related, also supporting a genetic basis. this is the first report of familial ppdh in cats. keywords: cats, congenital, peritoneopericardial diaphragmatic hernia, inbreeding, pregnancy clinical theriogenology • volume 10, number 3 • september 2018 306 spermatic cord enlargement due to lymphangioma spermatic cord enlargement due to lymphangioma breanthony baker, charles love department of large animal clinical sciences college of veterinary medicine, college station, tx enlargements of spermatic cord are rare, but have the potential to affect testes health and fertility. a 4 year old american quarter horse stallion presented for semen collection and freezing in january of 2014. evaluation of sperm quality indicated that the stallion was a good candidate for semen cryopreservation. as part of the manual evaluation of the scrotum and contents, a mass was identified in spermatic cord, just dorsal to right testis. ultrasonographic assessment revealed a fluid filled, multiloculated structure. in addition, a polycystic pattern characterized the cranial portion of parenchyma within right testis. since this lesion was not impairing sperm quality at that time, client decided to take the stallion home to breed during the 2014 breeding season. four months after discharge, the stallion was readmitted because of poor sperm quality, which was confirmed following 3 semen collections (total motility 48 57%; progressive motility 38 51%; normal morphology 28 40%). ultrasonography revealed the polycystic structure had enlarged in both spermatic cords and testis parenchyma, causing compression of left testis. right spermatic cord and testis were removed surgically and submitted for histopathologic evaluation. lymphangioma of the spermatic cord was diagnosed, an epithelial growth characterized by rapidly dividing lymphatic vessels within the connective tissue.1 following orchiectomy, the stallion recovered. although inguinal and axillary regions have been cited as lymphangioma predilection sites in horses,2 no known report of localization within the spermatic cord has been identified.3 while benign, most lymphangiomas tend to be locally invasive and interfere with health of associated structures,2,4 such as occurred in this case. due to its physiologic impact, spermatic cord lymphangioma should be considered when polycystic structures are identified in the region of the spermatic cord. keywords: stallion, lymphangioma, spermatic cord references 1. gehlen h, wohlsein p: cutaneous lymphangioma in a young standardbred mare. beva equine vet j 2010;32:86-88. 2. knottenbelt dc, snalune k, patterson-kane j: clinical equine oncology. amsterdam: elsevier; 2015. p. 337. 3. voge jl, edwards jf, love cc, et al: theriogenology question of the month. j am vet med assoc 2015;246:847850. 4. savage vl, cudmore la, russell cm, et al: intra�abdominal cystic lymphangiomatosis in a thoroughbred foal. beva equine vet educ 2018;30:403-408. 509 clinical theriogenology • volume 11, number 3 • september 2019 510clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2018: tranrectal massage of the accessory glands in bulls prior to electroejaculation does not affect interval to penile extension and ejaculation tranrectal massage of the accessory glands in bulls prior to electroejaculation does not affect interval to penile extension and ejaculation jennifer h. koziol,a george e. mooreb adepartment of clinical sciences, college of veterinary medicine, purdue university, west lafayette, in; bdepartment of veterinary administration, college of veterinary medicine, purdue university, west lafayette, in abstract transrectal massage of the accessory sex glands for 15-60 seconds in bulls prior to electroejaculation has long been advocated by theriogenologists as this procedure has been anecdotally stated to initiate sexual stimulation in the bull thereby decreasing time to extension of the penis and ejaculation consequently decreasing the amount of electrical stimulation needed for ejaculation. the present study was performed to test the hypothesis that transrectal massage reduces the time for extension of the penis and ejaculation to occur and to determine the optimal length of stimulation between 15 and 60 seconds. one hundred and twenty-two bos taurus bulls ranging in age from 12-14 months-of-age were randomly allocated into four groups:1) routine examination of accessory sex glands and inguinal rings only; 2) examination of accessory sex glands and inguinal rings followed by 15 seconds of massage of the ampullae, prostate, and urethralis muscle; 3) examination of accessory sex glands and inguinal rings followed by 30 seconds of massage of the ampullae, prostate, and urethralis muscle; 4) examination of accessory sex glands and inguinal rings followed by 60 seconds of massage of the ampullae, prostate, and urethralis muscle. groups did not differ significantly in mean extension time (p=0.76) or mean time to ejaculation (p=0.072). in conclusion, transrectal massage of the ampullae, prostate and urethralis muscle for 15-60 seconds prior to electroejaculation did not significantly reduce intervals to penile extension or ejaculation in bulls. keywords: bull, bovine, transrectal massage, semen collection, electroejaculation introduction bull breeding soundness examinations should be an integral part of every cattle operation’s management plan as it represents the best currently available predictor of bull breeding potential for pasture breeding. electroejaculation has long been utilized as a reliable method of collecting semen for performing breeding soundness evaluations. however, this procedure has been discouraged in the uk and banned in several european countries.1,2 it has been stated that since electroejaculation without anesthesia is painful to humans therefore it is assumed to be painful to bulls.1-4 however, bulls that were electroejaculated two to three times a week for six weeks did not show aversion to handlers or the restraint over the course of the study.5 furthermore, a lack of difference between plasma concentrations of substance p, a specific target for assessing pain, before and after electroejaculation was interpreted as a lack of pain associated with nociception. the authors concluded that the increase in vocalization, plasma cortisol and progesterone in bulls following electroejaculation was likely due to acute stress secondary to being handled and restrained in the chute.6 despite these findings one cannot disagree that the vocalization, muscular contractions, struggling and lying down experienced by some bulls during the electroejaculation process raises welfare concerns. consequently, multiple studies have attempted to reduce the perceived associated pain and stress from electroejaculation.5,7 transrectal massage alone8 and artificial vaginas2,9 are not as efficacious as electroejaculation for obtaining a semen sample and drugs used to facilitate electroejaculation have not been efficacious enough to warrant use.1,10,11 if electroejaculation of bulls is to remain an accepted procedure, veterinarians must strive to collect semen by using the gentlest methods and least amount of electrical stimulation possible. it has often been stated that transrectal massage of the ampullae, prostrate, and urethralis muscle for approximately 10-60 seconds will decrease the duration of the subsequent electroejaculation by sexually exciting the bull aiding in quicker penile extension and ejaculation.2,10,12,13 however, no field studies have clinical theriogenology • volume 10, number 2 • june 201891 determined the ideal length of time for transrectal massage before electroejaculation and if transrectal massage truly hastens the onset of penile extension and ejaculation. with the knowledge that the acute stress bulls experience during the electroejaculation process is likely due to handling and restraint suggests that there may be some benefit to decreasing the amount of time the bull is restrained in the chute.6 therefore, the objective of this study was determine if transrectal massage of the ampullae, prostate, and urethralis muscle prior to electroejaculation truly reduces the interval to penile protrusion or collection of the sperm-rich fraction of the ejaculate and if so, what is the ideal duration of transrectal massage. materials and methods bulls and data collection breeding soundness examinations were performed on performance tested bulls, 12-14 months-ofage, as part of the routine end of test protocol. bulls were randomly assigned, utilizing a random number generator, to one of the four treatment groups: 1) routine examination of accessory sex glands and inguinal rings only; 2) examination of accessory sex glands and inguinal rings followed by 15 seconds of massage of the ampullae, prostate, and urethralis muscle; 3) examination of accessory sex glands and inguinal rings followed by 30 seconds of massage of the ampullae, prostate, and urethralis muscle; 4) examination of accessory sex glands and inguinal rings followed by 60 seconds of massage of the ampullae, prostate, and urethralis muscle. bulls were initially assigned into equal sized groups however individuals were lost from each group due to conformational issues or insufficient scrotal circumference. for semen collection bulls were restrained in an indoor chute system. their forward and backward movement was limited by the use of a pole placed immediately behind them while their heads remained free. to aid palpation and suitable contact of the electroejaculator electrodes with the rectal mucosa, feces was evacuated manually from the rectum by insertion and removal of the gloved hand. examination of the accessory sex organs and the inguinal rings was completed according to the guidelines of the society for theriogenology for routine breeding soundness examinations. the accessory sex gland examination and transrectal massage was performed by the same veterinarian. massage consisted of vigorous longitudinal massage over the ampullae, prostate, and urethra. the veterinarian was instructed to start and stop massage by an official time-keeper. immediately following the end of the transrectal palpation a lubricated, 64 mm diameter rectal probe with three ventrally oriented electrodes was inserted into the rectum and the process of electroejaculation was accomplished using an automatic setting that provides a series of 40 cycles. the cycle begins at 0.5v and each subsequent cycle increases by 0.5v; each cycle lasts two seconds followed by a two second pause (electrojac, neogen, lexington, ky). the time to protrusion of the penis (approximately >10 cm of the penis visible) was recorded as well as the time to ejaculation of the spermrich fraction. the sperm-rich fraction was defined as a minimum of 0.5 ml of cloudy, white seminal fluid. once this was achieved electrical stimulation was ceased and the number of stimuli required was recorded. following the successful collection of a semen sample the rectal probe was removed and cleaned. disposable rectal sleeves were changed between every bull. a subjective scoring system was used to determine whether there was a difference in behavioral response to electroejaculation amongst the four treatment groups. subjective scores were categorized as follows: 0 – no vocalization and no reaction; 1 – one low vocalization or partial collapse; 2 – two or three moderately low bellows, or going down on carpal joints; 3 – four or more loud bellows, or complete collapse. the seminal characteristics evaluated were: percent progressively motile sperm, and percent live sperm. percent progressively motile was determined with bright field microscope at 400x magnification after placing a coverslip over a 2-4mm drop of semen on a warmed microscope slide. the percent live sperm was determined by identifying the percentage of sperm that did not take up eosin stain in an eosinnigrosin smear using bright field microscopy at 1000x. clinical theriogenology • volume 10, number 2 • june 2018 92 statistical analysis statistical analyses comparing the four treatment groups in age, weight, frame size, extension time (seconds), ejaculation time (seconds), and number of electrical stimuli were performed using a one way analysis of variance (anova) with scheffe’s adjustment for pairwise comparisons. fisher’s exact test was used to compare breed and behavior score distributions across groups. a p-value of less than 0.05 was considered statistically significant. data are reported as mean ± standard deviation (sd). data are graphically displayed in box-and-whisker plots to demonstrate data distribution, including range of observed values. all statistical analyses were performed with commercial software (stata se, v.14.2, statacorp, college station, tx). results a total of 122 bulls completed the study and included the following breeds: angus (89), red angus (2) charolais (4), hereford (7), simmental (3), simmental-angus (16) and simmental-cross (1). in the test groups of 0, 15, 30, and 60 seconds of massage, there were 26, 31, 31, and 34 bulls, respectively. age variation among the four treatment groups, from 12.8 ± 0.9 months to 13.1 ± 0.7 months, did not significantly differ (p=0.525). weight and frame size did not significantly differ among groups (p=0.972 and 0.505, respectively). breed distribution across groups was not significantly different (p=0.896). every bull extended the penis and ejaculated on the first attempt of electroejaculation, bulls were only recollected if poor semen motility or morphology were noted. time to protrusion of the penis averaged 21.0 ± 8.4 seconds across all 4 groups, and group means ranged from 20.1 ± 6.5 to 21.7 ± 8.4 seconds. groups did not differ significantly in mean extension time (p=0.76). time to ejaculation averaged 43.7 ± 13.2 seconds across all groups, and groups did not significantly differ in mean time to ejaculation (p=0.072; figure). mean times (seconds) to ejaculation were 48.7 ± 15.8 (0 second massage), 40.1 ± 13.4 (15 second massage), 41.8 ± 10.6 (30 second massage), and 44.9 ± 12.1 (60 second massage). mean time to ejaculation also did not differ if bull with 0 seconds massage where compared to all other group (≥ 15 second massage). the number of stimuli required for ejaculation averaged 11.0 ± 2.5 across all groups, and group mean number of stimuli ranged from 10.5 ± 2.8 to 11.3 ± 2.3. the number of stimuli required for ejaculation did not differ significantly differ among groups (p=0.63). behavior scores (p=0.55; table), percent progressively motile sperm and percent live sperm did not differ among groups. discussion this study could not demonstrate that transrectal massage of the ampullae, prostate, and urethralis muscle for a period of time between 15-60 seconds prior to electroejaculation significantly reduced the time to extension of the penis and ejaculation in bulls. group mean times in this study differed by less than 10 seconds. most veterinarians can agree that bulls often show some signs of sexual stimulation during the act of transrectal massage with the relaxation of the prepuce, pulsing of the urethralis muscle and emission of pre-seminal fluids. however, contrary to popular belief our study demonstrates that transrectal massage of the accessory sex glands and urethralis muscle for 15-60 seconds prior to electroejaculation does not significantly decrease the number of stimuli nor the time to extension of the penis and electroejaculation in the bull. furthermore, there is no change in behavioral scores amongst the groups. similarly, in a previous study, transrectal massage of the prostate, ampullae, and urethralis muscle for 2 minutes prior to electroejaculation did not significantly decrease interval from onset of stimulation to ejaculation.10 perhaps this lack of reduction in time and number of stimuli needed to reach electroejaculation can be attributed to the lack of oxytocin release secondary to transrectal massage. oxytocin has long been implicated in the process of sperm emission and ejaculation in multiple species.14-17 sharma and hays determined that the action of manually massaging the accessory sex glands per rectum for five minutes resulted in an increase in oxytocin one to four minutes after the end of the manual stimulation or six to nine minutes after the start. due to the prolonged nature of the rise in oxytocin following transrectal clinical theriogenology • volume 10, number 2 • june 201893 massage it is likely that bulls within this study did not realize an increase in endogenous oxytocin until after the electroejaculation process was completed. it was stated by palmer et al. that an average of 67.5 seconds of transrectal massage directed specifically at the ampullae hastened the time to ejaculation by perhaps inducing a greater endogenous oxytocin release than transrectal massage over the prostate ampullae, and urethralis muscle.10 research suggests that while electroejaculation may not be a painful procedure the procedure combined with handling and restraint may be stressful to animals.6 this would suggest that a shorter time of electroejaculation and quicker release from restraint would decrease the stress level of the animal consequently increasing the overall well-being of the animal. despite the statistical insignificance of varying times of transrectal massage prior to electroejaculation, 15 seconds of massage decreased the time to ejaculation by eight and one half seconds allowing for an average reduction of the number of stimuli needed to collect an adequate semen sample for analysis by two. while not substantial, 15 seconds of manual stimulation, shorter than the anecdotally described 30-60 seconds, reduces the amount of time and number of stimuli needed to collect a semen sample for breeding soundness examination. acknowledgements: the authors thank dr. dwight wolfe for his editorial comments of this manuscript and ms. crystal hagan for her technical support of this project. the authors have no conflicts of interest to report. references 1. mosure wl, meyer ra, gudmundson j, et al: evaluation of possible methods to reduce pain associated with electroejaculation in bulls. can vet j 1998;39:504-506. 2. palmer cw: welfare aspects of theriogenology: investigating alternatives to electroejaculation of bulls. theriogenology 2005;64:469-479. 3. ohl d: electroejaculation. urol clin north am 1993;20:181-188. 4. stafford k: electroejaculation: a welfare issue. surveillance 1995;22:15-17. 5. barth ad, bowman pa: the sequential appearance of sperm abnormalities after scrotal insulation or dexamethasone treatment in bulls. can vet j 1994;35:93-102. 6. whitlock bk, coffman ea, coetzee jf, et al: electroejaculation increased vocalization and plasma concentrations of cortisol and progesterone, but not substance p, in beef bulls. theriogenology 2012;78:737-746. 7. falk aj, waldner cl, cotter bs, et al: effects of epidural lidocaine anesthesia on bulls during electroejaculation. can vet j 2001;42:116-120. 8. palmer cw, brito lfc, arteaga aa, et al: comparison of electroejaculation and transrectal massage for semen collection in range and yearling feedlot beef bulls. anim reprod sci 2005;87:25-31. 9. barth ad, arteaga aa, brito lf, et al: use of internal artificial vaginas for breeding soundness evaluation in range bulls: an alternative for electroejaculation allowing observation of sex drive and mating ability. anim reprod sci 2004;84:315-325. 10. palmer cw, amundson sd, brito lf, et al: use of oxytocin and cloprostenol to facilitate semen collection by electroejaculation or transrectal massage in bulls. anim reprod sci 2004;80:213-223. 11. berndtson w, chenoweth p, seidel g, et al: influence of prostaglandin f2α on spermatogenesis, spermatozoal output, seminal quality, testosterone levels and libido of yearling beef bulls. j anim sci 1979;49:736-742. 12. barth a: evaluation of potential breeding soundness of the bull. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. philadelphia: wb saunders co; 2007. p. 228-240. 13. schrag n, larson rl: yearling bull breeding soundness examination: special considerations. vet clin north am food anim pract 2016;32:465-478. 14. sharma op, hays rl: release of an oxytocic substance following genital stimulation in bulls. j reprod fertil 1973;35:359-362. 15. murphy mr, seckl jr, burton s, et al: changes in oxytocin and vasopressin secretion during sexual activity in men. j clin endocrinol metab 1987;65:738-741. 16. ogawa s, kudo s, kitsunai y, et al: increase in oxytocin secretion at ejaculation in male. clin endocrinol 1980;13:9597. 17. debackere m, peeters g, tuyttens n: reflex release of an oxytocic hormone by stimulation of genital organs in male and female sheep studied by a cross-circulation technique. j endocrinol 1961;22:321-334. clinical theriogenology • volume 10, number 2 • june 2018 94 figure. box-and whisker plots of time to ejaculation in 122 twelve-to-fourteen month old bulls following transrectal massage of accessory sex glands for 0 (n=26), 15 (n=31), 30 (n=31), or 60 seconds (n=34) prior to electroejaculation. outlier (●) indicates value >1.5xiqr beyond the 75th percentile. table. behavior score distribution by group and total, represented by both number of bulls per group that received each score (top number) and percentage of each group per score. no bull in this study received a behavior score of 3. behavior score 0 1 2 group 1 0 seconds 25 96.2% 1 3.8% 0 0% group 2 15 seconds 27 87.1% 2 6.5% 2 6.4% group 3 30 seconds 26 83.9% 4 12.9% 1 3.2% group 4 60 seconds 32 94.2% 2 5.8% 0 0% total 110 90.2% 9 7.4% 3 2.4% 0 10 20 30 40 50 60 70 80 90 10 0 ti m e to e ja cu la tio n (s ec ) 0 sec 15 sec 30 sec 60 sec transrectal massage time clinical theriogenology • volume 10, number 2 • june 201895 hatching artefacts in hermann™s tortoise (testudo hermanni) after artificial incubation hatching artefacts in hermann’s tortoise (testudo hermanni) after artificial incubation mateja stvarnik,a igor klobu� ar,a renata knific,b gordana gra� ner,c alenka dov� b a clinic for reproduction and large animals, veterinary faculty, university of ljubljana, slovenia binstitute for poultry, birds, small mammals and reptiles, veterinary faculty university of ljubljana, ljubljana, slovenia cdepartment of hygiene, behaviour and animal welfare faculty of veterinary medicine, university of zagreb, zagreb, croatia after end of a hatching season for hermann's tortoise (testudo hermanni), from end of may to beginning of september, all unhatched eggs were examined. hatching remainder represented 19.2% (3,557) of all eggs (18,520). viability rate for incubated eggs was 80.8%. eggs were harvested at the farm and immediately put into hatcheries. temperature of hatching varied from 31 to 32°c. moisture was maintained by placing the eggs on substrate (vermiculite), which was occasionally moistened. on average, moisture was 80%. average hatching time was 67 days. data on embryonated and unfertilized eggs were analyzed. of all unhatched eggs, 61.0% were unfertilized, 52.5% were infected, and 19.1% was in various stages of dehydration including mummification; there was a percentage overlap as 2 or more causes (unfertilized, infected, dried) were identified in some eggs. fertilization and development were not confirmed in 24.1%. main reason for presence of multiple abnormalities was infection with various pathogens, with most common bacterial and fungal infections due to pseudomonas aeruginosa, bacillus sp. and purpureocillium lilacinum. unfortunately, in some cases, bacteria and fungus remained undetermined. data on fertilized eggs were further classified into 3 groups according to stage of fetal development. embryos in first third of development phase were present in 2.2%, in second third in 5.4% and in last third phase in 7.3% of total number of unhatched eggs. most tortoises died before hatching, whereas only a few remained alive (0.7%). in all 3 groups, only 14.9% embryos were confirmed. from these embryos, one-fourth belonged to the second development stage and threefourths to third stage. some were completely developed and just unable to peck the shell. dead and alive creatures were identified in 0.6% of total number of fertilized eggs. misshapen carapace and tail, lack of pigment, open coelomic cavity, missing eye or other parts of the body, and conjoined twins were the most commonly abnormalities. keywords: testudo hermanni, eggs, artificial incubation, hatching artifacts 479 clinical theriogenology • volume 11, number 3 • september 2019 480clinical theriogenology • volume 11, number 3 • september 2019 omniblank: a review of the equine oviduct: pathology, evaluation, and current treatments a review of the equine oviduct: pathology, evaluation, and current treatments maria schnobrich rood and riddle equine hospital, lexington ky abstract the equine oviduct has a vital role in fertilization and early embryonic support in the mare. its anatomy is unique compared to other domestic species, with a muscular sphincter at the uterotubal junction that allows selective transport of embryos but not unfertilized oocytes. although oviductal pathology is believed to be an infrequent cause of subfertility, it remains a potential differential when other causes for pregnancy failure have been ruled out. this paper is a brief review of equine oviduct anatomy and functional characteristics, common pathologies, current diagnostic tools and treatments for clinically relevant pathology. keywords: oviduct, pge2, paraovarian cyst, hysteroscopic hydrotubation introduction oviducts (uterine tubes) develop from the most anterior segment of fetal paramesonephric or müllerian ducts.1-2 concentric tissue layers of the oviduct from the external surface, to the oviductal lumen consist of: serosal surface, muscularis (longitudinal and circular muscular layers), submucosa, and mucosa. in a 500 kg mare, they are approximately 20 30 cm in length and travel a tortuous path in the mesosalpinx from the ovary to the tip of the uterine horn.1-2 the oviduct is divided anatomically into 3 sections: infundibulum, ampulla and isthmus. infundibulum’s funnel-shaped dilation at the proximal aspect of the oviduct covers the ovary’s ovulation fossa and transports the oocyte through ciliary action to the ampullary region. ampulla is the site of fertilization, comprising approximately half the length of the oviduct and is ~ 6 mm in diameter.1-2 following fertilization, conceptus passes through the isthmus (2 3 mm in diameter) and enters the uterus at the uterotubal junction.1-4 luminal epithelium of the oviduct is lined with a simple columnar epithelium with varying degrees of ciliated and nonciliated cells.5 the branched folding of the mucosal folds is most elaborate and extensive in the ampullary region, where the isthmus has a more narrow lumen with short and non-branched mucosa folds. regional differences in cilia density, mucosal folding, cytoplasmic granule characteristics and secretions have been reported. unique anatomical and epithelial characteristics of each section of the oviductal lining suggest that each region of the oviduct likely has a distinctive microenvironment related to its functional role.5 the most unique aspect of the mare’s oviduct is the muscular sphincter present at the uterotubal junction of the mare that selectively allows embryos, but not unfertilized oocytes, to pass into the uterus at ~ 5.6 6 days postovulation.6 elegant work by woods et al. demonstrated that prostaglandin e2 (pge2) secreted by the embryo facilitated passage of embryo into the uterus.7-8 this muscular constriction at the uterotubal junction makes salpingitis rare in the mare compared to other species (e.g. cattle and swine). it is hypothesized that the decreased incidence of clinically recognized salpingitis in the mare is due to this sphincter preventing uterine infections from gaining access to the oviduct. function fertilization the infundibulum sweeps the ovulated oocyte towards the ampullary region through concerted ciliary action and muscular contractions. sperm are believed to gain access to the oviduct within seconds after intrauterine insemination, but must undergo capacitation before fertilization. sperm entering the oviduct is believed to bind oviductal epithelium of the isthmus and subsequently be released in waves. upon release, the oviductal environment facilitates capacitation. the specific environmental trigger within the oviduct responsible for sperm capacitation is not known, but the alkaline micro-environment of the oviduct may aid in sperm preparation, as well as specific secretions released from the oviductal 379 clinical theriogenology • volume 11, number 3 • september 2019 epithelium9-11 role of uncharacterized capacitation triggers present in the equine oviduct continues to be an important area of research, as elucidation will increase efficiency of equine in vitro fertilization. transportation following fertilization, the embryo must travel through the isthmus into the uterus. this movement is believed to be facilitated by ciliary action and oviductal contractions mediated by embryonic secretion of pge and likely other factors. as mentioned, the early embryo passes into the uterus on days 5.5 6 and only through continuous application of pge2 to the oviduct, could earlier recovery of embryos from the uterus be achieved.7-8 this work was key to understanding the mechanism of the selective transport of the mare’s oviduct and also potential for oviductal treatment. pge2 has 4 known receptors with biological activity: e1, e2, e3, and e4. induction of ep1 and ep3 induce smooth muscle contraction, whereas ep2 and ep4 induce smooth muscle relaxation. strong expression of the receptor ep2 and ep4 was demonstrated in the luminal epithelium of both the isthmic and ampullar regions of the horse oviduct, with less expression noted within the smooth muscle of these regions.12 exact role of how pge2 interacts with these receptors is not known. it is hypothesized to induce relaxation of the circular smooth muscle fibers and contraction of the longitudinal smooth muscle fibers in the wall of the oviduct. delayed entry of the equine conceptus into the uterus allows the practitioner time to improve the uterine environment following ovulation without jeopardizing the health of the embryo. in cases of severe postmating induced endometritis, mares can be given intrauterine lavages and treatments up to 3 days post ovulation, with usually minimal adverse effects. pregnancy support oviductal secretions during pregnancy have been evaluated and there is clear evidence that the presence of an embryo changes the oviductal environment to facilitate its support. gene expression and protein expression patterns, as well as, secretory composition are altered by the presence of an embryo in the oviduct.13-14 the preimplantation embryo can utilize fatty acids produced by the oviduct through direct incorporation into lipid bilayers of the cell membrane and for energy production via ß oxidation. concentration and composition of fatty acids of the oviduct was higher than in plasma for arachidonic, eicosapentaenoic and dihomo-γ-linolenic acid, implying oviductal nutritional support for the early embryo.15 these studies continue to evaluate the role of the oviduct in early pregnancy support and provide information regarding the microenvironment required for the early embryo. this understanding may improve techniques for in vitro embryo production. pathology of the oviduct as a practitioner, oviduct pathology is rarely recognized as a clinical factor causing infertility or during routine reproductive examination of the mare. despite this, postmortem evaluations of the oviducts of many mares revealed a high prevalence of macroscopic and microscopic lesions. in an early study by salteil involving postmortem evaluation of oviducts from 325 mares, 87.7% (285/325) had at least 1 macroscopic lesion and 93.5% (116/124) had at least 1 microscopic lesion.16 macroscopic lesions were predominantly adhesions, paraovarian cysts and thick fibrous bands. microscopically, lesions included intraepithelial cysts, slight, focal lymphocytic infiltration in the infundibular-ampullary region and proteinoid material in the oviductal lumen. proximal segments of the oviduct were more affected than distal segments. these findings are supported by others who demonstrated that salpingitis was common in the infundibular region (37%), compared to the ampullary (21%), or isthmic region (9%) had isthmitis.17-18 increasing evidence for a higher incidence of salpingitis in the mare is emerging. in a recent study evaluating abattoir-derived tissues from criollo mares, most (64%) had some degree of lymphocytic infiltration in the oviduct mucosa, with a lower incidence of neutrophilic (8%) and eosinophilic (4%) infiltration in oviducts. additionally, this study demonstrated that oviductal inflammation can be present without endometrial inflammation.19 both mild salpingitis and adhesions are difficult to detect with transrectal palpation and are likely under reported in clinical literature. whereas the presence of oviductal pathology appears common in the literature, the presence of these lesions does not usually affect fertility. in saltiel’s abattoir study, 90.74% (40/54) of the pairs of oviducts collected from pregnant mares had at 380clinical theriogenology • volume 11, number 3 • september 2019 https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/preimplantation-embryo https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/lipid-bilayer https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/energy-yield https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/beta-oxidation https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/dihomo-gamma-linolenic-acid https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/mucous-membrane least 1 macroscopic or microscopic lesion.16 this finding parallels clinical observations that oviducts are rarely identified as a cause of subfertility in the mare. the most common pathology identified on reproductive examination in the oviductal region are cystic remnants referred to as fimbrial cysts (hydatid of morgagni), and paraovarian or uterotubal cysts. fimbrial cysts are believed to be caused by formation of more than 1 ostium from the coelomic epithelium in fetal life.20 paraovarian cysts located closed to the uterus are believed to be remnants of the mesonephric ducts and tubules and are thin walled cysts lined by a columnar epithelium. they are usually abaxial to the uterus and oviduct (paraovarian cysts) or within the cranial edge of the fimbrae of the oviductal infundibulum (fimbrial cysts).3,20-22 these appear on transrectal ultrasonography as round to tubular, occasionally multi-loculated, thin-walled, anechoic to hypoechoic cysts, ranging from several millimeters to several centimeters in diameter. most commonly they are identified between the tip of the uterine horn and the ovary itself and should be noted as they can often be mistaken for a dominant follicle when adjacent to the ovary or present on the infundibulum as it drapes over the ovulation fossa. those large enough to displace the infundibulum from covering the ovulation fossa have been hypothesized by some authors to interfere with oocyte transport and removal has been recommended. while reports exist of these interfering with oocyte transport, they are usually considered an incidental finding when diagnosed. 2325 monitoring size and character of the cysts is recommended, and only when failure to achieve pregnancy from that side is repeatedly noted, would intervention be recommended. a large body of research has demonstrated the presence of gelatinous and proteinaceous accumulations in the oviductal lumen with variable adhesions to the oviductal epithelium.26-29 these masses are believed to occur due to the retention of ovulated material (oocytes, fibroblasts, gelatinous matrix) within the oviduct, and salpingitis. what effect these accumulations have on fertility is a topic of debate based on postmortem frequency and the seemingly minimal effect on mare fertility. what is interesting is that subfertile mares suspected to have oviduct dysfunction, can have their fertility improved by treatment with various methods to restore oviduct “patency.” this begs the question whether these accumulations are pathologic and if so, when and at what point do they become an issue. hydrosalpinx is a rarely reported pathology of the equine oviduct. hydrosalpinx is usually caused by stricture or congenital failure of normal formation of the oviduct. in 1 report, histological evaluation of the distended tube in a mare revealed a dilated lumen, a decreased in-folding of the wall into the lumen, and a lack of a defined muscle layer. it was hypothesized that the cause of hydrosalpinx in this case was a congenital absence of the infundibulum, resulting in a blind ampullar terminus.30 abscess formation, or severe salpingitis that is recognized on transrectal ultrasonography, palpation or laparoscopy is rare and usually requires treatment and/or removal of the affected oviduct or ovary. oviductal neoplasia is rare, with adenoma and leiomyomas described.31 these cases usually include a history of infertility when ovulations occur from the affected side. removal of the ovary is recommended unless oocyte recovery will be attempted to circumvent the oviductal pathology. diagnostic approach and treatment paraovarian cyst tentative diagnosis of a fimbrial or paraovarian cysts is usually made by serial transrectal palpation and ultrasonography. these are usually small (~ 1 8 cm), round to tubular, thin-walled, soft to firm, anechoic to hypoechoic fluid-filled structures, which are non painful on palpation. evaluation with color flow doppler should show no evidence of blood flow. laparoscopic evaluation can be used to confirm diagnosis, but due to their low clinical significance, is usually not recommended. any similar appearing structure found within the mesovarium or any region from the tip of the uterine horn extending cranial to the ovary within the supporting tissues are considered. serial ultrasonography over several weeks usually detects little change in the structure with regards to size, though at times the segmentation or tortuosity of the structure may appear to change. laparoscopic evaluation of the structures reveals a clear to orange filled, thin-walled structure, well circumscribed on the surface of the mesovarium or arising from the infundibular mucosa. laparoscopic assessment is only recommended in cases where the 381 clinical theriogenology • volume 11, number 3 • september 2019 degree of interference of the cyst with the infundibulum’s normal anatomy and function is suspected (failure to conceive with ovulations from that side). if severe adhesions and disruption of the normal anatomy is seen, and the mare has a confirmed history of pregnancy failure following ovulation from that side, surgery can be attempted to restore anatomy, reduce the cyst or remove the affected ovary. removal of the ovary is recommended to increase efficiency of breeding as a dominant follicle can then only arise from the unaffected ovary and valuable time is not wasted. if oocyte aspiration is an option for the mare, the ovary is left and oocytes can be harvested, bypassing the affected oviduct. adhesions adhesions between the ovary and infundibulum are not easily identified by transrectal palpation or ultrasonography. laparoscopic evaluation of the ovary and oviduct is often the best way to diagnose this pathology. adhesions between the ovary and surrounding structures (gi tract, pelvic wall) are diagnosed through identifying abnormal anatomy on palpation. indication for laparoscopic evaluation would be abnormal palpation and suspicion of adhesions with a history of infertility. often mares with severe adhesions have a history of colic surgery, peritonitis, hemoabdomen or dystocia. these adhesions can form from the process of ovulation, salpingitis, peritonitis, trauma to the reproductive tract and unknown causes. they can vary from simple, string like adhesions, to thick fibrous bands that distort the relationship of the infundibulum to the ovulation fossa, which some speculate causes oocytes to be lost in the abdominal cavity.22 the decision to surgically remove or breakdown adhesions is based on how much these change the anatomy of the oviduct and reproductive history. recently, mares presented for infertility and laparoscopic pge2 application to the oviducts had oviductal adhesions removed as part of their treatment with good success.32 the role of adhesion breakdown in restoring fertility needs further work and has not yet been critically assessed. salpingitis inflammation of the oviduct (salpingitis) is rarely diagnosed during a reproductive examination of the mare. severe salpingitis may be recognized as a thickening and dilation of the oviduct on transrectal palpation and ultrasonography, or laparoscopically. hysteroscopic examination is usually unrewarding due to the muscular sphincter and variable appearance of the mare’s oviductal papilla. even in mares’ oviducts that have ultrasonographic appearance of dilation and luminal changes, the oviductal papilla often appears normal on hysteroscopy. subclinical salpingitis is difficult to definitively diagnose without biopsy of the tissue, or postmortem evaluation. in most patients, this is a presumptive diagnosis based on transrectal palpation and ultrasonography of the oviduct. the oviduct is not easily identifiable with transrectal ultrasonography, so any thickening, intraluminal fluid, or discrepancy in size from the contralateral oviduct is suspicious. these findings in conjunction with unilateral infertility should increase suspicion of pathology in this region. treatment may involve systemic antibiotics with good tissue penetration (enrofloxacin, oxytetracycline or chloramphenicol) following culture and sensitivity of fluid or tissue from the affected region. this may be obtained laparoscopically or empiric treatment may be elected to avoid peritonitis. flushing of the oviduct surgically or through a hysteroscopic approach can be attempted, but in the case of infection, peritonitis must be considered potential sequelae. severe salpingitis usually results in fibrosis and loss of oviduct function and the owner may choose unilateral ovariectomy. loss of oviductal function/patency often the loss of oviduct function is suspected when all other causes of fertility have been ruled out and the history suggests oviduct dysfunction. a history that is suspicious for oviductal pathology includes loss of ability to produce a pregnancy from unilateral or bilateral ovulations, despite ruling out all other causes of infertility (endometritis, adhesions, semen quality and sire, breeding management and timely ovulation relative to breeding, endocrine abnormality, genetic abnormality). occasionally the patient has a history of dystocia, peritonitis, colic, or trauma in the reproductive tract or pelvic area. there have been several studies that have described methods of assessing oviductal patency, but due to the fine, 382clinical theriogenology • volume 11, number 3 • september 2019 sensitive structure of the oviductal lumen, it is not clear if diagnostic methods for assessing patency could potentially cause a salpingitis and even create a problem. initial evaluations of patency described surgical exteriorization of the uterine horn and oviduct and flushing the oviduct (normograde and retrograde) with saline, air or new methylene blue to determine patency.22,25,32,34 these are invasive procedures and often the flushing was considered therapeutic. complications associated with this procedure included oviductal rupture as it was believed that the flushing from oviduct to uterus (normograde) and as the luminal size narrowed, excessive pressure would cause a rupture, potentially permanently damaging the oviduct. if rupture occurred or surgical evaluation of the affected ovary revealed severe, unfixable pathology, the ovary was often removed. another method for patency determination has been attempting to recover small starch or colored microsphere beads applied over the infundibulum. these beads are inserted over the infundibulum through either a vaginal or laparoscopic approach. love et al. used 15 µm microsphere colored beads (1 color for each side) applied laparoscopically over the infundibulum of the oviduct and then recovered uterine contents by uterine lavage 48 hours later. the oviducts were evaluated microscopically and compared to results of the beads recovered. the test was considered positive for oviductal intraluminal accumulations when no beads were recovered from that side in uterine lavage. sensitivity and specificity of the test were 71.4 and 85.7% when compared to postmortem evaluation of the oviducts.35 although this approach is helpful in confirming suspicions, it is costly. the downfall of these diagnostic tests for oviduct patency is the definition of “patency” or “normal function” is not clear. dye and fluorescent microsphere beads do not mimic sperm entering the oviduct, the oviductal environment required for embryo growth, nor the coordinated passage from the ampulla to uterine lumen. in clinical practice, oviductal patency, or loss of function, is often a diagnosis of exclusion. this suspicion is reached when all the more common causes for subfertility/infertility have been ruled out (endometritis, ovulatory failure, semen quality, genetic abnormality). several studies have demonstrated that treatment for oviductal “dysfunction,” usually attributed to lack of patency or “plugs,” have improved fertility in a specific subset of mares. because the treatment has been successful when case selection is properly performed, many owners and clinicians elect to treat without a confirmed diagnosis prior to treatment. allen et al. demonstrated that laparoscopic application of pge2 gel to the oviducts of mares presented for chronic subfertility significantly increased fertility.36 other studies have echoed the benefit of this treatment in similar subsets of mares. it is believed that the pge2 causes contraction of the oviduct and opening of the oviductal muscular sphincter which allows accumulations to be expelled. lavages of mares following treatment have recovered similar structures to the gelatinous oviductal plugs described. hysteroscopic application of pge2 directly to the oviductal papillae using deep horn insemination technique did not seem to enhance embryo transport into the uterus, though some are using this approach instead of the laparoscopic approach with reported anecdotal success.37 dr. inoue developed a hysteroscopic flushing technique that was first described in 2013, whereby a small catheter was placed in the oviductal papilla and sterile saline flushed retrograde.38 this procedure resulted in similarly positive effects as laparoscopically administered pge2 in mares with suspected pathology. in a study comparing the 2 techniques, there was no significant difference in pregnancy rates following laparoscopic pge2 application (65%, 13/20) and hysteroscopic hydrotubation (71%, 35/49), in a similar subset of previously subfertile mares.39 the hysteroscopic approach offers the advantage of not having to perform surgery and avoids the cost of the pge2 gel, but the disadvantage is inflammation caused by hysteroscopy and potential mechanical trauma to the oviduct from the procedure. recently, in 2018, alvarenga’s group in brazil demonstrated efficacy of an even simpler procedure.40 in this, study pge1 instead of pge2 was used without surgery. specifically, 1 tablet (200 µg of misoprostol, cytotec® pfizer, lake forest, il) was diluted in 3 ml of sterile water and is applied to the tip of the uterine horn lumen using a deep-horn insemination pipette, during diestrus. the mares were bred on the subsequent cycle. within 2 cycles after the procedure, 68% (15/22) produced a pregnancy. this treatment provides a new and easy to use approach with minimal downsides for the practitioner in the field, does not risk peritonitis or trauma and is very cost effective. 383 clinical theriogenology • volume 11, number 3 • september 2019 conclusion the equine oviduct is imperative for successful fertilization, initial embryonic support, and selective transport of the early embryo into the mares’ uterus. due to the unique structure of the equine oviduct, salpingitis is rare, but other pathologies are potential differentials for subfertility. conflict of interest author claims no direct or indirect affiliation with any of the manufacturers listed in the text. information regarding the equipment and various manufacturers is solely based on experience and is for the readers’ reference only. references 1. dyce km, sack wo, wensing cjg: textbook of veterinary anatomy. philadelphia, wb saunders. 1987. 2. sisson s: female genital organs. in sisson and grossman’s the anatomy of domestic animals. volume 1. 5th edition, philadelphia: wb saunders 1975, p.542-549. 3. kenney, rm: a review of the pathology of the equine oviduct. 1993 equine vet j 1993 l25:s15, p. 42-46. 4. mcentee k: the uterine tube. reproductive pathology of domestic mammals, 1990, p. 94-109. 5. desantis s, zizza s, accogli g, et al.: morphometric and ultrastructural features of the mare oviduct epithelium during oestrus. theriogenology, 2011:75:671. 6. freeman da, weber ja, geary rt, et al: time of embryo transport through the mare oviduct. 1991, theriogenology 1991:36;823-830. 7. weber ja, freeman da, vanderwall dk, et al: prostaglandin e2 secretion by oviductal transport stage equine embryos. biol reprod 1991:45;540-543. 8. weber, ja, woods gl, lichtenwalner ab: relaxatory effect of prostaglandin e2 on circular smooth muscle isolated from the equine oviductal isthmus. biol reprod monograph series 1, 1995;125-130. 9. gonzalez-fernandez l, macias-garcia b, velez ic, et al: calcium calmodulin and ph regulate protein tyrosine phosphorylation in stallion sperm. reproduction 2012:144;411-422. 10. aalberts m, sostaric e, wubbolts r, et al: spermatozoa recruit proteasomes in response to capacitation induction. 2013 biochimica et biophysica acta 1834;2326-2335. 11. leemans b, gadella bm, sostaric e, et al: oviduct binding and elevated environmental ph induce protein tyrosine phosphorylation in stallion spermatozoa. biol reprod 2014:91;1-12. 12. ball bb, scoggin ke, troedsson mh, et al: characterization of prostaglandin e2 receptors (ep2, ep4) in the horse oviduct. anim reprod sci 2013;142:35-41. 13. smits k, de coninck di, van nieuwerburgh f, et al: the equine embryo influences immune-related gene expression in the oviduct. biol reprod 2016;94. 14. smits k, nelis h, van steendam k, et al: a proteome of equine oviductal fluid effects of ovulation and pregnancy. reprod fertil dev 2017:29;1085-1095. 15. drews b, milojevic v, giller k, et al: fatty acid profile of blood plasma and oviduct and uterine fluid during early and late luteal phase in the horse. theriogenology 2018;114:258-265. 16. saltiel a, paramo r, murcia c, et al.: pathologic findings in the oviducts of mares. am j vet res 1986;47:594-597. 17. kenneth mcentee: cysts in and around the ovary. reproductive pathology of domestic mammals, 1990 p. 52-68. 18. kenneth mcentee:the uterine tube. reproductive pathology of domestic mammals, 1990 p. 94-109. 19. fiala-rechsteiner m, amaral mg, augusto cruz, et al: inflammatory lesions in the oviducts and its relationship with endometrial inflammation and ovarian activity in criollo mares. j eq vet sci 2015:35;731-734 20. kenney rm: a review of the pathology of the equine oviduct. in: antzcak a, oriole b: editors. equine embryo transfer iii. eq vet j suppl 15;1993. 21. ginther oj: reproductive biology of the mare: basic and applied aspects. 2nd edition. cross plains: equiservices, 1992. 22. sertich pl: clinical management if the equine oviduct. clinical theriogenology 2013;5:523-529. 23. blue mg: a tubo-ovarian cyst, paraovarian cysts and lesions of the oviduct in the mare. n z vet j 1985;33:8-10. 24. archibald lf, mather ec, mcclure jr jr: fimbrial cysts as a cause of infertility in the mare. vet med small anim clin 1974;69:1163-1165. 25. bennett sd, griffin rl, rhoads ws: surgical evaluation of oviduct disease and patency in the mare. proc am assoc equine pract 2002;48:347-349. 26. liu ikm, lantz kc, schlafke s, et al: clinical observations of oviductal masses in the mare, in proceedings. 36th annu conv am assoc equine pract 1991;41-45. 27. saltiel a, paramo r, murcia c, et al: pathologic findings in the oviducts of mares. am j vet res 1986;47:594-597. 384clinical theriogenology • volume 11, number 3 • september 2019 28. tsutsumi y, suzuki it, takeda t, et al: evidence of the origin of gelatinous masses in the oviducts of mares. j reprod fertil 1979;57:287-290. 29. oguri n, tsutsumi y: studies on lodging of equine unfertilized ova in fallopian tubes. res bull livestock farm hokkaido u 1972;6:32-43. 30. hinrichs k, kenney rm, hurtgen jp: unilateral hydrosalpinx and absence of the infundibulum in a mare. theriogenology 1984; 22:571-577. 31. nelis h, leemans b, de vries c, et al: oviductal and uterine leiomyomata in mares. vlaams diergeneeskundig tijdschrift 2013; 82:81-86. 32. kollman m, rottig a, heberling a, et al: laparoscopic techniques for investigating the equine oviduct. equine vet j 2011;43:106-111. 33. pye, j, clulow, j, adkins, a: laparoscopic transection of restrictive bands of the mesosalpinx as adjunct to the use of prostaglandin e2 for mares with suspected uterine tubal blockage. aust vet j 2018;96:252-256. 34. zent ww, liu ik, spirito ma: oviductal flushing as a treatment for infertility in the mare. third international symposium on equine embryo transfer. buenos aires, 1993. 35. arnold ce, love cc: laparoscopic evaluation of oviductal patency in the standing mare. theriogenology 2013;79:905-910. 36. allen we, kessy bm, noakes de: laparoscopic application of pge2 to re-establish oviductal patency and fertility in infertile mares: a preliminary study. equine vet j 2006;38:454-459. 37. schnobrich mr, vanderwall dk, bradecamp ea, et al: clinical theriogenology 2014.6:390. 38. inoue y. hysteroscopic hydrotubation of the equine oviduct. eq vet j 2013;45:761-765. 39. walbornn rs, schnobrich mr, bradecamp ea, et al: pregnancy rates after laparoscopic application of pge2 or hysteroscopic hydrotubation of the uterine tubes. aaep proc 2018;64:245. 40. alvarenga ma, segabinazzi lg: application of misoprostol as a treatment of unexplained infertility in mares. j equine vet sci 2018;71:46-50. 385 clinical theriogenology • volume 11, number 3 • september 2019 386clinical theriogenology • volume 11, number 3 • september 2019 omniblank: reproductive remnants, mammary sequelae, and renal agenesis in a domestic short hair cat reproductive remnants, mammary sequelae, and renal agenesis in a domestic short hair cat audrey kelleman university of florida, department of large animal clinical sciences college of veterinary medicine, gainesville, fl a 10 year old domestic short hair cat was evaluated for chronic recurrent estrous behavior. reportedly, the cat had been spayed prior to adoption as a kitten. approximately 2.5 years prior, during an exploratory surgery, tissues obtained from the right side of the abdomen were excised; there was histopathological confirmation of a portion of uterus, an ovary and also a mammary nodule consistent with hyperplasia. two weeks prior to presentation, the cat was in estrus. cat was bright and alert, but fractious and therefore was sedated. body condition, rectal temperature, pulse, and respiration were normal. all mammary glands were severely affected by multifocal large fluid filled (cystic) structures cranially and solid nodules caudally. abdominal palpation was normal and vaginal epithelium was noncornified based on cytological examination. complete blood work and chemistry were normal. serum progesterone was 11.4 ng/ml. abdominal ultrasonography revealed a uterine stump, segmental cranial left uterine horn remnant, left ovary, and mammary cysts and nodules. right kidney was identified, but not the left. ultrasound guided aspiration cytology of a solid mammary nodule was non diagnostic. full body radiography findings were normal, except for the mammary pathology. surgical management was performed by the oncology service and included exploratory laparotomy with surgical excision of the left ovary and segment of adjacent left uterine horn and initial unilateral right sided radical mastectomy. subsequently, left radical mastectomy was performed. histopathological evaluation confirmed uterine tissues affected by lymphohistiocytic endometritis and hyperplasia, a left ovary containing corpora lutea and follicles, and multifocal mammary adenocarcinoma with adjacent cystic and fibroadenomatous changes with glandular hyperplasia. three days after surgery, the cat returned for suture line bandage removal and there was evidence of adequate recuperation. the owner failed to return the cat for the left side mastectomy. ipsilateral presence of an ovary, but with renal agenesis, has previously been reported in cats with discovery of uterine anatomic anomalies at laparotomy, e.g. segmental aplasia or hypoplasia.1 identification and removal of the ovary is important in these cases, so that ovarian remnant syndrome does not occur. keywords: feline, ovarian remnant, segmental aplasia, renal agenesis, mammary neoplasia reference 1. mcintyre rl, levy jk, roberts jf, et al: developmental uterine anomalies in cats and dogs undergoing elective ovariohysterectomy. j am vet med assoc 2010;237:542-546. 471 clinical theriogenology • volume 11, number 3 • september 2019 472clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2015: diagnosis and clinical management of a spontaneous pre-partum uterine rupture in an angus heifer carrying twins diagnosis and clinical management of a spontaneous pre-partum uterine rupture in an angus heifer carrying twins p.e. phillips, p.j. plummer, a.c. krull, t.a. dohlman, a.j. kreuder college of veterinary medicine, iowa state university, ames, ia summary a two year-old, nulliparous angus heifer was presented because of reduced appetite, bloat and lethargy of one day duration. the heifer was estimated to be 7.5 months pregnant. physical examination, trans-abdominal ultrasound, blood chemistry analysis, complete blood count and abdominocentesis were performed to identify the possible cause. based on lack of response to therapy, changes in uterine tone on palpation per rectum, abdominal fluid cytology and trans-abdominal ultrasound, uterine rupture was suspected and an abdominal exploratory laparotomy was performed. an extra-utero, deceased calf was identified along with a second deceased calf partially within the uterus. exteriorization of the right uterine horn revealed an approximately 40 cm rent extending from the utero-tubal junction to the body of the uterus, which was surgically repaired. preand post-operative medical therapy included intravenous fluids, antibiotics, and anti-inflammatories. subsequent follow-up revealed that the cow had resumed normal estrous cycles and is being used as an embryo donor. keywords: uterine rupture, twinning, ultrasound, abdominocentesis, cesarean section background little information is available concerning pre-partum uterine rupture in the bovine. while periparturient uterine rupture is common, especially during stage ii of labor, pre-partum rupture is rare and usually from an unknown cause.1 when pre-partum uterine rupture occurs, it is often diagnosed at necropsy, rather than antemortem. when diagnosed early, however, the prognosis for survival can be good. case presentation the patient, a 545 kg, two year-old nulliparous angus heifer, was purchased three months prior to presentation and according to the current owner had been otherwise healthy until the day prior to admission into the iowa state uuniversity veterinary teaching hospital. the heifer had been restrained in the owner’s manual chute for pre-calving scours vaccination and final pregnancy confirmation four days earlier and was currently grazing on alfalfa pasture. no abnormalities with the pregnancy were noted by the referring veterinarian at the time of pregnancy confirmation. the owner first noted the heifer to be severely bloated the day prior to admission (see figure 1) and at that time passed an orogastric tube to deliver 50 g of poloxalene orally to relieve what was suspected to be frothy bloat. no change was noted in the bloat after poloxalene administration; however, a second attempt at passage of an orogastric tube by the owner yielded expulsion of a large amount of gas and fluid from the rumen. upon initial physical examination by the referring veterinarian, it was noted that rumen motility was decreased and the abdomen remained distended. blood chemistry analysis was performed on the farm and blood gas and electrolyte parameters were within normal limits. a jugular catheter was placed and 600 mg (1.1 mg/kg) of flunixin meglumine was administered iv and 6 g (11 mg/kg) of ampicillin trihydrate was given im prior to being referred to the hospital for suspected vagal indigestion and/or traumatic reticulitis. upon examination at the hospital, the heifer was noted to be quiet, alert and responsive with a pear-shaped abdomen and normal temperature, pulse and respiratory rate. based on skin tent and eye position, she was estimated to be five to seven percent dehydrated. a fluid wave could be elicited upon ballotment of the abdomen, however, no gas or fluid was removed from the rumen following passage of a large orogastric tube. palpation per rectum revealed a small rumen and a calf palpable within the uterus; a moderate amount of udder development was also noted. a brief trans-abdominal ultrasound examination, using a 3.5 mhz curvilinear probe, revealed a large amount of free fluid within the abdomen and floating objects suggestive of fibrin accumulation and peritonitis. thirty g of sulfadimethoxine (55 clinical theriogenology • volume 7 number 2 • june 2015109 mg/kg) was given iv to address the peritonitis and 40l of normal saline was administered iv to correct dehydration followed by balanced polyionic maintenance fluids at a rate of 50 ml/kg/day. based on these clinical findings, the differential list included vagal indigestion, traumatic reticulitis and peritonitis of unknown cause. the following day, further diagnostic tests were performed including a complete blood count (cbc) and abdominocentesis. the cbc revealed a low plasma protein (5.4gm/dl) and elevated fibrinogen (600mg/dl; plasma protein:fibrinogen ratio of 9 suggestive of an inflammatory process. interpretation of the abdominal fluid cytology and cell count showed a transudative process (protein of 0.1 gm/dl) with mild neutrophilic inflammation and a mild pathologic hemorrhage with phagocytized red blood cells. the abdominal fluid was orange in color and had a thick consistency. following the abdominocentesis, palpation per rectum and trans-abdominal ultrasound were repeated. a fetal heartbeat could not be identified via trans-abdominal ultrasound and within the free abdominal fluid in the lower right abdomen a region suspected to be free-floating placenta with cotyledons was now visible (see figure 2). upon palpation per rectum, thickened myometrial ridges near the body of the uterus could be identified and a calf was no longer palpable. a tentative diagnosis of uterine rupture was made and 40l of a balanced polyionic solution was administered prevent hypovolemic shock due to anticipated fluid loss during surgery. treatment following local anestheisia (inverted l) with 2% lidocaine, a right paralumbar flank approach was used to open the abdomen. entry into the abdomen allowed approximately 50l of fluid to be drained from the incision and free floating placenta was observed (figure 3). exploration of the abdomen revealed a free floating calf and completely detached placenta within the abdominal cavity. the body wall incision was extended to accommodate removal of the calf which was deceased. further examination revealed a large rent in the right horn of the uterus. within the rent, a second deceased calf was found and was subsequently extracted from the abdomen along with its placenta (figure 4). the age of the fetuses was estimated to be approximately 7.5 months based on physical characteristics and previous estimations. further evaluation of the uterus confirmed a long dorsolateral rent measuring 40cm and extending from the tip of the right uterine horn to near the uterine bifurcation (figure 5). the rent was cleaned of remaining placental tissue and two layers of modified utrecht pattern, using 2 polyglactin 910 was used to close the uterus. six liters of normal saline was used to lavage the uterine closure as well as the abdominal cavity, and a sterile orogastric tube placed on the floor of the abdomen was used to syphon a majority of the fluid from the abdomen. exploration of the remainder of the abdomen revealed no other abnormalities. the abdominal musculature and peritoneum were then closed using 2 polyglactin 910 in a simple continuous pattern in two layers. the skin was closed with 3 polymerized caprolactam using a ford’s interlocking pattern. balanced polyionic iv fluids were continued at 50 ml/kg/day as well as antibiotic and anti-inflammatory treatments of 1.1 mg/kg of flunixin meglumine iv bid, 11 mg/kg of ampicillin trihydrate im bid, and 27.5 mg/kg of sulfadimethoxine iv bid. the fetuses and placenta were submitted for necropsy with no fetal abnormalities found. outcome recovery was uneventful; the heifer was dismissed two days following surgery and was maintained on 22,000 iu/kg penicillin g im bid and 1 mg/kg of meloxicam po every other day for one week following discharge. uterine involution was monitored periodically by the referring veterinarian and mild adhesions to the pelvic floor and thickening of the right uterine horn were noted that have improved over time. the heifer was noted to resume normal estrous cycles and is enrolled as an embryo donor. clinical theriogenology • volume 7 number 2 • june 2015 110 discussion spontaneous pre-partum uterine rupture is a relatively rare event in cattle.1 in this case uterine rupture was not high on the initial list of differential diagnoses, but it was suggested by further diagnostic tests. the lack of resolution of abdominal distention indicated that problems could exist beyond the gastro-intestinal system. reports of uterine rupture in single calf pregnancies have been predominant in the literature.1,2 thirty-seven of the reported 39 uterine ruptures were reported in cows carrying a single fetus.1-5 two of the 39 cases involved twin fetuses.6,7 of the 39 cases of spontaneous uterine rupture, 27 occurred in nulliparous females three years of age or less.1,3,5,7 the remaining 12 animals that were older than three years ranged from four to eight years of age.1,3,5,7 as in the present case, a nutritional or gastrointestinal problem was the primary complaint in ten of the 39 reported cases.1,2,3,5 the remaining 29 cases were presented with abnormalities associated with the second stage of labor and/or dystocia.1,4,6,7 in previously reported cases survival of the fetus is greatly impacted by the timing of intervention relative to the occurrence of the rupture and age of the fetus at time of delivery.1 both fetuses were delivered dead in this case and it was estimated that rupture and subsequent death occurred greater than 48 hours prior to admission. previously published cases showed 30 of the 41 calves were dead at the time of delivery. of the 11 calves born alive, seven were singles and four were from twin pregnancies.1-7 survival of the dam was greatly impacted if the rupture occurred pre-partum and was not associated with concurrent dystocia. a major factor in outcome of the present case was that the cervix was completely closed at time of presentation and aided in maintaining a sterile abdominal environment, thus allowing time for intervention and subsequent normal healing following cesarean section. review of the literature showed that 13 of the 39 cows (33%) presented for uterine rupture died following treatment.1-7 all 13 dams that died had predisposing factors including uterine torsion, oversized fetus, and fetal malformations associated with dystocia that led to further debilitation and subsequent death or euthanasia. diagnostic evaluation of this patient revealed numerous abnormal findings that were helpful in diagnosis and subsequent treatment for spontaneous uterine rupture. trans-abdominal ultrasound with a 3.5 mhz convex probe revealed large amounts of fluid within the abdomen along with a region suspected of being free-floating placenta with cotyledons and attached placental chorioallantois. analysis of the abdominocentesis revealed a mild neutrophilic inflammation along with a mild pathologic hemorrhage. the existence of phagocytosed red blood cells within free abdominal fluid without evidence of gastrointestinal tract contents was suggestive of trauma or rupture of a non-gastrointestinal organ within the abdominal cavity. an additional significant finding was the protein level of the abdominal fluid which was 0.1 gm/dl (normal range 0.1-2.5 gm/dl) which is consistent with a transudative process associated with a large volume of fluid.8 abnormal thickening of uterus near the cervix was noted which also suggested of uterine pathology. early diagnosis and initiation of therapy in the case of uterine rupture is critical for survival of the dam and the fetus. due to the sterile environment of the abdomen, corrective surgery, removal of the fetuses and placenta and supportive care allowed a good prognosis for recovery and a guarded prognosis for future reproductive usage in this animal. it is unknown what caused the uterine rupture to occur, however, the history of bloat and removal of large amounts of gas and fluid from the rumen on-farm prior to referral is suggestive of a digestive problem leading to increased intra-abdominal pressure. carriage of twin fetuses in a single uterine horn may have also played a role, along with a recent history of being worked through a chute. as the fetuses and placenta were found to be normal at necropsy, the likelihood that the dam was experiencing hydrops prior to rupture of the uterus is low but could not be completely ruled out. it is also important to note that this event was not associated with parturition at the time of rupture. clinical theriogenology • volume 7 number 2 • june 2015111 learning points • while not a common occurrence, spontaneous pre-partum uterine rupture should be considered as a differential diagnosis during the third trimester of pregnancy in animals presented with vague clinical signs referable to the abdomen. • abdominal ultrasonography and abdominocentesis can be useful diagnostic aides in addition to palpation per rectum in determining if abdominal exploratory surgery is warranted to confirm rupture of the uterus. • if diagnosed early, the prognosis for life can be good and return to reproductive soundness is possible. references 1. pearson h, denny hr: spontaneous uterine rupture in cattle: a review of 26 cases. vet rec1975;97:240-244. 2. blenkhorn gw, adams nc: uterine rupture: a coincidence? vet rec 1974; 452. 3. drogt j: uterusruptuur bij het rund tijdens de dracht. tijdichr. diergeneeck 1976;101:674-675. 4. denooli pp: laparohysterectomy as a treatment for uterine rupture in the cow. can vet j 1982;23:37-38. 5. reddy b, khan n, yadgirkar g: a rare case of uterine rupture and escape of conceptus into the abdominal cavity in a pre-parturient buffalo-cow. vet rec 1966;79,540-541. 6. azawi oi, ali aj, naoman ut: uterine rupture with alive twins in a holstein frisian cow: a case report. iraqi j vet sci 2012;26:27-28. 7. oehme fw: hydrops allantois associated with twin pregnancy and uterine rupture in a cow. j am vet med assoc 1964;145,688-691. 8. anitha a, thangavel a: biochemical profile of ovine amniotic and allantoic fluids. tamilnadu j vet anim sci 2011;7:262-267. figure 1: image of the heifer taken by the owner prior to intervention. clinical theriogenology • volume 7 number 2 • june 2015 112 figure 2: abdominal ultrasound image taken in the lower right midsection of the abdomen (a = body wall; b = suspected cotyledon; c = free fluid in abdomen; d = suspected free-floating placenta). figure 3: free abdominal fluid draining from abdomen immediately upon entry into the peritoneal cavity during exploratory surgery. clinical theriogenology • volume 7 number 2 • june 2015113 figure 4: both calves and placentas after removal from abdomen. figure 5: image of rent in uterus after removal of calves. (editor’s note: the photographs in this manuscript are available in color in the online edition of clinical theriotenology.) clinical theriogenology • volume 7 number 2 • june 2015 114 2018: seminomas and an interstitial cell tumor in an 8 year old male husky seminomas and an interstitial cell tumor in an 8 year old male husky mary ontiveros, dave hanlon, alice anderson, fiona hollinshead matamata veterinary services, matamata, nz summary an eight year old male siberian husky was presented for semen collection and assessment. gross testicular asymmetry with an enlarged, firm, oval-shaped left testis and a small, atrophied right testis was found on palpation. there were no scrotal lesions or a history of any trauma involving the scrotal contents. the proportion of motile sperm in the ejaculate was 30%, and only 25% of his sperm were classified as morphologically normal. the predominant morphological defect present was proximal droplets. testicular ultrasonography revealed a large mottled mass of mixed echogenicity in the left testis and a smaller mass in the right testis. a routine, bilateral closed castration procedure was performed and the testes were submitted for histopathologic assessment. the mass in the left testis was confirmed to be a seminoma and the mass in the right testis was diagnosed as an interstitial cell tumor. an intra-tubular seminoma was microscopically present as well in the right testis. evidence of secondary testicular degeneration adjacent to both tumors was also observed. background this case report describes a classic seminoma with an incidental finding of an interstitial cell tumor (ict) in a dog. although the presence of multiple tumors is not uncommon in the dog, this particular case had a different form of seminoma in each testis, in addition to an ict. although this case is not a novel topic, this is the first published indepth case report of multiple testicular tumors in bilaterally descended testes in a dog. other thorough reports have been either retrospective histopathologic studies,1-6 lesions in retained testes,7-10 or single testicular tumors.11 there is significant variation in the literature in regards to the incidence rate of the different types of canine testicular tumors. the term “leydig cell tumor” can be used interchangeably with ict, but for the remainder of this paper, the tumor will be referred to as an ict. in a study by grieco et al, icts were the most commonly reported testicular tumors followed by seminomas.2 d’angelo et al reported that the seminoma was the most commonly diagnosed canine testicular tumor type followed by sertoli cell tumors.6 another retrospective study by liao et al found that seminomas were the most common testicular tumor diagnosed in dogs followed by icts.3 the reason for this variation may be due to geographic differences and also breed and genetic differences between the populations involved in these studies. it can be concluded from all these reports and previous studies that the seminoma, sertoli cell tumor, and interstitial cell tumor are the three most commonly diagnosed canine testicular neoplasms.24,6,12-14 seminomas originate from the spermatogenic cells of the seminiferous tubules. they are typically a soft tumor, measuring from one to ten cm in diameter.13 usually they are benign, but 5-10% can metastasize to inguinal, iliac, and sub-lumbar lymph nodes.13 other sites of metastasis include the lungs, liver, spleen, kidneys and pancreas with one case reported with aberrant metastases to the skin.11,13 seminomas in the dog are reported to be found more often in scrotal testes than retained testes, but in regards to retained testes, they are more commonly found abdominally than inguinally. on the cut surface, they are colored cream to pinkish gray to tan-colored.12 although an uncommon presentation, paraneoplastic syndromes of seminomas consist of progressive, non-pruritic alopecia with hyperpigmentation of the trunk, prostate disease, and non-insulin dependent diabetes mellitus.12,13 seminomas frequently begin as the intratubular form, in which the seminiferous tubules are overgrown with seminoma cells and multiple layers of neoplastic sertoli cells may stretch across the tubule. this was the presentation of the seminoma found in the right testis in our case study. they can also spread to the “diffuse form,” in which tumor cells are not confined to the seminiferous tubules and instead form broad sheets.4,15 this form of seminoma was found in the left testis of our case report. in human literature, seminomas are classified as “classical” or “spermatogenic” based on morphology, histochemistry, and clinical theriogenology • volume 10, number 2 • june 201897 immunohistochemistry findings. this classification system has not been uniformly adopted in the veterinary field but has been advocated as beneficial by some pathologists.1,15 canine seminomas tend to resemble the human “spermatogenic” seminoma in that they are often benign and originate from postpubertal germ cells, but they can also have the “classical” characteristics of cancerous in situ cells and inflammatory lymphocytic infiltrates, as seen in the left testis of this case.15 due to the incongruity of the human literature, this paper will refer to seminomas as intratubular or diffuse. interstitial cell tumors are derived from the interstitial cells adjacent to the seminiferous tubules in the testes. they are typically small, discrete (0.1-0.2 cm in diameter) masses that are difficult to palpate and are often an incidental finding. when palpation is possible, they are soft and nodular. they are typically yellow to brown in color. they are almost always benign.13,14 unlike some of the other testicular tumors, cryptorchidism is not a predisposing factor for icts. an associated paraneoplastic syndrome has been reported with the presence of icts. clinical signs seen are associated with hyperestrogenism (bone marrow suppression, pale mucus membranes, prolonged bleeding, bilateral symmetric flank alopecia) or hypertestosteronism but most often, clinical signs are absent.12 case presentation an eight year old maie siberian husky was presented for semen collection and assessment for artificial insemination at glenbred in matamata, new zealand. he was a reproductively proven dog, having sired a litter three years previously. on physical examination, he was bright, alert, and responsive with a body condition score of 5/9. he had pink mucous membranes with a capillary refill time of less than two seconds. skin and coat were clean and in good condition. chest auscultation was within normal limits. no enlarged peripheral lymph nodes were palpated. abdominal palpation and the rest of the physical examination were unremarkable. there was no evidence of paraneoplastic syndrome. on reproductive examination, a significantly enlarged left testis and a soft, smaller atrophied right testis were detected on palpation. the entire scrotal circumference measured nine cm. the left testis was large, firm in consistency, and oval-shaped. the left epididymal tail and spermatic cord were difficult to palpate. the right testis was soft in consistency and smaller in size compared to the left testis. a prominent but not enlarged right epididymal tail was palpated. there were no scrotal lesions or evidence of trauma. his non-erect penis and prepuce appeared anatomically normal with no evidence of inflammation, masses or trauma. manual collection of an ejaculate was performed using the open hand technique. the ejaculate was fractionated into the pre-sperm, sperm-rich and post-sperm fractions. the dog demonstrated a good libido with the presence of a bitch in standing heat. samples were taken from the spermrich (“raw”) fraction of the ejaculate immediately after collection for microscopic assessment. total motility was subjectively assessed on a warm stage under x100, x200 and x400 magnification. this evaluation was repeated after tris-based extender was added as well. sperm motility was classified as poor with only 30% of sperm in the ejaculate demonstrating progressive motility. a dry smear was made from a semen sample taken from the sperm-rich fraction and was stained using wright’s giemsa stain (diff-quik: new zealand veterinary pathology, hamilton nz). assessment under x1000 magnification with oil emersion and differential interference contrast (dic) found only 25% of sperm were classified as morphologically normal with 49% of the abnormal sperm having proximal droplets, 15% with coiled tails, 8% with detached heads, and 3% with distal midpiece reflex tail defects. less than five inflammatory cells (neutrophils) per high-powered field (hpf) were seen and no abnormal cells (round, sertoli, leydig, or epithelial cells) were detected. the sample volume was 1.5 ml with a concentration of 105 x 106 spermatozoa/ml for a total of only 157.5 million spermatozoa in the ejaculate. ultrasonographic examination of the scrotal contents was carried out in b-mode using an esaote mylabtm30 vet gold ultrasound with a variable frequency (9-3mhz) micro-convex transducer. sedation with acepromazine (0.01 mg/kg sq) and morphine (0.4 mg/kg sq) was given prior to the ultrasonographic examination. the smaller right testis dimensions were 4.22 cm x 1.62cm x 1.93cm. an ovoid mass with the dimensions 1.10 cm x 0.76 cm was visible within the testicular parenchyma at the caudal pole of the testis (figures 1a and 1b). the mass had smooth borders and was homogenous and clinical theriogenology • volume 10, number 2 • june 2018 98 hypoechoic compared to the surrounding normal homogenous testicular parenchyma. dilation of the vessels of the right pampiniform plexus was apparent. there were no obvious changes to the right epididymis. the larger left testis dimensions were 5.31 cm x 3.48 cm x 3.61 cm. a large mass with the dimensions 4.11 cm x 3.44 cm x 3.54 cm was visualized within the testis. its size was such that only a thin rim of normal homogenous appearing testicular tissue was present around the periphery of the mass (figures 2a and 2b). the mass had irregular borders and a mixed echogenicity but it was mostly hypoechoic. there was significant dilation of the vessels of the left pampiniform plexus. there were no apparent ultrasonographic changes to the left epididymis. an exfoliative cytology of the preputial mucosa was taken for cytological assessment. noncornified, nucleated squamous epithelial cells were seen. there was no cytological evidence of the presence of estrogen. measurements of plasma estrogen and inhibin concentrations were not carried out. thoracic and abdominal radiographs and bloodwork were not performed due to financial constraints and the reported low incidence of metastatic spread of testicular neoplasia in the dog.12-15 as both testes had detectable masses on ultrasonography the owner elected to carry out a bilateral castration. a closed castration procedure was performed, leaving the parietal vaginal tunic intact as to avoid potential seeding of the tumor and not incise through neoplastic tissue. a local incisional block of bupivacaine was injected and a subcutaneous meloxicam injection (0.2 mg/kg) was given. the dog recovered uneventfully from anesthesia. he was sent home on meloxicam 0.1 mg/kg po every 24 hours for pain management. differentials differentials for testicular asymmetry include neoplasia, trauma, inflammation, infection, spermatoceles, and sperm granuloma.12,13 after ultrasonographic examination of the testes, neoplasia was the most likely cause of the testicular asymmetry palpated with the most likely type of tumor present being a seminoma, sertoli cell tumor, and/or interstitial cell tumor. other tumors considered but less likely included benign epidermoid cysts, fibrosarcoma, hemangioma, neurofibrosarcoma, anaplastic carcinoma, gonadoblastoma, embryonal carcinoma, sarcoma, lymphoma, and granulosa cell tumor.12,13,15 treatment the treatment of choice for a canine testicular neoplasm is orchiectomy, which was performed in this case. a unilateral orchiectomy or hemicastration was considered as he was a valuable breeding dog.1214 however, the presence of multiple tumors affecting both testes, his older age, poor semen quality, and the degree of testicular degeneration present on ultrasound made hemi-castration a poor treatment option. bilateral orchiectomy is the treatment of choice in cases with bilateral neoplasia.12 however, unilateral orchiectomies have resulted in litters even with persistently low total sperm numbers ejaculated with the aid of breeding management and artificial reproductive technologies.12 chemotherapy is adjunct therapy for seminomas if metastasis is present, which is uncommon.12-15 while bilateral or unilateral orchiectomy is the treatment of choice,12-15 an area that potentially could be explored further for poor anesthetic candidates would be chemical ablation. intra-testicular injections exist currently for chemical castration16 and may provide an option in the future for testicular tumor treatment though an obvious concern would be leaving behind cancerous cells. outcome gross analysis of both the testes was performed immediately after surgical removal (figure 3). both testes were then sent to the pathology laboratory in a 10% formalin solution for histopathology. microscopically, the left testis contained a discrete, round, fleshy white mass measuring approximately 35 mm x 25 mm in diameter. within the testis was a discrete, expansile, partially encapsulated nodular proliferation of neoplastic round cells that formed sheets and lobules and were supported by fine, fibrovascular stroma. the cells had discrete cell borders and moderate amounts of eosinophilic cytoplasm and a central nucleus with stippled to vesicular chromatin and a single nucleolus. clinical theriogenology • volume 10, number 2 • june 201899 there were frequently multinucleated cells with up to five nuclei and scattered aggregates of lymphocytes. there was moderate anisocytosis and anisokaryosis and the mitotic rate ranged from 68/hpf. surrounding seminiferous tubules had reduced numbers of spermatids and there were rare spermatid multinucleated giant cells (figures 4a and 4b). the right testis contained a discrete, multilobulated, tan nodule within the proximal part of the testes that was approximately 10 x 7 mm in diameter. within the testis was a discrete, multinodular proliferation of neoplastic polygonal cells forming nests and cords, entrapping atrophic seminiferous tubules and supported by a fine, fibrovascular stroma. the cells had moderate to large amounts of pale eosinophilic granular to vacuolated cytoplasm and a central nucleus with stippled chromatin and a single nucleolus. there were occasional intranuclear cytoplasmic invaginations. there was mild to moderate anisocytosis and anisokaryosis and the mitotic rate was approximately 1/5 hpfs. adjacent to the neoplastic proliferation were a few, closely associated seminiferous tubules that were effaced by a population of neoplastic cells morphologically similar to those in the left testis. surrounding seminiferous tubules had reduced numbers of spermatids (figures 5a and 5b). a morphologic diagnosis of a seminoma in the left testis and an interstitial cell tumor with an intra-tubular seminoma in the right testis was reported. since surgery, the dog has recovered well with no postsurgical complications. discussion this case report highlights the importance of carrying out a complete diagnostic workup if a testicular tumor or tumors are suspected. the dog in this case report initially presented for semen collection for an artificial insemination, and the owner had hopes of future breeding. the owner had no previous concerns for the dog. when a tumor was suspected after testicular palpation and semen evaluation, serious consideration was given to carrying out a hemicastration procedure with the objective of restoring his inherent fertility. however, further diagnostics (testicular ultrasonography) revealed a non-palpable tumor present in the contralateral testis, leading to bilateral castration as the best treatment option for the dog’s future health combined with the poor prognosis for future fertility. had further investigation not been performed after finding the enlarged left testis, and removal of only the left testis by hemicastration was performed in an attempt to preserve fertility, there would have been considerable frustration and potential economic loss for the owner to find an ict detectable by ultrasonography as well as a microscopic seminoma present months later. although this case is not a novel topic, this is the first published in depth case report of multiple testicular tumors in bilaterally descended testes in a dog. canine testicular tumors are the second most common type of tumor to occur in the dog second to skin tumors.12 they are generally a disease of older dogs with different ages reported at the time of first diagnosis (mean age range: 9-11 years old; actual range: 2-19 years).12 testicular tumors occur more often in undescended testes and retained testes are 1013 times more likely to develop neoplasia.12,13 tumors can present unilaterally or bilaterally. the presence of multiple cell types as in our case is not uncommon with the occurrence of two or more tumor types reported in 11-35% of cases.4,12,13 a study by peters et al found a 30% incidence in apparently normal dogs that was not palpable on examination.4 often the only clinical sign associated with testicular neoplasia is testicular asymmetry. atrophy in the contralateral testis is common, due to negative hypothalamic-pituitary-testicular feedback from hormone production by the tumor of estrogens or androgens. furthermore, increased intrascrotal temperature, and pressure atrophy inflicted on the contralateral testis can result in asymmetry.12,13 with multiple potential causes of asymmetry, further diagnostic testing should be performed, especially if the owner is considering hemicastration as clearly demonstrated in this case study. learning points  palpate testes at every semen collection as owners are often unaware of any abnormalities.  in all cases of testicular asymmetry an ultrasonographic examination should be performed as neoplastic lesions may be undetectable by palpation alone. clinical theriogenology • volume 10, number 2 • june 2018 100  submit testes for histopathology as there may be multiple tumor types that are not appreciable on ultrasonography or grossly visible.  malignancy for canine testicular tumors is low.  if hemicastration is performed due to the value of the breeding stud, a recheck semen evaluation every 6 to 12 months should be performed to assess return to fertility given the potential for microscopic tumors to be present. references 1. grieco v, riccardi e, rondena m, et al: classical and spermatocytic seminoma in dog: histochemical and inmunohistochemical findings. j comp pathol 2007;137:41-46. 2. grieco v, riccardi e, greppi gf, et al: canine testicular tumours: a study on 232 dogs. j comp pathol 2008;138:8689. 3. liao at, chu py, yeh ls, et al: a 12-year retrospective study of canine testicular tumors. j vet med sci 2009;71:919923. 4. peters ma, de rooij dg, teerds kj, et al: spermatogenesis and testicular tumours in ageing dogs. j reprod fertil 2000;120:443-452. 5. masserdotti c, bonfanti u, de lorenzi d, et al: cytologic features of testicular tumours in dog. transbound emerg dis 2005;52:339-346. 6. d’angelo ar, vita s, marruchella g, et al: canine testicular tumours: a retrospective investigation in abruzzo and molise, italy. vet ital 2012;48:329-333. 7. herndon am, casal ml, jaques jt: testicular neoplasia in the retained testicles of an intersex male dog. j am anim hosp assoc 2012;48:118-124. 8. dugat dr, medici el, rochat mc, et al: an unusual case of metastatic seminoma in a dog. j am anim hosp assoc 2015;51:401-406. 9. aboh iku k, ndumari w, ternenge thaddaeus a, et al: sertoli cell tumor in a cryptorchid dog. j adv vet anim res 2017;4:394-398. 10. quartuccio m, marino g, garufi g, et al: sertoli cell tumors associated with feminizing syndrome and spermatic cord torsion in two cryptorchid dogs. j vet sci 2012;13:207-209. 11. takiguchi m, iida t, kudo t, et al: malignant seminoma with systemic metastases in a dog. j small anim pract 2001;42:360-362. 12. johnston sd, root kustritz mv, olson pns: canine and feline theriogenology. philadelphia: saunders; 2001. p. 313327. 13. feldman ec, nelson rw: canine and feline endocrinology and reproduction. 3rd ed. philadelphia: saunders; 2004, p. 964-967. 14. lopate c: the problem stud dog. vet clin north am small anim pract 2012;42:469-488. 15. agnew dw, maclachlan nj: tumors of genital tract. in: meuten dj, editor. tumors in domestic animals. 5th ed. ames: john wiley & son; 2017, p. 707-711. 16. kutzler m, wood a: non-surgical methods of contraception and sterilization. theriogenology 2006;66:514-525. clinical theriogenology • volume 10, number 2 • june 2018101 a     b   figure 1a. ultrasonographic image of the right testicle with a hypoechoic mass (ict) seen at the caudal pole of testicle. figure 1b. gross image of the cut surface of the interstitial cell tumor found in the right testicle. note the classic small, tan colored mass protruding from the interstitium of the testicle. a  ict clinical theriogenology • volume 10, number 2 • june 2018 102 a b figure 2a. ultrasonographic image of the left testicle which contains a large, irregular hypoechoic mass centrally (seminoma). only a small rim of normal testicular tissue remains (black arrows). figure 2b. gross image of the cut surface of the seminoma located in the left testicle. note the classic pink colored, large protruding mass encompassing the center of the testicle. a  seminoma  clinical theriogenology • volume 10, number 2 • june 2018103 figure 3. gross image of the two adjacent testicles. note that the left testicle containing the seminoma is larger than the right testicle containing the ict and microscopic seminoma.     clinical theriogenology • volume 10, number 2 • june 2018 104 a b   figure 4a. histologic image (h&e; ×4 magnification) of a sample taken from the left testicle which contained the seminoma. note the surrounding seminiferous tubules containing reduced numbers of spermatids. there is proliferation of neoplastic round cells forming sheets and lobules which are supported by fine, fibrovascular stroma. figure 4b. histologic image (h&e; ×40 magnification) of a sample taken from the left testicle containing the seminoma. note the eosinophilic cytoplasm and multinucleated cells. the cells have a central nucleus with stippled to vesicular chromatin and a single nucleolus.   clinical theriogenology • volume 10, number 2 • june 2018105 a   b   figure 5a. histologic image (h&e; ×4 magnification) of a sample from the right testicle containing the interstitial cell tumor and intra-tubular seminoma. note the unencapsulated, multinodular proliferation of neoplastic polygonal cells forming nests and cords, entrapping atrophic seminiferous tubules and supported by a fine, fibrovascular stroma. adjacent to the neoplastic proliferation were a few, closely associated seminiferous tubules (arrows) that were invaded by a population of neoplastic cells morphologically similar to those in the left testicle. figure 5b. histologic image (h&e; ×40 magnification) of a sample from the right testicle containing the interstitial cell tumor and intra-tubular seminoma. the neoplastic interstitial cells had moderate to large amounts of pale eosinophilic granular to vacuolated cytoplasm and a central nucleus with stippled chromatin and a single nucleolus. there were occasional intra-nuclear cytoplasmic invaginations. (editor’s note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 10, number 2 • june 2018 106 2018: nerve dysfunction in the penis of an angus bull nerve dysfunction in the penis of an angus bull college of veterinary medicine, auburn university, auburn, al a 4-year-old angus bull was presented for spiral deviation of the penis and inability to impregnate cows. at the end of the previous breeding season, the bull was removed from the breeding herd for seven months of sexual rest with no health concerns. a routine breeding soundness examination performed by the referring veterinarian revealed a corkscrew deviation of the penis during electroejaculation. the owner noticed during natural service attempts that the penis spiraled and coitus could not be achieved. the theriogenology service performed a breeding soundness examination. during electroejaculation, spiraling was observed and mild blushing was seen on the free portion of the penis just proximal to the glans penis. a test mating was observed and upon mounting the corkscrew deviation was noted. a cavernosagram was performed to evaluate blood flow within the penis. the results did not definitively indicate the presence of a shunt. a nerve stimulation test to evaluate nerve function in the penis determined no sensation to be present in the glans penis. even though the bull was able to achieve an erection, lack of sensation in the glans penis would not allow the bull to achieve intromission. the stimuli in the glans penis are conducted over the dorsal nerves of the penis to the pudendal nerves, which connect to the spinal cord and transfer stimulation to the brain. if 75% of the sensory fibers in the dorsal nerves are damaged, the bull usually cannot ejaculate.1 the best way to evaluate function of the dorsal nerve is to observe the bull successfully achieve intermission and ejaculation.1 intromission was not achieved by this bull and did not evoke action potentials nor sensory nerve or motor nerve conduction velocities as determined by the nerve stimulation test. this test provides consistent and highly reproducible measurement of sensory-system function of the penis.1 reference 1. wolfe df, moll hd: examination and special diagnostic procedures of the penis and prepuce. in: wolfe df, moll hd, editors. large animal urogenital surgery. baltimore: williams and wilkins; 1998. p. 221-231. clinical theriogenology • volume 10, number 3 • september 2018353 m. pelletier, m. edmondson, j. gard equine placentitis caused by stenotrophomonas maltophilia, a multiple drug resistant organism equine placentitis caused by stenotrophomonas maltophilia, a multiple drug resistant organism viviane gomes, fabio piero,a luis aguiar, amanda anderson, jennifer sones, carlos pinto department of veterinary clinical sciences, adepartment of pathobiological sciences louisiana state university, baton rouge, la bacterial placentitis, leading to late term abortions, premature parturitions and delivery of compromised foals, is often caused by opportunistic pathogens such as streptococcus equi subsp. zooepidemicus and escherichia coli. stenotrophomonas maltophilia, a free living, multiple drug resistant, non-fermentative, gram-negative bacillus, has emerged as an important human nosocomial pathogen, being mainly associated with respiratory infections and endocarditis in humans. s. maltophilia has been associated with chronic respiratory disease in 10 horses. in the reproductive tract, s. maltophilia has been isolated from a clinical and an experimental case of focal mucoid placentitis in mares affected by equine amnionitis and fetal loss syndrome, associated with ingestion of processionary caterpillars (ochrogaster lunifer) or their exoskeletons.1 a 10 year old thoroughbred mare failed to carry pregnancy past 7 months of gestation for 2 consecutive years. after the second abortion, the mare had a grade i endometrial biopsy, noninflammatory endometrial cytology and light growth of streptococcus sp. on uterine culture. the mare became pregnant by natural service after treatment with intrauterine infusion of amikacin. supplementation with long acting altrenogest (225 mg q 12 days, im) was performed throughout pregnancy. fetal heart rate was 105 beats per minute and the combined thickness of the uterus and placenta measured transrectally was 8.5 mm on the last pregnancy monitoring examination. twelve days later, the mare and her newborn filly were presented to our hospital 4 hours after premature delivery at 293 days of gestation. fetal membranes representing the uterine body and nonpregnant horn weighed ~ 23.7% of the filly’s body weight. chorioallantois of the pregnant uterine horn was retained. filly was severely compromised and was humanely euthanized. cervical star was grossly within normal limits. a friable mass of exudate (~ 30 x 40 cm) was present on the chorionic aspect of the chorioallantois, at the uterine body. the periphery of the mass was firm and green to grey, whereas the inner portion was yellow and soft. the chorion underneath the mass was avillous and yellow/pale. histopathologic findings included chronic, fibrosing, necrotizing allantochorionitis, with lymphocytes, plasma cells, neutrophils, and very abundant necrotic exudate; adenomatous hyperplasia of the allantois, with numerous cysts filled with neutrophils; and chronic peri funisitis, with neutrophilic, necrotic exudate. heavy growth of s. maltophilia was obtained on culture and myriads of this gram negative bacilli were seen using giemsa, steiner silver and hematoxylin eosin stain preparations. retained chorioallantois was expelled 3 days later following 2 uterine lavages and ecbolic therapy. the mare was treated with antibiotic, anti inflammatories and prophylactic cryotherapy for laminitis, and was discharged 4 days later with no complications. etiopathogenesis of placentitis induced by s. maltophilia and its relevance as an emerging pathogen in equine reproduction remains to be further elucidated. keywords: chorioallantois, nosocomial bacteria, multiple drug resistant organisms reference 1. todhunter, k. h. pathology of equine amnionitis and foetal loss: clinical and experimental studies. https://doi.org/10.14264/uql.2015.310 493 clinical theriogenology • volume 11, number 3 • september 2019 494clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2013: management of the pregnant doe: general caprine health management of the pregnant doe: general caprine health d.g. pugh college of veterinary medicine, auburn university, auburn, al management practices bio-security should be the first and most important part of a herd health program for goat health care maintenance. no new animals should be brought to the farm and introduced into the general population of the farm unless they first have undergone a complete examination and have been quarantined for a minimum of 30 days with no contact with the remainder of the flock. strict measures to minimize the introduction of diseases foreign to a farm must be observed. the dietary/nutrition program is critical for maintaining optimal health, productivity, and fecundity in a herd of goats. the body condition should be assessed on all farm animals on a bi-yearly to monthly basis. scores of 2.5 to 3.5 (on a 5-point system) are desirable. body condition scoring is the best tool for assessing long term energy adequacy. animals with optimal body condition score will more likely become pregnant, and be less likely to succumb to pregnancy toxemia. prevent over-conditioning and provide plenty of exercise for parturient does. obesity should be avoided as it is associated with both a greater incidence of dystocia, some forms of pregnancy toxemia, and increased production costs. the clinician should be aware that extra-label use of pharmaceuticals and vaccines in food animals is ‘frowned upon’ by the food and drug administration and united states department of agriculture, respectively. goats, whether used for meat, milk, pets, or fiber are considered food animals. all animals on the farm should be given yearly vaccinations and boosters for toxoids for clostridium perfringens type c and d and c. tetani. pregnant animals should be vaccinated 30 days before kidding. this will help protect the doe and ensures high levels of antibodies to protect the newborn kid. if the herd has a history of abortion and the causative agent has been confirmed, and if vaccines for those diseases exist, then pregnant animals should be vaccinated for those particular forms of abortion causing diseases one month before the start of breeding. bucks, yearlings, and other adults should receive annual boosters at the same time to streamline animal handling. kids from non-immunized dams should be vaccinated at one to three weeks and given a booster three to four weeks later. kids from immunized dams should be vaccinated at one to two months and given a booster three to four weeks later. multivalent clostridial vaccines, including those against blackleg, malignant edema, and bacillary hemoglobinuria, rabies and leptospirosis are occasionally used, but these are uncommon diseases in goats. vaccination to prevent such diseases may not economically justifiable when these diseases are endemic on certain farms the extra-label use of cow vaccines is commonly employed. vaccination sites should be chosen in order to minimize blemishes, lameness, and site reactions that require trimming at slaughter or poor performance in the show ring. subcutaneous injection behind the elbow or in the caudolateral neck region are preferred, with a third choice for injection being over the ribs. usually, one to two months prior to the breeding season, bucks used for breeding should be identified, examined to ensure good overall health, and a breeding soundness examination performed. all suspect bucks should be culled or removed from the farm, and only those found to be healthy and free of congenital defects, and those which pass the breeding soundness examination used. perform pregnancy diagnosis on all bred does 45 to 60 days after breeding to also enhance overall productivity. animals not found to be pregnant should be culled or removed from the herd/flock. animals confirmed pregnant should not be co-mingled with new or non-pregnant animals. keywords: goat herd health, parasite control, vaccination recommendations parasite control internal parasitism of meat goats is the most significant health risk affecting production and can result in serious economic losses for producers. financial losses are a result of decreases in growth, milk and fiber production, as well as increased treatment and prophylaxis costs. parasitic infections can also 479 clinical theriogenology • volume 5, number 4 • december 2013 result in death and/or loss of function. although a major concern in production animals, this can be quiet devastating for pet animal owners. feeding practices which increase stocking rates but also increase pasture contamination with nematode parasite eggs magnify internal parasite infection in grazing goats. care should be taken to minimize intake of infective nematode parasite larvae when feeding goats. animals with limited nutrient intake or those offered diets deficient in one or more nutrients are more likely to suffer losses from internal parasitism than those fed a balanced diet. meat goats infected with internal nematode parasites quickly contaminate pastures through their manure. others then become infected as they graze on the same pasture. small ruminant or camelid production systems that use grazing without pasture rotation, particularly in areas of high rainfall and ground moisture, are more likely to have infected animals. droughts, inclement weather or lack of adequate forage (or pasture) may result in increased animal concentration which can also equate to an increase in parasitism. because of the increase in parasite egg production and fecal contamination around late pregnancy/early postpostpartum, breeding herds usually experience more gastrointestinal parasite problems than mature non-breeding animals. this periparturient rise in nematode egg output in goat feces can readily lead to pasture contamination and an increased risk of infection in kids living on a heavily contaminated pasture. routine deworming 30 days prior to kidding and continued deworming through the birthing season for meat goats will help reduce the pasture contamination associated with this periparturient rise in egg shedding. fecal egg count should be monitored during this period. gastrointestinal parasites appear to have a negative effect on protein metabolism and, to a lesser extent, on energy metabolism and increase requirements of these nutrients in goats. increasing dietary protein (particularly proteins rich in sulfur-containing amino acids) intake will aid in overcoming some of the clinical signs seen with parasitism. protein quality appears to have a more significant affect than the quantity. supplemental soybean meal and energy can improve resilience. dietary supplementation appears is more effective when targeted (i.e., when specific nutrients are deficient and the goat’s requirements for those nutrients are greatest). given the complicated nature of parasite control and recent documentation of widespread resistance to deworming compounds in north america, meat goat producers must take a multi-pronged approach to parasite control: (1) employ husbandry and feeding practices that minimize parasite infection; (2) use novel deworming practices that maximize endectocide efficacy and minimize parasite resistance; (3) consult with the local veterinarian and/or a state agricultural extension specialist. strategic deworming strategic deworming is an effective control regimen if reinfection from pasture grazing is minimal. using a strategic program, producers should deworm animals: (1) just before they are placed on dry lot feeding prior to winter management; (2) during winter when freezing conditions kill infective parasite larvae, lowering the incidence of reinfection from pasture grazing; and/or (3) prior to the peripaturient rise in parasite eggs (one month pre-birthing). after deworming, meat goats should be moved, if possible, to a pasture with a low level of infective parasite larvae to help minimize the potential of reinfection. those pastures are typically used for small grain or hay production and are grazed by horses or cattle, llamas and alpacas. treating young animals at weaning and moving them to a “safe pasture” is also a form of strategic deworming. tactical deworming using a tactical program, a de-wormer is administered during the period of greatest pasture contamination. for example, animals are treated 10 to 14 days following a rain, especially if it follows a drought. tactical deworming can also be used in response to an increase in fecal egg counts. systems such as the mcmaster’s fecal flotation quantify the number of nematode parasite eggs per gram of feces, allowing producers to identify and address parasitic infections before they become serious. strategic and 480clinical theriogenology • volume 5, number 4 • december 2013 tactical regimens may also be combined, but doing so will result in a greater chance of recruiting anthelmintic resistance. suppressive deworming while suppressive deworming programs may be appropriate in certain situations and on some farms, many meat goat operations face other challenges as a result of using this regimen. suppressive programs call for deworming at regular intervals, usually two weeks to three months apart, which is expensive, labor intensive, and fails to use or take into account animals’ natural resistance to parasites. using these programs reduces the farm/flock’s natural refugia and enhances onset of anthelmintic resistance. if all animals are dewormed routinely without regard to weather or production status meat goats with some natural resistance to internal parasites cannot be identified, and parasite resistance to deworming compounds is magnified. with the exception of strategic and tactical deworming, routine deworming should be used cautiously, as it may result in animals building resistance to anthelmintics more rapidly. additional reading pugh dg, baird an: sheep and goat medicine, 2nd ed. philadelphia: elsevier; 2011. smith mc, sherman dm: goat medicine, 2nd ed. ames (ia): wiley-blackwell; 2009 481 clinical theriogenology • volume 5, number 4 • december 2013 482clinical theriogenology • volume 5, number 4 • december 2013 omniblank: factors associated with bulls not classified as satisfactory potential breeders factors associated with bulls not classified as satisfactory potential breeders nancy conkey,a chika okafor,b lew strickland,a,c brian whitlocka departments of alarge animal clinical sciences, and bbiomedical and diagnostic sciences, college of veterinary medicine cdepartment of animal science university of tennessee, knoxville, tn bull breeding soundness evaluation (bse) is an economically important component of cattle herd management. breeding soundness depends on adequate semen quality, physical soundness, and serving capacity. most extrinsic factors affecting bse classification are unknown. objective was to determine the prevalence of and factors associated with unsatisfactory and decision deferred bse classifications at our veterinary medical center. data were obtained on all bull bse conducted by clinical veterinarians between 2008 and 2018. data included date of evaluation, bull age, breed, body condition, physical abnormalities of feet and legs, reproductive tract, examining veterinarian, and previous bse. others were scrotal circumference (sc) and semen quality traits (progressive motility, percentage of normal, primary, and secondary morphologic defects). bulls were classified as satisfactory, decision deferred, or unsatisfactory. for analysis, bull bse classification was further categorized as pass (satisfactory) or failed (a combination of decision deferred and unsatisfactory). cochran-armitage test for trend was used to assess yearly trends in temporal distribution of apparent prevalence of bse failure. both univariable and multivariable logistic regression analyses were performed in sas9.4 to test possible associations among various factors and bse failure. of 1,405 records of bse recorded between 2008 and 2018, 30 were deleted due to missing information for year of examination, bse results, or attending veterinarian. out of 1,375 bulls, 1,049 (76.29%) were satisfactory and 326 (23.71%) failed. out of failed, 239 (73.31%) were because of abnormal sperm morphology and 28 (8.59%) because of sc. trend in the prevalence of failed bse significantly decreased from 18.6% in 2008 to 7.8% in 2017. in univariable logistic regression analysis, bulls were more likely to have a failed bse if they had reduced gross and progressive motility, abnormal sperm morphology, or previous bse. other significant factors were breed, body condition, examining veterinarian, year and quarter of the year bse was completed. both age and sc followed a cubic effect pattern. thus, the likelihood of failed bse was only evident in young and old bulls and in those with small and extremely large sc. bulls in between these extremes had reduced likelihood of failed bse. so far, this study has demonstrated the possible human bias of the examining veterinarian on the outcome of bull bse. if consistent in the multivariable analysis, this shows that bull bse should be performed by veterinarians with training in theriogenology. keywords: bull, breeding soundness evaluation 447 clinical theriogenology • volume 11, number 3 • september 2019 448clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2013: rhodococcus equi infections in foals rhodococcus equi infections in foals steeve giguère college of veterinary medicine, university of georgia, athens, ga introduction rhodococcus equi, a gram-positive facultative intracellular pathogen replicating in macrophages, is one of the most important causes of disease in foals between three weeks and six months of age, with most foals showing clinical signs before the age of four months. r. equi has also been increasingly recognized as an important cause of pneumonia in immunosuppressed people, especially those infected with human immunodeficiency virus. r. equi is considered to be a saprophytic inhabitant of the soil. although all horse farms are likely to be infected to various degrees with r. equi and antibody is widespread in the horse population, the clinical disease is enzootic and devastating on some farms, is sporadic on others, and is unrecognized on most. this probably reflects differences in environmental (temperature, dust, soil ph, soil type) and management conditions as well as differences in virulence of isolates. on enzootic farms the disease leads to significant financial loss because of the cost of therapy and occasional death of foals. although the virulence of r. equi likely depends on many determinants, isolates from pneumonic foals characteristically contain a large plasmid which encodes a gene responsible for the expression of a virulence associated protein (vapa) on the surface of the bacteria at temperatures > 34°c. foals experimentally infected with virulent (plasmid containing) r. equi develop severe pneumonia whereas plasmid cured derivatives are rapidly cleared and fail to induce lesions.1 inhalation of dust particles laden with virulent r. equi is the most important route of pneumonic infection in foals. ingestion of the organism is a significant route of exposure but does not lead to hematogenously acquired pneumonia unless the foal has multiple exposure to very large numbers of bacteria clinical manifestations the most common manifestation of r. equi infections in foals is a chronic suppurative bronchopneumonia with extensive abscessation. the slow spread of the lung infection combined with the remarkable ability of foals to compensate for the progressive loss of functional lung, make early clinical diagnosis difficult. early clinical signs often only consist of a mild fever or a slight increase in respiratory rate that may not be apparent unless foals are exercised or stressed by handling. as the disease progresses, clinical signs may include decreased appetite, lethargy, cough, fever, tachypnea, and labored breathing. nasal discharge is an inconsistent finding. because ultrasonographic screening for early detection has become routine practice at many farms endemic for pneumonia caused by r. equi, the most frequently recognized form of r. equi infection on those farms is a subclinical form in which foals develop ultrasonographic evidence of peripheral pulmonary consolidation or abscessation without manifesting clinical signs.2,3 on those farms, the cumulative frequency of sonographically visible areas of focal pulmonary consolidation or abscessation considerably exceeds the historical frequency of clinical pneumonia attributed to r. equi indicating that many subclinically affected foals might spontaneously recover without therapy. in two independent studies at endemic farms, 80% to 90% of foals with ultrasonographic lesion recovered without antimicrobial therapy.4,5 the proportion of such subclinically affected foals that progress to clinicallyapparent disease might vary by farm, geographical region, and age at which foals are examined. extrapulmonary manifestations of rhodococcal infections are common. in a retrospective study of 150 foals with r. equi infections, 111 (74%) had at least one of 39 extrapulmonary disorders.6 survival was significantly higher among foals without extrapulmonary disorders (32/39 [82%]) than among foals with extrapulmonary disorders (48/111 [43%]), but many such disorders were only recognized after death.6 intestinal lesions are present in approximately 50% of foals with r. equi pneumonia presented for necropsy.7 however, the majority of foals with r. equi pneumonia do not show clinical signs of intestinal disease. abdominal lesions may include ulcerative enterocolitis and typhlitis over the area of the peyer's patches, granulomatous or suppurative inflammation of the mesenteric and/or 537 clinical theriogenology • volume 5, number 4 • december 2013 colonic lymph nodes, or in some cases a single large abdominal abscess may be the only lesion.7 polysynovitis is present in approximately 25-30% of cases with r. equi infections. the degree of joint effusion is variable and, in most cases, lameness is mild or absent. cytological examination of the synovial fluid usually reveals a non-septic mononuclear pleocytosis and bacteriologic culture of the synovial fluid is negative.8 immune-mediated processes may also contribute to the development of uveitis, anemia, and thrombocytopenia in some foals infected with r. equi. bacteremic spread of the organism from the lungs or gastrointestinal tract may occasionally result in septic arthritis and, more commonly, osteomyelitis. however, foals can occasionally develop r. equi septic arthritis or osteomyelitis without apparent lung involvement or other source of infection. r. equi vertebral osteomyelitis or diskospondylitis resulting in spinal cord compression has also been reported. other rare extrapulmonary manifestations of r. equi infections in foals include panophthalmitis, guttural pouch empyema, sinusitis, pericarditis, nephritis, and hepatic, renal, and intracranial abscessation.6 diagnosis the distinction between lower respiratory tract infections caused by r. equi and that caused by other pathogens is problematic especially at farms without previous history of r. equi infections. diagnostic tests, including white blood cell concentration, measurement of fibrinogen concentrations, ultrasonography, and radiography, may help raise the degree of suspicion that pneumonia in a given foal may be caused by r. equi rather than by another microorganism. however, the definitive diagnosis of bronchopneumonia caused by r. equi should be based on bacteriologic culture or amplification of the vapa gene by polymerase chain reaction (pcr) from a tracheobronchial aspirate (tba) obtained from a foal with: 1) clinical signs of lower respiratory tract disease, 2) cytological evidence of septic airway inflammation, and/or 3) radiographic or ultrasonographic evidence of bronchopneumonia. amplification of vapa by pcr may be done in conjunction with, but should not replace bacterial culture because it does not permit identification of other bacterial pathogens and in vitro antimicrobial susceptibility testing of r. equi isolates.9 the definitive diagnosis of extrapulmonary infections (e.g. abdominal abscess, osteomyelitis) caused by r. equi must rely on bacteriologic culture or pcr amplification of vapa from samples from the site of infection. the diagnosis of extrapulmonary disorders from sites at which r. equi cannot be detected (e.g. uveitis or polysynovitis) should be based on isolation of r. equi from a tba or other primary sites of infection. the diagnosis of enterocolitis caused by r. equi is problematic because isolation of r. equi from feces cannot be taken as evidence of enterocolitis caused by r. equi.9 hyperfibrinogenemia is the most consistent laboratory finding in foals with r. equi pneumonia, although rare cases may have normal fibrinogen concentrations. neutrophilic leukocytosis with or without monocytosis is also common. one study showed significantly higher fibrinogen concentrations and white blood cell counts (wbc) in non-survivors than in survivors whereas other studies showed no difference between the 2 groups. in one study, wbc >20,000 cells/µl, fibrinogen concentration >700 mg/dl, and evidence of pulmonary abscessation were more likely to be found in foals with pneumonia caused by r. equi than in foals with pneumonia caused by other bacteria.10 however, there is a considerable overlap in distributions, which precludes the use of fibrinogen concentrations and wbc for diagnosis or prognosis for an individual foal. thoracic radiography is useful in evaluating the severity of pneumonia and in assessing response to therapy. a prominent alveolar pattern characterized by ill-defined regional consolidation is the most common radiographic abnormality. the consolidated lesions are often seen as more discrete nodular and cavitary lesions consistent with pulmonary abscessation. although non-survivors tend to have more severe radiographic lesions than survivors, many survivors have very severe radiographic lesions thus radiographs should not be used as the sole criterion for prognostication and euthanasia.11 ultrasonography is a helpful diagnostic tool when lung involvement includes peripheral areas but may not be as useful as radiography to evaluate the full extent of lung lesions since abscesses with overlying aerated lung will not be detected. however, in most horses and foals with pulmonary abscessation the periphery of the lung is affected, enabling the ultrasonographer to successfully image some of the abscesses. early ultrasonographic lesions are non-specific and may only include irregularities of the 538clinical theriogenology • volume 5, number 4 • december 2013 pleural surface. these lesions may progress to form focal areas of consolidation of various sizes. in more chronic cases, well circumscribed, encapsulated abscesses can be detected. ultrasonography is very useful in evaluating the severity of pneumonia and in assessing response to therapy especially for equine practitioners who do not have access to thoracic radiography. ultrasonography is also a useful tool for detection of some abdominal abscesses and for screening for r. equi-infected foals on farms where the disease is endemic (see section on control). ultrasonographic or radiographic detection of lung abscesses raises the degree of suspicion that pneumonia in a given foal is caused by r. equi. however, detection of pulmonary abscesses, while commonly used as a screening test (see below), is not a definitive diagnostic test. independent studies evaluating the performance of serological tests available for diagnosis of infection caused by r. equi at endemic farms have demonstrated these tests have either low sensitivity, low specificity, or both.12-15 improving either sensitivity or specificity of elisa assays by changing the cut-off value of the tests could only be done to the detriment of the other. the presence of antibodies indicates exposure, subclinical infection, or maternal transfer of antibodies but it does not necessarily indicate infection leading to clinical disease. the current state of knowledge precludes serology being used as a diagnostic test for r. equi pneumonia. treatment a wide variety of antimicrobial agents are active against r. equi in vitro. however, because r. equi is a facultative intracellular pathogen surviving and replicating in macrophages and therefore causes granulomatous lesions with thick caseous material, many of these drugs are ineffective in vivo. for example, in one study all 17 foals with r. equi pneumonia treated with the combination of penicillin and gentamicin died despite the fact that all isolates were sensitive to gentamicin.8 the combination of rifampin and erythromycin became the treatment of choice in the 1980s and has dramatically reduced foal mortality since its introduction. in recent years, clarithromycin or azithromycin, two newer generation macrolides, often replace erythromycin in the combination with rifampin. macrolides and rifampin are highly active against r. equi in vitro but only exert bacteriostatic activity. of the three macrolides listed above clarithromycin is the most active against r. equi in vitro. the combination of a macrolide and rifampin is synergistic both in vitro and in vivo and the use of the two classes of drugs in combination reduces the likelihood of r. equi resistance to either drug. rifampin and macrolides are lipid soluble, allowing them to penetrate cell membranes and caseous material. advantages of azithromycin and clarithromycin over erythromycin in foals include enhanced oral bioavailability, prolonged half-lives, and much higher concentrations in bronchoalveolar cells and pulmonary epithelial lining fluid.16 these properties of the newer generation macrolides contribute to their lower dosages and longer dosing intervals. concentrations of clarithromycin in pulmonary epithelial lining fluid and bronchoalveolar cells of foals at steady state are considerably higher than concentrations reported following daily administration of azithromycin to foals. however, clarithromycin concentrations at these sites decrease rapidly, whereas the release of azithromycin from cells is much slower, resulting in sustained concentrations of azithromycin in tissues for days following discontinuation of therapy. in a retrospective study, the combination clarithromycin-rifampin was significantly more effective than erythromycin-rifampin or azithromycin-rifampin, especially in foals with severe radiographic lesions.17 although well-tolerated by most foals, macrolides commonly cause diarrhea. most of the time the diarrhea is self-limiting and does not necessitate cessation of therapy but affected foals should be monitored carefully because some may develop severe diarrhea, leading to dehydration and electrolyte loss that necessitate intensive fluid therapy and cessation of oral macrolides. the incidence of diarrhea in foals treated with erythromycin-rifampin has ranged between 17 and 36%. in most cases, diarrhea was mild and self-limiting. in the same study, the incidence of severe diarrhea necessitating administration of iv fluids was not significantly different between groups of foals treated with azithromycin-rifampin, clarithromycin-rifampin or erythromycin-rifampin. during surges of very hot weather an idiosyncratic reaction characterized by severe hyperthermia and tachypnea has been described in foals treated with erythromycin. anecdotal reports suggest that these reactions may occasionally occur with newer 539 clinical theriogenology • volume 5, number 4 • december 2013 macrolides as well. administration of antipyretic drugs and placing the foal in a cool environment will treat this problem. severe enterocolitis has also been reported in mares whose foals are being treated with erythromycin, presumably due to disruption of the mare's normal colonic microflora following ingestion of small amounts of active drug during coprophagia or from contamination of feeders or water buckets with drug present on the foal's muzzle. prognosis prior to the introduction of the combination erythromycin-rifampin as the recommended treatment, the prognosis of r. equi pneumonia was poor with reported mortality rate as high as 80%. using erythromycin and rifampin a successful outcome (as assessed by survival) in 50 (88%) of 57 foals with confirmed r. equi pneumonia has been reported. however, until recently there was no information on the impact of r. equi infections on future athletic performance. recently, a large collaborative study involving several major veterinary hospitals was conducted in order to definitively assess the influence of prior r. equi pneumonia on racing performance. the records of 115 foals (49 thoroughbreds [tb] and 66 standardbreds [sb]) that had chest radiographs and were diagnosed with r. equi pneumonia based on culture of a tba were reviewed.18 all cases were treated with erythromycin and rifampin between 1984 and 1994. the survival rate was significantly higher in sb (80%) than in tb (61%). death was more likely in foals presented with respiratory distress and the non-survivors had a higher radiographic score on admission than survivors. of the survivors 54% (for both sb and tb) had at least one racing start as opposed to 65% for the control population suggesting that horses contracting r. equi pneumonia as foals are slightly less likely to race. however, those foals that raced performed as well as expected. prevention screening r. equi pneumonia is often not recognized until it is well advanced and, therefore, difficult to treat. even severely affected foals may appear to suckle and behave normally to a casual observer. the rationale for screening is the assumption that detecting foals in the early stages of disease along with appropriate treatment of affected foals will improve outcome. it is important to emphasize that screening methods are not diagnostic tests. a useful screening test is one in which the probability of disease is high with a positive test result (high positive predictive value) and very low with a negative test result (high negative predictive value). the higher the prevalence of disease at a given farm, the higher the positive predictive value of a given test will be. therefore, depending on the prevalence of r. equi infections on a given farm, a positive result of a screening test could be a basis to perform a diagnostic test (low to moderate prevalence) or to initiate therapy (high prevalence). a variety of screening techniques performed serially have been described, including visual inspection of foals for clinical signs of pneumonia, monitoring rectal temperatures, hematological parameters, serology, and thoracic imaging using either radiography or ultrasonography, with empiric recommendation that screening begin around three weeks of age. systematic comparisons of these tests have not been performed. thus, a specific recommendation for any particular screening test cannot be made, and it is likely that the optimal approach for screening may vary among farms on the basis of cumulative incidence of disease, resources available for control and prevention, and preferences of the attending veterinarian(s) and farm management. over the past decade, control of r. equi infections at many farms where the disease is endemic has relied on early detection of subclinical pulmonary disease using thoracic ultrasonography and initiation of treatment with antimicrobial agents prior to development of clinical signs.2,3,19 ultrasonography of the chest offers several advantages over other screening tests: 1) results are specific for the presence of pulmonary pathology; 2) the procedure can be performed relatively quickly for an individual foal; 3) results are available immediately; and 4) the procedure may be more sensitive than radiography for detecting lesions in their early stages of development or in certain regions where soft tissue structures are superimposed over regions of the lung. although controlled studies are lacking 540clinical theriogenology • volume 5, number 4 • december 2013 periodic ultrasonography of the chest appears to have decreased mortality due to r. equi pneumonia at some farms. 2,3,19 however, recent double blinded randomized placebo controlled studies documented that approximately 88% of foals with small pulmonary lesions (sum of lesion diameters [or abscess score] of 1-10 cm) recover without antimicrobial therapy.4,20 in addition, antimicrobial treatment of foals with small ultrasonographic lesions did not significantly hasten lesion resolution compared to administration of a placebo.4,20 the use of ultrasonography for screening to detect r. equi pneumonia was recently evaluated at an endemic farm with personnel and veterinarians blinded to screening results.5 of 270 foals enrolled in the study, 216 (80%) developed sonographically visible pulmonary consolidation whereas only 17% of foals developed clinically apparent r. equi pneumonia.5 pulmonary lesions resolved without clinically apparent illness or antimicrobial therapy in 79% of foals with ultrasonographic lesions.5 in the aforementioned study, the cumulative sensitivity of ultrasonography was very good (89%) but cumulative specificity was low (62%).5 because it is impossible to know which specific foals might recover spontaneously from subclinical disease, and because r. equi infections can cause severe disease, many breeding farms elect to treat all foals with ultrasonographic lesions. this approach has resulted in an increased number of foals treated for presumptive r. equi pneumonia. the temporal association between this widespread use of macrolides and rifampin as a result of ultrasonographic screening and a perceived increase in the frequency of detection of resistant isolates in the last decade21 suggest that this practice may not be innocuous. emergence of widespread macrolideand rifampin-resistance at a farm after widespread use of these drugs was instituted as part of an ultrasonographic screening program has been documented.22 on that particular farm, 20%-40% of r. equi isolates from pneumonic foals were resistant to macrolides and rifampin.22 currently, it is unknown if macrolideand rifampinresistance is widespread on many horse farms relying on mass antimicrobial therapy of foals with ultrasonographic lesions or if it is an isolated problem. in addition to the cost and risk for selection for resistant bacteria, unnecessary mass antimicrobial therapy of foals with pulmonary lesions might lead to development of life threatening adverse reactions (e.g. diarrhea or hyperthermia) in some foals. the spontaneous resolution of ultrasonographic lesions in a large proportion of foals with ultrasonographic lesions,4,5,20 combined with the apparent increase in macrolideand rifampin-resistance at some farms22 support the need to stop the practice of mass macrolide treatment of all subclinically affected foals with ultrasonographic lesions. the goal should be to more accurately identify, of the many subclinically infected foals, which few are likely to go on to develop disease and hence require treatment. the degree of severity and the number of ultrasonographic lesions that warrant therapy are unknown and may vary by farm, geographical region, and age at which lesions are detected. additional studies are needed to establish better criteria to determine the need for therapy in subclinically affected foals and to better quantify the risks versus benefits of treating foals with subclinical ultrasonographic lesions. passive immunization intravenous administration of hyperimmune plasma (hip) obtained from horses vaccinated against r. equi using various antigens has generally proved effective in significantly reducing the severity of r. equi pneumonia in foals following experimental challenge. however, studies evaluating the efficacy of various hip preparations under field conditions have given equivocal results. although the data are conflicting and not all trials have shown a statistically significant reduction in the cumulative incidence of r. equi pneumonia, five of seven studies have demonstrated reduction of relative risk, suggesting some benefit of hip.23 use of hip licensed as an aid in the control of r. equi pneumonia is recommended (rather than plasma simply obtained from horses hyperimmunized against r. equi) because licensure ensures standard of potency, purity, and safety. currently, there is insufficient information to recommend one brand of licensed antibody product over another. the optimal amount of plasma to be transfused and the optimal age at which transfusion should occur remain to be determined. administration of hip nine days after aerosol infection of foals with r. equi did not confer protection,24 suggesting that administration of hip prior to infection is important. 541 clinical theriogenology • volume 5, number 4 • december 2013 because of evidence that many foals become infected early in life, it is commonly recommended that foals receive transfusion of at leastone liter of hip no later than the second day of life. because early administration may result in the decline of passively transferred antibody to a non-protective level at a time when foals are still susceptible to r. equi and when environmental challenge is high, it is common practice to administer a second dose of hip at two to four weeks of age. transfusion of hip is not completely effective and therefore does not eliminate the need for careful monitoring of foals at risk. in addition to being incompletely effective, transfusion of hip carries some risk to foals, both in terms of trauma that may occur during handling and adverse reactions to transfusions. the process is also timeand labor-intensive, and expensive. the cost effectiveness of transfusion depends on the value of the foals and the prevalence of disease at a given farm. references 1. gigu�re s, hondalus mk, yager ja, et al: role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein a in intracellular survival and virulence of rhodococcus equi. infect immun 1999;67:3548-3557. 2. slovis nm, mccracken jl, mundy g: how to use thoracic ultrasound to screen foals for rhodococcus equi at affected farms. proc annu conv am assoc equine pract 2005. p. 274-278. 3. venner m, kerth r, klug e: evaluation of tulathromycin in the treatment of pulmonary abscesses in foals. vet j 2007;174:418-421. 4. venner m, astheimer k, lammer m, et al: efficacy of mass antimicrobial treatment of foals with subclinical pulmonary abscesses associated with rhodococcus equi. j vet intern med 2013;27:171-176. 5. chaffin mk, cohen nd, blodgett gp, et al: evaluation of ultrasonographic screening methods for early detection of rhodococcus equi pneumonia in foals. j equine vet sci 2012;32:s20-s21. 6. reuss sm, chaffin mk, cohen nd: extrapulmonary disorders associated with rhodococcus equi infection in foals: 150 cases (1987-2007). j am vet med assoc 2009;235:855-863. 7. zink mc, yager ja, smart nl: corynebacterium equi infections in horses, 1958-1984: a review of 131 cases. can j vet res 1986;27:213-217. 8. sweeney cr, sweeney rw, divers tj: rhodococcus equi pneumonia in 48 foals: response to antimicrobial therapy. vet microbiol 1987;14:329-336. 9. gigu�re s, cohen nd, keith cm, et al: diagnosis, treatment, control, and prevention of infections caused by rhodococcus equi in foals. j vet intern med 2011;25:1209-1220. 10. leclere m, magdesian kg, kass ph, et al: comparison of the clinical, microbiological, radiological and haematological features of foals with pneumonia caused by rhodococcus equi and other bacteria. vet j 2011;187:109112. 11. gigu�re s, roberts gd: association between radiographic pattern and outcome in foals with pneumonia caused by rhodococcus equi. vet radiol ultrasound 2012;53:601-604. 12. gigu�re s, hernandez j, gaskin jm, et al: evaluation of wbc concentration, plasma fibrinogen concentration, and an agar gel immunodiffusion test for early identification of foals with rhodococcus equi pneumonia. j am vet med assoc 2003;222:775-781. 13. gigu�re s, hernandez j, gaskin j, et al: performance of five serological assays for diagnosis of rhodococcus equi pneumonia in foals. clin diagn lab immunol 2003;10:241-245. 14. phumoonna t, muscatello g, chicken c, et al: clinical evaluation of a peptide-elisa based upon n-terminal b-cell epitope of the vapa protein for diagnosis of rhodococcus equi pneumonia in foals. j vet med b infect dis vet public health 2006;53:126-132. 15. martens rj, cohen nd, chaffin mk, et al: evaluation of 5 serologic assays to detect rhodococcus equi pneumonia in foals. j am vet med assoc 2002;221:825-833. 16. suarez-mier g, gigu�re s, lee ea: pulmonary disposition of erythromycin, azithromycin, and clarithromycin in foals. j vet pharmacol ther 2007;30:109-115. 17. gigu�re s, jacks s, roberts gd, et al. retrospective comparison of azithromycin, clarithromycin, and erythromycin for the treatment of foals with rhodococcus equi pneumonia. j vet intern med 2004;18:568-573. 18. ainsworth dm, eicker sw, yeagar ae, et al. associations between physical examination, laboratory, and radiographic findings and outcome and subsequent racing performance of foals with rhodococcus equi infection: 115 cases (1984-1992). j am vet med assoc 1998;213:510-515. 19. mccracken jl, slovis nm. use of thoracic ultrasound for the prevention of rhodococcus equi pneumonia on endemic farms. proc am assoc equine pract 2009;55:38-44. 20. venner m, rodiger a, laemmer m, et al. failure of antimicrobial therapy to accelerate spontaneous healing of subclinical pulmonary abscesses on a farm with endemic infections caused by rhodococcus equi. vet j 2012;192:293298. 542clinical theriogenology • volume 5, number 4 • december 2013 21. gigu�re s, lee e, williams e, et al. determination of the prevalence of antimicrobial resistance to macrolide antimicrobials or rifampin in rhodococcus equi isolates and treatment outcome in foals infected with antimicrobialresistant isolates of r equi. j am vet med assoc 2010;237:74-81. 22. burton aj, gigu�re s, sturgill tl, et al. macrolideand rifampin-resistant rhodococcus equi on horse breeding farm, kentucky, usa. emerg infect dis 2013;19:282-285. 23. giguere s, cohen nd, keith cm, et al. diagnosis, treatment, control, and prevention of infections caused by rhodococcus equi in foals. j vet intern med 2011;25:1209-1220. 24. chaffin mk, martins r.j., martens j.g. therapeutic effects of immune plasma in foals with rhodococcus equi pneumonia. equine vet j 1991;12 (suppl):23-29. 543 clinical theriogenology • volume 5, number 4 • december 2013 544clinical theriogenology • volume 5, number 4 • december 2013 omniblank: 2013: current genomic applications for improved reproductive performance in beef and dairy current genomic applications for improved reproductive performance in beef and dairy kent j. andersen, jason b. osterstock zoetis centennial, co abstract genetic improvement in beef and dairy cattle reproductive traits has historically been hampered by the availability and accuracy of applicable genetic predictions. reproductive trait phenotypes are generally time-consuming to measure, categorical and/or threshold in nature, often are not collected in the context of inventory-based performance recording systems, and typically of lower relative heritability. introduction of the illumina® bovine snp50 beadchip, and subsequently other related genotyping platforms, has provided useful marker information for inclusion in beef and dairy genetic evaluations and performance programs. increasingly, marker information is used to authenticate recorded pedigree information or assign parentage, enhance the non-parent accuracy of expected progeny differences (epd) and predicted transmitting abilities (pta), derive and manage haplotypes affecting fertility, as well as help manage inbreeding. in angus seedstock, genomic-enhanced (ge) epd and/or genomic predictions for the following reproductive traits are computed weekly by angus genetics inc. (agi): calving ease direct (ced), scrotal circumference (sc), heifer pregnancy (hp), and calving ease maternal (cem). the average ge accuracy values associated with angus non-parent ge-epd for ced and sc are .31 to .36, respectively. in holsteins, genomic-enhanced pta (gpta) computed by the council on dairy cattle breeding (cdcb) are available for the following major reproductive traits and indexes: daughter pregnancy rate (dpr), productive life (pl), sire calving ease (sce), daughter calving ease (dce), and net merit (nm$). the reliability values for non-parent dairy reproductive trait gpta range from .49 to .66 for tested holstein heifers, as compared to .23 to .28 for untested animals. enhanced accuracy and reliability values enable increased response to selection and commercial productivity. keywords: angus, holstein, genomics, accuracy, reliability introduction notable production and economic losses are incurred across the beef and dairy industries due to females with untimely or failed conception and/or calving difficulty. in beef cattle, the calving percentage ─ proportion of exposed females that calve ─ is documented as 91.5 percent.1 in beef herds with 200 or more cows, pregnancy status and other reproductive problems accounted for 47.9 percent of cow culling decisions, and 20.0 percent of cull cows were sold because of reproductive failure at less than five years of age.1 among beef heifers and cows, 11.6 and 4.3 percent, respectively, required some level assistance during calving.1 reproductive problems were the highest ranking category of reasons why dairy cows were permanently culled, and accounted for 26.3 percent of removals.2 almost one in five (17.2%) dairy calves required assistance during delivery, and 6.5% were either born dead or died prior to 48 hours of age.2 both environmental and genetic factors contribute to reproductive outcomes in beef and dairy cattle. increasingly, genomic technology is available to enhance the dependability of genetic predictions for reproductive and calving performance, to help reduce associated economic losses. since shortly after introduction of the illumina® bovinesnp50 genotyping beadchip3 in 2008, leading beef and dairy cattle breed associations and genetic evaluation service providers have incorporated marker information into their genetic evaluations and performance programs. specifically, of the various beef and dairy breeds, such integration of genomic information is most advanced and has been of most commercial impact in angus and holstein, respectively. while initial use of genomicenhanced breeding information was centered on seedstock, there is now growing adoption by commercial users of angus genetics and dairy producers with non-registered holsteins. the purpose of this paper is to describe current use of marker information to improve the accuracy of selection, mating and marketing decisions for improved reproductive performance and calving ease in these breeds. 473 clinical theriogenology • volume 5, number 4 • december 2013 materials and methods collaboration among respective breed associations, genetic evaluation service providers, research and development personnel and genomic companies was and remains the key to unlocking the benefits of genomic technology. cooperation among these entities was essential for appropriate technical integration of evolving marker information, along with continuously accumulating pedigree and performance data, into the existing beef and dairy genetic evaluation infrastructure. this integration into evaluation systems used and trusted by breeders and commercial producers was critical for adoption. while the illumina® bovinesnp50 genotyping beadchip that included just over 54,000 markers (typically referred to as 50k) was the foundational technology from which current genomic-enhanced predictions were developed, other lower density commercial genotyping platforms ranging from roughly 3,000 to 20,000 markers are now widely used in the dairy industry, particularly as applied to females that are less influential in the population. a process called imputation is then employed, whereby lower density marker information from tested animals is used to effectively infer their unknown genotypes to the higher 50k density. the impact of this strategy is that the costs of acquiring genotypes can be reduced while still affording the benefits of higher density genomic data; namely, greater accuracy of genomic predictions. as of july 29, 2013, across all dairy breeds and genotyping platforms, marker information from 439,528 animals contributed to the most recent dairy evaluations, with holstein comprising 382,386 head of that total.4 in contrast, while the total number of beef animals commercially genotyped across major breeds on comparable platforms is not documented, approximately 35,000 angus animals are known to have been commercially genotyped on the 50k platform. there are a number of different methods currently used to integrate genomic information into beef and dairy cattle genetic evaluations. in angus, externally calibrated molecular breeding values (mbv) are incorporated as correlated traits. the higher the correlation between the mbv for a given trait and performance phenotypes, the greater the boost in accuracy associated with ge-epd versus traditional epd. in dairy genetic evaluations including holstein, marker genotypes are primarily used along with recorded and verified parentage and production data to more precisely quantify pedigree relationships and gene sampling in the computation of genomic predicted transmitting abilities (gpta). it follows that a valuable feature of genomic testing in beef and dairy cattle includes the verification or correction of recorded parentage, as well as the assignment of candidate parents, as is the case in multiple-sire breeding schemes. as well, primarily for more informed mating decisions, marker information is used in dairy to quantify inbreeding and assess carrier status for important haplotypes affecting fertility. results and discussion angus genetics inc. computes ge-epd on a weekly basis for 14 traits, including the reproductive measures ced and sc5 (table 1). the correlations between mbv from 50k and expressed performance for ced and sc, estimated and used in genetic evaluation by agi, are .61 and .73, respectively. these correlations indicate that 37 percent of the additive genetic variation for ced and 45 percent for sc, are explained by the marker information. when 50k marker information is incorporated into ge-epd along with verified parentage, the average accuracy achieved in non-parent animals is .31 and .36 for ced and sc, respectively. in contrast, animals with only pedigree information are assigned an accuracy of .05 in the genetic evaluation conducted by agi. to help angus seedstock producers and commercial users of angus genetics better understand the increased accuracy due to integration of 50k information and verified parentage, agi and zoetis approximated the number of progeny with associated performance information in genetic evaluation that would be required to achieve equivalent accuracy.6 the number of progeny equivalents associated with the accuracy of non-parent ge-epds from hd 50k tested animals for ced and sc was approximated to be 21 and 11, progeny respectively. it follows that this added accuracy from 50k marker information is roughly equivalent to a typical yearling bull’s first calf crop with calving ease score information and sons with yearling scrotal circumference data incorporated into traditional epds. 474clinical theriogenology • volume 5, number 4 • december 2013 in addition to ge-epds for ced and sc, genomic predictions for hp and cem were developed through collaborations between agi and genomic companies, and are provided for 50k tested animals. genomic predictions for these traits are expressed as percent ranks, benchmarked against tested angus reference populations. lower percent ranks indicate more desirable genetic merit for likelihood of successful hp and unassisted cem. eventually, it is anticipated that the epd that are also available for these traits will be genomic enhanced. in the meantime, selection of young animals for improved hp and cem must be based on pedigree-based epds and separate genomic percent ranks for these traits. genetic evaluations that incorporate genomic information for dairy cattle are provided on a monthly basis by the cdcb for the following reproductive and calving traits: pl, hcr, cow conception rate (ccr), daughter pregnancy rate (dpr), sire calving ease (sce), daughter calving ease (dce), sire still birth (ssb), daughter still birth (dsb), and sire conception rate (scr). along with predictions for other economically relevant production, health and type traits, pta for these traits also contribute to a variety of bio-economic selection indexes, including: nm$, cheese merit (cm$), fluid merit (fm$) and calving ability (ca$). the usda-animal improvement programs laboratory routinely provides comparative average reliability values associated with gpta from traditional versus genomic evaluations for a subset of these traits and indexes, including nm$, pl, dpr, sce and dce.7 accuracy and reliability values associated with beef epd and dairy pta, respectively, quantify the relationship between predicted and actual genetic merit. while calculated differently and possessing different statistical properties (given similar amounts of information, accuracy is lower than reliability), each range from zero to .99. the higher the accuracy or reliability value, the less likely an epd or pta is to change as additional individual or progeny performance information accumulates. following each official dairy genetic evaluation provided by the cdcb, comparisons of average percent reliability associated with certain trait pta for traditional as compared to genomic enhanced predictions are published for various classes of animals.7 for the major reproductive traits (pl, dpr) and nm$ index, on average the reliability values associated with genomic pta (gpta) were .41 units higher in holstein heifers (.62 to .66 versus .22 to .25, depending on the trait/index) than traditional pta based on parent average (table 2). relative to sire and daughter calving ease (sce and dce), the gpta for these animals had average associated reliability values that were .26 units higher (.28 and .23 versus .54 and .49, for sce and dce, respectively). in addition to gpta for reproductive traits, important haplotypes impacting fertility in holstein, jersey, brown swiss and ayrshire have been identified through high density genomic testing. a haplotype is a segment of the single strand of dna that parents pass to offspring through oocyte and sperm cells, detected by combining genomic marker information from progeny and parents.8 nine different haplotypes, or chromosome segments, that are not found in the homozygous state have been identified (table 3). inheritance of two copies of the detrimental version of these haplotypes, one from each parent, results in failed conception or early embryonic loss. the exact biological pathways related to fertilization and embryo development impacted by these haplotypes are unknown. five of these haplotypes were found in holstein (frequency of .7 to 4.8 percent).9 fertility haplotype information is reported to customers, included in gpta for associated traits and indexes, and included in computerized mating programs. conclusion genomic technology for improved reproductive performance and calving ease in beef and dairy cattle has increased the accuracy and reliability values associated with genetic predictions for young, nonparent animals. in beef cattle, adoption of genomic technology by angus seedstock producers has increased in response to demand from commercial users of angus genetics for yearling bulls that have more dependable ge-epd. early and aggressive adoption of genomic technology in dairy cattle by the a.i. industry has now spread to increased use by commercial producers for improved accuracy of replacement heifer selection, mating, and application of various reproductive technologies for increased productivity. 475 clinical theriogenology • volume 5, number 4 • december 2013 table 1. high density 50k for angus impact on epd, accuracy values, associated progeny equivalents and interpretation of hd 50k percentile ranks. trait correlation (%gv) for ge-epds1 average (+/-) epd change2 average accuracy3 progeny equivalents4 hd 50k percent rank interpretation calving ease direct (ced) .61 (37) 3.0 .31 21 lower number, easier calving birth weight (bw) .64 (41) 0.8 .35 11 lower number, lower bw weaning weight (ww) .54 (29) 3.0 .29 19 lower number, heavier ww yearling weight (yw) .66 (44) 5.0 .32 22 lower number, heavier yw dry matter intake (dmi) component of residual average daily gain (radg) .59 (35) 0.03 .26 10 lower number, higher radg residual feed intake (rfi) na na na na lower number, more efficient yearling height (yh) .70 (49) 0.13 .35 9 lower number, taller yh scrotal circumference (sc) .73 (53) 0.23 .36 11 lower number, larger sc docility (doc) .67 (45) 5.0 .30 10 lower number, calmer doc heifer pregnancy (hp) na na na na lower number, higher hp calving ease maternal (cem) na na na na lower number, easier calving milking ability (milk) .38 (14) 2.0 .20 15 lower number, more milk mature weight (mw) .51 (26) 11.0 .25 7 lower number, heavier mw mature height (mh) na na na na lower number, taller mh carcass weight (cw) .57 (32) 5.0 .19 6 lower number, heavier cw marbling score (marb) .63 (40) 0.12 .31 16 lower number, more marb ribeye area (re) .63 (40) 0.10 .25 9 lower number, larger rea fat thickness (fat) .53 (28) 0.01 .25 11 lower number, less fat tenderness (tend) na na na na lower number, more tender 1correlation between hd 50k genomic predictions and expressed performance, and associated percent explained additive genetic variation (gv) – genomic update january 2013, american angus association/angus genetics inc. 2average absolute change in epd versus ge-epd from 50k for non-parent animals – fast forward epds, accuracy values quick tips, zoetis and angus genetics inc. 3average accuracy of ge-epds from 50k based on verified parentage and 50k information – fast forward epds, accuracy values quick tips, zoetis and angus genetics inc. 4approximate progeny equivalents associated with accuracy of ge-epds based on pedigree and 50k information. progeny equivalents for carcass traits are actual progeny carcass records – equates to ultrasound scans from over 30 progeny na (not available) – designates that either an epd is not available for the trait (rfi, tend) or that 50k information for the trait is not yet directly integrated into the epd (hp, cem, mh) 476clinical theriogenology • volume 5, number 4 • december 2013 table 2. comparison of percent reliability values associated with pta from the april 2013 genomic and traditional evaluation of holstein heifers (n=174,003). trait reliability (%) genomic traditional difference1 average average net merit (nm$) 66 25 41 productive life (pl) 63 22 41 daughter pregnancy rate (dpr) 62 22 40 sire calving ease (sce) 54 28 26 daughter calving ease (dce) 49 23 26 1genomic minus traditional. table 3. identified haplotypes impacting fertility in holstein, jersey, brown swiss and ayrshire breeds. breed haplotype name carrier frequency impact on conception rate impact on return rate earliest known ancestor(s) holstein hh1 4.5% -3.1% -1.1% pawnee farm arlinda chief hh2 4.6% -3.0% -1.7% willowholme mark anthony hh3 4.7% -3.2% -3.1% grey view skyliner & glendell arlinda chief hh4 0.7% -3.0% besne buck hh5 4.8% -3.5% thornlea texal supreme jersey jh1 23.4% -3.7% -3.7% observer chocolate soldier brown swiss bh1 14.0% -3.4% -2.5% west lawn stretch improver bh2 20.5% 0.3% rancho rustic my design ayrshire ah1 26.1% -4.4% selwood betty's commander references 1. united states department of agriculture animal and plant health inspection service veterinary services national animal health monitoring system february 2010 beef 2007–08 part iv: reference of beef cow-calf management practices in the united states, 2007–08 2. united states department of agriculture animal and plant health inspection service veterinary services national animal health monitoring system march 2007 dairy 2007 part ii: changes in the u.s. dairy cattle industry, 1991– 2007 3. illumina data sheet: dna analysis. bovinesnp50 genotyping beadchip. 2011-2012. http://res.illumina.com/documents/products/datasheets/datasheet_bovine_snp5o.pdf 4. http://aipl.arsusda.gov/genotype/cur_freq.html 5. genomic update january 2013, american angus association/angus genetics inc. 6. fast forward epds, accuracy values quick tips, zoetis and angus genetics inc. 7. http://aipl.arsusda.gov/eval/summary/comparexml_menu.cfm?r_menu=v_ 1304.v_heifers.v_holstein_wddx#startbody 8. cassell b: new use for genomics: haplotypes affecting fertility. hoard’s dairyman august 25, 2011. 9. http://www.holstein usa.com/news/press_release2013.jsp#pr2013_20 477 clinical theriogenology • volume 5, number 4 • december 2013 478clinical theriogenology • volume 5, number 4 • december 2013 omniblank: introduction the zoos in the us, accredited by the association of zoos and aquariums (aza), maintain 17 species of cats in managed breeding programs called species survival plans (ssps). the goal of these ssps is to maintain genetically diverse populations (> 90% of the diversity present in the founder population), for 100 – 200 years, that could be used to supplement or reestablish wild populations that have significantly declined or gone extinct.1 given the small size of zoo populations that is dictated by the number of zoos exhibiting the species, reproduction is carefully managed to avoid inbreeding and maximize genetic diversity. in addition, periodic infusion of new genetics from wild individuals or unrelated captive animals from other regional populations (e.g. european zoos) is necessary. unfortunately, animals recommended for breeding are not always located at the same zoo and, following transport, genetically matched individuals are not always behaviorally compatible. similarly, the acquisition of new genetics is complicated by extensive regulations and permit restrictions even for international transport from 1 zoo to another. assisted reproductive technologies (art) could be used to overcome many of these challenges, but our understanding of basic reproductive biology in nondomestic species is severely lacking and access to the animals needed to conduct the necessary studies is limited.2 the felids are 1 of the most well-studied groups of zoo species, but our understanding of feline biology and the protocols for art in cats are still far assisted reproductive technologies for endangered felids jason herrick department of reproductive sciences, omaha’s henry doorly zoo and aquarium abstract assisted reproductive technologies (art) could overcome many of the challenges associated with zoo-based conservation programs for endangered felids. although felids are 1 of the most well-studied groups of zoo species, our understanding of feline biology and the protocols for art in cats are still far behind what is available for rodents, livestock, and humans. the dangerous nature of these animals has shaped experimental designs to study nondomestic cats and determined which art are the most relevant. domestic cats have been produced by artificial insemination, in vitro fertilization, and embryo transfer, providing basic protocols that can be extrapolated to nondomestic cats. variation in estrous cycle dynamics among species and variable rates of spontaneous ovulation between individuals necessitate the use of exogenous hormones for most art procedures. for artificial insemination, pregnancies have been produced by vaginal, uterine, and oviductal inseminations. oviductal inseminations may be the most broadly applicable based on the minimal number of sperm required, but these procedures are logistically challenging for large cats. in vitro fertilization is effective for producing viable, early-stage embryos, but culture conditions for blastocyst production remain suboptimal. in addition, performing long-term culture outside of the appropriate laboratory environment is problematic. further optimization of oocyte and embryo vitrification would expand the applicability of these technologies. other research priorities are the development of methods for real-time cycle monitoring and alternative gonadotropin products for stimulation of follicular development and ovulation. finally, theriogenologists are encouraged to contact their local zoos if they are interested in providing their expertise to these efforts. keywords: cat, artificial insemination, in vitro fertilization, embryo transfer behind what is available for rodents, livestock, and humans. many species of wild canids are housed in zoological institutions, with some species having endangered status. captive breeding programs aim to maintain a healthy captive population and some work with wildlife agencies to support wild populations. whereas some species reproduce well in captivity, others are less consistently successful. some species have a predilection for pyometra and require methods to prevent, diagnose, and treat this potentially fatal condition. breeding recommendations from species survival plan (ssp) committees sometimes involve older individuals that have not been reproductive in years or involve individuals that are not actively in a social pair bond. techniques used in domestic dog theriogenology practice have provided a useful framework for addressing these challenges in captive wild canids. as nondomestic felids are dangerous carnivores and routine handling for research is not possible due to both their size (amur tiger, panthera tigris altaica; 175 kg) and general temperament (black-footed cat, felis nigripes, 3 kg), the domestic cat has proven to be an invaluable model for studies of basic feline biology.3,4 as a result, most of what we know about reproduction in domestic cats, as well as many of the ‘world’s first’ births from various art in domestic cats, have come out of zoo-based clinical theriogenology 2021; 13: 378 research programs interested in nondomestic cats.5,6 estrous cycle dynamics repeated collection of blood samples for hormone monitoring is not possible in nondomestic felids, so virtually all that is known about estrous cycle dynamics in nondomestic felids is based on longitudinal monitoring of hormone metabolites in fecal samples.7-11 the opportunity to noninvasively collect daily samples during routine maintenance of the animal’s holding and exhibit areas has provided information on the estrous cycles of > 12 species of cat and revealed a surprising degree of diversity. perhaps most surprising was the realization that spontaneous ovulation occurs in at least 8 species of cats, including the domestic cat,12 with the frequency varying between both species and individuals of the same species.7-10,12 in addition, some species exhibit prolonged periods of ovarian inactivity that may be influenced by environmental factors.13 the most extreme example of novel reproductive mechanisms within felids is the female lynx that appears to be seasonally monoestrous with the corpora lutea resulting from induced ovulation after breeding or spontaneous ovulation persisting and producing progesterone, well-beyond the length of pregnancy and continuing until the following breeding season.14 ejaculate quality ejaculates from male cats are characterized by a high proportion (> 40%) of sperm with abnormal morphology, including several species in which the proportion of sperm with abnormal morphology, can exceed 60% (teratospermia).3,15,16 high incidence of abnormal sperm morphology does not seem to affect fertility following natural breeding, at least in the cheetah.17 in most cat species, males and females breed repeatedly during estrus perhaps making up for deficiencies in quality with quantity. for example, male lions have been reported to breed females > 40 times per day during estrus.18 however, teratospermia likely has a more pronounced effect on art, especially when cryopreserved sperm are used, since teratospermia has been correlated with functional differences in capacitation, zona pellucida penetration, tolerance of thermal and osmotic shock, and cellular metabolism.19,20 although a low incidence of sperm with normal morphology is prevalent in many felid species, this condition can be further exacerbated by both genetic and environmental factors. ejaculate traits in florida panthers21 and lions from genetically-isolated populations22 suggest that reduced genetic heterozygosity is associated with an increased incidence of abnormal sperm forms. similarly, a recent study of snow leopards indicated that ejaculate traits were correlated with the dietary content of several nutrients.23 exogenous hormones given the variability of the feline estrous cycle, arts require exogenous hormones to stimulate follicular development and ovulation at specific times. follicular development is most often stimulated with equine chorionic gonadotropin (ecg, pregnant mare’s serum gonadotropin) because this hormone has an extended half-life and is effective following a single dose, compared to follicle stimulating hormone (fsh) preparations that often require multiple, sometimes daily, injections.24-26 effective doses of ecg have been identified for most cat species and are not related to body size.24 following ecg treatment, ovulation can be induced with either human chorionic gonadotropin (hcg) or porcine luteinizing hormone (plh), but fertility is greatly improved when plh is used.27 recent studies used oral progestins (altrenogest, regumate®, merck animal health, madison, nj) to down-regulate endogenous ovarian activity prior to gonadotropin treatments. this treatment facilitates planning and allows ecg treatment in the absence of large follicles or functional corpora lutea.28 the combined protocol of altrenogest, ecg, and plh has resulted in excellent pregnancy rates in domestic cats following ai, as well as pregnancies in several nondomestic cat species.25 however, the dose and duration of altrenogest, and the period between its cessation and the injection of ecg, may need to be optimized for each species.29 artificial insemination for ai in cats, the 2 big questions have been when and where. in ecg-hcg treated domestic cats, that were inseminated (laparoscopic uterine insemination) prior to ovulation, 14% became pregnant, and of those inseminated following ovulation, 50% became pregnant.30 the conclusion was that periovulatory anesthesia may interfere with ovulation and/or sperm transport. this led further studies to focus almost exclusively on identifying the relative time of ovulation in each species, so that ai could be performed postovulation.24 however, pregnancy rates in domestic cats induced to ovulate with hcg in a natural cycle were higher when ai was done preovulation.31 lions given a gonadotropin releasing hormone (gnrh) analogue (burserelin-acetate, receptal®, intervet, south africa) to stimulate ovulation during natural estrus had excellent pregnancy rates (~ 33%) with preovulatory inseminations.32 pregnancies have been produced in domestic cats following inseminations in which sperm were deposited in the vagina, uterus, and oviduct.24,25,31,33 most of the ‘world’s first’ pregnancies in nondomestic cats were accomplished with laparoscopic uterine inseminations, but that was due to the almost exclusive use of that technique at the time.24 recently, oviductal inseminations were more widely used that resulted in higher success rates than previous uterine ai studies, due in part to the use of the improved ecg-plh protocol.25,34 although direct comparisons of various insemination sites were not conducted in nondomestic cats, domestic cat studies suggest that acceptable (> 50%) pregnancy rates can be achieved with any of the possible sites of sperm deposition. from a technical perspective, intravaginal ais are the easiest to perform and, with appropriate training, could even be performed without sedation. in contrast, oviductal inseminations may be among the most challenging given the difficulty of catheterizing a ~ 1 mm opening via laparoscopy. uterine inseminations, whether laparoscopic or transcervical also require extensive experience, clinical theriogenology 2021; 13: 379 although that experience may be more widely available given the popularity of these techniques in small ruminants and domestic dogs. for the large cat species, there are logistical advantages to a transcervical procedure that could be performed in the female’s holding area compared to a laparoscopic procedure that most often involves transport to the zoo hospital’s surgical suite. in contrast, the need to transport females for laparoscopic procedures is a negligible factor for many of the small cats. ultimately, the most appropriate site of sperm deposition may be determined by the quantity and quality of sperm available. in domestic cats, intravaginal inseminations require ~ 80 x 106 sperm compared to uterine inseminations in which 8 x 106 sperm are required and oviductal inseminations in which 2 4 x 106 sperm are effective.25,31,35 similar numbers of sperm appear to be effective for oviductal ais in nondomestic cats.36,37 in contrast, the limited data available on intravaginal or transcervical ais in nondomestic cats suggest insemination doses require at least 100 x 106 sperm, with most pregnancies resulting from the use of > 500 x 106 sperm.32,38 for many of the cat species, especially the small cats, ejaculates rarely contain > 100 x 106 sperm.7,15,16 snow leopards averaged 120 x 106 sperm per ejaculate that is sufficient for just a single transcervical ai compared to ~ 24 oviductal inseminations.23 if cryopreserved sperm are used, the number of ais per ejaculate would be even smaller. additional studies are necessary to better define minimum sperm numbers needed for pregnancy that may be dependent on further refinements to synchronization protocols in vitro fertilization and embryo transfer with limited numbers of sperm available from a single ejaculate, ivf and embryo transfer (et) could be a valuable art for population management.39 offspring have been produced using these procedures in domestic cats, as well as several species of small cats, tigers, and cheetahs.40-43 exogenous hormone protocols stimulating follicular development can be easily adapted for oocyte collections and the laparoscopic follicular aspirations can be used to collect oocytes.44,45 sperm capacitation occurs readily in various culture media, fertilization rates are high, and resulting embryos will progress to the blastocyst stage.41,46,47 most pregnancies have resulted from the laparoscopic transfer of early cleavage stage embryos into the oviduct. pregnancies from the transfer of later stage morulae or blastocysts into the uterus are far less common, which may be related to the reduced rate of cell division for in vitro cultured embryos compared to their in vivo counterparts.41,47 further optimization of culture conditions to support the development of viable blastocysts could facilitate transcervical embryo transfers. in addition to the biological challenges, ivf and et present a unique set of logistical challenges. first, few zoos in the us are equipped with suitable laboratories for ivf and embryo culture. in most cases, portable laboratory equipment was used to create ‘mobile labs’ that can be set-up in the corner of a zoo’s veterinary hospital.48 this approach has been successful, especially for short-term culture to cleavage stages,42,48 but far from an ideal environment for such sensitive procedures.49 second, most zoos only maintain a single female of each species. two immobilizations of the same female for the oocyte collection and the subsequent embryo transfer may be overly stressful on the females (and staff) and the uterine environment of a gonadotropin-stimulated cycle is not always ideal for implantation.50 portable incubators may allow embryos to be transported short distances to other zoos for transfers of fresh embryos, but in most cases, ivf and et will involve embryo cryopreservation. recent advancements with oocyte and embryo vitrification in humans suggest it may be possible to vitrify oocytes immediately after collection at 1 zoo, warm those oocytes for ivf and embryo culture in a suitable laboratory, vitrify the resulting embryos, and warm just prior to transfer.51 domestic cat kittens were produced from vitrified oocytes and vitrified embryos, but further research is necessary to improve these procedures.52,53. future directions every aspect of art in felids, even domestic cats, requires additional optimization. however, a few areas that would be particularly valuable. the first is improved methods for real-time monitoring of estrous cycles in females that do not exhibit overt behavioral changes. fecal hormone monitoring is a great method for cycle characterization, but it only provides a retrospective indication of when a female was in estrus. currently, vaginal cytology is the only method that can be used to determine cycle status in real-time, but it requires extensive training with the animal.32,54 behavioral training for voluntary blood collections (e.g. tails accessed under cage bars) would also allow the analysis of concentrations of hormones in serum that can be determined faster and with less ‘noise’ than a fecal hormone assay. importantly, these techniques would be equally valuable to managers attempting to time animal introductions as it would be for researchers hoping to perform art. although behavioral training and real-time cycle monitoring may allow for more art procedures to be completed in the context of a natural cycle, there will still be a need for exogenous gonadotropins in some cases. unfortunately, the 2 hormones that are the most widely used currently, ecg and plh, are difficult to acquire. to the best of author’s knowledge, ecg (pmsg) is only available from a single commercial source (prospec, east brunswick, nj) in the us and there are no longer any commercial sources for plh, at least not in the quantities needed for in vivo use. in the very near future, new hormones have to be identified and tested for efficacy in felids. a variety of hormones are used within the human fertility industry, but many of these are designed for repeated, daily injections. longer acting preparations would be ideal for cats. however, behavioral training for voluntary injections is becoming more common in zoos that could provide additional options that are already commercially available. finally, there is a need for more personnel to conduct art procedures in cats. currently there are only 3 research groups clinical theriogenology 2021; 13: 380 in the us working on art in nondomestic cats (smithsonian conservation biology institute, cincinnati zoo and botanical gardens’ center for conservation and research of endangered wildlife, and omaha’s henry doorly zoo and aquarium). application of art to cats housed in all of the zoos in the us requires extensive travel by these scientists, greatly increasing the costs of the research. however, it is possible that university and/or private practice-based veterinarians and theriogenologists could be trained for these procedures and assist their local zoos. in addition, many of these theriogenologists already have extensive experience with techniques, such as vaginal cytology and endoscopic tci, that are still new to many zoo-based researchers. collaborations are common between zoo veterinarians and local specialists in veterinary dentistry, ophthalmology, orthopedics, etc. expanding these collaborations between zoos and private and university-based theriogenologists would provide zoos, especially those without staff scientists, with valuable expertise to improve breeding efforts and advance the development of art for felids and other endangered species. conflict of interest there are no conflicts of interest to declare. acknowledgement thank you to all of the animal care staff, zoo veterinarians, and ssp managers that have been so supportive of reproductive research over the years. references 1. soulé m, gilpin m, conway w, et al: the millenium ark: how long a voyage, how many staterooms, how many passengers? zoo biol 1986;5:101-113. 2. herrick jr: assisted reproductive technologies for endangered species conservation: developing sophisticated protocols with limited access to animals with unique reproductive mechanisms. biol reprod 2019;100:1158-1170. 3. howard jg, brown jl, bush m, et al: teratospermic and normospermic domestic cats: ejaculate traits, pituitary-gonadal hormones, and improvement of spermatozoal motility and morphology after swim-up processing. j androl 1990;11:204-215. 4. swanson wf, roth tl, wildt de: in vivo embryogenesis, embryo migration, and embryonic mortality in the domestic cat. biol reprod 1994;51:452-464. 5. goodrowe kl, wall rj, o’brien sj, et al: developmental competence of domestic cat follicular oocytes after fertilization in vitro. biol reprod 1988;39:355-372. 6. pope ce, mcrae ma, plair bl, et al: in vitro and in vivo development of embryos produced by in vitro maturation and in vitro fertilization of cat oocytes. j reprod fertil 1997;51(suppl):69-82. 7. herrick jr, bond jb, campbell m, et al: fecal endocrine profiles and ejaculate traits in black-footed cats (felis nigripes) and sand cats (felis margarita). gen comp endocrinol 2010;165:204-214. 8. brown jl: female reproductive cycles of wild female felids. anim reprod sci 2011;124:155-162. 9. putman sb, brown jl, franklin ad, et al: characterization of ovarian steroid patterns in female african lions (panthera leo), and the effects of contraception on reproductive function. plos one 2015;10:e0140373. 10. barnes sa, andrew teare j, staaden s, et al: characterization and manipulation of reproductive cycles in the jaguar (panthera onca). gen comp endocrinol 2016;225:95-103. 11. reichert-stewart jl, santymire rm, armstrong d, et al: fecal endocrine monitoring of reproduction in female snow leopards (uncia uncia). theriogenology 2014;82:17-26. 12. pelican km, brown jl, wildt de, et al: short term suppression of follicular recruitment and spontaneous ovulation in the cat using levonorgestrel versus a gnrh antagonist. gen comp endocrinol 2005;144:110-121. 13. wielebnowski nc, ziegler k, wildt de, et al: impact of social management on reproductive, adrenal and behavioural activity in the cheetah (acinonyx jubatus). animal conservation 2002;5:291-301. 14. jewgenow k, painer j, amelkina o, et al: lynx reproduction--long-lasting life cycle of corpora lutea in a feline species. reprod biol 2014;14:83-88. 15. pukazhenthi b, laroe d, crosier a, et al: challenges in cryopreservation of clouded leopard (neofelis nebulosa) spermatozoa. theriogenology 2006;66:1790-1796. 16. crosier ae, marker l, howard j, et al: ejaculate traits in the namibian cheetah (acinonyx jubatus): influence of age, season and captivity. reprod fertil dev 2007;19:370-382. 17. lindburg dg, durrant bs, millard se, et al: fertility assessment of cheetah males with poor quality semen. zoo biol 1993;12:97-103. 18. sunquist m, sunquist f: wild cats of the world. chicago, il: the university of chicago press, 2002. 19. pukazhenthi bs, neubauer k, jewgenow k, et al: the impact and potential etiology of teratospermia in the domestic cat and its wild relatives. theriogenology 2006;66:112-121. 20. terrell ka, wildt de, anthony nm, et al: evidence for compromised metabolic function and limited glucose uptake in spermatozoa from the teratospermic domestic cat (felis catus) and cheetah (acinonyx jubatus). biol reprod 2010;83:833-841. 21. roelke me, martenson js, o’brien sj: the consequences of demographic reduction and genetic depletion in the endangered florida panther. curr biol 1993;3:340-350. 22. brown jl, bush m, packer c, et al: developmental changes in pituitary-gonadal function in free-ranging lions (panthera leo leo) of the serengeti plains and ngorongoro crater. j reprod fertil 1991;91:29-40. 23. herrick jr, iske cj, santymire rm, et al: factors affecting reproductive traits in male snow leopards (uncia uncia). reprod fertil 2020;1:35-49. 24. howard jg, wildt de: approaches and efficacy of artificial insemination in felids and mustelids. theriogenology 2009;71:130-148. 25. swanson wf: practical application of laparoscopic oviductal insemination for the propagation of domestic cats and wild felids. reprod fertil dev 2019;31:27-39. 26. thongphakdee a, tipkantha w, punkong c, et al: monitoring and controlling ovarian activity in wild felids. theriogenology 2018;109:14-21. 27. conforti va, bateman hl, schook mw, et al: laparoscopic oviductal artificial insemination improves pregnancy success in exogenous gonadotropin-treated domestic cats as a model for endangered felids. biol reprod 2013;89:4. clinical theriogenology 2021; 13: 381 28. stewart ra, pelican km, brown jl, et al: oral progestin induces rapid, reversible suppression of ovarian activity in the cat. gen comp endocrinol 2010;166:409-416. 29. crosier ae, comizzoli p, koester dc, et al: circumventing the natural, frequent oestrogen waves of the female cheetah (acinonyx jubatus) using oral progestin (altrenogest). reprod fertil dev 2017;29:1486-1498. 30. howard jg, barone ma, donoghue am, et al: the effect of pre-ovulatory anaesthesia on ovulation in laparoscopically inseminated domestic cats. j reprod fertil 1992;96:175-186. 31. tsutsui t, tanaka a, takagi y, et al: unilateral intrauterine horn insemination of fresh semen in cats. j vet med sci 2000;62:1241-1245. 32. callealta i, ganswindt a, malan m, et al: non-surgical artificial insemination using a gnrh analogue for ovulation induction during natural oestrus in african lions (panthera leo). theriogenology 2019;139:28-35. 33. chatdarong k, axner e, manee-in s, et al: pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen. theriogenology 2007;68:1326-1333. 34. tipkantha w, thuwanut p, maikeaw u, et al: successful laparoscopic oviductal artificial insemination in the clouded leopard (neofelis nebulosa) in thailand. j zoo wildl med 2017;48:804-812. 35. tanaka a, takagi y, nakagawa k, et al: artificial intravaginal insemination using fresh semen in cats. j vet med sci 2000;62:1163-1167. 36. lambo ca, bateman hl, swanson wf: application of laparoscopic oviductal artificial insemination for conservation management of brazilian ocelots and amur tigers. reprod fertil develop 2014;26:116. 37. vansandt lm, adania ch, yanai pr, et al: ovarian synchronization, ovulation induction, and successful artificial insemination in the jaguar (panthera onca). 51st annual conference of the american association of zoo veterinarians 2019. 38. silva jnc, leitão rm, lapão ne, et al: birth of siberian tiger (panthera tigris altaica) cubs after transvaginal artificial insemination. j zoo wildl med 2000;31:566-569. 39. swanson wf, johnson we, cambre rc, et al: reproductive status of endemic felid species in latin american zoos and implications for ex situ conservation. zoo biol 2003;22:421-441. 40. donoghue am, johnston la, seal us, et al: in vitro fertilization and embryo development in vitro and in vivo in the tiger (panthera tigris). biol reprod 1990;43:733-744. 41. pope ce, gomez mc, dresser bl: in vitro production and transfer of cat embryos in the 21st century. theriogenology 2006;66:59-71. 42. herrick jr, mehrdadfar f, campbell m, et al: birth of sand cat (felis margarita) kittens following in vitro fertilization and embryo transfer. biol reprod 2010:6-7. 43. pope ce: aspects of in vivo oocyte production, blastocyst development, and embryo transfer in the cat. theriogenology 2014;81:126-137. 44. herrick jr, campbell m, levens g, et al: in vitro fertilization and sperm cryopreservation in the black-footed cat (felis nigripes) and sand cat (felis margarita). biol reprod 2010;82:552-562. 45. crosier ae, lamy j, bapodra p, et al: first birth of cheetah cubs from in vitro fertilization and embryo transfer. animals (basel) 2020;10. 46. murakami m, dong yj, suzuki t, et al: development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media. cryobiology 2011;63:170-174. 47. herrick jr, lyons sm, greene-ermisch af, et al: a carnivore embryo’s perspective on essential amino acids and ammonium in culture medium: effects on the development of feline embryos. biol reprod 2018;99:1070-1081. 48. swanson wf: research in nondomestic species: experiences in reproductive physiology research for conservation of endangered felids. ilar j 2003;44:307-316. 49. wale pl, gardner dk: the effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. hum reprod update 2016;22:2-22. 50. shapiro bs, daneshmand st, garner fc, et al: evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfer in normal responders. fertil steril 2011;96:344-348. 51. cobo a, castello d, vallejo b, et al: outcome of cryotransfer of embryos developed from vitrified oocytes: double vitrification has no impact on delivery rates. fertil steril 2013;99:1623-1630. 52. tharasanit t, manee-in s, buarpung s, et al: successful pregnancy following transfer of feline embryos derived from vitrified immature cat oocytes using ‘stepwise’ cryoprotectant exposure technique. theriogenology 2011;76:1442-1449. 53. pope ce, gomez mc, galiguis j, et al: applying embryo cryopreservation technologies to the production of domestic and black-footed cats. reprod domest anim 2012;47(suppl 6):125-129. 54. asa cs, junge re, bircher s, et al: assessing reproductive cycles and pregnancy in cheetahs (acinonyx jubatus) by vaginal cytology. zoo biol 1992;11:139-151. clinical theriogenology 2021; 13: 382 2015: hemoperitoneum in a mare following rupture of a granulosa-theca cell tumor hemoperitoneum in a mare following rupture of a granulosa-theca cell tumor camilla j. scott,a bruce w. christensen,b sarah n. graya awilliam r. pritchard veterinary medical teaching hospital and bdepartment of population health and reproduction, school of veterinary medicine, university of california-davis, davis, ca summary an 18-year old quarter horse mare with a five-month history of suspected granulosa-theca cell tumor on the right ovary was presented with acute abdominal pain. the mare was displaying signs of cardiovascular shock on arrival so attempts were made to stabilize her while performing further diagnostic tests. transabdominal ultrasound examination revealed a large, well-demarcated mass in the right caudal abdomen and a significant peritoneal effusion, which was confirmed to be hemorrhagic following abdominocentesis. transrectal ultrasound examination confirmed a large, multi-loculated right ovarian mass. despite aggressive medical therapy the mare continued to show signs of cardiovascular deterioration. serial blood samples and repeated abdominal ultrasonography were consistent with uncontrolled intra-abdominal hemorrhage. based on the mare’s clinical deterioration euthanasia was elected. postmortem examination confirmed rupture of a granulosa-theca cell tumor on the right ovary associated with profuse intraabdominal hemorrhage. case presentation an 18-year-old quarter horse mare was presented to the veterinary medical teaching hospital at the university of california davis for evaluation of colic. the mare had been examined and treated by the referring veterinarian but no improvement was seen so the mare was referred for further diagnostics and treatment. the mare had a history of displaying stallion-like behavior and five months prior to presentation a presumptive diagnosis of a granulosa-theca cell tumor on the right ovary had been made based on transrectal palpation and ultrasound examination. the diagnosis was not confirmed by endocrine diagnostic tests and financial limitations had precluded surgical excision at that time. on presentation the mare displayed signs of depression and cardiovascular shock. based on history and initial clinical examination, differential diagnoses included gastrointestinal volvulus/strangulation, visceral rupture, and hemoperitoneum following rupture of the previously diagnosed ovarian mass. a complete blood cell count (cbc), serum biochemistry and venous blood gas analysis were performed. the cbc revealed reduced hematocrit (22.6% [reference range 30-46%]), red blood cell count was 4.15 m/ul (reference range 6.2-10.2) and hemoglobin was 7.8 gm/dl (reference range 11.217.2) which were consistent with anemia. the platelet count was 110,000 u/l (reference range100,000225,000) but a coagulation panel was not performed. serum biochemistry revealed hypoproteinemia (3.7 g/dl [reference range 5.8-8.7]), and hyperlactemia (12.7 mmol/l [reference range<2mmol/l]). transabdominal ultrasound examination revealed a significant peritoneal effusion, with a classical echogenic swirling consistency, suggestive of a hemoabdomen. a large, (approx. 19 x 16 cm), well-demarcated mass was visualized in right caudal abdomen. based on ultrasound findings abdominocentesis was performed to characterize the peritoneal effusion. upon entry into the peritoneal cavity a large volume of hemorrhagic fluid flowed from the cannula. the fluid was collected and submitted for evaluation, which confirmed it to be frank blood. transrectal palpation and ultrasound examination revealed a solid, large (>15 cm) mass on the right ovary, which was well demarcated, circular/oval in shape, with a multi-loculated appearance. it was not possible to obtain exact measurements of the mass as both the length and width exceeded the measuring capabilities of a 7.5 mhz transrectal probe. an area of approximately 7 cm on the ventromedial aspect of the mass had a homogenous echogenicity, consistent with fluid/hemorrhage (figure 1). the mass appeared to be suspended by the broad ligament, but it was not possible to determine whether the source of hemorrhage was from the ovarian parenchyma or pedicle and ovarian clinical theriogenology • volume 7, number 4 • december 2015425 vessels. based on the severity of the clinical signs displayed and diagnosis of active intra-abdominal hemorrhage, anemia and hypoproteinemia, a cross-match was performed. three available blood donors were tested at the teaching hospital and a suitable donor selected. two intravenous catheters had been placed upon presentation to facilitate intravenous fluid therapy with a balanced electrolyte solution (plasma-lyte a, abbott animal health, abbott park, il) and 4 l of commercial plasma (equiplas j, plasvacc usa inc., templeton, ca) both at a rate of 2-4 ml/kg/hr. treatment with aminocaproic acid was also started, with a loading dose of 40 g in 1 l saline iv followed by 20 g in 1 l saline iv, q 6 h. following cross-matching and identification of a suitable donor, 4 l of blood was transfused initially at a rate of 0.1 ml/kg/hr; no transfusion reactions were noted so the rate was increased to 15 ml/kg/hr. a further 4 l of blood was transfused over the course of the treatment. flunixin meglumine (1.1mg/kg, iv, q 24 h) was continued for its anti-endotoxic, anti-inflammatory, and analgesic properties. the goal was to perform surgical resection once the mare was hemodynamically stable enough for surgery, however despite aggressive treatment the mare continued to show signs of cardiovascular deterioration. the mare remained persistently tachycardic and became tachypneic. serial blood samples revealed both the hematocrit (13%) and total protein levels (3.1 g/dl) to be falling and repeat abdominal ultrasonography was consistent with uncontrolled intra-abdominal hemorrhage. further treatment options were discussed with the owner, including laparoscopic exploration with the hope to ligate the ovarian pedicle and a second blood transfusion, but based on the mare’s clinical deterioration despite aggressive medical therapy, the fact that she was a poor surgical candidate due to cardiovascular instability, and the financial implications of continued treatment, euthanasia was elected. postmortem examination revealed the right ovary consisted of a large (~20 cm diameter), spherical, encapsulated mass (b). on cut section, the mass was composed of dozens of variably-sized (1 to 11 cm diameter), cystic spaces separated by dense bands of firm, pale yellow to white, fibrous tissue (figure 3). the cysts were filled with yellow, watery fluid and blood clots. a 0.5 cm thick, fibrous, white capsule circumscribed the majority of the ovarian mass. at the rostral aspect of the mass, however, a 10 x 5 cm region of the capsule overlying a large blood-filled cyst was ruptured. this was presumed to be the site of profuse hemorrhage into the abdominal cavity, as was severe, hemorrhagic effusion within the peritoneal cavity (figure 4). histopathological examination confirmed a granulosa-theca cell tumor of the right ovary with an atrophied left ovary (figure 5). discussion granulosa cell tumors are the most commonly reported ovarian neoplasia in the mare1-3 and account for more than 85% of equine reproductive tract tumors.4 they are typically diagnosed based on a combination of clinical signs, transrectal palpation and ultrasound examination, and endocrine assays. histopathological examination of the ovary provides a definitive diagnosis. clinical signs reported include stallion-like behavior, aggression and persistent or irregular estrous cycles.3,5 taken alone these signs can be misleading, as they may be manifestations of other problems such as musculoskeletal pain or behavioral issues. transrectal palpation and examination via ultrasound cannot be used to definitely diagnose a granulosa cell tumor, as tumor appearance can be quite variable and not always consistent with the classical mixed “honey-combed” echogenicity on ultrasound examination. other causes of ovarian enlargement such as an ovarian hematoma, teratoma or hemangiosarcoma may have a similar appearance and so cannot be ruled out based on ultrasound examination alone.1,4-6 in contrast to granulosa cell tumors, ovarian hematomas typically regress rather than grow with time, the ovulation fossa will still be palpable on the ovary, and the mare will continue to cycle.7 endocrine assays used to diagnose granulosa cell tumors include inhibin, testosterone, progesterone, and anti-müllerian hormone (amh). antimüllerian hormone in particular has been shown to be highly sensitive for the detection of granulosa cell tumors, being elevated in 98% of cases compared to elevations in inhibin (80% of cases), testosterone (48% of cases) or a combination of both inhibin and testosterone (84% of cases).3 endocrine diagnostic clinical theriogenology • volume 7, number 4 • december 2015 426 tests were not carried out in this case due initially to financial constrictions and later because concerns regarding treatment of the hemoperitoneum outweighed the necessity to diagnose the tumor type. although reports exist,8 colic is not a typical sign associated with granulosa cell tumors. in a review of 67 cases of hemoperitoneum, however, colic was the most reported associated clinical sign (79% of cases)9 and hence in addition to the mare’s history, hemoperitoneum was high on the list of concerns. while a granulosa cell tumor was suspected based on clinical signs and the external appearance of the mass, histopathological diagnosis was performed for a definitive diagnosis and to rule out other causes of ovarian enlargement and hemorrhage, such as hemangiosarcoma or hematoma. treatment was initiated with the aims of restoring circulating fluid/blood volume, enhancing coagulation, controlling pain and reducing the effects of endotoxemia. in spite of aggressive treatment, however, the degree of hemodynamic compromise was irreversible, hence the decision to euthanize the mare. interestingly a significant difference between survivors and non-survivors in terms of treatment was not reported in two reviews of hemoperitoneum.9,10 the prognosis for both survival and future fertility following surgical excision of granulosa cell tumors is good.1,6 this is in contrast to reported short-term survival rates in cases of hemoperitoneum.9,10 in a review by dechant et al. approximately three quarters of the horses that did not survive were euthanized because of the severity of hemodynamic compromise (as in this case) or due to the underlying disease process.9 unfortunately it was not possible in this case to stabilize the mare’s cardiovascular parameters to a level where she would have been a good surgical candidate. while the vast majority of cases of granulosa cell tumors in the horse are benign neoplasms, the growth rate of these tumors cannot be predicted. this case highlights the importance of surgical removal following initial detection to avoid catastrophic complications. hemoperitoneum following rupture of a granulosa cell tumor is a rarely reported but life-threatening complication associated with this neoplasm6,9,11-13 that could have been avoided in this case with early and appropriate treatment. learning points • growth of granulosa-theca cell tumors is unpredictable • surgical removal following detection should not be delayed as potential complications are lifethreatening acknowledgments the authors wish to thank dr. andrew cartoceti for pathology consultation. references 1. mccue pm, roser jf, munro cj et al: granulosa cell tumors of the equine ovary. vet clin north am equine pract 2006;22:799-817. 2. ball ba, conley aj, almeida j, et al: a retrospective analysis of 2,253 cases submitted for endocrine diagnosis of possible granulosa cell tumors in mares. j equine vet sci. 2014;34:307-313. 3. ball ba, almeida j, conley aj: determination of serum anti-mullerian hormone concentrations for the diagnosis of granulosa-cell tumours in mares. equine vet j 2013; 45:199-203. 4. pauwels fe, wigley sj, munday js, et al: bilateral ovarian adenocarcinoma in a mare causing haemoperitoneum and colic. n z vet j 2012;60:198-202. 5. cameron ij: granulosa cell tumours diagnostic considerations. equine vet educ 2014;26:242-243. 6. harper j, stewart aj, kuhnt l, et al: ultrasonographic appearance and abdominal haemorrhage associated with a juvenile granulosa cell tumour in a foal. equine vet educ 2010;22:115-120. 7. watson ed: granulosa cell tumors in the mare: a review of 9 cases. equine vet educ 1999;11:136-142. 8. van der zaag ej, rijkenhuizen ab, kalsbee hc, et al: a mare with colic caused by an ovarian tumour. vet q 1996: 18: 60-62. 9. dechant je, nieto je, le jeune ss: hemoperitoneum in horses: 67 cases (1989–2004). j am vet med assoc 2006;229:253-258. 10. conwell rc, hillyer mh, mair ts, et al: haemoperitoneum in horses: a retrospective review of 54 cases. vet rec 2010; 167: 14 514-518. 11. alexander gr, tweedie ma, lescun tb, et al: haemoperitoneum secondary to granulosa cell tumour in two mares. aust vet j 2004; 82: 481-484. clinical theriogenology • volume 7, number 4 • december 2015427 12. green sl, dowling sc, nixon aj, et al: hemoperitoneum caused by rupture of a juvenile granulosa cell tumor in an equine neonate. j am vet med assoc 1988;193:1417-1419. 13. gatewood dm, douglass jp, cox jh, et al: intra-abdominal hemorrhage associated with a granulosa-thecal cell neoplasm in a mare. j am vet med assoc 1990;196:1827-1828. figure 1. transrectal ultrasound image of the right ovary revealing echogenic fluid consistent with hemorrhage both within the mass and the peritoneal cavity. figure 2. photograph of the right ovarian mass revealing the ruptured capsule. figure 3. photograph of the cut surface of granulosa-theca cell tumor of right ovary revealing multiple cystic structures filled with blood clots. clinical theriogenology • volume 7, number 4 • december 2015 428 figure 4. photograph at necropsy of the hemoabdomen following rupture of the granulosa-theca cell tumor. figure 5. photograph at necropsy of the granulosa-theca cell tumor of the right ovary and atrophied left ovary in situ. (editor’s note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 7, number 4 • december 2015429 clinical theriogenology • volume 7, number 4 • december 2015 430 omniblank: uterine perforation secondary to metritis and placenta percreta in a labradoodle bitch uterine perforation secondary to metritis and placenta percreta in a labradoodle bitch lacey rosenberg,a jacqueline marinoff,b esther crouch,b dominick valenzano,a jeanine peters-kennedy,b soon cheong,a mariana amorima adepartment of clinical sciences and bdepartment of biomedical sciences cornell university, ithaca, ny placenta accreta is a spectrum of conditions characterized by abnormal trophoblast invasion into the myometrium. depending on the level of trophoblast invasion, it is subcategorized into 3 types: accreta, increta, and percreta (the latter being the highest degree of trophoblast invasion, with cells penetrating through myometrium into serosa). this is a rare condition in humans and has apparently not been reported in dogs. a 3 year old female intact primiparous labradoodle presented as an emergency due to fever, lethargy, and increased serosanguinous vaginal discharge 4 days postpartum. history included a natural whelping of 7 puppies without veterinary intervention, but closely monitored by the breeder, who reported a long, protracted labor in which at least 1 neonate had to be extracted manually. the breeder was unsure if any or all fetal membranes were delivered. on initial evaluation, the patient was dull but responsive, dehydrated, and in poor body condition (bcs of 2/9). there was foul-smelling brownish vaginal discharge and pain on abdominal palpation. mammary glands were subjectively milkdepleted but were expressible, and puppies were briefly evaluated and were in good health and body condition. bloodwork revealed a moderate normocytic, normochromic, regenerative anemia (pcv of 28%), leukocytosis with neutrophilia characterized by a left shift (wbc 24.3 x 103/µl, ri 5.7 14.2; segmented neutrophils 17.5 x 103/µl, ri 2.7 9.4; band neutrophils 3.9 x 103/µl, ri 0.0 0.1), and hypoalbuminemia (2.3 g/dl, ri 3.2 4.1). abdominal ultrasonography and radiography indicated an enlarged, fluid-filled uterus with a small amount of gas. a presumptive diagnosis of metritis was made; due to clinical condition, dog was stabilized overnight, with an ovariohysterectomy performed the following morning. abdominal exploration revealed mild peritoneal effusion and moderate uterine distension. on the ventral surface of the right uterine horn, 3 discrete, 5 mm diameter uterine perforations with exuding purulent material, were noted. submitted cultures identified staphylococcus pseudintermedius, enterococcus faecalis, and fusobacterium necrophorum, which were sensitive to a combination of enrofloxacin and amoxicillin/clavulanate. histological examination revealed trophoblastic cells penetrating deep into myometrium to serosa, and severe necrosuppurative inflammation of the uterus with intralesional bacteria. findings were consistent with placenta percreta, necrosuppurative metritis with focal rupture, and locally extensive mesovaritis with vasculitis. the patient recovered well from surgery and was discharged 48 hours after surgery with oral antibiotics and pain medications; she and her puppies were reported to be doing well 3 months later. trophoblastic invasion through the myometrium predisposed to uterine friability and likely caused weakened contractions, leading to prolonged and difficult labor. these uterine conditions, in combination with the patient’s poor body condition as well as fetal manipulation at parturition, could have led to impaired uterine clearance, bacterial invasion and eventual postpartum metritis with subsequent perforation. to the authors’ knowledge, this is a first report of a case of placenta percreta in a dog. keywords: canine, dystocia, metritis, placenta percreta, uterine perforation 475 clinical theriogenology • volume 11, number 3 • september 2019 476clinical theriogenology • volume 11, number 3 • september 2019 omniblank: prolonged estrus in an intact doberman prolonged estrus in an intact doberman gabrielle montone, robyn wilborn, rachel moon college of veterinary medicine, auburn university, auburn, al a 17 month old intact female doberman presented with serosanguinous vulvar discharge of 2 months duration. on physical examination, vulva was moderately swollen with a notable amount of serosanguinous discharge. vaginal cytology revealed 100% cornification of vaginal epithelial cells, confirming the influence of estrogen and serum progesterone concentrations were < 0.02 ng/ml. on ultrasonography, a 2.0 x 3.0 cm fluid filled structure was identified on the cranial aspect of left ovary. differential diagnoses included follicular cyst, exogenous estrogen exposure and neoplasia. based on history, physical examination and diagnostics, a follicular cyst was placed at top of the differential list. follicular cysts are well recognized in association with prolongation of clinical signs of proestrus or estrus. most follicular cysts have a granulosa cell lining, are anovulatory, and secrete substantial amounts of estrogen, which explains the clinical signs commonly associated with this condition. these cysts are usually solitary, occur in bitches < 3 years of age, and measure 1 1.5 cm in diameter, but some cysts can be as large as 5 cm.1 most common reason owners seek veterinary care is vaginal bleeding that persists for weeks, rather than the expected 7 10 days. diagnosis of follicular cysts is based on observation of prolonged vulvar discharge, persistent estrous behavior, increased blood estrogen concentrations as evidenced by vaginal cytology findings, and abdominal ultrasonography.2 ovariohysterectomy was performed. left ovary and uterine horn appeared enlarged. histopathology of ovary confirmed a follicular cyst. surgical recovery and healing were uneventful and the patient’s clinical signs resolved. this case demonstrated a practical approach to the diagnosis and treatment of canine follicular cyst. keywords: follicular cyst, prolonged estrus, estrogen references 1. feldman, ec, nelson rw: canine and feline endocrinology and reproduction. 3rd edition, saunders, 2004. 2. pasquini c, pasquini s: guide to small animal clinics. 3 edition, sudz publishing, 2011.rd 503 clinical theriogenology • volume 11, number 3 • september 2019 3 edition 504clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2011: epididymal sperm harvest and freezing epididymal sperm harvest and freezing a. jacques fuselier department of rural animal health management, college of veterinary medicine, university of illinois, urbana, il introduction artificial insemination of captive cervids is becoming more popular. as with raising traditional domestic species, deer farmers strive to improve the genetics of their herd in order to increase the value of their animals. using advanced reproductive technologies is often a more economical way of accomplishing this without incurring the tremendous expense of purchasing a high quality breeding buck. having the ability to harvest and preserve epididymal sperm from a deceased valuable breeding buck is of great value to a deer farmer. moreover, being able to preserve sperm from a hunter-killed trophy buck can prove to be an economical method of adding quality genetics to a deer herd. it has been shown that successful pregnancies have been achieved by using epididymal sperm for artificial insemination.1 this manuscript describes methods of shipping testicles, harvesting epididymal sperm, sperm analysis, and sperm processing. keywords: deer, sperm, epididymal, artificial insemination shipping and handling harvesting sperm immediately after death of the animal would be ideal. however, because veterinarians, technicians, and lab equipment are not usually present at the time of death, the specimens must be shipped to the appropriate location in a short period of time. studies involving iberian red deer have shown that cooling sperm maintained within the epididymis to 5oc, as soon as possible, keeps sperm viable up to four days.1-3 maintaining this temperature minimizes the deleterious ph and osmolality changes associated with decay.1 investigators have also found that the percentage of high quality sperm decreases by half every 24 hours that sperm are stored in refrigeration.1 therefore, processing the semen as soon as possible increases the chance for success. an important consideration when harvesting epididymes is the time of year. most cervid species exhibit strict seasonal reproductive patterns based on photoperiod.4 it has been discovered that males display drastic changes in testicular size and function based on photoperiod changes.4 this change in testicular size produces alternating periods of fertility and infertility. additionally, aspermatogenesis is often found during the spring or summer antler regeneration period.4 in short, sperm will be found from antler hardening to antler shedding. additionally, if the sperm will undergo cryopreservation, epididymes should not be harvested from the animal longer than two hours post-mortem, but in many cases this is not possible.3 in such situations, it is still better to harvest the epididymides rather than not to. the entire scrotum containing the testes and their attached epididymides should be removed from the animal, placed in a closeable plastic bag, and put on ice or ice packs in a cooler. including as much of the vas deferens as possible may yield additional mature sperm. this package should be overnight shipped to the laboratory. the scrotum helps to insulate the testes and epididymides from direct contact with the ice and allows gradual cooling. an alternative method is to remove the testes with attached epididymides from the scrotum, place them into a plastic bag, and use a towel (paper or cloth) as insulation from the ice. methods of sperm collection the three most commonly described methods for obtaining sperm from the cauda epididymis include the flotation method, the cut method, and the flush method. the flotation method is performed by slicing or mincing the cauda in a buffered medium. the minced pieces are left in the medium for several minutes to allow the spermatozoa to swim into the medium.5 this medium is then filtered to recover the spermatozoa. clinical theriogenology • volume 3 number 4 • december 2011503 the cut method involves making several cuts with a blade or multiple punctures with a needle into the cauda epididymis and gently squeezing the spermatic fluid out of the cut tubules. the recovered sample is then diluted with extender.5 flushing the cuts with extender may help with sperm recovery. this author’s preferred method is the flush method. the epididymis and extender are allowed to warm to room temperature. once cleaned, the epididymis is carefully dissected away from the testicle and transected near the junction of the corpus epididymis and the proximal cauda. with experience, one will adjust the exact location of the transection based on the amount of sperm recovered. a plastic, latexfree conical tube is placed on a stable surface in an upright position. the cauda epididymis is placed into the opening of the plastic conical tube and the cut end of the vas deferens is catheterized. depending on the lumen size of the vas deferens, one can use a blunted 25 gauge x 5/8 inch needle up to a 21 gauge x 1 inch needle. a tom cat catheter or a teflon iv catheter may also be used. the catheter that is used must fit tightly to prevent any back-flushing. a 3-6 cc, latex-free syringe containing freezing extender (triladyl®, minitube, verona, wi) is used to retrograde flush the spermatic fluid out of the cauda epididymis into the collection tube. more extender may be used if necessary to complete the flushing process. additional cuts also may be needed in the cauda epididymis to facilitate flushing. an immediate benefit to this method is that the sample is already diluted with extender. a study was performed to compare the cut method and the flush method.5 it was discovered that a greater percentage of the flushed samples were free of red blood cells and less contaminated than the cut samples. furthermore, the pre-freezing and post-thawing analysis revealed better motility with the flushed samples than with the cut samples. evaluation of the sperm once the sperm sample has been obtained and diluted with extender, a drop is placed on a warm slide and examined for motility and morphology under 200x magnification. although the sperm stored in the tubules of the cauda epididymis are capable of fertilizing an oocyte, the motility and morphology of the sperm will appear considerably worse than an ejaculated sample. without undergoing normal ejaculation, the majority of the spermatozoa do not display progressive motility. if the sperm show any movement (wiggling), it is considered acceptable, but the more motility the better. total motility improves as the sperm sample warms. however, one study in iberian red deer indicated that harvested epididymal sperm refrigerated for 24 hours had 75% progressive motility. morphology will not resemble that of a normally ejaculated sample. it is not uncommon for the spermatozoa to retain their cytoplasmic droplets. bent mid-piece is another fairly common abnormality. one study discovered that the percentage of spermatozoa with bent mid-piece increased drastically after three days of storage in the epididymis at 5oc.2 this phenomenon is thought to be a result of thermotropic phase transitions in the plasma membrane lipids due to prolonged exposure to low temperatures.2 it is imperative that owners and breeders are aware of these differences from ejaculated sperm. this author recommends providing a sperm data sheet which describes the motility and morphology findings of the respective sample. this may help whomever buys or uses the semen realize these differences. freezing once motility and morphology data have been collected, concentration of the diluted sample is determined by using a hemocytometer. this is accomplished by placing drop of the diluted sample at the designated well on the hemocytometer and allowing the sample to spread across the gridded area. it is important to wait about 10 minutes before counting to allow all of the sperm to settle onto the hemocytometer. the count from the hemocytometer indicates the concentration of sperm (x 106) per milliliter of sample. the desired concentration of harvested epididymal sperm intended for cryopreservation is 75-100 x 106 sperm/half-milliliter straw. therefore, this equates to 150-200 x 106 sperm/milliliter of total sample. it is usually required to quantitatively adjust the sample to reach the desired concentration by adding more extender. once the desired concentration has been achieved, the entire sample is placed into the refrigerator for three to four hours to cool to 5oc. the cooled sample is clinical theriogenology • volume 3 number 4 • december 2011 504 then loaded into pre-labelled straws, which have also been cooled to 5oc. filling of the straws should be done under refrigeration. all straws should be pre-labeled with the buck’s name, species, owner/farm name, date, and name of the person or lab that processed the semen. it is also a good idea to have straws available with a pre-printed code (i.e., wtd 1234) that can be used if testicles arrive unexpectedly. this code can then be designated to that particular buck, owner, and date. filled and sealed straws are then frozen in nitrogen vapor by placing them onto a rack 5 cm above the level of the liquid nitrogen for 15 minutes. it is common practice at the author’s institution to plunge the vapor-frozen straws into the liquid nitrogen immediately prior to loading them into their respective storage canes. the loaded canes, which should be accurately labeled with the buck’s name, owner’s name and date, are then placed into a liquid nitrogen tank for long-term storage. summary epididymal sperm can be used to preserve genetic material from deceased males. although proper handling of the epididymides and sperm is important, acceptable pregnancy rates can be achieved using harvested epididymal sperm. acknowledgements i thank clifford shipley, dvm, dact and kim kensell, bs, cvt for support in this manuscript preparation. references 1. martinez-pastor f, guerra c, kaabi m, et al: decay of sperm obtained from epididymes of wild ruminants depending on postmortem time. theriogenology 2005;63:24-40. 2. soler aj, perez-guzman md, garde jj: storage of red deer epididymides for four days at 5oc: effects on sperm motility, viability, and morphological integrity. j exp zool 2003;295a:188-199. 3. fernandez-santos mr, martinez-pastor f, dominguez-rebolledo ae, et al: effects of long-term chilled storage of red deer epididymides on dna integrity and motility of thawed spermatozoa. anim reprod sci 2009;111:93-104 4. asher gw, berg dk, evans g: storage of semen and artificial insemination in deer. anim reprod sci 2000;62:195211. 5. martinez-pastor f, garcia-macias v, alvarez m, et al: comparison of two methods for obtaining spermatozoa from the cauda epididymis of iberian red deer. theriogenology 2006;65:471-485. clinical theriogenology • volume 3 number 4 • december 2011505 clinical theriogenology • volume 3 number 4 • december 2011 506 omniblank: 2011: efficacy of lyophilized deslorelin on induction of ovulation in mares efficacy of lyophilized deslorelin on induction of ovulation in mares ryan a. ferris, alicia r.g. lindholm, david b. scofield, patrick m. mccue equine reproduction laboratory, colorado state university, fort collins, co abstract options for induction of ovulation in mares include human chorionic gonadotropin (hcg) and potent agonists of gonadotropin-releasing hormone (gnrh) such as deslorelin acetate. when no commercially produced gnrh agonists are available, gnrh agonists can be acquired through compounding pharmacies. the objective of this project was to evaluate the efficacy of lyophilized deslorelin acetate to induce ovulation and to determine if the efficacy was adversely affected after 12 months of storage. mares in estrus were administered 1.5 mg of deslorelin (lyophilized2009, lyophilized2010, or aqueous deslorelin) as an intramuscular injection once a follicle  35 mm was detected in the presence of uterine edema. mares were subsequently examined once per day to determine the day of ovulation. a total of 66 mares were administered lyophilized deslorelin in 2009 and 135 mares were treated in 2010 with lyophilized deslorelin from the same initial lot stored for 12 months. thirty mares were administered liquid suspension deslorelin in 2010. there was no significant difference in mean days to ovulation among treatment groups with majority of treated mares (89.6%) ovulating within 48 hours after deslorelin administration. results of this study indicate that lyophilized deslorelin stored for 12 months, reconstituted in sterile saline, is equally as effective at inducing a timed ovulation in estrual mares as deslorelin in a liquid suspension. in conclusion, non-reconstituted vials of lyophilized deslorelin may be stored for at least 12 months without a significant loss in efficacy. keywords: ovulation, induction, mare, deslorelin, gnrh agonist introduction options for induction of ovulation in mares include hcg and potent agonists of gnrh such as deslorelin acetate.1-3 deslorelin and hcg are equally effective in inducing a timed ovulation when administered to mares in estrus with a follicle  35 mm in diameter.4 the interval from administration to ovulation has been reported to be approximately 36 hours for hcg and 40 hours for deslorelin.5 one advantage of deslorelin is that efficacy in inducing a timed ovulation is not reduced after multiple doses are administered during a single breeding season.6 in contrast, efficacy of hcg has been reported to decrease if used multiple times in a single breeding season.2,3 the implant version of deslorelin acetate (ovuplant™, fort dodge animal health, fort dodge, ia) is no longer commercially available in the united states. however, the gnrh agonist can be acquired through a variety of compounding pharmacies. this project was designed and completed prior to the release of a fda approved deslorelin product. the goals of the current study were to: 1) evaluate the efficacy of a lyophilized deslorelin preparation at inducing ovulation and 2) determine if lyophilized deslorelin could be stored at room temperature for 12 months and still retain clinical efficacy. materials and methods deslorelin an allotment of lyophilized deslorelin acetate produced in a single lot was acquired from a compounding pharmacy (applied pharmacy services, mobile, al) in april 2009. the allotment consisted of multiple glass vials each containing 15 mg of lyophilized deslorelin acetate. the shipment was divided into two batches, one batch was used beginning in april 2009 for the 2009 breeding season and the other batch was stored at room temperature and used beginning in april 2010 for the 2010 breeding season. individual vials of deslorelin were reconstituted with 10 ml of sterile 0.9 % saline as needed to yield 10 doses of 1.5 mg each. 99 clinical theriogenology • volume 3 number 2 • june 2011 a compounded preparation of deslorelin acetate in aqueous suspension (franck’s pharmacy, ocala, fl) was acquired in april 2010 for comparison to the lyophilized form. the dose of liquid suspension deslorelin was also 1.5 mg. mares mares utilized in this study were 2-12 years of age and were of light stock-type breeds. mares were examined by transrectal ultrasound to monitor follicular development and the quantity and degree of uterine edema. the diameter of the largest follicle was measured with electronic calipers and the edema score evaluated on a scale of 0 to 3, with 0 being no edema and 3 being maximal edema.7 once a follicle  35 mm was detected in the presence of uterine edema, mares were randomly assigned to receive 1.5 mg of deslorelin as an intramuscular injection. mares were subsequently examined once per day to determine the day of ovulation. statistical analysis comparison of follicular size at the time of treatment, uterine edema at the time of treatment, interval to ovulation and the percentage of mares ovulating within 48 hours were made by use of a oneway anova with post-hoc analysis by tukey’s test. values were considered to be statistically different at p < 0.05. all data are presented as the mean ± standard deviation. results a total of 66 mares received lyophilized deslorelin in 2009 and 135 mares were treated with lyophilized deslorelin in 2010 from the same initial lot and stored for 12 months. thirty mares were administered liquid suspension deslorelin in 2010. there was no significant difference (p >0.05) between 2009 and 2010 mares treated with lyophilized deslorelin in diameter of the largest follicle or uterine edema score at the time of deslorelin treatment (table). the 2009 lyophilized deslorelin treated mares had smaller follicles (p <0.05) at the time of treatment as compared to the mares administered liquid deslorelin. the 2010 lypholilized deslorelin treated mares had less uterine edema (p <0.05) at the time of treatment as compared to the mares administered liquid deslorelin. there was no significant difference in the interval to ovulation between mares treated with lyophilized deslorelin in 2009, mares treated with the same lot of lyophilized deslorelin in 2010, and mares treated with deslorelin in liquid suspension. there was also no difference in the percentage of mares ovulating within 48 hours after deslorelin administration between deslorelin treatment groups. fifteen of the 24 mares that failed to ovulate within 48 hours after administration of deslorelin did go on to ovulate within the subsequent 24 hours (within 72 hours after deslorelin treatment). these mares had significantly (p <0.05) smaller follicles (37.7  3.2 mm) at the time of deslorelin treatment than mares that ovulated by 48 hours (40.9  4.6 mm). there was no difference (p > 0.05) in uterine edema or month of year between mares that ovulated by 72 hours versus mares that ovulated by 48 hours post-treatment with deslorelin. combining all three deslorelin treatment groups, a total of 35 of 231 (15.2 %) treated mares ovulated within 24 hours while the majority of treated mares (172 of 231; 74.5 %) ovulated between 24 and 48 hours after deslorelin administration. mares that ovulated within 24 hours after deslorelin administration had significantly (p<0.05) larger follicles (45.3  4.6 mm) at the time of treatment as compared to mares that ovulated within 48 hours (40.9  4.6 mm). there was no difference in edema scores or month of year at the time of treatment between mares that ovulated by 24 or 48 hours after administration of deslorelin (data not shown). when the three deslorelin treatment groups were combined, 207 of 231 mares (89.6 %) ovulated within 48 hours of gnrh agonist administration (figure). of the remaining 24 mares, 15 (6.5 %) ovulated 3 days after treatment, 5 (2.2 %) developed hemorrhagic follicles, 3 (1.3 %) ovulated after hcg was administered (2 days after deslorelin treatment), and the dominant follicle of 1 (0.4 %) mare regressed. 100clinical theriogenology • volume 3 number 2 • june 2011 discussion deslorelin stimulates a prolonged release of luteinizing hormone (lh) from the anterior pituitary that induces ovulation of the dominant follicle in mares in estrus.8 mares may be administered deslorelin when a timed ovulation is desired, such as when stallion or semen availability is limited or in the use of cooled-transported semen or frozen semen. timing of deslorelin administration during the estrous cycle is critical for successful outcome. in light horse breeds, it is recommended that deslorelin be administered when the dominant follicle first develops to a diameter of 35 mm or greater and edema is present in the endometrium.4 with this treatment protocol, a majority of mares will ovulate within 48 hours after treatment. in the current study, the average interval from deslorelin treatment to ovulation, based on ultrasound examinations once per day, was 1.9  0.5 days. consistency in the interval to ovulation is an important attribute of an ovulation inducing agent. in the case of the lyophilized deslorelin, 90.0 % of treated mares ovulated within 48 hours after administration. mares that ovulated within 24 hours after treatment may have either responded exceptionally fast to the gnrh agonist or the dominant follicle may have already been under the influence of endogenous lh. the percentage of mares that ovulated within 24 hours after deslorelin treatment (15.2 %) was similar to the percentage of mares that ovulated within 24 hours after hcg administration (18.5 %) in a previous retrospective study.3 the larger follicle diameter in mares that ovulated within 24 hours (45.3  4.6 mm) as compared to mares that ovulated between 24 and 48 hours of the deslorelin administration (40.9  4.6 mm) suggests that mares that ovulated within 24 hours of treatment may have already been approaching ovulation secondary to a rise in endogenous lh. unfortunately, endogenous lh concentrations were not evaluated in this study. the obvious clinical implication of this observation is that the administration of deslorelin does not preclude a mare in estrus from ovulating within 24 hours. five of the 231 mares (2.2 %) treated with deslorelin developed hemorrhagic anovulatory follicles. this is lower than the percentage of anovulatory follicles reported in other studies9-11 and suggests that there is no causative relationship between deslorelin administration and formation of anovulatory follicles. results of this study indicate that lyophilized deslorelin, reconstituted in sterile saline, is equally as effective in inducing a timed ovulation in estrual mares as deslorelin in a liquid suspension. in addition, storage of lyophilized deslorelin at room temperature for 12 months did not adversely affect efficacy at inducing ovulation. this information is important in clinical practice, as non-reconstituted vials of lyophilized deslorelin may be stored until the next breeding season without a loss of efficacy. however, this study only evaluated efficacy at inducing ovulation after prolonged storage and did not address any potential loss of biological activity or potency. the dose of deslorelin used in the current study was 1.5 mg administered as an intramuscular injection. results of a recent study suggest that intramuscular doses of 1.5 mg, 1.0 mg and 0.5 mg are equally effective in inducing ovulation in mares.8 in summary, a dose of 1.5 mg of lyophilized deslorelin was effective in inducing ovulation in estrual mares. storage of lyophilized deslorelin at room temperature for 12 months was not associated with a decrease in efficacy. references 1. barbacini s, zavaglia g, gulden p, et al: retrospective study on the efficacy of hcg in an equine artificial insemination programme using frozen semen. equine vet educ 2000;12:312-317. 2. green jm, raz t, epp t, et al; relationship between utero-ovarian parameters in the ovulatory response to human chorionic gonadotropin in mares. proc annu conv am assoc equine pract 2007. p. 563-567. 3. mccue pm, hudson jj, bruemmer j, et al: efficacy of hcg at inducing ovulation: a new look at an old issue. proc annu conv am assoc equine pract 2004. p. 510-513. 4. mccue p, magee c, gee e: comparison of compounded deslorelin and hcg for induction of ovulation in mares. j equine vet sci 2007;27:58-61. 101 clinical theriogenology • volume 3 number 2 • june 2011 5. mckinnon ao, perriam wj, lescun tb, et al: effect of a gnrh analogue (ovuplant), hcg and dexamethasone on time to ovulation in cycling mares. world equine vet rev 1997;2:16-18. 6. mumford el, squires el, jochle e, et al: use of deslorelin short-term implants to induce ovulation in cycling mares during 3 consecutive estrous cycles. anim reprod sci 1995;39:129-140. 7. ferris ra, mccue pm: the effects of dexamethasone and prednisolone on pituitary and ovarian function in the mare. equine vet j 2010;42:438-443. 8. lindholm arg, bloemen ehg, brooks rm, et al: comparison of deslorelin and buserelin in mares: lh response and induction of ovulation. anim reprod sci 2010;121:68-70. 9. mccue pm, squires el: persistent anovulatory follicles. theriogenology 2002;58:541-543. 10. lefranc ac, allen wr: incidence and morphology of anovulatory haemorrhagic follicles in the mare. pferdeheilkunde 2003;19:611-612. 11. gastal el, gastal mo, ginther oj: the suitability of echotexture characteristics of the follicular wall for identifying the optimal breeding day in mares. theriogenology 1998;50:1025-1038. 102clinical theriogenology • volume 3 number 2 • june 2011 table. follicle diameter and edema score at time of treatment, interval to ovulation after treatment, and percentage of mares ovulating within 48 hours of deslorelin treatment. group (n) follicle size (mm) edema score interval to ovulation (days) % of mares ovulating within 48 hours lyophilized deslorelin (2009) 66 40.9 ± 4.4* 1.6 ± 0.7 1.9 ± 0.5 58/66 (87.9 %) lyophilized deslorelin (2010) 135 41.0 ± 5.0 1.4 ± 0.6* 1.9 ±0.5 123/135 (91.1 %) liquid suspension deslorelin (2010) 30 43.5 ± 4.9* 1.7 ± 0.6* 1.9 ± 0.4 28/32 (87.5 %) * within columns indicates a significant difference of p <0.05, ** indicates a significant difference of p <0.01) figure. interval from treatment to ovulation for mares administered deslorelin acetate (all treatment groups combined). 225 of the 231 mares are shown in the figure, 5 mares did not ovulate due to a hemorrhagic follicle, and the dominant follicle regressed in 1 mare. 103 clinical theriogenology • volume 3 number 2 • june 2011 2013: bovine leukemia virus in michigan beef bulls bovine leukemia virus in michigan beef bulls jessica zalucha,a daniel grooms,a ronald erskine,a bo norby,a paul coussens,b paul c. bartletta adepartment of large animal clinical sciences, bdepartment of animal sciences, michigan state university, east lansing, mi abstract bovine leukemia virus (blv) is a contagious retrovirus of cattle. little is known about the prevalence, risk factors, or potential health impact of blv in beef cattle. michigan beef bulls receiving breeding soundness examinations (bse) were tested for blv antibodies in serum using elisa to determine the crude and age-specific prevalence of blv in michigan beef bulls. our main objective was to measure the association between bse results and blv seropositivity. additionally, management surveys were administered to the bull owners to identify potential risk factors for blv infection. the crude blv prevalence was 24.7% with an age-specific prevalence increasing with age from 1.5% to 67%. this indicates that these animals have a very low rate of blv infection before they reach breeding age and then experience a very high rate of new infections once they begin natural breeding. fertility, as measured by a bse, did not appear to be affected by blv infection. scrotal circumference was the only measured bse component that was associated with blv infection. exposure to cows through natural breeding and the frequency of changing hypodermic needles were the only management risk factors associated with blv infection. the significant association between blv infection and the reuse of hypodermic needles supports the findings of others in suggesting that needle reuse may be an important means of blv transmission. keywords: cattle, leukosis, management, fertility, prevalence introduction enzootic bovine leukosis is a contagious retroviral disease of cattle caused by blv and is clinically characterized by a persistent lymphocytosis. in dairy cattle, infection becomes more prevalent with increasing age.1 most cattle remain asymptomatic, but between 30-40% of blv infected cattle will develop persistent leukocytosis. fewer than five percent of infected cattle develop malignant lymphosarcoma, which in the u.s. is the most common neoplastic disease of cattle discovered at slaughter.2 at least 20 nations have successfully eradicated blv from their cattle herds.3 surveys in locations outside of europe have reported a within-herd cow prevalence of 23-46% in dairy cattle.4-7 in michigan, the cow-level prevalence of blv in dairy cattle averaged 32.8%.1 few reports have studied the prevalence of blv in u.s. beef cattle, but they have estimated the animal-level prevalence to be 1.210.3%.8-11 in the 1960’s and1970’s when many european countries undertook blv eradication programs, animal health agencies in the u.s. and canada decided that blv was not a sufficiently costly disease to warrant national control. however, recent studies have suggested that blv prevalence has since increased dramatically, and that blv has a previously unappreciated subclinical economic impact on the cattle industry. according to roeber et al., 1.1% of market cow and bull carcasses were condemned at slaughter, and 14.9% of these were condemned due to malignant lymphosarcoma.12 in comparison, in 1979 only 0.8% of carcass condemnations were due to malignant lymphosarcoma.13 bovine leukemia virus infection has also been shown to decrease cow longevity and milk production in dairy cows.1,14 in addition, the presence of blv infection precludes the sale of exported animals, semen, embryos, and animal products to many countries. programs to control the spread of blv in cattle have largely focused on segregation of positive animals and other management interventions.15 however, management changes are only effective if they can be easily and inexpensively implemented by the producer and target modes of transmission that account for a significant proportion of the new infections. in a recent study of dairy cattle in michigan, reusing hypodermic needles, gouge dehorning, uncontrolled stable flies, reusing obstetrical (ob) sleeves, 451 clinical theriogenology • volume 5, number 4 • december 2013 and using natural breeding instead of artificial insemination (ai) were all found to be significantly associated with blv infection.15 these risk factors all relate to hematogenous transmission. trauma to the vagina and penis during natural breeding may facilitate blood-borne transmission of blv between cows and bulls and may be responsible for the decreased prevalence of blv in herds which only use ai for breeding.15 bulls used for natural breeding have the potential to spread blv to previously blv negative herds, and may also be responsible for increasing a herd’s overall blv prevalence. it has been reported that about one in three cow-calf operations (30.7%) borrowed, leased, or purchased a bull for use during the breeding season, and over half of the operations (53.3%) added bulls over 18 months of age; on average, 16.3 cows were mated to each yearling bull while 23.7 cows were mated to each mature bull.16 consequently, bulls could serve as a source of infection for cows if natural breeding is a common method of transmission. the objectives of this study were to determine the crude and age-specific prevalence of blv among beef bulls in michigan, to assess the possible impact of blv on bull fertility, and to identify risk factors for blv seroprevalence. materials and methods beef bulls that presented for bse’s as part of the michigan state university extension bse program were enrolled in this study. the bse’s were performed on 363 bulls from 113 owners in the spring of 2009 (156 bulls) and 2012 (207 bulls). bulls were categorized into six age groups (1-12 months, 13-24 months, 25-36 months, 37-48 months, 49-60 months, 61+ months) and six breed groups (angus, red angus, polled herefords, maine-anjou, simmental, other) for analysis. the bse’s were performed by one of the authors (dlg). established protocols for the bse’s were followed as described by hopkins and spitzer.17 a physical examination, scrotal circumference (sc) measurement, sperm motility, sperm morphology (normal, primary defects, and secondary defects), and the presence of white blood cells (wbc) in semen were recorded for each of the bulls. also indicated was whether the bull passed the bse, failed, or was deferred based on the specifications in hopkins and spitzer.17 blood samples from the median caudal vein (tail vein) were collected into serum separator tubes, centrifuged, and then submitted to the michigan state university diagnostic center for population and animal health. antibodies for blv were detected using a commercially available antibody capture elisa (bovine leukemia virus antibody test kit, veterinary medical research and development, pullman, wa). the optical density (od) for each sample was measured and recorded. bovine leukemia virus status was based on the od of the sample; those samples with an od value of greater than or equal to 1.0 were considered positive for blv. an eight question survey (appendix 1) was administered to the owner or manager of those bulls that were enrolled in the study in 2012 (n=207). the questions targeted previously identified risk factors for blv, including the number of heifers or cows to which the bull was exposed during the breeding season, whether the bull was tattooed or dehorned, whether fly control was utilized on the farm, and the extent of hypodermic needle reuse.15 statistical analysis was performed using sas (sas pc, cary, nc) to test for the association between blv status and od measurement and each of the management risk factors included on the questionnaire (type of fly control, number of herds serviced, number of cows serviced, whether the bull was polled, whether the cows to which the bulls were exposed were polled, the number of injections received per year, frequency of changing hypodermic needles, history of tattoos) as well as each of the measures of fertility comprising the bse (scrotal circumference, sperm motility, presence of wbc and normal, primary defect and secondary defect sperm counts). herd was included as a random effect in the analysis of each risk factor, and age group was also included as a confounder. the sas proc mixed was used for the analysis of blv elisa od and proc glimmix was used for the analysis of blv status. the age-specific prevalence of blv in beef bulls in this study was compared to that of dairy cows studied by bartlett et al.14 and fitted with a linear trendline (figure). the apparent difference in slope was 452clinical theriogenology • volume 5, number 4 • december 2013 tested using a glmmix model to predict blv status and a mixed model to predict od. age group was included as a significant predictor of blv status. results the overall prevalence of blv was 24.7% (95/385). age group was associated with blv status (p < 0.004; table 1), but breed group was not associated with blv status (p < 0.4505) or blv od (p < 0.9726; table 2). the age-specific prevalence of blv in beef bulls increased with each age group from 1.5% to 66.7% (figure). the sc was the only measured bse component associated with blv status (table 3). further analysis of sc was undertaken to adjust for breed group and to use age as a continuous variable instead of age group. regardless, sc continued to be significantly associated with both blv status and od. exposure of bulls to cows for natural breeding and frequency of changing hypodermic needles were the only studied management risk factors significantly associated with blv status (table 4). all but one of the bulls in the youngest age group had not yet been exposed to cows, and all of the older bulls had been exposed to cows. therefore, age and exposure to cows were inseparably confounded. frequency of changing hypodermic needles was analyzed to adjust for breed and age groups and was still found to be significant. bulls whose owners changed hypodermic needles between every animal had no blv (0/19) while those that changed needles about every ten animals had a prevalence of 17.5% (10/57) and those that changed about every 20 animals had a prevalence of 43.5% (37/85; p < 0.0317). the age-specific prevalence determined in this study does not directly reflect the rate of acquiring new infections because these data were collected at a single point in time rather than from a longitudinal study in which animals were followed forward in time and the rate of new infections directly measured. however, if transmission in the herds had reached an equilibrium ‘steady-state’, the slope of the lines in the figure suggest that the rate of acquiring new infections was almost twice as great among the beef bulls in this study as it was in dairy cows studied by bartlett et al.14 the interaction terms between animal type (beef or dairy) and age group were significant at p < 0.001 indicating that the relationship between age and blv in beef bulls was significantly different from that observed for dairy cows. table 1. age-specific prevalence of blv in michigan beef bulls age group* n prevalence (%) 0 65 1.5 1 140 14.3 2 75 32.0 3 35 40.0 4 18 50.0 5 30 66.7 * 0 = 1-12 months, 1 = 13-24 months, 2 = 25-36 months, 3 = 37-48 months, 4 = 49-60 months, 5 = 61+ months) table 2. breed group-specific prevalence of blv in michigan beef bulls breed group n prevalence (%) angus 178 29.2 red angus 68 22.1 polled hereford 33 0.00 maine-anjou 16 31.3 simmental 27 29.6 others 41 19.5 453 clinical theriogenology • volume 5, number 4 • december 2013 table 3. association between blv elisa results and measures of bull fertility. potential outcome blv positive blv negative p value for blv p value for od scrotal circumference (cm) 41.6 38.9 0.0198 0.0866 percent with fair or better motility 98.9 98.5 0.7570 0.8736 mean % normal sperm morphology 81.7 80.3 0.3823 0.8266 mean % sperm with primary defects 4.8 7.0 0.9989 0.9859 mean % sperm with secondary defects 13.1 12.4 0.4098 0.6926 mean wbc (#/hpf) 0.4 0.3 0.4055 0.5630 percent with passing bse 80.9 82.8 0.7296 0.8270 *above data are adjusted for age group and includes herd as a random effect variable table 4. association between blv elisa results and blv risk factors risk factor blv+ blvp value for blv p value for od percent bulls exposed to cows for breeding 97.8 53.3 <0.0001 <0.0001 mean number of herds to which the bull was exposed 1.0 0.7 0.3042 0.4316 percent using routine fly control 63.3 77.9 0.8668 0.8464 percent that reuse hypodermic needles 100.0 83.6 0.0317 0.0708 percent that used tattoos 49.1 70.9 0.4299 0.4682 figure. age-specific prevalence of blv in beef bulls and dairy cows *dairy cow data from bartlett et al.14 discussion in general, beef herds reportedly have a lower prevalence of blv than do dairy herds.4-10,18,19 a recent study of six michigan beef feedlot herds and six michigan dairy herds found that the blv seropositivity in dairy cattle was 48% while it was only 10% in beef cattle (cristina venegas, unpublished y = 12.594x 9.9947 y = 7.397x + 3.008 0 10 20 30 40 50 60 70 80 0 1 2 3 4 5 %  b lv  p o si ti ve age (years) percent of blv positive cattle in dairy and  beef herds beef bulls dairy cows 454clinical theriogenology • volume 5, number 4 • december 2013 data). this difference may be partially due to the younger average age of most feedlot beef cattle and to differences in management. regardless, since the prevalence of blv has generally been found to be lower in beef cows than in dairy cows, it is unexpected that the prevalence of blv in beef bulls is comparatively high (figure). the comparison of age-specific prevalence of blv in beef bulls compared to dairy cows outlined in the figure suggests that the rate of acquiring new infections was almost twice as great among beef bulls as it was in dairy cows. many possible reasons could exist for this apparent difference. beef bulls typically receive fewer injections with hypodermic needles and reproductive examinations with ob sleeves, which are both important risk factors for blv transmission in dairy cows.15 however, beef bulls might have greater exposure to biting flies and more natural sexual contacts than do dairy cows, both of which could have increased blv transmission in beef bulls. since blv is a retrovirus, prospects for an effective blv vaccine are problematic. due to the high prevalence of blv in the u.s., transmission of blv is probably best controlled by segregation and management interventions, as was done in europe, until the prevalence is sufficiently low that ‘test and removal’ procedures are economically feasible. therefore, identifying the most significant routes of blv transmission is important for establishing effective control measures. for example, one blv management program in a dairy herd involved the use of single-use needles and ob sleeves, disinfection of tattoo and dehorning equipment, and feeding of milk replacer and heat-treated colostrum.20 the prevalence of blv-infected heifers on the farm decreased from 44% to 17% in two years. several methods of hematogenous transmission have been suggested to have an association with blv prevalence, but it is difficult to determine the percent of new cases that can be attributed to each possible route of transmission.15, 21-25 this study did not find an association between blv and tattooing (table 4). this procedure is done at an early age, and transmission by such means should have been reflected by elisa positivity before one year of age. the blv prevalence in bulls that were one-yearold and younger was only 1.5% (table 1), so transmission occurring early in the bull’s life from tattooing, appears to be relatively rare. in addition, calves receiving colostrum from blv-positive cows have a greatly reduced risk of becoming infected with blv.26 this could further reduce their likelihood of contracting blv from procedures occurring early in life, such as tattooing. this study also failed to find an association between blv prevalence and the use of a fly control program as was found for a recent dairy cow study.15 the results of this study agreed with those of earlier studies in finding that the reuse of hypodermic needles was significantly associated with an increase in blv seropositivity.15,23 in addition, the dose-response demonstrated in this study supports the theory that this association might be causal in nature, as does the biological plausibility that blv-infected lymphocytes could be transferred via the immediate reuse of a hypodermic needle on multiple cattle. while it may be prudent to advise beef farmers to use new needles for every animal, acceptance of single-use needles may conflict with labor efficiency and economic return especially in larger herds that currently realize some economy of scale by vaccinating large numbers of cattle at the same time. further research will be required to determine if blv transmission via reuse of hypodermic needles is more expensive than the cost of labor and supplies needed for employing single-use needles. the bulls in this study had a very low prevalence of blv infection prior to reaching breeding age and then experienced a high rate of new infections once they begin natural breeding (table 1). therefore, natural breeding may play a significant role in blv transmission among beef bulls. this study found that blv prevalence was associated with increasing age in beef bulls, which may be due to the cumulative number of sexual contacts. however, other hematogenous exposures, such as biting stable flies and hypodermic needle use, also accumulate as the bull ages. therefore, this increasing rate of blv seropositivity in older cattle cannot necessarily be attributed to an increasing number of sexual contacts any more than to other exposures that also accumulate as a bull ages. however, this population of breeding bulls is unique in having a very large number of sexual contacts compared with either dairy or beef cows. natural breeding must be investigated as a potential route of transmission accounting for the 455 clinical theriogenology • volume 5, number 4 • december 2013 apparently high rate of new infections in beef bulls. attribution studies are needed to compare the relative importance of the various routes of blv transmission. erskine et al. found that natural service breeding of dairy heifers and cows was significantly associated with an increase in blv prevalence compared with herds that only utilized ai.15 according to the usda, an average of 16.3 beef cows are mated to each yearling bull while 23.7 cows are mated to each mature bull.16 as a beef bull grows older, he is exposed to more cows through natural service and, therefore, is potentially at a greater risk of contracting blv. if natural breeding was an important route of blv transmission within beef herds, one would expect much higher rates of infection among the beef bulls than among beef cows, because the bulls have many times more sexual partners than do the beef cows. this explanation is consistent with the relatively low rate of blv reported among the beef cow population. yeon choi, monke, and stott found that blv was not transmitted in the semen of seropositive bulls.27 however, penile and vaginal trauma could allow for hematogenous transmission during natural breeding. a previous study showed that the association between blv and natural breeding was more significant in heifers than in cows, which could be attributed to a greater likelihood of vaginal trauma in the smaller heifers.15 if future research confirms natural breeding as an important route of blv transmission, the increased use of ai on beef farms would be a potential disease control intervention. confirming that a breeding bull is blv-negative could also help to reduce transmission. since 30.7% of cow-calf operations borrow, lease, or purchase a bull for use during the breeding season, the use of a blv-infected bull for natural breeding could be a significant source of blv exposure to the herd.16 in addition, any bull owner wishing to maintain the blv-negative status of their bull may wish to prevent exposure via natural breeding to any blv-infected cows. an important objective of this study was to determine whether blv infection impacted the fertility of a bull as determined by a standard bse. the sc was significantly associated with blv positivity, and this association remained after adjusting for age and breed of the bull. those bulls that were positive for blv had a sc that averaged almost 3cm larger than those bulls that were negative. it is not clear why blv positive bulls may have a larger sc; this finding requires further investigation and analysis. further research into the bull’s ability to successfully breed a cow or heifer and produce a live calf is needed to more fully evaluate the impact of blv on fertility. bartlett et al. showed that dairy cow longevity was negatively impacted by blv infection.14 however, cows were not significantly impacted until after their first lactation when they were about three to four years of age. therefore, while young feedlot steers may not be significantly impacted by blv due to their truncated lifespan, older beef breeding cows and bulls may experience reduced longevity and productivity due to blv infection. similarly, impacts of blv on milk production are reportedly greater among older cows.28 if older beef cattle exhibit immune suppression, this might necessitate an increased rate of replacement. consequently, those farms that need to replace their bulls and cows more frequently may enjoy less discretionary culling.14 conclusion approximately 25% of beef bulls that presented for bse’s in michigan were blv positive. older bulls were more likely to be infected than younger bulls, and their apparent rate of new infections increased rapidly once they began functioning as breeding animals. bovine leukemia virus status did not appear to have a significant impact on fertility as measured by the bse. the reuse of hypodermic needles was associated with an increased prevalence of blv in beef bulls. acknowledgement this research was supported by funds from the michigan state university center for microbial pathogenesis. 456clinical theriogenology • volume 5, number 4 • december 2013 references 1. erskine rj, bartlett pc, byrem tm, et al: using a herd profile to determine age-specific prevalence of bovine leukemia virus in michigan dairy herds. vet med int 2012; doi: 10.1155/2012/350374 2. schwartz i, levy d: pathology of bovine leukemia virus. vet res 1994;25:521-536. 3. european commission: bovine and swine diseases–2011 annual report. section 3.3 enzootic bovine leucosis. accessed may 17, 2013; http://ec.europa.eu/food/animal/liveanimals/bovine/docs/final_report_2011_en.pdf 4. ott sl, johnson r, wells sj: association between bovine leukosis virus seroprevalence and herd-level productivity on u.s. dairy farms. j prev vet med 2003;61:249-262. 5. sargeant jm, kelton df, martin sw, et al: associations between farm management practices, productivity, and bovine leukemia virus infection in ontario dairy herds. j prev vet med 1997;31:211-221. 6. trono kg, perez-filgueira dm, duffy s, et al: seroprevalence of bovine leukemia virus in dairy cattle in argentina: comparison of sensitivity and specificity of different detection methods. vet microbiol 2001;83:235-248. 7. vanleeuwen ja, forsythe l, tiwari at, et al: seroprevalence of antibodies against bovine leukemia virus, bovine viral diarrhea virus, mycobacterium avium subspecies paratuberculosis, and neospora caninum in dairy cattle in saskatchewan. can vet j 2005;46:56-58. 8. baumgartener le, olson c, miller jm, et al: survey for antibodies to leukemia (c-type) virus in cattle. j am vet med assoc 1975;166:249-251. 9. burridge mj, puhr dm, hennemann jm: prevalence of bovine leukemia virus infection in florida. j am vet med assoc 1981;179:704-707. 10. gnad dp, sargeant jm, chenoweth pj, et al: prevalence of bovine leukemia virus in young, purebred beef bulls for sale in kansas. j appl res vet med 2004;2:215-219. 11. usda-aphis-veterinary services national animal health monitoring system: reference of 1997 beef cow-calf production management practices. colorado: centers for epidemiology and animal health; 1997. 12. roeber dl, mies pd, smith cd, et al: national market cow and bull beef quality audit–1999: a survey of producerrelated defects in market cows and bulls. j anim sci 2001;79:658-665. 13. thurmond mc, lapuz gr, farver tb, et al: retrospective study of four years of carcass condemnation rates for malignant lymphoma in california cows. am j vet res 1985;46:1387-1391. 14. bartlett pc, norby b, byrem tm, et al: bovine leukemia virus and cow longevity in michigan dairy herds. j dairy sci 2013; doi: 10.3168/jds.2012-5930. 15. erskine rj, bartlett pc, byrem tm, et al: herd-level determinants of bovine leukemia virus prevalence in dairy farms. j dairy res 2012;1-6. doi: 10.1017/s0022029912000520 16. usda-aphis-veterinary services national animal health monitoring system: part ii: reference of beef cow-calf management practices in the united states, 2007-2008. colorado: centers for epidemiology and animal health; 2009. 17. hopkins fm, spitzer jc: the new society for theriogenology breeding soundness evaluation system. vet clin north am food anim pract 1997;13:283-293. 18. usda-aphis-veterinary services national animal health monitoring system: bovine leukosis virus (blv) in u.s. beef cattle. colorado: centers for epidemiology and animal health; 1999. 19. usda-aphis-veterinary services national animal health monitoring system: bovine leukosis virus (blv) on us dairy operations, 2007; 2008. retrieved from aphis.usda.gov/dairy/dairy07/dairy2007_blv.pdf. 20. sprecher dj, pelzer kd, lessard p: possible effect of altered management practices on seroprevalence of bovine leukemia virus in heifers of a dairy herd with history of high prevalence of infection. j am vet med assoc 1991;199:584-588. 21. kobayashi s, tsutsui t, yamamoto t, et al: risk factors associated with within-herd transmission of bovine leukemia virus on dairy farms in japan. bmc vet res 2010;6.doi: 10.1186/1746-6148-6-1. 22. hasselschwert dl, french dd, hribar lj, et al: relative susceptibility of beef and dairy calves to infection by bovine leukemia virus via tabanid (diptera tabanidae) feeding. j med entomol 1993;30:472-473. 23. roberts d, lucas m, wibberley g, et al: an investigation into the susceptibility of cattle to bovine leukosis virus following inoculation by various routes. vet rec 1982;110:222-224. 24. weber af, moon rd, sorensen dk, et al: evaluation of the stable fly (stomoxys calcitrans) as a vector of enzootic bovine leukosis. am j vet res 1988;49:1543-1549. 25. hopkins s, digiacomo r, evermann j, et al: rectal palpation and transmission of bovine leukemia virus in dairy cattle. j am vet med assoc 1991;199:1035-1038. 26. nagy dw, tyler jw, kleiboeker sb: decreased periparturient transmission of bovine leucosis virus in colostrum-fed calves. j vet intern med 2007;21:1104-1107. 27. yeon choi k, monke d, stott jl: absence of bovine leukosis virus in semen of seropositive bulls. j vet diagn invest 2002;14:403-406. 28. erskine rj, bartlett pc, byrem tm, et al: association between bovine leukemia virus, production, and population age in michigan dairy herds. j dairy sci 2012;95:727-734. doi: 10.3168/jds.2011-4760. 457 clinical theriogenology • volume 5, number 4 • december 2013 appendix 2012 breeding soundness exams please give us you mailing address, email or fax so that we can send you the bovine leukemia virus serologic results when they are available in a few weeks. email: _______________________ fax: ____________________ (preferred) (preferred) mailing address: name__________________ address _______________________________ city _______________________________ state _____ zip _________________ 1. about how many cows or heifers has this bull serviced in its life? __ none __ 1-49 __ 50-199 __ 200-500 __ over 500 2. about how many herds of cattle has this bull serviced in its life? _____ herds 3. does this bull's current herd use a fly control program? __ no __yes (please describe what is used and how often) ____________________________________________________________ 4. this bull was: ___ polled (dehorning not needed) ___ gouge dehorned ___ burn dehorn ___ paste dehorn __ other (describe _____________) 5. the cows to which this bull is exposed are mostly: ___ no exposure yet ___ polled ___ gouge dehorned ___ burn dehorned ___ paste dehorned __ other (describe _____________) 6. describe how many total injections (vaccination, supplements, etc.) this bull has received in his lifetime about _____ per year for each of _____ years. 7. about how many tattoos has this bull received? ________ 458clinical theriogenology • volume 5, number 4 • december 2013 9 abstracts opening session cytoplasmic blebbing as an indicator of cryodamage in bovine embryos soon hon cheong, abdallah wagih abdelhady, yoke lee lee, mariana diel de amorim, luis henrique de aguiar department of clinical sciences, college of veterinary medicine, cornell university, ithaca, ny, usa cryopreservation is an important tool for bovine embryo transfer. however, cryopreservation results in 10 13% lower pregnancy rates compared to fresh embryo transfer. the mechanisms and cellular response of embryos to cryodamage have not been fully elucidated. most blastocysts have a collapsed blastocoel after cryopreservation that will reform if the blastocyst survives and undergoes hatching. using time-lapse videography, we observed a unique cellular response of blastocysts to cryopreservation, where the cytoplasm formed large clear blebs in many cryopreserved blastocysts. this cytoplasmic blebbing has been reported in human stem cells in response to dissociation and is thought to be a precursor to apoptosis but has not been reported in embryos or as a response to cryoinjury. objective was to determine if cytoplasmic blebbing is associated with poor postthaw outcomes in bovine embryos. we hypothesized that embryos with cytoplasmic blebbing have decreased likelihood of hatching and increased risk of degeneration. slaughterhouse-derived bovine embryos were randomly assigned to a sham treatment where the embryos were exposed to cryopreservation media and loaded into straws but not cryopreserved (no-freeze group), or cryopreserved using the direct-transfer slow cooling method (frozen) group. embryos were then placed in time-lapse culture dishes and observed for 48 hours for blebbing, hatching, or degeneration. differences in proportions between groups were evaluated using logistic regression anova in jmp pro v16. blebbing was observed in only 3/88 (3.4%) blastocysts in the no-freeze group, whereas most of the frozen blastocysts 77/102 (77.5%) had blebbing (p < 0.0001) suggesting strong collinearity between treatment group and blebbing. postthaw hatching rate was lower 63/102 (61.8%) for the frozen group compared to the no-freeze group 68/88 (77.3%; p = 0.0203). a similar outcome was observed for blebbing where hatching rate was lower in bleb 46/82 (56.1%) compared to the no-bleb 85/108 (78.7%; p = 0.0009). interaction between treatments and blebbing was different (p = 0.0204) with hatching rate. degeneration rate was higher (p = 0.0026) for the frozen group (26/102, 25.5%) compared to no-freeze group (8/88, 9.1%). a similar outcome was observed for blebbing where degeneration rate was higher (p = 0.0004) for bleb 24/82 (29.3%) compared to no-bleb 10/108 (9.3%). interaction between blebbing and treatment was not significant for degeneration. as treatment and blebbing were collinear, when both were offered to the model, treatment became not significant and was dropped. this is the first description of cytoplasmic blebbing as a response of embryos to cryodamage. blebbing was associated with lower hatching rates and higher degeneration rates. blebbing is thought to be a precursor to apoptosis; however, some embryos that exhibit blebbing can recover, hatch, and avoid degeneration. time-lapse monitoring of embryos for blebbing postthaw may be a useful and noninvasive indicator of cryodamage to select embryos for transfer. keywords: cytoplasmic blebbing, cryodamage, bovine, embryos shifts in uterine microbiome associated with pregnancy outcomes at first insemination and clinical cure in dairy cows with metritis caio figueiredo,a,b hugo monteiro,c federico cunha,a fábio lima,c klibs galvão,a rafael bisinottoa adepartment of large animal clinical sciences, d. h. barron reproductive and perinatal biology research program, university of florida, fl, usa bdepartment of veterinary clinical sciences, washington state university, wa, usa cdepartment of population health and reproduction, university of california, ca, usa reproductive efficiency is a key component for sustainability of dairy herds as daily costs associated with nonpregnant cows increase across lactation stages. metritis is a major contributor to reduced reproductive efficiency, as cows with metritis have reduced risk of pregnancy per artificial insemination (ai), greater risk of pregnancy loss, and require more time to achieve pregnancy than unaffected cows. despite the detrimental impacts on uterine health, cyclicity, and reproductive performance associated with metritis, 30% of cows with metritis establish pregnancy after first ai. these data suggest the existence of mechanisms linked to fertility loss that are not present in every cow with metritis. clinical cure failure of metritis has also been associated with negative impacts on subsequent uterine health and fertility. differences in uterine microbiome associated with clinical cure failure in cows with metritis were reported and shifts associated fertility outcomes in cows with endometritis. therefore, the hypotheses of the present study were: 1. the uterine microbiome in cows with metritis that become pregnant at first ai differs from cows with metritis that fail to become pregnant; 2. the microbiome of cows with metritis that become pregnant at first ai is similar to cows without metritis that achieve pregnancy; and 3. clinical cure failure is associated with differences in uterine microbiome. objectives were to assess differences in uterine microbiome associated with clinical cure failure and subsequent pregnancy outcome in dairy cows treated for metritis. lactating holstein cows diagnosed with metritis (n = 43) characterized by the © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.10001 10 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 presence of reddish-brownish, watery, and fetid vaginal discharge were paired with counterparts without metritis (n = 42) based on parity and days postpartum. uterine contents were collected through transcervical lavage at the time of diagnosis (day 0); 5 days later, after completion of antimicrobial therapy for cows with metritis (day 5); and at 40 days postpartum. vaginal discharge in cows with metritis was evaluated on day 5 for definition of clinical cure. uterine microbiome was evaluated by sequencing of the 16s rrna gene. despite similar alpha-diversity on day 0 based on chao1, shannon, and inverse simpson indexes, metritis was associated with differences in beta-diversity. prevalence of porphyromonas, bacteroides, and veillonella was higher in cows with metritis, whereas, streptococcus, sphingomonas, and ureaplasma among other genera were more prevalent in cows without metritis. differences in beta-diversity between cows with metritis and counterparts without metritis persisted on day 5; however, no differences in uterine microbiome were observed between cows with metritis that cured after antimicrobial therapy and those with clinical cure failure on day 0 or 5. although differences in richness and diversity in the uterine microbiome 40 days postpartum associated with metritis and pregnancy were observed, no differences in beta-diversity were associated with pregnancy outcomes or metritis 40 days postpartum. in summary, no relationship between the uterine microbiome and pregnancy outcomes or clinical cure was observed, indicating that there are other mechanisms associated with recovery of fertility, and  clinical cure aside from uterine changes in bacterial community. keywords: uterus, microbiome, fertility, cure, metritis, dairy increased piezo drilling intensity does not appear to adversely affect cleavage or blastocyst rates following equine intracytoplasmic sperm injection charles scoggin,a alaina barhorst,a etta bradecamp,a peter sheerin,a maria schnobrich,a stephanie walbornn,b erin lohbeck,a ashley buchanan,a anne sheerina arood and riddle equine hospital, lexington, ky, usa brood and riddle equine hospital, wellington, fl, usa piezo intracytoplasmic sperm injection (piezo-icsi) utilizes pulses generated from a piezo drill (pd) for capture and immobilization of sperm, and for breaching of the zona pellucida and oolemma to deposit a sperm into the ooplasm. a blunt and smooth micropipette is utilized during piezo-icsi that differs from conventional-icsi wherein a beveled and spiked injection tip is used. the boring power of a pd can be adjusted by changing the intensity, speed, and number of the pulses. studies in humans and horses suggest improved embryonic development rates using piezo-icsi compared to conventional-icsi. however, piezo-icsi requires added expenses, training, and fine-tuning. in our laboratory, the learning curve with the pd was steep and hampered by limited information on proper drill settings. we thus performed a retrospective study to evaluate cleavage and blastocyst rates using 2 different intensities (i) of piezo pulses for equine piezo-icsi using an eppendorf piezoxperttm pd. one setting (pi10) utilized i = 10 whereas the other (pi30) used i = 30. the speed (10) and number of pulses (infinity) remained constant across the 2 different settings. oocytes were collected via transvaginal aspiration (tva), held overnight in a commercial embryo holding media (emcaretm; icpbio reproduction, spring valley, wi, usa) and placed in a commercial maturation media (eq-ivm, ivf-biosciences, falmouth, cornwall, uk) for ~ 30 hours. oocytes were then denuded and evaluated for the presence of a polar body. mature oocytes were fertilized with a single, motile and morphologically normal sperm using either pi10 or pi30. these presumptive zygotes (pzs) were placed back in maturation media for ~ 40 minutes and transferred to 500 µl of 1 of 2 culture medias (eq-ivc-1, ivf biosciences; or global®, life global group) under oil at 38.3ºc in an atmosphere of 6% co2 and 6% o2 and 88% n2. on day 5, pzs were assessed for cleavage, and cleaved embryos were moved to 1 of 2 sequential culture medias (eq-ivc-2, ivf biosciences; or dmem/f-12 [sigma-aldrich #d6421] supplemented with 6 ml/liter 1 n naoh and 10% fbs) and cultured under oil and at the same temperature and atmosphere. blastocyst development was assessed from days 7 through 10. data were analyzed using chi-square. a total of 845 immature oocytes were collected from 1,422 follicles punctured (59.4% recovery rate). of these, 457 oocytes matured (54.0% maturation rate); 289 oocytes were fertilized using pi10, and 168 oocytes were fertilized using pi30. cleavage rates between pi10 (176/289; 60.9%) and pi30 (104/168; 61.9%) were similar (p = 0.91706). blastocyst rates were also similar between pi10 (24/289; 8.3%) and pi30 (17/168; 10.1%). in conclusion, increasing the intensity of piezo pulses during piezo-icsi may not hinder embryonic development rates; however, controlled studies are needed to determine the optimal settings of the pd during equine piezo-icsi. keywords: in vitro equine embryo, intracytoplasmic sperm injection, piezo drill horse (equus caballus) and mule (equus mulus) embryo morphometry giorgia podico, kianna spencer, igor canisso department of veterinary clinical medicine, university of illinois urbana-champaign, urbana, il, usa donkeys are used worldwide to produce mules that are considered prized animals for leisure, show, and fieldwork. the different number of chromosomes fits the hypothesis that aneuploidy and other nuclear abnormalities stand behind the higher rate of early pregnancy losses in mule pregnancies. this study aimed to compare the morphometry of equine and mule embryos. the hypothesis was that the micronuclei and nuclear fragmentation indexes are higher in mule embryos than in horse embryos. twenty-two mares were randomly assigned in a crossover design to receive semen from both species. twelve horse and 13 mule embryos were obtained from 44 estrous cycles of mares. all mares were bred by a fertile small standard jack or a fertile quarter horse stallion. embryos were recovered 8 days after ovulation and classified regarding the stage of development and quality (compactness of the blastomeres, size, appearance of perivitelline space, color, presence of extruded cells, cell granulation, and cytoplasmic fragmentation) with a score from 1 (excellent) to 5 (degenerate). embryos were stained with hoechst33342, and images were acquired with a fluorescence microscope coupled with a 60 x objective (keyence bz-x, keyence corporation of america, itasca, il, usa). nuclei were categorized as compact, mitotic (prophase, metaphase, anaphase of mitosis), or fragmented; micronuclei were identified as small extranuclear dna bodies within the same cell. the fragmented, mitotic, http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 11 and micronuclei indices were calculated based on their proportion over the total amount of nuclei counted. embryo size and nuclear morphometry were assessed through imagej. data were analyzed with graphpad using anova and student’s t-test; significance was set at p < 0.05. mule embryos were numerically higher (p > 0.05) than horse ones. one set of twins was recovered from a mare bred to the stallion that had a double ovulation; a mule and horse embryo were both recovered from 8 mares. there were no differences (p > 0.05) in size between mule and horse embryos (915.5 ± 288 versus 575.8 ± 69.6 µm). the mule embryos scored grade 1 (n = 8) or grade 2 (n = 5); similarly, the horse embryos scored grade 1 (n = 7) or grade 2 (n = 6). the evaluation of the nuclear morphometry revealed that horse and mule embryos had a similar number (p > 0.05) of compact nuclei per sector (148.7 ± 6.8 versus 156.5 ± 8.5); however, the number of mitotic nuclei tended to be higher in mule embryos (5.2 ± 0.82) than in horse embryos (3.3 ± 0.3). the fragmented nuclei index was similar (p = 0.3818) between mule (0.25 ± 0.1%) and horse (0.22 ± 0.1%) embryos, the mitotic nuclei index was higher (p  = 0.0209) in mule embryos (3.2 ± 0.4%) than in horse embryos (2.2 ± 0.2%) and the micronuclei index was similar (p = 0.7666) between mule (0.02 ± 0.02%) and horse (0.01 ± 0.01%) embryos. this is the first study to compare the embryo morphology of mares bred with donkeys and horses. equid embryos shared similar nuclear ultrastructure features, except that mule embryos had a higher mitotic index. keywords: hybrid embryos, nuclear morphometry, micronuclei index, mitotic index metabolome and proteome of the seminal plasma and semen of horses and donkeys giorgia podico, igor canisso department of veterinary clinical medicine, university of illinois urbana-champaign, urbana, il, usa clinical experience suggests that mares bred with donkey semen (donkey-s) develop a milder postbreeding inflammatory response and achieve higher pregnancy rates than when they are bred with horse semen (horse-s). the presence and effect on the endometrium of pro and antiinflammatory molecules (e.g. prostaglandins, prostaglandin e2, 19-hydroxypge, secretory leukocyte protease inhibitor) contained in the seminal plasma have been demonstrated in other species; however, literature about this topic in equids is scant. this study aimed to investigate the metabolome and proteome of donkey-s and seminal plasma (donkey-sp), and horse-s and seminal plasma (horse-sp). we hypothesized that donkey-sp contains more antiinflammatory molecules than horse-sp. semen (n = 9) and seminal plasma (n = 9) were collected from a single adult quarter horse stallion and a single adult jack; 3 pooled samples were submitted for extraction and quantification by liquid chromatography-tandem mass spectrometry. statistical analyses were carried out with metaboanalyst; data were normalized by sum, not transformed, and then auto-scaled. gene ontology was evaluated with strings and panther. a total of 118, 80, 333, and 251 proteins were identified in donkey and horse-sp, and donkeyand horse-s, respectively. there were 69 proteins in common between donkeyand horse-s, and 16 proteins in common between donkeyand horse-sp. noteworthy findings; there were 42 (52%) proteins in common between horse-s and horse-sp and 57 proteins (49%) in donkey-sp were in donkey-s. binding was the main molecular function in horse-s and horse-sp whereas catalytic activities were the main functions of proteins in donkey-s and donkey-sp. cluster analysis revealed that the metabolomic composition of donkeyand horse-s and donkeyand horse-sp was significantly different. metabolomic fingerprinting revealed a higher number of anti-inflammatory metabolites (e.g. choline, epinephrine, palmitoleic acid, and palmitic acid) in donkey-sp compared to horse-sp. further in vitro and in vivo studies are warranted to evaluate their interactions with the mare endometrium. keywords: horses, donkeys, seminal plasma, endometritis, proteins uterine edema and fluid accumulation associations with progesterone concentrations in early pregnant mares ilaria colombo,a,b humberto magalhaes,a giorgia podico,a gaetano mari,b igor canissoa adepartment of veterinary clinical medicine, university of illinois urbana-champaign, il, usa bdepartment of veterinary medical sciences, university of bologna, ozzano dell’emilia, italy uterine edema is a useful clinical parameter to determine the readiness of a follicle to respond to induction agents and as a uterine infection/inflammation parameter. the number of days in estrus promotes a favorable environment for the establishment of a pregnancy. conversely, uterine fluid accumulation 48 96 hours after breeding has been linked with persistent breeding induced endometritis in mares. progesterone has a pivotal role during early pregnancy in maintaining and preparing the uterus to support embryo growth and development. objective was to assess the relationships between uterine edema, and fluid accumulation in mares and progesterone in early pregnant mare. we hypothesized that uterine edema and fluid accumulation positively and negatively, respectively, affect progesterone concentrations in early pregnant mares. a retrospective study was conducted with 174 cycles. when in estrus, mares were assessed for the presence or absence of uterine edema and fluid accumulation, induced to ovulate with deslorelin acetate, and bred with cooled shipped semen. mares had pregnancy diagnosis at 15 days after ovulation, with measurements of the embryonic vesicles and assessment of progesterone concentrations with a chemiluminescent assay (immulite 1,000). mares were reexamined for pregnancy on days 25, 45, and 60 days after ovulation. end points considered during the retrospective analyses included, mare, number of days with uterine edema, uterine fluid accumulation after breeding, progesterone concentrations, and embryo size. linear mixed-effect models were used to determine the influence of all the factors on progesterone concentrations and embryo vesicle size. postbreeding (0 96 hours) uterine fluid accumulation was associated with a decrease (-1.13 ng/ ml; p = 0.009) in progesterone concentrations. in addition, mares experiencing early pregnancy loss (< 65 days) presented with lower (-2.96 ng/ml; p = 0.05) progesterone concentrations than mares with normal pregnancies. longer periods of uterine edema, were associated with higher (p = 0.0003) progesterone concentrations at the first pregnancy diagnosis interestingly, mares presenting with low endogenous http://dx.doi.org/10.58292/ct.v15.10001 12 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 progesterone concentrations (< 5 ng/ml) that were supplemented with progestins had an increase (p = 0.09) in average conceptus size (3.19 mm) from 10 to 45 days after ovulation. in conclusion, the number of days with uterine edema positively influenced progesterone concentrations in early pregnancy whereas uterine fluid accumulation negatively influenced progesterone concentrations. finally, these findings provided an intriguing observation on the positive impact of progestins on the embryo vesicle size that could be related to an improved uterine environment despite low endogenous progesterone concentrations. the results of this study need to be confirmed under controlled conditions. keywords: mares, estrus, endometritis, embryo, progestins evaluation of aerobic culture results and the expression of embryonic transcripts from in vivo derived embryos exposed to media with and without antibiotics jessica cowley,a rachel hollingsworth,b paul dyce,b jessica klabnik,a richard hopper,a lamia briand-amirat,c julie gard schnuellea adepartment of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa bdepartment of animal sciences, college of agriculture, auburn university, auburn, al, usa coniris, nantes-atlantic college of veterinary medicine and food science, nantes, france centers for disease control and prevention reports that more than 2.8 x 106 antibiotic-resistant infections occur in the united states each year leading to 35,000 deaths. usage of antibiotics in embryo collection media may be unnecessary and detrimental to in vivo-derived embryos. objective was to determine if embryo collection media with (ab+ treatment) or without antibiotics (abtreatment) impacted the number of embryos recovered, bacterial growth, and abundance of embryonic transcripts related to apoptosis and free radical scavenging. we hypothesized that there are no differences in embryo recovery with and without antibiotic treatments but an impact on embryonic transcripts regulating apoptosis and free radical scavenging. in a crossover study design, 6 superovulated angus cross cows underwent 2 conventional (day 7) uterine body embryo collections 77 days apart. commercial embryo flush media with antibiotics (abt complete flush, abt 360, pullman, wa, usa) was utilized when cows were assigned to the ab+ treatment. commercial flush media without antibiotics (abt complete flush without antibiotics, abt 360) was utilized for the abtreatment. during each embryo collection procedure, 5 samples were submitted to the thompson bishop sparks alabama state diagnostic lab for aerobic culture. the highest-grade embryos available were used for mrna rt-qpcr analysis. rna from individual embryos was isolated utilizing the picopure™ rna isolation kit (thermo fisher scientific, waltham, ma, usa) and quality was confirmed using an agilent 2100 bioanalyzer (agilent technologies, santa clara, ca, usa). rna samples, passing quality checks, were used to synthesize cdna using a qscript cdna synthesis kit (quantabio, beverly, ma, usa). subsequent molecular analysis using sybr based real-time rt pcr was completed (perfecta sybr, quantabio). the relative fold change was determined for abundance of superoxide dismutase 1 (sod-1), bcl2-associated x (bax) and tumor protein p53 (tp53) normalized to histone 2a (h2a). treatment differences were determined by unpaired student’s t-test with significance set at p < 0.05. total number of embryos recovered between treatments was not different (p = 0.09) nor the relative fold change of expression of sod-1, bax, and tp53 (p > 0.05). no bacterial growth was detected in samples, except for 1 sample from a single cow. this sample from the unopened bag of embryo flush media grew microbacterium testaceum, a suspected environmental contaminant. based on study findings, utilization of embryo flush media without added antibiotics may be a viable option for reducing antibiotic use in food producing species with no detrimental effects on embryo production. keywords: antibiotics, embryo, superovulation, cows hypoluteoidism in a pedigree of portuguese water dogs lily lewis, aime johnson, robyn wilborn college of veterinary medicine, auburn university, auburn, al, usa hypoluteoidism is a rare cause of pregnancy loss in dogs in which progesterone decreases to < 2 ng/ml during mid to late pregnancy with no apparent underlying cause. a heritable component has been theorized for the broad range of progesterone concentrations reported throughout normal pregnancy (e.g. sporting and working groups have lower concentrations throughout pregnancy).1 two portuguese water dog (pwd) dogs, a dam and female offspring, were diagnosed and treated for hypoluteoidism during 4 successful pregnancies (2 litters each). first dog was presented for a workup of unexplained infertility;2 breeding was attempted on 3 previous estrous cycles with no resulting pregnancy. serum progesterone concentrations (p4) and vaginal cytology were used to determine the day of ovulation and peak fertile window; transcervical insemination (tci) was performed on days 2 and 4 using fresh-extended semen. pregnancy was confirmed on day 26 via transabdominal ultrasonography. due to a family history of suspected hypoluteoidism, serum progesterone concentrations were obtained throughout the second half of pregnancy. twice weekly p4 values were obtained starting on day 26 (16.9 ng/ml), with a steady declined observed. supplementation with oral altrenogest (regu-mate®, merck animal health, usa) at 0.088 mg/kg once a day began on day 45 (6.5 ng/ml). serum p4 concentrations continued to decline, reaching a nadir of 1.4 ng/ml on day 53. cesarean surgery was performed on day 60 and a litter of 4 viable pups was delivered. dog was bred again the following year using fresh extended semen via vaginal insemination (on days 2 and 3) and tci (on day 4). serum p4 concentrations were monitored from day 33 (12 ng/ml), and a similar decreasing trend was observed. on day 43, p4 concentrations were 5.3 ng/ml and altrenogest treatment was initiated, with declining concentrations reaching a nadir of 2.3 ng/ml on day 47. cesarean surgery and ovariohysterectomy were performed on day 61, delivering a healthy litter of 6 pups. upon sexual maturity, a female from the second litter (previously mentioned) was presented for ovulation timing and breeding management. breeding was performed with cool-shipped semen via vaginal insemination (on day 1) and tci (on day 4). pregnancy was confirmed on day 32, and p4 http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 13 concentrations were 11.2 ng/ml. similar to the aforementioned pregnancies, p4 was monitored throughout the second half of pregnancy and altrenogest treatment was initiated on day 51 (p4 concentrations were 3.4 ng/ml). serum p4 concentrations continued to decline (1.6 ng/ml on day 56 and 0.7 ng/ml on day 60). a healthy litter of 9 pups was delivered via cesarean surgery on day 63. a subsequent litter was successfully managed in the same dog, with pregnancy confirmed on day 35 (p4 concentrations were 14.2 ng/ml). treatment with altrenogest began on day 47 (p4 5.9 ng/ml). during her final few days of pregnancy, p4 concentrations continued to decline with values reaching 2.3 ng/ml on day 60 and 1.1 ng/ ml on day 63. cesarean surgery was performed on day 63, delivering a litter of 8 pups. although other cases of hypoluteoidism have been reported,3-5 to our knowledge this is the first confirmation of hypoluteoidism diagnosed in first-degree related individuals. thus, investigation into the heritability of this condition should be strongly considered. keywords: hypoluteoidism, progesterone, pregnancy, abortion, pedigree references 1. hinderer j, ludeke j, riege l, et al: progesterone concentrations during canine pregnancy. animals (basel) 2021;11:7-9. 2. shumack b, wilborn rr, johnson ak: management of a high-risk pregnancy in a portuguese water dog. clinical theriogenology 2017;9:486. 3. görlinger s, galac, s, kooistra, hs, et al: hypoluteoidism in a bitch. theriogenology 2005;64:213-219. 4. zedda mt, bogliolo l, antuofermo e, et al: hypoluteoidism in a dog associated with recurrent mammary fibroadenoma stimulated by progestin therapy. acta vet scand 2017;59:1-6. 5. johnson ca: high-risk pregnancy and hypoluteoidism in the bitch. theriogenology 2008;70:1424-1430. bovine oviductal organoids and their extracellular vesicles as a biomimetic model to develop a novel contraceptive riley thompson,a ahmed gad,b mindy meyers,a nico menjivar,b dawit tesfaye,b fiona hollinsheada adepartment of clinical sciences, colorado state university, fort collins, co, usa bdepartment of biomedical sciences, colorado state university, fort collins, co, usa organoids are 3d, spherical cell clusters formed in vitro that are capable of regeneration and self-organization with similar function to their tissue of origin. extracellularvesicles (evs) are nanoparticles containing bioactive molecules (e.g. lipids, proteins, and nucleic acids) that are secreted by cells to induce molecular changes in recipient cells. evs produced by organoids are more similar to those produced in vivo because organoids retain more in vivo-like properties than other cell culture models. evs have been exploited for use in assisted reproductive technologies to create a more biomimetic environment for embryo production and for their natural delivery mechanisms by loading them with therapeutic cargo to treat various pathologies. evs loaded with crispr-cas9 ribonucleoproteins (rnps) may be a novel method for delivery of a permanent contraceptive by localized knockout of genes governing fertility. our objectives were to: 1. characterize bovine oviductal organoids and their secreted evs; and 2. load evs with rnps targeting the progesterone receptor (pgr) gene. we hypothesized that: 1. bovine oviductal organoids maintain similar protein expression and function as their in vivo counterpart; and 2. evs produced by oviductal organoids can be successfully loaded with specific crispr-cas9 rnps. bovine oviductal organoids (n = 13 cows) were cultured for up to 77 days (passaged every 7 14 days) and spent culture medium was collected for isolation of evs via differential ultracentrifugation. first, organoids were assessed via brightfield imaging, histology, and immunohistochemistry (ihc). evs were characterized using nanoparticle tracking analysis (nta), western blot (wb), transmission electron microscopy (tem), and uptake into epithelial cells of the oviductal organoids after fluorescently labelling evs with pkh26. then, evs were loaded with rnps targeting pgr using exofecttm transfection kit or electroporation, and size exclusion chromatography was performed to remove any unloaded rnps. wb was then performed to assess cas9 protein in the evs compared to pbs control. brightfield imaging and histology demonstrated round cellular clusters with a central lumen, consistent with other oviductal organoid reports. ihc findings were positive immunostaining for pgr, oviduct-specific glycoprotein (ovgp1), foxj1 (cell ciliation), and periodic acidschiff (secretory function). evs assessed by nta and tem were 50 150 nm in diameter, and wb indicated the presence of ev-specific surface proteins (cd63, flot1, tsg101) and the absence of cell-specific cytochrome c (cycs). ev uptake was confirmed by fluorescence signal in recipient organoid cells following co-culture of organoids with pkh26-labelled evs. furthermore, evs loaded with rnps using exofecttm demonstrated a dark band of cas9 protein on wb, indicating evs were loaded at a high rate, compared to low or absent expression in pbs control or after electroporation. these results demonstrated for the first time that bovine oviductal organoids display in vivo-like morphology, protein expression, and function, indicating they are a biomimetic model of the bovine oviduct to assess ev interaction with oviductal cells. additionally, evs secreted by organoids can be loaded with rnps to potentially facilitate transfection of oviductal cells as a novel next generation contraceptive in various species. keywords: cows, cell culture, in vitro, extracellular vesicles, crispr-cas9 http://dx.doi.org/10.58292/ct.v15.10001 competition session cryopreservation of stallion sperm using commercially available cryoprotectant combinations brittany middlebrooks,a james graham,b paula moffett,a patrick mccuea adepartment of clinical sciences, colorado state university, fort collins, co, usa bdepartment of biomedical sciences, colorado state university, fort collins, co, usa glycerol has been the sole sperm cryopreservation agent in several species. due to toxic effects of glycerol at high concentrations, researchers have evaluated alternate cryoprotectants (e.g. amides for stallion sperm). the use of amides, alone or in combination with glycerol, improved postthaw sperm motility and fertility compared to glycerol alone. objective was to compare 4 commercially available freezing extenders on postthaw semen parameters and sperm quality. we hypothesized that lactose-edta extenders containing the amides methylformamide (mf) or dimethylformamide (dmf), alone or in combination with a low concentration of glycerol, yield higher postthaw sperm motility and viability than lactoseedta extender containing only glycerol. ejaculates from 10 stallions were collected using a colorado model artificial vagina and diluted 1:1 in a skim milk glucose extender. diluted ejaculates were transferred to conical tubes and a centrifugation cushion was added below the extended semen. samples were centrifuged at 1,000 x g for 25 minutes. the supernatant was removed, and the sperm pellet was diluted to 200 x 106 sperm/ml in each of the 4 freezing extenders (le, 5% glycerol; cmle, 2% glycerol plus 3% mf; cmlef1, 5% mf; cmlef2, 5% dmf; animal reproduction systems, ontario, ca). extended samples were loaded in 0.5 ml straws, cooled to 5 8ºc for 30 minutes, floated over liquid nitrogen for 15 minutes, then plunged into liquid nitrogen. straws were stored in liquid nitrogen for a minimum of 24 hours prior to evaluation. three straws from each freezing extender for each 10 ejaculates were thawed in a 37ºc water bath for 30 seconds. the contents of the straws were transferred into individual test tubes and allowed to incubate at 37ºc for 10 minutes. the following parameters were evaluated by computer-assisted semen analysis (casa; spermvision; mofa, verona, wi): percent total motility (tm), percent progressive motility (pm), percent hyperactivity (hyp), curvilinear velocity (vcl), path velocity (vap), straight-line velocity (vsl), and linearity (lin). results for the 3 straws were averaged and recorded. data were analyzed using one-way anova, tukey’s hsd test, and chi-square analysis (jmp pro 16.2). data were considered significantly different at p < 0.05. mean tm and pm percent were higher in cmle (70.5 and 60.8%, respectively) and  cmle1 (70.3 and 59.2%, respectively) compared to le (60.2 and 49.6%, respectively). there were no differences in mean tm and pm between le and cmlef2 (66.6 and 54.6%, respectively). additionally, there were no differences between extenders regarding hyp, vcl, vap, vsl, and lin. commercially available freezing extenders with a combination of glycerol and amide (cmle) or an amide alone (cmlef1) had higher postthaw total and progressive motility compared to a freezing extender containing glycerol as the only cryoprotectant (le). it is recommended that a test freeze is performed on all stallions to determine the optimal extender prior to banking frozen semen. keywords: cryopreservation, stallion, sperm, motility, amide, glycerol is decline in prepartum progesterone concentrations associated with litter size in dogs? a retrospective study of 42 cases daniela cortes, robyn ellerbrock, patricia xavier, maria ferrer department of large animal medicine, college of veterinary medicine, university of georgia, athens, ga, usa accuracy to predict fetal readiness for birth is essential for proper timing of elective cesarean surgery and for subsequent increase in neonates’ survival rate. in clinical practice, transabdominal ultrasonography and determination of serum progesterone concentrations have been used to decide when to safely intervene during full term canine pregnancies. prepartum decreases in maternal serum progesterone concentrations is induced by prepartum elevation of maternal cortisol concentrations and is associated with fetal readiness for birth. aim of this retrospective study was to determine the effect of litter size on serum progesterone concentrations during late pregnancy in dogs undergoing cesarean surgery. it was hypothesized that maternal serum progesterone concentrations did not decrease below 2 ng/ml in dogs carrying 1 or 2 pups. medical records from 42 pregnant dogs presenting for elective or emergency cesarean surgery between 2014 and 2022 were evaluated. the information retrieved included age, pregnancy length, progesterone concentrations on the day of the cesarean surgery, and litter size. progesterone concentrations were determined using chemiluminescence immunoassay. dogs were assigned into 3 groups based on litter size as follows: 1. g1: 1 pup (n = 6), 2. g2: 2 pups (n = 4), and 3. g3: > 3 pups (n = 32). progesterone concentrations of < 2 ng/ml were considered to be associated with the prepartum decline and consistent with fetal readiness for birth. the frequency of dogs that experienced a prepartum progesterone decline prior to surgery was compared among groups using chi square analysis. pregnancy length was compared among groups using one-way anova. mean age of dogs was 2.9 years (range;1.5 8 years). pregnancy length at surgery was 63.2 ±1.5 days 14 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 15 (mean ± sd) after ovulation. pregnancy length did not differ (p > 0.05) between dogs with and without a decline in progesterone concentrations at surgery. eighty percent of dogs carrying 1 or 2 pups (g1 and g2) did not have a decrease in progesterone concentrations at surgery, whereas 81% of dogs with > 3 pups (g3) had marked decreases (p < 0.05) in progesterone concentrations < 2 ng/ml. all pups were considered mature at birth and there were no neonatal deaths associated with prematurity. these data suggested that the littler size affected prepartum progesterone concentrations during late pregnancy in dogs. furthermore, prepartum progesterone concentrations cannot be used reliably to predict fetal readiness for birth in dogs carrying 1 or 2 pups. keywords: dog, pregnancy, progesterone, litter size development of a porcine oocyte collection and maturation protocol for intracytoplasmic sperm injection utilizing stallion sperm elizabeth patton, joanne stokes, emily may and jennifer hatzel college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co, usa assisted reproductive technologies (art), such as intracytoplasmic sperm injection (icsi), are commonly utilized in clinical equine practice for infertile mares and stallions. in icsi, a mare’s oocyte is injected with 1 sperm from the desired stallion. embryonic development to the stage of transfer remains lower and research advancements are stifled due to lack of equine oocyte availability. we proposed using readily available porcine oocytes as a model to evaluate stallion fertility and semen processing techniques. we hypothesized that porcine oocyte reach maturity using media and protocols utilized for equine oocyte. we further hypothesized that early embryonic development is achieved with porcine oocytes after icsi with equine sperm. we collected porcine ovaries from a local abattoir, aspirated oocytes from the follicles using the vacuum suction technique previously validated for processing bovine ovaries, and evaluated various maturation process components to optimize maturation indicated with polar body (pb) extrusion. comparisons were made utilizing student’s t-test with significance established at p 0.05. follicular aspiration provided 760 total oocytes. each week, the total collection of oocytes was batched and evenly distributed into 2 groups: control maturation medium (cmm) utilized in the clinical equine program and the experimental maturation medium (either containing 10 or 20% fetal calf serum [fcs]; 0, 33, or 50% porcine follicular fluid [pff]). all experiments were conducted at 38.2ºc unless otherwise noted. initially, we determined difference (p = 0.004) in pb extrusion when oocytes were placed directly in cmm after aspiration compared to holding at room temperature for 24 hours in a commercial embryo holding medium (14/58 versus 2/60). a difference (p = 0.019) was also identified in pb formation when evaluating cmm containing 50% pff compared to cmm (10/38 versus 5/44). there were no differences (p = 0.938) in pb formation when only 33% pff was added to cmm compared to cmm (18/76 versus 14/58) or (p = 0.753) when cmm contained 20% fcs versus 10% fcs (17/89 versus 15/88). most promising results (p = 0.028) were obtained when 33% pff was added to cmm in comparison to cmm and both groups incubated at 39ºc (34/90 versus 25/91). finally, we injected 6 mature porcine oocytes with stallion sperm from the same frozen ejaculate and observed an 83% (5/6) cleavage rate and 17% (1/6) early embryonic development rate. additionally, 5 matured oocytes were selected for a sham injection resulting in a 20% (1/5) parthenogenetic activation. therefore, porcine oocytes can be collected and successfully matured in an equine-based maturation medium with the addition of porcine follicular fluid and incubated at a higher temperature in preparation for icsi. we achieved early embryonic development after stallion sperm injection. although we cannot rule out parthenogenic activation due to limitations of assessing pronuclear fusion, these pilot data were intended to determine the optimal conditions for establishing a porcine oocyte maturation protocol for future studies. keywords: equine oocyte, porcine oocyte, intracytoplasmic sperm injection, assisted reproductive technologies determining the most effective method to remove red blood cells in cases of hemospermia hannah maxwell,a carly turner-garcia,b dale kelleya adepartment of veterinary clinical sciences, college of veterinary medicine, oklahoma state university, stillwater, ok, usa blazy e ranch, guthrie, ok, usa hemospermia has been associated with infertility, subfertility, and poor semen longevity in stallions. regardless of the cause of hemospermia, there is lack of data regarding how to process semen containing red blood cells (rbcs). purpose of this study was to compare the efficacy of equipure™, cushion centrifugation, and sperm filter in removing rbcs from stallion semen. we hypothesized that equipure™ is the most effective method. six ejaculates were collected from 1 stallion; immediately after each collection, 3 ml of venous blood was drawn. blood was added to 12 ml of semen to create a standardized level of 20% hemospermia (%v/v). the initial concentration of the blood semen mixture was obtained via nucleocounter® sp-100™. blood-semen mixture was loaded into a microhematocrit tube and centrifuged at 10,030 x g for 5 minutes and packed cell volume (pcv) was determined using a hematocrit reader chart. the 20% hemospermic sample was divided into 3 aliquots, each extended 1:1 with inra 96. the pcv of the initial extended sample was measured as described. one aliquot of extended blood semen was added to an equal volume of equipure™ in a 15 ml conical tube and centrifuged at 400 x g for 20 minutes. pellet was suspended with inra to the preprocessed volume. second aliquot of extended blood semen was centrifuged with cushion for 20 minutes at 1,000 x g, and the pellet was resuspended to the initial volume with inra. third aliquot of extended blood semen was poured into the sperm filter and processed according to package directions. inra was then used to wash the filter and return the sample back to its original volume. for each of the processed aliquots, the resultant pcv was measured for comparison and the final concentration was obtained to determine recovery rate. sas (version 9.4) mixed procedure modeled the change in pcv and recovery rate, with ejaculate and treatment as independent variables. there were no effects of ejaculate on change in pcv or recovery, so data were combined for analysis. data http://dx.doi.org/10.58292/ct.v15.10001 16 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 are represented as ls means ± sem. equipure™ processing lowered (p < 0.0001) the pcv on an average of 4.8 ± 0.5%. cushion centrifugation lowered (p = 0.004) the pcv on an average of 1.7 ± 0.5%. sperm filter processing did not have an effect (p = 0.32) on pcv. there were differences (p = 0.0004) in pcv change of equipure™ compared to cushion and sperm filter (p < 0.0001) with no differences (p = 0.11) between cushion to sperm filter. recovery rates of cushion (94.3 ± 2.9%) and sperm filter (93.6 ± 3.2%) processing were comparable (p = 0.87). recovery rate of equipure™ was 12 ± 2.9% and was less (p = < 0.0001) compared to cushion and sperm filter. in conclusion, equipure™ was effective in removing rbcs from semen whereas cushion centrifugation and sperm filter were not effective. however, the low recovery rate of equipure™ processing on hemospermic ejaculates may render the method impractical for most situations. keywords: hemospermia, stallion, semen processing mammary gland secretions conductivity alone or coupled with ph is a novel and accurate predictor of foaling and its associations with electrolytes humberto magalhaes, ilaria colombo, kianna spencer, giorgia podico, igor canisso department of veterinary clinical medicine, university of illinois urbana-champaign, urbana, il, usa accurate prediction of parturition is paramount to providing assisted delivery and preventing foaling-induced complications. assessment of ph and electrolyte concentrations of mammary gland secretions (mgs) is useful to detect impending parturition. we hypothesized that changes in electrolytes and ph alter the conductivity of mgs, thus, conductivity is a more accurate foaling predictor than ph and electrolytes. in experiment 1, periparturient mares (n = 23) had mgs assessed daily for ph, conductivity, and electrolytes (ca2+, na+, and k+) using bench-top analyzers (laqua-twin d 771, horiba ltda, kyoto japan for ph and conductivity; and hitachi 911 roche diagnostics, basel switzerland for electrolytes) and handheld devices with the aim to validate portable handheld devices worldwide available from one manufacturer (laquatwin b-722 na+ meter, k+ meter laquatwin b-731 k+ meter, laquatwin b-751 ca2+ meter, laquatwin ph meter and conductivity; horiba, ltd., usa) laqua-twin, horiba). mixed-model analyses revealed significant reductions in ph, conductivity, na+, and increases in ca2+, and k+ for impending parturition. bland-altman analyses revealed no significant deviation from the linearity for all devices. in experiment 2, periparturient mares (n = 114) had daily mgs  assessed for conductivity, ph, and electrolytes using hand-held devices (laqua-twin). mixed-model analyses suggested effects of time (p > 0.05) or interaction (group*time) (p > 0.05) for all analytes. conductivity was strongly associated with ph (r = 0.90) and moderately associated with na+ (r = 0.77), ca2+ (r = -0.60), and k+ (r = -0.70). analyses of receiver operating characteristics (roc) were performed to determine the cutoffs for foaling in 24 hours for all analytes. then, the cutoff values were used to calculate the sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv). the results of conductivity for sensitivity, specificity, ppv, and npv (0.82, 0.80, 0.82, 0.60, respectively), ph (0.80, 0.78, 0.81, and 0.70, respectively), ca2+ (0.92, 0.35, 0.68, and 0.76, respectively), na+ (0.65, 0.28, 0.53, and 0.39, respectively), and k + (0.67, 0.25, 0.50, and 0.41, respectively). the sensitivity, specificity, ppv, and npv for conductivity coupled with ph, were 0.84, 0.85, 0.81, and 0.90, respectively. in conclusion, the handheld devices tested herein were as accurate as bench-top analyzers for ph, conductivity, and electrolytes. the conductivity of mgs presented similar results to ph to predict foaling, and the combination of ph and conductivity enhanced the prediction of parturition in mares. keywords: mares, parturition prediction, mammary gland, secretions, electrolytes improving the art of semen banking in amur leopard (panthera pardus orientalis) julie barnes, lindsey vansandt center for conservation and research of endangered wildlife, cincinnati zoo & botanical garden, cincinnati, oh, usa amur leopard (panthera pardus orientalis) is a critically endangered subspecies of the leopard taxon (panthera pardus). the iucn red list estimated1 that < 40 individuals are left in the fragmentated habitat, making in situ conservation efforts pivotal for this species. semen cryopreservation is a critical component of their conservation management strategy; however, current methods to collect and freeze sperm have yet to be assessed in this felid species. our objectives were as follows: 1. evaluate the ability of a newer alpha-2 agonist, medetomidine, to facilitate semen collection via urethral catheterization; 2. compare soy lecithin-based cryomedium to the standard egg yolk-based cryomedium for sperm cryopreservation; and 3. compare ultra-rapid freezing (urf) of semen to traditional straw-freezing. adult male amur leopards (n = 10) located in zoos (accredited by the association of zoos and aquariums) were anesthetized with various combination protocols that each included a moderate dose of medetomidine. at 20 40 minutes atter injection, a urinary catheter was inserted 20 cm into the urethra to recover semen (2 males were not able to be catheterized until 55 -75 minutes after injection). immediately after catheterization, males were subjected to 1 3 series of electroejaculation (eej). semen was either immediately placed in soy lecithin urf medium with 0.2 m sucrose (urf1) or split into 3 aliquots, centrifuged, and resuspended in soy lecithin cryomedium with 4% glycerol (soy), test-egg yolk cryomedium with 4% glycerol (tey), or soy lecithin urf medium with 0.2 m sucrose (urf2). sperm extended in glycerol-based cryomedia were sealed in liquid nitrogen vapor. sperm extended with urf were frozen by pipetting ~ 30 µl droplets directly into liquid nitrogen. samples from 5 males were thawed and assessed for percent progressive and rate of progressive motility (0 5 scale), and heterologous ivf was performed using in vitro matured domestic cat oocytes (n = 10; 24/replicate). at 48 hours after insemination, hoechst33342 staining was used to determine oocyte maturation stage. data were analyzed with either paired t-test or repeated measures anova and reported as mean ± sem. on average, 120.7 ± 41.5 x 106 total sperm were recovered via catheter; however, more sperm (285.8 ± 56.3 x 106) were recovered during successive eej. postthaw motility only differed (p < 0.05) between tey and urf2 and the rate did not differ among other groups (tey: 31.0 ± 3.3% motile, 1.8 ± 0.3 rate; soy: 16.0 ± 4.3%, 2.2 ± 0.1; urf1: 18.0 ± 5.6%, 2.0 ± 0.2, or urf2: 12.0 ± 2.6%, 2.0 ± 0.2. similarly, embryo cleavage percentage (tey: 37.4 ± 9.0; soy: 31.7 ± 8.9; urf1: 22.5 ± 7.2; http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 17 urf2: 10.9 ± 2.9) did not differ among treatments. in conclusion, medetomidine allowed recovery of a substantial number of sperm using urethral catheterization in amur leopards; however, (if the technology is accessible), eej can significantly increase the number of sperm collected. both soy and urf cryomedia were comparable animal protein-free alternatives to tey, with urf providing a more rapid, simplified cryopreservation option to standard straw freezing methods. we determined the current standards of cryopreservation for amur leopard semen that can aid in conservation management on the individual and the population level of this iconic imperiled felid. keywords: semen collection, cryopreservation, ultra-rapid freezing, in vitro fertilization, amur leopard reference 1. stein, ab, athreya, v, gerngross, p et al: panthera pardus (amended version of 2019 assessment). the iucn red list of threatened species: 2020. early diagnosis of ascending placentitis in mares lorena feijo,a karen wolfsdorf,b igor canisso,c julia felippea aequine immunology laboratory, college of veterinary medicine, cornell university, ithaca, ny, usa bhagyard equine medical institute, lexington, ky, usa ccollege of veterinary medicine, university of illinois, urbanachampaign, il, usa pregnancy loss and neonatal mortality have significant economic impact on the equine industry. placentitis is the most common cause of abortion, stillbirths, and neonatal infections.1 early diagnosis of placentitis allows prompt treatment intervention and increases the chances of term pregnancy. our study was designed to identify blood parameters that are diagnostic of imminent placentitis in natural conditions. we hypothesized that differences from physiological parameters of a healthy pregnancy indicate imminent placentitis. blood sample collections and reproductive examinations were prospectively performed monthly, from 180 to 320 days of pregnancy in mares from 2 large broodmare herds in lexington, ky. from this cohort, we analyzed samples collected from 12 mares before ultrasonographic detection of ascending placentitis (samples named preonset), and from the same mares when they developed signs of placentitis at subsequent examination (samples named early onset). in addition, samples from 12 mares with healthy pregnancies (pregnancy-matched controls) were collected at the same time points. blood concentrations of serum amyloid a (saa), alpha-fetoprotein (afp), and estradiol-17β were measured and the values were compared between groups and timepoints using parametric (student’s t-test) and nonparametric (mannwhitney u test) analyses, and significance was set at p < 0.05. based on the stage of pregnancy, the interval between preonset timepoint and the diagnosis of placentitis (early onset) was ~ 40 days (227 ± 6 days and 267 ± 5 days, respectively). blood concentrations of saa and afp in the maternal circulation were within their respective normal range with no differences between groups and timepoints. both proteins increased in  maternal circulation after induction of experimental placentitis.2,3 plasma estradiol-17 β concentrations were different (p = 0.08) in mares at early onset of placentitis (408 ± 35 pg/ml) compared to control group (498 ± 35 pg/ml) mares. in conclusion, early events of placentitis did not alter inflammatory and immunological parameters in the maternal circulation. plasma estradiol-17β concentrations may be further investigated as an early indicator of placental dysfunction in mares with naturally occurring placentitis. keywords: mare, pregnancy, placentitis, diagnosis, estradiol references 1. giles rc, donahue jm, hong cb, et al: causes of abortion, stillbirth, and perinatal death in horses: 3,527 cases (1986-1991). j am vet med assoc 1993;203:1170-1175. 2. canisso if, ball ba, scoggin ke, et al: alpha-fetoprotein is present in the fetal fluids and is increased in plasma of mares with experimentally induced ascending placentitis. anim reprod sci 2015;154:48-55. 3. canisso if, ball ba, cray c, et al: serum amyloid a and haptoglobin concentrations are increased in plasma of mares with ascending placentitis in the absence of changes in peripheral leukocyte counts or fibrinogen concentration. am j reprod immunol 2014;72:376-85. comparing sperm concentrations for cooled semen use in dogs nicole sugai, julie cecere, orsolya balogh department of small animal clinical sciences, virginia-maryland college of veterinary medicine, blacksburg, va, usa stallion semen is shipped cooled at sperm concentrations of 25 50 x 106/ml with a minimum of 1:3 (volume) dilution with an extender. optimal sperm concentration for cooled semen transport in the dog is unknown. canine semen is commonly diluted to a 1:3 5 (volume) without standardization for concentration. therefore, the purpose of this study was to compare various sperm concentrations during cooled shipment. we hypothesized that lower sperm concentrations result in higher sperm quality during cooled storage for 24 hours in dogs, similar to stallions. healthy, client-owned stud dogs (n = 7, 2 6 years of age, > 15 kg, various breeds) were collected using manual technique. only dogs with a negative brucella canis serology and an ejaculate with appropriate concentration, ≥ 70% total motility and ≥ 40% normal morphology, were included. ejaculates were divided into a control (con) aliquot (≥ 300 µl) that was extended with caniplus chill lt at 1:3 (volume) dilution. remaining sample was centrifuged at 720 g for 10 minutes in a 15 ml falcon conical tube, the sperm pellet extended to 200 x 106/ml (c200) with caniplus chill lt, followed by serial dilutions to arrive at 100, 50, and 25 x 106/ml sperm concentrations (c100-25). all aliquots were then cooled for 24 hours according to standard packaging technique in a minitube canine shipping box. afterwards, they were all recentrifuged and reextended to initial aliquot concentration. this last step was conducted to evaluate whether shipping at larger volumes with lower concentrations needing further processing for  insemination would give adequate results. semen evaluations for http://dx.doi.org/10.58292/ct.v15.10001 18 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 concentration and viability (plasma membrane integrity) by nucleocounter® sp-100™, subjective total motility (stm), total and progressive motility (tm, pm) by casa (spermvision™, minitube) were performed on the raw semen (t0), immediately after extension (t1), after 24 hours of cooling (t2), and after centrifugation and reextension at 24 hours (t3). data were analyzed by one-way anova or kruskal-wallis test to evaluate viability and motility (stm, tm, pm) at t1, t2, and t3 by treatment. viability was lower for c25 compared to the c200, c100 and con groups for time points t1 and t2 (p ≤ 0.04), and compared to c50 at t1 (p = 0.01). at time t1, viability for con was higher (p ≤ 0.03) among treatment groups. also, viability for con was higher compared to c50 and c25 at time t2 (p ≤ 0.01), and compared to c25 at time t3 (p = 0.01). for motility evaluations (stm, tm, and pm) there were no differences among treatment groups at time t1 (p ≥ 0.17) or t2 (p ≥ 0.18). at time t3, stm was lower (p = 0.001) for c25 compared to con and casa tm and pm were lower (p ≤ 0.02) for c25 compared to c200 and con. in conclusion, cooling canine semen for 24 hours at 100 200 x 106/ml concentration after processing or extending 1:3 (volume) without processing, resulted in better sperm viability compared to lower concentrations and having similar motility parameters. keywords: dogs, chilled semen, concentration, viability http://dx.doi.org/10.58292/ct.v15.10001 mixed species session canine endometrial organoid generation and response to exogenous hormones alexandria horner, riley thompson, mindy meyers, fiona hollinshead department of clinical sciences, colorado state university, fort collins, co, usa pyometra is a life-threatening reproductive pathology that commonly affects middle-aged to older, nulliparous, intact female dogs.1 no studies have yet focused on developing an in vitro 3d culture system to analyze this disease complex that would eliminate the need for an in vivo model to improve welfare. recent technological developments in 3d organoid cell culture may provide an innovative method for studying pyometra and other reproductive pathologies that affect dogs. unlike traditional 2d monolayer culture systems, organoids are capable of long-term growth while maintaining similar structure and function to their organ of origin.2 due to these features, organoids are an ideal in vitro model for evaluating disease processes and investigating potential therapeutic options that may improve the overall health and welfare of intact dogs, domestic and wild. therefore, our objectives were to: 1. generate organoids from endometrial tissue of diestrous dogs; and 2. evaluate the gene and protein expression response of these endometrial organoids to exogenous steroid hormones. we hypothesized that estrogen and progesterone receptor gene and protein expression increase in estradiol and decrease in progesterone presence.3,4 discarded diestrous canine reproductive tracts (n = 6) were collected after ovariohysterectomy at a local spay and neuter clinic. endometrial epithelial glands then were isolated and established in organoid culture by adapting a protocol described for equine endometrial organoids.2 after 20 days in culture (passaged every 6 days), the following treatments were initiated: 1. 30 ng/ml progesterone (p4) over 6 days; 2. 100 pg/ml estradiol (e2) over 6 days; 3. 100 pg/ml e2 for 2 days followed by 30 ng/ml p4 for 4 days; and 4. control (no steroid hormones added). after 6 days of hormonal treatments, organoids were removed from culture for transmission electron microscopy (tem), histology, immunohistochemistry (ihc), and gene expression analysis. brightfield imaging, histology, and tem demonstrated, for the first time, round cell clusters with a central lumen and intact tight junctions that is consistent with endometrial organoid reports of other species.2 ihc demonstrated positive immunostaining for cytokeratin, a marker for epithelial cells. preliminary findings reflected the structural similarity of canine endometrial organoids to their in vivo counterpart (with molecular and functional data pending). findings also indicated that organoids may be a functional model that can be used to study canine uterine diseases to reduce reliance on research animals and the associated financial and welfare costs. funding provided by the akc chf. keywords: dogs, cell culture, in vitro, endometrial, organoid references 1. verstegen j, dhaliwal g, verstegen-onclin k: mucometra, cystic endometrial hyperplasia, and pyometra in the bitch: advances in  treatment and assessment of future reproductive success. theriogenology 2008;70:364-374. 2. thompson re, johnson ak, dini p, et al: hormone-responsive organoids from domestic mare and endangered przewalski’s horse endometrium. reproduction 2020;160:819-831. 3. vermeirsch h, simoens p, hellemans a, et al: immunohistochemical detection of progesterone receptors in the canine uterus and their relation to sex steroid hormone levels. theriogenology 2000;53:773-788. 4. vermeirsch h, simoens p, lauwers h, et al: immunohistochemical detection of estrogen receptors in the canine uterus and their relation to sex steroid hormone levels. theriogenology 1999;51:729-743. epididymal sperm acquisition, cryopreservation, and use of epididymal frozen semen in armenian gampr dogs andrea hesser genesis canine reproduction, carrollton, tx, usa an intact 2-year, armenian gampr male, was presented for reproductive consultation and semen collection after initial diagnosis of cervical trauma by a local veterinarian. animal’s injury caused both cervical pain and rear limb ataxia. prognosis for return to full musculoskeletal and nervous function was poor. a working livestock dog that is not suitable for a companion lifestyle, the owner opted to pursue semen freezing prior to euthanasia. manual attempts to collect semen were unsuccessful. owner elected to castrate the dog and have an epididymal flush performed to obtain semen for freezing. an intravenous catheter was placed and the dog was anesthetized with intravenous propofol. animal was castrated, giving special attention to obtain maximum length of the vas deferens within the spermatic cord. after castration, patient was euthanized with pentobarbital. epididymes were dissected to remove all tissues except for the cauda epididymis. vas deferens was flushed in retrograde manner using canine semen extender to obtain an ejaculate in a conical tube. semen was citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 19 http://dx.doi.org/10.58292/ct.v15.10001 20 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 evaluated for sperm quantity, motility, quantity, and morphologic defects, and was in excellent form. semen was cryopreserved in straws, and the sample yielded multiple frozen semen doses. after several months, the same owner presented a 2-year, armenian gampr dog, in estrus and opted to use the frozen semen from the epididymal flush sample. ovulation timing was determined via vaginal cytology, progesterone concentrations, and vaginoscopy findings; dog was bred (when maximal vaginal crenulation was observed) with 200 x 106 progressively motile sperm by transcervical insemination twice at 24-hour intervals. after breeding, ultrasonography was not performed. near term radiographs revealed 2 fetuses. planned cesarian surgery was opted, and 2 healthy pups were delivered. this case demonstrated many procedures of advanced reproductive technology that could be applied in dogs. epididymal flush is performed in dogs by some practitioners, although successful pregnancy using epididymal semen in dogs has not yet been described. epididymal frozen semen success is still not well understood in dogs, and recommended insemination dosing of total progressively motile sperm may vary from the standard approaches in traditionally acquired frozen semen samples. keywords: epididymal flush, semen cryopreservation, transcervical insemination pregnancy outcome with uterine serosal inclusion cyst in a dog fabio pinaffi, jacob rother, sheila megehee department of veterinary clinical sciences, college of veterinary medicine, oklahoma state university, stillwater, ok, usa a 6-year, female german shepherd that had previously whelped 3 times, was presented for failure to conceive. she had her first litter via cesarean surgery, second by natural whelping, and third by a combination of natural whelping and emergency cesarean surgery. during her last cesarean surgery, an increased amount of space was noticed between pups that required excessive manipulation of the uterus to clear. outer (serosal) surface of the uterus had multiple cysts. thereafter, 2 breedings (transcervical insemination [tci] with > 300 x 106 progressively motile sperm [pms] from days 2 to 4 after ovulation) attempts were unsuccessful. ovulation was defined as the day when vaginal cytology reached > 90% superficial epithelial cells and serum progesterone concentrations were 4 to 10 ng/ml. transabdominal ultrasonography was performed on day 30 after ovulation that revealed a slight amount of fluid within her uterus with no fetuses and multiple cysts at the distal part of uterine horns. the third breeding management included 2 breedings by tci on days 2 and 4 after ovulation with > 300 x 106 pms, intravenous oxytocin treatment (2 iu) 6 hours after each breeding and repeated every 12 hours for 24 hours, and uterine lavage using 60 ml saline by tci scope the day after each breeding. oxytocin treatment and uterine lavage were aimed to improve uterine clearance by inducing uterine contractions and decreasing bacterial contamination, respectively. transabdominal ultrasonography performed on the day after breeding revealed no intraluminal uterine fluid. dog whelped 1 pup naturally and 7 pups via cesarean surgery. the smallest pup had difficulty breathing and failed to thrive. it received intensive care but was pronounced dead within a few hours. during cesarean surgery, multiple cysts were observed grossly on the serosal surface of the distal part of uterine horns. additionally, a uterine laceration that occurred during manipulation of the uterus (matched with the scar from the previous cesarean surgery incision) was repaired. uterine serosal inclusion cysts (sic) are incidental findings in multiple domestic animal species including pigs, cats, and dogs. clinical importance is still unknown (commonly localized at uterine horns’ tip), lack endocrine activity, and are not associated with the estrous cycle, and mostly occur in old multiparous dogs of large breeds. it is suggested that sic may cause adhesion of the uterus to other organs or peritonitis by rupturing. potential causes of sic are overstretching of the uterus which can occur in big litters, rapid uterine contractions of the myometrium during involution, genetic predisposition, and excessive handling of the uterine horns during cesarean surgery. in the present case, sic could have affected uterine contractility and, consequently, reduced uterine clearance after estrus and/or breeding. outcome of this case suggested that fertility could be achieved without a major decrease in litter size using tools to improve uterine clearance (e.g. oxytocin and uterine lavage). keywords: serosal inclusion cysts, uterine clearance, pregnancy, dog disorder of sexual development due to xx/xy chimerism in a siberian husky female dog jenna dockweiler, takeshi kawakami, adam boyko embark veterinary, inc., boston, ma, usa a 22-month, intact female siberian husky, underwent genetic testing as part of a prebreeding screen. her owner reported that she never exhibited signs of estrus. however, her littermate sister had experienced at least 2 apparently normal estrous cycles. a review of veterinary records indicated that her external genitalia appeared normal, albeit juvenile, female. she had no remarkable medical history other than an episode of nocturnal urinary incontinence that resolved without treatment. a commercially available direct-to-consumer saliva-based genetic test was performed (embark® breed & health dog dna test) at 3 separate times. genotyping samples were assayed using a canine-specific research-grade microarrayplatform with > 230,000 probes, including probes to detect loci on both the x and y chromosomes. all 3 samples failed, calling diploid genotypes across all chromosomes, suggesting that she was a chimeric dog by having 4 copies of each autosome. furthermore, probe signals on the x and y chromosomes suggested that she had 3 copies of x chromosome and 1 copy of y chromosome, the pattern expected in an xx/xy chimera. this chromosomal abnormality was the likely cause of her primary anestrus. this patient will be sterilized at 24 months of age, and her gonads will be submitted for histopathology at that time. although chimerism is not associated with known major health concerns in the dog, affected patients are likely infertile. therefore, this patient is no longer considered a breeding candidate. this case illustrated a cost-effective method for screening for chromosomal disorders that affect sexual development in the dog. keywords: genetics, disorder of sexual development, chimera, primary anestrus http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 21 medical management of follicular cysts in a dog lily lewis, candace lyman, robyn wilborn department of clinical sciences, college of veterinary medicine, auburn university, auburn, al a 14-year, nulliparous english setter dog, was presented for evaluation of persistent vulvar bleeding of 3 months duration. despite her geriatric age, no other health concerns were reported, previous estrous cycles and interestrus intervals were consistent. patient was bright, alert, and responsive, with an ideal body condition score and vital parameters within normal limits. vulva was moderately enlarged. a small amount of frank blood was dripping from the vulva intermittently throughout the examination, and excessive blood staining was noted on the hind limbs. a vaginal swab cytology was performed that revealed a population of 95% superficial and anuclear cells, suggesting cellular hyperplasia secondary to estrogen. serum progesterone concentrations were 0.2 ng/ml. based on these diagnostic tests and clinical findings, hyperestrogenism (exogenous or endogenous) was suspected. a more detailed history of the patient’s diet and environment made exogenous estrogen less likely as the cause. duration of vulvar discharge exceeded the timeframe of expected proestrus and estrus. thus, additional diagnostic testing was recommended to investigate an endogenous source of estrogen. abdominal ultrasonography revealed bilateral ovarian enlargement with cystic changes noting that the left side was more severely affected than the right. specifically, the left ovary had a large, hypoechoic, thin-walled structure within the mid ovarian body, measuring 2.25 x 1.47 cm. right ovary had a smaller hypoechoic structure, measuring 0.33 x 1.47 cm. these structures were determined to be cystic in nature as they measured larger than expected for a normal canine ovarian follicle (2 8 mm). other changes observed included generalized cystic endometrial hyperplasia and ill-defined hypoechoic splenic nodules within the splenic head. remarkably, no evidence of bone marrow suppression was evident on a complete blood count. thus, ovariohysterectomy was recommended, noting that a laparotomy would allow for evaluation of the splenic nodules, with a possibility for additional diagnostics or treatments (e.g. splenic biopsies, splenectomy). although the owners declined surgical treatment, they did inquire about medical management possibility. intramuscular human chorionic gonadotropin (chorulon®, merck animal health, usa) treatment was discussed, noting that the success rate could vary. patient was treated with 1,000 units every 24 hours for 3  days. seven days after treatment, the owner reported substantial decreases in the amount of vulvar bleeding. approximately 30 days after treatment, the owner reported that the bleeding had resolved completely. throughout this period, no adverse side effects were reported by the owner. a final follow-up was made ~ 60 days after treatment and no relapse of bleeding was reported. keywords: follicular cyst, estrogen, hyperestrogenism effects of pituitary abscess and subsequent treatment on cyclicity in a thoroughbred broodmare rachel doenges, maria schnobrich, brad tanner rood and riddle equine hospital, lexington, ky, usa a 7-year, thoroughbred broodmare, was presented for malodorous, left-sided nasal discharge in february. tooth 210 (left maxillary second molar) was nonvital with an open pulp horn. the tooth was extracted, and a sinus trephination performed. antibiotic therapy was started based on culture. mare developed intermittent fever, anisocoria, and ataxia. she was readmitted and was treated with intravenous potassium penicillin, gentamicin, flunixin meglumine, and dimethyl sulfoxide. signs persisted despite therapy. magnetic resonance imaging (mri) was performed that revealed an enlarged pituitary gland with heterogeneous signal intensity and irregular margins, indicative of a pituitary abscess. additionally, inspissated material was still present in the sinus, and there was increased signaling at the basisphenoid bone. horses and ruminants are prone to bacterial meningitis and brain abscess secondary to systemic infection. however, brain abscess secondary to local extension has also been reported in horses. main routes for local extension are as follows: via cranial nerves; direct inoculation, as observed with a penetrating wound; extension from adjacent structures; or hematogenous spread.1 based on mri findings, we speculated that local extension occurred from the left sinus through the basisphenoid bone to the pituitary gland. a right retrobulbar abscess developed, mri findings supported that this abscess was secondary to the pituitary abscess draining via the optic nerve. an enucleation was performed. the neurological deficits improved, and she was transitioned to oral chloramphenicol. an mri was repeated 2 months later. pituitary had returned to normal size, and other signs of pituitary abscess were resolved. prior to the mare’s initial presentation, the mare’s reproductive tract was monitored in preparation for breeding. transrectal ultrasonography findings were consistent with vernal transition until january 18th, when a normal ovulation was confirmed. during her hospitalization, regular reproductive ultrasonography was not performed. however, transrectal evaluation (performed after she was discharged) revealed small inactive ovaries with no uterine edema or cervical softening. dominant follicles did not develop. this was documented from late february until august. on august 17th, a dominant follicle (38 mm) with appropriate uterine edema and cervical softening was noted, indicating return to cyclicity. the following year, the mare conceived but pregnancy was lost between 60 and 120 days. she had a successful pregnancy the subsequent year. in human medicine, hypopituitarism is commonly observed with pituitary abscess and is treated with hormone replacement therapy, as needed. pituitary abscess in domestic animals is usually fatal. in human medicine, in addition to antibiotics and management of the primary infection site, neurosurgical drainage is normally performed.2 neurosurgical drainage is generally not feasible in large animals. however, in this case, apparently the drainage occurred via the optic nerve and may have contributed to the positive outcome. a bull was successfully treated for pituitary abscess; however, the bull was slaughtered due to low testosterone and teratozoospermia that did not resolve with treatment.3 this case is notable, not only in that the mare survived but she also returned to reproductive soundness. keywords: pituitary abscess, tooth root abscess, hypopituitarism, sinusitis http://dx.doi.org/10.58292/ct.v15.10001 22 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 references 1. smith jj, provost pj, paradis mr: bacterial meningitis and brain abscesses secondary to infectious disease processes involving the head in horses: seven cases (1980-2001). j am vet med assoc 2004;224,739-742. 2. zhu h, gu xm, hong j, et al: successful treatment of pituitary abscess with intravenous antibiotics: a case report. genet mol res 2014;13,10523-10528. 3. braun u, malbon a, kochan m, et al: computed tomographic findings and treatment of a bull with pituitary gland abscess. acta vet scand 2017;8,59. differences in uterine bacterial community compositions and gene expression between normal and metritic holstein dairy cows at 5 12 days postpartum tal raz,a,b ron sicsic,a shaked druker,c tamir goshen,c maor kedmi,c nahum shpigela akoret school of veterinary medicine; robert h. smith faculty of agriculture, food and environment, the hebrew university of jerusalem, rehovot, israel bkrieger school of arts & sciences, advanced academic programs (bioinformatics), johns hopkins university, baltimore, md, usa chachaklait, mutual society for veterinary services, caesarea industrial park, israel metritis is the most prevalent postpartum uterine disease in dairy cows that results in tremendous economic losses to farmers. postpartum contamination of the uterus probably occurs in all dairy cows; however, not all animals develop metritis. nevertheless, the pathophysiology of this disease is still not clear. in israel, cows are routinely examined during 5  to 12 ‘days in milk’ (dim) and may be diagnosed with metritis if clinical findings include fetid mucopurulent vaginal discharge and transrectal palpation findings include large, gas, or fluid-filled uterus. our objective was to utilize a large data set of 16s amplicon sequences and mrna-seq gene expression to: 1. describe and compare uterine bacterial load and community composition; and 2. analyze and compare endometrial gene expression, between cows with and without metritis, and between cows with different bacterial community compositions. postpartum holstein-frisian dairy cows (n  = 139) were examined routinely at 5 12 dim, and the luminal endometrium was sampled aseptically using a transcervical sterile double-guarded cotton swab. as expected, reproductive performance parameters after the voluntary waiting period were superior in healthy cows. uterine bacterial load (ubl), quantified by 16s rrna gene qpcr, did not differ between healthy (n = 70) and metritis cows (n = 59). endometrial cytological analysis revealed a bimodal distribution of polymorphonuclear cell (pmn) percentage within each group, with most cows having either very low or very high pmn (30% of cows < 4% pmn; 30 % of cows > 88% pmn), regardless of the disease status (healthy or metritis). however, the pmn percentage was correlated to the ubl. metagenomic analysis of 16s sequencing revealed that in metritis cows, there was a typical bacterial community with higher relative abundances of the phyla bacteroidetes (metritis: 28.1 ± 2.9%, healthy: 9.1 ± 1.6%; p < 0.001) and fusobacteria (metritis: 28.3 ± 2.4%, healthy: 19.0 ± 2.8%; p < 0.001). in contrast, the bacterial community composition of healthy cows was more diverse. however, some healthy cows had a ‘metritis-like’ bacterial community, with no clinical signs of metritis. analysis of gene expression by mrna-seq methods indicated 152 differentially-expressed genes (degs) when metritis (n = 5) and healthy cows (n = 10) were compared; most were immune-related degs. furthermore, comparing metritis cows (n = 5) with typical bacterial community composition to healthy cows (n = 4) with similar ‘metritis-like’ bacterial community composition indicated 27 degs, suggesting variation in uterine response to similar bacterial composition, resulting in different clinical outcomes. further understanding of the pathophysiology of uterine diseases may lead to new preventive and therapeutic approaches. keywords: metritis, dairy cows, 16s metagenomics, rna seq, gene expression spontaneous pneumothorax resolved via pneumocentesis in a neonatal alaskan malamute tiffany hoffman,a morgan agnewb arutgers university, new brunswick, nj, usa bphiladelphia animal hospital, philadelphia, pa, usa a neonatal male alaskan malamute pup was presented after developing acute respiratory distress shortly after delivery. pup was delivered via cesarean surgery (performed at 66 days after lh surge). five live pups were delivered; however, 1 with schistosomus reflexus was euthanized immediately after owner consent. physical examination was initially unremarkable; however, shortly after resuscitation, the patient’s mucous membranes and foot pads became pale blue, he started gasping and a small amount of clear fluid bubbled from his nose. initially the patient’s respiratory effort improved with oxygen supplementation and cpr. radiographs revealed severe pneumothorax displacing the patient’s heart cranially and laterally against the left lateral thorax. pneumocentesis was performed with a 25 gauge butterfly with a 20 ml syringe attached; 24 ml air was removed from the right lateral thorax. radiographs were repeated that revealed decreases in air in the pleural space and lungs appeared to reinflate. oxygen therapy was continued. gv26 was stimulated using a 25 gauge needle. cpr was discontinued to avoid a perceived risk of reintroduction of air into pleural space from the force used with cpr. radiographs were repeated (for the last time) that revealed further improvement with only a small amount of air visible around pericardium and diaphragm. patient’s clinical picture improved with a return to normal vocalization, respiratory rate, muscle tone, and mucous membrane color. patient was discharged with the instruction to keep him separated from other pups and to monitor closely for any signs of respiratory distress. patient was empirically treated with ceftiofur at a dose of 2.5 mg/kg, twice daily, for 7 days and has been doing well. although this condition is widely studied in human neonates, there is limited research on acute pneumothorax and treatment in neonatal pups, in humans, symptomatic spontaneous pneumothorax (sp) occurs in 0.05 1% of all live born infants, and presents within the first hours of life.1 in dogs, primary spontaneous pneumothorax (psp) is the most common form of spontaneous pneumothorax and is typically associated with the rupture of bullae or subpleural blebs.2 psp is the most commonly diagnosed in healthy, younger-middle aged dogs, and is overrepresented in siberian huskies.2 additional research on the resolution of psp in canine neonates is http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 23 warranted. this case illustrated the importance of radiographs in determining a cause for respiratory distress in neonates to help improve neonatal outcomes. keywords: pneumothorax, alaskan malamute, respiratory distress, neonatal, pneumocentesis references 1. clark s, saker f, schneeberger m, et al: administration of 100% oxygen does not hasten resolution of symptomatic spontaneous pneumothorax in neonates. j perinatol 2014;34: 528-531. 2. gilday c, odunayo a, hespel am: spontaneous pneumothorax: pathophysiology, clinical presentation and diagnosis. top companion anim med 2021;45:100563. http://dx.doi.org/10.58292/ct.v15.10001 production animal session reducing the incidence of endometritis in dairy cows by hormonal regimen given to shorten the interval from calving to the first postpartum ovulation tal raz,a,b shaked druker,a,c ron sicsic,a alon kaplan,c maor kedmic akoret school of veterinary medicine; robert h. smith faculty of agriculture, food and environment, the hebrew university of jerusalem, rehovot, israel bkrieger school of arts & sciences, advanced academic programs (bioinformatics), johns hopkins university, baltimore, md, usa chachaklait, mutual society for veterinary services, caesarea industrial park, israel postpartum uterine inflammatory diseases, such as metritis and endometritis, are prevalent in bovine dairy herds, resulting in major economic losses. endometritis can be diagnosed from 21 day-in-milk (dim) onward by evaluating the percentage of polymorphonuclear cells (pmn) in endometrial cytology, typically obtained by cytobrush and, therefore, is commonly referred to as cytological endometritis (cem).1 previous studies suggested that spontaneously ovulating cows early in lactation have reduced endometritis incidence later in their lactation. our objective was to evaluate the efficacy of 2 hormonal regimens for hastening ovulation in early lactation (treatment initiation at 24 27 dim) and explore the incidence of cem later in lactation. holstein dairy cows (n = 450) were included in a prospective cohort study. based on transrectal ultrasonographic examination (presence of corpus luteum, cl) combined with milk progesterone cow-side test (mp4) at 24 27 dim, cows were assigned; positive control: cows spontaneously ovulated by 24 27 dim, no treatment. cows that did not spontaneously ovulate were randomly assigned to following groups (n = 101 cows/group): select synch: gnrh analog and pgf2α 7 days later; select synch-cidr: gnrh analog and pgf2α 7 days later, with 7 day cidr; negative control: no hormonal treatment (2 saline injections 7 days apart). cows were examined by transrectal ultrasonography 5 times (24 27 dim; 31-34 dim; 38 41 dim; 45 49 dim; 66 69 dim). cem diagnosis was made based on endometrial cytobrush at 38 41 dim and 66 69 dim, according to the criteria we recently defined.1 at 24 27 dim, 92.7% (417/450) of the cows were clearly defined as ovulating or not (cl + high mp4 versus no cl + low mp4). of these, 114 (27%) spontaneously ovulated by 24 27 dim, with overall, a higher (p = 0.0003) proportion among multiparous versus primiparous cows (33 versus 17%; or = 2.44, 95% ci 1.49 4.00). both hormonal protocols efficiently induced ovulation and increased percentage of cows ovulating during the 70 dim voluntary waiting period (select synch 92.1, select synchcidr 89; negative control 71.3; p = 0.0466). furthermore, select synch was associated with a lower risk for cem than the negative control, particularly in cows without postpartum ketosis (18.6 versus 35.4%; or = 0.42, 95% ci 0.18 0.90; p = 0.0295). we concluded that simple hormonal treatments given at early lactation stimulated ovarian activity and reduced the risk for subsequent endometritis in postpartum holstein dairy cows. keywords: endometritis, dairy cows, postpartum, uterus, ovulation reference 1. druker sa, sicsic r, van straten m, et al: cytological endometritis diagnosis in primiparous versus multiparous dairy cows. j dairy sci 2022;105:665-683. induction of parturition in embryo recipient camels carrying calves produced by somatic cell nuclear transfer ahmed tibary,a irfan khan,b abdelhaq anouassib adepartment veterinary clinical science, college of veterinary medicine, washington state university, pullman, wa, usa badvanced scientific group, camel embryo transfer laboratory, sweihan, uae embryo transfer is an established reproductive technology in camels.1 pregnancies from in vitro produced embryos by somatic cell nuclear transfer (scnt) have been reported. however, very limited data are available regarding parturition complications and calf survival. pregnancy duration in camels is extremely variable and averages 386 days. perinatal mortality in camels is high, and dystocia is the major predisposing factor.2 monitoring of pregnant recipients during parturition is essential for early veterinary intervention and calf resuscitation if needed. induction of parturition is performed to ensure adequate monitoring during labor. here we report preliminary results on a protocol of induction of parturition in recipients carrying calves from embryos obtained by scnt. parturition was induced at 380 days in 22 pregnant embryo recipients. each recipient received an intramuscular injection of 750 µg of cloprostenol. the females were monitored hourly for onset of the second stage of parturition (appearance of the amniotic sac), duration of second stage of labor (fetal expulsion), and duration of the third stage of parturition (expulsion of the placenta). additionally, sex, weight and health of calves were recorded. all data are reported as mean ± sem. the interval from cloprostenol treatment to the beginning of the second stage of labor was 25.33 ± 2.18 hours (range 6 to 32 hours) with 70% of the recipients calving between 24 and 32 hours after treatment. duration of the second stage of parturition was 25.33 ± 4.49 minutes. the 24 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 25 duration of the 3rd stage of labor was 3.31 ± 0.81 hours. one female required obstetrical assistance and retained the fetal membranes (> 12 hours). the mean weight of calves was 34.23 ± 1.47 kg (33.54 ± 1.64 for males and 35.78 ± 3.2 for females). calves were standing and nursing withing 2.73 ± 0.6 hours after birth. the duration of the stages of parturition, the incidence of dystocia and of retained fetal membranes were similar to those reported previously by the authors for pregnancies from in vivo produced embryos.3 these preliminary results suggested that induction of parturition using cloprostenol is a good strategy to ensure the presence of veterinary help at parturition. the majority of females are expected to calve between 24 and 32 hours after induction. therefore, treatment should be performed early in the morning in order to obtain birth during the daylight. keywords: embryo, cloning, parturition, neonatal survival references 1. anouassi a, tibary a: development of a large commercial camel embryo transfer program: 20 years of scientific research. anim reprod sci 2013;136:211-221. 2. nagy p. reiczigel j, barua r. et al: pregnancy and parturition in dromedary camels iii. incidence, timing and factors affecting abortions and perinatal mortality under intensive management. theriogenology 2023;197:322-333. 3. tibary a, anouassi a: reproductive physiology in the female camelidae. in tibary a, anouassi a. theriogenology in camelidae, 1st edition, rabat; actes editions: 1997. p. 208-222. are leukocytes in semen and poor semen quality indicators of brucella ovis infection in rams? dinesh dadarwal, abby toews, roshan fernandopulle, fritz schumann, brooklin hunt, kamal gabadage large animal clinical sciences, western college of veterinary medicine, university of saskatchewan, saskatoon sk, canada brucella ovis (b. ovis) is a nonzoonotic, infectious reproductive pathogen of sheep known to cause subfertility and infertility in rams due to inflammation of genital organs and accessory sex glands. our objectives were to assess 1. the prevalence of b. ovis in rams in saskatchewan and 2. the association between naturally occurring b. ovis infection and leucocyte and/or semen quality parameters. fifty-eight rams (various breeds) from 5 producer-owned flocks in various locations in saskatchewan were enrolled for the study carried out in june 2022. on the day of the visit, a breeding soundness evaluation (bse) was performed. the bse included a brief physical examination (including body condition score, 5-point scale), scrotal content assessment, scrotal circumference measurement, and semen quality (motility and sperm morphology). gross motility of good and very good (10 x objective), individual progressive motility of > 60% (40 x objective), and > 70% morphologically normal sperm (using eosin-nigrosin stained semen smears under 100 x objective) indicated a satisfactory semen sample. semen samples were aliquoted and submitted to prairie diagnostic services for the culture of b. ovis. the b. ovis isolates were confirmed by whole genome sequencing. continuous and binomial outcomes were compared by linear and logistic  regressions (odds ratio), respectively, using stata/be17. p values < 0.05 were considered significant. a total of 15/58 (25.9%) rams from 2/5 flocks were positive for b. ovis on bacteriological culture. the scrotal circumference was, on average, 4.7 cm lower (p < 0.01, 95% confidence interval [ci] 2.2 5.7) in rams with bcs < 2.5 than in those with bcs > 2.5. the scrotal circumference tended to differ (p = 0.08) among breeds. rams with leukocytes in semen had 16 times higher odds (p = 0.01, 95% ci 1.9-133.5) of diagnosed with b. ovis than those with no leukocytes. the b. ovis positive rams had only 7.5 and 6.8% odds (p < 0.01) of having > 70% morphologically normal sperm and sperm motility characterized as fair to very good, respectively. further, the semen ejaculate of b. ovis positive rams had 29 times higher odds (p < 0.01, 95% ci 18.1 40.4) of having detached normal sperm. in conclusion, 1 in every 4 tested rams was positive for b. ovis on culture. leukocytes in semen and poor semen quality (motility and morphology) are valuable indicators to identify rams for b. ovis testing. keywords: brucellosis, ram, infectious epididymitis, infertility fetal ultrasonographic assessment to predict days prepartum in unsynchronized ewes jamie stewart, nicole sugai, sherrie clark-deener, kevin pelzer virginia-maryland college of veterinary medicine, blacksburg, va, usa parturition induction is the treatment of choice for ewes diagnosed with pregnancy toxemia. with unknown breeding dates, parturition induction is frequently delayed due to the owner’s desire to preserve fetuses’ life. there is a need to better predict fetal maturity, as this may hasten the decision to induce parturition. objective was to assess parameters that can predict when healthy ewes are near term. a group of 33 haired ewes were confirmed pregnant by transabdominal ultrasonography after an unsynchronized breeding to a single ram over a 30-day breeding period. sample collection and evaluation began at 4 days prior to the first possible due date. milk ph (digital ph-meter), milk protein percentage (brix refractometer), and serum progesterone concentrations (ria) were measured twice weekly until parturition. transabdominal ultrasonography to assess fetal heart rate (hr), renal corticomedullary distinction (cmd), and gastrointestinal peristalsis (gip) was performed once weekly until parturition. parturition dates for each ewe were used to calculate the days prepartum (dpp) for each parameter measured at each collection point. data were analyzed using r. continuous parameters (milk ph/protein, serum progesterone concentrations, and fetal hr) were analyzed by anova with a fixed variable of dpp grouped into ≤ 5, 6 10, 11 15, or 16 22 days. for categorical data (fetal gip and cmd), data on dpp were analyzed as a continuous variable using anova with the categorical score included as the fixed variable. ewe was included as a random variable for all analyses. milk ph did not differ (p = 0.72) in relation to dpp but tended to differ (p = 0.08) based on collection date which we speculate may have resulted from differing ambient temperatures. milk protein also did not differ (p = 0.12) in relation to dpp but there were no similar effects (p = 0.86) of collection date. fetal hr at 16 22 dpp (166 ± 7.2 bpm) was greater than at 6 10 dpp (140 ± 3.9 bpm; p = 0.007) and ≤ 5 dpp (138 ± 3.4 bpm; p = 0.001) and tended (p = 0.09) to be greater than at 11-15 dpp. fetal hr did not differ (p ≥ 0.35) among 11-15, 6 -10, or ≤ 5 dpp. mean dpp http://dx.doi.org/10.58292/ct.v15.10001 26 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 for absent/occasional fetal gip (13 ± 1.2 days) was greater than mean dpp for frequent (8.1 ± 0.8 d; p = 0.005) or continuous (5.4 ± 0.9 d; p < 0.01) gip, which did not differ (p = 0.15) from each other. mean dpp for absent fetal cmd (13 ± 2.0 days) was greater than mean dpp for distinct cmd (7.7 ± 0.9 d; mean dpp for indistinct cmd (8.6 ± 0.9 d) did not differ (p = 0.04) from absent (p = 0.13) or distinct (p = 0.81) cmd. results presented herein demonstrated that ultrasonographic evaluation of fetal hr, gip, and cmd can predict when healthy ewes are within ~ 1.5 2 weeks from their due dates. in a field setting, milk ph proved to be an unreliable measurement; however, further investigation is necessary to assess its measurement in controlled temperature (e.g. in a laboratory). keywords: ewe, fetus, milk ph, parturition, ultrasonography agreement between evaluators assessing bull sperm morphology by conventional methods versus video capture jenifer koziol,a laura waugh,a ryan williams,a leo brito,b colin palmer,c chance armstrongd aschool of veterinary medicine, texas tech university, amarillo, tx, usa bschool of veterinary medicine, university of pennsylvania, philadelphia, pa, usa cwestern college of veterinary medicine, university of saskatchewan, saskatoon, sk, canada dcollege of veterinary medicine, auburn university, auburn, al, usa sperm morphology assessment has been an important aspect of bull breeding soundness evaluation and has been reported as one of the most significant reasons that bulls fail evaluation. objective was to determine the agreement between skilled evaluators assessing bull sperm morphology by conventional methods versus video capture. the impetus behind this study was that though intra-evaluator and inter-evaluator experiments have been conducted, no studies have been reported comparing reading of stained morphology slides via microscope versus evaluation of video captured by microscopic evaluation at the same power. for this experiment semen was collected from 6 bulls and 5 eosin-nigrosin stained slides were made per bull. one slide was utilized for video capture at 1,000 x oil-immersion utilizing a nikon ds-fi3 high-definition color microscope camera taking care to make sure that the slide was in focus during image capture. once 100 cells had been video captured, the video captured sperm were labeled 1 100 ensuring that all sperm captured were clearly in focus and fully visible on the screen using the video capture software. care was taken to ensure that the video captured cells were representative of the entire slide. this process was repeated for 6 bulls. following imaging, 1 slide and 1 video per bull were sent to 4 independent board-certified theriogenologists with substantial expertise in evaluation of bovine sperm morphology. eosin-nigrosin slides and video were coded for anonymity so that one would not influence the evaluation of the other. the 4 evaluators were requested to review slides using their preferred individual methods for reviewing stained slides and assess 100 cells at 1,000 x magnification under oil-immersion and categorize per society for theriogenology morphology reporting standards. for the videos, cells were numbered as described above and evaluators were asked to categorize as they did for stained slides. evaluators had the opportunity to add additional comments to their evaluator sheets. data were analyzed utilizing r stats package utilizing a mixed model anova. there was a difference (p = 0.005) between evaluators evaluating the video captures versus using conventional methods. the coefficient value of -7.568 suggested that the video capture leads to less variability among the evaluators; with a standard error of 2.119, the results were proven to be precise. however, following comparison between conventional versus video capture, evaluators declared more cells as normal. this was most evident in 1 bull that suffered from high levels of diadem defects. this defect was described by evaluators during conventional evaluation but was not identified with the safe frequency on video capture which led to a large discrepancy in morphologic classification numbers. consequently, it could be inferred that although video capture offers a greater precision between evaluators, conventional methods provide increased accuracy as evaluators are able to fine focus on sperm defects enabling them to identify those defects that were not as easily identified (e.g. diadem defect) for correct categorization. keywords: bovine, bull, sperm morphology, assessment effect of season on gestation length and parturition interval in alpacas salman waqas, eduardo arroyo, ahmed tibary department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa, usa alpacas are considered nonseasonal breeders although nutritional deficiencies and harsh environment can dictate seasonality. gestation length in alpacas ranges from 320 to 390 days. objective was to analyze the hypothesis that mating season (spring: march may, summer: june august, fall: september november, winter: december january) affects gestation length and subsequent parturition interval in alpacas. records on 978 gestations from 339 huacaya females in a large pacific northwest herd were analyzed. all females started their reproductive career between 13 and 16 months of age and all postpartum breeding was initiated 2 to 3 weeks after parturition. only gestations that were between 320 and 390 days in length and resulted in an offspring that survived by 3 weeks postpartum were included. data were analyzed by descriptive statistics and one-way anova. the means were compared using tukey’s test and presented as mean ± sem. pregnancy rate of mated females was 82.4% with a mean number of services per pregnancy of 1.8 ± 0.2. the overall mean gestation length was 342.24 ± 0.31 days with a median of 341 days. fifty percent of gestation lengths were between 336-348 days. the season of breeding had effect (p < 0.0001) on the length of gestation. the gestation length for spring breeding (349.27 ± 1.07 days) was significantly longer than for summer breeding (342.94 ± 0.43 days), winter breeding (341.98 ± 1.33 days) and fall breeding (339.06 ± 0.43 days). gestation length for summer breeding and winter breeding did not differ significantly from each other. gestation length for fall breeding was the shortest but did not differ significantly from that of winter breeding. in multiparous females, the interval between parturitions ranged from 334 to 1,412 days with a median of 367 and mean of 418.67 ± 5.87 days. fifty percent of parturition intervals ranged from 355 to 407 days. the season of preceding parturition had no effect (p = 0.529) on subsequent http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 27 parturition interval. the subsequent parturition interval was 433.9 ± 11.6, 416.18 ± 9.07, 413.3 ± 12.2 and 413.46 ± 8.63 for the preceding fall, winter, spring and summer birthing, respectively. the record analysis from this alpaca herd provided information that can help in establishment of herd reproductive efficiency and productivity benchmarks. these data confirmed the effect of season on pregnancy length and illustrate that, in well managed herds, pregnancy rate and interval between parturitions are better than previously reported.1,2 a more complete analysis of reproductive records from several large herds will allow a better characterization of areas that theriogenologists can focus on to improve herd productivity. keywords: alpacas, pregnancy, parturition, seasonality, productivity references 1. cruz a, cervantes i, burgos a, et al: estimation of genetic parameters for reproductive traits in alpacas. anim reprod sci 2015;163:48-55. 2. davis gh, wuliji t, moore gh, et al: growth, reproduction and fiber production of alpacas imported from chile. proc nz soc anim prod 1991; p. 255-258. delayed insemination with sexed semen relative to estrus improved pregnancy in beef heifers rachel hanson,a,b ramanathan kasimanickam,b vanmathy kasimanickam,b,c kamron ratzburg,b,d kate madsenb,e aanimal health clinic, blackfoot, id, usa bcollege of veterinary medicine, washington state university, pullman, wa, usa caarvee animal biotech llc, corvallis, or, usa dmarias veterinary clinic, shelby, mt, usa emountain view veterinary service, twin bridges, mt, usa sexed semen (ss) has been designed for the deliberate production of genetically superior calves of the desired sex, thereby accelerating genetic progress in a cattle operation. initially, ss was recommended for inseminating nulliparous heifers at 12 hours after the first estrus sign. however, delayed (> 12 hours) insemination with ss relative to the onset of standing estrus resulted in an increased pregnancy rate/ai (pr/ai) in dairy cattle. in beef cattle, ovulation occurs at 32 hours after the onset of estrus. given that oocyte lifespan is constant (~ 10 hours after ovulation), the optimal interval between insemination and ovulation is vital to allow adequate time for sperm transport and capacitation in the female reproductive tract. objectives were to 1. determine the effect of timing of ss insemination after estrus onset and ai sire on pr/ai in beef heifers and 2. elucidate the sire differences in postthaw sperm dna fragmentation percentage (%sdf) at hours 0, 12, and 24. angus beef heifers (n = 891) from 3 locations were blocked by age, bcs, and rts. the cidr + select synch program was implemented to synchronize estrus in all heifers. on day 0, heifers received a 1.3 g progesterone intravaginal insert (cidr), plus 100 μg of intramuscular gnrh (gonadorelin diacetate tetrahydrate; 2 ml). seven days later cidr devices were removed, estrus detection patches were applied, and 25 mg of intramuscular pgf2α (high concentration dinoprost; 2 ml) was given. heifers were observed thrice daily for estrus (visual + estrus alert patch status) until 96 hours after pgf2α treatment. heifers that expressed estrus were randomly assigned to receive insemination at 12, 20, or 28 hours after the first estrus expression. ss (sexedultra™ 4m) containing y chromosome-bearing sperm from 3 bulls was utilized. pregnancy was diagnosed by ultrasonography between 30 and 60 days after ai. postthaw microscopic percentages sdf at 0, 12, and 24 hours were evaluated for each bull using the acridine orange staining method. data were analyzed by mixed model. the pr/ai percentage did differ (p  = 0.05) among insemination time, 12 hours: 46.5 (139/299), 20 hours: 47.7 (142/298), and 28 hours: 55.8 (164/294), respectively. the pr/ai between 12 and 28 hours and between 20 and 28 hours differed (p = 0.02) but did not differ (p = 0.78) between 12 and 20 hours. no differences (p = 0.20) in pr/ai percentages among bulls and locations (bull 1: 47.9 [138/288]; bull 2: 47.7 [143/300]; bull 3: 54.1 [164/303]). an interaction (p = 0.04) of time of insemination by bull for pr/ai was detected. for bull 1, percent sdf at 0 and 24 hours postthaw did differ (p < 0.05), 2.2 and 6.4. for bull 2, postthaw percent sdf at 24 hours (7.9) was greater (p < 0.05) compared to 0 hour (1.3) and 12 hours (4.6). for bull 3, postthaw percent sdf did not differ (p > 0.1) among 0, 12, and 24 hours (1.2, 1.9, and 2.4, respectively). in conclusion, the time of insemination with ss when delayed to 28 hours after the first estrus expression resulted in improved pregnancy. lower pr/ai for heifers in 12 and 20 hour groups compared to 28 hour group was plausibly due to an increase in bull’s postthaw percent sdf over time and the time interval between insemination and ovulation. keywords: beef heifers, sexed semen, estrus onset, insemination, sperm dna fragmentation, pregnancy periparturient plasma prolactin concentrations and maternal behavior in beef cattle rory nevard,a,b sameer pant,a,b john broster,a,b scott norman,a,b,c cyril stephena,b aschool of agricultural, environmental and veterinary sciences, charles sturt university, wagga wagga, nsw, australia bgulbali centre for agriculture, environment and water, wagga wagga, nsw, australia, ckallangur veterinary surgery, kallangur, queensland, australia maternal ability of a cow contributes to calf health and survival, particularly in extensive beef production systems. improving maternal behavior could mitigate calf wastage, and therefore, characterizing physiological drivers of superior maternal behavior, and its objective assessment thereof are of interest. whilst the hormonal milieu around calving is complex, past studies have demonstrated that periparturient blood prolactin concentrations are a key component. it is yet to be established whether plasma prolactin concentrations can be used to predict maternal behavior postcalving in beef cattle. use of radio telemetry and activity meters have afforded novel opportunities for objective behavioral quantification in extensive systems, and therefore the primary objective was to evaluate the relationship between periparturient prolactin concentrations (ppc) and maternal behavior of cows assessed both subjectively via maternal behavior scores (mbs), and objectively via measuring cow-calf contact using proximity loggers. measures of maternal behavior and ppc were also compared against transfer of passive immunity (tpi) and calf http://dx.doi.org/10.58292/ct.v15.10001 28 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 performance measures such as weaning weights (ww) and average daily gain (adg). it was predicted that periparturient plasma prolactin concentrations are correlated to cow-calf contact in the postpartum period and calf health parameters. thirty-one multiparous angus cows (age 9 11 years) were selected after a single-sire controlled mating period. a month prior to the expected calving dates, cows were fitted with ultrahigh frequency proximity logger collars provided by sirtrack® (now lotek, havelock north, nz) and had blood collected for assessment of ppc. on the day of calving, calves were fitted with proximity logger collars and mbs was recorded based on the cow’s response to calf handling. throughout the calving period, cows were yarded, and blood was continually collected every second or third day. a commercial bovine prolactin elisa kit (assaygenie) was used to measure ppc. area under the curve (auc) analysis was performed to quantify ppc, which was then used to assess correlation with mbs and cow-calf proximity logger contact data. the loggers recorded between 702 1,893 contacts (mean 1,394, sd 294, ± sem 54) and between 47,884 350,360 seconds of total contact time (mean 178,694, sd 97,366, ± sem 17,777), cow-calf contact over 12 days was negatively correlated with ppc (r = -0.45; p = 0.012). precalving ppc was also weakly negatively correlated with mbs (r = -0.31; p = 0.043). neither cow-calf contact nor mbs were correlated (p > 0.05) with calf performance (ww, adg) or health (calf vigour scores, serum total protein 24 72 hours postcalving). average weaning weights of calves grouped according to their dams’ assigned mbs at birth tended to be greater for higher mbs, although not significant (p > 0.05). overall, results indicated that ppc could influence cow-calf interactions and mbs. however, further research is warranted to validate these results, and to evaluate the efficacy of using cow-calf contact data as an objective measure of maternal behavior. keywords: maternal behavior, beef cattle, calving, prolactin, proximity logger, calf health http://dx.doi.org/10.58292/ct.v15.10001 equine session surgical excision of a leiomyoma in a subfertile morgan mare rachel doenges, maria schnobrich rood and riddle equine hospital, lexington, ky, usa a 6-year, morgan mare, was presented for subfertility. during the previous breeding season, the mare conceived but could not maintain a pregnancy. referring veterinarian felt a mass associated with left uterine horn prior to breeding and owner was not interested in referral. on transrectal ultrasonography, a 3.2 cm firm mass was noted at the left uterine horn tip (as an intramural mass) that was observed during hysteroscopy. owner opted for surgical resection in hopes of improving mare’s fertility. mare was placed in dorsal recumbency, and a midline incision was made. left uterine horn was exteriorized allowing visualization of the mass at the distal end. an incision was made over the mass. this incision extended through the serosa and the myometrium down to the mass. mass was well encapsulated, making it possible to easily dissect around it. mass was removed from the uterine wall, except for a small portion that required incision into the uterine horn for complete removal. histopathology confirmed the presumptive diagnosis of leiomyoma (spindle cells arranged in intersecting streams and bundles and low mitotic rate [1 per 10, 400 x fields]). histopathology also confirmed that clean margins were achieved with normal myometrium surrounding the mass without any evidence of infiltration of neoplastic cells. mare’s recovery was uneventful. mare was treated with intravenous flunixin meglumine, potassium penicillin, and gentamicin. she was discharged on sulfadiazine/trimethoprim. a full recovery was made with no reported complications. occurrence of uterine tumors is low in horses;1 however, leiomyoma is the most common uterine tumors in mares, accounting for 26% of uterine neoplasia.2,3 leiomyomas are usually singular masses ranging 2 5 cm in diameter, but multiple masses in the same mare can occur.1,2 surgical excision is usually curative; however, there have been instances of recurrence, including a report of 2 half-sibling appaloosa mares that developed cervical masses despite ovariohysterectomy.4 these cases also suggested a familial predisposition.4 leiomyomas are commonly pedunculated but can be intramural as in this case.3 although leiomyoma is a benign neoplasia, these masses can negatively affect fertility. for this reason, surgical excision is indicated in broodmares with fertility issues.3 in contrast, removal is not necessary in mares not used for breeding as long as there is not hemorrhage or recurrent secondary endometritis.3 with pedunculated leiomyomas, removal can be performed by incision at the base of the stalk. this has been accomplished intrauterine using a snare or electrosurgical loop.5 removal of intramural masses proves more difficult and often requires partial hysterectomy or ovariohysterectomy, both negatively impact reproductive potential. however, mares with partial hysterectomies have gone on to produce healthy foals.6 in this case, preservation of the left ovary and uterine horn is a positive prognostic indicator for future reproductive success. keywords: mare, leiomyoma, uterine tumor references 1. hurtgen jp: uterine abnormalities. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 2671. 2. card ce: endoscopic examination. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 1947-1948. 3. mccue pm: neoplasia of the female reproductive tract. vet clin north am equine pract 1998;14:510-511. 4. romagnoli we, momont hw, hilbert bj, et al: multiple recurring uterocervical leiomyomas in half-sibling appaloosa fillies. j am vet med assoc 1987;191:1449-1450. 5. leblanc mm: diseases of the reproductive system: the mare. in: colahan pt, mayhew ig, merritt ig, et al: editors. equine medicine and surgery, 4th edition, goleta: american veterinary publications: 1991. p. 1036. 6. santschi em, slone de: successful pregnancy after partial hysterectomy in two mares. j am vet med assoc 1994;205:1180-1181. determining the volume of blood in a stallion’s ejaculate dale kelley,a hannah maxwell,a carly turner-garcia,b reed holyoaka adepartment of veterinary clinical sciences, college of veterinary medicine, oklahoma state university, stillwater, ok, usa blazy e ranch, guthrie, ok, usa hemospermia can have a detrimental effect on fertility depending on the amount of blood present in an ejaculate, with 5% having no impact on fertility.1 estimating the amount of blood in an ejaculate may aid in decision making on whether to utilize the semen for breeding or attempt further processing. purpose of this experiment was to determine a method to estimate the percent of blood contamination in an ejaculate. first portion of the experiment used a mathematical simulation to predict the packed cell volume (pcv) of a hemospermic ejaculate based on the pcv of whole blood. the pcv of the blood was assumed to be either 30, 35, or 40% and range of blood and semen dilutions from 0 to 100% was modeled for each assumed blood pcv by multiplying the percent dilution of the blood semen combination by the blood pcv to give a predicted pcv for the semen. a regression line was citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 29 http://dx.doi.org/10.58292/ct.v15.10001 30 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 plotted for each simulation, and the equation of the line calculated. this produced the following equation: percent of blood in ejaculate = [pcv semen+(pcv blood/100)] ÷ (pcv blood/100) second portion of this experiment compared the actual percent of blood in the ejaculate to predicted percent of blood in the ejaculate based on pcv of blood and hemospermic sample. semen was collected from 1 stallion and blood was drawn from the jugular vein of 10 donor horses into 10-ml edta tubes using a vacutainer needle. blood was loaded into microcapillary tubes and centrifuged (10,030 x g for 5 minutes) and pcv was determined using hematocrit reader chart. blood from each donor was added to semen at 5, 10, 20, 40 and 50% (v/v) to create 0.5-ml aliquots. raw semen and blood semen dilutions were loaded into microcapillary tubes, centrifuged, and measured. the above equation was used to predict the percent of blood in the ejaculate based on the pcv of whole blood and blood-semen combination. sas (version 9.4) sgplot procedure was used to generate a blandaltman plot of the difference and mean of the actual percent of blood contamination and predicted percent of blood contamination. student’s t-test was performed for zero bias using actual and predicted values and independence of bias was tested using the corr procedure using the bias (difference between actual and predicted) and magnitude (average of actual and predicted). outliers (n = 2) were removed and data were reanalyzed when standard deviations > 3. semen had no evidence of hemospermia based on the color after collection and centrifugation of microcapillary tubes. the mean difference between the actual percent of blood and predicted was 0.59 ± 0.51. the test for zero bias (actual versus predicted values) was not different (p = 0.26) and there was no correlation (p = 0.80) in the independence of bias. these data suggested that the amount of blood contamination in semen can be estimated based on the pcv of the stallion’s blood and pcv of the ejaculate. keywords: stallion, ejaculate, hemospermia, packed cell volume reference 1. turner ce, walbornn sr, blanchard tl et al: the effect of two levels of hemospermia on stallion fertility. theriogenology 2016;86: 1399-1402. prepartum hemorrhage in a warmblood mare erin newkirk,a stephanie walbornnb awellgrove equine, loxahatchee, fl, usa brood and riddle equine hospital in wellington, wellington, fl, usa periparturient hemorrhage (pph) is a potentially serious, life-threatening complication of pregnancy in mares. hemorrhage usually occurs at foaling or within 48 hours. prepartum hemorrhage has been described to occur in up to 14% of cases, with fatality range of 40 100%.1,2 early recognition of hemorrhage with prompt aggressive medical management appears to be related to successful treatment of the condition. a case of prepartum hemorrhage in a 19-year, multiparous warmblood mare, is described. mare presented at 324 days of pregnancy for acute onset of colic that was poorly responded to routine medical management including nonsteroidals, antispasmodics, and sedation. transrectal palpation and nasogastric intubation at presentation were within normal limits for a late-pregnant mare. transabdominal ultrasonography using the flash protocol3 was performed due to lack of response to treatment. swirling echogenic free-fluid was noted in the left mid-abdomen that was consistent with hemoperitoneum. supportive care was instituted on farm that included intravenous fluids, aminocaproic acid and yunnan baiyao treatment. resolution of overt colic signs were noted within 12 hours of initial presentation. mare was started on trimethoprim sulfamethoxazole and gentamicin sulfate for antimicrobial coverage and maintained on flunixin meglumine every 12 hours. she received intravenous fluids supplemented with calcium, potassium, dextrose, and vitamin b12 at 48 hours after initial colic due to continued depression, poor appetite, intermittent fever, and abnormalities on blood chemistry. mare became colicky at 72 hours after initial presentation. ultrasonography revealed distended stomach and sluggish small intestinal motility with no free intraabdominal fluid and 28l of gastric reflux. fetus was active with heart rate between 90 -100 beats per minute. she was subsequently referred to rood and riddle equine hospital for further medical support and evaluation. on further workup, no hematoma was felt on transrectal palpation or observed via ultrasonography. transabdominal ultrasonography findings were within normal limits with fetal heart rate were ~ 90 beats per minute. mare was treated with intravenous fluids, gastroprotectants, and antibiotics were changed to ceftiofur and rifampin. she was discharged 5 days later. at 342 days of pregnancy, the mare entered labor and foaled without assistance or complications. no attempt was made to breed the mare the same season as foaling, however the following year the mare was managed via embryo transfer. keywords: prepartum hemorrhage, periparturient hemorrhage references 1. dechant je, nieto je, le jeune ss: hemoperitoneum in horses: 67 cases (1989-2004). j am vet med assoc 2006;229:253-258. 2. arnold ce, payne m, thompson ja, et al: periparturient hemorrhage in mares: 73 cases (1998-2005). j am vet med assoc 2008;232:1345-1351. 3. busoni v, de busscher v, lopez d, et al: evaluation of a protocol for fast localised abdominal sonography of horses (flash) admitted for colic. vet j 2011;188:77-82. http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 31 assessment of methods to activate epididymal sperm motility giorgia podico,a victor absalón-medina,b,c igor canissoa adepartment of veterinary clinical medicine, college of veterinary medicine, university of illinois urbana-champaign, urbana, il, usa bdepartment of animal science, college of food, agricultural, and environmental science, the ohio state university, columbus, oh, usa cst genetics, south charleston, oh, usa horse epididymal sperm have satisfactory motility activation (mot-act) after harvesting and cryopreservation; however, donkeys typically display poor mot-act despite high-plasma-membrane integrity (pmi) with high-mitochondrial-membrane-potential (δψm). mot-act can be achieved in murine epididymal sperm with centrifugation by removal of sperm-bound seminal plasma (sp) proteins; also, in other mammals, the dilution of epididymal sperm with sp promotes mot-act. dilution of cryopreserved sperm with an extender without cryoprotectants aids mot-act in other species. it is possible that these methods can enhance donkey and horse epididymal mot-act and that there might be positive interactions between methods; however, these hypotheses have yet to be tested. single-layer colloid-centrifugation (slc) is a powerful tool to remove sperm-bound particles. thus, we assessed methods and potential interactions to promote motact in epididymal sperm by comparing an equid with satisfactory mot-act (i.e. horse) with another poor mot-act (i.e. donkey). mot-act, pmi, and δψm were assessed by submitting horse and donkey cryopreserved sperm to reextension (1:3) in the same egg-yolk extender used for cryopreservation (bc), also the same extender lacking cryoprotectants (cpa), and donkeyand horse-sp. the mot-act, pmi, and δψm were carried out 10-, 30-, and 60-minutes after incubation. donkey and horse epididymal sperm had lower (p < 0.05) mot-act, pmi, and δψm when diluted with donkeyand horse-sp. conversely, mot-act was similar (p > 0.05) among horse-sp, bc, and cpa and increased (p < 0.05) after 10 minutes of incubation. pmi and δψm were not different (p > 0.05) among groups in horse epididymal sperm. similar processing methods were then applied before and after slc for cryopreserved horse and donkey epididymal sperm; mot-act, pmi, and δψm were assessed. donkey epididymal sperm had higher (p < 0.05) mot-act when diluted in the same egg-yolk extender compared to donkey-sp or cpa. horse epididymal sperm had lower (p < 0.05) pmi and δψm when diluted in donkey-sp compared to bc and horse-sp. sperm recovery after slc was similar (p > 0.05) between donkey and horse epididymal sperm. zona-pellucida binding was carried out as an in vitro functional assessment of mot-act, as it is necessary for fertilization. dilution with sp from either species’ sperm did not enhance (p > 0.05) zona-pellucida binding as in other mammals. previously, it was suggested that donkey sperm does not bind to heterologous zona-pellucida (i.e. bovine); however, herein, it did bind similarly to horse sperm (p < 0.05). findings suggested that donkey epididymal sperm can have enhanced mot-act by reextension with bc unlike other standard methods. furthermore, this is the first report to demonstrate that donkey sperm binds to heterologous zona-pellucida. keywords: equid epididymal sperm, single-layer centrifugation, zona-pellucida binding assay two manual noncommercial methods to prepare platelet-rich plasma in donkeys (equus asinus) lorenzo segabinazzi, cynthia xue, alexis hall, silvia marchi, patrice bernier, robert gilbert, hilari french ross university school of veterinary medicine, p.o. box 334, basseterre, saint kitts platelet-rich plasma (prp) is a biologic, whole blood (wb) derived, immunomodulatory therapy that has gained popularity in broodmare programs for its potential to mitigate postbreeding uterine inflammation/infection and enhance fertility rates. in jennies, increased susceptibility to postbreeding endometritis and poor fertility rates have been associated with insemination using frozen-thawed jack semen. because many donkey breeds are threatened with extinction, frozen semen technology offers a viable chance at recovery. thus, prp may have potential in similarly benefitting donkey breeding programs. however, the majority of prp protocols previously described in donkeys have not undergone critical assessment. commercial and noncommercial prp production methods exist, but the latter confers advantages (e.g. field applicability and decreased cost) for a species more likely to receive veterinary care in resource-limited settings. therefore, the objective was to evaluate 2 manual noncommercial methods of producing prp (double centrifugation [dc] and single centrifugation [sc]) in 6 healthy adult donkeys (n = 3 jennies; n = 3 johns; ages 4 8 years) undergoing 3 blood collections 3 weeks apart. for dc-prp, wb collected in a 150 ml blood transfusion bag containing citrate-phosphate-dextrose solution with adenine was split into 50 ml tubes and centrifuged at 400 × g for 15 minutes. plasma was transferred in 15 ml conical tubes for centrifugation at 1,000 × g for 10 minutes, then the bottom 2.5 ml of plasma fraction from the second centrifugation was preserved as dc-prp with the remaining plasma supernatant as dc platelet-poor plasma (ppp). for sc-prp, wb was collected in 2.7 ml sodium citrate vacutainer tubes, centrifuged at 120 × g for 10 minutes, and the top third supernatant plasma fraction subsequently deemed as sc-ppp with the remaining lower plasma fraction as sc-prp. concentrations of platelets, leukocytes (wbc), and erythrocytes (rbc), and platelet-derived growth factor-bb (pdgf-bb) were assessed in the wb and extracted prp and ppp. data were analyzed by friedman and dunn’s as a post hoc test with significance as p < 0.05. both protocols increased platelet concentration (1.7 5, 2-fold), reduced wbc (1.1 50, 4-fold), and decreased rbc (at least 829-fold) compared to wb (p < 0.0001). activity of pdgf-bb in dc-prp and sc-prp increased (p < 0.003) by 1.2  -7.0-fold compared to wb platelet concentration and pdgf-bb activity increased 3-fold (p < 0.0001) and 5.8-fold (p = 0.0007), respectively, in dc-prp than sc-prp. leukocyte concentrations were wider (p < 0.0001) in dc-prp than sc-prp but were not different (p = 0.61) between wb and dc-prp reductions in wbc and rbc were 42-fold and 22-fold greater, respectively, in sc-prp than in dc-prp (p < 0.0001). these results indicated that noncommercial methods of prp preparation described in horses are applicable to donkeys. double centrifugation yielded higher platelet concentrations and pdgf-bb activity despite greater wbc and rbc contamination than prp processing via sc. future studies are needed to determine in vivo efficacy of prp in donkeys. keywords: endometritis, jennies, frozen semen, blood therapy http://dx.doi.org/10.58292/ct.v15.10001 32 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 colostrum quality associations with passive immunity transfer in foals humberto magalhaes, gustavo araujo, ilaria colombo, kianna spencer, giorgia podico, igor canisso department of veterinary clinical medicine, university of illinois urbana-champaign, urbana, il, usa equine epitheliochorial placentation precludes the transfer of passive immunity (tpi) from mare to fetus; thus, colostrum serves as the sole source of tpi. colostrum assessment at foaling with a brix-refractometer is the primary method used in practice to determine colostrum quality and its potential for a complete tpi (> 800 mg/dl); however, guidelines have not been critically established. in addition, as new tools become available to monitor mammary gland secretions to detect impending foaling (e.g. handheld ph and conductivity devices), it would be helpful to determine if those devices can also add value to the colostrum evaluation at foaling. objective was to determine whether ph, conductivity, and brix-index at foaling can be used to predict tpi. colostrum of periparturient mares (n = 114) were assessed at foaling for ph, conductivity, and brix-refractometer (laquatwin-horiba, japan). foaling was assisted, and time to stand and time to the nurse were recorded. at 24 hours after foaling, foals had a complete blood cell count and tpi assessment using a commercial analyzer (ars, foal-igg-analyzer). foals were grouped based on igg concentrations as complete-tpi (i.e. ≥ 800 mg/dl) and failed-tpi (i.e. < 800 mg/dl). data were analyzed for normality with shapiro-wilk’s test and multiple logistic regression. cutoff values for the brixindex (> 24.4%), conductivity (< 4.5), and ph (< 6.4) were determined by receiver operating characteristics (roc). sensitivity, specificity, positive predictive value (ppp), and negative predictive value (npv) for the brix-index, conductivity, and ph of the colostrum at foaling were calculated to predict the tpi. of all foals, 74.5% (85/114) had complete tpi and 25.4% (29/114) failed. foals with complete-tpi had igg (1,912 ± 278.1mg/dl) four-fold higher (p < 0.0001) than those with failed-tpi (480.4 ± 260.6 mg/dl). their brixindexes were 26.5 ± 4.3 versus 21.4 ± 5.9 (p = 0.0007), respectively. conductivity and ph differed (p = 0.007 and p = 0.02, respectively) between completeand failed-tpi (conductivity 4.4 ± 0.6 versus 5.4 ± 11.7 and ph 6.2 ± 0.2 versus 6.5 ± 0.4, respectively). there were strong associations between conductivity and ph (r = 0.87) and a negative and strong correlation between ph, conductivity, and the brixindex (r = -0.87; r = -0.78). igg concentra rations were moderately correlated with rbc (r = 0.70) and the brix-index (r = 0.71). sensitivity, specificity, ppp, and npv of the brix-index, conductivity, and ph of colostrum at foaling to predict the tpi were 0.80, 0.76, 0.95, and 0.38; 0.77, 0.71, 0.93, and 0.38; 0.75, 0.37, 0.71, and 0.42, respectively. tpi of brixindex coupled with conductivity or ph presented a sensitivity, specificity, ppp, and npv of 0.85, 0.60, 0.89, and 0.40 (p = 0.02), and 0.83, 0.50, 0.89, and 0.37 (p = 0.01), respectively. combining conductivity and ph resulted in a sensitivity 0.91, specificity 0.91, ppp 0.33, and npv 0.33. in conclusion, colostrum’s brix-index, ph, and conductivity are closely linked with tpi in foals; roc-values could indicate if a foal could benefit from colostrum supplementation or will likely need hyperimmune plasma. keywords: equine colostrum, quality, immunoglobulin g commensal virome of the equine uterus lulu guo,a udaya desilva,b reed holyoaka acollege of veterinary medicine, oklahoma state university, stillwater, ok, usa bdepartment of animal and food science, oklahoma state university, stillwater, ok, usa our aim was to classify and characterize the resident uterine virome of healthy mares. we determined the presence of a resident virome in the healthy uterus with the objective to elucidate the association between the microbiome and the virome within the uterus. although the vaginal microbiome of mares has been well studied, very few investigations of the healthy uterine microbiome have been conducted due to the uterus long being considered a sterile environment. we recently established that the healthy equine uterus harbors a rich and diverse microbiome. as an obvious next step in elucidation of the equine uterine microbiome, we searched for the presence of an endometrial virome within healthy mares’ uterus. we were also interested in the presence of a bacteriophage community within the uterus that could be harnessed to control pathogenic bacteria in lieu of commercial antibiotics to which many organisms have developed a resistance. small volume lavage samples were filtered through 0.45and 0.22-μm filters to remove bacteria, dnase and rnase treated to remove free nucleic acids, and proteinase treated to dissolve viral capsids. the remaining nucleic acids were whole genome amplified and subjected to metagenomic next generation sequence analysis (mngs). subsequent bioinformatic analysis revealed the presence of a robust virome including several hitherto unknown bacteriophages. we also established some host microbe bacteriophage relationships. a preliminary analysis of the identified viruses is presented. our analysis demonstrated the presence of both rna and dna viruses belonging to 11 classes, 11 orders, 14 families, 8 genera, 7 phyla, and 29  species. the core host species predicted for the viruses identified in this study perfectly correspond to the uterine microbiome described by us in a previous study. our research not only revealed the existence of a native virome, but also the presence of antibiotic resistance genes involved with resistance to 1 or more antibiotics. we anticipate that our research initiative will provide a foundation for researchers to explore the reproductive tract virome in other mammalian species. keywords: virome, bacteriophage, horse, endometrium, uterus, microbiome antiluteogenic effects of cloprostenol in cycling mares sabrina cousseau,a erin oberhaus,b carlos pintoa adepartment of veterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la, usa bschool of animal sciences, college of agriculture, louisiana state university, baton rouge, la, usa the aim of this study was to assess the effects of serial treatment of the prostaglandin f2α (pgf2α) synthetic analogue, cloprostenol, on early luteal development and gonadotropins profiles. it was hypothesized that cloprostenol treatment would prevent http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 33 luteal development and affect gonadotropins concentrations. eight cycling mares were arranged in a crossover design being studied during 1 control (luteal) cycle (ctrl) and 1 aluteal cycle (al). serial transrectal ultrasound examinations were performed, mares were detected in estrus with ≥ 35 mm follicles, and ovulation was induced with intramuscular deslorelin acetate (1.8 mg). at detected ovulation (day 0), mares were randomly allotted to a ctrl group or an al group induced by 500 µg of cloprostenol treatment twice daily on days 0, 1, and 2 and once daily on days 3 and 4. daily blood samples for ria determination of plasma progesterone, luteinizing hormone (lh), and follicle-stimulating hormone (fsh) concentrations were collected. interovulatory intervals (ioi) were recorded and compared using a paired t-test. hormone plasma concentrations were compared between groups until seven days postovulation (day 7) using a repeated measures anova. the significance level was set as p ≤ 0.05. mean (± sd) ioi of al cycles was shorter (p < 0.0001) than ctrl cycles (12.88 ± 1.35 versus 21.38 ± 1.30 days). concentrations of plasma progesterone remained below 1 ng/ml in the al group throughout the period evaluated. aluteal cycles presented sustained lh concentrations that became higher (p ≤ 0.04) than ctrl cycles starting 6 days postovulation. day-by-day plasma fsh concentrations did not differ (p ≥ 0.1416) between groups at any of the time points evaluated. in conclusion, serial treatment of cloprostenol during the early stages of luteal development had a similar antiluteogenic effect to what was previously reported with the use of the pgf2α natural analogue dinoprost.1 cloprostenolinduced aluteal cycles were characterized by a shortened interovulatory period, persistently low (< 1 ng/ml) plasma progesterone concentrations, sustained elevated concentrations of lh, and no day-by-day differences on plasma fsh. keywords: mares, antiluteogenesis, pgf2α, lh, progesterone reference 1. coffman ea, pinto cr, snyder hk, et al: antiluteogenic effects of serial prostaglandin f2α administration in cycling mares. theriogenology 2014;82:1241-1245. http://dx.doi.org/10.58292/ct.v15.10001 case reports conservative medical management of a grade iii rectal tear in a mare alex wittorff, reed holyoak department of veterinary clinical sciences, college of veterinary medicine, oklahoma state university, stillwater, ok, usa a 9-year, american paint mare, was presented for routine breeding management; previous examination by a local veterinarian the week prior revealed that the mare was not in estrus. additionally, the owner noted that the mare appeared lethargic and was rapidly losing body condition. on visual examination the mare was quiet and alert. transrectal palpation quickly revealed an unanticipated grade iiia rectal tear (serosa intact with disruption of mucosal, submucosal, and muscularis layers). the defect was located laterally at 3 o’clock, 20 cm cranial to the anal sphincter and included a 30 cm diverticulum. a presumptive abscess was diagnosed via transrectal ultrasonography when a firm structure, 7 cm in diameter, was palpated perirectal and ventral to the diverticulum opening. edges of the diverticulum opening had begun to fibrose, but safe removal of inspissated purulent material, sloughing necrotic tissue, and impacted feces was achieved. due to the thinness of the cranial diverticulum wall and continued fecal accumulation with resulting diverticulum distention, rupture into the peritoneum was impending if not treated immediately. referral for surgical treatment was discussed with the owner, but due to financial constraints, the owner elected to donate her to the veterinary teaching program where she was treated with conservative medical treatment. mare received a dose of intramuscular ceftiofur crystalline free acid (excede® 6.6 mg/kg) on days 1 and 4 of treatment and a dose of intravenous n-butylscopolammonium bromide (buscopan, 0.3 mg/kg) and flunixin meglumine (0.25 mg/kg) on days 1 to 5. on day 1, manual removal of diverticulum contents was performed prior to lavaging the defect. a 3/4” diameter nasogastric tube was guided into the diverticulum and water infused by a stomach pump until the diverticulum was full, being careful not to overfill and stress the thin diverticulum wall. feces and debris were evacuated within the efflux. this was repeated until the efflux drained clear. packing of the diverticulum was performed with 6-inch brown gauze soaked in a 10% sugar-betadine solution to promote granulation tissue formation and for antimicrobial effects. lavaging and packing was repeated daily from days 2 to14, then every other day between days 14 and 21. early on during treatment, fecal and purulent material was adhered to the gauze, but by day 14 very little debris was present. by day 21, the diverticulum had reduced to a size too small for continued packing; localized treatment was discontinued at that point and a perirectal abscess could not be identified. examination of the remaining defect was performed every 2 3 days to ensure complete reduction of the diverticular space. scar formation and complete healing of the defect was achieved by week 4 of treatment. she was successfully utilized for transrectal palpation laboratories by veterinary students. this case demonstrated the resolution of a life-threatening, high-grade rectal tear with conservative medical treatment. survival rates for grade iii rectal tears vary from 75% (surgical treatment) to 38% (medical treatment). practitioners presented with grade iiia or b rectal tears can successfully implement conservative, medical treatment plans for these life-threatening injuries. keywords: rectal tear, medical management seminal vesiculitis in a zangersheide stallion anne sheerin, maria schnobrich, peter sheerin rood and riddle equine hospital, lexington, ky, usa seminal vesiculitis is the most common abnormality affecting accessory sex glands of the stallion. it is characterized by changes in seminal characteristics and inflammation of the glandular mucosa. it is typically caused by pathogenic bacteria resulting in purulent excretions. seminal vesiculitis has varying effects on the stallion’s fertility but typically results in reduced fertility, sperm longevity, and motility.1 this report describes a case of seminal vesiculitis in a 2009 zangersheide stallion. he was presented to the theriogenology service at rood and riddle equine hospital for routine semen freezing. the ejaculate from the first collection contained malodorous, thick, red tinged mucoid material. this material contained blood and neutrophils from the gel portion. a second collection was obtained 2 hours later, and the ejaculate was fractionated into the sperm-rich and sperm-poor fractions. due to the abnormal semen the stallion was examined via transrectal ultrasonography. transrectal ultrasonography of the accessory sex glands revealed thickened walls with edema and hyperechoic flecks in the seminal vesicles. it was determined that the purulent material was from the gel portion of the ejaculate. a culture was performed on the semen, which resulted in the growth of pseudomonas aeruginosa that was sensitive to amikacin, enrofloxacin, gentamicin, imipenem, polymyxin b, ticarcillin, and timentin. twice daily clean out collections and systemic enrofloxacin were recommended. however, the infection persisted. therefore, subsequent collections were obtained by fractionating the ejaculate to collect the sperm rich fraction and dispose of the gel fraction. further collections successfully obtained semen that had no growth in culture. fractionated ejaculates were frozen using a commercial extender containing antibiotics and no growth was noted in postthaw cultures. a total of 50.75 commercial quality doses of semen were collected in 9 collections. mean postthaw progressive motility was 62.1% (range 32.6 81%). stallion was able to continue his show career throughout collections and 34 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 35 his performance was not affected. seminal vesiculitis can presumptively be defined by semen characteristics, namely, pyospermia and hemospermia. further diagnostics include transrectal ultrasonography of the seminal vesicles, culture of semen, and endoscopy. most definitive diagnosis is by culturing seminal vesicle contents obtained via urethral endoscopy. various treatments for seminal vesiculitis exist, but most have limited success. systemic antimicrobial treatments often do not reach therapeutic concentrations within the glands. treatments frequently must be repeated as the infections can be recurrent.1 fractionating the ejaculate into sperm rich and gel portions is a useful technique to obtain a sperm rich fraction with no bacteria. keywords: stallion, seminal vesiculitis, semen collection reference 1. scheeren vfc, sancler-silva yfr, ali h, et al: update on seminal vesiculitis in stallions: j equine vet sci 2020;94:103234. neonatal pup death due to hyperthermia in a malfunctioning incubator bruce christensen, alyssa shelby kokopelli assisted reproductive services, sacramento, ca, usa a 4-year, female french bulldog, was presented for breeding with chilled, shipped semen from a proven male french bulldog. serum progesterone and lh assays determined the lh surge date and transcervical insemination was performed 5 days later with 600 x 106 sperm. at day 31 of pregnancy, transabdominal uranography revealed 3 embryos with heartbeats and appropriate development, and 1 site of embryonic resorption. serum progesterone concentrations were 33.94 ng/ml (tosoh aia-360 automated immunoassay analyzer; south san francisco, ca, usa). patient was given fenbendazole and amoxicillin/clavulanic acid. an evaluation conducted a week later confirmed no further signs of fetal losses. transabdominal ultrasonography (64 days after lh surge) revealed 3 fetuses with heart rates ranging from 167 188 bpm. all fetal kidneys had marked corticomedullary distinction. fetal intestines had mild degree of peristalsis. serum progesterone concentrations were 8.18 ng/ml. two female and 1 male neonate were delivered via elective cesarean surgery and resuscitated without complications; however, 1 of the females was markedly smaller than the other 2 neonates. each neonate was placed in an incubator with the thermostat set at 90ºf (32.2ºc) (puppywarmer proseries transport incubator [note: numbers not credible] (325 x 450 x 350 mm (battle creek, mi, usa). an oxygen concentrator was set to deliver 1 liter of oxygen/minute into the incubator (puppywarmer pro series, richland, mi, usa). after 30 minutes in the incubator, it was noticed that the pups had fluid pooling around their faces. temperature on the incubator thermostat was turned down to 85ºf (29.4ºc). pups were evaluated, they had good movement, were dried off and turned to their other sides. pups were visually monitored every few minutes through the incubator window and the temperature was observed on the thermostat that dropped to 85ºf (29.4ºc). labored breathing from pups was observed and 30 minutes after turning down the incubator to 85ºf, 2 pups ceased breathing. upon removal, it was noted that these 2 pups had dry mucous membranes. the incubator subjectively felt very warm. a rectal digital thermometer was placed in the anus of the remaining neonate that read > 109ºf (42.8ºc; maximum temperature capacity for the rectal thermometer). alcohol was placed on the ventrum and feet of the neonate, but it ceased breathing within 2 minutes. a digital thermometer placed in the incubator also read > 109ºf (42.8ºc). the incubator thermostat still read 85ºf (29.4ºc). following the death of the neonates, an independent mechanical engineer was able to repeat the malfunction. wide temperature fluctuations occurred independent of the positioning of the oxygen tube or oxygen flow. neonatal pups and kittens are poikilothermic and highly sensitive to environmental temperatures, unable to compensate using physiologic thermoregulatory mechanisms. much attention is placed on preventing hypothermia, but little has been focused on the dangers of hyperthermia. it is recommended that veterinarians and breeders do not rely on incubator thermostat readings alone, but should closely monitor environmental temperatures using independent devices, such as thermocouples, infrared sensors, and other thermometers. neonates should be closely monitored for signs of hypoor hyperthermia. keywords: neonate, hyperthermia, resuscitation, incubator, thermoregulation semen parameters, gross and histopathological findings of brucella ovis outbreak in rams devinda wickramasingha,a fritz schumann,a soraya sayi,b abby toews,a roshan fernandopulle,a kamal gabadage,a dinesh dadarwala alarge animal clinical sciences, western college of veterinary medicine, university of saskatchewan, saskatoon sk, canada b department of veterinary pathology, western college of veterinary medicine, university of saskatchewan, saskatoon sk, canada brucella ovis (b. ovis) is a gram-negative bacterium that primarily causes infectious epididymitis, mainly in mature rams. objective was to describe the clinical signs and pathological lesions in the reproductive tract of rams affected by a natural outbreak of b. ovis infection in a commercial sheep flock. eleven rams of various breeds, aged 1 5 years, were enrolled in a more extensive study on the breeding soundness evaluation (bse) criteria. rams were used for breeding in a multi-sire group during the 2021 2022 natural breeding season. during the summer of 2022, semen samples of all 11 rams were submitted to prairie diagnostic services, saskatoon for bacteriological culture. brucella ovis was isolated from all 11 rams and further confirmed by whole genome sequencing on the bacterial isolates. a complete bse was performed before euthanasia, followed by necropsy, giving special attention to reproductive organs. scrotal circumferences were satisfactory (36 ± 3 cm) for all rams. based on sperm morphology, 1 yearling ram was classified as questionable and others as unsatisfactory. two of 11 rams had unilateral palpable lesions in epididymis tail. rams that failed bse (n = 10) had good semen density but poor gross and individual motility (23.9 ± 17%). sperm morphology had low proportion of normal sperm (41 ± 12%) and high proportion of detached heads (40 ± 15%) and mid-piece defects (14.6 ± 14%). at necropsy, 6/11 rams had grossly visible lesions in testes (focal hemorrhages or mineralization), tunics (adhesions), and epididymides (epididymitis with purulent or caseous exudate). however, http://dx.doi.org/10.58292/ct.v15.10001 36 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 histopathological evaluation revealed inflammatory lesions in all rams in multiple reproductive organs, specifically, in epididymides (11/11, head n = 8, body n = 9, and tail n = 11), ductus deferentia (10/10), ampullae (11/11) and seminal vesicles (11/11) and to a lesser degree in testes (9/11, parenchyma n = 6 and rete testes n = 6). lesions in these tissues were mainly lymphoplasmacytic and neutrophilic infiltrates and fibrosis. other histopathologic lesions were vasculitis, mineralization, and spermatic granuloma formation. in conclusion, subclinical infections with b. ovis may be missed during a routine bse. this is evidenced by only 2 of 11 rams having abnormalities on physical examination whereas all rams had internal lesions. keywords: brucella ovis, rams, breeding soundness evaluation, epididymitis, histopathology testicular interstitial cell tumor in a cryptorchid horse eduardo arroyo,a salman waqas,a julie cecere,b thomas cecere,b phillip sponenberg,b ahmed tibarya acomparative theriogenology, department of veterinary clinical science, college of veterinary medicine, washington state university, pullman, wa, usa bvirginia-maryland regional college of veterinary medicine, virginia tech, blacksburg, va, usa a 6-year, american quarter horse gelding, was presented for evaluation of suspected cryptorchidism due to stallion-like behavior. the horse was purchased at an auction 10 months prior to presentation. owner reported occasional stallion-like behavior, going so far as to mount mares. referring veterinarian reported the presence of scrotal scar that suggested prior castration. anti-mullerian hormone serum concentrations were 10.2 ng/ml (geldings < 1 ng/ml) and testosterone serum concentrations were > 500 pg/ml (geldings < 100 pg/ml). the horse did not have any known medical problems, vaccination and deworming status were not known. physical examination was within normal limits on presentation. results of palpation of the inguinal area were compatible with those reported by the referring veterinarian. further diagnosis by transrectal palpation and ultrasonography revealed a soft mass that was suspicious of a testis in the right inguinal region. the mass was easily movable and located over the internal inguinal ring. percutaneous ultrasonography revealed a 5 cm diameter testis just cranial to the right external inguinal region. testicular parenchyma contained multiple 1 cm diameter echolucent areas that were suspected to be due to neoplasia. cryptorchidectomy was performed by laparoscopy in the standing horse under heavy sedation, and premedication with antibiotics (intravenous gentamicin 6.6 mg/kg, once daily and intravenous cefazolin 11 mg/kg, 3 or 4 times daily) and tetanus toxoid. retained testis was removed with the aid of a morcellator. mild emphyema was noted around the surgical sites, but no major complications were observed, and the horse was discharged 2 days after the surgery. histopathological examination of testis was inconclusive, but a diagnosis of interstitial cell tumor was made after consultation with a second group of pathologists. histomorphology features were different from the classic leydig cell tumor and included multinucleated spindleoid neoplastic cells that surrounded seminiferous tubules. the tumor was suspected to be hormonally inactive because leydig cells surrounding the sertoli cell-lined seminiferous tubules remote from the neoplasm were not atrophic. germinal and nongerminal cell tumors are rare, and the frequency of occurrence is difficult to ascertain in horse populations. most male horses are castrated at an early age. it is hypothesized that retained cryptorchid testes are more likely to develop a neoplasia due to the constant exposure to normal body temperature. most leydig cell tumors do not result in overproduction of steroidogenic hormones (estrogen and androgens). usually, these tumors do not metastasize, so prognosis is excellent after retained testis removal. this case highlighted the need of accurate diagnosis of location, size and echotexture of the testis in case of suspected cryptorchidism in hemicastrated horses. keywords: leydig cells, gelding, seminiferous tubule, hemicastration disorder of sexual development: 64 xx, sry negative phenotypical mare with bilateral retained testes gail mcrae,a erin runcan,a marco coutinho da silva,a christopher premanandanb adepartment of veterinary clinical sciences, college of veterinary medicine, the ohio state university, columbus, oh, usa bdepartment of veterinary biosciences, college of veterinary medicine, the ohio state university, columbus, oh, usa a 4.5-year, percheron mare, was presented for unilateral ovariectomy. referring veterinarian reported that the mare was displaying stallion-like behavior and had elevated serum testosterone (298.2 pg/ml) concentrations suggestive of a granulosa cell tumor (gct). examination of the mare’s external genitalia revealed a mildly elongated vulva and a slightly enlarged clitoris with no other abnormalities. vaginoscopy revealed normal vagina and cervix. complete palpation of her gonads was not possible due to the size of the mare. transrectal ultrasonography revealed gonads that had homogenous, moderately hyperechoic echotexture bilaterally. gonads measured 3 x 3 cm (left gonad) and 3 x 4 cm (right gonad). no follicles were observed within the parenchyma of the gonads and uterine horns were connected to the uterine body. additionally, the uterus was flaccid and contained a moderate amount of anechoic fluid within the lumen and absence of endometrial edema. due to the abnormal appearance of gonads and the owner’s eventual lack of desire to breed the mare, bilateral gonadectomy was recommended. surgery was performed laparoscopically with the mare sedated and restrained in stocks. after surgery, the gonads were transected along the sagittal plane, fixed in formalin, and submitted for histopathology. a sample of whole blood was submitted to the texas a&m molecular cytogenetics laboratory for karyotyping. grossly, the parenchyma of the gonads had a homogenous texture more consistent with testes, with no visible follicles. histopathological findings were sertoli-only tubules with tissue suggestive of ductus deferens and absence of ovarian tissue. karyotype results revealed a normal female genotype (i.e. 64, xx sry (-), inconsistent with the presence of testes. this case is a good demonstration of a disorder of sexual development (dsd) that is not characteristic of those that are more often described in the literature. in this case, the reason for the development of testes is unclear. there is evidence in other species that an upregulation of the sox9 gene may be involved.1 currently there is no clear answer, but this case exemplified an abnormal presentation of a dsd with an otherwise normal phenotypic presentation. http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 37 keywords: disorder of sexual development, mare, karyotyping reference 1. peretti v, katiuska s, ciotola f, et al: an unusual case of testicular disorder in sex development of arabian mare (64, xx sry negative). animals 2020;10:1963-1975. persistent endometrial cups in a thoroughbred mare mariah slack,a carol mcleod,b maria schnobricha arood & riddle equine hospital, lexington, ky, usa bcarol mcleod private practice, lexington, ky, usa endometrial cups in the mare are believed to regress by 140 days of pregnancy. these fetal tissues from the chorionic girdle invade the endometrium at ~ 35 days and secrete equine chorionic gonadotropin (ecg), which facilitates formation of accessory corpora lutea that provide progesterone support for pregnancy. maternal lymphocytic immune response is responsible for the natural destruction of endometrial cups from days 100 to 140 of pregnancy. failure of endometrial cup regression results in elevated ecg concentrations, prolonged diestrus, and continuous luteinization of follicles with failure of ovulation. this case describes a 12-year-old thoroughbred mare that had persistent endometrial cups for 2 years. initial confirmation of endometrial cups was performed after diagnosis of bilateral twin death at 53 days of pregnancy. transrectal ultrasonography of the reproductive tract revealed multiple corpora lutea on both ovaries and twin nonviable fetuses located at the base of each uterine horn. manual removal of the conceptuses was performed, and hysteroscopy confirmed numerous endometrial cups circumferentially located at the base of each uterine horn. serum ecg concentrations were 100.0 iu/ml. mare had numerous treatments to induce regression of the endometrial cups with marginal decreases in ecg each time. following treatments (ecg concentrations at treatment, day postovulation) were performed: 1 vial mycobacterium cell wall fraction immunostimulant (settle®, mcwfi) injected hysteroscopically (100 iu/ml, 53 days); 1 vial amnion product (stemwrap d™) injected hysteroscopically (12 iu/ml, 293 days); laser ablation (8.3 iu/ ml, 319 days); kerosene infusion (4.2 iu/ml, 335 days); 4 vials of mcwfi injected hysteroscopically in both uterine horns (3.4 iu/ml, 379 days; 4 vials intrauterine mcwfi of infusion (3.1 iu/ml, 410 days); and 4 vials of mcwfi injected in the region of previous cups hysteroscopically (1.3 iu/ml, 545 days). final regression of the cups was achieved at ~ 18 months after pregnancy loss. this case highlighted how endometrial cups can persist despite aggressive treatment. it is possible that the twin pregnancy and mechanism of removal was associated with the persistence of these tissues and further work is needed to identify why some mares have such difficulty with natural regression of endometrial cups. keywords: mare, endometrial cups, equine chorionic gonadotropin, persistent endometrial cups persistent müllerian duct syndrome and enlarged prostatic utricle in a male dog peter welsh,a kaylyn mcdaniel,a elizabeth goldsmith,b joshua ramsay,b eduardo arroyo,a alan conley,c tina owen,a yoko ambrosini,a michela ciccarellia aveterinary clinical sciences, washington state university, pullman, usa bveterinary microbiology and pathology, washington state university, pullman, usa cschool of veterinary medicine, university of california davis, davis, usa a 1-year, male intact miniature schnauzer mix, was presented for chronic intermittent hematuria. abdominal ultrasonography revealed a large, fluid-filled cystic structure dorsal to the prostate that extended cranially. computed tomography imaging revealed that the fluid-filled cavity resembled uterus, with both uterine horns entering scrotum through the inguinal canal adjacent to testes. dog had homozygote mutation of the anti-müllerian hormone receptor type 2 consistent with persistent müllerian duct syndrome (pmds). intraoperative fluoroscopy examination revealed a communication between uterus and urinary bladder via an enlarged utricle, explaining hematuria and urine in the reproductive tract (urometra). gonadohysterectomy was performed, and the tissue was submitted to the washington animal disease diagnostic laboratory for assessment. on histologic examination, the rudimentary uterine body and uterine horns (uterus masculinus) consisted of tubular structures lined by round to columnar epithelial cells and multifocal ulcerations. rudimentary vagina was lined by thick squamous epithelium. cross sections of rudimentary ductus deferens were randomly interspersed throughout the smooth muscle layers of the rudimentary uterus and vagina and within the connective tissue adjacent to right testis and epididymis. within the testes, < 10% of the seminiferous tubules had evidence of complete spermatogenesis, < 10% were atrophied, and the remaining had evidence of incomplete spermatogenesis. few mature and immature spermatids were within the epididymal lumen. several other cases of pmds in miniature schnauzers have been described in the literature, often being an accidental finding at cryptorchidectomy or associated with reproductive neoplasia like leiomyoma or sertoli cell tumors. to our knowledge, this is the first clinical report of a phenotypically intact male dog with pmds and urometra due to an enlarged prostatic utricle. this case illustrated a combination of a disorder of sex and urogenital sinus development. keywords: uterus masculinus, disorder of sex development, hematuria, urometra http://dx.doi.org/10.58292/ct.v15.10001 38 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 parturition induction in a late-term pregnant alpaca based on fetal maturity with unknown breeding timing nicole sugai, sherrie clark-deener, jamie stewart department of large animal clinical sciences, virginia-maryland college of veterinary medicine, blacksburg, va, usa a 4-year, intact female alpaca, was presented on 2/26/22 for pregnancy diagnosis and gestational aging due to unknown breeding history after the owner received her from a camelid rescue group. at this initial evaluation, transabdominal ultrasonography revealed a healthy fetus. thoracic height was measured 5.04 cm and fetal biparietal diameter measured 4.97 cm. using equations derived from a previous publication,1 the gestational age was determined to be between 229 and 247 days, with a due date between 8/1 and 8/20/22. reevaluation was performed on 8/3, due to the owner not observing any impending signs of parturition, such as behavioral alterations, vaginal discharge, or mammary gland development. both transabdominal and transrectal ultrasonography were performed to assess fetal viability and positioning. ability to visualize only caudal abdomen prevented diagnosis of a possible fetal posterior presentation. owner was instructed to wait another week before considering induction of parturition due to the unknown breeding date and lack of signs for impending parturition. client returned on 8/18 for a single lateral abdominal radiograph to better assess the fetus’ positioning. radiologist reported that the fetus was in posterior presentation with dorso-sacral posture, flexion of forelimbs and hindlimbs, and cervical ventroflexion. fetal viability appeared normal with an appropriate heartbeat visualized on ultrasonography, so the decision was made to continue monitoring. owner returned on 8/23 with concerns that the dam was acting lethargic and losing weight. the tail head ligament was slightly more relaxed than it was at the previous visit, and thick colostrum was expressed from the udder. using a hand-held ph meter, the milk ph was determined to be 6.44, consistent with impending parturition in mares.2 transabdominal ultrasonography revealed a strong fetal heartbeat (124 beats per minute) and good cortico-medullary distinction of the fetal kidney. at this time, the owner elected to induce parturition with 250 μg of intramuscular cloprostenol treatment at ~ 1:00 pm. owner took the dam home to monitor overnight with a plan to bring her back next day morning for further monitoring and parturition assistance in the clinic. dam began showing signs of active labor by ~ 9:30 am (~ 20.5 hours after induction). cria began to present cranially and dorsosacral with both front limbs extended by ~ 10:00 am. a healthy male cria (5.63 kg) was delivered with minimal assistance at 10:15 am. fetal membranes passed at 10:52 am and was evaluated to confirm complete expulsion. this case is important due to the ability to successfully induce parturition in an alpaca with an unknown breeding date when there was concern for the dam’s wellbeing. the information collected was extrapolated from other species and appeared to be an adequate predictor of fetal maturity and survival in this case. findings highlighted the need for further studies to better understand prepartum ultrasonographic and radiographic findings and milk composition changes in normal versus abnormal camelid pregnancies to facilitate such management decisions. keywords: alpaca, fetal maturity, parturition induction, gestational aging references 1. gazitúa fj, corradini p, ferrando g, et al: prediction of gestational age by ultrasonic fetometry in llamas (lama glama) and alpacas (lama pacos). anim reprod science 2001;66:81-92. 2. canisso if, ball ba, troedsson mh, et al: decreasing ph of mammary gland secretions is associated with parturition and is correlated with electrolyte concentrations in prefoaling mares. vet rec 2013;173:218. successful embryo transfer after large colon displacement laparotomy patricia xavier, maria ferrer, daniela cortes, amy brandon, naomi crabtree, robyn ellerbrock department of large animal medicine, college of veterinary medicine, university of georgia, athens, ga, usa an 8-year, appendix quarter horse mare, was presented in january 2023 for embryo transfer. mare had an extensive history of embryo transfer attempts, with no embryos recovered in 2021, and 4 embryos recovered in 2022 that were shipped and transferred, resulting in no pregnancies. on presentation, the mare had a no bacterial growth on uterine culture and was in diestrus. she received intramuscular dinoprost tromethamine (5 mg once), and ovarian activity was monitored via transrectal ultrasonography. ovulation was induced with deslorelin at insemination, when the mare had 4 follicles on the right ovary (29, 34, 43, and 44 mm), grade 3 uterine edema, and no intrauterine fluid. she was inseminated with 1.8 x 109 progressively motile sperm from a quarter horse stallion, and ovulated 2 follicles 44 hours later. due to a history of breeding-induced endometritis, the uterus was lavaged 6 hours after insemination with 0.9% saline and dmso, and was infused with 2 grams of ampicillin. the following day, the mare received an infusion with platelet rich plasma after uterine lavage. thereafter, she received intramuscular oxytocin (20  iu, once daily), oral firocoxib (0.1 mg/kg, once a day), oral altrenogest (0.44 mg/kg, once a day) and oral trimethoprim sulfamethoxazole (20 mg/kg, twice a day). at 60 hours after ovulation, the mare developed severe colic characterized by severe large intestinal gas distention, unresponsive to medical management (intravenous fluid therapy, flunixin meglumine [1.1 mg/kg, twice daily]), gastric decompression and lavage, sedation with detomidine and butorphanol, and 2 right-sided percutaneous large intestinal trocarisations). exploratory celiotomy via a ventral midline incision was elected 12 hours after onset of colic signs, and a right dorsal displacement of the large colon was diagnosed and corrected after pelvic flexure enterotomy, with a duration of 5 hours total anesthetic time. after surgery, the mare was managed with intravenous potassium penicillin g (20,000 iu/kg, once daily), intravenous gentamicin (6.5 mg/kg, once daily), intravenous flunixin meglumine (1.1 mg/kg, twice daily), and oral altrenogest (0.044 mg/kg, once daily) until embryo flush. she continued to colic, was febrile, and refluxed 24 32 liters/day for 48 hours after surgery, and was held off feed for 4 days after  surgery, during which time the colic signs and reflux resolved, although the mare remained intermittently febrile. embryo flush was performed 8 days after ovulation (5 days after surgery), and a grade 1 blastocyst was recovered. the embryo was immediately transferred to a 14-year, maiden warmblood mare. recipient mare ovulated 2 follicles 6 days http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 39 prior to transfer, and was treated with oral firocoxib (0.1 mg/ kg, once daily), oral trimethoprim sulfamethoxazole (20 mg/ kg, twice daily) and oral altrenogest (0.044 mg/kg, twice daily) starting at the time of transfer. three days after transfer, a 7 mm embryo was visualized via transrectal ultrasonography, and the mare is still pregnant at 60 days. several papers have described increased pregnancy loss after colic surgery or illness, and other retrospective studies have described a lower foaling rate when the mare underwent surgery before 40 days of pregnancy; however, there is no information on embryo recovery rates after colic surgery. to our knowledge, this is the first case report of embryo recovery from a mare after colic surgery, and successful pregnancy in a recipient mare. keywords: mares, embryo, colic surgery, endotoxemia, hyperthermia http://dx.doi.org/10.58292/ct.v15.10001 poster session progesterone concentrations in mares after follicular puncture alex wittorff,a candace lymanb adepartment of veterinary clinical sciences, college of veterinary medicine, oklahoma state university, stillwater, ok, usa bdepartment of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa transvaginal follicular aspiration of oocytes in mares may be performed during all stages of estrous cycle. interestingly, data are minimal, contradictory, and inconclusive when evaluating effects on serum progesterone (p4) concentrations. some literature suggested late formation of luteal tissue and rising p4 concentrations after follicular aspiration whereas others indicated that follicular aspiration had no effect on luteal formation and resulting p4 secretions. objective was to quantify serum p4 concentrations in unsynchronized mares undergoing follicular puncture for the purposes of either ablation or oocyte collection as part of a separate but concurrent research project. the concurrent ongoing project called for follicular puncture to take place in 6 healthy, young mares (3 10 years) 17 days apart. we hypothesized that luteal formation and resulting serum p4 concentrations are not affected by follicular puncture. all follicles > 5 mm were punctured and plasma samples were collected on day 0 (the day of follicular puncture), 3 or 4, and 6. serum p4 concentrations indicated that puncture of ovaries with mature luteal tissue present (n = 2) was not detrimental to the already elevated p4 concentrations present at follicular puncture. mares documented as having a recent ovulation and, therefore, with freshly developing luteal tissue (n = 4) had p4 concentrations > 2 ng/ml on day 3 and > 7 ng/ml on day 6, also indicating no detriment to the maturation and function of the luteal tissue. serum p4 concentrations in mares with a preovulatory (≥ 35mm) follicle (n = 4) were < 1 ng/ml on day 6, indicating stunted p4 concentrations. although testing and assay error should always be considered, delayed luteinization and corpus luteum (cl) formation due to follicular puncture must not be ruled out. all findings are further strengthened by the corresponding luteinizing hormone (lh) concentrations; if a numerical rise in lh concentrations was documented, it occurred alongside a numerical rise in p4 concentrations, with the exception of 1 mare. similarly, in the absence of quantitative increases in lh concentrations, p4 concentrations also did not rise to expected concentrations. in conclusion, follicular puncture in the presence of newly developing or mature cls did not compromise overall systemic impact of cls and p4 concentrations after 6 days in majority of mares (n = 8). two mares had a stunted lh concentrations rise, and similar inadequate p4 concentrations (p4 < 2 ng/ml) after 6 days. we suggest that more research is needed to determine whether delayed luteinization and stunted p4 concentrations of preovulatory follicles after follicle punction are inevitably linked with or occur independent of lh concentrations at puncture. keywords: mares, follicles, transvaginal follicular ablation, oocyte, corpus luteum immunohistological and molecular characterization of the hippo pathway in granulosa cell tumors of the mare aurore le breton,a mouhamadou diaw,a guillaume saintjeanb adepartment of clinical sciences, faculty of veterinary medicine, university of montreal, saint-hyacinthe, qc, canada, bdepartment of pathology and microbiology, faculty of veterinary medicine, university of montreal, saint-hyacinthe, qc, canada ovaries with honeycomb aspects are typical of granulosa cell tumors (gct) in the mare. they reflect abnormalities in the dynamic of the proliferation of granulosa cells, even if gcts are known to be benign neoplasms. to bring information on this poorly understood phenomenon, we focused on the hippo pathway, a series of oncoproteins (serine/threonine kinases) that have a key role in several cancers. hippo pathway is well known to control cell turnover in a physiological and pathological context. we hypothesized that this pathway is modified in the tumor compared to control tissues. therefore, we performed immunohistochemistry (ihc), western blot (wb), and polymerization chain reaction (pcr) analysis on paraffin-stored ovaries from mares with gct and controls (12 samples each). presence of yap and taz proteins (critical effectors of the hippo pathway) was evident in granulosa cells of both tumoral and control tissues. nevertheless, their intracellular repartition was significantly (anova) more cytoplasmic in gct suggesting their proproliferative action was repressed compared to controls for which the immunohistological signal was also unclear. the quantitative pcr can confirm this observation proving that target genes (ctgf, ankrd1, cyr61, and nov) are downregulated in gct in the mare. those findings suggested that gct in the mare has a regulation of granulosa cell proliferation by an antioncotic mechanism, resulting in the retention of the main effectors of the hippo pathway in the cytoplasm of the cell. this is an important argument to understand the benign character of equine gcts that are different from human (and most documented) ones, known to be malignant and have metastatic potential. keywords: mare, granulosa cell tumor, hippo pathway 40 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 41 clitoral adenocarcinoma in a mixed breed, spayed female dog christina moss,a nicole sugai,b rebecca persons,b brittany ciepluch,b julie cecereb apiedmont equine practice, the plains, va, usa bdepartment of small animal clinical sciences, virginiamaryland college of veterinary medicine, blacksburg, va, usa our case is a 9-year, mixed-breed, spayed female dog that was presented to the primary care veterinarian for dermatologic signs on the nasal planum consistent with pemphigus. a clitoral mass was an incidental finding at this examination and benign neglect was chosen as the mass was not currently causing any distress. multiple other masses were removed from the body at the initial visit with a high suspicion of neoplasia. subsequently, the patient was diagnosed with pemphigus foliaceus and was maintained on immunosuppressive doses of steroids. dog was presented to the emergency service after 5 weeks for prolapse of the clitoral mass and hemorrhagic discharge. in a follow up appointment, theriogenology department identified a 2 x 2 cm, multi-lobulated, highly vascular, nonexfoliating mass on the ventral wall of the vestibule around the clitoris; the urethra was not involved and could be easily catheterized. blood work revealed normal total calcium levels with evidence of hepatopathy attributed to the chronic steroid treatment for the dermatologic disease. no lymph node enlargement was detected on physical examination nor on abdominal ultrasonography. anal glands were of normal appearance during ultrasonographic examination. surgical removal of the mass was elected and performed. histopathology revealed an unencapsulated, infiltrative, densely cellular mass with cells arranged in sheets, ducts, and rosettes supported by a fibrous stroma. there were 15 mitotic figures identified per 10 fields (400 x). diagnosis was made of clitoral adenocarcinoma. a follow up examination with the primary care veterinarian ~ 1 month after mass removal revealed no gross evidence of local regrowth and no evidence of local lymph node metastasis. canine clitoral carcinoma is a rare neoplasm of the female genital tract. fewer than 12 cases have been reported and the first case was reported in 2010.1 clitoral carcinomas share a variety of clinical, histological, and immunohistochemical features with apocrine gland anal sac adenocarcinomas with normal anal glands.1-5 this suggests that the apocrine glands of the clitoris are the true primary site of the tumor versus metastasis from the anal glands. a review of 6 cases of clitoral adenocarcinoma described the condition as occurring in spayed females with a median age of 9.5 years.4 individual case reports of the condition involved animals of similar signalment.1-3,5 presenting clinical signs included vulvar licking, perineal swelling and irritation, hemorrhagic vulvar discharge, lower urinary tract signs, and presence of a visible or prolapsed soft tissue mass. clitoral masses have also been an incidental finding during physical examination, as observed in this case.4,5 previous case reports suggested that hypercalcemia of malignancy was present in up to half of the cases of canine clitoral carcinoma, potentially due to primary tumor production of parathyroid hormone-related protein.4 if not addressed, metastasis to locoregional lymph nodes may occur and has occurred, with histologic confirmation, in 60% of the previously reported cases.1,3-5 canine clitoral carcinoma remains a rarely reported condition requiring continued study and reports to determine nature, associated complications, and prognosis. keywords: clitoris, prolapse, neoplasia, adenocarcinoma, calcium, canine references 1. neihaus sa, winter je, goring rl, et al: primary clitoral adenocarcinoma with secondary hypercalcemia of malignancy in a dog. j am anim hosp assoc 2010;46:193-196. 2. mitchell ke, burgess dm, carrigan mj: clitoral adenocarcinoma and hypercalcemia in a dog. aust vet pract 2012;42:279-282. 3. rout ed, hoon-hanks ll, gustafson tl, et al: what is your diagnosis? clitoral mass in a dog. vet clin pathol 2016;45:197-198. 4. verin r, cian f, stewart j, et al: canine clitoral carcinoma: a clinical, cytologic, histopathologic, immunohistochemical, and ultrastructural study. vet path 2018;55:501-509. 5. mcaloney ca, taikowski kl, cronin jp, et al: pathology in practice. j am vet assoc 2021;259:1-3. vaginal lipoma in a cow eduardo prado, tulio prado, elizabeth whitt, alisson bradley, pierre-yves mulon department of veterinary clinical sciences, college of veterinary medicine, university of tennessee, knoxville, tn, usa lipoma is a benign tumor due to fat accumulation. it was observed in different parts of the body altering normal function of an affected organ. except for a report,1 vaginal lipoma in cows have not been documented. a 5-year, mixed breed cow, was presented on 01/09/2023 with cervicovaginal and rectal prolapse. on 12/2022, owner noticed a mass in the vaginal caudal aspect. cow last calved in august of 2022. on presentation, the cow was bright, alert, and responsive, and all vital parameters were within normal limits. cow had a body condition score of 8 out of 9. cow was restrained in a chute, and prolapses were washed with water and dilute iodine solution. rectal prolapse was resolved, and then the cervicovaginal prolapse was visualized. mass was observed in the vaginal left caudal aspect. attempts to replace the cervicovaginal prolapse were unsuccessful due to the mass blocking the ability of the reproductive tract to return to its normal anatomical position. on 01/10/2023, a resection of the mass was performed. mass was removed by electrocautery and closed in routine fashion. after mass removal, the cervicovaginal prolapse was replaced after several attempts and buhner stitch was applied. mass was submitted to histopathology for further evaluation and was interpreted to be a vaginal lipoma with secondary infection. keywords: cow, prolapse, lipoma, vagina reference 1. el-maghraby hm: a retrospective study on some surgical affections of the perineum and vagina in farm animals. zag vet j 2002;30:84-100. http://dx.doi.org/10.58292/ct.v15.10001 42 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 blastomycotic prostatitis marthina greer veterinary village and icsbwi/il/in lomira, wi, usa blastomycosis should be included in the differential diagnosis when a dog from a blastomycosis endemic area presents for signs and symptoms of prostate disease. prostate disease is a relatively common finding in intact male dogs. forms of prostate disease described in the literature include: benign prostatic hyperplasia/hypertrophy (bph), bacterial prostatitis, neoplasia of the prostate, and paraprostatic cyst(s). fungal prostatitis is not frequently described in the literature. of the fungal systemic infections noted, blastomycosis is the most commonly reported infection of the canine reproductive tract. blastomycosis is a fungal disease from soil fungus primarily in the upper midwest near a body of water and other regions in the united states. infrequently, prostate and testes are involved in blastomycosis making it as a systemic disease. organs more commonly affected by blastomycosis include lungs (in the form of fungal pneumonia), eyes, and skin. four dogs were diagnosed with blastomycosis prostatitis in our practice in 20 years. three of 4 dogs were presented for primary prostate disease whereas 1 was presented with a diagnosis of blastomycosis with primary ocular disease. consistent with reports in the literature, where sporting and hunting dogs are most commonly affected by blastomycosis, the dogs diagnosed were german shorthaired pointer, a boykin spaniel, a curly coated retriever, and the outlier, a samoyed. also consistent with the literature, 2 were under 3 years of age (curly coated retriever and samoyed) whereas the other 2 were > 7. first dog, the german shorthaired pointer, presented at age 7 with the only symptom of prostate disease. his ejaculate was submitted for bacterial culture. he returned 1 week later with respiratory distress and radiographs revealed evidence of fungal pneumonia. culture had grown blastomycosis; laboratory personnel were understandably distressed to find out that a fungal culture had been submitted. dog was euthanized due to the advanced form of pneumonia and lack of good treatment options at this time. second dog, a samoyed, presented as with the first, with the only symptoms being enlarged prostate. at surgery, a draining skin lesion was noted on a rear foot, suggesting blastomycosis on the differential list. third dog, a boykin spaniel, presented after examined by an ophthalmologist for retinal lesions, for castration and enucleation of both eyes. owner, a retired physician, elected to remove only the affected testes and both eyes so the dog could continue to serve as his hunting buddy. he was successfully treated with oral antifungal drugs. fourth dog, a curly coated retriever, was 3 years. his age was not consistent with the age most often associated with bph. he was recently seen enough that the mira vista urine blastomycosis test was used for diagnosis. he was treated with oral itraconazole, managed by an ophthalmologist to spare his vision, and was later castrated. he is still alive at this abstract submission. keywords: blastomycosis, fungus, prostate, prostatitis pregnancy in a warmblood mare inseminated with frozen-thawed-extended semen on-farm justin mcnaughten,a karen wolfsdorfb apalm beach equine clinic, wellington, fl, usa bhaygard’s equine medical institute, lexington, ky, usa frozen-thawed semen provides numerous advantages for breeders; however, for veterinarians this modality can create organizational and logistical challenges (more intensive mare management, specialized equipment to thaw and transport frozen semen, and the hazards of working with liquid nitrogen). to ameliorate these challenges, breeders are encouraged to utilize purpose-built facilities for breeding management and insemination. when a haul-in scenario is not an option, practitioners breeding onfarm with frozen-thawed semen must decide how to overcome the aforementioned challenges efficiently and safely. researchers have reported pregnancies after insemination with frozen semen that was thawed, extended, and cooled for 24 hours.1 in this case report, the authors describe the use of frozen-thawed-extended semen on-farm. a 12-year, multiparous warmblood mare (with a foal at foot), was examined on-farm for breeding management. owner requested that the mare is managed and inseminated on-farm with a single multi-straw dose of frozen semen. to manage expectations, the authors outlined a breeding management plan utilizing a timed-insemination protocol and splitting the multi-straw dose of frozen semen. serial reproductive examinations were performed. once a preovulatory follicle and moderate uterine edema were detected, the mare was given an intramuscular gnrh analogue (sucromate®, 1.8 deslorelin acetate mg/ml). treatment timing was selected ensuring the pre and postovulation inseminations were no more than 12 hours before and no more than 6 hours after ovulation, respectively. on the day of insemination, 0.5 ml straws were immersed in a water bath at 37ºc for 30 seconds. a commercial semen extender was added diluting the frozen-thawed semen to a final concentration of 50 x 106 total sperm/ml. to create equivalent insemination doses, the total volume was halved, transferred into 2 latex-free syringes, and packaged in individual styrofoam semen shipping containers with ice packs. first dose was utilized within 1 hour after thawing whereas the 2nd dose was chilled and stored at 5ºc for 14 hours. prior to each insemination, transrectal palpation and ultrasonography were performed confirming that the 1st and 2nd insemination occurred pre and postovulation, respectively. mare was assessed next day and postbreeding management was routine. transrectal ultrasonography on day 15 postovulation confirmed pregnancy. although the need for specialized equipment and the hazards of working with liquid nitrogen will not be eliminated, this case report demonstrated that some of the logistical issues may be overcome while breeding on-farm with frozen-thawed semen. keywords: equine, insemination, frozen, semen, breeding management reference 1. prell mj, mccue pm, moffett pd, et al: motility and fertility evaluation of thawed frozen stallion semen after 24 hours of cooled storage. j equine vet sci 2020;90:102983. http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 43 influence of season and plasma prolactin concentrations on fertility of bos taurus beef bulls rory nevard,a,b sameer pant,a,b john broster,a,b scott norman,a,b,c cyril stephena,b aschool of agricultural, environmental and veterinary sciences, charles sturt university, wagga wagga, nsw, australia bgulbali centre for agriculture, environment and water, wagga wagga, nsw, australia ckallangur veterinary surgery, kallangur, queensland, australia prolactin is known to influence testicular function in beef bulls, but the relationship between plasma prolactin concentrations and semen quality parameters (e.g. sperm motility and morphology) is unclear. furthermore, both seasonal variability and innate circadian rhythms can potentially influence plasma prolactin concentrations, complicating efforts to characterize the relationship between prolactin and bull semen quality parameters. objective was to assess seasonal variability in bull plasma prolactin concentrations and determine its relationship with semen quality parameters. twelve mixed-age beef bulls (7 angus and 5 limousin) were used. bulls were yarded, had blood collected, and examined for breeding soundness on the last week (± 1 week) of each month from april to december. reproductive parameters assessed included scrotal circumference (sc), progressive motility (pm) of sperm (assessed using computer assisted sperm analysis), and sperm morphology. plasma prolactin concentrations were quantified using a commercial bovine prolactin elisa kit (bprl, assaygenie). results indicated that plasma prolactin concentrations were lower (p = 0.043) in summer compared to winter in contrast to previous findings. plasma prolactin concentrations and daylength (measured in hours) were negatively correlated (r = -0.23, p = 0.040), but the correlations between plasma prolactin concentrations and reproductive parameters were not significant (prolactin versus sc: r = -0.041, p = 0.736; prolactin versus sperm morphology: r = -0.03, p = 0.789; prolactin versus pm: r = 0.02, p = 0.923). bull age had influence on both percentage normal sperm (p = 0.032) and sc measurements (p = 0.025). older bulls tended to have larger sc and more morphological sperm abnormalities. month of sampling significantly influenced sc. overall, plasma prolactin concentrations had influence on seasonal variability, and although no relationship was identified between plasma prolactin concentrations and male reproductive parameters, this could likely be attributed to the relatively small sample size, insufficient statistical power, and infrequent sampling. future studies investigating this relationship in larger populations, accounting also for seasonal variability, are warranted. keywords: bull, beef cattle, fertility, prolactin, season severe edema of the uteroplacental unit in a quarter horse mare stephanie walbornn,a erin newkirkb arood and riddle equine hospital in wellington, wellington, fl, usa bwellgrove equine, loxahatchee, fl, usa placentitisis described as inflammation of the placenta and is most commonly caused by an ascending infection through the cervix. clinical signs of placentitis include premature mammary development and vulvar discharge. ultrasonographic changes include thickening of the uteroplacental unit and separation between the placenta and the endometrium. several studies have described normal values for the combined thickness of the uteroplacental unit (ctup).1,2 routine monitoring throughout pregnancy via ultrasonographic examination can help to diagnose early cases of placentitis and implement treatment at the early stage of the disease process. a case of severe uteroplacental edema in a maiden 2008 quarter horse mare is described. mare was presented at 309 days of pregnancy for evaluation and monitoring after premature mammary development was observed and a higher ctup was diagnosed via transrectal ultrasonography. on presentation, mammary development was observed and vulvar discharge was not noted. on transrectal ultrasonography, ctup measured 2.5 3 cm and it was difficult to determine if the increase in thickness was present within the endometrium, chorion, or both. ctup was diffusely increased both dorsally and ventrally. a small area of separation was observed close to the internal cervical os. echogenicity of fetal fluids were within normal limits. allantoic fluid was slightly echogenic (1 out of 4) and the amniotic fluid was more echogenic (2 out of 4) and the amniotic membrane did not appear thickened. fetus was mobile and average fetal heart rate was 74 beats per minute. mare was given antibiotics, antiinflammatories, and regumate® and was monitored daily via ultrasonography for fetal and placental viability. next day, fetal heart rate increased to 120 beats per minute and remained elevated at subsequent examinations; therefore, the mare was started on intranasal oxygen at 10 liters/hour. no changes were observed in the placenta once treatment was implemented. mare entered spontaneous labor at 317 days of pregnancy and foaled with assistance. filly had signs of prematurity (floppy ears, domed head, silky haircoat, and incomplete ossification of the cuboidal bones). mare expelled most of fetal membranes within 3 hours after delivery and retained a piece in the nongravid uterine horn. treatment was implemented. examination of fetal membranes revealed thickening at the area of the cervical star and a few avillous areas on the chorion. fresh and fixed samples were submitted to the university of kentucky diagnostic laboratory for culture and histopathology. pathology report indicated chronic placentitis, but no etiologic agent was identified. postfoaling ultrasonography of the uterus revealed no severe edema within the endometrium; therefore, it was suspected that the edema was primarily within the chorion. the amount of edema present within the placenta raised the question of whether placental insufficiency or another placental abnormality also had a part in the premature foaling and the possibility of an infectious agent contributing to placentitis. keywords: placentitis, uteroplacental thickness, placental insufficiency http://dx.doi.org/10.58292/ct.v15.10001 44 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 references 1. bucca s, fogarty u, collins a, et al: assessment of feto-placental well-being in the mare from mid-gestation to term: transrectal and transabdominal ultrasonographic features. theriogenology 2005;64:542-557. 2. renaudin, cd, troedsson, mht, gillis, cl, et al: ultrasonographic evaluation of the equine placenta by transrectal and transabdominal approach in the normal pregnant mare. theriogenology 1997;47:559-573. fertility in a mare with pharyngeal lymphoma that was treated with doxorubicin stephanie walbornn,a justin mcnaughtenb arood and riddle equine hospital in wellington, wellington, fl, usa bpalm beach equine clinic, wellington, fl, usa doxorubicin is a chemotherapeutic agent utilized for several kinds of neoplasia including lymphoma. in mice, doxorubicin had a negative effect on granulosa cells.1 limited information is available on the effect of doxorubicin treatment on mares’ fertility. a case of a 12-year, irish draft maiden mare, with a history of doxorubicin treatment for localized pharyngeal lymphoma is described. last treatment was performed in september 2021. mare also had a history of recurrent respiratory infections and had been diagnosed with combined variable immunodeficiency. breeding management began in april 2022. mare was bred during 4 estrous cycles prior to recovery of an embryo. normal ovarian architecture with normal ovarian activity were present. mare was bred with frozen semen on the first estrous cycle and freshcooled semen on the subsequent 2 estrous cycles from 1 stallion and then switched to another stallion and bred over 1 estrous cycle with freshcooled semen. on day 7, grade 3 expanded blastocyst was recovered from an embryo flush. this embryo was transferred into a recipient mare on the same day and the mare was confirmed pregnant at 14 days. heart beat was also confirmed and to our knowledge the recipient mare is still pregnant. it is difficult to discern whether the difficulty obtaining an embryo was due to subfertility from the chemotherapeutic agent or systemic immunodeficiency predisposing the mare to a decreased immune response within the uterus. to our knowledge, this is the first report of breeding management in a mare treated with doxorubicin and successful recovery of an embryo with a viable pregnancy. keywords: subfertility, embryo transfer, doxorubicin reference 1. ting z, hong h, lin f, et al: effect of doxorubicin-induced ovarian toxicity on mouse ovarian granulosa cells. regul toxicol pharmacol 2017;86:1-10. diagnosis of canine herpesvirus preserves future neonatal viability tiffany hoffman,a casey rubin,b morgan agnewc arutgers university, new brunswick, nj, usa bphiladelphia animal hospital, philadelphia, pa, usa a 5-year, great dane female dog, was presented for right hind leg lameness, lethargy, and decreased appetite of 2 days of duration at 49 days of pregnancy. physical examination was unremarkable. transabdominal ultrasonography revealed that the fetus immediately cranial to the cervix had no heartbeat, decreased fetal fluids, and a thickened placenta. all other fetuses appeared normal. vaginal cytology revealed many cocci and neutrophils. owner declined for a culture. patient was empirically treated with 9 mg/ kg cefpodoxime once a day. patient’s initial serum progesterone concentrations were 28.54 ng/ml (in-house mini vidas® machine); high enough to maintain pregnancy. follow up ultrasonography (in 5 to 7 days) was recommended, but was not performed. patient was presented 8 days later with decreased appetite and blood-tinged purulent vulvar discharge. transabdominal ultrasonography was performed that revealed multiple deceased fetuses surrounded by echogenic fluid in both uterine horns. no viable fetuses were visualized. a complete blood count and serum chemistry were within normal limits; patient’s serum progesterone concentrations were 4.39 ng/ml. ovariohysterectomy was recommended and performed the same day. five fetuses were easily isolated from the uterus and it was diagnosed that they had died at different stages of development. based on the appearance of the fetuses, canine herpes virus (chv) was suspected, and a titer was submitted to cornell university. patient’s virus neutralization antibody titer was positive for chv. testing was recommended for other breeding female dogs in the same kennel, 2 of which also tested positive for chv. for all female dogs with positive titers, following recommendations were made for subsequent pregnancies: begin oral antiviral treatment starting 1 week prior to due date; give oral plasma from donor with a positive chv titer to all neonates at birth; give pups oral antivirals for 2 weeks after delivery. blood was drawn from female dogs in the kennel and plasma was stored for oral treatment to neonates at birth. cvh is 1 of the most common causes of viral abortion and neonatal death in canines whereas brucella canis and streptococcus spp. are the most common causes of bacterial abortion and neonatal death.1 naive female dogs exposed to chv during middle of pregnancy are at high risk for aborting underdeveloped fetuses.2 chv abortion is not common in subsequent litters after an initial exposure; however, viral recrudescence is possible.2 oral blood plasma (collected from patients with a positive chv titer) treatment may suppress viremia, help prevent chv infection, and decrease mortality in neonates.2 this case illustrated the importance of appropriate diagnostic testing to help preserve reproductive function in female dogs and neonatal viability. keywords: herpesvirus, abortion, vaginal cytology, neonatal viability references 1. lamm cg, njaa bl: clinical approach to abortion, stillbirth, and neonatal death in dogs and cats. vet clin north am small anim pract 2012;42:501-513. 2. decaro n, martella v, buonavoglia c: canine adenoviruses and herpesvirus, vet clin north am small anim pract 2008;38:799-814. http://dx.doi.org/10.58292/ct.v15.10001 student clinical case session ovarian teratoma in a quarter horse mare ashley beyer,a kylie roux,a allison salinger,b gabriel gonzalez,a christine lopp-schurter,c kevin walters,a heath king,a darcie sidelingera amississippi state university college of veterinary medicine, mississippi state, ms, usa bmid south equine sports medicine & surgery, tioga, tx, usa ciowa state university college of veterinary medicine, ames, ia, usa a 5-year, quarter horse, mare was presented for an ovarian mass. left ovary appeared enlarged with abnormal texture on transrectal palpation and measured ~ 8 cm in diameter with multiple hypoechoic, cystic areas along with several hyperechoic areas with shadowing on transrectal ultrasonography. right ovary was normal in size and contained multiple small to medium follicles. mare was diagnosed nonpregnant. based on the size and appearance of the left ovary, an ovarian tumor was suspected, and surgical removal was recommended. a standing-flank, hand-assisted, laparoscopic ovariectomy was performed. because of its size, the left ovary was removed from the abdomen using a sterile bag. on gross examination, tissues that resembled hair, adipose, bone, and cartilage were noticed, fitting the description of ovarian teratoma; however, owner declined histopathology. ovarian teratomas are reported to be the second most common ovarian tumor in mares, second only to granulosa cell tumors, and more commonly observed in younger mares.1 as a tumor of germ cell origin, teratoma must contain at least 2 of the mature form of germ cell tissues (endoderm, ectoderm, or mesoderm).2 prognosis for ovarian teratoma is good as they are typically benign and hormonally inactive.1 ovarian teratomas are often incidental findings with no associated clinical signs.3 contralateral ovary is often unaffected, continues cycling normally, and pregnancy rates are acceptable.2,4 removal of the affected ovary is the treatment of choice.1 mare recovered uneventfully from surgery and was discharged next day. owners report that the mare is doing well and they did not attempt breeding. keywords: mare, ovary, teratoma, laparoscopic ovariectomy references 1. catone g, marino g, mancuso r, et al: clinicopathological features of an equine ovarian teratoma. reprod domest anim 2004;39:65-69. 2. duarte l, allen wr, guimei m, et al: equine ovarian teratomas: diagnostic challenges illustrated by case reports. equine vet educ 2021;34:393-405. 3. singh h, singh n, jan mh, et al: clinicopathological features of ovarian teratoma presented as colic in mare. theriogenology insight 2019;9:7-11. 4. gündüz m, kaşikçi g, kiliçarslan r, et al: reproductive performance following unilateral ovariectomy for treatment of ovarian tumors in 7 mares. turk j vet anim sci 2010;34:283-287. outbreak of penile warts among a group of angus and charolais bulls thomas burke, manuel chamorro, thomas passler, miguel saucedo, chance armstrong department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa a group of angus and charolais bulls was presented for evaluation of penile masses. a total of 10% of the 300 bulls prepared for sale was affected. this group of bulls was reportedly eating and drinking normally and fed a diet of hay, 60% soy hull pellets, 20% distillers’ grain, and 20% ground corn. affected bulls were individually placed on a tilt table and stimulated rectally until erection was achieved. once extended, a towel clamp was placed under the dorsal apical ligament of the penis, to maintain extension, and the penis was examined. the masses appeared to be fibrous and varying in severity from small to large, single, coalescing, or pedunculated. masses were located laterally or circumferential with some invading towards or extending beyond the external urethral orifice on the distal third of the penis. three bulls had necrosis of the associated penile mass. bulls’ penises were cleaned thoroughly, and a dorsal penile nerve block was placed proximally with 3 5 ml of 2% lidocaine. masses were surgically resected using cautery and scalpel blades. an 8 french polypropylene catheter was placed in the urethra to prevent iatrogenic damage to urethra and urethral lumen compromise. absorbable sutures were placed in the surgical sites of 4 bulls. three bulls received mycobacterium cell wall fraction immunotherapeutic treatment after surgery and owner was instructed to give the injection to remaining bulls once supplies were restocked. samples from the resected masses were sent for recombinant vaccine production for the specific strain of papillomavirus that caused this outbreak. owner was instructed to monitor penis and prepuce for bleeding, swelling, and inflammation for 1 week and have their herd veterinarian reexamine these bulls for recurrence of the masses at 30 45 days after surgery. they were also informed that these bulls were not to be sold for meat for 21 days after the immunostimulant was given to allow for appropriate withdrawal time. penile warts or fibropapillomas are caused by bovine papilloma virus.1,2 bovine papilloma virus lesions typically citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 45 http://dx.doi.org/10.58292/ct.v15.10001 46 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 appear on areas of the body more prone to abrasion like head, neck, and legs.3 fibropapillomas of the penis occur in young bulls housed together and participating in homosexual behavior.1,3 the virus enters through small abrasions in the penile skin and causes neoplastic growth of fibroblasts on the distal portion or glans penis.1,3 penile warts are a common occurrence in young bulls, but the prevalence within a group tends to be low, 2 5%. however, some instances the prevalence was 10 30%.1-3 penile warts treatments include surgical removal via scalpel excision, co2 laser, or cryotherapy. these therapies may be performed alone or with an immunostimulant treatment to prevent recurrence.1 phimosis and paraphimosis may occur secondary to large penile fibropapillomas due to the distal portion of the penis becoming too large to pass though the preputial orifice.4 keywords: fibropapilloma, bull, penis, wart references 1. hopper rm, wolfe df: restorative surgery of the prepuce and penis. in: hopper rm: editor. bovine reproduction. 2nd edition, hoboken; wiley-blackwell: 2021. p. 210-211. 2. whittier wd, warnick ld, eller al: efficacy of bovine papilloma virus vaccination in preventing papillomas in yearling beef bulls. bov proc 1997;30:146-148. 3. dawson l, bierchwal cj: fibropapilloma of the bovine penis. bov prac 1983;18:184-187. 4. maxwell h: inability to breed due to injury or abnormality of the external genitalia of bulls. in: hopper rm: editor. bovine reproduction. 2nd edition, hoboken; wiley-blackwell: 2021. p. 161-166. monozygotic twin pregnancy in a quarter horse embryo recipient mare emma mullins, humberto magalhaes, kianna spencer, giorgia podico, igor canisso college of veterinary medicine, university of illinois urbanachampaign, urbana il, usa monozygotic twinning is a rare incidence compared to dizygotic twins.1 development of monozygotic twins has been associated with zona pellucida hatching during intracytoplasmic sperm injection and assisted hatching protocols with in vitro embryos.2 thus, in vivo embryos are less likely to develop monozygotic twins. occurrence of a monozygotic twin pregnancy in a quarter horse mare that was presented for premature udder development and lactation is described. mare had a nonsurgical embryo transfer performed 8 months before. embryo was produced in vivo and transferred immediately after collection from the donor mare. recipient mare was diagnosed pregnant with a singleton at the initial examination with no further examinations. at ~ 7 months of pregnancy, the mare developed an engorged udder with early milk production. premature udder development and lactation is often indicative of placentitis, a leading cause of infectious abortions in the horse.3 mare was given intramuscular penicillin g procaine (22,000 ui/kg, once every 12 hours), oral altrenogest (0.088 mg/kg, once daily), and intravenous flunixin meglumine (1.1 mg/kg, once every 12 hours) at the referring facility to treat the presumed placental disease. mare’s udder decreased in volume; however, purulent vulvar discharge developed, and the mare was referred. physical examination was unremarkable. transrectal ultrasonography revealed fetal fluids with floating hyperechoic materials. no fetal movements were detected on ballottement and transabdominal ultrasonography revealed a nonviable fetus. intramuscular prostaglandin f2α (cloprostenol 250 µg) was given to promote delivery of the fetus. however, 2 fetuses and 2 fetal membranes were delivered 30 minutes after treatment; the  smaller fetus presented signs of umbilical torsion. postabortion, the mare was given a large volume uterine lavage and flunixin meglumine with butorphanol to address her discomfort. as more complications occurred (i.e. metritis, laminitis), owners elected to euthanize due to poor prognosis and financial constraints. to prevent twinning complications, recipient mares should be examined between 25 and 30 days of pregnancy.4 although the initial pregnancy diagnosis determined a singleton, it is common that twins are not identified at the first examination, and a second examination should be conducted to confirm the diagnosis to prevent further development of both embryos.5 this case was unique as twinning is very unlikely in in vivo embryo transfers. studies are limited on monozygotic twinning in in vivo embryos due to reduced prevalence compared to in vitro embryos. keywords: monozygotic pregnancy, embryo transfer, noninfectious abortion references 1. roberts ma, london k, campos-chillon lf, et al: presumed monozygotic twins develop following transfer of an in vitro-produced equine embryo. j equine sci 2015;26:89-94. 2. hodder adj, liu ikm, ball ba: current methods for the diagnosis and management of twin pregnancy in the mare. equine vet educ 2008;20:493-502. 3. fedorka ce, scoggin ke, ruby re, et al: clinical, pathologic, and epidemiologic features of nocardioform placentitis in the mare. theriogenology 2021;171:155-161 4. crabtree jr: management of twin horses. br vet j 2018;40:66-74. 5. mancill ss, blodgett g, arnott rj, et al: description and genetic analysis of three sets of monozygotic twins resulting from transfers of single embryos to recipient mares. j am vet med assoc 2011;238:1040-1043. low conception rates in a breeding standardbred stallion due to spermiostasis and seminal plasma toxicity complicated by low libido and osteoarthritis hayley rossiano,a kianna spencer,a giorgia podico,a igor canissoa acollege of veterinary medicine, university of illinois urbanachampaign, urbana il, usa an 11-year, standardbred stallion, was presented for breeding soundness evaluation after a reported pregnancy rate of 5% per cycle and average of 7 jump attempts per collection. on presentation, he displayed signs of low libido and lameness localized bilaterally to both tarsi. semen was collected in 2 jumps and subsequently extended using a skim milk-based (sc) and an egg yolk-based (ey) extender. percentages of total motility (tm) and progressive motility (pm) were assessed with computer-assisted semen analyzer. on presentation, initial ejaculate (ei) had a volume of 45 ml with concentration of 328 x 106 sperm/ ml and 1.5 x 1010 total sperm. total motility was 64% and progressive motility was 52%. motility decreased after extension http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 47 with ey (tm 56%, pm 51%) and sc (tm 30%, pm 24%). motility decreased with ey (tm 54%, pm 50%) and sc (tm 47%, pm 39%) in 24-hour cooling test. on average, with serial collections, the first ejaculate (e1) had a volume of 73 ml, concentration of 218 x 106 ml, tm 48% (range: 10 68%) and pm 45% (range: 31 58%) and second ejaculate (e2) had a volume of 65 ml, concentration of 103 x 106 ml, tm 62% (range: 32 80%) and pm 53% (range: 25 67%). stallion was diagnosed with sperm accumulation, seminal plasma toxicity, low libido, and osteoarthritis. semen collection was performed, every other day, for 60 days, to control sperm accumulation.1 both tarsometatarsal joints and distal intertarsal joints received intra-articular corticosteroid injections. oral analgesics included firocoxib (0.1 mg/kg, once daily), intermittent oral phenylbutazone (2.2 mg/kg), as needed, and oral gabapentin (10 mg/kg, twice a day). stallion’s grain was supplemented with extra-virgin olive oil, vitamins e and c, dha, and alpha lipoic acid top dressing. vitamin c and e blended with polyunsaturated fatty acids (pufas) reduced sperm oxidative damage and a combination of pufas and antioxidants stimulated overall strengthening of membrane integrity in fresh, cooled, and frozen semen.2,3 pentoxifylline (8.5 mg/kg) was given twice daily to increase testicular perfusion, sperm production, and increase live capacitated sperm.4,5 imipramine (3 mg/kg) was given orally, 1 hour prior to collection to improve erection and to lower ejaculation threshold.6,7 an extended teasing protocol was utilized before mounting the phantom to minimize number of collection attempts and decrease orthopedic strain. omeprazole (1 mg/kg, once a day) was given adjunctively to prevent gastric ulcerations during hospitalization. lastly, cushion centrifugation was performed on all ejaculates to remove seminal plasma, increasing the percentage of progressively motile sperm to combat seminal plasma toxicity.8 semen collected after 60 days of treatment had a volume of 80 ml with concentration of 242 x 106 sperm/ml, 1.9 x 1010 total sperm, 73% tm and 68% pm. abnormal accumulation of sperm and seminal plasma toxicity has been anecdotally described in literature but there is a lack of research in the area on standards of treatment. this case also described the unique factor of low libido and osteoarthritis that complicated increased numbers of collections on the phantom needed to increase semen parameters. keywords: sperm accumulation, seminal plasma toxicity, low libido, osteoarthritis references 1. mccue pm, scoggin c, moffett p, et al: spermiostasis in stallions: a retrospective study of clinical cases. j equine vet sci 2014;34:47. 2. bazzano m, laus f, spaterna a, et al: use of nutraceuticals in the stallion: effects on semen quality and preservation. reprod in domest anim 2021;56:951-957. 3. freitas ml, bouéres cs, pignataro ta, et al: quality of fresh, cooled, and frozen semen from stallions supplemented with antioxidants and fatty acids. j equine vet sci 2016;46:1-6. 4. pozor ma, muehlhaus j, king a, et al: effect of pentoxifylline treatment on testicular perfusion and semen quality in miniature horse stallions. theriogenology 2011;76:1027-1035. 5. ortgies f, klewitz j, martinsson g, et al: effect of procaine, pentoxifylline and trolox on capacitation and hyperactivation of stallion spermatozoa. andrologia 2011;44:130-138. 6. mcdonnell sm, garcia mc, kenney rm, et al: imipramine-induced erection, masturbation, and ejaculation in male horses. pharmacol biochem behav 1987;27:187-191. 7. mccue pm: chemical ejaculation. in: dascanio jj, mccue pm: editors. equine reproductive procedures. 2nd edition, ames; wiley blackwell: 2021. p. 347-348. 8. brinsko sp, crockett ec, squires el: effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. theriogenology 2000;54:129-136. severe hind limb lameness in a female dog with closed-cervix pyometra jewel toenges,a michela ciccarellia,b acollege of veterinary medicine, washington state university, pullman, wa, usa bschool of molecular bioscience, washington state university, pullman, wa, usa pyometra is a life-threatening disease that is commonly presented in older intact female dogs with clinical signs of lethargy, anorexia, polyuria, polydipsia, and vaginal discharge in cases of open-cervix. severe cases of pyometra are often associated with other signs of systemic disease such as leukocytosis, neutrophilia with a left shift, anemia, hypoalbuminemia, and other changes in liver or kidney function. pyometra has been only reported concomitant to lameness in 2 other reports but may be an under-documented clinical sign.1,2 a 4-year female intact, english bulldog, was presented for sudden grade 6 left hind lameness, 20 days after transcervical insemination (tci). transabdominal ultrasonography revealed multiple vesicles without visible embryo proper and multiple corpora lutea on both ovaries. complete blood count, biochemistry, and blood progesterone were completed to rule out pyometra and recent pregnancy loss. results were unremarkable and blood progesterone concentrations were 21.2 ng/ml. she was prescribed carprofen and recommended strict kennel rest until an orthopedic examination appointment was available. six days after the initial presentation, she was evaluated by the orthopedic surgery service. musculoskeletal examination was performed along with stifle radiographs, both were within normal limits, presuming that the lameness was due to a soft-tissue injury. another transabdominal ultrasonography was performed that confirmed the lack of embryos and possible mucometra/pyometra. ovariohysterectomy (ohe) was recommended to resolve mucometra/pyometra and was performed the same day. after ohe, the patient’s lameness resolved completely. due to the sudden resolution of hindlimb lameness following surgery, it was assumed that the pyometra was the cause. several mechanisms may have had a role in the pathogenesis such as immune mediate arthritis due to bacterial infection or toxin-induced, visceral pain, pressure, and discomfort from uterine distension, but neuromuscular pathology was not ruled out. this case demonstrated that hindlimb lameness may be included as a rare clinical sign of pyometra. further research is needed to investigate the pathogenesis of pyometra and lameness. keywords: closed-cervix pyometra, hindlimb lameness http://dx.doi.org/10.58292/ct.v15.10001 48 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 references 1. klainbart s, ranen e, glikman g, et al: hindlimb lameness and gait abnormalities in bitches with pyometra. vet rec 2014;175:46. 2. jitpean s, ström-holst b, emanuelson u, et al: outcome of pyometra in female dogs and predictors of peritonitis and prolonged postoperative hospitalization in surgically treated cases. bmc vet res 2014;10:6. suspected penile spiral deviation secondary to penile denervation in a shorthorn bull cody davis, jessica cowley, amy yanke, katelyn waters, jessica klabnik department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa performing a thorough breeding soundness examination and test mating prior to surgical correction of penile or preputial abnormalities is recommended for diagnosis of comorbidities that may impact a bull’s return to reproductive soundness postoperatively. a 4-year, milking shorthorn bull, was presented for suspected spiral deviation of the penis. owner reported successful intromission in this bull during first breeding season but intromission failure during subsequent seasons. to assess bull’s reproductive soundness, test mating and breeding soundness examination were performed. during the initial test mating, bull was uninterested in mounting. breeding soundness examination revealed a visually normal erect penis (observed during electroejaculation) with normal internal genitalia, prepuce, scrotal contents, and scrotal circumference. progressive motility of the sperm was adequate with acceptable sperm morphology (70% normal, 11% head abnormalities and 19% midpiece abnormalities). during second test mating, bull failed to achieve intromission during 4 mountings. first mount involved a normal erect penis that spiraled upon contact with the female’s perineal area. latter 3 mountings resulted in an immediate spiral deviation. given the delayed spiral during the first mount and consistent inability to copulate (impotentia coeundi), damage to the dorsal nerve of the penis was suspected with secondary penile deviation. a primary spiral deviation was lower on the differential list because it is expected that the penis consistently spirals during live cover in primary spiral deviation. abnormal penile spiraling results from an abnormal apical ligament or premature high corpus cavernosum penis pressure.1 to further evaluate the differential diagnosis of penile denervation, a nerve conduction test was performed to quantitatively determine dorsal nerve function;2,3 no conduction velocity was measured from distal penis, and low conduction velocity was measured immediately distal to the sigmoid flexure. lack of innervation detected from the nerve conduction test is confirmatory for the diagnosis of penile denervation.2,3 damage to the dorsal nerve of the penis likely occurred due to trauma during bull’s initial breeding season. in this case, it is reasonable to conclude that the penile spiral deviation is observed secondary to penile denervation. penile denervation injuries are considered permanent injuries; therefore, the bull’s prognosis for natural service is poor. however, the bull could be utilized for semen collection via electroejaculation.3 owner opted to utilize this bull as a teaser animal. penile denervation should be considered as a differential or concurrent diagnosis for penile spiral deviation defects. keywords: penile denervation, spiral deviation, bull, nerve conduction references 1. hopper rm, wolfe df: restorative surgery of the prepuce and penis. in: hopper rm: editor. bovine reproduction. 2nd edition, hoboken; joe wiley & sons: 2021. p. 210-229. 2. wolfe df, moll hd: examination and special diagnostic procedures of the penis and prepuce. in: wolfe df, moll hd: editors. large animal urogenital surgery. 2nd edition, baltimore; williams  & wilkins: 1999. p. 221-231. 3. maxwell h: inability to breed due to injury or abnormality of the external genitalia of bulls. in: hopper rm: editor. bovine reproduction. 2nd edition, hoboken; joe wiley & sons: 2021. p. 155-172. unilateral segmental aplasia of epididymis tail in a bull keri shipman, jessica cowley, katelyn waters, lily lewis, jessica klabnik department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa epididymis is the final site of sperm maturation; therefore, aplasia of the epidydimal tract could decrease reproductive efficiency despite having a normal scrotal circumference.1 in september of 2022, an apparently healthy 2-year, aberdeen angus bull, was referred due to an abnormal right testis that was diagnosed during a routine bull breeding soundness examination. as per the owner, referring veterinarian noted a firmer right testis on palpation. medical record documented the following: ‘a mal-structured right testis’, very good sperm motility, 87% normal sperm morphology (7% abnormal heads and 6% tail defects) and a scrotal circumference of 39 cm. transscrotal ultrasonography revealed missing right epididymis tail. based on bull’s age and history, congenital unilateral segmental aplasia of the epididymis was suspected. congenital anomalies occurred secondary to defects during embryonic development. elongation and coiling of the mesonephric duct lead to epididymal development. only this bull’s left epididymis was competent to transport semen from testis to ductus deferens. therefore, bull’s ejaculate could only contain up to 50% of total possible sperm that could be produced, despite having an acceptable scrotal circumference. it was recommended to cull the bull due to poor reproductive efficiency and the possibility of transmitting an inherited autosomal recessive defect.2 however, owner elected to breed a few cows due to bull’s quality and pedigree despite the inheritable nature of this congenital defect. this case highlighted the need for thorough palpation of scrotal contents in addition to scrotal measurement. keywords: testis, epididymis, reproductive efficiency, congenital anomalies http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 49 references 1. james er, carrell dt, aston ki, et al: the role of the epididymis and the contribution of epididymosomes to mammalian reproduction. int j mol sci 2020;21:5377. 2. könig h, weber w, kupferschmied h: epididymal aplasia in bulls and boars. a. demonstration of 18 cases with recessive inheritance in the simmentaler spotted breed. b. occurrence of abnormalities in one boar and 3 offsprings. schweizer archiv für tierheilkunde1972;114:73-82. http://dx.doi.org/10.58292/ct.v15.10001 student research semen collection in domestic cat: comparison of techniques kristyn burton,a aime johnsonb acollege of veterinary medicine, oklahoma state university, stillwater, ok, usa bcollege of veterinary medicine, auburn university, auburn, al, usa semen collection in cats in the clinic setting can be difficult. however, semen analysis is vital. electroejaculation (eej) is currently the most reliable semen collection method but requires specialized equipment. urethral catheterization is a technique that allows semen collection without special equipment a catheter is placed in the urethra of a sedated tom and semen is collected passively in the catheter. earlier studies used medetomidine at high doses. however, medetomidine has been replaced with dexmedetomidine in some countries. this study sought to compare the results of electroejaculation (eej) and urethral catheterization (uc) for semen collection in domestic cat using dexmedetomidine, a potent α2-adrenoceptor agonist (α2a) as a substitute for medetomidine at the equivalent dose used in earlier studies. twelve domestic cats were collected thrice at weekly intervals. all cats received intramuscular ketamine (5 mg/kg) and intramuscular dexmedetomidine (30 µg/kg) for initial cleanout via eej, then randomly underwent either eej or uc 1 week apart. the eej was performed under the same anesthetic protocol as the initial cleanout. the uc was performed using intramuscular dexmedetomidine at a dose of 60 µg/kg. success of collection, total sperm number, and motility characteristics were analyzed. sperm was collected successfully from all 12 cats via eej and from 11/12 via uc. there were no significant differences in the percentage of total motile, progressively motile, or morphologically normal sperm between ejaculate types when averaged across all cats or individual cats. although uc yielded a lower volume and higher concentration ejaculate, it consistently produced a lower total sperm number than ejaculates retrieved via eej (17.91 x 106 total sperm for uc versus 46.51 x 106 total sperm for eej). these results indicated that dexmedetomidine is a very effective α2a and performed satisfactorily in both procedures at the doses used in this study. it was also safe with no adverse effects on toms. eej remained the most reliable in terms of assessing semen quality and retrieving semen with adequate number of sperm for breeding purposes. however, uc with dexmedetomidine at this dose can be a remarkably consistent alternative that demonstrated 92% success rate. keywords: cats, tom, sperm, electroejaculation, catheterization, dexmedetomidine effect of age on serum anti-müllerian hormone concentrations in female alpacas dane schwartz,a cristian patino,a salman waqas,a eduardo arroyo,a alan conley,b ahmed tibarya adepartment veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, usa bdepartment of population health and reproduction, school of veterinary medicine, university of california, davis, usa anti-müllerian hormone (amh) is produced by the granulosa cells of early antral follicles. multiples studies identified that circulating amh concentrations are reliable markers of the antral follicle population. in ruminants, serum amh concentrations are well correlated with the response to superovulation treatment. similar results were obtained for alpacas. in mares and women, serum amh concentrations decline after 20 years of age indicating depletion of antral follicle reserves. we hypothesized that a similar effect of age on serum amh concentrations exists in alpacas. objective of this study was to compare serum amh concentrations in 3 age groups. twenty healthy, multiparous huacaya female alpacas of 3 age groups were used (group 1: 4 8 years, n = 6; group 2: 9-13 years, n = 7; and group 3: 16 21 years, n = 7). blood samples were collected by jugular venipuncture for determination of serum amh concentrations. blood samples were allowed to clot then centrifuged to harvest serum that was stored at -20°c until analysis. serum amh concentrations were determined in duplicate according to the manufacturer’s instructions assay (rat and mouse amh elisa al-113, ansh labs, webster, tx, usa). the monoclonal antibody pairs used in the amh assays bind to the noncovalent amh complex and do not detect other related members of the tgfβ superfamily. samples from each female were included in the same analytical run. intra-assay coefficients of variation for serum pools with high (13.5 ng/ml, n = 14), medium (6.2 ng/ml, n = 14) and low concentrations (2.3 ng/ml, n = 14) were 2.6, 5.5 and 4.1%, respectively. the lowest calibrator was 0.41 ng/ml, below which concentrations were estimated by extrapolation. serum amh concentrations among 3 groups were compared by anova, with age as the main factor. serum amh concentration (mean ± sem) was 2.61 ± 0.66, 1.22 ± 0.39, and 0.47 ± 0.23 for group 1, group 2, and group 3, respectively. serum amh concentrations were lower (p < 0.05) in group 3 females than in groups 1 and 2. there were no significant differences between group 1 and group 2 in serum amh concentrations. in conclusion, these preliminary results indicated that serum amh concentrations decreased with advanced age and may reflect ovarian senescence in female alpacas. a study on a larger number of females of various ages is needed to confirm these results and their effects on reproductive efficiency in camelids. keywords: ovary, aging, camelid, ovarian reserve, fertility 50 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 51 molecular mechanisms underlying endometrial cup immunotolerance: expression of fas/fasl and tnfr2 genes kassandra crissman,a jenny sones,a viviane gomesa,b aschool of veterinary medicine, louisiana state university, baton rouge, la, usa bcollege of veterinary medicine, michigan state university, east lansing, mi, usa endometrial cups are trophoblast-derived structures that have an important role in equine pregnancy maintenance. however, mechanisms underlying maternal immunotolerance to these semiallogenic structures is incompletely understood. a precisely coordinated cellular response has been previously described in the maternal endometrium-cup interface. namely, marked recruitment of maternal mononuclear leukocytes to the adjacent endometrial stroma occurs early in the endometrial cup development. composed primarily of maternal cd4+ and cd8+ t lymphocytes, this leukocyte population is locally regulated by trophoblast and regulatory t cell-derived signaling molecules, preventing cytotoxicity and endometrial cup destruction. interestingly, trophoblast cell secretion of fas ligand (fasl), a type ii transmembrane protein of the tumor necrosis factor (tnf) family, and indoleamine 2,3 disoxygenase 1 (ido1) has been reported in other species and may provide modulation of the cell mediated immune response. additionally, tnf receptor (tnfr2) has been associated with fas/fasl-induced cd8+ t cell death. it is hypothesized that fas/fasl, ido1, tnf-α, and tnfr2 have an immunomodulatory role during endometrial cup development, leading to lymphocyte apoptosis and prevention of cytotoxicity. endometrial cup samples were dissected from the maternal endometrium of pony mares carrying viable pregnancies during early development, between 42 to 47 days after ovulation, and regression, between 96 to 120 days after ovulation (n = 5 per group). gene expression of fas, fasl, ido1, tnf-α and tnfr2 was assessed via rt-pcr using validated primers. gel electrophoresis was then performed to verify amplification of the expected targets. relative mrna quantification was calculated using the delta-delta ct method and normalized based on the expression of equine glyceraldehyde3-phosphate dehydrogenase. data distribution was assessed by shapiro wilk tests and comparisons were determined using unpaired student’s t-tests. significance was set at p < 0.05 (graphpad 9.0). when comparing developing to regressing endometrial cups, fasl gene expression was 2.7-fold higher (p = 0.034) whereas there were no differences on fas levels. gene expression of tnfr2 was 4-fold higher (p = 0.013) in developing endometrial cups versus demise. expression of ido1 and tnf-α were similar between timepoints. therefore, further investigation of fas/fasl-tnfr2 signaling in the equine endometrial cup-maternal interface is warranted. keywords: mare, endometrial cups, immunotolerance, lymphocytes, apoptosis http://dx.doi.org/10.58292/ct.v15.10001 student poster ovarian hematoma in a tennessee walking horse/ friesian cross foal andrew kirk, robyn wilborn, lindsey boone, kara lascola, anne wooldridge, rachel pfeifle, patricia egli department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa on june 12, 2022, a 5-day, filly was presented at the equine emergency service for lethargy and for an apparent dehydration secondary to decreased milk production by the dam. on initial physical examination, filly appeared quiet, alert, and responsive; a strong latch and suckle ability of the foal was noted. mucous membranes were pink, but slightly dry, with all other vitals in appropriate parameters. patent urachus was noted on physical examination. a minimum database panel revealed a mild, mature neutrophilia, hyponatremia, and a marked metabolic acidosis, having bicarbonate measured at 12.3 mmol/l. a subsequent nova panel was elected and performed, confirming the hyponatremia and metabolic acidosis (bicarbonate 11.6 mmol/l, ph 7.2). filly was sedated for thoracic and abdominal ultrasonography thoracic ultrasonography had no significant findings. however, a large mass was discovered in the cranioventral abdomen adjacent to the liver that was best viewed from the right side. patent urachus was examined via abdominal ultrasonography, but the umbilical artery and vein were noted as normal. no other abnormal findings were observed. hospitalization with supportive care was elected. repeat abdominal ultrasonography was performed the next day by a board-certified radiologist, along with abdominal computed tomography. etiologies of the mass and metabolic acidosis were not determined, but the structure of the mass suggested resection was warranted. exploratory laparotomy, along with surgical correction of the patent urachus was elected and performed on june 16, 2022. surgical visualization revealed a large, cystic right ovary with abnormal attachment of the ovarian pedicle. remainder of the reproductive organs appeared normal, but complete ovariohysterectomy was performed. patent urachus was resected at spay. potassium penicillin and amikacin (broad spectrum antibiotics), along with flunixin meglumine (nonsteroidal antiinflammatory) were prescribed for 3 days postoperatively. tissues were sent to histopathology for a confirmatory diagnosis. results showed a thin capsule over the mass, along with a layer of smooth muscle resembling ovarian tissue. the center was filled with edematous connective tissue and a dense central region of uniform cells. however, abundant hemorrhage, edema, and fibrin accumulations completely prevented any diagnostic description of cellular features. multifocal, variably dilated vessels were noted to have fibrin accumulations, described as fibrin thrombi. the center of the mass contained several cavitations filled with blood (hematoma). the histopathological diagnosis was limited in detail due to the obstructed view of the cells, but a broad diagnosis of hematoma with multifocal vascular thrombosis was made. no abnormalities were observed on the left ovary or uterus. postoperatively, the filly recovered exceptionally. during postoperative monitoring, physical examination parameters appeared normal, and the foal continued to gain weight and remain hydrated. foal continued to thrive after a 3-day course of antibiotics and nonsteroidal antiinflammatory medications. on june 22, 2022, the foal was discharged. keywords: hematoma, multifocal vascular thrombosis, filly, urachus pyometra and dilated cardiomyopathy phenotype in a great dane female dog dane schwartz,a lynne nelson,a cynthia carlson,a michela ciccarellia,b acollege of veterinary medicine, washington state university, pullman, wa, usa bschool of molecular bioscience, washington state university, pullman, wa, usa a 5-year, nulliparous great dane female dog, was presented in october 2022 for suspected pyometra (bloody mucopurulent vaginal discharge, anorexia, and anxious behavior). one month before presentation, she was diagnosed with dilated cardiomyopathy (dcm) via echocardiography. her dcm was managed with oral pimobendan, given twice daily, and a cardiac prescription diet. on physical examination, dog appeared bright, alert, and responsive and had normal heart and respiratory rates with normal mucous membranes. she had elevated temperature (104.1ºf) with bloody mucoid vaginal discharge and soft enlarged nodular mammary glands that expressed milk. vaginal cytology had red blood cells with a few neutrophils and blood progesterone concentrations were 0.447 ng/ml. transabdominal ultrasonography findings were suggestive of cystic endometrial hyperplasia. ultrasonography of the mammary glands revealed signs of galactostasis. hematology had marked leukocytosis (marked neutrophilia, with monocytosis and eosinophilia), and nonregenerative anemia. marked hyperglobulinemia was identified in blood chemistry. ovariohysterectomy was recommended as soon as possible as the diagnostic findings were suggestive of ceh/ pyometra syndrome. clavamox and carprofen were prescribed until further evaluation by the small animal surgery service. ovariohysterectomy was performed with no complications. uterine cultures isolated 2 distinct morphologic types of escherichia coli. revaluation with cardiology service in december 2022 determined that the dog had a resolution of dcm with normal heart size and function on echocardiogram. the prior systolic dysfunction was considered most likely due to an inflammatory condition causing myocardial 52 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 53 suppression by systemic cytokines. dcm phenotype is mostly caused by nutritional deficiency, hereditary (primary), secondary to severe hypothyroidism, or systemic inflammation. a full thyroid panel had normal values. the potential cause of ceh, in this case, was repeated irregular estrous cycles without pregnancy. secondary (chronic) infection led to a dcm-like condition that was reversed with therapy for pyometra. keywords: cystic endometrial hyperplasia, echocardiogram, ultrasonography, ovariohysterectomy chronic hydrometra in a goat daniella adams,a andrew muir,a shannon dehghanpir,a emi sasaki,b robert foster,c nadia richmond,a naomi falconnier,b jenny sones,a clare scullya adepartment of veterinary clinical sciences bdepartment of pathobiological sciences, school of veterinary medicine, louisiana state university, baton rouge, la, usa cdepartment of pathobiology, ontario veterinary college, university of guelph, guelph, on, canada hydrometra is an important cause of subfertility in goats. it is characterized by accumulation of sterile clear fluid in the uterus with a persistent corpus luteum.1 a 2-year, nubian doe, was presented for a 90day pregnancy examination. abdominal ultrasonography revealed a bifurcated fluid-filled uterus indicative of hydrometra. she was treated with 2 doses of intramuscular dinoprost tromethamine (lutalyse®), 1 in hospital and 1 at home. ten months later, she was presented for laparoscopic artificial insemination. no abnormalities were noticed on physical examination. patient was anesthetized with ketamine, butorphanol, midazolam, and lidocaine locally, placed in dorsal recumbency, and clipped and prepped. two small incisions were made in the skin and a blunted end teat cannula was introduced into abdomen for insufflation. brown fluid filled the co2 delivery tube. another attempt at the other incision was performed with similar result. after this, an exploratory laparotomy and ovariohysterectomy was performed and the abdomen was flushed with sterile saline and procaine penicillin was injected into abdomen. patient was treated with subcutaneous ceftiofur sodium and intravenous flunixin meglumine. cytology of the uterine fluid suggested chronic hemorrhage with possible low-grade inflammation. uterine fluid had a low number of nucleated cells, several erythrocytes, and birefringent amorphous crystals. although no infectious organisms were lymphocytes (6%), and eosinophils(1%) were present. macroscopic evaluation and histopathology of the uterus revealed chronic hydrometra with endometrial, myometrial, and perimetral fibrosis, myometrial hypertrophy and bilateral ovarian hypoplasia. uterus measured 20 x 14 x 6 cm and the uterine wall was expanded up to 2 cm thick and firm with a light tan/white and glistening mucinous endometrium. endometrium was largely replaced with concentric fibrosis that was up to 1.25 mm thick and had no surface epithelial cells. fluid dilation of the uterus indicated an outflow obstruction that may be due to segmental aplasia of the cervix or cranial vagina. both ovaries lacked follicles, there were 1 primordial and 1 secondary follicle on the left ovary and several tertiary follicles in the right ovary. patient was discharged with meloxicam and naxcel along with stall rest. she reportedly recovered without complication. this case was unique in its presentation of chronic hydrometra without a corpus luteum. it also highlighted the impact on doe fertility in cases of chronic hydrometra and the good prognosis of life with treatment. serial monitoring of hydrometra is recommended after initial treatment. keywords: chronic hydrometra, fibrosis, corpus luteum references 1. kornalijnslijper, je bevers mm, van oord ha et al: induction of hydrometra in goats by means of active immunization against prostaglandin f2α. anim reprod sci 1997;46:109-122. gonads to no ‘nads: ovariohysterectomy after chronic pyometra in a mare ellianna blair, ahmed mosallam, jessica klabnik, lindsey boone, candace lyman department of clinical sciences, college of veterinary medicine, auburn university, auburn al, usa a 10-year, tennessee walking horse mare, was previously presented to her primary practitioner for multiple episodes of white vulvar discharge and perceived hind end discomfort. examinations frequently revealed pooling of uterine fluid and uterine lavage therapy was provided on numerous occasions. after 2 years of recurring episodes, the mare was referred to university teaching hospital where intermittent reproductive examinations were performed during the following 18 months. examination findings included anywhere from no uterine fluid or vulvar discharge to pyometra with 5 cm of uterine fluid pooling with white or serosanguinous vulvar discharge. scant growth of escherichia coli, susceptible to all antibiotics tested, was obtained but only in enhancement broth. eventually, the mare was presented with intensely foul-smelling vulvar discharge and a uterine culture swab had heavy growth of escherichia coli, klebsiella pneumoniae, and streptococcus equi subsp. zooepidemicus. hysteroscopy revealed vaginal and endometrial adhesions pocketing fluid within the uterus, preventing fluid evacuation. the owner inquired about cervical wedge resection: however, as no cervical pathology existed and the cervix was capable of relaxation, this was not determined to be a curative solution. ovariohysterectomy was suggested, many months later the owner elected to pursue surgery. preparation for ovariohysterectomy included uterine lavages for 2 days to remove hyperechoic fluid to minimize the risk of abdominal contamination during uterine removal. gentamicin and lidocaine were infused into uterine lumen for relaxation. a standing laparoscopic approach for ovarian pedicle transection using cautery was planned; thereafter, prolapsing the uterus through the cervix was attempted as an alternative to laparotomy under general anesthesia. in standing stocks, the sedated mare’s ovarian pedicles and broad ligament were visualized with laparoscopy equipment, injected with lidocaine, then cauterized and transected with no complications. unfortunately, the attempt to manually prolapse the uterus into the vagina and through the vulva was unsuccessful, as the cervix did not dilate wide enough to allow introduction of a hand into uterus while the ovarian pedicle transection was occurring. at that point, the laparoscopic portals were closed and the mare was anesthetized for placement in dorsal recumbency. a caudal ventral midline incision allowed exteriorization of the ovaries and uterus. soon after, substantial amount of abdominal bleeding was noticed but the origin could not be located; uterine transection and http://dx.doi.org/10.58292/ct.v15.10001 54 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 abdominal closure were performed. before recovery, the mare received an infusion of aminocaproic acid to minimize excessive bleeding by way of preventing blood clot breakdown. postoperative transabdominal ultrasonography revealed a moderate hemoabdomen but the blood appeared clotted rather than fluid, suggesting active hemorrhage had stopped. free blood within the abdomen substantially decreased over the first 12 hours postoperatively, likely due to autotransfusion. five days of systemic procaine penicillin and gentamicin were provided before transitioning to oral trimethoprim/sulfamethoxazole and metronidazole. a tapering course of flunixin meglumine was provided for pain and inflammation. two weeks following surgery, the referring veterinarian reported that the mare was healing well with no vulvar discharge. keywords: pyometra, ovariohysterectomy, equine spay, manual uterine prolapse induction of abortion in a mare with hydroallantois kristyn burton, reed holyoak, dale kelley department of veterinary clinical sciences, college of veterinary medicine, oklahoma state university, stillwater, ok, usa hydroallantois is a rare condition characterized by a sudden increase in allantoic fluid volume leading to marked abdominal distention. this occurs quickly over the course of several days, typically between 6 and 10 months of pregnancy. the condition is serious, as it can lead to prepubic tendon rupture a complication that seriously impacts the reproductive future of a mare. a 14-year, american quarter horse mare, was presented at 301 days pregnancy with abdominal distention and a history of treatment for placentitis. transrectal palpation and ultrasonography revealed that the uterus was above the pelvic brim, and the allantoic fluid was at a higher volume than normal. fetus could not be visualized. due to the duration of pregnancy remaining and concern over the mare’s future fertility it was decided to terminate pregnancy. an intravenous catheter was placed and 1 liter of lactated ringer’s solution was given and heart rate monitored. a sterile 21 inch ch18 foley catheter was inserted through the cervix into the allantois, and over a period of 2.5 hours a total volume of 45.4 liters was drained. after draining the allantoic fluid, parturition was induced by giving 60 units of oxytocin in 1 liter of 0.9% nacl solution intravenously over 60 minutes. at 45 minutes the front limbs were visualized in the birth canal, 10 minutes later hind limbs appeared near the right shoulder, indicating that the fetus was malpositioned. digital palpation revealed extended fetal left hind limb with right hind limb crossing over the left hindlimb. intravenous detomidine and ketamine were given to place the mare under anesthesia in order to facilitate repositioning and extraction of the fetus. foal was stillborn and between 4 and 6 weeks premature. mare was given 1 liter of intravenous lactated ringer’s solution during recovery, followed by flunixin and sulfadiazine ~ 2 hours after procedure. fetal membranes passed 3 hours after recovery. mare recovered uneventfully and it was advised rest for at least 2 estrous cycles prior to breeding, with a breeding soundness examination. keywords: hydroallantois, mare, abortion unilateral testicular enlargement in a quarter horse stallion lexie oncale, dale kelley department of veterinary clinical sciences, college of veterinary medicine, oklahoma state university stillwater, ok, usa a 16-year, american quarter horse stallion, was presented in june 2022 with an enlarged scrotum and poor fertility documented during the previous breeding season. on presentation, a breeding soundness examination was performed. on semen evaluation, the ejaculate appeared slightly opaque and had a total sperm number of 159 x 106 sperm, 50% total motility and 30% progressive motility. sperm morphology evaluation using differential interference microscopy showed 38% normal sperm morphology, with the primary abnormality of abnormal heads (43%). palpation of scrotum identified the right testis as noticeably large and firm, with a marked indentation on the ventral aspect. left testis was soft on palpation, substantially smaller than the right testis, and was high in the scrotum. on ultrasonography right testis measured 9.6 x 9.4 x 13.8 cm (width x height x length) with a volume of 655 cubic cm (cc) and had a heterogenous echotexture. left testis measured 3 x 4.2 x 6.4 cm with a volume of 81 cc and had a homogenous echotexture. no other abnormalities were noticed during palpation or ultrasonography; notably, the lymph nodes and accessory sex glands appeared normal. based on the diagnostic findings, the stallion was presumed to have right testicular neoplasia. major differentials included: sertoli cell tumor, leydig cell tumor, seminoma, and teratoma. hemicastration was the recommended treatment to potentially preserve fertility and for definitive diagnosis of the testicular enlargement by histopathology. stallion underwent semi-closed hemicastration of the enlarged testis. testis was cut into 4 sections and fixed in a formaldehyde solution and submitted for histopathology. testicular tissue had aggressive invasion into the capsular stroma with lymphatic and vascular infiltration consistent with seminoma. semen was collected 8 months after hemicastration. ejaculate was slightly opaque with a total sperm number of 92 x 106 sperm, 20% total motility and 7% progressive motility. sperm morphology evaluation showed 43% normal sperm, with the primary abnormality being abnormal heads (26%). although histopathology determined the tumor to be malignant, there was no evidence of metastasis on the breeding soundness examination. stallion’s low total number of sperm and low progressive motility could suggest deeper issues beyond the tumor such as testicular degeneration and intrinsic subfertility due to genetics. most literature highlighted seminomas as one of the most common tumors of stallions. however, there is limited information and studies on the effects of testicular neoplasms on fertility. this case emphasized a need for more research to better understand the impact of testicular neoplasms on sperm development and how to best manage them to maintain the stallion’s reproductive life. keywords: stallion, testicular neoplasia, hemicastration http://dx.doi.org/10.58292/ct.v15.10001 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 55 leiomyosarcoma associated with uterine rupture in an american quarter horse matthew rafferty,a salman waqas,a denise mcsweeney,a steven edmonds,b nicholas muir,a michela ciccarellia adepartment of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa, usa bdepartment of veterinary microbiology and pathology, college of veterinary medicine, washington state university, pullman, wa, usa a 13-year, maiden quarter horse mare, was referred to the university teaching hospital in november 2022 for possible surgical removal of a uterine mass. in august 2022, mare was diagnosed pregnant (~ 45 days) by the referring veterinarian but in september she was diagnosed not pregnant. in october, mare was again evaluated for a slight intermittent vaginal discharge and transrectal ultrasonography revealed a mass in the left uterine horn. on arrival, fresh blood and a large clot were noticed on trailer floor. additionally, mare exhibited colic signs including belly kicking, rolling, tachycardia, tachypnea, and hypothermia. mare was given a dose of intravenous flunixin meglumine and placed in a dark stall isolated from other horses to calm her before further assessment. in the stall, the mare continued to roll while expelling profuse bloody discharge and clots from the vulva. due to poor prognosis and grave future fertility even if she recovered, owner opted for euthanasia. on necropsy, a large (14 x 12 x 8 cm) pedunculated mass extending from the uterine body to the left uterine horn was noticed. a 6-cm fullthickness uterine wall tear was identified on the greater curvature of the left uterine horn where the mass was pressuring. a large clot occupied the entire left uterine horn, and concurrent mild hemoabdomen was noticed. on histological evaluation, the mass was diagnosed as leiomyosarcoma. tumors of the urogenital tract of the mare are rare, with most arising from the ovary. the most common uterine tumor is leiomyoma. although there have been 2 previous reports of equine uterine leiomyosarcoma in the last 40 years,1,2 none have been larger than 4-cm in diameter or associated with uterine rupture. this case highlighted the importance of prompt evaluation and diagnosis of reproductive issues in horses, particularly in the presence of pregnancy loss, abnormal masses, and vaginal bleeding. keywords: uterine tumor, reproductive emergencies, vaginal bleeding, mare references 1. lofstedt rm, spurlock g, williams r: leiomyosarcoma in the uterus of a mare. compend contin educ pract vet 1987;9:93. 2. hinojosa am, holyoak gr, moll d, et al: theriogenology question of the month. leiomyosarcoma. j am vet med assoc 2003;223:786-788. a case report of feed and water contamination causing poor breeding season pregnancy rate in beef heifers matthew rafferty, ramanathan kasimanickam veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa, usa selenium is an essential trace mineral of livestock that is responsible for many functions, including antioxidant defense, immune function, thyroid hormone synthesis, muscle function, and fertility. while many regions of the united states contain an adequate amount of selenium in the soil, the pacific northwest has been historically known to be deficient in this mineral in several counties. in grant county, washington, soil levels of selenium are 0.12 ± 0.04 ppm, as reported by the united states geological survey, with concentrations in plants being roughly the same or less than that of the soil, in nonaccumulator plants. in 2014, a poor breeding season pregnancy rate (74.2% [72/92]) in a well-managed herd of angus heifers was realized at fall pregnancy diagnosis. the ranch generally averages breeding season pregnancy rates of > 90%. through the investigative process, blood, well water, and feed (both baled and on pasture) samples were submitted for analysis. results from the blood mineral panel analysis revealed selenium values ranging 0.71 3.1 ppm (~ reference range is 0.12 0.3 ppm), slightly elevated zinc concentrations, and copper values at the low end of the reference range. it was noticed that the heifers had been grazing separately from the rest of the herd on a pasture with a well containing water that had extremely high levels of iron, very high levels of manganese, and extremely hard. the feed analysis report revealed selenium values of up to 30.9 ppm on a dry matter basis. no other signs of mineral imbalance at the animal level were noted. it was also made known that there was a potential or mixing of sewage water with the pasture that has been documented as a source of selenium environmental contamination. to address the investigation’s findings, the herd of heifers was removed from the pasture, open heifers were culled, mineral supplementation was stopped, monthly blood mineral panel monitoring was employed, and a new well was installed. since excess iron concentration may reduce copper absorption in cattle leading to copper deficiency causing impaired reproduction, slow-release copper bolus was given to all heifers before the following breeding season. reproduction rates of the following 2 years were 92.6% (87/94) and 90.6% (87/96). this case highlighted the importance of prompt investigation of low pregnancy rates in well-managed herds, testing of feed and water sources for herds, and herd management to correct the issues before the next breeding season. keywords: trace minerals, environmental contamination, herd management http://dx.doi.org/10.58292/ct.v15.10001 56 citation line: clinical theriogenology 2023, 15, 10001, http://dx.doi.org/10.58292/ct.v15.10001 one placenta’s-itis: stable for two? sierra ott, robyn wilborn, peyton draheim, candace lyman department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa a 17-year, thoroughbred mare, was presented to a veterinarian for concerns of premature udder development and streaming of milk at 300 days pregnancy. veterinarian noted an elongated vulva and an abnormal combined thickness of uterus and placenta (ctup) of 19 mm via transrectal ultrasonography. treatment for ascending placentitis was initiated that included altrenogest, trimethoprim sulfa, and flunixin meglumine. mare was referred to the university teaching hospital for evaluation. at presentation the mare was bright with a heart rate of 64 beats per minute (bpm) and a respiratory rate of 16 bpm. an external reproductive examination was performed and findings were unremarkable except for milk actively dripping from the mare’s teats. transrectal ultrasonography confirmed a thickened ctup of 21 mm with corrugated chorioallantoic margins and suspicion of placental detachment near cervix. fetal heart rate was not obtained, despite after many attempts at stimulation, responsive movement of the fetus was observed. mare was hospitalized and therapy was changed to intravenous potassium penicillin, gentamicin, and flunixin meglumine; oral altrenogest was continued as before. on day 2, the udder was no longer streaming milk, and transabdominal ultrasonography revealed a fetal rib cage with no heartbeat. however, fetal movement was felt on transrectal palpation; oral pentoxifylline treatment was added for improved uterine blood flow and to further support pregnancy. repeat transabdominal ultrasonography led to visualization of a fetal heart rate of 80 bpm in the left hemi-abdomen; a right hemi-abdominal scan revealed a rib cage with no heartbeat, indicating that the mare was experiencing a twin pregnancy with a deceased fetus. on day 307 of pregnancy the mare’s intravenous catheter was removed, mare was transitioned to oral broad-spectrum antibiotics and flunixin meglumine, and altrenogest was discontinued from day 315. during next 2 weeks mare was observed in hospital. early morning on day 325, mare entered stage ii labor. a fetus presented cranially but ventral deviation of the head prevented delivery; assisted vaginal delivery and correction of the malposture resulted in quick delivery of a live filly that died shortly afterwards from respiratory complications. following delivery of the filly, the chest of a second fetus was identified with flexed forelimbs and a neck and head turned backwards. an underdeveloped, deceased colt was delivered after manual manipulation while the mare was standing; mare remained bright and alert throughout delivery. a manual vaginal examination revealed a shallow laceration, starting 10 cm cranial to the dorsal commissure of the vulva, and was 5 cm in length; this was expected to heal without complication. fetal membranes were passed within 3 hours after delivery and were normal. placentitis and termination of a late term twin pregnancy can have similar clinical presentations in both stage of gestation and premature udder development – particularly following demise of 1 twin fetus. transabdominal ultrasonography is required to determine the presence of a twin pregnancy. keywords: placentitis, mare, twins, dystocia, pregnancy http://dx.doi.org/10.58292/ct.v15.10001 57 index abby toews, 25, 35 abdallah wagih abdelhady, 9 abdelhaq anouassi, 24 adam boyko, 20 ahmed gad, 13 ahmed mosallam, 53 ahmed tibary, 24, 26, 36, 50 aime johnson, 12, 50 alaina barhorst, 10 alan conley, 37, 50 alex wittorff, 34, 40 alexandria horner, 19 alexis hall, 31 alisson bradley, 41 allison salinger, 45 alon kaplan, 24 alyssa shelby, 35 amy brandon, 38 amy yanke, 48 andrea hesser, 19 andrew kirk, 52 andrew muir, 53 anne sheerin, 10, 34 anne wooldridge, 52 ashley beyer, 45 ashley buchanan, 10 aurore le breton, 40 brad tanner, 21 brittany ciepluch, 41 brittany middlebrooks, 14 brooklin hunt, 25 bruce christensen, 35 caio figueiredo, 9 candace lyman, 21, 40, 53, 56 carlos pinto, 32 carly turner-garcia, 15, 29 carol mcleod, 37 casey rubin, 44 chance armstrong, 26, 45 charles scoggin, 10 christina moss, 41 christine lopp-schurter, 45 christopher premanandan, 36 clare scully, 53 cody davis, 48 colin palmer, 26 cristian patino, 50 cynthia carlson, 52 cynthia xue, 31 cyril stephen, 27, 43 dale kelley, 15, 29, 54 dane schwartz, 50, 52 daniela cortes, 14, 38 daniella adams, 53 darcie sidelinger, 45 dawit tesfaye, 13 denise mcsweeney, 55 devinda wickramasingha, 35 dinesh dadarwal, 25, 35 eduardo arroyo, 26, 36, 37, 50 eduardo prado, 41 elizabeth goldsmith, 37 elizabeth patton, 15 elizabeth whitt, 41 ellianna blair, 53 emi sasaki, 53 emily may, 15 emma mullins, 46 erin lohbeck, 10 erin newkirk, 30, 43 erin oberhaus, 32 erin runcan, 36 etta bradecamp, 10 fábio lima, 9 fabio pinaffi, 20 federico cunha, 9 fiona hollinshead, 13, 19 fritz schumann, 25, 35 gabriel gonzalez, 45 gaetano mari, 11 gail mcrae, 36 giorgia podico, 10, 11, 12, 16, 31, 32, 46 guillaume saint-jean, 40 gustavo araujo, 32 hannah maxwell, 15, 29 hayley rossiano, 46 heath king, 45 hilari french, 31 hugo monteiro, 9 humberto magalhaes, 11, 16, 32, 46 igor canisso, 10, 11, 16, 17, 31, 32, 46 ilaria colombo, 11, 16, 32 irfan khan, 24 58 jacob rother, 20 james graham, 14 jamie stewart, 25, 38 jenifer koziol, 26 jenna dockweiler, 20 jennifer hatzel, 15 jenny sones, 51, 53 jessica cowley, 12, 48 jessica klabnik, 12, 48, 53 jewel toenges, 47 joanne stokes, 15 john broster, 27, 43 joshua ramsay, 37 julia felippe, 17 julie barnes, 16 julie cecere, 17, 36, 41 julie gard schnuelle, 12 justin mcnaughten, 42, 44 kamal gabadage, 25, 35 kamron ratzburg, 27 kara lascola, 52 karen wolfsdorf, 17, 42 kassandra crissman, 51 kate madsen, 27 katelyn waters, 48 kaylyn mcdaniel, 37 keri shipman, 48 kevin pelzer, 25 kevin walters, 45 kianna spencer, 10, 16, 32, 46 klibs galvão, 9 kristyn burton, 50, 54 kylie roux, 45 lamia briand-amirat, 12 laura waugh, 26 leo brito, 26 lexie oncale, 54 lily lewis, 12, 21, 48 lindsey boone, 52, 53 lindsey vansandt, 16 lorena feijo, 17 lorenzo segabinazzi, 31 luis henrique de aguiar, 9 lulu guo, 32 lynne nelson, 52 manuel chamorro, 45 maor kedmi, 22, 24 marco coutinho da silva, 36 maria ferrer, 14, 38 maria schnobrich, 10, 21, 29, 34, 37 mariah slack, 37 mariana diel de amorim, 9 marthina greer, 42 matthew rafferty, 55 michela ciccarelli, 37, 47, 52, 55 miguel saucedo, 45 mindy meyers, 13, 19 morgan agnew, 22, 44 mouhamadou diaw, 40 nadia richmond, 53 nahum shpigel, 22 naomi crabtree, 38 naomi falconnier, 53 nicholas muir, 55 nico menjivar, 13 nicole sugai, 17, 25, 38, 41 orsolya balogh, 17 patrice bernier, 31 patricia egli, 52 patricia xavier, 14, 38 patrick mccue, 14 paul dyce, 12 paula moffett, 14 peter sheerin, 10, 34 peter welsh, 37 peyton draheim, 56 phillip sponenberg, 36 pierre-yves mulon, 41 rachel doenges, 21, 29 rachel hanson, 27 rachel hollingsworth, 12 rachel pfeifle, 52 rafael bisinotto, 9 ramanathan kasimanickam, 27, 55 rebecca persons, 41 reed holyoak, 29, 32, 34, 54 richard hopper, 12 riley thompson, 13, 19 robert foster, 53 robert gilbert, 31 robyn ellerbrock, 14, 38 robyn wilborn, 12, 21, 52, 56 ron sicsic, 22, 24 rory nevard, 27, 43 roshan fernandopulle, 25, 35 ryan williams, 26 sabrina cousseau, 32 salman waqas, 26, 36, 50, 55 sameer pant, 27, 43 scott norman, 27, 43 shaked druker, 22, 24 shannon dehghanpir, 53 sheila megehee, 20 sherrie clark-deener, 25, 38 sierra ott, 56 silvia marchi, 31 soon hon cheong, 9 soraya sayi, 35 stephanie walbornn, 10, 30, 43, 44 steven edmonds, 55 takeshi kawakami, 20 tal raz, 22, 24 59 tamir goshen, 22 thomas burke, 45 thomas cecere, 36 thomas passler, 45 tiffany hoffman, 22, 44 tina owen, 37 tulio prado, 41 udaya desilva, 32 vanmathy kasimanickam, 27 victor absalón-medina, 31 viviane gomes, 51 yoke lee lee, 9 yoko ambrosini, 37 2017: pyometra and cervical wedge resection in a mare   pyometra and cervical wedge resection in a mare lauren r. pasch, eric j. parente, tamara dobbie department of clinical studies, new bolton center, school of veterinary medicine, university of pennsylvania, kennett square pa summary a 21-year-old mare evaluated for inappetence and lethargy was diagnosed with pyometra secondary to cervical fibrosis and adhesions. palliative treatment consisted of uterine evacuation and lavage, however the chronic fibrosis and recurrence of cervical adhesions precluded complete resolution of the condition. a cervical wedge resection was performed to reestablish cervical patency and facilitate uterine drainage. following surgery, the cervix was manipulated regularly to prevent stenosis and adhesion formation. eleven weeks after surgery, the mare developed a dilation of the cranial cervical canal and stenosis of the internal cervical os. the stenotic internal cervical os allowed a small amount of malodorous purulent fluid to drain from the mare’s uterus but the opening was too small to permit uterine lavage. ovariohysterectomy was offered as a curative solution for the mare’s recurrent pyometra; the owner elected conservative management. keywords: mare, pyometra, cervical wedge resection, ovariohysterectomy background pyometra is an infrequent diagnosis in the mare and is rarely associated with systemic illness, however mares may become uncomfortable and colicky when the uterus becomes grossly distended. pyometra occurs secondary to cervical adhesions and fibrosis, but has also been associated with retained endometrial cups1 or a closed cervix secondary to progesterone from a persistent corpus luteum.2 pyometra may be managed medically with frequent uterine evacuation and lavage or surgically with cervical wedge resection, which reestablishes cervical patency and allows for uterine drainage. ovariohysterectomy is a surgical option that is infrequently performed given the difficulty in gaining access to the mare’s reproductive tract and the risk of complications (ie. peritonitis and hemorrhage) associated with the surgery. this case represents a spectrum of the severity and treatments existing for pyometra in the mare. case presentation a 21-year-old irish sport horse mare was evaluated for inappetence of one day’s duration. the mare was dull and lethargic and appeared to have lost weight despite abdominal distension. the mare was ridden daily and had competed two weeks prior to presentation. the mare had never carried a foal to term but had produced multiple offspring via embryo transfer; the last embryo was recovered from the mare in 2013. in 2014 the owner planned to perform embryo transfer one final time, however the mare maintained sterile, mucoid fluid in the uterine lumen despite repeated treatment. in addition, the mare’s cervix was difficult to navigate due to its length, tightness and tortuous nature, which may have been exacerbated by repeated cervical manipulation during embryo transfer and likely contributed to the accumulation of mucoid fluid within the uterus. the owner elected to retire the mare from breeding. on presentation, the mare was quiet, alert and responsive with thin body condition (bcs 4/9) and abdominal distension. the mare’s perineal conformation was unremarkable and a vulvoplasty was in place. palpation per rectum revealed a grossly distended uterus, mimicking the size of a late-gestation pregnancy; echogenic intrauterine fluid was visible via transrectal (figure 1) and transabdominal ultrasound. the ovaries could not be imaged at this time due to marked uterine distension causing the uterus to expand dorsally above the pelvic brim and into the abdomen. a manual vaginal examination revealed a tightly closed tortuous cervix, with transluminal adhesions. the mare was administered intravenous detomidine hydrochloride (0.01 mg/kg [0.005 mg/lb], iv), butorphanol tartrate (0.01 mg/kg [0.005 mg/lb], iv), and n-butylscopolammonium bromide (0.1 mg/kg [0.05 mg/lb], iv). manual cervical dilation was performed to allow passage of a small bore nasogastric tube into the uterine lumen to facilitate the drainage of approximately 115 liters of thick, nonclinical theriogenology • volume 9, number 2 • june 2017217   odorous, purulent material. the uterus was lavaged with ten liters of 0.9% nacl following uterine evacuation. the mare was administered flunixin meglamine (1 mg/kg [0.5 mg/lb], iv q 24 h), gentamicin (6.6 mg/kg [3 mg/lb], iv q 24 h), penicillin g procaine (20,000 iu/kg [9,091 iu/lb], im q 12 h), and oxytocin (10 iu/animal, im q 8 h). the following day the mare was started on a ten day course of sulfamethoxazole/trimethoprim (30 mg/kg [13.6 mg/lb], po q 12 h) to provide broad spectrum antimicrobial coverage. treatment the mare was presented to the university of pennsylvania school of veterinary medicine, new bolton center hospital for large animals for standing cervical wedge resection eight days following initial examination. immediately prior to surgery, 55 liters of thin orange fluid was drained from the mare’s uterus. following evacuation, the uterus was lavaged with four liters of 0.05% povidone-iodine solution, which can be used in the management of microbial infections to suppress the growth of bacteria without detrimental effects on the uterus. point of care bloodwork (packed cell volume, total protein, creatinine) was within normal reference ranges. the mare was placed in standing stocks prior to sedation with detomidine hydrochloride (0.01 mg/kg [0.005 mg/lb], iv) and butorphanol tartrate (0.01 mg/kg [0.005 mg/lb], iv). caudal epidural anesthesia was achieved with the administration of 2% lidocaine (60 mg) and xylazine (60 mg) at the first intercoccygeal space. adhesions within the cervical canal were broken down (figure 2) and a full-thickness wedge-shaped defect was created in the cervix from the nine to 12 o’clock position, with the apex pointed cranially.3 the entire portio vaginalis was surgically removed. the mare was administered phenylbutazone (4 mg/kg [1.82 mg/lb], iv once) and started on a course of sulfamethoxazole/trimethoprim (30 mg/kg [13.6 mg/lb], po q 12 h for 10 days) and phenylbutazone (2.2 mg/kg [1 mg/lb], po q 24 h for 3 days). the mare was evaluated once daily for three days after surgery. the first assessment revealed a patent cervix that easily admitted two fingers into the external os and one finger into the internal os. the internal cervical os was gently dilated using two fingers and a nystatin-neomycin sulfate-thiostreptontriamcinolone acetate ointment (quadritop™ ointment, henry schein animal health, dublin, oh) was applied topically to the cervical openings and canal to prevent adhesion formation. the mare was administered oxytocin (10 iu/animal, im q 8 h) that day to stimulate uterine contractions and drainage. prostaglandin (or a synthetic analogue), another commonly used ecbolic, was not selected due to the potential for mare discomfort. the following day, ultrasonographic examination of the mare’s reproductive tract per rectum revealed several centimeters of echogenic intrauterine fluid. the mare’s uterus was lavaged with four liters of 0.9% nacl and the initial effluent recovered was watery and pink. the cervix was gently dilated and steroid ointment was applied. three days after surgery, ultrasonographic examination per rectum of the mare’s reproductive tract was repeated. approximately 2.5 cm of slightly echogenic fluid was present in the uterine body and horns. multiple medium sized follicles were noted on both ovaries. the cervix was once again dilated and steroid ointment applied. the owner continued to manually dilate the mare’s cervix and apply topical steroid ointment to the cervical canal and openings. for the first four weeks, the procedure was performed daily then every other day for two weeks, and compliance was confirmed via frequent communication with the owner. six weeks after surgery, the mare’s reproductive tract was examined via transrectal ultrasonography. the uterine wall was moderately thickened throughout the uterine body and horns. approximately 5 cm of echogenic fluid was visible at the uterine bifurcation. manual cervical examination revealed a patent cervix that permitted two fingers into the external cervical os and one finger into the internal cervical os. the cervical canal was short and straight. the owner was instructed to continue with manual dilation once every three days for the next four weeks. nine weeks after surgery, the owner noted a malodorous white-yellow vulvar discharge. the mare was examined the following week by transrectal palpation and ultrasonography. the mare’s uterine body and uterine horns were distended with moderately echogenic fluid (greater than 10 cm). manual cervical examination revealed a straight cervical canal that could easily admit two fingers. a double guarded culture swab was passed through the cervix in an effort to obtain a sample of uterine fluid for clinical theriogenology • volume 9, number 2 • june 2017 218   bacterial culture and sensitivity. following passage of the swab, no purulent material was evident on the swab or gloved hand. careful cervical palpation revealed a large dilation, approximately 5 to 6 cm in length, cranial to the cervical canal. the exact location of the dilation was difficult to ascertain. two scenarios were possible: the circumferential dilation was in the cranial cervical canal and developed secondary to repeated stretching of the internal cervical os and cranial cervical canal, or the dilation was a fibrous adhesion in the caudal uterine body created by repeat trauma to the internal cervical os. the former seemed to be the most plausible, however endoscopy was recommended to characterize the exact location of the dilation. eleven weeks after surgery, the mare presented to the university of pennsylvania school of veterinary medicine, new bolton center hospital for large animals for endoscopic evaluation. she was bright, alert and responsive and vital parameters were within normal limits. the mare was placed in standing stocks and administered detomidine hydrochloride (0.01 mg/kg [0.005 mg/lb], iv), butorphanol tartrate (0.01 mg/kg [0.005 mg/lb], iv), n-butylscopolammonium bromide (0.1 mg/kg [0.05 mg/lb], iv) and flunixin meglamine (1 mg/kg [0.45 mg/lb], iv). transrectal ultrasonographic evaluation of the mare’s reproductive tract revealed greater than 10 cm of echogenic fluid within the uterine body and horns. air was visualized in the vagina, caudal uterine body and cervical canal, indicating some degree of cervical patency. the mare’s vulvoplasty had been previously opened but she maintained adequate apposition of vulvar lips. manual examination of the cervix confirmed the presence of a large, circumferential dilation cranial to the cervical canal. no obvious opening was palpable along the cranial borders of the dilation. a small amount of purulent malodorous fluid was present in the dilation and a sample was obtained for aerobic culture and sensitivity. examination of the caudal reproductive tract was performed using a flexible videoendoscope (gif-q160 gastroscope, clv-180 light source, cv-180 image processor, olympus, center valley, pa). a circumferential dilation was visualized cranial to the cervical canal. the tissue lining the dilation was pink and smooth and a small opening (3-4 mm in diameter) was visualized in the cranial wall of the dilation. purulent fluid was visualized emanating from the opening. the opening appeared to be most consistent with a stenotic internal cervical os, but was too small to permit advancement of the endoscope into the uterus. it was not possible to advance a small gauge catheter through the biopsy channel of the endoscope given the tortuous nature of the canal cranial to the opening. because uterine lavage was no longer feasible, the owner was advised of the options of conservative treatment with systemic antibiotics for a finite period of time (to combat the production of purulent fluid within the uterus) or standing hand-assisted laparoscopic ovariohysterectomy. systemic antibiotic treatment, rather than intrauterine therapy, was proposed as a means to establish better uterine distribution given the presence of purulent fluid within the uterus. ovariohysterectomy was proposed as a curative, surgical option recognizing the risk of surgical complications and the large financial commitment. aerobic culture of the purulent uterine fluid yielded a light growth of actinobacillus rossii, a gram negative coccobacillus species with in vitro sensitivity to the following antimicrobials: chloramphenicol, doxycycline, ciprofloxacin, ticarcillin and amikacin. the owner declined both surgical and antimicrobial therapy. outcome the mare continues to drain approximately 240 ml (one cup) of malodorous, purulent fluid from the vulva each day with no signs of systemic illness. the owner is hesitant to pursue further treatment at this time given that the mare’s uterus is draining consistently. the mare’s reproductive tract is examined once monthly by transrectal and transabdominal ultrasonography to ensure the uterus does not become markedly distended. currently the mare continues to be ridden daily with plans for competition in the near future. at the time of writing, the mare remains comfortable with markedly reduced uterine distension and continued vulvar drainage from the uterus. clinical theriogenology • volume 9, number 2 • june 2017219   discussion pyometra in the mare is seldom accompanied by systemic clinical signs2,4,5 and mares with transluminal cervical adhesions and concurrent pyometra are often clinically stable for years until the uterus becomes severely distended. if the cervix is closed due to fibrosis and/or adhesions, vulvar discharge will not be visualized. pyometra commonly results from cervical incompetence secondary to parturition, attempts to resolve dystocia, or repeated cervical manipulation during intrauterine treatments or embryo transfer. transluminal cervical adhesions interfere with normal uterine clearance mechanisms2,3,5 leading to accumulation of fluid within the uterus and ultimately pyometra. the most common organism associated with pyometra in the mare is streptococcus equi ssp. zooepidemicus; escherichia coli, actinomyces sp., pasteurella sp., pseudomonas sp., proprionobacterium sp., and candida rugosa have been reported as well.2,4 equine pyometra is typically managed with uterine lavage and ecbolics such as oxytocin or synthetic prostaglandin analogues, with appropriate antimicrobial therapy helping to facilitate treatment.2,4 surgical treatment is considered when severe cervical adhesions or fibrosis prevents adequate drainage. cervical wedge resection3 is one treatment option for mares with pyometra secondary to cervical adhesions and fibrosis. in a report involving six mares with cervical defects and chronic pyometra, a fullthickness wedge-shaped defect was created in the dorsolateral aspect of the cervix to create a permanent opening to the uterus. in that report, the wedge was approximately 3-4 cm wide at its base and narrowed to a 2-3 cm diameter lumen near the internal cervical os. post-operative care consisted of daily treatment with topical steroids and antibiotics for two weeks, then once weekly treatment for the following two weeks. resolution of pyometra was achieved in five of six mares. the one mare that did not resolve the pyometra and required periodic lavage was presumed to have poor drainage subsequent to ventral displacement of the uterus secondary to a large accumulation of uterine fluid. ovariohysterectomy is a treatment option for refractory cases of pyometra in which uterine lavage is ineffective or impossible due to cervical or serosal adhesions or the dependent, ventral location of the uterus. ovariohysterectomy in the mare is an infrequently performed procedure.6 surgical access to the uterus with the mare under general anesthesia in dorsal recumbency is difficult, and ovariohysterectomy with this approach has resulted in more postoperative complications than more recent laparoscopic assisted approaches.7,8 laparoscopic standing transection of the dorsal uterine attachments followed by general anesthesia and resection of the uterus at the caudal body was successful without any significant complications in three mares.6 surgical approaches with the mare standing and the use of improved laparoscopic techniques make this approach a more viable option with decreased risks. in the present case, uterine lavage was difficult given the tight, tortuous nature of the mare’s cervix and the cervical adhesions. the owner elected cervical wedge resection as a treatment for the mare’s pyometra, allowing the mare to maintain quality of life and athletic aspirations without the complications and costs associated with ovariohysterectomy. the large volume of uterine fluid at presentation may have had a negative influence on the outcome by decreasing the mare’s ability to actively expel uterine fluids after the wedge resection was performed. that is, the cranioventral distension of the uterus may have contributed to decreased uterine clearance even after the establishment of cervical patency. in cases of a ventrally-angled, pendulous uterus, uteropexy has been reported as a technique to facilitate drainage and improve pregnancy rates, but has not been reported as a treatment for advanced pyometra and was not pursued in this mare.9 learning points  pyometra in the mare is rarely associated with systemic illness.  cervical wedge resection is a surgical option for pyometra for cases in which uterine evacuation and lavage is insufficient.  ovariohysterectomy is an infrequently performed procedure in the mare but if successful, is a curative option for refractory cases of pyometra. clinical theriogenology • volume 9, number 2 • june 2017 220    significant cervical remodeling may occur following cervical wedge resection, and repeated evaluation is recommended following surgery. references 1. vandeplassche m, spincemaille j: advanced pyometra with intact endometrial cups in a mare. equine vet j 1979;11:122-123. 2. lu kg: pyometra. in: ao mckinnon, el squires, we vaala , et al, editors. equine reproduction. 2nd ed. philadelphia: blackwell publishing, 2011; p. 2652-2654. 3. arnold ce, brinsko sp, varner dd: cervical wedge resection for treatment of pyometra secondary to transluminal cervical adhesions in six mares. j am vet med assoc 2015;246:1354-1357. 4. sertich pl: cervix adhesions. in: ao mckinnon, el squires, we vaala , et al, editors. equine reproduction. 2nd ed. philadelphia: blackwell publishing, 2011; p. 2721-2723. 5. hughes jp, stabenfeldt gh, kindahl h, et al: pyometra in the mare. j reprod fertil suppl 1979;27:321-329. 6. woodford ns, payne rj, mccluskie lk: laparoscopically-assisted ovariohysterectomy in three mares with pyometra. equine vet educ 2014;26:75-78. 7. santschi em, adams sb, robertson jt, et al: ovariohysterectomy in six mares. vet surg 1995;24:165-171. 8. rotting ak, freeman de, doyle aj, et al: total and partial ovariohysterectomy in seven mares. equine vet j 2003;36:29-33. 9. brink p, schumacher j, schumacher j: elevating the uterus (uteropexy) of five mares by laparoscopically imbricating the mesometrium. equine vet j 2010;42:675-679. figure 1. transrectal ultrasound image of the uterus illustrating echogenic material within the caudal uterine body. marks are at intervals of 5 mm. clinical theriogenology • volume 9, number 2 • june 2017221   figure 2. transluminal cervical adhesions prior to cervical wedge resection. (editor’s note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 9, number 2 • june 2017 222 2011: relationships among morphologic characteristics and membrane integrity of stallion sperm relationships among morphologic characteristics and membrane integrity of stallion sperm* steven p. brinsko, jennifer a. spooner,† terry l. blanchard, charles c. love, dickson d. varner department of large animal clinical sciences, texas a&m university, college station, tx abstract various measures of semen quality and fertility often yield either nonexistent or weak correlations. while it is generally assumed that morphologically normal sperm are progressively motile and capable of fertilization, some sperm may have membrane damage that could limit their fertilizing capacity. this study examined the relationship between morphology and membrane integrity of stallion sperm. semen from 22 sexually active stallions underwent standard evaluation as part of a breeding soundness examination. aliquots of gel-free semen were diluted to 200 x106 sperm/ml in semen extender then stained with sybr-14 and propidium iodide (pi). random fields were observed and digitally captured under 1250x magnification using epifluorescence, followed by differential interference contrast (dic) microscopy to assess morphologic characteristics of sperm in the same microscopic field. captured images were evaluated side by side to compare fluorescent staining to sperm morphology. membrane integrity of sperm was based on sybr-14 (green = membrane-intact) or pi (red = membranedamaged) labeling of the sperm head. bivariate analyses were performed to determine the odds ratios (or) of sperm having intact membranes within each morphologic classification compared to sperm without that morphologic characteristic. morphologically normal sperm were equally likely to be membrane-damaged as membrane-intact (or = 1.015). while sperm with abnormal heads (or = 3.26, p<0.0001) were more likely to be membrane-damaged, 39% of sperm with abnormal heads were membrane-intact. sperm with proximal droplets (or = 4.95) and distal droplets (or = 5.75) were more likely to be membrane-intact than sperm without droplets. keywords: stallion, sperm, morphology, membrane integrity introduction evaluation of semen quality is integral to stallion breeding soundness examinations and in the calculation of insemination doses. studies attempting to correlate various measures of semen quality with fertility have yielded mixed results, with some of these studies yielding either nonexistent or weak correlations.1,2 early on, it was noted that poor fertility can exist even in the presence of sufficient numbers of morphologically normal sperm and it was suggested that additional tests might more accurately predict fertility.1 however, total sperm numbers, percentage of progressively motile sperm and percentage of morphologically normal sperm remain significant, albeit weak, predictors of fertility as well as principal components of semen evaluation.2-4 integrity of the plasma membrane is essential to proper sperm functions which lead to capacitation, the acrosome reaction and fertilization.5 however, samper found that membrane integrity was poorly correlated with motility.6 while it is generally assumed that morphologically normal sperm will be both progressively motile and capable of fertilization, it is conceivable that some morphologically normal and motile sperm may have some degree of membrane damage that could limit their survivability or otherwise render them incapable of fertilization. in the boar for example, sperm membrane integrity is reported to be a more accurate predictor of fertility than motility.7 to date, there have been no published reports of the association between membrane integrity and morphology in equine sperm. the purpose of this study was to determine if relationships exist among sperm morphologic characteristics and membrane integrity in fresh ejaculated semen of sexually active stallions. * portions of these data were presented in abstract form in 2004 at the annual meetings of the society for theriogenology and the american association of equine practitioners. † current address: oklahoma state university, center for veterinary health sciences, stillwater, ok 55 materials and methods semen was obtained from 22 stallions of varying fertility (10 to 79% per-cycle pregnancy rate) and semen quality (22 to 83% morphologically normal sperm) using a missouri model artificial vagina (nasco, ft. atkinson, wi) equipped with an in-line nylon micromesh filter (animal reproduction systems, chino, ca). an ovariectomized mare was used for sexual stimulation and breeding phantom was used as a mount source. following at least seven days of sexual rest, two ejaculates were collected one hour apart. a third ejaculate was collected the following day to examine the association between sperm morphologic characteristics and membrane integrity. ejaculates underwent standard evaluation as described by kenney et al.8 sperm concentration in ejaculates was determined photometrically using a densimeter (model 543a, animal reproduction systems). an aliquot of raw semen was diluted to 25 x 106 sperm/ml in semen extender (ez-mixin cst®, animal reproduction systems) and sperm motion characteristics were determined using a computer-assisted sperm motion analysis system (casma; ivos version 12.2l, hamilton thorne biosciences, beverly, ma). separate aliquots of gel-free semen were extended to 200 x 106 sperm/ml in semen extender prior to fluorescent staining with pi and sybr-14 (live/dead® sperm viability kit, molecular probes, eugene, or), and jc-1. stock solutions of sybr-14, pi and jc-1 were prepared as previously described by garner and johnson.9 although included in protocol, jc-1 staining characteristics were not included in the analyses. samples were incubated at 37° c for 20-30 minutes then, immediately prior to analysis, 0.5 μl of 2% parafomaldehyde solution was added to 100 μl of fluorescently labeled samples to immobilize the cells and facilitate microscopic imaging and digital capture without adversely affecting membrane integrity.10 however, because the sperm were immobilized to facilitate imaging and capture, jc-1 staining characteristics of the mitochondrial membrane potentials in the midpiece were not included in the analyses. two μl of the stained sample was placed on a microscope slide and covered with a 22 x 22 mm coverslip. random fields were observed under 1250x magnification using epifluorescence to assess fluorescent staining patterns of 100 sperm followed by dic microscopy of the same sperm to assess morphologic characteristics. each microscopic field was captured under both epifluorescence and dic using a digital camera (optronics, goleta, ca) interfaced to an olympus bx3 microscope (olympus america, inc., center valley, pa). time required to capture images encompassed approximately one hour per sample. preliminary data indicated that addition of paraformaldehyde did not alter membrane staining characteristics over time (p > 0.1). captured images were later evaluated side by side on a computer screen to compare the fluorescent staining patterns to the morphologic characteristics of individual sperm cells (figure). morphologic classification was based on the system established by kenney et al,8 and all abnormalities present on an individual sperm were recorded. the order in which the sperm images were captured was recorded to determine if time to evaluation affected membrane integrity. only sperm cells visualized throughout their entire length were included for analysis. premature germ cells were not enumerated or evaluated. membrane integrity of sperm was based on the labeling of the sperm head with either sybr-14 (green = membrane-intact) or pi (red = membranedamaged). sperm cells considered membrane-intact had heads that fluoresced entirely green, while heads of sperm with some mixture of red and green or entirely red were considered membrane-damaged. bivariate analyses were performed to determine the odds ratios of sperm having intact membranes within each morphologic classification by comparing them to sperm not having that morphologic characteristic (e.g. proportion of membrane-intact sperm with an abnormal head compared to sperm not having an abnormal head). a p-value of < 0.05 was used to determine if significant odds ratios existed. effect of time to evaluation (approximately one hour to capture images of 100 sperm) on membrane integrity was examined using paired t-tests to determine if overall sperm membrane integrity and membrane integrity of sperm within each morphologic classification differed (p< 0.05) between the first 50 and last 50 sperm evaluated. 56 results percentages of membrane-intact and membrane-damaged sperm, and odds ratios for sperm within morphologic classifications, are presented in the table. more of the first 50 sperm per sample evaluated were membrane-intact (66.6%) compared to the second 50 (56.7%; p < 0.01). however, pair wise comparisons within morphologic classifications demonstrated that this difference was only due to a reduction in membrane integrity of morphologically normal sperm (68% vs 54.5%; p < 0.01) and coiled tails (68% vs 44%; p = 0.014) over time. of the 2200 sperm evaluated, 58% were morphologically normal, and 5% had coiled tails. surprisingly, morphologically normal sperm were just as likely to be membrane-damaged as they were to be membrane-intact. as expected, all sperm with detached heads allowed permeation of pi and were considered membrane-damaged. sperm with abnormal heads were 3.26 times more likely to be membrane-damaged than sperm without abnormal heads; yet, 39% of sperm with abnormal heads were membrane-intact. only 18% sperm with abnormal acrosomes had damaged plasma membranes. sperm with abnormal acrosomes were almost three times more likely to be membrane-intact than sperm with normal appearing acrosomes. sperm with proximal droplets were almost five times more likely, and sperm with distal droplets were almost six times more likely, to be membrane-intact than sperm without droplets. discussion these data demonstrate that morphologically normal sperm are equally likely to be membranedamaged as membrane-intact. associations with fertility were not assessed in the present study because insufficient data were available to make valid comparisons11 and any attempt to do so would only add to the confusion that already exists in the literature regarding semen quality and fertility. assessment of stallion fertility is a multifactorial process that encompasses far more than simply correlating semen quality with pregnancy rates. in addition to semen characteristics, valid comparisons would also require not only data regarding how the stallions were bred, i.e. natural cover, artificial insemination, fresh semen, cooled semen, frozen semen, but also a multitude of other factors which are difficult if not impossible to control retrospectively, including, but certainly not limited to, the inherent fertility of the mares and their breeding management. even so, the data presented here may help to explain results of earlier studies where correlations between fertility and motility,2 or fertility and morphology,1,12 were poor or nonexistent. these findings also raise concerns over using only the number of progressively motile sperm or number of morphologically normal sperm in an ejaculate as an assessment of semen quality. this would be especially worrisome if these methods were employed on the first ejaculate obtained after sexual rest to determine its suitability for cooling or freezing as the discrepancy between normal morphology and membrane integrity is magnified in ejaculates from sexually rested stallions.13 stresses associated with cooling or freezing could amplify any membrane damage that existed prior to processing. when this occurs, fertility could be much lower than would be expected based upon the morphologic and motion characteristics of the sample. approximately 14% of the normal sperm in this study appeared to lose membrane integrity within one hour, suggesting that longevity tests should be performed on stallion semen, especially when initial assessments of sperm motility and morphology do not coincide with a stallion’s reduced fertility. of particular interest is the finding that sperm with droplets were almost five to six times more likely to be membrane-intact than sperm without droplets. the biological significance of this is unclear, but may be related to biochemical or structural properties of droplets that are not found in non-droplet bearing sperm.14 cytoplasmic droplets are usually shed in the cauda epididymis or upon ejaculation,15,16 and ejaculates containing a high proportion of spermatozoa with attached droplets have been associated with altered epididymal function and reduced fertility,17-19 although the exact mechanism by which cytoplasmic droplets adversely affect fertility has yet to be elucidated. in contrast, using dismount samples and logistic regression to examine the relationship of specific sperm morphologic characteristics on the pregnancy outcome, love et al., found that the presence of proximal or distal droplets in thoroughbred ejaculates did not have significant influence on the odds of pregnancy.3 though not confirmed in the stallion, in other species sperm with droplets have been shown 57 to be incapable of capacitation, binding to the zona pellucida and undergoing the membrane fusion events of the acrosome reaction.20 it would seem unlikely that the cytoplasmic droplets of stallion sperm would be that much different than those of other species. rathi et al., reported that bicarbonate-induced merocyanin responses associated with capacitation were absent in equine sperm with cytoplasmic droplets.21 it is interesting to note that in the current study, sperm with abnormal acrosomes were almost three times more likely to be membrane-intact than sperm with normal appearing acrosomes. both the acrosome and cytoplasmic droplets contain lysosomal enzymes and have an intimate association with the golgi apparatus during their formation. in addition, the acrosomal membrane, plasma membrane and cytoplasmic droplets all undergo significant modifications as sperm traverse the length of the epididymis.13,22-24 in light of this and the above findings, determining whether or not the retention of cytoplasmic droplets and altered acrosomal function share common mechanistic influences on fertility, as well as the significance of cytoplasmic droplets in equine ejaculates, warrant further investigation. references 1. kenney rm, kingston rs, rajamannon ah: stallion semen characteristics for predicting fertility. proc annu conv am assoc equine pract 1971.p 53-67. 2. jasko dj, little tv, lein dh, et al: comparison of spermatozoal movement and semen characteristics with fertility in stallions: 64 cases (1987-1988). j am vet med assoc 1992;200:979-985. 3. love cc, varner dd, thompson ja: intraand inter-stallion variation in sperm morphology and their relationship with fertility. j reprod fertil suppl 2000;56:93-100. 4. blanchard tl, brinsko sp, varner dd, et al: relationships between stallion age, book size, number of matings (covers), breeding soundness examination findings, and fertility parameters in 15 thoroughbred stallions (34 stallion years). clin therio 2010;2:78-89. 5. brito lfc, barth ad, bilodeau-goeseels s, et al: comparison of methods to evaluate the plasmalemma of bovine sperm and their relationship with in vitro fertilization rate. theriogenology 2003;60:1539-1551. 6. samper jc: evaluation of cryopreserved semen: an alternative assay. acta vet scand 1992;88:59-65. 7. fraser l, gorszczaruk k, strzezek j: relationship between motility and membrane integrity of boar spermatozoa in media varying in osmolality. reprod domest anim 2001;36: 325-329. 8. kenney rm, hurtgen j, pierson r, et al: clinical fertility evaluation of the stallion. society for theriogenology manual. j soc therio 1983;vol ix, part ii. 9. garner d, johnson la: viability assessment of mammalian sperm using sybr-14 and propidium iodide. biol reprod 1995;53:276-284. 10. haas gg, debault le, d’cruz o, et al.: the effect of fixatives and/or air-drying on the plasma and acrosomal membranes of human sperm. fertil steril 1988;50:487-492. 11. amann rp: weaknesses in reports of “fertility” for horses and other species. theriogenology 2005;63:698-715. 12. voss jl, pickett bw, squires el: stallion spermatozoal morphology and motility and their relationship to fertility. j am vet med assoc 1981;178:287-289. 13. brinsko sp, spooner ja, blanchard tl, et al: relationships among sperm membrane integrity, motility and morphology in first and third ejaculates of sexually rested stallions. proc annu conv am assoc equine pract 2004. p. 502-504. 14. coratadellas n, durfort m: fate and composition of cytoplasmic droplet of hamster epididymal spermatozoa. j morphol 1994;221:199-210. 15. dott hm, dingle jt: distribution of lysosomal enzymes in the spermatozoa and cytoplasmic droplets of bull and ram. exp cell res 1968;52:523-540. 16. amann, rp, hay sr, hammersted rh: yield, characteristics, motility and camp content of sperm isolated from seven regions of ram epididymis. biol reprod 1982;27:723-733. 17. cummins jm: the effects of artificial cryptorchidism in the rabbit on the transport and survival of spermatozoa in the female reproductive tract. j reprod fertil 1973;33:469-479. 18. bedford jm: maturation, transport and fate of spermatozoa in the epididymis. in: hamilton dw, greep ro, editors. handbook of physiology, section 7, endocrinology, vol 5. male reproductive system. washington, dc: american physiological society, 1975. p. 303-317. 19. amann rp, seidel ge, mortimer rg: fertilizing potential of semen from young beef bulls containing a high or low percentage of sperm with a proximal droplet. theriogenology 2000;54:1499-1515. 20. peña ai, barrio m, becerra jj, et al: infertility in a dog due to proximal cytoplasmic droplets in the ejaculate: investigation of the significance for sperm functionality in vitro. reprod domest anim 2007;42:471-478. 21. rathi r, colenbrander b, bevers mm, et al: evaluation of in vitro capacitation of stallion spermatozoa. biol reprod 2001;65:462-470. 58 22. jones r: plasma membrane structure and remodeling during sperm maturation in the epididymis. j reprod fertil suppl 1998;53:73-81. 23. cooper tg, yeung c-h: acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet. microsc res tech 2003;61:28-38. 24. toshimori k: biology of spermatozoa maturation: an overview with an introduction to this issue. microsc res tech 2003;61:1-6. figure. digital images of the same sperm viewed under dic and epiflourescence microscopy for side by-side comparison of morphologic and membrane integrity staining characteristics using sybr-14 and pi. sperm with heads fluorescing green (bright in black and white image) are considered membraneintact and sperm with heads fluorescing red (arrows; pale in black and white image) are considered membrane-damaged. (for color rendition of the image, please consult the on-line version of clinical theriogenology at http://www.therio.org) 59 table. mean percentages of and odds ratios for sperm (n = 2200) with intact or damaged membranes within morphologic classifications in the third ejaculate of 22 stallions (n = 100 sperm evaluated per ejaculate). morphologic characteristic % of total sperm evaluated % membrane intact % membrane damaged p-value odds ratio to be (i = intact; d =damaged) normal 57.9 61 39 0.60 1.015i non-normal 42.1 63 37 proximal droplet 5.8 89 11 <0.0001 4.95i no proximal droplet 94.2 60 40 distal droplet 5.2 89 11 <0.0001 5.75i no distal droplet 94.8 60 40 abnormal head 8.3 39 61 <0.0001 3.26d no abnormal head 91.7 64 36 bent midpiece 13 60 40 0.40 1.15d no bent midpiece 87 62 38 abnormal midpiece 5.4 57 43 0.29 1.51d no abnormal midpiece 94.6 62 38 bent tail 6 65 35 0.43 1.13i no bent tail 94 62 38 detached head 1.5 0 100 <0.0001 - no detached head 98.5 63 37 abnormal acrosome 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/allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2015: sonographic changes during gestation in primiparous chinchilla rabbit does sonographic changes during gestation in primiparous chinchilla rabbit does adetola ajadi,a temitope ajadi,b iriangbonse eweka,c frederick smithc adepartment of veterinary medicine and surgery, federal university of agriculture, abeokuta;bdepartment of veterinary public health and reproduction, federal university of agriculture, abeokuta;cdepartment of animal physiology, federal university of agriculture, abeokuta abstract sonographic changes during gestation were evaluated in ten primiparous female chinchilla rabbits with mean weight 1.9±0.1kg. the rabbits were synchronized with 2mg/kg intramuscular injections of prostaglandinf2α, mated naturally and thereafter examined sonographically on day 7, 14, 21 and 27 after mating using an ultrasound machine. measurements of diameters of gestational sac, embryonal sac and fetal sac were obtained. heart beats of at least two fetuses were also counted manually and the average determined. descriptive statistics were used to analyze the sonographic measurements. the results showed that the earliest sonographic feature of pregnancy observed in the rabbit was the gestational sac observed by day 7 after mating and was characterized by oval shaped anechoic sac containing bipolar hyperechoic band. the mean diameters of the gestational sacs in the rabbits were 7.3±0.71mm. the embryonal sac was observed by day 14 after mating, which was characterized by an oval shaped hyperechoic band with central anechoic cavity and some hyperechoic structures. the mean diameter of the embryonal sacs was 16.1±1.79mm. fetal sac, fetal skeletal structures, fetal movement and fetal heartbeat were first detected on day 21 and 27 after mating. the mean diameter of the fetal sac on day 21 and 27 were 21.8±1.39mm and 36.4±3.20mm respectively, the mean fetal heart rate on day 27 after mating was 125.3±14.3beats per minute. it was thus concluded that sonographic changes during pregnancy in rabbits is valuable in predicting gestational length and fetal viability. the values obtained in this study can serve as baseline reference for comparison with other breeds. keywords: sonographic changes, fetal sac, hyperechoic, gestation, chinchilla introduction accurate prediction of the date of parturition in the rabbit is clinically useful to prevent or minimize reproductive losses by timely intervention.1 this is because accurate parturition dates may assist with monitoring animals at high risk for dystocia, thus allowing for intervention and decreased kit mortality. for rabbits with a history of abortion, embryonic resorption, or insufficient luteal phase, accurate assessment of gestational age can assist in therapeutic decision making. finally, progress in assisted reproductive techniques in rabbits, such as estrus synchronization and embryo transfer, require accurate prediction of ovulation, gestational age, and parturition date.2 undoubtedly, the conventional method of pregnancy diagnosis in rabbit is digital palpation of the abdomen. abdominal palpation reportedly is accurate in a window from 14to 21-days after mating.3 prior to about seven days, the individual amniotic gestational sacs are small and difficult to palpate, especially in obese or tense rabbits. in dogs, palpation is accurate for positive pregnancy diagnosis 87-88 percent of the time during the second trimester of pregnancy and is 73 percent accurate for negative pregnancy diagnosis; with veterinarians more likely to err with a false positive diagnosis.4 b-mode ultrasonography has been reported in rabbits to be 94-98 percent accurate for pregnancy diagnosis when used after 14-21 days of gestation, and 99 percent accurate for pregnancy diagnosis at greater than 22 days from the last breeding.5 fetal heartbeats have been reported visible from 15 days of gestation, while fetal movement has been reported to be visible from 12 days of gestation.5 measurement of bi-parietal head diameter of fetuses, with or without measurement of dorso-ventral trunk diameter, has been demonstrated to be accurate for the estimation of gestational age in dogs,6 however this is yet to be determined in rabbits. also, the sonographic measurements of different gestational structures in the rabbit clinical theriogenology • volume 7 number 2 • june 2015103 are yet to be reported in the literature. the aim of this study was to determine the sonographic features and measurements at different stages of gestation in primiparous chinchilla rabbits. materials and methods animals ten nulliparous female chinchilla rabbits mean weight (1.9±0.1kg) and four sexually mature chinchilla male rabbits with mean weight (2.1±0.1kg) were used. they were purchased from breeders located within the abeokuta metropolis and housed singly in wooden cages. the animals were fed with pelleted grower ration (guinea feeds ltd, benin, nigeria) ad-libitum and forages (tridax procumbens), while water was provided ad-libitum. they were maintained on twelve hours of daylight and darkness cycle. prior to commencement of the study, they were dewormed with 2.5% albendazole syrup (shanuzole, jawa international limited, lagos, nigeria) at 22 mg/kg. all the rabbits were judged to be in good general health based on the result of complete blood counts and physical examination. experimental procedure the rabbits were weighed and thereafter they were synchronized with intramuscular injections of prostaglandin f2α (lutalyse®, upjohn pharmaceutical limited, crawley, sussex, uk) at the rate of 2mg/kg. three days after treatment, all the rabbit does were mated naturally by introducing each doe to a proven stud four times, with an interval of two hours between each breeding to ensure maximum conception. thus day 0 of breeding was taken to be the time the doe was first introduced to the buck. ultrasound examination all the rabbits were examined sonographically on day 7, 14, 21 and 27 after mating using a portable ultrasound machine with a 10.0 mhz transducer (kaixin kx 2000®, xuzhou, china). the machine was fitted with a 3.5mhz curvilinear and 7.0mhz linear transducers. each transducer has four windows of frequency range. in addition, the machine was programmed with software for obstetric and cardiac calculations, as well as having a digital imaging and communication in medicine (dicom) facility for image storage. all the rabbits were imaged in right dorsal recumbency with leg up without prior administration of intravenous fluid. basic preparation included clipping of the hair around the ventral abdomen and application of acoustic gel. once the images were clear, they were frozen and measurements of the diameters of the gestational sac, embryonal sac and fetal sac were obtained. in addition, the heartbeat at least two fetuses were counted manually for each rabbit doe and the average determined. all images were saved on the ultrasound machine for further review. ethical approval for this study was obtained from the research ethics committee, college of animal science, federal university of agriculture, abeokuta, ogun state, nigeria. results the earliest sonographic feature of pregnancy observed in the rabbit was the gestational sac. gestational sac was observed by day 7 after mating and was characterized by oval shaped anechoic sac containing bipolar hyperechoic band (fig. 1). gestational sac was observed in nine of the ten rabbit does giving detection rate of 90 percent (table 1). the mean diameters of the gestational sacs in the rabbits were 7.3 ± 0.95mm, while the values ranged from 6.0 to 8.0 mm (table 2). embryonal sac was observed by day 14 after mating. this was characterized by an oval shaped hyperechoic band with central anechoic cavity and some hyperechoic structures (fig.2). embryonal sac was detected in all the rabbits giving a detection rate of 100 percent (table 1). the mean diameter of the embryonal sacs was 16.1 ± 1.79mm, while the values ranged from 12 to18mm (table 2). fetal sac, fetal skeletal structures, fetal movement and fetal heartbeats were first detected on day 21 ater mating (fig. 3) and again at day 27 after mating. both fetal sac and fetal skeletal structures were detected in all the rabbits, while fetal heartbeat was only measurable in eight out of the ten rabbits, (table 1). the mean diameters of the fetal sac on day 21 and 27 after mating were respectively 21.8 ± 1.39mm and 36.4 ± 3.20mm (table 2). also, the mean fetal heart rate on day 27 after mating was 125.3 ± 14.1 beats per minute (table 2). clinical theriogenology • volume 7 number 2 • june 2015 104 discussion different sonographic criteria have been developed in dogs and cats for pregnancy detection and estimation of fetal age. these include differential features of fetal organ development that occur in early and mid-pregnancy and the use of various ultrasonography measurement to predict gestational age.1,7 ultrasonographic measurements that have been described for pregnancy detection in rabbit include embryonal vesicle at day 7, fetal sac at day 20 and fetal skeletal structures by day 27 and 29.5 in this study, the gestational sac was the earliest observable sonographic evidence of pregnancy. other features observed include embryonal sac on day 14, fetal movement and fetal sac from day 21, fetal skeletal structures and fetal heart beat also observed from day 21 after mating. this study showed that the gestational sac was the earliest recognizable sonographic evidence of pregnancy in rabbit and was observed by the seventh day after mating. the high detection rate of the gestational sac at day seven post mating offers advantage over traditional abdominal palpation method. the study also showed that both embryonal and fetal skeletal structures were observed as from fourteenth day after mating. the 100 percent detection rate of these structures will suggest that it may be better to check rabbits for pregnancy using ultrasound as from day 14 after mating . even though early detection by checking for gestational sacs is possible, this requires more expertise and time so as to reduce the number of false positives. the mean values of the measurement of the sonographic parameters in this study was similar to that reported in earlier study involving new zealand, california and chinchilla breeds.5 this suggests that the values obtained in the study can form a reference for use in rabbits. fetal heartbeat was first detected as an anechoic, pulsating oval shaped structure starting from day 21. recognition of the fetal heartbeat is useful in the assessment of fetal viability and to detect fetal distress. in this study, fetal heartbeat was only detected in eight out of ten rabbits, this was probably due to uncontrolled abdominal movements of the two does because of their temperaments which made detection of the heartbeats difficult. the mean heart rate of the rabbit fetuses was lower compared with dogs, although there is no record in the literature regarding the normal heart rate of rabbit fetuses. this result of this study also confirms the reliability of brightness (bmode) ultrasound in the prediction of pregnancy, all the rabbits were accurately predicted as pregnant. in addition the predicted parturition date was within a window of ± 3days from the actual date of delivery. in conclusion, the application of low intensity ultrasound is useful in rabbit production for pregnancy confirmation and prediction of fetal age. it also could help in detecting early embryonic losses, a common problem in rabbit production. in addition, the technique is not injurious to either the fetus or the doe, and can help maximize the reproductive efficiency in breeding programs by helping to access fetal viability, planning for parturition and estimation of the likelihood of fetal survival during pregnancy. references 1. kim bs, son ch: time of initial detection of fetal and extra-fetal structures by ultrasonographic examination in miniatureschnauzer bitches. j vet sci 2007;8:289-293. 2. baracaldo m: advanced techniques in rabbit production. clin therio 2013;5:152-162. 3. ypsilantis p, saratsis ph: early pregnancy diagnosis in the rabbit by real time ultrasonography. world rabbit sci j 1999;7:93-99. 4. kim y, travis aj, meyers-wallen vn: parturition prediction and timing of canine pregnancy. theriogenology 2007;68:1177-1182. 5. gutierrez he, zamora fmm: ultrasonography study of rabbit pregnancy. proc 8th world rabbit cong; 2004. p. 276280. 6. beccaglia m, luvoni gc: comparison of the accuracy of two ultrasonographic measurements in predicting the parturition date in the bitch. j small anim pract 2006;47: 670-673. 7. lenard zm, hopper bj, lester nv et al: accuracy of prediction of canine litter size and gestational age with ultrasound. aust vet j 2007;85:222-225. clinical theriogenology • volume 7 number 2 • june 2015105 table 1: sonographic changes during gestation in chinchilla rabbits animals gsd7 esd14 fsd21 fmd21 fskd21 fskd27 fmd27 fhbd27 1 2 3 4 5 6 7 8 9 10 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + total (%) 9 (90%) 10(100%) 10(100%) 10(100%) 10(100%) 10(100%) 10(100%) 8(80%) key: gsd7: gestational sac day 7 fskd21: fetal skeletal structure day 21 esd14: embryonal sac day 14 fskd27: fetal skeletal structure day 27 fsd21: fetal sac day 21 fhbd27: fetal heart beat day 27 fmd21: fetal movement day 21 fmd27: fetal movement day 27 table 2: sonographic parameters during gestation in chinchilla rabbits animals gsld7 esld14 fsld21 fsld27 fhbd27 1 2 3 4 5 6 7 8 9 10 7 15 22 33 120 8 16 20 40 110 7 17 22 36 124 6 17 24 42 114 8 18 22 38 nd 7 16 22 32 156 nd 15 19 33 120 8 12 23 36 nd 7 17 22 36 124 8 18 22 38 134 range mean ± sd 6-8 12-18 19-24 32-42 110-156 7.3 ± 0.71 16.1 ± 1.79 21.8 ± 1.39 36.4 ± 3.20 125.3 ± 14.3 key: gsld7: gestational sac length (mm) day 7 esld14: embryonal sac length (mm) day 14 fsld21: fetal sac length (mm) day 21 fsld27: fetal sac length (mm) day 27 fhbd27: fetal heart rate (beats/min) day 27 clinical theriogenology • volume 7 number 2 • june 2015 106 figure. 1: gestational sac of a 7 month old chinchilla doe at 7 days after mating. figure 2: embryonal sac of a 7 month old chinchilla doe at 14 days after mating. clinical theriogenology • volume 7 number 2 • june 2015107 figure 3: fetal sac of a seven month old chinchilla doe at 21 days after mating. clinical theriogenology • volume 7 number 2 • june 2015 108 2016: improvements in equine semen processing techniques that aid optimal fertility improvements in equine semen processing techniques that aid optimal fertility david b. scofield, julie baumber-skaife, paul r. loomis select breeders services inc., chesapeake city, md technology is constantly changing in all aspects of our lives. although developments in the equine breeding industry do not move as fast as those in computers or cell phone technology, there is nonetheless a slow and steady progress forward with respect to the diagnostic tools and equipment veterinarians have available, as well as improved protocols for semen processing and breeding management of the mare. improved success with assisted reproductive technologies like embryo transfer and intracytoplasmic sperm injection (icsi), have led to increased utilization by breeders in recent years, and frozen semen has allowed growth in the global genetics market. some of the advances in semen processing techniques shall be reviewed here; however, whether these advances in technology have resulted in an overall improvement in fertility rate is tough to say. aspects of semen processing are just a small piece of the puzzle when it comes to achieving a pregnancy in the mare; there are so many other variables that can impact the final result. nonetheless, every little bit helps and only by optimizing all the pieces do we give ourselves the best opportunity for success. therefore, it is in our best interest to utilize those semen processing techniques that are available in order to prepare a breeding dose that will provide the most optimal chances of conception. centrifugation cushion centrifugation cushions, as their name suggests, provide a soft pillow at the bottom of the centrifugation tube upon which the sperm come to rest during the process of centrifugation. the use of the cushion allows longer centrifugation times at a higher g force, resulting in optimal sperm recovery. whereas standard centrifugation protocols for semen processing result in recovery rates of 70-80%, the centrifugation cushion can result in recovery rates of 90-95%. although the use of a centrifugation cushion is unlikely to result in a direct impact on fertility, improved sperm recovery allows optimization of the ejaculate potentially increasing the number of breeding doses available. in addition, improved sperm motility due to reduced cellular stress during the centrifugation process may benefit sperm longevity during cooled transport or improve post-thaw motility after cryopreservation. sperm enrichment protocols one of the most significant applications for semen processing that may actually impact fertility has been the introduction of sperm enrichment protocols like density gradient centrifugation. commercially available products include equipure™ (nidacon international ab, mölndal, sweden) and androcoll® (minitube international, tiefenbach, germany). during centrifugation the density gradient selectively separates and enriches a population of sperm with improved morphology and motility. it has also been reported to remove bacteria and viruses. the cost of the product and the limited sperm recovery generally rule out routine application, and there is still some debate about whether the removal of dead or dying sperm from a normal ejaculate offers any benefit to fertility. however, the technique has particular application for those stallions with poor initial semen quality or sub-fertility. sperm enrichment offers the possibility of transported cooled semen for those stallions whose sperm did not have the longevity of motility after standard processing protocols, or allows the option of semen cryopreservation. semen processing with density gradient centrifugation has resulted in an improvement in pregnancy rates, primarily for those stallions with previous sub-fertility. sperm filter developed by botupharma (botucatu, sao paulo, brazil) the spermfilter™ is a new alternative for centrifugation. the filter is composed of a synthetic hydrophobic membrane that essentially separates sperm cells from seminal plasma or the dilution medium. comparative studies with cushioned centrifugation have not demonstrated significant improvement in semen quality. however, it is a quick, clinical theriogenology • volume 8, number 4 • december 2016465 simple procedure that may offer benefit for breeding farms that do not have a centrifuge, for stallions whose semen does not respond well to centrifugation protocols or for low concentration samples where optimal recovery is desirable. changes in extender formulation although numerous commercial extenders are available for diluting stallion semen for cooling, the majority of them are skim milk-sugar based with the most commonly used being a skim milk-glucose based extender known as the kenney extender, named for its developer dr. bob kenney. this extender along with slight variations of the original formula is used by many laboratories processing cooled semen today and is available under numerous commercial brands. more recently extenders that contain defined milk protein rather than complete milk powder have been introduced for diluting and cooling equine semen. these extenders were developed to allow for a more chemically defined media that contains only the native phosphocasienates from milk which were determined to be the milk fraction that provides the most benefit to sperm survival. vendors may claim to have developed the latest and best semen extender, with new and improved formulas or additives that improve semen quality. however, one must look critically at the supporting literature to determine if appropriately controlled studies have been performed and if the increase in reported semen quality actually correlated with increased fertility. in our experience there are some individual stallions that benefit from use of these extenders over standard skim milk-glucose extenders and some that do not. it is therefore essential to test the longevity of sperm motility in split ejaculates for each stallion in a variety of available extenders to determine which one provides the best retention of sperm quality after storage for 24-48 hours. there are many commercial extenders available for semen freezing, but most of these are variations on a few basic formulations. most all freezing extenders have a buffered salt solution as a base, they may include non-fat dried skim milk powder or milk components, a combination of sugars and they almost always contain egg yolk and a cryoprotective agent (cpa) or agents. the most common cpa added to freezing extenders for sperm is glycerol and is typically included in extenders for stallion sperm at concentrations of 2 to 5%. alternative cpa’s including ethylene glycol, propylene glycol, dmso and amides such as methyl formamide and dimethyl formamide have also been used to successfully to cryopreserve stallion sperm, as well as sugars like lactose and mannose that are nonmembrane permeable cpas. a search of the literature will find many journal articles reporting the results of various trials related to extender composition, and/or the addition of different additives to the semen extender in the hopes of improving post-thaw semen quality and fertility. many of these studies investigate the addition of antioxidants, like vitamin e and glutathione. there is a growing amount of evidence for a role of reactive oxygen species (ros) and oxidative stress in the promotion of cellular damage that occurs during cryopreservation and thawing. addition of antioxidants to semen extenders, as well as oral supplementation of antibiotics has been demonstrated to show improvements in semen quality for humans and food animal species. the reports for equine semen are variable, with possibly the same number of manuscripts reporting a positive effect as those that report no impact at all. nonetheless, it is an active avenue for research. it is important to remember that no one extender is going to provide an optimal result in every stallion. each stallion is an individual and they likely have their own preference for a particular brand or extender formulation or freezing protocol. this is likely related to differences in their sperm membrane composition, components of their seminal plasma and their inherent ability to withstand cellular stress. the best way to determine the right protocol for your stallion is to perform a split-ejaculate test freeze with a selection of available extenders. improved accuracy in determination of cell concentration most methods for determination of cell concentration are indirect and rely upon densitometry or photometry. the main disadvantage of these methods is that any contaminating debris in the ejaculate clinical theriogenology • volume 8, number 4 • december 2016 466 contributes to the sperm count. in contrast, for sperm counting with the nucleocounter sp-100 (chemometec, denmark) semen is diluted in a detergent solution and then loaded into a cassette through a microfluidic network containing the dna stain, propidium iodide. internal fluorescence detection by the software enables measurement of an accurate sperm count, as only the sperm heads are fluorescently labeled by the propidium idodide. consequently, the sperm count obtained by the nucleocounter may be 15-20% less than a traditional densimeter, because of the exclusion of debris. in some stallions known to have a lot of background debris in their ejaculates, we have observed the count on the nucleocounter to be almost 50% less than the densimeter. this means that breeding doses can be more accurately proportioned. despite being more expensive, the nucleocounter sp-100 has been utilized by institutions, veterinary clinics and breeding farms alike throughout the equine industry for the reliable, repeatable and accurate determination of sperm cell concentration. other advantages are that it is quick and easy to use, and that it can be used to measure sperm concentration when semen has been diluted into extender. stallion selection in the dairy industry young bulls are fertility tested by inseminating thousands of cows with their frozen semen to establish the level of fertility that can be expected before the frozen semen is sold on the commercial market. furthermore, the difference between a low fertility bull and a high fertility bull due to genetic selection for fertility and “freezability” over many decades is just a few percentage points per cycle. this is vastly different from the situation for stallions where most will never breed “thousands” of mares over the course of a lifetime in order to demonstrate a reliable estimate of expected fertility, let alone before the semen is marketed. also as pleasure and competition animals the selection for fertility is not as relevant to equines as it is for food animals, instead the temperament, pedigree or performance of an individual is more likely to influence his selection as a breeding stallion. nonetheless, there is a growing research interest focused at developing better tools for the prediction of fertility in a breeding stallion or for success in a frozen semen breeding program. we have limited assays available to measure semen quality in the average semen processing laboratory-primarily sperm motility, viability and morphology. sperm motility is the sperm attribute most readily applied for the assessment of semen quality, and industry recommended minimums for a breeding dose of cooled semen (500 million progressively motile sperm (pms)/dose at 24hr) or frozen semen (200 million pms/dose and a minimum of 30% post-thaw progressive motility) are based upon the number of pms in the breeding dose. however, motility by itself is an indicator of relative cell health but fertilization is a complex process that requires numerous functional attributes of both sperm and egg. there may be dysfunction of one of these attributes that renders the sperm incapable of fertilization even in the presence of “acceptable” motility. it is unlikely that we will have the capability in the near future to measure all the components a sperm cell needs in order to effect fertilization, however access to better markers of sperm function, those that actually correlate with fertility, would be advantageous. flow cytometry offers a wider range of semen analysis options, and the general practitioner can usually access these tools through university laboratries. one such test is the sperm chromatin structure assay (scsa) that can detect sperm samples that have a high degree of dna fragmentation (small breaks in the sperm chromosomes). other flow cytometry tests available include assessment of sperm mitochondrial function, capacitation and the acrosome reaction, membrane fluidity and integrity, apoptosis, and measures of ros production, oxidative stress and lipid peroxidation; although the application of many of these tests to select for or predict fertility has still to be determined. the scsa is one of the few sperm analysis tools that has actually been correlated to fertility in humans, bulls, stallions and other species. genetic markers for fertility with the completion of the equine genome sequence in 2009 we now have new tools to determine the genetic components of complex conditions like disease resistance, athletic performance, or potentially fertility. genetic screening panels are already available for the identification of multiple genetic disorders, for several coat color traits, and there are even genetic tests related to gene markers for speed clinical theriogenology • volume 8, number 4 • december 2016467 and gait. genetic mechanisms have been determined in other species to be related to the variability in fertility traits of males and females, and genetic markers for components of seminal plasma and semen quality traits proposed as predictors of fertility. discovery of genetic markers impacting fertility in the stallion could be applied immediately for genotype assisted selection of breeding stallions. one promising example are the genes coding for equine cysteine-rich secretory proteins (crisps); previous studies have shown that crisps play a role in the fertilizing ability of male animals. crisp-3 has been proposed as a potential marker for freezability in stallions, and a polymorphism in the crisp-3 gene has been reported to correlate with fertility in hanoverian stallions. sources of variability and limitations despite many advances in semen processing techniques there are still many potential sources of variability that may influence the result when it comes to obtaining a pregnancy, aside from the inherent fertility of the mare or stallion. although new tools and techniques offer many advantages, they are only as accurate or successful as the technician that is using them. the skill and experience of the clinician processing the semen or breeding the mare can ultimately impact the potential fertility of any breeding. for example, when it comes to semen quality at the time of insemination many factors may have come in to play during the collection, processing and packaging of the breeding dose including: ‐ stallion collection frequency and the number of mares booked to the stallion that day may influence the number of sperm in the breeding dose. ‐ the accuracy of preparation of that breeding dose may be influenced by errors in the determination of sperm concentration, measurement of volume or insufficient mixing of the semen prior to dividing up the breeding doses. ‐ the semen quality may be negatively impacted by inappropriate semen collection and handling techniques, from temperature deviations to poor hygiene practices. ‐ the semen may not have been packaged appropriately in the shipping container, the shipping container may not hold temperature as it should, or it may have been tampered with or compromised during shipment. no single advancement in semen handling protocols is going to guarantee fertility when so many potential variables exist to impact the final result. therefore, a clinician must educate their clients about these sources of variability and encourage them to purchase semen or breedings from reputable breeding farms or processing facilities with demonstrated experience and success. one of the many challenges within the equine breeding industry is that there are very few regulations concerning the collection, processing, distribution and insemination of stallion semen in the us. any owner who can manage to extract semen from his or her stallion can without any prior training, experience, certification or license, sell semen from that stallion without restriction of any kind. there are also no regulations concerning standards for semen quality, e.g., the number of sperm in a dose, or minimum recommended values for motility or morphology, or for the health status of the stallion. the world breeding federation for sport horses proposed semen standards several years ago, and all registries (and thus their members) that are part of the wbfsh are encouraged to adopt these standards. member laboratories of the select breeders affiliate laboratory network have also agreed to follow a set of self-regulated guidelines with respect to quality standards for frozen semen. the american association of equine practitioners (aaep) has published “biosecurity guidelines for control of venereally transmitted diseases” that provides recommendations for equine viral arteritis and other potential venereal diseases. whether controls should be put in place to regulate semen collection and processing facilities in the us is a topic beyond the scope of this review, nonetheless it is mentioned here simply as a possible means to improve reproductive outcomes. progress is inevitable and the future looks bright, particularly in the field of genetics and in the burgeoning tools available for analysis of sperm function. it is likely the next decade will produce amazing new technologies that will benefit the equine breeding industry. clinical theriogenology • volume 8, number 4 • december 2016 468 suggested reading aitken rj, de iuliis gn, gibb z, et al: the simmet lecture: new horizons on an old landscape -oxidative stress, dna damage and apoptosis in the male germ line. reprod domest anim 2012;47 suppl 4:7-14. alvarenga ma, papa fo, landim-alvarenga fc, et al: amides as cryoprotectants for freezing stallion semen: a review. anim reprod sci 2005;89:105-113. anzar m, kroetsch t, buhr mm: comparison of different methods for assessment of sperm concentration and membrane integrity with bull semen. j androl 2009;30:661-668. aurich c: recent advances in cooled-semen technology. anim reprod sci 2008;107:268-275. ball ba: oxidative stress, osmotic stress and apoptosis: impacts on sperm function and preservation in the horse. anim reprod sci 2008;107:257-267. barrier battut i, kempfer a, becker j, et al: development of a new fertility prediction model for stallion semen, including flow cytometry. theriogenology 2016: epub ahead of print. batellier f, gérard n, courtens jl, et al: preservation of stallion sperm quality by native phosphocaseinate: a direct or indirect effect? j reprod fertil suppl 2000;56:69-77. batellier f, magistrini m, fauquant j, et al: effect of milk fractions on survival of equine spermatozoa. theriogenology 1997;48:391-410. bliss sb, voge jl, hayden ss, et al: the impact of cushioned centrifugation protocols on semen quality of stallions. theriogenology 2012;77:1232-1239. carmo mt, papa fo, medeiros as, et al: improvement of stallion semen post-thaw motility with the association dimethyl formamide and methyl formamide as cryoprotectors. anim reprod sci 2005;89:286-288. giesecke k, sieme h, distl o: infertility and candidate gene markers for fertility in stallions: a review. vet j 2010;185:265-271. hamann h, jude r, sieme h, et al: a polymorphism within the equine crisp3 gene is associated with stallion fertility in hanoverian warmblood horses. anim genet 2007;38:259-264. hansen c, vermeiden t, vermeiden jp, et al: comparison of facscount af system, improved neubauer hemocytometer, corning 254 photometer, spermvision, ultimate and nucleocounter sp-100 for determination of sperm concentration of boar semen. theriogenology 2006;66:2188-2194. jobim mi, trein c, zirkler h, et al: two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their relation with semen freezability. theriogenology 2011;76:765-771. loomis pr: advanced methods for handling and preparation of stallion semen. vet clin north am equine pract 2006;22:663667. leeb t, sieme h, töpfer-petersen e: genetic markers for stallion fertility lessons from humans and mice. anim reprod sci 2005;89:21-29. morrell jm, richter j, martinsson g, et al: pregnancy rates after artificial insemination with cooled stallion spermatozoa either with or without single layer centrifugation. theriogenology 2014;82:1102-1105. morrell jm, klein c, lundeheim n, et al: removal of bacteria from stallion semen by colloid centrifugation. anim reprod sci 2014;145:47-53. morrell jm, timoney p, klein c, et al: single-layer centrifugation reduces equine arteritis virus titre in the semen of shedding stallions. reprod domest anim 2013;48:604-612. morrell jm, pihl j, dalin am, et al: restoration of seminal plasma to stallion spermatozoa selected by colloid centrifugation increases sperm progressive motility but is detrimental to chromatin integrity. theriogenology 2012;78:345-352. pagl r, aurich je, müller-schlösser fet al: comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 degrees c. theriogenology 2006;66:1115-1122. pagl r, aurich c, kankofer m: anti-oxidative status and semen quality during cooled storage in stallions. j vet med a physiol pathol clin med 2006;53:486-489. papa fo, felício gb, melo-oña cm, et al: replacing egg yolk with soybean lecithin in the cryopreservation of stallion semen. anim reprod sci 2011;129:73-77. peña fj, ortega ferrusola c, martín muñoz p: new flow cytometry approaches in equine andrology. theriogenology 2016;86:366-372. peña fj, plaza davila m, ball ba, et al: the impact of reproductive technologies on stallion mitochondrial function. reprod domest anim 2015;50:529-537. ramires neto c, monteiro ga, soares rf, et al: new seminal plasma removal method for freezing stallion semen. theriogenology 2013;79:1120-1123. roach j, schnobrich m, ellerbrock r, et al: comparison of cushioned centrifugation and spermfilter filtration on longevity and morphology of cooled-stored equine semen. vet rec 2016;178:241. squires el: integration of future biotechnologies into the equine industry. anim reprod sci 2005;89:187-198. stuhtmann g, oldenhof h, peters p, et al: iodixanol density gradient centrifugation for selecting stallion sperm for cold storage and cryopreservation. anim reprod sci 2012;133:184-190. töpfer-petersen e, ekhlasi-hundrieser m, kirchhoff c, et al: the role of stallion seminal proteins in fertilisation. anim reprod sci 2005;89:159-170. waite ja, love cc, brinsko sp, et al: factors impacting equine sperm recovery rate and quality following cushioned centrifugation. theriogenology 2008;70:704-714. clinical theriogenology • volume 8, number 4 • december 2016469 clinical theriogenology • volume 8, number 4 • december 2016 470 omniblank: 2011: synchronization of estrus of whitetail deer and other select cervids, sheep & goats   synchronization of estrus of whitetail deer and other select cervids, sheep & goats manoel tamassia northstar vets, robbinsville, nj abstract estrus synchronization in small ruminants is achieved by controlling the luteal phase of the estrous cycle during the breeding season or by inducing premature heat cycles during the anovulatory season. anestous animals can be induced into cycling with hormonal treatment or manipulation of the environment. the most commonly used protocol for synchronization of heat in small ruminants involves the use of controlled internal drug release (cidr) devices, intravaginal sponge impregnated with progestagens, equine chorionic gonadotropin (ecg), and prostaglandin. prostaglandin based protocols are used during the breeding season. the sudden introduction of a male can also be used to induce and synchronize fertile heat in sheep and goats. estrus induction and synchronization in deer use similar techniques. this article reviews some successful protocols used to induce heat, synchronize estrus, induce ovulation, and enhance ovulation in small ruminants and deer. keywords: estrus, synchronization, reproduction, small ruminants, cervids the recent increase in interest in small ruminants and the high value of deer has raised the demand for veterinary reproductive services for those species. the development of synchronization protocols has allowed other advanced reproductive techniques such as artificial insemination (ai) and embryo transfer to flourish making valuable genetic material more easily available. producers are requesting reproductive services that involve intensive reproductive management including synchronization of estrus, off season breeding, ai and embryo transfer. this paper will review available tools that can be used to improve reproductive efficiency in small ruminants and deer. estrus induction protocols have the objective of synchronizing heat, advancing the breeding season and both induce and maintain off season cyclic activity. estrus synchronization techniques in small ruminants must not only establish a tight synchrony, but also provide an acceptable level of fertility upon ai or natural mating during the natural mating season and also the non-breeding season. small ruminants and cervids are seasonal polyestrous breeders with reproductive activity centered during the fall and winter. the reproduction of these species is controlled by the variations in daylight (long vs. short days). the effect of light on reproduction is influenced by the number of hours of light and by the time of exposure. short day breeders can be induced into cycling by reducing the exposure to light (confinement inside a dark building) or by the administration of melatonin implants.1 progesterone implants can also be used for induction of fertile heat out of the breeding season2,3 by mimicking the function of the corpus luteum (cl). the male effect the sudden introduction of a male or androgen-treated castrated male can induce and synchronize heat in anestrous ewes and does.4,5 this is known as the “male effect”. this synchronization is less predictable than that achieved by hormone treatment but is economical and fertility rates are high. the effects are mediated through changes in pulsatile gonadotropin releasing hormone (gnrh) release followed by an increase in luteinizing hormone (lh). this will cause ovulation (silent heat) in two to four days that is characterized by low fertility and premature regression of the cl. the second ovulation (five to seven days later) is of normal fertility and followed by a normal luteal phase. olfactory response may be a leading stimulus for estrus induction but other factors like tactile, auditory and visual stimuli are also involved. in sheep, preventing the vomeronasal organ from functioning does not affect the female responses to male odor suggesting that the accessory olfactory system does not play a major role in estrus induction. female responses also seem to depend on previous experience. the responsible pheromone is present in wool and buck hair clippings but not in urine and it is not associated with the buck odor during the breeding season.6 clinical theriogenology • volume 3 number 4 • december 2011531   the male effect can be used successfully in estrus synchronization programs that use exogenous progestins with or without the use of ecg. the introduction of a male in association with estrus induction protocols using progesterone will increase the number of females bred/marked. the addition of exogenous progesterone at the time of male introduction can reduce significantly the number of short estrous cycles and extended the period from male induction to ovulation in sheep increasing ovulation rate in goats.4 the uterus is also implicated in the premature regression of the cl after the male effect as both hysterectomy and inhibitors of prostaglandin f2α suppressed short cycles.4 estrus synchronization during the breeding season estrus synchronization is easily accomplished during the breeding season. the opportunity for control is greater during the luteal phase, which is longer and more responsive to manipulation than the follicular phase. synchronization protocols during the natural breeding season are based on either the prolongation or shortening of the progesterone phase of the cycle. prolongation of the cycle is usually accomplished by supplying exogenous progesterone with the use of vaginal implants or oral progesterone. alternatively, the cycle can be cut short prematurely by regressing existing cls with the use of prostaglandin.7 use of prostaglandin for estrus synchronization a cl must be present in the ovary for prostaglandin-based protocols to work. the control of the estrous cycle with prostaglandin is achieved by termination of the luteal phase through regression of the cl. natural prostaglandin and its analogs can be used alone or in combination with progesterone or male-effect for estrus synchronization during the breeding season. a typical protocol for synchronization with prostaglandin is to give two injections of prostaglandin nine days apart for sheep and eleven days apart for goats. the typical dose for dinoprost tromethamine is 10-15 mg im and for cloprostenol the commonly used dose is 50-125 mcg im. ewes should be bred 10-12 hours after being marked by teaser ram (around 42-54 hours after last injection).8 does should be in heat and ready to be inseminated at 4852 hours after the last treatment with prostaglandin.9,10 prostaglandins are not expensive and provide an economical method of shortening the breeding season in natural mating situations. the addition of progesterone and ecg (250-500 iu im) to prostaglandin treatments shortens the interval to heat when compared with females not receiving these supplemental drugs. breeding should be adjusted to this variation and the use of teaser males is strongly recommended. the ovsynch protocol, similar to the program used in cattle, has proven to be successful in inducing synchronized fertile heat in does. it allows fixed-time insemination in goats during the breeding season. the typical protocol involves the injection of gnrh (50-100 mcg im; considered day zero) followed by a luteolytic dose of prostaglandin (e.g. 125 mcg of cloprostenol im) on day seven and another dose of gnrh on day nine. males may be introduced and breeding allowed take place on day eight. breeding by ai is performed 16 hours after the second gnrh dose.11 estrus induction with progesterone impregnated sponges or cidrs is done by placing the progesterone carrying device in the cranial vagina of the doe for 12 days. on day 10 the animals are injected with a luteolytic dose of prostaglandin and receive 250-500 iu of ecg. artificial insemination is performed 42-46 hours after sponge removal. in sheep the sponge or cidr is left in place for 14 days and ecg is given at progesterone removal. animals should be exposed to fertile males after progesterone withdrawal.10 estrus induction and synchronization outside the breeding season several methods have been used to induce and synchronize heat during the off season. the male effect, artificial lighting (to stimulate decreasing day length) and melatonin are successful in inducing heat in anestrous females. these techniques do not provide tight synchrony, but they permit out of season breeding, are readily available and economical. prostaglandin-based treatments do not work in noncycling animals (off season) and are not recommended. the most effective treatments are those using progesterone releasing devices in conjunction with gonadotropin. the use of the male effect in clinical theriogenology • volume 3 number 4 • december 2011 532   conjunction with these protocols will affect the time to ovulation and may increase the ovulation rate in goats. this effect needs to be taken into consideration if breeding is done by timed ai.4,12 melatonin implants mimic short days and are used to induce out of season cycles. they are commercialized in europe under the trade names of regulin® and melovine®. they are used after at least two months of increasing daylight and will advance the breeding season by 30-45 days. it increases the ovulation rate and prolificacy by as much as 25%. melatonin treatment can be used at any time during the anestrous season in breeds less influenced by season. during seasonal anestrous the average ewe/doe is not cycling and serum progesterone is less than 1 ng/ml with negligible ovarian activity. progesterone is the natural hormone that sustains pregnancy and is produced by the cl in the ovary following ovulation. artificially introduced progesterone will mimic pregnancy and will keep the animal in artificial anestrous, preventing ovulation and keeping the female out of heat. when the progesterone source is suddenly removed, the animal will ovulate within a very predictable period even during seasonal anestrous. the effectiveness of a progesterone to serve as a synchronization agent is related to its ability to delay estrus and ovulation and is influenced by the route of administration, duration of treatment and the speed of withdrawal. alternatively, hormones may be used in various combinations and dosages to induce follicle development and ovulation. a typical protocol involves the use of a progesterone releasing device (e.g., vaginal sponge impregnated with 45 mg of fluorogestone acetate) that is left in place for 11 days and ecg (250-500 iu) 48 hours prior to sponge removal. insemination with frozen-thawed semen should be performed 24 h after the onset of estrus.13 several studies have been conducted to evaluate the usefulness of a combination of human chorionic gonadotropin (hcg) and ecg (pg 600, intervet/schering-plough animal health, summit, nj) as a source of ecg. pg 600 is the only veterinary-grade source of ecg readily available in the us and is marketed to induce cycling in prepubertal gilts. the product is a mixture of chorionic gonadotropins with 200 iu of hcg and 400 iu of ecg/5 ml. the typical extra-label use is based on the amount of ecg that is desired.14-17 lambing rates of 36 to 59% have been reported when pg 600 was used to replace ecg. time to estrus and ovulation in ewes treated with pg 600 in conjunction with intravaginal sponge is 47 h (24-60 h) and 83 h (60-112 h), respectively15. the typical dose is 400 iu of ecg. superovulation has been reported when1200 iu of ecg was used. both ecg and pg 600 have the potential to induce the formation of ovarian cysts.18 the oral progestagen melengestrol acetate (mga) has also been used to synchronize heat and induce cyclicity outside the natural breeding season. melengestrol acetate can be used in combination with gonadotropins, male effect and prostaglandin (during the breeding season) and is fed in doses of 0.125 to 0.3mg/ewe/d for at least seven days prior to introduction of fertile rams. treatment with mga increases the percentage of ewes conceiving early in the breeding period and lambing rates.3,17 estrus synchronization in deer it is unwise to try to group all cervids for the purpose of estrus synchronization. there is no “typical” deer which we could use as an example for the purpose of our discussion in this paper. we strongly encourage the reader to search specific information for the species and subspecies that you will be working with. however, some similarities will be used to describe the procedures being used currently to induce and synchronize estrus in deer. cervids are represented by over 40 species with 200 subspecies. they are characterized by a huge diversity in morphology, physiology, ecology and geographic distribution. most cervid species give birth at the time of the year that provides the best chance for the survival and growth of offspring. this adaptation to the natural environment has exerted considerable influence on their reproductive physiology. species adapted to the northern temperate or arctic regions typically conceive in autumn and calve in the following summer. species located in the tropics often exhibit limited seasonality or are completely aseasonal. it is accepted that seasonality is affected by endogenous recognition of photoperiodic changes with the majority of species cycling during decreasing day length (short day breeders).19 clinical theriogenology • volume 3 number 4 • december 2011533   for best results and survival of the offspring breading should follow the natural cycle. the doe’s first ovulation of the year is characterized by silent ovulations and short-lived (8-10 days) cl. a true first cycle follows this first ovulation. males should be placed with females the as they will continue to cycle until pregnant. farmers may require buck removal to avoid late breeding in the season. these decisions are made based on management and financial considerations and not on the physiological potential of the specie. estrus in the white-tailed doe lasts approximately 24 hours and, if not bred, she will cycle again in 21-29 days. female deer do not show overt signs of receptivity when in heat in the absence of a male, challenging the producer’s task of keeping accurate reproductive records. controlled breeding is possible with the best time to breed being between 18 and 36 hours after the beginning of a standing heat. due to these challenges, the best chance to have successful breeding with ai is to breed by appointment. in many ways, the cervid estrous cycle is similar to that of domestic ruminant species. thus, artificial estrus synchronization is done in a similar way as it is performed is small ruminants and cattle. drugs used for manipulation of the reproductive cycle in small ruminants and cattle can be used in a similar manner in deer. heat synchronization is done using prostaglandin, gnrh, gonadotropins, mga, vaginal sponges, cidrs, or ear implants as described above.20 prostaglandin is effective if used during the breeding season when cls are present and active in the ovaries. cloprostenol is used at 125-250 mcg/doe im at cidr removal after 8-12 days or it is given as two injections 11 days apart.21 timed ai should be done following proven protocols. the most reliable protocols rely on the use of an exogenous source of progesterone in the form of a cidr or progesterone impregnated sponge. the amount of progesterone and the size of the cidr are chosen based on the species and size of the doe. for larger species the cidr-b (1.9 g progesterone) is utilized, whereas the cidr-g (0.33 g progesterone) is used for smaller deer. the time of exposure to the progesterone delivering device also varies depending on the species. the device is left in place between 8 and 14 days and then removed manually.21-25 equine chorionic gonadotropin is often used at cidr removal with the final dose depending on the species. it provides for better synchronization of estrus and ovulation (see table). higher doses may result in superovulation and excessive number of fetuses. conclusion the ultimate aim of any estrus synchronization method is to allow ai at a predetermined time after the end of treatment. this requires a very tight synchronization of estrus which can be achieved after careful protocol selection and taking into consideration herd composition, location, level of management, health and nutrition of each individual farm operation. table: recommendations for estrus synchronization in various deer species22 species progestin device cidr duration ecg dose (i.u.) time for ai after cidr removal wapiti cidr-b 12–14 days 190–200 60–66 hr red deer 1 or 2 cidr-g 10–12 days 150–200 56–60 hr sika deer cidr-g 12–14 days 50 58–62 hr fallow deer cidr-g 12–14 days 0 63–67 hr white-tailed deer cidr-g 12–14 days 100–150 60–65 hr references 1. chemineau p, malpaux b: [melatonin and reproduction in domestic farm animals]. therapie 1998;53:445-452. 2. iida k, kobayashi n, kohno h, et al: a comparative study of induction of estrus and ovulation by three different intravaginal devices in ewes during the non-breeding season. j reprod dev 2004;50:63-69. 3. daniel ja, sterle sw, mcfadin-buff el, et al: breeding ewes out-of-season using melengestrol acetate, one injection of progesterone, or a controlled internal drug releasing device. theriogenology 2001;56:105-110. 4. chemineau p, pellicer-rubio mt, lassoued n, et al: male-induced short oestrous and ovarian cycles in sheep and goats: a working hypothesis. reprod nutr dev 2006;46:417-429. clinical theriogenology • volume 3 number 4 • december 2011 534   5. veliz fg, moreno s, duarte g, et al: male effect in seasonally anovulatory lactating goats depends on the presence of sexually active bucks, but not estrous females. anim reprod sci 2002;72:197-207. 6. gelez h, fabre-nys c: the "male effect" in sheep and goats: a review of the respective roles of the two olfactory systems. horm behav 2004;46:257-271. 7. chemineau p, gauthier d, poirier jc, et al: plasma levels of lh, fsh, prolactin, oestradiol-17beta and progesterone during natural and induced oestrus in the dairy goat. theriogenology 1982;17:313-323. 8. olivera-muzante j, fierro s, lopez v, et al: comparison of prostaglandinand progesterone-based protocols for timed artificial insemination in sheep. theriogenology;75:1232-1238. 9. lopez-sebastian a, gonzalez-bulnes a, carrizosa ja, et al: new estrus synchronization and artificial insemination protocol for goats based on male exposure, progesterone and cloprostenol during the non-breeding season. theriogenology 2007;68:1081-1087. 10. martemucci g, d'alessandro ag: synchronization of oestrus and ovulation by short time combined fga, pgf(2alpha), gnrh, ecg treatments for natural service or ai fixed-time. anim reprod sci;123:32-39. 11. holtz w, sohnrey b, gerland m, et al: ovsynch synchronization and fixed-time insemination in goats. theriogenology 2008;69:785-792. 12. lassoued n, khaldi g, cognie y, et al: [effect of progesterone on ovulation length and duration of the ovarian cycle induced by the male effect in the barbarine ewe and the local tunisian goat]. reprod nutr dev 1995;35:415-426. 13. freitas vj, baril g, saumande j: estrus synchronization in dairy goats: use of fluorogestone acetate vaginal sponges or norgestomet ear implants. anim reprod sci 1997;46:237-244. 14. windorski ej, schauer cs, wurst ak, et al: effects of melengestrol acetate and p.g. 600 on fertility in rambouillet ewes outside the natural breeding season. theriogenology 2008;70:227-232. 15. cline ma, ralston jn, seals rc, et al: intervals from norgestomet withdrawal and injection of equine chorionic gonadotropin or p.g. 600 to estrus and ovulation in ewes. j anim sci 2001;79:589-594. 16. safranski tj, lamberson wr, keisler dh: use of melengestrol acetate and gonadotropins to induce fertile estrus in seasonally anestrous ewes. j anim sci 1992;70:2935-2941. 17. jabbar g, umberger sh, lewis gs: melengestrol acetate and norgestomet for the induction of synchronized estrus in seasonally anovular ewes. j anim sci 1994;72:3049-3054. 18. christman sa, bailey mt, head wa, et al: induction of ovarian cystic follicles in sheep. domest anim endocrinol 2000;19:133-146. 19. asher gw: reproductive cycles of deer. anim reprod sci 2011;124:170-175. 20. umapathy g, sontakke sd, reddy a, et al: seasonal variations in semen characteristics, semen cryopreservation, estrus synchronization, and successful artificial insemination in the spotted deer (axis axis). theriogenology 2007;67:1371-1378. 21. zanetti edos s, polegato bf, duarte jm: comparison of two methods of synchronization of estrus in brown brocket deer (mazama gouazoubira). anim reprod sci 2010;117:266-274. 22. plante c, pollard j, bringans mj, et al: estrous synchronization and artificial insemination in domesticated cervids. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: wb saunders; 2007. p. 987. 23. osborn da, gassett jw, miller kv, et al: out-of-season breeding of captive white-tailed deer. theriogenology 2000;54:611-619. 24. hosack da, miller kv, ware lh, et al. stag exposure advances the lh surge and behavioral estrus in eld's deer hinds after cidr device synchronization of estrus. theriogenology 1999;51:1333-1342. 25. morrow cj, asher gw, macmillan kl: oestrous synchronisation in farmed fallow deer (dama dama): effects of season, treatment duration and the male on the efficacy of the intravaginal cidr device. anim reprod sci 1995;37:159-174. clinical theriogenology • volume 3 number 4 • december 2011535 clinical theriogenology • volume 3 number 4 • december 2011 536 omniblank: 2013: update on antimicrobial therapy in mare reproduction update on antimicrobial therapy in mare reproduction kristina g. lu hagyard equine medical institute, lexington, ky bacterial uterine infections are associated with significant time and monetary loss in the equine breeding industry. the incidence of identified bacterial uterine infection is estimated at 25 to 60% of barren mares.1 treatment and resolution of mare urogenital infection involves a holistic approach which must include consideration of the systemic health of the mare, correction of urogenital anatomic defects, enhancement of uterine clearance in non-pregnant mares, identification of specific infections, and appropriate antimicrobial selection and administration. the following is a brief review of antimicrobial properties and classes, and discussion regarding antimicrobial selection for use in specific mare reproductive disease states. excellent additional resources are available.2 in this age of increasing bacterial and fungal antimicrobial resistance and heightened awareness of antimicrobial use in animals, judicious and wise employment of antimicrobials should be emphasized. keywords: antibiotics, antifungals, chelators, endometritis, placentitis general antimicrobial properties bactericidal antimicrobials a) time dependent (there is no benefit in increasing the drug concentration above the minimum inhibitory concentration (mic); instead the time the drug concentration is above mic is most important); examples are beta-lactams and trimethoprim-sulfonamide b) concentration dependent (rate of bacterial killing increases as the peak concentration increases above the pathogen’s mic rather than the duration of the drug’s concentration); examples are aminoglycosides, fluoroquinolones, metronidazole, and peptides bacteriostatic antimicrobials concentration dependent; examples are macrolides, tetracyclines, and chloramphenicol antimicrobial classes beta-lactam: penicillin commonly used formulations are procaine penicillin, aqueous penicillin salts, ampicillin, ticarcillin; those less commonly used in equine reproduction include carbapenems (imipenem, meropenem) mechanism of action. beta-lactams are time-dependent antimicrobials that interfere with cell wall formation by inhibiting the penicillin-binding proteins that catalyze polymer cross-linking necessary for cell wall formation. beta-lactams are bactericidal for replicating cells, thus clinical benefit is reduced when used concurrently with a bacteriostatic drug. penicillins are generally most effective against grampositive organisms, however they vary in efficacy against gram-negative organisms. differences in organism susceptibility are attributable to differences in penicillin-binding protein receptor sites, the amount of peptidoglycan present in the cell wall, drug penetration of the outer cell membrane of gramnegative bacteria, and beta-lactamase-induced resistance. distribution. penicillins are well distributed throughout the body, reaching mic in most organs including the reproductive tract. they are ionized, poorly lipid soluble, and tend to cross biological membranes poorly. they are excreted almost entirely by the kidney and thus can be useful to treat urinary tract infections caused by sensitive organisms. 503 clinical theriogenology • volume 5, number 4 • december 2013 synergism. beta lactams are synergistic with aminoglycosides evaluation of endometrial ampicillin concentration 24 hours after a three-gram intrauterine infusion of ampicillin revealed drug concentrations above mic for investigated isolates 24 hours after infusion.3 intrauterine infusion of 6.2-g ticarcillin/clavulanic acid resulted in endometrial tissue concentrations of 150-424 micrograms/g 60 minutes after infusion. clearance is rapid, potentially necessitating frequent administration to maintain concentrations.4 systemically administered potassium penicillin achieved mic for streptococcus equi subspecies zooepidemicus (hereafter s. zooepidemicus) in allantoic fluid.5 penicillins are also used for treatment of leptospirosis. addition of timentin® (ticarcillin plus clavulanic acid) to inra96® semen extender to a final concentration of 0.5 to 1.5 mg/ml can limit the growth of taylorella equigenitalis.6 addition of timentin® to semen extender (1 mg/ml) did not impair sperm quality but did not provide added protection against bacteria commonly found in equine semen.7 beta-lactams: cephalosporins commonly used formulations are ceftiofur sodium, ceftiofur crystalline free acid, ceftiofur hydrochloride mechanism of action: cephalosporins are also beta-lactam antimicrobials, however the molecular structure of the cephalosporin group of antimicrobials make them inherently more resistant to betalactamases. cephalosporins are grouped in their order of development. ceftiofur is a third generation cephalosporin in common usage in equine medicine. ceftiofur is rapidly metabolized by plasma esterases to the active metabolite, desfuroylceftiofur. distribution. widespread distribution into tissues in extracellular fluid is achieved however passage across membranes and physiological barriers is poor. excretion is largely through the urinary tract. uses in reproduction. in mares administered ceftiofur sodium (2 mg/kg im bid), ceftiofur sodium was not detected in endometrial tissue after the fifth dose when steady state concentrations were achieved.8 in mares administered ceftiofur hydrochloride (2.2 mg/kg im), a ceftiofur derivative was detected in endometrial tissue 24 hours after administration at concentrations above reported mic for s. zooepidemicus and escherichia coli.9 in mares administered ceftiofur crystalline free acid (6.6 mg/kg im), endometrial drug concentrations remained above mic for s. zooepidemicus for 96 hours.10 in a study evaluating efficacy of ceftiofur crystalline free acid in equine placentitis, the drug was not found in effective concentrations in the placenta, fetal tissue, amnionic fluid or fetal serum and did not improve foal survival rates.11 intrauterine ceftiofur sodium (1 g in 100 ml saline) has been found to be safe and effective.12 beta-lactamase inhibitors clavulanic acid, a broad-spectrum beta-lactamase inhibitor, is used in combination with other antimicrobials such as ticarcillin. certain bacteria can resist the action of beta-lactam antimicrobials by producing beta-lactamases, enzymes that degrade beta-lactam antimicrobials by opening the beta-lactam ring. genes encoding beta-lactamase are transmitted through bacterial populations with plasmids and transposons as an important mechanism of spreading resistance. beta-lactamase inhibitors bind irreversibly to beta-lactamases allowing the accompanying beta-lactam antibiotic to bind to the penicillin binding protein. peptide antibiotics a commonly used formulationis polymyxin b 504clinical theriogenology • volume 5, number 4 • december 2013 mechanism of action. polymyxins are peptide antibiotic products of bacillus polymyxa. polymyxin b is toxic at systemic antimicrobial doses with nephrotoxic, neurotoxic, and neuromuscular blocking effects, thus it is used topically for its antimicrobial properties and used systemically at subantimicrobial doses for binding and inactivating endotoxin (lipopolysaccharide). when used as an antimicrobial, polymyxin b is bactericidal and concentration-dependent. this cationic peptide disrupts the outer membrane of gram-negative bacteria by binding lipopolysaccharide (lps), and increasing cell permeability. gram-positive bacteria are resistant. distribution. polymyxin diffuses poorly through biologic membranes and attains low concentrations in transcellular fluids. uses in reproduction. polymyxin b can be infused into the uterus for gram-negative bacterial infections and used systemically for endotoxemia as may be seen in post-foaling complications. macrolides macrolides (erythromycin, azithromycin, clarithromycin) are predominantly bacteriostatic antimicrobials that inhibit protein synthesis by reversibly binding to the 50s ribosomal subunit, and thus are specific for prokaryotic cells. in adult horses, macrolide use can be associated with severe diarrhea. macrolides are infrequently used in mare reproduction and will certainly be highlighted in discussion on antimicrobial use in foals and weanlings. aminoglycosides commonly used formulations are entamicin and amikacin; others include kanamycin, neomycin, streptomycin, dihydrostreptomycin, tobramycin. mechanism of action. aminoglycosides are bactericidal, concentration dependent antimicrobials used primarily to treat aerobic gram-negative bacteria and staphylococci. their bactericidal action is influenced by ph, being most active in alkaline ph. aminoglycosides must penetrate bacterial cell walls, with penetration enhanced when in combination with drugs that interfere with the cell wall (e.g., penicillins). once within the bacteria, they bind to the 30s ribosomal subunit (prokaryotic) and inhibit bacterial protein synthesis. the most clinically important resistance is due to enzymes from plasmids that are transferrable between bacteria. elimination is by renal excretion (glomerular filtration) and nephrotoxicity is an important side effect. risk factors for aminoglycoside toxicity include prolonged therapy (>7-10 days), multiple doses per day, acidosis, electrolyte disturbances, dehydration, concurrent nephrotoxic drug therapy, age, pre-existing renal disease, and elevated plasma trough concentrations. ototoxicity may also occur. amikacin has the broadest spectrum of the aminoglycosides, and is the least nephrotoxic. concurrent administration of gentamicin with phenylbutazone was found to decrease elimination half-life of gentamicin by 23% and decrease the volume of distribution by 26% while pharmacokinetics of phenylbutazone were not affected. aminoglycosides are pharmaceutically incompatible with many beta-lactams and should not be mixed in same syringe.2 distribution. aminoglycosides are large polycationic molecules that are poorly lipid soluble, with limited ability to enter cells and penetrate cellular barriers unaided. purulent or necrotic debris bind and inactivate aminoglycosides. synergism. aminoglycosides are synergistic with beta-lactams and trimethoprim-sulfonamide. antagonism. antagonism may occur with chloramphenicol, tetracycline, erythromycin; should not be mixed with penicillins in vitro.2 505 clinical theriogenology • volume 5, number 4 • december 2013 uses in reproduction. intravenous gentamicin (6.6 mg/kg iv sid) yielded concentrations in allantoic fluid above mic for potential gram-negative pathogens such as e. coli.5 amikacin is labeled for intrauterine use at a dose 2 g in 200 ml saline q 24 hrs for three consecutive days for endometritis, metritis, and pyometra in mares. gentamicin (2 g in 80 ml saline) intrauterine infusion has been associated with shorter duration histologic inflammation though more cellular changes were seen with scanning electron microscopy compared to saline controls.13,14 it is generally recommended that gentamicin be buffered or well diluted in saline to minimize irritation. tetracyclines commonly used formulations are oxytetracycline and doxycycline mechanism of action. tetracyclines are broad spectrum, bacteristatic antimicrobials that diffuse through the outer cell membrane to reversibly bind to the 30s ribosomal subunit to inhibit protein synthesis. they are strong chelating agents with the ability to chelate divalent and trivalent ions such as calcium, with potential to cause tooth discoloration. there is widespread acquired resistance particularly among gram-negative bacteria and variable susceptibility in many staphylococcus sp., streptococcus sp., enterococcus sp., e. coli, and klebsiella sp. resistance is rare in obligate intracellular pathogens such as ehrlichia sp. and anaplasma sp. doxycycline (semi-synthetic) is more lipid soluble than oxytetracycline. tetracyclines are relatively safe, with reported risks of enterocolitis, occasional fatal anaphylaxis, and collapse if intravenous administration is not slow. renal tubular damage may occur if outdated product used. distribution. tetracyclines are widely distributed. synergism. tetracyclines are synergistic with polymyxins by enhancing bacterial uptake uses in reproduction. after intragastric administration of doxycycline (five doses, 10 mg/kg bid), the endometrial concentration was approximately 1.3 micrograms/ml, above the mic for the evaluated strains of s. zooepidemicus and staphylococcus aureus.15 tetracyclines can be used systemically for treatment of leptospirosis. in a study evaluating the efficacy and safety of intrauterine oxytetracycline, six grams of oxytetracycline was infused daily for three days. endometrial oxytetracycline concentration remained above mic for eight hours after infusion for s. zooepidemicus, klebsiella pneumonia, pseudomonas aeruginosa, and e. coli, with observation of transient endometrial inflammation, and thus the recommendation for further evaluation prior to routine intrauterine use.16 chloramphenicol mechanism of action. chloramphenicol is a bacteriostatic, lipid soluble, neutral, broad spectrum antimicrobial. it irreversibly binds to the 50s subunit of bacterial ribosomes and also inhibits mitochondrial protein synthesis is mammalian bone marrow cells in a dose-dependent manner. resistance occurs via inactivation by chloramphenicol acetyltransferases (cats) shared via plasmids, transposons, and integrons. elimination occurs through the liver. chloramphenicol is associated with idiosyncratic aplastic anemia in humans and is restricted in some countries because of bacterial resistance and risk of aplastic anemia. distribution. chloramphenicol is widely distributed. uses in reproduction. anecdotally used for intrauterine infusion when proteinaceous debris is present (w. zent, personal comminication). systemic use is based on clinical necessity. sulfonamides 506clinical theriogenology • volume 5, number 4 • december 2013 commonly used formulations are sulfadiazine, sulfasalazine, and sulfamethoxazole mechanism of action. sulfonamides are bacteriostatic when used alone and bactericidal when combined with trimethoprim. they interfere with biosynthesis of folic acid in bacterial cells by competitively preventing para-aminobenzoic acid (paba) incorporation into folic acid molecule by competing with paba for dihydropteroate synthetase. bacteriostatic action depends on the requirement for folic acid; susceptible microorganisms must synthesize folic acid while mammalian cells use preformed folic acid. tissue exudate or necrotic tissue must be removed for penetration. bacterial resistance is extensive. elimination occurs through a combination of renal excretion and metabolic transformation. adverse side effects, though uncommon, may include urinary tract disturbances, hematopoietic disorders, dermatologic disorders, and keratoconjunctivitis sicca in dogs. distribution. sulfonamides are widely distributed. uses in reproduction. systemic administration of trimethoprim sulfamethoxazole (30 mg/kg po bid) in combination with pentoxifylline and altrenogest have been well evaluated in placentitis models (see below). diaminopyrimidines a commonly used formulation is trimethoprim in conjunction with sulfonamides. mechanism of action. trimethoprim interferes with folic acid production by inhibition of dihydrofolate reductase with greater affinity for bacterial versus mammalian enzymes, preventing synthesis of purines and thus dna. they are bacteriostatic when used alone. efficacy is decreased with plasmid associated resistance, and antagonism by tissue debris. fluoroquinolones commonly used formulations are enrofloxacin and ciprofloxacin. mechanism of action. fluoroquinolones are concentration dependent, bactericidal antimicrobials that bind dna-gyrase-dna complexes to impair negative supercoiling and target dna topoisomerase, disrupting replication. they have activity against a wide range of gram-negative bacteria, with ciprofloxacin being most potent against pseudomonas sp. the drug is concentrated within phagocytic cells and largely excreted in urine. protein synthesis inhibitors such as chloramphenicol and rna synthesis inhibitors such as rifampin may reduce efficacy. resistance is not thought to be plasmid associated. enrofloxacin inhibits cell proliferation, induces morphological changes and alters equine tendon structure, more pronounced in juvenile tendon cells. arthropathies have been documented in two-week old foals, though not in adults. fluoroquinolones are not recommended in pregnant animals, though they have been used in a pregnant mare with no known detrimental effect on the foal.2 transient neurologic signs have been seen with rapid administration. distribution. fluoroquinolones are widely distributed. synergism. fluoroquinolones are synergistic with beta-lactams, aminoglycosides, and vancomycin. antagonism. in vitro antagonism is seen between ciprofloxacin and chloramphenicol and ciprofloxacin and rifampin. 507 clinical theriogenology • volume 5, number 4 • december 2013 uses in reproduction. systemic enrofloxacin (5 mg/kg iv) achieved sufficient endometrial tissue concentrations to be used to treat endometritis caused by susceptible bacteria.17 enrofloxacin (5 mg/kg iv) has also been proposed as a therapeutic strategy for the prevention of endometritis is susceptible mares with a recommended dose (5 mg/kg iv) pre-breeding followed by two further doses 36 to 48 hours post-breeding.18 systemic ciprofloxacin (2.5 g po sid) and probenecid (1 g po sid) has been reported to effective against pseudomonas infection.19 intrauterine infusion of enrofloxacin has been evaluated with one author reporting enrofloxacin concentrations remaining above mic for susceptible bacteria 24 hours after infusion of 2.5 mg/kg enrofloxacin.20 a more recent study found endometrial biopsy grades worsening from kenney doig grade i to grade iii over a 60 day period following intrauterine infusion of 2.5 mg/kg enrofloxacin daily for three days and concluded that enrofloxacin is not suitable for conventional intrauterine infusion treatment in mares.21 nitroimidazole a commonly used formulation is metronidazole. mechanism of action. metronidazole undergoes reduction of the nitro group to yield unstable intermediates some of which interact with bacterial or protozoal dna; the reduction occurs under anaerobic conditions. resistance is rare among usually susceptible bacteria. anorexia can be seen with oral use. occasional neurological signs have been reported in other species with recovery facilitated by diazepam. distribution. metronidazole is rapidly and well absorbed and lipophilic. it is metabolized in the liver and eliminated in the urine and manure. hepatic metabolism may be decreased when concurrent with cimetidine, thus delaying elimination. uses in reproduction. uncommonly used unless an anaerobic infection in suspected. after systemic administration of metronidazole (15 mg/kg loading followed by 7.5 mg/kg q6h via nasogastric tube), the mean endometrial concentration of metronidazole was approximately 0.9 micrograms/ml.22 rifamycins a commonly used formulation is rifampin. mechanism of action. rifampin is bactericidal, broad spectrum, and active against both extracellular and intracellular pathogens. it inhibits dna-dependent rna polymerase. because of rapid development of resistance, rifampin is usually administered in conjunction with other antimicrobials. distribution. rifampin is very lipophilic and penetrates most tissues including abscesses, bone, milk and the central nervous system. rifampin crosses the placenta and is teratogenic in rodents. rifampin is eliminated via the liver. uses in reproduction. uncommonly used in reproduction but can be useful in reproductive tract associated abscesses (e.g. abscessed broad ligament hematomas or retroperitoneal abscesses). antifungals the main sites of action of antifungal drugs are the cytoplasmic membrane (polyenes, azoles), the cell wall, and dna or protein synthesis. polyenes commonly used formulations are amphotericin b, natamycin, and nystatin. 508clinical theriogenology • volume 5, number 4 • december 2013 mechanism of action. polyenes bind ergosterol, the principal sterol of the fungal cell membrane, causing leakage of cell contents. they also bind cholesterol in mammalian cell membranes (less avidly) which makes them the most toxic of clinically used antifungals. polyenes are associated with renal toxicity. amphotericin b is the mainstay for systemic fungicidal treatment of filamentous fungal infection. it is poorly soluble in water, unstable at 37°c, with maximal antifungal effects at ph 6-7.5, and decreased effects at low ph. amphotericin b is not well absorbed orally and is thus intravenously administered. nystatin has greater nephrotoxicity and is thus used topically (e.g. intrauterine). uses in reproduction. polyenes are a reasonable first choice for treatment of fungal endometritis caused by yeast. molds are most susceptible to polyenes and less susceptible to imidazoles and triazoles.23 azoles commonly used formulations are imidazoles (clotrimazole, ketoconazole, and miconazole) and triazoles (itraconazole, fluconazole, and voriconazole). mechanism of action. azoles inhibit cytochrome p450 dependent ergosterol synthesis leading to disruption of fungal membranes. azoles are fungistatic, but fungicidal at high concentrations. ketoconazole is poorly water soluble, lipophilic, requires acid ph, requires nasogastric tube administration (if administered in conjunction with hcl as recommended, the suspension can be irritating to the oral cavity and throat). oral administration at 30 mg/kg does not result in detectable serum concentrations.2 itraconazole is poorly water soluble, lipophilic, and requires acid ph for absorption. fluconazole is water soluble, oral absorption is unaffected by acid and it is well absorbed after oral administration, it distributes widely to tissues with the half-life in horses ~40 hours, and with 100% oral bioavailability. it can be administered orally or intravenously, though resistance is more frequent. voriconazole is a second generation triazole, with a wide spectrum, can be administered orally or intravenously, is metabolized by liver, with excellent tissue penetration, though with limited experience in horses. topical azoles commonly used formulations are clotrimazole and miconazole. uses in reproduction. ketoconazole and clotrimazole are reasonable empiric treatment choices for uterine yeast infections based on a susceptibility survey. fluconazole is popular due to its administration flexibility, however organisms’ resistance to this drug appears to be increasing.23 ketoconazole is not recommended in pregnant animals. iodine addition of povidone-iodine to infusions or uterine lavage solutions is common for management of microbial infections and is included here for completion rather than an update. one study found that intrauterine infusion of 1% povidone-iodine solution in mares causes chronic inflammatory changes in the endometrium.24 a concentration of 0.5% povidone-iodine still demonstrated suppression of bacterial growth and is a commonly recommended ‘safer’ dose.25 chelators chelators increase the permeability of the bacterial cell wall and cell membrane and are synergistic with antimicrobials including gentamicin, penicillin, oxytetracycline and chloramphenicol. in general, first and second generation chelators have a greater application in treating gram-negative infections. the third generation chelator, tricide®, appears to potentiate the effects of antimicrobials against gram-positive and gram-negative bacteria as well as yeast and fungi.26 509 clinical theriogenology • volume 5, number 4 • december 2013 topical antimicrobials topical antimicrobials are important components of therapy for vaginal, vestibular and vulvar inflammation and necrosis as most commonly occurs post-foaling. additionally, the clitoris can occasionally be found to harbor pathogenic organisms and maintenance of the mare’s uterine health may benefit from cleansing the clitoris. ointments that have anecdotal utility include nystatin, neomycin sulfate, thiostrepton and triamcinolone acetonide ointment (animax®), bovine intramammary treatments (cephapirin), and sodium hypochlorite hydrogel (anasept®). diagnostic methods for identifying infection the use of intrauterine or systemic antimicrobials for endometritis, the most common mare reproductive tract infection, is ideally preceded by organism identification and antimicrobial sensitivity. methods for diagnosing endometritis include a uterine swab for microbial culture, endometrial cytology to evaluate for the presence of inflammation, and an endometrial biopsy for histopathology and/or microbial culture. in a comparison of the uterine swab versus endometrial biopsy for assessment of any bacterial growth, one study found that in 2% of all positive cultures, bacterial growth was observed from the swab and not from the biopsy, while in 55% of positive cultures the swab was negative for bacterial growth and the biopsy positive. calculation of the sensitivity and specificity was performed in two scenarios: culture using a swab compared to culture from an endometrial biopsy as the gold standard, and culture from either a swab or a biopsy compared to inflammatory cells seen in the biopsy as the gold standard.27 swab with culture from biopsy as gold standard: sensitivity specificity positive predictive value negative predictive value swab culture 0.44 0.98 0.95 0.74 tests with inflammatory cells in the biopsy as the gold standard3: in an assessment of subclinical endometritis as defined by polymorphonuclear cells (pmns) present during diestrus in the stratum compactum of the endometrial biopsy in the absence of intrauterine fluid, another study compared the value of a uterine swab, a cytology brush, and an endometrial biopsy for cytological and bacteriological diagnosis of endometritis, with the following results:28 sensitivity specificity cytology bacteriology cytology bacteriology swab 0.00 0.33 0.93 0.83 brush 0.17 0.25 0.83 0.80 biopsy 0.25 0.25 0.85 0.95 brush: cytology+bacteriology 0.42 0.70 these studies suggest that a uterine swab for microbial culture used alone is insufficient for diagnosis of endometritis and that diagnostic ability is improved with addition of at least endometrial cytology. thus, if bacterial infection is suspected as an underlying cause of the inflammation, antimicrobial treatment may be empirical. sensitivity specificity positive predictive value negative predictive value swab culture 0.34 1.00 1.00 0.44 cytology 0.77 1.00 1.00 0.62 biopsy culture 0.82 0.92 0.97 0.67 510clinical theriogenology • volume 5, number 4 • december 2013 bacterial growth states it has been suggested that bacteria are able to survive for long periods in a “dormant” or “viable but non-culturable (vbnc)” state during which time neither plating onto solid media nor inoculation into liquid media lead to growth of cells. furthermore, bacteria in dormant states are resistant to antimicrobial therapy. testing for the presence of these bacteria requires fluorescence microscopy and fluorescence labeled antibodies, propidium iodide in conjunction with molecular probes, or amplification of bacterial mrna using rt-pcr techniques. some species of bacteria retain their pathogenic potential during dormancy. a human example of the medical importance of non-growth states is mycobacterium tuberculosis.29-31 research is actively targeting signals for bacteria to resume growth. an example of such a signal is the resuscitation-promoting factor (rpf), an enzyme that has been shown to increase the culturability of dormant bacteria. a mechanism by which this enzyme functions in conjunction with other bacterial proteins is under investigation.32 in a study to evaluate for a similar bacterial dormancy state in the equine endometrium, petersen et al used fluorescence in situ hybridization (fish) to evaluate s. zooepidemicus endometritis in mares treated with systemic antibiotics. despite systemic antibiotic treatment, streptococci could still be visualized deep within the endometrium.33 research into the effect of a proprietary “activation” signal for resumption of bacterial growth is ongoing.34 post-mating induced endometritis treatment of post-mating induced endometritis in the form of routine antimicrobial infusion postbreeding is common in the thoroughbred breeding industry. as in any healthy debate, there are at least two sides to the argument. on one hand, there is limited published, statistically significant evidence supporting this practice.35 copious use of antimicrobials is also scrutinized for potential antimicrobial resistance development. on the other hand, a study evaluating thoroughbred reproductive efficiency and financial value found that mares that were barren twice over a seven-year investment were not profitable. this taken together with drift (in the same study, drift was 13.4 +/23.2 days) demonstrates that the average mare needs to become pregnant within one to two estrous cycles every year.36 this may drive the broad treatment for even potential post-breeding infectious endometritis. in a recent study of s. zooepidemicus from horses conducted by the university of kentucky veterinary diagnostic laboratory, resistance of s. zooepidemicus has not developed.37 placentitis thirty-four percent of equine abortions or stillbirths were associated with feto-placental infection. of these, 17.8% had an identified bacterial etiology.38 antimicrobials are a critical component of bacterial placentitis treatment. antimicrobials that have evidence of reaching allantoic fluid include penicillin g (22,000 units/kg im bid), gentamicin (6.6 mg/kg iv sid) and trimethoprim sulfamethoxazole (15-30 mg/kg po bid).39,40 other antimicrobials are anecdotally used with effect. in an experimental ascending placentitis model with beta-streptococcus, the introduced organism was be cultured from the uterus post-foaling in both treated and untreated controls. the treated ponies received antimicrobials (trimethoprim sulfamethoxazole 30 mg/kg po bid) continuously until parturition.41 this information suggests that antimicrobial therapy in pregnant mares with placentitis may need to be prolonged and weighed against side effects of long-term antimicrobial therapy. references 1. causey rc, weber ja, emmans ee, et al: the equine immune response to streptococcus equi subspecies zooepidemicus during uterine infection. vet j 2006;172:248-257. 2. giguere s, prescott jf, baggot jd, et al, editors: antimicrobial therapy in veterinary medicine. 4th ed. ames (ia): blackwell publishing; 2006. 3. love cc, strzemienski pj, kenney rm: endometrial concentrations of ampicillin in mares after intrauterine infusion of the drug. am j vet res 1990;51:197-199. 511 clinical theriogenology • volume 5, number 4 • december 2013 4. van camp sd, papich mg, whitacre md: administration of ticarcillin in combination with clavulanic acid intravenously and intrauterinely to clinically normal oestrous mares. j vet pharmacol ther 2000;23:373-378. 5. murchie t, macpherson ml, leblanc m, et al: a microdialysis model to detect drugs in the allantoic fluid of pregnant pony mares. proc annu conv am assoc equine pract; 2003. p. 118-121. 6. olivieri bt, love bc, rezabek gb, et al: effect of antibiotic-containing extenders on taylorella equigenitalis contaminated semen. j equine vet sci 2011;31:655-660. 7. dean cj, hobgood am, blodgett gp, et al: the addition of ticarcillin-clavulanic acid to inra 96 extender for stallion semen cooling. equine vet j 2012;44:95-99. 8. cervantes cc, brown mp, gronwall r, et al: pharmacokinetics and concentrations of ceftiofur sodium in body fluids and endometrium after repeated intramuscular injections in mares. am j vet res 1993;54:573-575. 9. witte ts, bergwerff aa, scherpenisse p, et al. ceftiofur derivates in serum and endometrial tissue after intramuscular administration in healthy mares. theriogenology 2010;74:466-472. 10. scofield d, black j, wittenburg l, et al: endometrial tissue and blood plasma concentration of ceftiofur and metabolites after intramuscular administration of ceftiofur crystalline-free acid to mares. proc annu conv am assoc equine pract; 2012. p. 519-520. 11. hatzel jn, macpherson ml, giguere s, et al: administration of ceftiofur crystalline-free acid to pony mares with placentitis. proc annu conv am assoc equine pract; 2012. p. 521-522. 12. ricketts sw: treatment of equine endometritis with intrauterine irrigations of ceftiofur sodium: a comparison with mares treated in a similar manner with a mixture of sodium benzylpenicillin, neomycin sulphate, polymixin b sulphate and furaltadone hydrochloride. pferdeheilkunde 1997;13:486-489. 13. eilts be, mccoy dj, taylor h, et al: effect of repeated intrauterine infusions of gentamicin on the equine endometrium. theriogenology 1988;29:1253-1259. 14. al bagdadi fk, eilts be, richardson gf: scanning electron microscopy of the endometrium of mares infused with gentamicin. microsc microanal 2004;10:280-285. 15. bryant je, brown mp, gronwall rr, et al: study of intragastric administration of doxycycline: pharmacokinetics including body fluid, endometrial and minimum inhibitory concentrations. equine vet j 2000;32:233-238. 16. ros bd, willsallen cc, norman st: the renaissance of oxytetracycline-a treatment for equine endometritis? proc aust college vet sci annu conf 2010. p.70. 17. papich mg, van camp sd, cole ja, et al: pharmacokinetics and endometrial tissue concentrations of enrofloxacin and the metabolite ciprofloxacin after i.v. administration of enrofloxacin to mares. j vet pharmacol ther 2002;25:343-350. 18. gonzalez c, moreno l, fumuso e, et al. enrofloxacin-based therapeutic strategy for the prevention of endometritis in susceptible mares. j vet pharmacol ther 2010;33:287-294. 19. troedsson mh: treatment strategies in mares with endometritis: proc soc therio mare reprod symp 1996. p. 40-50. 20. fumuso e, checura c, losinno l, et al: endometrial tissue concentrations of enrofloxacin after intrauterine administration to mares. vet res commun 2002;26:371-380. 21. rodriguez js, han s, nielsen s, et al: consequences of intrauterine enrofloxacin infusion on mare endometrium. j equine vet sci 2012;32:106-111. 22. specht te, brown mp, gronwall rr, et al: pharmacokinetics of metronidazole and its concentration in body fluids and endometrial tissues of mares. am j vet res 1992;53:1807-1812. 23. beltaire ka, cheong sh, coutinho da silva ma: retrospective study on equine uterine fungal isolates and antifungal susceptibility patterns (1999-2011). equine vet j 2012;44:84-87. 24. olsen lm, al-bagdadi fk, richardson gf, et al: a histological study of the effect of saline and povidone-iodine infusions on the equine endometrium. theriogenology 1992;37:1311-1325. 25. fuursted k, hjort a, knudsen l: evaluation of bactericidal activity and lag of regrowth (postantibiotic effect) of five antiseptics on nine bacterial pathogens. j antimicrob chemother 1997;40:221-226. 26. ritchie bw, wooley re, kemp dt: use of potentiated antibiotics in wound management. vet clin north am exotic anim pract 2004;7:169-189. 27. nielsen jm: endometritis in the mare: a diagnostic study comparing cultures from swab and biopsy. theriogenology 2005;64(3):510-8. 28. overbeck w, witte ts, heuwieser w: comparison of three diagnostic methods to identify subclinical endometritis in mares. theriogenology 2011;75:1311-1318. 29. curras m, magarinos b, toranzo ae, et al: dormancy as a survival strategy of the fish pathogen streptococcus parauberis in the marine environment. dis aquat organ 2002;52:129-136. 30. oliver jd: recent findings on the viable but nonculturable state in pathogenic bacteria. fems microbiol rev 2010;34:415-425. 31. keep nh, ward jm, cohen-gonsaud m, et al: wake up! peptidoglycan lysis and bacterial non-growth states. trends microbiol 2006;14:271-276. 32. kana bd, mizrahi v: resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling. fems immunol med microbiol 2010;58:39-50. 33. petersen mr, lehn-jensen h, bojesen am: use of fluorescent in situ hybridization (fish) to identify endometritis pathogens in the mare (abstract). j anim sci 2010;88[e-suppl]:1097. 512clinical theriogenology • volume 5, number 4 • december 2013 34. petersen mr, lu kg, christoffersen m, et al: chronic streptococcus equi subspecies zooepidemicus endometritis in the mare-induction of active growth and treatment improves diagnosis and fertility in the subfertile thoroughbred mare. proc havemeyer workshop strangles and other streptococcal diseases 2012. 35. brinsko sp: common procedures in broodmare practice: what is the evidence? vet clin north am equine pract 2007;23:385-402. 36. bosh ka, powell d, neibergs js, et al: impact of reproductive efficiency over time and mare financial value on economic returns among thoroughbred mares in central kentucky. equine vet j 2009;41:889-894. 37. erol e, locke sj, donahoe jk, et al: beta-hemolytic streptococcus spp. from horses: a retrospective study (20002010). j vet diagn invest 2012;24:142-147. 38. giles rc, donahue jm, hong cb, et al: causes of abortion, stillbirth, and perinatal death in horses: 3,527 cases (19861991). j am vet med assoc 1993;203:1170-1175. 39. murchie ta, macpherson ml, leblanc mm, et al: continuous monitoring of penicillin g and gentamicin in allantoic fluid of pregnant pony mares by in vivo microdialysis. equine vet j 2006;38:520-525. 40. macpherson ml: treatment strategies for mares with placentitis. theriogenology 2005;64:528-534. 41. bailey cs, macpherson ml, pozor ma, et al. treatment efficacy of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest in experimentally induced equine placentitis. theriogenology 2010;74:402-412. 513 clinical theriogenology • volume 5, number 4 • december 2013 table 1. antimicrobial routes and doses antimicrobial systemic dose intrauterine dose procaine penicillin g 22,000 iu/kg 5 million iu potassium penicillin 22,000 iu/kg 5 million iu ampicillin 20-40 mg/kg iv tidqid 3 g ticarcillin and clavulanic acid 50-60 mg/kg iv tidqid 3.1-6.2 g 0.5 to 1.5 mg/ml in inra96 ceftiofur sodium 2.2 mg/kg iv/im sidbid 1 g marketed as naxcel in us ceftiofur crystalline free acid 6.6 mg/kg im 2 doses 4 days apart marketed as excede in us ceftiofur hydrochloride 1 g marketed as excenel in us polymyxin b 6000 u/kg iv bidtid (antiendotoxic) 1 million u amikacin 10-15 mg/kg iv sid (adults) 2 g gentamicin 6.6 mg/kg iv sid 500-2000 mg buffered or diluted oxytetracycline 6.6-10 mg/kg sidbid slow dilute iv (6 g)* *pending further study doxycycline 10 mg/kg po bid enrofloxacin 6 mg/kg iv sid ciprofloxacin 2.5 g po sid with 1 g probenecid po sid amphotericin b 0.3-0.5 mg/kg iv eod 100-200 mg nystatin 0.5-2.5 million u ketoconazole 10 mg/kg in 0.2n hcl ngtube bid clotrimazole 500-700 mg miconazole 400-700 mg itraconazole 6 mg/kg po sid fluconazole 5 mg/kg po sid 100 mg 514clinical theriogenology • volume 5, number 4 • december 2013 t ab le 2 . s en si ti vi ty o f ut er in e or ga ni sm s is ol at ed a t h ag ya rd l ab or to ry in 2 01 2. r es ul ts a re b as ed o n cu lt ur es c ol le ct ed b y h ag ya rd v et er in ar ia ns in c en tr al k en tu ck y. t ot al n um be r of e nd om et ri al c ul tu re s= 60 62 . * a nt ib io ti c no t r ec om m en de d fo r us e w it h th is o rg an is m p er c li ni ca l l ab or at or y s ta nd ar ds i ns ti tu te ( c l s i) g ui de li ne s. p en ic il li n su sc ep ti bl e be ta s tr ep to co cc us s pe ci es c an b e co ns id er ed s us ce pt ib le to a m pi ci ll in , ce fa zo li n an d im ip en em p er c l s i g ui de li ne s. o f th e 60 62 c ul tu re s, 3 0 ye as t s pe ci es a nd 1 7 fu ng al s pe ci es w er e id en ti fi ed . 515 clinical theriogenology • volume 5, number 4 • december 2013 516clinical theriogenology • volume 5, number 4 • december 2013 omniblank: 2017: preservation, handling, and common artifacts of the endometrial and testicular biopsy preservation, handling, and common artifacts of the endometrial and testicular biopsy christopher premanandan,a erin runcan,b marco coutinho da silvab adepartment of veterinary biosciences and bdepartment of veterinary clinical sciences, college of veterinary medicine, the ohio state university, columbus, oh introduction the value of histologic evaluation of tissues in theriogenology is two-fold: diagnostics utilized for autopsy evaluation (abortion cases) and assessment of changes in tissue structure through biopsy evaluation. abortion cases involve the evaluation of fetal membranes and fetal organs in addition to any required ancillary diagnostic testing. any tissue from the reproductive tract can be evaluated from the surgical biopsy perspective and these tissues are frequently evaluated at private diagnostic laboratories and state diagnostic laboratories in terms of general pathology. specimens in this category include whole testicles after orchiectomy and whole female tubular reproductive tracts cranial to the cervix, and ovaries. additionally vaginal or vestibular lesions as well as penile and preputial lesions may be evaluated. however, in addition to these commonly submitted surgical pathology specimens, the theriogenologist often submits a unique subset of specimens required to evaluate the potential fertility of a breeding animal, namely endometrial and testicular biopsies. equine and canine endometrial biopsies and canine testicular biopsies are the most common tissue submitted for evaluation to the ohio state university theriogenology and veterinary reproductive pathology service. each of these tissues has unique characteristics in terms of preservation and evaluation that can impact the histopathologic evaluation and results received by the submitting clinician. this review will cover the ideal methods of tissue fixation and preservation and will focus on the common artifacts, idiosyncracies and difficulties unique to the interpretation of endometrial biopsies and testicular biopsies. fixation, storage and shipping tissue fixation prior to processing for histologic sectioning is generally categorized into two methods: physical (cryopreservation, heat fixation) and chemical. while cryopreservation has a place in diagnostic histopathology, chemical preservation is by far the most common method of fixation. chemical fixation acts by inducing either protein denaturation (coagulation) or protein crosslinking. the most common cross-linking agent utilized for the fixation of surgical biopsy specimens from the reproductive system is 10% neutral buffered formalin (nbf). fixatives such as bouin’s solution and modified davidson’s medium induce protein denaturation (coagulation) by disruption of hydrogen and electrostatic charges. each of these fixatives has advantages and disadvantages (table). in the authors’ experience, there appears to be confusion regarding the best fixative to use for reproductive tissues. there is no ideal fixative that completely eliminates cellular preservation artifacts. preparation of tissue prior to fixation is often far more important for optimal results. ideal fixation takes place when a tissue is less than or equal to 0.5 cm in thickness in one dimension. the middle regions of excessively thick tissue can undergo autolysis while completely immersed in preservative. temperature can affect the rate of tissue fixation as well. environmental room temperature (20°c) is adequate for most purposes. increases in temperature can hasten the fixation process, however, this can also increase the rate of autolysis in tissue. conversely, refrigeration can impede the diffusion of fixative into the tissue. while variations in temperature have the clearest implications regarding tissue fixation in research, one must consider its implications for clinical samples when shipping tissues to a diagnostic facility. ten percent nbf is composed of 4% formaldehyde in addition to phosphate buffers that maintain ph around 7. the neutral ph prevents the formation of artifactual formalin pigments. neutral buffered formalin preserves tissue by forming stable covalent cross linkages (methylene bridges) between amino acids.1 there are several advantages to utilizing this fixative, the first being that the majority of pathologists are accustomed to evaluating tissues fixed in nbf. also, nbf is the most readily available preservative and often provided by commercial diagnostic laboratories for biopsy submissions. if properly stored, small quantities of nbf can be kept for long periods of time before it is utilized. in addition, although not ideal for some diagnostic and research purposes (such as immunohistochemistry), clinical theriogenology • volume 9, number 2 • june 2017159 tissue can be stored in nbf for long periods of time. for a majority of specimens submitted for evaluation by a reproductive pathologist, nbf is a suitable fixative. utilizing nbf does have disadvantages. immunoreactivity for immunohistochemical and immunofluorescence assays can be reduced with long periods of storage in nbf. in these situations, tissue should be transferred to 70% ethanol or phosphate buffered saline if additional storage time is necessary. alternatively, processing and storing the tissue as paraffin embedded blocks soon after the minimum fixation period helps to retain immunoreactivity. the accumulation of formic acid in nbf can result in deposits of an artifactual pigment in tissue known as acid hematin.2 it is commonly observed in autopsy specimens stored in nbf for extended periods of time, however, it is rarely an issue for biopsy specimens due to the rapid turnover of tissue for processing2. for practitioners specializing in reproduction, this is more of a concern for the aborted or stillborn fetus submitted for evaluation. formaldehyde does pose a health risk to personnel (both acute toxicity and as a carcinogen) but this risk can be easily minimized through proper storage and handling of the chemical. the quality of the histologic sections obtained with nbf fixation is adequate for most diagnostic purposes. however, tissue shrinkage can occur and, in some tissues, nuclear detail and to a lesser degree, cytoplasmic detail can be obscured. this tends particularly to be the case in thicker testicular biopsies in which separation of the spermatogenic epithelium from the underlying basement membrane can take place. in addition, the identification of the cells comprising the adluminal compartment can be difficult within the seminiferous tubules. bouin’s fixative is a formaldehyde based fixative that contains a small percentage of acetic acid (5%) and picric acid (0.9%). similar to nbf, bouin’s has advantages and disadvantages. bouin’s penetrates tissues rapidly and uniformly. because this fixative induces both protein crosslinking and protein coagulation, this fixative has a reputation for excellent preservation of cellular detail.3 this is particularly useful in testicular biopsies but is of less value in endometrial, ovarian and vaginal biopsies. however, bouin’s has some unique properties that present some disadvantages. a distinguishing component of the fixative is presence of picric acid, a compound that imparts the coagulative action of this particular fixative. however, picric acid is flammable and explosive in its dried form. excessive storage times in bouin’s can render tissues brittle and difficult to process, causing marked artifacts in histologic sections that can make interpretation of biopsies difficult (figure 1). tissues should be fixed in bouin’s for 24-48 hours and then transferred to 70% ethanol to prevent excessive hardening.4,7,8 occasionally, repeated rinses in 70% ethanol or saline may be required depending on the application, such as immunohistochemistry. davidson’s medium (also known as hartmann’s fixative and modified davidson’s fixative) is a formalin-based fixative that has additional components of glacial acetic acid (5%) and ethanol (14.25%). the distinction between davidson’s fixative and modified davidson’s fixative is unclear in histology references as the components of both are identical in most material safety data sheets. similar to bouin’s, this solution induces both protein coagulation and protein crosslinking. tissue fixation in this mediim is rapid and tissues (particularly small biopsies) should be transferred to 70% ethanol in 24-48 hours. davidson’s medium has a good reputation regarding preservation of cellular detail, particularly of the nucleus and chromatin patterns5. while this may not be an advantage for endometrial biopsies, it has been demonstrated as an advantage for testicular biopsies. rat testes fixed in modified davidson medium demonstrated less shrinkage artifact than 4% paraformaldehyde or bouin’s fixative, particularly in the central region of the testes.6 in addition, nuclear detail was better when tissue was fixed in modified davidson’s medium.6 in a similar study, rat, rabbit, dog and cynomologus macaque testes demonstrated less shrinkage artifact when fixed in modified davidson’s medium compared to bouin’s fixative.8 this study also compared acrosomal staining with periodic acid schiff. acrosomal staining in rat testes was judged to be subjectively more prominent when fixed in modified davidson’s medium.8 overall clarity of detail was also better with modified davidson’s medium in addition to immunohistochemical staining of proliferating cell nuclear antigen, androgen receptor and protein gene product 9.5.8 the society of toxicologic pathology (stp) has stated that modified davidson’s medium is recommended over bouin’s for regulatory studies involving fertility, and formalin should be avoided.9 clinical theriogenology • volume 9, number 2 • june 2017 160 numerous other types of fixatives, processing and techniques are useful for reproductive tissue particularly for research purposes, but a detailed overview of these modalities is out of the scope of this discussion. some of these are limited in their usage due to their availability, cost, or inherent safety concerns. for instance, zenker’s fixative is described as a fixative useful for testicular biopsies in particular,6 however, since the solution contains mercuric chloride, its usage in practice can be cumbersome in the event of spillage. plastic embedding of testicular tissue for histologic sections provides excellent cellular detail and minimal processing artifact but is costly and requires more technical expertise.10 other modalities of tissue processing and staining are available depending on the requirements of the tissue from the biopsy. general concepts and artifacts the female reproductive system and endometrial biopsies in general, most tissues from the female reproductive system are adequately fixed by immersion in nbf as long as care is taken to insure an adequate nbf to tissue ratio (10:1). normally sized canine and feline ovaries will typically fix well when immersed whole; however, if there is an excessive amount of adipose tissue in the mesosalpinx, mesovarium and ovarian bursa, exposing the ovary by opening the bursa (particularly in canines) is prudent. equine and bovine ovaries of normal size should be partially sectioned prior to immersion. enlarged ovaries (typically due to neoplastic disease) should be sectioned, but the practitioner who does not have the luxury of a pathology service directly attached to the clinical facility may be forced to obtain representative regions of the ovary rather than submitting the whole specimen. the canine and feline uterus can be submitted intact for evaluation; however, the practitioner must keep in mind that a relatively small proportion of the organ will be examined histologically. in our service, the standard protocol is to examine a cross section and longitudinal section of the both uterine horns and the uterine body. technical aspects regarding equine endometrial biopsy collection, fixation, processing and evaluation are well-described in the literature.11 endometrial biopsies from llamas and cattle are very similar to equine endometrial biopsies in terms of these factors.12,13 descriptions regarding the canine endometrial tissue biopsy in regards to collection have been described as well as lesions associated with infertility in the bitch.14,15,16 collection methods include punch biopsies, transcervical endometrial biopsies and wedge biopsies. many histologic artifacts are shared between endometrial biopsies of any species. numerous other general artifacts are present associated with fixation and tissue processing. since these artifacts are common among all organ systems, the more clinically relevant artifacts pertaining to reproduction are presented here. crush artifact is often present at the edges of a tissue section that manifests as nuclear stretching or “streaming” (figure 2a and b). hemorrhage is often present at the edge of a specimen associated with tissue sampling and care should be taken not to interpret this as a lesion. the presence of hemosiderin-laden macrophages can be helpful to determine if the hemorrhage was present in the tissue before the biopsy was taken. another common artifact takes place with the transient increase in pressure on tissue that takes place during the biopsy procedure that causes invagination or “telescoping” of endometrial glands into the adjacent segment (figure 2a). storage during shipping and the manner in which the specimen is processed can have a significant impact on interpretation and results. the typical equine endometrial biopsy can be immersed in fixative directly following the procedure. less discrimination regarding sectioning of the specimen is required as the typical biopsy does not include the myometrium. however, depending on the manner in which the canine endometrium is sampled, some care may need to be taken to insure adequate results are obtained. the transcervical biopsy typically results in the smallest tissue volume, similar to what is obtained in an endoscopic gastrointestinal biopsy. because of the size of the specimen, care should be taken with fixation and transport of the specimen. ideally, screw top containers of smaller volume (2-5 mls) should be utilized to minimize risk of tissue loss at the laboratory. specimens can be placed in mesh cassettes (figure 3) or, alternatively, placed in a small folded piece of paper towel prior to immersion. due to the small size of the specimens, crush artifact tends to be significant and can often prevent adequate clinical theriogenology • volume 9, number 2 • june 2017161 evaluation of the tissue. the typical punch biopsy used in canines ranges from 2-6 mm and the amount of endometrial tissue available for evaluation depends on the size of the punch, the amount of crush artifact present and surgical experience with this procedure. because of the disproportionate amount of endometrial tissue present compared to myometrial tissue present, orientation and embedding can cause issues with interpretation. these issues typically take place if the endometrial tissue is faced away from the cutting surface of the block and result in a section containing only myometrium. this can be easily remedied by requesting that the lab or pathologist melt the paraffin block and reorient the specimen (figure 4). this, of course, excludes transcervical biopsies that do not contain a portion of myometrium at all. it is helpful in both transcervical and punch biopsies to sample from both horns and the uterine body to improve the correlation between overall changes of the endometrium from a small tissue sample and the entire uterus. wedge biopsies provide the largest amount of endometrial tissue for evaluation and seldom present a problem with tissue orientation. however, this type of sample may present a problem in terms of tissue closure during surgery from a fully involuted uterus taken during anestrus. in all circumstances, care should be taken to minimize the amount of crush artifact in the tissue by avoiding manipulation of the tissue directly with fingers. fine forceps or needles should be used to gently tease the tissue into the container from the biopsy instrument. the male reproductive system and testicular biopsies similar to the female reproductive system, tissues from the male reproductive tract are adequately fixed in nbf, provided that the fixative to tissue ratio is optimal. testicles from adult dogs, horses, and ruminants should be sectioned prior to fixation. testicles from felines and young dogs can be immersed whole provided that the width of the tissue is less than 1 cm. testicular biopsy is an underutilized diagnostic tool for the assessment of infertility in domestic animals. as a consequence, it has been neglected by most veterinary pathologists in terms of understanding the nuances of the biology of the tissue reflected in the histology. in contrast, the role of the testicular biopsy is fairly well delineated in human medicine.17,18,19 confirmation of obstructive azoospermia in infertile men with normal testicular size and hormonal profiles are one indication for a testicular biopsy. another major indication is the therapeutic biopsy in which testicular spermatozoa extraction is utilized for intracytoplasmic sperm injection.17,18 in general, however, it is thought that the testicular biopsy has little value if a thorough clinical evaluation is performed as the underlying causes of abnormalities in spermatogenesis cannot be generally be determined histologically.19 considering that the needs of a theriogenologist can be much different than those of a human fertility specialist, and testicular causes of infertility can be much more different between domestic animals and in humans, it is worth investigating the diagnostic value of the testicular biopsy in veterinary medicine. the testicular biopsy has been established as safe in the dog, bull, and stallion and the procedure is covered in greater detail in many references.20-23 methods of sampling testicular parenchyma include open incisional/wedge biopsies, tru-cuttm needle biopsies and fine needle aspirates (fna). although wedge biopsy provides the most parenchyma for evaluation with the least amount of artifact, the potential complications (testicular hemorrhage, post-biopsy adhesions) generally outweigh its benefits. it is generally recommended that at least 100 seminiferous tubule cross sections are available for proper evaluation of spermatogenesis in a specimen.17 many tru-cuttm needle biopsies are incomplete to some degree and do not contain the recommended 100 cross sections. therefore, at least two samples from the same testis may be required to provide an adequate sample. crush artifacts tend to be the highest in this type of specimen (figure 2b). in addition, significant variation can occur in regards to the degree of testicular degeneration or hypospermatogenesis observed in different regions of the testes. therefore a single tru-cuttm needle biopsy may not reflect the overall changes present. fine needle aspirates of testicular parenchyma can provide an overall assessment of spermatogenesis. while the use of fna is well established for the diagnosis of testicular tumors,24 the diagnosis of underlying causes of infertility in male animals is likely a more valuable use of this technique. fine needles aspirates and large needle aspirates have been shown to be safe regarding future reproductive function of the testes.25,26 the cytologic characteristics of normal adult canine testicular clinical theriogenology • volume 9, number 2 • june 2017 162 fna as well as canines from four weeks old to 52 weeks old have been established. a sertoli cell index and sperm cell index are used to provide a reference point and a basis to evaluate spermatogenesis.27,28 however, in a similar fashion to the needle biopsy, an aspirate of a single region of the testicular parenchyma may not be reflective of the overall state of spermatogenesis in the organ. similar parameters have been studied in the stallion.29,30 conclusion endometrial biopsy remains an integral part of assessing fertility in domestic animals, particularly in the bitch and the mare. testicular biopsy and testicular fna are not yet as well established for assessing fertility in males. however, reference parameters exist to assist the clinician in investigating testicular causes of infertility. multiple methods of fixation exist for both types of samples, but the clinician must be aware that each solution has drawbacks and care should taken to insure that artifact that can impede interpretation is minimized. in terms of preservation prior to submission, endometrial biopsies can be fixed in nbf with little concern in terms of the quality of the histologic section obtained. although nbf will work well for the fixation of testicular biopsies in most cases, consideration can be given to utilizing bouin’s or modified davidson’s medium. however, care should be taken to either process the tissue soon after the recommended fixation period or transfer the tissue to 70% ethanol for shipping for longer term storage. references 1. thavarajah r, mudimbaimannar vk, elizabeth j, et al: chemical and physical basics of routine formaldehyde fixation. j oral maxillofac pathol 2012;16: 400-405. 2. pizzolato p: formalin pigment (acid hematin) and related pigments. am j med technol 1976;42:436-440. 3. gatta, lb, cadei, m., balzarini, p, et al: application of alternative fixatives to formalin in diagnostic pathology. europ j histochem�2012;56,e12:63-70. 4. harleman jh, nolte t: testicular toxicity: regulatory guidelines-the end of formaldehyde fixation? toxicol pathol 1997;25:414-417. 5. moore kl, graham ma, barr ml: the detection of chromosomal sex in hermaphrodites from a skin biopsy. surg gynecol obstet 1953;96:641-648. 6. tu l, yu l, zhang h: morphology of rat testis preserved in three different fixatives. j huazhong univ sci technolog med sci 2011;31:178-180. 7. howroyd p, hoyle-thacker r, lyght o, et al: morphology of the fetal rat testis preserved in different fixatives. toxicol pathol 2005;33:300-304. 8. latendresse jr, warbrittion ar, jonassen h, et al: fixation of testes and eyes using a modified davidson's fluid: comparison with bouin's fluid and conventional davidson's fluid. toxicol pathol 2002;30:524-533. 9. lanning ll, creasy dm, chapin re, et al: recommended approaches for the evaluation of testicular and epididymal toxicity. toxicol pathol 2002;30:507-520. 10. vukasovic a, kozina v, jezek d: histologic procedures and testicular biopsy freezing. in: jezek d, editor. atlas on the human testis. london: springer-verlag; 2013. p. 23-35. 11. snider ta, sepoy c, holyoak gr: equine endometrial biopsy reviewed: observation, interpretation, and application of histopathologic data. theriogenology 2011;75:1567-1581. 12. powers be, johnson lw, linton lb, et al: endometrial biopsy technique and uterine pathologic findings in llamas. j am vet med assoc 1990;197:1157-1162. 13. chapwanya a, meade kg, narciandi f, et al: endometrial biopsy: a valuable clinical and research tool in bovine reproduction. theriogenology 2010;15;73:988-994. 14. christensen bw, schlafer dh, agnew dw, et al: diagnostic value of transcervical endometrial biopsies in domestic dogs compared with full-thickness uterine sections. reprod domest anim 2012;47 suppl 6:342-46. 15. gifford at, scarlett jm, schlafer dh: histopathologic findings in uterine biopsy samples from subfertile bitches: 399 cases (1990-2005). j am vet med assoc 2014;244:180-186. 16. mir f, fontaine e, albaric o, et al: findings in uterine biopsies obtained by laparotomy from bitches with unexplained infertility or pregnancy loss: an observational study. theriogenology 2013;79:312-322. 17. dohle gr, elzanaty s, van casteren nj: testicular biopsy: clinical practice and interpretation. asian j androl 2012;14:88-93. 18. chan pt, schlegel pn: diagnostic and therapeutic testis biopsy. curr urol rep 2000;1:266-272. 19. schoor ra, elhanbly s, niederberger cs, et al: the role of testicular biopsy in the modern management of male infertility. j urol 2002;167:197-200. 20. attia ka, zaki aa, eilts be, et al: anti-sperm antibodies and seminal characteristics after testicular biopsy or epididymal aspiration in dogs. theriogenology 2000;53:1355-1363. clinical theriogenology • volume 9, number 2 • june 2017163 21. hunt, wl. foote, rh: effect of repeated testicular biopsy on testis function and semen quality in dogs. j androl 1997;18:740-744. 22. heath am, carson rl, purohit rc, et al: effects of testicular biopsy in clinically normal bulls. j am vet med assoc 2002;220:507-512. 23. pozor, ma, zambrano, gl, runcan, ee, et al: ultrasonographic and morphological sequelae to repeated testicular biopsies in stallions in relation to the lateral branching of the testicular artery. equine vet educ 2002;28: 696-704. 24. masserdotti c, bonfanti u, de lorenzi d, et al: cytologic features of testicular tumours in dog. j vet med a physiol pathol clin med 2005;52:339-346. 25. gouletsou pg, galatos ad, leontides ls, et al: impact of fineor large-needle aspiration on canine testes: clinical, in vivo ultrasonographic and seminological assessment. reprod domest anim 2011;46:712-719. 26. james rw, heywood r, fowler dj: serial percutaneous testicular biopsy in the beagle dog. j small anim pract 1979;20:219-228. 27. santos m, marcos r, caniatti m: cytologic study of normal canine testis.theriogenology 2010;73:208-214. 28. venianaki ap, galatos ad, doukas d, et al: cytological study of normal canine testis from birth to maturity iscfr viii international symposium on canine and feline reproduction, paris; june 22-25, 2016. 29. leme dp, papa fo, roser jf: reproductive characteristics of stallions during the breeding and non-breeding season in a tropical region. trop anim health prod 2012;44:1703-1707. 30. leme dp, papa fo: cytological identification and quantification of testicular cell types using fine needle aspiration in horses. equine vet j 2000;32:444-446. table. comparison between the most common fixation solutions for endometrial and testicular biopsies. 10% nbf (neutral buffered formalin bouin’s fixative modified davidson medium length of fixation indefinitely (unless immunohistochemistry is required). nbf penetrates tissue at a rate of 0.5-1 mm per hour 24-48 hours followed by transfer to 70% ethanol 24-48 hours followed by transfer to 70% ethanol safety issues carcinogen, ensure proper storage of solution similar to nbf. dried picric acid is an explosive hazard similar to nbf artifact potential mostly shrinkage artifact. acid hematin formation (artifactual pigment) can induce tissue hardening, erythrocyte lysis mostly shrinkage artifact clinical theriogenology • volume 9, number 2 • june 2017 164 figure 1. endometrial biopsies fixed in bouin’s solution (a) and 10% nbf (b) for approximately 10 days (hematoxylin and eosin staining). note the relative lack of differential staining in a as opposed to b. the star labels the myometrium in both images. bar = 200 µm. figure 2. examples of artifact in an endometrial biopsy and testicular biopsy. a. an equine endometrial biopsy exhibiting artifactual loss of surface epithelium (arrowheads) and invagination (telescoping) of endometrial glands (arrows). b. a canine testicular biopsy with marked crush artifact. seminiferous tubules are difficult to visualize and nuclear streaming (arrows) is prominent. bar = 50 µm. clinical theriogenology • volume 9, number 2 • june 2017165 figure 3. a. transcervical endometrial biopsies in 10% nbf. a mesh tissue cassette is adjacent to the container. b. transcervical endometrial biopsies in the mesh cassette and a standard histology tissue cassette. note the size of the endometrial biopsies compared to the size of the grid of the tissue cassette. the mesh cassette prevents tissue loss during processing. the mesh cassette (or a folded paper towel) can be used to insure that the specimens stay together during transport of the fixative container. figure 4. orientation of a uterine punch biopsy can affect what is visible on the slide to the pathologist. a. in this section, the endometrium is oriented away from the microtome blade, resulting in a section that contains only myometrium and perimetrium. note that the perimetrium is visible on both sides of the specimen (arrows). the star indicates the approximate location of the image shown in figure 2b. bar = 500 µm b. 200x magnification photomicrograph depicting the perimetrium and myometrium. bar = 50 µm c. the same specimen following melting of the paraffin block and reorientation of the specimen by 90 degrees. endometrium is now visible on the section. the star indicates the approximate location of the image shown in figure 2b. bar = 500 µm. d. 200x magnification photomicrograph depicting the endometrium. bar = 50 µm. (editor’s note: the photographs in this manuscript are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 9, number 2 • june 2017 166 enrofloxacin crosses equine placenta in early pregnancy without inducing gross lesions in fetus enrofloxacin crosses equine placenta in early pregnancy without inducing gross lesions in fetus robyn ellerbrock,a,c igor canisso,a patrick roady,a elizabeth uhl,c gioria podico,a zhong lib adepartment of veterinary clinical medicine broy carver biotechnology center, university of illinois, urbana il ccollege of veterinary medicine, university of georgia, athens, ga ongoing studies in our laboratory suggest that administration of enrofloxacin to late term pregnant mares does not induce lesions in the fetus or resulting foals. however, organogenesis occurs early in pregnancy and it is possible that fluoroquinolone exposure will affect initial cartilage and bone formation during this period. we hypothesized that enrofloxacin administration to early pregnant mares results in high concentrations of both enrofloxacin and its active metabolite ciprofloxacin in fetal fluids and that fluoroquinolone exposure in early pregnancy induces chondrotoxic lesions in the 60 day fetus. objectives were to: (i) determine enrofloxacin and ciprofloxacin concentrations in fetal fluids when enrofloxacin is administered at therapeutic doses during early pregnancy; and (ii) compare endochondral ossification of long bones from those fetuses with gestationally age matched controls. mares carrying normal pregnancies (45 days) were assigned to 2 groups, control (n = 7) or a therapeutic dose of enrofloxacin (7.5 mg/kg po, n = 6). enrofloxacin was administered orally every 24 hours for 14 days abortion was induced on day 15 by administration of 500 µg cloprostenol. fetal heart rate was monitored via transrectal ultrasonography every 6 hours and the fetus removed manually when no heartbeat was detected. after removal, samples of allantoic and amniotic fluids were aspirated with an 18 gauge needle and preserved at 80oc until analysis. limbs, heart, lung, liver, kidney, and placenta were fixed in 10% formalin for histological analysis. all tissues were stained with h&e. fetal limbs were sectioned in the sagittal plane to include the whole limb and were also stained with toluidine blue and masson’s trichrome to assess cartilage and extracellular matrix. slides were evaluated by a board-certified pathologist blinded to treatment group. enrofloxacin and ciprofloxacin concentrations in fetal fluids were measured by l ms/ms (5500 qtrap lc/ms/ms system sciex, framingham, ma). software analyst 1.6.2 was used for data acquisition and analysis. mares aborted at 62 ± 0.5 days of gestation, and mean time from last enrofloxacin treatment to abortion was 41 ± 7.5 hours, with no difference between treated and control mares. enrofloxacin and ciprofloxacin were detected in both amniotic (223.2 ± 160.6 and 58.5 ± 36.3 ng/ml, mean ± sem) and allantoic fluids (326 ± 118 and 102.5 ± 19.6 ng/ml) of treated mares. no differences were noted in histological features of the front or hind limbs of fetuses in the control and enrofloxacin exposed experimental groups. fetal stage of development and ossification of the bones of the limbs was comparable between groups. short term administration of enrofloxacin in early pregnancy did not result in apparent pathologic lesions in the equine fetus. although further research is needed to assess other stages of pregnancy, longer durations of treatment and long term foal outcomes, enrofloxacin may be useful to treat select severe bacterial infections in pregnant mares. keywords: ciprofloxacin, fetal, fluoroquinolone, cartilage 451 clinical theriogenology • volume 11, number 3 • september 2019 452clinical theriogenology • volume 11, number 3 • september 2019 omniblank: subfertility in a stallion caused by a genetic mutation affecting the acrosome reaction subfertility in a stallion caused by a genetic mutation affecting the acrosome reaction katelyn kimble, charles love college of veterinary medicine, texas a&m university, college station, tx stallion subfertility is a major source of reproductive and economic loss in the horse industry.1 diagnosis is often through conventional methods, e.g. evaluation of sperm motility and morphology, but occasionally routine testing is inadequate when stallions exhibit normal sperm quality and no apparent cause for subfertility.2 semen samples (fixed and frozen) of a 5 year old thoroughbred stallion were received by mail and evaluated for morphology and dna quality. approximately 6 days later, a coolshipped semen sample was assessed for sperm motility, morphology, and viability. breeding record evaluation (until may 7, 2018) indicated that the stallion impregnated 17/37 (46%) mares in 3.11 cycles/pregnancy (32% pregnancy rate per cycle). pregnancy rate was higher in march (6/14; 43%) when fewer mares were bred than april (8/25; 32%). results of initial sperm evaluation (normal: 69% and abnormal dna: 12%) and cool shipped sample (total motility: 76%, normal sperm: 54%, and viability: 72%) indicated good to excellent sperm quality, inconsistent with level of subfertility. initial diagnosis was overbreeding, supported by higher fertility of mares bred early in breeding season, compared to mares bred later in breeding season, when more mares were being bred. following breeding season, a hair sample from the stallion was analyzed and found to be positive for impaired acrosome reaction (iar) susceptibility gene (fkbp6). test for iar was not considered initially because the level of fertility in this individual, while subfertile, was higher than a previous report (< 20% per cycle).2 this gene (fkbp6) has implications as a potential location for single nucleotide polymorphisms associated with iar which can lead to stallion subfertility not explained by poor sperm quality.1,3,4 additionally, this stallion was unusual compared to previous stallions with this condition because his fertility was higher. keywords: stallion, impaired acrosomal reaction, fkbp6 references 1. schrimpf r, metzger j, martinsson g, et al: implication of fkbp6 for male fertility in horses. reprod dom anim 2014;50:195-199. 2. varner dd, brinsko sp, blanchard tl, et al: subfertility in stallions associated with spermatozoal acrosome dysfunction. proc annu conv am assoc equine pract 2001;47:227-228. 3. raudsepp t, mccue me, das pj, et al: genome-wide association study implicates testis-sperm specific fkbp6 as a susceptibility locus for impaired acrosome reaction in stallions. plos genet 2012;12: e1003139. 4. kjöllerström h, do mar oom m, chowdhary b, et al: fertility assessment in sorraia stallions by sperm‐fish and fkbp6 genotyping. reprod dom anim 2016;51:351-359. 499 clinical theriogenology • volume 11, number 3 • september 2019 500clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 1 contact stuart meyers smeyers@ucdavis.edu supplemental data for this article can be accessed here. © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10670, http://dx.doi.org/10.58292/ct.v16.10670 review report intracytoplasmic sperm injection zone: insights and applications from a university-based assisted reproduction laboratory katarzyna malin,a alejandro de la fuente,a,b thadeu de castro,a soledad martin-pelaez,a margo verstraete,a pouya dini,a stuart meyersb* adepartments of population health and reproduction, banatomy, physiology, and cell biology, school of veterinary medicine, university of california, davis, ca, usa abstract intracytoplasmic sperm injection (icsi) has become one of the most important tools used for in vitro production of embryos (ivp) in equine reproduction management programs around the world. this procedure is often performed in the us for several breeds and is used primarily to optimize foal production for broodmares and performance mares but also for stallions with limited semen availability or poor semen quality. as such, there are a limited number of us laboratories capable of using this method, which requires advanced training of personnel and specialized equipment. many veterinarians and breeding farms currently aspirate ovarian follicles in cycling mares and ship oocytes to icsi-capable laboratories, where embryos can be produced and typically vitrified for ultralow temperature storage and transport for transfer to recipient mares and establishment of surrogate pregnancies. the veterinary assisted reproduction laboratory in the school of veterinary medicine at the university of california, davis, maintains a 3-dimensional approach to equine assisted reproductive technology. we offer commercial solutions to breeders, educational advice and training to other laboratories, veterinarians, and visiting scholars. moreover, as a research laboratory, our objective is to analyze and apply observations from ivp to elucidation of complex developmental problems such as embryonic and fetal loss. in 2018, we reported the birth of the first foal produced at uc davis using icsi, and we have since developed a commercial icsi program for practitioners and horse breeders in the us. today, our laboratory receives thousands of immature oocytes for icsi sessions every year, with an average of 2 embryos per mare-session. our research is focused on molecular, cellular and genetic aspects of gamete biology, perifertilization events, and early equine development using an array of tools including advanced microscopy, sequencing, and time-lapse imaging of developing embryos. in this review, we have highlighted our laboratory’s current methods for commercial equine ivp and research and clinical studies conducted to optimize ivp in horses. keywords: equine embryo, intracytoplasmic sperm injection, noninvasive time-lapse microscopy introduction1 in the late nineteenth century, experiments in artificial insemination in animals were conducted in multiple research centers across europe. the pioneering work of ilya ivanov and walter heape inspired an emerging generation of scientists to further develop this and other gamete techniques, with the first american artificial insemination (ai) cooperative in cattle *recipient of 2023 bartlett award, editor appreciates author’s willingness to prepare and submit a manuscript for peer review; author’s bartlett address is posted in the following journal url. https://doi.org/10.58292/ ct.v15.9776 established in 1938.1 it took almost a century to commercialize embryo transfer and embryo transportation; embryo vitrification was introduced only in 1996.2,3 today, advanced technological solutions such as ivp, biopsy for preimplantation genetic diagnosis, embryo vitrification, and time-lapse microscopy have become routine procedures for equine embryologists  in their endeavors to optimize pregnancy outcomes and further understand equine development, with the potential for involvement of artificial intelligence in the near future.4 intracytoplasmic sperm injection (icsi) was initially developed in 19925 as a natural development of subzonal mailto:smeyers@ucdavis.edu http://dx.doi.org/10.58292/ct.v16.10670 http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10670 https://doi.org/10.58292/ct.v15.9776 https://doi.org/10.58292/ct.v15.9776 2 citation: clinical theriogenology 2024, 16, 10670, http://dx.doi.org/10.58292/ct.v16.10670 insemination.6 the resulting manuscript was published in the medical journal lancet, which highlighted the worldwide impact of advanced reproductive technologies. as of 2024, icsi remains the only commercially reliable method for ivp in horses.7-10 in this species, several laboratories in the us and europe routinely report ~ 20-25% success rate in blastocyst production with an average of ~ 2 blastocysts per successful icsi session for most mares.11-13 however, there has been at least one recent report regarding successful conventional in vitro fertilization (ivf) in horses with viable offspring obtained in a research setting.14 the veterinary assisted reproduction laboratory in the school of veterinary medicine at the university of california, davis maintains a 3-dimensional approach to equine assisted reproductive technology. first, as a clinical service, we offer reproductive solutions to horse breeders and veterinarians (transvaginal follicular aspiration, oocyte in vitro maturation, postmortem gamete retrieval, in vitro embryo production, embryo biopsy and vitrification, intracytoplasmic sperm injection, cell isolation and storage for posterior cloning, and embryo transfer services). secondly, as an academic institution, we provide educational support and training to veterinarians and visiting scholars. finally, as a research facility, we aim to apply conclusions and findings from in vitro production of embryos to the elucidation of complex developmental problems such as embryonic and fetal loss. in the last ~ 5 years, our laboratory has placed special emphasis on the study of embryo morphokinetics using noninvasive imaging incubation technology, which allowed us to monitor embryo development in near real-time without removing embryos from the incubator to visualize them.13 this innovation provides insight into not only fundamental equine biology, which is applicable to other species, but also allows us to optimize equine embryo production, serving the horse industry directly. in each icsi session, time-lapse imaging provides an insight into the development from zygote to blastocyst stage, when the embryo is transferable into a recipient mare, and predict which embryos are most likely to develop to a healthy foal. we have also been focusing on advanced microscopy techniques, such as conventional and laser-scanning confocal immunofluorescence as well as genomic and transcriptomic approaches to gametes, embryos, and single-cell dynamics. in 2018, we reported the birth of the first foal produced at uc davis using icsi, and we have since focused on developing a commercial icsi program for horse breeders in the us. today, our laboratory performs hundreds of icsi sessions every year, with an average of 2 embryos per mare-session. an essential value of icsi is that the shipment of oocytes and sperm does not require transportation of the animal, and valuable mares and stallions are not subjected to the risks and costs associated with transportation. moreover, multiple embryos can be obtained in a single icsi session and transferred into recipients located in different parts of the country. the demand for this service in the us and abroad is continuing to increase. in this review, we discuss our practical approach to icsi, lessons learned, and potential applications of this reproductive technology. laboratory protocols, general commentary embryologists in our laboratory are responsible for all media preparation, which is performed in a laminar-flow biosafety cabinet using reported preparations for maturation medium (discussed below).11,15 since our main interest is optimizing the use of gametes and ultimately producing more embryos, it is necessary to maintain the highest standard of hygiene as to not pose a threat to gamete, zygote, or embryo health. positive pressure ventilation in the icsi laboratory is useful to prevent the ingress of airborne particles when the entry door is opened. we refrain from the use of ethanol-containing solutions and instead, we use oosafe® disinfectant (oosafe, inc, hingham, ma 02043) for cleaning surfaces. access is restricted to essential personnel, who use dedicated sets of shoes, scrubs, and laboratory coats when working in the icsi room. commonly, we avoid using gloves; thus, rigorous hand-scrubbing is required before entering the room. use of scented soap, perfume, or lotions is forbidden, even when not working directly with embryos/gametes. we maintain a sticky floor mat by the door to control foot-carried lint and debris, and end-of-day daily sweeping for dust and debris using a swiffer floor mop with dry pads sprayed with oosafe® disinfectant after all incubators are closed for the night. when gametes or embryos are outside the incubator, dim light is used, and the door remains closed. science of intracytoplasmic sperm injection in broader terms, there is a universal scheme for icsi, regardless of the protocol or species specificity: gamete acquisition, in vitro maturation (ivm), sperm selection, fertilization using microinjection of a single sperm, in vitro culture (ivc) to the blastocyst stage, vitrification (optional), and embryo transfer into recipient mares. although commercial media and their strictly prescribed protocols are readily available, embryo production methods in our laboratory are based on previously reported equine culture systems.11,15 for purposes of this discussion, we refer to ‘holding’ medium as an oocyte transportation culture medium that is commercially available and used for oocyte shipping to our laboratory, and in which metabolic state is supported but does not allow oocytes to develop or otherwise mature. after a brief period, typically 24 hours in a commercial holding medium, the oocytes are transferred to a medium that supports ivm from the mi stage (no visible polar bodies) to the mii stage of gametogenesis, revealing a single polar body that indicates the resumption of meiosis. at this stage of development, the oocyte is prepared to undergo fertilization by a single sperm. although, many aspirations are performed in-house at uc davis, our labortory, like most icsi facilities, receives numerous shipped oocytes from referral practitioners performing aspirations in distant locations. we also obtain oocytes from postmortem ovarian retrieval. after ivm, both oocytes and sperm are evaluated. oocytes that extruded their first polar body and motile sperm of normal morphology are then moved into the icsi dish and the microinjection procedure is performed by an embryologist. in a typical session, the embryo is vitrified when it reaches the blastocyst stage of development at 7 to 10 days. vitrified embryos are then express-shipped to veterinarians managing recipient mares for thawing and embryo transfer. in special circumstances, alternative options are available depending on the animal’s status. although rare, we retrieve ovaries at the time of or shortly after euthanasia. inevitably, postmortem oocyte recovery has a negative impact on the quality of oocytes that can be reflected downstream on the success of the icsi procedure. the most critical factors for icsi success in such cases were the number of collected oocytes http://dx.doi.org/10.58292/ct.v16.10670 citation: clinical theriogenology 2024, 16, 10670, http://dx.doi.org/10.58292/ct.v16.10670 3 and euthanasia being due to a nonacute medical condition.16 furthermore, pentobarbital penetrates follicles (detectable soon after injection), and our research suggested that this may  impede both bovine and equine in vitro embryo production.17 acquiring gametes for intracytoplasmic sperm injection transvaginal aspiration of oocytes transvaginal aspiration (tva) of oocytes or ovum pick-up (opu) is necessary to obtain oocytes from ovarian follicles. it is performed on standing, sedated mares, using an ultrasound-guided transvaginal probe with needle channel attached to an exapad ultrasound scanner (imv technologies, inc.). our group uses a rocket craft duo-vac vacuum suction pump (rocket medical plc, uk) connected to a 12g x 25” double-lumen needle that is manually directed into each follicle (5-20 mm diameter) which is punctured and followed by manual needle agitation (necessary to scrape the follicular wall) accompanying follicular lavage. each follicle is lavaged by injecting a small volume adequate to visibly expand the follicle and subsequently draining the fluid while observing the ultrasound flat-screen display. although numerous factors contribute to variations in pump settings (length and diameter of tubing, needle length and inner diameter, and mare’s movement/sedation status), we typically set the pump to aspirate at a flow rate of 20 ml/40 seconds, but the pressure measurement can vary under those circumstances. follicles > 20 mm diameter are aspirated after the small to medium follicles (5-20 mm), and as the collapse of larger follicles distorts the ovarian size and shape, they are best aspirated last to optimize the smaller follicle aspiration. although mares with very active ovaries with small to medium antral follicles are recommended as oocyte donors (10-15 follicles of 5-20 mm diameter), this is not always possible. when less than an optimal number of follicles are visualized (typically < 5-10 visible follicles), we recommend postponing transvaginal aspiration of oocytes by 2 weeks to allow for the mare’s natural cycle to recover and generate a new cohort of aspiratable follicles, and then rechecking the mare’s ovarian activity.18-20 however, with owner consent, we occasionally schedule mares with ~ 3 immature follicles of 5-20 mm diameter; we make sure the owner understands that these mares typically yield lower numbers of transferrable embryos. for optimal results, which include at least 1 transferrable blastocyst, our recommendations are that between both ovaries, at least 12 follicles with 10-20 mm diameter be visualized at the day of aspiration. regardless of the outcome, at least 14 days between sequential aspirations are required for health and safety concerns for donor mares. during that period, a new wave of follicles can develop as well. we can obtain oocytes from antral follicles throughout the year, including fall and spring transition periods, but this  is not possible from most winter anestrous mares in northern california. for a tva procedure at our facilities, mares are placed into a well-padded stock for standing restraint. perineum, vulva, and tail base are aseptically scrubbed using a mild soap such as ivory soap. detomidine (0.01-0.02 mg/kg) and butorphanol (0.01-0.02 mg/kg) are used for standing sedation. intravenous buscopan (0.32 mg/kg) is given to further relax the rectum. the procedure takes 30-45 minutes, but mares are usually monitored overnight prior to release from the hospital. although safe, our research revealed that there was a significant transitory increase in serum lactate, total protein, and peritoneal neutrophil count after tva. furthermore, mares can develop low-grade fever, mild colic, and transient anorexia.21 from a safe practice perspective, we recommend 24 hours posttva monitoring of all mares undergoing the procedure for appetite, feed intake, output of feces and urine, and signs of discomfort. as a prophylactic method, we give oral equisul (sulfadiazine and trimethoprim, 24 mg/kg) prior to the procedure and 2 extra doses every 12 hours after the procedure (total of 3 doses). we also use flunixin meglumine (banamine®, 1.1 mg/kg) before the procedure and an extra dose 12 hours prior to a mare’s discharge from the veterinary medical teaching hospital. transvaginal aspiration of oocytes can be performed by a single highly experienced person; but as a teaching hospital, we prefer a team approach to provide training in all aspects of the process for residents and other veterinarians. our clinicians follow an established protocol, with 2 veterinarians operating the rectal and vaginal part of tva, respectively. first person maneuvers the ovaries transrectally and then manipulates 1 ovary to fix it against the transvaginal probe for optimal visualization. second person operates the needle and aspiration pump, being responsible for the puncture and scraping of each follicle (figure 1). the double-lumen needle allows a separate channel for inflow and outflow from follicles. the latter person typically operates the vacuum pump for follicular lavage using a foot pedal. a third person manually manages the fluid injection into the punctured follicle using equiplus opu recovery medium (minitube). all procedures are under strict aseptic conditions with care to maintain them at all times. typically, 5-20 oocytes are retrieved per tva session (12 ± 7 [average ± sd]; n = 136). in the case of a single mature preovulatory follicle (> 35 mm) present on 1 or both ovaries, we will aspirate that follicle and search for an oocyte. since such an oocyte has likely already started maturation in vivo, they generally have a higher developmental potential figure 1. illustration of a mare’s ovary having 2 immature antral follicles in addition to a corpus luteum. an aspiration needle tip is depicted within the larger follicle during aspiration and scraping (nonscale representation). http://dx.doi.org/10.58292/ct.v16.10670 4 citation: clinical theriogenology 2024, 16, 10670, http://dx.doi.org/10.58292/ct.v16.10670 with icsi injection of sperm, but the blastocyst yield per mare is slightly lower. after tva, the aspirated fluid is transported to laboratory at ambient temperature, filtered using opu recovery medium and an emcon embryo filter that is carefully rinsed and flushed into large petri dishes which are then searched using a bright-field stereomicroscope. identified oocytes are then placed into holding medium (see below) overnight (12-16 hours) in sterile glass vials at ambient temperature (22oc) until they are placed into maturation medium in a trigas environment for 27-30 hours in droplets of maturation medium at a ratio of 10 µl per oocyte under light paraffin oil at 38.2oc in a humidified atmosphere of 5.8% co2, 5%o2, and 89.2% n2. oocytes are then denuded from cumulus cells and evaluated for extrusion of the first polar body into the perivitelline space, indicating that prophase ii of meiosis has reached and oocytes are ready for fertilization by sperm microinjection. oocyte transport typically, > 80% of oocytes used for icsi are shipped to our facility from veterinarians in the us. our laboratory receives oocytes in sterile glass vials in 1 or 3 ml volume of emcare (icpbio reproduction, spring valley, wi) or equihold (minitube, verona, wi) commercial media, as they are available to most clinics shipping oocytes, and oocytes are stable in these media at ambient temperature for at least 24 hours.8,11 oocytes are express-shipped overnight in holding medium with minimal air in either a battery-powered shipping incubator (microq technologies, mesa, az 85204) or in the hamilton biovet equitainer or equocyte shippers (ipswich, ma) using 22oc insert cans (figure 2). generally speaking, isothermal devices maintain stable temperature that is critical to oocyte’s survival and further developmental competence. we recently observed that microq and equitainer supported oocyte stability during shipping and their subsequent maturation, cleavage, and blastocyst rates, comparing them to those held overnight in the laboratory in a battery-powered incubator. interestingly, the overall blastocyst rates were consistent across all devices, but oocytes transported in equocyte device had a lower maturation rate.22 cryopreservation of oocytes remains an active research area for several laboratories; however, vitrification of equine oocytes23–25 has not yet yielded commercially acceptable results. regardless, viable offspring from such a pregnancy was reported in 2018.26 sperm handling nearly all icsi sessions at our laboratory are performed with frozen/thawed sperm. although full motility cannot be restored from cryopreserved sperm, this technique requires a very small amount of thawed sperm for successful fertilization of all oocytes. this is especially important in cases where issues with collection or consistent low-quality sperm occur, and when limited numbers of straws are available (i.e. from deceased or castrated stallions). in our observations, some stallions perform better than others, having higher fertilization, cleavage, and blastocyst rates after icsi sessions. furthermore, certain mares paired with specific stallions achieve highly variable blastocyst rates, depending on the specific breeding cross. similarly, different batches of cryopreserved semen from the same stallion can be of dramatically different quality. typically, when possible, we request batches of frozen semen from stallion management that have known icsi success rates. our laboratory maintains a commercial sperm bank from highly desired sires for the mare clients’ convenience, but every icsi cycle requires legally binding contracts. our optimized technique allows us to perform straw cuts such that only 1/12 to 1/8 of a straw is used in each session, conserving semen samples that oftentimes are limited and highly valuable. for the same reason, we try to schedule oocyte collections on the same day so they can be fertilized with sperm from a single straw cut. the cutting is performed under liquid nitrogen to prevent thawing of the remaining straw segment. in our laboratory, we perform swim-up as the selection method for high-quality sperm. swim-up is performed by layering the thawed sample underneath 2.0 ml g-mops (vitrolife, inc. sweden) containing 10% fetal bovine serum (fbs) and incubated for 30 minutes at 37oc. the sperm are aspirated from the upper swim-up layer after incubation, and their quality is evaluated by the embryologist. if needed due to poor quality, another sample can be readily processed. selected sperm are suspended in the icsi dish in a droplet of polyvinylpyrrolidone (pvp, 7%), an inert macromolecule that helps prevent sperm agglutination and facilitates sperm aspiration into the injection needle. it is important to note that the semen is thawed and processed after oocytes are denuded and maturation is assessed. intracytoplasmic sperm injection procedures oocyte in vitro maturation immature oocytes are placed in droplets of maturation medium consisting of 54.0% dulbecco’s modified eagle’s a b c figure 2. isothermic shipping containers for equine oocyte transport to the icsi laborary. a. hamilton research equitainer (blue) and equocyte (red) transporters. b and c. microq battery-powered warmed transporter system. http://dx.doi.org/10.58292/ct.v16.10670 citation: clinical theriogenology 2024, 16, 10670, http://dx.doi.org/10.58292/ct.v16.10670 5 medium (dmem)/f-12 with 15 mm hepes, 25 mg ml-1 gentamicin, 36.0% global medium (lifeglobal), 0.1 mm sodium pyruvate, 6.0% fetal bovine serum (fbs; f2442; sigma), 10 ml ml-1 insulin–transferrin– selenium (its) solution, 10% dominant stimulated follicle follicular fluid, 8.8 mu ovine fsh (national hormone and peptide program) and 1.1 mu ml-1 porcine somatotropin (harborucla research and education institute), as described.11,13 the morning after aspiration and overnight holding, protected from light, oocytes are moved from holding medium in 22oc to 38.2oc in a triple gas incubator using n2 gas to lower oxygen tension (5.8% co2, 5%o2, and 89.2% n2) for maturation. oocytes remain in maturation medium for 27-30 hours, then cumulus oocyte complexes are denuded using hyaluronidase (hyaluronidase 0.2%/ml [sigma aldrich h3506-1g]; 2-minute incubation) and mechanical pipetting in a 4-well dish. the first polar body, if present, can then be visualized between the oolemma and zona pellucida, known as the perivitelline space, using a magnification of 20 x with brightfield optics on an olympus ix-70 microscope. denuding the surrounding cumulus cells is necessary to visualize the oolemma and perform icsi. this is in contrast to traditional ivf, in which the cumulus supports, and is necessary for fertilization, as it provides natural sperm selection and prevents polyspermy. after the maturation period of a batch of oocytes, they cannot be assumed to be in the same stage of meiosis. occasionally, we observe that even 10 minutes of additional ivm culture may be sufficient for the first polar body extrusion to occur if not observed during the initial assessment. on the other hand, some oocytes might already be atretic, and thus fail to extrude a polar body regardless of any additional time. the appearance of the first polar body defines the meiotic stage of the oocyte and consequently its readiness to be fertilized by injection of a single sperm. all oocytes presenting this morphology are injected with a single sperm (figure 3). our laboratory uses a time-lapse imaging system to observe oocyte maturation inside the incubator which has allowed us to observe cumulus expansion (figure 4) and polar body extrusion in near-real time. we have measured rates of cumulus-oocyte complex (coc) expansion. similarly, we elucidated the dynamics of genetic expression in the cumulus cells and oocytes during maturation in vitro and in vivo after induction with gnrh analogs. this unraveled important biological pathways critical for the proper development of the oocyte and provided some clues about the main difference between the in vitro and in vivo environments.27 intracytoplasmic sperm injection for every icsi procedure, we prepare a dish with microdroplets of g-mops-10% fbs for injection and pvp 7% for semen pickup; both of these media are buffered to maintain the ph while they are outside the incubator environment. although the micromanipulator-microscope system (figure 5) is equipped with a heating stage, each minute spent outside the incubator decreases the chances of obtaining blastocysts, thus this step needs to be performed efficiently and quickly. when the swim-up process ends and a good quality sample of sperm is obtained, mature oocytes are individually placed in microdroplets (4 μl) of g-mops 10% fbs that are laid out in the icsi dish, while 3 μl of semen is placed in the droplet of pvp (sperm droplet). the first step is the immobilization of the targeted sperm by pressing or trapping the midpiece/ tail with the injection needle against the bottom of the dish. this immobilizes the sperm and creates a visible bend in the tail. then, the sperm is picked up with the injection needle by aspirating the tail first. next, the dish is repositioned to center the image in a droplet containing one of the mii oocytes, and with the holding pipette, the oocyte is held from one side (9 o’clock). oocyte is rotated in a manner that the polar body is situated at either 6 or 12 o’clock position, leaving the other side (3 o’clock) free for inserting the injection needle and depositing the sperm. in our laboratory, we use conventional icsi that requires an injection needle sharp enough to penetrate the zona pellucida and oocyte plasma membrane (oolemma). some laboratories report improved embryo cleavage and subsequent development using a piezo drive that gently ‘drills’ the injection pipette into and through the zona pellucida while maintaining the membrane integrity of the oolemma and inducing activation of the oocyte to some extent.7,11 in our hands, the piezo drive has not resulted in improved blastocyst rates for equine zygotes and appears to be highly dependent on the skills of the embryologist performing icsi. after the sperm is injected, all steps are repeated for all oocytes in the dish; then, they can be moved into the embryo culture media dish in embryo culture medium (ec) and placed back in the incubator. it is critical that the injection itself is performed with a sense of urgency while still being careful to not cause mechanical damage to the oocyte, and the fertilized oocytes be placed in ec (54% dmem/f-12, 40% global, 6% fbs, 10 µl/ml1 its solution, and 0.1 mm sodium pyruvate) immediately after the microinjection. in vitro culture of equine embryos our laboratory uses time-lapse embryo imaging system (miri®tl, esco technologies) for observation of cell division timing and cellular integrity during embryo culture immediately after the icsi procedure. this noninvasive system captures bright-field images at 5-minutes intervals in 5 various focal planes using a single red light-emitting diode (635 nm), with total light exposure of 0.064 second per captured image. figure 3. mature oocyte held by holding pipette at the 9 o’clock position. first polar body is observed in the bottom part of the oocyte (6 o’clock), injection needle holding a single sperm about to be injected in the ooplasm. this image was obtained in an olympus microscope (ix-70) 20 x objective. http://dx.doi.org/10.58292/ct.v16.10670 6 citation: clinical theriogenology 2024, 16, 10670, http://dx.doi.org/10.58292/ct.v16.10670 images are then assembled into videos that can be used for retrospective morphokinetic analysis in near real-time. there are multiple benefits to time-lapse embryo culture, but the most important is that continuous embryo evaluation is possible without an interruption to remove embryos from the incubator environment, except for day 4, when the culture medium is replaced. embryos are vitrified when blastocysts are visualized to expand (zona thinning) along with the presence of pulsation (see below description and video). from our general observations, a normally developing embryo is expected to reach the blastocyst stage on day 7, and embryos that take longer have lower chances for pregnancy.28 the smaller the blastocyst, the higher the potential viability, thus throughout their monitoring, we make sure to vitrify as soon as possible once a blastocoel is observed in conjunction with pulsing. our laboratory has been researching time-lapse embryo evaluation since 2014, and in addition to documenting key cell division and mitotic events at specific hours after icsi a b c figure 4. cumulus expansion during the in vitro maturation culture for 27-30 hours. a. initial state of the cumulus surrounding the oocyte as a compacted group of cells known as the corona radiata (0 hours). b. cumulus expansion at mid-culture, or 11.5 hours expansion of cumulus layer during oocyte maturation. c. end of culture, at ~ 27 hours, demonstrates the maximum expansion of the cumulus cell layer. images were obtained in the miritl® time-lapse incubator. a b c figure 5. a. olympus ix-70 with digital eppendorf micromanipulators and microinjectors and brightfield optics and heated stage. b. microscope stage during setup for icsi with injection dish under sterile embryo grade mineral oil. c. injection dish ready for injection with oocytes in g-mops medium, swim-up-selected sperm in 7% pvp medium. http://dx.doi.org/10.58292/ct.v16.10670 citation: clinical theriogenology 2024, 16, 10670, http://dx.doi.org/10.58292/ct.v16.10670 7 (figure 6a-f), we have identified several morphokinetic parameters of successful blastocysts that are consistent with blastocyst formation and pregnancy success rates.13,29 we have described 2 cleavage-related phenomena that are closely associated with the completion of development to a blastocyst. first, a precleavage event known as cytoplasmic extrusion occurs in equine zygotes, whereby extraneous cytoplasmic zygotic material is released into the perivitelline space from the single-celled zygote just before cleavage division (figure 6b). embryos that fail to become blastocysts have a lower rate of this precleavage extrusion.13 second, as the embryo reaches the late morula and early blastocyst stage, we routinely observe the successful embryo begin a pulsation sequence30 at regular intervals (0.22 hour), in which an overall growth in embryo diameter occurs with expansion and subsequent contraction. the pulsation also results in thinning of the zona pellucida and can only be observed using the time-lapse videos rather than single observations. we have reported this dynamic phenomenon to occur in both in vitro and in vivo derived embryos (flushed from the uterus of mares at 6.5 days postovulation and cultured in the tl system).30 while irregularities in mitosis can figure 6. time-lapse development in an equine zygote after icsi (a) and progressing to blastocyst stage (b-f) as visualized in the miritl® incubator. a. equine oocyte immediately after sperm injection and placement into time-lapse incubator. we observe cellular extrusion (b) to occur soon before the first cleavage where the extruded material occupies the perivitelline space. in (c), 2 blastomeres are visualized alongside the extruded material, giving an impression of a third blastomere. d. formation of a 4-cell embryo. however, in contrast to human embryos, it quickly becomes impossible to judge the number of blastomeres beyond the 8-cell stage due to the equine blastomeres’ dark color (e, f). f. icsi-derived blastocyst. video. time-lapse development in an equine early embryo. to view the video, read the html version of the article where the video is embedded. http://dx.doi.org/10.58292/ct.v16.10670 8 citation: clinical theriogenology 2024, 16, 10670, http://dx.doi.org/10.58292/ct.v16.10670 often indicate aneuploidy or polyploidy in developing embryos, embryo pulsing is linked to successful implantation in human embryos.31,32 our observations are in agreement with these reports.29,30 the tl system has allowed researchers to document the embryo development timeline for each cell division of growing human, bovine, and equine embryos.13,31,33-35 as mentioned above, studies from our laboratory and others have determined the precise timing of embryo cleavage from a single cell fertilized zygote from the moment of sperm injection to 2-cell, 4-cell, 8-cell, morula and blastocyst.13 recent work from our laboratory has extended the observations of the first few equine cell divisions and their influence on blastocyst development to that of equine pregnancy outcomes. with ~ 70 pregnancies from transferred vitrified blastocysts, we observed that those stemming from embryos with low rates of abnormal early-stage cleavages (at the 2-cell stage) were sustained at a higher percentage. these pregnancies demonstrated a lower rate of pregnancy loss further on during pregnancy demonstrating that early cleavage abnormalities likely lead to subsequent pregnancy loss.28 we cannot, at this point, determine the cost-effectiveness of the time-lapse system as this new technology for equine development has not been fully evaluated for adequate numbers of clinical pregnancy outcomes. blastocyst vitrification and thawing for embryo transfer a great majority of the blastocysts produced in our laboratory are vitrified and stored in liquid nitrogen or shipped to other clinics. embryos are vitrified as soon as they reach the blastocyst stage (i.e. a clear trophectoderm and blastocoele structures, and pulsation can be observed), particularly if no recipient mares are available or the client desires storage of the embryos for a later transfer. we use a 2-step vitrification protocol with 1.5 m ethylene glycol (solution a) and 7.0 m ethylene glycol, and 0.6 m galactose (solution b). cryolock® vitrification devices (biotech, inc, alpharetta, ga) are used to store the frozen blastocysts which are then stored in individual canes in liquid nitrogen dewars. vitrified embryos, shipped overnight in dry shipping liquid nitrogen containers, are thawed and warmed after a simple 1-step, but effective, field protocol, which only requires the use of a commercial holding media by veterinarians in the field. a sterile, 30-mm culture dish is filled with room temperature holding media and the cryolock® is quickly moved from liquid nitrogen to the dish, where the embryo will thaw and remain protected from the light for ~ 5 minutes to allow reexpansion before loading into a 0.25-ml embryo transfer straw. then, the embryo can be loaded into an embryo transfer gun. summary and future plans the modern equine icsi laboratory has become essential for complete reproductive management of horses in the us and around the world. oocytes can be readily express-shipped to any icsi laboratory using a variety of shipping containers and media. the success of icsi depends on a multitude of factors discussed, including mare and stallion effects, media, culture conditions, environmental conditions, technical experience, knowledge, and hygiene. the time-lapse embryo imaging system used in our laboratory allows us to optimize our commercial and research programs, and it also delivers an abundance of embryo development data for individual embryo analysis. this allows us to track each embryo’s cell division timing that provides detailed data for each embryo’s success from fertilization to establishment of pregnancy. although there is no universally-agreed upon grading system for equine oocytes or in vitro produced embryos, recent research from our laboratory indicated that video morphokinetics may provide a novel method to dynamically grade equine embryos that correlates well with pregnancy rates after embryo transfer.13,28 our main field of interest for future research is the series of complex events taking place around the first cleavage division,13 such as cytoplasmic extrusion and pronuclei formation, which seem to be good predictors of the time of cleavage, aneuploidy, and overall developmental potential. in addition to time-lapse characterization of early embryonic events, we and others utilize immunofluorescence and transcriptomics13,25,27,36,37 to broaden our insight into this period of equine development. conflict of interest none to report references 1. ombelet w, van robays j: artificial insemination history: hurdles and milestones. facts views vis obgyn 2015;7:137-143. 2. hochi s, maruyama k, oguri n: direct transfer of equine blastocysts frozen-thawed in the presence of ethylene 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em, sessions dr: in vitro production of equine embryos. j equine vet sci 2012;32:367-371. doi: 10.1016/j.jevs. 2012.05.054 9. choi yh, roasa lm, love cc, et al: blastocyst formation rates in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. biol reprod 2004;70:12311238. doi: 10.1095/biolreprod.103.023903 10. hinrichs k, choi yh, walckenaer be, et al: in vitro-produced equine embryos: production of foals after transfer, assessment by differential staining and effect of medium calcium concentrations during culture. theriogenology 2007;68:521-529. doi: 10.1016/ j.theriogenology.2007.04.046 http://dx.doi.org/10.58292/ct.v16.10670 https://doi.org/10.1016/s0093-691x(96)00292-0 https://doi.org/10.1016/s0093-691x(96)00292-0 https://doi.org/10.1006/cryo.1996.0030 https://doi.org/10.1006/cryo.1996.0030 https://doi.org/10.1038/s41598-023-29319-z https://doi.org/10.1038/s41598-023-29319-z https://doi.org/10.1016/0140-6736(92)92425-f 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the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation†. biol reprod 2022;107:1551-1564. doi: 10.1093/biolre/ioac172 15. metcalf es, masterson kr, battaglia d, et al: conditions to optimise the developmental competence of immature equine oocytes. reprod fertil dev 2020;32:1012-1021. doi: 10.1071/rd19249 16. lorenzen e, carstensen a, winther m, et al: factors affecting post-mortem production of equine icsi blastocysts. j equine vet sci 2023;125:104660. doi: 10.1016/j.jevs.2023.104660 17. martin-pelaez s, rabow z, de la fuente a, et al: effect of pentobarbital as a euthanasia agent on equine in vitro embryo production. theriogenology 2023;205:1-8. doi: 10.1016/ j.theriogenology.2023.04.002 18. iacono e, merlo b, rizzato g, et al: effects of repeated transvaginal ultrasound-guided aspirations performed in anestrous and cyclic mares on p4 and e2 plasma levels and luteal function. theriogenology 2014;82:225-231. doi: 10.1016/ j.theriogenology.2014.03.025 19. velez ic, arnold c, jacobson cc, et al: effects of repeated transvaginal aspiration of immature follicles on mare health and ovarian status. equine vet j 2012;43:78-83. doi: 10.1111/j.2042-3306.2012.00606.x 20. cuervo-arango j, claes an, stout tae: mare and stallion effects on blastocyst production in a commercial equine ovum pick-up-intracytoplasmic sperm injection program. reprod fertil dev 2019;31:1894-1903. doi: 10.1071/rd19201 21. orellana-guerrero d, dini p, santos e, et al: effect of transvaginal aspiration of oocytes on blood and peritoneal fluid parameters in  mares. j equine vet sci 2022;114:103949. doi: 10.1016/ j.jevs.2022.103949 22. de la fuente a, martin-pelaez s, megehee s, et al: evaluating equine oocyte transportation techniques: a comparative study of maturation, fertilization, and blastocyst rates in equitainer, microq, and incubator systems. in: demetrio d, tesfaye d: editors. reprod fertil dev 2024:36. clayton: proc iets csiro publishing; 2024:1-274. 23. agnieszka n, joanna k, wojciech w, et al: in vitro maturation of  equine oocytes followed by two vitrification protocols and subjected to either intracytoplasmic sperm injection (icsi) or parthenogenic activation. theriogenology 2021;162:42-48. doi: 10.1016/j.theriogenology.2020.12.022 24. maclellan lj, albertini df, stokes je, et al: use of confocal microscopy and intracytoplasmic sperm injection (icsi) to assess viability of equine oocytes from young and old mares after vitrification. j assist reprod genet 2023;40:2565-2576. doi: 10.1007/ s10815-023-02935-4 25. angel-velez d, meese t, hedia m, et al: transcriptomics reveal molecular differences in equine oocytes vitrified before and after in vitro maturation. int j mol sci 2023;24(8):6915. doi: 10.3390/ ijms24086915 26. ortiz-escribano n, bogado pascottini o, woelders h, et al: an improved vitrification protocol for equine immature oocytes, resulting in a first live foal. equine vet j 2018;50:391-397. doi: 10.1111/evj.12747 27. de la fuente a, scoggin c, bradecamp e, et al: transcriptome signature of immature and in vitro-matured equine cumulus-oocytes complex. int j mol sci 2023;24(18):13718. doi: 10.3390/ ijms241813718 28. martin-pelaez s, de la fuente a, meyers s, et al: capturing the miracle: time-lapse imaging of equine embryos reveals cleavage patterns impact pregnancy success. in: demetrio d, tesfaye d: editors. reprod fertil dev 2024:36. proc iets csiro; 2024:1-274. available from: https://www.publish.csiro.au/rd/rdv36n2ab98 [cited 26 january 2024]. 29. burruel v, klooster k, barker cm, et al: abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage. sci rep 2014;4:6598. doi: 10.1038/ srep06598 30. de la fuente a, omyla k, cooper c, et al: embryo pulsing: repeated expansion and contraction of in vivo and in vitro equine blastocysts. j equine vet sci 2023;128:104891. doi: 10.1016/ j.jevs.2023.104891 31. gazzo e, peña f, valdéz f, et al: blastocyst contractions are strongly related with aneuploidy, lower implantation rates, and slow-cleaving embryos: a time lapse study. jbra assist reprod 2020;24:7781. doi: 10.5935/1518-0557.20190053 32. caprell h, connerney m, boylan c, et al: increased expansion and decreased contraction of embryos corresponds to increased clinical pregnancy rates in single fet cycles. reprod biomed online 2019;39:e11-e12. doi: 10.1016/j.rbmo.2019.07.023 33. sugimura s, akai t, imai k: selection of viable in vitro-fertilized bovine embryos using time-lapse monitoring in microwell culture dishes. j reprod dev 2017;63:353-357. doi: 10.1262/ jrd.2017-041 34. lewis n, schnauffer k, hinrichs k, et al: morphokinetics of early equine embryo development in vitro using time-lapse imaging, and use in selecting blastocysts for transfer. reprod fertil dev 2019;31:1851-1861. doi: 10.1071/rd19225 35. lewis n, canesin h, choi yh, et al: equine in vitro produced blastocysts: relationship of embryo morphology, stage and speed  of development to foaling rate. reprod fertil dev 2023;35:338-351. doi: 10.1071/rd22224 36. iqbal k, chitwood jl, meyers-brown ga, et al: rna-seq transcriptome profiling of equine inner cell mass and trophectoderm. biol reprod 2014;90:61. doi: 10.1095/biolreprod.113.113928 37. chitwood jl, burruel vr, halstead mm, et al: transcriptome profiling of individual rhesus macaque oocytes and preimplantation embryos. biol reprod 2017;97:353-364. doi: 10.1093/biolre/iox114 http://dx.doi.org/10.58292/ct.v16.10670 https://doi.org/10.1111/evj.12159 https://doi.org/10.1111/evj.12159 https://doi.org/10.1016/j.jevs.2020.103097 https://doi.org/10.1016/j.jevs.2020.103097 https://doi.org/10.1071/rd19260 https://doi.org/10.1093/biolre/ioac172 https://doi.org/10.1071/rd19249 https://doi.org/10.1016/j.jevs.2023.104660 https://doi.org/10.1016/j.theriogenology.2023.04.002 https://doi.org/10.1016/j.theriogenology.2023.04.002 https://doi.org/10.1016/j.theriogenology.2014.03.025 https://doi.org/10.1016/j.theriogenology.2014.03.025 https://doi.org/10.1111/j.2042-3306.2012.00606.x https://doi.org/10.1071/rd19201 https://doi.org/10.1016/j.jevs.2022.103949 https://doi.org/10.1016/j.jevs.2022.103949 https://doi.org/10.1016/j.theriogenology.2020.12.022 https://doi.org/10.1007/s10815-023-02935-4 https://doi.org/10.1007/s10815-023-02935-4 https://doi.org/10.3390/ijms24086915 https://doi.org/10.3390/ijms24086915 https://doi.org/10.1111/evj.12747 https://doi.org/10.3390/ijms241813718 https://doi.org/10.3390/ijms241813718 https://www.publish.csiro.au/rd/rdv36n2ab98 https://doi.org/10.1038/srep06598 https://doi.org/10.1038/srep06598 https://doi.org/10.1016/j.jevs.2023.104891 https://doi.org/10.1016/j.jevs.2023.104891 https://doi.org/10.5935/1518-0557.20190053 https://doi.org/10.1016/j.rbmo.2019.07.023 https://doi.org/10.1262/jrd.2017-041 https://doi.org/10.1262/jrd.2017-041 https://doi.org/10.1071/rd19225 https://doi.org/10.1071/rd22224 https://doi.org/10.1095/biolreprod.113.113928 https://doi.org/10.1093/biolre/iox114 2011: case report: do peripheral testosterone concentrations differ between dogs with abdominal testicles and scrotal testicles? clinical implications case report: do peripheral testosterone concentrations differ between dogs with abdominal testicles and scrotal testicles? clinical implications amy zwingelberg,a stephen heckler,a augustine peterb alake station pet clinic, lake station, in; bdepartment of veterinary clinical sciences, school of veterinary medicine, purdue university, west lafayette, in summary cryptorchidism is an important clinical problem and it is not fully known if testosterone production is affected because of the location of the testicles. hence the peripheral concentrations of testosterone in a cryptorchid dog were measured and compared to dogs with scrotal testicles. further, the response in testosterone production to human chorionic gonadotropin (hcg) was determined in this cryptorchid dog. baseline testosterone concentrations (209 ng/dl) in the cryptorchid dog were comparable to those in dogs with scrotal testicles (246 + 255.25 ng/dl). testosterone concentrations increased to 400 ng/dl after hcg injection in this cryptorchid dog. two structures that appeared to be testicles were removed during a laparatomy. histologic examination of these structures indicated that one of these structures did not contain testicular tissue, but did contain epididymal and rete testis tissues. peripheral testosterone concentrations determined five weeks after surgery were < 20 ng/dl suggesting that no testicular tissue remained. preand post-injection testosterone concentrations were also < 20 ng/dl during a subsequent hcg stimulation test. background leydig cells, also known as interstitial cells of leydig, are found adjacent to the seminiferous tubules in the testicles. they produce testosterone in the presence of luteinizing hormone (lh). leydig cells are polyhedral in shape, display a large prominent nucleus, and have an eosinophilic cytoplasm with numerous lipid-filled vesicles. mean baseline peripheral concentration of testosterone in non-castrated dogs is 2.44 + 0.41 ng/ml.1 testosterone production may be influenced by factors such as testicular pathology and the location of the testicle in the body.2-4 lower production of testosterone may result in reduced sexual function5 and possibly abnormal sperm production.6 researchers have reported that there is no compensatory production of testosterone by a normal testicle when the contralateral testicle was abnormal4 or absent7. testicular production of testosterone is measured by determining its concentration in peripheral blood.8 testosterone concentrations may be influenced by the time of day when a blood sample is obtained,9,10 the breed of dog,11 and the laboratory and type of assay utilized. gonadotropin releasing hormone (gnrh) or hcg can be administered to test the presence of testicular tissue since these compounds stimulate leydig cells to synthesize testosterone. gonadotropin releasing hormone elicits this response by its ability to stimulate release of lh from the anterior pituitary gland followed by lh action on leydig cells while hcg acts directly on the leydig cells since it has lh-like activity. however, the response in testosterone synthesis by the leydig cells to these compounds may be different12 and further, it is not known if the response is influenced by the testicular histology. cryptorchid conditions have been reviewed and identified as an important clinical problem.13 it is not fully known whether testosterone production is affected in testicles because of their location. some investigators found that the endocrine function in retained testicles is normal despite exposure to core body temperature.14,15 however, one study found that the testosterone concentrations were lower in unilateral cryptorchid dogs compared to dogs with scrotal testicles.16 later these researchers reported that the testosterone concentrations were also lower in bilateral cryptorchid dogs compared to dogs with scrotal testicles.17 it was suggested that the higher temperature due to location of the testicle might decrease the conversion of cholesterol to androgen at the testicular level. a recent study found lower concentrations of testosterone in a bilateral cryptorchid dog.4 hence in this present clinical study the peripheral concentrations of testosterone in a cryptorchid dog were measured and compared to dogs with scrotal testicles. further, the response in testosterone production to hcg stimulation was determined in this cryptorchid dog. clinical theriogenology • volume 3 number 4 • december 2011483 case presentation a five-year-old male boxer was adopted by a client three years prior to presentation from a local humane society. the original owner of the pet informed the humane society that the dog has not been castrated nor had they noticed evidence of testicular decent. after being adopted, the dog was given a complete physical examination in a local veterinary hospital and the client was informed about the potential complications of bilateral cryptorchidism. no further diagnostic tests were performed. the dog had normal behavior and there was no evidence of aggression. however, it was noted that the dog lifted one of its hind limbs while urinating and also marked the fence or the cage. the scrotal and inguinal areas were palpated while the dog was conscious and anesthetized and there was no evidence of testicle(s). the client was informed that abdominal testicle(s) can become neoplastic and also informed that a stimulation test is available to determine if a testicle is present. the client decided to have the test conducted. no attempt was made to collect semen for evaluation. the dog weighed 85 lbs. two blood samples were obtained in the early afternoon, one before and one 60 minutes after 1,000 iu hcg8,18 (chorulon®, intervet/schering-plough animal health, millsboro, de) was administered intravenously. serum testosterone concentrations were determined in a commercial laboratory (clinilab, valparaiso, in) by chemiluminescent enzyme immunoassay (immulite, diagnostic products corporation, los angeles, ca). the testosterone concentrations were 209 and 400 ng/dl before and after injection, respectively. treatment a decision was made to conduct an exploratory laparotomy. prior to induction of anesthesia, the patient was given atropine (1.6 mg sc) and carprofen (150 mg po). anesthesia was induced with ketamine (200 mg iv) and diazepam (10 mg iv). the dog was intubated and maintained on isoflurane for the duration of the procedure. a ventral midline incision was made from the umbilicus to the pubis. the right testicle was visible immediately when the abdomen was opened. the testicular vessels and ductus deferens were clamped and double ligated. tissue that appeared to be the left testicle was more difficult to locate but was visible after the urinary bladder was moved to the right and the intestines were moved cranially. the ductus deferens was visible along with a narrow small white to pale blue object (approximately 1.5 x 1.5 x 0.5 cm). it had a visible ductus deferens cranially and a fibrous band of tissue caudally. the fibrous tissue and ductus deferens were clamped and ligated and the tissue excised. the abdominal incision was closed routinely and postoperative recovery was uneventful. outcome the histopathology report indicated that the specimen obtained from the right side contained testicular, epididymal, and rete testis tissues. there was a diffuse loss of seminiferous epithelium and the tubules were lined by sertoli cells. the leydig cells were found to be mildly hyperplastic. the specimen from the left side contained epididymis and rete testis tissue but lacked testicular tissue. the testosterone concentration determined five weeks after surgery was < 20 ng/dl which was consistent with the absence of testicular tissue. the dog continued to lift its leg while urinating and also marked with urine. to compare baseline testosterone concentrations blood samples were obtained in the early afternoon from seven dogs with scrotal testicles. their ages ranged from 1.5 to 7 years and their weight ranged from 24 to 72 lbs. they belonged to different breeds (1 boxer, 1 beagle, 1 boston terrier, 1 pit bull and 3 mixed breed) and their average testosterone concentrations ranged from 50.3 to750 (mean 246 + 255.25) ng/dl. discussion to minimize the influence of diurnal variation in serum testosterone concentrations, blood samples were collected in the early afternoon. baseline serum concentrations in this cryptorchid dog and in the dogs with scrotal testicles were similar. in other words, the amount of testosterone produced by one abdominal testicle was similar to dogs that had two scrotal testicles. it should be pointed out that three of the control dogs had lower testosterone concentrations (50.3, 58.9, 80.8 ng/dl) than this clinical theriogenology • volume 3 number 4 • december 2011 484 cryptorchid dog. however, it is not known whether these dogs with lower testosterone production had normal testicular histology or poor sperm production. despite the abnormal histopathology of the abdominal testicle, testosterone production was similar to scrotal testicles. it is interesting to note that there is a possibility of reduction in testosterone production by an abdominal testicle19. the correlation between histopathological changes in leydig cells and testosterone production in abdominal testicles needs to be characterized. there was evidence of mild hyperplastic changes in leydig cells in the abdominal testicle. it is not known whether these changes contributed to normal baseline testosterone concentrations in this dog. since only one dog was used in the present study, cautious interpretation of findings is necessary. further studies with adequate number of cryptorchid dogs are warranted. the positive response in testosterone production to an hcg stimulation test suggested the presence of abdominal testicle(s). given the cost of testosterone assays, an hcg stimulation test was not conducted in control dogs. although this test has been used in studies,8,18 reports of its application to detect the presence of retained testicles in clinical cases are scarce. one study found that there was no difference in response to hcg in a bilateral cryptorchid dog.20 in another study gnrh was used in the place of hcg,21 however, the efficacy of one compound over the other is not known. it is not clear how much of an increase in testosterone concentrations one can expect and the best time to detect such an increase. further, it is not known whether this increase will be the same whether the animal has one or two testicles. a clinical investigation is necessary to address these issues. although we did not determine the type of sperm production in this dog prior to surgery, an existing report22 indicated that testosterone concentrations may be lower in azoospermic dogs. these workers reported that the response to gnrh stimulation was delayed and lower in azoospermic dogs compared to dogs with normal sperm production.23 since the histopathology findings of this abdominal testicle suggested that there was a diffuse loss of seminiferous epithelium one can speculate that there would not have been any sperm production. failure to notice changes in urinating and marking behavior despite the removal of testicle is an important clinical observation. while it is not known whether castrated dogs would continue to lift their legs while urinating it is known that some castrated dogs will continue to have marking behavior.24 learning points based on these findings it is evident that an abdominal testicle can produce testosterone concentrations similar to those produced by scrotal testicles even in the presence of histological evidence of lack of sperm production. it is possible to detect the presence of abdominal testicle(s) by determining testosterone concentrations before and after a challenge test using hcg. an important clinical observation was that the urination and marking behavior remained despite removal of the abdominal testicle and after testosterone concentrations reached those of neutered dogs. references 1. immegart hm, threlfall, wr: evaluation of intratesticular injection of glycerol for nonsurgical sterilization of dogs. am j vet res 2000;61:545-549. 2. kawakami e, yamada y, tsutsui t, et al: changes in plasma androgen levels and testicular histology with descent of the testis in the dog. j vet med sci 1993;55:931-935. 3. mischke r, meurer d, hoppen ho, et al: blood plasma concentrations of oestradiol-17 beta, testosterone and testosterone/oestradiol ratio in dogs with neoplastic and degenerative testicular diseases. res vet sci 2002;73:267-272. 4. ortega-pacheco a, rodrıguez-buenfil jc, segura-correa jc, et al: pathological conditions of the reproductive organs of male stray dogs in the tropics: prevalence, risk factors, morphological findings and testosterone concentrations. reprod dom anim 2006;41:429-437. 5. günzel-apel ar, hille p, hoppen ho: spontaneous and gnrh-induced pulsatile lh and testosterone release in pubertal, adult and aging male beagles. theriogenology 1994;413:737-745. 6. kawakami e, takemura a, sakuma m, et al: superoxide dismutase and catalase activities in the seminal plasma of normozoospermic and asthenozoospermic beagles. j vet med sci 2007;69:133-136. 7. garibyan h, de jong fh, klopper pj: effect of experimental microvascular orchidopexy on testosterone secretion in the dog. urol int 1983;38:337-339. 8. boyden tw, silvert ma, pamenter rw: chronic ethanol feeding impairs human chorionic gonadotropin-stimulated testicular testosterone responses of dogs. biol reprod 1982;27:652-657. clinical theriogenology • volume 3 number 4 • december 2011485 9. falvo re, depalatis lr, moore j, et al: annual variations in plasma levels of testosterone and luteinizing hormone in the laboratory male mongrel dog. j endocrinol 1980;86:425-430. 10. knol bw, dielman sj, bevers mm, et al: diurnal and seasonal variations in plasma luteinizing hormone and testosterone levels in the male dog [thesis]. utrecht: university of utrecht; 1989. 11. urhausen c, seefeldt a, eschricht fm, et al: concentrations of prolactin, lh, testosterone, tsh and in thyroxine normospermic dogs of different breeds. reprod domest anim 2009 ;44:279-272. 12. schorner g, choi hs, bamberg e: plasmatestosterongehald beim rüden nach behandlung mit pmsg, hcg und lhrh. wien tierarztl monatsschr 1977; 64:153-156. 13. birchard sj, nappier m: cryptorchidism. compend contin educ vet 2008;30:325-336. 14. mattheeuws d, comhaire d: concentration of oestradiol and testosterone in peripheral and spermatic venous blood of dogs with unilateral cryptorchidism. domest anim endocrinol 1989;16:203-209. 15. batista am, gonzalez vf, cabrera mf, et al: morphologic and endocrinologic characteristics of retained canine testicles. canine pract 2000;25:12-15. 16. kawakami e, tsutsui t, yamada y, et al: spermatogenesis and peripheral spermatic venous plasma androgen levels in unilateral cryptorchid dogs. jpn j vet sci 1987;49:349-356. 17. kawakami e, tsutsui t, saito s, et al: changes in peripheral plasma luteinizing hormone and testosterone concentrations and semen quality in normal and cryptorchid dogs during sexual maturation. lab anim sci 1995;45:258-263. 18. sundby a, ulstein t: plasma concentrations of testosterone in the male dog and plasma testosterone profile following single intramuscular injection of hcg. acta vet scand 1981;22:409-416. 19. kawakami e, hori t, tsutsui t: function of contralateral testis after artificial unilateral cryptorchidism. j vet med sci 1999;61:1107-1111. 20. cassata r, iannuzzi a, parma p, et al: clinical, cytogenetic and molecular evaluation in a dog with bilateral cryptorchidism and hypospadias. cytogenet genome res 2008;120:140-143. 21. munro ea, watson ed, van den broek ah: testosterone release in intact male dogs following the injection of a gnrh analogue. vet rec 1996;139:96. 22. kawakami e, kuroda d, hori t, et al: changes in the plasma testosterone level and testicular superoxide dismutase activity of 5 azoospermic beagles after gnrh analogue injections. j vet med sci 2007;69:561-562. 23. kawakami e, masaoka y, hirano t, et al: changes in plasma testosterone levels and semen quality after 3 injections of a gnrh analogue in 3 dogs with spermatogenic dysfunction. j vet med sci 2005;67:1249-1252. 24. neilson jc, eckstein ra, hart bl: effects of castration on problem behaviors in male dogs with reference to age and duration of behavior. j am vet med assoc 1997;15:180-182. clinical theriogenology • volume 3 number 4 • december 2011 486 1 contact rejean lefebvre rejean.lefebvre@umontreal.ca © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10309, http://dx.doi.org/10.58292/ct.v16.10309 research report diagnosis of clinical cervicitis and vaginitis in dairy cows in relation to various postpartum uterine disorders ali bazzazan,a dario antonio vallejo-timaran,b juan maldonado-estrada,c mariela segura,d rejean lefebvrea adepartment of clinical sciences, faculty of veterinary medicine, university of montréal, st-hyacinthe, quebec, canada bfaculty of veterinary medicine and animal sciences, research group in agricultural sciences (gisca), autonomous university foundation of the americas, medellin, colombia cdepartment of theriogenology, ohvri research group, school of veterinary medicine, faculty of agrarian sciences, university of antioquia, medellin, colombia dresearch group on infectious diseases in production animals, and swine and poultry infectious diseases research centre, faculty of veterinary medicine, university of montréal, saint-hyacinthe, quebec, canada abstract there is poor agreement between clinical findings and results of diagnostic tests in postpartum uterine diseases that may be explained at least in part by the presence of inflammation and infection at sites other than the endometrium. it was hypothesized that clinical cervicitis and vaginitis are components of reproductive tract inflammatory disorders in postpartum dairy cows. a total of 61 postpartum dairy cows were enrolled in a nested case-control design. periparturient disease occurrence in weeks 1 (1w), 3 (3w), and 5 (5w) postpartum were recorded. clinical cervicitis was diagnosed in 36.0, 40.1, and 31.1% of cows in 1w, 3w, and 5w postpartum, respectively. approximately 64% of cows with clinical endometritis in 5w postpartum also had clinical cervicitis (p ≤ 0.05). prevalence of clinical and cytological vaginitis was 0% in 5w postpartum. average days open: 110 for healthy cows and 117 for cows with clinical cervicitis (p ≤ 0.001), 145 for cows with clinical cervicitis and clinical endometritis (p < 0.005), and 199 for cows with clinical cervicitis and cytological endometritis (p < 0.001). contrary to vaginitis, high prevalence of clinical cervicitis and its association with clinical endometritis and longer average days open suggested a substantial role of the condition in postpartum uterine diseases. keywords: cervicitis, vaginitis, postpartum uterine diseases, neutrophils, inflammation introduction postparturient cows sustain substantial damage to endometrium along with uterine bacterial contamination, triggering an active inflammatory process that clears cellular debris and bacteria, as well as repairs the endometrium, as part of normal uterine involution. in healthy cows, uterine inflammation eventually subsides and bacteria are eliminated by 4 6 weeks postpartum.1 in scenario where pathogenic bacteria persist in uterus, reproductive tract inflammation is prolonged that has a substantial role in postpartum uterine diseases’ pathogenesis.2,3 in dairy cows, occurrence of postpartum uterine diseases (puds) is high (up to 40%) and include clinical conditions like retained fetal membranes, metritis, clinical endometritis, and pyometra, as well as nonclinically evident conditions (e.g. subclinical endometritis). these conditions have negative effects on fertility, causing decreases in pregnancy rates and increases in the number of services per pregnancy and days open.4 infertility is directly linked to changes in the uterine environment and ovarian function. uterine inflammatory response and the presence of bacterial toxins like lipopolysaccharides (escherichia coli [e. coil]) and pyolysin (trueperella pyogenes) have negative effects on ovarian function, resulting in the establishment of anovulatory conditions and postpartum anestrus.2,5 distinction between physiological and pathological uterine involution depends on the severity of the disorder, stage postpartum, duration of inflammatory process and, most importantly, whether it impairs cow’s fertility at the end of the voluntary waiting period. diagnosing puds is complicated by a lack of clarity in case definition, use of various gold standard tests, reduced sensitivity and specificity of diagnostic tests, and simply the difficulty mailto:rejean.lefebvre@umontreal.ca http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10309 2 citation line: clinical theriogenology 2024, 16, 10309, http://dx.doi.org/10.58292/ct.v16.10309 inherent in exploring bovine reproductive tract.6,7 several techniques used for diagnostic purposes are not suitable for widespread use in clinical practice because they are not rapid, neither easy to use nor cost-effective. sometimes there is poor agreement between clinical findings and diagnostic test results due to an inflammatory process at a site other than endometrium, such as cervix, vagina, or uterus. cervix is a self-contained organ of the reproductive tract that serves as an anatomical and functional barrier between vagina and uterus. however, our understanding of cervical inflammation and bacterial contamination, and its subsequent influence on reproductive efficiency in dairy cows, is incomplete. research has suggested that cervicitis is a distinct condition that has both a separate and additive effect on reproductive performance of dairy cows.8 cervicitis has a prevalence of 11 30% in dairy cows, and ~ 75% of cows with cervicitis also exhibit clinical or subclinical endometritis.9 in women of reproductive age, diagnosing cervicitis is difficult, partly because it is frequently asymptomatic and so remains undiagnosed.10,11 in 20 25% women cervicitis is associated with an increased risk of pelvic inflammatory disease and adverse pregnancy outcomes.10 vagina is the female copulatory organ that extends from cervical caudal segment of cervix to vestibular border at the external urethral orifice. one important function of vagina (similar to cervix) is to serve as a line of defence against bacterial invasion by secreting fluids that inhibit undesirable bacterial growth. very limited information is available on vaginitis in postpartum dairy cows. objective was to investigate whether cervicitis and vaginitis are potentially a substantial problem in postpartum dairy cows. we examined the occurrence of these 2 disorders during the postpartum period and their associations with other puds. materials and methods research was conducted in compliance with the experimental practices and standards approved by the animal care committee of the university of montréal (protocol no. 211-03), and efforts were made to minimize animal suffering in compliance with the canadian council on animal care guidelines. study population and experimental design this prospective observational longitudinal cohort study was carried out in postpartum holstein dairy cows (n = 61) between first and third lactation from 3 convenient commercial dairy herds. nested case-control design was used, cows were enrolled systematically based on calving date. cows were housed in tie-stall barns and milked twice daily. rolling herd average for milk production was 9,000 kg. cows were fed a total mixed ration of corn and hay silage to meet the nutrient requirements of dairy cows as recommended by the national research council (nrc, 2011). farms were visited weekly by the same veterinarian. cows were vaccinated intramuscularly twice against e. coli before calving (2 ml on day 40 and on day 26 before parturition; j-vac®, boehringer ingelheim, burlington, ontario, canada) and once against ibr, bvd (type 1 and 2), pi3, and brsv after calving (2 ml between days 10 and 30 postpartum; bovi-shield gold5® fptm 5 l5, zoetis, kirkland, quebec, canada). in addition, pregnant cows were injected with selenium 5.0 ml on day 60 before calving (mu-se, intervet, merk, kirkland, quebec, canada). cows were examined 3 times: week 1 postpartum (1w), week 3 postpartum (3w), and week 5 postpartum (5w) postpartum. at each examination, a complete clinical and reproductive examination was performed. reproductive assessment comprised: 1. vaginoscopy to assess cervical and vaginal appearance as well as the presence of purulent vaginal discharge and 2. transrectal ultrasonographic examination (7.5 mhz probe; ibex, e.i. medical imaging, co, usa) to assess uterine horn symmetry (2.5 cm beyond the external bifurcation). cytological sampling to diagnose subclinical conditions, cytobrush technique7 was used to collect samples of mucosa from vaginal fornix (1w, 3w, 5w), cervix (5w), and uterus (1w, 3w, 5w), cytobrush samples were collected using a modified casou cannula. double-protected casou containing cytobrush was inserted into vaginal fornix and uterus. at each location, cytobrush was exposed and rolled over the mucosa before being recovered in the double-protected casou. new brush and protective sheet were used for each sampling location. once withdrawn from the reproductive tract, cytobrush was rolled over a sterile slide, fixed, and stained. one smear was used per location and examined under the microscope (400 x magnification) by a reviewer without knowledge of the identity of the cows. total of 200 cells were counted and characterized.12 percentage of polymorphonuclear neutrophils (pmns) was determined for each 3 sampling sites (vagina, cervix, uterus). uterine bacterial culture and identification uterine cytobrush samples for bacteriological examination that were only collected in 3w after calving, were subjected to aerobic and anaerobic culturing using standard bacteriology testing methods (api system, biomérieux, marcy étoile, france). samples for bacteriology were stored in a culture tube on ice (starplex scientific inc, etobicoke, ontario, canada) and transported at room temperature to the faculty of veterinary medicine’s diagnostic laboratory within 3 hours. for microbiological analysis, brushes were plated onto sheep blood agar (soy agar with 5% sheep blood; becton, dickinson and co, sparks, maryland, usa) using a sterile disposable inoculating loop within 6 hours after arrival. plates were incubated for 48  hours at 35°c under aerobic conditions and examined. when growth was observed, colony type was identified based on morphology, pigmentation and hemolytic pattern. very small beta-hemolytic, catalase-negative colonies consisting of gram-positive coryneform rods were identified as trueperella pyogenes. swabs were quickly plated on brucella agar containing neomycin (100 g/ml) and incubated anaerobically at 35°c for 5 days. when gram-negative rods were observed, colonies were examined using analytical profile index 20 a gallery for identification of fusobacterium necrophorum and prevotella melaninogenica. for e. coli isolation, swabs were plated on blood agar and macconkey agar (oxoid, ottawa, ontario, canada) at 37°c at the woah reference laboratory for e. coli (faculty of veterinary medicine, university of montréal). for confirmation of e. coli, isolates were submitted to 3 biochemical tests: indole spot, simmons citrate, and motility. disease definitions and selection of cases and controls dairy cows were examined for 7 puds, defined as follows: 1. clinical metritis (met): an abnormally enlarged uterus and purulent uterine discharge in week 1 postpartum (1w)13; 2. http://dx.doi.org/10.58292/ct.v16.10309 citation line: clinical theriogenology 2024, 16, 10309, http://dx.doi.org/10.58292/ct.v16.10309 3 clinical endometritis (ce): purulent vaginal discharge (pvd) of uterine origin in weeks 3 postpartum (3w) with normal-sized uterus and no fluid14; 3. cytological endometritis (cytoe); absence of pvd (3w and 5w) plus pmns exceeding 5% on cytobrush examination, a normal-sized uterus and no fluid in the uterus15; 4. clinical cervicitis (cc): cervical folds on vaginoscopy in 5w were assessed, where grade 0 = normal cervical fold, grade 1 = second cervical fold was swollen without redness and protruding through first cervical fold, and grade 2 = second fold swollen with redness and protruding through first cervical fold9; 5. cytological cervicitis (cytoc): pmns exceeding 5% on cytobrush sampling between first and second fold in 5w; 6. cytological vaginitis (cytov): pmns exceeding 5% on cytobrush sampling of the vaginal fornix at 5w without purulent discharge; and 7. clinical vaginitis: red and edematous mucosa on vaginoscopy without purulent discharge. note that these postpartum reproductive tract diseases were not associated with systemic clinical signs. at each reproductive tract examination, body condition score was recorded. variable definitions following variables were included in the analysis: 1. herd variables; average parity and herd effect; 2. disease variables: metritis, clinical endometritis, cytological endometritis, clinical and cytological cervicitis (5w postpartum), and clinical and cytological vaginitis (1w, 3w, 5w); and 3. clinical variables: body condition score; cervical, vaginal fornix and uterine pmn counts, vaginal discharge grade, and uterine asymmetry grade. data analyses descriptive analysis was performed using frequency tables to determine the percentage of each variable category in the study population. percentage of cows affected by various postpartum conditions (met, ce, cytoe, cc, cytoc, and cytov) were calculated by dividing the number of cows with the condition by the number of cows studied. co-occurrence of cytov and cytoc, cytov and cytoe, and cytoc and cytoe were assessed using kappa coefficients. associations between clinical cervicitis and other postpartum diseases were assessed using contingency tables and chi-square tests. logistic regression model was used to explore the association between risk factors and occurrence of cc. univariate analysis was conducted first for each independent variable (herd, clinical, disease variables), with cc or cytov as the outcome variable (separate models for each outcome variable). variables with a p value < 0.15 were considered for the multivariate analysis that was conducted using backward selection with a p value of 0.05. confounding and interaction effects were examined via noncausal exposure-outcome associations.16 variable parity and body condition score were considered potential confounding variables. random herd effect in a mixed-effects logistic regression model was explored. hosmer– lemeshow test was used to determine the goodness of fit of the logistic regression models. analyses were conducted using stata® statistical software (release 15, statacorp llc, college station, texas, usa). results study population consisted of 85 cows, but an initial exclusion resulted in the removal of some cows due to culling (n = 5), use of antimicrobials (n = 4), metabolic disease (n = 5) or missing data (n = 10). a total of 61 cows were without symptoms related to systemic illness. clinical cervicitis was diagnosed in 36.7, 40.1, and 31.1% of cows in 1w, 3w, and 5w postpartum, respectively. many cases of cervicitis occurred concurrently with another postpartum condition. in total, 47% of cases of clinical cervicitis (cc) were associated with purulent vaginal discharge (pvd), and 21% with cytological endometritis (cytoe). only 32% of cases of cc did not co-occur with another clinical condition. association between cervicitis in 5w and other postpartum diseases in 1w, 3w and 5w is provided (table 1). sixty percent (n = 15) of cows with cc and 37.5% (n = 12) of cows with clinical endometritis in 3w postpartum had clinical cervicitis in 5w postpartum (table 1). however, the association was not significant. cows with clinical endometritis in 5w postpartum (64.2%, n=9) had clinical cervicitis (p = 0.002). cytov, cytoc and cytoe (3w and 5w) were not associated with cc in 5w (p > 0.05). average days open were: 110 for healthy cows, 117 for cows with cc, 145 for cows with cervicitis plus purulent vaginal discharge, and 199 for cows with clinical cervicitis plus cytoe. there were low to very low levels of co-occurrence between cc and cytov (kappa = 0.024), cytoc (kappa = 0.077) and cytoe (kappa = 0.132). the results were similar to the co-occurrence of cytoe and cytov (kappa = 0.041) and cytoc (kappa = 0.253). there was slight agreement between vaginal and cervical findings and the results of the cytological evaluation (kappa = 0.211). there was no significant herd effect when herd was used as a random effect in a mixed-effects logistic regression model. univariate logistic regression analysis concluded that the variable parity, body condition score in 1w postpartum, diagnosis of subclinical ketosis (bhb > 1.2 mmol/l) in 1w postpartum, high uterine pmn count in 1w postpartum (> 5%), and positive culture for t. pyogenes (15/61) in 3w postpartum were associated (p < 0.15) with clinical cervicitis on 5w postpartum. cows with clinical ketosis were removed from the study and no other bacteria were present in substantial amounts. univariate analysis did not detect any associations between independent variables and vaginitis (p < 0.15) and hence vaginitis was not included as an outcome variable in the multivariate analysis. results of the multivariate logistic regression model for cc are provided (table 2). using a backward elimination process in the final model, body condition score in 1w postpartum was a confounder and was forced in the model. in the model, subclinical ketosis in 1w postpartum increased the odds of cc compared to cervicitis-free cows (or: 5.2, 95% ic: 1.3 – 19.8), and a positive culture for t. pyogenes in 3w postpartum increased the odds of cervicitis compared to cows with a negative culture (or: 4.2, 95% ic: 1.0 – 16.7). discussion bovine cervix is a thick-walled cylindrical structure composed of 3 or 4 annular folds that separate the uterus from the vagina. it forms a recess in the vagina known as the fornix.17 because of its strategic position, it is exposed to changes that occur in the vagina or uterus, such as infection/inflammation. for that reason, fornix and cervical status can be a good indicator of puds. however, in this study, there were low levels of co-occurrence between cc and cytov and cytoe, and between cytoe and cytov that indicated a substantial compartmentalization of the reproductive tract in cows. based on the assumption that cervical involution is slower than the remainder of the reproductive tract, cervical status in 5w may http://dx.doi.org/10.58292/ct.v16.10309 4 citation line: clinical theriogenology 2024, 16, 10309, http://dx.doi.org/10.58292/ct.v16.10309 be an important element in attempts to develop more sensitive, specific and easier to use diagnostic tools for postpartum uterine diseases (e.g. cc and cytoe). weak associations between purulent vaginal discharge (resulting in colonization by putative bacteria), presence of endometrial inflammation (as measured by pmns in uterus), and absence of visible inflammation of vaginal mucosa (clinical vaginitis) in postpartum cows (unpublished data), coupled with the strong reproductive tract compartmentalization, point to the need for a better understanding of reproductive tract involution. this study examined cervicitis and vaginitis in postpartum dairy cows as diagnosed by visual and cytological examination, respectively, in established cases of ce and cytoe in the absence of exclusion criteria. occurrence of cc in postpartum dairy cows at 5w was 31.2% (35-39 dim) similar to a previous report (26.1% 42–50 dim).9 there was a decrease of ~ 22% in the percentage of cows with cc between 3w and 5w (table 1) but not as important as the decrease of ce (56%). similar decreases of ce in the same postpartum period were measured in other studies.12,18 our study is the first to report changes in the percentage of cows diagnosed with cc over the postpartum period: 36.7 in 1w, 40.1 in 3w, and 31.1% in 5w. due to the phenomenon of spontaneous clinical cure during physiological uterine involution, prevalence of postpartum reproductive tract diseases is expected to decrease with the number of days in milk (dim). however, as cervical involution is slower than uterine involution19 one would also expect the rate of spontaneous clinical recovery of the cervix to be lower than that of the uterine horn in postpartum cows. prevalence of cytoc in 5w in the present study was 6.6%, 42% lower than that reported8 for a similar postpartum period (< 35 dim). in the latter study, higher prevalence of cytoc may have been associated with undiagnosed ce given that no assessment of vaginal purulent discharge was performed. this could also explain the presence of bacteria on their endometrial cytology samples. similar to that study, we used a threshold of 5% pmns to diagnose cytoc. note that we performed cervical cytology only in 5w. clinical cervicitis occurred in conjunction with various puds and our final model revealed that cc cases increased (or = 3.8 p = 0.05) when cows had a positive culture for t. pyogenes. there was concomitant ce and cytoe in 47 and 21% of cc cases, respectively. in a previous study, ~ 12% of cows with cc between 42 and 50 days postpartum also had cytological endometritis.9 in that study, cows with ce and abnormal uterine content were excluded. in another study where no exclusion criteria were applied, 75% of cows with cytoe also had cytoc, and ~ 50% of cows with ce had cytoc.8 numerous risk factors have been documented for postpartum uterine diseases like met, ce and cytoe. however, because studies often have limited statistical power, as is true of this study, it is difficult to rank possible risk factors. in the case of table 1. contingency table and chi-square test results: clinical cervicitis diagnosed in 5 weeks postpartum in relation to postpartum uterine disorders diagnosed in 1, 3 and 5 weeks postpartum in dairy cows weeks postpartum disease clinical cervicitis (week 5) no yes 1 metritis no 26 (70.3%) 11 (29.7%) yes 15 (62.5%) 9 (37.5%) grade 2 cervix no 25 (65.8%) 13 /34.2%) yes 16 (69.6%) 7 (30.4%) 3 clinical endometritis no 22 (75.9%) 7 (24.1%) yes 20 (62.5%) 12 (37.5%) grade 2 cervix no 32 (88.9%) 4 (11.1%) yes 10 (40%) 15 (60%) cytov no 35 (68.6%) 16 (31.4%) yes 7 (70%) 3 (30%) cytoe no 21 (72.4%) 8 (27.6%) yes 21 (65.6%) 11 (31.4%) 5 clinical endometritis* no 37 (78.7%) 10 (21.3%) yes 5 (35.7%) 9 (64.3%) cytoc no 26 (66.7%) 13 (33.3%) yes 8 (36.4%) 14 (63.6%) cytoe no 39 (68.4%) 18 (31.6%) yes 3 (75%) 1 (25%) (%) = represent the percentage of cows in a row cytov = cytological vaginitis cytoe = cytological endometritis cytoc = cytological cervicitis *pearson chi-square ≤ 0.05 http://dx.doi.org/10.58292/ct.v16.10309 citation line: clinical theriogenology 2024, 16, 10309, http://dx.doi.org/10.58292/ct.v16.10309 5 ce, dystocia, twin calves, stillbirth, male calves, retained fetal membranes, and puerperal metritis have been reported as important risk factors.20 taking into account independent variables like herds, clinical variables, and disease variables, cc in 5w was significantly associated with ce in 5w, implicating the involution process. if cc is associated with trauma during calving, the condition should be visible earlier in the postpartum period (e.g. in 3w). additionally, pmns of > 5% in the uterus in 1w was associated (p < 0.001) with cc in 5w. a threshold of 18% may have been more appropriate in 1w and could have altered the results. similarly, t. pyogenes in 3w was associated (p < 0.001) with cc. however, presence of  pmns of > 5% in the vagina in 3w was not associated (p < 0.05) with cc in 5w. in the present study, cows with cc and cytoe had 89 additional days open compared to cows with only cc. effect of cc on reproductive efficiency is amplified (p < 0.001) when associated with other postpartum uterine diseases, thus supporting previous reports for various puds.20 this amplified effect may also be associated with the fact that involution is slower in the cervix than it is in the uterus. cows affected by ce were 1.7 times more likely to be culled compared to cows without endometritis.20 cows with postpartum clinical metritis exhibited reduced conception rates, and time to first insemination was extended by 7.2 days, ultimately leading to subfertility.21 in addition, postpartum dairy cows with more than 1 disease have poorer reproductive performance. cows with ce and cytoe had worse reproductive performance than those with only 1 pathological condition.20 similarly, cc reduced reproductive performance,9 and its negative effect is amplified when the disease is combined with other uterine diseases. it is believed that postpartum uterine infection/inflammation is caused by bacteria ascending from the vagina or travelling through the vagina from the outside environment when the cervix dilates during parturition,22 and that this state continues until the uterus returns to normal functioning. however, this concept is not necessarily associated with infection of the table 2. results for multivariate logistic regression model of clinical findings associated with clinical cervicitis to 35 days in milk in dairy cows disease variable or std. err. p 95% ci clinical cervicitis intercept 0.18 0.03 <0.001 (0.12 – 0.28) body condition score 1w < 3.0 / 5.0 ref. 3.0 – 3.25 / 5.0 0.51 0.46 0.468 (0.08 – 3.04) 3.5 – 3.75 / 5.0 0.19 0.16 0.062 (0.03 – 1.08) ≥ 4.0 / 5.0 0.08 0.12 0.079 (0.005 – 1.33) subclinical ketosis 1w no ref yes 5.22 3.55 0.015 (1.37 – 19.85) trueperella pyogenes 3w negative culture ref positive culture 4.25 2.96 0.038 (1.08 – 16.70) vagina as demonstrated (very low prevalence of cytov (3%) and absence of clinical vaginitis in the cows) in our study. vaginal microbiota in dairy cows harbour main uterine pathogens.23 in addition, it has been hypothesized that the vagina is responsible for a portion of the purulent vaginal discharge observed in postpartum endometritis in dairy cows; however, there was no visual evidence of this in the present study. conclusion prevalence of cc and cytoc in 5 weeks postpartum dairy cows was 31.1 and 6.6%, respectively. clinical cervicitis occurred concomitantly with other common uterine conditions like endometritis and metritis, with 32% of cows exhibiting only cc. high prevalence of cc with its association with clinical endometritis and longer average days open suggested that it would be advantageous to conduct a visual vaginal examination during 5 weeks postpartum as part of a complete reproductive examination. absence of clinical vaginitis and low prevalence of cytov in cows indicated that these conditions do not have an important role in puds in high-producing dairy cows. better understanding of the interaction between cc and other puds should be possible with larger studies. conflict of interest authors declare that they have no conflict of interest that would prejudice their impartiality in conducting this research or in publishing the results. references 1. dadarwal d, palmer c, griebel p: mucosal immunity of the postpartum bovine genital tract. theriogenology 2017;104:62-71. doi: 10.1016/j.theriogenology.2017.08.010 2. herath, s, lily st, fisher dp, et al: bacterial lipopolysaccharide induces an endocrine switch from prostaglandin f2α to http://dx.doi.org/10.58292/ct.v16.10309 https://doi.org/10.1016/j.theriogenology.2017.08.010 6 citation line: clinical theriogenology 2024, 16, 10309, http://dx.doi.org/10.58292/ct.v16.10309 prostaglandin e2 in bovine endometrium. endocrinology 2009;150:1912-1920. doi: 10.1210/en.2008-1379 3. carneiro lc, cronin jg, sheldon im. mechanisms linking bacterial infections of the bovine endometrium to disease and infertility. reprod biol 2016;16:1-7. doi: 10.1016/j. repbio.2015.12.002 4. williams ej, fisher dp, noakes de, et al: the relationship between uterine pathogen growth density and ovarian function in the postpartum dairy cow. theriogenology 2007;68:549-559. doi: 10.1016/j.theriogenology.2007.04.056 5. williams ej: drivers of post-partum uterine disease in dairy cattle. reprod domest anim 2013;48:53-58. doi: 10.1111/rda.12205 6. de boer mw, leblanc sj, dubuc j, et al: invited review: systematic review of diagnostic tests for reproductive-tract infection and inflammation in dairy cows. j dairy sci 2014;97:3983-3999. doi: 10.3168/jds.2013-7450 7. wagener k, gabler c, drillich m: a review of the ongoing discussion about definition, diagnosis and pathomechanism of subclinical endometritis in dairy cows. theriogenology 2017;94:21-30. doi: 10.1016/j.theriogenology.2017.02.005 8. deguillaume l, geffré a, desquibet l, et al: effect of endocervical inflammation on days to conception in dairy cows. j dairy sci 2012;95:1776-1783. doi: 10.3168/jds.2011-4602 9. hartmann d, rohkohl, merbach s, et al: prevalence of cervicitis in dairy cows and its effect on reproduction. theriogenology 2016;85:247-253. doi: 10.1016/j.theriogenology.2015.09.029 10. marrazzo jm, weisenfeld hc, murray pj, et al: risk factors for cervicitis among women with bacterial vaginosis. j infect dis 2006;193:617-624. doi: 10.1086/500149 11. li m, li l, wang r, et al: prevalence and risk factors for bacterial vaginosis and cervicitis among 511 female workers attending gynecological examination in changchun, china. taiwan. j obstet gynecol 2019;58:385-389. doi: 10.1016/j.tjog.2018.11.036 12. tison n, bouchard e, descôteaux, et al: effectiveness of intrauterine treatment with cephapirin in dairy cows with purulent vaginal discharge. theriogenology 2017;89:305-317. doi: 10.1016/j. theriogenology.2016.09.007 13. giuliodori mj, magnasca rp, becu-villalobos, et al: metritis in dairy cows: risk factors and reproductive performance. j dairy sci 2013;96:3621-3631. doi: 10.3168/jds.2012-5922 14. sheldon im, williams ej, miller ana, et al: uterine diseases in cattle after parturition. vet j 2008;176:115-121. doi: 10.1016/j. tvjl.2007.12.031 15. madoz lv, giuliodori mj, migliorisi al: endometrial cytology, biopsy, and bacteriology for the diagnosis of subclinical endometritis in grazing dairy cows. j dairy sci 2014;97:195-201. 16. dohoo i, martin w, stryhn h: veterinary epidemiology research. prev vet med 2005;68:289-292. 17. breeveld-dwarkasing vna, de boer-brouwer m, te koppele jm, et al: regional differences in water content, collagen content, and collagen degradation in the cervix of nonpregnant cows. biol reprod 2003;69:1600-1607. doi: 10.1095/biolreprod.102.012443 18. gautam g, nakao t, koike k, et al: spontaneous recovery or persistence of postpartum endometritis and risk factors for its persistence in holstein cows. theriogenology 2010;73:168-179. doi: 10.1016/j.theriogenology.2009.08.010 19. mortimer rg, farin pw, steven rd: reproductive examination of the non-pregnant cows. in: youngquist rs: editor. current therapy in large animal theriogenology. philadelphia, pa; wb saunders: 2006. p. 268-275. 20. dubuc j, duffield tf, leslie ke, et al: risk factors for postpartum uterine diseases in dairy cows. j dairy sci. 2010;93:5764-5771. doi: 10.3168/jds.2010-3429 21. leblanc sj, duffield tf, leslie ke, et al: defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows. j dairy sci 2002;85:2223-2236. doi: 10.3168/jds.s0022-0302(02)74302-6 22. leblanc sj: postpartum uterine disease and dairy herd reproductive performance: a review. vet j. 2008;176:102-114. doi: 10.1016/j. tvjl.2007.12.019 23. bicalho mls, santin t, rodrigues mx, et al: dynamics of the microbiota found in the vaginas of dairy cows during the transition period: associations with uterine diseases and reproductive outcome. j dairy sci 2017;100:3043-3058. doi: 10.3168/jds.2016-11623 http://dx.doi.org/10.58292/ct.v16.10309 https://doi.org/10.1210/en.2008-1379 https://doi.org/10.1016/j.repbio.2015.12.002 https://doi.org/10.1016/j.repbio.2015.12.002 https://doi.org/10.1016/j.theriogenology.2007.04.056 https://doi.org/10.1111/rda.12205 https://doi.org/10.3168/jds.2013-7450 https://doi.org/10.1016/j.theriogenology.2017.02.005 https://doi.org/10.3168/jds.2011-4602 https://doi.org/10.1016/j.theriogenology.2015.09.029 https://doi.org/10.1086/500149 https://doi.org/10.1016/j.tjog.2018.11.036 https://doi.org/10.1016/j.theriogenology.2016.09.007 https://doi.org/10.1016/j.theriogenology.2016.09.007 https://doi.org/10.3168/jds.2012-5922 https://doi.org/10.1016/j.tvjl.2007.12.031 https://doi.org/10.1016/j.tvjl.2007.12.031 https://doi.org/10.1095/biolreprod.102.012443 https://doi.org/10.1016/j.theriogenology.2009.08.010 https://doi.org/10.3168/jds.2010-3429 https://doi.org/10.3168/jds.s0022-0302(02)74302-6 https://doi.org/10.1016/j.tvjl.2007.12.019 https://doi.org/10.1016/j.tvjl.2007.12.019 https://doi.org/10.3168/jds.2016-11623 evaluation of commercial semen extenders and crystalloids for short term cooled extension of epididymal spermatozoa in squamates and chelonians evaluation of commercial semen extenders and crystalloids for short term cooled extension of epididymal spermatozoa in squamates and chelonians sean perry, sarah camlic, mark mitchell department of veterinary clinical sciences, louisiana state university, baton rouge, la our working knowledge of assisted reproduction in male reptiles is limited to our ability to collect semen from less than 0.2% reptile species. to date, cooled storage using egg yolk based semen extender has had some short term benefits for corn snakes (elaphe guttata), green iguanas (iguana iguana), and leopard tortoises (stigmochelys pardalis).1-3 our objective was to evaluate commercial semen extenders and crystalloid solutions utility to provide short-term sperm storage in squamates and chelonians. the hypothesis was that commercial semen extenders maintain motility of squamate and chelonian sperm, specifically the green anole (anolis carolinensis), the diamondback water snake (nerodia fasciata), and the red eared slider (trachemys scripta elegans). epididymal sperm from a. carolinensis (n = 6), n. fasciata (n = 6), and t. scripta elegans (n = 6) were collected post mortem. a. carolinensis and n. fasciata samples were aliquoted into 6 commercial extender treatments (ham’s f 10 without albumin, test yolk buffer, inra 96, sperm washing media, andro pro chill lt and hank’s balanced salt solution) and 3 crystalloid treatments (0.9% sodium chloride injection, phosphate buffered saline, and lactated ringer’s solution), whereas t. scripta elegans samples were aliquoted into 6 extender treatments (ham’s f 10 without albumin, test yolk buffer, inra 96, sperm washing media, electrolyte free media, and hank’s balanced salt solution). samples were stored at 4°c for 72 and 96 hours respectively. motility analysis was performed using a computer assisted sperm analysis system at time 0, 12, 24, 48, and 72 hours for a. carolinensis and n. fasciata and at 0, 6, 12, 24, 48, 72, and 96 hours for t. scripta elegans. linear mixed models were used for statistical analyses. epididymal sperm motility in a. carolinensis declined over time for each treatment over the first 24 hours (p < 0.001), with no significant difference in motility between commercial semen extenders and crystalloid solutions. epididymal sperm motility in n. fasciata remained high in commercial semen extenders compared to crystalloid solutions (p < 0.001) for more than 72 hours. sperm wash media, f 10, and inra 96 maintained highest sperm motility for n. fasciata. epididymal sperm motility in t. scripta elegans remained high over 96 hours in the sperm wash media, test yolk buffer, and inra 96 (p < 0.001). however, motility declined over time in all treatments except inra 96 (p < 0.001). we concluded that inra 96, sperm wash media, or f 10 were the best choices for handling and cool storing reptile sperm. keywords: semen extender, reptiles, trachemys scripta elegans, anolis carolinensis, nerodia fasciata references 1. zimmerman dm, mitchell ma: semen collection and ejaculate characteristics of the leopard tortoise (stigmochelys pardalis). cons physiol 5 2017. 2. zimmerman d, mitchell ma, perry bh: collection and characterization of semen from green iguanas (iguana iguana). am j vet res 2013;74:1536-1541. 3. fahrig b, mitchell ma, eilts be, et al: characterization and cooled storage of semen from corn snakes (elaphe guttata). j zoo wild med 2007;38:7-12. 421 clinical theriogenology • volume 11, number 3 • september 2019 422clinical theriogenology • volume 11, number 3 • september 2019 omniblank: delayed sperm production in a presumed sertoli cell only syndrome case in two boars affected by severe combined immunodeficiency delayed sperm production in a presumed sertoli cell only syndrome case in two boars affected by severe combined immunodeficiency malavika adur, adrianne kaiser, swanand sathe,a haibo liu, sara charley, yunsheng li, christopher tuggle, jason ross department of animal science, college of agriculture and life sciences aveterinary clinical sciences, college of veterinary medicine, iowa state university ames, ia a biomedical pig model of severe combined immunodeficiency, developed at iowa state university, lacks functional t and b lymphocytes due to 2 natural mutations in artemis (dclre1c), a gene encoding a dna repair enzyme.1 pigs born with this deficiency survive only after successful bone marrow transfer and engraftment of lymphocyte precursor cells, although after 11 bone marrow transfer attempts in boars, only 4 survived this process and achieved an age of expected sexual maturity. two such boars (full siblings from different litters) had severe reductions in testicular size and dilute semen samples, compared to a heterozygous littermate. microscopic evaluation of semen revealed absence of sperm and presence of large numbers of “round” epithelial like cells, possibly immature germ cells that are often released prematurely from their association with sertoli cells in non-obstructive azoospermia.2 histological examination of affected testis from 1 boar had markedly reduced numbers of seminiferous tubules with an abundance of leydig cells. extensive vacuolization was observed within tubules along with sertoli cells, possibly due to increased germ cell apoptosis and/or meiotic arrest. this was indicative of germ cell aplasia, also known as sertoli cell only syndrome (scos). spermatogenic arrest, a common cause of male infertility, results in low sperm count and motility, greater morphologically abnormal sperm and has been associated with genetic defects.3 motile sperm were observed in ejaculates from the other boar at 19 months of age. consecutive ejaculates from this boar were lower in sperm concentration and higher in abnormal sperm. focal spermatogenesis was reported in cases of maturation arrest and scos.3 to determine genomic regions of interest associated with observed phenotype, we used whole genome sequencing of parents, affected boars and 1 unaffected male sibling to identify candidate mutations and eliminate less likely variants for consideration of causality. variants with homozygosity in affected boars only were observed in a few candidate genes, such as racgap1, spata18, dzip3, tex13c, among others, which were shown to have vital roles in spermatogenesis in other species requiring further investigation in boars. keywords: porcine, severe combined immunodeficiency, sertoli cell only syndrome, germ cell aplasia acknowledgement funded by nih 1r24od019813-4 and iowa state university office of the vice president for research. references 1. waide eh, dekkers jcm, ross jw, et al: not all scid pigs are created equally: two independent mutations in the artemis gene cause severe combined immunodeficiency (scid) in pigs. j immunol 2015;195:3171-3179. 2. anniballo r, ubaldi f, cobellis l, et al: criteria predicting the absence of spermatozoa in the sertoli cell-only syndrome can be used to improve success rates of sperm retrieval. human reproduction 2000;15:2269-2277. 3. sironen a, uimari p, venhoranta h, et al: an exonic insertion within tex14 gene causes spermatogenic arrest in pigs. bmc genomics 2011;12:591-601. 495 clinical theriogenology • volume 11, number 3 • september 2019 496clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 1 contact michelle kutzler michelle.kutzler@oregonstate.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9745, http://dx.doi.org/10.58292/ct.v15.9745 research report onset of reproductive capacity in unowned free-roaming cats ellie bohrer,a devany billings,b kristin patton,c michelle kutzlerb avca north portland veterinary hospital, portland, oregon, usa bdepartment of animal and rangeland sciences, oregon state university, corvallis, oregon, usa cfour star quarter horses, ostrander, ohio, usa abstract purpose was to investigate if an underlying biological cause exists for the reproductive success in unowned free-roaming (ufr) cats. these cats were presented for surgical sterilization during late summer/early fall. after castration of ufr toms, presence of penile spines was recorded, slides were made from vas deferens secretions for sperm morphology analyses, and testes were hemi-sectioned, formalin-fixed, paraffin-embedded, sectioned, stained with hematoxylin and eosin, and seminiferous tubule diameters were measured. after ovariohysterectomy of ufr queens, total ovarian-uterine weights were recorded. ovaries were hemi-sectioned, formalin-fixed, paraffin-embedded, sectioned, stained, and follicles were counted. presence of penile spines did not differ (p = 0.07) between toms 2 6 (n = 13) and > 6 months (n = 16). percentage of normal sperm morphology did not differ (p = 0.39) between toms 2 6 (n = 11; 77 ± 11%) and > 6 months (n = 9; 81 ± 13%). seminiferous tubular diameter increased (p < 0.01) in toms 2 2.5 (n = 6; 88.1 ± 10.9 µm), 3 4 (n = 6; 109.8 ± 8.9 µm), 5 6 (n = 6; 142.2 ± 16.9 µm), and > 6 months (n = 6; 237.9 ± 52.5 µm). there was no association (r2 = 0.20; p = 0.07) between age and total ovarian-uterine weight of queens. number of primary, secondary, and tertiary follicles recorded did not differ (p = 0.08) between queens 2 4 (n = 13) and 5 6 (n = 4) months. the largest follicle diameter did not differ (p = 0.14) between queens 2 4 (581.6 ± 53.7 µm) and 5 6 months (469.4 ± 113.9 µm). results may explain why ufr cat populations continue to increase despite investments in extensive trap-neuter-return efforts. selective pressure responses to shortened lifespans may contribute to onset of earlier reproductive capacity in ufr cats. keywords: feral cats, fertility, puberty, reproduction introduction unowned free-roaming (ufr) cats are cats that live within managed outdoor colonies. these cats are ecological threats to native habitats and their inhabitants. they are considered so invasive in nature that they have been listed among the hundred worst nonnative invasive species in the world.1 a study2 that analyzed 229 cases of feline impacts on islands revealed that ufr cats caused or contributed to 33 of the bird, mammal, and reptile extinctions. in the contiguous united states, 89% of the 12.3 x 109 cat-caused mammal mortality is by ufr cats.3 these cats also serve as vectors for numerous diseases, including feline panleukopenia virus, feline leukemia virus, feline immunodeficiency virus, and rabies. additionally, toxoplasma gondii (t. gondii) from ufr cats indirectly impacts marine ecosystems as t. gondii from cat feces leeches into marine environments through freshwater sources.4 this has led to higher quantity of t. gondii in southern sea otters (enhydra lutris nereis) and hawaiian monk seals (monachus schauinslandi).4 problems associated with ufr cats are exacerbated by their sizable population. current population estimate of ufr cats in the united states is somewhere between 30 and 80 x 106 individuals.5 additionally, a sexually mature domestic female cat can produce up to 5 litters of kittens each year.5 the most publicly supported sterilization program in the united states, great britain, canada, the netherlands, and denmark is trap-neuter-return (tnr).6 even with the efforts placed into tnr programs, humane societies in the united states still cannot surgically sterilize ufr cats fast enough to control their numbers.7 additionally, these programs are expensive. various estimates place neutering costs of ufr cats in tnr programs around $70.77 ± 11.75 per cat nationally.8 with the population of ufr cats in the united states alone, it would cost ~ $4.2 x 109 to sterilize all individuals. since ufr cat populations continue to increase even with extensive tnr efforts, it is important to understand why ufr cats are so reproductively successful. a possible explanation mailto:michelle.kutzler@oregonstate.edu http://dx.doi.org/10.58292/ct.v15.9745 2 citation line: clinical theriogenology 2023, 15, 9745, http://dx.doi.org/10.58292/ct.v15.9745 for their reproductive success is an earlier onset of reproductive capacity. puberty in owned domestic cats occurs ~ 8 10 months in queens and 7 12 months in toms; however, this can vary depending on body condition, breed/body weight, and season.9,10 we hypothesized that ufr cats gain reproductive capacity earlier than 6 months. purpose was to analyze reproductive parameters of ufr toms and queens from 2 to 6 months of age. materials and methods tom study 1 unowned free-roaming toms were presented for castration at a humane society in oregon during august through october. age was estimated by dental eruption using silver’s chart.11 age groups were as follows: 2 2.5 months (n = 6), 3 4 months (n = 6), 5 6 months (n = 6), and > 6 months (n = 6). after general anesthesia was induced, scrotal hair was clipped and the skin was aseptically prepared for surgery. a routine open castration was performed, and both testes from each cat were obtained. all toms recovered from surgery uneventfully. tissues were hemi-sectioned, formalin-fixed, paraffin-embedded, cut into 6 µm sections, and stained with hematoxylin and eosin. seminiferous tubular diameter and presence of spermatogenesis within the seminiferous tubule lumen was determined at 200 x magnification using a bright field microscope with image capture capability. a single observer (eb) blinded to the tom’s age measured perpendicular diameters of 5 randomly selected seminiferous tubules for each tom. in addition, the spermatogenesis score and leydig cell density score for each tom was determined by a board-certified anatomic pathologist (kp) blinded to tom’s age. a modified version of the yoshida scoring method12 was used to determine seminiferous tubular scores (table 1). leydig cell density within the interstitium was scored on a 0 3 scale: 0 = no cells present; 1= scattered/few cells present; 2 = moderate number of cells present; and 3 = densely packed. tom study 2 unowned free-roaming toms were presented for castration at a feral cat neutering clinic in corvallis, oregon, during september. age was determined by dental eruption using silver’s chart.11 age groups were as follows: 2 6 months (n = 13) and > 6 months (n = 16). after general anesthesia was induced, scrotal hair was clipped and the skin was aseptically prepared for surgery. penis was examined to determine whether spines were present. a routine open castration was performed and vas deferens were used for further analyses. all toms recovered from surgery uneventfully. fluid contents from both vas deferens were digitally expressed onto a prewarmed microscope slide and mixed with 1 drop of eosin-nigrosin morphology stain, spread with a spreader slide, and allowed to air dry. sperm morphology analysis was performed under oil immersion (1000 x) using a bright field microscope with image capture capability. fifty sperm were evaluated per slide by a board-certified theriogenologist (mk) blinded to tom’s age, unless there were < 50 sperm present on the entire slide. these samples were not included in the sperm morphology evaluation. sperm were categorized as having normal or abnormal morphology. queen study unowned free-roaming queens were presented for ovariohysterectomy at a humane society in oregon during august and september. age was determined by dental eruption using silver’s chart.11 the queen ages ranged from 2 6 months of age and were grouped for comparison: 2 4 months (n = 13) and 5 6 months (n = 4). after general anesthesia was induced, a routine ovariohysterectomy was performed. all queens recovered from surgery uneventfully. the total ovarian-uterine weights from queens were recorded. both ovaries were hemi-sectioned, formalin-fixed, paraffin-embedded, cut into 6 µm sections, and 1 section from each ovary was stained with hematoxylin and eosin. slides were analyzed using bright field microscopy at 200 x by a single observer (eb) blinded to the queen’s age. follicles were classified as primary, secondary, or tertiary13 and the number of each follicle type was counted. the diameter of the largest tertiary (antral) follicle for each ovary was also recorded. data analyses data were analyzed using graphpad prism, version 8 (graphpad software inc, la jolla, ca). for all data, significance was defined as p < 0.05. tom study 1 diameters of 5 seminiferous tubules from each testis were averaged for each tom. mean ± sd seminiferous tubular diameter and spermatogenesis score for each age group (2 2.5, 3 4, 5 6, and > 6 months) were compared using one-way analysis of variance (anova), followed by a tukey’s post hoc test. presence of sperm in the seminiferous tubular lumen was compared among age groups (2 2.5, 3 4, 5 6, > 6 months) using a chi-square test. table 1. seminiferous tubular scores (modified from12) score criteria 12 many late spermatids or sperm (≥ 10) 11 only a few late spermatids or sperm (< 10) 10 no sperm or late spermatids, but many round spermatids (≥ 10) 9 no sperm or late spermatids, but only a few round spermatids (< 10) 8 no sperm or spermatids, but many secondary spermatocytes (≥ 10) 7 no sperm or spermatids, but only a few secondary spermatocytes (< 10) 6 no sperm, spermatids, or secondary spermatocytes, but many primary spermatocytes (≥ 10) 5 no sperm, spermatids, or secondary spermatocytes, but only a few primary spermatocytes (< 10) 4 no sperm, spermatids, or spermatocytes, but many spermatogonia (≥ 10) 3 only germ cells present are a few spermatogonia (< 10) 2 absence of germ cells, but sertoli cells are present 1 total absence of cells in tubular section http://dx.doi.org/10.58292/ct.v15.9745 citation line: clinical theriogenology 2023, 15, 9745, http://dx.doi.org/10.58292/ct.v15.9745 3 tom study 2 presence of penile spines was compared between age groups (2 6 and > 6 months) using a fisher’s exact test. mean ± sd percent normal sperm morphology for each age group (2 6 and > 6 months) was compared by an unpaired student’s t-test. queen study linear regression was used to determine the effect of age on total ovarian-uterine weight. the percentage of each follicle classification (primary, secondary, tertiary) was compared by a chi square test. the largest tertiary follicle diameter for each age group (2 4 and 5 6 months) was compared by a welch’s t-test. results tom study 1 seminiferous tubular diameters for 2 2.5, 3 4, 5 6, and > 6 months toms were 88.1 ± 10.9, 109.8 ± 8.9, 142.2 ± 16.9, 237.9 ± 52.5 µm, respectively (figure 1). seminiferous tubular diameter was larger (p < 0.01) in each successive age group. there was a 67% increase in seminiferous tubule diameter between toms 5 6 and > 6 months. spermatogenesis and leydig cell density scores were also different among age groups (p < 0.01 and p = 0.01, respectively; table 2). spermatogenesis score in toms > 6 months was higher than in 2 2.5 or 3 4 months (p < 0.01). in addition, leydig cell density in was higher (p < 0.01) in toms 2 2.5 months versus > 6 months. tom study 2 eight of 13 (62%) toms aged 2 6 months and 12 of 16 (75%) toms aged > 6 months had penile spines (p = 0.07). sperm were present in vas deferens secretions from all toms examined, regardless of age. percentage of normal sperm morphology (figure 2) for samples with > 50 sperm per slide was not different (p = 0.39) between toms aged 2 6 months (n = 11, 77 ± 11%) and > 6 months (n = 9, 81 ± 13%; figure 2). table 2. mean ± sd spermatogenesis and leydig cell density scores for unowned free-roaming toms. within a column, means without a common superscript are different (p < 0.05). age (months) spermatogenesis score leydig cell density score 2 2.5 5.0 ± 2.2a 1.8 ± 0.4a 3 4 4.8 ± 2.0 a 1.3 ± 0.5a,b 5 6 8.5 ± 3.2b 1.2 ± 0.4a,b > 6 12.0 ± 0.0b 1.0 ± 0.0b figure 1. representative cross-sectional images of seminiferous tubules in toms of various ages. a: 2 2.5, b: 4 5, c: 5 6, and d: > 6 months. magnification: 200 x. hematoxylin and eosin stain. bar = 100 µm. http://dx.doi.org/10.58292/ct.v15.9745 4 citation line: clinical theriogenology 2023, 15, 9745, http://dx.doi.org/10.58292/ct.v15.9745 queen study there was no association (r2 = 0.20, p = 0.07) between total ovarian-uterine weight of queens and age (figure 3). no differences (p = 0.08) were observed between 2 4 and 5 6 months queens in the numbers of primary, secondary, or tertiary follicles (table 3). average ovarian-uterine weight observed in 4 6 months queens was 1.18 ± 0.31 g. diameter of largest tertiary follicle diameter did not differ (p = 0.14) between queens 2 4 (581.6 ± 53.7 µm) and 5 6 months (469.4 ± 113.9 µm). a representative image of a tertiary follicle from a 2-month queen is illustrated (figure 4). discussion in the current study, ufr toms aged 3 4 months had significantly wider seminiferous tubular diameter (109.8 ± 8.89 µm) compared to what has been reported (86 µm)14 for domestic toms of similar ages. this finding was accompanied by most ufr toms 2 6 months and > 6 months having sperm in the vas deferens and a high percentage of these sperm with normal morphology (77 ± 11% and 81 ± 13%, respectively). percentage of sperm with normal morphology from ufr cats in the current study was higher than that reported in fertile domestic cats (> 55 70%).15–17 these data indicated that ufr toms as early as 2 months of age may be able to ejaculate sperm to achieve pregnancy. together, these data supported the hypothesis that spermatogenesis is occurring at an earlier age in ufr toms (< 6 months) compared to what was reported in domestic toms (8 10 months).10 although the 2 2.5 months ufr toms in the current study had the highest leydig cell density score, the average score was 1.8 out of 3, demonstrating that these males only had scattered cells throughout the seminiferous tubules. it is unclear why the other age groups in the current study did not demonstrate higher densities, especially considering that previous studies have observed adult leydig cells in domestic toms ~ 5 6 months of age.18 however, growth of penile spines is positively correlated with androgen-secretion and androgen-dependent mating activity.19 therefore, the presence of penile spines is a good indicator that toms have the capacity to copulate and sire kittens. fully matured penile spines are reported to not occur in domestic toms until ~ 8 months of age; however, they can first be detected ~ 6 7 months of age.20 in the current study, 1 and 2 toms, 4 and 5 months old, respectively, had fully matured penile spines, suggesting that even at these young ages, toms may be mating with estrous queens. in domestic nonpregnant queens, the average ovarian-uterine weight was reported to be 1.5 grams,21 similar to weights observed in queens 4 6 months (1.18 ± 0.31 g) in our study. young ufr queens had increased ovarian weights due to number and size of tertiary follicles as reported for prepubertal cats.22 average largest tertiary follicle diameter in the current study was observed in queens 2 4 months. although the tertiary follicles measured in the current study were smaller than reported for preovulatory follicles in queens (~ 3 mm), these findings suggest that ufr queens < 4 months age may have table 3. follicle classification (percent) in queens. age (months) primary secondary tertiary 2 4 47 23 30 5 6 35 20 45 b a figure 2. representative sperm morphology photomicrographs prepared from vas deferens secretions from toms of 2 6 (a) and > 6 months of age (b). magnification 1000 x. eosin-nigrosin stain. figure 3. relationship of ovarian-uterine weight and age in queens (2 6 months old). http://dx.doi.org/10.58292/ct.v15.9745 citation line: clinical theriogenology 2023, 15, 9745, http://dx.doi.org/10.58292/ct.v15.9745 5 the capacity to ovulate. this is supported by work in domestic queens that displayed pubertal estrus as early as 4.5 months.23 a limitation of the current study was that the exact age was not known due to the feral origins of the cats. for this reason, estimating age was based on dental eruption.11 however, using dentition to determine the age of kittens is somewhat imprecise. for example, using silver’s chart, there is some overlap in ages, with eruption of permanent canine teeth occurring between months 5.5 6.5. for the purposes of the current study, cats with 4 fully erupted canine teeth were estimated to be > 6 months of age. conclusion average lifespan of ufr cats is 2 years of age, with as many of 90% of kittens dying before 6 months of age.24 these cats may have adapted to selective pressures of early-age mortality by collectively decreasing the age at which they can reproduce, ensuring the propagation of their genetic material before their death. research presented in the current study supported a possible shift in puberty to a younger age in both male and female ufr cats. toms < 6 months of age had penile spines with morphologically normal sperm in the vas deferens secretions. queens as early as 2-4 months of age had small tertiary follicles. together, this evidence explains why even with the amount of effort placed into tnr programs, humane societies in the united states still cannot surgically sterilize ufr cats fast enough to control their numbers.7 to control ufr cat populations more effectively, sterilization efforts may need to include cats < 4 months of age. care and use of animals this research used tissues from animals that were not manipulated expressly for the purpose of obtaining those materials and therefore was exempt from iacuc review. author contributions conceptualization, mk and eb; methodology, mk and eb; formal analysis, mk, eb, and kp; investigation, mk and eb; resources, mk; data curation, mk, eb, and kp; writing—original draft preparation, mk, eb, kp, and db; writing—review and editing, mk, eb, kp, and db; supervision, mk; project administration, mk. all authors have read and agreed to the published version of the manuscript. funding research received no external funding. conflict of interest authors declare no conflict of interest. references 1. lowe s, browne m, boudjelas s: 100 of the worst invasive species: a selection from the global invasive species database. aliens 2000;1–12. 2. medina f, bonnaud e, vidal e, et al: a global review of the impacts of invasive cats on island endangered vertebrates. global change biol 2011;17:3503–3510. 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10.2460/javma.2004.225.1403 9. johnson ak: normal feline reproduction: the queen. j feline med surg 2022;24:204–211. doi: 10.1177/1098612x221079706 10. johnson ak: normal feline reproduction: the tom. j feline med surg 2022;24:212–220. doi: 10.1177/1098612x221079707 figure 4. ovarian cross-section from a 2-month queen; note tertiary (antral) follicle. magnification: 200 x. hematoxylin and eosin stain. http://dx.doi.org/10.58292/ct.v15.9745 https://doi.org/10.1111/j.1365-2486.2011.02464.x https://doi.org/10.1038/ncomms2380 https://doi.org/10.1371/journal.pntd.0001142 https://doi.org/10.1136/vr.114.18.447 https://doi.org/10.1126/science.325_1490 https://doi.org/10.2460/javma.2004.225.1403 https://doi.org/10.1177/1098612x221079706 https://doi.org/10.1177/1098612x221079707 6 citation line: clinical theriogenology 2023, 15, 9745, http://dx.doi.org/10.58292/ct.v15.9745 11. silver ia: the ageing of domestic animals. in: brothwell b, higgs e: editors. science in archaeology. new york; basic books: 1963. p. 250–268. 12. yoshida a, miura k, shirai: evaluation of seminiferous tubule scores obtained through testicular biopsy examinations of nonobstructive azoospermic men. fertil steril 1997;68:514–518. doi: 10.1016/s0015-0282(97)00239-2 13. araki y: formation and structure of mammalian ovaries. in tulsiani d: editor. introduction to mammalian reproduction. 1st edition, boston; kluwer academic publishers: 2003. p. 141–155. doi: 10.1007/978-1-4615-0273-9_9 14. sanchez b, pizzaro m, garcia p, et al: postnatal development of seminiferous tubules in the cat. j reprod fertil suppl 1993; 47:343–348. 15. axner e, linde forsberg c. sperm morphology in the domestic cat, and its relation with fertility: a retrospective study. reprod domest anim 2007;42:282–291. doi: 10.1111/j.1439-0531.2007.00780.x 16. howard jg, brown jl, bush m, et al: teratospermic and normospermic domestic cats: ejaculate traits, pituitary-gonadal hormones, and improvement of spermatozoal motility and morphology after swim-up processing. j androl 1990;11:204–215. 17. wildt d, bush m, howard j, et al: unique seminal quality in the south african cheetah and a comparative evaluation in the domestic cat. biol reprod 1983;29:1019–1025. doi: 10.1095/ biolreprod29.4.1019 18. sanchez b, pizzaro m, garcia p, et al: histological study of leydig cells in the cat from birth to sexual maturity. j reprod fertil suppl 1993;47:349–353. 19. aronson lr, cooper ml: penile spines of the domestic cat: their endocrine-behavior relations. anat rec 1967;157:71–78. doi: 10.1002/ar.1091570111 20. shille vm, stabenfeldt gh: current concepts in reproduction of the dog and cat. adv vet sci comp med 1980;24:211–243. 21. latimer hb. the prenatal growth of the cat. viii. the weights of the kidneys, bladder, gonads, and uterus, with the weights of the adult organs. growth 1939;3:89–108. 22. uchikura k, nagano m, hishinuma m: evaluation of follicular  development and oocyte quality in pre-pubertal cats. reprod  domest anim 2010;45:405–411. doi: 10.1111/ j. 1439 0531.2010.01590.x 23. jemmett je, evans jm: a survey of sexual behavior and reproduction of female cats. j small anim pract 1977;18:31–37. doi: 10.1111/j.1748-5827.1977.tb05821.x 24. van aarde rj: population of feral cats on marion island. acta zool fennica 1984;172:107–110. http://dx.doi.org/10.58292/ct.v15.9745 https://doi.org/10.1016/s0015-0282(97)00239-2 https://doi.org/10.1007/978-1-4615-0273-9_9 https://doi.org/10.1111/j.1439-0531.2007.00780.x https://doi.org/10.1095/biolreprod29.4.1019 https://doi.org/10.1095/biolreprod29.4.1019 https://doi.org/10.1002/ar.1091570111 https://doi.org/10.1111/j.1439-0531.2010.01590.x https://doi.org/10.1111/j.1439-0531.2010.01590.x https://doi.org/10.1111/j.1748-5827.1977.tb05821.x long term reproductive effects of 3 dogs given deslorelin acetate at birth long term reproductive effects of 3 dogs given deslorelin acetate at birth marcela faya,a,c cynthia marchetti,a marcelo priotto,a mariela grisolía,a,b camila lapuente,b thomas conlond, cristina gobellob,c acatholic university of córdoba, bnational university of la plata cnational research council, argentina although long term, detailed reproductive assessments of canine contraceptive treatments are scarce, they provide additional valuable information about protocol efficacy and safety. the aim was to describe the reproductive status of 2 males (dog 1 and dog 2) and 1 female. these were crossbred dogs, littermates, 28 months old, body weight 11.1 ± 1.7 kg and had just achieved puberty. within 24 hours after birth, they had been given subcutaneous implants of 18.8 mg of deslorelin acetate (suprelorin, virbac, france), for contraceptive purposes.1 all 3 dogs were exposed to other fertile dogs for mating throughout an entire estrous period. after blood sampling for hormone concentrations and pregnancy diagnosis, they were gonadectomized. the gonads were grossly and histomorphometrically (image pro plus v6.0 media cybernetics, silver spring, ma) examined. before surgery, serial physical examinations revealed a small vulva in the female as well as unilateral delayed (6 months old) testicular descent and bilateral inguinal cryptorchidism in dogs 1 and 2, respectively. in these 2 dogs, seminal samples could not be collected due to azoospermia and aspermia, despite their ability to achieve an erection. although the female ovulated (serum progesterone 15 ng/ml; elecsys, roche diagnostics, mannheim, germany) and became pregnant, the males did not achieve mating. serum anti müllerian hormone (amh-gen ii, beckman coulter, brea, ca) concentrations were 21.3 and 4.9 ng/ml in dogs 1 and 2, respectively and 0.35 ng/ml in the female. serum testosterone was normal in dog 1 but non detectable in dog 2, whereas estradiol 17² concentrations (elecsys, roche diagnostics, mannheim, germany) in the female were physiologic. gonadosomatic index was < 0.02 in the 3 dogs. the ovaries had 48% atretic, 44% primordial and 8% primary follicles, as well as corpora lutea. the high and low proportions of the 2 former follicles may indicate enhanced recruitment due to low amh concentrations. testes of dog 2 had small seminiferous tubules composed of spermatogonia and sertoli cells i.e. sertoli cell only syndrome. testes of dog 1 had disorganized spermatogenesis up to the spermatid level, vacuolization and a spermatids sertoli cell ratio of 3.07, much lower than in the less efficient species. the area of the leydig nuclei was 40% smaller in dog 2 than in dog 1, indicating abnormal steroideogenic function and correlating with the nondetectable serum testosterone concentrations. it was concluded that this contraceptive protocol caused delayed puberty, abnormalities in both testicular descent, and spermatogenesis, as well as ovarian and hormonal findings that could be associated with premature reproductive failure in the female. keywords: canine, gonad, neonatal, reproduction control, sterilization acknowledgement funded by michelson found animals foundation (mg13-s03), los angeles, ca reference 1. faya m, marchetti c, priotto m, et al: postponement of canine puberty by neonatal administration of a long term release gnrh superagonist. theriogenology 2018;15;118:190-195. 513 clinical theriogenology • volume 11, number 3 • september 2019 https://www.ncbi.nlm.nih.gov/pubmed/29913424 https://www.ncbi.nlm.nih.gov/pubmed/29913424 514clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2015: effects of different applications of pyrethrins and cyfluthrin, a synthetic pyrethroid, on bull reproductive parameters effects of different applications of pyrethrins and cyfluthrin, a synthetic pyrethroid, on bull reproductive parameters* jamie l. stewart,a,b clifford f. shipley,a,b frank a. ireland,b tara l. felix,b vickie l. jarrell,b sandra c. jimenez poveda,b daniel w. shikeb adepartment of veterinary clinical medicine, college of veterinary medicine, university of illinois, urbana il; bdepartment of animal sciences, college of agricultural, consumer and environmental sciences, university of illinois, urbana, il abstract the use of pyrethroid insecticides has been reported to affect semen quality and steroidogenesis in many mammals. recent experiments with bulls demonstrated no effects on semen quality when the pyrethroid insecticide, cyfluthrin, was applied at labeled dosages. the current study determined effects of cyfluthrin and pyrethrin spray products, used in combination with cyfluthrin pour-on and fly tags, on bull sperm motility and serum testosterone concentrations. angus x simmental bulls were assigned randomly to one of two treatment groups: 1) pour-on plus fly tags (cont; n = 12), or 2) pour-on, fly tags, premise spray plus fog spray (exp; n = 11). overall and progressive sperm motility and serum testosterone concentrations were measured weekly for nine weeks. there were treatment by week interactions (p < 0.01) for overall motility, progressive motility, and testosterone concentrations. at week two, overall motility was greater (p = 0.05) for cont than exp; however, there were no differences (p ≥ 0.12) at other times. progressive sperm motility in exp tended to be reduced (p = 0.07) at week two and increased (p = 0.08) at week seven, but did not differ (p ≥ 0.15) in other weeks. serum testosterone concentrations were reduced (p = 0.05) at week one in exp; however, concentrations did not differ again until week nine when exp bulls had a two-fold decrease (p < 0.01) in serum testosterone concentrations when compared to cont bulls. this steep decline suggests a delayed effect of the insecticide applications on blood testosterone concentrations. keywords: bull, fertility, insecticide, pyrethroid, reproduction introduction pest control is a crucial management strategy for cattle production facilities. insect control is needed because ectoparasite infestations can cause economic losses in affected cattle. production losses include poor performance of the animal, reduced value of the hides as a result of irritation, and transmission of a variety of diseases.1 the severity of these effects is strongly correlated with environmental conditions, with the summer months being the most devastating.1 pyrethroid insecticides have been implicated as a cause for depression of sperm motility in bulls.2 these claims were based on clinical observations by volkmann,3 who reported a correlation between pyrethroid insecticide use and poor semen quality of bulls and rams. negative effects were evident within a few days after the first exposure to the insecticides, improved approximately two to four weeks after its use was discontinued, and included a variety of applications, such as pour-on, premise sprays, and fogging.3 dosage or frequency of application was not reported.3 pyrethroid insecticides have been reported to negatively affect semen quality and steroid hormone production in vivo in many species such as man,4,5 dwarf goats,6 rats,7 and rabbits.8 semen quality and motility were hypothesized to be affected because pyrethroids are antiandrogenic endocrine disruptors in vitro.9-11 more recently, french et al12 * these data were presented at the 2013 midwest section meeting of the american society of animal science in des moines, ia and at the 2014 theriogenology conference in portland, or. clinical theriogenology • volume 7, number 1 • march 201527 found that commercial cyfluthrin (1%) products administered topically to bulls according to label directions (24 ml; or 0.3 mg/kg) had no effect on sperm motility, morphology, or peripheral testosterone concentrations when compared with bulls receiving no insecticide. similarly, cain et al13 found that topical applications of permethrin (1%) at 150% label dose (225 ml; or 3 mg/kg) did not affect the proportion of normal sperm cells in bulls. the contradictory results among these studies12,13 and other reports3 could be due to the route, dosage, and/or frequency of administration. topical/pour-on and fly tag applications at labeled dosages are intended to provide insecticidal properties without allowing absorption of these compounds; whereas in many of the reports in which detrimental results were observed,7,8 large, oral dosages (≥ 10 mg/kg) of compounds were administered. approved routes for direct administration of pyrethroids include pour-on (topicals) and/or fly tags. approved routes for indirect administration include premise sprays and foggers. misuse of these products, by more frequent or more concentrated applications than labeled, creates opportunities for inducing detrimental effects. as these insecticides must be diluted with water and then sprayed throughout the barn (premise) or over the entire animal (fogging), administering the correct dosage is reliant upon the accuracy of the applicator. improper dilutions or over-spraying could lead to inhalation or ingestion of large concentrations of pyrethroid compounds by the animals. these potential misuses of pyrethroid products could account for reported deleterious effects on reproductive parameters in bulls;3 however, controlled studies on the effects of spray applications have not been performed. therefore, the objectives of this study were to determine the effects of pyrethroid and pyrethrin sprays used in combination with pour-on and fly tags on bull sperm motility and serum testosterone concentrations. our hypothesis was that the addition of pyrethrin and pyrethroid spray applications would have a negative effect on bull reproductive parameters. materials and methods animals all procedures were approved by the university of illinois institutional animal care and use committee. angus x simmental crossbred bulls (n = 23) ranging from one to six years in age (average initial bw = 796 ± 160 kg) were used in this study and housed at the dixon springs agricultural center in simpson, il. seventeen of the bulls were owned by the university of illinois and six of the bulls were privately owned and leased for the purposes of this study. due to the need to isolate the leased bulls from the university-owned bulls, bulls were blocked by source and assigned randomly to one of two treatment groups: 1) pour-on plus fly tags (cont, n = 12), or 2) pour-on, fly tags, premise spray plus fog spray (exp, n = 11). the six leased bulls were housed in one barn (three per treatment group), and the seventeen university-owned bulls were housed in a second barn (eight or nine per treatment group). within each barn, treatment groups were separated by aisles to avoid cross-contamination of spray products. pyrethrin/pyrethroid insecticide treatments each bull in both the cont and exp groups was treated with 24 ml cyfluthrin 1% solution (average dose 0.3 mg cyfluthrin/kg bw; cylence®, bayer animal health, shawnee mission, ks; active ingredient cyfluthrin; 1%) poured along the topline at weeks zero and four, and each bull received one cyfluthrin-impregnated fly tag in each ear (cylence® ultra fly tags, bayer animal health, shawnee mission, ks; active ingredients beta-cyfluthrin; 8% and piperonyl butoxide; 20%) at week zero. additionally, the exp bulls were fogged daily with a pyrethrin spray (prozap® ld-44z, loveland industries, inc., greeley, co; active ingredients pyrethrins; 0.5% and piperonyl butoxide; 4%) at approximately 3 seconds per bull, and their stalls were treated with a beta-cyfluthrin premise spray (tempo®, bayer animal health, shawnee mission, ks; active ingredients beta-cyfluthrin, cyano and methyl 3; 11.8%) at 16 ml/1000 sq. ft. once weekly. all products were used according to label directions as approved for use in food-producing animals. clinical theriogenology • volume 7, number 1 • march 2015 28 sample collection the experiment was performed over nine weeks from may through july. a complete health and reproductive examination was performed before applying treatments at week zero and upon conclusion of the study at week nine. individual body weights were obtained and each bull was assigned a body condition score (bcs) of one to nine. scrotal circumference was measured using a scrotal tape. tone and symmetry were assessed in the testicles and epididymides. transrectal palpation was performed to assess the accessory sex glands. during semen collection the penis was examined for any abnormalities. samples were collected once weekly beginning at week zero (prior to receiving treatments), at similar times of day to account for daily hormonal fluctuations. to obtain semen samples, an electroejaculator was placed rectally and the programmed cycle was allowed to run. at least 3 ml of semen were collected from each bull before the program concluded and the electroejaculator probe removed. semen was collected into 15 ml conical tubes using collecting handles and sleeves. immediately after collection, tubes were transported in a warm water (37°c) bath to the laboratory. semen was diluted 1:60 in warm saline (37°c) and 2 µl of diluted semen was placed into 20 µm chamber slides (vitrolife, microcell counting chambers, san diego, ca). computer-assisted sperm analysis (casa) equipment (spermvision, minitube of america, inc., verona, wi) was used to obtain overall and progressive motility by averaging seven readings from various portions of the chambered slide. whole blood, as a source of serum for testosterone analysis, was obtained via the tail vein once per week immediately following electroejaculation. samples were obtained with 1 to 1.5 inch, 18 to 20 gauge needles into serum separating evacuated tubes. blood samples were centrifuged for ten minutes at 1200 rpm, serum was removed with a sterile pipette, and the sample was frozen at -20°c. testosterone concentrations were determined by double-antibody radioimmunoassay (coat-a-count®; diagnostics products corporation, los angeles, ca) at the animal health diagnostic center, cornell university, ithaca, ny. statistical analysis all data were analyzed in mixed procedure of sas as a randomized complete block. repeated measures, with compound symmetry covariance structure, were used to test the treatment by week interactions for reproductive parameters. the effect of block (barn) was tested in the model and found not to be significant. therefore, it was removed from the model. significance was declared at p ≤ 0.05 and trends were discussed at 0.05 < p ≤ 0.10. results physical examination bull weight and bcs were not different at the end of the experimental period (p ≥ 0.82; table). bull bcs remained adequate with an average score of 5.4 in both exp and cont groups. subjectively, there were no changes on physical examination, and no differences were observed in initial or final scrotal circumferences between cont and exp groups (p ≥ 0.22; table). reproductive parameters there were treatment by week interactions (p < 0.01) for overall motility (fig. 1), progressive motility (fig. 2), and serum testosterone concentrations (fig. 3). at week two, overall motility was greater (p = 0.05) for cont than exp; however, there were no differences (p ≥ 0.12) in overall motility at other times (fig. 1). progressive sperm motility in exp tended to be reduced (p = 0.07) at week two and increased (p = 0.08) at week seven compared to cont, but did not differ (p ≥ 0.15) in other weeks (fig. 2). serum testosterone concentrations were decreased (p = 0.05) by week one in exp bulls when compared to cont; however, concentrations did not differ again until week nine when exp bulls had a two-fold decrease (p < 0.01) in serum testosterone concentrations when compared to cont bulls (fig. 3). clinical theriogenology • volume 7, number 1 • march 201529 discussion the use of pyrethroid insecticides on breeding bulls has recently become a controversial topic in the cattle industry due to reports of negative effects on semen quality in other mammals.4-8 the route of exposure may be a crucial determinant in the manifestation of negative effects.15 dermal absorption of pyrethroids across intact skin was reported as very poor,15,16 and pour-on applications are considered safe for breeding cattle when administered at labeled dosages.12,13 concentrations of pyrethroids in body fluids were not directly measured in our previous study,12 nor were they quantified here; however, no effects were observed on serum testosterone or semen quality in bulls receiving both pour-on and fly tag applications.12 therefore, if dermal absorption of pyrethroid compounds did occur, it did not result in deleterious effects on the reproductive parameters measured.12, 13 in contrast to transdermal absorption, pyrethroid insecticides are easily absorbed through the gastrointestinal tract and pulmonary membranes,15 suggesting that routes of administration such as sprays may impart harmful effects on semen quality not observed with dermal applications. in the current study, weekly premise sprays with beta-cyfluthrin were applied while the exp bulls were housed in the barns. the application likely contaminated feed and surfaces to which bulls were exposed, allowing for potential oral ingestion. the exp bulls also received a daily pyrethrin fogging application, which presented opportunity for inhalation and oral exposures. spray applications are consistent with those reported by volkmann3 that correlated with the development of poor quality semen in bulls, including increased secondary sperm defects and diminished progressive motility. in one case,3 volkmann reported diminished semen quality in numerous ejaculates collected from a bull stud two days after application of a pyrethroid premise spray, and bifenthrin was detected both in serum and semen in two of the exposed bulls. in the current study, overall motility of both cont and exp groups did not decline below 70% and progressive motility did not decline below 50% at any week, both of which exceed the minimum recommendations set by the society for theriogenology for classification as satisfactory potential breeders (> 30% for both progressive and overall motility).14 although a reduction in overall and progressive motility was observed in exp bulls at week two in the present study, the percentages were still well above acceptable minimums and did not persist in following weeks, with progressive motility being greater in exp bulls at week seven. since this treatment effect was on the threshold of significance (p = 0.05), a larger sample size may be warranted to confirm its importance. additionally, scrotal circumference of cont and exp bulls did not differ and were also above the minimum recommended scrotal circumference set by the society for theriogenology for satisfactory potential breeders (≥ 34 cm).14 information regarding the amount of bifenthrin contained in the premise spray used in the case reported by volkmann3 is not available. the potentially higher dosage or the difference between pyrethroid products used (bifenthrin versus cyfluthrin/pyrethrin) in that report3 may account for contradictory results on semen motility reported here. further controlled studies would be needed to explore the effects of higher dosages or different pyrethroid products on bull reproductive parameters. the most striking finding in the current study was the significant decline in serum testosterone concentrations of exp bulls at week nine. this suggests that chronic exposure to cyfluthrin, betacyfluthrin, and/or pyrethrin may decrease testosterone production which would be consistent with pyrethroid effects observed in men,4 rabbits,8 and rats.17 while zhang11 observed rats treated with oral cyfluthrin and beta-cyfluthrin had serum testosterone levels which did not differ from controls, antiandrogenic properties were demonstrated. more in-depth toxicological studies are needed to explore these findings. the specific compound of pyrethroid, dose, and route of administration may each play a role. there are several hypotheses regarding how pyrethroid insecticides may affect steroidogenesis. cholesterol content in the testes, a necessary precursor for testosterone production, was reported to be significantly reduced in cyfluthrin-treated mice,18 which could inhibit steroidogenesis and cause a drastic decline in peripheral testosterone concentrations. during metabolism of pyrethroids, reactive oxygen clinical theriogenology • volume 7, number 1 • march 2015 30 species (ros) are generated, leading to oxidative stress.19,20 while the body is able to compensate for low-levels of ros produced naturally, an overproduction could have damaging effects on cell membranes in the testes.21 histological examination in mice given oral cyfluthrin showed various structural abnormalities in the testes,18 indicative of cell damage. oxidative damage to testosterone-producing leydig cells in the testes could result in a decline in peripheral testosterone levels. a decline in peripheral blood testosterone could impact steroidal feedback to the hypothalamus and cause increased production and secretion of luteinizing hormone from the anterior pituitary, as was demonstrated in both men4 and mice.22 the effects of pyrethroid insecticides on steroidogenesis in beef bulls have not been reported. we hypothesized that semen motility and testosterone concentrations would be affected by spray applications allowing for potential oral and/or inhalation exposures. however, testosterone concentrations did not decline until week nine and sperm motility was not adversely affected in the exp bulls. if a toxic level of insecticide were to accumulate over time, the decline in serum testosterone at week nine could be early signs of effects on steroidogenesis. if oxidative damages had occurred, they would likely affect both leydig and sertoli cells. sertoli cells are responsible for supporting developing germ cells; therefore, we would expect sperm motility to deteriorate following a decline in serum testosterone. zhang et al22 found that mice treated with oral cis-permethrin had decreased testosterone production due to mitochondrial damage to leydig cells, causing a dose-dependent negative effect on sperm motility. a decline in testosterone concentrations, as seen in the current study, could subsequently alter spermatogenesis and semen motility. since the current study ended after one spermatogenic cycle, while decreases in testosterone were not detected until the final week, it is not known if sperm quality would be affected at some later time. additional studies examining sperm motility and morphology at later time points following a decline in testosterone are therefore warranted. conclusion the current study examined the effects of commercially available pyrethroid (cyfluthrin and betacyfluthrin) and pyrethrin insecticide applications on bull reproductive parameters over one spermatogenic cycle (~60 days). these results showed no consistent deleterious effects on overall or progressive sperm motility in bulls which received fly tags, pour-on, daily fogging, and weekly premise spray applications (exp) compared with those which received only fly tags and pour-on products (cont). testosterone concentrations were significantly reduced in exp bulls when compared with cont bulls at week nine, suggesting a delayed effect of the beta-cyfluthrin and pyrethrin spray applications on testosterone production. while these results conclude that these spray applications do not adversely affect sperm motility in the short-term (nine weeks), additional studies are needed to determine the long-term effects of these application methods on semen quality. acknowledgements the authors would like to acknowledge erica navis, mary west, the staff at the dixon springs agricultural center, corinne bromfield, elena bichi, elsbeth grimmer, katie maples, ashley seder, and the university of illinois veterinary students for their assistance with the experimental process; and bayer animal health for the donation of the pyrethroid products used. references 1. peter rj, van den bossche p, penzhorn bl, et al: tick, fly, and mosquito control – lessons from the past, solutions for the future. vet parasitol 2005;132:205-215. 2. ishmael w: common insecticide can hurt bull breeding ability. beef magazine 2012. http://beefmagazine.com/genetics/common-insecticide-can-hurt-bull-breeding-ability. 30 mar 2012. 3. volkmann dh, voelkl dl: clinical observations on the effects of pyrethroid insecticides on bull semen quality. proc acad vet consult 2012. available at: http://www.avc-beef.org/proceedings/2012-2/volkman.pdf. 4. meeker jd, barr db, hauser r: pyrethroid insecticide metabolites are associated with serum hormone levels in adult men. reprod toxicol 2009;27:155-160. clinical theriogenology • volume 7, number 1 • march 201531 5. perry mj, venners sa, barr db, et al: environmental pyrethroid and organophosphorus insecticide exposures and sperm concentration. reprod toxicol 2007;23:113-118. 6. ahmad, m, hussain i, kahn a: deleterious effects of cypermethrin on semen characteristics and testes of dwarf goats (capra hircus). exp toxicol pathol 2009;61:339-346. 7. kim ss, lee rd, lim kj, et al: potential estrogenic and antiandrogenic effects of permethrin in rats. j reprod dev 2005;51:201-210. 8. yousef mi: vitamin e modulates reproductive toxicity of pyrethroid lambda-cyhalothrin in male rabbits. food chem toxicol 2010;48:1152-1159. 9. sun h, xu xl, xu lc, et al: antiandrogenic activity of pyrethroid pesticides and their metabolite in receptor gene assay. chemosphere 2007;66:474-479. 10. wang l, liu w, yang c, et al: enantioselectivity in estrogenic potential and uptake of bifenthrin. environ sci technol 2007;41:6124-6128. 11. zhang j, zhu w, zheng y, et al: the antiandrogenic activity of pyrethroid pesticides cyfluthrin and ß-cyfluthrin. reprod toxicol 2008;25;491-496. 12. french hm, shipley cf, ireland fa, et al: the effect of cyfluthrin, a commercially available synthetic pyrethroid, on bovine semen quality and pregnancy rates. clin therio 2014;6:33-39. 13. cain aj, king h, wills r, et al: evaluation of the effects of topical permethrin insecticide on bull semen quality. clin therio 2014;6:25-31. 14. chenoweth pj: a new bull breeding soundness form. proc soc therio 1993. p 63-70. 15. pyrethrins and pyrethroids. in: recognition and management of pesticide poisonings. 6th ed. washington (dc): united states environmental protection agency; 2013. p. 38-42. 16. arnold d: residues of some veterinary drugs in animals and food. fao food and nutrition paper 41/10: cyfluthrin 1997. available at: http://www.fao.org/docrep/w8338e/w8338e06.htm. 17. elbetieha a, da’as si, khamas w, et al: evaluation of the toxic potentials of cypermethrin pesticides on some reproductive and fertility parameters in the male rats. arch environ con tox 2001;41:522-528. 18. rajawat nk, soni i, mathur p, et al: cyfluthrin-induced toxicity on testes of swiss albino mice. int j curr microbiol appl sci 2014;3:334-343. 19. kale m, rathor n, john s, et al: lipid peroxidative damage on pyrethroid exposure and alterations in antioxidant status in rat erythrocytes: a possible involvement of reactive oxygen species. toxicol lett 1999;105:197-205. 20. sadowska-woda i, wójcik n, karowicz-bilińska a, et al: effect of selected antioxidants in β-cyfluthrin-induced oxidative stress in human erythrocytes in vitro. toxicol in vitro 2010;24:879-884. 21. sanocka d, maciej k: reactive oxygen species and sperm cells. reprod biol endocrinol 2004;2:1-7. 22. zhang wy, ito y, yamanoshita o, et al: permethrin may disrupt testosterone biosynthesis via mitochondrial membrane damage of leydig cells in adult male mouse. endocrinology 2007;148:3941-3949. table. effects of cyfluthrin and pyrethrin insecticides on bull bw, bcs, and scrotal circumference. treatment body weight (kg) conta expb sem p-value week 0 886 870 33 0.72 week 9 881 872 34 0.84 bcsc week 9 5.4 5.4 0.17 0.82 scrotal circumference (cm) week 0 41.11 42.28 0.71 0.22 week 9 41.71 41.61 0.73 0.92 acont: pour-on, fly tags bexp: pour-on, fly tags, weekly premise spray, daily fogging cbcs scores ranged from 1 to 9 clinical theriogenology • volume 7, number 1 • march 2015 32 figure 1. effects of cyfluthrin and pyrethrin insecticides on overall sperm motility (%) throughout nine weeks (one spermatogenic cycle) in bulls. cont=pour-on, fly tags; exp=pour-on, fly tags, weekly premise spray, daily fogging figure 2. effects of cyfluthrin and pyrethrin insecticides on progressive sperm motility (%) throughout nine weeks (one spermatogenic cycle) in bulls. cont=pour-on, fly tags; exp=pour-on, fly tags, weekly premise spray, daily fogging clinical theriogenology • volume 7, number 1 • march 201533 figure 3. effects of cyfluthrin and pyrethrin insecticides on serum testosterone concentrations (ng/ml) throughout nine weeks (one spermatogenic cycle) in bulls. cont=pour-on, fly tags; exp=pour-on, fly tags, weekly premise spray, daily fogging clinical theriogenology • volume 7, number 1 • march 2015 34 2011: effect of gnrh administration at day 5 or day 7 after ai on progesterone concentrations, corpus luteum volume and pregnancy in lactating dairy cows   effect of gnrh administration at day 5 or day 7 after ai on progesterone concentrations, corpus luteum volume and pregnancy in lactating dairy cows ramanathan k. kasimanickam,a mathew r. mink,b raymond l. nebelb* adepartment of veterinary clinical sciences, washington state university, pullman, wa; bdepartment of dairy sciences, virginia tech, blacksburg, va abstract the objective of the study was to determine the effect of gonadotropin releasing hormone (gnrh) administration on day 5 or day 7 after artificial insemination (ai) on progesterone concentration, total corpus luteum (cl) volume and pregnancy/ai (p/ai) in lactating dairy cows. multiparous dairy cows (n=1054) from two dairy farms, one in virginia and one in north carolina, were enrolled in this study. all cows were synchronized with presynch-co-synch protocol for first service. following ai (day 0), cows were randomly allocated to one of the following three treatment groups: cows in the gnrh5 group (n=335) received 100 µg of gnrh im five days after insemination; cows in the gnrh-7 group (n=390) received 100 µg of gnrh im seven days after insemination and cows in the control group (n=329) received no treatment. blood samples were collected from a subset of cows (n=52) at day -38, day -24, day -10, day 0 (day of ai) and day 14 for progesterone concentration determination. the progesterone concentrations at day -38, -28, and -10 determined ovarian cyclicity of the cows and the progesterone concentration at day14 (9 or 7 days following gnrh treatment) were used to detect differences in progesterone among treatment groups. the ovaries of the subset of cows were screened using ultrasonography for the formation of accessory cl and total volume of cls (primary + accessory cls) was recorded. cows were examined for pregnancy by transrectal ultrasonography at 30 days and again at 60 days after insemination. the p/ai for gnrh-5, gnrh-7 and control groups were 31.6% (106/335), 33.8% (132/390) and 31.9% (105/329), respectively (p > 0.1). the total cl volume differed among the treatment groups (p < 0.05) but day 14 progesterone concentration did not differ among the treatment groups (p > 0.1). the progesterone concentration in the control group (n=17) was 5.9 ± 0.3 ng/ ml and total cl volume was 8.3 ± 0.6 cm3. the progesterone concentration in the gnrh-5 group (n=15) was 6.3 ± 0.4 ng/ ml and total cl volume was 12.6 ± 0.7 cm3. the progesterone concentration in the gnrh-7 group (n=20) was 6.6 ± 0.4 ng/ ml and total cl volume was 12.0 ± 0.8 cm3. in conclusion, gnrh administration at day 5 or day 7 following insemination neither increased progesterone concentrations nor improved p/ai compared to controls. keywords: gnrh, progesterone, pregnancy rate, timed artificial insemination, dairy cattle, fertility introduction establishment and maintenance of pregnancy are complex processes that require precise communication between conceptus and dam. the role of progesterone is critical in these processes and insufficient luteal activity has been associated with infertility in cattle. conception failure is coincident with less than normal concentrations of progesterone as early as six days after ai.1 blood progesterone generally reaches greater concentrations earlier in pregnant than in non-pregnant cows suggesting that strategies to increase progesterone concentration earlier following insemination may be beneficial in reducing embryo loss. embryo development is related to concentrations of progesterone and the ability of the conceptus to secrete interferon-τ.2 mann et al tested the effect of timing of progesterone supplementation on embryo development and interferon-τ production in cows and concluded that both early (5 to 9 days) and later (12 to 16 days) progesterone supplementation resulted in significant increases in plasma progesterone. in addition they have found early progesterone supplementation resulted in a fourfold increase in trophoblast length (p < 0.01) and a six-fold increase in uterine concentration of interferon-τ (p < 0.05).3 hence more exposure of an embryo to progesterone may increase its chances of secreting interferon-τ and thus survive. exogenous progesterone can stimulate embryo development, so by manipulating post-insemination concentrations of progesterone, conception rates may ultimately improve.4 several strategies have been used to increase progesterone concentration after breeding.5 administration of gnrh and human chorionic gonadotropin (hcg) during the early luteal phase induces                                                              * current address: select sires inc., plain city, oh clinical theriogenology • volume 3 number 4 • december 2011455   ovulation of the dominant follicle from the first follicular wave and results in formation of an accessory cl.6 injection of gnrh or its agonists induced accessory cl when administered on days 5 to 12 of the estrous cycle.7,8 differences in response to gnrh administration from days 5 to 12 of the estrous cycle exist and may be due to the difference in the size of the follicle present at that time. the follicle size on day 5 of the estrous cycle is smaller in cows with a longer inter-wave interval (two follicular waves) and renders them non-responsive to gnrh when administered at day 5. however these follicles may responsive to gnrh on day 7. the objective of the study was to determine the effect of gnrh administration on day 5 or day 7 after ai on progesterone concentration, total cl volume and p/ai in lactating dairy cows. materials and methods multiparous dairy cows (n=1054) from two dairy farms (one in virginia and one in north carolina) were enrolled in this study. all cows were synchronized with presynch-co-synch protocol for first service (figure). briefly, cows received two injections of 25 mg prostaglandin f2α (pgf) im (lutalyse®, pfizer animal health, new york, ny) at 32 ± 3 days postpartum and 46 ± 3 days postpartum (presynch); 100 μg gnrh im (cystorelin®, merial ltd. duluth, ga) at 60 ± 3 days postpartum; 25 mg pgf at 67 ± 3 days postpartum; and 100 μg gnrh + timed ai (tai) at 70 ± 3 days postpartum (cosynch; day 0). body condition scores (1 to 5 scale; 1-emaciated; 5-obese) of all cows were recorded at the time of ai. following ai, cows were blocked by body condition score and parity, and were randomly allocated to one of the following three treatment groups: cows in the gnrh-5 group (n=335) received 100 µg of gnrh im on day 5 after insemination; cows in the gnrh-7 group (n=390) received 100 µg of gnrh im on day 7 after insemination and cows in the control group (n=329) received no treatment. blood samples were collected from a subset of cows (n=52) at 32, 46, 60, 70, and 84 days in milk (dim) for progesterone concentration determination and the ovaries were screened using ultrasonography. total volume of cls (primary and accessory cl) was recorded. the cl volume was calculated using the formula 4/3πr3. the progesterone concentration at 60 dim (at first gnrh the initiation of the ovsynch protocol) determined ovarian cyclicity of the cows. progesterone concentrations <1 ng/ml indicated a non-cycling cow. the progesterone concentration at 84 dim (14 days after insemination) was used to measure differences among treatment groups in progesterone concentrations following gnrh administration (7 or 9 days after gnrh injection). cows were examined for pregnancy by transrectal ultrasonography (sonosite 180 plus, sonosite inc., bothell, wa) at 30 days and again at 60 days after insemination. figure. pgf2 pgf2 gnrh pgf2 gnrh + tai 14 d 14 d 7 d 72 h 5/7 d 32 ± 3 46 ± 3 60 ± 3 67 ± 3 70 ± 3 75/77 (day -38) (day -24) (day -10) (day -3) (day 0) (day 5/7) progesterone assay blood samples were collected via coccygeal venipuncture into blood collection tubes (vacutainer®, becton dickinson, franklin lakes, nj), placed immediately on ice. the samples were brought to the laboratory and centrifuged at 3,000 x g for 15 minutes. harvested serum was stored at 20°c until further processing. progesterone concentration was determined using a coat-a-count kit (dpc diagnostic products inc., los angeles, ca) solid-phase 125i radioimmunoassay. the intra-assay and inter-assay coefficients of variation were 6.34% and 7.29%, respectively. the assay sensitivity was 0.03 ng/ml. control (n=17); gnrh-5 (n=15); gnrh-7 (n=20); clinical theriogenology • volume 3 number 4 • december 2011 456   statistical analyses data were analyzed by proc logistics procedure of sas (sas version 9.12, sas institute inc., cary, nc) to determine the difference in p/ai among treatment groups. the variables included in the model were parity, season, farm, treatment x parity, treatment x season, and treatment x farm interactions. glm procedure was used to determine the difference in progesterone and total cl volume among the treatment groups. progesterone and total cl volume were tested for normality using kolmogorov-smirnov test and observed to be normally distributed. p  0.05 was considered as significant. results the p/ai for gnrh-5, gnrh-7 and control groups were 31.6% (106/335), 33.8% (132/390) and 31.9% (105/329), respectively (table; p > 0.1). the p/ai was not different for parity, season, farms, treatment x parity, treatment x season, or treatment x farm interactions (p > 0.1). the mean (±sd) cl volume differed among the treatment groups (table; p < 0.05) but mean (±sd) progesterone concentration did not differ among the treatment groups (p > 0.1). the progesterone concentration for the control group (n=17) was 5.9 ± 0.3 ng/ ml and mean (±sd) total cl volume was 8.3±0.6 cm3. the progesterone concentration for the gnrh-5 group (n=15) was 6.3 ± 0.4 ng/ ml and total cl volume was 12.6±0.7 cm3. in the gnrh-5 group six cows failed to develop accessory cl. in the gnrh-5 group, the progesterone concentration and total cl volume for the cows that did not develop accessory cl were 4.7 ± 0.3 ng/ ml and 9.2±0.5 cm3, respectively, and for the cows that did develop accessory cl were 7.4 ± 0.3 ng/ ml and 14.8±0.7 cm3. the progesterone concentration for the gnrh-7 group (n=20) was 6.6 ± 0.4 ng/ ml and total cl volume was 12.0±0.8 cm3. in the gnrh-7 group ten animals failed to develop accessory cl. in the gnrh-7 group, the progesterone concentration and total cl volume for the cows that did not develop accessory cl were 6.6 ± 0.5 ηg/ ml and 7.7±0.8 cm3, respectively, and for the cows that did develop accessory cl were 6.5 ± 0.4 ηg/ ml and 16.2±0.9 cm3. the p/ai for the control, gnrh-5 and gnrh-7 groups at 30 days after insemination in the subset of cows was 76.5% (13/17), 80.0 % (12/15) and 65.0% (13/20), respectively. the p/ai in the subset of cows for the control, gnrh-5 and gnrh-7 groups at 60 days after insemination was 70.6% (12/17), 73.3 % (11/15) and 50.0% (10/20), respectively. one cow with accessory cl in gnrh-5 group and three cows with no accessory cl in the gnrh-7 group lost their pregnancies between 30 and 60 days of gestation. of the 52 cows, ovarian cyclicity affected the p/ai [cycling: 54.5% (43/79); non-cycling 15.6% (5/32); p < 0.02]. there was no effect of treatment or treatment x ovarian cyclicity on p/ai (p > 0.1). discussion treatment with gnrh on day 5 or day 7 following insemination neither increased progesterone concentrations nor improved p/ai compared to controls. corpus luteum formation and development is highly responsive to lh, and small luteal cells synthesize more progesterone when stimulated by lh.9 the administration of gnrh or lh during the early luteal phase induces ovulation of the first-wave dominant follicle and formation of a functional accessory cl. schmitt et al observed that most of the increase of progesterone production after hcg injection was due to the formation of accessory cl.10 furthermore, cows treated with hcg on day 5 after estrus had a greater increase in plasma progesterone from day 6 to 13 and greater circulating progesterone on day 13 of the estrous cycle. however, sterry et al observed that gnrh did not affect circulating progesterone concentrations from 5 to 19 days after tai.11 in this study, 40% and 50% of cows failed to develop accessory cl when gnrh was administered on day 5 and day 7, respectively. it should be noted that progesterone determination was from a subset of 52 cows only. interestingly, in the gnrh-5 group, the progesterone concentration was higher in cows that developed accessory cl compared to cows that did not develop accessory cl; however this difference was not observed in the gnrh-7 group. in both the gnrh-5 and the gnrh-7 group the total cl volume was higher in cows that developed accessory cl compared to cows that did not. these observations (increased total cl volume and similar progesterone concentrations in cows with accessory cl compared to cows with no accessory cl) indicate that individual variation in dairy cow metabolism might play a role in modulating circulating progesterone concentrations. higher producing cows generally consume more dry matter, and feed intake can affect plasma progesterone concentrations of dairy cows.12,13 clinical theriogenology • volume 3 number 4 • december 2011457   previous studies have found conflicting results on conception rates in cows receiving hcg.14-16 sianangama and rajamahendran and santos et al observed a remarkable increase in conception rate in cows receiving hcg on day 7 and day 5 after ai, respectively.16,17 however, other authors report no differences in conception rate between control and hcg-treated cows.14,15 in the latter studies, the conception rate of control cows (conception rate > 59%) was markedly similar to those in this study.13,14 it is likely that such highly fertile groups of cows might not benefit from hcg treatment. in addition, a limited number of cows and different protocols and dosage regimens were utilized, which might have affected the ability to detect any difference between treatment groups. sterry et al conducted experiments in which cows were assigned to either control (n = 223), intravaginal progesterone (n = 218), or gnrh 5 days after tai (n = 227) treatments in one experiment and in another cows were assigned to control (n = 160), gnrh 5 days after tai (n = 159), or gnrh 7 days after tai (n = 163).11 they observed that the treatment did not affect p/ai in these experiments; however, when data were combined to compare control animals (n = 383) with those treated with gnrh 5 days after tai (n = 386), p/ai tended to be greater in those treated with gnrh 5 days after tai (49.1%) than in control cows (45.8%). they also have observed that the effect resulted from a gnrh treatment x cyclicity status interaction in which p/ai for non-cycling cows receiving gnrh 5 days after tai was greater than for noncycling control cows (45.5 vs. 31.1%). no treatment x cyclicity interactions observed in the current study. blood progesterone (timing and magnitude) generally reaches greater concentrations earlier in pregnant than in non-pregnant cows suggesting that strategies to increase progesterone concentration earlier following insemination may be beneficial in reducing embryo loss. santos et al observed an increase in conception rate for cows receiving hcg on day 5 after ai.17 ginther et al indicated that deviation of the dominant follicle takes place two to three days after a new cohort of follicles is recruited.15 the dominant follicle acquires lh receptors between two and four days after wave emergence when it reaches 8 to 10 mm in diameter.18,19 therefore, on day 5 of the estrous cycle, dairy cows should have a dominant follicle that is responsive to lh and is capable of ovulating when ovulation is induced by hcg. it is reasonable to expect a similar response when gnrh is administered on day 5 or day 7. however, it is possible that the increased response to hcg administration on day 5 is due to its longer half-life compared to gnrh. in conclusion, gnrh administration on day 5 or day 7 following insemination neither increased progesterone concentrations nor improved p/ai. table. effect of treatment, parity, season and farms on the odds of pregnancy in lactating dairy cows predictor coefficient ± se p odds ratio 95% lower upper constant -0.6265 ± 0.3451 0.069 treatment† 0.1119 ± 0.2542 0.66 1.12 0.68 1.84 parity‡ -0.1481 ± 0.1073 0.168 0.86 0.7 1.06 season§ 0.0612 ± 0.0932 0.511 1.06 0.89 1.28 farm** -0.1894 ± 0.1431 0.462 0.89 0.71 1.05 treatment x parity 0.0176 ± 0.7945 0.824 1.02 0.87 1.19 treatment x season -0.0440 ± 0.0679 0.517 0.96 0.84 1.09 treatment x farm 0.0248 ± 0.0574 0.852 0.98 0.81 1.10 †treatment: gnrh administration on day 5 or day 7 or control; ‡parity categories: 1, 2 or 3; §season: fall, winter, spring or summer; **2 farms references 1. thatcher ww, guzeglu a, mattos r, et al: uterine conception interactions and reproductive failure in cattle. theriogenology 2001;56:1435-1450. 2. mann ge, lamming ge, robinson rs, et al: the regulation of interferon-τ production and uterine hormone receptors during early pregnancy. j reprod fertil 1999;54(suppl):317-328. clinical theriogenology • volume 3 number 4 • december 2011 458   3. mann ge, fray md, lamming ge: effects of time of progesterone supplementation on embryo development and interferon-τ production in the cow. vet j 2006;171:500-503. 4. thatcher ww, staples cr, danet-desnoyers g, et al: embryo health and mortality in sheep and cattle. j anim sci 1994;72:16-30. 5. binelli m, thatcher ww, mattos r, et al: antiluteolytic strategies to improve fertility in cattle. theriogenology 2001;56:1451-1463. 6. schmitt ejp, diaz t, barros cm, et al: differential response of the luteal phase and fertility in cattle following ovulation of the first-wave follicle with human chorionic gonadotropin or an agonist of gonadotropin-releasing hormone. j anim sci 1996;74:1074-1083. 7. pursley jr, mee mo, wiltbank mc: synchronization of ovulation in dariy cows using pgf2alpha and gnrh. theriogenology 1995;44:915-923 8. vasconcelos, jlm, silcox rw, rose gjm, et al: synchronization rate, size of ovulatory follicles, and conception rate after syncronization of ovulation beginning on different days of estrous cycle in lactating dairy cows. theriogenology 1999;52:1067-1078 9. hoyer pb, niswender gd: the regulation of steroidogenesis is different in the two types of ovine luteal cells. can j physiol pharmacol 1985;63:240-248. 10. schmitt ejp, barros cm, fields pa, et al: a cellular and endocrine characterization of the original and induced corpus luteum after administration of a gonadotropin-releasing hormone agonist or human chorionic gonadotropin on day five of the estrous cycle. j anim sci 1996;74:1915-1929. 11. sterry ra, welle ml, frickepm: treatment with gonadotropin-releasing hormone after first timed artificial insemination improves fertility in noncycling lactating dairy cows. j dairy sci 2006;89:4237-4245. 12. vasconcelos jl, bungert mka, tsai sj, et al: acute reduction in serum progesterone concentrations due to feed intake in pregnant lactating dairy cows [abstract]. j dairy sci 1998;81(suppl 1):226. 13. wiltbank m, fricke p, sangsritavong s, et al: mechanisms that prevent and produce double ovulations in dairy cattle. j dairy sci 2000;83:2998-3007. 14. eduvie lo, seguin be: corpus luteum function and pregnancy rate in lactating dairy cows given human chorionic gonadotropin at middiestrus. theriogenology 1982;17:415-422. 15. helmer sd, britt jh: fertility of dairy cattle treated with human chorionic gonadotropin (hcg) to stimulate progesterone secretion. theriogenology 1986;26:683-695. 16. sianangama pc, rajamahendran r: characteristics of corpus luteum formed from the first wave dominant follicle following hcg in cattle. theriogenology 1996;45:977-990. 17. santos jep, thatcher ww, pool l, et al: effect of human chorionic gonadotropin on luteal function and reproductive performance of high-producing lactating holstein dairy cows. j anim sci 2001;79:2881-2894. 18. ginther oj, wiltbank mc, fricke pm, et al: selection of the dominant follicle in cattle. biol reprod 1996;55:11871194. 19. xu z, garverick ha, smith gw, et al: expression of follicle-stimulating hormone and luteinizing hormone receptors messenger ribonucleic acids in bovine follicles during the first follicular wave. biol reprod 1995;53:951-957. clinical theriogenology • volume 3 number 4 • december 2011459 clinical theriogenology • volume 3 number 4 • december 2011 460 omniblank: 2011: reproductive anatomy and physiology of whitetail deer and other select cervids, sheep and goats reproductive anatomy and physiology of whitetail deer and other select cervids, sheep and goats sherrie g. clark college of veterinary medicine, university of illinois, urbana, il introduction understanding the basic anatomical and physiological characteristic of breeding stock of the cervid and small ruminant is essential for a successful breeding program. this knowledge will insure that the males and females are in the correct stage of their reproductive cycle in the proper season for the appropriate timing of an insemination. although this can be somewhat difficult in the cervids as there is such a large number of species and subspecies that vary in their physiologic nature. this article will concentrate on white tailed deer and elk as the major cervid species. keywords: cervid, sheep, goat, anatomy, physiology anatomy of the female cervid and small ruminant the reproductive anatomy of cervids and small ruminants overall are not that different. the major anatomical structures of the female cervid and small ruminant reproductive tract are quite similar.1 the major components include: the ovaries, oviducts, cervix, vagina, and vulva and clitoris.2 the external genitalia consists of the vulva, which has two labia that are marked by dorsal and ventral commissures, and the tissue that is similar to the erectile penis, the clitoris.1 just cranial to the clitoris is the vestibule. it is lined with stratified squamous epithelial cells and mucus producing glands located on the walls and sometimes floor of the vestibule.1,3 these glands are not present in the goat and are most times located near the urethral orifice in the ewe.1 another unique anatomical feature of the ruminant vestibule is the presence of the suburethral diverticulum that opens in the external urethral orifice. this blind sac is 2 cm deep and located behind the urethral orifice and can make catheterization of the urinary bladder difficult.3 the inside of the vagina is also lined with stratified squamous epithelium and measures approximately 10-15 cm in length in small ruminants. the cervix is located at the most cranial portion of the vagina. the small cervix has 5-8 (5-6 in the ewe and 5-8 in the doe; 4-6 in the deer and wapiti) irregular overlapping folds within the lumen and is not easily dilatable.4 the lumen can be quite narrow and tortuous to maneuver during assisted reproductive techniques.1,3 the uterus is bicornuate and composed of a short body and two uterine horns that range from 6.5 to 15 cm in length in the nongravid state in all of the small ruminants and cervids.3,4 the lumen of the uterus is lined with endometrium (mucosa) with folds that form convex caruncles that serve as attachment sites for the cotyledons of the fetal placenta.1,3 the uterine horns are connected to the ovaries by the oviducts. the ovaries are typically small (1-2 cm) and oval in shape. in the nonbreeding season, they can be somewhat circular and smooth on the surface. during the breeding season, the surface of the ovaries are uneven and rough due to the development of numerous follicles and corpora lutea (depending on the species) and sometimes double in their overall size.1,3,4 anatomy of the male the reproductive organs of the male consist of the testes, epididymides, ductus deferens, spermatic cords and tunics, accessory sex glands, penis and prepuce.1,3 the testes are oval to spherical in shape and can vary in size depending on season and hormonal influence. in the ram, the testes weigh between 200-250 g and the 130-160 g in the buck.3 the testes are located in the pendulous scrotum between the hind legs–they appeared to be held closer to the body in the cervid species compared to domestic small ruminants. the scrotum of the small ruminant can be covered by wool or hair and needs to be considered when examining these structures. the body of the epididymides is located laterally and ends caudomedially to the testes in small ruminants and is oriented so that the head of the epididymis is proximal and the tail is distal to the clinical theriogenology • volume 3 number 4 • december 2011517 corresponding pole of the testis.1,3 the tail of the epididymides flow into the ductus deferens and their distal ends enlarge to form the ampullae. the accessory sex glands of the small ruminant and cervid male include paired ampullae, paired vesicular glands, both a body and disseminate part of the prostate, and small bulbourethral glands.1,5 many of these glands will increase the height of the epithelial cells lining the mucosa in response to seasonal influences in preparation for the breeding season. the male cervid and small ruminant has a fibroelastic penis with a urethra that is covered by corpus spongiosum penis throughout its length and is composed of three parts.1 the base (root) of the penis is attached to the ischial arch, the shaft (main portion of the penis), and the glans penis. the shape of the penis is a simple rod shape that does not increase much in circumference during erection, but does increase in length by approximately 40%.5 when the penis is not erect, the ram or buck penis is normally held in a s-shaped bend (sigmoid flexure) by the retractor penis muscles.1 the penis of the wapiti bull does not contain a sigmoid flexure.5 in the ram and buck, the glans penis has a specialized distal end of the penis that contains a urethral process or vermiform appendage.2,3 the only unique feature to the wapiti bull penis is the upward pointing direction of the distal urethra used for directing urine onto the ventral abdomen, neck and throat regions during the rut season.5 physiology of cervids when referring to cervids in north america there tends to be a concentration on the species that are farmed: white tailed deer, wapiti (elk), red and sika deer. there are many species and numerous more subspecies of cervids that exhibit a variety of different physiologic behaviors and patterns for their reproductive cycles – “no one species can be considered to represent a ‘typical’ deer in terms of reproductive function”.6 depending on the classification of cervid, they could exhibit reproductive cycles in a seasonal pattern or are aseasonal and some can bear multiple offspring while others give birth to a single offspring. an example of the “rselected” species is the white tailed deer. these cervids are the small-bodied animals that are relatively territorial, live in the forest, exhibit a short breeding life (< 10 years), and produce multiple offspring. the “k-selected” species tend to be more seasonal in their breeding patterns and produce singletons and have a longer breeding life than the other species.6 examples of these cervids include red deer, wapiti, sika deer, and reindeer. there is more known about the reproductive cycles of the “k-selected” species due to their nature and ability to adapt to human management. seasonality the majority of cervids studied in north america follow similar patterns to other short day breeders like sheep and goats. they will conceive in the months where day length is decreasing and give birth in the months when the days are becoming longer.7 differences in the actual mating seasons can vary up to eight weeks and can be due to variations in the duration of gestation between various species. there are many other species of cervids that live in tropical climates and cycle throughout the year and do not follow the any seasonal pattern as there is less of a seasonal influence of the environment and available food supply for rearing offspring.6 physiology of the female cervid the white tailed deer is seasonally polyestrous and breeds during short day length. the decreasing day length sends a signal to the pineal gland to produce melatonin, which stimulates gonadotropin releasing hormone (gnrh) secretion.2 the gnrh via luteinizing hormone (lh) and follicle-stimulating hormone initiate follicular development and the one or two subsequent “silent” ovulations (ovulations not preceded by overt estrus) that are then followed by regular estrous cycles that will continue for the next four to six months if the female is not impregnated.8,9 the length of the estrous cycle is 25-28 days. the length of the estrous cycles for red deer is 18 days and for wapiti is 21 days.4 the period of rut is influenced by geographical regions and seasonal environment. doe fawns are influenced by their birth date and body weight as to the initial period when they enter puberty and an estrus period. if the nutrition is adequate for the does to reach 70% of their adult body weight in the first clinical theriogenology • volume 3 number 4 • december 2011 518 autumn of their life, then they will come into estrus in year one.4 additionally, they may not come into estrus during the first rut of the breeding season as the more dominant does will be bred during this time period and they are allowed to exhibit estrous behavior and be bred during the second rut of the season.* ovulation occurs approximately 24 hours after the onset of estrus and is very similar to the timing of sheep and other small ruminants (table). in the preovulatory period, the brain of the female needs to be “primed” with progesterone so that it is sensitive to the estrogen produced by the ovaries. as described previously, if this progesterone is not present prior to follicular development and ovulation, a “silent” ovulation will occur and no behavioral signs of estrus will be exhibited.2,9 once the female has formed mature corpora lutea and appropriate progesterone levels, the brain can respond to the estrogen and behavioral estrus can be exhibited. female cervids, particular hinds, do not show many of the same signs of overt estrus as is seen in ewes and does, there is little or no vulvar swelling.9 depending on the proximity to ovulation, the frequency of mounting activity can vary for each female, but hinds do not usually mount other hinds but will mount the stag. generally it is thought that the close proximity of a hind to the stag indicates her receptivity to be bred.9 estrus behavior terminates when the female is bred or when the day length begins to increase again. an anestrous period occurs annually for about four to six months after the spring equinox similar to other short day breeders such as sheep and goats. table. reproductive characteristics of select female cervids and sheep and goats species age at puberty length of estrus length of the estrous cycle gestational length ovine (ewe) 6-9 months 30 h (18-48 h) 17 d (13-19 d) 142-152 days caprine (doe) 6-8 months 36 h (24-72 h) 21 d (18-24 d) 145-150 days white tail deer (doe) 1 ½ years† 12-24 h 25-28 d 200 days wapiti (hind) 1 ½ years† 12-24 h 21 d 242-252 days red deer (hind) 1 ½ years† 12-24 h 18 d 230-236 days †age at puberty is dependent on nutrition and can be delayed until they are 2 ½ years old physiology of the female sheep or goat from a pure physiologic point of view, sheep and goats are quite similar to female cervids in their seasonality of breeding and responsiveness to decreased day length. the lengths of the estrous cycles are different: 17 days in the ewe and 21 days in the doe.1,2 they have a seasonal anestrous period that can vary amongst breed and climate. the majority of breeds will show a similar yearly transition from anestrus to seasonal cyclicity as the brain of the female undergoes “priming” by progesterone.2 there are some (as there are in various species of cervids) that are not influenced by season, and usually live in more tropical climates, that will continue to exhibit estrous cyclicity throughout the year.1 additionally, the first estrous cycles of each breeding season tend to be shorter than those at the middle to end. this is attributable to hormonal influence and stimulation by the decreased day length and melatonin production.1,2                                                              * shipley cf. college of veterinary medicine at the university of illinois at urbana-champaign, urbana, il, personal communication.   clinical theriogenology • volume 3 number 4 • december 2011519 estrus behavior has been observed and studied more in sheep and goats than in cervids, likely due to their proximity to humans. signs of estrus in the sheep include vulvar swelling with a copious thin vaginal mucous secretion and anorexia.1,10 ewes will ovulate 14-26 hours after the lh surge (toward the final third of estrus) and the length of estrus will likely be breed dependent – wood breeds having longer estrus than meat breeds.1 there does not seem to be a similar phenomenon in goats breeds – they are more influenced by nutrition and milk production (especially in dairy breeds) in the length of estrus.1 the length of estrus in the doe varies from 24 to 72 hours, with ovulation occurring 12-36 hours after the onset of standing heat. as with all of the females discussed, ovulation and expression of estrus can be hastened by the presence of a mature male. does may not exhibit homosexual mounting behavior, but they will have vulvar swelling and have a clear mucus discharge that becomes cloudy towards the end of estrus.1,11 they will exhibit unique tail flagging activity that is described as “rapid side-to-side and upand-down” movements of the tail that serves to spread pheromones from the doe’s vulva to any males that are nearby.11 additionally, they will vocalize more than usual and tend to be more restless and increase their urination frequency to help attract the male.1,11 physiology of the male puberty in the male is defined as “the point at which the male develops an interest in sexual activity and produces spermatozoa in sufficient numbers to achieve pregnancy”.1 the exact age at which this occurs is dependent on the age and breed of the male. in seasonal breeders, males that are born in the spring of the year tend to enter into puberty earlier than males born in the autumn. as the majority of cervid males and rams and bucks are seasonal breeders, they will exhibit signs of puberty in the first autumn when their body weight is 70% of their mature weight. additionally, males that are exposed to cycling females before their first puberty will exhibit signs earlier.1 rams are much more influenced by the season than are bucks, but they will tend to express a lower libido, less odor and reduced scrotal circumference in the fall of the year.12 a unique feature to the buck is that he will not be able to copulate with a female until his glans penis is free of attachments between the prepuce and the penis.1,12 in most bucks, these attachments are completely gone by four to five months of age. for wapiti and deer, puberty is not only influenced by season and reaching a critical weight, but also by growth of his first set of antlers. puberty will be halted during the growth of the antlers, but will resume again at about 13-14 months of age.4 puberty and the normal breeding season are all influenced by decreasing day length in these species. as the length of the day decreases, lh levels rise and stimulate testicular development via growth of the leydig cells. the leydig cells in turn increase production of testosterone and influence spermatogenesis.2,8 the males will remain fertile throughout the breeding season and produce adequate levels of spermatozoa until the early spring when the days begin to increase in length as long as there are no other factors that affect the males’ fertility. antler growth is a unique feature of the cervid male and is under the influence of hormonal (testosterone) control. a young growing male has antlers that are covered with a highly vascularized and sensitive skin known as velvet. as the mating season approaches, serum testosterone levels increase and the antlers harden and the velvet dries. stags will rub their antlers against objects such as trees to remove the dried and flaking velvet. once the mating season has concluded, a layer of bone-dissolving cells invades the base of the antlers, causing them to fall off. each year the size and the complexity–depending on the species–increases until the stag reaches full maturity.8 sexual behavior of the male appears to be influenced by decreasing day length and is increased as the females begin exhibiting overt signs of estrus. most of the males will begin a courtship behavior with the female and will display a typical flehman response after sniffing the vulva region and urine of a female in estrus.1 the buck (or ram) will deposit semen in the anterior vagina of the female during a natural breeding event. clinical theriogenology • volume 3 number 4 • december 2011 520 references 1. mobini s, heath am, pugh dg: theriogenology of sheep and goats. in: pugh dg, editor. sheep and goat medicine. philadelphia: saunders; 2002. p. 129-148. 2. senger pl: pathways to pregnancy and parturition, 2nd ed. pullman (wa): current conceptions, inc.; 2003. p. 1-368. 3. constantinescu ia: developmental anatomy of reproductive organs. in: schatten h, constantinescu gm, editors. comparative reproductive biology. ames (ia): blackwell publishing; 2007. p. 33-48. 4. haigh jc, hudson rj: farming wapiti and red deer. st. louis: mosby-year book inc.; 1993. p. 36-66. 5. haigh jc: reproductive anatomy and physiology of male wapiti and red deer. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology, 2nd ed. st. louis: saunders; 2007. p. 932-936. 6. asher gw. reproductive cycles of deer. anim reprod sci 2011;24:170-175. 7. lincoln ga, short rv: seasonal breeding: nature’s contraceptive. recent prog horm res 1980, 36:1-52. 8. flach e: cervidae and tragulidae. in: fowler me, miller re, editors. zoo and wild animal medicine. st. louis: saunders; 2003. p. 634-649. 9. asher gw: reproductive cycles in female cervids. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology, 2nd ed. st. louis: saunders; 2007. p. 921-931. 10. rawlings nc, bartlewski m: clinical reproductive physiology of ewes. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology, 2nd ed. st. louis: saunders; 2007. p. 642-649. 11. smith mc: clinical reproductive physiology and endocrinology of does. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology, 2nd ed. st. louis: saunders; 2007. p. 535-537. 12. goyal ho, memon ma: clinical reproductive anatomy and physiology of the buck. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology, 2nd ed. st. louis: saunders; 2007. p. 511-514. clinical theriogenology • volume 3 number 4 • december 2011521 clinical theriogenology • volume 3 number 4 • december 2011 522 omniblank: 2016: fetal anasarca in an abyssinian kitten   fetal anasarca in an abyssinian kitten abigail rosemary brough, rachael-kate llewellyn, nima nakahara, philip george aveston thomas queensland veterinary specialists, brisbane, queensland, australia abstract case description a four-year old abyssinian queen was presented to a reproductive specialist for dystocia. clinical findings physical examination and history of the queen was consistent with stage ii labor that has not progressed normally. transabdominal ultrasound revealed an abnormal fetus with visible fluid accumulation in the thorax, abdomen and subcutaneous space, with no detectable heartbeat. heart rates of the other two visible fetuses were measured and were found to be low or not detectable. hematology and biochemistry analysis of the queen revealed mild decrease in the mean corpuscular hemoglobin concentration, mild monocytosis and mild elevation of creatinine kinase. treatment and outcome cesarean section was performed soon after admission. three kittens were born. the first kitten had no heartbeat and appeared grossly abnormal; significantly larger than the other two kittens, with marked accumulation of fluid subcutaneously, ascites and pleural effusion. two remaining kittens required intensive neonatal resuscitation but survived and were discharged on the same day with the queen. clinical relevance fetal anasarca or hydrops fetalis has been reported in humans, dogs and ruminants but there have been no reported cases in the domestic cat. keywords: anasarca, queen, cesarean, fetus, hydrops fetalis, feline introduction fetal anasarca, more commonly referred to as hydrops fetalis in human medicine, is defined as the accumulation of abnormal quantities of subcutaneous fluid in the fetus, with or without visceral cavity fluid.1 fetal anasarca has been described in sheep,2-4 cattle,5 buffalo,6 goats,7,8 and humans9 with limited reports in canines.1,10,11 in canines, a predisposition of brachycephalic breeds, including pugs, french and english bulldogs, has been established.1 one or several pups in a litter may be affected, with the remainder born clinically normal. the pathophysiology of anasarca in non-human species is broad and poorly defined. a number of processes, including cardiac malformations, infectious processes, trauma, genetic abnormalities, abnormal lymph node or kidney development, teratogens, immune mediated causes, placental and umbilical disorders have been have been suggested in the etiology. the functional disturbance associated with these processes can create an imbalance between plasma oncotic and hydrostatic pressure and/or lymph flow, resulting in interstitial fluid accumulation.12 in all species in which the abnormality is described the prognosis for the fetus is grave and dystocia is common, which has associated risk for the dam. while anecdotally fetal anasarca is understood to occur in felines, to date there are no case reports in the literature. the aim of this case report is to provide a detailed case description of fetal anasarca in an abyssinian kitten to establish a database and contribute to the understanding of the pathophysiology of the condition in cats. case report history and physical examination a four-year-old abyssinian queen was presented for dystocia. stage ii labor commenced four and one half hours prior to presentation, with production of placental fluid but with no evidence of any kittens. the queen had a body condition score (bcs) of four out of nine. she had a previous clinical theriogenology • volume 8, number 4 • december 2016425   pregnancy two years prior, and this litter had been born clinically normal and had not required veterinary intervention. physical examination of the queen was consistent with stage ii labor with slight tachypnea (36 breaths/min) and tachycardia (160 beats/min) noted, but was otherwise unremarkable. reproductive examination revealed a swollen vulva and a soft and dilated vagina with sanguinous discharge. no kitten was palpable on digital vaginal examination. transabdominal ultrasound revealed three kittens: two visible on the left side of the abdomen, and one on the right. the caudalmost kitten on the left side had no observable heartbeat and fluid accumulation was visible in the thorax, abdomen and subcutaneous space. the cranial kitten on the left had a visible heart beat with a heart rate of 150 beats/min. the kitten on the right had no sonographically visible heartbeat. procedure a cesarean section was performed under general anesthesia promptly after admission. general anesthesia was induced with alfaxalone iv to effect to facilitate intubation, followed by maintenance with isoflurane and 100% oxygen. midline laparotomy was performed to expose the uterus and an incision was made at the base of the left uterine horn. the first kitten removed was large, edematous and malformed in appearance and lacked an audible heartbeat. the two remaining live kittens were significantly bradycardic with severe respiratory depression and required intensive neonatal resuscitation with supplemental oxygen. the uterus was closed using a continuous utrecht suture pattern, followed by a three-layer closure of the abdomen. after fetal removal, the queen was given oxytocin (2 iu iv during surgery and 2 iu sc in recovery), and meloxicam (0.1mg/kg sc). recovery from anesthesia was uneventful. diagnostic testing all three kittens born were female. the two live kittens weighed 84.9 g and 89.9 g, respectively, and both were anatomically normal. necropsy examination of the deceased kitten was performed just after surgery in the hospital. the deceased kitten (figure 1 and 2) weighed 102.9g after the abdominal fluid was inadvertently removed prior to weighing (estimated to be approximately 107 g with fluid). the following gross abnormalities were noted: the skin and subcutis had generalized edema with a “jelly-like” consistency; the peritoneal cavity contained a large volume of serosanguinous fluid; and the liver was bright red in color with marked rounding of the lobes (figure 2). the kidneys and gastrointestinal tract appeared grossly normal. the pleural cavity contained serosanguinous fluid, but the heart and lungs were grossly normal, with no evidence of pericardial effusion. the brain did not have a formed structure, and the brain tissue was poorly formed and spontaneously exteriorized following craniotomy. the placental tissue appeared grossly normal. blood was taken from the queen for analysis. in-house blood glucose was 9.0 mmol/l on handheld glucometer, and blood ketones were 0.0 mmol/l. a feline total annual health profile was sent for analysis to idexx laboratories. notable abnormalities on hematology were a very mildly decreased mean corpuscular hemoglobin concentration (mchc) of 280g/l (reference range 282333g/l) and a mild monocytosis of 1.0x109/l (reference range 0.0-0.6x109/l). the only abnormality on biochemistry was a moderately elevated creatinine kinase (ck) of 725 u/l (reference range 64400 u/l). formalin fixed samples of placenta, heart, and lungs were submitted for gross examination and histopathology. histopathology results showed that the placenta had multifocal necrosis with infiltration by lymphocytes, plasma cells, macrophages, and neutrophils. the pulmonary airways and alveoli contained lymphocytes, macrophages and neutrophils, with an interstitial leukocytic infiltration. the myocardium had interstitial edema with leukocytic, predominantly lymphocytic, infiltration. there was concurrent mediastinal edema and lymphocytic infiltration. these results were consistent with placentitis, with fetal bronchopneumonia, myocarditis, and mediastinitis. these findings indicate an infectious process, possibly a low-grade bacterial infection. clinical theriogenology • volume 8, number 4 • december 2016 426   outcome the remaining clinically normal kittens were assisted to nurse on the queen and were discharged the same day of presentation. at 14 days after surgery the kittens and queen were doing well. discussion hydrops fetalis is a result of deranged fluid homeostasis. in humans, the underlying pathophysiology is broadly divided into two categories: immune mediated and non-immune mediated.12 immune mediated hydrops fetalis is understood to occur due to rhesus gene incompatibility whereby a rhesus-d (rh)-d negative mother delivers a (rh)-d positive fetus and develops autoantibodies against the (rh)-d positive blood type. any subsequent pregnancy with a (rh)-d positive fetus, in the presence of the maternal autoantibodies, results in severe hemolytic anemia in the fetus, as well as hypoproteinemia and multi-cavitary effusion.13 advancements in biomedical science have greatly reduced the incidence of immune mediated fetal anasarca in humans, with non-immune causes of hydrops fetalis accounting for 85-90% of all cases.14-16 non-immune hydrops fetalis in humans can be attributed to a variety of factors including teratogens, cardiovascular abnormalities, congenital fetal abnormalities, placental insufficiencies, infection, and miscellaneous causes.17 in non-immune mediated hydrops fetalis in humans, four mechanisms as per starlings’ forces have been identified. these include an increase in hydrostatic capillary pressure, a reduction of intravascular osmotic pressure, obstruction of lymphatic flow, and damage to peripheral capillary integrity. over 80 conditions associated with these four mechanisms have been identified to cause hydrops fetalis in humans.18 the pathophysiology of fetal anasarca in domestic animals remains largely undetermined when compared to human medicine. proposed causes of anasarca in domestic species include hereditary predisposition due to autosomal recessive genes,19 abnormal lymph node development,20 minute virus in canines,21 congenital renal dysfunction,22 and many others. the mechanisms of fluid accumulation in the subcutis and visceral cavities in fetal anasarca, as described by starling’s principles, is likely comparable to that seen in humans. the present case details placentitis, with fetal bronchopneumonia, myocarditis and mediastinitis consistent with a low-grade bacterial infection. both infectious processes, and placental insufficiency/disease have been postulated as potential underlying causes for fetal anasarca in domestic species. this may provide one possible explanation for the present case, however may also be an incidental finding in a kitten which may have been deceased in utero for >12h. in human reports, a wide variety of infectious agents have been associated with fetal anasarca.12 infections have been shown to preferentially target the fetal bone marrow, the myocardium, and vascular endothelium.12 subsequent congestive heart failure, anemia, fetal sepsis, anoxia, endothelial cell damage, and increased capillary permeability result in the fetal anasarca.23 the most likely route of infection in this case report would have been hematogenous spread or ascending placentitis. this therefore raises the question why not all of the kittens in the litter were affected, when in fact the remaining two kittens were anatomically normal, resuscitated successfully, and discharged the same day with the queen. furthermore, the queen had been clinically healthy throughout pregnancy. an important point to note is that the affected kitten was the caudal-most kitten in its respective uterine horn so this may fit the pattern of an ascending placentitis. a limitation of the present study was that external gross pathology and complete post mortem by a registered veterinary pathologist was not performed. it is plausible the fetus with anasarca may have had underlying congenital defects, which may have been the primary cause of the anasarca, or perhaps increased susceptibility to infection and subsequent fluid derangements. fetal anasarca, as in the present case, can result in dystocia. in canine breeds defined as having an increased risk for fetal anasarca, namely bulldogs, it has been suggested that preparturient diagnosis (transabdominal ultrasound) and planned cesarean section may help reduce risk for both the bitch and the remaining pups.11 cats are, in general, at low risk for dystocia, reported at 5.8%.24 this is one possible explanation for the lack of published literature in feline anasarca. it is likely that if the queen did not have an obstructive dystocia she may have not presented to the veterinary hospital. clinical theriogenology • volume 8, number 4 • december 2016427   the clinopathological findings of the queen in the present case report are most likely unremarkable. there was a moderate elevation in creatinine kinase, which may be explained by prolonged obstructive dystocia, or may be insignificant. the mild clinopathological findings in the queen would not account for the fetal anasarca. conclusions fetal anasarca is well described in human medicine and in several domestic species, notably ruminants and, to a lesser extent, canines. while anecdotally it is thought to occur in felines, there is presently no published literature. to the authors’ knowledge this is the first reported case of anasarca in a kitten, suggesting while an uncommon condition in cats, it can occur and may result in obstructive dystocia in the queen. learning points fetal anasarca is a common cause of dystocia in mammals. theoretically fetal anasarca can occur in any mammalian species, however there are no published reports documenting its occurrence in felines. fetal anasarca in this abyssinian kitten is believed to have occurred due to placentitis. acknowledgements the authors would like to acknowledge dr neill sullivan bvsc, mvsc, phd, facvsc, davcp of idexx laboratories for the histopathology interpretation in this case, and queensland veterinary specialists for access to clinical data and diagnostic testing. references 1. cunto m, zaembelli d, castagnetti c, et al: diagnosis and treatment of foetal anasarca in two english bulldog puppies. pak vet j 2015; 35: 251-252. 2. monteagudo l, luján, l, tejedor t, et al: fetal anasarca (hydrops foetalis) associated with lymphoid tissue agenesis possibly due to an autosomal recessive gene defect in sheep. theriogenology 2002;58:1219-1228 3. hailat n, lafi sq, al-darraji a, et al: foetal anasarca in awassi sheep. aust vet j 1997;75:257-259. 4. selvarju m, vijay d, chandrahasan c: a case of foetal anasarca in a sheep. indian vet j 2004; 81:590-591. 5. winsor pa, cavanagh jl, tammen i: hydrops fetalis associated with pulmonary hypoplasia in dexter calves. aust vet j 2006; 84:278 6. bijurkar rg, tandle mk, honnappagol ss, et al: dystocia due to foetal anasarca with ascites in a buffalo. indian vet j 2004;111:1276. 7. ayyappan s, balasubramanian s, david a: foetal hydroperitoneum with mild anasarca in doe-a case report. indian vet j 1993;70: 273-274. 8. philip lm, mohan mr, bastin pf: fetal anasarca twins with hydroallantois in malabari does. j indian vet assoc 2012;10:52-53. 9. having k, bullock s: fetal anasarca. j diagn med sonogr 2011;27:19-25. 10. gokulakrishnan m, vijayanand v, thirunavukkarasu p, et al: fetal anasarca in a dachshund puppy. indian j vet surg 2008;29:141. 11. latha c, radhakrishna r, mohan m: management of obstructive dystocia due to foetal anasarca in a bitch. intas polivet 2014;15:361-362. 12. bellini c, hennekam rcm, fulcheri e, et al: etiology of nonimmune hydrops fetalis: a systematic review. am j med genet 2009;149:844-851. 13. jojic d, predojevic-samardzic j, petrovic-tepic s, et al: immune hydrops fetalis. scripta medica 2015;46:80-83. 14. huang h,tsay p, chiang m, et al: prognostic factors and clinical features in liveborn neonates with hydrops fetalis. am j perinatol 2007;24:33-38. 15. ismail kmk, martin wl, ghosh s, et al: etiology and outcome of hydrops fetalis. j matern fetal med 2001;10:175-181. 16. prasad c, rupar ca: non-immune hydrops: genetic and metabolic causes. perinatology 2006;8:164-174. 17. norton me, chauhan sp, dashe js: society for maternal-fetal medicine (smfm) clinical guideline #7: nonimmune hydrops fetalis. am j obstet gynecol 2015;212:127-139. 18. hutchison aa, drew jh, yu vy, et al. nonimmunologic hydrops fetalis: a review of 61 cases. obstet gynecol 1982;59:347-352. 19. arthur gh, noakes de, pearson h, et al: veterinary reproduction and obstetrics, 7th edition. philadelphia: w.b.saunders co., ltd:1996. p. 131. 20. tamizharasan s, babu prasath n, balachandran c, et al: fetal anasarca in sheep. indian vet j 2008;85:897-898. 21. carmichael le, schlafer dh, hashimoto a: pathogenicity of minute virus of canines (mvc) for the canine fetus. cornell vet 1991;81:151-171. clinical theriogenology • volume 8, number 4 • december 2016 428   22. vander-linde sipam ts, mullnik jwma: hydrops fetalis in the dog: a record of ten cases. zentralblatt fur veterinar medizin reihe a 1972;19:8-14. 23. barron sd, pass rf. infectious causes of hydrops fetalis. semin perinatol 1995;19:493-501. 24. gunn-moore da, thrusfield mv: feline dystocia: prevalence, and association with cranial conformation and breed. vet rec 1995;136:350-353. figure 1. fetal anasarca in a stillborn female abyssinian kitten. figure 2. postmortem examination of a kitten with fetal anasarca showing fluid in pleural, peritoneal and subcutaneous spaces. (editor’s note: photographs in this paper are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 8, number 4 • december 2016429 clinical theriogenology • volume 8, number 4 • december 2016 430 omniblank: 2017: complications associated with the use of intrauterine glass marbles to suppress estrus in a standardbred mare complications associated with the use of intrauterine glass marbles to suppress estrus in a standardbred mare erin e. runcan, john burnett, gabriela c. de sousa, marco a. coutinho da silva department of veterinary clinical sciences, college of veterinary medicine, the ohio state university, columbus, oh abstract an 11-year-old maiden standardbred mare was presented to the ohio state university veterinary medical center (osu-vmc) theriogenology service for treatment of endometritis and breeding management. it was suspected that pieces of a marble, placed in the uterus to suppress estrous behavior, remained in the mare’s reproductive tract following removal. the clear glass marble was reported to appear “flakey” upon removal and had been present for an unknown period of time. reproductive ultrasonography showed luteal tissue, endometrial edema, hyperechoic free intrauterine fluid and small echogenic fragments in the endometrium. hysteroscopy showed free glass shards in the uterus. treatment of the endometritis included: acetylcysteine, uterine lavages, ceftiofur, and ecbolics. intensive breeding management over two estrous cycles and two embryo recovery attempts were unsuccessful. this report describes potential complications with the use of an intrauterine glass marble and provides additional evidence that serious complications such as embedded glass shards in the endometrium, persistent endometritis and infertility may occur. glass marbles are not recommended for estrus suppression in mares. keywords: mare, estrus suppression, marble, endometritis background estrus suppression in mares, particularly performance mares, is desirable for owners who report that their mare is difficult to manage when signs of behavioral estrus are present. some owners report their mare may experience ovarian or back pain during estrus, which is perceived to affect the mare’s performance. some signs of behavioral estrus include leaning toward the stallion, lifting of the tail, squatting stance, clitoral eversion, and urinating.1 however many times the mare’s performance issues may not be related to estrus in the mare, because the these signs fail to occur about every fourteen days and do not last for six days. in addition, in mares that are not under lights, the signs persist when the mare is in anestrus in the winter. thorough examination of the mare by the veterinarian, and the chief complaint is important to determine whether the mare’s behavior is reproductive in origin, or if these perceived signs are related to other causes.1 there are numerous documented methods for suppressing estrus including: administration of exogenous progesterone or progestins, induction of a prolonged luteal phase, suppression of the follicular activity of the ovaries, and ovariectomy.1,2 of these four possible methods to suppress estrus, administration of exogenous progesterone/progestins appears to be used most frequently.2 oral administration of altrenogest, a synthetic progestin, is considered by most veterinarians to be the “gold standard” for preventing behavioral estrus in mares, but the expense of the medication and the frequency at which it must be administered are just two of its potential drawbacks for owners. due to the drawbacks of progesterone/progestin administration, some owners may elect to suppress estrus by prolonging the luteal phase, which results in continued secretion of progesterone, which inhibits estrus behavior. there are numerous methods for prolonging the luteal phase such as the use of exogenous oxytocin, or human chorionic gonadotropin (hcg).1 one method involves the use of an intrauterine device (iud) in the form of a glass ball or marble, placed into the uterus immediately after ovulation.2-5 common sizes reported in recent literature include 25mm and 35 mm diameter glass marbles3 while another study used a 20mm polypropylene ball filled with water.4 in the 2003 study by nie et al, 25mm and 35mm glass marbles were placed in the uterus immediately following ovulation. in that study, 50%, or 6/12 mares spontaneously expelled the 25 mm marble. none of the 35 mm marbles were expelled. their results indicated that in the mares which did not expel their glass marbles (18/24), luteal secretion of progesterone persisted for 35 days for 39% (7/18) of mares. in the control mares prolonged luteal function was noted in 13% (4/32) of mares. they also found that of those seven mares showing persistent luteal function, four showed persistent luteal function following the first normal estrous cycle after marble placement. the remaining three mares clinical theriogenology • volume 9 number 1 • march 201781 showed persistent luteal function following multiple normal estrous cycles.3 these results appear to indicate that use of the iud glass marble is somewhat efficacious for causing persistent luteal function and estrus suppression. however, the application of intrauterine marbles produced varying results in other studies. in a study conducted by rivera del alamo et al, 75% of mares showed prolonged luteal phases. the iud was smaller (20mm), made of polypropylene, and filled with water.4 argo and turnbull investigated iud glass marbles in pony mares. mares in both the study group and control group showed normal estrus behavior throughout the duration of the study. however, even though the study failed to show any measurable change in estrus behavior, owner perception of improved behavior was greater than 60%.5 recently, there have been multiple reports of complications related to the use of intrauterine glass marbles. in 2015, turner et al reported fragmentation of a glass marble in the uterus of a mare after placement of two glass marbles in her uterus. following uterine lavage and treatment, the pieces of the marble were removed from the uterus but the mare failed to become pregnant after multiple breeding attempts.2 diel de amorim et al reported pyometra, infertility, and embedded glass in the endometrium of mares who had intra-uterine glass marbles(s) for longer than one year.6 another report by freeman and lyle described a case of intermittent signs of colic in a mare, associated with the presence of two round glass marbles in the uterus. after cervical dilation, the two marbles were removed from the uterus. follow-up with the owner revealed that the mare had not had any signs of colic during the 12 months following marble removal.7 here we report the complications related to the use of a single glass marble in the uterus of a mare. these complications include: marble fragmentation and embedded glass fragments within the endometrium, endometritis, and infertility. case presentation an 11-year-old maiden standardbred mare was referred to osu-vmc) for evaluation of a potential uterine foreign body from a previously placed uterine marble used to suppress estrus. the mare’s primary care veterinarian removed the marble one month prior to presentation and the marble was reported as appearing “flakey” by the owner. for an undetermined period of time, the mare was observed urinating frequently, straining to urinate, and producing either small quantities or nothing at each urination. at presentation, her physical examination parameters were within normal limits and she appeared adequately hydrated. transrectal ultrasonographic examination of the reproductive tract revealed medium sized follicles ranging in size from 20-27mm and a corpus luteum on both ovaries. a moderate amount of uterine edema (grade 2 of 3) was noted despite the mare being in diestrus, and the cervix was closed at the time of examination. a moderate amount (ranging from 1-5 cm in depth) of mildly hyperechoic (grade 3) uterine fluid was visualized and multiple multifocal hyperechoic areas were visualized in the uterine lumen on ultrasonography. digital palpation of the cervix revealed a 2cm x 1cm linear diverticulum within the body of the cervical lumen. both the inner and outer cervical os were tightly closed. the mare was sedated with 8 mg detomidine hydrochloride (dormosedan®; zoetis inc. kalamazoo, mi ) and hysteroscopy was performed in an effort to remove the hyperechoic structures believed to be remnants of the uterine marble. following sedation, purulent material and mucous were identified on endoscopic examination, but the glass fragments could not be detected due to a large amount of fluid. following the initial examination, 30 milliliters of 20% acetylcysteine (20% acetylcysteine solution, usp, hospira, inc., lake forest, il) was infused into the uterus to help to break up the mucopurulent material that was present. approximately three hours later, a uterine lavage was performed using three liters of lactated ringer’s solution (lrs). a sample collected from the uterine lavage was submitted for antimicrobial culture and sensitivity testing. results showed a heavy pure growth of staphylococcus warneri. sensitive to cephalosporins. cytology of the efflux revealed a high number of degenerative neutrophils with intracellular bacteria. the mare was treated with 6.6 mg/kg ceftiofur crystalline free acid (excede®; zoetis, florham park, nj) given intramuscularly. no episodes of straining to urinate were noted during her hospitalization and urine produced was normal in color and quantity. it was determined that the previous urinary issues noted by the owner were related to estrus behavior. clinical theriogenology • volume 9 number 1 • march 2017 82 the following day, a second uterine lavage was performed also using three liters of lrs. at this lavage, numerous glass fragments were retrieved along with the efflux (figure 1). following the lavage, 20 international units of oxytocin were administered and repeated every six hours to promote uterine clearance. on the forth day of hospitalization, an additional uterine lavage with lrs was performed in an effort to remove any remaining glass fragments. no additional fragments were recovered but on ultrasonographic examination, multiple hyperechoic areas were still visualized within the uterine lumen (figure 2). a second hysteroscopic examination was elected to attempt to directly visualize and remove the remaining glass pieces, but despite the lack of intrauterine fluid, they could not be seen. since they could not be visualized, it was assumed the hyperechoic areas seen within the endometrium on ultrasound examination were pieces of glass from the marble that were embedded in the uterine endometrium. a decision was made to attempt to breed the mare following the removal of the uterine debris and treatment of endometritis. on the fifth day of hospitalization, two preovulatory follicles (37 and 36mm) were visualized on the left ovary, and the mare was administered 1.8 mg of deslorelin acetate (sucromate™; thorn bioscience, llc, louisville, ky) iv to induce ovulation. approximately six hours later, uterine lavage was again performed and the mare was artificially inseminated using cooled semen. on the sixth day of hospitalization, ultrasound revealed multiple large follicles on the right ovary. uterine lavage was again performed to remove inflammatory debris, and she was administered 250 micrograms of cloprostenol sodium (estrumate®; schering plough animal health, union, nj) to promote uterine clearance. the following day, ovulation of two follicles was confirmed via ultrasound. repeated transrectal ultrasonographic examinations were performed throughout the mare’s hospitalization to monitor ovarian structures, uterine edema, uterine fluid, cervical tone, and to evaluate the multiple multifocal hyperechoic areas in her uterus. uterine lavage was performed as needed according to uterine fluid accumulation. intramuscular injections of 6.6 mg/kg ceftiofur crystalline free acid were performed every 96 hours during hospitalization. additional intramuscular injections of oxytocin were administered every 4-6 hours following uterine lavage. prednisolone acetate (prednistab®, phoenix, clipper distributing company, llc, st. joseph, mo), 0.1 mg/kg by mouth every 12 hours, was administered on days 5-9 of hospitalization in an effort to decrease postbreeding uterine inflammation. intrauterine fluid was noted for three days following ovulation, gradually decreasing in volume through response to lavage and ecbolic therapy. on the tenth day of hospitalization, the mare was discharged to the owner with a recommendation to have an ultrasound performed 14 days following insemination to evaluate the mare for pregnancy and the presence of twins. there was no free intrauterine fluid noted at this time. the owner was advised that the mare’s prognosis for fertility was guarded at the time of discharge, due to the prolonged fluid accumulation after breeding. thirteen days following discharge, the mare presented again to the osu-vmc for suspected endometritis and uterine fluid accumulation noted at the time of her pregnancy evaluation. she was found to not be pregnant by her primary care veterinarian with a shortened inter-estrus interval. a reproductive examination showed that the mare had a 31mm follicle on the right ovary and one 42mm dominant follicle and a 27mm follicle on the left ovary. a marked amount (4-5cm in depth) of moderately hyperechoic (grade 3) uterine fluid was also seen on ultrasound. uterine lavage with three liters of lrs was performed and the fluid collected appeared cloudy. a sample of the uterine fluid was submitted for culture and sensitivity testing and results showed no bacterial growth at that time. numerous neutrophils were seen on cytology. following uterine lavage, the mare was administered 2500 iu hcg iv to induce ovulation. she was also administered 6.6 mg/kg ceftiofur crystalline free acid for presumptive endometritis, ecbolics, and oral phenylbutazone (2.2 mg/kg) for mild trauma sustained during transport. phenylbutazone was continued for four days for pain management. two days following presentation, an anovulatory follicle was noted on ultrasound and it was decided that she should not be inseminated at that time. hospitalization was continued in an effort to breed on the next estrous cycle with the plan to enroll the mare in an embryo transfer protocol to a recipient mare. on day thirteen of hospitalization, a large, 40 mm follicle was visualized by ultrasound. a marked amount of uterine fluid (4-5cm in clinical theriogenology • volume 9 number 1 • march 201783 depth) was also seen on ultrasound. uterine lavage was performed using four liters of lrs, followed by artificial insemination using cooled semen. she was also administered oxytocin and oral prednisolone acetate. lavage, ecbolic therapy, and prednisolone were continued as needed to remove uterine fluid and debris. ovulation was confirmed with visualization of a corpus luteum using ultrasound 48 hours later. seven days following artificial insemination, the mare began oral altrenogest (regu-mate® 0.22%; intervet, inc. millsboro, de) at a dosage of 0.044 mg/kg. on day seven following ovulation, an embryo recovery procedure was performed but no embryo was recovered. the mare was administered 250ug cloprostenol intramuscularly in order to advance the onset of next estrus for a final attempt at embryo transfer immediately after uterine lavage. the mare was monitored via ultrasound until a preovulatory follicle was noted and administered 1.8 mg deslorelin acetate iv to induce ovulation. she was inseminated again with cooled semen from the same stallion. after breeding uterine lavage was performed using six liters of lrs. oxytocin and prednisolone were administered at previously recorded doses for uterine clearance and mitigation of inflammation. prednisolone was administered twice daily for six days following insemination and oral altrenogest was administered daily at the labeled dosage. she was flushed seven days after ovulation, and no embryo was recovered. the mare was then discharged. outcome during hospitalization at osu-vmc, the mare was bred three times on three consecutive cycles via artificial insemination. unfortunately, pregnancy was never achieved despite intensive breeding management. prognosis for fertility was guarded due to the presence of a cervical diverticulum, persistent uterine fluid accumulation, post-breeding endometritis and embedded shards of glass in the mare’s endoemtrium. after the mare’s second visit and failure of embryo recovery on two consecutive cycles, it was recommended that the owner consider oocyte transfer as a final option to achieve a foal. discussion intra-uterine glass marbles are not approved medical devices. as seen in this case, one of the disadvantages of intrauterine glass marbles is fragmentation. in 2013, vanderwall described reports of spontaneous fragmentation of a glass ball in the uterus.8 dascanio also indicated that a low quality glass ball could fracture, leaving glass shards present in the uterine lumen and making recovery of the remaining pieces of glass difficult.9 one recent report evaluating five mares showed 5/5 (100%) of mares had their glass marbles present for longer than one year and of those five mares, two (40%) had chronic endometritis, three (60%) had pyometra, and three (60%) had fragmented marbles with glass shards embedded in the endometrium.6 in another recent study, fragmentation of two intrauterine glass marbles was reported in a mare that presented to the authors for removal of both glass marbles.2 one marble was found to have “pitting” on the marble surface, while the second marble was found to be shattered. following repeated uterine lavage, multiple glass shards were recovered, but collection of every piece of glass could not be definitively determined. the authors indicated that additional pieces could have been embedded in the uterine wall. it was presumed that repeated contact between the marbles caused the fragmentation.2 in the case of the mare in the current report; only one intrauterine glass marble was placed in the uterus and subsequently removed by the primary care veterinarian. the owner’s report that the marble appeared “flakey” is consistent with the “pitting” reported by tuner and associates.2 following repeated uterine lavage, numerous glass shards were recovered, but multiple hyperechoic areas continued to be visualized on ultrasonography, indicating foreign bodies such as glass shards were embedded in the uterine endometrium. these glass shards if embedded in the endometrium, could act as a nidus for inflammation and decrease fertility. glass is a porous substance and body fluids and inflammatory debris likely act to break down the surface of the glass. this would lead to fragmentation of the surface and the release of glass shards into the lumen that may then become embedded in the endometrium over time. after culture and cytology of the purulent discharge, a bacterial infection was identified as staphylococcus warneri. this species of bacteria is often considered a contaminant, however with the heavy pure growth and positive cytology seen, it is believed that this was in fact a true infection. it is possible the bacterium could have been introduced during manual marble removal. while reports clinical theriogenology • volume 9 number 1 • march 2017 84 describing uterine infection following placement or long term retention of an intrauterine glass marble are limited, reports have been made describing the possibility of intrauterine glass marbles causing pyometra.10 in one case report, a mare was found to have pyometra following presentation for foul smelling uterine discharge. a glass marble was found in the uterine lumen that the owners indicated had been present for at least two years. the authors suspected the marble acted as a nidus for infection following diestrus. the authors did not evaluate long-term fertility in that case.10 additionally, nie et al describe infusing ticarcillin sodium into the uterus following placement of an intrauterine glass marble to prevent bacterial infection and endometritis.3 in that study, some uterine fluid accumulation was seen following glass marble placement and removal, but none of the mares in the study were shown to develop pyometra or endometritis. these data indicate placement of an iud glass marble poses some risks as this procedure has the potential for introducing bacteria into the uterus. cervical incompetence is one predisposing factor to persistent fluid accumulation as well as reduced myometrial contractions, poor lymphatic drainage, and degenerative uterine changes.11 during placement or removal of an intrauterine glass marble, the cervix may be damaged if it is not relaxed, such as during diestrus, or if proper medications have not been administered to induce cervical relaxation. lacerations, or other injury to the cervix, are possible and can lead to cervical adhesions when healing. when the cervix is adhered to another tissue layer, it becomes more difficult, or impossible, to relax appropriately.12 during the initial examination, it was noted that the mare in our report had a cervical diverticulum upon palpation. the cause of the diverticulum could not be established, but the presence of the diverticulum could lead to impairment in cervical function. transrectal ultrasonography of the uterus is an extremely valuable tool for estimating quality and quantity of fluid in the uterine lumen.13 the mare in this case report was monitored daily as needed for evaluation and treatment. in this case, the mare usually presented with trace amounts of hypoechoic uterine fluid before breeding and moderate to large amounts of slightly echoic fluid after breeding. post-breeding uterine fluid accumulation is a physiologic inflammatory response that may help to clear the uterus of foreign material such as excess spermatozoa, seminal plasma and bacteria.11,14 however, persistent post-breeding uterine accumulation has been associated with a decrease in fertility after natural mating or artificial insemination of fresh semen.15,16 chronic uterine fluid accumulation after breeding is typically managed with uterine lavage and ecbolic therapy to remove inflammatory products. ecbolic therapy such as oxytocin increases myometrial contractions and aids expulsion of material.17,18 the aim of therapies in the immediate period after breeding is to reduce infection and inflammation to create a uterine environment capable of supporting pregnancy and prevent further contamination. spermatozoa are safely in the oviduct within four hours of breeding and are protected from inflammatory products in the uterus and/or uterine treatments by the utero-tubal junction.13 in this case, uterine lavage was instituted prior to insemination and again, along with ecbolic therapy, following insemination. this was in an effort to clear the uterus of fluid and any foreign material present, allow the semen to migrate to the oviduct, and aid in decreasing inflammation of the endometrium in preparation for pregnancy. however, in this case, the mare did not become pregnant despite the appropriate measures taken. embryo transfer now lends itself to obtaining foals from performance and show mares, multiple foals from the same mare in one year, foals from mare with non-reproductive health or musculoskeletal problems, and foals from mares with reproductive problems.19 embryo transfer may be a good alternative for the mare in this case if she is found to be unable to carry a foal to term. glass shards embedded in the endometrium may function as a nidus for inflammation and/or infection and affect the mobility phase of early gestation or implantation.2 however, if the oocyte is able to be fertilized in the oviduct, embryo transfer into a recipient mare with a normal, healthy uterus may be an attractive alternative in this case. carnevale and coworkers analyzed the effects of repeated inseminations and embryo transfer attempts on uterine health and found an association with increased chronic inflammatory changes. these authors concluded that mares were susceptible to the additive effects of repeated uterine insults. in clinical practice, there is substantial evidence that repeated breeding and embryo transfer attempts yield an increased likelihood of acute bacterial endometritis, in addition to chronic inflammation.20 there is a concern that glass shards embedded in the endometrium and repeated attempts at embryo transfer may lead to additive effects and decrease fertility in this mare. however, in this case, chronic clinical theriogenology • volume 9 number 1 • march 201785 endometritis has already been documented. assisted reproductive procedures may be necessary to allow this mare to reproduce. oocyte transfer can be used to bypass many of the fertility problems that render an older mare unsuitable as an embryo transfer donor. these include failure of ovulation, chronic endometritis, pyometra, cervical scarring or severe lacerations and unexplained infertility.21 in this case, repeated breeding and embryo transfer attempts were unsuccessful, but the mare continued cycling normally. oocyte transfer into a recipient mare with a normal, healthy uterus may allow this mare to reproduce since embryo transfer was unsuccessful. in conclusion, this case supports that severe complications such as chronic endometritis and embedded shards of glass leading to infertility, may occur when an intrauterine glass marble is inserted into a mare’s uterus to suppress estrus. intra-uterine placement of glass marbles is not recommended for estrus suppression in mares. learning points intra-uterine marbles are not medical devices and are not currently recommended for estrus suppression in mares. complications of intra-uterine marble use include loss, fragmentation, chronic inflammation, endometritis, embedded shards of glass in the endometrium and infertility. references 1. vanderwall dk, nie gj: estrous suppression. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. ames (ia): wiley-blackwell; 2011. p. 1845-1853. 2. turner rm, vanderwall dk, stawicki r: complications associated with the presence of two intrauterine glass balls used for oestrus suppression in a mare. equine vet educ 2015;27:340-343. 3. nie gj, johnson ke, braden td, et al: use of an intra-uterine glass ball protocol to extend luteal function in mares. j equine vet sci 2003;23(6):266-273. 4. rivera del alamo mm, reilas t, kindahl h, et al: mechanisms behind intrauterine device-induced luteal persistence in mares. anim reprod sci 2008;107:94-106. 5. argo cm, turnbull eb: the effect of intra-uterine devices on the reproductive physiology and behavior of pony mares. vet j. 2010;186:39-46. 6. diel de amorim m., chenier t., nairn, et al: complications related to the use of intrauterine glass marbles in mares. proc annu meet am assoc of equine pract 2014;60:317. 7. freeman ce, lyle sk: chronic intermittent colic in a mare attributed to uterine marbles. equine vet educ 2015;27:469-473. 8. vanderwall dk: prolonging function of the corpus luteum to suppress estrus in mares. proc annu meet am assoc equine pract 2013;59:342-349. 9. dascanio jj: use of an intrauterine ball in estrus suppression. in: dascanio j, mccue p, editors. equine reproductive procedures. ames (ia): wiley blackwell; 2014. p. 158-160. 10. klabnik-bradford j, ferrer ms, blevins c, et al: marble-induced pyometra in an appaloosa mare. clin therio 2013;5:410. 11. troedsson mht: therapeutic considerations for mating induced endometritis. pferdeheilkunde 1997;13:516–20. 12. sertich pl: cervix adhesions. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. ames (ia): wiley-blackwell; 2011. p. 2721-2723. 13. mckinnon ao, mccue pm: uterine abnormalities. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction., ames (ia): wiley-blackwell; 2011. p. 2137, 2145-2146. 14. katila t: interaction of the uterus and semen. pferdeheilkunde 1997;13:508-511. 15. adams gp, kastelic jp, bergfetlt dr, et al: effect of uterine inflammation and ultrasonically detected fluid. j reprod fertil 1987;35:445-454 16. pycock jf, newcombe jr: assessment of the effect of three treatments to remove intrauterine fluid on pregnancy rate in the mare. vet rec 1996;138:320-323. 17. jones dm, fielden ed, carr dh: some physiological and pharmacological factors affecting uterine motility as measured by electromyography in the mare. j reprod fertil suppl 1991;44:357-368. 18. liu ikm, troedsson mht, williams dc, et al: electromyography of uterine activity in the mare: comparison of single vs multiple recording sites. j reprod fertil suppl 1991;44:744 19. hartman dl: embryo transfer. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. ames (ia): wiley-blackwell; 2011. p. 2871-2874. 20. carnevale em, beisner ae, mccue pm, et al: uterine changes associated with repeated inseminations and embryo collections in mares. proc annu meet am assoc equine pract 2005. 21. madill s: management of the geriatric mare. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. ames (ia): wiley-blackwell; 2011. p. 2815. clinical theriogenology • volume 9 number 1 • march 2017 86 figure 1. multiple glass fragments retrieved from the uterus of an 11 year old standardbred mare via uterine lavage following marble removal; no. 22 scalpel blade provided for size. figure 2. ultrasonographic image of the uterus of an 11 year old standardbred mare showing hyperechoic areas consistent with embedded glass fragments in the endometrium. clinical theriogenology • volume 9 number 1 • march 201787 clinical theriogenology • volume 9 number 1 • march 2017 88 omniblank: 2018: editorial editorial the june 2018 issue of clinical theriogenology is the last issue i will have the privilege of editing. when dr. patrick hearn called me now over a decade ago and told me that the society for theriogenology executive board was considering establishing a new journal for the purpose of publishing clinically relevant papers and the proceedings of the annual meeting and asked if i would consider being the editor i had no concept that the adventure would last this long. a five-year commitment seemed reasonable at the time but we are now publishing volume 10. this endeavor was a perfect transition from being an active faculty member and clinician to a stint as a college administrator and finally a professor emeritus and has allowed me to continue contact with the members of the society for theriogenology and the american college of theriogenologists and provide extraordinary continuing education. i would like to express my sincere appreciation to the executive boards and management of the society and the college for allowing me to participate in this endeavor and to the authors who submitted manuscripts for publication. i would also like to give special recognition to all of the colleagues on whose time and patience i have imposed by requesting that they serve as manuscript reviewers. this support is often invisible and insufficiently recognized but is essential to insure the quality of the information presented. dr. augustine peter has been appointed as the new editor of clinical theriogenology and i ask that all of the members support him as he brings new ideas and energy to clinical theriogenology and takes the journal to the next level of excellence. i could not have asked for a more rewarding career than to have been a veterinarian and a theriogenologist. but now it is time to step away and become a full-time grandfather and toy maker. so in the words of one of my favorite non-theriogenologist authors, douglas adams, “so long and thanks for all the fish.” best regards to all. bob youngquist clinical theriogenology • volume 10, number 2 • june 201889 clinical theriogenology • volume 10, number 2 • june 2018 90 omniblank: induction of parturition in a goat with pregnancy toxemia using aglepristone induction of parturition in a goat with pregnancy toxemia using aglepristone vanessa peixoto de souza, juan guerra, william whitler, erica mckenzie, charles estill oregon state university, corvallis, or pregnancy toxemia is a metabolic disease in sheep and goats caused by negative energy balance and impaired gluconeogenesis resulting in hypoglycemia, lipid mobilization, ketonemia and ketonuria. it occurs due to increased metabolic demand of late pregnancy and is more prevalent when multiple fetuses are present. pregnancy termination by cesarean section or induction of parturition is often required as part of the treatment. aglepristone is a progesterone antagonist which acts by competitively binding to progesterone receptors. we hypothesized that aglepristone can safely induce parturition in goats with pregnancy toxemia with reduced likelihood of adverse reactions. we used aglepristone to induce parturition in a doe pregnant with triplets that was presented to the oregon state university, veterinary teaching hospital with pregnancy toxemia. a 7 year old boer goat was presented with 5 days history of lethargy, prolonged recumbency, and partial anorexia. the doe was 145 days pregnant and presumed to be carrying triplets based on prior ultrasonography. doe had a low body condition score (2/5), tachypnea, left pelvic limb grade 3 lameness, rumen hypomotility, and mild diarrhea. serum chemistry analysis revealed hyperglycemia (95 mg/dl), hypocalcemia (7.8 meq/l), hypomagnesemia (2.0 mg/dl) and elevated creatine kinase (682 u/l), aspartate transaminase (180 u/l), gamma-glutamyl transferase (33 u/l), and beta-hydroxybutyrate (58.38 mg/dl or 5.6 mmol/l) with low bicarbonate (tco2 19.3 meq/l). results were consistent with pregnancy toxemia, elevated muscle enzymes due to prolonged recumbency and metabolic acidosis. presence of 3 viable fetus was established via transabdominal ultrasonography. doe was given iv fluids (lactated ringer’s solution and calcium gluconate), amino acids, vitamin b complex, and dextrose in addition to oral propylene glycol and iv flunixin meglumine. given the value of kids and the importance of their survival, parturition was induced. a sc injection of aglepristone (2.5 mg/kg) was given on day 145 of pregnancy, with signs of parturition (abdominal contractions) 40 hours later. a digital vaginal examination identified adequate cervical dilation and all 3 kids were delivered alive and active with obstetrical assistance. within 10 hours after stage 2 of labor, all 3 sets of fetal membranes were expelled. systemic signs of pregnancy toxemia started decreasing although she still had mild inappetence. interval from injection to parturition was similar to that in marjorera goats (36 42 hours) induced with a similar dose of aglepristone.1 in conclusion, aglepristone was used with satisfactory efficacy to induce parturition in a goat, similar to that observed in goats induced with natural prostaglandins and analogues commonly used in veterinary practice. onset of parturition occurred during a predictable interval after a single dose of aglepristone, without any additional treatment before the start of the expulsion phase. keywords: caprine, parturition, inducing, aglepristone, ru 46534 reference: 1. batista m, reyes r, santana m, et al: induction of parturition with aglepristone in the majorera goat. reprod dom anim 2011;46:882-888. 483 clinical theriogenology • volume 11, number 3 • september 2019 484clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2015: preputial stoma: a novel surgical approach for semen collection in bulls with phimosis due to preputial stenosis; three cases (2007-2014) preputial stoma: a novel surgical approach for semen collection in bulls with phimosis due to preputial stenosis; three cases (2007–2014) chance l. armstrong, dwight f. wolfe, jennifer h. koziol, robert l. carson college of veterinary medicine, auburn university, auburn, al summary three bulls were presented to the auburn university large animal teaching hospital food animal section for inability to extend the penis following prior preputial injury. each bull had stenosis of the prepuce from excessive scar tissue from previous preputial laceration. in each bull the extent and location of the stenosis prevented circumcision or preputial scar revision to return the bull to breeding soundness. the goal of the owners was either to allow the bull to live comfortably or to be able to collect semen for cryopreservation. this report describes a surgical technique to satisfy those goals. keywords: bovine, preputial stoma surgery, prepuce, semen collection, preputial laceration background preputial lacerations most often occur during coitus in breeding bulls and these injuries may result in significant financial loss to both beef and dairy producers.1 these injuries usually require prolonged sexual rest and medical and or surgical treatment options are often expensive and prolonged in nature or may result in culling an animal that may have significant genetic and monetary value. severe preputial injuries or frostbite are more common in bos indicus breeds and crosses of those breeds but may also occur in bulls of bos taurus breeds.1 the preputial anatomy of bos indicus bulls increases their risk of trauma to the area compared to bulls of bos taurus breeds. this is due to the fact that bulls of b. indicus breeds have a more pendulous sheath with a larger preputial orifice, a longer prepuce relative to the length of the free portion of the penis, and a lower preputial angle in relation to the ventrum of the bull.1 naturally polled bulls are also at increased risk for developing preputial disease because of lack of retractor preputial muscles that prevent chronic eversion of the prepuce.2 bulls with preputial injuries often require a combination of medical and surgical management to correct pathology of the prepuce. preputial injuries most often occur in the mid-preputial area or near the attachment of the prepuce to the free portion of the penis.1 the most common injuries are preputial laceration with cellulitis or perhaps secondary to retropreputial abscess or frostbite of the prepuce. the prognosis for return to breeding soundness is dependent on many factors including the degree in which the epithelial covering of the prepuce is damaged, the extent of damage to the peripenile elastic tissue, the amount of normal prepuce remaining, and location of the injury within the prepuce; all determine the prognosis for successful return to breeding soundness.1 bulls with a preputial injury or frostbite often develop extensive scar tissue such that there is insufficient length of healthy prepuce for scar revision or preputial reconstruction or circumcision.1 the creation of a stoma of the preputial cavity through the sheath may allow sufficient penile protrusion for semen collection for cryopreservation or fresh transfer. additionally, in the authors’ experience two bulls successfully achieved coitus following this surgical procedure. surgical creation of a preputial stoma can be accomplished with the bull in lateral recumbency either under general anesthesia or with light sedation in combination with an internal pudendal nerve block. an internal pudendal nerve block can be performed by first clipping and surgically preparing the skin at the ischiorectal fossa on both sides. a 14 gauge, 1.25 cm needle is inserted through the desensitized skin at the ischiorectal fossa to serve as a cannula. an 18 gauge, 10-15 cm spinal needle is then directed through the cannula to the pudendal nerve. the operator’s left hand is placed in the rectum to the level of the wrist and the fingers are directed laterally and ventrally to identify the right lesser sacrosciatic foramen. the lesser sciatic foramen is first identified and the internal pudendal nerve can be readily identified lying on the ligament immediately cranial and dorsal to the foramen and approximately one finger’s width dorsal to the pudendal artery. this structure can be readily palpated a finger’s width clinical theriogenology • volume 7, number 4 • december 2015419 ventral to the nerve. once the pudendal nerve is located, 20 ml of local anesthetic is deposited around the nerve. the procedure is repeated for the left internal pudendal nerve using the right hand. clip and prepare the sheath for aseptic surgery and make a longitudinal elliptical incision through the skin on the ventral aspect of the sheath. this incision should be just proximal to the stenotic scar tissue in the preputial cavity. the diameter of the stoma should be sufficiently large to allow the free portion of the penis to extend but not large enough that the remaining prepuce could prolapse through the opening. discard the excised elliptical skin and continue dissection through the peripenile elastic layers to the internal lamina of the prepuce (figure 1). elevate the prepuce through the opening in the sheath and incise the prepuce near the distal end of the elliptical incision. exteriorize the free portion of the penis through the incision by grasping the glans penis with sponge forceps. using #0 absorbable suture appose the preputial epithelium to the skin of the sheath around the periphery of the stoma with a simple interrupted suture pattern (figure 2). finally, suture a 2.5cm wide by 10cm long penrose drain over the free portion of the penis to allow post-operative urine drainage away from the incision (figure 3). remove the drain ten days postoperatively and ensure the bull has 60 days of strict sexual rest prior to an attempt at semen collection. case presentation case 1 a ten year-old brahman bull was presented with a history of preputial laceration approximately six months previously that had been managed medically by the referring veterinarian. the bull was referred to the teaching hospital at auburn university because of preputial swelling that impeded urination. upon presentation the bull had phimosis and the lumen of the prepuce was one cm in diameter due to excessive scar tissue near the end of the sheath. the bull underwent a resection and anastomosis procedure to remove the stenotic area of prepuce. the bull recovered from the surgery uneventfully, but later scar tissue developed at the surgical site once again creating stenosis that threatened to impede the bull’s ability to void urine. surgery for creation of the preputial stoma was performed under light sedation using 0.1 mg/kg xylazine with the bull in lateral recumbency with the aid of a pudendal nerve block as previously described.4 case 2 a two year old angus bull was presented with a history of a preputial laceration that had been repaired by resection and anastomosis of the prepuce by the referring veterinarian. the bull later developed a preputial stricture that precluded extension of the penis. the stricture was located approximately eight cm inside the preputial opening and had a luminal diameter of one cm. the bull displayed retention of urine in the preputial cavity proximal to the stricture. surgery for creation of the preputial stoma was performed under general anesthesia. the anesthetic protocol involved a preanesthetic of 50mg xylazine im, followed by induction with 1000 mg ketamine and 50 mg xylazine im, and the patient was maintained on isoflurane in lateral recumbency for the entirety of the surgery. case 3 a three year-old santa gertrudis bull presented with severe preputial prolapse and upon examination a preputial laceration was identified. following medical management to reduce swelling and cellulitis the damaged preputial tissue was removed using a preputial resection and anastomosis procedure and the bull was discharged following the surgery. the bull returned after 60 days of sexual rest for evaluation of the surgical site and a breeding soundness evaluation. at that time it was discovered that a stricture had formed that precluded the bull from fully extending the penis. due to the previous procedure the prepuce lacked sufficient length to perform a second resection and anastomosis (figures 3 and 4). this bull had surgery performed under light sedation and an internal pudendal nerve block as described previously. clinical theriogenology • volume 7, number 4 • december 2015 420 treatment and outcome each of these bulls suffered severe phimosis due to stenosis of the preputial cavity following injury or surgical repair of preputial lacerations by resection and anastomosis of the damaged preputial tissue. for each of these cases the owners wished to either allow the bull to live without difficult urination or to continue to collect semen. each of the bulls underwent either general or local anesthesia for the creation of the preputial stoma. in each case the bull recovered from anesthesia and surgery uneventfully. this procedure and the following outcomes are the first to be reported to our knowledge and have to date been the only attempts made by the authors for the described method. the goal for the bull in case 1 was for him to live without the complication of urine accumulation within the sheath proximal to the stenotic area of the prepuce. the bull underwent surgery under light sedation and a pudendal nerve block. there were no complications during or following this procedure. the bull was able to urinate unimpeded and the owner reported that the bull unexpectedly bred two cows resulting in one pregnancy. the goal for the bull in case 2 was to collect semen for cryopreservation at a semen processing facility. after the stoma site healed semen from the bull was collected successfully via electroejaculation. semen collected met the standards set forth by the society for theriogenology prior to cryopreservation. the goal for the bull in case 3 was to collect semen for cryopreservation. the goal was acheived and the owner was able to bank several thousand straws of semen following collection. it was later reported by the referring veterinarian that the bull achieved intromission with several cows that pregnancies resulted. discussion acquired preputial injuries in bulls are a common problem encountered in clinical practice. stricture formation, a complication following correction with medical and/or surgical procedures, is not uncommon. it is the observation at the auburn university large animal teaching hospital that a significant increase in post-operative complications are seen in the summer months when the ambient temperature and humidity are both high. historically, bulls have gone to slaughter following the formation of a stricture within the prepuce that was not correctable by other means. often these bulls are not candidates for a second surgical procedure because of inadequate preputial length that would preclude the necessary minimum of two times the length of the free portion of the penis to achieve full extension.3 the initial injury (trauma, frostbite, etc.) may also preclude an initial surgery to correct the fibrotic area because of the length of the prepuce. the authors have demonstrated that the creation of a preputial stoma in cases of stricture formation may be not only a salvage procedure, but also extend the breeding life of some sires through cryopreservation of semen and in some cases live cover. this procedure may be considered as the primary surgical procedure depending on wishes of the client and financial considerations as this procedure typically cost one-half to one-third of resection and anastomosis. it should be noted that bulls that achieve intromission following this procedure will likely be those with excellent libido. semen collection via artificial vagina and electroejaculation are both possibilities after healing of the surgical site. learning points • preputial injuries are common in bulls in pasture breeding systems. • preputial stoma surgery should be considered as an option in bulls with preputial stricture precluding extension of the penis. • the genetics of bulls can be preserved following this procedure via cryopreservation or by natural breeding. clinical theriogenology • volume 7, number 4 • december 2015421 figure 1. schematic demonstrating stoma placement on the ventral aspect of sheath. figure 2. immediate postoperative image of the preputial stoma created in case 1. clinical theriogenology • volume 7, number 4 • december 2015 422 figure 3. penrose drain exiting stoma allowing urine drainage away from incision site. figure 4. note position of stoma along the ventral sheath in bull described in case 3. references 1. wolfe df, beckett sd, carson rl: acquired conditions of penis and prepuce. in: wolfe df, moll hd, editors. large animal urogenital surgery. 2nd ed. baltimore: williams & wilkins; 1999. p. 237-272. 2. long se: eversion of the preputial epithelium in bulls at artificial insemination centres. vet rec 1969;84:495-499. 3. walker df, vaughan jt: bovine and equine urogenital surgery. philadelphia: lea & febiger; 1980. p. 277. 4. lin hc, walz ph: farm animal anesthesia. hoboken (nj): john wiley & sons; 2014. p. 278. (editor's note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 7, number 4 • december 2015423 clinical theriogenology • volume 7, number 4 • december 2015 424 omniblank: 2011: the interaction of blood urea nitrogen and semen type in a fixed time ai program in angus cross beef cows-a pilot study the interaction of blood urea nitrogen and semen type in a fixed time ai program in angus cross beef cows–a pilot study ramanathan k. kasimanickam,a william d. whittierb adepartment of veterinary clinical sciences, washington state university, pullman, wa; bdepartment of large animal clinical sciences, virginia polytechnic institute and state university, blacksburg, va abstract cows fed excess dietary protein had increased blood urea (bun), altered uterine fluid composition, decreased uterine ph, and reduced fertility. the objective of the study was to examine the effect of bun concentrations at the day of breeding on the fixed time artificial insemination (ai) pregnancy rate in angus cross beef cows inseminated with fresh extended or frozen thawed semen. angus cross beef cows (n=266) from seven locations were synchronized with a co-synch + cidr protocol for fixed time ai. cows were inseminated with either fresh extended (3 million sperm, caprogen® extender) or frozen thawed semen (20 million sperm; egg yolk extender) from two ai sires. on the day of ai, blood samples were collected for bun analysis. in the study population, there were 165 cows with  18 mg/dl (mean 11.6) and 101 cows with > 18 mg/dl (mean 23.2) bun concentrations. accounting for significant variables such as ai sire (p<0.001) and semen type by bun level interaction (p<0.05) in the model, there were no differences in the fixed time ai pregnancy between bun groups (p>0.05). however, the fixed time ai pregnancy rate was different between fresh and frozen semen, 61.1% and 52.4%, respectively (p<0.05). the ai pregnancy rates were significantly different between sires 1 and 2, 66.7% and 44.0%, respectively (p<0.05). a semen type by bun concentration interaction for ai pregnancy was recorded. no ai sire by bun concentration or ai sire by semen type interactions were recorded. there was a quadratic relationship observed for locations’ mean bun concentrations with locations’ mean ai pregnancy rates. lower and higher mean location bun concentrations had reduced ai pregnancy. cows with >18% mg/dl bun concentrations inseminated with fresh semen had a 61.2% ai pregnancy rate. our findings suggest that the negative effect of increased bun concentrations may be overcome by utilizing fresh semen extended in an extender with catalase (caprogen®) in a fixed time ai programs. introduction previous research has shown that cows fed excess dietary protein had increased blood urea, altered uterine fluid composition, decreased uterine ph, and reduced fertility.1-4 dairy cows with bun and milk urea nitrogen (mun) >19 mg/dl showed a 20 percentage point decrease in ai pregnancy rate.2 rhoads et al showed that short-term increase in plasma urea nitrogen (pun) can exert direct effects on the uterine environment by decreasing uterine ph.4 nitrogenous waste products (e.g., nh3 and urea) are toxic to bovine gametes and/or embryos and have been suggested to play a role in reproductive inefficiency.1 high pun concentrations changed the follicular, oviductal and uterine environment, and decreased embryo viability through effects exerted on the oocyte or embryo.3 also the pun status of the donor cows affected pregnancy rate following embryo transfer.3 cows grazing spring pastures in virginia are exposed to very high protein diets which may have the potential to decrease pregnancy rates (http://pubs.ext.vt.edu/news/livestock/2010/06-07/lu_06-01-10-3.html). the objective of the study was to examine the effect of bun concentrations at the day of breeding on the fixed time ai pregnancy rate in angus cross beef cows inseminated with fresh and frozen semen. materials and methods angus cross beef cows (n=266) from seven locations were synchronized with a co-synch + cidr protocol (figure 1) for fixed time ai (ftai). briefly, cows enrolled in the study received 100 g of gonadorelin diacetate tetrahydrate im (gnrh; cystorelin sterile solution, merial, athens, ga) and 1.3 g progesterone in an intra-vaginal insert (cidr; eazi-breedtm cidr cattle insert®, pfizer animal health, new york, ny) on day 0. additionally, body condition scores (bcs) of cows in the study were recorded 93 clinical theriogenology • volume 3 number 2 • june 2011 on day 0. on day 7, the cidr was removed, and 25 mg of dinoprost (pgf2; lutalyse sterile solution, pfizer animal health, new york, ny) was administered im. cows were inseminated with either fresh extended or frozen thawed semen, at a fixed-time on day 10, 66 h after cidr removal and received 100 g of gnrh at this time. two mature, known highly fertile angus bulls from a commercial stud center were used in this study. prior to initiation of the study, semen samples of each sire were screened for presence of mycoplasma sp. weekly for three consecutive weeks according industry guidelines (certified semen services, columbia, mo). on collection day two ejaculates were collected serially by artificial vagina and pooled within bull. the pooled semen from each bull was assessed for motility and concentration and then divided into two aliquots. aliquot one was extended using caprogen diluent (lic, hamilton, new zealand) to a concentration of 3 × 106/ 0.25 ml french straw. fresh semen samples were packaged in 0.25 ml french straws that are specially designed for use with non-frozen semen (imv technologies, l'aigle, france). straws were stored in polystyrene foam boxes at ambient temperature (approximately 20 to 25c) until insemination. aliquot two was extended using an egg-yolk-glycerol extender to a concentration of 20 × 106/ 0.5 ml french straw and frozen in liquid nitrogen vapor (-196c). semen straws were shipped from the stud center to the breeding locations and cows were artificially inseminated within 48 hours of collection. to meet the stud center standard, fresh semen samples were evaluated each morning to confirm sufficient maintenance of motility (≥ 60 %) and acrosomal integrity (≥ 70 %) prior to use in ai. frozen semen was assessed for post-thaw motility after 0 and 3 h of incubation at 37c (> 60 % and > 25 %, respectively) and for the percentage of intact acrosomal membranes after 3 h of incubation at 37c (> 60%). cows were randomly assigned to fresh semen and frozen semen groups and two ai sires. blood samples were collected by coccygeal venipuncture for bun analysis on the day of ftai. pregnancy diagnosis was performed 60 days after insemination using transrectal ultrasonography (sonosite 180 plus, sonosite inc., bothell, wa). artificial insemination pregnancy rate was calculated by number of cows diagnosed pregnant divided by number of cows inseminated. figure 1. statistics mixed model (glimmix procedure of sas version 9.12, sas institute inc., cary, nc) was used to determine the effect bun concentration categories ( 18 mg/dl vs. >18 mg/dl) on ftai. other variables included in the model were semen type (fresh, 3×106 vs. frozen, 20×106), ai sires (1 vs. 2), bcs (<5, 5 to 6, >6; 1 = emaciated; 9 = obese), days postpartum at initiation of synchronization (30 to 60, 61 to 80, >80), age of dam (2, 3 to 6 and > 6 years). locations were treated as random effect. a p value of  0.05 was considered significant. the model was created by manual backward stepwise elimination procedure (p > 0.10 for exclusion). only variables of interest (bun categories) and statistically significant variables were retained in the final model. results among the study population, 132 cows were inseminated with semen from sire 1 and 134 cows were inseminated with semen from sire 2. there were 130 and 136 cows inseminated with fresh and frozen semen, respectively. in the study population, there were 165 cows with  18 mg/dl (mean 11.6) and 101 cows with > 18 mg/dl (mean 23.2) bun concentrations. accounting for significant variables 94clinical theriogenology • volume 3 number 2 • june 2011 such as ai sire (p<0.001), and semen type  bun level interaction (p<0.05) in the model, there were no differences in the ftai pregnancy rate between bun groups (p>0.05). however, the ftai pregnancy rate was different between semen types (p<0.05; table 1). the ai pregnancy rates were significantly different between sire 1 and 2, 64.7% and 45.4%, respectively (p<0.05). a semen type  bun concentration interaction for ai pregnancy was recorded (figure 2). there were no ai sire  bun concentration or ai sire  semen type interactions observed. the bun concentration affected the ai pregnancy rate for frozen semen (figure 2). there was a quadratic relationship observed for mean location bun concentrations and location ai pregnancy rates (figure 3). lower and higher mean location bun concentrations had reduced ai pregnancy rates. discussion in the present study, the pregnancy rate following insemination with fresh semen and frozen semen was not different despite differences in average sperm concentration in fresh versus frozen straws. the similarity in pregnancy rates may be attributable to both survivability of fresh sperm and the impaired functional and structural parameters of sperm after the freeze-thaw process.5 it is possible that frozen semen is less likely either to survive long enough in the female reproductive tract or to maintain competency in fertilization, as compared to fresh extended semen.6 a plateau theory for the impact of increasing sperm numbers for most ai sires on fertility has been proposed.7 beyond this plateau number no increase in fertility results. our results suggest that this plateau had been reached with both fresh and frozen semen. the results from this study indicated that cows with altered bun concentrations inseminated with frozen semen had numerically reduced ai pregnancy rates. sperm binding to oviductal epithelium preserves their fertility during storage in the sperm reservoir.8 cows with an increased number of sperm in the sperm reservoir resulted in increased risk of becoming pregnant.9 it has been demonstrated that sperm viability is maintained by preventing premature capacitation and the suggested mechanism is presence of catalase in the bovine oviduct.10,11 caprogen diluent is enriched with catalase. therefore it is reasonable to expect that the fresh semen extended with caprogen diluent provided a better environment which facilitated a higher number of sperm in the sperm reservoir.12 it has been demonstrated that nitrogenous waste products (e.g., nh3 and urea) are toxic to bovine gametes and/or embryos and have been suggested to play a role in reproductive inefficiency. cows in this study with > 18 mg/dl bun concentrations inseminated with fresh semen had a 61.2% ai pregnancy rate. so it is notable that the negative effect of increased bun concentrations may be overcome by utilizing fresh semen extended in an extender with catalase (caprogen®) in ftai programs. 95 clinical theriogenology • volume 3 number 2 • june 2011 table 1. the effect of semen type and bun level on ftai pregnancy rate in beef cows synchronized with co-synch+cidr protocol* parameter n treatment groups mean ai-pr† bun levels 165  18 mg/dl (mean 11.6) 55.0 a 101 > 18 mg/dl (mean 23.2) 58.6 a semen type 136 fresh semen (3×10 6 /straw) 61.1 a 130 frozen semen (20×10 6 /straw) 52.4 b sires 132 1 45.4 a 134 2 64.7 b *refer to figure 1 for protocol; †accounted semen type by bun level interaction (p<0.05) a,bnumbers with no common superscript were different (p < 0.05) ai-pr – pregnancy rate for fixed time ai. cidr – controlled internal drug release (intravaginal progesterone insert). bun – blood urea nitrogen. figure 2. fixed time ai pregnancy rate among semen type and bun concentration (mg/dl) groups in beef cows abbars with no common superscript were different (p < 0.05) 96clinical theriogenology • volume 3 number 2 • june 2011 figure 3: association of locations’ mean bun concentration and mean ai pregnancy rate. references 1. butler wr: review: effect of protein nutrition on ovarian and uterine physiology in dairy cattle. j dairy sci 1988;81:2533-2539. 2. butler wr, calaman jj, beam sw: plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. j anim sci 1996;74:858-865. 3. rhoads ml, rhoads rp, gilbert ro, et al: detrimental effects of high plasma urea nitrogen levels on viability of embryos from lactating dairy cows. anim reprod sci 2006;91:1-10. 4. rhoads ml, gilbert ro, lucy mc, et al: effects of urea infusion on the uterine luminal environment of dairy cows. j dairy sci 2004;87:2896-2901. 5. parks je, graham jk: effects of cryopreservation procedures on sperm membranes. theriogenology 1992;38:209-222. 6. vishwanath r, pitt cj, shannon p: sperm numbers, semen age and fertility in fresh and frozen bovine semen. proc nz soc anim prod 1996;56:31-34. 7. saacke rg: sperm morphology: its relevance to compensable and uncompensable traits in semen. theriogenology 2008;70:473-478. 8. lefebvre r, chenoweth pj, drost m, et al: characterization of the oviductal sperm reservoir in cattle. biol reprod 1995;53:1066-1074. 9. hunter rhf: sperm transport and reservoir in the pig oviduct in relation to time of ovulation. j reprod fertil 1981;63:109-117. 10. lapointe s, sullivan r, sirard m-a: binding of a bovine oviductal fluid catalase to mammalian spermatozoa. biol reprod 1998;58:747-753. 11. lapointe, s., sirard m-a: catalase and oviductal fluid reverse the decreased motility of bovine sperm in culture medium containing specific amino acids. j androl 1998;19:31-36. 12. vishwanath r, shannon p: do sperm cells age? a review of the physiological changes in sperm during storage at ambient temperature. reprod fertil dev 1997;9:321-331. 97 clinical theriogenology • volume 3 number 2 • june 2011 1 contact mariana diel de amorim md649@cornell.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10439, http://dx.doi.org/10.58292/ct.v16.10439 case report sexual development disorder in a dog sarah wright,a yamilka lago–alvarez,a christopher champion,b christian folk,a benjamin rivard,a bradley back,a luis henrique de aguiar,a mariana diel de amorima adepartment of clinical sciences, college of veterinary medicine, cornell university, ithaca, ny, usa bdepartment of population medicine and diagnostic sciences, college of veterinary medicine, cornell university, ithaca, ny, usa abstract a 1-year intact, phenotypic female doberman pinscher dog, was evaluated for suspected sexual development disorder. patient had a history of white mucoid vaginal discharge with no estrous signs or behavior. clitoral hypertrophy with a palpable os clitoris were noted. transabodminal ultrasonography revealed a right gonad and tubular structures (possibly uterus). exploratory laparotomy revealed 2 underdeveloped gonads in caudodorsal abdomen and a markedly underdeveloped right uterine horn. histopathology confirmed bilateral ovotestes. karyotyping (number and morphology of chromosomes) and fluorescence in situ hybridization (xx and xy cells) results were normal. samples were pcr positive for sex-determining region y (sry) and x-linked androgen receptor gene. we concluded that the patient was either a mosaic or chimera with normal female 78,xx and normal male 78,xy cells and genetically a mix of male and female. final diagnosis was sex chromosome (78,xx/xy), sry-positive, ovotesticular, disorder of sexual development with female phenotype. keywords: abnormality of chromosomal sex, chimera, mosaic, os clitoris, ovotestes background disorders of sexual development (dsds) include congenital reproductive tract abnormalities.1 abnormalities in chromosomal sex, gonadal differentiation, and phenotypic differentiation of the external genitalia result in a variety of phenotypic presentations. therefore, determining both gonadal sex and chromosomal sex is critical to allow for precise diagnosis and to understand underlying pathophysiology.2,3 in y chromosome presence, sex-determining region y (sry) gene acts on sry-box transcription factor 9 (sox9) that is supported by positive feed-forward loops with the fibroblast growth factor 9 (fgf9) gene.4 potentially, steroidogenic factor 1 (sf-1) also acts to initiate activation of sox9;5 sox9 expression allows sertoli cell development and inhibits expression of wingless-related mmtv integration site 4 (wnt4) and forkhead box l2 (foxl2) genes, important for ovarian development.6 sertoli cells continue to express sox9 throughout life7 and also secrete antimüllerian hormone that regresses müllerian ducts and stimulates differentiation of leydig cells.6 in contrast, absence of sry expression results in differentiation of bipotential gonads into ovaries. expression of r-spondin family member 1 (rspo1) activates wnt48 upregulating dosage-sensitive sex reversal-adrenal hypoplasia congenital critical region on x chromosome, gene 1 (dax1), an x chromosome gene that inhibits sertoli cell development by suppressing sf-1 activation of sox9.9 rspo1 also activates the β-catenin signaling pathway that further drives expression of wnt4 along with other genes associated with female differentiation.8 failure in 1 or more of these pathways, led by a multitude of causes, might lead to an ambiguous phenotype. chromosomal abnormalities have been reported in dogs, but are considered uncommon disorders.10,11 chimerism originates through the combination of 2 differing zygotes whereas mosaicism originates from a mitotic error in a single zygote, making 2 or more genetically varying type of cells 78,xx and 78,xy. chromosomal mosaicism may take place at any stage of development.12 chimeras that occur in early development are defined as primary chimeras, but if they occur postimplantation are called secondary (e.g. blood chimeras, which originate due to the exchange of blood cells between twins). freemartinism is a common condition in cattle where there is exchange of blood between twin male and female through placental anastomosis. affected females may present with differing degrees of reproductive tract masculinization.13 female or male dogs with xx/xy leukocyte chimerism is uncommon, mailto:md649@cornell.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10439 2 citation line: clinical theriogenology 2024, 16, 10439, http://dx.doi.org/10.58292/ct.v16.10439 may present with ambigious phenotype, and sterility of the individual is common.14–16 a dog diagnosed with a sex chromosome dsd is presented. case presentation a 1-year intact, phenotypic female doberman pinscher dog, was referred for evaluation of a suspected dsd due to an enlarged clitoris. owner reported that the dog had multiple episodes of white mucoid vaginal discharge with no evidence of estrus. dog’s dam was not supplemented with progesterone during pregnancy and it was unknown if any of the other littermates were affected. physical examination findings were normal, except for slight increase in anogenital distance, clitoral hypertrophy, and palpable os clitoris (figure 1a). urethral groove extended from os clitoris base to ~ ¾ distance to distal clitoral tip. no other phenotypic abnormalities were noted. serum progesterone concentrations (0.25 ng/ml) were consistent with lack of luteal activity and anestrus. right gonad was idenitified via transabdominal ultrasonogrpaphy. it was located craniomedial to right kidney, adjacent to multiple mesenteric lymph nodes, and was 2.7 x 1.6 cm, circumscribed, heteroechoic, with small, round, cyst-like hypoechoic to anechoic structures within it that were interpreted to be ovarian follicles. a hyperechoic, relatively linear structure was observed in gonadal center that was suspected to be rete testis (figure 1b). left gonad could not be detected via transabdominal ultrasonogrpaphy. additionally, small, tubular, hypoechoic structure with hyperechoic parallel walls were observed in the expected region of uterus; structure appeared to bifurcate right and left at the caudal aspect of urinary bladder into small, tubular, structures that abruptly terminated. abnormally developed uterus or vas deferens was suspected. treatment abdominal exploratory surgery identified 2 small, underdeveloped gonads in the caudodorsal abdomen between kidneys’ caudal pole and bladder. there was evidence of a markedly underdeveloped right uterine horn. uterine body and left uterine horn were not palpable. reproductive tissue was surgically removed and histopathology of gonads revealed bilateral ovotestes. these ovotestes consisted of a central testicular component and peripheral ovarian component surrounded by bursa (figure 2a). adjacent to gonad were numerous closely tortuous arteries and veins (resembling pampiniform plexus) as well as several tortuous mesonephric ducts (resembling epididymis) surrounded by dense fibrous connective tissue (figure 2b). central testicular component was composed of large numbers of variably pigmented leydig cells and irregular seminiferous tubules that consisted  solely of sertoli cells  with no evidence of spermatogonia, spermatocytes, spermatids, or sperm (figure 2c). peripheral ovarian component lacked  overt follicular structures and was merely composed of irregular and tortuous rete ovarii and abundant interstitial cells (figure 2d). outcome cytogenetic analysis (texas a&m university) revealed normal number and morphology of chromosomes. approximately 60% of the cells had 2 x chromosomes and ~ 40% of the cells were xy. fluorescent in situ hybridization (fish) assay was performed using y chromosome-specific probes. pcr analyses were performed to identify sry gene presence and an x-linked androgen receptor gene. results indicated the presence of both xx figure 1. a. os clitoris exposed for size visualization; b. ultrasonographic image (craniocaudal view) of suspected right gonad, note: multifocal hypoechoic follicles and suspected rete testis (hyperechoic band [arrows]). http://dx.doi.org/10.58292/ct.v16.10439 citation line: clinical theriogenology 2024, 16, 10439, http://dx.doi.org/10.58292/ct.v16.10439 3 and xy cells, confirming the final diagnosis of sex chromosome (78,xx/xy), sry-positive, ovotesticular, disorder of sex development with a female phenotype. discussion phenotypic abnormality with chromosomal anomaly (xx/xy) and bilateral ovotestis suggested a development disorder due to either 1 or both of them. abnormalities of chromosomal sex are caused by errors in the number or structure of x and y chromosomes. additional sex chromosomes or missing sex chromosome can affect phenotype. additionally, errors in chromosomes’ structures such as insertions, deletions, or translocations can disrupt the expression of genes that reside on altered chromosomes and can result in ambiguous sex phenotype.17,18 this patient was diagnosed with a disorder of sexual development that included clitoral hypertrophy, bilateral ovotestes, underdeveloped uterus, and a chromosome disturbance consistent with chimerism/mosaicism. chimerism occurs when different zygotes or cells from different zygotes fuse, whereas mosaicism is due to nondisjunction in a single zygote or cells derived from a single zygote.3,19 in this case, cytogenetic and fish analyses were performed only in blood. various nonhemapoetic tissues along with blood and other genetic diagnostic tests such as dna microsatellite, short tandem repeats (str) and/or single nucleotide polymorphism (snp) microarray are required to differentiate the underlying mechanism of mosaicism/chimerism.20,21 amount of testicular gonadal tissue and its corresponding hormonal activity determines the animal’s phenotype22 and may include unilateral or bilateral ovotestes as in this patient.23–27 chimerism in a dog was previously reported wherein both blood leukocyte and skin cytogenetic analyses were performed.28 a limitation of the present case report was that the cytogenetic analysis was performed only on blood leukocytes. inability to indentify left gonad via transabdominal ultrasonography was most likely due to postioning of animal and transducer angle. additionally, follicle-like structures identified via transabdominal ultrasongraphy were likely distended mesonephric ducts within the structure resembling epididymis, as follicles were not identified on histopathology. this case is unique because the subset of chimeric and mosaic dogs is rare,2 with only a few case reports of abnormalities of chromosomal sex in dogs.27,28 conflict of interest authors have no conflict of interest or funding to declare. figure 2. light microscopic images of gonad stained with hematoxylin and eosin: a. central testicular component and peripheral ovarian component surrounded by bursa (1x); b. tortuous arteries and veins (resembling the pampiniform plexus) and several tortuous mesonephric ducts (resembling the epididymis) surrounded by dense fibrous connective tissue (2x); c. large numbers of variably pigmented leydig cells and irregular seminiferous tubules that consist solely of sertoli cells with no evidence of spermatogonia, spermatocytes, spermatids, or sperm (20x), and d. tortuous rete ovarii and abundant interstitial cells (20x). http://dx.doi.org/10.58292/ct.v16.10439 4 citation line: clinical theriogenology 2024, 16, 10439, http://dx.doi.org/10.58292/ct.v16.10439 learning points • mosaicism occurs when there is nondisjunction in a single zygote or cells derived from a single zygote. • chimerism occurs when different zygotes or cells from differening zygotes fuse. • sex chromosome disorders of sexual development are uncommon in dogs. references 1. foster ra: male genital system: disorders of sexual development. in: grant m: editor. jubb, kennedy, and palmer’s pathology of domestic animals. 6th edition, elsevier: st. louis; 2016. p. 468-471. 2. poth t, breuer w, walter b, et al: disorders of sex development in the dog-adoption of a new nomenclature and reclassification of reported cases. anim reprod sci 2010;121:197-207. doi: 10.1016/j. anireprosci.2010.04.011 3. meyers-wallen vn, patterson df: sexual differentiation and inherited disorders of sexual development in the dog. j reprod fertil 1989;39:57-64. 4. sekido r, lovell-badge r: sex determination and sry: down to a wink and a nudge? trends genet 2009;25:19-29. doi: 10.1016/j. tig.2008.10.008 5. jakob s, lovell-badge r: sex determination and the control of sox9 expression in mammals. febs j 2011;278:1002-1009. doi: 10.1111/j.1742-4658.2011.08029.x 6. barrionuevo f, bagheri-fam s, klattig j, et al: homozygous inactivation of sox9 causes complete xy sex reversal in mice. biol reprod 2006;74:195-201. doi: 10.1095/biolreprod.105.045930 7. christensen bw: disorders of sexual development in dogs and cats. vet clin north am small anim pract 2012;42:515-526. doi: 10.1016/j.cvsm.2012.01.008 8. chassot aa, gregoire ep, magliano m, et al: genetics of ovarian differentiation: rspo1, a major player. sex dev 2008;2:219-227. doi: 10.1159/000152038 9. vilain e, mccabe erb: mammalian sex determination: from gonads to brain. mol genet metab 1998;65:74-84. doi: 10.1006/ mgme.1998.2749 10. raudsepp t, chowdhary bp: chromosome aberrations and fertility disorders in domestic animals. annu rev anim biosci 2016;4:15-43. doi: 10.1146/annurev-animal-021815-111239 11. meyers-wallen v: gonadal and sex differentiation abnormalities of dogs and cats. sex dev 2012;6:46-60. 12. west jd, everett ca: preimplantation chromosomal mosaics, chimaeras and confined placental mosaicism. reprod fertil 2022;3:r66. doi: 10.1530/raf-21-0095 13. padula am: the freemartin syndrome: an update. anim reprod sci 2005;87:93-109. doi: 10.1016/j.anireprosci.2004.09.008 14. szczerbal i, switonski m: clinical cytogenetics of the dog: a review. animals (basel) 2021;11:947. doi: 10.3390/ani11040947 15. dockweiler j, kawakami t, boyko a: disorder of sexual development due to xx/xy chimerism in a siberian husky female dog. clin theriogenol 2023;15:18. 16. szczerbal i, nowacka-woszuk j, nizanski w, et al: a case of leucocyte chimerism (78,xx/78,xy) in a dog with a disorder of sexual development. reprod domest anim 2014;49(3):e31-e34. doi: 10.1111/rda.12318 17. swain a, narvaez v, burgoyne p, et al: dax1 antagonizes sry action in mammalian sex determination. nature 1998;391:761-767. doi: 10.1038/35799 18. romagnoli s, schlafer dh: disorders of sexual differentiation in puppies and kittens: a diagnostic and clinical approach. vet clin north am small anim pract 2006;36:573-606. doi: 10.1016/j. cvsm.2005.12.007 19. meyers-wallen vn: inherited disorders in sexual development. j  hered 1999;90:93–95. doi: 10.1093/jhered/90.1.93. pmid: 9987911 20. madan k: natural human chimeras: a review. eur j med genet 2020;63:103971. doi: 10.1016/j.ejmg.2020.103971 21. charalsawadi c, jaruratanasirikul s, hnoonual a, et al: case report: molecular analysis of a 47,xy,+21/46,xx chimera using snp microarray and review of literature. front genet 2022;13:802362. doi: 10.3389/fgene.2022.802362/full 22. feldman e, nelson rw: infertility, associated breeding disorders, and disorders of sexual development. in: canine and feline endocrinology and reproduction. 3rd edition, saunders: philadelphia; 2004. p. 868-900. 23. bosu wtk, chick bf, basrur pk: clinical, pathologic and cytogenic observations on two intersex dogs. cornell vet 1978;68:375-390. 24. hare wc: intersexuality in the dog. can vet j 1976;17:7. 25. dain ar, walker rg: two intersex dogs with mosaicism. j reprod fertil 1979;56:239-242. doi: 10.1530/jrf.0.0560239 26. johnston sd: premature gonadal failure in female dogs and cats. j reprod fertil 1989;39:65-72. 27. szczerbal i, nizanski w, dzimira s, et al: chromosome abnormalities in dogs with disorders of sex development (dsd). anim reprod sci 2021;230:106771. doi: 10.1016/j. anireprosci.2021.106771 28. schwartz r, sugai nj, eden k, et al: case report: disorder of sexual development in a chinese crested dog with xx/xy leukocyte chimerism and mixed cell testicular tumors. front vet sci 2022;9:981. doi: 10.3389/fvets.2022.937991/bibtex http://dx.doi.org/10.58292/ct.v16.10439 https://doi.org/10.1016/j.anireprosci.2010.04.011 https://doi.org/10.1016/j.anireprosci.2010.04.011 https://doi.org/10.1016/j.tig.2008.10.008 https://doi.org/10.1016/j.tig.2008.10.008 https://doi.org/10.1111/j.1742-4658.2011.08029.x https://doi.org/10.1095/biolreprod.105.045930 https://doi.org/10.1016/j.cvsm.2012.01.008 https://doi.org/10.1159/000152038 https://doi.org/10.1006/mgme.1998.2749 https://doi.org/10.1006/mgme.1998.2749 https://doi.org/10.1146/annurev-animal-021815-111239 https://doi.org/10.1530/raf-21-0095 https://doi.org/10.1016/j.anireprosci.2004.09.008 https://doi.org/10.3390/ani11040947 https://doi.org/10.1111/rda.12318 https://doi.org/10.1038/35799 https://doi.org/10.1016/j.cvsm.2005.12.007 https://doi.org/10.1016/j.cvsm.2005.12.007 https://doi.org/10.1093/jhered/90.1.93 https://doi.org/10.1016/j.ejmg.2020.103971 https://doi.org/10.3389/fgene.2022.802362/full https://doi.org/10.1530/jrf.0.0560239 https://doi.org/10.1016/j.anireprosci.2021.106771 https://doi.org/10.1016/j.anireprosci.2021.106771 https://doi.org/10.3389/fvets.2022.937991/bibtex 2017: hormonal, biochemical and hematological changes during gestation in rabbit does synchronized with prostaglandin f2 alpha hormonal, biochemical and hematological changes during gestation in rabbit does synchronized with prostaglandin f2 alpha temitope a. ajadi, a monsuru o. abioja, b mercy ndehedehe, b olusiji f. smith b a department of veterinary public health and reproduction, federal university of agriculture, abeokuta; b department of animal physiology, federal university of agriculture, abeokuta abstract assessment of physiological parameters such as hormonal, biochemistry and hematology of animals at different stages of gestation are helpful to monitor the health and nutritional status of animals. this study therefore, evaluated the changes in hormonal levels, biochemical and hematological parameters during gestation in domestic rabbit (oryctolagus cuniculus) does following estrous synchronization with prostaglandin f2 alpha (pgf2α). eight nulliparous, sexually mature intact new zealand rabbit does with mean weight of 1.9±0.1kg were used for the study. they were distributed into eight hutches, and were synchronized with 0.7 mg/kg bw im injection of pgf2α prior to mating. after 48 hours, the eight does were naturally mated with four bucks each (within two hours each doe was allowed to mate with four bucks to maximize chances of pregnancy). does were examined for pregnancy using ultrasonography seven days after mating. blood was sampled from the jugular vein before mating (bm), seven days after mating (7dam), 14 days after mating (14dam), 21 days after mating (21dam), 28 days after mating (28dam) and three days post-parturition (3dpp), respectively. blood plasma progesterone, follicle stimulating hormone (fsh), estrogen and prolactin were assayed using enzyme linked immunosorbent assay. hematological and biochemical parameters determined were packed cell volume (pcv), hemoglobin (hb) concentration, red blood cell (rbc) count, white blood cell (wbc) count, cholesterol, triglycerides, high density lipoproteins (hdl) and low density lipoproteins (ldl). data obtained for hormone and serum biochemistry were subjected to descriptive statistics, while other data were subjected to analysis of variance using general linear model procedure of statistical software. results revealed that mean values for progesterone, fsh, estrogen and prolactin during gestation significantly varied at different periods of the experiment (p<0.05). progesterone secretion during gestation peaked at 14dam (32.1 ± 0.27 ng/ml). estrogen secretion was 857.2 ± 3.22 ng/ml bm, 857.5 ± 3.80 ng/ml 14dam and 866.6 ± 2.17 ng/ml at 28dam but subsequently declined to 850.7 ± 6.04 ng/ml at 3dpp. prolactin increased from 92.3± 0.13 ng/ml at bm to 92.8 ± 0.06 ng/ml at 7dam, but decreased to 91.8 ± 0.36 ng/ml at 14dam then increased to 92.5±0.20 ng/ml at 3dpp. cholesterol, triglyceride and ldl were not significantly (p>0.05) influenced by the period of sampling. the pcv, rbc, hb, and wbc significantly varied from bm to 3dpp (p<0.05). the rbc, pvc, hb and wbc counts decreased gradually from bm to 28dam and subsequently increased until 3dpp. the study concluded that there were changes in hormonal parameters, pcv, rbc, hb, wbc, and ldl while cholesterol, triglyceride, hdl and the wbc differential counts showed no changes during the study period. keywords: gestation, new zealand white, progesterone, mating, parturition introduction developing countries including nigeria are rapidly growing in human population, and as such the demand for protein source is increasing. most of the world population is fed on small farm products which are becoming insufficient as the human population pressure increases.1 this has led to the need for alternative protein sources that are cheap, readily available and pose minimal competition to man. 2 rabbits are small mammals in the family leporidae of the order lagomorpha, found in several parts of the world. domestic rabbits have been used as sources of food and wool, research subjects, and as pets. some rabbits are bred mainly for meat production, while others are bred for laboratory and exhibition purposes. rabbits compared to other laboratory animals are more advantageous as research subjects because of their unique lipid metabolism which is similar to that of humans. 3 they have a relatively short clinical theriogenology • volume 9 number 1 • march 201725 http://en.wikipedia.org/wiki/animal_testing http://en.wikipedia.org/wiki/animal_testing http://en.wikipedia.org/wiki/house_rabbit http://en.wikipedia.org/wiki/house_rabbit gestation period averaging 30-31 days, early maturity, fast growth rate, high genetic selection potential, high feed conversion efficiency, high prolificacy and economic utilization of space. 1,4 the rabbit’s adaptation to tropical regions, their feeding and low costs of production are also added advantages which could make their use for research economical and easily affordable. 5 successful reproduction is an orderly sequence of events involving puberty, cyclicity, copulation, pregnancy, postpartum, lactation and recovery. 6 maintenance of pregnancy as a reproductive event and the initiation of parturition are under endocrine control. these involves the secretion of reproductive hormones progesterone, estrogen, luteinizing hormone (lh), follicle stimulating hormone (fsh) and prolactin. during the pregnancy period, metabolic changes may occur that may alter physiological range of blood constituents in the animal’s body. 6 therefore, assessment of the physiological parameters such as hormonal, biochemistry and hematology of animals at different stages of gestation are helpful to monitor the health and nutritional status of animals. different authors have worked on the reproductive hormone profile of rabbit does during mating and during pregnancy. 7-9 the significance and variation in the biochemical and hematological indices observed among breeds of rabbits were also described by different researchers. 10,11 however, it appears that little or no work has been carried out on the variation in the hormonal, biochemical and hematological indices all through the different stages of gestation in rabbits. therefore, this study was designed to investigate the effect of gestation on different levels of hormones, some biochemical parameters and hematology of rabbit does. materials and methods animals eight nulliparous sexually mature intact non-pregnant new zealand rabbit does with mean weight of 1.9 ± 0.1kg and four sexually mature bucks with mean weight of 1.9 ± 0.1kg were used for the experiment. they were purchased from a breeder located within the abeokuta metropolis. each of the rabbits was kept individually in a wooden hutch, housed in a naturally ventilated building. the experimental animals were fed a pelleted grower ration in the morning and thereafter with forage (tridax procumbens), and water was supplied ad-libitum. prior to commencement of study, all the animals were acclimatized for two weeks during which they were dewormed with albendazole syrup at 22 mg/kg/bw. all the animals were confirmed healthy based on the result of complete blood count and physical examination before the commencement of the study. ethical approval for this study was obtained from the research ethics committee, college of animal science, federal university of agriculture, abeokuta, ogun state, nigeria. experimental design and procedure the does were synchronized with 0.7mg/kgbw single im injection of pgf2α (lutalyse®, upjohn pharmaceutical limited, crawley, sussex, uk) administered at the follicular phase of their cycle when they were non-receptive to bucks. all the does were thereafter naturally mated with four bucks 48 hours after synchronizing them, during which all of them became receptive to the bucks. on the day of mating, the does were transferred to the cage of the bucks, two does were introduced to one buck for a period of 30 minutes during which mating was observed. this procedure was repeated amongst all the does while rotating them with each of the bucks ensuring that all the bucks bred all the does, the whole breeding lasted for 24 hours. pregnancy diagnosis does were examined for pregnancy seven days after mating with the use of portable ultrasound machine with a 7.5 mhz transducer (kaixin kx 2000®, xuzhou, china). the ultrasonography was performed transcutaneously. prior to scanning, the doe was restrained manually in dorsal recumbency to prevent her from moving. the hair on the abdominal region was clipped and scanning gel was applied at the examination site so that the space between the probe and the clinical theriogenology • volume 9 number 1 • march 2017 26 animal’s skin is free of air. the probe was positioned externally against the abdominal wall, and moved from right to left with the probe in the sagittal orientation and the image was viewed on the screen. blood sampling three milliliters of blood were obtained from each rabbit doe via the jugular vein before mating (bm), seven days after mating (7dam), 14 days after mating (14dam), 21 days after mating (21dam), 28 days after mating (28dam) and three days postpartum (3dpp) from all does. blood samples meant for hematological analysis were collected into plastic bottles containing ethylenediamine-tetraacetic acid (edta) while those that were meant for biochemical and hormonal assays were collected into lithium heparin bottles and taken to the laboratory for analysis. the plasma was obtained by centrifugation at 3000rpm in a refrigerated centrifuge, (4°c) for 15 minutes and stored at 20°c for hormone assay. hematological determination hematological analysis was carried out using mindray® bc-2800vet auto hematology analyzer. this machine operates on the principle of measuring the impedance or light dispersion of edta blood (1ml). the parameters measured include rbc count, hb concentration, pcv, wbc count and wbc differentials. plasma biochemistry and hormonal assay (progesterone, fsh, estrogen and prolactin) the plasma obtained was analyzed for concentration of cholesterol, triglycerides and lipoproteins using automated spectrophotometer (agappe diagnostics®, switzerland) using a conventional method. plasma concentrations of progesterone, fsh, total estrogen and prolactin were however carried out using enzyme l inked immunosorbent assay (elisa, mybiosource) as previously described. 12 statistical analysis data obtained were presented as m ean ± s tandard deviation and compared both between and within groups using anova for repeated measures, with significance set at p=0.05. data analysis was performed with statistical analysis system (sas institute, 2000). results all the eight does that were mated were confirmed pregnant with gestational sacs observed at day 7 after mating. the gestational sacs were characterized by oval shaped anechoic sac containing bipolar hyperechoic bands (plate 1). the mean plasma progesterone concentrations varied from bm until three days pp (figure 1). the plasma progesterone was at the lowest value (30.2± 0.11 ng/ml) bm, rose after mating until it reached the highest value of 32.1± 0.27 ng/ml at 14dam and thereafter dropped significantly (p<0.05) from 28dam until three days pp (figure 1). changes in mean plasma estrogen in rabbit does from before mating to three days pp is shown in figure 2. there was no significant difference (p>0.05) in mean plasma estrogen concentration from bm up to 14dam. it thereafter increased significantly (p<0.05) from 14dam to 21dam after which it significantly reduced from 28dam to three days pp (figure 2). the mean plasma fsh concentration had an initial significant increase (p<0.05) bm and 7dam, it thereafter stabilized until 3 days pp (figure 3). figure 4 shows the changes in mean plasma prolactin bm to three days pp in rabbit does. following an initial slight increase in mean plasma prolactin concentration after mating, it gradually reduced (p<0.05) between 7dam and 14dam. changes in cholesterol level during gestation in rabbit does are shown in figure 5. the highest cholesterol level was at 14dam, all the other values obtained throughout the period of study were not significantly (p>0.05) different. figure 6 shows the mean triglyceride changes as obtained in the rabbit does before they were mated up to three days pp. the result showed no significant (p>0.05) difference throughout the period of study. there was a significant increase (p<0.05) in the mean plasma hdl changes in rabbit does from before mating to 24dam following which it significantly reduced until clinical theriogenology • volume 9 number 1 • march 201727 28dam (figure 7). the changes in mean plasma ldl are presented in figure 8 which showed that all the mean values obtained from before mating until 3 days pp were not significantly (p>0.05) different. the hematological changes from bm through mating until three days pp in the rabbit does a r e presented in table 1. the mean values obtained for pcv, rbc, hb, wbc and neut differ significantly (p<0.05) between days of sampling, while the mean values obtained for lymphocytes, monocytes, eosinophils and basophils showed no significant (p>0.05) differences between the days of sampling. red blood cells, hb, pcv, wbc and neutrophils significantly decreased during the last week of gestation. hemoglobin increased significantly from bm to 7dam and subsequently decreased till 28dam. packed cell volume increased from bm until 7dam and subsequently decreased until 28dam and then increased until 3 days pp. discussion pregnancy in rabbits has three stages: the period of fertilization and implantation, organogenesis and fetal growth. 13 during these periods, different hormones are secreted in pulses, which play a role in the maintenance of the pregnancy and also in preparing the mammary gland for lactation. 13 progesterone action on the endometrium is essential for embryo implantation and pregnancy maintenance. the progressive increase in progesterone secretion obtained in this study after mating to mid-gestation could be attributed to the important role of progesterone in maintenance of pregnancy. 14 follicle stimulating hormone plays a key role in the development and functions of the reproductive system. it is necessary for the follicular selection and growth and for the production of estrogens from androgen substrates. 14 there was a decrease in fsh synthesis from 7dam which almost remained the same until three days pp. there was a slight increase in estrogen production at 14dam which was maintained at a plateau until 28dam. the main role of this estrogen could be attributed to its indispensable luteotropic role in the pregnant rabbit. 15 prolactin levels observed in this study were high at the first and last stage of pregnancy. the initial gradual increase may be attributed to its central role in the development of the mammary glands and in the initiation and maintenance of lactation after parturition. 16 cholesterol levels recorded in the present study increased progressively from 7dam to 14dam and then decreased to 28dam, and triglyceride increased gradually to 14dam then slightly reduced until 28dam during the period of gestation. this result agrees with that reported by wells et al during the gestation period in new zealand whites, although they recorded an outstanding increase on day 19 compared to the result observed in this study. 17 hematological parameters pass through a series of changes and are helpful to determine the health and nutritional status of animals. 18 the reduction in rbc, pcv and hb up to 28dam could be attributed to physiological anemia that might occur due to hemodilution. 19 the reduction in the hb may be due to mobilization of the dam’s hemoglobin into fetal circulation and also due to dilution of blood which occurs sequel to plasma volume increase. 19 the rbc and hb results correlate with that reported by wells et al, 17 who recorded downward trends in rbc and hb towards the end of organogenesis. the results for eosinophils, basophils and monocytes obtained in this study agrees with those reported by kim et al who reported an increase in these parameters from day 0-12 and then decreased to its lowest level on day 24 of gestation. 20 in conclusion, the results obtained in this study have shown that physiological parameters (hormonal, biochemical and hematological) are at variance during gestation as compared to before mating. the data obtained in this study could serve as useful tool to accurately assess and adjudge the health status of rabbit does during pregnancy. references 1. ajala mk, balogun jk: economics of rabbit production in zaria. kaduna state. trop j anim sci 2004;7:1-10. 2. mailafia s, onakpa mm, owoleke oe: problems and prospects of rabbit production in nigeria. a review. bayero j pure appl sci 2010;3:20-25. 3. fan j, watanabe t: transgenic rabbits as therapeutic protein bioreactors and human disease models. pharmacol 2003;99:261-262. 4. hassan he, elamin km, yousif ia, et al: evaluation of body weight and some morphometric traits at various ages in local rabbits of sudan. j anim sci adv 2012;2:407-415. clinical theriogenology • volume 9 number 1 • march 2017 28 5. ajayi, fo, balogun oo, ovuni ss: reproductive performance of rabbits fed maize-milling waste based diet. afr j biotechnol 2005;4:439-443. 6. khan jr, ludri rs: changes in blood glucose, plasma non-esterified fatty acids and insulin in pregnant and nonpregnant goats. trop anim health, pro 2002;34:81-90. 7. ubilla e, alvariño jmr, esquifito a: effects of induction of parturition by administration of a prostaglandin f2α analogue in rabbits: possible modification of prolactin, lh and fsh secretion patterns. anim reprod sci 1992; 27:1320. 8. ubilla e, rebollar pg, pazo d: effects of doe-litter on endocrinological and productivity variables in lactating does. livest prod sci 2000;67:67-74. 9. ubilla e, rebollar pg, pazo d: pituitary and ovarian response to transient doe litter separation in nursing rabbits. j reprod fert, 2000;118:361-366. 10. ajadi ta, bello aa, ekunleye ed, et al: changes in blood glucose and plasma lipids during gestation in chinchilla rabbits synchronised with pregnant mare serum gonadotropins. b anim health prod afr 2015; 63:27-32. 11. mizoguchi y, matsuoka t, mizuguchi h, et al: changes in blood parameters in new zealand white rabbits during pregnancy.lab anim 44: 33-39. 12. bliedtner a, zierau o, albrecht s, et al: effects of genistein and estrogen receptor subtype-specific agonistsin arko mice following different administration routes. mol cell endocrinol 2009;314:41-52. 13. fortun-lamothe l: effects of pre-mating energy intake on reproductive performance of rabbit does. anim sci 1998;66:263-269. 14. gilbert m, hauguel s, boulsset m: uterine blood flow and substrate update in conscious rabbit during late gestation. endocrinol metab 1984;16:574-580. 15. gadsby je, keyes pl, bill ch: control of corpus luteum function in the pregnant rabbit: role of estrogen and lack of a direct luteotropic role of the placenta. endocrinology 1983;113:2255-2262. 16. frederick wm: prolactin and luteinizing hormone cells of pregnant and lactating rats as studied by immunohistochemistry and radioimmunoassay. j anim anat 1999; 139:245-268. 17. wells my, decobecq cp, decouvelaere dm, et al: changes in clinical pathology parameters during gestation in the new zealand white rabbit. toxicol pathol 1999;27:370-379. 18. gupta ar, putra rc, saini m: haematology and serum biochemistry of chital (axis axis) and barking deer (muntiacus muntjak) reared in semi-captivity. vet res commun 2007;31:801-808. 19. ozegbe pc: influence of on some erythrocyte biochemical profiles the rabbits. afr j biomed res 2001;4:276 20. kim jc, yun hi, cha sw: hematological changes during pregnancy in new zealand white rabbits: a longitudinal study. comp clin pathol 2002;11:98-106. clinical theriogenology • volume 9 number 1 • march 201729 plate 1: 7 days old gestational sac of pregnant doe clinical theriogenology • volume 9 number 1 • march 2017 30 figure 1: plasma progesterone changes from before mating (bm) through post mating (pm) to three days post parturition (pp). means with different letters are significantly (p<0.05) different figure 2: plasma estrogen changes from before mating (bm) through post mating (pm) to three days post parturition (pp). means with different letters are significantly (p<0.05) different c abc a ab bc bc 30 30.5 31 31.5 32 32.5 bm 7dpm 14dpm 21dpm 28dpm 3dpp m ea n pl as m a pr og es te ro ne (n g/ m l) time of sampling ab ab ab a a b 840 845 850 855 860 865 870 bm 7dpm 14dpm 21dpm 28dpm 3dpp m ea n pl as m a o es tr og en (p m ol /l ) time of sampling clinical theriogenology • volume 9 number 1 • march 201731 figure 3: plasma follicle stimulating hormone (fsh) changes from before mating (bm) through post mating (pm) to three days post parturition (pp). means with different letters are significantly (p<0.05) different figure 4: plasma prolactin changes from before mating (bm) through post mating (pm) to three days post parturition (pp). means with different letters are significantly (p<0.05) different b a ab ab ab ab 176 178 180 182 184 186 188 190 bm 7dpm 14dpm 21dpm 28dpm 3dpp m ea n pl as m a fs h (m lu /m l) time of sampling abc a c bc bc ab 90.5 91 91.5 92 92.5 93 bm 7dpm 14dpm 21dpm 28dpm 3dpp m ea n pl as m a pr ol ac tin (n g/ m l) time of sampling clinical theriogenology • volume 9 number 1 • march 2017 32 figure 5: plasma cholesterol (chol) changes from before mating (bm) through post mating (pm) to three days post parturition (pp). figure 6: plasma triglyceride (trig) changes from before mating (bm) through post mating (pm) to three days post parturition (pp). 0 20 40 60 80 100 120 140 bm 7dpm 14dpm 21dpm 28dpm 3dpp m ea n pl as m a c h o l ( m g/ dl ) time of sampling 0 10 20 30 40 50 60 70 80 90 bm 7dpm 14dpm 21dpm 28dpm 3dpp m ea n pl as m a t ri g (m g/ dl ) time of sampling clinical theriogenology • volume 9 number 1 • march 201733 figure 7: changes in mean serum high density lipoprotein (hdl) before mating (bm) through post mating (pm) to three days post parturition (pp). means with different letters are significantly (p<0.05) different figure 8: changes in mean low density lipoprotein (ldl) before mating (bm) through post mating (pm) to three days post parturition (pp). means with different letters are significantly (p<0.05) different b ab a a ab a 0 5 10 15 20 25 30 35 40 bm 7dpm 14dpm 21dpm 28dpm 3dpp m ea n pl as m a h d l( m g/ dl ) time of sampling 0 10 20 30 40 50 60 70 80 bm 7dpm 14dpm 21dpm 28dpm 3dpp m ea n pl as m a ld l( m g/ dl ) time of sampling clinical theriogenology • volume 9 number 1 • march 2017 34 table 1: hematological changes from before mating (bm) through post mating (pm) to three days post parturition (pp). means with different superscripts are significantly different. parameter bm 7dpm 14dpm 21dpm 28dpm 3dp rbc (x 10 6 /ul) 5.40±0.11 ab 5.47±0.18 a 5.35±0.23 ab 5.08±0.21 ab 4.94±0.14 b 5.54±0.09 a pcv (%) 34.63±0.73 ab 35.13±0.90 a 33.00±1.57 ab 32.38±1.15 ab 31.63±0.84 b 34.25±0.59 ab hb (g/dl) 11.58±0.22 ab 11.81±0.33 a 11.59±0.52 ab 10.88±0.39 ab 10.60±0.31 b 11.44±0.19 ab wbc (x 10 3 /ul) 10.03±0,76 bc 10.60±0.61 b 9.66±0.59 bc 7.81±0.90 c 7.90±0.35 c 12.91±0.79 a lymphocyte (%) 68.63±3.59 56.88±5.89 61.38±6.65 62.88±6.08 74.57±3.53 57.00±6.90 monocyte (%) 1.75±0.31 0.75±0.31 2.13±0.79 1.25±0.45 2.00±0.46 1.63±0.50 eosinophil (%) 0.75±0.31 1.00±0.38 1.25±0.41 1.13±0.30 0.75±0.31 0.63±0.26 basinophil (%) 0.00±0.00 0.13±0.13 0.13±0.13 0.00±0.00 0.00±0.00 0.00±0.00 neutrophil (%) 28.00±3.51 ab 41.25±5.83 a 34.88±6.99 ab 35.00±6.06 ab 22.63±3.65 b 40.75±6.94 a clinical theriogenology • volume 9 number 1 • march 201735 clinical theriogenology • volume 9 number 1 • march 2017 36 omniblank: 1 contact paul mennick pacintgen@gmail.com © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommerical use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9784, http://dx.doi.org/10.58292/ct.v15.9784 technique report recovery, storage, and preparation of epididymal and vas deferens sperm for insemination, cryopreservation, and shipping paul mennick pacific international genetics, los molinos, ca, usa abstract the current technology allows preservation of sperm from testes and the distal reproductive tract if harvested after death determined largely by environmental and handling conditions (e.g. how long sperm remain viable within their nurturing structures postmortem) after death. pregnancies are established using non-ejaculated and even immotile sperm with appropriate preparation. there are very simple techniques available that can be employed by any practitioner to recover and prepare sperm from testes and the distal reproductive tract. this paper focuses on techniques for recovery, storage, and preparation of epididymal and vas deferens sperm, and techniques for shipping and cryopreservation. keywords: epididymis, vas deferens, postmortem, preparation, sperm, storage introduction one of the more rewarding reproductive services that practitioners can offer their clients at the difficult time of euthanasia is sperm recovery from a valuable stud for cryopreservation. under certain circumstances, this can also be successful after an untimely death. for example, goat sperm were still viable 3 days postmortem.1 general practitioners do not need any specialized equipment, extender, or supplies in order to provide this service. intact epididymides and vas deferens can be surgically removed easily and shipped to other facilities set up for cryopreservation or insemination. in many cases, sperm can be extended and cooled or held within these structures without processing for insemination a week later without cryopreservation. excellent reviews on epididymal sperm preservation and fertility are available.2,3 pregnancies have been obtained using cryopreserved epididymal sperm in bulls and stallions, and embryos from cryopreserved epididymal boar  sperm.4–6 in european mouflon rams, sperm obtained  from the cauda epididymis resulted in higher pregnancy rates and embryo survival compared with ejaculated sperm.7 sperm can also be obtained from testes; however, they are immotile. sperm within the caput epididymis of most mammals are also immotile. intracytoplasmic sperm injection (icsi) is a technique wherein a single sperm is directly injected into the oocyte cytoplasm. for icsi, sperm need not be motile to achieve pregnancies.8 in humans, testicular sperm obtained via biopsy are used to establish pregnancies following icsi.9 human sperm recovered via percutaneous epididymal aspiration has also resulted in pregnancies after icsi.10 in veterinary practice, unejaculated sperm can be obtained after removal of the entire testis or from the accessory glands, particularly, the ampullae of stallions or ruminants. epididymal sperm maturation sperm mature as they are transported through the epididymis (transit takes ~ 10 days for most species). during epididymal transit, sperm become motile, shed cytoplasmic droplets, undergo acrosomal membrane change, and other osmotic and chemical changes.11,12 although the capability of sperm to fertilize an oocyte increases as they enter the cauda epididymis, the author does not discriminate between specific regions of the epididymis during sperm recovery and combines all epididymal sperm (cauda, corpus, and caput). *presented at the 2022 society for theriogenology conference (canine symposium), published after peer-review. mailto:pacintgen@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9784 2 citation line: clinical theriogenology 2023, 15, 9784, http://dx.doi.org/10.58292/ct.v15.9784 sperm recovery the method selected for sperm recovery depends upon: 1. intended disposition of the sperm (e.g. preservation, immediate insemination); 2. client’s desire for obtaining the most insemination doses possible (e.g. including immotile sperm from testes for icsi); and 3. the amount of sperm present within the reproductive tract. the procedure to recover sperm from the reproductive tract begins with a modified castration procedure. special care is necessary to remove the entire epididymis attached to each testis and dissect up into the inguinal canal to retrieve as much of the vas deferens as possible in one piece. the distal cut end of the vas deferens should be ligated to prevent sperm loss. for large animals, the proximal end of the caput epididymis should also be ligated and the testis separated. for small animals, testes can remain attached provided they will fit in the transport container. if there is no attempt to preserve testicular sperm for icsi, testes are not shipped. sperm recovery from vas deferens recovery of sperm from vas deferens can be accomplished using manual expression (figures 1–3). after removing small blood vessels that run closely parallel to the vas deferens, the vas deferens is cut into ~ 5 cm segments in a petri dish. the sterile cover of a culture plate can be used if a petri dish is not available. holding one end of the vas deferens segment closed, the contents from the vas deferens are stripped out of the other end into the dish. the vas deferens contents are thick (like batter). semen extender should be used to rinse the vas deferens contents out of the dish and into a centrifuge tube. if sperm cryopreservation is planned, the vas deferens contents should be diluted directly with an extender containing a cryoprotectant. this approach would not stimulate motility prior to freezing. however, there is an experimental possibility of stimulating sperm motility by adding heterologous seminal plasma as in other species (e.g. ram).13 sperm recovery from epididymis sperm recovery from the epididymis is more challenging given its convoluted tubule construction. manual expression can be accomplished but requires more patience and repeatedly stripping the epididymis from the proximal to distal direction for several minutes. this method leaves a substantial number of sperm in epididymis but the majority of sperm recovered will be from the cauda epididymis. after manual expression, the epididymis can be flushed by catheterizing a tubule in the caput epididymis using an appropriately sized needle and all-plastic syringe containing semen extender. cauda epididymis should be held vertically with the tip of a mosquito forceps over a petri dish. after flushing, repeating manual expression of the epididymis might recover more sperm. if more complete recovery is desired, the epididymis can then be minced in a petri dish. epididymis should not be minced until the serous membrane covering the epididymis and any prominent blood vessels adjacent to the epididymis are removed. while blood itself is not spermicidal, breakdown products of blood (e.g. degraded hemoglobin) is spermicidal, so efforts should be made to minimize blood contamination. the epididymis is then minced into 2–3 mm segments. in author’s experience, a stiff-backed office razor blade is more effective at mincing than a scalpel blade. extender is then added to epididymal segments and the mixture is stirred for a couple of minutes. the mixture should be poured through a semen filter into a graduated cylinder to filter out as much of the nonsperm cellular components as possible. additional extender should then be used to rinse the petri dish and rinse the filter to recover remaining sperm. finally, the recovered sperm in extender should be centrifuged. it is important to note that the figure 1. intact pair of canine testes and intact spermatic cords resected to the level of the abdominal wall from a deceased patient. note the thin, pale vas deferens parallel to the rest of the spermatic cord, and the suture tying off its distal end to prevent sperm leakage. http://dx.doi.org/10.58292/ct.v15.9784 citation line: clinical theriogenology 2023, 15, 9784, http://dx.doi.org/10.58292/ct.v15.9784 3 addition of prostatic fluid to epididymal canine sperm prior to freezing can significantly increase pregnancy rates.14 shipping tissues for delayed sperm recovery if a practitioner is not able to perform sperm recovery, vas deferens, epididimydes, and testes can be shipped to another facility for sperm recovery. as mentioned, the distal cut end of vas deferens should be ligated to prevent sperm loss. for large animals, epididymis proximal end should also be ligated and the testis separated. prior to shipping, isolated vas deferens and epididymides should be rinsed with sterile saline and packaged inside a moist whirl-pak or ziploc bag excluding excess air. reproductive organs should be then packed in a semen shipping container or, if unavailable, a styrofoam vaccine shipping container with a frozen ice pack on both the top and bottom. reproductive organs should be wrapped figure 2. intact vas deferens (horizontal structure in foreground) as it is stripped away from the rest of the spermatic cord. paper towel can be used to absorb any blood from the surface of the vas deferens upon removal. intact spermatic cord with attached vas deferens clearly visible in the background awaits processing. figure 3. drops of expressed vas deferens and epididymal sperm with and without blood contamination in a petri dish. an extender of choice is added to centrifuge away as much blood as possible (if necessary) and a glycerol-containing freeze medium is then used for cryopreservation. http://dx.doi.org/10.58292/ct.v15.9784 4 citation line: clinical theriogenology 2023, 15, 9784, http://dx.doi.org/10.58292/ct.v15.9784 in ~ 1 inch of bubble wrap or roll cotton to insulate tissues from direct contact with ice packs. if immediate shipping is not possible, then reproductive organs should be stored in a refrigerator and prepared for shipping (as described earlier). if the receiving organization has specific instructions for shipping the reproductive organ, those should be followed. sperm recovery for cryopreservation and insemination can then be undertaken upon receipt. preparation for insemination without shipping or preservation if sperm can be used for insemination within a week after recovery, isolated vas deferens and epididymides should be rinsed off with sterile saline after closing the ends with mosquito forceps. use a litter bag with either saline or lactated ringer’s solution for placing the organs. cut the top of the bag, expel some fluid to accommodate organs, and add twice the recommended dose (mg/kg) of antibiotic (either ceftiofur or penicillin/gentamicin), place the organs, roll down top and hold shut with mosquito forceps and stand upright in back of a refrigerator. the litter bag provides good insulation, allowing for a slow, steady cooling rate. saline also prevents excessive desiccation of the tissues. antibiotics inhibit bacterial growth and prevent premature tissue decomposition. tissues should be placed in a refrigerator as soon as possible to start the cooling process. because sperm are still within the reproductive tract, their very low metabolic state is not disturbed. when needed for insemination, sperms are removed from the vas deferens and epididymides, diluted with an extender appropriate for the species and evaluated (for motility and concentration) to determine the volume needed for each insemination dose. it is important to note that the motility of the unextended caudal epididymal/vas deferens sperm is not progressive, but rather has a slight vibratory motion. sperm will become motile after dilution with semen extenders or homologous seminal plasma. in author’s experience, motility development might be almost instantaneous or may take several minutes. the author has used this method to produce foals from a stallion that died a week before mares were to be inseminated. it is possible that sperm may retain their fertilization ability for substantially longer using this in situ storage method. semen extenders for flushing, extending, filtering, and centrifuging the collected sperm, any cryoprotectant-free medium (homemade or commercial) that is appropriate for the given species can be used. this is also true for the extender containing a cryoprotectant.15 there are individual biological differences in post-thaw motility among various extenders. for example, this author recently collected and processed sperm from a 25-year-old arabian stallion that was euthanized because of a shattered hock. the stallion had small, firm testes at euthanasia and had not bred or been collected in more than 3 years. as described earlier, sperm were collected from vas deferens and epididymides and pooled together. sperm sample was divided into equal aliquots and extended in two commonly used equine centrifuging media. extended sperm were slow-cooled over 90 min and then gradually mixed with the freezing medium (the same freezing medium used for both aliquots). aliquots were frozen simultaneously on the same rack. post-thaw motility of the two aliquots differed significantly, with < 1% progressive motility (still presumably useful for icsi) in one aliquot and 35% progressive motility (also vigorous) in the other. the latter aliquot yielded more than 11 commercial quality doses of at least 300 × 106 postthaw motile sperm. conclusion postmortem preservation of sperm is not only feasible but also pregnancies are possible using available recovery and shipping techniques that any practitioner can perform. when confronted with an impending or unexpected loss of a valuable stud, practitioners should inform their clients that sperm collection and preservation are possible. conflict of interest none to declare. references 1. pangetsu m: viability of goat epididymal sperm up to three days after death. proceedings of the 4th international meeting biotechnology animal reproduction, bogor, indonesia 1997; ps-07. 2. stilley k: survival of canine epididymal sperm under cooled and frozen-thawed conditions. louisiana state university master’s thesis 3830. agricultural and mechanical college; 2001. 3. hassan h, domain g, luvoni g, et al: canine and feline epididymal semen – a plentiful source of gametes. animals (basel) 2021;11:2961. https://doi.org/10.3390/ani11102961 4. barker ca: low temperature preservation of bovine epididymal spermatozoa. can j comp med vet sci 1954;18:390–393. 5. barker ca, gandier jc: pregnancy in a mare resulting from frozen epididymal spermatozoa. can j comp med vet sci 1957;21:47–51. 6. nagai t, takahashi t, masuda h, et al: in-vitro fertilization of pig oocytes by frozen boar spermatozoa. j reprod fertil 1988;84: 585–591. https://doi.org/10.1530/jrf.0.0840585 7. martinez-fresneda l, castano c, boveda p, et al: epididymal and ejaculated sperm differ on their response to the cryopreservation and capacitation processes in mouflon (ovis musimon). sci rep 2019;9:15659. https://doi.org/10.1038/s41598-019-52057-0 8. nagy zp, liu j, joris h, et al: the result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. hum reprod 1995;10:1123–1129. https://doi.org/10.1093/ oxfordjournals.humrep.a136104 9. bourne h, watkins w, speirs a, et al: pregnancies after intracytoplasmic injection of sperm collected by fine needle biopsy of the testis. fertil steril 1995;64:433–436. https://doi.org/10.1016/ s0015-0282(16)57747-4 10. lin y, huang l, seow k, et al: intentional cryopreservation of epididymal spermatozoa from percutaneous aspiration for dissociated intracytoplasmic sperm injection cycles. acta obstet gynecol scand 2004;83:745–750. https://doi. org/10.1111/j.0001-6349.2004.00374.x 11. franca l, avelar g, almeida f: spermatogenesis and sperm transit through the epididymis in mammals with emphasis on pigs. theriogenology 2005;63: 300–318. https://doi.org/10.1016/j. theriogenology.2004.09.014 http://dx.doi.org/10.58292/ct.v15.9784 https://doi.org/10.3390/ani11102961 https://doi.org/10.1530/jrf.0.0840585 https://doi.org/10.1038/s41598-019-52057-0 https://doi.org/10.1093/oxfordjournals.humrep.a136104 https://doi.org/10.1093/oxfordjournals.humrep.a136104 https://doi.org/10.1016/s0015-0282(16)57747-4 https://doi.org/10.1016/s0015-0282(16)57747-4 https://doi.org/10.1111/j.0001-6349.2004.00374.x https://doi.org/10.1111/j.0001-6349.2004.00374.x https://doi.org/10.1016/j.theriogenology.2004.09.014 https://doi.org/10.1016/j.theriogenology.2004.09.014 citation line: clinical theriogenology 2023, 15, 9784, http://dx.doi.org/10.58292/ct.v15.9784 5 12. marchiani s, tamburrino l, muratopri m, et al: epididymal sperm transport and fertilization. in: simoni m, huhtaniemi it, editors. endocrinology of the testis and male reproduction. new york city; springer publishing; 2017, p. 457–478. 13. mataveia ga, terblanche sj, nöthling jo, et al: effect of heterologous seminal plasma and semen extenders on motility of frozen-thawed ram spermatozoa. j s afr vet assoc. 2010;81:139–142. https://doi.org/10.4102/jsava.v81i3.135 14. hori t, hagiuda k, kawakami e, et al: unilateral intrauterine insemination with prostatic fluid-sensitized frozen caudal epididymal sperm in beagle dogs. theriogenology 2005;63:1573–1583. https://doi.org/10.1016/j.theriogenology.2004.05.027 15. galarza d, landi g, mejia e, et al: cryopreservation of dog epididymal spermatozoa by conventional freezing or ultra rapid freezing with nonpermeable cryoprotectant. cryobiology 2021;103:15–21. https://doi.org/10.1016/j.cryobiol.2021.10.002 http://dx.doi.org/10.58292/ct.v15.9784 https://doi.org/10.4102/jsava.v81i3.135 https://doi.org/10.1016/j.theriogenology.2004.05.027 https://doi.org/10.1016/j.cryobiol.2021.10.002 2011: quality breeding practices quality breeding practices margret l. casal school of veterinary medicine, university of pennsylvania, philadelphia, pa the ultimate goal of breeding is to produce the best dog or cat that fits the breed standard or the desired purpose. in cats that may mean hair length or quality, eye color, hair color or even body structure. in dogs, we expect perfection not only in physical traits but also in their abilities to hunt, retrieve, point, show, herd, race and more. as most breeders know, producing these high quality animals requires hard work and dedication that starts well before the actual breeding. the environment in which the breeding animals are to be raised and bred is very important. breeding rooms and areas should be kept quiet, bright, clean, and well ventilated. lighting is especially important when breeding cats, as insufficient lighting leads to suppression of heat cycles. overcrowding leads to disease outbreaks because the animals are stressed. cleaning agents need to be carefully evaluated, as they may be toxic, especially for puppies and kittens. beds, bins, cages, runs, and whelping boxes are chosen that can be sanitized easily and that are safe with no possibility of injuring the mother or the offspring. breeding animals should not be exposed to feral, wild or homeless animals (e.g., for runs that are outdoors, an extra chain linked fence about two yards away from the actual enclosure prevents wild animals from coming into contact with the breeding dogs and their offspring). especially during whelping or queening and when puppies and kittens are present, visitors should be kept at a minimum, as people may unintentionally track diseases in with them. lastly, when breeding dogs, it is important to know the state laws. some states have caps on how many dogs are allowed in a breeding kennel; some states have flooring requirements (grates vs. no grates); and some states have different designations for breeders depending on how many litters they produce per year (commercial vs. hobby breeder). the law school of michigan state university has a handy online tool to look at state laws:(www.animallaw.info/articles/armpusbreedinglaws.htm). puppies and kittens destined to become breeding animals should be raised with good quality american association of feed control officers-approved foods or foods that have been formulated to fit these high standards. it is sometimes difficult to understand pet food labels, even for those who are trained to do so. explanations can be found on the food and drug administrations webpage: www.fda.gov/animalveterinary/resourcesforyou/ucm047113.htm. vaccinations and treatment for parasites, both internal (roundworms, tape worms, etc) and external (fleas, heartworm, ticks, etc) are important to prevent serious diseases and to maintain a healthy breeding stock. vaccination guidelines are listed on the american animal hospital association’s website (www.aaha.org) for dogs and on the american association of feline practitioners’ site (www.aafp.org) for cats. these are all issues that should be discussed with your veterinarian. next, we want to make sure that we begin with high quality breeding material. all of the breed specific health checks should be performed before planning to breed. for example, most larger breeds of dogs should have their hips checked by either the orthopedic foundation for animals or pennhip; dobermans would be screened for von willebrands disease; portuguese waterdog dna is checked for juvenile dilated cardiomyopathy; dna is submitted from a variety of breeds for different forms of progressive retinal atrophy; and the list goes on and on. veterinarians cannot be expected to know all of the diseases that each breed should be screened for, but breeders generally are aware of their breedspecific diseases. for up-to-date information, there are resources for breed specific recommendations, such as the american kennel club’s website (www.akc.org) or the orthopedic foundation for animal’s site (www.offa.org) for dogs and the cat fanciers association (www.cfa.org) or the dna testing laboratory at texas a&m (www.catdnatest.org) for cats. now that we have successfully raised a female or male that we would like to breed and it has reached sexual maturity, a pre-breeding examination should be performed. this consists of testing for brucellosis in dogs (a disease that can shut down an entire breeding facility) and a good physical examination with special attention to the external reproductive organs. in male dogs, semen should be evaluated well before the dog is bred for the first time and around the time of breeding again. in female clinical theriogenology • volume 3 number 4 • december 2011537 dogs, breeding timing is a must, as improper timing is the most common cause of the bitches failing to “take”. our motto is: “put excellent quality semen into a fertile female at the right time for the best results.” accurate breeding timing is achieved through vaginal cytology, progesterone measurements, and luteinizing hormone measurements if needed. once the bitch or queen has been bred, if desired, pregnancy can be determined after about 21 days by palpation, ultrasound or in dogs by measuring relaxin levels, although this may not be accurate this early in pregnancy or if only one pup is present. after 28 days, palpation is not easily possible in dogs, as the single fetal units can no longer be distinguished and the uterus “just feels big”. however, at this time the relaxin test becomes much more sensitive and ultrasound is much easier. after 45 days of pregnancy, radiographs will help determine how many puppies or kittens are present, which is not readily possible with ultrasound. during pregnancy medications should not be given unless absolutely necessary. towards the second half of pregnancy, the bitch’s and queen’s diet should be supplemented with puppy and kitten food, respectively, to cover the increasing needs of the mothers and their offspring, as well as future milk production. raw food diets may not be the ideal choice, as parasites and bacteria may be present leading to fetal malformations, disease and death. lastly, keeping track of the offspring long after they have been sold or transferred provides useful feedback on soundness of the animals. if genetic diseases are detected down the road, it is helpful to seek discuss with a veterinary genetic counselor to avoid this from happening again. most important though, finding that the animals that were bred produced a healthy and long-lived pet that has become someone’s family member and has provided the family with years of happiness is priceless. clinical theriogenology • volume 3 number 4 • december 2011 538 2013: balanoposthitis and paraphimosis in the stallion. a novel support for an inflamed penis and prepuce balanoposthitis and paraphimosis in the stallion. a novel support for an inflamed penis and prepuce a.j. gunn,a,e v.j. brookes,a a.d.j. hodder,b j. rodger,c j.b. chopind agunn and brookes veterinary service, scone, nsw, australia; bedgewater equine clinic, santa cruz, ca; crodger and associates, jerry’s plains, nsw, australia; dtamarang veterinary breeding services, tamworth, nsw, australia; eschool of animal and veterinary science, charles sturt university, wagga wagga, nsw, australia abstract balanoposthitis with associated paraphimosis is an uncommon medical emergency in a stallion. immediate support of the prolapsed tissues in conjunction with associated medical and physical treatments is paramount for a successful outcome. this reliable method of support that can be readily, quickly, easily and inexpensively applied to all cases of prolapsed preputial and penile tissues by a practitioner in the field. this report illustrates a method using a pair of underpants, baler twine and other readily available materials to support the swollen prepuce and penis of four stallions that suffered traumatic injury to their external reproductive organs. keywords: balanoposthitis, paraphimosis, inflammation, penis, prepuce, stallion introduction a pendulous and protruding penis and/or prepuce in the horse is typically the result of inflammation. inflammation of the free body or glans of the penis is known as balanitis, and inflammation of the laminae of the prepuce is known as posthitis.1 as they often appear as a combined entity, the condition often presents clinically as balanoposthitis.2,3 priapism is characterized by an erection that persists in the absence of sexual stimulation while paraphimosis is characterized by the inability to retract the penis into the preputial cavity. all have a similar clinical presentation. due to the different treatment options, the definitive diagnosis of a pendulous penis and/or prepuce is more than an academic exercise.4 to prevent the inevitable cycle of inflammation that is likely to occur, supporting the organs is an important part of the treatment regimen. balanoposthitis, as a result of the associated swelling of the penis and decrease in size of the preputial orifice, results in paraphimosis. prolonged protrusion impairs venous and lymphatic drainage exacerbating preputial edema, particularly swelling of the relatively inelastic internal preputial ring. an inflammatory cycle is induced.3 the primary aim in the treatment of balanoposthitis is to reduce inflammation. ensuring the continued integrity of the urogenital tract including urethra and integument, and prevention or control of any associated infection are ancillary objectives. the normal anatomy is illustrated in figure 1.5 attempts to limit potential behavioral aberrations induced by a traumatic event is an ancillary aim in a breeding stallion. diazepam has been reported to reduce anxiety, possibly induced by traumatic incidents in some breeding stallions.6 there are a number of etiologies of balanoposthitis. by far the most common is acute and often blunt trauma from breeding incidents,2,7 typically a kick by the mare. there are reports of methods of supporting the pendulous organs including nylon mesh, firm support methods such as the use of modified plastic bottles, pantyhose, proprietary supports,2 purse-string sutures8 and the use of a probing.9 beltaire et al report the application of a non-adherent dressing, wrapped by elastic bandage material supported by the application of a mesh sling.10 this report describes the use of underpants (briefs, ‘y’-fronts’), baler (or other ) twine, rubber tubing, and a neck strap or surcingle to quickly, easily, safely and effectively support a stallion’s swollen penis and prepuce. this support procedure was used on four stallions described in the following case summaries. 45 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 figure 1. diagram illustrating the normal sagittal cross-section of the prepuce and penis. (from o’brien et al.5; used with permission) case reports case 1 a ten year old thoroughbred stallion was kicked by a mare in a breeding barn accident. the penis and prepuce were immediately treated with cold hydrotherapy for 20 minutes. on examination the penis and prepuce were obviously swollen and protruding from the preputial orifice. other clinical parameters were within normal limits. the stallion was sedated with intravenous xylazine (200 mg) and butorphanol (20 mg), and the swollen area more closely examined (figure 2). edema was visually and palpably obvious, which was reduced with manual pressure. there was no obvious free fluid present. the area was scanned ultrasonographically using a 5 mhz linear probe. no obvious abnormalities were detected in the integrity of the penis, no free fluid was detected, and preputial edema was visualized. the stallion was treated with intravenous flunixin meglumine (600 mg), and intramuscular procaine penicillin (15,000,000 iu). manual reduction of the swelling was initiated after a further 10 minutes of cold hydrotherapy. the swelling reduced appreciably after massage, but was insufficient to permit return the penis and remaining prepuce through the preputial orifice. lanolin ointment was liberally applied to the affected area. a support mechanism was difficult to obtain quickly and easily. the use of underpants, supported by soft bandaging and attached to a crudely fashioned neck strap was instigated (figure 3) and the stallion was re-examined the following day. the following day the horse was again sedated and his condition reassessed. the balanoposthitis was no worse. the prolapsed tissues were treated with cold hydrotherapy and massaged to reduce the dependent edema; emollient ointment was liberally applied to the exposed skin, and the “underpants support mechanism” reapplied. medical treatment consisted of procaine penicillin (as above) and oral phenylbutazone (2 g) twice daily. the horse was hand walked for 10 minutes at a time four times daily, ensuring the support mechanism remained in the correct position. he was not exposed to mares. the support was removed in the evening, followed by cold hydrotherapy, massage, emollient ointment application, and replacement of the support mechanism. on the third day, the swelling was minimal. after hydrotherapy and massage, the penis and prepuce were retracted through the preputial orifice by the stallion. the support was not replaced during the day, and the stallion was hand walked as on the previous day. in the evening, hydrotherapy, massage, emollient application, and replacement of the support was carried out in conjunction with continued medical treatment. by the morning of the fourth day, the swelling was markedly reduced, and paraphimosis was no longer evident. hydrotherapy, massage and emollient application were carried out twice daily. 46clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 the stallion was allowed into his small yard for the day, and stabled overnight. no further paraphimosis occurred. the antibiotic treatment was discontinued, and the dose of phenylbutazone decreased to 1 g twice daily. hydrotherapy and emollient application continued twice daily. on the fifth day erection was induced under veterinary supervision in the presence of a mare. the penis, although slightly swollen was easily protruded and retracted. erection was induced in the presence of a mare for another five days, with continued improvement in the integrity of the penis and prepuce. medical treatment terminated at this point. ten days after the initial insult the stallion served a mare with no detrimental effects. dismount samples11 to confirm ejaculation contained motile spermatozoa. two days later he was serving twice daily, and two days after that he was serving as necessary. two weeks later pregnancy rates of approximately 65% per cycle, similar to those prior to the incident, were detected in the mares he had served. figure 2: left side view of case 1 showing the enlarged and swollen penis and prepuce protruding from the preputial orifice. figure 3. view from the front left side of the horse in case 1. the supporting bandages are attached to the neck strap. the horse is shown urinating through the underpants. 47 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 case 2 in 2005, a maiden four year old thoroughbred stallion that was being schooled for the breeding season was presented due to the sudden onset of swelling of the penis and prepuce after mating. the stallion had not been kicked and the swelling was noticed immediately after copulation. a putative diagnosis of superficial blood vessel rupture or shearing trauma to the erect penis was made. cold hydrotherapy, application of ice packs, and massage with emollient antibiotic ointment (nitrofurazone cream, ranvetpl, botany, nsw) was instigated immediately. the stallion was treated with diazepam (20 mg) and butorphanol (20 mg) within 20 minutes of the occurrence. ultrasonographic examination of the penis did not reveal any free fluid, and the penile tunica albuginea was intact. edema and swelling were obvious on ultrasonography. flunixin meglumine (750 mg) was administered intravenously, and procaine penicillin (15,000,000 iu) intramuscularly. due to the balanoposthitis, associated paraphimosis was evident, which was not reduced after treatment. the “underpants support mechanism” was applied. on this occasion it was attached to a surcingle with a breast plate. the stallion was able to retract the penis and prepuce within the preputial cavity without assistance on the third day. on the fifth day erection was induced under veterinary supervision in the presence of an estrus mare. similar to case 1, supervised erection was induced for another four days. the stallion was serving mares ten days after the original incident. there was no recurrence of balanoposthitis. pregnancy rates 14 days after mating were 50% per cycle. dismount samples revealed the presence of motile spermatozoa. case 3 a pony teaser stallion was kicked on the erect penis by a maiden broodmare while she was being assessed for suitability for breeding. this resulted in the sudden onset balanoposthitis and associated paraphimosis. he was immediately treated with 20 mg diazepam and 350 mg flunixin meglumine intravenously. the penis and prepuce were treated with cold hydrotherapy for 15 minutes. examination revealed a swollen and edematous penis and prepuce. ultrasonography confirmed the presence of preputial edema, absence of free fluid and lack of damage to the penis. massage, application of emollient antibiotic ointment, and application of the “underpants support mechanism” followed the initial examination and treatment. systemic antibiotic and anti-inflammatory drugs were administered. hydrotherapy, massage, and application of the support mechanism continued in conjunction with medical therapy for three days. on the fourth day preputial and penile swelling was minimal, and the stallion was able to fully retract the penis and prepuce into the preputial cavity. he was able to obtain an erection and to retract his penis unsupported. after a two week break, the teaser returned to his duties with no apparent physical or behavioral abnormalities. case 4 an approximately five year old 500 kg australian stock horse stallion was presented having been found in the paddock adjoining an “in season” mare, with a very large and swollen penis and prepuce(figure 4). there was also an obvious wound on the left dorsal mid-shaft of the penis. the stallion appeared to have jumped into the paddock adjoining the mare, and attempted to serve her through the fence. 48clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 figure 4: left view of the inguinal area of case 4 following cold hydrotherapy. there is obvious swelling of the penis and prepuce. there are signs of abrasions and hemorrhage. the swollen penis and prepuce could not be retracted through the preputial orifice. initial treatment was cold hydrotherapy for approximately 20 minutes. when examined approximately three hours later, the stallion was bright, alert and responsive with obvious balanoposthitis resulting in paraphimosis. clinical examination was otherwise unremarkable. the stallion was sedated with 20 mg romifidine and 20 mg butorphanol, and 750 mg flunixin meglumine was administered intravenously. procaine penicillin (10,500,000 iu) was administered intramuscularly. the penis was extended with manual traction, palpated, examined visually, and scanned ultrasonographically (figure 5). there was an obvious full-thickness wound on the left dorso-lateral aspect of the internal preputial lamina at the level of the internal preputial ring. the wound was left to heal by second intention. ultrasonography did not reveal any free fluid, the penile tunica albuginea was intact, and edema was obvious. massage was instigated to decrease the edema and assist in returning the penis to the prepuce. the penis was cleaned with water and very dilute iodine surgical scrub, rinsed and dried. emollient antibiotic ointment was applied to the swollen area. figure 5: ultrasonogram of a cross section of the protruding penis and prepuce of case 4 at initial presentation (top is ventral.) the integrity of the penile tunica albuginea appears intact. there is obvious preputial edema evident between the calipers3.24 cm. skin urethra tunica albuginea of penis 49 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 the “underpants support mechanism” was applied to this horse. he was hand walked four times daily, and treatment was similar to that in the cases described above. the support was deviated to the side twice daily for hydrotherapy, and application of the antibiotic ointment. the support was changed daily for three days. on the fourth day, the stallion was able to retract his penis and prepuce into the preputial cavity. the support was removed as it was no longer required. daily hydrotherapy of the penis and prepuce, and antibiotic ointment application continued for seven days. after obtaining an erection the stallion was able to retract his penis into the preputial cavity. the wound had granulated, epithelialization was occurring, and contraction of the area was evident. a triangular wound without any adhesions was evident (figure 6). erection was induced daily for two weeks. the penile wound was substantially reduced and hemorrhage was not evident when the stallion served a mare that was used successfully as an embryo donor. figure 6: extended penis of case 4 approximately two weeks after the insult. the wound at the distal end of the inner preputial fold is showing signs of granulation, epithelialization and contraction. materials and methods • pair of men’s (or possibly ladies’) underpants (briefs/y-fronts) • baler or other twine or rope (approximately 9 m depending on the size of the horse) • a surcingle with chest strap or a broad neck strap • rubber tubing (optional) • scissors • elastic bandage (optional) (figure 7) 50clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 figure 7: materials (top to bottom, left to right): girth strap or surcingle; chest straps; underpants, scissors, elastic bandage (optional); baler (or other) twine; rubber tubing (optional). four small holes are cut through the material of the underpants. one hole on each side of the ‘gusset’ between the leg openings of the underpants, ensuring the stronger elastic material is preserved. the cord is attached to this stronger material. one hole is made on each outside edge of the waistband, maintaining the integrity of the reinforced elastic such that the baler twine can be attached to the four corners of the underpants. the strings attached to the gusset area are longer (approx. 2.5 m) than those attached to the waist (approx. 2 m; figure 8). figure 8: underpants laid out with bandaging as straps. 51 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 the surcingle and breast band or neck strap, are then applied to the stallion. the gusset strings are then pulled caudally and dorsally between the legs of the stallion, with one string on each side of the tail, along the dorsal midline, and loosely tied to the surcingle or neck strap. the gusset of the underpants should be approximately at the level of the scrotum. the strings from the waist part of the underpants are attached to either side of the surcingle or neck strap, travelling cranio-dorsally from the caudal-ventral abdominal area. this is usually well tolerated by the horse, however safety of the personnel attaching the support is paramount. the position of attachment and the length of the strings are altered as deemed necessary for the underpants to provide the required support for the prolapsed penis and prepuce. with correct manipulation the underpants can be made to fit snugly under the swollen organs. the strings from between the legs that run cranially along the dorsum can be tied together along the midline. some rubber tubing can be used to attach the cranial diagonal supports to the dorsal supports or to the girth strap to alter the angle, thereby enhancing support. this also allows flexibility of the supporting mechanism (figure 9). the knots are then secured with the underpants in the desired position (figures 10 and 11). figure 9: illustrating the position of the gussett straps between the legs, over the dorsum to be attached to the girth strap. 52clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 figure 10: attached “underpants support mechanism”. figure 11: close-up of the applied underpants showing the snug and uplifting support. discussion protrusion of the penis and/or prepuce from the preputial orifice is a medical emergency in the stallion. authors of published reports1-4,8-10 are unanimous in recommending immediate support of the inflamed penis and prepuce when balanoposthitis with associated paraphimosis is evident, especially as a result of a traumatic incident. fortunately these incidents are not common in breeding stallions or other intact males. supporting the swollen penis and prepuce is paramount in decreasing the inflammation that occurs with stagnation of edema and other fluids.2,3 supporting the organs decreases the chances of further trauma to the exposed and swollen tissues. balanoposthitis is uncommon in neutered males, however this support mechanism could be used in geldings with a protruding and/or pendulous penis. this support mechanism could also be used in a horse with priapism. appropriate medical treatment is essential when dealing with paraphimosis. the primary benefit of the support mechanism used in the cases described in this report is the ease and speed with which it was made. support can be applied immediately using materials readily available on the farm (and personnel). the underpants material is usually cotton or cotton based and is soft and porous. this allows the stallion to urinate unhindered, but the material is sufficiently resilient to support the 53 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 swollen organs without any apparent discomfort to the stallion.2 the material is relatively inexpensive, readily available and replaceable with a clean new support as often as required. urine scalding and abrasions were not noticed in the cases in which it was used. the size of underpants is not important, however medium or large size tends to offer the best compromise between support and stability. the potential effects of inflammation and hyperthermia of the testes is another important consideration regarding the future fertility of the stallion.12 twelve hours of hyperthermia have been shown to affect spermatozoal function. limiting inflammation in the surrounding tissues diminishes the likelihood of testicular hyperthermia. hydrotherapy, physiotherapy and the use of hypertonic (hyperosmotic) agents such as sugar, salt or glycerin may assist in reducing edema.3 the use of these agents would not preclude the use of the “underpants support mechanism”. purse-string sutures around the preputial orifice have been advocated by some authors,8 whereas others2 advise caution as sutures may induce further inflammation and possibly result in abscesses. the probang device9 is limited to cases in which the penis and prepuce can be replaced through the preputial orifice. this method could be used after preputial swelling has been reduced following the use of the support mechanism. the success of retention bandaging10 may be limited depending on the size and temperament of the stallion. the “underpants support mechanism” was well tolerated by the patients described in this report. importantly, it does not appear to require full-time surveillance, although frequent surveillance is recommended. the device appears to maintain its position regardless of stallion behavior such as rolling. if the device does happen to move, it is unlikely to have any detrimental effects on the stallion, or the penis and prepuce. mesh support mechanisms2 are reportedly very useful, and can be purchased or homemade. however the materials are not always readily available at the time and place of need. immediate support is paramount for successful of treatment. the use of pantyhose has many advantages, including availability and they are made of porous non-irritant material. however, the material is slippery and tends to evolve into a rope-like structure when placed under tension to elicit support of the protruding penis and/or prepuce. as a result they tend to slip off to the side of the swollen organs. firmer materials such as modified plastic bottles or planks of wood, have the disadvantage of easily slipping to one side when the stallion moves. the above mentioned drawbacks are minimal when using the “underpants support mechanism”, with all of the advantages of a snug and uplifting fit. treatment with diazepam as soon as possible (preferably within 20 minutes) after a traumatic occurrence is reported to alleviate or decrease aberrant behavior that may be induced in the stallion.6 this has been used in conjunction with butorphanol and/or alpha 2 adrenoceptor agonists to assist in managing stallions for examination and treatment. application of the support, especially the caudal strings, have potential risks to personnel which requires judicious management. the “underpants support mechanism” is an effective, inexpensive, readily available resource for a clinician dealing with a stallion with balanoposthitis and paraphimosis, or any protrusion from the preputial orifice requiring support. acknowledgement katrina partridge for most of the photographs. references 1 frazer gs: stallion reproductive emergencies. proc north am vet conf; 2008. p. 106-109. 2 vaughan jt: penis and prepuce. in: mckinnon ao, voss jl, editors. equine reproduction. philadelphia: lea and febiger; 1992. p. 885-894. 3 schumacher j, varner dd: abnormalities of the penis and prepuce. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. hoboken: wiley-blackwell; 2011. vol 1 p. 1130-1144. 4 mcdonnell sm: managing the paralysed penis, priapism or paraphimosis in the horse. equine vet educ 2005;17:310-311. 5 o’brien t, koch c, livesey ma, et al: direct preputial hernia associated with a ventral abdominal wall defect in a two-year-old gelding . equine vet educ2010; 22:321-325. 6 mcdonnell sm, kenney rm, meckley pe, et al: conditioned suppression of sexual behavior in stallions and reversal with diazepam. physiol behav 1985;34:951-956. 54clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 7 neely dp: physical examination and genital disease of the stallion. in: morrow da, ed. current therapy in theriogenology. philadelphia: wb saunders; 1980. p. 694. 8 boero mj: a simple technique for conservative therapy of acute traumatic paraphimosis in the hose. proc am assoc equine pract; 1990. p. 625-628. 9 koch c, o’brien t, livesey ma: how to construct and apply a penile repulsion device (probang) to manage paraphimosis. proc am assoc equine pract; 2009. p. 338-341. 10 beltaire ka, tanco vm, bedford-guaus sj: theriogenology question of the month. trauma-induced paraphimosis. j am vet med assoc 2011; 238:161-164. 11 gunn aj, brookes vj: neutrophil prevalence in dismount semen samples of thoroughbred stallions. clin therio 2011;3:477-481. 12 johnson l, blanchard tl, varner dd, et al: factors affecting spermatogenesis in the stallion. theriogenology 1997;48:1199-1216. (editor’s note: the photographs in this paper appear in color in the online edition of clinical theriogenology.) 55 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 2017: vaginal stricture as an unusual case of dystocia in a dairy cow   vaginal stricture as an unusual case of dystocia in a dairy cow allan j. gunn school of animal and veterinary sciences, and the graham centre for agricultural innovation, charles sturt university, wagga wagga, nsw, australia abstract dystocia (difficult birth) is a relatively common occurrence in bovine reproduction. in one study on dairy farms in the usa 51.2% heifers and 29.4% of cows required assistance at parturition.1 causes include abnormalities of the birth canal such as a narrow vagina. this is a report of a case of a stricture in the caudal vagina in a multiparous friesian/holstein cow leading to dystocia and delivery of the fetus by cesarean section. to the author’s knowledge, a vaginal stricture (‘pseudocervix’) has not been reported as a cause of dystocia in cattle. keywords: dystocia, cow, multiparous, vaginal stricture, pseudocervix case report a multiparous friesian/holstein cow in good body condition (3/5) had failed to deliver a calf after approximately 24 hours of what appeared to be the second stage of parturition. she had been bred by artificial insemination approximately 280 days prior to presentation. on examination it was not possible to palpate the calf due to a complete obstruction/stricture of the reproductive tract. the stricture was cranial to the vestibulo-vaginal sphincter, and was impenetrable despite considerable manual force and digital manipulation. fetal membranes and fluid were absent from the caudal reproductive tract. a small (approximately 1-2mm in diameter) aperture was palpable in the central region of the obstruction. per rectum examination confirmed the presence of a calf in the uterus; ultrasonographic evaluation was not performed in this case because of unavailability. it was not possible to determine the viability of the calf. initially it was assumed the stricture was due to an incompletely dilated cervix, as the calf was not palpable per vaginum. unfortunately, a speculum examination was not performed. in retrospect, it is unlikely that a speculum examination would have been beneficial at presentation. however, a vestibulo-vaginal examination postpartum or prior to breeding, would more likely have led to detection of this potential problem prior to impregnation of the cow. a decision was made to undertake a cesarean section in order to deliver the fetus, and determine the reason for the obstruction. a dead, but anatomically normal, fetus was removed via laparotomy and hysterotomy. gross external signs (cloudy cornea) suggested the fetus had been dead for longer than 12 hours based on reports on the changes associated with autolysis.2 the cow was subjected to euthanasia, as the prognosis for a live cow after caesarean section with a dead fetus is not good,3 and she was unlikely to conceive again. postmortem the reproductive tract was removed from the cow, and examined (figure). the cervix was open with the internal cervical aperture being approximately 5 cm in diameter, and the vagina was stained brown. it was coated by a brown colored amorphous substance that was easily removed. this material, presumed to be meconium, suggested that the fetal membranes had ruptured during dystocia. approximately 0.5cm cranial to the external urethral opening was a small (1-2mm) aperture within a thickened fibrotic vestibule caudal to the vestibulo-vaginal sphincter. the cranial vestibule was obviously bruised, probably as a result of iatrogenic trauma associated with the dystocia. grossly, sectioning the vestibule and the area of the stricture revealed firm, white, nonyielding tissue suggestive of fibrous tissue, approximately 5 mm thick in most places (figure 1). this stricture at the caudal vagina was the reason for dystocia in this cow. cervical causes of dystocia have been reported in the doe,4 the ewe,5 and in cattle.6-8 typically they are a manifestation of dystocia due to some other primary cause such as uterine inertia, malposition, malpresentation, or malposture of the fetus.9 relaxation of the vagina is an important part of the preparation for birth in the cow. the presence of scar tissue from a previous calving injury, congenital stenosis of the vagina, obstruction by embryonic remnants, a hymen, abscess or cyst formation have been reported as vaginal causes of dystocia.8 unfortunately the owner was unable to verify if the cow had a dystocia previously, and/or was difficult to inseminate for this pregnancy. clinical theriogenology • volume 9 number 1 • march 201789   to the author’s knowledge this is the first reported case of an impenetrable fibrotic vaginal stricture resulting in dystocia in a dairy cow. the reason for the stricture could not be ascertained by gross examination of the reproductive tract. histology was not carried out on this occasion and is unlikely to have elucidated the etiology. previous trauma to the caudal reproductive tract, such as from a previous dystocia, with fibrosis and contraction of the caudal vestibule is presumed to be the most likely cause of the stricture. this report adds another previously unreported differential diagnosis to the list of reasons for dystocia due to caudal reproductive tract obstruction in the cow. acknowledgements the author thanks dr. stewart scott and the owner for their assistance in this case; rob lofstedt and scott norman for assistance in assessing the article; and lee-anne mcinerney for her library assistance in preparing the document. no funding was required for this report, nor has it been presented at any meetings/conferences. references 1. lombard je, garry fb, tomlinson sm, et al: impacts of dystocia on health and survival of dairy calves. j dairy sci 2007;90:1751-1760. 2. dillman rc, dennis sm: sequential sterile autolysis in the ovine fetus: macroscopic changes. am j vet res 1976;37:403-407. 3. lyons na, karvountzis s, knight-jones tjd: aspects of bovine caesarean section associated with calf mortality, dam survival and subsequent fertility. vet j 2013;197:342-350. 4. braun jr w: parturition and dystocia in the goat. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: wb saunders; 2007. p. 555-558. 5. menzies pi: lambing management and neonatal care. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: wb saunders; 2007. p. 680-695. 6. wehrend a, bostedt h: species-specific significance of the cervix as a cause of dystocia. tierarztliche umschau. 2005;60:7-12. 7. azawi oi, naoman ut, al-kass zm, et al: misoprostol treatment of dystocia due to incomplete dilatation of the cervix in a cow: a case report. iraqi j vet sci 2011;25:97-98. 8. jackson pgg: handbook of veterinary obstetrics. 2nd ed. edinburgh: wb saunders ltd.; 2004. 9. norman s, youngquist r: parturition and dystocia. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: wb saunders; 2007. p. 310-335. figure. a photograph illustrating the caudal vaginal stricture (‘pseudocervix’) of the reproductive tract of the case presented with dystocia. there is obvious fibrosis of the vestibular walls and constriction of the caudal vagina. (left is cranial [uterine]). vulva: limits of vestibule: limits of vagina (cranial vagina): limits of the cervix: placentome (caruncle covered with cotyledon): internal opening of the stricture: urethral opening: thickened fibrotic wall of the vestibule: urinary bladder: (editor’s note: the photograph in this manuscript is available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 9 number 1 • march 2017 90 contact stuart meyers smeyers@ucdavis.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9221, http://dx.doi.org/10.58292/ct.v15.9776 the 2023 bartlett address the journey of a theriogenologist stuart meyers school of veterinary medicine, university of california, davis, ca usa it is truly an honor to be recognized by my professional association for my efforts in theriogenology. i thank my nominators and the boards of act, sft, and the foundation for this amazing honor and i humbly accept this year’s bartlett award, knowing that many other deserving members continue to humble me and amaze us all. i follow in the footsteps of many of my theriogenology heroes whom i have long admired and respected. [caution to the reader: from here forward, the reader should only continue if interested in excruciating detail about one person’s life story! i have detailed my journey in theriogenology for posterity but not for the typical reader.] my journey in theriogenology has been a convoluted trip with roots in my undergraduate studies at michigan state university in the state where i grew up. the trip is marked by many meaningful relationships and opportunities in which i received advice and opinions about career routes and strategies for success. the journey is also one of great humility. veterinary practice is a great humbler. some failures also marked the journey but i feel strongly that the roads not taken gave me as much meaning and experience as the roads i took. for some milestones, i had opportunities denied and for other milestones, my choices led me to great opportunity, random and deliberate choices, but at every step the people i have met turned into wonderful experiences, lifelong friendships, and deep rewards. it’s all about the people for me in theriogenology. friendships. great advice that bends the road traveled and not traveled. did i mention lots of opportunities for laughing? i have never been disappointed by the genuine warmth and support from our profession. my journey is partly marked by  the  faculty positions and residency opportunities that didn’t happen! this led me to a pivotal texas a&m university (tamu) opportunity and lifelong acquaintances, friendships and collaborations. faculty positions at csu, georgia, auburn, kentucky, and wisconsin, also didn’t pan out but in each case i met wonderful people in our profession. i didn’t realize it at the time but these roads not taken served to broaden and deepen my network in theriogenology for the long haul. getting into the therio zone … medicine my journey begins as an undergraduate student at michigan state university, where as relatively unfocused undergraduate i enrolled in a dairy reproduction course in animal science and i was hooked on reproduction. i spent a year as an undergraduate senior in a research laboratory in anthony hall where i learned about working with cattle and hormones. memorably, my mentors made me stand up and give an intimidating seminar presentation that buckled my knees but had to have built character. that opportunity allowed me to land at oregon state university where my mentors, fred stormshak, don holtan, and lloyd swanson taught me, and my graduate student colleagues, about management and physiology of cattle, sheep, and horses. although these animal scientists were (probably) disappointed i wanted to be a vet, i was accepted into the college of veterinary medicine at michigan state. when i finally got to 3rd year, i was excited for theriogenology. i had bought the textbook (current therapy in theriogenology edited by david morrow) a year earlier! on the first day of theriogenology class, dave morrow knew us all by name and he called on me minutes into the first class, gulp. i grew to know and respect dr. morrow, and i also learned (and got awesome advice) from him and ed mather, carla carleton, resident cindy smith and rick bridges, and dean neely, the latter stoking my equine interests. dave morrow was among the nerdiest people i’ve known, in the best way, but he was mailto:smeyers@ucdavis.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9776 citation line: clinical theriogenology 2023, 15, 9776, http://dx.doi.org/10.58292/ct.v15.9776 3 genuine and loved students! he always wore a tie, even when palpating. one day, when i was a senior student in the la clinic, a young veterinarian named dickson varner, gave an interview seminar in semen freezing in stallions. i was impressed, of course, but didn’t know the impact of this for a few years yet to come. also, during my time as a vet student, i had an opportunity as a research intern at the san diego zoo where i was involved in an ivf project with barbara durrant in goats as a model for african and sub-saharan antelope. this fed my passion for wildlife and conservation reproductive biology. i also met and became friends with vic shille who was spending a sabbatical year at the zoo. vic was a wonderful and brilliant mentor and theriogenologist and i benefitted from much advice and vic’s warm personality. vic also had ties to uc davis and that impacted later. possibly the most valued advice i received about my future career was from dave morrow who told me i must get out into vet practice if i wanted to be a resident and ultimately a professor of theriogenology. i joined a 5-person dairy practice on the eastern shore of maryland in which i had my own herds and relationships with farmers. except for this one guy who shall remain nameless. that is, until the day he had a cow with an rda and uterine torsion, and then he needed me and gave me a chance. sadly, i lost that cow, but the dairyman saw how hard i worked to resolve the problem, and we went on to several years of humbling vet work. during my time in maryland, while being a dairy herd vet, i also filled in evenings in the small animal clinic. a nice young woman came in one evening with two baby rabbits. would i tell her if they were sisters or brothers? no idea. i called my boss for advice and he suggested i look at their bottoms and if they looked the same, just say they were sisters. it seemed reasonable. of course, i ran into that woman in a grocery store a few weeks later and she told me the bunnies’ babies were doing fine, and did i want one or two? humbled again. now i knew i needed advanced training! it was in that practice that i met my colleague and wonderful friend, peyton jones, who in turn, introduced me to tom bowman, maryland thoroughbred breeding, winfield farm and northern dancer, and other notable thoroughbred farms. tom was a presence at winfield’s (later northview stallion station) and, at that time, was just adapting ultrasound and twin management into equine practice. tom was incidentally the first repro resident at new bolton center so there was lineage in common. i fell in love with horse breeding and peyton introduced me to dale paccamonte, then a resident at university of florida. dale, also an msu grad, had previously been in the same practice. we still talk about the stories from that dairy practice! when i interviewed at florida for a residency, dale hosted and we became friends and colleagues. this was the first opportunity i didn’t get (i wasn’t offered the  residency) but gained some wonderful friends in theriogenology with michelle leblanc and dale. a few months later, michelle called me to tell there was an out-ofcycle residency available at texas a&m university and i immediately called ron elmore, and four weeks later, i arrived in college station. college station, as a yankee, opened my eyes to the joys of the south – food, cattle, horses, open space, two-stepping and waltzing, chicken-fried steak, bbq to name a few. literally millions of stories about texas texas a&m residency, college station, tx livestock, people and ranches! i also met my wonderful wife, becky, who was a 3rd year tamu vet student at the time. she later provided another journey for me in reproduction, ultimately making me a father of two boys, but that is a story for another time… it was here that i was mentored by an amazing team consisting of dickson varner, terry blanchard, ron elmore, and kathy bretzlaff. surgeons david morris, tex taylor and jeff watkins offered huge learning opportunities and an amazingly colorful field service team. humbled along the way, of course. dave morris was a surgeon, but he was also a theriogenologist and asuper nice person and great mentor. here we learned about bull penile surgeries, rodeo bulls, and vaginal prolapse management. dave was also a role model for the hopelessly disorganized. i also bonded with my fellow residents, lloyd kloppe, steve brinsko and pat meyers. i was replacing lloyd but in the few months we overlapped, i learned so much from this master of equine therio and we were friends for years. sadly, lloyd recently passed and this devastated many colleagues and friends. hats off to lloyd! jim rugila was also a latter resident and officemate. chickens in the office! pat and i were the ‘meyers bros’ – unrelated but put together by personality and as northerners out of water. both lloyd and pat finished their residencies in my first year but steve and i held the fort for better than a year and a half and he has been a close friend to this day. steve was truly one of the funniest people i’d ever known. steve had file folders of jokes-printed and organized by topic. either every friday afternoon, or whenever we had downtime, out came the files. the laughing and biting sarcasm about situations and ultimate ironies almost never stopped to this day. texas pasture skiing, whereby one was towed after roping an unruly cow that broke loose, took shape as a potential olympic event. when i began to study for act boards, i realized that i needed to have “the red book”, dr. steve roberts’ massive state-of-theart of theriogenology sprung from his decades at cornell, http://dx.doi.org/10.58292/ct.v15.9776 4 citation line: clinical theriogenology 2023, 15, 9776, http://dx.doi.org/10.58292/ct.v15.9776 i  couldn’t afford to buy the book on a resident’s salary. in those days before email, i sent off a letter (using stamps and an envelope, of course) to steve roberts, then ‘retired’ in woodstock vermont, explaining that i really wanted and needed the book. in pretty short time, i received the book still in its wrapper compliments of dr. roberts along with a warm personal note that he hoped it would help. a few years later, i would face dr. roberts across a table as an examiner for the act board practical exam. at texas a&m, i developed a special and long-lasting relationship and collaboration with dickson who mentored me as an texas a&m: “i’ve been everywhere, man.” older than typical resident. dickson was a master of modern therio and a great doctor but also had a keen eye for historical context. terry blanchard was a superb theriogenologist but also an amazing vet. he had a wealth of practice and life experience, which was incredibly valuable as a mentor to me. we spent most time on stallions and mares but also beef cattle and tropical breeds: bos indicus and taurus. some of our learning (about life and vet practice) was done after hours in the field service trucks on our own with the humbling presence of students. the tamu experience was hard and gritty and the caseload was sometimes crippling. but we learned and taught from all the richness. i had opportunities to work on rhino and giraffe reproduction with dr. dewey kramer and his group in the reproductive sciences laboratory. add to the mix, the theriogenology work at the texas prison system for bull bses and horse breeding. this was an exceedingly rich mixture of clinical experience and teaching opportunities that set the stage for many years of wide-eyed memories and stories. steve, dickson, and i also played music together and (deservedly) won a talent contest at tamu! after the tamu residency, i became a resident veterinarian at a large standardbred breeding farm in northern illinois where i put to use my tamu training. a texas beach girl, becky was really looking forward to living in real winter and snow in northern illinois but was definitely ready to move on to the next adventure, in california, a few years later. what i suspected but had not appreciated, or prepared for, was that foals were everything and that repro was just a means to get foals on the ground! i learned an amazing amount of foal medicine along with practical obstetrics and neonatology – we delivered approximately 250 foals per year, so there was always something. during my third breeding season, the state had a large strangles outbreak and many horses were sick. a few died. i learned to perform field tracheotomies for the severe cases and had numerous sick adult and juvenile horses. this was above and beyond the endemic levels of rhodococcus equi infections existing on the farm. it was time to refocus and pursue research and teaching… dr. john hughes at uc davis offered me an opportunity to run the california side of a now-famous synthetic gnrh implant study for fda approval as a funded phd student opportunity. i was accepted as a doctoral student at the university of california. the project was a multi-center collaboration between colorado state and uc davis with a large number of mares at each institution to gain approval for clinical use of deslorelin or ovuplant. this experience allowed me to learn about the fda approval process and do graduate studies with john hughes, george stabenfeldt, irwin liu, bob bondurant, jan roser, along with many others at ucd. my graduate student friends were pat mccue, mats troedsson, and peter daels; under the training of george and john we became lifelong colleagues and friends. i can never forget my first meeting with george as he played jazz piano beautifully for me in his office and provided tremendous mentoring. i channeled his coolness and have always kept a guitar in my office. dr. hughes was also a wonderful mentor and we became good friends with lavonne and john. phd graduate student, uc davis as a graduate student, my first course was an amazing fertilization biology class taught by a team of amazing and wellknown reproductive biologists. i developed a deep interest in andrology and male subfertility in horses and i was focused on the cell and molecular biology of sperm. studies focused on acrosomal function of horse sperm and interaction with oocytes and we developed the beginnings of a program to understand the role of sperm membrane function in fertility. i developed experience working with fluorescence microscopy, along with acrosomal biology and the sperm surface membrane enzyme system. in the context of fertilization, we began to think of sperm function in livestock related to sperm–egg interaction, of which the acrosomal reaction was the first step. http://dx.doi.org/10.58292/ct.v15.9776 citation line: clinical theriogenology 2023, 15, 9776, http://dx.doi.org/10.58292/ct.v15.9776 5 being in the therio zone … science and life after a postdoctoral fellowship on contraceptive studies with non-human primates with the human andrology and fertility group at uc davis school of medicine, i joined the faculty as assistant professor at new bolton center, in the school of veterinary medicine, university of pennsylvania. in addition to a large equine caseload that included a state-of-the-art neonatal icu, i joined a well-established team that performed more stallion bses and semen shipping than most places around the world. bob kenney had recently retired, and it turned out, bob was still around for another couple years and always could be counted on for wisdom, burning sarcastic wit, and knowledge. pat sertich, sue mcdonnel, dick mcfeely, and regina turner (graduate student at the time) rounded out a great team, along with amazing and several hard-working selfless residents including tamara dobbie and sylvia bedford. john hurtgen, another master of the game, was nearby in new freedom and i never failed to learn from many things about stallions from john along the way. ina dobrinski joined the faculty ‘in town’ (philadelphia) in ralph brinster’s lab and we developed a long-term friendship as she began to master mammalian germ cell biology, which began in this unequaled lab at penn. i was able to obtain external funding (usda, grayson) for stallions and the lab published a few articles on acrosomal function and hyaluronidase of the sperm surface membrane in stallion sperm function and capacitation. here, pep rutllant from spain joined the lab as postdoc initially (and then as lecturer at uc davis) and we collaborated for many years with a deep friendship. philadelphia zoo zebra mare bse. after four years as assistant professor at new bolton center, i accepted an offer to join a basic science department in the veterinary school at the university of california, davis. this opportunity would lead me out of clinical medicine but allowed a scientific career to expand. it was at this time my academic and personal life began to take shape. our toddlers grew into teens, and then young men. numerous generations of dogs and cats…we made a life in davis california and now have many close friends and colleagues! a lifetime of wonderful theriogenology colleagues and students met me: al conley, barry ball, bob bondurant, linda munson, jim maclachlan, irwin liu, joannie rowe, countless residents and graduate students who are now accomplished theriogenologists and reproductive biologists around the world. in addition, many international colleagues and collaborators in germany, australia, italy, france, uk, the netherlands, belgium, japan, and spain. [honaramooz, li mw, penedo mc, et al: accelerated maturation of primate testis by xenografting into mice. biol reprod. 2004 may;70(5):1500–1503.] at ucd, my research and teaching took hold. in addition to theriogenology teaching, i took on gross anatomy teaching and i surprisingly developed a love and expertise for this fundamental discipline’s ability to reach 1st and 2nd year vet students. most theriogenology programs don’t historically engage these early years of the vet curriculum and i really enjoyed the refreshing enthusiasm of first year dvm students. i was course leader for many years of the basic gross anatomy course and i was recently awarded a school of veterinary medicine faculty teaching award (2022) and a uc davis (2023) distinguished teaching award for professional and graduate education. i also began serving the act first as board member in the mid-2000s and then as treasurer for six years during this time. many more act and sft friendships! being in the research zone… my research journey is a bit more convoluted. at upenn and at uc davis, my lab’s work on acrosomal function and sperm membrane biology focused on stallion sperm function. we developed a record of publications and presentations in this area that eventually encompassed sperm mitochondrial function and mitochondrial genetics as related to oxidative metabolism in sperm from stallions, dogs, and, more recently, fish. our research in sperm physiology and function has been focused on oxidative stress due to cryopreservation conditions and aging in stallions, dogs, and bulls for the past 20+ years. in our canine work, the lab has collaborated with guide dogs for the blind and we’ve received generous funding from the great dane club of america for the canine aspects of this work in which we began to explore the effects from male age and sperm function in large populations of breeding labradors and danes. along the way, we realized that sperm function must really put into context of fertilization to more fully understand the role of this remarkable and unique cell. for this reason, the lab http://dx.doi.org/10.58292/ct.v15.9776 6 citation line: clinical theriogenology 2023, 15, 9776, http://dx.doi.org/10.58292/ct.v15.9776 began moving into the direction of the sperm’s role in fertilization physiology and this drew us into the sperm’s role in early embryo development. several studies focused on oxidative stress in stallion and rhesus monkey sperm with collaborations with barry ball’s lab at uc davis and peter sutovsky’s lab at the university of missouri. however, i will highlight some of the unique opportunities we’ve had in recent years. as in life, the failures, or at least the roads not taken (or unable to be taken) were also important. many side roads emerged and i will tell a few stories about that. in the early years as faculty member at uc davis, my lab collaborated with the uc davis lab of the esteemed dr. john crowe, a world-renowned lyobiologist (freeze drying). together, we put a strong effort forward on attempts to understand cryobiology of sperm (which was new to john) through membrane phase behavior that led to attempts to freeze-dry sperm using the intracellular sugar trehalose, which is used naturally by many desert creatures to withstand seasonal and decades-long drought. since sperm cells are known to already have the one of the lowest water contents of any mammalian cell, it made sense to try to load these cells with trehalose and freeze-dry them for long-lasting preservation. in the end, we never were able to recover live or motile sperm, but we did determine that stallion and monkey sperm could be dried to zero water content and maintain good morphology and even could be used to fertilize eggs using intracytoplasmic sperm injection (icsi). additionally, we reported that immature rhesus monkey spermatogenesis could be re-established when testicular explants were transferred to immunocompromised mice in an acceleration of spermatogenesisearlier than the donor was entering puberty. the mice hosted testicular explants that grew and maintained monkey spermatogenesis! furthermore, we were able to then freeze-dry the matured sperm harvested from the mice and used these sperm for icsi to make viable monkey embryos. since then, numerous species have successfully reported icsi embryos fertilized with dried sperm. someday, the ‘code’ will be cracked to preserve sperm without liquid nitrogen! a side project from this collaboration, set our lab on the path to understand phase transitions in stallion sperm during cooling. this work had not been performed previously in horses and allowed us to begin to think about stallion sperm membranes existing in multiple phase states, which include crystalline and gel states with varying lipid melting points in a single cell’s membrane that opened an understanding of how these cells withstand, or do not withstand, low temperature. perhaps some males have differing success in cryopreservation due to their membrane phase traits? another offshoot of our work was the use of oxidative stress in stallion and dog sperm, which we then applied to zebrafish. we demonstrated that zebrafish sperm were equally susceptible to oxidative stress as mammalian sperm. this was particularly interesting because fish sperm are exposed routinely to highly varying osmotic and oxidative conditions (and temperatures) in which these cells can persist with high success rates. lessons such as these can be applied to our growing understanding of livestock sperm stress. several years ago, we participated in a series of studies of the marine invertebrate, elkhorn coral in the caribbean. the latter are struggling around the world to survive as the great coral reefs are under increasing global pressure primarily due to warming and pollution. the elkhorn coral (acropora palmata) of the caribbean are highly endangered and form the basis of complex habitat for countless ocean animals and plants. beginning in the 1980s, the elkhorn coral population declined 97 percent from warming-related white band disease. my lab had the opportunity to participate in a cryopreservation project conducted by the smithsonian institution along with several major international aquariums for a field study in puerto rico. in this international collaboration, we performed the cryopreservation studies of sperm and accompanying studies on embryos for this endangered hermaphroditic species. again, collaborations and friendships became long-lasting with several national zoo and smthsonian investigators. we were able to send several uc davis veterinary students to the national zoo in washington, dc over the years for research internships. elkhorn coral are known to broadcast spawn naturally only during a few days per year at night after a full moon so the team needed to be ready to jump into action when the spawn began. when they spawn, the hermaphroditic coral simultaneously release sperm and eggs (they do not self-fertilize), which float to the surface and mix. the fertilized motile zygotes then sink to the bottom and begin to attach to rocks and substrate. these organisms are deeply susceptible at this specific time to predation and hostile environmental conditions. so there is a tiny window in which to collect the gametes. this required a team of professional night divers to monitor for the beginning spawn each night around the second week of august. when the spawning coral were spotted, the lab teams sprung into action and retrieved samples of the rising sperm and eggs released by each hermaphroditic individual, which are released and mix at the surface to provide genetic diversity. our goal was to cryopreserve as much genetic material as possible, to optimize the freezing process so that the aquariums and conservation biologists could propagate the baby coral under controlled conditions. the project is ongoing and successful for in vitro management, but still remains unknown if repopulation is possible in the caribbean reefs. as sperm function needed to be understood during sperm– oocyte interaction, our lab developed a non-human primate model for early events following icsi fertilization and combined the oxidative stress model for early embryo development. a. normal 4-cell rhesus monkey embryo showing intact nuclei and cytoskeleton. b. abnormal 4-cell embryo resulting from icsi using oxidatively stressed sperm. note multiple abnormal micronuclei and chromatin fragmentation. confocal fluorescence image (600x); [burruel v, klooster k, barker cm, et  al: abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage. sci rep 2014 oct 13;4:6598.] http://dx.doi.org/10.58292/ct.v15.9776 citation line: clinical theriogenology 2023, 15, 9776, http://dx.doi.org/10.58292/ct.v15.9776 7 we induced oxidative stress on monkey sperm and then used icsi to inject sperm into mature meiosis ii oocytes. we found that oxidative damaged sperm showed somewhat normal cleavage division but improper nuclear structure and function evidenced by fragmented nuclei and micronuclei: a condition that mimicked embryo development resulting from the use of sperm from aging men. we began to incorporate time-lapse imaging in near real-time for monkey embryos, as had been reported for human in vitro embryos in which we defined the timing and durations of each mitotic division in early embryos leading to blastocysts. this led to equine embryo time-lapse studies. as the lab became more experienced with icsi in non-human primates, we began to experiment with icsi in horses and our program has now developed into a robust equine research and commercial icsi program. we reported the first all-uc davis icsi foal in 2018 as the program was begun. the birth of uc davis’s first icsi foal, petri. although monkey embryos are more or less transparent and easy to monitor in vitro, horse oocytes and embryos are very optically dense and cell divisions (in addition to being barely able to see sperm during icsi) are quite challenging to observe and record due to this dark appearance under most light microscopes. a new optical imaging system allowed us to perform kinetic monitoring of horse embryos in near real-time. dr. pouya dini joined our faculty in 2020 and together we have developed a research and clinical icsi program in the past several years along with a great collaboration and friendship! in our lab at uc davis, we developed a state-of-the-art commercial icsi laboratory to complement our research efforts that has performed hundreds of icsi sessions annually for shipped and in-house aspirated immature oocytes for horse breeders from around the us. although not every icsi session results in a frozen embryo, we can report encouraging results both commercially and for research that have allowed us to collect real-time imaging data for each embryo growing in the lab. knowing the timeline for certain specific cellular events to occur has allowed us to gain confidence in predicting which embryos are most likely to survive vitrification and result in a viable and healthy foal. using imaging and molecular techniques our lab is studying the events that occur in the oocyte prior to fertilization, the zygote immediately after fertilization, and the resulting embryo. further studies have focused recently on using the non-invasive time-lapse imaging system to measure and monitor oocyte maturation and cumulus expansion and we are able to predict which oocytes will most likely succeed following maturation and icsi. we are extending these studies into singe cell transcriptomics to understand the difference between in vitro-produced and in vivo-produced embryos, the embryonic capsule, embryo pulsation: all the while teaching an old dog new tricks along the way! conclusions to conclude, i have had a lifetime of amazing experiences in theriogenology, friendships and collaborations and life lessons. i hope my impact on each has been as positive for others as it has been for me. i am indebted to my theriogenology colleagues for many years of sharing expertise in reproduction and for long-lasting, and new, friendships. a few assorted lessons to pass on don’t speak too loudly or enthusiastically about reproductive biology and pathology in restaurants-not everyone appreciates what we do! don’t hesitate to talk to the leaders and founders of our specialty! there is a wealth of information and you will not find nicer people in the world. do a better job listening than talking. it’s been said your ears will never get you in trouble! http://dx.doi.org/10.58292/ct.v15.9776 1 contact julie cecere juliet@vt.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 9916, http://dx.doi.org/10.58292/ct.v15.9916 review report hypothyroidism in canine reproduction† julie cecere department of small animal clinical sciences, virginia-maryland college of veterinary medicine, blacksburg, va, usa abstract hypothyroidism is one of the most commonly diagnosed endocrine diseases in dogs. it has been implicated for many reproductive difficulties in breeding dogs; however, only a few studies have investigated the involvement of thyroid hormones in reproduction. due to intricacies related to appropriate testing, breeding animals may be placed on thyroid supplementation without justification. the disease, testing, heritability, and animals that may need supplementation are discussed. additionally, the assumption/appropriateness of breeding animals that are truly hypothyroid is questioned. keywords: hypothyroid, reproduction, endocrine, infertility pathophysiology and diagnostic challenges hypothyroidism is the most common endocrine disease diagnosed in canine practice, although true disease prevalence is unknown.1,2 over 90% of cases are due to an immune-mediated pathogenesis characterized by lymphocytic thyroiditis or idiopathic follicular atrophy.1,3 rarely hypothyroidism is due to a secondary or tertiary pathogenesis, but those account for  < 5% of cases.3 gonadectomized animals appear to be over-represented and in euthyroid animals circulating thyroid hormone concentrations decrease as dogs age. disease onset is insidious and can take months to years before any clinical signs develop.3 average age at onset of clinical signs is 7 years and clinical signs overlap with other endocrinopathies in many cases.1,3 obesity is present in ~ 50% of cases, followed by dermatologic aberrations (present in 60 80% of dogs).3 dermatologic conditions commonly include alopecia, seborrhea, poor hair coat, and lethargy or a dull mentation.1,3 several breeds are at an increased risk of developing hypothyroidism, mainly, golden retrievers and doberman pinschers. beagles and borzois have heritable lymphocytic thyroiditis.4,5 breeds that have a high incidence of thyroglobulin autoantibodies (tgaa) (antithyroglobulin antibodies) include boxers, dalmatians, giant schnauzers, great danes, setters, and old english sheepdogs, among others.1,3–7 diagnosis should be based on serologic testing and not on clinical signs alone as there are many diseases and medications, or even breed differences, that contribute to altered circulating thyroid hormone concentrations. accurate testing should be utilized before supplementation is considered. both 3,5,3’-triiodothyronine (t3) and thyroxine (t4) are highly protein bound and subject to drastic changes in concentrations of albumin and thyroxin-binding globulin.1 many diseases can also falsely lower circulating t4 concentrations and yield a diagnosis of euthyroid-sick syndrome. specific drugs can alter circulating serum concentrations of thyroid hormones; therefore, any animals being treated with sulfonamides, glucocorticoids, and/or phenobarbital should be tested after a ‘washout’ period, or if this is not practical, the results should be interpreted carefully.1,3 there are lower circulating t4 concentrations in specific breeds, including greyhounds, whippets, scottish deerhounds, basenji, alaskan sled dogs, and sloughis; these breeds have breed-specific reference ranges1,3,8–11 importantly, euthyroid dogs have lower t4 concentrations at some time point during the day, a major consideration when testing is recommended.1 thyroxine concentrations are lower in > 90% of hypothyroid dogs. most general practitioners can use ‘in-house’ serum t4 concentration tests. this test is sensitive, but is not specific for canine hypothyroidism and should be avoided as a single test.1,3 the most accurate single test to diagnose hypothyroidism is free thyroxine (ft4) by equilibrium dialysis.3 thyroid †presented at the 2023 society for theriogenology conference, published after peer review. mailto:juliet@vt.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.9916 2 citation line: clinical theriogenology 2023, 15, 9916, http://dx.doi.org/10.58292/ct.v15.9916 autoantibodies, and to a lesser extent nonthyroidal illness, do not affect ft4 concentrations. combining serum concentrations of thyroid hormones increases success in identifying animals with true hypothyroidism. using thyrotropin (tsh) information with t4 or ft4 typically yields a 98% specificity.1,3 in sighthounds, t3 concentrations may be useful because their t4 concentrations are typically outside reference ranges.3 thyroglobulin autoantibodies (tgaa) form as the dog’s immune system reacts to their thyroglobulin and are present in approximately 50% of all hypothyroid dogs.3 animals that are positive for tgaa but with normal thyroid function tests should be retested annually, as they have a higher risk of actually developing clinical disease than do euthyroid dogs with negative tgaa.12 reproductive manifestations hypothyroidism in humans has been studied extensively and is currently ongoing. there are known reproductive manifestations that affect both women and men. reports include abnormal cyclicity or anovulation, infertility, loss of pregnancy in women, and loss of libido or fertility in men.13,14 women who did not receive adequate supplementation of thyroid hormones or who have had impaired iodine intake during pregnancy had children with substantial effects,15,16 particularly impaired cognitive function. the relation between thyroid hormone supplementation and diet is being elucidated in women. the effect of thyroid hormones on both in vitro and in vivo pregnancy rates are also being studied. men who were > 35 years and subclinically hypothyroid had decreased clinical pregnancy rate when compared with their euthyroid counterparts.14 although these findings are valid, it is important to notice that human and canine pregnancies are very different. humans have higher concentrations of thyroxine binding globulins (tbg) and placental deiodinases; both drastically increase the metabolic demand for thyroid hormones during pregnancy.17 humans also produce human chorionic gonadotropin (hcg) in pregnancy, which has substantial thyrotropic activity.17 broad assumptions based on human literature surrounding hypothyroid disease and its reproductive effects in dogs should be avoided due to these differences. fewer studies were performed on dogs, but despite the lack of data, a plethora of reproductive diseases are ‘blamed’ on hypothyroidism. reports have implicated hypothyroidism for cycle aberrations, abortion, infertility, and stillbirth.3,18–20 to date only a handful of prospective studies have been performed specifically highlighting the reproductive manifestations of hypothyroidism in female dogs. a review of 204 dog breeds suspected to have heritable hypothyroidism failed to demonstrate any significant difference in circulating thyroid hormone concentrations among normal, infertile, and subfertile male and female individuals.7 additionally, a large prospective study that utilized fertile, previously proven mongrel female dogs, in which hypothyroidism was induced with 131i treatment, shed some light on pregnancy and hypothyroidism. during the short-term portion of the study, all dogs cycled normally, became pregnant and carried their litters to term.21 furthermore, there was no difference in litter size, although pups born to hypothyroid dogs weighed less and experienced more periparturient mortality. weight gain of pups was equal between the groups to day 28 postpartum. the increased periparturient mortality was attributed to possible prolonged uterine contractions that could be due to generalized muscle weakness in hypothyroid patients.21 a second article, from the same cohort, evaluated the effects of prolonged experimentally induced hypothyroidism on reproduction. all experimental animals were profoundly hypothyroid and had typical clinical manifestations of weight gain, hair coat changes, weakness, and lethargy.22 control and hypothyroid female dogs were bred twice during this portion of the study, once without supplementation and once with levothyroxine supplementation. many variables were monitored to document the effects of long-term uncontrolled hypothyroidism during pregnancy (2nd breeding) and the effects of supplementation and pregnancy after clinical signs were controlled (3rd breeding). the interestrus interval (iei), pregnancy rates, and duration of pregnancy were not different between groups.22 following the second breeding, only half of the hypothyroid dogs became pregnant, highlighting profound long-term effects of hypothyroidism.22 ultimately, levothyroxine supplementation eliminated any differences between the groups at the 3rd breeding.22 a final study evaluated circulating thyroid hormones present in the hypothyroid female dogs from the 3rd breeding of the same cohort. no gestational adjustments in thyroid supplementation were required for inducing a euthyroid state due to no difference in thyroid hormone concentrations or progesterone concentrations between groups, which is drastically different from hypothyroid women.17 another study evaluated dogs that had aborted their litters after 4th week of pregnancy. this study measured only t4 and progesterone and failed to conduct further diagnostics to work up the cause of abortion. this leads to only a weak argument for a direct effect of t4 being a singular cause of abortion.23 conflicting reports exist regarding hypothyroidism and its effects on male dog fertility. specific colonies containing similar genetic makeup had both hypothyroidism and infertility2,5,24 due to the specifics of genetic makeup of these colonies, one might argue that the infertility observed may be due to limited genetic diversity rather than a direct effect of low circulating thyroid hormones. one 1997 study reported that induced hypothyroidism did affect male dog fertility but a later (2009) in vivo study reported no statistical difference in thyroid function between fertile and infertile dogs.7,25 another 1999 study utilized induced hypothyroidic dogs and noted no change in libido, daily sperm output, motility parameters, nor sperm morphology between control and hypothyroid dogs.26 a few major points from these studies are important. no experimental study to date has reproduced reproductive failure following an immunogenic hypothyroidism mechanism, the naturally occurring disease in canine populations. manifestations of any reproductive failure have required elimination of all functional thyroid tissue within the animal. this raises the question of whether the reproductive effects observed are due to the lack of circulating hormones or a chronic inflammatory state produced by profound endocrine disease and associated endocrine disruption. furthermore, many of the animals in the studies were obese, which is an independent cause of reproductive failure in many species. despite these drawbacks, it is fair to conclude that primary hypothyroidism and its effects on reproduction in canine patients are only weakly associated. hypothyroidism should be approached cautiously as a cause of clinical infertility or subfertility. heritability and testing breeding animals many breeds are labeled as ‘at risk’ for developing hypothyroidism. consequently, there is a high suspicion that a  heritable http://dx.doi.org/10.58292/ct.v15.9916 citation line: clinical theriogenology 2023, 15, 9916, http://dx.doi.org/10.58292/ct.v15.9916 3 and genetic component may be a factor in developing the disease. the insidious nature of the disease and difficulties surrounding its definitive diagnosis have left heritability studies as inconclusive. this leads to the conclusion that the underlying genetics are likely a polygenic trait that varies from one breed to another and that developing hypothyroidism is also heavily influenced by environmental factors.6 penetrance of the offending alleles is also likely to have a role in the expression of clinical disease. despite these drawbacks, there are testing mechanisms that help screen potential breeding stock and assist breeders and veterinarians in choosing animals less likely to pass hypothyroidism on to their offspring. the use of specific, accurate testing mechanisms is very important. the use of a single t4 or even t4 combined with tsh should be avoided in screening broodstock. a full panel including ft4 by equilibrium dialysis, t4, ctsh, tgaa, and t3/ft3 should be used. animals that are exhibiting signs of concurrent disease should not be tested until the disease state has been resolved. testing should begin around 1 year of age and should continue every 1–2 years while the animal is breeding or until the animal reaches 8–10 years of age. this allows for identification of animals that may develop subclinical disease over the course of their life, despite never having fulminant clinical signs. it is important to remember that hypothyroidism is a disease that is easily and successfully treated. animals that may be tgaa positive and remain in the normal range of other thyroid function tests should not be eliminated from the breeding pool, because eliminating them can lead to diminished genetic diversity for many other traits. breeding animals must be taken as a whole, and breeders should avoid ‘throwing the baby out with the bathwater’, especially for a disease that is so easily treated. levothyroxine supplementation only animals that had appropriate testing and elimination of all other potential causes of infertility should be considered for levothyroxine supplementation. typically, these dogs have been bred several times, conceived, and failed to deliver or lost a large portion of their litter to resorption during the 4th to 6th week of pregnancy. documentation of similar history with appropriate diagnostics, serial ultrasonographic examinations of the reproductive tract, infectious disease testing, complete blood count and biochemical profiles are warranted before considering supplementation. this includes a thorough history including pharmaceuticals used (preventatives and antibiotics), appropriate iei, breeding method(s) and timing, fertility of the sire, genetic evaluation of the breeding pair, and any other comorbidities. intrauterine cytology, culture and/or biopsy should be performed in early proestrus, positive results should be taken seriously (addressed and discussed), and automatic pharmaceutical treatment is avoided. if the animal is timed appropriately, bred to a proven, fertile sire and again fails to carry to term, then it is time to consider supplementation. supplementing typically causes female dogs to be hyperthyroid, and this should only be done if a clear justification exists. supplementation needs to be fully discussed prior to implementation. this discussion should include the genetic possibility of needing exogenous hormone therapies to maintain pregnancy in their lines. terminal, performance breeding is well accepted, but a kennel that is experiencing reproductive failure should carefully consider eliminating this type of animal as a brood female dog. once daily treatment of oral levothyroxine (0.02 mg/kg) is sufficient for supplementation. treatment for female dogs should start as soon as the veterinarian feels that they fit the criteria discussed above (during pregnancy) or at the beginning of her next estrous cycle and will be bred. all pregnancies in these dogs are considered high risk and should be monitored carefully and often. a tapered withdrawal over 2 weeks should be initiated immediately after whelping. typically, these dogs do not have any other confounding factors, and are without evidence of endometritis, pyometra, or metritis, hypoluteoidism, or other causes of pregnancy failure. breeders often demand supplementation well before a clear cause is identified; however, there is sufficient evidence that the thyroid gland must be profoundly affected before severe reproductive problems manifest. this provides a clear path to appropriate and science-based supplementation. there may be another mechanism in which thyroid supplementation will assist these specific cases, but the exact role has not been elucidated and is the very reason for caution. this calls for the judicious use of thyroid supplementation despite what is known in breeder community as ‘breeder lore’. conclusion hypothyroidism is a common disease of our canine population and is difficult to definitively diagnose. furthermore, evidence of direct cause and effect of hypothyroidism and reproductive failure in a natural disease state is lacking. reproductive veterinarians need to weigh all infectious, genetic, and husbandry causes of reproductive failure diligently and in a systematic approach before diagnosis and supplementation of thyroid hormones are implemented. conflict of interest the author declares that she has no conflict of interest. references 1. bruyette ds: canine hypothyroidism. in: bruyette ds, bexfield n, chretin jd, et al., editors. clinical small animal internal medicine,  vol. 1, john wiley & sons, 2020, p. 71–74. doi: 10.1002/9781119501237 2. fritz te, lombard ls, tyler sa, et al: pathology and familial incidence of orchitis and its relation to thyroiditis in a closed beagle colony. exp mol pathol 1976;24:142–158. doi: 10.1016/0014-4800(76)90002-2 3. panciera d: hypothyroidism in dogs. in: j rand, editor. clinical endocrinology of companion animals. john wiley & sons, 2013, p. 263–272. doi: 10.1002/9781118997093 4. benjamin s, stephens l, hamilton b, et al: associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles. vet pathol 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http://dx.doi.org/10.58292/ct.v15.9916 8. gaughan kr, bruyette ds: thyroid function testing in greyhounds. am j vet res 2001;62:1130–1133. doi: 10.2460/ajvr.2001.62.1130 9. shiel re, sist m, nachreiner rf, et al: assessment of criteria used by veterinary practitioners to diagnose hypothyroidism in sighthounds and investigation of serum thyroid hormone concentrations in healthy salukis. j am vet med assoc 2010;236:302–308. doi: 10.2460/javma.236.3.302 10. hegstad-davies rl, torres sm, sharkey lc, et al: breed-specific reference intervals for assessing thyroid function in seven dog breeds. j vet diagn invest 2015;27:716–727. doi: 10.1177/1040638715606953 11. panakova l, koch h, kolb s, et al: thyroid testing in sloughis.  j  vet  intern med 2008;22:1144–1148. doi: 10.1111/ j.1939-1676.2008.0155.x 12. graham pa, refsal kr, nachreiner rf: etiopathologic findings of canine hypothyroidism. vet clin north am small anim pract 2007;37:617–631. doi: 10.1016/j.cvsm.2007.05.002 13. silva jf, ocarino nm, serakides r: thyroid hormones and female reproduction. biol reprod 2018;99:907–921. doi: 10.1093/biolre/ioy115 14. rao m, yang z, su c, et al: paternal subclinical hypothyroidism affects the clinical outcomes of in vitro fertilization/intracytoplasmic sperm injection. thyroid 2021;31:12–22. doi: 10.1089/ thy.2020.0154 15. monaghan am, mulhern ms, mc sorley em, et al: associations between maternal thyroid function in pregnancy and child neurodevelopmental outcomes at 20 months in the seychelles child development study, nutrition cohort 2 (scds nc2). j nutr sci 2021;10:e71. doi: 10.1017/jns.2021.66 16. chen j, zhu j, huang x, et al: subclinical hypothyroidism with negative for thyroid peroxidase antibodies in pregnancy: intellectual development of offspring. thyroid 2022;32:449–458. doi: 10.1089/thy.2021.0374 17. cecere j, purswell b, panciera d: levothyroxine supplementation in hypothyroid bitches during pregnancy. theriogenology 2020;142:48–53. doi: 10.1016/j.theriogenology.2019.09.036 18. johnson ca: thyroid issues in reproduction. clin tech small anim pract 2002;17:129–132. doi: 10.1053/svms.2002.34643 19. david lp: conditions associated with canine hypothyroidism. vet clin small am pract 2001;31:935–950. doi: 10.1016/ s0195-5616(01)50006-6 20. wilborn rr, maxwell hs: clinical approaches to infertility in the bitch. vet clin small am pract 2012;42:457–468. doi: 10.1016/j. cvsm.2012.01.016 21. panciera d, purswell b, kolster k: effect of short-term hypothyroidism on reproduction in the bitch. theriogenology 2007;68:316–321. doi: 10.1016/j.theriogenology.2007.04.026 22. panciera d, purswell b, kolster k, et al: reproductive effects of prolonged experimentally induced hypothyroidism in bitches. j vet intern med 2012;26:326–333. doi: 10.1111/ j.1939-1676.2011.00872.x 23. thuróczy j, müller l, kollár e, et al: thyroxin and progesterone concentrations in pregnant, nonpregnant bitches, and bitches during abortion. theriogenology 2016;85:1186–1191. doi: 10.1016/j. theriogenology.2015.11.035 24. conaway d, padgett g, bunton t, et al: clinical and histological features of primary progressive, familial thyroiditis in a colony of  borzoi dogs. vet pathol 1985;22:439–446. doi: 10.1177/ 030098588502200502 25. johnson c, nachreiner r, mullaney t, et al: reproductive manifestations of hypothyroidism. santa barbara, ca: canine practice, 1997, p. 29–30. 26. johnson c, olivier nb, nachreiner r, et al: effect of 131 i-induced hypothyroidism on indices of reproductive function in adult male dogs. j vet intern med 1999;13:104–110. doi: 10.1111/j.1939-1676.1999.tb01137.x http://dx.doi.org/10.58292/ct.v15.9916 https://doi.org/10.2460/ajvr.2001.62.1130 https://doi.org/10.2460/javma.236.3.302 https://doi.org/10.1177/1040638715606953 https://doi.org/10.1111/​j.1939-1676.2008.0155.x https://doi.org/10.1111/​j.1939-1676.2008.0155.x https://doi.org/10.1016/j.cvsm.2007.05.002 https://doi.org/10.1093/biolre/ioy115 https://doi.org/10.1089/thy.2020.0154 https://doi.org/10.1089/thy.2020.0154 https://doi.org/10.1017/jns.2021.66 https://doi.org/10.1089/thy.2021.0374 https://doi.org/10.1016/j.theriogenology.2019.09.036 https://doi.org/10.1053/svms.2002.34643 https://doi.org/10.1016/s0195-5616(01)50006-6 https://doi.org/10.1016/s0195-5616(01)50006-6 https://doi.org/10.1016/j.cvsm.2012.01.016 https://doi.org/10.1016/j.cvsm.2012.01.016 https://doi.org/10.1016/j.theriogenology.2007.04.026 https://doi.org/10.1111/j.1939-1676.2011.00872.x https://doi.org/10.1016/j.theriogenology.2015.11.035 https://doi.org/10.1016/j.theriogenology.2015.11.035 https://doi.org/10.1177/030098588502200502 https://doi.org/10.1177/030098588502200502 https://doi.org/10.1111/j.1939-1676.1999.tb01137.x 2015: cattle handling pointers: stockmanship and low-stress handling cattle handling pointers: stockmanship and low-stress handling ron gill, rick machen texas a&m agrilife extension, texas a&m university, college station, tx understanding cattle behavior there are three basic means of communicating with livestock. very simply they are: • sight • sound • touch cattle prefer to communicate through line of sight. good stockmanship and low-stress handling can only be accomplished when a complete understanding of how a prey animal responds to line of sight and adoption of these in livestock handling are in place. understanding the link between cattle’s eyesight and their movement and behavior is critical in handling and in facility design. noise of any kind, but in particular the human voice, is usually stressful and marginally successful in getting the desired result. sound should be used as a secondary method of communication and preferably only used when sight and position is not adequate. distracting sounds shift cattle’s focus away from the desired direction. touch is really only useful in situations where animals are confined and additional stimulus is needed to get cattle to move or respond. effective touch does not include the use of driving aids such as hotshots or sorting sticks or paddles. there are five basic principles of cattle behavior that when used properly can improve the ease and speed of working cattle while reducing stress and increasing efficiency. those principles are: 1. cattle want to see you. understanding vision is foundational to handler positioning and cattle response. cattle have excellent peripheral vision with the exceptions of blind spots directly behind (large) and in front of (small) them. when working from behind and to keep cattle from turning, it is important to stay in their sight by moving from side to side. 2. cattle want to go around you. this is also related to the desire to maintain visual contact allowing the handler to get in a position such that, when cattle do go around them, the cattle are pointed directly at the intended gate or destination. they’ll think it was their idea to go there. 3. cattle want to be with and will go to other cattle. a herding instinct is natural among ‘prey’ animals. stockmen can take advantage of this natural instinct as they work from the front of cattle. start the front the back will follow. 4. cattle want to remove pressure. the natural instinct of a cow is to return to the last known safe or comfortable place. this behavior is in response to pressure and their desire to remove pressure. handlers use this to their advantage when sorting and moving cattle from one corral to another. the simple principle of the return box or “bud box” takes advantage of this instinct. 5. cattle can only process one main thought at a time. if cattle are thinking about anything other than what you are asking them to do, change their focus before putting pressure on them. handling cattle in corrals handling cattle in corrals is somewhat different than handling cattle in open pastures or large feeding pens. the main difference is the cattle’s inability to remove pressure by moving away from human pressure. because the entire basis of stockmanship and low-stress handling is pressure and release the handler must be aware that cattle confined in corrals may not be able to move far enough away from the handler to completely remove pressure. if they cannot then the stress level increases in the cattle. effective stockmanship skills are based on pressure and release. an animal will quickly learn to tolerate pressure and not develop stress if they perceive a way for pressure to be released. it is critical clinical theriogenology • volume 7, number 4 • december 2015441 that cattle are trained while in a pasture setting or at least in a large corral until the flight zone is reduced to a point the cattle can become content while confined in a corral. cattle are intelligent and usually do what they are asked to do. however, if asked incorrectly cattle will likely not respond as the handler intended. when this happens we have come to rely on facilities, equipment or manpower to force cattle to do what is needed. this results in increased stress on cattle and handlers and results in cattle becoming more and more difficult to handle. the job of a handler is to teach an animal to tolerate pressure and stress for short periods of time. the role of a handler in stockmanship is to create movement in cattle and then use position to control and manage that movement to the desired result. when cattle loose movement they become reluctant to work. when movement is lost, excessive pressure, force and driving aids are more likely to be used. creating and managing movement is key to achieving effective stockmanship. however, when cattle are confined into crowded corrals there is an inherent loss in movement that makes stockmanship and handling somewhat more difficult. although working pens are smaller there is more than adequate room to get cattle to establish some movement as a group. it is important to not overcrowd any corral, pen, or crowding area with too many cattle. the key will be to work cattle in smaller groups as you get into smaller pens and processing areas. understanding behavior and handler position can make this much less of a problem when moving cattle out of holding pens and to processing and shipping facilities. these same principles apply when pulling one animal from the pen or when sorting cattle out of pens. the entire premise of low-stress handling is keeping stress to a minimum. in a very simple explanation of stress… if you decide to do something it is not stressful; if you are forced to do something it will be stressful. sound stockmanship involves convincing an animal the intended movement is their idea. force is avoided and stress is reduced. the handler has to understand behavior before this can work. to understand behavior a sound understanding of flight zone and point of balance is needed. flight zone the flight zone or “pressure zone” refers to the area around an animal where it begins to feel uncomfortable and perceives pressure. movement by animal or human into that zone will elicit a response away from that intrusion. use of the zone allows humans to manage movement in cattle. the most common figure depicting the concept of flight zone and point of balance is shown below. the most important point to remember about the flight zone is not the zone; it is the area immediately outside the flight zone. stockmen must learn to anticipate, read and manage this ‘boundary’ area. when approaching an animal it is important to predict the response to your approaching the flight zone. if the desired movement is not going to occur, the handler should retreat, reposition and return from a different angle. clinical theriogenology • volume 7, number 4 • december 2015 442 point of balance another key part of effective stockmanship is understanding and manipulating the point of balance. the diagram above indicates the point of balance to be the point of the shoulder. point of balance varies greatly among animals and is influenced by pressure from front or behind, draw of cattle ahead, push of cattle behind and whether or not they are comfortable going by the handler. suffice it to say that the point of balance on any given animal is not necessarily where it is drawn on the diagram above. the point of balance is not static and is actually related to handler position relative to the animal’s eye. flight zone and point of balance are not static and can be manipulated and changed by human management. flight zones need to be reduced on wild or nervous cattle and point of balance needs to be moved forward. both can and should be done with proper handling. handling pointers keeping these behavioral principles and methods of communicating in mind, following is a list of ten handling pointers to keep in mind and a few suggestions that will improve the ease of handling cattle, whether they are being gathered from the pasture or processed through the corrals. 1. slow down so you can be fast. “never mistake motion for accomplishment” patience is a great virtue when moving or working cattle. when handlers get in a hurry, inevitably excessive or incorrect pressure is placed on cattle, which usually results in an unintended reaction from the cattle that must be corrected before work can continue. most handlers have the mind set that as they go to a pen they are going through the gate and to the back of the pen to push the cattle out. often little attention is paid as they enter the gate or move to the back of the pen. nothing could be further from what needs to be done when handling cattle effectively. it is critical that handlers slow down as they approach cattle. pay attention to cattle’s reaction to your presence and use that to set up the next move. 2. work from the front to draw cattle to you. this goes back to the basic principle #1. cattle can be easily controlled from the front if they are not afraid of a human. (if they are afraid you are a long way from being able to handle cattle using low stress principles). working from the front maintains their focus on the intended direction of movement. by moving in and out of the flight zone and across the point of balance, cattle can be easily drawn forward and past the handler. this is a key point in working with cattle in confinement. pushing cattle out of confinement pens can be difficult and stressful on cattle and handlers. when moving cattle from a pen work from the front and draw the cattle toward the gate or opening. start flow out into the alleyway and then work from the side of the group to keep flow going out the gate. 3. cattle must be comfortable to go by you and stay straight. if cattle are not comfortable going by the handler, they will not work very well. working from the front requires cattle to be comfortable passing by without balking or spooking. this simple principle facilitates penning, sorting and processing cattle. as point of balance moves forward (with training), moving, sorting and working cattle gets easier. thus using the draw of other cattle makes it easier to work and sort cattle in an alley or from one corral to another. 4. apply pressure when cattle have a place to go. success of handling cattle depends on knowing when and where to apply pressure and how much pressure to apply. the other key component to effective stockmanship is setting the cattle up to go where you want them to go before you apply pressure. equally important is the release of pressure as soon as the desired result is achieved. low stress livestock handling is not about handling cattle without pressure. in reality it often requires a lot of pressure for a short period of time. 5. pressure cattle from behind only when absolutely necessary. clinical theriogenology • volume 7, number 4 • december 2015443 like any ‘prey’ animal, cattle cannot see directly behind. if you assume a position directly behind cattle (in their blind spot), they will turn to one side or the other in order to see you. to ‘drive’ cattle in a straight line, assume a position behind their point of balance (shoulder) and off to either side. you can also work in a zigzag fashion behind the cattle causing them to switch eyes and move straight forward. note: move cattle in smaller groups. larger groups are difficult to drive behind when motion is lost in the front of the cattle. excess pressure has to be place on the cattle in the rear in order to force movement to resume throughout the group. 6. pressure from the side. this relates back to working from the front and down the side of an animal and not working from directly behind (in their largest blind spot). by working from the side the eye can be manipulated as needed to move an animal in any direction 7. going with the flow of cattle slows them down or stops their movement. it’s all about that point of balance – as you move in the same direction cattle are traveling, when you approach a position parallel to their point of balance, they will slow down, and as you pass the point of balance they will stop. the important part in this process is to get the cattle to stop without reversing their direction. teach them to stop and stay pointed in the direction they were headed. 8. going against the flow of cattle initiates or accelerates their movement. using the point of balance as the tool to initiate movement passing from the front to the back signals an animal to move forward. once movement is initiated it will normally continue until it is stopped by someone passing the point of balance by moving in front of the point of balance. the ability to start and stop movement works whether in a pasture setting or in the confinement of a crowd alley. 9. when working cattle, move in triangles. working in an arch pattern around cattle will simulate movements of a predator, which will elicit a response of fight or flight. move in straight lines when asking for a response from cattle. move straight toward a point on an animal to get a response. once movement is initiated the handlers’ next movement to reposition needs to be in a straight line at an angle away from the movement. handler movement in the same direction as cattle flow will stop the movement just gained. once repositioned the handler can then take a straight direct path back to the cattle to change movement. move into their flight zone to create or correct movement. retreating straight away from the flight zone slows or stops movement. 10. cattle work best when they are ready you have to get them there. cattle have to be taught, conditioned and prepared to work. unfortunately, today’s cattle owners are short on time and experienced labor, and consequently, don’t spend time acclimating cattle to new production settings. it is a process that will pay dividends for those who do spend the time. numerous others will handle your cattle after they have left your care. bad habits and unruly behavior in cattle and humans is learned. shouting, whistling, poking and prodding cattle is unnecessary and counterproductive. in fact, they distract cattle from the intended movement. development of effective stockmanship skills improves worker safety, animal performance and potentially increases income on each individual operation. facilities in working cattle in any processing facility it is important to keep the principles of behavior in mind as facilities are designed. anytime we can create cattle flow where they can go past where we need them to end up it will make handling and processing easier. also remember cattle do not like being moved toward a solid sided or closed in area, as they do not perceive a way out of. if it is necessary or desirable to use closed sided processing areas then the design must be large enough for cattle to go past where they need to come back to without putting too much pressure on the cattle. many current designs have short changed that last requirement and simply try to rely on forcing cattle to enter the crowding area and using a forcing gate to push them around to the opening into the processing lead up. clinical theriogenology • volume 7, number 4 • december 2015 444 there are two basic designs that allow cattle flow to work correctly into the processing area. one is designed using a forcing pen commonly called a circular tub or simply “tub” design. there are literally dozens of variations of tub designs however few work as smoothly as the two below. the other design is a “bud box”. the bud box is the simplest to design but requires the better understanding of cattle behavior because there is no way to force an animal out of the box and into the crowd alley. if handlers/processors of cattle are unwilling or unable to develop and adopt this understanding they should not build or try to use a bud box. they should stick to the more expensive designs that will allow people who do not completely understand behavior to get cattle through the facility. there is nothing magical or mystical about a bud box. it is a facility design that allows the handlers to position themselves correctly to facilitate cattle flow out of the box into either the crowd alley leading to a chute or to a trailer load out. dimensions are important to successful use of a box but not as critical as handler position in relation to the stock leaving the box. without proper position and attention to detail a box will only confuse the stock and frustrate the handler. always keep in mind that the tub and box are a flowthrough part of the facility. cattle should never be stored in a tub or box waiting to be sent into the crowd alley or to a trailer. bring them in and let them flow back out immediately. the tub or box should be large enough to accommodate a volume of cattle adequate to fill the crowd alley or fill a trailer compartment. a crowd alley to a squeeze chute should hold a minimum of four cows and might need to hold 20 head depending on the speed of processing. crowd alleys on cowcalf operations will typically hold five to six cows. facilities working calves or yearlings routinely need crowd alleys for 12 to 20 head of cattle. remember, the crowd alley will normally not be empty when additional cattle are brought through the tub or box. to maintain flow it will be necessary to add additional cattle while one or two still stand in the crowd alley waiting processing. consequently the length of the crowd alley is important. ideally the crowd alley would be long enough to hold an adequate number of cattle for processing while more cattle are brought through the tub or box without disrupting flow. a short crowd alley may result in frequent interruptions of cattle flow and processing. for some reason the industry has migrated toward the crowd alley starting to curve at the entrance from the tub or box. the exit from a tub or a box and entrance into the crowd alley should be straight for at least two mature cow body lengths. this allows flow to become established without the appearance of entering a dead end crowd alley. keep it straight for at least 12 feet and then start a curve if warranted (ex. space is limited). otherwise a long straight crowd alley works very well for processing cattle. most operations will need a box that is at least 12 feet wide and 20 feet deep. it can be 14 feet wide and should be if the handler will be horseback. depending on the size of the cattle being worked it could be 16 feet wide if the handler in the box will always be horseback. both the 14 and 16-foot widths are too wide for comfortably working most stock on foot. a box can certainly be wider than an alley leading up to it. in fact, going from a 10 or 12-foot alleyway into a wider box will normally allow the cattle entering the box to do so faster setting up the transition even better. do not let the width of an alley dictate the width of the box. the length/depth needed is determined by the size of the group handled. again, group size is dictated by the capacity of the crowd alley or trailer compartment being loaded. the box needs to be deep enough to allow the cattle to flow to the back of the box, let the handler close the gate and get in position before the cattle transition out of the back of the box. just like a tub system never overfill the box. success depends on the flow into, transition, and flow out of the box. for most crowd alleys a 20 to 24 foot box is adequate depth. any deeper may force the handler working in the box to move too deep in the box to initiate flow. as the handler returns to the correct position, their movement with the cattle will stop flow and turn the cattle back. going with movement slows it or stops it. neither response is desirable in getting cattle to flow out of the box. other aspects of a box design that are critical to success relate to whether or not the sides are enclosed. it is absolutely essential to have the end of the box open sided so cattle are going to light and clinical theriogenology • volume 7, number 4 • december 2015445 will build speed as they enter the box. entry speed facilitates the transition and correct flow out of the box. solid (opaque) panels should be limited to the box’s entry gate and the sides of the box closest to the crowd alley and load out exits. note: solid sides in these areas are not required but may minimize distractions. load out and crowd alley exit gates must open back flat against the sides of the box. a box used in loading semi-trailers may require additional depth (30 feet maximum) to facilitate filling compartments quickly. if using this same large box for a crowd alley, the addition of a block gate in the box to shorten it might be a good solution. in summary, a box needs to be 12 to 14 feet wide for most operations and 20 to 30 feet deep depending on the number of cattle needed to flow through the system at any given time. leave the back open (translucent); cover the sides and entrance gate if necessary. resources continually look for ways and opportunities to improve your skills as a stockman. for more information and additional training opportunities go to: • hands on and live demonstrations and trainings at http://www.effectivestockmanship.com • videos demonstrating these principles found on at: https://www.youtube.com/user/ronaldjgill o on the stockmanship playlist. • publications can be found on resource page of http://www.effectivestockmanship.com o designing a bud box o cattle handling pointers contact information ron gill, ph.d., college station, texas: email: effectivestockmanship@gmail.com, or rgill@ag.tamu.edu rick machen, phd, uvalde, texas, email: rmachen@ag.tamu.edu clinical theriogenology • volume 7, number 4 • december 2015 446 2016: sexually transmitted disease monitoring, surveillance and impact on breeding farms in the usa sexually transmitted disease monitoring, surveillance and impact on breeding farms in the usa charles f. scoggin rood and riddle equine hospital, lexington, ky introduction sexually transmitted (stds) or venereal disease in horses is of significant concern. emergence of diseases within breeding operations can restrict or close breeding sheds, leading to significant economic loss. transmission can occur during various steps in the breeding process, whether from a mare or fomite. many stds are of importance with respect to the international transportation of horses. outbreaks of stds here or abroad can have far-reaching implications, leading to restrictions in trade or closure of borders. for these reasons, stallions and broodmares originating from outside north america are required to undergo testing before and/or after importation. these requirements are established and enforced by both the usda-aphis and state agricultural officials and will be reviewed below. stallions can be asymptomatic carriers of stds, which can makes diagnosis of subfertility difficult and time-consuming. instead, some clinicians will choose prophylactic antimicrobial treatment (local, systemically or both) in broodmares during reproductive management. however, this practice is controversial and potentially misguided as evidenced by the emergence of multi-drug resistant organisms.1 it should be noted that the presence of microorganisms on the external genitalia of stallions is not uncommon.2 they are usually nonpathogenic and instead represent the natural flora and fauna of the external reproductive tract. there are instances when potentially pathogenic bacteria are isolated, raising concern over shedding of these organisms to mares; for example, certain strains of klebsiella pneumoniae isolated from stallions has been shown to cause metritis and reduced pregnancy rates in mares.3 however, their presence should be interpreted in light of relative fertility.4 this paper will provide background information regarding current breeding practices in the usa, as well as commonly encountered stds in the usa. specific monitoring and surveillance programs will then be discussed, concluding with a discussion on general consideration to reduce the risk of stds in breeding sheds. overview of equine sexually transmitted diseases contagious equine metritis this particular form of metritis is caused by the bacterium taylorella equigenitalis, a gramnegative coccobacillus that is considered highly contagious.5 transmission via fomites also appears to be likely and was implicated as a cause for an outbreak in the usa in 2008-2010.6 clinical signs vary from mild to copious vulvar discharge originating from the uterus, as well as infertility.7 the majority of cem-affected mares are otherwise clinically normal and will resolve the infection within 2-4 weeks. unfortunately, by the time clinical signs are observed at least one cycle is lost to endometritis and spread of the disease is rapidly occurring. inapparent or “silent” carrier stallions are the most common means of transmission because they are much more sexually active. what ensues is a significant drop in productivity—as measured by per cycle pregnancy rates—and increased costs associated with management and veterinary care. treatment for t. equigenitalis is straightforward and consists of careful cleansing of the internal and external genitalia, application of local anti-infective agents and, in some cases, systemic antibiotics. equine viral arteritis equine viral arteritis (eva) is another disease that has important regulatory implications. the causative agent is equine arteritisvirus (eav), which is an enveloped, single-stranded rna virus.8 clinical signs include: vasculitis, distal limb edema, pyrexia, rhinitis, conjunctivitis and anorexia. the clinical theriogenology • volume 8, number 4 • december 2016471 virus can also cause abortion outbreaks and severe neonatal pneumonia in naïve herds. it is transmitted via respiratory secretions and other body secretions, including semen, fetal fluids/tissues, and fomites. equine herpesvirus type 3 equine herpesvirus type 3 (ehv-3) is the causative agent of equine coital exanthema, characterized by vesicular or pox-like lesions on the genitalia. it is not currently considered a reportable disease in the usa. transmission occurs most commonly via coitus, but can also occur via fomites. clinical disease is primarily manifest as vesicular and painful lesions on the external genitalia of both mares and stallions. during the acute phase, these lesions can cause discomfort during reproductive evaluations and during live-cover matings, but infection has no lasting or impressionable effects on fertility.9 dourine dourine is a venereal disease of significance due to both its high level of morbidity and its effect on international transport of equids. the etiologic agent is the protozoa trypanosoma equiperdum. clinical signs include intermittent fever, swelling of and discharge from the genitalia, cutaneous plaques, and neurologic signs.10 generally considered a chronic and progressive disease, the onset of recumbancy usually indicates terminal disease. this disease has been eradicated in the usa (and thus considered a foreign animal disease [fad]), but is still present in africa, the middle east, and south and central america. sporadic outbreaks have been reported in asia and germany.5 breeding management in north america live cover live cover refers to the process whereby a stallion actively mounts and breeds a mare. different variations exist, but the two most common methods are: in-hand mating, which is common in commercial thoroughbred studs; and pasture mating, which is used in large ranch horse herds in the western part of the usa. past reviews on the venereal disease suggests that both methods pose a higher risk for transmission compared to artificial insemination (ai).11 in-hand mating this method is commonly used in commercial thoroughbred studs. prior to mating, mares are typically exposed to a teaser stallion to determine if the mare is in estrus. they are then prepared for breeding by wrapping the tail and cleansing of the perineum. the breeding process involves restraint of the mare with some or all of the following: lead shank, nose twitch, leather cape, kick boots and/or a leg strap. the stallion is then introduced to the mare. when properly aroused, the stallion will mount and breed the mare. it is common practice to collect the drippings from the stallion as he dismounts for microscopic evaluation of live sperm. finally, the stallion’s external genitalia is cleansed with warm water and practical cotton. pregnancy examinations are typically performed 14-16 days after the last breeding, and a stallion’s performance is judged based upon both early and later (e.g., 45 day) pregnancy and live-foal rates. risk factors include nose-to-nose exposure of teaser and mare, expulsion of bodily fluids during teasing and fomite transmission. pasture mating this breeding method is fairly self-explanatory and usually involves running a stallion with a harem of mares during the physiologic breeding season. the stallion is usually turned out for a set period of time and will thus breed mares at his leisure. pasture breeding is much less controlled and carries inherent risks, such as injury to the stallion or mare. however, management not as intense, requires fewer personnel and resources, and deemed highly effective when managing a large band of broodmares on clinical theriogenology • volume 8, number 4 • december 2016 472 open ranges. pregnancy rates are evaluated by the number of mares pregnant following removal of the stallion. risk factors include introduction of a new horse into a herd, significant social and sexual interaction leading to rapid exchange of bodily fluids and an uncontrolled environment. artificial insemination artificial insemination consists of collecting a stallion’s semen with an artificial vagina (av) followed by extension of the ejaculate into semen extender. the sample can then be prepared for immediate insemination or processed for cooling and shipping. alternatively, the sample can be frozen and stored for future use. collection usually occurs off a phantom mount in the presence of a tease mare. when breeding a mare, the sample is deposited manually via a transcervical approach. risk factors include fomite transmission, interstate (and potentially international) spread of disease and exposure of tease mare to different horses monitoring venereal disease in equine breeding programs from an epidemiologic standpoint, monitoring systems are used to identify changes in disease status of a particular population. collection and organization of these data can then be used to evaluate productivity, such as a stallion’s breeding efficiency. aerobic cultures of the external genitalia of stallions swabs of the penis, prepuce, distal urethra (both preand post-ejaculation), and urethral fossa and sinus are routinely performed in thoroughbred breeding sheds. they are commonly done as part of a preseason stallion reproductive examination and will continue through the breeding season (note: frequency and specific sites will vary based upon individual farms). isolation of potentially pathogenic bacteria, especially ß-streptococci and escherichia coli, in otherwise clinically normal and apparently fertile breeding stallions is not uncommon in live-cover breeding sheds in central kentucky. in a recent clinical study involving 15 stallions and 206 mares, the presence of these organisms on the external genitalia of stallions did not appear to transmit to mares as judged by post-breeding uterine cultures taken ~24 hours after mating.4 this study also suggested that stallions with positive cultures do not necessarily experience a reduction in fertility. consequently, treatment of the stallion and mare is not always indicated, which is important due to the potential costs and side effects of therapy. the environment in which a stallion is housed may influence the type of microorganisms harbored on the external genitalia, and should also be considered. when there is concern about the presence of transmission of potentially pathogenic bacteria, breeding strategies can be implemented to abate the risk. in the case of stallions used for live-cover, semen extender containing an appropriate antibiotic can be infused into the uterus prior to or after mating. semen from stallions used for ai should be screened for microorganisms and extended in an appropriate semen extender. for mares bred to these stallions, post-breeding management could entail uterine lavages, infusions and ecbolics. surveillance programs in equine breeding programs surveillance programs differ from monitoring systems in that they entail an ongoing evaluation of data upon which strategies can be established and implemented to mitigate disease. aerobic cultures and cytology of the endometrium commercial stud farms may require a negative or “clean” uterine culture prior to breeding a mare to one of their stallions. clinical bacterial endometritis is a common cause of subfertility in broodmares,12 and identification of these mares prior to mating can prevent a potentially wasted breeding. contemporary data in thoroughbreds suggest that a culture should be combined with a cytology to identify the presence and degree of inflammation, since severe inflammation was associated with a reduction in fertility.13 clinical theriogenology • volume 8, number 4 • december 2016473 prophylactic treatment with a single infusion of an antibiotic after mating has been used in certain areas of high breeding intensity. a pilot-study conducted in 2015 in 39 maiden thoroughbred mares in central kentucky demonstrated a first cycle pregnancy rate of 83.3% in mares infused with 35 ml of saline and 65.0% in those receiving 5 million units of penicillin g (ppg) qs to 35 ml with saline. interestingly, early pregnancy loss—as defined by the initial detection of a vesicle 14-15 days after ovulation but subsequent death or absence of a fetus 42-45 days after ovulation—was 9.5% in mares infused with saline, but 0.0% in mares receiving ppg infusions. screening programs for contagious equine metritis strategies to prevent cem in a particular locale depend on that country’s disease status. for disease-free countries, horses are screened prior to importation and again upon arrival. infected animals must be treated and test negative prior to entering the breeding population. in cem-infected countries, control measures include the following: ‐ screening all stallions prior to the beginning of the breeding season ‐ screening certain mares based upon risk, such as those with copious vulvar discharge and a premature return to estrus ‐ proper hygiene of horses and humans ‐ sanitation of fomites in some countries, this disease has been eradicated through surveillance, quarantine and excluding infected animals from the breeding population. to the authors’ knowledge, no vaccine exists for this disease. due to the fairly recent outbreak of cem, the usa has undergone a reclassification in its status as a cem-free country. as of this writing (june 2016), international transport of horses to and from the usa was permissible by most agricultural ministries provided proper screening and prophylactic treatments were performed prior to introduction of the animal into the country’s breeding population. with respect to testing, culture swabs of the clitoral fossa, clitoral sinus and endometrium should be placed directly into amies with charcoal transport medium. regulations often require serial cultures before declaring an animal cem-free. in addition, a serum complement fixation (cf) test can be performed on mares two to three weeks after mating to determine if they are producing antibodies against t. equigenitalis. these cf tests are commonly done in central kentucky breeding sheds for mares bred by a stallion that had also covered a newly imported mare or filly. similar screening measures can be employed in other breeding programs, such as those utilizing ai. in fact, it may be as if not more important to utilize a similar protocol in these particular sheds. doing so may prevent another cem outbreak like that which occurred in 2008 in the usa. mitigation of equine viral arteritis current estimates suggest that 30-70% of exposed stallions will become persistently infected when exposed to the virus, and are referred to as “shedder” stallions.14 consequently, prevention of the disease is key and the disease has been nearly eradicated in north american thoroughbreds by screening and vaccination. a modified-live virus vaccine is commercially available in the usa (arvac®, zoetis, florham park, nj) and approved for use in breeding stallions. in some breeds that allow ai, breeding with eav-infected semen is permissible with proper documentation of the mare’s vaccination status. certain breed-registries and state agricultural departments have restrictions regarding vaccination of breeding stock and use of eav-infected semen. these requirements can be obtained by contacting the breed registry and local regulatory officials. it appears that the most useful means of mitigating spread of eva are proper hygiene, evaluating the serologic status of breeding animals and targeted vaccination. an excellent description for control of eva is provided by the american association of equine practitioners (aaep),15 and are summarized below: ‐ seronegative stallions ‐ seropositive stallions clinical theriogenology • volume 8, number 4 • december 2016 474 ‐ seronegative mares ‐ seropositive mares ‐ teaser stallions and nurse mares ‐ colt foals and weanlings abatement of coital exanthema (ehv-3) active lesions resulting from ehv-3 infections are readily recognized a small blistering of the mucocutaneous portion of the genitals. lesions take four to six weeks to heal and are characterized by focal areas of depigmentation. gentile cleansing of active lesions may soothe and reduce inflammation. reports of antiviral treatments are sparse. the author has used docanosol cream (abreva®, glaxo-smithkline, philadelphia, pa) in four cases with apparently good results in resolving the acute inflammation associated with the lesions and prevention of future lesions. however, no comparison was made regarding the length of the convalescent period between treated and control animals. transmission to other animals is a concern. due to the contagious nature of this disease, a period of four to six weeks of sexual rest is recommended to prevent further transmission and allow the lesions to heal. resumption of breeding activity can occur once the lesions have healed. they often leave rather characteristic areas of hypopigmentation on the external genitalia. recrudescence of this particular herpes virus is unknown,16 but experimental administration of dexamethasone has been shown to cause reactivation of the virus.17 control is aimed at early detection and withhold from breeding until lesions have subsided. there is no commercially available vaccine against ehv-3 in the usa. dourine diagnosis is made via compatible clinical signs and laboratory testing. the complement fixation test is prone to false positives from other species of trypanosoma, and newer methods of diagnosis, including elisa and pcr technologies are currently being explored for both testing and screening purposes. infected animals should be removed from breeding and culled. treatment with anti-protozoal drugs has been performed in some endemic areas, but they are unproven and even discouraged owing to concern about inapparent shedding animals. suspected cases of dourine should be reported immediately to local usda-aphis officials. impact of stds on north american breeding operations in 2006, eva spread from new mexico to 18 states via either cooled-shipped semen or horse transport, resulting in numerous cases of respiratory illness, abortion, neonatal pneumonia and death.18 the consequences were significant, resulting in substantial economic loss in both the breeding and performance sectors for the affected operations. as described above, surveillance for cem is fairly rigorous. however, an outbreak was triggered in 2008 from a stallion in kentucky. significant resources were expended during the investigation, which included over 1,000 horses screened for cem in 48 states.6 ripple effects were noteworthy, leading to a change in cem-status for the usa and altering import and export regulations. anecdotal reports exist of coital exanthema emerging on stud farms serving a large number of mares. infections with ehv-3 can be problematic because they can temporarily halt a stallion’s breeding activity due to their highly contagious nature. assuming lesions take three to four weeks to heal, the stallion could miss several cycles, causing mares to switch off the stallion. the end result is a reduction in stud fee revenues, which, for some stallions commanding large stud fees (e.g., $30,000+) can translate into a substantial reduction in income. because of the various ramifications of stds, clinicians should be increasingly vigilant and maintain proper protocols for minimizing risk of disease transmission and outbreak. strategies to reduce the risk of stds in equine breeding operations in addition to the monitoring and surveillance strategies listed above, there are other steps that can be taking to prevent stds in breeding operations. clinical theriogenology • volume 8, number 4 • december 2016475 employing experienced and conscientious personnel these are the first-line of defense in breeding sheds. handlers should be vigilant for any signs of disease and alert the stallion or breeding manager with any concerns. maintaining a veterinary presence in breeding sheds among its many advantages, a veterinarian can implement biosecurity strategies and facilitate communication with usda-aphis should any concerns arise. introduction of a new horse to the herd infectious disease testing prior to introduction accompanied by surveillance measures after introduction prevent nose-to-nose contact of teaser stallion with mare or vice versa instead, position mare so the stallion can sniff the flank. expulsion of bodily fluids during teasing use ample and absorbent bedding and remove waste as quickly possible help maintain a clean environment. washing of the mare many stds have characteristic lesions. personnel responsible for preparing the mare should be observant of any abnormalities of the external genitalia, including growths, wounds or discharge. used materials should be discarded and clean ones used prior to washing another mare. suitable methods of washing include the use of a water hose and/or water bucket; practical cotton can be used to cleanse and rinse the mare mild detergent soaps, such as ivorytm, are most commonly used. handling of the mare and stallion proper hand hygiene is instrumental in reducing the risk of disease transmission. all handlers should wear gloves and clean attire. handling of semen every effort should be made to handle semen cleanly and gently to avoid contamination and thermal stress. fomite transmission the various resources used to restrain the horses should have either protective barriers (e.g., sleeves or cellophane wrap on breeding rolls) or cleaned and disinfected (e.g., rinsing chains and twitches in tap water and soaking in dilute [1% chlorhexidine solution]). the lower end of the phantom can be wrapped in cellophane and changed in between horses to prevent contamination with bodily fluids. prophylactic treatment with antimicrobial agents this practice is controversial and the pros and cons carefully weighed prior to implementing due to concerns over antimicrobial resistance. in operations utilizing ai, many semen extenders contain antibiotic agents. however, their use should not preclude proper semen handling and sample preparation. the same is true for keeping the collection area and laboratory clean and organized. clinical theriogenology • volume 8, number 4 • december 2016 476 conclusion venereal disease is an important consideration in equine breeding operations. there are several diseases of known importance, and outbreaks can lead to significant economic and emotional loss. monitoring and surveillance programs should be utilized to screen for diseases, and clinicians should either be familiar with regulations governing their particular locale. finally, risk of transmission can be mitigated to a large degree by proper hygiene and breeding management practices. references 1. cooke c: prophylactic intra-uterine antibacterial therapy. equine vet educ 2015;27:554-555. 2. rota a, calicchio e, nardoni s, et al: presence and distribution of fungi and bacteria in the reproductive tract of healthy stallions. theriogenology 2011;76:464-470. 3. kikuchi n, iguchi i, hiramune t: capsule types of klebsiella pneumoniae isolated from the genital tract of mares with metritis, extra-genital sites of healthy mares and the genital tract of stallions. vet microbiol 1987;15:219-228. 4. cerny k, little t, scoggin c, et al: presence of bacteria on the external genitalia of healthy stallions and its transmission to the mare at the time of breeding by live cover. j equine vet sci 2014;34:369-374. 5. metcalf e: venereal disease. in: mckinnon a, squires e, vaala w, et al., eds. equine reproduction. 2nd ed. ames(ia): wiley-blackwell, 2011. p. 1250-1258. 6. erdman m, creekmore l, fox p: diagnostic and epidemiologic analysis of the 2008-2010 investigation of a multi-year outbreak of contagious equine metritis in the united states. prevent vet med 2011;101:219-228. 7. timoney p: contagious equine metritis. comp immunol microb infect dis 1996;19:199. 8. balasuriya u, snijder e, heidner h, et al: development and characterization of an infectious cdna clone of the virulent bucyrus strain of equine arteritis virus. j gen virol 2007;88:918-924. 9. pascoe r: the effect of equine coital exanthema on the fertility of mares covered by stallions exhibiting the clinical disease. aust vet j 1981;57:111-144. 10. dourine. available at: http://web.oie.int/eng/maladies/technical%20disease%20cards/dourine_final.pdf. 11. couto m, hughes j: sexually transmitted (venereal) diseases of horses in: mckinnon a,voss j, eds. equine reproduction. philadelphia: wiley; 1993. p. 845-854. 12. leblanc m, causey r: clinical and subclinical endometritis in the mare: both threats to fertility. reprod domest anim 2009;44:10-22. 13. riddle w, leblanc m, stromberg a: relationships between uterine culture, cytology, and pregnancy rates in a thoroughbred practice. theriogenology 2007;68:395-402. 14. holyoak gr, broaddus cc, timoney pj: equine viral arteritis: current status and prevention. theriogenology 2008;70:403-414. 15. aaep: biosecurity guidelines for control of venereally transmitted diseases. am assoc equine pract. 16. lu k, moerresey p: infectious diseases in breeding stallions. clin tech equine pract 2007;6:285-290. 17. barrandeguy m, vissani a, olguin c: experimental reactivation of equine herpesvirus-3 following corticosteroid treatment. equine vet j 2008;40:593-595. 18. timoney p, creekmore l, meade b, et al: multi-state occurrence of eva. proc annu meet us anim health assoc 2006;354-362. clinical theriogenology • volume 8, number 4 • december 2016477 clinical theriogenology • volume 8, number 4 • december 2016 478 omniblank: 2011: evaluation of endometrial thickness near timed ai using ultrasound evaluation of endometrial thickness near timed ai using ultrasound alexandre h. souza, henderson ayres, julio o. giordano, roberta m. ferreira, paulo d. carvalho, jerry n. guenther, milo c. wiltbank department of dairy science, university of wisconsin, madison, wi in cattle and other mammalian species, an ideal uterine environment is essential to prepare the uterus for embryonic implantation and growth.1 many factors such as hormonal milieu, nutritional status, clinical or subclinical uterine infections, and potentially other factors may alter uterine environment resulting in decreased fertility. methods to accurately evaluate uterine status prior to fertilization could be useful for predicting fertility, validly determining optimal treatments for individual cows, and further understanding factors that regulate fertility in cattle or other species. some techniques such as uterine cytology, biopsy, reproductive tract scoring (rts),2 and ultrasound examinations have been used to assess fertility in many species including humans.3 in bovines, ultrasound evaluation of the reproductive tract has some advantages over the other techniques because it provides a rapid, noninvasive, and accurate diagnosis of uterine status. although, rts has been shown to be an important tool that can be used to predict fertility performance of heifers,2 it tends to be a more subjective technique as compared to ultrasound measurement of uterine and ovarian structures. interestingly, in women, several studies have reported the use of ultrasound as a tool for predicting fertility. for example, ultrasonographic evaluation of the endometrial thickness (et) has been used for over 20 years to evaluate endometrial receptivity during assisted reproduction programs for humans.3 nevertheless, there is surprisingly little information in the scientific literature relating ultrasound measurement of et to fertility in cattle. recently we described a technique to evaluate endometrial thickness as a predictor of fertility in lactating dairy cows.4 this manuscript is a further analysis of this ultrasound technique to evaluate endometrial thickness. johnson et al5 studied the influence of steroid hormones in ewes. they observed that uterine weights were greater near estrus compared to mid-luteal phase, and that this increase in weight was due to estrogen-induced endometrial tissue hypertrophy rather than hyperplasia. this is consistent with data from other species showing hormonal regulation of uterine morphology and function.6 thus, ultrasonographic measurement of et near ovulation might be a good indicator of hormonal environment (low progesterone [p4] and/or high estradiol [e2]) and could be used to estimate whether the uterus has been exposed to adequate concentrations of hormones compatible with optimal fertility. although cows do not lose endometrial tissue due to menses, the thickness of the endometrium7 and certain histological aspects8,9 vary considerably during the estrous cycle. an early study7 using holstein heifers described remarkable ultrasonographic changes in the endometrium of the uterine body near ovulation. these authors reported an increase in et at the time of expected luteolysis, with maximal et found on the day before ovulation. a schematic representation of their results is shown in figure 1. a number of studies have described the decrease in et and changes in endometrial echotexture after ovulation, probably due to increasing circulating p4.7,10,11 we observed substantial variation in et in lactating dairy cows during the ovsynch protocol when we were doing routine ultrasonographic examination of cows in the university of wisconsin herd. clinical theriogenology • volume 3 number 4 • december 2011547 day of the estrous cycle -3 0 3 6 9 12 15 18 21 e nd o m e tr ia l t hi ck n es s (m m ) 8 10 12 14 16 18 ovulation ovulation figure 1. schematic representation of the changes in endometrial thickness in the uterine body of heifers throughout the estrous cycle. data from pierson and ginther.7 figure 2. schematic of the method used for measuring et. endometrial thickness was determined by ultrasound, with measurements done using electronic calipers in a 90-degree cross-sectional frozen image acquired at about 2 cm from the internal uterine body bifurcation. minimum pressure was applied with the ultrasound probe on top of the uterus to avoid deformation of the uterine horns when performing ultrasound measurements of et. endometrial thickness was defined as the distance between the edge of the endometrial lumen to the visualized interface between the endometrium and myometrium. the above results by pierson and ginther7 were obtained by et measurements on the body of the uterus. we decided to measure et in the uterine horns in all of our experiments.4 the uterine horns were an excellent location for validly measuring et since the internal uterine bifurcation could be used as a guide to provide a consistent location for obtaining measurements from both horns for each animal (see figure 2). within an animal, the et was similar between horns, presumably because cows enrolled in the myometrium perimetrium90o endometrium clinical theriogenology • volume 3 number 4 • december 2011 548 study were far enough in lactation to allow for full regression of the previously gravid horn.4 the average for the two horns was used as a final score for et. further, the uterine horns were chosen for measurements in these experiments since a 90o cross-sectional ultrasound image was easier to obtain from uterine structures that are deeper in the abdominal cavity due to the physical space needed for perpendicular placement of the probe on the uterus (see figure 2 for schematic of measurement method). finally, the distance between the uterine lumen and myometrium seemed easier to define for measurements using the uterine horns than the uterine body. endometrial area11 or endometritis12 have previously been examined by ultrasound of the uterine horns in cattle. our first experiment evaluated the changes in et associated with the periovulatory period (figure 3). eight lactating holstein cows (four multiparous and four primiparous) were housed in tie-stall barns. cows were 150.9 ± 1.4 dim and had milk production of 30.0 ± 0.3 kg/day. all cows received the ovsynch protocol (gonadotropin releasing hormone (gnrh) 7d – prostaglandin f2α (pgf2α) 72h gnrh). based on weekly ultrasound examinations at the university herd, all animals were cycling regularly and presented no uterine disorders prior to ovsynch. in addition, all cows had at least one corpus luteum (cl) and circulating progesterone > 1ng/ml at the beginning of ovsynch. all cows had cl regression following pgf2α with circulating p4 decreasing to less than 1 ng/ml within 24 h. as shown in figure 1, circulating e2 averaged 3.1 pg/ml at the time of pgf2α treatment and was similar at 1 d after pgf2α before increasing to 3.7 pg/ml at 2 d after pgf2α and to 5.1 pg/ml at 3 d after pgf2α, near the time of last gnrh treatment. at 1 d after the final gnrh treatment, circulating e2 decreased from 2.7 pg/ml to 1.2 pg/ml at 2 d after gnrh treatment. the et increased rapidly after pgf2α treatment from ~ 7 mm to ~ 9.5 mm by 24 h after pgf2α. the et remained similar at 2 d (9.2 mm) and 3 d (9.1 mm) after pgf2α. however, following the final gnrh treatment there was a decrease to 8.0 mm at 1 d after gnrh and 7.4 mm at 2 d after gnrh (figure 1). using data from each individual cow on each day, we found a negative correlation between circulating p4 and et (r = 0.28; p = 0.05); whereas, circulating e2 was positively correlated with et (r = 0.33; p = 0.02). we found a more acute increase in et within 24 h after induced luteolysis than described by pierson and ginther7 during the expected time of luteolysis in natural estrous cycles. the increase in et from 6.9 mm to 9.4 mm corresponds to a 2.5-fold increase in endometrial tissue volume, if calculated as a spherical measurement. this is an astounding increase in volume of endometrial tissue and probably primarily accounted for by increases in endometrial blood flow. the decrease in p4 is the primary hormonal value associated with this dramatic increase in et. an increase in circulating e2 was not observed during the initial rise in et; however, it seems likely that elevated circulating e2 needs to be present to facilitate the increase in et. the reduction in et near the time of ovulation corresponds to a decrease in e2 with no detected change in p4, providing further evidence that elevated e2 is important for elevated et. clinical theriogenology • volume 3 number 4 • december 2011549 days after pgf2 treatment 0 1 2 3 4 5 6 e nd om et ria l t hi ck ne ss ( m m ) 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 pgf2 gnrh 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 c irculating p 4 ( ng/m l) 3.1 3.1 3.7 5.1 2.7 1.2 circulating e2 ( pg/ml) 1.7 0.7 0.4 0.3 0.2 0.6 6.9 9.4 9.2 9.1 8.0 7.4 a b b b b b x x x y y y c c cd d c e figure 3. profile of endometrial thickness (mm), circulating p4 (ng/ml), and circulating e2 (pg/ml) from pgf2α to 2nd gnrh treatment during ovsynch in lactating dairy cows (n = 8). within each profile, averages without common superscript letters are different; p<0.05. our major experiment was performed with lactating holstein cows, in a total of 942 breedings (581 multiparous and 361 primiparous). there were no significant differences between primiparous vs. multiparous cows for distribution of et (figure 4). endometrial thickness (mm) 7 8 9 10 11 12 13 f re q ue nc y (% ) 0 5 10 15 20 25 30 35 primiparous (n = 361) multiparous (n = 581) figure 4. distribution of endometrial thickness in primiparous vs. multiparous dairy cows. probably the most interesting findings in our study are illustrated in figure 5. even though changes in et during the estrous cycle in cattle were first described more than two decades ago,7 we found only clinical theriogenology • volume 3 number 4 • december 2011 550 two studies12,13 that attempted to associate ultrasound measurements of the bovine endometrium with fertility. both of these studies evaluated the endometrium of holstein cows in an attempt to diagnose subclinical endometritis in early lactation (40 to 60 days). uterine cytology was used to define endometritis with results compared with ultrasound findings. overall, maximal et was associated with presence of endometritis. however, cows in these previous studies were not at the same stage of the estrous cycle and therefore the wide variation in et throughout the estrous cycle that was described previously7 and that we observed in our study could not be taken into account. in the current study, we did not enroll cows with detectable uterine disorders in the ovsynch program making the fundamental questions addressed in our study different from the questions addressed in previous studies. our results showed a linear increase in pregnancies per artificial insemination (ai) from less than 20% to more than 40% when et increased from 6 to 10 mm (figure 5b). these differences in fertility were associated with differences in circulating e2 and p4 concentrations. the p4 concentrations were greater in cows with et of < 7 or 8 mm. this is due to a lack of luteolysis in a greater percentage of cows that were found to have lower et (see table). this is further illustrated by evaluating the circulating p4 after removal of cows that did not undergo luteolysis (cows with p4 > 0.5 ng/ml). although there is a difference between cows with thinner vs. thicker et prior to removing these cows (see table), there was no difference in p4 for cows with thinner et (0.127 ± 0.008 ng/ml; n = 147) vs. thicker et (0.134 ± 0.004 ng/ml; n = 482; p > 0.10) after removal of cows without luteolysis. this demonstrates that the reason for higher p4 in cows with thinner et in the original comparison (prior to removal of cows with p4 > 0.5 ng/ml) was due to differences in percentage of cows without complete luteal regression and not due to higher p4 in all cows in this group. we also found that lower fertility in ovsynch-treated cows with lower et seemed to be related to size of the ovulatory follicle. cows ovulating smaller follicles after ovsynch might have lower fertility due to lower ovulation rates, due to lower circulating e2 near ovulation, and due to impaired cl function after ovulation. for instance, vasconcelos et al14 observed that cows that were intentionally induced to ovulate smaller follicles had lower circulating e2 prior to ai, had smaller cl after ai, lower circulating p4 after ai, an increase in short luteal phases, and poorer fertility. any of these same problems could be present in cows with lower et since they ovulated smaller follicles, had lower circulating e2 prior to ai, and had lower fertility. cows with lower et also had about 12% fewer cows that ovulated to the second gnrh of ovsynch. this would clearly lower fertility. the luteolysis rate was also lower in the cows with lower et resulting in an elevated circulating p4. thus, the lower et is indicative of cows that were not properly synchronized by the ovsynch protocol and do not have the proper environment for optimal fertility. clinical theriogenology • volume 3 number 4 • december 2011551 circulating e2 endometrial thickness (mm) 7 8 9 10 11 12 13 c irc ul at in g p 4 (n g/ m l) 0.1 0.2 0.3 0.4 0.5 0.6 circulating p4 1.5 2.0 2.5 1.0 3.0 3.5 c irculating e 2 (pg/m l) * * ** ** + + + + + a) circulating e2 and p4 endometrial thickness (mm) 7 8 9 10 11 12 13 p /a i ( % ) 0 10 20 30 40 50 n=90 n=105 n=105 n=260 n=189 n=129 n=64 a b c c c c bc b) pregnancy per ai figure 5. a) relationship between endometrial thickness (et) and circulating concentrations of e2 and p4 at 48 h after the pgf2α treatment of the ovsynch protocol. lower et (< 8 mm) was associated with lower e2 and greater p4 concentrations. b) relationship between et and pregnancies per ai (p/ai). lower et (< 8 mm) was associated with lower p/ai. clinical theriogenology • volume 3 number 4 • december 2011 552 table. differences in various measures in lactating dairy cows with thinner (< 8 mm) or thicker (>8 mm) endometrium. endometrial thickness (mm) ≤ 8 > 8 p-value bcs (scale 1-5) 2.79±0.03 2.84±0.02 0.19 (174) (703) anovular before 1st ai (%) 24.7 15.3 <0.01 (21/85) (67/438) ovulation to 2nd gnrh (%) 85.1 97.6 <0.01 (166/195) (729/747) p/ai (%)* 24. 44.0 <0.01 (48/195) (329/747) luteolysis rate (%)** 89.2 96.1 <0.01 (174/195) (718/747) circulating p4 (ng/ml) 0.32±0.05 0.18±0.01 <0.01 (177) (678) circulating e2 (pg/ml) 1.6±0.2 2.5±0.1 <0.01 (151) (606) * pregnant per ai ** circulating p4 < 0.5ng/ml at tai our results in cattle are surprisingly similar to reported results in humans. most of the studies done in women indicated that et of less than 7 mm on the day of ovulation is not likely to result in a pregnancy. for example, some authors15-19 have reported a minimal threshold of 5 to 8 mm, below which no pregnancies are likely to occur in women. another study20 found that women with greater et have greater fertility. interestingly, baerwald et al3 described a positive association between follicular waves and endometrial growth, assessed by ultrasound, in women. in contrast, some studies have found that ultrasound evaluations of the endometrium are not specific enough to accurately predict future fertility in women.20,21 these conflicting results might be related to the day of the ovulation cycle that the endometrium was measured, accuracy and repeatability of et measurements with ultrasound within and between different technicians, or even to the measurement techniques used. nevertheless, the strong relationship with fertility that we found in cattle, as well as the previous relationship found between et and fertility in humans supports the need for future research in this area. from a practical standpoint studies need to be performed that evaluate the variation among technicians, farms, and types of ultrasound machines and probes used for evaluations. in addition, it is not clear whether et measurements will be of utility for predicting fertility in cows bred to a natural estrus due to potentially greater synchrony of hormonal concentrations during natural estrus. in conclusion, assessment of et near ovulation in a timed ai program was a surprisingly good predictor of ovulation failures and pregnancy success. this measurement can be performed routinely in order to detect cows with lower fertility. this finding may open the way for a broader use of ultrasound for measuring et in practical cattle reproductive management. in addition, fertility research could be enhanced by using this technique to develop more optimized timed ai programs and for understanding clinical theriogenology • volume 3 number 4 • december 2011553 the molecular and physiological mechanisms that underlie optimal uterine fertility. after concluding these studies, it remains to be determined what hormonal, cellular, and molecular mechanisms underlie the reduction in fertility in cows with lower et. references 1. gray ca, taylor km, ramsey ws, et al: endometrial glands are required for preimplantation conceptus elongation and survival. biol reprod 2001;64:1608-1613. 2. holm de, thompson pn, irons pc: the value of reproductive tract scoring as a predictor of fertility and production outcomes in beef heifers. j anim sci 2009;87:1934-1940. 3. baerwald ar, pierson ra: endometrial development in association with ovarian follicular waves during the menstrual cycle. ultrasound obstet gynecol 2004;24:453-460. 4. souza ah, silva epb, cunha ap, et al: ultrasonographic evaluation of endometrial thickness near timed ai as a predictor of fertility in high-producing dairy cows. theriogenology 2011;75:722-733. 5. johnson ml, redmer da, reynolds lp: effects of ovarian steroids on uterine growth, morphology, and cell proliferation in ovariectomized, steroid-treated ewes. biol reprod 1997;57:588-596. 6. nayak nr, brenner rm: vascular proliferation and vascular endothelial growth factor expression in the rhesus macaque endometrium. j clin endocrinol metab 2002;87:1845-1855. 7. pierson ra, ginther oj: ultrasonographic appearance of the bovine uterus during the estrous-cycle. j am vet med assoc 1987;190:995-1001. 8. dhaliwal gs, murray rd, rees em, et al: quantitative unbiased estimates of endometrial gland surface area and volume in cycling cows and heifers. res vet sci 2002;73:259-265. 9. wang ck, robinson rs, flint apf, et al: quantitative analysis of changes in endometrial gland morphology during the bovine oestrous cycle and their association with progesterone levels. reproduction 2007;134:365-371. 10. bonafos ld, kot k, ginther oj: physical characteristics of the uterus during the bovine estrous cycle and early pregnancy. theriogenology 1995;43:713-721. 11. jimenez-krassel f, folger jk, ireland jlh, et al: evidence that high variation in ovarian reserves of healthy young adults has a negative impact on the corpus luteum and endometrium during estrous cycles in cattle. biol reprod 2009;80:1272-1281. 12. barlund cs, carruthers td, waldner cl, et al: a comparison of diagnostic techniques for postpartum endometritis in dairy cattle. theriogenology 2008;69:714-723. 13. haskell jk, hammon ds, holyoak gr:. ultrasonographic characteristics of the uterus of holstein cows with late endometritis. j dairy sci (suppl 1) 2004;87:183-183. 14. vasconcelos jlm, sartori r, oliveira hn, et al: reduction in size of the ovulatory follicle reduces subsequent luteal size and pregnancy rate. theriogenology 2001;56:307-314. 15. abdalla hi, brooks aa, johnson mr, et al: endometrial thickness a predictor of implantation in ovum recipients. hum reprod 1994;9:363-365. 16. alam v, bernardini l, gonzales j, et al: a prospective-study of echographic endometrial characteristics and pregnancy rates during hormonal replacement cycles. j assist reprod genet 1993;10:215-219. 17. check jh, nowroozi k, jung c, et al: influence of endometrial thickness and echo patterns on pregnancy rates during in vitro fertilization. fertil steril 1991;56:1173-1175. 18. coulam cb, bustillo m, soenksen dm, et al: ultrasonographic predictors of implantation after assisted reproduction. fertil steril 1994;62:1004-1010. 19. gonen y, casper rf, jacobson w, et al: endometrial thickness and growth during ovarian stimulation a possible predictor of implantation in vitro fertilization. fertil steril 1989;52:446-450. 20. glissant a, demouzon j, frydman r: ultrasound study of the endometrium during in vitro fertilization cycles. fertil steril 1985;44:786-790. 21. yuval y, lipitz s, dor j, et al: the relationships between endometrial thickness, and blood flow and pregnancy rates in in-vitro fertilization. hum reprod 1999;14:1067-1071. clinical theriogenology • volume 3 number 4 • december 2011 554 2017: a field study to compare post-thaw sperm progressive motility and pregnancy rate using brahman bull semen frozen in milk based extender containing egg yolk or soybean lipids extract   a field study to compare post-thaw sperm progressive motility and pregnancy rate using brahman bull semen frozen in milk based extender containing egg yolk or soybean lipids extract fernando perea,a roberto a. palomares,b,c maria s. ferrer,c rumualdo gonzález,d,e andrzej t. palasz,f jormaryory rosales,a h. hector nava,d alejandro hoyos-jaramillo,b eleazar soto-bellosod,e adepartamento de ciencias agrarias. universidad de los andes, trujillo, venezuela;bdepartments of population health and clarge animal medicine, college of veterinary medicine university of georgia, athens ga; dfacultad de ciencias veterinarias, universidad del zulia, maracaibo, venezuela; evenezolana de inseminación artificial y transplante de embriones (viateca), villa del rosario, venezuela; fdepartamento de reproducción animal. inia, madrid, spain abstract the objective of this study was to compare post-thaw sperm progressive motility and pregnancy rate using brahman bull semen frozen in milk based extender containing egg yolk or soybean lipids extract. three semen ejaculates from two brahman bulls were divided into three aliquots for dilution with one of three milk-based semen extenders: complete egg yolk (ce), processed egg yolk (pe) or soybean extract (se) before freezing. mean post-thaw progressive motility was not different between soybean extract and processed egg yolk treatments, except for two time points (0.5 and 1 h) when soybean extract was superior to processed egg yolk (p <0.01). post-thaw progressive motility was consistently lower in frozen semen in complete egg yolk during the 2.5-h incubation period compared to processed egg yolk and soybean extract (p<0.05). pregnancy rate was not significantly different among extenders, with values of 35.7, 40.1 and 40.6 % for complete egg yolk, processed egg yolk and soybean extract, respectively. in conclusion, the use of semen extenders containing processed egg yolk or soybean extract resulted in higher post-thaw sperm progressive motility than complete egg yolk based extender. pregnancy rates using brahman bull frozen semen containing complete egg yolk, processed egg yolk or soybean extract, did not differ. procedures to obtain the processed egg yolk seem to diminish the detrimental compounds of egg yolk. the use of soybean extract represents a promising alternative for milk-based bull semen extender and provides additional benefits reducing the potential risk for microbial contamination. keywords: semen extenders, egg yolk, soybean extract, sperm progressive motility, pregnancy rate introduction the quality of frozen semen used for artificial insemination (ai) is a critical point that affects cattle reproductive performance and profitability of the farms.1,2 the cryopreservation process has been shown to reduce sperm viability and impair sperm function. it is commonly accepted that a substantial number of sperm are damaged during cryopreservation.3 cold shock, osmotic stress, ice crystal formation and oxidative damage are considered main sources of sperm cryoinjury.4 therefore, the freezing protocol and selection of semen extender are key steps to maintain optimal quality of frozen semen. egg yolk has been used as an extender component extensively. it has been categorized as a biological compound that supports sperm viability and fertilization capacity of frozen bull semen.5 it is considered the most effective agent to protect sperm against cold shock during cryopreservation.6 egg yolk and particularly the low-density lipoprotein fraction (ldl) are reported to have a cryoprotectant effect, since it prevents damage of membrane phospholipids, thus increasing the sperm tolerance to cold shock.6,7 in addition, it has been suggested that egg yolk lipoproteins bind bovine seminal plasma (bsp) proteins,8 which contain factors that are detrimental for sperm fertilization ability. the effect of bsp on sperm function is time and concentration dependent; therefore, continuous exposure of sperm to bsp may damage the cell membrane by increasing cholesterol efflux.6,9 however, the precise mechanism through which ldl protects the spermatozoa from cold shock remains unclear. previous studies have reported that the beneficial effects of ldl during the cryopreservation process depend on lipoprotein concentration. moussa et al10 reported that the optimal concentration of clinical theriogenology • volume 9 number 1 • march 201737   ldl in semen extender is 8%, and that an increase to 10% may impair post-thaw sperm function. other components in egg yolk, such as high-density lipoprotein (hdl) affect cell respiratory activity and sperm motility.3,11-13 moreover, the use of non-pasteurized egg yolk in semen extender represents a risk of bacterial contamination,14,15 in addition to the lack of consistency in the individual quality standards due to differences inherent to egg yolk composition depending on the storage period after laying.16 the use of chemically defined semen extenders (not originated from animal tissues) containing only the protecting fraction of egg yolk (e.g. ldl) could be a valuable alternative for the ai industry.17 another alternative is the use of extenders containing soybean extract in substitution for egg yolk. semen extenders based on soybean extract provide a higher consistency of the components and eliminate the risk for bacterial contamination.16 in previous reports, soybean extenders have shown higher post-thaw motility compared to egg yolk based diluent.2,17 some studies have reported a higher non-return rate after using semen that contains a soybean extract based extender.18 however, other studies did not show a positive effect of soybean extract on reproductive performance.15,19-21 in the present study we hypothesized that the use of semen extenders containing egg yolk or soybean extract will result in different post-thaw sperm progressive motility and pregnancy rate after artificial insemination using semen from brahman bulls. the aim of this study was to compare post-thaw sperm progressive motility and pregnancy rate using brahman bull semen frozen in milk based extender containing egg yolk or soybean lipids extract. materials and methods semen collection and processing semen samples were obtained from two brahman bulls (bulls 34 and 56), which were housed on an ai center, located in the county rosario de perija, zulia state, venezuela, under dry tropical forest conditions (mean rainfall: 1147 ml/year; temperature: 27.9°c; relative humidity: 74 %, and wind velocity: 4.6 km/h). bulls had four days of sexual rest between collections. semen was collected using artificial vagina (av) and a steer for sexual stimulation. the inner rubber liner of the av was filled with warm water (45°c). a small amount of non-spermicidal lubricant was applied to the inner liner before the collection was performed. after 3-4 false mounts for sexual stimulation, the penis was grasped through the sheath and directedt to the opening of the lubricated av. immediately after collection, the semen samples were assessed for volume, sperm concentration and percentage of sperm progressive motility and percentage of morphologically normal sperm by an experienced technician according to the guidance by the society for theriogenology (sft).22 volume was assessed using a 10 ml tube. individual sperm progressive motility was visually evaluated using a phase contrast microscope (olympus, bx20) under 400x magnification. sperm concentration was determined using a spectrophotometer following instructions from the manufacturer (spermacue, minitube, germany). ejaculates with >75% progressive motility, >85% morphologically normal sperm, and > 750 million sperm/ ml were used for this study. the research protocols used in this study were reviewed and approved by the scientific and animal care committee of the consejo de desarrollo científico, humanístico, tecnológico y de las artes (cdchta), universidad de los andes. three semen ejaculates from each bull that met the minimum requirements for cryopreservation, were divided into three aliquots for dilution with the experimental semen extenders: complete egg yolk (ce), processed egg yolk (pe) or soybean extract (se). each extended semen aliquot was frozen using a conventional two-step freezing protocol. briefly, a solution a (without glycerol) which was maintained at 37°c, was added and then refrigerated at 5°c for 60 minutes (equilibration period). after that, precooled solution b (containing 14% glycerol) was added at a 1:1 (v:v) ratio in four fractions at 15 minute intervals at 5°c (within a refrigerator) to reach a final glycerol concentration of 7%. the solutions a and b were added to obtain a final sperm concentration of 30 x 106 spermatozoa/ml. extended semen was packed in pre-cooled 0.54 ml straws, so that each straw contained 15 x 106 spermatozoa. straws were frozen in liquid nitrogen vapors at -120°c for 5 minutes and then plunged in liquid nitrogen at -196°c. straws were stored at -196 c until use. clinical theriogenology • volume 9 number 1 • march 2017 38   extender preparation semen was frozen in three different low-fat milk (1%) based extenders containing 10 mg/ml of fructose and supplemented with: 8% of whole egg yolk (ce extender), 8% processed egg yolk (pe extender), and 10% (7.3 mg/ml) of phospholipids of soybean-origin rich in phosphatidylcholine (bioniche inc., beleville, ontario, canada; se extender). egg yolks from fresh chicken eggs were carefully separated from albumin and then homogenized by blending and refrigerated at 7°c. egg yolk was not further processed for the ce extender. for the pe extender, egg yolk was diluted 1:1 (v:v) in an isotonic saline solution (0.9% sodium chloride; w/v). after homogenization, egg yolk solution was centrifuged at 3,000 rpm for one hour at 5°c to remove the egg yolk granules. the resulting supernatant was transferred to a new tube and again centrifuged at 3,000 rpm for one hour at 5°c. the new supernatant fraction was then used to prepare the pe extender. all three extenders were supplemented with 1000 iu of penicillin, 1 mg/ml of streptomycin and 150 μg/ml of lincomycin. post-thaw progressive sperm motility evaluation seventy two hours after freezing, six straws per extender [one of each extender (ce, pe, and se), ejaculate (three different ejaculates) and bull (two bulls, 34 and 56)] were individually thawed in a water bath at 37ºc for 30 seconds. the thawed semen samples were transferred into 1 ml eppendorf tubes and incubated in a water bath at 37°c for 2.5 h. semen from each straw was evaluated twice at 0, 0.5, 1.0, 1.5, 2.0 and 2.5 hours after thawing to determine the progressive sperm motility visually using a phasecontrast microscope (400x, olympus, bx20) equipped with a heating plate (37°c). sperm progressive motility was determine as the percentage of sperm crossing the microscopic field linearly, according to the sft guidance.22 one experienced technician, who was blinded to the treatments (semen extenders) evaluated all samples. field study. location and cattle management six hundred and sixteen multiparous crossbred brahman cows were included in this study. cows were artificially inseminated on detected estrus as described below. each cow was inseminated once with frozen semen using one of the three semen extenders. allocation to extender treatment was done randomly. the number of cows allocated into each treatment was distributed as follows: ce= 207 cows; pe= 212 cows; se= 197 cows. only cows that received their first artificial insemination (first service cows) were considered in this study. artificial inseminations were performed during all year round. seasons were classified according to the accumulative rainfall; dry: december-march (rainfall: 131.6 ml; temperature: 28°c; relative humidity: 71.5 %; wind velocity: 6.0 km/h); intermediate: april-july (rainfall: 637.1 ml; temperature: 28.6°c; relative humidity: 73.6 %; wind velocity: 5.3 km/h); humid: augustnovember (rainfall: 881.3 ml; temperature: 28°c; relative humidity: 77.1 %; wind velocity: 4.5 km/h). cows were housed in a commercial farm located in machiques de perijá county, zulia state, venezuela; in an area classified as sub-humid tropical forest (mean daily temperature: 28.3°c, relative humidity: 6580% and mean rainfall: 1650 ml/year), characterized by a bimodal rainfall pattern with two high rain peaks in may and october. cows were milked by hand twice daily. after each milking, they were suckled by their calf for 1 h, until the calf was weaned at seven months of age. daily average milk production of the cows was 5.4 kg/day. cows grazed german grass (echinochloa polystachya). water and mineral supplementation were offered ad libitum and during the dry season, cows received supplementation (2 kg/cow) of 50% guinea hay, 30% chicken litter, 10% corn flour, and 10% molassesurea mix (16% crude protein). the herd health program was based on diagnostic tests, periodic deworming, vaccinations and biosecurity measures to prevent common diseases that affect the herds at the maracaibo lake basin in venezuela, including brucellosis, leptospirosis, infectious bovine rhinotracheitis, bovine viral diarrhea virus, campylobacteriosis, and clostridial diseases. additionally, it also included the prevention of mastitis through cleaning and disinfection protocols during milking. clinical theriogenology • volume 9 number 1 • march 201739   estrus detection, artificial insemination and pregnancy diagnosis estrus detection was done by visual observation of estrous behavior for a period of 1 h each morning (06:00–07:00) and afternoon (18:00–19:00) and with the help of teaser bulls that were surgically altered with resection of both epididymis tails and penile deviation, so that intromission could not occur. cows were artificially inseminated by two experienced technicians (a and b) 12 h after first detected in estrus according to the am-pm insemination rule. pregnancy diagnosis was performed by an experienced veterinarian by palpation per rectum 45 to 60 d after service. after calving, the voluntary waiting period was 30 days. cows were inseminated after 30 days postpartum if uterine involution was completed and there were no signs of any ovarian or uterine pathology. pregnancy rate was calculated as the number of pregnant cows 45 to 60 days after ai divided by the number of cows inseminated, multiplied by 100. statistical analysis data were analyzed using the statistical analysis system (sas, version 9.2). analysis of variance was performed to compare post-thaw progressive sperm motility among groups for each day. differences among means were compared by the lsd procedure of sas. pregnancy rate was analyzed using proc logistic and differences were detected using chi-square test from sas. the following explanatory variables were considered: semen extender (ce, pe and se), bull (34, 56), inseminator (a, b) and season (dry, intermediate and humid). results post-thaw sperm progressive motility mean post-thaw sperm progressive motility was not different between se and pe treatments, except for two time points (0.5 and 1 h) when se was superior to pe (p < 0.01; figure 1). post-thaw progressive motility was consistently lower in semen frozen in ce at all time points during the 2.5-h incubation period compared to pe and se (p < 0.01 from 0 to 1.5 hours and p < 0.05 at 2 and 2.5 hours; figure 1). moreover, the decline in sperm motility after 2.5 hours of incubation was more pronounced in semen with ce (26.6 %) compared to pe (20 %) and se (13.4 %; figure 1). consequently, after 2.5 h incubation sperm motility was greater in se (56.6 %, p < 0.01) and pe (48.3 %, p < 0.05) than in ce (35.0 %) extender (figure 1). the effect of semen extender on sperm motility was observed in both bulls. no differences were found between bulls (figure 2). pregnancy rate pregnancy rate was not significantly different among extenders with values of 35.7 % (74/207), 40.1 % (85/212) and 40.6 % (80/197) for ce, pe and se, respectively. there was no significant effect of bull or the interaction bull x treatment on pregnancy rate (p > 0.05; table 1). inseminator b had a higher pregnancy rate than inseminator a (44.5 vs 33.2 % p < 0.01; table 1). for inseminator b, pregnancy rate tended to be greater when using semen containing pe (48.6 % p ≤ 0.05) and se (50.0 % p ≤ 0.02) compared to ce (35.9 %; table 1). in contrast, pregnancy rate did not differ among extenders for inseminator a. for all treatments, cows inseminated during the dry season had a higher pregnancy rate than those inseminated during the humid season, (46 vs 28 %, respectively; p < 0.01; table 2). there was no significant effect of the interaction season x treatment on pregnancy rate. discussion under the conditions of this study, post-thaw sperm progressive motility was significantly higher for extenders containing processed egg yolk or soybean compared to extender containing complete egg yolk. previous reports have demonstrated that deleterious components in egg yolk can negatively affect bull sperm motility.4,23 phospholipids, lecithin and lipoprotein fractions contained in egg yolk might form yolk granules that impair sperm movement, resulting in lower post-thaw sperm progressive motility when semen is evaluated in vitro.11,13 in the present study, dilution and processing of egg yolk apparently clinical theriogenology • volume 9 number 1 • march 2017 40   decreased the concentration of harmful components or yolk granules that mechanically altered in vitro motility patterns. however, it appears that the remaining ldl present in the processed egg yolk extender provided appropriate conditions to improve post-thaw sperm motility, as previously reported.10 the positive effects of ldl on sperm motility might be associated with sequestration of proteins in seminal plasma6,8 or adhesion to the sperm membrane.24,25 in the present study, the use of soybean based extender resulted in an improved post-thaw sperm motility of brahman bull semen when compared to the extender containing complete egg yolk. these results might imply a great potential of chemically defined semen extenders with vegetal additives as an alternative to substitute semen extenders of animal origin which are associated with biosafety issues.14,15 in a previous study, substitution of soy milk based extender for egg yolk based extender for bovine semen cryopreservation did not result in significant differences in post-thaw motility, viability, membrane integrity, acrosome integrity and cryo-capacitation.26 further, aires et al18 reported similar sperm-zona pellucida binding capacity and progesterone-induced acrosome reaction for egg yolk and soybean extenders. however, in that study post-thaw sperm motility was significantly lower when semen was extended in the tris-egg yolk diluent. in the present study pregnancy rate was not affected by the semen extenders used for cryopreservation. the results indicate that frozen semen extended with soybean or egg yolk based extenders had similar fertilization ability in vivo, resulting in similar pregnancy outcomes in crossbred brahman cows under tropical conditions. in spite of the observed in vitro differences in mean motility among extenders, post-thaw motility was acceptable under commercial standards with all treatments. moreover, if the lowered post-thaw motility were associated with mechanical interference of yolk granules on motility, this effect could be counteracted during dilution of semen with female genital tract secretions. this might have possibly contributed to the lack of difference in fertility. similar to the results of the present study, a field insemination trial using frozen semen from holstein bulls revealed that extenders containing egg yolk or soybean extract showed similar non-return rate at 56 days after ai.27 in contrast, another field study reported a higher pregnancy outcome for semen extended in soybean lecithin based extender compared to that for the egg yolk extender.18 the results presented here also contrasts with previous studies performed by van watgtendonk et al.17 where semen extended with a commercial soybean extract-containing extender resulted in a significant reduction in the fertility (56-day non-return, estimated conception and calving rates) of bovine frozen semen. in subsequent trials, the authors observed that the sperm motility and survival were significantly lower for semen frozen in a soybean-based extender than in egg yolk-based extender.17,27 comparable results have been previously reported where progressive motility of semen containing the soybean-based extender, biociphos® was lower compared to a tris-egg yolk diluent.2,19,21 additionally, in the study performed by thun et al,2 the use of a soybean-based extender resulted in increased proportion of secondary sperm defects and decreased osmotic resistance. amirat et al23 observed that both extenders containing egg yolk or soybean were relatively non-toxic to cells during the first hour of incubation. however, acrosome and plasma membrane alterations occurred after 4 h of incubation in semen diluted with soybean-based extender. the differences observed between the results of the current trial and previous studies could be associated with individual variation, differences in the method to assess progressive motility (visual vs computerized), breed of bulls (b. taurus vs b. indicus) and frequency of semen collections. a major limitation of this study is the low number of bulls included. in the present trial, for all semen extenders pregnancy rate was higher during the dry season compared to the humid season. previous studies to evaluate the monthly variation of fertility (n= 8,308 ai) and estrus frequency in crossbred dual-purpose cows (n= 2,960 cows) in three agroecological areas of the south american tropics revealed that the pregnancy rate of first serviced cows was the highest during the cooler and drier months of the year (decemberapril), while the lowest fertility was obtained during the months of highest rainfall and humidity (augustnovember).28,29 clinical theriogenology • volume 9 number 1 • march 201741   conclusions the use of semen extenders containing processed egg yolk or soybean extract resulted in higher post-thaw sperm progressive motility than complete egg yolk based extender. pregnancy rates after artificial insemination of crossbred brahman cows using frozen semen containing complete egg yolk, processed egg yolk or soybean did not differ. procedures to obtain the processed egg yolk are easy and inexpensive and seem to be able to diminish the detrimental compounds of egg yolk. the use of soybean extract represents a promising alternative for milk-based bull semen extender and provides additional benefits reducing the potential risk for microbial contamination. conflicts of interest the authors of this article have no conflicts of interest. acknowledgements the authors thank consejo de desarrollo científico, humanístico, tecnológico y de las artes (cdchta), universidad de los andes, venezuela (project nurr-c-321-02-03-f) for financial support. the authors extend their gratitude to venezolana de inseminación artificial y transplante de embriones (viateca) for providing the facilities, bulls and equipment to conduct this research. thanks to agropecuaria santa ana farm for providing support on experimental units, animal husbandry, facilities and records during this study. references 1. de leeuw fe, de leeuw am, den daas jhg, et al: effects of various cryoprotective agents and membrane-stabilizing compounds on bull sperm membrane integrity alters cooling and freezing. cryobology 1993;30:32-44. 2. thun r, hurtado m, janett f: comparison of biociphos-plus and tris-egg yolk extender for cryopreservation of bull semen. theriogenology 2002;57:1087-1094. 3. watson pj, martin ca: the influence of some fractions of egg yolk on the survival of ram spermatozoa at 5°c. aust j biol sci 1975;28:145-152. 4. amirat l, tainturier d, jaenneau l, et al: bull semen in vitro fertility after cryopreservation using egg yolk ldl: a comparison with optidyl ®, a commercial egg yolk extender. theriogenology 2004;61:895-907. 5. phillips ph, lardy ha: a yolk buffer pabulum for the presentation of bull semen. j dairy sci 1940;23:399. 6. bergeron a, crete mh, brindle y, et al: low-density lipoprotein fraction from hen’s egg yolk decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane. biol reprod 2004;70:708-717. 7. parks je, graham jk: effects of cryopreservation procedures on sperm membranes. theriogenology 1992;38:209-222. 8. manjunath p, nauc v, bergeron a, et al: major proteins of bovine seminal plasma bind to the low-density lipoprotein reaction of hen's egg yolk. biol reprod 2002;67:1250-1258. 9. vishwanath r, shannon p, cursen b: cationic extracts of egg yolk and theirs effects on motility, survival and fertilizing ability of bull sperm. anim reprod sci 1992;29:185-194. 10. moussa m, martinet v, trimeche a, et al: low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen. theriogenology 2002;57:1695-1706. 11. pace mm, graham ef: components in egg yolk which protect bovine spermatozoa during freezing. j anim sci 1974;39:1144-1149. 12. kampschmidt rf, mayer dt, herman ha: lipid and lipoprotein constituents of egg yolk in the resistance and storage of bull spermatozoa. j dairy sci 1953;36:733-742. 13. demaniowicz w, strzezek j: the effect of lipoprotein fraction from egg yolk on some of the biological properties of boar spermatozoa during storage of the semen in liquid state. reprod domest anim 1996;31:279-280. 14. bousseau s, brillard jp: in vitro and in vivo results of fertility in cattle, following inseminations performed with semen diluted and frozen in an extender free of animal origin products (biociphos-plus®). proc 6th eur ai vets meet 1994. 15. bousseau s, brillard jp, marguant-le guienne b, et al: comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. theriogenology 1998;50:699-706. 16. fukui y, kohno h, togari t, et al: fertility after artificial insemination using a soybean-based semen extender in sheep. j reprod devel 2008;54:286-289. 17. van wagtendonk-de leeuw am, haring rm, kaal-lansbergen lmte, et al: fertility results using bovine semen cryopreserved with extenders based on egg yolk and soybean extract. theriogenology 2000;54:57-67. 18. aires va, hinsch kd, mueller-schloesser f, et al: in vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen. theriogenology 2003;60:269-79. clinical theriogenology • volume 9 number 1 • march 2017 42   19. gil j, januskauskas a, haard mch, et al: functional sperm parameters and fertility of bull semen extended in biociphos plus® and triladyl®. reprod domest anim 2000;35:69-77. 20. decuadro-hansen g, delhomme a, camus s, et al: in vitro and in vivo fertility results of bovine frozen semen with ready to extender free of egg yolk. 13th intern congr anim reprod 1996; p 2-15. 21. hinsch e, hinsch k-d, boehm jg, et al: functional parameters and fertilization success of bovine semen cryopreserved in egg-yolk free and egg-yolk containing extenders. reprod domest anim 1997; 32:143-149. 22. chenoweth pj, spitzer jc, hopkins fm: a new bull breeding soundness evaluation form. proc soc therio 1992; 6370. 23. amirat l, anton m, tainturier d, et al: modifications of bull spermatozoa induced by three extenders: biociphos, low density lipoprotein and triladyl, before, during and after freezing and thawing. reproduction 2005; 129:535-543. 24. foulkes ja: the separation of lipoproteins from egg yolk and their effect on the motility and integrity of bovine spermatozoa. j reprod fertil 1977;49:277-284. 25. graham jk, foote rh: effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull spermatozoa alter cold shock and freezing. cryobiology 1987;24:42-52. 26. singh vk, singh ak, kumar r, et al: development of soya milk extender for semen cryopreservation of karan fries (crossbreed cattle). cryoletters 2013;34: 52-61. 27. christensen p, boelling d, pedersen km, et al: relationship between sperm viability as determined by flow cytometry and non-return rate of dairy bulls. j androl 2005;26:98-106. 28. perea f, soto e, hernández h, et al: monthly variation of fertility and oestrus frequency in crossbred dual-purpose cows in three agroecological areas of the south american tropics. trop anim health prod 2006;38:353-363. 29. perea-ganchou f, soto belloso e, gonzález stagnaro c, et al: factors affecting fertility according to the postpartum period in crossbred dual-purpose suckling cows in the tropics. trop anim health prod 2005;37:559-572.     figure 1. post-thaw sperm motility during 2.5 hour incubation period of brahman bull semen frozen with complete egg yolk(– ■–), processes egg yolk(–○–) or soybean (–▲–) extract-containing diluent. values are mean ± sem of 6 ejaculates (three ejaculates × two bulls). a,b,c p < 0.01; d,e p < 0.05.   30 40 50 60 70 80 0 0.5 1 1.5 2 2.5 p os tth aw s pe rm m ot il it y (% ) incubation period (h) a a a a d d a d b a d b b c c b e e clinical theriogenology • volume 9 number 1 • march 201743     figure 2. average of post-thaw sperm progressive motility during 2.5 h of incubation of semen from two brahman bulls frozen in milk-based extender containing complete egg yolk (ce), processed egg yolk (pe) or soybean extract (se). no significant differences between bulls. extender variable ce pe se overall n % n % n % bull 34 56 inseminator a b 40/109 34/98 32/92 41/114 36.7 a,1 34.7 a,1 34.8 a,1 35.9 a,1 34/96 51/116 32/96 59/109 35.4 a,1 43.9 a,1 33.3 a,1 48.6 b,2 39/99 41/98 32/101 48/96 39.4 a,1 41.8 a,1 31.7 a,2 50.0 b,c,3 37.8 1 40.4 1 33.2 2 44.5 3 overall 74/207 35.7 a 85/212 40.1 a 80/197 40.6 a values with different superscript letters in the same row differ significantly: a,b p ≤ 0.05; a,c p ≤ 0.02 values with different superscript numbers in same column differ significantly: 1,2 p ≤ 0.02; 2,3 p 0.01; table 1. effects of bull and inseminator on pregnancy rate in cows inseminated with semen frozen in milk-based extenders containing complete egg yolk (ce), processes egg yolk (pe) or soybean extract (se). values with different superscript letters in the same row differ significantly: a,b p ≤ 0.05; a,c p ≤ 0.02. values with different superscript numbers in same column differ significantly: 1,2 p ≤ 0.02; 2,3 p 0.01; 0 10 20 30 40 50 60 70 80 ce pe se p os tth aw s pe rm m ot il it y (% ) extender bull 34 bull 56 clinical theriogenology • volume 9 number 1 • march 2017 44   extender season ce pe se overall n % n % n % dry intermediate humid 46/107 16/49 12/51 43.0 a,1 32.6 a,1,2 23.5 a,2 44/95 25/60 16/57 46.3 a,1 41.6 a,1,2 28.1 a,2 43/87 18/50 19/60 49.4 a,1 31.4 a,1,2 31.6 a,2 46.0 3 37.1 4 28.0 5 values with different superscript letters in same row differ significantly a,b p 0.05 values with different superscript numbers in same column differ significantly: 1,2 p 0.05; 3,4 p = 0.06; 4,5 p = 0.06; 3,5 p 0.001 table 2. effects of season on pregnancy rate in cows inseminated with semen frozen in milk-based extender containing complete egg yolk (ce), processes egg yolk (ce) or soybean extract (se). values with different superscript letters in same row differ significantly a,b p 0.05. values with different superscript numbers in same column differ significantly: 1,2 p 0.05; 3,4 p = 0.06; 4,5 p = 0.06; 3,5 p 0.001 clinical theriogenology • volume 9 number 1 • march 201745 clinical theriogenology • volume 9 number 1 • march 2017 46 omniblank: 2013: buck evaluation and semen handling buck evaluation and semen handling misty a. edmondson department of clinical sciences, college of veterinary medicine, auburn university, auburn, al abstract buck selection and management are critical to maintaining or improving the performance of the herd. the buck’s health and breeding soundness should be evaluated regularly to ensure his ability to successfully breed and/or be collected for semen preservation. the ability to collect and cryopreserve semen has greatly changed the goat industry. artificial insemination (ai) allows better control of reproduction, control of sexually transmitted diseases, and the rapid distribution of valuable genetics. this lecture will review the selection parameters for bucks and will discuss important aspects of handling preserved semen. keywords: buck, selection, semen, evaluation, cryopreservation introduction buck selection and management are vital to maintaining or improving the performance of the herd. bucks should be routinely evaluated as part of the farm’s herd health program and receive annual breeding soundness evaluations. the ability to collect and cryopreserve semen has greatly changed the goat industry. artificial insemination allows better control of reproduction, control of sexually transmitted diseases, and the rapid distribution of valuable genetics. satisfactory fertility to ai requires careful attention to semen storage, handling and cryopreservation. buck evaluation buck selection bucks are selected based on their individual performance or progeny testing for traits such as meat traits, adaptability, twinning rates, and milk production. obviously, prolific bucks are preferred. in addition, birth, weaning, and yearling information are valuable when evaluating bucks for suitability as potential sires. bucks should be large with good musculature and have good conformation. bucks with any conformational and genital tract abnormalities should be avoided. because the intersex condition has been linked to the polled gene, the use of phenotypically polled bucks should be avoided. changing bucks every two years will help preserve vigor and reduces inbreeding in the herd. scrotal circumference should also be evaluated as bucks with large scrotal circumference usually produce the highest-quality sperm.1,2 serving capacity tests may be used to help determine how many does a buck can be expected to service. serving capacity tests are performed to measure how many times a buck services does during a defined period. typically, the buck is placed into a pen (3 m by 5 m) with two to four cycling, unrestrained does for a period of 20 to 40 minutes. all sexual behavior is monitored and recorded with an emphasis on the number of breedings. these tests are the best predictor of the adequacy of an animal’s libido. using bucks identified as high-performing or having a high degree of libido, in comparison with low-libido bucks, will result in higher kidding percentages and greater numbers of live-born kids per exposed doe.1 serving capacity tests may also be used to determine proper buck-to-doe stocking ratios. these tests may help produce a shorter, more uniform kidding season. adult bucks achieving four to six or more breedings during 30 minutes are preferred. breeding soundness evaluation all breeding bucks should be evaluated for breeding soundness three to four weeks prior to the breeding season. the examination of the buck should include a physical examination, reproductive examination, measurement of scrotal circumference, and semen collection and evaluation. breeding soundness examinations are only able to evaluate the physical soundness and semen quality of the buck. the breeding soundness examination does not evaluate the buck’s libido or ability to breed a doe. 495 clinical theriogenology • volume 5, number 4 • december 2013 therefore all bucks that pass a breeding soundness examination with a satisfactory rating should also be observed for ability to breed a doe and to assess libido. the physical examination of the buck includes a general examination for health which includes an assessment of body condition and musculoskeletal condition of the feet and legs. a satisfactory buck should be in good body condition; thin or obese animals should be avoided.1,3,4 the buck should be free of genetic defects such as hernias, cryptorchidism, supernumerary teats, musculoskeletal defects and intersex condition (phenotypically polled). the examination of the reproductive tract includes evaluation of the testes, epididymis, spermatic cord, and penis. the testes should be evaluated for size, symmetry, and consistency. a buck should have two large, oval-shaped testes of equal size. the testes should be firm during the breeding season and slightly softer during the non-breeding season. ultrasound examination may be beneficial confirming suspected abnormalities.1,3,4 gross changes in the epididymis are fairly rare in goats. the penis should be examined for abnormalities when collecting semen. the penis must be manually extended from the sheath so that the entire length of the extended penis can be evaluated. the absence of the urethral process in bucks with a history of obstructive urolithiasis is of no real concern as it appears to have no detrimental effect on the buck’s fertility.4 because of the high association between testicular size and capacity for sperm production, scrotal circumference is an important measure to evaluate in the buck. the scrotal circumference in 45 kg dairy goats has been reported to be 25 to 28 cm with larger bucks having scrotal circumferences of 34 to 36 cm.3,4 no age or breed standards exist for scrotal circumference in meat goats. one evaluation of meat goats found that the scrotal circumference in 45 kg, seven month old kiko and boer bucks averaged 26 to 29 cm.5 semen collection may be performed with an artificial vagina (av) with a trained buck or by electroejaculation with the buck restrained. the normal buck ejaculate is 0.5 to 1.5 ml in volume. semen is evaluated for color, concentration and sperm characteristics such as gross and progressive motility and morphology (figure).1,4 semen quality and quantity may vary with age, season, temperature, and breed. the volume of semen is of little value when collected by electroejaculation as this is not a physiologic ejaculation; volume collected by av is of some value. the color of semen depends on the concentration and can range from milky to creamy. gross motility and progressive motility are examined microscopically. figure. minimum acceptable reproductive criteria for a satisfactory potential breeder buck volume 0.5 ml progressive motility 70% concentration 2 billion morphology 80% normal modified from edmondson ma, roberts jf, baird an, et al. theriogenology of sheep and goats. in: pugh dg, an, editors. sheep and goat medicine. 2nd edition. st. louis: elsevier saunders; 2012. p. 150-230. management of bucks bucks are often overlooked and neglected from many herd health management practices. urolithiasis, although more common in castrated males, is still one of the most important diseases seen in bucks and can be quite detrimental to the breeding potential of a buck. providing fresh clean water, increasing salt concentration in the diet (2% to 4%), sufficient trace mineral intake, maintain a 2:1 of dietary calcium to phosphorus ratio and using urinary acidifiers (1% to 2% ammonium chloride) are important steps in the herd health management of bucks. bucks should be in good body condition (bcs 3-3.5) before the onset of the breeding season without excessive fat.6 bucks should be maintained at a pre-breeding body condition score of 3 to 4 because they may lose more than 10% to 12% of their body weight in 1.5 months of a breeding season.7 it is usually beneficial to feed a concentrated energy-protein supplement to bucks approximately four to six weeks prior to the breeding season. depending on the body condition and size of the buck and the quality of forages, a daily ration of six to eight pounds of forage and one to two pounds of 12 to 14% 496clinical theriogenology • volume 5, number 4 • december 2013 crude protein concentrate usually is sufficient.7 after the breeding season, it may be necessary to feed some concentrate to help the buck regain adequate body condition. the buck can be maintained on good quality hay during the remainder of the year. in addition, bucks should be evaluated regularly for routine hoof trimming and level or degree of gastrointestinal parasite burden. before the breeding season, the buck should undergo a breeding soundness evaluation as previously described. breed, age and nutrition play a major role in the onset of sexual maturity in the buck. the age at puberty with pygmy breeds ranges from two to three months and in nubian and boer bucks ranges from four to five months. most goats raised in the northern hemisphere will have spermatozoa present in the ejaculate at four to five months of age. however at this age, semen quality it poor, and these young bucks are not suitable for breeding.2 nubian and boer bucks begin to exhibit sexual behavior at ten to 12 weeks and start producing good quality semen at eight months of age.1 natural adhesions of the urethral process and glans penis to the prepuce make the immature buck incapable of copulation. this attachment begins to break down or separate at three months of age. fertile mating is possible at four to five months of age.2 fast-growing, well-fed and well-managed kids are able to breed sooner than malnourished males of the same age. semen handling semen collection and storage the collection and storage of semen form mature, healthy bucks can be a valuable component of a reproductive program that maximizes the overall fertility of a herd and emphasizes genetic progress.5 the two most common methods of semen collection include the use of an electroejaculator and or an av, respectively. the use of a warm av (39°c) is the preferred method for semen collection as it is faster and less stressfull for a trained male and produces a more physiologically normal sample. the electroejaculation method may be necessary for single collections. while samples obtained by electroejaculation tend to have a larger volume, they are inconsistent and often contain a large amount of seminal fluid and a lower concentration of spermatozoa than samples collected with an av. in order to maintain good concentrations and adequate libido, collections in bucks are often performed two to three times daily on alternate days with intervals of 30 minutes to one hour between collections. however, the timing of collections is often dependent on the age, condition and temperament of each buck.1 semen quality is also dependent on the season of collection with the frequency of morphologically abnormal spermatozoa more common during the spring and summer. upon collection, the ejaculate is placed into a warm waterbath (37°c) and evaluated immediately. the evaluation consists of a macroscopic examination (volume, consistency, and color) and a microscopic examination (gross motility, individual motility, morphology, and sperm concentration). the same parameters for semen evaluation for a breeding soundness evaluation are used. the normal color of semen is off-white to milky. other colors may be seen as well: hints of pink (blood contamination), brown (infection of the reproductive tract), and yellow (urine contamination). all of these color variations indicate a poor quality ejaculate.1 a two hour semen stress test should also be performed prior to cryopreservation. the semen is collected and evaluated immediately for progressive motility. the sample is then incubated at room temperature for two hours. progressive motility is then reevaluated. the incubated sample should maintain 20% of the motility of the initial sample. the fertility potential of cryopreserved semen is dependent on or influenced by the natural variation in semen quality among bucks, management, nutrition, and environmental stressors. once semen has been collected, semen can be used raw or undiluted, extended and chilled, or frozen and thawed. when using raw semen, females can be inseminated with approximately 0.1 ml of normal good quality semen immediately after collection. after evaluation, semen is diluted with appropriate extenders to achieve final semen-to-extender ratios of 1:1 to 1:4, depending on the sperm concentration of the ejaculate. if necessary, semen can be diluted with the extender at 30°c and cooled to 4°c and then kept at this temperature for up to 24 hours. 497 clinical theriogenology • volume 5, number 4 • december 2013 freezing and thawing semen a variety of methods of freezing semen have been used. semen that is intended for freezing should have a concentration greater than 3 x 109/ml and a motility rate greater than 70% of the ejaculate. normal concentrations range from 2.5 to 5.0 x 109 ml. traditionally, buck semen has been centrifuged to remove the seminal plasma which enhances freezability. however if semen is adequately diluted, centrifugation may be excluded. semen extenders are commercially available with most formulated to enhance sperm cell maintenance by providing energy, isotonic osmotic pressure, a buffering system and protection from cold shock.1 temperature control is extremely important when successfully freezing semen. the semen should be placed in an incubator or waterbath (30°c). semen should be diluted as appropriate using a warm extender (30°c). for freezing semen, several packaging systems have been developed for cryopreservation of semen (straws, pellets, ampules). the type of packaging system used for cryopreservation is dependent on the dilution of extender and semen.1 the most popular way of freezing semen is to use plastic straws (0.25 to 0.5 ml). the thawing and handling procedures for semen must be consistent with on-farm recommendations. improper thawing procedure as well as other factors can degrade the semen and affect fertility. appropriate semen handling techniques are critical in helping to maximize conception rates. handling of frozen semen frozen semen for bucks is stored in liquid nitrogen (-196°c). any deviation from this storage temperature can alter semen quality. the straw is placed into the holding goblet with two goblets attached to a cane. multiple canes are set inside a canister which is submerged and maintained in a liquid nitrogen tank. the liquid nitrogen tank should be stored in a cool, well-ventilated room. the lid of the liquid nitrogen tank should kept closed at all times. the liquid nitrogen level should be checked and maintained at an adequate level with regularly planned tank maintenance. it is important to maintain accurate records so that the straws are easily to locate which will expedite semen transfer and retrieval. the straws should be thawed with extreme care. once the desired straw is correctly identified, the canister is raised until the cane tops can be seen (5 to 7 cm below the mouth of the tank). straws should be maintained below the “frost line” in the neck of the tank at all times. using a light source to identify the correct cane, the straw is removed from the cane with tweezers or forceps. the cane is immediately replaced into the canister which is then re-immersed in the liquid nitrogen. straws should not be touched by the handler’s hands. the straw is then quickly immersed in a water bath(33 to 35°c). thawing procedures should follow manufacturer’s recommendations. however, general thawing procedures require only 30 to 40 seconds for 0.5 ml straws and 20 to 30 seconds for 0.25 ml straws. only as many straws as can be used in 10 to 15 minutes should be thawed at one time. if at all possible, no more than three straws should be thawed at one time in order to avoid lowering the thaw water temperature. the straw should then be thoroughly dried, the air bubble shaken to the crimped end, and the straw opened.1 if pellets are used, then the pellets should be removed from liquid nitrogen storage and two or three placed into a dry, sterile tube. the tube is kept in a warm waterbath (37°c). the thawed semen should be pulled into a pipette and used for ai immediately. alternatively, some processing procedures may require the addition of a warm diluent to the frozen pellets.1 evaluation of thawed semen for the evaluation of frozen semen samples, the semen should be thawed using the recommendations that were made by the company where it was originally processed or frozen. the straw should be properly identified, thoroughly dried, the semen examined for motility and morphology and a two hour semen stress test performed. the society for theriogenology has set the following expected standards for ram spermatozoa which appear to be applicable to the buck as well: 70% normal, 50% intact acrosomes, initial motility of 25%, and a two hour percent motility of 15%. the society also recommends a motile sperm dose of 25 x 106.1 498clinical theriogenology • volume 5, number 4 • december 2013 conclusion buck selection and management are critical to maintaining or improving the performance of the herd. the buck’s health and breeding soundness should be evaluated regularly to ensure his ability to successfully breed and/or be collected for semen preservation. the ability to collect and cryopreserve semen has greatly changed the goat industry. artificial insemination allows better control of reproduction, control of sexually transmitted diseases, and the rapid distribution of valuable genetics. satisfactory fertility to ai requires careful attention to semen storage, handling and cryopreservation. references 1. edmondson ma, roberts jf, baird an, et al: theriogenology of sheep and goats. in: pugh dg, baird an, editors. sheep and goat medicine. 2nd edition. st. louis: elsevier saunders; 2012. p. 150-230. 2. goyal ho, memon ma: clinical reproductive anatomy and physiology of the buck. in: youngquist rs, threllfall wr, editors. current therapy in large animal theriogenology. 2nd edition. st. louis: wb saunders; 2007. p. 511-514. 3. pugh dg: breeding soundness examination in male goats. proc seventh annu hudson-walker/vaughan theriogenology conference, auburn university college of veterinary medicine; 1996. p. 29. 4. memon ma, mickelsen wd, goyal ho: examination of the reproductive tract and evaluation of potential breeding soundness in the buck. in: youngquist rs, threllfall wr, editors. current therapy in large animal theriogenology. 2nd edition. st. luis: wb saunders; 2007. p. 515-518. 5. mohbini s: reproductive management in goats. proc north am vete conf, vol. 14, orlando, fl, 2000. 6. scharko p, johnson j, mobini s, et al: flock and herd health. in: pugh dg, baird an, editors. sheep and goat medicine. 2nd edition. st. louis: elsevier saunders; 2012. p. 539-556. 7. rankins dl, pugh dg: feeding and nutrition. in: pugh dg, baird an, editors. sheep and goat medicine. 2nd edition. st. louis: elsevier saunders; 2012. p. 18-49. 499 clinical theriogenology • volume 5, number 4 • december 2013 500clinical theriogenology • volume 5, number 4 • december 2013 omniblank: 2011: erratum erratum overton mw, heins bd: concepts and considerations for successful reproductive management and montoring. clin therio 2011;3:249-263. figure 1. a schematic representation of some of the predominant tai protocols in use on dairies around the world.   a. ovsynch gnrh pgf2α gnrh tai │ │ 48-60 hrs │ 12-16 hrs │ b. cosynch-72 gnrh pgf2α gnrh + tai │ │ │ c. cidr synch gnrh + cidr in pgf2α + cidr out gnrh tai │ │ 48-60 hrs │ 12-16 hrs │ d. presynch-ovsynch pgf2α pgf2α gnrh pgf2α gnrh tai │ │ │ │ 48-60 hrs │ 12-16 hrs │ e. double ovsynch gnrh pgf2α gnrh gnrh pgf2α gnrh tai │ 7 d │ 72 hrs │ │ │ 48-60 hrs │ 12-16 hrs │ f. pg-ovsynch pgf2α gnrh pgf2α gnrh tai │ 2-3 d │ │ │ 48-60 hrs │ 12-16 hrs │ 72 hrs 7 d 7 d14 d 10-14 d 7 d 7 d 7 d 6-7 d gnrh 7 d 7 d clinical theriogenology • volume 3 number 4 • december 2011603 clinical theriogenology • volume 3 number 4 • december 2011 604 omniblank: 2011: effect of resynchronization with intravaginal progesterone device from 14 to 21 days post-ai on estrus synchrony and pregnancy in lactating dairy cows effect of resynchronization with intravaginal progesterone device from 14 to 21 days post-ai on estrus synchrony and pregnancy in lactating dairy cows r. kasimanickam,a j.m. cornwell,b r.l. nebelb* adepartment of veterinary clinical sciences, washington state university, pullman, wa; bdepartment of dairy science, virginia polytechnic institute and state university, blacksburg, va abstract resynchronization in dairy cows provides tighter synchrony of follicular dynamics and estrus, and facilitates submission for subsequent artificial insemination (ai) in a timely manner. intravaginal progesterone devices (ipd) are considered safe for the use in lactating dairy cows and have been granted zero milk withdrawal and zero milk discard time. (http://www.fda.gov/downloads/animalveterinary/products/approvedanimaldrugproducts/foiadrugs ummaries/ucm235348.pdf). the objective of this study was to evaluate whether resynchronization of cows with ipd from day 14 to 21 post-ai provides tighter synchrony of estrus and improved pregnancy/ai for subsequent ai. lactating dairy cows (n=1132) were randomly assigned to two groups. group 1 (ipd group, n=532) received an ipd (eazi-breed cidr, pfizer animal health, new york ny) for seven days starting from day 14 to 21 post-ai. group 2 (con group, n=600) received no treatment. cows in the ipd group were observed for estrus three times daily from day 20 to 42 after ai and those in the con group from day 18 to 42 after ai. cows observed in estrus (ipd: n=352 and con: n=403) were inseminated by the am-pm rule. pregnancy examination was performed approximately 40 days after insemination by per-rectal palpation. estrus frequency distribution revealed no difference between the ipd and con groups (p>0.1). accounting for season (p<0.05), there was no difference in pregnancy/ai between ipd and con groups, 20.2 % (71/352) and 23.3% (94/403), respectively (p>0.05). in conclusion, ipd treatment for seven days from day 14 to 21 post-ai did not improve estrus synchrony and pregnancy compared to controls. keywords: dairy cows, progesterone, resynchronization, estrus, pregnancy introduction in lactating dairy cows, different strategies are used to resynchronize return to estrus in order to increase the number of cows re-inseminated in a timely manner. initiation of ovsynch/heatsynch/cosynch at or seven days prior to pregnancy diagnosis,1,2 presynch with gonadotropin releasing hormone (gnrh) seven days prior to pregnancy diagnosis and initiation of ovsynch/co-synch in non-pregnant cows,3-5 insertion of an ipd, or a combination of a gnrh injection at ipd insertion and/or after ipd removal at or seven days prior to pregnancy diagnosis1 resynchronizes the return to estrus following a prior insemination. these strategies require compliance since several injections need to be administered over a period of ten days. also these strategies are advocated for timed ai protocols and hence useful on farms with heat detection concerns. in these protocols cows will be submitted for re-insemination from 35 to 45 days after the previous ai. alternatively a strategy that involves insertion of ipd from day 14 to 21 would minimize the interval between inseminations. upon removal of the ipd on day 21, nonpregnant cows will return to estrus. using this strategy cows that show estrus would be re-inseminated within 28 days after ai. the objective of this study was to evaluate if resynchronization of cows with an ipd from day 14 to 21 post-ai would provide tighter synchrony and improved pregnancy/ai for subsequent ai. materials and methods lactating dairy cows (n=755) from two herds located in north carolina were enrolled in this study. cows were housed in free-stall barns and bedded on rubber mats or sand. cows received a total mixed ration balanced to meet or exceed the requirements for lactating dairy cows. cows were randomly assigned to one of two groups. cows in group 1 (ipd group, n=532) received an ipd for seven days * present address: select sires inc., plain city, oh clinical theriogenology • volume 3 number 4 • december 2011473 starting from day 14 to 21 post-ai. cows in group 2 (con group, n=600) received no treatment. cows were observed for estrus three times daily from day 21 to 42 after ai. cows observed in estrus (ipd: n=352 and con: n=403) were inseminated by the am-pm rule. pregnancy examination was performed 40 days after ai by per-rectal palpation. cows were synchronized with presynch(14-14 d)-ovsynch for the previous insemination. statistical analysis logistic model (proc logistic procedure of sas version 9.12, sas institute inc., cary, nc) was used to determine the effect of the ipd on pregnancy/ai and submission rates. variables included in the model were treatment (ipd vs. control), season, location, technician and appropriate interactions. the model was created by manual backward stepwise elimination procedure (p > 0.10 for exclusion). a p value of  0.05 was considered as significant. results there was no difference in the submission rate between ipd and con groups, 66.2% (352/532) and 67.2% (403/600), respectively (p>0.1). estrus frequency distribution revealed no difference between the ipd and con groups (p>0.05; figure), except on d 21 more cows were detected in estrus in the con group and on d 23 more cows were detected in estrus in the ipd group. accounting for season (p<0.05), there was no difference in pregnancy/ai between the ipd and con groups, 20.2 % (71/352) and 23.3% (94/403), respectively (p>0.05; table). discussion in this study, 66.2% (352/532) of cows in the ipd group and 67.2% (403/600) in the control group were inseminated. bartolome et al treated cows with an ipd from 14 to 23 days after ai. in that study 27.9% cows in the ipd group and 49.2% in the control group were inseminated at observed estrus.1 the authors have claimed that administration of the ipd between days 14 and 23 after first service reduced the frequency of cows re-inseminated at a detected estrus. they observed for estrus from 23 to 30 days after ai compared to 21 to 42 days in the current study. interestingly, in contrast, estrus distribution from 21 to 28 days was higher for the ipd group compared to the con group, 65.2% (230/353) and 56.6% (228/403), respectively (p<0.05). it should be noted that about 8.7% (35/403) of cows in the con group in the present study showed estrus from d 18 to 20 post-ai, a plausible reason for the observed difference in estrus distribution during 21 to 28 days after ai. galvao et al inserted an ipd from 14 to 21 d after ai. the cows were observed for estrus and inseminated using the am-pm rule.7 cows in the control group were inseminated at detected estrus from d 14 to 31. the estrus distribution during 21 to 24 d after ai was 25.1% (54/215) for controls compared to 39.1% (86/220) for the ipd group (p<0.05). however, the estrus distribution during 14 to 24 days after ai was 42.8% (92/215) for the control group compared to 40.9% (90/220) for the ipd group (p>0.1). in the present study, there was no difference in pregnancy/ai between ipd and con groups, 20.2 % (71/352) and 23.3% (94/403), respectively (p>0.05). even though cows observed in estrus were different during the seven day period from 21 to 28 days after ai there was no difference in pregnancy/ai (p>0.1). similarly, in a previous study pregnancy/ai at 30 d gestation was not different for both groups, 28.6% (61/213) for the control group and 24.7% (48/194) for the ipd group.1 in conclusion, ipd treatment for seven days from day 14 to 21 post-ai did not improve estrus synchrony and pregnancy compared to control groups. acknowledgements the authors thank select sires, inc. and pfizer animal health for partial financial support. the authors also extend their gratitude to ethan myers (myers family farm) and dr. ben shelton (rocky creek dairy) for their participation in this study. references clinical theriogenology • volume 3 number 4 • december 2011 474 1. b da 2. b da th 3. b iii th 4. d ut re 5. ch in 6. a no 7. g un 8. ch in figure table. pre of pregnan predicto constant treatment season †ipd–intra-v se–standar ci–confide artolome ja, van airy cows with th artolome ja, sil airy cows i: use heriogenology 2 artolome ja, so i. administration heriogenology 2 ewey st, mend tilizing a presync esponses. j dairy hebel rc, santo nsemination on p alkar a, tibary a ot improve conce alvão kn, santo nknown pregnan henault jr, bou nseminated dairy edictors of pre ncy at re-inse or coeffic -1.684 t -0.17 0.195 vaginal progeste rd error nce interval n leeuwen jj, t he cidr insert a lvestre ft, kam of the ovsynch 2005;63:1617-16 ozzi a, mchale j n of gnrh 23 d 2005;63:1643-16 donça lg, lopes chronization inje y sci 2010;93:40 os jep, cerri rl pregnancy rate an a, wenz jr, et a eption rate in lac os je, cerri rl, ncy status. j dair ucher jf, dame k y cows. j dairy s egnancy/ai, o emination for cient se 443 847 767 erone device (ea thieme m, et al: and the ovsynch mimura s, et al: r and heatsynch p 627. j, et al: resynch days post ai and 658. s g jr, et al: resy ection of gnrh 086-4095. la, et al: effect o nd pregnancy los al: presynchroniz ctating dairy cow et al: evaluation ry sci 2007;90:4 kj, et al: intrava sci 2000;86:203 odds ratio, 95 dairy cows in e coefficient 0.273545 0.177539 0.094667 azi-breed cid synchronization h protocol. theri resynchronizatio protocols after n hronization of ov ultrasonography ynchronization s or supplemental of resynchroniza ss in lactating da zation with gnr ws. theriogenolo n of methods of 4240-4252. aginal progestero 39-2049. 5% confidenc n the ipd† or t p 0.0001 0.315 0.039 r, pfizer anim n and resynchron iogenology 2009 on of ovulation a non-pregnancy di vulation and time y for nonpregnan strategies to imp l progesterone: i ation with gnrh airy cows. theri rh 7 days prior to ogy 2011;76:103 resynchronizatio one insert to syn e interval and control group odds r 1 0.8 1.2 mal health) nization of insem 9;72:869-878. and timed insem iagnosis by ultra ed insemination ncy diagnosis on prove fertility in i. pregnancy rate h on day 21 after iogenology 2003 o resynchronizat 36-1041. on for inseminat nchronize return d level of sign ps ratio l 4 0 22 1 minations in lact ination in lactati asonography. in lactating dair n day 30. lactating dairy c es and ovarian r artificial 3;60:1389-1399. tion with ovsyn tion in cows of to estrus if prev nificance for r 95% ci lower up 0.59 1 1.01 1 tating ing ry cows cows nch did viously risk pper .18 .46 clinical theriogenology • volume 3 number 4 • december 2011475 clinical theriogenology • volume 3 number 4 • december 2011 476 omniblank: 2011: anesthesia of white-tailed deer and other cervids anesthesia of white-tailed deer and other cervids jordyn m. boesch college of veterinary medicine, university of illinois, urbana, il introduction white-tailed deer (odocoileus virginianus) and other cervids such as mule deer (odocoileus hemionus) and elk (cervus elaphus) may require sedation or anesthesia for a variety of procedures in theriogenology practice, such as semen collection and standing or laparoscopic artificial insemination. this lecture will focus on field anesthesia but will touch on anesthesia of hospitalized cervids where applicable. keywords: anesthesia, cervids, deer, elk, pre-anesthetic preparation anesthesia of cervids often requires that veterinarians ignore principles of safe anesthetic practice, such as a thorough pre-anesthetic history, physical examination, and bloodwork. a reasonable history might be obtained from the owner or farm employees. there are some uncommon situations in which an examination and venipuncture may be possible, such as if a cervid is hospitalized or sick or tame enough to handle. packed cell volume, total protein, electrolytes, acid-base status, blood glucose, lactate, and creatinine are important values to obtain, if possible, prior to anesthesia, as they are frequently dramatically deranged by illness, labor, or stress of capture.1 if at all possible, deer with fluid, acid-base, or electrolyte derangements should be stabilized before inducing general anesthesia. the jugular veins are prominent and easily used for intravenous (iv) injections or catheterization. in a field situation, preanesthetic stabilization is often impossible, and veterinarians must be prepared to treat preexisting problems during anesthesia. deer are, of course, prey species and are remarkably stoic and adept at hiding clinical signs of illness and pain until they are near death. prior to elective anesthesia, healthy cervids should be fasted for 24-36 hours to reduce the risk of ruminal tympany, and water should be withheld for 12 hours. pediatric cervids that are still nursing should not be fasted. field capture should be reserved for the coolest part of the day, as these species are prone to hyperthermia. a field anesthetic should be thoroughly planned, with consideration given to such variables as weather, terrain, and available personnel and equipment. drug delivery cervids can be injected intramuscularly (im) with immobilizing anesthetic drugs in a two general ways: 1) physical restraint followed by handor pole syringe-injection and 2) remote drug delivery. the method selected depends on many factors such as the temperament of the patient and available personnel, facilities, and equipment. there is evidence that physical restraint/struggling and prolonged pursuit times (longer than five minutes) result in severe metabolic perturbations, including myocardial damage, and may predispose to capture myopathy.1,2 the cranial and caudal thigh muscles are often used for remote drug delivery because they are the largest muscle groups and further away from the thoracic cage than neck or proximal thoracic limb muscles; however, the sciatic nerve can be damaged by darting the caudal thigh. except for perhaps the largest males, smaller cervid species such as white-tailed deer and mule deer have delicate musculoskeletal systems and can easily be seriously injured by darts. the weight of the patient must first be estimated to calculate drug doses if a scale is not available; mature white-tailed deer weigh approximately 60-150 kg, mule deer 75-135 kg, and elk 230-318 kg.5 small and/or tame cervids can sometimes be moved into a smaller enclosure, then manually caught, restrained, and hand-injected. deer can seriously injure handlers with their antlers and by kicking. larger and/or more fractious cervids can be run through a chute or similar physical restraint device and hand-injected. pole syringes are designed for use in animals that have been restrained in a small, enclosed area. the pole syringe affords less control than hand injection but may be safer for both animal and veterinarian in larger, more fractious animals. clinical theriogenology • volume 3 number 4 • december 2011511 deer may be injected remotely with a blow dart using a blowpipe. blow darts are clear plastic, twochambered darts that use compressed gas added to the posterior chamber to push drug(s) added to the anterior chamber into the animal; because of their sharp needles and light weight, they are meant to be used with blowpipes.3 blowpipes are long, lightweight tubes with a mouthpiece on one end; the dart is placed in one end, and the operator blows into the tube to propel the dart.3 they are inexpensive and very quiet. maximum effective range is about 10-15 m.4 greatest accuracy is obtained when used indoors. practice is required to attain proficiency. due to their light weight, limited mass, and low velocity, blow darts cause minimal tissue trauma.3,4 power projection systems must be used to dart deer at greater distances (up to 40 m) after stalking on foot or from a vehicle or helicopter.4 blowgun projectors (e.g., dan-inject of north america or telinject usa, inc.), which add an external source of compressed gas (e.g., air from a foot pump or compressed carbon dioxide [co2] from a portable bottle), are the type used most commonly in zoological parks for deer.3,4 they are quiet and reasonably accurate. they are used most often with molded nylon, two-chambered plastic darts which also use compressed gas to push drug(s) into the animals.3 these darts are heavier, more robust, have better ballistic properties, and can withstand greater impact forces than blow darts.3 they thus have a greater range and can be used outdoors.3 they usually cause only minor tissue trauma unless fired at close range; it is still possible to fracture long bones with these darts, however. darts that use an explosive discharge mechanism can induce severe tissue damage in small, lean animals and are not recommended. drug combinations the selection of drugs should take into consideration such factors as the patient itself (age, species, temperament—which can range from tame and easily approachable to wild and unapproachable, and preexisting health problems); onset and duration of action of drugs, their side effects, and whether or not they can be antagonized; the concentration of drug available; intended duration and type of procedure; and the animal’s enclosure. if a procedure will inflict pain (e.g., laparoscopy, cesarean section) then a plan for preemptive and postoperative analgesia should be made. the theriogenologist should be aware of the advantages and disadvantages/side effects of each drug class. advantages of ultrapotent opioids such as carfentanil include rapid onset, reliability, potency, and reversibility with opioid antagonists like naltrexone.4 carfentanil is usually combined with a sedative like xylazine to decrease muscle rigidity and cns excitement; however, carfentanil-based protocols can still cause respiratory depression, hypoxemia, hypertension, cns excitation, and hyperthermia and present significant human safety risks.4 cyclohexamines (e.g., ketamine, tiletamine [in combination with zolazepam as telazol®]) have a rapid onset and a wide margin of safety with only moderate cardiopulmonary changes, preserve laryngeal reflexes somewhat, and provide some analgesia, but when used alone, can cause hyperthermia, hypersalivation, convulsions, and catecholamine release.4 alpha2agonists are analgesic and useful in combination with ultrapotent opioids or cyclohexamines but are unreliable when used alone in wild cervids. at the doses required in cervids, they significantly depress respiratory and cardiovascular function; common side effects include hypoxemia, hypertension, bradycardia, rumenal tympany, and disruption of thermoregulation.4 highly concentrated forms of these drugs, which will allow the use of small volumes in a dart, are available commercially (e.g., zoopharm, inc., ft. collins, co); all must be handled carefully to avoid accidental human injection. following administration of any drug(s), cervids should be left undisturbed, with stimulation kept to a minimum. however, the veterinarian should observe the animal for problems during induction. once the animal has become recumbent, then wait another five minutes or so, then approach cautiously and apply blindfold and hobbles. cervids often have very different drug requirements depending on their temperaments. excited animals will likely require higher doses. tame white-tailed deer and mule deer confined in an enclosure can be injected with 2-3 mg/kg of xylazine to produce recumbent sedation; elk require about 1 mg/kg.5 elk can also be sedated with 1.5-2 mg/kg of xylazine administered intranasally.6 cervids administered xylazine or other α2-agonists alone may appear to be immobilized but can rouse suddenly if stimulated. using α2clinical theriogenology • volume 3 number 4 • december 2011 512 agonists alone is not recommended for wild cervids and will likely not provide adequate immobilization for more invasive procedures such as laparoscopy or semen collection. brief, non-invasive procedures such as physical examination, weighing, and venipuncture may be possible. adding 1-3 mg/kg ketamine im to the xylazine will decrease the chance of sudden arousal and is necessary for more invasive procedures such as laparoscopy or semen collection.5 excited white-tailed deer that are not approachable will require higher doses of ketamine (up to 6 mg/kg).7 ketamine plus xylazine induces immobilization in less than 15 minutes provides approximately 30 minutes of immobilization after approach.7 the more specific α2-agonist, medetomidine (0.1 mg/kg) lasts longer than xylazine and permits the use of lower doses of ketamine (2.5 mg/kg).4,8 xylazine plus telazol® is another effective combination for invasive procedures. white-tailed deer and mule deer can be immobilized with 1.5 mg/kg xylazine plus 3 mg/kg telazol®, while elk can be immobilized with 1 mg/kg xylazine plus 2 mg/kg telazol®.5 this combination lasts about 45 minutes to one hour.5 carfentanil (10 µg/kg) plus xylazine (0.3 mg/kg) has been used in white-tailed deer and mule deer/white-tailed deer hybrids; the quality of immobilization was considered inferior to medetomidine plus ketamine because of more frequent patient movement.8 onset of action is at least as fast as ketamine plus an α2-agonist and provides at least an hour of immobilization.8 elk have also been immobilized with carfentanil (10 µg/kg) and xylazine (0.1 mg/kg).5 a new drug combination consisting of butorphanol, azaperone, and medetomidine (bam) was introduced by zoopharm several years ago and has been evaluated in white-tailed deer undergoing noninvasive procedures.9 concentrated formulations of butorphanol, azaperone, and medetomidine are mixed to create a solution of 27.3 mg/ml butorphanol, 18.2 mg/ml azaperone, and 10.9 mg/ml medetomidine, which is dosed at 0.3-0.5 ml/fawn, 1-1.5 ml/adult doe, and 2-2.25 ml/buck. this is equivalent to a very high medetomidine dose of 190-250 µg/kg. induction times can be quite variable, and white-tailed deer will still sometimes move when stimulated.9 immobilization will last for several hours unless the butorphanol and medetomidine are antagonized with naltrexone and atipamezole; the azaperone component is not reversible. whether or not bam is suitable for common theriogenology procedures in the field has not been extensively studied. for cesarean sections, propofol is the recommended induction drug in dogs and domestic small ruminants, as it is rapidly metabolized and results in increased neonatal vigor, and may be the best option for hospitalized cervids that can be injected iv.10 anesthesia can then be maintained with an inhalant such as isoflurane delivered via endotracheal tube. xylazine can be antagonized using the α2-antagonist yohimbine at 0.1-0.2 mg/kg im.4 atipamezole, a specific α2-antagonist, is used to antagonize medetomidine typically at three to five times the medetomidine dose im.4,5 the dose of an antagonist should be based on the length of time that has elapsed since the agonist was injected. ultrapotent opioids should be antagonized with naltrexone im.4,5 remember that antagonizing α2-agonists and opioids also antagonizes the analgesia provided by these drugs. neuroleptic agents can be useful for tranquilizing cervids for non-painful procedures. azaperone, a butyrophenone tranquilizer similar to acepromazine, may reduce stress in tame cervids for non-painful procedures. it has no analgesic effects and is not pharmacologically reversible. a dose of 0.2 mg/kg im will last up to six hours.5 anesthetic complications hypoxemia (arterial blood partial pressure of oxygen [pao2] less than 80 mm hg) is probably the most common complication in deer and can be caused by a combination of 1) hypoventilation caused by anesthetic drug effects on the medullary ventilatory center and respiratory muscle relaxation and 2) ventilation-perfusion (v:q) mismatching. in lateral and especially dorsal recumbency, the rumen and other abdominal organs splint the diaphragm and prevent full contraction and thus, alveolar expansion, resulting in progressive atelectasis, especially of dependent lung regions. this is compounded by gravitational redistribution of blood flow in the lungs and potentially by the procedure itself. insufflation of the peritoneal cavity for laparoscopy may splint the diaphragm as well if insufflation pressure is too high. intranasal oxygen supplementation has been proven effective at raising the fraction of inspired oxygen and pao2, and a readily available source of oxygen is recommended.4,5 pulse oximetry is highly recommended. clinical theriogenology • volume 3 number 4 • december 2011513 because the machine relies on the detection of a pulse to provide a reading, it may not work reliably in animals with poor peripheral perfusion from hypertension or hypovolemia. the veterinarian must ensure the immobilized cervid has a patent airway and is ventilating adequately. if the airway is obstructed, positioning the animal in sternal recumbency and extending the neck may help, but the veterinarian should be prepared to intubate with a cuffed endotracheal tube. a long laryngoscope, a stylet to stiffen the tube, or a plastic guide tube will facilitate intubation. cervids are most easily intubated in sternal recumbency with neck extended and nose pointed upward. they may salivate profusely, necessitating swabbing or suctioning, or, if they are too lightly anesthetized or if the pharynx is stimulated during repeated intubation attempts, may actively regurgitate and aspirate rumen contents. (they may also passively regurgitate at any time.) intubation must be swift, and the cuff must be inflated immediately to help protect against aspiration. cervids are not routinely intubated in the field for brief procedures (< 20 minutes), which is acceptable as long as a patent airway is maintained, the rate and pattern of breathing appears to be adequate, and the patient is positioned properly. sternal recumbency is safest, but if the patient is in lateral recumbency, the neck should be extended and a rolled towel placed under the poll to elevate the head above the level of the rumen with the nose tipped down toward the ground. it is recommended that cervids in dorsal recumbency be intubated, and they may require positive pressure ventilation (ppv). patients undergoing laparoscopy may also require ppv. a mildly elevated arterial carbon dioxide (co2) partial pressure (paco2, > 60 mm hg) is usually well-tolerated, but a higher paco2 may prolong recovery and cause a dangerously low blood ph (< 7.2). monitoring end-tidal co2 to assess ventilation can also be performed in the field using portable capnometers in intubated animals. both oxygenation and ventilation are most accurately assessed by monitoring arterial blood gases, which can be done in the field using portable analyzers. the auricular and femoral arteries are easy to access in most cervids. cervids are also prone to hyperthermia. a rectal temperature should be monitored every five minutes. a rectal temperature higher than 41°c is an emergency, and the animal must be aggressively cooled (e.g., using cool iv fluids) or the anesthetic drugs pharmacologically reversed. hyperthermia in the face of hypoxemia is particularly dangerous because it increases metabolic oxygen requirement, especially in metabolically active tissues such as the brain and myocardium. capture myopathy is a potentially serious or even fatal complication that results from massive sympathetic outflow from the stress of capture. four forms have been recognized: the acute death syndrome, delayed peracute death syndrome, ataxic-myoglobinuric syndrome, and muscle rupture syndrome.4 common clinical signs of the acute form include hyperthermia, tachycardia, tachypnea, cyanotic mucous membranes, metabolic acidemia, and hypotension.4 prevention (e.g., by limiting chase times and physical restraint and avoiding capture in warm weather) is easier than treatment, which consists of supportive care such as oxygen supplementation, iv fluids, and anti-inflammatory drugs. ruminal tympany and dart impact trauma are other potential complications. ruminal tympany can be relieved by sitting the animal up in sternal recumbency to facilitate eructation, passing an orogastric tube, trocarizing the rumen (as a last resort), or antagonizing anesthetic drugs such as α2-agonists that decrease rumen motility. pain management very little information is available in the literature on effective, safe analgesia in cervids; extrapolation from domestic sheep and goats is usually necessary. injectable non-steroidal antiinflammatory drugs (nsaids, such as flunixin, ketoprofen, or carprofen) are excellent choices for mild to moderate pain as long as the patient has no specific contraindications for their use. local anesthetics such as bupivacaine combined with a vasopressor such as epinephrine or an α2-agonist to prolong duration of effect are useful for procedures such as ring blocks for velvet antler removal or line blocks for cesarean section. preservative-free morphine and α2-agonists can be administered epidurally for cesarean sections. zoopharm, inc. now also manufactures sustained-release buprenorphine that may prove useful in cervids. more information on analgesia is available from the author on request. clinical theriogenology • volume 3 number 4 • december 2011 514 references 1. boesch jb, boulanger jr, curtis pd, et al: biochemical variables in free-ranging white-tailed deer (odocoileus virginianus) after chemical immobilization in clover traps or via ground-darting. j zoo wildl med 2011;42:18-28. 2. paterson j: capture myopathy. in: west g, heard d, caulkett n, editors. zoo animal and wildlife immobilization and anesthesia. ames (ia): blackwell publishing; 2007. p. 115-121. 3. isaza r: remote drug delivery. in: west g, heard d, caulkett n, editors. zoo animal and wildlife immobilization and anesthesia. ames (ia): blackwell publishing; 2007. p. 61-74. 4. caulkett na, arnemo jm: chemical immobilization of free-ranging terrestrial mammals. in: tranquilli wj, thurmon jc, grimm ka, editors. lumb and jones veterinary anesthesia and analgesia. 4th ed. ames (ia): blackwell publishing; 2007. p. 807-831. 5. caulkett n, haigh jc: deer (cervids). in: west g, heard d, caulkett n, editors. zoo animal and wildlife immobilization and anesthesia. ames (ia): blackwell publishing; 2007. p. 607-612. 6. cattet mrl, caulkett na, wilson c, et al: intranasal administration of xylazine to reduce stress in elk captured by net gun. j wildl dis 2004;40:562-565. 7. amass k: chemical immobilization of animals: technical field notes: 2004-2006. mt. horeb (wi): safe-capture international, inc. 8. caulkett na, cribb ph, haigh jc: comparative cardiopulmonary effects of carfentanil-xylazine and medetomidineketamine for immobilization of mule deer and mule deer/white-tailed deer hybrids. can j vet res 2000;64:64-68. 9. miller bf, osborn da, lance wr, et al: butorphanol-azaperone-medetomidine for immobilization of captive white-tailed deer. j wildl dis 2009;45:457-467. 10. raffe mr, carpenter re: anesthetic management of cesarean section patients. in: tranquilli wj, thurmon jc, grimm ka, editors. lumb and jones veterinary anesthesia and analgesia. 4th ed. ames (ia): blackwell publishing; 2007. p. 955967. 11. delguidice gd, mech jd, paul wj, et al: effects on fawn survival of multiple immobilizations of captive pregnant whitetailed deer. j wildl dis 1986;22:245-248. clinical theriogenology • volume 3 number 4 • december 2011515 clinical theriogenology • volume 3 number 4 • december 2011 516 omniblank: 2012: unilateral epididymitis associated with salmonella bacteremia in a dog unilateral epididymitis associated with salmonella bacteremia in a dog cristina pérez,a* michael d. whitacre,a siddhartha thakur,b shivaramu keelara,b megan fauls,b edward b. breitschwerdta departments of clinical sciencesa and population health and pathobiology,b college of veterinary medicine, north carolina state university, raleigh, nc summary this report describes the clinical features, diagnosis and treatment of a dog with unilateral epididymitis associated with salmonella spp. bacteremia. fever and an enlarged and painful testicle were the main clinical signs that resulted in referral for diagnostic evaluation. unilateral septic epididymitis was diagnosed via ultrasonography of the genitourinary tract and aerobic culture of scrotal fluid, urine and blood, which yielded heavy growth of salmonella spp. pulsed-field gel electrophoresis (pfge) confirmed the presence of salmonella javiana. following antibiotic therapy there was total resolution of clinical signs, and no salmonella was isolated from a post-treatment urine culture. the source of infection was unknown, however an environmental exposure was suspected. although infrequent, infection with salmonella spp. should be included in the differential diagnosis of canine epididymitis. given the major zoonotic importance of salmonellosis, and to prevent re-infection after treatment, the source of the infection should be investigated and eliminated, if possible. keywords: bacteremia, diagnosis, bacteria, blood culture case presentation a four-year-old, 42 kg, intact-male american bulldog was referred to the north carolina state university veterinary teaching hospital (ncsu-vth) for evaluation of fever, lethargy and unilateral testicular enlargement of 24 hours duration. after the primary veterinarian identified fever (102.8°f) and an enlarged and painful right testicle, the dog was referred for further evaluation. the owners lived on a large rural cattle and horse farm, and owned four other american bulldogs, three intact males and one spayed female. the dogs remained mostly indoors, but were occasionally left in the yard which shared a common fence with a horse pasture. the dogs had occasional contact with farm animals and many canada geese were seen around the farm in september when the dog became ill. according to the owners, the dogs sporadically ingested horse feces. travel history was limited to visits to the primary veterinarian. flea or tick preventatives were not used, but vaccination status was current and all dogs received year-round heartworm prevention medication (ivermectin). diet consisted predominantly of dry commercial dog food (pedigree adult complete nutrition small crunchy bites); however, table food was offered sporadically. according to the owners, the dog was previously healthy and had never been used for breeding purposes. on admission to ncsu-vth, the dog was depressed and quiet, body condition score was estimated at 5/9, mucus membranes were pale pink and the capillary refill time was 1 second. rectal temperature was 103.5°f and the heart rate was 84 beats/min. femoral arterial pulses were strong and no heart murmur was auscultated. the right testicle and epididymis were markedly enlarged and painful and the scrotum was warm and hyperemic. the left testicle and epididymis were palpably normal. doppler systolic blood pressures ranged between 100 to 140 mmhg. hematological abnormalities included leukocytosis characterized by neutrophilia (29.92 x 103/μl; reference intervals, 4.39-11.61 x 103/μl) and a left shift (bands 598 x 103/μl) with mild neutrophil toxicity and a monocytosis (1.197 x 103/μl; reference intervals, 0.21-1.05 x 103/μl). the dog was mildly thrombocytopenic (178 x 103/μl; reference intervals, 190-468 x 103/μl), mildly hyperglycemic (7.4 mmol/l; reference intervals, 4-6.4 mmol/l), hypokalemic (3.9 mmol/l; reference intervals, 4-5.3 mmol/l) and alt activity was mildly increased (83 iu/l; reference range, 12-54 iu/l]). prothrombin and activated partial thromboplastin times were normal, *current address: department of clinical veterinary sciences veterinary faculty, university of helsinki, finland. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201261 clinical theriogenology • volume 4 number 1 • march 2012 however, d-dimers were moderately increased (500-1000), potentially reflecting a hypercoagulable state. as bacteremia was suspected, three 10-ml blood samples for bacteriologic culture were collected aseptically at 1-hour intervals from the left jugular vein. urinalysis findings included a urine specific gravity of 1.056 and pyuria (5-10 wbc/hpf), which prompted a urine culture. due to the thrombocytopenia, a snap 4dx (idexx laboratories, westbrook, me) for detection of anaplasma spp., borrelia burgdorferi, ehrlichia canis antibodies and dirofilaria immitis antigen was performed, as well as indirect fluorescent antibody testing using babesia canis, ehrlichia canis, ricketsia rickettsii, bartonella henselae, bartonella vinsonii subsp. berkhoffii antigens. all serological results were negative. also, a rapid slide agglutination test (rsat) was negative for brucella canis antibodies. ultrasound examination of the genitourinary tract demonstrated marked right epididymal swelling with decreased regional blood flow on doppler examination, suggestive of thrombosis involving the vessels to the epididymis. there was a large amount of scrotal edema and anechoic fluid surrounding the right testicle and epididymis (figure 1). the echogenicity of both testicles appeared normal. the prostate was heperechoic and measured 38.5 mm, which was consistent with benign prostatic hypertrophy. with the dog under sedation, free scrotal fluid (3 ml) was obtained for analysis and aerobic culture, using a 22gauge needle connected to a 6 ml syringe, under ultrasound guidance. the dog did not show discomfort during or after the procedure. cytopathology of the scrotal fluid and fine needle aspiration cytology of the right epididymis were consistent with septic suppurative inflammation, with intraand extracellular bacilli visualized in both samples. based on these findings, septic epididymitis with possible thrombosis was diagnosed. after the aforementioned samples were obtained for aerobic and anaerobic bacterial culture, ampicillin-sulbactam (22 mg/kg intravenously q 8 h) and enrofloxacin (5 mg/kg intravenously q 24 h) were administered in conjunction with maintenance fluid therapy, ondansetron (0.2 mg/kg intravenously q 8 h) for nausea and hydromorphone (0.5 mg/kg intravenously q 8 h) for pain control. within 18 hours of incubation, all three blood cultures, the urine culture and the scrotal fluid culture yielded a heavy growth and pure culture of salmonella spp. based on the sensitivity results, the organism was susceptible to cephalosporins, aminopenicillins, and resistant to aminoglycosides. pulsed-field gel electrophoresis confirmed the presence of salmonella javiana. the dog was immediately transferred to the isolation ward, where special handling and personal protective measures were instituted according to ncsu-vth infection control policies. because hydromorphone alone failed to control testicular pain, fentanyl was administered at a constant rate of infusion (cri) at 2 µg/kg intravenously. the dog developed stranguria, but continued to completely empty the urinary bladder after diazepam (0.3 mg/kg intravenously q 12 h) and phenoxybenzamine (0.25 mg/kg orally q 12 h) were prescribed for possible urethral sphincter dyssynergia. a complete blood count 48 hours after admission identified persistent leukocytosis characterized by neutrophilia (30.76 x 103/μl), with resolution of the left shift and neutrophil toxicity and increased monocytosis (3.076 x 103/μl). thrombocytopenia persisted (172 x 103/μl; reference intervals, 190-468 x 103/μl) and d-dimers remained unchanged. by the next day, the dog’s attitude and appetite had improved and the testicular region was less painful, but remained very swollen. a fentanyl patch (100 µg/h) was placed and tramadol (5 mg/kg orally q 8-12h) was prescribed. the fentanyl cri was gradually decreased and the dog was discharged from isolation to the owners with instructions to administer the following medications: enrofloxacin 5mg/kg orally q 24 h for six weeks, tramadol 5 mg/kg orally q 8 h until recheck, carprofen 2 mg/kg orally q 12 h for five days, and phenoxybenzamine 0.25 mg/kg orally q 12 h until urination was normal. specific precautions including limiting exposure to other dogs and avoiding contact with urine and feces to prevent zoonotic infection were provided to the owners. a sample of the dog´s food (approximately100 g) cultured negative for salmonella spp. additionally, the owners were asked to bring a small, fresh sample of feces from each of the other dogs in the household (properly identified) for culture and pfge analysis. samples from three dogs were negative by culture for salmonella spp., whereas the fecal culture from one dog was positive, so antibiotic therapy was instituted for that housemate. pulsed-field gel electrophoresis was used to determine the clonal relationship among the salmonella isolates from this dog and the housemate using the pulsenet protocol.1 clonal relationships among the isolates were analyzed using bionumerics version 6.1 (applied maths, austin, tx) by clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 62clinical theriogenology • volume 4 number 1 • march 2012 upgma method with tolerance and optimization of 1.5% each. the genotyping profiles of the salmonella isolates from the patient (blood, urine, scrotal fluid) and the fecal isolate from the housemate matched 100%, which suggested infection with the same strain of salmonella in both dogs (figure 2). on subsequent examination ten days following diagnosis, the dog was urinating normally and his right testicle and epididymis had decreased in size. a complete blood count documented resolution of the thrombocytopenia (398 x 103/μl) and diminished leukocytosis (12.72 x 103/μl). the rsat was again negative for b. canis antibodies. despite the recommendation for bilateral castration, the owners elected unilateral castration so as to maintain the dog for breeding purposes. unilateral orchiectomy was preformed two weeks later while the dog continued receiving antibiotics. pre-operatively, urine and blood cultures were negative. histopathology of the testicle and epididymis was consistent with chronic moderate neutrophilic and lymphohistiocytic epidydimitis with secondary diffuse, marked hypospermatogenesis. aerobic culture of the testicle was negative. on reevaluation at ncsu-vth four months later, the findings of the physical examination were unremarkable. following manual ejaculation, 5.5 ml of bloodstained semen was obtained. total spermatozoa count was 347.16 x 106. total motility was 80% with 70% progressively motile sperm, resulting in 263 x 106 total motile spermatozoa. morphologically 79% of the sperm were anatomically normal, with 15% primary abnormalities and 6% secondary abnormalities. given the presence of blood in the ejaculate and one remaining testicle the dog was considered questionable as a potential breeding animal. the owners declined repeat blood culture, aerobic culture of sperm or urine, prostatic evaluation, and surgical removal of the remaining testicle. six months later, the owners called the ncsu-vth to report that the dog had successfully bred a bitch that subsequently delivered 12 puppies. discussion this report describes the clinical and pathological features of a dog with unilateral epididymitis associated with salmonella spp. bacteremia. the authors were unable to find other case reports describing salmonella bacteremia with concurrent genito-urinary involvement in dogs. epididymitis is an inflammatory reaction of the epididymis, which can be associated with an infectious or non-infectious etiology.2,3 the epididymes may become infected by direct penetrating wounds, by hematogenous or lymphatic spread from distant sites, or via the ductus deferens. the diagnosis is based on the clinical signs, which most often include a painful, swollen scrotum, and fever. ultrasonography of the testes in this dog identified unilateral swelling of the tail of the epididymis and spermatic cord, with diffuse or focal hypoechoic areas which helped exclude neoplasia and testicular torsion as differential diagnoses.3 the right epididymis was markedly enlarged and the diameter in the transverse plane was almost equal to the diameter of the testicle. the diameter of a normal canine epididymis should not be more than 28% of the testis at its greatest width.4 salmonella sp. are gram-negative facultative intracellular anaerobes that cause a wide spectrum of disease.5 although classically considered intestinal bacteria, salmonellosis may manifest clinically as acute enterocolitis, extra-intestinal localized disease, bacteremia, or endotoxemia. localized infections involving specific tissue sites may occur in association with clinical or subclinical bacteremia.6 epididymitis associated with salmonella species has been rarely reported in domestic animals. in humans, involvement of the genito-urinary tract is also rare, but can occur as a complication of enteric salmonellosis, or in association with congenital urogenital abnormalities, malignancy or immune suppression.6-8 in a review of more than 700 cases of human extra-intestinal infections caused by salmonella spp., cohen et al found only 12 cases (1.4%) of orchitis and epididymitis.6 in dogs, clinical salmonellosis is rare compared to the prevalence of subclinical intestinal infection, as salmonella spp. have been isolated from the feces of up to 36% of healthy dogs.5 ingestion of contaminated food, water or fomites are the most common sources of salmonella.5 raw and improperly cooked cat or dog food products prepared from contaminated meat and meat by-products have a higher prevalence of bacterial contamination than heat-processed foods. however, even commercially processed foods can become contaminated with salmonella spp. if exposed to feces from infected animals or fomites.3,9 in our case, the source of infection was unknown, however, the dog most likely acquired the infection through clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201263 clinical theriogenology • volume 4 number 1 • march 2012 environmental exposure to cattle, horses and geese, known to be salmonella sp carriers.9,10 geese in particular can be reservoirs of multidrug resistant s. enterica typhimurium strains.9 whether our patient acquired the infection from his housemate, or both dogs became infected from the same environmental source remains unknown. the most important agents of canine infectious epididymitis/orcho-epididymitis include brucella canis and pseudomonas malei; however, other bacteria such as staphylococcus, streptococcus, e. coli. mycoplasma, ureasplama and chlamydia have also been reported.1 orchitis was diagnosed after experimental infection of trypanosoma brucei in one dog,11 and in association with rickettsia rickettsii infection in two dogs.12 investigation of b. canis should always be pursued, since it can be transmitted sexually to other dogs and is a zoonotic pathogen. in our case, rsat for b. canis was negative twice, and salmonella was the only organism isolated from blood, scrotal fluid and urine. antibiotics and antiinflammatory drugs are generally recommended for the treatment of epididymitis. castration is usually advised, as the effects of the inflammation may likely have a permanent detrimental effect on spermatogenesis.3 in our case, four months after the diagnosis, the dog was healthy and subsequently successfully sired a litter. in conclusion, although infrequent, infection with salmonella spp. should be included in the differential diagnosis of canine epididymitis. considering the major zoonotic importance of salmonellosis, and to prevent re-infection following antibiotic treatment, the source of the infection should be investigated and eliminated, if possible. learning points • salmonella spp is a gram negative organism that can cause a variety of extra-gastrointestinal signs in dogs, including epididymitis. • ultrasonography of the genitourinary tract is helpful in order to exclude other diagnostic differentials for testicular enlargement, such as testicular torsion or neoplasia. • dogs can suffer from salmonella bacteriemia without any history of gastrointestinal signs or ingestion of raw meat. references 1. ribot em, fair ma, gautom r, et al: standardization of pulsed-field gel electrophoresis protocols for the subtyping of escherichia coli o157:h7, salmonella, and shigella for pulsenet. foodborne pathog dis 2006;3:59-67. 2. flanders ja, chlafer dh, yeager ae: diseases of the canine testes. in: bonagura jd, editor. kirk's current veterinary therapy xiii. philadelphia: w.b. saunders; 2000. p. 941. 3. johnston sd, root k, olson pns: disorders of the canine testes and epididymes. canine and feline theriogenology. philadelphia, pa: w.b. saunders co; 2001. 4. england gcw: relationship between ultrasonographic appearance, testicular size, spermatozoa output and testicular lesions in the dog. j sm anim pract 1991;32:306-311. 5. greene ce: salmonellosis. in: greene ce, editor. infectious diseases of the dog and cat, 3rd edition. philadelphia: w.b. saunders; 2006. p. 355-360. 6. cohen ji, bartlett ja, corey gr: extra-intestinal manifestations of salmonella infections. medicine (baltimore) 1987;66:349-388. 7. hakim a, bradley h: salmonella epididymo-orchitis in infancy and childhood. clin pediatr (phila) 1992;31:120-122. 8. al-obeid k, al khalifan nn, jamal w, et al: epididymo-orchitis and testicular abscess caused by salmonella enteritidis in immunocompromised patients in kuwait. med princ pract 2006;15:305-308. 9. yu cy, chou sj, yeh cm, et al: prevalence and characterization of multidrug-resistant (type acssut) salmonella enterica serovar typhimurium strains in isolates from four gosling farms and a hatchery farm. j clin microbiol 2008;46:522-526. 10. aceto h, miller sa, smith g: onset of diarrhea and pyrexia and time to detection of salmonella enterica subsp enterica in feces in experimental studies of cattle, horses, goats, and sheep after infection per os. j am vet med assoc 2011;238:1333-1339. 11. morrison wi, murray m, sayer pd, et al.: the pathogenesis of experimentally induced trypanosoma brucei infection in the dog. i. tissue and organ damage. am j pathol 1981;102:168-181. 12. ober cp, spaulding k, breitschwerdt eb, et al: orchitis in two dogs with rocky mountain spotted fever. vet radiol ultrasound 2004;45:458-465. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 64clinical theriogenology • volume 4 number 1 • march 2012 figure 1: ultrasound image of the right testicle and epididymis. the epididymal diameter in the transverse plane is almost equal to the diameter of the testicle. figure 2. dendrogram showing the pfge profiles of the dog salmonella isolates obtained in this study using xbai enzyme. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201265 clinical theriogenology • volume 4 number 1 • march 2012 clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 66clinical theriogenology • volume 4 number 1 • march 2012 omniblank: improvement of canine semen vitrification using sucrose and soy lecithin extender improvement of canine semen vitrification using sucrose and soy lecithin extender maja pipan,a margret casal,b janko mrkun,a irma klunc aclinic for reproduction and large animals, veterinary faculty, university of ljubljana, slovenia bclinical sciences and advanced medicine, university of pennsylvania school of veterinary medicine, pa cuniversity medical centre ljubljana, ljubljana, slovenia major challenge in freezing semen for short or long term availability is avoiding damage to intact spermatozoa caused by freezing. freezing-induced impairment becomes particularly problematic for lowquality semen. most freezing methods involve tedious slow cooling, which requires increased processing time and specialized media. although vitrification protocols are faster (no slow cooling) and provide improved results, they are not commonly used for canine semen. our objective was to improve vitrification methods for canine semen by adding sucrose to a basic canine semen extender not containing animal proteins. twenty ejaculates were obtained from 19 dogs. semen volume, concentration, sperm motility, progressive motility, morphology, tail membrane integrity and acrosomal defects were determined on the day of collection. each ejaculate was divided into 9 aliquots and diluated in semen extenders labeled a g. control extender consisted of tris, citric acid, fructose and antibiotics, with soy bean lecithin and sucrose added to create test extenders. for extender a, only soy lecithin (1%) was added. extenders b, c, and d contained 0.2, 0.25, and 0.3 m of sucrose, respectively, but no soy lecithin, whereas extenders e, f, and g contained corresponding concentrations of sucrose, plus 1% soy lecithin. from each group, a 33 µl suspension of cells was dropped directly into liquid nitrogen. after at least 48 hours, frozen drops were placed in 5 ml of devitrification medium (control medium containing 1% bovine serum albumin) that has been prewarmed to 37ºc. the after thaw suspension was maintained at 37ºc for 10 minutes and was then centrifuged at 300 g for 5 minutes. the cell pellet was finally resuspended in 50 µl of devetrification medium only for sperm evaluation. after thaw evaluation included motility, progressive motility, morphology, viability, plasma membrane modifications, acrosomal defects, and dna fragmentation. neither soy bean lecithin (a) or sucrose alone (b, c, d) were sufficient for preserving thawed sperm quality. however, extenders containing soy lecithin and various concentrations of sucrose (e, f, g) resulted in significantly better semen quality than all other extenders. in particular, extender f (2.5 m sucrose) had a higher proportion of motile (p < 0.001), progressively motile (p < 0.001), morphologically normal (p < 0.05) and viable (p < 0.01) spermatozoa compared to extenders e and g. there was also a higher proportion of spermatozoa with intact dna (p < 0.01) and normal plasma membrane integrity (p < 0.05) in group f. vitrification of canine spermatozoa in extenders containing soy bean lecithin and sucrose preserved important physiological attributes, including viability, motility, progressive motility, sperm tail membrane integrity and dna integrity during. we concluded that this method was simple, fast and effective for vitrification of canine semen. use of soy lecithin in lieu of animal proteins (e.g. egg yolk) facilitates ease of shipping to countries with strict import requirements. keywords: vitricification, semen preservation, canine 515 clinical theriogenology • volume 11, number 3 • september 2019 516clinical theriogenology • volume 11, number 3 • september 2019 omniblank: historyitem_v1 trimandshift range: all pages trim: none shift: move down by 40.50 points normalise (advanced option): 'improved' keep bleed margin: no d:20190702122647 32 1 0 0 full 1521 302 fixed down 40.5000 -20.2536 both 1 alldoc 2 currentavdoc none 0.0000 top qite_quiteimposingplus4 quite imposing plus 4.0m quite imposing plus 4 1 0 1 0 1 1 historylist_v1 qi2base 2018: case report: successful foaling in a warmblood mare with uterus unicornis case report: successful foaling in a warmblood mare with uterus unicornis kirsty gallacher,ᵃ robert o. gilbertᵇ ᵃschool of animal and veterinary sciences, the university of adelaide, roseworthy, sa, australia; ᵇross university school of veterinary medicine, basseterre, federation of st. kitts and nevis, west indies summary uterus unicornis is a rare congenital condition in the horse. there are limited cases documented in horses and little data on the outcome of pregnancy. this report describes the birth of a small filly foal (35kg) at 360 days gestation. postpartum examination of the mare and placenta confirmed absence of the right uterine horn. keywords: mare, uterus, unicornis, pregnancy, placenta case description history a 6 year old maiden warmblood mare was admitted to the cornell university equine park, at 306 days after ovulation for supervised foaling. she had been bred with cooled shipped semen and ovulation date and pregnancy diagnosis were confirmed by trans-rectal palpation and ultrasound by the referring veterinarian. clinical findings upon arrival the mare was bright, alert and responsive. her vital parameters were within normal limits. she had no udder development and was in good body condition. her pregnancy status was confirmed using transrectal palpation and ultrasound and the fetus was confirmed to be alive and in anterior presentation. the combined thickness of uterus and placenta (ctup) was nine mm and so within normal range for this stage of gestation (306 days from ovulation).1 the mare’s pregnancy status was confirmed regularly, and the placental thickness and fetal heart rate remained consistently within normal limits. a foal alarm system was used for monitoring the mare (foalert, abfohlsystem, germany). mares at the center for supervised foaling were also under continuous video surveillance and were visually monitored by a student foal watch crew who covered overnight periods. the mare was slow to develop pre-partum mammary tissue, which is not unusual for maiden mares. she was also reluctant to allow any mammary secretions to be milked from her udder to check electrolyte values for prediction of readiness for birth. eventually, her pelvic ligaments started showing signs of relaxation in the week preceding parturition. outcome the mare foaled spontaneously at 360 days of gestation. the foaling was supervised and uncomplicated with stage two labor lasting around 15 minutes, and stage three was complete at 30 minutes after delivery of the 35kg filly foal. routine examination of the placenta revealed that it had only occupied one uterine horn, otherwise it was normal in consistency and villous coverage. the mare’s colostrum was checked with a brix refractometer and deemed to be of good quality (26%). it soon became evident that the foal was impaired and most likely dysmature. the filly foal was small in stature and thin with curling of its ears and a silk-like texture to its hair coat. the foal had a suckle reflex but was weak and unable to stand or nurse by three hours of age. vital parameters were within normal limits and there was no evidence of any deformities. the foal had poor maternal recognition and environmental awareness. colostrum (200mls) was stripped from the mare and fed to the foal by means of a nasogastric tube. as the foal was unable to stand unsupported and had not managed to make any successful suckling attempts from the mare, an indwelling nasogastric tube was sutured in place. the foal was supplemented with mare’s milk at regular intervals and observed for evidence of postprandial colic. attempts were made to assist the foal to stand and try to nurse directly from the mare. from 12 hours of age the foal was able to periodically nurse from the mare if assistance was given with clinical theriogenology • volume 10 number 1 • march 201851 standing. if the foal was not witnessed to suckle then she was given additional milk. the foal was small in size for its breed and weighed approximately 35kg (foal was weighed on scales with person holding it) at 24 hours of age. at nine hours of age the foal’s igg concentration was assessed using a snap test assay and it had a serum igg concentration of greater than 800 mg/dl, indicating adequate transfer of passive immunity. over the next 24 hours the foal required less assistance to stand and suckle, but required help to lie down as it would fall asleep standing. the nasogastric tube was removed at 48 hours and the foal became progressively stronger and was able to stand, nurse and lie down unaided. the foal’s initial depression and weakness were attributed to in-utero growth retardation (iugr) owing to placental insufficiency. the placenta occupied only one uterine horn (fig. 1). in effect, the foal had the same disadvantage as an equine twin, with less than complete uterine surface available for placentation. no postpartum abnormalities were noted on physical examination of the mare and there were no signs of trauma within the vaginal vault. two days after foaling, trans-rectal palpation revealed that the uterus was involuting well with good tone. the uterine body, left uterine horn and both ovaries could be palpated. the right uterine horn was not palpable and instead a membranous band of tissue was identified, at the approximate location where the missing right horn would be expected, along the cranial edge of the broad ligament, and slightly thicker than the broad ligament. this band of membranous tissue originated from the base of the left horn and coursed to and apparently attached to the mesovarium of the right ovary. trans-rectal ultrasonographic imaging using a 10 5.0 mhz linear array transducer revealed follicular activity on both ovaries (fig. 2). the uterine body and left uterine horn had mild endometrial edema (fig. 3; consistent in size and echotexture with two days postpartum). the thin band of membranous tissue that was identified in place of the right uterine horn was imaged as a small linear structure and color doppler revealed prominent vasculature (fig. 4). on palpation there was an obvious difference in size, shape and texture between the mesometrium on the right hand side and the existing left uterine horn. an additional trans-rectal palpation and ultrasound examination of the uterus at seven days postpartum confirmed that the uterus was involuting appropriately and there was no evidence of a right uterine horn. three different clinicians all reached the same conclusion. discussion uterus unicornis is a rare congenital condition in which there is absence of one uterine horn. the condition has been reported in many domestic species including mares, cows, ewes, bitches and queens.2-7 pregnancies in mares, cows and ewes with uterus unicornis are reported and reproductive inefficiency caused by ovulation ipsilateral to the missing uterine horn have also been observed.3-5 it has also been suggested that fetal growth or litter size is restricted in uterus unicornis due to limitations in the uterine surface area available for placentation.4,5 in one case report, uterine unicornis was diagnosed as occurring on the right side in two mares.5 one of the mares previously had a mule foal that the owner had described as “small” at the time of birth. both of these mares failed to conceive during the breeding season that was documented. newcombe et al, described two cases involving aplasia of the left uterine horn; one mare also had a missing left ovary.3 both of these mares managed to conceive but there are no data on the gestation length and the weight of foals produced was reported. uterus unicornis is recognized as the most common congenital abnormality of the ovine uterus, with one survey reporting 20 cases among 30,000 abattoir specimens examined.4 a large multicenter study describing the characteristics and frequency of gross uterine anomalies in cats and dogs undergoing elective ovariohysterectomy, uterine anomalies were more than twice as common in cats (approx. 10/10,000 cats) than in dogs (approx. 5/10,000 dogs). more than half of cats and dogs with uterine anomalies also had ipsilateral renal agenesis, but both ovaries were identified in most affected animals, although the ipsilateral ovary was sometimes malpositioned.6 concurrent uterine and renal anomalies can arise as a result of the interdependence between the tracts during embryological development.8 both tracts are derived from a common embryonic intermediate mesoderm. the embryonic mesonephric urinary system develops first, followed by the definitive kidney and reproductive tract. the müllerian (paramesonephric) duct system gives rise to the clinical theriogenology • volume 10 number 1 • march 2018 52 uterine tubes, uterus, and vagina. it is believed that the mesonephric ducts induce müllerian duct formation and guide the ducts in their growth caudally.9-11 damage to the mesonephric duct may affect the development of the associated müllerian duct.9 in the case described in this report it may have been informative to assess the urinary tract and visualise if the right kidney was indeed intact. the horse has a diffuse epitheliochorial placenta that attaches to the entire endometrium. the size of the uterus is governed by the size of the mare and this, in turn, will directly control the available area for placentation and, hence, fetal growth.12 in the studies by allen et al, transfer of equine embryos between breeds led to either restricted thoroughbred-in-pony (tb-in-p) or enhanced pony-inthoroughbred (p-in-tb) growth of the foal, which was directly determined by the growth of the allantochorion. the mean birth weight of the tb-in-p foals was significantly lower than that of the tb-intb controls. the growth retardation and dysmaturity of the tb-in-p foals and the overgrowth of the p-intb foals may be a good model for comparison of mares with developmental conditions such as unicornuate uteri versus the normal bicornuate structure. twinning is another example in which the allantochorions of the two foals compete for the limited surface area of endometrium. occasionally, in the case of bicornuate twins, each allantochorion achieves attachment to roughly half the uterus and live, but smaller than normal, foals are born.13 twin foals which do survive to term are usually of small size and body weight, emaciated and have a very high rate of mortality compared to singleton foals.14 twin placentae are divided into three morphological groups according to the disposition of the chorionic sacs within the uterus. in type a, one fetus occupies one horn and most of the body (a mean 68 % of the total functional surface area),14 this may be comparable to the total placental area engaged with unicornuate pregnancy. it has been shown in another study that placental weights (which also include amnion and umbilical cord) up to 6.5kg are rate-limiting for foal birthweight.15 clearly in the case of uterus unicornis placental weight and also surface area would be reduced. the average gestation length of mares is approximately 340 days, and the range of normal gestations is considered to be 320 to 360 days.16 a gestation length of <320 days may be associated with delivery of a premature foal. a gestation of >360 days is considered prolonged. in the case described, the gestation length was on the cusp of prolonged at 360 days. the clinical examination of the case described confirmed uterus unicornis given the findings of trans-rectal palpation and ultrasonography of the mare’s reproductive tract and the passage of a placenta lacking one horn. a considerable palpable difference in size, shape and texture between the right mesometrium and existing left uterine horn could be appreciated. further diagnostic investigation could have been performed in the case presented. this may have included hysteroscopy of the uterus, fluid distention of the uterus combined with trans-rectal palpation and ultrasonography or laparoscopic imaging of the reproductive tract. the mare described in this case study had a breeding soundness examination performed prior to purchase and had been examined by trans-rectal palpation and ultrasound multiple times prior to foaling. if the absence of one uterine horn is suspected, the diagnosis should be confirmed by serial examinations or backed up by a second experienced clinician, especially if the mare is in anestrus. mating should then be delayed until ovulation is anticipated from the ovary contralateral to the absent horn. other forms of treatment which might be considered would include ovariectomy of the ipsilateral ovary. assisted reproductive technology, such as embryo transfer would be a valid recommendation for mares with uterus unicornis. learning points  uterus unicornis is an important condition requiring accurate diagnosis so that appropriate advice can be given to mare owners regarding potential reproductive inefficiency  the diagnosis of uterus unicornis in mares may be challenging depending upon the phase of the estrous cycle. if there are any doubts, serial examinations would be encouraged and also having a second experienced clinician involved would be beneficial clinical theriogenology • volume 10 number 1 • march 201853  birthweight of the foal is determined by the size of the mare’s uterus which it inhabits during gestation. one would therefore expect that mares with uterus unicornis would deliver smaller foals than bicornuate mares carrying fetuses with similar genetic potential. in this case the foal weighed only 35kg and an average warmblood foal would be 50-60kg  developmental uterine anomalies are uncommon. however, when anomalies are present in the small animals and humans, they were commonly associated with renal anomalies. therefore, the finding of unicornuate uterus should trigger an assessment of both kidneys because associated ipsilateral renal agenesis may require specific management practices for future health of the mare conflicts of interests no conflicts of interest to declare sources of funding none references 1. renaudin cd, troedsson mht, gillis cl, et al: ultrasonographic evaluation of the equine placenta by transrectal and transabdominal approach in the normal pregnant mare. theriogenology 1997;47:559-573. 2. thursby-pelham rh: uterus unicornis in mares. vet rec 1997;141:132. 3. newcombe jr: uterus unicornis in two mares. vet rec 1997;141:21. 4. smith kc, long se, parkinson tj: abattoir survey of congenital reproductive abnormalities in ewes. vet rec 1998;143:679-685. 5. brown ja, hodder adj, benak j, et al: uterus unicornis in two mares. aust vet j 2007;85:371-374. 6. mcintyre rl, levy jk, roberts jf, et al: developmental uterine anomalies in cats and dogs undergoing elective ovariohysterectomy. j am vet med assoc 2010;237:542-546. 7. rousset n, abbondati e, posch b, et al: unilateral hydronephrosis and hydroureter secondary to ureteric atresia, and uterus unicornis in a young terrier. j small anim pract 2011;52:440-443. 8. taylor e, gomel v: the uterus and fertility. fertil steril 2008;89:1-16. 9. li sy, qayyum a, coakley fv, et al: association of renal agenesis and mullerian duct anomalies. j comput assist tomogr 2000;24:829-834 10. romagnoli s, schlafer dh: disorders of sexual differentiation in puppies and kittens: a diagnostic and clinical approach. vet clin north am small anim pract 2006;36:573. 11. christensen bw: disorders of sexual development in dogs and cats. vet clin north am small anim pract 2012;42:515-526. 12. allen wr, wilsher s, turnbull c, et al: influence of maternal size on placental, fetal and postnatal growth in the horse. i. development in utero. reproduction 2002;123,445-453. 13. allen wr, stewart f: equine placentation. reprod fertil dev 2001;13:623-634. 14. jeffcott lb, whitwell ke: twinning as a cause of fetal and neonatal loss in thoroughbred mare. j comp pathol 1973;83:91-106. 15. elliott c, morton j, chopin j: factors affecting foal birth weight in thoroughbred horses. theriogenology 2009;71:683-689. 16. rossdale, pd: clinical view of disturbances in equine foetal maturation. equine vet j 1993;14,3-7. clinical theriogenology • volume 10 number 1 • march 2018 54 fig. 1. (a) placenta with allantoic surface imaged, this demonstrates only the uterine body and one uterine horn attachment. fig. 1. (b). placenta with chorionic surface photographed. this demonstrates only contact with one uterine horn and uterine body. there is no evidence of a twin membrane. clinical theriogenology • volume 10 number 1 • march 201855 fig. 2. the left and right ovaries with follicular activity. clinical theriogenology • volume 10 number 1 • march 2018 56 fig. 3. the left uterine horn and uterine body showing mild endometrial edema clinical theriogenology • volume 10 number 1 • march 201857 fig. 4. ultrasound and color doppler imaging displaying mesometrial vessels on the right side and no evidence of a right uterine horn. 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/preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] /pagelayout /singlepage >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1 contact kaylin mcnulty kaylin.mcnulty@msstate.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10328, http://dx.doi.org/10.58292/ct.v16.10328 case report amorphus globosus kaylin mcnulty,a michael jaffe,b joanne yib adepartment of pathobiology and population medicine, college of veterinary medicine, mississippi state university, mississippi state, ms, usa bdepartment of clinical sciences, college of veterinary medicine, mississippi state university, mississippi state, ms, usa abstract amorphus globosus is a rare malformation that is defined as an acardiac asymmetrical rough spherical mass of tissue covered in skin. to authors’ knowledge, amorphus globosus has not been previously reported in canids. herein, we describe amorphus globosus in an american bulldog pup with accompanying radiograph, gross images, and histopathology. additionally, a systematic review (preferred reporting items for systematic reviews and meta-analyses protocol) was performed for veterinary species; amorphus globosus has been reported in 22 bovids, 3 caprids, 1 ovid, and 1 equid. keywords: canine, dystocia, amorphus globosus, acardiac monster background fetal monsters are a rare occurrence in all species that may result in complications during pregnancy and parturition (e.g. dystocia). amorphus globosus1–5 is a type of acardiac fetal monster that is an asymmetrical rough spherical mass of tissue covered in skin. these monsters are associated with a cotwin and are considered a parasite twin, as the amorphus globosus individual’s blood supply relies solely on normal cotwin circulation.6 case presentation a 2.5-year, female intact american bulldog, was presented at the university emergency service with the primary complaint of dystocia. the dog had already delivered 4 healthy pups during a 9-hour period; no active labor was observed for 3 hours prior to presentation. duration of pregnancy was unknown; however, pups appeared normal and were nursing well. seven months before, the dog had dystocia and delivered live pups. appropriate mammary gland development with normal milk production was observed and abdominal distention with a brown vaginal discharge was noted. fetuses could be palpated within the abdomen. an abdominal and thoracic focused assessment revealed 2 fetuses with heart rates of 184 and 160 beats/minute, respectively. abdominal radiographs revealed 5 fetuses. bloodwork revealed normal ionized calcium and glucose concentrations. treatment cesarean surgery was performed, and 5 additional viable male pups and 1 nonviable pup of an unknown sex were removed. an uncomplicated ovariohysterectomy was performed before closure and recovery. all 5 viable pups were successfully resuscitated. the nonviable pup was within a grossly normal amniotic sac and placenta; the pup was examined and there were no appendages, head, or obvious body orifices noted. this pup was placed in 10% neutral-buffered formalin and submitted for postmortem examination and histopathology. outcome the dog recovered uneventfully from anesthesia and surgery, and was discharged from the hospital with 5 healthy, nursing male pups along with 4 healthy pups (2 male, 2 female) that were delivered before presentation. the nonviable pup weighed 67 grams and measured 6 x 4.5 x 3 cm. it was composed of a portion of brown to tan haired skin that measured 4.6 x 3.2 x 2 cm and a cystic portion that measured 5.5 x 3.7 x 2.1 cm. there was a remnant ear with an adjacent indentation interpreted as a remnant orbit. there was also a nearby indented puckered area that resembled a malformed nose and a 0.5 cm long tail-like structure with a nearby indentation that resembled an imperforate anus. there were no discernible limbs (figure 1). sectioning revealed no discernible consistent anatomy, much of the tissue was mailto:kaylin.mcnulty@msstate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10328 2 citation line: clinical theriogenology 2024, 16, 10328, http://dx.doi.org/10.58292/ct.v16.10328 gelatinous, and there was no heart (figure 2). sections were taken from what grossly appeared as remnants/portions of the gastrointestinal tract, a large vessel, and fetal bone, and these were submitted for routine histopathology. the remaining tissue was then sutured back together for preservation as a teaching specimen. a radiograph identified a mass of tissue containing irregular radiopaque fragments of partially formed bony structures and multifocal variably sized radiolucent fluid-filled spaces (figure 3). on histopathological examination, the tissue had multifocal luminal structures lined by short villi with an organized layer of epithelial cells that multifocally contained finely stippled large clear cytoplasmic vacuoles resembling goblet cells. submucosa of these luminal structures was filled with mixed inflammatory cells with a preponderance of lymphocytes and plasma cells. there were also scattered variably sized vessels within the submucosa and muscularis. muscularis layers had luminal structure and were variably sized (figure 4a). surrounding tissue contained edema and variably sized vascular structures that occasionally coalesced to form irregular amorphous channels with multifocal aggregates of reticular fibers reminiscent of splenic parenchyma. tissue also contained scattered mixed inflammatory cells, variably sized nerve bundles, and multifocal islands of variably vacuolated polygonal cells organized into cords reminiscent of hepatic cords. there was also a much larger section of these polygonal cells that formed a rudimentary liver including haphazardly arranged portal and central regions and sinusoid-like structures (figure 4b). focally, cartilaginous fragments surrounded a luminal space lined by cuboidal to columnar ciliated epithelium reminiscent of a trachea. in another area, bone with marrow elements and nearby large bundles of nerve cell bodies were observed (figure 4c). adjacent to this was figure 1. gross images of the nonviable pup; a. note structures reminiscent of an ear (black arrowhead), closed orbit (white arrowhead), nose (asterisk), tail (open black arrow), and imperforate anus (open white arrow); b. note prominent irregular vasculature in the cystic portion (arrows). figure 2. cut sections of nonviable pup; note the amorphous cystic fluid-filled structures and edematous tissue without heart. figure 3. radiograph of nonviable pup after sectioned for histopathology; note multifocal irregular bony fragments and multifocal fluid-filled spaces. horizontal defects are artifacts from sectioning for histopathology. http://dx.doi.org/10.58292/ct.v16.10328 citation line: clinical theriogenology 2024, 16, 10328, http://dx.doi.org/10.58292/ct.v16.10328 3 striated skeletal muscle and a section of bone undergoing bony maturation (figure 4d). discussion to assess the occurrence of this malformation in veterinary literature, a systematic review of case reports published prior to september 30, 2023 was performed. the framework as highlighted in ‘preferred reporting items for systemic review and meta-analysis protocols (prisma-p)’ was used.7 pubmed and the mississippi state university library system (http://library.msstate.edu/) were searched using terms ‘amorphus globosus’ and ‘amorphous globosus’ that yielded 102 results. with duplications and non-english papers removed, this left a total of 20 publications. publications were assessed and compiled (table). a systematic review identified reports in the following species: 22 bovids, 3 caprids, 1 ovid, and 1 equid (table). to author’s knowledge based on this review, there are no current reports of amorphus globosus in canids. amorphus globosus most commonly occurs in bovids, with the most common occurrence in holsteins (~ 1 in every 3,500 pregnancies).23 in humans, it occurs in 1 out of every 35,000 births.9 the most commonly accepted theory for the cause of this malformation is twin reverse arterial perfusion sequence (trap). in trap, there is a countercurrent flow of deoxygenated blood from the umbilical cord arteries of the cotwin and the amorphus globosus individual with return along the umbilical cord vein back to the placenta. this increases the cardiac demand on the cotwin.10 however, most cotwins in veterinary species die due to dystocia or dam-related disease from multiple fetal burden rather than heart failure.10,17 anastomosis of arteries or veins was lacking in one report of malformed fetus;22 it was speculated in that instance that insufficient blood supply led to complete destruction of a past existing heart structure.22 amorphus globosus has reportedly never been observed as a single birth.14 however, there is a single case report in a buffalo that presented in dystocia with an amorphus globosus structure and no cotwin.20 whether a cotwin was born out of sight or this entity can truly occur without a cotwin, is uncertain. there are reports of both monozygotic and dizygotic twins.6 interestingly, 2 freemartin cotwins have been reported in cattle.11,17 in these cases, the amorphus globosus individuals contained identifiable testicular tissue on histopathology. in another case, a normal heifer cotwin was identified with a male amorphus globosus.13 affected dams of all species are usually multiparous, with only 2 reported cases of uniparity in a japanese black heifer and a buffalo heifer.16,20 there is no sex predilection for amorphus globosus individuals or the associated cotwins, although there may be a trend towards male cotwins according to this review. there is no evidence to support culling or spaying of affected dams, as there are no reports of this condition occurring more than once in the same dam. future research can focus on pregnancy management of identified amorphus globosus dogs including early identification and treatment. another avenue to explore is the long-term effects on surviving cotwins. figure 4. histopathology (h&e): a. luminal structure resembling intestines with villous mucosa, submucosa, and distinct muscularis layers including mixed inflammatory cells and vessels, 4 x. inset: prominent goblet cells in villi, 40 x. b. large expanse of tissue resembles liver with hepatocytes forming irregular cords with interspersing sinusoid-like blood channels, 20 x. c. organized bundle of nerve cell bodies, 20 x. d. remnant of organized pale basophilic cartilage with appropriate maturation to bone and marrow resembles a neonatal growth plate; note structure surrounded by amorphous edematous tissue with admixed blood cells, 4 x. http://dx.doi.org/10.58292/ct.v16.10328 http://library.msstate.edu/ 4 citation line: clinical theriogenology 2024, 16, 10328, http://dx.doi.org/10.58292/ct.v16.10328 learning points • amorphus globosus is defined as an acardiac asymmetrical roughly spherical mass of tissue covered in skin • this disorder may increase the incidence of birth complications, most commonly dystocia and retention of fetal membranes • this malformation has the potential to cause cotwin mortality due to dystocia and potentially cardiac compromise • early identification and treatment of amorphus globosus individuals may decrease cotwin mortality and birth complications • there is no evidence to support culling or spaying of affected dams, as there are no reports of this condition occurring more than once in the same dam conflict of interest none to report. acknowledgement radiograph is courtesy of the university radiology service. references 1. anwar mt, khan ai, balasubramanian s, et al: a rare case of globosus amorphus in a goat. can vet j 2009;50:854–856. 2. baghbadorani mk, oghadam mf: histological investigation and karyotyping of amorphous globosus cases occurred in a dairy herd throughout one year observation. reprod domest anim 2017;52:100.  3. citek j, rehout v, hajkova j: congenital disorders in the cattle population of the czech republic. czech j anim sci 2009;54: 55–64. doi: 10.17221/1668-cjas 4. crossman jp, dickens psem: amorphus globosus in the mare. vet rec 1974;95:22. doi: 10.1136/vr.95.1.22-b 5. duncan: monstrosity in a cow – amorphous globosus. vet j 1891;33:163–164. doi: 10.1016/s2543-3377(17)35545–0 6. dunn ho, lein dh. kenney rm. the cytological sex of a bovine anidian (amorphous) twin monster. cytogenetics 1967;6:412–419. doi: 10.1159/000129959 7. shamseer l, moher d, clarke m, et al: preferred reporting items for systemic review and meta-analysis protocols (prisma-p): elaboration and explanation. bmj 2015;350:g7647. doi: 10.1136/ bmj.g7647 8. dunn ho, roberts sj. chromosome studies of an ovine acephalic-acardiac monster. cornell vet 1971;62:425–431. 9. fischer ur, hofmeister b: abnormal multiple pregnancy with  septuplets in a cow a case report. tierärztl prax ausg g  grosstiere nutztiere 2018;41:253–256. doi: 10.1055/s-0038 1623180 10. gehrke m, blaszak b, stachowiak m, et al: amorphus globosus foetuses in polish holstein cattle: anatomical, histological, and genetic studies. j vet res 2019;63:391–398. doi: 10.2478/ jvetres-2019-0057 11. gobikrushanth m, zachar ek, singh n, et al: theriogenology question of the month. j am vet med assoc 2022;260:170–173. doi: 10.2460/javma.21.06.0285 table. summary of published amorphus globosus cases in animal species species breeds reported (no. animals) sex ratio of ag (f:m) twin birth status (no. animals) sex ratio of twins(f:m) complications (no. animals) refs. bovine (bovidae family) holstein (10); unspecified (5); buffalo (2); aryshire (1); brown swiss (1); japanese black (1); jersey-dutch (1); white galloway (1) 2:3 (plus 18 unspecified) live (13); stillborn (12); nr (3) 3:4 (plus 13 unspecified) dystocia (5); none (6); nr (4); retained placenta (3); death (2); peri-parturient paralysis (1) 2,3,5,6,9, 10–14, 16–18,20,21–23 caprine (capra sp.) unspecified (2); kani-adu (1) 3 unspecified stillborn (4); live (2) 6 unspecified dystocia (3) 1,15,19 equine (equus caballus) unspecified (1) 1 unspecified live (1) 1 unspecified none (1) 4 ovine (ovis sp.) corriedale (1) 1 unspecified live (2) 0:2 nr (1) 8 canine (canis familiaris) american bulldog (1) 1 unspecified live (9) 2:7 dystocia (1) f = female; m = male; nr = not reported; ag = amorphus globosus. http://dx.doi.org/10.58292/ct.v16.10328 https://doi.org/10.17221/1668-cjas https://doi.org/10.1136/vr.95.1.22-b https://doi.org/10.1016/s2543-3377(17)35545-0 https://doi.org/10.1159/000129959 https://doi.org/10.1136/bmj.g7647 https://doi.org/10.1136/bmj.g7647 https://doi.org/10.1055/s-0038-1623180 https://doi.org/10.1055/s-0038-1623180 https://doi.org/10.2478/jvetres-2019-0057 https://doi.org/10.2478/jvetres-2019-0057 https://doi.org/10.2460/javma.21.06.0285 citation line: clinical theriogenology 2024, 16, 10328, http://dx.doi.org/10.58292/ct.v16.10328 5 12. hartigan pj: an acardius amorphus from a cow. irish vet j 1972; 26:133–136. 13. hishinuma m, hoshi n, takahashi y, et al: vasculature and  chromosomal composition in a bovine acardius amorphus. jpn j vet sci 1988;50:1139–1141. doi: 10.1292/jvms1939. 50.1139 14. jena d, khatti a, singh sk, et al. amorphous globosus in a cow: a  case report. theriogenol insight 2017;7:79–81. doi: 10.5958/ 2277-3371.2017.00022.5 15. john d, narasimhan ks: amorphus globosus in a she goat. cheiron 1980;9:129–131. 16. kamimura s, enomoto s, goto k, at al: a globosus amorphus from an in vitro fertilized embryo transferred to a japanese black cow. theriogenology 1993;40:853–858. doi: 10.1016/0093-691x (93)90220-y 17. nascimento lc, nakagaki kyr, orlando dr, et al: amorphus globosus in bovine. arch vet sci 2013;18:19–20. 18. pearson lk, rodriguez js, tibary a: theriogenology question of the month. j am vet med assoc 2011;238:1261–1263. doi: 10.2460/javma.238.10.1261 19. purohit gn, gupta ak, gupta ka, et al: amorphus globosus monster in a goat. indian vet j 2000;77:79. 20. pushp mk, purohit gn, tripathi a, et al: amorphus globosus monster removed by laparohysterotomy in a she buffalo – a case  report. theriogenol insight 2015;5:153–156. doi: 10.5958/2277-3371.2015.00017.0 21. singh ak, brar ps, pandey ak, et al: amorphous globosus monster in a buffalo. indian vet j 2009;86:846–847. 22. usta z, jacobsen b, gottschalk m, et al: amorphus globosus in  a  white galloway cattle. kafkas univ vet fak derg 2016;22:627–630. 23. weber j, rudolph n, freick m: facets of clinical appearance and aetiology in an unusual bovine amorphus globosus. anat histol embryol 2017;46:502-506. doi: 10.1111/ahe.12286 http://dx.doi.org/10.58292/ct.v16.10328 https://doi.org/10.1292/jvms1939.50.1139 https://doi.org/10.1292/jvms1939.50.1139 https://doi.org/10.5958/2277-3371.2017.00022.5 https://doi.org/10.5958/2277-3371.2017.00022.5 https://doi.org/10.1016/0093-691x(93)90220-y https://doi.org/10.1016/0093-691x(93)90220-y https://doi.org/10.2460/javma.238.10.1261 https://doi.org/10.5958/2277-3371.2015.00017.0 https://doi.org/10.1111/ahe.12286 ovarian abscess caused by transvaginal oocyte aspiration in a quarter horse mare ovarian abscess caused by transvaginal oocyte aspiration in a quarter horse mare stephanie grissom, carolyn arnold, dustin major, john edwards, a. perry, charles love department of large animal clinical sciences, college of veterinary medicine texas a&m university, college station, tx assisted reproductive technologies, such as transvaginal oocyte aspiration (tva), are common in the equine industry. complications are rare, but include internal hemorrhage and ovarian abscess formation.1,2 an apparently healthy, 15 year old quarter horse mare was referred for transvaginal oocyte aspiration. she had a history of a previous tva. transrectal palpation and ultrasonography revealed a not freely movable left ovary that was firm, with no palpable ovarian fossa, and a 4.9 x 2.2 cm structure with a fluid-filled center. her right ovary had a corpora hemorrhagica, 20 and15 mm follicles and 3 follicles that were 5 mm. she had 1.5 cc of fluid in the body of her uterus and a tight cervix. due to history, elevated temperature (39.6oc), and findings of left ovary, an ovarian abscess was suspected, and tva was not performed. owners elected to remove left ovary via a laparoscopic ovariectomy. during the procedure, ovarian abscess ruptured prior to removal from abdomen, requiring placement of an abdominal drain and continued lavages with sterile saline and infusions of cefazolin over the next 24 hours. ovary was submitted for histopathology and cultures from the abscess were submitted to clinical pathology. immediate postoperative treatment included potassium penicillin, saccharomyces, gentamicin, and flunixin meglumine. subsequently, klebsiella pneumoniae, resistant to potassium penicillin and gentamicin was cultured. those drugs were discontinued and she was then treated with chloramphenicol. histopathology of the ovary indicated a focal, chronic, marked, suppurative oophoritis with fibrosis (abscess). this mare required substantial, continued, postoperative medical management due to several complications but did eventually recover after more than 6 months in the hospital. this case highlighted a potential complication of a frequent reproductive procedure, as well as the presentation of an ovarian abscesses. keywords: mare, ovarian, abscess, transvaginal, oocyte, aspiration references 1. vanderwall dk, woods gl: severe internal hemorrhage resulting from transvaginal ultrasound-guided follicle aspiration in the mare. j equine vet sci 2002;22:84-86. 2. velez ic, arnold c, jacobson cc, et al.: effects of repeated transvaginal aspiration of immature follicles on mare health and ovarian status. equine vet j 2012;44:78-83. 535 clinical theriogenology • volume 11, number 3 • september 2019 536clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2016: review of breeding shed biosecurity and disinfection agents review of breeding shed biosecurity and disinfection agents david b. scofield, julie baumber-skaife, paul r. loomis select breeders services inc., chesapeake city, md introduction a complete biosecurity plan for an equine facility hinges upon a current understanding of disease risk, disease entrance, disease propagation, and disease recognition. all reproduction facilities need to be aware of the acute disease risks for our patients, areas of disease entrance and incubation, protocols for disease testing, routine disease mitigation, and specific action protocols to follow in the face of a disease breakthrough. furthermore, an adequate biosecurity plan and strategy requires 100% buy-in from staff, technicians, veterinarians, and front office personnel. this buy-in includes client understanding of the risks to their horses and their willingness to help provide a safe and disease free veterinary or breeding center. clients, veterinarians, and veterinary staff are ultimately responsible for adhering to adequate disease testing schedules, routine and specific facility cleaning, horse movement management, monitoring for disease breakthrough, and providing information for traceability in the event of biosecurity breakdown. currently in the us and other north american countries, there are no regulations, certifications or licenses required to collect, process, and distribute semen and to operate a breeding or boarding center; as such, the possibility for venereal disease transmission as well as contagious disease transfer between equines, remains a major veterinary and economic threat. recent occurrences of both equine viral arteritis (eva) and contagious equine metritis (cem) in the us have illustrated the potential for the spread of such venereal diseases in the equine breeding population. moreover, a lack of sufficient quarantine and entrance requirements for many breeding farms allows for a high likelihood of contagious disease to be spread rapidly as numbers of breeding horses spike during the busy times of the year. areas of concern this author identifies biosecurity risks to our facility into three distinct categories. there is certainly crossover between all categories, but this classification scheme helps to outline the best management strategies to implement and to control exposure. in all areas, the goal is to limit the possibility of direct (horse-to-horse contact and venereal transmission) and indirect (environmental and fomites) passage of disease in the following categories: 1. facility oriented 2. venereal transmitted 3. contagious etiologies briefly, facility oriented diseases come from an individual location and disease history for a farm. is rhodococcus present? is there history of clostridium in foals? is rabies prevalent in the area? is leptospirosis or tickborn disease a concern? others might be concerned about transmission of equine protozoal myeloencephalitis and viral encephalopathy in some areas of the country. possible venereal diseases of concern include:  equine viral arteritis virus  contagious equine metritis  equine herpes virus 3 (coital exanthema)  west nile virus  equine nfectious anemia  piroplasmosis  vesicular stomatitis  african horse sickness venereal diseases of the horse are discussed elsewhere, see metcalf and timoney in suggested readings. exposure to these venereal diseases can be from outside stallions or mares shipped into the facility, cooled or frozen semen sources, horizontal transmission from fomites, and from horses housed at clinical theriogenology • volume 8, number 4 • december 2016479 the facility such as tease mares and recipient mares. contagious areas of concern include equine herpes virus, streptococcus equi, influenza, and respiratory or gastroenterological disease of various etiologies that remain a concern for many breeding farms and stallion stations throughout the country. specific action areas each area of the facility has unique and specific requirements in the overall biosecurity plan. however, every facility has the following areas to address in a biosecurity plan: 1. long term horse population a. resident horses, tease mares, recipients, and stallions 2. incoming/departing horses a. short term boarders, foaling mares/foals, and non-resident stallions 3. barn and outdoor environment a. pasture turnouts, communal areas, and round pen/exercise area b. environmental concerns c. vectors 4. breeding shed a. phantom b. tease mare c. equipment 5. laboratory a. collection supplies b. outside sample handling from other centers c. microbiology 6. quarantine a. import and export qualification b. sick or hospitalized animals this presentation will focus primarily on a biosecurity plan on the laboratory and breeding shed. collection equipment should be new and disposable for each individual stallion. if reusable artificial vaginas (av) are utilized, each stallion should have a specific av that is properly cleaned and disinfected prior to each use or undergo appropriate disinfection protocols as outlined in the american association of equine practitioners guidelines in between stallions. other types of avs can be utilized but must undergo a specific and thorough cleaning, disinfection, and drying period in between uses. wash buckets, lip chains, twitches, and breeding halters should be cleaned and disinfected between uses as well as having appropriate disposable barrier protection applied to the rear of the breeding phantom and appropriate cleaning of the entire phantom, especially near the bite strap, before each use. shed floors should be washable and sprayed with environmental disinfectants to keep contaminant and infectious agents at manageable levels. collection and processing personnel should wear appropriate barrier protection including smock, two layers of disposable gloves, and use appropriate footbaths placed at the entrance/exit of the breeding shed and laboratory entrances, as well as wash hands and use alcohol based sanitizing hand wash frequently and before handling collected semen or laboratory equipment. lastly, laboratory areas should be routinely disinfected with appropriate tabletop laboratory sprays, and areas of semen processing, water baths, and sinks/water supplies, should be cleaned and monitored as areas for possible bacterial and fungal contamination. a common source of contamination is laboratory water baths. easily contaminated with bacteria from hands, collection bottles, tubes, and labware, these water baths must be cleaned and disinfected regularly. for a semen collection shed and laboratory, there is the added concern of residues that could have a toxic on the semen. therefore, it is imperative to have protocols in place with agents that leave no detergent, soap, or antimicrobial residue that may have an undesired effect on the collection equipment or areas in close proximity to the collection vessel. it is important to have an in-house quality control program to document disinfection protocols. it should also include periodic surveillance cultures of clinical theriogenology • volume 8, number 4 • december 2016 480 surfaces, water baths, counters and tap water used to wash the stallion. hoses attached to water faucets can also contain pseudomonas spp. and other bacteria and when used to wash the stallion’s penis can result in repeated inoculation of the penis just prior to collection. overview of common disinfectants antimicrobial pesticides as defined by the environmental protection agency can be very effective agents to control and sometimes to destroy microorganisms on inanimate and hard, non-porous surfaces. classified as a sanitizer, disinfectant or sterilant, certain agents have patterns of efficacy that can be used to help develop appropriate biosecurity measures. sanitizers reduce the microorganism load, but do not eliminate all microorganisms. sanitizers help bring bacterial or viral load into a more manageable level, but do not act as a germicide that removes all microorganisms. a disinfectant, when applied to surfaces, destroys or inactivates most bacteria and some viruses. spores and hardy viruses remain viable even in the presence of a disinfection protocol. the most extreme agents act as sterilants, and by definition, remove all forms of microbial life. using extreme heat or chemicals, surfaces are sterilized of all microbial life. detergents are additives that help disinfectants and sanitizers remove organic debris and dirt to allow access of the disinfectant to the microorganisms. detergents, based upon their cationic or anionic nature have different properties for use in disinfectants, and can affect their use in a breeding shed environment. the following table provides a basic outline for choosing a disinfectant for various locations throughout the breeding shed and stallion center. of note, specific concerns such as relative humidity, application temperature, hard/soft water, and ph can affect the efficacy of each agent. all agents require that the surface be free of organic debris, therefore adequate washing with water for equipment and pressurized hose for the shed is required to remove the organic debris and dirt that will prevent the mechanism of action of the applied disinfectant. implementation and conclusions overhauling or implementing a new biosecurity plan is not without difficulty. however, a biosecurity plan is one of the most important plans in place at the clinic, hospital, or collection center. the cost of routine and effective biosecurity is far less than the cost of a total shutdown of operations and cleanup following a disease outbreak or biosecurity breach. to successfully implement an effective biosecurity plan, each member of the team has an important and definable role, from the receptionist confirming vaccination history of a patient, to the technician performing a visual inspection of the horse, to the attending veterinarian providing physical examinations, and to the barn staff who interact with the animals daily. staff must follow checklists, between collections, daily, weekly, and monthly to insure the biosecurity plan is being followed. sometimes, it takes a dedicated staff to slow down during the busiest times of the year and perform extensive cleaning to prevent any disease spread and to create a “go-back point” to know that the facility was adequately cleaned and disinfected. with the advancements in reproductive technology and the increased use of cooled and frozen semen, the potential for devastating consequences from a disease breakthrough is real. as seen recently, a disease outbreak can easily spread to multiple breeding centers very quickly even when separated by vast physical distances. simple biosecurity steps can help mitigate and reduce these risks, but only when all staff follow proper cleaning techniques, use effective disinfectants, and appropriate application technique. suggested reading timoney pj: diseases potentially transmitted with frozen or cooled semen. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. hoboken: blackwell publishing ltd; 2011. biosecurity guidelines for control of venereally transmitted diseases. american association of equine practitioners. available at: http://www.aaep.org/info/breeding-guidelines metcalf es: venereal disease. in: mckinnon ao, squires el, vaala we, et al, editors. equine eproduction. 2nd ed. hoboken: blackwell publishing ltd; 2011. dvorak g: disinfection 101. may 2008. http://cfsph.iastate.edu clinical theriogenology • volume 8, number 4 • december 2016481 d isi nf ec ta nt al co ho l al de hy de s bi gu an id es h yp oc hl or ite io di ne co m po un ds o xi di zin g ag en ts ph en ol s q ua te rn ar y am m on iu m co m po un ds (q ac ) t ra de n am e 70 % i so pr op yl a lc oh ol fo rm al de hy de g lu ta ra ld eh yd e c hl or oh ex id in e n ol va sa n b le ac h b et ad in e p ro vi do ne h yd ro ge n p er ox id e v ir ko ns o ne -s tr ok e e nv ir on t ek -t ro l r oc al d iq ua t m ec ha ni sm o f a ct io n (m o a ) -p ro te in pr ec ip it at io n -d en at ur es li pi ds -d en at ur es p ro te in s -a lk yl at es nu cl ei c ac id s -a lt er s m em br an e pe rm ea bi lit y -d en at ur es pr ot ei ns -d en at ur es pr ot ei ns -d en at ur es li pi ds an d pr ot ei ns -a lt er s m em br an e pe rm ea bi lit y -d en at ur es pr ot ei ns -d en at ur es pr ot ei ns -b in ds c el l m em br an es a dv an ta ge -s ho rt c on ta ct ti m e -n o r es id ue s -b ro ad e ff ic ac y -n on c or ro si ve -b ro ad e ff ic ac y -b ro ad e ff ic ac y -c os t -s ho rt c on ta ct ti m e -s af et y -b ro ad e ff ic ac y -a ct iv e w it h or ga ni c m at te r -n on -c or ro si ve -n on ir ri ta ti ng -e ff ic ac y at h ig h te m p an d hi gh ph d is ad va nt ag e -r ap id ev ap or at io n -d am ag e to so m e ru bb er -s af et y (c ar ci no ge ns ) -w ea r pr ot ec ti ve eq ui pm en t -i rr it at in g to m uc us m em br an es -s pe ci fi c ph re qu ir ed -l im it ed u se w it h fu ng i, vi ru es , a nd sp or es -i na ct iv at ed b y su nl ig ht -c or ro de s m et al -t is su e ir ri ta nt -i na ct iv at ed b y q a c co m po un ds -s ta in in g -c or ro de s m et al s -c or ro de s m et al s -e ye ir ri ta nt pr ec au tio ns f ir e r is k c ar ci no ge ns c hl or in e ga s ta bl e. s um m ar y of co m m on d is in fe ct an ts clinical theriogenology • volume 8, number 4 • december 2016 482 1 contact scott bailey csvetconsulting@gmail.com © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10536, http://dx.doi.org/10.58292/ct.v16.10536 research report reproductive and health outcomes of horses affected by focal mucoid placentitis scott bailey,a,b,c thalita leite,a mats troedssonb aclaiborne farm, paris, ky, usa bdepartment of animal science, university of kentucky, lexington, ky, usa ccollege of veterinary medicine, north carolina state university, raleigh, nc, usa abstract focal mucoid (nocardioform) placentitis occurs sporadically worldwide and occasionally has been associated with local epidemics of disease in central kentucky. objective was to quantify the effects of focal mucoid placentitis on a farm during spring 2020 outbreak. outcomes were prospectively identified early in the spring and data were collected in a single cohort of mares during 20202021 and on their foals during 2020-2022. focal mucoid placentitis was diagnosed in 36 of 162 mares; 6 foals died at birth and 30 foals (83%) were viable compared to 119 of 126 viable foals (94%) from normal mares. foals born to mares with placentitis weighed 10 lb less compared to those from healthy mares, received colostrum that was slightly lower in quality and had ~ 30% lower blood igg concentrations. however, postnatal foal survival, incidence of infectious or orthopedic foal diseases, sale values, and racing performances were not different between these 2 groups. focal mucoid placentitis did not recur in subsequent years and the reproductive performance of mares with this form of placentitis was not affected in the subsequent breeding season. study suggested that although the disease may be underdiagnosed via farm-submitted samples, surviving foals and affected mares were able to overcome the effects of this condition and their performance was comparable to their peers in the absence of additional treatment. keywords: focal mucoid placentitis, nocardioform, mare, pregnancy loss, foal health introduction focal mucoid, or nocardioform placentitis is a well-recognized cause of equine perinatal disease that has been most often described in central kentucky and has occurred sporadically worldwide.1,2 focal mucoid placentitis is characterized by acute abortion or birth of small, thin foals that may be precociously developed, but can also go undetected with normal pregnancy length and the delivery of healthy foals.3 gross examination of the chorioallantois frequently reveals a thick mucoid discharge on the chorionic surface that is characteristically at the base of the gravid uterine horn or occasionally extending into the gravid or nongravid uterine horns.4 ‘nocardioform’ organisms, including crossiella equi, amycolatopsis spp., and streptomyces spp. are frequently detected on cytologic evaluation of a touch prep from the lesion margin or identified by lesion culture and/or pcr.5 the condition has been reported in the us, europe, and south africa and several ‘outbreaks’ have been documented in the equine population of central kentucky.1-10 the objective was to monitor all pregnant mares in a farm in central kentucky for clinical signs of focal mucoid placentitis, neonatal health, and gross and microscopic/microbiological changes to fetal membranes during a recognized regional outbreak in 2020 spring. subsequently, breeding performance of mares, incidence of infectious and orthopedic diseases of foals, sale values, and racing performance as 2-years old were compared between animals affected by this form of placentitis and those that were not affected. materials and methods pregnancy monitoring and treatment in a thoroughbred farm in central kentucky, 167 pregnant mares were managed for foaling. mares ranged from 3-23 years (median 15 years) in age and all mares had been bred by natural cover in the preceding breeding season (february 15-july 4, 2019). for the purposes of this study, data were mailto:csvetconsulting@gmail.com http://dx.doi.org/10.58292/ct.v16.10536 2 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10536 collected from routine clinical procedures on a private commercial farm and no experimental treatments or procedures were performed. institutional animal care and use committee approval is not required, and the study is exempt. mares were monitored for evidence of impending parturition and brought to a designated foaling barn when parturition was deemed imminent. at the foaling barn, mares were observed throughout the day and night by an experienced foaling team. at the onset of parturition, the caslick vulvoplasty (if present) was incised with metzenbaum scissors and normal presentation and positioning of the foal were confirmed by palpation. in normal foaling, minimal intervention was provided other than amnion removal from the foal’s nostrils and prophylactic application of iodine on the umbilical stump after umbilical rupture. in instances of dystocia, a veterinarian was called and the team provided immediate assistance to the mare. if delivery of the foal was not accomplished in 30-45 minutes, mares were transported to a referral hospital for further care. only mares that foaled successfully on-site were included in this analysis (n = 162). preparturient milk production, placental weight and colostrum quality (via refractometer) were recorded at foaling. complete examination of the neonate and fetal membranes was performed within 12-24 hours after foaling. on the morning after foaling, blood was collected from foals (> 2 hours old) for white blood cell count (wbc) and igg assay and foals were given 1.5 liters of plasma for prevention of rhodococcus pneumonia. examination and plasma infusion of foals that were born between 4 and 6 am was delayed until 2-4 pm of the same day. foal and fetal membrane abnormalities and foal weight were recorded. at fetal membranes examination, samples were collected from any suspicious lesions for cytologic examination of a touch prep, pcr for nocardioform organisms, and aerobic culture. cytologic examination of the touch prep was performed on-site by an experienced practitioner, blood samples were submitted to the diagnostic laboratory of the rood and riddle equine hospital, lexington, ky, and samples of the fetal membranes were refrigerated and sent to university of kentucky veterinary diagnostic laboratory (ukvdl), lexington, ky within 24 hours after collection. a diagnosis of focal mucoid placentitis was made if any of the following occurred: evidence of branching, chain-forming bacteria on cytologic examination; histologic diagnosis of focal placentitis on necropsy; positive diagnosis of crossiella equi, amycolatopsis spp. or streptomyces spp. on pcr; or culture of gram-positive branching bacilli on aerobic culture. in the absence of additional clinical disease, mares and foals with diagnoses of focal mucoid placentitis received no additional treatment based on this diagnosis. comparisons were made between mares and foals with a diagnosis of focal mucoid placentitis and those which had no detectable periparturient disease (normal mares), for the parameters outlined in table 1. data analysis data were analyzed using publicly available free software (www.socscistatistics.com). duration of pregnancy at foaling, foal weight, placental weight, igg, wbc, sale value, and racing income were analyzed using a student’s t-test and nonparametric data were analyzed using a pearson’s chi-square test. results during 2020 breeding season, 167 pregnant mares were maintained on the farm for foaling management. of those, 5 mares experienced dystocia or delivered a severely compromised foal that was immediately transferred to a referral hospital without further diagnostics. six mares with focal mucoid placentitis experienced abortion or stillbirth. three mares delivered foals with traumatic injuries that necessitated immediate euthanasia, and 2 foals were medically compromised at birth and immediately transferred to a referral hospital without further diagnostics. mares (n = 162) and foals born to them were included in the analysis; 151 (93%) mares delivered live foals that stood and nursed. examination of fetal membranes revealed 36 cases (22% of foaling mares) with gross abnormalities of placental chorionic surface, characterized by a thickened chorioallantois with blunted or denuded villi and variable amounts of mucoid exudate adhered to chorion and were diagnosed with focal mucoid (nocardioform) placentitis. no cases of ascending placentitis were diagnosed, and focal mucoid placentitis was diagnosed more commonly earlier in the year than later during the same foaling season (figure). thirty of 36 grossly abnormal fetal membranes were also examined on farm cytologically, whereas 22 of 36 were submitted to the ukvdl for aerobic culture and pcr assay (table 2). characteristic chain-forming branching bacteria were observed in 23 of 30 (76%) of examined smears and culture or pcr revealed the presence of nocardioform placentitis in 17 of 22 (77%) of submitted samples. two of 22 (9%) cases that were grossly consistent with focal mucoid placentitis had no detectable organisms on microscopic examination of a touch prep slide, but were pcr positive for nocardioform organisms. in 5 of 22 (23%) cases, chain-forming organisms were observed cytologically, but pcr was negative. however, in 3 of those cases, culture and histology were most consistent with focal mucoid placentitis. in total, onsite cytologic findings matched advanced diagnostic results from the diagnostic laboratory in 16 of 22 (73%), table 1. parameters for comparison between groups mares foals incidence of postpartum reproductive disease incidence of neonatal disease number of breeding attempts per pregnancy incidence of hernias incidence of pregnancy loss incidence of surgical developmental joint disease (osteochondritis dissecans) number of mares foaling in subsequent season sale value (highest numerical bid recorded by sales agency, independent of reserve) incidence of placentitis in subsequent season racing performance during first 3 months in 2-year-old foals http://dx.doi.org/10.58292/ct.v16.10536 http://www.socscistatistics.com citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10536 3 whereas 6 cases with gross lesions consistent with focal placentitis but which were cytologically negative were confirmed to have focal mucoid placentitis by the diagnostic lab (27%). in 7 cases (32%), culture and pcr did not identify nocardioform organisms, whereas cytologic or histologic examination of grossly abnormal tissues confirmed a diagnosis of focal mucoid placentitis. retrospective comparison of pregnancy outcomes mares diagnosed with focal mucoid placentitis after foaling had a slightly shorter (p = 0.0009) pregnancy length on average than mares with normal uncomplicated pregnancies (325.4 ± 15 versus 331.9 ± 9 days). thirty of 36 mares (83%) with focal mucoid placentitis produced live foals (p = 0.0015) compared to 119 of 126 (94%) mares without placentitis. mares that foaled with gross lesions of mucoid placentitis were not more likely to drip milk (p = 0.18) during prefoaling (20 versus 11%). however, colostrum quality was assayed in 117 foaling mares and was lower (p = 0.0015) in foaling mares with a diagnosis of placentitis than normal mares (25 ± 6.3 versus 28 ± 3.3%). duration of pregnancy at foaling for mares with focal mucoid placentitis that produced live foals was similar (p = 0.054) to normal mares (328.8 ± 10 versus 331.9 ± 9 days). birthweights of 30 live foals born to mares with placentitis were ~ 10 lb lower (p = 0.001) on average than those of 113 live foals from normal mares (112.7 ± 19.7l versus 123 ± 14.9 lbs). fetal membrane weights were not different (18.9 ± 3.5 versus 18.5 ± 3.6 lbs; p = 0.29). however, the relative fetal membrane weight in relation to foal weight was greater (p = 0.002) in cases of focal mucoid placentitis (16.7 ± 3.1 versus 15 ± 2.6%). immunoglobulin g of foals born to mares with placentitis was lower (p = 0.0003) at the first examination than that of normal mares (1,498 ± 1,005 mg/dl versus 2,295 ± 1,134 mg/dl); however, this did not translate to a difference in the incidence of failure of passive transfer (igg < 400: 4 and 8 respectively) or partial failure of passive transfer (igg 400800: 2 and 7 respectively in foals born to mares with placentitis compared to normal mares; p > 0.1). foals’ wbc at the first examination was not different (p > 0.1) between groups (11 ± 2.9e3/ml versus 11.2 ± 2.5e3/ml). subsequent mare reproductive performance none of the 36 mares with placentitis experienced periparturient complications (e.g. retained fetal membranes or metritis) or significant vaginal or vulvar trauma that required intervention. consequently, in accordance with our protocols, mares diagnosed with focal mucoid placentitis did not receive any systemic or local treatment at foaling. during 2020 breeding season, 156 foaling mares were bred again. there were no differences (p > 0.1) between foaling outcomes of mares that had experienced focal mucoid placentitis in the previous year and those that had not; 33 of 35 mares (94%) that had experienced focal mucoid placentitis were bred again and became pregnant with an average of 1.5 cycles/pregnancy, and 113 of 121 mares (93%) that had foaled normally and were bred back became pregnant (p > 0.1) with an average of 1.5 cycles/ pregnancy. no mares in either group were diagnosed with focal mucoid placentitis in 2021. three mares that had experienced placentitis and 8 normal foaling mares experienced embryonic loss (p > 0.1) before 45 days of pregnancy. in these groups, 128 mares were maintained on the farm until foaling in 2021. of those, 1 pregnant mare that had experienced placentitis and 9 normal mares aborted in the 2021 foaling season (p > 0.1; table 3). neonatal and developmental disease incidences of broken ribs, neonatal developmental limb contracture and infectious disease broadly were not different (p > 0.1) in foals born to mares with focal mucoid placentitis than normal mares; however, foals born to mares with placentitis had a slightly higher risk (p = 0.04) of developing umbilical hernias that required surgical repair (table 4). foals (n = 139) that were kept beyond 12 months of age received full radiographic examination for developmental orthopedic disease (survey radiographs) with evaluation by an experienced practitioner blinded to foals’ status. there was no difference (p > 0.1) in the number of yearlings requiring arthroscopic surgery from mares with placentitis (8/29; 29%) compared to those from normal mares (39/110; 35%). sale and racing performance foals of mares with placentitis (n = 28) and 116 foals born to normal mares survived until december 2022 and were included in analysis of performance. there were no differences (p > 0.1) in either sales value or racing income of horses born to mares with focal mucoid placentitis compared to normal mares. nine of 28 (32%) foals of mares with placentitis were sold for an average of $106,670 (range: 10,000-425,000; median 9 13 9 4 0 9 38 43 24 7 0 10 20 30 40 50 60 january february march april may number of foals born with and without placentitis by month placentitis normal figure. number of foalings with and without gross or histologic evidence of focal mucoid placentitis during each month of the 2020 foaling season. http://dx.doi.org/10.58292/ct.v16.10536 4 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10536 50,000) whereas 42 of 116 (36%) foals of healthy mares were sold for an average of $130,200 (range 1,000-950,000; median 75,000; p = 0.7). fifteen of 28 (54%) foals with placentitis were started in races with an average of 1.67 starts per horse, whereas 39 of 124 (33%) foals of healthy mares started with an average of 0.84 starts per horse (p < 0.05). foals of mares with placentitis (n = 28) produced $353,549 in earnings with an average of $12,626.75 for each surviving horse. foals (n = 116) of table 3. pregnancy outcomes of mares with and without evidence of focal mucoid placentitis in the 2021 foaling season foaled barren early embryonic death aborted sold normal (n = 121) 76 (63%) 8 (6.6%) 8 (6.6%) 9 (7.4%) 23 focal mucoid placentitis (n = 35) 25 (71%) 2 (5.9%) 3 (8.6%) 1 (2.9%) 5 table 2. diagnostics performed and outcomes for mares diagnosed with focal mucoid placentitis mare # histologic diagnosis cytologic diagnosis culture results pcr results outcome 1 placentitis not performed g+ branching bacillus amycolatopsis sp. and crosiella equi abortion 2 chronic focal placentitis branching bacilli nonpath not performed normal foal 3 placentitis branching bacilli not diagnostic crosiella equi abortion 4 not performed not performed g+ branching bacillus crosiella equi stillbirth 5 placentitis branching bacilli g+ branching bacillus amycolatopsis sp. normal foal 6 placentitis branching bacilli g+ branching bacillus amycolatopsis sp. normal foal 7 avillous placenta normal no growth not performed normal foal 8 placentitis not performed g+ branching bacillus crosiella equi abortion 9 focal placentitis normal nonpath not detected normal foal 10 placentitis branching bacilli g+ branching bacillus amycolatopsis sp. normal foal 11 placentitis not performed g+ branching bacillus crosiella equi abortion 12 placentitis branching bacilli g+ branching bacillus amycolatopsis sp. abortion 13 chronic focal placentitis normal nonpath not performed normal foal 14 not performed branching bacilli nonpath amycolatopsis sp. normal foal 15 not performed branching bacilli g+ branching bacillus crosiella equi normal foal 16 not performed branching bacilli nonpath not detected normal foal 17 not performed branching bacilli nonpath not detected normal foal 18 not performed branching bacilli g+ branching bacillus crosiella equi normal foal 19 not performed branching bacilli not performed not performed normal foal 20 placentitis normal nonpath not detected normal foal 21 not performed branching bacilli g+ branching bacillus not detected normal foal 22 not performed normal not performed not performed normal foal 23 not performed normal nonpath crosiella equi normal foal 24 not performed branching bacilli g+ branching bacillus crosiella equi normal foal 25 not performed branching bacilli not performed not performed normal foal 26 not performed branching bacilli g+ branching bacillus amycolatopsis sp. normal foal 27 not performed branching bacilli not performed not performed normal foal 28 not performed normal acinetobacter wolfii not performed normal foal 29 not performed branching bacilli not performed not performed normal foal 30 not performed branching bacilli not performed not performed normal foal 31 not performed branching bacilli nonpath crosiella equi normal foal 32 not performed branching bacilli rhizobium radiobacter not detected normal foal 33 not performed branching bacilli nonpath not detected normal foal 34 not performed branching bacilli not performed not performed normal foal 35 not performed branching bacilli g+ branching bacillus amycolatopsis sp. normal foal 36 not performed normal not performed not performed normal foal http://dx.doi.org/10.58292/ct.v16.10536 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10536 5 healthy mares produced $886,073 with an average of $7,638.5 for each horse that survived to december 2022 (p = 0.26). discussion results of this observational field study on the incidence of focal mucoid (nocardioform) placentitis in a single farm during the 2020 outbreak in central ky suggested that subclinical focal mucoid placentitis may be more common than previously recognized during an outbreak. in the farm studied 36/162 (22%) of foaling mares examined had gross lesions consistent with this form of placentitis. as reported,3 most cases of focal mucoid placentitis were associated with the growth of nocardioform actinomycetes, most commonly crosiella equi, amycolatopsis spp., and streptomycetes spp. however, as described,3 nocardioform actinomycetes were not isolated on culture or pcr in all clinical cases with typical pathological lesions. when combined with results of prospective inoculation studies that failed to induce disease with intrauterine, oral, respiratory, and intravenous inoculation of these microorganisms,11 raises the question whether nocardioform actinomycetes caused the disease or if their frequent identification merely represents an opportunistic consequence of the disease. clinically, these findings underscore the importance of detailed gross and cytologic evaluations of fetal membranes on-site that may be more sensitive in detecting placentitis than laboratory results of submitted samples. furthermore, it is unknown with what frequency positive pcr or culture results for nocardioform actinomycetes might be obtained from normal foaling mares. mares with focal placentitis had a slightly shorter duration of pregnancy than normal mares (325 days versus 331 days) and focal mucoid placentitis had substantial effects on foal survival and birthweight of affected foals, with a 17% mortality and 10 lb lower birthweight than foals from normal mares. this can likely be explained by a reduction in attachment of healthy chorion to the endometrium with consequences on prepartum nutritional fetal support; however, nocardioform placentitis was not associated with any detected clinical changes in these mares. incidence of prepartum milk production was not different and there were no reports of vulvar discharge or precocious mammary development in this group. routine transabdominal ultrasonographic examinations and endocrine monitoring were not performed in this farm and it is unknown whether such procedures could have accurately diagnosed focal mucoid placentitis or whether treatment of affected mares might have altered pregnancy outcomes. however, mares with focal mucoid placentitis had 83% foal survival rate and surviving foals from mares with focal mucoid placentitis were of normal age of pregnancy at parturition and did not experience neonatal health issues commonly observed in foals from mares with ascending placentitis. furthermore, the risk of surgical osteoarthritis was not increased in foals born to mares with focal mucoid placentitis and they performed similar as yearlings and 2-year old race-horses. statistical increase in umbilical hernias in this group of foals was surprising and should be confirmed with additional observations. however, hernial size was similar and all were repaired surgically with no clinical impact. it was interesting to observe that foals born to mares with focal mucoid placentitis were more likely to race and had more starts per horse than the cohort of foals that was unaffected by placentitis. these findings suggested that in foals surviving to term or born with precocious joint and gastrointestinal maturation sufficient to allow normal neonatal development, any effect of focal mucoid placentitis related to fetal starvation can successfully be compensated after birth. focal mucoid placentitis in the absence of other perinatal disease did not negatively affect the subsequent mare’s reproductive performance or foal health and development. the per cycle and seasonal pregnancy rates in mares with placentitis were nearly identical to those of normal-foaling mares on the same farm in the absence of any treatment, and none of the affected mares developed placentitis during the subsequent pregnancy. this demonstrated that mares had the capacity for uterine clearance during postpartum and supported a conservative treatment approach in foaling mares with placentitis. in conclusion, under the conditions of a regionally recognized outbreak, focal mucoid (nocardioform) placentitis occurred on this farm more frequently than has been reported elsewhere. most cases were subtle, with relatively small placental lesions that would have gone undetected without close monitoring for typical focal mucoid lesions of the chorioallantois. postpartum treatment was not necessary for mares with uncomplicated cases of this form of placentitis and their future reproductive performance was not affected by the condition. although surviving foals were smaller, this disadvantage was effectively corrected during very early stages of their lives, and focal mucoid placentitis did not affect athletic performance or sale prices of these foals. conflict of interest none to declare. references 1. hong cb, donahue jm, giles jr. rc, et al: equine abortion and stillbirth in central kentucky during 1988 and 1989 foaling seasons. j vet diagn invest 1993;5:560-566. doi: 10.1177/ 104063879300500410 2. erol e,  sells sf,  williams nm,  et al: an investigation on of a recent outbreak of nocardioform placentitis caused abortions in horses. vet microbiol 2012;158:425-430. doi: 10.1016/j.vetmic. 2012.02.023 3. fedorka ce, scoggin ke, ruby re, et al: clinical, pathologic, and epidemiologic features of nocardioform placentitis in the mare. theriogenology 2021;171:155-161. doi: 10.1016/j.theriogenology. 2021.05.023 table 4. incidence of neonatal disease in foals from mares with or without evidence of focal mucoid placentitis placentitis (n = 30) normal (n = 119) none 27 92 infectious disease 1 15 fractured ribs 2 4 limb contracture 2 8 hernia 6 9 http://dx.doi.org/10.58292/ct.v16.10536 https://doi.org/10.1177/104063879300500410 https://doi.org/10.1177/104063879300500410 https://doi.org/10.1016/j.vetmic.2012.02.023 https://doi.org/10.1016/j.vetmic.2012.02.023 https://doi.org/10.1016/j.theriogenology.2021.05.023 https://doi.org/10.1016/j.theriogenology.2021.05.023 6 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10536 4. donahue jm, williams nm: emergent causes of placentitis and abortion. vet clin north am equine pract 2000;16:433-456. doi: 10.1016/s0749-0739(17)30088-3 5. erol e, williams nm, sells sf, et al: antibiotic susceptibility patterns of crosiella equi and amycolatopsis species causing nocardioform placentitis in horses. j vet diagn invest 2012;24:1158-1161. doi: 10.1177/1040638712462377 6. cattoli g, vascellari m, corro m, et al: first case of equine nocardioform placentitis caused by crosiella equi in europe. vet rec 2004;154:730-731. doi: 10.1136/vr.154.23.730 7. christensen bw, roberts jf, pozor ma, et al: nocardioform placentitis with isolation of amycolatopsis spp in a florida-bred mare. j am vet med assoc 2006;228:1234-1239. doi: 10.2460/javma.228.8.1234 8. donahue jm, williams nm, sells sf, et al: crosiella equi sp. nov., isolated from equine placentas, int syst evol microbiol 2006;52:2169-2173. doi: 10.1099/ijs.0.02257-0 9. hanlon dw, mclachlan d, gibson i: the first reported case of equine nocardioform placentitis in new zealand. n z vet j 2016;64:198-199. doi: 10.1080/00480169.2015.1120166 10. volkmann dh, williams jh, henton jh, et al: the first reported case of equine nocardioform placentitis in south africa. j s afr vet assoc 2001;72:235-238. doi: 10.4102/jsava. v72i4.659 11. canisso i, ball ba, erol e, et al: attempts to induce nocardioform placentitis (crossiela equi) experimentally in mares. equine vet j 2015;47:91-95. doi: 10.1111/evj.12249 http://dx.doi.org/10.58292/ct.v16.10536 https://doi.org/10.1016/s0749-0739(17)30088-3 https://doi.org/10.1177/1040638712462377 https://doi.org/10.1136/vr.154.23.730 https://doi.org/10.2460/javma.228.8.1234 https://doi.org/10.1099/ijs.0.02257-0 https://doi.org/10.1080/00480169.2015.1120166 https://doi.org/10.4102/jsava.v72i4.659 https://doi.org/10.4102/jsava.v72i4.659 https://doi.org/10.1111/evj.12249 direct effects of nerve growth factor ?, purified from bull seminal plasma, on steroidogenesis and angiogenic markers of the bovine preovulatory follicle direct effects of nerve growth factor β, purified from bull seminal plasma, on steroidogenesis and angiogenic markers of the bovine preovulatory follicle jamie stewart,a,b liying gao,a jodi flaws,a vitor mercadante,c nicholas dias,c igor canisso,a fabio limaa acollege of veterinary medicine, university of illinois, urbana, il bvirginia-maryland college of veterinary medicine virginia polytechnic institute and state university, blacksburg, va ccollege of agricultural and life sciences virginia polytechnic institute and state university blacksburg, va nerve growth factor β (ngf) is a seminal plasma protein that induces ovulation and has a luteotrophic effect in camelids. in spontaneously ovulating species, ngf signaling in the ovary is critical for the first ovulation, but little is known regarding interactions of seminal plasma derived ngf on the preovulatory follicle. objectives were to assess direct effects of purified bovine ngf on steroidogenesis and angiogenic markers in the bovine preovulatory follicle. our hypothesis was that ngf administration stimulates steroidogenesis and angiogenic markers in thecal and granulosa cells from the bovine preovulatory follicle. two holstein heifers were synchronized using a 5 day cidr synch (day 0; gnrh + cidr insert, day 5; pgf2α + cidr removal, day 6; pgf2α). ovariectomy was performed via colpotomy 48 hours after the second pgf2α injection when a pre-ovulatory follicle > 12 mm was present. preovulatory follicle was excised from the ovary, fluid was aspirated and tissue dissected into quarters. theca interna with adherent granulosa cells was peeled from the theca externa and surrounding stromal tissue and cut into small pieces (average weight: 5.3 ± 0.7 mg). follicle tissue pieces were incubated (37oc, 5% co2:95% air) in 0.5 ml of eagle’s mem culture media supplemented with 1% l glutamine, 1% nonessential amino acids, 1% penicillin streptomycin, 1% insulin-transferrin-selenium, 10% fetal bovine serum, 40 ng/ml cortisol, 4 ng/ml lh, and 4 ng/ml fsh. culture wells were either supplemented with 100 ng/ml ngf (n = 12) or left as an untreated control (n = 12). media was withdrawn and replaced with fresh media at 3, 6, 12, 24, 48, and 72 hours of culture and frozen at -80oc pending hormone analyses. after 72 hours, follicle tissue pieces were flash frozen to measure steroidogenic and angiogenic gene expression using qpcr. analysis of variance was applied to parametric data using a general linear mixed model, with repeated measures used for hormone data. a kruskal wallis rank sum test was performed on non-parametric data. treatment with ngf increased (p < 0.01) testosterone production and upregulated (p = 0.04) steroidogenic enzyme 17 beta-hydroxysteroid dehydrogenase gene expression at 72 hours in follicle-wall extracts. there was no effect of ngf treatment on production (p ≥ 0.14) of progesterone or estradiol or gene expression (p ≥ 0.31) of other steroidogenic enzymes in the follicle. additionally, treatment with ngf downregulated (p = 0.02) gene expression of the angiogenic fibroblast growth factor 2, but did not alter expression (p ≥ 0.44) of other angiogenic factors. increased androgen production with ngf treatment may be secondary to theca cell proliferation. based on reduced expression of fibroblast growth factor 2 in ngf treated cells, perhaps there was an early onset of cell remodeling during early corpus luteum development. keywords: angiogenesis, cattle, follicle, nerve growth factor β, steroidogenesis acknowledgement this project was funded by the united states department of agriculture national institute of food and agriculture (hatch funds # 1014712). 443 clinical theriogenology • volume 11, number 3 • september 2019 444clinical theriogenology • volume 11, number 3 • september 2019 untitled untitled untitled omniblank: effect of semen collection method on semen quality in developing bulls effect of semen collection method on semen quality in developing bulls jarret proctor, nathan long, connor biehler, justin rhinehart, lewrell strickland, jason smith department of animal science, university of tennessee institute of agriculture, knoxville, tn breeding soundness examinations (bse) are commonly utilized to provide an objective indication of sire fertility. although semen collection method is thought to influence bse outcomes, limited evidence supports the use of 1 method over another. the objective of this experiment was to quantify the effects of 2 common semen collection methods on semen quality, and test the hypothesis that semen quality and bse outcomes are affected by semen collection method. angus bulls (n = 48; 332 ± 47 days of age) enrolled in an 84 day development program were randomly assigned to 1 of 2 initial collection methods [transrectal manual stimulation (man; n = 24) or electroejaculation (ee; n = 24)] performed by a trained technician. semen was collected on day 21 utilizing the initial assigned method. thereafter, semen collection method was alternated within bull for each of 3 subsequent semen collections that occurred on days 42, 63, and 84 (416 ± 47 days of age). duration of each collection procedure was standardized at 120 seconds of transrectal stimulation for man and 15 seconds of pulsed electrostimulation utilizing a programmed electroejaculator for ee. immediately following collection, progressive motility was determined via light microscopy. duplicate slides were prepared for morphological analysis and analyzed in duplicate by each of 2 technicians. semen samples were weighed and sperm quantified via photometry using a standard sample volume and expressed as total sperm per ejaculate. data were analyzed using analysis of variance and logistic regression to determine the main effect of collection method on all response variables and likelihood of bse failure, respectively. day of experiment and bull were included as fixed blocking factors in each model and independently accounted for a significant (p ≤ 0.01) portion of the variation in all response variables. electroejaculation resulted in less total volume (4.6 versus 8.0 ml x collection-1 for ee versus man, respectively; sem = 0.4; p < 0.0001) and fewer total sperm (2.4 x 109 versus 3.0 x 109 sperm x ejaculate-1 for ee verus man, respectively; sem = 0.2; p < 0.05), but a greater percentage of progressively motile sperm (52.9 versus 46.9% for ee versus man, respectively; sem = 1.2; p < 0.001). electroejaculation also decreased the percentage of abnormal sperm (38.4 versus 43.3% for ee versus man, respectively; sem = 1.5; p = 0.02), which corresponded with an increased percentage of normal sperm (61.6 versus 56.8% for ee versus man, respectively; sem = 1.5; p < 0.05). semen collection method altered total abnormal sperm (0.7 x 109 versus 1.2 x 109 sperm x ejaculate-1 for ee versus man, respectively; sem = 0.1; p < 0.0001) but not total normal sperm (1.6 x 109 versus 1.7 x 109 sperm x ejaculate-1 for ee versus man, respectively; sem = 0.13; p = 0.51) or the likelihood of bse failure (x2 = 1.83; p = 0.18). although collection method affected semen quality parameters to a small degree, bse passage rate was not affected by semen collection method. therefore, it is concluded that semen collection method should not influence bse outcomes. keywords: breeding soundness examination, bull, semen 437 clinical theriogenology • volume 11, number 3 • september 2019 438clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2017: determining optimal age for gonadectomy in the dog: a critical review of the literature to guide decision making determining optimal age for gonadectomy in the dog: a critical review of the literature to guide decision making margaret v. root kustritz,a margaret r. slater,b g. robert weedon,c philip a. bushbyd acollege of veterinary medicine, university of minnesota, st. paul, mn; bamerican society for the prevention of cruelty to animals, northampton, ma; ccollege of veterinary medicine, university of illinois, urbana, il; dcollege of veterinary medicine, mississippi state university, starkville, ms abstract gonadectomy is the most common elective surgery performed on dogs in the united states. concerns have been expressed by veterinarians, dog breeders, and pet owners or guardians about the need to better understand effects of gonadectomy on individual animal health. many studies to date on this topic have been performed on small or unique populations of dogs and data from those studies may or may not readily be extrapolated to dogs seen by veterinarians in private practice. veterinarians should be careful to read manuscripts in their entirely whenever possible rather than to rely on brief summaries that do not permit the reader to make their own decisions regarding value of the data as presented to their clinical practice. keywords: gonadectomy, ovariohysterectomy, castration, lifespan, neoplasia, orthopedics introduction proposed benefits and detriments of spay and castration surgeries in dogs have been reported in a growing body of literature over the last 20 years. some of the data are contradictory or are difficult to reconcile with known pathophysiology. some of the papers have received a great deal of coverage in the popular press, leading to inappropriate attention to limited data points. this manuscript is a review of the current literature on this topic, an argument for better attention to detail about published studies by veterinarians before extrapolating small data sets to all populations, and a discussion of some options about how best to present information from the literature to clients. gonadectomy is the most common elective surgery performed on dogs in the united states, with reported prevalence of 67% in female dogs and 61% in male dogs.1 it is recommended that all animals surrendered to humane organizations should undergo gonadectomy prior to adoption for purposes of population control.2 large review articles on the topic of determining optimal age of spay / castration surgery in dogs have been published over the last ten years.3-6 concerns have been expressed by veterinarians, dog breeders, and pet owners or guardians about the need to better understand effects of gonadectomy on individual animal health. a review of pet owners’ needs for information regarding the decision whether or not to neuter their animal demonstrated that clients desire information regarding positive and negative pet health and welfare outcomes.7 to best meet needs of dog owners, veterinarians must maintain an awareness of studies published and, most critically, the value they bring to the question of suitable age for gonadectomy in dogs. the question about impact of gonadectomy on health is one of causation: does gonadectomy at certain ages cause or prevent specific health issues? answering this type of question requires a large body of high quality data of varying types ranging from laboratory studies to clinical trials and observational studies of populations. no single study can prove causation. many studies to date on this topic have been performed on small or unique populations of dogs and data from those studies may or may not readily be extrapolated to dogs seen by veterinarians in private practice. conclusions about causation are less likely to be true when there are few studies in a given field, when samples sizes are very small, when differences between groups are small, when the study subjects are not similar to animals seen in private veterinary practice, when not enough attention is paid to confounding effects, and when measurement error is present (table 1).8,9 researchers, manuscript reviewers, and readers of publications must be aware of possible biases in measurement and must pay attention to whether or not findings that are statistically significant have any practical implications in veterinary practice. peer-reviewed clinical theriogenology • volume 9, number 2 • june 2017167 publications should include an in-depth assessment of the study limitations and generalizability; studies which do not provide this should be assessed with extra caution. veterinarians should be careful to read manuscripts in their entirely whenever possible rather than to rely on brief summaries that do not permit the reader to make their own decisions regarding value of the data as presented to their clinical practice. current information from the literature the association between intact status and behavior in dogs is complex. this review will focus on medical concerns and will not address effect of gonadectomy on behavior. data from the studies below may be described as relative risk, hazard ratios or odds ratios (or). a relative risk is the likelihood that an event will occur after a specific exposure. interpretation is the following: a relative risk of 2.0 means that a dog that has been gonadectomized is twice as likely to develop the condition as is an intact dog. specifically, a relative risk is the ratio of the risk of developing the condition in gonadectomized dogs to the risk of developing the condition in intact dogs. the odds ratio is typically interpreted similarly to the relative risk; specifically the odds ratio is the odds of the condition in the gonadectomized dogs divided by the odds in intact dogs. hazard ratios are similar but compare the hazard of an event or death and assume that risk is constant over time. put another way, we can say that if the relative risk or the odds ratio is 1.0, the risk (odds) in the exposed animals equals the risk (odds) in the non-exposed animals. if the rr (or) is greater than 1.0, the risk (odds) in exposed animals is greater than the risk (odds) in non-exposed animals. if the rr (or) is less than 1.0, the risk (odds) in exposed animals is less than the risk (odds) in non-exposed animals. all of these values have 1 as the “no difference” value and should be accompanied by a confidence interval, which is the range of values defined with a specific probability that the “real” value lies within that range. mammary neoplasia reported incidence of mammary gland tumors in dogs is 3.4% with some select populations reporting incidence as high as 13% by ten years of age.10,11 mammary neoplasia is the most common form of cancer in female dogs based on data from several large european cancer registries.12-15 increasing age and breed are risk factors for development of mammary neoplasia.10,16-19 age at time of diagnosis does not vary between spayed and intact dogs.20 being thin as a young dog is reported to be protective.20 high fat diet and obesity have not been demonstrated to increase risk of development of mammary neoplasia in dogs.20 there are six studies in the literature reporting an association between gonadectomy and mammary neoplasia in dogs, with five of those studies documenting decreased incidence (table 2). these studies report that bitches that are spayed are one-third to one-half less likely to develop tumors than are bitches that are left intact, and that bitches that remain intact are seven times more likely to develop tumors than are spayed bitches.17,19 benefit of spaying is increased with younger age or decreasing number of heat cycles prior to spay, with a classic paper describing female dogs as having 0.5% the risk of intact dogs if spayed before their first heat, 8% the risk if spayed between their first and second heat, and 26% if spayed after their second heat.20,21 in general, greatest benefit is described for any bitch spayed before 2.5 years of age with a decreasing benefit up to nine years of age.20,22 no difference was shown between intact and spayed dogs in one study, which evaluated dogs of one breed presented to a specialty hospital.23 a systematic review of effect of gonadectomy on incidence of mammary neoplasia concluded that there was insufficient evidence to support any connection.24 systematic reviews are analyses of all of the published literature on a given topic performed in a repeatable and objective fashion and strive to identify bias that would nullify results of published studies.25 of the 13 studies they chose to include in their initial analysis, nine were deemed to have a high risk of bias and four to have a moderate risk of bias. the inability to identify a connection in that systematic review was more a reflection of the lack of depth in the veterinary literature on this topic than a true evaluation of the association between gonadectomy and mammary neoplasia. hormonal stimulation is assumed to be the cause of mammary neoplasia in dogs. eighty percent of mammary tumors and 95% of normal mammary tissues have estrogen receptors, progesterone clinical theriogenology • volume 9, number 2 • june 2017 168 receptors, or both.26 studies differ regarding correlation of estrogen and progesterone receptors with type of tissue, with some studies suggesting that there are fewer receptors as tumors become less differentiated.26,27 an association has been reported between treatment with exogenous progestins and increased incidence of mammary tumors.18 mammary neoplasia may be more common in bitches that had multiple episodes of overt pseudopregnancy with lactation, suggesting either a hormonal effect or the possibility of malignant transformation of mammary tissue that is metabolically active and undergoes the physical changes associated with lactation.22 prostatic neoplasia reported incidence of prostatic carcinoma is 0.2-0.6%.28,29 it is difficult for pathologists to differentiate prostatic adenocarcinoma, which arises directly from glandular tissue, from invasion of transitional cell carcinoma from the prostatic urethra into the prostatic parenchyma. data presented encompasses both forms of prostatic carcinoma, a limitation in understanding the disease process. incidence is reported to be increased in some breeds.28,30 evaluation of other factors for association with incidence of prostatic carcinoma, including diet, activity level, housing, and exposure to tobacco smoke, failed to demonstrate any significant associations.31 there are five studies in the literature evaluating association between gonadectomy and prostatic neoplasia, with four showing increased incidence with gonadectomy (table 3). mean and median age at the time of diagnosis may vary between castrated and intact dogs, with one study demonstrating disease in castrated dogs at an older age compared to intact dogs.30 no study to date has demonstrated any sparing effect dependent on age at the time of gonadectomy.28-33 the cause-and-effect relationship between gonadectomy and prostatic carcinoma has not been defined. prostatic tumors in dogs arise from ductal / urothelial tissues, which are androgen-independent.28 it may be that testosterone exerts a carcinogenic effect earlier in life.29 because of the great variability reported in time from castration to diagnosis of prostatic neoplasia, it has been suggested that castration does not promote tumor formation but instead promotes tumor progression.28 it may be that lack of testosterone after castration is associated with atrophy of androgen-dependent tissues in the prostate and less inhibition of growth of androgen-independent tissues.34 epigenetic effects associated with silencing of genes associated with function of androgen receptors have been demonstrated in humans with prostatic neoplasia.30 finally, superoxide dismutase activity declines in prostatic tissue by six months after castration, suggesting a lack of response to the toxic effects of reactive oxygen species, which could be associated with oncogenesis.35 the authors are unaware of any published research documenting these phenomena in dogs. transitional cell carcinoma (non-prostatic) incidence of transitional cell carcinoma is at most 1%.36 predisposing factors previously reported include increasing age, breed, and exposure to environmental herbicides and insecticides.37,38 there are two studies documenting increased incidence of transitional cell carcinoma related to neutering in dogs (table 4).30,37,39 one study evaluated dogs of only one breed and that breed has a hereditary predisposition to transitional cell carcinoma, making extrapolation to other populations of dogs difficult.37 the cause-and-effect relationship between gonadectomy and transitional cell carcinoma has not been defined. osteosarcoma incidence of canine osteosarcoma is <0.1%.19 factors reported to be associated with incidence of osteosarcoma include increasing age, breed and increasing size.40-42 there are four studies in the veterinary literature specifically addressing effect of gonadectomy on incidence of osteosarcoma with three showing increased incidence and one showing no effect (table 5).23,40,42,140 two studies evaluated dogs of only one breed, and one evaluated rottweilers a breed that has a hereditary predisposition to osteosarcoma, making extrapolation to other populations of dogs difficult.42 the cause-and-effect relationship between gonadectomy and osteosarcoma has not been defined. clinical theriogenology • volume 9, number 2 • june 2017169 hemangiosarcoma reported incidence of hemangiosarcoma is 0.2%43. predisposing factors previously reported include increasing age and breed; there is no variation in prevalence between female and male dogs.43-46 there are seven studies describing relationship between gonadectomy and hemangiosarcoma (table 6). splenic and cardiac hemangioma and hemangiosarcoma were reported to be more common in spayed females than in intact females.43,45,140 castrated males had a slightly increased risk of developing a heart based tumor compared to intact males.43 hemangiosarcoma was reported to occur more commonly in females spayed after 12 months of age than in females spayed early or left intact in one study and to be more common in females spayed at less than six months or more than 12 months in another study; there was no reported effect of castration in male dogs in either study.47,48 several studies have reported raw data showing numbers of affected spayed females, intact females, castrated males, and intact males with hemangiosarcoma in specific breeds using data drawn from records of a referral hospital, with varying amounts of statistical analysis.23,47,49 it can be difficult to assess the value of raw data without more complete information about the population of dogs presented to that hospital. referral hospitals see a unique population of dogs, generally those with non-standard or complicated disorders, often owned by individuals who have disposable income available for advanced care for their dog, and perhaps more likely to be related as they tend to be drawn from a specific geographic region.50 the cause-and-effect relationship between gonadectomy and hemangiosarcoma has not been defined in dogs. one hypothesized association is alteration in immune function with gonadectomy including decreased immune surveillance for cancer cells due to lack of sex steroids.48 the authors are unaware of any published research documenting this phenomenon in dogs. lymphoma reported incidence of lymphoma in dogs is 1.1%.15 there is a hereditary component, increasing age is a risk factor, and environmental factors also may play a role in tumor development.15,51 there are six reports evaluating the effect of gonadectomy on incidence of lymphoma with conflicting results (table 7). five studies showed increased risk with two studies showing increased risk in females only and two reports showing a difference in males only.47-49,51 several studies have reported raw data showing numbers of affected spayed females, intact females, castrated males, and intact males with lymphoma in specific breeds using data drawn from records of a referral hospital, with varying amounts of statistical analysis.23,47,49 concerns are as described for hemangiosarcoma. the cause-and-effect relationship between gonadectomy and lymphoma has not been defined in dogs. hypothesized associations include a possible protective effect of estrogens, and alterations in immune function with gonadectomy including decreased immune surveillance for cancer cells due to lack of sex steroids.48,51 the authors are unaware of any published research documenting these phenomena. cutaneous mast cell tumor prevalence of mast cell tumors in a study evaluating information submitted to an electronic database by a large number of private practices was 0.27%.52 some breeds are reported to be at increased risk.52,53 there are seven reports evaluating association between gonadectomy and mast cell tumors, with conflicting conclusions. five reported increased incidence with gonadectomy (table 8). two studies demonstrated increased incidence with gonadectomy only in females.49,53 several studies have reported raw data showing numbers of affected spayed females, intact females, castrated males, and intact males with mast cell tumor in specific breeds using data drawn from records of a referral hospital, with varying amounts of statistical analysis.23,47,49 concerns are as described for hemangiosarcoma. the cause-and-effect relationship between gonadectomy and mast cell tumor has not been defined in dogs. hypothesized associations include alterations in immune function with gonadectomy including decreased immune surveillance for cancer cells due to lack of sex steroids, and increased attention from clinical theriogenology • volume 9, number 2 • june 2017 170 owners who have already had their dogs spayed or castrated.48,53 the authors are unaware of any published research documenting these phenomena. testicular neoplasia reported incidence of testicular neoplasia in dogs is 0.9%, with increasing age a reported risk factor.54 castration early in life obviously is protective in all cases and castration at the time of diagnosis is reported to be curative in the majority of cases.25 obesity reported incidence of obesity varies from 21.4-44.4%.55-58 some studies report higher incidence in females than in males and a breed predisposition is described.55-57 other factors reported to be associated with obesity in dogs include housing, increasing age, and ownership by an overweight person or a person ≥ 40 years old.56,57,59-61 although many studies report percent of a population of dogs that is reported to be obese, either by veterinarians or by pet owners, only three studies with veterinarian determined body condition have been published specifically addressing the association between gonadectomy and obesity, with all three studies showing increased incidence with gonadectomy (table 9). risk for development of obesity after neutering was reported to be greatest for the first two years after surgery, with no difference in likelihood of becoming obese when comparing neutered to intact dogs over ten0 years.62 the cause-and-effect relationship between gonadectomy and obesity has not been defined in dogs. in cats, decline in metabolic rate after gonadectomy has been demonstrated.63,64 estrogen has been reported to effect satiety in women, perhaps through modulation of cholecystokinin.65 studies disagree regarding effect of estrogen on satiety in dogs. in a study comparing dogs gonadectomized at seven weeks of age or seven months of age, or left intact, the dogs showed no difference in food intake or depth of back fat at 15 months of age.66 in other studies, spayed female dogs have been demonstrated to have an increase in food intake and increase in indiscriminate appetite after spaying compared with shamoperated or age-matched control dogs.67,68 testosterone may effect satiety in men through secretion of gastric ghrelin.69 castration was associated with increased appetite and subsequent weight gain in one study in dogs.70 cranial cruciate ligament injury the incidence of cranial cruciate ligament injury was reported to be 0.6-3.5%.71-74 the disorder may or may not be more common in females than in males and is more prevalent in some breeds.71-77 other factors reported to be positively associated with incidence of cranial cruciate ligament injury are age and weight, as well as obesity.73,74,76,78 association of cranial cruciate ligament with conformation of the pelvic limb and with exercise are controversial, with conflicting reports in the literature.79 there are ten studies evaluating the effect of gonadectomy on incidence of cranial cruciate ligament injury, with nine showing increased incidence with gonadectomy (table 10). some factors may be confounded; in one study, intact dogs that developed cranial cruciate ligament injury did so at a younger age than did neutered dogs.77 several studies have reported raw data showing numbers of affected spayed females, intact females, castrated males, and intact males with cranial cruciate ligament injury in specific breeds using data drawn from records of a referral hospital, with varying amounts of statistical analysis.23,47-49 concerns are as described for hemangiosarcoma. in another study, it was suggested that incidence in neutered dogs was artificially elevated because owners who had paid to have their dog spayed or castrated were perhaps more attentive or more willing to bring their dog in for orthopedic treatment.72 the cause-and-effect relationship between gonadectomy and cranial cruciate ligament injury has not been defined. because cranial cruciate ligament injury occurs more commonly in women than in men with incidence associated with phases of the menstrual cycle, it may be that hormonal changes alter the properties or function of the ligament.71,80 closure of the growth plate in long bones may or may not be delayed if dogs undergo gonadectomy prior to puberty. a study evaluating long bone length up to six clinical theriogenology • volume 9, number 2 • june 2017171 months of age after ovariohysterectomy failed to show statistical significance between groups, while a study evaluating long bone length at 15 months of age did demonstrate such a difference.66,81 it has been hypothesized that delayed closure of growth plates may be associated with variation in length of long bones and subsequent misalignment of joints. specific attention has been paid to possible delay in closure of femoral physes compared to tibial physes, such that there is relative overgrowth of the femur and accentuation of the tibial plateau angle. several studies have refuted the connection of degree of the tibial plateau angle and incidence of cranial cruciate ligament injury.82-84 the authors are unaware of any published research documenting development of cranial cruciate ligament injury due to asynchrony of long bone growth and subsequent anatomic abnormalities of the stifle. patellar luxation reported incidence of patellar luxation was 1.3% in one study.85 patellar luxation is a hereditary disorder that is equally prevalent in males and females and is reported to be more prevalent in some breeds.85,86 incidence is reported to be positively correlated with age and body weight.85,86 there are three studies describing a possible link between gonadectomy and patellar luxation; two showed an increased incidence with spay or castration (table 11). one study sampled dogs from 119 clinics across england, which minimizes the bias that may be present in studies performed at single locations.85 however, there may be differences in the breed genetics of dogs from different countries that could influence the generalizability of results. the cause-and-effect relationship between gonadectomy and patellar luxation has not been defined. age may be a confounding factor, as increased age, which also was shown to be correlated with increased incidence of patellar luxation, may be associated with increasing laxity of the stifle joint.86 the association with increased body weight in these studies was not a reflection of increased incidence in obese dogs but rather an increased incidence in dogs with higher than average adult bodyweight for their breed and sex.85 the authors of one of these studies concluded that the available data was insufficient to permit any deductions about effect of removal of sex hormones on development of patellar luxation.86 hip dysplasia reported incidence of hip dysplasia is 1.7-3.5%.72,87 this is a hereditary condition that is equally prevalent in males and females and is more prevalent in some breeds.72,87 environmental influences, including diet, exercise, and housing, also play a role in clinical manifestations of the disorder.88-92 effect of gonadectomy on incidence of hip dysplasia is well described in six studies. incidence was increased with gonadectomy in five of those studies; no change in incidence in at least one sex was noted in three studies (table 12). several published studies described incidence relative to gonadectomy in specific breeds, automatically making the data less valuable for wide extrapolation due to the hereditary component of this disorder.23,47,49,93 in addition, these studies did not include any genetic analysis of dogs in the study so the influence on genetics even within the breed could not be evaluated. in one study, incidence of hip dysplasia was reported to be higher in dogs neutered before 5.5 months of age but dogs neutered early that developed hip dysplasia were less likely to be euthanized for it than dogs neutered between six and 12 months, suggesting variation in clinical manifestation of the disorder as well as possible owner preferences for various treatment options.58 it was not clear in that study whether or not all dogs defined as having hip dysplasia had been diagnosed with the disorder by a veterinarian.58 similarly, it is not clear in all studies at what age hip dysplasia was diagnosed, if dogs were presented for routine screening or were clinical for hip dysplasia, and if the diagnosis was made by a private practitioner or a specialist, or verified by the orthopedic foundation for animals or other external reviewer. several studies have reported raw data showing numbers of affected spayed females, intact females, castrated males, and intact males with canine hip dysplasia in specific breeds using data drawn from records of a referral hospital, with varying amounts of statistical analysis.23,47,49 concerns are as described for hemangiosarcoma. the cause-and-effect relationship between gonadectomy and canine hip dysplasia has not been defined. as with cranial cruciate ligament injury, it has been hypothesized that delayed closure of growth clinical theriogenology • volume 9, number 2 • june 2017 172 plates may be associated with variation in length of long bones and subsequent misalignment of joints. the authors are unaware of any published research documenting misalignment of joints. benign prostatic hypertrophy/hyperplasia benign prostatic hypertrophy/hyperplasia (bph) is very common in middle-aged to older dogs, with reported incidence of 50% by 2.4 years of age and 75-80% by six years of age.94-96 development is androgen-dependent so castration early in life is protective in all cases. surgical castration at the time of diagnosis is curative.97 castration also is reported to increase effectiveness of treatment and long-term resolution of bacterial prostatitis in dogs, most likely through resolution of underlying bph.98 pyometra incidence of pyometra in bitches increases with age, with reported incidence of 24-25% by ten years of age.99 ovariohysterectomy early in life obviously is protective in all cases. reported mortality associated with ovariohysterectomy at the time of diagnosis is 0-17%.100,141 urinary incontinence urethral sphincter mechanism incompetence, formerly known as estrogen-responsive urinary incontinence, is reported to occur in 4.9-20.0% of spayed dogs.58,101-105 the disorder has been reported to be more common in some breeds.103,106 other factors reported to be associated with increasing incidence include increasing age and body weight, and tail docking.103,105-107 there are ten studies specifically documenting the association between gonadectomy and urethral sphincter mechanism incompetence, with all four showing increased incidence with spaying (table 13). incidence may vary with age or physiologic status; studies have demonstrated increased incidence when bitches were spayed at fewer than three to five months, greater than six months, or before their first estrus.23,58,104,108, 142-144 others have refuted any connection between incidence and age at time of spaying.105 a systematic review was performed to evaluate this topic. as with mammary neoplasia, the veterinary literature is not deep enough to permit this type of analysis and the authors were forced to conclude that currently available data is insufficient for determination of the effect of neutering on the risk of urinary incontinence.109 the cause-and-effect relationship between urethral sphincter mechanism incompetence and gonadectomy has not been defined. it has been demonstrated that maximum urethral closure pressure and functional urethral length are decreased after spaying.110 serum concentrations of follicle stimulating hormone (fsh) and luteinizing hormone (lh) from the pituitary are persistently elevated after spaying, most likely due to lack of negative feedback to the hypothalamus and pituitary from the ovaries.111 elevated concentration of serum fsh has been associated with incontinence but effect to changes in gonadotropin concentration on changes in urethral closure pressure and subsequent incontinence are not clear.111,112, 144 urolithiasis – calcium oxalate and struvite prevalence of first time calcium oxalate urolithiasis in one study was reported to be 0.01%.113 incidence of struvite urolithiasis was reported to be 0.004%.114 calcium oxalate urolithiasis is reported to be more common in males than in females and to be more common in smaller breeds of dog.113,115,116 struvite urolithiasis is reported to be more common in females than in males and to be more common in small breeds.114,116 in one study, calcium oxalate urolithiasis was reported to be more common than struvite urolithiasis in males, older dogs, and smaller breed dogs.117 association between urolithiasis and gonadectomy has been reported in four studies, three of which showed increased incidence in gonadectomized dogs (table 14). incidence of calcium oxalate urolithiasis is reported to be increased after castration in males and incidence of struvite urolithiasis is reported to be increased after spaying in females.114,117 again, it is possible that owners who have had their dogs gonadectomized are more observant and/or more willing to seek veterinary care and treatment. the cause-and-effect relationship between urolithiasis and gonadectomy has not been defined. clinical theriogenology • volume 9, number 2 • june 2017173 diabetes mellitus reported incidence of diabetes mellitus in dogs is 0.5-0.6%.118,119 it has been reported to occur more commonly in females than in males and a higher prevalence is reported in some breeds.119,120 other factors associated with increased incidence of diabetes mellitus are increasing age and obesity.119,121,122 there are four studies evaluating the possible connection between gonadectomy and incidence of diabetes mellitus (table 15). two studies showed no change for females but an increased risk for diabetes mellitus in castrated males.119,121 one study showed no change in either gender.58 the most likely causeand-effect relationships between gonadectomy and diabetes mellitus are through effect of progestogens in insulin resistance and increased likelihood of spayed or castrated dogs being obese.119,121,122,146 chronic kidney disease prevalence of chronic kidney disease in the single study evaluating association with gonadectomy was 0.37%.123 incidence is increased with increasing age and some breeds are at increased risk. gonadectomy was associated with increased incidence in one of two studies (table 16). this study sampled 107,214 dogs from 89 clinics across england, which minimizes the bias that may be present in studies performed at single locations.123 the cause-and-effect relationship between chronic kidney disease and gonadectomy has not been defined. atopic dermatitis incidence of atopic dermatitis is 1.7%.124 factors associated with incidence include age, breed, sex, and geographical location.124 there are only three studies documenting the association between gonadectomy and atopic dermatitis, with two showing increased incidence and one showing no association (table 17).58,124,125 all studies evaluated large populations of dogs, with one describing 90,090 dogs seen at a referral hospital, one describing 22,280 dogs seen at a corporate practice, and the third describing 1842 dogs adopted from a humane organization. the first and largest of these studies also demonstrated that gonadectomized dogs were more likely to be diagnosed with other autoimmune disorders, including autoimmune hemolytic anemia (or 1.67±0.28 in females, 1.76±0.31 in males), hypoadrenocorticism (or 1.49±0.32 in females, 2.07±0.54 in males), hypothyroidism (or 3.03±0.39 in females, 1.29±0.11 in males), immune-mediated thrombocytopenia (or 3.14±0.73 in females, 2.05±0.42 in males), inflammatory bowel disease (or 2.2±0.54 in females, 1.43±0.23 in males) and lupus erythematosus (systemic and discoid, or 2.64±1.24 in females, not significant in males).125 the causeand-effect relationship between autoimmune disorders, including atopic dermatitis, and gonadectomy has not been defined. idiopathic epilepsy/cluster seizures incidence of idiopathic epilepsy is reported to be 0.6%.126 one study reported increased incidence in male dogs and some breeds are at increased risk.126,127 there are three studies evaluating association between gonadectomy and idiopathic epilepsy (table 18). two studies showed increased incidence in spayed or castrated dogs and one study showed no change in incidence with gonadectomy.126128 regarding cluster seizures, intact males were reported to be more likely to have cluster seizures than neutered males and intact females had cluster seizures with greater frequency than did spayed females.128 the cause-and-effect relationship between idiopathic epilepsy and gonadectomy has not been defined. one study suggested that hormones may play a role, perhaps by having an excitatory effect, but the infrequent cycling of female dogs would argue against an effect of such a stimulus throughout the normal prolonged anestrus.128 one author suggested that any association between gonadectomy and idiopathic epilepsy may be artefactual if owners of dogs with epilepsy present their dogs for gonadectomy as a way of managing their seizures or to prevent them from reproducing.127 clinical theriogenology • volume 9, number 2 • june 2017 174 lifespan gonadectomy has been associated with increased lifespan in most studies where this was evaluated (table 19). spayed dogs have been reported to live 23.0-26.3% longer than intact bitches and castrated males have been reported to live 13.8-18.0% longer than intact males.129-133 therefore, any health problem that tends to occur in older dogs could be more common in gonadectomized animals than in intact animals, simply because gonadectomized animals may live long enough to develop the health problem. suggested reasons for this increased longevity include increased attention from owners of dogs they have had neutered, and changes in behavior, for example decreased roaming for breeding purposes, which minimize risks to the animal. the latter hypothesis is supported by data demonstrating that neutered dogs were less likely to die of infectious disease or trauma and were more likely to die of cancer and immune-mediated disease.130 one study evaluating a group of exceptionally long-live bitches of one breed demonstrated increased lifespan associated with increased ovary exposure over their lifetime, suggesting benefit of exposure to compounds secreted directly from the ovary or compounds for which synthesis or secretion is modulated by the ovary.134 an example of one such compound is lh, for which concentrations are persistently elevated after ovariectomy and for which many tissues have receptors.135 interpretation and explanation of data when reading any scientific study, the reader is recommended to ask questions to help them interpret the data. suggested concerns include: what kind of study was performed? ideally, to study causation, the animals should be newly diagnosed with the disease of interest (incident cases). studies that identify animals for inclusion based on the outcome (the health problem rather than on gonadectomy) are more prone to bias than those studies which sample based on gonadectomy status or age. studies sampling on the outcome may include errors from missing records and the need for dog owners and veterinarians to rely on memory.50 for these reasons, studies that do not rely on historical data or memory (prospective studies) generally are considered to generate more accurate information. a study where the dogs were randomly assigned to age of gonadectomy (clinical trial) would be the best design to decrease bias. however, such studies are rare and expensive. other studies that do not sample based on outcome and are prospective are the next best option (cohort designs). for all studies, the choice of controls is crucial for an unbiased study. a control for studies which select dogs based on the outcome (case control study) is an individual selected in an unbiased manner from those individuals who would have been included in the case series had they developed the disorder under study.136 sampling for controls within a referral hospital is plagued with the problems described above but sampling for controls in the general population outside of the referral hospital also may introduce error. those dogs never would have been in the case series precisely because they would not have been presented to the referral hospital. for these reasons and others, studies including dogs evaluated at a large number of private veterinary hospitals across a geographic region may be of great value and be less prone to bias. from what population was the sample drawn? very small samples may not be representative of the population.8 very small sample size may also not permit the investigator to demonstrate an effect statistically even if an effect is truly present.8 very large studies may show statistical significance of a very small difference between groups that is not large enough to be relevant scientifically or biologically. as previously mentioned, samples drawn from referral hospitals, including veterinary teaching hospitals, may not be an accurate reflection of the larger population. samples collected by evaluation of dog breeders also may not be reflective of the large dog-owning public as dog breeders often have a different level of knowledge and different goals for their animals compared to pet owners.50 how do we know what the statistics are telling us? statistically significant results can be due to errors in the study design or in the interpretation of the results. in addition, statistical significance is only one criterion for determining a cause-and-effect sequence. the lack of a defined cause-and-effect for gonadectomy and many of the disorders described is troubling for this reason. when looking at odds or hazard ratios or relative risks which provide an estimate of the magnitude of the risk, always consider whether the range includes unity; if so, the author is stating that there is a statistical chance that incidence clinical theriogenology • volume 9, number 2 • june 2017175 in neutered animals is equivalent to incidence in intact animals. for that reason, the range of an odds ratio or similar statistical construct is more valid to assess than is any single value, including a p value. in addition, when applying these results to a clinical situation, consider the actual incidence of the problem. if the problem is very rare, even a large relative risk from a good study does not mean that the problem is a major concern. another consideration with statistical analysis and publications, especially when performing systematic analyses is the “file drawer problem”, or lack of publication of negative data. a few of the studies described above did include negative data, which helps create a more balanced overall picture. finally, when looking at the statistical analysis, readers should be cautious not to draw conclusions solely on the apparent complexity of the analysis. simple statistics may be superior to very complex statistics, which sometimes reveal mathematically valid evidence for statistical significance that is not supported scientifically. conversely, more complex statistics may be necessary to evaluate confounding factors within a study, for example the association between gonadectomy and any disorder to which animals are predisposed by increasing age, sex, heredity, or other factors.8 statistical significance demonstrating associations is only one small piece of evidence for causation. sir austin bradford hill established in 1965 what have become known as hill’s criteria of causation (table 20). these provide a framework to help the reader understand the complexities of determining causation. they also put into context data that is statistically significant but may not make an obvious contribution to demonstrating causation. veterinarians are strongly encouraged to read manuscripts completely, not just to read the abstract or short summaries of studies, and to consider criteria such as these when evaluating information. one of the authors (mvrk) used data from a veterinary teaching hospital to try to illustrate the above points. for one calendar year, information was gathered regarding how many unique spayed female, intact female, castrated male, and intact male dogs were seen overall and how many dogs in each category were diagnosed with a variety of disorders, as coded in the electronic medical record, a crosssectional study (table 21). two-way contingency table analysis was performed. as is obvious from the table, very few of the disorders described in this paper were supported by data from this unique population. specific errors that arose in this small study were lack of data due to inadequate completion of medical records by clinicians and subsequent lack of coded data; low number of animals admitted to the referral hospital for common conditions such as mammary neoplasia; and lack of consideration for confounding factors where appropriate. some examples demonstrate finding of p values that are misleading when looking at the single odds ratio estimate. for example, the odds ratio for transitional cell carcinoma is 4.1; this is a rather large odds ratio (far from unity). however, the p value is not significant and the range for the odds ratio includes unity, due in part to a small sample size. if only the single value for the odds ratio had been provided, a high risk if gonadectomized would appear to be present using these data. without the p value or confidence interval, statistical significance cannot be determined. for osteosarcoma, the p value is just significant (near the cutoff of 0.05) but the range for the odds ratio just includes unity. if only the single p value or the single value for the odds ratio had been provided, a significant risk would appear to exist that is not clearly supported if all the data are presented. these data demonstrate the value of confidence intervals and how they can be used to identify significant risk when single values are near the cutoff point. it is not uncommon for practicing veterinarians to contact one of the authors and ask for a onesentence response to their question of the best age at which to spay or castrate dogs. there is no appropriate simple response as there is a great amount of information available, all of which will be assessed slightly differently by each person reading those studies, and non-medical considerations must be taken into account, including population control, effects of gonadectomy on behavior, and the wishes of and ability to provide care by the owner or guardian of the dog. general recommendations are the following: for populations of unowned dogs, for example at humane organizations, population control concerns outweigh concerns about individual animals. animals that leave the humane organization intact clinical theriogenology • volume 9, number 2 • june 2017 176 may repopulate that shelter with their offspring, and may well be returned to the shelter themselves, as it has been demonstrated that being intact is a risk factor for surrender.137 for animals with a responsible owner or guardian, veterinarians need to develop a means of communicating this large amount of contradictory data. one way to help clients think through the information is to provide them with an idea of the varying impacts of the disorders the veterinarian believes to be associated with gonadectomy. in one example of this method, veterinarians generated a morbidity score for various disorders that was multiplied by incidence of those disorders to create an impact score (table 22).138 positive impact scores were associated with better health after gonadectomy and negative impact scores with worse health after gonadectomy. for female dogs, benefits of ovariohysterectomy outweighed detriments and prepubertal spay was recommended; for male dogs, benefits of castration did not outweigh detriments until the animal was likely to develop age-related, benign diseases of the reproductive tract, or about 2.5 years of age.138 use of this kind of tool also gives veterinarians an opportunity to talk with clients about other topics in preventive medicine. for example, this provided a very real way to demonstrate to clients the concerns associated with obesity, which had a high impact score in both females and males.138 another technique would be to provide clients with some idea of positives and negatives for their specific animal. this requires specific knowledge of predispositions due to age, breed, and other factors for all of the disorders described. an example previously described is the following:4 you are a veterinarian speaking to the owner of an eight-week-old female labrador retriever that is not intended for breeding. this dog would benefit greatly from spaying before her first estrus as a means of preventing mammary gland tumors, which are extremely common relative to other diseases of concern and cause substantial morbidity. because of her breed, reported detriments of spaying include an increased predisposition to cranial cruciate ligament injury, hemangiosarcoma, and obesity. however, there is a low incidence of hemangiosarcoma, and obesity can be readily controlled with diet and exercise, which leaves cranial cruciate ligament injury as the most important possible detriment. because the incidence of cranial cruciate ligament rupture is lower than that of mammary gland neoplasia, you choose to recommend spaying and educate the owner about maintenance of optimal body condition and other management techniques that will minimize potential for cranial cruciate ligament injury. the best age at which to perform the spay must include considerations of when the dog’s first estrus is likely to occur and how young one can spay a dog before greatly increasing risk of urethral sphincter mechanism incompetence. the likely recommendation for this bitch would be ovariohysterectomy at about five to six months of age. all of these methods rely on the veterinarian having taken the time to assess the information available and to use their knowledge of the subject to educate clients as they collaborate on the best decision for a specific individual animal. research 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<https://www.banfield.com/getmedia/34ffd1bf-65e24d73-9d81-493231df3d91/4_2-the-epidemiology-of-atopic-dermatitis>. 125. sundburg cr, belanger jm, bannasch dl, et al: gonadectomy effects on the risk of immune disorders in the dog: a retrospective study. bmc vet res 2016;12:278-287. 126. kearsley-fleet l, o’neill dg, volk ha, et al: prevalence and risk factors for canine epilepsy of unknown origin in the uk. vet rec 2013;172:338-342. 127. short ad, dunne a, lohi h, et al: characteristics of epileptic episodes in uk dog breeds: an epidemiological approach. vet rec 2011;169:48-51. 128. monteiro r, adams v, keys d, et al: canine idiopathic epilepsy: prevalence, risk factors and outcome associated with cluster seizures and status epilepticus. j small anim pract 2012;53:526-530. 129. banfield state of pet health 2013 report: available at: <www.banfield.com/banfield/media/pdf/downloads/soph/banfield-state-of-pet-health-report_2013.pdf>. 130. hoffman jm, creevy ke, promislow de: reproductive capability is associated with lifespan and cause of death in companion dogs. plos one 2013;8:e61082. doi:10-1371/journal.pone.0061082. 131. moore ge, burkman kd, carter mn, et al: causes of death or reasons for euthanasia in military working dogs: 927 cases (1993-1996). j am vet med assoc 2001;219:209-214. 132. bronson rt: variation in age at death of dogs of different sexes and breed. am j vet res 1982;43:2057-2059. 133. michell ar: longevity of british breeds of dog and its relationship with sex, size, cardiovascular variables and disease. vet rec 1999;145:625-629. 134. waters dj, kengeri ss, clever b, et al: exploring mechanisms of sex differences in longevity: lifetime ovary exposure and exceptional longevity in dogs. aging cell 2009;8:752-755. 135. zwida k, kutzler ma: non-reproductive long-term health complications of gonad removal in dogs as well as possible causal relationships with post-gonadectomy elevated luteinizing hormone (lh) concentrations. j etiology anim hlth 2016;1:1-11. clinical theriogenology • volume 9, number 2 • june 2017181 136. kelsey jl, moore as, glickman lt: epidemiologic studies of risk factors for cancer in pet dogs. epidemiol rev 1998;20:204-217. 137. new jc, salman md, king m, et al: characteristics of shelter-relinquished animals and their owners compared with animals and their owners in u.s. pet-owning households. j appl anim welfare sci 2000; 3:179-201. 138. root kustritz mv: use of an impact score to guide client decision-making about timing of spay-castration of dogs and cats. clin therio 2012;4:481-485. 139. duerr fm, duncan cg, savicky rs, et al: risk factors for excessive tibial plateau angle in large-breed dogs with cranial cruciate ligament disease. j am vet med assoc 2007;231:1688-1691. 140. gruntzig k, graf r, boo g, et al: swiss canine cancer registry 1955-2008: occurrence of the most common tumour diagnoses and influence of age, breed, body size, sex and neutering status on tumour development. j comp pathol 2016;155:156-170. 141. jitpean s, strom-holst b, emanuelson u, et al: outcome of pyometra in female dogs and predictors of peritonitis and prolonged postoperative hospitalization in surgically treated cases. bmc vet res 2014;10:6-17. 142. byron jk, taylor kj, phillips gs, et al: urethral sphincter mechanism incompetence in 163 neutered female dogs: diagnosis, treatment, and relationship of weight and age at neuter to development of disease. j vet intern med 2017;31:442-448. 143. forsee km, davis gj, mouat ee, et al: evaluation of the prevalence of urinary incontinence in spayed female dogs: 566 cases (2003-2008). j am vet med assoc 2013;242:959-962. 144. reichler im, hung e, jochle w, et al: fsh and lh plasma levels in bitches with differences in risk for urinary incontinence. theriogenology 2005;63:2164-2180. 145. marmor m, willeberg p, glickman lt, et al: epizootologic patterns of diabetes mellitus in dogs. am j vet res 1982;43:465-470. 146. poppl ag, mottin ts, gonzalez fh: diabetes mellitus remission after resolution of inflammatory and progesteronerelated conditions in bitches. res vet sci 2013;94:471-473. clinical theriogenology • volume 9, number 2 • june 2017 182 t ab le 1 . k ey c ri te ri a to c on si de r w he n re vi ew in g st ud ie s ab ou t g on ad ec to m y an d di se as e ca us at io n or p re ve nt io n b es t s tu dy d es ig n fo r ev id en ce f or c au sa tio n, w he n do ne w el l: ra nd om iz ed c lin ic al tr ia ls p ro vi de th e st ro ng es t l in k be tw ee n po te nt ia l c au se a nd p ot en tia l e ff ec t. c oh or t s tu di es a re n ex t, fo ll ow ed b y ca se -c on tr ol a nd th en c as e se ri es . e xp er t o pi ni on a nd n on -s ys te m at ic r ev ie w s pr ov id e th e w ea ke st e vi de nc e fo r ca us at io n. u nb ia se d su bj ec t s el ec tio n (h ow w er e th e do gs s el ec te d an d fr om w he re ): o ne s ou rc e of p ot en tia l b ia s is if th er e ar e m an y m is si ng d at a po in ts o r la rg e lo ss es to f ol lo w -u p. i n ad di tio n, w he th er th e do gs w er e re cr ui te d fr om p ri va te pr ac ti ce , r ef er ra l o r sp ec ia lt y ho sp it al s, b re ed c lu bs o r pa th ol og y se rv ic es c an a ls o in fl ue nc e th e di se as e di ag no si s fr eq ue nc y, s ev er it y an d ag e. d og s se en a t r ef er ra l h os pi ta ls a re n ot li ke ly to b e re pr es en ta tiv e of d og s se en in p ri va te pr ac ti ce s. f ac to rs s uc h as c os t, di st an ce to tr av el , s ev er it y of th e di se as e or c om pl ex ity o f th e tr ea tm en t, in te re st o f fa cu lty a nd a va ila bi lit y of f un di ng to p ay f or tr ea tm en t a ll in fl ue nc e w hi ch d og s ar e re fe rr ed a nd a t w ha t s ta ge o f di se as e. a de qu at e sa m pl e siz e: a la rg e en ou gh s am pl e he lp s to e ns ur e th at th e an im al s in th e st ud y re pr es en t t he a ni m al s to w ho m w e w ou ld li ke to e xt ra po la te th e re su lts . a ls o, a la rg e en ou gh s am pl e is n ee de d to b e su re th at th e st ud y ha d en ou gh a ni m al s to b e ab le to f in d a di ff er en ce s ta tis tic al ly if o ne r ea ll y ex is te d (p ow er c al cu la ti on s ca n en su re th is ). c on ve rs el y, a n ex tr em el y la rg e sa m pl e m ay id en ti fy s ta ti st ic al s ig ni fi ca nc e th at is m at he m at ic al in n at ur e an d no t re fl ec tiv e of u nd er ly in g sc ie nc e. a cc ur at e an d pr ec ise m ea su re m en t o f th e ch ar ac te ri st ic s of in te re st : in th is c as e, a cc ur at e as se ss m en t o f th e ag e of g on ad ec to m y is c ri ti ca l. d at a fr om th e ve te ri na ri an p er fo rm in g th e su rg er y is li ke ly m os t a cc ur at e. o w ne r re co lle ct io n of th e da te is p ro ne to d if fi cu lty in r ec al l, pa rt ic ul ar ly f or o ld er d og s. d og s w ho w er e ac qu ir ed a s ad ul ts an d w er e al re ad y go na de ct om iz ed m ay n ot h av e a kn ow n da te o f su rg er y an d th er ef or e w ill b e sy st em at ic al ly ex cl ud ed f ro m th e da ta s et . a de qu at e co nt ro l o f c on fo un di ng fa ct or s: a li st in g th e co nf ou nd er s, w hy e ac h w as in cl ud ed a nd h ow e ac h w as ad dr es se d is c ri ti ca l. i n th is c as e, a ge , s ex , b re ed , g en et ic s, b od y co nd iti on s co re , d ie t a nd e xe rc is e m ay b e im po rt an t fo r th e di se as es o f in te re st . c au tio us a nd c ri tic al a ss es sm en t o f r es ul ts b y th e au th or s: t he re s ho ul d be a s ec tio n in th e di sc us si on a bo ut th e li m it at io ns o f th e st ud y as w el l a s th e in te rp re ta ti on o f th e re su lts in li gh t o f th e ob je ct iv es , l im ita ti on s, a na ly si s, ot he r pu bl is he d w or k an d ot he r re le va nt e vi de nc e th at h ig hl ig ht s th e st re ng th s an d w ea kn es se s of th e cu rr en t s tu dy .       clinical theriogenology • volume 9, number 2 • june 2017183 t ab le 2 . l it er at ur e re vi ew – m am m ar y ne op la si a r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s fo r c o n fo u n d in g f a c t o r s 17 d ec re as ed n eu te re d bi tc he s 1/ 3 as li ke ly a s in ta ct bi tc he s to de ve lo p co nd iti on c as eco nt ro l st ud y d ia gn os ti c la bo ra to ry su bm is si on s 20 75 tu m or s n ot a va ila bl e in a ll m ed ic al r ec or ds ac ce ss ed a ge -m at ch ed c on tr ol s, br ee d va ri at io n id en tif ie d in st ud y bu t n ot in co rp or at ed in to s ta tis tic al a na ly si s 20 d ec re as ed o r 0 .0 1 if sp ay ed b ef or e 1 ye ar o f ag e, 0 .1 1 if s pa ye d 12. 5 ye ar s of a ge c as eco nt ro l st ud y d ia gn os ti c la bo ra to ry su bm is si on s 15 0 af fe ct ed do gs o w ne r qu es tio nn ai re c on tr ol s m at ch ed f or a ge (n = 14 7) a nd s iz e (n = 13 1) ; ob es ity a nd h ig h fa t d ie t w er e in cl ud ed in s ta tis tic al an al ys is 18 d ec re as ed p< 0. 00 01 c as e c on tr ol st ud y p at ie nt s ad m it te d to pr iv at e pr ac ti ce s an d sh ar ed w it h a ca nc er r eg is tr y 57 6 af fe ct ed do gs r ev ie w o f ho sp ita l re co rd s c on tr ol s (n = 14 55 ) an d hi st or y of p ro ge st in tr ea tm en t, ag e w er e ev al ua te d se pa ra te ly 19 d ec re as ed 2x g re at er in ci de nc e in in ta ct f em al es , p< 0. 00 01 c as eco nt ro l st ud y c an ce r re gi st ry su bm is si on s - n ot c on si de re d c on tr ol s fr om p ri va te pr ac ti ce s, a ge , s ex a nd br ee d in cl ud ed in s ta ti st ic al an al ys is 58 u nc ha ng ed - r et ro sp ec tiv e co ho rt s tu dy a ni m al s ad op te d fr om hu m an e or ga ni za tio n 18 42 d og s o w ne r qu es tio nn ai re , re vi ew o f ho sp ita l re co rd s p ur eb re d or n ot , o w ne r su rr en de r or s tr ay in ta ke ty pe , t im e in s he lte r an d ot he r do gs in h ou se ho ld w er e in cl ud ed in s ta tis tic al an al ys is , l ow n um be r of af fe ct ed a ni m al s m ay h av e clinical theriogenology • volume 9, number 2 • june 2017 184 de cr ea se d st at is ti ca l p ow er 23 u nc ha ng ed - r et ro sp ec tiv e co ho rt s tu dy g er m an sh ep he rd d og s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 70 5 fe m al e do gs r ev ie w o f ho sp ita l re co rd s in cl ud in g re fe rr in g ve te ri na ri an s s m al l s am pl e si ze , b re ed sp ec if ic s tu dy ( g er m an sh ep he rd d og s) , s ex in cl ud ed in s ta tis ti ca l an al ys is 14 0 d ec re as ed o r 0 .4 ( ex v iv o or p os tm or te m sa m pl es ) or 0 .6 (p os tm or te m sa m pl es ) co m pa re d to o r = 1 fo r in ta ct fe m al es r et ro sp ec ti ve st ud y c an ce r re gi st ry su bm is si on s e xa ct nu m be r of tu m or s in fe m al e do gs n ot pr ov id ed n ot c on si de re d s ex a nd a ge in cl ud ed in st at is ti ca l a na ly si s. t um or ty pe s no t a lw ay s se pa ra te d by a na to m ic lo ca tio n. clinical theriogenology • volume 9, number 2 • june 2017185 t ab le 3 . l it er at ur e re vi ew – p ro st at ic n eo pl as ia r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 33 in cr ea se d o r 3 .9 ( 2. 36. 8) c as eco nt ro l st ud y d ia gn os ti c la bo ra to ry su bm is si on s 70 af fe ct ed do gs r ev ie w o f ho sp ita l re co rd s, n ec ro ps y re po rt s, o w ne r / d v m qu es tio nn ai re /in te rv ie w , ju st g on ad ec to m iz ed ye s or n o a ge -m at ch ed c on tr ol s 28 in cr ea se d o r 4 .3 ( 2. 57. 6) c as e co nt ro l st ud y p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 56 af fe ct ed do gs r ev ie w o f ho sp ita l re co rd s, o w ne r in te rv ie w , j us t go na de ct om iz ed y es o r no b re ed v ar ia tio n id en tif ie d in s tu dy b ut n ot in co rp or at ed in to st at is ti ca l a na ly si s 29 in cr ea se d o r 2 .4 ( 1. 09. 6) c as e se ri es p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 31 af fe ct ed do gs r ev ie w o f ho sp ita l re co rd s, ju st go na de ct om iz ed y es o r no n o co nt ro ls 30 in cr ea se d o r 2 .8 ( 2. 63. 1) c as eco nt ro l st ud y p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l ( v m d b re gi st ry ) 68 1 af fe ct ed do gs n ot a va ila bl e th ro ug h re gi st ry ; j us t go na de ct om iz ed y es o r no a ge -m at ch ed c on tr ol s, br ee d in cl ud ed in s ep ar at e an al ys is 32 u nc ha ng ed -- c as e se ri es p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 43 af fe ct ed do gs r ev ie w o f ho sp ita l re co rd s n o co nt ro ls , s m al l s am pl e si ze o r = o dd s ra ti o clinical theriogenology • volume 9, number 2 • june 2017 186 t ab le 4 . l it er at ur e re vi ew – tr an si ti on al c el l c ar ci no m a r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s fo r c o n fo u n d in g f a c t o r s 37 in cr ea se d o r 4 .4 ( 1. 413 .8 ) c as eco nt ro l st ud y s co tti sh te rr ie rs re cr ui te d th ro ug h sp ec ia lt y/ re fe rr al ho sp ita l, br ee d cl ub 83 af fe ct ed do gs o w ne r qu es tio nn ai re a ge -m at ch ed c on tr ol s, br ee dsp ec if ic s tu dy 39 in cr ea se d o r 2 .0 ( no c i re po rt ed ), p= 0. 03 c as eco nt ro l st ud y d ia gn os ti c la bo ra to ry su bm is si on s 11 5 tu m or s r ev ie w o f ho sp ita l re co rd s c on tr ol p op ul at io n fr om sp ec ia lt y/ re fe rr al h os pi ta l, ag e, b re ed , g en de r, w ei gh t w er e an al yz ed s ep ar at el y o r = o dd s ra ti o clinical theriogenology • volume 9, number 2 • june 2017187 t ab le 5 . l it er at ur e re vi ew – o st eo sa rc om a r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 42 in cr ea se d r r 3 .8 ( 1. 59. 2) f em al es ; r r 3 .1 ( 1. 18. 3) m al es r et ro sp ec ti ve co ho rt s tu dy r ec ru it ed th ro ug h br ee d cl ub s 86 a ff ec te d do gs , 59 7c on tr ol s w ith ou t co nd iti on , o w ne r qu es tio nn ai re /in te rv ie w fo r ag e at g on ad ec to m y 59 7c on tr ol s w ith ou t c on di tio n, br ee dsp ec if ic st ud y (r ot tw ei le r) , he ig ht , w ei gh t in cl ud ed in st at is ti ca l a na ly si s 23 u nc ha ng ed - r et ro sp ec tiv e co ho rt s tu dy g er m an sh ep he rd d og s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 46 5 m al e do gs , 7 05 fe m al e do gs r ev ie w o f ho sp ita l re co rd s in cl ud in g re fe rr in g ve te ri na ri an s s m al l s am pl e si ze , br ee dsp ec if ic st ud y (g er m an sh ep he rd d og s) , se x in cl ud ed in st at is ti ca l a na ly si s 14 0 in cr ea se d o r 1 .6 ( ex vi vo o r ne cr op sy sa m pl es ) or 2 .0 (n ec ro ps y sa m pl es ) co m pa re d to o r = 1 fo r in ta ct m al es , no s ig ni fi ca nt ef fe ct in fe m al es r et ro sp ec ti ve st ud y c an ce r re gi st ry su bm is si on s e xa ct nu m be r of tu m or s in m al e do gs no t pr ov id ed n ot c on si de re d s ex a nd a ge in cl ud ed in st at is ti ca l a na ly si s. r r = r el at iv e ri sk clinical theriogenology • volume 9, number 2 • june 2017 188 t ab le 6 . l it er at ur e re vi ew – h em an gi os ar co m a r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 45 in cr ea se d (f em al es ) o r 2 .2 ( 1. 24. 1) c as eco nt ro l st ud y d ia gn os ti c la bo ra to ry su bm is si on s 59 af fe ct ed do gs d v m q ue st io nn ai re , ju st g on ad ec to m iz ed ye s or n o r an do m s am pl e of bi op sy s ub m is si on s , a ge in cl ud ed in s ta tis ti ca l an al ys is 43 in cr ea se d r r 5 .3 ( 4. 07. 2) fe m al es ; 1 .6 ti m es m or e lik el y if ca st ra te d c as e co nt ro l st ud y p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l ( v m d b re gi st ry ) 63 3 af fe ct ed do gs r ev ie w o f re gi st ry re co rd s, ju st go na de ct om iz ed y es o r no s ex , b re ed a nd y ea r of di ag no si s w er e ev al ua te d se pa ra te ly 47 in cr ea se d (f em al es ); u nc ha ng ed ( m al es ) r r 6 .1 ( 1. 231 .4 ) fo r fe m al es s pa ye d af te r 1 ye ar o f ag e r et ro sp ec ti ve co ho rt s tu dy g ol de n re tr ie ve rs ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 36 4 fe m al e do gs , 3 95 m al e do gs r ev ie w o f ho sp ita l re co rd s b re ed -s pe ci fi c st ud y (g ol de n re tr ie ve rs ), s ex w as in cl ud ed in s ta ti st ic al an al ys is , a ge w as an al yz ed s ep ar at el y 23 u nc ha ng ed - r et ro sp ec tiv e co ho rt s tu dy g er m an sh ep he rd d og s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 46 5 m al e do gs , 7 05 fe m al e do gs r ev ie w o f ho sp ita l re co rd s in cl ud in g re fe rr in g ve te ri na ri an s s m al l s am pl e si ze , b re ed sp ec if ic s tu dy ( g er m an sh ep he rd d og s) , s ex in cl ud ed in s ta tis ti ca l an al ys is 49 u nc ha ng ed -- r et ro sp ec tiv e co ho rt s tu dy l ab ra do r an d g ol de n re tr ie ve rs ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 11 47 fe m al e do gs , 14 35 m al e do gs r ev ie w o f ho sp ita l re co rd s b re ed -s pe ci fi c st ud y (g ol de n an d l ab ra do r re tr ie ve rs ), s ex w as in cl ud ed in s ta tis ti ca l an al ys is clinical theriogenology • volume 9, number 2 • june 2017189 48 in cr ea se d (f em al es ); u nc ha ng ed ( m al es ) o r 9 .0 ( 2. 829 .4 ) r et ro sp ec ti ve co ho rt s tu dy v iz sl as r ec ru ite d th ro ug h br ee d cl ub s 25 05 do gs o w ne r qu es ti on na ir e b re ed -s pe ci fi c st ud y (v iz sl as ), s ex w as in cl ud ed in s ta tis ti ca l an al ys is 14 0 in cr ea se d o r 1 .6 ( ex v iv o or p os tm or te m sa m pl es ) or 2 .4 (p os tm or te m sa m pl es ) co m pa re d to o r = 1 fo r in ta ct fe m al es r et ro sp ec ti ve st ud y c an ce r re gi st ry su bm is si on s e xa ct nu m be r of tu m or s in f em al e do gs n ot pr ov id ed n ot c on si de re d s ex a nd a ge in cl ud ed in st at is ti ca l a na ly si s. t um or ty pe s no t a lw ay s se pa ra te d by a na to m ic lo ca tio n. o r = o dd s ra ti o, r r = r el at iv e ri sk clinical theriogenology • volume 9, number 2 • june 2017 190 t ab le 7 . l it er at ur e re vi ew – ly m ph os ar co m a r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 51 in cr ea se d (f em al es ) o r 1 .5 ( 1. 41. 6) g ol de n an d l ab ra do r re tr ie ve rs , g er m an sh ep he rd s an d bo xe rs w er e at in cr ea se d ri sk c as eco nt ro l st ud y p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l ( v m d b re gi st ry ) 14 ,5 73 do gs r ev ie w o f re gi st ry re co rd s, go na de ct om iz ed y es or n o a ge an d br ee dm at ch ed co nt ro ls f ro m r eg is tr y, al so s ex , b re ed a nd a ge w er e in cl ud ed in st at is ti ca l a na ly si s 47 u nc ha ng ed (f em al es ); in cr ea se d (m al es ) p< 0. 05 f or m al es c as tr at ed ea rl y r et ro sp ec ti ve co ho rt s tu dy g ol de n re tr ie ve rs ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 36 4 fe m al e do gs , 3 95 m al e do gs r ev ie w o f ho sp ita l re co rd s b re ed -s pe ci fi c st ud y (g ol de n re tr ie ve rs ), s ex w as in cl ud ed in st at is ti ca l a na ly si s, a ge w as a na ly ze d se pa ra te ly 23 u nc ha ng ed - r et ro sp ec tiv e co ho rt s tu dy g er m an sh ep he rd d og s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 46 5 m al e do gs , 7 05 fe m al e do gs r ev ie w o f ho sp ita l re co rd s in cl ud in g re fe rr in g ve te ri na ri an s s m al l s am pl e si ze , br ee dsp ec if ic s tu dy (g er m an s he ph er d do gs ), s ex in cl ud ed in st at is ti ca l a na ly si s 49 in cr ea se d (f em al e g ol de ns ); in cr ea se d (m al e g ol de ns ); u nc ha ng ed ( l ab s) p= 0. 01 f or fe m al es s pa ye d fr om 6 -1 1 m on th s of a ge ; p= 0. 00 7 fo r m al es c as tr at ed fr om 6 -1 1 r et ro sp ec ti ve co ho rt s tu dy l ab ra do r an d g ol de n re tr ie ve rs ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 11 47 fe m al e do gs , 14 35 m al e do gs r ev ie w o f ho sp ita l re co rd s b re ed -s pe ci fi c st ud y (g ol de n an d l ab ra do r re tr ie ve rs ), s ex w as in cl ud ed in s ta tis ti ca l an al ys is , a ge w as an al yz ed s ep ar at el y clinical theriogenology • volume 9, number 2 • june 2017191 m on th s of a ge 48 in cr ea se d o r 4 .3 ( 1. 99. 7) r et ro sp ec ti ve co ho rt s tu dy v iz sl as r ec ru ite d th ro ug h br ee d cl ub s 25 05 do gs o w ne r qu es ti on na ir e b re ed -s pe ci fi c st ud y (v iz sl as ), s ex w as in cl ud ed in s ta tis ti ca l an al ys is 14 0 in cr ea se d o r 1 .1 ( ex v iv o or p os tm or te m sa m pl es ) or 1 .6 (p os tm or te m sa m pl es ) co m pa re d to o r = 1 fo r in ta ct fe m al es ; o r 1 .3 (e x vi vo o r po st -m or te m sa m pl es ) or 2 .3 (p os tm or te m sa m pl es ) co m pa re d to o r = 1 fo r in ta ct m al es r et ro sp ec ti ve st ud y c an ce r re gi st ry su bm is si on s e xa ct nu m be r of tu m or s in f em al e ve rs us m al e do gs no t pr ov id ed n ot c on si de re d s ex a nd a ge in cl ud ed in st at is ti ca l a na ly si s. o r = o dd s ra tio clinical theriogenology • volume 9, number 2 • june 2017 192 t ab le 8 . l it er at ur e re vi ew – m as t c el l t um or r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 53 in cr ea se d (f em al es ) o r 4 .1 ( 2. 27. 7) c as eco nt ro l st ud y p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 25 2 af fe ct ed do gs r ev ie w o f ho sp ita l re co rd s, ju st go na de ct om iz ed y es or n o c on tr ol s fr om ho sp ita l p op ul at io n w ith ou t c on di tio n, ag e, s ex a nd b re ed w er e in cl ud ed in st at is ti ca l a na ly si s 52 u nc ha ng ed in cr ea se d o r 0 .1 ( 0. 10. 2) a ll ne ut er ed co m pa re d to a ll in ta ct , n o in fl ue nc e of s ex c as eco nt ro l st ud y r eg is tr y fr om pr iv at e pr ac ti ce su bm is si on s 45 3 af fe ct ed do gs r ev ie w o f re gi st ry re co rd s, ju st go na de ct om iz ed y es or n o a ge , s ex , i ns ur an ce st at us , b re ed , w ei gh t an d br ee d ty pe w er e in cl ud ed in st at is ti ca l a na ly si s 47 in cr ea se d (f em al es ); u nc ha ng ed ( m al es ) c ou ld n ot co m pl et e st at is tic al an al ys is r et ro sp ec ti ve co ho rt s tu dy g ol de n re tr ie ve rs ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 36 4 fe m al e do gs , 3 95 m al e do gs r ev ie w o f ho sp ita l re co rd s b re ed -s pe ci fi c st ud y (g ol de n re tr ie ve rs ), se x w as in cl ud ed in st at is ti ca l a na ly si s, ag e w as a na ly ze d se pa ra te ly 23 u nc ha ng ed - r et ro sp ec tiv e co ho rt s tu dy g er m an sh ep he rd d og s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 46 5 m al e do gs , 7 05 fe m al e do gs r ev ie w o f ho sp ita l re co rd s in cl ud in g re fe rr in g ve te ri na ri an s s m al l s am pl e si ze , br ee dsp ec if ic s tu dy (g er m an s he ph er d do gs ), s ex in cl ud ed in s ta tis tic al a na ly si s 49 in cr ea se d (f em al e g ol de ns ); u nc ha ng ed ( m al e p= 0. 01 f or fe m al es s pa ye d at 2 -8 y ea rs o f r et ro sp ec ti ve co ho rt s tu dy l ab ra do r an d g ol de n re tr ie ve rs 11 47 fe m al e do gs , r ev ie w o f ho sp ita l re co rd s b re ed -s pe ci fi c st ud y (g ol de n an d l ab ra do r re tr ie ve rs ), clinical theriogenology • volume 9, number 2 • june 2017193 g ol de ns ); u nc ha ng ed ( l ab s) ag e ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 14 35 m al e do gs se x in cl ud ed in st at is ti ca l a na ly si s, ag e w as a na ly ze d se pa ra te ly 48 in cr ea se d o r 3 .5 ( 2. 35. 4) r et ro sp ec ti ve co ho rt s tu dy v iz sl as r ec ru ite d th ro ug h br ee d cl ub s 25 05 do gs o w ne r qu es ti on na ir e b re ed -s pe ci fi c st ud y (v iz sl as ), s ex w as in cl ud ed in st at is ti ca l a na ly si s, ag e w as a na ly ze d se pa ra te ly 14 0 in cr ea se d o r 1 .2 ( ex vi vo o r po st m or te m sa m pl es ) or 3 .7 (p os tm or te m sa m pl es ) co m pa re d to o r = 1 fo r in ta ct fe m al es ; 1 .2 (e x vi vs o or po st -m or te m sa m pl es ) or 3 .0 (p os tm or te m sa m pl es ) co m pa re d to o r = 1 fo r in ta ct m al es r et ro sp ec ti ve st ud y c an ce r re gi st ry su bm is si on s e xa ct nu m be r of tu m or s in m al e do gs n ot pr ov id ed n ot c on si de re d s ex a nd a ge in cl ud ed in st at is ti ca l a na ly si s. o r = o dd s ra tio clinical theriogenology • volume 9, number 2 • june 2017 194 t ab le 9 . l it er at ur e re vi ew – o be si ty r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 55 in cr ea se d o r 1 .6 ( 1. 22. 1) c as eco nt ro l st ud y p at ie nt s ad m it te d to pr iv at e pr ac ti ce s in c hi na ; d v m s de te rm in ed b od y co nd iti on s co re (b c s ) 23 91 do gs o w ne r qu es tio nn ai re /in te rv ie w , ju st g on ad ec to m iz ed ye s or n o a ge , b re ed , s ex a nd m an y ot he r va ri ab le s in cl ud ed in s ta tis ti ca l an al ys is 57 in cr ea se d g on ad ec to m iz ed do gs 2 ti m es m or e lik el y to be o be se th an in ta ct d og s c ro ss se ct io na l st ud y p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita ls a nd pr iv at e pr ac ti ce s in e ng la nd ; d v m s de te rm in ed b c s 82 68 do gs d v m q ue st io nn ai re , ju st g on ad ec to m iz ed ye s or n o b re ed , s ex a nd a ge w er e in cl ud e in s ta tis tic al an al ys is 62 in cr ea se d g on ad ec to m iz ed do gs ( ye s/ no ) h r ~ 2 fo r 1 to 2 ye ar s af te r go na de ct om y; no d if fe re nc e fo r ag e at go na de ct om y r et ro sp ec ti ve co ho rt s tu dy c or po ra te pr iv at e pr ac ti ce ; ho sp ita l s ta ff de te rm in ed b c s 25 99 do gs r ev ie w o f ho sp ita l re co rd s a ge -m at ch ed c on tr ol s, br ee d si ze , s ex , a nd o th er va ri ab le s in cl ud ed in st at is ti ca l a na ly si s o r = o dd s ra ti o clinical theriogenology • volume 9, number 2 • june 2017195 t ab le 1 0. l ite ra tu re r ev ie w – c ra ni al c ru ci at e li ga m en t i nj ur y r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 71 in cr ea se d o r 2 .2 ( 1. 33. 8) f em al es ; o r 1 .6 ( 0. 83. 0) m al es c as eco nt ro l s tu dy p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 11 2 af fe ct ed do gs r ev ie w o f ho sp ita l re co rd s, ju st go na de ct om iz ed y es o r no b re ed , s iz e an d se x w er e in cl ud ed in st at is ti ca l a na ly si s 72 in cr ea se d ca st ra te d m al es o r sp ay ed fe m al es d ec re as ed in ta ct m al es o r in ta ct fe m al es c as tr at ed m al es co m pa re d to a ll ot he r do gs 1 .6 8 (1 .6 -1 .7 ) s pa ye d fe m al es co m pa re d to a ll do gs o r 2 .3 5 (2 .3 -2 .4 ) in ta ct m al es co m pa re d to a ll ot he r do gs o r 0. 47 ( 0. 46 -0 .4 9) in ta ct f em al es co m pa re d to a ll ot he r do gs o r 0. 51 ( 0. 49 -0 .5 3) c ro ss -s ec tio na l st ud y p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l ( v m d b re gi st ry ) 11 93 af fe ct ed do gs r ev ie w o f re gi st ry re co rd s, ju st go na de ct om iz ed y es o r no b re ed , s ex a nd a ge w er e an al yz ed se pa ra te ly 13 9 in cr ea se d if go na de ct om iz ed be fo re 6 m on th s o r 3 .0 ( 1. 28. 0) if go na de ct om iz ed pr io r to 6 c as eco nt ro l s tu dy r ec ru ite d fr om sp ec ia li st s in pr ac tic e 38 af fe ct ed do gs o w ne r qu es tio nn ai re /in te rv ie w 42 c on tr ol s m at ch ed f or b od y w ei gh t a nd ti bi al pl at ea u an gl e, br ee d, w ei gh t, clinical theriogenology • volume 9, number 2 • june 2017 196 m on th s of a ge bo dy c on di tio n an d ot he r va ri ab le s an al yz ed s ep ar at el y 78 in cr ea se d p< 0. 00 01 c as eco nt ro l s tu dy p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l ( v m d b re gi st ry ) 10 ,7 69 af fe ct ed do gs r ev ie w o f re gi st ry re co rd s b re ed , a ge , s ex a nd w ei gh t w er e an al yz ed s ep ar at el y 73 u nc ha ng ed -- c as eco nt ro l s tu dy p at ie nt s ad m it te d to a pr iv at e pr ac ti ce in e ng la nd 18 9 af fe ct ed do gs r ev ie w o f ho sp ita l re co rd s, ju st go na de ct om iz ed y es o r no c on tr ol g ro up f ro m sa m e ho sp it al , b re ed , s ex , a ge a nd ob es ity w er e st at is ti ca l a na ly si s 76 in cr ea se d d at a no t pr ov id ed c as eco nt ro l s tu dy p at ie nt s ad m it te d to th re e sp ec ia lt y/ re fe rr al ho sp ita l 20 1 af fe ct ed do gs r ev ie w o f ho sp ita l re co rd s, ju st go na de ct om iz ed y es o r no a ge -m at ch ed co nt ro ls , b re ed , s ex an d w ei gh t w er e an al yz ed s ep ar at el y 74 in cr ea se d (f em al es ) o r 2 .1 ( 1. 62. 9) c as eco nt ro l s tu dy r eg is tr y su bm is si on s fr om p ri va te pr ac ti ce in e ng la nd 95 3 do gs r ev ie w o f re gi st ry re co rd s c on tr ol s w ith in re gi st ry p op ul at io n, br ee d, p ur eb re d or no t, se x, in su ra nc e, ag e, b od y w ei gh t w er e in cl ud ed in st at is ti ca l a na ly si s 47 in cr ea se d p< 0. 05 f or d og s go na de ct om iz ed at le ss th an 1 ye ar o f ag e r et ro sp ec ti ve co ho rt s tu dy g ol de n re tr ie ve rs ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 36 4 fe m al e do gs , 3 95 m al e do gs r ev ie w o f ho sp ita l re co rd s b re ed -s pe ci fi c st ud y (g ol de n re tr ie ve rs ), s ex w as in cl ud ed in st at is ti ca l a na ly si s, bo dy c on di tio n sc or e an al yz ed clinical theriogenology • volume 9, number 2 • june 2017197 se pa ra te ly 23 in cr ea se d h r 9 .5 ( 1. 274 .9 ) fe m al es ; h r 2 6. 2 (5 .6 12 3. 3) m al es r et ro sp ec ti ve co ho rt s tu dy g er m an sh ep he rd d og s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 46 5 m al e do gs , 7 05 fe m al e do gs r ev ie w o f ho sp ita l re co rd s in cl ud in g re fe rr in g ve te ri na ri an s b re ed -s pe ci fi c st ud y (g er m an sh ep he rd d og s) , se x w as in cl ud ed in st at is ti ca l a na ly si s, bo dy c on di tio n sc or e w as a na ly ze d se pa ra te ly 49 in cr ea se d (f em al e g ol de ns ); in cr ea se d (m al e g ol de ns ); u nc ha ng ed (f em al e l ab s) ; in cr ea se d (m al e l ab s) p= 0. 03 ( fe m al e g ol de ns ), p< 0. 00 1 fo r m al e g ol de ns ca st ra te d at le ss th an 6 m on th s of a ge , p = 0. 00 4 fo r m al e g ol de ns ca st ra te d at 6 11 m on th s of ag e; p = 0. 02 f or m al e l ab s ca st ra te d at le ss th an 6 m on th s of a ge r et ro sp ec ti ve co ho rt s tu dy l ab ra do r an d g ol de n re tr ie ve rs ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 11 47 fe m al e do gs , 14 35 m al e do gs r ev ie w o f ho sp ita l re co rd s b re ed -s pe ci fi c st ud y (g ol de n an d l ab ra do r re tr ie ve rs ), s ex w as in cl ud ed in st at is ti ca l a na ly si s, bo dy c on di tio n sc or e an al yz ed se pa ra te ly o r = o dd s ra tio , h r = h az ar d ra tio clinical theriogenology • volume 9, number 2 • june 2017 198 t ab le 1 1. l ite ra tu re r ev ie w – p at el la r lu xa ti on r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 86 in cr ea se d o r 3 .1 ( 1. 37. 5) , bu t c on fo un de d by o ld er a ge as so ci at ed w it h go na de ct om y c as eco nt ro l st ud y p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l, re cr ui te d th ro ug h br ee d cl ub s (s m al l a nd m in ia tu re b re ed s on ly in a us tr ia ) 43 2 af fe ct ed do gs r ev ie w o f ho sp ita l re co rd s, ju st go na de ct om iz ed y es or n o 26 2 un af fe ct ed d og s as co nt ro ls , b re ed , a ge , s ex an d w ei gh t w er e in cl ud ed in s ta tis ti ca l an al ys is 85 in cr ea se d o r 2 .4 ( 1. 83. 2) , fe m al es a ls o at in cr ea se d ri sk o r 1 .3 ( 1. 11. 5) c as eco nt ro l st ud y r eg is tr y su bm is si on s fr om p ri va te pr ac ti ce s in e ng la nd 75 1 af fe ct ed do gs r ev ie w o f re gi st ry re co rd s, ju st go na de ct om iz ed y es or n o b re ed , p ur eb re d or n ot , bo dy w ei gh t, ag e, s ex an d in su re d or n ot w er e in cl ud ed in s ta tis ti ca l an al ys is 58 u nc ha ng ed -- r et ro sp ec tiv e co ho rt s tu dy a ni m al s ad op te d fr om h um an e or ga ni za tio n 18 42 d og s o w ne r qu es tio nn ai re , re vi ew o f ho sp ita l re co rd s p ur eb re d or n ot , o w ne r su rr en de r or s tr ay in ta ke ty pe , t im e in s he lte r an d ot he r do gs in h ou se ho ld w er e in cl ud ed in st at is ti ca l a na ly si s, lo w nu m be r of a ff ec te d an im al s m ay h av e de cr ea se d st at is ti ca l po w er o r = o dd s ra ti o clinical theriogenology • volume 9, number 2 • june 2017199 t ab le 1 2. l ite ra tu re r ev ie w – h ip d ys pl as ia r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 93 in cr ea se d g on ad ec to m iz ed do gs 1 .5 x m or e lik el y to d ev el op co nd iti on th an ar e in ta ct d og s n es te d ca se co nt ro l s tu dy d og s re gi st er ed w ith n at io na l br ee d cl ub 97 af fe ct ed do gs o w ne r qu es tio nn ai re , ju st g on ad ec to m iz ed ye s or n o b re ed -s pe ci fi c st ud y, ag e, s ex , s li pp er y fl oo r co ve ri ng o r no t a nd ot he r va ri ab le s in cl ud ed in s ta tis tic al a na ly si s 72 in cr ea se d ca st ra te d m al es d ec re as ed in ta ct fe m al es a nd s pa ye d fe m al es c as tr at ed m al es co m pa re d to a ll ot he r do gs 1. 2 (1 .1 81. 24 ) in ta ct f em al es co m pa re d to a ll ot he r do gs o r 0. 93 ( 0. 91 -0 .9 6) s pa ye d fe m al es co m pa re d to a ll do gs o r 0 .9 7 (0 .9 50. 99 ) c ro ss se ct io na l st ud y p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l ( v m d b re gi st ry ) 11 93 af fe ct ed do gs r ev ie w o f re gi st ry re co rd s, ju st go na de ct om iz ed y es or n o b re ed , s ex a nd a ge w er e an al yz ed se pa ra te ly 58 in cr ea se d h r 1 .7 ( 1. 02. 8) > 5. 5 m on th s at ne ut er r et ro sp ec ti ve co ho rt s tu dy a ni m al s ad op te d fr om h um an e or ga ni za tio n 18 42 do gs o w ne r qu es tio nn ai re , re vi ew o f ho sp ita l re co rd s p ur eb re d or n ot , o w ne r su rr en de r or s tr ay in ta ke ty pe , t im e in sh el te r an d ot he r do gs in h ou se ho ld w er e in cl ud ed in s ta tis ti ca l an al ys is ,, no t c le ar if a ll di ag no se s m ad e by ve te ri na ri an clinical theriogenology • volume 9, number 2 • june 2017 200 47 in cr ea se d (m al es ) p< 0. 01 r et ro sp ec ti ve co ho rt s tu dy g ol de n re tr ie ve rs ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 36 4 fe m al e do gs , 3 95 m al e do gs r ev ie w o f ho sp ita l re co rd s b re ed -s pe ci fi c st ud y (g ol de n re tr ie ve rs ), s ex w as in cl ud ed in st at is ti ca l a na ly si s, bo dy c on di tio n sc or e an al yz ed s ep ar at el y 23 u nc ha ng ed - r et ro sp ec tiv e co ho rt s tu dy g er m an sh ep he rd d og s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 46 5 m al e do gs , 7 05 fe m al e do gs r ev ie w o f ho sp ita l re co rd s in cl ud in g re fe rr in g ve te ri na ri an s s m al l s am pl e si ze , br ee dsp ec if ic s tu dy (g er m an s he ph er d do gs ), s ex w as in cl ud ed in s ta ti st ic al a na ly si s, bo dy c on di tio n sc or e an al yz ed s ep ar at el y 49 u nc ha ng ed (f em al e g ol de ns ); in cr ea se d (m al e g ol de ns ); u nc ha ng ed (f em al e l ab s) ; u nc ha ng ed ( m al e l ab s) p< 0. 00 1 fo r m al es c as tr at ed at le ss th an 6 m on th s of a ge , p< 0. 05 f or m al es c as tr at ed 611 m on th s of ag e r et ro sp ec ti ve co ho rt s tu dy l ab ra do r an d g ol de n re tr ie ve rs ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 11 47 fe m al e do gs , 14 35 m al e do gs r ev ie w o f ho sp ita l re co rd s b re ed -s pe ci fi c st ud y (g ol de n an d l ab ra do r re tr ie ve rs ), s ex w as in cl ud ed in s ta tis ti ca l an al ys is , b od y co nd iti on s co re an al yz ed s ep ar at el y o r = o dd s ra tio , h r = h az ar d ra tio clinical theriogenology • volume 9, number 2 • june 2017201 t ab le 1 3. l ite ra tu re r ev ie w – u ri na ry in co nt in en ce r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 10 4 in cr ea se d r r 7 .8 ( 2. 631 .5 ) p ro sp ec tiv e co ho rt s tu dy p at ie nt s ad m it te d to pr iv at e pr ac ti ce s 80 9 fe m al e do gs r ec or ds c om pl et ed b y pr iv at e pr ac tit io ne rs c on tr ol s w ith in h os pi ta l po pu la tio n, a ge , b re ed , es tr us in fl ue nc in g dr ug s w er e in cl ud ed in st at is ti ca l a na ly si s 10 8 in cr ea se d o r 4 .9 ( 3. 17. 8) r et ro sp ec tiv e co ho rt p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 32 60 fe m al e do gs r ev ie w o f ho sp ita l re co rd s o nl y in cl ud ed in ta ct a nd sp ay ed a t 6 m on th s an d ol de r, n o co nf ou nd in g fa ct or s in cl ud ed 58 in cr ea se d h r 1 .2 ( 1. 11. 3) ov er al l, h r 3 .5 if s pa ye d be fo re 3 m on th s of a ge r et ro sp ec ti ve co ho rt s tu dy a ni m al s ad op te d fr om hu m an e or ga ni za tio n 98 3 fe m al e do gs o w ne r qu es tio nn ai re , re vi ew o f ho sp ita l re co rd s p ur eb re d or n ot , o w ne r su rr en de r or s tr ay in ta ke ty pe , t im e in s he lte r an d ot he r do gs in h ou se ho ld w er e in cl ud ed in st at is ti ca l a na ly si s 23 in cr ea se d p< 0. 05 f or d og s sp ay ed a t 6 -1 1 m on th s of a ge r et ro sp ec ti ve co ho rt s tu dy g er m an sh ep he rd d og s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l 70 5 fe m al e do gs r ev ie w o f ho sp ita l re co rd s in cl ud in g re fe rr in g ve te ri na ri an s s m al l s am pl e si ze , br ee dsp ec if ic s tu dy (g er m an s he ph er d do gs ), a ge w as a na ly ze d se pa ra te ly 14 2 in te ra ct io n be tw ee n ag e at ne ut er a nd w ei gh t w it h lo w er r is k of in co nt in en ce f or do gs > 25 k g fo r h r 0 .8 9 (0 .8 20. 97 ) r et ro sp ec ti ve co ho rt s tu dy p at ie nt s ad m it te d to a m er ic an a ni m al h os pi ta l a ss oc ia ti on ac cr ed it ed u s 35 6 fe m al e do gs r ev ie w o f ho sp ita l re co rd s c on tr ol s w ith in h os pi ta l po pu la tio n, a ge a t pr es en ta tio n an d bo dy co nd iti on s co re w er e an al yz ed s ep ar at el y; w ei gh t w as in cl ud ed in clinical theriogenology • volume 9, number 2 • june 2017 202 ev er y m on th o ld er ne ut er in g w as d on e ve te ri na ry cl in ic s st at is ti ca l a na ly si s 14 3 in cr ea se d fo r do gs > = 15 k g, a ge o f ne ut er w as n ot si gn if ic an t o r d og s > = 1 5 kg 7 ( 2. 521 ) r et ro sp ec ti ve co ho rt s tu dy p at ie nt s ad m it te d to a m ul ti -p ra ct ic e ge ne ra l a nd sp ec ia lt y ve te ri na ry c li ni c 56 6 fe m al e do gs r ev ie w o f ho sp ita l re co rd s an d in te rv ie w s of o w ne rs c on tr ol s w ith in h os pi ta l po pu la tio n, b od y w ei gh t in c at eg or ie s, n um be r of li tt er s, e le ct iv e or em er ge nc y ne ut er w er e in cl ud ed in s ta tis ti ca l an al ys is 14 4 in cr ea se d w ith de cr ea si ng f s h pl as m a co nc en tr at io ns in cr ea se d w ith in cr ea si ng a ge a t sa m pl in g o r 0 .5 ( 0. 24 0. 91 ) fs h a ll br ee ds e xc ep t bo xe rs a nd g er m an sh ep he rd s o r 2 .7 ( 1. 35. 9) ag e at s am pl in g c ro ss se ct io na l st ud y in di vi du al p ur e br ed d og s, o ft en at d og s ho w s 49 9 fe m al e do gs in te rv ie w s w ith ow ne rs b ox er s an d g er m an s he ph er ds a na ly ze d se pa ra te ly , t im e si nc e ne ut er a nd b ef or e or af te r fi rs t e st ru s, a ge a t sa m pl in g, b od y w ei gh t, br ee d, l h a nd f s h le ve ls w er e st at is tic al ly an al yz ed . o r = o dd s ra tio , h r = h az ar d ra ti o, r r = r el at iv e ri sk clinical theriogenology • volume 9, number 2 • june 2017203 t ab le 1 4. l ite ra tu re r ev ie w – u ro li th ia si s r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 11 3 in cr ea se d, in fl ue nc ed b y ag e an d se x o r 2 .6 ( 1. 44. 6) c as eco nt ro l st ud y p at ie nt s ad m it te d to c or po ra te ve te ri na ry pr ac tic e 45 2 af fe ct ed do gs w ith st ru vi te ur ol ith s r ev ie w o f ho sp ita l re co rd s, ju st go na de ct om iz ed y es or n o c on tr ol s fr om s am e ho sp ita l p op ul at io n, di et , b re ed s iz e, a ge , se x, h os pi ta l l oc at io n an d m or e in cl ud ed in st at is ti ca l a na ly si s 11 7 u nc ha ng ed o r 1 .4 ( 0. 99 1. 8) , p = 0. 06 c as eco nt ro l (o xa la te v s st ru vi te ) u ro lit h ce nt er su bm is si on s 38 67 ca lc iu m ox al at e ur ol ith s, 34 30 st ru vi te ur ol ith s r ev ie w o f re co rd s ac co m pa ny in g su bm is si on s, ju st go na de ct om iz ed y es or n o n o co nt ro ls , a ge , s ex , br ee d, b od y co nd iti on , di et w er e in cl ud ed in st at is ti ca l a na ly si s 11 4 in cr ea se d, in fl ue nc ed b y ag e an d se x o r 2 .2 ( 1. 63. 0) c as eco nt ro l st ud y p at ie nt s ad m it te d to c or po ra te ve te ri na ry pr ac tic e 50 8 af fe ct ed do gs w ith ca lc iu m ox al at e ur ol ith ia si s r ev ie w o f ho sp ita l re co rd s, ju st go na de ct om iz ed y es or n o c on tr ol s fr om s am e ho sp ita l p op ul at io n, di et , b re ed s iz e, a ge , se x, h os pi ta l l oc at io n an d m or e in cl ud ed in st at is ti ca l a na ly si s 58 u nc ha ng ed -- r et ro sp ec tiv e co ho rt s tu dy a ni m al s ad op te d fr om h um an e or ga ni za tio n 18 42 d og s o w ne r qu es tio nn ai re , re vi ew o f ho sp ita l re co rd s p ur eb re d or n ot , o w ne r su rr en de r or s tr ay in ta ke ty pe , t im e in s he lte r an d ot he r do gs in h ou se ho ld w er e in cl ud ed in st at is ti ca l a na ly si s, , l ow nu m be r of a ff ec te d an im al s m ay h av e de cr ea se d st at is ti ca l po w er o r = o dd s ra ti o   clinical theriogenology • volume 9, number 2 • june 2017 204 t ab le 1 5. l ite ra tu re r ev ie w – d ia be te s m el li tu s r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 11 9 u nc ha ng ed (f em al es ); i nc re as ed (m al es ) o r 1 .9 ( 1. 72. 2) c as eco nt ro l st ud y p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l ( v m d b re gi st ry ) 68 60 af fe ct ed do gs r ev ie w o f re gi st ry re co rd s, ju st go na de ct om iz ed y es or n o a ge an d in st itu tio nm at ch ed c on tr ol s, br ee d, s ex a nd w ei gh t in cl ud ed in s ta tis ti ca l an al ys is 12 1 u nc ha ng ed (f em al es ); i nc re as ed (m al es ) o r 2 .5 ( 1. 54. 3) c as eco nt ro l st ud y r eg is tr y su bm is si on s fr om p ri va te pr ac ti ce s 20 9 do gs r ev ie w o f re gi st ry re co rd s, ju st go na de ct om iz ed y es or n o a ge -m at ch ed c on tr ol s, w ei gh t, br ee d an d ot he r va ri ab le s w er e in cl ud ed in s ta ti st ic al a na ly si s 58 u nc ha ng ed -- r et ro sp ec tiv e co ho rt s tu dy a ni m al s ad op te d fr om h um an e or ga ni za tio n 18 42 d og s o w ne r qu es tio nn ai re , re vi ew o f ho sp ita l re co rd s p ur eb re d or n ot , o w ne r su rr en de r or s tr ay in ta ke ty pe , t im e in s he lte r an d ot he r do gs in h ou se ho ld w er e in cl ud ed in st at is ti ca l a na ly si s, lo w nu m be r of a ff ec te d an im al s m ay h av e de cr ea se d st at is ti ca l po w er 14 5 in cr ea se d (f em al es an d ne ut er ed m al es co m pa re d to in ta ct m al e) n o di ff er en ce be tw ee n ne ut er ed an d in ta ct f em al es r r 2 .2 ( 1. 53. 0) ne ut er ed m al e re la ti ve to in ta ct m al e r r 2 .5 ( 2. 12. 9) ne ut er ed f em al e co m pa re d to in ta ct m al e c as e co nt ro l st ud y p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita l ( v m d b re gi st ry a nd a ni m al m ed ic al c en te r) 14 68 d og s r ev ie w o f ho sp ita l re co rd s s ev er al d if fe re nt c on tr ol gr ou ps u se d, go na de ct om iz ed o r no t, ag e an d br ee d in cl ud ed in s ta ti st ic al a na ly si s, m on th o f di ag no si s an al yz ed s ep ar at el y. o r = o dd s ra ti o   clinical theriogenology • volume 9, number 2 • june 2017205 t ab le 1 6. l it er at ur e re vi ew – c hr on ic k id ne y di se as e r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 12 3 in cr ea se d o r 3 .2 ( 2. 14. 7) , p< 0. 00 1 c as eco nt ro l st ud y r eg is tr y su bm is si on s fr om p ri va te pr ac ti ce s in th e u k 22 8 af fe ct ed do gs r ev ie w o f re gi st ry re co rd s, ju st go na de ct om y ye s or no c on tr ol s fr om r eg is tr y po pu la tio n, a ge , b re ed , se x, w ei gh t, in su ra nc e an d ot he rs w er e in cl ud ed in s ta tis ti ca l an al ys is 58 u nc ha ng ed -- r et ro sp ec tiv e co ho rt s tu dy a ni m al s ad op te d fr om h um an e or ga ni za tio n 18 42 d og s o w ne r qu es tio nn ai re , re vi ew o f ho sp ita l re co rd s p ur eb re d or n ot , o w ne r su rr en de r or s tr ay in ta ke ty pe , t im e in s he lte r an d ot he r do gs in h ou se ho ld w er e in cl ud ed in st at is ti ca l a na ly si s , l ow nu m be r of a ff ec te d an im al s m ay h av e de cr ea se d st at is ti ca l po w er o r = o dd s ra ti o clinical theriogenology • volume 9, number 2 • june 2017 206 t ab le 1 7. l ite ra tu re r ev ie w – a to pi c de rm at it is r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 12 4 in cr ea se d r r 3 .2 ( no c i re po rt ed ) c as eco nt ro l st ud y p at ie nt s ad m it te d to c or po ra te ve te ri na ry pr ac tic e 22 ,2 80 af fe ct ed do gs r ev ie w o f ho sp ita l re co rd s c on tr ol s fr om s am e ho sp ita l p op ul at io n, ag e, b re ed , s ex , r eg io n, ot he r di ag no se s in cl ud ed in s ta tis ti ca l an al ys is 58 u nc ha ng ed -- r et ro sp ec tiv e co ho rt s tu dy a ni m al s ad op te d fr om h um an e or ga ni za tio n 18 42 d og s o w ne r qu es tio nn ai re , re vi ew o f ho sp ita l re co rd s p ur eb re d or n ot , o w ne r su rr en de r or s tr ay in ta ke ty pe , t im e in s he lte r an d ot he r do gs in h ou se ho ld w er e in cl ud ed in st at is ti ca l a na ly si s , l ow nu m be r of a ff ec te d an im al s m ay h av e de cr ea se d st at is ti ca l po w er 12 5 in cr ea se d o r 2 .2 ( 2. 02. 5) fe m al es ; o r 1 .5 (1 .2 -1 .8 ) m al es r et ro sp ec ti ve co ho rt s tu dy p at ie nt s ad m it te d to a s pe ci al ty / re fe rr al h os pi ta l 90 ,0 90 do gs r ev ie w o f ho sp ita l re co rd s l ar ge s am pl e si ze a nd lo w p re va le nc e of di so rd er s in s am pl e po pu la tio n m ay h av e pr om ot ed a rt if ic ia l de si gn at io n of si gn if ic an ce . r r = r el at iv e ri sk , c i = c on fi de nc e in te rv al     clinical theriogenology • volume 9, number 2 • june 2017207 t ab le 1 8. l ite ra tu re r ev ie w – e pi le ps y r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 12 6 u nc ha ng ed - c as eco nt ro l st ud y r eg is tr y su bm is si on s fr om p ri va te pr ac ti ce s in th e u k 53 9 af fe ct ed do gs r ev ie w o f re gi st ry re co rd s, ju st go na de ct om y ye s or no a ge , s ex , b re ed , c ol or an d w ei gh t w er e in cl ud ed in s ta tis ti ca l an al ys is 12 8 d ec re as ed fr eq ue nc y (f em al es ), de cr ea se in c lu st er se iz ur es ( m al es ) p= 0. 00 7 in cr ea se d fr eq ue nc y of cl us te r se iz ur es in in ta ct f em al es , in ta ct m al es m or e lik el y to ha ve c lu st er se iz ur es o r 2 .3 (1 .4 -3 .9 ) cl us te r se iz ur es co m pa re d to ne ut er ed c as e se ri es c ha ri ty ve te ri na ry pr ac ti ce in u k 15 9 af fe ct ed do gs (f re qu en c y an al ys is ), 15 8 af fe ct ed do gs (s ev er it y an al ys is ) r ev ie w o f ho sp ita l re co rd s, ju st go na de ct om y ye s or no c on tr ol s un cl ea r, ju st co m pa re d ty pe s of se iz ur es ; a ge , b re ed , s ex w er e in cl ud ed in st at is ti ca l a na ly si s 12 7 in cr ea se d o r 1 .6 ( 1. 41. 9) , m al es a ls o at in cr ea se d ri sk c as eco nt ro l st ud y p at ie nt s er a su bm it te d fo r dr ug a na ly si s 12 60 d og s r ev ie w o f do cu m en ts ac co m pa ny in g su bm is si on , d v m in te rv ie w , j us t go na de ct om y ye s or no c on tr ol s fr om la rg e pr iv at e pr ac ti ce r eg is tr y, ag e, s ex a nd b re ed w er e an al yz ed s ep ar at el y o r = o dd s ra ti o   clinical theriogenology • volume 9, number 2 • june 2017 208 t ab le 1 9. l ite ra tu re r ev ie w – li fe sp an r e f e r e n c e n u m b e r v a r ia t io n w it h g o n a d e c t o m y s ig n if ic a n t r e s u l t s s t u d y t y p e s u b je c t s e l e c t io n s a m p l e s iz e d e t e r m in a t io n o f a g e a t g o n a d e c t o m y c o n t r o l s / a n a l y si s f o r c o n fo u n d in g f a c t o r s 12 9 in cr ea se d li fe sp an 23 % lo ng er (f em al es ), 1 8% lo ng er ( m al es ) c ro ss se ct io na l s tu dy p at ie nt s ad m it te d to c or po ra te ve te ri na ry pr ac tic e 2. 2 m il lio n do gs r ev ie w o f ho sp ita l re co rd s, ju st go na de ct om y ye s or no d es cr ip ti ve s ta ti st ic s on ly 13 0 in cr ea se d li fe sp an 26 .3 % lo ng er (f em al es ), 1 3. 8% lo ng er ( m al es ) r et ro sp ec ti ve co ho rt s tu dy h os pi ta ls p at ie nt s ad m it te d to sp ec ia lt y/ re fe rr al ho sp ita ls (v m d b r eg is tr y) - r ev ie w o f re gi st ry re co rd s a ge in cl ud ed in st at is ti ca l a na ly si s, br ee d an d w ei gh t ev al ua te d se pa ra te ly 13 4 d ec re as ed li fe sp an - c as eco nt ro l st ud y s pe ci fi c po pu la tio n of ex ce pt io na lly lo ng -l iv ed fe m al e do gs o f on e br ee d 83 lo ng liv ed d og s q ue st io nn ai re /in te rv i ew w ith o w ne r/ d v m 10 0 br ee dm at ch ed co nt ro ls w ith a ve ra ge li fe sp an , b re ed -s pe ci fi c st ud y (r ot tw ei le rs ) t ab le 2 0. h il ls c ri te ri a of c au sa ti on s tr en gt h – t he la rg er th e as so ci at io n, th e m or e li ke ly th er e is a tr ue c au se -a nd -e ff ec t. c on si st en cy – s tu di es p er fo rm ed b y di ff er en t p eo pl e lo ok in g at d if fe re nt p op ul at io ns h av e co ns is te nt fi nd in gs . s pe ci fi ci ty – t he re is n o co nf ou nd in g fa ct or th at is e qu al ly o r m or e li ke ly to h av e ca us ed th e ef fe ct . t em po ra li ty – t he e ff ec t a lw ay s oc cu rs a ft er th e ca us e. * b io lo gi ca l g ra di en t – g re at er e xp os ur e le ad s to g re at er in ci de nc e of th e ef fe ct . p la us ib il ity – t he p ro po se d ca us e an d ef fe ct m ak e se ns e w it h cu rr en t k no w le dg e. c oh er en ce – a ll f in di ng s (e pi de m io lo gi c, la bo ra to ry te st in g, e tc .) s up po rt th e ca us e an d ef fe ct . e xp er im en t – t he e ff ec t c an b e al te re d th ro ug h ju di ci ou s ex pe ri m en ta l t es ti ng o f th e ca us e. a na lo gy – a ll ot he r po ss ib le c au se s ha ve b ee n ta ke n in to c on si de ra ti on . * t hi s cr it er io n m us t a lw ay s be p re se nt to d em on st ra te c au sa ti on . clinical theriogenology • volume 9, number 2 • june 2017209 t ab le 2 1. d em on st ra ti on o f po te nt ia l i n fa ul ts o f da ta a na ly si s by e va lu at io n of e ff ec t o f go na de ct om y on a v ar ie ty o f di so rd er s di ag no se d at a ve te ri na ry te ac hi ng r ef er ra l h os pi ta l d is o r d e r p v a l u e o d d s r a t io m a m m a r y n e o p l a s ia 0. 7 1. 2 (0 .4 -5 .2 ) p r o s t a t ic n e o p l a s ia 0. 9 1. 1 (0 .1 -2 3. 4) t r a n s it io n a l c e l l c a r c in o m a 0. 1 4. 2 (0 .6 -8 2. 9) o s t e o s a r c o m a 0. 03 4. 1 (1 .0 -2 4. 4) h e m a n g io s a r c o m a 0. 1 2. 0 (0 .8 -5 .7 ) l y m p h o m a < 0 .0 01 4. 0 (1 .7 -1 0. 1) c u t a n e o u s m a s t c e l l t u m o r 0. 00 2 5. 4 (1 .6 -1 9. 9) o b e s it y 0. 04 3. 2 (1 .0 -1 2. 8) c r a n ia l c r u c ia t e l ig a m e n t i n ju r y < 0 .0 01 4. 8 (2 .1 -1 2. 0) p a t e l l a r l u x a t io n 0. 06 2. 6 (0 .9 -8 .3 ) h ip d y s p l a s ia 0. 9 1. 0 (0 .4 -3 .0 ) u r in a r y in c o n t in e n c e 0. 5 1. 3 (0 .6 -3 .2 ) d ia b e t e s m e l l it u s 0. 3 1. 5 (0 .6 -3 .9 ) c h r o n ic k id n e y d is e a s e 0. 6 1. 3 (0 .6 -3 .0 ) a t o p ic d e r m a t it is < 0 .0 01 3. 2 (1 .7 -5 .9 ) id io p a t h ic e p il e p s y / c l u s t e r s e iz u r e s 0. 6 1. 3 (0 .5 -4 .4 ) u r o l it h ia s is – c a l c iu m o x a l a t e a n d s t r u v it e 0. 07 2. 9 (0 .9 -1 1. 4)   clinical theriogenology • volume 9, number 2 • june 2017 210 t ab le 2 2. i m pa ct * on h ea lth o f m al e an d fe m al e do gs a ft er g on ad ec to m y (u se d w it h pe rm is si on ; r oo t k us tr it z m v . u se o f an im pa ct s co re to gu id e cl ie nt d ec is io nm ak in g ab ou t t im in g of s pa yca st ra ti on o f do gs a nd c at s. c li n t he ri o 20 12 ;4 :4 81 -4 85 ) d is o r d e r f e m a l e d o g m a l e d o g m am m ar y ne op la si a + 24 p yo m et ra + 10 0 s ur gi ca l c om pl ic at io ns -2 0 -1 6 o st eo sa rc om a -2 -2 h em an gi os ar co m a -2 -2 t ra ns it io na l c el l c ar ci no m a -7 -7 p ro st at e ne op la si a -3 t es ti cu la r ne op la si a + 5 u re th ra l s ph in ct er m ec ha ni sm in co m pe te nc e -6 6 b en ig n pr os ta ti c hy pe rt ro ph y + 36 8 r up tu re o f th e cr an ia l c ru ci at e li ga m en t -1 1 -1 1 o be si ty -1 4 -1 3 *i m pa ct s co re is d er iv ed f ro m in ci de nc e an d se ve ri ty o f di se as e. p os it iv e im pa ct s co re = b en ef it f ro m g on ad ec to m y, n eg at iv e im pa ct s co re = de tr im en t f ro m g on ad ec to m y clinical theriogenology • volume 9, number 2 • june 2017211 2017: major infectious abortion diseases of domestic animals and their zoonotic implications   major infectious abortion diseases of domestic animals and their zoonotic implications augustine t. peter department of veterinary clinical sciences, college of veterinary medicine, purdue university, west lafayette, in abstract convergence of people, animals, and our environment is inevitable in the present world creating a new dynamic. a dynamic in which the health of humans and animals and the wellbeing of the environment are inextricably linked carrying both benefits and risks. one such risk includes the impact of animal abortion diseases on human health. abortions that occur in animals not only result in severe economic loss but also pose a threat to people if these agents have zoonotic implications. knowledge of ‘nonabortion pathology’ of animals is important since these are the major lesions, more than the incidence of abortion, observed in people following infection by these agents. major infectious abortion diseases of domestic animals that have zoonotic importance are discussed. the list includes brucellosis, leptospirosis, coxiellosis, listeriosis, chlamydophiliosis and waddliosis, toxoplasmosis, and campylobacteriosis. this review summarizes the information on etiological agents, major clinical signs in animals and humans, and available vaccinations for animals. four key future directives are suggested to address the domestic animal abortion diseases from the perspective of their impact on human health. the directives are establishment of programs in comparative medicine, initiation of further research in the disease process in animals, continuation of spatial and temporal analyses of the diseases for a region, and finally completion of epidemiological representation of the disease. keywords: domestic animals; infectious abortion; bacterial agents; protozoan agents; zoonotic importance introduction human contact with animals has grown in part due to increases in demand for production of food of animal origin and in part due to increases in desire for animal companionship, and finally increases in participation in animal sporting events. undoubtedly, the importance of animal value in the current global economy forces more people to work with them in intensive animal agriculture programs and thereby increasing the chances of exposure to diseases of animal origin. zooeyia, the positive benefits on human health resulting from people interacting with pets, is well established. furthermore, there is a growing body of evidence documenting the improved physical, social, and mental health in people who share their households and environment with pets.1 these findings have created a mindset in more people to own pets and interact with them regularly. in summary, convergence of people, animals, and our environment is inevitable which creates a new dynamic in which the health of humans and animals and the well-being of the environment are inextricably linked2 which incidentally carries both benefits and risks.3 the above new dynamic brings challenges to human health, specifically, increasing the risk of zoonotic problems. rudolf virchow, a 19th century german physician, coined the term zoonosis4 and indicated that there is no dividing line between animal and human medicine and nor should there be one, although the object is different but the experience obtained constitutes the basis of all medicine. james law in 18784,5 mentioned that physicians are not to be left ignorant of the affection in the beast, and veterinarians of the same in man, they each miss the golden link which would reveal the true nature and dangers of the disease, and enable them to contend with it successfully; both branches of medicine (veterinary and human) suffer from separation; each is necessary to the rapid progress and highest advancement of the other. furthermore, it has become evident that our increasing interdependence with animals and their products has spurred the medical and veterinary professions to readdress the approach to veterinary medicine and human health.6 besides the existing zoonotic diseases there is also a possibility of emerging zoonotic pathogens. among the infectious agents considered to be emerging, 175 species from 96 genera have been documented, of which 132 (75%) species from 76 genera are zoonotic. clinical theriogenology • volume 9 number 1 • march 201759   the importance of zoonotic problems is further strengthened by the fact that zoonotic pathogens are twice more likely to be emerging than their non-zoonotic counterparts.7 miscarriage or abortion in people and animals has been with us since the dawn of civilization and one can find it mentioned in the pages of faith; ‘no one will suffer miscarriage’ (exodus 23:26) and ‘their cows calve without miscarriage’ (job 21:10). abortions that occur in animals not only result in economic loss but also pose a threat to people if these agents have zoonotic implications. besides causing abortion these agents also inflict damage to other organs in animals. knowledge of nonabortion pathology of animals is equally important since these are the major lesions recognized in people due to infection by these agents more than abortion. hence the purpose of this review is to address specifically the major abortion conditions that occur in domestic animals due to bacterial and protozoal agents of zoonotic importance. first, the organism, clinical signs of the condition, lesions, and preventive and diagnostic measures including the handling of these cases are reviewed. second, the clinical ramification observed in people due to these specific zoonotic pathogens is considered. finally, future directions in the study of ‘one health’ in the area of infectious abortion in animals are proposed. brucellosis animals brucellosis is caused by the microorganisms of the genus brucella which are gram-negative facultative intracellular bacteria that infect phagocytes. transmission of these organisms in general occurs via contact with infected fetal membranes, fetuses, fetal fluids, and vaginal discharges, with subsequent bacterial entry via ingestion, through mucous membranes of the oropharynx, upper respiratory tract, and conjunctiva or abraded skin. although there are many brucella organisms that currently exist, the focus of the discussion will be on four brucella organisms that cause abortion in domestic animals, hence, nonabortion causing brucella organisms in domestic and brucella organisms affecting nondomestic species will not be discussed. brucella melitensis contains three biovars, all of which can cause disease in goats and sheep despite their variable geographic distributions. the primary clinical signs in naturally infected sheep and goats include abortions, stillbirths, and birth of weak neonates. the condition significantly reduces production yields and can cause widespread infertility, posing extensive economic loss for producers on account of infected flocks. sheep and goats become infectious following abortion or parturition and can shed the organism for weeks (sheep) to months (goats) in vaginal discharges, semen, and milk. lifelong shedding in milk can occur, particularly, in goats. nursing offspring can become infected and shed organisms in their feces. in utero transmission is also possible. postmortem lesions include granulomatous inflammation of the reproductive tract, udder, supramammary lymph nodes, joints, and synovial membranes. necrotizing orchitis, epididymitis, seminal vesiculitis, and prostatitis are some of the lesions in males. aborted fetuses may be normal, autolyzed, or may have excess bloodstained fluids in the body cavities along with splenomegaly and hepatomegaly. inflammation of fetal membranes with edema and/or cotyledonary necrosis and thickened/leathery intercotyledonary spaces are some of the lesions. vaccines (e.g., rev 1 strain vaccine) are available and have been successfully used in control programs. in general, serological or intra-eyelid allergy testing followed by slaughter protocols are essential to control the disease. serological testing is primarily used for a presumptive diagnosis as they are not very specific. the card, rose bengal plate agglutination, and complement fixation tests are commonly used. gel diffusion or radial immunodiffusion tests can be used to distinguish vaccination from true infection. definitive diagnosis is by the isolation of the organism from the animal. samples from live animals should include milk samples, vaginal swabs, or semen and should be handled with precaution given the high pathogenicity in humans. brucella canis displays low mortality rates but high morbidity in breeding kennels or where large numbers of dogs are kept in close quarters. subclinical infections characterized by lymphadenopathy are clinical theriogenology • volume 9 number 1 • march 2017 60   very common despite brucella canis being the leading cause of spontaneous late term abortions in dogs. infection causes decreased fertility, early embryonic death, death of the fetus at virtually any part of gestation followed by abortion, stillbirths, and birth of weak puppies. in male dogs the pathogenesis includes orchitis, epididymitis, and testicular degeneration and atrophy. less common presentations include diskospondylitis, pyogranulomatous dermatitis, osteomyelitis, and endophthalmitis. serological tests used in the dog include 1) rapid-slide agglutination test (rsat), a highly sensitive test used for screening, 2) mercaptoethanol rsat, a specific test, 3) tube agglutination test, and 4) agar gel immunodiffusion (agid). a positive agid result is considered diagnostic,8 however, animals are often not positive on this test until approximately 12 weeks postinfection. two case reports8,9 described adequately the practical course of action when dealing with the type of tests, implication of test findings, handling of positive cases and kennel, and quarantine measures. the infected dog should be euthanized, other dogs in the kennel should be quarantined, and dogs that had contact with the female dog, contaminated tissues, or fluids (including urine, vaginal discharges, and aborted materials) should be serologically tested by the rsat.9 it is imperative that the kennel be quarantined during an outbreak of brucellosis and no dogs should enter or leave the premises until the disease is eradicated. space will not allow elaboration on the details of the comprehensive information provided in these case-reports and readers are encouraged to go over the excellent discussion points provided in these reports. since brucella canis is an intracellular organism it is difficult to eliminate it by administration of antimicrobials.10-12 furthermore, the bacteria is shed in large numbers in milk, as well as in post-abortion discharges for up to six weeks following abortion, which provides a continual source of infective material for other dogs.11 it is recommended that these dogs not be sold or placed in homes as pets.13 brucellosis is a reportable disease in some states.14 specific regulations can be obtained by contacting the department of agriculture and state veterinarian in the usa or similar regulatory offices in other countries. brucella abortus infection in cattle usually results in abortion after five months of pregnancy and occasionally in the birth of weak offspring. retained fetal membranes and post-abortion metritis are common. lesions include inflammation of fetal membranes, focal necrosis of thickened cotyledons with or without a yellow exudate. the fetus may show no gross lesions or may be autolyzed and in some cases there may be evidence of bronchopneumonia. calves vertically infected (or from milk) may remain serologically negative and show no sign of disease but serve to maintain the disease in the herd. infected cattle also show reduced milk production with or without an increased milk somatic cell count. other clinical signs include infertility in bulls and cows, and possible systemic illness in bulls characterized by anorexia, fever, depression, and/or orchitis. brucella ovis causes abortion in sheep15,16 albeit to a lesser degree. a yellowish purulent exudate may be seen, particularly, in the intercotyledonary areas. the fetal membrane is edematous, gelatinous, and thickened with the adherence of amnion to chorioallantois. the intercotyledonary areas are thickened with multifocal to coalescent areas of plaque-like thickening resembling yellow-white chamois leather. the cotyledons can be found in various stages of necrosis and detachment.17 typically brucella ovis infection in rams results in epididymitis, or orchitis. immunization of rams with brucella melitensis rev1 vaccine has been recommended for control of brucella ovis in some countries. diagnosis is via culture and serology although serology cannot distinguish animals exposed to infection or responding to vaccination. information on maternal serology is most useful from a nonvaccinated animal. the screening tests include the buffered acidified plate antigen test and the milk ring test, both of which are highly sensitive. important samples to test include fetal membranes, uterine fluid, milk, fetal lung/abomasa fluid of infected/aborted animals. modified live vaccine of the rough strain rb 51 interfere with serological testing. humans to this day, brucellosis remains a significant zoonosis in the developing world18-20 and is emerging or reemerging in many parts of the world.17 consumption of sheep or goat milk containing brucella melitensis is an important source of human brucellosis worldwide and has caused several clinical theriogenology • volume 9 number 1 • march 201761   outbreaks.21-24 human outbreaks related to aerosol transmission in laboratories have also been reported.25 although brucella melitensis infection is frequent among people (farmers and veterinarians) in developing countries who have close contact with infected animals, human outbreaks attributed to contact with infected animals have been rarely reported. the majority of human cases, and the more severe ones, are caused by brucella melitensis, although it has a more limited geographical distribution than brucella abortus. transmission of brucella melitensis from infected animals to humans may be either direct or indirect. the first mechanism involves the respiratory, conjunctival and cutaneous routes, and is more important in people in close contact with infected goats26 or sheep. conversely, indirect transmission is made possible in people by the consumption of unpasteurized goat milk or cheese by consumers who are not familiar with this type of infection. infection may result in encephalitis, spondylitis, endocarditis, and arthritis. an unusually high number of abortions has occurred among goats bred on a farm in argentina. shortly afterwards an outbreak of human brucellosis took place among farm workers. it was established that epidemic human infections by brucella melitensis may develop among people in contact with infected goat herds or goat manure and not necessarily consuming goat products such as milk and cheese.27 although still considered uncommon, increasing numbers of reports over the past decade have documented human infection with brucella canis causing endocarditis and peritonitis. the most common routes of transmission to humans are through contact with infected dogs or their secretions, fetal membranes or aborted puppies. diagnosis of brucella canis infection in humans is likely underdiagnosed due to a low index of suspicion, nonspecific symptoms (e.g., fever and fatigue), and lack of cross-reaction on common serologic tests using smooth brucella antigens. brucella canis infection in humans appears to be an emerging problem primarily in people of low socioeconomic status living in urban slums where there are high numbers of free-roaming dogs. epidemiologic investigations of a human brucella canis infection in a slum environment in argentina revealed 29 of 97 (29.9%) neighborhood dogs and 13 of 69 (18.6%) neighbors of the index case were serologically positive for brucella canis. likewise, a serologic survey in turkey documented the highest prevalence (7.8%) in patients from an urban area where stray dogs outnumbered pet dogs. some reports suggest that there may be a link between brucella canis infection and patients with immunosuppressive, inflammatory, or metabolic disorders, such as endocarditis, gaucher disease, human immunodeficiency virus, and guillain-barre´ syndrome.17 there is a possibility of clinical brucellosis in workers in vaccine production wherein m-strain of brucella canis is used.28 finally, it should be remembered that infected offspring from breeding facilities may result in widespread exposures to humans and other dogs and breeding facilities.29 infection is possible for people working in the plants making vaccine for brucella abortus30 which may result in prepatellar bursitits.31 brucella ovis is widespread in its occurrence in sheep but very much limited to this species and its zoonotic implications is limited. it is also possible for people to have meningoencephalitis following brucella suis infection.32 the diagnosis is made difficult since literally all the organs and systems are affected. the suspicion of infection by the above organisms, apart from epidemiological history and clinical symptoms, must therefore be supported by laboratory diagnostics that were reviewed recently.33 as with many classic zoonotic diseases, the role of veterinarians is critical for the detection and continued prevention and control. this role remains vital since there has been difficulty among human health-care professional in recognizing and diagnosing brucellosis in humans in nonendemic areas.34 leptospirosis leptospires are gram-negative, highly motile, obligate-aerobic spirochetes; these organisms are tightly coiled with characteristic hooked ends and are 0.2 µm in diameter and 6 to 20 µm in length; their taxonomy has been continually evolving. there are many different serovars associated with the leptospira interrogans, each having one or more maintenance hosts. pathogenic serovars include hardjo, grippotyphosa, pomona, canicola, and icterohemorrhagiae. these organisms can survive for prolonged periods of time in warm/wet/humid environments, especially in a neutral to alkaline environment like soil. while they are robust in the right kind of environment, they will not replicate outside of a host. clinical theriogenology • volume 9 number 1 • march 2017 62   transmission occurs via direct and indirect routes. direct transmission occurs through contact of mucous membranes, abraded skin, or even intact skin with infected urine, vaginal discharge, or other tissues from infected animals. however, indirect transmission from contact with contaminated soil, water, food or bedding is likely more common. in direct transmission entry of the organism into the blood stream is followed by rapid replication resulting in bacteremia and hematogenous spread to other organs including kidney, liver, spleen, central nervous system, eyes, and genital tract. despite positive immune response by the host, the bacteria can persist within the renal tubules, evading host immunity which results in persistent shedding of leptospires in the urine for weeks. in general, acute leptospirosis in animals is associated with fever, hemolytic anemia, hemoglobinuria, icterus, and high mortality. animals in ruminants, particularly in cattle, a range of reproductive disease may result in anything from infertility to abortion or birth of weak calves. abortion is a manifestation of chronic infection in adult cattle and usually occurs between four months of gestation to term. serovars hardjo, grippotyphosa, and pomona can cause abortions. abortion lesions include diffuse inflammation of fetal membranes described as avascular light tan cotyledons with yellowish intercotyledonary spaces and in most instances the fetus is autolyzed. small ruminants are less susceptible to leptospirosis, however, chronic infection can cause impaired fertility, abortions, neonatal deaths, and decreased production. a good control program can increase the productivity of small ruminants in developing tropical countries.35 leptospirosis is a common disease of swine throughout the world and can be a significant cause of reproductive loss36 with serovar pomona causing abortion, stillbirth or the birth of infected piglets.36 other serovars such as grippotyphosa, pomona, canicola, and icterohemorrhagiae can cause abortion. in companion animals such as cats and dogs,37 the clinical signs depend on whether the infection is acute, peracute or chronic. subacute infections are the most common and the animal is present with fever, anorexia, emesis, dehydration, and polyuric renal failure. reproductive problems are very minimal while the major serovars of leptospira interrogans have been isolated.38 the standard serological test used for diagnosis is the microscopic agglutination test and is considered positive when there is a fourfold rise in antibody titer over serial sampling for three weeks or a single high titer, if the animal has not been vaccinated in the last three months. without elevation of titer the animal may be shedding organisms hence caution is necessary while interpreting serology. in addition the vaccination status of the animal has to be considered. treatment involves early and aggressive antimicrobial therapy, particularly with penicillin or alternatively with doxycycline along with supportive care in accordance with the signs present. annual vaccination can prevent leptospirosis caused by serovars included in the vaccine and is recommended for dogs at risk of infection.39 humans leptospirosis is a worldwide zoonotic disease and its transmission is linked through multiple factors in the animal-human-ecosystem interface,40 chief among them is the local/regional animal demand for production.41,42 leptospira interrogans serovar canicola has been isolated in people among other serovars.42 data emerging from prospective surveillance studies suggest that most human leptospiral infections in endemic areas are mild or asymptomatic. development of more severe outcomes likely depends on three factors: epidemiological conditions, host susceptibility, and pathogen virulence.43 a zoonosis outbreak summary44 provided detailed information on the transmission, epidemiology, clinical signs, control and prevention, and public health implications. leptospirosis in humans is characterized by a wide variety of symptoms and a biphasic course of illness. the first phase corresponds to the multiplication and spread of the organism throughout the body. the second phase is characterized by the development of circulating antibodies and the detection of leptospires in the urine. the incubation period is typically one to two weeks (range, two to 30 days). most infections appear to be subclinical or so mild that they are never reported. among clinical cases, initial influenza-like signs during the first phase include fever, headache, chills, myalgia, and occasionally a maculopapular skin rash and conjunctival clinical theriogenology • volume 9 number 1 • march 201763   suffusion (i.e., redness of the conjunctiva without inflammatory exudates). this phase is followed by a one to three day period of defervescence and symptomatic improvement. leptospirosis is considered the most widespread zoonosis in the world. it has a global distribution with a higher incidence in the tropics and subtropics, ranging from 10 to 100 human cases per 100,000 individuals.45 in addition to their involvement in the prevention, diagnosis and treatment of leptospirosis in animals, veterinarians have an important role in public health by providing guidance and information on risk factors, prevention, and control measures.46 coxiellosis coxiellosis is caused by the gram-negative, obligate intracellular bacterium coxiella burnetii. the term ‘q fever’ has been adapted in veterinary medicine although the ‘query fever’ or ‘q fever’ refers to a febrile illness originally observed in abattoir workers in australia. this terminology has been maintained but the more appropriate term might be coxiellosis since fever is only one of the clinical signs.47 the diagnosis is often overlooked due to its non-specific presentation both in animals and humans. as a result the true worldwide incidence is not known. coxiella appears in three different morphological forms: large cell variants, small cell variants, and small dense cells which can be differentiated by their morphology, physical and chemical resistance, and antigenic and metabolic characteristics. the last two are considered to be the persistent forms in the host and are responsible for the high resistance of the organism to environmental stress. in addition, the organism displays antigenic variation by appearing in two infectious forms, phase i, a highly virulent one with lipopolysaccharide and phase ii, a less virulent form with atrophied lipopolysaccharide. animals coxiella burnetii infection is reported worldwide with the apparent prevalence higher in cattle than in small ruminants.48 however, the presence of the organism in dairy herds has not been clearly demonstrated to negatively affect reproductive performance.49 it should be noted that cattle can be one of the important reservoirs for this organism in some parts of the world.50 the problems in small ruminants have been reviewed51 and it is established that infections in goats and sheep are usually asymptomatic during the non-breeding period. the most common clinical manifestations appear during late pregnancy until lambing, i.e., abortion, stillbirth, and the delivery of weak offspring. other symptoms such as pneumonia, conjunctivitis, and hepatitis occur rarely in infected animals. infected female animals shed large quantities of bacteria into the environment in the course of abortion or normal delivery, not only through the allantoic and amniotic membranes and their fluids but also through urine and feces. shedding occurs via vaginal mucus and milk.52 in contrast to ewes, goats may experience reproductive failure and they shed the organism in two successive parturitions after an infection. goats remain chronically infected. diagnosis is generally undertaken only if abortions are observed. the detection of coxiella burnetii dna in the placenta and vaginal mucus by pcr has radically changed the diagnosis of coxiellosis. several pcr kits are available and provide a specific, sensitive, and rapid tool for the detection of the organism in various clinical samples. real-time pcr kits are available for quantification and identifying animals shedding the organism.53 the phase 1 vaccination has potential for the vaccination of not only infected flocks, but also nearby flocks, to reduce the risk of disease dissemination; prevent abortion and greatly decrease the shedding of the organism in milk.53 recombinant vaccines have been developed in experimental conditions and have great potential for the future.54 humans the organism posed a considerable risk to people because of its environmental stability, aerosol transmission possibility, and its capacity to use animal reservoirs. it has become a major health problem in certain parts of the world55-59 and a relatively minor problem in other parts. inhaling aerosols, which have been contaminated with the organisms, is the most important route of human infection. it is also transmitted, less commonly, through the consumption of raw milk and dairy products. clinical signs include fever,60 pneumonia, headache, and weakness. chronic infection can result in severe valvular clinical theriogenology • volume 9 number 1 • march 2017 64   endocarditis, granulomatous hepatitis, and rarely in osteomyelitits. human-to-human transmission (following child birth/autopsy) has been reported, as has infection following tick bites. studies confirmed the association between coxiellosis and spontaneous abortion.61,62 although seropositivity is observed and the condition has accounted for a notable proportion of undifferentiated febrile episodes and infective endocarditis in humans in africa, further studies are needed to determine the exact incidence and its impact in animals and humans.63 recently coxiellosis has become a threat to usa military personnel who are deployed in the middle east64 due to possible exposure to livestock. the signs include flu-like illness that responds to antibiotic therapy. there is a potential for infection among farm workers dealing with dairy cattle and individuals working in the milk processing industry with dairy products. listeriosis listeriosis is caused by listeria monocytogenes, a saprophytic gram-positive facultative anaerobic and intracellular rod-shaped bacterium. the bacterium can be classified into four genetic lineages (i-iv) and 13 serotypes and is ubiquitous in the environment. listeria monocytogenes is among the most versatile, adaptive, and widely distributed pathogenic bacteria.65 animals in general listeriosis causes abortion in animals. bovine listeriosis is usually acquired by ingestion of poorly fermented silage or by ingestion of organisms from aborted tissues. infection can also arise from ingestion of contaminated soil, milk, silage, feces and the organism can survive in the proper environment for years. the lineage of the organism may determine whether it can cause abortion since certain lineages are associated with the encephalitis while other lineages can cause major forms of listeriosis in cattle including abortion.66 clinical presentation involves abortion, usually, occurring in the third trimester. abortions are sporadic but abortion storms may be noticed in some herds. aborting cattle may or may not show non-specific signs of systemic illness, however, retained fetal membranes are common. fetuses are autolyzed and display a fibrinous polyserositis and white necrotic foci in liver and/or the cotyledons. similar to cattle, listeriosis infections in small ruminants occur mostly due to contaminated feed. acidified silage provides an excellent survival medium and a substrate for multiplication of the organism. feeding of contaminated grass silage in sheep resulted in abortive, encephalitic, and septicemic forms of listeriosis.67 listeriosis is rarely identified in cats and dogs, however, it can infect these species. clinical disease is characterized by nonspecific signs such as vomiting, diarrhea, and fever with neurologic and dermatologic signs in rare instances. abortion has been reported in a dog.68 humans in people, it is recognized as a life-threatening pathogen, poses severe risk during pregnancy causing abortion, fetal death or neonatal morbidity as a result of septicemia and meningitis. nearly all (99%) cases of listeriosis in humans in the usa are thought to result from the consumption of contaminated foods.69,70 the chance of humans contacting infection via the biological materials of an aborted animal is very minimal. the most common route of infection for people is via ingestion of contaminated ready-to-eat food products, soft cheeses, and deli meats. these observations may be true for all over the world. clinical listeriosis is associated with high mortality, especially in immunocompromised patients, pregnant women, neonates, and the elderly. a recent review summarized listeriosis in humans and domestic animals during pregnancy and described the animal models used to study the pathogenesis and immune response to listeria monocytogenes infection.71 the health concern of listeriosis during pregnancy has been reviewed.72 the organism has been isolated from cervical and vaginal discharges of women with a history of abortions, miscarriages, stillbirths or neonatal deaths. the organism has the ability to induce its own entry into host cells such as macrophages, epithelial cells, and endothelial cells of the gastrointestinal tract.73 cells surviving the low ph of the stomach, pass through the small intestine and once the organisms arrive at the mesenteric lymph nodes they disseminate to the clinical theriogenology • volume 9 number 1 • march 201765   spleen and liver. from these organs the organism can reach the brain or the placenta, causing, respectively, infections of the central nervous system (cns), mostly in immunocompromised patients, and intrauterine/cervical infections in pregnant women.74 encephalitis, ataxia, septicemia, and meningitis are other clinical signs. diagnosis of listeriosis abortion both in animals and in humans is based on the isolation of the organism and with pcr. currently, the real-time pcr diagnosis has become much easier further strategies for vaccine development and novel therapies for control of listeriosis have been proposed.75 chlamydophilosis and waddliosis as per the recent taxonomy,76 organisms belonging to the order chlamydiales are responsible for causing abortion in domestic animals and these agents have zoonotic implications. the first organism, chlamydophila abortus, belongs to the family chlamydiaceae and genus chlamydophila and causes abortion in cattle,76 sheep,77 goats, horses, and pigs. chlamydophila abortus is a major cause of abortion in small ruminant-rearing regions of the world, with the exception of australia and new zealand.78 the second organism is waddlia chondrophila that belongs to the family waddliaceae and genus waddlia causes abortion in cattle. animals usually, abortion in small ruminants is associated primarily with chlamydophila abortus and rarely with chlamydophila pecorum.79 lesions caused by the latter organism to the fetus included moderate fetal anasarca, intermuscular edema in the hindquarters, brachygnathia, and palatoschisis. placental lesions included fibrinosuppurative exudate that covered the cotyledons and extended to the inter cotyledonary areas.79 early investigations into the pathogenesis of this disease in sheep revealed that sheep typically aborted late in gestation or gave birth to weak or stillborn lambs.80,81 progression of the disease follows placental colonization with subsequent inflammation and necrosis of the maternal-fetal junctions.82 in both natural and experimental infections, ewes that experienced abortion or the birth of weak lambs were shown to be protected against further pregnancy failure due to this organism81,83 although apparently protected, such ewes continued to excrete the organism during the peri-ovulatory stage of subsequent estrous cycles84 and venereal transmission is possible.84 the most noticeable lesions included placentitis with a variable number of necrotic cotyledons.80-85 one report documented that the pathologic changes in the placenta were evident after 90 days gestation.82 these changes seldom affected the entire placenta and the peripheral regions were delineated by zones of hyperemia and hemorrhage. it has been suggested that infection may precipitate premature labor by altering placental steroid and prostaglandin release.86 chlamydophilal infections in cattle cause abortion, polyarthritis, encephalomyelitis, keratoconjuctivits, pneumonia, fever, depression, nasal secretion, coughing, dyspnea, enteritis, hepatitis, vaginitis, endometritis, infertility, and chronic mastitis.87 organisms responsible are chlamydophila abortus, c. pecorum, and c. psitacci. data published in the last two decades suggest a high seroprevalance of chlamydial infection (primarily for c. abortus and c. pecorum) in herds worldwide with seropositivity at a herd level ranging from 45% to 100%.87 c. abortus is primarily associated with the genital tract and mammary tissue; c. pecorum with pulmonary, joint, intestinal, ocular, and central nervous system disease and c. psittaci has been identified in samples from the respiratory and genital tracts. a key advance in the laboratory diagnosis of chlamydophilal infections has been the development of tests that do not require the agent to be viable,87 the detection of chlamydophilal non-replicating sporelike elementary bodies of approximately 0.3 µm diameter. the standard method for detecting antibodies is the complement fixation test using crude or partially purified preparations of the organism-specific lipopolysaccharide. numerous elisa methods have been used but due to cross-reactivity between species no commercially available test is able to differentiate antibodies at the species level. for example, the direct fluorescent antibody test cannot distinguish between c. pecorum and c. abortus because of the antigenic cross reactivity.79 clinical theriogenology • volume 9 number 1 • march 2017 66   humans infection with chlamydophila abortus can result in acute and sometimes life-threatening illness.87 similar to ewes, persistent infection leading to infertility in women has been recognized.88 it is recognized as an occupational hazard. women who work with sheep have suffered sporadic, documented cases of abortion76 and the presence of antibody has been characterized. 89 although there is anecdotal evidence linking atypical respiratory disorders or asthma-like symptoms in farmers working with chlamydia-infected herds, proof of cause and effect remains elusive. based on the current evidence, the zoonotic potential of chlamydophilal found in cattle would appear minimal.87 toxoplasmosis toxoplasma gondii is an extremely successful obligate intracellular protozoan parasite that infects almost all species of mammals and birds on all continents. historical facts have been reviewed.90 a key factor to understand the biology and clinical relevance of this organism is to know its parasitic life cycle. toxoplasma gondii exists in different stages: oocysts are the parasite’s sexual cycle in the intestine of its definitive host, the felidae (cat family). oocysts are excreted in cat feces and following sporulation in the environment, sporozoites become infective. upon oral uptake of sporulated oocysts by new hosts, sporozoites transform to the invasive tachyzoites. tachyzoites actively penetrate all nucleated cells and replicate rapidly in an intracytoplasmic vacuole. following repeated intravacuolar replication, host cells are disrupted and tachyzoites invade neighboring cells. the tachyzoites cause tissue destruction and are therefore responsible for clinical manifestations of the disease including abortion. the ensuing immune response is accompanied by the transformation of tachyzoites into slowly replicating intracellular bradyzoites that form persisting cysts. tissue cysts become infective to intermediate and definitive hosts via their consumption in meat and tissue. so, entry of sporulated cysts from the environment and tissue cysts from the consumption of tissue of animals enable the parasite to maintain its sexual cycle and incidentally be responsible for the clinical disease including abortion. animals toxoplasma gondii causes abortion in cats and small ruminants, and particularly, in immune compromised animals. severe disease may be seen in transplacentally or lactationally infected kittens. general clinical manifestations include fever, lethargy, anorexia, pneumonia, hepatitis, stiff gait, lameness, or cns signs from encephalitis. toxoplasma gondii has caused considerable reproductive wastage in small ruminants in many parts of the world, particularly, in developing countries.91,92 its importance became known when it was found to cause abortion storms in sheep in 1957.93 most sheep and goats acquire infections via ingestion of forage contaminated with cat feces (sporulated oocysts). while these animals’ fetuses can be infected transplacentally, transmission via this route is much less common. under most circumstances, clinical disease in infected/exposed dams is rare with the only observable manifestation being abortion. these animals are typically asymptomatic before and after abortion. abortion can be sporadic among a few animals to a herd-wide abortion storm characterized by mummification, stillbirths, and birth of weak progeny in those dams infected later in gestation. small white necrotic foci are noticed in the cotyledons and typical fetal brain lesions (gliosis of cerebral white matter) are recognized. in small ruminants, control can be instituted through prophylactic feeding of medications like monensin during gestation or through use of the modified live vaccine toxovax (s48 strain). its use results in reduction of tissue cyst development. humans toxoplasma gondii’s human importance remained unknown until 1939 when the organism was identified in tissues of a congenitally infected infant.93 animals are key to human toxoplasmosis.94 its ubiquitous distribution and its high prevalence makes it an important zoonotic agent. approximately 2530% of the human population is infected with toxoplasma gondii. humans get infected by consumption of undercooked cyst-contaminated meat products or by sporulated cysts. relatively high seroprevalences in animals meant for human consumption suggests the potential risk factor posed by food animals. clinical theriogenology • volume 9 number 1 • march 201767   immunity to toxoplasma gondii, congenital toxoplasmosis, ocular toxoplasmosis, and the impact of the strain on the course of human toxoplasmosis have been adequately described.94 latent toxoplasmosis is characterized by the life-long presence of cysts in different host tissues, including the nervous system, and by the presence of anamnestic toxoplasma igg antibodies in the serum. latent toxoplasmosis is believed to increase the risk of conditions such as schizophrenia and parkinson’s disease.95 the prevalence of latent toxoplasmosis is found to be higher in female (23.7%) than in male (10.9%) patients in a study conducted in prague96 and this difference was speculated to be due to the difference in the gender specific hormones. accidental ingestion of toxoplasma gondii infected meat products from the domestic animals by women during pregnancy can cause abortion and congenital defects.97 the risk of ingestion is higher from uncontrolled water supplies from the wells and springs98 and from the soil contamination.99 there is a possibility of sexual transmission.100 the incidence in pregnant women may be influenced by factors such as the trimester of pregnancy.101 high prevalence is noticed in women who had a repeated history of abortion.102-104 estimation of serorprevalence of anti-toxoplasma gondii antibodies and dna of women with spontaneous abortion may be a way to determine the impact of this disease on abortion.105 only 10-15% of human cases are associated with clinical signs. immunocompetent individuals are usually asymptomatic but may present with clinical signs such as lymphadenomegaly, fever, or malaise. more severe forms of disease include encephalitis, myocarditis, hepatitis, and reticulocarditis and these are typically manifested in immunocompromised individuals as latent infections. transplacental transmission results in severe neurological or ocular disease in the developing fetus that may not be apparent at birth. as the infant matures clinical signs develop and may involve blindness, retinochorditis, deafness, mental retardation, learning disabilities, and psychomotor deficiencies. abortions106,107 and neonatal deaths occur in 10% of the cases in pregnant women. toxoplasmosis continues to be a significant public health problem all over the world including the usa.108 what was said earlier is also true today the major ways to reduce the transmission of toxoplasma gondii in people is to reduce fecal shedding by cats and by killing tissue cysts in meat.109 the new trend in the production of free-range organically raised meat may increase the risk of toxoplasma gondii contamination of meat. foodborne transmission can be prevented by production practices that reduce toxoplasma gondii in meat,110 adequate cooking of meat, washing of raw fruits and vegetables, prevention of cross contamination in the kitchen, and measures that decrease spread of viable oocysts into the environment.111 diagnostic tests include the sabin-feldman dye test, igm antibody detection by elisa test, direct agglutination test, and detection of toxoplasma gondii dna test.93 campylobacteriosis campylobacter organisms are gram-negative, curved, slender, comma or spiral shaped rods which appear as ‘seagull wings’ in silhouette. ultrastructurally, this bacterium has a double layered outer membrane with loosely packed hexagonal subunits and a three-layered cell wall compromising an outer lipoprotein, a middle lipopolysaccharide and an inner mucopeptide layer. campylobacter fetus compromises two subspecies, campylobacter fetus subspecies venerealis and campylobacter fetus subspecies fetus. these two subspecies mainly differ in epidemiological, phenotypic, and molecular characteristics including molecular mechanisms.112 these two campylobacters along with campylobacter jejuni are responsible for causing abortion in domestic animals. animals in cattle, the organism campylobacter fetus subspecies venerealis is highly adapted to the genital tract and is transmitted venereally by carrier bulls. the cattle industry refers to this venereal disease caused by this organism as ‘vibrio’.113 the other subspecies of campylobacter fetus (campylobacter fetus subspecies fetus) and campylobacter jejuni are also responsible for causing abortion. in addition to causing abortion these agents are associated with lowered fertility, embryo mortality, repeated return to service, reduced pregnancy rates, and extended calving intervals. the incidence is highest in developing countries where natural breeding of cattle is widely practiced.114 although vaccine against campylobacter fetus subspecies venerealis is available115,116 the major focus is to provide pathogen-free clinical theriogenology • volume 9 number 1 • march 2017 68   semen for the ai industry. successful diagnosis and mitigation are dependent on isolation of infected animals and consistent reporting activity and diagnostic tests include culture and pcr.116 humans campylobacter fetus subspecies venerealis has caused abortion in people117 and campylobacter fetus has caused bacterial meningitis.118 apart from causing abortion in animals and rarely in people, the intestinal campylobacter species are relevant from the aspect of public health since they cause gastroenteritis in people. discussion of all the campylobacters is beyond the scope of this review. however, it has to be pointed out that there is a greater need for a ‘one health’ approach119 to combat campylobacteriosis in general and it will continue to challenge the global heath in the years to come.120 conclusions zoonotic infectious diseases are an important concern to humankind due to the ever increasing contact between animals and humans. today, approximately 75% of newly emerging infectious diseases (eids) are zoonoses that result from various anthropogenic, genetic, ecologic, socioeconomic, and climatic factors. these interrelated driving forces make it difficult to predict and to prevent zoonotic eids. although significant improvements in environmental and medical surveillance, clinical diagnostic methods and medical practices have been achieved in recent years, zoonotic eids remain a major global concern, and such threats are expanding, especially in less developed regions.121 five decades ago infectious causes of abortion common in animals and humans were reviewed.122 however, currently, most of the agents that cause abortion in domestic animals cause systemic diseases in humans and abortion only in some instances. the bacterial and protozoan agents that cause abortion in domestic animals were identified and immunization methods were developed for some of them. the table summarizes the salient points of these agents and their clinical impacts on animals and people. as new information emerge this table may have to be updated. research has clearly and convincingly established that most of the abortion agents of animals are capable of establishing the disease process in immunocompromised people; a plausible reason for higher incidence of these animal illnesses in people in the developing world. improvement of global health depends on addressing and remedying this very important factor. further efforts are needed to translate our knowledge on abortion causing agents in animals to improve the health management of animals and people. in this regard, certain steps are suggested. first, the concept of ‘one health’ has to move further by breaking down the historical separation of humans and animals and embrace the development of comparative medicine when it comes to pathology123 of abortion diseases. second, the government agencies must continue to provide financial support for the colleges of veterinary medicine and other agencies for studies to be continued in animal abortion diseases. this is one of the ways to realize the far-reaching benefits of the investments made in veterinary medical education and research. such benefits include the well-being of people, families, and society as well as the economy.124 third, a spatial and temporal analyses of a particular zoonotic abortion disease for a region can help to fill in the current gaps in knowledge for public health practitioners to provide vaccination strategies.125 fourth, the most complete epidemiological representation of any reemerging threat to animals and people by the agents that cause abortion in animals is necessary to combat any disaster that may be on the horizon.126 in this context creation of a ‘global signaling surveillance system’ is worthy of consideration.127 furthermore, bringing together the policy makers and the scholars who have identified the ‘local spatial heterogeneity’ in the distribution of zoonotic diseases is paramount in128 one health initiatives. conflict of interest the author declares that he has no conflict of interest nor he should be perceived as prejudicing the impartiality of research literature in not citing all publications in this review. this article does not contain any studies with human participants or animals performed by the author. clinical theriogenology • volume 9 number 1 • march 201769   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infected with an ovine abortion strain of chlamydia psittaci. prostaglandins leukot essent fatty acids 1989:37;203-211. 87. reinhold p, sachse k, kalteannboeck b: chlamydiaceae in cattle: commensals, trigger organisms, or pathogens? vet j 2011;189:257-267. 88. papp jr, shewen pe: chlamydia psittaci infection in sheep: a paradigm for human reproductive tract infection. j reprod immunol 1997;34:185-202. 89. smith iw, morrison cl, lee rj et al: serological survey of chlamydial antibody in post-natal sera. j infect 1997;35:277-282. 90. innes ea: a brief history and overview of toxoplasma gondii. zoonoses public health 2010;57:1-7. 91. onyiche te, ademola io: seroprevalence of anti-toxoplasma gondii antibodies in cattle and pigs in ibadan, nigeria. j parasit dis 2015;39:309-314. 92. rahimi mt, daryani a, sarvi s, et al: cats and toxoplasma gondii: a systematic review and meta-analysis in iran. onderstepoort j vet res 2015;82:823, doi:10.4102/ojvr.v82i1.823 93. dubey jp: the history of toxoplasma gondii--the first 100 years. j eukaryot microbiol 2008;55:467-475. 94. schlüter d, däubener w, schares g, et al: animals are key to human toxoplasmosis. int j med microbiol 2014;304:917-929. 95. flegr j: how and why toxoplasma makes us crazy. trends parasitol 2013;29:156-163. 96. flegr j, stříž i: potential immunomodulatory effects of latent toxoplasmosis in humans. bmc infect dis 2011;11:274, doi: 10.1186/1471-2334-11-274. 97. shahiduzzaman m, islam r, khatun mm, et al: toxoplasma gondii seroprevalence in domestic animals and humans in mymensingh district, bangladesh. j vet med sci 2011;73:1375-1376. 98. ertug s, okyay p, turkmen m, et al: seroprevalence and risk factors for toxoplasma infection among pregnant women in aydin province, turkey bmc public health 2005;5:66, doi: 10.1186/1471-2458-5-66. 99. gotteland c, gilot-fromont e, aubert d, et al: i spatial distribution of toxoplasma gondii oocysts in soil in a rural area: influence of cats and land use. vet parasitol 2014;205;629-637. 100. flegr j, klapilová k, kaňková s: toxoplasmosis can be a sexually transmitted infection with serious clinical consequences. not all routes of infection are created equal. med hypotheses 2014;83l:286-289. 101. findal g, barlinn r, sandven i, et al: toxoplasma prevalence among pregnant women in norway: a cross-sectional study. apmis. 2015;123:321-325. 102. altintas n, kuman ha, akisu c, et al: toxoplasmosis in last four years in agean region, turkey. j egypt soc parasitol1997;27:439-443. 103. zargar ah, wani ai, masoodi sr, et al: seroprevalence of toxoplasmosis in women with recurrent abortions/neonatal deaths and its treatment outcome. indian j pathol microbiol 1999;42:483-486. clinical theriogenology • volume 9 number 1 • march 2017 72   104. qublan hs, jumaian n, abu-salem a, et al: toxoplasmosis and habitual abortion. j obstet gynaecol 2002;22:296298. 105. vado-solís ia, suárez-solís v, jiménez-delgadillo b, et al: toxoplasma gondii presence in women with spontaneous abortion in yucatan, mexico. j parasitol 2013;99:38-35. 106. jenum pa, stray-pedersen b, melby kk, et al: incidence of toxoplasma gondii infection in 35,940 pregnant women in norway and pregnancy outcome for infected women. j clin microbiol 1998;36;2900-2929. 107. zhou p, chen z, li hl, et al: toxoplasma gondii infection in humans in china. parasit vectors 2011;4:165. 108. hill de, dubey jp: toxoplasma gondii prevalence in farm animals in the united states. int j parasitol 2013;43:107113. 109. dubey jp: strategies to reduce transmission of toxoplasma gondii to animals and humans. vet parasitol 1996;64:6570. 110. paştiu ai, ajzenberg d, györke a, et al: traditional goat husbandry may substantially contribute to human toxoplasmo sis exposure. j parasitol 2015;101:45-49. 111. jones jl, dubey jp: foodborne toxoplasmosis. clin infect dis 2012;55:845-851. 112. sprenger h, zechner el, gorkiewicz g: so close and yet so far-molecular microbiology of campylobacter fetus subspecies. eur j microbiol immunol (bp) 2012;2:66-75. 113. bondurant rh: venereal diseases of cattle: natural history, diagnosis, and the role of vaccines in their control. vet clin north am food anim pract 2005;21:383-408. 114. mshelia gd, amin jd, woldehiwet z, et al: epidemiology of bovine venereal campylobacteriosis: geographic distribution and recent advances in molecular diagnostic techniques. reprod domest anim 2015;45:221-230. 115. givens md: a clinical, evidence-based approach to infectious causes of infertility in beef cattle. theriogenology 2006;66:648-654. 116. michi an,favetto ph, kastelic j, cobo er: a review of sexually transmitted bovine trichomoniasis and campylobacteriosis affecting cattle reproductive health. theriogenology 2016;85:781-791. 117. sauerwein rw, bisseling j, horrevorts am: septic abortion associated with campylobacter fetus subspecies fetus infection: case report and review of literature. infection 1993;21:331-333. 118. samakar av, brouwer mc, van der ende a, et al: campylobacter fetus meningitis in adults. report of 21 cases and review of literature. systemic review and meta-analysis. 2016;95:1-5. 119. gölz g, rosner b, hofreuter d, et al: relevance of campylobacter to public health--the need for a one health approach. int j med microbiol 2014;304:817-823. 120. kaakoush no, castaño-rodríguez n, mitchell hm, et al: global epidemiology of campylobacter infection. clin microbiol rev 2015;28:687-720. 121. gebreyes wa, dupouy-camet j, newport mj, et al: the global one health paradigm: challenges and opportunities for tackling infectious diseases at the human, animal, and environment interface in low-resource settings. plos negl trop dis. 2014;8: e3257. doi: 10.1371/journal.pntd.0003257. 122. dennis sm: comparative aspects of infectious abortion diseases common to animals and man. int j fertil 1968;13:191-197. 123. sundberg jp, schofield pn: one medicine, one pathology, and the one health concept. j am vet med assoc 2009;234:1530-1531. 124. moore rm, hubbell ja, king lj: the role of the colleges of veterinary medicine in realizing the research mission of land-grant institutions to promote animal, human, and environmental health. j am vet med assoc 2012;241:869-874. 125. hennebelle jh, sykes je, carpenter te, et al: spatial and temporal patterns of leptospira infection in dogs from northern california: 67 cases (2001-2010). j am vet med assoc 2013:242;941-947. 126. rodríguez-prieto v, vicente-rubiano m, sánchez-matamoros a, et al: systematic review of surveillance systems and methods for early detection of exotic, new and re-emerging diseases in animal populations. epidemiol infect 2015;143:2018-2042. 127. asokan gv, kasimanickam rk, asokan v: surveillance, response systems, and evidence updates on emerging zoonoses: the role of one health. infect ecol epidemiol 2013:3, doi: 10.3402/iee.v3i0.21386. 128. blackburn jk,kracalik it,fair jm: applying science: opportunities to inform disease management policy with cooperative research within a one health framework. front public health 2016 jan 8;3:276. doi: 10.3389/fpubh.2015.00276. ecollection 2015. clinical theriogenology • volume 9 number 1 • march 201773   table. summary of major infectious abortion diseases of domestic animals: etiological agents, major clinical signs in animals and humans, and available vaccinations for animals. name of the disease organism(s) responsible major clinical signs in animals major clinical signs in humans animal vaccinations brucellosis brucella melitensis small ruminants abortion, stillbirths, birth of weak neonates. epididymitis, orchitis, prostatitis, and seminal vesiculitis encephalitis, spondylitis, endocarditis, and arthritis rev 1 strain brucella canis dogs decreased fertility, early embryonic death, abortion, stillbirth, birth of weak puppies. orchitis, epididymitis, testicular degeneration and atrophy. fever, fatigue, endocarditis, peritonitis brucella abortus cattle abortion, retained fetal membranes, and metritis. infertility, systemic illness, and orchitis. prepatellar bursitis modified live vaccine of the rough strain rb 51 brucella ovis abortion. epididymitis and orchitis. meningoencephalitis rev 1 strain leptospirosis leptospira hardjo, l. grippotyphosa, l. pomona, l. canicola, l. icterohemorrhagiae cattle infertility, abortion, birth of weak calves sheep and goat impaired fertility, abortion, neonatal death, decreased production dogs and cats fever, anorexia, emesis, dehydration, and polyuric renal failure fever, headache, chills, myalgia, conjunctivitis, flulike symptoms, liver or kidney failure food animals (cattle, swine, small ruminants) are vaccinated and annual vaccination is recommended for at-risk dogs. commercial vaccines are available and the serotypes in the vaccine differ between species. coxiellosis coxiella burnetii sheep abortion, stillbirth, weak offspring, pneumonia, conjunctivitis, hepatitis goat reproductive failure fever, pneumonia, headache, weakness, endocarditis, hepatitis. listeriosis listeria monocytogenes cattle abortion, retained fetal membranes dogs vomiting, diarrhea, fever, abortion abortion, intrauterine/cervical infections, encephalitis, ataxia, septicemia, meningitis chlamydophilosis and waddliosis small ruminants chlamydophila abortus waddlia chondrophila small ruminants late term abortion, weak or stillborn lambs acute, some-times lifethreatening illness, respiratory disorders, and commercial vaccines are available clinical theriogenology • volume 9 number 1 • march 2017 74   cattle chlamydophila abortus, c. pecorum, c. psitacci, and chlamydia suis. cattle abortion, polyarthritis, encephalomyelitis, keratoconjunctivitis, pneumonia, fever, depression, nasal secretion, coughing and dyspnea, enteritis, hepatitis, vaginitis and endometritis, and infertility and chronic mastitis abortion toxoplasmosis toxoplasma gondii general clinical manifestations include fever, lethargy, anorexia, pneumonia, hepatitis, stiff gait, lameness, or cns signs from encephalitis. abortion storms in small ruminants lymphadenomegaly, fever, or malaise, encephalitis, myocarditis, hepatitis, and reticulocarditis, neurological or ocular disease in the developing fetus, abortions small ruminants modified live vaccine toxovax (s48 strain) campylobacteriosis campylobacter fetus subspecies venerealis, campylobacter fetus subspecies fetus, campylobacter jejuni cattle abortion, lowered fertility, embryo mortality, repeated return to service, reduced pregnancy rate, extended calving intervals. gastroenteritis clinical theriogenology • volume 9 number 1 • march 201775 clinical theriogenology • volume 9 number 1 • march 2017 76 omniblank: 2011: granulosa cell tumor in a heifer: clinical, ultrasonographic, endocrine and pathologic findings granulosa cell tumor in a heifer: clinical, ultrasonographic, endocrine and pathologic findings marsha m. severt,a,* juan e. romano,a john f. edwards,b timothy e. eberhart,c r. neil hoopera alarge animal clinical sciences, college of veterinary medicine and biomedical sciences, texas a&m university, college station, tx; bveterinary pathobiology, college of veterinary medicine and biomedical sciences, texas a&m university, college station, tx; ccanton veterinary clinic, canton, tx summary an 18-month-old 652-kg black limousin heifer was evaluated because of an enlarged left ovary and infertility following herd pregnancy examination. the heifer had been treated for cystic ovarian disease, however the left ovary remained enlarged. palpation per rectum revealed a firm, multilobulated mass in the region of the left ovary. transrectal ultrasonography confirmed a large, hypoto anechoic polycystic left ovary. hormone assays showed an increase in estrogens and inhibin, normal or slightly elevated concentration of testosterone, and a low concentration of progesterone. a left ovariectomy via ventral midline celiotomy was performed. gross and histopathologic evaluation of the mass confirmed a granulosa cell tumor (gct). six months after surgery, the heifer was found dead in the pasture due to causes unrelated to surgery. necropsy revealed the remaining right ovary had grown in size, suggesting the beginning of follicular activity. background granulosa cell tumors appear to be the most common ovarian neoplasm affecting cattle.1 affected cattle can display altered behavior and decreased fertility due to hormones secreted by the tumor. in particular, inhibin is a glycoprotein hormone secreted by granulosa cells that suppresses follicle stimulating hormone (fsh) secretion from the anterior lobe of the pituitary gland. therefore, inhibin is one of the hormones responsible for infertility due to suppression of fsh and inactivity of the contralateral ovary.2 alterations in the hormone profile of cattle during the estrous cycle can result in conception failure, which represent an economic loss to the producer. therefore, identifying cattle with ovarian abnormalities such as gcts is important to the productivity of a herd. if the female is of sufficient value to the producer, the gct can be surgically removed. once the hormone-producing tumor, and thus the source of excess inhibin is removed, the contralateral ovary may regain normal function. case presentation an 18-month-old 652-kg black limousin heifer was referred to the texas a&m large animal hospital for evaluation of an enlarged left ovary. the heifer was the only non-pregnant heifer in a group that had been pastured with a bull during the previous summer. an enlarged left ovary was identified on palpation per rectum by the referring veterinarian and the heifer was treated with gonadotropin releasing hormone for cystic ovarian disease. after the next breeding, the heifer still did not become pregnant and the ovary remained enlarged. blood was collected and submitted for measurement of progesterone and estradiol 17-ß concentrations. the progesterone concentration was <0.20 ng/ml indicating that the heifer did not have a functional corpus luteum (cl) on either ovary. estradiol 17-ß concentration was 0.00 pg/ml. the heifer was then referred for further evaluation of the enlarged left ovary. initial physical examination revealed a temperature of 101.4 f, pulse of 80 bpm, and a respiratory rate of 40 bpm. body condition of the heifer was scored 8/9. the heifer was noted to have a masculinized appearance due to well developed neck musculature. the ranch manager stated that the heifer had not displayed any bull-like behavior. there were no other significant findings. vaginoscopy was performed and revealed a normal vagina with a closed cervix. palpation per rectum revealed a ‘basketball’ sized, firm, multilobulated mass at the distal end of the left uterine horn. the uterine horns were flaccid and the entirety of the mass could not be appreciated due to inability to retract the uterus because of the size and weight of the mass. transrectal ultrasonography with a 7.5 mhz * current address: brown creek equine hospital, polkton, nc 61 linear transducer revealed a large, multilobulated, hypoto anechoic polycystic ovary (figure 1). many vessels were observed suggesting high vascularization of the mass. differential diagnosis a presumptive diagnosis of a left ovarian gct was made. differential diagnoses included atypical cystic ovarian degeneration, abscess, lipomatosis, and lymphoma. treatment the owner elected surgical removal of the left ovary. a preoperative hemogram revealed a mild stress leukogram and no clinically significant abnormalities of serum chemistry. blood was also submitted for measurement of progesterone, estrogens, testosterone, and inhibin concentrations (table). food and water were withheld for 48 hours in preparation for a left ovariectomy via ventral midline celiotomy. a catheter was placed in the left jugular vein. ceftiofur sodium (3.0 mg/kg iv and sq), flunixin meglumine (2.2 mg/kg iv), and tetanus toxoid were administered prior to surgery. in the event of excessive blood loss during surgery, eight l of fresh, whole blood was collected from a donor cow. table. serum concentrations of progesterone, estradiol 17-β, testosterone and inhibin. progesterone 0.3 ng/ml estradiol 17-ß 21.5 ng/ml testosterone 111.6 pg/ml inhibin 4.56 ng/ml the patient was anesthetized and positioned in dorsal recumbency on a padded surgical table. the ventral abdomen was clipped and prepared for aseptic surgery. a 30 cm incision was made on the ventral midline beginning 10 cm cranial to the umbilicus and ending 20 cm caudal to the umbilicus. the left uterine horn was approximately 5 cm in diameter. multiple vessels estimated to be 5-10 mm in diameter were palpable in the left ovarian pedicle. the left ovary was not adhered to other structures in the abdomen, but could not be completely exteriorized (figure 2). the right uterine horn was approximately 1 cm in diameter and the right ovary was approximately 2 mm in diameter and 5 mm in length. an unsuccessful attempt was made to reduce the size of the mass using a 14-gauge needle. only a small amount of viscid, black material could be removed by suction. using hemostats, openings were created in the mesovarium on either side of the vessels supplying the ovarian mass. ligation was accomplished with sterile umbilical tape. a vessel sealing device (ligasure™, covidien, mansfield, ma) was used to create a path of sealed tissue between the ligatures and the base of the ovary. the ovarian mass was removed by cutting along the path of the sealed tissue with surgical scissors. the ovarian mass was separated from the uterine horn without entering the lumen, however a large vessel was encountered in the mesovarium which bled profusely. a whole blood transfusion was initiated as attempts to identify and ligate the vessels were made. as the serosal surface of the left uterine horn was removed with the ovary, the tip of the left horn was oversewn with a single layer of size 1 polyglactin 910 in a lembert pattern. the linea alba, subcutaneous space and skin were closed in a routine fashion. outcome postoperatively, the heifer appeared anxious and was tachypneic (80 bpm). additional butorphanol tartrate (0.1 mg/kg, sq) was administered every six hours for analgesia. the heifer appeared comfortable and grain and coastal hay were offered. the third day postoperatively the heifer showed signs of colic and became anorexic. butorphanol tartrate and flunixin meglumine were administered. a hemogram and serum chemistry panel were performed and the results were within reference ranges. signs of colic subsided following treatment. the heifer’s appetite remained reduced, and two days later the heifer’s rectal temperature was 103.0° f. differential diagnoses included septic peritonitis, incisional infection, aspiration pneumonia or pleuritis, and endogenous pyrogen release from tissue destruction 62 associated with surgery. the heifer continued to pass normal amounts of feces and lung sounds were normal. the incision was not painful, warm, and did not have any drainage. edema of the ventral midline continued to subside. the heifer was again treated with a single dose of flunixin meglumine and tulathromycin. over the next two days the rectal temperature steadily declined to within normal range and the heifer’s appetite improved. the heifer remained hospitalized for 10 days postoperatively. the heifer was discharged with instructions to keep her confined to a small paddock separate from other cattle for an additional 90 days to allow her incision to fully heal. the left ovary weighed 3.6 kg and measured 28 x 27 x 18 cm with multiple dark red to tan 6-8 cm nodules (figure 3). sectioning of the mass revealed two (2 cm and 4 cm) cysts filled with clear fluid. numerous nodules of dry, necrotic tissue were surrounded by hemorrhage. histological evaluation revealed a gct with viable ovarian tissue mixed with areas of subacute, coagulative necrosis and pools of fresh hemorrhage. several vessels contained thrombi. proliferating, cuboidal epithelial cells were piled 2-3 cells deep on a fine fibrovascular stroma (figure 4). the cells had round, hyperchromatic nuclei of variable diameter (anisokaryosis), coarsely clumped chromatin, and ample, purple cytoplasm. groups of cells were subtended by thick, connective tissue bands. mitoses were rare, and necrosis occupied 60% of the sections examined. discussion various ovarian neoplasms have been described in cattle, and although rare, gcts appear to be the most common.1 one of the authors (jfe) routinely encounters gcts in two to three per 1000 mature beef cows at slaughter in texas. from 20,913 reproductive examinations in dairy cattle from minnesota and wisconsin, 0.1% were diagnosed with gcts.3 the prevalence of gcts in cattle varies, with a recent report of 11 gcts in 1,489 (0.74%) of slaughtered cattle.4 although most authors report that gcts in all species are rarely malignant, one study5 found nine of 13 bovine gcts had implanted in the abdomen and metastasized and in another case6 a malignant gct in an angus cow with peritoneal implantation and distant metastasis was reported. granulosa cell tumors are most commonly characterized by unilateral ovarian enlargement. the enlarged ovary may be smooth or coarsely lobulated and the contralateral ovary may be small. behavior of affected cows can range from anestrus, to nymphomania, to bull-like behavior. affected cattle can have a masculinized appearance. udder development and lactation may occur in affected heifers with hyperestrogenism.7 occasionally, gcts are found in pregnant animals.8 a presumptive diagnosis is made by palpation per rectum coupled with behavioral abnormalities. transrectal ultrasonography can provide additional evidence for the diagnosis of gcts.9 ultrasonic examination of the mass often reveals a multicystic or honeycombed structure. in the present case, transrectal ultrasonography revealed a large, lobulated ovary with multiple hypoto anechoic regions within the neoplasm (figure 1). hormone profiles can provide supporting evidence for the diagnosis of gcts. in horses these tumors are hormonally active and assays for the detection of a gct include measurements of inhibin, testosterone, and progesterone.19,20 estrogen, testosterone, and progesterone concentrations in cattle affected by gcts have been reported.9,21-23 however, a review of the literature did not reveal published concentrations of inhibin in cattle affected by gcts. the hormone profile in the present case showed a significant increase in the concentrations of estrogens and inhibin, a normal to slightly elevated concentration of testosterone, and a low concentration of progesterone. the concentration of estrogen in the present case was higher than those reported in females during the estrous cycle10,11 or cows with follicular ovarian cysts.12 the concentration of testosterone in blood was similar to11 or slightly higher than those reported for cows during the estrous cycle,13 but lower than the values reported for bulls.14,15 a slight increase in testosterone can exert anabolic effects such as an increase in neck musculature as seen in this heifer. the concentration of progesterone indicated an absence of luteal tissue.10 the plasma concentration of inhibin was more than ten times higher than basal levels in healthy cattle reported previously.16,17 inhibin is secreted mainly by the granulosa cells of antral follicles and has the ability to suppress fsh secretion in mammals.18 estrogens are also produced by the granulosa cells.10 the increased plasma concentrations of estrogen 63 and inhibin found in this case suggest an up-regulation of granulosa cellular function. unfortunately, no other published reports of the concentrations of inhibin in cattle with gcts were found. inhibin is elevated in approximately 90% of mares with gct.19 inhibin produced by the gct is thought to be responsible for the inactivity of the contralateral ovary by suppressing fsh release. serum testosterone is elevated in approximately 50-60% of mares and is associated with stallion-like behavior.20 progesterone concentrations are low, usually less than 1 ng/ml, since ovulation and corpus luteum formation do not occur.20 one report21 described elevated plasma estrogen and testosterone concentrations and a low progesterone concentration in a cow with a gct. after surgical removal of the affected ovary, estrogen and testosterone levels fell and estrous cycles were re-established three weeks after surgery. another author9 reported elevated testosterone and progesterone concentrations in a heifer with a malignant gct. definitive diagnosis of gct requires histopathologic confirmation. sections of the tumor often reveal neoplastic changes such as anisokaryosis, as evident in this case, along with variable amounts of thrombosis and necrosis. much of the neoplasm in this case was undergoing ischemic necrosis, presumably due to compromise of the blood supply.22 though most bovine gcts are found at slaughter, surgical removal of the affected ovary is a viable treatment option. due to the genetic value of the female of this report, a unilateral ovariectomy was recommended. this advice was based on previously published information that showed that animals can resume estrous cycles and can become pregnant between two and 12 months after surgery.9,21,23,25-28 unfortunately, in the present case the animal was found dead but a necropsy showed that the remnant ovary began to show signs of follicular activity six months after surgery. ovariectomy via a ventral midline approach as a treatment for a gct in a 15-month-old holstein heifer has been described.26 the heifer was bred and delivered a healthy heifer calf approximately one year after surgery. surgical approaches for ovariectomy in cases of gcts include colpotomy, flank laparotomy, and ventral midline celiotomy9,25-27,29. due to the size of this heifer, position of the mass, and inability to palpate the full extent of the ovary per rectum, a ventral midline approach was selected in this case. during surgery, the tumor could not be completely exteriorized preventing full visualization of the left ovarian pedicle. in some cases including the present case, gcts are highly vascular. the risk of hemorrhage is high,28 and in this case, there was hemorrhage from the left ovarian pedicle, necessitating a blood transfusion. the heifer in this case was hospitalized for a total of ten days postoperatively and was discharged with instructions to keep her separate from other cattle for a total of 90 days to allow her incision to fully heal. a subsequent report from the ranch manager indicated that the heifer was noted to stand for service by a bull. in summary, gcts are a common ovarian neoplasm in multiple species, including cattle. this report describes a clinical case of a gct in a heifer in which hormone assays coupled with palpation per rectum and ultrasongography supported the histopathological diagnosis. though concentrations of inhibin have not been reported previously in cases of gcts in cattle, this information provided valuable supporting evidence for the diagnosis in this case and should be considered when selecting diagnostic tests. more work is needed to develop reference ranges for inhibin cattle. as demonstrated by this case and others reported previously, treatment of gcts by surgical removal is a viable option in cattle. learning points  this is the first report describing the measurement of inhibin concentration determination to aid in the diagnosis of gcts in cattle.  future work is needed to develop bovine-specific reference ranges for hormone concentrations associated with gcts.  surgical removal of the affected ovary remains a viable treatment option in cattle with gcts. references 1. troedsson mht, christensen bw: diseases of the reproductive system. in: smith bp, editor. large animal internal medicine. 4th ed. st. louis: mosby elsevier; 2009. p. 1419-1483. 64 2. stevenson js: clinical reproductive physiology of the cow. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: saunders elsevier; 2007. p. 258-270. 3. zemjanis r, larson ll, bhalla rps: clinical incidence of genital abnormalities in the cow. j am vet med assoc 1961;139:1015-1018. 4. perez-martinez c, duran-navarrete aj, garcia-fernandez ra, et al: biological characterization of ovarian granulose cell tumours of slaughtered cattle: assessment of cell proliferation and oestrogen receptors. j comp pathol 2004;130:117-123. 5. norris hj, taylor hb, garner fm: comparative pathology of ovarian neoplasms. ii. gonadal stromal tumors of bovine species. pathol vet 1969;6:45-58. 6. zachary jf, haliburton jc: malignant granulosa cell tumor in an angus cow. vet pathol 1983;20:506-509. 7. short rv, shorter dr, linzell jl. granulosa cell tumour of the ovary in a virgin heifer. j endocrinol 1963;27:327-332. 8. tontis a, könig h, luginbühl h: granulosaund thekazelltumor beim rind. schweiz arch tierheilkd 1982;124:233243. 9. hostetler de, sprecher dj, yamini b, et al. diagnosis and management of a malignant granulosa cell tumor in a holstein nulligravida: a case study. theriogenology 1997;48:11-17. 10. dieleman sj, bevers mm, van tol htm, et al: peripheral plasma concentrations of oestradiol, progesterone, cortisol, lh and prolactin during the oestrous cycle in the cow, with emphasis on the peri-oestrous period. anim reprod sci 1986;10:275-292. 11. nix jp, spitzer jc, chenoweth pj: serum testosterone concentration, efficiency of estrus detection and libido expression in androgenized beef cows. theriogenology 1998;49:1195-1207. 12. nakao t: response of ovaries and serum steroid levels after treatment of cystic ovarian disease in dairy cattle. i. treatment with corticosteroids and a combination of human chorionic gonadotropin and progesterone. theriogenology 1977;7:53-62. 13. shemesh m, hansel w: measurement of bovine plasma testosterone by radioimmunoassay (ria) and by a rapid competitive protein binding (cpb) assay. j anim sci 1974;39:720-724. 14. kiser te, milvae ra, hafs hd, e al: comparison of testosterone and androstenedione secretion in bulls given prostaglandin f2α or luteinizing hormone. j anim sci 1978;46:436-442. 15. welsh th, randel rd, johnson bh: temporal relationship among peripheral blood concentrations of corticosteroids, luteinizing hormone and testosterone in bulls. theriogenology 1982;12:169-179. 16. tohei a, shi f, ozawa m, et al: dynamic changes in plasma concentrations of gonadotropins, inhibin, estradiol-17β and progesterone in cows with ultrasound-guided follicular aspiration. j vet med sci 2001;63:45-50. 17. parker ki, robertson dm, groome np, et al: plasma concentrations of inhibin a and follicle-stimulating hormone differ between cows with two or three waves of ovarian follicular development in a single estrous cycle. biol reprod 2003;68:822-828. 18. dejong fh: inhibin. physiol rev 1988;68:555-607. 19. mccue pm: equine granulosa cell tumors. proc annu conv am assoc equine pract 1992. p. 587-593. 20. mccue pm: review of ovarian abnormalities in the mare. proc annu conv am assoc equine pract 1998. p. 125-133. 21. bosu wtk: granulosa cell tumor in a cow: clinical, hormonal, and histopathological observations. theriogenology 1977;8:119-128. 22. mcentee k: reproductive pathology of domestic animals. san diego: academic press, 1990. p. 83. 23. descôteaux l, harvey d, girard c: tumeur des cellules de la granulose chez une taure: observations cliniques, endocrinologiques et post-mortem. can vet j 1989;30:501-503. 24. jorritsma r, antonise afg, stockhofe n, et al: case report of a granulosa-theca tumour in a cow. tijdschr diergeneeskd 2002;127:286-288. 25. plourde v, martineau r, harvey d: surgical removal of a granulosa cell tumor from a heifer. can vet j 1984;25:12-13. 26. leder rr, lane vm, barrett dp: ovariectomy as treatment for granulosa cell tumor in a heifer. j am vet med assoc 1988;192:1299-1300. 27. pugh dg, elmore rg: granulosa cell tumor in a cow. compend contin educ pract vet 1987;9: f327-f330. 28. masseau i, fecteau g, desrochers a, et al: hemoperitoneum caused by the rupture of a granulosa cell tumor in a holstein heifer. can vet j 2004;45:504-506. 29. elmore rg: focus on bovine reproductive disorders: granulosa cell tumors. vet med small anim clin 1992;87:744746. 65 figure 1. transrectal ultrasonography with a 7.5 mhz linear transducer revealed a large, multilobulated mass with multiple hypoto anechoic cystic areas. figure 2. the left ovary during surgery prior to removal. the ovary was not adhered to other structures in the abdomen, but could not be completely exteriorized. the ovary is supported by the surgeon’s arm. 66 figure 3. gross appearance of a left ovarian mass. the 3.6 kg neoplasm measured 28 x 27 x 18 cm with multiple dark red to tan, 6-8 cm nodules. figure 4. photomicrograph of a section of the viable tissue within the gct at 200x magnification. areas of proliferating, cuboidal epithelial cells piled 2-3 cells deep on a fine fibrovascular stroma. 67 68 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /usedeviceindependentcolor /dothumbnails false /embedallfonts true /embedopentype true /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false 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/usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2018: corynebacterium pseudotuberculosis as a pathogen of the reproductive tract of male small ruminants: case study and review   corynebacterium pseudotuberculosis as a pathogen of the reproductive tract of male small ruminants: case study and review jamie l. stewart, miranda d. vieson, clifford f. shipley department of veterinary clinical medicine, college of veterinary medicine, university of illinois urbana-champaign, urbana, il summary three cases are presented herein describing manifestations of corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis (cl), in the reproductive tract of two bucks and a ram. the two bucks were presented for poor sperm quality and were subsequently diagnosed with bilateral suppurative orchitis (case 1) and epididymitis (case 2) from which c. pseudotuberculosis was cultured. case 3 is a ram that was presented for semen cryopreservation followed by euthanasia. electroejaculation produced a poor quality ejaculate, whereas epididymal sperm harvest yielded a better quality sample. abscesses located adjacent to the epididymis and sigmoid flexure of the penis from cl may have prevented normal ejaculation. all cases presented progressive testicular degeneration, which could result indirectly from exotoxins produced by c. pseudotuberculosis or directly from abscess formation. these cases highlight the need to test for and eradicate cl, as it may have deleterious effects on reproductive success in small ruminant breeding farms. keywords: caprine, epididymitis, infertility, oligozoospermia, orchitis, ovine background corynebacterium pseudotuberculosis is a contagious bacterial agent that causes cl in both sheep and goats.1 this disease has long been known to cause significant financial loss due to increased mortality related to internal lesions and carcass condemnation related to external lesions.1 it is both time consuming and costly to eradicate cl from a herd, and therefore, many owners chose to live with it. an early study reported an 8% prevalence of cl abscesses confined to the scrotal fascia of rams with no effect on semen quality.2 however, the effect of this pathogen on reproductive soundness is becoming increasingly more obvious, even when no abscesses are present.3 this report includes three cases of cl in small ruminants that highlights the need to eradicate c. pseudotuberculosis within small ruminant breeding facilities due to its potentially dire herd health consequences and widespread effects throughout the reproductive tract. case 1: orchitis in a buck case presentation an 8-year-old boer buck was presented to the university of illinois veterinary teaching hospital with a one month history of an enlarged left testicle and oligozoospermia noted by the owners during routine semen collection. the owners also stated that the buck had a chronic cough for an unknown amount of time. on physical examination, generalized muscle atrophy was apparent with a body condition score of 2/9.4,5 bilateral mucoid nasal discharge was present, and increased intensity of lung sounds were audible throughout the entire left thorax. the left testicle was noticeably and asymmetrically larger than the right testicle and firm on palpation (figure 1). both epididymides were palpably normal. a complete blood cell count revealed active suppurative inflammation (severe neutrophilia: 24.3×103 /µl, reference range 1.2-7.2×103/µl; hyperfibrinoginemia: 504 mg/dl, reference range 100-400 mg/dl). serum chemistry revealed hperglobulinemia (6.0 g/dl, reference range 2.7-4.1 g/dl). semen was collected by electroejaculation for analysis. a 31.75 mm diameter, three-electrode ram probe (lane manufacturing, inc., denver, co) was placed rectally, and a programmed cycle was run on the electroejaculator unit (pulsator iv, lane manufacturing, inc., denver, co). grossly, the semen appeared to be mildly cloudy and yellow-colored. microscopic evaluation revealed oligozoospermia and asthenozoospermia with a subjective total motility of 10% and negligible progressive motility. clinical theriogenology • volume 10, number 2 • june 2018107   morphologic analysis of the spermatozoa revealed 57% normal sperm cells, 7% simple bent tails, 14% detached heads, 6% proximal droplets, 3% mid-piece defects, and 12% strongly folded tails. thoracic ultrasonography revealed mild pleural and pericardial effusion (figure 2) with a normal heart. cytological analysis of the pleural fluid revealed a modified transudate with no visible bacteria. ultrasonography of the right testicle detected 5 and 15-mm round hypoechoic nodules filled with numerous hyperechoic foci (figures 3a and b). multiple similar hypoechoic nodules with multiple hyperechoic foci were also visible within the left testicle (figures 3c and d). these nodules likely represent abscesses consistent with multifocal suppurative orchitis, whereas the hyperechoic foci within the parenchyma suggest mineralization and testicular degeneration.6 differentials for suppurative orchitis in small ruminants included chlamydiosis (chlamydophilosis),7 specific pyogenic bacteria (e.g., trueperella pyogenes), opportunitistic bacteria (e.g., actinobacillus seminis, mannheimia haemolytica, bibersteinia trehalosi, pasteurella multocida), brucellosis, (brucella mellitensis or brucella ovis), and viruses (sheep or goat pox viruses, lentiviruses, bluetongue virus).8 brucella mellitensis has been reported once as a cause of orchitis in a buck determined by serum antibodies and testicular culture.9 while brucella ovis commonly causes epididymitis in rams, experimental infection has led to the development of titers and excretion of the bacteria in semen of goats.10 due to herd health concerns, the owner elected to have a brucella ovis serum elisa performed, which was negative. fine needle aspiration was performed on both testicles for culture and cytology. cytology revealed suppurative inflammation with marked necrosis within both testicles. bacterial culture revealed growths of staphylococcus xylosus from the left testicle and corynebacterium pseudotuberculosis from the right testicle. bacterial culture of the semen yielded s. xylosus and bacillus sp. both s. xylosus and bacillus sp. isolates in this case were considered incidental contaminants, as they have previously been isolated from both scrotal skin and prepuce in rams without clinical disease.11 outcome this buck was given a grave prognosis for recovery of breeding ability based on ultrasonographic findings that indicated the efferent ducts were likely destroyed by the abscesses. the overall poor condition of the buck also warranted euthanasia due to welfare concerns. the owner wished to pursue cloning options for the buck, so he was discharged to be euthanized on the farm by the referring veterinarian. case 2: epididymitis in a buck case presentation a 4-year-old boer buck was presented to the university of illinois veterinary teaching hospital with a primary complaint of asthenozoospermia and subfertility. the buck was purchased by the owners four months previously, but the owners noticed that most does he was observed breeding did not become pregnant. the owners did a semen evaluation at home and estimated ~30% motility. generalized physical examination was unremarkable with an adequate body condition score of 5/9.4,5 examination and palpation of the scrotum revealed bilaterally enlarged epididymides and small, soft testicles. ultrasonography revealed flocculent material within the epididymides with multiple, small, hyperechoic foci (figure 4). the testicles appeared to be undergoing bilateral degeneration and mineralization as determined by the presence of multiple hyperechoic foci within the testicular parenchyma (figure 4).6 transrectal ultrasonography was used to evaluate the accessory sex glands and did not reveal any abnormalities.12 differentials for epididymitis in small ruminants include those described above for orchitis, with brucellosis, a. seminis, and histophilus somni reported most commonly.13 semen was collected using electroejaculation as described in case 1. the ejaculate was clear and tested weakly positive (ca. 25) on a leukocyte esterase strip, indicating the presence of leukocytes. the sample was smeared on a slide, stained with a modified wright giemsa stain (jorvet dip quick stain, clinical theriogenology • volume 10, number 2 • june 2018 108   jorgensen labs, loveland co)), and evaluated with light microscopy. no spermatozoa were seen, but small numbers of neutrophils were noted (figure 5). the remainder of the ejaculate was submitted for aerobic and anaerobic culture and brucella spp. multiplex pcr. brucella pcr was negative, and culture results are summarized in thetable. the most relevant findings were isolation of both corynebacterium pseudotuberculosis and fusobacterium necrophorum from the ejaculate. while the latter has rarely been implicated as a cause of male reproductive disease,11 it has been associated with late term abortion in does and ewes.14,15 outcome due to the severity of the epididymal lesions and testicular degeneration, this buck was given a poor prognosis for recovery of breeding ability. the growth of two significant pathogens from the ejaculate raised the concern for venereal transmission. since the buck serviced several does, recommendations were made to test the herd for cl and cull any positives. due to the high-quality genetic line, the owners instead elected to monitor the herd and opt to test if further attempts to breed the does were unsuccessful. case 3: peri-sigmoid flexure abscess in a ram case presentation a 2.5-year-old montadale ram presented for semen collection and subsequent euthanasia due to progressive and severe musculoskeletal defects. necropsy later defined these defects as degenerative joint disease (multiple joints) and ankylosing spondylosis of the thoracic vertebrate. semen collection was performed using electroejaculation as described in the previous cases. the buck produced 3 ml of moderately cloudy ejaculate with a concentration of 163 million spermatozoa/ml. microscopic evaluation revealed 50% overall motility. morphological assessment using a phase contrast microscope at 100× determined that there were 33% normal spermatozoa, 27% primary defects (13% proximal droplets, 9% strongly folded, 5% midpiece defects), and 40% secondary defects (19% simple bent, 12% distal droplets, 8% tailless, 1% abnormal acrosome). six 0.5-ml straws were produced from this collection with a dose of 81.5 million sperm/straw. after euthanasia, the testicles were removed and submitted for epididymal sperm harvesting. the caudal epididymis and remaining vas deferens were excised from the tract. the cauda epididymis and vas deferens were flushed with optixcell extender (imv technologies, maple grove, mn). a total of 3 ml of well-concentrated (1810 mill/ml) semen was obtained with motility of 90%. since this was an epididymal collection, morphological analysis was not performed. the ejaculate was extended to a final dose of 200 million sperm/straw and yielded 23 0.5-ml straws. one straw was thawed in a 37°c water bath for 30 sec and found to have a post-thaw motility of 70%. during this procedure, an abscess was observed around one cauda epididymis, but did not penetrate into the lumen (figure 6a). the caseous material was submitted for aerobic culture, and no bacteria were recovered. on postmortem evaluation, a 4×3×3 cm abscess was identified adjacent to the sigmoid flexure of the penis, which yielded a moderate growth of corynebacterium pseudotuberculosis (figure 6b). grossly, the testicles were asymmetrical. the cut surface of the smaller testicle revealed multifocal yellow to light brown, granular, slightly firm areas scattered throughout the testicle and a thickened, firm epididymis (figure 7a). in the smaller testis, histopathology revealed testicular atrophy and degeneration with fibrosis, ductular loss, and ectasia of the epididymis (figure 7b). brucella spp. multiplex pcr of the testis and epididymis were negative, and culture yielded light to very rare growths of gram negative bacteria interpreted as contaminants (enterococcs hirae, escherichia coli, beta escherichia coli). discussion caseous lymphadenitis is a disease of sheep and goats characterized by abscess formation and caused by corynebacterium pseudotuberculosis. corynebacterium pseudotuberculosis has also been diagnosed as a cause of unilateral orchitis and epididymitis in a stallion.16 while the decreased clinical theriogenology • volume 10, number 2 • june 2018109   reproductive fitness in these three cases seem to be related to the cl abscesses, mahmood et al. recently reported that an exotoxin produced by c. pseudotuberculosis, phospholipase d, is also a major contributor to reduced semen quality, independent of abscess formation.3 bucks inoculated with c. pseudotuberulosis developed abscesses and enlarged lymph nodes at the site of inoculation (subaxillary region), as expected.3 in bucks treated with only phospholipase d endotoxin, there was a lack of abscess formation.3 however, despite the lack of gross lesions, there was a significant decrease in systemic testosterone and an increase in dead and abnormal sperm cells similar to that seen in the bucks inoculated with c. pseudotuberculosis.3 furthermore, these findings in both treatment groups, when compared to untreated controls, coincided with varying degrees of testicular degeneration and necrosis after three months.3 based on this study, it is possible that the testicular degeneration observed in all three cases were primarily a sequela to phospholipase d endotoxin release. additionally, though post-thaw sperm motility in case 3 was acceptable, in vivo fertility should be evaluated as phospholipase d may prematurely induce sperm hyperactivation.17 these results, in addition to the findings in this case series, highlight a major concern for breeding populations with endemic cl, whether or not there are external lesions. bacterial culture of the ejaculate in case 2 yielded for c. pseudotuberculosis, which raises concerns about potential venereal transmission. as is evident in case 3, clinicians are commonly presented with animals for semen cryopreservation with unknown disease status. due to the addition of broad-spectrum antibiotics to extenders, bacterial culture is not routinely performed on semen intended for artificial insemination. though the prevalence of cl varies greatly by region, information on its prevalence and transmission through semen is lacking.18,19 though the semen itself was not cultured, necropsy results indicated that no significant pathogens were isolated from the epididymal mass, testis, or epididymis in case 3, nor were pathogens isolated in case 1. regardless, the pathogen isolation observed in case 2 should present concerns in regards to transmission of this pathogen when used for artificial insemination. judson and songer reported the in vitro susceptibility of c. pseudotuberculosis to a variety of commonly used antibiotics that include penicillins, macrolides, tetracyclines, cephalosprins, lincomycin, and rifampicin.20 though the incorporation of broad-spectrum antibiotics into commercial semen extenders should mitigate transmission concerns, further work may be necessary to ensure that it is sufficient to prevent transmission in vivo. additionally, future studies need evaluate the concentration of phospholipase d in semen of cl-positive animals and ascertain if venereal transmission of these exotoxins can be harmful within the female. caseous lymphadenitis has been implicated as a cause of abortion and neonate illness in horses by poonacha and donahue21 and in sheep by dennis and bamford.22 in boer does, both intranasal and oral inoculation with c. pseudotuberculosis altered progesterone and estrogen production following estrus synchronization23 which may contribute to infertility. a subsequent study by this group found that 30 days after intradermal, intranasal, or oral inoculation, there were varying degrees of necrosis, congestion, inflammatory cell infiltration, and edema within the ovaries, uterus, and iliac lymph nodes.24 though he long-term effects of inoculation on female fertility have not been evaluated, they do present concern for a potential naïve herd bred by a new, infected male. alonso et al evaluated timing of inoculations in manchega sheep and found that three of four ewes inoculated seven days before mating had normal pregnancies.25 the remaining ewe had a prolonged gestation with a lamb that died at 26 days of age with c. pseudotuberculosis isolated from its liver, heart, and kidney.25 in all ewes inoculated during the embryonic stage (gestational day 23), abortions, stillborns, or ill-thrift/weak lambs were reported.25 the last group was inoculated during the fetal stage (gestational day 103) and produced apparently normal lambs.25 these results provide further evidence that introduction of this contagious pathogen either during mating or in the early embryonic stages can be detrimental to herd reproductive success. further studies are warranted to better understand the pathology of different strains of c. pseudotuberculosis within the female reproductive tract and determine if phospholipase d production is responsible for interfering with conception and pregnancy. understanding these underlying mechanisms is crucial for developing better preventative and intervention strategies for this highly infectious and detrimental disease. clinical theriogenology • volume 10, number 2 • june 2018 110   in conclusion, this case report series describes three separate presentations of cl that adversely affected the reproductive potential of bucks and a ram. the findings of testicular degeneration described herein are consistent with those reported previously3 and raise concerns regarding the pathogenesis of cl in the reproductive fitness of small ruminants. future studies need to focus on the role of phospholipase d exotoxin on both male and female reproduction and its transmission to develop effective vaccines to assist with eradication efforts.26 learning points  corynebacterium pseudotuberculosis may cause male infertility directly (abscesses) or indirectly (exotoxins).  abscess formation associated with cl may present as orchitis, epididymitis, or incidentally.  testicular degeneration, either bilateral or unilateral, was reported in all three cases associated with cl.  all forms of cl-induced reproductive disease may not be detected solely by semen culture. acknowledgement we thank the university of illinois diagnostic imaging service for providing ultrasonographic images for case 1 and the veterinary diagnostic laboratory for providing necropsy images for case 3. no financial support was received for this project and none of the authors have any conflict of interest or bias for the publication of this report. references 1. williamson l: caseous lymphadenitis in small ruminants. vet clin north am food anim pract 2001;17:359-371. 2. williamson p, nairn me: lesions caused by corynebacterium pseudotuberculosis in the scrotum of rams. aust vet j 1980;56:496-498. 3. mahmood zk, jin zam, jesse ff, et al: relationship between the corynebacterium pseudotuberculosis, phospholipase d inoculation and the fertility characteristics of crossbred boer bucks. livest sci 2016;191:12-21. 4. mortimer rg, boyd gw, morris dl: evaluating the impact of body condition on production parameters in beef cows. vet med 1991;86:1030-1036. 5. mellado m, vera a, loera h: reproductive performance of crossbred goats in good or poor body condition exposed to bucks before breeding. small rumin res 1994;14:45-48. 6. ahmad n, noakes de, middleton dj: use of ultrasound to diagnose testicular degeneration in a goat. vet rec 1993;132:436-439. 7. appleyard wt, aitken id, anderson ie: attempted venereal transmission of chlamydia psittaci in sheep. vet rec 1985;116:535-538. 8. gouletsou pg, fthenakis gc: microbial diseases of the genital system of rams or bucks. vet microbiol 2015;181:130135. 9. ahmad r, niaz b: orchitis due to brucellosis in a buck. pak vet j 1998;18:108-109. 10. burgess gw, spencer tl, norris mj: experimental infection of goats with brucella ovis. aust vet j 1985;62:262-264. 11. gouletsou pg, fthenakis gc, tzora a, et al: isolation of arcanobacterium pyogenes from the scrotal skin and the prepuce of healthy rams or from rams with testicular abnormalities. small rumin res 2006;63:177-182. 12. gonzalez-bulnes a, pallares p, vazquez mi: ultrasonographic imaging in small ruminant reproduction. reprod domest anim 2010;45:9-20. 13. gouletsou pg, fthenakis gc: microbial diseases of the genital system of rams or bucks. vet microbiol 2015;181:130135. 14. moeller rb: causes of caprine abortion: diagnostic assessment of 211 cases (1991-1998). j vet diagn invest 2001;13:265-270. 15. agerholm js, aalbæk b, fog-larsen am, et al: veterinary and medical aspects of abortion in danish sheep. apmis 2006;114:146-152. 16. gonzalez m, tibary a, sellon dc, et al: unilateral orchitis and epididymitis caused by corynebacterium pseudotuberculosis in a stallion. equine vet educ 2008;20:30-36. 17. gangwar dk, atreja sk: signalling events and associated pathways related to the mammalian sperm capacitation. reprod domest anim 2015;50:705-711. 18. stoops sg, renshaw hw, thilsted j: ovine caseous lymphadenitis: disease prevalence, lesion distribution, and thoracic manifestations in a population of mature culled sheep from western united states. am j vet res 1984;45:557-561. 19. paton mw, walker sb, rose ir, et al: prevalence of caseous lymphadenitis and usage of caseous lymphadenitis vaccines in sheep flocks. aust vet j 2003;81:91-95. clinical theriogenology • volume 10, number 2 • june 2018111   20. judson r, glenn songer j: corynebacterium pseudotuberculosis: in vitro susceptibility to 39 antimicrobial agents. vet microbiol 1991;27:145-150. 21. poonacha kb, donahue jm: abortion in a mare associated with corynebacterium pseudotuberculosis infection. j vet diagnostic investig 1995;7:563-564. 22. dennis sm, bamford vw: the role of corynebacteria in perinatal lamb mortality. vet rec 1966;79:105-108. 23. othman am, jesse ffa, adamu l, et al: changes in serum progesterone and estrogen concentrations in non-pregnant boer does following experimental infection with corynebacterium pseudotuberculosis. j vet adv 2014;4:524-528. 24. othman am, abba y, jesse ffa, et al: reproductive pathological changes associated with experimental subchronic corynebacterium pseudotuberculosis infection in nonpregnant boer does. j pathog 2016:1-7. pmc4781953 25. alonso jl, simon mc, girones o, et al: the effect of experimental infection with corynebacterium pseudotuberculosis on reproduction in adult ewes. res vet sci 1992;52:267-272. 26. windsor pa, bush rd: caseous lymphadenitis: present and near forgotten from persistent vaccination? small rumin res 2016;142:6-10. table. culture results from ejaculate of a boer buck with infertility (case 2). culture isolate # isolate level aerobic 1 proteus mirabilis tntc aerobic 2 staphylococcus aureus tntc aerobic 4 streptococcus oralis tntc aerobic 5 corynebacterium sp. 60 aerobic 6 staphylococcus warneri 200 aerobic 3 corynebacterium pseudotuberculosis tntc anaerobic 51 bacteroides fragilis tntc anaerobic 55 bacteroides pyogenes tntc anaerobic 54 fusobacterium necrophorum tntc figure 1. asymmetrically enlarged left testicle in a boer buck associated with cl orchitis (case 1). clinical theriogenology • volume 10, number 2 • june 2018 112   figure 2. ultrasonographic images from the thoracic cavity of a boer buck with thoracic disease presumptively associated with cl (case 1). note the pleural (a) and pericardial (b) effusions denoted by the white arrowheads, respectively. clinical theriogenology • volume 10, number 2 • june 2018113   figure 3. ultrasonographic images of the right (a, b) and left (c, d) testicles associated with cl orchitis in a boer buck (case 1). large 5and 15-mm abscesses are apparent in the right testicle (arrowheads). multiple hyperechoic foci (arrows) are consistent with mineralization and testicular degeneration bilaterally. clinical theriogenology • volume 10, number 2 • june 2018 114   figure 4. bilateral epididymitis and testicular degeneration in a boer buck affected with cl (case 2). multiple hyperechoic foci (arrows) were noted within the testicular parenchyma of both testicles and are consistent with mineralization and testicular degeneration. the cauda epididymides were both large, distended, and filled with hyperechoic material. figure 5. positive leukocyte esterase test and confirmation of neutrophil presence (arrowhead) and asthenozoospermia in an ejaculate from a boer buck with bilateral cl epididymitis (case 2). clinical theriogenology • volume 10, number 2 • june 2018115   figure 6. gross images representing cl abscesses associated with the genitourinary tract in a montadale ram (case 3). (a) image of a caseous peri-epididymal abscess found incidentally during epididymal sperm harvest. abscess did not communicate with the lumen of the cauda epididymis. (b) image of a cl abscess (arrow) that is lifted from its location adjacent to the sigmoid flexure of the penile urethra. clinical theriogenology • volume 10, number 2 • june 2018 116   figure 7. images representing testicular degeneration associated with cl diagnosis in a montadale ram (case 3). (a) gross image comparing the testicles. the testicle on the right is approximately 2/3 the size of the contralateral testicle and is firm, pale, and contains multiple white flecks (arrowheads) throughout the parenchyma. the cranial aspect of the epididymis is also markedly thickened. (b) photomicrograph of the smaller testicle from a ram with cl demonstrating testicular degeneration. there is a marked decrease in spermatids and a moderate decrease in spermatogonia within seminiferous tubules that are often lined by vacuolated sertoli cells and supported by a thickened, hyalinized tubular wall. there are also small numbers of multinucleated (degenerated) spermatogonia within the lumen of seminiferous tubules. occasional tubules are occluded by dense aggregates of spermatids (spermatozoal stasis). h&e stain. bar = 50 um. (editor’s note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 10, number 2 • june 2018117 2013: antimicrobial therapy in foals antimicrobial therapy in foals steeve giguère college of veterinary medicine, university of georgia, athens, ga antimicrobial therapy has long played a vital role in the treatment of equine bacterial diseases. however, the arsenal of equine-approved antimicrobial agents available to veterinarians is limited. few antimicrobial drugs are licensed for use in horses, often requiring the clinician to resort to extra-label use. unfortunately, many human drugs are not appropriate for use in horses because of poor oral bioavailability, cost, lack of species-specific pharmacokinetic data, or risk of serious adverse effects such as enterocolitis. while development of new antimicrobials initially outpaced development of bacterial resistance, the trend has reversed over the last 10 years. this has resulted in emergence of resistant bacteria as a major problem worldwide. although any use of antimicrobials has the potential to contribute to development of resistance, antimicrobial therapy is vital for controlling equine infectious disease. thus, it is the veterinarian’s responsibility to use antimicrobials judiciously. in general terms, judicious use is defined as the optimal selection of a drug, dose, dosing interval, and duration of therapy along with reduction of inappropriate or excessive use of antimicrobials. judicious use of antimicrobials is essential to maximize treatment efficacy while minimizing development of bacterial resistance, occurrence of adverse reactions, and relapses. this technical bulletin provides an overview of current concepts that guide rational antimicrobial therapy in horses. general principles of antimicrobial drug use in horses a variety of veterinary organizations have developed generic recommendations for judicious antimicrobial drug use. the most comprehensive guidelines for equine practitioners is a consensus statement issued by the american college of veterinary internal medicine.1 based on these guidelines, key prudent-use principles are:  antimicrobials should only be used when there is a reasonable likelihood that a bacterial infection is present.  whenever possible, antimicrobial therapy should be based on culture and in vitro susceptibility results.  a drug with a spectrum of activity as narrow as possible should be selected.  extra-label use should be avoided when on-label options are available.  antimicrobial agents should be used for as short a period as necessary.  antimicrobials that are important for treating refractory or serious infections in humans should be reserved and only used after careful consideration, and only when culture and susceptibility results indicate that there are no alternatives.  methods to decrease the risk and incidence of infection should be emphasized in order to decrease the need for antimicrobials. to underscore the last point, antimicrobial therapy should never be used as a substitute for good disease control and hygiene, appropriate medical and surgical practices, or as an alternative to sound animal husbandry. in choosing the appropriate antimicrobial agent, the clinician must consider a variety of factors including: (1) the likely identity of the infecting microorganism(s), (2) their typical in vitro susceptibility patterns or the clinical response in equine patients infected with the same pathogens, (3) the nature and site of the infectious disease process, (4) the pharmacokinetic characteristics of the chosen antimicrobial agent in horses such as bioavailability, tissue distribution, and rate of elimination, (5) the pharmacodynamic properties of the antimicrobial agent selected, (6) its safety in horses, and (7) the cost of therapy. in all situations, therapy should be adjusted based on initial clinical response and, when available, results of culture and susceptibility testing. 517 clinical theriogenology • volume 5, number 4 • december 2013 common bacterial pathogens of horses and their typical susceptibility patterns because the identity and in vitro susceptibility of an infecting microorganism are rarely known when therapy is begun, initial therapy is usually empirical and based on knowledge of the agents likely to be present and their historical susceptibility. in some cases, the most likely etiologic agent is highly predictable based on the clinical presentation and the horse’s history. for example, abscessation of the submandibular and retropharyngeal lymph nodes is most likely caused by streptococcus equi subspecies equi (s. equi). lower respiratory tract infection in adult horses is most commonly associated with s. equi subspecies zooepidemicus (s. zooepidemicus), followed by non-enteric gram-negative bacteria such as pasteurella spp. on the other hand, pleuropneumonia in an adult horse may be caused by any one or combinations of a number of bacteria and thus requires bacteriologic culture of a tracheobronchial aspirate and pleural fluid to determine etiology. similarly, peritonitis, urinary tract infections, musculoskeletal infections, cellulitis, and mastitis may be caused by a variety of bacteria, so that initial therapy with a broadspectrum antimicrobial agent is recommended. the selection of the antimicrobial agent and route of administration will depend on severity of disease and site of infection. many infectious diseases involving the neonatal foal such as pneumonia, peritonitis, meningitis, osteomyelitis, septic arthritis, and omphalophlebitis are the sequelae of bacteremia. gram-negative bacteria account for 70% to 95% of the microorganisms isolated from cultures of blood samples in equine neonates, with escherichia coli being by far the most common isolate.2,3 gram-positive cocci account for approximately 25% of isolates.4 treatment protocols for equine neonates must include antimicrobials with a high level of activity against enteric gram-negative bacteria while providing adequate coverage against gram-positive microorganisms. bacteria that are typically considered to be contaminants or part of the normal microflora need not be tested for susceptibility. however, when pathogenic bacteria are identified, selection of an antimicrobial agent is often simplified because some common equine pathogens have predictable in vitro susceptibility profiles. for example, β-hemolytic streptococci and pasteurella spp. are almost invariably susceptible to penicillin, ampicillin, and ceftiofur. in contrast, enterobacteraceae, pseudomonas spp., enterococcus spp. and staphylococcus spp. have unpredictable susceptibility profiles. in vitro susceptibility testing is particularly important for these bacterial species. causes of treatment failure therapeutic failure may occur when the disease process does not have a bacterial etiology, when there is a change in the bacterial population at the site of infection, or when the pathogens have become resistant to the chosen antimicrobial agent. therapeutic failure may also result from pharmacokinetic factors that are often underappreciated by clinicians, for example when there is poor diffusion of the drug at the site of infection. the rate and extent of penetration of a drug into most sites outside the vascular space are determined by the drug’s concentration in plasma, molecular charge and size, extent of plasma protein binding, and blood flow. thus, effective antimicrobial drug concentrations may not be attained in tissues that are poorly vascularized (e.g., abscess, sequestered bone). in other tissues such as the central nervous system, the eye, and the prostate, a lipid membrane provides a barrier to drug diffusion. lipophilic drugs such as macrolides, fluoroquinolones, tetracyclines, rifampin, trimethoprim, and chloramphenicol are more likely to diffuse across lipid membranes and reach therapeutic concentrations in these tissues. these drugs are also more likely to accumulate within cells and represent an advantage for the treatment of susceptible intracellular bacterial pathogens. therapeutic failure may occur when the microenvironment at the site of infection is not conductive to antimicrobial activity. for example, gentamicin requires an oxidative transport system to penetrate the bacterial membrane. therefore, a given microorganism may be susceptible to gentamicin in vitro but the drug may be ineffective in an anaerobic microenvironment. similarly, the acidic environment of infected tissues may reduce the efficacy of macrolides, fluoroquinolones, and aminoglycosides. thus, the goal of antimicrobial therapy is to select an antibiotic that, in addition to 518clinical theriogenology • volume 5, number 4 • december 2013 exhibiting good antimicrobial activity against the infecting microorganism, will achieve therapeutic concentrations in the infected area. of course, noncompliance is a major cause of treatment failure. this is particularly true for antimicrobials used in an unapproved manner, in which case data may not be available to determine an appropriate dosage regimen. nor can compliance be assumed when a horse owner treats an animal at a veterinarian’s direction. there is general consensus that noncompliance occurs in a high percentage of treatment regimens in human and veterinary medicine. for example, compliance rates of 44% to 64% for canine antimicrobial treatment have been reported.5,6 the reasons for noncompliance are manifold, and range from fractious animals to simple oversight on the part of the owner. minimum inhibitory concentration versus breakpoint – what is the difference? in vitro bacterial susceptibility is determined by disk diffusion, concentration-gradient, or dilution methodologies. disk diffusion provides qualitative susceptibility data whereas broth-dilution methods and the concentration-gradient test (e test) generate a minimum inhibitory concentration (mic) expressed quantitatively in µg/ml. all of the tests assess inhibition of bacterial growth rather than killing of the pathogen as the endpoint. susceptibility designations are determined by comparing the microorganism's mic (or zone of inhibition if the disk diffusion method is used) to clinical breakpoints established by the clinical laboratory standards institute (clsi). simply stated, an antimicrobial’s clinical breakpoint is the concentration above and below which specific bacterial isolates are categorized as susceptible, intermediate, or resistant. clinical breakpoints take an antimicrobial’s mic into consideration, but are based on additional interpretive criteria. specifically, clinical breakpoints are determined by (1) the range of in vitro mics of an antimicrobial for representative populations of specific bacterial pathogens, (2) pharmacokinetic parameters of the antimicrobial in target animal species (e.g., drug distribution at the site of infection), and when available, (3) results of clinical trials in the target species, the ultimate standard of efficacy. clinical breakpoints are relevant for the specific bacteria, specific drug, and specific organ system infected only. as an example, the breakpoint for ceftiofur in horses is only relevant to s. zooepidemicus in equine lung, and infections in other organs caused by aberrant s. zooepidemicus infections would not necessarily have the same clinical breakpoint. results of in vitro susceptibility tests are presented to the clinician by designating the pathogen as susceptible, intermediate, or resistant. the clsi defines the three susceptibility designations as follows:  susceptible – an infection caused by the specific isolate can be effectively treated with the recommended antimicrobial and dosage regimen; clsi generally requires clinical response rates of at least 80% at a specific mic before organisms are categorized as susceptible.  intermediate – an infection by the isolate can be treated at body sites where drugs are physiologically concentrated or when a high dosage can be used; also indicates a “buffer zone” that should prevent minor technical factors from causing major discrepancies in interpretations.  resistant – an infecting isolate is not inhibited by achievable concentrations of the drug with normal dosage schedules; clinical efficacy has not been reliable in treatment studies. interpreting mic values when species-specific breakpoints are used, pathogens with an mic below an antimicrobial’s susceptibility breakpoint have a higher probability for treatment success, and organisms with an mic above the resistance breakpoint have a lower probability of treatment success. however, there is no evidence that efficacy increases the further the mic is below the breakpoint. conversely, it should be noted that a relatively high mic in itself is not necessarily an indicator of resistance. some resistance breakpoints have been set at >32 µg/ml or higher (e.g., the resistance breakpoint for pasteurella multocida against tulathromycin for bovine respiratory disease is 64 µg/ml). for the equine practitioner, an important limitation in interpreting the results of in vitro susceptibility data is that breakpoints for only a small number of drugs (ceftiofur, gentamicin, ampicillin) have been established for specific infections in horses. for all other antimicrobials, the breakpoints have 519 clinical theriogenology • volume 5, number 4 • december 2013 been adapted from human or other domestic animal species data. for these antimicrobials, a result indicating susceptibility is unquestionably preferable to one indicating resistance. however, there are no data correlating the results to clinical efficacy and there is no guarantee that the breakpoint is valid for a given pathogen or site of infection in horses. for example, the clsi breakpoint for susceptibility to doxycycline is ≤4 µg/ml based on human pharmacokinetic and clinical efficacy data. administration of oral doxycyline to an adult horse at the recommended dosage of 10 mg/kg results in peak serum, synovial fluid and peritoneal fluid concentrations of approximately 0.5 µg/ml.7 a pathogen isolated from the synovial fluid of a horse with a mic of 4 µg/ml would be reported as susceptible even though such concentrations are far from achievable in horses. based on pharmacokinetic data in horses, a breakpoint of ≤0.25 µg/ml would be more appropriate as a susceptibility standard for doxycycline.7,8 thus the lack of equineand disease-specific interpretive criteria is one factor that may explain discrepancies between in vitro susceptibility and clinical response. by itself, in vitro susceptibility of a specific pathogen does not guarantee clinical outcome. other factors, such as the host animal’s age, immune status, and presence of mixed infections can contribute to individual clinical response. pharmacokinetic-pharmacodynamic data determine optimal dosage determination of the appropriate dose and dosing interval of an antimicrobial agent requires knowledge and integration of its pharmacokinetics and pharmacodynamic properties. the pharmacokinetic properties of a drug describe its disposition within the body and include drug absorption, distribution, metabolism, and excretion. pharmacodynamic properties address the relationship between drug concentration and antimicrobial activity. drug pharmacokinetic features, such as plasma concentrations over time and area under the serum concentration-time curve (auc), when integrated with mic values, can predict the probability of bacterial eradication and clinical success. these pharmacokinetic and pharmacodynamic relationships may also play an important role in preventing the genetic selection and spread of resistant strains. the most significant factor determining the efficacy of -lactams, trimethoprim-sulfonamide combinations, and most bacteriostatic agents such as macrolides, tetracyclines, and chloramphenicol is the length of time that serum concentrations exceed the mic of the pathogen.9 increasing the concentration of the drug several-fold above the mic does not significantly increase the rate of microbial killing. rather, it is the length of time that bacteria are exposed to concentrations of these drugs above the mic that dictates killing effect. therefore, optimal dosing of such antimicrobial agents typically involves frequent administration. other antimicrobial agents such as the aminoglycosides, fluoroquinolones, and metronidazole exert concentration-dependent killing characteristics. their rate of killing increases as the drug concentration increases above the mic for the pathogen, so it is not necessary or even beneficial to maintain drug levels above the mic between doses. thus, optimal dosing of concentration-dependant drugs involves administration of high doses with long dosing intervals. with aminoglycosides and fluoroquinolones, the optimal cmax-to-mic ratio is 8-10. some drugs exert characteristics of both time and concentration dependent activity. the best predictor of efficacy for these drugs is the 24-hour aucto-mic ratio. glycopeptides, rifampin and, to some extent, fluoroquinolones fall into this category.10 conclusion judicious use of antimicrobials consists of a discriminating approach to selection and administration of anti-infective agents that maximizes treatment efficacy while minimizing adverse effects and treatment failures. perhaps the most important aspects of judicious use are to follow initial empirical treatment with culture and susceptibility testing by a diagnostic facility that uses clsi standards, and then administer an antimicrobial agent with an indication and activity at the site of infection against the target pathogen that has been identified. 520clinical theriogenology • volume 5, number 4 • december 2013 references 1. morley ps, apley md, besser te, et al: antimicrobial drug use in veterinary medicine. j vet intern med 2005;19:617629. 2. marsh ps, palmer je: bacterial isolates from blood and their susceptibility patterns in critically ill foals: 543 cases (1991-1998). j am vet med assoc 2001;218:1608-1610. 3. wilson wd, madigan je: comparison of bacteriologic culture of blood and necropsy specimens for determining the cause of foal septicemia: 47 cases (1978-1987). j am vet med assoc 1989;195:1759-1763. 4. sanchez lc, giguère s, lester gd: factors associated with survival of neonatal foals with bacteremia and racing performance of surviving thoroughbreds: 423 cases (1982-2007). j am vet med assoc 2008; 233:1446-1452. 5. grave k, tanem h: compliance with short-term oral antibacterial drug treatment in dogs. j small anim pract 1999;40:158-162. 6. adams vj, campbell jr, waldner cl, et al: evaluation of client compliance with short-term administration of antimicrobials in dogs. j am vet med assoc 2005;226:567-574. 7. bryant je, brown mp, gronwall rr, et al: study of intragastric administration of doxycycline: pharmacokinetics including body fluid, endometrial and minimum inhibitory concentrations. equine vet j 2000;32:233-238. 8. davis jl, salmon jh, papich mg: pharmacokinetics and tissue distribution of doxycycline after oral administration of single and multiple doses in horses. am j vet res 2006;67:310-316. 9. andes d, anon j, jacobs mr, et al: application of pharmacokinetics and pharmacodynamics to antimicrobial therapy of respiratory tract infections. clin lab med 2004;24:477-502. 10. craig wa: basic pharmacodynamics of antibacterials with clinical applications to the use of beta-lactams, glycopeptides, and linezolid. infect dis clin north am 2003;17:479-501. additional reading papich mg, davis jl: antimicrobial therapy. in: sellon dc, long mt, eds. equine infectious diseases. st louis: saunders-elsevier; 2007. p. 578-591. weese s: prudent use of antimicrobials. in: giguère s, prescott jf, baggot jd, et al, eds. antimicrobial therapy in veterinary medicine, 4th ed. ames(ia): blackwell publishing; 2006. p. 437-446. wilson wd: rational selection of antimicrobials for use in horses. proc annu conv am assoc equine pract; 2001. p. 75-93. 521 clinical theriogenology • volume 5, number 4 • december 2013 522clinical theriogenology • volume 5, number 4 • december 2013 omniblank: luteinizing hormone receptor mediated proliferation of isolated canine t lymphoma cells luteinizing hormone receptor mediated proliferation of isolated canine t lymphoma cells camryn flint, sabrina gust, michelle kutzler department of animal and rangeland sciences, oregon state university, corvallis, or luteinizing hormone receptor (lhr) have been identified in nonreproductive tissues, including in canine lymphoma and isolated canine t lymphoma cells.1 the objective was to determine if the lhr in canine t lymphoma cells was functional. it was hypothesized that stimulation of the lhr by human chorionic gonadotropin (hcg) or canine lh (clh) induces proliferation of canine t lymphoma cells in vitro. immortalized t cell lines from 3 dogs with multicentric t cell lymphoma donated from dr. takuya mizuno at yamaguchi university, japan were plated in 96 well plates in rpmi 1640 (phenol and protein free) media (#02-0105, vwr life science, sanborn, ny) at 370c with 5% co2. for each cell line, standard curves from 10 to 500 x 103 cells/well were plated in triplicate. increasing concentrations of hcg (4 40,000 u/ml, chorulon®, merck animal health, madison, nj) or canine lh (0.002 to 20 ng/ml, #qp604-ec, enquire bioreagents, denver, co) were added to wells containing 100 x 103 cells plated in triplicate. plates were incubated for 24, 48, and 72 hours for hcg or 48, 72, 96, and 120 hours for clh before cells were counted using an mtt cell proliferation assay kit (#10009365, cayman chemical, ann arbor, mi) following manufacturer's instructions. average ± sd cell number was compared among hormone concentrations using one way anova. significance was defined as p < 0.05. activation of lhr in isolated canine t lymphoma cells induced significant cell proliferation in all 3 cell lines with both hcg and clh, but varied among concentrations and incubation times. greatest proliferation from hcg occurred with 40,000 u/ml after 72 hours of incubation in all 3 cell lines. greatest proliferation from clh occurred with 2 ng/ml after 96 hours of incubation in all 3 cell lines. however, there was a significant decrease in cell counts following administration of clh at the highest concentration (20 ng/ml) at all times of incubation. to the author's knowledge, this was the first study to provide evidence that lhr in nonreproductive tissues is functional. these findings provided an explanation for why gonadectomized dogs are 3 4 times more likely to develop lymphoma.2,3 clinical trials are planned to include lh downregulation with conventional chemotherapy in efforts to prolong survival times in dogs. keywords: cancer, dog, human chorionic gonadotropin, lymphocyte, mtt assay references 1. ettinger a, gust s, kutzler ma: luteinizing hormone receptor expression in normal and neoplastic canine lymphocytes. am j vet res (accepted 9/13/2018). 2. torres de la riva g, hart bl, farver tb, et al: neutering dogs: effects on joint disorders and cancers in golden retrievers. plos one. 2013;8(2). 3. zink mc, farhoody p, elser se, et al: evaluation of the risk and age of onset of cancer and behavioral disorders in gonadectomized vizslas. j am vet med assoc 2014;244:309-319. 477 clinical theriogenology • volume 11, number 3 • september 2019 478clinical theriogenology • volume 11, number 3 • september 2019 omniblank: four healthy canine litters from sperm retrieved from urine after retrograde ejaculation four healthy canine litters from sperm retrieved from urine after retrograde ejaculation mateja stvarnik, maja pipan clinic for reproduction and large animals, veterinary faculty university of ljubljana, slovenia retrograde ejaculation is a medical condition in which semen enters urinary bladder instead of traveling through the urethra after ejaculation. in some cases, a limited amount of semen appears in the ejaculate. complete retrograde ejaculation causes male infertility. a 6 year old portuguese water dog male was presented for fertility assessment. after siring a few successful litters, he became infertile. on clinical and neurological examination, no abnormalities were noticed. there was no history of trauma or any surgical procedure. good libido was observed during the semen collection, but only 0.2 ml of semen was obtained with really low sperm concentration (15 x 106/ml), with 80% progressively motile sperm. following the second semen collection, urine was obtained via cystocentesis. urinalysis after ejaculation revealed a total of 640 million sperm with 4% motility. treatment with phenylpropanolamine for 7 days slightly increased ejaculate volume (first semen collection 0.5 ml, second semen collection 0.4 ml). since number of sperm was too low for insemination (48 x 106/ml and 53 x 106/ml, respectively), a decision was made to retrieve sperm from urine. the dog was given 650 mg of sodium bicarbonate, twice 12 hours apart, prior to semen collection in order to alkalize his urine. after centrifugation of the urine and washing the precipitate, extracted sperm were used for transcervical insemination in 2 bitches (both became pregnant). the procedure was repeated twice in 3 months; 3 more bitches were inseminated and 2 became pregnant. keywords: retrograde ejaculation, portuguese water dog, pregnancy 517 clinical theriogenology • volume 11, number 3 • september 2019 518clinical theriogenology • volume 11, number 3 • september 2019 omniblank: historyitem_v1 trimandshift range: all pages trim: none shift: move down by 13.50 points normalise (advanced option): 'improved' keep bleed margin: no d:20190702122621 32 1 0 0 full 1521 302 fixed down 13.5000 -20.2536 both 1 alldoc 2 currentavdoc none 0.0000 top qite_quiteimposingplus4 quite imposing plus 4.0m quite imposing plus 4 1 0 1 0 1 1 historylist_v1 qi2base life threatening complications of a third degree perineal laceration in a quarter horse mare life threatening complications of a third degree perineal laceration in a quarter horse mare viviane gomes, natalia bernstein,a nathan souza, hugo de almeida, clara mota, poliana botelho veterinary teaching hospital renato de andrade, faculdades integradas do norte de minas montes claros, mg, brazil aprivate practitioner, curvelo, mg, brazil third degree perineal laceration involves complete disruption of the rectovestibular shelf, perineal body and anal sphincter, resulting in a common vault between the rectum and the vaginal vestibule. it is generally agreed that immediate attempts to repair the laceration are often unsuccessful and surgical reconstruction should be delayed for at least 3 6 weeks to allow second intention healing. however, there is limited information regarding potential life threatening complications in the presurgical second intention healing phase. a 3 year old primiparous quarter horse mare was presented for a third degree perineal laceration, extending approximately 20 cm cranially into the perineal body. foaling was unattended and a live foal delivered. at 24 hours postpartum, there was severe inflammation and infection of involved tissues, but the vestibulovaginal fold remained functional. treatment included tetanus prophylaxis, flunixin meglumine (1.1 mg/kg iv sid for 7 days) and sulfamethoxazole/trimethoprim (30 mg/kg po sid for 30 days), as well as daily wound cleaning, topical antibiotic ointment, and dietary changes to soften feces. at 47 days postpartum, the mare had intense abdominal contractions (1 2 contractions per second) eversion of the vestibulovaginal floor and external urethral sphincter. these clinical signs did not subside after initial treatment with n butylscopolammonium bromide (160 mg iv) and flunixin meglumine (500 mg iv) administered on farm. the mare was transported to a referral equine service at the veterinary teaching hospital renato de andrade (minas gerais, brazil). upon arrival, contractions were suppressed by epidural anesthesia with a combination of 5 ml of lidocaine 2% and 1 ml of xylazine 10%. the uterus was involuted, with no edema or intraluminal fluid. no abnormalities were identified in the cervix via a vaginal speculum exam. external urethral sphincter was severely inflamed and assumed to be the inciting factor for signs of discomfort and recurrent abdominal contractions. straining resumed approximately 5 hours after epidural anesthesia and the mare presented intense pollakiuria. epidural anesthesia was given every time the abdominal contractions and vestibulovaginal eversions became more frequent and severe (~ twice daily for the first 5 days, then once daily for 3 days). a course of enrofloxacin (5.5 mg/kg im sid) and flunixin meglumine (1.1 mg/kg iv sid) was implemented. additionally, temporary relief was achieved for: 1) 2 3 hours with topical lidocaine spray on the external urethral sphincter; 2) 7 hours with phenazopyridine (4 mg/kg po tid); and 3) 4 5 hours after voiding urine from the bladder with a foley catheter and infusing 200 ml of cold lactate ringer solution with 10 ml of lidocaine 2%. eight days after presentation to the referral center, the urethritis seemed to be resolving; however, the mare still had mild contractions 1 2 times a day, which precluded any attempt to perform a surgical perineal reconstruction. at 16 days, despite responding moderately to the palliative care, the mare suddenly exhibited severe contractions that led to bladder eversion and rupture, accompanied by evisceration and sudden death. this report highlighted the need for frequent medical monitoring during second intention healing phase to address any potential life threatening complications that may arise secondary to perineal lacerations. keywords: third degree perineal laceration, urethritis 491 clinical theriogenology • volume 11, number 3 • september 2019 492clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2016: canine inherited myopathy as a translational model canine inherited myopathy as a translational model martin k. childers,a,b mushtaq memon,c joe n. kornegay,d david l. macka,b adepartment of rehabilitation medicine, and binstitute for stem cell and regenerative medicine, university of washington, seattle, wa; cdepartment of veterinary clincial sciences, washington state university, pullman, wa,; ddepartment of veterinary integrative biosciences, college of veterinary medicine, texas a&m university, college station, tx abstract the inherited myopathies include channelopathies, myotonias, metabolic and mitochondrial conditions, congenital myopathies, and the muscular dystrophies. this review focuses on a single centronuclear (congenital) myopathy, termed x-linked myotubular myopathy (xlmtm). x-linked myotubular myopathy of labrador retrievers have recently become a valuable model for preclinical studies. in developing preclinical trials in dogs, the clinical course of the xlmtm syndrome in labrador retrievers evolves rapidly, so affected dogs must be treated and assessed as early as ten weeks of age. although interventional therapeutic studies in xlmtm dogs are in the early preclinical stages, promising results have already been seen. having a dependable breeding program is absolutely essential to the success of these preclinical experiments. future studies using this canine model should shed further light on the human disease. keywords: inherited myopathies, centronuclear myopathies, x-linked myotubular myopathy (xlmtm), myotubularin, preclinical trials introduction inherited and acquired myopathies in dogs are becoming more widely recognized for how well they recapitulate their respective human conditions. from clinical presentation to the underlying ultrastructural and biochemical etiology of disease initiation and progression, canine muscle resembles human muscle to a remarkable degree. smaller laboratory animal models, like mice, have provided a powerful tool to study the molecular pathogenesis of disease for decades, partly because transgenic or gene knock-down/out strains can be created quickly and relatively inexpensively. however, it is becoming clear that drug trials tested exclusively in mice often fail to be predictive of efficacy and/or toxicity in human clinical trials.1 this dependence on model species that do not extrapolate to humans with high fidelity, because of differences in size, drug biodistribution, anatomy or physiology,2, 3 has resulted in an expensive and time-consuming “fail late” phenomenon in the drug discovery pipeline.4 these shortcomings are mostly avoided by utilizing canine models because of their similarity to humans in metabolic, physiological and anatomical characteristics.5 in addition, our understanding of the genetic similarities between dogs and humans is growing, augmented by the sequencing of the canine genome more than ten years ago.6 therefore, the translational value of naturally occurring canine models of disease will likely increase as more studies corroborate their ability to predict therapeutic efficacy and safety in humans.7 several sporadic and inherited myopathies have been described in dogs and have provided the opportunity to study the underlying disease mechanisms that are analogous in human conditions and to develop preclinical models. acquired myopathies include both infectious8 and immune-mediated inflammatory conditions,9 as well as metabolic and endocrine diseases.10 the inherited myopathies include channelopathies, myotonias, metabolic and mitochondrial conditions, congenital myopathies, and the muscular dystrophies.11 inherited muscular dystrophies and congenital myopathies, arising from mutations in genes coding for several components of the dystrophin-glycoprotein complex and proteins involved in membrane remodeling, respectively, have been described in dogs (table 1).12-15 this review focuses on the condition homologous to a centronuclear myopathy (cnm) of human beings--specifically,xlmtm. at this stage, a clear distinction should be made between the centronuclear myopathies and the more familiar muscular dystrophies. dystrophies typically present with cycles of degeneration and attempted regeneration, leading to myofiber necrosis, fibrosis and fatty deposition. clinical theriogenology • volume 8, number 4 • december 2016449 historically, muscular dystrophies were classified based primarily on their pattern of inheritance and the particular muscle groups involved.16, 17 over the last decade, distinct subtypes of muscular dystrophies have arisen as specific mutations are identified, primarily in the proteins that make up the dystrophinglycoprotein complex.17 in contrast, the centronuclear myopathies are caused by mutations in genes whose proteins are involved in membrane remodeling within the myofibers.18,19 in general terms, myopathies are distinguished from dystrophies because of their relatively non-progressive histologic nature and the presence of specific structures, like cores or nemalin rods within muscle fibers.20 canine model of x-linked myotubular myopathy the diagnosis of centronuclear myopathy refers to a subset of congenital myopathies characterized by skeletal muscle weakness and a majority of muscle fibers with centrally located nuclei and/or cores on biopsy. among the congenital myopathies, murine and canine models exist for most of the genetically distinct cnms, including dynamin2, protein tyrosine phosphatase-like member a (ptpla),21,22 amphiphysin (bin1)23,24 titin and the ryanodine receptor,25 (table 2). the most common cnm, xlmtm, is caused by mutations in the myotubularin 1 gene (mtm1) and affects about 1 in 50,000 male births.26 newborn males with xlmtm show a diverse clinical presentation that includes decreased fetal movement, low apgar scores, large head circumference, areflexia, cryptorchidism, a need for immediate ventilator support and, of course, profound hypotonia.26-28 boys with severe mutations who survive their first two years will never walk and will be dependent on a ventilator so development of speech is precluded. two murine models have been valuable in studying initiation and progression of the pathology and have served as a proof-of-concept platform for gene therapy efficacy and safety, but have inherent limitations for preclinical testing.29-32 the labrador retriever model of xlmtm, with a p.n155k mutation in the mtm1 gene,33,34 has proven enormously valuable in that it recapitulates the pathology observed in human patients and is a similar size to affected infants and young boys. this model will be the focus of this review, but it should be noted that a recent paper identified a second mutation, p.q384p in a family of australian rottweilers that showed similar generalized, progressive and fatal myopathy in the second to third month of life.35 to appreciate the pre-clinical utility of the canine model of xlmtm, it is important to understand the disease onset and progression from birth to humane euthanasia in the laboratory setting, and how it compares to patients with this devastating disease. a comprehensive natural history study from our laboratory36 documented the decline in limb strength, walking gait and respiratory function in young dogs as a consequence of the appearance of typical cnm histological biomarkers. these results corroborated and expanded on earlier studies of this particular p.n155k mutation.33,34,37-39 labrador retriever puppies with xlmtm have lower birth weight, grow more slowly and display mild muscle atrophy and pelvic limb weakness up to about seven weeks of age.33,34 patellar hyporeflexia, dysphagia, dropped jaw and a hoarse bark gradually appear over time. we have been struck by how little phenotypic variation occurs in our laboratory colony, even after outcrossing labrador female carriers with beagle males in an attempt to interject genetic diversity and decrease the size of the dogs.36 by ten weeks of age clear signs of the myopathy are present, and muscle weakness and atrophy progress rapidly leading to loss of ambulation, necessitating euthanasia by four to six months of age. an age equivalency paradigm used for a canine muscular dystrophy model was proposed.40 assuming a parallel between the first three months of a labrador retriever’s life and first five years of a human’s, the four to six month course of xlmtm in labradors is somewhat delayed compared to the progression of xlmtm in humans.28 our principal outcome measure has always been limb strength, because assessing muscle function during the downward progression of the disease and to test potential therapeutic interventions should have the greatest impact on quality of life of the patients. using physiology instrumentation to measure joint forces, torque generated across tibiotarsal or metatarsal joints in the hindlimbs or forelimbs, respectively by xlmtm dogs at ten weeks is ~40% lower than normal. by 18 weeks-of-age, torque generated by affected dogs is only ~15% of that of wild-type dogs.36,39 subjecting the dogs to a series of eccentric contractions and testing their force output gives us a glimpse into the underlying pathophysiology of xlmtm. in contrast to the progressive force drop observed in a canine muscular dystrophy model, clinical theriogenology • volume 8, number 4 • december 2016 450 xlmtm dogs display a progressive increase (summation) in the isometric phase. despite a progressive increase during repeated contractions, maximum isometric torque never exceeds values observed in normal controls or carriers.39 mutant dogs generate a gradual increase in isometric torque during a series of eccentric contractions until activations cease. following a brief rest, the next eccentric contraction begins at a lower initial torque. the pronounced torque increase following repeated stretch activations in xlmtm dogs is in keeping with findings in ryanodine receptor (ryr1) mutant mice.39,41 both mtm1 mutant dogs and mtm1 mutant mice display similar increases in force summation with repetitive nerve stimulation. while the precise molecular mechanism underlying these observations is unknown, a defect in excitation-contraction (e-c) coupling is likely responsible. in addition, affected dogs exhibit rightshifted torque-frequency curves compared to those of the normal and carrier dogs indicating that an e-c coupling defect is a central feature of this mutant canine model. the single most burdensome aspect of xlmtm for young male patients, their families and fulltime nurses is the absolute dependency on ventilator support. therefore, careful assessment of respiratory indices has been a primary component of the phenotypic characterization of the canine model.32,38 affected dogs demonstrate paradoxical breathing at ~10 weeks and, when compared to wild-type dogs on serial measurements, have reduced inspiratory and expiratory flow rates before and after dosing with doxapram, suggesting involvement of the diaphragm. ultrasonography images of the diaphragm were acquired from normal and affected xlmtm dogs42,43 and showed that the normal diaphragm appeared thicker and less echogenic than in the xlmtm dog. lastly, walking gait of the xlmtm dogs was assessed using video-based motion capture analysis and an instrumented carpet.36,37 instrumented carpets with imbedded sensors coupled to customized software allows investigators to measure spatiotemporal gait characteristics, such as overall speed, step length, step width and stride length. however, spatio-temporal data alone do not capture postural changes or range-of-motion (rom) excursion; kinematic (motion-capture) analysis is required to assess these parameters. x-linked myotubular myopathy dogs appear to maintain relatively normal joint range-of-motion, but walk slower than normal with shorter stilted strides. these differences become greater over time, suggesting that slowing of walking gait generally correlates with progressive limb weakness. production of forelimb and hindlimb torque, diaphragmatic strength as assessed by peak inspiratory flow and walking gait speed and stride length constitute the core outcome measures used to track disease progression. the fact that these assessments are sensitive, repeatable and non-invasive make them well suited to test for correction of the disease phenotype after systemic treatment with aav8-mtm1 gene therapy. to summarize briefly, p.n155k mutant dogs were assessed at nine weeks to establish baseline readouts, then given approximately 1x1014 of an adeno-associated viral vector carrying the normal copy of the mtm1 transgene at ten weeks of age.44 two dogs treated with this regimen showed normalized tibiotarsal joint torque and peak inspiratory flow rates six to seven weeks after infusion. moreover, all treated dogs remained ambulatory for more than three years, well beyond the age of 18 weeks when untreated xlmtm dogs can no longer walk. these two treated males remain in the xlmtm colony and have successfully bred to carrier females producing several litters of dogs, indicating the sustainability of gene replacement therapy. xlmtm canine colony breeding program these experiments, plus a much larger dose-finding study involving 22 dogs could not have been completed without a successful breeding program. to our knowledge, this is the first instance in which two dogs, affected with a fatal monogenic disease of muscle, have been treated and successfully bred to carrier females to produce multiple offspring. this is important given the x-linked mode of inheritance of myotubular myopathy. before this breakthrough, carrier females were bred with normal males, producing one-quarter carrier females and one-quarter affected males. however, now by breeding affected-treated males with carrier females we can produce one-quarter affected males, but also carrier and homozygous affected females, thus enabling us to keep breeding and colony management costs down. in addition, xlmtm human females have been reported, although they are rare but having affected clinical theriogenology • volume 8, number 4 • december 2016451 female dogs in our colony allows us to test the gene therapy on the opposite sex, thus fulfilling another food and drug administration requirement for approval in clinical trials. because of the central importance of the xlmtm dog colony breeding program, the remainder of this review will focus on the reproductive methods employed to increase the likelihood of a successful natural coupling, and guarantee the long-term survival and inexpensive dissemination of this valuable model through semen preservation. the following summary is adapted from the following book chapters and journal articles.45-50 female reproduction the estrous cycle in the female dog is unique and different from other domestic animals. it starts with proestrus. most breeders refer proestrus as start of the ‘season’. bitch owners notice spotting or bleeding from the vagina. during proestrus, the bitch attracts a male but will not allow him to breed. estrogen produced by ovarian follicles causes change in the epithelial cells of the vaginal mucosa, which we will discuss in great detail. estrus or the time of receptivity follows the proestrus, when the bitch accepts the male for breeding. the duration of proestrus and estrus cited in textbooks is nine days each. the authors agree with these times as an average but unfortunately they have not found many bitches following the textbook and have a nine-day schedule. the authors have seen bitches with a range of 2-21 days of each phase. during proestrus, the vulvar lips of the bitch become turgid or firm which become soft and ‘wrinkly’ towards receptivity phase of the bitch. the bloody discharge of the proestrus also changes to straw colored discharge towards estrus in most bitches but some bitches bleed all the way through estrus. the production of progesterone (p4) is also unique during estrous cycle of the bitch. the early rise of the p4 during estrus is from the luteinized ovarian follicles and is useful in breeding management which will be discussed later. the bitch ovulates with the help of luteinizing hormone (lh). ovulation occurs about three days after the lh peak. after ovulation, the follicles are replaced by corpora lutea (cls) which produce p4. the production of p4 continues throughout diestrus, the next phase of estrous cycle. the duration of diestrus is the same as the pregnancy, about 62-63 days from the lh peak. even the bitch is not pregnant; p4 will be produced for the duration of diestrus. it appears that there is no production of prostaglandin f2alpha (pgf2alpha; as in other domestic animals) from the endometrium to cause luteolysis (cl regression). during diestrus, many bitches go through ‘pseudopregnancy’. the bitch may gain weight with an enlarged abdomen and start showing overt signs of pseudopregnancy during late diestrus, including mammary gland enlargement, nesting, adopting toys and shoes, and giving impression that she is about to whelp. pseudopregnancy (or diestrus) is considered a normal occurrence, and may not need any treatment. ovulation the ova released from the ovary during ovulation in the bitch are at the primary oocyte stage, i.e. the first polar body has not come out of the ovum and sperm are not able to penetrate the ovum. it takes about three days for the ovum to become a secondary oocyte and ready to be penetrated by sperm. dog sperm can survive up to ten days in the bitch’s reproductive tract. it can become a diagnostic challenge when a bitch’s owner asks for a cesarean section 62 days after breeding. theoretically that pregnancy (62 d after breeding) could be as early as 52 days. a few tools are avilable to predict whelping. hypothermia (2-3°f) or a decrease in rectal temperature, caused by p4 decrease 12 to 36 hours before whelping is fairly reliable to predict delivery. the indication of diestrus day 1 (d1) by vaginal cytology is another reliable way to predict whelping 57-58 days from d1. following is a discussion of the use of vaginal cytology in breeding management. reproductive anatomy. for successful vaginal swabbing technique, the operator needs to appreciate the unique reproductive anatomy of the bitch. the vagina of the bitch is very long, about 20 cm (~9 inches) in a medium size bitch. the cervix in the bitch is located in the abdominal cavity and is different than other domestic animals (e.g. cow, mare) where cervix is located at the pelvic inlet. therefore, cervix in the bitch cannot be visualized by a speculum examination but can be visualized with a flexible endoscope and light source. this becomes important when performing artificial insemination clinical theriogenology • volume 8, number 4 • december 2016 452 (ai) and wish to deposit semen transcervically into the uterus. artificial insemination and semen deposition will be discussed in more detail below. the size of the uterus and ovaries vary considerably with the breed. vaginal cytology. a clean cotton tipped swab is commonly used for swabbing the vagina for the cytology sample. one can use vaginal speculum to guide the swab. a right-handed operator holds the bitch’s vulva with the gloved left hand, opening the vulvar lips with the thumb and the middle finger, while placing the index finger behind the vulva to support it. the swab is inserted with the right hand almost vertically into the vagina avoiding the clitoral area, dorsally and over the brim of the pelvis. the swab is moistened with warm tap water before use which is helpful to advance the swab especially in small size bitches and bitches without a serosangious discharge. if the swab is ‘stuck’ in the vaginal folds, it is slightly withdrawn, redirected and advanced. once the swab is in the vagina for at least 6-10 cm (2-4 inches), the swab is rolled a few times in one direction (if rotated back and forth, the cotton may unroll and drop in the vagina). the authors recommend advancing the swab as far as possible as there is less debris on swabs taken from the cranial vagina compared to the caudal vagina. getting into the habit of reaching the cranial vagina is also be helpful for taking vaginal cultures and for ai. the swab is rolled on a clean microscope slide, air-dried and stained with romanowski stain (diff-quick, harleco, gibbstown, nj). other stains used by practitioners include methlyene blue, eosin-nigrosin, gram stain, etc but the authors have found romanowski stain satisfactory for staining epithelial cells as well as white blood cells (wbcs). the slides with smears are dipped five to seven times in each solution, rinsed with tap water, air-dried and examined under the microscope. the authors start under 200x magnification to get an overall impression and distribution of the cells before moving on to 400x magnification to closely examine the desired field. various theriogenologists, clinical pathologists and other practitioners have interpreted vaginal cytology of the bitch differently but for this presentation discussion will be limited to that previously published.51 the vaginal mucosa is responsive primarily to estrogens (e2), and vaginal cytology is useful only during estrogenic phase of the cycle. parabasal cells are small and round with large and distinct nuclei. the total area of the cytoplasm of the cell is smaller than the nucleus. these cells (along with red blood cells; rbcs) are present during proestrus. superficial intermediate cells are larger than parabasal cells with small nuclei and irregular/folded borders. large numbers of these cells are observed during late proestrus to early estrus. superficial cells, also called cornfield or anucleated, are the largest of the epithelial cells present during estrus. under a simple microscope, the nuclei of these cells appear faded or absent. the cells also appear ‘light in weight’, multi-layered, and have folded borders. the appearance of 80-90% of these cells in the smear is used as an indication to start breeding. a series of vaginal cytology samples is needed to observe the progressive change in epithelial cells. a single sample is unreliable. some bitches display same type of epithelial cells for many days, whereas others change within 24 hours. many bitches reach to 80-90% cornification during estrus but many bitches never go above 70% cornification. obviously, if one waits for 80-90% cornification to breed, these bitches will be missed. the other tool useful in managing breeding of these bitches is to measure blood p4, which will be discussed below. if multiple cytology samples are obtained, neutrophils appear and this is the first day of the diestrus or d1. this finding is important because the bitch is going to whelp 57-58 days after d1. the authors recommend taking cytology every day, because in some bitches the change from cornfield to superficial intermediate and the appearance of neutrophils happens within 24 hours. most breeders and bitch owners are able to obtain vaginal cytology samples at home after brief training. the swabs are brought to the clinic, stained and evaluated. progesterone assay. as discussed above, in addition to the cl, p4 also is secreted by luteinized follicles in the ovaries. the early p4 rise can be used to determine ovulation in the bitch. ovulation takes place at about three days after the lh peak which is very short. luteinizing hormone assays are available and it is recommended to measure lh daily when ovulation is expected. progesterone assays are commonly used for canine breeding management. enzyme linked immunosorbent assay (elisa) kits (synbiotics corp., san diego, ca) are available to determine p4. these are qualitative tests based upon a color change which is interpreted as a range of a p4 concentration. many clinicians prefer to send the clinical theriogenology • volume 8, number 4 • december 2016453 blood samples to an endocrinology laboratory for p4 determination by radioimmunoassay (ria). progesterone is the same hormone in all species, including humans; therefore blood samples can be analyzed for p4 in veterinary or in human hospital laboratories. progesterone assays are an excellent tool to determine ovulation time for breeding at the appropriate time. this becomes critical when using chilled transported or frozen-thawed semen for ai. like vaginal cytology, p4 concentration is also monitored starting few days after the start of proestrus; p4 concentrations in range of 2-10 ng/ml, with 2-2.9 ng/ml indicative of ovulation in about two days, 3-3.9 in one day, and 4-10 ng/ml indicative of the ovulation day. basic understanding of canine semen preservation and artificial insemination for semen preservation and ai, semen is collected and evaluated in the same manner as for male breeding soundness examination (bse). depending upon the use of semen fresh, chilled or frozen semen is processed accordingly. ai with fresh semen artificial insemiation is performed when the male and the female are at the same location but are unable to breed. the semen is usually deposited immediately after collection without adding any extender. two to five ml of semen is inseminated. some of the reasons that bitch and the stud dog would not breed and may require ai include:. the bitch is not ready. the most common reason for fresh semen ai is when the dog owner ‘believes’ the bitch is ready but she does not stand for the male. it is important that the veterinarian examine the bitch including vaginal cytology to determine if she is ready. in few instances, the authors have performed ai when cytology showed only 50-60% cornification (superficial anucleated vaginal epithelial cells). the reason is that some bitches don’t reach more than 70% cornification and they ovulate. progesterone assay would be helpful to confirm this, but for the owner of the bitch and the stud dog, it is a less expensive option to have an ‘extra’ ai performed. for ai, the authors use half of a cow ai pipette attached to a rubber connector, and other end of the connector is attached to the syringe with semen. canine ai pipettes are available commercially. the pipette is inserted into the vagina in manner similar to that used for taking vaginal smear. the authors prefer to pass the pipette up and over the brim of the pelvis, depending on the size of the bitch, at least four inches, and deposit the semen in the cranial part of the vagina. the bitch’s rear legs are raised for about 20 minutes to prevent semen back-flow and encourage the sperm transport through the cervix. ai with chilled transported semen this is a common procedure used when male and female are separated by long distances. frequent airline transportation has made it possible to ship semen across the country, and in many locations between north america and western europe within 24 hours. with strict airline regulations related to animal transport, it has become very difficult or impossible to ship a dog by air. appropriate coordination and communication between the owners of the female and male is important for success of ai with transported semen. the bitch owner works with a veterinarian to manage the breeding by monitoring the bitch’s vaginal cytology and blood p4. the authors recommend that examinations of the bitch begin two to three days after onset of proestrus (spotting or bleeding) and that monitoring continues at one to two day intervals. steps in shipping semen collect semen dilute the semen by adding semen extender. dilution rate will depend upon the total number motile and morphologically normal sperm in the ejaculate (refer to discussion of dog bse below). it is a common practice to dilute semen 1:2 (1 part semen: 2 parts extender). the basic concept of a semen extender is to provide appropriate environment and energy for the sperm to survive. skim milk, tris-egg clinical theriogenology • volume 8, number 4 • december 2016 454 yolk and many other extenders have been utilized for dog semen. if using a commercial semen extender, follow the manufacturer’s directions very closely. semen packaging and shipping. semen cooling rate and temperature control are two important factors when selecting a shipping container. numerous closed-cell extruded polystyrene foam boxes and other containers are available commercially. the authors’ experience when receiving semen in homemade containers has been mixed with sperm motility ranging from 5 to 70%. in many cases, semen is packed touching the ice packs, resulting in cold shock to sperm. the semen is shipped for next day delivery. insemination timing. as discussed above, proper insemination timing is crucial for successful outcome of ai with chilled or with frozen-thawed semen. in addition to vaginal cytology, blood p4 assay (preferably by ria) is very helpful to monitor the time of ovulation. like vaginal cytology, p4 concentration is monitored starting few days after start of proestrus, using p4 concentrations in range of 2-10 ng/ml, with 2-2.9 ng/ml indicative of ovulation in about two days, 3-3.9 ng/ml in one day, and 4-10 ng/ml indicative of the day of ovulation. as epithelial cells change from parabasal to intermediate superficial cells, p4 is measured to obtain a baseline of 2ng/ml and repeated every 24 hours, until concentrations reach 5 ng/ml. insemination may be performed two to three days later. ai with frozen-thawed semen semen freezing technology was developed in the 1960’s to preserve desirable genetic material (semen) from bulls for future use. since then this technology has been perfected to the point that more than 60% of the dairy cows in the us and canada are bred with frozen-thawed semen. it is also interesting to note that proven (progeny tested) bulls in the us are kept in a few locations, semen is collected, frozen, stored and sold almost all over the world. even though reports of freezing dog semen were published in late 1960’s, it was not until recently that frozen-thawed dog semen has been used on commercial basis. obvious advantages of the frozen semen are that semen can be collected from a valuable dog (preferably at a young age), frozen and kept for very long time (almost forever) and used even after the dog’s death. semen is collected (as discussed below) and extended. semen extender used for freezing has an additional component of cryoprotectant (e.g., glycerol) to protect the sperm membrane from injury by ice crystals formed during freezing and thawing process. semen is commonly packed in mini straws (0.25-0.5 ml), frozen in liquid nitrogen (ln) vapor and stored in ln. the procedure for thawing semen varies depending upon the freezing rate of the semen. therefore, it is important to follow the thawing instruction received from the semen supplier. one common thawing technique used is to thaw frozen semen in straws at 370c in a water bath for 60 seconds. it is important to note that semen and water do not mix. make sure water does not come into contact with semen during the thawing procedure. artificial insemination with frozen-thawed semen requires deposition of semen into the uterus of the bitch at the appropriate time. even though the dog deposits semen in the vagina during copulation, and chilled semen is deposited in the vagina, frozen-thawed sperm lives for a much shorter time therefore it is desirable to deposit semen directly intp the uterus. some practitioners have been successful getting bitches pregnant by vaginal deposition two to three times with frozen-thawed semen and monitoring vaginal cytology and blood p4. the following techniques are commonly used for ai with frozen-thawed semen. surgical ai. a 4-6cm midline incision over the pubis is made in an anesthetized bitch after routine surgical preparation for a laparotomy. the uterus is exteriorized and the semen is deposited into the lumen of the uterine body at a 450 angle with the bevel of the needle facing up. the operator should be able to see the distension of the uterine horns as the semen in injected, otherwise needle should be repositioned. saline moistened gauze is held over the injection site for a minute or so after the needle is withdrawn. the uterus is replaced in the abdomen and the incision is closed. clinical theriogenology • volume 8, number 4 • december 2016455 norwegian catheter. a stainless steel catheter protected by a plastic sheath is used for transcervical intrauterine ai. three different length catheters are available for various size dogs. the cervix is fixed between operator’s fingers through the abdominal wall of the standing bitch. the catheter is passed through the vagina and the tip of the catheter is passed through the cervix and the frozen thawed semen is deposited. the authors’ experience based upon few bitches is that practice is required to locate the cervix and pass the catheter tip through the cervix. the technique is commonly used in scandinavian countries with successful outcomes. transcervical insemination. transcervical insemination (tci) is primarily used to inseminate female dogs with frozen-thawed semen. however, it can be used for fresh chilled semen, or semen of poor quality. a special endoscope is used in a bitch during “standing estrous” which avoids the need to anesthetize the bitch for intrauterine insemination by laparotomy. the long vagina in the female dog requires an elongated endoscope. in addition, the cervix appears to exit the vagina at a right angle to the lumen and there is a blind cul-de-sac just cranial to the external vaginal os. therefore, for tci the cervix is directly visualized. a specialized 29 cm long cystoscope with a biopsy channel for the insemination catheter is used. the commonly used equipment is a rigid cysto-urethroscope from storz. it includes a telescope, a sheath, bridge, xenon light source, and a video camera. insemination timing (breeding management), semen handling and appropriate insemination technique are key factors for a success. pregnancy diagnosis the three most commonly used pregnancy detection techniques include abdominal palpation, ultrasonography and radiography. other less commonly used techniques are relaxin and other assays. how early after breeding these techniques can be used can be highly variable depending upon ovulation time, sperm survival (dog sperm can live up to ten days in the bitch’s reproductive tract), etc. abdominal palpation this method is relatively easy to learn, inexpensive and requires some experience to be reliable but can be difficult if the bitch is obese or her abdomen is tense. reliable results are obtained during 2030 days after breeding. embryonic vesicles are felt as ‘little marbles’. before 20 days and after 30 days after breeding, it is difficult to be certain because uterus feels like a tubular structure full of uterine fluid, which could be confusing and may mimic pyometra. other limitations of this technique include inability to assess the fetal health. ultrasonography (us) this is an excellent technique to confirm pregnancy, determine embryo viability and diagnose abnormalities of the pregnancy. familiarity with us equipment and the image interpretation is critical for correct diagnosis. depending upon the time of fertilization, which is difficult to determine, this techniques reliably can be used 25 days after breeding. at this time, fetal heartbeat can be detected in most bitches. in the authors’ study involving ultrasonography of six german shepherd bitches during pregnancy the stage of pregnancy was determined retrospectively, counting back from whelping (63 days). pregnancy duration is more reliable if based upon the lh surge which causes ovulation. embryonic tissue and heartbeats were first detected between 23 and 27 days after the lh surge in 10 bitches. in the pregnant queen, we were able visualize fetal heartbeats on days 21-23 after breeding. pregnancy duration variability in the queen is less than in the bitch because cats are induced ovulators and breeding takes place within few hours compared to many days in the bitch. radiography this is the best technique for determining the number (litter size) of developing fetuses. the technique is useful after fetal mineralization, which takes place after 42 days after breeding. the limitations are the inability to assess fetal health and having to wait until last trimester of pregnancy. clinical theriogenology • volume 8, number 4 • december 2016 456 whelping pregnancy in the female dog lasts about 65 days, but predicting the timing of whelping can be difficult. a drop in rectal temperature (hypothermia) usually precedes delivery by about eight to 24 hours, which coincides with a decrease in blood p4. labor and delivery in dogs is divided into three stages. stage i lasts 12 to 24 hours. during this stage uterine contractions begin, but are not visible externally. the cervix also begins to dilate. during stage ii, abdominal contractions can be seen and the puppies are delivered. the puppies are usually delivered at intervals of one to two hours, but that can vary considerably. stage ii can last up to 24 hours. stage iii is defined as the delivery of the placenta. dogs typically alternate between stages ii and iii until delivery is complete. male breeding soundness examination the purposes of semen collection and evaluation are to assess an animal for breeding soundness; to perform artificial insemination; to preserve semen; to attempt to localize the site of disorder in some infertile dogs; and to determine treatment response in infertile dogs. collection of the prostatic fluid and submission for cytology and bacterial culture may help differentiate prostatic disorders from other problems. semen evaluation may be performed before a dog is purchased, before he is first used for breeding, or if he shows signs of infertility. semen collection and evaluation are relatively easy and can be done at any small animal practitice. the results should be recorded on the canine breeding soundness examination (bse) form, which was devised by the society for theriogenology. semen collection equipment. semen is easily collected from the dog using an artificial vagina (av), a device composed of a latex rubber cone (reproduction resources, hebson, il) connected to a 15 ml calibrated plastic centrifuge tube. the av should be washed with plain tap water (no soap, detergent, or other cleansing agent is used), rinsed thoroughly with distilled water, and air dried. gas sterilization, if available, may be used on the av. some dogs may ejaculate into other containers under the pressure of hand massage alone. however, semen is collected successfully from most dogs with an av because the av simulates the pressure normally felt by the male during copulation. the calibrated collection tubes used in the procedure should also be washed with tap water, rinsed with distilled water, and sterilized with gas or autoclaved before use to prevent the formation of formaldehyde residues which are harmful to sperm. for optimum results, collection should be done in a quiet room with the dog on a nonslip surface and with the owner present. semen may be collected before other examinations are performed, including the physical examination. for better responsiveness, the semen can be collected in the presence of a teaser bitch in proestrus or estrus. if a bitch in estrus is not available, a commercially available pheromone (eau d' estrus, international canine genetics, malvern, pa; or methylparaben; sigma chemical, saint louis, mo) or vaginal swabs from a disease-free estral bitch can be used. the top of the av should be folded down, and a scant amount of lubricant should be applied to facilitate later removal of the device from the dog's erect penis. procedure. the male and female are brought on leashes into a collecting room and allowed to play for a few minutes. the semen collector, if right handed, kneels at the dog's left side and, holding the av in the left hand, uses the right hand to move the prepuce up and down the penile shaft to achieve protrusion of the penis into the av. (left-handed collectors may reverse the sides described above.) as the male begins to thrust the penis into the av, the collector moves the sheath above the engorging bulbus glandis, keeping firm, steady pressure around the circumference of the penis to maintain the erection. most male dogs ejaculate the presperm (clear) and sperm-rich fractions (cloudy) of the semen during pelvic thrusting; they then dismount and lift one hind leg as though trying to step over the bitch and achieve the tie. at this point the penis should be rotated 180° so that it is directed caudally and held still with tight pressure maintained around the circumference until the third prostatic fraction of semen has been ejaculated. the prostatic fluid is ejaculated after a short pause in association with anal contractions and rhythmic pulsations of the penile urethra, which the collector can detect. after a few milliliters of prostatic fluid have been collected, the av may be removed from the penis; the dog may clinical theriogenology • volume 8, number 4 • december 2016457 continue to ejaculate prostatic fluid for some time after removal of the av. the dog should not be kenneled or sent home until the erection has subsided completely and the entire penis is back in the preputial sheath. normally, this takes about five to ten0 minutes after semen collection. evaluation five parameters should be evaluated and the results recorded for each semen collection: color and volume of the sample, motility, concentration and total number of sperm, and morphology of the sperm. color. the color of the sample is observed grossly in the collection tube. the first fraction should be clear, whereas the second (sperm-rich) fraction should be milky white. the third fraction (prostatic fluid) should be clear. yellow discoloration may indicate urine contamination or the presence of purulent exudate. a red or brown color may indicate prostatic disease, trauma, urethritis, or ulceration. volume. the volume of semen can be read directly from the calibrated collecting tube. volume varies among breeds and among individual dogs. semen volume is not an important parameter with regard to the bse, but it is necessary for determining the number of sperm per ejaculate. the volume of the prostatic fluid need not be recorded because it varies according to the length of time prostatic fluid is collected. sperm motility. motility testing should be performed immediately after collection. the examiner places a drop of undiluted semen on a slide with a cover slip, examines the specimen at 100x to 200x magnification (because canine spermatozoa are resistant to cold shock, the slide need not be warmed), and estimates the percentage of sperm cells moving in a progressive and forward manner across the field. although sperm motility estimation is not precise, it is a quick functional test. concentration/total number of sperm. the total number of sperm in the ejaculate is more important than the concentration of sperm per milliliter because the concentration changes dramatically depending on the amount of prostatic fluid collected. the sperm concentration can be measured with a commercial wbc unopett system (becton dickinson, rutherford, nj). the semen sample is stirred gently, drawn up into the capillary pipette provided with the kit, and dispensed into the diluent-containing reservoir chamber. diluted semen then is dispensed into both chambers of a neubauer hemocytometer (becton dickinson) and the contents of the 1 mm central square (composed of 25 small squares) are counted on each side of the hemocytometer and averaged (the central square fills the field of a light microscope when the 10x objective is used). the number of spermatozoa in the central square is the concentration in millions of spermatozoa per milliliter of semen. the total number of sperm in the ejaculate equals the concentration multiplied by the volume. the normal total number of sperm ranges from 300 to 2000 x 106; large breed dogs ejaculate more sperm than small breed dogs (direct cell counts done with the hematocytometer are time-consuming; quicker methods used to determine the sperm concentration are an electronic blood cell counter or spectrophotometer.) the total number of sperm may be decreased in young, old, and inbred dogs. morphology. sperm morphology is usually assessed by staining the semen sample and observing the sperm under oil immersion (1,000x) objective. various stain techniques may cause some artifactual changes in sperm morphology. defects vary with the stain used, but the percentage of morphologically normal sperm should be fairly consistent regardless of method. eosin/nigrosin stain and romanowski stain (diff quik) are the stains most commonly used. for staining with eosin-nigrosin, a drop of semen and a similar-sized drop of stain are placed on one end of a clean glass slide and mixed gently with a wooden applicator. a second slide then is used as a spreader slide. a thin film, similar to a blood smear, is made and air-dried. for staining with rapid romanowski stain, the semen smear is made as for a blood smear, air-dried, and immersed in each of the three solutions for five minutes each. the slide then is rinsed and allowed to dry. one hundred sperm cells should be examined and the findings recorded, including normal sperm, primary abnormalities (all head defects, proximally coiled tails, proximal cytoplasmic droplets) and secondary abnormalities (detached heads, bent tails, and distal cytoplasmic droplets). primary abnormalities are abnormalities that occur in the testis during spermatogenesis. secondary abnormalities occur during epididymal transport or as a result of sample-handling techniques. most dogs have greater than 80% normal sperm per ejaculation. clinical theriogenology • volume 8, number 4 • december 2016 458 conclusions inherited myopathies cause debilitating limb weakness and respiratory dysfunction in affected humans. animal models provide platforms to study underlying disease mechanisms and test potential therapies. the clinical courses of spontaneous canine conditions, such as xlmtm, largely parallel those of the human disease. this makes the canine myopathies particularly attractive models for studying their human counterparts making tried-and-true breeding methodologies essential to experimental success. in the context of preclinical trials, particular attention must be paid to onset and progression of the disease. studies in xlmtm dogs have provided insight on the efficacy of treatments intended for human patients and hold great promise for future investigation. references 1. perel p, roberts i, sena e, et al: comparison of treatment effects between animal experiments and clinical trials: systematic review. bmj 2007;334:197. pubmed pmid: 17175568. pubmed central pmcid: 1781970. 2. lin jh: applications and limitations of genetically modified mouse models in drug discovery and development. curr drug metab 2008;9:419-438. pubmed pmid: 18537578. epub 2008/06/10. eng. 3. partridge ta: the mdx mouse model as a surrogate for duchenne muscular dystrophy. febs j 2013;280:4177-4186. pubmed pmid: 23551987. pubmed central pmcid: 4147949. 4. rocha dn, carvalho ed, pego ap: high-throughput platforms for the screening of new therapeutic targets for neurodegenerative diseases. drug discov today 2016;9:1355-1366. pubmed pmid: 27178019. 5. tsai kl, clark la, murphy ke: understanding hereditary diseases using the dog and human as companion model systems. mamm genome 2007;18:444-451. pubmed pmid: 17653794. pubmed central pmcid: 1998873. 6. lindblad-toh k, wade cm, mikkelsen ts, et al: genome sequence, comparative analysis and haplotype structure of the domestic dog. nature 2005;438:803-819. pubmed pmid: 16341006. epub 2005/12/13. eng. 7. lawlor mw, beggs ah, buj-bello a, et al: skeletal muscle pathology in x-linked myotubular myopathy: review with cross-species comparisons. j neuropatho exp neuro 2016;75:102-110. pubmed pmid: 26823526. 8. reichel mp, ellis jt, dubey jp: neosporosis and hammondiosis in dogs. j small anim pract 2007;48:308-312. pubmed pmid: 17547641. epub 2007/06/06. eng. 9. shelton gd: from dog to man: the broad spectrum of inflammatory myopathies. neuromuscul gisord 2007;17:663670. pubmed pmid: 17629703. epub 2007/07/17. eng. 10. platt sr: neuromuscular complications in endocrine and metabolic disorders. vet clin north am small anim pract 2002;32:125-146. pubmed pmid: 11785726. epub 2002/01/12. eng. 11. guan x, goddard ma, mack dl, et al: gene therapy in monogenic congenital myopathies. methods 2016;99:91-98. pubmed pmid: 26454198. pubmed central pmcid: 4826856. epub 2015/10/11. eng. 12. shelton gd, engvall e: canine and feline models of human inherited muscle diseases. neuromuscul disor 2005;15:127-138. pubmed pmid: 15694134. epub 2005/02/08. eng. 13. yu x, bao b, echigoya y, et al: dystrophin-deficient large animal models: translational research and exon skipping. am j transl res 2015;7:1314-1331. pubmed pmid: 26396664. pubmed central pmcid: 4568789. epub 2015/09/24. eng. 14. mcgreevy jw, hakim ch, mcintosh ma, et al: animal models of duchenne muscular dystrophy: from basic mechanisms to gene therapy. dis model mech 2015;8:195-213. pubmed pmid: 25740330. pubmed central pmcid: 4348559. epub 2015/03/06. eng. 15. kornegay jn, bogan jr, bogan dj, et al: canine models of duchenne muscular dystrophy and their use in therapeutic strategies. mammal genome 2012;23:85-108. pubmed pmid: 22218699. pubmed central pmcid: 3911884. epub 2012/01/06. eng. 16. wicklund mp: the muscular dystrophies. continuum 2013;19:1535-70. pubmed pmid: 24305447. epub 2013/12/07. eng. 17. amato aa, griggs rc: overview of the muscular dystrophies. handb clin neurol 2011;101:1-9. pubmed pmid: 21496621. epub 2011/04/19. eng. 18. cowling bs, toussaint a, muller j, et al: defective membrane remodeling in neuromuscular diseases: insights from animal models. plos genet 2012;8:e1002595. pubmed pmid: 22496665. pubmed central pmcid: 3320571. epub 2012/04/13. eng. 19. amoasii l, bertazzi dl, tronchere h, et al: phosphatase-dead myotubularin ameliorates x-linked centronuclear myopathy phenotypes in mice. plos genet 2012;8:e1002965. pubmed pmid: 23071445. pubmed central pmcid: 3469422. epub 2012/10/17. eng. 20. gilbreath hr, castro d, iannaccone st: congenital myopathies and muscular dystrophies. neurol clin 2014;32:689703, viii. pubmed pmid: 25037085. epub 2014/07/20. eng. clinical theriogenology • volume 8, number 4 • december 2016459 21. gentilini f, zambon e, gandini g, et al: frequency of the allelic variant of the ptpla gene responsible for centronuclear myopathy in labrador retriever dogs as assessed in italy. j vet diagn invest 2011;23:124-126. pubmed pmid: 21217042. epub 2011/01/11. eng. 22. maurer m, mary j, guillaud l, et al: centronuclear myopathy in labrador retrievers: a recent founder mutation in the ptpla gene has rapidly disseminated worldwide. plos one 2012;7:e46408. pubmed pmid: 23071563. pubmed central pmcid: 3465307. epub 2012/10/17. eng. 23. bohm j, vasli n, maurer m, et al: altered splicing of the bin1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy. plos genet 2013;9:e1003430. pubmed pmid: 23754947. pubmed central pmcid: 3675003. epub 2013/06/12. eng. 24. lujan feliu-pascual a, shelton gd, targett mp, et al: inherited myopathy of great danes. j small anim pract 2006;47:249-254. pubmed pmid: 16674719. epub 2006/05/06. eng. 25. romero nb, bitoun m: centronuclear myopathies. semin pediat neurol 2011;18:250-256. pubmed pmid: 22172420. 26. laporte j, kress w, mandel jl: diagnosis of x-linked myotubular myopathy by detection of myotubularin. annal neurol 2001;50:42-46. pubmed pmid: 11456308. 27. herman ge, finegold m, zhao w, et al: medical complications in long-term survivors with x-linked myotubular myopathy. j pediat 1999;134:206-214. pubmed pmid: 9931531. 28. jungbluth h, wallgren-pettersson c, laporte j: centronuclear (myotubular) myopathy. orphanet j rare dis 2008;3:26. pubmed pmid: 18817572. pubmed central pmcid: 2572588. 29. buj-bello a, laugel v, messaddeq n, et al: the lipid phosphatase myotubularin is essential for skeletal muscle maintenance but not for myogenesis in mice. proc nat acad sci usa 2002;99:15060-15065. pubmed pmid: 12391329. pubmed central pmcid: 524320. 30. cowling bs, toussaint a, muller j, et al: defective membrane remodeling in neuromuscular diseases: insights from animal models. plos genet 2012;8:e1002595. pubmed pmid: 22496665. pubmed central pmcid: 3320571. 31. pierson cr, dulin-smith an, durban an, et al: modeling the human mtm1 p.r69c mutation in murine mtm1 results in exon 4 skipping and a less severe myotubular myopathy phenotype. hum mol genet 2012;21:811-825. pubmed pmid: 22068590. pubmed central pmcid: 3263994. 32. smith bk, goddard m, childers mk: respiratory assessment in centronuclear myopathies. muscle nerve 2014;50:315-326. pubmed pmid: 24668768. pubmed central pmcid: 4140950. 33. beggs ah, bohm j, snead e, et al: mtm1 mutation associated with x-linked myotubular myopathy in labrador retrievers. proc nat acad sci usa 2010;107(33):14697-14702. pubmed pmid: 20682747. pubmed central pmcid: 2930454. 34. cosford kl, taylor sm, thompson l, et al: a possible new inherited myopathy in a young labrador retriever. can vet j 2008;49:393-397. pubmed pmid: 18481550. pubmed central pmcid: 2275345. 35. shelton gd, rider be, child g, et al: x-linked myotubular myopathy in rottweiler dogs is caused by a missense mutation in exon 11 of the mtm1 gene. skelet muscle 2015;5:1. pubmed pmid: 25664165. pubmed central pmcid: 4320619. 36. goddard ma, mack dl, czerniecki sm, et al: muscle pathology, limb strength, walking gait, respiratory function and neurological impairment establish disease progression in the p.n155k canine model of x-linked myotubular myopathy. ann transl med 2015;3:262. pubmed pmid: 26605308. pubmed central pmcid: 4630545. 37. goddard ma, burlingame e, beggs ah, et al: gait characteristics in a canine model of x-linked myotubular myopathy. j neurol sci 2014;346:221-226. pubmed pmid: 25281397. pubmed central pmcid: 4253544. 38. goddard ma, mitchell el, smith bk, et al: establishing clinical end points of respiratory function in large animals for clinical translation. phys med rehab clin n am 2012;23:75-94, xi. pubmed pmid: 22239876. 39. grange rw, doering j, mitchell e, et al: muscle function in a canine model of x-linked myotubular myopathy. muscle nerve 2012;46:588-591. pubmed pmid: 22987702. pubmed central pmcid: 3448125. 40. patronek gj, waters dj, glickman lt: comparative longevity of pet dogs and humans: implications for gerontology research. j gerontol a biol sci med sci 1997;52:b171-178. pubmed pmid: 9158552. epub 1997/05/01. eng. 41. yamaguchi n, prosser bl, ghassemi f, et al: modulation of sarcoplasmic reticulum ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and s100a1. am j physiol cell physiol 2011;300:c998-c1012. pubmed pmid: 21289290. pubmed central pmcid: 3093939. 42. sarwal a, cartwright ms, mitchell e, et al: guiding intramuscular diaphragm injections using real-time ultrasound and electromyography. muscle nerve 2015;51:287-289. pubmed pmid: 25354257. pubmed central pmcid: 4293336. epub 2014/10/30. eng. 43. sarwal a, cartwright ms, walker fo, et al: ultrasound assessment of the diaphragm: preliminary study of a canine model of x-linked myotubular myopathy. muscle nerve 2014;50:607-609. pubmed pmid: 24861988. pubmed central pmcid: 4175136. epub 2014/05/28. eng. 44. childers mk, joubert r, poulard k, et al: gene therapy prolongs survival and restores function in murine and canine models of myotubular myopathy. sci transl med 2014;6:220ra10. pubmed pmid: 24452262. pubmed central pmcid: 4105197. 45. johnson ca, memon ma: management of reproduction in dogs. in: the merck veterinary manual. kenilworth (nj): merck sharp and dohme; 2011. clinical theriogenology • volume 8, number 4 • december 2016 460 46. memon ma: reproductive diseases of the female – small animals. in: kahn c, editor. the merck veterinary manual. 9th edition. kenilworth (nj): merck & co., inc; 2005. 47. memon ma, kaitkanoke s: semen evaluation, canine male infertility, common disorders of the male. feldman see, editor. philadelphia wb saunders; 2005. 48. memon ma: common causes of male dog infertility. theriogenology 2007;68:322-328. pubmed pmid: 17512045. 49. memon ma, kutzler ma: normal parturition. in: cote e, editor. clinical veterinary advisor –dogs and cats. philadelphia: mosby-elsevier; 2007. 50. memon ma: i did everything right to breed my bitch – what went wrong! clin therio 2010;2:501-507. 51. olson pn: vaginal cytology i. a useful tool for staging the canine estrous cycle. compend contin educ pract vet 1984;6: 288-297. clinical theriogenology • volume 8, number 4 • december 2016461 table 1. inherited canine dystrophinopathies and centronuclear myopathies with confirmed mutations disease breed gene/mutation clinical signs reference(s) x-linked dystrophinopathy golden retriever dmd – base change (a-g) in the 3' splice site of intron 6, with deletion of exon 7 in the mrna transcript weakness and respiratory difficulty possible at birth; stunted growth; progressive weakness and contractures, especially over the ages of 3 to 6 months; respiratory and cardiac involvement. kornegay jn, et al. 2012; sharp njh, et al. 1992. german shorthaired pointer dmd – large deletion encompassing entire gene schatzberg sj, et al. 1999. pembroke welsh corgi dmd – repetitive element-1 (line-1) insertion in intron 13 smith bf, et al. 2011. cavalier king charles spaniel dmd – base change (g-t) in the 5’ splice site of intron 50, with deletion of exon 50 in the mrna transcript walmsley gl, et al. 2010. rottweiler dmd – base change (g-t) in exon 58 resulting in stop codon winand n, et al. 1994. cocker spaniel dmd – deletion of four nucleotides in exon 65, resulting in stop codon kornegay et al. 2012. tibetan terrier dmd – deletion of exons 8-29 kornegay et al. 2012. labrador retriever dmd – 184 nucleotide [pseudoexon] insertion between exons 19 and 20, resulting in stop codon kornegay et al. 2012. x linked myotubularin myopathy labrador retriever mtm1 – base change (c-a) in exon 7; p.n155k stunted growth; muscle atrophy, and pelvic limb weakness at 7 weeks. patellar hyporeflexia, dysphagia, and hoarse bark; paradoxical respiration at 10 weeks. progressive weakness and muscle atrophy, with loss of ambulation, by 4 to 6 months. beggs et al. 2010; cosford et al. 2008 hereditary (centronuclear) myopathy of labradors labrador retriever ptpla – short interspersed repeat element (sine) insertion in exon 2 abnormal head and neck posture; stiff, hopping gait; muscle atrophy; signs stabilize somewhat at 1 year. blot et al. 2002; kramer et al. 1976; maurer et al. 2012 inherited (centronuclear) myopathy of great danes great dane bin1 – base change (a-g) in the 3' splice site of intron 10, with deletion of exon 11 in the mrna transcript weakness, muscle atrophy, exercise intolerance, trembling, and characteristic posture with the pelvic limbs held under the body. onset of signs at 6 to 19 months (median of 7), with variable progression. böhm et al. 2013; lujánfeliu-pascual et al. 2006 clinical theriogenology • volume 8, number 4 • december 2016 462 table 2. centronuclear myopathies structural features disease gene protein mammalian models reference(s) congenital myopathies with central nuclei myotubular myopathy mtm1 myotubularin mtm1 ko mouse r69c mouse xlmtm dog p.n155k xlmtm dog p.q384p buj-bello et al, 2002. pierson et al, 2012. beggs et al, 2010. shelton et al, 2015 centronuclear myopathy dnm2 dynamin 2 r465w mouse durieux et al, 2010. ptpla protein tyrosine phosphatase-like member a ptpla dog gentilini et al, 2011. bin1 amphiphysin shrna-bin1 knockdown mouse imgd dog tjondrokoesoemo et al, 2011. bohm et al, 2013. ryr1 ryanodine receptor ryr1i4895t/wt (it/+) mouse zvaritch et al, 2009. ttn titin ttn(mdm) mouse garvey et al, 2002. clinical theriogenology • volume 8, number 4 • december 2016463 clinical theriogenology • volume 8, number 4 • december 2016 464 omniblank: 2015: use of etonogestrel implants to suppress estrous behavior in mares use of etonogestrel implants to suppress estrous behavior in mares* ghislaine a. dujovne,a aime k. johnson,b robyn r. wilborn,b roberto a. palomares,c anne a. wooldridge,b tim d. bradend apopulation health and reproduction, school of veterinary medicine, university of california, davis, ca; bdepartment of clinical sciences, college of veterinary medicine, auburn university, auburn, al; cdepartment of population health, college of veterinary medicine, university of georgia, athens, ga; ddepartment of anatomy, physiology and pharmacology, college of veterinary medicine, auburn university, auburn, al abstract the objective of this study was to evaluate a human contraceptive subcutaneous implant (implanon®) as a method to suppress behavioral estrus in mares. twenty mares were randomly assigned to 4 groups (n=5). group c was the control group, group t1 received one implanon® implant (68 mg etonogestrel), group t2 received two implanon® implants (136 mg etonogestrel), and group r was the positive control, receiving 0.044 mg/kg altrenogest daily. estrous behavior was evaluated twice weekly and was graded by a blinded observer. estrous cycles were monitored during three months using progesterone levels and transrectal examinations. interestrus interval (iei) was measured based on both behavioral estrus (teasing scores) and serum progesterone concentration (below 1.0 ng/ml). mean interestrus interval ± sem per group, based on teasing and progesterone levels, respectively, were as follows: group c 21±0.3 and 21±0.4 days; group t1: 34±8.2 and 31±6.4 days; group t2: 42±14.1 and 41±14.4 days; and group r: 111±1.3 and 48±0.9. group t2 had an interestrus interval twice as long as the control group, however, no statistical difference was found between groups c, t1 and t2. group r positive control) was different from all other groups (p<0.05) based on teasing observations. etonogestrel was not consistently effective for estrus suppression in mares, however, it did produce an iei twice as long as the negative control at the highest dose used in this study (136mg). further studies with a higher dose would be necessary to determine whether or not etonogestrel can be used to fully suppress estrus in mares. keywords: mare, estrus suppression, etonogestrel, implanon introduction estrous behavior (“heat”) is a problem in many performance mares. animals have temperament changes and become more difficult to handle during the estrus period. some mares show aggression, pain, and reduced performance while in heat.1 because of performance problems associated with estrus in mares, several treatments have been evaluated to find an ideal method for behavioral estrus suppression. currently no single treatment has been shown to be effective, easy to use, and safe for prolonged estrus suppression. the most common and consistently effective treatments include oral altrenogest and natural progesterone injections. however, both methods necessitate repeated administration, which makes them costly and impractical in many situations. the injections can be painful and may induce swelling at the injection site leading to injection aversion or sore muscles that affect performance. daily oral treatment with altrenogest may make the mare reluctant to be handled around the mouth and must be administered every day for complete effectiveness.2 other methods that have been tested with limited success include the use of intrauterine devices, repeated oxytocin injections, manual reduction of an established conceptus, herbal supplements, and induction of a diestral ovulation.3-8 synthetic progestins other than altrenogest (medroxyprogesterone, norgestomet, megestrol acetate), completely failed to suppress estrous behavior or maintain pregnancy in mares presumably because of a failure of these compounds to bind to the equine progesterone receptors.9-11 there are conflicting studies *these data were presented, in part, at the 2011 annual meeting of the society for theriogenology. clinical theriogenology • volume 7, number 1 • march 201535 about the use of gonadotropin releasing hormone (gnrh) immunization to suppress estrous behavior. some studies report inconsistent results while others show promising results for reducing or eliminating behavioral signs.12-14 this treatment is currently unavailable in the united states. implanon® (organon laboratories ltd, science park, cambridge, uk) is a subcutaneous implantable contraceptive containing 68 mg of the progestin etonogestrel. this implant is formulated for long-term birth control in women.15,16 etonogestrel is a progestin with proven efficacy and safety in women, and also has been used in a contraceptive vaginal ring (nuvaring®, organon, usa inc).16 in women, a release rate of 25-35 µg/day of etonogestrel is required to suppress ovulation and prevent pregnancy.17-18 etonogestrel in implanon® is released at an initial rate of 60 µg/day, which then decreases to 30 µg/day. this release rate results in maintaining a sufficient plasma concentration of the progestin to inhibit ovulation for up to three years in women.17,18 the objective of this study was to evaluate this synthetic progestin (etonogestrel) as a reliable method to suppress behavioral estrus in mares. if etonogestrel is effective at inhibiting estrous behavior in mares, the implant may provide a long-term, safe, easy, and potentially reversible treatment to suppress estrous behavior in performance mares. materials and methods twenty healthy mares between the ages of 6 and 20 years old with normal estrous cycles were used. the mares were part of the auburn university equine reproduction center teaching herd and auburn university animal science horse center. all the animals were average size for adult horses (approximately 500 kg body weight); they were kept under similar husbandry conditions (free choice hay with grain supplementation as needed) and were housed as groups in paddocks or pasture. the study took place between june and september. follicular growth, ovulation, and estrous behavior were monitored prior to each mare’s enrollment into the study, and only animals demonstrating regular estrous cycles and normal estrous behavior in response to a teaser stallion were included. the animals were randomly allocated to 4 groups of 5 animals each using a random number generator (research randomizer, www.randomizer.org). treatments group c (control): negative control; animals did not receive any treatment and were allowed to cycle normally. group t1 (treatment dose 1): one implanon® implant containing 68 mg of etonogestrel. group t2 (treatment dose 2): two implanon® implants containing 136 mg etonogestrel. group r: positive control; 0.044 mg/kg altrenogest orally once daily. implant application was performed using aseptic technique. the mare’s movement was restricted by placing her in stocks and sedating lightly with xylazine (0.2-0.4 mg/kg iv). two milliliters of 2% lidocaine was used as a local anesthetic at the site of insertion. the implants were introduced subcutaneously into the vulvar lips, parallel to the vulvar opening, and 5 to 10 centimeters below the dorsal vulvar commisure. introduction was performed using the applicator provided by the manufacturer (figure 1). for mares in group t2, one implant was applied in each vulvar lip. the removal of the implants at the conclusion of the study was performed using local anesthetic (same protocol as insertion). a 2 to 3 mm incision was made at the tip of the implant followed by gentle manual manipulation of the implant toward the incision until it was externally visible through the incision. the implant was then removed and discarded. the small incision was allowed to heal by second intention. altrenogest was used for the positive control group because of its proven effectiveness to suppress behavioral estrus (0.044mg/kg orally once daily).11,19 all treatments started in mid-diestrus, seven days after ovulation was detected by transrectal ultrasonography. the study ended with removal of the implant (groups t1 and t2) or discontinuation of altrenogest (group r) after 90 days of treatment, and mare’s reproductive cycles were followed until their first ovulation after the treatment withdrawal was detected. clinical theriogenology • volume 7, number 1 • march 2015 36 data collection teasing. behavioral estrus was evaluated twice weekly by an experienced observer who was blinded to the treatment groups. a teaser stallion was led to the paddock, and the mares were allowed fence line contact. if a mare did not present at the fence, she was captured with a halter and lead rope and teased individually across the fence. behavior was scored 1 to 4 using the following scale: 1: mare completely rejects the stallion, presenting one or more of the following refusal manifestations: squealing, pawing, kicking, switching tail, holding ears back; 2: mare is indifferent to the presence of the stallion; she does not move away, but does not lift the tail or wink the clitoris (rhythmic eversion of the labia and exposure of the clitoris); 3: mare is interested in the stallion and approaches him, raising the tail, urinating and/or winking the clitoris; 4: full estrous behavior (posture change facilitating copulation), the mare will present similar behavioral signs as score 3 (clitoris eversion (winking), elevation of the tail and urination in presence of the stallion) plus a change in the mare’s posture to one that facilitates copulation (arched tail, flexed stifles and hocks, abducted rear limbs and tipped pelvis with associated lowering of the perineal area). mares scored with a one or two were considered not in estrus, and those scored three or four were considered to be in estrus (behavior absent or present, respectively). progesterone. to further evaluate each mare’s hormonal profile, a baseline blood sample was collected immediately prior to treatment (day seven post-ovulation), 24 and 48 hrs after treatment onset, and then weekly to evaluate plasma progesterone concentrations. the samples were processed with a progesterone radioimmunoassay (ria) kit (coat-a-count®, siemens, los angeles, ca) which has previously been evaluated for use in this species (intra-assay inter-assay coefficient of variation 2.46% and 17.9%).20 supplemental altrenogest does not affect the ability to quantify natural progesterone levels.21 palpation and ultrasonography. twice weekly examinations using transrectal ultrasonography were performed for estrous cycle monitoring and to evaluate the correlation between progesterone concentration, ovarian structures, and teasing behavior in this group of mares. an ultrasonography system (micromaxx®, sonosite inc, bothell, wa) with an l52e transducer (5mhz) was used for all examinations. data analysis interestrus interval was calculated based on both behavioral estrus (teasing) and progesterone levels. plasma samples with progesterone concentrations below 1.0 ng/ml, and teasing score of 3 or 4 were considered consistent with estrus. the iei was used as an indicator of estrus suppression duration, and is counted as the time in days between periods of behavioral estrus. data were analyzed using the statistical software statistical analysis system (sas version 9.1, sas institute, cary, nc). sample size of the groups was determined through the use of proc power based on the expected means, standard deviations and a statistical power of 95%. significant differences were identified with p < 0.05. results are reported as mean +/standard error of the mean (sem). data were transformed using a logarithmic scale in order to achieve statistical assumptions (normality and constant variance). descriptive statistics (means, sem and coefficient of variation) were determined using proc univariate. in order to assess the effect of the treatment on estrus suppression, iei were analyzed using the analysis of variance through the general linear model (proc glm). comparisons among group means were performed using the least significant difference test (lsd). pearson’s correlation analysis was performed using proc corr. animal welfare this study was performed under the regulations of the institutional animal care and use committee (iacuc), office of animal resources, auburn university. clinical theriogenology • volume 7, number 1 • march 201537 results clinical observation no abnormalities at the site of implant application were observed in any of the treated mares. implant application and removal was easily performed, and mares showed no evidence of pain or discomfort during the procedure. one mare in group r was euthanized due to a severe colic episode (unrelated to the study) during the first month of the study; her data were not considered for analysis. ovulation (detected by transrectal palpation and ultrasonography) was observed in all five mares in groups c, t1 and t2. in group c, ovulation occurred regularly in all mares as expected. however, in groups t1 and t2, ovulations were erratic, without an established pattern. in group r (positive control), three out of four mares ovulated. the mares that ovulated in group r also showed inconsistent ovulation intervals. after treatment withdrawal, all mares returned to normal cyclicity, and ovulation occurred within 3 to 46 days. after the 90 day study period, ovulation in group c occurred in a mean ± sem of 10 ± 1.1 days (range 7 to 14). in group t1 and t2, ovulation occurred in a mean of 14 ± 5.3days (range 3 to 30) and 23 ± 6.2 days (range 10 to 46), respectively. in group r, ovulation occurred in a mean of 9 ± 0.7 days after the treatment withdrawal (range 8 to 11). no significant differences were found among groups. differences approaching significance were identified between groups c and t2 (p=0.06), and also between t2 and r (p=0.06); which may indicate an association between etonogestrel (2 implant dose) treatment removal and days to ovulation. interestrus interval mean iei based on teasing scores ± sem was 21 ± 0.3 days for group c, 34 ± 8.2 days for group t1, 42 ± 14.1 days for group t2 and 111 ± 1.3 days for group r. mean iei based on progesterone values ± sem were as follows: 21 ± 0.4 days for group c, 31 ± 6.4 days for group t1, 41 ± 14.4 days for group t2 and 48 ± 0.9 days for group r (fig. 2). mean iei of each individual mare during the 90 day study period are shown (table). no statistical difference was found between groups c, t1 and t2 based on teasing or progesterone levels. as expected, iei in group r (positive control) was longer than all other groups (p<0.001) based on teasing observations. behavioral estrus in this group was suppressed during the entire study period. based on progesterone concentrations, the iei of group r was different from group c (p=0.049). no significant differences were found among the other groups (figs. 3-6). a significant difference in the response to etonogestrel was observed among mares within the same group in both t1 and t2. the coefficient of variation (cv) for group t1 was 53%. in group t2 the variation was even greater with a cv of 73% with 3/5 (60%) of the mares experiencing a prolonged iei (two mares showed 35 days, and one mare failed to show estrus behavior the entire study period). the other two mares in the group, however, showed no effects and experienced a normal estrus period. the variation was greater in both implant groups compared with the control groups. the cv in groups c and r (negative and positive control) was 3.6% and 2.3% respectively. the two methods used to determine iei (progesterone and teasing score) provided similar results. the iei determined by teasing and progesterone levels was highly correlated (r=0.91). there was moderate correlation in group c (r=0.58), strong correlation in groups t1 and t2 (r= 0.99), and very low correlation in group r (r=0.05), as expected due the fact that erratic ovulations occurred in this group independent of the total suppression of the behavioral estrus. in this study, the correlation coefficient demonstrates the correlation of the iei based on progesterone and teasing, showing no differences in the iei as determined by each method. discussion at the dose used in this study, etonogestrel was not effective for complete behavioral estrus suppression. however, the behavioral iei for group t2 (136 mg etonogestrel) averaged twice as long as group c (control) indicating some clinical effect. clinical theriogenology • volume 7, number 1 • march 2015 38 the use of the implant was determined to be safe in mares for at least 90 days. none of the ten treated mares in our study showed adverse reactions to the implants during the study period which implies that the implants are safe to use in the mare at least for this period of time. this is consistent with a safety study performed in humans using implanon® implants. in that study, very few abnormalities or complications were observed at the implant site in 474 women (<3% complication rate).22 ovulation was detected in 95% (18/19) of all mares, during the treatment period. ovulation occurred at regular intervals (21 to 23 days) in the control group (group c). irregular ovulations occurred in groups t1 and t2 which may indicate that etonogestrel, at the dose used, was unable to completely block ovulation, but did have some effect on cyclicity. ovulation was detected in 75% (3 out of 4) of mares in group r (regumate®), with one mare showing only a single ovulation during the entire observation period. this is consistent with previous studies that documented that altrenogest was not always able to exert predictable control over the estrous cycle in the mare and completely suppress ovulation.23 group t2 displayed an iei that was twice as long as the control group c. although not statistically significant, this difference may be clinically relevant. the fact that the difference was not significant statistically could be explained by the high individual variation within the group t2 (cv=73%). a larger number of animals per group could have reduced this variation. prior to this study, the sample size was determined by the proc power test based on the expected means, standard deviations and a statistical power of 95%. this power was calculated based on expected means and standard deviations of previous studies where the variation was lower than that obtained in this experiment. based on the results obtained in this study, the power was recalculated. in order to maintain a power of 95% 18 mares per group would be required. therefore, in future studies, it seems likely that a larger sample size may be needed to reduce variation. as expected, group r (positive control) had an iei significantly longer than all other groups. estrous behavior in these mares was completely suppressed during the entire study period which confirms altrenogest as a reliable positive control for suppressing behavioral estrus. this is consistent with several previous studies where altrenogest effectively suppressed estrous behavior in mares.9,11,19,24 when iei was calculated based on progesterone concentrations, only groups r and c were significantly different (positive and negative control groups, respectively). this is because, as previously mentioned, altrenogest failed to completely suppress ovulation. progesterone concentrations in mares receiving altrenogest may be unpredictable, sometimes rising over or decreasing below 1 ng/ml without being accompanied by estrous behavior, indicating ovulations are occurring.23 the difference between iei based on teasing and progesterone concentrations in group r (111 days versus 48 days, respectively) can be attributed to the fact that behavioral estrus is suppressed in mares receiving altrenogest even in the presence of follicular growth or ovulation. this explains the low correlation (r=0.05) between the two parameters (iei based on teasing scores and progesterone levels) in group r when compared with the general correlation of teasing to progesterone (r=0.91). if the group r is excluded from the correlation test, the correlation coefficient increases to r=0.98, almost perfect correlation. etonogestrel determination in plasma was not possible in our study mares due to technical limitations to measure the small concentrations of the drug expected in circulating blood (picograms/ml). in women, the maximum serum concentrations detected were 813 pg/ml four days after implant insertion, and declined to 156 pg/ml at the end of the three years.25 in that study, body weight was related to overall serum concentrations of etonogestrel where the highest levels were found in women weighing less than 5 0kg (≈200 pg/ml) and the lowest concentration was found in women weighing over 70 kg (≈150 pg/ml).25 based on this information, it is possible to speculate that the expected concentrations of etonogestrel in the horse should be five to ten times less than the serum levels obtained in women, considering an average woman’s weight of 65 kg compared with a 500 kg body weight in the mare. this study used a dose of 0.13 to 0.27 mg/kg, compared with 1.05 mg/kg used in the commercial dose for women (assuming an average body weight of 65 kg). for this study we chose a lower dose in order to make this treatment financially comparable with daily altrenogest administration during a period of 90 days, clinical theriogenology • volume 7, number 1 • march 201539 knowing that a higher dose would be more adequate but not financially practical for estrus suppression use in mares based on the commercial cost of the individual implants. based on previous information, the fact that behavioral estrus was completely suppressed in one of the study mares throughout the treatment period, and iei was twice as long in the t2 (two implant) group compared with the control mares, we speculate that etonogestrel in the correct dose may suppress estrous behavior more consistently. further studies using a weight-adjusted dose (525 mg per 500 kg horse) and a greater number of animals per group to reduce the effect of individual variability are warranted to determine whether or not etonogestrel is a reliable alternative for estrus suppression in the mare. conclusions the high correlation of the two parameters to determine iei (teasing and progesterone) validates the teasing score as a reliable method to detect estrus in mares. this may only be valid if the teasing is performed by an experienced observer and the mare normally exhibits estrus signs in presence of a stallion. at the dose used in this study, etonogestrel was not effective for complete behavioral estrus suppression. however, the behavioral iei for group t2 (136mg etonogestrel) was twice as long as group c (control) which suggests that future studies with a higher dose are warranted to determine whether or not etonogestrel can be used to fully suppress estrous behavior in mares. acknowledgments the authors thank the united states equestrian federation (usef) for their financial support; ms. marti mccoy and dr. barbara schmidt for their assistance with mare handling during this project; and dr. sue duran, auburn university large animal pharmacy, for her assistance in obtaining the implants. the usef had no involvement in the study design, data collection, or preparation of this report. references 1. hinrichs k: irregularities of the estrous cycle and ovulation in mares (including seasonal transition). in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd edition. st louis: wb saunders; 2007. p. 150-152. 2. pryor p, tibary a: management of estrus in the performance mare. clin tech equine pract 2005;4:197-209. 3. nie gj, johnson ke, braden td, et al: use of an intra-uterine glass ball protocol to extend luteal function in mares. j equine vet sci 2003;23:266-273. 4. rivera del alamo mm, reilas t, kindahl h, et al: mechanisms behind intrauterine device-induced luteal persistence in mares. anim reprod sci 2008;107:94-106. 5. vanderwall dk, rasmussen dm, woods gl: effect of repeated administration of oxytocin during diestrus on duration of function of corpora lutea in mares. j am vet med assoc 2007;231:1864-1867. 6. lefranc a: nonpharmacological suppression of oestrus in the mare. equine vet j 2004;36:183-185. 7. mccue pm: estrus suppression in performance horses. j equine vet sci 2003;23:342-344. 8. hedberg y, dalin a, santesson m, et al: a preliminary study on the induction of dioestrous ovulation in the mare-a possible method for inducing prolonged luteal phase. acta vet scand 2007;48:12-18. 9. gee ek, deluca c, stylski jl, et al: efficacy of medroxyprogesterone acetate in suppression of estrus in cycling mares. j equine vet sci 2009;29:140-145. 10. mckinnon ao, lescun tb, walker jh, et al: the inability of some synthetic progestagens to maintain pregnancy in the mare. equine vet j 2000;32:83-85. 11. wiepz gj, squires el, chapman pl: effects of norgestomet, altrenogest, and/or estradiol on follicular and hormonal characteristics of late transitional mares. theriogenology 1988;30:181-193. 12. dalin am, andresen ø, malmgren l: immunization against gnrh in mature mares: antibody titres, ovarian function, hormonal levels and oestrous behaviour. j vet med a 2002;49:125-131. 13. imboden i, janett f, burger d, et al: influence of immunization against gnrh on reproductive cyclicity and estrous behavior in the mare. theriogenology 2006;66:1866-1875. 14. elhay m, newbold a, britton a, et al: suppression of behavioural and physiological oestrus in the mare by vaccination against gnrh. aust vet j 2007;85:39-45. 15. glasier a: implantable contraceptives for women: effectiveness, discontinuation rates, return of fertility, and outcome of pregnancies. contraception 2002;65:29-37. clinical theriogenology • volume 7, number 1 • march 2015 40 16. croxatto hb, mäkäräinen l: the pharmacodynamics and efficacy of implanon® 1: an overview of the data. contraception 1998;58:91-97. 17. wenzl r, van beek a, schnabel p, et al: pharmacokinetics of etonogestrel released from the contraceptive implant implanon®. contraception 1998;58:283-288. 18. makarainen md: ovarian function during the use of a single contraceptive implant: implanon compared with norplant. fertil steril 1998;69:714-721. 19. daels pf, mccue pm, demoraes mj, et al: persistence of the luteal phase following ovulation during altrenogest treatment in mares. theriogenology 1996;46:799-811. 20. ball ba, wilker c, daels pf, et al: use of progesterone in microspheres for maintenance of pregnancy in mares. am j vet res 1992;53:1294-1297. 21. squires el, heesemann cp, webel sk, et al: relationship of altrenogest to ovarian activity, hormone concentrations and fertility of mares. j anim sci 1983;56:901-910. 22. funk s, miller mm, mishell dr: safety and efficacy of implanon (tm), a single-rod implantable contraceptive containing etonogestrel. contraception 2005;71:319-326. 23. lofstedt rm, patel jh: evaluation of the ability of altrenogest to control the equine estrous cycle. j am vet med assoc 1989;194:361-364. 24. squires el: use of progestins in open and pregnant mares. anim reprod sci 1993;33:183-193. 25. huber j: pharmacokinetics of implanon®: an integrated analysis. contraception 1998;58:85-90. table. average iei of each mare based on 90-day observation period; iei was calculated based on twice weekly teasing observations and once weekly progesterone samples. treatment group interestrus interval (days) mare teasing progesterone group c c.1 21.75 22.75 c.2 20 21 c.3 21.75 21 c.4 21 21 c.5 21.75 22.75 group t1 t1.1 21.6 21 t1.2 21.75 21 t1.3 21 21 t1.4 49 47 t1.5 59.5 47 group t2 t2.1 21 21 t2.2 24.5 21 t2.3 35 30.7 t2.4 98 98 t2.5 35.3 34.3 group r r.1 110 49 r.2 111 45.5 r.3 115 49 r.4 109 49 clinical theriogenology • volume 7, number 1 • march 201541 figure 1. implant application. the implant is being placed in the right vulvar lip using the applicator provided by the manufacturer. local anesthesia was used prior to implant placement. figure 2. interestrus interval based on teasing scores and progesterone levels ± sem. different letters indicate significant differences between groups (p<0.05). uppercase letters compare iei based on teasing score and lowercase letters based on progesterone levels. clinical theriogenology • volume 7, number 1 • march 2015 42 figure 3. progesterone concentration in animals within the control group (no treatment). figure 4. progesterone concentration in animals within the one implant group (t1). clinical theriogenology • volume 7, number 1 • march 201543 figure 5. progesterone concentration in animals within the two implant group (t2). figure 6. progesterone concentrations in animals within the altrenogest group (r). clinical theriogenology • volume 7, number 1 • march 2015 44 2011: case report: gestational diabetes mellitus in a seven year-old german shorthair pointer bitch case report: gestational diabetes mellitus in a seven year-old german shorthair pointer bitch clinton yudelman,a courtney north,b phillip g.a. thomasc aadvanced vetcare, kensington, vic, australia; bcapital area veterinary specialists, austin, tx; cqueensland veterinary specialists, stafford heights, qld, australia summary a seven year-old pregnant female german shorthair pointer presented to the florida veterinary referral center on june 28, 2010 for reduced appetite, polydipsia and lethargy. the bitch was 53 days pregnant (based on date of mating) at presentation. serum biochemistry, blood glucose measurement and urinalysis were performed. the results indicated hyperglycemia and glycosuria. an abdominal radiograph confirmed the presence of seven fetuses. the bitch was diagnosed with the rare condition gestational diabetes mellitus (gdm) and managed medically with neutral protamine hagedom (nph) (novolintm, novo nordisk, bagsværd, denmark) insulin at increasing doses towards the end of gestation. an elective cesarean section and ovariohysterectomy were performed on day 63 after breeding. seven live puppies were delivered. three died within 15 days of birth and four puppies were alive at 24 weeks postpartum. the dog remained insulin dependent. keywords: gestational diabetes mellitus, insulin, insulin resistance, neonatal mortality, pregnancy background gestational diabetes mellitus is an extremely rare condition in bitches with only 15 reported cases in the veterinary literature.1,2 the 15 reported cases come from two studies. the first was a study published in 2006 as a case report of two dogs. in these two cases, seven puppies were born and all died within five days. the other study was a retrospective study involving 230 clinics across sweden. it described 13 cases of gdm. in this study bitches were separated into two treatment groups: termination of pregnancy (achieved through chemical abortion, ovariohysterectomy and cesarean section) or insulin therapy until the end of gestation (intermediate acting forms of human and porcine insulin). the results showed that termination of pregnancy at the time of diagnosis was associated with the bitch having a lower risk of developing permanent diabetes mellitus (dm).1 bitches with gdm display the classical quartet of clinical signs of dm: weight loss, polyphagia, polydipsia and polyurea. typically gdm occurs 50 days after mating in the last trimester of gestation and in the third or greater parity.1 gestational diabetes mellitus occurs as a result of insulin resistance mediated by progesterone and growth hormone (gh).3,4 these two hormones combine to have a synergistic antagonistic effect causing peripheral insulin resistance and producing hyperglycemia. in gdm, a post-receptor deficit results in an interruption of the normal intracellular signal transduction pathway preventing the normal action of insulin binding to its receptor.5 the beta cells of the islets of langerhans are subjected to glucotoxicity (blood glucose greater than 14 mmol/l, [252mg/dl])2,3 whereby irreversible damage occurs. subsequently the bitch has an increased susceptibility to sustaining permanent dm after parturition. these affects are related to the severity and duration of hyperglycemia.1 termination of pregnancy and ovariohysterectomy are advised once a diagnosis is confirmed to reduce the risk of the bitch developing permanent dm. additionally, the offspring may develop juvenile diabetes and other complications since the risk of fetal macrosomia increases as the length of gdm increases.5 although pregnancy termination at the time of diagnosis is the therapy most likely to prevent permanent dm in the bitch, each individual case is unique and there are multiple variables that need to be taken into account. these include the desire of the breeder and the potential monetary value of the litter. this case report and literature review describes the management of a bitch with gdm and supports the approach of pregnancy termination at time of diagnosis for treatment. furthermore, this case illustrates the lack of data pertaining to gdm and highlights the need for further research into the insulin clinical theriogenology • volume 3 number 4 • december 2011487 management requirements of bitches with gdm in order to reduce the associated morbidity and mortality. case presentation a seven year-old intact female german shorthair pointer presented to the florida veterinary referral center for reduced appetite, polyuria and lethargy on june 28, 2010. no abnormalities apart from tacky mucous membranes were noted on physical examination. the bitch was heavily pregnant with seven fetuses confirmed by abdominal radiographs. hematology, serum chemistry and urinalysis showed mild anemia (hematocrit 33.2%), neutrophilia (13.6x109/l), eosinophilia (2.96x109/l), hyperglycemia (26.5 mmol/l, [477 mg/dl]), high normal blood urea nitrogen (bun; 27 mmol/l) and 2+ glycosuria. there were no ketones in the urine. the serum progesterone concentration was 7.5 ng/ml and abdominal ultrasonography showed fetal heart rates ranging from 220 to 240 bpm. this was the third litter and the bitch had no history of these clinical signs during any previous gestation. a diagnosis of gdm was made and managed medically with twice daily subcutaneous injections of nph insulin commencing at 17 units. the intentions were to manage the pregnancy and control the clinical signs prior to an elective cesarean section closer to the due date. the dog was discharged with instructions to measure blood glucose three times daily; once before each insulin injection and once in the middle of the day. the dog was fed three times daily using the same food. blood glucose levels were measured at 09:00, 14:00 and 21:00 each day. the insulin dose was increased as the length of gestation increased, with insulin requirements increasing from 17 units bid to 30 units bid within five days. despite the increased dose, glycemic control was not achieved, and the bitch experienced increased levels of hyperglycemia. figure. blood glucose levels and insulin doses pre-partum the owner reported that seven days after presentation, the bitch began to display nesting behavior and the rectal temperature dropped from 37.7°c to 36.9°c. fetal ultrasound examination revealed heart rates of 160 to 180 bpm, a placental width of 0.8 cm, clear amniotic fluid and no evidence of fetal intestinal peristalsis. the plan was to examine the bitch with ultrasound the following day to determine if a cesarean section was required. the following day fetal ultrasound examination revealed heart rates as low as 130 bpm. an elective cesarean section and ovariohysterectomy were performed. the uterus contained seven fetuses, all of which were delivered alive. the dog’s blood glucose postoperatively was 35 mmol/l (630 mg/dl) and the dog was given a dose of short acting insulin. the following day the bitch was discharged with instructions to monitor blood glucose every two to three hours. the night after surgery the blood glucose clinical theriogenology • volume 3 number 4 • december 2011 488 was 13.1 mmol/l (235 mg/dl) and no insulin was given. the following day the blood glucose was 33.3 mmol/l (>600 mg/dl) and one of the puppies was not feeding well, did not gain weight and subsequently died that day. in the days following surgery the dog was maintained on 15 units of insulin bid and achieving glycemic control remained difficult with sporadic episodes of hyperglycemia. on day four postpartum another puppy was vocalizing and refused to suckle. the neonate died at four days of age. two weeks after surgery the owner reported that two of the seven puppies had died and the following day, the third pup died. the four remaining pups were doing well, suckling and gaining weight. twenty-five days after surgery, the bitch represented with hematuria. urinalysis was performed and the results showed increased microalbuminuria (16.4 mg/dl), urine specific gravity of 1.022, 2+ protein, 3+ blood and rod-shaped bacteria were seen on sediment examination. there was no glycosuria. bacterial cultured yielded a moderate growth of e. coli that was sensitive to amoxicillin/clavulanic acid. the bitch was treated with 250 mg amoxicillin trihydrate/clavulanate potassium (clavamox®, pfizer, new york, ny) bid for 14 days. one week later a urine culture was repeated and no bacterial growth was detected. control of the dog’s diabetes improved after the urinary tract infection resolved. the bitch was maintained on a dose of 12 units of nph insulin bid with four remaining healthy pups at the time of writing. this dose of insulin, being the lowest the bitch received, supports the presence of type ii insulindependent dm. insulin resistance mediated dm should be suspected in a bitch with supranormal blood insulin concentrations in the presence of normal or increased blood glucose concentrations. demonstrating the absence of islet-related autoantibodies along with supranormal insulin levels would be further evidence to support an insulin resistance mediated dm. this, along with serial measurement of serum progesterone and growth hormone concentrations would have been necessary to unequivocally support the diagnosis of gdm. despite the lack of irrefutable evidence to demonstrate pregnancy as the cause of gylcemic control, the previous history of the bitch in combination with the coincidence of clinical signs with gestation and improvement in glycemic control postpartum supports the diagnosis of gdm. the current recommendation is for bitches with diestrus diabetes to be spayed prior to their next cycle due to high risk of sustaining permanent dm if they go through another estrous cycle.3 a similar recommendation is made for cases of gdm. the longer pregnant bitches are subjected to insulin resistance, the more likely they are to develop permanent dm. although there is no biochemical evidence to support the presence of type ii diabetes in these cases, anecdotal evidence is strongly suggestive. discussion pregnancy is a diabetogenic condition. the fetus requires growth factors in-utero to mediate its growth and development. the hormones of pregnancy coalesce to cause peripheral insulin resistance which facilitates fetal growth and development in-utero. the two most notable hormones of pregnancy contributing to peripheral insulin resistance and gdm are progesterone and gh. in the bitch, progesterone is produced exclusively by the corpora lutea during pregnancy.6 in the absence of progesterone, gh production by the pituitary gland is pulsatile. however, under the influence of progesterone, higher concentrations of gh are constantly secreted by the hyperplastic ductular epithelium of the mammary gland.7 the combination of the progesterone and increased levels of gh have a strong synergistic effect resulting in significant peripheral insulin resistance. this combination provides the fetuses with glucose to support growth. evidence relating to the severity of insulin resistance under the influence of progesterone developed during the 1980’s. pioneering work by eigenmann demonstrated the relationship between progesterone and diabetes and that mean insulin requirements were 4.9 iu/kg in dogs with diestrus diabetes compared with much lower levels in diabetic dogs that were not in diestrus.8 this finding paved the way for the investigation into pregnancy-related insulin resistance and gdm. the increase in insulin resistance and concurrent reduction in insulin sensitivity is vital to the development of gdm. in humans, insulin action during the third trimester of pregnancy is reduced by 45 clinical theriogenology • volume 3 number 4 • december 2011489 to 70% compared with that in non-pregnant women.9 similarly, in pregnant bitches a 43% reduction in peripheral insulin sensitivity has been demonstrated.9 the bitch has often been used as a subject to investigate carbohydrate metabolism and is described as “an excellent biological model for the understanding of carbohydrate metabolism in late pregnancy”.1 the major tissues responsible for the insulin resistance in bitches are skeletal muscle and adipose tissue. the liver retains some insulin sensitivity during pregnancy.9 the pathophysiology of insulin resistance during diestrus diabetes is identical to gdm,4 yet the insulin resistance during pregnancy is much greater compared to that during diestrus diabetes.3 even though the insulin resistance during pregnancy is much greater, the incidence of gdm is negligible compared to diestrus dm. in the non-pregnant diestrus bitch, despite lower insulin resistance, the absence of the fetal drain of maternal glucose predisposes to development of diabetes more frequently and earlier than in pregnant dogs.2 bitches with diestrus dm develop the condition three to five weeks after estrus, which is much earlier than the average of seven weeks of gestation in bitches with gdm. the fetus plays an important role in draining maternal blood glucose and negating development of gdm. although gdm is rare, diestrus diabetes is common and is responsible for the higher incidence of dm in females than in males. in populations where juvenile females are not spayed, the incidence of diabetes in females is twice that of males while in populations where juvenile females are spayed, the incidence in females approaches that in males.5 in an uncomplicated case of gdm where minimal glucotoxicity occurs and pregnancy is terminated at the time of diagnosis, there is an abrupt cessation of progesterone secretion and progesterone-mediated gh concentrations, restoring glucose metabolism to normal.10 bitches return to normoglycemia within seven1 to 215 days postpartum. carbohydrate intolerance resolves once gh concentrations return to normal, whether through ovariohysterectomy, spontaneous regression of corpora lutea or withdrawal of progesterone.12 in this case, hyperglycemia exceeded the glucotoxic levels and consequently there was irreversible beta cell damage resulting in permanent dm. women with gdm are seven times more likely to develop permanent type ii dm postpartum compared to those who have a nondiabetic pregnancy.11 the same phenomenon occurs in the bitch and her offspring are at a greater risk of developing dm later in life.4 termination of pregnancy is reported to be the best option to reduce the likelihood of the bitch developing permanent dm.1 in that study bitches in which pregnancy was terminated did not go on to develop dm. eighty percent of the bitches treated with insulin developed permanent dm.1 one bitch that did not develop dm was the only one to receive an insulin dose > 2 iu/kg. despite insulin therapy (mean 1.3 iu/kg) during pregnancy, the mean blood glucose concentration was 28 mmol/l (504 mg/dl). in the case reported here, an insulin dose of >60 iu would have been required to achieve similar glucose concentrations. the highest dose of insulin given was 30 iu (approximately 1 iu/kg). in that study the bitches that were treated with insulin were on average older than those in which pregnancy was terminated. aging might be expected to make development of permanent dm more likely since amongst humans, aging is associated with decreased insulin secretory capacity.12 drawing a conclusion between treatment and outcome is thus difficult because of the confounding effect of age in addition to the low power of the study. gestational diabetes mellitus in the bitch is associated with a high neonatal morbidity and mortality rate. three of this bitch’s seven pups died before 15 days of age and at six months after delivery there are four surviving puppies. estimates of neonatal mortality following natural birth are 10 to 15% and 20% following cesarean section.13 in cases of gdm, a neonatal mortality rate of 27% is reported.1 in the case reported here the neonatal mortality rate was 43%. as in humans with gdm, if canine neonates do survive, they are at increased risk of developing dm. in a previous study, three of 60 pups born to bitches with gdm developed dm.1 the incidence of five percent found in that study is higher than the incidence of diabetes in the general population (0.31.3%).14 in another study, the incidence of stillborn pups, fetal macrosomia and associated complications was higher in pups born to bitches with experimentally induced dm.15 eighty percent of bitches with clinical theriogenology • volume 3 number 4 • december 2011 490 gdm that go to term experience dystocia from fetal macrosomia3 which contributes to the reduced survival rate of neonates. bitches are also subject to higher morbidity when pregnancy is complicated by gdm. bacterial urinary tract infections are common in dogs with dm. a decrease in immunity and increased glucose in the urine allow growth of bacteria and development of urinary tract infections. the effect of diabetes on the risk of infection is well-documented in humans. diabetic dogs suffer a similar increased susceptibility to infection. proposed mechanisms in humans include decreased blood supply due to microangiopathy and atherosclerosis leading to decreased phagocytes and antibodies, impaired humoral and cell mediated immunity and abnormal chemotaxis.16 in this report the bitch developed a postpartum urinary tract infection which complicated control of diabetes. after the effect of pregnancy on insulin resistance had subsided, the presence of a urinary tract infection contributed to ongoing insulin resistance making it difficult to find a consistent insulin dose to control the diabetes. following recovery from the infection, the bitch was maintained on a low dose of insulin (12 iu) suggesting that insulin resistance declined and was related to progesterone and gh concentrations and the infection. learning points  consider gdm as a differential for any pregnant bitch with hyperglycaemia. despite pregnancy, bitches should maintain normoglycemia.  pregnancy termination at the time of diagnosis of gdm is associated with the most favorable outcome and reduces the bitch’s chance of developing permanent dm.  the neonatal mortality and morbidity rates are higher in pups born to bitches with gdm.  medical management of the bitch with insulin requires doses to be >2 iu/kg.  much more information is required regarding the dose and frequency of insulin required to manage bitches with gdm. references 1. fall t, kreuger sj, juberget a, et al: gestational diabetes mellitus in 13 dogs. j vet intern med 2008;22:1296-1300. 2. norman ej, wolsky kg, mackay ga: pregnancy-related diabetes mellitus in two dogs. nz vet j 2006;54:360-64. 3. concannon pw: canine pregnancy and parturition. vet clin north am small anim pract 1986;16:453-475. 4. connolly cc, papa t, smith ms, et al: hepatic and muscle insulin action during late pregnancy in the dog. am j physiol regul integr comp physiol 2007;292:447-452. 5. feldman ec, nelson rw: canine and feline endocrinology and reproduction. 3rd ed. philadelphia: w.b. saunders; 2004. p. 486-535. 6. verstegen-onclin k, verstegen j: endocrinology of pregnancy in the dog: a review. theriogenology 2008;70:291-299. 7. kooistra hs, okkens ac: secretion of prolactin and growth hormone in relation to ovarian activity in the dog. reprod domest anim 2001;36:115-119. 8. eigenmann je, eigenmann ry, rjinberk a, et al: progesterone-controlled growth hormone overproduction and naturally occurring canine diabetes and acromegaly. acta endocrinol (copenh) 1983;104:167-176. 9. connolly cc, aglione ln, smith ms, et al: insulin action during late pregnancy in the conscious dog. am j physiol endocrinol metab 2004;286:909-915. 10. hackendahl n, schaer m, brown r: insulin resistance in diabetic patients: causes and management. compend contin educ vet 2006;28:271-283. 11. hod m, jovanovic l, carlo di renzo g, et al: textbook of diabetes and pregnancy. london: informa healthcare; 2008. 12. akehi y, oketa a, mitsoyushi y, et al: the effects of age on insulin sensitivity and insulin secretion in japanese subjects with normal glucose tolerance. rinsho byori 2007;57:901-905. 13. moon pf, erb hn, ludders jw, et al: properative risk factors for puppies delivered by cesarean sections in the united states and canada. j am anim hosp assoc. 2000;36:359-368. 14. fall t: characterisation of diabetes mellitus in dogs. acta univ agric (sueciae) 2009;45. 15. kleigman rm, miettinen el: fetal and neonatal metabolism in the pup of a canine diabetic mother. diabetes 1983;32:360-367. 16. radaelli t, varesthopour a, catalano p, et al: gestational diabetes induces placental genes from chronic stress and inflammatory pathways. diabetes 2003;52:2951-2958. clinical theriogenology • volume 3 number 4 • december 2011491 clinical theriogenology • volume 3 number 4 • december 2011 492 omniblank: 2014: conception following endoscopic removal of endometrial cysts in a mare � conception following endoscopic removal of endometrial cysts in a mare l.m.j. miller, s.m ferrer veterinary health center, college of veterinary medicine, kansas state university, manhattan, ks summary a 14-year-old appaloosa mare presented for breeding management. she was in diestrus with a history of failure to conceive that breeding season. in addition to poor vulvar conformation, the mare was determined to have a multi-loculated uterine cyst measuring 27 mm x 70 mm in her right uterine horn. a uterine culture yielded growth of streptococcus zooepidemicus. uterine biopsy was graded iia, with chronic endometritis being the main finding. at hysteroscopy, approximately seven endometrial cysts were seen in the right uterine horn in close approximation to each other and were ruptured using an endoscopic biopsy tool. endometritis treatment and breeding management were performed during the next estrus. uterine lavage and infusion of potassium penicillin was performed daily for five days. ultrasography was performed daily, and the mare was artificially inseminated on day three of treatment. the mare was determined to be pregnant at 60 days and at nine months of gestation. keywords: endometrial cysts, endometritis, hysteroscopy, pregnancy failure background while endometrial cysts are fairly common in middle-aged to older mares, this case is notable due to the successful treatment of intrauterine cysts and endometritis and conception in the same estrous cycle. it also shows that rupture with an endoscopic biopsy tool is sufficient to clear the uterus enough to succeed in conception in the same cycle. case presentation a 14-year-old appaloosa mare that had been shown as a halter horse was referred to the kansas state university veterinary health center (ksu-vhc) for failure to conceive and evaluation of an endometrial cyst. her reproductive history included having had one foal two years prior to presentation and she was artificially inseminated with cooled, fresh semen twice during the current breeding season. following the first breeding of the season, she was determined to be pregnant at 45 days post-ovulation, but was reported to have subsequently lost her pregnancy and returned to estrus. she was bred a second time by the referring veterinarian, presented to the ksu-vhc 23 days post-ovulation and was found to be not pregnant using transrectal ultrasonography. the referring veterinarian reported a 30 mm endometrial cyst in the right uterine horn that was thought to be affecting conceptus movement. on initial breeding soundness examination at ksu-vhc the mare was determined to have poor vulvar conformation (figure 1), and upon transrectal ultrasound evaluation the mare was determined to have a very large multiloculated endometrial cyst measuring 27 mm x 70 mm in her right uterine horn (figure 2). the endometrial cyst appeared to occupy approximately two thirds of the right uterine horn. the left ovary had a corpus luteum present. there was no edema present in the endometrium nor was there fluid present within the uterine lumen. the cervix measured two fingers wide. the mare was given 2.5 mg dinoprost tromethamine intramuscularly once per day for two days to induce corpus luteum regression and return to estrus for collection of uterine samples. the mare returned to ksu-vhc two days after the final dinoprost tromethamine treatment. a uterine culture and biopsy were performed. streptococcus zooepidemicus was isolated from the uterine culture. the uterine biopsy showed widespread infiltration with neutrophils and lymphocytes, scattered mild periglandular fibrosis, cystic glands and dilated lymphatics. the mare was diagnosed with endometritis and the biopsy was graded as iia according to kenney’s classification.1 the owner was given a prognosis for carrying a foal to term of 50-80%. treatment the mare returned 14 days after the previous visit during diestrus for treatment of the endometrial cysts via hysteroscopy. on hysteroscopy, it was noted that there was reddening of the endometrium clinical theriogenology • volume 6, number 1 • march 201441 41 � (figure 3), likely to be an area of endometritis. furthermore, approximately seven endometrial cysts were found in the right uterine horn in very close proximity to one another (figure 4). following rupture of the cysts using an endoscopic biopsy tool, transrectal ultrasography was performed. there was a corpus luteum present on the right ovary, a moderate amount of fluid present within the uterine lumen, and no remaining cysts were detected. a uterine lavage was performed using 3 l of 0.9% sodium chloride solution to remove the fluid and debris remaining following cyst removal. the mare was treated with 2.5 mg dinoprost tromethamine intramuscularly once per day for two days to induce corpus luteum regression and return to estrus. the mare returned three days after the final dinoprost tromethamine treatment for treatment of endometritis and breeding management. on presentation transrectal ultrasography was performed. the left ovary had a follicle measuring 31 mm x 30 mm, uterine edema grade 3, the cervix was measured two fingers wide, and there was a moderate amount of free fluid within the uterus. a uterine lavage was performed with 4 l of 0.9% saline, followed by infusion of 2,000,000 units of potassium penicillin into the uterus for two days. on day three of treatment, the mare had a 34 mm follicle on the left ovary, moderate endometrial edema, and the cervix measured two fingers wide. at that time, she was artificially inseminated with cooled semen containing 1 x 109 morphologically normal progressively motile spermatozoa. the mare received 2500 iu human chorionic gonadotropin intravenously at the time of insemination. six hours following insemination, transrectal ultrasonographic examination revealed severe endometrial edema and fluid accumulation in the uterus indicating postbreeding endometritis. the uterus was lavaged with 1 l of 13% dmso and 0.3% povidone iodine solution in sterile saline, followed by 2 l of sterile saline. penicillin (2,000,000 iu) was infused into the uterus after the lavage. at this time, the mare’s cervix was still very tightly closed, so 200 µg of misoprostol was placed within the cervical lumen to relax the cervix and aid in clearance of the uterine fluid. intrauterine infusions with penicillin continued for two more days for a total of five days of antibiotic treatment. oxytocin (20 iu) was given intramuscularly six hours after treatment and every two hours thereafter for six treatments. ovulation was detected with transrectal ultrasonography approximately 36 hours after insemination. the day following ovulation, a uterine infusion of 1 gram of ampicillin was administered and caslick’s vulvoplasty was performed. outcome the mare was determined to be pregnant at approximately 90 days post-ovulation. pregnancy was uneventful. however, at approximately 9.5 months of gestation the mare presented at the ksu-vhc with signs of colic. colon displacement was diagnosed, and surgery was performed. unfortunately, the mare had an adverse event while recovering from anesthesia and was euthanized. discussion in the case described here, the mare presented with multiple inciting causes of infertility. the most obvious on initial external examination was the poor vulvar conformation. her vulvar lips folded in along the sides as well as at the dorsal commissure with the rectum (figure 1). in addition, her vulva was also tilted cranially above the pelvic rim allowing contamination of the vestibule and vagina with feces. this poor conformation made this mare prone to pneumovagina and ascending endometritis.2,3 another possible cause of subfertility was presence of endometrial cysts. it is estimated that 55% of subfertile mares over the age of ten have endometrial cysts, which has an economic impact on the breeding industry in the form of additional breeding cycles, barren seasons, and misdiagnosis of pregnancy.4-6 endometrial cysts can be an indicator of underlying fibrosis and/or decreased myometrial contractility. the cysts tend to occur at the base of the uterine horns near the uterine body,7 which can interfere with implantation of the embryo. cysts can negatively affect pregnancy in many ways such as blocking conceptus movement, altering histotroph production, and causing insufficient endometrial contact with the conceptus,7,8 all of which contribute to embryonic death.9 cysts have been shown to reduce uterine vascular perfusion with a positive association between cyst area and disturbed uterine hemodynamics.6 clinical theriogenology • volume 6, number 1 • march 2014 42 42 � cysts can mimic signs of early pregnancy on ultrasographic examination, so it is suggested that a recent accurate map of any uterine cysts is prepared prior to breeding to decrease the probability of misdiagnosis of pregnancy. categorization of cysts is by size and location within uterine tissue. the three types are glandular, lymphatic, and transmural.10,11 lymphatic cysts or lymphatic lacunae are greater than 1 cm in diameter and can be up to 20 cm.11 these cysts are dilated areas of lymphatic tissue caused by uterine fibrosis and decreased myometrial contractility or may be caused by poor drainage due to gravity in a large pendulous uterus.5 mares with lymphatic cysts typically, but not always, have poor uterine biopsy scores.12 both glandular and lymphatic cysts are luminal, so their presence can affect conceptus migration and placentation since microcotyledons cannot be formed on the endometrium or the allantochorion in apposition to the cystic regions. both glandular and lymphatic cysts require numerous or very large cysts to compromise fertility and because of the typically larger size of lymphatic cysts, they are more commonly associated with fertility problems. histologically cysts are lined by columnar epithelium and lamina propria. they may contain normal or atrophied endometrial glands and within the cyst wall there is an inner lining of cuboidal cells and hyaline.4 a decision to treat uterine cysts should be based on the size, number and location of the cysts, and is typically reserved for mares in which they have a negative effect on fertility. a uterine biopsy is recommended prior to treatment of cysts to determine the prognosis for return to fertility after treatment. although mares are not typically treated and bred in the same estrous cycle, recovery from treatment is short, so a mare can be treated during the breeding season and returned to service that same year. the treatment in this mare consisted of rupture of the cysts using hysteroscopy and a uterine biopsy tool. other treatment options are to ablate the cysts using laser photoablation or loop electrocautery. neodymium/yttrium (nd:yag) laser irradiation is used in photoablation.4 loop electrocautery has the advantages of reduced equipment costs and reduced thermal trauma to the endometrium compared to photoablation.13 the advantages to using hysteroscopy and a uterine biopsy tool alone include minimal equipment needed and absence of thermal trauma to the endometrium. although the recurrence of cysts using this method is unknown, the cysts are likely to recur as a result of continuing fibrosis within the endometrium. while this treatment was successful in this case, there is not enough evidence to determine if this is a suitable treatment option for all mares or how it would compare with other treatment options. in addition to poor vulvar conformation and endometrial cysts, this mare also had bacterial endometritis (figure 3). it is unknown if the endometritis was secondary to her poor vulvar conformation or to overbreeding in previous estrous cycles. although it would have been preferable to treat the bacterial endometritis during the first estrus following hysteroscopy and wait until the following estrus to breed the mare, it was quite late in the breeding season and the owner elected not to wait. nevertheless, concurrent treatment of endometritis and insemination was successful. learning points � a mare can be successfully treated for endometrial cysts and endometritis and be successfully bred during the same estrous cycle. � it is important to evaluate an endometrial biopsy to determine the prognosis for an individual animal. large endometrial cysts are not always associated with fibrosis and poor prognosis for pregnancy. � lymphatic endometrial cysts can be treated by rupturing the cysts with biopsy instruments via endoscopy, followed by successful pregnancy in one cycle. references 1. kenney rm: cyclic and pathologic changes of the mare endometrium as detected by biopsy, with a note on early embryonic death. j am vet med assoc 1978;172:241-262. 2. hemberg e, lundeheim n, einarsson s: retrospective study on vulvar conformation in relation to endometrial cytology and fertility in thoroughbred mares. j vet med a physiol pathol clin med 2005;52:474-477. 3. heilkenbrinker t, schubert ts, oetjen j, et al: consequences of a surgical correction of an insufficient closure of the vulva on clinical theriogenology • volume 6, number 1 • march 201443 43 � genital flora and conception rate in mares. dtsch tierarztl wochenschr 1995;102:183-187. 4. blikslager at, tate lpj, weinstock d: effects of neodymium:yttrium aluminum garnet laser irradiation on endometrium and on endometrial cysts in six mares. vet surg 1993;22:351-356. 5. bracher v, mathias s, allen wr: videoendoscopic evaluation of the mare's uterus: ii. findings in subfertile mares. equine vet j 1992;24:279-284. 6. ferreira jc, gastal el, ginther oj: uterine blood flow and perfusion in mares with uterine cysts: effect of the size of the cystic area and age. reproduction. 2008;135:541-550. 7. tannus rj, thun r: influence of endometrial cysts on conception rate of mares. zentralbl veterinarmed a 1995;42:275-283. 8. mcdowell kj, sharp dc, grubaugh w, et al: restricted conceptus mobility results in failure of pregnancy maintenance in mares. biol reprod 1988;39:340-348. 9. yang y-j, cho g-j: factors concerning early embryonic death in thoroughbred mares in south korea. j vet med sci 2007;69:787792. 10. rambags bpb, stout tae: transcervical endoscope-guided emptying of a transmural uterine cyst in a mare. vet rec 2005;156:679-682. 11. wilson dl: diagnostic and therapeutic hysteroscopy for endometrial cysts in mares. vet med 1985;80:59-63. 12. adams gp, kastelic jp, bergfelt dr, et al: effect of uterine inflammation and ultrasonically-detected uterine pathology on fertility in the mare. j reprod fertil suppl 1987;35:445-454. 13. bartmann cp, stief b, schoon ha: thermal injury and wound healing of the endometrium subsequent to minimally invasive transendoscopic use of nd:yag-laser and electrosurgery in horses. dtsch tierarztl wochenschr 2003;110:271-280. ������� ���� ������������������������������� clinical theriogenology • volume 6, number 1 • march 2014 44 44 � (editor’s note: the photographs in this manuscript appear in color in the online version of clinical theriogenology.) figure 3. inflamed area of the��������������������� ����������������������������������������������� figure 4. uterine cysts as seen on hysteroscopy. figure 2. large, multiloculated intrauterine cyst in the right horn. the cyst appeared to involve two-thirds of the uterine horn. shown in cross-section (a) and length-wise (b). � �� clinical theriogenology • volume 6, number 1 • march 201445 45 46 can we preserve immunoglobulin g in frozen colostrum of mares for three years? can we preserve immunoglobulin g in frozen colostrum of mares for three years? thibaut lemirre, anne josson-schramme, samuel buff université de lyon, vetagro sup, veterinary campus of lyon, marcy-l’étoile, france foals need good quality colostrum at birth, containing large amounts of immunoglobulins to have adequate passive transfer for early life protection, as antibodies do not cross placenta during pregnancy. colostrum is therefore a key factor for immunity of foals, as they are born almost without immunoglobulins. colostrum banking is routinely used by breeders all over the world to overcome failure of passive transfer in the newborn foal. however, to our knowledge, there are no data in the literature regarding long term preservation of immunoglobulin g (igg) in frozen mare colostrum. radial immunodiffusion (rid) is the gold standard method to measure igg concentrations in colostrum of mares. objective was to evaluate igg concentrations in frozen colostrum and to compare rid and refractometry methods to measure igg in frozen colostrum. we hypothesized that igg concentrations remain stable for 3 years and that refractometry is a reliable method to evaluate the quality of frozen colostrum. during the foaling season of 2014, colostrum was collected from 36 mares of several breeds in normandie, france. after a simple filtration, igg concentrations were measured by rid and refractometry. all samples were divided into 2 ml plastic eppendorf microtubes before freezing. at 3, 6, 9, 12, and 36 months, 2 samples from each mare were thawed in a water bath at 37°c. immunoglobulin g concentrations were measured using both methods. colostrum from 22 thoroughbreds, 12 standardbreds, 1 warmblood, and 1 pony were collected immediately post foaling and prior to a foal suckling its dam. before freezing, igg concentrations were measured. they were higher (p < 0.005) in the colostrum (thick yellow) of standardbreds compared to the colostrum (watery and white) of thoroughbreds. there was no significant difference between the 2 methods. after 12 months, igg concentrations remained stable and there was no difference between methods. at 36 months, igg concentrations was significantly higher than before freezing when evaluated by rid and stable by refractometry. in a mixed model, the ‘mare’ factor was the only significant effect on colostrum concentration. the rid method had higher variation within the same sample compared to refractometry, whereas refractometry results were highly repeatable. compared to rid, refractometry was a practical and reliable method to evaluate colostrum quality of frozen thawed colostrum. furthermore, igg concentrations in frozen colostrum were stable after 36 months. keywords: horse, colostrum, freezing, immunoglobulins 465 clinical theriogenology • volume 11, number 3 • september 2019 466clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 1 contact maria ferrer msferrer@uga.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10351, http://dx.doi.org/10.58292/ct.v16.10351 review report role of trace minerals in bull reproductive physiology and semen quality* maria ferrer,a roberto palomares,b juan maldonado-estradac adepartment of large animal medicine, college of veterinary medicine, university of georgia, athens, ga, usa bdepartment of population health, college of veterinary medicine, university of georgia, athens, ga, usa cschool of veterinary medicine, university of antioquia, medellín, colombia abstract testicular dysfunction is often associated with an imbalance in antioxidant/oxidant homeostasis, leading to negative effects of oxidative stress on germ cell proliferation, steroidogenesis, and sperm function. trace minerals (tm) are involved in basic homeostatic and enzymatic processes like free radical detoxification, cellular respiration, carbohydrates, lipids, nucleic acids, synthesis and metabolism of proteins, and stabilization of membranes and dna. dietary source of tm is necessary to support these processes. supplementing cattle with tm is nowadays a common practice to help support growth, reproduction, and immunity. this review provides information on roles of tm in bulls testicular and sperm functions, and effects of tm mineral supplementation on bull reproductive parameters. keywords: cattle, sperm, zinc, selenium, copper, manganese introduction* every year, 30% of breeding bulls should be culled due to poor semen quality.1 the most common cause of declining semen quality is testicular degeneration that is often associated with an imbalance in antioxidant/oxidant homeostasis; this leads to increased concentrations of reactive oxygen species (ros) and oxidative stress that induces germ cell apoptosis and decreases germ cell proliferation.2–4 in young bulls, sexual immaturity is also often associated with poor semen quality. trace minerals, e.g. selenium (se), zinc (zn), copper (cu), and manganese (mn), are cofactors for antioxidant enzymes like superoxide dismutase (sod) and glutathione peroxidase (gpx). thus, trace minerals have an important role in preventing oxidative damage on cell membranes and nucleic acids in germ cells, and supporting spermatogenesis and steroidogenesis.5,6 this review provides information on trace minerals’ roles in bulls testicular and sperm function, and the effects of trace mineral supplementation on bull reproductive parameters. *presented at the 2023 society for theriogenology conference, published after peer review. oxidant-antioxidant balance in male reproductive tract and gametes oxygen-derived free radicals are molecules that contain unpaired electrons in their outermost orbits. these molecules are highly reactive and have a very short half-life since they tend to stabilize rapidly by yielding or accepting electrons from other molecules. reactive oxygen species (ros) include superoxide anions (o2−), hydrogen peroxide (h2o2), proxyl (·roo) and hydroxyl (·oh). low concentrations of endogenous ros are involved in spermatogenesis, sperm modification within the epididymis, and sperm function leading to fertilization.7 within testis, ros can act as second messengers regulating cell-signaling pathways involved in spermatogonia self-renewal and proliferation.7 they are also involved in regulating apoptosis that is fundamental to maintain an adequate germ cell to sertoli cell ratio, and to eliminate defective sperm.8 in epididymis, ros participate in the formation of sperm nuclear disulphide bridges required for chromatin condensation,  and in phosphorylation and dephosphorylation of proteins in the fibrous sheath of the flagellum, activating progressive motility.7 in female tract, ros are necessary for sperm capacitation, hyperactivation, acrosome reaction, and fertilization. mailto:msferrer@uga.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10351 2 citation line: clinical theriogenology 2024, 16, 10351, http://dx.doi.org/10.58292/ct.v16.10351 sperm-specific physiologic ros production occurs mainly in the mitochondrial electron transport system.9 bull sperm contain ~ 72 mitochondria located within 3 spirals around midpiece.10 oxidative phosphorylation in these mitochondria is essential for sperm motility, but generates hydroxyl radicals, superoxide anion, h2o2, and nitric oxide.8 intracellular ros are also produced through enzymes such as β-nicotinamide adenine dinucleotide phosphate (nadph) oxidases in the cell membrane, and cytochrome p450-dependent oxygenases in the mitochondria and endoplasmic reticulum.9 although lower concentrations of ros have a physiological role in male fertility, higher concentrations of ros overwhelm the endogenous antioxidant system and cause oxidative stress. endogenous sources of pathologically increased ros production in the ejaculate include immature germ cells, leucocytes, dead or abnormal sperm, and epithelial cells.9 immature germ cells in the ejaculate contain excess cytoplasm. additional cytoplasm contains glucose-6-phosphate dehydrogenase enzyme that controls intracellular nadph production. the nadph fuels the production of ros via nadph oxidase.11 leukocytes contain peroxidase and can produce 1,000 times more ros than sperm. activation of the myeloperoxidase system from neutrophils and macrophages leads to respiratory burst (the increase in cell metabolism and oxygen consumption during phagocytosis, coupled with the release of reactive oxygen species [ros], and increased ros products). increased ros production in the ejaculate is present in cases of inflammation, heat stress or pollutants such as phthalates and heavy metals.11 a low antioxidant activity in seminal plasma can also lead to imbalance and accumulation of seminal ros.7 sperm damage by ros results in alterations in nuclear and mitochondrial dna, plasma membrane lipid peroxidation, loss of atp, and axonemal injury.11 reactive oxygen species can also affect leydig cells and decrease testosterone production.11 oxidation of dna bases occurs more commonly in areas with less protamine. guanine base in dna is oxidized by free radicals and converted to 8-hydroxyguanine (8-ohg). sperm have 8-oxoguanine dna glycosylase enzymes that can remove 8ohdg. however, sperm lack the downstream components of the repair pathways and are unable to repair the dna at the sites of the base excisions. result is fragmentation of dna, altered gene expression, and activation of apoptosis and autophagy.7,11 epididymal or seminal ros can oxidize free thiol groups, inhibiting formation of disulphide bridges in sperm proteins within the flagellum that causes sperm morphological defects, such as bent tails.7,9 oxidation of sulfhydryl groups decreases the phosphorylation of axonemal proteins, decreasing sperm motility.8 in addition, sperm plasma membrane is rich in polyunsaturated fatty acids with double bonds separated by methylene groups that render them particularly vulnerable to oxidation. lipid peroxidation generates lipid radicals that propagate oxidation across plasma membrane; this process leads to generation of by-products such as malondialdehyde (mda), 4hydroxynonenal (4hne) and acrolein. malondialdehyde is mutagenic, whereas 4nhe and acrolein have a direct genotoxic effect on dna, and increase mitochondrial ros production.8 membrane lipid peroxidation reduces plasma membrane integrity and fluidity, with alterations in ion transport, receptors and enzymes; this affects membrane potential, sperm motility, viability and capacitation, and induces a premature acrosome reaction.7,9 higher concentrations of ros can also alter mitochondrial membrane, resulting in caspase activation and apoptosis.11 to keep the amount of ros under control, sperm have endogenous intracellular antioxidant enzymes, such as superoxide dismutase (sod), glutathione peroxidase (gpx), glutathione reductase, and catalase (cat). seminal plasma is also rich in enzymatic and nonenzymatic antioxidants.8,12 in addition, compartmentalization of mitochondria in midpiece provides a physical separation from nuclear dna, thus preventing possible damage in case of ros overproduction.8 superoxide dismutase spontaneously dismutates o2− to form oxygen (o2) and h2o2. superoxide dismutase is the most important antioxidant enzyme in bovine sperm and seminal plasma, and controls mitochondrial and plasma membrane o2production. intracellular sod-1 or cuznsod, contains copper and zinc in its active center, and is present in sperm cytoplasm. intracellular sod-2 or mnsod is located in the mitochondrial matrix, and contains manganese in the active center.13,14 extracellular form of sod or sod-3 also contains zinc and copper in its active center and originates in prostate.12–13 due to sod activity, h2o2 is produced that is eliminated by cat or gpx. glutathione peroxidase in sperm and semen has various isoforms, with most of them containing selenium. the gpx4 is located on the postacrosomal and apical head region, and within sperm nucleus, whereas gpx5 is expressed in the accessory sex glands, testis, and epididymal epithelium, and is present in seminal plasma.12 glutathione reductase is present in the flagellum and seminal plasma. catalase catalyzes the decomposition of h2o2 into o2 and h2o. bull sperm may acquire cat from extracellular milieu, and this membrane-bound cat may have an important role in protecting these cells during maturation and transport in the female reproductive tract. although some investigators have not detected cat activity in bull sperm, cat activity remains higher in bovine seminal plasma that originate from prostate.12 peroxiredoxins are nonselenium enzymes capable of scavenging h2o2, organic hydroperoxides, and onoo–, and are widely distributed in all compartments of bovine sperm.14 nonenzymatic antioxidants in bovine seminal plasma include glutathione, melatonin, ergothionine, carnitines, ascorbic acid, α-tocopherol, pyruvate, taurine/hypotaurine, retinol, and urate.12 oxidative damage can also occur due to a deficiency in enzymatic antioxidants, generated by a genetic mutation, incorrect posttranslational modification, posttranslational deactivation, nutritional deficiency, disease states, or toxicities.12 nutritional sources of zn, cu, mn, and se are required for sufficient sod and gpx production and activity.12 deficiencies in these trace minerals can result in inadequate production of these enzymes because they are integral parts of their active core. sperm from zn and cu deficient animals have reduced longevity, viability, acrosome integrity, and motility.12 similarly, gpx deficiency has been associated with se deficiency, and these sperm have incomplete dna condensation, dna fragmentation, and abnormal morphology.12 role of copper in male reproductive function copper is mainly in the highly soluble cupric (cu2+) oxidized state. dietary cu absorption is reduced in the rumen due to interactions with other minerals, mostly molybdenum and zn. organic cu has better absorption than the inorganic form. absorbed dietary cu is bound to albumin and is carried to liver. small amount of cu is excreted in the bile, whereas the majority binds to ceruloplasmin and is released into bloodstream. within testes, ~ 80% of cu is bound to http://dx.doi.org/10.58292/ct.v16.10351 citation line: clinical theriogenology 2024, 16, 10351, http://dx.doi.org/10.58292/ct.v16.10351 3 ceruloplasmin in sertoli cells. the remaining cu is bound to metallothioneins in sertoli and spermatogenic cells.15 copper is an essential element since it participates in electron transfer reactions within a large number of proteins involved in basic homeostatic processes, such as cellular respiration (cytochrome c oxidase), free radical detoxification (sod), iron transport (ceruloplasmin), neuropeptide synthesis (peptidylglycine α-amidating monooxygenase), connective tissue formation (lysyl oxidase), and pigment synthesis (tyrosinase). dietary sources of cu are needed for proper synthesis and function of these enzymes. sperm from cu-deficient ruminants and rodents have reduced concentrations, longevity, viability, acrosome integrity, and motility as a result of reduced sod activity and oxidative stress.12,15 in addition, cu is a cofactor in the mitochondrial cytochrome c oxidase, and is required for aerobic atp production by sperm. copper is also a cofactor for the enzymes involved in prostaglandin and steroid synthesis.15 daily dietary supplementation in nondeficient bucks with 12.5 or 25 mg/kg of cu proteinate increased sperm concentration, motility, membrane integrity, acrosome integrity, and cryosurvival.16–19 this was associated with increased expression and activity of sod-1, cat, and gpx4 in sperm and decreased lipid peroxidation.17,18 supplementation of bucks with cu also increased plasma testosterone concentrations, sexual behavior, scrotal circumference, and advanced puberty ~ 30 days.16,18,20 although cu is essential for spermatogenesis and sperm function, hypercuprosis can be toxic, especially in sheep. highly reactive ‘free’ cuprous ions (cu+) react with h2o2, generating hydroxyl radicals via fenton and haber-weiss reactions.15 copper can also bind directly to free thiols of cysteines, leading to oxidation and crosslinks between proteins, thus inactivating enzymes or impairing structural proteins. dietary supplementation of higher concentrations of cu to rodents and pigs decreased spermatogenesis, testicular weight, sod, and gpx expression and increased germ cell apoptosis and autophagy, and testicular ros production and lipid peroxidation.15,21–23 sperm count, motility, viability, and morphology, as well as testosterone production, were negatively affected with hypercuprosis.21,23 role of zinc in male reproduction zinc can be supplemented in inorganic forms, such as sulphate (znso4) or oxide (zno), or in organic forms, such as propionate or proteinate. bioavailability of organic forms is higher than that of inorganic zn. tryptophan content in the diet is important for absorption of dietary zn. tryptophan is metabolized to picolinic acid by pancreatic exocrine cells of that is secreted into the intestinal lumen, where it combines with zn, with the picolinic acid–zn complex absorbed by the intestine.24 zinc is involved in several enzymatic reactions, and is closely associated with carbohydrate, lipid, nucleic acid, and protein metabolism. apart from its involvement in the antioxidant system, zn directly stabilizes membranes, lysosomes, ribosomes, rna and dna; it is also involved in dna replication, transcription, and translation into proteins.16,18 within testis, zn is critical for germ cell proliferation, spermiogenesis and histone substitution by protamine, and regulates testosterone synthesis.25 in post-testicular sperm, zn is essential for production and function of numerous enzymes that are important in normal sperm function and prevention of sperm damage, and has a regulatory role in sperm capacitation and acrosome reaction. zinc controls sperm motility by controlling energy utilization through zn-containing enzymes like sorbitol dehydrogenase and lactate dehydrogenase.6 in addition, proteins in the outer dense fiber (odf) incorporate and release zn depending on the external environment.26 zinc in chromatin and the odf forms salt bridges with thiols, thus preventing oxidation of thiols. prostatic fluid is rich in zn that likely contributes to protecting sperm chromatin and odf from shear forces at ejaculation.26,27 after ejaculation, zn from chromatin and odf is rapidly lost, probably after the contribution of chelating agents from vesicular glands. removal of zn from chromatin and odf allows for formation of disulfide bridges and stiffening of these structures. stiffening of the odf results in small flagellar wave amplitudes characteristic of progressive motility. in contrast, incorporation of zn causes softening, leading to hyperactivated motility. therefore, chelation of zn has a role in inducing progressive motility and preventing premature hyperactivation. zinc is also important in preventing sperm dna damage since it inhibits dnaase.26,27 zinc deficiency resulted in spermatogenic arrest, germ cell apoptosis and degenerative changes in seminiferous tubules, extracellular matrix, blood-testis barrier, and leydig cells.28 testicular changes resulted in decreased sperm concentration and motility, with an increase in sperm acrosome, and head and mitochondrial abnormalities.28 supplementation of zn proteinate in nondeficient bucks increased sperm concentration, motility, and acrosome and plasma membrane integrity.16,18–20 this was associated with decreased oxidation of sperm lipids and proteins due to increased total antioxidant capacity and activity of seminal plasma sod, cat, and gpx.16,19,29 however, the beneficial effects of supplementation were only observed when zn was supplemented at low (20 mg/ kg of dry matter-dm) or medium (40 mg/kg dm) levels. feeding a higher level of zn proteinate (60 mg/kg dm) had detrimental effects on antioxidant capacity, oxidative stress and sperm parameters.16,18–20,29 feeding 50 200 mg of znso4 to goats also improved semen volume, sperm motility, and sod and gpx activity in seminal plasma.30 in 2-year old normospermic beef bulls, feeding organic or inorganic forms of zn for 6 months increased sperm count, motility, viability, acrosome integrity, membrane functionality, and mucous penetration.6 zn supplementation also increased blood testosterone concentrations.6 role of manganese in male reproduction manganese is a component of several enzymes, particularly mnsod, and it scavenges free oxygen radicals and stabilizes cell membrane.31 manganese is also a potent stimulator of sperm motility through the stimulation of adenylate cyclase activity. supplementation with lower concentrations of mn in rats increased lh, fsh, and testosterone production, increased daily sperm production and efficiency of spermatogenesis, and accelerated puberty.32 lower concentrations of mn also prevented negative effects of formalin on testicular weight, and sperm motility, viability, morphology, and count in mice.31 oral supplementation in mature dairy bulls with mnso4 (30 ppm), coclo2 (0.22 ppm) and crcl2 (2 ppm) for 120 days increased sperm motility, viability, membrane functionality, and acrosome integrity in fresh and frozen semen. this was associated with increased sod activity in blood and decreased lipid peroxidation in semen.33 http://dx.doi.org/10.58292/ct.v16.10351 4 citation line: clinical theriogenology 2024, 16, 10351, http://dx.doi.org/10.58292/ct.v16.10351 however, high doses of mn can be detrimental for fertility due to a decrease in sperm concentration, motility and viability.34 these changes were associated with oxidative stress within testis. even though the activity of sod increased, higher mn concentrations decreased cat activity. increased sod activity can eliminate superoxide radicals by dismuting them to h2o2; however, reduction in cat activity affected the detoxification of h2o2. 34 in bulls, mn supplementation at ≥ 1,300 mg/kg of mineral mix reduced sperm membrane and acrosome integrity, and even the small amount of mn contained in the control diet (540 mg/kg of mineral mix) had a negative effect on sperm mitochondrial membrane potential.35 thus, supplementation effect with mn on fertility is not entirely clear. role of selenium in male reproduction selenium is a component of several enzymes and has a role in many biological processes including antioxidant defense, fertility, immunity, thyroid metabolism, endocrine function, carcinogenesis, and muscle function. inorganic se is present in 3 oxidation states: selenite (se4+), selenate (se6+), and selenide (se2−). plants obtain se4+or se6+ from soil to synthesize se-containing amino acids. sodium selenite is the inorganic form most commonly used in animal diets, whereas organic se is supplemented as selenized yeast or selenomethionine. organic se was more effective at improving sperm morphology and viability in buffaloes than inorganic se.36 dietary se is transported to testis by plasma selenoprotein p. in testis, sertoli cells se uptake is by the apolipoprotein e receptor-2 (apoer2), and is utilized for the synthesis of selenoproteins. the main testicular selenoprotein is gpx4, expressed primarily in sperm. the gpx4 is present in 3 isoforms: mitochondrial, nuclear, and cytosolic, each carrying out separate functions but encoded by a single gene. mitochondrial gpx4 is involved in enzymatic defense against oxidative damage to mitochondria, and becomes active in spermatocytes and spermatids. mitochondrial gpx4 also maintains the form and structure of the mitochondrial capsule by constructing cross-linkages with itself and other proteins. nuclear gpx4 stabilizes condensed chromatin during sperm maturation. lastly, both gpx1 and cytosolic gpx4 are expressed in the epididymal epithelium.37 fate of se after injection was studied in bulls using radioactive 75se. injection of 75se into bulls resulted in an increase in 75se concentrations in the blood within 6 hours, followed by a decline within 48 hours.38,39 thereafter, 75se distributed primarily to the liver, kidneys, and reproductive tract, main storage sites for this element. concentrations of 75se in seminal plasma exceeded the concentrations in blood due to accumulation and secretion from prostate and seminal vesicles. 75se in seminal plasma reached peak concentrations 5 days after injection and declined after 12 days. seminal plasma was the only source of 75se in semen during the first 2 weeks after injection.38,39 starting from 12 to 15 days after injection, 75se appeared in ejaculated sperm. sperm 75se accumulation peaked ~ on day 20 and, after plateauing, declined rapidly after 40 days.38,39 timing of 75se appearance in sperm is due to the fact that se is incorporated by the spermatid but not during passage through epididymis.38 effect of se supplementation on reproductive parameters were studied in se-deficient bulls, buffaloes, boars, rams, and mice.36,40–43 although supplementation increased concentrations of se and gpx activity in reproductive tissues and semen, effect on semen quality was variable. effects ranged from no changes40,42 to increased ejaculate volume, sperm concentration, motility, morphology, and viability.36,41,43 se supplementation also increased blood testosterone concentrations, and the number of round and elongated spermatids.36,40,43 effects of supplementing nondeficient males with se are less well characterized. although providing an excessive amount of se to deficient mice improved semen quality compared to control animals, it did not provide any additional benefits of feeding above an adequate dose.43 role of iron in male reproduction iron (fe) is essential for oxygen transport and exchange, intracellular enzyme function and mitochondrial electron transport chain, and has a dual role in regulating redox reactions. iron is the bioactive center of catalase and superoxide dismutase. within testis, fe is essential to support mitosis and spermatogenesis since it is needed for dna synthesis, protein synthesis, cellular respiration, proliferation, and differentiation.44 circulating fe is transported through the blood-testis barrier by sertoli cells bound to testicular transferrin to be taken up by germ cells. mitochondria of spermatids are also engulfed by sertoli cells that recycle fe back to primary spermatocytes in the basal compartment. thus, sertoli cells modulate fe homeostasis within the seminiferous tubule, protecting germ cells from iron fluctuations.44 ferritin is an important source of iron during sperm development and provides an additional layer of protection for testicular tissues. peritubular myoid cells and sertoli cells are the main locations of testicular cytosolic ferritin storage, whereas mitochondrial ferritin is mainly expressed in germ cells and leydig cells. mitochondrial ferritin insulates excessive iron, making it less susceptible to fenton and haberweiss reactions and protecting mitochondria from oxidative damage.44 iron deficiency decreased testosterone synthesis, and glutathione peroxidase and catalase activity, whereas it increased cleaved-caspase 8 and caspase 3 expression in fe-deficient rats.45 anemia caused by severe fe deficiency creates a hypoxic environment for testis, impairing spermatogenesis.44 men with sickle cell disease had impaired sperm production, motility, and morphology associated with testicular failure and lower testosterone concentrations.15 iron aids in sperm motility and energy metabolism. concentrations of fe in bovine seminal plasma were positively correlated with sperm motility, membrane integrity and dna integrity.46 supplementation of the medium with lower concentrations of fe improved motility of bovine sperm.47 however, supplementation with higher concentrations were toxic and increased ros production.47 other studies have reported increased intracellular fe concentrations in sperm from bulls with low fertility, associated with dna damage and decreased motility.48 this dual action is due to excess fe ability to aggravate the generation of ros via the fenton and haber–weiss reactions. thus, iron homeostasis is critical for testicular and sperm function. iron overload was associated with hypogonadism in boars, bulls, and men.15 exposure of sperm to higher fe concentrations reduced motility and viability, decreased mitochondrial membrane potential, increased intracellular ros generation, and impaired sperm fertilizing ability.15 http://dx.doi.org/10.58292/ct.v16.10351 citation line: clinical theriogenology 2024, 16, 10351, http://dx.doi.org/10.58292/ct.v16.10351 5 associations between the concentrations of trace minerals in bull semen and semen quality most abundant tms in semen samples from fertile normospermic bulls are mg, zn, se, and fe.49 however, the literature reports contradictory findings regarding the association between concentrations of tms in semen samples and semen quality. in bovine seminal plasma, the concentrations of zn and fe were positively correlated with sperm motility, viability, acrosome integrity and dna integrity in fresh and frozen semen.46,50,51 some authors reported no correlation between cu concentrations in seminal plasma and sperm motility, whereas others reported a positive correlation between these 2 parameters.50,51 conversely, concentrations of se in seminal plasma were negatively correlated with sperm motility, viability, acrosome integrity, and dna integrity.46 intracellular concentrations of zn and fe in bovine sperm were negatively correlated with sperm dna integrity and motility parameters, and were lower in bulls with high fertility.46 however, another study reported a positive correlation between bovine sperm motility and intracellular concentrations of zn, fe, and cu.47 intracellular concentrations of mn was negatively correlated with sperm motility, acrosome integrity, atp content, and dna integrity.46 supplementing bulls with trace mineral mixes trace minerals supplementation in cattle is nowadays a common practice to help support growth, reproduction, and immunity. primary source of trace minerals for grazing cattle is forage, grain, and feed. forage uptakes trace minerals from the soil. in some geographical areas or conditions, the soil is deficient in some trace minerals, and the trace minerals provided during grazing are not enough, requiring supplementation. oral supplements are commonly provided in the form of loose mineral mixes, fortified salt blocks, and fortified energy or protein supplements. in addition, trace minerals can be given in injectable forms that bypass gut-level antagonisms and overcome poor intestinal absorption and the associated variation in trace-mineral intake. trace mineral mixes supplementation were used in beef bull calves in an attempt to hasten puberty and to improve reproductive parameters in peripubertal animals.48–55 trace mineral-depleted 9-month old calves were fed a supplement containing various concentrations of complexed or sulfated zn, mn, cu, and co for 100 days.52 trace minerals supplementation did not improve scrotal circumference, sperm motility, morphology, or concentration. it also did not increase blood concentrations of luteinizing hormone, follicular stimulating hormone, testosterone, or seminal plasma concentrations of trace minerals.52 however, a higher percentage of bulls reached puberty after 42 days of treatment when fed complexed trace minerals (79%) compared to those fed sulfated forms (47%).52 moreover, feeding high concentrations of sulfated trace minerals delayed puberty. this was presumably because of the higher sulphur content that may affect se and cu metabolism.52 similarly, feeding trace mineral-depleted 9-month old calves with 641 mg/day of zn hydroxychloride, 193 mg/day of cu chloride, or their combination for 84 days did not improve body weight, time to puberty, breeding soundness examination (bse) classification, scrotal circumference, or sperm motility, morphology, and concentration.53 however, feeding zn and cu combination improved sperm viability after 3 hours of incubation, and mitochondrial membrane potential and acrosome integrity.53 reproductive benefit of supplementing nondeficient calves with trace minerals is less clear. treatment of nondeficient 9-month old calves with a single dose of 1 ml/45 kg of injectable trace minerals (cu 15 mg/ml, mn 10 mg/ml, se 5 mg/ ml, and zn 60 mg/ml) did not improve semen quality, scrotal circumference or the percentage of bulls classified as satisfactory breeders as yearlings 91 days after injection.54 however, 2 doses of injectable trace minerals at 7 months (1 ml/54 kg) and 10 months of age (1 ml/68 kg) resulted in mild increases in sperm motility 2 months after second injection.55 injectable trace minerals did not improve the percentage of bulls classified as satisfactory breeders, scrotal circumference, or sperm morphology, but sperm motility was slightly higher during the second bse at 12 months of age in trace mineral-treated bulls (42%) compared to control bulls (40%).55 of the bulls classified as nonsatisfactory breeders during the first bse at 10 months of age, there was a tendency (p = 0.1) for a higher percentage of bulls to be reclassified as satisfactory breeders 2 months later when treated with trace minerals (98%) compared to control bulls (94%).55 mature trace mineral-depleted bulls benefited from supplementation with cu (83.3 ppm), zn (233.3 ppm), and mg (300 ppm) sulfate or chloride for 71 days.53 although scrotal circumference and sperm morphology did not differ from bulls fed a trace mineral-deficient control diet, sperm motility and concentration were higher in trace mineral-supplemented bulls. moreover, bulls fed trace minerals in chloride form had higher sperm motility and concentration than bulls fed trace minerals in sulfate form. this was likely due to a higher bioavailability of chloride forms, resulting in higher liver concentrations of zn and cu.53 nondeficient mature normospermic bulls fed organic amino acid complexed zn, cu, co, and mn for 123 days had higher total and progressive sperm motility than bulls fed inorganic sulfated forms.56 however, the benefit of feeding trace minerals to these bulls cannot be assessed since there was no control group without trace mineral supplementation. in addition, although the improvement in sperm motility was significantly different, the effect of a small numerical difference on fertility of normospermic bulls has not been critically evaluated. conclusion trace minerals have an important role in male reproductive function. deficiencies in trace minerals can impact testicular and sperm function, affecting semen quality and fertility. nutritional supplementation of trace mineral-deficient males can improve semen quality. benefits of supplementing nondeficient normospermic bulls on fertility are poorly understood. additionally, effects of trace mineral supplementation on testicular function and semen quality have not been evaluated in bulls with heat stress, testicular dysfunction, or reproductive disease. any recommendations for this purpose remain empirical. conflict of interest none to declare. http://dx.doi.org/10.58292/ct.v16.10351 6 citation line: clinical theriogenology 2024, 16, 10351, http://dx.doi.org/10.58292/ct.v16.10351 references 1. usda report – national animal health monitoring system, beef 2007-08 part ii: reference of beef cow-calf 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class. j dairy sci 2012;95:7066–7073. doi: 10.3168/ jds.2012-5515 50. khaki a, araghi a, lotfi m, et al: differences between some biochemical components in seminal plasma of first and second ejaculations in dual-purpose simmental (fleckvieh) bulls and their relationships with semen quality parameters. vet res forum 2021;12:39–46. 51. tvrda e, lukac n, schneridgenova m, et al: impact of seminal chemical elements on the oxidative balance in bovine seminal plasma and spermatozoa. j vet med 2013;125096. doi: 10.1155/2013/125096 52. geary tw, kelly wl, spickard ds, et al: effect of supplemental trace mineral level and form on peripubertal bulls. anim reprod sci 2016;168:1–9. doi: 10.1016/j.anireprosci.2016.02.018 53. geary tw, waterman rc, van emon ml, et al: effect of supplemental trace minerals on standard and novel measures of bull fertility. theriogenology 2021;172:307–314. doi: 10.1016/j. theriogenology.2021.07.006 54. kirchhoff aa, fike ke, breiner r: an injectable trace mineral supplement in yearling bulls causes a short-term increase in circulating trace mineral levels but does not improve sperm quality. kansas agric exp station res rep 2015;1:1. doi: 10.4148/2378-5977.1024 55. preedy gw, hill sl, stevenson js, et al: injectable trace-mineral supplementation improves sperm motility and morphology of young beef bulls. anim sci 2018;34:1–9. doi: 10.15232/ pas.2017-01667 56. rowe mp, powell jg, kegley eb, et al: influence of supplemental trace-mineral source on bull semen quality. anim sci 2014;30: 68–73. doi: 10.15232/s1080-7446(15)30085-1 http://dx.doi.org/10.58292/ct.v16.10351 https://doi.org/10.1111/and.12069 https://doi.org/10.1016/j.reprotox.2006.03.011 https://doi.org/10.1016/j.reprotox.2006.03.011 https://doi.org/10.18805/ijar.b-3870 https://doi.org/10.18805/ijar.b-3870 https://doi.org/10.1016/j.tox.2020.152504 https://doi.org/10.1016/j.anireprosci.2014.06.033 https://doi.org/10.1111/asj.12736 https://doi.org/10.1016/j.anireprosci.2014.01.009 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medicine: the future of veterinary medicine? personalized medicine: the future of veterinary medicine? margret casal, petra werner school of veterinary medicine, university of pennsylvania, philadelphia, pa a patient presents with hyperglycemia and polyuria/polydipsia. a physical examination and basic bloodwork are performed. the diagnosis is diabetes mellitus and the patient treated accordingly. clinical signs of disease may be similar in most patients with diabetes mellitus; however, the underlying cause can differ. therefore, one standard therapy will not always result in a successful outcome. also, adverse effects of treatment may occur due to differences in each patient’s physiology or genetic makeup. personalized medicine implies not just treating clinical signs (i.e. reducing blood glucose concentrations to a physiologic range, as in this case), but genetically dissecting cause of disease, considering genomic variability and t developing a more focused treatment. this has already been successfully applied in the field of cancer; for example, adoptive t cell immunotherapy using autologous t cells for non hodgkin’s b cell lymphoma in dogs, developed at pennvet, is an example of personalized veterinary medicine. rather than “blasting” a patient with systemic chemotherapy, the patient’s own t cells are genetically modified to specifically recognize b cells and eliminate them without affecting other cell lineages. personalized or precision medicine can be divided into 3 basic categories: cause of disease (diagnostics/genomics), responses to drugs (pharmacogenetics) and oncogenomics. diagnostics do not just include familiar technologies, e.g. radiographs and bloodwork, to characterize phenotypic presentation of disease, but also description of a possible underlying genetic cause through genome wide association studies and sequencing individual genes. identifying specific gene defects can direct treatment or assist in evading triggers for disease manifestation, e.g. avoiding ivermectins in mdr1 positive collies. a newer method of discovering potential mechanisms or indicators of disease is presenting data from diseased individuals to computers capable of deep learning to develop algorithms for these specific diseases. once algorithms are established, an individual patient can be assessed for their location within the spectrum of disease and personalized therapy instituted. in reproduction, an exciting example is to load videos of sperm movement from thousands of individuals into a computer that then learn to identify sperm that fail to conform to what has been established as normal, identifying a possible cause of infertility in a patient. although this method does not allow for determination of what the underlying defect is, it may have identified an individual with a disease process that our normal methods of investigation may not have identified. pharmacogenetics is another up and coming aspect of personalized medicine, in which genome variations are identified that determine how a patient might react to a particular medication. as veterinarians, we recognized that specific drugs are not always effective in or tolerated by various species. however, even within a species, there are breeds or even individual animals that cannot process certain drugs or have side effects not present in others. lastly, as briefly mentioned above, cancer genetics has been at the forefront of personalized medicine, both in humans and animals, particularly dogs. with widespread genetic screening in dogs, disease mechanisms and drug actions/interactions can be elucidated, enabling personalized therapies immediately or in the future. in the meantime, we as veterinarians need experts in genetics to help us interpret results and counsel our clients. keywords: personalized medicine, genetic screening, precision medicine 511 clinical theriogenology • volume 11, number 3 • september 2019 512clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 1 contact roberto palomares palomnr@uga.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10529, http://dx.doi.org/10.58292/ct.v16.10529 review report role of trace minerals in cow’s reproductive function and performance: a clinical theriogenology perspective* roberto palomares,a,b maria ferrer,a,c lee jonesd agroup for reproduction in animals, vaccinology & infectious diseases (gravid™), college of veterinary medicine, university of georgia, athens, ga, usa bdepartment of population health, college of veterinary medicine, university of georgia, athens, ga, usa cdepartment of large animal medicine and surgery, college of veterinary medicine, university of georgia, athens, ga, usa dboehringer-ingelheim animal health, duluth ga, usa abstract trace minerals (tm) have a crucial role in cattle reproduction. although microelements are required in small amounts, their bioavailability is essential for the cow reproductive physiology, and for adequate fertility and productivity. they are particularly important for antioxidant protection against cellular damage (e.g. gametes and embryonic cells), hormone synthesis, and pregnancy maintenance. oral tm supplementation is a common and highly recommended management practice in cattle operations. however, there is substantial variability in tm bioavailability in animals receiving oral tm supplementation. the strategic use of injectable tm supplementation (without replacing traditional oral tm supplements) before episodes of marked stress (e.g. parturition), higher metabolic demand with tm depletion (e.g. last trimester of pregnancy), active immune response (e.g. uterine involution or vaccination), and before breeding helps to maintain adequate tm and oxidative status during critical points of the reproductive program. this manuscript reviews the research-based evidence regarding the effects of tm supplementation on bovine reproduction and its impact on beef and dairy cattle reproductive performance. keywords: trace minerals, supplementation, cattle, reproduction introduction1 reproductive efficiency is essential to achieve the productive goals in dairy and beef cow-calf operations. multiple factors such as subnutrition, stress, poor management of periparturient cows, along with inadequate breeding programs (e.g. failure of heat detection, inefficient artificial insemination [ai], and bull subfertility) are associated with low reproductive performance. therefore, a successful reproductive program should be supported by the application of management practices directed to: 1. provide adequate nutrition; 2. minimize stress and control oxidative imbalance; 3. apply adequate breeding management tools represented by effective ovulation synchronization protocols and ai technique, and/or the use of reproductively sound bulls; 4. reduce the exposure to pathogens; and 5. generate protective immunity through vaccination against main pathogens that cause infertility. * presented at the 2023 society for theriogenology conference, published after peer review. nutritional management of transition cows, comprising adequate supply of energy, protein, minerals, and vitamins, is one of the pillars to achieve adequate reproductive performance in cattle operations.1 reproductive function and particularly resumption of ovarian cyclicity and maintenance of pregnancy are lower-level priorities for nutrient distribution in cattle. maintenance and lactation represent the main priorities for the use of carbohydrates, proteins, fatty acids, minerals and vitamins in the lactating cow. moreover, energy and protein demands increase substantially after parturition; therefore, they are commonly the focus of any nutritional plan.1 nonetheless, the importance of mineral and vitamin supplementation on reproductive function and performance is commonly overlooked. mineral status and particularly trace minerals (tm; copper [cu], zinc [zn], selenium [se], and manganese [mn]) have important roles in cattle reproductive function and therefore in herd fertility. although these elements are required in small amounts, their bioavailability is essential for many mailto:palomnr@uga.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10529 2 citation: clinical theriogenology 2024, 16, 10529, http://dx.doi.org/10.58292/ct.v16.10529 physiological processes, including several metabolic pathways, antioxidant protection against cellular damage, signal transduction, nucleic acid replication and transcription, among others.2,3 additionally, tm have specific functions in oocyte health, hormone production, fertilization, embryo and fetal development, and uterine environment to support pregnancy, parturition, and uterine involution.4-8 thus, appropriate tm supplementation is crucial for the achievement of optimal reproductive performance in beef and dairy cattle. several factors affect the tm status and requirements of cattle, including: the mineral composition of feedstuffs, forages, and soils; pregnancy and production stage; dry matter intake; the source tm formulations; and the presence of antagonists in water, forages and feed.9 assessment of the herd’s tm status is a critical point of control before supplementing tm to attempt  covering the needs for health and reproduction. supplementation with tm may be provided by free-choice oral mix, salt blocks, drench, rumen boluses, and injectable formulations that vary in their tm bioavailability and absorption. research evidence supports the strategic tm supplementation (combining both oral and injectable tm) as a promising aid to improve cattle reproductive performance.10-15 however, other studies had no benefits of tm supplementation on cattle fertility.16,17 this may be attributed to differences in baseline diets and tm formulations, tm deficit, animal type, and experimental designs, among others. this article summarizes the effects of tm supplementation on reproductive function in the bovine female and the impacts on dairy and beef cattle reproductive efficiency. such effects will be discussed from a clinical theriogenology standpoint, based on clinically relevant aspects of reproductive physiology, pathology, and applied biotechnology.  role of trace minerals in immunity and reproductive health of cattle various reproductive pathways, including uterine involution,18,19 ovulation and cl development,20 embryo-maternal interactions,21 and vaginal-uterine mucosal defenses,22 rely on the effective function of both branches of the immune system, namely, the innate and adaptive immunity. a compromised immune response can have detrimental effects on reproductive function, including postpartum complications and delayed uterine regeneration, late resumption of ovarian cyclicity, failure of fertilization, and pregnancy loss.23,24 it is well documented that tm (cu, se, zn, and mn) have a direct role in mucosal integrity and immunity, neutrophil and macrophage migration, phagocytosis and bacteria killing, antibody production, and cell mediated immunity.25 hence, maintaining an optimal balance of tm that supports a robust immunity is crucial for proper function of a healthy reproductive system. furthermore, prevalent reproductive disorders affecting dairy cattle productivity, such as retention of fetal membranes (rfm), puerperal metritis, endometritis, and ovarian cysts, along with other health issues like lameness and mastitis that indirectly affect cattle reproduction, are linked to oxidative stress.23,24,26 this condition is defined as an imbalance in the generation and neutralization of prooxidant substances, specifically reactive oxygen species (ros). these free radicals are normal byproducts of cellular oxidative metabolism and form when 1 atom of any molecule loses or gains 1 electron, making them unstable and extremely reactive with organic molecules like lipids.27 the extent of the impact of oxidative stress on cow reproduction remains unclear. the excessive amount of these free radicals induces notable cell damage through lipid cell membrane peroxidation,26,28 a process particularly impactful on leukocytes, gametes, and embryonic cells.8 consequently, the immune and reproductive functions are compromised during oxidative stress, resulting in increased susceptibility to diseases (e.g. mastitis, metritis, endometritis) and subfertility.26,29,30 antioxidants exert influence on the reproductive system in several ways. particularly, cu, zn, se, and mn serve as integral components of enzymes responsible for ros neutralization such as superoxide dismutase and glutathione peroxidase.31-33 these structural and functional roles help mitigate the adverse impacts of stress on both the immune system and the reproductive function (figure 1). zinc has an essential role as a structural element in the antioxidant enzyme superoxide dismutase (zn-sod), responsible for converting superoxide radicals into hydrogen peroxide.34 preventing extensive cell membrane and dna damage. additionally, zn has a crucial function in tissues that require active cell mitosis such as lymphoid tissue, and mucous membranes. therefore, zn contributes to integrity and healing of the epithelial lining of the endometrium and the mammary gland, particularly important during postpartum uterine involution and prevention of mastitis in dairy cows, respectively,35 due to its involvement in regulatory processes of constant regeneration.36,37 its ability to affect endometrial cells proliferation and remodeling may be based on its direct function during nucleic acid replication, transcription, and translation.38 zinc is also essential for lymphocyte proliferation and  differentiation before acquiring effector functions figure 1. role of trace minerals (se, cu, zn, and mn) as antioxidants in cattle http://dx.doi.org/10.58292/ct.v16.10529 citation: clinical theriogenology 2024, 16, 10529, http://dx.doi.org/10.58292/ct.v16.10529 3 (figure 2).39,40,41 additionally, zn influences the function of macrophages by impacting toll-like receptor-triggered proinflammatory and type i interferon responses and nitric oxide synthesis.34,42,43 copper has a vital role in mitochondrial energy production and various enzymatic reactions crucial for basal metabolism.40 similar to zn, it is integral to structure and function of the enzyme superoxide dismutase (cu-sod; figure 1). this is essential for regulating the production of free radicals by phagocytic cells to exert their bacterial-killing function in the uterine lumen (figure 2). additionally, this enzyme has a key role in mitigating oxidative stress by scavenging excess superoxide radicals, avoiding their accumulation and thereby preventing damage to cell membranes. furthermore, ceruloplasmin, a copper-containing acute-phase protein, has antiinflammatory properties.44 selenium has a major role in migration of polymorphonuclear phagocytes from the blood stream into tissues and subsequent inflammation, via e-selectin and icam-1 molecules.45 this is particularly important to control mammary gland and uterine inflammation during the early postpartum period (figure 2). additionally, se functions as a fundamental component of the enzyme glutathione peroxidase; this enzyme contributes to reduction in hydrogen peroxide (h2o2) by producing h2o and o2, and preventing harmful effects to white blood cells (figure 1). both se and glutathione peroxidase are crucial for normal detachment and expulsion of fetal membranes, uterine involution, and prevention of metritis and mastitis in cattle.46 manganese has a role as a component in numerous enzymes participating in diverse metabolic pathways, including arginase, pyruvate carboxylase, and superoxide dismutase. although evidence of manganese’s function in the immune response is somewhat limited, it serves as a structural component in the mn-sod enzyme, also crucial in neutralizing ros produced by phagocytic cells (figure 1).2 role of trace minerals in cow’s reproductive function trace minerals including zn, se, cu, and mn are involved in several reproductive functions including granulosa cell proliferation and steroidogenesis,47 corpus luteum (cl) function,48 oocyte quality,49-52 embryo development51,53 and implantation, production of interferon tau, fetal development and organogenesis,54 placentation, oxygen transportation, parturition, and uterine involution (figure 3).55 zinc governs appetite through the central nervous system, influencing dry matter intake, energy balance, and accumulation of fat reserves that are fundamental for resumption of postpartum ovarian cyclicity. as previously stated, zn serves as an essential element in enzymes and transcription factors,  contributing catalytic, structural, and regulatory functions.56 therefore, zn is a substantial factor for the basal metabolism. zinc is an important element affecting pregnancy rate in cattle57 due to its direct effects on hormone secretion and function (e.g. progesterone, insulin, pgf2α, among others), oocyte, and embryo viability58 and fetal development,59 contributing to maintain a viable pregnancy. zinc also modulates igf-1 secretion, bioavailability, and function.8,60,61 this growth factor is involved in key reproductive functions including uterine involution, embryo implantation, and fetal development.8 zinc is critical for endometrium remodeling due to its action in nucleic acid replication, transcription, and translation during cell division. zinc fingers are essential to binding of figure 2. role of trace minerals (se, cu, zn, and mn) in bovine immunity and reproductive health http://dx.doi.org/10.58292/ct.v16.10529 4 citation: clinical theriogenology 2024, 16, 10529, http://dx.doi.org/10.58292/ct.v16.10529 estrogensand progesterone-receptor complexes to dna to exert their actions.62 the antioxidant properties of zn as structural part of the zn-sod enzyme have a relevant role in protecting oocytes and embryonic cells from oxidative damage by superoxide free radicals.63 zinc also affects fetal growth and pregnancy due to its involvement in the metabolism of calcified bone matrix.59 a recent discovery about involvement of zn in oocyte activation and early embryo development is a sequence of zn exocytic events upon fertilization, known as ‘zinc spark,’ which leads to a reduction in the total amount of intracellular zn that is necessary and sufficient to induce oocyte activation. moreover, this zn release has a role in preventing polyspermy by modifying zona pellucida. this is a remarkably conserved event across various mammalian species including cattle. in humans, it is being studied as a promising biomarker associated with embryo quality.64,65 cows with adequate cl function, higher progesterone concentrations and pregnant status had higher zn and cu concentrations and sod, gpx antioxidant capacity at tai than cows with abnormal luteal phase length, delayed ovulation, and pregnancy failure.57 there was a clear association between concentrations of antioxidant and cu and zn, and both cl function and fertility.57 copper serves as a component in a variety of crucial metallo-enzymes and contributes to various physiologic pathways essential for reproduction, such as cellular respiration and mitochondrial energy production (e.g. via cytochrome oxidase), regulation of inflammation (e.g. ceruloplasmin), and antioxidant system (e.g. cu-sod). copper exerts its influence to enhance pregnancy maintenance in cattle by affecting various reproductive processes, including enhancing oocyte viability and embryogenesis.49,51,52,55 additionally, cu targets several other cell types such as endometrial cells, luteal cells, figure 3. role of trace minerals (se, cu, zn, and mn) in cattle reproductive functions http://dx.doi.org/10.58292/ct.v16.10529 citation: clinical theriogenology 2024, 16, 10529, http://dx.doi.org/10.58292/ct.v16.10529 5 and cumulus cells that have substantial impact on early stages of embryogenesis.55 copper has a direct involvement in fetal development, particularly due to its participation in the immune system, bone and connective tissue formation, cardiac function, keratinization, and spinal cord myelination.32 fetal demands of cu especially increase during the last trimester of pregnancy, when there is the most substantial fetal development.55 selenium is critical for the proper function of numerous seleno-proteins (glutathione peroxidase, iodothyronine deiodinase, and thioredoxin reductase). by contributing to these enzymes, se participates in neutralizing peroxides produced during lipid peroxidation.33,66 consequently, se has a crucial function in maintaining the integrity of cell membranes and organelles. through this mechanism, se protects oocyte, early embryo, and fetus from mortality caused by oxidative damage. employing x-ray fluorescence imaging, a study determined se concentrates in large ovarian follicles’ granulosa cells in cattle.67 additionally, the study indicated a significant increase in the expression of the selenoprotein gene gpx-1 in granulosa cells of the large healthy follicles. this particular positioning of se in large follicles, coupled with its recognized role in antioxidant defense, implies a potential role in protecting oocyte from oxidative stress and cell damage throughout follicular development. additionally, se directly influences granulosa cells, promoting their proliferation and enhancing estradiol synthesis,47 which is essential for oocyte maturation and lh surge, and ovulation. therefore, se has a dual role, stimulating folliculogenesis and estradiol secretion and shielding oocyte and embryo from cell damage due to its antioxidant function. supplementation of selenium in dairy cows was associated with a healthy postpartum period, normal fetal membrane expulsion and greater pregnancy outcomes.28,68 moreover, se supplementation has been associated to a potential decrease in the occurrence of postpartum metritis and ovarian cysts.35 a strong correlation in se concentrations between maternal and fetal liver was demonstrated69 suggesting an efficient se transfer from maternal circulation via placenta for supplying the higher fetal demands. manganese has a vital function in embryo development.50,53,70 bovine cumulus oocyte complexes (cocs) and preimplantation embryos actively transcribe and express mn-sod.70 this enzyme promotes cellular differentiation and embryo viability via antioxidant protection against cell damage. manganese might contribute to cl function, given that concentrations of mn and mn-sod in the cl of ewes increased between 4 and 11 days.71 therefore, it is conceivable that mn promotes pregnancy by enhancing progesterone secretion by the cl. additionally, a study reported improved pregnancy rates in cattle following dietary mn supplementation.72 however, precise mechanisms underlying the positive impacts of mn on reproductive performance remain unclear. a potential explanation is that manganese’s involvement in cholesterol production could govern steroidogenesis throughout pregnancy.73 cholesterol is essential for synthesizing steroid hormones like progesterone that has a central role in maintaining an adequate uterine environment for embryo implantation and pregnancy. suboptimal progesterone concentrations have been implicated as a potential factor in early embryonic loss.74 manganese also has a pivotal role in the formation of fetal bones during organogenesis, since it is crucial for the synthesis of chondroitin sulfate, a component of mucopolysaccharides within the organic bone matrix.75 a study that investigated the impact of dietary mn on the growth and reproductive performance of beef heifers suggested that 15.8 mg of mn/kg of diet dry matter is deemed sufficient for supporting growth, cyclicity, and conception of beef heifers.76 however, another report indicated that this diet may be inadequate for optimal fetal development in pregnant heifers, resulting in fetal structural deformities.77,78 trace mineral status and requirements in cattle before defining a tm supplementation regime, it is highly recommended to determine the herd tm status, as well as the mineral content of forages and feedstuffs the animals will consume. this will help to determine the baselines to ensure that supplements provide optimal amounts of tm for animal production, health, and reproduction. this tm assessment is also important for preventing se toxicity in some areas where se concentrations in soil and forages are high, so that additional se supplementation may not be needed. the best indicator of the tm status in cattle is the hepatic concentration. liver serves as the storage site, where tm are integrated into enzymes and released as needed. to determine the tm concentrations in liver, it is necessary to collect a liver biopsy sample. liver biopsies (~ 10 mg of liver tissue) are collected after local anesthesia in the 10th intercostal space (right side) using a sterile biopsy needle (14 g x 4 or 6”). blood concentrations of tm may exhibit variability, inconsistency, and have limited predictive value. however, blood se is very stable and is a useful tool to promptly diagnose se toxicity (> 2.5 µg/ml). copper concentrations in liver samples should exceed 75-90 ppm,79 within a range of 50-600 μg/g on a dry matter basis.80 a marginal deficiency of manganese (mn) is indicated when liver concentrations are below 9 ppm, within a range of 5-15 μg/g.80 adequate selenium (se) concentrations in the liver are between 0.8-1.0 ppm, with a range of 0.7-2.5 μg/g and concentrations below 0.2 ppm are deemed critically deficient.79 for zn, hepatic concentrations below 80-100 ppm are considered marginal or deficient,79 with a range of 90-400 μg/g.80 the tm status of cattle is influenced by various factors, encompassing the mineral composition of soils, forages, and feedstuffs, high variability in mineral intake, the existence of mineral antagonists in water, forages, and feedstuffs, and fluctuating tm requirements. the tm requirements have been instituted as the principles that define animal necessities, and help assess the formulation of tm supplementation. these requirements are derived from research findings reported established by the national academies of sciences, engineering, and medicine (nasem, 2016), board on agriculture subcommittee on beef cattle nutrition.81 these tm requirements are not a ‘one-size-fits-all’ parameter, as they vary considerably and are affected by factors such as breed, physiological status (e.g. lactation and pregnancy), health status, environmental conditions, management practices, and level of stress. a rule of thumb for adequate nutritional management is to plan the tm supplementation regime based on information of tm requirements, status, and content in forage, feedstuffs, and soil. a comprehensive study regarding the tm composition of forage samples collected from cow/calf operations across 18 states in the us indicated tm deficiencies in a substantial number of the samples.79 merely 2.5% of the forage samples exhibited adequate zn concentrations. copper concentrations http://dx.doi.org/10.58292/ct.v16.10529 6 citation: clinical theriogenology 2024, 16, 10529, http://dx.doi.org/10.58292/ct.v16.10529 were deemed sufficient in 36% of the samples.79 moreover, 50% of the samples were considered marginally deficient in copper, with 14% classified as critically deficient. another report, encompassing 23 states in the us, yielded comparable findings of tm deficiencies. in midwest us, only 4, 15, and 26% of fescue samples had adequate concentrations of se, zn, cu, respectively.82 additionally, 18, 24, and 24% of native forage samples exhibited adequacy in cu, se, and zn, respectively.82 notably, in both surveys, a substantial proportion of the samples had elevated or marginally high concentrations of iron (fe), molybdenum (mo), and sulfur (s).79,82 the observed cu deficiency in the tested forage samples is likely linked to antagonistic effects of fe, mo, and s that impede copper absorption through gastrointestinal tract. these antagonistic interactions could contribute to insufficient availability of cu in the diet, further highlighting the complexity of tm dynamics in forage and its impact on cattle nutrition. based on the authors’ experience, the observed variability in tm requirements and the difficulties to maintain an adequate herd tm status make it indispensable to establish a ‘strategic tm supplementation plan’ that supports herd’s health and reproductive performance. this plan needs to be specifically adapted to each particular production system and should combine permanent traditional free-choice oral supplementation plus pulse-dose tm injections during critical points of the production system when tm demands are greater (e.g. 30 days before parturition and 30 days before breeding). trace mineral deficiencies on cattle health and reproduction deficiencies in tm have a gradual impact, progressively affecting various physiological and metabolic functions with multifaceted implications for overall health, reproduction, and performance. commonly, the lack of specific clinical signs makes tm deficiency easily overlooked, with borderline concentrations possibly continuing to decrease, affecting first reproductive function, embryo viability, and immune response. this results in low reproductive performance (e.g. high incidence of early embryonic mortality) and increased susceptibility to health disorders, such as lameness, metritis, and mastitis (figure 4). overall, tm deficiency impairs the immune response by affecting neutrophil and macrophage migration, phagocytic function, and killing activity, as well as the mechanisms of mucous membranes integrity. tm deficiency also affects lymphocyte activation, proliferation, and differentiation, cytokine secretion, and antibody production.44,83,84 when tm concentrations decrease to a severe extent, clinical deficiencies can manifest with distinct clinical signs, such as increased incidence of rfm and muscle degeneration, both associated with severe se deficiency. regularly assessing herd’s mineral profile and animals’ tm intake is crucial to identify and address their specific tm needs. zn deficiency is associated with various conditions including anorexia, reduced igf-1 concentrations, cl insufficiency, low progesterone concentrations, higher risk of abortion, fetal teratogenesis, prolonged pregnancy, calving difficulties, fetal growth retardation, low birth weight, weak offspring, skin parakeratosis, and neonatal calf diarrhea (figures 4 and 5).85,86 deficiencies of cu in cattle may occur due to low dietary intake or consumption of antagonists (e.g. mo, fe, and s), and figure 4. effects of trace mineral deficiencies on cow production and reproduction http://dx.doi.org/10.58292/ct.v16.10529 citation: clinical theriogenology 2024, 16, 10529, http://dx.doi.org/10.58292/ct.v16.10529 7 are associated with low pregnancy rate, embryonic and fetal death, anestrus and repeat breeding, stillbirth, immunosuppression, and poor calf health and performance (mainly diarrhea, growth retardation, and mortality).85 reproductive disorders related to se deficiency encompass low fertility87 and abortion occurrences.88,89 low concentrations of se and glutathione peroxidase (gshpx) in plasma have been associated with a higher incidence of rfm (figure 4).46 further studies supported this, revealing that animals supplemented with se experienced none or few cases of rfm, whereas the control group receiving a se deficient basal diet had a rfm incidence between 17 and 32%.90-92 moreover, se deficiency has also been identified as a factor that increases the likelihood of puerperal metritis in dairy cattle.28,93 as mentioned above, se deficient calves may have muscular degeneration known as nutritional muscular dystrophy or white muscle disease (figure 5) that results in heart degeneration and death, the predominant pathology in neonatal calves. marginally se deficient calves may have weakness and low vigor. insufficient mn leads to suboptimal growth and compromised reproduction, characterized by impaired ovulation, silent estrus, diminished conception rates, occurrences of abortions, and decreased birth weight.94 a deficiency in dietary manganese generally manifests with relatively mild effects on immunity, except for a notable impact on the humoral immune response.95 manganese deficiency has been linked to adverse outcomes such as abortion and congenital deformities in long bones among newborn calves. these deformities include enlarged joints, stiffness, twisted legs, shorter bones, dwarfism, brachygnathism, chondrodysplasia, and overall physical weakness after birth.77,78,96,97 a retrospective study to determine the association between plasma tm (cu, zn, and se glutathione peroxidase) status on reproductive performance and health of commercial dairy and beef herds (n = 2,080) in france revealed that cu, zn, and se deficiencies may be important risk factors for impaired production, reproduction, and health in both beef and dairy herds.85 copper deficiency had negative effects on health and  performance of calves and cows, whereas, zn and se deficiencies substantially affected adults. selenium deficient status was associated with increased risk of infectious abortion, poor immune response, and higher calf morbidity and mortality. zinc deficiency was highly correlated with low milk yield and lameness in dairy herds, as well as neonatal calf diarrhea and poor growth. further, in that study, se deficiency in cows was associated with increased incidence of rfm.85 oral trace minerals supplementation: effects on cows’ reproductive performance oral mineral supplements can be provided in various forms, including free-choice blends of mineralized salt, blocks, or alongside energy and/or protein supplements within the total mixed ration. there are various tm sources for cattle supplementation, categorized into organic or inorganic compositions. organic sources, including chelates, amino acid or polysaccharide complexes, proteinates, propionates, and yeast derivatives, are more biologically available for absorption and utilization. this makes them particularly advantageous for improving health and reproductive performance compared to inorganic forms,98,99 such as sulfates, oxides, carbonates, and chlorides that are structured with sulfur, oxygen, or chloride molecules. sulfate forms of zn, cu, and mn are the most bioavailable inorganic sources. although inorganic tm may prevent severe deficiencies, supplementation with organic forms such as se yeast could offer additional benefits by providing enhanced tm status and antioxidant activity during periods of heightened stress, such as after parturition. over the past decades, numerous studies have highlighted the value of oral tm supplementation in enhancing reproductive function and efficiency in cows.98-100 a recent meta-analysis considering 20 research articles assessed the impacts of organic tm supplementation on milk yield and composition, and reproductive performance in dairy cows.100 the results indicated that organic tm supplementation increased daily milk production, milk fat, and milk protein, with no effects on milk somatic cell count. organic tm supplementation reduced days open, number of services per conception, and increased the risk of pregnancy on day 150 of figure 5. effects of trace mineral deficiencies on calves’ health and performance http://dx.doi.org/10.58292/ct.v16.10529 8 citation: clinical theriogenology 2024, 16, 10529, http://dx.doi.org/10.58292/ct.v16.10529 lactation. this study suggested that organic tm supplementation could enhance production and reproduction in lactating dairy cows.100 other researchers have reported similar improvements in conception rates and days to first service in dairy cows when substituting inorganic sulfates with organic sources of cu, zn, mn, and co, although the precise mode of action remains unclear.99 an additional effect of tm supplementation (particularly the use of organic forms) on dairy herds is the reduction in the occurrence of clinical mastitis, somatic cell counts,35 and increase in milk yield.101 another study was performed using crossbred, multiparous grazing beef cows to assess the impacts of cu, zn, and mn supplementation and the mineral source on reproduction and performance over a 2-year period.102 the cows received either organic (50% organic and 50% inorganic cu, zn, and mn); inorganic (100% inorganic cuso4, znso4, and mnso4) or no supplemental cu, zn, or mn from 82 days (year 1) and 81 days (year 2) before calving through 110 days (year 1) and 135 days (year 2) after calving. in year 1, pregnancy rate to ai in control cows did not differ from supplemented cows, but there was a trend for higher pregnancy rate in organic compared to inorganic tm supplementation cows. in year 2, supplemented cows had a higher pregnancy rate to ai than controls. in both years, when animals were inseminated after observed estrus, supplemented cows had a higher pregnancy rate than controls. overall, 60-day pregnancy rate tended to be higher for supplemented cows. this study indicated that tm supplementation and the tm source can improve the pregnancy rate to ai.102 contrasting findings have emerged from other studies, indicating marginal or negligible benefits of oral tm supplementation on reproduction of dairy103 and beef cattle.104–106 effects of organic or inorganic tm supplementation on ovarian follicular dynamics, ovulation, and embryo production in angus heifers submitted to hormonal protocols for superovulation were determined.106 in that study, dietary tm supplementation had no influence on the number of ova/embryos recovered from beef heifers or the cl counts, indicating no difference between control, organic or inorganic tm supplementation.106 this suggested that the source of tm supplementation did not produce a substantial impact on the number or quality of embryos in angus heifers and it is unlikely to have a direct impact on follicular dynamics and ovulation.106 however, it is important to notice in that study that tm supplementation tended to reduce the number of degenerated embryos and unfertilized ova, supporting the role of tm on enhancing fertilization and embryo quality.106 the inconsistency in results across studies on tm supplementation can be attributed to various factors, including differences in the forms of minerals supplemented (organic versus inorganic), high variability in tm concentration in control diets, variations in animals’ tm requirements and initial tm status, differences in intake and absorption, negative mineral interactions and antagonisms, varying levels of stress, and discrepancies in experimental models and endpoints. these diverse factors make the interpretation and comparison of data from several studies particularly challenging. strategic injectable trace minerals supplementation on reproductive performance as mentioned above, offering tm through free-choice oral supplementation may often result in tm deficiencies due to considerable variability in mineral composition and intake, binding of minerals to undigested feed particles, negative interactions and diminished absorption in the digestive tract, and antagonistic effects. there is a rising trend in cattle operations to adopt strategic extra supplementation using single treatment, pulse-dose trace mineral products. whether given orally or through injections, this approach aims to prevent tm deficiencies or enhance the overall tm status. it ensures that the necessary tm amounts are provided during crucial stages of the production cycle when animals experience heightened tm demands. good examples of such critical times may be: 1. the last trimester of pregnancy, when there is substantial tm transfer from the dam to the fetus due to increased fetal growth and its tm demand; and 2. at parturition, which is accompanied by substantial oxidative stress and a decrease in plasma zn concentrations,107,108 or before the breeding season in preparation for the upcoming reproductive cycle and pregnancy. it is important to note that although this strategic supplementation is valuable, it is not meant to replace the regular supply of oral minerals. injectable tm (itm) treatment in angus-cross steers with sufficient tm status led to a quicker and more substantial increase in plasma concentrations of zn, se, and mn through the first 24 hours, compared to other single-dose supplementation methods including drench, paste, or boluses.109 additionally, hepatic se concentrations remained higher through day 29 in steers treated with itm in comparison to the groups receiving tm paste or drench.109 this study demonstrated that itm is the most effective way to provide additional tm to be available during specific times when the animal’s needs are greater. utilization of itm ensures the delivery of a known and well-controlled quantity of tm that is rapidly and efficiently absorbed and stored after injection.110 this becomes particularly crucial for cattle experiencing reduced dry matter intake and mineral consumption, such as after vaccination or before parturition. it is also beneficial in situations where cattle have limited access to free-choice mineral supplements, as is common in extensive rangeland systems or flooded pastures. treatment of itm reduces the variability in tm status observed in animals with free-choice mineral intake.111 a commercially available itm product, multimin® 90 from axiota animal health® that contains zn (60 mg/ml), cu (15 mg/ml), mn (10 mg/ml), and se (5 mg/ml) has demonstrated rapid absorption, leading to an increase in blood tm concentrations within 8 hours after injection, maintaining elevated circulating concentrations for 24 hours, after which they are stored in liver.110,112 multiple studies have been performed to assess the impact of supplementation with itm on production and reproduction of dairy cows. in a trial using 1,416 multiparous dairy cows that received itm or not at 230 and 260 days of pregnancy, as well as 35 days postpartum, itm supplementation led to a decrease in the incidence of periparturient fetal deaths and cases of endometritis.10 however, it is noteworthy that this treatment did not have any marked effects on reproductive performance, milk yield, or other health variables. furthermore, treatment of itm yielded positive outcomes for udder health with a reduction in linear somatic cell count http://dx.doi.org/10.58292/ct.v16.10529 citation: clinical theriogenology 2024, 16, 10529, http://dx.doi.org/10.58292/ct.v16.10529 9 scores, and the occurrence of mastitis.10 in another report in dairy cows, tm supplementation increased serum sod activity, without affecting leukocyte function.11 a subsequent trial using 923 multiparous cows from 2 commercial dairy farms treated with itm or not, revealed that itm supplementation had a tendency to lower the incidence of metritis and stillbirths. additionally, it exhibited improvements in polymorphonuclear leukocyte function and contributed to a better inflammatory status in dairy cows undergoing the transition period, under high temperature-humidity conditions.12 treatment of itm 30 days before calving and 30 days before breeding in beef cows synchronized using a 5-day co-synch + cidr protocol, and tai at 72 hours after cidr removal resulted in higher p/tai (60.2%) compared to control cows (51.2%). final pregnancy rate was not different but the calving distribution for itm-treated cows improved (12.5% more early calving), with a greater proportion of cows becoming pregnant (77.5%) during the first 20 days of the breeding season compared to control cows (65.0%).13 effects of itm treatment at the beginning of the tai protocols (11 days before ai) have been evaluated in several trials. a study in bos indicus cows14 demonstrated that itm improved cl function, resulting in decreased cl diameter, although progesterone production was more efficient, having similar concentrations to control group.14 this might be due to the observed increase in antioxidant enzymes sod and gpx, reduced ros, and improved steroidogenesis. in the same study, itm improved overall pregnancy rate ~ 5% points higher (particularly in cows with low bcs; 53.7 versus 48.1% for itm and control cows, respectively), probably by enhancing oocyte quality and cl function.14 moreover, in another trial, angus heifers treated with itm at the initiation of a 14-day cidr protocol tended to have an increased ai pregnancy rate (62 versus 45%) compared to control heifers, despite no difference in overall pregnancy rate.17 the strategic supplementation with itm at ovulation synchronization in dual purpose and beef cows under tropical grazing conditions in mexico confirmed these findings, consistently reaching 5-8% greater (p = 0.05) pregnancy rate at tai in treated versus control cattle (58.1 versus 50.9%).113 in a trial performed in brazil, crossbred heifers underwent treatment with itm (itm 100 mg zn, 100 mg mn, 50 mg cu, and 25 mg se; multimin®, minerthal, brazil) 17 days before timed embryo transfer (tet). subcutaneous treatment of itm (n = 219) resulted in 1.58-fold and 1.72-fold higher odds of being pregnant 23 and 48 days after tet compared to control group (n  =  276), respectively. therefore, itm treatment 17 days before tet did not enhance the synchronization success, but it did result in increased conception rates (embryo survival) at both 23 and 48 days after tet.15 in contrast, other studies suggested that supplementation beyond an animal’s nutritional requirements may not provide benefits to reproductive performance.16,114 a research trial17 stated that repeated treatment with itm before ai did not have substantial impact on pregnancy rates in beef heifers. another study in dairy cattle reported that animals undergoing a 2-dose itm protocol, first given before calving and second before breeding, exhibited lower conception rates at the first service.16 trace mineral supplementation and in vitro production of bovine embryos supplementation with tm has been used to improve in vitro embryo production (ivp), during both in vivo oocyte collection (ovum pick-up, opu) and in vitro maturation (ivm) and culture (ivc). benefits and impacts of oral organic (amino acid complex) tm supplementation on opu and in vitro embryo production in lactating beef cows submitted to opu on days 52 and 67 of pregnancy were demonstrated.115 oral supplementation with organic tm increased total cocs recovery and ivm compared to unsupplemented cows. the production of transferable embryos tended to be greater for tm supplemented cows. additionally, the ratio of recovered cocs meeting maturation per transferable embryo was enhanced (lower) for cows receiving tm supplementation. therefore, in tm supplemented cows, ivp was more efficient, needing fewer mature cocs to produce a transferable embryo. replacing inorganic trace minerals with an organic source improved not only the number of recovered cocs via opu, and the ability to reach maturation, but also the in vitro embryo production in beef cows.115 addition of tm to ivm and ivc media have provided benefits to the production of bovine embryos. adding cu during oocyte maturation (4-6 µg/ml of ivm medium) notably increased both intracellular glutathione content and dna integrity of cumulus cells,49 decreased apoptosis, and improved subsequent embryo development up to blastocyst stage.49,51,116 in a similar fashion, supplementation of the ivm medium with zn (1.1-1.5 µg/ml) decreased dna damage and apoptosis in cumulus cells. this supplementation also led to increased cleavage and blastocyst rates, along with an increase in the inner cell mass and total cell numbers.49,58,117 manganese supplementation in the ivm medium (6 ng/ml), regardless of cumulus cells presence, also augmented the cocs quality (reduced % of apoptosis, and improved sod activity), enhancing subsequent embryo development.53 similar findings were observed when bovine oocytes were matured in ivm medium containing se (10 ng/ml); there was a reduction in the number of apoptotic cells and increased embryo hatching rates compared to the control oocytes.118 in a more recent study, supplementation of ivm medium with a combination of mn, se, cu, and zn, (6, 100, 600, and 400 ng/ml, respectively) had increased blastocyst rates and cell number, and decreased ros and lipid content.119 these studies demonstrated that addition of tm to the ivm and/or ivc may be a promising strategy to enhance the efficiency of the bovine ivp systems. conclusion trace minerals supplementation is a key support for the success of any dairy or beef herd breeding program. oral tm supplementation is a common and highly recommended practice in cattle operations. however, evidence indicated substantial variability in tm bioavailability in animals receiving oral tm supplementation. research and clinical evidence support the strategic use of itm supplementation (without replacing traditional oral tm supplements) before episodes of: substantial stress (e.g. parturition), higher metabolic demand and tm depletion (e.g. the last trimester of pregnancy), active immune response (e.g. uterine involution or vaccination) and before breeding. an effective protocol has suggested treatment of itm 30 days before parturition and 30 days postpartum or ~ 10 or 14 days before breeding (e.g. at starting ovulation http://dx.doi.org/10.58292/ct.v16.10529 10 citation: clinical theriogenology 2024, 16, 10529, http://dx.doi.org/10.58292/ct.v16.10529 synchronization protocol). this approach has enhanced herd’s oxidative balance, protective immunity, and fertility; reduced the occurrence of postpartum disorders in dairy cows (rfm, metritis, and mastitis) and enhanced pregnancy outcomes (5% greater pregnancy risk and 12% more calvings at the beginning of the season) with the final benefit of producing more uniform and heavier calf crops. implementation of tm supplementation plan should be preceded by assessment of the herd’s tm status by collecting liver biopsies in a representative number of various animal groups (e.g. calves, heifers, pregnant, and nonpregnant cows) and determination of the tm content of forages and feedstuffs. conflict of interest none to report. references 1. lean ij, van saun 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et al: trace mineral mixture supplemented to in vitro maturation medium improves subsequent embryo development and embryo quality in cattle. vet res com 2022;46:1111-1119. doi: 10.1007/s11259-022-09982-9 http://dx.doi.org/10.58292/ct.v16.10529 https://doi.org/10.1155/2013/154045 https://doi.org/10.2307/1293356 https://doi.org/10.15232/s1080-7446(15)31524-2 https://doi.org/10.3168/jds.2010-3058 https://doi.org/10.2527/2004.8282375x https://doi.org/10.3168/jds.s0022-0302(99)75322-1 https://doi.org/10.3168/jds.s0022-0302(99)75322-1 https://doi.org/10.15232/s1080-7446(15)31907-0 https://doi.org/10.15232/s1080-7446(15)31907-0 https://doi.org/10.2527/2001.7971650x https://doi.org/10.1016/j.anireprosci.2007.04.007 https://doi.org/10.1016/j.anireprosci.2007.04.007 https://doi.org/10.1111/j.1751-0813.1977.tb07935.x https://doi.org/10.1080/00480169.1976.34283 https://doi.org/10.1080/00480169.1976.34283 https://doi.org/10.15232/aas.2019-01856 https://doi.org/10.2527/jas.2012-4482 https://doi.org/10.15232/s1080-7446(15)31290-0 https://doi.org/10.15232/s1080-7446(15)31290-0 https://doi.org/10.2527/jas.2013-7066 https://doi.org/10.1093/jas/skab160 https://doi.org/10.1093/jas/skz005 https://doi.org/10.1262/jrd.18109 https://doi.org/10.1042/cbi20100507 https://doi.org/10.1007/s12011-019-01966-2 https://doi.org/10.1007/s12011-019-01966-2 https://doi.org/10.1007/s11259-022-09982-9 1 case report laparoscopic artificial insemination of white-tailed deer with fresh sex-sorted semen nicole sugai,a clifford shipley,b lindsey rothrock-gensler,b robyn ellerbrock,c jamie stewarta adepartment of large animal clinical medicine, virginia-maryland college of veterinary medicine, virginia polytechnic institute and state university, blacksburg, va, usa bdepartment of veterinary clinical medicine, college of veterinary medicine, university of illinois urbanachampaign, urbana, il, usa cdepartment of large animal medicine, college of veterinary medicine, university of georgia, athens, ga, usa abstract this case study describes the feasibility of using chilled, sex-sorted semen for laparoscopic artificial insemination (ai) in the captive white-tailed deer (odocoileus virginianus) industry. ejaculates from 3 bucks were collected, extended, and shipped overnight for flow cytometric sex-sorting. sex-sorted semen was chilled and shipped back overnight. synchronized does were bred using chilled, sexsorted (n = 30, farm 1; n = 10, farm 2) or conventional frozen (n = 7, farm 2) semen by laparoscopic ai. does were subsequently turned out with bucks. percentage of bucks born via ai with sex-sorted semen was 85% (28/33) for farm 1 and 100% (16/16) for farm 2. percentage of bucks born by natural cover was 50% (12/24) for farm 1 and percentage of bucks born by using either conventional semen or live cover was 56% (9/14) for farm 2. this case study demonstrated acceptable results using chilled, sex-sorted semen in white-tailed deer herds as a technique to manipulate the sex proportions per breeding season. keywords: cervids, flow cytometry, sex-sorted semen, white-tailed deer background in north america, white-tailed deer (wtd) farming is an industry generating an estimated $7.9 billion annually as of 2017.1 the breeding stock represents the major component of this industry (> $900 million) as it involves all the animals raised and sold.1 however, since trophy hunting and venison products are the end market for the breeding industry, male trophies are valued more than females for having larger carcasses and impressive antlers.2 of interest, ai with frozen or fresh semen is a widely used tool to select for desired features (e.g. fast growth, well-branched and broad antlers) in deer herds.2 the high demand for producing more terminal bucks per season leads to increased demands for implementing the development of more advanced reproductive technologies to alter the sex proportion in the offspring. more specifically, the use of sex-sorted semen with laparoscopic ai has been gaining popularity in the wtd industry as a way to increase buck production and improve profitability for farmers. here, we describe a case study to evaluate the feasibility of using chilled, sex-sorted semen for laparoscopic ai in captive herds of wtd. since data collected from routine assisted reproduction procedures from private farms for commercial purposes were used, institutional animal care and use committee approval is not required, and this case study is exempt. feeding and vaccination animals used were from privately-owned herds located in central illinois, ~ 50 miles apart from each other. farm 1 housed 3 bucks (buck 1: 1 year; buck 2: 2 years; buck 3: 3 years); collected semen was used to breed 34 does (mean age: 2.9 ± 0.3 years, ranging from 1 to 5 years) housed in farm 1 and 17 does (mean age: 3.1 ± 0.3 years, ranging from 2 to 6 years) housed in farm 2. animals were maintained in pastures with trees and shade cloths. animals were fed 18% protein textured feed with vitamin and mineral mix in farm 1 and 14% protein textured feed with vitamin and mineral mix in farm 2. animals were given free choice alfalfa hay and were on clover grass pasture. the vaccination protocol for both farms included a custom-made epizootic hemorrhagic disease (ehd)/bluetongue virus (btv) contact jamie stewart jlstewart13@vt.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10513, http://dx.doi.org/10.58292/ct.v16.10513 mailto:jlstewart13@vt.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10513 2 citation line: clinical theriogenology 2024, 16, 10513, http://dx.doi.org/10.58292/ct.v16.10513 9-way combination vaccine (contains antigens for ehd 1,2,6, btv 3, fusobacteria sp., pasteurella multocida a, escherichia coli, trueperella pyogenes, clostridium perfringens type a, and bibersteinia sp.) from newport laboratory (worthington, mn). additionally, red ridge autogenous vaccine for mycoplasma sp. were given (red ridge whitetails, williamsport, pa). animals from both farms were dewormed with topical moxidectin (cydectin® 0.5 mg/kg, boehringer ingelheim, st. joseph, mo). semen collection and processing ejaculates were obtained from 3 white-tailed deer bucks housed in farm 1. anesthesia and animal handling were performed by a team of experienced veterinarians. bucks were anesthetized using a combination of 0.88 mg/kg tiletamine-zolazepam (telazol®, zoetis, florham park, nj) and 2.2 mg/kg xylazine (cervicine®, zoopharm, windsor, co) given intramuscularly via projector. a 2.2 mg/kg dose of ketamine (ketaset®, zoetis, florham park, nj) was given intravenously if needed for additional anesthesia. this anesthesia protocol has been used extensively for semen collection of wtd in both clinical medicine and research.3-6 following semen collection, anesthesia was reversed with an intramuscular injection of 4.4 mg/kg tolazoline (tolazine®, lloyd, shenandoa, ia). once bucks were anesthetized, the penis was manually exteriorized and held with gauze to keep extended during collection. semen was obtained using an electroejaculator and 3-electrode ram probe (31.75 mm diameter) under manual pulse stimulation (pulsator iv lane manufacturing, denver, co). semen was collected in a 15 ml conical tube submerged in a 37°c water bath. raw volume was estimated by visual examination of the conical tubes and measured to be 1.7 ml (buck 1), 2.8 ml (buck 2), and 1.9 ml (buck 3). an aliquot of raw semen from each buck was diluted in lysis buffer at a 1:201 ratio for measurement of concentration using a nucleocounter® sp-100™ system (chemometec, somerville, ma). concentrations of raw ejaculates were measured to be 800 x 106/ml (buck 1), 545 x 106/ml (buck 2), and 800 x 106/ml (buck 3). an aliquot of each ejaculate was also evaluated via light microscopy on farm and determined to have a total motility of > 70% before proceeding with processing. a final aliquot of each sample was formalin-fixed for evaluation of morphological assessment as a wet mount at 1,000 x magnification using a phase-contrast microscope. ejaculates were determined to have acceptable morphology with ≤ 15% head defects, ≤ 15% tail defects, and < 25% total morphological defects. ejaculates were extended at 1:1-1:2 ratio with warmed tris a+ extender containing 20% egg yolk (provided by st genetics) to a final volume of ~ 5.6 (buck 1), 4.8 (buck 2), and 4.1 ml (buck 3) and were shipped overnight on ice to st genetics, previously known as sexing technologies (st genetics, fond du lac, wi). data provided by st genetics indicated that initial motility on arrival was ~ 80% for all samples and total concentrations were 643 x 106/ml (buck 1), 316 x 106/ml (buck 2), and 390 x 106/ml (buck 3) prior to sex-sorting. sex-sorting was performed by st technologies using proprietary methodologies. sorted semen assessments provided by st technologies are summarized (table 1). semen was packaged into 0.25-ml straws and either kept chilled or cryopreserved. only y-sorted (male) sperm were maintained as chilled samples for use within this case report. the x-sorted (female) sperm from bucks 1 and 2 and excess y-sorted (male) sperm from buck 1 were cryopreserved, producing a total of 18 frozen straws with x-sorted sperm and 20 straws with y-sorted sperm from buck 1 and 17 frozen x-sorted sperm from buck 2. the frozen sex-sorted semen samples were not used in this case study and kept by the farmer. chilled samples were stored in the dark at 18°c and transported overnight back to illinois. these fresh, chilled straws were used for laparoscopic ai ~ 48 hours (farm 1) or 72 hours (farm 2) after semen collection. doe synchronization and laparoscopic ai with sex-sorted semen does from both farms were synchronized to allow for timed laparoscopic ai. to begin synchronization (day 0), does on each farm were guided through a chute system to allow for placement of intravaginal cidr inserts containing 0.3 g of progesterone (eazi-breedtm cidr® sheep insert, zoetis, parsippany-troy hills, nj). at cidr insertion, the does were vaccinated with ehd/btv 9-way-combination vaccine (newport labs) and dewormed with topical moxidectin (cydectin). on day 14, cidr inserts were removed and does each received 150-200 iu of ecg (equine chorionic gonadotropin, folligon, merck animal health, intervet canada, kirkland, qc, canada). laparoscopic ai was scheduled for 55-60 hours after cidr removal and ecg treatment. does from farm 1 (n = 34) were synchronized for laparoscopic ai to be performed on the day that sex-sorted semen arrived, ~ 48 hours after semen collection, using only the chilled, sex-sorted semen. there were 4 does from farm 1 that were excluded from ai due to losing cidrs during synchronization but were turned out with bucks at the same time as the does that underwent ai for live cover. does from farm 2 (n = 17) were synchronized for laparoscopic ai to be performed the following day, ~ 72 hours after semen collection. a total of 10 does from farm 2 were inseminated with chilled, sex-sorted semen, whereas the remaining 7 does received conventional, frozen-thawed semen provided by the owner. table 1. semen quality and straw dosages in fresh and sex-sorted semen as provided by st technologies percent total motility day 0 percent progressive motility day 0 percent intact acrosome purity percent total motility day 1 sperm dose (106/ straw) total fresh male straws buck 1 91.6 77.4 91 0.88 88 4.79 19 buck 2 89.6 61 88 0.87 86.2 4.99 24 buck 3 85.9 65.6 85 0.9 68.8 4.59 13 http://dx.doi.org/10.58292/ct.v16.10513 citation line: clinical theriogenology 2024, 16, 10513, http://dx.doi.org/10.58292/ct.v16.10513 3 does were anesthetized to allow for low stress handling throughout the laparoscopic ai procedure, as described.7 does were guided through the chute and hand injected with tiletamine-zolazepam (0.88 mg/kg each) and xylazine (2.2 mg/ kg). following injection with the anesthetics, does were released into a small pasture until anesthetized, after which farm personnel placed a face mask on the doe and used a gurney to bring the doe back to the barn for the procedure. anesthetized does were placed into a cradle in dorsal recumbency, and the entire abdomen caudal to the umbilicus was clipped and aseptically prepared. the 2 laparoscopic port sites were identified (~ 6-8 cm cranial to the udder and ~ 6-8 cm lateral to midline) and desensitized with an infusion of 1 ml 2% lidocaine hydrochloride. an experienced veterinarian performed the laparoscopic ai procedure. a small incision (~ 1 cm) was first made into each of the blocked sites. a teat cannula attached to a co2 regulator was inserted into the peritoneum to allow for manual insufflation with medical grade co2. a 15-mm trocar was used to place the first cannula that allowed for placement of the laparoscope. on visualization of the uterus (confirming correct placement), a 5-mm trocar was used to place the second cannula. a 0.25-ml semen straw was loaded into an aspic device (imv technologies, maple grove, mn) that was placed through the second cannula. semen was injected directly into the greater curvature of the uterus, with half of the straw inserted into each of the 2 uterine horns. after manually deflating co2 from the peritoneum and removing the cannulas, the incision sites were closed using 1 or 2 staples per site. does each received 50 µg of gnrh agonist (cystorelin, boehringer ingelheim, ridgefield, ct) and a combined subcutaneous injection containing 2 mg/kg flunixin meglumine and 30 mg/kg oxytetracycline dihydrate (hexasol, norbrook, overland park, ks) in the axillary region. anesthesia was reversed with an intramuscular injection of 4.4 mg/kg tolazoline. does from both farms were turned out with an intact buck at ~10 to 15 days after laparoscopic artificial insemination. all does fawned in the spring of 2017. fawning data were recorded by the owners of each farm and provided for use in this case study. outcomes the fawning data are summarized (table 2). both farms had 100% total pregnancy rate for the herds (ai plus natural cover). ai pregnancy rates were determined using fawning dates. any deer that underwent ai but surpassed a 201-day pregnancy length was assumed to be pregnant by the herd buck rather than by ai (n = 10 for farm 1; n = 4 for farm 2). the 4 deer from farm 1 that lost their cidrs and did not undergo ai were also counted as pregnant by the herd buck, making a total of n = 14 counted as live cover for farm 1. the ai pregnancy rates for both sex-sorted and conventional semen ranged from 67 to 80%, which is consistent with industry standards. on average, there were ~ 1.6-2 fawns born per doe. the percentage of males born to sex-sorted semen was 85-100%, whereas the percentage of males born to live cover was 50% and born to conventional semen was 83%. the distribution of the specific bucks used for fresh, sex-sorted semen and pregnancy per ai rates between 2 farms are summarized (table 3). discussion semen sex-sorting has been used in domestic mammals (e.g. pigs, sheep, horses, cattle, and cervids) to obtain a high proportion of sperm carrying a chromosome of the desired sex.8-10 to date, sex-sorted semen has gained the most commercial popularity in the dairy cattle industry that is attributed to ability of the bovine sperm to tolerate the sex-sorting process and subsequent cryopreservation.8-10 in other species, more specifically pigs and horses, the high number of sperm needed to attain an optimal breeding dose and more fragile sperm have hindered the large-scale use of this technology.8 in small ruminants, the use of sex-sorted semen is feasible due to the satisfactory results reported with low doses (1-5 x 106 motile sperm) when laparoscopically inseminated directly into the uterus.11,12 sex-sorting semen could be an extremely beneficial tool for wtd producers to obtain a high proportion of male offspring. sales of bucks to hunting farms across north america result in better prices, and slaughtered males yield more meat than females. white-tailed deer have an advantage over domestic animals, with excellent semen quality that has table 2. fawning data from does bred by laparoscopic ai using sex-sorted semen, conventional semen, or live cover farm 1 farm 2 sex-sorted semen pregnant/ai (%) 20/30 (67) 8/10 (80) total fawns 33 16 fawns per doe 1.7 2 male fawns/total (%) 28/33 (85) 16/16 (100) conventional semen pregnant/ai (%)   5/7 (71) total fawns   8 fawns per doe   1.6 male fawns/total (%)   4/8 (50) live cover pregnant/bred (%) 14/14 (100) 4/4 (100) total fawns 24 6 fawns per doe 1.7 1.6 male fawns/total (%) 12/24 (50) 5/6 (83) live cover plus conventional semen male fawns/total (%)   9/14 (56) http://dx.doi.org/10.58292/ct.v16.10513 4 citation line: clinical theriogenology 2024, 16, 10513, http://dx.doi.org/10.58292/ct.v16.10513 a strong tolerance to cryopreservation when collected during rut.4,5,6 authors of an in vitro study reported that sex-sorting wtd semen using flow cytometry resulted in increased postthaw motilities and decreased dna fragmentation compared to conventional cryopreserved semen.13 despite this information on in vitro frozen-thawed sperm characteristics, there is limited information on field pregnancy rates in wtd using either fresh or frozen-thawed sex-sorted semen. the case study presented herein is the first to report the feasibility and usefulness of using fresh, sex-sorted semen in commercial wtd farms. a recent review cited anecdotal data that reported pregnancy rates of 93-95% using cryopreserved sex-sorted cervine doses of 8-9 x 106 sperm.14 with fresh sperm, doses of 4 x 106 sperm (used in the current report) have also reportedly produced better conception rates than conventional semen that is typically dosed at ~ 20 x 106 sperm per straw.14 in the current report, pregnancy per ai was numerically less for the conventional cryopreserved semen used (71%) compared to the chilled, sex-sorted semen (80%) for farm 2. for farm 1, we also report a lesser pregnancy rate (67%) than those reported anecdotally. in a large, controlled study using red deer (cervus elaphus), pregnancy rates were greater for conventional frozen-thawed semen (~ 78%) versus y sex sorted (50%) and control sorted semen (51%) when inseminated transcervically.15 in dairy heifers, ai pregnancy rates did not differ between conventional frozen-thawed semen (60.9%) and fresh, sex-sorted semen (54.2-53.5%), but was greater than frozen, sex-sorted semen (52.8%).16 on the contrary, cows inseminated with conventional frozen-thawed semen (48%) had greater ai pregnancy rates than those inseminated with sex-sorted semen, regardless if it was frozen or fresh (37.640.6%).16 based on these species differences, there is a distinct need to collect and publish data from a higher number of wtd to better determine if there are any relevant differences among conventional frozen-thawed, sex-sorted fresh, and sexsorted frozen thawed semen that will allow us to make accurate recommendations to farmers on the best method to use to achieve acceptable ai pregnancy rates. it is interesting to note that the pregnancy per ai was greater in farm 2 (~ 80%) from fresh semen that was 72 hours old compared to farm 1 (67%) where the semen was only 48 hours old, despite using similar synchronization protocols. there are few studies available that have investigated different dispatch-to-ai intervals and the potential effects on pregnancy per ai when using fresh, sex-sorted semen. in dairy cows, the dispatch-to-ai interval of fresh, sex-sorted semen did not affect pregnancy per ai, but did have an interaction with the bull.16 although pregnancy per ai was constant for most bulls, it actually increased with greater dispatch-to-ai intervals for 2 bulls.16 this interaction highlighted the need to investigate variability in male semen characteristics and fertility when using fresh, sex-sorted semen. in an in vitro study using frozen-thawed semen from wtd, authors reported the sexsorted semen had a decreased number of sperm containing fragmented dna and improved dna longevity over 72 hours compared to conventionally frozen sperm.13 sperm dna fragmentation is apparently a useful predictor of male fertility, and future studies are needed to better evaluate its changes over time in fresh, sorted samples to better understand the in vivo results presented herein. the purity of sex-sorted semen in cervids have been reported more recently to range from 92 to 95%.14 the fawn crop produced from ai with sex-sorted semen herein reported 85% bucks born on farm 1 and 100% bucks born on farm 2. interestingly, farm 2 also had a higher percentage of bucks born by live cover (83%) than farm 1 (50%). though a low number of animals may likely have a role in this finding, combining those bred by conventional semen and live cover for farm 2 still produced a greater number of bucks born than expected (56%). it is worth noting that though diets and pasture were similar between these farms, farm 2 also used an experimental feed additive from adm (adm animal nutrition, quincy, il) that is formulated to increase the sex ratio for male fawns. farm 2 used the feed additive until november 2016, encompassing 2 estrous cycles for the does on farm. studies in both roe deer17 and red deer18 have linked higher energy diets and increased male offspring production, but additional studies on this management strategy are lacking. further studies that compare the use of similar feed additives in conjunction with sex-sorted semen would be needed to better assess its usefulness in altering male offspring ratios in captive deer herds. this case study demonstrated the feasibility of using fresh, sexsorted semen for intrauterine laparoscopic artificial insemination in captive wtd farms. the semen from these 3 bucks survived transport and the sex-sorting processing. the sexsorted semen was still viable for insemination at 48-72 hours after initial collection. additionally, samples not needed for fresh insemination were cryopreserved and saved for use at a later date. this is the first published report for in vivo use of sex-sorted semen in wtd, though anecdotally, its use has table 3. does bred and pregnant per ai on each farm among 3 bucks collected for sex-sorted semen buck farm does bred percent does bred (bred/total) percent pregnant (pregnant/ai) 1 1 10 33 (10/30) 60 (6/10) 2 5 50 (5/10) 100 (5/5) combined 15 73 (11/15) 2 1 13 43 (13/30) 69 (9/13) 2 2 20 (2/10) 50 (1/2) combined 15 67 (10/15) 3 1 7 23 (7/30) 71 (5/7) 2 3 30 (3/10) 67 (2/3) combined 10 70 (7/10) http://dx.doi.org/10.58292/ct.v16.10513 citation line: clinical theriogenology 2024, 16, 10513, http://dx.doi.org/10.58292/ct.v16.10513 5 been gaining popularity throughout the past decade. future controlled studies would be useful to help determine ideal dosages, timing of ai in synchronized does, and interactions with feed additives. in red deer, frozen, y-sorted semen produced better ai rates when insemination occurred between 55 and 56 hours after ecg treatment (80-83%) compared to between 56 and 57 hours (< 33%).15 therefore, additional work at timing of insemination with frozen, sex-sorted semen in wtd is also needed. the use of sex-sorted semen with other advanced reproductive technologies, such as embryo flushing and in vitro fertilization, should also be critically analyzed to understand its usefulness in clinical practice. learning points • semen from white-tailed deer can easily withstand sex-sorting for use in clinical practice with acceptable viability after sorting. • the ai pregnancy rates using sex-sorted semen in whitetailed deer were consistent with industry standards. • selecting for y-sorted sperm resulted in an increased percentage of males in captive white-tailed deer herds. acknowledgment we thank sexing technologies genetics for working with us and our clients, and for providing in vitro semen data for the semen sorted. the clients, involved in this case study paid for all clinical services provided and described herein. conflict of interest none to report references 1. anderson dp, outlaw jl, earle ml, et al: economic impact of the us deer breeding and hunting operations. college station, texas: agricultural and food policy center; 2017. 2. bringans m: the captive deer industry in the usa and mexico: details on breeding, nutrition, and handling. in: j. fletcher ( editor) the management of enclosed and domesticated deer: international husbandry systems and diseases. cham: springer; 2022. p. 267-282. doi: 10.1007/978-3-031-05386-3_15/cover 3. kirschner sm, rodenkirch r: assessment of butorphanol-azaperone-medetomidine combination as anesthesia for semen collection and evaluation of semen quality in white-tailed deer (odocoileus virginianus). anim reprod sci 2017;184:196-203. doi: 10.1016/j.anireprosci.2017.07.016 4. stewart jl, shipley cf, ellerbrock re, et al: variation in post-thaw sperm quality of white tailed deer bucks (odocoileus virginianus) during rut. anim reprod sci 2018;195:121-130. doi: 10.1016/j. anireprosci.2018.05.014 5. stewart jl, shipley cf, ellerbrock re, et al: physiological variations in reproductive and metabolic features of white-tailed deer (odocoileus virginianus) bucks throughout the rutting season. theriogenology 2018;114:308-316. doi: 10.1016/j.theriogenology. 2018.04.015 6. stewart jl, shipley cf, katich as, et al: cryopreservation of whitetailed deer (odocoileus virginianus) semen using soybean-, liposome-, and egg yolk-based extenders. anim reprod sci 2016;171:7-16. doi: 10.1016/j.anireprosci.2016.05.006 7. shipley cf: equipment and techniques for transcervical artificial insemination and laparoscopic insemination in small ruminants. clin theriogenol 2011;3:525-530. 8. rath d, johnson la: application and commercialization of flow cytometrically sex-sorted semen. reprod domest anim 2008;43:338-346. doi: 10.1111/j.1439-0531.2008.01182.x 9. garner dl, seidel ge: history of commercializing sexed semen for cattle. theriogenology 2008;69:886-895. doi: 10.1016/j. theriogenology.2008.01.006 10. brito l, vishwanath r, heuer c, et al: bovine sexed semen production and utilization. clin theriogenol 2019;11:297-315. doi: 10.58292/ct.v11.9526 11. de graaf sp, beilby kh, underwood sl, et al: sperm sexing in sheep and cattle: the exception and the rule. theriogenology 2009;71:89-97. doi: 10.1016/j.theriogenology.2008.09.014 12. gonzález-marín c: evolution of cervine, caprine and ovine sexsorted semen processing. clin theriogenol 2018;10:205-209. doi: 10.58292/ct.v10.9914 13. kjelland me, gonzález-marín c, gosálvez j, et al: dna fragmentation kinetics and postthaw motility of flow cytometric-sorted white-tailed deer sperm. j anim sci 2011;89:3996-4006. doi: 10.2527/jas.2011-4014 14. gonzález-marín c, góngora ce, moreno jf, et al: small ruminant sexedultratm sperm sex-sorting: status report and recent developments. theriogenology 2021;162:67-73. doi: 10.1016/j. theriogenology.2020.12.028 15. anel-lópez l, garcia-álvarez o, tarantini t, et al: influence of insemination time on the fertility of sex sorted frozen-thawed y-sperm in red deer. theriogenology 2018;113:171-175. doi: 10.1016/j.theriogenology.2018.03.005 16. maicas c, hutchinson ia, kenneally j, et al: fertility of fresh and frozen sex-sorted semen in dairy cows and heifers in seasonal-calving pasture-based herds. j dairy sci 2019;102:10530-10542. doi: 10.3168/jds.2019-16740 17. wauters la, de crombrugghe sa, nour n, et al: do female roe deer in good condition produce more sons than daughters. behav ecol sociobiol  1995;37:189-193. doi: 10.1007/bf00176716/ metrics 18. luna-estrada aa, vera-avila hr, mora o, et al: effect of pre-mating nutritional status in red deer (cervus elaphus scoticus) hinds on the sex ratio of their offspring. small rumi res 2006;65:154-160. doi: 10.1016/j.smallrumres.2005.05.039 http://dx.doi.org/10.58292/ct.v16.10513 https://doi.org/10.1007/978-3-031-05386-3_15/cover https://doi.org/10.1016/j.anireprosci.2017.07.016 https://doi.org/10.1016/j.anireprosci.2018.05.014 https://doi.org/10.1016/j.anireprosci.2018.05.014 https://doi.org/10.1016/j.theriogenology.2018.04.015 https://doi.org/10.1016/j.theriogenology.2018.04.015 https://doi.org/10.1016/j.anireprosci.2016.05.006 https://doi.org/10.1111/j.1439-0531.2008.01182.x https://doi.org/10.1016/j.theriogenology.2008.01.006 https://doi.org/10.1016/j.theriogenology.2008.01.006 https://doi.org/10.58292/ct.v11.9526 https://doi.org/10.1016/j.theriogenology.2008.09.014 https://doi.org/10.58292/ct.v10.9914 https://doi.org/10.2527/jas.2011-4014 https://doi.org/10.1016/j.theriogenology.2020.12.028 https://doi.org/10.1016/j.theriogenology.2020.12.028 https://doi.org/10.1016/j.theriogenology.2018.03.005 https://doi.org/10.3168/jds.2019-16740 https://doi.org/10.1007/bf00176716/metrics https://doi.org/10.1007/bf00176716/metrics https://doi.org/10.1016/j.smallrumres.2005.05.039 is stallion age related to dystocia or neonatal foal morbidity? is stallion age related to dystocia or neonatal foal morbidity? james thompson, steven brinsko department of large animal clinical sciences veterinary college of veterinary medicine and biomedical science texas a&m university, college station, tx with advancing age, males have a nonlinear increase in germ line mutations related to cumulative changes in spermatagonial stem cells. these effects result from age related changes that compromise dna replication, dna repair, cell cycle control and epigenetic modifications. these errors accumulate with successive mitotic divisions and contribute to de novo mutations, affecting genetic traits in a variety of ways. objective was to evaluate if older stallions were more likely to sire foals involved in dystocia or affected by neonatal morbidity. records from 2014 to 2018 were retrieved from 2 veterinary referral practices (hagyard equine medical institute and rood & riddle equine hospital). thoroughbred mares admitted for dystocia and thoroughbred neonatal foals admitted for any illness were identified. expected age-related admission rates were estimated using state wide thoroughbred foaling records. stallion age was divided into 4 groups: < 10, 10 14, 15 19, and 20 plus years. odds ratios for admission by stallion age group were estimated using a bayesian modeling approach, with stallion age of < 10 years as baseline. neonatal foals sired by stallions aged 15 19 years and 20 plus years were significantly more likely to be admitted to veterinary hospitals with odds ratios (95% credibility intervals) 1.28 (1.05, 1.56) and 1.65 (1.04, 2.51), respectively. dams carrying foals sired by stallions aged 20 plus were significantly more likely to be admitted for dystocia with odds ratio (95% credibility interval) of 2.07 (1.10, 3.58). there is considerable potential to study both dystocia and neonatal foal morbidity as functions of stallion age and, more specifically, male germ line mutations. an equine model could provide considerable potential to study biology of male germ line mutations, as stallions often continue to breed well into advanced age; furthermore, accurate records are available to identify large numbers of specific stallion offspring (large families). keywords: stallion age, dystocia, foal morbidity 455 clinical theriogenology • volume 11, number 3 • september 2019 http://vetmed.tamu.edu/vlcs 456clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2013: clinical management of the equine oviduct clinical management of the equine oviduct patricia l. sertich university of pennsylvania, school of veterinary medicine, new bolton center, kennett square, pa introduction macroscopic and microscopic anomalies of the oviducts are common (87.69%), but infertility is rarely attributed to oviductal problems.1 one should consider evaluation of the oviducts after other genital tract abnormalities have been dismissed as the cause of a mare’s infertility and the mare has been properly bred with adequate amounts of good quality semen at the optimal time over several estrus cycles. certain oviductal problems may be treated to allow a mare to become pregnant and carry a foal to term. assisted reproductive techniques that involve harvesting oocytes may be required to produce foals from mares that have permanent bilateral oviduct abnormalities. keywords: mare, oviduct, oviductal patency, pge2 oviductal anomalies hydatid of morgagni (accessory uterine tube) hydatid of morgagni cysts are the most common structural oviductal anomaly and are usually present on the cranial edge of the infundibulum. these cysts result from more than one ostium forming from the coelomic epithelium in fetal life. most fimbrial cysts are small and an incidental finding in fertile mares that require no treatment.2 although not proven to cause infertility a large cyst could have the potential to prevent the fimbria from covering the ovulation fossa at the time of ovulation and interfere with the transport of the oocyte. a nd:yag laser has been used to remove large fimbrial cysts and was found to maintain hemostasis and prevent the formation of postoperative adhesions.3 cystic remnant of the mesonephric duct mesonephric duct remnants are commonly seen and usually occur unilaterally. these paraovarian cysts are frequently seen in pregnant mares, and not thought to contribute to infertility.1 adhesions the most common (66.5%) macroscopic abnormality of the oviduct is thin strands of fibrous tissue that extend from the infundibulum to the ovary or serosal surface of the genital tract and can be seen at postmortem in mares that have produced many foals. less commonly (26.8%), bands of thick strands of fibrous tissue are seen traversing the oviduct in a superficial, perpendicular and nonocclusive fashion.1 although the etiology is not completely understood the thin strands are often seen with blood clots and may be associated with ovulation. the thicker (> 4 mm thick) bands have not been seen to be associated with blood clots. transluminal adhesions of the oviduct are rarely seen.2 hydrosalpinx hydrosalpinx in the mare is rare and reports include both unilateral and bilateral cases. hydrosalpinx can be congenital and due to segmental aplasia of the tubular tract of the infundibulum.2,4 one reported case was bilateral and related to an ascending infection. the author has seen one multiparous mare with a history of pelvic fracture that spent an extended period of time in a sling and subsequently developed bilateral hydrosalpinx. if the hydrosalpinx is unilateral one may consider removal of its ipsilateral ovary to force the mare to cycle from the remaining ovary that is adjacent to the normal oviduct. otherwise the mare’s follicular activity can be managed carefully and insemination only performed when the preovulatory follicle is ipsilateral to the normal oviduct. a mare with bilateral hydrosalpinx would require an assisted reproductive technique that entailed oocytes to be harvested to produce a foal. 523 clinical theriogenology • volume 5, number 4 • december 2013 salpingitis in spite of the frequent occurrence of endometritis it has been assumed that the incidence of salpingitis is low in the mare because the oviducts are positioned dorsally to the uterus and the prominent oviduct papillae at the utero tubal junction prevent a uterine infection from ascending into the oviducts.5,6 in two abattoir studies, 7 to 37% of the samples evaluated had salpingitis. rather than a focal lesion in the oviduct the inflammation seen tended to be a slight but widespread infiltration of lymphocytes (and occasionally eosinophils). rarely was there occlusion of the oviduct associated with the inflammation.1 although cases of infundibulitis were seen without associated endometritis most (80%) cases of isthmic salpingitis occurred concurrently with endometritis. although specific therapy for salpingitis and for that matter even cervicitis is rarely instituted, clinical dogma has been that resolution of chronic endometritis is necessary for inflammation in the oviduct to subside. unfortunately a definitive and fail-safe antemortem diagnostic method for salpingitis is not available at this time. globular masses accumulations of type i collagen fibers have been reported in the lumen of the oviduct of 18.5 87% of mares.1,7 these globular masses are not cellular and the oviductal epithelium adjacent to them is healthy and intact. masses may be bilateral or unilateral and are seen in multiparous, pregnant, primiparous and maiden mares. the incidence is greater in older (> 7 years) mares than mares two to seven years of age. globular masses tend to lodge at the bends of the tortuous oviduct and at the ampullary-isthmus junction. interestingly unfertilized ova may be present proximally and distally to the collagen mass.7 occasionally collagen fibers occupy the entire luminal diameter of the oviduct and may even distend the lumen. the discovery that prostaglandin e2 (pge2) was responsible for the transport of embryos through the isthmus demonstrated that oviduct transport is not a passive event.8 it is not surprising that material may accumulate in the oviduct since unfertilized oocytes are retained. obviously some luminal content in the oviduct is not pathological as unfertilized oocytes are commonly seen in uterine lavage effluent when an embryo is recovered. obstructions due to an excessive accumulation of globular masses have been cleared by direct flushing or application of pge2 to the oviduct.3,9,10 history and physical examination considering that significant abnormalities of the oviduct are not common and since the diagnostic tests are time consuming, possibly inconclusive, invasive and not without risk to the mare, it is recommended that a thorough evaluation of the mare’s genital tract and management be made before pursuing specific oviductal diagnostic tests and treatment. all breeding records should be evaluated to determine a history of prolonged infertility in spite of repeat breeding with adequate doses of good quality semen from fertile stallions just prior to documented ovulations. a thorough evaluation of the genital tract should be made that includes visual examination of the external genitalia, palpation and ultrasonography per rectum, direct palpation of the cervix per vaginum, cytological examination of an endometrial swab or low volume lavage, aerobic culture of an endometrial swab, histological evaluation of an endometrial biopsy sample and hysteroscopy. a karyotype should be performed to rule out a chromosome abnormality. if the barren mare has been properly bred repeatedly, has no genital tract abnormalities and has a normal karyotype, one might then consider evaluation of the oviducts. diagnostic tests although none of the diagnostic tests to evaluate a mare’s oviduct are fail-safe the following are techniques that have been used with varying degrees of success. starch grain test the starch grain test involves the intraperitoneal deposition of a starch suspension onto the ovary. if the oviduct is patent the starch granules are transported through the oviduct into the uterus.11 the procedure is performed on the day of ovulation or on day seven of diestrus. it is recommended that one 524clinical theriogenology • volume 5, number 4 • december 2013 oviduct be evaluated and then the procedure be repeated on the other oviduct the next cycle. sterile soluble starch powder (1 gm) is mixed in 10 ml of cool sterile water. the mare’s paralumbar fossa is clipped and aseptically prepared. the genital tract is palpated per rectum and one ovary is manually positioned adjacent to the paralumbar fossa. five ml of 2% lidocaine are infused subcutaneously and deep into the flank muscles at the site over the ovary. a stab incision is made through the skin. a 12 cm 18 gauge spinal needle is directed toward the ovulation fossa of the hand-held ovary and passed through the flank. care should be taken to not lance the ovary with spinal needle. the cool starch solution (5 ml) is then infused over the surface of the ovary. the procedure is repeated on the other ovary the next estrous cycle. the uterus is then lavaged with a small volume (20-30 ml) of sterile saline daily for seven days or until starch is identified in the uterine lavage fluid. each day a drop of the effluent is mixed with 1-2 drops of 2% lugol’s iodine on a microscope slide, compressed with a cover slip and examined on a bright light microscope (10 x objective). the starch granules stain blue. when the procedure is performed on the day of ovulation in a mare with patent oviducts the starch granules are expected to be seen in the uterus on day four to seven of diestrus. when the procedure is performed on day seven of diestrus the starch is expected to be in the uterus within 24 hours.11 transvaginal application of fluorescent microspheres after cleansing the perineum the vagina is douched with 500 ml sterile saline containing 0.05% inorganic iodine and then 50 mls of 2% lidocaine diluted with 450 ml sterile saline. a cold-sterilized transvaginal ultrasound probe is used to visualize one ovary that is held in position by direct palpation per rectum. using a 60 cm, 18 gauge, single channel, sterile needle in the biopsy channel of the ultrasound probe, two million red, 15 μm microspheres (fluoresbrite® flow cytometry microspheres, polysciences inc., warrenton, pa) suspended in 5 ml of sterile saline are deposited over the surface of one ovary. two million green, 15 μm microspheres are suspended in 5 ml of sterile saline and then deposited over the surface of the other ovary. the mare’s uterus is lavaged with 1 l sterile saline at 24 and 48 hours after microsphere deposition. the saline lavage effluent can be either centrifuged at 1500 rpm for 10 minutes, the supernatant decanted and the pellet suspended in 5 ml sterile saline or filtered through a 2-μm filter to separate microspheres and other particulate debris from the larger volume of fluid. the centrifuged or filtered samples are then examined by flow cytometry for the presence of microspheres and fluorescence pattern (green and red) which indicate that the oviducts of each respective oviduct are patent. microspheres did not pass though the oviducts of 23% of the barren mares (3/13) examined using this technique.12 laparoscopic application of fluorescent microspheres each paralumbar fossa is clipped and surgically prepared. an operating laparoscope and oviductal grasping forceps are used to place an 8 fr polypropylene catheter through the flank and into the oviduct of a sedated diestrus mare. two million colored fluorescent microspheres suspended in 0.5 ml of a 1.5% solution of carboxymethylcellulose are inserted into one oviduct. microspheres of a different color are then laparoscopically guided into the other oviduct via the other flank. microspheres passed into the uterus of 43% of healthy diestrus mares examined in a study indicating that those mare’s oviducts were patent. mares that did not have the microspheres pass into the uterus were found to have fimbrial adhesions, oviductal plug formations, unilateral hydrosalpinx and cysts of the infundibulum or oviduct.13 laparoscopic oviduct infusion after a 24 hr feed restriction, mares are sedated and their paralumbar fossae are aseptically prepared and infiltrated with 2% lidocaine for laparoscopy. a rigid laparoscope with a 30° viewing angle is positioned through an instrument portal located at the ventral level of the tuber coxae, midway between the tuber coxae and the last rib. three other portals are created, one located 10 cm ventral and 2 located 5 cm ventral and 5 cm cranial or caudal to the laparoscope portal. visualization of the entire length of the oviduct is possible with this flank approach. two laparoscopic babcock forceps are used to grasp each 525 clinical theriogenology • volume 5, number 4 • december 2013 side of the infundibulum to expose the abdominal ostium in the center of the infundibulum. a 7 ch. balloon catheter is passed through the lower instrument portal and inserted 2 cm into the ampulla. the catheter cuff is inflated with 2 ml air. one babcock forceps is then positioned over the abdominal ostium and catheter to secure the catheter in place. sterile methylene blue solution (20 ml) is flushed through the oviduct. the procedure is repeated on the other side. presence of the methylene blue solution is determined hysteroscopically 15 minutes post-operatively. catheterization of the ampulla was successful in 63.7% (7/11) of the mares. in 71% of these mares methylene blue was found in the uterus indicating the oviducts were patent.14 therapeutic procedures the following techniques have been used to treat mares suspected of having oviduct dysfunction. retrograde flush general anesthesia is administered, and a diestrous mare is placed in dorsal recumbency and surgically prepared for a paramedial incision on the left and right side. one ovary and the tip of the ipsilateral uterine horn are exteriorized through a paramedical incision. the oviduct is examined and the uterotubal junction palpated. an incision is made in the tip of the uterine horn adjacent to the oviduct papilla. the endometrium is everted so that the papilla is exposed. a 0.5 inch 24 gauge blunt ended catheter is placed into the papilla and 12 ml of saline containing a fluorescein dye is gently flushed through the oviduct. the effluent is collected at the ostium in the infundibulum and examined for the presence of masses. the uterine incision is closed with a double row of mattress sutures, the ovary and uterus returned to the abdomen and the procedure is repeated on the opposite oviduct. using this procedure, oviductal flushing was successful and oviductal masses were expelled in five of eight infertile mares. three of the five mares produced foals subsequently.9 normograde flush after a 24 hr fast a mare is administered general anesthesia and placed in dorsal recumbency. a pelvic tilt apparatus is used to flex the mare at the lumbo-sacral junction to better exteriorize the genital tract through a 10 cm ventral midline incision just cranial to the mammary gland. the ovary and ipsilateral uterine horn are carefully examined for fimbrial adhesions, parafimbrial cysts and obvious oviduct abnormalities. a doyen intestinal forceps is placed 5 cm from the cranial tip of the uterine horn. an 8 fr foley 30 cm 5 ml balloon catheter is inserted into the ampulla and the balloon was inflated with 1.0-1.5 ml of air. strong digital pressure is applied over the catheter cranial to the balloon and 20 ml of 5% solution of new methylene blue dye in normal saline is slowly injected through the catheter. confirmation of patency is ascertained by aspirating 10-20 ml of normal saline from the lumen of the tip of the occluded uterine horn. the normograde flush procedure is then repeated on the opposite oviduct. if an oviduct is occluded the serosal surface of the oviduct is saturated with 2% lidocaine and carefully flushed. care must be taken to avoid rupturing the oviduct. in the study that reports this technique, if only one oviduct was occluded the ipsilateral ovary was removed. three of 31 barren mares with no other known cause for the infertility had patent oviducts. thirteen (13/31) mares had bilateral oviductal occlusion. the blockage was relieved in twelve of these occluded mares and 67% (8/12) became pregnant after their next breeding. of the mares with unilateral oviductal blockage (6/31) that were unilaterally ovariectomized and then bred, 83% (5/6) became pregnant after their first breeding.3 laparoscopic application of pge2 feed is withheld for 24 hours prior to laparoscopic surgery. each flank is clipped and surgically prepared. the standing mare is sedated by administration of detomidine hydrochloride (10 μg/kg) iv and butorphanol tartrate (10 μg/kg) iv. the skin and underlying musculature of the portal sites are infiltrated with 2% lidocaine. an 11.5 mm (o.d.) trocar and cannula are passed through a 1 cm skin incision 15 cm below the dorsal aspect of the tuber coxae. the abdomen is insufflated with co2 gas. a 526clinical theriogenology • volume 5, number 4 • december 2013 10 mm o.d. rigid laparoscope with a 30° viewing angle is used to view the ovary, oviducts and tip of the ipsilateral uterine horn. a 7.5 mm o.d. trocar and cannula is inserted through the abdominal muscles approximately 5 cm dorsal to the laparoscope. an atraumatic forceps is passed through this cannula to manipulate the ovary and mesovarium and position the oviduct so that the infundibulum, ampulla and isthmus can be visualized. the forceps are withdrawn and a plastic insemination pipette is used to apply 0.5 ml of a commercial preparation of triacetic gel containing 0.2 mg pge2 to the serosal surface of the ampulla and isthmus. the co2 gas is released from the abdomen, cannulae are withdrawn and the skin incisions closed. this procedure is then repeated on the opposite flank.15 in a study using this technique 14 of 15 chronically barren mares that were treated with pge2 conceived when subsequently bred.10 conclusions many anomalies of the oviduct do not interfere with fertility. a few diagnostic tests have been used to detect problems of the oviduct but a definitive ante-mortem diagnosis of oviduct dysfunction may be difficult to ascertain. if the oviduct is thought to be the cause of the infertility a procedure may be available to correct the underlying abnormality. the laparoscopic application of pge2 to the oviduct seems effective for oviducts that have luminal occlusions. if the oviductal problem is unilateral and irreversible the ipsilateral ovary can be removed to force the mare to cycle on the remaining ovary that is adjacent to the normal oviduct. alternatively the mare’s ovarian activity can be manipulated and breeding scheduled only when the ovary adjacent to a normal oviduct contains the preovulatory follicle. foals can be produced if the oviduct problem is bilateral and irreversible in breeds that permit artificial reproductive techniques. an oocyte can be harvested and transferred to the oviduct of an inseminated recipient mare, where fertilization can occur and the recipient mare can then carry the pregnancy to term.16 another option for the oocyte would be intracytoplasmic sperm injection (icsi) with the resulting embryo cultured in the laboratory and then transferred into a recipient mare that would carry the pregnancy to term.17 one should remember to consider the oviduct when other causes of infertility have been eliminated in the chronically barren mare. references 1. saltiel a, páramo r, murcia c, et al: pathologic findings in the oviducts of mares. am j vet res 1986;47:594-597. 2. kenney rm: a review of the pathology of the equine oviduct. equine vet j suppl 1993;15:42-46. 3. bennett sd, griffin rl, rhoads ws: surgical evaluation of oviduct disease and patency in the mare. proc annu conv am assoc equine pract 2002. p. 347-349. 4. hinrichs k, kenney rm, hurtgen jp: unilateral hydrosalpinx and absence of the infundibulum in a mare. theriogenology 1984;22:571-577. 5. dimock ws, edwards pr: the pathology and bacteriology of the reproductive organs of mares in relation to sterility. ky agric exp stn res bull 1928;286. 6. vandeplassche m, henry m: salpingitis in the mare. proc annu conv am assoc equine pract 1977. p. 123-131. 7. liu ikm, lantz kc, schlafke s, et al: clinical observation of oviductal masses in the mare. proc annu conv am assoc equine pract 1990. p. 41-45. 8. weber ja, freeman da, vanderwall dk, et al: prostaglandin e2 hastens oviductal transport of equine embryos. biol reprod 1991;45:544-546. 9. zent ww, liu ikm, spirito ma: oviduct flushing as a treatment for infertility in the mare. embryo transfer iii, equine vet j, suppl 1993;15:47-48. 10. allen wr, wilsher s, morris l, et al: laparoscopic application of pge2 to re-establish oviducal patency and fertility in infertile mares: a preliminary study. equine vet j 2006;38:454-459. 11. allen, we, kessy bm, noakes de: evaluation of uterine tube function in pony mares. vet rec 1979;105:364-366. 12. ley wb, bowen jm, purswell bj, et al: modified technique to evaluate uterine tubal patency in the mare. proc annu conv am assoc equine pract 1998. p. 46-59. 13. arnold ce, love cc: laparoscopic evaluation of oviductal patency in the standing mare. anim reprod sci 2010;121s:s86. 14. köllmann m, rötting a, heberling a, et al: laparoscopic techniques for investigating the equine oviduct. equine vet j 2011;43:106-111. 15. robinson sj, neal h, allen wr: modulation of oviductal transport in mares by local application of prostaglandin e2. j reprod fertil suppl 2000;56:587-592. 527 clinical theriogenology • volume 5, number 4 • december 2013 16. carnevale em, squires el, maclellan lj, et al: use of oocyte transfer in a commercial breeding program for mares with reproductive abnormalities. j am med vet assoc 2001;218:87-91. 17. choi yh, hinrichs k: intracytoplasmic sperm injection (icsi). in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. west sussex (uk): wiley-blackwell; 2011. p. 2948-2952. 528clinical theriogenology • volume 5, number 4 • december 2013 management of dystocia in the mare management of dystocia in the mare patrick mccue department of clinical sciences, colorado state university, fort collins, co abstract a dystocia is an emergency condition in the mare. the goals of dystocia management are to save the life of the foal, save the life of the mare, and to preserve future fertility of the mare. early detection and rapid appropriate intervention are critical for foal survival. the most common cause of an equine dystocia is an abnormality of fetal posture. keywords: dystocia, foaling, mare, obstetrics introduction the term dystocia refers to an abnormal or difficult birth.1 dystocia or difficult birth occurs in approximately 4% of thoroughbred foalings.2 abnormal orientation of the fetus in the birth canal (i.e. postural abnormalities) is the most common cause of dystocia in the horse. dystocia is more common in mares during their first foaling (i.e. maiden mares) than in mares that have previously given birth to 1 or more foals. the ultimate goals in assisting in an equine dystocia are to save the life of the foal, save the life of the mare and to preserve the future fertility of the mare. time management during a dystocia active labor (stage ii) is a rapid event in most mares, with most foals delivered within 20 30 minutes after rupture of the chorioallantoic membrane.3 the average length of stage ii for mares in 1 study was 16.7 minutes, with 71.7% of foals born in less than 20 minutes.4 an owner or foaling attendant should be concerned if a foal has not been born or if significant progress toward birth has not occurred within 20 minutes after rupture of the outer chorioallantoic membrane and subsequent release of allantoic fluid (i.e. when a mare ‘breaks her water’). dystocia and foal survival dystocia in mares is a medical emergency and time is critical.4-6 it has been estimated that each 10 minutes increase in the duration of stage ii labor beyond 30 minutes is associated with a 10% increase in the existing risk of a foal being born dead and a 16% increase in risk of the foal not surviving to discharge from a referral hospital or clinic.5 an additional study reported that the interval from rupture of the chorioallantoic membrane to delivery of the fetus was 13.6 minutes shorter for foals that were born alive and survived to discharge than for foals born dead or that did not survive to discharge.7 a third study noted a significant increase in stillbirth, foal morbidity and foal mortality occurred when the duration of stage ii of labor was greater than 40 minutes (table 1).4 combined, these statistics reinforce the concept that early accurate detection of a foaling problem and early effective intervention is critical to foal health and survival. early detection of an equine dystocia and rapid appropriate intervention in problem cases are critical for foal survival. placental separation and subsequent decrease in oxygen supply in utero are likely the primary factors that lead to fetal death during a prolonged dystocia. if the fetus is still within the uterus (i.e. not fully engaged into the birth canal) and the placenta is still attached and the umbilical cord is not compressed or disrupted, the oxygen supply to the fetus may be sufficient for short-term survival. however, it is critical that the fetus be delivered as safely and expediently as possible. dystocia management plan it is strongly recommended that an emergency plan be formulated prior to the foaling season to prepare for a difficult birth, a medical problem with a newborn foal, or medical issues with the mare. the plan should be the result of a conversation among the mare owner, breeding farm/ranch manager, clinical theriogenology • volume 12 number 3 • september 2020346 table 1. stillbirth, foal morbidity, and foal mortality associated with duration of stage ii of labor duration stage ii (minutes) (n) stillbirth (%) morbidity (%) mortality (%) 0 10 173 1.7a 4.6a 4.1a 11 20 549 0.4a 3.3a 2.2a 21 30 211 2.8a 6.6a 5.2a 31 40 45 0a 2.2a 4.4a 41 50 13 7.7b 7.7b 7.7b > 50 18 22.2b 11.1b 27.8b a,bwithin a column, percentages without a common superscript differed (p < 0.05) foaling attendant and the veterinarian(s). all personnel actively involved in foaling mares should be trained and ready to assist. a foaling kit should be readily available near the foaling stall. management of a dystocia is dependent on training, experience and availability of farm personnel, as well as the proximity of veterinary services. early communication in the event of a dystocia it is recommended that a foaling attendant call for assistance (on-farm personnel or veterinary assistance, as appropriate) in the following circumstances:  if there has been no progress toward delivery after 15 20 minutes after rupture of the chorioallantois  if progress toward delivery abruptly stops  if the mare becomes acutely painful or exhibits signs of shock  if the attendant is confident that a problem with foaling exists  if the attendant is unsure if a problem with foaling exists  if the attendant does not have the knowledge, training, or ability to identify or correct the problem causes of dystocia in the mare the most common causes of dystocia in the mare categorized by difficulty of management or correction are listed (table 2). mild dystocias are commonly managed on the farm by foaling personnel. more complicated dystocias may require advanced training or experience and often require veterinary intervention. veterinary advice or assistance should immediately be sought if the problem cannot be safely and quickly corrected by on-farm/ranch personnel. fetal alignment in the birth canal during foaling is described in obstetrical terms as:  presentation relationship of the long axis of the fetus to the dam’s birth canal  position relationship of the dorsum of the fetus to the quadrants of the maternal pelvis  posture relationship of the fetal extremities to the fetus itself alignment of the fetus in the birth canal during a normal delivery is described as:  anterior presentation, dorsal-sacral position, with both forelimbs extended and the head extended failure of the fetus to be properly oriented in the birth canal as foaling progresses will almost always prevent normal passage and result in dystocia. the most common causes of dystocia in the mare are abnormalities of fetal posture (i.e. abnormal alignment of the head or forelimbs).8 a retrospective study of over 1,000 equine births noted that abnormalities of fetal posture were associated with 37.7% of dystocias, including 1 or both front limbs retained (30.1%), retained fetal head (3.8%) or both limb(s) and clinical theriogenology • volume 12 number 3 • september 2020 347 head retained (3.8%).4 a 0.8% incidence rate of ‘hip-lock’ (feto-pelvic disproportion) was recorded. premature placental separation was recorded in 1.6% of foalings. a periparturient hemorrhage event was noted in 0.8% of foalings. table 2. common causes of dystocia in mares, arranged by degree of difficulty in correction correction difficulty foaling complication or issue mild elbow lock upside-down foal backwards foal uterine inertia ‘red-bag’ (premature placental separation) moderate front leg(s) flexed at the knee (carpus) neck flexed ventrally; muzzle below pelvic brim feto-pelvic disproportion or ‘hip-lock’ difficult front leg(s) flexed at shoulder neck flexed to side; muzzle not reachable backwards presentation, hind leg(s) flexed at hip transverse presentation twins (when both entering birth canal simultaneously) initial physical examination of the mare during a dystocia a brief physical examination should be performed to evaluate the health status of the mare.9 it is recommended that the examination be performed in a large stall or other open area or in stocks that can quickly and completely be taken apart with removal of a few pins. it is not recommended to examine a pregnant mare experiencing a dystocia in solid-sided stocks, because many foaling mares will attempt to lie down during vaginal examination or fetal manipulation. in addition, young inexperienced foaling mares may be exceptionally nervous, excited or scared during their first delivery and these behaviors may be even more pronounced during a difficult birth. safety of personnel should be of paramount concern during all examinations and obstetrical procedures. consequently, it may be necessary to sedate the mare, apply a twitch or perform an epidural to facilitate examination, provide pain relief and/or decrease straining. intervention in an equine dystocia choices for relieving an equine dystocia include an assisted or controlled vaginal delivery, cesarean surgery (cesarean section) or fetotomy (figure 1). the final decision may depend on the status of the fetus, duration and severity of the dystocia, economic value of the mare and fetus, clinician expertise, client preference, facilities available and other considerations. intervention by trained personnel may save the life of a foal in the event of a dystocia. however, the undisputed key factors in foal survival are early recognition of a foaling difficulty by breeding farm personnel and an early call for assistance. assisted vaginal delivery a reproductive examination is performed to identify the obstetrical problem, determine if the fetus is alive and to formulate a plan. if possible, the tail of the mare is quickly wrapped or placed in a plastic sleeve and held out of the way. a brief wash and rinse of the perineal area is performed to remove debris and manure. a manual vaginal examination is subsequently performed, with or without application of a sterile obstetrical sleeve. the goals are to determine:  if the chorioallantoic membrane had indeed ruptured (i.e. did the mare really ‘break her water’). in some cases, the mare may not actually be in stage ii of labor or the signs of abdominal pain may be colic in nature and not associated with uterine contractions. clinical theriogenology • volume 12 number 3 • september 2020348 figure 1. general plan for dystocia management in a mare  dilation status of the cervix  presentation, position and posture of the fetus (i.e. orientation of the fetus in the birth canal)  live/dead status of the fetus (if possible). accurate assessment of fetal viability may be critical to subsequent obstetrical decisions. in some cases, fetal viability status cannot be determined and one should proceed assuming that the fetus is viable. evaluation for fetal viability may include: o reflex reactions upon application of pressure to the tongue or the eye o detection of a suckle response after insertion of finger into mouth o detection of a heart-beat in the chest (if reachable) o detection of a pulse within the umbilical cord (if reachable) o detection of fetal movements, etc.  if perineal, vaginal, cervical or other problems have already occurred if only legs or feet are presented at the vulva, one needs to determine if they are front legs or hind legs, as subsequent management decisions are dramatically different. the best way to differentiate front legs from hind legs is to assess the direction of flexion of the 2 largest moveable joints above the foot.  in front legs, the 2 joints (fetlock and carpus) flex in the same direction  in hind legs, the 2 joints (fetlock and hock) flex in opposite directions. a plan for intervention (modified from a predetermined dystocia plan) should be developed for each specific dystocia, based on the information above, the health status of the mare, duration of dystocia, economic value of the mare and foal and other factors. in addition, a backup plan should always be discussed in the event that the initial plan is not successful. normal fetal orientation in birth canal a vaginal delivery may be attempted with the mare awake and either standing or recumbent if the fetus is lined up or oriented normally in the birth canal (anterior presentation, dorsal-sacral position, with both forelimbs and the head extended) or in posterior presentation with both hind limbs extended. a normal fetal orientation may be present in cases of uterine inertia or in cases of fetal oversize relative to the birth canal of the mare (feto-pelvic disproportion; ‘hip-lock’). the following guidelines are recommended when assisting delivery:  if the mare is straining and will not allow a vaginal examination, consider sedation or administration of an epidural o options for an epidural using an 18 gauge, 3 inch needle include:  lidocaine (2%) 5 8 ml  xylazine (0.17 mg/kg) diluted in 10 ml of 0.9% sterile saline  combination of lidocaine (0.22 mg/kg), plus xylazine (0.17 mg/kg) o pump obstetrical lubricant into the uterus around the fetus. the commercial product ‘j-lube’ (jorgensen laboratories, loveland, co) is an excellent obstetrical lubricant, clinical theriogenology • volume 12 number 3 • september 2020 349 but should be used with caution if a uterine tear is present or suspected or if a cesarean surgery is being considered. j-lube has been reported to cause severe peritonitis if leaked into the abdominal cavity.10  repel the fetus back into the abdominal cavity (if necessary) to correct any abnormalities of presentation, position or posture  confirm that the fetus is correctly lined up in the birth canal  place obstetrical chains or foaling straps on each front leg above the fetlocks o obstetrical chains are advantageous in that they are easy to place, a hook or handle can be attached at any location on the chain and the chain and hooks can be easily cleaned and either cold sterilized or autoclaved o wide-webbed nylon obstetrical straps can be advantageous in that a loop can be made on each end and the strap itself becomes the handle; the disadvantage is that they are not as easy to clean or sterilize as chains o thin rope, baler twine or other narrow devices should not be used on foals due to the potential for damage to skin, tendons or other structures  pull when the mare has a uterine contraction; stop pulling when the mare relaxes in-between contractions  do not exceed the force of 2 people when assisting  avoid use mechanical devices to pull a foal  pull in an outward (initially) and then slightly downward direction  ‘walk’ the shoulders of the foal through the birth canal, 1 at a time uterine inertia in some cases, a mare may not provide sufficient uterine contractions (or any apparent uterine contractions) to deliver a fetus, despite the fact that the fetus is aligned properly in the birth canal. in other cases, the mare may become exhausted and unable to muster additional uterine contractions. in these instances, the following guidelines should be followed:  confirm that the foal is in correct presentation, position and posture  apply obstetrical lubricant around the fetus (if needed)  attach obstetrical chains or straps  provide traction to deliver the fetus abnormal fetal presentation, position or posture if the fetus is not in a normal orientation in the birth canal, the abnormality must be corrected prior to any attempt to apply traction (i.e. do not pull on the fetus). it is imperative to accurately assess the abnormality so that proper and expedient intervention can be initiated. the most common disorders of presentation, position or posture in an equine dystocia include:  elbow lock  front leg(s) flexed at the knee or shoulder  head flexed laterally or (less commonly) flexed ventrally  posterior presentation elbow lock elbow lock is a common and relatively mild abnormality requiring minimal intervention by the foaling attendant. the foal will be in anterior presentation, usually a dorsal-sacral position, with both front feet and a nose or the cranial portion of the head visible. however, 1 front leg is clearly protruding further than the other, which may be visible only to the fetlock. the nose of the foal is positioned near the midcannon bone area of the most advanced front leg. only 1 leg and the head advances with each uterine contraction; the second front leg does not advance because the elbow is ‘locked against’ or ‘caught on’ the pelvic brim. when ‘elbow lock’ is recognized, the foaling attendant should immediately intervene. traction is applied to the retained limb when the mare relaxes between uterine contractions. a ‘pop’ is clinical theriogenology • volume 12 number 3 • september 2020350 often felt when the retained elbow is freed from the pelvis. the foal is usually delivered without further assistance with subsequent contractions. posterior presentation foals in a posterior presentation may be impeded from a smooth delivery if the tail-head becomes wedged on the dorsal pelvis of the mare. in addition, there is a possibility that the umbilical cord may be compressed on the ventral pelvis of the mare, potentially reducing blood flow and decreasing oxygen transport to the fetus. in contrast to an anterior presented foal, there is no possibility that a foal in a posterior presentation could breathe on its own if progress halts during delivery. for all of the above reasons, it is generally recommended that a posterior presented fetus be delivered as quickly and efficiently as possible, with assistance provided as needed. feto-pelvic disproportion feto-pelvic disproportion or ‘hip-lock’ is much less common in horses than cattle. however, it is a significant event when it does occur. normally the ‘test for delivery’ in a foal in anterior presentation is passage of the second shoulder through the pelvis. in a majority of equine deliveries, if the shoulders fit through, the hips will pass uneventfully. unfortunately, a mild to severe form of ‘hip-lock‘ occurs in ~ 0.5 1.0% of foalings.7 a recommended strategy for cases of ‘hip-lock’ is to infuse a generous volume of obstetrical lubricant around the fetus and into the uterus followed by rotation of the fetus ~ 30 45° to take advantage of a slightly wider internal pelvic diameter at that position and then apply traction. general anesthesia may be necessary if the mare becomes intolerant of the procedures. developmental abnormalities such as contracted tendons, wry neck or wry nose, a twisted neck or other conditions may prevent or at least delay correction of fetal posture. controlled vaginal delivery if significant progress is not made within 15 20 minutes with the mare awake, the mare may be placed under general anesthesia and a controlled vaginal delivery attempted. general anesthesia is used to facilitate safe evaluation of the mare and to reposition the fetus into the correct orientation to allow for a controlled vaginal delivery. induction of anesthesia is advantageous to eliminate uterine contractions and straining by the mare. however, lack of contractions means that the fetus must be delivered by traction applied from the outside. in the field, general anesthesia can be accomplished by premedication with xylazine (1.1 mg/kg, iv) with or without addition of butorphanol tartrate (0.01 mg/kg, iv), followed by induction with ketamine (2.2 mg/kg, iv), with or without addition of diazepam or midazolam (0.05 0.1 mg/kg, iv). additional doses of xylazine/ketamine may be administered as needed to maintain general anesthesia while the dystocia is being resolved. elevation of the hindquarters of an anesthetized mare may be beneficial to increase space in the caudal abdominal cavity and therefore make it easier to reposition fetal body parts that are not aligned properly in the birth canal. liberal application of obstetrical lubricants along all sides of the fetus and within the uterine cavity will greatly enhance repositioning and subsequent extraction of the fetus. in summary, a controlled vaginal delivery with the mare under general anesthesia, if successful, obviates the need for more invasive and expensive procedures such as a cesarean surgery or a fetotomy. the mare is allowed to recover and is reunited with the foal (if alive) when appropriate. cesarean surgery if significant progress toward an assisted or controlled vaginal delivery is not forthcoming, a cesarean surgery or other procedures may be indicated. in the horse, a cesarean surgery is almost exclusively performed in a designated operating room at a veterinary hospital or other referral center.11,12 unlike the situation in cows, cesareans are almost never performed in the field or other open areas. a brief description of the cesarean surgery is as follows:  the mare is anesthetized and placed in dorsal recumbency clinical theriogenology • volume 12 number 3 • september 2020 351  the ventral abdomen is clipped, scrubbed and draped in preparation for surgery  an incision is made along the ventral midline through which the uterus is subsequently exteriorized  an incision is made into the uterus and the foal is delivered and attended to by medical staff  hemorrhage from the uterine incision is controlled using a synthetic absorbable suture in a simple continuous pattern followed by an inverting cushing pattern  the linea alba is closed in a simple continuous pattern; the subcutaneous tissue is closed using synthetic absorbable suture; the skin is subsequently closed with sutures or surgical staples  the mare is subsequently moved to a recovery stall terminal cesarean surgery in exceedingly rare emergency circumstances, a pregnant mare may be placed under general anesthesia, a cesarean surgery performed and the foal delivered, and the mare subsequently euthanized while still under anesthesia. situations may include severe musculoskeletal issues, ruptured prepubic tendon or other conditions. it is recommended that the breeding history and prospective due date be reviewed and milk samples be collected and calcium concentration measured to evaluate fetal maturation. if time permits, dexamethasone (i.e. 100 mg, as an intramuscular injection for a 450 kg mare) may be administered once daily for up to 3 days to promote fetal maturation prior to the cesarean surgery. fetotomy the term fetotomy refers to the surgical dissection of a dead fetus in utero.13,14 the procedure is performed in the event of a dystocia that cannot be resolved by standard assisted or controlled vaginal delivery techniques and when a cesarean surgery is not an option. the goal is to save the life of the mare and protect her future reproductive potential. specialized obstetrical instruments are used to perform a fetotomy, the most common of which is a fetotome and obstetrical wire. the wire is passed around a retained fetal body part and is used to dissociate that part from the rest of the fetus to allow for delivery of the fetus. examples of when a fetotomy might be performed on a dead fetus include:  breech presentation in which a fetus is in posterior presentation with both hind legs flexed at the hip  flexed neck that cannot be extended into a normal position  front leg(s) retained/flexed at the shoulder and cannot be extended foal care following a dystocia resuscitation equipment should be available on farms that foal out a lot of mares and farm personnel should be trained in the proper care and use of the equipment. a resuscitation bag (i.e. ambu bag) attached to a face mask is a simple-to-use device that can be safely applied to foals by farm personnel to help stimulate breathing in a newborn foal. alternatively, a device called the ‘foal resuscitator’ may be used. special immediate attention must be given to the foal following a dystocia. the foal is at high risk of many neonatal diseases including neonatal maladjustment syndrome, failure of passive transfer, ruptured bladder and trauma, e.g. rib fractures. mare care following a dystocia the mare should also be examined in due course following resolution of a dystocia. it is common for the placenta to be retained following a dystocia and preventative treatments are often instituted after a dystocia has been relieved. finally, the reproductive tract of the mare (i.e. perineum, vagina, cervix and uterus) should be carefully examined for trauma that may lead to more severe medical issues and/or limit her future reproductive performance. clinical theriogenology • volume 12 number 3 • september 2020352 conclusion dystocia is a medical emergency and time is critical to optimize foal survival. the key factors in foal survival are early recognition of a foaling difficulty by breeding farm personnel and early appropriate intervention. conflict of interest there are no conflicts of interest to declare. references 1. robert sj: veterinary obstetrics and genital diseases (theriogenology). north pomfret, vermont; david and charles: 1986. p. 277-286. 2. rossdale pd, ricketts sw: the practice of equine stud medicine. baltimore; williams and wilkins: 1974. p. 98-146. 3. frazer g: dystocia management. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 2479-2496. 4. mccue pm, ferris ra: parturition, dystocia and foal survival – a retrospective study of 1,047 births. equine vet j 2012;44(supplement 41):22-25. 5. norton jl, dallap bl, johnston jk, et al: retrospective study of dystocia in mares at a referral hospital. equine vet j 2007;39:37-41. 6. lu kg, barr bs, embertson r, et al: dystocia a true equine emergency. clin tech equine pract 2006;5:145-153. 7. byron cr, embertson rm, wv bernard wv, et al: dystocia in a referral hospital setting: approach and results. equine vet j 2002;35:82-85. 8. ginther oj, williams d: on-the-farm incidence and nature of equine dystocias. j equine vet sci 1996;6:159-164. 9. frazer gs, perkins nr, embertson rm: normal parturition and evaluation of the mare in dystocia. equine vet educ 1999;11:41-46. 10. frazer g, beard wl, abrahamsen e, et al: systemic effects of peritoneal instillation of a polyethylene polymer based obstetrical lubricant in horses. proc annu conf soc theriogenol 2004; p. 93-97. 11. embertson rm, bernard wv, hance sr, et al: hospital approach to dystocia in the mare. proc annu conv am assoc equine pract 1995; p. 13-14. 12. freeman de, hungerford ll, schaeffer d, et al: caesarean section and other methods for assisted delivery: comparison of effects on mare mortality and complications. equine vet j 1999;31:203-207. 13. frazer gs: review of the use of fetotomy to resolve dystocia in the mare. proc annu conv am assoc equine pract 1997; p. 262-268. 14. carluccio a, contri a, tosi u, et al: survival rate and short-term fertility rate associated with the use of fetotomy for resolution of dystocia in mares: 72 cases (1991–2005). j am vet med a 2007;230:1502-1505. clinical theriogenology • volume 12 number 3 • september 2020 353 025_ms-025 mccue 2016: theriogenologist of the year theriogenologist of the year richard hopper department of pathobiology and population medicine, college of veterinary medicine, mississippi state university, mississippi state, ms i appreciate our clinical theriogenology editor, dr. robert youngquist, for allowing me to provide the text of my acceptance of the theriogenologist of the year award. this format enables me to expand on my original comments in a couple of important ways. i can thank specifically those that nominated me or supplied supporting information for the selection committee. it also allows me the opportunity to edit out mistakes or embarrassing faux paus. thus the following is not an exact transcript of my talk, but instead reflects perhaps a better version of the feelings i wished to convey. first of all i would like to thank those that placed my name in nomination for this (drs. augustine peter, dwight wolfe, david hardin, mike thompson, and peter ryan), the awards committee, the act board and especially universal ultrasound for their sponsorship of the award. despite being someone who believes that he doesn’t necessarily need a lot of external validation, i am nevertheless very honored by this and i cannot tell you how encouraging this recognition is to me coming now, at the midpoint juncture of my career. seriously, to best describe my feelings, specifically those experienced upon receiving the call from dr. kasimanickam, i have to use a term from my christian faith-the simple word“grace”. of course it has supreme significance to me in that context, but quite simply in a general contextgrace is defined as “unmerited favor” or “an undeserved reward”. now, following notification from ram, i was contacted by dr. franz with some specifics–which include this address–time limit 20 minutes, topic to be of my choosing–a biographical sketch or a summary of my achievements or an overview of my research; perhaps impart some wisdom. i quickly realized i could do all of this and still have time to spare. born in paducah, ky; our family lived in a suburb outside of chicago for a few years. my dad despite a promising business career held a strong desire that he rear his boys in a rural setting, preferably in the south, so during my third grade year we moved south to brandon, ms. i had a horse, bird dogs and at different times dabbled in cattle, hog raising ventures. i also worked for our local veterinarian–clyde taylor. i attended mississippi state for my undergraduate requirements and then auburn for veterinary school. while i am now employed at mississippi state and both hold special places in my heart i will forever be held by the sway of auburn, both as a place and as a time in my life and this is an appropriate spot to acknowledge my professors at auburn to whom i owe so much – doctors donald walker and robert hudson, john winkler, harold kjar, an instructor who had returned from practice for a phd, howard jones, ram purohit and a young newly minted faculty member named bob carson who arrived as i was starting my beef theriogenology rotation. all of these guys were great veterinarians and teachers, but dr. hudson was my favorite. in fact, i so wanted to emulate bob hudson, that i have often thanked the good lord that dr. hudson was not a chicken veterinarian or a pathologist. now during a quarter break in the fall of my senior year, i headed off with a classmate, james fullerton to visit clinics where we might arrange our preceptorships and perhaps future employment. my focus was central tennessee and mixed practices that were predominantly large animal. we also visited practices in north alabama as we headed back to auburn and when we stopped in cleveland, al to say hello to steve stewart, who had finished one year ahead, he suggested that instead of heading back to auburn through birmingham we detour and go to oneonta and visit the practice there. now there are four ways in which you can enter oneonta, al-hwy 75 east or west or hwy 231 from the south or northfrom the vantage of the first three it appears to be much like any other small town, but coming in from the north, as i did-coming off a hill (i later found out was called shuff mountain) looking down on this beautiful hamlet-like town of oneonta is a memory forever etched in my mind. the county, technically in the foothills of appalachia, still has several covered bridges. we stopped by the animal hospital of oneonta, owned by ed murray and the late charles payton, they offered me a job and i worked for them for the next four years. they were both great veterinarians and mentors. the little town of oneonta, alabama was also the setting for my all-time best achievement. several years ago i first heard the term clinical theriogenology • volume 8, number 4 • december 2016431 “trophy wife”–i learned that it refers to a beautiful, accomplished, intelligent mate–i guess you could say the whole package. anyway i can proudly say that i procured a trophy wife on the first try. i’d like to introduce her and also share the obligatory family pics (childrenmolly, mcrae, tricia & her husband caleb butts. the next teo slides are the grand kidsabigail, joshua, & ella). now my practice time was doing what i loved, a mixed “country” practice. i had some wonderful clients and, of course, some “not so wonderful” ones as well; but through the rear view mirror and with the passing of time; it was a great experience. bulls with seven-day-old preputial wounds, 23 year old mares with cervices that it seemed you could walk through, and cows with near circumferential uterine tears were all frustrating cases, but great teachers. i guess my best and coincidently my worst trait seems to have always been a ridiculous and common sense over-riding form of optimism when it came to the intersection of a tough case and my perception of my ability. on the financial side of things, my business plan of achieving a professional income was dependent on working 75-80 hr weeks-which meant 100+/week from marchjune. we (donna more than me) were active in church and community; although a lot of those things and more importantly family time, things like vacations or attending my children’s activities; seemed always erratic. so, after almost 15 years of practice, when i received a call inviting me to visit mississippi state about an extension position, it seemed like a good idea. working only 45-50 hours a week at mississippi state, in addition to allowing more family time, also allowed me the time to prepare to sit our boards through the alternate route. dr. fred lehman was my mentor and in addition to him, i need to thank drs. tom bailey, bruce clark, chuck estill, and al rathwell for their help and advice; advice which many of you of my vintage heard“read roberts, read ginther’s books, read all of the sft proceedings, morrow’s current therapy is a good overviewbe sure and read it”. also, during this time my friend herris maxwell introduced me to gary greene, who was also preparing to take the boards. we exchanged notes, etc and i am appreciative of the help he provided as a “study partner”. now during the fall of 1994, a few things happened that impacted the trajectory of my career. filling in for someone, on an equine rotation teaching laboratory, a student mentioned that it looked like they would graduate without learning to palpate a mare. this spurred me to action and with rathwell and estill, we organized an elective, which we actually ran with the help of dr. doss brodnax, a retired ag econ professor that had 28 mares that he was breeding to a jack. i could now spend 3-4 hours regaling the sort of mayhem that ensued on a daily basis associated with this activity and i also could explain why i have a complete understanding as to how the name “jackass” could come to have a derogatory meaning, but needless to say we began producing some veterinarians with good reproduction skills, some of whom are now in our group. now in my role as an extension veterinarian, i was still speaking at 30-40 cattlemen’s meetings/yr and several county agents contacted me to start doing meeting for their horse owners. i hit the road on that and spoke primarily on reproduction topics, which in retrospect amounted to a series of statewide infomercials. to summarize the next two years-we saw an equine theriogenology caseload that went from 100 to 900/year and i moved down to a clinical teaching position. we started a residency program and something else that had a significant impact on my career occurred, the arrival of peter ryan. we began collaborating on mare projects; fescue toxicity, placentitis, and fetal maturation. we not only incorporated the research into the residency/graduate program, but also involved the students that were taking the equine reproduction elective. the first residents, i personally recruited from practice and they were jack smith and david christiansen, followed by kevin walters and joy mordecai. all were mississippi state graduates with jack, david, and kevin later joining our faculty. two more mississippi state gradutess, heath king and craig easley stayed on for internships and residencies; with heath also joining our faculty. other residents coming through our program were renee jaklitsch, rana dutta, and kathryn bass. most of these guys had heavy equine interests, which allowed me to return to an emphasis on beef cattle. that and with peter’s ascendancy into the ranks of administration (dr. ryan is now an associate provost), have resulted in a different direction in my research focus, as well; bull fertility and fetal programing projects with dvm-phd graduate student-amanda cain. which brings me to the present. clinical theriogenology • volume 8, number 4 • december 2016 432 so, the theriogenologist of the year award, based on the description from our website, informs one that it recognizes recent activity (not lifetime but instead from the last five years), so i suspect the basis for this honor would be service on the society for theriogenology and therio foundation boards, and also the publication of bovine reproduction. well i would like to discuss the book and how that transpired some, first because i am very proud of it and because i believe the process provided me some insight on a few things. the aspect i am most proud of and inversely so humbled by, was the support i received. several of the contributors, for example maarten drost, al barth, dwight wolfe, and several others had either retired or were near retirement and had no likely personal (career related) motivation to participate in a project such as this, other than the completely altruistic desire to be of assistance to me as well as gifting our profession with their insight, experience, and knowledge. many of the people in this room, all very busy, took time to help create the book. thank you so very much. now, what would i like to share? what did i learn? would i undertake something like this again? was it more involved, more effort than i thought? well, i’ll begin with a quoteone that i will ascribe to myself: “there is a certain virtue to be found in fool-hardiness. there are so many things that would go undone if a rational assessment of the real cost & effort necessary to complete the task was done beforehand.” i’ll let you decide if that’s wisdom or a rationalization. either way this train of thought was perhaps better and more succinctly stated by my favorite author-malcolm gladwell, columnist for the new yorker, in a question “is ignorance an impediment to progress or a precondition for it?” so yes, while i routinely questioned the wisdom of embarking on the “i’ll edit a text”, and most often during the hours of 10:00 pm and 3:00am, i’m now glad that i didn’t let reason/wisdom/common sense or a realistic appraisal of the necessary work; stand in the way of this effort. so, insight gained from this, would not necessarily be “leap before you look”, but sometimes you need to bet on yourself and take a chance. an experience many of us have endured as a veterinarian that helps with a project like this is that late spring, summer fetotomy. the foal, dead of course, and you are thinking, after two to three hours of drudgery, arms/shoulders cramping or numb, that it looks likely the mare might not make it either and then you think–“i’m not giving up, i’m not going to quit”. well, i had that feeling sometimes with the book. so, just plain “not giving up” is a habit that comes in handy sometimes. with respect to my next advice i will relate some thoughts on our veterinary school at mississippi state in 1993 when i arrived. faculty turnover, reoccurring budget issues, and even after being in existence 15+ years there were still legislative threats of closure. not the place to start a career, build a program? right? well with every disadvantage there are usually pluses. for one thing, the turnover of faculty, allowed me to make a transfer from an extension position down to the clinics. a transition like that after three to four years if possible, would in most cases be a death knell for promotion and tenure. but, that was taken under consideration when i went up. i doubt my academic record at the time would have garnered me tenure at most any other college of veterinary medicine. also, i could focus on students and our referring dvm/client service and building a program. if you, and this is to the young people in the group: students, residents, junior faculty, or first years of practice; if you are looking for the perfect situation, perfect job; you’re going to get discouraged and frustrated. make your job what you want it to be. create or transform things. my advice quite simply then-“bloom where you’re-planted”. my final bit of advice, and i’ll close on this, comes from my favorite movie quote. it’s from the old black and white jimmy stewart film harvey. a woman was remarking to jimmy’s character about what a likable, kind person he was, to which stewart’s character replied “my mother once told me that to get on in this world, one had to either be very smart, very clever, or to be very nice. well, for many, many years, most of my life, i’ve tried to be clever. i recommend nice”. i again want to extend a sincere thanks to all of you who played a role in my receiving this award, for attending this ceremony, and universal ultrasound, specifically mr. peter brunelli, for his sponsorship of the award. clinical theriogenology • volume 8, number 4 • december 2016433 clinical theriogenology • volume 8, number 4 • december 2016 434 omniblank: fetal and maternal immune response to ascending placentitis fetal and maternal immune response to ascending placentitis carleigh fedorka, barry ball, kirsten scoggin, shavahn loux, mats troedsson, amanda adams department of veterinary science, university of kentucky, lexington, ky ascending placentitis is a leading cause of abortion in mares, resulting in considerable economic wastage. substantial work has been done on disease pathology and diagnostics, but minimal on the role of the fetal immune system in utero and its effect on fetal fluids. therefore, objectives were to assess cytokine protein synthesis in fetal fluids and circulation after induction of disease and to determine fetal immune system responses to this reproductive tract inflammation. we hypothesized that induction of ascending placentitis lead to a significant increase in inflammatory markers in fetal fluid compartments and associated tissues. ascending placentitis was induced via transcervical inoculation of streptococcus equi subspecies zooepidemicus between 275 285 days of pregnancy, with tissues/fluids retrieved at euthanasia 4 6 days after inoculation. cytokine protein concentrations were detected using luminex xmap® technology within fetal fluids (amniotic and allantoic) and serum (maternal and fetal) with experimentally induced placentitis (n = 5) and controls (n = 5). in addition, tissues from fetal (spleen, lung, liver and umbilicus) and maternal (spleen, lung, liver, amnion, chorioallantois and endometrium) origin were analyzed with qpcr for mrna expression of inflammatory markers. analysis was performed using sas® 9.4 utilizing an unequal variances t test to evaluate comparisons between treatments for cytokine concentrations. data on qpcr were analyzed utilizing a mixed general linear model, with treatment as a fixed effect. of the 23 cytokines analyzed, only il1ß, il6, il8, il10, ip10, and gro consistently exceeded limit of detection within fetal fluids. of the 4 fluids studied, there was only an effect of inoculation on amniotic fluid, with increases in il1ß, il6, il10, and gro (p < 0.05) in the inoculated group. these cytokines were then investigated in reproductive and immune system tissues. in reproductive tissues, there were increased expressions of il1ß and il8 in endometrium and chorioallantois in addition to an increase in il1ß and il6 in amnion of inoculated mares. in tissues associated with the immune system, il1ß was upregulated in the maternal spleen, whereas fetal spleens had increased expression of il1ß, gro, and il6 after inoculation. overall, fetal spleens had higher mrna expression of the anti-inflammatory cytokine il10 in comparison to both inoculated and control mares, whereas maternal spleen had heightened expression of il1ß. there was an increase in expression of il6 in fetal liver after induction of placentitis but no effect in maternal liver. no change was noted in the fetal lung. we inferred that inflammation caused by placentitis was fairly localized to the amniotic fluid, with minimal effect on the allantoic fluid or serum of inoculated animals. although the maternal response to placentitis was proinflammatory, the fetus appeared to have a regulatory role in this inflammation. in addition, increases in amniotic il6 and il10 were intriguing; these are currently diagnostic predictors for microbial invasion of the amniotic cavity in humans and indicate that amniotic fluid sampling may be more predicative of placentitis than serum or allantoic biomarkers. keywords: placentitis, cytokine, fetal fluid, inflammation, biomarker 433 clinical theriogenology • volume 11, number 3 • september 2019 434clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2016: reproductive evaluations and clinical interventions in beef herds reproductive evaluations and clinical interventions in beef herds ramanathan k. kasimanickam,a vanmathy r. kasimanickam,a john p. kastelic,b william d. whittierc aveterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa; bproduction animal health, faculty of veterinary medicine, university of calgary,calgary, ab, canada; clarge animal clinical sciences, va-md regional college of veterinary medicine,virginia tech, blacksburg, va abstract cow-calf and purebred beef producers need to operate at high efficiency to have profitable, sustainable operations. although beef cattle veterinarians are ideally positioned to support this, producers are often initially reluctant to purchase the broad range of services that can be provided. therefore, practitioners must demonstrate a sound knowledge of principal factors affecting beef cow herd productivity and their ability to translate that knowledge into effective herd investigative strategies and planned intervention programs. the goal of this review is to provide guidance for practitioners wishing to embrace this opportunity. introduction the single most important parameter in measuring production efficiency of a beef cow herd is maximum output of marketable pounds of beef produced per cow exposed in a year. consequently, there is a clear need for effective reproductive management to achieve production potential. reproductive herd health programs should focus primarily on: control of infectious and noninfectious diseases of the reproductive tract; bull fertility or synchronization and detection of estrus (or synchronization of ovulation), or breeding technique, semen quality and handling; and endocrine imbalances. given the importance of dietary energy and its effects on reproduction, fertility examinations should include nutrition monitoring and body condition scoring (bcs). although bovine veterinarians have critical roles in reproductive management of dairy operations, making them well positioned to assume the same role in beef herds, few beef herd farmers actively seek veterinary input regarding this aspect of herd management.1 one of the main reasons is that few farms have data in a readily useable format (e.g., excel, cowcalf5). in that regard, up to 17.7% of beef cow-calf operations had no records, whereas 78.6% kept records by hand.1 consequently, this is an opportunity for beef cattle practitioners to offer record keeping services to beef producers (on a fee-for-service basis). although data essential to basic fertility analysis and benchmarking are not complex (table 1), without recording and analyzing these data, it is difficult to identify deficits and recommend changes to improve performance. given the low input and high output nature of beef production, there is a great need for beef farmers to closely examine their production system, with the objective of optimizing reproductive efficiency. key skills for a practitioner to provide appropriate service is the ability to demonstrate an understanding of reproduction in the beef herd, investigate herd-level reproductive performance, establish goals, and recommend changes needed to achieve those goals. therefore, a thorough understanding of factors that determine reproductive performance in beef herds is required, along with an effective investigative strategy. determinants of reproductive performance the principal measure of output for a beef cow herd is weight of calf weaned per cow exposed or breeding. at the most basic level, this is determined by number of cows that conceived and the rate at which that happens. for an individual cow to achieve maximum reproductive efficiency, a calf must be produced every 12 months. the gestation length for commonly used terminal sire breeds is approximately 285 days, leaving only 80 days for uterine involution, resumption of ovarian cyclicity and conception. this restricts the breeding season to only nine weeks. however, extending the breeding season can conceal both poor conception rates and prolonged anestrus. even though this increases number of calves born, they are younger and therefore lighter weight at weaning or sale. clinical theriogenology • volume 8, number 4 • december 2016407 four weeks postcalving is widely accepted as the average interval from calving to complete uterine involution in suckled beef cows.2 however, resumption of ovarian cyclicity in beef cattle is more complex. suckling has an inhibitory effect on the hypothalamic-pituitary axis and prolongs postpartum anestrus compared to milked cows. postpartum anestrus in suckled beef cows (29 to 67 days) is longer than in milked dairy cows (15 to 21 days).3-5 duration of postpartum anestrus after calving influences cows’ opportunities to get re-bred.6 cows 50 days after calving and in moderate body condition had an increase from 45% cycling with no treatment to 79% cycling with treatment (calf removal coupled with nutritional flushing). however, anestrus is prolonged if there is a combination of low body condition and inadequate energy intake. although suckling prolongs postpartum anestrus, removing calves from cows for 48 h at the beginning of the breeding season, coupled with a nutritional flushing program, significantly improved pregnancy rates. calves experienced almost no ill effects when given access to feed and water during the 48-hour interval. it should be noted that even young cows, when given the opportunity, will achieve calving intervals < 365 days and repeat this for the next breeding season. consequently, despite obvious constraints, the physiology of beef cows is not an inherent barrier to achieving the requisite level of reproductive performance in a nine-week breeding season. in a 63-day breeding season with a 70% conception rate, a 98% pregnancy rate could theoretically be achieved if all cows were cycling at the onset of the season. it should be noted that under us management, only 30% of mature cows are cycling at 40 days postpartum, although up to 95% are cycling by 90 days postpartum.6 cows with a good bcs that calve in the first month of the calving season are highly likely to be cycling by the onset of bull exposure and thus have three or more chances of having a fertile estrus during the breeding period. in contrast, thin cows, even if they calve in first four weeks of the calving season, are unlikely to start cycling until after they are exposed to bulls. furthermore, thin cows that calve late in the calving period may only start cycling in the last few weeks of the mating period and are at increased risk of failing to conceive. however, if the average cow does not begin cycling until 45 days into the breeding season, a maximum pregnancy rate of only 81% would be expected. clearly, early calving is especially important, as it influences both resumption of ovarian cyclicity and probability of conception. key management areas for a beef herd to reach its reproductive potential, critical issues include: management of heifers nutritional management of cows prevention of dystocia management and assessment of bull fertility management of heifers heifers that have not reached mature bodyweight must be fed for growth as well as maintenance and pregnancy. the target breeding weight is 65% of mature weight at a body condition score of 5. these cattle should reach 85% of their mature weight at the start of the following breeding season. recent research suggests that development of beef heifers to approximately 55-60% of mature body weight at breeding may provide economic benefit in comparison to the previously recommended 65% of mature weight, if post-breeding nutrition is good.7-9 although, there is new evidence that 55% mature weight is adequate, it should be adopted with the caution. even though it is attractive from the perspective of reduced feed and costs, the following issues have not been addressed: economics of feeding pregnant heifers to gain from 65 to 85% versus 55 to 85% consequence of fetal programming in adult life for offspring born to heifers with 65 versus 55% bw at breeding lifetime productivity differences between heifers at 65 and 55% mature bw at breeding. feeding to gain mature weight can be difficult to achieve, as heifers and two-year old females that have calved are very susceptible to poor-quality rations and competition if rations are restricted. on clinical theriogenology • volume 8, number 4 • december 2016 408 average, the first estrus after the first calving is 20 days later than in mature cows. after the second calving, effects are similar, but the delay is less pronounced. one way to manage this is to breed heifers 30 days before cows. it should be noted that if replacement heifers are raised on farm, insufficient numbers may have reached target weight at that time. in order to overcome this, it was suggested to expose 20% more heifers to bulls and conduct pregnancy diagnosis soon enough to accurately stage pregnancies. alternatively, synchronization of estrus or ovulation and use of artificial insemination (ai) in heifers 30 days prior to the start of the cow’s breeding season is recommended. with a well-managed synchronization program, >50% should become pregnant. consequently, they will calve early and have a longer interval to recover. a good program should employ heifer selection based on body weight, reproductive tract development and pelvic area.10 producing replacement heifers within a commercial beef cow herd cannot be achieved in all situations. unfortunately, there is a conflict between growth characteristics of the terminal generation and maternal characteristics necessary for optimal heifer replacements. to prevent genetics of the cow herd from becoming progressively more dominated by those of the terminal sire breeds, a crisscross mating system has been recommended.11,12 the essential element is that bulls from two breeds with complementary characteristics are used. female offspring from one breed are then mated to bulls of the other breed. consequently, such herds have groups of cows that must be managed separately at breeding, which can be difficult in herds with <150 cows. consequently, smaller herds may have to purchase breeding replacements and forego biosecurity advantages offered by a closed herd. when purchasing replacement heifers, it is important to ensure that they are purchased at an optimal time and bodyweight that will allow them to reach the target bodyweight of 55 to 65% of mature weight at the onset of the mating season. irrespective of which heifer program is followed, it is vital that both virgin and first-calf heifers receive preferential nutritional management to ensure they continue to grow and achieve the target body condition score at calving. older cows in poor body condition can be included in this group for feeding. nutritional management of cows feed accounts for the largest percentage of input costs in both cow-calf and stocker operations.13 nutritional needs of beef cattle differ by age, weight, production stage and performance. physiological and environmental stressors may also impact nutritional requirements. it is imperative to understand beef cattle nutrient requirements for designing an effective and efficient grazing and supplementation program. comprehensive beef cattle nutrient requirements and ration formulation are available.14-17 a cow requires energy for eating, moving, fetal development, milk production, temperature maintenance, reproduction, etc. in addition, firstand second-calf cows require additional energy for growth until they mature. factors that influence energy requirements are: body weight, rate of gain, lactation, and fetal development. lactation represents the greatest need for additional energy beyond that needed for maintenance. on average, a lactating beef cow requires nearly 50% more net energy than she does when nonlactating. first-calf heifers represent a special challenge to maintain successful reproductive performance. their postpartum energy requirements exceed those of mature cows, because energy is needed for growth, in addition to body maintenance and lactation. inadequate energy during the last third of gestation and from calving to rebreeding can prolong anestrus (on average, they take longer than mature cows to resume cyclicity). in addition to demands that all beef cows have, these heifers are continuing to grow, but they have less body capacity and therefore consume less energy when fed high-roughage rations. consequently, without special management considerations, an excessive number of these heifers are likely to be culled for reproductive inefficiency when they fail to conceive during a controlled breeding season. body size affects energy requirements; as expected, large cows require more energy than small cows. for example, a 1,300 lb nonlactating pregnant cow in the middle third of pregnancy requires 32% more net energy than a 900 lb cow at the same stage of production. clinical theriogenology • volume 8, number 4 • december 2016409 a net energy deficit delays return to ovarian cyclicity; this can be either due to a failure to match nutritional input to production requirements and/or poor quality diet. for beef cows, the critical measure of nutritional adequacy is body condition score at calving. a system of scoring cows for body condition based on examination of the spine, short ribs and appearance of the tail head area has been developed (table 2). cows calving below target body condition score for the production system often have a delayed return to cyclicity, which is difficult to overcome with dietary manipulation immediately before or after calving or during the breeding season. in the us, the target condition score for beef cows at calving is 5, on a scale of 1 (emaciated) to 9 (obese). at breeding, they should be in moderate to good condition (5 to 7 bcs). influence of body condition on estrus expression, and ai and breeding season pregnancy rates are shown (figure 1). prevention of dystocia high rates of dystocia will delay conception and an increase percentage of cows not pregnant at the end of a restricted breeding season. dystocia also has an adverse effect on calf survival. the benchmark for dystocia in a beef farm is ≤ 3%; therefore, reducing the incidence of this condition is vital. rates of dystocia are highest in beef heifers and thereafter decline with age, indicating the importance of fetal-maternal disproportion.18,19 a heifer selection program based on bodyweight and pelvimetry (pelvic area measurement) will be beneficial, but is not a complete solution.20 paternal influence on dystocia can be managed by careful bull selection based on expected progeny difference (epd). the epd is a prediction of how future progeny of each animal are expected to perform relative to progeny of other animals in the database, expressed in units of measure for a trait (plus or minus). for example, to reduce dystocia, bulls with a negative epd for gestation length and a positive epd for calving ease should be selected. gestation length epd predicts difference in gestation length (in days) for progeny of a bull. bulls with lower gestation length epds are expected to sire calves that are born earlier. in that regard, shorter gestation lengths are associated with slight decreases in birth weights and improvements in calving ease. breeds that report epds for both calving ease and gestation length generally include effects of gestation length in calving ease epd. calving ease epd is reported as deviations in percentage of unassisted births. in the above example, if bulls a (0) and b (+5) were mated to the same set of heifers, we would expect heifers bred to bull b to have 5% more unassisted births. in other words, we would expect fewer calving problems when bull b was mated to heifers. by using ai in the heifer program, more extreme bulls can be selected for the greatest effect. this principle should also be applied, albeit to a lesser degree, when selecting bulls for cows. it should be noted that selection of sires should focus on other economically important traits other than calving ease in order to maintain a balance. management and assessment of bull fertility the minimum standard used to define a bull as fertile is the one that is expected to get 90% of 50 normal cycling disease-free females pregnant within nine weeks and 60% of these should be pregnant within the first three weeks after the onset of bull exposure. the emphasis is on the minimum standard and many bulls achieve the same or a better result with more cows. this is in contrast to the longstanding advice that one bull should be mated with 25 cows.21 the reason for this difference in approach can be related to the often-quoted figure that one in five bulls is subfertile.22 therefore, by recommending a group size well below the potential of a fertile bull, a subfertile bull has more chance of being accommodated. however, with subfertility being so prevalent, it is likely that at least several bulls and, in some cases, most or all bulls will be subfertile in many bull batteries. rotation of bulls every three weeks between breeding groups is commonly practiced, but further obscures effects of subfertile bulls on herd productivity. however, this inefficiency can be reduced by conducting breeding soundness examinations on the bull battery each year prior to the breeding season, removing infertile bulls and perhaps exposing subfertile bulls to fewer cows. semen evaluation is a critical part of the breeding soundness examination. however, facilities on most farms are not suitable for using an artificial vagina to collect semen. fortunately, electroejaculation has been refined and is now well tolerated by most bulls. semen evaluation using electroejaculation clinical theriogenology • volume 8, number 4 • december 2016 410 along with a clinical examination can usually be completed within eight to ten minutes per bull. although it is not commonly done, assessment of libido and service action on restrained, estrous cows, provides further information regarding breeding potential. consequently, it is suggested that bulls that have not been tested for libido and serving capacity should be restricted to 25 cows, whereas bulls that meet standards can be exposed to up to 50 cows.23 notwithstanding, even bulls that have met standards before the breeding season can become lame or have reduced fertility during the breeding season. observation of the breeding activity of bulls is therefore important. for herds with an established nineweek breeding season, >80% of cows should be bred in the first three weeks and no more than 40% in the second three weeks. observations not consistent with these standards should prompt re-evaluation of bull fertility and appropriate corrective action. indices of reproductive performance production indices are essential to properly assess reproductive performance in beef herds. the single most important denominator is the number of females that are bred; thereafter, the number of cows not pregnant at pregnancy diagnosis, abort, or produce a stillborn calf, or give birth to a live calf, can be compared to this denominator. analyses should be subdivided into separate groups for heifers, first calvers, and mature cows. thereafter, the second denominator is the number of breeding females that calve. again this also can be subdivided by parity. number of females that produce a stillborn calf, suffer from dystocia or have a calf that dies within 48 hours can be divide by this denominator. describing distribution of cows calving in relation to the breeding season is not as straightforward as it would seem. the start of the calving season starts, on average, 285 days after the bulls are exposed to cows (average gestation length for beef breeds). however, there is a natural variation in gestation length of ~10 days, even for cows mated to the same bull; therefore, all cows calving before the expected date are included in the first period. the second period commences 21 days later (which is the mean length of the estrous period) and the third 42 days later. this methodology inflates the number of cows estimated to be conceiving in the first three weeks, but providing it is consistently applied, meaningful data can still be generated. targets for reproductive performance the benchmark figure for reproductive performance in beef cow herds in north american production system is 95 cows pregnant per 100 cows mated. to achieve this, it is clear that every factor must be well managed. benchmark figures are outlined in table 3.24,25 investigative strategy investigating infertility in the beef herd can appear daunting. compared to dairy cattle, there is often no or poor records and, in most cases, the breeding season is over before the problem is recognized. a common response is to screen for infectious disease or assess trace element status (typically by collecting liver or blood samples in a subgroup of females and males) or evaluate breeding potential of bulls. however, an approach focused on these aspects is likely to yield little useful information, and may result in inappropriate advice. the preferred approach is to carry out a full herd reproductive evaluation in a systematic manner to minimize missing important factors. the key to resolving many fertility problems lies in details of the previous calving season; therefore, this should be the starting point for any investigation. basic information required for an investigation includes calving dates from the previous calving season and pregnancy diagnosis data from the previous breeding season (for each breeding group). most producers record this information. collection of data in an appropriate format should be delegated to the producer, but can be facilitated by providing appropriate tables. information required for each separate breeding group is: pregnancy rate per cow exposed (based on pregnancy data) length of current breeding season (i.e., one under investigation) length of previous calving season clinical theriogenology • volume 8, number 4 • december 2016411 length of previous breeding season (bull entry to bull exit) calving distribution (by date) of previous season number of calvings for each 3-week period of previous season number of assisted calvings in previous season age distribution of herd number of bulls used, their ages, and epd if available nonpregnant cow investigation the distribution of nonpregnant cows should be compared to that of pregnant cows on the basis of age, degree of dystocia (current and previous calving), body condition26,27 and temperament.28,29 a breed comparison should also be done to establish whether the herd is comprised of more than one breed of cows. this is readily achieved if tables are kept simple and distributions are expressed as a percentage. in excel, a pivot table may be used to explore this (https://www.youtube.com/watch?v=vx-fuw46vby). where doubt remains over the significance of any observed difference, a chi-square test (http://vassarstats.net/tab2x2.html) should be used. nonpregnant cows are frequently over-represented by the following: young or old cows that calved towards the end of the calving season cows assisted at calving cows with poor body condition cows with an excitable temperament a comprehensive program review should be performed when there are too many young cows in the nonpregnant group; information regarding their body weight, reproductive tract scores,30 pelvic measurements, and vaccination and breeding programs should be evaluated. however, if bovine virus diarrhea (bvd) or venereal diseases (trichomoniasis and/or campylobacteriosis) is endemic in a herd, heifers and first-calvers are also predominantly affected. trace mineral status of sub groups of females and males (liver or blood samples) should also be investigated.31 (it is suggested 10 to 15% of herd or animal populations should be sampled for screening. for accurate sample size calculations refer to reference32 and http://epitools.ausvet.com.au/content.php?page=surveytoolbox). excessive numbers of old cows indicate that the replacement strategy should be reviewed. over-representation of cows that calved late in the season points either to a failure to achieve the target body condition score at calving26,27 or to provide an adequate plane of nutrition after calving. if none of the above apply, then the following should be suspected: bull infertility, recent introduction of bvd or venereal diseases to a naive herd, or involvement of toxins. bull battery evaluation a bull battery evaluation is simply an assessment of the cow to bull ratio in the breeding groups by age of bulls. health and previous fertility record of bulls is also relevant. at this initial stage, a clinical examination of all breeding bulls should be done to ensure that they are not lame and have normal external genitalia. history of testing for and/or vaccination against venereal diseases should be included. biosecurity protecting cattle from infectious disease is generally cost-effective. biosecurity involves management practices that prevent diseases from infecting a herd. beef operation biosecurity centers around preventing introduction of disease into the operation and developing adequate herd immunity. a biosecurity inspection focuses on animals added to a herd (whether bulls are purchased, rented or shared) and whether animals are grazed on community pastures.24 farm boundaries are also important considerations, as heifers or bulls commonly break into or out of premises.27 on farms that use reproductive technologies such as ai and embryo transfer, sources of semen/embryos should also be recorded. records of vehicle and personnel entry should be maintained.33 clinical theriogenology • volume 8, number 4 • december 2016 412 for successful biosecurity measures, beef producers should be advised to: properly identify cattle and maintain accurate and complete records. monitor cattle for adverse health symptoms or behavior. test and remove animals that harbor certain diseases (e.g. johne’s, trichomoniasis or bvd). if these animals are not removed, they can shed these pathogens and infect other animals. minimize contact with wildlife that may harbor disease. disinfect/sterilize reusable equipment. develop a carcass disposal plan. minimize fecal and urine contamination of feed and water sources. control pest populations and limit access to feeds. create an emergency contact list of community resource people. health and nutrition status assessment a health and nutrition investigation covers vaccination history and any diagnostic testing during the previous two years. the ration in the past year should also be considered, particularly with regard to pattern of feeding and the adequacy of energy and protein provision. securing a good history of nutritional provision can be a major challenge. however, analysis of the ongoing nutrition in an operation can often elucidate routine management errors that negatively affect reproductive performance. deficiencies of energy and protein have major effects on reproductive outcomes.34 energy nutrition has been documented to influence beef cow pregnancy rates in two ways. long-term energy balance is the major determinant of body condition score which has been extensively researched and exerts a major influence on return to cyclicity postpartum.35 a report on the nutritional outcomes of medium sized (1,175 pounds) cows that were fed rations (hay) with three different nutritional characteristics showed different energy and protein balances during breeding times (60 and 90 days after calving).36 table 4 show cows fed rations with 50 % tdn and 7.9 % crude protein are in both negative energy and protein balance during this critical time; cows fed rations with 60 % tdn and 7.8 % crude protein rations are about neutral for energy and protein; cows fed rations with a 70 % tdn and 9.1 % crude protein ration are in positive protein and energy balances. energy and protein values of feeds, especially forages, can only be accurately known if feed analysis is conducted. there is much more variation in the nutrient values of forages than concentrates so using book values will result in large errors in estimating forage quality. correct procedures for feed sampling and storage prior to analysis are crucial. for hay sampling, bale corers and sampling a random group of bales must be used in order to get a true estimate of the nutritional value of forages. trace element supplementation should be examined in the light of the history of trace element problems in the herd or area. trace element supplementation is a complex subject and evaluation should include copper, zinc, manganese, iodine, cobalt, and selenium. furthermore, minerals interact with each other, antagonistically or synergistically (directly, or indirectly via other elements such as minerals or hormones) and such interactions should be anticipated during supplementation.37 assessment of reproductive technologies used in beef herds artificial insemination although ai is much more widely used in the dairy industry compared to the beef industry, its use in beef cattle has increased in recent years. historically, detection of estrus was done to determine when to inseminate. commercially available prostaglandins create a means to induce luteolysis, provided cattle are cycling and at the appropriate stage of the estrous cycle. a major challenge with prostaglandins alone for synchronization is that the interval from treatment to estrus/ovulation is variable.38-40 however, there are current protocols that control ovarian follicular development and luteal function, resulting in reasonably synchronous ovulation in a high percentage of cattle, with acceptable fertility (often 50 to clinical theriogenology • volume 8, number 4 • december 2016413 60%) to fixed-time insemination.41-43 with ongoing improvements in reliable characterization of genetic merit in beef cattle, ai facilitates introduction of specific genetics (e.g. calving ease sire for heifers, good maternal characteristics for generating future brood cows, and specific feedlot performance and carcass characteristics for production of feeder cattle). many current progesterone-based synchronization protocols for fixed-time ai will achieve a satisfactory pregnancy rate in pre-pubertal heifers and anestrus postpartum cows that are close to spontaneous ovulation. in addition to commercial frozen semen, cattle can be inseminated with fresh semen,44 including semen collected from one or more herd sires, or semen can be collected on-farm and frozen. in that regard, minimal specialized equipment is needed to successfully collect, extend and freeze bull semen and achieve good fertility. in case of reduced success of ai program, estrous and synchronization programs, semen handling and insemination techniques, body condition and temperament of cattle should be evaluated. as mentioned earlier, the investigation should also include screening for reproductive diseases and evaluating mineral status of the cattle. sexed semen the technology to produce gender-selected semen has improved dramatically, facilitating large shifts in gender ratios.45 gender-selected semen is much more widely used in the dairy industry, but gender-selected beef semen is becoming more widely available, and producers are starting to use this technology. the broadest use for gender-selected semen in cow-calf production is to produce maternal lines to be mated to terminal lines (using xand y-sorted sperm, respectively). furthermore, with artificial insemination and bull selection, a producer could create a marketing advantage by generating uniform market steers with specific characteristics. seed-stock applications include using y-sorted semen for bull production and x-sorted semen for replacement heifer production or enhancing female lines. currently, the only practical, proven method for producing gender-selected semen is flow cytometry to exploit the small difference in dna content between x and y chromosomes.46 despite substantial development and refinement, sperm sorting remains relatively slow and inefficient. therefore, insemination of a suboptimal number of ~2 million gender-selected sperm is commonly used as a compromise between cost and acceptable conception rate.47 in addition, the sorting procedure inflicts damage to sperm, especially to the dna.46,48,49 as a consequence of both a suboptimal dose and damage to sperm, conception rate with frozen gender-selected semen is typically 70 to 80% of that from conventional semen at the typical dose rate.45,46 furthermore, due to the uncompensable nature of sperm damage, the reduction in conception rate cannot be fully overcome by increasing insemination dose.50,51 unfortunately, reduced fertility with frozen gender-selected semen in seasonal production systems is directly at odds with cows needing to conceive within a short interval. improvements in fertility would make gender selected sperm more appealing. freezing and thawing damage sperm, with more profound effects on gender-selected sperm.52,53 in dairy cattle, the fertility of liquid (never frozen) gender-selected semen was >94% that of conventional semen, a two-fold efficiency gain relative to frozen, genderselected semen.54 seasonal beef production systems with short breeding periods would enable the benefits offered by liquid gender-selected semen to be efficiently exploited. however, it should be noted that success in terms of pregnancy and sex ratio varies depending upon breeding management (table 5). embryo technologies and application of genomics embryo transfer involves harvesting one or more embryos from a donor female and transferring them to recipient females. the primary reason most cattle producers utilize embryo transfer is to enhance genetic improvement in their herds. by collecting embryos from genetically elite females and transferring the harvested embryos into females of lesser genetic merit, it is possible to produce more calves from genetically superior females and fewer calves from genetically less valuable females. the result is an increase in the rate of genetic improvement. transfer of embryos harvested from genetically elite donor females also enables those genetically elite females to produce more calves in a single year than they would produce in their normal reproductive lifetime. currently, embryo recovery and transfer are done nonsurgically.55 improvements in protocols for superovulation of donors and synchronization of recipients have made this technology economical, and clinical theriogenology • volume 8, number 4 • december 2016 414 increased efficiency and availablity.56 furthermore, advances in embryo freezing have facilitated global distribution of frozen embryos and the ability to implant them under field conditions. one limitation of embryo transfer is that the interval between successive superovulation and embryo collections is a minimum of ~ four to eight weeks.56 however, with ultrasonographic imaging and transvaginal aspiration of ovarian follicles (ovum pick up; opu), oocytes can be retrieved every few days (in cycling, nonpregnant cattle, but even from pre-pubertal heifers and cows prior to mid-pregnancy) and fertilized in vitro, enabling production of large numbers of embryos in a short interval.57 this technology has great potential. a number of private companies and some embryo transfer firms have added this procedure to their list of commercially available services. the thrust for using these procedures in beef cattle include producing hundreds of offspring from a single genetically superior female, producing embryos from prepubertal or pregnant females, producing offspring from clinically infertile females, and ultimately obtaining more productive livestock at a lower cost. advances in genomics have led to identification of genes or markers associated with genes that influence traits. in association with classical genetic selection, genomics can be used to promote genetic improvement in cattle. female reproductive technologies could be readily combined with genomic selection in beef cattle breeding programs.58 depending on goals of the beef operation, application of these technologies may differ. with genomics technology, breeding females could be categorized as top 20%, middle 50% and bottom 30%, with these groups designated for embryo production, breeding (ai or natural service) and embryo recipients, respectively. in general, the genomic application should be focused on improving production parameters that are economically important. further the application should focus on improving reproductive performance, minimizing reproductive losses, and disease resistance. investigation of success of embryo transfer there are numerous factors influence the success of embryo transfer. even though several factors are beyond the control of the practitioner, the most important factor affecting the success is management of donors and recipients. breed, age, nutritional status, and reproductive history of donors and recipients, superovulation treatment of donors and synchronization treatment of recipients, hormones and drugs used, timing of insemination, method of collection and transfer, stress and biosecurity are some factors that influence the success. individual variation among females in response to superovulation remains the largest and least understood variable. some females consistently produce large numbers of embryos in response to superovulation, while other female cohort perform poorly. superovulation success is related to parity, virgin heifers and cows > eight years of age produce fewer embryos. elevated temperaturehumidity index are detrimental to superovualtion success. thin cows produced fewer transferable embryos compared to cows with moderate to good body conditions. cows in poor metabolic status (abnormal non-esterified fatty acid or blood urea nitrogen concentrations) had fewer transferable embryos. increased pregnancy losses associated with in vitro produced (ivp) embryos, increased risk of omphalophlebitis/arteritis in ivp calves, and the wide range of birth weights (some excessively large) of ivp calves should also be evaluated. a practitioner could make a meaningful contribution to improve success by providing advice on donor and recipient selection and better management options. focused investigation focused investigation is warranted in poor reproductive performance of a beef operation. this may include one or more of the following: bull breeding soundness examination (for all bulls); screening for infectious disease (e.g., venereal diseases, bvd, infectious bovine rhinotracheitis, leptospira borgpetersenii serovar hardjo type hardjo-bovis); review of nutritional management (e.g. ration and trace mineral evaluation and analysis, feeding groups and current body condition). determination of potential exposure to toxins clinical theriogenology • volume 8, number 4 • december 2016415 report once the investigation is complete, it should be possible to identify a specific problem or at least narrow down the investigation to allow a more focused review. finally, based on findings, a report for producers is prepared, including suggestions for changes in management. it should be noted that the report should include economic advantages of the management plan, as this is a key element that will convince the producer to implement changes detailed in the report. key actions for success continued success is vital for long term viability of a beef operation. in order for continued success it is important that required outcomes are achieved. key action for success includes: evaluate current performance of your client’s herd set clear and achievable goals and objectives for your clients explore alternative strategies, evaluate benefits, feasibility and risks, prioritize and recommend the best options implement the new strategy, maintain accurate records and regularly re-evaluate performance with expected targets future directions it is predicted that world’s population will reach 9.1 billion, 34% more than today and 70% of the world’s population will be urban compared to 49% today.59,60 in order to feed this larger, more urban and affluent population, food production must increase by 70%. meat production will need to rise by >200 million tons to reach 470 million tons.59,60 in comparison to the past 50 years, the rate at which pressures are building up on natural resources such as land, water, and biodiversity, will be increased during the next 50 years.61 furthermore, much of the natural resource base already in use worldwide shows worrying signs of degradation. the surge in livestock production that took place over the last 40 years resulted largely from an increase in the overall number of animals being raised. it is hard to envisage meeting projected demand using the same level of natural resources currently available. regardless, increases in production will need to come from improvements in efficiency of livestock systems in converting natural resources into food and reducing waste. veterinary input will be essential to develop systems and knowledge that will continuously improve production. the beef industry is a moving target. cattle owners have to balance their focus on management, genomics and biosecurity. farm animal genomics is of interest currently because of the usefulness derived from understanding how genomics and proteomics function in various organisms. genetic improvement in cattle populations mainly involves selection of males and females that, when mated, are expected to produce progeny that perform better than the average of the current generation. applications such as increased livestock productivity is one of several reasons that farm animal genome activity is thriving. given current genomic advancement, perhaps the top 10% of cattle today would only be the average in the next 10 to 20 years. consequently, it is critical to have knowledge of genomic applications and how targeted selection could improve economically important traits. the future of agriculture and the ability of the world food system to ensure food security for a growing world population are closely tied to improved stewardship of natural resources. major changes and investments are needed in all regions to cope with rising scarcity and degradation of land, water and biodiversity and with added pressures resulting from climate change and energy demands. the encouraging signs are: the beef industry today uses significantly less water and land than 30 years ago to produce each pound of beef; the industry has also reduced its carbon footprint by 16.3% for every 109 kg of beef produced.62 in addition, since 1992, the u.s. beef cow and overall beef cattle inventories have continually dropped an average of 1 to 1.25% annually,63 meaning more pounds of beef produced with fewer numbers of cattle, due to advances in pharmaceuticals, reproductive management, genetics and clinical theriogenology • volume 8, number 4 • december 2016 416 nutrition. clearly, comprehensive knowledge on reduced use of resources and reducing carbon footprint of the beef operation without affecting production is important. conclusions the approach described above can be used to provide routine services for reproductive management of the beef herd. data are collected and analyzed at pregnancy diagnosis and compared to set targets, with nutritional management and body condition scores concurrently reviewed. the bull battery should be examined at least one month prior to the start of the breeding season. appropriate monitoring and intervention will help herds achieve performance targets. references 1. nahms: part i: reference of beef cow-calf management practices in united states; 2007-2008. p. 8. 2. spicer lj, leung k, convey em, et al: anovulation in postpartum suckled beef cows. i. associations among size and numbers of ovarian follicles, uterine involution, and hormones in serum and follicular fluid. j anim sci 1986;62:734741. 3. callaghan cj, erb re, surve ah, et al: variables influencing ovarian cycles in postpartum dairy cows. j anim sci, 1971:33:1053-1059. 4. montgomery gw, scott ic, hudson n: an interaction between season of calving and nutrition on the resumption of ovarian cycles in postpartum beef cattle. j reprod fertil 1985:73:45-50. 5. yavas y, walton js: postpartum acyclicity in suckled beef cows: a review. theriogenology 2000;54:25-55. 6. williams gl: suckling as a regulator of postpartum rebreeding in 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two-sire systems. j anim sci 1989;67:40-47. 13. commodity costs and return. economic research service, usda. http://www.ers.usda.gov/data-products/commoditycosts-and-returns.aspx accessed jan 07, 2016. 14. gadberry s: beef cattle nutrition series part 1: nutrition basics. https://www.uaex.edu/publications/pdf/fsa-3078.pdf. last accessed dec 17, 2015. 15. gadberry s: beef cattle nutrition series part 2: establishing nutritional requirements. https://www.uaex.edu/publications/pdf/fsa-3079.pdf. last accessed december 17, 2015. 16. gadberry s: beef cattle nutrition series part 3: nutrient requirement tables. http://www.uaex.edu/publications/pdf/mp391.pdf, last accessed december 17, 2015. 17. gadberry s: beef cattle nutrition series part 4: formulating rations. https://www.uaex.edu/publications/pdf/fsa3080.pdf. last accessed december 17, 2015. 18. colburn dj, deutscher gh, nielsen mk, et al: effects of sire, dam traits, calf traits, and environment on dystocia and subsequent reproduction of two-year-old heifers. j anim sci 1997;75:1452-1460. 19. hickson re, morris st, kenyon pr, et al: dystocia in beef heifers: a review of genetic and nutritional influences. n z vet j 2006;54:256-264. 20. cook br, tess mw, kress dd: effects of selection strategies using heifer pelvic area and sire birth weight expected progeny difference on dystocia in first-calf heifers. j anim sci 1993;71:602-607. 21. rupp gp, ball l, shoop mc, et al: reproductive efficiency of bulls in natural service: effects of male to female ratio and singlevs multiple-sire breeding groups. j am vet med assoc 1977:171:639-642. 22. barth ad: bull breeding soundness evaluation manual. saskatoon: the western canadian association of bovine practitioners; 1994. p. 24-38. 23. chenoweth pj: bull libido/serving capacity. vet clin north am food anim prac 1997;13:331-344. 24. cammack km, thomas mg, enns rm: review: reproductive traits and their heritabilities in beef cattle. prof anim sci 2009:25;517-528. 25. rae do: assessing performance of cow-calf operations using epidemiology. vet clin north am food anim pract. 2006;22:53-74. 26. kasimanickam r, firth p, asay m, et al: impact of body condition change post-breeding on reproductive performance of beef cows. clin therio 2012;4:469-476. clinical theriogenology • volume 8, number 4 • december 2016417 27. kasimanickam r, whittier wd, currin jf, et al: effect of body condition at initiation of synchronization on estrus expression, pregnancy rates to ai and breeding season in beef cows. clin therio 2011;3:29-41. 28. kasimanickam r: effect of beef cow temperament at mid-gestation on reproductive performance. clin therio 2014;6:453-458. 29. kasimanickam r, asay m, schroeder s, et al: calm temperament improves reproductive performance in beef cows. reprod domest anim 2014;49:1063-1067. 30. gutierrez k, kasimanickam r, tibary a, et al: effect of reproductive tract scoring on reproductive efficiency in beef heifers bred by timed insemination and natural service versus only natural service. theriogenology 2014;81:918-924. 31. ahola jk, baker ds, burns pd, et al: effect of copper, zinc, and manganese supplementation and source on reproduction, mineral status, and performance in grazing beef cattle over a two-year period. j anim sci 2004;82:23752383. 32. fosgate gt: practical sample size calculations for surveillance and diagnostic investigations. j vet diagn invest 2009;21:3-14. 33. biosecurity on u.s. beef cow-calf operations, december 2009. https://www.aphis.usda.gov/animal_health/nahms/beefcowcalf/downloads/beef0708/beef0708_is_biosecurity.pdf. last accessed oct 22, 2015. 34. randel rd: nutrition and postpartum rebreeding in cattle. j anim sci 1990;68:853-862. 35. osoro k, wright ia: the effect of body condition, live weight, breed, age, calf performance, and calving date on reproductive performance of spring-calving beef cows. j anim sci 1992;70:1661-1666. 36. nutrient requirements of beef cattle: seventh revised edition: subcommittee on beef cattle nutrition, committee on animal nutrition, national research council; 2000. p. 109. 37. underwood ej: trace elements in human and animal nutrition. 3rd. ed. new york: academic press; 1971. 38. rowson lea, tervit hr, brand a: synchronization of oestrus in cattle using a prostaglandin f2α analog. j reprod fertil 1972;34:179-181. 39. lauderdale jw: effects of pgf2α on pregnancy and estrous cycle of cattle. j. animal sci 1972;35:246. 40. lauderdale jw, seguin bw, stellfiug jn, et al: fertility of cattle following pgf2α injection. j anim sci 1974;38:964967. 41. pursley jr, kosorok mr, wiltbank mc: reproductive management of lactating dairy cows using synchronization of ovulation. j dairy sci 1997;80:301-306. 42. whittier wd, currin jf, schramm h, et al: fertility in angus cross beef cows following 5-day co-synch + cidr or 7-day co-synch + cidr estrus synchronization and timed artificial insemination. theriogenology 2013;80:963-969. 43. kasimanickam r, schroeder s, hall jb, et al: fertility after implementation of longand short-term progesterone-based ovulation synchronization protocols for fixed-time artificial insemination in beef heifers. theriogenology 2015;83:1226-1232. 44. bucher a, kasimanickam r, hall jb, et al: fixed-time ai pregnancy rate following insemination with frozen-thawed or fresh-extended semen in progesterone supplemented co-synch protocol in beef cows. theriogenology 2009;71:1180-1185. 45. hall jb: current uses of gender-selected semen. bif 45th annu res symp conv; 2013. 46. garner dl, seidel jr ge: history of commercializing sexed semen for cattle. theriogenology 2008;69:886-895. 47. seidel jr ge: overview of sexing sperm. theriogenology 2007;68:443-446. 48. dejarnette jm, leach ma, nebel rl, et al: effects of sex-sorting and sperm dosage on conception rates of holstein heifers: is comparable fertility of sex-sorted and conventional semen plausible? j dairy sci 2011; 94:3477-3483. 49. frijters acj, mullaart e, roelofs rm, et al: what affects fertility of sexed bull semen more, low sperm dosage or the sorting process? theriogenology 2009;71:64-67. 50. dejarnette jm, mccleary cr, leach ma, et al: effects of 2.1 and 3.5 × 106 sex-sorted sperm dosages on conception rates of holstein cows and heifers. j dairy sci 2010;93:4079-4085. 51. lucena ja , kenyon ag, reynolds jp, et al: comparison between low-dose, high-sort and high-dose, low-sort semen on conception and calf sex ratio in jersey heifers and cows. j dairy sci 2014;97:1782-1789. 52. gosálvez j, ramirez ma, lópez-fernández c, et al: sex-sorted bovine spermatozoa and dna damage: i. static features. theriogenology 2011;75:197-205. 53. gosálvez j, ramirez ma, lópez-fernández c, et al: sex-sorted bovine spermatozoa and dna damage: ii. dynamic features. theriogenology 2011;75:206-211. 54. xu zz: application of liquid semen technology improves conception rate of sex-sorted semen in lactating dairy cows. j dairy sci 2014;97:7298-7304. 55. rowe rf, del campo mr, critser jk, et al: embryo transfer in cattle: nonsurgical transfer. am j vet res 1980;41:1024-1028. 56. hasler jf: forty years of embryo transfer in cattle: a review focusing on the journal theriogenology, the growth of the industry in north america, and personal reminisces. theriogenology 2014;81:152-169. 57. galli c, duchi r, colleoni s, et al: ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: from the research laboratory to clinical practice. theriogenology 2014;81:138-151. clinical theriogenology • volume 8, number 4 • december 2016 418 58. granleese t, clark sa, swan aa, et al: increased genetic gains in sheep, beef and dairy breeding programs from using female reproductive technologies combined with optimal contribution selection and genomic breeding values. genet sel evol 2015;47:70. 59. http://www.fao.org/fileadmin/templates/wsfs/docs/expert_paper/how_to_feed_the_world_in_2050.pdf 60. us census bureau: 2008. total midyear population for the world: 1950–2050. us census bureau, washington, dc. 61. changing disease landscapes. world livestock 2014. http://www.fao.org/docrep/019/i3440e/i3440e.pdf. 62. capper jl: the environmental impact of beef production in the united states: 1977 compared with 2007. j anim sci 2011;89:4249-4261. 63. lardy gp: trends to watch in cattle nutrition. midwest asas/adsa scientific session: 2015. clinical theriogenology • volume 8, number 4 • december 2016419 table 1. beef performance records birth birth weight birth date sex parentage calving ease color markings horned/polled weaning weaning weight weaning date hip height or frame environmental management code: contemporary group creep feeding embryo transfer yearling yearling weight yearling date hip height or frame pelvic area scrotal circumference reproductive tract score/age at puberty ultrasound (carcass) health vaccination dates vaccine codes health examination treatments temperament preconditioning procedures early weaning castrated – bunk trained – dehorned reproductive breeding dates breeding soundness exams pregnancy rate body condition score temperament cow age     parameters contributing to reproductive evaluation are in italics. to produce a comprehensive report, it is important to collect all required information. table 2. characteristics of body condition scores score category characteristics 1,2 thin spine and short ribs are sharp to the touch and can be distinguished visually. 3,4 borderline processes of the spine can be identified individually by touch. they feel rounded-not sharp and spaces between processes are less pronounced. 5 moderate processes can be felt with slight pressure and ends feel rounded. spaces between processes can be distinguished only with firm pressure. areas on either side of tail head are filled. 6,7 good ends of the processes can be felt only with firm pressure. spaces between processes cannot be distinguished at all. abundant fat cover around tail head, with some patchiness. 8,9 obese bone structure cannot be felt at all. tail head is buried in fat. clinical theriogenology • volume 8, number 4 • december 2016 420 table 3. benchmarks for beef herd reproductive performance parameter numerator denominator target (%) cyclicity rate* cycling cows total cows >85 ai pregnancy rate cows pregnant to ai total cows inseminated >50 breeding season pregnancy rate cows pregnant during breeding season total cows exposed >90 abortion rate gestational losses total females pregnant <2 early calving rate cows calving in 1st 21-day period total cows calving >65 stillbirth rate perinatal loses total females calving <2 weaning rate weaned calves total females pregnant/calved >85 replacement rate replacement heifers total females pregnant 15 *at beginning of breeding season references22,23 table 4. energy and protein balance for cows at typical rebreeding times (days 60 and 90 days after calving) ration tdn % (dm) crude protein % (dm) energy 60 d after calving (mcal/d) energy 90 d after calving (mcal/d) protein 60 d after calving (mp g/d) protein 90 d after calving (mp g/d) a 50 7.9 -4.59 -3.31 -216 -136 b 60 7.8 0.47 1.69 -38 35 c 70 9.1 6.48 7.65 187 256 reference 36 clinical theriogenology • volume 8, number 4 • december 2016421 t ab le 5 . s ex -r at io f ol lo w in g ar ti fi ci al in se m in at io n‡ a t a n ob se rv ed e st ru s or a t a f ix ed ti m e us in g co nv en ti on al o r se xe d (f em al e) s em en in b ee f he if er s p ar am et er s e st ru sa i f ix ed ti m e a i c on ve nt io na l s em en s ex ed s em en c on ve nt io na l s em en s ex ed s em en n um be r of h ei fe rs 10 0 10 0 10 0 10 0 e st ru s de te ct io n ra te ( % )§ 85 85 10 0 10 0 c on ce pt io n ra te ( % )† 60 48 a i pr eg na nc y ra te 51 43 60 48 s ex -r at io f ro m a i (f em al e: m al e)  26 :2 5 39 :4 30 :3 0 43 :5 r em ai ni ng o pe n he if er s pr eg na nt to c le an -u p bu ll 43 51 34 46 s ex -r at io f ro m c le an -u p bu ll b re ed in g 21 :2 2 25 :2 6 17 :1 7 23 :2 3 t ot al f em al e: m al e ca lv es ( a i + c le an -u p bu ll br ee di ng ) 47 :4 7 64 :3 0 47 :4 7 66 :3 8 a i a rt if ic ia l i ns em in at io n; a ss um pt io ns : ‡ b ee f ca tt le a re a rt if ic ia ll y in se m in at ed o nc e an d re m in de r of o pe n co w s ar e br ed b y cl ea nup b ul ls ; § e st ru s de te ct io n ra te e st ru sa i is 8 5% a nd f or f t a i is 1 00 % ; † 20 % r ed uc ti on in f er ti li ty f or s ex ed s em en ; n ot e: p ro ge st er on e ba se d es tr us s yn ch ro ni za ti on u se d fo r es tr us a i.  s ex r at io f or c on ve nt io na l s em en is 1 :1 a nd f or s ex ed s em en is 9 :1 ; b re ed in g se as on p re gn an cy r at e is 9 4% . clinical theriogenology • volume 8, number 4 • december 2016 422 figure 1. influence of body condition score on estrus expression, ai pregnancy and breeding season pregnancy rates body condition score 1emaciated; 9obese; ‘n’ is given in parentheses; reference (22), updated data figure. dotted lines represent associations between body condition score and estrus expression, ai pregnancy and breeding season pregnancy (r2=0.86, 0.97 and 0.89, respectively). 54.2 60.1 82.1 85.4 84.6 65.6 42.1 4… 5… 5… 5… 5… 76.5 80.8 91.3 94.3 94.8 84.6 0 20 40 60 80 100 3 (208) 4 (963) 5 (5236) 6 (6435) 7 (3426) 8 (468) p er ce nt ag e body condition score estrus expression ai pregnancy breeding season pregnancy breeding season pregnancy rate estrus expression rate ai pregnancy rate clinical theriogenology • volume 8, number 4 • december 2016423 clinical theriogenology • volume 8, number 4 • december 2016 424 omniblank: umbilical vein catheterization as an option for intravenous access in neonatal puppies soon hon cheong,a nicholas walsh,a allison miller,b marian diel de amorim,a ian porter,a peter scrivania adepartment of clinical sciences, bdepartment of biomedical sciences, college of veterinary medicine, cornell university, ithaca, ny intravenous access is essential to manage critically ill neonatal puppies and is difficult to achieve. intravenous access is achieved most commonly using intraosseous catheters but potential damage to the bone and growth plate is possible. in human neonatology, umbilical vein catheterization is the primary route of intravenous access in infants. this procedure has not been widely adopted in dogs. in hopes of using this procedure in neonatal puppies, the study objective was to determine the success rate of umbilical vein catheterization evaluated by gross dissection and imaging. six neonatal puppies were used for the study (size range: saint bernard to shih tzu) and were either stillborn or died shortly after birth. a 26-gauge intravenous catheter was used to achieve vascular access by holding the umbilicus at a 45-degree angle from a dorsal plane through the abdomen. entry from the ventral side of the umbilicus allowed a shallow puncture to enter the umbilical vein and easy sliding of the catheter into the vessel. a mixture of 4:1 latex:60% w/v barium sulfate suspension was administered into the catheter (total volume: saint bernard, 2 ml; shih tzu, 1 ml). latex aided in vascular dissection and barium provided contrast during computed tomography that was performed using a 16-slice scanner (aquilion lb, toshiba/canon america medical systems, tustin, ca) using these parameters: sternal recumbency; slice thickness, 0.5 mm; reconstruction interval, 1.0 mm; 120 kvp; 50 mas. based on dissection and imaging, umbilical vein catheterization was successful in 4/6 puppies; 1/6 (injections) was into the umbilical artery and 1/6 was extravascular (figure). umbilical vein catheterization was successful 67% of the time and may be a worthwhile means for vascular access in critically ill neonatal puppies. future studies using live puppies and maintaining catheter patency over time are warranted. keywords: puppy, umbilicus, catheterization, imaging, neonatology figure. catheter in umbilical artery and vein confirmed using computed tomography with barium contrast enhancement abstracts opening session clinical theriogenology 2021; 13: 299 temporal associations of b-mode, power doppler, and ovarian steroid changes of the periovulatory follicle and corpus luteum during luteogenesis and luteolysis in jennies humberto magalhaes,a,b jose dell-aqua jr,b igor canissoa adepartment of veterinary clinical medicine, university of illinois, urbana, il; bdepartment of animal reproduction, são paulo state university (unesp), brazil assessing the relationships between b-mode and power doppler ultrasonography of the periovulatory donkey follicle and respective corpus luteum (cl) could prove useful in clinical practice to predict impending ovulation and determining cl viability. this study’s objectives were to assess the associations between b-mode and power doppler ultrasonography and ovarian steroids of the periovulatory follicle and respective cl during luteogenesis and luteolysis in jennies. we hypothesized that information on changes in the granulosa are useful to detect impending ovulation and on blood flow are useful to differentiate an active versus inactive cl. one inter-ovulatory interval between 2 subsequent ovulations of 12 jennies (144 ± 22.5 kg; height 95.5 ± 113 cm) was used. jennies were teased daily to a mature jack. b-mode ultrasonography was carried out until the detection of a periovulatory follicle (≥ 28 mm, endometrial edema, and estrous signs). thereafter, jennies were monitored every 4 hours by b-mode and power doppler transrectal ultrasonography. once presumed signs of impending ovulation (thickened and irregular follicular wall, hyper-echogenicity of granulosa layer in transrectal ultrasonography and softened follicle in transrectal palpation) were detected, jennies were reexamined at 1 hour intervals until ovulation. after ovulation, the cl was examined daily until the completion of luteolysis (progesterone < 1.5 ng/ml). plasma estradiol and progesterone concentrations were assessed daily with chemiluminescence assays (immulite 1000, siemens, us). data were analyzed using rm-anova followed by tukey`s test (steroid concentrations, follicle and corpus luteum parame-ters), friedman test adjusted by dunn’s test (edema score and behavior), and pearson’s coeffi-cient correlations (thickness and echogenicity of the granulosa layer). mouth-clapping, a species-specific estrous sign, was the first and the last sign to be detected (± 24 hours postovulation). the diameter of the ovulatory follicle was 34.6 ± 3.3 mm (31 38 mm). the echogenicity and the thickness of the granulosa layer increased (p < 0.05) from 36 to 1 hour before ovulation in 70% of jennies; strong correlations between thickness (r = 0.70), granulosa echogenicity (r = 0.80), and impend-ing ovulation were noted. follicular wall blood flow increased (p < 0.05) from 72 to 24 hours before ovulation and estra-diol concentrations declined from 42 pg/ml at 72 hours to 31.6 pg/ml at 24 hours before ovulation. vascularization of periovulatory follicle decreased (p < 0.05) from 62% (36 hours before ovulation) to 37% (1 hour before ovulation); 75% of the jennies had a homogenous cl echogenicity with a white hyperechogenic central lacuna. the maximum cl size represented 76% of the periovulatory follicle diameter. vascularization of the cl and progesterone concentrations had a gradual rise, reaching the peak at 11 and 10 days after the ovulation, respectively (p < 0.05). luteal echo-genicity increased (p < 0.05) 4 days after luteolysis as a consequence of corpus albicans formation). vascularization of the cl started to decline (p < 0.05) 3 days before luteolysis and progesterone concentrations had a sharp reduction (p < 0.05) for 4 days before luteolysis. in conclusion, the structural changes of the periovulatory follicle detected on b-mode can be used to detec impending ovulation in donkeys; however, b-mode ultrasonography cannot be used to assess cl functionality. conversely, power doppler can be used to differentiate a functional versus nonfunctional cl in jennies. keywords: periovulatory period, luteogenesis, luteolysis, steroid suitability of noncycling recipient mares for in vitro produced equine embryos charles scoggin, etta bradecamp, jamie kaczor, erin lohbeck, crystal howard, holly hersey, alaina broach rood and riddle equine hospital, lexington, ky suitable cycling recipient mares are not always available at an equine embryo transfer station (ets). noncycling recipients primed with estrogen followed by progesterone before transfer of in vivo embryos can have similar pregnancy rates as those that are cycling. however, in vitro produced (ivp) embryos may be less hardy and require a more precise window of synchrony with recipient mares compared to in vivo embryos.1 to our knowledge, no data exist on the suitability of noncycling recipient mares when transferring ivp embryos. a retrospective study was conducted whereby data from a single ets during the 2020 breeding season were analyzed and pregnancy rates were compared between cycling and noncycling recipients receiving ivp embryos. all embryos were derived from commercial donors and transferred at the ets. cycling recipients (crs) were examined via transrectal ultrasonography until emergence of a dominant follicle, then evaluated daily until ovulation and subsequently received an ivp embryo 4 days postovulation. noncycling recipients (ncrs) were determined to be anestrus or transitional by a lack of luteal tissue and the presence of small follicles based on transrectal ultrasonography and serum progesterone concentrations. initially, ncrs were treated intramuscularly with 10 mg estradiol-17β in oil (e2) on the first day, 6.6 mg on the second day, and 3.3 mg e2 on the third day. the day after the last e2 treatment, ncrs were treated intramuscularly with 200 mg progesterone in oil (p4); once a day for 3 consecutive days. on the fourth day (at transfer), ncrs received intramuscularly 500 mg of p4 and were supplemented with p4 at least until their first pregnancy examination. data were analyzed using chi-square or fisher’s exact tests, and values considered significant at p < 0.05. during the study, 78 ivp embryos were shipped to the ets. of these, 60.3% (41/78) were transferred into crs and 39.7% (31/78) were transferred into ncrs. overall transfer rate for ivp embryos was 52.6% (41/78). day 42 pregnancy and embryo loss rates were 30.8% (24/78) and clinical theriogenology 2021; 13: 300 41.5% (17/78), respectively. there were no differences between crs and ncrs in rates of initial transfer (55.3 versus 48.4%, p = 0.5485), pregnancy at day 42 (34.0 versus 25.8%, p = 0.1367) or embryo loss (38.5% versus 46.7%, p = 0.7449) rates. results suggest that ncrs treated with e2 and p4 prior to transfer may be suitable recipients for ivp embryos. keywords: embryo transfer, ivp embryos, recipients reference 1.cuervo-arango j, claes an, stout tae: in vitro-produced horse embryos exhibit a very narrow window of acceptable recipient mare uterine synchrony compared with in vivo-derived embryos. reprod fertil dev 2019;31:1904-1911. serum prostaglandin e metabolite in diestrous and pregnant mares mariana diel de amorim,a michael byron,a claire cardb adepartment of clinical sciences, college of veterinary medicine, cornell university, ithaca, ny; bdepartment of large animal clinical sciences; western college of veterinary medicine, university of saskatchewan, saskatoon, canada in the nonpregnant mare, prostaglandin f2α (pgf2α) is luteolytic, and in early pregnancy prostaglandin e2 is reported to be luteotrophic. however, there is a lack of information on circulating concentrations of prostaglandin metabolite (pgem) during diestrus and pregnancy. we hypothesized that circulating concentrations of pgem increase in pregnant mares during the expected time of pregnancy recognition day (d) d13 d15 compared to diestrous mares. our objective was to compare daily/hourly plasma pgem concentrations and secretion profiles in pregnant and diestrous cycles using a randomized cross over design (n = 4 mares), with 1 cycle in between study periods. transrectal ultrasonography was used to detect estrus, day of ovulation (d0), and pregnancy. mares were bred to a fertile stallion during estrus. blood was sampled on d0, 4, 8, 12, 18, and 20. a jugular catheter was used to obtain hourly blood samples from d13 through d16. blood was placed in chilled edta tubes and immediately centrifuged at 4°c. plasma was separated, placed in cryovials, frozen in liquid nitrogen, and stored at -80°c until assayed. pgem was measured using commercial enzyme-linked immunosorbent assays (elisa) (cayman chemical, ann arbor, mi) validated in our laboratory according to the manufacturer’s instructions. progesterone concentrations were determined every 6 hours from d13 to d16 (siemen’s immulite, los angeles, ca). both assays had an intraand inter-assay coefficient of variation (cv’s) of < 15.8%. statistical analysis was performed on jmp® pro 15 at p < 0.05 using wilcoxon tests to compare differences between plasma pgem in diestrus and pregnant cycles. differences between days and times were compared individually by student’s t-test. one mare failed to become pregnant. diestrous mares had higher (p < 0.0001) overall plasma pgem concentrations from d0 to 20 compared to pregnant mares (mean ± sd) (30.7 ± 15 pg/ml and 17 ± 6 pg/ml, respectively) pgem concentrations were also higher (p < 0.0002) in diestrous mares compared to pregnant mares for d13, 14, 15 and 16. the pgem secretion profile was substantially different than that previously reported for pgfm. pregnant mares had small peaks of pgem that were different (p < 0.05) from diestrous mares on d13 and d14. this study is novel and demonstrated that plasma pgem concentrations in diestrous mares are higher than in pregnant counterparts. however, a larger number of estrous cycles has to be studied to characterize the pgem profile during early pregnancy. further investigation of pgfm and pgem in pregnancy is warranted to understand the importance of circulating concentrations and if the ratio or pattern of pge:pgf may be altered during the expected period of luteolysis and maternal recognition of pregnancy. keywords: equine, pregnancy, progesterone, prostaglandin cholesterol-loaded cyclodextrin improves cooling and fertility of donkey semen lorenzo segabinazzi,a,b veronica scheeren,b camila freitas-dell’aqua,b jose dell’aqua jr,b frederico papa,b marco alvarenga,b igor canisso adepartment of veterinary clinical medicine, university of illinois, urbana, il; bdepartment of animal reproduction, são paulo state university, botucatu, brazil the donkey and mule show industry is an ever-growing industry. high-performance mules drive the stud fee values and increase their demand as a sire to breed mares and jennies. cooledshipped semen is the primary approach used by the industry. skim milk-based (skm) extenders are most used to cool and ship equid semen. however, donkey semen does not tolerate cooling with a skm extender unless the seminal plasma is removed by centrifugation or 2% egg yolk is included as an additional cholesterol source. neither approach is practical in ambulatory conditions; thus, alternatives must be identified. inclusion of cholesterol-loaded cyclodextrin (clc) in freezing extender improves post-thaw semen quality of donkeys; however, clc has not been tested for cooling donkey semen. this study’s objective was to compare semen parameters and fertility of cooled donkey semen extended in a commercially available skm with and without clc (skm-clc). we hypothesized that clc enhances semen cooling and fertility of donkey semen. in the first experiment, 35 ejaculates from 7 mature jacks were split into skm (botusemen, botupharma) and skm-clc (botuspecial, botupharma) groups and extended at 50 x 106 sperm/ml. after extension, samples were stored in a passive semen cooling container (botuflex, botupharma) at 5°c for 48 hours. total motility (tm), progressive motility (pm), and percentage of sperm with rapid motility (rap) were assessed with casa (i.v.o.s. 12, hamilton thorne, beverly, ma). plasma membrane integrity (pmi), and mitochondrial membrane potential (mmp) were assessed with the combination of yo-pro® and mitostatusred with flow cytometry (lsr-fortessa, becton dickinson, mountain view, ca). semen was assessed clinical theriogenology 2021; 13: 301 before (time 0), 24, and 48 hours after cooling. in the second experiment, 2 estrous cycles of 15 mares were used for fertility assessment. mares were examined every other day by transrectal ultrasonography (sonoscape a6®, china). once a preovulatory follicle was detected (i.e. ≥ 35 mm in the presence of endometrial edema > 1, 0 absent and 3 max), ovulation was induced with 250 µg of histrelin acetate. at induction, semen from 1 jack was collected (n = 28), extended in either skm or skm-clc, and cooled for 24 hours. mares were randomly and equally assigned in a crossover for breeding with either extender 24 hours after induction of ovulation. thereafter, mares were examined daily to detect intrauterine fluid accumulation and ovulation. mares received oxytocin (20 units) to prevent intrauterine fluid accumulation. pregnancy diagnosis was carried out on day 15 day after ovulation and mares received dinoprost (5 mg) intramuscularly to induce estrus. data were analyzed with graphpad pris 8.0.1. (graphpad, san diego, ca). semen parameters were analyzed with a mixed model and tukey’s as posthoc. pregnancy diagnosis was assessed with fisher’s exact test. significance was set at p ≤ 0.05. there were no differences (p > 0.05) in tm, pm, rap, pmi, and mmp for semen extended in either extender at time 0. there was a reduction in tm, pm, rap, pmi, and mmp over time across groups; however, semen extended with skm-clc had superior (p < 0.05) tm, pm, rap, pmi, and mmp than semen extended in skm at 24 and 48 hours postcooling. mares bred with semen extended in skm had lower (p < 0.05) conception rate (13%, 2/15 cycles) than mares bred with skm-clc (47%, 7/15 cycles). incorporating clc to skm extender improved semen parameters and fertility of cooled donkey semen keywords: donkey, semen cryopreservation, extenders, chilling, cholesterol luteinizing hormone receptor activation stimulates endothelial adhesion of neoplastic canine t-lymphocytes alexa dietz,a michelle kutzlerb acollege of engineering, oregon state university, or; bdepartment of animal and rangeland sciences, college of agricultural sciences, oregon state university, or previous research from our laboratory has demonstrated that luteinizing hormone receptors (lhr) are expressed in neoplastic lymphocytes in canine lymph nodes and that activation of the lhr with human chorionic gonadotropin (hcg) increases lhr gene expression and cell proliferation in isolated neoplastic t-lymphocytes. the objective of the current study was to determine if hcg activation of lhr in neoplastic t-lymphocytes increase their adhesion to an endothelial cell monolayer. the hypothesis was that increasing hcg concentration induces a dose-dependent increase in neoplastic t-lymphocyte adhesion. canine aortic endothelial cells (#cn304-05, cell applications, inc.) were cultured to form a monolayer. endothelial cells were activated with tumor necrosis factor-alpha for 12 hours. immortalized t-cell lines isolated from 3 dogs (clc, ema, clk) with multi-centric lymphoma were cultured for 72 hours with increasing concentrations of hcg (from 4 4,000 iu/ ml). neoplastic t-lymphocytes were then fluorescently labeled (cytoselect leukotracker, cell biolabs, inc.) and added to the endothelial monolayer. after a 2-hour incubation, non-adherent cells were removed by washing. images of adherent cells were digitally captured (#qic-f-m-12-c, qimaging) at 400 x magnification using fluorescent microscopy (#dm4000b, leica microsystems). adherent cells were then quantified on a fluorescence plate reader (synergy 2, biotek) using 50% gain. four replicates of each t-cell line were used for each assay and the assays were repeated 3 times. results (mean ± sem) were expressed as a fold of baseline and compared between different hcg concentrations using a one-way analysis of variance (graphpad prism). significance was defined as p < 0.05. activation of lhr in neoplastic lymphocytes increased cell adhesion in a dose-dependent manner in all 3 cell lines (clc: p = 0.030; ema: p = 0.016; clk: p = 0.004). increases in hcg concentrations stimulated more neoplastic t-lymphocyte adhesion (figure). this is the first study to demonstrate that activation of lhr in neoplastic canine lymphocytes increases endothelial cell adhesion. these results could explain why gonadectomized dogs with elevated circulating lh concentrations develop lymphoma at higher rates than intact dogs. keywords: cancer, dog, human chorionic gonadotropin, lymphoma figure. neoplastic t-lymphocyte adhesion a: hcg 0 iu/ml; b: hcg 4 iu/ml; c: hcg 40 iu/ml; d: hcg 400 iu/ml; e: hcg 4000 iu/ml. clinical theriogenology 2021; 13: 302 characterization of reproductive parameters from pennsylvania bull elk (cervus canadensis) luisa ramírez-agámez,a camilo hernández-avíles,a jeremiah banfield,b michael o’connor,c mariah pearson,a sheila spacek,a sheila teague,a charles lovea acollege of veterinary medicine, texas a&m university, college station, tx; bpennsylvania game commission, harrisburg, pa; cdepartment of animal science, pennsylvania state university, university park, pa pennsylvania (pa) elk (cervus canadensis) are a population of free-ranging cervids that live in north central pa, and were initially reintroduced in 1913 as a measure to recover the elk population that was extirpated by uncontrolled hunting. currently, the herd size of pa elk approaches ~ 1,400 individuals. during the 2019 calving season, a reduction in the number of calves was noticed. gross anatomic inspection from the uteruses and ovaries harvested from selected cows revealed evidence suggestive of embryonic death whereas conventional sperm parameters (e.g. morphology, viability) analyzed from selected bulls were largely normal. this study was conducted to characterize certain sperm quality parameters from bulls (n = 24) harvested during the 2020 hunting season, to determine potential causes for reduced fertility. testes and epididymides were harvested from hunted animals, immediately cooled at 5°c and shipped from pa to texas within 24 hours. upon arrival, each testis and epididymis were isolated and weighed. tissue wedges were obtained from testes and fixed in davidson’s fixative. each caudal epididymis was flushed with 5 ml of inra-96® extender and total sperm numbers (tsn; 109), total and progressive motility (tmot, pmot; %), curvilinear velocity (vcl; µm/s), plasma membrane/acrosomal intactness (vai; %), dna integrity (compα-t; %), and sperm morphology (normal; %) were determined by casa, flow cytometry and dic microscopy, respectively. overall, the sperm quality was consistent with parameters from other species (e.g. stallion, bull; table). the % (mean ± sd) of morphologically normal sperm was low due to the incidence of proximal, distal droplets (13 ± 11, 43 ± 21; respectively), and bent midpieces (14 ± 11). surprisingly, the % compα-t was high (mean ± sd 45 ± 17; range: 3 71). spearman’s correlation analysis revealed no linear relationship (p > 0.05 [positive or negative]) among testes and epididymides sizes, sperm morphology, or compα-tvalues. at this point, it can be speculated that the observed values of compα-t (susceptibility of dna to denaturation) in the wild pa elk population may partially explain the reduced fertility recently observed. further studies are warranted to confirm this hypothesis and to determine a potential cause for such increased values of sperm dna damage in the absence of other sperm quality abnormalities. keywords: pennsylvania elk, cervus canadensis, sperm quality, fertility, dna integrity parameter testis epid. tsn tmot pmot vcl vai compα-t nortmal (mean ± sd) 83 ±21 21 ± 5 0.6 ± 1 55 ± 20 34 ± 15 162 ± 51 87 ± 6 45 ± 17 34 ± 14 table. sperm quality parameters from 24 pa elks obtained after epididymal harvest expression and abundance of prostaglandins in healthy and fibrotic mare endometrium michael byron,a jennine lection,a bettina wagner,b andrew miller,c tracey chenier,d soon hon cheong,a mariana diel de amorima adepartment of clinical sciences, bdepartment of population medicine and diagnostic sciences, cdepartment of biomedical sciences, college of veterinary medicine, cornell university, ithaca, ny; ddepartment of population medicine, ontario veterinary college, university of guelph, canada inflammation and fibrosis of the endometrium is a major cause of infertility in the mare. a better understanding of the mechanisms of degenerative diseases of the endometrium can help to improve diagnosis and clinical outcomes in the mare. prostaglandins have a wide range of roles including in tissue remodeling, inflammation, and fibrosis in the endometrium. we hypothesized that expression of prostaglandin-related genes in the endometrium and abundance of prostaglandins in low-volume uterine lavage is different in healthy mares compared to mares diagnosed with fibrosed endometrium. our objective was to discern any changes in prostaglandin abundance and gene expression among healthy and fibrosed endometrium in order to better understand the mechanisms of endometrial inflammation and fibrosis. a total of 27 estrous mares were enrolled in this study. a uterine lavage using 250 ml of 0.9% nacl solution was performed with a sterile bivona catheter, and the concentration of prostaglandins in the fluid was measured using enzyme-linked immunosorbent assays (elisas) for pge2 and pgf2α. an endometrial biopsy was collected and sectioned in half. one half of the endometrial biopsy was fixed in 10% formalin, sectioned, stained with hematoxylin & eosin and graded according to the kenney-doig system by a board-certified pathologist; the second half was snap frozen in liquid nitrogen and stored at -80°c until analysis. mares were assigned to either the healthy group (n = 15) if the endometrial biopsy score was i or iia, or to the fibrosed endometrium group (n = 12) if the endometrium biopsy score was iib or iii. total rna was extracted from endometrial biopsies and real-time pcr was performed to evaluate the relative abundance of the following genes: ptges, ptgs2, ptgfr, ptger2, ptger4, cbr1, and slco2a1. mean threshold cycle (cq) was determined and then normalized to the reference gene (gapdh) (δct). statistical analyses were performed with jmp® software using p < 0.05. data were clinical theriogenology 2021; 13: 303 analyzed for normality and rank transformed when necessary. student’s t-test was used to compare differences between healthy and fibrosed endometrium groups. healthy endometrium had higher expression of ptges (p < 0.03) and slco2a1 (p < 0.05) compared to fibrosed ones. there was no difference (p > 0.05) in abundance of pge2 or pgf2α in low-volume lavage between the 2 groups. in endometrial fibrosis, degenerative inflammatory conditions can increase cytokine production and disrupt normal cellular function that may impact pathways including prostaglandin synthesis and prostaglandin uptake/transport. slco2a1 is involved in the transport of pge2 and pgf2α that are present during pregnancy and luteolysis. a disruption of prostaglandin e-synthase could similarly affect prostaglandin concentrations, altering the pge/pgf ratio and resulting in a deficient environment for development and maintenance of conceptus and corpus luteum. keywords: endometrial biopsy, fibrosis, equine, prostaglandin clinical theriogenology 2021; 13: 304 epididymal sperm granulomas are associated with antisperm antibodies in frozen-thawed donkey semen giorgia podico,a yamilka lago-alvarez,a maria ferrer,b igor canissoa adepartment of veterinary clinical medicine, college of veterinary medicine, university of illinois urbana-champaign, urbana, il; bdepartment of large animal medicine, college of veterinary medicine, university of georgia, athens ga prior to ejaculation, sperm is stored in the epididymis for a variable time; the blood-epididymis barrier regulates exchanges of nourishment and hormones while maintaining sperm isolation. the innate and adaptive immune system intervenes when sperm is extravasated in the interstitium. although several conditions are known to affect the integrity of the blood-epididymis barrier (i.e. trauma, toxicants, parasites, and infections) in domesticated mammals, epididymal sperm granulomas and antisperm antibodies are a rare finding and have never been described in donkeys. this study aimed to describe and compare semen parameters (preand post-freezing) and antisperm antibodies of donkeys with epididymal sperm granuloma (granuloma) and healthy controls (control). feral donkeys (n = 10) castrated in a concurrent study were enrolled. three donkeys had unilateral granulomas, 2 donkeys had bilateral granulomas, whereas the remaining 5 were grossly normal. the granulomas were either single or multiple, firm, well-circumscribed, tan to red, and 1 5 mm in size. upon incision, abundant, thick, tan to white-yellow fluid was recovered. histopathology revealed epithelioid macrophages, multinucleated giant cells, and abundant sperm cell fragments with mineralized cellular debris. semen was harvested for cryopreservation through retrograde flushing of the cauda epididymis. sperm concentration and motility parameters (total motility, tm; progressive motility, pm) were assessed with an automated sperm analyzer; plasma membrane integrity (pmi), and mitochondrial membrane potential (hmmp) were assessed with flow cytometry preand post-freezing. postfreezing semen was assessed through flow cytometry for the presence of antisperm antibodies (igg and iga). statistical analysis was performed with the wilcoxon matched-pairs signed-rank test. significance was set at p < 0.05. the total sperm yield did not differ (p > 0.05) between groups (control 11.0 ± 2.0, granuloma 9.0 ± 0.4 x 109). tm did not change (p > 0.05) after freezing in the granuloma group (tm prefreezing 29 ± 6%, postfreezing 18 ± 3%). after freezing, pm and pmi of donkeys with sperm granuloma were lower (p < 0.05) than healthy ones (pm control 15 ± 2%, granuloma 7 ± 2%; pmi control 51 ± 4%, granuloma 36 ±5 %). preand post-freezing hmmp did not differ (p > 0.05) among groups. three of the 5 donkeys with granuloma had a percentage of igg and iga-bound sperm above the maximum value observed in control donkeys. mean percentage of iggand iga-bound sperm did not differ (p > 0.05) among groups (igg-bound control 2 ± 0.4%, granuloma 16 ± 10%; iga-bound control 0.1 ± 0.1%, granuloma 0.5 ± 0.4%). in conclusion, sperm granulomas only marginally affected sperm quality and resulted in igg and iga antisperm antibodies binding to sperm. it remains to be determined if sperm granuloma and antisperm antibodies affect fertility in donkeys. keywords: antisperm antibodies, epididymis, blood-epididymis barrier, epididymitis laser ablation of the equine oviductal papilla as a novel contraceptive technique grace edwards, robyn ellerbrock, randall eggleston, maria ferrer college of veterinary medicine, university of georgia, athens ga reproductive control of wild horse populations has frustrated the bureau of land management for years. gnrh and zona pellucida vaccines have a limited duration of efficacy, intrauterine devices are unpredictably retained, and attempts to ovariectomize mares were met with public outcry in 2018. the development of a nonsurgical permanent sterilization technique has the potential to revolutionize management of wild horses and burros on public lands. the objectives of this study were to develop a safe and efficient sterilization technique, and to demonstrate technique effectiveness in reproductively healthy mares. we hypothesized that laser ablation of the oviductal papillae would be an effective method of permanent sterilization. seven light breed reproductively healthy mares (5 21 years) were enrolled in the study after pregnancy confirmation at 14 days. mares were given prostaglandin to induce abortion and within 14 days were sedated with xylazine hydrochloride for hysteroscopy. examination of the endometrium and oviductal papillae was accomplished using a 103 cm flexible endoscope (olympus gif-160 gastroscope, center valley, pa) attached to an olympus evis exera cv-160 video processor. the endoscope competition session clinical theriogenology 2021; 13: 305 was guided through the cervix and the uterus insufflated with room air. in 5 mares, a 600 μm laser fiber was advanced through the biopsy channel, and using a diode laser (dornier medilas d, dornier medtech america, inc., kenneasaw, ga), set at 20 w, 2 6 direct contact pulses of 3 5 seconds in duration were delivered at the oviductal papilla. the endoscope was then guided up the contralateral uterine horn and the process repeated. total energy delivered ranged from 700 to 1500 j. in 2 control mares, the oviductal papillae were visualized, but no laser ablation was performed. all mares received 5 mg dinoprost im, and a uterine lavage was performed using 1 3 liters of lactated ringer’s solution within 4 hours postprocedure. transrectal ultrasonography was performed every 2 3 days until a 35 mm follicle was detected, and then mares were bred using a semen sample from a fertile stallion with a minimum of 500 x 109 progressively motile sperm. mares were bred every 48 hours until ovulation, and pregnancy status was determined by transrectal ultrasonography 14 days postovulation. after examination, mares were treated intramuscularly with 5 mg of dinoprost to induce luteolysis and were rebred on 2 4 consecutive estrous cycles. control mares conceived on 6 out of 9 cycles (67% pregnancy rate). pregnancy rate was lower (p = 0.003) in the treatment group (5%, 1 out of 20 estrous cycles). the first mare that was laser ablated conceived on the 4th cycle, and repeat hysteroscopy determined that the left oviduct was not effectively ablated. the procedure was repeated, and the mare reenrolled in the breeding trial. the third mare enrolled in the study developed fever and tachycardia 6 hours after hysteroscopy and was diagnosed with peritonitis via abdominocentesis. the mare was treated with broad spectrum antibiotics and recovered uneventfully. the mare that developed postprocedure complications was believed to be due to equipment difficulties early in the development of the procedure, and the 3 final mares included in the trial had no inflammation on peritoneal fluid evaluation after oviductal ablation, and no change in physical exam parameters. in conclusion, when the laser ablation of the oviductal papillae was appropriately performed, scar tissue formation effectively prevented pregnancy for a minimum of 4 months postprocedure. laser ablation of the oviductal papillae is a promising technique for permanent sterilization of the mare and a follow-up long-term fertility study is warranted. keywords: antisperm antibodies, epididymis, blood-epididymis barrier, epididymitis mammary gland electrolytes and ph to detect impending parturition in jennies humberto magalhaes, lucas canuto, igor canisso department of veterinary clinical medicine, university of illinois, urbana, il assisted foaling is ideal for maximum foal survival, as it allows for early intervention to cope with simple dystocia or referral to appropriate tertiary veterinary care facilities. unfortunately, most jennies foal unattended, and a high incidence of donkey foal mortality is a common problem in the breeding industry. the prediction of parturition is critical to ensure assisted delivery. because equids have a prolonged pregnancy and foal during the night, continuous foaling monitoring is not feasible on small farms. thus, serial assessment of mammary gland electrolytes and ph are used to circumvent this issue to detect impending parturition in mares but not in jennies. major objective was to determine the usefulness of serial assessment of mammary gland electrolytes and correspondent ph to detect impending parturition in jennies. in addition, the relationships between maternal, fetal membranes, and foal birth weight were investigated. we hypothesized that serial assessment of mammary gland ph predicts foaling in jennies. multiparous jennies (n = 37) were monitored daily starting from 350 to 355 days of pregnancy until foaling. the ph of mammary gland se-cretions was assessed daily with a hand-held device (laqua twin ph meter, horiba, irvine, ca). aliquots of mammary secretions were frozen daily and then assessed retrospectively for electrolyte concentrations (ca2+, mg2+, k+, and na+) (beckman coulter, switzerland) starting 5 days before foaling. electrolyte concentrations and ph values were analyzed with a mixed-effect model. student’s t-test was performed to compare dependent variables (pregnancy length, birth weight, placental weight, and umbilical cord length) according to foal sex as an independent variable. sensitivity, specificity, and negative predictive value (npv) and positive predictive values (ppv) were evaluated using a cut-off value for ph ≤ 6.4 and ca2+ >10 mmol/l. most foalings (91.9%) were during the night. the overall pregnancy length was 374 ± 8.7 days (range 357 390 days). there were no differences (p > 0.05) in pregnacy length for colts (374 ± 2.1 range 357-385 days) and fillies (373 ± 2.3 range 358 390 days). colts and fillies were 61.8 and 38.2%, respectively. fetal membranes weighed 3.4 ± 0.1 kg (range 1.9 4.7 kg). foals at birth weighed 31.1 ± 2.5 kg (range 26.5 37.5 kg), with no differences (p > 0.05) in birth weights for colts (31.1 ± 2; range 26.5 37.5 kg) and fillies (30.8 ± 2.2; range 26.5 34 kg). the ratio of foal birth weight with the dam’s bodyweight was 9.7%, and the ratio with fetal membranes was 11%. there was a significant reduction in na+ and an increase in ca2+, mg2+, and k+ concentrations leading to foaling. the ph of mammary secretions glands decreased during the 5 days preceding parturition. additionally, 2 distinct profiles for ph reduction were recorded, with 32% of the jennies displaying a fast reduction in ph values (profile 1) and 65% presenting a slow reduction in ph (profile 2) from the mammary gland secretions; 3% foaled with high and alkaline ph (ph = 7.5). the ph had a 90% sensitivity for foaling within 24 hours, whereas the specificity was 70%, and the ppv and npv values were 40 and 97%, respectively. of interest, ca2+ (> 10 mmol/l) had a sensitivity and specificity of 71 and 85%, respectively, whereas the ppv and npv were 72 and 84%, respectively. in conclusion, daily measurements of the ph of mammary gland secretion can predict foaling in jennies, whereas ca2+ is a useful marker to determine when parturition will not occur. therefore, ca2+ needs to be associated clinical theriogenology 2021; 13: 306 with ph to predict donkey parturition. jennies had a high placental efficiency, as demonstrated by the high placental, dam, and foal ratios. keywords: predicting parturition, foaling, periparturition, donkey, ph, electrolytes clinical and physiological ultrasonography of normal and abnormal donkey pregnancies humberto magalhaes, lucas canuto, igor canisso department of veterinary clinical medicine, university of illinois, urbana, il ultrasonography of the fetoplacental unit is carried out to detect abnormalities and to stage pregnancy. transrectal and transabdominal ultrasonography have well-established physiological parameters and clinical applications in horses, but these techniques have not been characterized or established in donkeys. season and sex of the foal are known to affect pregnancy length in horses but their effects are not known in donkeys. pregnancy loss in horses is associated with abnormal progestin profiles but is not studied in donkeys. major objective was to establish clinical and physiological ultrasonographic parameters of jennies carrying and delivering normal pregnancies and jennies undergoing premature delivery of stillborn foals. additionally, effects of season and sex of the foal on gestational length (gl) were assessed. we hypothesized that season and sex of the foal affect gl, and pregnancy loss results in abnormal ultrasonography parameters and progestin profiles. multiparous jennies (n = 140) ranging 4 16 years in age were enrolled by 120 days of pregnancy. jennies were artificially inseminated with fresh semen during the spring, summer, and fall, in a single calendar year, all on 1 farm. all jennies were submitted to transrectal ultrasonography (well. d, medical electronics co., shenzhen, china) coupled with a 7.5 mhz linear transducer at 15 day intervals until delivery. a subset of jennies (n = 50) had transabdominal ultrasonography (well. d, medical electronics co.) coupled with a 3.5 mhz sectorial convex transducer, also performed at 15 day intervals until delivery. parameters assessed during each evaluation included combined thickness of uterus and placenta (ctup) and fetal parameters (eyeball diameter, thorax, heartbeat, and aortic diameter). serum samples were collected from each jenny during each evaluation for the determination of progesterone concentrations by ria. foals were weighed after birth. data were assessed for normality with shapiro-wilk’s test, and then anova and tukey’s (aortic diameter, heartbeat, and thorax) or kruskal-wallis followed by dunn’s (eyeball and ctup). mixed models were used to assess the effects of season and interactions with foal sex and gl. statistical significance was set at p < 0.05. the incidence of late pregnancy loss was 3.5% (5/140 jennies). the gl was 365.4 ± 10.4 days (range; 345 390 days) for jennies carrying and delivering normal pregnancies and was 345 ± 32.3 days (range; 290 352 days) for the group experiencing pregnancy loss. spring bred jennies had the longest (p < 0.05) gl (375 ± 8.7 days), followed by summer bred (360 ± 32.3 days) and then fall bred (358.6 ± 5.8 days). colts had longer gl than fillies (363 ± 10.2 versus 358.5 ± 9.3 days). there was no effect of gl on the foal’s birth weight. there were significant associations between gl with eye orbit diameter (r = 0.70), fetal thorax (r = 0.80), fetal aortic diameter (0.60) and ctup (r = 0.60). fetal heartbeat (r = -0.9) was negatively correlated with gl. ctup significantly increased from 150 days of pregnancy to term. two jennies with premature deliveries had ctup outside normal ranges and placental separation consistent with ascending placentitis; before abortion, these jennies also had an increase in progesterone concentrations in comparison to other jennies. the remaining 3 jennies undergoing premature delivery did not experience these changes. in conclusion, the study established clinical, physiological, and ultrasonographic parameters for donkey pregnancy. the incidence of late pregnancy loss was 3.5%. spring-bred had the longest gl in jennies and colt-bearing pregnancies resulted in the longer gl than fillies; 40% of the abnormal pregnancies had abnormal ctup, placental separation, and abnormal progesterone profiles. keywords: fetoplacental unit ultrasonography, pregnancy loss, ctup no adverse effect of air exposure on stallion sperm motility after 48 hours of cooled storage emily may, christian bisiau, paula moffett, jennifer hatzel, patrick mccue department of clinical sciences, college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co stallion semen may be collected, extended, and cooled for up to 48 hours prior to insemination. it is common practice to remove all the air from the package of extended semen prior to cooled storage. the aim of this pilot study was to assess the effects of air exposure on sperm motility parameters during 48 hours of cooled storage. we hypothesized that air exposure is associated with decreased sperm motility after 48 hours of cooled storage. a total of 12 ejaculates were collected (4 ejaculates from each of 3 stallions) using an artificial vagina. semen was evaluated and diluted with a commercial extender (inra 96, imv technologies, maple grove, mn) to a concentration of 25 x 106 progressively motile sperm per ml. the extended semen was aliquoted into 3 treatment groups. group a: 40 ml of extended semen was placed into a 50-ml all-plastic syringe with all air removed (henke-ject®, air tite-products co., inc., virginia beach, va). group b: 20 ml of extended semen was placed into a 50-ml syringe with all air removed. group c: 20 ml of extended semen was placed into a 50-ml syringe along with 20 ml of air. the loaded syringes were placed into passive cooling containers (equine express ii™ cooled semen shipper™ boxes, nasco, fort atkinson, wi) along with a frozen ice pack (polarpack®, sonoco, hayward, ca). an aliquot (1 ml) of clinical theriogenology 2021; 13: 307 semen was removed after 24 and 48 hours of cooled storage and warmed for 10 minutes at 37°c prior to evaluation of sperm motility parameters using a computer assisted sperm analysis unit (spermvision®, minitube of america, inc., verona, wi). data are presented as a mean ± sd. a mixed model was fit to each response variable separately (sas institute, carey, nc). treatment (40 ml, 20 ml, or 20 ml plus air) was included as a fixed effect. sample id was included as a random effect to account for repeat observations on each sample. tukey adjusted pairwise comparisons were also performed. data were considered different at p < 0.05. total sperm motility values for groups a, b, and c after 24 hours of cooled storage were 71.9 ± 14.3, 73.3 ± 13.3 and 76.3 ± 12.5 %, respectively. total sperm motility values after 48 hours of cooled storage for groups a, b, and c were 65.6 ± 14.1, 65.8 ± 17.3 and 70.9 ± 12.8%, respectively. progressive sperm motility values for groups a, b, and c after 24 hours of cooled storage were 66.7 ± 14.9, 67.8 ± 14.4 and 71.7 ± 14.3%, respectively. finally, progressive sperm motility values for groups a, b, and c after 48 hours of cooled storage were 60.3 ± 13.6, 60.8 ± 17.8 and 65.7 ± 14.3%, respectively. a difference (p < 0.05) in total and progressive motility was detected between group a and group c after 24 hours of cooled storage. there were no differences (p > 0.05) in total or progressive sperm motility values between aliquots of extended stallion semen in the presence or absence of air after 48 hours of cooled storage. these pilot data suggest that the necessity of removing all air during preparation of a cooled semen dose may not be as absolute as previously considered. keywords: equine, cooled semen, air, sperm, motility comparison of nanoparticles and single-layer centrifugation for separation of dead from live stallion sperm christian bisiau,a paula moffett,a james graham,b patrick mccuea adepartment of clinical sciences; bdepartment of biomedical sciences, college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co artificial insemination with fresh, cooled or frozen semen is commonly used in the equine breeding industry. poor quality semen with a reduced number of live, motile sperm can lead to lower per cycle pregnancy rates. a study using boar sperm showed improved sperm motility when nanoparticles were used to separate dead from live sperm.¹ the objective was to determine if nanoparticles could separate dead from live stallion sperm. our hypothesis was that iron-core nanoparticles bind to dead sperm and allow for subsequent separation from live sperm using a magnet. experiment 1 compared 2 extenders (inra 96 and talp-e), 2 incubation temperatures (22 and 37°c) and 6 nanoparticle:sperm ratios (50, 100, 200, 400, 600, and 800 µl of nanoparticle working solution per 100 x 106 sperm) using magnetic nanoparticles (st genetics, navasota, tx, us). a research model to mimic a poor-quality ejaculate was made by killing 50% of the sperm by submersion into liquid nitrogen. experiment 2 compared sperm separation using single-layer centrifugation (slc) with equipure™ (nidacon international ab, mölndal, sweden) versus nanoparticle separation. in both experiments, total and progressive sperm motility, morphology, viability and acrosome status were evaluated. statistical analysis was performed using one-way anova (data presented as mean ± sd). values were considered different at p < 0.05. results of experiment 1: total and progressive sperm motility were not different between inra 96 and talp-e extenders or when incubated at either 22 or 37°c or when using 400 or 600 µl of nanoparticle solution per 100 x 109 sperm. results for experiment 2: progressive sperm motility was higher (p < 0.05) after slc (76 ± 9%) than after either nanoparticle treatment (59 ± 12%) or an untreated control (47 ± 5%). in addition, the percentage of viable and acrosome intact sperm was higher after slc (61 ± 11%) than after nanoparticle treatment (43 ± 3%) or an untreated control (35 ± 3%). there was no statistical difference in sperm morphology among groups. in summary, under the current study conditions based on an induced sperm damage model, single-layer centrifugation performed better than nanoparticles for separating dead from live stallion sperm. keywords: stallion, sperm, nanoparticles, single-layer centrifugation reference 1. durfey cl, burnett dd, liao sf, et al: nanotechnology-based selection of boar spermatozoa: growth development and health assessments of produced offspring. livest sci 2017;205;137-142. comparing serum progesterone measurements by a pointof-care analyzer with a chemiluminescent immunoassay in bitch breeding management of the bitch julia zuercher,a katie boes,b orsolya balogh,c alyssa helms,c julie cecerec advm candidate 2021, bdepartment of biomedical sciences and pathobiology, cdepartment of small animal clinical sciences, virginia-maryland college of veterinary medicine, blacksburg, va accurate serum progesterone measurements for timing bitches during breeding management is critical for reproductive practice. by monitoring the rise in progesterone during estrus, it is possible to predict the date of ovulation and the peak window of fertility, which is especially important as artificial insemination has become routine to facilitate breeding of animals that are geographically or temporally separated. although progesterone is a highly conserved molecule across species, laboratory methods for measuring serum progesterone concentrations in the dog vary in accuracy and precision. to measure serum progesterone, chemiluminescent immunoassay (clia) has replaced radioimmunoassay as the current standard in the bitch, due to its high correlation and increased practicality. in january 2019, a colorimetric point-of-care (poc) immunoassay was released as an in-clinic diagnostic for quantitative canine clinical theriogenology 2021; 13: 308 serum progesterone measurements in less than 30 minutes. this study provides an independent comparison of the poc (catalyst one, idexx) to the current industry standard, clia (immulite-2000, siemens), used by most veterinary reference laboratories. to assess inter-assay imprecision of poc and agreement of the poc and clia results, 100 canine serum samples were analyzed on 3 analyzers (poc-1, poc-2, and clia), of which, 74 (poc-1) and 75 (poc-2) results were within pocs’ reportable range of 0.2-20 ng/ml and included in the study. to assess intra-assay imprecision, pooled canine serum samples at low (l1), intermediate (l2), and high (l3) progesterone concentrations were analyzed 10 times each on poc-1 and clia. relative to clia, poc values had good correlation (poc-1, r = 0.9366; poc-2, r = 0.9438, p < 0.0001) and significant positive proportional bias at values > 2 ng/ ml. the poc inter-assay coefficients of variation (cvs) were 13.2% (0.2-2.9 ng/ml, 0.6-9.2 nmol/l, l1), 10.0% (3.0-9.9 ng/ ml, 9.5-31.5 nmol/l, l2), 7.1% (10.0-20.0 ng/ml, 31.8-63.6 nmol/l, l3), and 11.2% (all samples). the intra-assay cvs for poc (l1, 15.3%; l2, 7.0%; l3, 4.7%) were higher than those for clia (l1, 5.89%; l2, 4.89%; l3, 3.44%). the poc had a more rapid increase in serial serum progesterone concentrations in ovulating bitches and had greater imprecision than clia. therefore, caution should be used when interpreting the clinical significance of serum progesterone measurements by the poc as they relate to canine breeding management. keywords: dog, commercial assays, catalyst, accuracy, imprecision kisspeptin-10 on in vitro migration of equine chorionic girdle trophoblast cells viviane gomes,a kassandra crissman,b olivia geels,a victoria bailey,b caroline camp,a chin-chi liu,a christianne magee,c jenny sonesa aschool of veterinary medicine, bcollege of agriculture, louisiana state university, baton rouge, la, ccollege of veterinary medicine and biomedical sciencescolorado state university, fort collins, co chorionic girdle (cg) is a specialized component of the equine extraembryonic membranes, composed of rapidly proliferating uninucleated and terminally differentiated binucleated trophoblast cells (utcs and btcs, respectively). gonadotropin-secreting btcs invade the maternal endometrium to form key structures for pregnancy maintenance known as endometrial cups. mechanisms that regulate btc migration and invasion remain elusive. kisspeptins (kps), a family of small peptides with 10 (kp-10) to 54 (kp-54) amino acids, are highly expressed at the maternal-fetal interface during human and rodent placentation and may inhibit excessive tc invasion. hence, we aimed to investigate the effect of the equine kp decapeptide (ekp-10) on cg cell in vitro migration using gap closure and vesicle expansion assays. it was hypothesized that ekp-10 inhibit cg vesicle expansion and the closure of utc/btc monolayer gaps. chorionic girdle was isolated from embryos collected transcervically at 33 34 days postovulation (n = 5 mares). following mechanical dissociation, cg cells were cultured at 37°c in 8% co2 on serum-supplemented (sm) or serum-free (sfm) medium containing dulbecco’s-modified eagle’s medium. approximately 500 µm cell-free gaps were formed using silicone inserts (ibidi®). once ~ 90% confluency was achieved on both sides of the gap, cells were treated with 0 (control), 1, 10, and 100 µm of ekp-10 and photomicrographs were taken at 0, 6, 12, and 24 hours (n = 8 wells/group) with a nikon inverted microscope. concurrently, individualized cg vesicles were transferred from sfm to sm and treated with 0, 0.1, 10, and 100 µm of ekp-10 (n = 20 vesicles/group). photomicrographs were taken at 0, 12, 24, 36, and 48 hours. gap widths and vesicle areas were measured by observers blinded to the experimental design using image j®. data were assessed via mixed anova and post-hoc tukey’s tests. significance was set at p < 0.05 (jmp pro 15). gap closure was slower (p = 0.04) in wells treated with 100 µm of ekp-10 compared to control, whereas there was no difference (p > 0.05) in closure among control and groups treated with 1 µm or 10 µm of ekp-10). vesicle expansion occurred in all treatment groups and there was an interaction (p = 0.0008) between time and treatment. within each time point, compared to control, vesicle expansion rate was lower in 0.1 (p = 0.002) and 10 µm ekp-10 (p = 0.03) at 24 hours, and was also lower (p = 0.007) at 36 hours in 0.1 µm and ekp-10. interestingly, compared to control, expansion rate was higher (p = 0.04) in groups treated with 100 µm ekp-10 at 48 hours. therefore, ekp-10 may affect the migration of subpopulations of cg cells dynamically and in a concentrationand time-dependent manner. further investigations of kp expression in the equine maternal-fetal interface and potential role in endometrial cup formation are needed. keywords: mare, endometrial cups, binucleated, trophoblast cells, invasion. clinical theriogenology 2021; 13: 309 electroejaculation and breeding soundness examination on a clouded leopard taylor lashlee,a adrienne difoggio,a pablo jarrin,a heather coarsey schwartz,b margarita colburn,b andrew cushing,a tulio pradoa acollege of veterinary medicine, university of tennessee, knoxville, tn, bnashville zoo, nashville, tn in previous studies with clouded leopard electroejaculation, a variety of techniques have been performed, from varying probe sizes to varying stimulation procedures.1-5 in 2018, the smithsonian conservative biology institute released a felid semen collection, evaluation, and freezing procedure that can be used in clouded leopards. this procedure uses a 1.6 or a 1.9 cm probe.1 however, in this case, we used a 10 12 cm probe with 2 electrodes. this probe is commonly used in small ruminants, and therefore, more widely available to veterinarians than a probe specifically used for wild felids. the purpose of using the pulsator iv electro-ejaculator with a ram probe was to determine if a more widely available probe could be used to collect semen from a clouded leopard or other species of similar anatomic size. a 3-year-old, intact male clouded leopard (neofelis nebulosa) from the nashville zoo was presented to the university of tennessee college of veterinary medicine theriogenology service for a breeding soundness examination. on physical and reproductive ultrasonographic examinations, the clouded leopard appeared healthy with no physical abnormalities aside from an abnormally small left testis. semen was collected with the pulsator iv electro-ejaculator with a ram probe which is commonly used to collect semen in small ruminants. approximately 0.2 0.3 ml of semen was collected using said device during this procedure. collected semen was then examined for motility and morphology to determine the viability of the leopard’s semen. semen was determined to have 40% motility and 66% normal morphology. thus, the breeding soundness exam concluded this leopard had viable semen and a good chance to impregnate a female, and that the pulsator iv electro-ejaculator with a ram probe can be used to collect semen sample for evaluation in clouded leopards. keywords: electroejaculator, ram probe, clouded leopard references 1. smithsonian conservation biology institute. felid semen collection, evaluation, and freezing procedures. 2018. 2. zainuddin z, tarmizi mm, yap k, et al: first evaluations and cryopreservation of semen samples from sunda clouded leopards (neofelis diardi). animals 2020;10:1072. doi:10.3390/ani10061072 3. wildt de, howard jg, hall, ll, et al: reproductive physiology of the clouded leopard: i. electroejaculates contain high proportions of pleiomorphic spermatozoa throughout the year. biol reprod 1986;34:937-947. doi:10.1095/biolreprod34.5.937 4. durrant bs: semen collection, evaluation, and cryopreservation in exotic animal species: maximizing reproductive potential. ilar journal 1990;32;2-10. doi:10.1093/ilar.32.1.2 5. howard jg, roth tl, byers, et al: sensitivity to exogenous gonadotropins for ovulation induction and laparoscopic artificial insemination in the cheetah and clouded leopard. biol reprod 1997;56:1059-1068. doi:10.1095/biolreprod56.4.1059 preputial prolapse and injury in a brahman bull maryanna hudson, carolyn chisholm, kevin walters college of veterinary medicine, mississippi state university, mississippi state, ms a 3-year-old brahman, bull presented with preputial prolapse secondary to multiple preputial injuries. extensive swelling and edema engorged the prepuce in its entirety, with necrotic debris present along the ventral and left lateral aspects. the necrotic tissue was debrided and the prepuce was scrubbed with betadine before placing a pessary tube. medical grade honey and petercillin were applied to the edematous preputial tissue with appropriate bandaging to hold the tube in place and promote reduction of edema. a support sling was applied, along with daily osmotic and hydrotherapy for prolapse reduction. numerous tissue defects were allowed to heal by second intention before a reefing procedure was performed. bos indicus bulls have a pendulous sheath, redundant preputial tissue, enlarged preputial orifices, and lack retractor prepuce muscles in homozygous polled bovids, predisposing them to preputial prolapse and injury.1,2 most preputial injuries occur at the ejaculatory lunge when epithelial and underlying elastic tissues of the bunched prepuce are damaged due to compressive forces between the bull’s abdomen and the female’s pelvis.1 these tissue disruptions lead to edema, inflammation, further prolapse of preputial tissues, subsequent injury and fibrotic scar formation upon healing.1,2 whereas medical management is sufficient for returning tissues to the preputial cavity, surgical intervention is paramount for returning to breeding function.1,4 a reefing (circumcision) procedure facilitates full extension of the penis and student clinical case session clinical theriogenology 2021; 13: 310 prepuce to allow excision of ancillary and scarred preputial tissue,3,4 ensuring 1.5 times the length of the free portion of the penis of preputial tissue is left to reduce the risk of phimosis and recurrent prolapse.1,2 prognosis following recovery from this procedure is good to excellent provided hemorrhage is minimized, urine flow is diverted away from the surgical sites, and extension of the penis remains achievable.3,4 keywords: prepuce, prolapse, injury, bos indicus, reefing references 1. maxwell h: inability to breed due to injury or abnormality of the external genitalia of bulls. in: hopper rm, editor. bovine reproduction. hoboken: joe wiley & sons; 2015. p.115-116. 2. gill ms: surgery of the prepuce. in: gill ms: editor. penile and preputial problems in the bull. 2003. 3. wolfe df: restorative surgery of the prepuce. in: hopper rm, editor. bovine reproduction. hoboken: joe wiley & sons; 2015. p. 142-150. 4. hopper r: breeding bull injuries. 2015. atypical mammary mass in an intact geriatric female labrador retriever colleen kutzler,a margaret root kustritz,a michelle kutzlerb acollege of veterinary medicine, university of minnesota, st. paul, mn; bcollege of agricultural sciences, oregon state university, corvallis, or mammary pathology is a significant finding in any bitch. published research suggests intact bitches are at a greater risk of developing mammary neoplasia than those ovariectomized before their first heat cycle.1 a 12-year-old intact female black labrador retriever was examined in february 2021 for a rapidly growing mammary mass. the mammary mass was first noticed by the owner in july 2020 and grew slowly until december when it began to rapidly increase in size following standing estrus in late november. at presentation, a fluctuant mass (15 cm) extended from the cranial left abdominal mammary gland to the caudal abdominal mammary gland. the left inguinal lymph node was also enlarged and painful during palpation. all other lymph nodes palpated normally. the patient was over-conditioned, had age-related dental attrition, bilateral nuclear sclerosis, and a soft, free-moving subcutaneous mass medial to the right cranial abdominal mammary gland. all other physical exam findings were within normal limits. an ultrasonographic exam was performed on the mammary mass that revealed an accumulation of hypoechoic, heterogenic fluid with tags of tissue extending from the margins of the mass into the fluid filled center. hair over the mass was clipped and skin was prepared for a fine needle aspirate which retrieved a hemopurulent, thin and nonfetid fluid. few epithelial cells and red blood cells, moderate neutrophils, and no bacteria were evident on cytology. an aerobic bacterial culture of the fluid yielded no growth after 4 days. the mass was diagnosed as a sterile intramammary abscess. the patient was sedated with iv fentanyl and medetomidine then standard surgical preparation and anesthetic monitoring were used throughout the procedure. stab incisions were made on the cranioventral and caudoventral aspects of the abscess. compression was applied on the skin over the abscess to facilitate drainage. a gloved finger was used to digitally probe the abscess and debride tissue within the cavity. the cavity was lavaged repeatedly with dilute chlorhexidine solution and 2 penrose drains were placed. the patient was reversed with intramuscular atipamezole. recovery was uneventful and the patient was discharged on oral trimethoprim sulfa (960 mg twice daily for 2 weeks) and oral meloxicam (once daily for 5 days). the patient removed 1 drain tube 5 days after the procedure. reevaluation was performed at 7 and 14 days with normal healing and minimal discharge. the second drain tube was removed 14 days after the procedure. at this abstract submission, the patient has not had recurrence of the mammary swelling. the cause of this sterile abscess is not known. why the sterile intramammary abscess increased in size exponentially during diestrus is also unknown, though it may have to do with increased blood supply to the mammary glands during this stage of the estrous cycle. due to financial constraints, concurrent mammary pathology could not be ruled-out histologically. although the draining lymph node was enlarged, the pain and enlargement were likely due to reactivity from the abscess rather than neoplasia. additional diagnostics were not performed. in addition, thoracic radiographs were not performed. this case illustrated that not all mammary masses are neoplastic in geriatric intact bitches. keywords: canine, intramammary, sterile abscess, diestrus, geriatric reference 1. schneider r, dorn cr, taylor do: factors influencing canine mammary cancer development and postsurgical survival. j natl cancer inst 1969;43:1249-1261. abnormal mobility in neonatal labrador retrievers jordan farrell,a robyn wilborn,a pamela haney,b jamie douglas,a aime johnsona adepartment of clinical sciences, bcanine performance sciences, college of veterinary medicine, auburn university, auburn, al a litter of 3-week-old labrador retrievers (n = 5) were evaluated for abnormal ambulation that was noted in 1 puppy. upon presentation all puppies were evaluated for mobility on a blanket to improve traction. it was determined that the puppy in question had abnormal movement of the rear limbs compared to the remainder of the litter. in addition to this finding, a second puppy was determined to have an abnormally flat chest with decreased ambulation compared to littermates. the owner provided a daily weight chart and records of developmental milestones, such as opening eyes and first steps. upon review clinical theriogenology 2021; 13: 311 of this information, it was determined that the average daily gain of this litter was 3.37 ounces (95.5 grams) per day and that the 2 puppies exhibiting abnormalities had indeed met all developmental milestones within the same timeframe as littermates. despite receiving no nutrition beyond milk supplied by the dam, all 5 puppies were considered obese. of note, milk supply was plentiful, presumably due to the relatively small litter size. prior to pursuing further diagnostics, the owner elected to complete at-home physical therapy and dietary management for both affected puppies and monitor for improvement. physical therapy consisted of passive range of motion of both hind limbs and climbing over rolled towels for 15 minutes twice daily. after 1 week of physical therapy and dietary restriction, both puppies had improved overall mobility consistent with littermates and the puppy with altered hindlimb gait had a more consistent pattern of normal ambulation. physical therapy was performed until 6 weeks of age with continued improvement of ambulation. at 13 weeks of age, both affected puppies successfully entered training to become working dogs. this case illustrated the importance of monitoring puppy weights and the detection of early abnormalities to prepare the animal for the best outcome keywords: neonatal, canine, conformation, working dog references 1. peterson me, kutzler ma: small animal pediatrics: the first 12 months of life. 1st edition, saunders;2011. 2. aggouni c, albaret a, anne j: neocare5 years of science to improve the health of newborn puppies. royal canin 2015. 3. karcher de, costa rc, prada tc: a modified technique for treating swimmer puppy syndrome. veterinarni medicina. 2018;63. .recurrent uterine torsion in an arabian mare vaiva palunas, eduardo arroyo, cristian patiño, michela ciccarelli, ahmed tibary college of veterinary medicine, washington state university, pullman, wa an 11-year-old multiparous 270 days pregnant arabian mare was referred for a suspected uterine torsion. on arrival the mare was colicky, tachycardic, and normothermic. a 270 degree clockwise uterine torsion was diagnosed by transrectal palpation. she was anesthetized using a triple drip regimen and rolled 5 times using the flank plank technique. the mare recovered uneventfully. two days after uterine torsion resolution the ctup (6 mm) was within normal limits. the fetus was alive, and the mare was discharged. ten days later, the mare returned with another colic episode. she was examined and a 90° clockwise torsion was identified. the mare was rolled again and recovered uneventfully. one day after resolution of the torsion, serum progesterone concentrations were normal (9.88 ng/ml) but serum estrogen concentrations (765.12 pg/ml) were lower than expected at 280 days of pregnancy. there was a concern about this as estrogens are a good indicator of fetal viability and tend to decline over the last trimester of pregnancy.1 the mare delivered a healthy colt at home at 344 days. uterine torsion in the mare is most common in mid to late pregnancy. suspected causes include vigorous fetal movement, rolling, a large fetus in a relatively small volume of fetal fluid, lack of uterine tone, and a deep abdomen in larger breeds.2 transrectal palpation is the best way to diagnose uterine torsion and identify its direction that is essential for treatment. repeated uterine torsions during the same pregnancy are rare. this case is important to the field of theriogenology because uterine torsion in the mare is an emergency and presents with nonspecific clinical signs. client education regarding the urgency of colic signs or discomfort in late pregnancy is essential. early diagnosis and treatment improve outcome for the mare and foal. close monitoring until foaling should be recommended. keywords: high risk, plank flank technique, endocrinology, colic references 1. curcio br, canisso if, pazinato fm, et al: estradiol cypionate aided treatment for experimentally induced ascending placentitis in mares. theriogenology 2017;102:98-107. 2. martens ka, govaere j l, hoogewijs mk, et al: uterine torsion in the mare: a review and three case reports. vlaams diergeneeskundig tijdschrift 2008;77:397-405. .dystocia and diaphragmatic hernia in a quarter horse mare carrying twins vaiva palunas, cristian patiño, eduardo arroyo, michela ciccarelli, ahmed tibary college of veterinary medicine, washington state university, pullman, wa twin pregnancy is one of the major noninfectious causes of abortion and may jeopardize the health and welfare of the mare. a 16-year-old primiparous quarter horse mare was referred for dystocia due to abortion at ~ 10 months of pregnancy. she was depressed, tachycardic, tachypneic, and hypothermic. rectum and vulva were edematous and malodorous; fetal membranes were protruding from the vulva. due to signs of septic shock, she was treated with intravenous fluid and corticosteroids prior to attempting controlled vaginal delivery. the fetus was emphysematous and in anterior presentation with bilateral shoulder flexion and lateral deviation of the neck. due to poor prognosis, humane euthanasia of the mare was performed. on necropsy, the uterus was friable and edematous with a partial thickness tear. dysmature, autolyzed twins were removed from the uterus. an acute diaphragmatic hernia of the large colon was also diagnosed, which may have been a complication of straining efforts and the enlarged uterus. incidence of double ovulation ranges from 8 21% in quarter horses, with an incidence of twin pregnancy ranging from 8 11%.1 spontaneous twin reduction occurs frequently by day 40 of pregnancy in clinical theriogenology 2021; 13: 312 unilaterally fixed embryos; probability of twin maintenance increases substantially with bilateral fixation.2 the standard practice is to attempt to reduce the pregnancy to 1 vesicle after pregnancy diagnosis at days 14 15, prior to embryonic fixation. in this case, pregnancy diagnosis was not performed after natural breeding. the prolonged duration of the dystocia and autolysis of the fetuses resulted in severe deterioration of the mare’s health. the majority (64.5%) of twin pregnancies maintained past 42 days result in late term abortion.3 this case illustrated the importance of close monitoring of follicular dynamics during breeding, early pregnancy diagnosis, and the need for client education on the importance of adequate breeding management for the welfare of the mare. keywords: twins, abortion, dystocia, diaphragmatic hernia, breeding management references 1. chavatte p. twinning in the mare. equine vet educ 1997;9:286-292. 2. ginther oj: relationships among number of days between multiple ovulations, number of embryos and type of embryo fixation in mares. j equine vet sci 1987;7:82-88. 3. jeffcott lb, whitwell ke: twinning as a cause of foetal and neonatal loss in the thoroughbred mare. j comp pathol 1973;83:91-106. vulvar discharge associated with exogenous estrogen exposure in a spayed weimaraner bitch camille ogdon, francesca ivaldi, firdous khan school of veterinary medicine, st. george’s university, grenada, west indies vulvar discharge in spayed bitches is often associated with infections, chemical irritation, foreign bodies, anatomical defects, neoplasia, or an ovarian remnant.1 rarely, the discharge is associated with exposure to exogenous hormones.2 a 4-year-old spayed weimaraner bitch was presented for evaluation of inappetence and intermittent sanguineous vulvar discharge. the patient had elevated rectal temperature and respiration rate. physical examination indicated presence of vulvar edema and a sanguineous vulvar discharge. vaginal cytology revealed mainly parabasal cells, occasional intermediate cells, and abundant neutrophils and red blood cells. ultrasonographic findings were suggestive of an enlarged, fluid-filled uterine stump, and a complete blood count (cbc) indicated leukocytosis, neutrophilia, and monocytosis. a uterine stump pyometra due to ovarian remnant syndrome was suspected and celiotomy performed. the uterine stump appeared grossly cystic and thickened. histopathological evaluation of the removed uterine stump and ovarian pedicles revealed cystic endometrial hyperplasia and no ovarian tissue. fifteen days after surgery, the patient presented again with a sanguineous vulvar discharge. vaginal cytology revealed predominantly superficial cells, indicating estrogen influence.3 differential diagnoses included ectopic ovarian tissue, exogenous estrogen exposure, or an adrenal tumor. further questioning of the owner revealed long-term use of a topical estrogen cream by a member of the household. serial examinations were performed, and the cytology remained uniform, with predominantly superficial cells, indicating continued estrogen influence.3 progesterone and anti-müllerian hormone concentrations were determined to rule out ectopic ovarian tissue. both tests came back negative. the absence of 4,5 any clinical signs of adrenal disease coupled with the history of topical estrogen cream use in the household suggested that the patient’s clinical signs were likely due to exogenous estrogen exposure. several recommendations were made to prevent the exposure. follow-up vaginal cytology and cbc evaluations were also recommended to monitor future estrogen exposure and possible adverse effects on the patient’s health. keywords: dog, cystic endometrial hyperplasia, exogenous estrogen, vaginal cytology references 1. lopate, c: reproductive disorders of the spayed bitch. clinical theriogenology 2014;6:205-217. 2. sterman aa, mankin kt, barton cl: stump pyometra secondary to human topical estrogen hormone exposure in a spayed female chihuahua. j am anim hosp assoc 2019;55:e556-04. 3. feldman ec, nelson rw: ovarian cycle and vaginal cytology. in: feldman ec, nelson rw: editors. canine and feline endocrinology and reproduction, 3rd edition, st. louis; saunders: 2004. p. 752-774. 4. wallace ms. the ovarian remnant syndrome in the bitch and queen. vet clin north am small anim pract 1991;21:501-507. 5. turna yilmaz ö, toydemir ts, kirsan i, et al: anti-müllerian hormone as a diagnostic tool for ovarian remnant syndrome in bitches. vet res commun 2015;39:159-162. phimosis and preputial abscessation with draining tract in an angus bull rachel doenges, heath king, darcie sidelinger mississippi state college of veterinary medicine, mississippi state, ms a 22-month-old, angus bull presented for preputial laceration and phimosis with secondary cellulitis and ventral swelling cranial to the scrotum. the bull received 2 doses of ceftiofur crystalline free acid, 1 dose of transdermal banamine, and a 5 day course of penicillin. a preputial abscess was suspected. preputial lacerations commonly occur during breeding generally due to tissue rupture secondary to compressive force.1 preputial laceration and subsequent prolapse is more common in bos indicus breeds due to do their redundant preputial tissue and pendulous sheaths. however, bos taurus bulls are often capable of fully retracting preputial injuries leading to subsequent abscessation.1 prognosis is poor to guarded for return to breeding soundness.2,3 the bull was treated daily with hydrotherapy focused on the swelling. epsom salt poultice was applied over the swelling and a sweat wrap was applied using a support sling. after 6 days of treatment, the abscess ruptured along a draining tract that terminated near the preputial orifice. hydrotherapy, epsom salt clinical theriogenology 2021; 13: 313 poultice application, and the support sling were continued. the draining tract was lavaged daily through a catheter with a 10 liter carboy of normal saline containing 0.5% betadine solution. silver sulfadiazine was infused along the draining tract daily. the draining tract improved briefly but the abscess recurred and ruptured a second time. the silver sulfadiazine infusions were discontinued and the lavage frequency was reduced to every other day until the draining tract could no longer be accessed. after 33 days in clinic, the bull was discharged. the bull returned 6 weeks after discharge for evaluation and breeding soundness examination. on electroejaculation, erection, protrusion, and ejaculation were successful. his semen was of good quality with 70% motility and 83% normal morphology. this case demonstrated successful medical management of a condition that commonly has a poor clinical outcome. keywords: cattle, preputial laceration, retropreputial abscess references 1. maxwell h: inability to breed due to injury or abnormality of the external genitalia of bulls. in: hopper rm, editor. bovine reproduction. ames: wiley; 2015. p. 117-118. 2. memon ma, dawson lj, usenik ea, et al: preputial injuries in beef bulls: 172 cases (1980-1985). j am vet med assoc 1988;193:481-485. 3. wolfe d, beckett s, carson r: acquired conditions of the penis and prepuce (bulls, rams, and bucks). in: wolfe df, moll hd: editor. large animal urogenital surgery. baltimore; williams and wilkins: 1998, p. 237-272. spermatic cord torsion in an arabian stallion hannah neer, eduardo santos villanueva, lauren huggins, thomas bergstrom, isabelle kilcoyne, marcos perez-nogues, ghislaine dujovne, pouya dini pritchard veterinary teaching hospital, school of veterinary medicine, university of california-davis, davis, ca spermatic cord torsions greater than 180 degrees compromise the vascular supply to the affected testis and are a medical emergency in stallions.1 a 5-year-old arabian stallion presented for an episode of acute colic signs. diagnostic work-up including physical examination, bloodwork, abdominal ultrasonography and transrectal examination did not reveal a cause for colic signs. no sign of pain or discomfort was noted on or after admission. the left testis felt enlarged on palpation and a pronounced tubal structure was noted in the left inguinal canal during transrectal palpation. however, the scrotal structures were in the correct orientation on palpation and ultrasonography. scrotal ultrasonographic examination had an increased left spermatic cord diameter (6.5 cm) compared to right spermatic cord (3 cm). doppler demonstrated a reduction in blood flow through the left spermatic cord and testis compared to contralateral structures. although suggestive, the ultrasonographic findings did not confirm spermatic cord torsion (complete occlusion of the cord). therefore, it was elected to continue monitoring the stallion’s condition. on the following day, scrotal ultrasonography demonstrated changes to the left testicular parenchyma with minimal to no blood flow through the left spermatic cord. spermatic cord torsion of ≥ 360-degrees was diagnosed based on these findings and a closed hemicastration was performed. although potentially at higher risk for contralateral torsion,2 the owner elected hemicastration to preserve the breeding potential of the stallion. the stallion had an uneventful recovery and was discharged 5 days following presentation. ischemic (coagulative) necrosis was observed on histopathological analysis of the testicular parenchyma. although 360-degree spermatic cord torsions occur infrequently, torsion should always be considered for stallions presenting with symptoms of colic. it is important to note that colic signs and pain can subside due to the necrotic/ ischemic changes in ≥ 360-degree torsions. keywords: stallion, spermatic cord torsion, hemicastration references 1. perkins nr: reproductive emergencies in the stallion. vet clin north am equine pract 1994;10:671-683. 2. threlfall wr, carleton cl, robertson j, et al: recurrent torsion of the spermatic cord and scrotal testis in a stallion. j am vet med assoc 1990;196:1641-1643. clinical theriogenology 2021; 13: 314 student research session incidence of and factors affecting congenital defects in miniature dachshunds colleen kutzler,a michele kutzlerb acollege of veterinary medicine, university of minnesota, st. paul, mn, bcollege of agricultural sciences, oregon state university, corvallis, or a congenital defect is a malformation that is present and identifiable at birth. congenital defects may result from genetic and/or environmental influences. in a review of several dog breeds, the most common defects reported were cleft palate and hydrocephalus.1 in this review, dachshunds had a relatively low frequency of congenital defects compared to other breeds.1 however, the specific congenital defects in dachshunds were not stated. as such, the current study aimed to investigate the incidence of congenital defects in miniature dachshunds, along with the potential influences of neonatal sex, maternal age, and sire-dam consanguinity. descriptive statistics with an odds ratio was performed on the data. one hundred and nineteen puppies from 33 litters from a single kennel were examined at birth. fifty-nine were male and 60 were female pups. the mean ± sd litter size was 3.6 ± 1.3 pups. there were 3 stillborn pups (2.5%) including a schistosomus reflexus.2 additional congenital defects included severe retrognathism with class ii distocclusion (n = 2; 1.6%), hydrocephalus with occipital dysplasia (n = 1; 0.8%), and complete cleft palate (n = 1; 0.8%). one puppy with retrognathism also had congenital hindlimb rigidity that resolved spontaneously over the first 3 weeks of life. the overall frequency of congenital defects was 4.2%. within this kennel, the odds of having a congenital defect were 1.6 times larger in male than female pups. the incidence of congenital defects appeared (p = 0.334) to increase with maternal age (≤ 2 years old: 1 out of 56 pups [1.8%]; 3 5 years old: 2 out of 41 pups [4.9%]; ≥ 6 years old: 2 out of 22 pups [9.1%]) but this was not significant with this sample size using a chi square. none of the bitches with malformed pups were primiparous. one litter of consanguineal mating between half siblings did not result in malformed pups; however,1 litter with a consanguineal mating between mother and son and another litter between great grandmother and son resulted in 2 malformed pups (1 per litter). these results confirmed previous findings that dachshunds have a relatively low frequency of congenital defects.1 both cleft palate and hydrocephalus had been reported in dachshunds.1,3 in addition, the results from this study indicated that advancing maternal age may increase the incidence of congenital defects in dogs but more research with a larger sample size is needed to confirm this observation. keywords: dachshund, congenital, defects, factors references 1. nobre pacifico pereira kh, cruz dos santos correia le, ritir oliveira el, et al: incidence of congenital malformations and impact on the mortality of neonatal canines. theriogenology 2019;140:52-57. 2. kutzler c, root-kustritz m, kutzler m: dystocia due to a schistosomus reflexus in a miniature dachshund. clinical theriogenology 2019;11:507. 3. kobatake y, miyabayashi t, yada n, et al: magnetic resonance imaging diagnosis of dandy-walker-like syndrome in a wire-haired miniature dachshund. j vet med sci 2013;75:1379-1381. clinical theriogenology 2021; 13: 315 proteomic analysis of sperm with impaired acrosomal exocytosis from a subfertile stallion camilo hernández-avilés,a susan weintraub,b luisa ramírez-agámez,a brian davis,a terje raudsepp,a dickson varner,a charles lovea acollege of veterinary medicine, texas a&m university, college station, tx, buniversity of texas health science center, san antonio, tx stallion subfertility due to impaired acrosomal exocytosis has been reported in thoroughbred stallions that carry the genotype a/a-a/a for 2 snps in exon 5 of the fkbp6 gene (eca13). these animals are subfertile despite having otherwise normal sperm quality and good breeding management. mass spectrometry-based technologies are a powerful tool to investigate the potential causes of unexplained subfertility in the stallion and might further improve our understanding of the molecular processes that take place during fertilization. herein, we describe a preliminary experiment conducted to compare the sperm proteome from a fertile stallion (percycle pregnancy rate = 60%) and a subfertile stallion that carries the susceptibility genotype for iae (percycle pregnancy rate = 30%). fresh semen from each stallion was processed to induce spontaneous acrosomal exocytosis (ae) using a lactate-only-containing modified whitten’s medium (lac-mw). at 0, 4, and 6 hours of incubation, sperm aliquots were analyzed for sperm viability (viab) and the rate of ae in viable sperm (ae/viab) via flow cytometry (fitc-psa and fixable live/dead red stain). also, at each period, the sperm proteomes from each stallion were analyzed via data-independent acquisition mass spectrometry. student’s t-test was used to assess differences between experimental groups. during incubation in lac-mw, viab was similar (p > 0.05) between both stallions and was not affected by incubation time. ae/viab increased (p < 0.05) over time (0 hour: 3%, 4 hours: 32%, and 6 hours: 56%) for the fertile stallion, but not (p > 0.05) for the subfertile stallion (0 hour: 3%, 4 hours: 5%, and 6 hours: 5%). mass spectrometry analysis detected a total of 2,252 proteins in sperm (false discovery rate < 1.0%). of these, 144 proteins exhibited differences in relative quantity between the fertile and subfertile stallion (log2 fold change; p < 0.01). data analysis using the panther protein class annotation system revealed that most of the proteins with lower abundance (▼) in the subfertile stallion belonged to the calcium-binding protein (p = 3.36 x 10-5), and the metabolite interconversion enzyme (p = 1.11 x 10-7) groups, whereas proteins with higher abundance (▲) included those of the chaperonin (p = 8.25 x 10-4), protease (p = 5.21 x 10-4) and histone (p = 3.22 x 10-11) groups. among these, proteins of interest that were identified and are known to be related to sperm capacitation/acrosomal exocytosis and stallion fertility included: adam7 (▼), annexin-a2, -a4, and -a5 (▼), calpain-5 (▼), kallikrein-1e2(▼), and crisp-2 (▼). current experiments are focused on determining the potential relation between the fkbp6 gene genotype and the changes observed on the sperm proteome of more iae-affected stallions. keywords: stallion subfertility, acrosomal exocytosis, fkbp6, proteome thoraco-omphalopagus conjoined twins in a standardbred mare matthew cochran, patrick foth, melissa leonard, christopher premanandan, erin runcan, marco coutinho da silva ohio state university college of veterinary medicine, columbus, oh an eight-year-old standardbred mare presented to the ohio state university college of veterinary medicine in dystocia of 30 minutes duration. no breeding records were provided at presentation. pregnancy had been confirmed in this mare at 14 days postovulation with 1 embryonic vesicle visible. on vaginal palpation, the fetus was in posterior presentation, dorsosacral position with hindlimbs extended and there were no signs of fetal viability. general anesthesia was induced to allow for better manipulation of the fetus for extraction. during controlled vaginal delivery, 2 hind limbs were identified and a fetotomy was performed at the level of the tibiotarsal joint. following removal of 2 hind limbs, another hind limb was identified on palpation and removed via fetotomy at the level of mid-femur. following removal of 3 hind limbs, 2 distinct pelvises were identified on palpation. retropulsion of 1 pelvis was attempted, however no progress could be made to separate the suspected twins. caesarean section was recommended, but humane euthanasia was elected due to financial constraints. postmortem examination revealed a thoraco-omphalopagus conjoined twin following a ventral midline approach to the uterus. prior to necropsy of the conjoined twin, a computed tomography scan was performed. the calvarium of the conjoined twin was fused at the level of the facial crest caudal to the orbit. two independent vertebral columns with separate spinal cords were present connecting at a single sternum. one thoracic limb was present on clinical case session clinical theriogenology 2021; 13: 316 either side of the joined sternum. there was another fused thoracic limb that formed a bipedal hoof below the metacarpophalangeal joint with the metacarpal bones fused. there were 2 prepuces and 2 anuses present with meconium present in both anuses. two tails were also present. there was an umbilical hernia present with small intestines protruding through the hernia. respiratory and digestive tracts were fused at the larynx with a single trachea and esophagus. there was a single pair of lungs along with a single heart with 2 descending aortas. the single esophagus entered into a single stomach. the jejunum was divided into 2 ~ 90 cm oral to the ileocecocolic junctions. caudal to that point, there were 2 ilea, ceca, and large intestines present with fecal balls in both small colons. thoraco-omphalopagus conjoined twins have been reported in other species, including humans. to our knowledge, this is the first reported case of thoraco-omphalopagus conjoined twins in the horse. keywords: mare, dystocia, twins, thoraco-omphalopagus anaphylactic reaction following intrauterine administration of misoprostol in a mare jordan kiviniemi-moore rood & riddle equine hospital, lexington, ky deep horn intrauterine application of misoprostol, a synthetic prostaglandin e1 (pge1), has been demonstrated to improve fertility in mares suspected of oviductal dysfunction.1 adverse reactions in horses included mild abdominal discomfort and soft feces after oral misoprostol and in women anaphylactic reactions were observed after oral and vaginal treatment. an 18-year-old friesian mare weighing ~ 650 kg was presented for unexplained infertility of 3 years duration. misoprostol (600 µg, greenstone llc, peapack, nj) dissolved in sterile water was deposited at the tip of each uterine horn.1 ten minutes after treatment the mare collapsed in the stall. her mucous membranes appeared dark red with a prolonged capillary refill time of 4 seconds and she demonstrated tachycardia of 100 beats per minute, cool extremities, tachypnea of 60 breaths per minute, and was minimally responsive. dexamethasone (50 mg) and flunixin meglumine (750 mg) were given intravenously in addition to 6 mg epinephrine. a 14-gauge intravenous catheter was placed and 5 liters of lactated ringer’s saline (lrs) was given. while the mare was laterally recumbent, a cuffed intrauterine catheter was inserted and the uterus lavaged with 9 liters of lrs in 3 liter aliquots. fifteen minutes after the onset of treatment, the mare’s heart rate and respiratory rate improved and she was able to achieve sternal recumbency and stand with encouragement. however, 15 minutes later she again became tachycardic and tachypneic and collapsed a second time. an additional 6 mg of epinephrine was given intravenously in conjunction with continuous bolus fluids and the uterus was again lavaged with 9 liters of lrs. ten minutes after the second epinephrine the mare improved and regained ability to stand. her vital signs gradually normalized, she passed normal manure and began to graze. over the course of the 1.5-hour treatment window the mare received intravenously 50 mg dexamethasone, 750 mg flunixin meglumine, 12 liters of lrs, 2 doses (6 mg each) epinephrine, and twice 9 liters of uterine lavage with lrs. following recovery, no further ill effects of the incident were noted. the mare was bred with fresh cooled semen 2 weeks later but failed to become pregnant. she had no previously reported medication allergies or history of drug reaction. this is the first reported adverse event of its kind associated with intrauterine pge1 keywords: misoprostol, intrauterine, adverse reaction, anaphylaxis reference 1. alvarenga ma, segabinazzi lg: application of misoprostol as a treatment of unexplained infertility in mares. j equine vet sci 2018;71:46-50. next generation sequencing in deciding to discontinue antibiotic treatment in a stallion soon hon cheong, yamilka lago-alvarez, jennine lection, mariana diel de amorim department of clinical sciences, college of veterinary medicine, cornell university, ithaca, ny a 10-year-old morgan stallion presented for test cooling of semen with a history of mares not conceiving from cooled shipped semen. five weeks prior, the stallion was collected for semen shipment. the semen was greyish in color and had 60% progressively motile sperm (pms), mild teratozoospermia, 58% morphologically normal sperm, and 4.48 x 109 total number of sperm. penis was washed thoroughly before collection to minimize debris in the semen sample from the penis as a possible cause of semen discoloration. semen was collected using a missouri artificial vagina, and the semen sample was again greyish in color and had 3.4 x 109 total number of sperm, 60% pms, and 66% morphologically normal sperm. cytological examination of the semen sample with diff-quik stain had no leukocytes or germ cells. large numbers of branching rods were observed on cytology and an aerobic culture was performed. a fastidious and slow-growing actinomyces species was identified by culture. antibiotic susceptibility was not possible due to the bacteria’s slow-growing nature. actinomyces has been reported to infect the testes and accessory sex glands in humans. empirical antibiotic selection was based on published reports and oral doxycycline (10 mg/kg) was prescribed for 8 weeks. stallion was presented again 43 days later. semen appeared normal with improved number of total sperm (7.45 x 109), 60% pms, and 90% normal morphology. a culture was performed, and the sample was submitted for next generation sequencing (ngs) of 16s rrna for bacteria and its for fungi. the culture was negative, and ngs had 59% klebsiella oxytoca, 18% petrimonas sp., 7% streptococcus uberis, 3% corynebacterium kroppenstedii, 2% luteococcus sp., and 2% proteiniphilum sp. for bacteria and 91% clinical theriogenology 2021; 13: 317 of its reads were mucor circinelloides and 8% pleospora herbarum. traditional culture method is of limited use to identify when antibiotic treatments can be discontinued due to antibiotics’ inhibitory effects on culture results. the ngs results had several mixed bacteria and fungi that are more resistant to antibiotics. actinomyces species was not identified, which we interpreted as a positive sign. the improved spermiogram, negative culture, and lack of actinomyces detected by ngs were indications that antibiotic treatment could be discontinued. ideally, ngs should have been performed prior to the start of antibiotic treatment to be used as a baseline, and further studies are indicated to determine thresholds of potentially pathogenic bacteria on fertility. availability of a commercial clinical ngs laboratory service for the veterinary industry is a potentially groundbreaking advancement. one potential use of this technology may be to identify when long-term antibiotic treatments can be safely discontinued. keywords: stallion, actinomyces species, next generation sequencing thromboembolic disorder in a postcesarean section bitch yamilka lago-alvarez, hailey rose, denae campanale, ian porter, kenneth simpson, soon hon cheong, mariana diel de amorim department of clinical science, college of veterinary medicine, cornell university, ithaca, ny pregnancy and the postpartum period substantially increase the risk for venous thromboembolism (vte) in women, a disease that leads to pulmonary embolism and deep venous thrombosis. the most important risk factor contributing to a thrombotic event is the hypercoagulable state that occurs during a normal pregnancy, with the highest risk occurring the first 6 weeks postpartum. risk factors among women are age (> 35-years-old), cesarean delivery, hypertension, heart disease, obesity, and the presence of infection postpartum. to the authors’ knowledge, this condition has not been reported in dogs. a 3-year-old female intact primiparous labrador retriever presented as an emergency due to fever (104.3°f), lethargy, and anorexia. history included dystocia due to secondary uterine inertia with fetal distress (stillborn fetus) that was resolved by emergency cesarean section 4 days prior to presentation. on initial evaluation, the patient was quiet and responsive, obese with a body condition score of 8/9, and had a moderate amount of lochia with no foul-odor. bloodwork revealed a normocytic, normochromic, regenerative anemia (hematocrit of 27%, absolute reticulocytes 99.6 x 103/μl), leukocytosis with a neutrophilia characterized by a left shift and monocytosis (wbc 43.6 x 103/μl, reference interval [ri] 5.7 14.2; segmented neutrophils 35.8 x 103/μl, ri 2.7 9.4; band neutrophils 0.4 x 103/μl, ri 0.0 0.1; monocytes 2.6 x 103/μl, ri 0.1 1.3), hyperproteinemia (8.0 g/dl, ri 5.9 7.8) and hypoalbuminemia (1.8 g/dl, ri 3.2 4.1). considering the clinical condition, medical intervention and hospitalization for further diagnostic testing were pursued. medical management consisted of intravenous plasma-lyte a fluids (60 ml/kg/ day), ampicillin/sulbactam (unasyn, 30 mg/kg every 8 hours), and maropitant citrate (cerenia®, 1 mg/kg every 24 hours). a disseminated intravascular coagulation panel indicated significantly increased d-dimers (4965 ng/ml, ri 0 575), decreased antithrombin iii activity (50%, ri 65 145), and an increased aptt (19.6 seconds, ri 8.5 -15.5). abdominal ultrasonography revealed a diffusely, severely mottled spleen with innumerable hypoechoic regions of acute, multifocal infarction, suggesting the presence of splenic infarcts and no evidence of peritonitis. that evening, the patient developed labored breathing with a respiratory rate of 36 breaths per minute and pulse oxygenation of 93% (ri > 95%). an arterial blood gas revealed an alveolar-arterial oxygen gradient of 30 mmhg (ri 10 25). these findings were concerning for pulmonary thromboembolism. the patient improved after 24 hours of supplemental oxygen, and the supplementation was discontinued. subsequent echocardiogram revealed no evidence of pulmonary hypertension. the patient was discharged 48 hours after hospitalization with oral amoxicillin/clavulanate (13.75 mg/kg every 12 hours for 14 days). considering the clinical manifestation and increased risk reported with cesarean delivery and peripartum obesity in women, it was presumed that the pregnancy-related hypercoagulable state and postpartum period led to the development of a vte in the postcesarean section bitch. keywords: mare, pregnancy, placenta, cervix, ultrasonography funding: cesarean section, hypercoagulable, pulmonary embolism, venous thromboembolism polled intersex syndrome in a finnish landrace lamb katelyn waters, jenna stockler, richard hopper, ester malmström, yatta boakari, julie schnuelle department of clinical sciences, college of veterinary medicine, auburn university, auburn, al an apparently healthy, 4-month-old female finnish landrace lamb was presented for the presence of female and male external genitalia. the lamb was polled and a twin to a normal female. on initial examination the lamb had a short anogenital distance of 3.5 cm and a normal appearing vulva with a mildly enlarged clitoris. left gonad was descended and palpable in the inguinal area, whereas the other gonad was suspected to be retained intra-abdominally. left inguinal ring was severely dilated and the descended gonad could be moved freely intra and extra-abdominally. abdominal ultrasonography revealed a retained intra-abdominal right gonad that appeared testicular in origin with a hyperechoic mediastinum testis and a less echogenic parenchyma as seen in a normal testis. a fluid filled uterus was also identified, but ovaries were unable to be identified. lamb underwent a midline exploratory laparotomy with a bilateral castration/hysterectomy. uterus was identified and the uterine horns were followed to the location of the ovaries where testicular appearing structures with a pampiniform plexus clinical theriogenology 2021; 13: 318 and epididymis were identified. the testicular tissue was attached to the broad ligament as the uterus and ovaries would be. both testes were ligated and removed, the uterus was removed via a routine hysterectomy. incision was closed in a routine fashion, the lamb recovered from anesthesia with no complications. lamb was euthanized at a later time. uterus and gonads were submitted for histopathology. histopathology revealed that gonads contained hypoplastic testicular tissue. seminiferous tubules were diffusely hypoplastic with a complete lack of spermatogenesis. epididymis was hypoplastic with compressed ducts. there was marked congestion in the uterus with the lumen diffusely filled with erythrocytes consistent with intraluminal hemorrhage and the endometrial lamina propria was expanded by edema. no ovarian tissue was identified in the sections of the testis that were evaluated. however, due to the difficulty associated with identifying ovarian tissue, the presence of ovotesticular tissue could not be ruled out. blood was submitted to the texas a&m college of veterinary medicine, molecular cytogenetics laboratory for karyotyping. the results revealed a genetically female sheep with a normal sheep karyotype (54 xx). on polymerase chain reaction, the lamb was negative for the presence of the y-linked sry gene and positive for the x-linked androgen receptor gene. no chromosomal abnormalities were observed. intersex conditions in goats and in sheep are believed to be linked with the polled gene. affected animals are genetically female (xx), sry negative and believed to be homozygous for the polled gene. in this case the lamb is an intersex, consistent with polled intersex syndrome sex reversal but, may be a true hermaphrodite. keywords: sheep, polled, intersex, sex reversal, chromosome, hermaphrodite reduction of equine monozygotic twins using craniocervical dislocation via colpotomy adam bassett,a kendra rock,b carl propp,c dee gragg,d reed holyoaka acollege of veterinary medicine, oklahoma state university, stillwater, ok, bcross country genetics, manhattan, ks, cpryor veterinary hospital, pryor, ok, dgragg ranch equine reproduction center, guthrie, ok twin management is a very important facet of equine reproduction management. compared to dizygotic twins, monozygotic twins are a relatively rare occurrence in mares. management of monozygotic twins is considerably more complex when it comes to preserving the safety and welfare of the mare and developing pregnancy. of the options of twin reduction after day 50 of pregnancy, craniocervical dislocation (ccd) has been reported to be a superior choice.1 we have modified the surgical approach for a ccd from the reported flank laparotomy to colpotomy. in our hands, successful reduction of dizogotic twins by ccd via a colpotomy approach has been 71% (n = 29). to-date we have managed 2 cases of monozygotic derived twins with a 50% success rate. our hypothesis for the current case was that ccd via colpotomy is a successful technique for monozygotic twin management given its successful application on our first case of monozygotic twin reduction. an embryo recipient mare that received 1 embryo was presented at 65 days of pregnancy for the reduction of 1 fetus after diagnosed to have monozygotic twins via ultrasonography by the referring veterinarian. the referring veterinarian was actively monitoring the unilateral twin pregnancy for natural reduction. once the pregnancy reached 60 days with no reduction, the case was referred to us for ccd. after confirmation of monozygotic twins (i.e. 2 separate amnions were observed within 1 allantoic sac), a ccd was performed on 1 fetus via a colpotomy approach. unfortunately, both fetal heartbeats were lost on detection by the referring veterinarian and presumably aborted between 4 and 6 weeks after ccd procedure. other cases of monozygotic twins have been reported in recipient mares that received a single embryo.2-4 several management techniques were employed in each of these cases with varying outcomes. ccd via colpotomy is a novel approach and was chosen in this case due to the age of the fetuses and a desire to reduce the most craniad that we presumed would have less chorionic attachment and lower development capacity as the pregnancy progressed.1 the importance of this case is in demonstrating that ccd via colpotomy can be a useful method for management of monozygotic twins; however, prognosis for successful development of the remaining fetus is guarded due to the orientation of the twin fetuses enclosed within a single allantochorion. keywords: twins, monozygotic twins, twin management, embryo recipient, embryo transfer references 1. wolfsdorf, ke: management of postfixation twins in mares. vet clin north am equine pract 2006;22:713-725. 2. mancill ss, blodgett g, arnott rj, et al: description and genetic analysis of three sets of monozygotic twins resulting from transfers of single embryos to recipient mares. j am vet med assoc 2011;238:10401043. 3. roberts ma, london k, campos-chillon lf, et al: presumed monozygotic twins develop following transfer of an in vitro-produced equine embryo. j equine vet sci 2015;26:89-94. 4. sper rb, whitacre md, bailey cs, et al: successful reduction of a monozygotic equine twin pregnancy via transabdominal ultrasound-guided cardiac puncture. equine vet educ 2012;24:55-59. chronic seminal vesiculitis and blocked ampullae in a stallion lauren huggins, ghislaine dujovne, eduardo santos, pouya dini vmth, school of veterinary medicine, university of california, davis, ca seminal vesiculitis is a rare condition in the stallion; however, it can result in blocked ampullae. a 12-year-old gypsy vanner stallion with a previous successful breeding history was presented for persistent polyspermia, manifesting as an abnormal grey clinical theriogenology 2021; 13: 319 ejaculate. semen collected at presentation had pus with no sperm. testes felt normal but were mildly enlarged with firm epididymides. ultrasonography findings were normal. transrectal palpation of the ampullae and seminal vesicles (sv) elicited a painful response. transrectal ultrasonography revealed fluidfilled ampullae and sv with thickened walls (1.5 cm). stallion demonstrated normal libido during 3 collections and did not exhibit pain. cultures were obtained from the unwashed urethral fossa, the distal urethra before and after ejaculation, and from the gel fraction. streptococcus equi subspecies zooepidemicus was isolated, confirming the presumptive diagnosis of seminal vesiculitis. microscopic examination of fresh ejaculate and diff-quik stained smears revealed azoospermia, high number of neutrophils, and bacteria in both fractions of the ejaculate in all 3 collections. transrectal massage of the ampullae and oxytocin treatment immediately prior to the third collection did not alter the ejaculate. the diagnosis of blocked ampullae was confirmed by azoospermia and low concentration of alkaline phosphatase (30 iu/l) in the filtered fraction. semen was collected 3 times a day and the stallion was treated orally twice a day with trimethoprim/sulfamethoxazole (tms; 30 mg/kg) for 3 weeks at the owner’s facility. stallion was reexamined at the hospital. there were no changes to ampulla and sv. the first collection had > 70 ml heterogenous gel fraction and concentrated sperm-rich fraction (917 x 106 sperm/ml, 14.5 x 109 total) with marked number of detached heads and dead sperm (< 1% motility). sequential collections had decreasing volume of gel, increasing number of sperm (up to 83.2 x 109), and an increasing trend in the motility rate in the ejaculate. due to lack of complete response to tms, local treatment of the sv was initiated using video endoscopy (5.9 mm x 110 cm). the local treatment consisted of vigorous lavage (500 ml lactate ringer’s saline per gland) followed by local infusion of ampicillin (2 gram in 18 ml) initially for 2 days followed by a compounded penicillin gel (5 miu in 20 ml) every other day for a total of 3 treatments. purulent material within the seminal vesicles markedly decreased throughout treatment. stallion was discharged with instructions to have twice daily collections and continued tms treatment at the owner’s facility before readmission for follow-up. this case highlighted the importance of managing seminal vesiculitis aggressively, and the possible sequela of blocked ampullae due to local inflammation and accumulation of pus and sperm. keywords: stallion, seminal vesiculitis, blocked ampullae clinical theriogenology 2021; 13: 320 low volume uterine lavage: advantages for use in problem mares christina divine, christian bisiau, julie storme, melissa prell, jennifer morrissey, jennifer hatzel, patrick mccue college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co infectious endometritis is a leading cause of subfertility in broodmares. the standard sample collection technique for diagnosis of bacterial and fungal endometritis is a guarded swab for uterine culture and a guarded swab or brush for uterine cytology. however, these methods only sample a very small area of the uterus and may not always yield microbial growth in mares with infectious endometritis. low volume lavage (lvl) is an alternate diagnostic technique that samples the entire uterine lumen. aim of this study was to compare the results of microbial culture and cytologic evaluation from samples collected using a guarded swab and brush versus samples subsequently collected using a lal technique in mares suspected with infectious endometritis. reproductive records from 27 mares managed at colorado state university were analyzed retrospectively. transrectal ultrasonography examination and a standard uterine swab/brush were performed prior to lvl procedure. low volume lavage was performed using 250 ml of sterile lactated ringer’s solution and the effluent fluid was then transferred into a pair of 50 ml conical tubes for centrifugation. microbial culture was performed on swabs and lvl pellets using spectrumtm 4-part (colorado) microbial agar plates (vetlab supply, palmetto bay, fl). glass slides for cytologic evaluation were stained with a modified wright-giemsa stain (mwi animal healthtm, boise, id) and evaluated under 400 and 1000 x microscopy. percentages of mares with microbial growth of a uterine pathogen and presence of white blood cells on cytologic evaluation were compared by chi square analysis; data were considered different at p < 0.05. initial examination results determined that all mares were in estrus at reproductive evaluation based on the presence of mild to moderate uterine edema and a dominant follicle. an increased volume (> 2 cm depth) of uterine fluid was noted in 13 of the 27 mares (48.1%) and increased echogenicity of uterine fluid was noted in 12 of the 27 mares (44.4%) on initial transrectal ultrasonography examination. cytologic evaluation of the guarded brush sample revealed the presence of white blood cells and/or the presence of increased background debris in 12 of the 27 (44.4%) mares. microbial growth of 1 or more pathogenic bacteria was detected in the culture of guarded swabs collected from 4 of 27 mares (14.8%). overall, 21 of 27 mares (77.8%), had 1 or more factors that were suggestive of endometritis. culture from lvl pellets yielded growth of pathogenic bacterial in 15 of the 27 samples (55.6%) that was higher (p = 0.002) than guarded uterine swabs. cytology samples from lvl pellets had white blood cells in 24 out of the 27 mares (88.9%) that was higher (p = 0.005) than guarded uterine brushes. in summary, the lvl technique is a valuable procedure in the detection of uterine inflammation and microbial infection in problem mares. keywords: mare, uterus, low volume lavage, problem mare, endometritis use of progesterone and estradiol-17ß prior to transvaginal aspiration of oocytes peyton draheim,a cory anderson,b paul dyce,c candace lymana acollege of veterinary medicine, auburn university, auburn, al; bcountryside veterinary clinic, st. anthony, id; cdepartment of animal sciences, auburn university, auburn, al this pilot study assessed the impact of progesterone and estradiol-17ß treatment on ovarian follicle number. objective was to determine whether 150 mg/ml progesterone + 5 mg/ml estradiol 17ß (biorelease p&e; bet pharmacy, lexington, ky) treatment can impact mare ovarian follicle numbers to increase the odds of having more ovarian follicles on a predicted day. to increase foal production, transvaginal aspiration (tva) of mare oocytes is followed by in vitro maturation, fertilization (via intracytoplasmic sperm injection), and embryo culture. in efforts to aspirate several oocytes during 1 tva, common practice is to repeat ultrasonographic examinations of the ovaries on multiple sequential days and schedule the procedure on the day when the mare is expected to have her ‘peak’ number of suitable follicles. such a high maintenance management routine increases mare owner costs on a per foal basis. it also allows only ~ 1 2 days’ advance notice to the practitioner performing the tva procedure, due to the variability in follicular growth dynamics. currently, there are no accepted treatment regimens for advanced scheduling/planning of oocyte collection via tva in the mare. a validated estrous cycle management protocol would allow practitioners and mare owners to equine session clinical theriogenology 2021; 13: 321 plan multiple days in advance and also increasing the number of follicles that could be aspirated. furthermore, it would be highly beneficial to the equine industry and scheduling tva procedures. we hypothesized that a single injection of p&e result in synchronization and optimization of the number of follicles to occur at least 9 10 days after treatment. eight mares underwent initial ovarian follicles counting (i.e. follicular mapping) via transrectal ultrasonography and were given 10 ml of biorelease p&e intramuscularly on day 0; transrectal palpation and ultrasonographic examination, and follicular mapping was repeated for each mare 3 times (i.e., on days 3 and 6, in addition to days 9, 10, or 11, based on mare availability). serum was collected on the days of ultrasonographic examinations and frozen for determining concentrations of anti-mullerian hormone (amh), luteinizing hormone (lh), and follicle stimulating hormone (fsh). data on follicle count data from this study were combined with previously obtained pilot data, for a total of 24 mares. data on follicle count were analyzed by unpaired two-tailed student’s t-test and amh concentrations by a multiple comparison test. follicle counts increased (p = 0.024) by a mean of 4.13 follicles/mare between the first and last follicular mapping sessions. amh concentrations on day 3 (0.8473 ng/ml ± 0.1699, mean ± sem) were significantly higher compared to day 6 (0.7033 ng/ml ± 0.1737, mean ± sem). keywords: mare, progesterone, estradiol, follicle, transvaginal aspiration, oocyte prepartum amniotic rupture in a thoroughbred mare david alexander, kindra orr, kate christie, peter morresey, christine bartley, maria schnobrich rood & riddle equine hospital, lexington, ky prepartum amniotic rupture has been described in humans.1 in mares, there are reports of prepartum placental disruption associated with hydropsical conditions. this report describes a full thickness prepartum amniotic tear in a thoroughbred mare, initially diagnosed by transrectal ultrasonography at 236 days prepartum. a 7-year-old thoroughbred mare was pregnant with her first foal. transrectal ultrasonography (15, 17, 28, 42, 60, 89, 119, 148, and 183 days) findings were normal and the mare apparently had a filly. transrectal ultrasonography evaluation on 211 days revealed a normal amnion and a combined thickness of the uterus and placenta (ctup) of 0.6 cm. at 236 days, transrectal ultrasonography was performed as part of routine screening and no amnion could be identified. allantoic fluid was moderately and uniformly increased in echogenicity and the fetus was visualized with no amnion separating the fetus from the allantoic membrane. no precocious mammary gland development was noted, but substantial placental edema was observed in the chorioallantois and endometrium and ctup was 2 cm. serum amyloid a was slightly elevated (32 µg/ml) with normal white blood cell count (7.6 x103), and fibrinogen (200 mg/dl). total estrogens were 1,040.04 pg/ml and progesterone concentrations were 4.51 ng/ml. treatment for placentitis (once every 24 hours for altrenogest [0.044 mg/kg], 57 mg of firocoxib and 500 mg of flunixin meglumine, and once every 12 hours for pentoxifylline [8.5 mg/kg] and doxycycline [10 mg/kg]) was initiated. serial transrectal and transabdominal ultrasonography were performed and the chorioallantois continued to thicken and was edematous. amnion was intermittently viewed in transrectal ultrasonography, but in an abnormal and wrinkled appearance. at all times, fetal heartbeat was within 80 100 bpm. at 313 days of pregnancy, the mare was admitted for monitoring, and altrenogest, firocoxib and pentoxyfiline treatment continued. in addition, once every 24 hours, 2,660 mg of aspirin was given. at day 317, the mare foaled. no amnion was visible after observing the mare’s water breaking, foaling required manual assistance, and the amnion was not noted. fetus was born alive but euthanized due to 90° contracture of both front fetlocks. fetus and fetal membranes were submitted for histopathology analysis and revealed an amniotic rent measuring 10 cm with rolled edged and a diffusely thickened amnion. allantois was thickened with areas of adenomatous hyperplasia and embedded hair shafts. to our knowledge this is the first reported case of prepartum amniotic rupture diagnosed by transrectal ultrasonography in the mare keywords: mare, amniotic, rupture, prepartum reference 1. medina tm, hill da: preterm premature rupture of membranes: diagnosis and management. am fam physician 2006;73:659-664. evaluation of passively acquired rabies antibody titers and immune responses in healthy foals vaccinated against rabies at 4 or 6 months of age karen wolfsdorf,a luke fallon,a jeanette mccracken,a deb amodie,b jacquelin boggs,b bobby cowles,b nathan vorisb ahagyard equine medical institute, lexington, ky; bzoetis, parsippany, nj passively acquired antibodies provide protection against disease in foals prior to completion of a primary immunization series. postvaccination immunologic protection against rabies is expected with titers ≥ 0.5 iu/ml, but information on the lifespan of passively acquired antibodies and influence on immune response is lacking. in the spring of 2020, the american association of equine practitioners updated vaccination guideline recommendations to begin rabies vaccination at 4 to 6 months of age instead of initiating at 6 months of age. the update made initiation of foal vaccination recommendations consistent for all 5 core equine diseases (eastern equine encephalomyelitis [eee], western equine encephalomyelitis [wee], west nile virus [wnv], tetanus, and rabies). the aim was to evaluate the rate of passively acquired rabies antibody decline and compare the immune response to vaccination when initiated at 4 or 6 months of age. forty-nine foals, in 3 farms, born to mares vaccinated against eee, wee, wnv, tetanus, rabies, equine influenza virus, clinical theriogenology 2021; 13: 322 equine herpesvirus 1/4, botulism, rotavirus, plus or minus autogenous salmonella typhimurium and clostridium perfringens in the last 6 weeks of pregnancy, were enrolled. all foals were determined healthy and had adequate postnursing igg. none received hyperimmunized plasma. blood was drawn monthly and submitted to kansas state university rabies laboratory to quantify antirabies neutralizing antibody via the rapid fluorescent focus inhibition test. foals were allocated with a generalized, randomized block design, based on rabies titer at 1 month of age. foals received a commercial combination eee, wee, wnv, tetanus, rabies vaccine beginning at either 4 or 6 months of age. foals received a booster 30 days after their initial injection. rabies antibody concentrations were analyzed to establish the rate of decline prior to and after vaccination immune responses. prior to vaccination, geometric mean rabies titers were 3.5, 1.1, 0.6, and 0.4 iu/ml at 1, 2, 3, and 4 months of age, respectively. through the first 4 months of life, antibody titer decline was 90%. at 4 months of age, 33/49 (67%) foals had rabies titer ≤ 0.5 iu/ml. the geometric mean postvaccination rabies titers were 0.8 and 1.0 iu/ml in foals that began initial vaccination series at 4 and 6 months of age, respectively. postvaccination, 20/25 (80%) foals in the 4-months age group and 20/24 (83%) foals in the 6-months age group had rabies titer ≥ 0.5 iu/ml. in our study, the rate of rabies antibody declined over the first 4 months of life, indicating that most foals are expected to have a titer below 0.5 iu/ml prior to 4 months of age. the immune response to vaccination was similar between foals that started their immunization series at 4 months of age compared to those started at 6 months of age. future is unknown. keywords: rabies, vaccination, titers, immunization, immunity prospective ultrasonographic evaluation of caudal placenta and cervix in pregnant mares in relationship to foaling outcomes and placental abnormalities kristina lu,a karen von dollen,a karen wolfsdorf,a samantha levkulic,a barry ballb ahagyard equine medical institute, lexington, ky; bgluck equine research center, university of kentucky, lexington, ky clinical diagnostic procedures to evaluate the late pregnant mare are largely limited to physical examination and ultrasonographic evaluation of the fetus as well as the combined thickness of the uterus and placenta (ctup). the cervix of a pregnant mare is a key physical and immune barrier to contamination of the pregnant uterus from the vagina yet there is limited research that evaluated changes in the cervix, which might be indicative of potential problems during equine pregnancy. objective was to evaluate changes in ctup and mean cervical diameter (cx) determined by transrectal ultrasonography (sonoscape s9 with 9.5-15 mhz linear probe; seattle, wa), and the relationship of these parameters to subsequent foaling outcomes and placental abnormalities. the study was conducted in thoroughbred mares in central kentucky during 2017 (n = 112 mares), 2018 (n =109 mares), and 2019 (n = 139 mares). mares were examined by 1 of 2 examiners on a monthly basis from 4 months gestational age (ga) until term (total examinations; n = 1810). at term, outcomes were classified as normal or abnormal foal and normal or abnormal placenta (based on observation of a fetal membranes inspection at the farm). data were analyzed by a random-effects mixed model including mare as the random effect, gestational age as a covariate and foaling outcome, placenta as well as examiner as a fixed-effects (jmp ver 14.0). correlations were evaluated by a pearson’s coefficient. the ctup was higher (p = 0.001) in mares with abnormal placenta at term but was not related (p = 0.3) to foal outcome. the ctup increased (p < 0.001) with ga and was affected (p < 0.001) by examiner. the cx increased (p = 0.05) in mares with abnormal foaling outcome but was not related (p = 0.2) to abnormal placenta at term. again, cx increased (p < 0.001) with ga and varied (p < 0.001) with examiner. as noted, cx and ctup increased with ga and were positively correlated (r = 0.26; p < 0.01). our findings suggested that measurement of cx and ctup in mares are related to foaling outcome and placental abnormalities at term, respectively. future studies should examine the predictability of foaling outcomes in mares based upon prospective evaluation of these parameters. keywords: mare, pregnancy, placenta, cervix, ultrasonography funding: theriogenology foundation and the albert g. clay endowment of the university of kentucky oocyte collection rate and in vitro embryonic development with low dose deslorelin in mares jennifer hatzel,a delfina rodriguez,b christian bisiau,a julhiano rossini,c joanne stokesa acollege of veterinary medicine and biomedical sciences, colorado state university, fort collins, co; bla adela equine reproduction center, chascomus, buenos aires, argentina, cjbr – assisted equine reproduction, matão, sp, brazil equine assisted reproductive technologies have become increasingly popular throughout the past 20 years, especially ovum pick up (opu) and intracytoplasmic sperm injection (icsi). successful production of in vitro embryos through opu/icsi often relates to the number of oocytes, but limited research has been devoted towards ovarian super stimulation for this purpose. this study aimed to determine if low doses of the gonadotropin releasing hormone agonist, deslorelin, (ldd) would increase number of follicles, increase number of oocytes collected or affect oocyte quality. mares (n = 11, 5 13 years) were assigned for this study. each mare served as her own control in a cross over design. all estrous cycles were monitored through routine transrectal ultrasonography and all visible follicles recorded. routine transrectal vaginal aspiration was performed on mares in both groups at ~ 20 hours after ovulation induction treatment. for treated estrous cycles, when at least clinical theriogenology 2021; 13: 323 1 follicle measured ≥ 15mm in diameter, 50 µg deslorelin in aqueous solution (precision pharmacy, bakersfield, ca) was given intramuscularly every 12 hours until at least 1 follicle reached 32 mm in diameter with uterine edema. subsequently, the ldd protocol was discontinued, allowing for 24 hours without hormone treatment. after collection, oocytes deemed mature (expanded cumulus) or immature (compact cumulus) were handled accordingly, prior to icsi with frozen semen from a single ejaculate. presumptive zygotes were evaluated at 24 hours for evidence of cleavage and again at 4, 6, and 8 days. comparisons were made utilizing a student’s and welch’s t-tests with significance established at p ≤ 0.05 and reported as the mean ± standard deviation. in total, 41 estrous cycles were included (24 control and 17 treated). on average, 7.41 ± 2.3 injections of ldd were given during treated estrous cycles. average number of follicles observed or number of follicles > 25 mm in diameter at opu did not differ between groups (p = 0.13 and p = 0.46, respectively). average number of follicles aspirated in the treated group (2.1 ± 1.9) tended to be higher (p = 0.09) than control (1.9 ± 1.4); however, the number of oocytes collected in treated cycles (1.2 ± 1.4) compared to control (0.75 ± 1.0) did not differ (p = 0.28). a total of 36 oocytes were subjected to icsi from the treated and 38 from control estrous cycles. the cleavage rate was similar (p = 0.89) for treated (56%; 20/36) compared to control (61%; 23/38). finally, the blastocyst rate for treated was similar (p = 0.66) (19%; 7/36) compared to control (24%; 9/38). in conclusion, the utilization of a low dose deslorelin protocol did not affect the number of follicles or oocytes collected within estrous cycles for opu/icsi. additionally, cleavage and blastocyst rates were similar between the 2 groups indicating that this protocol did not affect the quality of oocytes. more research is necessary to identify an ovarian superstimulation protocol effective for opu/icsi. keywords: mare, oocytes, follicles, deslorelin pregnancy rates and ovarian activity after uterotubal infusion of n-butyl cyanoacrylate via a hysteroscopic approach in mares: a pilot study yuji inoue,a irwin liu,b j. vazquez,c j. nieto,d claire carde ainoue equine clinic, shin-hidaka, hokkaido, japan; bdepartment of population health and reproduction; ddepartment of radiology and surgical sciences, university of california davis, davis, ca; chospital veterinario lasilla, monterrey, mexico; edepartment of large animal clinical science, western college of veterinary medicine, university of saskatchewan, saskatoon, sk, canada sterilization of mares requires surgical removal of ovaries or ligation of uterine tubes and is performed for behavioral reasons, treatment of ovarian pathology, and preparation of tease or mount mares. however, it is invasive, costly, and may be associated with adverse outcomes. objective was to determine the effect of bilateral uterine tube infusion with n-butyl cyanoacrylate on fertility and estrous cycle activity of mares. we hypothesized that bilateral infusion of n-butyl cyanoacrylate in the uterine tubes causes infertility but does not affect mare’s estrous cycle activity. light horse mares (n = 8) ranging in age from 5 23 years, were selected for potential fertility using transrectal ultrasonography examination, uterine culture and cytology. a proven fertile thoroughbred stallion that successfully bred mares in 2015 was used. mares were sedated, perineum cleansed, and the uterine tubes of treated mares (n = 6) were infused with 0.5 ml n-butyl cyanoacrylate, and control mares (n = 2) were infused with 10 ml saline, using the endoscopic hydrotubation method.1 treated mares were hand mated through 2 or 3 breeding seasons (n = 78 estrous cycles), and control mares were hand mated through 1 breeding season (n = 4 estrous cycles). biweekly blood samples were obtained from mares during the breeding season for progesterone (p4) concentrations by ria. per cycle pregnancy rate, established by transrectal ultrasonography of the uterus and interestrus intervals, were recorded. stallion’s seasonal pregnancy rate (spr) in 2015 (n = 24 mares) was compared to the spr of treated (n = 6) and control mares (n = 2) for years 2016 2018. data were evaluated using fisher’s exact test at p < 0.05. per cycle pregnancy rates of treated (0/78) and control mares (1/4) were different (p = 0.048). spr for the stallion in 2015 prestudy breeding season (20/24 mares pregnant) was different (p < 0.001) than the spr of treated mares, wherein 0/6 became pregnant in each year. interestrus intervals of 6 treated mares averaged 20 days (range 18 22 days), and serum p4 profiles were consistent with estrous cycle activity. the n-butyl cyanoacrylate may function to physically obstruct sperm, oocyte and embryo movement in the uterotubal junction or uterine tube, disturb the milieu required for sperm capacitation, oocyte fertilization, and embryonic development, or a combination of these. uterotubal infusion of n-butyl cyanoacrylate via a hysteroscopic approach may serve as an effective management strategy for induced infertility in mares of at least 3 years duration and may cause permanent sterilization. keywords: mare, infertility, n-butyl cyanoacrylate, uterine tube, progesterone, estrous cycle reference 1. inoue y, sekiguchi m: clinical application of hysteroscopic hydrotubation for unexplained infertility in the mare. equine vet j 2018;50:470-473. daily sperm output, spermatogenic efficiency, and sexual behavior of donkey jacks mounting jennies in estrus humberto magalhaes, lucas canuto, igor canisso department of veterinary clinical medicine, university of illinois, urbana, il assessment of daily sperm output (dso) and spermatogenic efficiency are critical components of breeding soundness evaluation clinical theriogenology 2021; 13: 324 to diagnose reproductive diseases and estimate the number of females that may be bred in a season. however, there is no consensus on how to determine these parameters in stallions or donkeys. part of the discrepancies is due to estimation of testicular volume (tv) by several ways. additionally, donkeys are thought to be less consistent breeders when required to collect daily. we hypothesized that a donkey’s dso varies with the equation utilized for calculation of tv and his behavior parameters are influenced by increased numbers of collection days. aim was to assess the sexual behavior of jacks mounting jennies in estrus and determine the dso and spermatogenic efficiency. eight sexually rested jacks had semen collected once a day for 10 consecutive days using jennies in good standing estrus for mounting. sexual behavior and semen parameters were assessed during each collection. testicular measurements of height, width, and length were taken immediately before the first semen collection, and these measurements were used to calculate the tv using a nonellipsoid equation (tv1 [cm3]: 33.57 × height [in cm] – 56.57) and an ellipsoid equation (tv2 [cm3]: 4/3π length/2 x height/2 x width/2) used for stallions. after that, the tvs were used to predict the dso. the average total sperm number (tsn) obtained on days 8 -10 was deemed the actual dso. differences in the predicted versus the actual dso were used to calculate the spermatogenic efficiency. in addition, the actual dso was also used to calculate the number of inseminating doses a jack could produce for both onand off-site breeding. data were analyzed with the shapiro-wilk test and then with anova followed by tukey’s test or kruskalwallis. sexual rest did not affect (p < 0.05) sperm motility. jack’s sexual behavior did not vary across collection days (p < 0.05). sperm concentration and tsn reduced (p < 0.05) over time. the actual dso was 9.1 ± 4.1 x 109, and the predicted dso varied from 4.7 to 18 x 109. spermatogenic efficiency ranged from 50 to 150% based on the jack and the equation used to calculate tv. the predicted-dso obtained with tv1 demonstrated a strong and positive correlation with the actual-dso (r = 0.76, p < 0.05); however, there was no significant correlation between predicted-dso and tv2 (p > 0.05). the number of inseminating doses ranged from 15 to 47 at 300 500 x 106 progressively motile sperm (pms) /dose for on-site breeding. in contrast, the number of breeding doses with cooled-shipped semen (1 x 109 pms/dose) varied from 4 to 14 doses across donkeys. in conclusion, sexual behavior was not affected by daily semen collections. sexual rest did not affect sperm motility. the predicted dso varied with the equation used to determine tv. clinically normal donkeys have high spermatogenic efficiency that confirms previous histology reports. a distinct approach to calculate dso in donkeys is described. further studies are warranted to apply the findings of the present study in reproductively abnormal donkeys. keywords: sexual behavior, daily sperm output, spermatogenic efficiency clinical theriogenology 2021; 13: 325 infertility, pregnancy, and concurrent pyometra in a dog brittany shumack village center veterinary care, colorado springs, co a 3-year old greater swiss mountain dog was presented for breeding management. the patient had a history of 2 previous failed pregnancy attempts, 1 with ovulation timing and vaginal insemination, and the other with ovulation timing and transcervical insemination. regardless of failure to become pregnant, the owner wanted to breed once more due to high sentimental value. the patient was placed on enrofloxacin and carprofen at lh surge until 6 days postovulation. the patient had a negative vaginal culture at last breeding. based on ovulation timing, 2 transcervical inseminations were performed with fresh collected semen. at 25 days postovulation, the patient was confirmed pregnant on abdominal ultrasonography. only 1 fetus appeared to be viable, 1 fetus appeared to be in the early stages of resorption with no heartbeat, and 5 other resorptive sites were identified. the patient was placed on amoxicillin. taking into account the patient’s history of infertility, a concern for hypoluteoidism prompted us to assess the patient’s serum progesterone concentrations and were elevated (31.42 ng/ml). due to owner’s concern, progesterone concentrations were determined 3 days later. progesterone concentration decreased to 14.38 ng/ml. due to this unexpected degree of decline in 3 days, we continued to monitor progesterone concentrations throughout pregnancy. progesterone concentrations remained adequate for the rest of pregnancy. at 48 days postovulation, ultrasonography revealed 1 viable fetus and a fluid-filled uterus. the patient was asymptomatic for a pyometra, and the owner elected to maintain the pregnancy. the antibiotics were switched to amoxicillin/clavulanate potassium; patient was continually monitored via rectal temperature assessment and ultrasonographic examinations, and for behavioral changes. at 63 days postovulation, an elective cesarean section was performed. the fetus was viable and resuscitated quickly. substantial amount of purulent material was present in the uterus (> 1 liter). the serosal surface of the uterus did not have any defects and appeared normal. pathological diagnosis, based on a tissues sample from the right uterine horn, was ‘focal extensive eosinophilic endometritis and multifocal moderate lymphoplasmacytic endometritis/myometritis with multifocal mild cysts endometrial hyperplasia.’ infertility in this bitch was accompanied by multiple lesions and this case demonstrated that most ‘missed breeding’ may be due to an underlying cause, even in a young, otherwise healthy bitch. keywords: pyometra, infertility, cystic endometrial hyperplasia, pregnancy factors affecting survival and future foaling rates in thoroughbred mares with hydrops louise lemonnier,a karen wolfsdorf,b nicole kreutzfeldt,c rana bozorgmaneshb adepartment of equine internal medicine, university animal hospital, oniris, nantes, france; bhagyard equine medical institute, lexington, ky; cpeasebrook equine clinic, broadway, united kingdom prognosis for life and future fertility in broodmares following hydrops is reportedly good, and the role of inheritance in the development of hydrops has been suggested but lacks large-scale evidence. aims of this case series were to evaluate the prognosis for survival and fertility in mares following hydrops diagnosis and to attempt to identify if inheritance is a predisposing factor. thirty mares presented with hydrops were included. data collected included history (pregnancy and sire of the foal), clinical findings at presentation and throughout hospitalization (complications, treatments provided, and survival to discharge) and future foaling rates. ninety percent (90%) of mares survived (hydrallantois: 94.7%; hydramnios: 75%) and 95.2% of mares successfully had a future foal, of which 75% foaled the first year following hydrops. no reoccurrence of hydrops was identified, despite being bred back to the same stallion. transcervical gradual fluid drainage was associated with improved (p = 0.05) survival. complications associated with poor survival and decreased future foaling rates included hypovolemic shock (p < 0.005 and p = 0.010, respectively), hemorrhage (p < 0.005 and p = 0.025, respectively), peritonitis (p < 0.005 and p = 0.01, respectively) and abdominal wall rupture (p = 0.01 and p = 0.005, respectively). laminitis was associated with poor survival (p < 0.05). one mare suffered a uterine tear and was euthanized. these results suggest that prognosis for survival, future breeding and fertility following a diagnosis of hydrops is good, provided the hydrops is diagnosed and treated appropriately and no damage to the reproductive tract or body wall occurs. these data did not provide evidence for heritability and further investigation is required. keywords: mare, pregnancy, hydramnios, hydrallantois, fluid drainage mixed animal session clinical theriogenology 2021; 13: 326 comparison of 2d and 3d ultrasonography for gestational aging in dogs sabina pestelacci,a nikolaos tzanidakis,a iris reichler,a orsolya balogha,b aclinic of reproductive medicine, vetsuisse faculty, university of zurich, zurich, switzerland; bdepartment of small animal clinical sciences, virginia-maryland college of veterinary medicine, blacksburg, va gestational aging in the bitch by two-dimensional (2d) ultrasonographic measurements of fetal and extra-fetal structures has been well studied. in early pregnancy, the inner chorionic cavity (icc) diameter was the most reliable parameter, but its accuracy for parturition day prediction within 1 day error decreased from 81% in week 4 to 67.7% in week 5 of pregnancy.1 we hypothesized that three-dimensional (3d) volume ultrasonography, which has previously been studied only in a limited number of bitches, improves gestational age determination in dogs. the aim of our study was to compare embryonic vesicle measurements by 3d to 2d ultrasonography for gestational aging in early to mid-pregnant bitches. thirty-two pregnancy examinations were performed in 25 bitches of several breeds by abdominal ultrasound between 21 and 34 days after the first mating. icc diameter and length were measured by 2d, and icc volume by 3d ultrasonography (voluson® i, ge healthcare). icc volume was calculated using the virtual organ computer-aided analysis (vocal™) software with 30° rotational angle. measurements on 2 embryonic vesicles per examinations were averaged for statistical calculations (icc volume was available from only 1 embryo in 5 examinations). the associations between the dependent variables icc diameter, length and volume, and the independent variable time, calculated as either ‘days from ovulation’ (n = 15 dogs, ovulation day based on serum progesterone concentrations) or ‘days before parturition’ (n = 22 dogs, planned cesarean sections or bitches under progesterone supplementation excluded) were analyzed by linear or exponential regression using ibm® spss® statistics v. 26.0.0.0. statistical significance was set at p < 0.05. counting from the day of ovulation, regression lines for icc diameter, icc length and icc volume had a good fit (p < 0.001) with our data points (r2 = 0.707, r2 = 0.728 and r2 = 0.718, respectively). regression curves had improved fit (p < 0.001) for all icc measurements when time ‘days before parturition’ was used; icc length and icc volume gave slightly better estimates than icc diameter (r2 = 0.810, r2 = 0.818 and r2 = 0.800, respectively. in conclusion, regression analysis of conceptus size measured by icc length and icc volume were slightly more accurate to estimate conceptus age than icc diameter. furthermore, on the basis of icc volume and icc length regression curves, it is apparent that the development of canine conceptus follows an exponential growth curve already during the early stages of pregnancy that is not reflected by icc diameter measurements. icc length measured by 2d ultrasonography may represent a more feasible target for improving accuracy of canine parturition date prediction than 3d volume calculations, which require specialized, expensive equipment and more time to process data. keywords: dog, 3d ultrasonography, embryo, pregnancy, parturition prediction reference 1. beccaglia m, luvoni gc: prediction of parturition in dogs and cats: accuracy at different gestational ages. reprod dom anim 2012;47(suppl. 6):194-196. vaginal vault diverticulum causing functional urinary obstruction in a maiden bitch marthina greer veterinary village/international canine semen bank wi/il/in, lomira, wi a 14-month-old german shepherd bitch presented with a bloody vaginal discharge, followed by an inability to void her bladder. approximately 6 weeks prior, she had a dark colored vaginal discharge interpreted as her first estrus. the vaginal discharge at presentation was initially assumed to be associated with the development of a pyometra. ultrasonography revealed normal (no fluid) uterus and an overly distended urinary bladder. work up included comprehensive physical examination, radiographs, contrast study of her bladder and urethra, cbc, chemistry panel, urinalysis, vaginal cytology, vaginoscopy, culture of the vaginal discharge, and ultrasonography by a board-certified radiologist. no cystic calculi were detected in any imaging. fluid was present caudal to the cervix. her bladder required multiple episodes of catheterization to manage her functional urinary obstruction, pending surgery. exploratory laparotomy was performed to assess the structural abnormality. the urinary bladder, uterus and ovaries were normal. no other abnormalities were observed in the abdominal cavity. a large diverticulum containing dark red blood-tinged fluid was the only structural abnormality detected at exploratory. the ventral aspect of the diverticulum was incised and evaluated. the cervix appeared ~ 4 times the size of a normal cervical os at this stage of estrus. the dorsal median fold was pronounced. the remaining vaginal vault was normal with the exception of the diameter. redundant vaginal tissue was excised, and the vaginal vault was closed with a two-layer continuous absorbable suture. redundant tissues were submitted for histopathology. celiotomy incision was closed routinely after the area was explored and lavaged. bitch recovered unremarkably and was subsequently able to void her urinary bladder voluntarily. her uterus and ovaries were spared with the anticipated use as a brood bitch. as of this report, she remains clinically normal. she has not yet had a subsequent estrus and her reproductive future is unknown. keywords: vaginal diverticulum, functional urinary obstruction, vaginal distension, excess vaginal fluid clinical theriogenology 2021; 13: 327 screening canine sera for smooth brucella strain antibodies via brucella abortus fluorescent polarization assay alyssa helms,a orsolya balogh,a rebecca franklin-guild,b kevin lahmers,c clayton caswell,c julie cecerea adepartment of small animal clinical sciences, virginia-maryland college of veterinary medicine, blacksburg, va; bdepartment of population medicine and diagnostic sciences, college of veterinary medicine cornell university, ithaca, ny; cdepartment of biomedical sciences and pathobiology, virginia-maryland college of veterinary medicine, blacksburg, va dogs can serve as hosts for all 4 of the most common zoonotic strains of brucella: the smooth strains (i.e. brucella abortus [b. abortus], b. melitensis, b. suis, and the rough strains [i.e. b. canis]). however, due to differences in cell wall morphologies between the smooth and rough strains, the only validated serologic test currently available for brucellosis screening in dogs is limited to the identification of the rough strains (i.e. b. canis) alone. recently, the usda added the fluorescent polarization assay (fpa) as an approved test for the confirmation of brucellosis infection in cattle, bison, and pigs, due to the demonstration of sufficient cross reactivity between b. abortus, b. melitensis, and b. suis. thus, the goal of our study was to utilize the fpa test to identify antibodies to smooth brucella strains in canine sera, and to compare the results of the fpa test to the commonly utilized b. abortus card agglutination (bca) test. sera from 95 clinically healthy dogs, including 45 hog hunting dogs, were screened for circulating antibodies utilizing a combination of canine brucella slide agglutination test (cbsa), brucella canis agar gel immunodiffusion ii test (agid), and bca and fpa tests. suggested test interpretation results yielded a 0% (0/95) smooth brucella strain seropositivity rate, with 38/95 (40%) bca positive results, and 0/95 (0%) fpa positive results. rough brucella strain serology yielded an inconclusive result (0 2% rough strain seropositivity rate) in 2% (2/95) of dogs. additionally, a retrospective portion of the study was performed to identify sera containing circulating antibodies to any of the smooth strains of brucella via brucella abortus fpa by testing banked canine serum samples that had been submitted to cornell’s veterinary diagnostic laboratory between 2018 2019 and previously screened by cbsa and agid for b. canis with a positive or inconclusive test interpretation result. of the 769 serum samples tested, 30/769 (4%) yielded a positive fpa test result, 13/769 (1.7%) yielded an inconclusive result, 725/769 (94.2%) were negative, and 1/769 (0.1%) sample excluded due to insufficient sample remaining to perform the diagnostic test. of the 30 fpa suspect positive canine serum samples, 97% (29/30) also tested positive on cbsa. additionally, the signalment of fpa suspect positive dogs was much more likely (p < 0.0001) to be spayed or neutered compared to intact, and mixed breed compared to purebred. keywords: brucellosis, canine, abortion, smooth strain, fluorescence polarization test fetal loss at time of elective cesarean section in a dog brittany shumack village center veterinary care, colorado springs, co a 3-year-old greater swiss mountain dog was presented exhibiting prolonged signs of stage 1 labor. abdominal radiographs obtained 6 days prior revealed 8 fetuses appearing viable with heartbeats >180 bpm on ultrasonographic examination. owner elected for cesarean section. surgical site was prepared while the patient was given supplemental oxygen. an anesthetic protocol of alfaxalone for induction and isoflurane for maintenance were utilized. time from anesthetic induction to removal of the first puppy from the uterus was ~ 8 minutes. the first, fourth, and seventh puppy delivered resuscitated in < 2 minutes. five remaining puppies did not respond to any resuscitation efforts including accordion technique, gv26 acupuncture point, and epinephrine. when gv26 acupuncture points were performed substantial bleeding was noted from the site. on physical examination the 5 puppies were also judged to have distended abdomens. paracentesis was performed and abdomen of each puppy was full of what was suspected to be frank blood. blood was sent for cbc/coagulations panels. radiographic images of the neonates were obtained and 4 of 5 puppies had fluid in their lungs. three of the 5 struggling puppies had faint heartbeats. after 40 minutes of resuscitation efforts with no improvement the owner elected to discontinue the efforts. blood had markedly decreased platelets with very minor platelet clumping observed. many of the platelets were enlarged, suggesting accelerated platelet production of the bone marrow. a neutropenia was present. prothrombin time and aptt were markedly increased, fibrinogen was markedly decreased, and d-dimer was severely elevated. three fetuses that resuscitated quickly had no abnormalities on physical examination and there were no concerns going forward. multiple consults and discussions about this case did not provide answers as to why the fetal loss occurred. p2y12 is a platelet disorder present in greater swiss mountain dogs. the sire was negative, and the dam was negative or a carrier. it is unclear if a natural whelping would have yielded better results. the purpose of this case is to present an occurrence of multiple unexplained fetal losses and bring awareness to a possible phenomenon. despite much criticism from the greater swiss mountain dog breed club for isoflurane use during cesarean sections due to a supposed breed sensitivity, we have not changed our anesthetic protocol. multiple cesareans have been performed on greater swiss mountain dogs since this case with a 100% fetal resuscitation rate. keywords: fetal loss, cesarean section, clotting disorder clinical theriogenology 2021; 13: 328 using reflection to optimize student engagement during theriogenology practical classes: the benefits of mentoring and peer support in teaching allan gunn,a,b john harper,c jason condonb,c aschool of animal and veterinary sciences, faculty of science, bgraham centre for agricultural innovation (csu and nsw dpi), albert pugsley place, cschool of agricultural and wine sciences, faculty of science, sturt university, wagga wagga, nsw 2678, australia veterinary training is a resource consuming process. practical sessions throughout the curriculum are considered vital aspects of professional training and consume large amounts of time, space and human resources and are not without risks to human sperm acrosome associated 3 protein expression in the feline ovary decreases with age ann ramsey, michelle kutzler department animal and rangeland sciences, college of agricultural sciences,oregon state university, or sperm acrosome associated 3 (spaca3) is a lysozyme-like protein previously identified in 5 to 7.5-month-old cat ovarian follicles.1 the objective of this research was to compare spaca3 expression in young (2 months [n = 3]) and adult (>12 months [n = 3]) queens in different follicular stages. we hypothesized that spaca3 expression does not differ by age. routine immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections, followed by image analysis using fiji software. heat-induced epitope retrieval with sodium citrate (ph 6.1) was used. antispaca3 polyclonal antibody (#hpa023633, atlas antibodies) was applied at 1:200 dilution and immunostaining specificity was verified by replacing the primary antibody with negative control rabbit serum on adjacent sections. sections were then reacted with one-step horseradish peroxidase-conjugated polymer antirabbit igg (ih-8064-custom-orsu, immunobioscience), followed by a novared peroxidase substrate (#sk-4800, vector labs). representative images of each follicle stage from each ovary were digitally captured using qcapturepro image capture software by a single observer at 200 x magnification. cellular expression of spaca3 was then quantified in primordial (0o), primary (1o), secondary (2o), and tertiary (3o) follicles using fiji software with rgb stack and manual thresholding to isolate areas of staining. the oocyte nucleus, granulosa cells, and theca cells were outlined using the freehand selection tool and mean gray value was measured. results (mean ± sem) were compared between young and adult queens using a student’s t-test and significance was defined as p < 0.05. there was greater spaca3 expression in young compared to adult queens (figure) in the oocyte nucleus of 0o (p < 0.001), 1o (p = 0.006), 2o follicles (p < 0.001), in granulosa cells of 1o (p = 0.016), 2o (p < 0.001), 3o follicles (p = 0.004), in theca cells of 2o (p = 0.006), and in 3o follicles (p = 0.043). this is the first study to evaluate differences in spaca3 expression by age in any species. more research is needed on the mechanisms that regulate ovarian spaca3 expression and its role in female fertility. keywords: cat, granulosa cell, oocyte nucleus, ovary, theca cell reference 1. wagner a, holland oj, tong m, et al: the role of sprasa in female fertility. reprod sci 2015;22:452-461. and animal health.1 it is therefore, important that the educational experience is optimized during practical sessions. mentee dialogue with mentors, as part of institution peer teaching support, is aimed to optimize the practical sessions in order to develop professional expertise (knowledge and skills) of veterinary students learning theriogenology. we used the theory of experiential learning and reflective observation as a means of enhancing learning during the practical session.2 we attempted to do this by discussing abstract concepts associated with the authentic learning tasks covered in each practical session. anonymous end-of-course student feed-back revealed that the process encouraged in depth and alternative critical thinking and discussion in the groups that was a fun way for them to embed the knowledge and develop the skills being taught. the use of ‘abstract reflection’ appears to be a really useful and efficient clinical theriogenology 2021; 13: 329 category comparison % n % ah % aa % pd % dd % amp % bmp % ct high dic vs. en -4 (-15, 7) 11 (7, 16) 4 (-5, 15) -2 (-10, 7) -1 (-17, 15) 4 (-3, 11) 4 (-6, 13) 0 (-4, 3) dic vs. dq -5 (-14, 5) 12 (3, 20) 4 (-5, 14) 4 (-12, 4) 3 (-11, 18) 5 (-3, 12) 3 (-8, 13) 0 (-3, 3) dic vs. ph 0 (-7, 8) 4 (-19, 10) 4 (-5, 12) 2 (-10, 13) 6 (-10, 22) -1 (-13, 11) 3 (-6, 11) 0 (-2, 3) average dic vs. en -11 (-29, -6) 12 (6, 18) 3 (-3, 8) 6 (-17, 28) -1 (-11, 9) 8 (-9, 24) 4 (-7, 15) 0 (-2, 2) dic vs. dq -7 (-23, 9) 4 (-4, 12) 3 (-4, 10) 3 (-13, 18) 0 (-14, 14) 3 (-15, 21) 5 (-6, 16) 0 (-3, 3) dic vs. ph -1 (-15, 13) 3 (-22, 27) 1 (-10, 12) 1 (-15, 16) 0 (-10, 10) -4 (-20, 13) 1 (-8, 11) 0 (-2, 3) low dic vs. en -11 (-25, -5) 20 (17, 24) 5 (-7, 17) 6 (-22, 34) -2 (-11, 9) 7 (-7, 20) 7 (-12, 25) 0 (-8, 9) dic vs. dq -9 (-20, 3) 19 (14, 24) 3 (-3, 10) 13 (-12, 40) 0 (-20, 21) 2 (-14, 17) 7 (-9, 23) -1 (-8, 6) dic vs. ph -2 (-10, 7) 6 (-26, 38) 1 (-16, 17) 5 (-22, 33) -2 (-17, 12) -3 (-23, 17) 5 (-14, 24) -1 (-8, 10) way of enhancing the value of laboratory practical teaching and learning resources within the veterinary theriogenology curriculum. interestingly, a few of the reflective questions were formulated so as to not have answers that were known by the academic staff. the dissonance was relished by some of the cohort, those excited by investigation and research, but appeared to induce chagrin in a minority that were uncomfortable with the unknown. this was despite the whole group being made aware that this was a safe and fun environment that developed during the session. an important aspect of the peer review process has been the continued discussion between mentors and mentees in relation to enhancing teaching in general, and practical sessions in particular. the vibrancy associated with collegiate interactions between academic staff members and educational designers results in a more enthusiastic and beneficial teaching and learning environment. keywords: abstract reflection, experiential learning, peer support references 1. root kustritz mv, chenoweth pj, tibary a: j am vet med assoc 2006;229:514-521. 2. kolb ay, kolb da: acad manag learn educ 2005;4:193-212. agreement level among 4 techniques for analysis of stallion sperm morphology camilo hernández-avilés, luisa ramírez-agámez, mariah pearson, anne beckham, sheila teague, katrina lacaze, dickson varner, charles love college of veterinary medicine, texas a&m university, college station, tx analysis of sperm morphology is important for assessment of sperm quality and prognostication of fertility potential of stallions. previous studies have compared various techniques for morphologic analysis of stallion sperm using correlation analysis or mean comparisons. we hypothesized that such approaches might lead to erroneous interpretations when comparing different methods of sperm morphologic analysis. the present study sought to determine the agreement level among four techniques for analysis of stallion sperm morphology: eosin/nigrosin staining (en); diff-quick staining (dq); wet mount using phase-contrast microscopy (ph); or wet mount using differential interference contrast microscopy (dic – gold standard). ejaculates from 12 sexually active stallions (n = 36) were collected and analyzed using each technique. a total of 100 sperm were observed under 1,000 x magnification and classified as described by the society for theriogenology (normal [n], abnormal head [ah], abnormal acrosome [aa], proximal droplet [pd], distal droplet [dd], abnormal midpiece [amp], bent midpiece [bmp], and coiled tail [ct]). based on the percent normal sperm observed by dic microscopy, sperm morphology was categorized in each of 3 categories (n = 12/category) as high (h): > 57% normal sperm, average (a): 23 56% normal sperm, or low (l): < 23% normal sperm. within each morphology category (h, a, l), the agreement level (bias, 95% lower and upper limits of agreement) was determined (table). the results from this study indicate that the use of en or dq leads to an overestimation (negative bias value) of normal sperm, as well as an underestimation (positive bias value) of morphological defects that are known to impact stallion fertility, such as ah or amp (values in bold). most discrepancies among methods were observed in categories a and l. these results may affect the interpretation of sperm morphology evaluation and, thus, the estimation of stallion potential fertility. keywords: stallion sperm, sperm morphology, stained smear, wet mount, agreement clinical theriogenology 2021; 13: 330 preliminary report on bovine prenatal sex determination using pcr in maternal peripheral blood navgeet singh,a champika fernando,b janet hill,b john campbell,a dinesh dadarwala alarge animal clinical sciences, bveterinary microbiology, western college of veterinary medicine university of saskatchewan, saskatoon, canada fetal sex identification at pregnancy diagnosis may prove beneficial to beef producers as they could make herd management decisions in an organized manner well in advance. realtime ultrasonography is an ideal tool to obtain immediate information about the pregnancy status and fetal sex; however, the method is time-consuming, needs expertise, and lacks accuracy beyond 90 days of pregnancy. alternatively, sex-chromosome-specific genes of the fetal dna circulating in maternal peripheral blood can be targeted using polymerase chain reaction (pcr), but the technique needs further validation before its commercialization. therefore, the objective of the present study was to identify an appropriate pcr methodology to target sex-chromosome-specific genes to detect fetal dna in maternal plasma for determining fetal sex in pregnant cows. we hypothesized that both bovine amelogenin (baml) and y-chromosome specific genes are targets via pcr to predict fetal sex in cattle. in this initial experiment, we enrolled dairy cows (n = 5; 4 5 months pregnant) carrying male fetuses (confirmed by ultrasonography) from the rayner dairy research and teaching centre at the university of saskatchewan, saskatoon, canada. we collected 18 ml blood from each cow in k2 edta vials to harvest plasma. fresh plasma and frozen aliquots were processed for dna extraction using dneasy blood and tissue, magmax cfdna isolation, kapa express extract, nucleomag cfdna isolation, qiaamp dna blood midi, and qiaamp dsp virus kits. in addition, blood cells from bulls (n = 5) and nonpregnant heifers (n = 5) were processed for dna isolation and subjected to pcr to validate primers and identify the pcr conditions. isolated dna from the plasma of pregnant cows was used as a template for baml and y-specific gene pcr to predict fetal sex. no statistical comparisons were carried out due to the small sample size. the experiment results indicated that when frozen plasma samples were processed for dna isolation (dneasy blood and tissue kit, magmax cfdna isolation kit), pcr failed to predict fetal sex. however, pcr on dna isolated from fresh maternal plasma using the qiaamp dsp virus, dneasy blood and tissue, nucleomag cfdna isolation, and magmax cfdna isolation kits correctly predicted the presence of male fetii in 3/5, 2/5, 2/5, and 2/5 cows, respectively. however, pcr on the dna obtained from both qiaamp dna midi and kapa express kits failed to predict male fetus in all 5 cows. in conclusion, the dna isolation methods compared so far had variation in their ability to isolate fetal dna from the maternal plasma of pregnant cows. we are pursuing spiking experiments with fetal dna from aborted fetuses to further validate dna extraction methodology for cell-free dna from maternal plasma. keywords: bovine amelogenin, fetal dna, y-chromosome specific gene, fetal sex, pregnant cattle melatonin and l-arginine mitigate heat stress-induced reductions in quality of frozen-thawed ram sperm abdallah shahat,a,b jacob thundathil,a john kastelica adepartment of production animal health, faculty of veterinary medicine, university of calgary, calgary, ab, canada; bdepartment of theriogenology, faculty of veterinary medicine, cairo university, giza, egypt adding melatonin or l-arginine to semen extender enhanced postthaw sperm quality and protected against cryopreservation-induced oxidative stress.1,2 the objective was to determine the effects of melatonin or l-arginine on quality of frozen-thawed sperm from rams subjected to heat stress (hs). we hypothesized that addition of melatonin or l-arginine mitigates the effects of heat stress on frozen-thawed ram sperm. ten dorset rams with good semen quality were group-housed indoors (~ 18˚c), randomly allocated into 2 equal groups and subjected to either whole-scrotum insulation for 96 hours or placed in a warm room (28˚c, 30 34% relative humidity) for 8 hours per day for 4 consecutive days. semen was collected weekly for 1 5 weeks after hs, extended (steridyl css one step®) and divided into 5 aliquots: no additives (control) or 0.5 or 1 mm of either melatonin or l-arginine. for cryopreservation, semen was refrigerated for 2 3 hours, cooled to 5˚c, then loaded into 0.5 ml straws that were placed in straw racks and held horizontally in a styrofoam box, 5 cm above liquid n2 for 10 minutes and then plunged into liquid n2. straws were subsequently thawed at 37˚c for 35 seconds and immediately evaluated for postthaw motility using casa (sperm vision®), production animal session clinical theriogenology 2021; 13: 331 morphology using eosin-nigrosin, and acrosome integrity using fitc-psa. data were analyzed using repeated measures, with a post-hoc bonferroni test. for total and progressive motility, there were effects of group (p = 0.023 and p = 0.0008, respectively); for total abnormalities, there were effects of group (p = 0.001 and a group*week interaction (p = 0.003); and for acrosome integrity, there were effects of group (p = 0.046) and week (p = 0.0001). on all days, all end points were significantly improved for all treatments compared to control. all 4 treatments improved motility, whereas improvements in total abnormalities and acrosomal integrity were dose-dependent (greatest improvement with 1 mm). total and progressive motility were improved by ~ 5 10 percentage points, whereas total abnormalities and intact acrosomes were improved by ~ 7 and 12 percentage points, respectively. bowed midpiece, ruffled acrosome and distal midpiece reflex were highest in the control group. in summary, exogenous melatonin or l-arginine in semen extender mitigated hs-induced reductions in quality of frozen-thawed ram sperm by improving motility and acrosome integrity and reducing total abnormalities. keywords: ram, sperm, melatonin, l-arginine, heat stress references 1. ashrafi i, kohram h, ardabili ff: antioxidative effects of melatonin on kinetics, microscopic and oxidative parameters of cryopreserved bull spermatozoa. anim reprod sci 2013;139:25-30. 2. özer kaya, gür s, kaya e: effect of l-arginine addition on long-term storability of ram semen. andrologia 2018;50:1-5. an example of incorrect storage of bull semen samples on spermiogram assessment allan gunn,a,b mark bakerc aschool of animal and veterinary sciences, faculty of science, charles sturt university, wagga wagga, nsw, australia; bgraham centre for agricultural innovation, albert pugsley place, wagga wagga nsw, australia; cschool of environmental and life sciences, university of newcastle, callaghan, nsw, australia sperm morphology assessment and interpretation is an integral part of bull breeding soundness evaluation. historically the spermiogram has been classified into primary, secondary, and tertiary; major or minor; compensable or uncompensable abnormalities; and reporting individual sperm defects. abnormalities that might occur after semen collection (tertiary abnormalities) are often discussed. however, these abnormalities are generally not well defined nor explained. we report tertiary abnormalities that were detected because of incorrect samples submitted for morphological assessment. samples from a cohort of bulls were collected for morphological assessment as part of breeding soundness examination. the samples were examined grossly, crush side motility was assessed by diluting the sample in isotonic phosphate buffered saline (pbs). an ejaculate aliquot was placed in 10% buffered formol saline (bfs) for morphological assessment via differential interference contrast microscopy at 1000 x magnification. in this case, some of the pbs diluted samples were inadvertently sent for morphological assessment in the first instance. the results appeared aberrant, with a large proportion of loose and detached heads, and abnormal tails. the correctly stored samples were located and subsequently assessed. pbs and bfs samples (n = 13) had substantial differences in spermiograms between the storage methods. the bfs samples had 12/13 spermiograms with ≥ 68% morphologically normal sperm. by comparison, 1/13 of the pbs samples had ≥ 68%, with 5/13 having fewer than 20% normal sperm. there were 2 samples in the bfs cohort that had ≥ 19% loose or detached heads, compared to 12 in the pbs cohort that had ≥ 35%, 7 of which had ≥ 55% loose or detached heads. most of the abnormalities detected in the pbs samples were a combination of loose and detached heads. interestingly, the tails, particularly the detached tails, were noticeably devoid of the plasma membrane for some or most of the principal piece and other parts of the tail. these were typically not documented in the interpretation of the spermiogram, as most were recorded as detached heads. particular tertiary abnormalities are not often described in the literature. the inadvertent error of assessing the incorrect samples has given an opportunity to report abnormalities that are most likely due to incorrect storage of samples for morphological assessment of an ejaculate. it is clear from these observations that appropriate collection and storage of samples for morphological assessment is carried out when assessing spermiograms. incorrect sample preparation and storage should be considered as a reason for an abnormal spermiogram, especially when a large proportion of detached heads, with or without tail plasma membrane abnormalities, are detected in a semen sample. keywords: bull, spermiogram, tertiary morphological abnormalities, detached heads obstructive urolithiasis in a dromedary camel yatta boakari, katelyn waters, richard hopper, julie schnuelle adepartment of clinical sciences, college of veterinary medicine, auburn university, auburn, al a 9-year-old, castrated, male dromedary camel was presented with an inability to urinate for 1.5 days. the camel had a history of severe malnutrition and had been castrated prior to onset of puberty. the camel was maintained in a petting zoo and had received an excessive amount of grain prior to his presentation. the animal was bright, alert and responsive with moist and pink mucous membranes, but had mild icteric sclera and was posturing to urinate. there were perineal urethral pulsations accompanied by tail flagging. severe enlargement of the bladder was diagnosed via transrectal palpation and ultrasonography. the bladder was ~ 12 inches in diameter with a thickened wall and mucus debris within clinical theriogenology 2021; 13: 332 the fluid. on abdominal ultrasonography, there was no free abdominal fluid observed. ultrasonography of the penis and urethra revealed a hyperechoic structure proximal to the glans penis, and the urethra was intact. the penis could not be extended for examination due to nonseparation of the penis from the prepuce as a result of early castration. the urine ph was 6.0 and specific gravity was 1.028. on microscopic examination of the urine sample urate crystals were observed. serum chemistry had an elevated bun (52.3 mg/dl; normal range 11 30), creatinine (7.6 mg/dl; normal range 1-2.3), ast (79 u/l) and serum iron (22 µg/dl). the diagnosis was obstructive urolithiasis. a tube cystotomy surgery was done in a 'cushed' position using the following intravenous anesthetic protocol: detomidine (0.03 mg/kg), torbugesic (0.06 mg/kg) and ketamine (2.5 mg/kg). the bladder was accessed with a blind stick using a scalpel blade. a foley catheter with a stylet was inserted through the incision and the bladder was flushed with saline solution. the tube was sutured in place to allow for urine expression. antibiotic therapy with ceftiofur crystalline free acid (6.6 mg/kg subcutaneous, daily) and sulfadimethoxine (55 mg/kg initial dose, 27.5 mg/kg subsequent doses, intravenously, daily) and an antiinflammatory (flunixin meglumine 1.1 mg/kg, intravenously, once every 12 hours) was started. acepromazine (20 mg intravenously) to cause urinary tract relaxation and fluid therapy (5 liter bolus, once every 12 hours) were done. there was a continuous and steady urine dripping from the foley catheter after the surgery and during the next 3 days, so the treatment plan was continued. however, on the fourth day after surgery, the animal was seen posturing to urinate and the bladder was lavaged with 5 liters of saline solution, during which bloody drops were noted in the prepuce. urine had a ph of 7 and struvite crystals were seen. hence, a total of 120 ml of walpol’s solution was placed in the bladder. then, 30 minutes later, another 5 liters lavage was done. the next day, the foley catheter had become occluded due to fibrin deposition. the animal was posturing more frequently, although the bladder was small on ultrasound. animal was sedated in accordance with the previous surgery and an epidural was given with 3 ml of lidocaine. a perineal urethrostomy and penile amputation was performed while the animal was in a cushed position. the urethra was then spatulated at the level of the skin incision and a catheter was placed. the camel continued to urinate successfully, and treatment was discontinued. the animal was sent home on allopurinol to be given every other day to aid in reduction of uric acid and was instructed to receive a low grain diet. early castration in camelids can predispose to a narrowed urethra that coupled with a high grain diet may lead to urolithiasis. keywords: camelid, urogenital system, bladder, urethra evaluation of ovarian response to pg600 in alpacas eduardo arroyo, cristian patino, michella ciccarelli, ahmed tibary acomparative theriogenology, department of veterinary clinical science, college of veterinary medicine, center for reproductive biology, washington state university, pullman, wa the hormonal preparation pg600, a combination of equine chorionic gonadotropin (ecg) and human chorionic gonadotropin (hcg), is commonly used for swine estrus synchronization. however, it is often used off-label in sheep and goats to replace the ecg formulation not available in the us. in alpacas, ecg has been used alone or in combination with fsh to induce superovulation, with variable results. the aim of this preliminary trial was to investigate the effect of pg600 on follicular dynamics in alpacas. we hypothesized that a single treatment of pg600 (800 iu ecg and 400 iu hcg in 5 ml) 2 days after ovulation results in ovarian follicular superstimulation in alpacas. adult multiparous alpacas (n = 9) were used in the experiment. ovarian follicular activity was monitored by transrectal ultrasonography. ovulation was induced with gnrh (100 µg) given intramuscularly when a dominant follicle reached at least 8 mm in diameter and uterine tone and edema were present. all females received 5 ml pg600 intramuscularly 2 days after induction of ovulation. ovarian follicular response was assessed by transrectal ultrasonography on day 7 after pg600 treatment and the follicles were recorded and counted by 2 clinicians. ovulation was induced with gnrh (100 µg) given intramuscularly. the mean number of follicles between 7 and 12 mm in diameter present in the ovaries after the treatment was 14.9 ± 13.8 (mean ± sd). there was a large variation among females in the number of follicles, which ranged from 1 to 38. the ovulation rate (number of corpora lutea) following induction was very low 1.1 ± 1.6 (mean ± sd). the maximum ovulation rate (5) was observed in a female that had 6 follicles after stimulation. all females presented anovulatory hemorrhagic follicles 2 days after induction of ovulation (10.0 ± 11.5, mean ± sd). in conclusion, pg600 induced ovarian follicular stimulation (> 2 mature follicles) in 7 out 9 alpacas. however, the follicular response had large individual variability. the ovulation rate after ovarian superstimulation with pg600 was poor. however, this poor ovulatory response has been also observed with ecg and fsh. this preliminary trial suggests that pg600 may not be appropriate for ovarian superstimulation in multiple ovulation embryo transfer programs in alpacas but could be used for oocyte recovery within other advanced reproductive techniques. keywords: ovulation, superovulation, camelids, anovulatory follicles clinical theriogenology 2021; 13: 333 morphometric characteristics, testicular histology, and semen parameters in mature hybrid bucks born from white-tailed deer dams sired by a mule deer buck giorgia podico, clifford shipley, igor canisso department of veterinary clinical medicine, university of illinois, urbana, il despite white-tailed deer (wtd) and mule deer (md) sharing similar number of chromosomes (n = 70), the 2 species have distinct features making the crossing of species unsuccessful in wild or captive conditions. across mammals, hybrids are regarded as infertile, yet anecdotal reports suggest that wtd-md hybrids are fertile. however, there have been limited reports of hybrid animals producing fully formed sperm, though none on hybrids of wtd-md. we aimed to describe the somatic morphometric features, testicular histology and semen features of captive hybrids of wtd-md. four 1.5-year-old bucks were enrolled in this study, 2 hybrids wtd-md, 1 wtd, and 1 md from a captive farm. the hybrid animals were born from different wtd, but were sired by the same md. morphometric profile included thorax circumference, crown-rump length, metacarpus and metatarsus diameter, tail length, tail color, location of the metatarsal gland, antler configuration, antler inside spread, ear length, metatarsal tuft color, scrotal circumference and length of the penis. semen collected via electroejaculation was evaluated for the presence of sperm and the concentration of alkaline phosphatase (alp). testicular biopsies were collected from both testes using a split needle biopsy tool. the scrotal circumference was 22 cm for the wtd, 19 cm for the md, and both hybrids measured 12 and 14 cm, respectively. the penis length was 28.6 for the wtd, 27.4 for the md and 12 and 14 cm for the hybrids. it has been suggested that testosterone regulates penis growth after puberty; perhaps these animals had lower testosterone production hence the shorter penis. semen collection yielded ~1 ml of yellow and viscous fluid. no sperm were visualized under the phase-contrast microscope. histologic evaluation revealed the presence of hypoplastic seminiferous tubules in both animals populated with spermatogonia in the basal compartment and normal sertoli cells. one animal had primary spermatocytes in the adluminal compartment and scattered spermatids could be seen in a few seminiferous tubules. the basal membrane of the seminiferous tubules was surrounded by dense, irregular connective tissue. the leydig cells were present in the interstitium and appeared morphologically normal; this explains why the hybrids were able to produce intermediate sized antlers that hardened at the peak of rut. concentrations of alp in hybrid 2 was 1620 u/l. the color of the tail of the hybrids was brown on the dorsal surface, but white on the ventral part and resembled 1 of the wtd. the metatarsal gland was in the proximal segment of the metatarsus in the hybrids and wtd, whereas it was below in the md. only the md presented dichotomous antlers. the metatarsal tuft color was brown in the hybrids and md, but white in the wtd. in conclusion, hybrid 1 male was unable to complete spermatocytogenesis, and the second could not complete spermiogenesis, making then unable to fully form sperm. the high concentrations of alp confirmed ejaculation in these 2 animals; however, they were deemed infertile. keywords: azoospermia, infertility, mule deer, white-tailed deer, hybrids association of metabolic status with uterine diseases and reproductive outcomes in lactating holstein dairy cows andrew hubner,a phillip peixoto,a wilson coelho jr.,a leonardo ribeiro,a brian aldridge,a igor canisso,a fabio limaa,b adepartment of veterinary clinical medicine, university of illinois, urbana, il; bdepartment of population health and reproduction, university of california, davis, ca selection of high yielding dairy cows has predisposed them to develop metabolic and uterine diseases. while the association of hyperketonemia (hrk) with metritis and decreased reproductive performance is known, little data exists examining the association of hrk and concurrent hypoglycemia with metritis and reproductive outcomes. our hypothesis was that cows with hrk have a higher incidence of puerperal metritis and poorer reproductive outcomes than cows without hrk and those effects are more profound in cows with hypoglycemia. the objectives of this study were to analyze the association of metabolic status with metritis incidence and reproductive outcomes in lactating dairy cows. cows (n = 2651) had blood samples collected between 3 9 days postpartum (dpp) and whole blood beta-hydroxybutyrate (bhba) and whole blood glucose was measured using a hand-held cow-side device validated in dairy cows (precision xtra, abbott, mississauga, on, canada). hyperketonemia was defined as bhba ≥ 1.2 mmol/ liter and hypoglycemia was defined as glucose ≤ 2.2 mmol/ liter. cows were then categorized into the following 4 groups: first those having no metabolic abnormality (norm, n = 1996), those having hrk only (bhba, n = 260), those having hypoglycemia only (hg, n = 181), and those having both hrk and hypoglycemia (bhba + hg, n = 214). incidence of puerperal metritis (defined as watery, fetid discharge present at time of blood collection), and the reproductive outcomes first insemination pregnancy per ai (p/ai), pregnancy loss, average days open (dopn), and proportion of cows pregnant at 150 dim (p150) were compared for the 4 metabolic statuses enrolled in the study. the cow-level prevalence of hyperketonemia was 17.9% (474/2651), and the cow-level prevalence of hypoglycemia was 14.9% (395/2651). the cow-level prevalence for each metabolic category was as follows: norm, 75.3% (1996/2651); bhba, 9.8% (260/2651); hg, 6.8% (181/2651); and bhba + hg, 8.1% (214/2651). statistical analysis was performed using anova and logistic regression with jmp pro 13 (sas institute inc. cary, nc, us). parity (parity 1 (p1) versus parity ≥ 2 (p2)), season, and farm were retained in the model. p2 prevalence of puerperal metritis was significantly less for cows in the hg group compared to cows in the bhba and bhba+hg groups clinical theriogenology 2021; 13: 334 (hg, 1.9%; bhba, 16.8%; bhba+hg, 9.4%). p2 prevalence of puerperal metritis was significantly less in the norm group compared to cows in the bhba group but similar to cows in hg group (norm, 6.7%; bhba, 16.8%; hg, 1.9%). for primiparous cows there was no difference in incidence of puerperal metritis amongst metabolic categories. there were no differences between metabolic groups for p/ai, pregnancy loss, average dopn, or p150. in conclusion, hyperketonemia of multiparous cows was associated with increased puerperal metritis; however, hypoglycemia alone was associated with decreased puerperal metritis compared to cows with elevated bhba with or without concurrent hypoglycemia. keywords: hyperketonemia, hypoglycemia, dairy cows, uterine disease effect of gnrh at artificial insemination for dairy cows detected in estrus by an activity monitoring system or by conventional estrus detection andrew hubner,a phillip peixoto,a igor canisso,a fabio limaa,b adepartment of veterinary clinical medicine, university of illinois, urbana, il; bdepartment of population health and reproduction, university of california, davis, ca artificial insemination (ai) after detected estrus constitutes a substantial proportion of ai’s that occur in the us. moreover, ai after detected estrus may be increasing with the use of activity monitors. we hypothesized that gnrh treatment at ai increases both ovulation and circulating progesterone concentrations, thereby improving pregnancies per ai (p/ai); additionally, we hypothesized that this effect is higher in farms using activity monitors. the objectives of the study were to determine if gnrh treatment at ai increases p/ai for lactating dairy cows detected in estrus on farms using activity monitors (am) or not (nam). holstein cows were blocked by parity and randomly assigned to receive an injection of gnrh at ai (g-ai) or to receive no injection of gnrh (ng-ai) at ai on a farm using am for estrus detection and on a group of 4 farms using nam. on the farm with am, 409 cows were enrolled (g-ai, n = 207; ng-ai, n = 202) and for the farms using nam, 398 cows were enrolled (g-ai, n = 197; ng-ai, n = 201). ovarian structures and plasma progesterone concentrations were assessed in a subset of cows (g-ai, n = 52; ng-ai, n = 55) detected in estrus by conventional methods at the time of ai and 7 days later. data were categorized by milk production quartiles, genomic daughter pregnancy rate (high: > the median, low: < the median), activity level (al) for the farm using am (high: al > the median al versus low: al < the median al) and dim (> 150 dim versus < 150 dim). statistical analyses were performed using logistic regression and a student’s t-test. there were no differences in ovulation rate (g-ai = 83.2 ± 6.1%; ng-ai = 77.9 ± 5.5%) between g-ai and ng-ai. there were no differences in plasma progesterone concentrations at day of estrus detection (day 0) (g-ai = 0.16 ± 0.11 ng/ml; ng-ai = 0.09 ± 0.10 ng/ml) nor at day 7 after enrollment between g-ai and ng-ai (g-ai = 2.17 ± 0.15 ng/ml; ng-ai = 2.04 ± 0.15 ng/ml). data for all farms were analyzed together for p/ai; no difference for p/ai at first pregnancy diagnosis (g-ai = 38.7 ± 3.9%; ng-ai = 40.9 ± 3.9%) or second pregnancy diagnosis (g-ai = 35.1 ± 4.1%; ng-ai = 35.7 ± 4.2%) was identified. no difference in p/ai between g-ai and ng-ai when farms were analyzed separately based on estrus detection method (am separate from nam) at first pregnancy diagnosis (am: g-ai = 39.1 ± 5.0%; ng-ai = 38.6 ± 5.1%; nam: g-ai = 38.3 ± 5.2%; ng-ai = 43.3 ± 5.2%) or second pregnancy diagnosis (am: g-ai = 36.3 ± 5.1%; ng-ai = 33.8 ± 5.2%; nam: g-ai = 33.8 ± 5.3%; ng-ai = 37.8 ± 5.2%) was identified. there was no interaction between treatment and method of estrus detection. for the farm using am, there was a significant interaction between treatment and al with the injection of gnrh having a greater impact on cows with high al. in conclusion, gnrh treatment did not enhance p/ai. additional studies are warranted to understand the interaction between treating cows with gnrh at ai and al in herds using activity monitoring systems. keywords: dairy cows, estrus detection, activity monitors, gnrh clinical theriogenology 2021; 13: 335 uterus unicornis in a maiden mare stephanie walbornn,a nathaniel newton,b maria schnobrichb arood and riddle hospital in wellington, wellington, fl, bleblanc center for reproduction, rood and riddle equine hospital, lexington, ky uterus unicornis is a congenital abnormality that has been reported in several domestic species, but occurs uncommonly in mares. a previous report described a successful foaling in a warmblood mare with this condition, but the foal was small and dysmature.1 two additional cases reported in american paint mares have been described.2,3 the case reported herein corresponded with a maiden standardbred mare that was imported from europe. breeding management was attempted over 1 estrous cycle but the mare did not become pregnant. she was referred for a breeding soundness evaluation, as an abnormality was suspected on transrectal palpation of her reproductive tract. palpation and transrectal ultrasonography of the reproductive tract revealed the absence of a normal left uterine horn. both ovaries were normal in shape and size and ovary was active. the left ureter appeared normal and was traced to the left kidney. urine was visualized moving through the left ureter. the right uterine horn appeared normal and no uterine edema or fluid was observed. cervix was toned and had a competent canal. hysteroscopic examination was performed and a normal uterine body, right uterine horn, and right oviductal papilla were observed. there was no evidence of a left uterine horn presence. based on these findings, a diagnosis of uterus unicornis or segmental aplasia of the müllerian ducts was made. this abnormality has been observed in other species. in cattle, it is associated with a white coat phenotype in breeds such as shorthorn and belgian blue.4 this condition is associated with failure of the development of the mullerian ducts and appears to be genetically inherited. breeding this mare to carry to term was discouraged and assisted reproductive techniques such as embryo transfer and ovum pick up were recommended. the client decided not to utilize this mare for breeding. this is the first report of uterus unicornis in a standardbred mare. although rare, this condition can cause infertility in mares and may accompany other genetic or congenital abnormalities. it is important to pursue genetic testing to ensure the mare does not have karyotypic abnormalities. further diagnostic procedures to assess genitourinary function are also important as renal agenesis and ureteral abnormalities can occur ipsilateral to the affected uterine horn.3 keywords: uterus unicornis, congenital abnormality, mare infertility references 1. gallacher k, gilbert r: case report: successful foaling in a warmblood mare with uterus unicornis. clinical theriogenology 2018;10:51-58. 2. brown ja, hodder ad, benak j, et al: uterus unicornis in two mares. aust vet j 2007;85:371-374. 3. li s, qayyum a, coakley fv, et al: association of renal agenesis and mullerian duct anomalies. j comput assist tomogr 2000;24:829-834. 4. 1gallacher k, gilbert r. case report: successful foaling in a warmblood mare with uterus unicornis. clinical theriogenology. 2018;10:51-58. 2brown ja, hodder ad, benak j, et al: uterus unicornis in two mares. aust vet j. 2007;85:371-374. 3li s, qayyum a, coakley fv, et al: association of renal agenesis and mullerian duct anomalies. j comput 4rendel jm: white heifer disease in a herd of dairy shorthorns. j genet 1952;51:89-94. a congenital abnormality in a mare: nonpatent uterus christine bartley, maria schnobrich rood and riddle equine hospital, lexington, ky failure of normal uterine development was diagnosed in in a 4-year-old thoroughbred mare. congenital abnormalities of mares have been described; segmental aplasia in other species, such as cattle, is often associated with genetic abnormalities. this mare had no history of uterine infusions, breeding, or vaginal examination prior to initial evaluation performed at 3 years of age (in winter) while in training. transrectal palpation and speculum examination revealed no abnormalities. mare was presented again 2 months later for a prebreeding evaluation. mare had normal overall appearance, body condition score 6/9, normal mammae and external genitalia with a caslick’s in place. transrectal palpation and ultrasonography revealed a mildly flaccid uterus, normal shape and size to the ovaries with multiple 20 mm follicles. scant free fluid was observed in the uterine horns with swirling heterogeneous echogenic material and with no endometrial edema. cervix was short and flaccid. vaginoscopy revealed a small hypoplastic cervix poster session clinical theriogenology 2021; 13: 336 and external os and digital evaluation revealed a patent but short canal with no communication into uterine lumen. hysteroscopy was performed and the cranial cervical canal failed to connect to uterine lumen. fine spiderweb trabeculae of tissue traversed the area cranial to the cervix, consistent with subepithelial tissue that was distended with air. however, no uterine lumen and there was no evidence of normal endometrium. chromosomal analysis of the mare revealed normal female karyotype. these findings are consistent with a congenital abnormality and canalization failure of a normal uterine lumen. in some cases, uterine exposure to caustic substances can result in this condition; however, this mare had also had normal endometrium. as diagnosed in this case, congenital malformation of the uterus (that prevented normal ability to carry a pregnancy) can be missed with routine evaluation. keywords: mare, segmental aplasia, uterine and cervical hypoplasia, congenital abnormality ovine male pseudohermaphrodite with testes adjacent to mammary gland in a sheep alyssa ball,a brett woodie,a john hubbell,a laura kennedy,b terje raudsepp,c maria schnobricha arood & riddle equine hospital, lexington, ky; buniversity of kentucky veterinary diagnostic laboratory, lexington; ctexas a& m university molecular cytogenetics laboratory, college station, tx male pseudohermaphrodite sheep have been reported with variations in genotype and phenotype.1 a katahdin sheep was born as a triplet with 1 male and 1 female siblings. at birth, the sheep had what was considered normal ewe lamb phenotype. at 1.5 years of age the phenotype of the sheep described included a long-haired mane and heavy muscling. the lamb siblings developed normal ewe and ram phenotypes. the sheep’s vulva had a prominent ventral bulge with increased hair and a prominent clitoris, consistent in appearance with a glans penis. tip of the glans penis had a very short urethral process. the animal demonstrated behavior consistent with a male including protective ‘ramming‘ and mounting of its pasture mates. transabdominal ultrasonography could not confirm structures consistent with testicular parenchyma. serum testosterone concentrations were 5.0 pg/ml. based on the unwanted aggressive behavior, an exploratory laparoscopy was performed in an attempt to identify any testicular tissue, and if present, remove it. exploratory laparoscopy revealed bilateral tubular structures consistent with the vas deferens originating from the inguinal canal and reaching the dorsal aspect of the urinary bladder. external palpation identified 2 ovoid structures (~ 5 x 3 cm) located between mammary tissue and body wall. skin consistent with scrotal skin (wrinkled and slightly red) was observed in bilateral regions 2 cm in diameter caudal to the mammary gland. these 2 structures were removed via a 3 cm incision made lateral to the mammary tissue. histopathology revealed testicular tissue with abortive spermatic tubules, lined by sertoli cells without germinal cells, and bilateral suppurative epididymitis. no female gonadal tissue was identified by light microscopy. karyotype revealed a mixed population of genetically female 54, xx (80%), and male xy (20%) lymphocytes. this may indicate blood chimerism or true somatic mosaicism and dna analysis from an ear punch is pending. a case of an unusual location of extra-abdominal but undescended testes in a male pseudohermaphrodite katahdin sheep is described. keywords: male pseudohermaphrodite, sheep, bilateral epididymitis, triplets reference 1. breuere, an, mcdonald mf, marshall rb: cytogenetic analysis of an ovine male pseudohermaphrodite and the possible role of the y chromosome in cryptorchidism of sheep. cytogenetics 1969;8:148-157. effect of hyaluronic acid on fresh-cooled extended equine semen: sperm motility rachel fladung,a maria schnobrich,b christine bartley,b modesty burleson,a charlie love,c lauren moshier,b crystal howard,b alaina broach,b stephanie walbornnb aspy coast farm llc, lexington, ky; bleblanc center for reproduction, rood & riddle equine hospital, lexington, ky; ctexas a & m university college of veterinary medicine, college station, tx; drood & riddle equine hospital in wellington, wellington, fl hyaluronic acid (ha) is a glycosaminoglycan, has a role in in vitro sperm-oocyte binding and exerts antioxidant properties.1,2 conflicting results regarding the benefit of addition of ha to freezing extender on postthaw motility exist.2 this study determined whether addition of sodium hyaluronate (hytryl®, 10 mg/ml, kineticvet, lexington, ky) at varying concentrations to 2 milk-based extenders affected motility parameters of fresh-cooled equine semen stored at 5°c for up to 72 hours. we hypothesized that the addition of hytryl®, 10 mg/ml to fresh-cooled equine semen increases total and/or progressive motility, benefitting the longevity and quality of equine freshcooled semen. ejaculates from 8 stallions were extended in either inra (imv technologies, imv technologies, l’aigle, france) or cst (animal reproduction systems, chino, ca) with no or differing concentrations of hytryl® at 0, 100, and 1,000 µg/ml. the samples were stored in a passive cooling device (equisaver, integritemp, omaha, ne) and cooled to 5°c for up to 72 hours and aliquots were incubated at 37°c for 5 10 minutes prior to motility evaluations with a computer assisted sperm analysis (casa; spermvision®, minitube, verona, wi). sperm motility of each treatment group was compared at time 0, 24, 48, and 72 hours postcollection. means of total and progressive motility parameters were subjected to the mixed procedure of sas® for statistical analysis. means were compared using tukey’s range test at a significance level of alpha = 0.05. there were no significant differences in total or progressive motility clinical theriogenology 2021; 13: 337 among treatments at any time point. in conclusion, addition of hyaluronic acid to 2 milk-based extenders did not affect motility parameters of fresh-cooled equine semen. additional work is necessary to determine whether there is any benefit to stallion fertility with ha in semen extenders. keywords: stallions, sperm, motility, fertility, hyaluronic acid references 1. bruemmer j, wilson c, da silva mc et al: effects of hyaluronan supplementation on cryopreserved equine spermatozoa hyaluronan and cryopreserved equine spermatozoa. j equine vet sci 2009;29:223-228. 2. talbot p, shur bd, myles dg: cell adhesion and fertilization: steps in oocyte transport, sperm-zona pellucida interactions, and sperm-egg fusion. biol reprod 2003;68:1-9. methods to prepare platelet-rich plasma lorenzo segabinazzi,a,c giorgia podico,a michael rosser,a som nanjappa,b marco alvarenga,c igor canissoa adepartment of veterinary clinical medicine, bdepartment of pathobiology, college of veterinary medicine, university of illinois urbana-champaign, urbana, il; csão paulo state university (unesp), school of veterinary medicine and animal science, botucatu, sp, brazil platelet-rich plasma (prp) is a biological by-product commonly used in clinical practice to treat orthopedic and dermatologic conditions. recently, use of prp has become popular in management of a mare’s reproduction to mitigate postbreeding induced endometritis and improve fertility. currently, there are no standardized methods to prepare prp for intrauterine use in mares. the aim of this study was to compare 3 methods of prp preparation. eighteen clinically healthy mares had blood collected via venipuncture in a blood transfusion bag (method 1), blood tubes (method 2), and a syringe (method 3). in method 1, blood was collected in a 150 ml blood transfusion bag containing 21 ml of citrate-phosphate-dextrose solution with adenine as an anticoagulant (cpd-a). after collection, blood was divided into 50 ml conical tubes and centrifuged at 400 x g for 10 minutes. resulting plasma was split into 15 ml conical tubes and subjected to centrifugation at 1000 x g for 20 minutes. after second centrifugation, the 2.5 ml in the bottom of each tube was considered prp, and the remaining plasma as platelet-poor-plasma (ppp). method 2 involved centrifugation of blood collected in 4.5 ml vacutainer tubes containing 3.2% sodium citrate at 120 x g for 10 minutes. the top third layer of the plasma was deemed as ppp, while the remaining portion was considered prp. in method 3, blood was collected in a 60 ml syringe containing 7 ml of cpd-a; after collection, each syringe was wrapped in aluminum foil and placed in an upright position for 4 hours. the top 10 ml of plasma was considered ppp, and the remaining plasma (including sedimented blood cells) was deemed prp. after processing by 3 methods, prp and ppp were extracted and assessed for red and white blood cell counts, platelet counts, and viability. in a subset of mares (n = 6), samples of prp were also evaluated at 6 and 24 hours postcooling at 5°c. method 1 resulted in the highest, and method 3 in the lowest, platelet concentrations; the latter had higher (p < 0.05) wbc than others. platelet viability was similar among treatments. the recovery factor (i.e. the ratio of the prp volume to the whole blood volume) of plasma recovered as prp was different (p < 0.0001) among methods; method 1, 10.5%; method 2, 33.1%; method 3, 27.2%). cooling for 24 hours did not affect (p > 0.05). platelet counts. however, platelet viability was reduced (p < 0.05) after cooling in prp produced by method 3, and agglutination increased over time among methods. in conclusion, the 3 methods resulted in satisfactory prp yield without compromising platelet viability. method 1 (i.e. involving double centrifugation) resulted in the greatest platelet concentrations whereas method 3 (sedimentation) resulted in the lowest platelet concentration and tended to be more contaminated with leukocytes. cooling affected platelet viability in prp obtained by method 3 and increased platelet agglutination over time among methods. clinical efficacy of prp with these methods of cooling remains to be determined. funding: cesarean section, hypercoagulable, pulmonary embolism, venous thromboembolism keywords: platelet concentrates, horse, endometritis, tissue regeneration fetal bones in the uterus of a thoroughbred mare ethan sutherland,a stephanie walbornn,a james morehead,b maria schnobrichc aequine medical associates, lexington, ky; brood and riddle equine hospital in wellington, wellington, fl; cleblanc center for reproduction, rood and riddle equine hospital, lexington, ky fetal mummification has been reported occasionally in domestic species. it is more common in polytocous species, but has been observed in the horse, most commonly in twin pregnancies. mummification is typically a sterile process in which fetal death occurs, the conceptus dehydrates, and is retained within the uterus. this describes a case in a 7-year-old thoroughbred mare diagnosed pregnant with 1 conceptus (evaluations normal with transrectal ultrasonography at 15, 17, 29, and 42 days postovulation). transrectal palpation confirmed pregnancy at 5 months of pregnancy. at 7 months of pregnancy, ultrasonography revealed that the mare was not pregnant. two uterine lavages were performed and 6 grams timentin was infused into the uterus after lavage. two months later, uterine cytology and aerobic culture performed prior to breeding season revealed severe inflammation and moderate growth of escherichia coli and enterobacter aerogenes. mare’s uterus was lavaged for 4 days and infused with 2 grams amikacin, and a caslick’s was placed. transrectal ultrasonography of the uterus performed 1 month later revealed multiple small (2 cm) hyperechoic linear structures in the uterine lumen at the base of the uterine horns extending clinical theriogenology 2021; 13: 338 into the right uterine horn. uterine lavage was performed to aid in the removal of these structures with no success. a repeat uterine culture and cytology revealed moderate inflammation and a light growth of escherichia coli. hysteroscopy revealed several bony fragments within the right uterine horn and were extracted. there were in total 7 fetal bones, ranging from 1.5 to 2 cm consistent with 2 scapula, 2 pelvic bones, and 3 long bones. mare was given a dose of broad-spectrum systemic antibiotics, the uterus was lavaged, and acetylcysteine was infused. uterine lavage was continued for 3 more days. two weeks later, a culture and cytology were performed and were negative. mare was bred over 2 estrous cycles (~ 30 and 55 days after the procedure) and became pregnant with twins after the second estrus. one embryonic vesicle was successfully reduced and the mare was confirmed in foal with 1 fetus at 49 days of pregnancy. due to the low prevalence of fetal mummification in the horse, the underlying cause of this phenomenon has been difficult to discern. when twins are present, placental insufficiency is typically the cause of fetal demise of 1 fetus followed by fetal fluid resorption. in singleton pregnancies, there has been no established cause for fetal mummification and why they are retained within the uterus. this case demonstrated the future fertility of mares after fetal mummification treatment. keywords: mare, fetal mummy, uterine foreign body, endometritis reference 1. lefebvre rc: fetal mummification in the major domestic species: current perspectives on causes and management. vet med (auckl) 2015;6:233-244. intra-uterine injection of amnion-derived acellular bioscaffold product in mares: systemic and intra-uterine effects over 21 days nathaniel newton,a stephanie walbornn,a christine bartley,a jamie kaczor,a lauren moshier,a holly hersey,a dale kelley,b robert holland,c maria schnobricha arood & riddle equine hospital, lexington, ky; boklahoma state university, college of veterinary medicine, stillwater, ok; cholland management services inc., lexington, ky amnion-derived acellular bioscaffold product (adabp) has been used as an antiinflammatory agent to promote healing in human and veterinary medicine. proteins and cytokines present in adabp are reported to decrease fibroblast formation and fibrosis.1 thus, adabp may be beneficial in the treatment of uterine adhesions, uterine cyst ablations and remodeling of scar tissue. the safety of uterine injection of adabp is unknown. we studied the systemic and uterine effects after uterine injection. twelve clinically healthy light-breed mares (mean age 11.5 years; range 5 22) were the subjects. rectal temperature and behavior were recorded for the duration of the study. on day 0, all mares underwent a hysteroscopic examination, control mares (n = 3) received 3 ml injection of sterile saline in the base of 1 uterine horn, and anicell mares (n = 9) received 3 ml of adabp (equuscell stemwrap d™, anicell biotech, arizona) in the base of 1 uterine horn. blood (for serum amyloid a [saa], fibrinogen [fib], and white blood cell count [wbc]), endometrial cytology and aerobic cultures were obtained prior to hysteroscopy. four days (day 4) after injection, mares were evaluated via transrectal ultrasonography and blood was obtained. twenty-one days (day 21) after injection, endometrial cytology, aerobic culture, and hysteroscopy were performed. continuous data were analyzed to determine the main effects of group, day and their interaction using the sas mixed procedure with a repeated statement. categorical data were analyzed using the sas logistic procedure. no mares experienced an elevation in rectal temperature during the 21 days after injection. there were no differences in bloodwork for markers of inflammation (saa, fib, wbc) from day 0 to day 4 either in the control or anicell group. similarly, there were no differences in uterine cytology and culture results between groups or among days within groups. hysteroscopy following injection demonstrated no gross evidence of detrimental effects in any mare examined. in 1 mare that received a saline injection, a small 1 cm bleb of fibrous tissue was noticed and that remained for 21 days after injection. this study demonstrated that adabp had no detrimental effect on the systemic health of the mare and it is as safe as hysteroscopy and saline intrauterine injection up to 21 days after injection. further work is continuing, evaluating histological changes in the mares’ endometrium after injection and in clinical cases where injection is performed into uterine tissue, as adabp may be a useful tool to promote endometrial healing in the mare. keywords: amnion-derived cell product, endometritis, hysteroscopy, uterine injection reference 1. mamede ac, carvalho mj, abrantes am, et al: amniotic membrane: from structure and functions to clinical application. cell and tissue res 2012;349:447-458. luteal blood flow and side effects of luteolytic doses of dinoprost tromethamine and cloprostenol sodium in jennies humberto magalhaes,a,b felipe medrado,b jose dell-aqua jr,b igor canissoa adepartment of veterinary clinical medicine, university of illinois, urbana, il; bdepartment of animal reproduction, são paulo state university, botucatu, brazil exogenous prostaglandin f2α (pgf2α) treatment revolutionized the breeding management of livestock and horses. however, despite 4 decades of its continued use in theriogenology, the optimal luteolytic dose for donkeys has not been determined. clinical theriogenology 2021; 13: 339 cloprostenol sodium and dinoprost tromethamine are 2 most widely used and available pgf2α, with the former being a natural and the latter a synthetic prostaglandin. label dose treatment of pgf2α results in colic-like signs in mares, but the impact is unknown in donkeys. the latter species are thought to be more pain-tolerant than horses. this study aimed to objectively assess luteolysis and side effects of jennies receiving standard horse doses of cloprostenol and dinoprost. we hypothesized that the luteolytic doses widely recommended for horses have no side effects in donkeys and both types of pgf2α have equivalent luteolytic properties. eight jennies (144 ± 22.5 kg; height 95.5 ± 113 cm) were used. five days after ovulation, jennies were randomly assigned in a cross-over design and received either intramuscular dinoprost (5 mg) or cloprostenol (250 µg). b-mode and doppler ultrasonography were performed starting 15 minutes before pgf2α, and then repeated at 15 minute intervals until 1 hour after pgf2α and then at 2, 3, 4, 5, 6, 7, 8, 12, and 24 hours. at these times, serum samples were collected for progesterone concentrations by ria (beckman coulter, us). animals were observed from a distance for side effects (sweating, abdominal discomfort, and diarrhea) at 15 minute intervals starting before and for 1 hour after pgf2α. data normality was assessed with the shapiro-wilk’s test and comparisons of the cl area and luteal blood flow were performed using proc mixed of sas 9.4. the study was approved by the ethics committee on the use of animals – ceua (unesp, brazil) under protocol 0028/2019. jennies were accounted as random effect whereas time and luteolytic agent were fixed effects. interactions of fixed effects were also assessed. significance was considered as p ≤ 0.05. an increase (p < 0.05) in cl blood flow was observed 60 minutes and 45 minutes after treatment with dinoprost and cloprostenol, respectively. there was an increase (p < 0.05) in cl blood flow at 4 hours after dinoprost compared to cloprostenol treatment. however, at hours 5, 6, and 7, jennies that received cloprostenol had higher cl vascularity than dinoprost-treated cycles. blood flow and cl area decreased gradually during the first 24 hours in both groups. both prostaglandins reduced (p < 0.005) serum progesterone concentrations within 30 minutes after treatment with no differences (p > 0.05) between groups. dinoprost resulted in major score of sweating (p ≤ 0.05) whereas higher (p ≤ 0.05) abdominal discomfort and diarrhea were detected in cloprostenol. in conclusion, both prostaglandins and doses used were equivalent in inducing luteolysis in donkeys. however, both prostaglandins caused adverse reactions, leading us to believe that horse doses used are inappropriate for small-frame donkeys. keywords: jennies, corpus luteum, doppler ultrasonography, progesterone, side effects sperm-filter enhanced semen parameters and fertility of stallion poor cooled semen lorenzo segabinazzi,a,b igor canisso,b marcio carmo,a camila dell’aqua,a jose dell’aqua jr.,a fredrico papa,a marco alvarengaa adepartment of animal reproduction, são paulo state university botucatu, brazil; bdepartment of veterinary clinical medicine, university of illinois, urbana, il cooled-shipped semen is the horse industry’s primary approach to breed mares. whereas most stallion ejaculates tolerate cooling some have inadequate responses to cooling. despite the development of new extenders in the past 2 decades, some stallions still have poor semen cooling quality. therefore, there is a critical need to develop tools to process semen for stallions with a poor response to cooling. sperm-filter® (sf, botupharma) is a porous membrane used as an alternative to centrifugation. this technology has yet to be tested in stallions with poor semen cooling. therefore, this study’s objective was to assess semen parameters and fertility of cooled-stored stallion semen processed with sf or conventional centrifugation ([c] 600 x g for 10 minutes) and reextended in 3 commercial extenders. we hypothesized that sf enhances semen parameters and improves the fertility of stallions with poor semen cooling ability. the ejaculates were obtained from 7 stallions known to have poor semen cooling ability (i.e. < 25% total motility (tm) 24 hours postcooling at 5°c). after collection, semen was extended to 50 x 106 sperm/ml with a skim milk-based extender ([sm] botusemen, botupharma) and stored at 5°c for 24 hours. at 24 hours postcooling, samples were split into 7 groups. control (ct) consisted of cooled semen with no further processing and the remaining 6 groups were submitted to sf or c, then resuspended in either sm, sm containing pentoxifylline ([p] botuturbo, botupharma), or an egg yolk-based extender ([ey] botucrio, botupharma). total and progressive motility (pm) and percentage of sperm with rapid motility (rap) were assessed with casa (ivos 12, hamilton thorne, beverly, ma). plasma membrane integrity (pmi), and mitochondrial membrane potential (mmp) were assessed with the combination of yo-pro® and mitostatusred with flow cytometry (lsr-fortessa, becton dickinson, mountain view, ca). five stallions (4 8 ejaculates) were used for breeding mares (ct, n = 19; sf-sm-p, n = 9; sf-ey, n = 18 estrous cycles). data were analyzed with graphpad pris 8.0.1. (graphpad, san diego, ca). parametric data were analyzed with anova-rm with tukey’s as post-hoc. nonparametric data were analyzed by kruskal-wallis followed by wilcoxon–mann–whitney. pregnancy rates were compared by multivariate regression. significance was set at p ≤ 0.05. sperm kinetics (tm, pm, and rap) increased (p < 0.05) in all samples resuspended ey compared to ct, sm, and semen centrifuged and resuspended in sm-p. semen processed by sf and resuspended in sm-p was similar (p > 0.05) to ey groups. sm-p had superior (p < 0.05) results in all processed semen by sf compared to ct, whereas centrifuged semen had intermediate values (p > 0.05). there were no differences (p > 0.05) in pmi between ct and semen processed by sf. however, centrifuged semen had less (p > 0.05) pmi than sf processed semen. additionally, mares inseminated with sf-sm-p (66%) or sf-ey (67%) had higher (p < 0.05) pregnancy rates than mares inseminated with ct (13%). in conclusion, sperm parameters of stallions with poor semen cooling ability were enhanced by clinical theriogenology 2021; 13: 340 the removal of the supernatant and sperm resuspension with yg or sm-p. additionally, processing semen by spermfilter enhanced pmi compared to centrifuged semen. fertility rates of poor cooled semen improved by semen processing by sf and resuspended in sm-p or ey. keywords: stallion, extender, sperm kinetics, bad cooler pregnancy rates and subsequent pregnancy losses of in vitro produced embryos from oocytes etta bradecamp,a carly garcia,b charlie scoggin,a maria schnobrich,a crystal howard,a jamie kaczor,a erin lohbeck,a holly herseya arood and riddle equine hospital, lexington, ky; blazy e ranch, guthrie, ok over the past 5 years there has been an increased utilization of transvaginal aspiration of oocytes and intracytoplasmic sperm injection (icsi) to produce equine embryos. with this increase in demand has come the increase in the number of commercial icsi labs and in the number of icsi-produced embryos being shipped to commercial recipient herds for transfer. there are limited data in the literature describing the pregnancy rates and any subsequent pregnancy losses associated with these shipped icsi-produced embryos. data were collected from 2 facilities that performed a total of 572 aspirations over 3 breeding seasons and shipped the oocytes to 5 commercial icsi labs. embryos produced were shipped to 2 commercial recipient herds. pregnancy rates and subsequent losses were calculated for 3 of the icsi labs; 2 of the labs were not included due to a very small number of embryos transferred from these facilities. in total, 208 fresh embryos were shipped for transfer. fourteen-day pregnancy rates ranged from 41 to 75%; pregnancy loss rates tva facility/icsi lab/year embryos transferred 14-day pregnancies pregnancies lost tva facility 1/icsi lab a 2018 21 10 (47%) 6 (60%) tva facility 1/icsi lab a 2019 12 9 (75%) 2 (22%) 1 due to twins tva facility 1/icsi lab a 2020 53 29 (55%) 8 (27.5%) 3 due to twins tva facility 1/icsi lab b 2019 31 15 (48%) 8 (53%) tva facility 1/icsi lab b 2020 24 10 (41%) 5 (50%) tva facility 2/icsi lab a 2020 21 14 (66%) 8 (57%) plus 2 late term tva facility 2/icsi lab b 2019 13 8 (61%) 2 (25%) tva facility 2/icsi lab b 2020 14 8 (57%) 1 (12.8%) tva facility 2/icsi lab c 2020 19 13 (68%) 2 (15%) varied from 12.8 to 60% depending on the tva facility/icsi lab combination and year. due to the variability in transfer results both between and within the same facilities, more in-depth research needs to be performed to identify the ideal shipping conditions (media, time in transport, etc.) to maximize pregnancy rates and minimize subsequent pregnancy losses. keywords: mare, embryo, icsi, trans-vaginal aspiration, pregnancy incidence rate of reproductive problems in nonpregnant mares patrick mccue, melissa prell, christian bisiau, christina divine, jennifer hatzel acollege of veterinary medicine and biomedical sciences, colorado state university, fort collins, co broodmares may be affected by a variety of reproductive issues. the goal of this retrospective study was to document reproductive abnormalities encountered in broodmare veterinary practice. reproductive records of mares managed at colorado state university were evaluated retrospectively. reproductive issues were broadly categorized into abnormalities of the ovary, oviduct, uterus, cervix, vagina, perineum, mammary gland, and behavioral concerns. the abnormalities were then assigned to specific subcategories within each broad category. reproductive records were evaluated for 636 individual mares over a 3-year period (2018 2020). a mare was considered positive for an abnormality if the issue was noted at least once during a breeding season. the incidence rate (ir) was calculated as the percentage of mares with a specific abnormality compared to the overall population of mares. data are presented as the mean ± standard deviation. a total of 862 mare-years were evaluated, as some mares were evaluated over multiple breeding seasons. the average age of the mare population was 11.9 ± 4.8 years and ranged from 3 to 26 years. the most common breeds were american quarter horse (383 mares, 60.2% of total), warmbloods (all breeds combined) (55 mares, 8.6%) and arabians (26 mares, 4.1%). most common ovarian issues noted were after 250 µg of cloprostenol treatment were, persistence of luteal tissue (62 cases; 7.2% ir) and hemorrhagic anovulatory follicles clinical theriogenology 2021; 13: 341 (43 cases; 5.0% ir). most common oviductal abnormalities were presumptive blocked oviducts (28 cases; 3.2% ir) and parovarian cysts (10 cases; 1.2% ir). uterine issues comprised the greatest number of abnormalities, including persistent breeding-induced endometritis (pbie; 189 cases; 21.9% ir), endometrial cysts (137 cases; 15.9% ir), presence of excessive fluid prior to breeding (78 cases; 9.0% ir) and bacterial endometritis (70 cases; 8.1% ir). most common abnormalities of the caudal reproductive tract were failure of cervical relaxation (36 cases; 4.2% ir), urovagina (6 cases; 0.7% ir), and poor perineal conformation or tone (13 cases; 1.5% ir). mammary abnormalities were uncommon, with 3 cases of galactorrhea and 2 cases of mastitis. most common behavioral issues were stallion-like or aggressive behavior (3 cases), recurrent colic or pain (3 cases) that an owner was associating with the reproductive tract and persistent estrus (2 cases). issues with a higher incidence in mares > 15 years of age included hemorrhagic anovulatory follicles, uterine cysts, persistent breeding-induced endometritis and bacterial endometritis. mares with a tight cervix or excessive uterine fluid on initial examination had an increased incidence of pbie (75 and 39.7%, respectively). in conclusion, persistent breeding-induced endometritis was the most common reproductive abnormality and the incidence of reproductive issues increased with advanced age. keywords: equine, mare, reproductive, problems, pathology induction of parturition in a late pregnant mare with large colon displacement lauren pasch rhinebeck equine llp, rhinebeck, ny an 11-year-old multiparous pregnant (324 days) thoroughbred broodmare was presented to rhinebeck equine llp for treatment of right dorsal displacement of the large colon and associated abdominal discomfort. mare had minimal mammary development. no surgical option was available for the mare; foal survival was the owner’s priority. transrectal and transabdominal ultrasonography examinations revealed a viable fetus in anterior longitudinal presentation with a fetal heart rate of 72 bpm (reference range: 80 120 bpm). mare was treated conservatively with isotonic intravenous fluids (lactated ringer’s 1-2 liters/hour) and intravenous flunixin meglumine (1.1 mg/kg twice daily). dexamethasone (100 mg once daily) was given intramuscularly at days 325, 326, and 327 to stimulate fetal maturation. induction of parturition was proposed to allow for delivery of a live foal and possible improvement of colonic displacement postpartum. at 328 days of pregnancy the mare’s discomfort persisted and colon displacement was unresolved. induction of parturition using a low-dose oxytocin protocol was elected. mare was treated intramuscularly with 5 iu oxytocin; after 25 minutes, a vaginal examination confirmed cervical relaxation. mare was then treated intravenously with 5 iu oxytocin and had behavioral signs consistent with stage i labor. following an additional 25 minutes interval, the mare was treated intravenously with 5 iu oxytocin. seventy minutes after the first oxytocin treatment, stage ii labor was initiated with spontaneous rupture of the chorioallantois. duration of stage ii labor was 15 minutes, and the mare delivered a live colt with minimal assistance. complete and grossly normal fetal membranes were passed within 30 minutes. foal was given 36 ounces of frozen thawed colostrum via nasogastric intubation, and no gross signs of dysmaturity were noted. the colt received intravenous hyperimmunized plasma and was supplemented with stored mare’s milk via esophageal feeding tube for 5 days and remained clinically normal. mare was started on oral domperidone (1.1 mg/kg, once every 12 hours) to promote mammary development. colic signs ultimately resolved, and both mare and foal were discharged and remained healthy on farm. elective induction of parturition in the mare is uncommonly performed due to the marked variation in equine pregnancy length and the relatively late ability of the equine fetal adrenal gland to respond to acth. the criteria typically used to assess fetal readiness are length of pregnancy, cervical relaxation, and the presence of colostrum within the mammae. this case highlighted the successful use of oxytocin to induce parturition in a mare despite meeting only 1 of the 3 criteria for fetal readiness. additionally, dexamethasone was utilized to stimulate precocious fetal maturation prior to induction of parturition. keywords: mare, induction, parturition, oxytocin clinical theriogenology 2021; 13: 342 cover 4 cover 1 society for theriogenology “veterinary professionals dedicated to animal reproduction” p.o.box 168 mathews, al 36052 334-395-4666 (voice) 334-270-3399 (fax) http://www.therio.org c lin ical t h erio g en o lo g y • vo lu m e 13, n u m b er 3 • s ep tem b er 2021 clinical theriogenology issn: 2154-3968 official journal of the society for theriogenology the american college of theriogenologists volume 13, number 3 september 2021 c ya n m a g e n ta y e l l o w b l a c k 72881_st_vol 13 number 3_cover.indd 172881_st_vol 13 number 3_cover.indd 1 7/9/2021 12:15:47 pm7/9/2021 12:15:47 pm 014_proceeding 2021_color 015_proceeding 2021 2016: is per rectum palpation for pregnancy diagnosis deleterious for the conceptus? new information about an old quandary is per rectum palpation for pregnancy diagnosis deleterious for the conceptus? new information about an old quandary juan e. romano large animal clinical sciences, college of veterinary medicine and biomedical sciences, texas a&m university, college station, tx introduction in bovine practice, per rectum palpation of uterine contents is one of the most frequent methods used for pregnancy diagnosis by veterinarians around the world.1-7 it is generally understood that a trained veterinarian is able to correctly detect pregnant and nonpregnant animals by days 35 after breeding.1-11 the importance of a systematic and nontraumatic technique of per rectum palpation (prp) cannot be overemphasized, as it is well known that embryonic or fetal deaths can be induced accidentally or iatrogenically by prp of the uterus.12-14 an important step in intensive cattle production systems (e.g., dairy farms, embryo transfer programs, and pure breed farms) is to perform a pregnancy diagnosis within 45 days after breeding.10,15 the main purpose of examining cows or heifers early after insemination is not only to correctly detect pregnant cows but also to detect with confidence nonpregnant females in order to manage, treat, cull, or synchronize estrus.10,16 early pregnancy diagnosis can assist the producer in managing nonpregnant females and improving reproductive performance and economics of their herd.17 there is contradictory information about the potential deleterious effect of prp of the uterus for early pregnancy diagnosis on embryo or fetus viability. some studies have suggested an adverse effect of prp.18-23 in contrast, other studies24-26 have suggested little effect of the time at which the first prp is performed after insemination on calving rate. previous investigators diagnosed pregnant females by prp,18-20 progesterone concentrations21,23 or pregnancy-specific proteins.26,27 however, these methods are not able to assess the viability of the embryo or fetus. most published studies lack a positive control group of contemporaneous “pregnant non-per rectum palpated group”,13-15 to differentiate the effects of prp from the spontaneous pregnancy loss occurring during early pregnancy. the interval between prp and reevaluation varied from 30 to 90 days,13 44 to 48 days,16 at calving14 or flexible depending if the prp was performed before or after 40 days of pregnancy.15 a high level of peripheral progesterone as an indication of pregnant females is not a completely accurate method of pregnancy diagnosis. progesterone level is high in conditions other than pregnancy, as in presence of luteal cysts, long estrous cycles, short estrous cycle, sampling during luteal phase, pyometra28 as well as in a pregnant female with embryo or fetus death or in process of degeneration.15,29 progesterone level is an excellent indicator of nonpregnancy status rather than pregnancy status.30,31 the pregnant bovine female produces different kinds of psp such as protein b. bovine pregnancy-specific protein b (bpsp-b), a glycoprotein produced by the giant cells of the trophoblast, however, remains elevated despite death or degeneration of the embryo or fetus.15,27,32 in induced embryo/fetal death, elevated levels of progesterone29or psp-b,27 as well as positive signs of pregnancy, persisted for several days despite the embryo or fetal death14,29 in a recent study of spontaneous pregnancy loss in dairy cattle, elevated levels of psp-b persisted15 differences among farms in pregnancy loss are well established and are related more to management factors than to infectious diseases.33 this was not taken into consideration in some studies. most of the previous investigations pool heifers with cows. one study showed that pregnant heifers have lower pregnancy loss than cows;34 however, these data were retrospective and lacked a control group. twin pregnancies increase the risk of pregnancy loss.7,35 interestingly, in most of the studies dealing with pregnancy loss, the number of twin pregnancies was not reported.7 studies in the united states and in other areas of the world have found an association between amniotic sac per rectum palpation (asp) during the embryonic period for pregnancy diagnosis until day 45 of gestation36 and an increased risk of atresia coli/jejuni in newborn calves.37-41 in atresia coli/jejuni, a section of the large bowel or jejunum is absent, resulting in a blind intestinal tube. this clinical congenital condition is lethal, and surgical correction is the only treatment available.42-45 atresia clinical theriogenology • volume 8, number 4 • december 2016435 coli/jejuni has been reported in different countries and in more than ten breeds of cattle, with a marked predominance in holstein calves.46 based on those findings, some authors have recommended avoiding prp of the uterus during the first 45 days of gestation.36-39,47,48 intestinal atresia was also reported to be inherited as an autosomal-recessive trait in jersey and swedish highland cattle.49,50 intestinal atresia could develop either from imperfect canalization, of the gut or from insufficient blood supply to the affected portion of the intestine51and asp was suggested to act for this latter mechanism.39,46 nevertheless, the cause of atresia coli/jejuni remains controversial and not completely understood.45,46,51 in the author’s practice, asp is not routinely used for early pregnancy diagnosis; however, cases of atresia coli or jejuni were detected. interestingly, cases of atresia coli/jejuni were also diagnosed in newborn calves from dams that underwent prp only by detection of allantochorion membrane during the first trimester of gestation, either during the late embryonic period or fetal period, during the second trimester of gestation; females were diagnosed as pregnant only by transrectal ultrasonography (trus; romano, unpublished data). finally, in most of these previous studies, real practice conditions for prp were not followed; that is only one veterinarian palpated per rectum each cow or heifer once looking for positive signs of pregnancy. in those studies, females were palpated by more than one person successively or different techniques were used in the same animal by more than one person13-16 in those cases, the procedure of prp appears more invasive than that used for pregnancy diagnosis in a regular practice. confirmation of pregnancy status before or at the time of prp by another method allows for the differentiation of spontaneous pregnancy loss from pregnancy loss potentially induced by prp. the use of transrectal ultrasonography permits an earlier pregnancy diagnosis than prp, gives immediate information about the presence of positive signs of pregnancy as well as embryo or fetus viability, and reduces the potential misdiagnosis of pregnancy.11,52 in addition, reports about the use of ultrasonography during pregnancy have shown that it is a safe technique that does not affect embryo or fetus viability.53-57 the objective of this manuscript is to cover five studies, the first of which evaluated the effects of prp using the fetal membrane slip technique for early pregnancy diagnosis during late embryonic period on pregnancy loss. the second study evaluated the degree of invasiveness of prp on pregnant females on the proportion of pregnancy loss. the third study compared the effect of prp using the fetal membrane slip technique, not only on proportion of pregnancy loss but also in the type of pregnancy loss. the fourth study evaluated the effect of detection of asp during the late embryonic period on pregnancy loss. the fifth study assessed the effect of detection of asp during the late embryonic period for pregnancy diagnosis on pregnancy loss, calving rates, and abnormalities in newborn calves. materials and methods first study a controlled randomized block design with two blocks, one by category and the other by number of embryos, was performed.7 the categories were cows and heifers. single and twin pregnancies were determined based on number of embryos. five hundred and twenty clinically healthy pregnant females (360 cows and 160 heifers) with a viable embryo detected by transrectal ultrasonography between days 29 and 32 after artificial insemination were included. the pregnant females were randomly divided into two equals groups: per rectum palpation (pal group) and no per rectum palpation (npal group). the pal group was subjected to prp using the fetal membrane slip (fms) technique (detection of allantochorion membrane) once between days 34 and 41 of pregnancy.58 all prps were performed by the same person who avoided palpating the amniotic sac. throughout the investigation period the females did not undergo any other prp. both groups were subjected to additional transrectal ultrasonography at days 45 and 60 of pregnancy. day 45 was used to monitor spontaneous late embryonic death for the control group and the potential immediate deleterious effects of prp on embryo viability. day 60 was used to monitor spontaneous early fetal death for the control group and the potential late deleterious effect of prp on viability of the fetus. all trus procedures were performed in the morning, by the same operator, using an aloka ssd 500 ultrasound machine equipped with a 5 mhz linear transducer. the diagnosis of clinical theriogenology • volume 8, number 4 • december 2016 436 pregnancy loss was made when there was no embryonic or fetal heart beat; signs of embryonic/fetal degeneration were observed, or when the positive signs of pregnancy were absent in a cow or heifer previously diagnosed as pregnant by trus. second study a controlled randomized block design was performed.59 t he blocking was performed by farms, categories, and number of embryos. nine hundred twenty eight pregnant females with a viable embryo between 28 and 34 days detected by trus were included. two farms were used. the categories were cows and heifers. the number of embryos determined single and twin pregnancies. the females were divided in two groups: control (con; n= 476) and per rectum palpation (pal; n= 452). in addition, the prp groups were randomly divided in per rectum palpation 1 (pal-1; n= 230) and per rectum palpation 2 (pal-2; n=222). the control group was not subjected to prp. the pal-1 and pal-2 groups were subjected to prp using the fms technique once or twice at the same examination between days 34 and 43 of pregnancy, respectively. further, these groups were subjected to two additional trus procedures at days 45 and 60 as described in the first study and the same criteria of pregnancy and pregnancy loss were used. third study in this study, 580 pregnant females with a viable embryo diagnosed by trus (as studies 1 and 2) were used.60 two hundred seventy-two females were palpated using the fms technique once between 34 and 41 days of pregnancy, and 308 females not palpated were included. these animals were evaluated again by trus at 45 and 60 days later as in studies 1 and 2. the use of prp for embryonic or fetal attrition (induced abortion) produced a pregnancy loss that is characterized by persistence of a positive fms detected by prp for two weeks, signs of conceptus degeneration for three weeks detected by trus (three weeks), and maintenance of a functional corpus luteum for around four weeks12,14,29 as described for spontaneous pregnancy loss type i.15 therefore, if prp for pregnancy diagnosis is deleterious for the embryo or fetus, the proportion of pregnancy loss should be higher than in a nonpalpated pregnant group (positive control group), and the pregnancy loss will be characterized by persistence of positive signs of pregnancy, signs of conceptus degeneration, and persistence of a functional corpus luteum and should be higher than the non-per rectum palpated pregnant group. fourth study a controlled randomized block design that included 347 clinically healthy lactating pregnant cattle was performed.61 all cattle were detected pregnant by use of trus at approximately day 29 after estrus and randomly allocated into two groups (control group [con; n = 167 cows] and amniotic sac per rectum palpation group [asp; n= 180]). the con group was not subjected to pregnancy diagnosis via prp. the asp group involved the compression of the pregnant uterine horn by prp and detection of the amniotic sac vesicle between the hand and the fingers.62 the entire asp was performed by one trained veterinarian between days 34 and 43 after estrus. all cattle were reevaluated by trus on days 45, 60, and 90 to determine viability of conceptus. the same criteria of pregnancy and pregnancy loss were used as in the previous studies. fifth study the present investigation was a randomized controlled blocked double-blind design that included 680 pregnant lactating dairy cows.63 the blocks for farms and two farms (farms a and b) were used. from each farm, the females were diagnosed as pregnant based on the presence of a viable embryo by trus between days 28 and 45 after breeding by the same veterinarian. then, the pregnant females were randomly assigned to control (con group, no prp) and treatment groups (asp group, per rectum amniotic sac palpation). the con group did not receive any prp of the uterus. all the asps were performed only once between days 34 and 45 after artificial insemination by a board-certified theriogenologist. after being subjected to their respective treatments, each female was reevaluated for clinical theriogenology • volume 8, number 4 • december 2016437 pregnancy only by trus again between two and four weeks later in both farms. in farm a, the same veterinarian was involved in the initial treatment and reexamination; however, he was blind to the treatment of each cow at the time of reexamination. in farm b, pregnancy reexamination was performed by two different veterinarians who were blind to the treatment but were aware about the project. then, all of the females pregnant at reexamination were followed until calving. every abortion, premature calf, or stillbirth was submitted for necropsy to determine the type of abnormalities, if any. the veterinarians in charge of the necropsy were different from the initial prp and from the reexaminations. all the calves born alive were maintained for observation for three to five days postpartum to detect any of abnormalities. statistical analysis the proportion of females suffering pregnancy loss was compared between treatment groups using chi-square analysis or fisher exact test as appropriate. in addition, binary logistic regression analysis was used to detect the effect of farm (second and fifth study), categories (first, second and third studies) and the number of embryos (first, second, third, and fourth studies). a difference was considered statistically significant at p<0.05.64,65 a software program was used to analyze all data sets.66 results first study the overall pregnancy loss between days 30 and 60 was 14.0%. late embryonic pregnancy loss (from 30 to 45 days; 10.0%) was significantly higher than fetal death (from 46 to 60 days; 4.5%; p<0.001). pregnancy loss between the pal group (14.7%) and the npal group (13.4%) was not significantly different (p>0.05). pregnancy loss was higher in cows (16.4%) than in heifers (8.8%; p<0.025) and in females pregnant with twins (25.5 %) compared with single pregnancies (12.9%; p<0.025). second study the overall percent of pregnancy loss between days 28-34 and 60 was 14.1% (131/928). late embryonic pregnancy loss for con, pal-1 and pal-2 groups was 12.6% (60/477), 9.1% (21/230), and 9.5% (21/221), respectively (p=0.28). fetal pregnancy loss for the same groups was 2.4% (10/417), 3.8% (8/209), and 5.5% (11/200), respectively (p=0.15). the overall percentage of pregnancy loss for con, pal-1, and pal-2 from days 28 to 34 to 60 was 14.7% (70/477), 12.6% (29/230), and 14.5% (32/221), respectively (p=0.74). no statistical differences were detected among groups in all the possible comparisons. third study out of 272 pregnant females in the prp group, 41 were found with pregnancy loss (15.1%), while out of the 308 females pregnant in the non-prp group, 46 were found with pregnancy loss (14.9%) at day 60 after artificial insemination (p>0.05). pregnancy loss characterized by persistence of positive signs of pregnancy was 17 (6.3%) from the prp group and 26 (8.4%) from the non-prp group, respectively (p>0.05). no statistical differences between groups either in the proportions and type of pregnancy loss were detected. fourth study overall pregnancy loss between days 29 and 90 was 12.7% (44/347). overall pregnancy loss for late embryonic period (days 29 to 45; 9.5%) was significantly (p=0.001) higher than for early (days 46 to 60; 2.5%) or late (days 61 to 90; 1%) fetal periods. the pregnancy loss between days 29 and 90 between con and asp groups was 13.2% and 12.2%, respectively (p=0.79). late embryonic pregnancy loss for the con and asp groups was 10.8% and 8.3%, respectively (p=0.44). early fetal pregnancy loss for con and asp groups was 1.3% and 3.6%, respectively (p=0.93), and late fetal pregnancy loss for the clinical theriogenology • volume 8, number 4 • december 2016 438 same groups was 1.4% and 0.6%, respectively (p=79). no differences were detected between early and late fetal period (p=0.24). the percentage of cows with twins at initial pregnancy diagnosis by trus was 7.5%. the pregnancy loss (days 29 to 90) in single pregnancies was 12.2%, and in twin pregnancies, it was 19.2% (p=0.29). fifth study in farm a, the early pregnancy loss rate for the con group was 11.5% (19/165), and it was 13.2% (24/182; p=0.64) for the asp group. in farm b, the early pregnancy loss rate for the con group was 11.2% (19/170), and it was 8.8% (14/159; p=0.48) for the asp group. the early pregnancy loss between rate for farm a (12.4%; 43/347) and farm b (10.0%; 33/329) was not different (p=0.33). in farm a, the late pregnancy loss rate for the con group was 7.6% (11/145), and it was 5.2% (8/155; p=0.39) for the asp group. in farm b, the late pregnancy loss rate for the con group was 3.7% (5/137), and it was 6.3% (8/127; p = 0.32) for the asp group. the late pregnancy loss rate between farm a (6.3%; 19/300) and farm b (4.9%; 13/264) was not different (p=0.47). in farm a, the early pregnancy loss rate was higher than the late pregnancy loss rate (12.4% vs 6.3%; p=0.01). in farm b, the same results were detected (10.0% vs 4.9%; p=0.02). in both farms, the overall early pregnancy loss rate was higher (11.2%; 76/676) than late pregnancy loss rate (5.7%; 32/564; p = 0.0005). in farm a, calving rate one for the con group was 81.2% (134/165), and it was 80.8% (147/182; p=0.92) for the asp group; calving rate two for the same groups was 92.4% (134/145) and 94.8% (147/155), respectively (p=0.68). in farm b, calving rate one for the con group was 77.7% (132/170), and it was 74.8% (119/159; p = 0.55) for the asp group; calving rate two for the same groups was 87.4% (132/151) and 82.1% (119/145), respectively (p=0.20). the calving rate one for farm a was 80.9% (281/347), and it was 75.4% (251/333; p=0.14) for farm b; calving rate two for farm a was 92.4% (281/304), and it was 84.8% (251/296; p=0.0005) for farm b. two calves with atresia coli were diagnosed by necropsy only in the con group in farm a (p=0.23). these calves were singleton females born alive from holstein dams and sires. the gestation length was 273 and 288 days. all the fetuses or calves born dead and necropsied were negative for intestinal atresia. discussion these five independent studies showed that per rectum palpation for pregnancy diagnosis using either the fms or amniotic sac detection did not increase the pregnancy loss.7,59-61,63 the experimental design allowed differentiation between the effects of prp from the spontaneous pregnancy losses, because a positive control group of contemporaneous pregnant females non-per rectum palpated was included. this approach was only possible at this time, because trus permitted detection of pregnant females and the assessment of conceptus viability at an earlier stage of pregnancy. in addition, trus excluded pregnant females with an embryo already dead or in process of degeneration from all these investigations.7,52,59-61,63 in the current studies, the prp technique used was similar to those employed by most veterinarians in private practice for pregnancy diagnosis. each female was subjected to prp by only one person a single time while trying to find a positive sign of pregnancy. in earlier reports, realistic conditions were not followed, because the females were evaluated by more than one person successively; different techniques were utilized, or various procedures were applied successively to the same female by more than one person.18-21 previous investigators of prp and pregnancy loss did not report the number of twin pregnancies. factoring twins into the result is important, as it can skew the results.7,15,59 in some of the present studies, the numbers of twins were balanced between groups; therefore, the high inherent risk of pregnancy loss of twin pregnancies was equalized between treatments. the risk of pregnancy loss is more than double in twin pregnancies compared with single pregnancies.7,15,59 pregnancy loss in cows was almost double than in heifers at the same stage of pregnancy. in most previous studies of prp, heifers and cows were not separated.18,19 a study, in which prp was used between 30 and 70 days after breeding, showed that pregnancy loss was almost three times higher in cows clinical theriogenology • volume 8, number 4 • december 2016439 than in heifers.34 the reasons why cows lose more pregnancies than heifers are unknown. twinning rate can be one of these factors. however, in the present experiments, when the number of cows with twins was excluded from the statistical analysis a reduction in the percentage of pregnancy loss was observed. nevertheless, pregnancy loss continued to be higher in cows than heifers. therefore, factors other than twin pregnancy are implicated in such pregnancy loss. stress of lactation, subclinical mastitis, deficient nutritional support, and insufficient hormonal levels could be other causes for this increased mortality rate. all of the studies agreed that examinations during the embryonic period detected more spontaneous pregnancy loss compared with later periods.7,59-61,63 therefore, it is recommended that any female detected as pregnant during late embryonic period will require a further reexamination to reduce the chances of potentially maintaining a nonpregnant female in the production system. in the second study, the use of more than one fms at the same examination for pregnancy diagnosis as a measure of invasiveness of the technique did not increase the proportion of pregnancy loss.59 the proportion of pregnancy loss was not different among the groups, with one or two fetal membrane slips at the same examination compared to the positive control group of non-palpated per rectum pregnant females. these results showed that prp was not deleterious for the conceptus under the same experimental conditions. the third investigation not only confirmed the results of the first two studies, but also supports with additional evidence that prp was not harmful to the conceptus.61 if prp produced embryonic or fetal attrition, the pregnancy loss would be characterized by persistence of positive signs of pregnancy by per rectum palpation, signs of embryo/fetus degeneration and functional corpus lutem by trus.12-15,29 consequently, this type of pregnancy loss would be significantly higher compared with the positive control group of non-per rectum palpated pregnant females. however, no difference in the proportion of this type of pregnancy loss between treatments in this investigation was detected, therefore, showing with this new approach that prp was not detrimental for the conceptus. the fourth experiment showed that asp for pregnancy diagnosis was a safe procedure for the conceptus compared with the con group, because no increase in pregnancy loss in the three periods of assessment was detected.61 the three check points at the end of the embryonic period (day 45), early fetal period (day 60), and late fetal period (day 90) were designed to estimate the potential immediate, late, and delayed effect of asp on pregnancy loss, respectively. this new study showed that by using a different positive sign of pregnancy, such as the amniotic sac detection, was not harmful for the pregnancy as it was also demonstrated when allantochorion membrane detection was used. this investigation adds new information and expands our knowledge that when using either allantochorion membrane or amniotic sac detection, no increase in pregnancy loss was observed. the fifth study showed no increase of pregnancy loss in the asp group compared with the con group and is in agreement with the fourth report.63 in the current study, the two calves with atresia coli were diagnosed only in the con group, which is in contrast to the previous studies that associated asp during the late embryonic period with atresia coli or atresia jejuni.37-48 the asp group included 341 pregnant females that underwent prp between days 34 and 45 and from which 285 were assessed between days 35 and 42 of pregnancy, which was considered the period of highest risk of producing atresia coli. based on the prevalence of atresia coli from previous studies, it was expected in the present study that there would be between nine and 32 calves with atresia coli in the asp group; however, no cases of atresia coli were produced. this is strong evidence against the deleterious effect of asp on the conceptus. in general dairy practices, the veterinarian performs the prp for early pregnancy diagnosis during the late embryonic period before the potential second estrus (45 days)10,36 for technical and economic reasons.7,10,17 thus, it is very important to have accurate evidence-based medicine about the potential deleterious effect of prp on pregnancy loss, calving rates, or abnormalities in newborn calves. this knowledge will not only affect the way that the veterinarians practice but also how the owner or manager will perceive the use of this procedure for reproductive management. some authors previously recommended avoiding prp of the uterus during the first 45 days of gestation.30,37-39,41,42,47,48 however, clinical theriogenology • volume 8, number 4 • december 2016 440 the present investigation supports the theory that asp for pregnancy diagnosis, when performed by a trained veterinarian, was a safe procedure for the conceptus using the three assessment points: at reexamination, calving, and evaluation of newborn calves. in summary, from these five independent investigations, it was concluded that per rectum palpation for early pregnancy diagnosis using either the fms technique or asp did not increase the proportion of pregnancy loss, decrease the calving rates, or increase the abnormalities in newborn dairy calves. in addition, an increased risk of spontaneous pregnancy loss was detected in cows versus heifers, during late embryonic period compared with the fetal period, in females carrying twins as opposed to singletons, and differences in pregnancy loss among farms were also noticed. references 1. roberts sj: veterinary obstetrics and genital diseases. ithaca(ny): published by the author; 1971. 2. momont h: rectal palpation: safety issues. bov pract 1990;25:122-123. 3. youngquist rs: pregnancy diagnosis. in: youngquist rs, editor. current therapy in large animal theriogenology. philadelphia: wb saunders co; 1997. p. 295-303. 4. vandeplassche m: reproductive efficiency in cattle: a guideline for project in developing countries. rome, italy. f.a.o. no 25; 1982. 5. usda dairy. part iv: reference of dairy cattle health and management practices in the united states; 2007. 6. cowie at: pregnancy diagnosis tests: a review. commonwealth agricultural bureaux joint publication, number 13; 1948. p. 11. 7. romano je, thompson ja, kraemer dc, et al: early pregnancy diagnosis by palpation per rectum: influence on embryo/fetal viability in dairy cattle. theriogenology 2007;67:486-493. 8. euler v: die klinischen erkennungsmerkmale der frühgraviditat beim rind und ihre verwertbarkeit für die praxis. berl tierärztl wschr 1930;30: 477-481. 9. götze r: die feststellung der schwangerschaft beim rinde. dtsch tierarztl wschr 1940;48:183-185. 10. zemjanis r: diagnostic and therapeutic techniques in animal reproduction. baltimore: the williams and wilkins co; 1962. 11. romano je, fahning ml: accuracy of per rectum palpation for pregnancy diagnosis in cattle. proc xxviii world buiatrics congress; 2014. p. 68. 12. ball l, carroll ej: induction of fetal death in cattle by manual rupture of the amniotic vesicle. j am vet med assoc 1963;142:373-374. 13. dawson flm: methods for early termination of pregnancy in the 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26. alexander bm, johnson ms, guardia ro, et al: embryonic loss from 30 to 60 days post breeding and the effect of palpation per rectum on pregnancy. theriogenology 1995;43:551-556. 27. humblot p, camous s, martal j,et al: pregnancy-specific protein b, progesterone concentrations and embryonic mortality during early pregnancy in dairy cows. j reprod fertil 1988;83:215-223. clinical theriogenology • volume 8, number 4 • december 2016441 28. pennington ja, spahr sl, lodge jr: factors affecting progesterone in milk for pregnancy diagnosis in dairy cattle. brit vet j 1976;132:487-495. 29. kassam a, bondurant rh, basu s, et al: clinical and endocrine responses to embryonic and fetal death induced by manual rupture of the amniotic vesicle during early pregnancy in cows. j am vet med assoc 1987;191:417-420. 30. shemesh m, ayalon n, shalev e, et al: milk progesterone measurement in dairy cows: correlation with estrus and pregnancy determination. theriogenology 1978;9:343-353. 31. laing ja, gibbs ha, eastman sak: a herd test for pregnancy in cattle based on progesterone levels in milk. brit vet j 1976;132:204-209. 32. maurer rr, ruder ca, sasser rg: effectiveness of the protein b radioimmunoassay to diagnose pregnancy in beef cattle[abstract]. j anim sci 1985:61(suppl 1):390. 33. thompson ja, marsh we, etherington wg, et al: evaluation of the benefits of the timing of pregnancy testing by transrectal palpation in dairy attle. j am vet med assoc 1995;207:1462-1465. 34. labernia j, lopez-gatius f, santolaria p, et al: influence of management factors on pregnancy attrition in dairy cattle. theriogenology 1996;45:1247-1253. 35 day jd, weaver ld, franti ce: twin pregnancy diagnosis in holstein cows: discriminatory powers and accuracy of diagnosis by transrectal palpation and the outcome of twin pregnancies. can vet j 1995;36:93-97. 36. committee on bovine reproductive nomenclature. recommendations for standardizing bovine reproductive terms. cornell vet 1972;62:216-237. 37. bellows ra, rumsey ts, kasson cw, et al: effects of organic phosphate systemic insecticides on bovine embryonic survival and development. am j vet res 1975;36:1113-1140. 38. ness vh, leopold g, müller w: zur genese des angeborenen darmverschlusses (atresia coli et jejuni) des kalbes. mh vet med 1982;37:89-92. 39. müller w, kelker l, wünsche k, et al: derzeitiger stand der ermittlungen zum vorkommen und zur ätiologies angeborenen darm verschlusses bei kälbern im bezirk dresden. mh vet-med 1982;37:84-89. 40. schlegel f, müller w, willer s, et al: die rektale frühträchtigkeitsuntersuchung als auslösender faktor der partiellen kolonaplasie beim rind. mh vet-med 1986;41:377-382. 41. brenner j, orgad u: epidemiological investigations of an outbreak of intestinal atresia in two israeli dairy herds. j vet med sci 2003;65:141-143. 42. ducharme nf, arighi m, horney d, et al: colonic atresia in cattle: a prospective study of 43 cases. can vet j 1988;29:818-824. 43. constable pd, rings dm, hull bl, et al: atresia coli in calves; 26 cases (1977-1987). j am vet med assoc 1989;195:118-123. 44. smith df, ducharme ng, fubini sl, et al: clinical management and surgical repair of atresia coli in calves: 66 cases (1977-1988). j am vet med assoc 1991;199;1185-1190. 45. constable pd, huhn jc, morin de, et al: atresia coli in calves: etiopahogenesis and surgical management. bov pract 1999;33:70-73. 46. constable pd, shanks rd, huhn j, et al: evaluation of breed as a risk factor for atresia coli in cattle. theriogenology 1997;48:775-790. 47. bellows ra, patterson dj, burfering pj, et al: occurrence of neonatal and postnatal mortality in range beef cattle. ii. factors contributing to calf death. theriogenology 1987;28:573-586. 48. ducharme n, gilbert r, smith df: atresia coli: genetics or iatrogenics. proc annu meet soc therio; 1990. p 112-117. 49. jubb kvp, kennedy pc, palmer n: pathology of domestic animals. vol 2. 4th ed. new york: academic press; 1992. 50. nihleen b, eriksson k. a hereditary lethal defect in calves – atresia ilei. nord vet med 1958;10;113-127. 51. johnson r. intestinal atresia and stenosis: a review comparing its etiopathogenesis. vet res comm 1986;10:95-104. 52. romano je, magee d: applications of trans-rectal ultrasonography in cow/heifer reproduction. in: annual food conference. conception to parturition: fertility in texas beef cattle. college of veterinary medicine. texas a & m university; 2001. p. 99-104. 53. reece ea, assimkopoulos e, zheng xz, et al: the safety of obstetric ultrasonography: concern for the fetus. obstet gynecol 1990;76:139-146. 54. kähn w. ultrasonography as a diagnostic tool in female animal reproduction. anim reprod sci 1992;28:1-10. 55. ball pjh, logue ddn: ultrasound diagnosis of pregnancy in cattle. vet rec 1994;34:532. 56. baxter sj, ward wr: incidence of fetal loss in dairy cattle after pregnancy diagnosis using a ultrasound scanner. vet rec 1997;140:287-288. 57. miller dl. safety assurance in obstetrical ultrasound. semin ultrasound ct mr 2008;29:156-64. 58. abelein v. frühdiagnose der gravidität beim rind. münch tierärztl wschr 1928;79:4. 59. romano je, thompson ja, kraemer dc, et al: early pregnancy diagnosis by palpation per rectum on pregnancy loss in dairy cattle. j am vet med assoc 2011;239:668-673. 60. romano je. further evidence that per rectum palpation for early pregnancy diagnosis is not harmful for the conceptus. in preparation 61. romano je, fahning ml: effects of early pregnancy diagnosis by palpation of amniotic sac per rectum on pregnancy loss in dairy cattle. j am vet med assoc 2013;243:1462-1467. 62. pissl v: zur frühdiagnose der trächtigkeit beim rind. tierärztliche rundschau 1923;29:370-371. clinical theriogenology • volume 8, number 4 • december 2016 442 63. romano je, bryan k, ramos r, et al: effect of early pregnancy diagnosis by per rectum amniotic sac palpation on pregnancy loss, calving rates, and abnormalities in newborn dairy calves. theriogenology 2016;85:419-427. 64. devore j, peck r. statistics: the exploration and analysis of data. 2nd ed. belmont(ca): duxbury press , 1993. 65. hulley sb, cummings sr, browner gd, editors: designing clinical research: an epidemiologic approach. 2nd ed. philadelphia: lippincott williams & wilkins; 2001. 66. minitab 16: minitab inc. state college, pa clinical theriogenology • volume 8, number 4 • december 2016443 clinical theriogenology • volume 8, number 4 • december 2016 444 omniblank: 2011: equipment and techniques for transcervical artificial insemination and laparoscopic insemination in small ruminants equipment and techniques for transcervical artificial insemination and laparoscopic insemination in small ruminants clifford f. shipley department of rural animal health management, college of veterinary medicine, university of illinois, urbana, il equipment needs for transcervical insemination (tci) of whitetail deer and goats are essentially the same. transcervical insemination of sheep can use the same equipment, but pregnancy rates are low in this species with tci and the author does not recommend this technique for use in sheep, especially with frozen-thawed semen. a list of the equipment needed and comments concerning the equipment is as follows. vaginal speculum(s) a wide range of speculums have been used and are commercially available through a variety of sources. due to the range in size in goats and the difference between non-parous females and multiparous females, you will need at least three different sizes of vaginal speculums to visualize the cervix. canine speculums will work very well for some occasions and goat speculums are available through many goat specialty catalogs. deer speculums are available from several sources commercially although the goat speculums will work just as well in most cases. a human proctoscope, works well, but many will find it too long to use with the shorter goat artificial insemination (ai) equipment. homemade speculums using syringe cases may also work well for some. light source for tci many of the speculums, especially commercially made ones, come with a light source that may consist of a flashlight to connect to a fiber optic transducer to a simple flashlight adapted to attach to the speculum in some manner. whatever the source, it needs to be small so that space is available to pass the insemination rod, durable to stand up to the water, lubricant and abuse it will take, and have batteries or other power source that is easily portable and usable. the author prefers a very small (micro) flashlight clipped to the inside of the speculum. head lamps are also of value and there are a variety of available models to choose from. you should have several different light sources available to you, spare batteries and spare bulbs. insemination gun there are a variety of commercially made insemination guns available for small ruminants. a cassou gun for cattle ai may also be used. goat supply catalogs, veterinary reproductive companies and the internet are all good sources for this equipment. personal preference and differences in the cervix may dictate what gun works best in the species you are working with. lubricant there are several different companies that make lubricants that are appropriate for use on the vaginal speculum to make insertion easier. one should try to use a lubricant that is sterile, but more importantly, one that is free of spermicidal ingredients. make sure to check the ingredient list and read the fine print as many of the products on the market contain chlorhexidine or other spermicidal agent. thaw unit commercially available units to thaw semen are available through a variety of sources. you should probably obtain one that will work off of ac or dc or have an adaptor or converter so that you can work in a remote location or on farms that do not have electricity available. clinical theriogenology • volume 3 number 4 • december 2011525 generator in some remote locations or on farms that do not have electricity, a small generator to run your electrical equipment may be important. the author has also had several experiences where the electric service went out or fuses blew and to complete a job, had to rely on generator power. straw cutter commercially available from a variety of suppliers or one can use a razor blade, sharp knife or scissors to cut the end of the sealed straw. using a straw cutter will probably distort the end of the straw the least to assure a good seal when loaded in the insemination gun but the other methods will work, especially if one pays attention to the end of the straw prior to loading and straightens out any imperfections in the straw. extender many producers will want the straw “split” (especially whitetail producers) two, three or even more times to inseminate multiple animals with. you may choose to inseminate using the insemination gun and either calibrating it and using the appropriate fraction of the straw, or you may choose to thaw the straw and extend it and re-load straws from the extended semen. this method is probably best in most situations (especially deer), as the does being bred to that particular straw of semen may not be back to back in the breeding order, or you may have to hold the semen for a long period of time between does. straws empty straws for re-loading extended semen or in cases where the original straw is either flawed or explodes but the semen can still be recovered and used are essential. having both 0.25 cc and 0.50 cc straws available may save the day. permanent marker an ultrafine permanent marker will be useful for identifying straws and other equipment. it is also essential when writing on cuvettes. records keeping track of which doe is inseminated with what semen and when inseminated is essential. some producers will lose track of animals or change their minds after inseminations have started. most are now using dna to keep track of sire and dam as well. giving them the empty straws to confirm dna and what they have bred with is also valuable. microscope a good quality microscope is essential. one with a slide warmer is nice but not essential as one can use heat packs, warm “cool” packs or other creative ways to warm slides for semen evaluation. it will be impossible in most field situations to do anything other than a gross motility evaluation while doing ai. as most whitetails are bred with “split” straws, most of these owners expect the semen to be evaluated prior to insemination for determination of dilution rate. this is especially true when they have paid a very high price for the semen and want some sort of assurance that it is good prior to insemination. with experience, one can usually tell concentration and motility quickly so that the ai process is not slowed down much if any with this quick analysis. cuvettes since most semen for whitetails is split, using cuvettes or other similar tube to “hold” semen in is essential. most semen comes in 0.50 cc straws and is thawed and then expelled with a plunger or the other end cut to allow gravity flow of the semen into the warmed cuvette suspended in a warm water bath. to this, the proper amount of extender is added and a sample of the warmed, extended semen is placed on a warmed slide for the quick evaluation under the microscope. clinical theriogenology • volume 3 number 4 • december 2011 526 slide warmer while one can get around using a slide warmer, i find it essential for warming slides, straws, aspics and cuvettes especially in colder weather. holding multiple samples of semen in cuvettes can get confusing yet they can be laid out in good order and labeled on the slide warmer. floating cuvette holder this piece of equipment makes it handy for holding cuvettes in the water bath. it is simply a piece of polystyrene foam (styrofoam®, dow chemical co., midland, mi) with holes cut in it to hold the cuvettes in the water bath. there may be other methods to accomplish this, but this is cheap, easy and requires no specialized equipment to make. straw loading equipment this can be very easily done by taking a tom cat catheter and cutting it to fit the end of a 0.25 or 0.50 cc straw and attaching the other end to a syringe. simply attach to the plug end of the straw and plunge the open end into the extended semen and aspirate the semen up to the plug on the end of the straw. there is commercially available equipment for this that is also very easy to use. insufflation equipment some sort of gas and a delivery system is necessary for laparoscopic insemination (lai). some people use room air, filtered room air, or a commercially available gas system using oxygen or carbon dioxide. medical grade co2 is the preferred gas in most insufflation systems but it may be difficult to obtain and transport in some situations. there are insufflators that automatically maintain pressure and insufflate the abdomen but are expensive and too slow (in the author’s hands) for lai in the small ruminants, especially in a field situation. in most cases, silastic tubing attached to a co2 regulator and a teat cannula is the preferred method although other people may use another system and feel comfortable with it. some people use an assistant to turn the gas on and off and others use a foot pedal set up so the surgeon can regulate the amount of insufflation. trocars these will depend on the size of laparoscope selected, but 10 mm and 5 mm are very commonly used for most lai. there are reusable and disposable trocars as well as cutting and non-cutting available as well. the aspic fits the 5 mm trocar and the author prefers a 10 mm laparoscope so these are what he uses. other people may prefer other equipment or purchase used equipment that will work equally well for the purpose. laparoscope the laparoscope can be either 5 or 10 mm in diameter and 30 cm long rigid laparoscope or other length or size as the surgeon sees fit. a 0 degree head is preferable although some prefer ahead angled of 20 degrees. light source for laparoscope generally a high intensity fiberoptic cord is attached to the laparoscope and a high intensity variable power light source is used to illuminate the abdomen. battery powered sources may work and should be available for backup as well as extra bulbs and a backup lighting system if going on extended trips or in case of equipment failure. if using a flashlight, make sure to have extra batteries or that it is able to be charged from a dc or ac power source or inverter. one needs to make sure the fittings for the light source fit the laparoscope or an adapter is available for it. clinical theriogenology • volume 3 number 4 • december 2011527 cradle/surgical table a lai cradle makes the process much easier although animals can be restrained and moved without one. depending on the number of animals and facilities, it is usually best to have two or three cradles so animals can be prepared, moved and inseminated without interruption. the cradle should have fairly large wheels so that it can be rolled on uneven surfaces and locking wheels are a plus to keep the cradle from rolling on uneven surfaces as does are inseminated or prepared. it should be light enough that it can be moved easily and have adequate restraint of the legs and head and provide support for the animal at the same time. it is imperative that the rear legs be restrained so that the animal does not slip from the restraints when it is inverted for the lai. there are several designs on the market but the author has custom aluminum cradles that lengthen and collapse to some degree so they do not take up as much room and can adjust to different animal sizes and species. monitor some people use a monitor with a camera set up to view through the laparoscope. this could be a computer screen or a small television monitor. this will be a personal preference for the surgeon but it will allow all people watching to see what is going on. an excellent teaching tool, it may also be stressful for some to know that everybody is watching their every move and may subject them to even more stress in an already stressful situation. procedure for tci with the doe restrained either in a chute, standing or anesthetized, the vulvar area is cleaned and dried prior to insertion of the speculum. most deer are clean enough that they require little or no cleaning and using soap and water or just water or disinfectant can contaminate the speculum and vagina and kill sperm. some prefer that the rear quarters of the animal be raised to make it easier to visualize the cervix and inseminate. it will depend on the species and restraint method used as to whether this is advantageous. using a non-spermicidal lubricant on the vulva or the speculum, the speculum is inserted into the vagina until reaching the cervix. choosing the proper size speculum will depend on experience and the size and previous birth status of the doe. after visualizing the cervix, the insemination rod is passed through the speculum and an attempt is made to pass the rod through the cervix into the uterine body. in some animals the cervix will be wide open and the rod readily passed, in others it may be difficult if not impossible to pass the rod through any or all of the cervical rings. in either case, once the insemination rod is passed to the point where the inseminator is either satisfied that they can go no further without doing more harm than good, the semen may be deposited in a slow and deliberate manner. the author prefers to withdraw the speculum partially prior to semen deposition to allow the vaginal wall to collapse over the end of the insemination rod. one can also use a “split” speculum so that complete withdrawal of the speculum can be done prior to insemination. once the semen is deposited, the doe is given any medications necessary and released or the anesthetic reversed after transport to an appropriate place for recovery. procedure for lai the animal is prepared for surgery either with light to heavy sedation (sheep and goats) or with full anesthesia (whitetail deer and other cervids) by clipping the area cranial to the udder and extending almost to the navel and to the flank and doing a surgical scrub on the area. this is usually done while the animal is restrained in a lai cradle or other device used for restraint and tilting the animal for the surgery. two spots approximately 6-8 cm (a hand’s width) cranial to the udder and approximately 6-8 cm lateral to the midline are located and a local block with 2% lidocaine injected subcutaneously and intramuscularly in the abdominal wall. this takes approximately 2-4 cc of lidocaine and can be marked with surgical ink or scratched with the needle used for the injection so that the surgeon may easily see the place that has been blocked. care must be taken to avoid the major vessels in the area so that they are not cut and bleeding is kept to a minimum. this will lead to blood running down the laparoscope and obscuring the view of the internal organs as well as cause concern to the owners. the animal is tilted on clinical theriogenology • volume 3 number 4 • december 2011 528 the table and two small incisions (10 mm) are made in the skin taking great care to not cut into the abdomen (it will be next to impossible to maintain insufflation if the hole is very large) and at the same time to make sure that the skin is incised completely and underlying fascia has been exposed so that the insufflation needle (teat tube) can easily penetrate the musculature and the peritoneum without excessive pressure being applied to it. as soon as the skin is incised properly, the animal is tilted rear end up to approximately a 45 degree angle or more depending on surgeon preference. the insufflation needle is then inserted into the abdomen with a sharp thrust taking care to angle away from all internal organs (i angle the needle slightly caudal and lateral so that i miss the bladder and intestines) and gas is pumped into the abdomen until it is slightly taunt. at this point the trocar for the laparoscope is inserted using the same angle as for the insufflation needle, the dilation or cutting tip pulled, and the laparoscope inserted and visualization of the abdomen, bladder and uterus attempted. occasionally the omentum will be trapped by the gas, insufflation needle or the trocar and will impair visualization of the abdomen. the omentum must be swept away from the trocar or needle by gentle use of the laparoscope or movement of the needle so that the gas may push the omentum out of the way to allow visualization and manipulation of the uterus for insemination. once the abdomen has been evaluated and the uterus identified, the insemination trocar is inserted and the aspic introduced with the needle withdrawn into the aspic gun. the uterus is gently manipulated with the aspic gun until the gun and the uterus are at a 90 degree angle and then the inner sheath of the apsic protruded and the uterus pierced with the needle and the semen deposited into the uterine horn. usually one-half of the straw (dose) is injected into one horn and the other half injected into the other horn. once the semen is deposited, the insemination gun and the laparoscope are withdrawn and some, if not all, the gas is allowed to escape from the abdomen via the trocar’s gas valves and the skin incisions closed in some manner. staples, suture or glue are all used successfully and in some instances, no attempt to close the incision is made. however, the aesthetics are usually such that this would only be done in a very high speed sheep breeding situation. the animal is usually given antibiotics and an anti-inflammatory and returned to the recovery area. the laparoscope, trocars and scalpel are usually stored between animals in a tray with 70% alcohol or rinsed with alcohol and wiped dry with gauze sponges between animals to keep alcohol from running down the laparoscope and trocar which will impair visualization of the abdomen. other disinfectants could be used as well, but something that is easy on the equipment and will not irritate the abdominal cavity is essential. grasping the trocar and the laparoscope with the same hand and using the thumb to slide the laparoscope up and down and the hand to control the depth of the trocar and laparoscope is perhaps the most difficult to master. there will also be times when the bladder is obscuring access to the uterus and it must be expressed or drained. with practice, the laparoscope can be used to hold the bladder out of the way while manipulation and insemination take place. some surgeons wear surgical gloves, others use examination gloves or no gloves. the procedure is clean, fast and has little risk. most complications are due to handling (capture myopathy), anesthesia, or disease from stress. generally speaking, these skills can be mastered with moderate practice. hand-eye coordination can be developed using such simple tools as a cardboard box with objects inside to visualize and manipulate. using cull animals for practice on doing the procedure on owners’ animals with their full knowledge that you are just starting may be acceptable. much of the equipment can be purchased new or used through medical suppliers or dealers. when on the road, it is imperative that you have parts for equipment, spare equipment and a plan to deal with emergency situations that may arise. electrical cords and power strips, paper towels, scrub, gauze 4x4s, tool boxes to carry and organize equipment, tables to work on, semen tanks and other miscellaneous items can make or break you. checks off lists of equipment are helpful and highly recommended. clinical theriogenology • volume 3 number 4 • december 2011529 clinical theriogenology • volume 3 number 4 • december 2011 530 omniblank: a case of polioencephalomalacia in a breeding bull a case of polioencephalomalacia in a breeding bull madelyn pelletier, james wenzel, m edmondson, j rush, j stockler, thomas passler, julie gard college of veterinary medicine, auburn university, auburn, al a 16 month old angus bull was presented with a history of being off feed. bull was blind and diagnosed as having polioencephalomalacia due to a feed change, with consumption of a high concentrate feed resulting in ruminal acidosis and an overgrowth of thiaminase-producing bacteria. following treatment for polioencephalomalacia, including administration of thiamine and transfaunation, an initial breeding soundness exam (bse) was performed. he was determined to be an unsatisfactory breeder due to absence of menace response (indicative of central blindness) and high numbers of primary sperm morphological abnormalities. a follow up bse was performed 1 month later. bull had regained his menace reflex and percentage of morphologically normal sperm had increased to almost normal levels. at a third bse, 2 months following initial treatment, he was determined to be a satisfactory breeder. since he was a calving-ease bull, he was exposed to breeding age heifers. he successfully bred all heifers and was utilized for 2 additional breeding seasons. inciting factors responsible for polioencephalomalacia can result in severe clinical disease involving neurological and reproductive systems. however, recovery may be possible if the disease is identified quickly and treated appropriately. keywords: bull, breeding soundness examination, polioencephalaomalacia 531 clinical theriogenology • volume 11, number 3 • september 2019 532clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2011: development and validation of testicular biopsy for diagnosis of infertility in camels (camelus dromedarius) development and validation of testicular biopsy for diagnosis of infertility in camels (camelus dromedarius) m.m. waheed,a i.m. ghoneim,a m.m. hassiebb departments of clinical studiesa and pathology,b faculty of veterinary medicine and animal resources, king faisal university, alhasa, kingdom of saudi arabia abstract male camel infertility is a heterogeneous disorder. a variety of factors may adversely impact sperm production and function and impair fertility. twenty-six male dromedaries (4–14 years old) were used in this study during the rutting season. four sexually mature male camels were used to develop and validate a procedure for testicular biopsy in dromedary camels. no undesirable side effects in testicular function were apparent after use of an automatic biopsy gun. in 22 infertile male dromedaries, an algorithmic approach based on information collected during a careful history, clinical examination, testicular evaluation, testicular ultrasonography, the results of the semen analyses, and testicular biopsy was used to diagnose infertility. in diagnosis of male camel infertility, the results of testicular ultrasonography and testicular biopsy were correlated identically. testicular biopsy is a promising method that, along with carefully performed history, appropriate testicular ultrasonography procedure and semen analysis, can afford the veterinarian the opportunity for more precise diagnosis and treatment of male dromedary infertility disorders. keywords: dromedary camels, infertility, testicular biopsy, testicular ultrasonography. introduction the reproductive rate in camelidae has been described as low.1,2 many theories have been advanced to explain this low reproduction rate based on physiological characteristics of these species.3 management may explain some of the reproductive failures but a substantial number of infertility problems are due to pathological processes in the male genitalia. there are limited reports on the pathology of the male reproductive tract in camelidae. although the guidelines for breeding soundness examination of male camelids are in their infancy, there is evidence that thorough examination of males may help to identify many infertility problems.1 it is very important that all studs be examined for at least the following parameters: testicular size, testicular consistency, ability to extend the penis and ability to ejaculate and produce viable semen. the average testicular length and width are 9.1 cm and 5.1 cm, respectively, for male dromedaries three years and older during the breeding season.1 the testes should be resilient and non-painful. each part of the genital tract can be the site of congenital or acquired lesions that need to be identified.4 scrotal and testicular pathology in the camelid can be acquired (trauma ,orchitis, hydrocele, degeneration) or congenital (hypoplasia, cryptorchidism).1 several other reproductive problems are reported in the camelidae but their exact etiology is not known. amongst the most commonly reported problems are absent or reduced libido, ejaculatory problems and unexplained subfertility or infertility.3 testicular ultrasonography in camels is a very important technique for the evaluation of the testicular parenchyma as well as the surrounding tissues.1 moreover, testicular biopsies are useful procedures in the evaluation of testicular function in llamas.5 this study was designed to develop and validate testicular biopsy for assessment of the reproductive status of infertile male camels. materials and methods a total of twenty-six male dromedary camels (4–14 years old) were used in this study during the rutting season (november to may).6 four dromedaries were used as controls for development and validation of testicular biopsy in male camels. these animals were housed at the camel research center at king faisal university. the animals were healthy, weighed 400–700 kg with a history of normal fertility. they were maintained under standard conditions of feeding and management. each male camel was housed separately in a 12 x 6 m paddock and fed 3-4kg barley per day. rhodes grass and water were provided ad libitum. twenty-two infertile male camels were admitted to the 105 clinical theriogenology • volume 3 number 2 • june 2011 veterinary teaching hospital, college of veterinary medicine and animal resources, king faisal university and were subjected to the following examinations: reproductive history a full reproductive history of the animal was recorded which included the breed, age, management, libido and the owner’s complaint. general physical examination a general physical examination was conducted with emphasis on systemic problems or poor body condition score. testicular evaluation testicular size, mobility and consistency were examined carefully by palpation. any morphological, congenital or pathological lesions of the testes were recorded. the length and width of each testis were measured using vernier calipers. testicular ultrasonography ultrasonographic examination of the scrotum and its contents was conducted subsequent to scrotal palpation. ultrasonography of the testes was performed by direct manual manipulation of a convex multi-frequency transducer (1–15 mhz; prosound ssd-3500sx, aloka co., ltd; tokyo, japan). the testis was examined by placing the probe in a vertical position then in a horizontal position and sweeping it along the whole surface of the organ. testicular ultrasonography used for the evaluation of the testicular parenchyma as well as the surrounding tissues. collection and evaluation of semen collection of semen from male camels was carried out using an electro-ejaculator (electrojac® 5; ideal instruments, neogen co., lansing, mi) as described in an earlier study.7 immediately after collection, semen samples were evaluated for physical, morphological and physiological characteristics. testicular biopsy development of the procedure. testicular biopsy was developed in two stages. in the first stage, the suitability of the instruments and the size of the biopsy specimens for histopathological study were tested. to achieve this goal, biopsies were performed on ten camel testes in the laboratory using a disposable automatic gun (14, 16, and 18 gauge; speedy-ram®, ri.mos, mirandola, italy). the biopsy tissues were placed in 10% formaldehyde solution and processed for histopathological examination. in second stage, potential damage to testicular function was assessed. four sexually mature male camels with a history of normal fertility were used. results of the physical examination and testicular ultrasonography were within normal limits and semen was of acceptable quality (figure 1; tables 1 and 2). prior to surgery, camels were sedated with a mixture of xylazine (0.15 mg/kg) and ketamine hydrochloride (2.5 mg/kg) administered intravenously8 while restrained in sternal recumbency. the scrotal surface was prepared for aseptic surgery and painted with a local anesthetic gel (lidocaine 5%, julphar, u.a.e.). the area for puncture was covered with a sterile towel with a 12 cm x 6 cm fenestration. the testis was held firmly in the scrotum so as to tighten the scrotal skin over the testis. the biopsy sample was obtained using the 14 and 16 gauge automatic gun at the midpoint of the posterior border of the testis. the tissues were placed in 10% formaldehyde solution and processed for histopathological examination (figure 5). the animals were observed for seven days after surgery to detect any detrimental effects of the procedure. seventy-eight days after the biopsy, physical examinations, testicular ultrasonography and semen analysis were performed. evaluation of reproductive dysfunction. testicular biopsies were performed in the infertile male dromedaries with a 16-gauge needle when the results of the physical and ultrasonographic examinations did not yield a clear diagnosis of the cause of infertility or in cases of unacceptable semen quality. 106clinical theriogenology • volume 3 number 2 • june 2011 statistical analysis data are presented as means ± sem and the analysis was conducted using stastical package for social sciences, version 16.0.9 results during development of the biopsy procedure it was found that the use of the 18-gauge needle did not yield a tissue sample containing a sufficient number of tubules to permit a histopathologic diagnosis. when biopsies were performed in the control animals, a small amount of hemorrhage was observed immediately after the samples were obtained in three of four camels (six of eight testes; 75%). hemorrhage persisted for three minutes after the sample was obtained followed by slight testicular swelling for five days in one testis (one of eight; 12.5%). no other clinical changes were observed in any of the control animals. when physical and ultrasonographic examinations and semen evaluation were performed 78 days after the biopsies were performed, abnormalities were not observed and semen quality was acceptable (table 2). results of examination of the testes of the infertile animals are shown in table 3. testicular texture the texture of testes was palpably normal in four cases (18.2%), soft in 14 cases (63.6%) and hard in four cases (18.2%). testicular size testicular length and width (means ± s.e.m.) of the control and infertile dromedaries were 9.6 ± 0.2 and 4.8 ± 0.1 cm; and 9.3 ± 1.1 and 4.7 ± 0.6 cm, respectively. however, in cases of testicular hypoplasia, these measurements were 5.5 ± 1.5 and 3.1 ± 0.7 cm, respectively (table 1). testicular ultrasonography the image obtained from normal testes showed a peripheral area of homogenous tissue corresponding to the testicular parenchyma and a central echogenic area corresponding to the fibrous rete testis (figure 1).1 in the majority of the infertile animals (19 of 22; 86.4%), mild to severe degeneration of the testicular parenchyma characterized by loss of its homogeneous appearance due to the presence of localized lesions such as cysts or fibrotic nests was observd during the ultrasonographic examination (figure 2) testicular hypoplasia was diagnosed in three (13.6%) infertile animals on the basis of measurements obtained with electric calipers (table 3; figure 3).1 testicular fibrosis was characterized by hyper-echoic areas in the testicular parenchyma (figure 4). semen evaluation evaluation of semen samples from the infertile male dromedaries showed seven cases (31.8%) of oligoasthenozoospermia, six cases (27.3%) of athenozoospermia, six cases (27.3%) of azoospermia, two cases (9.1%) of oligozoospermia and one (4.5%) case of teratozoospermia (table 3). histopathology histopathologic examination of the testicular biopsy specimens from infertile camels showed mildly degenerated semineferous tubules containing multiple cell layers of scattered spermatocytic cells with pyknotic nuclei in addition to few spermatids and surrounded by excess numbers of leydig cells (figure 6). severely degenerated seminiferous tubules were lined by only one layer of sertoli cells and contained primary spermatocytic cells with pyknotic nuclei (figure 7). some testicular biopsy specimens from infertile dromedaries contained interstitial fibrosis accompanied by mildly degenerated seminiferous tubules that contained vacuolated sertoli cells and few spermatids (figure 8). histopathologic findings were compared to the abnormalities observed with ultrasonography and were found to be correlated in all cases of infertility (table 3). discussion under the conditions of this study, the disposable automatic gun with an 18-gauge needle was found to yield tissue samples with an insufficient number of seminiferous tubules. biopsy 107 clinical theriogenology • volume 3 number 2 • june 2011 instruments used in stallions,10 llamas5 and bulls11 utilized a 14-gauge needle and resulted in no difference between the biopsied and the non-biopsied testis. other investigators have used a 12-gauge split needle biopsy needle in boars, rams, bulls and pony stallions with no deleterious effects.12 in the present experiment, short-term changes appeared in the biopsied testes. similar findings were reported in stallions13 and bulls.11 no untoward effects were observed in the testicular parenchyma or semen characteristics when control animals were examined 78 days after tissue samples were obtained. the mean number of progressively motile and morphologically normal sperm was not significantly different before and after the biopsy procedure. similarly, there was no reduction in the number of normal seminiferous tubules seen in histologic sections obtained from llamas six weeks after a needle biopsy5 and there was no evidence of testicular atrophy in men following a testicular biopsy.14 the safety of needle biopsies has been demonstrated in dogs,15 humans16 and stallions.17 the testicular length and width in infertile dromedaries were similar to those reported for normal adult (three years and older) dromedary camels1 which indicates that no changes in testicular measurements occur in the presence of testicular degeneration and fibrosis. ultrasonograpic imaging systems have provided new methods for diagnostic examinations of pathological conditions of the genital system of domestic animals.18,19 the similarity found between the results of testicular biopsies and testicular ultrasonography supports the use of biopsies as a diagnostic tool in the evaluation of testicular function. biopsies could provide a clinically useful tool in the evaluation of potential breeding ability in dromedaries as well as aid in the investigation of infertility as has been described in other animals.12,20,21 taken together, semen analyses and either testicular ultrasonography or testicular biopsy could be used for predicting infertility in male dromedaries. conclusion testicular biopsy should probably be reserved for cases where a fertility problem is suspected but cannot be confirmed through more conventional means of fertility evaluation, or in cases where a problem has been identified and a definitive diagnosis is desired. acknowledgements this project was funded by the deanship of scientific research, king faisal university, kingdom of saudi arabia. the authors thank mr. a.a. alsumait for his technical assistance. references 1. tibary a, anouassi a: theriogenology in camelidae: anatomy ,physiology, pathology and artificial breeding. actes editions: institut agronomique et vétérinaire hassan ii; 1997. 2. kaufmann ba: reproductive performance of camels (camelus dromedarius) under pastoral management and its influence on herd development. livest prod sci 2005;92:17-29. 3. tibary a, anouassi a: reproductive disorders in the male camelid. in: skidmore ja, adams gp, editors. recent advances in camelid reproduction. ithica (ny): int vet inform serv 2000 nov [cited 2010 jan 12]; [about 4 p.]. available from: www.ivis.org 4. tibary a, memon ma: reproduction in the male south american camelidae. j camel pract res 1999;6:235-248. 5. heath am, pugh dg, sartin ea, et al: evaluation of the safety and efficacy of testicular biopsies in llamas. theriogenology 2002;58:1125-1130. 6. arthur hg, rahim at, hindi a: reproduction and genital diseases of the camel. br vet j 1985;141:650-659. 7. tingari md, manna mm, rahim at, et al: studies on camel semen. i. electroejaculation and some aspects of semen characteristics. anim reprod sci 1986;12:213-222. 8. al-mubarak ai, abdin-bey mr, ramadan ro: a retrospective clinical evaluation of xylazine-ketamine total intravenous anesthesia (tiva) in dromedary camels. j camel pract res 2008;15:201-203. 9. spss: statistical package for social science. spss inc, chicago, il, usa copyright© for windows 2007; version 16.0. 10. faber nf, roser jf: testicular biopsy in stallions: diagnostic potential and effects on prospective fertility. j reprod fert suppl 2000;56:31-42. 11. heath am, carson rl, purohit rc, et al: effects of testicular biopsy in clinically normal bulls. j am vet med assoc 2002;220:507-514. 12. galina cs: an evaluation of testicular biopsy in farm animals. vet rec 1971;88:628-631. 13. delvinto vr, amann rp, trotter gw, et al: ultrasonographic and quantitative histologic assessment of sequelae to testicular biopsy in stallions. am j vet res 1992;53:2094-2101. 108clinical theriogenology • volume 3 number 2 • june 2011 14. steele ek, ellis pk, lewis sem, et al: ultrasound, antisperm antibody, and hormone profiles after testicular trucut biopsy. fertil steril 2001;75:423-428. 15. lopate c, threlfall w, rusol t: histopathologic and gross effects of testicular biopsy in the dog. theriogenology 1987;32:585-602. 16. mallidis c, baker h: fine needle tissue aspiration biopsy of the testicle. fertil steril 1994;61:367-75. 17. blanchard tl, varner dd: evaluating breeding soundness in stallions-4: hormonal assay and testicular biopsy. vet med 1996;91:358-365. 18. kahn w: veterinary reproductive ultrasonography: horse, cattle,sheep, goat, pig, dog, cat. hanover: schlutersche verlagsgesellschaft mbh & co; 1994. 19. brook fm, kinoshita r, brown b, et al: ultrasonographic imaging of the testis and epididymis of the bottlenose dolphin, tursiops truncatus aduncas. j reprod fertil 2000; 119:233-240. 20. veeramachaneni dnr, ott rs, heath eh: pathophysiology of small testes in beef bulls: relationship between scrotal circumference, histopathologic features of the testes and epididymides, seminal characteristics, and endocrine profiles. am j vet res 1986; 47:1988-99. 21. meyers-wallen vn: clinical approach to infertile male dogs with sperm in the ejaculate. vet clin north am small anim pract 1991;21:609-633. table 1: testicular length and width (mean ± sem) in male dromedaries. testicular state length (cm) range width (cm) range control (normal testes) 9.6a ± 0.2 (n=8) 8.4 – 10.4 4.8a ± 0.1 (n=8) 4.4 – 5.2 degeneration and fibrosis 9.3a ± 1.1 (n=37) 7.4 – 12.0 4.7a ± 0.6 (n=37) 3.8 – 6.2 hypoplasia 5.5b ± 1.5 (n=6) 3.5 – 7.4 3.1a ± 0.7 (n=6) 2.3 – 4.1 means with dissimilar superscripts in the same column are significantly different at p<0.05 table 2: semen analysis of the control dromedary camels before and 78 days after testicular biopsy (mean ± sem). semen parameters before testicular biopsy 78 days after testicular biopsy motility % 77.5 ± 6.23 73.50 ± 8.29 sperm concentration ( x106 /ml) 364.75 ± 58.08 350.00 ± 79.06 sperm abnormalities % 18.75 ± 7.14 16.25 ± 10.78 109 clinical theriogenology • volume 3 number 2 • june 2011 table 3: testicular examination of the infertile male dromedaries. camel number external testicular evaluation testicular length x width (cm) testicular ultrasonography semen analysis histopathologic diagnosis 1 soft texture left: 6.2 x 3 right: 8.4 x 4.4 testicular hypoplasia, sever degeneration teratozoospermia mot1. 0 conc2. 6 abnorm3. 92 testicular hypoplasia, sever degeneration 2 hard texture left: 9.6 x 4.4 right: 10.1 x 5.1 fibrosis and degeneration azoospermia fibrosis and degeneration 3 slightly soft left: 8.0 x 4.0 right: 8.7 x 4.9 mild degeneration asthenozoospermia mot1. 30 conc2. 420 abnorm3. 57 mild degeneration 4 soft texture left: 7.6 x 4.1 right: 9.0 x 4.3 mild severe degeneration asthenozoospermia mot1. 25 conc2. 214 abnorm3. 43 mild severe degeneration 5 soft texture left: 9.8 x 4.1 right: 10.1 x 5.9 moderate degeneration azoospermia moderate degeneration 6 soft texture left: 7.4 x 4.1 right: 7.0 x 4.1 testicular hypoplasia and degeneration oligoasthenozoospermia mot1. 10 conc2. 26 abnorm3. 48 testicular hypoplasia and degeneration 7 soft texture left: 10.5 x 5.1 right: 9.3 x 5.0 moderate degeneration asthenozoospermia mot1. 20 conc2. 18 abnorm3. 65 moderate degeneration 8 small and hard left: 3.5 x 2.3 right: 4.3 x 2.4 testicular hypoplasia azoospermia severe testicular hypoplasia 9 soft texture left: 8.2 x 4.0 right: 10.0 x 4.9 mild moderate degeneration azoospermia mild moderate degeneration 10 normal firm texture left: 9.2 x 4.3 right: 9.2 x 4.5 mild moderate degeneration asthenozoospermia mot1. 5 conc2. 394 abnorm3. 75 mild moderate degeneration 11 soft texture left: 12.0 x 6.2 right: 11.5 x 5.6 mild moderate degeneration oligoasthenozoospermia mot1. 20 conc2. 10 abnorm3. 25 mild moderate degeneration 12 slightly soft left: 8.0 x 3.8 right: 7.5 x 4.4 moderate degeneration oligoasthenozoospermia mot1. 10 conc2. 34 abnorm3. 62 moderate degeneration 13 unilateral aplasia, soft texture left: 9.9 x 4.7 right: aplasia moderate degeneration oligoasthenozoospermia mot1. 0 conc2.53 abnorm3. 57 moderate degeneration 14 normal firm texture left: 7.6 x 4.0 right: 7.8 x 4.9 mild degeneration asthenozoospermia mot1. 55 conc2. 47 abnorm3. 39 mild degeneration 15 normal firm texture left: 8.5 x 4.4 right: 10.0 x 4.8 mild degeneration azoospermia mild degeneration 16 slightly soft left: 7.4 x 4.1 right: 8.7 x 3.9 mild degeneration oligoasthenozoospermia mot1. 10 conc2. 12 abnorm3. 68 mild degeneration 17 slightly soft left: 9.9 x 4.7 right: 10.4 x 5.5 mild degeneration oligoasthenozoospermia mot1. 30 conc2. 26 abnorm3. 16 mild degeneration 18 hard texture left: 9.5 x 5.3 right: 9.4 x 4.6 mild degeneration oligozoospermia mot1. 10 conc2. 12 abnorm3. 70 mild degeneration 19 slightly soft left: 9.6 x 3.9 right: 9.7 x 4.3 moderate severe degeneration severe oligozoospermia mot1. 0 conc2. 9 abnorm3. 74 moderate severe degeneration 20 unilateral hypoplasia, slightly hard texture left: 4.4. x 2.9 right: 10.7 x 5.8 mild moderate degeneration asthenozoospermia mot1. 15 conc2. 18 abnorm3. 66 mild moderate degeneration 21 slightly soft left: 8.5 x 5.1 right: 8.6 x 5.0 mild degeneration azoospermia mild degeneration 22 slightly soft left: 10.0 x 4.3 right: 9.9 x 5.4 moderate degeneration oligoasthenozoospermia mot1. 10 conc2. 24 abnorm3. 38 moderate degeneration mot1 = sperm motility % conc2 = sperm cell concentration ( x106 /ml) abnorm3 = sperm abnormalities % 110clinical theriogenology • volume 3 number 2 • june 2011 figure 1: ultrasonography of a testis in a control dromedary showing a homogenous peripheral area corresponding to the testicular parenchyma and a central more echogenic area corresponding to the fibrous rete testis. figure 2: testicular degeneration in a camel (hypo-echoic areas in the testicular parenchyma). 111 clinical theriogenology • volume 3 number 2 • june 2011 figure 3: testicular hypoplasia in a camel (hypo-echoic parenchyma) figure 4: testicular fibrosis in a camel (hyper-echoic parenchyma) 112clinical theriogenology • volume 3 number 2 • june 2011 figure 5: testicular biopsy of a camel showing a normal seminiferous tubule that contains multiple cell layers of spermatocytic cells in addition to spermatids and surrounded by leydig cells. h and e x=400. figure 6: testicular biopsy of a camel showing a mildly degenerated seminiferous tubule (st) that contains multiple cell layers of scattered spermatocytic cells with pyknotic nuclei (sc), in addition to few numbers of spermatids (sd) and surrounded by excess numbers of leydig cells (l). h and e x=400. 113 clinical theriogenology • volume 3 number 2 • june 2011 figure 7: testicular biopsy of a camel showing a severely degenerated seminiferous tubule lined by only one layer of sertoli cells (white arrow) that contains primary spermatocytic cells with pyknotic nuclei (black arrow) and associated with hyperplastic interstitial leydig cells (l) and congested capillaries (c). h and e x=400. figure 8: testicular biopsy of a camel showing a mildly degenerated seminiferous tubule that contains vacuolated sertoli cells (black arrow) and few spermatids (white arrow), in addition to an excess of interstitial leydig cells (l) and interstitial fibrosis (i). h and e x=400. 114clinical theriogenology • volume 3 number 2 • june 2011 1 contact sara lyle saraklyle3@gmail.com ‡current affiliation: veterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la, usa © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10491, http://dx.doi.org/10.58292/ct.v16.10491 research report pharmacokinetics of carprofen in lactating dogs after intravenous treatment amber nebel-karp,a mark papich,b kristen messenger,b sara lylec‡ aanimal hospital of east davie, advance, nc, usa bdepartment of molecular biomedical sciences, college of veterinary medicine, north carolina state university, raleigh, nc, usa cdepartment of clinical sciences, college of veterinary medicine, north carolina state university, raleigh, nc, usa abstract analgesia is an important component of veterinary care with nonsteroidal antiinflammatory drugs, particularly carprofen (rimadyl®) commonly used for dogs. pharmacokinetic studies of carprofen in lactating dogs have not been examined fully, questioning the use and safety of carprofen. the extent to which lactation changes the pharmacokinetics is unknown, nor is the extent of transfer into milk of lactating dogs and their neonates or pups. we hypothesized that after a single dose of intravenous (4.4 mg/kg) to dogs, concentrations in milk are low and not high enough to produce harmful exposure in pups. we tested our hypothesis in healthy, adult lactating dogs (n = 4) using pharmacokinetic methods and nonchiral and chiral specific assays measuring total, r-, and s-enantiomer carprofen concentrations with high performance liquid chromatography in maternal plasma, milk, and neonatal plasma samples. the cmax, elimination half-life, and clearance for maternal plasma were 9.09 and 7.3 µg/ml (r-, s+), 6.82 and 6.22 hours (r-, s+), and a higher clearance rate of 95.81 ml/hr/kg (r-) and 73.87 ml/hr/kg (s+) than reported. none of the neonatal plasma concentrations at any time point were more than 10% of the total maternal plasma concentrations and milk:plasma ratio was < 1. this study confirmed the approved dose is appropriate for perioperative and chronic pain in lactating dogs and is safe with reduced exposure to nursing pups. this study suggested that the pharmacokinetics of carprofen in lactating dogs may be different from their nonlactating counterparts. however, further pharmacokinetic studies with more dogs are needed to confirm these findings. keywords: lactation, analgesia, carprofen, neonate, pup, pharmacokinetics introduction carprofen, a preferential cox-2 (ptgs2; cyclooxygenase-2 or prostaglandin-endoperoxide synthase-2 enzyme) inhibitor first evaluated in dogs in 1990,1 is approved for perioperative and chronic pain management in dogs.2-5 carprofen is a racemic nonsteroidal antiinflammatory drug (nsaid) that has antiinflammatory, analgesic, and antipyretic properties. carprofen consists of 2 enantiomers r(-) and s(+), with the s(+) enantiomer having greater antiinflammatory activity compared to the r(-) enantiomer. carprofen ordinarily has a good safety profile;3,6-8 however, it has adverse effects common to all nsaids, such as kidney, liver, and gastrointestinal injury.1,2,6-9 an adverse reaction specific to dogs is an idiosyncratic acute hepatopathy, with an incidence ranging from < 0.05 to 1.6%.9,10 pharmacokinetics for carprofen have been reported in normal, healthy, nonlactating dogs1 and in canine induced-pain models.5,9,11-14 following cesarean surgery, some dogs experienced pain or had preexisting problems (e.g. osteoarthritis)15 that warranted analgesia. carprofen has been used anecdotally in dogs undergoing cesarean surgery as a single subcutaneous dose without adverse effects reported in either dogs or neonates.16-18 the fda approved label for carprofen states, “the safe use of rimadyl® in animals less than 6 weeks of age, pregnant dogs, dogs used for breeding purposes, or in lactating bitches has not been established.”9 this labeling may discourage effective pain treatment and control in dogs having recently whelped or are lactating. studies in women report that medications with greater than 85% protein binding produce low concentrations in breast milk.19-25 carprofen is 99% protein-bound in canine plasma and is a weak acid (pka = 4.3);1 therefore, it is less likely to pass from the plasma to milk and is not a candidate for ion trapping via the ph-partition theory. it has a low volume of distribution making it less likely to diffuse out of the mailto:saraklyle3@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10491 2 citation: clinical theriogenology 2024, 16, 10491, http://dx.doi.org/10.58292/ct.v16.10491 intravascular space into the body tissue, even in inflammatory conditions.12,13 in a study, dogs that received a 5-day course of carprofen following cesarean surgery were assigned to 3 groups: ‘normal’ (88), ‘mastitis’ (4), and ‘generalized inflammation’ (8).18 maternal plasma and milk sample concentrations were compared, and dogs with mastitis had higher concentrations of carprofen in the milk compared to the normal and generalized inflammation groups. predicted expected neonatal exposure was < 1 (milk:plasma ratio) in all groups.18 these authors concluded that carprofen was safe to treat lactating dogs; however, no pharmacokinetics analysis was performed in that study to provide further information on the disposition of carprofen in lactating dogs. our hypothesis is based on the work performed with other nsaids in people and other animals and results from carprofen concentrations in canine milk. minimal data are available regarding milk concentrations for carprofen in dogs,17,18 but no pharmacokinetic studies have been reported that provide direct measurements of neonatal plasma concentrations, or to examine if the pharmacokinetics in lactating dogs are different from other dogs. the purpose of our descriptive study was to determine the pharmacokinetic disposition of carprofen in plasma of lactating dogs, measure concentrations in canine milk of treated dogs, and measure concentrations in plasma of nursing pups. we predicted that a single intravenous dose of carprofen in dogs will fail to produce milk concentrations in lactating dogs that exceed the limit of quantification in milk.18 we also hypothesized that pups nursing from dogs after a single intravenous carprofen treatment will not have detectable concentrations of carprofen in their plasma. materials and methods exclusion/inclusion criteria seven dogs were enrolled in the study and recruited from privately owned kennels with approval via consent form and a factual sheet on carprofen provided from the manufacturer. the ages of the dogs were 2.5-6.0 (average 4.34) years and weighing between 8.91 and 27.27 kg (average 21.03 kg). litters ranged from 16-39 days of age (average 28 days); pups weighed more than 300 grams and were being fed exclusively maternal milk. dogs and litters consisted of 2 breeds: american foxhound (n = 6) and cavalier king charles spaniel (n = 1). enrollment criteria were: a. healthy based on physical examination; b. uneventful whelping or delivery; c. at least 14 days postpartum; and d. had no exposure to nsaids in the 30 days prior to enrollment. physical examinations of dogs and visual assessment of pups were performed at the start of the study prior to enrollment to determine whether they were healthy enough for the study. daily weight gains were not tracked during the study, but pups were weighed and monitored by the breeders prior to the start of the study to ensure they were over 300 grams. dogs were housed in their normal kennels with their owners/breeders and samples were taken on-site without transportation of any animal; dogs met enrollment criteria. three dogs were excluded shortly after samples were started due to: a case of mastitis present at milk collection, difficulty in venous access for sampling, and inability to collect milk from a third dog. four out of 7 litters successfully provided complete sets (maternal plasma, milk, and pup plasma). all 4 litters sampled ranged from 16-24 days old, with the majority of the pup diet consisting of milk, but food was available for dogs at all times. the study and experimental design were reviewed and approved by the institutional animal care and use committee at north carolina state university (protocol 21-382). experimental design each dog received a single intravenous carprofen at the fda approved subcutaneous/oral dose of 4.4 mg/kg between 14-39 days postpartum after blank plasma and milk samples were collected from each dog (time = 0). although the intravenous route of treatment is not included on the approved label for dogs in the us, we chose an intravenous route (extra-label use) because: a. there were no reported adverse effects observed in dogs from past experimental studies1,8,13 and in the uk; b. intravenous dose ensures 100% bioavailability that is important for pharmacokinetic calculations of clearance and volume of distribution; and c. our aim was to calculate transfer of drug from plasma to milk, irrespective of treatment route. a single milk and plasma sample prior to treatment was collected and measured to serve as a control for each female, and then a series of milk and simultaneous plasma samples were collected and measured following treatment at predetermined time points: 0, 0.5, 1, 1.5, 2, 8, 12, 24, 36, 48, and 72 hours.13 repeated blood sampling can be stressful to young pups; therefore, a sparse sampling strategy was used for pups in the same litters that were at least 14-days-old. this involved taking a maximum volume of 0.17 ml. this volume would not exceed a 10% blood loss over the entire collection period for a pup weighing 300 grams. for each time point, blood from at least 3 pups within the same litter was pooled that provided sufficient sample for that time point for analysis. the pharmacokinetic analysis performed used a naïve averaged data approach on a rotational basis to minimize the handling and venipuncture of each pup.25 milk was collected via manual expression into sterile 2-ml cryovials, with volumes measuring 20-1,000 µl, and stored at –80°c until analysis. blood was collected via venipuncture into sterile 2-ml heparinized tubes at a maximum of 4 attempts per dog or pup. the samples were immediately placed on ice following collection, centrifuged (20,000 x g 10 minutes) within 30-90 minutes after acquisition, the plasma was pipetted off and placed into sterile 2-ml cryovials, and plasma stored at –80°c until analysis. high performance liquid chromatography high performance liquid chromatography (hplc; or high pressure liquid chromatography) was performed on all samples using validated methods developed in our laboratory.13,26 maternal plasma, milk, and pooled neonatal plasma samples were analyzed via hplc to determine concentrations of carprofen in plasma and milk using methods modified from previous studies.10,13,26 the hplc system consisted of a 4-solvent delivery system, an autosampler, and fluorescent detection with an emission wavelength set at 310 nm and detection wavelength set at 375 nm (agilent 1100 series: agilent technologies, wilmington, de, usa).13 the chromatogram was integrated with proprietary software (openlab cds, agilent technologies, wilmington, de, usa). two columns were used for analysis, depending on the sample analyzed. all maternal plasma was analyzed with a chiral column (ultron es-ovm) in a column oven of 40°c that produced separation of the rand s-enantiomers with the r-enantiomer eluting from the column first. because of the smaller volume available for analysis, all milk and neonatal samples were analyzed with a nonchiral column (zorbax eclipse xdb-c18 column, agilent technologies). the mobile phase for the chiral http://dx.doi.org/10.58292/ct.v16.10491 citation: clinical theriogenology 2024, 16, 10491, http://dx.doi.org/10.58292/ct.v16.10491 3 column was isocratic at 85% 0.02 m potassium phosphate in water and 15% acetonitrile at a flow rate of 1 ml/minute. the mobile phase for the nonchiral column was set to 55% 0.02 m potassium phosphate in water and 45% acetonitrile at a flow rate of 1 ml/minute. the methods used were validated in our laboratory for canine plasma. a fresh calibration curve (described below) and quality control (qc) samples were analyzed with each batch of samples to ensure that the assay met our acceptance criteria. calibration standards a stock solution of carprofen was prepared using a racemic reference standard from the us pharmacopeial convention and dissolved in methanol to 1,000 μg/ml this was stored at refrigeration temperature in a closed container and used to fortify blank the matrix for standard calibration samples for all 3 sampling groups.26-28 the calibration curve for maternal plasma consisted of 6 standards ranging from 0.1-50 μg/ml and 5 standards ranging from 0.05-1 μg/ml for both milk and pup plasma of the racemic mixture. since specific enantiomer concentrations were being measured for maternal plasma, enantiomer specific calibration curves were based on rand s+ concentrations of 0.05-25 µg/ml as rand s+ are in a 50:50 ratio in the racemic mixture. calibration curves were accepted if the linear coefficient of determination (r2) was ≥ 0.99. the limit of detection (lod) was 0.01 μg/ml and the limit of quantification (loq) was 0.03 μg/ml, based on the lowest point on a linear calibration curve that met our acceptance criteria. sample preparation preparation for all plasma, milk, calibration, and blank samples were identical aside from volume of sample and 4% phosphoric acid due to sample volume available. samples were thawed at room temperature the day of preparation, and 500 μl was diluted in a 1:1 ratio with an equivalent volume of 4% phosphoric acid in water and then centrifuged for 5 minutes at 20,124 x g. the diluted  sample was loaded into labelled preconditioned solid-phase extraction cartridges (oasis prime hlb 1 ml; waters corporation, milford, ma, usa), and washed with a 5% methanol solution. carprofen was eluted into clean tubes with 1 ml of a 90% acetonitrile and 10% methanol solution. the eluates were evaporated at 40°c and 20 psi for 25 minutes. the dry residue was reconstituted with 250  μl of the mobile phase. the samples were vortexed briefly and 200 μl was transferred to hplc injection vials, and 20 μl was injected onto the column. pharmacokinetics the concentrations measured for maternal and neonatal plasma were averaged at each timepoint and plotted on nonlogarithmic and logarithmic scale against time to initially select the best model for pharmacokinetic analysis. pharmacokinetic analysis was performed with commercial pharmacokinetic software (phoenix® winnonlin™ and nlme®, certara, st. louis mo). pharmacokinetic models were fitted to the data to determine the best fit.25,26,29-31 two compartment modeling is ideal for intravenous treatment as the central compartment, or first compartment, is the intravenous space and the second compartment is the extravascular volume. this type of modeling considers the movement between the 2 compartments. one dog was excluded from the pharmacokinetic analysis (dog 4) because of inadequate samples collected during the first 24 hours, resulting in only a few concentrations in the elimination phase of the curve. because there were only 4 litter samples and 1 milk sample being above the loq, there were not enough data to perform a pharmacokinetic analysis on dog milk or pup plasma. results all maternal plasma carprofen concentrations were below the lod by 24 hours. postcarprofen treatment (0-72 h) concentrations ranged from 0 μg/ml to 6.11 µg/ml for the r(-) enantiomer and 0 µg/ml to 10.85 μg/ml for the s(+) enantiomer (figure 1). figure 1. pharmacokinetics of carprofen in dog’s plasma after a single intravenous (4.4 mg/kg) carprofen treatment between days 16 and 24 postpartum. a 2-compartment pharmacokinetic modeling system on a nonlogarithmic scale was used to graph the r-enantiomer concentrations of carprofen (black circles) compared to the s-enantiomer concentrations of carprofen (purple squares) against time for plasma samples from 3 dogs (between days 16 and 24 postpartum). http://dx.doi.org/10.58292/ct.v16.10491 4 citation: clinical theriogenology 2024, 16, 10491, http://dx.doi.org/10.58292/ct.v16.10491 maternal carprofen concentrations in plasma averaged for each timepoint out to 24-hours on a logarithmic scale broken into rand s-enantiomers concentrations, total carprofen concentrations in neonatal plasma, and the single milk sample above loq. litters’ concentrations were also averaged together for each timepoint. s-enantiomer concentrations in red circles, r-enantiomer concentrations in blue squares, pup plasma concentrations in green diamonds, and milk as a black triangle. most pup samples were below the lod for most time points with only 17 out of 35 samples being above the lod. out of these 17 samples; however, only 4 were above the loq. the  majority were in litter 1 (0.5, 1.5, and 12 hours postmaternal treatment) and the last was at the first time point for litter 2 (0.5 hour postmaternal treatment). milk carprofen concentrations were above the loq and lod only at a single timepoint (12 hours) in dog 1. none of the neonatal plasma concentrations at any time point were more than 10% of the total maternal plasma concentrations (figure 2). twenty-one concentration-time samples (time 0.5-24 hours) from 3 dogs (16, 22, and 24 days postpartum for dogs 1, 2, and 3 respectively) were included into pharmacokinetic modeling for both rand s-enantiomer of carprofen. our study identified multiple differences in the pharmacokinetic parameter estimates compared to studies. we observed a higher clearance rate compared to studies in healthy and pain-induced dogs.8,11,13,31,32 we also observed a higher volume of distribution in our dogs compared to studies – the volume of distribution for the central compartment was 2-3 times higher than reported. enantiomer-specific primary parameter estimates for total (protein bound and unbound carprofen) are provided (table). discussion because of uncertainty regarding the safety of carprofen to nursing pups after carprofen treatment to lactating dogs, we studied the disposition of carprofen in dogs’ plasma and milk, and in nursing pups’ plasma. this information was needed because carprofen is one of the most prescribed analgesics for perioperative use, mastitis, metritis, and osteoarthritis in dogs that may be lactating. although we were unable to analyze 0 2 4 6 8 10 12 14 16 18 20 22 24 0.001 0.01 0.1 1 10 ( noitartnecnoc )l m/g time (hour) s carprofen maternal r carprofen maternal rs carprofen puppy milk concentration rs carprofen pup r carprofen maternal s carprofen maternal µ figure 2. carprofen concentrations in maternal plasma, maternal milk, and pup’s plasma after a single intravenous carprofen (4.4 mg/kg) treatment to lactating dogs between days 16 and 24 postpartum. table. pharmacokinetic parameters for carprofen r(-) and s(+) enantiomers of maternal plasma in a 2-compartment model in 3 dogs after an intravenous dose of 4.4 mg/kg r(-) enantiomer s(+) enantiomer parameter units median range median range a µg/ml 7.7 (3.3-374.5) 6.0 (2.9-12.8) α 1/hour 1.2 (0.8-11.3) 0.7 (0.4-1.3) b µg/ml 1.4 (0.3-3.1) 3.0 (1.3-3.1) β 1/hour 0.1 (0.1-0.2) 0.1 (0.1-0.1) αhl hour 0.6 (0.1-0.8) 1.0 (0.5-1.6) βhl hour 6.8 (4.0-12.6) 6.2 (5.2-6.8) auc hour*µg/ml 23.0 (7.5-51.5) 29.8 (15.9-47.9) aumc hour*µg/ml 106.5 (86.4-147.2) 192.3 (105.4-311.7) cl ml/hr/kg 95.8 (43.0-292.9) 73.9 (46.0-138.7) cmax µg/ml 9.1 (3.6-377.6) 7.3 (5.8-15.7) k10 1/hour 0.5 (0.4-7.4) 0.3 (0.2-0.5) k12 1/hour 0.6 (0.3-3.8) 0.2 (0.1-0.7) k21 1/hour 0.2 (0.1-0.3) 0.3 (0.2-0.3) mrt hour 6.4 (2.1-11.5) 6.5 (6.5-6.7) v1 ml/kg 242.1 (5.8-611.8) 301.3 (139.8-378.0) a and b: y-axis intercepts for the phases of distribution and elimination; α and β: fractional elimination rate constants; k10, k12, k21: elimination and compartmental rate constants; mrt: mean residence time; k10 hl: elimination half-life; α-hl: distribution half-life; β-hl: terminal half-life; auc, area-under-the-curve; v1, volume of the central compartment; cl, total plasma clearance. estimates are based on a 2-compartment model. http://dx.doi.org/10.58292/ct.v16.10491 citation: clinical theriogenology 2024, 16, 10491, http://dx.doi.org/10.58292/ct.v16.10491 5 pharmacokinetics for all testing groups, these results supported our hypothesis that carprofen is safe to treat lactating dogs because there is low observed milk concentrations and negligible risk to pups. concentrations of carprofen in the maternal plasma of lactating dogs were below the lod by 24 hours that was expected for the dose (for 24 hours at 4.4 mg/kg) given and comparable to studies in healthy (intravenous, subcutaneous, and oral routes) and pain-induced (subcutaneous and oral routes) dogs.1,14,15,33 plasma concentrations in lactating dogs were different than published for nonlactating dogs in both enantiomers.1,10,14 the major differences observed between our results and earlier studies were higher clearance rates and volumes of distribution and lower aucs. the maximum concentration, movement between both compartments and elimination rates were similar. reasons for these differences are undetermined, but it may be due to higher plasma volume during lactation and underlying physiological inflammation associated with uterine involution.34 changes in plasma volume have been documented to increase by 23% in lactation for sheep35 and 45% in pregnant women.32 in women, plasma concentrations of some drugs have been reported to change during pregnancy and albumin concentrations undergo a steady decline to ~ 70-80% of normal values at the time of delivery.32 when albumin concentrations decreased, there is a significant increase in the auc of unbound drugs that can increase the pharmacological effect. in addition, estradiol and progesterone increased hepatic metabolism via xenobiotics as well as cy-p450 enzyme activity.32 evidence suggested that an increased clearance and decreased concentrations of some drugs may be the results of these physiologic changes.32 only a few studies described physiological changes that occur during canine pregnancy with limited studies during lactation. most reported hematologic and serum biochemical differences in pregnant versus nonpregnant dogs having anemia, thrombocytosis, leukocytosis, hypoalbuminemia, hypoproteinemia, and changes in cholesterol amongst other findings in late pregnancy.31 an increase in plasma volume during pregnancy in dogs has been noted,36 but an exact percentage increase has not been calculated to the authors’ knowledge. with this increase in plasma volume, increased renal blood flow and glomerular filtration rate have been observed.37 we theorized that changes in women and other species in pregnancy and lactation are similar in dogs and can explain our observations. the low detection of carprofen in the milk in our study was consistent with our predictions of low transfer from mother to milk, due to the partition theory as well as carprofen’s high percentage of protein binding in the plasma (> 98%),1 most of which is bound to albumin. the single milk sample with concentrations above the loq occurred at 12 hours after treatment in dog 1 coinciding with litter 1 which measured above the loq at that timepoint as well. this mother was the heaviest dog (23.3 kg) in our study; she was also the oldest and one of the better milk producers in the kennel having raised several litters (exact number unknown). she was of similar lactation stage as the other dogs at 22 days into milk with a similar litter size of 7 pups. without further investigation, we cannot speculate if dog size and/or milk production alone contributes to the extent of disposition of carprofen in milk and pup exposure. we were not able to accurately measure total milk ingestion in pups nor daily milk production in the dog during our study. there were only 4 timepoints in pups from 2 litters with carprofen concentrations above the loq. this agreed with our hypothesis that carprofen distribution to milk is low and exposure to nursing pups is too low to produce measurable pup plasma concentrations. most of the samples with concentrations above the loq were from litter 1. overall, pup plasma concentrations were less than 10% of maternal concentrations, consistent with our hypothesis and observation from human infant exposure.19-22 despite our small sample size, the low and unquantifiable concentrations of carprofen in milk were similar to the low concentrations reported18 after a 5-day oral treatment of carprofen; that study had slightly higher concentrations of carprofen in the milk compared to our study that may be attributed to the difference in the stage of lactation during the sampling period. at the beginning of lactation, the volume of milk produced is smaller and colostrum is excreted. therefore, larger-sized proteins will pass more readily through the mammary gland barrier in the first 24-hours as well as a higher concentration due to the smaller volume of milk produced at this time. in our study, dogs were at 2-4 weeks of lactation in which the mammary epithelial tight junctions are more restrictive and higher milk volume produces a dilution factor. other differences are the sampling and analyzing technique. we chose an intensive sampling model after a single dose of carprofen for both maternal plasma and milk along with plasma from nursing puppies, whereas once daily sampling of maternal milk over a 5-day course of oral carprofen (2 mg/kg twice per day) was chosen.18 by only collecting milk samples once a day, changes in milk concentrations (foremilk versus hindmilk and interglandular variations) may be missed that has been described in human lactation studies.38 milk samples were analyzed by liquid chromatography18 as in the current study. the main difference in analyzing techniques involved the maternal and neonatal plasma that were calculated via concentration-time course simulation models.18 versus in real time in the current study. limitations a major limitation of our study was the small sample size that resulted in high inter-individual variation among dogs. clearly, a larger study with more dogs is needed to confirm our results. aside from the small sample size, another limitation was the single carprofen treatment before steady-state conditions were met. because we used dogs recruited from client-owned kennels there were limitations for the number of doses and samples from dogs and pups. we were restricted in the volume of whole blood that could be collected for an individual pup during the sampling period for that litter. although we used an intravenous treatment that is not included in approved us label, this was the preferred route for calculating pharmacokinetic parameters such as clearance and volume of distribution. furthermore, intravenous route studies in dogs had no adverse effects in dogs.1,13 however, there was not enough data from the milk and pups to calculate pharmacokinetic parameters from these samples. despite these limitations, this is the first study to our knowledge that has measured carprofen in neonatal plasma and canine milk at multiple timepoints after maternal treatment, an advance over studies in dogs. in conclusion, carprofen treatment to lactating dogs has low transference to milk and a low risk of neonatal exposure in midlactation with an intact mammary epithelium barrier. http://dx.doi.org/10.58292/ct.v16.10491 6 citation: clinical theriogenology 2024, 16, 10491, http://dx.doi.org/10.58292/ct.v16.10491 carprofen disposition observed in lactating dogs in our study had a higher clearance rate and volume of distribution that may be attributed to the unique physiology in this group compared to nonlactating dogs. our study supported our hypothesis that carprofen is safe for lactating dogs as there is low transfer into their milk and to their pups, which is similar to reports.16,18 funding north carolina state university college of veterinary medicine intramural grants. conflicts of interest amber nebel-karp: no conflicts of interest to disclose. mark papich has received gifts, honoraria, consulting fees, and research support from zoetis, the sponsor of carprofen, but did not receive any payment or research support for the work on this study. kristen messenger has received gifts, honoraria, and research support from zoetis, but did not receive any payment or research support for the work on this study. sara lyle: no conflicts of interest to disclose. references 1. schmitt m, guentert tw: biopharmaceutical evaluation of carprofen following single intravenous, oral, and rectal doses in dogs. biopharm drug dispos 1990;11:585-594. doi: 10.1002/ bdd.2510110704 2. the united states pharmacopeial convention. (2007, january 1). carprofen (veterinary-systemic). available from: https://cdn. ymaws.com/www.aavpt.org/resource/resmgr/imported/carprofen.pdf [cited 19 january 2023]. 3. kukanich b, bidgood t, knesl o: clinical pharmacology of non-steroidal anti-inflammatory drugs in dogs. vet anesth analg 2012;39:69-90. doi: 10.1111/j.1467-2995.2011.00675.x 4. ricketts ap, lundy km, seibel sb: evaluation of selective inhibition of canine cyclooxygenase 1 and 2 by carprofen and other nonsteroidal anti-inflammatory drugs. am j vet res 1998;59: 1441-1446. doi: 10.2460/ajvr.1998.59.11.1441 5. wilson je, chandrasekharan nv, westover kd, et al: determination of expression of cyclooxygenase-1 and -2 isozymes in canine tissues and their differential sensitivity to nonsteroidal anti-inflammatory drugs. am j vet res 2004;65:820-818. doi: 10.2460/ajvr.2004.65.810 6. raekallio mr, hielm-björkman ak, kejonen j, et al: evaluation of adverse effects of long-term orally administered carprofen in dogs. j am vet med assoc 2006;228:876-880. doi: 10.2460/ javma.228.6.876 7. lascelles bdx, blikslager at, fox sm, et al: gastrointestinal tract perforation in dogs treated with a selective cyclooxygenase-2 inhibitor: 29 cases (2002–2003). j am vet med assoc 2005;7: 1112-1117. doi: 10.2460/javma.2005.227.1112 8. mckellar qa, delatour p, lees p: stereospecific pharmacokinetics and pharmacodynamics of carprofen in the dog. j vet pharmacol ther 1994;17:447-454. doi: 10.1111/j.1365-2885.1994.tb00276.x 9. rimadyl® package insert. rimadyl® (carprofen) sterile injectable solution 50 mg/ml. new york, ny; zoetis inc.: 2013. available from: https://www/zoetis.com/products/pages/rimadyldvm/docs/rimadyl_injectable_pi.pdf [cited 29 december 2022]. 10. macphail cm, lappin mr, meyer dj, et al: hepatocellular toxicosis associated with administration of carprofen in 21 dogs. j am vet med assoc 1998;212:1895-1901. doi: 10.2460/javma.1998.212.12.1895 11. lascelles bdx, mcfarland jm, swann h: guidelines for safe and effective use of nsaids in dogs. vet therap 2005;6:237-251. 12. priymenko n, garnier f, ferre jp, et al: enantioselectivity of the enterohepatic recycling of carprofen in the dog. drug metab dispos 1998;26:170-176. 13. messenger km, wofford ja, papich mg: carprofen pharmacokinetics in plasma and in control and inflamed canine tissue fluid using in vivo ultrafiltration. j vet pharmacol ther 2015;39:32-39. doi: 10.1111/jvp.12233 14. clark tp, chieffo c, huhn jc, et al: the steady state pharmacokinetics and bioequivalence of carprofen administered orally and subcutaneously in dogs. j vet pharmacol ther 2003;26:187192. doi: 10.1046/j.1365-2885.2003.00475.x 15. lipscomb vj, aliabadi fs, lees p, et al: clinical efficacy and pharmacokinetics of carprofen in the treatment of dogs with osteoarthritis. vet rec 2002;150:684-689. doi: 10.1136/vr.150.22.684 16. escabor sr, kolster ka: analgesia for canine cesarean section recovery. clinical theriogenology 2016;8:191-195. 17. matthews ka: pain management for the pregnant, lactating, and neonatal to pediatric cat and dog. vet clin north am small anim pract 2008;38:1291-1308. doi: 10.1016/j.cvsm.2008.07.001 18. ferrari d, lundgren s, holmberg j, et al: concentration of carprofen in the milk of lactating bitches after cesarean section and during inflammatory conditions.  theriogenology 2022;181: 59-68. doi: 10.1016/j.theriogenology.2022.01.016 19. national center for biotechnology information. drugs and lactation database (lactmed) ncbi bookshelf: 2011, march 28. available from: https://www.ncbi.nlm.nih.gov/books/ nbk501922/ [cited 31 december 2022]. 20. anderson po, sauberan jb: modeling drug passage into human milk. online wiley library 2016;100:42-52. doi: 10.1002/cpt.377 21. begg ej, atkinson hc: modelling of the passage of drugs into milk. pharmacol ther 1993;59:301-310. doi: 10.1016/ 0163-7258(93)90072-l 22. atkinson hc, begg ej: prediction of drug distribution into human milk from physicochemical characteristics. clin pharmacol 1990;18:151-167. doi: 10.2165/00003088-199018020-00005 23. stebler t, guentert tw: binding of drugs in milk: the role of casein in milk protein binding. pharmacol res 1990;7:633-637. doi: 10.1023/a:1015826413335 24. stelwagen k, singh k: the role of tight junctions in mammary gland function. j mammary gland biol neoplasia 2014;19: 131-138. doi: 10.1007/s10911-013-9309-1 25. ette ei, williams pj: population pharmacokinetics ii: estimation methods. ann pharm 2004;38:1907-1915. doi: 10.1345/ aph.1e259 http://dx.doi.org/10.58292/ct.v16.10491 https://doi.org/10.1002/bdd.2510110704 https://doi.org/10.1002/bdd.2510110704 https://cdn.ymaws.com/www.aavpt.org/resource/resmgr/imported/carprofen.pdf https://cdn.ymaws.com/www.aavpt.org/resource/resmgr/imported/carprofen.pdf https://cdn.ymaws.com/www.aavpt.org/resource/resmgr/imported/carprofen.pdf https://doi.org/10.1111/j.1467-2995.2011.00675.x https://doi.org/10.2460/ajvr.1998.59.11.1441 https://doi.org/10.2460/ajvr.2004.65.810 https://doi.org/10.2460/javma.228.6.876 https://doi.org/10.2460/javma.228.6.876 https://doi.org/10.2460/javma.2005.227.1112 https://doi.org/10.1111/j.1365-2885.1994.tb00276.x http://zoetis.com/products/pages/rimadyldvm/docs/rimadyl_injectable_pi.pdf http://zoetis.com/products/pages/rimadyldvm/docs/rimadyl_injectable_pi.pdf https://doi.org/10.2460/javma.1998.212.12.1895 https://doi.org/10.1111/jvp.12233 https://doi.org/10.1046/j.1365-2885.2003.00475.x https://doi.org/10.1136/vr.150.22.684 https://doi.org/10.1016/j.cvsm.2008.07.001 https://doi.org/10.1016/j.theriogenology.2022.01.016 https://www.ncbi.nlm.nih.gov/books/nbk501922/ https://www.ncbi.nlm.nih.gov/books/nbk501922/ https://doi.org/10.1002/cpt.377 https://doi.org/10.1016/0163-7258(93)90072-l https://doi.org/10.1016/0163-7258(93)90072-l https://doi.org/10.2165/00003088-199018020-00005 https://doi.org/10.1023/a:1015826413335 https://doi.org/10.1007/s10911-013-9309-1 https://doi.org/10.1345/aph.1e259 https://doi.org/10.1345/aph.1e259 citation: clinical theriogenology 2024, 16, 10491, http://dx.doi.org/10.58292/ct.v16.10491 7 26. xu jj, renner dm, lester pa: strength and sterility of stock and diluted carprofen over time. j am assoc lab anim sci 2021;60: 470-474. doi: 10.30802/aalas-jaalas-20-000102 27. united states pharmacopeia (usp-nf). usp 34-nf29. general chapter on validation of compendial methods <1225>. rockville, md; united states pharmacopeial convention: 2011. available from: https://cdn.ymaws.com/www.aavpt.org/resource/resmgr/ imported/carprofen.pdf [cited 4 february 2023]. 28. derendorf h, meibohm a: modeling of pharmacokinetic/pharmacodynamic (pk/pd) relationships: concepts and perspectives. pharmacol res 1999;16:176-185. doi: 10.1023/a:1011907920641 29. wright jd, boudinot fd, ujhelyi mr: measurement and analysis of unbound drug concentrations. clin pharmacol 1996;30: 445-462. doi: 10.2165/00003088-199630060-00003 30. bidgood t, papich mg: plasma pharmacokinetics and tissue fluid concentrations of meropenem after intravenous and subcutaneous administration in dogs. am j vet res 2002;63:1622-1628. doi: 10.2460/ajvr.2002.63.1622 31. kimura t, kotani k: perinatal veterinary medicine-related evaluation in hematological and serum biochemical profiles of experimental beagles throughout pregnancy and parturition. animal model exp med 2018;1:282-294. doi: 10.1002/ame2.12043 32. jeong h: altered drug metabolism during pregnancy: hormonal regulation of drug-metabolizing enzymes. expert opin drug metab toxicol 2010;6:689-699. doi: 10.1517/17425251003677755 33. lascelles bdx, cripps pj, jones a, et al: efficacy and kinetics of carprofen, administered preoperatively or postoperatively, for the prevention of pain in dogs undergoing ovariohysterectomy. vet surg 1998;27:568-582. doi: 10.1111/j.1532-950x.1998. tb00533.x 34. johnston sd, root kustritz mv, olson ps: canine and feline theriogenology. 1st edition, philadelphia; saunders: 2001. p. 139. 35. oukessou m, benlamith s: benzylpenicillin kinetics in the ewe: influence of pregnancy and lactation. res vet sci 1990;49: 190-193. doi: 10.1016/s0034-5288(18)31075-0 36. johnston sd, root kustritz mv, olson ps: canine and feline theriogenology. 1st edition, philadelphia; saunders: 2001. p. 71-72. 37. messenger km, papich mg: the influence of cefovecin on the enantioselective pharmacokinetics of carprofen in dogs. j vet intern med 2013;27:749. 38. ling b, alcorn j: lactation stage influences drug milk-to-serum values and neonatal exposure risk. int j toxicol 2010;29:411-417. doi: 10.1177/1091581810367949 http://dx.doi.org/10.58292/ct.v16.10491 https://doi.org/10.30802/aalas-jaalas-20-000102 https://cdn.ymaws.com/www.aavpt.org/resource/resmgr/imported/carprofen.pdf https://cdn.ymaws.com/www.aavpt.org/resource/resmgr/imported/carprofen.pdf https://doi.org/10.1023/a:1011907920641 https://doi.org/10.2165/00003088-199630060-00003 https://doi.org/10.2460/ajvr.2002.63.1622 https://doi.org/10.1002/ame2.12043 https://doi.org/10.1517/17425251003677755 https://doi.org/10.1111/j.1532-950x.1998.tb00533.x https://doi.org/10.1111/j.1532-950x.1998.tb00533.x https://doi.org/10.1016/s0034-5288(18)31075-0 https://doi.org/10.1177/1091581810367949 2015: use of hormones in equine breeding management use of hormones in equine breeding management carlos r. pinto,a elizabeth a. coffmanb adepartment of veterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la; bdepartment of veterinary clinical sciences, college of veterinary medicine, north carolina state university, raleigh, nc introduction the administration of hormones to mares during breeding management is an essential tool for equine practitioners. proper and timely administration of specific hormones to broodmares may be targeted: 1) to prevent reproductive disorders, 2) to aid in treating reproductive disorders or hormonal imbalances, and 3) to optimize reproductive efficiency, for example, induction of estrus or ovulation. the most common applications of exogenous hormone administration include controlling the duration and onset of the different stages of the estrous cycle (specifically by affecting duration of luteal function or hastening ovulation) and stimulating myometrial activity in mares susceptible to or showing delayed uterine clearance. in this discussion, we will address the effects and potential indications for the different hormones available to the equine practitioner working with broodmare reproduction. keywords: pgf2α, hcg, gnrh, mare, corticosteroids prostaglandin f2α (pgf2α) the administration of natural or synthetic pgf2α analogues causes interruption of luteal function by inducing luteolysis. the diestrus stage of the estrous cycle is then shortened, hastening onset of estrus. characteristics of estrus and ovulation (e.g., estrus behavior, follicle growth rate, ovulation, fertility) are not different from those occurring in natural cycles.1-3 however, there is some evidence of increased incidence of hemorrhagic anovulatory follicles (haf) following exogenous administration of the synthetic pgf2α analogue cloprostenol.4-6 the potential association between pgf2α administration and the incidence of haf has not been reported in mares treated with the natural analogue dinoprost tromethamine. duration of diestrus and interovulatory intervals are shortened following pgf2α administration.7-18 the equine corpus luteum (cl) is responsive to pgf2α luteolytic effects any day following ovulation; however, only cl >5 days are consistently responsive to the luteolytic effects of one bolus injection of pgf2α.15, 19 luteolysis or antiluteogenesis can be reliably achieved in cl <5 days only if multiple pgf treatments are administered.14,15,20 for that reason, it is widespread practice to administer pgf2α as a single bolus injection (sc or im) at least five to six days following ovulation. this common practice perhaps contributed to the prevailing assumption that pgf2α treatments only have significant effects in mares with cl >5 days old. mares with a cl >5 days old undergo luteolysis when treated with a single injection (sc or im) of 5 to 10 mg of dinoprost tromethamine or 250 to 500 ug of cloprostenol (synthetic pgf2α analogue). in the usa, dinoprost tromethamine* is the only pgf2α drug approved by the food and drug administration (fda) for use in horses although the use of the racemic formulation of d,l cloprostenol† is fairly common among practitioners working with horse breeding, especially owing to its longer half-life than dinoprost tromethamine salt preparations, hours vs. minutes, respectively.21 conversely, in other countries in europe, and south america for example, formulations of the synthetic analogue cloprostenol are approved and available for use in horses. in those countries, the racemic d,l cloprostenol and the more potent formulation containing only the d-enantiomer cloprostenol‡ are available; only 25 to 37.5 ug per mare of d-cloprostenol is needed to induce luteolysis in mares at least five days after ovulation.18 induction of ovulation the ability to induce ovulation is one of the most important strategies utilized in breeding management. ovulation-inducing agents typically trigger ovulation within 48 hours. in mares monitored closely via palpation per rectum and ultrasonography, ovulation occurs mainly within 36 to 48 hours after systemic administration of these agents.22,23 this fact is particularly important for mares being managed clinical theriogenology • volume 7, number 1 • march 201545 for artificial insemination with shipped cooled semen or frozen-thawed semen. for optimal chances to predict outcome and increase efficacy of the treatment, regardless of the ovulation-inducing agent being used, induction of ovulation should be attempted only in mares found unquestionably to be in estrus and with a growing follicle ≥30 mm in diameter. the most common hormones used to induce ovulation include human chorionic gonadotropin (hcg) and the gonadotropin-releasing hormone agonists (gnrh), which will be discussed below. human chorionic gonadotropin human chorionic gonadotropin§ has been widely and extensively used as an ovulation-inducing agent since the 1930’s and 1940’s.24-27 the luteinizing hormone (lh)-like activity of hcg is explained by its 85% homology in amino acid sequence in the β-subunit and by its ability to bind to the lh receptor.28 chemically, hcg is a glycoprotein composed of an α and a β subunit; the α-subunit is similar to that of human follicle stimulating hormone (fsh) and lh; however, the hcg β-subunit, despite its homology with the lh β-subunit, does have an additional carboxyl-terminal group with 31 amino acids and a high content of sialic acid that provides a substrate for glycosylation. glycosylation is the posttranslational modification of the hcg molecule that confers it a longer half-life (several hours) than that of lh (20 to 30 minutes). for many years, the hormone hcg was only produced after being extracted from urine of pregnant women, lyophilized and marketed in the usa in vials containing 10,000 international units (iu) available for clinical use in human and veterinary medicine. since the early 2000’s a recombinant form of hcg produced from genetically modified chinese hamster ovary cells is also available commercially in the usa for treatment of women undergoing fertility treatments. there are no reports of use of the recombinant hcg in the horse. despite reports of mares being treated with hcg since 1939,26 hcg is not approved by the fda for use in horses. nevertheless, a single bolus dose of 750 to 3300 iu of urinary hcg is typically sufficient to induce ovulation in mares during estrus, with distinct estrous uterine edema and with a viable, growing follicle >30 mm.29,30 mares with more than one presumptive preovulatory follicle do not need a double dose or a second injection during that estrus. in our experience, mares that repeatedly ovulate two or three follicles respond to a single hcg treatment similarly to mares ovulating only one follicle during estrus. because hcg is available in 10,000 iu vials, it is common for equine practitioners to administer bolus doses of 2,000 or 2,500 iu to optimize the use of each vial. although the authors have successfully used vials that have been reconstituted for several weeks, it is recommended that reconstituted vials be used within two weeks and kept refrigerated during that period. alternatively, unused reconstituted hcg can be frozen in separate doses for future use as repeated freezing-thawing reconstituted vials is not recommended. intramuscular or intravenous routes can be used to administer hcg in mares but it seems, anecdotally, that fewer ovulation failures are seen in mares receiving hcg intravenously than in mares receiving it intramuscularly. for example, intramuscular doses of 5,000 to 10,000 iu should be avoided. mares do develop antibodies to the human hormone but the presence of antibodies does not seem to interfere with ovulation.31 nevertheless some authors do recommend not treating mares with hcg more than once or twice during the same breeding season33 whereas others (including the current authors) have used it successfully (ovulation within 48 hours from administration) in mares treated four to seven times during the same breeding season. gonadotropin-releasing hormones administrations of simple (relative potency = 1) gnrh analogues (equivalent to the natural decapeptide gnrh) to cycling mares have failed to consistently induce ovulation. whereas doses of 50 to 100 ug of gnrh are sufficient to induce ovulation in cattle, doses as high as 1 to 4 mg of gnrh have failed to reliably induce ovulation in mares when administered once or twice daily during estrus. furthermore, only pulsatile administration (every hour) of gnrh has been found to be effective in inducing ovulation in the mare;33 this method is, however, not feasible for widespread clinical application. since the chemical structure and peptide identification was reported in 1971,34-36 more than 2,000 gnrh analogues were synthesized in the following 15 years.37 scientists working in research and private pharmaceutical institutions shared two main aims: (1) to create an analogue with high affinity for gnrh clinical theriogenology • volume 7, number 1 • march 2015 46 receptor binding; and (2) to avoid or minimize enzymatic degradation by proteolysis. the gnrh molecule is susceptible to cleavage and inactivation by hypophyseal endopeptidases especially between amino acids in position 5 and 6, and between 6 and 7. another enzyme, carboxypeptidase targets the bond between amino acids in positions 9 and 10. for these reasons, several gnrh synthetic analogues used in human and veterinary medicine have 1) d-amino acids substitutions in position 6 (glycine), and 2) the amino acid in position 10 (another glycine) and its amide terminal replaced by an ethylamide residue that is linked to the aminoacid 9 proline.38 deslorelin. in contrast with the poor or limited efficacy of simple gnrh analogues, more potent gnrh synthetic analogues such as deslorelin have a predictable effect in inducing ovulation within 48 hours when administered as intramuscular injections23,39 or subcutaneous implants.40-42 deslorelin differs from the natural decapeptide gnrh in two amino acid substitutions: in amino acid position 6 where lglycine has been replaced with the aminoacid d-tryptophan and in position 10 where glycine and its amino terminal have been replaced by n-ethylamide. ovulation rates following deslorelin administration are comparable with those resulting from hcg administration.41 commercially, deslorelin became available as implants containing 2.1 mg of deslorelin acetate.¶ after being first available in australia and other countries, ovuplant® became fdaapproved and available in the usa in 1998. after being on the market for approximately two breeding seasons, it was anecdotally reported that, occasionally, mares treated with the implant had prolonged interovulatory intervals and some actually had their reproductive cyclicity downregulated for months; this effect was confirmed in a controlled study.43 when investigating this issue, mccue et al reported normal interovulatory intervals in mares that had their implant removed once ovulation occurred.44 this phenomenon was later elucidated by the fact that prolonged delivery of deslorelin would induce ovulation but its continuous release would down regulate fsh and lh secretion from the pituitary,45 resulting in prolonged interestrous intervals. removing the implant after ovulation thus prevented sustained release of deslorelin. reportedly owing to problems in manufacturing logistics, ovuplant® has not been commercially available in the usa since the early 2000’s. interestingly, side effects related to sustained deslorelin release have not been reported in australia, where the drug originated and still remains available (as well as in other countries). for several years, deslorelin was available either via importation from countries that manufactured ovuplant® or via compounding of injectable formulations of deslorelin (1.5 mg/ ml; dose 1 ml per mare administered intramuscularly). recently, an injectable controlled release form of deslorelin acetate¶ (sucromatetm;1.8 mg/ml) was approved by the fda in 2010 for induction of ovulation in mares and became commercially available in the usa in 2011. sucromatetm is a product of and manufactured by thorn bioscience, louisville, ky, a division of creosalus and it is marketed by bioniche animal health usa, inc., athens, ga. it contains 1.8 mg of deslorelin acetate equivalent to 1.7 mg of deslorelin in a sucrose acetate isobutyrate and propylene carbonate matrix that promotes controlled release of the drug. this formulation has been shown to induce ovulation at comparable rates of those from hcg or deslorelin implant administrations. in the only published peer-reviewed report on the efficacy of this fda-approved deslorelin injectable formulation, 151 out of 168 (~ 90%) mares treated in estrus with sucromatetm (1 ml, i.m.) ovulated on average 41 hours after treatment whereas, 111 out of 134 mares (~83%) treated with hcg (2,500 iu, i.v.) ovulated on average 44 hours after treatment.39 histrelin. another synthetic gnrh analogue, histrelin (or historelin) is available in the usa from compounding pharmacies. histrelin is an even more potent gnrh analogue than deslorelin (table), but with similar clinical efficacy in inducing ovulation in horses as deslorelin or hcg.46,47 histrelin differs from the natural decapeptide gnrh in two amino acid substitutions: in amino acid position 6 where l-glycine has been replaced with the amino acid d-histidine and in position 10 where glycine and its amino terminal have been replaced by n-ethylamide. clinical theriogenology • volume 7, number 1 • march 201547 table. the comparative rank order potency (affinity for gnrh receptor) of synthetic gnrh analogues in relation to natural gnrh gnrh buserelin deslorelin histrelin relative potency 1 20 144 210 recently, a sustained release (biorelease) formulation histrelin was found to effectively induce ovulation within two days following administration.47 doses of 0.25-1.0 mg of a sustained release (biorelease) formulation histrelin effectively induce ovulation within two days following administration.47-49 in a more recent study,48 there were no differences among maiden, barren and foaling mares treated throughout the ovulatory seasons; similarly there were no significant differences for the two doses used in that study, 0.25 and 0.5 mg, administered as single intramuscular bolus injections to mares in estrus. the overall ovulation rate at 48 hours was 91% (42/46) and 85% (44/52) for doses of 0.5 mg and 0.25 mg of histrelin, respectively. in summary, there does not appear to be differences in the efficacy, relative to ability to induce ovulation, among deslorelin, histrelin and hcg. buserelin. buserelin is another gnrh analogue that also contains modification in the amino acid position 6 by having the natural occurring l-glycine replaced by a tertiary butylated d-serine and the amino acid 10 glycine and its amino terminal replaced by n-ethylamide, which is similar to the amino terminal modification present in deslorelin and histrelin. the moderate potency of buserelin (see table) perhaps may explain why a single administration of a labeled dose of buserelin (40 to 100 ug per mare) is not sufficient to induce ovulation in mares. for many years, buserelin (not available in the usa, except via compounding pharmacies) has had limited use and efficacy in inducing ovulation only when used in daily or twice daily injections for two to four days.49,50 this protocol likely has prevented veterinarians and horse owners from favoring its use in brood mare management. in contrast with this knowledge, levy and duchamp recently showed that much higher doses than those originally used resulted in ovulation rates comparable to those of hcg.51 in that study, 6 mg of buserelin were injected subcutaneously to cyclic mares, resulting in 89% to 95% ovulation rates that did not differ from hcg administration (86%). considering the relative potencies of deslorelin, histrelin and buserelin (table), we calculate that buserelin is at least 7 and 10 times less potent than deslorelin and histrelin, respectively. this may partially explain why in this study a buserelin dose ≈ 60 times greater (6000 ug vs. 100 ug) than that recommended in commercial preparations of buserelin (receptal) was able to effectively promote ovulation in treated mares at a similar rate reported for hcg, deslorelin and histrelin. ecbolic hormones for treatment of delayed uterine clearance in general, the use of ecbolic hormones to correct deficiencies or weakness in myometrial contractions commonly seen in mares with delayed uterine clearance and susceptible to endometritis are safely initiated at four to eight hours following artificial or natural insemination to avoid interferences with sperm transport that could potentially affect fertility.52,53 these treatments are then continued at the discretion of the prescribing veterinarian; ecbolics are often administered twice daily or more often if necessary and depending on the recorded response for an individual mare. commonly used ecbolics in broodmares include oxytocin and prostaglandin-f2α. oxytocin oxytocin is a nonapeptide synthetized in the magnocelullar neurons in the hypothalamus that extend their axons into the neurohypohysis where it is stored until released into the blood stream for biological action in the mare. oxytocin has several known physiologic effects, such as milk ejection, promotion of maternal behavior, and induction of myometrial contractions. because delayed uterine clearance is commonly seen in mares experiencing persistent mating-induced endometritis, oxytocin is one important therapeutic option for treating abnormal intrauterine accumulation of fluid. despite clinical theriogenology • volume 7, number 1 • march 2015 48 oxytocin’s relatively short-life of ≈ 7 minutes,54 it seems that its biological effect far exceeds this time. for that reason, treatment protocols varying from two to four daily treatments have been shown to be relatively efficacious in promoting uterine clearance. boluses doses of 10 to 20 iu administered subcutaneously, intramuscularly or intravenously are frequently used to treat mares with mating-induced intrauterine fluid accumulation. oxytocin treatments are administered during estrus before or after ovulation, with treatments scheduled to not coincide with breeding to avoid interfering with sperm transport, especially if given shortly after artificial insemination, i.e., < 4 hours.55 recently, our laboratory reported on the pharmacokinetics of carbetocin, a long-acting oxytocin analogue.56 carbetocin has a half-life of ≈ 17 minutes. carbetocin is available and approved for use in horses in several countries but not in the usa. despite its longer half-life than oxytocin, multiple daily treatments may still be required for optimal effects in promoting uterine clearance as is required with oxytocin or pgf2α. a direct comparison between the ability of oxytocin and carbetocin to promote uterine clearance has not been reported for in vivo treatment in mares. currently, it is not known whether the reported prolonged half-life of carbetocin would translate into a greater and/or more productive biologic response than oxytocin in mares that may require prolonged myometrial stimulation. steckler et al have recently reported on the results of a comparison of oxytocin vs. carbetocin in eliciting contractions in ex vivo uterine tissues collected from mares at different reproductive stages (e.g., anestrus, estrus and diestrus).57 the results indicated that the effect of oxytocin on equine myometrial tissue was greater during anestrus and diestrus than during estrus, whereas carbetocin seemed to elicit myometrial contractions independent of the reproductive status; however, there were no differences between their ability to induce myometrial contractions in the ex vivo strips of myometrial tissues. prostaglandin f2α in addition to being known for its luteolytic actions, pgf2α is also an effective promoter of myometrial contractions. owing to its longer half-life than oxytocin, especially the synthetic pgf2α analogue cloprostenol (≈ 2-3 hours), pgf2α has been favored by equine practitioners, especially for use in mares that anecdotally seem refractory or do not respond appropriately to oxytocin treatment. cloprostenol does elicit less vigorous myometrial contractions than oxytocin but for prolonged periods. for example, one study found that oxytocin induced strong myometrial contractions that lasted approximately one hour; whereas, cloprostenol induced low-amplitude myometrial contractions that lasted four to five hours.59 this sustained effect is particularly desirable to enhance lymph flow and consistent intraluminal evacuation of accumulated fluid (inflammatory transudate). despite its indisputable efficacy as an ecbolic hormone, the use of pgf2α during estrus, especially in the early postovulatory period, was found to transiently affect luteal function followed by resurgence in cl function.59,60 however, these studies had conflicting results on the effect of such treatment on the resulting fertility of treated mares. for this reason, if pgf2α is to be used as an ecbolic to treat delayed clearance during pre-ovulatory estrus and early postovulatory period, it is important that equine practitioners remain cognizant of its effects on luteal function. most mares so treated should experience resurgence in luteal function61 and may not suffer deleterious effects on their long-term luteal function and subsequent fertility. moreover, our laboratory has recently reported that the effect of pgf2α administration on early luteal function can induce luteolysis or antiluteogenesis, depending on the duration, timing, and dosage utilized.15,20 for these reasons, treatments with pgf2α during estrus should not continue after ovulation. blood sampling for monitoring of progesterone secretion and supplementation with exogenous progestagens may be considered during that period. modulation of mating-induced endometritis with steroids endometritis following artificial insemination or mating is one important cause of infertility especially in mares susceptible to persistent mating-induced endometritis. in addition to therapies aiming at combating endometritis by utilization of intrauterine or systemic antibiotic treatment and stimulation of myometrial function via administration of ecbolic hormones (mainly oxytocin and pgf2α), another strategy is to control or modulate the exacerbated inflammation response seen in these mares. dell’aqua clinical theriogenology • volume 7, number 1 • march 201549 et al first reported on the efficacy of prednisolone treatment of mares during estrus that benefited from the glucocorticoid modulatory effects on barren mares afflicted with endometritis.62 a significantly higher pregnancy rate was seen in barren mares treated five times with 0.1 mg/kg of prednisolone acetate (≈ 50 mg per mare) administered every 12 hours with four treatments occurring before artificial insemination and one at the time of insemination. more recently, bucca et al reported the use dexamethasone given as a single bolus administration of 50 mg per mare at time of breeding.63 in that study, the authors concluded that dexamethasone administered at the time of artificial insemination was safe and effective for modulating persistent mating-induced endometritis in susceptible mares; the glucocorticoid treatment decreased endometrial edema, accumulation of intraluminal uterine fluid and its turbidity, without altering the amount of polymorphonuclear cells seen in preparations obtained from endometrial cytology and increased fertility in mares displaying multiple risk factors for developing post-breeding inflammation. ferris and mccue studied the effects of multiple (twice daily for five days) glucocorticoid treatments (prednisolone or dexamethasone) in mares during early estrus.64 they reported that mares treated with dexamethasone showed reduced uterine edema and ovulation rate (40% vs. 83%, respectively) than mares treated with prednisolone. in addition, only two out five dexamethasone-treated mares had unaltered lh hormonal profiles, whereas five out of six mares treated with prednisolone had lh surges within normal limits. the differences noted between dexamethasone and prednisolone may derive from their differences in relative potencies in relation to cortisol (dexamethasone = 30; prednisolone = 4). nevertheless, it is unlikely that a single treatment with dexamethasone as prescribed by bucca and carli would result in ovulation failure as only mares with multiple, daily treatments with dexamethasone have been shown to have altered endogenous lh release that resulted in significant ovulation failures.65 it is important, however, to ensure that mares being treated with glucocorticoids during estrus are also treated with ovulation-inducing agents to prevent any potential negative effects of glucorticoid agents on the hypothalamic-hypophyseal axis that may result in anovulation. bucca et al provided supporting evidence to the efficacy of associating induction of ovulation with glucocorticoid treatment in mares susceptible to persistent post-mating endometritis.65 mares treated with a single bolus intravenous injection of 50 mg of dexamethasone within one hour of breeding and concurrent induction of ovulation with 1,500 iu of hcg exhibited normal ovulation rates (≈ 97% of mares ovulated within 48 hours). the minimal dose of prednisolone and dexamethasone to combat inflammation in mares during estrus has yet to be determined. conclusions several pharmacologic agents are now available for use in the breeding management of broodmares. shortening of the diestrus stage and induction of estrus with pgf2α can be routinely achieved at virtually any stage of the cycle. strategic utilization of hormones to induce estrus and ovulation and to modulate/prevent inflammatory processes in mares susceptible to mating-induced endometritis and delayed uterine clearance can significantly impact the outcome of breedings by increasing the odds of timed ovulation and pregnancy. footnotes * dinoprost tromethamine; lutalyse®; pfizer animal health, kalamazoo, mi † d,l cloprostenol sodium; estrumate®; merck animal health, union, nj ‡ d, cloprostenol; genestran®; forte healthcare limited; naul, dublin, republic of ireland § human chorionic gonadotropin; chorulon®; merck animal health, millsboro, de ¶ deslorelin acetate; sucromatetm equine; manufactured by thorn bioscience, louisville, ky; marketed by bioniche animal health usa, inc., athens, ga references 1. allen wr, cooper mj: use of synthetic analogues of prostaglandins for inducing luteolysis in mares. ann biol anim bioch 1975;15:461-469. 2. nelson am: the therapeutic activity, post-treatment fertility and safety of prostaglandin f2alpha-tham salt in clinically anoestrous mares: a review. equine vet j 1976;8:75-77. 3. burns sj, irvine ch, amoss ms: fertility of prostaglandin-induced oestrus compared to normal post-partum oestrus. j reprod fertil suppl 1979; 27:245-250. clinical theriogenology • volume 7, number 1 • march 2015 50 4. ginther oj, jacob jc, gastal mo, et al: follicle and systemic hormone interrelationships during spontaneous and ablation-induced ovulatory waves in mares. anim reprod sci 2008;106:181-187. 5. ginther oj, al-mamun m: increased frequency of double ovulations after induction of luteolysis with exogenous prostaglandin f2 alpha. j equine vet sci 2009;29:581-583. 6. cuervo-arango j, newcombe jr: the effect of hormone treatments (hcg and cloprostenol) and season on the incidence of hemorrhagic anovulatory follicles in the mare: a field study. theriogenology 2009;72:1262-1267. 7. oxender wd, noden pa, louis tm, et al: a review of prostaglandin f2alpha for ovulation control in cows and mares. am j vet res 1974;35:997-1001. 8. douglas rh, ginther oj: effect of prostaglandin f2alpha on length of diestrus in mares. prostaglandins 1972;2:265268. 9. douglas rh, ginther oj: route of prostaglandin f2alpha injection and luteolysis in mares (38519). proc soc exp biol med 1975;148:263-269. 10. kiefer bl, roser jf, evans jw, et al: progesterone patterns observed with multiple injections of a pgf-2 alpha analogue in the cyclic mare. j reprod fertil suppl 1979; 27:237-244. 11. gastal el, rodrigues bl, gastal mo, et al: responsiveness of the early corpus luteum to pgf2 alpha and resulting progesterone, lh, and fsh interrelationships in mares. anim reprod 2005;2:240-249. 12. ginther oj, gastal el, gastal mo, et al: effect of prostaglandin f2alpha on ovarian, adrenal, and pituitary hormones and on luteal blood flow in mares. domest anim endocrinol 2007;32:315-328. 13. utt md, acosta tj, wiltbank mc, et al: acute effects of prostaglandin f-2alpha on systemic oxytocin and progesterone concentrations during the midor late-luteal phase in mares. anim reprod sci 2007;97:63-73. 14. holland be, pinto crf: luteal function and ovulation in mares treated with pgf2alpha during early and mid-diestrus. reprod domest anim 2008;43:111. 15. rubio c, pinto cr, holland be, et al: anti-luteogenic and luteolytic effects of pgf2a during the post-ovulatory period in mares. theriogenology 2008;70:587. 16. samper jc: induction of estrus and ovulation: why some mares respond and others do not. theriogenology 2008;70:445-447. 17. ginther oj, siddiqui ma, beg ma: physiologic and nonphysiologic effects of exogenous prostaglandin f2alpha on reproductive hormones in mares. theriogenology 2009;72:417-424. 18. cuervo-arango j, newcombe jr: relationship between dose of cloprostenol and age of corpus luteum on the luteolytic response of early dioestrous mares: a field study. reprod domest anim 2012;47:660-665. 19. barker c, echeverria k, davis m, et al: effects of different doses of pgf2alpha on luteal function and on the subsequent estrous cycle. anim reprod sci 2006;94:207-209. 20. coffman ea, pinto cr, snyder hk, et al: antiluteogenic effects of serial prostaglandin f administration in cycling mares. theriogenology 2014;82:1241-1245. 21. shrestha hk, beg ma, burnette rr, et al: plasma clearance and half-life of prostaglandin f2alpha: a comparison between mares and heifers. biol reprod 2012;87:18. 22. mccue pm, hudson jj, bruemmer je, et al: efficacy of hcg at inducing ovulation: a new look at an old issue. proc am assoc equine pract; 2004. 23. mccue pm, magee c, gee ek: comparison of compounded deslorelin and hcg for induction of ovulation in mares. j equine vet sci 2007;27:58-61. 24. day ft: clinical and experimental observations on reproduction in the mare. j agr sci. 1940;30:244-261. 25. katzman pa, doisy ea: preparation of extracts of the anterior pituitary-like substance of urine of pregnancy. j biol chem 1932;98:739-754. 26. day ft: ovulation and the descent of the ovum in the fallopian tube of the mare after treatment with gonadotrophic hormones. j agr sci 1939;29:459-469. 27. hamilton wj, day ft: cleavage stages of the ova of the horse, with notes on ovulation. j anat 1945;79:127-130. 28. ascoli m, segaloff dl: on the structure of the luteinizing-hormone chorionic-gonadotropin receptor. endocrinol rev 1989;10:27-44. 29. bollwein h, braun j: follicular dynamics after treatment with hcg for ovulation induction in mares. tierarztl prax ausg g grosstiere nutztiere 1999;27:47-51. 30. morel mcgd, newcombe jr: the efficacy of different hcg dose rates and the effect of hcg treatment on ovarian activity: ovulation, multiple ovulation, pregnancy, multiple pregnancy, synchrony of multiple ovulation; in the mare. anim reprod sci 2008;109:189-199. 31. roser jf, kiefer bl, evans jw, et al: the development of antibodies to human chorionic gonadotrophin following its repeated injection in the cyclic mare. j reprod fertil suppl 1979;27:173-179. 32. chavatte p, palmer e: induction of ovulation in the mare. equine vet educ 1998;10:26-30. 33. johnson al: pulsatile administration of gonadotropin-releasing-hormone advances ovulation in cycling mares. biol reprod 1986;35:1123-1130. 34. burgus r, butcher m, ling n, et al: [molecular structure of the hypothalamic factor (lrf) of ovine origin monitoring the secretion of pituitary gonadotropic hormone of luteinization (lh)]. comptes rendus hebdomadaires des seances de l academie des scienc 1971;273:1611-1613. clinical theriogenology • volume 7, number 1 • march 201551 35. matsuo h, baba y, nair rm, et al: structure of the porcine lhand fsh-releasing hormone. i. the proposed amino acid sequence. biochem biophys res commun 1971;43:1334-1339. 36. nair rm, kastin aj, schally av: isolation and structure of hypothalamic msh release-inhibition hormone. biochem biophys res commun 1971;43:1376-1381. 37. padula am: gnrh analogues agonists and antagonists. anim reprod sci 2005;88:115-126. 38. andreyko jl, marshall la, dumesic da, et al: therapeutic uses of gonadotropin-releasing hormone analogs. obstet gynecol surv 1987;42:1-21. 39. ferris ra, hatzel jn, lindholm arg, et al: efficacy of deslorelin acetate (sucromate) on induction of ovulation in american quarter horse mares. j equine vet sci 2012;32:285-288. 40. mckinnon ao, nobelius am, figueroa std, et al: predictable ovulation in mares treated with an implant of the gnrh analog deslorelin. equine vet j 1993;25:321-323. 41. meinert c, silva jf, kroetz i, et al: advancing the time of ovulation in the mare with a short-term implant releasing the gnrh analogue deslorelin. equine vet j 1993;25:65-68. 42. jochle w, trigg te: control of ovulation in the mare with ovuplanttm a short-term release implant (sti) containing the gnrh analog deslorelin acetate studies from 1990 to 1994. j equine vet sci 1994;14:632-644. 43. johnson ca, thompson dl, kulinski km, et al: prolonged interovulatory interval and hormonal changes in mares following the use of ovuplanttm to hasten ovulation. j equine vet sci 2000;20:331-336. 44. mccue pm, farquhar vj, carnevale em, et al: removal of deslorelin (ovuplant) implant 48 h after administration results in normal interovulatory intervals in mares. theriogenology 2002;58:865-870. 45. johnson ca, thompson dl, cartmill ja: pituitary responsiveness to gnrh in mares following deslorelin acetate implantation to hasten ovulation. j anim sci 2002;80:2681-7. 46. voge jl, sudderth ak, brinkso sp, et al: comparison of the efficacy of two dose rates of histrelin to human chorionic gonadotropin for inducing ovulation in broodmares. j equine vet sci 2012;32:208-210. 47. lindholm arg, ferris ra, scofield rb, et al: comparison of deslorelin and histrelin for induction of ovulation in mares. j equine vet sci 2011;31:312-313. 48. kiser am, sudderth ak, brinsko sp, et al: comparison of efficacy of two dose rates of histrelin for inducing ovulation in broodmares. j equine vet sci 2013;33:820-822. 49. barrier-battut i, le poutre n, trocherie e, et al: use of buserelin to induce ovulation in the cyclic mare. theriogenology 2001;55:1679-1695. 50. harrison la, squires el, mckinnon ao: comparison of hcg, buserelin and luprostiol for induction of ovulation in cycling mares. j equine vet sci 1991;11:163-166. 51. levy i, duchamp g: a single subcutaneous administration of buserelin induces ovulation in the mare: field data. reprod domest anim 2007;42:550-554. 52. brinsko sp, varner dd, blanchard tl: the effect of uterine lavage performed 4 hours post insemination on pregnancy rate in mares. theriogenology 1991;35:1111-1119. 53. troedsson mht, liu ikm, crabo bg: sperm transport and survival in the mare: a review. theriogenology 1998;50:807-818. 54. paccamonti dl, pycock jf, taverne ma, et al: pgfm response to exogenous oxytocin and determination of the halflife of oxytocin in nonpregnant mares. equine vet j 1999;31:285-288. 55. bader h: an investigation of sperm migration into the oviducts of the mare. j reprod fertil suppl 1982;32:59-64. 56. schramme ar, pinto cr, davis j, et al: pharmacokinetics of carbetocin, a long-acting oxytocin analogue, following intravenous administration in horses. equine vet j 2008;40:658-661. 57. steckler d, naidoo v, gerber d, et al: ex vivo influence of carbetocin on equine myometrial muscles and comparison with oxytocin. theriogenology 2012;78:502-509. 58. troedsson mht, liu ikm: managing mares with chronic uterine inflammation. j equine vet sci 1995;15:305-306. 59. troedsson mh, ababneh mm, ohlgren af, et al: effect of periovulatory prostaglandin f2alpha on pregnancy rates and luteal function in the mare. theriogenology 2001;55:1891-899. 60. nie gj, johnson ke, wenzel jg, et al: luteal function in mares following administration of oxytocin, cloprostenol or saline on day 0, 1 or 2 post-ovulation. theriogenology 2003;60:1119-1125. 61. bergfelt dr, pierson ra, ginther oj: regression and resurgence of the cl following pgf2alpha treatment 3 days after ovulation in mares. theriogenology 2006;65:1605-1619. 62. dell'aqua ja, papa fo, lopes md, et al: modulation of acute uterine inflammatory response after artificial insemiation with equine frozen semen. anim reprod sci 2006;94:270-273. 63. bucca s, carli a, buckley t, et al: the use of dexamethasone administered to mares at breeding time in the modulation of persistent mating induced endometritis. theriogenology 2008;70:1093-1100. 64. ferris ra, mccue pm: the effects of dexamethasone and prednisolone on pituitary and ovarian function in the mare. equine vet j 2010;42:438-443. 65. bucca s, carli a: efficacy of human chorionic gonadotropin to induce ovulation in the mare, when associated with a single dose of dexamethasone administered at breeding time. equine vet j suppl 2011;40:32-34. clinical theriogenology • volume 7, number 1 • march 2015 52 clinical assessment and treatment of infertility in a greater one horned rhinoceros clinical assessment and treatment of infertility in a greater one horned rhinoceros monica stoops,a,b anneke moresco,a scott larsena adenver zoo, denver, co, bcincinnati zoo & botanical garden center for conservation and research of endangered wildlife, cincinnati, oh a 7 year old, nulliparous greater one-horned rhinoceros (rhinoceros unicornis) was housed with a younger male without breeding success. under full immobilization (3.5 mg etorphine, 100 mg azaperone and 15 mg detomidine), a complete reproductive system examination was done and the hymen broken. during the next 4 years, under standing sedation (combinations of 100 150 mg of butorphanol and 12.5 -100 mg of midazolam, with either 4 5 mg medetomidine or 125 150 mg of azaperone), artificial insemination (ai) was performed 10 times with frozen-thawed sperm from different males. estrous cycle monitoring and ai follow up was based on analysis of urinary estrogen and progesterone metabolite concentrations. ovulation was successfully induced with 1 im injection of 3.6 mg of deslorelin (sucromate™, thornbio science, louisville, ky). last year, the female was trained for voluntary transrectal ultrasonography (evo, curved linear array 3 6 mhz). ovarian dynamics and uterine appearance were monitored 2 times/week and urinary hormones were measured 3 times/week. a follicle that attained 21 cm and had cloudy follicular fluid was designated an anovulatory follicle additionally, significant persistent luminal fluid and moderate endometrial hyperplasia were present. ovulation was induced on the day of ai and based on confirmation by urinary hormone concentrations, occurred 48 hours later. oxytocin (25 usp) was given 1 day before and 1 day after ai. ultrasonography confirmed lack of intraluminal fluid and no accumulation. urine creatinine concentrations were increased (attributed to exogenous oxytocin) 1 day after each oxytocin injection. keywords: rhinoceros, gnrh, oxytocin, infertility, subfertility 497 clinical theriogenology • volume 11, number 3 • september 2019 498clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2018: hemorrhagic anovulatory follicle in a tropical jenny   hemorrhagic anovulatory follicle in a tropical jenny e. hughes, r. ambrosia, r.o. gilbert, h. french ross university school of veterinary medicine, st. kitts, west indies summary an approximately nine-year-old tropical jenny was being routinely examined via transrectal ultrasonography as part of a follicular dynamics research study. on day 26 after her first documented ovulation, a 42.75-mm diameter presumptive preovulatory follicle developed an internal structure typical of a hemorrhagic anovulatory follicle. this ovarian structure remained ultrasonographically evident for 37 days. the interovulatory period of the cycle characterized by the hemorrhagic anovulatory follicle was shorter than the preceding cycle and two subsequent cycles that followed. this jenny continued to return to normal behavioral estrus each cycle. the progesterone concentrations measured during the cycle dominated by the hemorrhagic anovulatory follicle were numerically lower than those of the presumably normal ovulatory cycles of this jenny, but not statistically significant. the affected jenny was mated with a reproductively sound jack and became pregnant on the first attempt at the fourth recorded ovulation. background there are no reports of hemorrhagic anovulatory follicle (haf) development in tropical jennies and it is believed that this is the first documented case. a study published in 2008 reported one catalonian jenny having similar hemorrhagic follicle patterns during two of 14 estrous cycles.1 the follicles showed an interior fibrin network and reached 46and 61-mm in size, similar to what was observed in this tropical jenny. these cases could provide further insight into the reproductive cycle of donkeys and the potential effects of haf formation, if any. controlling feral populations of donkeys is becoming a topic of interest in some tropical regions and in others, increased reproductive efficiency is desired. for both goals, a better understanding of reproductive pathophysiology of the species is required. these cases also serve as a comparison for other species in which hemorrhagic or luteinized anovulatory follicles occur, including women.2 case presentation a group of eight mature, cycling jennies was included in a trial designed to characterize ovarian follicular dynamics in tropical donkeys. donkeys were housed in a dry paddock with unlimited access to water and local cane grass. following protocols approved by the institutional animal care and use committee (iacuc) of ross university school of veterinary medicine, the jennies were examined by transrectal ultrasonography using a 5-7.5 mhz linear probe daily at approximately the same time to track follicular development on both ovaries. once a preovulatory follicle of 25to 30-mm or larger was identified, they were examined by ultrasonography every six hours until ovulation. these jennies were followed through four cycles and subsequently removed from the study. the jenny in question had a 42.75-mm diameter follicle on day 26 after the previous ovulation when this follicle developed an internal structure typical of a haf. this structure was monitored daily and ultrasound images of its progression are depicted in figure 1. the presumptive haf remained ultrasonographically evident for 37 days and ovulation during the third cycle occurred on the contralateral ovary after 19 days. differential diagnosis the differential diagnoses considered in this case were a haf, an ovulatory corpus luteum (cl) of anomalous structure, and ovarian hematoma. based on this jenny’s continued cyclicity, the progesterone assay data, and the information collected from similar cases in the literature, it is believed that this was a haf. treatment no treatment was administered to this jenny. she was allowed to cycle naturally without the influence of exogenous hormones or medical treatment while on this study. clinical theriogenology • volume 10, number 2 • june 2018119   outcome the length of this jenny’s second estrous cycle with persistence of the haf was shorter by six days relative to her first documented cycle on the study and the jenny continued to return to normal behavioral estrus. the third and fourth cycles were 23 days in length, four days longer than her second. these interovulatory periods are presented in the table. during the span of all four cycles, blood samples were collected on mondays, wednesdays, and fridays to run serum progesterone assays. this jenny’s fluctuations in progesterone levels were not statistically significant between cycles, but were seemingly lower during the cycle dominated by the haf. a graph of the progesterone concentrations can be viewed in figure 2. no other significant changes to this jenny’s cycles were seen and no other jennies on this study had any similar ovarian abnormalities over the course of these four cycles. once the eight jennies in this study had been observed through four complete estrous cycles, they were bred using live cover by a reproductively sound jack. the jenny that had a haf was successfully bred and maintained the pregnancy through 30 days. discussion little is known about the mechanisms affecting the incidence of hafs in equids.2,3 as previously mentioned, this phenomenon has been documented in a catalonian donkey, but all other information found on haf development in equids was for mares.1 formation of hafs in pony mares was reported to be accompanied by elevated plasma estradiol for a few days before expected ovulation, but no significant differences in systemic progesterone, luteinizing hormone (lh), or follicle stimulating hormone (fsh) levels were seen. 2,4,5 there was also greater vascularity and thickening of the follicle within a few days before expected ovulation associated with the conversion of the viable follicle into a haf.3-5 the ultrasonographic appearance of the structure in question more closely resembled a haf than an ovulatory structure, in part, because of the thickness of the luteal border. in mares, the luteal border of an anovulatory structure with central lacunae usually exceeds 5-mm.5,6 although measurements were not taken of the border of the haf in this jenny, thickening of the wall is visible in figure 1 as the follicle progressed and developed the fibrin network. no other jenny in this study had a cl with morphology similar to that described here. ovarian hematoma was ruled out because the central part of the ovarian structure had an irregular echogenicity in contrast to more uniform echogenicity of most hematomata, the affected ovary was not enlarged, and the progesterone profile was not consistent with diagnosis of a hematoma.4 another study in 2007 determined that hafs are more common in older mares (>20 years).2,4,7 this research also defined that a distinguishing feature between a corpus hemorrhagicum (ch) and haf is the clotting of blood immediately during extravasation into the evacuated antrum versus delayed clotting when blood enters follicular fluid, respectively.4,5,7 hemorrhage into the evacuated follicle has been described as scattered free-floating echogenic spots within the follicular fluid, which could explain the follicular appearance viewed on ultrasound near ovulation and before the fibrin network appeared in the jenny of interest in this case.3 other factors that may contribute to incidence of hafs is time of season and the use of prostaglandin to induce ovulation. in one study from 2008, only one out of seven mares studied over ten years had no recorded haf.2 the other six ranged from 2.8% to as high as 25.3% incidence.2 use of prostaglandin, especially in higher doses during months of high follicular activity, increased the likelihood of induction and recurrence of a haf.2,3 the mechanisms by which prostaglandin increases the likelihood of developing hafs is still unclear, but it may be related to lh influence after administration when only small immature follicles are present on the ovaries.2 neither the season nor the use of prostaglandin to control cyclicity were explored in this study, but all jennies were cycling and were not treated with prostaglandin for the duration of the project. ultimately, pregnancy is often the end goal for owners and veterinarians that are regularly tracking follicular development in donkeys or mares. the most relevant information found with respect to clinical theriogenology • volume 10, number 2 • june 2018 120   haf formation is that failure of ovulation or collapse of the dominant follicle can inhibit release of the oocyte. if this occurs, pregnancy cannot be achieved unless it is accompanied by normal ovulation of another follicle.2,3 the jenny in question was not bred on the haf cycle, so it cannot be determined if she would or would not have become pregnant. however, once the haf regressed, she was successfully bred and produced a pregnancy that was detected through day 30. learning points  the estrous cycle following development and persistence of a haf in this tropical jenny was decreased by several days, but it did not appear to have any other significant effects on behavioral estrus or her ability to continue cycling during and after its regression. this contradicts previous findings in the literature regarding mares that had significantly prolonged inter-ovulatory periods, but the ability to continue cycling is consistent.3  serum progesterone levels were not significantly altered by the presence of the haf in this case, but it cannot be determined at this time if preovulatory plasma levels of estradiol correlate with the production of a haf as has been shown in the mare.4,5,7 the lower progesterone levels measured in the haf cycle mentioned above, while not statistically significant, may have allowed for earlier dominance of another follicle on the contralateral ovary.  while the formation of a haf was only documented during one cycle for this tropical jenny, more research would need to be done to determine if recurrence would occur within this individual. this has been documented in a catalonian donkey and in mares.1,2,4,7  a persistent haf does not appear to impair the ability of this tropical jenny to become pregnant following its regression, even without hormone treatment or other therapy. based on evidence described in the mare, it is hypothesized that the jenny would likely not have conceived if she had been bred on the cycles when the haf was present.2,3 references 1. taberner e, medrano a, peña a et al: oestrus cycle characteristics and prediction of ovulation in catalonian jennies. theriogenology 2008;70:1489-1497. 2. cuervo-arango j, newcombe j: risk factors for the development of haemorrhagic anovulatory follicles in the mare. reprod domest anim 2008;45:473-480. 3. mccue p: review of ovarian abnormalities in the mare. proc annu conv am assoc equine pract 1998;44:125-133. 4. ginther o, gastal e, gastal m et al: incidence, endocrinology, vascularity, and morphology of hemorrhagic anovulatory follicles in mares. j equine vet sci 2007;27:130-139. 5. ginther o, gastal e, gastal m et al: conversion of a viable preovulatory follicle into a hemorrhagic anovulatory follicle in mares. anim reprod 2006;3:29-40. 6. cuervo-arango j, newcombe j: ultrasound confirmation of ovulation in mares: a normal corpus luteum or a haemorrhagic anovulatory follicle? reprod domest anim 2012;48:105-111. 7. hyat j, hollinshead f, hanlon d: common causes of ovarian enlargement in the mare. centre for veterinary education, control and therapy series 2017;287:10-12. clinical theriogenology • volume 10, number 2 • june 2018121   estrous cycle length of cycle (days) 1 25 2 (haf) 19 3 23 4 23 table. interovulatory periods. the length, in days, of each of the four estrous cycles that were documented during this study. clinical theriogenology • volume 10, number 2 • june 2018 122     figure 2. graph of progesterone concentrations by estrous cycle. each of the serum progesterone concentrations for samples collected over the course of this study are graphed based on the estrous cycle and days after ovulation. c1, c2, and c3 correspond to the first, second, and fourth cycles that were normal and not dominated by the presence of the haf. haf (the solid line) graphs progesterone concentrations during the cycle dominated by the haf. clinical theriogenology • volume 10, number 2 • june 2018123 clinical theriogenology • volume 10, number 2 • june 2018 124 omniblank: 2015: foundered! why many practices never reach their full potential and how to ensure yours does foundered! why many practices never reach their full potential and how to ensure yours does d. kirk eddleman weatherford, tx let’s face it, great businesses are managed differently than the majority of veterinary practices. why is that? a veterinary practice is a business, right? so what is blocking so many veterinary practices from reaching their full potential? be brutally honest with yourself. does your practice seem to constantly be looking for solutions to reoccurring problems? is it weighed down by a general lack of funds, high staff turnover, low morale and the tendency towards crisis management? if any of these apply, your practice may be suffering from a common condition known as founder’s syndrome, or fs, a serious and life-threatening disease affecting thousands of businesses each year. founder’s syndrome can affect businesses of any size but is extremely common in small to mid-sized companies, including veterinary practices, which have grown rapidly. if left untreated, fs can cause otherwise successful practices to stagnate, decline and even fail. in most cases the symptoms of fs are painfully obvious to everyone inside the practice except those afflicted. what is founder’s syndrome? carter mcnamara, mba, phd, in his book leadership and supervision in business, describes fs as a condition in which an organization operates primarily according to the personalities of one or more members, usually the founders, rather than by the organization’s mission, policies and systems. put another way, fs is basically the absence, or more appropriately, the lack of use of organizational systems and structures to manage a practice. founder’s syndrome may involve an individual or a small group of individuals who helped to bring the practice through tough times such as the start-up or a period of rapid growth. these situations require strong passionate personalities who can make fast decisions and motivate people to action. most veterinary practices are founded by energetic, dynamic entrepreneurs who, while extremely skilled at building a clientele, may not be the best candidates for leading a practice as it grows into a larger organization. how founder’s syndrome develops for a practice to grow it must continually evolve to meet the ever-changing needs of its customers. as the practice evolves it must progress through several organizational or life stages. each life stage entails a different style of management. in the beginning stages, the founders usually control everything, often making highly reactive, seat-of-their-pants decisions and using their own intuition to address issues. this style of management works well when the practice is in its infancy. however, as the practice matures, the decision-making process must change to a more proactive, consensus-building style of governance. control systems, formal lines of authority, policies, procedures, budgets and strategic plans must be established and followed for the practice to progress towards its full potential. this transition can be very distressing for the charismatic and visionary founders who fear that losing control of their organization means certain failure. it is at this point the practice becomes most vulnerable to fs. symptoms of foundered practice founder’s syndrome is generally characterized by a crisis management approach to problem solving, high staff turnover, nepotism, a general lack of funds, strong resistance to change in the way decisions are made and continual struggles around the same reoccurring set of issues. founder’s syndrome is frequently seen in practices that have grown quickly into large community powerhouses. as the practice grows the demands for additional services, more efficiency and more resources increase and problems begin to compound. as a result, the founders demand more from their employees who take on additional roles and responsibilities. but the problems are not due to a lack of effort or ambition. the problems are caused by the employees’ confusion about their purpose and their roles within the organization, and from the constant pressure from the founders that does not seem to solve any clinical theriogenology • volume 7, number 4 • december 2015435 of the problems facing the practice. the end result is that no one really knows what is going on or who is in charge. the good employees leave and those that remain take on the traits of the crisis driven founders, or even worse, just “numb out.” they continue to look to the frustrated founders for direction and the practice struggles from one crisis to the next. in these practices, the founders may see the practice as a big “family” (with them at the head). whenever a crisis arises, the founders make decisions as if they are gathered around someone’s dining room table trying to hold everything together. the practice may have policies and procedures in place but because the founders have more confidence in their own intuition and judgment these formalities are often ignored or viewed as “unnecessary paperwork”. it is important to note that fs is no one’s fault. the founders do not intentionally set out to harm their practices. further, fs is not an individual problem. it is an organizational condition that sets in because the practice is dependent upon the founders for decision making rather than organizational structures and systems to manage the practice. not sure if your practice is suffering from fs? read the characteristics of fs below or, even better yet, give a copy of this article to your staff. then hide and watch to see if their heads bob up and down in agreement. in a foundered practice the leaders, often: • make reactive, crisis driven decisions, often without seeking input from others. • motivate through fear or guilt, sometimes without realizing it. • handpick their employees, often family members or like-minded friends for key roles. • count on and promote those who seem loyal and accessible rather than on those with the best qualifications or talent. • see their employees as working for themselves rather than working for the organization’s mission. • inject themselves into routine operational decisions. • seem highly skeptical about planning, policies and procedures. • sometimes criticize those who suggest the need for rules and systems. • have a difficult time letting go of the strategies that helped to quickly grow the practice. treating founder’s syndrome if left untreated fs can become a chronic and reoccurring condition that simmers for years, slowly deteriorating the practice until either the founders leave or the practice folds. the good news is fs is not always fatal if treated aggressively. below are five steps for treating fs: 1. recognize the symptoms. the first and hardest part of treatment is extreme realization. the founders must first realize the need to change the way they manage their practice. this means changing from within to develop their own leadership to the next level. 2. ask for and accept help. founder’s syndrome is the result of doing what comes naturally. changing one’s own leadership style can be very difficult because it means changing what comes naturally. seek an outside mentor. having a mentor outside the organization will help the founders gain a new perspective about their own leadership style. further, an outside mentor may be able to point out how the founders’ actions affect the company. 3. develop the board of directors. as a whole your board of directors can be the greatest source of support in overcoming fs if they fully understand and take full responsibility for the role of board member. board members must understand their authority lies in the board room. once outside the board meeting, established lines of authority must be followed. provide board members with job descriptions and annual training sessions focused specifically on the responsibilities governing the board. a. develop strategic plans. strategic planning is the best way to engage the board members and key employees in setting the course for the practice. focus on two or three clinical theriogenology • volume 7, number 4 • december 2015 436 issues facing the practice in the next three months, six months, and 12 months and set realistic goals for the practice to achieve. b. conduct risk management exercises. pretend one or more of the founders suddenly left the practice. how would operations be affected? what actions would management take? what would happen to cash flow? who could step in? this kind of “what if?” scenario will help establish systems to manage the practice. 4. organize systems to meet goals. in any organization, systems (policies and procedures) govern. use strategic plans and budgets to develop systems that guide management and operations. then the management team creates the systems to manage the business. 5. keep morale high. using appropriate channels, remind employees that the problems associated with fs are the result of the practice’s success and growth. once employees perceive stability and progress, morale will improve. curing fs can be a painful process but the benefits of recognizing and handling the disease far outweigh the pain and hassle of treatment. once healed of fs the practice will advance once again towards its’ full potential, customer service and the quality of care will improve, profits will grow, and the founders will find they sleep better, have more money in their pockets and more balance in their lives. take home message the ultimate goal of this presentation is to share concepts with veterinarians that can help them improve the business management of their practice. the take home message is that fs is a very common condition affecting veterinary practices of all sizes and that once healed of fs the practice will advance once again towards its’ full potential. clinical theriogenology • volume 7, number 4 • december 2015437 clinical theriogenology • volume 7, number 4 • december 2015 438 omniblank: 1 contact marco coutinho da silva coutinhodasilva.1@osu.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10452, http://dx.doi.org/10.58292/ct.v16.10452 case report congenital hydrocephalus in a stillborn haflinger foal gail mcrae,a marco coutinho da silva,a erin runcan,a joanna koilpillai,a christopher premanandanb adepartment of veterinary clinical sciences, college of veterinary medicine, the ohio state university, columbus, oh, usa bdepartment of veterinary biosciences, college of veterinary medicine, the ohio state university, columbus, oh, usa abstract a 14-year, multiparous haflinger mare, apparently fullterm (unknown breeding or ovulation date), was presented for dystocia. mare was transvaginally palpated at the farm by the referring veterinarian; foal was in craniolongitudinal presentation, dorsosacral position with extended forelimbs in the vaginal vault and head just cranial to cervix, and a cranial abnormality of the foal prevented assisted vaginal delivery. mare was referred. foal was not alive at presentation and was undeliverable with assistance because of congenital hydrocephalus. anesthetized mare was placed in trendelenburg position, controlled vaginal delivery was elected and foal was removed via fetotomy. mare was negative for β1, 3- n-acetylgalactosaminyltransferase 2 (b3galnt2) nonsense mutation (reported to be responsible for hydrocephalus). dystocia in a haflinger mare because of hydrocephalus is reported for the first time. keywords: mare, haflinger fetus, congenital hydrocephalus, b3galnt2 gene introduction congenital hydrocephalus (ch) is an inborn abnormality where cerebrospinal fluid (csf) accumulates and dilates brain ventricles.1–5 congenital hydrocephalus can be internal, external, communicating, or noncommunicating depending on the location of csf accumulation in brain. dilation occurs either from increased production or decreased absorption of csf. in internal hydrocephalus, csf accumulates within ventricles whereas in external hydrocephalus, csf accumulates outside ventricles. communicating hydrocephalus is defined as a problem with absorption, and noncommunicating hydrocephalus occurs when there is an obstruction to absorption of csf.2,4,6 congenital hydrocephalus has been noted in multiple species including humans,7 rats,8 rabbits, ruminants, birds, cats, dogs,6 and many horse breeds (friesian, standardbred, belgian, quarter horse, and miniature horse).9 in horses, hydrocephalus is still a rare occurrence with an incidence of 0.6 per 1,000 births.6 equine ch has been studied in the friesian population where the average inbreeding coefficient is higher. for this breed, there is a commercial test of the nonsense mutation of β1, 3- n-acetylgalactosaminyltra nsferase 2 (b3galnt2)1,2,4 that identifies carriers of the hydrocephalus inducing autosomal recessive gene.1,2,4 mutation of b3galnt2 gene has also been detected in a belgian draft horse.4 in humans, only 40% of hydrocephalus cases are believed to be genetically related to an x-linked recessive trait7,10 other known causes of hydrocephalus in other species (dogs, cats, cattle, and pigs) include viral and bacterial causes.6 there are 2 reports in the same publication of 2 aborted halfinger foals that had hydranencephaly. these foals were sired by the same stallion.10 to date, there is no published case report of a presumed fullterm hydrocephalus foal in a haflinger mare. case presentation case history a 14-year, multiparous haflinger mare, apparently fullterm (unknown breeding or ovulation date), was presented for dystocia. mare had 6 foals before and last foaling was a normal delivery. mare was transvaginally palpated at the farm by the referring veterinarian; foal was in craniolongitudinal in presentation, dorsosacral in position with extended forelimbs in the vaginal vault and head just cranial to cervix, and a cranial abnormality of the foal prevented assisted vaginal delivery. mare was referred. mailto:coutinhodasilva.1@osu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10452 2 citation line: clinical theriogenology 2024, 16, 10452, http://dx.doi.org/10.58292/ct.v16.10452 clinical examination at presentation, mare had been in stage 2 of parturition for ~ 6 hours. initial examination revealed that the mare was tachycardic, tachypneic, had a small amount of dried blood on the right nostril, and had a mild amount of hemorrhagic discharge from the vulva. mare’s body condition was scored as 7/9. total protein was 6.4 g/dl, pcv was 38%, and lactate was 1.6 mmol/l. transabdominal ultrasonography was performed during left jugular vein catheterization; fetus was confirmed dead (visualization of lack of heartbeat). mare was given 350 mg of intravenous xylazine. tail was wrapped, and vulva was washed with a nonresidue soap. two minutes later, mare was given 1,200 mg of intravenous ketamine and 200 mg of intravenous propofol for induction of anesthesia. an endotracheal tube (26 mm) was placed, and mare was maintained on sevoflurane gas mixed with 100% oxygen, with lidocaine and dobutamine as a constant rate infusion. after being initially placed in the trendelenburg position, where the mare was in dorsal recumbency with the hind legs elevated via hoist, mare’s blood pressure increased which indicated that anesthetic plane became light, so a bolus of 30 mg of intravenous butorphanol was given over 10 minutes. fetus was transrectally palpated and was in craniolongitudinal presentation, dorsosacral position with extended forelimbs in the vaginal vault. foal’s head was extended but positioned just cranial to the pelvic brim, and an irregularly-shaped cranium was palpated. cesarean surgery was offered and declined. treatment due to enlarged cranium and the inability to perform cesarean surgery, a partial fetotomy was elected to extract the foal. first cut was made transversely through the enlarged cranium, and the second was made between c1 and c2 vertebrae (figures 1 and 2). fetal skull had an incomplete boney cranium with the bone forming around the lateral aspects and a 1-inch strip of bone forming down the sagittal midline on the dorsal aspect (figure 3). after the cranial cuts were made, resultant fragments were removed. chains were placed on the forelimbs with a loop above fetlock and half hitch below fetlock joints (chain running dorsally on the forelimbs) and foal was delivered with manual traction. mare’s nongravid uterine horn intussuscepted during delivery and was promptly replaced. fetal membranes remained tightly adhered to the endometrium. mare recovered from anesthesia and tetanus toxoid vaccine was given. outcome foal had a cranium volume of 3,721 cm3 that equates to ~ 1 gallon (3.71 liters). dimensions of foal’s cranium were as follows: 20.95 cm (rostral to caudal), 18.4 cm (width), and 18.4 cm (dorsal to ventral) (figure 4). foal’s brain was ~ 3 mm in thickness (figure 5). primary differential diagnosis was an internal congenital hydrocephalus. mare was routinely treated for retained fetal membranes with a combination of antibiotics, antiinflammatory agents, ecbolics, and uterine lavage. uterine lavage was performed twice daily with 5-10 liters of lactated ringer’s solution. intramuscular oxytocin (5 iu) was given every 2 hours until fetal membranes were expelled. antibiotics (intravenous potassium penicillin 22,000 iu/kg every 6 hours and intravenous gentamicin 6.6 mg/kg once daily) and an antiinflammatory (intravenous flunixin meglumine 1.1 mg/kg every 12 hours). mare shed fetal membranes 3 days after dystocia and was discharged from the hospital without any further complication. owners elected not to breed the mare during the subsequent season. genetic testing was authorized by the owner. since b3galnt2 mutation has previously been reported in a belgian mare and there are no other genetic variants that have currently been identified, 20-30 hairs with roots were submitted to uc davis veterinary genetics laboratory for analysis. this gene is autosomal recessive, and the stallion was not available for testing. final results indicated that the mare did not carry the recessive trait for this mutation. discussion congenital hydrocephalus is reported to affect 0.6 out of 1,000 births in horses.6 currently, there is only 1 commercial test available for friesian mares with the identified nonsense mutation of b3galnt2.1,2,4 congenital hydrocephalus is present in multiple species, with 40% of human cases stemming from a genetic cause.7 in ruminants, hydrocephalus is often associated with teratogenic viruses, whereas in felids and birds there are reports associating hydrocephalus with vitamin a deficiency. additionally in dogs, ch has been linked to anatomic defects of the skull, vertebral growth, and craniovertebral junction.6 in contrast, there have been no reports of vitamin deficiency or figure 1. location of fetotomy first cut. http://dx.doi.org/10.58292/ct.v16.10452 citation line: clinical theriogenology 2024, 16, 10452, http://dx.doi.org/10.58292/ct.v16.10452 3 viral causes of hydrocephalus in horses and the ultimate cause is not well described in the literature.8 to authors’ knowledge, this is the first case report of a haflinger mare with a hydrocephalus foal at term. a belgian draft horse had a hydrocephalic foal that was positive for b3galnt2 mutation.4 in our case, the haflinger mare did not carry the b3galnt2 mutation, suggesting that there is either another genetic mutation or another cause of hydrocephalus that has not yet been identified. there are no reported cases in horses of viral illness causing hydrocephalus.6 further research is needed to provide insight into different genetic and nongenetic causes of hydrocephalus in horses and provide broader diagnostic tests and possible preventions for this fatal condition. learning points • congenital hydrocephalus in horses is rare; however, in certain breeds can be associated with a mutation in b3galnt2 gene. • not all cases of fetal hydrocephalus are caused by a mutation in b3galnt2 gene; apparently, there are other causes. • hydrocephalic fetuses can complete pregnancy; however, will likely result in dystocia. figure 2. location of fetotomy second cut. figure 3. lateral formation of cranium with a boney strap down the sagittal midline. figure 4. dimensions of foal’s cranium; a. rostral to caudal, b. width, c. dorsal to ventral. http://dx.doi.org/10.58292/ct.v16.10452 4 citation line: clinical theriogenology 2024, 16, 10452, http://dx.doi.org/10.58292/ct.v16.10452 conflict of interest none to report. references 1. ayala-valdovinos ma, galindo-garcía j, sánchez-chiprés d, et al: genotyping of friesian horses to detect a hydrocephalus-associated c.1423c>t mutation in b3galnt2 using pcr-rflp and pcr-pira methods: frequency in stallion horses in méxico. mol cell probes 2017;32:69–71. doi: 10.1016/j.mcp.2016.12.005  figure 5. brain matter from foal’s cranium. 2. ducro bj, schurink a, bastiaansen jw, et al: a nonsense mutation in b3galnt2 is concordant with hydrocephalus in friesian horses. bmc genomics 2015;16:761. doi: 10.1186/s12864-015-1936-z 3. hodder adj, ball ba: theriogenology question of the month. j am vet med assoc 2008;232:211–213. doi: 10.2460/javma. 232.2.211 4. kolb ds, klein c: congenital hydrocephalus in a belgian draft horse associated with a nonsense mutation in b3galnt2. can vet j 2019;60:197–198. 5. ojala m, ala-huikku j: inheritance of hydrocephalus in horses. equine vet j 1992;24:140–143. doi: 10.1111/j.2042-3306.1992. tb02799.x 6. schmidt m, ondreka n: hydrocephalus in animals. pediatric hydrocephalus 2019;53–95. doi: 10.1007/978-3-319-27250-4_36 7. haverkamp f, wölfle j, aretz m, et al: congenital hydrocephalus internus and aqueduct stenosis: aetiology and implications for genetic counselling. eur j pediatr 1999;158:474–478. doi: 10.1007/s004310051123 8. jones hc, bucknall rm: inherited prenatal hydrocephalus in the h-tx rat: a morphological study. neuropathol appl neurobiol 1988;14:263–274. doi: 10.1111/j.1365-2990.1988.tb00887.x 9. edwards l, finno cj: genetics of equine neurologic disease. vet clin north am equine pract 2020;36:255–272. doi: 10.1016/j. cveq.2020.03.006 10. waelchli ro, ehrensperger f: two related cases of cerebellar abnormality in equine fetuses associated with hydrops of fetal membranes. vet rec 1988;123:513–514. doi: 10.1136/vr.123.20.513 http://dx.doi.org/10.58292/ct.v16.10452 https://doi.org/10.1016/j.mcp.2016.12.005  https://doi.org/10.1186/s12864-015-1936-z https://doi.org/10.2460/javma.232.2.211 https://doi.org/10.2460/javma.232.2.211 https://doi.org/10.1111/j.2042-3306.1992.tb02799.x https://doi.org/10.1111/j.2042-3306.1992.tb02799.x https://doi.org/10.1007/978-3-319-27250-4_36 https://doi.org/10.1007/s004310051123 https://doi.org/10.1111/j.1365-2990.1988.tb00887.x https://doi.org/10.1016/j.cveq.2020.03.006 https://doi.org/10.1016/j.cveq.2020.03.006 https://doi.org/10.1136/vr.123.20.513 _hlk156854374 2015: pregnancy toxemia in small ruminants: a review pregnancy toxemia in small ruminants: a review a.j. campbell, l.k. pearson, a. tibary department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa abstract pregnancy toxemia is a metabolic disease of ewes and does with multiple fetuses in late gestation. while pregnancy toxemia has been well described in the literature for decades, a thorough understanding of the pathogenesis, individual predisposing factors, and treatment options still remains limited. recent research has contributed to our understanding of the pathogenesis of this disorder and developing an improved diagnostic approach. in particular, an understanding of insulin regulation and development of insulin resistance, along with identification of markers of oxidative stress, has generated additional information in understanding individual animal variation for predisposition to development of the disease. the objective of this review is to present the current understanding of pregnancy toxemia including pathogenesis, various predisposing factors, clinical signs, available diagnostics, available treatment options, and discuss the current and future research which may result in an increased understanding of the disease, improved diagnostic efficiency, and provide better flock or herd management for prevention. keywords: small ruminants, metabolic, insulin, pregnancy, diagnosis introduction pregnancy toxemia, also referred to as pregnancy ketosis, pregnancy disease, twinning disease, and fatty liver syndrome, occurs in all countries worldwide. it is most commonly found in multiparous ewes or does carrying multiple fetuses. the disease occurs more frequently in ewes than in does.1 the condition is generally brought on by a competition between the dam and her fetuses for glucose.2,3 pregnancy toxemia is a metabolic disease that affects pregnant ewes and does during the last third of gestation, especially the last four to six weeks.4 this is the period of rapid fetal growth. susceptibility is widely variable, with some individuals being more predisposed than others. small ruminants are an important income source in numerous countries and are gaining popularity in the united states. economic losses associated with pregnancy toxemia could have lasting effects, especially in countries were nutritional support is limited.4,5 increased industry drive for higher prolificacy combined with increasing feed costs and decreasing profit margins can also result in development of pregnancy toxemia within a given flock or herd.6 the objective of this review is to present the current understanding of pregnancy toxemia including pathogenesis, various predisposing factors, clinical signs, available diagnostics, and available treatment options, as well as discuss the current and future research which may result in an increased understanding of the disease, improved diagnostic efficiency, and provide better flock or herd management for prevention. pathogenesis pregnancy toxemia is a complex metabolic disorder, and the full etiopathogenesis is not completely understood. pregnancy toxemia is a severe form of ketosis, characterized by low serum glucose concentrations and high ketone body concentrations.6 individual variation appears to be related to hepatic metabolic efficiency6 and ability to maintain functional insulin concentrations and maternal tissue responses,7 with some breeds (mainly those consistently delivering two or three offspring per litter) more predisposed to the condition. however, some highly prolific breeds, such as finnish landrace and booroola merino sheep, do not experience an increased incidence of pregnancy toxemia.8,9 maintenance of adequate concentrations of serum glucose is critical to brain function, fetal growth, and milk production.2 in ruminants, limited dietary glucose is absorbed because the ruminal flora is proficient at fermenting glucose. the majority of ruminant energy is derived from short-chain fatty acids including acetate, proprionate, and butyrate, which are produced by the ruminal flora. proprionate clinical theriogenology • volume 7, number 4 • december 2015407 is the most important glucose precursor in ruminant gluconeogenesis and derives from ruminal fermentation of high-carbohydrate-content feed. ruminal fermentation of forage produces acetate, which is the precursor of long-chain fatty acids for fat storage.2,4 serum glucose concentrations are largely maintained by gluconeogenesis, 85% of which occurs in the liver.4 excess glucose is stored in the liver as glycogen but these stores are inadequate to supply the dam’s needs in late gestation2. there is an increased sensitivity to hypoglycemic stress, such as prolonged feeding intervals, in ewes and does with multiple fetuses in late gestation. hypoglycemia in these animals results from a reduction in the glucose production rate.10 in late pregnancy ewes and does with multiple fetuses may become unable to maintain glucose homeostasis, resulting in a state of negative energy balance. this metabolic state results in a decreased ratio of insulin to glucagon,2 and insulin resistance may result.11 it has been demonstrated that insulin plasma concentrations are reduced in ewes carrying more than three fetuses.12 the ability to maintain functional insulin concentrations and maternal tissue responses appears to be key to an appropriate metabolic adaptation to a negative energy balance where pregnancy toxemia does not develop.4,7 in a state of negative energy balance, glucogenic precursors from the diet or endogenous sources are not able to meet the total energy requirements, and the body shifts to other sources for energy maintenance during late gestation. fat mobilization is initiated in an attempt to compensate according to the degree of the negative energy balance.7 lipases are activated that convert tissue fat into fatty acids and glycerol. the glycerol can then be converted into glucose in the liver, through hepatocyte mitochondria.2 non-esterified fatty acids (nefas) are the major component of triglycerides (fat stored in the body) and are either used for production of energy via the krebs cycle or are converted into ketone bodies (acetone, acetoacetate, and β-hydroxybutyrate [bhb]).2 the excessive release of nefas may overwhelm the liver’s capacity, in turn generating large quantities of ketone bodies, resulting in fat infiltration of the hepatocytes.7 furthermore, the direction of nefa metabolism is dependent on availability of oxaloacetate.2 oxaloacetate, a substrate for gluconeogenesis in states of negative energy balance, is an intermediate in the krebs cycle. if supplies of oxaloacetate are low, ketone formation results.13 if production of ketones predominates, the animal’s appetite is depressed, further reducing feed intake. it has also been demonstrated that hyperketonemia significantly depresses hepatic glucose production, contributing to the pathogenesis of the disease.14 metabolic acidosis develops from the excessive ketone body production.7,15 blood cortisol levels rise as a result of reduced hepatic metabolism, hypoglycemia, and continued stress levels.6 the severity of the acidosis can be demonstrated by the plasma total co2.15 it has also been suggested that cell-mediated and humoral immunity are altered in subclinical and clinical cases of pregnancy toxemia, where plasma nefa concentrations are correlated with decreased antigen-specific igg production in affected animals.16 acetoacetate, a ketone body, has been reported to generate superoxide radicals that form hydroxyl radicals. these free radicals exert a cytotoxic effect resulting in the accumulation of the final products of lipid peroxidation, such as thiobarbituric acid reactive substances (tbars). these products can cause alterations in biomembrane permeability and lipid organization, as well as cellular dysfunction and membrane damage,17 ultimately leading to cellular death.18 markers of oxidative stress have been demonstrated to be elevated in cows with subclinical ketosis.17 al-qudah et al. examined the role of hyperketonemia in initiating lipid peroxidation and evaluated the status of enzymatic and non-enzymatic antioxidant defense systems in ewes affected by pregnancy toxemia.17 an increased plasma concentration of tbars suggested a significant increase in lipid peroxidation in ewes with pregnancy toxemia compared with healthy pregnant and healthy non-pregnant ewes.17 a decrease in antioxidant enzyme activity was also observed and may be associated with an imbalance between lipid peroxidation and antioxidant capacity in pregnant ewes. in that study, oxidative stress was determined to be caused by four factors: negative energy balance and ketone formation, increased free radical production resulting from increased metabolic activity during pregnancy, reduction of antioxidant reserve during pregnancy, and physiological adaptation of pregnant animals to lactation.17 clinical theriogenology • volume 7, number 4 • december 2015 408 predisposing factors in the last four to six weeks of gestation, the ewe or doe experiences the most rapid fetal growth and demands from the pregnancy, while at the same time experiencing a decline in nutritional intake, predisposing the ewe or doe to a ketosis-prone metabolic state.2,7,19,20 in well-managed flocks or herds, individual variation may result in sporadic occurrence and low morbidity (<3%) that may be linked to genetic variation.4 in contrast, flocks or herds with improper nutritional management could experience outbreaks with morbidity rates exceeding 10% of the ewes or does.7 mortality rates of affected animals can exceed 80%, especially if treatment is delayed.4 pregnancy toxemia can be classified into two general categories: primary and secondary. primary pregnancy toxemia results from a combination of poor nutrition and a sharp increase in energy needs during late gestation, and most commonly affects older animals.2,7,20 it can be precipitated by other management or environmental factors. factors that might predispose a ewe or doe to development of pregnancy toxemia include lack of feed availability, decreased feed quality, sudden feed changes, transportation during late pregnancy, shearing, vaccinating, and heat or cold stress.2,7,19 these factors, either singly or in combination, result in decreased feed intake, development of a negative energy balance, and result in the progression to a ketotic state.7 decreased feed intake, even if only temporarily, can be detrimental because there is already limited abdominal space as a result of increased fetal size, and thus protracted limited feed intake6 multiparous, thin ewes in their last trimester are at increased risk. overconditioned animals (body condition score >4/5) are at an elevated risk of pregnancy toxemia due to increased fat storage for mobilization to the liver,7 along with reduced abdominal space as a result of increased intra-abdominal adipose tissue.6 secondary pregnancy toxemia is a result of conditions that cause reduced feed intake and increased energy needs. concurrent disease, such as acidosis, water deprivation, hypocalcemia, infectious pododermatitis, pneumonia, heavy parasite infestation, vaginal prolapse, or chronic wasting diseases can result in development of secondary pregnancy toxemia.2,5,19 while it has long been demonstrated that a lack of energy supply is responsible for the development of pregnancy toxemia, it is not likely to be the only major factor. considering that pregnancy toxemia develops during late gestation and not during lactation when the energy demands are the highest, and that there are known breed-dependent differences, insufficient energy utilization rather than a complete lack of energy supply may be a contributing factor.21 it is suspected that marked metabolic changes in late pregnancy may be the result of modified hormonal responses.22 harmeyer et al. experimentally induced ketosis in texel and german blackface ewes that were over three years of age during different reproductive stages (late gestation, early lactation, and non-pregnancy/non-lactation) to measure the turnover rate of ketone bodies during hyperketonemia in a steady state condition.22 β-hydroxybutyrate concentrations in the blood were elevated through a continuous iv infusion of dl-β-hydroxybutyrate (d-bhb) to maintain levels similar to concentration usually present in ewes with clinical signs of pregnancy toxemia.22 significant differences existed in maximal d-bhb turnover between reproductive states, with limited turnover observed during late gestation. the indication that ewes in late gestation, especially those carrying twins, have reduced utility of ketone bodies is surprising since these ewes undoubtedly rely to a greater extent on ketone bodies to satisfy energy needs, especially compared to ewes that are not pregnant or lactating.22 reduced disposal of bhb during late gestation along with an increased rate of hepatic ketogenesis promotes hyperketonemia, facilitating development of pregnancy toxemia. theoretically, several hormonal and metabolic factors could be contributing to this factor, with one possible candidate being insulin.22 in recent years, insulin resistance during late gestation has been evaluated as a potential predisposing factor to development of pregnancy toxemia.21 insulin appears to be less responsive in ruminants than in monogastric species, however it is still important for glucose homeostasis and also has anti-lipolytic activity in adipocytes and depresses ketone body formation. in cases of pregnancy toxemia where hypo/hyperglycemia, lipemia, and hyperketonemia are common, insulin resistance may be a major predisposing factor.21 duehlmeier et al. evaluated metabolic adaptations to pregnancy and lactation in ewes.23 ewes were separated into two groups (high and low risk). the high-risk group consisted of 4.5 to 6.5 year old clinical theriogenology • volume 7, number 4 • december 2015409 german blackheaded mutton ewes, a breed that has been recognized as being highly susceptible to pregnancy toxemia.24 the low risk group consisted of 2.5 year old finnish landrace ewes, a breed that has been recognized for high prolificacy8 and low incidence of pregnancy toxemia. blood samples were evaluated for glucose, insulin, nefas, and bhb concentrations at differing times during gestation and following parturition. the study revealed that high-risk ewes had decreased insulin and glucose concentrations during late gestation in comparison to low-risk ewes and ewes during lactation. nonesterified fatty acid and bhb concentrations were elevated during late gestation in comparison to low-risk ewes.23 in a follow-up study, the same parameters were evaluated after the administration of an intravenous glucose tolerance test (ivgtt) at differing times during gestation and following parturition.21 high-risk ewes exhibited a lower insulin response and reduced glucose elimination following each ivgtt. increased lipolysis in late gestation was only observed in the high-risk ewes. β-hydroxybutyrate concentrations were only elevated in late gestation in the high-risk ewes compared to the low-risk ewes, and during late gestation the bhb concentrations did not decrease at all after ivgtts in the high-risk ewes.21 it is suspected that termination of lipolysis relies on the inhibition of hormone-sensitive lipase (hsl) and activation of lipoprotein lipase (lpl) by insulin, which is secreted in response to glucose. it is also suspected that control of ketogenesis is regulated in part by insulin. insulin resistance in both of these scenarios could result in dysfunction of these pathways as observed in this study.21 clinical signs, diagnosis, and postmortem findings clinical signs in ewes and does vary in severity and progression of the metabolic derangements. initially the clinical signs associated with pregnancy toxemia are vague, with affected animals isolating themselves from the remainder of the flock or herd.7 affected animals will appear listless and obtunded and when moved to food sources will stand with the other animals, but do not eat.5,15 with the progression of hypoglycemia and ketonemia, neurological clinical signs (head pressing, circling, muscle tremors) will become apparent within one to two days with the resultant development of hypoglycemic encephalopathy.2,19,25 they are often hyperesthetic to tactile or auditory stimuli and are difficult to restrain once caught.6 ewes and does may appear blind (lack of a menace or “star-gazing” appearance), ataxic, or disoriented.6 feed intake decreases further, perpetuating the cycle. the ewe or doe will develop tachypnea and dyspnea with continued progression of neurologic signs to recumbency.7 once recumbent, there is often a lack of abdominal muscle tone and urine scalding can be observed on the abdomen and hind legs.6 chewing, teeth grinding, excessive salivation, and excessive licking may also be observed.2,5-7 in the advanced stages, severe neurologic signs, potential convulsions, and coma can be observed, followed by death. in some instances there may be a transient improvement if fetal death occurs, however more profound depression generally follows.6,15 death is usually the result of renal failure or toxemia following death of the fetuses.7 renal failure is thought to be the result of systemic hypertension caused by ketoacidemia. the onset of hypertension in affected animals results in renal dysfunction due to a decreased glomerular filtration rate.2 progression of clinical signs generally ranges from 12 hours to seven days, but most commonly occurs over three to four days.5,7 differential diagnoses should include hypocalcemia, polioencephalomalacia, listeriosis, impending abortion, copper toxicity, and ruminal acidosis.5,7,15 diagnosis of pregnancy toxemia is generally based on history and clinical signs. ketosis is generally diagnosed by identification of excessive ketone production in the urine. dipsticks and ketone powder both use the nitroprusside test to semi-quantify acetoacetate concentrations.7,19 serum or plasmabhb concentrations can also be predictive of pregnancy toxemia. normal bhb concentrations are below 8 mg/dl, whereas in cases of moderate ketosis associated with subclinical disease bhb concentrations are >15 mg/dl. clinical ketosis is generally associated with bhb levels in excess of 25 mg/dl.7 several studies demonstrated that increased bhb concentrations were a sensitive indicator for the complex alteration of glucose and lipid metabolism in pregnancy toxemia.25 other laboratory findings that might indicate the presence of pregnancy toxemia include elevated nefa levels (>0.4 meq/l), hypoglycemia, and in some cases elevated liver enzymes and hypocholesterolemia.7 in recent studies, hypotriglyceridemia and hypocholesterolemia have been clinical theriogenology • volume 7, number 4 • december 2015 410 observed in subclinical and clinical cases of pregnancy toxemia, when compared to control groups, and in association with increased fetal numbers.12,25 hypoproteinemia (hypoalbuminemia and hypoglobulinemia) can be observed in clinical cases of pregnancy toxemia, and could potentially be attributed to hepatic and/or renal failure.25,26 in the clinical or later stages of pregnancy toxemia, hyperglycemia (often associated with fetal death), hypokalemia, hypocalcemia, elevated creatinine, and elevated blood urea nitrogen (bun) may be evident.25,26 decreased serum fructosamine represents a persistent hypoglycemia and could also be used as an indicator of early pregnancy toxemia. fructosamine, a ketoamine compound formed when glucose reacts with amino groups on proteins, is related to the average glucose concentration over a period of several weeks.4 recently levels of the antioxidant ceruloplasmin were shown to be a good indicator of feed deficiency in late pregnancy in chios ewes.18 ceruloplasmin is an acute phase protein that functions as a copper transporter, but also acts as an intravascular antioxidant in response to tissue damage and inflammation.18 results from the study indicated that mean serum ceruloplasmin levels were not significantly different between groups until day 148 of gestation. at day 148 of gestation, representative of the last week of gestation, a significant rise in ceruloplasmin was observed in the feed deficient group of ewes.18 it is important to recognize, however, that ceruloplasmin at that stage of gestation could have also be elevated due to other factors including elevations in cortisol, acth, and estradiol-17β, which have been demonstrated to cause elevations in ceruloplasmin in pregnant females in other species.18 it is therefore important to recognize that further research is needed to establish elevated ceruloplasmin levels as an indicator of feed deficiency in pregnant small ruminants and as a marker for severity of pregnancy toxemia in clinical cases. due to expense of treatment and lack of success, especially in advanced cases, recent studies have evaluated diagnostic tools, which can be used in the field. many producers and veterinarians use urine ketone test strips for the diagnosis of ketoacidosis. in dairy cattle ketone strip accuracy is reportedly variable due to factors including the nature of handling, interval from testing to reading, environmental conditions, and pigmenturia.27 additionally, urine collection may be difficult. recently a point of care (poc) meter designed to measure blood glucose and bhb concentrations in humans was evaluated in ewes.28 comparison of poc bhb concentrations with reference laboratory concentrations showed that 93% of poc bhb concentrations were within 20% of the reference laboratory concentrations. results for blood glucose concentrations revealed that only 68% of poc glucose concentrations were within 20% of the reference laboratory concentrations. these results suggest that the poc meter is clinically acceptable for measuring blood bhb concentrations, but not glucose concentrations.28 in general the poc meter demonstrated low blood glucose concentrations in comparison to reference laboratory results, and could result in overtreatment with administration of glucose substrates to increase blood glucose concentrations unnecessarily.28 it was noted that the laboratory’s reference limits for adult sheep might not have been appropriate for pregnant ewes. overall human-derived devices, such as the poc meter, could be beneficial in evaluating bhb concentrations, becoming a useful on-farm diagnostic tool for pregnancy toxemia.28 also, considerations for usefulness on-farm would need to be made with respect to animal handling facilities and operator training to perform venipuncture. on postmortem examination, the carcass is either emaciated or in good condition with large quantities of intra-abdominal and subcutaneous fat.6,15 there can be differing degrees of fatty liver infiltration, with the liver generally enlarged, pale and friable (figure 1). on histological evaluation of liver samples obtained from ewes suffering from pregnancy toxemia, microvesicular steatosis with lipid droplet accumulation was reported (figure 2).29 the presence of multiple fetuses is also observed grossly, and depending on when fetal death occurred, autolysis may be present.6,7,19 other gross abnormalities that might be identified include enlarged, hemorrhagic adrenal glands.4 renal and cerebral lesions are often not identified on gross examination, but might reveal changes on histological examination.6 aqueous humor concentrations of bhb can correspond to antemortem serum concentrations and help diagnose the condition in the absence of significant lesions grossly.5 clinical theriogenology • volume 7, number 4 • december 2015411 treatment, flock/herd management, prevention treatment and prognosis for recovery of pregnancy toxemia are highly dependent on the stage of the disease at time of diagnosis and initiation of treatment. current treatment protocols are based on two general principles: administration of energy sources, and removal of factors that increase energy requirements of affected animals.1 early intervention may be effective; however, in general, response to treatment is poor because the disease becomes irreversible in its later stages. if the ewe or doe is in the early stages of the disease and is still responsive and willing to eat, dietary modification and supplementation with glucogenic precursors may be sufficient.7 affected animals should be separated from the remainder of the flock or herd and provided adequate shelter. it is important to ensure that high quality forage be provided along with an increased energy source in the form of easily digested concentrates. in addition to dietary modification, glucogenic precursors may be orally administered.7 oral glucogenic precursors include propylene glycol (60-200 ml po q12 hr for 6 days)1 or glycerol (60 ml q12 hr for 3-6 days).1 other sources such as calcium proprionate, sodium proprionate, liquid molasses, sodium lactate, or ammonium lactate may be used as glucose sources, but are not metabolized as quickly as propylene glycol.1 oral administration of a concentrated dextrose and electrolyte solution may also be effective. there are a wide variety of commercially available products containing the above precursors that have been used clinically with varied results.7 calf electrolytes for neonatal diarrhea (po q12 hr for 1-2 days) can combat acidosis and dehydration.2,4 if the ewe or doe is in a more advanced stage of the disease, more aggressive supportive therapy is necessary and the prognosis becomes more guarded. generally in these cases, unless the animal has high reproductive value, treatment is not attempted due to cost limitations. the hypoglycemia should be corrected by oral and/or intravenous administration of a glucose solution, such as 5% dextrose.7 it is important to recognize that hyperglycemia can result in later stages of the disease, and glucose levels should be evaluated before initiation of treatment.7 in combination with glucose treatment, insulin therapy should be initiated and monitored closely. insulin therapy facilitates tissue glucose uptake and inhibits fatty acid mobilization.7 acid-base status, degree of dehydration, associated electrolyte abnormalities, and renal function should be evaluated and appropriate treatment and fluid therapy provided in clinical cases of pregnancy toxemia. fluids with electrolytes can be administered either orally or intravenously depending on the condition of the animal. additional treatment modalities that might prove to be beneficial include administration of b-complex vitamins to stimulate the appetite7 and administration of flunixin meglumine.1,30 offering a variety of feeds and fresh cut grass may also help to stimulate the appetite. in some situations commercially concentrated pellets can be soaked and administered as a gruel via a stomach tube to provide supportive enteral nutrition7 or transfaunation could be considered if a source of rumen fluid is available.5 due to the potential correlation between ketonemia and oxidative stress in ewes with pregnancy toxemia, it may also be imperative to consider antioxidants as a useful addition to protocols for treatment (and potentially prevention) of pregnancy toxemia.17 in cases where high parasite loads are diagnosed, it is recommended to consider administration of a broad-spectrum anthelmintic for treatment of gastrointestinal nematodes and liver trematode parasites.1 it is important to consider the choice of anthelmintic when administering to a pregnant animal. recombinant bovine somatotropin has been administered experimentally to ewes with naturally occurring pregnancy toxemia by daily subcutaneous injection (0.15 mg/kg body weight)31 or a single subcutaneous injection of a slow release formulation (160 mg).32 it appears to improve the efficiency of glucose and ketone body usage at the cellular level, thereby reducing mortality of both the dam and the fetuses, although there was no statistical significance between treated and placebo groups warranting further studies.31,32 however, recombinant bovine somatotropin is currently not approved for extra-label use in small ruminants in the united states. to reduce the glucose demand and effects of negative energy balance, as well as to improve the potential response to therapy, induction of parturition is often recommended in affected females. transabdominal ultrasonography to assess fetal viability can be of benefit when considering induction of parturition if the animal is > 140 days in gestation.1 various protocols have been proposed for induction clinical theriogenology • volume 7, number 4 • december 2015 412 of parturition or termination of pregnancy in small ruminants. induction of parturition or termination of pregnancy in the ewe can be achieved by administering dexamethasone (10-20 mg im), betamethasone (10 mg im), or flumethazone (2.5 mg im) with parturition generally occurring 36-48 hours following administration.1 occasionally if the ewe is < 140 days gestation, a second dose of dexamethasone may be necessary.2 the placenta maintains pregnancy in late-term ewes, so it is important to note that administration of prostaglandin f2α (pgf) alone will not result in induction of parturition or termination of pregnancy. in does, induction of parturition or termination of pregnancy can be achieved by administration of pgf, or an analogue, with or without corticosteroid administration,7,19 because the doe is corpus luteum (cl) dependent throughout the entire pregnancy. dexamethasone administration can hasten fetal maturation if the pregnancy is not at term through stimulation of the fetal pituitary-adrenal axis. administration of dinoprost tromethamine (2.5-15 mg im)33, cloprostenol (75-125 μg im),34 or luprostiol (3.75-7.5 mg im)34 will result in parturition occurring 30-48 hours following administration. administration of dexamethasone alone in does will result in induction of parturition or termination of pregnancy, but generally occurs 48-72 hours following administration.1 it has been demonstrated previously that doses of pgf as low as 1.25 mg are luteolytic,35 although the higher doses of pgf result in a more predictable time of parturition.33 alternatively, cloprostenol (31.25-125 μg) administered by intravulvo-submucosal injection has also been shown to be luteolytic36 and may be considered for induction of parturition or termination of pregnancy in goats. if induction of parturition or termination of pregnancy in small ruminants does not result in delivery of the fetuses within 72 hours, or the animal is severely compromised, a cesarean section should be considered.7 animals that are recumbent, obtunded, have neurologic signs, or develop renal failure have a very poor prognosis for recovery. in severe cases of pregnancy toxemia a cesarean section may be considered without induction of parturition, although dam survival rate is guarded to grave and the cost of surgery must be considered (figure 3). neonatal survival rate is guarded in most of these cases, however delivery of live, viable lambs or kids can result (figure 4). females that are managed with induction of parturition/termination of pregnancy or cesarean section should be treated with broad-spectrum antimicrobials, a non-steroidal anti-inflammatory, oxytocin, and supportive care to facilitate passage of the placenta and prevent metritis.1 it is important for owners to understand the implications of advanced treatment options, as economic issues may limit treatment strategies.7 in cases where the animal is recumbent and unable to stand (figure 5), euthanasia may be indicated for welfare reasons.1,5 ewes or does that do recover from pregnancy toxemia without resultant fetal death often experience dystocia at lambing or kidding, develop a retained placenta, and have reduced lactation. lambs or kids are frequently born dead or weak, and must be managed for potential development of hypothermia and failure of passive transfer.6 pregnancy toxemia should be approached as a flock or herd problem. clinically healthy animals in a flock or herd with clinically ill animals should be assessed for potential risk factors that might predispose them to subclinical disease and a prophylactic treatment plan implemented to prevent clinical disease from developing.1 examination of 10-15% of the animals in the flock or herd may be more financially achievable and still provide valuable information. where possible, blood samples should be collected to evaluate bhb levels.1 animals with elevated bhb levels should be separated and monitored closely. prevention of pregnancy toxemia relies on good feeding practices and husbandry. because pregnancy toxemia occurs during late gestation, nutritional management during all stages (breeding, early gestation, and late gestation) can impact the disease.4 prior to breeding, ewes and does should be maintained on a rising plane of nutrition (flushing) from four weeks prior to breeding to six weeks following breeding.6,37 ideally ewes and does will be a body condition score of 3 or 3.5 out of 5 at the beginning of the breeding season.4,37 in the second and third month of gestation, ewes and does should maintain a consistent body weight and not be overfed. in the fourth and fifth month of gestation fetal growth is at its greatest,6 with fetuses acquiring up to 75-80% of their future body weight.18,37 an adequate nutritional program should be constructed in which adequate rumen-fermentable carbohydrate, clinical theriogenology • volume 7, number 4 • december 2015413 degradable protein, and fiber is provided to support rumen microbial growth.7,37 body condition scores one month prior to parturition should be between 2.5 and 3.5.4,37 lower quality hay can be fed in midpregnancy, or animals may be grazed without additional supplementation, but it should be replaced with high quality hay (ideally analyzed for nutritional content) and concentrates during late pregnancy.6,37 the addition of ionophores to the feed may also be considered during late gestation to encourage glucose production from proprionate.2 body condition should also be monitored and appropriate dietary changes made to ensure that animals are maintained at an ideal body condition score during pregnancy. in some cases forced exercise by enlarging the distance between feed and water sources could be instituted if animals are determined to be over conditioned.2,19 multiparous females with two or more fetuses will have a greater than 200% increase in caloric requirements compared to females with singleton pregnancies.3,37,38 it is therefore important that ewes be evaluated in early gestation for number of fetuses and separated and fed accordingly during late gestation. there are several methods for determining the presence of pregnancy including non-return of mated ewes to estrus, transabdominal palpation, udder examination, biochemical tests (progesterone measurements, ovine interferon tau, pregnancy-associated glycoproteins) and ultrasonographic examination.37 in previous years, serum progesterone concentrations were evaluated for pregnancy diagnosis and determination of fetal numbers in ewes.39,40 rawlings et al. demonstrated that serum progesterone concentrations varied significantly with the number of fetuses present, and that average serum progesterone concentrations for ewes bearing one (5.5±0.3 ng/ml), two (8.0±0.4 ng/ml), or more than two (12.4±2.1 ng/ml) fetuses were significantly different between 94 to 95 days of gestation. evaluation of the average serum concentrations for ewes carrying a single fetus or twins revealed incorrect diagnosis in 18% and 30% of the ewes, respectively. additionally, evaluation of the average serum concentrations for ewes carrying twins or more than two fetuses revealed incorrect diagnosis of fetal numbers in 31% and 59% of the ewes, respectively.40 transabdominal ultrasonography allows for a more accurate determination of fetal numbers,37 as well as diagnosis of viability, growth, size, and age of fetuses. transabdominal ultrasonography can be useful in early gestation, days 30-70, in determining the number of fetuses in order to separate animals into appropriate groups for nutritional management.37 prior to 30 days the uterus is located in the pelvic inlet preventing good visualization using transabdominal ultrasonography.41 as gestation advances, the increase in fetal size and normal reduction of the amount of amnio-allantoic fluid prevents reliable assessment of fetal numbers.42 dawson et al. evaluated alpine does at 35 and 49 days of gestation for pregnancy diagnosis and determination of fetal numbers. pregnant does were accurately differentiated from open does in 100% of the examinations. sensitivity of transabdominal ultrasonography for diagnosis of single kids was 44% and 82% respectively, diagnosis of twins was 73% and 89% respectively, and diagnosis of triplets was 67% and 100% respectively indicating that transabdominal ultrasonography was more accurate at 49 days than at 35 days of gestation.41 these findings were consistent with a previous study performed in sheep.43 gearhart et al. demonstrated that differentiating fetal numbers using transabdominal ultrasonography in suffolk and dorset ewes between 51 and 75 days of gestation was 100% for ewes carrying a single lamb and 97.3% for ewes carrying twins.42 while transabdominal ultrasonography is an accurate method for pregnancy diagnosis and determining fetal numbers, its accuracy can be strongly influenced by technical inexperience.38 veterinarians must gain proper skills for use of ultrasonography to ensure that ewes or does are placed on the right nutritional support during late pregnancy.38 other management issues such as feed bunk space, feed availability, abrupt dietary changes, transportation, processing, weather stressors, and parasite control need to addressed and managed as well as possible.7 conclusion in conclusion, pregnancy toxemia is a complex, multi-factorial disease affecting both sheep and goats. the pathogenesis of the disease is still not well understood, and additional studies are necessary to clinical theriogenology • volume 7, number 4 • december 2015 414 further develop an understanding behind individual variations, genetic links, and metabolic processes that correlate to the disease. while pregnancy toxemia has been described in the literature for decades, response to treatment remains variable. development of precise and economic diagnostic tools will allow the clinician to adjust treatment to individual cases. the cornerstone to reducing economic losses is prevention. it is imperative for veterinarians and producers to work together to manage flocks and herds to prevent the development of pregnancy toxemia in order to reduce economic losses associated with development of the disease. references 1. brozos c, mavrogianni vs, fthenakis gc.: treatment and control of peri-parturient metabolic diseases: pregnancy toxemia, hypocalcemia, hypomagnesemia. vet clin north am food anim pract 2011;27:105-113. 2. bulgin m: diseases of the periparturient ewe. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd edition. st. louis: w.b. saunders; 2007. p. 695-700. 3. lima ms, pascoal ra, stilwell gt: glycemia as a sign of the viability of the foetuses in the last days of gestation in dairy goats with pregnancy toxaemia. ir vet j. 2012;65. 4. rook js: pregnancy toxemia of ewes, does, and beef cows. vet clin north am food anim pract 2000;16:293-317. 5. scott pr: sheep medicine. london: mason publishing; 2010. p. 279-296. 6. andrews a: pregnancy toxaemia in the ewe. in practice 1997;19:306-312. 7. van saun rj: puerperal nutrition and metabolic diseases. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd edition. st. louis: w.b. saunders; 2007. p. 562-572. 8. oltenacu eab, boylan wj: productivity of purebred and crossbred finn sheep. i. reproductive traits of ewes and lamb survival. j anim sci 1981;52:989-997. 9. parr ra, campbell ip, cahill lp, et al: flock glucose profiles and productivity in booroola and random bred control merino ewes. proc aust soc anim prod 1984;15:517-520. 10. schlumbohm c, harmeyer j: twin-pregnancy increases susceptibility of ewes to hypoglycaemic stress and pregnancy toxaemia. res vet sci 2008;84:286-299. 11. duehlmeier r, fluegge i, schwert b, et al: insulin sensitivity during late gestation in ewes affected by pregnancy toxemia and in ewes with high and low susceptibility to this disorder. j vet intern med 2013;27:359-366. 12. moallem u, rozov a, gootwine e, et al: plasma concentrations of key metabolites and insulin in late-pregnant ewes carrying 1 to 5 fetuses. j anim sci 2012;90:318-324. 13. herdt th, sayegh ai: postabsorptive nutrient utilization. in: klein bg, editor. cunningham's textbook of veterinary physiology. 5th edition. st. louis : elsevier saunders; 2013. p. 342-358. 14. schlumbohm c, harmeyer j: hyperketonemia impairs glucose metabolism in pregnant and nonpregnant ewes. j dairy sci 2004;87:350-358. 15. marteniuk jv, herdt th: pregnancy toxemia and ketosis of ewes and does. vet clin north am food anim pract 1988;4:307-315. 16. lacetera n, bernabucci u, ronchi b, et al: effects of subclinical pregnancy toxemia on immune responses in sheep. am j vet res 2001;62:1020-1024. 17. al-qudah km: oxidant and antioxidant profile of hyperketonemic ewes affected by pregnancy toxemia. vet clin pathol 2011;40:60-65. 18. gursel fe, durak mh, alti�ner a: serum ceruloplasmin levels in ewes fed deficient-energy during late pregnancy. j anim vet adv 2010;9:820-825. 19. matthews j: diseases of the goat. 3rd edition. chelmsford (uk): wiley-blackwell; 2009. p. 130-132. 20. boileau mj: pregnancy toxemia in small ruminants. proc j vet intern med 2008:265-267. 21. duehlmeier r, fluegge i, schwert b, et al: post-glucose load changes of plasma key metabolite and insulin concentrations during pregnancy and lactation in ewes with different susceptibility to pregnancy toxaemia. j anim physiol anim nutr 2013;97:971-985. 22. harmeyer j, schlumbohm c: pregnancy impairs ketone body disposal in late gestating ewes: implications for onset of pregnancy toxaemia. res vet sci 2006;81:254-264. 23. duehlmeier r, fluegge i, schwert b, et al: metabolic adaptations to pregnancy and lactation in german blackheaded mutton and finn sheep ewes with different susceptibilities to pregnancy toxaemia. small rumin res 2011;96:178-184. 24. bickhardt k, henze p, ganter m: clinical findings and differential diagnosis of ketosis and hypocalcaemia in sheep. deutsche tierärztliche wochenschrift 1998;105:413-9. 25. bali�kci� e, yi�ldi�z a, gurdogan f: investigation on some biochemical and clinical parameters for pregnancy toxemia in akkaraman ewes. j anim vet adv 2009;8:1268-1273. 26. yari�m gf, ci�ftci� g: serum protein pattern in ewe with pregnancy toxemia. vet res commun 2009;33:431-438. 27. oetzel gr: monitoring and testing dairy herds for metabolic disease. vet clin north am food anim pract 2004;20:651-674. 28. hornig kj, byers sr, callan rj, et al: evaluation of a point-of-care glucose and β-hydroxybutyrate meter operated in various environmental conditions in prepartum and postpartum sheep. am j vet res 2013;74:1059-1065. clinical theriogenology • volume 7, number 4 • december 2015415 29. cal l, borteiro c, benech a, et al: histological changes of the liver and metabolic correlates in ewes with pregnancy toxemia. arq bras med vet zootec 2009;61:306-312. 30. zamir s, rozov a, gootwine e: treatment of pregnancy toxaemia in sheep with flunixin meglumine. vet rec 2009;165:265-266. 31. scott pr, sargison nd, penny cd: evaluation of recombinant bovine somatotropin in the treatment of ovine pregnancy toxaemia. vet j 1998;155:197-199. 32. andrews ah, holland-howes ve, wilkinson jid: naturally occurring pregnancy toxaemia in the ewe and treatment with recombinant bovine somatotropin. small rumin res 1997;23:191-197. 33. bretzlaff kn, ott rs: doses of prostaglandin f2α effective for induction of parturition in goats. theriogenology. 1983;19:849-853. 34. batista m, niño t, alamo d, et al: use of luprostiol and cloprostenol for induction of parturition in pregnant goats. reprod domest anim 2009;44:83-87. 35. bretzlaff kn, ott rs, weston pg, et al: doses of prostaglandin f2 alpha effective for induction of estrus in goats. theriogenology 1981;16:587-591. 36. mgongo fok: doses of prostaglandin analogue "cloprostenol" by intravulvo-submucosal (ivsm) injections effective for the induction of oestrus in goats. anim reprod sci 1987;14:139-146. 37. fthenakis gc, arsenos g, brozos c, et al: health management of ewes during pregnancy. anim reprod sci 2012;130:198-212. 38. alidadi n, rafia s, moaddab h: outbreak of primary pregnancy toxemia in fat tailed ewes due to ultrasonographic misdiagnosis of pregnancy. iran j vet res 2012;13:72-74. 39. gadsby je, heap rb, powell dg, et al: diagnosis of pregnancy and of the number of foetuses in sheep from plasma progesterone concentrations. vet rec 1972;90:339-342. 40. rawlings nc, jeffcoate is, savage nc, et al: pregnancy diagnosis and assessment of fetal numbers in the ewe in a commercial setting. theriogenology 1983;19:655-663. 41. dawson lj, sahlu t, hart sp, et al: determination of fetal numbers in alpine does by real-time ultrasonography. small rumin res 1994;14:225-231. 42. gearhart ma, wingfield we, knight ap, et al: real-time ultrasonography for determining pregnancy status and viable fetal numbers in ewes. theriogenology 1988;30:323-337. 43. white ir, russel ajf, fowler dg: real-time ultrasonic scanning in the diagnosis of pregnancy and the determination of fetal numbers in sheep. vet rec 1984;115:140-143. figure 1. pregnancy toxemia in a ewe: postmortem examination of the sectioned liver with multifocal pale, friable areas demonstrating the typical gross appearance of fatty infiltration. clinical theriogenology • volume 7, number 4 • december 2015 416 figure 2. pregnancy toxemia in a ewe: histologic section of liver demonstrating mild hepatocellular degeneration with lipid infiltration (arrows). figure 3. pregnancy toxemia in a recumbent obtunded targhee ewe at term: parturition was induced with administration of dexamethasone and cesarean section was scheduled. clinical theriogenology • volume 7, number 4 • december 2015417 figure 4. three crossbred lambs delivered through cesarean section from a ewe with pregnancy toxemia following induction of parturition. figure 5. pregnancy toxemia in a ewe: parturition was induced with administration of dexamethasone. postpartum the ewe was managed for recumbency and recovered following one week of supportive medical therapy. (editor’s note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 7, number 4 • december 2015 418 2014: bovine sperm abnormalities: prevalence, etiology and mechanisms leading to infertility bovine sperm abnormalities: prevalence, etiology and mechanisms leading to infertility jacob c. thundathil, alysha l. dance, john p. kastelic faculty of veterinary medicine, department of production animal health, university of calgary, calgary, ab, canada introduction sperm morphology is an excellent predictor of the outcome of natural mating1,2 and artificial insemination (ai),3,4 and success of in vitro fertilization5 in animals and humans. bovine sperm abnormalities are classified based on origin (primary, secondary or tertiary), effect on fertility (major vs minor), location of the sperm defect (head, midpiece, or tail), or whether its effects on fertility can be ameliorated by increasing the number of sperm in the inseminate (compensable vs uncompensable).6 although prevalence of various types of sperm abnormalities is low in the ejaculates and ~ 83% of bulls subjected to a traditional breeding soundness examination are classified as satisfactory breeders exclusively based on sperm morphology, it is noteworthy that 17% of bulls subjected to breeding soundness evaluation were designated unsatisfactory solely on the basis of sperm morphology, highlighting its importance in alberta.7 although an association between sperm abnormalities and infertility has been established and functional impairments of sperm abnormalities have been documented,8 the molecular basis of impaired function of abnormal sperm remains unknown. our recent studies demonstrated that expression of sperm proteins are altered in abnormal sperm, thereby affecting fertility. since sperm dna is transcriptionally inactive, content of sperm proteins in the mature sperm influences fertilizing ability. this paper summarizes current knowledge regarding the prevalence, etiology and mechanisms of infertility due to abnormal bovine sperm. prevalence of abnormal sperm morphology in alberta beef bulls we recently conducted a survey to determine the prevalence of sperm abnormalities in beef bulls in alberta, canada, and the percentage of satisfactory potential breeders based solely on normal sperm morphology. eosin-nigrosin stained semen smears and evaluation reports of 1642 bull breeding soundness evaluations were procured from six veterinary clinics in alberta. sperm morphology was determined for at least 100 sperm per bull. the most common defects were detached head (4.86±5.71%; mean±sd), distal midpiece reflex (6.19±9.13%), and bent tail (1.01±1.54%). overall, solely on the basis of sperm morphology, 1363 (83.0%) bulls were classified as satisfactory potential breeders, with the remainder 279 (17.0%) deemed unsatisfactory (>30% abnormal sperm, >20% defective heads, or both). that 17% of bulls subjected to breeding soundness evaluation were designated unsatisfactory solely on the basis of sperm morphology highlighted its importance.7 abnormal sperm morphology causes of abnormal sperm morphology the most common causes of abnormal spermatogenesis in bulls include: 1) abnormal thermoregulation of the testes due to heat, frostbite or fat deposition around the scrotal neck; 2) hormonal imbalances associated with stress; and 3) effects of toxins and expression of deleterious genes.9 consistent with this, chenoweth et al10 listed environmental factors as the most common cause of abnormal sperm structures; however, they also noted that increasing numbers of defects are being considered as of a genetic origin.10 increased scrotal temperature impairs spermatogenesis by increasing the metabolic rate and the oxygen demand. however, since the blood flow to the testis does not increase in response to this increased metabolic demand, this causes hypoxia of the testicular tissue, disrupts spermatogenesis, and can lead to abnormal sperm production.9 stress can have numerous causes, including the environment, illness, or injury. stress typically results in elevated systemic cortisol concentrations, decreased secretion of luteinizing hormone and testosterone, which cause hormonal imbalances and thus affect spermatogenesis.11 reported effects of specific genes on sperm morphology are reviewed under specific sperm abnormalities. 525 clinical theriogenology • volume 6, number 4 • december 2014 acrosomal abnormalities bull sperm (fresh or frozen-thawed) must have a tightly adherent, intact acrosome with a smooth surface and a distinct, uniformly shaped apical ridge.12 during spermiogenesis, an acrosome arises from the golgi complex and spreads over the anterior surface of the spermatid nucleus. the abundance of microtubules in the sertoli cell cytoplasmic processes surrounding the elongating spermatids suggests involvement of microtubules in sperm head shaping and transport of sperm organelle. microtubules are believed to have a major role in trafficking acrosomal vesicles from the golgi apparatus to the sperm nucleus, a critical step in acrosome development.13 the acrosome contains a variety of enzymes that facilitate fertilization by digesting the oocyte zona pellucida. microtubules are present within this region, and inhibitors interfere with acrosome biogenesis by inhibiting the ability of golgi-derived acrosomal vesicles to dock onto the nuclear membrane and coalesce into the pro-acrosomal vesicle.14,15 any aberrations including incomplete or irregular apical ridge, irregular acrosomal surface, grossly distorted acrosomes (swollen, ruffled or vacuolated) are classified as acrosomal abnormalities.12 these researchers demonstrated that the percentage of sperm with normal acrosomal morphology improved more slowly and had not reached mature levels by 16 wk after puberty (based on a study in beef bulls conducted almost 30 years ago). therefore, further studies are warranted as changes in genetics, environment and nutrition may have influenced spermatogenesis, influencing the time interval between puberty and age at which bulls produce sperm with normal acrosomal morphology. although acrosomal morphology may be improved during the peri-pubertal period, various acrosomal abnormalities have also been reported in mature bulls. classical knobbed acrosome defect in mature bulls has been described as a beaded appearance at the apex of the affected sperm.6 however, the more common form of this acrosomal defect is characterized by either flattened or indented sperm apex. although stress, abnormal thermoregulation of testes or genetic causes are potential causes for abnormal spermatogenesis, the specific causes leading to flattened or indented acrosomes remain unknown. concurrent appearance of a variety of sperm abnormalities, along with acrosomal abnormalities, are considered to be due to adverse environmental influence (stress or injury). however, a genetic cause should be considered when a high proportion of the sperm have acrosomal abnormalities and this defect is persistent in the absence of other sperm defects.6,10 the knobbed acrosome sperm defect was associated with an autosomal sex-linked recessive mode of genetic transmission in friesian breed16 and in boars.17,18 based on a canadian study on beef bulls used for natural breeding, 0.53% bulls (n =1331) had sperm with knobbed acrosomes, ranging from 25 to 100% sperm, and 1.6 % of beef and dairy bulls (n = 371) with low nonreturn rates to artificial insemination had high proportions of knobbed acrosomes (25 to 95% of affected sperm)6 due to unknown causes. in contrast, chenoweth10 reported an estimated genetic prevalence of 6.74% in an angus herd. a deletion in the long arm of y chromosome has been shown to be involved in acrosomal abnormalities described as "flat heads" in mice where acrosomes appeared damaged and often contained a vesicle, but lacked acrosomal proteinase. in mice deficient for tlf (tbplike factor), a protein that is critical for spermatogenesis, acrosomal granules did not coalesce in stage ii– iii spermatids, but were present in the cytoplasm, on the outer membrane of the nucleus, or invaginated in the nucleus, or elongating spermatids at stages ix-xi. in these cases, acrosomes were vacuolated or not properly associated with the head. during normal spermiogenesis, acrosomal vesicular contents should be processed (for example, removal of lysosomal proteins) for normal acrosomal biogenesis. any impairment in these maturational changes in the acrosome may lead to the development of an abnormal acrosome.19,20 acrosomal abnormalities due to toxins have been reported. subcutaneous administration of bisphenol a for six days at 20 or 200 μg/kg per injection induced a variety of acrosomal abnormalities affecting the acrosomal vesicle and acrosomal cap.19 the knobbed acrosome defect has been associated with infertility in many species, including pigs,21,22 horses23 and sheep.24 bulls with a high percentage of sperm with indented acrosomes may have normal fertility when used for ai or single-sire mating; however, their fertility may be low when breeding 526clinical theriogenology • volume 6, number 4 • december 2014 competitively with bulls with normal spermiograms.25 we used an ivf and culture system to determine the effect of bovine sperm with flattened or indented acrosome on fertilization and early embryonic development.26 results indicated that sperm with acrosomal abnormalities had a reduced ability to bind to the zona pellucida, depending upon the severity of the defect, and that these aberrant spermatozoa did not penetrate the zona pellucida. furthermore, apparently normal spermatozoa co-existing in the inseminate of bulls with a high percentage of sperm with acrosomal abnormalities were also functionally deficient; oocytes penetrated by these spermatozoa had a reduced potential for fertilization, and resulting zygotes had a reduced ability for cleavage and embryonic development to the blastocyst stage. we subsequently reported that bovine sperm with flattened and indented acrosomes had altered plasma membrane functional integrity, which predisposed them to premature capacitation and acrosome reaction, contributing to their inability to interact with the oocyte.27 although proteolytic activity of the acrosomal contents in the affected sperm appeared to be normal, sperm with the flattened or indented acrosomes also appeared to have a reduced ability to fuse with oolemma (demonstrated by a zona free oocyte-sperm fusion assay, coupled with confocal microscopy), impairing the ability of sperm with flattened and indented acrosome to penetrate ooplasm and undergo sperm chromatin decondensation.28 abnormal sperm head shape during spermatogenesis, haploid spermatids undergo a series of differentiation processes to achieve species-specific sperm morphology. although chromatin compaction is achieved by replacement of nuclear histones with protamines, sperm head shaping is facilitated by the manchette, a cuff-like sleeve of microtubules emerging from a perinuclear ring located at the junction of the acrosome-postacrosomal region during the acrosome phase of spermiogenesis. since the manchette moves caudally as sperm head elongates, it is believed that manchette sculpts sperm head shape.13 therefore, any impairments in development and function of this structure may lead to abnormal sperm head shape. in addition, the extensive and dynamic microtubule network existing in the sertoli cells is involved in regulating sperm head shape.13 sertoli cell microtubules have dramatic changes in their microtubule patterns as the germ cells progress through spermiogenesis.13 the microtubules in the apical processes of sertoli cells mediates shape changes in sertoli cells as they respond to changes in the germ cells (migration and cell shape). therefore, impaired function of sertoli cells may have a major role in development of abnormal sperm head shape. pyriform sperm has been reported as the most common sperm defect in bulls.6 a classical pyriform sperm have a pear-shaped head, a normal acrosome,6,29-31 and a narrow postacrosomal region.30 in canadian beef bulls, the overall incidence of the pyriform defect was 10%, with 1.3% of the bulls having more than half of their sperm affected with this defect.6 several variants of pyriform sperm have also been reported, from almost normal to varying degrees of narrowness to sperm head to severely pyriform sperm and their effects on fertility have been documented through comprehensive breeding trials.32 based on this study it was concluded that a moderate degree of sperm head narrowness, in the absence of other seminal signs of a disturbance of spermatogenesis, is not detrimental to fertility. however, extreme narrowness of the postacrosomal region of the sperm head of most sperm, as present in two bulls without other substantial signs of a disturbance of spermatogenesis, resulted in significantly reduced fertility. we evaluated the effects of the pyriform defect on fertility in vitro and determined that they had a reduced ability to bind to and penetrate the zona pellucida. if fertilization does occur, the resulting zygotes had a reduced ability to initiate cleavage.33 it is likely that pyriform sperm have a defective centrosome, affecting defective development of sperm aster following fertilization, blocking syngamy, cleavage or defective embryos leading to early embryonic loss as reported in humans.34 since sperm dna is transcriptionally inactive, sperm functions are regulated by proteins already present in sperm (without additional protein synthesis). therefore, we conducted proteomic analyses of pyriform sperm to elucidate molecular basis of functional impairments in this defect. expression of several proteins involved in sperm capacitation, sperm-egg interaction and sperm cytoskeletal structure were decreased in pyriform sperm, whereas proteins regulating antioxidant activity, apoptosis and 527 clinical theriogenology • volume 6, number 4 • december 2014 metabolic activity were increased. furthermore, contents of reactive oxygen species and ubiquitinated proteins were higher in pyriform sperm.35 nuclear vacuoles nuclear vacuoles (pouches, craters, diadems) have been described in bull sperm as well as in the sperm of other species.36-40 this is a narrow-mouthed invagination of the nuclear membrane into the nucleoplasm of the sperm head.41 the incidence of vacuoles in the spermatozoa of affected bulls varies from less than 1% to nearly 100%. however, a definitive cause for nuclear vacuolation of spermatozoa has not been detected.6 based on electron microscopic studies, vacuole formation in bulls begins as early as step 6 of spermiogenesis.42 nuclear vacuoles appear in bull sperm in two forms; single apical vacuoles, which can be present anywhere in the sperm head, and multiple nuclear vacuoles at the acrosome post-acrosomal junction,43-46 appearing as a string of beads.47 these vacuoles are formed during the early stages of spermatid elongation by invagination of the inner nuclear membrane into the condensing nucleus.8 breeding trials demonstrated that sperm with nuclear vacuoles caused infertility in the bull.45,46 superovulated cows were bred with semen from a bull with 80% nuclear vacuoles; the fertilization rate was 18% compared to 72% for control bulls.45 the diadem/crater defect was studied over several months in two related 20month-old angus bulls. in bull 1, diadem/crater defects were present in 2-99% of ejaculated spermatozoa at various times during the evaluation period. in bull 2, affected cells varied from 20 to 94%, with other abnormalities (head and acrosome defects, coiled tails, proximal cytoplasmic droplets) also common. single-sire mating trials conducted over 26 days during an apparent recovery phase yielded normal fertility. both resting and gonadotropin-releasing hormone-stimulated testosterone concentrations were within normal limits. histopathological evaluation of testes showed no obvious hypoplastic, inflammatory, or degenerative condition. however, electron microscopy of ejaculated spermatozoa demonstrated the characteristic diadem pattern of craters in the equatorial region of the head. many cells from bull 2 contained large craters in other regions of the nucleus. electron microscopy of testicular tissue demonstrated nuclear invaginations lined by a single unit membrane in round spermatids. lesions in elongated spermatids were more pronounced, with curling of the nucleus and large membrane-filled cavities in the chromatin occurring in addition to craters in the equatorial region of the nucleus.48 saacke et al49 investigated efficiency of barriers in the female tract against spermatozoa with abnormal heads. in experiment 1, day 6 ova/embryos were recovered nonsurgically from superovulated and single-ovulating cows following artificial insemination with semen of bulls selected for normal spermatozoal motility (≥50%) and high content (>30%) of spermatozoa with misshapen heads, random nuclear vacuoles, or the diadem defect. to assess characteristics of spermatozoa capable of traversing barriers in the female tract, accessory spermatozoa were classified morphologically (x 1250) and compared with those in the inseminate. accessory spermatozoa from 31 ova/embryos recovered from 44 cows were more normal in head shape than those in the inseminate (76 vs 62%; p < 0.05). however, spermatozoa with normal head shape, but with nuclear vacuoles, appeared as accessory spermatozoa at the same frequency as they were found in the inseminate. we used semen containing high percentage of sperm with multiple nuclear vacuoles in ivf and to determine subsequent embryo development. vacuolated sperm were deficient in their ability to bind to and penetrate the zona pellucida of ivm bovine oocytes. results of embryo culture suggested that vacuolated sperm which gained access to the ooplasm participated normally in fertilization and that the zygotes resulting from the fertilization of ivm oocytes by vacuolated sperm developed normally through morulae to blastocysts. the infertility caused by vacuolated sperm in vivo would appear to be due to a reduced ability to pass through female reproductive tract, reduced zona binding and reduced penetration of the zona pellucid.8 however, a semen sample with high percentage of spermatozoa with multiple nuclear vacuoles can be used in an ivf system for the successful production of embryos, although their developmental competence beyond blastocysts remains to be elucidated. 528clinical theriogenology • volume 6, number 4 • december 2014 detached heads a small percentage of detached heads are routinely identified in the semen of bulls with normal fertility. a specific defect, known as the decapitated sperm defect, was inherited in the guernsey breed, probably as a sex-limited recessive gene; affected bulls produce semen with an increased percentage of detached heads, resulting in low fertility or sterility.50 evidence for the inherited nature of this defect was also noticed in a group of eight hereford bulls diagnosed with testicular hypoplasia with no presenting genital abnormalities and maintained under ideal managemental conditions; however, they were traced back to a common ancestral origin.51 blom and birch-anderson52 suggested that the head and tail separation was due to defective formation of the basal plate, resulting in severe instability of the implantation fossa; the defect became evident in the caput epididymal sperm as the cells begin to initiate motility. conditions such as testicular degeneration or inflammation of the seminal vesicles, ampullae and epididymis have been associated with higher numbers of detached heads.6 bulls that were maintained in a sexually inactive state for a prolonged interval had a higher percentage of detached heads that subsequently returned to normal percentages following successive ejaculation. in a study conducted on 1642 alberta beef bulls (ranging from 11 to 26 months, with angus, simmental, charolais, hereford, and limousin bulls), the incidence of detached heads was 4.86 ± 5.71 % (mean ± sem). similarly, in another study by arteaga et al53 on 1641 yearling canadian beef bulls, the prevalence of detached heads decreased from 4.69 to 2.89% as the age of the bulls increased from 11 to 15 months. infertility in bulls may arise, depending upon the percentage of sperm that are affected by this defect. in a report by cooper and peet54 on a previously fertile hereford bull, testicular degeneration secondary to toxemia and laminitis lead to 91% detached heads in the ejaculate and infertility. tail defects mitochondrial sheath defects. sperm mitochondrial sheaths have a major role during motility. any impairment in this structure at any given point in the tail could lead to splitting and disruption of axial fibers at that level, leading to local damage of the tail.6 missing mitochondria or mitochondrial aplasia (gaps in the mitochondrial sheath) is the most common mitochondrial sheath defect in bulls. bulls with this defect seem to have normal progressive motility, with no negative effects on pregnancy rates of inseminated cows,55 as reported in charolais bulls. however, large gaps in the mitochondrial sheath result in weakness and can predispose sperm to fracture and separation of the principal piece from the midpiece, thereby reducing motility and fertility. proximal droplets. cytoplasmic droplets are commonly located either in the midpiece (proximal droplets) or the principal piece of the sperm tail (distal droplets).56 these masses of residual spherical cytoplasm should be normally released down the tail during spermiogenesis and shed when sperm are exposed to seminal plasma during ejaculation.8,56 presence of a high percentage of sperm with the cytoplasmic droplets is a sign of failure of maturation, abnormal spermiogenesis, or abnormal epididymal function.6,57 peripubertal bulls typically have a higher percentage of sperm with proximal droplets;6,57-61 however, as bulls mature, the percentage of proximal droplets in their semen declines to a normal level.6 based on a study in beef bulls, 67.1% of the bulls produced sperm with proximal droplets (average percentage 2.7 ± 0.25%; 8). there is general consensus that a high percentage of sperm with proximal droplets in a semen sample has a negative impact on fertility.62-65 furthermore, when sperm with a high prevalence of proximal droplets are used for ivf, zona binding and cleavage rates were poor.8,66 dag defect. this is a severe deformity of the midpiece, characterized by fractures of the axonemal fibers and mitochondrial sheath disruption. it has been reported that this sperm morphological defect has a heritable basis in jersey and hereford (up to 100% of sperm can be affected). defective formation of the mitochondrial sheath during spermiogenesis is considered the primary cause. distal midpiece reflex. distal midpiece reflex is the most common sperm tail abnormality in bovine sperm. this defect is characterized by a bend in the distal region of the midpiece in the shape of the letter “j”. a cytoplasmic droplet may be trapped within the bend. this sperm defect is believed to be epididymal in origin. affected sperm have impaired motility. since this defect is often stress-induced, the prognosis for improvement is generally good. therefore, bulls classified as unsatisfactory due to high 529 clinical theriogenology • volume 6, number 4 • december 2014 percentage of sperm with distal midpiece reflex in their semen should be re-evaluated on a monthly basis to establish breeding soundness. distal droplets. distal droplets do not cause substantial impairment of fertility even at high percentages67 and therefore sperm with normal morphology retaining distal droplets should not be considered as abnormal.68 this was supported in a recent study on swedish bulls which had higher percentages of distal droplets, although the defect was not significantly correlated with fertility status as determined by non-return rate.69 in another study of zebu bulls, it was suggested that there is an agedependent relationship of the sire to the occurrence of the distal droplets, with immature bulls displaying high percentages, whereas mature and older bulls had a low prevalence.70 we recently demonstrated that a high percentage of sperm recovered from cauda epididymis of bison bulls contain distal droplets. however, incubation of these sperm in the talph buffer resulted in shedding of these droplets.71 therefore, it is very likely that sperm with distal droplets may loose their droplets in the female reproductive tract immediately after ejaculation without affecting their subsequent migration in the female reproductive tract. abaxial and accessory tail. the abaxial tail defect is observed when the tail is attached to the head at an angle and is often accompanied by accessory tails, 2-3 µm long stumps arising from a secondary implantation region lateral to the main tail. in normal spermiogenesis, suppression of the centriole replication is apparent, so that sperm develop only one axoneme. in abnormal sperm, it is probable that there is failure of suppression or partial suppression of centriole replication in the spermatid leading to development of the accessory tails.6 bulls having such tail defects do not have any testicular abnormalities and do not appear affected by external factors that can adversely affect spermatogenesis. therefore, it is assumed that the defect is due to the genetic makeup of the bull. bulls affected with accessory tails may have impaired fertility. in contrast, abaxial tails do not have any detrimental effects on fertility and therefore they should be considered as a normal variation of bovine sperm morphology.6 in extreme conditions, an abaxial sperm is held at an acute angle and therefore affected sperm swim in tight circles; such cases are classified as abnormal sperm.67 summary in general, bovine sperm with abnormal morphology are discriminated against during zona binding and penetration, suggesting that impairment in molecular mechanisms of sperm-oocyte interaction may be similar among various types of aberrant sperm morphologies. therefore, abnormal morphology may be considered a marker for an accompanying functional deficiency. sperm with abnormal morphology have impaired ability to interact with oocyte or initiate embryonic development. in addition, deleterious effects of abnormal sperm may compromise the fertilizing ability of normal sperm co-existing in the ejaculate. therefore, abnormal sperm may be a symptom of accompanying functional deficiencies of the entire sperm population. semen samples classified as satisfactory should contain at least 70% morphologically normal sperm, with no more than 20% of sperm having defective heads. acknowledgement this manuscript is a revised version of the paper published in the proceedings of the annual meeting of the national association of animal breeders 2012 (thundathil j, et al: test your knowledge: evaluation of bovine sperm morphology. proc. 24th naab technical conference on ai and reprod; 2012. p. 86-93). references 1. bonde jpe, ernst e, jensen tk, et al: relation between semen quality and fertility: a population-based study of 430 firstpregnancy planners. lancet 1998;352:1172-1177. 2. wiltbank jn, parish nr: pregnancy rate in cows and heifers bred to bulls selected for semen quality. theriogenology 1986;25:779-783. 3. karabinus ds, gelety tj: the impact of sperm morphology evaluated by strict criteria on intrauterine insemination success. fertil steril 1997;67:536-541. 4. saacke rg, dalton jc, nadie s, et al: relationship of seminal traits and insemination time to fertilization rate and embryo quality. anim reprod sci 2000;60-61:663-677. 530clinical theriogenology • volume 6, number 4 • december 2014 5. coetzee k, kruge t, lombard c: predictive value of normal sperm morphology: a structured literature review. hum reprod update 1998;4:73-82. 6. barth ad, oko rj: abnormal morphology of bovine spermatozoa. ames: iowa state university press; 1989. p. 1-285. 7. menon ag, barkema hw, wilde r, et al: associations between sperm abnormalities, breed, age, and scrotal circumference in beef bulls. can j vet res 2011;75:241-247. 8. thundathil jc: in vitro fertilizing characteristics of bovine sperm with abnormal morphology [dissertation]. saskatoon (sk): university of saskatoon; 2001. 9. barth ad, bowmane pa: the sequential appearance of sperm abnormalities after scrotal insulation of dexamethasone treatment in bulls. can vet j 1994;34:93-102. 10. chenoweth pj: genetic sperm defects. theriogenology 2005;64:457-468. 11. welsh th, johnson bh: stress induced alteration in secretion of corticosteroids, progesterone, luteinizing hormone and testosterone in bulls. endocrinology 1981;109:185-190. 12. lunstra dd, echternkamp se: puberty in beef bulls: acrosome morphology and semen quality in bulls of different breeds. j anim sci 1982;55:638-648. 13. o’donell l. o’bryan mk: microtubules and spermatogenesis. semin cell dev biol. in press 2014. 14. ventela s, mulari m, okabe m, et al: regulation of acrosome formation in mice expressing green fluorescent proteins as a marker. tissue cell 2000;32:501-507. 15. huang wp, ho hc: role of microtubule-dependent membrane trafficking in acrosomal biogenesis. cell tissue res 2006;323:495-503. 16. eddy em. the spermatozoon. in: knobil e, neill j. editors. the physiology of reproduction. new york: ravon press; 1988. p. 106-108. 17. wohlfarth e: beitrag zum akrosom-defekt im eberspermia. zuchthyg fortpflstor besam haustiere 1961;5:268. 18. bishop mwh: genetically determined abnormalities of the reproductive system. j reprod fertil suppl 1972;15:51. 19. toshimori k, ito c, maekawa m, et al: impairment of spermatogenesis leading to infertility. anat sci int 2004;79:101-111. 20. ramalho-santos j, schatten g, moreno rd: control of membrane fusion during spermiogenesis and the acrosome reaction. biol reprod 2002;67:1043-1051. 21. buttle hrl, hancock jl: sterile boars with “knobbed” spermatozoa. j agric sci 1965;65:255-260. 22. bane a, nicander l: electron and light microscopical studies on spermateliosis in a boar with acrosome abnormalities. j reprod fertil 1966;11:133-138. 23. hurtgen, jp, johnson la: fertility of stallions with abnormalities of sperm acrosome. j reprod fertil 1982;32(suppl):15-20. 24. savage nc: infertility in a ram associate with a knobbed acrosome abnormality of the spermatozoa. can vet j 1984;25:126127. 25. meyer ra, barth ad: effect of acrosomal defects on fertility of bulls used in artificial insemination and natural breeding. can vet j 2001;42:627,630-634. 26. thundathil j, meyer r, palasz at, et al: effect of the knobbed acrosome defect in bovine sperm on ivf and embryo production. theriogenology 2000;54:921-934 27. thundathil j, palasz at, barth ad, et al: plasma membrane and acrosomal integrity in bovine spermatozoa with the knobbed acrosome defect. theriogenology 2002;1:58:87-102. 28. thundathil j, palomino j, barth a, et al: fertilizing characteristics of bovine sperm with flattened or indented acrosomes. anim reprod sci 2001;15:67:231-243. 29. liu dy, barker hw: morphology of spermatozoa bound to the zona pellucida of human oocytes that failed to fertilize in vitro. j reprod fertil 1992;94:71-84. 30. menkveld r, holleboom cag, rhemrev jpt: measurement and significance of sperm morphology. asian j androl 2011;13:59-68. 31. walters ah, eyestone we, saacke rg, et al: bovine embryo development after ivf with spermatozoa having abnormal morphology. theriogenology 2005;63:1925-1937. 32. barth ad, bowman pa, bow ga, et al: effect of narrow sperm head on fertility. can vet j 1992;33:31-39. 33. thundathil j, palasz at, mapletoft rj, et al: an investigation on the fertilizing characteristics of pyriform-shaped bovine spermatozoa. anim reprod sci 1999;57:35-50. 34. rawe vy, terada y, nakamura s, et al: a pathology of the sperm centriole responsible for defective sperm aster formation, syngamy and cleavage. hum reprod 2002;17:2344-2349. 35. shojaei saadi ha, van riemsdijk e, dance al, et al: proteins association with critical sperm functions and sperm head shape are differentially expressed in morphologically abnormal bovine sperm induced by scrotal insulation. j proteomics 2013;82:64-80. 36. truirr-gilbert aj, johnson la: the crater defect in boar spermatozoa: a correlative study with transmission electron microscopy, scanning electron microscopy and light microscopy. gamete res 1980;3:259-266. 37. fawcett dw, anderson wa, phillips dm: morphogenic factors influencing the shape of the sperm head. dev biol 1971;26:220-251. 38. bedford jm: fine structure of sperm head in ejaculate and uterine spermatozoa of the rabbit. j reprod fertil 1964;7:221-228. 39. bellve ar, anderson e, l. hanley-bowdoin l: synthesis and amino acid composition of basic protein in mammalian sperm nuclei. dev biol 1975;47:349-365. 40. anberg a: the ultrastructure of the human spermatozoa. acta obstet gynecol scand 1957;36(suppl 2):1-133. 41. bane a, nicander l: electron and light microscopical studies on spermateliosis in a boar with acrosome abnormalities j reprod fertil 1965;11:133-138. 42. oko rj. normal and defective bovine spermatogenesis [thesis]. calgary: university of calgary; 1977. 43. blom e, birch-andersen a: the ultrastructure of the bull sperm. ii. the sperm head. nord vet med 1965;17:193-212. 531 clinical theriogenology • volume 6, number 4 • december 2014 44. jiranek e, rob o: examination of vacuoles in the bull sperm nucleoplasma from fresh and deep frozen semen. vet med (praha) 1971;44:495-500. 45. miller dm, cates wf, mapletoft rj: infertility in a bull with a nuclear sperm defect: a case report. theriogenology 1982;17:611-621. 46. barth ad: the effect of nuclear vacuoles in bovine spermatozoa on fertility in superovulated heifers. proc can west soc reprod biol; 1984. p. 4-5. 47. bane a, nicander l: pouch formations by invaginations of the nuclear envelope of bovine and porcine sperm as a sign of disturbed spermiogenesis. nord vet med 1965;17:628-632. 48. larsen re, chenoweth pj: diadem/crater defects in spermatozoa from two related angus bulls. mol reprod dev 1990;25:8796. 49. saacke rg, dejarnette jm, bame jh, et al: can spermatozoa with abnormal heads gain access to the ovum in artificially inseminated superand single-ovulating cattle? theriogenology 1998;50:117-128. 50. alun-jones w: abnormal morphology of the spermatozoa in guernsey bulls. brit vet j 1962;118:257-261. 51. williams g: an abnormality of the spermatozoa of some hereford bulls. vet rec 1965;77:1204-1206. 52. blom e, birch-anderson a: ultrastructure of the “decapitated sperm defect” in guernsey bulls. j reprod fertil 1970;23:6772. 53. arteaga a, baracaldo m, barth ad. the proportion of beef bulls in western canada with mature spermiograms at 11 to 15 months of age. can vet j 2011;42:783-787. 54. cooper am, peet rl: infertility in a hereford bull associated with increased numbers of detached sperm heads in his ejaculate. aust vet j 1983;60:225-226. 55. rocha a, oliveira e,vilhena mj, et al: a novel apical midpiece defect in the spermatozoa of a bull without an apparent decrease in motility and fertility-a case study. theriogenology 2006;66:913-922. 56. pesch s, bergman m: structure of mammalian spermatozoa in respect to viability, fertility and cryopreservation. micron 2006;37:597-612. 57. johnson kr, dewy ce, bobo jk, et al: prevalence of morphologic defects in spermatozoa from beef bulls. j am vet med assoc 1998;213:1468-1471. 58. lagerlof n: morphological studies on the changes in sperm structure and in the testes of bulls with decreased or abolished fertility. acta pathol microbiol scand 1934;19:254-266. 59. ruttle jl, ezaz z, sceery ej: some factors influencing the semen characteristics in range bulls. j anim sci 1975;41:10691076. 60. saacke rg: morphology of the sperm and its relationship to fertility. proc tech conf ai reprod nat assoc anim breed; 1970. p. 17-29. 61. smith mf, morris dl, amoss ms: relationships among fertility, scrotal circumference, seminal quality, and libido in santa gertrudis bulls. theriogenology 1981;16:379-397. 62. blom e: sperm morphology with reference to bull infertility. first all-india symp anim reprod ludhina; 1977. p 61-81. 63. dott hm, dingle jt: distribution of lysosomal enzymes in the spermatozoa and cytoplasmic droplets of bull and ram. exp cell res 1968;52:523-540. 64. soderquist l, janson l, larsson k, et al: sperm morphology and fertility in ai bulls. zentralbl veterinarmed a 1991;38:534543. 65. mortimer rg, seidel ge, amann rp, et al: use of in viro fertilization to evaluate spermatozoa with proximal droplets in young beef bulls [abstract]. theriogenology 1991;35:247. 66. amann rp, seidel ge, mortimer rg: fertilizing potential in vitro of semen from young beef bulls containing a high or low percentage of sperm with a proximal droplet. theriogenology 2000;54:1499-1515. 67. barth ad: evaluation of potential breeding soundness of the bull. in: youngquist rs, editor. current therapy in large animal theriogenology. philadelphia: wb saunders; 1997. p 222-236. 68. barth ad: bull breeding soundness evaluation. proc western can assoc bovine pract; 2000. p. 1-68. 69. al-makhzoomi a, lundeheim n, haard m, et al: sperm morphology and fertility of progeny-tested ai dairy bulls in sweden. theriogenology 2008;70:682-691. 70. kashoma ipb, luziga c, mgongo fok: prevalence of spermatozoa morphologic defects from zebu bulls under free mating system. tanzania vet j 2009;26:1323. 71. aurini lc, whiteside dp, elkin bt, et al: recovery and cryopreservation of epididymal sperm of plains bison (bison bison bison) as a model for salvaging the genetics of wood bison (bison bison athabascae). reprod domest anim 2009;44:815-822. 532clinical theriogenology • volume 6, number 4 • december 2014 2013: diet and parasite association (in 15 minutes or less) diet and parasite association (in 15 minutes or less) d.g. pugh southern traxx vet services waverly, al internal parasitism is the most significant health risk affecting production and causes economic losses for producers. parasitic infections can cause death, loss of function, poor reproductive performance, depressed weaning weights, decreased milk production, poor fiber/wool/mohair production, etc. due to the difficulty in parasite control and the widespread resistance to deworming compounds, clinicians should focus their efforts on management, and away from simply ‘deworming’. the clinician should attempt to utilize some of the following management practices: (1) employ husbandry and feeding practices that minimize parasite infection. increasing the intake of dietary protein rich in sulfur-containing amino acids will help prevent clinical signs seen with parasitism particularly in those animals in greatest need. supplemental soybean meal and energy can improve resilience after animals are dewormed. providing good forages and meeting protein, energy, mineral and vitamin requirements are critical to maximizing flock or individual goat production. insuring adequate macro and trace minerals (with emphasis on ca, p, zn, cu, and se) and allowing access to condensed tannin containing forages will aid in the reduction of internal parasite burdens. animals fed to improve health are usually prone to have better resistance and resilience. providing supplemental protein, particularly with protected protein, improves resistance and resilience. feeding forages with condensed tannins may help prevent parasitic disease. some dietary/herbal modifications of the diet include: feeding small ruminants sericea lespedeza hay or pelleted sericea lespedeza. feeding birdsfoot trefoil and sainfoin for ≈ two weeks appears to reduce the fecal egg count (fec). feeding 600 mg/kg bw of orange oil emulsion can reduce the fec by over 90%. feeding albizia anthelmintica bark has some effect by reducing the fec by nearly 80% in some studies. lippia sioides essential oil may also reduce the fec by over 50%. ethanolic extracts of iris hookeriana rhizome may reduce the fec by about 45%, whereas feeding azadirachta indica (neem) leaves does not affect fec when studied. feeding azadirachta indica seed may reduce fec by up to 40%. rotate pastures, allowing small ruminants access to tannin and/or protein rich forages (legumes); use proper pasture fertilization techniques, and administer dewormers to maximize herd production while minimizing the harmful effects of internal parasites. animals fed to meet nutritional requirements for optimal growth/production are more prone to have better resistance and resilience. (2) identify the goat, sheep, or camelids that need to be dewormed and only treat only those animals. this will help maintain refugia, thus diminishing or slowing the onset of anthelmintic resistance in parasite populations in that flock or herd. (note: famacha is the most popular of these targeted deworming systems). (3) ensure adequate intake of all deworming products by weighing each animal; avoid administering an “average body weight” dose, and properly use administration equipment. dosing or administering an anthelmintic based on average body weight will result inappropriate drug use by underdosing some animals over-dosing others. (4) check the efficacy of deworming agents yearly via fecal egg reduction testing. when a class of anthelmintic is no longer efficacious, switch to another class. other reading pugh dg, baird an: sheep and goat medicine, 2nd ed. philadelphia: elsevier; 2011. smith mc, sherman dm: goat medicine, 2nd ed. ames (ia): wiley-blackwell; 2009 committee on the nutrient requirements of small ruminants: nutrient requirements of small ruminants: sheep, goats, cervids, and new world camelids: animal nutrition series. washington dc: the national academies press; 2007. 501 clinical theriogenology • volume 5, number 4 • december 2013 502clinical theriogenology • volume 5, number 4 • december 2013 omniblank: 2011: in search of a strategic disturbance: some thoughts on the timing of spaying in search of a strategic disturbance: some thoughts on the timing of spaying david j. waters department of veterinary clinical sciences, purdue university, and the gerald p. murphy cancer foundation, west lafayette, in abstract the aim of this paper is to provoke a thoughtful re-evaluation of our assumptions regarding the health consequences of elective ovariohysterectomy in pet dogs. it proposes that we might re-shape our thinking about the physiological impact of the timing of spaying and commit ourselves to making spaying a strategic physiological disturbance. several observations that may help to guide this re-shaping are offered. first, the scholarly manuscripts of the veterinary profession routinely describe study populations in terms of male versus female, as well as neuter status. this implies that sex differences in biology can lead to clinically significant differences between males and females, and that gonad removal might re-set the system in important ways. taken together, this suggests that the concept of ovary removal as a physiological disturbance, although not talked about, is already deeply rooted in the minds of veterinarians. second, we have an incomplete understanding of the association between the diagnosis of mammary cancer or pyometra and the life expectancy of female dogs. this lack of data regarding the impact that these two health hazards of ovary retention exert on overall longevity points to a high research priority. further, a more rigorous evaluation of the impact that the timing of spaying has on health will require investigators to revise their vocabulary, replacing the categories “spayed” or “intact” with measures of actual lifetime ovary exposure. finally, we might do well to mimic the mindset of scientists in the field of evolutionary developmental biology who know that timing is everything when it comes to discovering what strategic disturbances can nudge a biological process closer to an intended outcome. optimistically, the author posits that on the horizon sits a new set of research questions that will drive a revised conception about what we should want to know about the timing of spaying and health. keywords: aging, longevity, mammary cancer, neutering, ovarian conservation, ovariohysterectomy, sex differences, spay the tacit truth: ovary removal as a physiological disturbance in my reading of the veterinary literature, very seldom do i come across a clinical case series in which the investigator does not inform the reader that “there were 48 females and 26 males”. in most instances, i can also count on the author to advance the description to reveal that “28% of the females were intact, 72% were spayed”. evidently this means that authors (and presumably readers) acknowledge the possibility that being male or female might influence health outcomes – whether it be an increase in disease incidence or a poorer prognosis following treatment. further, this sort of discourse implies that gonads might be the germ of such sex differences and that disposal of these endocrine organs might disturb the organism in a clinically significant way. it is ironic that virtually every author busies themselves in this inventory of gonadal whereabouts, yet no papers i can find refer to spaying as a physiological disturbance. this surprising omission, however, is not that difficult to explain if one accepts either of two possibilities: (1) authors and readers do not really believe that gonads exert clinically relevant effects; or (2) authors and readers do believe that gonads exert clinically relevant effects, and consider it so intuitively obvious that they do not feel compelled to question the proposition. i do not accept the first explanation. for if this is indeed the belief held by so many, then why are the scholarly manuscripts of our profession written to include such descriptions as standard fare? my hunch is that, among veterinarians, view number two is the prevailing belief. apparently the relationship between gonads and health outcomes falls under the category of tacit knowledge – a feeling for how mammals work that requires no explicit explanation. it follows that if you remove gonads, you might reset the system in important ways – in ways important enough to affect a diverse collection of outcomes ranging from the incidence or lethality of neoplastic diseases, to the way an individual responds to an immune challenge. this possibility is so deeply rooted that it is not talked about. therefore it is clinical theriogenology • volume 3 number 4 • december 2011433 somewhat surprising that, despite these deep-seated beliefs, a physiological disturbance like spaying immature pet dogs is so widely advocated for its health-promoting attributes.1 expanding our thinking about ovaries beyond reproduction: sex and gonadal differences in health outcomes as signposts that can advance our understanding of biological systems sex-differences in biology and medicine go far beyond the obvious–beyond breast cancer and menstrual cycles. i believe that studying sex differences in health can open important doors, providing us with a more expansive view of how the body works. as a member of the organization for sex-specific differences, my research group has been exploring sex differences in healthspan as a possible tool to deepen our understanding of underlying factors that significantly impact who does or does not get disease.2 we need a better handle on the triggers and physiological contexts that determine health, and dissecting out the underpinnings of sex differences in biology could go far to meet this need. for example, in one study, sexually intact female dogs diagnosed with epilepsy lived for an average of six years after diagnosis; in contrast, males with epilepsy lived only two years after diagnosis.3 several possible explanations for these provocative observations come quickly to mind. do females get a less severe form of epilepsy? does being female increase the likelihood of response to treatment with anticonvulsant drugs? do the factors that predispose males to epilepsy also predispose them to other lifeshortening diseases or accelerated aging? perhaps being female has no influence on the severity of disease, but for any given severity of disease, females are less likely to succumb because they are overall more robust than males. taken together, there seems to be good rationale for exploring further the biology behind the apparent sex difference in the survival of canine epileptics. amidst all the attention paid to faithfully reporting sex and neuter status, why is there so little attention paid to the details of gonadal exposure? in a nationwide study examining the factors that influence longevity in rottweilers, we discovered a female longevity advantage over males.4 moreover, in females there was a direct relationship between actual years of ovary exposure and likelihood of reaching exceptional longevity.4 but as we looked more deeply we made another important discovery: no previous peer-reviewed paper in the veterinary literature had evaluated the association between longevity and actual number of years of ovary exposure. instead, reports exposed readers to data on how long two groups of female dogs lived –“spayed” and “intact”.5,6 “spayed” was the name given to bitches that lost their ovaries at some undetermined time during their lives. “intact” was the name given to bitches that were still intact at the time of death. no wonder our results in female rottweilers were considered so novel. no one had ever studied that relationship before. research priority: clarifying the health hazards of ovary retention to better understand the lifelong health consequences of ovariohysterectomy, we need studies situating the health hazards of keeping ovaries – in particular the risk of developing and dying from mammary cancer and pyometra – within the context of duration of lifetime ovary exposure and longevity. we need studies that will provide insights on how the timing of spaying effects trade-offs that impact overall health. studies of bitches that make use of longevity as an outcome can do just that – integrating the risk and lethality of mammary cancer and pyometra with the risk and lethality of every other disease, as well as each individual’s rate of physiologic decline. to what extent does the diagnosis of mammary cancer or pyometra adversely influence life expectancy? this is a critical question which no previous report has ventured to answer. as a result of this knowledge gap, we cannot confidently say right now whether keeping ovaries for any particular duration of adult life cuts short the longevity of female dogs as a consequence of mammary cancer-related mortality. it is quite possible that, after one accounts for competing disease conditions and mortalities, a diagnosis of mammary cancer does not cut short the average dog’s life expectancy. it seems that generating this kind of data could significantly change our perceptions and re-shape the kind of questions we are asking about the pros and cons of keeping or losing ovaries. ultimately, this information would enable us to see more clearly what we might expect in terms of health outcomes if we delay ovariohysterectomy until a particular age during adulthood. clinical theriogenology • volume 3 number 4 • december 2011 434 a need for new vocabulary: evaluating the impact of timing of spaying will require replacing “spayed” or “intact” with measures of lifetime ovary exposure if clinicians believe that gonads, as endocrine organs, might exert system-wide effects (for example, influencing the brain and other body parts of the aforementioned canine epileptics), then someone should be looking more closely at dose-response – paying attention to the timing of spaying. few investigators have made much effort in defining their study populations in terms of actual years of ovary exposure. instead they fall back on naming females as “spayed” or “intact” based upon gonadal status at the time of death, so-called dichotomous binning. all of us, as authors and readers of scientific reports, have grown accustomed to this lazy method of expressing lifetime ovary exposure. by ignoring actual years of ovary exposure, could we be developing misleading assumptions about the timing of spaying and its health consequences? i have come to believe that this dichotomous binning approach creates more confusion than clarity. my research group has documented this potential for distorting inferences in a recent peerreviewed publication.7 analytically, the categorizing of bitches as spayed or intact at the time of death is a blunt instrument that lacks rigor, falling far short of reliably representing biologically important differences in lifetime gonad exposure. what we need to probe more deeply is dose-response. for it is the timing of spaying that dictates the “window” during the life course when the body is under the influence of intact ovaries. this means not only the direct effects attributable to circulating factors produced by the ovary, such as estrogen, but also changes in pituitary function, such as the sustained increase in gonadotropin levels documented in spayed bitches.8 if we can agree that gonad removal disturbs normal physiology, it follows that we might tap into spaying as a strategic disturbance – one that would optimize the organism for healthy longevity. the challenge could be considerable, the payoff even greater. finding the optimal window of ovary exposure for different breeds and different environmental contexts will be a challenging goal indeed. but the sooner we as clinicians fully embrace the notion that adult health outcomes are profoundly influenced by early life events,9,10 the better we will be at helping pet owners make better decisions. with a deeper sense of life course perspective, the practice of “geriatric medicine” will grow beyond making recommendations about what are the best dietary supplements to give a dog after she has reached a ripe old age, to include making superior early choices that promote healthy longevity.11 our profession is in need of such chronological crusaders, schooled in the value of timing and life course perspective. it is logical that theriogenologists should play a leadership role in this evolutionary thinking. all about timing: jack horner’s dangerous idea the world-renowned paleontologist jack horner believes it’s time to make a strategic disturbance.12 his endgame is not the healthy longevity of pet dogs. instead, his goal is to grow a dinosaur. paleontologists know that birds are the only living descendants of the dinosaurs. so by carefully studying the developmental biology of the chicken, horner plans to disturb the process at a key moment, biochemically nudging the avian developmental program toward hatching a beast with tail, teeth, and grasping forearms. he has a clear picture of what he wants to create – a living dinosaur – and he’s betting that a strategic disturbance will make that goal a reality. some see horner’s directive as misinformed, even crazy. but i see a bigger picture in horner’s ponderings. he’s not all that far out on a limb. his radical hypothesis fits squarely under the umbrella of one of science’s hottest fields of inquiry known as evolutionary developmental biology or “evo-devo”.13,14 investigators in this field are probing the process of evolution by studying the process of change in developing embryos. it turns out that not-so-subtle differences – the difference between hatching a beast with a front leg instead of a wing – are under the control of relatively few genes and whether those genes are switched on or switched off. horner is obsessed with timing and i think we should follow his lead. horner knows that he will need to be very precise if he is to discover the critical sequence of developmental events that creates dinosaur-like, rather than chicken-like, traits. without this attention to detail, he will surely fail. clinical theriogenology • volume 3 number 4 • december 2011435 likewise, if our goal is to better understand the differences in health outcomes of female dogs with or without gonads, we will need to exercise similar precision. we will need to employ a vocabulary that best reflects the timing of critical life events, such as gonadectomy. we will need to learn how to strategically disturb the system. instead of horner’s window of embryonic development, our challenge will be to explore more fully the post-natal windows during the life course when key exposures – such as ovary exposure – dictate the differences we are striving to achieve, differences that favor longevity and disease avoidance in our pets and in ourselves. if, in the spirit of jack horner, i were permitted to put forward my own radical hypothesis it would be this. i would predict that, in large breed dogs prone to highly lethal forms of non-epithelial cancers (e.g. lymphoma, osteosarcoma, hemangiosarcoma) and whose body size means that cruciate ligament injury frequently leads to mobility issues that can precipitate earlier euthanasia, the age at spaying that optimizes longevity will be closer to six years than six months. just the other day, i spoke with a small animal practitioner who had read about our work on ovaries and sex differences in longevity.15 he commented: “i’m not so sure about what you’re saying about ovaries promoting longevity. i’ve been practicing for 25 years and i can’t honestly say that i appreciate a longevity difference between female and male dogs.” i asked him: “do you observe very many females who spend a sizeable percentage of their adult life with intact ovaries?” “no,” he said, “we advise our clients to spay at six months.” enough said. my colleague’s observations fit precisely within my emerging view of canine longevity – early ovary removal as the great homogenizer. i’m beginning to see ovaries as part of a system that promotes longevity.16-18 time for a new set of questions perhaps the most prized products of research are the new questions that come. the astrophysicist sir arthur eddington stated: “progress is marked not so much by the problems we are able to solve as by the questions we are enabled to ask.”19 the biochemist n.w. pirie took the notion one step further: “clearly the process of formulating a question has a great psychological effect on the questioner; it focuses his attention on what he thinks he wants to know.”19 reflecting on the last four decades, it is difficult to describe our profession as marching briskly toward a more complete understanding of the health consequences of retaining ovaries. instead, i sense we have sunk into a somnambulistic state, passively accepting report after clinical report of dogs as “spayed” or “intact” as if this dichotomous substitute for actual years of lifetime ovary exposure was informative. i know – i wrote some of those reports. i took part in the sleepwalking for more than 20 years, waking up just a few years ago. surely, this sleepy, indoctrinated behavior has shielded us from probing more deeply the question: what effect does the timing of spaying have on adult health outcomes? for many of us, our argument supporting the health benefits of ovariohysterectomy prior to adulthood has its foundation in the trusted mantra: “if we don’t spay early, we won’t prevent mammary cancer”. you may find yourself imprisoned by this notion that was instilled in you during your training; indeed the idea that early spaying protects against mammary cancer figured prominently in my own training.20 but just how confident are we that the diagnosis of mammary cancer in female dogs is a harbinger of premature death? i keep hearing the echo of pirie’s words. if we are willing to begin to ask new questions, we will discover there is much more that we should want to know about the timing of spaying and health. if we can openly acknowledge ovariohysterectomy as a physiological disturbance, we can begin to more rigorously explore its consequences, which may be linked to health outcomes yet unseen. by shifting the debate away from “should we spay, should we not?” toward a new question “what age at spaying best promotes healthy longevity”, we prepare the necessary ground for making spaying a strategic disturbance. references 1. root kustritz mv: determining the optimal age for gonadectomy of dogs and cats j am vet med assoc 2007;231:1665-1675. clinical theriogenology • volume 3 number 4 • december 2011 436 2. waters dj, chiang ec, cooley dm, et al: making sense of sex and supplements: differences in the anticarcinogenic effects of selenium in men and women. mutation res 2004;551:91-107. 3. berendt m, gredal h, ersboll ak, et al: premature death, risk factors, and life patterns in dogs with epilepsy. j vet intern med 2007;21:754-759. 4. waters dj, kengeri ss, clever b et al: exploring mechanisms of sex differences in longevity: lifetime ovary exposure and exceptional longevity in dogs. aging cell 2009;8:752-755. 5. bronson rt: variation in age at death of dogs of different sexes and breeds. am j vet res 1982;43:2057-2059. 6. michell ar: longevity of british breeds of dog and its relationships with sex, size, cardiovascular variables and disease. vet rec 1999;145:625-629. 7. waters dj, kengeri ss, maras ah et al: probing the perils of dichotomous binning: how categorizing female dogs as spayed or intact can misinform our assumptions about the lifelong health consequences of ovariohysterectomy. theriogenology 2011;76:1496-1500. 8. reichler im, hung e, jochle w et al: fsh and lh plasma levels in bitches with differences in risk for urinary incontinence. theriogenology 2005;63:2164-2180. 9. brown dw, anda rf, tiemeirer h et al: adverse childhood experiences and the risk of premature mortality. am j prev med 2009;37:389-396. 10. brown dw, anda rf, felliti vj et al: adverse childhood experiences are associated with the risk of lung cancer: a prospective cohort. bmc public health 2010;10:20. 11. waters dj: cellular and organismal aspects of senescence and longevity. in: wilmoth jm, ferraro kf, eds. gerontology perspectives and issues. new york: springer publishing company; 2007. p. 63-87. 12. horner j, gorman j: how to build a dinosaur. new york: dutton; 2009. 13. wagner gp: what is the promise of developmental evolution? part i: why is developmental biology necessary to explain evolutionary innovations? j exp zool 2000;288:95-98. 14. wagner gp: what is the promise of developmental evolution? part ii: a causal explanation of evolutionary innovations may be impossible. j exp zool 2001;291:305-309. 15. nolen rs: rottweiler study links ovaries with exceptional longevity. j am vet med assoc 2010;236:496. 16. parker wh, broder ms, chang e et al: ovarian conservation at the time of hysterectomy and long-term health outcomes in the nurses' health study. obstet gynecol 2009;113:1027-1037. 17. mason jb, cargill sl, anderson gb, et al: transplantation of young ovaries to old mice increased life span in transplant recipients. j gerontol a biol sci med sci 2009; 64:1207-1211. 18. waters dj: aging research 2011: exploring the pet dog paradigm. ilar j 2011;52:97-105. 19. pirie nw: selecting facts and avoiding assumptions. in: berthoff ae, ed. reclaiming the imagination. upper montclair (nj): boynton/cook publishers inc.; 1984. p. 203-211. 20. schneider r, dorn cr, taylor don: factors influencing canine mammary cancer development and postsurgical survival. j natl cancer inst 1969;43:1249-1261. clinical theriogenology • volume 3 number 4 • december 2011437 clinical theriogenology • volume 3 number 4 • december 2011 438 omniblank: 2011: accuracy of diagnosis of clinical endometritis with metrichecktm in postpartum dairy cows accuracy of diagnosis of clinical endometritis with metricheck™ in postpartum dairy cows augustine t. peter,a greg m. jarratt,b david w. hanlonb adepartment of veterinary clinical sciences, purdue university, west lafayette, in, bmatamata veterinary services ltd, matamata 3400, new zealand summary clinical endometritis, an important condition during the postpartum period in dairy cows, is diagnosed by different methods. a recently-developed vaginal device (metricheck™, simcro tech limited, hamilton, nz) utilizes information on the nature of vaginal exudate in diagnosis. in the cytobrush method, information about endometrial inflammation as determined by endometrial cytology is used in diagnosis. in this clinical investigation, the accuracy of metricheck™ for diagnosis was compared to that of the cytobrush method. specifically, this study tested the hypothesis that vaginal exudate is not always accompanied by endometrial inflammation. one hundred twenty two cows were chosen at random from a population of postpartum dairy cows that were between 28 and 41 days postpartum. the prevalence of clinical endometritis as diagnosed by metricheck™ and cytobrush was 15.6% and 13.1%, respectively. the percent positive agreement was 36.8% and the percent negative agreement was 91.3% between these tests. the sensitivity and specificity measures of metricheck™ were 43.8% and 88.7%, respectively. these findings support the hypothesis that a vaginal exudate is not always accompanied by endometrial inflammation. only 37% of cows diagnosed positive for clinical endometritis by the metricheck™ method had evidence of endometrial inflammation. keywords: dairy cow, postpartum, clinical endometritis, metricheck™, cytobrush background early resumption of continuous cyclic ovarian activity in postpartum cows is desirable since it distinctly enhances reproductive efficiency. one of the major impediments to the early resumption of cyclic ovarian activity is postpartum uterine health that is less than ideal.1-6 postpartum endometritis is an important condition amongst postpartum uterine health issues and is influenced by peripartum nutrition and other factors.6 evidence of inflammation in the absence of systemic clinical signs is defined as endometritis.7 for clarity, endometritis is further classified as clinical or subclinical. clinical endometritis is characterized by the presence of a purulent or mucopurulent uterine discharge after 21 or 26 days postpartum7-9 while subclinical endometritis is characterized by the presence of >18% polymorphonuclear inflammatory cells (pmn) in uterine (endometrial) cytology samples collected 21 to 33 days postpartum, or >10% pmn in samples collected at days 34 to 47.7,10 cows with subclinical endometritis do not have a uterine discharge; however, it is very important to recognize that the severity of the disease is still considered sufficient to impair reproductive performance.7 the prevalence of endometritis varies depending on the management of the farm, the time the diagnosis is made during the postpartum period, and the method employed for diagnosis. for example, a study conducted in southern ontario involving 228 cows in two tie-stall dairy farms and using the endometrial cytology technique as a diagnostic procedure, found that the prevalence of endometritis was 41.4% at 34 to47 days postpartum.11 another study, conducted in central new york involving 141 holstein cows in five commercial dairy herds that utilized samples obtained by uterine lavage for diagnosis concluded that at 40 to 60 days postpartum the prevalence of endometritis was 53% and among herds the range was 37 to 74%.12 these percentages are influenced by the cut-off points adopted for number of pmn. the former study calculated different cut-off points for different days postpartum using receiver operating characteristic (roc curves) while the latter study arbitrarily selected a cut-off point of 5%. a subsequent study13selected a cut-off point based upon the use of sequential model to detect the least level of pmns between 5 and 15% that produced a significant decrease in pregnancy rate. recent studies14,15 followed the first approach.11 endometritis how ever defined or diagnosed has been shown to have a significant detrimental effect on subsequent reproductive performance. affected cows are at a clinical theriogenology • volume 3 number 4 • december 2011461 higher risk of not being detected in estrus before the breeding season, have a lower first service conception rate and a lower pregnancy rate, and are more likely to be culled.9,11-13,16 historically, clinical endometritis has been diagnosed and scored by various methods and observations, namely, manual vaginal examination, vaginoscopy, transrectal palpation, transrectal ultrasound examination of the uterus, cytological evidence of inflammation, bacterial culture, and detection of cervical and vulvar discharges.9,11-13,17-20 recently an intravaginal device* been used in a few studies.16,21 other studies11,13,22 have used a cytobrush technique to document the cytological evidence of endometrial inflammation. the objective of the present study was to evaluate the efficacy of metricheck™ in the diagnosis of clinical endometritis. information on the vaginal exudate and its nature is used in the metricheck™ method to conclude whether or not the cow has clinical endometritis. however, diagnosis based on evidence of endometrial inflammation as determined by endometrial cytology11,13,22 appears to be sensitive provided the right cut-off value for pmns is adopted. it has also been suggested that cytobrush is a good reference diagnostic criterion and reliable test for clinical endometritis because of its greater intra-observer repeatability as evidenced by its higher concordance correlation coefficient.13 hence in the present study, observations made by the metricheck™ technique were evaluated in comparison to those of the of cytobrush method findings. the study tested the hypothesis that vaginal exudate is not always accompanied by endometrial inflammation. case presentation animals this clinical investigation was conducted in september of 2008 and involved 122 cows from 11 seasonally calving dairy herds in the matamata region of new zealand. herd size ranged from 300 to 1500 lactating cows maintained on pasture and the percentage of cows chosen for the study from these herds ranged from 1.5 to 3.3 %. as part of reproductive health management, each cow was tested for the presence of vaginal exudate by metricheck™. every fourth cow was moved into the chute for the cytobrush procedure. the cows were within 28 to 41 days postpartum with a median of 34 days and their ages ranged from three to seven years with a median of five years. the project was conducted in accordance with the new zealand animal welfare act, national animal welfare advisory committee 1999 stipulations. vaginal fluid samples samples of vaginal fluid were collected by metricheck™ and evaluated immediately as described previously.16 this intravaginal device consists of a 4 cm silicon hemisphere with its concave surface attached (facing) the tip of a 50 cm stainless steel rod. briefly, the metricheck™ was inserted through the cleaned vulvar lips, advanced to the cranial extent of the vaginal fornix, and with the handle slightly elevated the device was retracted caudally. material adhering to the surfaces of the silicon hemisphere was assessed visually. this procedure was performed by four veterinarians from matamata veterinary services ltd. endometrial samples after the collection of vaginal fluid samples, the selected animals were moved into a chute to obtain an endometrial sample. the procedure was conducted solely by the senior author. an endometrial sample for cytological examination was collected using a non-sterile brush (cytobrush® plus, gt cooper surgical company, trumbull, ct) modified for use in cattle as described previously.11 before threading the brush into the stainless steel rod (65 cm long, 4 mm outside diameter) and its stainless steel sheath (50 cm long, 6 mm outside diameter) both were cleaned with alcohol. the handle of the brush (17 cm) was shortened to 3 cm and was threaded into the hole at the tip of the stainless steel rod. the stainless steel * http://www.simcro.com/products/other_accessories/metricheck.html clinical theriogenology • volume 3 number 4 • december 2011 462 rod was retracted into the stainless steel sheath until the brush was hidden completely inside the sheath. the vulva and perineum were cleaned with a paper towel. the assembled device was covered by a plastic sanitary chemise (imv technologies, maple grove, mn), and introduced into the vagina. next, a sleeved arm was introduced into the rectum to facilitate passage of the instrument through the cranial vagina and cervix. at the external os of the cervix, the plastic sleeve was perforated and the metal sheath was manipulated through the cervix into the body of the uterus and the brush was exposed by pushing the metal rod forward and the rod was turned clockwise approximately one quarter turn for the brush to gather cellular material from the adjacent endometrium. the rod was withdrawn into the sheath to prevent the brush from collecting cellular material from the cervical and vaginal area. a slide was prepared immediately and the slides collected at each visit were stained within two h of collection. endometrial cytology slides were stained using a romanowsky stain (diff-quick; fisher diagnostics, middletown, va). after drying, each slide was examined using 400 x magnification by the senior author, and a differential count using a minimum of 100 cells (endometrial cells, pmn, and squamous cells) was obtained to provide a quantitative assessment of endometrial inflammation. analysis material gathered by the metricheck™ was scored as follows: 0 = no discharge, 1 = clear mucus, 2 = flecks of purulent material within otherwise clear mucus, 3 = mucopurulent but <50% purulent material, 4 = mucopurulent with >50% purulent material and 5 = mucopurulent with >50% purulent material and an odor. for the purpose of data analysis, scores of 0 and 1 were recorded as negative and the rest (2 and greater) were recorded as positive. for the cytobrush, the presence of 8% or more pmn in the endometrial sample was recorded as positive. this pmn cut-off point was chosen based on the findings of a recent study in which endometritis was diagnosed based on the lowest percentage of pmn that was significantly associated with the time to pregnancy.13 the mcnemar’s test was applied to the data to determine whether differences existed between expected results between the two tests. to assess the agreement (percent positive and percent negative agreement) between these tests and to determine the sensitivity and the specificity measures using the bayesian theory,23 a specific analysis developed for this purpose was used (sas version 9.1 for windows, sas institute, cary, nc). outcome the prevalence of clinical endometritis as diagnosed by metricheck™ and cytobrush was 15.6% and 13.1%, respectively. the mcnemar’s test showed that the different categories which defined the contingency table did not significantly differ from what was expected, an observation necessary to proceed further with subsequent analysis of data. there was a fair agreement between the two tests (kappa = 0.30). in 94 negative cases and seven positive cases metricheck™ and cytobrush agreed. twelve cases that were positive by metricheck™ were negative by cytobrush and nine cases that were positive by cytobrush were negative by metricheck™. the findings are summarized in the table. the percent positive agreement was 36.8% and the percent negative agreement was 91.3%. in other words, only 37% of the cows that had a vaginal exudate showed evidence of endometrial inflammation. the sensitivity and specificity measures of metricheck™ were 43.8% and 88.7%, respectively compared to cytobrush. discussion the tests used in the present study may result in over-diagnosis of endometritis because all cows have some degree of endometritis associated with normal uterine involution. hence the timing when these tests are performed during the postpartum period is very critical. it has been established very clearly that a test conducted after day 28 postpartum has merit in accurately diagnosing the condition9,11,13 and furthermore cows that were found positive at this time had lower reproductive performance in the ensuing period compared to negative cases.13 hence, it is suggested that besides using the pmn cut-off points in clinical theriogenology • volume 3 number 4 • december 2011463 defining metritis the effect of metritis in lowering the reproductive performance such as pregnancy rate should also be considered. the findings of this study support the hypothesis that cows can have a vaginal exudate without evidence of endometrial inflammation. presence of vaginal exudate in the absence of endometrial inflammation suggests that these cows may have had any of the following conditions during the postpartum period: vaginitis, cervicitis, and possibly resolving endometirits. thus it is possible that some of the cows that had a purulent exudate in the vagina could have had a healthy endometrium. it is important to note that only 37% of the cows that had a vaginal exudate showed evidence of endometrial inflammation which demonstrated the test’s reduced specificity in diagnosing subclinical endometritis. further studies are required to investigate the economic relevance of this finding. these studies should focus on the impact of treatment on subsequent fertility. learning points findings of this study suggest that a vaginal exudate as diagnosed by an intravaginal device is not always accompanied by endometrial inflammation. this may result in the over-diagnosis of endometritis when using this technique. acknowledgements major support for this investigation was provided by the theriogenolgy program incentive fund and by the purdue university school of veterinary medicine faculty enrichment fund. administrative help provided by katherine mora of matamata veterinary services, ltd. is appreciated and the valuable assistance given by the participating herd owners is recognized. the authors thank raju gautam for his help with statistical analysis. references 1. bosu wtk, peter at: evidence for a role of intrauterine infections in the pathogenesis of cystic ovaries in postpartum dairy cows. theriogenology 1987;28:725-736. 2. peter at, bosu wtk: effects of intrauterine infection on the function of the corpora lutea formed after first postpartum ovulations in dairy cows. theriogenoloy 1987;27:593-603. 3. sheldon im, dobson h: postpartum uterine health in cattle. anim reprod sci 2004;82-83:295-306. 4. földi j, kulcsár m, pécsi a, et al: bacterial complications of postpartum uterine involution in cattle. anim reprod sci 2006;96: 265-281. 5. leblanc sj: postpartum uterine disease and dairy herd reproductive performance: a review. vet j 2008;176:102-114. 6. peter at, vos plam, ambrose dj: postpartum anestrus in dairy cattle. theriogenology 2009;71:1333-1342. 7. sheldon im, lewis gs, leblanc s, et al: defining postpartum uterine disease in cattle. theriogenology 2006;65:15161530. 8. sheldon im, noakes de: comparison of three treatments for bovine endometritis. vet rec 1998;142:575-579. 9. leblanc sj, duffield tf, leslie ke, et al: defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows. j dairy sci 2002;85:2223-2236. 10. gilbert ro, shin st, guard cl, et al: incidence of endometritis and effects on reproductive performance of dairy cows. theriogenolgy 1998;49:251. 11. kasimanickam r, duffield tf, foster ra, et al: endometrial cytology and ultrasonography for the detection of subclinical endometritis in postpartum dairy cows. therigenology 2004;62:9-23. 12. gilbert ro, shin st, guard cl, et al: prevalence of endometritis and its effects on reproductive performance of dairy cows. theriogenology 2005;64: 1879-1888. 13. barlund cs, carruthers td, waldner cl, et al: a comparison of diagnostic techniques for postpartum endometritis in dairy cattle. theriogenology 2008;69:714-723. 14. santos nr, lamb gc, brown dr, et al: postpartum endometrial cytology in beef cows. theriogenology 2009;71:739745. 15. galvão kn, frajblat m, brittin sb, et al: effect of prostaglandin f2alpha on subclinical endometritis and fertility in dairy cows. j dairy sci 2009;92:4906-4913. 16. mcdougall s, macaulay r, compton c: association between endometritis diagnosis using a novel intravaginal device and reproductive performance in dairy cattle. anim reprod sci 2007;99:9-23. 17. sheldon im, noakes de, rycroft an, et al: effect of postpartum manual examination of the vagina on uterine bacterial contamination in cows. vet rec 2002;151:531-534. 18. williams ej, fischer dp, pfeiffer du, et al: clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle. theriogenology 2005;63:102-117. clinical theriogenology • volume 3 number 4 • december 2011 464 19. runciman dj, anderson ga, malmo j, et al: use of postpartum vaginoscopic (visual vaginal) examination of dairy cows for the diagnosis of endometritis and the association of endometritis with reduced reproductive performance. aust vet j 2008;86: 205-213. 20. sheldon im, williams ej, miller an, et al: uterine diseases in cattle after parturition. vet j 2008;176:115-121. 21. pleticha s, drillich m, heuwieser w: evaluation of the metricheck device and the gloved hand for the diagnosis of clinical endometritis in dairy cows. j dairy sci 2009;92:5429-5435. 22. kaufmann tb, drillich m, tenhagen ba, et al: prevalence of bovine subclinical endometritis 4h after insemination and its effects on first service conception rate. theriogenology 2009;71:385-391. 23. martin s, meek a, willeberg p: veterinary epidemiology. ames (ia): iowa state university press, 1987. p. 343-354. table: prevalence of clinical endometritis as diagnosed by metricheck™ and cytobrush in 122 cows 2841 days postpartum. metricheck™ cytobrush negative positive total negative 94 12 106 positive 9 7 16 total 103 19 122 clinical theriogenology • volume 3 number 4 • december 2011465 clinical theriogenology • volume 3 number 4 • december 2011 466 omniblank: metritis in early lactation in dairy cows: energy balance, ovarian function, incidence of subclinical endometritis and reproductive performance metritis in early lactation in dairy cows: energy balance, ovarian function, incidence of subclinical endometritis and reproductive performance shaked druker,a ron sicsic,a tamir goshen,a,b moran lifshitz,a tal raza akoret school of veterinary medicine, robert h. smith faculty of agriculture food and environment, the hebrew university of jerusalem, rehovot, israel bthe veterinary services, ministry of agriculture and rural development rishonlesion, israel uterine inflammatory diseases are prevalent in bovine dairy herds and responsible for major economic losses. in israel, dairy cows are routinely examined by farm veterinarians at 5 12 days in milk (dim); metritis is defined as presence of a fetid, watery to purulent vaginal discharge and an enlarged uterus. objective was to compare healthy cows to those with metritis, based on indicators of energy balance, ovarian function, incidence of subclinical endometritis and reproductive performance. holstein-friesian cows (n = 66) were included in a case-control study [healthy (n = 33) and metritis (n = 33)]. clinical examinations (transrectal and vaginal examinations) and sampling (endometrial cytology and blood) were performed at 5 12, 30 37 and 60 70 dim. ovarian function was assessed by neck tag activity records (scr heat-detection) and serum concentrations of progesterone and anti mullerian hormone (amh). body condition scores (bcs) and serum concentration of nonesterified fatty acids (nefa) and beta-hydroxybutyrate (bhba) were analyzed. compared to healthy cows, metritis cows had reduced (p < 0.05) reproductive performance (pregnancy rate at 150 dim: 60 versus 33.3%; interval from ai to conception: 47 ± 9 versus 64 ± 10 days); and a trend (p = 0.08) towards less milk yield by 90 dim (4062 ± 134 versus 3883 ± 108 liters). among primiparous cows, those with metritis at 5 12 dim had higher (p < 0.05) incidence of cytological endometritis at 30 37 dim (healthy: 8% versus metritis: 58%), whereas among multiparous cows there was no difference. overall, serum nefa, bhba and amh did not differ between healthy and metritis groups; however, amh was higher (p < 0.05) in multiparous cows compared to primiparous cows. among primiparous cows, the first detected estrus tended to be earlier in healthy compared to metritis cows (45 ± 6 versus 73 ± 12 dim), with more estruses detected by 70 dim (1.5 ± 0.3 versus 0.8 ± 0.3). overall, cows that had estrus prior to 50 dim had higher (p < 0.05) pregnancy rate at 150 dim than cows that had estrus later (63.6 versus 25%). in summary, early metritis (5 12 dim) was associated with alteration of ovarian activity, higher incidence of subclinical endometritis and poor reproductive performance later in lactation. keywords: bovine, metritis, endometritis, energy balance 525 clinical theriogenology • volume 11, number 3 • september 2019 526clinical theriogenology • volume 11, number 3 • september 2019 omniblank: a preliminary study comparing pregnancy rates and live foal rates using individually sheathed or nonsheathed insemination pipettes a preliminary study comparing pregnancy rates and live foal rates using individually sheathed or nonsheathed insemination pipettes justin mcnaughten,a ross wallaceb arhinebeck equine l.l.p, rhinebeck, ny bmurray veterinary services coolup, western australia, australia sterile, disposable, plastic equipment is often recommended for artificial insemination of the mare.1 once utilized, these plastics are discarded contributing to landfill waste and possibly increasing environmental pollution. objective was to investigate if the amount of plastic waste generated during the artificial insemination of mares could be decreased without negatively affecting pregnancy rate. twenty eight standardbred mares were bred over 40 estrous cycles. mares were randomly to assigned to the following 2 groups: high plastic (hp); individually sheathed insemination pipette or low plastic (lp); nonsheathed insemination pipette. mare reproductive cycles were routinely monitored by transrectal palpation and ultrasonography. deslorelin acetate was administered when a mare was in estrus with a preovulatory follicle greater > 35 mm in diameter. each mare was inseminated with a minimum of 500 x 106 progressively motile sperm from 1 standardbred stallion. pregnancy diagnosis (transrectal ultrasonography) was performed between 14 and 16 days after ovulation and repeated on days 28 and 45 of pregnancy. live foal rates were recorded. first and per cycle pregnancy rates for hp were 50.0 and 64.3%, respectively and for lp were 56.5 and 70.5%. live-foal rates were 92.3% (12/13) for hp compared to 83.3% (10/12) for lp. data were analyzed using two tailed fishers exact tests for small counts, with significance defined at p < 0.05. there were no differences between hp and lp groups for first cycle (p = 0.704) or per cycle (p = 0.512) pregnancy rates, or live foal (p = 1.0) rates. we concluded that nonsheathed pipettes (less plastic waste) can be used without negatively affecting pregnancy. keywords: equine, breeding management, artificial insemination, pregnancy rates references 1. brinsko sp: semen collection techniques and insemination procedures. in: mckinnon a, squires e, vaala w, et al: editors. equine reproduction. 2nd edition, ames: wiley blackwell; 2011. p. 1268–1277. 459 clinical theriogenology • volume 11, number 3 • september 2019 460clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2013: parasite control strategies for broodmares and foals parasite control strategies for broodmares and foals wendy vaala merck animal health, alma, wi abstract drug resistance among cyathostomin and ascarid populations is a growing concern for breeding operations. an effective deworming program for broodmares focuses on control of cyathostomes using targeted treatments based on an individual’s egg shedding potential. shedding status is based on fecs performed during periods of parasite transmission. efficacy of drug classes is determined using the fecrt. mares classified as low shedders benefit from two to three treatments per year, while high shedders may require four to five treatments that include at least one larvicidal treatment effective against encysted cyathostomins administered at the end of the grazing season. parascaris equorum is the focus of deworming recommendations for young foals up until weaning. due to increasing prevalence of macrocyclic lactone-resistant isolates of p equorum, it is strongly encouraged that the first treatment be delayed until foals are at least eight weeks of age and prior to weaning foals should receive a minimum of two anthelmintic treatments using either fenbendazole at 10 mg/kg and/or pyrantel. fecal egg counts should be performed at weaning to determine if the worm burden is primarily ascarids or strongyles. after weaning anthelmintic treatments should target cyathostomins and tapeworms. environmental control strategies should be included as an integral, non-chemical part of any parasite control regimen. keywords: anthelmintic resistance, parascaris equorum, cyathostomins, fecal egg count reduction test, strongyle shedding potential introduction anthelmintic resistance in equine parasites has been reported for more than five decades. there are numerous reports of confirmed or suspected resistance in small strongyles to piperazine,1 benzimidazoles,2-5 pyrantel salts,6-9 and more recently, macrocyclic lactones (e.g., ivermectin, moxidectin).10-12 the fact that cyathostomina are not usually serious pathogens in well-managed, healthy adult horses has, until recently, left practitioners fairly complacent that such resistance, although warranting caution, was manageable. that comfort level quickly dissipated with the first published reports of anthelmintic resistance among populations of p equorum in young horses.13,14 anecdotal reports of anthelmintic lack of efficacy also exist for other less pathogenic parasites including pinworms and habronema. interestingly there are no documented cases of drug resistance among large strongyle species including strongylus vulgaris. since an in-depth discussion of equine parasite biology, anthelmintic resistance and target deworming strategies is beyond the scope of this article, the reader is referred other review articles15 as well as the current and comprehensive set recommendations for equine parasite control strategies is available on the american association of equine practitioners website.16 when designing a parasite control program for a breeding operation there are at least two populations of horses to consider: 1) adults, including broodmares and stallions and 2) foals, weanlings and yearlings. parasites of concern and risk of infection vary considerably between these two populations and necessitate different monitoring and control strategies. among well-managed adult horse populations strongylus vulgaris infections are rare and cyathostomins (small strongyles) are now recognized as the major parasite of concern. deworming recommendations for this population are tailored first to the individual’s innate susceptibility to cyathostomes followed by their risk of infection from other parasites including large strongyles, tapeworms, pinworms, bots, habronema and strongyloides. young horses, less than three years of age, are more susceptible to parasites in general and are more likely to demonstrate parasite associated clinical disease. this age group requires a more regimented deworming protocol based on parasite biology and risk of exposure. foals and weanlings are uniquely susceptible to parascaris equorum infections and anthelmintic selection and frequency of administration prior to weaning are focused on control of this potentially lethal pathogen. 529 clinical theriogenology • volume 5, number 4 • december 2013 useful terminology and concepts to understand anthelmintic resistance is an inherited trait and is defined as the ability of worms to survive treatments that are considered effective against that species and stage of infection.17 in order for anthelmintic resistance to develop on a farm, the gene mutations that confer drug resistance must already be present within the resident worm population. frequent drug therapy subjects parasite populations to selective pressure and allows only resistant isolates to survive, reproduce and pass on their resistance genes to future generations of parasites. without routine fecal testing and drug monitoring it is easy to see how intensively managed breeding operations, accustomed to following deworming strategies based on tradition rather than science, can become permissive breeding grounds for resistant parasite populations. the history of drug use on a farm plays a major role in how quickly resistance develops and to which drugs. the fecal egg count reduction test (fecrt) is the only method currently available to determine if strongyles or ascarids are resistant to a particular anthelmintic. results should be interpreted cautiously. to perform a fecrt a fecal sample is collected prior to deworming and 14 days following treatment. the numbers of eggs per gram (epg) in the pre-treatment and post-treatment samples are used to calculate the percent reduction in fecal egg count (fec) using the following equation: epg (pre-treatment) – epg (14 days post-treatment) x 100 = fecr (%) epg (pre-treatment) the fecrt can be used for each horse individually, but in the interest of economics and labor, it is often sufficient to test those horses previously identified as moderate to high shedders. the fecr values obtained from those groups are used to infer drug efficacy for the farm. horses should not have received anthelmintic treatments for at least eight weeks prior to performing the fecrt. if moxidectin was the last drug administered it is preferable to wait at least 12 weeks before collecting a pre-treatment sample. the fecrt can also be used to screen newcomers arriving on the farm. only foals old enough to be shedding parasite eggs can be included in the screening. shedding of (small) strongyle eggs typically does not commence until foals are six weeks of age or older and ascarid eggs usually do appear in fecals until foals are at least eight to ten weeks of age. guidelines for interpreting fecrt results are presented in table 1. fecal egg counts (fec) are the only method currently available for evaluating drug efficacy and egg shedding status. it is important to recognize the inherent short-comings of the fecal examination while trying to optimize results. the fec does not accurately reflect the total adult strongyle, paracaris equorum or cestode burden of an individual horse. fecal examinations do not detect immature, larval or migrating stages of strongyles and ascarids. fecal examinations tend to miss or underestimate anoplocephala perfoliata eggs, although collecting a fecal sample 18-24 hours after administering a cestocide will increase the likelihood of finding cestode eggs if a patent infection is present. when using collecting a fecal sample to characterize an adult horse’s strongyle shedding potential it is best to analyze a fresh sample collected during a season of parasite transmission (e.g., not during cold, freezing winters or hot, dry summers). it is also important to wait a suitable period of time after the last anthelmintic was administered before collecting the sample to ensure the fec obtained reflects the horse’s innate immunity rather than the lingering effects of the last drug used. egg reappearance period (erp) is defined as the time interval between the last effective deworming treatment and the resumption of significant egg shedding. table 2 lists the erp for commonly used equine dewormers. occasionally monitoring of the erp for a given drug class on a farm is a reasonable way to determine if resistance is beginning to develop against drugs previously considered effective. a shortening of the erp is a precursor to the development of full resistance. when monitoring the erp it is only necessary to collect fecal samples from a subset of the resident population (often the horses classified as higher egg shedders). if ivermectin was last used, the expected erp is six to eight weeks. collecting fecal samples from a group of high shedders four weeks after ivermectin treatment will help determine if the drug is still suppressing strongyle egg counts as long as expected. 530clinical theriogenology • volume 5, number 4 • december 2013 the strongyle egg shedding potential varies among adult horses older than three years of age and is considered an innate trait for that individual. within bands of mature broodmares strongyle egg counts are often concentrated in a small percentage of the herd leading to the commonly quoted statistic: “twenty to 30% of adult horses shed approximately 80% of the eggs”. over-dispersion is the term used to describe this distribution of egg shedding within a group of animals. in some populations high egg counts may be concentrated in as few as 10 to 15% of the herd. this egg shedding potential tends to remain stable for a healthy horse over time18,19 unless the original classification was based on an improperly collected or performed fecal examination or the horse’s immune status has changed due to disease or other factors. a fecal sample being used to determine shedding status should be collected during seasons of optimal parasite exposure/transmission and long after any residual effects from the last drug treatment are gone. since there are little data available to scientifically establish the fec thresholds used to classify horses as low, moderate and high shedders, the author prefers not to use black and white cut-off values, but rather generate fec data for a specific herd and classify animals within the farm’s low and high fec range. high shedders are generally considered to be animals with fec >500 epg. suggested guidelines for classifying the contaminating potential are discussed elsewhere.15 the two most popular fecal egg counting techniques are the modified wisconsin method that involves centrifugation and flotation and can detect egg counts as low as 1-5 epg and the modified mcmasters technique that relies on flotation and the use of calibrated counting chamber and has a lower limit of detection of 25-50 epg. the modified wisconsin method is preferred when performing a fecrt and offers an increased likelihood of observing cestode eggs if present. the reader is referred to other references for details regarding sample handling and storage as well as detailed testing procedures.16 parasite control strategies for mares focus first on cyathostomin control. as mature horses, mares and stallions vary in their innate susceptibility to cyathostomin infection and their strongyle egg shedding potential. consequently, deworming regimens can be individualized among adults (>3 years of age) once their shedding status is determined using properly performed fecs. the vast majority of horses in a herd shedding low to moderate numbers of strongyle eggs can be managed with two to three anthelmintic treatments per year. the small percentage of horses classified as high contaminators benefit from more frequent deworming with as many as four or more treatments annually. herd size, pasture availability, general husbandry practices, prevalence of other parasite species and climate also impact specific deworming recommendations and anthelmintic selection. use the fecrt on a farm-by-farm basis to determine which drug classes are still effective against cyathostomins and ascarids. specific treatments for other parasites of interest, including anoplocephala perfoliata, oxyuris equi, habronema spp., strongyloides westeri and strongylus vulgaris can be incorporated into the annual treatment plan. one or two treatments a year are sufficient to prevent occurrence of large strongyles. all drug classes are effective against adult stages, while ivermectin, moxidectin and larvicidal doses of fenbendazole are effective against larval stages. ideally, anthelmintic treatments should be concentrated during seasons of parasite transmission, typically spring and fall in most regions, as well as during winter months in warmer climates and temperate summers in cooler regions. consider including a treatment against encysted cyathostomins (e.g., larvicidal dose of fenbendazole or moxidectin) at a time when the mucosal burden is expected to be at its peak. this is typically at the end of the grazing season (i.e., late fall / early winter in northern climates and late spring/early summer in warmer climates). if tapeworms are a concern in your region incorporate a cestocidal treatment at least once a year and preferable at the end of the grazing seasons. broodmares that are low shedders typically receive a treatment in the fall and again in the spring prior to foaling or immediately post-partum. ivermectin, with or without praziquantel, remains a popular post-delivery treatment. the incidence of clinical neonatal disease associated with strongyloides westeri, a parasite that can be transferred to the foal via the milk, is extremely low on most well-managed farms. therefore the practice of routinely deworming young foals at two to four weeks of age to prevent strongyloides-associated diarrhea is no longer warranted. maintaining the broodmare on a good 531 clinical theriogenology • volume 5, number 4 • december 2013 deworming regimen should continue to control this parasite. even though some anthelmintics have a “safe in pregnant mare” claim and all commonly used drug classes have been administered throughout pregnancy, the author still prefers to avoid minimize the administration of unnecessary drugs to the broodmare during the early period of fetal organogenesis. parasite control strategies for foals and weanlings foals and weanlings are susceptible to a wide range of parasites including ascarids, cyathostomes, large strongyles, tapeworms, pinworms and thread worms. the most pathogenic of these parasites is parascaris equorum. p. equorum can cause respiratory disease, poor growth, colic and death subsequent to intestinal impaction or perforation.20-22 the pre-patent period for p. equorum is 10-15 weeks. both adult and larval stages of p. equorum are pathogenic. following ingestion, infective, larvated eggs hatch and larvae emerge within the alimentary tract and migrate through the liver and lungs before returning to the small intestine approximately four weeks later as fourth stage larvae. ascarids mature in the small intestine and achieve patency approximately 75-80 days post-infection.20 larvated ascarid eggs are extremely persistent and can survive in the environment for up to ten years, although composting at temperatures >45-60°c does reduce egg viablility.23 although acquired immunity develops in most horses by eight to 18 months of age, effective larvicidal anthelmintics are needed to ensure adequate control of all stages of ascarid infections in juvenile equids. failure of macrocyclic lactone (ml) anthelmintics to reduce p. equorum egg counts in foals has been reported in north and south america and europe.24-34 more recently, there are reports of populations of p. equorum resistant to tetrahydropyrimidines.35 many farms with documented ml resistant (ml-r) ascarids share several common management practices: 1) foals often receive their first anthelmintic treatment prior of 60 days of age (often as early as 14-30 days of age), 2) deworming intervals are often ≤30-60 days, 3) ivermectin is used frequently, if not exclusively, and 4) prior to the discovery of clinical disease due to ascarid infection, fec monitoring had not been routinely performed. more than two decades ago, larvicidal doses of fenbendazole were proven to exert a larvicidal effect on migrating p. equorum larvae in experimentally infected pony foals.35 a more recent trial reconfirmed the efficacy of a larvicidal dose of fenbendazole (10 mg/kg once daily for five consecutive days) to control all stages of p. equorum infections in weanling horses, including those caused by ml-r isolates.36 this study validates the use of larvicidal fenbendazole, based on its documented efficacy against all stages of ascarids, as an integral component of the deworming regimen for foals and weanlings during the first year of life. during the first year of life foals should receive a minimum of four to five deworming treatments. the first treatment should be performed no earlier than two months of age, unless there is a documented medical indication to do treat at a younger age. a benzimidazole drug is recommended to ensure efficacy against ascarids. the label dose of fenbendazole for juvenile horses less than 18 months of age is 10 mg/kg. this is double the label dose for mature horses. the higher drug dose in young animals is critical to ensure efficacy against ascarids, the dose-limiting parasite for most equine anthelmintics. one or two additional treatments are recommended prior to weaning. pyrantel is another drug class with good efficacy against ascarids. the average interval between these early anthelmintic treatments should range between eight and 12 weeks with the goal of reducing the number of patent ascarid infections among the foal population while minimizing the drug selection pressure on resistant p. equorum isolates. moxidectin is not approved or recommended for young foals less than six months of age. at weaning, a fec should be performed to determine whether a foal’s worm burdens are primarily strongyles or ascarids. at least two additional treatments should be administered between weaning and 12 months of age. unless ascarid infection remains a persistent problem, these later drug treatments should target cyathostomins. a tapeworm treatment should be included as part of one of these later treatments. include a larvicidal treatment for encysted small strongyles at the end of the grazing season. the author prefers larvicidal fenbendazole for this purpose in older foals. perform yearly fecrt on foals and weanlings to monitor drug efficacy against cyathostomins and ascarids. save the cleanest pastures for the youngest foals. 532clinical theriogenology • volume 5, number 4 • december 2013 environmental control strategies the goals of any deworming program include prevention of parasite-related disease in the individual animal as well as reduction of egg contamination of the environment. while strategic use of effective anthelmintics is usually the backbone of most parasite control programs, it is important to remember good general husbandry is vital to the success of any deworming regimen. an “open” breeding farm should have biosecurity protocols for new and returning mares and foals that include fecal examinations and prophylactic therapy targeted against potentially drug-resistant cyathostomins and ascarids acquired during recent visits to other farms. new arrivals should be treated prior to gaining access to home pastures. strategies to reduce parasite egg and larvae build-up in the environment include cross-grazing pastures with other ruminant species, preferably sheep, and keeping pastures mowed and/or resting pastures for at least several months during periods of warm ambient temperatures sustained above 85°f to optimize dessication of vulnerable strongyle larvae. harvesting a hay crop off pastures helps reduce parasite burdens. regular manure removal from paddocks and pastures at least twice weekly, while labor intensive and unpopular, can greatly reduce mare and foal exposure to infective stages of strongyle and ascarid eggs and larvae. a testimony to the impact of a clean environment is the observation that most stallions, usually confined to individual, well-groomed paddocks and pastures, typically have low to negligible fecal egg counts. it is unlikely that all stallions are genetically more resistant to strongyle parasites and serves as a reminder that good husbandry practices are among the best parasite control measures. if pastures are harrowed, this procedure should only be done during hot, dry periods and pastures rested for three to four weeks or longer before re-introducing mares and foals. young foals are the most susceptible age group and ideally should be turned out on the farm’s “cleanest” pastures. overcrowding and limited pastures often contribute to the accumulation of heavy parasite burdens by late spring which will be a challenge for any deworming program to control. fresh manure should never be spread on active pastures. properly composted manure can attain high enough temperatures to kill both strongyle larvae and ascarid eggs. 23 references 1. drudge jh, lyons et, tolliver sc, et al: piperazine resistance in population-b equine strongyles: a study of selection in thoroughbreds in kentucky form 1966 through 1983. am j vet res 1988;49:986-994. 2. kaplan rm, klei tr, lyons et, et al: prevalence of anthelmintic resistant cyathostomes on horse farms. j am vet med assoc 2004; 225:903-910. 3. woods tf, lane tj, zeng qy, et al: anthelmintic resistance on horse farms in north central florida. equine pract 1998;20:14-17. 4. bjorn h, sommer c, schougard h, et al. resistance to benzimidazole anthelmintics in small strongyles (cyathostominae) of horses in denmark. acta vet scand 1991;32:253-260. 5. uhlinger c, johnstone c: prevalence of benzimidazole-resistant small strongyles in horses in a southeastern pennsylvania practice. j am vet med assoc 1985;187:1362-1366. 6. tarigo-martinie jl, wyatt ar, kaplan rm: prevalence and clinical implications of anthelmintic resistance in cyathostomes of horses. j am vet med assoc 2001;218:1957-1960. 7. lyons et, tolliver sc, drudge jh, et al: continuance of studies on population s benzimidazole-resistant small strongyles in a shetland pony herd in kentucky: effect of pyrantel pamoate (1992-1999). vet parasitol 2001;94:247256. 8. chapman mr, french dd, monahan cm, et al: identification and characterization of a pyrantel pamoate resistant cyathostome population. vet parasitol 1996; 66:205-212. 9. brazik el, luquire jt, little d: pyrantel pamoate resistance in horses receiving daily administration of pyrantel tartrate. j am vet med assoc 2006;288:101-103. 10. von samson-himmelstjerna g, fritzen b, demeler j, et al: cases of reduced cyathostomin egg-reappearance period and failure of parascaris equorum egg count reduction following ivermectin treatment as well as survey on pyrantel efficacy on german horse farms. vet parasitol 2007;144:74-80. 11. lyons et, tolliver sc, inonita m, et al : field studies indicating reduced activity of ivermectin on small strongyles in horses on a farm in central kentucky. parasitol res 2008;103:209-215. 12. molento m, antunes j, bentes rn: anthelmintic resistance in brazilian horses. vet rec 2008;162:384-385. 13. boersema jh, eysker m, nas jw: apparent resistance of parascaris equorum to macrocyclic-lactones. vet rec 2002;150:279-281. 533 clinical theriogenology • volume 5, number 4 • december 2013 14. hearn fp, peregrine as: identification of foals infected with parascaris equorum apparently resistant to ivermectin. j am vet med assoc. 2003;223:482-485. 15. kaplan rm, nielsen mk: an evidence-based approach to equine parasite control: it ain’t the 60s anymore. equine vet educ 2010; 22:306-316. 16. aaep equine parasite control guidelines: developed by the aaep parasite control subcommittee. www.aaep.org/images/files/parasitecontrolguidelinesfinal.pdf. 17. sangster nc: pharmacology of anthelmintic resistanace in cyathostomes: will it occur with the avermectin/milbemycins? vet parasitol 1999;85:189-201. 18. becher a, mahling m, nielsen mk, et al: selective anthelmintic therapy of horses in the federal states of bavaria (germany) and salzburg (austria): an investigation into strongyle egg shedding consistency. vet parasitol 2010;171:116-122. 19. nielsen mk, haaning n, olsen sn: strongyle egg shedding consistency in horses on farms using selective therapy in denmark. vet parasitol 2006;135:333-335. 20. clayton hm: ascarids: recent advances. vet clin north am equine prac 1986;2:313-328. 21. austin sm, dipietro ja, foreman jh et al: parascaris equorum infections in horses. compend contin educ pract vet 1990;12:1110-1118. 22. cribb nc, cote nm, boure lp, et al: acute small intestinal obstruction associated with parascaris equorum infection in young horses: 25 cases (1985-2004). n z vet j 2006;54:338-343. 23. hebert l, cauchard j, doligez p, et al: viability of rhodococcus equi and parascaris equorum eggs exposed to high temperatures. curr microbiol 2010;60:38-41. 24. slocombe jo, degannes rv, lake mc: macrocyclic lactone-resistant parascaris equorum on stud farms in canada and effectiveness of fenbendazole and pyrantel pamoate. vet parasitol 2007;145:371-376. 25. hearn fpd, peregrine as: identification of foals infected with parascaris equorum apparently resistant to ivermectin. j am vet med assoc 2003;223:482-485. 26. boersema jh, eysker m, nas jwm: apparent resistance of parascaris equorum to macrocyclic lactones. vet rec 2002;150: 279-281. 27. craig tm, diamond pl, ferwerda ns, et al. evidence of ivermectin resistance by parascaris equorum on a texas horse farm. j equine vet sci 2007;27:67-71. 28. lyons et, tolliver sc, ionita m, et al: evaluation of parasiticidal activity of fenbendazole, ivermectin, oxibendazole, and pyrantel pamoate in horse foals with emphasis on ascarids (parascaris equorum) in field studies on five farms in central kentucky in 2007. parasitol res 2008;103:287-291. 29. schougaard h, nielsen mk: apparent ivermectin resistance of parascaris equorum in foals in denmark. vet rec 2007;160: 439-440. 30. molento m, antunes j, bentes rn: anthelmintic resistance in brazilian horses. vet rec 2008;162:384-385. 31. veronesi f, moretta i, moretti a: field effectiveness of pyrantel and failure of parascaris equorum egg count reduction following ivermectin treatment in italian horse farms. vet parasitol 2009;161:138-141. 32. van doorn dck, lems s, weteling a, et al: resistance of parascaris equorum against ivermectin due to frequent anthelmintic treatment of foals in the netherlands. proc world assoc adv vet parasitol; 2007. p.138. 33. lind eo, christensson: anthelmintic efficacy on parascaris equorum in foals on swedish studs. acta vet scand 2009;51:45-49. 34. kaplan rm, reinemeyer cr, slocombe jo, et al: confirmation of ivermectin resistance in a purportedly resistant canadian isolate of parascaris equorum in foals. proc am assoc vet parasitol; 2006. p. 69-70. 35. vandermyde cr, dipietro ja, todd ks, et al: evaluation of fenbendazole for larvicidal effect in experimentally induced parascaris equorum infections in pony foals. j am vet med assoc 1987;190:1548-15499. 36. reinemeyer cr, prado jc, vaala we: larvicidal efficacy of fenbendazole against a macrocyclic lactone-resistant isolate of parascaris equorum in foals. proc am assoc vet parasitol; 2010. 534clinical theriogenology • volume 5, number 4 • december 2013 table 1: anthelmintic efficacy: interpretation of percent (%) fecal egg count reduction (fecr) values used to determine anthelmintic efficacy.16 anthelmintic expected efficacy if no resistance present susceptible (no evidence of resistance) suspected resistance resistance present benzamidazoles 99% > 95% 90 95% < 90% pyrantel 94 99% > 90% 85 – 90% < 85% ivermectin / moxidectin 99.9% > 98% 95 98% < 95% table 2: cyathostomin egg reappearance periods (erp) for commonly used equine anthelmintics.16 anthelmintic expected erp when drug is still effective erp when drug was first introduced benzimidazoles 4 – 5 weeks 6 weeks pyrantel 4 – 5 weeks 5 – 6 weeks ivermectin 6 – 8 weeks 9 – 13 weeks moxidectin 10 – 12 weeks 16 – 22 weeks 535 clinical theriogenology • volume 5, number 4 • december 2013 536clinical theriogenology • volume 5, number 4 • december 2013 omniblank: 2010: pathology of the mare reproductive system pathology of the mare reproductive system fabio del piero new bolton center, school of veterinary medicine, university of pennsylvania, kennett square, pa anomalies of phenotypic and genotypic sex pseudohermaphrodites are rare in the equine species. in these animals the chromosome constitution and gonadal sex are in agreement, yet the internal or external genitalia are ambiguous. there is one type of gonad, by which the individual is classified, and ducts modified toward those of the other gender. in animals, male pseudohermaphrodites are more common than female pseudohermaphrodites which are rare; for many of these abnormalities the mechanisms are not understood. hermaphrodites are also very rare. both types of gonadal tissue are present; male and female cells coexist in the same individual. the chromosomal make-up is a chimera, mosaic, xx with or without sry gene, or unknown. in unilateral hermaphrodites an ovotestis is on one side while bilateral hermaphrodites have an ovotestis on both sides. lateral hermaphrodites have an ovary on one side and a testis on the other and the ducts are abnormal. one or both testes may or may not be palpable, but are undescended. the diagnosis can be confirmed via karyotyping and histopathology. in species other than the horse possible causes include congenital adrenal gland hyperplasia, fetal exposure to sex hormones, testicular feminization syndrome, xy gonadal dysgenesis, xy gonadal agenesis, and chromosomal abnormalities. sry is present in many but not all cases in humans. ovarian lesions agenesis is the complete absence of the ovary and its associated primordium. it is very rare in horses. hypoplasia is the incomplete development or underdevelopment of the ovary with decreased numbers of cells. follicles are usually absent. aggressive testosterone treatment in racing mares can mimic this condition. in these cases the ovaries are both smaller than normal with minimal gross evidence of follicular activity, though histologically follicles are evident. treated mares may present clitoral hypertrophy. turner syndrome is characterized by partial or complete absence of one x chromosome (xo gonadal dysgenesis). the turner mare is affected by hypogonadism (small fibrotic ovaries). they may also have short stature, a broad-based neck, and narrowing of the aorta. external genitalia tend to be small and the tubular tract is small and flaccid. mares with this disease lack one of the pair of sex chromosomes resulting in a 63,x karyotype. the origin of the defect may be an accident in development, not an inherited problem. the syndrome has been diagnosed in various horse breeds throughout the world including thoroughbred, arabian, welsh pony, tennessee walking horse, standardbred, american saddlebred, paso fino, belgian, quarter horse, appaloosa, as well as in grade horses. cysts may arise from both mesonephric and paramesonephric embryonic duct systems. there are cysts adjacent to the ovary and cysts within the ovary. cysts within the ovary include non-gonadal stromal cysts (inclusion cysts, cystic rete ovarii), gonadal stromal cysts (from corpora lutea and follicles), anovulatory luteinized cysts, cystic corpora lutea, and neoplastic cysts. it is debated if cystic graafian follicles occur in mares. oophoritis is the inflammation of the ovary. it is a rare event. follicle atresia is a degenerative breakdown of follicles. excessive atresia can ultimately result in infertility. the selective loss of ovarian follicles by atresia occurs via apoptosis and this process is most evident within the granulosa cell layer. hemorrhage is secondary to trauma, vasculitis and neoplasia. hematomas may develop near ovulation. ovarian neoplasms are comprised of germ cell tumors (teratoma, dysgerminoma), gonadal stromal tumors and surface tumors (cystadenoma, carcinoma, mesothelioma). teratoma is rare, usually well-differentiated, and benign, exhibiting at least two of the three embryonic germ layers. it is generally comprised of nonproliferating somatic tissues. 545 dysgerminoma is even more rare than teratoma, analogous to seminoma and generally as malignant as this sporadic tumor in stallions. the neoplastic parenchyma is pale brown. the gross and microscopic appearance is essentially the same as that of a seminoma. granulosa cell tumor is a very common tumor in mares. it is rarely malignant in large animals. the gross appearance may be multicystic, unilocular cystic, or solid. forty to fifty percent produce androgens resulting in stallion-like behavior and ninety percent produce inhibin causing regression of the contralateral ovary. the nymphomania reported in mares seems to be due to lack of progesterone (not elevated estrogens) caused by increased inhibin production by the neoplastic granulosa cells (80-90 % of the cases). anestrus due to failure to cycle occurs. granulosa cell tumors can also occur in pregnant mares. cystadenoma, carcinoma and mesothelioma are rare. cystadenoma is unilateral. uterine (fallopian) tube lesions hydrosalpinx is the distention of the uterine tube by clear fluid. salpingitis is the inflammation of the uterine tube due to bacterial and viral infections. pyosalpinx is characterized by gathering of pus within the oviductal lumen predisposed by bacterial infection. all these salpingeal lesions are rare in mares. uterine lesions segmental aplasia is rare and characterized by the failure of müllerian ducts to fuse with the urogenital sinus. prostaglandin produced in the blind uterine horn can cause luteolysis in the contralateral ovary during pregnancy. long-term distention can cause inability to produce endometrial prostaglandin with failure of regression of the corpus luteum. imperfect fusion of primordial ducts may lead to the formation of a double uterine fundus. the normal sequence of fusion of the paired ducts is from posterior to anterior. uterine torsion is predisposed by pyometra, mucometra, and pregnancy and leads to hypoxia, infarction, and shock. prolapse may involve pregnant uterine horn, body, urinary bladder and intestine, also leading to hypoxia, infarction, and shock. endometrial atrophy follows loss of ovarian function in cases of inhibin secreting neoplasia or very old age. adenomyosis is characterized by ectopic endometrial glands within the myometrium; it is very rare in horses. endometritis is the inflammation of the mucosal uterine layer and is related to introduction of semen, pregnancy, parturition and postpartum involution. most infections start within the endometrium. several factors may play a role. progesterone may make the uterus more susceptible to infection. estrogens increase leukocytic phagocytosis and induce cervical opening with consequent drainage and/or exposure. prostaglandins trigger uterine motility. usually, in mares with endometritis, after a short diestrus due to early prostaglandin f2alpha release there is an early return to estrus. in cases of chronic endometritis there is decreased capability to produce prostaglandin and a corpus luteum and progesterone production may persist. the etiologic agents inducing endometritis can also cause abortion (e.g. streptococcus equi equi and s. equi zooepidemicus). occasionally the process can spread to the wall, leading to metritis. pyometra is the acute to chronic suppurative inflammation of the uterus with pus accumulation within the uterine lumen. endometritis is followed by fibrosis (transluminal fibrous adhesions may occasionally occur), which can impair the uterine gland functions and predispose to the formation of cystic glands. glandular cysts are frequent microscopic findings and lymphatic cysts are the most common macroscopic cyst observed in mares. lymphatic cysts occur in multiparous mares over 14 years of age and can be single or multiloculated. persistent endometritis can be induced by mating. breeding is followed by inflammation, decrease of mechanical clearance, increase of intraluminal fluid, increase of inflammation, 546 interstitial edema and fibrosis and cyclic endometrial irritation. poor conformation and perhaps abnormal hormonal release may decrease the mechanical clearance. perimetrial adhesions and abscesses are associated with trauma. uterine foreign bodies may include marbles, placental remnants, and cotton from swab tips. uterine primary and metastatic neoplasms are rare in the mare. in cases of lymphoma often the pregnant horn is less involved. endometrial carcinoma is very rare. leiomyomas and fibroleiomyomas are likewise rarely observed. cervical lesions double cervix is very rare and caused by the failure of fusion of the müllerian ducts. other malformations of the cervix are also very rare, including hypertrophy, hypoplasia, and diverticula. all these changes can create problems for artificial insemination. cervicitis is caused by trauma and infectious agents. vulvar and vaginal lesions vulvo-vaginitis is caused by viruses, bacteria, fungi, chemicals and trauma. pneumovagina and urovagina can be associated with conformation anomalies. melanoma, lymphoma and squamous cell carcinoma are the most common vulvar neoplasms. solar radiation and papillomavirus may be triggering factors of vulvar squamous cell carcinomas. complications arising from parturition are numerous. rectal prolapse, rupture of small colon mesentery, bladder eversion, prolapse and rupture, and cervical, vaginal and perineal lacerations may occur. postpartum hemorrhage in the broodmare is a common problem. vessel rupture usually involves the uterine artery, but the ovarian and iliac arteries may also be affected. retained fetal membranes can be associated with fescue toxicity, abortion, dystocia, placentitis, uterine inertia, and twin pregnancy, but can also be seen following an otherwise normal delivery. keywords: mare, pathology, uterus, ovary, uterine tube, cervix 547 548 omniblank: localized uterine body pyometra causing urinary obstruction in an american bully bitch localized uterine body pyometra causing urinary obstruction in an american bully bitch lacey rosenberg,a rocio barrientos,b daniel lopez,a anna mitchell,a sarah robbins,a sean mcdonough,b soon cheong,a mariana amorima adepartment of clinical sciences bdepartment of biomedical sciences, cornell university, ithaca, ny a 10 month old female intact american bully was presented as an emergency case with a 3 day history of stranguria, hyporexia, and vomiting. her owner had noticed his male dogs being interested in the female ~ 1 week prior to presentation and serosanguineous vulvar discharge was noted the day before arrival. the patient was housed separate from male dogs and the owner believed it was not possible that she had been mated. on presentation the patient was tense on abdominal palpation and the urinary bladder was enlarged and firm. stranguria and vulvar enlargement were noted on examination. the dog also had profuse foul smelling vaginal discharge. vaginal cytology revealed predominantly superficial and intermediate cells, heavy bacterial load and many degenerate neutrophils. focused caudal abdominal ultrasonography showed a markedly enlarged bladder and an echodense, fluid filled uterus. a presumptive diagnosis of pyometra was made and ovariohysterectomy recommended. the patient was treated supportively with fluids, antibiotics, analgesics, and gastroprotectants in preparation for surgery. exploratory laparotomy revealed hemoabdomen and a turgid, enlarged bladder with focal ulceration and necrosis. the uterine body was extremely enlarged caudal to the bifurcation and extending into the vaginal canal, which was suspected to cause a functional urethral obstruction. the urinary bladder was drained with suction and a bilateral ovariectomy and partial hysterectomy (focally dilated uterine body was not removed due to inability to excise) were performed. the remaining portion of the uterus was sampled which revealed the presence of pyohemorrhagic fluid. the fluid was drained via suction and a catheter was passed to ensure patency to the vagina. the ulcerated portion of the urinary bladder was resected and another catheter was passed to ensure urethral patency. due to surgical findings being atypical for the presumptive diagnosis of pyometra, the owner was contacted by the surgeon and elected to euthanize due to guarded prognosis. the patient was submitted for postmortem examination along with the surgically resected uterine horns and ovaries. on gross evaluation the mucosal folds of the vagina, cervix and uterine body were severely thickened (edema), the mucosal surface was diffusely white and rough, resembling the keratinized epithelium of the vagina. in the most cranial aspect of the body of the uterus, mucosa was diffusely gray to pink, with multifocal pinpoint areas of hemorrhage and three well demarcated, irregularly shaped, depressed, dark red ulcers from 1 1.5 cm in diameter and a larger defect that was 3 x 3 cm, with a pale, depressed center and a black rim around the edges. the uterine horns were small with thickened walls and a reduced luminal space. histological evaluation revealed complete absence of glandular structures and a severe subacute to chronic ulcerative process in the endometrium of the uterine body, most likely as a result of active inflammation and structural damage to the blood vessels (vasculitis) in this area. this inflammatory process extended rostrally to the bifurcation with moderate edema and inflammation in the uterine horns and caudally to the cervix. inflammation also extended to the serosal surface of the urinary bladder. this case presentation was not typical for pyometra; specifically, being in estrus and the localized accumulation of pus within the uterine body. it can be presumed the focal uterine enlargement led to functional urethral obstruction. keywords: canine, pyometra, ulcerative endometritis, urinary obstruction, vasculitis 469 clinical theriogenology • volume 11, number 3 • september 2019 470clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2010: overview of canine artificial insemination for the breeder overview of canine artificial insemination for the breeder harmon rogers veterinary teaching hospital, washington state university, pullman, wa insemination is the introduction of semen into the reproductive tract of a female. in mammals natural insemination follows copulation. artificial insemination (ai) is insemination by other means. the first successful artificial insemination reported in any species was performed for a bitch in italy in 1784. today ai is essential in animal agriculture. it is critical for efforts to prevent species extinction, and it now is performed commonly in the pure bred dog fancy. this is an overview of items of importance when ai is considered. keywords: artificial insemination, canine, first it is important to know that for most breeds and lines the greatest number of offspring come from natural cover of a healthy female by a proven sire repeated every two days during standing heat. when it is all said and done it is hard to improve on nature. artificial insemination provides for conception when physical problems or circumstances prevent natural breeding of otherwise healthy and genetically sound animals. it overcomes barriers of space and time so that regardless of the location of a male his genes can be brought to a female at the precise moment necessary for fertilization of her ova. artificial insemination combined with methods for collection, preservation, and shipping of semen can eliminate the need for travel of animals for breeding; ai can reduce risks of injury and infection of the bitch during natural breeding; and it can entirely eliminate these risks for the male. artificial insemination should not be used to overcome genetic problems which prevent natural breeding such as the inability to mount because of heritable joint disease. likewise, stored semen should not be used for ai if the male has evidence of heritable disease at any time of his life even if he appeared completely healthy at the time the semen was collected. because ai can facilitate multiple inseminations from a single dog it can maximize the number of puppies born to a desirable male. this is a two-sided sword and can have negative consequences long term should a male be an inadvertent source of a genetic defect. this is a good reason for preserving semen from many healthy males. a breed will be better able to recover from a widespread defect if there is available stored semen from many males who are unlikely to have been affected. when ai is done, compliance with registry requirements is important. some registries control the breeding of all registered dogs and must give their approval before a breeding can be performed. other registries require dna testing for breeding performed with shipped or stored semen. shortcuts for testing, incomplete animal and semen identification and failure to comply with registry rules can result in questioning parentage and is to be avoided always. in a number of states artificial insemination is considered an animal husbandry task and an inseminator does not have to be a licensed veterinarian. for success and to avoid serious injury to the bitch the inseminator must understand semen processing, instrument handling, and infection control. the inseminator must have very specific knowledge of anatomy, estrous cycles, and tissue manipulation in the species they inseminate. in the bitch the reproductive tract and surrounding tissues are complex structures which vary in size and position from breed to breed and female to female. a bitch in standing heat is remarkably tolerant of manipulation, but her tissues are delicate and easily injured. major nerves, blood vessels and the urinary tract are close at hand. inadvertent perforation of the vagina can occur, and insemination into the abdominal cavity rather than the vagina is life threatening. the estrous cycle of the bitch is unique among domestic animal species. a bitch may be in season for three weeks or longer, but the time when conception can occur lasts only two days. artificial insemination can be quite successful when it is done every other day throughout the 497 entire standing heat. in some bitches this may require four or more inseminations. when the number of inseminations is limited then ovulation timing to identify the fertile period is necessary. behavior changes, vaginal swelling, vaginal discharge, and vaginal cytology can verify when a bitch is in standing heat and when she goes out, but they cannot identify ovulation and predict the fertile period. that requires blood tests for progesterone and possibly luteinizing hormone. semen used for ai can be freshly collected and used right away. it can be extended and chilled for short term storage or shipment. it can be frozen for many years and then thawed and used. if all other factors are equal, fresh collected semen is preferred to extended chilled semen, and extended chilled semen is preferred to frozen then thawed semen. collection of semen is by manual manipulation. it is best done at a location where the male feels at ease. an in-heat teaser bitch for stimulation of the male is desirable, but she is not essential if the male has been trained for collection. semen can be collected into a variety of containers including artificial vaginas, plastic bags, and funnels with plastic tubes. semen should be collected with clean processes and held in very clean if not sterile containers from time of collection until it is used. semen used for insemination must be assessed for quality when collected and at the time of insemination. quality is determined by sperm count, sperm motility, and sperm morphology. one hundred million to two hundred million highly motile and morphologically normal sperm are minimum numbers needed for a single vaginal insemination. conception can occur with fewer sperm with intrauterine insemination. fresh collected semen generally has very high total sperm numbers with good motility and long lifespan. chilled and extended semen is similar except sperm motility and longevity are diminished by chilling and shipping or storage time. frozen thawed semen has limited number of live sperm, has greatly reduced motility and sperm have a lifespan of only about a day after thawing. semen from an infected male can be a source of infection for the bitch. infection may be suspected or confirmed by a medical history, physical examination, and testing of the male. infection should be considered if semen has unusual numbers of red and white blood cells with or without bacteria. compared with semen from many other species, canine semen is not degraded by short term exposure to room temperature. nevertheless, it should not be left at room temperature for long periods. semen is ejaculated in several fractions some of which contain sperm and some of which do not. the sperm rich fraction should be separated from other fractions, and dilution of extended chilled semen or frozen thawed semen with prostatic fluid is not desirable. if semen is collected from more than one male or if it is stored or shipped to another location, semen containers must be always be marked with positive identification of the male from which it was collected so no misuse will occur. techniques for ai in the canine place sperm in the forward part of the vagina or in the uterus. the vaginal environment is not hospitable to sperm. sperm which are left there survive for only a few hours. in contrast, the inside of the uterus is supportive. so a goal of insemination is to get a large number of motile sperm inside the uterus. during natural breeding semen is ejaculated into the forward part of the vagina. as mating begins the penis becomes engorged sealing the vagina so semen cannot escape. ejaculation of a sperm rich fluid fraction is followed by ejaculation of a greater volume of prostatic fluid. pressure from the continuing ejaculation forces some semen through the cervical canal and into the uterus. sperm motility also causes movement from the vagina and into the uterus. vaginal ai with a standard plastic insemination pipette is a decades old method for insemination of the bitch. this process deposits semen in the forward part of the vaginal space. after insemination, the bitch’s rear quarters are elevated so that gravity can assist in keeping the 498 semen forward and near the cervix. even though fluid pressure is not great some of the millions of motile sperm will find their way through the cervical canal and into the uterus. a recent development for vaginal ai is the mavic ® vaginal insemination catheter minitube, verona, wi). this device simulates an inside tie and provides a way to create and hold pressure to enhance movement of semen through the cervical canal. the catheter is currently available in three sizes for use in most breeds. a long, soft plastic outer catheter contains a small diameter flexible metal tube. the plastic catheter has an inflatable bulb at its forward end. after placement into the vagina the bulb is inflated and forms a temporary seal between the catheter and the wall of the vagina. semen is inseminated through the internal metal tube and reflux is prevented by a check valve. with proper size, placement and inflation, the bitch appears quite comfortable during breeding. following insemination the catheter is secured with wraps and left in place. after 15 to 30 minutes the wraps are taken off, the rubber bulb deflated and the catheter removed. with good quality fresh or chilled semen the preferred method for ai is vaginal insemination with a mavic ® catheter or similar device. when semen is poor quality or if it has been frozen and thawed, semen must be placed directly into the uterus by vaginal transcervical catheterization or by surgical intrauterine insemination. transcervical insemination (tci) is the placement of a catheter from the vagina through the cervix and into the uterus. it is best done with a small flexible catheter under direct endoscopic visualization. a non-visual method using a rigid insemination catheter is not recommended. because tci is well tolerated by the bitch and does not require sedation, anesthesia, or surgery it is the method of choice for intrauterine insemination. the bitch is gently restrained in a standing position on a table while an endoscope is advanced vaginally so that the cervix comes into view. the end of the scope is carefully manipulated and a flexible plastic catheter is advanced into the opening, through the cervical canal and into the uterus. semen is inseminated through the catheter into the uterus. transcervical insemination is a challenging procedure, and placement of a catheter is not always possible. in that case the alternative is insemination by surgical means. for the veterinary surgeon surgical insemination is a procedure somewhat similar to ovariohysterectomy surgery. of course, rather than removing the ovaries and uterus the surgeon places a small catheter thorough the wall of the uterus and semen is placed inside. regardless whether insemination is by tci or by surgical insemination the semen is placed inside the uterus so conception rates should be identical between the two procedures. recovery from surgical insemination is very rapid because other than a minor incision in the skin and abdominal wall no other tissues are disrupted. surgical insemination can be done only once during a single heat so ovulation timing is very important. transcervical insemination can be done more than once during a single heat, but cervical swelling following catheterization can make multiple inseminations difficult. 499 500 omniblank: 2016: breeding soundness examination of the male ovine, caprine and cervidae species breeding soundness examination of the male ovine, caprine and cervidae species clifford f. shipley college of veterinary medicine, university of illinois, urbana, il summary breeding soundness examination (bse) should be undertaken as a routine procedure to ensure that a fertile male is available to impregnate fertile females during the breeding season, to assess fertility prior to purchase, to diagnose non-pregnancy or return to estrus and to make sure the male is producing normal spermatozoa prior to cryopreservation. keywords: breeding soundness examination, ram, buck, cervidae the breeding soundness examination may consist of a variety of serological or other tests depending on species, a physical examination to make sure that the animal is in overall good health, and an in-depth examination of the reproductive tract and semen. a complete history that includes information about previous reproduction as well as purchase information, health and vaccination is also very important. intended use and expectations for the male should also be discussed. the bse is generally performed as part of a prepurchase examination for breeding animals to protect both buyer and seller. scrotal circumference has been linked with age of puberty onset as well as fecundity of offspring and is an important part of the bse. pregnancy rates can be affected as well as length of parturition season, uniformity of offspring, ease of management due to similar ages of offspring and nutrition and vaccination programs. a prolonged parturition season may also affect the next breeding season due to uterine involution, lactation and decreased body condition score. this may severely impact the producer from an economic standpoint. most prebreeding bse’s are conducted 30 to 60 days prior to breeding season but due to seasonality issues in these species (especially cerivdae), it may be better to wait until just prior to the breeding season. however, if conducted too close to breeding season, it may be hard if not impossible to obtain a replacement male of similar quality if they do not pass the bse or there may not be time for them to recover or improve to pass the bse. in cervidae, especially whitetail and mule deer, restraint, capture and anesthesia issues all enter into the bse conundrum. in cervidae, the bse results may also change dramatically over the course of a few weeks, especially early in the breeding season. post-breeding season bse’s are generally indicative of a disaster where very low or no conceptions occurred, lambs, kids, calves or fawns failed to make their appearance or observant livestock managers notice return to estrus, lack of abdominal fill or lack of udder development. this kind of bse may not tell you what you need or want to know due to seasonal changes in these animals (especially ceridae) and the fact that it may not be indicative of what was transpiring during the breeding season. the lost production may also be so much of a financial burden that the farm may not survive. the steps that are typically followed in doing a bse would include identification of the animal by some means that is permanent and unique. tattoos, microchips, tamper-proof ear tags, electronic identification, photographs, nose prints, dna and other methods may all be acceptable. a complete history of the animal is also important. origin, age, vaccination status, deworming, etc., as well as past breeding history provide important clues in regard to tests to run (brucella ovis on western origin rams for instance) or provide clinically relevant history. a physical examination (pe) of the subject should also be done. this data set could include anything from an observation that the animal appears healthy to getting a complete data set of information up to and including blood work, radiographs, serological tests, other laboratory tests as appropriate for species, location, state and federal requirements and the wishes of purchaser or owner. this should be discussed so that all involved are comfortable with the testing and physical examination. some animals may not be able to be handled except under anesthesia which may make a routine pe difficult. as part of the pe, on all species that it can be done, a rectal examination of the internal genitalia should be attempted. even if one cannot do a complete internal examination, a digital exammination may clinical theriogenology • volume 8, number 4 • december 2016445 reveal some alterations in the accessory sex glands and should be noted as such on the bse form. external genitalia should be closely examined in a systematic manner so that nothing is missed. scrotum, penis, testicles, head, body and tail of the epididymides as well as the prepuce and spermatic cord should all be palpated and visually inspected for lesions, swelling, symmetry or any other deformation. scrotal circumference should be measured by pushing the testicles into the distal part of the scrotum and measuring the greatest circumference. it is important that this be done with a slight amount of pressure that just indents the skin so that measurements by different individuals may be within agreement between evaluators. cutoffs for scrotal size are in tables 1-3 for sheep and goats. there are no published measurements for cervidae, but yearling whitetail deer generally have scrotal circumference in the 15-18 cm category and older whitetails are generally in the 18-24 cm range. wapiti are generally in the 24 cm range and, based on a limited number of measurements by the author, red stag appear to be about the same as wapiti. table 1. recommended minimum scrotal circumference in rams 8-14 months of age. size rating <24 cm questionable 28-36 cm satisfactory >36 cm exceptional table 2. recommended minimum scrotal circumference in rams >14 months of age. size rating <32 cm questionable 32-40 cm satisfactory >40 cm exceptional table 3. recommended minimum scrotal circumference in bucks. 45 kg dairy breeds 25-28 cm >45 kg dairy breeds 34-36 cm 45 kg 7 month-old kiko and boer goars 26-29 cm the penis should be examined, especially on sheep and goats. the urethral process is commonly damaged or cut off due to urolithiasis and should be noted. for sheep, it is relatively easy to set them up on their dock and exteriorize the penis. male goats tend to resist more than rams and may need sedation for examining the penis. whitetail and mule deer bucks are usually anesthetized and the penis can be extruded from the prepuce with careful manipulation of the prepuce and penis. grasping the glans with a gauze sponge is usually necessary as the penis is very difficult to hold without it. it is very important to note the anatomical differences in species, especially with sheep and goats, so that misdiagnosis of penile abnormalities are avoided. semen collection can be done with an artificial vagina (av), an intravaginal condom or other device or on a “dummy” fitted with an av depending on species. an av generally requires training and a jump animal. depending on the species, this can be a dangerous undertaking or the male may not perform due to temperament issues (wild). this does give a better evaluation of libido than does collection via electroejaculation (eej) however. it also allows the male to demonstrate erection, protrusion and intromission for evaluation. most males have not been trained, there is no available “jump” animal or “dummy” around or the temperament and danger make it untenable for av collection. clinical theriogenology • volume 8, number 4 • december 2016 446 most semen collection is done by eej using a variety of different machines that have been manufactured for bovine, sheep or goat use. electroejaculation for the ram generally consists of manual restraint with the animal standing in a chute or pushed up against a wall or gate. the rectum is cleared of feces and the internal sex glands massaged with the probe that has been properly lubricated. a series of short bursts from the eej consisting of 2-4 seconds on and 2-4 seconds off, generally result in ejaculation after 2-6 bursts. some rams may require more stimulation. the ejaculate is small in volume (1-2 ml) but is very concentrated. a collection cone or “baggie” is generally placed over the prepuce and observed for successful ejaculation. male goats may be collected in the same manner but using a lower power setting if possible. they also tend to vocalize to an extreme and as such, it may be appropriate to either warn the owner/buyer or ask them to wait in an appropriate area. studies have been done that show the procedure is not detrimental to the animal’s health or reproductive tract. in cold weather, appropriate care must be taken to control thermal effects on semen. the author usually collects semen into a container surrounded by a water jacket of the appropriate temperature. if collecting for semen freezing, the animal is usually laid in lateral recumbency and the penis exteriorized for a cleaner sample and maximum semen production. this may be facilitated by tranquilization or sedation. wapiti and red stag may be collected standing using an appropriate “squeeze” chute or box. some may benefit from mild sedation during the process, usually a very low dose of xylazine as they may lay down if given too much (25-50 mg im for a mature wapiti). they also may be collected under anesthesia if no restraint facilities are available. almost all whitetail and mule deer bucks are collected under anesthesia. a variety of drugs are available that work well, ranging from masking them down in a chute with gas to drugs or combinations of drugs given via remote injection or hand injection while in a chute or restraint box. telazol® (4.4 mg/kg); telazol®(1.6 mg/kg-2.5 mg/kg) plus xylazine (2.2 mg/kg); xylazine (1-3 mg/kg) plus ketamine (2-3 mg/kg); meditomidine (0.1 mg/kg) plus ketamine (3 mg/kg). bam (butorphanol + azaperone + meditomidine) is not appropriate for semen collection in the cervid because azaperone interferes with ejaculation. electroejaculation in a chute has been done with whitetail deer under tranquilization with haloperidol (murray woodbury personal communication). semen should be evaluated for motility under a microscope. the author generally evaluates gross motility under 10x or 20x first without a cover slip and then with a cover slip. most small ruminant semen is too concentrated to observe individual motility unless diluted with an extender or saline. some operators rate the percent progressive as well as assign a “speed” rating of from 1-5 with 5 being the fastest. many things may affect motility so temperature and concentration need to be taken into account. the author usually examines multiple areas (at least five) on the slide and computes the average motility. morphology of the sperm cells also needs to be taken into account. generally, sperm are divided into normal and abnormal classifications with primary and secondary abnormalities or major and minor defects. generally speaking, a ram would be rated as an unsatisfactory breeder with less than 50% normal sperm and less than 30% motility. for a buck goat, less than 70% motility and less than 80% normal morphology would be unsatisfactory. for cervidae, the author generally ranks using goat/sheep parameters as guidelines noting that there is much seasonality to consider in these animals. unsatisfactory ratings may also be given due to physical examination abnormalities such as scrotal, testicular or spermatic cord defects. eyes, feet, legs and other abnormalities should also be taken into account and weighted accordingly. some animals may have their classification deferred or may be referred to as questionable breeders. this is especially difficult in the very seasonal cervidae group. semen may also have been mishandled or exposed to agents that affect their motility. reexamining these animals is important so that a breeding season is not lost. reexamination these animals in one to two weeks may be appropriate but will subject them to another anesthesia and handling event that is very stressful. clinical theriogenology • volume 8, number 4 • december 2016447 clinical theriogenology • volume 8, number 4 • december 2016 448 omniblank: 2010: i did everything right to breed my bitch-what went wrong! i did everything right to breed my bitch–what went wrong! mushtaq a. memon department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa abstract there are many reasons for a bitch not getting pregnant after breeding. breeding soundness examination (bse) of the stud dog would establish if the dog used for natural service is infertile or sterile. improper breeding management for artificial insemination (ai) is a major cause for a bitch not getting pregnant. progesterone (p4) assay, vaginal cytology and other related tools are utilized for ai at the appropriate time. a bitch with a split heat shows proestral bleeding, vulvar swelling but does not ovulate. affected bitches go out of proestrus and come back into proestrus again two to ten weeks later. the second half of the split heat is usually normal. follicular cysts produce estrogen and cause prolonged proestrus or estrus. ovarian follicles develop normally but do not ovulate. luteal cysts produce p4. persistent and prolonged p4 production may cause infertility or predispose the bitch to cystic endometrial hyperplasia and pyometra. ovariohysterectomy is the treatment of choice for bitches not intended for breeding. valuable breeding bitches with follicular cysts are treated with gonadotropin-releasing hormone (gnrh) or human chorionic gonadotropin. hypothyroidism is the most common endocrine disorder, and is considered a hereditary condition in the dog. dogs suspected of hypothyroidism are diagnosed by measurement of both thyroxine (t4) and thyroid stimulating hormone (tsh). the condition is treated by oral supplementation with thyroid hormone. dogs without a confirmed diagnosis of hypothyroidism should not be given thyroid supplement. early embryonic loss and resorption may be caused by infectious agents, including bacteria, parasites and viruses. non-infectious causes of resorption include endocrine abnormalities, certain drugs given to the bitch during pregnancy and genetic factors. hypoluteoidism, an endocrine abnormality, can cause embryonic death and resorption. monitoring progesterone in a bitch with suspected hypoluteoidism once or twice weekly allows accurate diagnosis of this problem. hypoluteoidism is treated by p4 supplementation of the bitch. keywords: dog, breeding management, breeding soundness examination, early embryonic death, ovarian cysts introduction there are many reasons for a bitch not getting pregnant after breeding. it may be due to problems related to the male or the female. the male used for natural services may be infertile or sterile. for ai, improper breeding management and issues surrounding ai may be the problem. other reasons for the bitch not getting pregnant may include split heat, ovarian cysts, and early embryonic death. this paper will discuss briefly each topic to understand possible reasons for a bitch to fail to conceive after breeding. the male factor whether natural breeding or ai is used, a stud dog with desirable breeding potential is important. many dog owners and breeders assume that all males are fertile. stud dog owners and breeders may invest a lot of time and money showing the dog and may receive awards, only to find that the stud cannot get a bitch pregnant. it is relatively inexpensive to have the stud examined for breeding soundness before putting him on the show circuit or using him for breeding. breeding soundness examination the author recommends having a bse performed by a veterinarian. one must remember that the bse is valid only at the time of examination. the bse findings frequently cannot explain the reason for infertility and cannot guarantee the future performance of the stud. the examining veterinarian will ask for a general medical history which may include vaccinations and past illnesses. a detailed reproductive history would include the results of any previous semen evaluation if available and brucella canis test 501 results. if the male was used for breeding, it would be important to know the results such as litter size, the number of bitches bred by the male, and the method of pregnancy diagnosis used. if a bitch does not become pregnant after breeding, some dog owners and breeders assume the bitch was pregnant and reabsorbed the litter based upon weight gain and enlarged mammary glands. bitches in diestrus or pseudopregnancy also show these changes, so these signs cannot be used to conclude that the bitch was pregnant. special attention is given to abnormalities which may interfere with the expression of male sexual desire (libido) or interfere with mating ability (e.g., lameness). the examination of the reproductive organs includes the testes, epididymides and prostate. depending upon the temperament of the male, especially in the case of anxious dogs, it may be better to wait and do this part of the examination after semen has been collected when most of the studs are easily manageable. owners may be interested knowing about the hormones responsible for male reproductive functions including sperm production. the hypothalamus, which is located at the base of the brain releases gnrh, which stimulates release of two important reproductive hormones from the pituitary gland (located below the hypothalamus). these hormones have same names in males and females, follicle stimulating hormone (fsh) and luteinizing hormone (lh). the testes produce sperm and the male sex hormone, testosterone. both fsh and lh affect the functions of the testes. sperm production and sexual desire as mentioned above, the testes perform a dual function–producing sperm and testosterone. at the time of ejaculation, sperm are transported from the epididymis to the pelvic urethra by the ductus deferens. the sperm are mixed with fluid (seminal plasma produced primarily by the prostate gland) and released from the penis during ejaculation. it takes about 60 days to complete a cycle of sperm production (spermatogenesis), 45 days in the testes and 15 days to travel through the epididymis. in other words, the semen collected today was made about 60 days ago. this fact is important because if a semen sample collected today shows poor quality one must wait for about 60 days to collect semen containing a new ‘crop’ of sperm. testosterone therapy for dogs with poor sexual desire. some dog owners request testosterone therapy for their dogs with poor sexual desire. it has been shown in horses that testosterone therapy decreases testicular size and increases sperm abnormalities in the ejaculate. this is due to negative feedback on the pituitary gland. most experts agree that libido is inborn characteristic of the male. unique reproductive anatomy some unique features of dog reproductive anatomy include the presence of a bone in the penis (os penis or baculum) and the presence of a well-developed accessory sex gland, the prostate. the author considers the presence of two palpable testes in the scrotum a requirement for a sound breeding dog. the absence of one or both testes is unilateral or bilateral cryptorchidism, respectively, which is an undesirable hereditary condition. normal testes should be symmetrical and have a firm consistency. even though there is limited information available regarding testicular size in different breeds, most experts agree that there is a positive correlation between testicular size and sperm production. in other words, larger testes are associated with more the sperm production, assuming the testes are normal and not enlarged due to the presence of abnormalities. semen collection and evaluation the characteristics of normal canine semen are shown in table 1. semen can be collected by digital manipulation of the penis with the help of an artificial vagina (av; a latex rubber cone attached to a 15 ml calibrated plastic centrifuge tube). another technique is to utilize a gloved hand with semen collected into color-coded funnels. the author prefers the funnel technique because of the ease in collecting semen fractions separately. most experienced dogs will ejaculate with simple hand 502 stimulation, whereas inexperienced or younger dogs may require a teaser bitch in heat. some dogs do not care if the bitch is in heat or not–they just like the company. the semen collection environment, the collector’s experience and the dog’s temperament influence the outcome of collection. a door mat or a carpet for good footing is helpful. attempts to try to collect semen on a stainless steel examination table are almost always unsuccessful. the author gets on his knees on the floor at the dog’s level to collect semen. if a teaser bitch is used and is not familiar with the male, it is useful to give them some time to become acquainted with each other. most dogs ejaculate the pre-sperm (clear) and sperm rich fraction (cloudy) of semen during the time of most rapid pelvic thrusting, then dismount and lift one hind leg as though trying to step over the bitch and achieve a tie. at this time, the penis is rotated nearly 180 degrees and directed caudally while maintaining tight pressure around the bulbus glandis until the prostatic fraction of semen is ejaculated. prostate examination. the prostate is the only accessory sex gland present in the dog and it contributes its secretion as part of the ejaculate. a lubricated gloved index finger is inserted into the rectum to palpate the walnut-shaped, bi-lobed prostate on the floor of the pelvis. in medium to large dogs, the examiner has to push the ventral abdomen upward to facilitate feeling the whole prostate gland. benign prostatic hypertrophy is a common condition, especially in older dogs. semen evaluation. the color of normal semen should be cloudy to milky white. a red or pinkish ejaculate may be indicative of a prostate problem. a yellow semen color indicates urine contamination or, less commonly, purulent exudate or other foreign debris originating from the prostate or epididymis. semen volume can be read from the calibrated collection tube. semen volume varies with the breed, size of the dog and the amount of prostatic fluid collected. sperm motility is evaluated within a few minutes of semen collection by placing a drop of semen on a warm (37 °c) microscope slide, placing a glass cover slip over the drop and examining under the microscope. the percentage of motile sperm, those moving in a progressive forward manner, is estimated. sperm moving in circles or wiggling in one spot can indicate chemical or cold shock. even though sperm motility is a subjective test, it is a quick functional test of semen quality. expensive automated semen analyzers and computer-based equipment are available to assess sperm motility. about 80% or greater sperm motility is expected in a normal dog ejaculate. low sperm motility may be an indication of incomplete ejaculation, infectious diseases involving the reproductive system or increased temperature of the scrotum (e.g., fever). the total number of sperm in the entire ejaculate is more important than the number of sperm per milliliter of semen because the total volume depends upon how much prostate fluid is collected. again, expensive automated semen analyzers are available for this purpose, but the most common technique used is to count sperm using a hemocytometer. sperm morphology (normal and abnormal sperm) is assessed by examination of a stained slide of semen, and 100-200 sperm are examined. the cells are classified as normal sperm, those with primary abnormalities (e.g., head abnormalities, proximally coiled tails, proximal cytoplasmic droplets), or those with secondary abnormalities (e.g., detached heads, bent tails, distal cytoplasmic droplets). table 1. characteristics of normal canine semen parameter unfractionated ejaculate seminal fraction 1 2 3 color opaque to white clear milky-white clear volume (ml) ~30 0.1-2 0.1-4 1-25+ sperm motility >80% morphologically normal sperm >80% 503 the female factor improper timing of breeding is the major cause of conception failure in bitches bred by ai. basic understanding of the reproductive cycle of the bitch is essential for proper breeding management and expectation of successful ai. estrous cycle bitches cycle twice a year, with a minimum of a four month inter-estrus interval. bitches with less than four months between successive cycles usually do not get pregnant. some breeds (e.g., basenji, wolf and wild-dog crosses) may cycle every 9-12 months with normal fertility. bitches ovulate spontaneously, which means that they ovulate without any stimulus from breeding. the bitch starts cycling due to the action of hormones released from the hypothalamus and pituitary gland. gonadotropin releasing hormone, released from the hypothalamus stimulates the release of fsh and lh from the pituitary gland. as the names imply, fsh is primarily responsible for growth of follicles on the ovaries whereas lh causes ovulation of the follicles. the estrous cycle in the bitch is unique and is different from other domestic animals. it starts with proestrus. most breeders refer proestrus as start of the ‘season’. bitch owners notice spotting or bleeding from the vulva. during proestrus, the bitch attracts males but does not allow mating. estrogen produced by ovarian follicles causes a change in the epithelial cells of the vaginal mucosa, which will be discussed in detail below. estrus or the time of receptivity follows proestrus and the bitch accepts mating. the average duration of proestrus and estrus cited in most textbooks is nine days each. this average may be correct but unfortunately many bitches do not follow the textbook and each phase may range from two to 21 days. during proestrus, the vulvar lips are turgid or firm and become soft and wrinkled as the bitch approaches the receptive phase of the cycle. the bloody discharge characteristic of proestrus usually changes to a straw color near estrus in most bitches but some bitches continue to bleed throughout estrus. a graph depicting the hormone pattern during the estrous cycle of bitches can be found at http://www.peteducation.com/article.cfm?c=2+2109&aid=3201.1 the production of p4 is also unique during estrous cycle of the bitch. the early increase in p4 during estrus is due to luteinization of follicles on the ovaries. this increase in p4 can be useful in breeding management and will be discussed in detail below. the bitch ovulates in response to lh. ovulation occurs about three days after the lh peak. after ovulation, the follicles are replaced by corpora lutea (cls) which secrete p4. the production of p4 continues throughout diestrus, the next phase of cycle. the duration of diestrus is the same as pregnancy, about 62-63 days after the lh peak. even if the bitch is not pregnant p4 is produced for the duration of diestrus. it appears that there is no production of prostaglandin f2alpha (as in other domestic animals) from the endometrium (inner lining of the uterus) to cause luteolysis (cl regression). during diestrus, many bitches display variable signs of ‘pseudopregnancy’. the bitch may experience weight gain and an enlarged abdomen and may show more overt signs of pseudopregnancy including mammary gland enlargement, nesting, adoption of toys (or shoes), and give the impression she is about to whelp. pseudopregnancy (or diestrus) is considered a normal occurrence and may not require treatment. the ova released during ovulation in the bitch are primary oocytes; the first polar body has not been shed, and sperm are not able to penetrate the ovum. three days are required for the ovum to become a secondary oocyte and ready to be penetrated by the sperm. dog sperm can survive up to ten days in a bitch’s reproductive tract. it can be a diagnostic challenge for a veterinarian when a bitch’s owner requests a cesarean section 62 days after breeding. theoretically a pregnancy at 62 days after breeding could be as early as 52 days. a few tools are available to predict whelping. hypothermia or a decrease in rectal temperature of two to three degrees f, caused by a decrease in p4 12 to 36 hours before whelping is a fairly reliable predictor of whelping. the indication of diestrus day 1 (d1) by vaginal cytology is another reliable way to predict whelping. bitches whelp about 57-58 days after d1. 504 vaginal cytology in breeding management. for successful vaginal cytology, the examiner needs to appreciate the unique reproductive anatomy of the bitch. the vagina of the bitch is very long, about 20 cm (~9 inches) in a medium size bitch. the cervix in the bitch is located in the abdominal cavity, whereas in other domestic animals (e.g., cow and mare) the cervix is located at the pelvic inlet. therefore, in most bitches the cervix cannot be palpated or visualized with a speculum but can be visualized with a flexible endoscope with a light source. this becomes important when frozen-thawed semen needs to be deposited in the uterus via the cervix. a clean cotton-tipped swab is commonly used to obtain a sample for vaginal cytology. it is most convenient for a right-handed examiner to hold the bitch’s vulva with their gloved left hand and open the vulvar lips with the thumb and the middle finger, while using the index finger behind the vulva to support it. the swab is inserted with the right hand in a nearly vertical position into the vagina, avoiding the clitoral area, and directed up and over the brim of the pelvis. the swab is moistened with warm tap water before being inserted. if the swab sticks in the vaginal folds, pull it back slightly, redirect it and proceed. once the swab has been inserted at least 6-10 cm (2-4 inches), the swab is rolled a few times in one direction (if rotated back and forth, the cotton may unroll and drop in the vagina) and removed. the author recommends inserting the swab as deep as possible, as there is less debris on samples taken from the cranial vagina. getting into the habit of inserting the swab deep into the vagina is also helpful for ai. the swab is rolled on a clean microscope slide, air-dried and stained with romanowsky’s stain (diffquick®, dade behring, inc., newark, de). the author recommends making two slides as insurance against accidental breakage of a single slide. other stains used by veterinary practitioners include methlyne blue, eosin-nigrosin, gram stain, and others. romanowsky’s stain is suitable for staining epithelial cells and white blood cells. after the slides have been air-dried, they are dipped five to seven times in each solution, rinsed with tap water, air-dried and examined under a microscope. the author recommends an initial examination with the 10x objective to get an overall impression and distribution of the cells before moving on to the 20x lens to closely view the desired field. various theriogenologists (veterinary reproduction specialists), clinical pathologists and other veterinary practitioners interpret vaginal cytology differently. for this presentation, the author will use the definitions described by olson, et al.2 it is important to remember that the vaginal mucosa is responsive primarily to estrogen, thus vaginal cytology is useful only during estrogenic phase of the cycle. parabasal cells are small and round with large and distinct nuclei. the total area of the cytoplasmic portion of the cell is smaller than the nucleus. these cells (along with red blood cells) are present during proestrus. superficial intermediate cells are larger than parabasal cells with small nuclei and irregular or folded borders. large numbers of these cells are observed during late proestrus to early estrus. superficial cells, the anucleated cells also called cornified or keratinized cells, are the largest of the epithelial cells present during estrus. under the microscope, the nuclei of these cells appear faded or absent. the cells also appear thin, multi-layered and have folded borders. the appearance of 80-90% of these cells in the smear is used as an indication to start breeding. two observations are important here. first, the examiner must analyze a series of vaginal cytology samples to observe the progressive change in epithelial cells. a single smear is unreliable. some bitches maintain the same type of epithelial cells for many days, where as others change within 24 hours. second, in the author’s experience many bitches reach 80-90% cornification during estrus but others never go above 70% cornification. obviously, if one waits for 80-90% cornification to breed, the opportunity to breed will be missed in these bitches. another tool help manage breeding of these bitches is to measure blood p4, which will be discussed below. if vaginal cytology is continued, the appearance of neutrophils (a type of white blood cell) is detected; this is considered to be the first day of the diestrus or d1. this finding is important because the bitch is expected to whelp 57-58 days after d1. it is recommended that vaginal swabs be taken after breeding every day, because in some bitches the change from cornified to superficial intermediate cells and the appearance of neutrophils may occur within 24 hours. many breeders find it convenient to take vaginal 505 cytology samples at home. after a brief training, they prepare slides at home for the recommended time. the slides are then brought to the clinic, stained and evaluated. as discussed above, in addition to the cls, p4 also is secreted before ovulation by luteinized follicles on the ovaries. the early p4 rise can be used to determine ovulation in the bitch. recall that ovulation takes place about three days after the lh peak, which is very short. kits to measure lh are available, and it is recommended that lh be measured every day during the expected time of ovulation. progesterone assays are commonly used for canine breeding management. enzyme-linked immunoabsorbent assays are available to measure p4. these are qualitative tests based upon a color change and indicate a range of p4 concentrations. many clinicians prefer to send the blood samples to an endocrinology laboratory for p4 determination by radioimmunoassay. progesterone is the same hormone in all mammals, including humans; therefore blood samples can be analyzed for p4 in veterinary or human hospital laboratories. progesterone assays are a useful tool to determine the time of ovulation so breeding can take place at the appropriate time. this becomes very critical when using chilled transported or frozen-thawed semen for ai. like vaginal cytology, p4 concentrations are monitored starting a few days after the onset of proestrus. progesterone concentrations in the range of 2 to 2.9 ng/ml are indicative of ovulation in about two days; concentrations from 3 to 3.9 ng/ml indicate ovulation in one day, and those from 4 to 10 ng/ml are found on the day of ovulation.3 other causes of failure to conceive split heat. split heats are seen in bitches that have proestral bleeding, vulvar swelling and attract males, but do not progress to ovulation. in other words, the bitch has ovarian follicle growth, production of estrogen but ovulation does not take place. affected bitches go out of proestrus, but then come back into proestrus again two to ten weeks later. the second half of the split heat is usually normal. the causes of split heat are not known and the condition is not associated with infertility. however, bitches with a split heat cause problems in breeding management. ovarian cysts. ovarian cysts are characterized by prolonged proestrus or estrus and are seen mostly in younger bitches. the ovarian follicles develop normally but do not ovulate. as in normal follicles, estrogen is produced from the granulosa cells of the follicle lining. however, the granulosa cells do not go through the normal change to produce progesterone. cystic follicles may be induced by estrogen injections. bitches with ovarian cysts that produce estrogen have prolonged proestrus or estrus and may attract males. less common are luteal cysts which secrete progesterone. persistent and prolonged progesterone production may cause infertility or predispose the bitch to cystic endometrial hyperplasia and pyometra. follicular cysts may be diagnosed by series of vaginal cytology examinations as discussed above. animals with follicular cysts produce estrogen and vaginal cytology would show cornification of the epithelial cells. ultrasonography may detect fluid-filled cystic structures in the ovaries. ovariohysterectomy is the treatment of choice for bitches not intended for breeding. valuable breeding bitches with follicular cysts are treated with gnrh, which stimulates the release of lh from the pituitary gland to cause luteinization or ovulation of the cystic follicles. another option for treatment is human chorionic gonadotropin, which mimics the action of lh and causes luteinization or ovulation of the cystic follicles. even though there is no documented evidence that ovarian cysts are hereditary in the bitch, ovariohysterectomy is recommended in those with reoccurrence of ovarian cysts. ovarian cysts have been shown to be hereditary in dairy cattle. hypothyroidism. hypothyroidism is a common endocrine disorder and is considered to be a hereditary condition in the dog. even though dogs with hypothyroidism often have poor reproductive performance, an exact link between hypothyroidism and reproductive function has not been established. reproductive disorders may include irregular cycles, small litter size and poor semen quality in males. 506 dogs with hypothyroidism have a lower metabolic rate than normal dogs. owners may notice less tolerance for exercise, weight gain, dry hair coat and eventually hair loss. it is recommended that both t4 and tsh be measured in dogs suspected of hypothyroidism. the condition is easily treated by oral supplementation with thyroid hormone. dogs without a confirmed diagnosis of hypothyroidism should not be given thyroid supplement. dogs receiving the supplement should be tested periodically to confirm appropriate supplementation. excessive thyroid hormone supplementation may result in hyperthyroidism and can cause an increase in metabolic and heart rates and eventually weight loss. in humans, hyperthyroidism has been linked with reproductive dysfunction. early embryonic loss. early embryonic loss and resorption of embryos can be a diagnostic challenge. some owners assume a bitch was pregnant based upon outward signs of weight gain, but do not whelp. pregnancy should be confirmed before suspecting a pregnancy loss. pregnancy in the bitch can be reliably diagnosed by ultrasonography 25 days after the last breeding. if early embryonic death occurs, clinical signs are usually not observed. there are no abdominal contractions and embryos are not seen to pass. embryonic death and resorption is caused by infectious and noninfectious agents. infectious agents suspected causing embryonic death include bacteria (e.g., mycoplasma, brucella canis, campylobacter, e. coli, and b-hemolytic streptococci), parasites (e.g., toxoplasma gondii and neospera caninum) and viruses (e.g., canine herpesvirus, canine parvovirus type 1, and canine distemper virus). non-infectious causes of resorption may include endocrine abnormalities, certain drugs given to the bitch during pregnancy and genetic factors. hypoluteoidism. hypoluteoidism is an endocrine abnormality and can cause embryonic death and resorption. for pregnancy maintenance, p4 must be secreted from the cls throughout the pregnancy. if luteal tissue fails to secrete progesterone at any stage, the pregnancy is lost. monitoring p4 in a bitch with suspected hypoluteoidism once or twice weekly allows accurate diagnosis of this problem. greater than 2 ng/ml p4 is required to maintain pregnancy. in a normal pregnancy, p4 reaches peak levels (15-90 ng/ml) by 15-30 days after the lh peak. during the last semester, p4 decreases (4-16 ng/ml) before dropping below 2 ng/ml about one day before parturition. hypoluteoidism is treated by supplementing the bitch with p4. conclusion there are many reasons for failure to conceive after breeding. infertile or sterile males used for natural service and improper breeding management for artificial insemination are the most common. other causes may include split heat, ovarian cysts, and early embryonic death. appropriate examinations and diagnostic tests performed by a veterinarian specializing in reproduction may provide answers. references 1. hormone levels: determining breeding times and whelping dates. available from: http://www.peteducation.com/article.cfm?c=2+2109&aid=3201. 2. olson pn, thrall ma, wykes pm, et al: vaginal cytology. part 1. a useful tool for staging the canine estrous cycle. compend contin educ pract vet 1984;6:288-298. 3. root kustritz mv: breeding management. in: the dog breeder’s guide to successful breeding and health management. st. louis: saunders elsevier; 2006. p. 126-146. 507 508 omniblank: 1 contact edgar garrett efg@illinois.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12181, http://dx.doi.org/10.58292/ct.v17.12181 case report sperm granuloma in a buck seiya kanzawa, jonathan samuelson, edgar garrett department of veterinary clinical medicine, university of illinois, urbana, il, usa abstract a 7-month, saanen buck was presented for evaluation after failing to produce sperm during 2 attempts. physical examination revealed a firm and smooth mass, dorsal to each testis, with no other abnormalities. semen sample obtained with an electroejaculator had very small number of nonmotile sperm. ultrasonographic examination revealed a predominantly hypoechoic mass on the testis’ dorsal aspect and a normal echogenic pattern of the testicular parenchyma. future breeding potential of the buck was deemed to be poor; therefore, the buck was castrated. epididymal heads were occupied by sperm granuloma and the remainder of the testes was normal. keywords: oligospermia, granuloma, epididymis, goat background physical examination, including examination of external reproductive organs, is integral in breeding soundness examination. gross testicular abnormalities detected during a physical examination can enable clinicians to decide on proper diagnostic procedures. the work up for the present case was guided by physical examination findings (firm and smooth swelling immediately dorsal to both testes). a range of differential diagnoses were considered for a young buck that was not producing sperm; however, we were able to quickly move towards a diagnosis of an extragonadal obstruction. buck was castrated; final diagnosis was sperm granuloma (based on gross testicular lesions and histopathology). case presentation a 7-month, saanen buck was presented that was diagnosed azoospermic 1 month prior to presentation; buck was born on the farm and had no history of health problems. on general physical examination, buck had normal attitude, gait, and rectal temperature; weighed 69 kg and was in moderate body condition (bcs = 3/5). scrotal circumference (26.5 cm) was normal. palpation of the scrotum revealed a firm, ~ 3 x 1 cm, smooth swelling immediately dorsal to both testes. digital rectal examination was unremarkable. semen was collected with an electroejaculator (lane pulsator iv, lane manufacturing, denver, usa); ~ 2 ml of clear fluid was obtained. examination of a wet mount at 400 x with phase contrast (olympus bx50) revealed a very small number of nonmotile sperm (< 50 total cells per 20 µl). given the paucity of sperm in the sample, further evaluation of the morphology of the sperm present was not pursued. testes ultrasonography (sonoscape s8 exp, 3.2 mhz probe) revealed a parenchyma that was normal, homogeneous, moderate echogenic in appearance; epididymal heads were dilated and predominantly anechoic (figure 1). epididymal tails were small and predominantly anechoic. brucellosis card agglutination test was negative. differential diagnoses were epididymitis, sperm granuloma, ampullary blockage, orchitis, testicular congenital abnormality, testicular degeneration, and sexual immaturity. treatment and outcome based on palpable and ultrasonographic abnormalities and the paucity of sperm in the ejaculate, a presumptive diagnosis of an extragonadal duct obstruction was made. buck was surgically castrated because it was deemed unsuitable for breeding. testes were of similar size and there was a prominent, raised, white, moderately firm ~ 5 x 2 cm swelling involving epididymal heads (figure 2). on cut section, epididymal head was expanded by yellow, caseous material contained by tough fibrous tissue (figure 3). testes and epididymides were submitted for histologic examination; representative histology images are presented (figure 4). yellow caseous material observed grossly was innumerable sperm mixed with and bordered by epithelioid macrophages and multinucleate giant cells. the mixed population (sperm granuloma) was mailto:efg@illinois.edu http://dx.doi.org/10.58292/ct.v16.10416 2 citation: clinical theriogenology 2025, 17, 12181, http://dx.doi.org/10.58292/ct.v17.12181 figure 1. ultrasonogram of left epididymal head; note anechoic dilation figure 2. gross appearance of granulomatous left epididymal head; note marked and asymmetrical expansion http://dx.doi.org/10.58292/ct.v15.9221 citation: clinical theriogenology 2025, 17, 12181, http://dx.doi.org/10.58292/ct.v17.12181 3 encapsulated by a segmentally mineralized band of fibrosis that separated the granuloma from the adjacent atrophic epididymal tissue. both testes had mild to moderate germinal atrophy but were otherwise unremarkable. epididymal heads’ content was negative for brucella spp. and mycoplasma spp. but yielded a light growth of staphylococcus auricularis. discussion sperm granulomas occur at a low frequency in a wide variety of breeds and ages of sheep and goats.1,2 in observational studies, sperm granuloma was diagnosed in 0.3% of 1,0001 and 1.5% of 4042 bucks; this condition can be unilateral or bilateral. it can be a heritable trait and is associated with the polled condition in goats but also occurs in horned animals.3 efferent ducts join to form epididymal head and obstruction of the ducts would lead to accumulation of sperm in the area and eventual rupture followed by the formation of the sperm granuloma due to the inflammatory reaction in response to sperm presence in the epididymal stroma.3 an extragonadal duct obstruction such as sperm granuloma should be considered a differential diagnosis when oligospermia or azoospermia is encountered during a breeding figure 3. cut section left epididymal head; note abundant, yellow, caseous material figure 4. light microscopic image of left epididymal head; sperm granuloma (black star) and remaining epididymal tissue (white star), bordered by a thick band of fibrosis (red asterisk) that contains some mineralization (hematoxylin and eosin stain; 40 x). inset: interface is segmentally lined by epithelioid macrophages and giant cells; evidence of active phagocytosis of sperm (hematoxylin and eosin stain; 200 x) http://dx.doi.org/10.58292/ct.v15.9221 4 citation: clinical theriogenology 2025, 17, 12181, http://dx.doi.org/10.58292/ct.v17.12181 soundness examination. palpation of the scrotum and testes is a critical part of the routine breeding soundness examination. abnormal size, variation in size between testes, change in contour of the testis, or mobility of the testis within the scrotum are reasons for more detailed examination. ultrasonography of the scrotum and testes is a very useful and accessible diagnostic modality when abnormalities in the scrotum or testes have been identified on physical examination. the parenchyma, epididymis, and vaginal space should be evaluated. in this case, the ultrasonographic appearance of testicular parenchyma was normal and epididymal heads had the characteristic appearance of a sperm granuloma.4 there can be changes in testicular parenchyma or the mediastinum of the testis secondary to the occlusion of the outflow obstruction caused by the sperm granuloma.4 the lack of changes in the parenchyma and mediastinum was likely due to the young age and therefore relatively short duration of the condition. bilateral sperm granuloma affecting epididymal heads at a young age suggested a development abnormality, but this could not be proven. trauma could not be ruled out; however, lack of evidence of trauma, lack of history of trauma and the bilateral, symmetrical nature of the changes made trauma unlikely. epididymal head obstruction is associated with polled phenotype in goats3 but this was ruled out as a contributing factor because the buck was horned. it is possible that an infectious process contributed to the development of the sperm granuloma. bacterial epididymitis has been reported in bucks but is more common in rams.2,3 although only staphylococcus auricularis grew in the culture, it was possible that other bacteria failed to grow. actinobacillus seminis has been reported as a cause of unilateral epididymitis in a buck in a herd of comingled sheep and goats.5 brucella spp. (b. abortus, b. ovis and b. melitensis) and mycoplasma were ruled out with pcr. bilateral, symmetrical nature of the granulomas, negative tests for brucella spp., and mycoplasma spp., low level of growth of bacteria from the granuloma and lack of evidence of bacterial involvement in the granuloma on histology made an infectious cause unlikely. there is no treatment for sperm granuloma so the only option for a production animal is culling. in this case, the buck was castrated prior to culling to increase the market value of the animal. learning points • a careful examination of the scrotum, testes, epididymides, and spermatic cords is an important part of the reproductive examination of the male • abnormalities of the epididymis can reasonably be detected on physical examination • extragonadal obstruction is a reasonable differential diagnosis in cases of oligospermia or azoospermia • sperm granuloma is an uncommon finding in bucks and more likely to be encountered in rams conflict of interest none to report. references 1. tarigan s, ladds pw, foster ra: genital pathology of feral male goats. aust vet j 1990;67:286-290. doi: 10.1111/j.1751-0813.1990. tb07798.x 2. regassa f, terefe f, bekana m: abnormalities of the testes and epididymis in bucks and rams slaughtered at debre zeit abattoir, ethiopia. trop anim health prod 2003;35:541-549. doi: 10.1023/ a:1027348900286 3. mickelsen wd, memon ma: infertility and diseases of the reproductive organs of bucks. in: youngquist rs, threlfall wr: editors. current therapy in large animal theriogenology. 2nd edition, st. louis; saunders: 2007. p. 519-523. doi: 10.1016/b978-0-72169323-1.x5001-6 4. karaca f, aksoy m, kaya a, et al: spermatic granuloma in the ram: diagnosis by ultrasonography and semen characteristics. vet radiol ultrasound 1999;40:402-406. doi: 10.1111/j.1740-8261. 1999.tb02132.x 5. santos f, azevedo e, azevedo s, et al: isolation of actinobacillus seminis from a goat with clinical epididymo-orchitis in brazil. braz j microbiol 2014;45:205-209. doi: 10.1590/s1517-838220140 05000024 http://dx.doi.org/10.58292/ct.v15.9221 https://doi.org/10.1111/j.1751-0813.1990.tb07798.x https://doi.org/10.1111/j.1751-0813.1990.tb07798.x https://doi.org/10.1023/a:1027348900286 https://doi.org/10.1023/a:1027348900286 https://doi.org/10.1016/b978-0-7216-9323-1.x5001-6 https://doi.org/10.1016/b978-0-7216-9323-1.x5001-6 https://doi.org/10.1111/j.1740-8261.1999.tb02132.x https://doi.org/10.1111/j.1740-8261.1999.tb02132.x https://doi.org/10.1590/s1517-83822014005000024 https://doi.org/10.1590/s1517-83822014005000024 2010: porcine abortions: overview of prrsv, pcv2, ppv and other causes porcine abortions: overview of prrsv, pcv2, ppv and other causes t. opriessnig, m. j. yaeger college of veterinary medicine, iowa state university, ames, ia abstract reproductive failure in pregnant sows continues to present challenges for practitioners and diagnosticians. the diagnostic success rate on abortion cases is lower in comparison to many other disease syndromes and is estimated to be around 30-40% for pigs. increased numbers of abortions in breeding facilities can often be linked to viral pathogens with the economically most important virus to date being porcine reproductive and respiratory syndrome virus (prrsv). besides prrsv, other viruses such as porcine circovirus type 2 (pcv2), porcine parvovirus (ppv), swine influenza virus (siv) and others continue to contribute to abortion outbreaks. pseudorabies virus (prv) and hog cholera virus (classical swine fever) may also play a significant role in reproductive disease in countries where these diseases have not yet been eradicated. identification of a cause depends largely on correct sample collection strategies and use of appropriate diagnostic tests and accurate interpretation of test results. keywords: abortion, porcine, viruses introduction reproductive failure in a pregnant sow may be associated with acute systemic illness of the dam, abortion, increased numbers of non-viable fetuses at parturition or increased numbers of weakborn piglets. abortion is defined as termination of pregnancy after or accompanied by fetal death. reproductive failure and abortions in pigs are often of great economic importance and can be caused by infectious pathogens, hormonal processes, nutritional or genetic events, toxicoses, traumatic reasons or other causes. in modern swine production, different production stages (breeding-gestation, farrowing, nursery, grow-finishing) are typically separated on distant sites. breeding herds are usually comprised of a large population of mature females that are synchronized so that at any time point there are mature females in estrus, gestation or in the process of farrowing. there are also a small number of boars in these facilities that are used to make heat detection and artificial insemination easier. although modern production facilities often have superior biosecurity barriers and protocols, the dense population of animals in these facilities may facilitate rapid dissemination of reproductive pathogens that are introduced potentially causing great loses in naïve populations. investigations should be conducted when any of the following conditions are met: the abortion rate exceeds 3% or when there are clusters of abortions over a short time period or if they are concentrated in one area of the facility. early detection of reproductive pathogens in breeding herds is therefore crucial to prevent economic losses. diagnostic challenges especially with viral infection are related to the fact that (1) not all fetuses in a litter are typically infected and (2) sows can abort due to maternal illness and thus, the virus may not be detected in fetal material. therefore, collecting the appropriate number and types of samples is important to an accurate diagnosis in a timely fashion. we have attempted to provide a more detailed description of the most important viruses associated with porcine reproductive failure below. porcine reproductive and respiratory syndrome virus (prrsv) etiology. prrsv is an enveloped, positive-sense, single-stranded rna virus in the family arteriviridae.1,2 prrsv can infect pigs of any age resulting in reproductive failure in breeding animals, respiratory disease in growing pigs, and diarrhea in neonates. clinical disease can occur in previously seropositive or vaccinated herds.3 clinical signs in the breeding herd. clinical signs are variable and in general sows will exhibit mild clinical illness when initially exposed to the virus. clinical signs include slight depression, anorexia, and a mild fever for one to two days.4 sows will occasionally abort during this initial febrile phase of the illness.1 more typically, animals abort two to four weeks following the initial illness. prrsv classically 549 causes late gestation reproductive disease manifest as abortions, stillbirths, premature farrowing, and the birth of weakborn piglets.1,2 exposure of naïve sows to a virulent strain early in gestation (less than 45 days) often fails to result in fetal infection while exposure late in gestation (more than 70 days) leads to abortion.5,6 gross and microscopic lesions. a typical prrsv litter will have one to two, dark, autolyzed fetuses with abundant fluid in the body cavities and several fetuses with meconium staining of fetal skin.1,2,7 the remainder of the litter may appear essentially normal. additional gross changes reported with prrsv abortion include umbilical cord edema with segmental umbilical cord hemorrhage, perirenal edema, mesocolonic edema and increased amounts of clear to serosanguinous fluid in the body cavities.7 microscopic lesions appear to be less common than gross changes. umbilical cords that are grossly edematous with segmental hemorrhage will typically have necrotizing arteritis with periarterial hemorrhage.7 arteritis is occasionally observed in a variety of tissues. additional microscopic lesions reported in fetuses include nonsuppurative encephalitis, myocarditis and interstitial pneumonia.2,8,9 diagnosis. on an individual animal basis, examination of fetuses alone may be insufficient to establish a diagnosis of prrsv abortion. the percentage of infected fetuses is reported to vary from 18.8% to 73.8%, with an overall rate of approximately 50% reported in two large studies.7,9,10 assuming a 50% fetal infection rate, material from four to six fetuses should be pooled to achieve 90-95% confidence that at least one infected fetus is represented in a submission.10 if sows are exhibiting a mild febrile illness at the time of abortion, they may be aborting due to maternal illness. in these cases, fetal infection will generally not be demonstrated. sow serum should be submitted for prrsv pcr as these animals are typically viremic. the diagnostic test of choice for demonstration of prrsv-associated abortion is pcr which is extremely sensitive and is impacted little by autolysis. since all fetuses in a litter are typically not infected, it is critical to test multiple fetuses. with pcr, material from one positive fetus can be pooled with several negative fetuses without apparent loss of sensitivity.4 typically, pooled fetal thoracic fluid or lung tissues are used. it needs to be considered that marked strain variation can result in substantial genetic and antigenic differences between isolates11-15 and therefore false negative results if the pcr does not detect all known prrsv strains. other diagnostic tools with less sensitivity include immunohistochemistry (ihc) stains on formalin fixed tissue or indirect immunofluorescence assay (ifa) on frozen tissue to demonstrate prrsv antigen in fetal tissues; however, the sensitivity of this test is not great. if abortion due to maternal illness is suspected, seroconversion (by prrsv elisa) can be demonstrated on paired serum samples taken at the time of abortion and two weeks later. however, in the vast majority of prrsv abortions, the sow will have seroconverted at the time of abortion and a single sample, collected at the time of abortion, will demonstrate high prrsv titers. results can be difficult to interpret in vaccinated or known seropositive herds. if a sow is seronegative at the time of abortion and remains seronegative, prrsv can be ruled out with relative certainty. prevention and treatment. in order to prevent prrsv-associated abortions, the establishment of naïve herds and implementation of biosecurity barriers and protocols to keep those herds negative is the best solution. should this not be possible or economically feasible, practitioners may chose to do planned exposure of the population to prrsv on a routine basis in order to develop immunity. commercially available live prrsv vaccines and autogenous farm-specific prrsv isolates are commonly used for this purpose. porcine circovirus type 2 (pcv2) etiology. porcine circovirus (pcv) is a small circular single-stranded, non-enveloped dna virus of approximately 1.7 kb. there are two recognized types of pcv: pcv type 1 (pcv1) and pcv type 2 (pcv2). pcv1 was first discovered as a contaminate of a porcine kidney cell line (pk-15) in 1974, but has since been determined to be non-pathogenic in pigs.16,17 in contrast, pcv2 is pathogenic and has been linked to many disease entities in growing swine as well as to reproductive failure in mature animals. two main genotypes of pcv2 are currently recognized: pcv2a and pcv2b. globally, most swine herds have evidence of exposure to pcv2 based on the presence of antibodies. 550 clinical signs in the breeding herd. clinical signs of pcv2 infection in the dam are generally absent. dams may abort following infection due to systemic illness and abortions are often seen in a low percentage of females with the exception of rare cases of pcv2-infection of naïve populations. pyrexia and anorexia are frequently observed in aborting dams.18,19 delayed farrowing (more than118 days of gestation)20 or pseudopregnancy21 may be additional clinical features in affected dams. gross and microscopic lesions. gross lesions in dams are usually absent. individual fetuses may have lesions of heart failure characterized by dilated and hypertrophied myocardium, plural effusion, ascites, and enlarged, congested livers.22-24 gross heart lesions are infrequently present and are typically seen in stillborns or late gestational mummified fetuses. late gestational fetuses may exhibit generalized anasarca. perirenal and mesocolonic edema may be evident in some stillborn pigs. pcv2-associated microscopic lesions commonly seen in affected growing pigs (lymphoid depletion; granulomatous inflammation of organ systems) have not typically been reported in dams with pcv2-associated reproductive failure. fetal microscopic lesions can be absent or vary depending on stage of development in which the fetuses was infected. the most consistent microscopic lesions associated with pcv2infection include myocardial degeneration and necrosis, nonsuppurative myocarditis, fibrosis and mineralization with occasional amphophilic to basophilic intranuclear inclusion bodies.22,24 liver congestion with heptocellular atrophy may also be present along with nonsuppurative pneumonia.19,24 follicular hyperplasia and lymphocyte depletion with macrophagic replacement and rare multinucleated giant cells has been reported in lymphoid tissues.25,26 diagnosis. sow serology is difficult to interpret because pcv2 is ubiquitous and most dams have been previously exposed to pcv2 and will have a positive anti-pcv2-antibody titer. pcv2 viremia in the sow at parturition is also not diagnostic for pcv2-associated reproductive failure due the ubiquitous nature of pcv2. ihc27,28 and in situ hybridization (ish)29 are considered to be the gold standard diagnostic assay for confirmation of pcv2-associated abortion by detection of pcv2 antigen or dna in formalin fixed, paraffin embedded tissues from affected fetuses. myocardium readily stains if pcv2 is present and the antigen can be seen in the cytoplasm as well as the nucleus of infected cells. pcv2 antigen or dna can also be present in thymus, spleen, tonsil, and lung. demonstrating the presence of pcv2 in presuckle live-born piglet sera, aborted, mummified or stillborn fetuses can also be accomplished by other techniques including pcr,30,31 virus isolation,27,32 elisa,33 ifa,34 and immunoperoxidase monolayer assay (ipma).35 prevention and treatment. pcv2-associated abortion is usually an individual sow problem and treatment of the herd is usually not necessary. incoming gilts can be exposed to pcv2 prior to breeding to guarantee exposure prior to breeding. this can be done by using commercially available killed vaccines or by comingling animals from different sources. porcine parvovirus (ppv) etiology. porcine parvovirus is a non-enveloped, single-stranded dna virus.36 all ppv isolates appear to be antigenically similar, if not identical.36,37 ppv is endemic in most swine herds.38,39 the virus is shed in secretions for several weeks and then survives for four months in the environment.40 contaminated premises are likely the major reservoir of the virus. clinical signs in the breeding herd. ppv infection of naïve dams is characterized by increased numbers of mummified fetuses, increased numbers of sows that return to estrus, small litters, and decreased farrowing rates. abortions are not typically observed following in utero ppv infection. gross and microscopic lesions. in natural infections the primary gross lesion is fetal mummification. fetuses are dark in color, markedly dehydrated and the skin is dry and leathery. mummified fetuses are typically 3 to 16 cm in crown to rump length. due to in utero spread of the virus, fetuses are often mummified at different stages of gestation resulting in mummies of varying sizes. fetal infection during the first 35 days of gestation causes death and reabsorption of embryos, resulting in irregular return to estrus or reduced litter size. infection between 35 and 70 days of gestation results in fetal death and mummification. fetuses infected after 70 days of gestation develop an antibody response and may successfully resist infection and are born normal. microscopic lesions in fetuses are rarely 551 observed but most commonly consist of perivascular accumulations of mononuclear inflammatory cells in both grey and white matter of the cerebrum and meninges, necrotizing and nonsuppurative hepatitis, interstitial nephritis and placentitis with calcification.41,42 diagnosis. ppv antigen can be easily demonstrated in heart or lung tissues of affected fetuses either by ifa or ihc. pcr can also be used to demonstrate ppv dna in these tissues. hemagglutination inhibition (hi) is done to confirm if antibodies against ppv in pre-suckle blood are present which is indicative of intrauterine exposure to the virus. unlike several reproductive diseases of swine, serology is typically of little value. interpretation of antibody titers is difficult because a majority of farms vaccinate against ppv, the virus is ubiquitous and natural exposure is common prior to introduction into the breeding herd, there is evidence for an anamnestic response in sows that do not develop reproductive disease, and high hi titers can occur in a herd without any ppv-related symptoms.38 serology on stillborn pigs or neonates can provide useful information. the presence of ppv antibodies in the presuckle serum or thoracic fluid of stillborn pigs indicates in utero exposure to the virus. prevention and treatment. in order to prevent ppv-associated abortions, many producers vaccinate gilts on a routine basis using commercially available killed products. feedback of mummified fetuses to gilts in isolation and acclimatization has also shown to be beneficial in some situations. pseudorabies virus (prv) etiology. pseudorabies virus (prv) (suid herpesvirus-1) is an alphaherpesvirus.43 prv has been eradicated from commercial swine operations in the us and throughout many intensive swine rearing regions worldwide; however, it is still present in the feral swine population and transmission to the commercial populations could potentially occur. clinical signs in the breeding herd. sows may develop fever, become anorectic, and exhibit mild respiratory signs which may include sneezing and coughing.44,45 a generalized pruritis is occasionally observed.45 prv-associated maternal illness may result in abortion or fetal mummification (depending on the stage of gestation) without evidence of fetal infection due to the non-specific effects of maternal pyrexia.45 similar to what is seen with ppv, fetal prv infection during the later stages gestation leads to fetal mummification, abortion, stillborn, and weakborn pigs.43 pigs are usually weak, develop neurologic signs, and die within one to two days. suckling piglets will develop a high fever, neurologic signs (trembling, incoordination, ataxia and seizures) and shortly after onset of clinical signs, mortality in suckling pigs born to naïve dams may reach 100%.43 gross and microscopic lesions. fetal prv infection results in the development of 1-2 mm foci of necrosis.44,46 lesions are most readily visualized in liver and spleen, with occasional lesions observed in the lungs. it needs to be noted that fetal bacterial septicemia may occasionally cause similar gross changes. microscopically, prv infection causes multifocal random necrotizing lesions in many fetal organs including; liver, adrenal, spleen, lung, lymph node and placenta.44,46 eosinophilic to amphophilic intranuclear inclusions are occasionally observed in cells adjacent to foci of necrosis. brain lesions typical of a viral encephalitis may be identified in weakborn pigs and include nonsuppurative perivascular cuffs, multiple foci of gliosis and neuronal necrosis. diagnosis. the most common methods for detecting prv in aborted fetuses include fa, vi and pcr. because of the regulatory implications, multiple tests should be undertaken to establish a definitive diagnosis. fetal tissues of choice include lung, liver and spleen.44 as commercial operations are considered to be free of prv and are presently naïve (free of dna and antibody), serology on sow or pig serum and fetal thoracic fluid can also be used to confirm prv–associated abortions. prevention and treatment. highly effective vaccines are available for prv. additional viral causes of porcine abortion other potential viral causes of abortion are many and include viruses indigenous to the united states (siv, enteroviruses, teschoviruses and others) and a variety of foreign animal diseases (classical swine fever, african swine fever and other). some of these pathogens (i.e. siv) only induce disease in 552 the dam and rarely if ever cross the placental barrier. for these pathogens, diagnostics need to focus on demonstration of the pathogen in the dam. conclusion: suggested sampling protocol and testing if a viral agent is suspected sickness of the dam is present. any sampling strategies and diagnostic tests must focus on the dam. if the animal is alive, nasal swabs can be tested by pcr for presence of siv rna and serum of the dam can be tested by pcr for the presence of prrsv rna. if the dam is dead, lung tissues can be tested for presence of siv or prrsv rna. in addition, bacteriology on major organ systems should be conducted to rule out bacterial septicemia (salmonella sp., actinobacillus suis and others). serum samples from affected and non-affected animals can be tested for seroconversion to prrsv or siv. the dam appears normal. sampling and diagnostics should focus on the fetuses and piglets. more than one pig per litter needs to be examined. hearts (including hearts from mummies) should be collected in formalin to test for presence of pcv2 antigen by ihc or ish. fresh hearts can also be tested by fa or pcr for ppv. pooled fetal thoracic fluid or pooled fetal lungs (five fetuses and more) should be tested for presence of prrsv rna. if presuckle serum from weakborn pigs is available it can be used to test for presence of antibodies against ppv, prrsv or pcv2 to rule out or confirm intrauterine exposure. alternatively, serology can also be conducted on fetal thoracic fluid. fetal kidneys can be tested by pcr or fa for presence of leptospira sp. pooled fetal stomach contents, fetal livers and fetal lungs should be cultured for bacteria. references 1. zimmerman j., benfield da, murtaugh mp, et al: porcine reproductive and respiratory syndrome virus (porcine arterivirus). in: straw b, zimmerman j, d'allaire s, et al., eds. diseases of swine. 9th ed. ames (ia): blackwell; 2006. p. 387-417. 2. rossow kd: porcine reproductive and respiratory syndrome. vet pathol 1998;35:1-20. 3. meng xj: heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development. vet microbiol 2000;74:309-329. 4. benson je, yaeger mj, lager km: effect of porcine reproductive and respiratory syndrome virus (prrsv) exposure dose on fetal infection in vaccinated and nonvaccinated swine. swine health prod 2000;8:155-160. 5. kranker s, nielsen j, bille-hansen v, et al: experimental inoculation of swine at various stages of gestation with a danish isolate of porcine reproductive and respiratory syndrome virus (prrsv). vet microbiol 1998;61:21-31. 6. mengeling wl, lager km, vorwald ac: clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (prrs) virus isolated from field cases of "atypical" prrs. am j vet res 1998;59:1540-1544. 7. lager km, halbur pg: gross and microscopic lesions in porcine fetuses infected with porcine reproductive and respiratory syndrome virus. j vet diagn invest 1996;8:275-282. 8. rossow kd, laube kl, goyal sm, et al: fetal microscopic lesions in porcine reproductive and respiratory syndrome virus-induced abortion. vet pathol 1996;33:95-99. 9. cheon ds, chae c: comparison of the pathogenicity of two strains (wild type and vaccine-like) of porcine reproductive and respiratory syndrome virus (prrsv) in experimentally infected sows. j comp pathol 2004;130:105-111. 10. benson je, yaeger mj, christopher-hennings j, et al: a comparison of virus isolation, immunohistochemistry, fetal serology, and reverse-transcription polymerase chain reaction assay for the identification of porcine reproductive and respiratory syndrome virus transplacental infection in the fetus. j vet diagn invest 2002;14:8-14. 11. kapur v, elam mr, pawlovich tm, et al. genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern united states. j gen virol 1996;77(pt 6):1271-1276. 12. andreyev vg, wesley rd, mengeling wl, et al: genetic variation and phylogenetic relationships of 22 porcine reproductive and respiratory syndrome virus (prrsv) field strains based on sequence analysis of open reading frame 5. arch virol 1997;142:993-1001. 13. thanawongnuwech r, amonsin a, tatsanakit a, et al: genetics and geographical variation of porcine reproductive and respiratory syndrome virus (prrsv) in thailand. vet microbiol 2004;101: 9-21. 14. dea s, gagnon ca, mardassi h, et al: antigenic variability among north american and european strains of porcine reproductive and respiratory syndrome virus as defined by monoclonal antibodies to the matrix protein. j clin microbiol 1996;34:1488-1493. 15. nelson ea, christopher-hennings j, drew t, et al: differentiation of u.s. and european isolates of porcine reproductive and respiratory syndrome virus by monoclonal antibodies. j clin microbiol 1993;31:3184-3189. 16. tischer i, rasch r, tochtermann g: characterization of papovavirus-and picornavirus-like particles in permanent pig kidney cell lines. zentralbl bakteriol [orig a] 1974;226:153-167. 553 17. tischer i, mields w, wolff d, et al: studies on epidemiology and pathogenicity of porcine circovirus. arch virol 1986;91:271-276. 18. cariolet r, blanchard p, le dimna m, et al: experimental infection of pregnant spf sows with pcv2 through tracheal and muscular routes. proc eur soc vet virol 2001; p. 128. 19. park j-s, kim j, ha y, et al: birth abnormalities in pregnant sows infected intranasally with porcine circovirus 2. j comp pathol 2005;132:139-144. 20. ladekjær-mikkelsen as, nielsen j, storgaard t, et al: transplacental infection with pcv-2 associated with reproductive failure in a gilt. vet rec 2001;148:759-760. 21. josephson g, charbonneau g: case report of reproductive problem in a new startup operation. swine health prod 2001;9:258-259. 22. o'connor b, gauvreau h, west k, et al: multiple porcine circovirus 2-associated abortions and reproductive failure in a multisite swine production unit. can vet j 2001;42:551-553. 23. sanchez re, jr., nauwynck hj, mcneilly f, et al: porcine circovirus 2 infection in swine foetuses inoculated at different stages of gestation. vet microbiol 2001;83:169-176. 24. west kh, bystrom jm, wojnarowicz c, et al: myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus 2. j vet diagn invest 1999;11:530-532. 25. brunborg im, jonassen cm, moldal t, et al: association of myocarditis with high viral load of porcine circovirus type 2 in several tissues in cases of fetal death and high mortality in piglets. a case study. j vet diagn invest 2007;19:368-375. 26. mikami o, nakajima h, kawashima k, et al: nonsuppurative myocarditis caused by porcine circovirus type 2 in a weakborn piglet. j vet med sci 2005;67:735-738. 27. ellis j, hassard l, clark e, et al: isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. can vet j 1998;39:44-51. 28. sorden sd, harms pa, nawagitgul p, et al: development of a polyclonal-antibody-based immunohistochemical method for the detection of type 2 porcine circovirus in formalin-fixed, paraffin-embedded tissue. j vet diagn invest 1999;11:528-530. 29. kim j, chae c: optimal enhancement of in situ hybridization for the detection of porcine circovirus 2 in formalin-fixed, paraffin-wax-embedded tissues using a combined pretreatment of thermocycler and proteinase k. res vet sci 2003;74:235-240. 30. ellis j, krakowka s, lairmore m, et al: reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. j vet diagn invest 1999;11:3-14. 31. opriessnig t, yu s, gallup jm, et al: effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus. vet pathol 2003;40:521-529. 32. kim j, chae c: a comparison of virus isolation, polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine circovirus 2 and porcine parvovirus in experimentally and naturally coinfected pigs. j vet diagn invest 2004;16:45-50. 33. nawagitgul p, harms pa, morozov i, et al: modified indirect porcine circovirus (pcv) type 2-based and recombinant capsid protein (orf2)-based enzyme-linked immunosorbent assays for detection of antibodies to pcv. clin diagn lab immunol 2002;9:33-40. 34. pogranichnyy rm, yoon kj, harms pa, et al: characterization of immune response of young pigs to porcine circovirus type 2 infection. viral immunol 2000;13:143-153. 35. labarque gg, nauwynck hj, mesu ap, et al: seroprevalence of porcine circovirus types 1 and 2 in the belgian pig population. vet q 2000;22:234-236. 36. mengeling wl, lager km, vorwald ac: the effect of porcine parvovirus and porcine reproductive and respiratory syndrome virus on porcine reproductive performance. anim reprod sci 2000;60-61:199-210. 37. cartwright sf, lucas m, huck ra: a small haemagglutinating porcine dna virus. i. isolation and properties. j comp pathol 1969;79:371-377. 38. oravainen j, heinonen m, tast a, et al: high porcine parvovirus antibodies in sow herds: prevalence and associated factors. reprod domest anim 2005;40:57-61. 39. redman dr, bohl eh, ferguson lc: porcine parvovirus: natural and experimental infections of the porcine fetus and prevalence in mature swine. infect immun 1974;10:718-723. 40. mengeling wl, paul ps. interepizootic survival of porcine parvovirus. j am vet med assoc 1986;188: 1293-1295 41. joo hs, donaldson-wood cr, johnson rh, et al: pathogenesis of porcine parvovirus infection: pathology and immunofluorescence in the foetus. j comp pathol 1977;87:383-391. 42. narita m, inui s, kawakami y, et al: histopathological changes of the brain in swine fetuses naturally infected with procine parvovirus. natl inst anim health q (tokyo) 1975;15:24-28. 43. pejsak zk, truszczynski mj: aujeszky's disease (pseudorabies). in: straw b, zimmerman j, d'allaire s, et al., eds. diseases of swine. 9th ed. ames (ia): blackwell; 2006. p. 419-433. 44. wohlgemuth k, leslie pf, reed de, et al: pseudorabies virus associated with abortion in swine. j am vet med assoc 1978;172:478-479. 45. kluge jp, mare cj: swine pseudorabies: abortion, clinical disease, and lesions in pregnant gilts infected with pseudorabies virus (aujeszky's disease). am j vet res 1974;35:991-995. 46. hsu fs, chu rm, lee rc, et al: placental lesions caused by pseudorabies virus in pregnant sows. j am vet med assoc 1980;177:636-641. 554 maternal serum and allantoic fluid concentrations of activin a in experimentally induced equine placentitis maternal serum and allantoic fluid concentrations of activin a in experimentally induced equine placentitis harutaka murase, kristen scoggin, barry ball gluck equine research center, university of kentucky, lexington, ky activins belong to transforming growth factor ² superfamily. activins have a common ² subunit and are homodimers with 3 isoforms (a, b, and ab). although activins are principally gonadal hormones, they are also secreted from other reproductive tissues with functions such as placental development, immunity, or inflammation. intrauterine infection is suggested to elevate placental mrna expression and amniotic fluid concentration of activin a in pregnant women. we hypothesized that activin a is increased in serum and fetal fluids of pregnant mares with placentitis. objective was to determine maternal serum and allantoic fluid concentrations of activin a in mares with experimentally induced placentitis. at ~ 270 days of pregnancy, placentitis was induced in mares (n = 14) via intracervical inoculation of streptococcus equi zooepidemicus (n = 14 mares), whereas uninoculated mares (n = 15) served as controls. serum was collected daily and allantoic fluid was collected by transabdominal ultrasound-guided centesis in a subset of mares 5 days after inoculation. serum concentrations of activin a were determined at -8, -6, -4, -2, -1, and 0 days preceding abortion and in allantoic fluid by elisa (#dac00b, r&d systems, inc., minneapolis, mn). differences in serum activin a concentrations were determined by a random effects mixed model anova and differences in allantoic fluid concentrations were compared by a one tailed student’s t test (jmp 14). there were effects of time (days from abortion) and group by time interactions (p < 0.01 for each) on serum concentrations of activin a, with greater concentrations in placentitis mares compared to controls, beginning on day 6 prior to abortion. allantoic fluid concentrations of activin a were higher (p < 0.05) in mares with placentitis compared to control mares at day 5 after inoculation. activin a increased in maternal serum and in allantoic fluid from mares with experimentally induced placentitis secondary to s. equi zooepidemicus. activin a has been described as an acute mediator of inflammation in horses and other species. although source of increased activin a in mares with induced placentitis is unknown, it is likely derived from uteroplacental unit. keywords: activin a, chorioallantois, equine, placentitis acknowledgement supported by albert g. clay endowment and the clay fellowship of university of kentucky and authors thank dr. igor canisso for providing some samples. 457 clinical theriogenology • volume 11, number 3 • september 2019 458clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 1 contact peter chenoweth pchenoweth@hotmail.com © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/ct.v16.10600 review report andrology laboratory review: evaluation of sperm morphology peter chenoweth,a leo brito,b augustine peter,c dagmar waberski,d gary althouse,b christine aurich,e gaia luvoni,f regina turner,b natalie fraser,g cheryl lopateh aschool of veterinary sciences, college of public health medical & veterinary sciences, james cook university, townsville, queensland, australia bdepartment of clinical studies, new bolton center, school of veterinary medicine, university of pennsylvania, kennett square, pa, usa cdepartment of veterinary clinical sciences, college of veterinary medicine, purdue university, west lafayette, in, usa dunit for reproductive medicine of clinics/clinic for pigs and small ruminants, university of veterinary medicine, hannover, germany ecentre for artificial insemination and embryo transfer, university of veterinary sciences, vienna, austria fdepartment of veterinary medicine and animal science, università degli studi di milano, milan, italy gschool of veterinary medicine, university of queensland, gatton, australia hreproductive revolutions, case road ne, aurora, or, usa abstract sperm morphology assessment has an important role in male fertility diagnosis and prognosis, both for humans and animals. thus, it is important that relevant results are comparable and consistent. to achieve these aims, the following procedures are recommended: a. semen sample is suitably ‘fixed’ (e.g. in isotonic buffered formal-saline); b. sperm are examined at 1,000 x (phase or dic microscopy); c.at least 200 sperm are counted; d. each sperm is placed into 1 category, only (e.g. normal, head, midpiece etc), and e. 70% ‘normal’ sperm is the threshold for a satisfactory sample. in addition, morphologists should be provided with relevant continuing education, upskilling, and monitoring programs. this review provides guidelines for the best performance of this assessment, as well as for avoiding pitfalls. keywords: sperm, morphology, fertility, animal introduction a taskforce, representing the association of applied animal  andrology, american college of theriogenologists, european college of animal reproduction, and society for theriogenology, was given the task of providing recommendations on best methods of evaluating domestic animal semen quality. this is the third publication in a series, being preceded by reviews on sperm concentration1 and motility.2 initial evaluation of semen is generally performed macroscopically and usually includes volume, color, and consistency. semen quality is a term that usually includes sperm motility, viability, morphology, concentration, and seminal fluid composition,2 some of which were discussed.1,2 of these attributes, sperm morphology is widely recognized as the semen characteristic most directly associated with fertility,3 despite the latter often being ill defined.4 indeed, “the assessment of sperm morphology is probably the most useful and important aspect of the semen examination.”5 the microscopic assessment of sperm morphology is based on the premise that sperm shape is linked with sperm function,6 which is reinforced by evidence that abnormal sperm head shape is due to damaged dna/chromatin.7 and thus represents a ‘useful tool’ for assessing potential male fertility.8 the procedure itself is simple to perform, and the results are considered to reflect sperm fertility, at least to a degree, and particularly where a large proportion of sperm are abnormal.9 despite such considerations, wider acceptance of this procedure is constrained by variations in results, leading to a lack of confidence in their accuracy and relevance and reduced ability to directly compare data. for accuracy to be consistently achieved, sample handling, fixation and/or staining must all be optimal10 and standardized, as sperm morphology can be influenced by semen handling and observer variations,11,12 thus challenging consistency and objectivity. thus, this review aims to provide current guidelines for this procedure in order to help achieve greater consistency in its application, leading to improved acceptance of this important component of male fertility assessment. mailto:pchenoweth@hotmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10600 2 citation: clinical theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/ct.v16.10600 equipment to record sperm morphological defects, they must first be observed and recognized. the first consideration requires appropriate magnification. a variety of methods are available, with the decision to use a particular procedure often being influenced by its relative complexity, cost effectiveness, expedience and the degree of fine detail required. equipment list for a basic andrology laboratory is provided.13 here, it is incontestable that optimal results depend upon using the best available equipment available in concert with appropriate semen handling and sample preparation. in turn, recognition of particular sperm defects, and an understanding of their relative significance, benefits from a good understanding of current knowledge and developments in physiology and cell biology. equipment used for the microscopic examination of sperm includes brightfield microscopy, ordinary phase-contrast microscopy, differential interference contrast phase microscopy (dic), computer-assisted sperm analysis (casa) configured for morphology, and electron microscopy. brightfield microscopy has traditionally been used for routine andrological work as it has the advantages of relatively low cost and ease of use. it is, however, best suited for use with fixed, stained specimens, whereas phase microscopy and dic are usually preferred for unstained specimens.14 an earlier review15 provided commonly used semen stains and a recommended equipment list for a basic andrology laboratory was suggested.13 applications using casa systems for sperm morphology assessment (i.e. automated sperm morphology analyses) are rapidly gaining traction.11,16,17 these have advantages of highspeed, evaluation of large numbers of sperm, providing consistent, easily quantifiable results and thus reducing the significant variation that can occur between technicians and laboratories. however, the current lack of standardization methods makes comparisons difficult.18 in addition, casa employs negative phase-contrast microscopy that is less than ideal for the recognition of nuclear diadems and vacuoles. electron microscopy, although complex and expensive, is a most useful tool for depicting sperm ultrastructure, although this technique does not lend itself to quantitative applications.19,20 both transmission and scanning electron microscopy are now widely used in andrology.6 the generally recommended magnification of 1,000 x for the microscopical assessment of sperm morphology can be achieved using bright-field microscopy with an oil immersion objective and stained slides (e.g. using an eosin-nigrosin stain). however, it is considered preferable to use either ordinary phase or dic microscopy in conjunction with a ‘fixed’ (i.e. ‘wet’) semen sample. buffered formal saline,21 is widely used as a fixative for this purpose. in routine clinical applications, it is often expedient to ‘count’ 100 sperm per sample, although this number is at the lower end of recommended estimates of sample size, especially when confidence limits are taken into account.12,22,23 for example, world health organization (who) recommends counting at least 200 sperm for human semen assessment.24 however, counting more sperm did not lead to a change in bull semen morphology classification, even when as many as 400 sperm were counted.12 thus, this review supports the conventional approach of characterizing 100 to 200 sperm for routine bull sperm morphology assessment. sperm morphology categorization following systems categorize animal sperm morphology:25 1. origin of the defect (e.g. primary and secondary abnormalities26) 2. potential impact on fertility (e.g. major and minor abnormalities27) 3. localization of defect on sperm (e.g. head, midpiece, and tail defects28,29) 4. compensable and uncompensable sperm defects30 5. systematic sperm defects31,32 6. genetic sperm defects33 the most widely used system, at least in animal andrology laboratories, is considered to be number 3, above,24,28,29 which is also the simplest and least ambiguous system of those above. interpreting and reporting sperm morphology sperm morphology reports should include such details as: a. the criteria used to categorize different defects;25 b. the materials and methods used to prepare the samples for examination (e.g. fresh or frozen-thawed semen, fixatives, dilutions, and staining); c. relevant microscopic and/or imaging details; and d. the reference values used for final summation and conclusions.34 standardization of sperm morphology assessment it would be very useful to achieve consensus on sperm morphology techniques and interpretations to facilitate research and to reduce misunderstandings and differences that can result in economic loss, conflict, and personal distress. adoption of standardized procedures for semen analysis, including sperm morphology, would allow objective comparison of results, in turn improving confidence in the process.35 despite this, various attempts to standardize sperm morphology have not been widely adopted. this is probably due to difficulties in harmonizing differences in semen preparation and staining, microscope systems and optics and differences among technicians in their training, competence, and interpretation of results. this was illustrated that morphology evaluations of stallion sperm varied with both technician and methodology.36 here, wet-mount preparations examined by phase-microscopy produced better results than stained smears examined with bright-field microscopy. two techniques (‘wet’ preparation using dic phase-contrast microscopy and eosin nigrosin stained smears) were compared using microscopy and it was concluded that, although the results had some qualitative differences, the final breeding soundness examination classification of bulls did not differ.37 it is a reassuring fact that veterinary practitioners were 92% in accordance when categorizing bull semen morphology38 and there was little difference in the types of sperm defects observed in tropical bos indicus bulls compared to temperate bos indicus bulls.39 despite this, there remains a relative lack of confidence in sperm morphology results from both animal and human andrology laboratories.18,40,41 sperm abnormality thresholds an early observation was that there was a ‘threshold’ of observable sperm morphological abnormalities above which http://dx.doi.org/10.58292/ct.v16.10600 citation: clinical theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/ct.v16.10600 3 fertility became compromised. this threshold, ~ 30%, has remained remarkably consistent. in bulls, this was illustrated in natural mating trials in texas,42 where bulls preselected for good sperm morphology (> 70% normal sperm) achieved significantly more pregnancies than those that were unselected for sperm morphology. fertility is compromised by morphological anomalies by themselves or due to the correlation of sperm morphology to other variables (e.g. dna integrity in boars43). in felids, a threshold should be set lower than in other species, because domestic and wild are generally affected by teratozoospermia (< 40% morphologically normal sperm).44 since then, similar conclusions have been reached from trials in areas as diverse as in vitro fertilization and intrauterine insemination and studies on sperm dna damage. the pathogenesis of this relationship has not yet been well elucidated. in pigs, most breeding organizations have defined thresholds between 15 and 30% for abnormal sperm, with or without specification of thresholds for sperm with cytoplasmic droplets (15-30%).45 it is important to note that such thresholds only apply when the observed spermiogram is representative of normal, generalized spermatogenic stress. in some situations, such as immaturity or gossypol toxicity, the particular abnormalities encountered may be more indicative. stains and preparations various stains and preparations have been used in evaluating human and animal sperm microscopically and a representative list is provided (table 1 in appendix). there are useful references.15,46 supra-vital stains (e.g. eosin nigrosin) are commonly used for semen staining in the field as they are simple to use, and they can depict sperm morphology reasonably well in addition to providing an insight into sperm vitality.47,48 however, differential-interference phase contrast microscopy of ‘fixed’ (i.e. unstained) samples at 1,000 x is regarded as the ‘gold standard’ for depicting certain types of sperm abnormalities, particularly acrosomal,49,50 as well more subtle sperm head or midpiece defects. however, either stained or unstained methods produced similar results in terms of bull classification.51 here, it is considered that some approaches recommended for human semen assessment, such as using the papanicolaou stain and strict criteria for morphology categorization,3 are not easily adopted for animal semen assessment, due to problems of logistics and complexity. despite this, morphological indices for canine sperm have been developed52 based upon those described in the who laboratory manual for the examination and processing of human semen.24 it is important to note that such thresholds only apply when the observed spermiogram is representative of normal, generalized spermatogenic stress. in some situations, such as immaturity or gossypol toxicity, the particular abnormalities encountered may be more indicative. semen handling and preparation sperm morphological defects may occur both preand postejaculation,53 with the latter including collection, handling and cryopreservation procedures,54 as well as the staining method employed.10 semen collection methods as well as collection frequency can influence sperm morphology, and this can vary with species.55 in addition, sperm morphology can be influenced by environmental factors such as ph,56 bacteria, and inflammatory products,57–59 and age of the donor; sperm morphology declined in stallions after 11-14 years of age60 and in dogs after 7 years.61,62 if a representative semen sample has been obtained, then care should be taken to avoid subsequent sperm damage by protecting sperm viability and/or integrity during handling and processing. factors that can affect results include sample preparation, species, extender or medium, objective magnification and ‘quality’ as well as operator knowledge and experience.12 defects that can be attributed to poor semen handling and those linked with poor preparation of semen smears are listed (tables 2 and 3, respectively, in appendix). a checklist of factors that could adversely affect sperm morphology, as well as proposed solutions, is also available in the who laboratory manual for the examination and processing of human semen.24 other cells in semen semen consists of a fluid medium in which sperm are suspended. however, other cells and organisms can be present in semen, with many of these able to be observed microscopically. occasional sightings of other cells do not necessarily indicate that a problem exists, although some are more clinically relevant than others.63 for example, increased numbers of leukocytes in semen can indicate infection, whilst being capable of directly causing sperm oxidative damage.64 round spermatids, spermatocytes, and spermatogonia may indicate stress or damage to the spermatogenic epithelium. bacteria are commonly observed in both fresh and stained semen,13,58 and are, in themselves, capable of causing alterations to sperm dna and morphology.59,65 slide preparation, proper staining and appropriate microscopy are all important considerations for the recognition of nonsperm cells in semen.24 the recognition of major nonsperm inclusions (i.e. epithelial cells, macrophages, red blood cells, white blood cells, spermatogenic precursors, ‘round cells,’ epididymal cells, bacteria, contaminants, and debris), as well as appreciating their relative significance, should be an essential part of the sperm morphologist’s skillset. conclusion competent and accurate assessment of sperm morphology is an important component of male fertility diagnosis and prognosis. for sperm morphology assessment to be as useful as possible, it is important that results are comparable and consistent among and within veterinarians, technicians, and morphologists. this requires the combination of optimal technique with good equipment and its application within the context of relevant animal history, supported by appropriate knowledge of male physiology and pathology. to encourage greater consistency, the following procedures are suggested as a basis for sample comparisons, within and among species: a. semen sample in a suitable fixative; b. use of phase/dic microscopy at 1,000 x; and c. count of 200 sperm, and d; a 30% threshold for ‘abnormal’ sperm. it is also important that animal sperm morphologists have ongoing access to relevant continuing education, upskilling, and monitoring programs. conflict of interest none to report. references 1. brito lfc, althouse ga, aurich c, et al: andrology laboratory review: evaluation of sperm concentration. theriogenology 2016;85:1507-1527. doi: 10.1016/j.theriogenology.2016.01.002 http://dx.doi.org/10.58292/ct.v16.10600 https://doi.org/10.1016/j.theriogenology.2016.01.002 4 citation: clinical theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/ct.v16.10600 2. peter at, brito lf, althouse gc, et al: andrology laboratory review: evaluation of sperm motility. clinical theriogenology 2021;13:297-315. doi: 10.58292/ct.v13.9359 3. mortimer d, menkveld r: 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https://doi.org/10.1136/vr.97.1.12 https://doi.org/10.1136/vr.97.1.12 https://doi.org/10.1016/s0015-0282(99)00201-0 https://doi.org/10.1093/humrep/den365 6 citation: clinical theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/ct.v16.10600 73. pozor ma, zambrano gl, runcin e, et al: usefulness of dip quick stain in evaluating sperm morphology in stallions. proc am assoc equine pract 2012;58:506-510. 74. barth ad, oko rj: abnormal morphology of bovine spermatozoa. 1st edition, ames, ia; iowa state university press: 1989. p. 302. 75. erenpreiss j, jepson k, giwercman a, et al: toluidine blue cytometry test for sperm dna conformation: comparison with the flow cytometric sperm chromatin structure and tunel assays. hum reprod 2004;19:2277-2282. doi: 10.1093/humrep/deh417 76. tsarev i, bungum m, giwercman a, et al: evaluation of male fertility potential by toludine blue test for sperm chromatin structural assessment. hum reprod 2009;24:1569-1574. doi: 10.1093/ humrep/dep068 77. kovacs a, foote rh: viability and acrosome staining of bull, boar and rabbit spermatozoa. biotech histochem 1992;67:119124. doi: 10.3109/10520299209110020 78. casarett gw: a one-solution stain for spermatozoa. stain  technol  1953;28:125-127. doi: 10.3109/10520295 309105113 http://dx.doi.org/10.58292/ct.v16.10600 https://doi.org/10.1093/humrep/deh417 https://doi.org/10.1093/humrep/dep068 https://doi.org/10.1093/humrep/dep068 https://doi.org/10.3109/10520299209110020 https://doi.org/10.3109/10520295 309105113 https://doi.org/10.3109/10520295 309105113 citation: clinical theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/ct.v16.10600 7 table 1. stains and preparations used for assessing sperm morphology in animals stain details and source uses, advantages, and disadvantages eosin nigrosin sources lane manufacturing, usa minitube international www.minitube.com references15,66 a one-step differential membranedependent ‘supra-vital’ stain, easy to keep and simple to use. employed for ‘live/ dead’ estimation as well as sperm morphology, although finer sperm structures may not be obvious. recommended by the society for theriogenology. modified giemsa sources www.sigmaaldrich.com www.fischersci.com references15,67 commonly used in hematology for cellular depiction. also useful for sperm morphology, particularly for acrosome definition. williams stain reagents from www.sigmaaldrich.com. www.fischersci.com references68,69 double-stain method (carbol-fuchsin eosin counterstained with methylene blue). good sperm morphology stain, despite requiring several steps. spermblue® spermblue® prestained slides sources microptic sl, barcelona, spain.   www.micropticsl.com fertility technology resources. www.fertilitystuff.com youning biotech co. ltd. www.youning.com reference10 a one-step stain for human or animal sperm that can be used on fresh, frozen and extended semen, as well as for automated sperm morphology analyses. spermac® sources spermac laboratories www.spermac.com fertipro nv www.fertipro.com, minitube international www.minitube.com references70,71 versatile, rapid, dual stain allowing separate visualization of the nucleus and cytoplasm, as well as good acrosome definition. diff-quik, dip quick® sources microptic sl, barcelona, spain. www.micropticsl.com proiser r&d sl info@proiser.com references72,73 a romanowsky stain used widely in clinical cytology. commonly available in clinical settings. can be used for combined assessment of morphology (recommended by who), seminal cytology and sperm dna/chromatin stability. aniline blue reagents from www.sigmaaldrich.com www.fischersci.com reference74 histology stain. also differentiates histones and protamines, and is a simple procedure. (continued) appendix http://dx.doi.org/10.58292/ct.v16.10600 http://www.minitube.com http://www.sigmaaldrich.com http://www.fischersci.com http://www.sigmaaldrich.com http://www.fischersci.com http://www.micropticsl.com http://www.fertilitystuff.com http://www.youning.com http://www.spermac.com http://www.fertipro.com http://www.minitube.com http://www.micropticsl.com mailto:info@proiser.com http://www.sigmaaldrich.com http://www.fischersci.com 8 citation: clinical theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/ct.v16.10600 table 1. (continued) stain details and source uses, advantages, and disadvantages toluidine blue reagents from www.sigmaaldrich.com www.fischersci.com references75,76 useful to detect sperm chromatin abnormalities, as well as to depict morphology. trypan blue reagents from www.sigmaaldrich.com www.fischersci.com membrane dependent ‘vital’ stain, used for ‘live-dead’ estimation as well as sperm morphology. can be used with ‘fixed’ semen. also useful for hematology and cell cultures; one-step procedure. farrelly stain source minitube international www.minitube.com a 2-step contrast stain, useful for sperm morphology in samples which do not contain glycerol. kovacs stain reagents (trypan blue, congo-red and giemsa) from www.sigmaaldrich.com reference77 combined sperm viability and acrosome stain. casaretts stain reagents (aniline blue, eosin b, phenol) from www.sigmaaldrich.com. reference78 one step stain which is useful for depicting different structures in human and dog sperm. cell-vu® morphology slides source tekevent pty ltd info@tekevent.com prestained slides, simple procedure. can be used with undiluted semen. it provides good depiction of head, acrosome and tail. modified papanicolaou stain reagents www.sigmaaldrich.com www.fischersci.com reference13 a versatile cytology stain with a modified version used for sperm morphology, particularly human, and which is also useful for seminal cytology, including round cells in semen. commonly available in clinics. the procedure is relatively complex and time consuming. sperm stain ready to use source microptic sl, barcelona, spain. www.micropticsl.com a rapid, 2-step romanowsky stain used in human andrology. it is also useful for differential blood cell staining. table 2. sperm morphology problems associated with semen handling causes outcomes nonphysiologic temperatures contamination rough handling inappropriate extender nonisotonic media reduced percent intact acrosomes (pia) increased numbers of bacteria, sperm clumping detached sperm heads reduced pia, increased crystal formation reduced pia, increased ‘bent’ midpieces and tails http://dx.doi.org/10.58292/ct.v16.10600 http://www.sigmaaldrich.com http://www.fischersci.com http://www.sigmaaldrich.com http://www.fischersci.com http://www.minitube.com http://www.sigmaaldrich.com http://www.sigmaaldrich.com mailto:info@tekevent.com http://www.sigmaaldrich.com http://www.fischersci.com http://www.micropticsl.com citation: clinical theriogenology 2024, 16, 10600, http://dx.doi.org/10.58292/ct.v16.10600 9 table 3. sperm morphology problems associated with the preparation of semen slides contributing factor causes sperm are disrupted and/or have signs of mechanical damage rough smearing technique (including mixing using the edge of a glass slide) or the coverslip was disturbed prematurely sperm are too sparse on the slide poor mixing of sample over-dilution of sample excess stain the dried smear shows the appearance of ‘cracking’ over-thick smear slide exposed to excess heat while drying areas of excessive stain accumulation occur on the slide aged and/or unmixed stain poor technique in making the smear sperm are stained too darkly stain was too thick or strong staining time was too long sperm are stained too lightly stain was too thin or weak staining time was too short a clear area resembling a ‘halo’ is observed above sperm heads sperm movement before stain dried http://dx.doi.org/10.58292/ct.v16.10600 2011: commercial application and cost analysis of a 5 day co-synch synchronization protocol     commercial application and cost analysis of a 5 day co-synch synchronization protocol s.e. davis, e. curry, and j.r. gibbons animal and veterinary sciences department, clemson university, clemson, sc abstract artificial insemination (ai) is a tool available to progressive cattle producers to assist them in reaching reproductive, genetic and financial goals. fixed-time ai programs decrease labor costs for estrus detection and ensure every cow is inseminated. co-synch + cidr (controlled internal drug release) is a widely used ovulation synchronization protocol within the beef industry. the objective of the experiment within this technical report was to determine if the shortened 5 day co-synch + cidr sufficiently increased pregnancy rates to compensate for the added expense and increased labor of an additional injection and additional chute processing. mature, multiparous angus and angus-cross cows ranging in age from 3-15 years averaging 500 kg bodyweight were synchronized using the 5 day cosynch + cidr protocol. (day 0: administer 100 µg gonadotropin releasing hormone (gnrh) and insert cidr, day 5: remove cidr and administer 25 mg prostaglandin f2α (pgf2α), day 5 + 8 hours: second injection of 25 mg pgf2α, day 8 (72 hours after cidr removal): administer 100 µg gnrh and fixedtime artificially inseminate). the pregnancy rate achieved using the 5 day co-synch protocol was 53.7%, which was comparable to pregnancy rate (51.3%) achieved within the same herd using a modified ovsynch protocol (day 0: 100 µg gnrh, day 7: 25 mg pgf2α, day 9: 100 µg gnrh, day 9 +12 hours: fixed-time ai) the previous year. although comparable to other ai systems in efficiency (pregnancy rates) the success of this approach did not compensate for the additional financial and labor input. however, dependent upon management system, this program can sufficiently decrease estrus detection cost and labor associated with an estrus synchronization program to make this fixed-timed ai protocol economically feasible. keywords: cattle, co-synch + cidr, estrus synchronization, ovulation synchronization introduction artificial insemination is an important tool available to cattle producers that will facilitate increased genetic gain, propagation of more elite genetics from outstanding bulls, rapid improvement of economic traits, increased efficiency of sire selection and progeny testing and a decreased number of bulls that need to be maintained on an operation.1 from a management standpoint, for a cow to maintain a yearly calving interval, she must conceive again within 85 days post-calving.2 artificial insemination implementation can reduce the amount of time necessary to breed cows, shorten the calving season and produce a more uniform calf crop at weaning and provide more predictable calving ease.1,3 acceptable pregnancy rates using ai are dependent on several management factors such as proper nutrition before, during and after breeding, proper health status, accurate record keeping, organization and planning of the breeding program, estrus detection (if applicable), semen quality, storage and handling, adequate working facilities and skilled technicians.2 several uncontrollable factors such as the weather, latitude and daylight can contribute to ai program efficiency as well. artificial insemination is currently used to breed 72.5% of dairy cattle, with timed ai (tai) programs used in 58.2% of dairy operations, and 7.6% of beef cattle in the united states.4,5 there are many factors that contribute to the less frequent use of ai in beef cattle operations. producers list labor, time and cost as the main reasons not to implement ai followed by too difficult or complicated, other various reasons, lack of facilities and lower confidence in effectiveness.5 however, under the proper management strategy, ai can be used to add value to a calf crop to sufficiently cover added expense. estrus synchronization can reduce the amount of time and labor needed for estrus detection and ai. the success of estrus synchronization programs relies heavily on proper estrus detection, as the detection efficiency correlates to pregnancy rates following ai or embryo transfer (et). synchronization programs call for estrus detection anywhere from three to eight days. this is labor intensive and in some cases cost prohibitive, as estrus detection costs range from $15-50 per cow per synchronization cycle.6 there are many factors that influence detection efficiency which directly 129 clinical theriogenology • volume 3 number 2 • june 2011     affect the cost associated with detection. these factors include the days needed for detection, the amount of time allowed for estrus detection per session and the frequency of detection per day. others factors such as labor availability, facilities and management systems contribute to the ideal frequency and efficiency of detection. fixed time ai is utilized in an ovulation synchronization program which further reduces the amount of labor needed for estrus detection because cattle are not examined for estrus and this also ensures that every cow is inseminated. inseminating every cow adds the possible advantage of getting cows pregnant that might not have been observed in estrus with an estrus detection protocol, but do ovulate and may become pregnant. artificial insemination along with ovulation synchronization facilitates the use of a timed ai program, which are now achieving pregnancy rates comparable to those achieved by estrus detection programs. the used of a cidr (controlled internal drug release; a progesterone releasing device) can further increase the efficiency of an estrus synchronization protocol by inducing cyclicity in anestrous cows. an alternative method of delivering exogenous progesterone is the feed additive melangesterol acetate (mga). this method of progesterone delivery is inexpensive (about $0.02/head/day); however, consistent administration is difficult to control because of inconsistencies with feeding habits and irregular feed intake. the addition of a cidr has been shown to increase pregnancy rates by 10%, (58% vs. 48%) using a conventional co-synch protocol.7 a commonly used fixed timed ai program in beef cattle is co-synch + cidr. average conception rates using this program in postpartum beef cattle are approximately 55%, with reported rates ranging from 31-80%.7,9-15 the conventional 7 day co-synch + cidr ai program is initiated by administration of 100 µg gnrh, on day 0 along with the insertion of a cidr. on day 7 the cidr is removed and 25 mg pgf2α is administered. sixty to sixty-six hours after cidr removal all cattle receive a second injection of 100 µg gnrh and are fixed-timed artificially inseminated. a modification to the conventional 7 day co-synch + cidr is to shorten the exogenous progesterone delivery to 5 days and administer two injections of pgf2α. reported pregnancy rates using this protocol range from 55-80%.15-17 the 5 day co-synch + cidr is initiated following the same protocol as the 7 day co-synch + cidr with administration of 100 µg gnrh and insertion of a cidr at day 0. a novel approach to the conventional system is to shorten the 7 day cidr interval to 5 days. on day 5, the cidr is removed and an initial injection of 25 mg pgf2α is administered. eight hours following the initial injection of pgf2α a second dose of 25 mg is administered, which is necessary for the success of the shortened protocol. ideally, the added advantages in terms of increase in conception rates of using the 5 day co-snych + cidr program should compensate for the added expense of an extra injection and labor associated with an additional chute processing. the primary goals of this technical report were to evaluate expenses and economical advantages or disadvantages with using a particular fixed timed ai protocol within a particular cowherd. materials and methods mature, multiparous angus, angus cross and hereford cattle (n=123) ranging in age from 3-15 years and averaging 500 kg body weight, were administered 100 µg of gnrh and a cidr (eazi-breed™ cidr®, pfizer animal health, new york, ny) was inserted on day 0. cidr removal and administration of 25 mg pgf2α occurred simultaneously on day 5. approximately 7 hours after cidr removal, all cattle received a second dose of 25 mg pgf2α. on day 8, each cow received 100 µg gnrh and were inseminated with frozen-thawed semen from several angus (n=4) or hereford (n=2) bulls (figure). cattle exhibiting signs of estrus the next day after fixed-timed ai were eliminated. approximately two weeks after tai, bulls were placed with the cows for 45 days. all cows were subjected to transrectal ultrasonic evaluation of their reproductive tracts to determine pregnancy 30 days after insemination. cattle determined to have conceived from ai were again evaluated ultrasonically on day 70 to determine fetal sex. in another study cattle from a similar origin and environmental conditions were synchronized with a modified ovsynch program. on day 0 cows (n=152) were administered 200 µg gnrh. on day 7, cows were given 25 mg pgf2α and on day 9, given 100 µg gnrh. any cows that were observed in estrus before day 9 were eliminated. twelve hours after the second gnrh injection, the cattle were randomly divided into control (n=75) and heiferplus™ (hp; n=77) groups.18 the control group was 130clinical theriogenology • volume 3 number 2 • june 2011     inseminated with 0.5 ml of frozen-thawed semen which had been incubated at 37°c for 20 min and the hp group was inseminated with 0.5 ml frozen-thawed semen heiferplus™ treated semen.19,20 pregnancy was determined via ultrasonic evaluation 36-38 days after ai. fetal sex was determined via ultrasonic evaluation 55-58 days after ai. figure: timeline for ovulation synchronization protocols cost analysis all prices used to calculate $/pregnancy and $/female calf are subject to the assumption that all drugs were purchased from the same vendor (valley vet supply; marysville, ks21). it is a realistic assumption that producers would purchase their drug inputs from a single supplier and price fluctuations for different inputs could be substituted in the calculations. also, even though two year’s worth of data were used, the price of inputs would carry minimal variation from year to year and assigning a dollar amount for each protocol was critical for economic evaluation. the prices for injections on a per cow basis were: gnrh = $2.90/dose pgf2α = $2.67/dose cidr = $10/cidr mga = $0.02/head/day20 chute processing = $1/head/time through the chute; chute charges were calculated based on $12.48 per hour wage ($10.00 + 28% benefits) for four individuals for two hours on a hundred cow basis. estrus detection = $15/head/synchronization; estrus detection fees were based on checking for estrus for four days with two checks per day for 30 minutes and two laborers at $12.48/hour ($10.00 + 28% benefits) on a 100 cow basis. 131 clinical theriogenology • volume 3 number 2 • june 2011     results the pregnancy rate achieved with the 5 day co-synch + cidr was 53.7%. the pregnancy rate achieved using the modified ovsynch protocol within the same herd in a previous year was 48% (36/75) for the control group and 54.5% (42/77) for the hp group. when these two groups were combined the total herd pregnancy rate was 51.3% (78/152). the pregnancy rate data were combined in the treatment and control groups because there was no treatment effect within the study. also, this protocol did not affect fertility within the herd from previous years or when compared to the present study. the pregnancy rate for the 5 day co-synch was not statistically different from either the control group pregnancy rate from the previous study or for the control and hp groups combined (there was no treatment effect within study). table: comparison of $/ai, $/pregnancy and $/female calf for different protocols synchronization protocol $/ai $/pregnancy $/female calf traditional 7 day co-synch + cidr $21.67 $39.40 * 5 day co-synch + cidr $25.34 $47.19 $111.32 modified ov-synch $12.47 $24.30 $52.65 2 injection pgf2α $22.91 $65.43 * select-synch $23.24 $51.64 * mga + 2 injection pgf2α $23.55 $33.64 * assumptions: gnrh = $2.90/dose pgf2α = $2.67/dose cidr = $10/cidr mga = $0.02/head/day fed chute processing= $1/head/time through the chute estrus detection = $35/head/synchronization cycle * not analyzed in study. calculations for $/ai and $/pregnancy for the traditional co-synch used a pregnancy rate of 55%. the modified ov-synch and 5 day co-synch + cidr were calculated using data collected, finding 51.3% and 53.7% pregnancy rates, respectively. calculations were based on field trial studies by patterson and smith which found the estrus response rate for the 2 injection pgf2α, the select-synch and the mga + 2 shot to be 57%, 67% and 93%, respectively. also, pregnancy rates were 35%, 45% and 70%, respectively. discussion although the conception rates achieved in this study were slightly lower than other published data using the same protocol,15-17 the pregnancy rate of 53.7% is still an acceptable value for a fixed-timed ai protocol. although not demonstrated in the current report, an increase in cost per pregnancy might be accompanied by an increase in pregnancy rate which would enhance economic viability of this approach. however, an increase in the cost of inputs (labor, pharmaceuticals, etc.) may not be associated with an increase in pregnancy rates, so producers should evaluate all protocols to determine the improvements needed within their herd and if these improvements are cost effective within their production system. using a pregnancy rate of 55% for the conventional 7 day co-synch protocol, and all posted assumptions (table), within this herd, a pregnancy rate of 64.5% would be needed to make the shortened 5 day cosynch protocol more financially advantageous. although pregnancy rates in this study did not approach this value, it is however within the range of published conception rates for the 5 day co-synch protocol.16 this further shows the need for a classic control group beyond the scope of this technical report. on farm, similar pregnancy rates were achieved using the modified ov-synch protocol without a cidr and 132clinical theriogenology • volume 3 number 2 • june 2011     cost significantly less. the three estrus detection protocols were comparable to the fixed-timed $/ai protocols; however, when comparing the $/pregnancy, an increase in cost was observed when estrus response was lower. the mga 2 injection pgf2α was the least expensive in terms of $/pregnancy with excellent estrus response and pregnancy rates; however, supplementing mga requires a feed or protein carrier and consistent delivery on a per head basis is difficult to control. the cost per female calf was outside of the scope of this project; however, was included in the calculations to show that the two protocols studied within this herd did not alter sex ratio and this dollar amount could potentially be of valuable to certain producers. also, estrus detection costs were calculated on the minimal end of the price spectrum (ranges from $15-50 per cow per synchronization cycle) and increased estrus detection intensity would increase costs and labor associated and efficacy of detection would also impact the number of cattle observed in estrus and subsequently artificially inseminated. labor costs are also impacted by herd size, which should be taken into account when determining which, if any, estrus synchronization program would be the most economically feasible. the larger the herd size, the less viable the more intensive programs that require several trips through the chute will be. alternatively, a smaller herd size could potentially be cost-prohibitive because price could increase to a $/head amount that is not financially feasible. herd size should be an important consideration when developing a proper synchronization protocol. in conclusion, the 5 day co-synch protocol produces acceptable pregnancy rates for a tai protocol, but within this herd, did not increase the pregnancy rates to sufficiently compensate for the added expense. references 1. a.i. management manual: deforest (wi): american breeders services; 1983. p. 169-177. 2. beef cattle management in south carolina and ipm approach: clemson (sc): clemson extension; 2001. p 91. 3. johnson sk, jones r: costs and comparisons of estrous synchronization systems. research and extension bulletin(s). manhattan (ks): kansas state university; 2003. 66506-4011. 4. info sheet. feb 2009. fort collins (co): usda-aphis-vs:ceah. p 2-3. 5. national animal health monitoring system. 2007-2008 part ii: reference of beef cow-calf management practices in the united states. fort collins (co): usda-aphis-vs. p 18-21. 6. gibbons j: economics of beef cattle reproductive decisions. in: haskell srr, ed. blackwells’s five minute veterinary consult: ruminant. ames: wiley-blackwell; 2008. p. 314-315. 7. lamb gc, stevenson js, kesler dj, et al: inclusion of an intravaginal progesterone insert plus gnrh and prostaglandinf2α for ovulation control in postpartum suckled beef cows. j anim sci 2001;79:2253-2259. 8. busch dc, schafer dj, wilson dl, et al: timing of artificial insemination in post-partum beef cows following the administration of the co-synch + cidr protocol. j anim sci 2008;86:1519-1525. 9. geary tw, whittier jc: effects of a timed insemination following synchronization of ovulation using the ovsynch or co-synch protocol in beef cows. prof anim sci 1998;14:217-220. 10. geary tw, salverson rr, whittier jc: synchronization of ovulation using gnrh or hcg with the co-synch protocol in suckled beef cows. j anim sci 2001;79:2536-2541. 11. martinez mf, kastelic jp, adams gp, et al: the use of progestins in regimens for fixed-time artificial insemination in beef cattle. theriogenology 2002;57:1049-1059. 12. stevenson js, lamb gc, johnson sk, et al: supplemental norgestomet, progesterone, or melengesterol acetate increases pregnancy rates in suckled beef cows after timed inseminations. j anim sci 2003;81:571-586. 13. larson je, lamb gc, stevenson js, et al: synchronization of estrus in suckled beef cows for detected estrus and artificial insemination and timed artificial insemination using gonadotropin-releasing hormone, prostaglandin f2α, and progesterone. j anim sci 2006;84:332-342. 14. kasimanickam r, collins jc, wuenschell j, et al: effect of timing of prostaglandin administration, controlled internal drug release removal and gonadotropin releasing hormone administration on pregnancy in fixed-time ai protocols in crossbred angus cows. theriogenology 2006;66:166-172. 15. beef reproduction task force: protocols for synchronization of estrus and ovulation. lincoln: university of nebraskalincoln; 2009. http://beefrepro.unl.edu 16. bridges ga, helser la, grum de, et al: decreasing the interval between gnrh and pgf2α from 7 to 5 days and lengthening proestrus increases timed-ai pregnancy rates in beef cows. theriogenology 2008;69:843-851. 17. kasimanickam r, day ml, rudolph js, et al: two doses of prostaglandin improve pregnancy rates to timed-ai in a 5 day progesterone-based synchronization protocol in beef cows. theriogenology 2009;71:762-767. 18. williams t: the use of heiferplus in superovulated heifers; 2007; http://emlabgenetics.com/documents/heiferplus%20superovulated%20donors%20data.htm 133 clinical theriogenology • volume 3 number 2 • june 2011     19. curry e, pratt sl, lapin dr, et al: efficacy of a commercially available post-thaw bovine semen sexing kit in both single-ovulating and hyperstimulated cows. anim reprod sci 2009; 116: 376380. 20. curry e, davis se, gibbons jr: advances in sex ratio management in cattle. clin therio 2010;2:29-40. 21. valley vet supply. ames, iowa. www.valleyvet.com 22. patterson dj, smith mf: progesterone-based estrus synchronization for beef replacement heifers and cows. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. st. louis: saunders; 2007. p. 278-286. 134clinical theriogenology • volume 3 number 2 • june 2011 metastatic ovarian stromal tumor in a young bitch metastatic ovarian stromal tumor in a young bitch alyssa helms,a elizabeth frieden,a kayla fowler,a phillip sponenberg,b nam baik,a kemba clapp,a julie cecerea adepartment of small animal clinical sciences bdepartment of biomedical sciences and pathobiology, college of veterinary medicine virginia-maryland, blacksburg, va a 2 year old, nulliparous, intact female mixed breed dog was initially presented to the primary care veterinarian for evaluation of 2 week history of hyporexia, abdominal distension, lethargy and acute weight loss. the bitch’s most recent estrous cycle was 2 weeks prior to presentation. physical examination and complete blood count revealed a mild nonregenerative anemia, leukocytosis and hyperthermia. the patient was diagnosed with presumptive pyometra and placed on an unrecorded form and dose of antibiotics. two weeks later, the patient was presented after hours to an emergency clinic for continued symptoms. abdominal ultrasonography revealed an abdominal mass. physical examination findings at our hospital were: body condition score of 4/9, tachycardia, tachypnea, grossly distended and painful abdomen, and a firm tubular structure palpable ventrally per rectum. all other physical examination parameters were within normal limits, including a lack of vulvar discharge. complete blood count displayed an inflammatory leukogram with a neutrophilic leukocytosis and left shift. partial thromboplastin clotting time was also slightly elevated. abdominal ultrasonography revealed a large, complex, heterogeneous mass in the right cranial abdomen that was closely associated with the right ovary though its origin from a specific organ was not identifiable. a smaller, ovoid mass was identified causing dorsal displacement of the right cranial aspect of liver. moderate pleural and peritoneal effusion and enlarged right medial iliac and portal lymph nodes were also identified. ultrasound guided abdominocentesis produced a serosanguinous transudate. uterus was not clearly appreciated. primary differential diagnoses included cystic peritoneal neoplasia of unknown origin or disseminated granulomatous disease. due to poor prognosis, the owners elected humane euthanasia. necropsy findings included a 20 x 20 x 20 cm lobulated mass originating from the right ovary with multiple corpora lutea. multiple tan, firm and round soft tissue nodules of varying size were observed within the diaphragm, at the level of thoracic inlet and adjacent to the pancreas. histology of the primary ovarian mass, pancreatic and diaphragmatic nodules revealed a large neoplastic population of cells with variable cellular detail. cells varied from plump, eosinophilic and foamy (luteal cells) to slender fusiform cells (theca cells) to rounder or cuboidal cells with moderate cytoplasm that tended to surround fluid filled spaces (granulosa cells). final diagnosis was malignant ovarian stromal tumor with a tendency of differentiation towards luteal cells. to the authors’ knowledge this is the first report of a mixed cell type metastatic ovarian stromal cell tumor in a young dog. keywords: ovarian, stromal tumor, metastatic, canine, luteal, neoplasia 467 clinical theriogenology • volume 11, number 3 • september 2019 468clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2015: clinical teaching paradigms at the university of minnesota college of veterinary medicine clinical teaching paradigms at the university of minnesota college of veterinary medicine margaret v. root kustritz, robert j. washabau college of veterinary medicine, university of minnesota, st. paul, mn abstract course coordinators were surveyed and course syllabi reviewed at the university of minnesota college of veterinary medicine for types of teaching paradigms used for clinical training and extent of those varying types of teaching in large animal, small animal, and non-species-based rotations. showand-tell, or apprenticeship, was the most commonly used teaching paradigm. other teaching paradigms, in decreasing order, included topic rounds, case rounds, critical scientific reading/evidence-based medicine, assignments and other assessments, games, and discussion of roles in society/ethics. apprenticeship teaching was more common in small animal rotations and topic rounds was more common in large animal rotations; this may be a reflection of caseload. specific teaching methods, tools, and unique learning opportunities are described. keyword: clinical teaching introduction accreditation by the american veterinary medical association council on education requires that students experience at least one year of clinical training. this generally is accepted to be the capstone experience for students, during which they integrate the basic science and clinical didactic training they have received and start to practice as a professional with clinical oversight. kolb described learning as being driven by concrete experience, with reflection, abstract conceptualization, and an opportunity to try again being the path to deep understanding.1 the kolb model clearly describes learning in a clinical environment. historically, medical training has been accomplished through a show-and-tell, or apprenticeship, model. apprenticeship learning includes the type practiced historically in the arts where a master worked with a group of students who strove to emulate his work, and the type common to artisans, where a product was manufactured under guidance of a master until the protégé demonstrated competence. efficiency is one of the great advantages of the apprenticeship model since the clinician is already seeing cases and can readily take students along into the clinic. it uses client-owned animals for teaching purposes, minimizing the expense and animal welfare concerns of housing teaching animals. finally, it is multi-faceted, permitting students to practice communications, technical skills, problem-solving, and other higher level competencies within one authentic experience. students benefit from seeing cases with a variety of clinicians, with their varying backgrounds, knowledge, perspectives, and teaching styles.2 inconsistency is one of the disadvantages of the apprenticeship model since there is a potential for limited hands-on experience by students. this can be true if the caseload and subsequent demands on clinician time are high or if the caseload is very low, such that inadequate case material for teaching is present. competition often exists for cases between students and other trainees (graduate students and house officers). finally, the need to balance student learning opportunities with risk assessment may decrease student learning opportunities. student:faculty ratios must be appropriate to permit students opportunities to interact directly with clinical instructors. clinical teaching is challenging. clinicians are required to create a supportive learning environment for students of varying ability while seeing cases in a timely manner.3-5 caseload may vary, requiring the instructor to be flexible in considering what information will be covered by seeing cases and what should be presented in more formalized rounds or seminars.4 in one survey, veterinary clinical instructors cited their greatest challenges as managing individual learners and groups of learners, and balancing their clinical workload and teaching responsibilities.3 several studies have identified a common concern held by faculty that provision of clinical care often supersedes teaching.6,7 increasingly complicated electronic medical record systems, and the time it takes to master them, are a further drain on clinical teaching time. clinical theriogenology • volume 7, number 4 • december 2015447 at the university of minnesota, students enter clinics during the spring semester of their third year in the curriculum, such that they complete slightly more than one year of clinical training. all clinical rotations are two weeks in length. required rotations vary by track (table 1). the number of rotations available to students varies slightly annually, as not all rotations are offered every year, and as faculty members work with the collegiate curriculum committee to provide appropriate breadth and depth of clinical experiences in a wide variety of species. this study evaluated teaching paradigms used on clinical rotations at one point in time at this institution. materials and methods course coordinators of all clinical rotations were sent an electronic survey and solicited for input via repeated email messaging (appendix 1). those who did not respond initially also received a hard copy of the survey in their mailbox. syllabi for all clinical rotations were evaluated for data by the primary author and those data verified by communications with the course coordinator or course instructors. statistical analysis was not performed due to the subjective nature of the data. results response rate was 100%. information for 43 rotations (78%) was gathered by completion of the survey of the course coordinator. information for the other 12 rotations was taken from syllabi and verified by the course coordinator or instructors. teaching paradigms are as described in the survey in appendix 1. show-and-tell was the primary instructional paradigm overall (figure 1). rotations were split into large animal clinical rotations (n=27), small animal clinical rotations (n=23), and “other” rotations (n=5; table 2). show-and-tell was the primary instructional paradigm for large animal and small animal clinical rotations, while other teaching paradigms predominated for those rotations that were not strictly species-based. variance in use of all teaching paradigms is demonstrated graphically in figure 2. games described included card and board games, crossword puzzles and jeopardy-type games, and the poultry olympics quiz bowl. a specific example is a board game used to help students think through thoracic anatomy, and to link radiographically visible abnormalities with that anatomy and relate it to disease conditions (http://www.cvm.umn.edu/prod/groups/cvm/@pub/@cvm/@education/documents/asset/adrthoraxposter_ 2015.pdf). assignments and assessments include openor closed-book take-home examinations, review of instructional videos with an associated quiz, student-created examinations, practical examinations of technical skills, practical examinations by direct oversight of independent case management, case studies, and on-line quizzes or cases. specific examples include the small animal theriogenology content covered on the primary care rotation. students are required to assess vaginal cytology images and use their interpretation of those images to manage cases. on the second week of the rotation, students choose a case, are given the signalment, history, and physical examination findings, and are required to generate for later small-group discussion with an instructor a problem list, rule-outs, and diagnostic and treatment plans, with an estimate of cost (https://sites.google.com/a/umn.edu/margaret-v-peggy-root-kustritz/home). examples of “other” teaching paradigms included: • field trips • teaching laboratories or lectures • completion of on-line coursework created by the instructor or drawn from other institutions or from industry • student presentations • student attendance at collegiate or departmental rounds including grand rounds, cytology rounds, and morbidity and mortality rounds • student review of unknowns • student and faculty attendance at outside venues, for example stateor industrysponsored continuing education • language classes clinical theriogenology • volume 7, number 4 • december 2015 448 faculty also described experiences they believed to be unique and of great value to students. students on one service act as primary clinicians with a peer as the secondary clinician and the house officer or faculty member providing oversight. on the biosecurity rotation, students complete a biosecurity audit of a production animal facility, with written report, as they would in practice. on the equine ambulatory service, students create a formulary; this helps them think through what medications they need to have on hand for ambulatory equine practice, including considering costs of medications, deciding which drugs from a given class they wish to use most often, learning inventory control, and managing controlled substances. fourth-year students on the nutrition service assist in teaching a nutrition laboratory in the first-year preventive medicine course. students on that service also act as peer reviewers for each other’s discharge notes before final review by a faculty member. students on the general practice rotation are videotaped during a client encounter with client permission, and those tapes reviewed with the student by a faculty member for communications training. the miracle of birth rotation is a joint venture with the minnesota veterinary medical association where students provide veterinary care and educate the public about cattle, sheep, and pigs as those animals give birth in a supervised facility during the state fair. the dairy education center is a public-private partnership providing students from minnesota and other schools with extensive hands-on training in dairy herd management. finally, the college has partnered with a large local animal humane society to provide students with surgical training and experience in small animal population medicine. the primary barrier described by course coordinators in developing new teaching paradigms was lack of time. faculty members also described lack of knowledge for accessing case materials for teaching through the electronic medical record and imaging archive system and lack of time and knowledge for creating a game or setting up a quiz on-line. faculty expressed concern about lack of continuity between rotations and subsequent inability to build on prior learning when using some teaching paradigms that require demonstration of steps in competence. caseload was described as a constraint, either because it was very high, making it difficult to see all the cases and still find time for teaching, or because it was very low. caseload is also seasonal in some species, necessitating wise scheduling of those clinical experiences for students. course coordinators expressed concern about using teaching animals and the necessary paperwork, expense, and concerns about animal welfare. finally, biosecurity constraints sometimes made it difficult to schedule field trips in a timely fashion. discussion the show-and-tell, or apprenticeship, model is the most commonly used teaching paradigm at this institution. this traditional method of teaching has its advantages as delineated previously and, if used judiciously, is a valuable teaching method. effective clinical teaching in the apprenticeship model requires structured interaction between the student and clinician. many models for case review exist. these include the aunt minnie model of pattern recognition, the one-minute preceptor model, and the snapps model.5,8,9 all of these models require the clinician to ask focused questions of the student, to verify the students’ strengths and weaknesses, and to model their own thought process. variation in types of teaching paradigm used by type of rotation was most evident when comparing the clinical rotations with the “other” rotations, which was expected. the show-and-tell model was used slightly more of the time on small animal clinical rotations than on large animal clinical rotations (49 versus 42%), most likely due to the larger caseload in the small animal hospital. conversely, topic rounds were more commonly employed in large animal than in small animal (20 versus 11%, respectively). evidence-based medicine and critical reading were much more common on the clinical rotations than on the “other” rotations and usually were described as a requirement by faculty for students to demonstrate use of the primary literature for case management. some clinical rotations also held a regular journal club for faculty, house officers, and staff, with some permitting and some requiring student participation. the whole topic of ethics and roles in society was somewhat perplexing to coordinators responding to this survey. they discussed situations as they arose but all assumed that students had received more formalized training earlier in the curriculum and no attempt was made to recreate that on any service. students on services that required or encouraged them to attend departmental morbidity and mortality rounds would hear ethical discussions through that venue. clinical theriogenology • volume 7, number 4 • december 2015449 knowledge of specific barriers preventing r faculty members from considering alternative teaching paradigms is valuable to collegiate administration. a college of veterinary medicine education support group has been formed to provide support for faculty members with an interesting idea who need help finding materials or enacting it, especially if that requires technology support. the veterinary medical center (vmc) and college are continuing to work with practitioners and industry partners to provide students with exposure to individual animals and animal populations for clinical training. attention also is being paid to how improving patient care in the vmc can become a part of the curriculum; teamstepps, an evidence-based teamwork communications system for healthcare professions, is being instituted in the hospital and will be used throughout the curriculum to help build student communication and teamwork skills.10 some of the “other” experiences included use of teaching animals or other laboratory experiences, either to provide more training before students were exposed to client-owned animals or to replace that experience when caseload was too low or did not include specific experiences deemed to be of value. caution must be taken not just to provide clinical skills training without placing it in a larger framework.11 one author describes a professional as someone who can complete a technical task within a larger context of knowledge, leading to growing mastery and lifelong learning.12 use of simulations and models is well described.13-15 models and simulations and those providing assessment should be validated.13,14,16 models and simulations should not take the place of learning necessary hands-on techniques but instead should be stepping-stones on the way to student exposure to authentic experiences.17,18 some non-case-based learning methods are well proven clinical teaching methodologies. for example, review of authentic videotaped client encounters has been well demonstrated to aid students in understanding their verbal and non-verbal communications and in increasing student competence in this discrete set of skills.19-22 good clinical training supports students’ internal motivation and is associated with deep learning, and with improved performance and enhanced well-being of the learner.23 students benefit greatly from appropriate feedback and reflection. feedback can be brief, with short, concrete suggestions; formal, with time set aside to talk through a specific encounter or concern; or major, for example in a final evaluation at the conclusion of a rotation.24 students benefit most from timely feedback that makes clear to them what should be done differently the next time they face that situation, and ideally are provided with an opportunity for them to remediate and practice.25 faculty members often fail to give feedback because they are not observing the learners firsthand, they feel like the student is doing fairly well and do not recognize the value of reinforcing those behaviors, or they fear student reactions to negative feedback, either as an emotional response or as poor instructor evaluations.25 reflection is another valuable component of learning that often is not stressed during clinical training. this may be because time is insufficient or because the clinicians are not capable of being reflective themselves while busy on clinics.3,24 it is valuable to build in time for students to reflect on what they have seen and done in a clinical service and for faculty members to model such reflection. psychological growth within an educational program requires reflection as described by kolb.1,26 a systematic review of the literature defined three things required by students to build internal motivation and provide an optimal clinical learning experience.27 students desire autonomy, or ability to make their own decisions. clinicians can foster this by promoting student participation in case management, by providing different learning approaches, and by giving students choices in their learning. while students desire autonomy, they also greatly value accessibility of instructors as they encounter individual problems.27 students also desire competence, or capability of performing tasks that are challenging but not unreachable. clinicians can foster this by providing structured learning opportunities that provide challenge, by valuing student work, and by providing feedback. finally, students desire relatedness, or a sense of belonging to their professional community. clinicians can foster this by respecting students, acknowledging their own mistakes or concerns, providing emotional support, and by being an open-minded and accepting member of the healthcare team of which they and the student are members.23 students generally rate their clinical instructors more highly than those instructors rate themselves.28 specific attributes students find most compelling in a clinical instructor include knowledge, clinical theriogenology • volume 7, number 4 • december 2015 450 enthusiasm, and respect for student independence.29 good clinical teachers do not view teaching as an opportunity to display their own knowledge but rather as a process of identifying where students need help and creating interventions that permit them to discover information themselves.30 clinicians should be cautious when self-assessing their shortcomings as a clinical instructor and should recognize that students highly value their passion and knowledge of their subject. conclusion show-and-tell is the primary teaching methodology employed on clinical rotations at the university of minnesota but many other teaching methods are used. students benefit from being presented with a variety of learning experiences. clinicians struggle to find time to enhance clinical teaching while managing caseload. use of proven teaching methodologies and provision of support by the college should lessen faculty concerns while improving clinical experiences for the students. references 1. kolb da. experiential learning: experience as the source of learning and development. englewood cliffs (nj): prentice-hall, 1984. 2. lane ea: problem-based learning in veterinary education. j vet med educ 2008;35:631-636. 3. magnier km, wang r, dale vhm, et al: challenges and responsibilities of clinical teachers in the workplace: an ethnographic approach. j vet med educ 2014;41:155-161. 4. ramani s, leinster s: amee guide no. 34: teaching in the clinical environment. med teach 2008;30:347-364. 5. smith jr, lane if: making the most of five minutes: the clinical teaching moment. j vet med educ 2015. doi: 10.3138/jvme.0015-004r. 6. hays r: teaching and learning in clinical settings. in: hays r, editor. teaching and learning in clinical settings. oxford: radcliffe publishing, 2006:51-79. 7. magnier k, wang r, dale vhm, et al: enhancing clinical learning in the workplace: a qualitative study. vet rec 2011;169:682-686. 8. neher jo, stevens ng: the one-minute preceptor: shaping the teaching conversation. fam med 2003;35:391-393. 9. cunningham as, blatt sd, fuller pg, et al: the art of precepting: socrates or aunt millie? arch pediatr adolesc med 1999;153:114-116. 10. united states department of health and human services: team stepps. available at: teamstepps.ahrq.gov. accessed 08-19-15. 11. kneebone r, baillie s: contextualized simulation and procedural skills: a view from medical education. j vet med educ 2008;35:595-598. 12. schoen da. educating the reflective practitioner. san francisco: jossey-bass, 1987. 13. nibblett bmd, mauldin pereira m, williamson ja, et al: validation of a model for teaching canine fundoscopy. j vet med educ 2015;42:133-139. 14. capile kv, campos gmb, stedile r, et al: canine prostate palpation simulator as a teaching tool in veterinary education. j vet med educ 2015;42:146-150. 15. root kustritz mv: simulations in veterinary education. clin therio 2014;6:607-609. 16. artemiou e, adams cl, hecker kg, et al: standardised clients as assessors in a veterinary communication osce: a reliability and validity study. vet rec 2014. doi: 10.1136/vr.102633. 17. stewart sm, dowers kl, cerda jr, et al: microscope use in clinical veterinary practice and potential implications for veterinary school curricula. j vet med educ 2014;41:331-336. 18. sachana m, theodoridis a, cortinovis c, et al: student perspectives on the use of alternative methods for teaching in veterinary faculties. atla 2014;42:223-233. 19. paskins z, mchugh g, hassell ab: getting under the skin of the primary care consultation using video stimulated recall: a systematic review. bmc med res methodol 2014;14:1-18. 20. latham ce, morris a: effects of formal training in communication skills on the ability of veterinary students to communicate with clients. vet rec 2007;160:181-186. 21. hafen m, siquiera drake aa, rush br, et al: using authentic client interactions in communication skills training: predictors of proficiency. j vet med educ 2013;40:318-326. 22. meehan mp, menniti mf: final-year veterinary students’ perceptions of their communication competencies and a communication skills training program delivered in a primary care setting and based on kolb’s experiential learning theory. j vet med educ 2014;41:371-383. 23. orsini c, evans p, jerez o: how to encourage intrinsic motivation in the clinical teaching environment?: a systematic review from the self-determination theory. j educ eval health prof 2015;12:1-10. 24. branch wt, paranjape a: feedback and reflection: teaching methods for clinical settings. acad med 2002;77:11851188. 25. ende j: feedback in clinical medical education. j am med assoc 1983;250:777-781. clinical theriogenology • volume 7, number 4 • december 2015451 26. sprinthall na: counseling and social role taking: promoting moral and ego development. in: rest jr, editor. moral development in the professions: psychology and applied ethics. hillsdale (nj): lawrence erlbaum associates; 1994. p. 55-100. 27. wood akw, lublin jr, hoffmann kl, et al: alternatives for improving veterinary medical students’ learning of clinical sonography. vet radiol ultrasound 2000;41:433-436. 28. windish dm, knight am, wright sm: clinician-teachers’ self-assessment versus learners’ perceptions. j gen intern med 2004;19:554-557. 29. bolt dm, witte th, lygo-baker s: the complex role of veterinary clinical teachers: how is their role perceived and what is expected of them? j vet med educ 2010;37:388-394. 30. ursano am, kartheiser ph, ursano rj: the teaching alliance: a perspective on the good teacher and effective learning. psychiatry 2007;70:187-194. figure 1. teaching methodologies for all rotations (percentage of total) 0 5 10 15 20 25 30 35 40 45 50 clinical theriogenology • volume 7, number 4 • december 2015 452 figure 2. teaching methodologies by type of rotation teaching paradigm variation by type of rotation show-and-tell topic rounds evidence-based medicine / critical reading 0 20 40 60 la clinical sa clinical other 0 5 10 15 20 25 la clinical sa clinical other 0 2 4 6 8 10 la clinical sa clinical other clinical theriogenology • volume 7, number 4 • december 2015453 roles in society / ethics case rounds games 0 1 2 3 4 5 la clinical sa clinical other 0 10 20 30 40 la clinical sa clinical other 0 1 2 3 4 la clinical sa clinical other clinical theriogenology • volume 7, number 4 • december 2015 454 assignments / assessments other table 1. rotation requirements by track rotation requirements by track small animal food animal equine mixed animal / interdisciplinary research orientation to clinics orientation to clinics orientation to clinics orientation to clinics orientation to clinics 4 sam (1 sam-a + 3 sam-b) 1 lam + 3 lam or dofc or swppdxt or camelid or feedlot or smrum or raois with ambulatory service 3 lam + 1 lam or sam or raoi 4 medicines 4 medicines 2 surgeries total: 1 sas + 1 esas or shelter or shelter raoi 2 las or 1 las + 1 bov sx or raoi 2 las 2 surgeries 2 surgeries 1 public health 1 public health 1 public health 1 public health 1 public health 1 necropsy 1 necropsy 1 necropsy 1 necropsy 1 necropsy 1 anesthesiology 1 anesthesiology 1 anesthesiology 1 anesthesiology 1 anesthesiology 1 clinical labs 1 clinical labs 1 clinical labs 1 clinical labs 1 clinical labs 1 radiology 1 radiology or 1 radiology or 1 radiology 1 radiology 0 5 10 15 la clinical sa clinical other 0 20 40 60 la clinical sa clinical other clinical theriogenology • volume 7, number 4 • december 2015455 imaging raoi radiology raoi 2 gp + 1 dent + 1 ecc + 1 derm or derm raoi dofc or swhpi + 6 fa rotations 1 elamp + 1 eqamb + eqthi + 3 equine rotations 1 gp + 1 ecc + 4 fa, eq or sa rotations 3-5 externships*/raois 3-5 externships*/raois 3-5 externships*/raois 3-5 externships*†/raois 6-8 elective rotations 4-6 elective rotations 4-6 elective rotations 4-6 elective rotations 16 elective rotations, externships*, or raois 28 total 28 total 28 total 28 total 28 total * one externship must be a general practice-type experience in a revenue-generating for-profit facility † an extra 2 weeks of externship may be taken in place of one elective rotation with mentor approval for externships requiring longer blocks of attendance bov sx = bovine surgery, dent = small animal dentistry, derm = comparative dermatology, dofc = dairy on-farm campus (dec), ecc = small animal emergency/critical care, elamp = equine lameness and podiatry, eq = equine, eqamb = equine ambulatory, eqthi = introductory equine theriogenology, esas = elective small animal surgery, fa = food animal, gp = general practice, lam = large animal medicine, las = large animal surgery, raoi = rotation at an outside institution, sa = small animal, sam = small animal medicine, sas = small animal surgery, smrum = small ruminant, swhpi = introduction to swine health and production, swppdxt = swine diseases, diagnostics, therapeutics, and prevention table 2. clinical rotations large animal clinical rotations small animal clinical rotations other • beef production cow-calf • biosecurity for food animals • bovine surgery • camelid • dairy on-farm clinic • dairy production medicine i-iv • dairy theriogenology palpation / podiatry • equine ambulatory • equine dentistry and nutrition • equine lameness and podiatry • equine sports medicine • equine theriogenology advanced • equine theriogenology intro • feedlot • food animal disease and diagnostics • fresh doe and goat management • large animal medicine • large animal surgery • minnesota zoo • miracle of birth • mixed animal radiology • overview of dairy production medicine • poultry medicine clerkship • small ruminant • swine diseases • swine health and production advanced • swine health and production intro • acupuncture • advanced sa theriogenology • banfield • behavior • cardiology • companion bird • comparative anesthesiology • dermatology • emergency critical care • exotic animal medicine • general practice • neurology • nutrition • oncology • ophthalmology • raptor • sa dentistry • sa medicine a and b • sa radiology • sa rehabilitation • sa surgery • sa ultrasound • umn spay/neuter • advanced veterinary public health • clinical laboratories • exotic animal necropsy • necropsy • public health clinical theriogenology • volume 7, number 4 • december 2015 456 appendix 1. survey instrument clinical ng at umn cvm 2015 please complete a separate survey for each rotation you coordinate. for each of the listed teaching paradigms, please estimate the percentage of time over two weeks spent on the activity by the students. the "comments" section gives you a place to describe those activities in detail; this is helpful particularly if you think they work very well or if you see ways to improve use of this activity. 1. rotation acupuncture advanced veterinary public health comparative anesthesiology exotic animal medicine exotic animal necropsy laboratory medicine minnesota zoo necropsy ophthalmology public health mixed radiology sa radiology equine dentistry and nutrition equine lameness and podiatry equine sports medicine equine theriogenology advanced equine theriogenology intro equine ambulatory biosecurity for fa bovine surgery beef production medicine cow calf camelid dairy on farm clinic dairy production medicine 1 dairy production medicine 2 dairy production medicine 3 dairy production medicine 4 dairy theriogenology palpation / podiatry clinical theriogenology • volume 7, number 4 • december 2015457 fresh doe and goat management fa disease and diagnostics feedlot miracle of birth overview of dairy production medicine poultry medicine clerkship swine disease small ruminants swine health and production, introductory swine health and production, advanced la medicine la surgery advanced sa theriogenology banfield behavior cardiology companion birds dermatology emergency critical care general practice neurology nutrition oncology raptor sa dentistry sa medicine a sa medicine b sa rehabilitation sa surgery sa ultrasound umn spay neuter clinical theriogenology • volume 7, number 4 • december 2015 458 2. show-and-tell / apprenticeship = student does some hands-on case management, much observation of faculty and house officers, relatively little independent decision-making percentage time spent over two weeks comments 3. topic rounds = discussion between students and faculty on a given topic, chosen by the faculty for completeness or by the students for interest percentage time spent over two weeks comments 4. evidence-based medicine / critical reading = faculty-led discussion of current literature and value in case management percentage time spent over two weeks comments 5. roles in society / ethics = discussion of ethical concerns about specific cases, larger role of veterinarians in health provision and in society percentage time spent over two weeks comments 6. case rounds = daily review of cases in the hospital or facility, discussion between students and faculty regarding case management percentage time spent over two weeks comments 7. games = games used for education, played independently or in groups, or with faculty facilitation percentage time spent over two weeks comments 8. assignments / assessments / examinations = independent study work that is structured and submitted for evaluation / grading. evaluations for self-assessment or submitted for grading. percentage time spent over two weeks comments 9. other (please describe) = required attendance at grand rounds, cytology rounds, m&m rounds, peer teaching, specific projects, etc. percentage time spent over two weeks comments 10. please describe one activity that is particularly successful on your rotation and one activity that you hope to add or improve upon in the next 1-2 years (include information about barriers to implementation if applicable). clinical theriogenology • volume 7, number 4 • december 2015459 clinical theriogenology • volume 7, number 4 • december 2015 460 omniblank: a case of testicular torsion in an intact cane corso a case of testicular torsion in an intact cane corso nichole whitfill, aime johnson, robyn wilborn, marty greera college of veterinary medicine, auburn university, auburn, al aveterinary village, lomira, wi a 1.5 year old intact male cane corso presented with bilateral hind limb lameness of 3 days duration and scrotal swelling of 1.5 days duration. on examination, moderate scrotal swelling was appreciable and right testis was twice the size of left. increased turgidity and pain were present on palpation of right testis, whereas left testis was normal on palpation. differential diagnoses included testicular torsion, trauma, brucellosis, and neoplasia. based on clinical presentation and the acute nature of symptoms in this case, a testicular torsion was placed at top of the differential list. canine testicular torsion most commonly results from cryptorchidism with associated neoplasia. testicular torsions of scrotal testes are less common.1 regardless of origin, rotation of testis compromises venous return and leads to interstitial hemorrhage, local ischemia, testicular infarction, and eventual necrosis of the gonad.2 clinical signs of testicular torsion can include lethargy, stiffness or lameness of the hind limbs, pyrexia, pain, enlarged testis, and visible scrotal swelling.2,3 ultrasonography with doppler is useful in diagnosing testicular torsion via identification of compromised blood flow to affected testis.1-3 client expressed no interest in preserving the patient’s breeding capacity and a bilateral orchiectomy was performed. right testis was enlarged and dark purple, suggestive of congestion, ischemia, and edema. gross appearance of left testis was normal. histopathology of right testis had marked congestion with hemorrhage, edema, and thrombosis within spermatic cord to the level of epididymis with marked interstitial edema in seminiferous tubules. surgical recovery and healing were uneventful and the patient achieved complete resolution of clinical signs. this case demonstrated the diagnosis and treatment of scrotal testicular torsion, considered a rare event in dogs. keywords: canine, testicle, torsion, intact, scrotum, swelling references 1. young, ac: two cases of intrascrotal torsion of a normal testicle. journal of small animal practice 1979; 20:229-231. 2. viliotti, tatiane alexandre azevedo, et al: testicular torsion in the scrotum of a young canine. acta scientiae veterinariae 2018;46:6. 3. boothe hw: diseases of the testes and epididymides. in: handbook of small animal practice, 5th edition, philadelphia: saunders elsevier; 2008. p. 575. 505 clinical theriogenology • volume 11, number 3 • september 2019 506clinical theriogenology • volume 11, number 3 • september 2019 omniblank: introduction pregnancy loss reduces reproductive efficiency in cattle.1-3 implications of this loss are not only technical but also economical.4,5 additionally, some agents are zoonotic and hence a public health concern. the estimated annual cost of pregnancy loss was ~ $1.4b in the us6 and ~ £250m in the uk.7 the cost of each case of abortion in the us was $640,8 averaging $600 in the us. average calving rate in dairy cows for each insemination is ~ 50%, and in large commercial dairy operations in the us it is frequently 30 40% or lower.10,11 since fertility rate in dairy cattle has declined considerably, each pregnancy is now even more valuable.11 every factor that affects the wellbeing of a pregnant female is a potential cause of pregnancy loss, acting either directly or indirectly on the conceptus. pregnancy status is a clinically dynamic condition, with an inherent risk of pregnancy loss. multiple risk factors are associated with pregnancy loss (time of pregnancy, twin pregnancy, body condition score change, lameness, mastitis, medications during pregnancy, and vaccinations). the objective of this paper is to review the common causes of pregnancy loss in cattle, with an emphasis on infectious causes and to supplement information provided in the proceedings.12-14 prevalence in 2 us studies,15,16 a cause for abortion was detected in 23.3 pregnancy loss in cattle andrés de la concha-bermejillo,a juan romanob atexas a&m veterinary medical diagnostic laboratory blarge animal clinical sciences, college of veterinary medicine & biomedical sciences texas a&m university, college station, tx abstract pregnancy loss in cattle has a major economic impact on livestock producers. additionally, some agents are zoonotic and therefore are also a public health concern. determining the cause of pregnancy loss in cattle is often unsuccessful. in only ~ 30% of midand late-term pregnancy losses is a specific cause identified. pregnancy loss is higher during the embryonic period compared to the fetal period, and causes are noninfectious or infectious. noninfectious causes include genetic defects (arthrogryposis multiplex, bovine arachnomelia syndrome, bovine citrullinemia, and chromosomal abnormalities), toxic plants and toxins (lupine, hemlock, locoweed, pine needles, ergot alkaloids, certain molds, and nitrates), nutritional deficiencies (vitamin a and e, selenium, and thyroid hormones), environmental factors (heat stress), and medications (prostaglandin f2α and glucocorticoids). infectious causes include bacterial (brucellosis, chlamydiosis, coxiellosis, foothill abortion, leptospirosis, listeriosis, and ureaplasma diversum), fungal (aspergillus fumigatus), viral (bluetongue virus, bovine viral diarrhea, cache valley virus, and infectious bovine rhinotracheitis), and protozoal (neosporosis and trichomoniasis). keywords: cattle, abortion diseases, pregnancy loss, embryonic mortality and 35.3% of cases out of a total of 3,812 and 2,544 cases, respectively. from 1983 to 2001, 1618 aborted fetuses from northcentral us farms were examined and a specific cause was reported in 592 cases (36.59%).17 in australia, the cause of pregnancy loss was detected in 37% of 265 case submissions18 and in canada the cause was detected in 23% of 227 cases.19 in england, the cause of abortion remained unknown in several cases.20 two aspects are noteworthy; only aborted fetuses of a recognizable size are submitted to laboratories; hence most of the fetuses are > 4 5 months of age. consequently, reports are biased toward the latter half or third of pregnancy.23 secondly, the organism isolated may not have been the cause of abortion; especially, if the sample was highly contaminated as is often the case with fecal contamination and the consequent isolation of coliform bacteria. noninfectious causes of pregnancy loss noninfectious causes of pregnancy loss include genetic defects (arthrogryposis multiplex, bovine arachnomelia syndrome, bovine citrullinemia, and chromosomal abnormalities), toxic plants and toxins (lupine, hemlock, locoweed, pine needles, ergot alkaloids, certain molds, and nitrates), nutritional deficiencies (vitamin a and e, selenium, and thyroid hormones), environmental factors (heat stress), and medications (prostaglandin f2α and glucocorticoids). clinical theriogenology 2021; 13: 167 infectious causes of pregnancy loss infectious causes of pregnancy loss include bacterial (brucellosis, chlamydiosis, coxiellosis, foothill abortion, leptospirosis, listeriosis, and ureaplasma diversum), fungal (aspergillus fumigatus), viral (bluetongue virus, bovine viral diarrhea, cache valley virus, and infectious bovine rhinotracheitis), and protozoal (neosporosis and trichomoniasis) causes. some viral agents (schmallenberg virus, akabane virus, and aino virus) are exotic to north america.24 risk factors associated with noninfectious causes period of pregnancy pregnancy loss decreased as the interval between breeding and pregnancy diagnosis increased.25,26 in general, 4 of 5 lactating dairy females diagnosed pregnant at ~ day 30 had a viable fetus at the end of first trimester.25,27,28 herd size influenced both frequency of veterinary visits and the timing of pregnancy diagnosis with larger herds generally having more frequent assessments of reproductive performance. as frequency of farm visits increased, the average days pregnant at diagnosis decreased,29 therefore, the likelihood of finding pregnancy loss is higher in larger herds compared to smaller herds. cows first diagnosed pregnant at day < 41 were less likely to have a calf than cows diagnosed later.30 pregnancy loss was higher when pregnancy diagnosis was performed before day 48 of pregnancy.29 recent studies, involving only lactating cows reported an increase in pregnancy loss when transrectal ultrasonography was used for pregnancy diagnosis. this could be due to earlier and perhaps more accurate pregnancy diagnosis compared to transrectal palpation. in california, embryo/fetal mortality was 19% when diagnosed between days 28 90.28 in texas dairy farms, embryo/fetal mortality was 19.2% when diagnosed at ~ days 30 -120.25 herds from central utah and california that used fixed time artificial insemination had 24% pregnancy loss between days 28 98.27 risk of pregnancy loss is higher during the embryonic period compared to the fetal period. therefore, every female diagnosed pregnant during the embryonic period should have a follow-up examination during the fetal period to verify pregnancy status. lactation pregnancy loss was lower in heifers than cows25,31-32 however, age of cow was not a risk factor for pregnancy loss.8 a gradual increase in pregnancy loss, as estimated by progesterone profiles, was observed, particularly beyond the fourth lactation.34 in lactating cows, the risk of pregnancy loss was similar among parities.32 higher rates of late embryo/fetus mortality in older compared to younger females have been reported in many species.34-37 cited reason include reduced oocyte fertilization capability, and the inability of the uterus to provide adequate gestational support.35,36 number of embryos the number of embryos increased the risk of pregnancy loss.25,33,38 twin pregnancies had 2.5 3 times higher possibility of pregnancy loss compared to a singleton.25,33 the majority of embryo/fetal mortality observed in twins was type i characterized by positive fetal membrane slip, embryo/fetal degeneration, a functional corpus luteum, and prolonged uterine clearance.25,39 reasons for the risk of higher pregnancy loss in twin pregnancy is unknown. however, competition between embryos or fetuses for nutrition, space, or both could account for some embryo/fetal losses. in singleton pregnancies, higher pregnancy loss occurred during the embryonic period compared to the fetal period.25 however, for twin pregnancies, pregnancy loss during the first 4 months of pregnancy was similar at each evaluation.25 body condition score a 1-point reduction in body condition score from calving to 30 days postpartum increased pregnancy loss in a subsequent pregnancy by 2.4-fold.40 in dairy cows, under pasture-based milk production systems, cows that lost body condition during days 28 56 of pregnancy had a higher rate (11.6%) of embryonic loss compared to cows that either maintained (4.7%) or gained (5.7%) body condition during this period.41 furthermore, cattle with lower body condition scores (< 2.5; scale 1 5) at pregnancy diagnosis had higher chances of pregnancy loss than those with higher body condition scores (romano, unpublished observations). bull sire had a substantial effect on the rate of embryonic death.42 a higher incidence of pregnancy loss was observed in cows inseminated with semen from 1 of 6 bulls. this particular bull increased the rate of pregnancy loss by 3.4 times.40 increased risks of abortion were associated with 8 sires out of 233 (odds ratios of mates to abortion ranged from 1.9 to 3.9).32 sire affected pregnancy loss and therefore selection of bulls according to this criterion might result in higher calving rates in lactating holstein cows.43 artificial insemination insemination of pregnant cows resulted in pregnancy loss. two factors might be associated with this circumstance; observation of primary signs of estrus (standing to be mounted) in pregnant cows and incorrect estrus detection. fifteen percent of pregnant females had signs of estrus during the first half of pregnancy.44-47accuracy of estrus detection ranged from 3 to 26% in females (had higher concentrations of p4 at ai),48-54 and 60% farms faced this problem.49,50 confirmed pregnant females after insemination had conceptus death and consequent pregnancy loss55-59 with abortion some weeks later.60 clinical theriogenology 2021; 13: 168 transrectal palpation transrectal palpation continues to be the most frequent method for pregnancy diagnosis.61-64 although 100 years have passed from the first report,65 its impact on safety and accuracy of conceptus detection are still not clearly established.62-64 besides pregnancy diagnosis, this technique was also used to rupture the amniotic sac or crush the embryo or fetus in unwanted pregnancies prior to the availability of prostaglandin f2α. 37,60,66 safety of the conceptus remains controversial;64 pregnancy loss was not reported by some4,67,68 whereas others30,69-73 suggested increases. palpation of the allantochorion or amniotic sac during the embryonic period did not increase pregnancy loss as confirmed by subsequent transrectal ultrasonography in the fetal period.64,74,75 however, calving rates or clinical status of newborn calves were not reported. amniotic sac palpation during the embryonic period (until day 45) for pregnancy diagnosis,76 especially between 36 and 42 days, increased the risk of atresia coli/jejuni in newborn calves.77-82 in atresia coli/ jejuni, a section of the large bowel or jejunum is absent, resulting in a blind-ending intestine. this congenital condition is lethal, and surgical correction is the only effective treatment.83-87 late embryonic or early fetal period palpation of the allantochorion membrane or amniotic sac did not increase pregnancy loss nor affected calving rates or incidence of calves with congenital abnormalities.25,26,88 false-negative diagnoses of pregnancy increased the probability of culling or submission to an immediate or delayed estrus synchronization (protocols use at least 1 dose of prostaglandin f2α or its analogs) treatment protocol.89,90 prostaglandin f2α is a potent luteolytic agent that induces immediate abortion during early stages of pregnancy.91,92 iatrogenic abortion will most often go undetected25 yet will still have a negative effect on herd economics.9,93 a false-positive diagnosis resulted in an animal coming into estrus or not calving at the expected time, ultimately, increasing the probability of culling. mastitis mastitis increased the risk of pregnancy loss.94-96 exposure to clinical mastitis during the first 45 days of pregnancy was associated with loss of pregnancy during the next 90 days.97 pregnancy loss was higher (9.7 11.8%) in cows that were affected with clinical mastitis compared to unaffected cows (5.8%).95 defining a clear risk period for pregnancy loss in clinical mastitis is critical to determine the true impact.98 after controlling for breeding type and lameness, the odds ratio for pregnancy loss was 2.21 times higher (95% ci = 1.01 4.83) in cows that had clinical mastitis.99 higher rates of pregnancy loss were associated with the occurrence of clinical mastitis (hazard ratio =1.57) in pasture-fed dairy cattle.100 furthermore, subclinical mastitis prior to artificial insemination was associated with subsequent pregnancy loss during the late embryonic period.96 odds ratio for pregnancy loss was 20% higher in cows affected with subclinical mastitis. the impact of mastitis on pregnancy loss was higher in older cows (parity ≥ 3).101 cows with a linear somatic cell count (score > 4.5 before ai) were twice more likely to lose the embryo from 28 to 41 days.96 inbreeding pregnancy loss was higher in inbred systems in heifers (2.5%) and cows (13.0%).98 at 150 days, inbred dams had a higher pregnancy loss (28.4 versus 19.2%).42 inbreeding increased the risk of deleterious lethal disorders (e.g. bovine leukocyte adhesion deficiency),103 or uridine monophosphate synthase deficiency,104 recessive deleterious haplotypes,105 and accumulation or interaction of genes with small negative effects on fertility.106 noninfectious causes of pregnancy loss genetic diseases with increased use of artificial insemination and reproductive techniques a growing concern worldwide is the emergence and widespread dissemination of hereditary diseases. a nonsense mutation in the apaf1 gene created a lethal effect (holstein haplotype1) responsible for 525,000 spontaneous abortions worldwide over the past 35 years, accounting for ~ $420m in losses. this disease-associated haplotype was traced to the ancestor holstein sire pawnee farm arlinda chief born in 1962, a bull considered the second most influential sire in the holstein breed history.107 arthrogryposis multiplex congenita (commonly known as curly calf syndrome) is a lethal autosomal recessive genetic disorder of aberdeen angus, originating in the bull rito 9j9 of b156 7t26 and distributed widely through the bull gar precision 1680. the condition was characterized by fetal musculoskeletal malformations including severe muscular atrophy, arthogryposis, scoliosis, and torticollis.108 arachnomelia syndrome is an autosomal recessive inherited disease in cattle. affected calves have skeletal malformations mainly affecting legs, spinal column and skull, and die around birth. the disease has been reported in holstein friesian, red holstein, and simmental cattle.109 although a description of bovine congenital abnormalities is beyond the scope of this paper, heritable bovine fetal abnormalities were reviewed.110. toxic plants and toxins pregnancy loss and teratogenesis were attributed to numerous plants and toxins. however, with only limited studies, and with methodological limitations and small sample sizes, results must be interpreted with caution. some toxic plants contain compounds that might cause death, reproductive problems, teratogenesis, and neurological or digestive disorders. late-term abortion and fetal membrane clinical theriogenology 2021; 13: 169 retention were attributed to consumption of juniper (juniperus communis). chronic ingestion of some species of locoweed (oxytropis and astragalus) during pregnancy resulted in joint malformation and abortion. consumption of pinus ponderosa needles caused third trimester abortion and fetal membrane retention. perennial broomweed (gutierrezia microcephala) consumption caused abortion, premature delivery, birth of weak offspring, and fetal membrane retention.111 as the amount of nitrate needed to cause abortion is close to the lethal dose, it is uncommon to have abortions without some fatalities. nitrate toxicity occurs when high nitrate concentrations in the feed overwhelm the capacity of the animal’s digestive system to the extent that the rate of conversion of nitrate to nitrite is faster than the conversion of nitrite to ammonia that is incorporated into amino acids and proteins.112 mycotoxins are secondary metabolites of fungi that contaminate feed and have substantial negative impacts on animal health and productivity. feed contamination of zearalenone, an important mycotoxin produced by fungi of fusarium genera, caused hepatotoxicity, hematotoxicity, immunotoxicity, and genotoxicity. zearalenone and its major metabolites α-zearalenol and β-zearalenol, mimic17β-estradiol and elicit substantial estrogenic activity. although cattle are resistant, increased consumption of zearalenone was associated with infertility, enlargement of the mammary gland, reduced milk production, vaginitis, and early pregnancy loss.112 vitamin e and selenium deficiency congenital nutritional muscular dystrophy caused by vitamin e and selenium deficiency is uncommon but has been reported in beef cattle.114 affected calves were in lateral recumbency, unable to move at birth, and with no suckling reflex. serum creatine kinase and aspartate aminotransferase were elevated with lower vitamin e and selenium concentrations. affected calves responded adequately to supportive therapy and with vitamin e and selenium treatment. animals that died had pale to white skeletal muscles. histological examination revealed swollen skeletal muscle fibers with fragmented sarcoplasm and mineralization. vitamin a deficiency deficiency during pregnancy was suspected in cases of perinatal calf mortalities.115 birth of hypovitaminosis a calves to animals fed a deficient ration is less well documented; calves delivered were dead or weak, lacked coordination and were blind.116 medications prostaglandin f2α treatment during the first 4 5 months of pregnancy caused luteolysis, resulting in immediate abortion.91,92 therefore, an accurate diagnosis of nonpregnancy is essential, if the female is submitted to an estrus synchronization protocol that used prostaglandin f2α. glucocorticoids use during pregnancy have an inherent possibility to provoke abortion. use of corticosteroids, depending on the dose, duration, and specified steroid, might result in inhibition of endogenous steroid production following drug withdrawal. use of glucocorticoids in smaller pregnant females was associated with cleft palate. use of sodium iodine and pregnancy loss was inconclusive;117,118 therefore, further investigation is necessary.119 environmental heat stress had major effects on fertility and embryonic survival in lactating dairy cows.120,121 compromised endometrial function and secretory activity, smaller follicles, and suppressed dominance of large follicle were noticed.122 decreased serum estradiol concentrations, decreased plasma concentrations of lh, and decreased progesterone secretion were documented. furthermore, oocyte quality, embryo development, and embryo survival were impaired by heat stress. oocyte and earlier stage embryos were highly sensitive, whereas day-3 or older embryos appeared resistant.120,123 absence of heat stress had similar results for artificial insemination and embryotransfer.124 higher pregnancy loss was observed in dairy cattle if heat stress was experienced at artificial insemination.125 day 7 embryos without heat stress were more capable of establishing pregnancy. infectious causes of pregnancy loss brucellosis brucellosis is a zoonotic disease (notifiable in us) caused by several brucella species. brucellosis in cattle is caused by brucella abortus and could result in abortion, birth of weak calves, retention of fetal membranes and decreased milk production. abortion generally occurred in the last trimester of pregnancy.126 bison and cervids also are susceptible.127 currently in the us, individual states are designated brucellosis free when none of their cattle are infected for 12 consecutive months under an active surveillance program.128 as of august 1, 2020, all states are considered free of cattle brucellosis (aphis. usda.gov); however, presence of infected free-ranging bison and elk in the greater yellowstone area, yellowstone national park and grand teton national park threatens surrounding states’ brucellosis status. brucellosis is transmitted to susceptible animals mainly by direct contact with infected animals or by fomites. fluids and fetal membranes from infected fetuses and vaginal discharges from cows that have aborted contain large numbers of organisms, an important source of infection. infected wildlife could transmit the disease to domestic livestock. fetal membranes might have macroscopic evidence of inflammation, hard to distinguish from inflammation caused by other bacteria or fungi. gross lesions are not uniform throughout clinical theriogenology 2021; 13: 170 the fetal membranes. some cotyledons are swollen and necrotic and others might have mild lesions or appear normal. intercotyledonary spaces might have extensive ill-defined thick areas of yellow discoloration. histologically, there is sloughing of chorionic epithelial cells in the intercotyledonary spaces and infiltration of large numbers of mononuclear leukocytes and some neutrophils in stroma. large numbers of bacteria are observed in the cytoplasm of chorionic epithelial cells and in the exudate. aborted fetuses generally exhibit advanced autolysis characterized by extensive blood-tinged subcutaneous edema and blood-tinged fluid in the thoracic and abdominal cavities. fetal lung might have microscopic evidence of bronchopneumonia characterized by infiltration of mononuclear leukocytes and some mature and immature neutrophils. interlobular septae are expanded with edema and leukocytes. organisms can be demonstrated in tissues, secretions, and exudates using modified ziehl-neelsen staining.130 diagnosis of brucellosis is by isolation of the organism from fetal membranes, fetal tissues, and stomach content. brucella polymerase chain reaction is generally used to identify the organism in cultures and less often directly from tissue of infected fetuses. brucella antigen tests (rose bengal test and buffered plate agglutination test), complement fixation, indirect or competitive enzyme-linked immunosorbent assay (elisa) and the fluorescence polarization assay are the most common serological tests that demonstrate the presence of antibodies in maternal serum. antibodies in milk are detected by elisa test and the brucella milk ring test.127 bovine brucellosis can be prevented by vaccination with rb51 vaccine, a live vaccine that should not be used in pregnant animals. heifers should be vaccinated between 4 and 12 months of age. adult cattle might be vaccinated in selected high-risk situations.131 the vaccine is not 100% effective and usually protects between 70 80% of vaccinated animals. b. abortus strain rb51, is a rough rifampicin-resistant strain that lacks the expression of lipopolysaccharide o-side chain and does not induce antibodies against this chain detectable by routine serological tests, therefore, allowing vaccination and test-andslaughter policies to be performed at any age.132 campylobacteriosis campylobacter spp. are important animal pathogens and opportunistic human pathogens. several species and subspecies of campylobacter cause pregnancy loss and infertility in ruminants.133 mammal-associated campylobacter fetus is comprised of 2 subspecies: c. fetus subsp. venerealis and c. fetus subsp. fetus, with both being well-known causes of reproductive failure in ruminants.134 c. fetus subsp. venerealis causes bovine genital campylobacteriosis, characterized by infertility and abortion. chlamydiosis chlamydia abortus, formerly known as chlamydophila abortus or chlamydia psittaci serovar 1, is an obligate intracellular organism that causes abortions in sheep, goats, and occasionally in deer, cattle or llamas. subclinical infection with c. abortus might severely affect bovine herd health and production.135 coxiellosis coxiellosis, often referred to as q (query) fever, is a highly infectious zoonotic disease caused by the intracellular bacterium coxiella burnetii that primarily affects goats and sheep, and less often cattle.136-138 other species less commonly affected include dogs, cats, rabbits, a variety of wild and domestic mammals, and birds. in cattle, abortion by c. burnetti is generally sporadic and only rarely occurs in clusters. fetal membranes might have diffuse reddening of the cotyledons and loss of translucency of the intercotyledonary areas; however, in some cases, there are no gross lesions. microscopically, there is fibrinonecrotic placentitis with large numbers of intratrophoblastic gram-negative coccobacilli.139 foothill abortion foothill abortion, also known as epizootic bovine abortion, is a tick-borne disease caused by the bacterium pajaroellobacter abortibovis, transmitted by the bite of the pajaroello tick. it is a substantial problem for beef producers in the foothills and mountainous regions of california, northern nevada and southern oregon. abortion or birth of weak offspring occurs only in pregnant naive heifers or cows that are introduced to endemic areas 100 145 days before calving. abortion occurs in the last trimester of pregnancy. it is common for affected fetuses to induce their own delivery, but often die during calving or shortly after birth.140 some of the aborted fetuses have severe abdominal distention caused by ascites. there is severe, generalized fetal lymphadenomegaly and splenomegaly. numerous petechial hemorrhages are present in mucous membranes; the thymus is generally small with areas of severe hemorrhage and edema; and the liver is swollen and nodular. multifocal, areas of pale discoloration are observed in many organs but especially in the heart and kidney. the most characteristic microscopic lesion is inflammation of the thymus with attenuation of the cortex, loss of thymocytes and diffuse infiltration of macrophages in the medulla and septae. microscopic lesions in the liver consist of distention of the central veins and attenuation of the hepatic plates. there are large areas of hepatic granulomatous inflammation. alveolar walls of the lung are distended with histiocytes. in the brain, there is histiocytic inflammation of the meninges and multifocal areas of vasculitis.141,142 leptospirosis leptospirosis is a global zoonosis that causes significant economic losses for cattle production. leptospirosis is an important cause of abortion in cattle, as well as septicemia, hepatitis, nephritis, clinical theriogenology 2021; 13: 171 and meningitis, particularly, in young animals. leptospirosis is caused by > 260 antigenically distinct serovars belonging to 25 serogroups grouped in 9 pathogenic species, 5 intermediate and 6 saprophytic species of leptospira, and a gram-negative bacterium belonging to the spirochaetales order.143 transmission most commonly occurs by contact with urine of infected animals, postabortion discharges, milk, and contaminated water. infected bulls may transmit during coitus and pregnant cows may transmit organisms transplacentally to their fetus. leptospira serovars hardjo and pomona cause endemic reproductive problems in cattle manifested as abortions, fetal mummification, stillbirth, retained fetal membranes, premature births and the birth of weak and/or low-weight calves. in addition, the disease has been associated with a subtler syndrome characterized by early embryonic death and subfertility.144 the majority of abortions occur in the last trimester of pregnancy, but some serovars cause second trimester abortion, fetal mummification or embryonic mortality. abortion rate vary from 3 10% with l. hardjo to 50% with l. pomona. fetuses are generally autolyzed and do not have specific gross lesions. histologically, some cases have mild inflammation of fetal membranes and the presence of the organism might be identified (not in all cases) by special silver stains. some fetuses have renal tubular necrosis and interstitial nephritis.130 demonstration of leptospira dna by reverse transcription polymerase chain reaction (rt-pcr) in the kidney of aborted fetuses is the preferred diagnostic test. on a herd basis, serologic diagnosis of leptospirosis could be challenging in vaccinated animals. microscopic agglutination tests, used commonly, measures the antibody titer in maternal serum at abortion and again 2 3 weeks later. a 4-fold increase in paired samples is considered diagnostic. in some serovars such as pomona, grippotyphosa, icterohaemorrhagiae and canicola, maternal serum antibody titers ≥ 1600 appear to correlate with abortion. however, maternal antibodies in cases of abortion caused by serovar hardjo often are low or negative at the time of abortion; therefore, a low antibody titer does not rule out leptospirosis as the cause.145 salmonellosis salmonella enterica subsp. enterica serovar dublin might, in the course of a systemic infection, colonize the placenta and fetus and cause placentitis, abortion, and stillbirth.146 the organism could be isolated from fetal membranes and fetal abomasal contents. ureaplasma diversum ureaplasma diversum is a common inhabitant of vagina and prepuce of clinically normal cattle, associated with infertility, endometritis, salpingitis, and abortions. abortions are usually sporadic; however, severe outbreaks, involving multiple animals, occur occasionally in the second and third trimester of pregnancy.147 mycotic abortion mycotic abortion in cattle is most often caused by aspergillus fumigatus infection. other causes of mycotic abortion include other fungi of the genera absidia spp., mucor spp., rhizopus spp., mortierella wolfii, candida spp., and torulopsis.148 in cattle, fetal infection generally occurs through the hematogenous route to the placentomes by extension from maternal fore stomachs or respiratory infections. mycotic infections of the fetus often result in sporadic, late-term abortions between the 6th and 8th month of pregnancy. fetal infection is characterized by severe fetal membrane inflammation and fetal dermatitis, the latter characterized by raised circular epidermal plaques. histologically, mycotic fetal membrane inflammation is characterized by severe necrotizing and suppurative inflammation with thrombosis. presence of fungal hyphae in the lesions could be apparent in h&e-stained sections or with gomori’s methenamine silver and periodic acid–schiff stains. confirmation of the diagnosis could be done by culture. the genus and species of the fungus also could be identified in fresh tissues, or in paraffin embedded sections by panfungal pcr.149,150 bluetongue virus bluetongue is an arthropod-borne, non-contagious viral infection of domestic and wild ruminants, and less often south american camelids caused by bluetongue virus (btv). btv is the type-species of the genus orbivirus in the family reoviridae. currently, 28 serotypes of btv are recognized worldwide. in the us, btv 2, 10, 11, 13, and 17 are endemic, but infections with other serotypes occur sporadically.151 transmission of btv among susceptible hosts occurs through the bite of certain species of infected culicoides or biting midges. the main vector of btv endemic serotypes in us is culicoides sonorensis (c sonorensis; previously known as c varipennis). c. insignis is also identified in the southeastern us. more recently, new btv serotypes (btv-25, btv-26, and btv-27) were horizontally transmitted without vector involvement.152 bluetongue virus abortion occurs most commonly in sheep and deer and sporadically in cattle and goats. before the recent btv-8 outbreak in europe, the ability of btv to cross the placenta and cause congenital infection in cattle had been largely limited to cell-adapted btv strains (i.e. live attenuated vaccine strains).153,154 in endemic areas, the epidemiological relevance of naturally and congenitally infected calves is believed to be negligible. btv-induced brain malformations still occur infrequently among aborted bovine fetuses in california, but it is assumed that those bovine btv-induced abortions are the result of infection with live-attenuated btv strains present in vaccines licensed only for sheep.153 clinical theriogenology 2021; 13: 172 one of the characteristics of wild-type btv-8 that circulated in northern europe is its ability to cross the placenta. transplacental transmission of wild-type btv-8 is ~ 10 41.7% in cows and up to 69% in sheep.155 infection of pregnant cows with wildtype btv-8 caused abortion, stillbirth, and fetal malformations (hydranencephaly).156 serological assays available for demonstration of btv antibodies include complement fixation, virus neutralization, the agar gel immunodiffusion test, and several elisa formats. presence of btv antibodies in maternal serum indicates exposure at any given time and does not indicate that the abortion was caused by btv.151 assays based on rt-pcr are used to detect btv rna in clinical samples (e.g. blood or spleen).157 infectious bovine rhinotracheitis infectious bovine rhinotracheitis (ibr) is a major cause of viral abortion in cattle with abortion rates of 5 60% in naïve unvaccinated herds; it is caused by bovine herpesvirus-1 (bhv1), a member of herpesviridae family in the alphaherpesvirinae subfamily.158 bhv-1 is ubiquitous in cattle populations and is the cause of several clinical syndromes including abortion, vulvovaginitis, balanoposthitis, respiratory disease, conjunctivitis, encephalomyelitis, and fatal systemic infections in neonates.159 all bhv-1 strains are capable of becoming latent infections. stressful situations including transportation, calving, treatment with corticosteroids and other stressful situations could induce recrudescence of the infection and shedding of the virus in respiratory and reproductive secretions, or semen of latently infected animals. after infection in pregnant cows, bhv-1 may remain latent in the placenta and only invade the fetus after several weeks. once the fetus is infected, it dies quickly and remains in utero for several days resulting in autolysis before expulsion. the subcutis is edematous and red-tinged. large amounts of red-tinged fluid are present in the thoracic and abdominal cavities and in the pericardium.130 in some cases, small, discrete white nodules are observed in the liver. histologically, there are discrete areas of necrosis in the liver, kidneys, spleen, lungs and adrenal glands. intranuclear inclusions characteristic of herpesvirus infections are difficult to find in hematoxylin and eosin-stained tissue sections but are most likely to be found in the adrenal glands. lesions in the placenta consist of necrosis and vasculitis. abortion generally occurs between 5 8 months of pregnancy. bovine herpesvirus-4 has been detected in tissues of aborted fetuses.160 in general, the use of inactive vaccines is safer for pregnant females as well as in females of unknown pregnancy status or during early stages of pregnancy. in an experiment in which an inactivated bhv-1 vaccine was used to immunize females prior to breeding protection against pregnancy loss was similar to that of modified-live bhv-1 vaccines following substantial challenge infection performed around 180 days of pregnancy.161use of modified live vaccines in naïve pregnant females can increase the risk of pregnancy loss. therefore, the importance of a reliable clinical history is of paramount importance. a number of bovine abortions have occurred following administration of bhv-1 vaccines in the us, which was in part due to confusion about the appropriate use of modified-live virus products. although most postvaccination abortions appear to reflect inadvertent extra label use of bhv-1 vaccines by owners and veterinarians (i.e. vaccinating pregnant cattle during pregnancy that were not vaccinated in the previous 12 months with an appropriate modified-live bhv-1 vaccine), some involve appropriately vaccinated heifers. until there is a method to distinguish vaccine strains from field strains of bhv-1, diagnosticians will be unable to confirm whether the virus is from vaccine or wild type origin.162-163 a retrospective study examined data on bovine abortion submissions from 5 veterinary diagnostic laboratories from 2000 to 2011 (ia, ca, wa, mn, and sd) and history of vaccination against bhv-1 in the herd was associated with reduced detection of bhv-1 positive abortion submissions.164 in one study, several bhv-1 strains were sequenced using whole-genome sequencing technologies and the data analyzed to identify single nucleotide polymorphisms (snps). the outcome of this investigation showed promise for the differentiation of viral vaccine virus from field strains; however, more research is required.165 a recent study involving bhv-1 field strains from pennsylvania and minnesota reported a novel snp-based pcr assay that could allow differentiation of vaccine and clinical strains and accurately determine the incidence of bhv-1 and the association of mlvs with clinical disease in cattle.166 the prevalence of abortion declines in regions with a decline in bhv-1 naïve populations. detection of bhv-1 in semen and elimination of contaminated samples is the most important procedure for controlling transmission. detection of bhv-1 could be by real-time pcr or virus isolation (vi) from fetal lung, liver or other tissues, in whole-blood samples with edta, or semen of adult animals. diagnostic tests revealed a moderate degree of agreement the prevalence of abortion declines in regions with a decline in bhv-1 naïve populations. detection of bhv-1 in semen and elimination of contaminated samples is the most important procedure for controlling transmission. detection of bhv-1 could be by real-time pcr or virus isolation (vi) from fetal lung, liver or other tissues, in whole-blood samples with edta, or semen of adult animals. diagnostic tests revealed a moderate degree of agreement (kappa value = 0.498) between pcr and vi, with pcr being a more sensitive and specific technique for the diagnosis of ibr.167 bovine viral diarrhea virus bovine viral diarrhea virus (bvdv) is an economically important pathogen of cattle worldwide. it is estimated that the cost of infection on productivity in cattle ranges from 0.50 to us$ 687.80 per animal.168 bovine viral diarrhea is caused by several different strains of bovine viral diarrhea virus, singlestranded rna viruses belonging to the pestivirus genus in the family flaviviridae. based on the genotype, bvdv is grouped clinical theriogenology 2021; 13: 173 into type 1 and type 2 with multiple subtypes. bovine viral diarrhea viruses also are classified into cytopathic (cp) and noncytopathic (ncp) biotypes.169,170 interspecies transmission between sheep and cattle with border disease virus (bdv), the small ruminant pestivirus is common and might represent a challenge in herds with a bvdv eradication programs because sheep are not included in eradication schemes.171 infection with bvdv in cattle could result in various clinical manifestations. acute disease in adult immunocompetent cattle could result in subclinical infection causing mild transient infection (ti), or in respiratory signs that might last 2 3 weeks and include fever, nasal discharge, pneumonia and death. animals that recover of this form of bvd develop lifelong immunity. the infection also has been associated with diarrhea and a hemorrhagic syndrome.172 the outcome of bvdv fetal infections in susceptible heifers and cows is dependent on the age of the fetus when exposed.173 fetal infection up to 45 days of pregnancy may result in embryonic death. abortion may occur following cpbvdv infection between 45 and 175 days of pregnancy. when fetal infection with ncpbvdv occurs prior to fetal development of immunocompetence (usually between day 45 and 145 of pregnancy), the fetus may survive and become persistently infected (pi). these pi animals generally do not develop bvdv antibodies and shed the virus through a variety of body fluids including semen, and are the source of the majority of new acute and fetal infections. the majority of pi animals die before two years of age, often of mucosal disease that occurs when ncpbvdv mutates into cpbvdv causing super infection. bvdv superinfection also can result when pi animals are infected with cpbvdv or vaccinated with modified live virus vaccine containing a cytopathic strain. fetal infection between 100 and 150 days of pregnancy may also result in the birth of a live calf expressing any number of fetal abnormalities including cerebellar hypoplasia, microencephalopathy, cataracts, microophtalmia, and thymic aplasia. when fetal infection occurs after 150 days of pregnancy, the fetus is generally capable of developing antibodies and clearing the virus. these fetuses are born clinically normal and have bvdv precolostral serum antibodies.174 the following diagnostic tests identify bvdv-infected cattle.175-176 1. bvdv pooled ear notch rt-pcr this test is done using ear skin biopsies samples > 3 mm. samples should be collected and labeled with the individual animal identification. if pooling is requested, the diagnostic laboratory will pool the samples after submission 2. rt-pcr assay is used to detect bvdv nucleic acid in fetal tissues, including spleen, lung, liver, and lymph node. this test is also used in semen and unclotted blood (with edta) from adult cattle, mostly for export purposes. 3. bvdv comprehensive serology. includes bvd 1a (virus neutralization – vn), bvd1b (vn), and bvd 2 (vn). 4. bvd pi immunohistochemistry (ihc) utilizes ear skin biopsies fixed in 10% buffered formalin to detect bvdv pi cattle. 5. bvdv antigen capture elisa. this test is used for detection of bvdv antigen in serum or ear skin biopsies to identify pi animals. 6. bvdv type 1 (singerstrain). detects serum antibodies to bvdv type 1 (singer strain) by virus neutralization. 7. bovine abortion panel. includes bacterial abortion culture, histopathology, bovine herpesvirus 1 rt-pcr, bvdv rt-pcr, leptospira spp. rt-pcr, and neospora caninum rt-pcr. control of bvd should include detection of pis, implementation of biosecurity measures and vaccination. bvdv vaccines available in the us include killed virus and modified live virus vaccines.177 cache valley virus cache valley is an arthropod-borne viral infection, afflicting a variety of domestic and wild ruminants and humans. the majority of infections are subclinical, but embryonic mortality, fetal teratogenesis, abortion, and stillbirth might be common in sheep, goats and less common in other ruminants. many other bunyaviruses prevalent in north america including main drain virus, san angelo virus and lacrosse virus can cross the placenta and result in similar fetal lesions as cache valley virus.178,179 other viruses in the family bunyaviridae, including schmallenberg virus, akabane virus, rift valley fever virus, and aino virus that are exotic to north america also could cause fetal teratogenesis, abortion, and stillbirth in cattle and other ruminants.178,180 neospora neosporosis is caused by the protozoan parasite, neospora caninum, an obligate intracellular coccidian parasite. until 1988, n caninum was confused with a closely related parasite, toxoplasma gondii. n. caninum is regarded as an important infectious cause of pregnancy loss in cattle.181 approximately 10 to 20% of all cattle throughout the world are infected with n. caninum and it is recognized as the cause of ~ 20% of bovine abortions. both endemic and epidemic patterns of abortion can occur in herds. abortion rate in the endemic form is usually > 5% per year and persists year after year. in the epidemic form, > 30% of pregnant heifers and cows may abort over several months. dogs and coyotes are the definitive host for n. caninum, whereas cattle and other ruminants are intermediate hosts.181 in cattle, n. caninum transmission might occur by 1 of 2 routes: horizontally by ingestion of feed or water contaminated with sporulated coccidial oocysts shed by the definitive host; or by vertical transmission through the placenta to the fetus from acutely or persistently infected dams. fetal infection could clinical theriogenology 2021; 13: 174 result in abortion (usually between 5 7 months of pregnancy), mummification, birth of weak compromised calves, or the birth of clinically normal infected neonates that preserve the infection in the herd. vertical transmission occurs because fetal infection does not always result in abortion, but rather the fetus survives and becomes a persistently infected animal. congenitally infected heifer calves remain persistently infected and are capable of passing the infection on to their offspring, thereby maintaining the infection in the herd. the clinical outcome of transplacental fetal infection with n. caninum is likely determined by the maternal and fetal humoral and cellular immune status. cows that abort have no other clinical signs. apparently, risk of abortion in subsequent pregnancies is lower.181 aborted fetuses are generally autolyzed. pale areas are observed in the myocardium and skeletal muscle of some affected fetuses. there are no macroscopic lesions in fetal membranes. microscopic lesions are more frequently observed in the brain and consist of areas of necrosis in the neuropile often surrounded by a rim of mononuclear leukocytes and gliosis. occasionally, parasite cysts not associated with inflammation are observed in the brain of affected fetuses. other fetal lesions consist of necrotizing myositis, multifocal epicarditis, nonsuppurative myocarditis, lymphocytic portal hepatitis, and necrotizing placentitis.159 numerous tests should be used concurrently for the accurate diagnosis of neosporosis in aborted fetuses including rt-pcr and histopathology of fetal membranes, brain, liver, lung or heart, and fetal serology from blood clots in the heart or fetal fluid in cavities. elisa testing of maternal serum is used to determine the infectious status of individual animals; however, on their own, the presence of neospora maternal antibodies does not prove neosporosis as the cause of the abortion.181 control of neosporosis in cattle herds should focus on reducing the number of congenitally infected heifers retained in the herd and by minimizing the likelihood of postnatal transmission from definitive hosts.183 sarcocystosis infection with sarcocystis spp is very common in cattle but abortion is rare. cattle acquire infection through ingestion of feed or water contaminated with feces of canids containing infective parasites. lesions in aborted fetuses consist of granulomas and gliosis in the brain and infiltration of mononuclear leukocytes in kidneys, liver, and heart. the organism can be identified by immunofluorescence in frozen sections of tissues.184 trichomoniasis bovine trichomoniasis is a venereal disease caused by tritrichomonas foetus, a flagellate protozoan. the parasite is more efficiently transmitted from an infected bull to susceptible cows than vice versa. in that regard, 95% of susceptible nulliparous cows became infected after a single mating with a t. foetus-positive bull.185 infection might be asymptomatic or involve a transient balanoposthitis in bulls and vaginitis, cervicitis, and endometritis or pyometra in cows. in pregnant cows, infection is more often characterized by early embryonic death and less often by abortion. most abortions occur during the first half of pregnancy, with a few as late as the 7th month. large numbers of organisms might be observed microscopically in h&e-stained sections of fetal membranes. there is edema of the placental stroma , necrosis of the chorionic 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tritrichomonas foetus. vet pathol 1988;25:350-355. https://doi.org/10.1177/030098588802500503. 187. anderson m: disorders of cattle. in: njaa bl: editor. kirkbride’s diagnosis of abortion and neonatal loss in animals. 4th edition, oxford; wiley-blackwell: 2012. p. 13-48. https://doi. org/10.1002/9781119949053.ch3 clinical theriogenology 2021; 13: 180 pregnancy loss in cattle microsatellite-based characterization of nigerian indigenous dogs (canis lupus familiaris) in southwest nigeria microsatellite-based characterization of nigerian indigenous dogs (canis lupus familiaris) in southwest nigeria oluwatoyin ajala,a d. osunkoya,a o. fagbohun,b a. olumidec adepartments of theriogenology and bveterinary microbiology, university of ibadan, ibadan, nigeria cafrican bioscience ltd, ibadan, nigeria microsatellite-based genetic diversity studies are important for breed identification, disease mapping, parentage analysis and forensic medicine. genetic diversity studies of a vast number of exotic breeds of dogs have been conducted and documented. however, there is limited information on genetic variability in nigerian indigenous dogs (nids). objective was to identify genetic diversity of indigenous dogs in southwest nigeria. thirty nids were used for genotyping. polymerase chain reaction was carried out using 2 sets of microsatellite markers (h 2305 and fh 2361) previously used to examine genetic variability in dogs. total analysis of molecular variance for both microsatellites showed 26% variability among populations, 4% among individuals and 70% within individuals. the mean number of different alleles (na) was 2.90; number of effective alleles (ne) 2.40; mean observed heterozygosity (ho) 0.52; and expected heterozygosity (he) was 0.48. the polymorphic information content (pic) for locus fh 2305 and fh 2361 was 0.69 and 0.54, respectively. it was concluded that genetic variation existed in the nids in southwest nigeria, perhaps due to deletions, insertions or substitutions in their genomes. keywords: microsatellite, genetic variability, southwest nigeria 527 clinical theriogenology • volume 11, number 3 • september 2019 528clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 1 contact julie cecere juliet@vt.edu © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 10227, http://dx.doi.org/10.58292/ct.v15.10227 technique report canine semen processing for chilled shipment julie cecere, nicole sugai department of small animal clinical sciences, virginia-maryland college of veterinary medicine, blacksburg, va, usa abstract advanced reproductive techniques for dogs have become commonplace in general practice during last decade. most procedures performed in these practices are based on what is known for semen processing and shipping in the equine industry. this presentation reviews current literature of what is known on canine semen processing and attempts to standardize techniques to maximize their successful use in clinical settings. keywords: canine, chilled semen, processing, shipping, veterinarian role importance of semen shipment as an advanced reproductive technique over the last decade, advanced reproductive techniques (art) have blossomed in multiple species, especially in equine and canine patients. the shipping of gametes throughout the world aids in diversifying genetic pools and overcoming geographical or time-related challenges. specifically for dogs, semen processing after collection has become commonplace in both general and reproductive practices. however, techniques that are used and the process by which semen is handled and shipped is not standardized. due to the uniqueness of the canine reproductive cycle and the distinct disadvantage of resistance to in vitro manipulation, fertility related to semen processing or shipping are difficult to predict.1 many practitioners are using techniques from equine semen processing and shipment and applying those concepts to canine semen. current research areas of canine art are numerous and geared toward minimizing the knowledge gap between canine and equine semen processing and handling in order to assure better fertility outcomes. importance of semen evaluation at collection semen evaluation is still the primary step of any semen processing. using semen from animals that have not had semen evaluation can lead to missed pregnancies due to poor concentration, motility, viability, or morphologic defects. semen that is frozen without a full evaluation may lead to future genetic losses due to unproductive frozen-thawed semen used for breeding. over the past several years, there have been presentations and publications related to canine semen evaluation and also in the context of infertile or subfertile male dogs.2-4 referring to those resources will be helpful to practitioners conducting canine andrology work and details will not be discussed. a dog that is collected for the purpose of a current or future breeding, shipping, or frozen semen should have a full semen evaluation. a single evaluation parameter is not accurate in predicting the performance of a stud dog.5 in the event that semen from the dog is shipped or frozen, the semen should be evaluated in the same manner after shipping or thawing to document the performance of that individual collection. these evaluation(s) should include the following: • full physical examination, paying attention to possible systemic, congenital or heritable diseases that can be observed, heard, or palpated. ° examples: heart murmurs, ocular abnormalities, pain or reduced range of motion in joints during the examination, or endocrinopathies. • evaluation of libido and gross appearance of the collection that may lead to diagnosis of disease specific to penis, urethra, or prostate. ° examples: hemospermia, penile lesions, persistent frenulum • a full spermiogram to include concentration, motility, and morphology. ancillary tests such as viability (plasma *presented at the 2023 society for theriogenology conference, published after peer review. mailto:juliet@vt.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.10227 2 citation line: clinical theriogenology 2023, 15, 10227, http://dx.doi.org/10.58292/ct.v15.10227 membrane integrity), hypo-osmotic swelling test (host), or computer-assisted sperm analysis may also be used to further describe sperm functionality. ° these will require additional training or equipment to benefit from their advanced evaluation capabilities. examples include a phase contrast microscope, flow cytometer-based machine, or additional reagents needed to perform additional tests. ° these tests will identify extremely poor or excellent semen, although there are no assurance of fertilizing capability based on a single evaluation.6 ° the host test is positively correlated with normal progressive motility and normal morphology in fresh semen.7 ° in stallions, the phase contrast technique used for morphologic evaluations resulted in better agreement between evaluators when compared to other morphologic evaluation methods.8 comparison (canine and equine) and application of semen processing studies canine andrology work is currently in its infancy compared to what is known for other species. specifically, the equine and porcine industries use chilled semen commonly in routine breeding management, so dog breeders have some ‘catching up’ to do. there are several experiments that have been performed over the past 15–20 years that shed some light on what is known and that can be implemented in practices to use chilled semen as a routine technique in routine breeding management. currently, what is used as a standard for semen evaluation, centrifugation, processing, and dilution for shipping is based on what is known from stallions and a handful of studies in dogs. due to the size disparity in both the animal and testicular volume between stallions and dogs, andrology research about processing and shipment is difficult in dogs due to their smaller size and semen volume. most studies that have evaluated canine semen for centrifugation, processing, chilling, or freezing have used pooled samples. this is an astute way to maneuver around small volumes from each individual dog collected; however, has the weakness of making it impossible to measure the effect of the experimental parameter on an individual dog.6 this must be kept in mind when discussing papers moving forward. stallions produce large volumes of ejaculate and sperm (20–160 ml and 0.08–0.35 x 109/ml) in a collection.9 this lends itself to splitting a single ejaculate and using that ejaculate for multiple breeding doses. there are also many advances related to reducing the number of nonmotile or abnormal sperm during processing, so only superior sperm are contained within the shipment, such as cushions, filters, or sperm washing, to name a few.9 currently these technologies can be used in canine collections for the same reason, but they have several disadvantages: • they drastically reduce the number of sperm within the sample that can reduce the sample to be below a normal breeding dose. • the process may be cost prohibitive to a typical client as the price of the apparatus may be as much as the appointment to collect and process the semen without advanced processing. currently, equine semen is processed in the following manner:9 • collected, extended at a 1:1 volume:volume ratio and centrifuged at a speed of 400–900 g for 9–15 minutes. • removal of seminal fluid to 5–20% of the original volume and diluted with extender to 25–50 x 106/ml.10,11 • packaged in unique containers to slow cooling at a rate of 1.8–6°c/hour down to 4–5°c for holding. semen retains its fertilization capacity for 24–48 hours. • newer research currently evaluating the practicality of higher temperature storage of stallions (17°c) for 7 days appears promising.9 many decades of research have gone into optimizing the conditions in which stallions are collected, the semen processed, and then chilled for shipping or short-term storage. these studies are ongoing and constantly changing. in contrast, canine studies evaluating different parameters of semen collection and processing have been limited. centrifugation speeds and amounts of seminal plasma have been represented.12–14 more recently, studies compared individual performance of canine semen, rather than pooled samples. these results will possibly shed light on the future of canine semen shipping. centrifugation speeds have varied for canine semen processing; however, for pooled samples and fertile individuals 400–900 g for 5–10 minutes is adequate for sperm recovery rate, minimized sperm damage, and viability at 24 hours after cooling with standard procedures.15 an ongoing study is determining the optimal concentration that canine semen requires for cooled shipping. higher concentration(s) (compared to equine semen) is necessary during cooling. dilution and cooling at 100–200 x 106/ml rather than 25–50 x 106/ml had significant effects on the 24 hour evaluations.16 the current recommendations for canine semen processing in practice based on these studies and others are as follows: • collection with immediate evaluation and processing of the collection if the total volume is > 2 ml. this will reduce the seminal plasma volume that has been, similar to stallions, detrimental at high concentrations in chilled or frozen semen.13 • centrifugation speeds of 400–900 g for 5–10 minutes are acceptable for processing. • dilution at a final concentration of 200 x 106/ml, or dilution at a 1:3 volume:volume, for cooling and shipping. • for quality assurance and liability purposes, retaining a small aliquot of the processed and extended semen sample at the facility for assessment at 24 hours. this could help assess if handling had a factor in poor arrival quality. insemination with any cooled semen at 24 hours after collection and no longer. further studies are needed to optimize the extender types or additives, practical semen filtration procedures, and optimization of in vivo fertility parameters. owner-collected semen may affect fertility outcomes those breeders that are bypassing semen collection, evaluation, and processing for shipment at a veterinary clinic may be placing their dogs at risk for missed pregnancies or reduced litter sizes without warning. failure to detect changes in their stud dog’s systemic status that may affect semen quality and/or concentration, poor performance with certain types of http://dx.doi.org/10.58292/ct.v15.10227 citation line: clinical theriogenology 2023, 15, 10227, http://dx.doi.org/10.58292/ct.v15.10227 3 extenders or lack of formal processing prior to shipping may negatively affect a planned breeding or kennel’s breeding plan. discussing the importance of the following with clients is key to achieving success with stud dog management: • all canine collections should contain the first and second or second fractions only.12 this allows for reduced volumes and easier dilutions for shipment as well as reduction of possible detrimental components within the other fractions. remember that higher volumes do not mean that more sperm have been collected. • evaluation of collected semen allows the veterinarian and owner to determine the best breeding practices for the stud dog and/or if adjustments are necessary.2,4 for example, an aging stud dog may not be able to adequately provide semen for 2 female dogs in a single collection due to declining concentrations as testicular degeneration progresses. communicating this to prospective female dog owners is key to transparency when managing a stud dog, but if routine spermiograms are not performed, an owner may be unaware of declining numbers or quality. • processing should occur to reduce the amount of seminal plasma that may be detrimental to sperm during shipping and to achieve the appropriate shipping concentration. the ideal concentration of seminal plasma should be ~ 10–20% of initial volume. centrifugation is the only way to achieve this reduction in seminal plasma and overall volume. volume to volume dilutions is known to be inadequate, since canine sperm needs more concentrated shipping conditions, so collection and addition of extender without processing and evaluation should be avoided. role of veterinarians in assisting clients the main mission of reproductive veterinarians is to help clients achieve their goals for their breeding programs while educating them on the current, best practices necessary to accomplish those goals. to achieve the results that a breeder wants from their stud dog(s) program, it is necessary to ensure that proper collection and evaluation techniques are used every time. this allows for real time adjustments to occur and to prevent any unwanted or surprising outcomes. test shipments for any stud dog that is offering chilled semen as a method of breeding should be performed. collection, evaluation, and processing of the semen occurs in a standard format, then the collection is divided between different extenders, at an appropriate dilution, packaged for shipment and evaluated at 24–72 hours. if adequate performance is not achieved with standard processing technique, altering centrifugation speed, final concentration, or seminal plasma concentration can be adjusted until either the animal is deemed inappropriate for chilled semen shipment or the adjustment(s) result in acceptable quality after the test shipment is concluded. this allows for optimization of semen processing parameters (centrifugation speeds and time, appropriate concentration and preferred semen extender for each individual) and performance metrics when the shipment arrives at its intended destination. it also allows the breeder some evidence that due diligence occurred as steps were performed to ensure the best quality product was provided to the female dog owner. extension and outreach to local clubs is essential. education and current literature dissemination is extremely helpful to breeders, and aids in preventing the dissemination of ‘breeder lore.’ these types of events reach a large cohort of breeders and ensure that they are receiving correct information that is science based. they may also boost client numbers for clinics, so it is a ‘win’ for both veterinarian and breeder. it is important to support research that is ongoing for canine reproductive studies. referrals to programs conducting research are essential for the growth of the knowledge base, which will translate into real time positive change in the field of canine reproduction. practitioners should start conversations with universities on things that are wanted or needed and discuss if a study is necessary or feasible. many private practices can perform or assist in studies without having to refer the animal to a different facility. also request continuing education in the form of wet labs or didactic instruction to learn new techniques or refresh on things that are not often performed or that are additions to services that a practice offers. conclusion canine semen processing standards are rapidly changing as scientific data highlight best practices. as animals are continued to be bred globally, with exchange of genetic material over vast distances and generations, it is important to continue to research and adjust to current trends in semen processing. discussing these trends often with breeders and their clubs will assure that the veterinary and breeding communities are maximizing their breeding potential over large distances. conflict of interest authors report no conflict of interest. funding nicole sugai’s theriogenology residency program is funded by the american kennel club/canine health foundation/ theriogenology foundation grant. references 1. de los reyes m, songsasen n: new insights in canine reproduction. animals (basel) 2021;11:2021. doi: 10.3390/ani11072021. 2. randall j: “this stud’sa dud!”-canine semen evaluation protocols and pitfalls. clinical theriogenology 2020;12:204–209. 3. randall j: “boys in a box”-chilled and frozen canine semen shipments. clinical theriogenology 2020;12:210–214. 4. kolster ka: evaluation of canine sperm and management of semen disorders. vet clin north am small anim pract 2018;48:533–545. doi: 10.1016/j.cvsm.2018.02.003 5. hesser a, darr c, gonzales k, et al: semen evaluation and fertility assessment in a purebred dog breeding facility. theriogenology 2017;87:115–123. doi: 10.1016/j.theriogenology. 2016.08.012 6. arlt sp, reichler im, herbel j, et al: diagnostic tests in canine andrology-what do they really tell us about fertility? theriogenology 2023;196:150–156. doi: 10.1016/j.theriogenology.2022.11.008 7. karger s, geiser b, grau m, et al: prognostic value of a pre-freeze hypo-osmotic swelling test on the post-thaw quality of dog semen. anim reprod sci 2016;166:141–147. doi: 10.1016/j. anireprosci.2016.01.015 http://dx.doi.org/10.58292/ct.v15.10227 https://doi.org/10.3390/ani11072021. https://doi.org/10.1016/j.cvsm.2018.02.003 https://doi.org/10.1016/j.theriogenology.2016.08.012 https://doi.org/10.1016/j.theriogenology.2016.08.012 https://doi.org/10.1016/j.theriogenology.2022.11.008 https://doi.org/10.1016/j.anireprosci.2016.01.015 https://doi.org/10.1016/j.anireprosci.2016.01.015 4 citation line: clinical theriogenology 2023, 15, 10227, http://dx.doi.org/10.58292/ct.v15.10227 8. hernández-avilés c, ramírez-agámez l, pearson m, et al: a matter of agreement: the effect of the technique and evaluator on the analysis of morphologic defects in stallion sperm. theriogenology 2023;202:74–83. doi: 10.1016/j.theriogenology.2023.02.025 9. wiebke m, hensel b, nitsche-melkus e, et al: cooled storage of semen from livestock animals (part i): boar, bull, and stallion. anim reprod sci 2022;246:106822. doi: 10.1016/j.anireprosci.2021.106822 10. ferrer m, canisso i, ellerbrock r, et al: optimization of cryopreservation protocols for cooled-transported stallion semen. anim reprod sci 2020;221:106581. doi: 10.1016/j. anireprosci.2020.106581 11. len j, beehan d, eilts b, et al: stallion sperm integrity after centrifugation to reduce seminal plasma concentration and cool storage for 4 days. j equine vet sci 2020;85:102819. doi: 10.1016/j. jevs.2019.102819 12. england g, allen w: factors affecting the viability of canine spermatozoa: ii. effects of seminal plasma and blood. theriogenology 1992;37:373–381. doi: 10.1016/0093-691x(92)90195-w 13. pan c, wu y, yang q, et al: effects of seminal plasma concentration on sperm motility and plasma and acrosome membrane integrity in chilled canine spermatozoa. pol j vet sci 2018;21:133–138. 14. rijsselaere t, van soom a, maes d, et al: effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa. theriogenology 2002;57:1669–1681. doi: 10.1016/s0093-691x(02)00663-5 15. sugai n, cecere j, balogh o: defining an optimal range of centrifugation parameters for canine semen processing animals (basel) 2023;13:1421. doi: 10.3390/ani13081421 16. sugai n, cecere j, balogh o: comparing different sperm concentrations for cooled semen shipment in dogs reprod domest anim 2023;58:184–198. http://dx.doi.org/10.58292/ct.v15.10227 https://doi.org/10.1016/j.theriogenology.2023.02.025 https://doi.org/10.1016/j.anireprosci.2021.106822 https://doi.org/10.1016/j.anireprosci.2020.106581 https://doi.org/10.1016/j.anireprosci.2020.106581 https://doi.org/10.1016/j.jevs.2019.102819 https://doi.org/10.1016/j.jevs.2019.102819 https://doi.org/10.1016/0093-691x(92)90195-w https://doi.org/10.1016/s0093-691x(02)00663-5 https://doi.org/10.3390/ani13081421 induction of ovulation in donkeys using gnrh or hcg induction of ovulation in donkeys using gnrh or hcg ava kent, shelby nester, erik peterson, robert gilbert, hilari french ross university school of veterinary medicine, st. kitts, west indies ovulation induction agents are commonly used to manipulate reproductive cycles in equine practice to optimize breeding management. although this procedure is routinely used in horses, limited information exists to support its use in donkeys. this has posed a challenge to conservation efforts depending on improved reproductive efficiency in endangered donkey species. a pilot study was designed to develop a protocol for inducing ovulation in jennies to determine the interval from administration of induction agents, hcg (chorulon®) and gnrh analogue (sucromatetm) to observed ovulation. eight reproductively sound, non-pregnant caribbean jennies between 3 and 12 years old were examined daily via transrectal ultrasonography to monitor ovarian activity and follicular growth. jennies were randomly assigned to each of 3 treatment groups (hcg, gnrh analogue, and control) and injected (hcg iv, gnrh analogue im) at a follicular diameter of 27 or 30 mm. starting 24 hours after treatment, jennies were monitored every 6 hours via transrectal ultrasonography until ovulation. number of jennies ovulating within 48 hours after treatment (or assignment to control group) for jennies treated at a follicular diameter 27 mm was 1/8, 5/8 and 4/8 for control, gnrh and hcg, respectively. mixed effects logistic regression with jenny as random variable indicated a near significant effect of gnrh (p = 0.059) but not of hcg (p = 0.17). results for induction at 30 mm follicular diameter were similar (2/8, 5/8, and 3/8, respectively). mean interval to ovulation was shorter after induction at 30 mm and variation was less. for this group, interval to ovulation was 81 ± 40 (control), 50 ± 22 (gnrh) and 56 ± 25 hours (hcg) with a p = 0.03 for gnrh and 0.05 for hcg by multiple logistic regression, with jenny as random variable. results indicated that ovulation can be induced in jennies. gnrh induced ovulation within 48 hours more consistently than hcg, but at a follicular diameter of 27 or 30 mm, the effect was not as predictable as it is in horse mares induced at a follicular diameter of 35 mm. waiting until the dominant follicle was larger may improve response to induction agents, but increases the risk of spontaneous ovulation. keywords: donkey, ovulation, induction, hcg, gnrh analogue 519 clinical theriogenology • volume 11, number 3 • september 2019 520clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2014: breeding soundness examination, semen freezing and evaluation of frozen-thawed semen in native omani bulls – preliminary study breeding soundness examination, semen freezing and evaluation of frozen-thawed semen in native omani bulls – preliminary study n. pratap,a m.a. memon,b o. mahgoub,c y. al-shikaili,d r.s. al-habsi,d b.e. hagoa alaboratories and animal research center, dg of veterinary services, royal court affairs, muscat, oman; bdepartment of veterinary clinical science, washington state university, pullman, wa; cdepartment of animal and veterinary sciences, college of agriculture and marine sciences, sultan qaboos university, muscat, oman; danimal production center, rumais, ministry of agriculture, muscat, oman abstract the present study was undertaken as part of a project on ‘characterization, evaluation and conservation of indigenous animal genetic resources in the sultanate of oman’. the objective of this study was to conduct breeding soundness examinations (bse) and determine the characteristics of frozenthawed semen of native omani bulls. breeding soundness examinations were carried out in twelve bulls age three to four years; six bulls were found to be satisfactory, while the remaining six were unsatisfactory as potential breeders. semen was collected using an electroejaculator (ee) in the six bulls once per week for two consecutive weeks. the volume of the ejaculates and the initial sperm concentration were measured. nine ejaculates were qualified for freezing. semen was diluted in tris egg yolk extender and frozen in liquid nitrogen vapor. frozen semen straws were thawed at 37°c for at least 30 seconds in a water bath for assessment of post-thaw semen quality. the mean volume of semen and sperm concentration was 9.08 ± 0.9 ml and 284.6 ± 24.8 x 106/ml, respectively. the post-thaw total sperm motility was 64.6 ± 5.2% and progressive motility was 34.2 ± 4.1%, as evaluated by computer assisted sperm analysis. the hypo-osmatic swelling test positive spermatozoa was 27.1 ± 3.5%, intact acrosome was 90.7 ± 1.41% and sperm abnormalities were 8.3 ± 1.0% in post-thawed semen. overall, the quality of the post-thawed semen in the present study was similar to that obtained from crossbred and exotic bulls. in conclusion, bse was beneficial in bull selection, semen was collected successfully using an ee and frozen in native omani bulls. the frozen-thawed semen was suitable for artificial insemination procedures. keywords: native omani bull, breeding soundness examination, semen evaluation, semen freezing, computer assisted semen analysis, hypo-osmotic swelling test. introduction the importance of animal biodiversity was recognized by the united nations convention on biological diversity for the integrity and sustainability of the biosphere and human society.1 conservation of indigenous genetic resources is vital for the development of new animal species and improvement of existing animal species. oman has noticeable biodiversity in local animals that are adapted to the unique and diverse topographic and climatic conditions of the sultanate. limited work has been carried out to identify or conserve these breeds, which are well-adapted to omani conditions. at present, native omani breeding bulls are selected on the basis of subjective evaluation of physical appearance and visual observation of the testes. however, no semen collection and evaluation is usually performed before animals are used for breeding purposes. accurate and reliable information on omani breeds is essential in order to make optimal use of breeding programs. the current study was a part of a project entitled: ‘characterization, evaluation and conservation of indigenous animal genetic resources in the sultanate of oman’. in the wider context of the project, the current study aimed to evaluate the reproductive efficiency of omani breeding animals and establish a semen bank for future genetic improvement programs. the cattle population in the sultanate of oman was estimated at 326,240 head. based on the distribution and phenotypic appearance of omani cattle, they are classified into north omani cattle and south omani cattle (dofari) breed.2 north omani cattle are small in size with short horns, brown to dark brown color, and are found in batina coastal planes, interior sharqiyah and dhahira regions of oman. clinical theriogenology • volume 6 number 2 • june 201485 they are highly aggressive and disease resistant and used mainly for meat production and traditional bull fighting. collection and cryopreservation of semen is an effective means of conservation of dwindling native omani cattle breeds. the most commonly used methods for successful semen collection in cattle are the use of an artificial vagina and an ee. considering the aggressive nature of the omani bulls, semen collection with an ee was utilized. to our knowledge, there are no reports available on semen collection and cryopreservation in native omani bulls. the objective of the present study was to conduct bses, freeze semen, and determine the characteristics of frozen-thawed semen of native omani bulls. materials and methods breeding soundness evaluation breeding soundness examinations were carried out in twelve bulls age three to four years, according to the methods described by barth.3 bulls were evaluated for their body condition score (bcs) on a scale of 1 to 5 (1= thin, 5 = fat)4 and weighed on an electronic balance. scrotal circumference (sc) was measured by a standard scrotal tape at the widest mid-scrotal point. testes were palpated, accessory sex glands were evaluated per rectum, and semen was collected by an ee. initial semen quality was evaluated by assessing volume, mass activity, sperm motility and sperm concentration. based upon the established bse standards,3 the bulls were classified as satisfactory or unsatisfactory potential breeders. animals and semen collection six native omani bulls, age three to four years, qualified as satisfactory potential breeders3 were selected for this study. the study was conducted at the agricultural research centre, rumais station, ministry of agriculture and fisheries, sultanate of oman (latitude 23° 36ʹ ʹ n: longitude 58° 37 e). the bulls were maintained with an average body condition score of 3.0 through intensive feeding and management conditions and received 3 kg of concentrates daily, with green fodder and water ad libitum as well as mineral supplementation. these bulls were aggressive and not trained to donate semen by artificial vagina. therefore, the bulls were adequately restrained in a cattle chute and semen collection was performed using an ee (electro ejaculator e320, minitube, tiefenbach, germany). electrical pulses of increasing intensity were started slowly untill a response was observed. the frequency and duration of electrical stimuli and the voltage level were similar for all bulls. penile protrusion was followed by ejaculation of semen, which was collected in a hand-held collection device. semen was immediately transferred to a 37°c water bath. semen was collected once per week from each bull for two consecutive weeks. semen processing and cryopreservation the volume of the ejaculates was measured in a conical graduated tube and the sperm concentration was measured using a photometer (spermacue, minitube). initial sperm motility of the fresh semen was evaluated subjectively by phase contrast microscope, as well as objectively using computer assisted semen analysis (casa). only ejaculates with > 70% initial total motility (> 30% progressive motile sperm) were used in the study (n = 9). semen was diluted in an egg yolk extender containing tris, fructose, citric acid, egg yolk, streptomycin, penicillin and glycerol to give a concentration of 50x106 sperm/ml. diluted semen was cooled to 4°c over 2 h, equilibrated for 4 h at 4°c, and placed in 0.5 ml straws with a suction pump at 4°c in a cold cabinet unit (minitube, germany). the equilibrated straws were subsequently frozen using a computerized programmable freezer (ice cube 1810, sy-lab, neupurkersdorf, austria) by reducing the temperature initially at a rate of 5°c/min from + 4 to -12°c, then 40°c/min from -12 to -100°c and finally at a rate of 20°c/min from -100 to -140°c. straws were then plunged into and stored in liquid nitrogen (-196°c). clinical theriogenology • volume 6 number 2 • june 2014 86 assessment of post-thawed semen frozen semen straws were thawed at 37°c for at least 30 sec in a water bath for assessment of post-thaw semen quality. assessment of sperm motility by computer assisted sperm analysis. the sperm motion characteristics were evaluated using casa (animal version 12.3, ceros, hamilton thorne biosciences, ma). a 3µl drop of semen was placed in a 20µm standard count analysis chamber (leja, nieuw-vennep, the netherlands). the loaded chamber was placed on the thermal plate of the microscope (37°c) for 3 min before being analyzed according to the methods of anzar et al.5 the instrument settings for the hamilton thorne ceros animal software (version, 12) used to assess sperm kinetics were 30 frames at a rate of 60 frames/sec, minimum contrast of 80, and minimum cell size (pixels) of 5. for progressive cells, the velocity average pathway (vap) was 70µm/s and straightness was 75%. spermatozoa with a vap of more than 30µm/s were considered motile. five randomly selected microscopic fields were scanned five times each. the mean of these 25 scans was used for statistical analysis. the following parameters were analyzed: total motile spermatozoa (%); spermatozoa with progressive motility (%); velocity average pathway (vap, µm/s, the average velocity of the spermatozoon through a smoothed cell path); velocity straight line (vsl, µm/s, the minimum distance a spermatozoon travelled during the time between two scans); velocity curvilinear (vcl, µm/s, the mean of the sperm’s velocity measured over the actual tracks followed); amplitude of lateral head displacement (alh, µm, the mean width of the sperm’s head oscillation); beat cross frequency (bcf, hz, a measure of the frequency of lateral movement, causing a sperm head to cross the average path in either direction); straightness of the sperm’s movement (str, %, vsl/vap); linearity of the sperm movement (vsl/ vcl = lin, %). furthermore, spermatozoa were classified into four groups based on their velocity of movement, viz: rapid (vap > 50 µm/s, %), medium (30 µm/s < vap < 50 µm/s, %), and slow (vap < 30 µm/s, or vsl < 15 µm/s, %) moving spermatozoa and static spermatozoa. hypo-osmotic swelling test sperm plasma membrane integrity was evaluated using the hypo-osmotic swelling test (host) according to the methods of correa and zavos6. a hypo-osmotic swelling solution (100 mosm/kg) was prepared by dissolving 0.49 g sodium citrate and 0.9 g fructose in 100 ml distilled water. for the host test, 100 µl of semen was added to 1ml of pre-warmed host medium and incubated at 37°c for 1 h. after incubation, a 5 µl drop from each sample was transferred to a warm, clean microscope slide and covered with a coverslip. this preparation was examined microscopically using a warm stage at 400x magnification and phase contrast optics. two hundred spermatozoa were counted per sample and the number of spermatozoa showing characteristic swelling of the tail, indicative of an intact plasma membrane, was recorded. sperm acrosomal integrity and sperm abnormalities the sperm acrosomal integrity and abnormalities were assessed according to the methods of watson.7 a 100µl sample of semen was fixed in 500µl of 1% formal citrate (2.9 g tri-sodium citrate dihydrate, 1 ml of 37% solution of formaldehyde, dissolved in 100 ml of distilled water); one hundred spermatozoa were examined under phase contrast microscopy at 1000x magnification. normal acrosomes were characterized by a normal apical ridge. sperm abnormalities were recorded as abnormalities of the sperm head, mid piece and tail. statistical analysis the semen characteristics were analyzed using descriptive statistics and all results were expressed as a mean ± sem. results table1 summarizes the mean (±se) of the bse in the native omani bulls (n=12), namely, bcs, body weight, sc and semen evaluation parameters (volume, mass activity, initial motility, sperm clinical theriogenology • volume 6 number 2 • june 201487 concentration, normal sperm, primary and secondary abnormalities). fifty percent of the bulls were found to be satisfactory potential breeders, while the remaining six bulls were declared unsatisfactory due to several factors such as, low sc (26 to 27.5 cm), azoospermia, poor sperm motility (30 to 60%) or high percent (15 to 22%) of morphological abnormalities. the mean volume of semen and the sperm concentration observed during the study were 9.08 ± 0.9 ml and 284.6 ± 24.8 x 106/ml, respectively. the mean values of sperm motion characteristics observed during the present study for diluted semen and frozenthawed sperm are presented in table 2. the percentage of host-positive spermatozoa was 27.1 ± 3.5, intact acrosomes was 90.7 ± 1.41 and sperm abnormalities was 8.3 ± 1.0 % in post-thawed semen. discussion breeding soundness examination and semen collection satisfactory potential breeding bulls play an important role in successful breeding outcomes and a positive impact on overall reproductive efficiency. there are obvious economic losses when bulls with potentially low fertility are used for artificial insemination. the bcs of bulls in the present study (3 ± 0.07 v/s 3.2±0.5) was similar to that reported by chacon et al,8 however, the average sc in the present study was much lower than their study (28.3 ± 0.47 v/s 36.2±4) in bos indicus bulls. native omani bulls are a beef breed with a small frame and body weights ranging between 276 and 431kg. bulls with lower body weights (276 to 358 kg) had a smaller scrotal circumference (26 to 27.5 cm); however, bulls found to be satisfactory as potential breeders (n=6), had greater body weights (364 to 431kg) and sc (29.5 to 30.5 cm). the present study demonstrated the successful collection and cryopreservation of semen in native omani bulls. an ee is usually used to collect semen from animals that are unwilling or not trained to mount and ejaculate semen into an artificial vagina in this study, 90% of the ejaculates collected by an ee were qualified for freezing. the semen volume collected in the present study was comparable but sperm concentrations were lower than that reported for curraleiro bulls.9 however, the sperm concentrations obtained in the present study were higher than in beef bulls reported by palmer et al.10 these findings indicate that an ee can be used as a reliable method to collect semen in the aggressive native omani bulls. semen freezing and thawing artificial insemination has made a profound contribution to genetic improvement, particularly in cattle, and this impact would not have been possible without successful freezing of bull semen. the process of cryopreservation causes damage to sperm membranes, cytoskeleton, and motile apparatus and nucleus, and can alter cell metabolism.7,11 the post-thaw quality of spermatozoa is of prime interest for the ai industry, since it can provide insights into the fertilizing capacity of the cryopreserved spermatozoa. sperm motility is one of the commonly used criteria to assess sperm quality, as it is essential for sperm transport and fertilization in the female reproductive tract. however, subjective or visual assessment of post-thaw sperm motility has been reported to be poorly correlated with fertility. variations of 30 to 60% have been reported in the estimation of motion parameters of the same ejaculate.12 in the past several years, new criteria for evaluating spermatozoa have emerged, made possible by the development of imaging equipment for performing casa. at present, casa has been widely used in most species to obtain accurate and objective kinetic sperm measurements. the sperm motion parameters assessed by casa in the present study were comparable to the previous report in bulls.13 the motility of freshly diluted semen in the present study with tris extender (74.1% vs 73.93%) was similar to that reported by vera munoz et al14 using a commercially available extender, triladyl. however, the post-thaw motility (50.6% vs 46.4%) was higher in the present study. the total motility, vsl, bcf, lin and str in the current study with tris extender were higher (50.4, 58, 32, 60.8, 85.6 v/s 41, 55.8, 22, 48.6, 79.6, respectively) than those reported by leite et al15 using tris extender with four hour equilibration in cryopreserved semen from gyr bulls. however, the progressive motility and alh were lower in our study (27.4, 4.8 v/s 30.8, 6.0). the total and progressive motility of post-thawed semen clinical theriogenology • volume 6 number 2 • june 2014 88 in the present study were higher than those reported for hf bulls.14 the sperm velocity parameters (vap, vsl and vcl) were lower in the current study than those reported by chaverio et al.16 these variations could be attributed to different settings of the casa system. semen evaluation the sperm plasma membrane is the primary site where damage occurs during freezing and thawing of semen.11,17 an intact and functionally active plasma membrane is essential for the spermatozoon to sustain metabolism, undergo capacitation and acrosome reaction and to attach to and penetrate the oocyte zona pellucid.18,19 the host has been successfully employed for evaluating the plasma membrane integrity of spermatozoa in bulls.6,14,20-22 there was a positive correlation between host reacted sperms and progressive motility (r = 0.705).21 the mean percentage of host positive spermatozoa in the current study was similar to that reported in previous studies;14,22 however, it was lower than those reported by some authors.6,20,21 the percentage of intact acrosomes in the current study was similar to those reported in the studies of tuncer et al.23 the percentage of sperm abnormalities was less than the values reported for jersey, tharparkar, and holstein-friesian bulls.24-27 overall, the quality of the post-thawed semen in the present study was similar to that obtained from crossbred and exotic bulls. in conclusion, bse was essential in bull selection, with an average or above average body weight and sc recommended for potential breeders. semen was collected successfully using an ee and frozen in native omani bulls. the frozen-thawed semen was suitable for ai procedures. acknowledgements this research study was conducted as a part of the project entitled “characterization, evaluation and conservation of indigenous animal genetic resources in the sultanate of oman”. the project was funded by hm research fund at sultan qaboos university (pi: o. mahgoub). the authors would like to thank his excellency nasr bin humood al kindi, minister of royal court affairs and engineer mohamood bin badr al abri, director general, dg of veterinary services, rca, muscat sultanate of oman, for providing the facilities and support to conduct this research. references 1. anonymous: implications of the convention on biological diversity. in: strauss ms, editor. management of animal genetic resources and the conservation of domestic animal diversity.. rome: animal production and health division un fao; 1994. 2. anonymous: annual agriculture census 2008-09. sultanate of oman: department of statistics and information; ministry of agriculture and fisheries; 2009. 3. barth ad: evaluation of potential breeding soundness of the bull. in: youngquist rs and threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: saunders-elsevier; 2007. p 228-240. 4. barth ad, waldner cl: factors affecting breeding soundness classification of beef bulls examined at the western college of veterinary medicine. can vet j 2002;4:274-284. 5. anzar m, hassan mm, graham ef, et al: efficiency of the hamilton thorn motility analyser (htm-2030) for the evaluation of bovine semen. theriogenology 1991; 36: 307-317. 6. correa jr, zavos pm: the hypo-osmotic swelling test: its employment as an assay to evaluate the functional integrity of the frozen thawed bovine sperm membrane. theriogenology 1994;42:351-360. 7. watson pf: recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post thawing function. reprod fertil dev 1995;7:871-891. 8. chacon j, perez e, muller e, et al: breeding soundness evaluation of extensively managed bulls in costa rica. theriogenology 1999;52:221-231. 9. teixeira hca, nascimento nv, mcmanus c, et al: seasonal influence on semen traits and freezability from locally adapted curraleiro bulls. anim reprod sci 2011;125:56-61. 10. palmer cw, britoa lfc, arteaga aa, et al: comparison of electroejaculation and transrectal massage for semen collection in range and yearling feedlot beef bulls. anim reprod sci 2005;87:25-31. 11. hammerstedt rh, graham jk, nolan jp: cryopreservation of mammalian sperm: what we ask them to survive. j androl 1990;11:73-88. 12. budworth pr, ammann rp, chapman pl: relationships between computerized measurements of motion of frozen thawed bull spermatozoa and fertility. j androl 1988;9:41-54. 13. awad mm: effect of some permeating cryoprotectants on casa motility results in cryopreserved bull spermatozoa. anim reprpd sci 2011;123:157-162. clinical theriogenology • volume 6 number 2 • june 201489 14. vera munoz o, amirat briand l, diaz t, et al: effect of semen dilution to low sperm number per dose on motility and functionality of cryopreserved bovine spermatozoa using low density lipoproteins extender: comparison to triladyl and bioxcell. theriogenology 2009;71:895-900. 15. leite tg, do vale filho vr, de arrud rp, et al: effects of extender and equilibration time on post-thaw motility and membrane integrity of cryopreserved gyr bull semen evaluated by casa and flow cytometry. anim reprod sci 2010;120:31-38. 16. chaverio a, machado l, frijters a, et al: improvement of parameters of freezing medium and freezing protocols for bull sperm using two osmotic supports. theriogenology 2006;65:1875-1890. 17. krogenæs a, andersen-berg k, hafne al, et al: membrane alterations in bull spermatozoa after freezing and thawing and after in vitro fertilization. acta vet scand 1994;35:17-26. 18. jeyendran rs., van der ven hh, perez-pelaez m, et al: development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. j reprod fertil 1984;70:219-228. 19. burks dj, sailing pm: molecular mechanisms of fertilization and activation of development. anim reprod sci 1992;28:79-86. 20. zavos pm, correa jr, zarmakoupis-zavos pn: measurement of the sperm motility index via the sperm quality analyzer and its relationship to other qualitative sperm parameters. theriogenology 1996; 46:421-427 21. kathiravan p, kalatharan j, edwin mj et al: computer automated motion analysis of crossbred bull spermatozoa and its relationship with invitro fertility in zona free hamster oocytes. anim reprod sci 2008;104:9-17. 22. gholami h, chamani h, towhidi a, et al: effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in holstein bulls. theriogenology 2010;74:1548-1558. 23. tuncer pb, serpil s, mustafa nb, et al: effect of glutamine and sugars after bull spermatozoa cryopreservation. theriogenology 2011;75:1459-1465. 24. verma hk, singh gd, pant hc: andrological evaluation of jersey bulls. indian j anim sci 1992;62:405-409. 25. rao mr, rao a: studies on semen characteristics of tharparkar and jersey bulls. indian vet j 1975;52:889-890. 26. prasad jk, kumar s, mohan g: sperm head biometry and acrosomal integrity in freezable and non-freezable semen of crossbred bulls: effects of dilutors and raffinose. indian vet med j 1999;23:113-118. 27. sharma ml, greesh mohan, sahni kl: characteristics and cryopreservation of semen of holstein-friesian bulls under tropics. indian j anim health 1991;61:977-999. table 1: breeding soundness examination (bse) parameters of native omani bulls (n=12). parameter s of bse bcs bw sc semen volume ma initial motility sperm conc. normal sperm abnormal sperm primary secondary kg cm cc + % m/ml % % % mean±se 3 ± 0.07 360.5 ±11.4 28.3 ± 0.4 4.6 ± 0.7 1.7 ± 0.3 55.4 ± 4 869.8 ± 119.5 89 ± 4.1 3 ± 1.9 8 ± 2.6 range (min – max) 2.53.0 276 – 431 26 30.5 1.5 9.5 0-3 30 to 80 364 1856 57-99 0-21 1-22 bscbody condition score (1= thin, 5= too fat or obese), bw – body weight, scscrotal circumference, ma sperm mass activity (semen wave motion observed under microscope, 0= no ma to 4= max) clinical theriogenology • volume 6 number 2 • june 2014 90 table 2: sperm motion characteristics of freshly diluted and frozen-thawed native omani bull semen measured by casa. (mean ± sem). parameters freshly diluted frozen thawed velocity average pathway (vap, µm/s) 71.3±4.7 67.9±6 velocity straight line (vsl, µm/s) 57.7±3.3 58± 4.6 velocity curvilinear (vcl, µm/s) 111.2±8 101 ± 10.6 amplitude of lateral head displacement (alh, µm) 5.8±0.3 4.8 ± 0.4 beat cross frequency (bcf, hz) 27.9±1.1 32 ± 0.9 straightness (str, %) 81.7±1.3 85.6 ± 1.6 linearity (lin, %) 54.9±1.6 60.6 ± 2.2 total motility (%) 74.1±5.4 64.6 ± 5.2 progressive motility (%) 38.3±4.5 34.2 ± 4.1 rapid moving spermatozoa (%) 50.8±6.1 33.1 ± 6.3 medium moving spermatozoa (%) 23.1±3.9 17.2 ± 3.8 slow moving spermatozoa (%) 16.6±2.6 15.7 ± 2.3 static spermatozoa (%) 9.4±3 33.5 ± 9.2 clinical theriogenology • volume 6 number 2 • june 201491 pregnancy outcome of a mare with renal failure pregnancy outcome of a mare with renal failure luis de aguiar, viviane gomes, linda paul, frank andrews, jenny sones veterinary clinical sciences, school of veterinary medicine louisiana state university, baton rouge la a 15 year old quarter horse mare was in late-term pregnancy (~ 310 days) with azotemia (creatinine 8.0 mg/dl [reference range 0.5 2.0 mg/dl]) and poor body condition. renal disease was suspected. mare was bright, alert and responsive with severe pitting ventral edema and excellent appetite. serum chemistry panel, urinalysis and urinary indices confirmed renal azotemia. initial treatment consisted of intravenous fluids (3 liter/hour), furosemide (0.5 mg/kg, iv and 0.5 mg/kg im) to initiate urination and omeprazole (4.0 mg/kg, po, q24 hours). transabdominal ultrasonography showed a viable pregnancy with a fetal heart rate of 71 beats per minute (bpm). fetal membranes were assessed via transabdominal ultrasound and were edematous with a combined thickness of uterus and placenta (ctup) of 17.7 and 16.0 mm on the cranial and mid ventral abdomen, respectively. transrectal ultrasonography showed 12.8 mm ctup at the cervical star area, with no placental separation noted. udder development was difficult to assess due to profound ventral edema. right kidney was enlarged and cystic and left kidney appeared shrunken. mare was given altrenogest (0.044 mg/kg, po, q24 hours) and pentoxifylline (7.5 mg/kg, q12 hours). after 24 hours, the ctup was reassessed transabdominally and values decreased in the cranial and mid ventral abdomen (13.4 and 12 mm, respectively). average fetal heart rate was 62 bpm. mare’s creatinine values ranged from 5.5 9.6 mg/dl and was fed a low protein diet (grass hay and concentrates) and diuresed with intravenous fluids (3 liter/hour) and furosemide. six days later, mare showed signs of labor, including restlessness and discomfort. milk ph was 6.2 and calcium was 200 ppm (foal watch, chemetrics inc, midland, va) consistent with eminent parturition (within 24 48 hours). approximately 42 hours later (~ 318 days of pregnancy) the mare foaled a healthy filly. foaling was uneventful, fetal membranes were passed 1 hour after foaling and the foal stood and nursed within 2 hours. fetal membranes weighed 5.5 kg with moderate edema in the amnion and areas of the pregnant and nonpregnant horns. placental thickening was compatible with abnormalities detected during the ultrasound examinations before parturition. histopathologic findings of fetal membranes were consistent with normal pregnancy and uterine edema was not apparent microscopically in either horn, as thickness between pregnant and nonpregnant horns were within reference range. postpartum rectal and vaginal speculum examinations revealed no abnormalities. foal’s creatinine (2.67 mg/dl) was within reference range for a day old foal. although mare’s azotemia and body condition failed to improve after foaling, supportive care enabled the mare to have normal foaling and a viable foal. keywords: pregnancy, renal failure, mares 481 clinical theriogenology • volume 11, number 3 • september 2019 482clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2014: evaluation of the effects of topical permethrin insecticide on bull semen quality evaluation of the effects of topical permethrin insecticide on bull semen quality amanda j. cain, heath king, robert wills, whitney agee, sara howard, richard hopper department of pathobiology and population medicine, college of veterinary medicine, mississippi state university, mississippi state, ms abstract a number of different chemicals, including synthetic pyrethroids are used to treat and prevent ectoparasitic infestations in cattle. permethrin is a type i pyrethroid insecticide that is commonly used to treat cattle for lice and flies via topical application. concerns have recently been raised as to the effects of pyrethroid insecticides, including permethrin, on male fertility and semen quality. however, no controlled studies have been conducted to evaluate the effects of type i pyrethroids on beef bull semen quality. to this end, 15 purebred beef bulls were assigned to either a permethrin treatment group (per; n=8) or control group (con; n=7). bulls in the per group were treated with permethrin 1% (control solutions inc., pasadena, tx) at 150% label dose on d 0 and d 14. sperm motility and morphology were analyzed via light microscopy on d 0, 7, 14, 21, 35, 49, 63, and 84. dermal application of a permethrin insecticide did not affect the proportion of normal sperm cells even at 150% label dose. thus, it was concluded that permethrin parasiticide labeled for livestock use did not increase the risk of producing morphologically abnormal sperm cells. keywords: permethrin, pyrethroid, insecticide, bull fertility, bull breeding soundness introduction pyrethrins and their synthetic derivatives, pyrethroids, have been widely used as insecticides for over 40 years and currently account for over 25% of worldwide insecticide use.1 these chemicals target voltage gated sodium and chloride channels to kill external parasites, but are not considered a toxicological threat to humans or wildlife due to their high insect to mammal toxicity ratio.2,3 nevertheless, concerns have been raised that pyrethroid insecticides may act as potentially harmful endocrine disrupters and cause reproductive dysfunction by altering hormonal pathways. however, in vivo experimental findings as to the endocrine effects of pyrethroid chemicals have been largely inconclusive.4,5 the toxic effects of pyrethroids on mammals, including potential reproductive effects, were reviewed by tsuji et al.6 the majority of studies conducted thus far on the effects of pyrethroids on fertility use rodent models and relatively large, highly concentrated, oral doses of pesticide. thus, these studies do not accurately model a livestock production scenario of pyrethroid exposure. in one recent study, researchers examined the effects of cyfluthrin and beta-cyfluthrin (type ii pyrethroids) pour-on and impregnated eartags at label dosages on bull fertility. there were no significant differences in sperm motility, morphology, and testosterone concentration between pyrethroid exposed and control bulls.7 synthetic pyrethroids have been implicated as the cause of transient male infertility in many livestock species. in case reports, drastic increases in secondary sperm abnormalities, particularly distal midpiece reflexes, were observed in bulls and rams mere days after initial pyrethroid exposure and persisted for up to four weeks after initial insecticide contact.8,9 however, these observations have not been validated in any livestock species in a controlled experimental setting. permethrin is a type i pyrethroid insecticide that is commonly used to treat cattle for ectoparasites, particularly lice and flies, via dermal application. as most currently published studies on permethrin altered fertility focus on teratogenicity and the female reproductive system, there has been much debate as to the effects of permethrin on the male reproductive system. oral administration of permethrin was reported to have had antiandrogenic effects in male rats.5 in another study, sub-chronic dermal exposure of pubescent male rats to permethrin resulted in arrested spermatogenesis and altered testosterone concentrations, however both interactions were dependent on dosage and duration of exposure.10 bull fertility and breeding readiness is commonly evaluated by breeding soundness examinations (bse). the society for theriogenology (sft) has published guidelines designed to assess bull reproductive performance. these standards set thresholds for scrotal circumference (sc), percent of clinical theriogenology • volume 6, number 1 • march 201425 25 morphologically normal sperm, and percent of progressively motile sperm and are used to identify whether or not a bull is a satisfactory potential breeder, physiologically capable of impregnating �25 normally cycling females in a 65 to 70 d breeding season.11,12 thus, bull bses may be used to identify subfertile or transiently infertile males under traditional cattle production scenarios. the objective of the present experiment was to determine the effects of a topical permethrin insecticide on bull semen quality. as pyrethroid chemicals have been used for ectoparasite control in breeding males with no obvious adverse effects for decades, it was hypothesized that dermal application of permethrin would not have a significant deleterious effect on the semen quality and fertility of breeding age beef bulls. materials and methods all procedures were approved by mississippi state university institutional animal care and use committee (protocol #13-031). this project was conducted in the spring of 2013 at h. h. leveck animal research center at mississippi state university. early spring was chosen to initiate this study in attempt to avoid confounded results due to heat and insect stress. animals and experimental design purebred bos taurus beef bulls (n=15) aged 14 months to 4 years were stratified by scrotal circumference (sc) and assigned to either a permethrin treatment group (per; n=8) or control group (con; n=7). bulls in the per group were treated with permethrin 1% (control solutions inc., pasadena, tx) at 150% label dose (225 ml) based on body weight on d 0 and d 14. all bulls received a complete bse11 on d 0 including sc measurement, transrectal palpation of accessory sex glands, and semen evaluation. on d 0, 7, 14, 21, 35, 49, 63, and 84, semen was collected via electroejaculation and sperm gross motility and morphology were analyzed using light microscopy. for morphological analysis, 100 sperm cells were assessed. all samples were analyzed by the same veterinarian who was blinded to treatment group assignments. statistical analyses sas for windows 9.3 (sas institute inc., cary, nc) was used for all statistical analyses. based on a histogram generated with the univariate procedure, the data for initial sc were deemed approximately normal. therefore, the ttest procedure was used to compare initial (d 0) mean sc between the two treatment groups. the proportion of normal sperm cells on d 0 in each of the two groups was compared with logistic regression using the glimmix procedure. the glimmix procedure was also used to analyze dependent variables repeated over time. multivariable repeated measures logistic regression was used to analyze the effects of treatment, time, and their interactions on the proportion of normal sperm cells, primary abnormalities, and secondary abnormalities. events/trials syntax was used in the logistic regression model statements. the residual option and a first-order autoregressive covariance structure, in the case of repeated measures, were specified in the random statements. p-values less than 0.05 were considered significant. data are presented as means standard deviation, unless otherwise noted. results initial fertility average sc on d 0 was 36.1±3.45 cm and 35.5±3.07 cm for per and con bulls, respectively, and was not significantly different between groups (p=0.55). furthermore, there was no significant difference in proportions of morphologically normal sperm on d 0 (p=0.54). thus, initial fertility between bulls in the con and per groups was regarded as not different. additionally, all bulls were body condition score (bcs) 6 at d 0 of the study. table 1 provides descriptive information on the bulls used in the study. after d 35, three bulls (two per and one con) were removed from the study in order to be used for breeding purposes, thus semen quality data from those individuals were not available for inclusion in d 49, 63, and 84 analyses. clinical theriogenology • volume 6, number 1 • march 2014 26 26 table 1. breed composition, age, body condition score (bcs), and initial fertility metrics for permethrin treated (per) and control (con) bulls. group n #angus #charolais #hereford age (mo)* bcs sc (cm) %normal** per 8 7 1 0 17.2 6 36.1±3.45 79.3±10.99 con 7 4 2 1 16.7 6 35.5±3.07 75.4±12.35 *median age in months **mean (±standard deviation) percentage morphologically normal sperm on d 0 critical period for permethrin induced infertility days 0, 14, and 35 were chosen as critical points a priori for sperm morphological analysis. clinically observed pyrethroid-induced fertility changes had rapid onset and persisted for up to four weeks.8,9 if treatment with topical permethrin were to have a deleterious effect on fertility, it should have been clearly evident on both d 14 (14 d after initial application) and d 35 of the study (35 d after the first application and 21 d after the second parasiticide treatment). the results of repeated measures logistic regression for the effects of treatment, day, and treatment x day interactions on sperm morphology measures are presented in table 2. table 2. results of multivariable logistic regression for d 0, 14, 35 for treatment, day, and treatment*day interactions on proportions of morphologically normal sperm and sperm cells with primary or secondary defects from permethrin treated bulls (per) and control bulls (con). outcome variable comparison odds ratio 95% ci of or* p-value normal treatment per vs. con 1.41 0.80, 2.48 0.208 day 0.098 d 35 vs. d 0 1.5049 1.070, 2.0921 d 14 vs. d 0 1.07 0.80, 1.43 treatment*day 0.264 primary treatment per vs. con 0.68 0.34, 1.35 0.242 day 0.152 d 35 vs. d 0 0.67 0.44, 1.01 d 14 vs. d 0 0.84 0.63, 1.11 treatment*day 0.034 per vs. con d 35 0.38 0.030 per vs. con d 14 0.70 0.326 per vs. con d 0 1.16 0.663 secondary treatment per vs. con 0.77 0.44, 1.33 0.315 day 0.106 d 35 vs. d 0 0.74 0.49, 1.12 d 14 vs. d 0 1.07 0.78, 1.45 treatment*day 0.144 *95% confidence interval of the odds ratio proportion of normal sperm cells did not decrease over time as a result of permethrin exposure, and there were no significant differences in sperm morphology between groups. no detrimental effects on semen quality were observed in bulls treated with permethrin. there was a significant treatment x day interaction on the incidence of primary abnormalities due to the apparent protective effect of permethrin against primary defects on d 35 of the study. clinical theriogenology • volume 6, number 1 • march 201427 27 sperm morphology over time bulls in both groups increased the mean percent of morphologically normal sperm as the study progressed. consequently the proportion of sperm cells with abnormal morphology tended to decrease over time. the results of sperm morphology analyses for d 0-84 are illustrated in figures 1-3. the incidence of sperm cells displaying a primary defect is shown in figure 2. as previous studies observed an increase in the number of sperm cells with defects of epididymal origin (i.e. secondary abnormalities), the proportion of secondary defects over time was of particular interest and is shown in figure 3. there was no significant difference in the incidence of secondary abnormalities between per and con during the critical period, and a graph of secondary abnormalities between groups reveals a slight, gradual decrease over time for bulls in both groups. figure 1. mean proportions (±standard deviation) of morphologically normal sperm from d 0-84 from permethrin treated bulls (per) and control bulls (con). the threshold line represents the minimum percent normal sperm cells (70%) in order to achieve satisfactory potential breeder status per sft standards. clinical theriogenology • volume 6, number 1 • march 2014 28 28 * figure 2. mean proportions (±standard deviation) of sperm with primary defects from d 0-84 from permethrin treated bulls (per) and control bulls (con). figure 3. mean proportions (±standard deviation) of sperm with secondary defects from d 0-84 from permethrin treated bulls (per) and control bulls (con). gross motility progressive motility was not compromised by permethrin exposure as all bulls maintained acceptable gross motility (>30% progressively motile, per sft standards) for the entirety of the study. clinical theriogenology • volume 6, number 1 • march 201429 29 discussion results of the present study suggest that topical permethrin does not adversely affect the semen quality of beef bulls. the average proportion of normal sperm cells increased slightly over the course of the study (see figure 1). keeping in mind that the median age at the start of the study was 16.8 months this improvement is consistent with earlier findings in which bull semen quality and percent normal sperm morphology improved in the months following attainment of puberty.13-15 the results of the present study do not agree with the findings of issam et al, in which dermal administration of permethrin led to arrest of spermatogenesis in male rats.10 our results also do not corroborate clinical observations in which the percentage of morphologically normal sperm in bulls and rams was drastically reduced for weeks after pyrethroid exposure. however, the specific types of pyrethroid insecticide implicated in these instances of transient infertility were not specified in all case reports. in at least one instance, the synthetic pyrethroid bifenthrin was presumed responsible for observed teratospermia.8,9 like permethrin, bifenthrin is a type i pyrethroid.16 unlike permethrin, however, bifenthrin is primarily marketed as a premise insect treatment and is not commonly found in parasiticides labeled for animal use. the results of the present study are, however, consistent with the findings of french et al in which cyfluthrin and beta-cyfluthrin insecticides were found to have no deleterious effects on bull fertility.7 however, it should be noted that cyfluthrin and beta-cyfluthrin are type ii pyrethroids, whereas permethrin is a type i pyrethroid. also, unlike the french et al study, the present study applied a greater dose of parasiticide (150% label dose). it should be noted that the present study measured neither androgen hormone concentrations nor libido. it is possible that a chemically induced alteration in either could detrimentally affect the ability of a bull to service and impregnate the cowherd. there is evidence that permethrin may disrupt hormone pathways and alter testosterone concentrations in male rodents.5,10 conclusion in the present study, no detrimental effects on bull semen quality were observed even at 150% of label dose of topical permethrin labeled for livestock use. acknowledgements the authors would like to thank mississippi agriculture and forestry experiment station for study resource support and dr. david smith for assistance with statistical analyses. conflicts of interest the authors declare no conflicts of interest. references 1. schafer tj, meyer da, crofton km: developmental neurotoxicity of pyrethroid insecticides: critical review and future research needs. environ health perspect 2005;113:123-136. 2. miyamoto j, kaneko h, tsuji r: pyrethroids, nerve poisons: how their risks to human health should be assessed. toxicol lett 1995;82-83:933-940. 3. elliot m, janes nf: synthetic pyrethroids-a new class of insecticide. chem soc rev 1978;7: 473-480. 4. kunimatsu t, yamada t, ose k, et al: lack of anti-androgenic or estrogenic effects of three pyrethroids (esfenvalerate, fenvalerate, and permethrin) in the hershberger and uterotrophic assays. regul toxicol pharmacol 2002;35:227-237. 5. kim ss, lee rd, lim kj, et al: potential estrogenic and antiandrogenic effects of permethrin in rats. j reprod dev 2005;51:201-210. 6. tsuji r, yamada t, kawamura s. mammal toxicology of synthetic pyrethroids. top curr chem 2012;314:83-112. 7. french hm, ireland fa, shipley cf, et al: effects of pyrethroid insecticides on cattle fertility. 62nd annu florida beef cattle short course 2013. p. 53. 8. volkmann dh: bull management for optimal reproductive performance. proc appl reprod strategies beef cattle; 2011. p. 297-302. 9. ishmael w. common insecticide may affect bull fertility. beef 2012. clinical theriogenology • volume 6, number 1 • march 2014 30 30 10. issam c, zohara h, monia z, et al: effects of dermal sub-chronic exposure of pubescent male rats to permethrin (prmt) on the histological structures of genital tract, testosterone and lipoperoxidation. exp toxicol pathol 2011;63:393-400. 11. chenoweth pj, hopkins fm, spitzer jc, et al: guidelines for using the bull breeding soundness evaluation form. theriogenology handbook b-10. hastings (ne): society for theriogenology, 1993. p. 1-5. 12. kastelic jp, thundathil jc: breeding soundness evaluation and semen analysis for predicting bull fertility. reprod domest anim 2008;43(suppl 2):368-373. 13. lunstra dd, echternkamp se: puberty in beef bulls: acrosome morphology and semen quality in bulls of different breeds. j anim sci 1982;638-648. 14. almquist jo, amann rp: reproductive capacity of dairy bulls. xi. puberal characteristics and postpuberal changes in production of semen and sexual activity of holstein bulls ejaculated frequently. j dairy sci 1976;59:986-991. 15. martig rc, almquist jo: reproductive capacity of beef bulls. iii. postpuberal changes in fertility and sperm morphology at different ejaculation frequencies. j anim sci 1969;375-378. 16. johnson m, luukinen b, gervais j, et al: bifenthrin technical fact sheet; national pesticide information center. oregon state university extension services 2010. clinical theriogenology • volume 6, number 1 • march 201431 31 32 2010: relationships between stallion age, book size, number of matings (covers), breeding soundness examination findings, and fertility parameters in 15 thoroughbred stallions (34 stallion years) relationships between stallion age, book size, number of matings (covers), breeding soundness examination findings, and fertility parameters in 15 thoroughbred stallions (34 stallion years) t. l. blanchard,a s. p. brinsko,a d. d. varner,a c. c. love,a a. o’meara,b and j. ramseyb adepartment of large animal clinical sciences, college of veterinary medicine and biomedical sciences, texas a&m university, college station, tx; bhill ‘n’ dale farm, lexington, ky abstract pre-season breeding soundness examinations were performed on 15 stallions at one breeding farm in central kentucky during the years 2005–2007 (34 stallion years). pregnancy rates per cycle, seasonal pregnancy rates, and pregnancy loss rates were determined from breeding and foaling reports. spearman rank order correlations or pearson’s product moment correlations between semen quality in second ejaculates and fertility parameters were calculated, as well as between fertility parameters and stallion age, stud fee, testicular size, mare age, book size, and number of covers required to service the mare book. stallion age was negatively correlated to both pregnancy rate per cycle (r = -0.462; p < 0.01) and seasonal pregnancy rate (r = -0.412; p < 0.02). seasonal pregnancy rate was positively correlated to both book size (r = 0.584; p < 0.001) and number of covers required to service the mare book (r = 0.552; p < 0.001). pregnancy rate per cycle was linearly correlated to percent (r = 0.495; p < 0.01) and total number (r = 0.396; p = 0.02) of progressively motile sperm, and percent (r = 0.484; p < 0.01) and total number (r = 0.437; p = 0.01) of morphologically normal sperm, in second ejaculates. pregnancy rate per cycle and mean-αt determined in the sperm chromatin structure assay of second ejaculates were negatively correlated (r = -0.398; p < 0.02). these data support breeding soundness examination findings as beneficial for assessing potential fertility of thoroughbred stallions prior to the onset of the breeding season. keywords: equine, stallion, thoroughbred, breeding soundness examination, fertility introduction performance of breeding soundness examinations prior to onset of the breeding season has been recommended for most domestic large animal species, including the horse.1-3 breeding soundness examinations of the stallion typically include physical examination of the genitalia (including measurement of testicular size), assessment of mating ability, and evaluation of at least two ejaculates collected an hour or more apart.1,2 aerobic culture of urethral swabbings procured after washing, and prior to and after ejaculation, is also recommended. in the last 15 years, performance of the sperm chromatin structure assay has become an integral component of the semen evaluation, as it may detect aberrations in dna integrity that are related to subfertility/infertility, even when sperm quality is sometimes within normal limits.4 when assessing fertility of a given stallion, it should be remembered that a number of factors besides inherent fertility of the stallion contribute to pregnancy outcome, such as number of mares bred in a season,5,6 daily mating frequency,7 and inherent fertility of mares bred (affected by age and/or beginning status [maiden, foaling, barren, etc.]).8-15 it has also been postulated that higher fertility is achieved by stallions with higher stud fees,* perhaps due to more fertile mares comprising their book (i.e., younger, fewer problem breeding mares, etc.) or a higher level of management being involved due to the increased financial risk.6 these factors could preclude finding significant relationships between semen quality and fertility. the purpose of this study was to examine the relationships between stallion age, stud fee, mare age, number of mares bred, number of matings (covers) required to service the mare book, and breeding soundness examination findings to fertility of thoroughbred stallions. 91 materials and methods fifteen stallions at one farm in central kentucky had breeding soundness examinations performed prior to the onset of the 2005-2007 breeding seasons (34 stallion years). breeding soundness examinations were performed according to the guidelines of the society for theriogenology,1 with total testicular volumes predicted from individual caliper measurements of the testes according to love et al.16 a minimum of two ejaculates were collected in an artificial vagina using a mare in estrus as the mount source. swabbings were procured from the urethra after washing, and prior to and after ejaculation, as well as a swabbing of the raw filtered semen, for aerobic bacterial culture on blood and macconkey’s agar. volume of gel-free semen was measured in a graduated cylinder. sperm concentration in gel-free semen was determined spectrophotometrically (590b equine densimeter, animal reproduction systems, chino, ca). an aliquot of gel-free semen was diluted in warm semen extender (inra 96 semen extender, imv technologies, maple grove, mn) to a concentration of 25 million sperm/ml. a 5 µl drop of extended semen was placed on a warm microscope slide and cover slipped, and sperm motility (total/progressive) was assessed by one examiner using a phase contrast microscope (250x) with warming stage. an aliquot of raw semen was mixed in 2% buffered formal saline, and a 5 µl drop was placed on a microscope slide and cover slipped, and examined under oil immersion (1250x) using differential contrast microscopy to assess sperm morphologic defects in 100 sperm. the examiner (tlb) of sperm motility and morphology was not blinded to the stallion. an aliquot of raw gel-free semen was pipetted into a cryovial and snap frozen in liquid nitrogen until assessed by sperm chromatin structure assay. after staining with acridine orange, a minimum of 5000 sperm cells were evaluated for dna fluorescent patterns by flow cytometry according to the method of kenney et al.4 data retrieved from records included results of the pre-season breeding soundness examinations (total testicular volume, total number of sperm in second ejaculate, percentage and total number of progressively motile sperm in second ejaculate, percentage and total number of morphologically normal sperm in second ejaculate, total number of progressively motile, morphologically normal sperm (product of total number of sperm, percent progressively motile sperm, and percent morphologically normal sperm) in second ejaculate, and results of the sperm chromatin structure assay from sperm in second ejaculate (mean-αt, standard deviation-αt, and comp-αt). breeding records from this farm were examined to retrieve data on pregnancy rate per cycle, seasonal pregnancy rate, and pregnancy loss (at any time after establishment of pregnancy) rate for the years 2005 to 2007. additional data retrieved for comparisons included age of the stallion, book size (number of mares bred) for each given year, number of matings (covers) required to service the book of mares for each given year, mean mare age, and stud fee for each given year. the shapiro-wilk statistic was used to test normality of the data. data for fertility parameters (pregnancy rate per cycle, seasonal pregnancy rate, and pregnancy loss rate) were normally distributed (p > 0.01). pearson product moment correlations were performed to determine linear relationships among normally distributed data (book size, number of covers, mean mare age, total testicular volume, percentage and number of progressively motile sperm in second ejaculates, percentage of morphologically normal sperm in second ejaculates, number of progressively motile, morphologically normal sperm in second ejaculates, and mean-αt and standard deviation-αt in second ejaculates, and fertility parameters. spearman rank order correlations were determined to examine linear relationships among fertility parameters and non-normally distributed data (stallion stud fee, stallion age, total sperm number in second ejaculates, total number of morphologically normal sperm in second ejaculates, and comp-αt of sperm in second ejaculates; p < 0.01). results stallion ages during the study ranged from 4 to 25 years. stud fees for stallions ranged from $5,000 to $60,000. the range in book size and number of covers per year for stallions were 23 to 199 mares, and 27 to 305 covers per year, respectively. overall pregnancy rates per cycle achieved by stallions during this study ranged from 50 to 73%. yearly pregnancy rates per cycle for individual stallions varied from none (0%) to as much as 15% 92 between years (table 1). stallion age was negatively correlated to both pregnancy rate per cycle (r = 0.462; p < 0.01) and seasonal pregnancy rate (r = -0.412; p < 0.02). no relationship (p > 0.05) existed between pregnancy rate per cycle or pregnancy loss rate and stud fee, book size, or number of covers required to service the mare book. while stud fee was not correlated with seasonal pregnancy rate (p > 0.05), book size (r = 0.584; p < 0.001) and number of covers (r = 0.552; p < 0.001) were correlated to seasonal pregnancy rate. mean mare age for the mares within a stallion’s yearly book was not correlated (p > 0.05) with pregnancy rate per cycle or per season, or with pregnancy loss rate. however, stallion stud fee was positively correlated to book size (r = 0.409; p < 0.02), and was negatively correlated with mean mare age (r = -.520; p < 0.01). no stallion had pseudomonas aeruginosa or klebsiella pneumoniae (considered potential venereal pathogens) isolated from urethral swabbings procured before or after ejaculation, nor from swabbings of the raw semen. no stallions had neutrophils present in semen, suggesting internal genital infections did not exist in these stallions. using guidelines proposed by kenney et al.,1 all stallions had normal total testicular size (except for two stallions, stallions 10 and 11, each having only one scrotal testis of normal size). total testicular volumes in stallions with two scrotal testes ranged from 271 cc to 536 cc. no correlations between total testicular volume and fertility parameters were detected (p > 0.05). per cycle pregnancy rate was positively correlated to percentage of progressively motile sperm (r = 0.495; p < 0.01), total number of progressively motile sperm (r = 0.396; p = 0.02), percent morphologically normal sperm (r = 0.484; p < 0.01), total number of morphologically normal sperm (r = 0.437; p < 0.01), and total number of progressively motile, morphologically normal sperm (r = 0.402; p < 0.02) in second ejaculates. pregnancy rate per cycle was negatively correlated with mean-αt of second ejaculates determined in the sperm chromatin structure assay (r = -0.398; p < 0.02), but no correlation (p > 0.05) was detected among any fertility parameters and standard deviation-αt or comp-αt. no correlations (p > 0.05) were detected between semen quality parameters and seasonal pregnancy rate. pregnancy loss rate was not correlated with any of the parameters recorded/measured (p > 0.05). discussion it has been postulated that higher fertility is achieved by those stallions with higher stud fees. reasoning provided for a relationship between stud fee and fertility is that more fertile, better managed mares will comprise the mare book when stud fees are high because of the increased financial risk. however, within the range of stud fees for stallions standing at this farm, we found no significant linear relationship between stud fee and fertility parameters. stud fees for thoroughbred stallions standing in central kentucky can exceed $500,000. perhaps if more stallions with very high stud fees were included in the data, significant correlations between stud fee and fertility might have existed. alternatively, the management level of mares bred at thoroughbred farms in central kentucky may be optimal (e.g., negative cultures were required from all but first cycle foaling mares at this farm; use of ovulationinducing drugs to promote ovulation within two days of breeding and examining dismount samples to ensure the stallion ejaculated during mating are common practices at most thoroughbred farms in central kentucky), regardless of the stud fee for the stallion used for mating, thereby negating influence of management on overall fertility. the range in pregnancy rates per cycle included in this study was similar to that described by others when surveying fertility of large groups of thoroughbreds, and were within ranges reported as normal.10,15,17 ages for stallions during the study ranged from four to 25 years. the negative correlation between stallion age and pregnancy rate per cycle could be explained, in part, by some age-related decline in testicular function that typically occurs in older stallions. others have described age-related decline in fertility in stallions,7,12,18 as well as individual stallion variation in fertility.7,10,12,19,20 no attempt was made to determine sources of variation in fertility among years within stallions, as the beginning status and age of a stallion’s book of mares can vary significantly from year to year based on his popularity as a sire. in one report of a thoroughbred stallion experiencing an 11% decline in 93 pregnancy rate per cycle and per season between two successive years of breeding, the percentage of barren mares comprising the mare book more than tripled to 25%, and the associated low pregnancy rate in this group of mares accounted for most of the decline in fertility achieved between years.3 however, how popularity of a stallion influences the age and beginning status of mares within the mare book has not been well described. the finding of positive correlations between seasonal pregnancy rate and book size, and between seasonal pregnancy rate and number of covers required to service a stallion’s book of mares, is interesting. baker et al.5 reviewed jockey club foaling records for stallions standing in kentucky during 1987, and found reproductive success increased as book size increased, with foaling rates being 47% for stallions with the smallest book sizes and 70% for stallions with the largest book sizes. turner and mcdonnell6 confirmed similar findings in a retrospective study of jockey club records for thoroughbred stallions of various sized mare books during 2003–2005.6 baker et al.5 postulated that mares on large farms were managed better so that they had increased opportunity to become pregnant. they theorized the primary reason mares on larger farms had a better chance of becoming pregnant in a season was that a higher level of management resulted in earlier detection of failure to become pregnant, and thus more chances for rebreeding. our finding that pregnancy rates per cycle were higher in stallions with larger books would suggest that either more fertile mares were presented to these stallions, or the mares were managed better to increase their initial opportunity to become pregnant. stallion stud fee was positively correlated to book size, indicating that at this farm the stallions with higher stud fees were more popular. also of interest was our finding that stallion stud fee was significantly negatively correlated with mean mare age. it is well known that younger mares are more fertile than older mares,8-15 so perhaps more young, fertile mares are presented to stallions with higher stud fees because more investment is at risk. this could explain some of the increase in fertility of the more popular stallions. we were not surprised that no correlations were detected between total testicular volume and fertility parameters. poor semen quality due to testicular dysfunction is commonly associated with small testes, and none of the stallions in this study had smaller than normal testicular volumes.20,21 semen parameters often reported to be correlated with fertility in stallions include percent progressively motile sperm and percent morphologically normal sperm.4,22-25 our finding that percentage and number of progressively motile sperm, or percentage and number of morphologically normal sperm, in second ejaculates were significantly correlated with pregnancy rate per cycle supports earlier findings. so, preseason breeding soundness examination findings were associated with fertility parameters in this group of fertile stallions. perhaps if some stallions at this farm were subfertile, more variation in semen quality parameters would have existed, thereby resulting in greater correlations among semen characteristics and fertility parameters. however, the fact that correlations could be found in this small subset of fertile stallions suggests that the breeding soundness examination could be even more useful to separate fertile from subfertile/infertile stallions. of additional interest is the finding of significant correlations of semen quality parameters with pregnancy rate per cycle, but not with seasonal pregnancy rate. pregnancy rate per cycle is probably a more representative indicator of stallion fertility than seasonal pregnancy rate. stallions with low pregnancy rates per cycle can achieve satisfactory seasonal pregnancy rates if mares that do not become pregnant on the first estrous cycle of breeding have sufficient opportunity to be rebred promptly on subsequent estrous cycles.5 we expected comp-αt to be significantly associated with fertility parameters, as it has been cited as the sperm chromatin assay statistic of semen quality either better correlated with infertility in the horse,4,26 or showing a greater percentage of change in cooled semen quality between fertile and subfertile stallions.27 perhaps the reason it was not associated with fertility parameters in this study was that only three stallions (stallion 9 in 2006 [18%], stallion 12 in 2006 [18%], and stallion 7 in 2005 [27%]) had values elevated over 16% comp-αt, the maximum value reported as normal.4 kenney et al.4 reported all scsa statistics were elevated in subfertile stallions achieving a 38% seasonal pregnancy rate when compared to normal stallions achieving a 70% seasonal pregnancy rate. the finding of a negative association between mean-αt and pregnancy rate per cycle in this group of normally fertile stallions could 94 suggest that it is a more sensitive indicator of unstable sperm chromatin. a shift in mean-αt represents a shift in the chromatin stability of the entire population of sperm in the ejaculate. we were also interested in investigating the relationship between sperm chromatin integrity and pregnancy loss rate. if sperm with lowered chromatin integrity resulted in fertilization, it is possible the developing conceptus would be compromised and thus more prone to loss. however, scsa statistics were not significantly related to pregnancy loss rates in this study. summary our findings suggest seasonal pregnancy rate is improved in thoroughbred stallions breeding large books of mares, requiring more covers to service the mare book, than in those stallions breeding smaller books of mares. whether this relationship is due to increased level of management could not be determined from this study. our finding of linear relationships between pregnancy rate per cycle and progressive sperm motility, morphologically normal sperm, and mean-αt, of sperm confirms the value of these semen parameters for evaluating stallions for potential breeding soundness. since pregnancy rate per cycle was significantly correlated with several semen quality parameters while seasonal pregnancy rate was not, it may be a more sensitive indicator of fertility of a stallion. *pickett bw, personal communication, 1982. references 1. kenney rm, hurtgen j, pierson r, et al: theriogenology and the equine. part ii. the stallion. j soc therio 1983; vol ix. p. 1-100. 2. varner dd, schumacher j, blanchard tl, et al: diseases and management of breeding stallions. santa barbara: american veterinary publications; 1991. p. 61-96. 3. blanchard tl: management of subfertile stallions in natural service programs. in: mckinnon ao, samper j, pycock jc, et al., editors. equine reproduction. 2nd ed. philadelphia: lea and febiger; in press. 4. kenney rm, evenson d, garcia mc, et al: relationships between sperm chromatin structure, motility, and morphology of ejaculated sperm, and seasonal pregnancy rate. biol reprod 1995; mono 1:647-653. 5. baker cb, little tv, mcdowell kj: normal reproductive success rates in thoroughbreds. proc annu meet soc therio 1992; p.71-78. 6. turner rm, mcdonnell sm: mounting expectations for thoroughbred stallions. j am vet med assoc 2007;230:1458-1460. 7. merkt h, jacobs k–o, klug e, et al: an analysis of stallion fertility rates (foals born alive) from the breeding documents of the landgestűt celle over a 158-year period. j reprod fertil 1979; suppl 27:73-77. 8. sullivan jj, turner pc, self lc, et al: survey of reproductive efficiency in the quarter-horse and thoroughbred. j reprod fertil 1975; suppl 23;315-318. 9. laing ja, leech fb: the frequency of infertility in thoroughbred mares. j reprod fertil 1975; suppl 23;307-310. 10. baker cb, little tv, mcdowell kj: the live foaling rate per cycle in mares. equine vet j 1993; suppl 15;28-30. 11. brűck i, angerson ga, hyland jh: reproductive performance of thoroughbred mares on six commercial stud farms. aust vet j 1993;70:299-303. 12. davies morel mcg, gunnarsson v: a survey of the fertility of icelandic stallions. anim reprod sci 2000;64:49-64. 13. amann rp: the fertility dilemma: perception vs. actuality. equine vet educ 2006;june:203-208. 14. blanchard tl, thompson ja, love cc, et al: role of reinforcement breeding in a natural service mating program. proc annu conv am assoc equine pract 2006; p. 384-386. 15. allen wr, brown l, wright m, et al: reproductive efficiency of flatrace and national hunt thoroughbred mares and stallions in england. equine vet j 2007;39:438-445. 16. love cc, garcia mc, riera fr, et al; evaluation of measures taken by ultrasonography and caliper to estimate testicular volume and predict daily sperm output in the stallion. j reprod fertil 1991; suppl 44:99-105. 17. morris lha, allen wr: reproductive efficiency of intensively managed thoroughbred mares in newmarket. equine vet j 2002;34:51-60. 18. douglas rh, umphenour n: endocrine abnormalities and hormonal therapy. vet clin north am equine pract 1992;8:237-250. 19. dusek j, munk z: the effect of the age of stallions and mares on their fertility. vet med (praha) 1980;25:437-448. 20. blanchard tl, brinsko sp, love cc, et al. how to use testicular measurements for first-season subfertility insurance considerations in thoroughbred stallions. proc annu conv am assoc equine pract 2008; p. 374-379. 21. blanchard tl, johnson l, varner dd, et al: low daily sperm output per ml of testis as a diagnostic criteria for testicular degeneration in stallions. j equine vet sci 2001;21:11, 33-35. 95 22. voss jl, pickett bw, squires el: stallion spermatozoal morphology and motility and their relationship to fertility. j am vet med assoc 1981;178:287-289. 23. dowsett kf, pattie wa: characteristics and fertility of stallion semen. j reprod fertil 1982; suppl 32:1-8. 24. jasko dj, lein dh, foote rh: determination of the relationship between sperm morphologic classifications and fertility in stallions: 66 cases (1987-1988). j am vet med assoc 1990;197:389-394. 25. love cc, varner dd, thompson ja: intraand inter-stallion variation in sperm morphology and their relationship with fertility. j reprod fertil 2000; suppl 56:93-100. 26. love cc, kenney rm: the relationship of increased susceptibility of sperm dna to denaturation and fertility in the stallion. theriogenology 1998;50:955-972. 27. love cc, thompson ja, lowry vk, et al: the relationship between chromatin quality and fertility of chilled stallion semen. proc annu conv am assoc equine pract 2001; p. 229-231. 96 ta bl e 1. s em en q ua lit y fin di ng s in s ec on d ej ac ul at es o bt ai ne d du rin g br ee di ng s ou nd ne ss e xa m in at io ns a nd p re gn an cy ra te p er c yc le (p r /c yc le ), se as on al p re gn an cy ra te (s p r ). an d pr eg na nc y lo ss ra te (p lr ), ac hi ev ed b y 15 t ho ro ug hb re d st al lio ns o n on e fa rm in c en tra l k en tu ck y du rin g 20 05 – 2 00 7 (3 4 st al lio n ye ar s) . s ta lli on y ea r a ge b oo k a m a ge n o. s pe rm % p m s % m n s n o. p m m n s m ea nα t s d α t % c o m p -α t p r /c yc le s p r p lr 1 20 05 6 14 2 9. 31 5. 92 1 65 % 77 % 2. 96 3 23 8 54 5% 64 .8 % 90 .1 % 18 .8 % 1 20 06 7 17 2 9. 33 8. 72 0 55 % 69 % 3. 30 9 19 8 40 3% 69 .9 % 90 .1 % 18 .2 % 1 20 07 8 33 9. 62 11 .4 18 65 % 71 % 3. 19 7 24 7 45 7% 76 .9 % 90 .9 % 3. 3% 2 20 05 6 17 0 10 .5 6 10 .8 16 50 % 56 % 3. 02 8 24 0 23 5% 68 .7 % 93 .5 % 24 .3 % 2 20 06 7 14 6 10 .0 2 7. 05 6 40 % 46 % 1. 29 8 22 9 70 13 % 66 .5 % 85 .6 % 25 .2 % 2 20 07 8 14 0 10 .1 6 8. 35 2 50 % 55 % 2. 29 7 23 8 51 13 % 78 .4 % 92 .9 % 22 .1 % 3 20 05 9 64 10 .0 7 15 .1 20 65 % 56 % 5. 50 4 23 3 34 5% 64 .2 % 84 .4 % 13 .1 % 4 20 05 8 12 1 9. 51 12 .2 50 55 % 72 % 4. 89 1 24 0 44 7% 67 .7 % 90 .9 % 14 .2 % 4 20 06 9 19 5 9. 26 31 .6 80 35 % 51 % 5. 65 5 23 3 69 14 % 69 .8 % 93 .9 % 17 .9 % 4 20 07 10 11 5 9. 63 7. 22 0 60 % 63 % 2. 72 9 21 5 45 8% 67 .1 % 85 .2 % 11 .0 % 5 22 6 5 18 7 9. 99 6. 81 6 45 % 42 % 1. 28 8 24 4 42 5% 55 .4 % 86 .1 % 16 .0 % 5 20 07 6 10 1 10 .5 7 8. 20 0 30 % 25 % 0. 65 6 22 6 35 7% 69 .7 % 91 .1 % 16 .2 % 6 20 05 21 27 11 .9 1 5. 53 9 45 % 48 % 1. 19 6 24 8 56 12 % 59 .1 % 92 .6 % 19 .2 % 6 20 06 22 28 10 .6 8 6. 68 5 30 % 32 % 0. 64 2 25 9 53 13 % 59 .5 % 78 .6 % 13 .6 % 7 20 05 18 88 11 .0 8 8. 58 8 50 % 52 % 1. 47 5 27 2 49 27 % 53 .2 % 81 .2 % 15 .7 % 8 20 05 6 18 4 9. 89 8. 58 8 65 % 67 % 3. 74 0 25 5 30 8% 67 .9 % 94 .6 % 11 .1 % 9 20 05 14 11 3 9. 58 11 .0 88 60 % 70 % 4. 65 7 25 8 38 7% 58 .9 % 89 .4 % 25 .7 % 9 20 06 15 65 10 .0 2 14 .1 88 60 % 56 % 4. 76 7 25 9 88 18 % 70 .7 % 93 .9 % 24 .6 % 9 20 07 16 11 4 9. 69 12 .7 80 70 % 55 % 4. 92 0 25 0 42 8% 58 .0 % 85 .1 % 17 .1 % 10 20 06 4 19 7 9. 68 6. 03 1 55 % 51 % 1. 69 2 23 1 49 7% 70 .1 % 93 .4 % 17 .4 % 10 20 07 5 11 2 10 .2 5 11 .6 61 60 % 68 % 4. 75 8 23 0 44 4% 78 .1 % 90 .2 % 21 .1 % 11 20 05 6 12 0 9. 74 3. 22 5 45 % 38 % 0. 55 1 24 0 55 11 % 54 .8 % 90 .0 % 11 .4 % 12 20 05 11 15 9 8. 96 12 .2 08 60 % 61 % 4. 50 5 25 3 60 13 % 63 .9 % 89 .9 % 22 .7 % 12 20 06 12 19 1 9. 36 8. 72 0 35 % 50 % 1. 52 6 23 8 67 18 % 61 .2 % 98 .5 % 16 .9 % 12 20 07 13 19 9 9. 70 6. 83 6 50 % 48 % 1. 64 1 25 4 50 10 % 68 .1 % 91 .5 % 23 .4 % 13 20 05 23 42 8. 44 16 .4 40 35 % 47 % 2. 70 4 25 8 38 7% 58 .3 % 88 .1 % 14 .3 % 13 20 06 24 50 10 .3 5 11 .3 30 25 % 35 % 0. 99 1 24 4 39 9% 52 .3 % 76 .0 % 15 .6 % 13 20 07 25 36 9. 88 10 .2 12 20 % 35 % 0. 71 5 25 7 40 10 % 35 .7 % 77 .8 % 25 .0 % 14 20 05 5 13 6 9. 14 3. 27 6 65 % 58 % 1. 23 5 24 6 51 10 % 66 .0 % 92 .7 % 20 .1 % 14 20 06 6 13 6 9. 76 7. 71 0 60 % 52 % 2. 40 6 24 1 68 16 % 64 .2 % 87 .5 % 16 .3 % 14 20 07 7 12 7 9. 71 8. 15 4 55 % 57 % 2. 55 6 25 5 59 13 % 72 .2 % 94 .5 % 16 .9 % 15 20 05 9 40 10 .3 7 10 .9 48 60 % 64 % 4. 20 4 23 8 54 8% 61 .4 % 77 .5 % 14 .3 % 15 20 06 10 35 9. 98 11 .2 69 55 % 55 % 3. 40 9 23 2 66 10 % 67 .3 % 88 .6 % 21 .2 % 15 20 07 11 23 11 .5 9 13 .1 13 65 % 64 % 5. 45 5 24 1 39 6% 70 .4 % 82 .6 % 15 .8 % k ey : a ge = y ea rs , b oo k = nu m be r o f m ar es b re d in s ea so n; n o. s pe rm = to ta l n um be r o f s pe rm in b ill io ns ; a m ag e = av er ag e ag e in y ea rs o f m ar es in b oo k; 9 .7 4% p m s = pe rc en ta ge o f p ro gr es si ve ly m ot ile s pe rm ; % m n s = pe rc en ta ge o f m or ph ol og ic al ly n or m al s pe rm ; n o. p m m n s = to ta l n um be r o f p ro gr es si ve ly m ot ile , m or ph ol og ic al ly n or m al s pe rm in b ill io ns ; m ea nα t = m ea n ch an ne l o f e nt ire d is tri bu tio n of s pe rm c el ls s ta in ed w ith a cr id in e or an ge th at fl uo re sc e gr ee n or re d de pe nd in g on e xt en t o f d n a d en at ur at io n as de te rm in ed b y flo w c yt om et er in s pe rm c hr om at in s tru ct ur e a ss ay ; s d -α t = s ta nd ar d de vi at io n of s pe rm c el ls s ta in ed w ith a cr id in e or an ge th at fl uo re sc e gr ee n or re d de pe nd in g on ex te nt o f d n a d en at ur at io n as d et er m in ed b y flo w c yt om et er in s pe rm c hr om at in s tru ct ur e a ss ay ; % c o m p -α t = p er ce nt ag e of s pe rm c el ls s ta in ed w ith a cr id in e or an ge o ut si de m ai n po pu la tio n de pe nd in g on e xt en t o f d n a d en at ur at io n as d et er m in ed b y flo w c yt om et er in s pe rm c hr om at in s tru ct ur e a ss ay ; p r /c yc le = p re gn an cy ra te p er c yc le ; s p r = se as on al p re gn an cy ra te ; p lr = p re gn an cy lo ss ra te (i nc lu de s pr eg na nc ie s lo st d ur in g an y st ag e of g es ta tio n) . 97 98 omniblank: 2018: seasonality affects semen cryopreservation of white-tailed deer bucks (odocoileus virginianus) throughout rut seasonality affects semen cryopreservation of white-tailed deer bucks (odocoileus virginianus) throughout rut jamie l. stewart,a clifford f. shipley,a robyn e. ellerbrock,ab fabio s. lima,a igor f. canissoa acollege of veterinary medicine, university of illinois, urbana, il; bcollege of veterinary medicine, university of georgia, athens, ga white-tailed deer farming is a growing industry in the united states, with breeding operations contributing significantly to the industry’s economic impact. artificial insemination with frozen semen allows for selection and dissemination of valuable genetics, yet surprisingly little information is available regarding the best time throughout rut to perform semen cryopreservation. the objective of this study was to compare the efficacy of semen cryopreservation of white-tailed deer bucks collected early in the breeding season (september), at peak rut (december), and late season (march). we hypothesized that semen freezing ability would be enhanced at peak rut. mature bucks (n = 7-11) were anesthetized with tiletamine-zolazepam (0.88 mg/kg) and xylazine (2.2 mg/kg) administered intramuscularly via projector. the penis was manually exteriorized, and semen was collected by electroejaculation (pulsator iv, lane manufacturing, inc). an aliquot of the ejaculate was diluted 1:60 in warm (37°c) optixcell extender (imv technologies) and placed into a pre-warmed 20 µm chamber slides (vitrolife, microcell counting chambers) for assessment of motility via computer-automated sperm analysis (spermvision ii, minitube of america). the remainder of each sample was extended with optixcell to a final concentration of 120 million sperm/ml. ejaculates were cooled to 5°c over 1 h, loaded into 0.5-ml straws, and incubated for 3 to 5 h. straws were frozen manually in liquid nitrogen vapors at 4 cm above the liquid nitrogen level for 15 m before submerging them for final freezing and storage. each semen straw was thawed in a 37°c water bath for 30 s for post-thaw analyses. overall and progressive sperm motilities were evaluated in each sample using computer-automated semen analysis. additionally, samples were stained with fluorescent probes and subjected to flow cytometry for characterization of sperm viability (sybr-14/pi), acrosome integrity (fitc-pna/pi), and chromatin stability (acridine orange). data were analyzed using a kruskal-wallis chi-squared test with a dunn posthoc test (non-parametric) or anova with repeated measures and tukey post-hoc test (parametric) in r version 3.2.2 (https://www.r-project.org/). pre-freeze overall and progressive sperm motilities were lowest in march and highest in december (p≤0.04). however, post-thaw overall and progressive motilities were lowest in september and highest in december (p=0.01). the dna fragmentation index was lowest in december (p<0.01), but did not differ between march and september (p=0.23). the percentage of live sperm cells was highest in december (p=0.03), and the percentage of intact acrosomes per live sperm cell was highest in september (p=0.02). this study confirms that bucks have superior semen quality when in peak rut (december). though semen collected early or late in rut may present acceptable motility, dna stability appears to be impaired, which could adversely affect fertility rates. therefore, we suggest that semen cryopreservation be performed closer to peak rut to optimize post-thaw semen quality and ensure successful artificial insemination outcomes. keywords: andrology, cervid, fluorescent probes, semen cryopreservation clinical theriogenology • volume 10, number 3 • september 2018 304 successful treatment of presumptive cystic ovarian disease in okapi (okapia johnstonii) successful treatment of presumptive cystic ovarian disease in okapi (okapia johnstonii) anneke moresco,a gwen jankowski,a loren berry,a jacobo rodríguezb adenver zoo, denver co, bvista equine colorado, fort collins, co a 7 year old parous, female okapi was presumptively diagnosed with cystic ovarian disease (cod) based on persistent estrus behavior and a lack of conception, despite continued breeding. in nondomestics, routine diagnostics (serum hormones and transrectal ultrasonography) are challenging, even with intense behavioral training and may still require general anesthesia. diagnosis of cod is even more difficult in giraffids, as inter estrus intervals are only 14 days, duration of estrus is 2 3 days, estrus signs can be subtle and breeding behavior can be related to dominance. since evidence is obtained mainly by observation, experienced keepers and meticulous recordkeeping are key for a presumptive diagnosis. this female was trained for tactile contact, but not for transrectal ultrasonography or blood collection. during full anesthesia (medetomidine, ketamine and thiafentanil), on transrecal ultrasonography, the right ovary contained a large (34 x 29 mm) thick-walled (9 mm) structure containing hyperechoic fluid. the left ovary appeared normal in size without large antral follicles and the uterus lacked endometrial edema. serum progesterone (2.9 ng/ml) was consistent with active luteal tissue and 2 doses of dinoprost (12.5 mg) were given im 48 hours apart. three days after the second dose she displayed estrus behavior. four weeks after initial examination, a 12 mm follicle with clear fluid and thin walls, was present on the right ovary and the uterine horns were edematous. ovulation was induced with im deslorelin (3.4 mg) and hcg (5,000 iu). she went through 3 apparently normal cycles without conceiving, whereas during the fourth cycle, estrus lasted 7 days. ovulation was induced again and she conceived as documented by increased and sustained fecal progesterone metabolites. fetal sex (male) was determined at ~ 7 months gestation via transabdominal ultrasonography. keywords: okapia johnstonii, cystic ovarian disease, ultrasonography, ovulation, induction 523 clinical theriogenology • volume 11, number 3 • september 2019 524clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 1 contact sara lyle saraklyle3@gmail.com © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10705, http://dx.doi.org/10.58292/ct.v16.10705 case report hemoperitoneum secondary to bilateral ovarian enlargement due to undiagnosed male cotwin pregnancy in a mare natalie orner,a michael whitacre,b megan burke,b amber nebel-karp,c jose len,d sara lyleb‡ asouthern oaks animal hospital, hope mills, nc, usa bdepartment of clinical sciences, college of veterinary medicine, north carolina state university, raleigh, nc, usa canimal hospital of east davie, advance, nc, usa dveterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la, usa ‡current affiliation: veterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la, usa abstract a 17-year, quarter horse mare, was presented for colic symptoms that began the day prior to presentation; patient had no breeding history. severe bilateral ovarian enlargement precluded transrectal palpation of the gastrointestinal tract and much of the uterus. transrectal ultrasonography revealed severely enlarged ovaries with multiple, large, ovarian follicles, and structures with an echotexture consistent with hemorrhagic anovulatory follicles, corpora hemorrhagica, and hematoma formation. transcutaneous abdominal ultrasonography revealed a large volume of peritoneal fluid. abdominocentesis was performed that identified fluid consistent with frank blood (packed cell volume of 40% consistent with intraabdominal hemorrhage). initial differential diagnoses included bilateral granulosa-theca cell tumors, other ovarian neoplasia, or ruptured ovarian corpora hemorrhagicum/hematoma resulting in hemoperitoneum. repeat abdominal ultrasonography revealed a viable pregnancy (~ 70 days). additional diagnostics obtained 5 days after admission had severely elevated serum testosterone (517.8 pg/ml), elevated inhibin b (160.7 pg/ml), and normal antimüllerian hormone (0.12 ng/ml) concentrations. after misoprostal and dinoprost tromethamine treatment, manual termination was performed that resulted in the removal of 2 male cotwins. ovarian size markedly reduced soon after pregnancy termination and serum hormonal concentrations decreased 1 week later to concentrations approaching the reference range for a mare in the first trimester of pregnancy. keywords: mares, twin pregnancy, peritoneal fluid, pregnancy termination background cases of hemoperitoneum secondary to ovarian pathology are rare but have been reported.1,2 if an ovarian cause of hemoperitoneum is suspected, the primary rule out is granulosa-theca cell tumor (gct); other ovarian neoplasia and ruptured corpus hemorrhagicum have also been reported.1-4 other causes of hemoperitoneum secondary to gastrointestinal conditions or vascular anomalies such as rupture of hepatobiliary vasculature, neoplasms, traumatic injury to the pelvis, coagulopathy, and foaling or pregnancy complications.3,4 because of nonspecific clinical symptoms including colic and lethargy, multimodal approach was necessary for diagnosis and treatment. case presentation a 17-year, 461 kg, quarter horse mare, was presented for evaluation of mild colic symptoms that began a day before and were not responsive to 200 mg of flunixin meglumine treatment. according to the owner, the mare had no breeding history within the past year and was used for competitive barrel racing. the mare was examined by the primary veterinarian on the farm who diagnosed a potential large colon impaction and recommended referral. on initial examination by the emergency service, the mare was observed to be bright and alert with mild tachycardia (60 beats per minute [bpm]); had normal respiratory rate and temperature. the mare’s mucous membranes were pink and tacky with capillary refill time mailto:saraklyle3@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10705 2 citation: clinical theriogenology 2024, 16, 10705, http://dx.doi.org/10.58292/ct.v16.10705 between 1-2 seconds. gastrointestinal borborygmi were normal in all 4 abdominal quadrants and colic symptoms were absent on presentation. transrectal palpation revealed 2 severely enlarged ovaries that limited palpation of the remainder of the gastrointestinal tract. transrectal ultrasonography confirmed enlarged ovaries (> 10 cm) with multiple large structures with an echotexture consistent with corpora hemorrhagica, hematomas, or hemorrhagic anovulatory follicles (figure 1). images of the uterine body also confirmed a large volume of hypoechoic fluid. due to ovarian and uterine size, the entire reproductive tract was not palpable transrectally. transabdominal ultrasonography revearled a large volume of swirling free peritoneal fluid; abdominocentesis was performed. fluid obtained via abdominocentesis was consistent with frank blood (6.3 mmol/l lactate, 40% packed cell volume [pcv], 4.5 g/dl total protein). comparison of peripheral pcv (33%) and total protein (4.6 g/dl) confirmed acute hemorrhage into the peritoneal cavity. elevation of abdominal and peripheral lactate was presumably a result of combination of dehydration and acute blood loss into the peritoneal cavity leading to decreased tissue perfusion. hemoperitoneum can also be responsible for intense local inflammation that may lead to hyperlactatemia.5 although physical examination parameters did not suggest that the patient experienced hemorrhagic shock due to blood loss, the decision to stabilize the patient using whole blood was made by the attending emergency clinician out of concern for the potential for further losses and resultant destabilization of the patient. initial stabilization was performed prior to a theriogenology consult and included an unmatched 8.5 liters whole blood transfusion and constant rate infusion of aminocaproic acid, followed by maintenance treatment of isotonic crystalloids to ensure adequate hydration. the patient’s heart rate returned to 46 bpm immediately after transfusion and serial pcvs confirmed that there was no continued blood loss. follow up focused abdominal ultrasonographic revealed resorption of free peritoneal fluid. repeat transrectal ultrasonography was performed, and ovarian findings and uterine echotexture were similar to that at presentation. hypoechoic fluid in the uterine lumen was similar in appearance to allantoic fluid, and amniotic membrane was suspected. extensive transabdominal ultrasonography revealed a viable fetus (~ 70 days).6 fetal heart rate was within normal limits, and blood flow to the umbilicus was confirmed using color doppler. serum was submitted (uc davis clinical endocrinology laboratory) for gct panel and results were: normal antimüllerian hormone (amh) [0.12 ng/ ml]), moderately elevated inhibin b (160.7 pg/ml), and markedly elevated testosterone (517.8 pg/ml) concentrations. differential diagnoses initial differentials included ovarian enlargement secondary to ecg production in pregnancy, ruptured ovarian hematomas, gct, or some combination thereof. based on confirmation of pregnancy and endocrinological findings of markedly elevated testosterone and moderately elevated inhibin concentrations, a provisional diagnosis (bilateral gct development during pregnancy) was made. since pregnancy was not planned, coupled with suspicion of a gct and risk for continued hemoperitoneum formation, decision was made to terminate the pregnancy. to confirm our diagnosis, we decided to repeat endocrinological testing after pregnancy termination. treatment the owners elected to terminate pregnancy and agreed to pursue potential ovariectomy once mare was stabilized. figure 1. transrectal ultrasonographic images of ovaries on initial presentation. (a) left ovary (note multiple large follicular like structures and fibrin strands). (b) right ovary (note a 7.7 cm structure [echotexture resembling corpus hemorrhagicum, hematoma, or hemorrhagic anovulatory follicle] and fibrin strands) ba http://dx.doi.org/10.58292/ct.v16.10705 citation: clinical theriogenology 2024, 16, 10705, http://dx.doi.org/10.58292/ct.v16.10705 3 intramuscular prostaglandin f2α (dinoprost tromethamine 5 mg, lutalyse®) was given twice, 24 hours apart. in the afternoon, the second pgf2α treatment was given and 200 µg of prostaglandin e1 (misoprostol) was applied topically to the cervix. manual dilation was successfully performed; termination of the pregnancy was completed by manual removal of the fetus and any unattached extra fetal membranes. after cervical dilation and further intrauterine examination, it became apparent that there was another fetus. after removal it was confirmed that both fetuses had male external genitalia (phenotypical male); however, karyotypes (for xy chromosomes) were not test. the next day morning, uterine lavage (sterile lactated ringer’s solution) was performed to remove debris and confirm complete expulsion of fetal membranes. there was no evidence (via transrectal ultrasonography) of retained fetal membranes. outcome transrectal ultrasonography was repeated the day after pregnancy termination; there was a reduction in ovarian sizes. the left ovary measured 7.19 cm in diameter, whereas the right measured 4.12 cm in diameter. repeat endocrinological testing was performed 1 week after discharge; amh (0.31 ng/ml) and inhibin b (64.8 pg/ml) concentrations were normal, and testosterone concentrations remained mildly elevated (73.4 pg/ ml). immediate reduction in ovarian sizes ovaries and echotexture after termination (consistent with early pregnancy) combined with a precipitous decrease in maternal testosterone concentrations confounded the diagnosis (gct during pregnancy). transrectal ultrasonography performed (no images were archived) by the referring veterinarian was reportedly normal. reevaluation (10 weeks after initial discharge) confirmed normal ovarian echotexture and size (5-7 cm in diameter). the left ovary had a single follicular structure (~ 55 mm) with smaller follicles and the right ovary had a structure (~ 55 mm) with an echotexture that resembled a corpus hemorrhagicum or hemorrhagic anovulatory follicle (figure 2). concentrations of ecg were 100 iu/ml consistent with the continued presence of endometrial cups and supported ultrasonographic findings. discussion an unusual case of a mare (not expected to be pregnant) having substantial ovarian enlargement and hematoma rupture, most likely secondary to equine chorionic gonadotropin (ecg) production is described. hemoperitoneum resulting from ovarian enlargement and hematoma during pregnancy has been reported.1 other differentials were eliminated after the response to pregnancy termination, also subsequent endocrine testing was not definitively compatible with a gct; although hemoperitoneum from a gct during pregnancy has been reported in other instances.7,8 in our patient, other causes of hemoperitoneum were ruled out using available imaging modalities. coagulopathy was also ruled out using thromboelastography and initial evaluation via transrectal ultrasonography was consistent with substantial ovarian stimulation, ovarian hematomas, or corpora hemorrhagica. we speculated that ovarian stimulation by high concentrations of ecg led to extensive growth and follicular luteinization, formation of multiple ovarian hematomas, rupture of ovarian hematoma(s), and ultimately accumulation of hemorrhagic peritoneal fluid. the authors are unaware of a reported case of hemoperitoneum during pregnancy sharing similar pathogenesis. in the normal equine ovary, antral follicles are composed of 3 layers (theca externa, theca interna, and granulosa cell layer). theca externa’s rich blood supply is responsible for the evacuation of serosanguinous fluid during ovulation. during pregnancy, ecg is produced after invasion of trophoblast cells into the endometrium beginning ~ on day 35 of pregnancy, reaching a maximum ~ on days 65-70 of pregnancy. the resulting structures are referred to as endometrial cups and are responsible for production of ecg that has both follicle stimulating hormone and luteinizing hormone activity in the mare. mares continue to have follicular waves during early pregnancy due to ecg secretion, resulting in ovarian enlargement because of growth of ovarian follicles; formation of secondary and figure 2. transrectal ultrasonographic images of left (a) and right (b) ovaries 10 weeks after manual termination of pregnancy (note the decrease in ovarian size) ba http://dx.doi.org/10.58292/ct.v16.10705 4 citation: clinical theriogenology 2024, 16, 10705, http://dx.doi.org/10.58292/ct.v16.10705 accessory corpora lutea, and corpora hemorrhagica. these luteal structures lead to additional progesterone and testosterone production.9-11 secretary pattern of testosterone in pregnant mares with functional corpora lutea having testosterone concentrations is markedly increased from prostaglandin-treated, altrenogest-supplemented pregnant mares without a functional corpus luteum.11 this process of excess steroid production occurs at a critical point in equine pregnancy, as the fetoplacental unit does not fully take over production of progestogens until ~ day 120 of pregnancy. due to severe, bilateral, ovarian enlargement and ultrasonographic appearance, the possibility of an existing gct should have been investigated as a cause of hemoperitoneum early in the case management. endocrine testing revealed extremely elevated testosterone and moderately elevated inhibin b concentrations in the absence of amh elevation. testosterone concentrations during the third trimester of pregnancy in mares consistently increase above that of a nongravid mare due to marked steroid hormone production by the fetal gonads. the combination of an elevated inhibin and excessively high testosterone concentrations (517 pg/ml) observed in this case are suggestive of an atypical or early gct due to the absence of amh elevation. analysis of endocrine findings were considered alongside the uc davis clinical endocrinology laboratory reference ranges for testosterone concentrations: 10-45 pg/ml for normal pregnancy, 45-70 pg/ml ‘marginally elevated’ 70-100 pg/ml, and > 100 pg/ml ‘severely elevated’. one week after pregnancy termination, testosterone concentrations were 74 pg/ml, still warranting a concern for a gct. equine fetal gonadal development begins ~ on day 40 of pregnancy and is complete by day 60. steroid hormone production by the formed fetal gonad increases thereafter to reach its peak in mid pregnancy. currently, there is no convincing literature to suggest that fetal androgen development can influence increases in maternal testosterone concentrations. although maternal testosterone concentrations during male cotwin pregnancy have not been investigated formally, it is unlikely that fetal testosterone would be produced in sufficient quantities to escape placental aromatization to estrogens. in this case it is possible that prostaglandin treatment prior to termination allowed some degree of luteolysis or ovulation of follicular structures present on both ovaries. this could cause a resultant drop in testosterone concentrations if production by luteal tissue had a substantial role in steroid elevation. in a case of confirmed gct, testosterone concentrations (> 1,000 pg/ml) decreased 10 fold within 30 days after the removal of affected ovary.12 currently, we can only speculate that the patient had ovarian pathology that contributed to normal physiologic rises in androgen production during early pregnancy. lack of ultrasonographic images and laboratory work after the 10 week follow up examination prevented us to further elucidate the endocrine findings. a follow up gct panel is highly desired as this is the only reported case of testosterone elevation to that degree during pregnancy in the absence of a gct. examination and aforementioned diagnostics performed after regression of the endometrial cups might have provided further endocrinological explanation to this unique case. learning points • pregnancy should always be considered in mares with ovarian enlargement despite no breeding history • hemoperitoneum is a rare complication arising from ovarian stimulation related to ecg secretion and presumed rupture of a corpus hemorrhagica/hematomas • androgen synthesis by luteal tissue as a result of ecg secretion may cause increases in maternal testosterone above concentrations expected of a pregnant mare conflict of interest none to declare. acknowledgement authors thank alan conley of uc davis clinical endocrinology laboratory for his invaluable assistance in the interpretation of endocrinological results. references 1. beachler tm, mckelvy ka, bailey cs, et al: haemoperitoneum in a pregnant mare with an ovarian haematoma. eq vet edu 2016;28:359-363. doi: 10.1111/eve.12211 2. pauwels fe, wigley sj, munday js, et al: bilateral ovarian adenocarcinoma in a mare causing haemoperitoneum and colic. n z vet j 2012;60:198-202. doi: 10.1080/00480169.2011.647607 3. conwell rc, hillyer mh, mair ts, et al: haemoperitoneum in horses: a retrospective review of 54 cases. vet rec 2010;167: 514-528. doi: 10.1136/vr.c4569 4. dechant je, nieto je, le jeune ss: hemoperitoneum in horses: 67  cases (1989-2004). j am vet med assoc 2006;229:253-258. doi: 10.2460/javma.229.2.253 5. murase h, endo y, tsuchiya t, et al: ultrasonographic evaluation of equine fetal growth throughout gestation in normal mares using a convex transducer. j med vet sci 2014;76:947-953. doi: 10.1292/jvms.13-0259 6. scott c, christensen b, pritchard w, et al: hemoperitoneum in a mare following rupture of a granulosa-theca cell tumor. clinical theriogenology 2015;7:425-429. 7. worsman fcf, barakzai sz, rubio-martinez lm, et al: treatment of haemoperitoneum secondary to ruptured granulosa theca cell tumors in two mares. equine vet edu 2020;32:71-77. doi: 10.1111/ eve.12929 8. squires el, ginther oj: follicular and luteal development in pregnant mares. j reprod fertil 1975;23:429-433 9. ginther oj: reproductive biology of the mare, basic and applied aspects. 2nd edition, cross plains; equiservices publishing: 1992. p. 237-240. 10. daels pf, chang gc, hansen b, et al: testosterone secretion during early pregnancy in mares. theriogenology 1996;45: 1211-1219. doi: 10.1016/0093-691x(96)00076-3 11. tennent-brown bs: lactate production and measurement in critically ill horses. compend contin educ vet 2011;33:e5. 12. gee ek, dicken m, archer rm, et al: granulosa theca cell tumour in a pregnant mare: concentrations of inhibin and testosterone in serum before and after surgery. n z vet j 2011;60:160-163. doi: 10.1080/00480169.2011.645776 http://dx.doi.org/10.58292/ct.v16.10705 https://doi.org/10.1111/eve.12211 https://doi.org/10.1080/00480169.2011.647607 https://doi.org/10.1136/vr.c4569 https://doi.org/10.2460/javma.229.2.253 https://doi.org/10.1292/jvms.13-0259 https://doi.org/10.1111/eve.12929 https://doi.org/10.1111/eve.12929 https://doi.org/10.1016/0093-691x(96)00076-3 https://doi.org/10.1080/00480169.2011.645776 _heading=h.gjdgxs 1 contact kristyn burton kristyn.burton@okstate.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12176, http://dx.doi.org/10.58292/ct.v17.12176 case report pregnancy termination in a mare with hydroallantois* kristyn burton, reed holyoak, adam bassett, dale kelley college of veterinary medicine, oklahoma state university, stillwater, ok, usa abstract we describe the successful management, resolution, and subsequent return to breeding of a mare with hydroallantois. a 14-year, american quarter horse mare in late pregnancy was referred for marked abdominal distention with a history of treatment for placentitis. physical examination findings were consistent with hydroallantois. it was decided to terminate pregnancy. during fetal delivery, dystocia was diagnosed and corrected under field anesthesia; recovery was uneventful. mare conceived the following season and delivered a healthy filly. keywords: hydroallantois, mare, termination of pregnancy background hydroallantois is a rare condition characterized by a sudden increase in allantoic fluid volume leading to marked abdominal distention, typically between 6-10 months of pregnancy. it occurs quickly over several days, with few to no notable abnormalities preceding its onset.1 initial signs typically include dramatic increase in abdominal distention, reduced locomotion, difficulty feeding, dyspnea, and obvious discomfort.1,2 the condition is considered a serious threat to mare and foal;1 without intervention, it can seriously impact mare’s reproductive future. diagnosis is made by transrectal palpation of the markedly distended fluid-filled uterus, combined with an inability to visualize the fetus on ultrasonography.3 in some cases, fluid excess of up to 100 liters has been reported.1 although the fetus is often developmentally normal, survival to term and successful delivery depends on the mare’s ability to support the pregnancy to the point of viability.2-4 therefore, probability of a live foal depends mostly on the interval from onset of symptoms to term. pregnancy termination is often elected to protect the mare’s future reproductive health.1,4,5 case presentation a 14-year, american quarter horse mare was presented on day 301 of pregnancy with abdominal distention (figures 1 and 2) and a history of treatment for placentitis. mare had a history of 5 full-term pregnancies; all were conceived via live cover and with no notable complications for the duration of pregnancy. the first 4 pregnancies resulted in live foals. the fifth was stillborn; it had an intact amniotic sac and visually normal fetal membranes. all deliveries were unassisted. mare was reportedly up to date on vaccinations (including pneumabort) and had a caslicks on presentation. mare was initially examined by the referring veterinarian on day 284 of pregnancy, after owner noticed premature lactation. mare received twice daily oral trimethoprim-sulfamethoxazole (30 mg/kg; amneal pharmaceutical bridgewater, nj), metronidazole (20 mg/kg; unichem pharmaceuticals inc, new brunswick, nj), and once daily oral firocoxib (0.1 mg/kg; equioxx®, boehringer ingelheim, st. joseph, mo) at home the night before. referring veterinarian felt a slightly thickened placenta with a combined uterus and placenta measurement of 15 mm. medications were continued, and once daily oral altrenogest (0.044 mg/kg; regu-mate®, merck animal health, intervet canada, kirkland, qc, canada) was added. mare was reexamined by the referring veterinarian on day 300; size of mare’s abdomen had increased dramatically, with notable distention. placenta was within normal limits, but due to the change in fluid volume a decision was made to refer. all medications were continued until arrival at the hospital. on physical examination, mare was bright and alert. transrectal palpation and ultrasonography revealed the uterus to be above the pelvic brim with allantoic fluid at a greater volume than normal. fetus could not be visualized with *poster presented at the society for theriogenology 2023 conference; abstract (https://doi.org/10.58292/ct.v15.10001) updated to reflect additional medical records and case report represents the newest and most complete information. mailto:kristyn.burton@okstate.edu http://dx.doi.org/10.58292/ct.v17.12176 https://doi.org/10.58292/ct.v15.10001 2 citation: clinical theriogenology 2025, 17, 12176, http://dx.doi.org/10.58292/ct.v17.12176 ultrasonography. due to the length of pregnancy remaining and concern over mare’s future fertility, it was decided to terminate the pregnancy. bloodwork on admission had creatinine kinase and calcium values within reference range, indicating the immediate lower risk of prepubic tendon rupture. because the mare was presented on a friday afternoon, mare was kept for 2 days prior to induction to have full staff availability during the procedure. mare was monitored closely during this time; condition remained stable for the duration. treatment an intravenous catheter was placed; 1 liter of lactated ringer’s solution (lrs, dechra veterinary products, overland park, ks) was given, and heart rate was monitored throughout the procedure. caslicks was removed and a sterile 21-inch ch18 foley catheter was inserted through the cervix into allantois; over a period of 2½ hours a total volume of 45.4 liters of allantoic fluid was drained (figure 3). after drainage, parturition was induced with 60 units of intravenous oxytocin (bimeda-mtc animal health, cambridge, on, canada) in 1 liter of 0.9% nacl solution (dechra) given over 60 minutes. at 45 minutes fetal front limbs appeared in the birth canal; 10 minutes later hind limbs were felt near the right shoulder, indicating malposture. palpation revealed fetus in anterior presentation, with the left hind limb extended and the right hind limb crossing over the left hindlimb; both were extended beneath the fetus (‘dogsitting’ posture). intravenous detomidine (0.02 mg/kg; dormosedan®, zoetis, parsipanny, nj) and ketamine (1.5 mg/kg; ketaset®, zoetis) were given to anesthetize the mare, along with 240 mg buscopan (0.35 mg/kg; boehringer ingelheim) to facilitate repositioning and delivery of the fetus; foal was stillborn (4-6 weeks premature). mare was given an additional liter of intravenous lrs during recovery, followed by intravenous flunixin (1.1 mg/kg; merck) and oral sulfathiamethoxine (2 mg/kg) ~ 2 hours after procedure. mare did not experience hypovolemic shock and expelled fetal membranes 3 hours after recovery. outcome mare recovered uneventfully; rest for at least 2 estrous cycles and a breeding soundness examination prior to breeding were recommended. referring veterinarian observed (transrectal ultrasonography) residual intrauterine fluid 2 weeks after discharge and antibiotic treatment was extended. mare’s uterus was normal at the next appointment. owner made a decision not to breed the mare until the next season due to an unrelated tendon injury. the following year, mare was bred via artificial insemination on a natural cycle, and the following spring delivered a healthy, figure 1. marked distention of the abdomen (lateral view) at presentation figure 2. marked distention of the abdomen (frontal view) at presentation figure 3. draining allantoic fluid http://dx.doi.org/10.58292/ct.v17.12176 citation: clinical theriogenology 2025, 17, 12176, http://dx.doi.org/10.58292/ct.v16.12176 3 live foal at full-term (figure 4) after a medically normal and uncomplicated pregnancy. discussion the full pathophysiology of hydroallantois remains poorly understood, although it is suspected to occur in cases of compromised placental function.2,4,5 however, placentitis is not predictive of hydrops, as there are more common etiologies.6 therefore, although placentitis often precedes a diagnosis of hydroallantois, identification of placentitis is not commonly the point at which hydroallantois is selected as a primary differential. contributing genetic and epigenetic factors have also been suggested.4 although findings of concurrent placentitis are not consistent,2 suspected placentitis and subsequent treatment were present in this mare’s recent medical history. at a rate of 10:1,1 hydroallantois is the more common form of hydrops in the mare compared to hydramnios. diagnostically, it may be differentiated by a more rapid rate of abdominal distention (typically in days), substantially higher volume of fluid (sometimes in excess of 100 liters), and increased inability to visualize the fetus on transabdominal and transrectal ultrasonography (although possible in both conditions). gross fetal deformities of the face and head are characteristic of hydramnios, and although fluid volumes of up to 50 liters have been reported, lesser amounts are more common and tend to accumulate over a longer interval.3 it is understood to be a disorder of fluid dynamics due to fetal inability to cycle amniotic fluid, compared to hydroallantois which is a primary dysfunction of the placenta and movement of allantoic fluid.1 although there are reports of hydrops pregnancies resulting in live foals,2,4 most cases resulted in foals that were either stillborn or euthanized soon after parturition.4,5 furthermore, dysmaturity and severe fetal abnormalities were identified;7 foals born alive received substantial medical support and intervention.4 in contrast, prognosis for the mare is generally good with prompt and appropriate intervention. unattended hydroallantois can quickly lead to herniation of the ventral abdominal musculature, prepubic tendon rupture,8 or eventual uterine rupture.1 given poor prognosis to mare and fetus, termination of pregnancy is commonly elected. the single most important factor in fetal viability appears to be the interval remaining to term when signs develop.1 overall prognosis for live delivery and survival of the foal is guarded to poor,2 and generally limited to mares that present after 330 days of pregnancy. these cases typically represent relatively low fluid volumes and often involve controlled partial drainage of the allantoic fluid.1,2 regardless of desired outcome or stage of pregnancy, a controlled delivery is important, as hypovolemic shock is well documented in mares after expulsion of fetal membranes.1 given the early stage of pregnancy and large volume of fluid in the present case, the likelihood of maintaining pregnancy to term was determined to be neither probable nor rewarding. in this case, fetal malposture complicated delivery of the fetus, necessitating anesthesia of the mare and manual repositioning of the fetus; thereafter, delivery and recovery were uneventful. fluids were given and an intravenous port was maintained for the procedure duration. similar to this case, a majority of mares conceived and had normal pregnancy after hydroallantois.1-5,7 although the mare was deemed sound for breeding later in the same season, breeding was delayed for treatment of an incidental tendon sheath injury. learning points • hydroallantois is a rare but serious complication of equine pregnancy. although full pathogenesis is unknown, it is closely correlated to placental dysfunction, and often identified in cases that have been treated for placentitis. • quick and effective intervention carries a favorable prognosis for the mare and full return to breeding soundness is expected. • prognosis for fetal viability is guarded to poor, heavily dependent on case presentation, and attempts to manage a hydrops pregnancy carry significant risks for the mare. acknowledgement authors thank trisha martin for her enthusiastic cooperation and willingness to provide information regarding the longterm outcome of this case. conflict of interest the authors report no conflict of interest. references 1. waelchli ro: hydrops. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wileyblackwell: 2011. p. 2368-2372. 2. mitchell arm, delvescovo b, tse m, et al: successful management of hydrallantois in a standardbred mare at term resulting in the birth of a live foal. can vet j 2019;60:495-501. pmc646 3767 3. wolfsdorf k: placental hydrops. hagyard equine medical institute. available from: http://www.hagyard.com/placental-hydrops 2018 [cited 14 july 2023]. 4. bocci c, sgorbini m, panzani d, et al: impact of managing presumptive fetal membrane hydrops in a mare on fetal livability. figure 4. filly delivered after uneventful pregnancy the following year http://dx.doi.org/10.58292/ct.v16.12176 http://www.hagyard.com/placental-hydrops 4 citation: clinical theriogenology 2025, 17, 12176, http://dx.doi.org/10.58292/ct.v17.12176 am j anim vet sci 2023;18:131-138. doi: 10.3844/ajavsp.2023. 131.138 5. govaere jlj, de schauwer c, hoogewijs mk, et al: hydrallantois in the mare – a report of five cases. reprod domest anim 2014;48:e1-e6. doi: 10.1111/j.1439-0531.2012.02013.x 6. johnson ak: equine placentitis: diagnosis and treatment options. proc auburn university cvm annual conference. 2023. 7. arroyo e, whitelock lm, stanton me, et al: hydroallantois in a mare associated with schistosomus and unilateral ovarian agenesis in the fetus. j equine vet sci 2025;144:105228. doi: 10.1016/j. jevs.2024.105228 8. morrison mjw, back b, mcclure jt: hydroallantois and prepubic tendon rupture in a standardbred mare. proc soc theriogenol 2016;8:359. http://dx.doi.org/10.58292/ct.v17.12176 https://doi.org/10.3844/ajavsp.2023.131.138 https://doi.org/10.3844/ajavsp.2023.131.138 https://doi.org/10.1111/j.1439-0531.2012.02013.x https://doi.org/10.1016/j.jevs.2024.105228 https://doi.org/10.1016/j.jevs.2024.105228 _int_ew7rysrz _int_fs7w8jdm _int_nksicshz _int_f79bgdys _int_coerjkj5 _int_vkefowgw _int_bmlweush _int_wcp1hch4 _int_46fwnjzj _int_sxpjzmga _int_xo8iaw2y 1 contact joaquin paredes bparedes@uoguelph.ca © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10690, http://dx.doi.org/10.58292/ct.v16.10690 case report a case of impaired fertility in a large white boar with sex chromosome chimerism joaquin paredes,a lauren lambert,b lukasz czochara,c patrick charagu,d darren griffin,e megan magee,c laura favetta,b allan king,b,c daniel villagomezb,f adepartment of population medicine bdepartment of biomedical sciences, ontario veterinary college, university of guelph, guelph, on, canada; ckaryotekk incorporated, guelph, on, canada dhypor incorporated, regina, sk canada eschool of biosciences, university of kent, canterbury, uk fdepartamento de producción animal, universidad de guadalajara, zapopan, mexico abstract chromosomal abnormalities are a well-established cause of fertility impairment in domestic animals. chromosomal abnormalities in pigs may result in low prolificacy and subsequent reduction in reproductive parameters (e.g. litter sizes and nonreturn rates) having substantial impact on swine industry. until now, the effect of sex chromosome abnormalities on fertility has not been thoroughly documented for male pigs. we report a case study involving a large white boar with sex chromosome chimerism (2n = 38, xx/xy) identified by conventional karyotyping and fluorescence in-situ hybridization. progeny of this boar was evaluated; there was reduction (p < 0.001) in litter size demonstrating the potential impact of sex chromosome chimerism on herd productivity in pigs. keywords: male pigs, chromosomal abnormalities, subfertility introduction chromosomal abnormalities are a common cause of infertility in mammals, including several species of domestic animals. in pigs, alterations in chromosome structure (mainly, reciprocal translocations and to a lesser extent inversions) and sex chromosome numerical abnormalities (usually mosaic)1,2 resulted in poor fertility due to embryo or fetal loss causing reduction in prolificacy and subsequent impact to farming industry.3 chromosome abnormalities that lead to low prolificacy can be compatible with a normal phenotype, albeit some can lead to demonstrable infertility (e.g. compromised semen parameters or complete sterility),4 whereas others are only identified when litter size or nonreturn rate data are analysed or through a proactive screening program.2,5 blood cell sex chromosome chimerism (xx/xy) originating due to vascular placental anastomosis in utero has been demonstrated as a cause of complete infertility in females of several species, including cattle,6 sheep,7 goats,8 and pigs.9 although controversial, xx/xy blood cell chimerism has been deemed not to produce substantial effect on fertility in males.10,11 however, a study in bulls suggested that chimeric males may have a reduction in fertility, potentially due to poor sperm quality.12 sex chromosome abnormalities have been related to subfertile phenotype in male pigs,1 although in several cases they might present a normal phenotype.10 interestingly, fertility data from affected boars are scarce and seldom reported. recently, an increased incidence of xx/xy blood chimerism in pigs has been observed and linked to the selection of highly prolific lines,9 making investigation into the effects of sex chromosome chimerism on male fertility crucial. we present a case of a large white xx/xy chimeric boar from a commercial herd whose reproductive parameters were tracked to assess the impact of this condition on progeny. materials and methods a total of 66 boars, from a large canadian commercial swine operation (hypor incorporated, regina, canada) were karyotyped by karyotekk incorporated (guelph, canada) as part of the prebreeding screening program (2021-2022). whole blood was collected by trained veterinarians and processed for karyotyping by standard procedures.3 this led to the identification of an xx/xy sex chromosome chimeric boar. additionally, fibroblast cultures from a ear skin biopsy sample from the chimeric boar, provided by the owner, were mailto:bparedes@uoguelph.ca http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10690 2 citation: clinical theriogenology 2024, 16, 10690, http://dx.doi.org/10.58292/ct.v16.10690 processed for cytological analysis by karyotekk incorporated by standard procedures.13 fluorescence in situ hybridization (fish) was performed using a x chromosome probe labelled with fluorescein isothiocyanate provided by cytoscreen solutions (university of kent, canterbury, uk) and applied to lymphocytes and skin fibroblasts14 to determine the degree of chimerism in hematopoietic and nonhematopoietic cells. reproductive parameter data from 66 boars, including 1 chimeric boar collected on the farm from january 2021 to january 2022 were analyzed. parameters of interest included litter size, total live born, and stillborn. boars (n = 65) with no known chromosome abnormality were assessed for mean litter size (total number of piglets divided by total number of litters), and mean live born and stillborn rates. mean litter sizes were compared using the r programming language (r core team, vienna, austria, v3.6.0). z-test was employed to compare the chimeric boar reproductive parameters with the farm population. in accordance with the canadian council for animal care and the university of guelph animal care committee a review of the research protocol was not required. results cytogenetic analysis examination of the gtg-banded karyotypes obtained from blood leukocytes of the proband revealed a male (2n = 38, xy) and a female (2n = 38, xx) karyotype (figure 1) and thus confirmed blood cell chimerism (2n = 38, xx/xy). in hematopoietic tissue (leukocytes), fish technique revealed  that 55% of cells with one fluorescence signal, suggesting a 2n = 38, xy karyotype (figure 2-a), and 45% of  leukocytes with 2 fluorescent signals suggesting 2n = 38, xx karyotype. in nonhematopoietic (fibroblasts) tissue (figure 2-b), a low proportion (< 7%) of 2n = 38, xx were detected (table 1). reproductive parameters litter sizes were different between the chimeric and control groups (11.91 versus 15.04, p < 0.0001) as were live born rates (10.32 versus 13.55, p < 0.0001), whereas stillborn rates were not different (table 2). discussion effect of blood chimerism (xx/xy) on male fertility has not been thoroughly documented in pigs. there were no reproductive impairments in 7 xx/xy ‘phenotypically normal boars’;10 however, anecdotally, reduced litter size has been observed.14 although this study was focussed on a single boar and is thus not a systematic overview, it provided an evidence that xx/xy chimerism can be related to subfertility. the exact mechanism responsible for reduced fertility in this case has not been identified; however, it could be similar to previous reports that described poor semen figure 1. karyotypes of lymphocyte metaphases from the chimeric boar (a. 2n = 38, xy and b. 2n = 38, xx) a b figure 2. fish-stained cells from the chimeric boar from a. lymphocytes and b. fibroblasts. white arrows indicate female (xx) nuclei and red arrows indicate the single x chromosome in an xy nucleus a b http://dx.doi.org/10.58292/ct.v16.10690 citation: clinical theriogenology 2024, 16, 10690, http://dx.doi.org/10.58292/ct.v16.10690 3 quality in chimeric bulls.12 in pigs, chimerism often extended to nonhematopoietic tissues, including, lung, hair, and skin, although at a low rate (< 8%).10 in agreement with that study, we identified a low percentage (7%) of xx cells in skin fibroblasts. chimerism in somatic tissues might relate with the reduction in fertility observed in this boar. sex chromosome chimerism is currently considered the most common intersex condition in pigs. it has been suggested to be related to large litter sizes where uterine crowding may cause an increase in placental anastomosis  between litter mates.9 large white boar is a prolific breed with an average litter size of 13.4 piglets.15 average litter size of the herd in this investigation was 15.04 total born and 13.55 born  alive  that included chimeric boar’s dam, whose average over 6 litters was 15.3 total born and 14.8 born alive suggesting that this factor might have a role in this case. to our knowledge, ours is among only a few cases that reported the fertility of a chimeric boar. this report demonstrated that chimerism might also have detrimental effects on male fertility in pigs. considering the available information, it may be advisable to pay particular attention to xx/xy chimeric boars in breeding programs to obtain additional data regarding the effects of this condition on fertility. proactive routine cytogenetic screening involving conventional karyotyping and fish,2,5 can identify these and other problems before an animal enters the breeding program. acknowledgement authors thank hypor incorporated for providing access to herd breeding records and cytogenetic screening data, and for giving permission to analyze data. conflict of interest none of the authors has any conflict of interest to disclose. author contributions jp performed data analysis and wrote the manuscript. lc contributed to data analysis and karyotyping. ll contributed to sample processing, images, and evaluation. mm contributed to manuscript preparation, dv performed karyotyping analysis. dg edited the manuscript and provided the fish probe. lf contributed to editing the manuscript and supervising students involved in the project. pc provided reproduction data. ak edited the manuscript, supervised students involved in the project, and provided funding. all authors have read and approved manuscript submission. references 1. quilter cr, wood d, southwood oi, et al: x/xy/xyy mosaicism as a cause of subfertility in boars: a single case study. anim genet 2003;34:51-54. doi: 10.1046/j.1365-2052.2003.00924.x 2. o’connor re, kiazim lg, rathje cc, et al: rapid multi-hybridisation fish screening for balanced porcine reciprocal translocations suggests a much higher abnormality rate than previously appreciated. cells 2021;10:1-7. doi: 10.3390/cells10020250 3. quach at, villagómez daf, coppola g, et al: a cytogenetic study of breeding boars in canada. cytogenet genome res 2010;126:271-280. doi: 10.1159/000251964 4. villagómez daf, parma p, radi o, et al: classical and molecular cytogenetics of disorders of sex development in domestic animals. cytogenet genome res 2009;126:110-131. doi: 10.1159/000245911 5. lewis nm, rathje cc, canedo-ribeiro c, et al: incidence, reproductive outcome, and economic impact of reciprocal translocations in the domestic pig. dna 2021;1:68-76. doi: 10.3390/ dna1020007 6. kozubska-sobocinska a, smołucha g, danielak-czech b: early diagnostics of freemartinism in polish holstein-friesian female calves. animals 2019;9:1-11. doi: 10.3390/ani9110971 7. brace md, peters o, menzies p, et al: sex chromosome chimerism and the freemartin syndrome in rideau arcott sheep. cytogenet genome res 2008;120:132-139. doi: 10.1159/000118752 8. szatkowska i, zychi s, udała j, et al: freemartinism: three cases in goats. acta vet brno 2004;73:375-378. doi: 10.2754/avb2004730 30375 9. szczerbal i, nowacka-woszuk j, dzimira s, et al: elevated incidence of freemartinism in pigs detected by droplet digital pcr and cytogenetic techniques. livest sci 2019;219:52-56. doi: 10.1016/j.livsci.2018.11.009 10. barasc h, ferchaud s, mary n, et al: cytogenetic analysis of somatic and germinal cells from 38,xx/38,xy phenotypically normal boars. theriogenology 2014;81:1-5. doi: 10.1016/j.theriogenology. 2013.10.006 11. padula am: the freemartin syndrome: an update. anim reprod sci 2005;87:93-109. doi: 10.1016/j.anireprosci.2004.09.008 12. dunn ho, mcentee k, hall ce, et al: cytogenetic and reproductive studies of bulls born co-twin with freemartins. j reprod fertil 1979;57:21-30. doi: 10.1530/jrf.0.0570021 table 1. distribution of xy and xx cells on hematopoietic and nonhematopoietic tissues type of cell 2n = 38, xy 2n = 38, xx percent xx hematopoietic (lymphocyte culture) 115 85 42.5 nonhematopoietic (fibroblast culture) 186 14 7 table 2. reproductive parameters of chimeric boar and the population of boars of the farm reproductive parameter all boars (n = 65) 1,243 litters chimeric boar 34 litters p value litter size (mean ± sem) 15.04 ± 0.103 11.91 ± 0.594 < 0.0001 live born (mean ± sem) 13.55 ± 0.098 10.32 ± 0.607 < 0.0001 stillborn (mean ± sem) 1.49 ± 0.051 1.58 ± 0.443 0.3775 http://dx.doi.org/10.58292/ct.v16.10690 https://doi.org/10.1046/j.1365-2052.2003.00924.x https://doi.org/10.3390/cells10020250 https://doi.org/10.1159/000251964 https://doi.org/10.1159/000245911 https://doi.org/10.3390/dna1020007 https://doi.org/10.3390/dna1020007 https://doi.org/10.3390/ani9110971 https://doi.org/10.1159/000118752 https://doi.org/10.2754/avb200473030375 https://doi.org/10.2754/avb200473030375 https://doi.org/10.1016/j.livsci.2018.11.009 https://doi.org/10.1016/j.theriogenology.2013.10.006 https://doi.org/10.1016/j.theriogenology.2013.10.006 https://doi.org/10.1016/j.anireprosci.2004.09.008 https://doi.org/10.1530/jrf.0.0570021 4 citation: clinical theriogenology 2024, 16, 10690, http://dx.doi.org/10.58292/ct.v16.10690 13. rezaei s, donaldson b, villagomez daf, et al: routine karyotyping reveals frequent mosaic reciprocal chromosome translocations in swine: prevalence, pedigree, and litter size. sci rep 2020;10:1-9. doi: 10.1038/s41598-020-64134-w 14. quach at, revay t, villagomez daf, et al: prevalence and consequences of chromosomal abnormalities in canadian commercial swine herds. genet sel evol 2016;48:1-7. doi: 10.1186/ s12711-016-0246-5 15. sell-kubiak e: selection for litter size and litter birthweight in large white pigs: maximum, mean and variability of reproduction traits. animal 2021;15:1-10. doi: 10.1016/j.animal.2021. 100352 http://dx.doi.org/10.58292/ct.v16.10690 https://doi.org/10.1038/s41598-020-64134-w https://doi.org/10.1186/s12711-016-0246-5 https://doi.org/10.1186/s12711-016-0246-5 https://doi.org/10.1016/j.animal.2021.100352 https://doi.org/10.1016/j.animal.2021.100352 1 contact dale kelley dale.kelley@okstate.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10782, http://dx.doi.org/10.58292/ct.v16.10782 research report pipette or vortex are equally effective for mixing stallion sperm after cushion centrifugation giovana catandi,a haley wilkerson,a malorie kittle,a kelli beavers,a maria schnobrich,b dale kelleya adepartment of veterinary clinical sciences, college of veterinary medicine, oklahoma state university, stillwater, ok, usa brood and riddle equine hospital, lexington, ky, usa abstract data on effects of mixing technique after cushion centrifugation on stallion sperm quality and longevity are limited. objective was to compare mixing sperm via pipette or vortex after centrifugation. in experiment 1, effects of 2 vortex speeds (minimum and maximum) and 3 vortex intervals (5, 15, and 30 seconds), and mixing (20 and 100 times) with a 1,000 μl pipette on sperm quality and sperm clumping were determined. there was no effect (p > 0.05) of treatment on total motility, progressive motility, curvilinear velocity, number of detached heads, or size of sperm clumps. there was an effect (p = 0.05) of treatment on number of sperm clumps, with fewer clumps in vortexed sample. in experiment 2, effects of minimum vortex speed for 15 seconds, maximum vortex speed for 15 seconds and pipetting 20 times with a 1,000 μl pipette on sperm parameters and sperm clumping and at 24 and 48 hours of cooled storage were determined. there was an effect (p = 0.0001) of treatment on sperm viability (higher immediately after collection) but no difference among treatments. there was no effect (p > 0.05) of treatment on total motility, progressive motility, curvilinear velocity, number of sperm clumps, nor size of sperm clumps at 24 or 48 hours. we concluded that either a vortex or pipette can be used to mix sperm after cushion centrifugation with no detrimental effect on sperm quality or longevity. keywords: stallion, sperm, centrifugation, mixing, vortex, pipette introduction cushion centrifugation is a useful technique to concentrate stallion semen and is routinely performed.1 generally, after cushion centrifugation the supernatant and cushion media are removed, and semen is reconstituted with extender. removal of cushion is optional as cushion in reextended sperm had no effect on sperm parameters and fertility.2 most laboratories resuspend sperm pellet in extender by mixing with a pipette; however, some use a vortex (schnobrich m., unpublished observation). there are limited data on the impact of various mixing methods on sperm quality after centrifugation. agitation methods are commonly used in biopharmaceutical industry for anything from mixing to mass transfer.3 various agitation methods can be used; however, they introduce mechanical stresses to container and sample. vortex mixers are common in laboratories and feature a rotary motor with an offset cupped rubber piece. when a tube is pressed into the cupped rubber piece, a motor is activated and because of offset rubber piece a swirling motion occurs that mixes the suspension. this introduces shear stresses into the system3 and mixing layers can potentially form at different velocities, resulting in turbulent flow.4 these factors could improve mixing but also could have a deleterious effect on sperm. objective was to compare mixing sperm via pipette or vortex after centrifugation. our hypothesis was that vortex reduces sperm clumps and increases tailless heads compared to pipette. materials and methods animal use was approved by the institutional animal care and use committee (iacuc 21-04-stw). experiment 1 a pilot study was conducted to determine effects of various vortex speeds and duration on sperm and sperm clumping. mailto:dale.kelley@okstate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10782 2 citation: clinical theriogenology 2024, 16, 10782, http://dx.doi.org/10.58292/ct.v16.10782 three ejaculates from 1 stallion were utilized; stallion was collected using a missouri artificial vagina, phantom, and tease mare. after the gel-free semen was collected the volume of semen, concentration (nucleocounter®, chemometec a/s, allerod, denmark), total motility, progressive motility, curvilinear velocity (vcl; ivosii, hamilton thorne, beverly, ma, usa) were determined.5 a sample from the initial ejaculate was diluted with 10% buffered formalin saline (bfs; formalin 10, animal reproduction systems, chino, ca, usa) to evaluate sperm morphology.6 semen was then extended 1:1 (inra96; imv technologies, osseo, mn, usa) and 10 ml was aliquoted to 8 conical tubes (15 ml; falcontm, fisher scientific, waltham, ma, usa). then, 1 ml of cushion centrifugation media (red cushion, botupharma usa, phoenix, az, usa) was layered underneath the semen-extender mix using a 3 ml syringe with a tom cat catheter (argyletm open end catheter with 14 cm adaptor, covidien, dublin, ireland) attached. conical tubes were balanced and centrifuged at 1,000 x g for 20 minutes. after centrifugation, a vacuum aspirator was used to remove the supernatant and a syringe with tom cat catheter was used to remove the cushion media. semen was resuspended with extender (inra96) to a concentration of ~ 25 x  106 sperm/ml and evaluated with a cell counter (nucleocounter®). conical tubes were allocated to the following groups to mix the resuspended sperm-extender solution: vortex minimum speed 5 seconds (min5s); vortex minimum speed 15 seconds (min15s); vortex minimum speed 30 seconds (min30s); vortex maximum speed 5 seconds (max5s); vortex maximum speed 15 seconds (max15s); vortex maximum speed 30 seconds (max30s); mixing via 1,000 μl pipette (eppendorf north america, enfield, ct, usa) 20 times (p20); and mixing via 1,000 μl pipette 100 times (p100). motility parameters were assessed after centrifugation and mixing, and a sample from each treatment was diluted with 10% buffered formalin saline to evaluate sperm morphology. for sperm clumping, samples were diluted 1:1 with 10% bfs. sperm morphological evaluation was performed using dic microscopy at 1,250 x magnification. sperm were classified as normal, abnormal heads, abnormal acrosomes, tailless heads, distal cytoplasmic droplets, proximal cytoplasmic droplets, abnormal midpieces, bent midpieces, bent tails, coiled tails, and premature germ cells.7 only the number of normal sperm and tailless heads were used for analysis. clumping was defined as 3 or more sperm aggregated together. a makler® counting chamber (sefi-medical instruments, ltd, santa ana, ca, usa) was utilized for counting clumps. after semen mixture was well mixed, a 1.5 μl drop was placed on the center of the disc area using a micropipette. a coverslip was then placed on the 4 pins and gently pressed down. sperm clumps were counted within each grid and those that touched the top or left lines, whereas those touching the bottom or right lines were not counted. a line of 10 squares were counted and multiplied by 2 (to account for dilution), this represented the sperm clump concentration (106/ml). a second strip of 10 squares was counted and sperm clump concentration was determined as before; an average of 2 counts was used to determine sperm clump concentration. each time a sperm clump was observed the number of sperm in the clump was counted and recorded. experiment 2 seven stallions were each collected between 1 to 3 times to produce a total of 12 collections. semen was initially analyzed as described above. a sample from the initial ejaculate was diluted with 10% buffered formalin saline (bfs) to evaluate sperm morphology. semen was then extended 1:1 (inra96) and aliquoted to 3 conical tubes (50 ml; falcontm, fisher scientific) and 1 ml of cushion centrifugation media (red cushion) was layered underneath the semen-extender mix using a 3 ml syringe with a tom cat catheter attached. conical tubes were balanced and centrifuged at 1,000 x g for 20 minutes. after centrifugation, a vacuum aspirator was used to remove the supernatant and a syringe with tom cat catheter was used to remove most of the cushion media. sperm pellet was resuspended with extender (inra96) to a concentration of ~ 200 x 106 sperm/ml and evaluated with a cell counter (nucleocounter®). conical tubes were allocated to the following groups to mix: minimum vortex speed for 15 seconds, maximum vortex speed for 15 seconds and pipetting 20 times (1,000 μl pipette). a sample from each group was then stored in a passive cooling system (equitainerò) for 48 hours. sperm concentration, viability and motility parameters were assessed at 24 hours and 48 hours of cooling after centrifugation. a sample from each treatment was taken at 24 and 48 hours and diluted with 10% bfs to evaluate sperm morphology. undiluted samples from 24 and 48 hours were evaluated for sperm clumping. sperm morphology was evaluated as described above. counting the number of sperm clumps was performed as described above, except for multiplication by 2, as the samples were not diluted in fbs as performed in experiment 1. additionally, clump sizes were categorized based on the number of sperm per clump: 3-5 sperm (1), 5-10 sperm (2), and > 10 sperm (3). data analyses for experiment 1, total motility, progressive motility, curvilinear velocity (vcl), number of sperm clumps, size of sperm clumps, sperm with normal morphology, and sperm with detached heads were analyzed using sas (9.4) mixed procedure with treatment and collection as fixed effects, and treatment and collection interaction as random effects. pairwise comparisons were made between treatments using pdiff option. data are expressed as lsmeans ± sem. for experiment 2, sperm viability, total motility, progressive motility, curvilinear velocity (vcl), straightness, number of sperm clumps, sperm with normal morphology, and sperm with detached heads were analyzed using sas (9.4) general linear model procedure with collection, stallion, treatment and time as fixed effects and interaction between time and treatment, stallion and treatment, stallion and collection, and stallion and time as random effects. means were determined and when there were interaction (p < 0.05) comparisons were made using a tukey test. clump size was analyzed using sas (9.4) logistic regression with collection, stallion, treatment and time as fixed effects and interaction between time and treatment, stallion and treatment, stallion and collection, and stallion and time as random effects. the proportion clump sizes by treatment and time by clump size by treatment were determined using the frequency procedure with a chi square procedure invoked. significance was set at p ≤ 0.05. results in experiment 1, there was no effect of treatment on total motility (p = 0.97), progressive motility (p = 0.62), vcl http://dx.doi.org/10.58292/ct.v16.10782 citation: clinical theriogenology 2024, 16, 10782, http://dx.doi.org/10.58292/ct.v16.10782 3 (p = 0.16), number of detached heads (p = 0.27) or size of sperm clumps (p = 0.16). there was an effect of treatment on the number of sperm clumps (p = 0.05; figure 1). the min15s and min30s treatments had fewest clumps (4.7 ± 2.0 and 4.7 ± 2.0, respectively), followed by max15s (5.7 ± 2.0), max30s (6.7 ± 2.0), max5s (11.7 ± 2.0), min5s (12.0 ±2.0), p100 (12.7 ± 2.0), and p20 (17.7 ± 2.0; figure 1). in experiment 2, there was an effect of stallion (p = 0.0062), treatment (p = 0.0001), and treatment and stallion interaction (p < 0.0001) on sperm concentration. pairwise comparisons indicated that the concentration immediately after collection was different (p < 0.05) than all treatment groups and there was no difference in sperm concentration between any treatment group. the mean sperm concentration for the combined treatments groups was 215.5 ± 3.4 x106 sperm/ml. there was effect of collection (p < 0.0001), stallion (p < 0.0001), treatment (p < 0.0001), and collection and stallion interaction (p < 0.0001) on sperm viability. sperm viability evaluated immediate post collection was higher (p < 0.05) than the treatment groups and there was no significant difference among treatments (figure 2a). figure 1. mean number of sperm clumps in centrifuged samples after vortexing at minimum (min) speed for 5, 15 or 30 seconds; at maximum speed for 5, 15 or 30 seconds; and after mixing with pipette for 20 or 100 aspirations. groups without common superscripts differed (p < 0.05). 0 5 10 15 20 25 min5s min15s min30s max5s max15s max30s 20 100 mean number of sperm clumps vortex pipe�e groups 01x(sp mulc mrepsforeb mun 6 pe r m l) i, j e d k c, e, f a, c, d, e b, f, g, h a, b figure 2. percentage of sperm viability (a); total motility (b); progressive motility (c); curvilinear velocity (vcl μm/second) (d) at time 0 (prior to centrifugation) and at 24 hours after mixing with a pipette (pt24); mixing at minimum vortex speed (mint24) and maximum vortex speed (maxt24), and 48 hours after mixing with a pipette (pt48); and mixing at minimum vortex speed (mint48) and maximum vortex speed (maxt48). groups in a without common superscripts differed (p < 0.05). http://dx.doi.org/10.58292/ct.v16.10782 4 citation: clinical theriogenology 2024, 16, 10782, http://dx.doi.org/10.58292/ct.v16.10782 there was effect (p < 0.05) of stallion and time (p = 0.05), and collection and stallion interaction (p < 0.05) on total and progressive motility. there was no treatment effect on total or progressive motility (figures 2b and 2c). there was effect of collection (p = 0.0195), stallion (p = 0.0049), and time (p = 0.0008), but no effect (p = 0.95) of treatment on vcl (figure 2d). there was effect (p = 0.005) of collection, stallion (p < 0.0001), and time (p = 0.0002), and collection and stallion interaction (p = 0.0189) on str. there was effect (p < 0.0001) of collection, stallion (p < 0.0001), time (p = 0.0075), and collection and stallion interaction (p < 0.0001) on percent morphologically normal sperm (figure 3a). there was effect (p < 0.0001) of stallion, time (p = 0.002), and stallion and time interaction (p = 0.0015) on the number of tailless heads (figure 3b). there were no significant effects in the models for number of sperm clumps or size of sperm clumps (figures 3c and 3d). discussion to authors’ knowledge, this is the first study to examine the effect of mixing technique on sperm quality, longevity, and clumping. there were no significant differences between mixing sperm via a pipette or vortex mixer after cushion centrifugation on sperm quality, longevity, or clumping. there was no effect of treatment on motility, detached heads or size of sperm clumps but there was a difference in the number of sperm clumps among treatments (experiment 1). both treatments mixed with the pipette had the most sperm clumps suggesting that in this method mixing was not adequate. increased number of pipette aspirations improved mixing; however, this still performed poorly compared to most vortex treatments. therefore, we inferred that pipetting did not sufficiently agitate the solution compared to vortex mixing. vortex mixing in experiment 1 had varying numbers of sperm clumps, depending on the vortex time, with vortexing for 5 seconds, regardless of speed, having the most clumps. at both the minimum and maximum vortex speeds, both 15 and 30 seconds had the fewest number of sperm clumps. based on these data, both minimum and maximum vortex speeds for a 15 seconds vortex time were chosen for experiment 2, since it performed better and was the shortest time that should minimize potential stresses on sperm. there was no effect of treatment on sperm quality, longevity or sperm clumping (experiment 2). these data suggested that mixing with either a vortex or pipette is a sufficient method after cushion centrifugation. the most interesting finding in experiment 1 was a significant reduction in the mean number of sperm clumps among the pipette groups and some of the vortex groups; however, this was not observed in experiment 2. this could be due to 2 factors; concentration of reconstituted semen and tube diameter. as concentration increases, the interparticular distance decreases, based on the equation: = λ c n 1 3 , where n ≈ 6.02 x 1023 is avogadro number and λ is the interparticle distance.8 thus, it seems reasonable to assume that after mixing a less concentrated solution there would be a lower likelihood of sperm interacting and forming aggregates (clumps) compared to a more concentrated solution. secondly, a smaller diameter vortex (i.e. 15 ml conical tubes) has the material closer to the center of the vortex and consequently the material is stretched faster, reducing mixing time.8 thus, larger diameter tubes may require longer mixing times compared to smaller diameter tubes to have equal dispersion figure 3. percentage of morphologically normal sperm (a); number of tailless sperm (b); number of sperm clumps (x 106) per ml (c) and size of sperm clumps (classified as 1, 2, and 3) (d) at time 0, 24, and 48 hours after mixing with a pipette (p); and mixing at minimum vortex speed (min) and maximum vortex speed (max). http://dx.doi.org/10.58292/ct.v16.10782 citation: clinical theriogenology 2024, 16, 10782, http://dx.doi.org/10.58292/ct.v16.10782 5 of sperm into a mixture. thus, in hindsight, using 15 ml conical tubes in experiment 1 to maximize the number of treatments per ejaculate, may not represent the ideal vortex times for a 50 ml conical tube to maximize mixing. fluid properties of protein solutions (which vary depending on the molecular weight and concentration of protein and type and concentration of liquid3) can alter the fluid stresses during mixing. thus, various extenders could experience different stresses, depending on the composition, during mixing that could impact sperm quality. a chemically defined semen extender (inra96)9 was used in the present study. thus, the findings herein reported are not necessarily applicable to other types of semen extenders, especially egg yolk based that have larger particles than skim milk based extenders.10 in summary, vortex mixing had the same impact on sperm (quality and longevity) as mixing with a pipette and may be a quick and applicable alternative for sperm pellet resuspension after centrifugation. degree of clumping between 2 mixing methods varied with the concentration of sperm and size of the container used. conflict of interest none to report. references 1. varner dd: strategies for processing semen from subfertile stallions for cooled transport. vet clin north am equine pract 2016;32:547-560. doi: 10.1016/j.cveq.2016.07.007 2. bradecamp ea: centrifugation of semen: cushion technique. in: dascanio j, mccue p: editors. equine reproductive procedures [internet]. 1st edition, hoboken; wiley: 2021. p. 557-560. available from: https://onlinelibrary.wiley.com/doi/10.1002/9781119556015. ch150 [cited 7 august 2024]. 3. bai g, bee js, biddlecombe jg, et al: computational fluid dynamics (cfd) insights into agitation stress methods in biopharmaceutical development. int j pharm 2012;423:264-280. doi: 10.1016/j. ijpharm.2011.11.044 4. winant cd, browand fk: vortex pairing: the mechanism of turbulent mixing-layer growth at moderate reynolds number. j fluid mech 1974;63:237-255. doi: 10.1017/s0022112074001121 5. waite ja, love cc, brinsko sp, et al: factors impacting equine sperm recovery rate and quality following cushioned centrifugation. theriogenology 2008;70:704-714. doi: 10.1016/j.theriogenology. 2008.04.047 6. kenney rm: clinical fertility evaluation of the stallion. annu meet am assoc equine pract 1976;336-355. 7. love cc: modern techniques for semen evaluation. vet clin north am equine pract 2016;32:531-546. doi: 10.1016/j.cveq.2016.07.006 8. villermaux e: mixing versus stirring. ann rev fluid mech 2019;51:245-273. doi: 10.1146/annurev-fluid-010518-040306 9. batellier f, duchamp g, vidament m, et al: delayed insemination is successful with a new extender for storing fresh equine semen at 15°c under aerobic conditions. theriogenology 1998;50:229236. doi: 10.1016/s0093-691x(98)00130-7 10. clulow j, gibb z: liquid storage of stallion spermatozoa – past, present and future. anim reprod sci 2022;247:107088. doi: 10.1016/j.anireprosci.2022.107088 http://dx.doi.org/10.58292/ct.v16.10782 https://doi.org/10.1016/j.cveq.2016.07.007 https://onlinelibrary.wiley.com/doi/10.1002/9781119556015.ch150 https://onlinelibrary.wiley.com/doi/10.1002/9781119556015.ch150 https://doi.org/10.1016/j.ijpharm.2011.11.044 https://doi.org/10.1016/j.ijpharm.2011.11.044 https://doi.org/10.1017/s0022112074001121 https://doi.org/10.1016/j.theriogenology.2008.04.047 https://doi.org/10.1016/j.theriogenology.2008.04.047 https://doi.org/10.1016/j.cveq.2016.07.006 https://doi.org/10.1146/annurev-fluid-010518-040306 https://doi.org/10.1016/s0093-691x(98)00130-7 https://doi.org/10.1016/j.anireprosci.2022.107088 2012: isolation and identification of taylorella asinigenitalis from a mare in oklahoma, usa   isolation and identification of taylorella asinigenitalis from a mare in oklahoma, usa thomas j. reilly,a,b michael j. calcutt,a irene k. ganjam,b matthew m. erdman,c alan m. aalsburg,c joshua f. blyden,d and william h. falesa,b college of veterinary medicine, adepartment of veterinary pathobiology, bveterinary medical diagnostic laboratory, university of missouri, columbia, mo; usda, canimal plant health inspection service, national veterinary services laboratories, ames, ia; dnorman ok abstract contagious equine metritis (cem) is a highly contagious venereal disease of horses whose etiologic agent has been identified as taylorella equigenitalis. the bacterium is a catalase and oxidase positive pleomorphic gram-negative coccobacillus which grows best in a humidified environment of reduced oxygen (95% air containing 5% co2) on eugon chocolate agar. during routine screening of an oklahoma equine breeding population slated for export, a t. equigenitalis-like organism was isolated. based on results from requisite conditions for cultivation, biochemical tests, 16s rdna and intergenic spacer region (isr) sequence analyses, the isolated organism was identified as taylorella asinigenitalis. as required by federal regulations, this identification was confirmed at the national veterinary services laboratories (nvsl) in ames, ia. pulsed-field gel electrophoresis (pfge) analysis revealed that the newly identified strain has a distinct genomic fingerprint from the two previously characterized t. asinigenitalis isolates from the united states and represents the second strain to be recovered from a horse in the us. keywords: contagious equine metritis, taylorella equigenitalis, taylorella asinigenitalis introduction contagious equine metritis is a bacterial infectious disease of horses caused by taylorella equigenitalis. five reported outbreaks of cem have occurred in the united states within the past 44 years including those in kentucky in 19781 and again in 1979 along with missouri,2 and a multi-state outbreak investigation from 2008 to 2010.3 incidents involving a single horse have also been reported in 2010 and 2011.4 while symptomatic infection in mares is characterized by discharge of copious amounts of mucopurulent fluid from the vagina, infertility, and/or early abortion, stallions appear to remain asymptomatic during infection and serve as a reservoir for infection.5 cases of cem are rarely, if ever, fatal to an adult animal and while no prolonged deleterious medical effects have been described, the presence of cem in a breeding equine population has a significant economic impact.6 to avert such financial losses, many countries require certified and validated cem-testing of equine breeding populations prior to import or export into or from cem-endemic countries. in the united states, testing is federally regulated and conducted exclusively by usda-approved laboratories and diagnosticians. while direct pcr methods exist to detect t. equigenitalis in genital swabs,7-9 bacteriologic examination of submitted samples is still regarded as the only definitive means of determining the presence or absence of t. equigenitalis. the etiologic agent of cem, t. equigenitalis, is a non-motile, gram-negative coccobacillus with fastidious growth requirements. the bacterium is microaerophilic, and grows best on a semi-solid medium of chocolatized eugon agar or timoney agar in a humidified environment maintained at 5-10% co2, 95-90% air, and 37 °c.(5,10 while members of the genus taylorella are extremely limited in the repertoire of easily detectable enzyme activities, all known isolates are biochemically positive for cytochrome c oxidase, catalase, and alkaline phosphatase. in addition, the bacterium is susceptible to most antimicrobial agents except lincosamides, sulphamethoxazole, and while streptomycin-susceptible strains have been recovered, streptomycin-resistant strains have been described.5 phylogenetic analysis of the 16s ribosomal dna sequence revealed that taylorella, while phenotypically similar to haemophilus, occupies a unique niche in the β-subclass of the proteobacteria most closely related to pelistega europaea11 and constitutes a new genus within the subclass. clinical theriogenology • volume 4 number 2 • june 2012163   in the late 1990’s, three atypical taylorella isolates were obtained in california and kentucky from the genital tract of donkey jacks (equus asinus). these three isolates were identified during routine regulatory testing for cem and were found to be nearly indistinguishable from t. equigenitalis when assessed phenotypically. results from 16s ribosomal dna sequence analysis however, together with genomic dna-dna hybridization and g+c composition studies between these t. equigenitalis-like organisms and t. equigenitalis suggested that the former were unique and constituted a new species within the genus which was named taylorella asinigenitalis.12 significantly, following natural breeding, disease in mares was not detected; however, clinical signs were noted in another study following experimental inoculation of mares with t. asinigenitalis.13 a recent publication on the containment of the 1998 outbreak in kentucky reported that seven mares and one stallion were infected.14 in 2006, båverud et al. reported the isolation and identification of t. asinigenitalis from a naturally infected stallion in sweden and found the 16s rrna sequence to be identical to that of the california ucd-1 type strain, first described in the 2001 report.15 in february 2008, franco et al. reported the isolation of taylorella asinigenitalis organisms, identical in 16s rdna sequence analysis (nucleotide 505-1120) to the t. asinigenitalis ucd-1 type strain (atcc 70093) strain from two donkey jacks of the martina franca endangered breed in apulia, italy.16 in 2011, breuil et al. reported the isolation of three french t. asinigenitalis strains from horses, each of which could be distinguished by a combination of profiling methods.17 interestingly, one of these isolates was collected in 1995 and was, retrospectively, the first t. asinigenitalis to be recovered. we report here the isolation and identification of taylorella asinigenitalis from a mare in the state of oklahoma. the isolate was discovered during routine testing of samples at a time concomitant with but independent of the united states 2008-2010 cem investigation. both phenotypic and genotypic properties were assessed in the identification of this isolate, designated t. asinigenitalis umc-1, which also demonstrated that the strain is distinct from that associated with the 1998 kentucky outbreak in horses and from the type strain identified in a california donkey jack. methods unless noted otherwise all strains used in this study were cultivated for 48-72 hr at 37 °c in a humidified environment containing 95% (v/v) air and 5% (v/v) co2 on chocolatized eugon and timoney agars (biomed diagnostics, white city, or) as previously described.10 additional media including tubed biochemical tests, blood and macconkey agars were supplied by remel® (lenexa, ks). initial oxidase and catalase tests were performed on colonies recovered from timoney media after at least 48-hr incubation. the presence of catalase activity was determined by exposing selected bacterial colonies to 3% (v/v) h2o2 and observing the generation of oxygen. the presence of oxidase was determined using oxoid® oxidase sticks (remel). the specific alkaline phosphatase (alkp) activity present in crude bacterial extracts was measured in quadruplicate using a 0.2 ml discontinuous colorimetric assay consisting of 100 mm tris ph 10.0, 1 mm mgcl2, 2 mm p-nitrophenylphosphate (pnpp), and varying amounts of suspended bacteria. the mixtures were incubated at 37 c for 15 min with constant agitation after which time the concentration of liberated p-nitrophenol (pnp) was determined at 405 nm using a model 680 microplate reader (biorad carlsbad, ca) and a standard curve of known concentrations of pnp. the protein concentration was determined using the bicinchoninic acid technique as previously described18 using a bsa standard. the conditions used for alkp determination were identical to those at which significantly enhanced alkp activity was noted for the purified recombinant t. equigenitalis alkp (unpublished results). chromosomal dna used for pcr and sequencing was initially isolated from 48-72 hr cultures using a wizard genomic dna purification kit according to the manufacturer’s protocol (promega, madison, wi). primers isr-f (5’ ctggggtgaagtcgtaacaag) and isr-r (5’ tgtgatcgccaaggcatccacc) were used for pcr amplification of isr dna as described by tazumi et al.19 briefly, 50 ng total genomic dna was used as template with 20 pmol of each primer in standard buffer conditions for phusion high-fidelity dna polymerase (new england biolabs, ipswich, ma). following an initial denaturation step of 98 c for 30 s, amplification was achieved by 30 cycles of clinical theriogenology • volume 4 number 2 • june 2012 164   the following parameters: 98 c for 10 s, 57 c for 30 s and 72 c for 30 s. the resulting amplicons were purified (qiaquick spin column, qiagen, valencia ca) and ligated into the positive selection vector pjet1.2 (fermentas, glen burnie, md). after transformation of escherichia coli dh10b competent cells (invitrogen, calsbad, ca), plasmid dna from individual colonies was prepared (qiagen) and used as template for dna sequencing using vector-derived primers and bigdye terminator chemistry. sequencing was performed at the dna core facility at the university of missouri-columbia on a pe biosystems 3730 capillary dna sequencer. the resulting dna sequences were queried against entries in genbank available through the national center for biotechnology information. the isr sequences reported herein have been deposited in genbank under accession numbers jq783350 and jq783351. additional confirmatory tests were performed at nvsl. these tests included 16s ribosomal dna (rdna) sequencing, slide agglutination test (monotayl, bionor laboratories as, skien, norway), and direct fluorescent antibody testing using polyclonal antibody conjugate. to assess for streptomycin susceptibility, bacterial isolates were plated on chocolated eugon agar with or without streptomycin at 200 μg/ml final concentration. plates were incubated at 37 °c in 5% (v/v) co2 for at least seven days. any growth in the presence of streptomycin indicated resistance. pulsed-field gel electrophoresis (pfge, chef-dr ii system, biorad, carlsbad, ca) techniques to determine the relatedness of taylorella strains have been described previously.4 briefly, cell suspensions in 100 mm tris hcl 100 mm edta, ph 8.0 from 72-96 hour cultures grown on timoney media were used to prepare agarose plugs containing genomic dna. the dna-containing plugs were incubated with the restriction enzyme not i (invitrogen, carlsbad, ca) at 37 °c. resulting pfge patterns were standardized with a universal sized standard salmonella enterica subsp. enterica serovar braenderup h9812 and analyzed (bionumerics, applied maths, austin tx). a dendogram was created using dice’s coefficient and upgma based on band position tolerance values of 1.5%. results and discussion assessed after 48-72 hours of incubation at 37 °c in 5% (v/v) co2, the umc-1 isolate along with both t. equigenitalis and t. asinigenitalis control strains were gram-negative, nonmotile coccobacillary organisms. all three isolates were strongly oxidase and catalase positive resulting in a rapid darkening color of the oxidase stick and generation of copious amounts of bubbles (oxygen) in the presence of hydrogen peroxide, respectively. the three isolates were deemed streptomycin resistant, fastidious, and co2-dependent as growth of the microbes was noted only on chocolated eugon and timoney agars in a humidified environment composed of 95% air and 5% co2. the growth of the isolates was barely perceptible on blood agar incubated in co2 and not at all on any medium cultured in an aerobic incubator. the isolates also failed to grow anaerobically or on macconkey agar. in addition, pre-incubation of the isolate for 24 hours on a rich medium in an air incubator prior to transfer to a co2 incubator failed to result in observable growth. biochemically, all three isolates were negative in all the tests included in a tsi series consisting of various tubed media including triple sugar iron agar, lysine iron agar, motilityindole-ornithine decarboxylase agar, citrate and urea agar. the specific alkaline phosphatase activity of the umc-1 isolate was 665 ± 64 nmole pnp produced/hr/mg protein; that of the t. asinigenitalis type strain was 637 ± 109 nmole pnp produced/hr/mg protein. the specific alkaline phosphatase activity of the t. equigenitalis type strain was 951 ± 107 nmole pnp produced/hr/mg protein. while certainly not definitive for identification, the alkaline phosphatase activity of umc-1 was consistently most similar to that of the reference t. asinigenitalis strain than to that of the t. equigenitalis strain. amplification with isr-flanking primers resulted in the generation of multiple amplicons in the 900-1000-bp approximate size range, as anticipated from a previously published study.19 after cloning into a positive selection vector, plasmid dnas corresponding to each of two differently sized amplicons were identified and subjected to dna sequence analysis. each isr sequence was closely related to those determined for other t. asinigenitalis isolates, and each contained coding sequences for trna ile and trna ala. sequence 1 was 922 bp in length and was most similar to isr c of strains uk-1 and uk-2 (97%) and slightly less similar to isr c of strain ucd-1 (93%) as described by tazumi et al .19 sequence 2 (1002 bp) was 99% identical to sequence 1 in regions of overlap between the two differently clinical theriogenology • volume 4 number 2 • june 2012165   sized isrs, and shared 99% identical residues with isr b of strains uk-1 and uk-2.19 both isr sequences determined in this study are also distinct from those present in the complete genome of t. asinigenitalis strain mce9 (isolated in 2004 from a donkey jack in france).20 importantly, both amplicons exhibited significantly less sequence identity to isr regions from t. equigenitalis. sequence 1 and 2 were only 72% and 70% identical, respectively, to t. equigenitalis eq59, the most closely related strain of this species. as each isr from the mare isolate (umc-1) had differences from the published sequences, an additional pcr/cloning was performed to eliminate the possibility of the introduction of inadvertent mutations during amplification. sequence analysis of independent plasmid clones verified the presence of characteristic patterns of snps as well as insertion/deletion (indels), which may prove useful in strain discrimination as additional t. asinigenitalis isolates are identified. pfge analysis of genomic dna from the umc-1 strain showed that it differed from both the uk isolate and the ucd-1 isolate when digested with not i (figure). the pfge profile of umc-1 was more similar (88%) to the uk isolate and less similar (59.4%) to the ucd-1 type strain. although the time between isolations of umc-1 and ucd-1/uk strains spans approximately ten years, these results indicate that the umc-1 isolate is not closely related to past strains of t. asinigenitalis found in the united states. in conclusion, small gram-negative coccobacilli possessing copious amounts of catalase, oxidase, and alkaline phosphatase activity were unexpectedly recovered from one horse during routine cem screening of eight oklahoma quarter horses for export. while this isolate was nearly indistinguishable from the etiologic agent of cem, t. equigenitalis phenotypically, results from genomic analysis found the t. asinigenitalis umc-1 isolate to be more closely related to, though distinct from the ucd-1 type strain identified in 1997. these findings highlight the need for further epidemiological studies of the prevalence and strain profiles of t. asinigenitalis isolates from equid species. figure: pfge analysis of taylorella asinigenitalis strains uk, umc-1, and ucd-1 after digestion of genomic dna with not i. dendrogram was created using dice’s coefficient and upgma and based on 1.5% band position tolerance values. acknowledgement this work was supported by usda animal health formula funds to the university of missouri college of veterinary medicine. references 1. swerzek tw: contagious equine metritis in usa. vet rec 1978;102:512-513. 2. fales wh, backburn, bo, youngquist, rs. et al: laboratory methodology for the diagnosis of contagious equine metritis in missouri. proc 22nd ann meeting amer assn vet lab diag 1979; p.187-197. 3. erdman, mm, creekmore lh, fox pe. et al: diagnostic and epidemiologic analysis of the 2008-2010 investigation of a multi-year outbreak of contagious equine metritis in the united states. prev vet med 2011;101:219-228. 4. aalsburg am, erdman mm: pulsed-field gel electrophoresis genotyping of taylorella equigenitalis isolates collected in the united states from 1978 to 2010. j clin microbiol 2011;829-833. 5. taylor ced, rosenthal ro, brown dfj: the causative organism of contagious equine metritis 1977: proposal for a new species to be known as haemophilus equigenitalis. equine vet j 1978;10:136-144. 6. matsuda m, moore je: recent advances in molecular epidemiology and detection of taylorella equigenitalis associated with contagious equine metritis (cem). vet microbiol 2003;97:111-122. 10 0 90807060 uk umc-1 ucd-1 clinical theriogenology • volume 4 number 2 • june 2012 166   7. niwa h, anzai t, hobo s: construction of a recombinant plasmid as reaction control in routine pcr for detection of contagious equine metritis (cem-pcr). j vet med sci 2007;69:1199-1201. 8. wakeley pr, errington j, hannon s, et al: development of a real time pcr for the detectin of taylorella equigenitalis directly from genital swabs and discrimination from taylorella asinigenitalis. vet micro 2006;118:247-254. 9. duquesne f, pronost s, laugier c, et al: identification of taylorella equigenitalis responsible for contagious equine metritis in equine genital swabs by direct polymerase reaction. res vet sci 2007;82:47-9. 10. timoney pj, shin sj, jacobson rh: improved selective medium for isolation of the contagious equine metritis organism. vet rec 1982;111:107-108. 11. sugimoto c, isayama y, sakazaki r et al: transfer of haemophilus equigenitalis taylor et al. 1978 to the genus taylorella gen. nov. as taylorella equigenitalis comb. nov. curr microiol 1983;9:155-162. 12. jang ss, donahue jm, arata ab, et al: taylorella asinigenitalis sp. nov., a bacterium isolated from the genital tract of male donkeys (equus asinus). int j sys evol microbiol. 2001;51:971-976. 13. katz, jb. evans, le, hutto, dl, et al. clinical bacteriologic pathogenic features of infections with atypical taylorella equigenitalis in mares. j am vet med assoc 2000:216:1945-1948. 14. meade, bj, timoney, pj, donahue, jm, et al: initial occurrence of taylorella asinigenitalis and its detection in nurse mares, a stallion and donkeys in kentucky. prev vet med 2010: 95:292-296. 15. båverud v, nyström c, johansson ke: isolation and identification of taylorella asinigenitalis from the genital tract of a stallion, first case of natural infection. vet microbiol 2006;116:294-300. 16. franco a, donati v, troiano p, et al: detection of taylorella asinigenitalis in donkey jacks in italy. vet rec 2009;165:540-541. 17. breuil mf, duquesne f, laugier c, et al: phenotypic and 16s ribosomal rna gene diversity of taylorella asinigenitalis strains isolated between 1995 and 2008. vet microbiol 2011;260-266. 18. smith pk, krohn rl, hemanson gt, et al: measurement of protein using bicinchoninic acid. anal bioch 1985;150:7685. 19. tazumi a, ono s, sekizuka t, et al: molecular characterization of the sequences of the 16s-23s rdna internal spacer region (isr) from isolates of taylorella asinigenitalis. bmc res notes. 2009; 2:33. 20. hébert l, mourmen b, pons n, et al. genomic characterization of the taylorella genus. plos one 201 e29953. clinical theriogenology • volume 4 number 2 • june 2012167 2016: a case report on the use of domperidone for management of agalactia in a queen a case report on the use of domperidone for management of agalactia in a queen rochelle l. jensen, aime k. johnson, robyn r. wilborn, natalie s. fraser college of veterinary medicine, auburn university, auburn, al summary a pregnant three year old himalayan queen was presented to auburn university college of veterinary medicine with her fourth pregnancy. she had lost three previous litters of kittens within two days of birth and had a history of minimal mammary development during the peri-parturient period. repeated sonographic measurements of fetal structures were used to determine gestational age. six days before parturition, domperidone was prescribed at a rate of 2.2mg/kg po q12 hours, and was continued for seven days postpartum. parturition was uneventful and the queen’s mammary development and milk production were adequate. four weeks postpartum, three of five kittens that survived appeared healthy. domperidone is a d2 receptor antagonist used anecdotally for induction of lactation in small animals but the efficacy for this purpose in the queen is not well described. this case demonstrates successful induction of lactation with domperidone in a previously agalactic queen. keywords: queen, agalactia, domperidone, mammary development background throughout gestation, proliferation of mammary epithelium is induced by the sex steroids 17βestradiol and progesterone.1 mammary glands begin to assume their functional secretory structure late in gestation.2 lactogenesis (production of colostrum and first milk) requires proper differentiation of secretory luminal epithelium during pregnancy. an additional requirement involves the coordinated contractions of the myoepithelial cells that surround the alveoli and duct system in order to transport secretory products.3,4 the essential role of gonadal, pituitary, and adrenal hormones on mammary development was investigated by nandi in 1958.5 this study investigated hormone therapies using hypophysectomized, ovariectomized, and adrenalectomized mice. that work revealed that both growth hormone and estrogen are required for mammary duct development. they also demonstrated that progesterone induced side branching of the ducts, and prolactin was associated with alveolar development.5 the exact role that growth hormone contributes to regulation of lactogenesis and lactation in small animals remains unclear, yet it is known that progestins stimulate local production of growth hormone in the mammary gland and that mammary derived growth hormone has a stimulatory effect on mammary development.6 during lactogenesis, prolactin and growth hormone play a significant role in the transition from a proliferative state to a fully functional milk secreting gland.4 in the pregnant bitch, a rapid decline in progesterone to <2 ng/ml usually precedes parturition and occurs concomitantly with or just before a prolactin surge.7 this rapid decline in progesterone prior to parturition is not a prerequisite for the initiation or completion of parturition in the queen as demonstrated by schmidt et al.8 in that study, seven of 12 queens had serum progesterone concentration of <1 ng/ml on the day of parturition. one queen had a serum progesterone concentration of 11.4 ng/ml on the day of parturition but had decreased to 2.6 ng/ml one day postpartum.8 failure of mammary development and milk production can account for neonatal losses due to inadequate colostral intake or starvation. agalactia may be primary or secondary, with most cases of agalactia occurring secondary to stress induced failure of the release of prolactin from the anterior pituitary. risk factors include systemic disease, genetic factors, environmental stressors or poor nutritional status. treatment is directed at treating the primary cause and providing supplementation to the neonates whilst encouraging suckling to promote milk let-down. primary agalactia may occur due to failure of endocrine mechanisms responsible for lactogenesis and galactopoesis.9 a diagnosis of primary agalactia can only be made when causes of secondary agalactia are ruled out. disturbances of the pituitary-ovarian-mammary axis and subsequent failure of an adequate rise in prolactin could explain primary agalactia, yet the exact pathophysiology in the queen remains unclear. despite a lack of understanding of the exact mechanisms behind this condition, there is anecdotal support for the use of antidopaminergic medications to improve lactation in the queen. two antidopaminergic clinical theriogenology • volume 8 number 2 • june 2016121 medications currently used for lactational management in small animals are metoclopramide and domperidone. metoclopramide is an anti-emetic agent but its antidopinergic effects for successful induction of lactation in small animals is anecdotally reported. it antagonizes d2-dopinergic receptors and 5-ht3 serotonergic receptors10 domperidone is another oral d2 receptor antagonist that is indicted for induction of lactation in mares with fescue toxicosis. published reports of the use of domperidone in the dog and cat are scarce. this case demonstrates successful induction of lactation in a previously agalactic queen utilizing domperidone. case presentation a two-year-old himalayan queen was initially presented to auburn university college of veterinary medicine small animal theriogenology service for a postpartum evaluation and assessment of her kittens. the queen had delivered three kittens approximately 8-12 hours before presentation. on presentation, the owner reported that kittens from two earlier litters had died within 24 hours of birth, but were not presented to a veterinary hospital. all litters were sired by the same himalayan tom. the tom had sired other litters with no reports of neonatal morbidity or mortality. the queen was negative for feline immunodeficiency virus antibody and feline leukemia virus antigen. the queen was clinically normal with all physical parameters within normal limits. her mammary glands were poorly developed and milk could not be expressed from any gland. the kittens had not been observed nursing, were lethargic, cold to touch, and had yellow staining around the perineum. the queen was administered metoclopramide (0.2 mg/kg sq q8hrs) in an effort to induce lactation. oxytocin (1-2 iu sq q8hrs) was administered to stimulate milk let-down. the kittens were cleaned, placed in a warmed incubator, and were administered milk replacer at a rate of 3.5ml/100mg every two hours via an orogastric tube. the sire and the dam were blood typed in order to rule out neonatal isoerythrolysis (ni) and both were found to be blood type a, thus excluding ni as a cause of the neonatal death from previous litters. the queen and her kittens were hospitalized overnight for continued treatment. two of three kittens died before morning and were submitted for postmortem examination. postmortem examination of both kittens revealed moderate pleural effusion and focally extensive atelectasis. on the liver, there were small pin-point, tan-white, multifocal to coalescing nodules randomly distributed over the surface of the liver. histopathological analysis of liver tissue showed multiple foci of lipid type vacuolar degeneration and glycogen type vacuolar degeneration. although bacterial culture from a swab of lung tissue from one of the kittens yielded a heavy growth of pasteurella stomatitis, there was no histological evidence of an inflammatory process in lung tissue. polymerase chain reaction assay was performed on kidney and spleen tissue for detection of feline coronavirus and was negative. postmortem findings suggested marked metabolic derangement and unknown pathology, possibly due to transient starvation but the exact etiology could not be confirmed. for the queen, metoclopramide and oxytocin treatments were continued every eight hours for a total of six treatments, with the last two treatments being administered by the client after discharge. at the time of discharge, the one remaining kitten was nursing and the queen was showing some slight improvement in mammary development. the client was instructed to monitor maternal behavior, nursing, and to perform twice daily weight checks on the kitten. furthermore, it was recommended that the queen undergo ovariohysterectomy based on the history of neonatal mortality and agalactia. despite treatment, the remaining kitten died two days following discharge. despite recommendations to the owner to not allow breeding again, the queen was again presented six months later for a pregnancy examination. on this visit, transabdominal ultrasonography was performed and pregnancy was confirmed by visualization of multiple viable conceptuses. using sonographic measurements of fetal and extra-fetal structures, it was estimated that the queen had been bred approximately 30 days previously. gestational aging was performed using a combination of measurements of biparietal, abdominal, and gastric diameter.11 additional estimates were based on sonographic evidence of fetal organ maturation.11 transabdominal ultrasound was performed every seven to ten days for the remainder of the pregnancy to monitor fetal growth and viability. clinical theriogenology • volume 8 number 2 • june 2016 122 treatment eight days prior to the queen’s estimated parturition date (six days prior to her actual delivery date), treatment with domperidone was initiated at a rate of 2.2mg/kg po q12 hours. the queen and five kittens were presented to the university within two hours of parturition. on examination of the queen, milk could be expressed from the caudal four mammary glands. all kittens were mildly dehydrated but their body temperatures were within normal limits. body weights ranged from 67 grams to 80 grams. the kittens appeared to have been cleaned and the dam was attentive to the kittens. within an hour of presentation, kittens were supplemented with milk replacer via an orogastric tube at a rate of 3.5ml/100g, and warmed subcutaneous fluid [plasmalyte®] was administered at a rate of 2-3ml per kitten. serum was harvested from the sire (blood type a) and was administered to each kitten at a rate of 150 ml/kg sq.12 outcome the queen and kittens were discharged with instructions to continue administration of domperidone to the queen at a dose rate of 2.2mg/kg po q12 hours for an additional seven days. the owner was instructed to weigh the kittens twice daily and supplement kittens with milk replacer via an orogastric tube if the kittens failed to gain weight. following discharge, the weakest and smallest two kittens failed to thrive and required additional supplementation. despite treatment, both died at home within the first week of life. a necropsy was not performed so the cause of death could not be determined. the surviving three kittens did not require additional supplementation following discharge. four weeks following discharge, the remaining three kittens were growing, appeared healthy, and were transitioning to solid food. further communication with the owner revealed that the three remaining kittens survived and at eight weeks of age, were placed into adoptive homes. discussion agalactia is reported occasionally in queens but the true prevalence of this condition remains unknown. the relative role of reproductive hormones, prolactin, and growth hormone in mammary development is likely species dependent.13 this species specific variation is important because the medications and doses currently used for induction of lactation in small animals are extrapolated from both human and large animal studies. in domestic mammals, prolactin is secreted by the anterior pituitary and is under inhibitory control by the hypothalamus, mediated in part by dopamine.14 in addition to its role in mammogenesis, lactogenesis and lactation, it also plays a central role in the development of maternal behavior.15 in the queen, prolactin production starts to increase around day 35 of gestation.16 it reaches a plateau around the third and fourth week of gestation and then gradually declines. it rises again relatively abruptly to 39-48 ng/ml a few days before parturition.8,16,17 the dopamine neurons that control prolactin secretion are located in the hypothalamus. they act as a synchronous network to release dopamine into the pituitary portal system in a pulsatile or phasic fashion.18 hypothalamic control of prolactin secretion differs from other pituitary hormones in that it is primarily inhibitory.18 dopamine acts on d2 receptors in the pituitary gland to inhibit lactotrophs. a unique feature of lactotrophs is that they display spontaneous electrical activity in the absence of dopaminergic activity and calcium influx stimulates prolactin secretion.18 dopamine exerts its effects by inhibiting calcium influx and induces a state of membrane hyperpolarization. the regulation of dopamine neurons is via a short feedback loop where prolactin itself stimulates hypothalamic dopamine synthesis.18 during pregnancy and early lactation, dopamine release from the hypothalamus reduces so that prolactin secretion remains high.18 a clearer understanding of the pathophysiology of primary agalactia in the queen is needed. knowing the unique species specific regulators of lactogenesis and galactogenesis would guide management of this condition. it is likely that primary agalactia has multiple etiologies which have complex interactions. much of the current research in mammary responses to prolactin in small animals revolves around identification of markers for mammary neoplasia.19,20 interestingly, the observed initial clinical theriogenology • volume 8 number 2 • june 2016123 rise in prolactin that occurs during pregnancy occurs relatively concomitantly or just after a rise in placental relaxin.21 placental relaxin is a peptide hormone that is first detectable in plasma of the pregnant queen between days 20 and 25 of gestation. concentrations increase and reach a plateau by days 30-35 and then decline in the last 10-15 days of gestation.21 studies investigating the association between mammary development and relaxin have produced conflicting data. some research supports a role of relaxin in mammary development while others do not support an association. a study by zhao et al. demonstrated that relaxin deficient mice were unable to develop mammary tissue. pups were unable to suckle and died within the first 24 hours of life.22 in contrast, more recent studies revealed that relaxin does not play a significant role in mammary development. mice with targeted gene deletion for relaxin or its receptor (rxfp1) had normal mammary development, evidenced by an absence of morphological or phenotypical defects in the lobuloalveoli or ductal system of the mammary gland during late pregnancy or early lactation.23 furthermore, a very recent study revealed that nipple size and lactation were normal in rxfp1 knockout mice.24 the anti-dopaminergic effect of metoclopramide has been utilized for lactation management in both small animals and women. it was originally marketed in europe as an antipsychotic for humans, and later in the united states as a gastrokinetic.14 its use for induction of lactation in the bitch and queen is anecdotal. romagnoli et al. reviewed current protocols in small animal veterinary medicine and reported ranges as low as 0.2-0.5 mg/kg sq or po twice or three times daily, to higher doses ranging from 1-5 mg/kg sq or po up to four times daily.9 administration of metoclopramide results in a subjective improvement in milk production in at least 50% of cases.9 there is no clear dose dependent relationship with efficacy, although high doses have been associated with extrapyradmidal signs in canines and should therefore be avoided.9 oxytocin is frequently used alone or in combination with metoclopramide for treatment of galactostasis and can be used at doses of 0.5 to 1 iu im 15-20 minutes before nursing.25 in this particular queen, administration of metoclopramide and oxytocin had only a minimal effect on milk production and milk let down. domperidone was first developed as an antiemetic agent in humans. it was withdrawn from the human market following the observation of cardiac arrhythmias in patients who were receiving very high doses when used concurrently with chemotherapy. it is still used widely in canada and europe for lactational management in women.9 in veterinary medicine, it is labeled as equidone® gel (dechra veterinary products, overland park, ks) and is used to treat agalactia associated with fescue toxicosis in periparturient mares.26 the drug is administered orally at a dose of 1.1 mg/kg once daily starting 10 to 15 days prior to the expected foaling date. treatment is continued for up to five days postpartum.26 there are anecdotal reports that domperidone is effective at improving milk yield in small animals at doses of 1.5-2.0 mg/kg in the queen and 2.2 mg/kg in the bitch.9 the most commonly reported side effect in the bitch is diarrhea.9 to our knowledge, this is the first publication reporting the use of domperidone for induction of lactation in the queen. the apparent improvement in treatment response to domperidone rather than metacloprmide may be attributable to the differences in pharmacological behavior of the two drugs. while both metoclopramide and domperidone increase prolactin by inhibition of dopaminergic activity, a notable difference between the two drugs is that the latter does not readily cross the blood brain barrier.27 domperidone acts on peripheral d2-dopinergic receptors, and therefore offers the advantage that it is less likely to cause extrapyradmidal signs when compared with metoclopramide.27 alternatively, had drug therapy using metoclopramide been initiated prepartum, as was domperidone, treatment response in this queen may have been equal or better. serial measurement of endogenous prolactin, and perhaps relaxin, could have provided valuable insight to this observation. two of the five kittens failed to survive despite treatment and supplementation with milk replacer. since these kittens died at home and necropsy was not performed, the cause of death could not be determined. the owner was performing the supplemental feedings as directed by clinicians during daily phone conversations. both kittens failed to gain weight, became progressively weaker, and died during the night when supplemental feedings were more sporadic. starvation is not likely a cause of clinical theriogenology • volume 8 number 2 • june 2016 124 death in these two kittens if the owner was complying with the scheduled feedings. underlying pathology of the gastrointestinal tract or other system is more likely, but since no necropsy was performed, cannot be ruled in or out as a cause of death. therefore, when treating agalactia in the mother, care should be taken to fully assess kittens for any signs of other pathology and rule out other common causes for neonatal loss. domperidone administered orally at a rate of 2.2mg/kg bid initiated approximately one week prior to parturition and continued for seven days postpartum was successful for induction of lactation in a previously agalactic queen, and was not associated with negative side effects. further research is warranted regarding exact dose, efficacy and toxicological effects. learning points • the pathogenesis of primary agalactia in the queen remains to be elucidated. • current, published estimates of sonographic measurements of fetal maturity are useful to time protocols for lactation induction in the queen when breeding dates are not known. • domperidone is a reasonable option for induction of lactation in the queen. references 1. clarke r: introduction and overview: sex steroids in the mammary gland. j mammary gland biol neoplasia 2000;5:245-250. 2. akers rm, denbow dm: lactation and animal agriculture in: akers rm, denbow dm, editors. anatomy and physiology in domestic animals. 2nd ed. oxford: john wiley & sons; 2013. p. 501-510. 3. plante i, stewart mk, laird dw: evaluation of mammary gland development and function in mouse models. j vis exp 2011;53:2828. 4. lamote i, meyer e, massart-leen am, et al: sex steroids and growth factors in the regulation of mammary gland proliferation, differentiation, and involution. steroids 2004;69:145-159. 5. nandi s: endocrine control of mammary gland development and function in the c3h/he crg1 mouse. j natl cancer inst 1958;21:1039-1063. 6. selman pj, mol ja, rutteman gr, et al: progestin-induced growth hormone excess in the dog originates in the mammary gland. endocrinology 1994;134:287-292. 7. concannon pw, butler wr, hansel w, et al: parturition and lactation in the bitch: serum progesterone, cortisol and prolactin. biol reprod 1978;19:1113-1118. 8. schmidt pm, chakraborty pk, wildt de: ovarian activity, circulating hormones and sexual behavior in the cat. ii. relationships during pregnancy, parturition, lactation and the postpartum estrus. biol reprod 1983;28:657-671. 9. romagnoli s, lopate c: control of mammary gland function in the bitch and queen: a review. clin therio 2012;4:196205. 10. german a, maddison j, guilford g: gastrointestinal drugs in: maddison j, page s, church d, editors. small animal clinical pharmacology. 2 ed. edinburgh: elsevier science; 2008. p. 469-497. 11. zambelli d, prati f: ultrasonography for pregnancy diagnosis and evaluation in queens. theriogenology 2006;66:135144. 12. levy jk, crawford pc, collante wr, et al: use of adult cat serum to correct failure of passive transfer in kittens. j am vet med assoc 2001;219:1401-1405. 13. akers rm: major advances associated with hormone and growth factor regulation of mammary growth and lactation in dairy cows. j dairy sci 2006;89:1222-1234. 14. valdes v: a critical review of pharmaceutical galactagogues. breastfeed med 2007;2:229-242. 15. bridges rs, numan m, ronsheim pm, et al: central prolactin infusions stimulate maternal behavior in steroid-treated, nulliparous female rats. proc natl acad sci usa 1990;87:8003-8007. 16. banks dr, paape sr, stabenfeldt gh: prolactin in the cat: i. pseudopregnancy, pregnancy and lactation. biol reprod 1983;28:923-932. 17. tsutsui t, stabenfeldt gh: biology of ovarian cycles, pregnancy and pseudopregnancy in the domestic cat. j reprod fertil suppl 1993;47:29-35. 18. grattan dr: 60 years of neuroendocrinology: the hypothalamo-prolactin axis. j endocrinol 2015;226:101-122. 19. michel e, rohrer bley c, kowalewski mp, et al: prolactin-to be reconsidered in canine mammary tumourigenesis? vet comp oncol 2014;12:93-105. 20. michel e, feldmann sk, kowalewski mp, et al: expression of prolactin receptors in normal canine mammary tissue, canine mammary adenomas and mammary adenocarcinomas. bmc vet res 2012;8:72-80. 21. stewart dr, stabenfeldt gh: relaxin activity in the pregnant cat. biol reprod 1985;32:848-854. 22. zhao l, roche pj, gunnersen jm, et al: mice without a functional relaxin gene are unable to deliver milk to their pups. endocrinology 1999;140:445-453. clinical theriogenology • volume 8 number 2 • june 2016125 23. parry lj, vodstrcil la, madden a, et al: normal mammary gland growth and lactation capacity in pregnant relaxindeficient mice. reprod fertil dev 2009;21:549-560. 24. kaftanovskaya em, huang z, lopez c, et al: conditional deletion of the relaxin receptor gene in cells of smooth muscle lineage affects lower reproductive tract in pregnant mice. biol reprod. 2015;92:1-9. 25. wiebe vj, howard jp: pharmacologic advances in canine and feline reproduction. topics in companion animal medicine 2009;24:71-99. 26. cross dl, reinemeyer cr, prado jc, et al: efficacy of domperidone gel in an induced model of fescue toxicosis in periparturient mares. theriogenology 2012;78:1361-1370. 27. jolliet pj, nion s, allain-veyrac g, et al: evidence of lowest brain penentration of an antiemetic drug, metopimazine, compared to domperidone, metoclopramide, and chlorpromazine, using an in invitro model of the blood-brain barrier. pharmacol res 2007;56:11-17. clinical theriogenology • volume 8 number 2 • june 2016 126 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype true /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 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minerals increase mrna expression of some toll like receptors in testes of bulls with heat induced testicular degeneration mohammad ibrahim,a maria ferrer,a roberto palomares,b annie bullington,a alejandro hoyos-jaramillo,b joão bittarb adepartment of large animal medicine, bdepartment of population health college of veterinary medicine, university of georgia, athens, ga activation of toll like receptors (tlrs) initiates an innate immune response. these receptors recognize pathogen-associated ligands in microorganisms and endogenous damage-associated molecular patterns (damps) released by damaged cells. treatment with 1 dose of injectable trace minerals (itm; 1 ml/150 lb, multimin 90, multimin north america, fort collins, co) resulted in a more pronounced decrease in semen quality in bulls with testicular degeneration. release of damps by degenerating germ cells may exacerbate testicular dysfunction via activation of testicular tlrs. since itm can upregulate immune function in cattle, we hypothesized that tlrs are expressed in bovine testis, and that tlr mrna transcript levels are affected by tms in bulls with testicular degeneration. objectives were to characterize mrna expression of tlrs in bulls with testicular degeneration and after treatment with itm. testicular degeneration was induced by scrotal insulation for on days 0 8. bulls (n = 9) were assigned to 3 groups; c: control without itm; tm1: received itm on day -21; and tm2: received itm on days -21 and 8. testicular biopsies were collected from 3 bulls in each group on day 110. testicular samples were frozen at -80ºc until rna extraction. samples were cryopulvurized and total rna was extracted using qiagen rneasy mini kit. total rna was subjected to reverse transcription and qpcr. level of gene transcription was estimated in comparison with standard curves generated from 10 fold serial dilutions of amplicons. expression values were normalized against gapdh as a housekeeping gene. expression of tlrs 1 10 was confirmed in testis of bulls with testicular degeneration. bulls in group tm1 had higher mrna expression of tlr1 (mean ± sem: c 0.032 ± 0.005, tm1 0.056 ± 0.006, tm2 0.04 ± 0.006) (p = 0.022, anova) than group c, but not tm2. a similar tendency was seen for increased transcripts levels of tlr4 (p = 0.057), tlr6 (p = 0.075) and tlr10 (p = 0.05) in group tm1. bulls in group tm1 also had a tendency for increased transcript levels of tlr1 (p = 0.089), 3 (p = 0.079), 4 (p = 0.076), 7 (p = 0.066) and 9 (p = 0.068) compared to group tm2. there were no differences in mrna expression between c and tm2. there was moderate to strong correlation (r2 > 0.3) between certain tlrs, which indicated that tlrs were expressed cooperatively to provide a robust innate immune response within bovine testes. we concluded that bovine testicular tissue expressed tlrs at the mrna level and expression of some tlrs was upregulated by a single dose of itm in bulls with testicular degeneration. keywords: testis, innate immunity, tlr, degeneration, bovine 435 clinical theriogenology • volume 11, number 3 • september 2019 436clinical theriogenology • volume 11, number 3 • september 2019 omniblank: body pregnancy twin reduction in the mare by colpotomy body pregnancy twin reduction in the mare by colpotomy sabrina hall, jessica klabnik-bradford, tulio prado department of veterinary clinical sciences, college of veterinary medicine university of tennessee, knoxville, tn previously established techniques for twin reduction are not well suited for pregnancies in the uterine body > 60 days of pregnancy. most techniques work optimally in conditions where the fetuses are located unilaterally or bilaterally in the base of the horn. this case presents a novel methodology of twin reduction, as colpotomy is most commonly performed for ovariectomy and not for twin reduction. a 15 year old american saddle horse mare was presented with twins past 60 days of pregnancy. ultrasonographic examination revealed the presence of one fetus in the uterine body and the other in the right uterine horn. previously established techniques for twin reduction could not be used due to the location of the fetus (uterine body). ultimately, cranio cervical dislocation (ccd) via colpotomy was performed on the twin located in the body of the uterus. ultrasonography indicated no heartbeat from that twin, with a strong and healthy heartbeat in the remaining twin. reduction of the fetus located in the body of the uterus was attempted earlier while it was a vesicle by pinching, but was unsuccessful. although ccd can be performed transrectally or intra abdominally, neither approach could be used for a body pregnancy.1 a colpotomy technique, used for ovariectomy, enabled ccd of a fetus. keywords: mare, colpotomy, cranio-cervical dislocation, twin, body pregnancy reference 1. wolfsdorf ke, rodgerson d, holder r, schoergendorf a: success rate of post-fixation twin reduction using cranio-cervical dislocation. proc annu conv am assoc equine pract 2009;55:257-261. 485 clinical theriogenology • volume 11, number 3 • september 2019 486clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2014: genetic improvement of feed efficiency genetic improvement of feed efficiency bob weaber department of animal sciences and industry, kansas state university, manhattan, ks improvement of the economic position of the farm or ranch is an ongoing process for many commercial cow-calf producers. profitability may be enhanced by increasing the volume of production (i.e. the pounds of calves you market) and/or the value of products you sell (improving quality). the reduction of production costs, and thus breakeven prices, can also improve profitability. for commercial beef producers, the implementation of technologies and breeding systems that increase the quality and volume of production and/or reduce input costs is essential to maintain or improve the competitive position of the operation. profitability is influenced by these factors concurrently. efficiency is the proportion of outputs to inputs and is frequently used by beef producers. there are many different ‘efficiencies’ that affect beef production, especially at the cow-calf level. some of these efficiencies are observed at the individual animal level and some observed at the system or herd level. the various efficiencies can be categorized into with measures of biological or economic efficiency. improvement in individual animal efficiency, especially during the post-weaning growing or finishing phases, may or may-not improve efficiency at the herd or system level, and may have undesirable correlated response in traits of cows. so, why is improvement in feed efficiency important and why does the beef industry focus on it? during the growing and finishing phase of production, a 1% improvement in feed efficiency has the same economic impact as a 3% increase in rate of gain. the traits that beef producers routinely record are outputs which determine the value of product sold and not the inputs defining the cost of beef production. the inability to routinely measure feed intake and feed efficiency on large numbers of cattle has precluded the efficient application of selection despite moderate heritabilities (h2 = 0.08-0.46). feed accounts for approximately 65% of total beef production costs and 60% of the total cost of calf and yearling finishing systems. the cow-calf segment consumes about 70% of the calories; 30% are used by growing and finishing systems. of the calories consumed in the cow-calf segment, more than half are used for maintenance. table 1 shows the potential cost savings to the us beef cattle industry that could occur with selection for feed intake, feed efficiency, growth, and carcass traits. calves and yearlings selected for residual feed intake (rfi) have the same average daily gain (adg) but eat less feed thus saving feedlot operators money. assuming 27 million cattle are fed per year and that 34% of cattle in the feedlot are calves and 66% are yearlings, the beef industry could save over a billion dollars annually by reducing daily feed intake by just 2 lb. per animal. table 1. estimated cost savings to the us beef cattle industry from selection for a 2 lb. reduction in residual feed intake. in wt. out wt. lb. gain adg days on feed rfi reduced feed intake feed cost savings % of fed mix feed cost savings calf feds 600 1,250 650 3.5 186 0.0 0 600 1,250 650 3.5 186 -2.0 -371 $ (54.72) 0.34 $ (502,620,656) yearling feds 775 1,300 525 4.0 131 0.0 0 775 1,300 525 4.0 131 -2.0 -263 $ (38.67) 0.66 $ (689,539,820) total savings: $(1,192,160,476) annual fed slaughter: 27 million head; delivered feed cost: $294.62 as fed weaber, 2011 543 clinical theriogenology • volume 6, number 4 • december 2014 different measures of efficiency there are a variety of measures of efficiency discussed and utilized in beef production. some may or may-not be important to cow-calf producers. for improvements in ‘efficiency’ to positively impact profitability of a cow-calf producer, the efficiency improvement must be realized prior to the marketing endpoint of progeny. while that may seem rather obvious, members of a production sector in the beef industry often get caught up in selection for outcomes for which they have no or limited opportunity to capture the value of the genetic gain. often that selection pressure is at the cost of selection for traits that are economically relevant to the enterprise’s market endpoint. in the following sections a variety of ‘efficiency’ measures are discussed including their applicability and limitations for improvement in efficiency of the cow herd. these measures or their component traits have been shown to be heritable, so selection for improvement is possible but anticipated to be slow, requiring a decade or more to move the population a meaningful distance. a number of the measures, especially measures of biological or economic efficiency are also favorably impacted, typically, by the improvements in lowly heritable traits like longevity and fertility due to heterosis generated in structured crossbreeding systems. system efficiency improvements due to crossbreeding can be realized in three to five years depending on replacement rate in the herd. feed efficiency or feed conversion ratio many cow-calf producers and, certainly cattle feeders, are familiar with the term feed efficiency (fe) or its reciprocal, feed conversion ratio (fcr). both of these measures are indicative of differences in the efficiency of feed utilization and are most commonly associated with animals during the growing or finishing phases. they represent a gross efficiency measure of the conversion of feed to gain. both measures are suitable for managerial use during feeding but are poor selection tools. their utility is limited in selection due to two issues. first, the measures are ratios of inputs and outputs, so improvement in the ratio can be achieved by changing the numerator, the denominator or both. therefore breeders don’t have control over which parameter in the ratio changes due to selection. in practice the parameter improved in selection tends to be the one with the largest genetic variance. selection tools like an index that consider each input and output separately are more effective. second, fcr or fe is strongly related to adg and composition of gain. leaner biological types and larger, faster growing animals tend to have better fe and fcr. selection based on fe or fcr results in larger, later maturing and leaner cows. this type of cow tends to have higher maintenance energy requirements. residual feed intake recently, rfi has been reintroduced as an efficiency measure for beef production. residual feed intake was first proposed by koch et al in 1963, so rfi is not a new idea. it is a residual computed by deviating actual average daily feed intake (afi) from the predicted daily dry-matter intake. predicted daily dry-matter intake is computed from a multiple regression model by regressing afi on adg and body weight (bw) scaled to the ¾ power (est. of metabolic weight). by regression, rfi is independent (i.e. zero correlation) from differences in adg and bw. recall the problems with fcr and fe centered around their undesirable association with other growth parameters. when rfi is computed on the phenotypic scale independence is assured for predictor variables. however, this does not assure genetic independence. in fact research shows underlying genetic correlations between rfi with fi, adg and bw as well as measures of composition. computing rfi on the genetic scale as an index of epds assures a selection tool with fewer antagonisms. that said, rfi is not a perfect tool. the data used to compute it are quite expensive to gather as it requires individual feed intake monitoring systems. additionally, rfi can and does identify efficient animals that also have slow growth and low feed intake making these candidates undesirable for selection and use in the commercial beef industry. so, rfi must be used with other measures like adg to assure that industry acceptable animals are selected. some research suggests that selection for rfi produces slightly larger and leaner cows over time and cows that have older ages at first calving. in general, selection for favorable (negative) rfi results in animals with equivalent performance, but achieves that output with less feed consumed. 544clinical theriogenology • volume 6, number 4 • december 2014 residual average daily gain a concept closely related to rfi is residual average daily gain (radg) which was proposed at the same time as rfi as a potential tool for selection for improved feed efficiency. it is the residual from regression of adg on afi and bw raise to the ¾ power (metabolic body weight). selection for radg seeks to find animals that consumed equivalent afi but resulted in better performance. radg, like rfi, is a transformation of the data and can be computed on either the phenotypic or genetic scales. differences in adg are controlled for differences in afi and bw. like rfi it is typically computed on growing animals and is indicative of difference in efficiency of feed utilization for growth. it may have limited utility for prediction of differences in maintenance efficiency of cows. residual average daily gain should not be used alone in selection for feed efficiency. data reveal that some animals with favorable radg have sub-par feed intake and consequently undesirable adg. feed intake and growth, not surprisingly, have a strong positive genetic association. input drives output. one additional challenge with radg, and rfi for that matter, is that these measures are computed on growing animals. in the case of cows, growth is not desired endpoint, reproduction, maintenance and lactation are the principle energy sinks. average daily feed intake also known as afi. average daily feed intake is a gross measure of nutrient input. while it cannot be used alone as a predictor of feed efficiency, it provides a useful data input for computation of selection index. feed intake represents an economically relevant measure of cost that can be associated with a variety of output or endpoint measures. average daily feed intake could be measured on animals during different phases of production and used to capture input:output (efficiency) information. a selection index for afi or an afi expected progeny difference (epd) can be reliably produced analyzing performance records for a variety of growth traits. an afi epd produced without actual feed records but based on genetic associations between growth and intake can account for nearly 75% of the variation in observed feed intake. weaning weight per cow exposed this is a gross measure of biological efficiency and relates the importance of reproductive success, longevity, calf survival and other factors on system output. improvements in maintenance efficiency of cows (or a reduction in maintenance or production requirement under stressful environments) would likely improve this efficiency metric. clearly, both production potential (growth and lactation) and heterosis from crossbreeding can substantially affect this measure. weaning weight per cow exposed per unit of energy consumed another measure of biological efficiency that includes accounting of nutrients consumed for both production and maintenance of cow and calf. this metric should point to best combination of genetic merit for economically relevant traits to a weaning market endpoint including calving ease, growth, lactation, and mature cow weight among other. researchers (ferrell and jenkins, 1994) have conducted a number of studies to evaluate different sire breeds for biological efficiency under low, average and high nutrient availability. in this experiment, they found little difference in efficiency across biological type (growth, lactation and leanness) at moderate nutrient availability. under low nutrient availability, smaller breeds with lower lactation potential were more efficient. at high nutrient availability, large, high milk breeds were more efficient. the primary difference was the impact of nutrient availability on fertility for a given biological type. value ($) output per $100 of total input this is a measure of economic efficiency and the results are highly dependent upon selection of appropriate endpoint. nielsen and colleagues (1993) demonstrated the differences in economic efficiency for three different levels of milk production from cows of three different breed crosses but of similar body 545 clinical theriogenology • volume 6, number 4 • december 2014 size. the weaning endpoint favored the low and medium lactation groups over the high milk group. if progeny were sold as finished calves the group ranks were the same, but the range between them widened. kress and others (1988) demonstrated the importance of longevity to both biological and economic efficiency. selecting to improve efficiency role of growth and lactation potential on me efficiency and me requirements mature cow weight and lactation potential play a key role in determining annual nutrient requirements for cows. increasing average cow mature weights from 1,000 lb to 1,400 lb, approximately the change we have observed over the last 30 years, increases nutrient requirements by 27%. increasing lactation potential from 10 lb. to 30 lb. per day at peak results in a 16% increase in nutrient requirements. these increases in potential have the opportunity to be associated with increases in output, but they also have the potential to undermine a cow’s fitness in a given production environment. increases in mature weight and lactation drive up maintenance requirements. optimization of growth and lactation genetics, and ultimately profitability, requires understanding the marginal revenues and marginal costs associated with these attributes. the associated change in maintenance requirement due to mature weight change is distinctly different from the change increased weight has on maintenance energy or metabolic efficiency. metabolic rate does not scale linearly with mass or weight. instead, it increases exponentially by the ¾ power. thus, warm blooded animals with larger mass are more metabolically efficient than ones of small mass. the principal reason for these phenomena is relationship between surface area of the animal and it’s mass. large animals have less surface area per unit mass enabling them to conserve heat more effectively. so, large cows are more efficient users of maintenance energy but have higher requirements. the key then is finding cows with appropriate levels of mature weight and lactation potential (or biological type) for your production environment. note that managerial (i.e., reducing supplemental feedstuffs) or environmental (i.e. drought) changes that alter nutrient availability may substantially change the fitness of your existing cows. care should be taken in sire selection for production of replacement females such that their growth, mature weight and lactation potential are appropriate. current tools at present several selection tools are available for selection to improve feed efficiency in beef cattle. these include the radg epd published by the american angus association. the epd leverages a variety of molecular and phenotypic data to produce a genetic prediction describing differences in expected post-weaning gain given some level of intake. more positive values are indicative of higher levels of efficiency. a number of other breeds, including simmental, gelbvieh, hereford and limousin have active breeding programs and data collection efforts to gather individual feed intake records with goals of producing genetic predictors for efficiency of gain. a large usda-funded integrated research and extension project is focused on the genetic improvement of feed efficiency in beef cattle and will leverage a variety of methods to achieve this goal. the american angus association and the red angus association of america both produce selection indexes that describe differences in maintenance energy requirements. these tools rely on the genetic associations between maintenance energy required with mature size and milk genetic predictors. as before, animals with higher potentials for these traits generally have higher maintenance energy requirements. unfortunately, little work has been done to address the additive genetic improvement of maintenance efficiency in beef cattle. researchers know differences exist across breeds and individuals but accumulation of a substantial number of records has been elusive. clearly this parameter would benefit from the development of genomic selection tools to enable genetic improvement. 546clinical theriogenology • volume 6, number 4 • december 2014 value of heterosis in improving biological efficiency one of the only, yet very effective ways, to improve biological efficiency of beef cattle production systems is through the use of planned crossbreeding systems to leverage heterosis, especially maternal heterosis, and breed complementarity. heterosis refers to the superiority of the crossbred animal relative to the average of its straight bred parents. heterosis results from the increase in the heterozygosity of a crossbred animal’s genetic makeup. heterozygosity refers to a state where an animal has two different forms of a gene. it is believed that heterosis is the result of gene dominance and the recovery from accumulated inbreeding depression of pure breeds. heterosis is, therefore, dependent on an animal having two different copies of a gene. the level of heterozygosity an animal has depends on the random inheritance of copies of genes from its parents. in general, animals which are crosses of unrelated breeds, such as angus and brahman, exhibit higher levels of heterosis, due to more heterozygosity, than do crosses of more genetically similar breeds such as a cross of angus and hereford. heterosis generates the largest improvement in lowly heritable traits. moderate improvements due to heterosis are seen in moderately heritable traits. little or no heterosis is observed in highly heritable traits. heritability is the proportion of the observable variation in a trait between animals that is due to the genetics that are passed between generations and the variation observed in the animal’s phenotypes, which are the result of genetic and environmental effects. traits such as reproduction and longevity have low heritability. these traits respond very slowly to selection since a large portion of the variation observed in them is due to environmental factors and a small percentage is due to genetic differences. heterosis generated through crossbreeding can significantly improve an animal’s performance for lowly heritable traits. crossbreeding has been shown to be an efficient method to improve reproductive efficiency and productivity in beef cattle. improvements in cow-calf production due to heterosis are attributable to having both a crossbred cow and a crossbred calf. the two tables below detail the individual (crossbred calf) and maternal (crossbred cow) heterosis observed for various important production traits. these heterosis estimates are adapted from a report by cundiff and gregory, 1999, and summarize crossbreeding experiments conducted in the south-eastern and mid-west areas of the us. the heterosis generated in calves that are the progeny of straight bred parents of different breeds or crossbred parents is called individual heterosis. while this type of heterosis has import effects on economically important traits, it only accounts for approximately one-third of the total economic benefits of having crossbred cows and calves. thus if you only have crossbred calves (i.e. straight bred cows) you’re missing the biggest share of economic benefit from crossbreeding. individual heterosis improves performance in a number of traits measured on calves including survival and growth (table 2.). for example, individual heterosis can improve weaning weights by nearly 4% which on a 500 lb. weaned calf is 20 lbs. table 2. effects of individual heterosis on performance of crossbred calves trait units % heterosis calving rate, % 3.2 4.4 survival to weaning, % 1.4 1.9 birth weight, lb. 1.7 2.4 weaning weight, lb. 16.3 3.9 yearling weight, lb. 29.1 3.8 average daily gain, lb./d 0.08 2.6 why is it so important to have crossbred cows? the production of crossbred calves yields advantages in both heterosis and the blending of desirable traits from two or more breeds. however, the largest economic benefit of crossbreeding to commercial producers comes from having crossbred cows. maternal heterosis improves both the environment a cow provides for her calf as well as improves the longevity and durability of the cow. the 547 clinical theriogenology • volume 6, number 4 • december 2014 improvement of the maternal environment a cow provides for her calf is manifested in the improvements in calf survivability to weaning and increased weaning weight. crossbred cows exhibit improvements in calving rate of nearly 4% and an increase in longevity of more than one year due to heterotic effects (table 3). heterosis results in increases in lifetime productivity of approximately one calf and 600 pounds of calf weaning weight over the lifetime of the cow (table 2). crossbreeding can have positive effects on a ranch’s bottom line by not only increasing the quality and gross pay weight of calves produced but also by increasing the durability and productivity of the cow factory. crossbred cows may be the only free lunch in the world. the effects of maternal heterosis on the economic measures of cow-calf production have been shown to be very positive. the added value of maternal heterosis ranges from approximately $50/cow/year to nearly $100/cow/year depending on the amount of maternal heterosis retained in the cowherd (ritchie, 1998). maternal heterosis accounted for an increase in net profit per cow of nearly $75/cow/year (davis et al., 1994). their results suggested that the benefits of maternal heterosis on profit were primarily the reduced cost per cow exposed. crossbred cows had higher reproductive rates, longer productive lives, and required fewer replacements than straight bred cows in their study. all of these factors contribute to reduced cost per cow exposed. further, they found increased outputs, including growth and milk yield, were offset by increased costs. table 3. effects of maternal heterosis on calf traits affected by maternal environment, cow productivity and longevity. trait units % heterosis calving rate, % 3.5 3.7 survival to weaning, % 0.8 1.5 birth weight, lb. 1.6 1.8 weaning weight, lb. 18.0 3.9 longevity, years 1.36 16.2 lifetime productivity number of calves 0.97 17.0 cumulative weaning wt., lb. 600 25.3 how can i harness the power of breed complementarity? breed complementarity is the effect of combining breeds that have different strengths. when considering crossbreeding from the standpoint of producing replacement females, one could select breeds that have complementary maternal traits such that females are most ideally matched to their production environment. matings to produce calves for market should focus on complementing the traits of the cows and fine tuning calf performance (growth and carcass traits) to the market place. there is an abundance of research that describes the core competencies (biological type) of many of today’s commonly used beef breeds. traits are typically combined into groupings such as maternal/reproduction, growth and carcass. when selecting animals for a crossbreeding system, their breed should be your first consideration. what breeds you select for inclusion in your mating program will be dependent on a number of factors including the current breed composition of your cow herd, your forage and production environment, your replacement female development system, and your calf marketing endpoint. all of these factors help determine the relative importance of traits for each production phase. what are the keys to successful crossbreeding programs? many of the challenges that have been associated with crossbreeding systems in the past are the result of undisciplined implementation of the system. with that in mind, one should be cautious to select a mating system that matches the amount of labor and expertise available to appropriately implement the system. crossbreeding systems range in complexity from very simple programs such as the use of hybrid 548clinical theriogenology • volume 6, number 4 • december 2014 genetics, which are as easy to use as straight breeding, to elaborate rotational crossbreeding systems with four or more breed inputs. the biggest keys to success are the thoughtful construction of a plan and the sticking to it! be sure to set attainable goals. discipline is essential. modify cows or modify environment? historically, supplemental feedstuffs have been relatively inexpensive compared to current costs. in fact, much of the early motivation to develop farmer owned confinement feeding systems, common in the midwest, was to add value to coarse grains by feeding it to cattle. present costs for supplemental feedstuffs, fertilizer and fuel inputs have many producers reconsidering their production model and moving towards systems with reduced inputs. indeed producers are evaluating modification of the cow rather than modification of the production environment. it seems that in the short run, the most effective way to improve efficiency at the production or herd level is through selection for cows of the appropriate biological type that fit their production environment. further, these cows should likely be crossbred cows to leverage the benefits of maternal heterosis and breed complementarity. in the intermediate to long run, seedstock and ultimately, commercial producers, should select for animals, via selection index, that optimize efficiency to the enterprise’s market endpoints. such a two pronged approach leverages efficiency gains due to additive and non-additive genetics that affect animal efficiency of feed utilization as well as biological efficiency, respectively. 549 clinical theriogenology • volume 6, number 4 • december 2014 550clinical theriogenology • volume 6, number 4 • december 2014 omniblank: 2010: feral cats: a review of concerns and control measures feral cats: a review of concerns and control measures margaret v. root kustritz college of veterinary medicine, university of minnesota, st paul, mn abstract feral cat populations exist worldwide. concerns include predation and spread of disease, both within and between species including humans. control measures described in the literature include depopulation, trap-adoption, trap-neuter-return, and medical reproductive control. keywords: feline, feral, trap-neuter-return, megestrol acetate introduction cats (felis catus) live outdoors on every continent except antarctica and on islands of every size, either as natural or introduced species.1 because cats are loosely domesticated, with freedom to roam considered acceptable by cat owners in many countries, it is difficult to classify cat populations as owned, stray, or feral. this classification becomes important when considering how best to control the various populations, as one must consider the welfare of the individual cats, public attitudes, and legal implications when designing control programs.2 a suggested scheme is the following: companion or owned cats are those who live the majority of the time with humans and are dependent on them for basic needs, stray cats are lost or abandoned companion cats that may live outside but do so near human habitation and rely on humans to some extent, and feral cats are those that live outside and are selfsustaining. feral cats may have been companion cats once and may choose to live near human habitation but do not rely on humans for shelter, food, or water.2 another definition for a feral cat would be one that cannot be handled by humans and would not be suitable for adoption into a home as a pet; this definition is complicated by lack of consistent guidelines within or between facilities regarding methods used to identify whether or not a given cat is adoptable.3,4 a colony of feral cats is defined as a group of three or more sexually intact cats living and feeding in close proximity.3 this paper is a review of concerns and reported control measures for feral cats living in colonies. concerns many papers cite the concerns associated with maintenance of large colonies of free-roaming cats. some suggest benefits as well. these include the social aspects of providing support for these cats and potential building of networks between caregivers.5,6 multiple stakeholders must be taken into consideration when evaluating control of feral cat colonies including humans who consider cats a nuisance, who are at risk of their own health from diseases carried or transmitted by cats, or who are concerned about welfare of wildlife or welfare of the cats, and the cats and other animal species themselves.7 society at large also has a stake with growing societal opposition to euthanasia and differing understanding of the role of humans in exerting dominion over animals.6,8 concerns about feral cat colonies include loss of wildlife from predation by feral cats, spread of disease from cats to other animals, and potential spread of disease from cats to humans. these concerns must be well understood before sound recommendations can be made regarding control measures. predation it is estimated that there as many feral cats as there are owned cats in the united states, with an estimated population of 73 million in the year 2000.6 cats hunt and kill small mammals, reptiles, amphibians, and birds, both adults and nestlings, with one estimate suggesting that over a billion small mammals and millions of birds are killed by cats each year.9,10 in wisconsin alone, it is estimated that one to two million free-roaming cats kill about 40 million birds annually.9 the large number of animals killed is due in part to the density of cats tolerated in a feral colony. one study demonstrated 35 feral cats in the same territory that would have supported only one or two pairs of larger natural predators, such as fox or coyotes.11 there is collateral damage from cat predation of small animals to these larger predators, 437 as cats decrease availability of natural food sources.9 to be complete, one must also recognize that not all decline in prey availability is due to predation from cats and that cats may help support prey populations in some instances by controlling intermediary predators, such as rats.12,13 intraspecific disease transfer there are no reports in the literature of feral cat colonies specifically spreading disease to owned cats. however, because feral cats do not receive regular veterinary care, including vaccinations, there is a greater risk of their contracting contagious diseases such as feline leukemia and rabies, and transmitting them if they interact with owned cats. there are documented reports of feral cats spreading disease to wild cat populations, including feline leukemia virus to mountain lions and panleukopenia to florida panthers.14,15 interspecific (zoonotic) disease transfer public health concerns center around spread from feral cats to humans of viral diseases, including rabies;5,16-19 protozoal infections, such as toxoplasma gondii;16,20-22 fungal infections, including dermatophytosis;5 and parasites.23-25 some might argue that management of feral cat colonies increases public health risk by exposing caregivers to disease and to direct injury; others suggest that risk is decreased by vaccination of cats returned and removal of diseased cats from managed colonies.6,26 rabies is a disease of great significance in all species, including humans. it is fatal to humans unless they are treated immediately after exposure. rabies is maintained in wildlife and the variant of rabies contracted by domestic animals varies by region, with raccoon, skunk, fox, and bat variants those most commonly seen in the united states. in the most recently published survey of rabies surveillance in the united states, rabid animals were identified in 49 states and on puerto rico, with 93% of cases occurring in wildlife and 7% in domestic animals; cats represented 4.3% of the total cases seen, a number nearly four times that reported for dogs.18 in one survey of feral cats in italy, two of eight rabid cats were from known feral cat colonies that were managed by human caregivers.5 concern exists even if feral cats are vaccinated; in one survey of 840 rabid cats in the united states, 22 (2.6%) had been previously vaccinated at some point in their lives, with three of those animals described as being current on their vaccinations.17 toxoplasmosis is caused by a protozoan organism carried asymptomatically by cats. in one survey of six feral cats trapped in mexico city, all were seropositive for toxoplasma gondii.16 in larger studies, prevalence of seropositive cats varied from 73.9 to 84.7%.21,22 because of constant exposure to toxoplasma through ingestion of prey species, toxoplasma gondii always is present in feral cat colonies, although prevalence may vary over time and may be higher in adults than in juveniles.20,22 intestinal parasites most commonly identified in feral cat populations are roundworms (toxocara cati), hookworms (ancylostoma tubaeforme) and several species of tapeworm.23-25 only a small percentage of cats were identified to be carrying those parasites most readily transmitted to people, including toxocari cati (0.8% of 658 cats) and ancylostoma tubaeforme (14.7% of 658 cats). reported percentages were higher in cats living on islands than on the mainland.24 passage of protozoa and parasites through feces is not the only public health concern. one investigator estimated that in a single community, the amount of feces deposited outdoors by the 2046 feral cats living there every year was about 29.5 tons.27 a study tracking sources of e. coli in storm sewers feeding rivers and streams demonstrated that the highest percentage from any one source came from cats.28 it may be that it is not exposure to the cats but rather exposure to their feces that is the greatest public health risk.26 control measures eradication eradication of feral cat populations has been demonstrated successful only on small islands.29 methods for eradication include trapping; hunting, with or without dogs; direct poisoning; indirect 438 poisoning by placement of poisoned rats or other food sources; and introduction of fatal viral disease, such as panleukopenia.29 any eradication method must take into account safety of the environment, of humans and of non-target species, and also consider expense, sustainability of population control, and public support of the eradication and of the method chosen.30 complete depopulation is not considered a viable option in most situations. trap-placement an alternative to eradication is depopulation by trapping and placement of all trapped cats into homes or sanctuaries. there is recognition that not all feral cats are adoptable; that is, in fact, one definition of feral.4 at present, millions of cats are euthanized in shelters in the united states each year because there are not enough homes for them all. addition of feral cats to this population would lead to euthanasia of even more cats, many of whom might have been adopted in the current system.30 creation of cat sanctuaries has not been demonstrated to be economically viable long-term and may actually be associated with increased abandonment of companion cats in the vicinity of the sanctuary.30 trap-neuter-return trap-neuter-return (tnr) programs are those best described in the veterinary literature. the reader should be aware that tnr may refer to any number of steps in the system including trapping; health evaluation with or without testing for feline leukemia and feline immunodeficiency virus and fecal flotation examination; vaccination; gonadectomy; decisions regarding adoptability and euthanasia, based on behavior evaluation and health status; and eventual return to the colony. in some countries, management of feral cat colonies is required because eradication by means of euthanasia is illegal.31 in other countries, the goal of management is to create a stable neutered population with the eventual goal of decreasing colony size, ideally to zero. some studies find that tnr programs are associated with a decrease in colony size, and a decrease in complaints about the feral cats as a public nuisance compared to eradication programs.32-36 how humane are tnr programs? it has been demonstrated that feral cats respond to vaccination as intended, becoming seropositive and maintaining detectable titers.37 feral cats that have been neutered undergo a decrease in metabolic rate and subsequent increase in body weight as do all neutered cats, but do not become obese, with a mean body condition score one year after neuter and return to the colony of 5 ± 1 on a 9–point scale, where 1 is emaciated and 9 is grossly obese.38-40 surgery does not appear to be a greater risk for these cats than for owned cats; one study revealed a surgical mortality rate of 0.4% in a large tnr program.41 kitten mortality rate in outdoor colonies is reported to be greater than 50%, suggesting that tnr, and subsequent decrease in kitten numbers, is humane because it prevents birth of kittens that would have died in the colony environment.42 the primary concerns about tnr programs are their cost and lack of sustainability. in order to maintain a stable population, models suggest that trapping and neutering must be performed on a large scale, sterilizing over 51% of all female cats in the colony that are over one year of age annually such that at least 71% of the queens in the colony are sterilized at any given time.7,43 in a survey of large managed colonies in italy, despite large scale trapping and neutering, about 21% of the population consisted of new cats every year.31 as long as humans continue to abandon cats as strays that can repopulate hospitable locations, the problem of feral cats cannot be completely controlled and it is wisest to assume that complete eradication of colonies will not occur and to understand that due to environmental variations, colony size may even increase with tnr management.30,44,45 trapping is labor intensive; one study documented that 8.9 ± 3.9 nights of trapping per cat were required to capture at least 90% of the cats in one colony.46 there also is the consideration of who bears the cost for the surgeries, which must be performed by veterinarians. it has been suggested that veterinary practitioners should subsidize these programs but that it is inappropriate to suggest that any single aspect of any profession bear the burden of a societal problem.47 439 medical control of reproduction megestrol acetate megestrol acetate (ma) is a synthetic progestogen approved for estrus suppression in bitches. no product containing this chemical ever has been approved by the food and drug administration (fda) for use in cats. extralabel use is described by the fda in the following way: extra-label use of drugs in treating animals is allowable only by licensed veterinarians within the context of a valid veterinarianclient-patient relationship, and does not include drug use in treating animals by the layman (except under the supervision of a licensed veterinarian).48 unapproved products containing very low doses of ma for oral distribution in feral cat colonies are commercially available. megestrol acetate has no anabolic or estrogenic properties and is reported to have no masculinizing effect if ingested by queens early in pregnancy.49,50 all studies reporting use of ma for reproduction control in feral cat colonies describe weekly oral doses of 2.5–5.0 mg. reported success rates in preventing conception vary from 57.1 to 100.0%; in at least one of these studies, cats were fed the drug by hand to ensure consistent dosing.51-53 male cats given 5 mg ma weekly showed a decline in sex-related behaviors; this effect was not seen at a dose of 2.5 mg weekly.52 in all reports, inconsistent dosing was associated with quick return to normal fertility in queens.51,52 side-effects of ma can be severe. reproductive tract abnormalities in treated females include cystic endometrial hyperplasia and pyometra.51,54 mammary hypertrophy has been reported as a sideeffect in treated male and female cats.53,55,56 mammary neoplasia (carcinoma or adenoma) also has been reported.53,56 endocrine abnormalities associated with treatment include adrenocortical suppression and diabetes mellitus.53,57-63 less severe side-effects include increased appetite, increased thirst and urination, lethargy, and aggression; the latter may be particularly concerning to caretakers of feral cat colonies.53,64 cabergoline cabergoline is a prolactin inhibitor that has been demonstrated to effect pregnancy termination in cats after mid-gestation.65 there is one report of successful termination of pregnancy and control of reproduction in a feral cat colony by hand-feeding of cabergoline to visibly pregnant queens.66 there are no reports documenting possible side-effects of cabergoline available in food and accidentally ingested by prepuberal cats, adult male cats, or queens early in pregnancy. conclusion at present, the preferred method for management of feral cat colonies is tnr. these programs require consistent funding and commitment and cannot be expected to lead to eradication as long as the environment is hospitable to cats and cats are available for immigration into the area. to address public health concerns, it must be remembered that exposure to feral cat feces may well be a greater risk than exposure to the cats themselves. medical therapy would be preferred to tnr programs if it could be shown to be effective and safe, and less labor-intensive. commercially available products containing ma at very low doses have not been shown to be either effective or safe and cannot be recommended. in the absence of a medical alternative, it is recommended that society invest in education of the public regarding responsible cat ownership, creation of appropriate public policy regarding maintenance of feral cat colonies and funding for tnr programs with the goal of decreasing feral cat populations, and support of research to identify safe and effective contraceptives and sterilants for cats.6 references 1. owen p: felis silvestris catus, digital morphology. http://digimorph.org/specimens/felis_sylvestris_catus/, accessed august 24, 2010. 2. farnworth mj, dye ng, keown n: the legal status of cats in new zealand: a perspective on the welfare of companion, stray, and feral domestic cats (felis catus). j appl anim welf sci 2010;13:180–188. 3. slater mr: the welfare of feral cats. in: rochlitz i, editor. the welfare of cats. dordrecht: springer, 2005. p.141–176. 440 4. slater mr, miller ka, weiss e, et al: a survey of the methods used in shelter and rescue programs to identify feral and frightened pet cats. j feline med surg 2010;12:592–600. 5. mutinelli f: rabies and feral cat colonies in italy. vet rec 2010;166:537–538. 6. richards jr: the 2004 american association of feline practitioners position statement on free-roaming, abandoned and feral cats. j feline med surg 2004;6:vii–ix. 7. robertson sa: a review of feral cat control. j feline med surg 2008;10:366–375. 8. hyland jr: the slaughter of terrified beasts: a biblical basis for the humane treatment of animals. sarasota: viatoris ministries; 1998. 9. edwards j: minnesota’s killer kitties should be kept indoors. minnesota department of natural resources, st paul, minnesota. http://news.dnr.state.mn.us/index.php/wp-admin/enforcement/ranges?paged=84, accessed august 17, 2010. 10. coleman js, temple sa, craven sr: facts on cats and wildlife: a conservation dilemma. usda cooperative extension, university of wisconsin, madison, wisconsin. http://wildlife.wisc.edu/extension/catfly3.htm, accessed august 25, 2010. 11. crooks kr, soule me: mesopredator release and avifaunal extinctions in a fragmented system. nature 1999;400:563– 566. 12. clark brittingham m, temple sa: have cowbirds caused forest songbirds to decline? bioscience 1983;33:31–35. 13. fan m, kuang y, feng z: cats protecting birds revisited. bull math biol 2005;67:1081–1106. 14. jessup da, pettan kc, lowenstine lj, et al: feline leukemia virus infection and renal spirotrichetosis in free-ranging cougar (felis concolor). j zoo wildl med 1993;24:73–79. 15. roelke me, forester dj, jacobson er, et al: seroprevalence of infectious disease agents in free-ranging florida panthers (felis concolor coryi). j wildl dis 1993;29:36–49. 16. suzan g, ceballos g: the role of feral mammals on wildlife infectious disease prevalence in two nature reserves within mexico city limits. j zoo wildl med 2005;36:479–484. 17. murray ko, holmes kc, hanlon ca: rabies in vaccinated dogs and cats in the united states, 1997-2001. j am vet med assoc 2009;235:691–695. 18. blanton jd, robertson k, palmer d, et al: rabies surveillance in the united states during 2008. j am vet med assoc 2009;235:677–689. 19. epstein jh, rahman sa, zambriski ja, et al: feral cats and risk for nipah virus transmission. emerg infect dis 2006;12:1178–1179. 20. mendes-de-almeida f, labarthe n, guerrero j, et al: follow-up of the health conditions of an urban colony of freeroaming cats (felis catus linnaeus, 1758) in the city of rio de 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thoughts on feral cat control [letter]. j am vet med assoc 2010;237:26–27. 27. dabritz ha, atwill er, gardner ia, et al: outdoor fecal deposition by free-roaming cats and attitudes of cat owners and non-owners toward stray pets, wildlife, and water pollution. j am vet med assoc 2006;229:74–81. 28. ram jl, thompson b, turner c, et al: identification of pets and raccoons as sources of bacterial contamination of urban storm sewers using a sequence-based bacterial source tracking method. water res 2007;41:3605–3614. 29. nogales m, martin a, tershy b, et al: a review of feral cat eradication on islands. conserv biol 2004;18:310–319. 30. levy jk, crawford pc: humane strategies for controlling feral cat populations. j am vet med assoc 2004;225:1354– 1360. 31. natoli e, maragliano l, cariola g, et al: management of feral domestic cats in the urban environment of rome (italy). prev vet med 2006;77:180–185. 32. hughes kl, slater mr: implementation of a feral cat management program on a university campus. j appl anim welf sci 2002;5:15–28. 33. hughes kl, slater mr, haller l: the effects of implementing a feral cat spay/neuter program in a florida county animal control service. j appl anim welf sci 2002;5:285–298. 34. neville pf, remfry j: effect of neutering on two groups of feral cats. vet rec 1984;114:447–450. 35. levy jk, gale dw, gale la: evaluation of the effect of a long-term trap-neuter-return and adoption program on a free-roaming cat population. j am vet med assoc 2003;222:42–46. 36. stoskopf mk, nutter fb: analyzing approaches to feral cat management–one size does not fit all. j am vet med assoc 2004;225:1361–1364. 441 37. fischer sm, quest cm, dubovi ej, et al: response of feral cats to vaccination at the time of neutering. j am vet med assoc 2007;230:52–58. 38. scott kc, levy jk, gorman sp, et al: body condition of feral cats and the effect of neutering. j appl anim welf sci 2002;5:203–213. 39. root mv, johnston sd, olson pn: effect of prepuberal and postpuberal gonadectomy on heat production measured by indirect calorimetry in male and female domestic cats. am j vet res 1996;57:371–374. 40. fettman mj, stanton ca, banks ll, et al: effects of neutering on body weight, metabolic rate and glucose tolerance of domestic cats. res vet sci 1997;62:131–136. 41. wallace jl, levy jk: population characteristics of feral cats admitted to seven trap-neuter-return programs in the united states. j feline med surg 2006;8:279–284. 42. nutter fb, levine jf, stoskopf mk: reproductive capacity of free-roaming domestic cats and kitten survival rate. j am vet med assoc 2004;225:1399–402. 43. budke cm, slater mr: utilization of matrix population models to assess a 3-year single treatment nonsurgical contraception program versus surgical sterilization in feral cat populations. j appl anim welf sci 2009;12:277–292. 44. longcore t, rich c, sullivan lm: critical assessment of claims regarding management of feral cats by trap-neuterreturn. conserv biol 2009;23:887–894. 45. foley p, foley je, levy jk, et al: analysis of the impact of trap-neuter-return programs on populations of feral cats. j am vet med assoc 2005;227:1775–1781. 46. nutter fb, stoskopf mk, levine jf: time and financial costs of programs for live trapping feral cats. j am vet med assoc 2004;225:1403–1405. 47. keyes m: additional views on the costs of feral cat control [letter]. j am vet med assoc 2010;237:257. 48. food and drug administration: extra-label use of fda approved drugs in animals. http://www.fda.gov/animalveterinary/resourcesforyou/fdaandtheveterinarian/ucm077390.htm, accessed august 26, 2010. 49. gupta c, bullock lp, bardin cw: further studies on the androgenic, anti-androgenic, and synandrogenic actions of progestins. endocrinology 1978;102:736–744. 50. david a, edwards k: anti-ovulatory and other biological properties of megestrol acetate. j reprod fertil 1963;5:331– 346. 51. remfry j: control of feral cat populations by long-term administration of megestrol acetate. vet rec 1978;103:403– 404. 52. jochle w, jochle m: reproductive and behavioral control in the male and female cat with progestins: long-term field observations in individual animals. theriogenology 1975;3:179–185. 53. romatowski j: use of megestrol acetate in cats. j am vet med assoc 1989;194:700–702. 54. bellenger cr, chen jc: effect of megestrol acetate on the endometrium of the prepubertally ovariectomised kitten. res vet sci 1990;48:112–118. 55. hayden dw, barnes dm, johnson kh: morphologic changes in the mammary gland of megestrol acetate-treated and untreated cats: a retrospective study. vet pathol 1989;26:104–113. 56. macdougal ld: mammary fibroadenomatous hyperplasia in a young cat attributed to treatment with megestrol acetate. can vet j 2003;44:227–229. 57. pukay bp: a hyperglycemia-glucosuria syndrome in cats following megestrol acetate therapy. can vet j 1979;20:117. 58. mccann tm, simpson ke, shaw dj, et al: feline diabetes mellitus in the uk: the prevalence within an insured cat population and a questionnaire-based putative risk factor analysis. j feline med surg 2007;9:289–299. 59. church db, watson adj, emslie dr, et al: effects of proligestone and megestrol on plasma adrenocorticotrophic hormone, insulin and insulin-like growth factor-1 concentration in cats. res vet sci 1994;56:175–178. 60. middleton dj, watson adj: glucose intolerance in cats given short-term therapies of prednisolone and megestrol acetate. am j vet res 1985;46:2623–2625. 61. weikel jh, nelson lw, reno fe: a four-year evaluation of the chronic toxicity of megestrol acetate in dogs. toxicol appl pharmacol 1975;33:414–426. 62. chastain cb, graham cl, nichols ce. adrenocortical suppression in cats given megestrol acetate. am j vet res 1981;42:2029–2035. 63. peterson me: effects of megestrol acetate on glucose tolerance and growth hormone secretion in the cat. res vet sci 1987;42:354–357. 64. henik ra, olson pn, rosychuk ra: progestogen therapy in cats. compend contin educ pract vet 1985;7:132–140. 65. root kustritz mv, johnston sd, olson pn: canine and feline theriogenology. philadelphia: wb saunders co; 2001. p. 450–451. 66. jochle w, jochle m: reproduction in a feral cat population and its control with a prolactin inhibitor, cabergoline. j reprod fertil 1993;suppl 47:419–424. 442 2011: semen handling for insemination in the field: splitting straws, loading the equipment and juggling eight straws at a time semen handling for insemination in the field: splitting straws, loading the equipment and juggling eight straws at a time kim kensell department of rural animal health management, college of veterinary medicine, university of illinois, urbana, il thawing semen in the field for cervid work presents unique challenges due to the time of year that breeding takes place. the months of october to december can range from a comfortable chilly range to whipping winds with very low temperatures. the ideal situation for performing surgical inseminations is an indoor facility with a heat source, cement floors and a semen breeding list that is filled out with the buck to be used and the order that the does will be presented. the reality is each farm is different and nothing is ever the same. the basic equipment for thawing semen is the same as with any species: thaw unit, slide warmer, microscope, slides, cover slips, and scissors or a straw cutting unit. in addition, you will need aspics (sheath with an attached needle) and two to three insemination guns (both from imv, l’aigle, france), 0.25 ml straws, microcentrifuge tubes (1 ml) with snap lids, extra extender for splitting straws, a stylet to empty straws, a device to hand fill the straws (a tb syringe with a tom cat catheter cut down to fit in the end of the 0.25 ml straw works well) and a fine tip marker. it is also helpful to have a thin piece of polystyrene foam that has two holes cut in it to fit the microcentrifuge tubes and allows them to float in the water bath. the slide warmer is set up and slides, cover slips, straws, aspics, and insemination guns are placed on it to help prevent cold shock to the sperm cells. as the does are brought in and prepared, each ear tag is checked against the breeding list. since most owners want to split straws, it is best to have all does being mated to the same buck come in together. this does not always happen due to poor planning by the owner or problems with anesthesia. when the doe is brought over to the insemination area, the ear tag is checked again. i find it helpful to have the breeding list arranged so that each doe is listed under the name of the buck being used for insemination. this will help you know if straws from a particular buck need to be split more than one time. it is also beneficial in cases where straws from a buck are unable to be split the way the owner originally intended and you need to see if a straw from another buck can be split one more way. once the incisions are made in the abdomen of the doe, the straw of semen is placed in the water bath for the recommended time (generally 45-60 seconds). the abdomen is inflated with co2 as the semen straw is thawing. the straw of semen is wiped off and the semen is placed in the warmed microcentrifuge tube in the water bath. a tiny drop of semen is placed on a warmed slide with a cover slip and evaluated on the microscope for movement and concentration. a judgement call based on that drop of semen is made about dividing the straws. the industry has been freezing at 50 million cells per 0.5 ml straw and most owners are using one straw for two does. if the owner wants to divide the straw and inseminate more than two doses, extra extender is added depending on the number of times the straw will be split. i find it easier to draw extra extender into 0.25 ml straws and have them on the slide warmer ready to use. if the owner wishes to divide it between three does, one straw of extender can be added and if four does are to be inseminated, two straws of extender are added. the semen is drawn into the 0.25 ml straw using the tb syringe and tom cat catheter. the lid of the microcentrifuge is snapped closed to prevent water from getting in the sample. the insemination gun and aspic are loaded with the straw. to prevent cooling, a hand can be cupped around the straw. the straw is placed on the pink plunger and slid halfway into the gun. the handle of the gun must be loosened to release pressure against the o-ring. the aspic is placed over the straw and inside the gun and slid down into the handle. once the aspic is seated in the handle, the gun is tightened down so the o-ring holds the aspic in place. the plunger is then used to push the straw up and seat it in the end of the aspic near the needle. sometimes the straws do not seat correctly and when the plunger is rolled the semen does not come out the needle but is dispensed into the aspic. if this happens, move the end of the aspic gently back and forth to re-seat the straw. the needle guard is removed and the guide is placed over the gun to cover the needle for insertion into the trocar. the semen is deposited into clinical theriogenology • volume 3 number 4 • december 2011523 the uterine horns and the gun is removed from the trocar. the guide is removed and the gun is loosened from the handle which allows the o-ring to release the aspic. the aspic is removed and the gun and guide are placed back on the slide warmer. the empty microcentrifuge tubes are thrown out so semen from different bucks is not accidently mixed. write down the insemination time on the breeding list for the owner. make any notes about the sample or the condition of the uterine horns on this paper as well. many times the does are not presented in any particular order. when this happens, you may end up with semen from five or more bucks thawed at the same time. our thaw unit will allow us to have semen from four bucks thawed and in microcentrifuge tubes at one time. the name of each buck is written on the lid of the tube with permanent marker and the lid securely shut. remember that the water bath will still be used to thaw semen straws and you don’t want to inadvertently drop water into any samples. at this time, you need to look at the breeding list and see which does need to be brought in to use the rest of the thawed semen. many times this cannot be done and you will need to remove a tube from the water bath. if i know a doe is going to be brought in soon i will draw the semen into the 0.25 ml straw and put it on the slide warmer with a 4 x 4 gauze square over it to prevent it from cooling down and being exposed to light. i still have to open the slide warmer to get other straws off the slide warmer so i put this straw towards the back of the warmer. if it is going to be longer than a few minutes i leave the sample in the tube and put it on the slide warmer. you will need to be able to multitask when you have semen from multiple bucks thawed. remember to check each ear tag as the doe is presented for insemination and keep track of how long each sample has been thawed. at 10 minutes after thawing i start reminding the manager of which doe is needed and at 20 minutes after thawing i start demanding those does. if you are doing a large number of does, it may not be possible to control the situation and you just do the best you can. i check a very small drop of semen if it has been sitting in the water bath or on the slide warmer more than 15 minutes. the longest i have had a sample be on the warmer or in the water bath is 50 minutes. this sample was still motile and the doe did conceive. this is in no way an ideal situation but it was unavoidable at this particular farm. in addition to the semen thawing, you will also need to be aware of what is going on in the preparation area and be able to troubleshoot problems that may occur. many times you have the owner and their assistants preparing the does and they will not have any idea how to care for clippers or how to change the blades. you also need to be watching as does come in to the preparation area so you can differentiate between the does that need more drugs and those that need more time to go down. the owners do not always remember in which order does were darted. also, make sure that the anesthetized does have their heads extended and their tongues are pulled to the side. most of all, have a great time and start writing a book with the first farm you visit! clinical theriogenology • volume 3 number 4 • december 2011 524 controlled internal drug release device, another tool for the transitional mare controlled internal drug release device, another tool for the transitional mare ashton broman, brad tanner, charlie scoggin rood and riddle equine hospital, lexington, ky several studies tried to identify strategies to hasten the first ovulation of the year to have foals born as close to the first of the year as possible in the northern hemisphere. despite many advances in our knowledge of the mare’s estrous cycle, the transitional phase continues to frustrate equine clinicians. multiple studies have shown that progesterone releasing intravaginal devices can be used to hasten the onset of estrus in the transitional mare without repeated daily hormone injections.1 however, only a few studies used a controlled internal drug release device (cidr). there were 2 objectives: 1) evaluate at which stage of transitional estrus the use of a cidr would be most effective; and 2) determine ovulation and pregnancy rates. a total of 24 mares were used. for each, the perineum was scrubbed aseptically with chlorhexidine and/or ivory soap. the cidr was placed intravaginally by hand and the plastic tail trimmed so it did not protrude from the vulva. after 7 days, the cidr was removed and mares concurrently received 250 µg cloprostenol im, followed by routine breeding management. mares were categorized by their relative stage of transition and defined as follows: anestrus (no luteal tissue and follicles < 10 mm), early transition (follicles 10 20 mm), mid transition (follicles 20 30 mm), and late transition (follicles > 30 mm). data regarding reproductive status at cidr placement (maiden, barren, and postpartum), culture results (pre and post cidr placement), comorbidities, and pregnancy rates from the cidr cycle were analyzed. of the 24 mares, 2 were in anestrus, 8 early transition, 13 mid transition and 1 in late transition. seventeen mares (65.4 %) responded to therapy as defined by subsequent breeding and ovulation within 10 days of cidr removal. of these, 5 (29.4%) were in early transition and 12 (70.6%) were in mid to late transition. ovulations following cidr removal appeared fairly predictable with 11 (65%) ovulating within 4 6 days after cidr removal. nine mares (53%) were diagnosed pregnant via transrectal ultrasonography at 14 day pregnancy diagnosis, whereas the remaining 8 mares that did not become pregnant continued to cycle and became pregnant on subsequent cycles within the respective breeding season. in conclusion, cidr devices were regarded as an effective alternative strategy for use in transitional mares, and response rates appeared better in mares more advanced in the transitional stage relative to anestrus and early transitional mares. keywords: mare, transitional mare, cidr, progesterone releasing intravaginal device reference 1. klug e, jöchle w: advances in synchronizing estrus and ovulations in the mare: a mini review. j equine vet sci 2001;21:474-479. 463 clinical theriogenology • volume 11, number 3 • september 2019 464clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 2017: side effects and occupational risks of using intrauterine kerosene in a mare   side effects and occupational risks of using intrauterine kerosene in a mare j. coyle, m. ferrer, h. fishman, e. eaton, v. gomes department of large animal medicine, college of veterinary medicine, university of georgia, athens, ga summary use of intrauterine kerosene is becoming popular for treatment of chronic endometritis. in this case, intrauterine kerosene was used to treat pyometra in a mare after failure of traditional treatments and adjuvants to resolve the condition. intrauterine infusion with kerosene was followed by uterine lavage the following morning. reflux of kerosene into the vagina resulted in chemical vaginitis with behavioral changes in the mare the morning after infusion. five minutes after uterine lavage, the mare developed severe signs of colic and pain associated with the vaginal procedures that exacerbated the irritation. side effects subsided with local instillation of 2% lidocaine into the vagina. in addition, kerosene eroded the obstetrical sleeve of the operator during uterine lavage, and resulted in chemical dermatitis with local redness, heat and pain for several hours. the severe side effects seen in this mare raise questions regarding animal welfare and ethical use of kerosene. the use of commercially available kerosene poses significant health risks to personnel and additional precautions are needed to minimize occupational exposure. intrauterine kerosene should be used with caution until controlled efficacy and safety studies become available and the benefits are proven to outweigh the risks. keywords: infertility, mare, kerosene, occupational hazards, safety background the use of intrauterine kerosene in mares is advocated by some clinicians as an adjuvant in treatment of chronic endometritis caused by gram negative and positive bacteria, yeast or fungi.1 use of adjuvants in the treatment of chronic endometritis becomes a necessity since traditional therapy with uterine lavage, antibiotics and oxytocin often fails to resolve the infection. treatment failure may occur due to recurrent contamination of the uterus, or failure to completely eliminate inflammatory exudates in mares with anatomical abnormalities or dysfunctional myometrial contractions, production of biofilm by bacteria or yeast, or abnormal mucus production by endometrial cells.1,2 biofilm is a complex structure of bacterial colonies, polymers and channels that results in a focal infection of non-dividing bacteria, covered by a matrix. this matrix confers bacteria protection from the environment and immune system, and structural support, and interferes with antibiotic penetration. furthermore, the lack of bacterial division results in inherent resistance to most antimicrobials. disruption of the biofilm is necessary to allow for antibiotic penetration, interaction of bacteria with the immune system and re-initiation of bacterial division. biofilm disruptors used in mares with endometritis include ethylene diaminetetraacetic acid-tromethamine (edta), tricide, n-acetyl-cysteine (nac), dimethyl sulfoxide (dmso) and hydrogen peroxide.1,2 the mucociliary apparatus also has an important role in preventing adhesion of bacteria to the endometrium and promoting physical clearance. in addition, endometrial mucus contains bacteriostatic products like lactoferrin or lyzozyme. normal mucus maintains the endometrial surface hydration and facilitates phagocytosis. changes in production, viscosity and elasticity of mucus can interfere with the ability of the mucociliary apparatus to remove bacteria or with antibiotic penetration. inspissated mucus results in accumulation of bacteria and inflammatory debris within the uterus, exacerbating the problem. therefore, mucolytics such as nac, dmso and kerosene are used to improve treatment success.1,2 treatment with intrauterine kerosene is becoming popular since it seems to improve pregnancy rates in chronically infertile mares. an intrauterine infusion of 50 ml of kerosene resulted in 50% and 45% foaling rate in mares with grade 2 and 3 endometrial biopsies, respectively.3 no control mares where included in the study. however, the findings were later interpreted as an improvement in fertility since foaling rates in mares with grade 3 biopsy are expected to be less than 10%.2 uterine lavage with 250 to 500 ml of kerosene also resulted in pregnancy in chronically infertile mares.4 activation of clinical theriogenology • volume 9 number 1 • march 201797   endometrial glands, reduction of mucus and exudate through desquamation and removal of the uterine epithelium, and clearance of inspissated glandular secretions were proposed as mechanisms by which kerosene seemed to improve pregnancy rate.1-4 histologically, intrauterine infusion with kerosene caused diffuse severe endometritis and edema, production of a serum-like exudate, and necrosis of luminal epithelium. however, these changes were transient and resolved within 14 days.3,4 loss of ciliated cells occurred 24 hours after treatment. however, a regenerated muco-ciliary apparatus was re-established by four days after infusion with kerosene.5 while the safety of kerosene on endometrial health has been demonstrated,3,5 no studies have evaluated the effect of this treatment on mare health and welfare. side effects have not been reported, although there is anecdotal evidence of transient vaginitis and discomfort immediately after treatment. this case report demonstrates the presence of severe side effects associated with the use of intrauterine kerosene in a mare and untoward effects in a clinician. these effects need to be taken into account if this treatment is to be used, and ways to prevent or mitigate them need to be installed taking patient welfare into consideration. case presentation a 12-year old multiparous quarter horse mare was presented in july 2016 to the veterinary medical center at the university of georgia with a complaint of infertility. the mare was recently acquired by the owner and her reproductive history was unknown. during the breeding season prior to presentation, she was mated to a stallion of unknown fertility on two estrous cycles. the owner was unable to provide information on breeding management (i.e. ovarian monitoring, timing of ovulation, number of matings, treatment of breeding-induced endometritis, semen quality, etc.), but wished to determine the reason for infertility and to artificially inseminate the mare with cooled shipped semen. on presentation, a breeding soundness examination was performed. the mare was bright, alert and responsive. her rectal temperature was 99.1f, pulse was 36 bpm and respiratory rate was 24 bpm. her general physical condition was within normal limits. perineal conformation was within normal limits except for a slight cranial tilt of the vulva (around 20). on palpation per rectum, the uterus was heavy and the cervix was tight. ultrasound per rectum revealed presence of a 24 mm follicle on the right ovary, small follicles on the left ovary and mild endometrial edema. no corpus luteum was noticed. the findings were consistent with estrus. there was also a large amount of hyperechoic fluid within the uterine lumen. upon vaginal speculum examination, a white-yellow fluid was noticed on the floor of the cranial vagina, and the cervix was closed. digital cervical examination revealed that the cervix was not dilated and only one finger could be introduced though the lumen. furthermore, a 2-cm cervical defect resembling a diverticulum was noted in the ventral portion of the cervical canal. the cervical lesion could also be identified on ultrasound as a hypoechoic irregular area (figure 1). the mare’s uterus was lavaged on presentation with 5 l of 0.9% sodium chloride (nacl) solution. a large amount of purulent material was recovered. on endometrial cytology, a large number of degenerate neutrophils were present. endometrial bacterial culture yielded growth of streptococcus equi subspecies zooepidemicus, streptococcus dysgalactiae and an unidentifiable gram variable rod. on endometrial biopsy, ulcerated areas of endometrial epithelium were present, with a thick layer of mucus covering the epithelium and diffuse inflammatory infiltrate. fibrosis was mild and sporadic (no glandular nests, no to one layer of periglandular fibrosis). the mare was diagnosed with pyometra. cervical laceration and fibrosis with failure to dilate was suspected to be the underlying predisposing factor. treatment for pyometra initially consisted on daily uterine lavage with 0.2% povidone iodine solution (v:v) in sterile 0.9% nacl solution for seven days during estrus to evacuate her uterus. ampicillin sodium, 2 g, was infused into the uterus daily for five days. antibiotic therapy continued systemically with procaine penicillin g, 22000 iu/kg im q12h, for five more days since the mare progressed into diestrus while still having evidence of intrauterine fluid accumulation. antimicrobials were selected based on the result of the antibiotic sensitivity test. the mare also received oxytocin 10-20 iu im every four h for the duration of antimicrobial treatment. clinical theriogenology • volume 9 number 1 • march 2017 98   n-acetyl-cysteine (3.3% solution, 150 ml) was used as a mucolytic and anti-inflammatory on the first day of treatment. on days 2 and 3, tricide (8mm disodium edta dehydrate and 20 mm 2-amino-2hydroxymethyl-1,2-propanediol, 500 ml intrauterine) was used as a biofilm disruptor. after ten0 days of antibiotic and oxytocin treatment, intrauterine fluid was no longer present and treatment was discontinued. however, intrauterine fluid started accumulating again 48 hours after discontinuation. treatment since medical treatment alone failed to resolve the condition, cervical wedge resection together with embryo technologies (embryo transfer or intracytoplasmic sperm injection) were recommended. these options were declined and the client insisted on having the mare carry the pregnancy. given the presence of a layer of mucus on biopsy and the failure of traditional treatment with adjuvants to clear the infection, chemical curettage with kerosene was elected as a last resort. commercial kerosene (klean-strip 1-k kerosene heater fuel), 240 ml, was infused into the uterus of the mare during estrus in the afternoon. uterine lavage to remove kerosene and debris was planned for the next morning. the following morning, it was noticed that the mare was showing behavioral changes. she had a decreased appetite, was reluctant to approach people and seemed restless and aggressive. ultrasonographic character of the uterine contents had changed from anechoic the day before infusion, to hyperechoic and heterogeneous the day after infusion (figure 2). uterine lavage was performed until the effluent was clear, requiring 8 l of 0.9% nacl. the effluent initially contained a large amount of debris and sloughed material (figure 3). the mare was restless in the stocks during the procedure. within five minutes of finalizing the uterine lavage and returning to the stall, the mare started showing signs of colic, lying down, rolling and flank watching. the signs were severe and did not subside after administration of flunixin meglumine, 1.1 mg/kg iv once. a complete colic examination was performed to rule out a gastrointestinal source of acute abdominal pain. this examination included palpation per rectum, percutaneous abdominal ultrasound and nasogastric intubation. findings were within normal limits and gastrointestinal pain was ruled out. chemical irritation of the vagina caused by reflux of kerosene through the cervix was suspected. it was thought that the vaginal and cervical manipulations done during the lavage may have worsened the discomfort. the mare’s vagina was lavaged with 1 l of 0.9% nacl to remove any residual kerosene, and 20 ml of 2% lidocaine were infused into the vagina with an insemination pipette to provide local analgesia. an additional unexpected finding was the development of skin redness, heat and pain in the arm of the operator performing the uterine lavage (figure 4). these signs became evident within five minutes of the uterine lavage while the mare’s colic was being monitored. during the uterine lavage, it was noticed that backflow of kerosene into the vagina resulted in erosion of the obstetrical sleeve that the operator was wearing, which had to be replaced. direct contact of kerosene with the operator’s skin resulted in chemical dermatitis. the skin was washed with abundant water and detergent. outcome the signs of discomfort resolved after vaginal infusion of lidocaine, and the mare remained comfortable thereafter. skin inflammation and pain in the operator’s arm also subsided within two hours with no further complications. uterine lavages continued daily with no further complications for three more days. on the third day, cellular debris were no longer present in the effluent and lavage was discontinued. oxytocin therapy with 20 iu im every four hours continued for one more day. intrauterine fluid was no further noticed at the end of treatment. however, anechoic intrauterine fluid was again present 48 h after discontinuing oxytocin injections (figure 2). a final uterine lavage was performed at that time to determine the character of the effluent. the effluent was mucoid in nature, and mucometra was suspected at that moment. uterine cytology was not performed to confirm the absence of inflammatory cells. due to recurrence of intrauterine fluid accumulation, hysteroscopy was performed to rule out a foreign body, endometrial adhesions or other pathology acting as a source for re-contamination or clinical theriogenology • volume 9 number 1 • march 201799   impeding physical clearance. the cervix was fibrotic, tortuous and closed, and there were focal areas of endometrial inflammation within the uterine body (figure 5). two blister-like structures were present at the uterine body and the bifurcation of the uterine horns (figure 5). the mare was discharged with the recommendation to perform cervical wedge resection and embryo technologies. oocyte aspiration and intracytoplasmic sperm injection was recommended since exposure of the embryo to an altered uterine environment would likely decrease the success rate of embryo transfer. discussion pyometra is the accumulation of purulent contents within the uterine lumen. this condition often results from failure of the cervix to dilate due to fibrosis, tortuosity or transluminal adhesions, or ineffective uterine contractions that fail to physically evacuate uterine contents.6 streptococcus zooepidemicus is the most commonly isolated bacterium, often in the context of mixed infections.6 treatment of pyometra consists of uterine lavage and intrauterine infusion with antibiotics. uterotonic drugs and exercise are used to encourage expulsion of uterine contents. unfortunately, medical treatment of pyometra is often unsuccessful due to persistence of the predisposing factor leading to mechanical impairment of uterine evacuation. cervical wedge resection has been used to facilitate uterine clearance and resolution of pyometra in mares.6 even though this treatment impairs the ability of the mare to carry a pregnancy, offspring could be produced using in vitro or in vivo embryo technologies.6 however, even when cervical patency was surgically re-established, fluid accumulation persisted in 20% (1/5) of the mares.6 the large and prolonged fluid accumulation prior to surgery could have led to stretching of the mesometrium and ventral positioning of the uterus, or stretching of the myometrium and inefficient uterine contractions, resulting in treatment failure. the cervical diverticulum could have also acted as a reservoir and source of re-contamination after treatment. surgical removal of the diverticulum was considered. however, due to the inability of the cervix to relax, cervical wedge resection was deemed to be a more appropriate option for this mare. in the case reported here, cervical laceration and fibrosis was suspected to be the predisposing cause. the duration of the lesions was unknown, but there was no recollection of presence of intrauterine fluid prior to mating. repeated mating likely resulted in bacterial contamination of the uterus. failure to clear bacteria resulted in persistent endometritis with infertility, which then progressed to pyometra. it could be speculated that more intensive involvement of a veterinarian in the breeding management or earlier referral of the mare could have allowed for an earlier diagnosis and treatment of the condition before it progressed into pyometra, potentially improving the prognosis for fertility. the chronic and severe endometrial irritation associated with pyometra possibly stimulated production of the mucus film seen on biopsy. it is also possible that denuded areas of epithelium allowed for bacterial adhesion and formation of biofilm. given the complex environment associated with pyometra, mucolytics (nac) and biofilm disruptors (tricide) were initially used as adjuvants together with traditional uterine lavage and antibiotics. the owner of this mare initially declined the use of embryo technologies or surgical treatment. due to the limitation to medical treatment and failure of traditional therapies to resolve the condition, use of non-traditional therapy was attempted. chemical curettage with kerosene was deemed to be the last resort, although it was acknowledged that its efficacy in treating pyometra was unknown. infusion of 240 ml during estrus was elected based on current practices.5 kerosene has been proposed for treatment of chronic endometritis caused by gram negative organisms, fungi or yeasts as it appears to improve pregnancy rates in persistently infected mares.1 however, the mechanism of action of kerosene is currently unknown. the presumed positive effect relates to its ability to stimulate a strong inflammatory response through chemical curettage and the activation of fibrosed endometrial glands, permitting the growth of new, functional endometrial tissue.1,3 additionally, kerosene reduced mucus production through the destruction of the uterine epithelium and clearance of inspissated secretions from dilated and cystic endometrial glands.1-4 clinical theriogenology • volume 9 number 1 • march 2017 100   uterine lavage with 250 to 500 ml of kerosene in subfertile mares caused diffuse, mild endometritis and edema. inflammation lasted one or two days, and was followed by expulsion of retained excretions from the endometrial glands and mucopurulent uterine discharge. one to three days after treatment, there was an influx of healthy leukocytes within the endometrium and uterine lumen. lavage was performed during diestrus and mares returned to estrus within two to five days of treatment presumably due to release of endometrial prostaglandin f2. 4 this information was presented as a summary of a symposium. the reviewing author commented that intrauterine kerosene markedly improved the fertility of these mares and that most conceived when mated in the estrus following treatment. it was also mentioned that kerosene was not suitable for treatment of cervical lacerations, uterine adhesions or fungal infections.4 unfortunately, no data are available from this study for thorough analysis and evaluation of the conclusions. in another study, 50 ml of commercial kerosene was infused into the uterus during diestrus to avoid reflux of the kerosene into the vagina. on day 1, all mares had a moderate-to-severe inflammation, with or without necrosis of the luminal epithelium. inflammation resolved by 14 days. a marked increase in the secretory activity and mitotic index was observed in the glandular epithelium on days 4 and 7, but there was no reduction in the number of glandular nests or overall improvement of the degree of endometrial degeneration after treatment with kerosene.3,4 loss of ciliated cells also occurred one day after kerosene infusion. however, a regenerated muco-ciliary apparatus was re-established by four days.5 these three studies provide evidence for a transient effect of kerosene on the endometrium, with resolution of inflammation, edema and necrosis by two weeks after treatment and regeneration of the muco-ciliary apparatus. therefore, there seem to be no long-term deleterious effects of intrauterine kerosene on the endometrium. on the other hand, there seem to be no beneficial effects or improvement of degenerative changes.3-4 with respect to pregnancy rates, kerosene was proposed to improve pregnancy rates in mares with chronic infertility. pregnancy rate after kerosene infusion was 50% in mares with category i and ii endometrium, and 82% (9/11 mares) in those with category iii endometrium. while pregnancy rate was high in mares with severe endometrosis, 44% (4/9) mares aborted between 40 and 100 days of gestation. foaling rate in these mares was 45% (5/11).3 this was later interpreted as an improvement in fertility since foaling rate in mares with category iii endometrium is expected to be less than 10%.2 it must be noted that another study reported a foaling rate of 35% in mares with category iii endometrium.7 it is unknown if a 10% increase in foaling rates would be statistically significant. in addition, use of kerosene in mares with category ii endometrium did not seem to provide an advantage, since foaling rate in mares with moderate endometrosis is expected to be around 50%.7,8 in a later symposium, bracher et al. commented that these findings tended to “confirm the rather short-term benefits of kerosene therapy, without a desired long-term improvement”.4 therefore, while use of kerosene may be of value treating chronic endometritis, controlled studies or large clinical trials with appropriate control groups are needed to determine the true efficacy of this treatment and its real impact on fertility. nevertheless, the use of kerosene has recently gained in popularity among clinicians due to the perceived improvement in pregnancy rates. however, there is little documentation of the risks of such a procedure beyond its effect on the endometrium. side effects have not been reported, although there is anecdotal evidence of transient vaginitis and discomfort immediately after treatment. the risks of occupational exposure to kerosene are also often overlooked. kerosene is an oil distillate that consists of 78 to 96% paraffin or naphthene, and 4 to 25% aromatic compounds, which act as mild irritants.4 according to the world health organization chemical hazards and poisons division, exposure to commercially available kerosene can have toxic effects on the dermis, mucous membranes, central nervous system and respiratory system.9 cardiac arrhythmias and ventricular fibrillation can also occur in some individuals after exposure to commercially available kerosene.9 one of the most frequent risks of exposure to kerosene is local skin irritation. a small percentage of individuals can sustain chemical burns following acute dermal exposure.9 in this case, reflux of kerosene into the vagina caused severe vaginal irritation. the behavioral changes noted the morning after kerosene infusion likely represented discomfort associated with vaginitis. clinical theriogenology • volume 9 number 1 • march 2017101   the vaginal manipulation and friction created during uterine lavage likely exacerbated the discomfort leading to severe pain that affected the mare’s welfare. while there is anecdotal evidence of discomfort caused by kerosene reflux, controlled studies are needed to evaluate the effect of this treatment on animal welfare and the individual variation in the severity of the response. additionally, treatments to mitigate pain and discomfort should be evaluated. in this mare, intravaginal irrigation with lidocaine was a suitable option for analgesia. also, while the current practice is to infuse 250 ml of kerosene during estrus,2,5 use of a lower volume during diestrus as reported in other studies3,4 may help prevent reflux into the vagina and minimize undesirable side effects. an often overlooked aspect of veterinary practice is occupational exposure to hazardous chemicals. in this case, the kerosene eroded the obstetric sleeve and caused contact dermatitis in the arm of the operator, also leading to pain for several hours. therefore, the risk of human exposure to such chemicals should also be considered when selecting treatments. the use of kerosene remains controversial, and the documentation of improved fertility requires the institution of controlled studies or large properly designed clinical trials. the side effects noted during this case were severe and call into question the welfare of the animals and personnel. selection of treatments should be based on solid evidence of their efficacy and lack of side effects. the definition of safety in reproductive medicine is often limited to the effect of chemicals on the endometrium or fertility. the welfare of animals and veterinarians is often overlooked and should be an integral part of a responsible decision making process. the use of intrauterine kerosene may be efficacious and with time it may prove to be a valuable adjuvant in the treatment of infertility. however, such use should be done with caution until controlled efficacy and safety studies become available and the benefits are proven to outweigh the risks. learning points  appropriate breeding management and early referral of infertile mares allow for an early diagnosis and treatment, likely improving the prognosis for fertility.  medical treatment alone, in spite of addition of adjuvants and non-traditional therapy, failed to improve the uterine condition in this mare with pyometra.  use of kerosene to treat chronic uterine infections in mares currently lacks appropriate efficacy and safety studies to be recommended.  the development of severe pain secondary to chemical vaginitis in this case raises questions regarding animal welfare and ethical use of kerosene.  the use of commercially available kerosene can poses significant health risks to the operator and additional precautions are needed to minimize occupational exposure. references 1. leblanc mm, causey rc: clinical and subclinical endometritis in the mare: both threats to fertility. reprod domest anim 2009; 44(s3):10-22. 2. leblanc mm: advances in the diagnosis and treatment of chronic infectious and post-mating induced endometritis in the mare. reprod domest anim 2010;45(s2):21-27. 3. bracher v, neuschaefer a, allen wr: the effect of intra-uterine infusion of kerosene on the endometrium of mares. j reprod fertil 1991;s44:706-707. 4. allen wr: proceedings of the john p. hughes international workshop on equine endometritis-summary by w. r. allen. equine vet j 1993;25:184-193. 5. bradecamp ea, ahlschwede sa, cook jl: the effects of intra-uterine kerosene infusion on endometrial epithelial cilia concentration. j equine vet sci 2014;34:134. 6. arnold ce, brinsko sp, varner dd: cervical wedge resection for treatment of pyometra secondary to transluminal cervical adhesions in six mares. j am vet med assoc 2015;246:1354–1357. 7. de la concha-bermejillo a, kennedy pc: prognostic value of endometrial biopsy in the mare: a retrospective analysis. j am vet med assoc 1982;181:680. 8. doig pa, mcknight jd, miller rb: the use of endometrial biopsy in the infertile mare. can vet j 1981;22:72-76. 9. chilcot rp: compendium of chemical hazards: kerosene (fuel oil). in: chemical hazards and poisons division (hw), world health organization. 2006. version 1. http://www.who.int/ipcs/emergencies/kerosene.pdf clinical theriogenology • volume 9 number 1 • march 2017 102   figure 1. ultrasonographic image of the cervical defect (a) delineated in white (b). figure 2. ultrasonographic image of the uterine contents one day before (a), one day after (b), and 6 days after (c) intrauterine infusion with kerosene. figure 3. debris and sloughed material present in the effluent recovered from the uterus the day after infusion with kerosene. a b c a b clinical theriogenology • volume 9 number 1 • march 2017103   figure 4. kerosene eroded the obstetric sleeve during the uterine lavage and caused contact dermatitis and pain in the clinician’s arm. figure 5. endoscopic view of focal areas of endometrial inflammation (arrows) within the uterine body (a,b) and blister-like structure within the uterine body (c). (editor’s note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) * a b c clinical theriogenology • volume 9 number 1 • march 2017 104 2010: diagnosis of infectious abortion in domestic ruminants diagnosis of infectious abortion in domestic ruminants mark l. anderson california animal health and food safety laboratory, college of veterinary medicine, university of california-davis, davis, ca abstract abortion problems in a client’s herd will often prompt the veterinarian to seek the assistance of a veterinary diagnostic laboratory. the chances of obtaining an answer for the abortions are improved when there is good communication between the veterinarian and diagnostician which leads to appropriate sampling and test selection. a persistent effort in abortion diagnosis is warranted as many surveys find that a cause is determined in less than half of abortion submissions to diagnostic laboratories. the majority of diagnosed abortions are attributed to infections by a moderate number of bacterial, viral, fungal and protozoal agents. the pathology and other findings used in the laboratory diagnosis of the major infectious agents causing abortion in the cattle, sheep and goats will be discussed. keywords: infectious abortion, diagnosis, domestic ruminants introduction veterinarians confronted by a herd abortion problem often seek assistance from a veterinary diagnostic laboratory that has the capacity to perform a variety of pathology, microbiology and immunology procedures to help identify the cause. abortion diagnosis is challenging as indicated by the fact that an etiology, most attributed to infections, is identified in less than half of submissions. in the past several decades there has been improvement in the diagnostic success in abortion cases, likely due to improvements in procedures and new knowledge about significant causes. there are regional differences in the infectious agents identified in various geographic locations, presumably reflecting various factors such as climate, production type, feeds, management practices, vaccination programs, as well as sampling and laboratory procedures available.1-5 abortion diagnosis protocol information provided with the submissions about the herd abortion problem may help suggest potential causes and exclude others. some useful information would include an estimate of the abortion rate in the herd, the duration of the problem, and the gestational ages of the abortions. other information to include with the submission would be whether the aborted fetuses are fresh or autolyzed, the age of the aborting dams, whether the placentas are retained, whether aborting animals appear sick, whether natural or artificial breeding is used, and the vaccination history. pathognomonic gross lesions are uncommon and may be obscured by autolysis. gross lesions are often undependable guides by which to select specific diagnostic tests so most diagnostic laboratories use a standardized diagnostic protocol containing a panel of diagnostic procedures aimed at identifying the relevant abortifacients known to be associated with abortions in their region. there are differences in the procedures selected as part of the abortion protocol among veterinary diagnostic laboratories depending on many factors. consult your diagnostic laboratory to determine the optimal samples and conditions to submit specimens in an abortion workup. the web sites that most laboratories maintain are an efficient way to obtain information on specific test and sample requirements. the following example of an abortion protocol can be modified depending on the condition and type of samples submitted, or other circumstances. the intact aborted fetus, placenta and a serum sample from the dam are the optimal specimens. when available, samples from several abortions are recommended. the placenta is often critical to successful determination of the cause of abortion, particularly in the sheep and goats where the placenta may be the primary tissue affected. a complete necropsy examination is performed on the fetus to identify any gross lesions and to estimate the fetal age and degree of autolysis. if the entire fetus cannot be submitted the preferred samples would include 555 complete selection of formalin fixed tissues and fresh lung, liver, kidney, placenta, fetal thoracic fluid, and abomasal fluid. these samples should be submitted chilled in separate sterile containers and the fluids in sterile tubes (not syringes). it is important that both the veterinarian and client are aware that there are numerous zoonotic infections associated with ruminant abortion (brucellosis, leptospirosis, listeriosis, salmonellosis, chlamydophila, coxiella and others), so appropriate precautions should be employed in sampling and shipping specimens. formalin fixed tissues for histopathology examination include brain, lung, heart, liver, kidney, adrenal, spleen, thymus, lymph node, skeletal muscle, abomasum, small intestine, eyelid and placenta.6,7 the routine microbiological diagnostic protocol includes an aerobic bacterial culture and brucella culture on the lung, an aerobic culture and campylobacter culture on the liver, and an aerobic culture, brucella culture, and campylobacter culture on the abomasal fluid. the abomasal fluid is also examined with a gram stain and a dark field examination for campylobacter and trichomonads. the abomasal fluid may be cultured for tritrichomonas foetus on appropriate media based on positive dark field examinations or other indications. a direct smear on the kidney is examined for leptospires by fluorescent antibody staining. bacteria cultures of the placenta are dependent on condition and gross lesions. fungal cultures are performed if there are suggestive placental or skin lesions. routine virology procedures vary among laboratories. routine virus isolation in tissue culture on an organ pool of fetal tissues, usually including lung, liver, spleen, kidney, adrenal and placenta, is performed by some diagnostic laboratories. other protocols may limit routine virology testing to bovine herpes virus (bhv-1) and bovine viral diarrhea virus (bvdv) fluorescent antibody stains in cattle and border disease virus (bdv) in sheep on frozen sections of fetal lung (or liver) and kidney. bhv-1 and bvdv virus immunohistochemistry procedures are relatively specific and sensitive with the advantage that the pathologist can retrospectively test for the presence of the virus based on the lesions observed the fetal tissues. polymerase chain reaction (pcr) assays for the dna of various bacterial, viral and protozoal agents are available for use in some diagnostic laboratories. an abortion serology panel on a single serum sample from an aborting dam may help determine if there had been exposure to an agent but usually cannot differentiate between vaccination and natural exposure, or between recent versus incidental previous exposure. paired acute and convalescent serum samples rarely can identify a significantly rising titer to a particular pathogen because maternal seroconversion often precedes abortion and paired serum samples collected at and following abortion may not demonstrate an increasing titer. maternal serology is most useful when serum from non-vaccinated animals is examined, when several animals from the herd are tested, and when history on each animal is provided. some advocate collecting and storing serum from select pregnant animals for use as a paired sample in the event of an abortion. routine serology on the bovine dam includes bhv-1, bvdv types i and 2, leptospira serovars canicola, grippotyphosa, hardjo, icterohamorrhagiae, and pomona , neospora caninum, and brucella serology. in sheep and goats usual dam serology tests may include bdv, leptospira serovars, bluetongue virus, brucella and toxoplasma gondii serology. immunologic procedures on the fetus depend on the post mortem condition and fetal age. a quantitative immunoglobulin assay for bovine igg can be performed on blood or thoracic fluid. if the fetal igg is elevated (>20 mg/dl), this is an indication of an active immune response by the fetus to a foreign antigen which is usually an infectious agent.8 if there is evidence of elevated igg, serology for ibr, bvd, leptospira, neospora, brucella abortus, bluetongue virus and parainfluenza 3 virus can be performed. when specific fetal titers are elevated, they suggest fetal exposure to that agent. on occasion, low titers to one or more of these agents may be present in a fetus without other evidence of infection so a cautious interpretation is warranted. the source of antibody in the fetal fluids, particularly when antibody to multiple agents is present, is not certain but possibly maternal antibodies may cross to the fetus due to placental lesions. when available, the placenta should be examined and sampled. in a fresh normal placenta the cotyledons are red with a clear, translucent intercotyledonary placenta although with autolysis the cotyledons become dull brown and the intercotyledonary placenta less translucent. intercotyledonary 556 opacity can be associated with edema, inflammation or fibrosis. exudate on the surface or thickening of the chorioallantois is evidence of inflammation. cotyledons whose surface is depressed relative to the surrounding intercotyledonary placenta (“cupping”) can be an indication of inflammation in the surrounding stroma. gross abnormalities in the cotyledons include adherent caruncular tissue, hemorrhage, necrosis and exudation. an estimation of the gestational age of the fetus can be made from the crown to rump length and the extent and distribution of hair development.9 potentially useful external changes include evidence of meconium staining implying fetal distress and round raised skin plaques that can be associated with mycotic infection. with the fetus opened, an estimation of the degree of autolysis can be made. evidence that the fetus may have been alive at the time of parturition such as lung inflation, hemorrhage surrounding the umbilical vessels or thrombosis of the umbilical arteries should be noted. a freshly aborted fetus will usually have clear amber colored fluid in the body cavities but within one to two days following death, serosanguinous fluid collects in the body cavities and subcutis. this process is followed by gradual dehydration of the tissues such that by a week following fetal death the fetus is dehydrated with no abomasal content. the selection of fetal tissues for histopathology examination varies among laboratories due to a variety of factors. while autolytic changes are common and can be severe, unless the fetus is mummified, histopathology will usually provide some information concerning any evidence of infectious disease and therefore help to evaluate whether the microbiology or immunology results are relevant. examination of a full selection of tissues is recommended whenever possible. although autolysis may render the fetal brain to consist of liquefied fragments, collection of this material into formalin is still recommended because the histologic detail is surprisingly preserved. a complete histological examination of the fetal tissues provides information about the distribution of lesions which may also suggest the route of fetal infection and potential causes. some infections proliferate in the placenta then enter the fetus through the umbilical vessels leading to hepatic and systemic changes. examples of this pattern of fetal injury can include such agents as listeria, salmonella, bhv-1 and neospora. in other bacterial and fungal infections which initiate a placental infection and then invade via the placental fluids surrounding the fetus the inflammatory lesions are usually associated with lungs, digestive tract or skin. histologic examination of the eyelid having both conjunctiva mucosa and skin can be a useful and sensitive indicator of infection.8 on occasion in situations with severe placentitis, there may not be any significant inflammatory lesions in the fetus. diagnostic features of infectious causes of abortion the following sections discuss the presentation and diagnosis of commonly diagnosed infectious causes of abortion in cattle, sheep and goats. bacterial infections sporadic abortions associated with opportunistic bacterial infections. a diverse group of bacterial species are associated with opportunistic infections of the placenta and fetus resulting in abortion. these bacteria are not contagious pathogens but are commonly found in the environment or on mucosal surfaces. a maternal bacteremia is the presumed means by which they reach the gravid uterus and subsequently infect the placenta. the specific reasons for the susceptibility of the placenta to colonization by these bacteria is uncertain but factors such as local oxygen and nutrient levels, decreased inflammatory responses and isolation from maternal immunity are likely involved. among the bacteria in this group arcanobacterium pyogenes is the most commonly identified but bacillus spp., e. coli, histophilus somni, pasteurella spp., pseudomonas spp., serratia marcescens, staphylococcus spp., streptococcus spp and many other bacteria species have been associated with sporadic placentitis and abortion.1,10,11 while opportunistic bacterial infections are a significant proportion of diagnosed abortions their occurrence is sporadic. abortion storms are not associated with this group of bacteria so if multiple abortions occur in a herd this might suggest maternal health issues that could enhance hematogenous 557 bacterial infections or there could be additional infectious agents involved in the abortion. a possible example is that up to a third of bovine abortions associated with arcanobacterium pyogenes, bacillus sp. or mycotic infections had concurrent bvdv virus infection in one large midwest abortion survey.12 sporadic bacterial abortions may occur at any stage of gestation, but most are identified in the second half of gestation. there are no specific signs in the dam although the placenta may be retained. the degree of fetal autolysis is variable. the placenta may have yellow to brown surface exudation. gross fetal lesions may infrequently include fibrin exudation in body cavities. histologic lesions include suppurative placentitis and neutrophilic fetal bronchopneumonia of varying severity. in arcanobacterium pyogenes infection large clusters of bacterial colonies may be present in the lung with minimal inflammation. as the bacteria involved are common in the environment or mucosa, their presence in fetal tissues might be due to incidental contamination. therefore in order to establish an etiologic diagnosis the bacteria should be isolated in pure or nearly pure culture from abomasal contents or tissues and there should be lesions consistent with a bacterial infection in the fetus or placenta.7 brucellosis. brucellosis in sheep, goats and cattle is caused by several brucella species, brucella ovis, brucella melitensis and brucella abortus. brucella ovis is a relatively newly recognized mutant that is predominately associated with epididymitis in the ram although the infection can be transmitted to the ewe at breeding resulting in infertility or on occasion, placentitis with abortion or premature live lambs. brucella melitensis is the major cause of brucellosis in sheep and goats although the disease is not currently present in north america. brucella abortus infection causes brucellosis in cattle worldwide. the infection is now rare in the united states and canada due to eradication programs but pockets of infection persist primarily in wildlife populations. infection is primarily through ingestion, the bacteria multiply in regional lymph nodes and then spread hematogenously to other organs, most importantly, the mammary gland, mammary lymph nodes and gravid uterus usually during the second trimester. bacteria invade the placental trophoblasts and cause chronic placentitis and fetal infection resulting in fetal death due to placental disruption and endotoxemia. fetuses abort 24 to 72 hours after in utero death and abortion usually occurs after the fifth month of pregnancy. metritis and retained placenta are common. grossly there is often a severe placentitis with edema, focal necrosis of cotyledons and thickened intercotyledonary areas with adherent yellowish exudate. the fetus is frequently autolyzed with no gross lesions. histologically, there is severe placentitis with numerous bacteria visible in chorionic epithelial cells. fetal lesions consist of bronchopneumonia which can vary in severity and character from acute neutrophilic bronchopneumonia to more chronic pleocellular bronchointerstital pneumonia with periairway infiltrate of mononuclear cells. bacterial isolation is necessary to confirm the diagnosis. b. abortus can be isolated from various sources including fetal abomasal fluid, lung, placenta, uterine fluid and milk. various serologic tests have been developed for governmental surveillance and detection of cattle exposed to b. abortus.13 listeriosis. listeria species are widespread in the environment and abortion due to listeria monocytogenes and l. ivanovii infections occur throughout the united states. most abortions have a sporadic occurrence but in some conditions abortion storms may occur. in some instances, there may be illness in aborting dams with fever and anorexia due to metritis. listeriosis can cause encephalitis but this is not commonly seen in association with abortion. the infection can be spread by ingestion from environmental contamination of the feces of carrier animals and discharges from aborting animals. outbreaks of listeria abortions have been associated with ingestion of poorly fermented silage which provides a favorable environment for the proliferation of the organism. fetuses are usually aborted in the third trimester and often markedly autolyzed. the placenta is usually retained at abortion. gross lesions are often absent or obscured by autolysis but in some, pinpoint white to yellow foci are present in the liver. additional gross findings may include small pale foci in placental cotyledons, and fibrin in body cavities. the histopathologic changes include suppurative placentitis and multifocal necrotizing or suppurative hepatitis. focal necrotizing mucosal lesions with 558 bacterial colonization may be present in the intestine. in addition, meningitis and intravascular bacterial colonization in many organs with or without associated inflammatory lesions is sometimes encountered. in most aborted fetuses listeria species are present in multiple tissues and usually does not require cold enhancement for successful isolation. in fresh tissues, liver impression smears or abomasal fluid can be gram stained to identify gram-positive coccobacilli. in fixed tissues, gram staining is often useful in multiple tissues particularly liver sections. immunohistochemistry stains for listeria monocytogenes utilizing commercially available antibody can assist the diagnosis when a positive culture result is unavailable. salmonellosis. in the united states, abortions attributed to salmonella species infections are not common and usually present as sporadic occurrences. although, in the united kingdom and some other regions of the world salmonella abortion is a significant cause of both enzootic and epizootic abortion in cattle and sheep. most cattle abortions are associated with salmonella dublin infections but other serotypes can be involved. in sheep, occasional abortion outbreaks in densely populated flocks have been associated with salmonella abortus-ovis and s. brandenburg. the infection is presumed to originate from the intestinal tract with bacteremic episodes leading to the localization and proliferation in the placentome. the abortions are usually in the second half of gestation and the placenta is often retained. the chorioallantois is thickened with fibrinous fluid with a diffusely grey to red chorionic surface. portions of caruncular tissue may be adherent to the cotyledons. the fetus is usually quite autolyzed and may be emphysematous. usually no remarkable gross lesions can be identified though indistinct pale foci may rarely be present in the liver. in the placenta there is neutrophilic placentitis and mineralization with bacterial proliferation in cotyledonary villi. in the liver there can be a multifocal suppurative hepatitis. lung lesions may be minimal consisting of neutrophilic bronchial exudate. salmonella and listeria abortions have similarities in that the bacterial infection proliferates in the placenta followed by massive infection of the fetal liver, septicemia and death, often without development of bronchopneumonia typical of some other bacterial infections of the placenta.14 leptospirosis. leptospirosis is likely an under-diagnosed cause of infertility and abortion. the most significant serovars of leptospira interrogans associated with bovine abortion are l. hardjo and l. pomona though rarely leptospira interrogans serovars icterohemorrhagiae and grippotyphosa have been associated with bovine abortion. in sheep, leptospirosis is not a common cause of late term abortion and stillbirth although outbreaks in intensively managed conditions may occur. the major serovar involved is l. hardjo. leptospira hardjo serovars are adapted to cattle who serve as the maintenance host while other serovars of leptospira involved in bovine abortions are maintained in other domestic or wildlife species. leptospires can be shed in urine for several weeks and more prolonged urine shedding can be observed with l. hardjo infections. leptospires can survive in wet environments for up to 30 days and can penetrate intact mucous membranes or abraded skin. abortion may be the only clinical sign observed although fever, hemolytic anemia, hemoglobinuria, icterus, and mortality can be seen in younger cattle. in lactating cattle, agalactia and mastitis can occur with flaccid udders and thick yellow to occasionally blood-tinged secretions.15-16 reproductive disease can occur one to three months following initial infection with l. hardjo and is associated with infertility, abortions four months to term, and weak calves. abortion due to l. pomona usually occurs in the last trimester. the herd abortion rate seldom exceeds 10% with l. hardjo infections but can be higher with herd infections of l. pomona. the aborted fetus is usually autolyzed. icterus may be seen in late gestation fetuses infected with l. pomona. histologic lesions may not be observed but in some cases renal tubular necrosis and interstitial nephritis is present. bile retention within liver canaliculi may be present. nonsuppurative meningitis has also been reported. because leptospires are labile and difficult to culture, bacterial isolation is impractical for routine diagnostics. identification of leptospires by darkfield microscopy of fetal fluids or silver stains of fetal tissues is rarely successful. fluorescent antibody examination of fetal kidney smears using multivalent 559 antisera is a convenient, rapid procedure although not highly sensitive and specific. the specific leptospira serovar involved cannot be determined with this procedure. immunohistochemistry staining is sometimes useful in identifying leptospires in bovine fetal tissues but autolytic fragmentation of the spirochetes can make interpretation difficult. pcr assays are available and used by some diagnostic laboratories to identify leptospira in fetal tissues. maternal serology using the microscopic agglutination microtiter test may be useful in the diagnosis of leptospirosis, though caution must be used to distinguish between vaccination, previous exposure, and recent infection. serology for l. hardjo is especially difficult to interpret since infected animals often have a low or negative titer at the time of abortion. higher serologic titers are associated with l. pomona infection at the time of abortion but seroconversion usually precedes abortion and the titer may be declining. it can be difficult to distinguish between vaccination and field exposure titers although multivalent vaccines usually produce low and short-lived titers compared to recent field exposures. in addition, the multivalent bacterins usually exhibit a pattern of elevated titers to multiple serovars as contrasted with elevated titers to a single serovar following field exposure. fetal titers of 1:40 or above would be supportive of a diagnosis but most infected fetuses have no detectable titer. chlamydophila abortus. chlamydophila (chlamydia) abortus is a significant cause of late term abortion in sheep and goats. herd outbreaks may occur in pregnant animals which acquire the infection orally from contaminated fecal or placental sources. intestinal colonization can be followed by blood borne infection of the placenta resulting in placentitis and abortion. the placental lesions are often grossly evident with cotyledonary necrosis and exudation with intercotyledonary opacity and thickening. the fetus is usually well preserved and only rarely there may be pinpoint necrotic foci visible in the liver. histologically placentitis is the most consistent feature with chorionic epithelial necrosis and pleocellular exudation. remaining trophoblasts may contain intracytoplasmic organisms. the stroma of the chorioallantois is expanded by edema and mixed inflammatory cells, frequently with fibrinoid vasculitis of major arteries. gimenez stained impression smears of placenta can demonstrate elementary bodies suggestive of chlamydophila although specific antibody utilizing either a fluorescent antibody stain on impressions smears or immunohistochemistry stains on histologic sections is needed for confirmation.17 ureaplasma diversum. ureaplasma diversum, in the mycoplasma family, is a common mucosal inhabitant of the reproductive and respiratory tract of cattle. there is evidence of considerable genetic variation among u. diversum isolates which could be associated with more pathologic potential in some stains. ureaplasma diversum has been associated with granular vulvitis, infertility, embryonic death, abortion, stillbirths and the birth of weak calves. in north america, the diagnosis of ureaplasma diversum as a cause of abortion in cattle is regional. in some northern regions a substantial portion of abortions are attributed to this infection while in other regions it is rarely identified. the differences may reflect differences in infection rate or perhaps a lack of appropriate microbiologic procedures regionally available by diagnostic laboratories by which to identify this fastidious organism.18 fetuses are usually aborted the last third of gestation and stillbirths or weak calves may occur. the aborted fetuses are often in fresh condition. retained placenta is frequently reported. there is placentitis in which the amnion is often most affected portion of the placental membranes. the amnion is thick and opaque with multifocal to extensive areas of hemorrhage, fibrin exudation, necrosis and fibrosis. similar changes may be present in the chorioallantois which are usually more severe on the allantoic surface. the cotyledons may be tan to dark red, cupped with adherent caruncular material. histologic changes in the stroma of the chorioallantois and amnion include fibrosis, necrosis and mineralization with macrophage and plasma cell infiltrates and mononuclear vasculitis. in the fetus the lungs may be swollen and firm grossly. histologically, the lung lesions consist of a nonsuppurative alveolitis and periairway mononuclear infiltrate. erosive lymphoplasmocytic conjunctivitis has been reported. 560 ureaplasma diversum is a common inhabitant of the upper respiratory tract and lower reproductive tract of cattle so contamination of placental and fetal tissues is a potential confounding factor in diagnosis. diagnosis of u. diversum as the cause of abortion can be supported by the isolation of the organism from the fetus or placenta, or by demonstration of specific u. diversum dna by pcr assay coupled with the presence of compatible lesions in the lung and placenta.19 campylobacter species. in sheep and goats, campylobacter fetus ss fetus and c. jejuni (ss jejuni) infections are a significant cause of late term abortion, stillbirths and weak lambs/kids. campylobacter abortions can occur as a flock outbreak in which the intestinal infection can spread orally through the flock resulting in new intestinal infection and subsequent hematogenous infection of the placenta in pregnant naive animals. these campylobacter species also cause sporadic abortions in cattle through a similar mechanism of hematogenous spread of an intestinal infection. however, cattle have another clinically important campylobacter species, campylobacter fetus ss venerealis, which is a venereal disease that causes infertility with early embryonic death and occasional abortions occurring at four to seven months gestation. in bovine campylobacter abortion it is important to identify the specific species involved as c. fetus ss. venerealis is venereally transmitted and must be differentiated from the other intestinal campylobacter species. gross lesions of campylobacter abortion are usually found in the placenta. the changes are not unique but usually consist of swelling, necrosis and yellow-brown discoloration of cotyledons with intercotyledonary thickening with variable exudation. aborted fetuses can be in variable post mortem condition ranging from fresh to autolyzed. serosanguinous to fibrinous exudation is often present in the pleural cavity, peritoneal cavity or pericardial sac. in small ruminants, a striking but inconsistent gross lesion can be present in the liver consisting of multiple white to brown circular to ring foci up to 4 cm in diameter. occasionally sporadic sheep abortions similar lesions can be associated with an anaerobic bacterium, flexispira rappini.20 splenomegaly is a variable feature and fetal serum immunoglobulin levels may be moderately elevated. histologic changes include fibrinous neutrophilic placentitis with chorionic necrosis, neutrophilic bronchopneumonia, fibrinous neutrophilic serositis and occasionally a lymphocytic abomasitis in small ruminants multifocal hepatic necrosis can be present depending on the post mortem condition and other factors. silver stains of fetal tissues with inflammation may assist in the diagnosis by identification of curved silver stained bacteria. immunohistochemistry has been used to detect campylobacter fetus in fetal tissues and differentiate it from other campylobacter species.21 in a fresh fetus, campylobacter may be identified in darkfield microscopic examination of abomasal fluid as small bacteria with darting motility. campylobacter fetus ss. venerealis is fastidious, requiring careful sample collection and proper transport. the bacteria can be cultured from lung, placenta, and abomasal fluid. the bacteria can also be identified in preputial samples and vaginal secretions. phenotypic characterization and molecular techniques have been used to differentiate campylobacter species.22 epizootic bovine abortion. epizootic bovine abortion (eba, also known as foothill abortion) is a cause of abortion and premature calving in cattle grazing foothill rangelands in california, nevada and oregon. the infection is transmitted to susceptible pregnant cattle by an argasid tick (ornithodorus coriaceus), that feeds on deer and cattle. the disease is seen in heifers or cows exposed to endemic areas for the first time while in the first trimester of pregnancy. abortions, either sporadic or as an outbreak, usually occur in the last trimester and premature weak calves may also occur. following an abortion affected cattle are resistant to repeat abortion.23 a currently unnamed and unculturable gram negative bacterium is presumed to be the etiology of eba based on molecular techniques on extracted 16s bacterial ribosomal dna fragments in infected fetal tissues and ticks. the dna sequences suggest that the bacterium is a delta-proteobacteria in the myxobacteria family. a modified steiner silver stain and immunohistochemistry can stain the bacterium in the thymus, spleen, lymph node and in other tissues in the sites of inflammatory lesions.24 561 the diagnosis of eba is based on the identification of characteristic gross and histologic lesions which are chronic, having developed over a period of three months or more. the fetus is usually fresh and may be born alive. petechiae are common in the mucosa of the conjunctiva and oral cavity. there is enlargement of peripheral and internal lymph nodes. enlarged superficial lymph nodes can be easily palpated through the skin. abdominal distension due to ascites and liver enlargement is often present. splenic enlargement and enlargement of internal lymph nodes is usual. the thymus may be reduced in size with interlobular or widespread hemorrhage and edema in the cranial portion. histologic examination of fetal tissues, particularly the lymphoid organs, is required to confirm the diagnosis. thymic lesions, which are unique in eba, develop late in the course of the disease and consist of a loss of cortical thymocytes and infiltration of the medullary region with macrophages. the thymic interlobular septa are distended with edema, fibrin, hemorrhage and cellular infiltrates consisting of macrophages and other mixed inflammatory cells. the gross enlargement of the lymph nodes is associated with lymphoid hyperplasia and widespread macrophage infiltration in the sinuses and medulla. there is lymphoid hyperplasia and histiocytic infiltration in the spleen. late in the course of disease, following the proliferative response, acute necrotic foci develop in lymphoid organs. there are widespread inflammatory lesions with a vascular orientation in most organs, including the brain, lung, heart, liver, kidney, skeletal muscle and other organs. fetal serum immunoglobulin levels are usually markedly elevated from 100 to 1000 mg/dl. coxiella burnetii. coxiella burnetii is an obligate intracellular coccobacilli in the rickettsiaceae family associated with late term abortion in sheep, goats, rarely cattle and is a zoonotic disease causing of q fever in humans. animals can be persistently infected resulting in environmental contamination from milk, feces, uterine discharges and placenta. new infections are by oral transmission and dust inhalation. problems of abortions, stillbirths or weak newborn animals tend to occur in newly infected animals. aborted fetuses are in variable post mortem condition but usually fresh. gross lesions are usually confined to the placenta where the intercotyledonary placenta is thick and covered with white to yellow exudate with variable circumferential infiltration of the cotyledons. histologically there is suppurative inflammation of the chorion with necrosis of villi and chorionic epithelium. remaining trophoblasts often have swollen foamy basophilic cytoplasm containing organisms. the chorioallantoic stroma is infiltrated with mononuclear cells and plasma cells. the organisms are usually plentiful and special stains on smears or histologic sections can help to visualized them but specific antibody stains either fluorescent antibody on smears or immunohistochemistry on sections, or pcr on placenta should be employed to differentiate coxiella from other similar organisms, especially chlamydophila.25,26 fungal infections mycotic abortion is reported worldwide with an overall low incidence in north america. the majority of mycotic abortions in cattle in the united states are associated with aspergillus fumigatus with other aspergillus spp., absidia spp., mucor spp., rhizopus spp., candida spp. and other fungi less commonly reported.27 the fungi responsible are ubiquitous saprophytes in the environment. the concentration of fungal conidia in the environment may increase the risk for fetal infection since more cases occur in the winter where cattle are housed and fed. injury to the respiratory or digestive tract of the dam may also enhance the entry of fungi into the bloodstream. localization in the uterus probably occurs by hematogenous spread from these sites of entry. experimentally, intravenous injection of a. fumigatus causes abortion 23 to 35 days post-infection. mortierella wolfii, a common abortifacient in the southern hemisphere, is rare in north america. mycotic abortions usually occur as sporadic third trimester abortions. clinical signs in the dam are infrequent aside from retained placentas. placentitis is the primary lesion so submission of the placenta is critical to the diagnosis. grossly there is often a severe placentitis involving both the cotyledons and intercotyledonary placenta results in a diffusely thickened, leathery placenta. cotyledons may have necrotic, hemorrhagic infarcts with adherent caruncular tissue. histologically, the placenta may 562 have suppurative inflammation, necrosis, and vasculitis with thrombosis associated with fungal invasion. fetal autolysis may be minimal, especially with aspergillus fumigatus infections. in the fetus lesions are variable and may be absent. in a minority of affected fetuses there may be raised circumscribed plaques on the skin. internally fetal lesions associated with mycotic infection may include bronchopneumonia or focal digestive tract inflammation associated with fungal invasion. a rapid presumptive diagnosis may be obtained by direct microscopic examinations of scrapings of placental or skin lesions after digestion with 10% potassium hydroxide (koh) solution. samples of the abomasal fluid can be examined in a similar manner. the fungi are not digested by the koh solution and can be visualized microscopically in the wet mount. histologic identification of fungi in tissue lesions utilizing histochemical stains is also effective. for isolation cultures of the placenta, abomasal fluid, or lung on fungal media with antibiotics to suppress bacterial growth can be employed. as fungi are ubiquitous in the environment their presence could be the result of contamination so the diagnosis of mycotic abortion requires compatible lesions in the placenta or fetus in addition to the microscopic demonstration or isolation of fungi. viral infections herpes virus infections. bovine herpesvirus type i is found worldwide although there has been successful eradication in some european countries. in the united states, it remains a significant cause of abortion which is frequently epizootic. infection at breeding can cause infertility and embryonic death may result from infections early in pregnancy. the virus may persist as a latent infection following acute infection. infection occurs through contact with infected cattle shedding virus from respiratory, ocular, and reproductive secretions. exposure of previously unexposed, non-vaccinated pregnant cattle can result in abortion storms with 25 to 60% of cows aborting. experimentally, abortion occurs at any stage of gestation, but in field conditions abortions are usually seen in the second half of gestation. most abortions occur several weeks (20 to 52 days) following initial infection of the dam that may not exhibit signs of illness other than abortion.28 aborted fetuses are usually five months to term and are autolyzed with red tinged fluid in body cavities and fascia. there are usually no gross lesions other than placental edema though rarely indistinct pinpoint white foci may be appreciated in the liver. a presumptive diagnosis can be made from histopathology of the liver which despite considerable autolysis usually has striking multifocal necrosis, best appreciated at low magnification. focal necrotizing lesions are usually present in other fetal tissues, especially lung, adrenal, spleen and lymph node. eosinophilic intranuclear inclusions are often difficult to recognize but may be identified in the adrenal cortical lesions. placentitis with necrosis and vasculitis in placenta villi is usually present associated with abundant viral antigen as detected with immunohistochemistry. diagnosis is confirmed by viral isolation, by detection of viral antigen in fetal tissues by immunofluorescent staining on frozen tissue sections (especially kidney) or by immunohistochemistry on formalin fixed tissues (especially liver, lung, kidney, adrenal, placenta) using monoclonal antibodies. caprine herpes virus is related to bhv-1 and in goats is associated with a similar array of diseases manifestations including late term abortion.29 pestivirus infections. bovine virus diarrhea virus (bvdv) and border disease virus (bdv) are common, closely related pestivirus species and some cross species infection may occur between cattle and sheep.30 bvdv virus infection is widespread in the cattle population and in susceptible pregnant animals fetal infection is likely to occur. fetal infections have a variable outcome depending on the timing of the infection, the biotype and other properties of the virus. an important aspect of fetal bvd virus infection is that noncytopathic bvd virus infections in fetuses prior to four months gestation can result in persistently infected live calves which are a major source of infection for other cattle. contact with infected animals shedding the virus and contaminated biologics are additional sources of infection. there are often no obvious clinical signs seen in herds with fetal losses due to bvd virus and abortions occur a few days or several weeks following maternal infection. fetal bvd virus infection can 563 have variable outcomes depending on the gestational age of the fetus infected and other factors. first trimester infections can cause infertility, embryonic death, fetal resorption, mummification, or abortion. however, infections with noncytopathic bvd virus between 18 to 125 days gestation may result in persistently infected live calves. fetal infections beyond approximately four months gestation often result in transient fetal infections, with the development of a fetal immune response, specific fetal antibody production, and elimination of the virus. however, abortions can also occur during later gestational infections. mid-gestational infections (from approximately 100 to 150 days gestation), can also result in the birth of term calves with congenital anomalies.31 the fetal lesions associated with abortions attributed to bvd virus infection are quite variable. the fetuses can be fresh or autolyzed and mummification can occur. the aborted fetuses may be small for their gestational age or premature small calves may be born. gross lesions that may suggest bvd virus infection in the nervous system includes microencephaly, cerebellar hypoplasia, hydranencephaly and hydrocephalus. ocular lesions including microphthalmia, cataracts, retinal dysplasia and optic neuritis have been described. alopecia may be present. thymic hypoplasia with histologic evidence of thymic cortical atrophy is associated with bvd virus infection. pulmonary and renal hypoplasia or dysplasia has been reported. bvd virus infection has been associated with a necrotizing myocarditis with nonsuppurative vasculitis in the heart and the affected fetuses may exhibit marked anasarca with a round dilated heart and chronic passive congestion of the liver. proof that bvd virus infection is the cause for an abortion is confounded by the fact that the virus may infect the fetus without causing abortion and there are diverse fetal lesions attributed to infection. to attribute bvd virus infection as the cause of abortion, evidence of infection needs to be combined with compatible fetal pathology and/or herd history. fetal infection can be determined by detection of the virus in fetal tissues by various methods.32 virus isolation on a pool of fetal tissues (most often lung, liver, kidney and/or lymphoid organs) is widely used in diagnostic laboratories although it is a relatively expensive and time-consuming procedure. virus isolation is specific but the sensitivity in diagnostic submissions is estimated to be reduced due to autolysis and other factors. fluorescent antibody staining for bvdv on frozen sections of either lung or liver and kidney is often used as it is a convenient and rapid procedure. however, there are significant false positives and false negative results with this procedure compared to either virus isolation or immunohistochemistry. bvdv immunohistochemistry using monoclonal antibody appears to be a sensitive and specific procedure capable of detecting a variety of virus isolates.33,34 useful tissues for bvdv immunohistochemistry staining include kidney, lung and placenta. in fetuses with vasculitis and necrotizing myocarditis immunohistochemistry may demonstrate the presence of bvdv antigens in the vessel wall and/or muscle. a number of bvdv pcr procedures are available in diagnostic laboratories that have been evaluated and appear highly sensitive in detecting bvdv in a variety of samples.35 after four months gestation the fetus may respond immunologically to bvdv infection so a positive bvdv titer in the fetal fluids is an indication of fetal infection. however, if earlier infection by noncytopathic bvdv induces persistent infection, the fetus may be immunotolerant so a negative fetal serology does not rule out infection. serology on the dam is of limited value in diagnosing bvdv abortion, particularly if animals are vaccinated. an important outcome of fetal infection with noncytopathic bvd virus is the birth of persistently infected calves which are a significant source of infection to the herd. virus isolation, serology, immunohistochemistry, polymerase chain reaction and antigen capture elisa procedures have been used to screen for the presence of persistently infected animals as a means to control this infection. the choice of the optimal screening procedure for a specific herd will depend on a number of factors which have been reviewed.36 skin ear notch biopsy procedures utilizing bvdv immunohistochemistry or antigen capture elisa to identify individual persistently infected animals have been evaluated.32-35 reproductive disease associated with bdv infection in sheep and goats has many similarities to bvdv infection in cattle. the infection is common in sheep, less so in goats. most bdv isolates are noncytopathogenic and the primary effects of the infection are seen in the fetus, influenced by the gestation age similar to bvdv infection in pregnant cattle. after about 90 days gestation the fetus is 564 immunocompetent to bdv infection and prior to this, infection may result in a variety of changes including fetal death, mummification, abortion, abnormal organ development, stillbirth or weak lambs/kids, or the birth of clinically normal, persistently infected offspring which are a major source of infection. a notable syndrome associated with bdv infection is the “hairy shaker syndrome” in which there is curly hair-like wool, frequently abnormally pigmented, with tremors or neurologic deficits in the affected live animal. various central nervous system dysplastic or hypoplastic lesions can be observed but hypomyelination is the most consistent change. an oligodendrocytic dysfunction is thought to be responsible either from direct viral infection of oligodendrocytes or due to a hormonal effect from low thyroxine activity due to viral infection of thyroid follicular cells. confirmation of bdv as the cause of reproductive problems in sheep and goats utilizes similar procedures as with bvdv in cattle.30 cache valley virus. cache valley virus is a bunya virus, endemic in north america and transmitted by arthropods including culicoides spp., gnats and mosquitoes, that is known to cause disease in pregnant sheep. depending on the stage of gestation at which the fetus is infected, fetal death, abortion or fetal abnormalities can occur. the most striking gross lesions associated with this teratogenic virus infection are arthrogryposis, vertebral abnormalities (scoliosis, torticollis) and various central nervous changes including hydranencephaly, porencephaly, hydrocephalus and micromyelia. diagnosis is difficult as the virus is usually eliminated earlier in gestation. a serology test is available which is best utilized on affected pre-colostral lambs. cases of congenital defects such as arthrogryposis/hydranencephaly suspected to be due to a teratogenic virus infection merit attention as similar lesions can be caused by another bunya virus, akabane, which is exotic to north america.37 bluetongue virus. bluetongue virus (btv) infection is a worldwide, arthropod (culicoides spp.) transmitted orbivirus virus infection with a seasonal occurrence. there are multiple btv serotypes which vary considerably in their virulence. the ability of the virus to cross the placenta and cause fetal infection is enhanced in egg or cell culture adapted vaccine strains. reproductive disease associated with btv is seen in sheep and cattle and is influenced by the stage of gestation that infection occurs and the serotype involved. in sheep, infection early in gestation can result in fetal death with resorption, mummification or abortion. between approximately 50 to 80 days gestation, the viral infection can induce cavitating brain lesions resulting in hydranencephaly or porencephaly in fetuses that survive to birth. nonsuppurative meningoencephalitis with cerebral cortical necrosis is present. cattle are frequently infected with btv and can maintain a prolonged viremia but reproductive disease associated with btv has historically been uncommon and sporadic apparently limited to certain cell-adapted strains of btv. however, recently btv serotype 8 as emerged in europe as a significant cause of both clinical disease in cattle and the infection has an enhanced ability to cause fetal infection. reproductive disease in the bovine is similar to that in sheep. in early gestation, less than 70 days, death with absorption or abortion can occur. between 70 and 130 days btv targets neuronal and glial cells progenitor cells in the subepidermal region of the cerebrum resulting in hydranencephaly. slightly later infections but prior to fetal immunocompetence at 150 days, can result in porencephaly and hydrocephalus. later infection may result in encephalitis without brain malformations. the diagnosis of btv infection can be difficult because the virus may longer be present in a term fetus presented with compatible brain lesions. fetal or precolostral antibodies can indicate exposure. btv pcr assays are available in many diagnostic laboratories.38,39 protozoal infections tritrichomonas foetus. trichomoniasis is a venereal disease that is primarily associated with early embryonic loss. however, occasional abortions may occur and submission of these may offer the first opportunity to identify this disease in some infected herds. abortions can occur from two months to late gestation with variable autolysis. in most cases there are no gross lesions other than placental edema. 565 histologic lesions consist of pleocellular placentitis and fetal bronchopneumonia occasionally with multinucleated giant cells in the lung airways.40 in fresh samples, trichomonads may be identified in abomasal fluid samples by darkfield microscopy followed by confirmation by culture of the fluid utilizing appropriate media. the trichomonads may be visualized in the placenta, lung and other tissues in routine hematoxylin-eosin stained sections. histochemical stains such as giemsa and bodian’s silver protargol are useful in identifying trichomonads in tissue sections. immunohistochemistry utilizing polyclonal and monoclonal antibodies is an effective method to detect tritrichomonas foetus in fetal and placental sections.41 neospora caninum. neospora infection is a major cause of abortion and fetal infection worldwide in beef and dairy cattle with only rare incidences in sheep and goats.42-43 this protozoan parasite has a complex life cycle utilizing both horizontal and vertical methods of transmission. in horizontal transmission the carnivore definitive host, identified as dogs and coyotes, can shed infective oocysts in their feces from which the intermediate ruminant host can acquire the infection. the other method of transmission, vertical transmission, is an important means of maintaining this infection. the protozoa can be maintained in the cow as a chronic infection which can be transplacentally transmitted to her fetus during pregnancy. some of these infected pregnancies may abort, but many will produce a congenitally infected calf and a congenitally infected heifer calf is capable of transmitting the infection onto the next generation when she becomes pregnant, thus maintaining the infection in the herd. cows that abort a neospora-infected fetus may have additional abortions or infected fetuses in subsequent pregnancies. cows and heifers that are seropositive are at an increased risk of abortion.44-45 there are no signs of clinical illness in cows that abort and while abortions have been diagnosed in both heifers and cows from three months gestation to term, but the majority of abortions occur in the second trimester of pregnancy, four to six months gestation, which is a distinctive feature of this disease. fetal mummification has been associated with neospora outbreaks. a rare outcome of fetal neospora infection is the birth of a full-term calf with central nervous system signs. however, the majority of calves that acquire a neospora infection during gestation are born clinically normal. these calves have a high precolostral antibody titer to neospora caninum which is useful in diagnosing in utero infection. the aborted fetuses are autolyzed with serosanguinous fluid in body cavities. subtle gross lesions, consisting of pale white foci or streaks in the skeletal muscles or the heart may be rarely observed. there are widespread histologic lesions in many organs, the most diagnostically significant in the brain consisting of scattered foci of cellular infiltrates and/or foci of necrosis. other routinely identified lesions include nonsuppurative epicarditis and/or myocarditis and myositis. liver lesions consist of portal hepatitis with foci of paracentral hepatic necrosis. lung and kidney often have scattered nonsuppurative interstitial infiltrates. placentitis varies in severity but may be the primary cause of fetal death and abortion. immunohistochemistry using antibodies raised against neospora caninum antigens is an effective method to identify the tachyzoite and tissue cyst stages of the parasite in fetal tissues. neospora immunohistochemistry is most successful in sections of brain, lung, kidney, skeletal muscle and placenta. the evidence of neospora infection in an aborted fetus as the cause of the abortion can be questioned because a fetus can be infected and not abort due to the infection. a diagnosis of neospora infection as the cause of abortion should take in consideration the gestational age, autolyzed condition, compatible disseminated inflammatory lesions, detectable parasites and no other identified causes. a fetus with mild lesions, often limited to focal encephalitis in late term fetuses, may likely have an incidental neospora infection so other causes for the abortion should be investigated. neospora pcr techniques have been used to identify fetal neospora caninum infection and are reported to be more sensitive than immunohistochemistry in identification of fetal infection.46 however, establishing neospora infection as the cause for the abortion on the basis of a positive neospora pcr should employ the same criteria for diagnostic significance as for a positive neospora immunohistochemistry test. 566 a variety of serologic tests are available to assist in the diagnosis of neosporosis. these include the indirect fluorescent antibody test, the modified agglutination test, and a number of enzyme-linked immunosorbent assays.47-48 the assays utilize neospora caninum tachyzoites or specific derived antigens. the specificity and sensitivity of the various serologic tests are comparable depending on the minimum antibody titer that has been established as the cut-off for a positive result. laboratories utilizing any of the serologic tests for neospora should establish appropriate cut-off titers using sera from known infected and noninfected cattle. in some tests, the positive cut-off titer has been selected based on the antibody titer in a cow that has aborted an infected fetus so this cut-off may not be the most appropriate for the serologic diagnosis of a chronic infection in cattle which vary in age and pregnancy status. a single serum sample from an individual cow may not accurately reflect her infection status since titers in known positive cattle fluctuate and may fall below the cut-off value for some period of time. in rare instances, cows that abort a neospora-infected fetus may not have a significantly elevated titer. also, previously elevated titers at abortion may decline over several months following abortion. in newborn calves, neospora serology on precolostral serum is an effective method to determine fetal exposure. in aborted fetuses, neospora serology is less useful in diagnosis. an infected fetus may have a negative titer because of the gestational age, duration of infection prior to death, or autolysis. a negative fetal titer does not rule-out infection nor does a positive titer prove that this infection caused the abortion. in the individual aborting cow, a positive serology result does not prove that the abortion was due to neosporosis but it can assist the diagnosis. serology can be used on a herd basis to investigate the association between seropositivity and abortion by comparing results among aborting and non-aborting cattle to estimate the extent that the abortions can be attributed to neospora infection.44 toxoplasma gondii. toxoplasma gondii, a protozoan parasite similar to neospora caninum, is an important cause of reproductive disease in sheep and goats. the parasite has a two-host life cycle with cats as the definitive host which shed infective oocysts following intestinal infection that are capable of infecting a wide variety of mammals and birds which serve as intermediate hosts. infection and/or exposure is not uncommon in small ruminants with little evidence of clinical disease other than during pregnancy. reproductive disease is most commonly seen in younger, previously unexposed animals. in the susceptible pregnant animal, transplacental infection can result in resorption, fetal death, mummification, abortion or stillbirth. the infection can also be congenitally transmitted to the fetus with the birth of healthy lambs that are capable of infecting their offspring although the significance of vertical transmission is still unclear. the various outcomes appear to be dependent on the stage of gestation at which infection occurs. aborted fetuses can vary from autolyzed to fresh, even among the same pregnancy. gross fetal lesions are usually not apparent. placenta lesions, if visible, are diagnostically useful. the cotyledons contain numerous small white foci with a grossly normal intercotyledonary placenta. on histologic examination there are foci of necrosis of the cotyledonary villi often with mineralization and mononuclear inflammation. rarely individual tachyzoites or tissue cysts can be identified in or adjacent these lesions with conventional histology. fetal lesions are variable and may be absent in some cases but the most distinctive lesion is found in the brain consisting of multifocal necrosis and gliosis. scattered mononuclear inflammation may be present in other organs including the lung, heart and liver. diagnosis most commonly is based on the presence of the placental and fetal lesions coupled with demonstration of the organism by specific antibody using an immunohistochemistry stain. toxoplasma dna pcr procedures have been used by some laboratories to detect infection with good results even on autolyzed specimens. serology on the aborting dam may be useful to help rule out toxoplasma infection if negative but a positive result is not diagnostic as exposure is common. fetal serology is occasionally useful in cases when the aborted fetus has sufficient maturity and duration of infection to mount an antibody response.49 sarcocystis. infection with sarcocystis species is very common in the muscular tissues of domestic ruminants but clinical disease or abortion is rare. the protozoal parasite has a two-host life 567 cycle with a sexual intestinal stage in the carnivore definitive host and an asexual tissue stage in the ruminant intermediate host. there are a number of sarcocystis species identified in ruminants but the most important are s. cruzi in cattle, s. tenella in sheep and s. capracanis in goats which all have canid definitive hosts. abortions are rare and sporadic. the fetus is usually autolyzed with no gross lesions. histologic examination reveals disseminated inflammation with some similarities to fetal neosporosis in cattle including encephalitis with necrosis and gliosis, myocarditis, myositis and hepatitis. the protozoal schizonts are associated with the endothelium of most organs. on occasion, a distinctive rosette formation of mature schizonts within endothelial cells can be identified which is useful in distinguishing this parasite from neospora and toxoplasma species.50 summary the laboratory results obtained from the abortion submissions should be interpreted by the veterinarian to determine whether they provide a sufficient answer to the herd abortion problem. on occasion, aborted fetuses may present with incidental infections, have multiple infections, or the fetus submitted may not be representative of the herd problem. when laboratory testing is completed, the clinical situation and laboratory diagnosis can be compared to assess whether the abortion submission is a representative sample and if the identified cause is a significant factor in the herd abortion problem. identification of an infectious cause in abortion submissions is usually successful in less than half of submissions. the failure to find an infectious cause may be a correct interpretation in cases in which there are abnormal genetic, hormonal, metabolic, developmental or other factors responsible which are often difficult or impossible to confirm in diagnostic laboratory submissions. the lack of significant findings in multiple fetuses submitted from a herd can be used as evidence by the veterinarian to investigate other possible noninfectious factors. among the bovine fetuses submitted to veterinary diagnostic laboratories for which no etiologic diagnosis was obtained a significant minority have inflammatory lesions compatible with an infectious etiology, suggesting that there is room for improvement. it is reasonable to presume that with improved detection methods and further investigations the diagnosis rate on abortion submissions can be improved and new causes identified. references 1. kirkbride ca: etiologic agents detected in a 10-year study of bovine abortions and stillbirths. j vet diagn invest 1992;4:175-80. 2. barr ba, anderson ml: infectious disease causing bovine abortion and fetal loss. vet clin north am food anim pract 1993;9:343-368. 3. moeller rb: causes of caprine abortion: diagnostic assessment of 211 cases 1991-1998. j vet diagn invest 2000;13:265-270. 4. kirkbride ca: diagnosis in 1784 ovine abortions and stillbirths. j vet diagn 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abortion. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology, 2nd ed. st. louis: wb saunders; 2007. p. 389-398. 12. kirkbride ca: viral agents and associated lesions detected in a 10-year study of bovine abortions and stillbirths. j vet diagn invest 1992;4:374-379. 13. nicoletti p: brucellosis. in: howard jl , smith ra, editors. current veterinary therapy food animal practice, 4th ed. st. louis: wb saunders; 1999. p. 364-368 14. hinton m: the diagnosis of salmonella abortion in cattle with particular reference to salmonella dublin. a review. j hyg (lond) 1977;79:25-38. 568 15. bolin ca, prescott jf. leptospirosis. in: howard jl , smith ra, editors. current veterinary therapy food animal practice, 4th ed. st. louis: wb saunders; 1999. p. 352-358. 16. ellis wa: leptospirosis as a cause of reproductive failure. vet clin north am food anim pract, 1994;10:463-478. 17. schlafer dh, miller rb: female genital system. in: maxie mg, editor. jubb, kennedy and palmer’s pathology of domestic animals. 5th ed. st. louis: saunders; 2007. vol 3. p. 429-564. 18. miller r, chelmonska-soyta a, smits b, et al: ureaplasma diversum as a cause of reproductive disease in cattle. vet clin north am food anim pract 1994;10:479-490. 19. cardoso mv, blanchard a, ferris s, et al: detection of ureaplasma diversum in cattle using a newly developed pcr based detection assay. vet microbiol 2000;72:241-250. 20. kirkbride ca, cates ce, collins je: abortions in sheep caused by a nonclassified anaerobic flagellated bacterium. am j vet res 1986,47:259-262. 21. campero cm, anderson ml, walker rl, et al: immunohistochemical identification of campylobacter fetus in natural cases of bovine and ovine abortions. j vet med b infect dis vet public health 2005;52:138-141. 22. walker rl: bovine venereal campylobacteriosis. in: howard jl , smith ra, editors. current veterinary therapy food animal practice, 4th ed. st. louis: wb saunders; 1999. p. 323-326. 23. bondurant bh, anderson ml, stott jl, et al: epizootic bovine abortion (foothill abortion). in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology, 2nd ed. st. louis: wb saunders; 2007. p. 413 416. 24. anderson ml, kennedy pc, blanchard mt, et al: histochemical and immunohistochemical evidence of a bacterium associated with lesions of epizootic bovine abortion. j vet diagn invest 2006;18:76-80. 25. moore jd, barr bc, daft bm, et al: pathology and diagnosis of coxiella burnetii infection in a goat herd. vet pathol 1991;28:81-84. 26. dilbeck pm, mcelwain tf: immunohistochemical detection of coxiella burnetii in formalin-fixed placenta. j vet diagn invest 1994;6:125-127. 27. walker rl: mycotic bovine abortion. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology, 2nd ed. st. louis: wb saunders; 2007. p. 417-419. 28. kelling cl: viral diseases of the fetus. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology, 2nd ed. st. louis: wb saunders; 2007. p. 399-408. 29. uzal fa, woods l, stillian m, et al: abortion and ulcerative posthitis associated with caprine herpesvirus-1 infections in goats in california. j vet diagn invest 2004;16:478-484. 30. nettelton pf, gilray ja, russo p, et al: border disease of sheep and goats. vet res 1998;29:327-340. 31. grooms dl: reproductive consequences of infection with bovine viral diarrhea virus. vet clin north am food anim pract 2004;20:5-19. 32. saliki jt, dubovi ej: laboratory diagnosis of bovine viral diarrhea virus infections. vet clin north am food anim pract 2004;20:69-83. 33. fulton rw, hessman b, johnson bj, et al: evaluation of diagnostic tests used for detection of bovine viral diarrhea virus and prevalence of subtypes 1a, 1b, and 2a in persistently infected cattle entering a feedlot. j am vet med assoc 2006;228:578-584. 34. cornish te, van olphen al, cavender jl, et al: comparison of ear notch immunohistochemistry, ear notch antigen capture elisa, and buffy coat virus isolation for detection of calves persistently infected with bovine viral diarrhea virus. j vet diagn invest 2005;17:110-107. 35. kennedy ja, mortimer rg, powers b: reverse transcription-polymerase chain reaction on pooled samples to detect bovine viral diarrhea virus by using fresh ear-notch-sample supernatants. j vet diagn invest 2006;18:89-93. 36. ellis ja, martin k, norman gr, et al: comparison of detection methods for bovine viral diarrhea virus in bovine abortions and neonatal death. j vet diagn invest 1995;7:433-436. 37. edwards jf: cache valley virus. vet clin north am food anim pract 10:515-524. 38. maclachlan nj, drew cp, darpel ke et al: the pathology and pathogenesis of bluetongue. j comp pathol 2009;141:1 16. 39. osburn bi: bluetongue virus. vet clin north am food anim pract 1994;10:547-560. 40. rhyan jc, stackhouse ll, quinn wj: fetal and placental lesions in bovine abortion due to tritrichomonas foetus. vet pathol 1988;25:350-355. 41. rhyan jc, wilson kl, burgess de, et al: immunohistochemical detection of tritrichomonas foetus in formalin-fixed, paraffin-embedded sections of bovine placenta and fetal lung. j vet diagn invest 1995;7:98-101. 42. anderson ml, andrianarivo ag, conrad pa: neosporosis in cattle. anim reprod sci 2000;60-61:417-431. 43. dubey jp, schares g: diagnosis of bovine neosporosis (review). vet parasitol 2006;140:1-34. 44. thurmond mc, hietala sk: neospora caninum infection and abortion in cattle. in: howard jl, smith ra, editors. current veterinary therapy: food animal practice; 1999. st. louis: w.b saunders. p. 425-431. 45. gay jm: neosporosis in dairy cattle. an update from an epidemiological perspective. theriogenology 2006;66:629 632. 46. baszler tv, gay ljc, long mt, et al: detection by pcr of neospora caninum in fetal tissues from spontaneous bovine abortions. j clin microbiol 1999;37:4059-4064. 569 47. pare’ j, hietala sk, thurmond mc: an enzyme-linked immunosorbent assay (elisa) for serological diagnosis of neospora infection in cattle. j vet diagn invest 1995;7:352-359. 48. bjorkman c, uggla a: serological diagnosis of neospora caninum infection. int j parasitol 1999;29:1497-1507. 49. dubey jp: toxoplasmosis in sheep-the last 20 years. vet parasitol 2009;163:1-14. 50. anderson ml, barr bc, conrad pa: protozoal causes of reproductive failure in domestic ruminants. vet clin north am food anim pract 1994;10:439-461. 570 1 contact ramanathan kasimanickam ramkasi@wsu.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10528, http://dx.doi.org/10.58292/ct.v16.10528 research report ovum pick up, in vitro embryo production, and embryo transfer as value-added theriogenology service in miniature hereford cattle operation randa boler, ramanathan kasimanickam college of veterinary medicine, washington state university, pullman, wa, usa abstract goal was to provide evidence for adopting assisted reproductive technologies (art), ovum pick up (opu), in vitro embryo production (ivp), and embryo transfer (et) in small beef cattle operations as value-added service. day 7 embryos (n = 14) were produced via ivp utilizing oocytes (n = 87) collected by opu from miniature hereford donor cows (n = 3; mean age: 7 years) and fertilized with semen from miniature hereford bulls. irish black (n = 8; 14 months) and hereford (n = 1; 20 months) recipient heifers were synchronized using 5-day select-synch + controlled internal drug release (cidr) protocol. on day 9 after cidr removal, heifers (n = 7) with a corpus luteum (> 2 cm in size) received an embryo in the ipsilateral uterine horn. pregnancy/et was 57.1% (4/7). economic advantage (increase in number of offspring) was realized comparing opu-ivp and artificial insemination procedures. these art technologies (opu, ivp, and et) can be utilized in genetically superior cows as value-added theriogenology service to increase their offspring. keywords: miniature hereford, synchronization, ovum pick-up, in vitro, embryo transfer introduction transvaginal ultrasound-guided follicular aspiration and oocyte retrieval are performed for ovum pick up (opu) and in vitro embryo production (ivp). ovum pick up technique was first described in humans in 1940s1 and was performed in cattle by the 1980s.1 collected oocytes are matured, fertilized, and cultured in vitro up to transferable embryo stage. follicle stimulating hormone (fsh) is commonly used to improve oocyte quality,2 by synchronizing follicular wave emergence and maximizing the number of competent oocytes. follicular stimulation and oocyte collection methods vary based on cattle breed, collection frequency, and veterinarian preference. there are several methods primarily used (e.g. a slow-release porcine-derived fsh formulation given as a single injection or traditional twice-daily fsh treatment regimen given over 4 days3). however, opu can also be performed at various stages of the estrous cycle without hormone use.4 use of opu-ivp technology has increased greatly in the last decade due to improved methods and ability to implement the procedure in the field. in recent years, over 1 x 106 embryos5 were produced per year via opu and ivp. number of ivp embryos became equal to or greater than those produced in vivo in 2016.5-7 globally, the total number of embryos produced has increased each year in the last decade.5-7 lately, more ivp embryos are produced compared to in vivo embryos as there are more preference and demand for ivp embryos in the field.5-7 in usa alone, 133,000 ivp and 301,000 in vivo embryos were produced in 2020.8 a huge advantage of opu-ivp is that numerous embryos can be produced from animals of various reproductive statuses, including pregnant cows up to 100 days in pregnancy, postpartum cows, and prepubertal heifers,9 making this technology a viable option in smaller cattle operations. several calves can be produced frequently from valuable animals, giving an economic advantage to the farm. our objective was to demonstrate the application of opu-ivf in the field as value added theriogenology service. materials and methods since data collected from routine assisted reproduction procedures from a private farm for commercial purposes were used, mailto:ramkasi@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10528 2 citation: clinical theriogenology 2024, 16, 10528, http://dx.doi.org/10.58292/ct.v16.10528 institutional animal care and use committee approval is not required, and the study is exempt. donor animals and opu-ivf method three donor animals, from a small, registered miniature hereford cattle herd (herd size; n = 40), were selected based on phenotype, genetic value, and a history of successfully delivering offspring. selected donors were multiparous miniature hereford cows (4, 7, and 10 years of age; 2 polled and 1 horned). each animal was subjected to an opu procedure after receiving 120 mg of intramuscular fsh [3.5 ml; 350 mg nih-fsh-p1 diluted in 10 ml of slow-release formula (20 mg/ ml hyaluronan); vetoquinol, tx, usa] 3 days prior to the day of follicle aspiration.10,11 before the procedure, epidural anesthesia was induced by injecting 5 ml of 2% lidocaine (vet one, boise, id, usa) into the sacrococcygeal epidural space. follicles > 2 mm were identified via transvaginal ultrasonography (sonoscape s8, universal imaging inc., bedford hills, ny, usa) with convex transducer probe holder (minitube usa inc., verona, wi, usa) after ovary was aligned against the transducer via transrectal manipulation. a long-beveled needle (18 g x 3” for bovine opu, minitube usa inc.) was used to puncture the vaginal wall at the anterior vaginal fornix and outer layers of the ovarian follicle. cumulus oophorus complexes were aspirated by an aspiration pump (cook® vacuum pump, cook medical llc, bloomington, in, usa) for opu after creating an optimal negative pressure (60 ± 5 mm hg), one end of a tube was connected to an aspiration needle and the other end to the vacuum pump, to facilitate collection of oocytes into a 50 ml collection tube containing transport medium (bo-hepes-ivm, agtech inc., manhattan, ks, usa). collected oocytes were graded and viable oocytes were transported to a private commercial ivf laboratory in a portable incubator. harvested oocytes were washed, graded, and allowed to mature in the maturation medium (bo-ivm, agtech inc.). sperm from conventional semen or reverse sexsorted semen from a miniature herford bull were used for fertilizing oocytes (day 0); oocytes were vortexed to remove their cumulus layers. conventional semen was used for fertilization of oocytes from 2 polled animals, whereas reverse x-sorted semen (x-rss) was used for fertilization of oocytes from the horned animal. after in vitro culture and daily assessment for embryo development, embryos were staged and graded12 on day 7. grades 1 and 2 embryos at developmental stages 5, 6, and 7 were selected and cryopreserved in ethylene glycol (eg) for direct transfer. briefly, embryos were placed into a controlled rate freezer at a temperature of –6°c. ice crystal formation was induced in eg surrounding embryo. crystallization increased eg concentration outside the embryo resulting in further cellular dehydration. embryos were cooled at a rate of 0.5°c/minute, enabling further dehydration, to a temperature of –34°c before plunging into liquid nitrogen (–196°c). recipient animals and embryo transfer procedure nine 14-month virgin irish black heifers and 1 20-month hereford heifer were enrolled as embryo recipients. heifers received primary and booster vaccinations against reproductive and respiratory pathogens (bovine rhinotracheitis, bovine virus diarrhea virus types 1 and 2, infectious bovine  rhinotracheitis virus, parainfluenza 3 virus, bovine respiratory syncytial virus, leptospira canicola, l. grippotyphosa, l. hardjo, l. icterohaemorrhagiae, and l. pomona [bovishield gold® fp® 5 vl5, zoetis]), 13 and 9 weeks prior to embryo transfer, respectively. they were concurrently vaccinated against clostridial species (chauvoei, septicum, haemolyticum, novyi, sordellii, perfringens type c and d, and tetani [ultrabac®  8, zoetis]). crystal-phos® 8 mineral (cyrstalyx, mankato, mn, usa) was available freely and heifers were maintained on pasture. reproductive tract score (rts),13 body condition score (bcs),14 pelvic area,15,16 and temperament score17 were assigned to recipients as part of farm heifer selection protocol. one irish black heifer was excluded due to advanced pregnancy at rts. estrus was synchronized in recipient heifers using the 5-day controlled internal drug release (cidr) select-synch18 protocol (figure). briefly, heifers received a cidr insert (eazibreed cidr®, 1.38 g of progesterone, intravaginal; zoetis animal health, parsippany, ny, usa) and 100 µg gonadorelin (intramuscular gnrh, factrel®, 2 ml; zoetis animal health) on day 0. on day 5, the cidr device was removed, and 500 µg cloprostenol (intramuscular pgf2α, estrumate®; 2 ml, merck animal health) was given. eight hours after cidr removal and initial pgf2α treatment, a second dose of pgf2α was given. it was predicted that heifers would exhibit estrous behavior near day 7 and ovulate ~ 30 hours after estrus onset.19 nine days after cidr removal, heifers were examined via transrectal palpation and transrectal ultrasonography (sonoscape s8) for corpus luteum. seven of 9 heifers had an active corpus luteum, deemed as functional based on a size > 2 cm. heifers were given 2% lidocaine (4 6 ml; vet one) epidurally between caudal vertebrae 1 and 2. each embryo was identified from the straw label with dam and sire information, stage, grade, and collection date. embryos selected were thawed in a water bath at 36oc for 30 seconds and were transferred as far cranial as possible into the horn ipsilateral to the identified corpus luteum. recipients age, embryo stage, and grade are listed (table 1). after embryo transfer, heifers were allowed back onto pasture with no further manipulation until pregnancy determination. pregnancy was determined by measuring pregnancy-specific protein b20 (biopryn®, biotracking, moscow, id, usa) 3 weeks after embryo transfer. pregnancy was confirmed again at 5 months of pregnancy via transrectal ultrasonography. economic analysis economic advantage was determined using a crude economic analysis with following assumptions. opu-ivp procedure 1. one opu procedure + drug @ $225 2. four transferable embryos 3. freezing + embryo transfer cost @ $170/embryo 4. four recipients (15 months) purchased @ $1,200/ recipient 5. et success at 50%, 4 embryo transfer result in two calves 6. newborn embryo calf market price @ 5,100/calf figure. select-synch + cidr protocol. http://dx.doi.org/10.58292/ct.v16.10528 citation: clinical theriogenology 2024, 16, 10528, http://dx.doi.org/10.58292/ct.v16.10528 3 7. $800 to cover expenses for a donor cow from 15 months 2 years (breeding to calving). 8. sale of 1 open cow at $1,200 artificial insemination procedure 1. $1,800 to cover expenses for 1 heifer from calf to 2 years (birth to calving) 2. estrus synchronization and ai success at 50%, 2 ais result in 1 calf 3. newborn ai calf market price @ 3,000/calf 4. sale of 1 open cow at $1,200 recipients purchase cost ($1,200), expenses from calf to 2 years ($1,800), and from breeding (15 months) to 2 years ($800) were determined considering feed cost, pasture cost, yardage, vaccine/drugs; veterinary expense, death loss, trucking, and interest. results from 3 donor animals, 87 cocs were harvested via opu for oocytes, and 14 transferrable embryos were produced from the ivp procedure (table 2). forty-seven oocytes (fertilized with conventional semen) from 2 polled donor cows resulted in 11 transferrable embryos, whereas 40 oocytes (fertilized with x-rss) from the horned donor cow resulted in 3 transferrable embryos. there was a trend toward a significance (fisher’s exact; p = 0.07) in blastocyst rate between conventional semen versus x-rss (table 3). four of the 7 recipient heifers were pregnant (57.1%). confirmation via transrectal ultrasonography was performed at 5 months of pregnancy (pregnant heifers had fetuses and placentomes of appropriate size). an association between bcs and rts to conception was observed. heifers with moderate to good body condition were more likely to have a cl at embryo transfer. heifers with an rts of 5 had higher heifer pregnancy rates/embryo transfer than their counterparts. economic analysis based on the assumption for opu-ivf and artificial insemination procedures is provided (table 4a and 4b). approximate economic advantage was estimated based on the costs of average embryo and calf value reported by the farm. economic advantage (revenue opu-ivf – revenue ai) = $ 3,469.00 – $ 432.00 = $ 3,037.00 table 1. recipients age, semen type used for in vitro fertilization (ivf), embryo stage, and grade heifer id age (months) semen type used for ivf embryo stage embryo grade 1 14 cs 5 1 2 14 cs 6 1 3 14 cs 7 2 5 14 cs 7 1 6 14 cs 6 1 8 14 cs 5 1 11 20 x-rss 6 2 cs: conventional semen; x-rss: reverse x-sorted sexed semen table 2. number of oocytes harvested and total and transferable embryos from 3 donor cows heifers oocytes recovered embryos blastocyst rate transferable numbers (stage, grade) 1 24 4 2 (6, 1) and 2 (5, 1) 16.7% (4/24) 2 23 7 2 (7, 1), 1 (7, 2), and 4 (6, 1) 30.4% (7/23) 3 40 3 1 (6, 2) and 1 (5, 2) 7.5% (3/40) table 3. embryo development from conventional semen and reverse x-sorted sexed semen semen type oocytes transferable embryos blastocyst rate* cs 47 11 23.4% (11/47) x-rss 40 3 7.5% (3/40) cs: conventional semen; rss: reverse x-sorted sexed semen *cs versus rss (p = 0.07) http://dx.doi.org/10.58292/ct.v16.10528 4 citation: clinical theriogenology 2024, 16, 10528, http://dx.doi.org/10.58292/ct.v16.10528 discussion advanced reproductive technologies can accelerate genetic gain by increasing the number of offspring within a shorter period from genetically superior dams.21 in this report, donor recipients were selected for desired phenotype and genetic importance within the herd, and opu-ivf technologies were utilized to maximize their reproductive efficiency. pharmaceutical manipulation of estrus or ovulation, ai application, and use of gender-selected semen are well-established methods for improving genetic potential in beef cattle herds;22 however, these technologies can only produce 1 calf a year per cow using a conventional approach. success of opu-ivf technologies, as noted in this report, provide an additional avenue for producers to potentiate the genetics of their most valuable animals. cows, similar to those included in this report, are limited to produce ~ 7 high value, show calves with ai technology during their lifetime; however, implementation of opu-ivp technologies significantly increased the number of offspring21 and consequently herd profit. three of the pregnancies in this study were from a single oocyte collection procedure from a cow with great genetic importance. this offered a monetary advantage to the operation by a three-fold increase in calves produced from this particular animal in a year. the 4th embryo pregnancy was achieved from an older cow that was retired from breeding due to predicted poor ability to produce good quality day 7 embryos or to carry pregnancy; however, use of opu-ivf technologies effectively lengthened cow’s reproductive life. to estimate the monetary benefit, we performed a crude economic analysis comparing opu-ivp and ai procedures. the opu-ivf procedures resulted in ~ $3,000 more profit than the ai procedure. readers should exert caution while interpreting economic analysis since economic benefit would vary due to year-to-year differences in costs and calf market value. in general, the breakeven cost for raising a replacement heifer can be met after production of 1.5 2 offspring by that heifer. therefore, the profit can be realized when a 3-year cow has a successful weaned second calf. it should be noted that the profit depends on the sale price of a calf; several factors including age of calf at sale, season of sale, genetic composition of calf, gender and sale options such as sale for slaughter, sale as table 4a. economic analysis for profit estimation generated after implementing opu-ivp procedures (expenses and revenues considered, based on the assumptions listed*) items opu-ivf total expenses donor opu procedure + drug cost freezing + embryo transfer $170 × 4 shipping labor ($15/hour × 1 hour) syringe and needle 225.00 680.00 40.00 15.00 2.50 recipient  purchase cost ($1,200/head × 4) expenses (15 month 2 year) $800 × 4 drug cost  cidr ($14.50 × 4) gnrh ($2.50 × 4) pgf2α × 2 ($5.50 × 4)  lidocaine (0.50 × 4) blood test (biopryn) ($5 × 4) ultrasonography ($6 × 4) labor ($15/hour × 2 hours) syringe and needle 4,800.00 3,200.00 58.00 10.00 22.00 2.00 20.00 24.00 30.00 2.50 total expenses 9,131.00 income calf sale value ($5,100 × 2) open cow sale ($1,200 × 2) 10,200.00 2,400.00 total income 12,600.00 profit 3,469.00 *refer materials and methods for the list of assumptions http://dx.doi.org/10.58292/ct.v16.10528 citation: clinical theriogenology 2024, 16, 10528, http://dx.doi.org/10.58292/ct.v16.10528 5 breeding bull or replacement heifer determines the sale price. the biggest loss to an operation is culling a 3-year open cow due to its reproductive failure. in this report, 3 calves were produced from 1 opu-ivf procedure from 1 animal, the breakeven cost was achieved much earlier. assessment of body condition score and reproductive status of recipients by evaluating the reproductive tract score can be valuable in recipient selection. in this report, moderate to good body condition and reproductively mature heifers were highly reflective of conception success. in general, a lower blastocyst rate with sex-sorted semen (2 x 106 sex-sorted sperm/straw) compared to cs has been reported.23 however, the pregnancy and efficiency were comparable between cs and sex-sorted sperm (sexedultra™ with 4 million sex-sorted sperm/straw).23 in the current study, reduced fertilization and embryo production were realized (p < 0.05) for rss compared to cs. although oocytes recovered were graded and selected for further procedures, variation in donor animals plausibly contributed to this difference in the outcome.24 reduced blastocyst rate for rss compared to cs, in 3 different bulls, 15 versus 35%, 34 versus 50%, and 34 versus 41%, respectively, whereas no differences were observed between rss and cs for pregnancy rate.25 decreased motility, viability, and speed of sperm were reported26 when rss were subjected to an additional freezing procedure. the potential of refreezing rss without losing sperm fertilization capability might overcome this limitation. conclusion reproductive technologies (opu-ivf) can be successfully utilized as a value-added theriogenology service to produce genetically superior offspring in smaller beef operations. use of advanced reproductive technologies in genetically superior cows can increase the herd value and profit via the production of an increased number of valuable offspring. conflict of interest none to report. references 1. pincus g, saunders b: the comparative behaviour of mammalian eggs in vivo and in vitro. vi. the maturation of human ovarian ova. anat rec 1939;75:537. doi: 10.1002/ar.1090750410 2. pieterse mc, kappen ka, kruip ta, et al: aspiration of bovine oocytes during transvaginal ultrasound scanning of the ovaries. theriogenology. 1988;30:751-762. doi: 10.1016/0093-691x (88)90310-x 3. ongaratto fl, cedeño av, rodriguez-villamil p, et al: effect of fsh treatment on cumulus oocyte complex recovery by ovum pick up and in vitro embryo production in beef donor cows. anim reprod sci 2020;214:106274. doi: 10.1016/j.anireprosci.2020.106274 4. bó ga, mapletoft rj: historical perspectives and recent research on superovulation in cattle. theriogenology 2014;81:38-48. doi: 10.1016/j.theriogenology.2013.09.020 5. egashira j, ihara y, khatun h, et al: efficient in vitro embryo production using in vivo-matured oocytes from superstimulated japanese black cows. j reprod dev 2019;65:183-190. doi: 10.1262/jrd.2018-155 6. ferré lb, kjelland me, strøbech lb, et al: review: recent advances in bovine in vitro embryo production: reproductive biotechnology table 4b. economic analysis for the estimation of profit generated using artificial insemination (expenses and revenues considered based on the assumptions listed*) items ai total expenses expenses (from birth to 2 years) × 2 drug cost cidr × 2 ($14.50 × 1) gnrh × 2 ($2.50 × 2) pgf2α × 2 ($5.50 × 2)  semen ($20/straw) × 2 ai fee ($7/head) × 2 syringe/needle labor ($15/hour × 2 hours) ultrasound ($6/head) × 2 3,600.00 29.00 10.00 11.00 40.00 14.00 10.00 30.00 24.00 total expenses 3,768.00 income calf sale value ($3,000 × 1) open cow sale ($1,200 × 1) 3,000.00 1,200.00 total income 4,200.00 profit 432.00 *refer materials and methods for the list of assumptions http://dx.doi.org/10.58292/ct.v16.10528 https://doi.org/10.1002/ar.1090750410 https://doi.org/10.1016/0093-691x​(88)90310-x https://doi.org/10.1016/0093-691x​(88)90310-x https://doi.org/10.1016/j.anireprosci.2020.106274 https://doi.org/10.1016/j.theriogenology.2013.09.020 https://doi.org/10.1262/jrd.2018-155 6 citation: clinical theriogenology 2024, 16, 10528, http://dx.doi.org/10.58292/ct.v16.10528 history and methods. animal;14:991-1004. doi: 10.1017/s1751731 119002775 7. viana jhm, figueiredo acs, siqueira lgb: brazilian embryo industry in context: pitfalls, lessons, and expectations for the future. anim reprod 2017;14:476-481. doi: 10.21451/1984-3143-ar989 8. viana jhm., siqueira lgb, palhao mp et al: features and perspectives of the brazilian in vitro embryo industry. anim reprod 2012;9:12-18. 9. demetrio d, demetrio c, oliveira m, et al: from oocyte to calf: practical aspects of in vitro embryo production. clinical theriogenology 2022;14:193-201. doi: 10.58292/ct.v14.9671 10. baruselli ps, batista eos, vieira lm, et al: factors that interfere with oocyte quality for in vitro production of cattle embryos: effects of different developmental & reproductive stages. anim reprod 2016;13:264-272. doi: 10.21451/1984-3143-ar861 11. bó ga, rogan dr, mapletoft rj: pursuit of a method for single administration of pfsh for superstimulation in cattle: what we have learned. theriogenology 2018;112:26-33. doi: 10.1016/j. theriogenology.2017.09.034 12. international embryo transfer society manual, 4th edition. available from: https://www.iets.org/.publications/iets-manual [cited 6 june 2023]. 13. gutierrez k, kasimanickam r, tibary a, et al: effect of reproductive tract scoring on reproductive efficiency in beef heifers bred by timed insemination and natural service versus only natural service. theriogenology 2014;81:918-924. doi: 10.1016/j.theriogenology. 2014.01.008 14. bellows ra, short re, richardson gv: effects of sire, age of dam and gestation feed level on dystocia and postpartum reproduction. j anim sci 1982;55:18-27. doi: 10.2527/jas1982.55118x 15. van donkersgoed j, ribble cs, booker cw, et al: the predictive value of pelvimetry in beef cattle. can j vet res 1993;57:170-175. 16. thompson kw: pelvic area as a tool for selection of beef replacement heifers. master’s thesis, university of tennessee, knoxville, tn, usa 1996. available from: https://trace.tennessee.edu/utk_ gradthes/6808 [cited 7 june 1983]. 17. kasimanickam r, schroeder s, assay m, et al: influence of temperament score and handling facility on stress, reproductive hormone concentrations, and fixed time ai pregnancy rates in beef heifers. reprod domest anim 2014;49:775-782. doi: 10.1111/rda.12368 18. leitman nr, busch dc, bader jf, et al: comparison of protocols to synchronize estrus and ovulation in estrous-cycling and prepubertal beef heifers. j anim sci 2008;86:1808-18. doi: 10.2527/jas.2008-0970 19. white fj, wettemann rp, looper ml, et al: seasonal effects on estrous behavior and time of ovulation in nonlactating beef cows. j anim sci 2002;80:3053-9. doi: 10.2527/2002.80123053x 20. sasser rg, branen j, howard j, et al: biopryn®, a measure of pregnancy-specific protein b for detection of pregnancy in ruminant animals. proc am assoc bovine pract 2009:42:38-47. doi: 10.21423/aabppro20094248 21. crowe ad, lonergan p, butler st: invited review: use of assisted reproduction techniques to accelerate genetic gain and increase value of beef production in dairy herds. j dairy sci 2021;104:12189121206. doi: 10.3168/jds.2021-20281 22. patterson dj, thomas jm, bishop jm, et al: control of estrus and ovulation in heifers. proceedings of applied reproductive strategies in beef cattle stillwater, ok. 2014, p. 75-109. 23. reese s, pirez mc, steele h, et al: the reproductive success of bovine sperm after sex-sorting: a meta-analysis. sci rep. 2021;11:17366. doi: 10.1038/s41598-021-96834-2 24. bó ga, cedeño a, mapletoft rj: strategies to increment in vivo and in vitro embryo production and transfer in cattle. anim reprod 2019;16:411-422. doi: 10.21451/1984-3143-ar2019-0042 25. morotti f, sanches bv, pontes jh, et al: pregnancy rate and birth rate of calves from a large-scale ivf program using reverse-sorted semen in bos indicus, bos indicus-taurus, and bos taurus cattle. theriogenology 2014;81:696-701. doi: 10.1016/j.theriogenology. 2013.12.002 26. underwood sl, bathgate r, pereira dc, et al: embryo production after in vitro fertilization with frozen-thawed, sex-sorted, re-frozen-thawed bull sperm. theriogenology 2010;73:97-102. doi:10.1016/j.theriogenology.2009.08.005 http://dx.doi.org/10.58292/ct.v16.10528 https://doi.org/10.1017/s1751731119002775 https://doi.org/10.1017/s1751731119002775 https://doi.org/10.21451/1984-3143-ar989 https://doi.org/10.58292/ct.v14.9671 https://doi.org/10.21451/1984-3143-ar861 https://doi.org/10.1016/j.theriogenology.2017.09.034 https://doi.org/10.1016/j.theriogenology.2017.09.034 https://www.iets.org/.publications/iets-manual https://doi.org/10.1016/j.theriogenology.2014.01.008 https://doi.org/10.1016/j.theriogenology.2014.01.008 https://doi.org/10.2527/jas1982.55118x https://trace.tennessee.edu/utk_gradthes/6808 https://trace.tennessee.edu/utk_gradthes/6808 https://doi.org/10.1111/rda.12368 https://doi.org/10.2527/jas.2008-0970 https://doi.org/10.2527/2002.80123053x https://doi.org/10.21423/aabppro20094248 https://doi.org/10.3168/jds.2021-20281 https://doi.org/10.1038/s41598-021-96834-2 https://doi.org/10.21451/1984-3143-ar2019-0042 https://doi.org/10.1016/j.theriogenology.2013.12.002 https://doi.org/10.1016/j.theriogenology.2013.12.002 2014: the effect of cyfluthrin, a commercially available synthetic pyrethroid, on bovine semen quality and pregnancy rates the effect of cyfluthrin, a commercially available synthetic pyrethroid, on bovine semen quality and pregnancy rates h.m. french,a* c.f. shipley,a f.a. ireland,b v.l. jarrell,b a.j. fuselier,a,† d.g. williams,a,‡ d.w. shikeb acollege of veterinary medicine, university of illinois, urbana, il; bdepartment of animal sciences, university of illinois, urbana, il abstract reports of research in laboratory animals and clinical case studies in livestock suggest pyrethroids may reduce fertility. the objective of the experiments reported here was to assess the effects of cyfluthrin on cattle reproduction. in experiment 1 angus x simmental bulls were allocated to the following treatments: control with no pesticide applied (n=5), cyfluthrin pour-on (n=5), cyfluthrin fly tag (n=7), and cyfluthrin pour-on plus fly tag (n=7). semen was collected weekly for nine weeks and analyzed for overall sperm motility, progressive sperm motility, and sperm morphology with the aid of computer-assisted semen analysis. blood samples were taken weekly at the time of semen collection and properly stored until testosterone concentrations could be assayed. there were no differences in overall motility (p=0.41), progressive motility (p=0.60) or morphology (p=0.41) among treatments. mean testosterone concentrations did not differ (p=0.16) between control and treated bulls. in experiment 2 angus and crossbred cows were synchronized for an artificial insemination (ai) program and randomly assigned to a control (n=61) or treated group (n=62). the treated group received both pour-on and fly tags at label doses. insecticide was applied to the treated group at the time of controlled internal drug release (cidr) insertion. blood samples for progesterone analysis were collected on days 10 and 17 following timed ai. pregnancy status was assessed 35 days after ai. the treated group had lower (p=0.02) progesterone concentrations on day 10 but not on day 17 (p=0.57). no differences (p=0.65) were observed in pregnancy rates between treatments. reproductive parameters measured in bulls were not affected by cyfluthrin pour-ons or fly tags, even when used in combination. transient effects were observed in the concentration of progesterone in the plasma of cows, but did not result in differences in pregnancy rates observed at 35 days of gestation. keywords: cyfluthrin, pyrethroid, insecticide, reproduction, cattle introduction pyrethroids are included in over 3500 registered insecticide products,1 many of which are commonly used on or around cattle. products such as ear tags, pour-ons and dusts combat pests such as flies, lice and ticks. pyrethroids prolong the opening of sodium channels when a nerve is excited, thereby paralyzing the insect. the use of pyrethroids has increased during the past decade as the more toxic organophosphate pesticides have been replaced.2 although pyrethroids are less toxic than organophosphates to mammals and birds, detrimental effects have been associated with pyrethroid containing products. pyrethroids have been implicated as reproductive toxicants in humans and several species of animals. the effects of pyrethroids on reproductive or endocrine function have been investigated in several studies using a variety of animals as well as humans. results from these studies have shown or suggested that these chemicals or their metabolites may disrupt endocrine function3-11 and affect semen quality.4,12-18 several studies in humans showed associations between pyrethroid metabolites and reduced sperm concentration, motility, and normal morphology.19-21 contamination of the environment with pyrethroids may also affect aquatic animal reproduction.22,23 one of the purported mechanisms for disruption of endocrine function by pesticides is through mimicking or blocking the actions of the steroid hormones via binding to hormone receptors.10 these *current address: ross university school of veterinary medicine, basseterre, st. kitts, west indies †current address: school of veterinary medicine, louisiana state university, baton rouge, la ‡ current address: vet ent, leeston 7632, nz clinical theriogenology • volume 6, number 1 • march 201433 33 receptors play critical roles in the differentiation, development and maintenance of male reproductive functions. liu et al demonstrated that permethrin and cypermethrin have anti-androgenic activity by interfering with the androgen receptor.18 a significant decrease in numbers of androgen receptors and in sperm production was found in rats treated with high levels of beta-cypermethrin; however, no changes were seen in the reproductive organs.18 in vitro studies have been conducted using rat and cattle ovarian tissues and cells to assess effects of pyrethrins on reproduction.11,24 in one study, treating luteal cells in culture with cypermethrin altered the production of progesterone and was toxic within 24 hours.11 several pyrethroids have also been reported to cause a decrease in testosterone production in male laboratory animals.4,13 anecdotal evidence incriminating pyrethroids as a cause for decreased fertility in bulls and rams has been reported.25 this report suggested that premise sprays and insecticide pour-ons may alter sperm motility and cause abnormal sperm morphology; however no controlled experiments were conducted. the implication that a commonly used insecticide may decrease productivity in animal agriculture is of high economic importance and interest to food animal industries. the goal of this study was to determine if the commercially available synthetic pyrethroid, cyfluthrin, used in single or combined applications would negatively affect reproductive parameters in cattle. the specific objectives were to determine the effects of cyfluthrin, a commercially available pyrethroid product used according to label instructions on 1) sperm motility and morphology and serum testosterone concentrations in bulls and 2) serum progesterone concentrations and pregnancy rates in cows. materials and methods the products used in this study were a synthetic pyrethroid pour-on (cyfluthrin; cylence®, bayer animal health, shawnee mission, ks) and fly tag (beta-cyfluthrin; cylence ultra® insecticide cattle ear tag, bayer animal health). all products were applied according to label directions and approved for use in food-producing animals. the fly tags also contain a synergist, piperonyl butoxide, which acts to enhance the pyrethroid properties but has no pesticidal effects. experiment one angus x simmental bulls (n=28) ranging from one to six years of age (bw=773.00±185.51 kg) were blocked by age and randomly assigned to one of four treatment groups. all bulls were owned by the university of illinois and had no opportunity for sexual activity for at least one month prior to the experiment. treatment groups consisted of: controls (con, n=5) that received no pesticides; pour-on (pour, n=5) that received 24 ml of cyfluthrin liquid applied along the topline; ear-tag (tag, n=7) that received an ear tag impregnated with beta-cyfluthrin in each ear; and a pour-on/ear-tag (pour + tag, n= 7) that received both treatments applied at the same time. all animals were housed in non-adjacent pastures with their cohorts to prevent cross-contamination of products from animal to animal. experiments were conducted from may through july. all bulls were maintained on endophyte infected tall fescue (festuca arundinacea), red clover (trifolium pratense), and white clover (trifolium repens) pastures. an initial physical examination including weight and body condition scoring was performed on each bull to establish health status. a reproductive examination was also performed to assess reproductive soundness prior to the experiment. scrotal circumference was measured using scrotal tape. tone and symmetry of each testicle and epididymis was assessed. accessory sex glands were assessed through transrectal palpation. an electroejaculator was inserted into the rectum and a pre-programmed cycle was used to obtain a semen sample. three or more ml of semen were collected from each bull. the penis was examined for abnormalities during the semen collection. semen was collected by electroejaculation once each week for the following nine weeks within the same 4.5 hours of the day to negate diurnal effects on hormonal fluctuations. semen was collected into 15 ml conical tubes using collecting handles and sleeves. immediately after collection, the tube containing semen was transported in a warm (37°c) water bath to the laboratory. semen was diluted 1:60 in saline (37°c) and 20 μl of clinical theriogenology • volume 6, number 1 • march 2014 34 34 diluted semen was placed into a 20 μl chamber slide (vitrolife, microcell counting chambers, san diego, ca). computer assisted sperm analysis (casa) (spermvision®, minitube of america, inc., verona, wi) was used to measure overall and progressive motility by averaging seven readings from various portions of the chambered slide. morphological abnormalities were assessed by examining high power images (100x with phase contrast lens) of multiple sections of the chambered slide. images were used to classify morphology as normal or abnormal. abnormalities were further defined as primary or secondary by the same observer for all samples on all collection days. following assessment of 100 sperm from each diluted semen sample the percentages of normal, primary, and secondary abnormalities were tabulated. blood was obtained for testosterone analysis prior to each weekly electroejaculation. samples were obtained from the tail vein through a one inch 18 gauge needle into a serum-separating evacuated tube. serum was removed after centrifugation 1200 rpm for 15 minutes with a sterile pipette and frozen at -20°c for later analysis. testosterone concentrations were determined by double-antibody radioimmunoassay (coat-a-count®; diagnostics products corporation, los angeles, ca) at the animal health diagnostic center, cornell university, ithaca, ny. statistical analysis the experiment was conducted in a completely randomized design. data were analyzed via the mixed procedure of sas (sas institute; cary, nc). repeated measures were used to analyze overall motility, progressive motility, normal morphology, and testosterone concentration using the covariate structure for compound symmetry. individual bull was the experimental unit. significance was declared at p�0.05. experiment two mature, reproductively sound angus and crossbred cows (n=123) were blocked by breeding date (april and july) and by breed, and randomly assigned to a control (con; n=61) or pyrethroid group (pour+tag; n=62). all cows were owned by the university of illinois, had a previous history of calving, and were in good body condition (4-6/9 bcs). the pour + tag group received both pour-on and fly tag cyfluthrin products at label doses. insecticide was applied to the pour+tag group at the time of cidr insertion. cows were synchronized with a seven day co-synch+cidr program.26 briefly, the co-synch+cidr program is comprised of a 100 mcg dose of gonadotropin releasing hormone (gnrh; cystorelin®, merial, duluth, ga) and a cidr (eazi-breedtm cidr®, zoetis, florham park, nj) insertion on day 0, followed by a seven day waiting period at which time the cidr is removed and the cow is treated with a 25 mg of prostaglandin f2alpha (pgf; lutalyse®, zoetis). cows were bred with semen of good quality by timed ai at 66-72 hours from cidr removal by the same technician. pregnancy was evaluated by transrectal palpation in combination with real-time ultrasound at day 35 after ai. blood was obtained on days 10 and 17 after ai for measurement of progesterone concentrations. samples were obtained from the tail vein through a one inch 18 gauge needle into a serum-separating evacuated tube. serum was removed with a sterile pipette after centrifugation 1200 rpm for 15 minutes and the sample was frozen at -20°c. concentrations of progesterone in the serum were determined by double-antibody radioimmunoassay (coat-a-count® kit; diagnostics products corporation) at the animal health diagnostic center, cornell university. statistical analysis the experimental design was a randomized complete block. treatments were randomly assigned to cows within blocks. individual cow was the experimental unit. non-categorical data were analyzed using the mixed procedure of sas. categorical data were analyzed using the genmod procedure of sas. significance was declared at p�0.05. clinical theriogenology • volume 6, number 1 • march 201435 35 results experiment one four bulls (two con and two pour) were excluded from the study due to medical conditions that required systemic antibiotic therapy. individual bull weights and body condition scores remained consistent throughout the experimental period. individual scrotal circumference measurements and assessment of accessory sex glands were also consistent throughout the experiment. there were no treatment by time interactions for variation in motility or morphology (motility p=0.13; morphology p=0.67). the overall means for each treatment group across time were compared (table 1). there were no differences in overall motility (p=0.41; figure), progressive motility (p=0.60) or in percent normal morphology (p=0.41) among treatments. serum samples for testosterone measurement were lost from one bull in the con group. testosterone concentrations did not differ (p=0.16) between con and pour + tag bulls. table 1. results of experiment one. least square means of overall motility, progressive motility, normal morphology, and testosterone concentration in serum of bulls. con=control; pour=pour-on cyfluthrin product; tag=ear tags impregnated with cyfluthrin; pour+tag=pour-on and ear tag cyfluthrin products; sem=standard error of means. treatment group con (n=5) pour (n=5) tag (n=7) pour+tag (n=7) sem p value observation overall motility (%) 81.67 80.54 84.75 86.01 2.81 0.41 progressive motility (%) 67.35 67.50 72.40 72.63 3.99 0.60 normal morphology (%) 80.96 80.12 80.04 71.27 5.23 0.41 testosterone (ng/ml) 8.17 (n=4) 14.35 3.22 0.16 figure. scatterplot of means of overall motility for all treatment groups. no statistical differences (p=0.41) were found between treatment groups. experiment two a significant difference in serum concentrations of progesterone was observed on day 10 but not on day 17 (table 2). no differences (p=0.65) were observed in ai pregnancy rates between pour+tag (45%) and con (40%) cows. clinical theriogenology • volume 6, number 1 • march 2014 36 36 table 2. results of experiment two. mean concentrations of progesterone in serum at day 10 and day 17 and pregnancy rates in con and pour+tag cows. con=control; pour+tag=pour-on and ear tag cyfluthrin products; sem=standard error of means. item con (n=61) pour+tag (n=62) sem p value day 10 progesterone (ng/ml) 6.4 5.5 0.3 0.02 day 17 progesterone (ng/ml) 6.2 6.5 0.4 0.57 ai pregnancy rate 40% 45% 0.3 0.65 discussion experiment one according to previous reports of the effects of pyrethroids on bull and ram fertility, a decrease in motility would be expected to result quickly after exposure to the pesticide.25 semen collections were performed weekly for nine weeks in order to account for early variation in motility and long-term variation in morphology. no treatment by time effects were detected throughout the experiment and no significant differences were noted in motility parameters. morphology was monitored weekly for 62 days to span the period of time necessary for completion of one spermatogenic cycle. no significant differences in morphologies were detected across all treatment groups for the duration of the experiment. the route of exposure could play a critical role in the amount of pyrethroid the animals absorb. a study using rats compared absorption of orally and dermally administered pyrethroids and found 75% of orally applied pyrethroids were absorbed compared to 20% following topical administration.27 another study in humans found absorption of 35% after an oral dose of permethrin compared to 2% after dermal application.28 limited residues of cyfluthrin were detected in whole blood and liver, and slight residues were detected in fat, muscle, kidney, and whole milk following dermal applications suggesting a low level of absorption transdermally.29 concentrations of cyfluthrin in the serum were not determined in the current study. results from animal experiments with male new zealand white rabbits showed that oral treatment with cypermethrin resulted in a significant decrease in ejaculate volume, sperm concentration, total sperm output, sperm motility, total motile sperm per ejaculate, and packed sperm volume.13 in a study of 30 sprague-dawley rats treated orally with 30 mg/kg of beta-cypermethrin for 15 days, a negative effect was seen in daily sperm production.18 in contrast, 135 wistar rats treated with 40 mg/kg doses of oral fenvalerate exhibited no detrimental effects in reproductive parameters.16 in experiments conducted on dwarf goats, the entire animal was dipped in varying percentages of cypermethrin at day 0 and day 15. a dose-dependent decline in semen quality was observed at day 30, however, semen quality returned to normal by day 75.17 results from these experiments suggest that decreases in fertility associated with pyrethroids may be dependent on type of pyrethroid, dosage, and route of exposure. the dosages used in many of these studies were commonly twice the recommended dosages and oral routes of administration were most commonly used. volkmann’s report on the effects of pyrethroids on bull and ram infertility involved a premise spray (bifenthrin) that could have saturated the entire environment, including water and feed containers. the current study found no effects on bull fertility when cyfluthrin was applied topically and at the labeled dose. animals were grouped in pastures based on their treatment groups and may have had the opportunity to ingest the product from a pasture-mate’s back. no detrimental effects were seen in this experiment, but animals in a more confined housing system may have the opportunity to ingest more product. pyrethroids have been reported to have antiandrogenic activity and may inhibit hormone production.3-11 testosterone concentrations were analyzed for the con and pour+tag treatment groups in this experiment and no significant differences were found. arena et al conducted experiments on rats using fenvalerate and found similar results with no changes in testosterone concentrations.16 in contrast, zhang et al found that oral administration of cis-permethrin (35 and 70 mg/kg) for 42 consecutive days in mice decreased testosterone concentrations.4 oral permethrin also decreased testosterone concentrations in rabbits after 16 weeks of administration.13 in experiments assessing clinical theriogenology • volume 6, number 1 • march 201437 37 hormone receptor activity, du et al10 found the relative potency of antagonistic activities descended in the following order: deltamethrin > cyfluthrin > fenvalerate >cyhalothrin > cypermethrin > permethrin > 3phenoxybenzoic acid. they also reported that antiandrogenic activities of deltamethrin and cyfluthrin were more potent than other compounds.10 differences in the concentrations of testosterone as reported by other investigators, like changes reported for motility and morphology, may be dependent on the type of pyrethroid used, the route of administration, and dose. experiment two several studies have suggested that pyrethroid products had toxic effects on cells of the corpus luteum which could alter the production of progesterone.5,11,24 in this study, progesterone was measured in serum collected on day 10 after ai when the ovaries of all cows (con and pour+tag) should have contained a functional corpus luteum regardless of their pregnancy status. progesterone analysis was also performed on day 17 after ai. as there were no differences in the rates of pregnancy between the two treatment groups, comparison of concentrations of progesterone at day 17 between pregnant and nonpregnant cows within each group was not pursued. cows in the pour+tag group had significantly lower concentrations of progesterone in their serum at day 10, but this difference was not seen at day 17. fei et al reported that the pyrethroid, fenvalerate, had concentration-related inhibitory effects on follicular growth characterized by low granulosa cell numbers with no significant effects on follicular survival rates.30 it is possible that pyrethroids used on the cows in the present study affected pre-ovulatory follicles, initially inhibiting cells that would later be responsible for progesterone production, but this inhibition could be overcome by day 17. another study using bovine luteal cells in vitro indicated that cypermethrin caused a transient decrease in progesterone production.11 if pyrethroid application similarly affected luteal cells in our study at day 10, the effect may have been overcome by day 17. the findings from this experiment suggest that pour-on and ear tags can have an early, time-dependent effect on serum progesterone concentrations. elucidating the absorption of cyfluthrin, the exposure to specific bovine ovarian tissues, and the possible mechanisms for decreased concentrations of progesterone will require additional experiments. results from experiments conducted on pregnant rats receiving oral administration of a synthetic pyrethroid, deltamethrin, indicated that a sublethal dose could produce qualitative and quantitative alterations in the blastocyst-endometrium interaction, compromising the implantation process.24 the results from the current study indicated no significant difference in conception rates between cows in the con and pour-tag treatment groups conclusion cyfluthrin pyrethroid insecticide ear tags and pour-on products did not result in negative effects on the male or female cattle reproductive parameters measured. a transient effect on the concentration of progesterone in the serum of cows was not associated with decreased pregnancy rates. acknowledgements the authors would like to acknowledge tim devalle and ashley kloth for their assistance with experimental process; drs. yvette johnson and tara felix as well as lindsay shoup for their assistance with statistical analysis; and bayer animal health for the donation of the pyrethroid products used. references 1. united states environmental protection agency: pyrethroid cumulative risk assessment. washington: office of chemical safety and pollution prevention; 2011. 2. atsdr: toxicological profile for pyrethrins and pyrethroids. atlanta: agency for toxic substances and disease registry; 2003. 3. sun h, xu xl, xu lc, et al: antiandrogenic activity of pyrethroid pesticides and their metabolite in reporter gene assay. chemosphere 2007;66:474-479. 4. zhang sy, ito y, yamanoshita o, et al: permethrin may disrupt testosterone biosynthesis via mitochondrial membrane damage of leydig cells in adult male mouse. endocrinology 2007;148:3941-3949. clinical theriogenology • volume 6, number 1 • march 2014 38 38 5. he j, chen j, liu r, et al: alterations of fsh stimulated progesterone production and calcium homeostasis in primarily cultured human luteinizing-granulosa cells induced by fenvalerate. toxicology 2004;203:61-68. 6. chen h, xiao j, hu g, et al: estrogenicity of organophosphorus and pyrethroid pesticides. j toxicol environ health a 2002;65:1419-1435. 7. hu jy, wang sl, zhao rc, et al: [effects of fenvalerate on reproductive and endocrine systems of male rats.] zhonghua nan ke xue 2002;8:18–21. chinese 8. mani u, islam f, prasad ak, et al: steroidogenic alterations in testes and sera of rats exposed to formulated fenvalerate by inhalation. hum exp toxicol 2002;21:593-597. 9. tyler cr, beresford n, van der woning m, et al: metabolism and environmental degradation of pyrethroid insecticides produce compounds with endocrine activities. environ toxicol chem 2000;19:801-809. 10. du g, shen o, sun h, et al: assessing hormone receptor activities of pyrethroid insecticides and their metabolites in reporter gene assays. toxicol sci 2010;116:58-66. 11. gill sa, rizvi f, khan mz, et al: toxic effects of cypermethrin and methamidophos on bovine corpus luteal cells and progesterone production. exp toxicol pathol 2011;63:131-135. 12. el-demerdash fm, yousef mi, kedwany fs, et al: role of alphatocopherol and beta-carotene in ameliorating the fenvalerate-induced changes in oxidative stress, hemato-biochemical parameters, and semen quality of male rats. j environ sci health b 2004;39:443-459. 13. yousef mi, el-demerdash fm, al-salhen ks: protective role of isoflavones against the toxic effect of cypermethrin on semen quality and testosterone levels of rabbits. j environ sci health b 2003;38:463-478. 14. elbetieha a, da’as si, khamas w, et al: evaluation of the toxic potentials of cypermethrin pesticide on some reproductive and fertility parameters in the male rats. arch environ contam toxicol 2001;41:522-528. 15. salem mh, abo-elezz z, abd-allah ga, et al: effect of organophosphorus (dimethoate) and pyrethroid (deltamethrin) pesticides on semen characteristics in rabbits. j environ sci health b 1988;23:279-290. 16. arena ac, fernandez cdb, porto em, et al: fenvalerate, a pyrethroid insecticide, adversely affects sperm production and storage in male rats. j toxicol environ health a 2008;71:1550-1558. 17. ahmad m, hussain i, khan a, et al: deleterious effects of cypermethrin on semen characteristics and testes of dwarf goats (capra hircus). exp toxicol pathol 2009;61:339-346. 18. liu l, hu j, wang h, et al effects of beta-cypermethrin on male rat reproductive system. environ toxicol pharmacol 2010;30:251-256. 19. meeker jd, barr db, hauser r: human semen quality and sperm dna damage in relation to urinary metabolites of pyrethroid insecticides. hum reprod 2008;23:1932-1940. 20. xia y, han y, wu b, et al: the relation between urinary metabolite of pyrethroid insecticides and semen quality in humans. fertil steril 2008;89:1743-1750. 21. perry mj, venners sa, barr db, et al: environmental pyrethroid and organophosphorus insecticide exposures and sperm concentration. reprod toxicol 2007;23:113-118. 22. moore a, waring c: the effects of a synthetic pyrethroid pesticide on some aspects of reproduction in atlantic salmon (salmo salar l). aquat toxicol 2001;52:1-12. 23. weston dp, asbell am, hecht sa, et al: pyrethoid insecticides in urban salmon streams of the pacific northwest. environ pollut 2011;159:3051-3056. 24. lemos aj, wanderley-teixeira v, teixeira aac, et al: response of blastocyst-endometrium interactions in albino rats to sublethal doses of biological and synthetic insecticides. food chem toxicol 2011; 49:2541-2547. 25. volkman dh, voelkl dl: clinical observations on the effects of pyrethroid insecticides on bull semen quality. proc acad vet consult: 2012. available at: http://www.avc-beef.org/proceedings/2012-2/volkman.pdf 26. lamb gc, stevenson js, kesler dj, et al: inclusion of an intravaginal progesterone insert plus gnrh and prostaglandin f2a for ovulation control in postpartum suckled beef cows. j anim sci 2001;79:2253-2259. 27. kaneko h, ohkawa h, miyamoto j: absorption and metabolism of dermally applied phenothrin to rats. j pestic sci 1981;6:169-182. 28. woollen b, marsh j, laird w, et al: the metabolism of cypermethrin in men: difference in urinary metabolite profiles following oral and dermal administration. xenobiotica 1992;22:983-991. 29. soderlund d: predicting the metabolic fate of cyfluthrin following dermal application to cattle. environmental protection agency reference 171-4. november 9, 1994. 30. fei j, qu jh, ding xl, et al: fenvalerate inhibits the growth of primary cultured rat preantral ovarian follicles. toxicology 2010:267:1-6. (editor’s note: the figure in this manuscript is available in color in the online edition of clinical thriogenology.) clinical theriogenology • volume 6, number 1 • march 201439 39 40 1 contact alexandria horner alexandria.horner@colostate.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10840, http://dx.doi.org/10.58292/ct.v16.10840 case report management of a granulosa-theca cell tumor in a female rottweiler alexandria horner,a greg burns,a mac harris,b jenny sones,a fiona hollinsheada adepartment of clinical sciences, college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co, usa bdepartment of microbiology, immunology & pathology, college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co, usa abstract a 4-year, multiparous rottweiler female dog, was presented for breeding management. dog had clinical signs of proestrus and estrus for > 6 weeks, including cornification of vaginal epithelium and low circulating progesterone concentrations (< 2 ng/ml). transabdominal ultrasonography of the reproductive tract and measurement of circulating hormones (antimüllerian hormone, inhibin-b, and progesterone) suggested granulosa-theca cell tumor. affected ovary was removed via laparoscopy. histopathology confirmed granulosa-theca cell tumor; dog resumed cyclicity 4 months after surgery, had normal estrus and was successfully mated with a young stud dog. dog was diagnosed pregnant (30 days after lh surge) via transabdominal ultrasonography; 4 amniotic sacs were detected, and 3 grew to term but were not viable at delivery (via cesarian surgery). keywords: persistent estrus, granulosa-theca cell tumor, laparoscopic ovariectomy background ovarian tumors are rare in mammals, but among domestic animals, they appear to be most common in female dogs and cows.1–5 primary ovarian tumors can be categorized into 3 groups; epithelial, germ cell, and sex cord-stromal. granulosatheca cell tumors (gct) are sex cord-stromal cell-based ovarian tumors that account for 0.5 to 1.2% of all canine tumors. gcts arise from granulosa cell cords of atretic follicles5 and are not locally invasive; however, in 10 to 20% of cases they can metastasize.2,5 a mass effect of the gct causing clinical signs is very uncommon. however, gcts can be challenging to diagnose as they do not always have the classical honeycomb-like appearance that is recognizable via ultrasonography.6 most gcts are unilateral in all species, although bilateral tumors were described in a female dog.5 the most common presentation associated with canine gcts are signs of estrus that persist for > 6 weeks.7 clinical signs of persistent estrus are associated with constant secretion of estrogen and include behavioral signs such as standing/sexual reflexes (flagging and postural changes), receptiveness to intact males, and anatomical changes, including vulvar swelling and serosanguineous vulvar discharges.3–5,8–12 however, clinical signs associated with estrogen can also be associated with normal physiologic cyclicity, ovarian remnant syndrome, ovarian cyst(s), ovulation failure, and exposure to exogenous sources of estrogen. diagnosis of gct has also been previously reported in ovariohysterectomized dogs with concurrent ovarian remnant syndrome.13-15 therefore, determination of when clinical signs of estrus begin, and ongoing vaginal cytology and serum progesterone measurements are required to facilitate differentiation of a gct from other causes of estrogen-associated conditions. long-term exposure to estrogen arising from continuous secretion of steroid hormones from neoplastic granulosa cells of a gct can have detrimental effects on bone marrow and uterine tissue, resulting in anemia and infertility, respectively.3-5,8-12 exposure to sustained elevated concentrations of estrogen affects the hematopoietic system in 3 stages: a brief increase in platelets followed by severe thrombocytopenia, then development of bone marrow granulocyte hyperplasia with neutrophilia and, finally, bone marrow recovery or aplasia, dependent on the magnitude of hormonal insult.16,17 gcts can also produce progesterone that in combination with estrogen, causes stimulation of endometrial growth, proliferation, and glandular secretions.8 direct exposure to these steroid hormones results in the development of cystic endometrial hyperplasia (ceh), which not only increases the risk of developing a life-threatening pyometra but can lead to sub/infertility through early embryonic loss and impaired placentation.8,18,19 bioassay panels of reproductive hormones in mares and cows are routinely used to help differentiate ovarian mailto:alexandria.horner@colostate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10840 2 citation: clinical theriogenology 2024, 16, 10840, http://dx.doi.org/10.58292/ct.v16.10840 pathology and diseases. increased serum concentrations of antimüllerian hormone (amh), inhibin b and/or testosterone, all produced by granulosa cells, have been demonstrated to be useful biomarkers for identifying functional gcts in cows and mares.20–25 in mares, amh secretion can be identified in normal growing and small antral follicles.26 however, studies comparing normal ovarian function with gcts determined that bioactive amh was more strongly expressed in mares with gct.20,26 elevated serum concentrations of inhibin and testosterone are present in 87 and 67% of mares with a gct, respectively.25–28 inhibin b isoform is the only isoform of inhibin that reliably indicates ovarian neoplasia in mares.22 when amh, inhibin b and testosterone are used together as a screening panel in mares, 100% sensitivity is achieved.23 unfortunately, a screening panel for canine gct has not yet been established. previous studies reported serum concentrations of amh in dogs with gcts to range from 1.12 to ≥ 23 ng/ml.21,29,30 at the time of this report, no studies were identified regarding the use of inhibin-b serum concentrations for canine gcts diagnosis. as a result, no reference range has been established. testosterone has also not been reported as a bioassay in the dog for gct diagnosis and was not measured in this case. currently, in dogs, measurement of amh and inhibin needs to be interpreted in conjunction with other clinical and diagnostic findings (e.g. ovarian anatomical changes on ultrasonography) for a presumptive diagnosis of gct.20,21 ultrasonographic examination of the ovaries is a valuable screening and diagnostic tool for determining the presence of pathological changes associated with ovarian parenchyma in many species.20 previous publications in dogs have demonstrated that ovarian tumors, particularly gcts, have a wide range of alterations to echogenicity and shape.6 in this case report, we discuss the steps taken to diagnose gct at a very early stage of development in a young, genetically valuable breeding female dog and a novel treatment approach to remove the ovarian tumor whilst maintaining fertility. case presentation a 4-year, multiparous rottweiler female dog, was presented 7 days after the onset of clinical signs associated with proestrus (i.e. bloody vulvar discharge and vulvar edema). a routine breeding examination was performed, which included a complete physical examination, screening for  brucella canis (brucella multiplex, cornell university, ithaca, ny). additionally, complete blood count (cbc) and chemistry analyses were performed. no abnormal findings were reported. starting on day 8 (from the first signs of proestrus observed by the owner), blood samples were obtained every other day to measure serum progesterone concentrations (immulite® 1000 immunoassay system, colorado state university, fort collins, co) and vaginal smears were taken for cytological evaluation of vaginal epithelial cells every 2nd or 3rd day to determine stage of estrus and detect lh surge (lh0). this was performed, as is routinely done, to facilitate optimal timing of insemination (> 90% superficial keratinized vaginal epithelial cells) with frozen-thawed canine semen.31–35 progesterone concentrations ranged from 0.90-1.85 ng/ml but never exceeded 2 ng/ml, and vaginal cytology never changed from > 90% superficial cell (table). table. serum progesterone profile and vaginal cytology day of cycle from onset of clinical signs progesterone concentrations (ng/ml) vaginal cytology (percent superficial cornification) 8 0.923 > 90 10 0.976 > 90 13 0.971 > 90 16 0.924 > 90 21 1.030 > 90 23 0.895 > 90 27 1.16 > 90 30 1.03 > 90 35 1.85 > 90 differential diagnoses differential diagnoses for persistent estrus included an anovulatory follicle/ovulation failure, an ovarian follicular cyst, exogenous exposure to estrogens through diet, oral treatment, or inadvertent exposure to human hormonal creams and lastly, hormone-secreting ovarian neoplasia (e.g. gct).6,7,32,36,37 intersex conditions were not considered as a differential diagnosis due to previous history of successful litters.38,39 diagnosis initial b-mode ultrasonographic examination (canon® tus-ai700 ultrasound machine with a multi-frequency micro-convex (8m1c) transducer) of the reproductive tract (ovaries, uterus) was performed on day 40 of estrus. enlarged (19-21 mm in diameter) left ovary had a heterogeneous hypoechoic region within the caudal pole (figure 1a). right ovary was normal in appearance and size (figure 1b). differentiation between an anovulatory follicle undergoing luteinization, an ovarian follicular cyst, or ovarian neoplasia was not possible. on day 57, progesterone concentrations remained lower (< 2 ng/ml) and > 90% vaginal epithelial cells were cornified. another transabdominal ultrasonography was performed; right ovary was still normal in appearance, size with ultrasonographic heterogenicity and had no follicular activity, left ovary had increased in size (19-21 to 20-22 mm) and the heterogeneous hypoechoic region within caudal pole was still present (suspected to be neoplastic). left ovary had begun to take on a honeycomb-like appearance (figure 2), a common finding with gcts in mares.40 using color doppler, moderate peripheral vascularity and a few small internal vessels associated with the heterogenous hypoechoic structure on left ovary were visualized. increased size, level, and location of angiogenesis and the honeycomb-like appearance of left ovary were all suggestive of gct. at each transabdominal ultrasonographic examination, uterus was notably enlarged (~ 10.4 mm diameter/width/thickness) for a young, multiparous dog, and echogenic changes (cystic structures) observed throughout the endometrium (figure 3), indicative of cystic endometrial hyperplasia (ceh).41 http://dx.doi.org/10.58292/ct.v16.10840 citation: clinical theriogenology 2024, 16, 10840, http://dx.doi.org/10.58292/ct.v16.10840 3 on day 57, serum was submitted (university of california davis, davis, ca) for inhibin-b (al163, ansh labs, webster, tx) and amh (canine amh al116, ansh labs, webster tx). inhibin concentrations were 182.4 pg/ml, and amh concentrations were 0.89 ng/ml. the laboratory reported that amh concentrations were within the normal reference range for an intact dog in estrus (0.12 to > 12 ng/ ml). however, there is currently no normal reference range for canine inhibin, but concentrations detected were considered highly elevated compared to equine normal reference range of 2 to 100 pg/ml. progesterone concentrations (enzyme immunoassay, university of california davis) were 9.3 ng/ml. cbc and biochemistry revealed nonregenerative anemia, severe thrombocytopenia, and leukopenia. prothrombin time and partial thromboplastin time were unremarkable, but fibrinogen was elevated. biochemistry markers, including fibrinogen, were consistent with chronic inflammation associated with myelosuppression. serum estrogen and estradiol concentrations were not measured as the cytological assessment of vaginal smears remained at > 90% superficial/cornified vaginal epithelial cells. based on these clinical, ultrasonographic, and endocrine results, a preliminary diagnosis of gct was made. due to the development of nonregenerative anemia, it was decided that immediate surgical removal of left ovary was necessary to eliminate the source of estrogen and to confirm our diagnosis. no additional measurements nor follicular activity of the contralateral ovary were determined due to the critical state of the patient (bone marrow suppression). treatment before surgery, blood crossmatching (centrifuged edta blood samples of donor and recipient) was carried out, and a unit of dea 1.1 (+) blood was given intraoperatively to improve low oxygen-carrying capacity caused by bone marrow suppression. unilateral ovariectomy (laparoscopic laparotomy) was performed. briefly, a 3 cm incision was made in caudal abdomen on the ventral midline to facilitate laparoscope introduction. abdomen was insufflated to 11 mm hg, and left ovary was visualized (figure 4) and transected using a ligasure™ (medtronic). incision site was widened to exteriorize left ovary from abdomen (figure 5). right ovary appeared small with no grossly evident follicular activity during the procedure. hemorrhage was minimal and controlled using monopolar electrocautery. abdominal wall was closed, and a tegaderm™ (nexcare™, 3m) bandage was placed over the incision site. dog recovered uneventfully and was discharged the following day. removed left ovary was submitted for histopathology. outcome left ovary (~ 8.5 x 4 cm) had prominent multinodular white to tan ovarian tissue with well-demarcated white, variably friable, and partially caveated mass (3 cm); expanding around the mass were regions of frank blood (hemorrhage). circumscribing the mass was white to yellow thick fibrous capsule. remainder of the ovarian tissue was tan with variably sized cystic structures (developing follicles; figure 6). histopathology confirmed that the tumor effaced over 60% of left ovary and was highly cellular, encapsulated and well-demarcated sex cord neoplasm. neoplastic cell population was arranged in a rare glandular/pseudo-rosette structure, with the remaining population located within interlacing streams supported by collagenous stroma. anisocytosis and anisokaryosis was moderate with 3 mitotic figures per mitotic area. histopathology findings were consistent with a poorly differentiated gct. remaining ovarian tissue within left ovary was histologically normal, with ovarian follicles observed in varying stages of maturation (figure 7). a portion of left uterine horn was also submitted for histologic evaluation, and endometrium was described as hyperplastic, measuring ~ 2 mm in thickness with variably ectatic endometrial glands containing scant a b figure 1. a. ultrasonographic image of ‘abnormal’ left ovary (note enlarged ovary with a heterogeneously hypoechoic region within the caudal pole) on day 40 of estrus; distance between calipers: 20.8 mm and arrow pointing cranial side. b. ultrasonographic image of ‘normal’ right ovary on day 40 of estrus and arrow pointing cranial side. figure 2. ultrasonographic image of ‘abnormal’ left ovary on day 57 of estrus (note: honeycomb-like appearance suggestive of early stages of differentiation of a gct) http://dx.doi.org/10.58292/ct.v16.10840 4 citation: clinical theriogenology 2024, 16, 10840, http://dx.doi.org/10.58292/ct.v16.10840 flocculent eosinophilic material. uterine findings were consistent with ceh. on day 14 after surgery, serum was collected and submitted (university of california davis) for measurement of progesterone, inhibin b, and amh concentrations. inhibin b concentrations had declined substantially to 8.4 pg/ml; progesterone was baseline (< 0.2 ng/ml), and amh concentrations remained within normal reference range, consistent with declining concentrations of inhibin after ovariectomy as observed in mares.22 amh and inhibin concentrations were not measured during the follow-up period prior to returning to cyclicity. dog returned to proestrus 4 months after unilateral ovariectomy. blood sample was submitted for cbc and chemistry analyses; nothing was remarkable. importantly, there was complete resolution of nonregenerative anemia, thrombocytopenia, and inflammatory profile. ultrasonographic examination of uterus demonstrated continued endometrial changes associated with ceh. there was no intrauterine fluid. to maximize chances of pregnancy, breeding management was performed, and the dog was naturally mated (young proven stud dog) on days 4 and 6 after lh0. transabdominal ultrasonography was performed on day 30 after lh0; 4 viable amniotic sacs and 1 resorption site (figure  8) were observed. a single lateral abdominal radiograph was obtained 1 week before calculated whelping date, and 3 fetal skeletons were identified (figure 9). due to unexpected complications, cesarian surgery was performed at a local veterinary clinic and deceased pups were removed. information on presurgical fetal viability (transabdominal ultrasonography) nor gross findings of pups were not provided and necropsy was not performed. discussion granulosa cell tumors are among the most common ovarian neoplasia identified in female dogs.42 however, literature regarding diagnosis and treatment options in dogs is limited. this is attributed to most female dogs, especially in north america (gonadectomized early in life), apparently resulting in a low incidence rate of this disease.15 however, as more female dogs remain intact for extended periods, either for figure 3. ultrasonographic images of uterus on day 30: a. transverse view of uterine body and b. uterine horn bifurcation (characteristic of cystic endometrial hyperplasia) cysts; note cysts (arrows) within uterine endometrial lining. a b figure 4. intraoperative image of left ovary (black arrow) before surgical resection (note abnormal gross appearance ovarian serosal surface with multilobulated darkened parenchyma). figure 5. gross image of left ovary immediately after surgical removal (note irregular nodular mass [black arrow] representing gct that is clearly visible through ovarian bursa). http://dx.doi.org/10.58292/ct.v16.10840 citation: clinical theriogenology 2024, 16, 10840, http://dx.doi.org/10.58292/ct.v16.10840 5 a b b figure 6. left ovary after surgical removal; a. gct along with intact oviduct and uterine horn tip (note cut surface of gct has well-demarcated white, variably friable, and partially cavitated mass [arrow]) and b. diameter of gct (~ 3 cm) mass. figure 7. histopathology of left ovary: a. subgross (10 x magnification); b. tissue margin (20 x magnification); c. neoplastic cells arranged in variably formed glandular structures that contain mucinous eosinophilic material (100 x magnification), and d. neoplastic cells are polygonal to rarely spindled with variably discrete cell borders and foamy eosinophilic cytoplasm, mild to moderate anisokaryosis and anisocytosis (di 200 x magnification and dii 400 x magnification). ba figure 8. ultrasonographic image of amniotic sacs (a) and reabsorption site (b) http://dx.doi.org/10.58292/ct.v16.10840 6 citation: clinical theriogenology 2024, 16, 10840, http://dx.doi.org/10.58292/ct.v16.10840 breeding purposes or for potential health benefits, the likelihood of diagnosing an ovarian tumor will become more common.43,44 therefore, understanding the current diagnostic tools available and the potential limitations of each method is important for clinicians to recognize when pursuing gct diagnosis. in mares, tests available for the diagnosis of a gct include hormonal bioassays or ‘gct panels’ (amh, inhibin, progesterone, and testosterone), ovarian ultrasonography, transrectal ovarian palpation and overt behavioral changes associated with increased concentrations of estrogen and/or testosterone through stallion-like or aggressive behavior.20 in dogs, a distinctive behavioral change, similar to stallion-like behavior observed in mares, has not been documented, and diagnosis can be more challenging because there is no pathognomonic finding, no consistent ultrasonographic appearance and no standardized hormone panel with reference ranges specific to dogs. hormonal findings associated with canine gcts can be unpredictable, especially when other physiological processes may present concurrently in the early stages of pathology.20 ovarian tumors, particularly gcts, alter normal ovarian echotexture and have an irregular shape in 69% of reported cases in dogs.6 however, there can be many variations in appearance; as in our case, only a change in echotexture of the ovarian parenchyma and a slight increase in ovarian size but normal shape was initially detected on ultrasonography. this is most likely attributed to the very early diagnosis of gct in our case. human medicine utilizes color doppler to differentiate between malignant and benign ovarian neoplasms by detecting abnormal changes to the intraovarian vasculature.45,46 we utilized this technique in our case to demonstrate an increase in the vasculature to and within the abnormal region that when combined with initial changes to a honeycomb-like appearance, aided in the presumptive diagnosis of gct. in mares, peripheral amh and inhibin b concentrations are routinely measured to diagnose gct.20 elevated amh concentrations ranging from 1.12 to ≥ 23 ng/ml have been reported in dogs diagnosed with gct.21 however, reference ranges can be > 12 ng/ml in clinically normal intact dogs.47 therefore, supportive clinical signs, vaginal cytological findings (indicative of estrogen), ultrasonography findings and ultimately, histopathology are required for the diagnosis of a gct in dogs. in this case, circulating amh concentrations were not high (possibly due to the early diagnosis of a gct). another endocrine marker used in large animals to diagnose gcts is inhibin b that was elevated in this case compared to mare’s reference range of 2 to 100 pg/ml.20 however, there is currently no reference range for inhibin in dogs, though high concentrations of inhibin, in this case, followed by substantial decline after removal of the ovary containing gct, helped support our other initial clinical findings suggestive of gct and confirm that a hormonally active tumor had successfully been resected. appearance of ovary on ultrasonography with the coinciding lack of contralateral ovarian follicular activity (acquired hypoplasia and suppression of normal positive and negative feedback mechanism of cyclicity) suggested gct; typical findings in other species with hormonally active gcts as in mares.17,48 lack of follicular activity by the unaffected ovary was hypothesized to be the result of negative feedback due to elevated inhibin secretion by the tumor, causing suppression of follicle-stimulating hormone (fsh).23,49 thus, it affects normal follicle recruitment, deviation, growth, and ovulation. ongoing suppression is likely caused by normal ovary’s acquired hypoplasia. we suspect a similar process was occurring in the present case, though data collection at that stage of the investigation was not a priority due to dog’s clinical well-being. however, further research is still required to fully understand the pathogenesis of unaffected ovary as there are discrepancies in literature, especially in mares.17 reports confirmed low and no differences in fsh concentrations in mares with gcts.49,50 definitive diagnosis of a gct was confirmed by histopathology. resection of part of uterine horn with laparoscopic laparotomy also allowed confirmation of ceh. this information allowed us to give the owner a more informed prognosis of future fertility. although gct, in this case, was easily identified by our pathologist through gross and histologic assessment, it is important to note that cytological features can be variable regarding cellularity and mitotic indices that ultimately reflects the wide variability in the functionality of gcts across species.20,51 unilateral ovariectomy is a standard treatment option for gcts in mares.52–54 however, to authors’ knowledge, there was no reported cases of pregnancy after unilateral ovariectomy via laparoscopy for gct treatment in dogs. laparoscopic approach is minimally invasive and has a lower postoperative complication rate and a shorter recovery time than traditional laparotomy methods.55 a minimally invasive surgery was particularly important in this case, as the patient had hematological clinical signs associated with bone marrow suppression due to prolonged endogenous estrogen exposure caused by gct secretions. although the objective of our treatment methodology was aimed to preserve dog’s reproductive potential, there were several concerns regarding reproductive performance after surgery that included: a) fewer oocytes would be ovulated with only 1 remaining ovary; and b) uterine ceh changes. however, remaining ovary reported to have compensated by undergoing hypertrophy and ovulated similar number of oocytes despite contralateral ovary removal.56 in this case, a possible slight figure 9. left lateral radiograph (note: 3 fetal skeletons) http://dx.doi.org/10.58292/ct.v16.10840 citation: clinical theriogenology 2024, 16, 10840, http://dx.doi.org/10.58292/ct.v16.10840 7 compensatory effect was appreciated with 5 amniotic sacs detected via ultrasonography on day 35 after lh0. many ovarian pathologies such as gcts and ovarian cysts, especially those processes that are hormonally active, are associated with concurrent uterine changes, specifically, ceh.9,36 repeated exposure of the endometrium to cycles of estrogen priming followed by 2 months of luteal/nonpregnant progesterone is a prominent contributing factor to the development of ceh especially in older, maiden dogs.6,57–59 ceh is associated with infertility in dogs as the changes induced can result in interference with embryonic attachment and increase the risk of pyometra.54–58 endometrial changes associated with ceh include hyperplasia of endometrial glands that become enlarged and cystic in appearance and result in delayed uterine clearance of ascending bacteria during estrus and dead sperm and extenders after intrauterine insemination. furthermore, the cystic endometrial environment is not conducive to implantation and normal placentation required for embryonic development.18,59 in mares, endometrial biopsies are graded using the kenny-doig system. this helps predict the potential likelihood of pregnancy, maintenance of pregnancy and ultimately, delivery of a live foal based on microscopic features of luminal and glandular epithelium, stratum compactum and spongiosum.60 unfortunately, we currently do not have biopsy ‘grades’ for dogs; furthermore, biopsies are obtained in diestrus (time for it to be diagnostic) can result in pyometra and loss of an estrous cycle due to the unique feature in dogs (no luteolytic mechanism if not pregnant). larger studies are required in dogs for the development of cytological and endometrial ultrasonographic grading parameters of ceh that are correlated to reproductive performance and allow prediction of the likelihood of fertility/pregnancy success after breeding, as in mares. in this case, despite dog’s young age, excessive exposure of endometrium to estrogen and progesterone produced by gct resulted in ceh development and was most likely the underlying cause of embryonic and fetal loss (detected via ultrasonography and radiography). ultrasonography is valuable not only to assist in gct diagnosis but also to assess any damage to uterus caused by chronic sex steroid hormone exposure. it is important to take into consideration uterine ‘health’ when offering treatment that also preserves fertility. loss of at-term fetuses in this case is unknown. despite loss of pups during whelping, this case had positive outcomes (successful treatment of gct and maintenance of breeding potential on a young and genetically valuable female dog). we discussed diagnostic tools and surgical techniques that are available to facilitate the diagnosis and treatment of gcts to allow a genetically valuable young dog to maintain fertility potential. we also highlighted the importance of assessing secondary uterine changes caused by excessive and constant hormone stimulation to predict future fertility after removal of gct. learning points • gct in dogs (unlike mares) have a wide range of undefined ultrasonographic appearances • measurement of serum concentrations of inhibin b and amh are a 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https://doi.org/10.1016/j.theriogenology.2006.03.030 https://doi.org/10.1016/j.theriogenology.2006.03.030 https://doi.org/10.​1111/j.1748-5827.2004.tb00200.x https://doi.org/10.​1111/j.1748-5827.2004.tb00200.x https://doi.org/10.1111/j.1532-950x.2011.00884.x https://doi.org/10.1111/j.1532-950x.2011.00884.x https://doi.org/10.1016/j.theriogenology.2003.09.015 https://doi.org/10.1016/j.theriogenology.2003.09.015 https://doi.org/10.1136/vr.160.21.740 https://doi.org/10.1136/vr.160.21.740 https://doi.org/10.1186/s12917-018-1658-y https://doi.org/10.1111/rda.12075 https://doi.org/10.1016/j.theriogenology.2012.07.009 https://doi.org/10.1016/j.theriogenology.2012.07.009 https://doi.org/10.1016/s0195-5616(91)50052-8 https://doi.org/10.1016/s0195-5616(91)50052-8 https://doi.org/10.1016/j.theriogenology.2016.03.022 https://doi.org/10.1016/j.theriogenology.2016.03.022 https://doi.org/10.1016/j.theriogenology.2010.12.013 https://doi.org/10.1016/j.theriogenology.2010.12.013 2013: fate of the unfertilized oocytes in alpacas fate of the unfertilized oocytes in alpacas yessenia picha,a julio sumar,b mushtaq a. memona adepartment of veterinary clinical sciences, washington state university, pullman, wa; bivita research station, la raya, san marcos university, cusco, peru abstract fourteen mature female alpacas between three and six years of age were assigned to two experiments that were designed to determine the fate of unfertilized alpaca ova. in experiment one, 12 alpacas were given 50 ug of gonadotropin releasing hormone (gnrh) to induce ovulation. ovulation was detected by transrectal ultrasonography (tus). alpacas were sacrificed, the oviducts and uterine horns were flushed and ova were identified under a stereo microscope. in experiment two, two alpacas having no dominant follicle, or corpus luteum, were given 400 iu of equine chorionic gonadotropin (ecg), monitored by tus, and when the follicles reached 6 mm diameter, 75 ug of gnrh was given to induce ovulation. animals were sacrificed seven days after ovulation induction and uterine horns were flushed. results from experiment one show that at one day after ovulation, oocytes in the oviduct were of normal morphology and surrounded by cumulus oophorus cells. at two days after ovulation, the oocyte showed a clear, shrunken, dissociated cytoplasm as well as unequal cell division. at day three after ovulation, ova were surrounded by cumulus oophorus cells similar to those observed on day one after ovulation, as well a completely disorganized and clear cytoplasm. by day six after ovulation, ova were shrunken with broken zona pellucida. results of experiment two show that all ova collected from the uterus were degenerated, but still surrounded by cumulus oophorus cells. in conclusion, unfertilized ova are not retained in the oviduct of the alpaca. keywords: alpaca, unfertilized ova; oocyte collection; ovulation induction. introduction female south american camelids (sac) present striking reproductive peculiarities compared to other domestic livestock: induced ovulation, short half-life of the corpus luteum (cl), and different luteolytic activity between the two uterine horns resulting in establishment of exclusively left horn pregnancies.1-3 scientific literature describing the fate of the unfertilized mammalian ova is scarce. degeneration and disappearance of the ova in the oviduct is considered a possibility. knowledge of the fate of unfertilized ova, including its morphological description, may shed some light on physiological processes. van nierke and gerneke4 first drew attention to the differential transport of oocytes and embryos in the equine oviduct. namely, if the freshly ovulated oocyte remains unfertilized, it passes down the oviduct only to the ampullary-isthmus junction where it remains lodged in the highly convoluted folds of oviductal mucosa and degenerate slowly over many months. bouker et al5 suggested that in llamas, the non-fertilized oocyte does not reach the uterus after ovulation implying that camelids are similar to horses with respect to selective embryo transport through the oviduct. in mares, embryo transport through the uterine tube and especially through the utero-tubal junction (utj) into the uterus seems to be under the influence of the embryo.6 the mechanisms controlling embryonic descent into the uterus are not understood in alpacas. our hypothesis was that unfertilized or defective oocytes are retained in the oviduct, and embryo hatching from the zona pellucida needs to occur within the utj in order to permit passage of the embryo. the main objective of the study was to determine the fate of unfertilized oocyte in alpacas. material and methods animal selection a total of fourteen female alpacas destined for meat production were selected for the study in experiment one (n=12) and for experiment two (n=2). all animals were between 3 and 6 years age (median = 5 years) and had a cria the previous year. the study was conducted during august (dry season clinical theriogenology • volume 5 number 2 • june 201393 in peru). animal selection criteria for inclusion in the study were good general health, no prior reproductive disorders, and a body condition score between 3 and 3.5 (1-very thin, 5-obese). all females were maintained at la raya research station, cusco, peru, and were maintained under natural pasture, supplemented with a mix of oat hay and dry corn straw (500 g per animal) for maintenance and had free access to fresh water. ovulation induction and ovarian superstimulation management all females were examined by transrectal palpation by one veterinarian with small hands to determine if there were any gross abnormalities,7 and to verify the uterine tone and edema. in experiment one, follicular activity was monitored daily by tus using 7.5 mhz linear transducer. when a dominant single follicle was between 7 and 10 mm in diameter, and the uterus showed maximum tone and edema, the females were treated with 50µg of gnrh for induction of ovulation. to determine the time of ovulation, females were examined by tus every four hours starting 24 hours after the administration of gnrh. after confirmation of ovulation (disappearance of the follicle), females were randomly assigned to a group for oocyte collection at day 1, 2, 3, 4, 5 and 6 postovulation (day 0 = ovulation). the numbers of animals for each collection day were two. the two females of experiment two were treated with a superovulation protocol. when the alpacas did not have any dominant follicle (smaller than 3 mm) and no cl present, were given a single dose of 400 ui of ecg the alpacas were monitored by tus to evaluate follicular development on day 2, 4 and 6 after ecg administration. when follicles reached 6 mm diameter, alpacas were given 75 ug of gnrh to induce multiple ovulations. animal were sacrificed seven days after ovulation. ovulation and corpus luteum formation in experiment one, eight females ovulated, and the cl was not detected before day 4 after ovulation. in our experience the cl can be imaged properly at four days after ovulation (five days after mating). in experiment two, there were three (10, 11, and 11 mm), and four corpora lutea (9, 10, 10 and 12 mm), in each of the females. in both cases the cls were imaged properly. postmortem reproductive tract collections and examination all females were slaughtered according to the approved methods set forth by the peruvian veterinary authorities. following slaughter, the entire reproductive tracts were removed en block and immediately transported to the laboratory. since the slaughter facility was located in the same building that the laboratory, transportation of the reproductive tracts, took approximately two minutes. the number of cls and the side of ovulation (left and right) were correlated with the number and side of the follicles observed in each animal by tus. after collapse of the ovulatory follicle on day 1, we observed reduced size of the newly-formed cl (4 mm), with a blood clot in the center corpora lutea were larger on the following days, and became round by day 4, and reached their mature size on days 6 and 7 (9-12 mm). after recording the ovarian structures, reproductive tracts from both experiments were submitted for oviductal and uterine flushing. oviductal flushing technique the uterine tube ipsilateral to the side of ovulation was dissected from the surrounding tissue in order to stretch the convoluted oviduct. the utj and its papillae were also removed. the uterine tube was flushed with 20 ml of phosphate buffered saline solution, containing 10% of fetal calf serum and 10,000 units/ml penicillin and 10,000 mcg/ml streptomycin. a 16 gauge blunt needle connected to a syringe was inserted 2 cm into the uterine tube through the infundibular ostium, and flushed normograde towards the papillae. the flushing medium was recovered in a watch glass and examined under a dissecting microscope for the presence of oocytes. clinical theriogenology • volume 5 number 2 • june 2013 94 uterine horn flushing technique the uterine horns from females in experiment one were flushed separately starting with the uterine horn ipsilateral to the cl-bearing ovary. in experiment two, both uterine horns were flushed separately. a 16 fr foley catheter was gently threaded through the cervix and placed at the base of the uterine horn. the catheter was maintained in place by filling the cuff balloon with 5 to 10 ml of flushing medium. the same procedure was applied to the uterine horn contralateral to the cl-bearing ovary. each uterine horn was flushed four times with 20 to 60 ml of warm (37°c) flushing medium. fluid was recovered into a graduated embryo filter and transferred to petri dishes containing commercial enriched holding medium (vigro holding plus® agtech, manhattan, ks), under a 4x and 10x stereomicroscope to determine the presence and condition of the oocytes. statistical analysis descriptive statistics were used to describe the oocytes on each collection date. results results from experiment one showed that the interval from gnrh treatment to ovulation was 27.89 ± 1.6 hours (range 26-32 h). the ovulation rate was 83.33% (10/12), and the collection rate was 66.66% (8/12). on day 1 post-ovulation, the two ova collected from oviducts of the two females showed normal morphology, surrounded by the cumulus oophorus. the ova were slightly smaller (150 µ) compared to the ova collected after normal mating (179.47 ± 9.47µ). in comparison the oocytes of llamas ranged from 172 to 200 µ (mean ± sd, 184±14).10 on day 2 post-ovulation, one ovum collected from two females contained clear, shrunken, and dissociated cytoplasm, and did not display normal cell division. on day 3 post-ovulation, one oocyte collected from oviducts of the two females showed the cumulus oophorus cells. this finding was similar to that observed on day 1 post-ovulation; a completely disorganized and clear cytoplasm. no oocytes were collected from tracts revovered on day 4 and day 5 post-ovulation. on day 6 post-ovulation, two oocytes were collected from the uterine horn ipsilateral to the side of ovulation in two females. the oocytes were shrunken with broken zona pellucida, and a few cells from the cumulus oopohorus were attached to the broken zona. in experiment two, two broken, shrunken and degenerated oocytes were collected from one alpaca with one cl on the left ovary and two cls on the right ovary. one oocyte was collected from the left uterine horn, and one was collected from the right uterine horn. the other female alpaca had two cls on the left ovary and two on the right ovary; two oocytes were collected from the left uterine horn and one oocyte was collected from the right uterine horn. the varying degrees of degeneration and disintegration of oocytes collected from the superovulated females were probably a result of different ovulation times and other unknown factors. discussion this is the first study that attempted to determine the fate of the unfertilized ova in alpacas. the interval between gnrh administration and ovulation (27.9 ± 1.6 hours) is similar to the results of previous studies in llamas and alpacas.10,11 time of ovulation following administration of gnrh remains quite variable which complicates the timing of oocyte collection based only on the time of administration of gnrh (24 to 36 hours after ovulation induction). the alpaca breeding season in peru, occurs during the rainy season (december to march) which provides abundant green grass. however, some studies have demonstrated that supplementing alpacas during the dry season when the food supply is low can yield the same results as in the wet season and show the importance of nutrition in regulating the breeding season.12,13 ovulation rates in experiments one and two were 83.33% and 100%, respectively, which confirms earlier reports that high ovulation rates can be achieved in this species with good nutrition and based on ultrasonographic detection of a mature follicle. the overall oocyte collection rates in experiment one and experiment two were 70% and 85%, respectively. in the current study, oocytes were found in the oviduct from day 1 to day 3 post-ovulation, were at different stages of degeneration, and they were found in the left and right uterine horns at 6 days postclinical theriogenology • volume 5 number 2 • june 201395 ovulation with degenerated or with broken zona pellucida. during the oviductal transport of the oocytes, they must be in the area of the isthmus on day 4 and 5 post-ovulation. the isthmus area which is highly convoluted narrows somewhat at the point of entry into the uterus may be the reason that we were not able to collect any oocytes on these days in experiment one or they were blocked at the utj. our results are in agreement with the observation that it is difficult to find embryos at day 6 and 7 after mating.8,9 therefore, the collection rate for females on days 4, 5 and 6 post-ovulation may underestimate the actual number of oocytes present in the uterine tubes. our findings contradict earlier hypothesis that camelids may have selective transport of fertilized ova through the utj as seen in the mare.4 the results show that unfertilized oocytes reach the uterine horns on day 6 after ovulation demonstrating that there is no selective transport. therefore, the hatching/hatched embryo may not be needed to open the utj. we hypothesize that as the embryo enlarges and develops into a blastocyst, passage through the utj is slowed and that the process of hatching from the zona pellucida may be the result of mechanical action of the strong utj papilla muscle. experiment two was performed in order to examine the hypothesis that in superovulated females the first embryo to descend to the uterus sends the signal to open the utj and enter the uterine cavity and, as a consequence, the remaining embryos as well unfertilized eggs enter the uterus. however, our results contradicte this hypothesis since unfertilized eggs were found in the uterine horns at day 6 post-ovulation in single ovulating and superovulated females. we conclude that in alpacas unfertilized eggs are located in the oviduct ipsilateral to the side of ovulation from day 1 to day 5 post-ovulation. they continue their journey and enter the uterine horns on day 6 after ovulation in both single ovulating and superovulated females. the unfertilized oocytes in alpacas are not retained in the utj as is the case in mares. we recommend further studies on the mechanism of the oocyte and embryo passage through utj in alpacas. references 1. fernandez-baca s, hansel w, saatman r, et al: differential luteolytic effects of right and left uterine horns in the alpaca. biol reprod 1979;20:586-595. 2. fernández-baca s, sumar j, novoa c, et al: relación entre la ubicación del cuerpo lúteo y la localización del embrión en la alpaca. rev inv pec (ivita). univ nac s marcos 2, 1973. p. 131-135. 3. sumar j, leyva v: relación entre la ubicación del cuerpo lúteo y la localización del embrión en la llama (lama glama). anales iii conv. int. sobre camélidos sudamericanos, viedma, argentina. 1979. p.124-127. 4. van nierkerk ch, gerneke wh: persistence and parthenogenetic cleavage of tubal ova in the mare. onderstepoort j vet res 1966;33:195-231. 5. bouker da, kyle ce, mcevoy tg, et al: superovulatory responses to ecg in llamas (lama glama). theriogenology 1995;44:255-268. 6. flood pf, jong a, betteridge kj: the location of eggs retained in the oviduct of mares. j reprod fertil 1979;57:291294. 7. sumar j: embryo transfer in domestic south american camelids. anim reprod sci 2013;136:170-177. 8. sumar j: studies on reproductive pathology in alpacas {thesis].uppsala: swedish university of agricultural sciences; 1983. 9. bravo, pw, moscoso j, ordoñez c, et al: transport of spermatozoa and ova in female alpaca. anim reprod sci 1996;43:173-179. 10. cárdenas h: desarrollo morfológico, transporte y supervivencia embrionaria en alpacas. xx reunión científica anual, asociación peruana de producción animal, univ nac agraria de la selva, tingo maría, perú. 1997. p. 64-66. 11. del campo mr, del campo ch, adams gp et al: the application of new reproductive technologies to south american camelids. theriogenology 1995;43:21-30. 12. ratto m, huanca w, adams jp: comparison of the effect of natural mating, lh, and gnrh on interval to ovulation and luteal function in llamas. anim reprod sci 2010;91:299-306. 13. rodriguez c, arellano p, torres r, et al: time interval between natural mating vasectomized mating and gnrh on ovulation in alpacas. icar proceedings satellite meeting on camelid reproduction, vancouver, canada 2012. p. 1518. clinical theriogenology • volume 5 number 2 • june 2013 96 2019 management of a large uterine cyst protruding through the cervix of a mare management of a large uterine cyst protruding through the cervix of a mare jason grady,a kelli almes,b warren bearda adepartment of clinical sciences and bdiagnostic medicine and pathology college of veterinary medicine, kansas state university, manhattan, ks abstract a 17-year-old, multiparous, american quarter horse mare was presented for breeding 7 days after foaling. transrectal ultrasonography revealed multiple endometrial lymphatic cysts; 2 at the base of the left uterine horn and 1 in the caudal uterine body that extended into the cervical lumen (the latter was surgically removed). the mare was artificially inseminated on the subsequent estrous cycle, confirmed in foal 30 days after ovulation and delivered a viable foal without complications 350 days after ovulation. keywords: lymphatic cyst, cervix, endometrial cyst, endometrium background a multiparous, american quarter horse mare was presented for artificial insemination at equine reproduction services at kansas state university in 2016. her last foal was born in 2015 and was diagnosed not-pregnant following artificial insemination that season. during the 2016 breeding season, a breeding soundness examination was performed. during an ultrasonographic examination, 2 endometrial lymphatic cysts were detected at the base of the left uterine horn; 1 was 15.7 x 8.7 mm and the other 9.6 x 9.0 mm. endometrial cytology and culture were performed. a non-inflammatory cytology and a mixed population of bacteria (streptococcus equinus, streptococcus dysgalactiae, and actinobacillus sp.) considered non-pathogens were recovered on enrichment only. no other abnormalities were detected on physical or vaginal examination or on transrectal palpation ultrasonography. on the following estrous cycle, the mare was given deslorelin acetate (sucromate™, thorn bioscience, louisville, ky), and then artificially inseminated once with fresh, cooled semen. using transrectal ultrasonography, the mare was confirmed in foal 16 days after ovulation. she was maintained at pasture with other brood mares and supplemented with grass hay and commercial feed. the mare had an unattended foaling on may 24, 2017 at 331 days gestation. the owner reported that the foal was found dead enclosed within the amnion. neither the fetus nor fetal membranes were examined or submitted for evaluation. case presentation the mare presented with a good body condition score (5/9) 7 days after foaling for breeding during foal heat. her perineal conformation was within normal limits and no vaginal discharge was present. transrectal palpation revealed a postpartum uterus that was involuting and of expected size for 7 days postpartum. transrectal ultrasonography revealed the presence of the previously detected endometrial lymphatic cysts at the base of the left uterine horn with no change in size. however, a large fluid-filled mass (figure 1) with mixed echodensity, 35.7 x 44.7 mm, was present at the caudal uterine body and projected into the cervical canal. a tail wrap was placed and the perineum cleansed with a gloved hand and cotton using warm water and a mild detergent soap. digital examination of the cervix revealed a fluid-filled lobulated structure originating within the caudal uterine body and extending into the cervical canal. the mass was attached to the dorsal wall of the uterine endometrium in a sessile manner with a broad fibrous base approximately 50 mm long and could not be reduced back into the uterine body. the mare was sedated with 0.01 mg/kg detomidine hydrochloride iv (dormosedan®, zoetis inc., kalamazoo, mi). hysteroscopy was done with a cold-sterilized video endoscope, confirming the presence of a mass in the cervical canal and caudal uterine body. differential diagnosis the differential diagnosis considered for a fluid-filled mass in this mare included a large endometrial lymphatic cyst, hematoma, abscess and neoplasia. endometrial cysts are commonly described as having anechoic fluid or appearing hypoechoic on transrectal ultrasonography.1 in this case, the mixed 149 clinical theriogenology • volume 11, number 2 • june 2019 echodensity of the fluid and the hyperechoic structure within the fluid were not typical for an endometrial lymphatic cyst. furthermore, the location of the mass at the caudal uterine body and within lumen of the cervix was atypical, as endometrial cysts are more commonly located at the base of uterine horns.2 based on the mixed echodensity, location, and recent foaling, it was hypothesized that the mass could be a hematoma or an abscess secondary to trauma during foaling. although uterine neoplasia (leiomyoma, leiomyosarcoma, fibroleiomyomas, and adenocarcinoma) was considered as an initial differential, it was considered unlikely, as the mass did not have the typical consolidated, hyperechoic appearance on transrectal ultrasonography and hysteroscopy that would be expected with any of the uterine neoplastic conditions.1 treatment the mare was sedated with 0.01 mg/kg detomidine hydrochloride iv (dormosedan®, zoetis inc.,). a tail wrap was placed and the perineum cleansed with a gloved hand and cotton, using warm water and a mild detergent soap. the uterine mass was removed using a snare technique.3,4 approximately, 150 cm of obstetric wire (ob saw wire, jorgensen laboratories, loveland, co) was passed through two sterilized artificial insemination pipettes to create a loop between them. a sterile obstetrical sleeve with sterile lubricant was placed on one arm and passed along with the loop through the vagina. the loop was placed around the mass, and tightened by pushing the pipettes inward against the base of the mass tightening the wire. the wire was then moved back and forth through the pipettes cutting through the base of the mass and the mass was subsequently removed per vaginum.3,4 once removed, a lobulated, fluid-filled mass measuring approximately 70 mm long and 50 mm in diameter was submitted for histopathology and cytology. uterine lavage with 2 liters of sterile lactated ringers solution was performed immediately following the mass removal and again the following day with 1 liter sterile lactate ringers solution. each uterine lavage was followed by 20 usp units of oxytocin im (oxytocin, bimeda, oakbrook terrace, il). the mare was given 1.1 mg/kg flunixin meglumine iv (banamine®, merck animal health, madison, nj) following mass removal and prescribed 2 grams phenylbutazone paste (phenylbute® paste, phoenix pharmaceutical, st, joseph, mo) to be given orally once daily for 2 days, starting the following day. diagnostic testing the mass was submitted to kansas state university veterinary diagnostic laboratory (1800 denison ave, manhattan, ks) for fluid analysis and histopathology. the fluid had low nuclear cellularity, with a low density of erythrocytes, occasional extracellular pink proteinaceous globules and pale basophilic backgrounds with multiple protein crescents. nucleated cell populations consisted of few nondegenerative neutrophils and foamy macrophages, some of which contained phagocytized protein material. no infectious agents were observed and was reported as a mixed cell infiltrate with protein debris supportive of cystic material. histopathology revealed a roughly round cystic structure composed of a large central cavity lined by attenuated cells that resembled lymphatic endothelium (figure 2). the cystic cavity contained eosinophilic fibrillary debris with scattered moderate numbers of macrophages, lymphocytes, and plasma cells that compressed the surrounding endometrial tissue (which was moderately to markedly edematous). there were multifocal areas of mild to moderate hemorrhage within the surrounding endometrial mucosa. throughout the mucosa and submucosa of the endometrium there was moderate to marked dilation of lymphatics and scattered inflammatory infiltrates of lymphocytes and plasma cells. these histopathologic findings were most consistent with an endometrial lymphatic cyst. outcome the mare ovulated the day after removal of the endometrial lymphatic cyst. five days after ovulation, cervical tone and function were assessed via vaginal examination and digital palpation. no abnormalities were detected, but potential for cervical adhesion formation was discussed with the owner, as well as incompetent cervical function throughout gestation, predisposing to placentitis. as the mare owner elected to rebreed the mare, 10 mg dinoprost tromethamine (lutalyse®, zoetis inc.,) was given im. 150clinical theriogenology • volume 11, number 2 • june 2019 deslorelin acetate (sucromate™, thorn bioscience, louisville, ky) was given to the mare while in estrus, with a 35 mm follicle present. the mare was artificially inseminated with 1 dose of fresh, cooled semen 12 days after cyst removal (21 days after foaling) with ovulation confirmed the following day. the mare was monitored for the presence of mating induced endometritis with transrectal ultrasound prior to discharge. she was confirmed in foal 14 and 30 days after ovulation with transrectal ultrasonography. a caslick procedure was performed following confirmation of pregnancy on day 14. recommendations were made to evaluate the mare’s pregnancy with transrectal palpation and transrectal ultrasonography at 60 70 days of gestation, and at monthly intervals starting at 7 months gestation to monitor for signs of placentitis. further recommendations were made to vaccinate the mare for equine herpes virus 1 at 5, 7, and 9 months in pregnancy and to give other pre-foaling vaccinations. in addition, she was to have the caslick opened 4 6 weeks prior to her expected due date. at 350 days after ovulation, the mare delivered a viable foal without assistance. discussion endometrial lymphatic cysts have been reported in a variety of species, including horses, cattle, ewes, pigs, cats, dogs, elephants and humans.5 uterine cysts are best described as anechoic, fluid-filled endometrial structures filled with yellow, watery fluid that generally increase in size over months to years. they may be seen as an individual, compartmentalized sacs, or as multilobulated structures divided by septa. they are often pedunculated, but may also be attached by a sessile base.5 if large enough or numerous they may be detected via transrectal palpation, but are best characterized with transrectal ultrasonography. the incidence of uterine cysts has been reported to be 22 55%, with an increasing incidence with mare age and parity.5-7 uterine cysts are more likely in mares 10 years of age or older, but have been detected in a mare as young as 3 years.6 in 1 study, mares > 11 years of age were 4.2 times more likely to have uterine cysts than mares < 11. in an earlier study with 259 mares, 22.4% had uterine cysts, although 73.1% of the uterine cysts were in mares > 14 years.7 in another study with 76 mares, 44.7% had uterine cysts, with 2 mares < 7 years of age, 12 were from 7 14 years, and 20 were > 14 years.5 number of cysts was not different between left versus right horns, but frequency of cysts was higher in area of vesicle implantation (left horn middle, right horn middle, uterine body middle, cornualbody junction, left horn posterior, right horn posterior, and anterior uterine body) compared to the extremities (left horn anterior, right horn anterior, and posterior uterine body).5,7 two types of uterine cysts have been described, namely glandular and lymphatic.2,3,5 glandular cysts, located in the lamina propria, are generally small, ranging from only a few millimeters up to 1 cm in diameter.5,6 these glandular cysts develop from distention of endometrial glands due to chronic endometrial changes and more specifically periglandular fibrosis.1,4-6 glandular distention then occurs from the strangulating effect of the periglandular fibrosis on endometrial glands and decreased uterine myometrial tone resulting in decreased flow of secretions.6 glandular cysts have been reported as a normal finding during pregnancy.5,6 the significance of glandular cysts in cases of infertility or subfertility is yet to be defined. lymphatic cysts, the most common form of uterine cysts, likely have more clinical relevance for fertility when compared to glandular cysts. lymphatic cysts, or pooling of lymphatic fluid, initially develop in the stroma of the endometrium as microscopic structures from obstructed lymphatic channels or potentially from the gravitational effects of an enlarged gravid uterus, or postpartum uterus.5-7 as they further develop and enlarge, due to continued pooling of lymphatic fluid, they distend and enter into the uterine lumen. diameters of lymphatic cysts have been reported to range between 2 48 mm, and appear to increase in size with age of the mare.5-7 clinically, lymphatic cysts may or may not have an impact on pregnancy rate and foaling rate. the presence of lymphatic cysts may make initial pregnancy detection challenging as they may be confused with an early embryonic vesicle, or they may make the loss of pregnancy difficult to confirm. the clinical impact of endometrial lymphatic cysts is dependent on their size, number and location. it was reported that when the number of cysts was > 5, there was a negative correlation with foaling rate.6 endometrial cysts may negatively affect fertility by impeding mobility of the embryonic vesicle and thus blocking maternal recognition.7 furthermore, direct contact between the 151 clinical theriogenology • volume 11, number 2 • june 2019 embryonic vesicle and the cyst, instead of endometrium, could negatively affect nutrient absorption by reducing the area of total placental contact and nutrient exchange.7 pregnancy rate at day 40 in mares without uterine cysts was 88% compared to 71.4% in mares with uterine cysts,7 although embryo loss between days 14 and 40 was not significantly different between the 2 groups.7 using transrectal ultrasonography, endometrial cysts should be mapped out and their size and location documented. depending on the number, size and location of endometrial cysts clinicians may elect to not initiate treatment or cyst removal, although removal of large or multiple cysts may increase fertility.3 multiple methods of cyst removal have been described, including a snare, laser hysteroscopy, loop cautery, and uterine biopsy or needle aspiration. the location of the caudal uterine body and involvement of the cervical canal was not a typical finding of endometrial lymphatic cysts. in the present case, it is unknown when the endometrial cyst at the level of the caudal uterine body and cervical canal developed, although it either developed during pregnancy or rapidly postpartum. it was not present or detectable via transrectal ultrasound when the mare was managed for breeding in 2016. due to delivery of a nonviable foal in 2017, it is possible that it developed during the pregnancy and jeopardized the pregnancy by affecting cervical function, perhaps predisposing the mare to an ascending placentitis. it is also possible that it rapidly developed and extended into the lumen after foaling. in order to restore cervical function and attempt to allow the mare to successfully carry a pregnancy to term, surgical removal was performed, carefully trying to not remove any portion of the cervical epithelium as the cyst was removed from the caudal uterine body. due to its proximity to the cervix, main complications considered following removal of the cyst included cervical adhesions leading to a future dystocia, inability of the cervix to function properly predisposing the mare to subfertility, early embryonic death, or placentitis. other complications considered less likely were intrauterine adhesions or cyst reoccurrence. if cervical adhesions were to occur, they were more likely to be linear and much less likely to be circumferential which should result in a lower occurrence of dystocia compared to circumferential adhesions. learning points • lymphatic cysts may make initial pregnancy detection challenging, as they may be confused with an embryonic vesicle, or a non-viable embryo. • the clinical impact on fertility of endometrial lymphatic cysts is dependent on their size, number, and location. • presence of > 5 endometrial lymphatic cysts can reduce foaling rate. • removal of the cyst is possible by a snare and methods such as laser hysteroscopy, loop cautery, uterine biopsy, or needle aspiration. references 1. rickets s, troedsson m: fertility expectations and management for optimal fertility. in: samper jc, pycock jf, mckinnon ao, editors. current therapy in equine reproduction. st louis: saunders elsevier; 2007. p. 53-69. 2. immegart h: infertility due to noninflammatory abnormalities of the tubular reproductive tract. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology 2nd edition. st louis: saunders elsevier; 2007. p. 153-157. 3. deluca ca, gee ek, mccue pm: how to remove large endometrial cysts with an improvised snare: a simple technique for practitioners: proc annu conv am assoc equine pract 2009;55:328-330. 4. dascanio j: endometrial cyst removal. in: dascanio j, mccue p, editors. equine reproductive procedures. ames: wiley blackwell; 2014. p. page 74-76. 5. ferreira jc, gastal el, ginther oj: uterine blood flow and perfusion in mares with uterine cysts: effect of the size of the cystic area and age. reproduction 2008;135:541-550. 6. stanton mb, steiner jv, pugh dg: endometrial cysts in the mare: j equine vet sci 2004;24:14-19. 7. tannus rj and thun r: influence of endometrial cysts on conception rate of mares: j vet med assoc 1995;42:275283. 152clinical theriogenology • volume 11, number 2 • june 2019 figure 1. ultrasound image of a fluid-filled mass with mixed echodensity, 35.7 x 44.7 mm, that extended from the posterior region of the uterine body into the cervical canal. figure 2. histologic image of an endometrial lymphatic cyst and adjacent endometrial tissue stained with hematoxylin and eosin at 20x magnification. the cyst wall was lined by attenuated endothelium and the lumen (star) contained eosinophilic fibrillary material (arrow). the endometrium had dilated lymphatics (diamond), mild infiltrates of lymphocytes and plasma cells, as well as mild multifocal hemorrhage. 153 clinical theriogenology • volume 11, number 2 • june 2019 omniblank: << /ascii85encodepages false /allowpsxobjects false /allowtransparency false /alwaysembed [ true ] /antialiascolorimages false /antialiasgrayimages false /antialiasmonoimages false /autofiltercolorimages true /autofiltergrayimages true /autopositionepsfiles true /autorotatepages /none /binding /left /calcmykprofile (u.s. web coated \050swop\051 v2) /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /checkcompliance [ /none ] /coloracsimagedict << /hsamples [ 1 1 1 1 ] /qfactor 0.15000 /vsamples [ 1 1 1 1 ] >> /colorconversionstrategy /usedeviceindependentcolor /colorimageautofilterstrategy /jpeg /colorimagedepth -1 /colorimagedict << 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affect the scrotal circumference of the bull and its impact on herd reproductive performance. a review. factors that affect the scrotal circumference of the bull and its impact on herd reproductive performance. a review. roberto a. palomares, dwight f. wolfe food animal section, department of clinical sciences; college of veterinary medicine, auburn university, auburn, al abstract scrotal circumference (sc) is an indirect measure of testicular weight and therefore of the amount of sperm-producing tissue in bulls. the seminiferous tubules, where spermatozoa are produced, constitute more than 80% of the testicular mass. scrotal circumference is positively correlated with important productive and reproductive traits such as age at puberty and semen quality in the bull. since sc has a high heritability estimate, it is highly correlated with age at puberty and fertility of the male and female offspring of the bull. therefore, bull selection based on sc will improve the reproductive traits of the offspring, with significant enhancement of the herd reproductive performance. this article will review the importance of sc to breeding soundness evaluation (bse) of the bull as a factor to increase the precocity and reproductive performance of the herd. keywords: scrotal circumference, bulls, puberty, fertility introduction reproductive performance is one of the important factors determining profitability of cow-calf production systems. to maximize reproductive performance each cow within the herd should raise a calf to weaning each year. this goal can be achieved if both the cows and bulls have optimal fertility during a limited breeding season (approximately 70 days). since a single bull is generally used to breed numerous cows, the evaluation of bull fertility prior to breeding is of supreme importance to reach breeding success. breeding soundness evaluation of bulls is a simple, systematic, and cost-effective procedure to estimate bulls’ reproductive soundness and classify their potential fertility. the evaluation of a bull’s breeding soundness potential consists of a general physical soundness examination, examination of both the external and internal genitalia, measurement of sc, and semen quality evaluation.1 the measurement of sc is an easy, reproducible and inexpensive method to determine testicular development. it is an indirect estimate of the amount of sperm-producing tissue, being directly correlated with semen quality and therefore with bull fertility.2-4 since testicular development is an excellent indicator of puberty and future breeding performance of the bull, producers should base early selection of young bulls on the measurement of sc to assure the value of future potential breeders. additionally, sc has a moderate to high heritability5,6 (0.38 to 0.68) and is strongly correlated with age at puberty and fertility of male and female progeny. thus, bull selection based on sc will improve the reproductive traits of the offspring, with significant improvement of herd reproductive performance. the society for theriogenology (sft) has established minimum values for sc according to the age of the bull to provide a useful and well-documented criterion to be used in conjunction with the physical examination and seminal evaluation to classify bulls as potential breeders.7 the objective of this article is to review the importance of sc as a selection factor to improve precocity and reproductive performance of the individual bull and the herd. we will also review the anatomic and physiological aspects of testicular development, how to measure sc, the factors that influence sc and the impact of sc on herd reproductive traits. scrotum and testes, anatomical aspects bull testes are glands that function as reproductive and endocrine organs producing sperm and secreting male sex hormones (mainly testosterone). testes are located in the scrotum, a protuberance of the ventral abdominal skin. the scrotum consists of skin, sweat glands, and the tunica dartos, and is lined 115 clinical theriogenology • volume 3 number 2 • june 2011 by the tunica vaginalis parietalis, an extension of the parietal peritoneum. the testes are maintained in this location as part of the system to maintain thermoregulation that is crucial for normal spermatogenesis. the testes must be cooler than core body temperature and there is a temperature gradient of 4 to 6° c from base to apex of the scrotum. the tunica dartos functions in conjunction with the cremaster muscle, part of the spermatic cord, to elevate the testes toward the abdomen to reduce heat loss in periods of cold, or to relax and provide greater surface for heat loss during times of normal or excess heat load within the scrotum.8,9 the spermatic cord exits the inguinal ring and consists of the testicular artery, vein and nerve, the ductus deferens, and the cremaster muscle. a few centimeters dorsal to the testis, the testicular artery forms an extensive pampiniform plexus with the testicular vein. the function of this plexus is for venous blood returning from the testis to cool arterial blood before the artery enters the testis proper. after leaving the pampiniform plexus, the testicular artery continues ventrally to enter the testis at its distal pole. the epididymis is the structure for sperm maturation and storage before ejaculation and consists of three areas. the head or caput epididymis is a turgid structure that lies on the dorsal pole of the testis and continues as the body or corpus epididymis located on the medial aspect of the testes. the tail or cauda epididymis is also turgid and prominent on the distal pole of the testes. these structures transport sperm from the testis to the ductus deferens and are major sites of sperm maturation. testicular development; hormonal aspects testicular development depends mainly on endocrine mechanisms (gonadotropin and metabolic hormone secretion) occurring around puberty. in bulls, luteinizing hormone (lh) and follicle-stimulating hormone (fsh) concentrations transiently rise between two and five months after birth (early gonadotropin rise) as a consequence of increased endogenous gonadotropin releasing-hormone (gnrh) pulse frequency.10,11 this hormonal secretion results in cellular mechanisms that lead to accelerated testicular growth (after gonadotropins concentrations decrease) and the beginning of spermatogenesis.10-13 delayed puberty in bulls of some breeds (e.g. bos indicus breeds such as brahman and its crosses) could be the result of delayed gonadotropin rise or slower initiation of spermatogenesis.14,15 in addition to breed variations there are within breed individual differences in gonadotropin secretory patterns (timing and magnitude of the rise) which may also be responsible for differences in testicular development and age at puberty between earlyand late-maturing bulls.11,16 accordingly, calves that have greater fsh and lh levels during calfhood would be expected to develop larger testes and possibly reach puberty at an earlier age.17 the nutritional level in young bulls also influences prepubertal gonadotropin secretion, testicular development and onset of puberty. in bulls, a significant peripubertal increase in the concentrations of the metabolic hormones such as leptin, insulin and insulin-like growth factor-i (igf-i) and decreased growth hormone (gh) concentrations, with no changes in gonadotropin concentrations is responsible for the increase in testosterone concentration and most of the variation in scrotal circumference and paired testes volume. this indicates that these hormones might be involved in a gonadotropin-independent mechanism regulating the testicular development.18 insulin-like growth factor-i and insulin stimulate the increase in testosterone by regulating the proliferation, differentiation and function of leydig cells and sertoli cells, which are associated with increased testicular development and spermatogenesis.12 leydig and sertoli cells produce igf-i, indicating the existence of a paracrine/autocrine mechanism of testicular regulation involving igf-i.19,20 examination of the scrotum, testes and scrotal circumference during the breeding soundness evaluation the bse is a simple and systematic procedure to estimate a bull’s reproductive potential. it is intended to improve herd fertility by defining thresholds above which bulls would be classified as satisfactory potential breeders.21 the bse procedures established by the sft consist of evaluating physical soundness, sc, semen quality, and overall health of the bull. 116clinical theriogenology • volume 3 number 2 • june 2011 during the physical examination of the reproductive organs, it is important to evaluate the scrotum for the presence of lacerations, scars or other pathology.7,22 the testes must move freely within the scrotum and there should be no more than approximately ten percent difference in size between the paired testes. careful palpation of the testes must be done to determine the consistency, which should be firmly resilient. additionally palpate each testis deeply to determine areas of firmness that might indicate granulomas, fibrosis, tumors, abscesses or calcification. alternatively, excessive softness is associated with testicular degeneration. there should be no adhesions or fluid accumulation between the tunica vaginalis parietalis lining the scrotum and the tunica vaginalis visceralis covering the testis. the head, body and tail of the epididymides must be palpated for the presence of normal development, or inflammation with pain and granulomas. palpate the spermatic cord for aneurysms, abscesses, granulomas, excess fat, or other anomalies.23 after physical examination of the scrotum and testes, it is crucial to measure sc. the sc is an indirect measurement of testicular weight and consequently sperm-producing tissue, because sperm are produced in the seminiferous tubules, which constitute 80-90% of the testicular weight.23 therefore, sc is a reliable indicator of the sperm producing tissue providing a useful tool for predicting bull fertility. scrotal circumference can be measured following the method described by the sft.7 the testes must be firmly forced ventrally in the scrotum. the thumb and fingers are placed on either side of the neck of the scrotum and a flexible, non-stretchable metric tape measure is placed around the largest circumference of the scrotum, pulled snugly to establish firm contact of the tape with the entire circumference. bulls with adequate sc for their age are more likely to become satisfactory sires than bulls with a small scrotal circumference.24 thus, selection for more sperm-producing tissue, as indicated by increased sc, may result in more male progeny passing a bse due to improved sperm quality.3 in contrast, small sc and high percentage of sperm abnormalities are the most likely reasons for classifying a bull as an unsatisfactory potential breeder.25-27 the sft guidelines require that all breeding bulls should have a minimum sc of 30 cm at 15 months of age.28 kasari, et al29 disagreed with this criterion, suggesting that slightly higher values (32–33 cm) should be used in some breeds with larger testes (e.g., simmental, angus and maine-anjou). conversely, hopkins and spitzer28 stated that no measurement less than 30 cm should be allowed, even for breeds that are of smaller stature. in addition, the sft guidelines require that the sc of bulls that are older than two years should be greater than 34 cm. changes in testis size after two years of age are breed dependent. chenoweth, et al30 reported that testis growth continues beyond two years in angus, but not in hereford bulls. the sc thresholds recommended by the sft for a satisfactory breeding bull based upon age are shown in table 1. scrotal circumference as a decision maker for early selection of bulls puberty the beginning of puberty is considered to occur when the bull’s first ejaculate has at least 50 x 106 sperm per ml with at least 10% progressive motility. measurement of sc is a good indicator of age at puberty in bulls. in general, puberty is achieved when scrotal circumference is between 28 and 30 cm. a previous study demonstrated that 52% of bulls are pubertal when their scrotal circumference reached 28 cm, while 97% have reached puberty by the time their sc was 30 cm.31 in spite of the dramatic changes in testicular development at puberty, sperm concentration, percentage of progressively motile sperm, proportion of sperm with normal structure and seminal protein concentration all increase significantly until at least four months after puberty.23 weanling bulls the first selection and culling of bulls is generally at weaning when they are seven to ten months old. the most important reproductive criterion for selection of bulls at this age is the measurement of sc. however, most culling at weaning is based on the breeder’s assessment of the bull’s growth potential. 117 clinical theriogenology • volume 3 number 2 • june 2011 the sc measurement in weaned bulls is helpful to predict the yearling sc. coe and gibson32 evaluated 264 bulls representing 13 beef breeds and found that at 200 days of age calves with sc >23 cm had a 95% probability of achieving sc >34 cm by 365 days of age whereas calves with sc <23 cm only had a 54% probability of achieving sc >34 cm by 365 days. young bulls with significantly small testes should be culled as they have a low probability of attaining adequate sc by one year of age. yearling bulls with small testes (sc below 30 cm) are not likely to catch up over time and will still have small testes at two years of age. culling at weaning minimizes costs related to maintaining cull bulls or inadvertently entering them in performance test programs.23 producers should measure the sc at weaning in order to have an early estimate of the future reproductive potential of young bulls. yearling bulls the final selection of bulls for breeding potential should be at 12 to 16 months of age. determination of sc is essential in the evaluation of yearling bulls because it is an excellent indicator of whether or not the animal is pubertal and at this age most conformational abnormalities are apparent.23,33 in yearling bulls, a large scrotal circumference (>30 cm) indicates early reproductive maturity which will be inherited by his progeny. studies in yearling bulls indicate drastic differences in semen quality according to age,34,35 which are summarized in table 2. clearly, after 12 months of age a few months make a tremendous difference in the amount and quality of semen a bull may produce despite the low testicular development rate evidenced by the sc. another study testing physical and semen traits in yearling beef bulls (angus, brahman, hereford senepol, romosinuano and nellore x brahman) in florida showed that qualitative semen traits increased with bull age, particularly from 12 to 18 months.36 bull age was positively associated with normal spermatozoa (p<0.01) and negatively with primary abnormalities (p<0.001), with breeds differing in their age curves for both traits. sperm motility increased and spermatozoa with primary abnormalities decreased with bull age. the authors stated that the use of sc thresholds linked more with growth traits than with calendar age would improve comparisons of relative reproductive development in young, pasture-raised bulls in semi-tropical environments, particularly in bos indicus breeds.36 one troublesome aspect of bse in yearling bulls is the presence of a high percentage of sperm with proximal cytoplasmic droplets. proximal cytoplasmic droplets are one of the most frequent sperm abnormalities in yearling bulls and may be associated with immaturity or testicular degeneration. since it is associated with immaturity and lower fertilization rates, the percentage of affected sperm declines as the bull completes puberty and most bulls will have satisfactory semen quality in the near future. fertilization rates are markedly lower for bulls with at least 30% sperm with proximal cytoplasmic droplets and as the percentage of droplets decreases, fertilization rates increase.37,38 therefore, according to accepted standards, if the yearling bull is physically sound and meets the other minimum requirements, the presence of proximal cytoplasmic droplets in combination with fewer than 70% normal sperm in the ejaculate would dictate that the bull be placed in the deferred category until further evaluation.39 factors influencing scrotal circumference. testicular growth and sc are influenced by several factors such as breed, age, nutrition and body weight, climate, dam parity, genetic value and testicular pathologies. age and breed age has a major effect on testicular development in young bulls from six months of age. there is rapid testicular growth in young bulls (six through 16 months of age) before and during puberty. after puberty, testes continue growing until sexual maturity is reached. differences in sc among breeds at different ages have been reported.30,36, 40-42 the sft recognized this wide variation of sc among breeds, suggesting lower thresholds for sc based on age groups but independent of breed.3 118clinical theriogenology • volume 3 number 2 • june 2011 values of sc in bulls of different breeds at one year of age are shown in table 3. simmental, gelbvieh and brown swiss bulls have the largest sc. on the other hand, limousin and blonde d'aquitane have markedly lower sc, with intermediate values for angus, charolais, maine anjou, hereford and shorthorn.42 evidence indicates that simmental, angus and zebu derived bulls (predominantly santa gertrudis), must have a minimum sc of 23 cm at 198-291 days of age to have a nearly 100% probability of attaining >30 cm sc by 365 days of age. other breeds, predominantly charolais, and polled hereford bulls require >26 cm sc at 198-291 days to reach >30 cm sc by 365 days of age.43 as yearlings, sc was significantly smaller in b. indicus sired breeds (brahman [30.7 cm], boran [31.3 cm], and tuli [30.2 cm]) than in b. taurus breeds (angus [34.4 cm], hereford [33.0 cm] and belgian blue [32.4 cm]). however, at 454 days of age, testes size was more similar among breeds (around 35 cm) than at previous time points. thus, b. indicus bulls have significantly smaller testes at seven months of age and display delayed testicular development compared with progeny of b. taurus bulls through one year of age. however, post-pubertal testes growth rate is faster in offspring of b. indicus sires compared with b. taurus breeds.14 a more recent study in italy during a ten-year period determined the mean sc and sperm concentration at 13 months of age for bulls of the chianina, marchigiana, and romagnola breeds.93 the sc of romagnola bulls (35.06 ± 2.22 cm) was significantly greater than those of chianina bulls (33.82 ± 2.31 cm) and marchigiana bulls (33.87 ± 2.41 cm). moreover, sperm concentration in romagnola bulls (875.89 ± 416.13 x 106 cells/ml) was higher than those in chianina bulls (751.63 ± 444.45 x 106 cells/ml) and marchigiana bulls (862.57 ± 421.87 x 106 cells/ml) and was positively correlated with sc.44 double-muscled breeds (e.g. belgian blue, piedmontese, pathenaise, blonde d’ aquataine and limousin) have later onset of puberty and smaller testes size at puberty and sexual maturity compared with other breeds.42 accordingly, hoflack, et al45 reported that approximately 44% of belgian blue bulls younger than four years had a sc below the minimum threshold, compared with only 17.6% of holstein bulls. two possible reasons for this difference were that sc threshold values established by the sft are too high for belgian blue bulls or these bulls are more prone to testicular hypoplasia or degeneration compared to bulls of other breeds.45 based on such breed differences, some authors have suggested the establishment of breed specific exceptions for sc thresholds, since some breeds have smaller testes.36,46,47 a requirement for allowing a lower sc threshold is that bulls of those breeds with smaller testes should still produce good quality semen. in the case of bulls with very small sc accompanied by poor sperm quality, one should suspect testicular hypoplasia or testicular degeneration and the bulls should be classified as unsatisfactory potential breeders.45 inbreeding is considered another possible factor associated with small sc and poor seminal quality in some cattle populations.48 in contrast, a high level of heterosis in some breed populations49-51 (e.g., b. indicus × b. taurus offspring) may be associated with improved testicular growth and seminal traits of the offspring.51 nutrition and body weight the level of nutrition in young growing bulls has a great influence on age at onset of puberty, testicular development and initiation of spermatogenesis. the nutritional management of the bull can be divided in two phases: calfhood and post-weaning. calfhood nutrition. good management practices that include high-energy diets with adequate protein balance, vitamins, and minerals during calfhood increase early gonadotropin secretion and hasten the onset of puberty resulting in larger testes at one year of age and earlier initiation of spermatogenesis. these high lh pulses can prime testicular development and augment testicular size at sexual maturity by stimulating the multiplication and differentiation of sertoli and leydig cells.17 on the other hand, nutritional restrictions early in life can cause permanent damage to neural centers that regulate the secretion of hypothalamic releasing factors, which negatively affects 119 clinical theriogenology • volume 3 number 2 • june 2011 gonadotropin secretion, the onset of puberty and testicular development at maturity.52 thus, nutritional deficiencies during calfhood can delay puberty, in spite of optimal post-weaning nutrition. therefore, adequate nutritional management during calfhood will provide greater benefits to reproductive function than nutritional strategies applied later during the post-weaning phase in bulls.17 post-weaning nutrition. most yearling bulls are fed high-energy diets in the post-weaning period to maximize rates of body weight gain. these high-energy diets after weaning might accelerate the beginning of puberty and result in a larger sc at one year of age; although, part of the increase in scrotal size may be due to scrotal fat. in one experiment, beef bulls were fed a high-energy diet (high concentrate) or a low energy diet (hay + one-half concentrate) from weaning for a 168-day test period.53 the calves on the high nutritional plane showed the largest sc, which could suggest accelerated pubertal development. however, the highenergy diet caused detrimental effects on semen quality.53 in another trial, beef bulls fed a diet based on 80% grain and 20% forage from weaning to 15 months of age had lower semen quality at 15 months than bulls on a medium-energy diet consisting of 100% forage.54 thus, high-energy rations continued past 12 months of age might cause detrimental effects on semen traits, possibly because of excessive scrotal fat with impaired testicular thermoregulation and altered spermatogenesis.17 furthermore, high-energy rations given after weaning may cause lameness (due to laminitis),55 rumen inflammation and liver abscesses affecting the general health status.17 in contrast, high-energy intake in the post-weaning period, up to approximately 12 months of age, does not affect future semen quality, if the diet does not result in excessive scrotal fat deposition.56 similarly, ohl, et al57 reported that beef bulls fed a high-gain ration at 11.6 to 15.3 months of age had increased testicular development compared with bulls fed a low-gain ration (sc: 34.0 vs 31.7 cm) without negative effects on sperm morphology. meacham, et al58 studied the effects of different protein levels on beef bull reproduction. testis, epididymis, and accessory sex gland weights as well as seminiferous tubule diameter and seminiferous epithelium thickness were significantly reduced in bulls fed protein-deficient rations (8.09, 5.10, and 1.35% crude protein [cp]) compared with bulls receiving diets containing 14.75% cp. sperm morphology and motility were negatively affected only in the bulls receiving 1.35% cp. in addition, rekwot, et al59 reported that sc, sperm concentration, and progressive motility were greater in bulls fed a diet with 14.5% cp compared with bulls fed a diet containing 8.5% cp. in summary, reproductive function in bulls could be improved by providing high-energy diets during calfhood and adequate nutrition in the post-weaning period.17 nutritional management must ensure optimal energy-protein balance, as well as vitamin and mineral supplements according to the animal’s needs during different growth stages. dam parity it has been demonstrated that sc is smaller in yearling bulls raised by first-parity dams, compared with bulls raised by older dams.60 in addition, parity of the dam also affected the lh concentrations in bull calves.60 the effect of parity on sc and lh levels could be due to lower milk production by firstparity dams, an in utero effect, or both. in beef cattle, milk yield is highly correlated with a calf’s weaning weight61 which has been associated with testicular development and age at puberty. in addition, primiparous dams are still growing during their first gestation so that there might be a lower availability of nutrients during pregnancy, giving rise to calves with lower birth weights compared with the calves born to multiparous dams.51 the prenatal nutrient restriction of calves born to primiparous dams may compromise development of the hypothalamic-pituitary-gonad axis,62,63 resulting in a low post-natal serum lh concentration, thereby affecting testicular development.60 genetic value some calves are genetically superior for sexual precocity than their herdmates (same breed and under the same nutritional management), and become earlier-maturing bulls. these bulls with larger 120clinical theriogenology • volume 3 number 2 • june 2011 testes have greater concentrations of lh during the period of the early gonadotropin rise (8–16 weeks of age) than later-maturing bulls.17 early selection of these bulls would contribute to preservation of such a genetic reproductive value within the cattle population. at present, many purebred cattle associations are using dna-marker technology to identify and select those individuals harboring genes associated with desirable productive and reproductive traits. dna-markers genetically associated with sc and sperm morphology have been identified and used experimentally as a tool for predicting early or late sexual maturity in b. indicus breeding programs.64 climate seasonal variations in semen quality and fertility of bulls have been reported in both temperate and tropical areas.65,66 in b. taurus bulls, the seasonal reduction in ejaculate volume and sperm output, and the increased prevalence of morphologically abnormal spermatozoa usually seen during the summer is associated with heat stress.67 prolonged exposure to extremely high temperature during summer months could result in testicular degeneration and impaired spermatogenesis in older b. taurus bulls.68 tropical adaptation (heat-tolerance) is associated with reduced testis size in b. indicus bulls.69-71 however, chacón, et al72 evaluating brahman bulls under tropical conditions did not find any relationship between the climatic variables and sc, sperm motility or morphology, which indicates that temperature is not a main factor influencing testicular development in b. indicus bulls. on the contrary, the differences in sc during the year suggested that nutrition might be a major factor affecting seasonal variations in male reproductive parameters, especially testicular size, in these sires. testicular pathologies some testicular disorders could result in altered sc. testicular hypoplasia is a heritable condition associated with a single recessive gene. it is characterized by insufficient testicular development with some derangement of germinal cells. one or both testes are smaller than normal for the bull’s age. a difference of more than 25% in size of either testis should be considered with suspicion.68 affected bulls may be subfertile or infertile because semen has poor quality (low concentration, progressive motility and percentage of normal cells), but libido might not be affected.73 when the condition is unilateral, it can be genetically transmitted causing testicular or ovarian hypoplasia in the bull’s progeny. cryptorchidism is another heritable trait which consists of failure of one or both testes to descend into the scrotum. weanling bulls that do not have both testes well descended into the scrotum should be culled regardless of sc.23 testicular degeneration is accompanied by reduced sc, testes that are palpably soft and reduced semen quality. it occurs as a consequence of infectious or other diseases that cause fever or other conditions that cause orchitis such as brucellosis, testicular trauma, heat stress, undernutrition, ischemia with degenerative vascular lesions, toxins and gonadotropin deficiency.74 other pathological conditions such as inguinal hernia and hydrocele result in abnormally large sc along with reduced thermoregulatory ability of the testes and scrotum and may cause testicular degeneration. prompt diagnosis with appropriate therapeutic intervention should be performed in order to optimize the chance for affected bulls to return to breeding soundness. effects of sc on reproductive and productive traits. as previously mentioned, sc is correlated with testes weight, age at puberty, semen quality75,76 and reproductive performance of the bulls’ daughters including age at puberty, first mating and calving7779 and fertility.80,81 moreover, the heritability for sc ranges between 0.38 and 0.68, indicating that it is a moderately to highly heritable trait.5,6 thus, selection of bulls on the basis of sc will improve the reproductive traits of the offspring, with significant improvement of herd reproductive performance. age at puberty of the bull and his sons scrotal circumference is strongly correlated (r = -0.64 to -0.80) with age at onset of puberty in beef bulls.82,83 that is, the more rapid the testicular development, the lower the age at which puberty is 121 clinical theriogenology • volume 3 number 2 • june 2011 reached. therefore, sc is a useful predictor of age at puberty among and within bulls of widely different breeds.84-86 interestingly, testis size at puberty does not vary among most breeds of bulls that exhibit marked differences in age at puberty. in general, for each 1 cm increase in bull’s sc, there will be a 0.31 cm increase in their sons’ scrotal circumference.86 puberty of the bull’s daughters age at puberty and sc in bulls are also correlated with ages at puberty (r = -0.71), first breeding (r = -0.77), and first calving (r = -0.66) of his female offspring.17,87 bulls that achieve puberty earlier and have a large scrotal circumference sire heifers that also have early puberty88 and first calving.89 it has been demonstrated that for each 1 cm increase in sc, offspring heifers achieve puberty 3.86 days earlier.90 this improvement is particularly important in cattle with delayed puberty, such as occurs in b. indicus herds under tropical conditions. these heifers calve at approximately at 38 months of age, representing a major problem that affects the reproductive performance and profitability of farms. thus, selection of bulls with early puberty, would reduce the age at puberty, first service and calving in heifers and improve the herd’s reproductive performance.86,91 semen quality semen analysis is crucial to predict potential fertility in bulls. there is a strong positive correlation between sc and semen quality,2,92 suggesting the use of sc as a predictor of bull fertility. the sc has a positive effect on sperm concentration and the proportion of spermatozoa with normal morphology.3 therefore, selection of bulls with larger sc at an early age will contribute to increasing semen production and subsequently improve cattle development programs. the use of sc as an early screening criterion prior to semen analysis would be an excellent management strategy for producers before the bse. if a bull meets the minimum standard for its age in sc, then semen tests can be used to make further selection decisions.44 kealey, et al4 reported positive estimates of genetic correlation for sc with semen color (0.73) and volume (0.20), sperm concentration (0.77), swirl (0.40), progressive motility (0.34), and percentages of live (0.63) and normal (0.33) sperm. similarly, favorable negative genetic correlations were found between sc and percentages of primary (-0.36), and secondary (-0.45) sperm abnormalities. the proportion of proximal cytoplasmic droplets is approximately 60% before puberty and decreases significantly after puberty.82 in general, for each 1.0 cm increase in sc a decrease of 1.51% and 1.42% in primary and secondary abnormalities, respectively, would be expected.4 thus, the reported negative correlation between percentage of proximal cytoplasmic droplets and sc might be an expression of the well-documented negative genetic correlation between sc and age at puberty.93 frequently, bulls with sc <30 cm have a low pregnancy rate after natural service.94 examination of the sperm of these bulls commonly shows a high percentage of head abnormalities, low progressive motility and concentration. bulls with small sc are subfertile especially when expected to mate a high number of cows during the breeding season. if selection pressure is applied to increase sc, semen characteristics (particularly sperm concentration and normal morphology) would be expected to improve, generating a positive impact on the fertility in subsequent breeding seasons. fertility of the bull’s daughters in addition to the positive association between testicular development and age at puberty in heifers, sc has been correlated with the subsequent fertility of the female progeny,80,81 specifically with conception and pregnancy rates.95-97 in contrast, other authors have reported that breeding soundness traits are poorly or not correlated with the female offspring fertility.98-100 some of the discrepancies between the effects of sc on heifer fertility can be explained by differences in the animal populations examined as well as dissimilar management conditions.101 it is important to emphasize that in most reports that have shown a positive correlation between sc and offspring fertility, the relationship could be considered too imprecise to permit the accurate 122clinical theriogenology • volume 3 number 2 • june 2011 prediction of actual pregnancy rates.101 makarechian and farid96 found a genetic correlation between sc and fertility; however, the relationship was not strong enough to predict the achieved pregnancy rates. coulter and kozub95 included sc into a multifactorial prediction model that was not capable of accurately predicting herd fertility results. one study estimated the genetic correlation between heifer pregnancy and sc using 18,145 records of nellore (b. indicus) heifers exposed to breeding at 14 months of age. heritability estimates were 0.69 for heifer pregnancy and 0.57 for sc. however, the genetic correlation estimates between the two traits was low (0.20), suggesting that bull selection based on both heifer pregnancy and sc would be more effective than selection based only on sc when the objective is to increase the accuracy of prediction for heifer pregnancy expected progeny difference (epd) of young bulls.102 although sc is an excellent predictor of age at puberty and seminal quality, the magnitude of the genetic association with heifer pregnancy needs further research. conclusion the sc of the bull is a valuable tool for predicting bull fertility before the breeding season. this trait has a high heritability and a strong correlation with the onset of puberty and fertility of the bull’s offspring. thus, selection of young bulls for precocious sexual maturation based on measurement of sc will reduce costs, shorten the generation interval, and improve the genetic value for herd reproductive efficiency. in addition to the heritable component of the bull’s sc, cattle producers must provide optimal nutrition, as well as an appropriate health and management program to enhance gene expression of superior bulls for precocity and reproductive performance, increasing productivity and profitability of the farm. references 1. bruner ka, van camp sd: assessment of the reproductive system of the male ruminant. vet clin north am food anim pract 1992; 8:331-345. 2. brito lf, silva ae, rodrigues lh, et al: 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barth ad: influence of abnormal sperm morphology on conception rates in cattle. proc annu mtg soc therio 1989; p. 13-40. 39. carson rl, wenzel jg: observations using the new bull-breeding soundness evaluation forms in adult and young bulls. vet clin north am food anim pract 1997;13:305-311. 40. coulter gh, mapletoft rj, kozub gc, et al: scrotal circumference of two-year-old bulls of several beef breeds. theriogenology 1987; 27:485-491. 41. bruner ka, mccraw rl, whitacre md, et al: breeding soundness examination of 1952 yearling beef bulls in north carolina. theriogenology 1995;44:129-145. 42. barth ad: evaluation of breeding soundness of the bull. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: saunders elsevier; 2007. p. 228-240. 43. pratt sl, spitzer jc, webster hw, et al: comparison of methods for predicting yearling scrotal circumference and correlations of scrotal circumference to growth traits in beef bulls. j anim sci 1991;69:2711-2720. 44. sylla l, stradaioli g, borgami s, monaci m: breeding soundness examination of chianina, marchigiana, and romagnola yearling bulls in performance tests over a 10-year period. theriogenology 2007;67:1351-1358. 45. hoflack g, van soom a, maes d, et al: breeding soundness and libido examination of belgian blue and holstein friesian artificial insemination bulls in belgium and the netherlands. theriogenology 2006;66:207-216. 46. keen je, rupp gp, wittenberg pa, et al: breeding soundness examination of north american bison bulls. j am vet med assoc 1999;214:1212-1217. 124clinical theriogenology • volume 3 number 2 • june 2011 47. sosa jm, senger pl, reeves jj: evaluation of american wagyu sires for scrotal circumference by age and body weight. j anim sci 2002;80:19-22. 48. smith ba, brinks js, richardson gv: estimation of genetic parameters among breeding soundness examination components and growth traits in yearling bulls. j anim sci 1989;67:2892-2896. 49. koger m: effective crossbreeding systems utilizing zebu cattle. j anim sci 1980;50:1215-1220. 50. green rd, cundiff lv, dickerson ge: life-cycle biological efficiency of bos indicus x bos taurus and bos taurus crossbred cow-calf production to weaning. j anim sci 1991;69:3544-3563. 51. gregory ke,. lunstra dd, cundiff lv, et al: breed effects and heterosis in advanced generations of composite populations for puberty and scrotal traits in beef cattle. j anim sci 1991;69:2795-2807. 52. brown bw: a review of nutritional influences on reproduction in boars, bulls and rams. reprod nutr dev 1994;34:89114. 53. mwansa pb, makarechian m: the effect of post weaning level of dietary energy on sex drive and semen quality of young beef bulls. theriogenology 1991;35:1169-1178. 54. coulter gh, carruthers td, amann rp, et al: testicular development, daily sperm production and epididymal sperm reserves in 15-mo-old angus and hereford bulls: effects of bull strain plus dietary energy. j anim sci 1987;64:254260. 55. greenough pr, vermunt jj, mckinnon jj, et al: laminitis-like changes in the claws of feedlot cattle. can vet j 1990; 31:202-208. 56. pruitt rj, corah lr, stevenson js, et al: effect of energy intake after weaning on the sexual development of beef bulls. ii age of first mating, age at puberty, testosterone and scrotal circumference. j anim sci 1986;63:579-585. 57. ohl mw, ott rs, faulkner db, et al: effects of rate of gain on scrotal circumference and histopathologic features of the testes of half-sibling yearling beef bulls. am j vet res 1996;57:844-847. 58. meacham tn, warnick ac, cunha tj, et al: hematological and histological changes in young beef bulls fed low protein rations. j anim sci 1964;23:380-384. 59. rekwot pi, oyedipe eo, akerejola oo, et al: the effect of protein intake on body weight, scrotal circumference and semen products of bunaji bulls and their friesian crosses in nigeria. anim reprod sci 1988;16:1-9. 60. bagu et, davies kl, epp t, et al: the effect of parity of the dam on sexual maturation, serum concentrations of metabolic hormones and the response to luteinizing hormone releasing hormone in bull calves. reprod domest anim 2009; in press. doi:10.1111/j.1439-0531.2009.01356. 61. miller sp, wilton jw: genetic relationships among direct and maternal components of milk yield and maternal weaning gain in a multibreed beef herd. j anim sci 1999;77:1155-1161. 62. bielli a, katz h, pedrana g, et al: nutritional management during fetal and postnatal life, and the influence on testicular stereology and sertoli cell numbers in corriedale ram lambs. small rumin res 2001;40:63-71. 63. leonhardt m, lesage j, dufourny l, et al: perinatal maternal food restriction induces alterations in hypothalamopituitaryadrenal axis activity and in plasma corticosterone-binding globulin capacity of weaning rat pups. neuroendocrinology 2002;75:45-54. 64. alves bca, unanian mm, silva e, et al: use of rapd markers for identifying nelore bulls with early reproductive maturation onset. anim reprod sci 2005;85: 183-191. 65. sekoni v, kumi-diaca j, saror d, et al: seasonal and monthly variations in the incidence of morphological abnormalities in bovine spermatozoa in shika zaria nothern nigeria. anim reprod sci 1988;17:61-67. 66. sordequist l, rodriguez-martinez h, haard m, et al: seasonal variation in sperm morphology in proven swedish dairy ai bulls. reprod domest anim 1997;32:263-265. 67. hardin d, chenoweth p, randel r, et al: effect of seasonal variation on seminal parameters and libido in angus and brahman bulls. j anim sci (abstr) 1981;53:326. 68. chenoweth pj: breeding bull selection, assessment and management. in: chenoweth pj, sanderson mw, editors. beef practice: cow-calf production medicine. ames: blackwell publishing; 2005. p. 151. 69. godfrey rw, lunstra dd, jenkins tg, et al: effect of location and season on body and testicular growth in brahman and hereford bulls. j anim sci 1990;68:1520-1529. 70. randel rd: reproductive characteristics of the brahman and brahman-based bull. in: fields mj, sand rs, editors. factors affecting calf crop. boca raton: crc press; 1994. p. 331. 71. chase jr cc, chenoweth pj, larsen re, et al: growth and reproductive development from weaning through 20 months of age among breeds of bulls in subtropical florida. theriogenology 1997; 47:723-745. 72. chacón j, pérez e, rodríguez-martínez h: seasonal variations in testicular consistency, scrotal circumference and spermiogramme parameters of extensively reared brahman (bos indicus) bulls in the tropics. theriogenology 2002; 58:41-50. 73. hopkins fm: diseases of the reproductive system of the bull. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: saunders elsevier; 2007. p. 240-243. 74. ladds pw: the male genital system. in: jubb kbf, kennedy pc, palmer n, editors. pathology of domestic animals. new york: academic press; 1985. p. 409. 75. coulter gh, foote rh: bovine testicular measurements as indicators of reproductive performance and their relationship to reproductive traits in cattle: a review. theriogenology 1979;11:297-311. 125 clinical theriogenology • volume 3 number 2 • june 2011 76. gipson ta, vogt dw, massey jw, et al: associations of scrotal circumference with semen traits in young beef bulls. theriogenology 1985;24:217-225. 77. toelle v, robison w: estimates of genetic correlation between testicular measurements and female reproductive traits in cattle. j anim sci 1985;60:89-99. 78. moser dw, bertrand jk, benyshek ll, et al: effects of selection for scrotal circumference in limousine bulls on reproductive and growth traits of progeny. j anim sci 1996;74:2052-2057. 79. vargas ca, elzo ma, chase jr cc, et al: estimation of genetic parameters for scrotal circumference, age at puberty in heifers, and hip height in brahman cattle. j anim sci 1998;76:2536-2541. 80. jakubiec, j: testicular size and consistency in bulls as a criterion for evaluation and selection with regard to fertility in cattle. proc annu meet eur assoc anim prod 1983; p. 428-429. 81. werre jf, binks js: relationships of age at puberty with subsequent productivity in beef heifers. proc west sect am soc anim sci 1986; p. 300. 82. lunstra dd, echternkamp se: puberty in beef bulls: acrosome morphology and semen quality in bulls of different breeds. j anim sci 1982; 55:638-648. 83. lunstra dd, gregory ke, cundiff lv: heritability estimates and adjustment factors for the effects of bull age and age of dam on yearling testicular size in breeds of beef bulls. theriogenology 1988;30:127-136. 84. lunstra dd, ford jj, echternkamp se: puberty in beef bulls: hormone concentrations, growth, testicular development, sperm production and sexual aggressiveness in bulls of different breeds. j anim sci 1978;46:1054-1062. 85. bourdon rm, brinks js: scrotal circumference in yearling hereford bulls: adjustment factor, heritabilities and genetics, environment and phenotypic relationship with growth traits. j anim sci 1986;62:958-967. 86. smith ba, brinks js, richardson gv: relationships of sire scrotal circumference to offspring reproduction and growth. j anim sci 1989; 67:2881-2885. 87. brinks js, mcinerney mj, chenoweth pj: relationship of age at puberty in heifers to reproductive traits in young bulls. proc west sect am soc anim sci 1978; p. 28-30. 88. king rg, kress dd, aderson dc, et al: genetics parameters in hereford for puberty in heifers and scrotal circumference in bulls. proc west sect am soc anim sci 1983; p. 11-19. 89. pereira e, eler jp, ferraz jbs:. correlacão genetica entre perımetro escrotal e algumas caracterısticas reprodutivas na raca nelore. rev bras zootec 2000; 29:1676-1683. 90. brinks js: genetics of fertility traits in bulls. proc annu meet soc therio 1985; p. 56-64. 91. morris ca, verkerk ga, wilson ja, et al: angus selection herd reproductive data: a genetic model for dairy cattle?. proc new zealand soc anim reprod 1999; p.169-172. 92. kastelic j, cook rb, pierson ra, et al: relationships among scrotal and testicular characteristics, sperm production and seminal quality in 129 beef bulls. can j vet res 2001;65:111-115. 93. beef improvement federation: guidelines for uniform beef improvement programs. 7th ed. bailey c, editor. colby (ks): beef improvement federation; 1996. 94. smith mf, morris dl, amoss ms, et al: relationship among scrotal circumference, fertility, seminal quality and libido in santa gertrudis bulls. theriogenology 1981;16:379-381. 95. coulter gh, kozub gc: efficacy of methods used to test fertility of beef bulls used for multiple-sire breeding under range conditions. j anim sci 1989;67:1757-1766. 96. makarechian m, farid a: the relationship between breeding soundness evaluation and fertility of beef bulls under group mating at pasture. theriogenology 1985;23:887-898. 97. mccosker th, turner af, mccool cj, et al: brahman bull fertility in a north australian rangeland herd. theriogenology 1989;32:285-300. 98. holroyd rg, doogan vj, de faveri j, et al: bull selection and use in northern australia. 4. calf output and predictors of fertility of bulls in multiple-sire herds. anim reprod sci 2002; 71:67-79. 99. evans jl, golden bl, bourdon rm, et al: additive genetic relationship between heifer pregnancy and scrotal circumference in hereford cattle. j anim sci 1999; 77:2621-2628. 100. thompson ja, johnson wh: scrotal size of yearling sires and early calving in beef herds: epidemiological investigation of possible causal pathways. theriogenology 1995;43:1279-1287. 101. parkinson tj: evaluation of fertility and infertility in natural service bulls. vet j 2004;168:215-229. 102. eler jp, silva jaiiv, evans jl, et al: additive genetic relationships between heifer pregnancy and scrotal circumference in nellore cattle. j anim sci 2004;82:2519-2527. 126clinical theriogenology • volume 3 number 2 • june 2011 table 1. minimum threshold for sc according to the age of the bull established by the society for theriogenology 7 age (months) threshold scrotal circumference <15 30 cm >15-18 31 cm >18-21 32 cm >21-24 33 cm >24 34 cm table 2. percentage of 254 yearling bulls of various beef breeds with satisfactory semen quality34,35 table 3. scrotal circumference by breed in bulls at 1 year of age*,42 *values corrected to 365 ± 14 days of age. data (for 6 studies in the us and canada) from barth ad: breeding soundness evaluation of bulls. the western canadian association of bovine practitioners. continuing education, western college of veterinary medicine, saskatoon, canada, 2000. age (mo+15d) n mean scrotal circumference (cm) % satisfactory semen quality 12 40 33.8 (28.5-39.5) 40.0 13 100 34.5 (28-41) 55.0 14 84 34.1 (28-45) 55.9 15 30 34.9 (27-41) 73.3 breed number of bulls mean scrotal circumference (cm) simmental 1246 34.7 gelbvieh 261 33.9 brown swiss 260 33.8 pinzgauer 144 33.7 angus 1051 33.2 charolais 1887 32.5 red poll 250 32.5 maine anjou 64 32.2 tarentais 14 32.0 hereford 1567 31.9 shorthorn 167 31.9 galloway 132 30.6 limousin 345 29.8 blonde d'aquitaine 15 29.7 salers 45 29.5 127 clinical theriogenology • volume 3 number 2 • june 2011 1 contact angelika stock therio.montreal@gmail.com © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10722, http://dx.doi.org/10.58292/ct.v16.10722 technique report handling techniques of frozen semen: storage, transfer, transport, and thawing* angelika stocka,b acentre dmvet-sud, saint hubert, qc, canada; bcanine semen bank chambly-montreal, chambly, qc, canada abstract enormous demand for assisted canine reproduction services in the last decade has increased the use of frozen semen in veterinary clinics. however, training and knowledge regarding frozen semen handling, as observed by the author, has been lagging. therefore, this report focuses on simple steps to avoid damage to frozen semen prior to its use. keywords: frozen semen handling, dryshipper, semen damage, semen storage introduction damage to frozen semen prior to artificial insemination by improper storage or handling are underestimated causes of pregnancy failure. since there is an increase in demand for frozen semen use in dogs, many general practitioners have added frozen semen usage in their practice. rapid and invisible damage may easily occur to cryopreserved sperm if storage, handling, and shipping procedures are performed by untrained personnel. the safest place for frozen semen is when they are immersed in liquid nitrogen at a constant temperature of –196°c. nitrogen vapor, alone, does not protect frozen semen from the harm due to temperature variations within the vapor. nitrogen vapor temperature varies depending on the surface area of evaporation and the distance from the liquid nitrogen surface. damage can occur to frozen semen when moved from one tank to another due to varying temperatures of the room environment air and the nitrogen vapor in the tank. studies in the early 70’s using bovine frozen semen demonstrated that recrystallization (a short thawing and refreezing of ice crystals that occur in a frozen semen sample when an inside-temperature of –130°c and higher is reached within the straw) can damage sperm.1,2 temperature of –130°c within a straw may be reached faster when semen is handled in vapor. this report focuses on describing possible errors while handling frozen semen and simple ways to prevent them in a veterinary practice setting. semen storage in liquid nitrogen tanks frozen semen is stored in liquid nitrogen at –196°c. a liquid nitrogen tank, also called a wet-tank or ln2 tank, is a double-walled container where the inner tank is suspended from the outer shell by a neck-like structure. vacuum between these 2 tanks helps to insulate the inner tank so that nitrogen in the inner tank holding the semen evaporates much slower. depending on its size, a tank in a veterinary clinic usually contains between 6-10 canisters that are held at the neck opening and carry canes with a lower and an upper goblet containing semen straws or vials of semen pellets. a great advantage of a liquid nitrogen tank is that it does not depend on electrical power, unlike other freezers. however, tanks need regular supervision since liquid nitrogen evaporates and the level of the liquid decreases over time. the plug is designed to loosely fit in the tank’s neck to allow vapor to escape. a plug should never get wet with water; otherwise, it may freeze to the tank’s neck. if capped too tightly, the gas builds pressure in the tank and causes it to crack open or even ‘explode’ eventually. since there is continuous evaporation, liquid level should be measured weekly with measuring sticks especially designed for that purpose. the measuring stick should be lowered to the tank bottom, allowed to cool, and then withdrawn. the level of nitrogen is represented by the frosty layer on the measuring stick. all measured levels and the amount of new nitrogen added should always be recorded not only for insurance purposes *presented at the 2023 society for theriogenology conference, published after peer review. mailto:therio.montreal@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10722 2 citation: clinical theriogenology 2024, 16, 10722, http://dx.doi.org/10.58292/ct.v16.10722 but also to assess the continued performance of the liquid nitrogen tank. a sudden increase in evaporation rate may be a sign that damage has occurred to the insulating properties of the tank. stored semen may be lost if tanks are not supervised regularly. semen tanks should be stored away from heavy traffic of personnel in a well-ventilated area due to the continual evaporating nitrogen gas. tanks should be placed on a smooth surface, ideally on a dry platform with rollers, so that they can be easily moved. liquid nitrogen tanks should always be carried by 2 people using both handles so that the tank remains in a vertical position to avoid shaking the canister rods inside that could potentially damage the fragile tank’s neck. at the same time, the tank should be visible enough every day so that any sudden vacuum loss and a higher evaporation rate can be noticed. typically, damage of the vacuum can be noticed by frost accumulation around the lid of the tank and, in severe cases, all over the area where the plug closes the tank. it is very important to maintain an accurate and updated semen inventory. special programs for inventory are offered by various companies but simple ‘excel sheets’ are sufficient and are easy to follow by personnel in private clinics. owner, tank and canister number, cane tag color and tag labeling, reception date, and removal dates must be entered. a good inventory saves time to find a specific semen dose; most importantly, it protects the quality of sperm. it must be kept in mind that each unnecessary lifting of a canister and searching for the right cane may cause damage to all semen in the canister over time.2 damage to frozen semen starts at inside straw temperatures as low as –130°c.1 recrystallization was definitely observed by –80°c, occurring within a matter of a few hours of exposure at –57°c, within a few minutes at –48°c and within a few seconds at –30°c. nitrogen vapor temperature can vary from –190 to ~ –30°c depending on how far from the liquid/vapor interface and how close to ambient air the measurement is taken (table 1). it took only 10-15 seconds for the temperature of a 0.5 cc straw in a vapor filled goblet to drop to –100°c when the goblet was raised to the upper third area in tank’s neck.3 semen exposed to reach an inside temperature of –45°c resulted in a dramatic decline in postthaw motility.1 invisible semen damage occurs before a drop in postthaw motility is noticeable during a routine semen evaluation, making preventive measures when handling semen so important.4 in large animal practices, where daily inseminations and embryo transfers are common, these findings are known as the reason for the so-called ‘8 second rule’, stating that semen should not be lifted beyond the frostline (table 1) and be limited to no more than 5-8 seconds in the neck of a tank during a search.4 obviously, this rule must be applied to frozen semen of all species. a warmer temperature is reached much faster when the liquid nitrogen level is low compared to searching for semen in a fully charged nitrogen tank. it is clearly demonstrated that repeated exposure of canes to the neck is more harmful in a tank that has a low level of nitrogen compared to a full tank and if this exposure is repeated the harm is additive and irreversible (figure 1). again, depending on the frequency and duration of damage due to these types of mishandling, a decrease in postthaw motility cannot always be observed; however, fertility decline has been noticed when frozen semen samples had been handled incorrectly in bovine practice. author has measured the vapor temperature at the neck of 6 semen tanks (all model mve xc 20) with a thermocouple device. a tank with a liquid nitrogen level of only 10 cm had a neck temperature of –54°c compared to –89°c in the same model tank with a level of 30 cm of liquid nitrogen. again, this simply means that search of a specific semen cane is safer when the tank has a high level of nitrogen. in addition, with a level of 30 cm, the upper goblet or cryovial in an mve-xc20 tank contains still liquid nitrogen that keeps the semen at –196°c when withdrawn or lifted for a short time. therefore, before searching for semen, it is safer to make sure that any tank has or is being topped up to a liquid nitrogen level that covers the upper goblets with semen portions.2 transfer of semen from one tank to another tank when transferring semen from one tank to another tank, semen passes shortly through nitrogen vapor and air and then back into vapor and/or liquid nitrogen; this must be done swiftly. again, a good inventory to locate the semen is crucial to avoid lifting canisters unnecessarily. the next important step is to have both tanks side by side to do the transfer. shipments often contain semen from several dogs with a varying number of doses per insemination. author usually transfers all received semen into a shallow styrofoam dish with liquid nitrogen so that the name and number of doses can be confirmed before transferring canes into the clinic’s tank. this is still the safest method. if just 1 dose on a cane is received, it is possible to transfer it swiftly from one tank to the other. although canes can be touched with a gloved hand, straws should not be handled with fingers but only with ln2 precooled forceps to avoid rising temperature within the straw.3 table 1. example of temperature range in the neck of a 20-liter semen storage tank filled up to 75% with liquid nitrogen (*frostline of the tank); adapted from stroud: 20134 depth in neck temp °celsius 0 inches 24.1 –1 inch 21.9 –2 inches 11.9 –3 inches –23.0 –4 inches* –47.1 –5 inches –73.6 –6 inches –110.5 –7 inches –160.4 –8 inches –181.2 figure 1. influence of level of liquid nitrogen on the temperature within semen straws (0.25 cc) during repeated lifting and exposure to the neck of a liquid nitrogen tank (picket et al., 19762) http://dx.doi.org/10.58292/ct.v16.10722 citation: clinical theriogenology 2024, 16, 10722, http://dx.doi.org/10.58292/ct.v16.10722 3 again, the ‘5-8 second rule’ applies for any transfer through vapor to avoid recrystallization of frozen semen. preparation and handling of frozen semen in a vapor shipper (commonly known as dryshipper) dryshippers were designed for the safe transportation of frozen semen or other biological samples at cryogenic (–150°c or colder) temperatures. they are manufactured from durable, lightweight aluminum and employ a hydrophobic and porous compound that absorbs liquid nitrogen to ensure dry, spill free vapor-phase shipments. these shippers are the ideal way to ship frozen semen safely and, since there is no liquid nitrogen inside, they are not considered ‘dangerous goods’ when transported on ground or by plane. as mentioned before, nitrogen vapor is not always a safe place for frozen semen. when dealing with dryshippers, meticulous preparation is required to ensure a damage free transport of frozen semen. it is important to follow the instructions of the manufacturer, especially when filling a tank that has no vapor left. whenever either a ln2 tank or a dryshipper is filled with liquid nitrogen, the liquid should be added slowly. personnel should wear protective glasses or a face shield and insulated gloves since a significant amount of gas is generated and liquid may reflux out towards the person when it comes in contact with the ambient temperature in the shipper. it is advisable to fill such tanks with small amounts at a time until the dryshipper is cool enough inside. this is also important to avoid any cracks inside the tank due to sudden cooling when initially loaded. the tank can be filled to the neck once well cooled, the cap replaced, and then it should be put aside for a few hours to let the liquid be absorbed. when all liquid is absorbed, new nitrogen may be added. if nitrogen liquid is bubbling in the dryshipper, it means that nitrogen is still being absorbed into the porous membrane. once the bubbling stops, it can be assumed that the porous membrane is saturated. the remaining liquid must be removed completely, and the dryshipper is ready to receive the frozen semen samples. since liquid nitrogen is classified as ‘dangerous goods’ by the international air transport association, all liquid nitrogen must be removed completely from the dryshipper so it can be identified as ‘not restricted’ as per special provision a152 on the shipping waybill. personnel must be trained to work safely with liquid nitrogen to avoid any accidents. liquid nitrogen that spills on the skin may cause severe burn wounds. therefore, violation of these regulations can result in civil or criminal charges. dryshippers are usually shipped in a protective case to avoid any damage during transport. author uses only the round, mushroom shaped protective case because the dryshipper is transported in an upright position. there are also rectangular protective cases. however, the author has received several dryshippers in these cases that were almost out of vapor, although the shipper confirmed adequate charge before shipment. when the dryshipper is transported or stored horizontally in these rectangular cases, there is a faster evaporation rate and the holding time may be shorter compared to tanks that are transported upright (personal observation). again, nitrogen vapor alone does not mean that the semen is at the right temperature. in the dryshipper, there is no level of nitrogen that we can measure with a measuring stick. however, the weight of a dryshipper is correlated with the absorbed liquid nitrogen in its inner porous membrane. manufacturers provide us with specifications for each nitrogen tank, i.e. with the specific weight when a dryshipper is fully loaded or when fully dry (table 2). table 2. specifications of various dry shippers as published online by the manufacturer of mve models vapor series sc 2/1v sc 4/2v sc 4/3v sc 20/12v xc 20/3v static holding time days 8 13 21 85 16 maximum storage capacity no. of canisters 1 1 1 6 4 +1 center no. of 1/2 cc straws 10/cane 280 120 540 2500/2000** no. of 1/2 cc straws 1 level bulk 88 440 210 780 3750/3000** no. of 1/4 cc straws 1 level bulk 182 938 452 1630 7410/6000 no. of 1.2 & 2.0 ml vials 5/cane 95 40 150 675/560** no. of 1.2 & 2.0 ml vials 6/cane 9 106 48 180 840/672** no. of blood bags stored 4r9953 performance ln2 capacity l 1.5 3.6 4.3 12.3 6.8 static evaporation rate l/day 0.19 0.26 0.20 0.09 0.35 unit dimensions neck opening in. (mm) 1.40 (35.0) 2.75 (70.0) 2.00 (51.0) 2.00 (51.0) 3.81 (96.7) overall height in. (mm) 13.5 (343) 18.4 (468) 19.4 (492) 25.7 (652) 25.0 (635) outer diameter in. (mm) 7.25 (184) 8.70 (222) 8.70 (222) 14.50 (368) 14.50 (368) canister height in. (mm) 5.0 (127) 11.0 (278) 11.0 (278) 11.0 (278) 11.0 (278) canister diameter in. (mm) 1.20 (31) 2.62 (67) 1.81 (46) 1.50 (38) 3.20 (80) weight empty lb. (kg) 6.0 (2.7) 11.0 (5.0) 13.0 (5.9) 30.0 (13.6) 28.4 (12.9) weight full lb. (kg) 8.8 (4.0) 17.0 (7.7) 20.6 (9.3) 52.0 (23.6) 38.4 (17.4) http://dx.doi.org/10.58292/ct.v16.10722 4 citation: clinical theriogenology 2024, 16, 10722, http://dx.doi.org/10.58292/ct.v16.10722 author regularly weighs a shipper before it leaves to confirm that it is fully loaded. author also weighs a dryshipper when it arrives in the clinic. it is highly recommended that semen arriving in a dryshipper be transferred to a wet tank upon arrival. author has recently experienced the loss of shipped semen due to a sudden malfunction in holding time of a dryshipper that had been fully loaded and was working well in the past. if the receiving clinic would have transferred the semen upon arrival and not have left the semen in the dryshipper for several days until the planned insemination, the semen could have been saved. author always transfers incoming semen from the dryshipper to a wet tank in her semen bank and records the weight of the arrived dryshipper before sending it back. again, if the temperature is around –130°c or warmer, there may be a problem with recrystallization of the received semen. author often observed personnel topping up the dryshipper with liquid nitrogen upon arrival, but then it is too late to know if semen arrived at a critical temperature or not. author has examined the bottom temperature using a thermocouple instrument (reproduction resources, wi, usa) of 2 dryshippers each weighing 7.7 kg when fully charged (vapor only). first dryshipper (empty weight considered 4.5 kg) had a temperature of –130°c and weighed 5.2 kg whereas the second dryshipper (empty weight considered 5.0 kg) had a temperature of –99.8°c and weighed 5.5 kg, suggesting that a weight difference of 300 grams resulted in a major difference in temperature when close to the empty weight. again, despite the presence of vapor, the author measures the bottom temperature of all dryshippers that arrive close to their empty weight with semen in it and transfers the semen to a wet tank upon arrival. most often, frozen straws or pellet vials are attached to metallic canes and easily retrieved from a dryshipper. it is, however, advisable to verify that all semen received correlates with the enclosed paperwork. the canister should be inspected if semen is missing as frozen straws or vials may have fallen off the cane to the bottom of the canister during transport. also, some european countries have a different way of packaging semen straws. sometimes, there are semen straws left loosely in the bottom within a big plastic goblet covered with cottonwool. in other cases, straws are not in goblets on canes but in cassettes in the very bottom of the canister. thawing but not ‘killing’ sperm thawing semen appears very easy but may be tricky. in a few seconds, a few straws of semen and thousands of dollars that client paid can be wasted. once a straw is removed from the water bath, water must be wiped off from the straw (water is spermicidal) before emptying the thawed semen into a tube where all semen is pooled. different methods of semen freezing may demand different protocols for thawing. so, it is crucial to keep thawing instructions with the inventory or inquire about thawing instructions from the shipper to have them available when insemination is performed. even a trained person can experience problems when thawing semen. some semen straws may ‘explode’ upon thawing and the semen straw may end up in the corner of the room. this is often due to incorrect filling technique prior to freezing. to apply a good freezing technique, it is essential to moisten the cotton end of the straw when filling the straw with the semen, so that no air space is possible between the media and the cotton plug because such air space draws liquid nitrogen into the straw (figure 2). warming expands liquid nitrogen, the pressure created pushes cotton plugs into water and propels the straws out of water bath. although metal or glass balls are good sealing methods for straws, the author has encountered most ‘explosions’ in straws closed with metal or glass balls. author, therefore, always keeps frozen semen straws in a styrofoam dish with liquid nitrogen before thawing to examine how straws are filled to avoid problems. author usually cuts straws off at the ball-sealed end under nitrogen to prevent any pressure problems to avoid semen loss. similarly, if the straw is fully filled without any visible air bubble within the straw and well-sealed at both ends; the author prefers cutting the sealed end under liquid nitrogen. straws are then thawed with an open end, as done with straws frozen by the clone technique. due to pressure changes in such incorrectly filled straws during thawing, cotton plugs are pushed out and water can enter the straw from the bottom and semen is wasted. once the sealed end is opened, this does not occur. care must be taken that no thawing water enters the upper, opened end in such cases. thawing pellets of frozen semen in a whirl pack is probably the easiest method. yet even here attention to detail is important. the whirl pack should be undamaged so that water cannot enter and mix with semen. after the semen vial is removed from the tank, it is crucial that all liquid nitrogen within the vial is allowed to evaporate prior to placing the pellets into the whirl pack. otherwise, any remaining liquid nitrogen kills sperm in the water bath. if thaw medium is necessary to thaw the semen, it must be ensured that the thaw medium had enough time to warm up to the desired thawing temperature. during transcervical or surgical insemination procedure, it is best to wait to thaw the semen until the uterus is entered. in general, the author never performs any transfer or thawing of frozen semen without having a shallow styrofoam dish with liquid nitrogen in proximity to keep semen safe until ready to thaw. figure 2. right versus wrong filling of a straw before freezing. upper straw has incomplete filling (air space close to the cotton plug; see arrow) compared to lower straw where semen is pulled up correctly to harden the polyvinyl powder between 2 cotton plugs, therefore avoiding entrance of liquid nitrogen http://dx.doi.org/10.58292/ct.v16.10722 citation: clinical theriogenology 2024, 16, 10722, http://dx.doi.org/10.58292/ct.v16.10722 5 conclusion this paper is meant to emphasize the importance of paying attention to details when working with frozen semen. simple techniques and preventive steps can ensure that avoidable damage to frozen semen was not the reason for pregnancy failure. conflict of interest none to report. acknowledgement this article is dedicated to late professor dr. w. leidl (19252023) who taught theriogenology while the author was a veterinary student at the university of munich, germany. his excellent lectures and rigorous training inspired the author to become and be an enthusiastic theriogenologist for the past 40 years. references 1. saacke rg: concepts in semen packaging and use. proc 8th naab conference on artificial insemination of beef cattle 1976; p. 54–75. 2. picket bw, berndtson we, rugg cd: semen handling in the field. proc 10th naab conference on artificial insemination of beef cattle 1976; p. 54–75. 3. picket bw, berndtson we: procedures for handling frozen semen in the field. in: morrow da: editor. current therapy in theriogenology. philadelphia; wb saunders: 1980. p. 354–370. 4. stroud b: consequences of mishandling frozen semen and embryos. in: proceedings: applied reproductive strategies in beef cattle. 2012; p.191–203. http://dx.doi.org/10.58292/ct.v16.10722 2013: neosporosis in cattle neosporosis in cattle david noall, ramanathan kasimanickam, mushtaq memon, john gay department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa abstract neospora caninum is an obligate intracellular coccidian parasite of cattle and dogs that infects 1020% of all cattle worldwide and causes up to 20% of bovine abortions. n. caninum cycles at low levels between the definitive host, canids, and a wide range of intermediate hosts, cattle being the most important. abortion is the only clinical sign observed in adult cattle. n. caninum most commonly causes an endemic increase in a herd’s annual abortion rate and infrequently causes epidemic abortion storms. rarely, congenitally infected calves are born with neurological disease. most infected calves are born clinically normal but with a titer. the parasite persists within cattle herds through vertical transmission via transplacental infection of successive pregnancies of chronically infected dams and by infrequent horizontal transmission. all infected cows are at increased risk of abortion. abortion diagnosis is challenging because neospora may be present but not the cause. ruling out other causes of abortion, detecting the characteristic fetal lesions, or establishing an association between abortion and infection within the herd is important. control options are limited. no antiparasitic drugs are approved or economical for treatment or prevention. although a killed vaccine is available commercially, it has questionable efficacy. in many instances, tolerating the infection is the most economical option. testand-cull strategies are often cost-prohibitive. for reducing a herd infection rate, precolostral testing of heifer calves and excluding those with titers from breeding is usually an economical option. introduction neospora caninum is an apicomplexan intracellular parasite, first described in 1984, that is a leading cause of bovine abortion worldwide.1,2 it is closely related to and for many years was misdiagnosed as toxoplasma gondii. domestic dogs and some wild canids are the definitive hosts. many species, including dogs, serve as intermediate hosts, cattle being the most important (figure 1).2 n. caninum is transmitted vertically (dam to fetus) remarkably efficiently with up to 95% of calves born to infected dams congenitally infected themselves. vertical transmission is the primary mechanism by which the parasite maintains itself within cattle herds.2,3 in utero infection appears key to establishing a chronic infection capable of infecting successive pregnancies of the infected dam.4 n. caninum appears highly tuned to the physiology of bovine pregnancy. experimental feeding of oocysts to cows does not reliably produce infection. it appears that horizontal transmission between cattle or between cattle and canids is uncommon and thus less important. although estimated losses to the beef and dairy industries are at hundreds of millions of dollars, effective and economical control strategies for neosporosis are lacking.5 understanding the adaptations that make n. caninum a successful parasite and a difficult control target will help practitioners to diagnose and manage this ubiquitous and expensive parasite. prevalence since its recognition in 1984, n. caninum has been detected in cattle populations worldwide. numerous seroprevalence surveys have documented specific antibody responses in cattle, canids, and a plethora of likely intermediate hosts, both wild and domestic (figure 2).6 studies employing molecular techniques have demonstrated n. caninum dna in neural tissues of various animals.2 while, these techniques document widespread exposure to and likely infection with n. caninum, they do not confirm infection or disease. the gold standard for demonstrating infection includes identifying tissue cysts histopathologically, identifying oocysts in feces, and isolating viable parasites. the successful isolations of viable parasites are limited to cattle, sheep, water buffalo, bison, white-tailed deer, and dogs.6 oocyst shedding has been detected in only a few naturally infected dogs.6 infrequently tissue cysts have been observed histopathologically in fetuses, calves, and experimentally infected mice but not in adult cattle.7 clinical theriogenology • volume 5 number 2 • june 2013109 for complete n. caninum epidemiology, see dubey et al.2 worldwide, approximately 15 to 25% of dairy cattle are seropositive and approximately 10 to 20% of beef cattle. some regions approach 50-60% seropositivity and some herds approach 100%. seropositivity among domestic dogs varies more widely among regions, ranging from 5% to 60% positive.2 a survey in northwestern u.s. found 24% (n=2585) of beef cows seropositive to n. caninum, with 100% of herds infected.8 a study including 20 eastern and midwestern states and puerto rico found 16% of beef and dairy cows were seropositive with 90% of herds infected.9 only 7% of 1,077 canine serum samples from pet dogs across 35 states submitted on suspicion of neosporosis were seropositive.10 due to the difficulty of definitively diagnosing n. caninum as causing an abortion, estimating the proportion of abortions it causes is challenging. a study of a large number of fetuses submitted from california cattle attributed approximately 20% of bovine abortions to n. caninum.2 although contact with dog feces potentially containing n. caninum oocysts and consumption of raw or undercooked beef potentially containing tissue cysts likely provide ample exposure opportunity for humans, the evidence supporting human infection is limited and inconclusive. one u.s. study using a low positive titer threshold found 7% seropositivity to n. caninum among healthy blood donors.11 the majority of titer positive people were negative for antibodies against t. gondii, indicating that crossreactivity with this pathogen is unlikely. a brazilian study found 18% and 38% seropositivity among neurologic and aids patients, respectively, but found rates similar to the u.s. study among healthy adults and newborns.12 in contrast to the u.s. study, nearly all the neospora positive samples were also positive for toxoplasma, suggesting that cross-reactivity and/or polyclonal immune stimulation may have occurred. a low positive threshold of 1:80 or 1:100 was used compared to the higher ones used for cattle and dogs (1:320 and 1:200, respectively). three serological surveys in england, france, and denmark found little or no evidence for neospora infection in humans.13-15 taken together, these data appear to support n. caninum exposure but not infection. further studies to detect parasites or parasite dna, most likely to be observed among immunocompromised individuals, are indicated. economic impact monetary losses from neosporosis are due to abortions and increased culling risk. losses may result from decreased milk production, increased services per conception, increased calving interval, and increased veterinary costs, but authors differ on these. neospora-induced abortion is estimated to cost the california cattle industry $35 million dollars annually.2 global losses, based on ten countries with sufficient published data, are estimated at $1.3 billion dollars annually.5 the us dairy industry is estimated to lose $546 million per year, an annual loss of $12,200 to the average dairy farm.5 the us beef industry is estimated to lose $111 million per year, an annual loss of $100 to the average beef producer.5 the impact to an individual producer can be devastating when abortions occur as epidemics in which more than10% of a herd aborts in a short period.5 life cycle a schematic presentation of neospora caninum life cycle is given in figure 3. neospora caninum is a member of the coccidia class containing the related genera eimeria, isospora, cryptosporidium, sarcocystis, and toxoplasma, all having a facultative heteroxenous life cycle. unsporulated 10-14 µm diameter oocysts are shed in the feces of the definitive host and have been found in the feces of dogs, coyotes, dingoes, and wolves.16-18 oocysts are highly resistant to environmental temperatures and common chemicals. treatments that inactivate cysts include 100˚c for one minute or 10% sodium hypochlorite for one hour, with less severe treatments failing to inactivate all cysts.19 within 24 hours, the oocysts sporulate to the infective form. each sporulated oocyst contains two sporocysts, each of which contains four sporozoites. when ingested by an intermediate host, eight sporozoites are released into the gut for each oocyst consumed.4 cattle, sheep, goats, horses, deer, dogs, and other animals may serve as intermediate hosts, but cattle are the most frequent and economically important.2 sporozoites differentiate into tachyzoites, which parasitize small intestinal epithelial cells. tachyzoites, 5-7 µm long and 1-2 µm wide, replicate rapidly by asexual endodyogeny, forming two clinical theriogenology • volume 5 number 2 • june 2013 110 daughter cells within the parent cell.1 these replicate in various tissues, including neural cells, vascular endothelial cells, myocytes, hepatocytes, renal cells, alveolar macrophages, maternal caruncular septal cells, and placental trophoblasts,7 and disseminate hematogenously, presumably within mononuclear phagocytes. n. caninum dna has been demonstrated in the leukocyte fraction but not the serum of blood from naturally infected cows.20 parasitemia is difficult to detect directly, suggesting that it may be of short duration and may wax and wane. tachyzoites differentiate into slowly-replicating bradyzoites, which are found within thickwalled, 100 µm cysts in the brain and spinal cord and rarely in muscle tissue. each cyst may contain hundreds of bradyzoites.4 bradyzoite cysts are thought to persist for the life of the host and to re-activate and differentiate back into tachyzoites, thereby establishing persistent infection. tissue cysts have been detected in congenitally infected calves and fetuses but not adult cattle.7 viable n. caninum have been isolated from the brains of adult cows that produced infected calves.21 the parasite has been consistently isolated from placental tissue of congenitally infected dams at calving. bradyzoite cysts may be present because dogs fed placental tissue from infected cows reliably shed oocysts.22,23 because bovine placental tissues are often readily available in the environment, they are likely are the principal infection source for dogs and other canids. the cycle repeats when tissue cysts are consumed by the definitive host. the sexual stages that lead to oocyst formation have not been demonstrated but are presumed to occur in the intestinal epithelial cells of dogs and other definitive hosts.4 transmission cattle become infected with n. caninum either by ingestion of sporulated oocysts postnatally (horizontal transmission) or congenitally by transplacental migration of tachyzoites from an infected dam to her fetus (vertical transmission). some evidence suggests that the immune responses provoked by horizontallyand vertically-acquired infections are different.2,24 cattle are apparently often infected for life, regularly or intermittently transmitting the infection to successive pregnancies.22 the majority of natural infections are thought to result from vertical transmission, which occurs with 75% to 100% efficiency.5 horizontal cow to cow transmission does not appear to occur.2 horizontal transmission evidence for horizontal transmission comes from observed abortion patterns, avidity-elisa serological studies, mathematical models, and risk-factor analyses. because vertical transmission risk is less than 100%, some horizontal transmission must occur to maintain the parasite within the herd. otherwise, within a herd prevalence would decline until the infection was eliminated.25 neosporaassociated abortions occur in two patterns: in concentrated epidemic outbreaks or as a higher endemic annual herd abortion rate. the occurrence of epidemics, or “abortion storms,” suggests a point-source; broad herd exposure to infective material and provides evidence of horizontal transmission.7 avidityelisa, based on an infected animal’s immunoglobulin antigen binding affinity increasing with time postinfection, has been applied to herds after epidemic neospora abortions. bjorkman et al described a beef herd abortion outbreak in which at the time of the outbreak seropositive cows had low-avidity antibody responses, suggesting that many of the infections were recent and were the result of horizontal transmission of n. caninum.26 the mean avidity of seropositive cows increased over the subsequent three years, suggesting the onset of chronic infections. this study also documented that calves born seronegative had a low risk of seroconversion and that vertical transmission occurs with 85% efficiency. observational studies of bovine neosporosis risk factors have frequently implicated dogs in neospora transmission, with their presence on farms, their number, their behavior (e.g., feeding on placentas and defecating near feeds), and their density in the region all being associated with increased risk.2 coyote abundance in an ecological area was identified as a risk factor in a texas study.27 a french study identified using pond water as a drinking water source as a risk factor, suggesting that oocysts may be disseminated in water.2 these studies indicate the importance of continuing horizontal neospora clinical theriogenology • volume 5 number 2 • june 2013111 oocyst transmission from definitive hosts for maintaining the parasite in cattle populations, despite the larger role played by transplacental vertical transmission. other routes of vertical and horizontal transmission are thought not to occur or to be rare. milk and colostrum containing added neospora caninum tachyzoites infected calves experimentally, but this is not thought to occur naturally to any significant extent.2 similarly, n. caninum dna was identified in an infected bull’s semen and semen containing added tachyzoites infected heifers but large numbers were needed and no evidence suggest that venereal transmission occurs naturally.2 the outcome of horizontal infection depends on the fetal gestational age in pregnant dams and presumably on the dose and virulence of the neospora strain involved. a non-pregnant animal will mount an immune response that either causes the parasite to form bradyzoite tissue cysts and become dormant or eliminates it.28 the parasite may or may not cross the placenta in the pregnant animal. if the dam’s immune response quickly controls the infection, transmission may not occur and the calf is born healthy and uninfected. if transplacental transmission occurs, the outcome depends on fetal gestational age, which determines its immunocompetence.4 the fetus may be killed, in which case resorption, mummification, abortion, or stillbirth occurs, depending on gestational age. it may survive to birth but be born with neurologic disease or develop it within four weeks of birth. it may be infected but mount an immune response that controls the infection and be born clinically normal but persistently infected. in all these scenarios, most dams are presumed to remain persistently infected.4 this is not well-established and some evidence suggests that horizontal transmission may not always result in persistent infection. vertical transmission vertical, or transplacental, transmission, the predominant mode in cattle, is divided into endogenous and exogenous forms. the possible outcomes for the developing fetus are the same for both. exogenous transplacental transmission occurs when tachyzoites cross the placenta and infect the fetus after a maternal parasitemia initiated by a naïve dam consuming infective oocysts. this form is primarily associated with epidemic abortion outbreaks. endogenous transplacental transmission occurs when the maternal parasitemia is initiated by the recrudescence of a persistent infection acquired before that pregnancy. this form is associated with an increased endemic abortion rate.29 it is believed to most commonly occur in the second or third trimesters when the fetus is more immunocompetent, usually resulting in the birth of a healthy but infected calf.4 the risk of endogenous transplacental transmission decreases with increasing parity,30 suggesting that cows may eventually develop sufficient immunity to reduce recrudescence and transplacental transmission. host immune response the immune response to n. caninum is characterized by a strong pro-inflammatory type 1 helper cells (th-1) response that promotes a specific cell-mediated immune response. cattle appear to have some innate immunity to n. caninum, as ntural killer (nk) cells have been shown to be able to lyse infected fibroblasts in culture.31 natural killer cells also secrete ifn-γ, a cytokine that plays an important role in a th-1 response. ifn-γ causes type 0 helper (th-0) cells to differentiate into th-1 cells, and suppresses type 2 helper (th-2) cells that would otherwise promote a humoral response and downregulate the cell-mediated response. type 1helper cells then secrete more ifn-γ, creating a positive reinforcement loop. ifn-γ has also been shown to inhibit parasite growth in cell culture and may play a role in causing the parasite to switch from the tachyzoite to the bradyzoite stage.31 type 1 helper cells coordinate a specific immune response that is mediated by cd4+ cytotoxic t-cells, which lyse infected host cells by a perforin-granzyme pathway.32 although humoral immunity plays a lesser role in n. caninum control, host antibodies generated against proteins on the tachyzoite surface block parasite entry into host cells.33 immunomodulation during pregnancy the immune system changes of pregnancy may be significantly related to the vertical transmission of n. caninum. a fetus represents a genetic foreign body to its dam, being essentially a clinical theriogenology • volume 5 number 2 • june 2013 112 semi-allogeneic tissue graft. to prevent the maternal immune system from rejecting the fetus, the cytokine balance at the materno-fetal interface is slightly tipped to favor “beneficial” th-2 regulatory cytokines, including interleukin 10 and tgf-β.31 th-2 cytokines inhibit cell-mediated immunity and promote a humoral immune response. innes et al observed a pregnancy-specific down-regulation of the maternal cell-mediated immune response, evidenced by decreased responsiveness of circulating lymphocytes to antigen that occurred regardless of n. caninum infection status.34 this decrease occurred between gestation weeks 12 and 18 after which the response slowly increased back to previous levels by gestation week 38,34 corresponding to the typical timing of neospora-induced abortions, which mainly occur at three to 8 months gestation (12-32 weeks).4 host-parasite interactions n. caninum vertical transmission appears to correspond with this pregnancy-associated immunomodulation. when pregnant cows were experimentally infected, the likelihood of exogenous transplacental transmission increased if the infection occurred later in gestation when the maternal cellmediated response was down-regulated.31 in naturally infected dams, the endogenous transplacental transmission is associated with a transient increase in circulating neospora-specific antibody.31 this increase is seen earlier in dams that abort and later in dams that give birth to congenitally infected calves. recrudescence later in pregnancy is beneficial to parasite survival because congenitally infected heifer calves are more likely to be produced that are capable of passing the infection on to their offspring.3 the mechanism by which n. caninum senses pregnancy-related immune down-regulation is unknown. in utero exposure may be required for persistent n. caninum infections to be capable of infecting offspring.4 research suggests that post-natal infection may not result in persistent infections while congenital infection increases the risk of endogenous transplacental transmission. the protective quality of the immune response from in utero exposure may be inferior to that from post-natal exposure.29 this suggests that in utero exposure produces immune tolerance somewhat similar to that which occurs with bovine viral diarrhea in which the congenitally infected animal’s immune system cannot mount a response equivalent to that of a post-natally exposed animal.2 due to fetal immune system immaturity, in utero exposure may be more likely to result in persistent infections characterized by bradyzoite tissue cysts than does postnatal exposure.7 evidence suggests that post-natal exposure rarely results in persistent infection capable of endogenous transmission to subsequent pregnancies. three studies have shown that naïve cattle experimentally infected six or ten weeks prior to pregnancy do not transmit the parasite to their fetuses, even when challenged by n. caninum in mid-gestation.34-36 williams et al infected naïve cows with a low-virulence strain ten weeks prior to insemination and at 70 days of gestation administered an intravenous fetopathic challenge dose of 107 live tachyzoites to this and a control group.34 in the treatment group, all six calves were born healthy with no signs of n. caninum infection while in the control group 82% (9 of 11) of the fetuses died.35 this study also demonstrated that cattle fail to become persistently infected after a post-natal challenge and that they develop a protective immune response against future challenge. pregnant congenitally infected animals challenged at mid-gestation with a fetopathic infection were also immune to the challenge dose and did not abort. later in gestation their persistent infection recrudesced and transmitted to their fetus, just as would have been expected with no intervention.29 this suggests that they were tolerant of their own infections but not of the new strains or that their bradyzoite cyst burden developed before they were completely immune-competent. the persistent cysts are apparently able to reactivate quietly enough and to disseminate to the placenta before maternal immune control. mccann et al orally infected naïve, pregnant cows and followed them through subsequent pregnancies.24 all five calves or fetuses were congenitally infected but not those of subsequent pregnancies. no signs of recrudescence appeared in the dams’ serology, indicating that they had not become persistently infected.24 clinical theriogenology • volume 5 number 2 • june 2013113 in a herd that experienced a mass seroconversion without increased abortions, dijkstra et al observed endogenous transmission in subsequent pregnancies.28 nine infected daughters were born to dams that seroconverted before their insemination dates, suggesting that persistent infection with subsequent endogenous transplacental transmission occurred due to the post-natal exposure.28 this interpretation depends on the nine dams being correctly classified as naïve based on serological test results and on the nine offspring not having seroconverted after birth but before they were tested at about four to six months of age after maternal antibody waned. the circulating antibody level of persistentlyinfected animals is known to periodically dip below the threshold of serological tests.7 more research is indicated to clarify this question, which has direct management implications. mechanism of abortion neosporosis is primarily a disease of the placenta and the fetus, the only clinical sign observed in naturally infected adult cattle being abortion.4 n. caninum is an example of a parasite that is highly adapted to persist within its host with minimal morbidity. several abortion mechanisms are hypothesized. parasite replication and the fetal response may destroy enough fetal tissue to kill the fetus outright or parasite replication and the maternal response may destroy enough placental tissue to kill the fetus by insufficient oxygenation/nutrition. damage to the placenta may release maternal prostaglandins that cause luteolysis and subsequent abortion. the strong th-1 type immune response stimulated by the parasite may disrupt the normal immunologic environment of the fetal-placental interface, leading to maternal immune rejection of the fetus.4 the maternal inflammatory infiltrate with nk cells, cytotoxic t-cells, and cells containing mrna for ifnγ surrounding areas of fetal villous necrosis present in the placenta of aborting dams support this hypothesis.37 relatively small parasite numbers in the placenta might tip the balance towards a damaging maternal immune response that causes immune rejection of the pregnancy. diagnosis because neospora is prevalent in cattle populations and may be present without causing abortion, definitive abortion diagnosis is difficult and depends on serology, molecular techniques, and histopathology. ruling out other causes of abortion is important. dubey and schares developed a diagnostic strategy that relies on the subjective impression of the histopathologist and/or on statistical analysis of herd serology.7 gross lesions are not usually present in the aborted fetus and they are commonly autolyzed or mummified.7 pale white foci may be observed in the heart or skeletal muscles and very small, pale to dark necrotic foci may be observed in the brain.7 hydrocephalus has been reported.38 the first laboratory submissions should include sera from aborted dams, fetal fluids, and tissues, including placenta if available. maternal serum and fetal fluids (e.g., serum, pleural, and peritoneal fluids) may contain neospora specific antibody detectable by serological assays that include competitive and indirect elisa and ifat.3 before acquiring and submitting samples, consult with the laboratory regarding the specimens they prefer, the tests they offer, and their diagnostic performance. because neospora may be present as a bystander, a positive serological result on a fetal or maternal sample indicates neospora may be associated with the abortion but does not provide a definitive diagnosis.3 because most dams aborting due to neospora are antibody-positive, a negative result on maternal serum indicates that neospora was unlikely the cause.7 rarely, infected calves are born to seronegative dams, likely due to fluctuating antibody levels in the dam.39 in contrast, a negative result on fetal submissions does not rule out neospora, because autolysis, immature fetal immunocompetence, or a short interval between infection and death may lead to false negative serological results.7 polymerase chain reaction (pcr)-based assays are available to detect parasite dna in fetal tissues, amniotic fluid, milk, semen, or dog feces. these tests are interpreted much like serology, positive results requiring the presence of lesions incompatible with life or a clear statistical association between abortions and test results.7 clinical theriogenology • volume 5 number 2 • june 2013 114 fetal tissues may be examined histopathologically for characteristic lesions and stained immunohistochemically to identify parasites in tissue sections. lesions are most commonly observed in the brain, spinal cord, heart, and liver, but may be present in other tissues.7, 40.41 the most characteristic lesions are multifocal encephalitis, myocarditis, and periportal hepatitis, with tachyzoites demonstrable immunohistochemically in up to 85% of brains.40 when lesions are present and associated with n. caninum, the pathologist must decide whether the severity of lesions is incompatible with life and, if so, concluding that n. caninum was the cause of the abortion is reasonable.7 herd-level serology and epidemiology provide additional evidence for diagnosing neosporainduced abortion, which is useful when serology and histopathology are inconclusive. gather information about herd reproduction and abortion occurrence timing to establish the abortion pattern, which also has direct control implications. a herd experiencing a neospora-associated abortion storm (abortions began recently and occurred over a time course of eight to ten weeks) was likely recently exposed to infective oocysts, most likely in the feed or water.7 a herd experiencing neospora-associated abortions sporadically over a longer time course suggests a high level of persistent infection with vertical transmission and possibly the presence of other factors that increase abortion risk, such as mycotoxins in moldy feed.3,7 once the duration of abortion is established, the ‘dams at risk’ can be determined, meaning all dams pregnant and therefore at risk of aborting during the period when the abortions occurred. if the first round of serology and histopathology are inconclusive, serologically test sufficient samples of ‘dams at risk,’ randomly selecting both aborting and non-aborting dams, and determine if a positive association exists between presence of n. caninum antibodies and abortion. a statistically significant positive association provides strong evidence for neospora-associated abortion.3,7 serological testing can also determine carrier status for embryo recipient selection, for test-andcull control strategies, and to aid in purchasing decisions. using a lower test cut-off and therefore higher sensitivity is desirable.7 verify with the laboratory that the test and protocol being used have been validated for determining infection status. bulk milk can be tested with elisa to detect those herd with a within herd seroprevalence greater than 15%, which are herds likely to benefit from a control program.42 control developing a neospora-related abortion control strategy starts with a cost-benefit analysis for the farm in question. due to regional differences in n. caninum epidemiology and in how economic losses are counted, researchers differ on the most cost-effective means of control.2 control strategies include no intervention, improving farm biosecurity, vaccination, test-and-cull, test-and-exclude-from-breeding, and use of beef bull semen to reduce abortions. where herd seropositivity is low, the most cost-effective option may be to do nothing. a decision-tree analysis of the australia and new zealand dairy industries determined that doing nothing was the most economical option for within-herd seroprevalence up to 18 to 21% and that vaccination with an efficacious product was the more economical option at higher prevalences.43 no protection is provided against epidemics; in the model it was assumed that only one abortion storm was likely to occur in the time frame considered, followed by an increased risk of sporadic abortion. farm biosecurity may be important in controlling bovine neosporosis, particularly for uninfected and low seroprevalence herds. maintaining a closed herd is the best way to avoid introducing the parasite. although avoiding the introduction of infected replacements into negative herds is important, occasional false negative results on individual animal tests are likely because antibody levels wax and wane. a better approach is to buy only from herds that are test-negative or that have an ongoing testing strategy.44 to reduce the risk of horizontal transmission, farm access to dogs and other canids should be minimized. dogs and other canids should not be allowed to feed on aborted fetuses, placentas, or dead stock or to defecate in feed bunks or feed storage areas and should be excluded from the premises.2 no pharmaceuticals are approved for treating bovine neosporosis but several compounds have shown promise in research trials. the coccidiostat toltrazuril has been shown to block exogenous transplacental transmission in mice infected early in gestation.45 congenitally infected calves treated with the compound had stronger humoral immune responses to the parasite at four to six months of age than clinical theriogenology • volume 5 number 2 • june 2013115 did untreated controls.46 the related compound ponazuril given to calves one day following experimental infection prevented them from developing an infection detectable by pcr.47 treatment expense and lengthy withholding times may make treatment uneconomical for all but exceptionally valuable cattle.2 an adjuvanted killed tachyzoite vaccine (bovilis neoguard®, merck animal health, millsboro, de) is commercially available. field efficacy data suggest that up to a 25-60%, reduction in abortions may be possible but negative effects have been detected in some herds. in a costa rican field trial bovilis neoguard® reduced abortion incidence by approximately half.48 in a new zealand field trial the vaccine had a statistically significant effect in one of five herds where it reduced abortions by about 60% but had little effect in the others and may have increased early embryonic death risk.49 vaccination results in seroconversion that cannot be differentiated from infection, eliminating the use of test-and-cull strategies in vaccinated animals. research on a subunit vaccine is underway. immunization with a surface protein involved in host cell attachment and invasion stimulated a humoral immune response that reduced transplacental transmission in mice.33 a protective immune response to vaccination is possible in non-congenitally infected animals. infection with nc-nowra (australian isolate), a naturally attenuated strain, before gestation protected 100% of fetuses from a lethal dose of a virulent strain delivered on gestation day 70. all fetuses were born uninfected and no parasite dna was detectable in the vaccinated dams.35,50 congenitally infected dams did not abort after a mid-gestation challenge but their chronic infection later recrudesced and infected their calves.29 this suggests that producing a protective vaccination response is much more difficult in congenitally infected animals. live vaccines present other challenges, including difficulty of production, stability, and concerns over reversion to virulence.50 several approaches to neospora control use whole herd serological testing to determine infection status of every animal. test-and-cull is the most straightforward but is not economical in most circumstances.51 a more economical approach is to test the herd, identify the daughters of seropositive cows, and exclude them from the replacement pool. two computer models identified this as the most economical approach, reducing herd prevalence by half within five years.51,52 these models do not account for any horizontal transmission. embryo transfer technology can preserve the genetics of high genetic merit but seropositive animals. embryos transferred following the international embryo transfer society protocol into seronegative dams remain uninfected, regardless of donor infection status.53,54 dairy operations can reduce neospora abortions by inseminating seropositive dams with beef breed semen. crossbreed pregnancies have a more robust placentation with higher levels of peripartum pregnancy associated glycoprotein-1 (pag-1), which is a marker of placental/fetal well-being. high pag-1 may represent a placental mechanism reducing maternal rejection, which is increased in crossbreed pregnancy because of increased genetic distance between dam and fetus.55 beef breeds in general and the limousin breed in particular are more resistant to n. caninum. in a study conducted in holstein-friesians using semen from four beef breeds, abortion rates were reduced by half, and rates in low-titer dams bred to limousin bulls were the same as those in seronegative animals.55 using beef semen also has the advantage of producing hybrid calves that are easily identified for removal and have a higher value for beef production. determining the serostatus of each animal at birth is valuable for selection decisions and ultimately for effective and economical control of neospora-induced abortion. dubey et al and mccann et al note that post-natal infections do not or rarely lead to persistent infections characterized by recrudescence and transmission during repeat pregnancies.2,24 if animals born seronegative are infected post-natally, they are unlikely to abort due to neospora or transmit the infection transplacentally more than once. thus, nothing is gained by culling post-natally infected dams after they have aborted or have seroconverted. congenitally infected animals should be identified and excluded from breeding, or bred only with beef semen in the case of dairy animals. if an attenuated live vaccine becomes available, vaccine costs could be reduced by not vaccinating congenitally infected animals identified through precolostral screening because vaccination is unlikely to be of benefit in them.29 elimination of congenitally infected females through precolostral screening and vaccination of naïve heifers before clinical theriogenology • volume 5 number 2 • june 2013 116 breeding with an attenuated live vaccine may dramatically reduce losses associated with neospora caninum. more research will be needed to clarify the differences between congenital versus post-natally acquired neosporosis with respect to endogenous transplacental transmission and the efficacy of live vaccines. conclusion neospora caninum is a highly successful, ubiquitous, intracellular parasite of cattle that is thought to be responsible for up to 20 % of all bovine abortions worldwide, incurring a global annual loss estimated at $1.3 billion. it persists within cattle populations primarily by evading its host’s immune system in thick-walled cysts until, by an unknown mechanism, it senses the conditions of second to third trimester pregnancy and recrudesces to cross the placenta and infect a new fetal host. it is challenging to diagnose and control; indeed, there is no indication that our efforts have reduced its prevalence since its discovery.7 the most successful control efforts involve vaccinating naive animals before gestation with live, naturally attenuated strains of the parasite, and identifying congenitally infected female progeny by precolostral serological testing to exclude them from becoming replacement animals. references 1. dubey jp: neosporosis in cattle. vet clin north am food anim pract 2005;21:473-483. 2. dubey jp, schares g, ortega-mora lm: epidemiology and control of neosporosis and neospora caninum. clin microbiol rev 2007;20:323-367. 3. wouda w: diagnosis and epidemiology of bovine neosporosis: a review. vet q 2000;22:71-74. 4. dubey jp, buxton d, wouda w: pathogenesis of bovine neosporosis. j comp pathol 2006;134:267-289. 5. reichel mp, alejandra ayanegui-alcérreca m, et al: what is the global economic impact of neospora caninum in cattle the billion dollar question. int j parasitol 2013;43:133-142. 6. dubey jp, schares g: neosporosis in animals-the last five years. vet parasitol 2011;180:90-108. 7. dubey jp, schares g. diagnosis of bovine neosporosis. vet parasitol 2006;140:1-34. 8. sanderson mw, gay jm, baszler tv: neospora caninum seroprevalence and associated risk factors in beef cattle in the northwestern united states. vet parasitol 2000;90:15-24. 9. rodriguez i, choromanski l, rodgers sj, et al: survey of neospora caninum antibodies in dairy and beef cattle from five regions of the united states. vet ther 2002;3:396-401. 10. cheadle ma, lindsay ds, blagburn bl: prevalence of antibodies to neospora caninum in dogs. vet parasitol 1999;85:325-330. 11. tranas j, heinzen ra, weiss lm, et al: serological evidence of human infection with the protozoan neospora caninum. clin diagn lab immunol 1999;6:765-767. 12. lobato j, silva da, mineo tw, et al detection of immunoglobulin g antibodies to neospora caninum in humans: high seropositivity rates in patients who are infected by human immunodeficiency virus or have neurological disorders. clin vaccine immunol 2006;13:84-89. 13. robert-gangneux f, klein f: serologic screening for neospora caninum, france. emerg infect dis 2009;15:987988. 14. petersen e, lebech m, jensen l, et al: neospora caninum infection and repeated abortions in humans. emerg infect dis 1999;5:278-280. 15. mccann cm, vyse aj, salmon rl, et al: lack of serologic evidence of neospora caninum in humans, england. emerg infect dis 2008;14:978-980. 16. mcallister mm, dubey jp, lindsay ds, et al: dogs are definitive hosts of neospora caninum. int j parasitol 1998;28:1473-1478. 17. dubey jp, jenkins mc, rajendran c, et al: gray wolf (canis lupus) is a natural definitive host for neospora caninum. vet parasitol 2011;181:382-387. 18. king js, slapeta j, jenkins dj, et al: australian dingoes are definitive hosts of neospora caninum. int j parasitol 2010;40:945-950. 19. alves neto af, bandini la, nishi sm, et al: viability of sporulated oocysts of neospora caninum after exposure to different physical and chemical treatments. j parasitol 2011;97:135-139. 20. okeoma cm, williamson nb, pomroy we, et al: the use of pcr to detect neospora caninum dna in the blood of naturally infected cows. vet parasitol 2004;122:307-315. 21. okeoma cm, williamson nb, pomroy we, et al: isolation and molecular characterisation of neospora caninum in cattle in new zealand. n z vet j 2004;52:364-370. 22. piergili fioretti d, pasquali p, diaferia m, et al: neospora caninum infection and congenital transmission: serological and parasitological study of cows up to the fourth gestation. j vet med b infect dis vet public health 2003;50:399-404. clinical theriogenology • volume 5 number 2 • june 2013117 23. dijkstra t, eysker m, schares g, et al: dogs shed neospora caninum oocysts after ingestion of naturally infected bovine placenta but not after ingestion of colostrum spiked with neospora caninum tachyzoites. int j parasitol 2001;31:747-752. 24. mccann cm, mcallister mm, gondim lf, et al: neospora caninum in cattle: experimental infection with oocysts can result in exogenous transplacental infection, but not endogenous transplacental infection in the subsequent pregnancy. int j parasitol 2007;37:1631-1639. 25. french np, clancy d, davison hc, et al: mathematical models of neospora caninum infection in dairy cattle: transmission and options for control. int j parasitol 1999;29:1691-1704. 26. björkman c, mcallister mm, frössling j, et al: application of the neospora caninum igg avidity elisa in assessment of chronic reproductive losses after an outbreak of neosporosis in a herd of beef cattle. j vet diagn invest 2003;15:3-7. 27. barling ks, sherman m, peterson mj, et al: spatial associations among density of cattle, abundance of wild canids, and seroprevalence to neospora caninum in a population of beef calves. j am vet med assoc 2000;217:1361-1365. 28. dijkstra t, lam tj, bartels cj, et al: natural postnatal neospora caninum infection in cattle can persist and lead to endogenous transplacental infection. vet parasitol 2008;152:220-225. 29. williams dj, hartley cs, björkman c, et al: endogenous and exogenous transplacental transmission of neospora caninum how the route of transmission impacts on epidemiology and control of disease. parasitology 2009;136:18951900. 30. dijkstra t, barkema hw, eysker m, et al: evaluation of a single serological screening of dairy herds for neospora caninum antibodies. vet parasitol 2003;110):161-169. 31. innes ea: the host-parasite relationship in pregnant cattle infected with neospora caninum. parasitology 2007;134(pt 13):1903-1910. 32. staska lm, mcguire tc, davies cj, et al: neospora caninum-infected cattle develop parasite-specific cd4+ cytotoxic t lymphocytes. infect immun 2003;71:3272-3279. 33. haldorson gj, mathison ba, wenberg k, et al: immunization with native surface protein ncsrs2 induces a th2 immune response and reduces congenital neospora caninum transmission in mice. int j parasitol 2005;35:1407-1415. 34. innes ea, wright se, maley s, et al: protection against vertical transmission in bovine neosporosis. int j parasitol 2001;31:1523-1534. 35. williams dj, guy cs, smith rf, et al: immunization of cattle with live tachyzoites of neospora caninum confers protection against fetal death. infect immun 2007; 75:1343-1348. 36. weber fh, jackson ja, sobecki b, et al: on the efficacy and safety of vaccination in cattle with live tachyzoites of neospora caninum for the prevention of neospora-associated fetal loss. clin vaccine immunol 2012 20:99-105. 37. maley sw, buxton d, macaldowie cn, et al: characterization of the immune response in the placenta of cattle experimentally infected with neospora caninum in early gestation. j comp pathol 2006;135:130-141. 38. dubey jp, abbitt b, topper mj, et al: hydrocephalus associated with neospora caninum infection in an aborted bovine fetus. j comp pathol 1998;118:169-173. 39. davison hc, otter a, trees aj: estimation of vertical and horizontal transmission parameters of neospora caninum infections in dairy cattle. int j parasitol 1999;29:1683-1689. 40. wouda w, moen ar, visser ij, et al: bovine fetal neosporosis: a comparison of epizootic and sporadic abortion cases and different age classes with regard to lesion severity and immunohistochemical identification of organisms in brain, heart, and liver. j vet diagn invest 1997;9:180-185. 41. anderson ml, blanchard pc, barr bc, et al: neospora-like protozoan infection as a major cause of abortion in california dairy cattle. j am vet med assoc 1991;198:241-244. 42. wapenaar w, barkema hw, o'handley rm, et al: use of an enzyme-linked immunosorbent assay in bulk milk to estimate the prevalence of neospora caninum on dairy farms in prince edward island, canada. can vet j 2007;48:493499. 43. reichel mp, ellis jt: if control of neospora caninum infection is technically feasible does it make economic sense? vet parasitol 2006;142:23-34. 44. haddad jp, dohoo ir, vanleewen ja: a review of neospora caninum in dairy and beef cattle--a canadian perspective. can vet j 2005;46:230-243. 45. gottstein b, razmi gr, ammann p, et al: toltrazuril treatment to control diaplacental neospora caninum transmission in experimentally infected pregnant mice. parasitology 2005;130(pt 1):41-48. 46. haerdi c, haessig m, sager h, et al: humoral immune reaction of newborn calves congenitally infected with neospora caninum and experimentally treated with toltrazuril. parasitol res 2006;99:534-540. 47. kritzner s, sager h, blum j, et al: an explorative study to assess the efficacy of toltrazuril-sulfone (ponazuril) in calves experimentally infected with neospora caninum. ann clin microbiol antimicrob 2002;1:4. 48. romero jj, pérez e, frankena k: effect of a killed whole neospora caninum tachyzoite vaccine on the crude abortion rate of costa rican dairy cows under field conditions. vet parasitol 2004;123:149-159. 49. weston jf, heuer c, williamson nb: efficacy of a neospora caninum killed tachyzoite vaccine in preventing abortion and vertical transmission in dairy cattle. prev vet med 2012;103:136-144. 50. reichel mp, ellis jt: neospora caninum-how close are we to development of an efficacious vaccine that prevents abortion in cattle? int j parasitol 2009;39:1173-1187. clinical theriogenology • volume 5 number 2 • june 2013 118 51. larson rl, hardin dk, pierce vl: economic considerations for diagnostic and control options for neospora caninuminduced abortions in endemically infected herds of beef cattle. j am vet med assoc 2004;224:1597-1604. 52. häsler b, stärk k, gottstein b, et al: epidemiological and financial considerations for the control of neospora caninum on swiss dairy farms. schweiz arch tierheilkd 2008;150:273-280. 53. baillargeon p, fecteau g, paré j, et al: evaluation of the embryo transfer procedure proposed by the international embryo transfer society as a method of controlling vertical transmission of neospora caninum in cattle. j am vet med assoc 2001;218:1803-1806. 54. landmann jk, jillella d, o'donoghue pj, et al: confirmation of the prevention of vertical transmission of neospora caninum in cattle by the use of embryo transfer. aust vet j 2002;80:502-503. 55. almería s, lópez-gatius f, garcía-ispierto i, et al: effects of crossbreed pregnancies on the abortion risk of neospora caninum-infected dairy cows. vet parasitol 2009;163:323-329. figure 1. animals reported as seropositive for neospora caninum australian dingoes bali cattle beef cattle bison black tailed deer camel capybara caribou cat chicken coyote dairy cattle dog egyptian mongoose eurasian badger eurasian lynx european lynx feral cat follow deer fox gaur grey wolf hare iberian lynx jaguar kuril harbor seal leopard lion llama moose mouflon mouse mule deer north american opossum panda pine martin pole cat puma rabbit rat raven red deer red panda roe deer sea otter spanish ibex sparrow stone martin tiger vietnam sika deer vole water buffalo water vole white rhinoceros white tailed deer yak clinical theriogenology • volume 5 number 2 • june 2013119 figure 2. countries reported seroprevalence of bovine neosporosis in the last 5 years algeria argentina australia brazil canada china czech republic egypt germany greece iran latvia mexico new zealand norway pakistan peru philippines romania spain sweden thailand turkey united kingdom united states of america vietnam clinical theriogenology • volume 5 number 2 • june 2013 120 figure 3. life cycle of neospora caninum clinical theriogenology • volume 5 number 2 • june 2013121 2015: pricing strategies and the perceptions that drive them pricing strategies and the perceptions that drive them d. kirk eddleman weatherford, tx in order to survive, let alone grow, a practice has to attract and retain loyal customers. a loyal customer is one who has a positive attitude about the practice, who buys frequently and who recommends the practice to friends and colleges. it costs more to acquire new customers than to retain current customers. this means loyal customers generate more profits each year they are retained. given that successful practices typically see 80% of their income come from just 20% of their customers, it is vital that we learn to focus on customer loyalty especially with our top customers. one of the three key determinates of customer loyalty is the total cost of using a practice’s products or services. customers will buy from the practice they see as offering the highest perceived value. customer perceived value, or cpv, is the difference between the customers’ perceived benefit derived from using a company’s product or service and the total costs of purchasing that product or service. therefore, pricing and other financial techniques can play a significant role in the customer’s perceived value and, ultimately, are a key factor in practice profitability. what is customer loyalty? the “loyalty effect” is a powerful profit generator because loyal customers spend more, refer others, and cost less to serve. a loyal customer is one who has a positive attitude about the practice, who buys frequently and who recommends the practice to friends and colleges. customer loyalty is about attracting the right customers and getting them to buy, buy often and bring you even more customers. businesses with loyal customers grow, on average more than twice as fast as the industry average. in fact, the probability of selling a product or service to loyal clients is much higher, 60% to 70%, compared to former client, 20% to 40%, and only 5% to 20% for a prospective client. often, veterinary practices will spend more of their time, energy and money on attracting new clients than on client loyalty programs. it’s time for a paradigm shift. our focus needs to be more on retaining customers rather than acquiring new ones. drivers of client loyalty in order to focus on loyalty we must understand what drives it. customers will buy from the practices they see as offering the highest perceived value. the cpv is the difference between the customer’s evaluation of all the benefits and all the costs of an offering and the perceived alternatives. cpv = total perceived benefit total cost the total customer value is the perceived monetary value of the bundle of economic, functional, and psychological benefits the customer expect from a given product or service. total customer cost is the total costs the customer expect to incur in evaluating, obtaining and using, a given product or service. several factors can influence the customer’s perceived value of a service. these include: • attitude about the brand, quality and customer focus • brand image such industry leader, trustworthy, innovative • customer experiences perceived quality, purchasing process, supportive, caring staff, and service provided • perceived value of the product or service received notice that the first thee have nothing to do with price. price is important but it is not the main thing that impact the customer perceived value. emotions play a much bigger role than price or even quality. yet, traditional customer loyalty programs have focused almost exclusively on lowering the price in the form of discounts. clinical theriogenology • volume 7, number 4 • december 2015439 pricing strategies for customer loyalty in veterinary medicine it is important to remember that while discounts have a high value for most customers (everyone loves to save money) they alone do not create loyalty. discounts attract bargain hunters who are easily lured away from the practice by someone offering a better discount. remember customer loyalty cannot be bought. it must be earned over time with good value for the money, superior products or services and the integrity of the practice. this means long-term loyalty must be developed at the emotional level. it must also be noted that giving a discount is giving away pure profit with no certainty that there will be an increase in volume to compensate for the discount given. therefore, smart practice managers will use more sophisticated pricing methods that make customers earn discounts rather simply giving them away. discounts should be used to reward customers and to direct their behavior. to achieve this there are several pricing strategies we can employ. the ones most commonly used in veterinary medicine described below: 1. multi-step quantity discounts this strategy offers increasing discounts at higher purchase levels. as the purchased quantity increases, the discount is also increased progressively. this encourages increased purchases, as the next discount level is easily attainable. however, because the discount starts lower and increases as the quantity increases the average discount given is lower than the current discount. 2. multi-product pricing there are two types of discounts in this category. the first is “tie-in sales” is where a strongly discounted sale of the main product/service is linked to a longerterm agreement to purchase complimentary products or services. the main product may even be sold as a loss leader. with this strategy a regular stream of income is offset the discounted product or service. this means the loyalty effect is very strong although additional action will be required at the end of the agreement. the second type, “price/product bundling,” combines products and services that are generally sold separately. the bundle is sold at a significant discount versus the sum of the prices for the individual products. 3. multi-person discounts with this strategy a discount is not offered to the main buyer but to an additional buyer if their purchases are tied together. here the loyalty program is aimed at the additional participants as well as the main purchaser. this creates a price advantage for the entire group. multi-person discounts are used for revenue maximization and are ideal for companies/industries with low variable cost and high fixed costs such as veterinary medicine. 4. the two-part tariff a mixture of an up-front flat payment and subsequent discounts spread out over a fixed period of time. each of these will be discussed in detail in the presentation with several examples of each for discussion purposes. determining price (how to properly set fees) before implementing any discount program it is critical a manager understands their cost of providing a good or service. the remainder of this presentation will focus on determining the total cost of providing goods and services and then setting fees based upon the actual cost plus a reasonable profit margin. this portion of talk is an interactive session designed to help veterinarians and practice managers better understand the true cost of doing business. we will use a hypothetical income statement to determine the direct variable and fixed costs that must calculated when setting fees. in addition, we will apply these concepts to set fees both professional service and product fees common in almost any veterinary practice. summary the ultimate goal of any customer loyalty program or pricing strategy is to maximize profits. in order to implement the right strategy in-depth analysis is needed. rather than cutting into profits by simply discounting this thorough preparation will be rewarded with higher profits and more loyal customers. value-oriented customer loyalty programs will help establish emotional relationships between the practice and the customer to create long-term loyalty. clinical theriogenology • volume 7, number 4 • december 2015 440 introduction domestic dogs have been theriogenology subjects for a very long time. some of the first artificial inseminations were on dogs in the 1700’s. breeding dogs is a widespread and active hobby and source of supplementary income for many dog owners. because dogs have various reproductive diseases and because breeding between males and females that cannot breed naturally is desired, veterinary assistance is often sought. clinicians who offer theriogenology services to dog breeders usually have a substantial portion of their practice devoted to these clients. diagnostic and treatment techniques have been developed that work very well in domestic dog theriogenology practice. many species of wild canids are housed in zoological institutions, with some species having endangered status. captive breeding programs aim to maintain a healthy captive population and some work with wildlife agencies to support wild populations. whereas some species reproduce well in captivity, others are less consistently successful. some species have a predilection for pyometra and require methods to prevent, diagnose, and treat this potentially fatal condition. breeding recommendations from species survival plan (ssp) committees sometimes involve older individuals that have not been reproductive in years or involve individuals that are not actively in a social pair bond. techniques used in domestic dog theriogenology practice have provided a useful framework for addressing these challenges in captive wild canids. application of domestic dog theriogenology practice in wild canids bruce christensen kokopelli assisted reproductive services, elk grove, ca abstract theriogenologists working in clinical practice with domestic dogs (canis familiaris) have experience in managing a wide variety of subfertility cases and several aspects of breeding. tools used include hormone monitoring, cycle manipulation, transcervical insemination, uterine and testicular biopsy, ultrasonography, and semen collection, analysis and storage. compared to some mammalian groups, members of the family canidae are relatively similar to each other anatomically. species vary in their reproductive physiology and much is still unknown about how disease processes differ among species. furthermore, susceptibility to certain diseases is not uniform throughout the clade. diagnostic and treatment techniques developed in domestic dog theriogenology have been used with variable success in wild canids. transcervical insemination has been used successfully in wolves and coyotes. transcervical endometrial biopsies have been obtained and interpreted from gray wolves (canis lupus), red wolves (canis rufus), and african wild dogs (lycaon pictus). semen has been collected via electroejaculation and chilled or cryopreserved from numerous species. successful use of frozen semen to obtain a pregnancy has been documented in gray wolves. ultrasonography has been used in many species to diagnose pregnancy. estrus induction has been used in wolves. areas that still require much investigation include cycle monitoring and manipulation, gestational aging with ultrasonography, and semen freezing techniques. keywords: canids, transcervical, cryopreservation, pyometra, ultrasonography breeding management most canids are seasonally monoestrous, with prolonged proestrus and estrus, followed by a long period of diestrus (regardless of pregnancy status), and then prolonged anestrus.1-4 the asian wild dog (cuon alpinus) is an exception as they have a seasonally polyestrous cycle.5 domestic dogs are nonseasonally monestrous.6 a common method of breeding management in domestic dogs is to monitor serum progesterone concentrations every 1 3 days. progesterone increases due to preovulatory luteinization correlate with the luteinizing hormone (lh) surge, ovulation, and the fertile period.6-7 this hormone monitoring allows accurate planning for natural breeding or artificial insemination. breeding recommendations have developed based on changes in serum progesterone, vaginal cytology, and vaginoscopy to maximize success when using fresh, chilled, or frozen semen.7 protocols are still being developed to monitor proestrus and estrus in wild canids to predict the optimal time for artificial insemination. the reproductive advisory group for the mexican wolf ssp has tried various protocols over the past 5 years in efforts to develop an effective way to utilize frozen semen for species propagation. logistical challenges are involved in capturing and restraining individual wolves for each blood collection, and each capture causes some degree of stress on the individual wolf. the effect of this stress on reproductive function and hormone concentrations is not known. developing these protocols involves balancing the perceived stress of capture with the need for relatively frequent samples in order to adequately clinical theriogenology 2021; 13: 375 monitor the dynamic changes during proestrus and estrus. even with the progesterone data, it is unknown how those concentrations actually correlate with the physiological events of the lh surge, ovulation, and fertile period. assumptions have been made that wolf profile will mirror domestic dog profile, but this has not been verified. artificial inseminations in wolves have had mixed results, and lower success than would have been expected in domestic dogs. over the past 5 years, many artificial inseminations with frozen semen have been performed, with only 1 verified pregnancy, resulting in a singleton birth from frozen semen at the endangered wolf center in eureka, mo in april, 2017.8 in a study investigating evolutionary relationships between wolves and coyotes, wherein 4 female coyotes were inseminated with chilled semen from gray wolves, 2 pregnancies resulted.9 work is ongoing to better understand wild canid cycles and how we might better time inseminations to coordinate with their fertile periods. protocols have been developed to induce domestic dogs to cycle, rather than waiting for natural proestrus, by either a dopamine agonist or a gonadotropin releasing hormone (gnrh) agonist treatment. these techniques have been used successfully in wolves to induce fertile heats, as demonstrated by a number of pregnancies resulting from both natural and artificial inseminations following induced cycles. results have been inconsistent, in part due to changing availability of gnrh products. estrus induction can be particularly beneficial in captive breeding programs as the insemination usually involves experts from various institutions, most of whom do not live close to the institution housing the wolves. each of these workers must take time away from their jobs and families to participate in the procedure. waiting for the wolf to come into estrus naturally would require frequent monitoring and then everyone must come immediately when she is ready. inducing the estrous cycle allows planning ahead of time. semen evaluation and preservation semen collection in domestic dogs is accomplished through manual stimulation of the penis. semen evaluation techniques mirror those for other species, including basic measurements of concentration, motility, and morphology. more advanced evaluation of parameters such as dna and membrane integrity have been established. chilling domestic dog semen and preserving sperm function is easily accomplished now through the use of multiple commercial extenders, including but not limited to caniplus chilltm (minitube, verona wi), botudogtm (botupharma, phoenix, az), canine-exttm (reproduction provisions, walworth, wi), and fresh express® (zoetis, parsippany, nj). cryopreservation of domestic dog sperm has more variable results, most likely due to individual biological variations among dogs and extender components (all of the aforementioned canine chilled semen extenders also have frozen semen extender media available). semen collection in wild canids is accomplished through electroejaculation under sedation. sperm collected manually in domestic dogs maintained better motility than semen collected via electroejaculation;10 similar effects may be present in wolf sperm, perhaps due to the prolonged exposure to prostatic fluid, which has a detrimental effect on canine sperm.11-12 centrifugation techniques developed in domestic dogs, using semen separators and cushions, may be of use in wild canid electroejaculated semen. centrifugation of electroejaculated wolf semen failed to yield improvements, but had a positive effect with some extenders.13 cryopreservation of wild canid semen has been practiced now for over 2 decades, but successful pregnancies using frozen semen are lacking. one litter of 6 viable gray wolf neonates and 1 stillborn was achieved following multiple vaginal inseminations with frozen semen in 1973. the cubs lived 7 days before dying from maternal neglect.14 no other successful pregnancies from frozen semen were reported until 2017 when the previously mentioned singleton pregnancy was achieved using transcervical insemination (tci) in a mexican gray wolf. this overall lack of success may largely be due to the need to develop better protocols for timing inseminations. postthaw motility is variable among males and ejaculates, but some had impressive postthaw motility percentages. pregnancy diagnosis and evaluation in domestic dogs, pregnancy diagnosis may be accomplished through transabdominal palpation, relaxin assay, transabdominal ultrasonography, or radiographs. all of these require some degree of restraint. with wild canids, a relaxin assay may be the most accessible, as it only requires a blood sample, but does not give any information regarding the size or health of the litter.15 in domestic dogs, measurements of fetal and extrafetal structures have been correlated to gestational age. various formulas are used, depending on breed size.16 however, these measurements have not yet been tested in wild canid species. subfertility evaluation evaluation of causes of subfertility in female domestic dogs often involves cranial vaginal cultures (using a double-guarded cultrate), transcervical endometrial biopsy17 and culture, and ultrasonographic evaluation. data derived from these procedures can be useful to detect inflammation, infection, fibrosis, and cystic endometrial hyperplasia. for the male dog, subfertility evaluation involves semen evaluation, prostatic fluid evaluation, ultrasonography of the testes and prostate, and testicular biopsy. these tests can help detect infection, degeneration, hyperplasia, and neoplasia. in wild canids, cranial vaginal and transcervical samples may also be obtained. this has been accomplished in some wild canid species, including mexican gray wolves, red wolves, and african wild dogs. histopathological changes appear similar among species. both african wild dogs and red wolves are predisposed clinical theriogenology 2021; 13: 376 to develop cystic endometrial hyperplasia and pyometra.18 early detection of cystic endometrial changes could be very helpful in management decisions. semen evaluation in wild canids can be done as in domestic dogs, with the exception that it is not possible to easily separate prostatic fluid from the sperm-rich fraction as the prostate continuously expresses fluid throughout the electroejaculation process. ultrasonographic evaluations of prostate and testes, and testicular biopsy, may also be similarly performed on any wild canid, though the author is not aware of their use to date. conclusion techniques developed for use in domestic dogs can sometimes be used in their wild counterparts. not all techniques are directly applicable, due to species differences and logistical challenges of handling wild species. work needs to continue to investigate basic reproductive endocrinology and how to effectively manipulate cycles to more effectively manage breeding. conflict of interest there are no conflicts of interest to declare. references 1. songasen n, rodden m, brown jl, et al: patterns of fecal gonadal hormone metabolites in the maned wolf (chrysocyon brachyurus). theriogenology 2006;66:1743-1750. 2. monfort sl, wasser sk, mashburn kl, et al: steroid metabolism and validation of noninvasive endocrine monitoring in the african wild dog (lycaon pictus). zoo biol 1997;16:533-548. 3. walker sl, waddell wt, goodrowe kl: reproductive endocrine patterns in captive female and male red wolves (canis rufus) assessed by fecal and serum hormone analysis. zoo biol 2002;21:321-335. 4. nagashima jb, songsasen n: canine reproductive biology: norm and unique aspects in strategies and mechanisms. animals (basel) 2021;11:653. 5. khonmee j, rojanasthien s, thitaram c, et al: non-invasive endocrine monitoring indicates seasonal variations in gonadal hormone metabolites in dholes (cuon alpinus). conserv physiol 2017;5:doi: 10.1093/conphys/cox001. 6. concannon pw: reproductive cycles of the domestic bitch. anim reprod sci 201;124:200-210. 7. root kustritz mv: managing the reproductive cycle in the bitch. vet clin north am small anim pract 2012;42:423-437. 8. greeley s: international studbook: mexican gray wolf, canis lupus baileyi. living desert zoo and gardens, palm desert; living desert zoo and gardens: 2017. 1604. 9. mech d, christensen bw, asa cs, et al: production of hybrids between western gray wolves and western coyotes. plos one 2014;9:e88861. 10. christensen bw, asa cs, wang c, et al: effect of semen collection method on sperm motility of gray wolves (canis lupus) and domestic dogs (c. l. familiaris). theriogenology 2011;76:975-980. 11. nizanski, w: intravaginal insemination of bitches with fresh and frozen-thawed semen with addition of prostatic fluid: use of an infusion pipette and the osiris catheter. theriogenology 2006;66:470-483. 12. korochkina e, johannisson a, goodla l, et al: effect of prostatic fluid on the quality of fresh and frozen-thawed canine epididymal spermatozoa. theriogenology 2014;82:1206-1211. 13. christensen bw, asa cs, wang c, et al: kinematic activity of gray wolf (canis lupus) sperm in different extenders, added before or after centrifugation. theriogenology 2013;79:953-960. 14. seager swj, platz cc, hodge w: successful pregnancy using frozen semen in the wolf. int zoo yearb 1975; 15,140-143. 15. bergfelt, dr, peter at, beg ma: relaxin: a hormonal aid to diagnose pregnancy status in wild mammalian species. theriogenology 2014;82:1187-1198. 16. lopate c: gestational aging and determination of parturition date in the bitch and queen using ultrasonography and radiography. vet clin north am small anim pract 2018;48:617-638. 17. christensen bw, schlafer dh, agnew dw, et al: diagnostic value of transcervical endometrial biopsies in domestic dogs compared with full-thickness uterine sections. reprod domest anim 2012;47 (suppl 6):342-346. 18. asa cs, bauman kl, devery s, et al: factors associated with uterine endometrial hyperplasia and pyometra in wild canids: implications for fertility. zoo biol 2014;33:8-19. clinical theriogenology 2021; 13: 377 2019 an ultrasonographic technique to locate retained testes in swine an ultrasonographic technique to locate retained testes in swine sherrie g. clark department of large animal medicine virginia-maryland college of veterinary medicine virginia tech, blacksburg, va abstract cryptorchidism is apparently more common in boars than most other domesticated mammals. this condition causes economic losses due to diminished carcass quality. furthermore, although rarely reported, it can be life-threatening to companion swine. surgical excision is usually the treatment of choice. this article describes the use of ultrasonography to diagnose cryptorchidism and locate retained testes, as well as a surgical approach to remove testes. keywords: cryptorchid, boar, swine, surgery, ultrasonography introduction cryptorchidism is commonly defined as failure of one or both of the testes to descend into the scrotum in the time frame typical for a particular species.1 the probability of cryptorchidism is higher in companion animals and pigs than in sheep or cattle.2 in pigs, retained or undescended testes are usually unilateral. cryptorchidism is further classified, based on testis location, as being abdominal, inguinal, or subcutaneous. approximately 1% of male pigs are born cryptorchid, with an increasing percentage in association with inbreeding.3 cryptorchidism is not considered a single disease, but rather, an early indication of testicular dysgenesis syndrome,1,2 with causes grouped into genetic, epigenetic, or environmental components.2 to recognize potential causes or timing of the insult that causes cryptorchidism, one must understand normal testicular development and descent. primordial germ cells migrate from the hindgut onto the gonadal ridge of the mesonephros during early embryogenesis. mesenchymal cells then migrate to the developing gonad, surround the primordial germ cells, differentiate into sertoli cells and produce anti-mullerian hormone, halting mullerian duct formation. further proliferation and differentiation of sertoli and primordial germ cells continue for the next 7 to 14 days, until the gonad becomes a functional testis. during this proliferation, other mesenchymal cells migrate into the spaces among the developing seminiferous tubules and become leydig cells. in the pig, differentiation of the gonad to a functional testis occurs around gestational day 35.2 during differentiation, the gubernaculum and cremaster muscles form from abdominal muscles and the vaginal process forms in the area of the internal inguinal ring as an evagination of the peritoneal lining.1,2 the genitofemoral nerve also begins to innervate the cremaster muscle.1 at this point in development, the testis is located between the neck of the bladder and the internal inguinal ring. it becomes anchored to the caudal abdomen by the gubernaculum. this anchor prevents the testis from migrating cranially while the fetal abdomen expands cranially. as the fetus and testis grow, the gubernaculum extends and vaginal process extend through the inguinal ring. leydig cells are important at this point, producing testosterone and insulin-like peptide-3,1,2 a peptide with an important role in increasing gubernaculum size. this enlarged gubernaculum dilates the inguinal canal to facilitate passage of the enlarging fetal testis.1,2 testosterone masculinizes the genitofemoral nerve, which then produces calcitonin gene-related peptide, a peptide that serves as a chemoattractant and directs migration of the gubernaculum and testis to its final scrotal location.1,2 testosterone also stimulates gubernacular regression once the testis reaches its scrotal location, as well as the inguinal canal to constrict or close.1 in the pig, this final scrotal descent occurs 14 days before to 14 days post-farrowing, whereas final testicular maturation occurs when testis is in the scrotum.1,4 cryptorchidism results when a problem occurs during one of the three phases of testicular descent and one or both testes do not reach a final scrotal location. in swine, cryptorchidism occurs in 1% of male offspring, with unilateral occurrence more common.3 in cryptorchid boars, the unilateral phenotype represents 59% of the cryptorchid cases.2 of the many theories regarding the cause of cryptorchidism in clinical theriogenology • volume 11 number 1 • march 201973 pigs, genetic predisposition remains a common concern. it has been suggested that there is incomplete penetrance, because not all males in an affected litter will be cryptorchid.2 another study indicated that cryptorchidism is a recessive trait with genes on at least two loci3 and inbreeding increased frequency of cryptorchidism to 3%. this is more likely in potbellied pigs compared to domestic pigs.3 failure of testicular descent could also be hormone related. increased estrogen production could limit insulin-like peptide-3 production and prevent gubernacular enlargement if exposure occurs just prior to testosterone dependency.1,5,6 without gubernacular enlargement, the developing fetal testis cannot fit through the inguinal ring and becomes retained. surgical correction removal of retained testis or testes is important for animal health and for economics of commercial pork production. it was estimated that testicular tumors occur 4 11 times more frequently in cryptorchid than non-cryptorchid males.2 the most commonly reported tumors in pigs are gonocytic seminomas and carcinoma in situ; these are more common in pet pig dues to their longer lifespan.2 furthermore, this condition results in economic losses to the commercial producer due to increased culling rates and market discounts. retained testes produce increased amounts of 5-androst-16-ene-3-one resulting in boar odor tainted meat, with lower consumer appeal.1,3,7 for pigs with abdominal retention of a testis, the most commonly described approach for surgical excision is inguinal or para-inguinal on the side of the retained testis.8,9,10 alternatively, a ventral midline approach is more likely if the side of retention is unknown or if retention is bilateral. male pigs are often presented to the veterinarian after the descended testis has been removed, assuming that the retained testis will descend at a later date.9 unfortunately, by the time the pig presents for cryptorchid surgery, the incision over the descended testicle has healed and a scar is not detectable. the literature suggests that retained testes are more common on the left side. in the author’s experience, >99% of retained testes are in the abdominal cavity. furthermore, the author proposes real-time b-mode ultrasonography to locate retained testes, followed by way a paramedian incision directly over the testis. the author prefers that pigs, especially companion pigs, weigh at least 4.5 5 kg. surgery is performed under general anesthesia (induced with a combination of telazol® reconstituted with 250 mg of xylazine (100 mg/ml) and 250 mg ketamine and then dosed at 4.4 mg/kg im of this mixed solution11) and positioned in dorsal recumbency. a 5 mhz frequency, linear-array ultrasound transducer is used to locate the abdominal testis. if the descended testis is still present or the surgical scar from the previous castration is evident, the probe is placed on the abdomen on the opposite side of the scar or testis. the testis is usually found by placing the transducer midway between the inguinal canal and the preputial orifice and midway between the flank fold and midline, with the tip angled toward the pig’s head (figure 1). it is not uncommon to need to adjust transducer position to detect the testis, depending on its general location within 1 2 cm below skin level. this transducer position usually provides a sagittal section of the retained testis. the ultrasonographic image of the testis is usually oblong, with the tunica albuginea producing a hyperechoic border. in addition, the mediastinum testis produces a hyperechoic band in the center of the testis that runs the entire length of the testis. testicular parenchyma has a homogenous, granular pattern with medium echodensity on both sides of the mediastinum testis, and within the tunica albuginea (figure 2).12,13 once the testis has been located, a paramedian incision is made the length of the testis and extended through the abdominal musculature and peritoneum. the surgeon inserts a gloved finger into the abdomen and locates the testis or epididymis by moving the finger in a sweeping motion on either side of the incision. once the structure is located, the surgeon hooks the structure with their finger and exteriorizes it through the incision (figure 3). it is often helpful at this point to use the thumb of the same hand to press downward on the skin near the incision to help “pop” the testis through the incision. once exteriorized, the surgeon must examine the structure to identify testis, epididymis, spermatic cord and cremaster muscle. the spermatic cord is separated from the cremaster and each structure is ligated individually. preferably ligatures are placed 5 mm apart and the structure transected between ligatures. following removal of the testis, the incision is closed in a two-layer closure. if there is a large amount of clinical theriogenology • volume 11 number 1 • march 2019 74 abdominal fat, a three-layer closure may be necessary to minimize dead space. the author prefers to close the skin using an intradermal suture pattern using poliglecaprone 25 (monocryl®; ethicon, bridgewater, nj) for cosmetic purposes. choice of suture is at surgeon’s discretion, but the author prefers polydioxanone (pds®ii, ethicon, brdigewater, nj) for ligation of the spermatic cord and closure of the body wall. conventional pigs are routinely given flunixin meglumine (2.2 mg/kg) intramuscularly and ceftiofur crystalline free acid (excede® for swine; zoetis, kalamazoo, mi) at a dose of 5 mg/kg im. for companion swine, carprofen (4.4 mg/kg orally once daily for 3 days) is often used in lieu of flunixin meglumine. conclusion use of ultrasonography is likely more common with companion swine, unless the domestic pig is considered valuable. the use of this approach facilitates locating a retained testis prior to surgery, thereby increasing successful procedures. in general, there are minimal complications with this surgical procedure. rare complications include excessive hemorrhage due to improper vessel ligation, incisional dehiscence, or incisional abscessation. references 1. amann rp, veeramachaneni dnr: cryptorchidism in common eutherian mammals. reproduction 2007;133:541-561. 2. amann rp, veeramachaneni dnr: cryptorchidism and associated problems in animals. anim reprod 2006;3:108120. 3. rothschild mf, christian ll, blanchard w: evidence for multigene control of cryptorchidism in swine. j hered 1988;79:313-314. 4. van straaten hwm: lack of a primary defect in maldescended testis of the neonatal pig. biol reprod 1978;19:994-998. 5. ryan pl, friendship rm, raeside ji: impaired estrogen production by leydig cells of the naturally retained testis in unilaterally cryptorchid boars and stallions. j androl 1986;7:100-104. 6. veeramachaneni dnr: germ cell atypia in undescended testes hinges on the aetiology of cryptorchidism but not the abdominal location per se. int j androl 2006;29:235-240. 7. liptrap rm, raeside ji: urinary steroid excretion in cryptorchidism in the pig. j reprod fert 1970;21:293-301. 8. st. jean, g: male reproductive surgery. vet clin north am food anim pract 1995;11:55-93. 9. st. jean g, anderson de: surgery of the swine reproductive system and urinary tract. in: fubini sl, ducharme ng, editors. farm animal surgery. ist edition. st. louis; elsevier; 2004. p. 565-575. 10. st. jean g, anderson de: anesthesia and surgical procedures in swine. in: straw be, d’allaine s, mengeling wl, taylor dj, editors. diseases of swine. 9thedtion, ames; blackwell; 2006, p. 114-1115. 11. reibold tw, geiser dr, goble do. large animal anesthesia: principles and techniques, 2nd edition. ames: iowa state press; 1995, p. 16-58. 12. pugh cr, konde lj, park rd. testicular ultrasound in the normal dog. vet radiol 1990; 31:195-199. 13. clark sg, schaeffer dj, althouse gc: b-mode ultrasonographic evaluation of paired testicular diameter of mature boars in relation to average total sperm numbers. theriogenology 2003;60:1011-1023. figure 1. transducer placement to locate abdominal testes in swine. clinical theriogenology • volume 11 number 1 • march 201975 figure 2. b-mode ultrasonographic image of a porcine abdominal testis, with key structures identified. figure 3. porcine testis exteriorized through a paramedian incision and key structures identified. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: neonatal and pediatric ultrasonographypart i neonatal and pediatric ultrasonographypart i 1 t.w. baker, a.p. davidson 2 department of medicine and epidemiology, school of veterinary medicine, university of 3 california, davis, ca, usa 4 5 introduction 6 small animal patients are commonly presented to the veterinarian because of 7 signs referable to the abdominal cavity due to congenital anomalies, dietary indiscretion, 8 parasitic infestation and infectious or inflammatory disease. abdominal ultrasound 9 provides valuable clinical information about the peritoneal cavity, great vessels, 10 abdominal viscera and lymph nodes obtained in a non invasive fashion, with no 11 confirmed adverse biologic effects, and usually not necessitating sedation or 12 anesthesia. this paper reviews the techniques for performing the pediatric abdominal 13 ultrasound scan. 14 abdominal ultrasound provides useful data in a short period of time. the normal 15 paucity of intra-abdominal fat in pediatric patients results in less informative abdominal 16 radiography, but actually improves ultrasonographic imaging. (abdominal fat attenuates 17 the ultrasound beam.)1 image quality is improved with small patient size as a higher 18 frequency scanhead can be employed. acquisition of special equipment for pediatric 19 ultrasonography is usually not necessary as scanheads selected for small animal 20 (especially feline) clinical use are appropriate for most pediatric cases. 21 small animal patients are best evaluated using an ultrasound machine equipped 22 with a curvilinear variable frequency scanhead (6.0-8.0 mhz). many portable machines 23 143 now have available a high frequency linear scanhead (8.0 -10.0 mhz) which will 24 improve quality and also allow evaluation of smaller regional anatomy (thyroid, 25 parathyroid, cryptorchid testes).2 26 keywords: ultrasound, pediatric, abdominal, neonatal 27 preparation 28 the small animal patient is placed in dorsal recumbency within a padded v-29 trough, and is gently restrained by assistant(s) holding the forelimbs and hindlimbs. 30 sedation is rarely required for the basic abdominal scan unless marked pain or 31 apprehension is present. allowing the patient to become accustomed to this restraint 32 before clipping the hair or initiating the scanning process usually minimizes struggling 33 and the resulting aerophagia.3 34 to prepare the pediatric patient for abdominal ultrasonography, the cranioventral 35 abdominal hair is clipped using a no. 40 blade. wetting the skin with water, tincture of 36 zephiran or 70% isopropyl alcohol, followed by a liberal application of ultrasound gel, 37 permits the best acoustic coupling of the scanhead to the patient and improves the 38 image obtained. some pediatric patients have scant ventral haircoats that do not 39 require clipping. care should be taken to avoid excessive chilling of pediatric patients. 40 chilling can occur secondary to the application and evaporation of room-temperature 41 wetting agents. electric warming devices (warm water blankets) may cause electronic 42 interference with the ultrasound equipment; warm water bottles or their equivalent are 43 superior.1 44 patient preparation includes fasting as much as is safely possible given the 45 animal’s age and size. fasting minimizes obscurement of the liver imaging by gastric 46 144 ingesta and of other abdominal viscera by gastrointestinal gas accumulation. preventing 47 urination immediately prior to the examination allows urine to accumulate within the 48 urinary bladder lumen and permits better evaluation of this structure. 4 49 serial evaluations can provide useful information when the clinical status of the 50 small animal patient has changed. indications for serial ultrasonographic evaluation 51 include clinicopathologic deterioration, progressive lethargy or obtundation, acute pain, 52 changes in abdominal palpation findings and refractory vomiting or diarrhea. all warrant 53 repeat evaluation for signs indicating the development of intussusception, perforation 54 and/or peritonitis. 55 the normal abdomen 56 regardless of the clinical history, the abdomen should be evaluated methodically 57 with the animal in dorsal decumbency. realize the sector image you are looking at on 58 the monitor screen is perpendicular to that ultrasound beam. you are viewing the 59 sagittal image from the side of the animal and the transverse image from the rear of the 60 animal. (fig 1a-c, fig 2a-c).1 61 place the scanhead under the xyphoid with the beam in sagittal plane. 62 visualization of the liver is achieved by fanning the beam from right to left. the gall 63 bladder is seen on the right; the left liver lobes are seen ventral and sometimes caudal to 64 the stomach. turning the beam to transverse allows for visualization of the liver between 65 stomach and gall bladder. this view is useful for evaluation of the hepatic border, 66 echogenicity of hepatic parenchyma and portal architecture. the portal vessels have very 67 echogenic walls.5 (fig 3) 68 145 resuming the sagittal plane, scan to the left of the dog past the stomach to the 69 spleen. the spleen will be visualized ventrally in the near field. splenic border, 70 parenchyma and shape should be evaluated.6 (fig 4) following the spleen transversely 71 down the left body wall, you will image the left kidney. (fig 5) 72 once visualization of the kidney is achieved, turn the transducer to the sagittal 73 plane and evaluate the renal border, cortical echogenicity and pelvic architecture. 74 dilatation of the renal pelvis is best seen in the transverse plane. normal ureters are not 75 usually visible ultrasonographically.1,7 the left adrenal gland is located medial to the 76 cranial pole of the kidney. in a sagittal plane, maintaining strong hand pressure, scan 77 medially to visualize the linear aorta and the renal artery. the left adrenal is located cranial 78 to the left renal artery and caudal to the left cranial mesenteric artery. the left adrenal 79 gland is imaged as a bi-lobed structure with the phrenicoabdominal vein at its waist.1 (fig 6) 80 with the transducer in a transverse plane in the midabdomen, scan caudally to 81 image a large hypoechoic structure, the urinary bladder. evaluate bladder wall and lumen 82 contents, and, dorsal to the bladder, the major vessels (caudal vena cava and aorta). 83 sublumbar lymph nodes will be seen at the aortic bifurcation into the iliac arteries, 84 adjacent to the bladder wall. (fig 7) sagittal scanning of the urinary bladder caudally will 85 allow visualization of the urethra (and prostate in the male).4 (fig 8) 86 continuing in methodic fashion, the right kidney will be imaged at the edge of the 87 right ribcage adjacent to the renal fossa of the liver. the right kidney should be evaluated 88 as was the left with respect to renal border, cortical echogenicity and pelvic architecture. 89 by scanning sagittally between the right kidney and the caudal vena cava with a fanning 90 146 technique, the right adrenal gland is visualized just lateral to the caudal vena cava. in a 91 transverse plane, the duodenum is imaged lateral to the right kidney.1 (fig 9) 92 at the cranial end of the right kidney medial to the duodenum will be the right limb 93 of the pancreas. the right pancreatic limb is identified by visualizing the caudal 94 pancreaticoduodenal vein within the structure. (fig 10) turning to the sagittal plane, follow 95 the pancreas, scanning medially to the angle of the body and left limb, or sagittally scan 96 the caudal border of the stomach. the pancreatic body is seen caudal to the stomach, 97 cranial to the splenic vein. the left pancreatic limb is found caudal to the splenic vein and 98 midline to the cranial pole of the left kidney.5 (fig 11) 99 returning to the transverse plane in midabdomen at the mesenteric root, scan for 100 mesenteric lymph nodes and small bowel wall changes. scanning in a uniform serpentine 101 fashion, 2-3 passes may be required to evaluate the entire bowel. normally, the small 102 bowel appears sonographically as four distinct layers. (fig 12, 13) the bowel lumen is 103 hyperechoic, as gas and ingesta are compressed. the layer just outside the lumen is 104 the mucosa; it is hypoechoic and normally the thickest appearing section. outside the 105 mucosa is the submucosa, it is hyperechoic to the mucosa and about one third the 106 thickness. the muscularis, the bowel muscle layer, is outside of the submucosa and 107 appears as a very thin hypoechoic black line. the outermost serosal layer is 108 hyperechoic.1 (fig 14) 109 references 110 111 147 1. baker tw: find your friends: location and appearance of normal abdominal 112 viscera, liver, spleen, kidney and urinary bladder. in: what’s that? a beginner’s 113 guide to veterinary abdominal ultrasound. lakewood, co: aaha press, 2009. 114 115 2. nyland tg, mattoon js, herrgesell ej, et al: physical principles, instrumentation, 116 and safety of diagnostic ultrasound. in: nyland tg, mattoon js, editors. small 117 animal diagnostic ultrasound, 2nd ed. philadelphia: wb saunders; 2002. p. 1-118 18. 119 120 3. penninck dg: artifacts. in: nyland tg, mattoon js, editors. small animal 121 diagnostic ultrasound, 2nd ed. philadelphia: wb saunders; 2002. p. 19-29. 122 123 4. nyland tg, mattoon js, herrgesell ej, et al: urinary tract. in: nyland tg, 124 mattoon js, editors. small animal diagnostic ultrasound, 2nd ed. philadelphia: 125 wb saunders; 2002. p.158-195. 126 127 5. penninck dg: gastrointestinal tract. in: nyland tg, mattoon js, editors. small 128 animal diagnostic ultrasound, 2nd ed. philadelphia: wb saunders; 2002, p.207-230. 129 130 6. nyland tg, hager da: sonography of the liver, gallbladder, and spleen. vet clin 131 north am sm anim pract 1985;15:1123-1148. 132 133 148 7. lamb cr: ultrasonography of the ureters. vet clin north am sm anim pract 134 1998; 28:823-848. 135 149 fig 1a the correct sagittal scanhead placement: button2: button4: button5: button6: button7: button8: button9: button10: button11: button12: button13: button15: button16: 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true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2013: incidence of post-surgical complications of ovariohysterectomy and orchiectomy in field conditions incidence of post-surgical complications of ovariohysterectomy and orchiectomy in field conditions laura a. baltodano,a mushtaq a. memon,a cathy kingb adepartment of veterinary clinical sciences, washington state university, pullman, wa; bworld vets, fargo, nd abstract in many developing countries, one of the major public health challenges is to control overpopulation of dogs and cats while considering the welfare of these animals. the objective of the current study was to determine the incidence of post-surgical complications arising from ovariohysterectomy and orchiectomy surgery in nicaragua under clean field conditions. the purpose of the study was to determine if described methods are a viable and safe option for performing sterilization surgeries on dogs and cats in situations where traditional surgery facilities and or/certain supplies are not available. a total of 181 ovariohysterectomies and orchiectomies were performed in may and june 2010. the animals ranged from 1.5 months to 13 years old. the first author performed a post-operative visit for a total of 132 animals four days following the procedure; 4.5% (6/132) of animals had redness of 3 or higher on a scale 0 to 5; 6.1% (8/132) of animals had swelling 3 or higher on a scale 0 to 5; 8.3% (11/132) of animals had skin dehiscence 1 or higher on a scale 0 to 5. no animal (0/132) had wound discharge and <1% (1/132) of animals died post-operatively due to ovariohysterectomy or orchiectomy. it is concluded that ovariohysterectomies and orchiectomies performed in field conditions have a similar rate of wound complications as elective surgeries performed in the united states in a clinical setting. keywords: spay, neuter, ovariohysterectomy, orchiectomy, complications, field conditions introduction as in many developing countries, in nicaragua one of the major public health challenges is to control overpopulation of dogs and cats while considering the welfare of these animals. in 2004, the panamerican health organization in conjunction with the nicaraguan government identified management of domestic and stray animal populations as a core domain of public health in the country. , traditionally, pet animals are maintained outdoors and are free to roam. consequently, intact animals are able to breed without restriction leading to canine overpopulation especially in the larger cities of the pacific coast. anemia is also endemic in children in nicaragua, the second poorest nation in the hemisphere after haiti, and high incidence of hookworm infestation has been identified as a confounding factor in this health problem. additionally, socioeconomic conditions result in limited resources for pet owners to seek medical care for their pets. unlike developed countries, there is no control of stray animals, and they roam freely in streets and many of them starve or are killed inhumanely by people that may consider them a nuisance. non-government organizations (ngo) with the help of volunteers and local partnering veterinarians perform ovariohysterectomies and orchiectomies to assist in population control of the stray animals. world vets (worldvets.org), a united states-based international ngo provides veterinary aid in many countries, including nicaragua. the objective of the current study was to determine the incidence of post-surgical complications arising from spay and neuter surgery in nicaragua under clean, field conditions. the outcome of the study will be used to determine if described methods are a viable and safe option for performing sterilization surgeries on dogs and cats in situations where traditional surgery facilities and or/certain supplies are not available. materials and methods field clinics and conditions field clinic 1 is a newly-built building located within a school of agriculture and cattle production. the facility has overhead lights, electricity and running water. surgery tables are made of tile. field clinic 2 is vacant building located at a local church. the facility has natural lighting, electricity, and running water. surgery tables are made of wood. 37 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 each field clinic had work stations for pre-surgical consultations; surgical preparation including induction, iv catheter placement, shaving and surgical scrub; surgery; recovery; and instrument cold sterilization. there was limited and unreliable availability of the electricity and running water in both field clinics. water was brought in bottles and jugs to be used onsite. ventilation was primarily accomplished by opening the doors and windows. fans were available and working when the electricity was on. there was no fly control. a new clean drape was utilized for each patient. the surgeon used a new pair of sterile gloves for each procedure, but no surgical masks or gowns were used. some surgeons used surgical caps during the procedure. prior to the start of surgery for the day, hands were scrubbed with chlorhexidine scrub and hand sanitizer was used between patients when changing sterile gloves. the surgical tables were sprayed with common household disinfectant spray between surgeries. all the surgical instruments were scrubbed with soap and water to remove any blood or debris and then rinsed in water. they were then cold sterilized by placing them in a disinfecting solution for a minimum of ten minutes. the cold sterilization was accomplished using steris amerse 2 (steris healthcare, mentor, oh), a one-step germicidal detergent with the active ingredients being o-phenylphenol and p-tertiaryamylphenol in a 1:128 usesolution. consultations and pre-surgical examination the owners were brought into an examination room with their pet for a pre-operative consultation and physical examination during which any unusual findings were noted and the animal’s weight was estimated. dogs were given injectable anesthetics, including a combination of acepromazine (dose range from 0.06-0.4 mg/kg), atropine (0.02-0.12mg/kg), and morphine (0.1-1.0 mg/kg). acepromazine, atropine and morphine were administered subcutaneously as a pre-anesthetic medication. for analgesia, cats were administered nalbuphine pre-operatively and dogs were administered ketoprofen postoperatively. the drug selection was based on limited availability in the country. cats were kept in a carrier until they were ready for induction of anesthesia. cats were given a combination that included ketamine, xylazine, acepromazine, and nalbuphine for induction. one bottle of ketamine, 50 mg xylazine, 2.5mg acepromazine, 10 mg nalbuphine where combined into one bottle. cats were dosed according to weight based on the following guidelines: <3 lbs 0.3 ml im, 3-4 lbs 0.5 ml im, 5-6 lbs 0.6 ml im, 79 lbs-0.7 ml im, 10-12 lbs -0.8 ml im, and >13lbs 0.9 ml im. this protocol maintained cats in an adequate plane of anesthesia throughout the surgical procedure for both ovariohysterectomies and orchiectomies. at the conclusion of the procedure, anesthesia was reversed using yohimbine (dosed at 0.1 mg/kg) intravenously. nitenpyram (capstar®, novartis animal health, basel switzerland)) was administered orally prior to surgery if the patient had an excessive number of fleas or ticks. a veterinary technician assisted in intake, premedication, and surgical preparation of patients. volunteer united states-based veterinarians and veterinary students from accredited veterinary colleges were involved in all procedures. the lead veterinarian, an experienced us-veterinarian, provided an orientation to the surgical team which included veterinarians and students involved in various aspects of animal handling and surgeries. surgical preparation an intravenous catheter was placed (most often in the cephalic vein) in all pre-medicated dogs. anesthesia for both ovariohysterectomies and orchiectomieswas maintained with a combination of ketamine and diazepam. the ventral abdomen of all animals was shaved and scrubbed with chlorhexidine. alcohol was applied to the skin prior to surgery for a contact time of three to five minutes. surgery a veterinarian performed surgeries with a veterinary student assisting and/or performing various aspects of the surgery under direct supervision. a variety of surgical techniques were used based on the surgeon’s preference but the fundamental procedure was removal of the ovaries and uterus with proper 38clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 hemostasis. absorbable sutures were used which consisted of poliglecaprone 25, polyglycolic acid, polydioxanone, and chromic catgut. after induction of anesthesia, patients were placed in dorsal recumbency and the ventral abdomen was clipped and prepared for an ovariohysterectomy or orchiectomy. ovariohysterectomies were performed via a ventral midline incision. the majority of the orchiectomies were performed using a prescrotal approach; however, one veterinarian used a scrotal approach. open and closed orchiectomy techniques were used at the discretion of individual veterinarians. for ovariohysterectomies, three layer closures were used: simple interrupted suture pattern in the linea alba, simple continuous in the subcutaneous layer, and intradermal in the dermal layer. tissue adhesive was placed on the incision to achieve further apposition. for ovariohysterectomies, tattoo ink was placed on or near the incision after closure in order to allow future identification of sterilized animals. lidocaine was injected on both sides of the incision following surgery to provide post-operative analgesia. total surgery time was between 5 and 115 minutes. an assistant administered additional anesthetic as needed throughout the surgery. recovery during recovery from anesthesia patients were placed in pre-designated areas monitored by students, volunteers, or technicians at all times. animals were discharged to owners after adequate recovery (standing and able to walk) from the anesthesia. prior to being discharged, patients received injections of benzathine penicillin g and procaine penicillin g and morphine. pyrantel pamoate was administered orally. insecticides for control fleas and ticks were administered topically. additional analgesics (tramadol), anti-inflammatories (carprofen) and antibiotics (doxycycline, cephalexin, or amoxicillin) were occasionally dispensed for patients at the veterinarian’s discretion. post-operative patient visits patients were visited at their homes four days after surgery to evaluate the animal and the surgical site. a questionnaire (appendix) was read in spanish to each owner by the first author. body condition score and wounds were assessed and the environment in which the animal was maintained after surgery was observed. some patients were lost to follow-up due to their remote location or inability to contact their owner. results animals were presented with a number of pre-existing conditions including, but not limited to pyometra, flea and tick infestation, biting louse infestation, pregnancy, transmissible venereal tumors, estrus, external and internal tumors, heart murmurs, probable ehrlichiosis, hepatomegaly, ascites, traumatic injury, bite wounds and cryptorchidism. surgical complications included three dropped ovarian pedicles and one torn uterus as well as hemorrhage in several patients (common with ehrlichiosis) and one incident of inadvertent partial thickness laceration of the serosa of the jejunum. bowel adhesions were also noted. anesthetic complications included seizures (n=2) and apnea (n=11). one patient was administered an incorrect dose and type of induction agent but recovered uneventfully. the post-operative home environment ranged from outdoors (either tied or free roaming) on dirt to indoors on tile floors. many pet owners did not have transportation. consequently, owners would walk the animal home, take them on a bicycle or cart or carry them. a few were transported by motor vehicle. 39 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 table 1. outcome of canine and feline ovariohysterectomies and orchiectomies under field conditions. week 1 week 2 week 3 week 4 total percent total number of animals seen for surgery and/or consultation 206 178 164 127 675 dogs spayed 26 30 16 17 89 dogs neutered 5 8 13 19 44 cats spayed 11 8 7 5 31 cats neutered 4 2 5 6 17 total post-operative evaluations 36 37 33 26 132 73 redness score 3-5* 1 1 2 2 6 4.5 inflammation score 3-5* 1 2 3 2 8 6.1 skin incision dehiscence 1 4 6 0 11 8.3 surgical site discharge score 3-5* 0 0 0 0 0 0 deaths associated with surgery† 0‡ 0 1§ 0** 1 <1 *assessment scores of 0-2 were common and were considered to be within normal limits. thus, these results were not included in the data reported in this table. †a total of four deaths occurred during this study. three were not related to the spay/neuter procedure and are not included in the data reported in this table. details of the unrelated deaths are included below. ‡one patient was a puppy that was presented with a proptosed eye following an attack by another dog. the eye was enucleated but the puppy died post-operatively. blood was found in the lungs upon intubation for cardiopulmonary resuscitation. death was attributed to trauma prior to presentation. §a dog was euthanized three days following orchiectomy due to poor recovery from surgery. a cat died pre-operatively after injection of anesthetic. **a dog had been struck by an automobile and was presented with a lacerated scrotum and an exposed testicle. the dog was neutered but died the day following surgery. death was attributed to trauma from the accident. table 2. body condition score§§ of canine and feline patients presented for ovariohysterectomies and orchiectomies in field conditions. §§hill’s pet nutrition body condition scoring chart was used. the score is based on a 5-point scale; 1=very thin and 5=obese. discussion the purpose of this study was to assess the incidence of post-surgical complications following ovariohysterectomy and orchiectomy of dogs and cats under clean but non-sterile field conditions. although surgical conditions were not ideal in that surgery was performed neither in aseptic conditions nor on ideal surgical candidates, the vast majority of animals recovered from surgery with minimal or no 1 9 11 14 70 18 6 1 2 0 10 20 30 40 50 60 70 80 1 1.5 2 2.5 3 3.5 4 4.5 5 body condition score body condition score assessment number of animals 40clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 complications. although post-operative care instructions were discussed with each owner, follow-up visits revealed a number of animals did not have appropriate post-operative care by the owner or caretaker which likely was a contributing factor to some of the complications. week 1. 1) a dog with redness had been licking her incision every day since surgery. 2) a cat with swelling had kittens that were still nursing. 3) a dog with minor skin dehiscence had been licking her incision. all of these complications were most likely due to inadequate post-operative care. week 2. skin dehiscence: surgery in three out of four animals with skin dehiscence was supervised by the same veterinarian. it is likely that the intradermal sutures were not closed with appropriate tension. the other case of skin dehiscence occurred in a cat that had been allowed to go outdoors too soon after surgery. week 3. skin dehiscence: five out of six animals with skin dehiscence did not have appropriate post-operative care. one of the orchiectomies had been performed through a scrotal approach which may have contributed to swelling and dehiscence. death: one patient was euthanized after an unsuccessful attempt to treat post-operative complications. this was the only death that occurred as a result of an ovariohysterectomy or orchiectomy. the patient was in poor condition pre-operatively and the death was likely due to the pre-existing health of the dog. a necropsy was not performed. week 4. an owner presented a number of dogs for surgery. the owner lived more than a mile away from the surgery center up a long, steep slope. only two of the dogs had body condition scores >2, one of which escaped and went running the following day. all of the other animals which received post-operative visits recovered uneventfully without further intervention or care needed. in conclusion, the majority of animals recovered well after surgery. the higher rate of skin dehiscence is most likely due to poor post-operative care. many of the animals presented for surgery were infested with fleas and ticks. these animals were likely positive for ehrlichiosis which made surgery and recovery more challenging owing to bleeding disorders caused by decreased platelet production thereby impairing adequate clotting. wound infection rates following similar surgeries performed in the united states typically range from 2.2% to 5.7%. the rates of wound infections following ovariohysterectomy and orchiectomy found in this study were similar. the skin dehiscence rates were higher than those seen in the united states. this is most likely due to the inadequate postoperative care provided by the owners. it is possible that although the surgeries were performed in field conditions, the population of animals has adapted to survive harsher environmental conditions than a similar population in the united states resulting in good surgical outcomes. although this study concluded that the described conditions resulted in acceptable outcomes, world vets’ standard of care now includes a new sterile drape for each procedure and mandatory surgical cap and mask. in many cases volatile anesthetics and steam-sterilized instruments are now the standard. references 1. anonymous: evaluation methodology: elimination of human and dog rabies in latin america. http://www.paho.org/english/ad/dpc/vp/rabia-prog-elim-cd.htm; accessed august 19, 2012. 2. schneider mc, belotto a, adé mp, et al: current status of human rabies transmitted by dogs in latin america. cad saúde pública 2007; 23: 2049-2063. 3. mora jo: integrated anemia control strategy has significantly reduced anemia in women and children in nicaragua. http://www.micronutrient.org/cmfiles/mi around the world/americas/nicaragua_anemiacontrolprog_finalrpt.pdf; accessed august 19, 2012. 4. chediek j, castrillo a, enriquez j, et al: informe de desarrollo humano 2005: las regiones autonomas de la costa caribe. in: builes ia, editor. programa de las naciones unidas para el desarrollo. managua: pnud; 2005. p. 66-68. 5. adin ca: complications of ovariohysterectomy and orchiectomy in companion animals. vet clin north am small anim pract 2011;41:1023-1039. 6. little se: ehrlichiosis and anaplasmosis in dogs and cats. vet clin north am small anim pract 2010;40:1121-1140. 41 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 appendix questionnaire/survey we are interested in knowing how your cat/dog has been doing since the surgery it received on [insert date]. this information will be helpful to our future efforts to provide spay and neutering for cats and dogs in your area. overall, how is your cat/dog doing? [enter respondent comments] death yes no [if the cat/dog has died, express your sympathy]. i do have just a few specific questions about what your cat/dog experienced in the days following the surgery that will be useful to our overall program. q1a. was your cat/dog lethargic (or use a term/phrase that would be more common such as: “more tired than usual”) 24 hours (or “about one day”) after the surgery? q1b. was your cat/dog lethargic 48 hours, or “about 2 days”, after the surgery? q1c. was your cat/dog lethargic 72 hours, or “about 3 days” after the surgery? attitude lethargic yes no 24 hrs post-op 48 hrs post-op 72 hrs post-op q2a. how was your cat/dog’s appetite 24 hours (or “about one day” ) after the surgery? would you say good, fair, poor, or not at all? q2b. how was your cat/dog’s appetite 48 hours (or “about 2 days”) after the surgery? would you say good, fair, poor, or not at all? q2c. how was your cat/dog’s appetite 72 hours (or “about 3 days”) after the surgery? would you say good, fair, poor, or not at all? appetite none 24 hrs post-op 48 hrs post-op 72 hrs post-op poor 24 hrs post-op 48 hrs post-op 72 hrs post-op fair 24 hrs post-op 48 hrs post-op 72 hrs post-op good 24 hrs post-op 48 hrs post-op 72 hrs post-op q3a. did your cat/dog lick or chew the incision 24 hours (or “about one day” ) after the surgery? q3b. did your cat/dog lick or chew the incision 48 hours (or “about 2 days”) after the surgery? q3c. did your cat/dog lick or chew the incision 72 hours (or “about 3 days”) after the surgery? licking/chewing at incision yes 24 hrs post-op 48 hrs post-op 72 hrs post-op no 24 hrs post-op 48 hrs post-op 72 hrs post-op we are also interested in knowing about other conditions your pet may be experiencing since the surgery. for each of the conditions i read, please tell me if the condition is very severe (5),moderately severe (4), moderately mild (3), very mild (2), or not at all (0). q4a. the first condition is redness, would you say your cat/dog’s condition is very severe, moderately severe, moderately mild, very mild or not at all? q4b. the second condition is swelling, would you say your cat/dog’s condition is very severe, moderately severe, moderately mild, very mild or not at all? q4c. the third condition is dehiscence [need to define for respondent], would you say your cat/dog’s condition is very severe, moderately severe, moderately mild, very mild or not at all? 42clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 q4d. the fourth condition is discharge, would you say your cat/dog’s condition is very severe, moderately severe, moderately mild, very mild or not at all? wound assessment none severe redness 0 1 2 3 4 5 swelling 0 1 2 3 4 5 dehiscence 0 1 2 3 4 5 discharge 0 1 2 3 4 5 total score: death yes no comments/ observations: body condition score 1 1.5 2 2.5 3 3.5 4 4.5 5 those are all the questions i have. thank you for taking the time to talk with me today. is there anything else you would like to tell with regard to your cat/dog’s surgery and recovery? [enter respondent comments] 43 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 64 contact karen wolfsdorf kwolfsdorf@hagyard.com © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11645, http://dx.doi.org/10.58292/ct.v17.11645 review report effects of buserelin on the reproductive performance in transitional and anestrous mares karen wolfsdorf hagyard equine medical institute, lexington, ky, usa abstract breeding mares early in the year can alleviate economic pressure by increasing the number of estrous cycles per season, recovery of embryos per season or recovery of embryos prior to the beginning of performance season. pharmacological manipulation of anestrus and transition periods is managed indirectly by increasing the photoperiod or directly using exogenous gnrh, gnrh agonists, pituitary extracts, gonadotropins, dopamine antagonists, progesterone, and estrogens. efforts to evaluate the effectiveness of gnrh and its agonists (goserelin, deslorelin, historelin, and buserelin) to induce ovarian follicular development and return to cyclicity in anestrous and transitional mares have had conflicting results. however, specific buserelin protocols appear to actively stimulate folliculogenesis under the influence of a short-duration lighting program. additionally, buserelin treatment is effective in producing adequate ovulatory, pregnancy, and return to estrus rates. keywords: gnrh agonist, mare, transition, anestrus, buserelin introduction the natural breeding season in the mare is timed to maximize the foal’s chances of survival during the winter. this adoption has led the mare to be a long-day, seasonally polyestrous animal with regular ovulatory cycles occurring in response to increasing day length.1,2 during winter, in response to an increased period of darkness, the pineal gland releases melatonin.3 melatonin has a pivotal role in seasonality of reproduction; however, the mechanism by which it exerts its action is still unclear.4 regardless, the mare’s hypothalamic-pituitary axis changes with decreased gnrh secretion, leading to baseline concentrations of lh and cessation of ovarian follicular waves in most mares.3,5 as day length and photoperiod increase the transitional period ensues; this lasts for 60-80 days and culminates in season’s first ovulation.1 as melatonin decreases, there is a concurrent increase in gnrh.1 the anterior pituitary increases secretion of gonadotropins, most importantly lh, since fsh concentrations do not change remarkably throughout the year.5 ovarian activity increases with growth and regression of follicles, causing erratic estrous behavior.1,6,7 there can be a great variation between and within mare groups regarding the onset of fall transition, duration of winter anestrus, and onset of spring transition due to variable latitude, climate, age, body condition, and nutrition.2,3,6,7 young mares, lower latitudes, warmer ambient temperatures, green grass, and an upward plane of nutrition appear to hasten transitioning out of the winter anestrous period.8–10 the early transition phase is characterized by the development of numerous smaller follicles (> 20 mm) that develop and become atretic.3,11 during late transition, 1-3 anovulatory follicular waves occur each with a larger dominant follicle (> 35 mm) that often regresses before, finally, a dominant follicle is selected and proceeds to ovulate.3,11 cyclicity returns with the first ovulation. in the horse industry there is economic pressure to produce foals early in the year since as weanlings, yearlings, and performance horses, they will have a developmental advantage compared to their younger counterparts. additionally, breeding mares early in the year increases the number of estrous cycles with potentially higher recovery of embryos per season or recovery of embryos prior to the beginning of a mare’s performance season. unfortunately, mares are not typically reproductively active at desired breeding (late winter); therefore, many resources have been invested in the effort to induce earlier cyclicity.4 pharmacological manipulation of anestrus and transition periods is managed indirectly by increasing the photoperiod12 or directly using exogenous gnrh, gnrh agonists, pituitary mailto:kwolfsdorf@hagyard.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11645 citation: clinical theriogenology 2025, 17, 11645, http://dx.doi.org/10.58292/ct.v17.11645 65 extracts, gonadotropins, dopamine antagonists, progesterone, progestagens, or estrogens.6 efforts to evaluate the effectiveness of gnrh and its agonists (goserelin, deslorelin, historelin, and buserelin) to induce ovarian follicular development and return to cyclicity in anestrous mares have had conflicting results.13–16 it appears that dose, route of treatment, frequency, and most importantly date of onset of treatment and the pretreatment status of the ovary have an influence on the efficacy of the treatment.17 this review focuses on the specific use of buserelin, as it appears to have clinical and economic benefits. in a study, 45 nonlactating, seasonally anestrous mares were assigned randomly to 1 of 3 groups (n = 15 per group): 1. untreated controls; 2. intramuscular buserelin at 12-hour intervals (40 pg/injection) for 28 days or until ovulation; 3. gnrh agonist (buserelin) as a subcutaneous implant designed to release ~ 100 pg/12 hours for 28 days.18 injected or implanted buserelin hastened ovulation in transitional mares compared to control mares. seven of 15 (47%) mares in group 2 responded to twice-daily injections of gnrh agonist (40 pg/ injection) and ovulated mean 18.3 ± 5.2 days after initiation of treatment. implant mares ovulated 60% (9/15) of the time, with a mean of 15.8 ± 9.2 days.18 response interval and number of mares responding to gnrh agonist treatment were not different (p > 0.05) between treated groups. no control mares versus 53% of treated mares ovulated within 30 days after initiation of treatment.18 an increased response to buserelin over time indicated that the gnrh agonist increased pituitary stores of lh by day 7 in mares given the implant and by day 21 for mares injected with gnrh agonist. the implant resulted in a better response than the twice-daily treatment, with greater lh peak and daily serum concentrations and a more rapid and larger decline in fsh response parameters. this correlated with mares’ response to gnrh-induced biphasic lh profiles with peaks at ~ days 6 and 16-20 after initiation of treatment, with ovulations occurring near the second peak.19 mares that failed to ovulate exhibited the first peak, but not the second, with lh declining to baseline concentrations. the implications from the above study were that buserelin can induce ovulation in anestrous mares more frequently than control mares; however, some mares did not respond. it was hypothesized that due to a failure to cause sufficient decline in fsh concentrations and/or sufficient increase in concentrations of lh failing to result in a reciprocal relationship between the gonadotropins. therefore, they concluded that the doses used may have been too high or low and further studies are needed to determine the best dose and treatment route.18 a further study in which subcutaneous buserelin (10 µg) was given twice daily in january, february, and march was compared to a native gnrh (10 µg/hour) pump in seasonally anestrous mares; both produced ovarian follicular growth and induced ovulation. additionally, buserelin was more effective in february and march.17 building on that study, 2 experiments were designed with 60 anestrous mares.20 the first utilized 26 primiparous or seasonally anestrous (follicles < 20 mm, no edema, small ovaries) maiden mares were assigned to 3 groups. group 1a (n = 9) received subcutaneous injections of 10 µg buserelin twice daily, beginning february 15 for a maximum of 28 days plus a single injection of hcg (2,500 iu) when a follicle > 35 mm in diameter was noted. group 1b (n = 9) mares received 1 intravenous hcg (2,500 iu) when a spontaneously developed follicle > 35 mm in diameter was detected. group 1c (n = 8) mares were untreated and not bred. mares were bred every other day until the day of ovulation. pregnancy was diagnosed (via ultrasonography) on days 14, 25, 35, 45, 60, and 180 after ovulation. the second experiment included a random assortment of 34 mares on private farms that were placed on buserelin twice daily beginning in early february for a maximum of 21 days, plus 1 hcg (2,500 iu) injection when a follicle > 35 mm was detected. group 2b (n = 18) mares were given hcg (2,500 iu) when a spontaneously developed follicle ~ 35 mm was detected. mares with a follicle > 35 mm were bred by natural service or inseminated (semen from a stallion of known fertility). pregnancy was diagnosed (via ultrasonography) on days 14, 25, and 40 after ovulation. both buserelin plus hcg treatment in anestrous mares and hcg treatment alone in transitional mares significantly advanced the date of the first ovulation of the year compared to untreated mares. the mean interval from onset of gnrh treatment and detection of a follicle > 35 mm was 10.6 ± 2.2 days. the percentage of mares ovulating in response to gnrh plus hcg in experiment 1 (7 of 9; 77.8%) and experiment 2 (11 of 16; 68.8%) was not significantly different. pregnancy rates for ovulations induced by gnrh plus hcg for experiments 1 (71.4%) and 2 (81.8%) were similar. no significant differences in pregnancy rates were noted between buserelin plus hcg (77.8%) or transitional mares treated only with hcg (85.2%),20 further supporting the concept that buserelin protocols are suitable for inducing fertile ovulations in anestrous mares early in the breeding season. most recently a larger study used 79 anestrous (< 20 mm follicles) or transitional (> 20 mm follicles without prior ovulation) mares with a specific buserelin treatment protocol and the efficacy of inducing ovarian follicular development, ovulation, pregnancy, and return to estrus were documented.21 mares were placed under 16 hours of day light for a minimum of 2 weeks. as this was a clinical situation, it was not possible to have a control group that was not placed under lights and therefore, if there was no follicular development, they were placed on 12.5 µg intramuscular buserelin twice daily. when a 35-40 mm follicle was present, ovulation was induced with intravenous hcg (3,000 iu), the mare was bred and buserelin treatment ceased after ovulation was identified. the average length of treatment was 10.42 days, with anestrous mares requiring longer treatment compared to transitional mares (12.20 versus 9.96 days, respectively). there was no significant effect of length of treatment on ovulation, pregnancy outcome, or twinning. overall, 71% of mares developed follicles large enough to induce ovulation. transitional mares were more likely to ovulate than anestrous mares (94 versus 64%, respectively).21 of the mares that ovulated, 72% became pregnant, 24% of which were twins.21 mares that did not become pregnant returned to estrus within the normal 18-21 days interval. the buserelin protocol described above appeared to actively stimulate folliculogenesis in both anestrous and transitional mares under the influence of a short-duration lighting program and regardless of length of treatment. the authors concluded that buserelin treatment at this dose should be considered effective in producing adequate ovulatory, pregnancy, and return to estrus rates in anestrous and transitional mares.21 conclusion buserelin, a gnrh agonist, can be used successfully to advance the onset of cyclicity during anestrus and transitional periods. http://dx.doi.org/10.58292/ct.v17.11645 66 citation: clinical theriogenology 2025, 17, 11645, http://dx.doi.org/10.58292/ct.v17.11645 conflict of interest none to declare. references 1. donadeu fx, watson ed: seasonal changes in ovarian activity: lessons learnt from the horse. anim reprod sci 2007;100:225-242. doi: 10.1016/j.anireprosci.2006.12.001 2. sharp dc: vernal transition into breeding season. in: mckinnon ao, squires el, vaala we et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 1704-1715. 3. ginther oj: reproductive biology of the mare. cross plains, wisconsin; equiservices: 1992. 4. oberhaus el, paccamonti d: review of management of anestrus and transitional mares. proc am assoc equin pract 2013;59:325-330. 5. thompson jr, dl, johnson l, st george rl, et al: concentrations of prolactin, luteinizing hormone and follicle stimulating hormone in pituitary and serum of horses: effect of sex, season and reproductive stat. j anim sci 1986;63:854-860. doi: 10.2527/ jas1986.633854x 6. nagy p, guillaume d, daels p: seasonality in mares. anim reprod sci 2000;60:245-262. doi: 10.1016/s0378-4320(00)00133-0 7. sharp dc: environmental influences on reproduction in horses. vet clin north am large anim pract 1980;2:207-223. doi: 10.1016/s0196-9846(17)30157-x 8. gentry lr, thompson jr, dl, gentry jr, gt, et al: the relationship between body condition, leptin, and reproductive hormonal characteristics of mares during the seasonal anovulatory period. j anim sci 2002;80:2695-2703. doi: 10.2527/2002.80102695x 9. van niekerk ch, van heerden js: nutrition and ovarian activity of mares early in the breeding season j s afr vet med assoc 1972;43:351-360. pmid: 4677611. 10. carnevale em, hermenet mj, ginther oj: age and pasture effects on vernal transition in mares. theriogenology 1997;47:1009-1018. doi: 10.1016/s0093-691x(97)00058-7 11. turner dd, garcia mc, ginther oj: follicular and gonadotropic changes throughout the year in pony mares. am j vet res 1979;40:1694-1700. pmid: 575024 12. sharp dc: photoperiod. in: mckinnon ao, squires el, vaala we et al: editors. equine reproduction. 2nd edition, ames; wileyblackwell: 2011. p. 1771-1777. 13. hyland jh, wright pj, clark ij et al: infusion of gonadotrophin-releasing hormone (gnrh) induces ovulation and fertile oestrus in mares during seasonal anoestrus. j reprod fertil suppl 1987;35:221-220. pmid: 3316638. 14. allen wr, sanderson mw, greenwood re, et al: induction of ovulation in anoestrous mares with a slow-release implant of a gnrh analogue (ici 118 630). j reprod fertil suppl 1987;35:469-478. pmid: 2960804 15. mumford el, squires el, peterson kd, et al: effect of various doses of a gonadotropin-releasing hormone analogue on induction of ovulation in anestrous mares. j anim sci 1994;72:178-183. doi: 10.2527/1994.721178x 16. johnson al: gonadotropin-releasing hormone treatment induced follicular growth and ovulation in seasonally anestrous mares.  biol reprod 1987;36:1199-1206. doi: 10.1095/biolreprod36. 5.1199 17. mccue pm, troedsson mht, liu ikm, et al: follicular and endocrine responses of anoestrous mares to administration of native gnrh or gnrh agonist. j reprod fert suppl 1991;44:227-233. pmid: 1795264 18. harrison la, squires el, nett tm, et al: use of gonadotropin-releasing hormone for hastening ovulation in transitional mares. j anim sci 1990;68:690-699. doi: 10.2527/ 1990. 683690x 19. hyland jh, wright pj, clarke j, et al: infusion of gonadotrophin-releasing hormone induces ovulation and fertile oestrus in mares during seasonal anestrus. j reprod fertil 1987;35:211-210. pmid: 3316638 20. mccue pm, warren rc, appel rd, et al: pregnancy rates following  administration of gnrh to anestrous mares. eq vet sci 1992;12:21-23. doi: 10.1016/s0737-0806(06)81379-1 21. wolfsdorf ke, fedorka ce, lu kj, et al: the effect of buserelin on reproductive performance in the transitional and anestrous  mare. j eq vet sci 2018;66:98. doi: 10.1016/j. jevs.2018.05.148 http://dx.doi.org/10.58292/ct.v17.11645 https://doi.org/10.1016/j.anireprosci.2006.12.001 https://doi.org/10.2527/jas1986.633854x https://doi.org/10.2527/jas1986.633854x https://doi.org/10.1016/s0378-4320(00)00133-0 https://doi.org/10.1016/s0196-9846(17)30157-x https://doi.org/10.2527/2002.80102695x https://doi.org/10.1016/s0093-691x(97)00058-7 https://doi.org/10.2527/1994.721178x https://doi.org/10.1095/biolreprod36.5.1199 https://doi.org/10.1095/biolreprod36.5.1199 https://doi.org/10.2527/1990.683690x https://doi.org/10.2527/1990.683690x https://doi.org/10.1016/s0737-0806(06)81379-1 https://doi.org/10.1016/j.jevs.2018.05.148 https://doi.org/10.1016/j.jevs.2018.05.148 2018: paraphimosis and partial phallectomy in a french bulldog paraphimosis and partial phallectomy in a french bulldog college of veterinary medicine, auburn university. auburn, al a 1.5-year old intact male french bulldog was presented with a four day history of paraphimosis. upon presentation, the distal penis was engorged and necrotic. the penis was able to be extended to the bulbus glandis, revealing a line of demarcation from necrotic tissue to the bulbus glandis of approximately 1 inch. urinary function remained normal. a partial phallectomy was recommended to remove the necrotic tissue and preserve reproductive function for possible future semen collection. a partial phallectomy was performed with a partial preputial resection. the patient experienced mild hematuria after surgery and was able to urinate normally. imaging of the bladder using ultrasound after the surgery revealed only moderate enlargement. treatment after surgery included a non-steriodal anti-inflammatory drug (carprofen® 2mg/kg orally twice daily) to alleviate swelling, an antibiotic (cefazolin® 4.2mg/kg intramuscularly) and an elizabethan collar to prevent self-mutilation. the patient’s reproductive viability remains unknown pending a future breeding soundness examination. paraphimosis is the inability to withdraw the non-erect penis completely into the prepuce. paraphimosis in the canine can occur after copulation or sexual arousal when hair around the preputial orifice strangulates the penis causing prolonged exteriorization, as well as due to trauma or neurological deficits.1 damage to the penial mucosa amplifies with sustained paraphimosis. conservative treatment includes cleaning, massaging, and lubricating the penis to reposition it in the prepuce, using a hypertonic dextrose solution, if needed, to reduce swelling.1 if the penis cannot be restored within the prepuce or if the trauma is severe, surgical therapy such as enlargement of the preputial orifice or partial phallectomy is indicated.2 this case demonstrates how canine paraphimosis is considered as an emergency condition to prevent severe necrotic damage to the penis requiring surgical correction. keywords: paraphimosis, canine, phallectomy references 1. greer ml: infertility and reproductive disorders in the valuable stud dog. in: canine reproduction and neonatology. jackson(wy): teton newmedia; 2015. p. 313. 2. kutzler ma: pathophysiology of the penis. in: bojrab mj, monnet e, editors. mechanisms of disease in small animal surgery. 3rd ed. jackson(wy): teton newmedia; 2010. p. 443-444. clinical theriogenology • volume 10, number 3 • september 2018 348 c.e. sanders, c. barstow, a.k. johnson, r.r. wilborn 1 contact rory nevard rory.nevard@sydney.edu.au © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10415, http://dx.doi.org/10.58292/ct.v16.10415 case report modified partial posthectomy surgery for chronic preputial prolapse in a bos indicus bull rory nevard,a,b marco lopes,b heidi lehmann,b chris quinn,b cassandra macdonalda asydney school of veterinary science, the university of sydney, camden, nsw, australia bveterinary clinical centre, school of agricultural, environmental and veterinary sciences charles sturt university, wagga wagga, nsw, australia abstract preputial prolapses in bulls are frequently encountered by bovine reproductive practitioners and are common physical conditions impairing a bull’s ability to copulate. marked chronicity or unsuccessful medical management warrant surgical correction. preputial resection/circumcision (also known as ‘reefing procedure’) or preputial amputation are 2 well-documented procedures performed to restore penile function. it is widely believed that preputial resection is considered superior to amputation, as with the latter procedure, strictures and wound contracture stenosis are common reported sequelae. therefore, surgery should aim at preserving tissue and resolving the prolapse without interference to complete penile extension. we adapted novel surgical features in a bull with a chronically prolapsed prepuce: an updated preputial amputation technique (i.e. modified posthectomy) and used a single-layer closure pattern with rapidly absorbable suture (not reported for bulls). there was total resolution and bull returned to service. this case illustrated that severe and chronically prolapsed prepuces can be successfully repaired using this updated modification of a historical technique. keywords: beef bull, chronic preputial prolapse, preputial amputation, modifed posthectomy background injuries to the prepuce of breeding beef bulls are common, particularly those of bos indicus influence as a result of certain anatomical arrangements.1,2 although the etiology of this condition may appear straightforward, a variety of factors have been linked to preputial prolapse occurrence in bulls: genotype,3,4 breed,5–7 innate sheath conformation,8–10 bovine herpes virus (bhv-1) associated balanoposthitis,9 and potentially bulls that may preferentially evert preputial skin when relaxed (remains speculative).11,12 in general, however, trauma usually occurs to prepuce during breeding1,3,13 or when tissue is not maintained within the sheath and therefore exposed to noncoital injury such as incidental lacerations and inadvertent selfinjury.3 bos indicus subspecies bulls are renowned for sustaining injuries to the prepuce at breeding due to anatomical traits such as pendulous sheaths and substantial preputial skin.1,4 some angus bulls may also have similar problems, with a horizontal sheath opening being another additional association.14–16 certain bos taurus breeds of the polled phenotype have also been historically linked to a higher predisposition of preputial injuries possibly due to incompletely developed caudal retractor preputial muscles.6,7 these anatomical associations may be unduly represented as the original paper that cited this association misinterpreted preputial eversion as preputial prolapse.16,17 since then, it has prevailed in literature. for instance, there is no completely conclusive evidence that preputial eversion is directly related to prolapses in bos taurus bulls specifically, despite seeming a plausible cause to practitioners and producers.14,16,18 also, it is generally considered that minor lacerations or injury to the prepuce in bos taurus breeds seldom leads to fulminant preputial prolapse as in bos indicus type.13 depending on the chronicity and extent of injury, either medical management or surgical correction may be attempted.4,19 a scale initially developed by wolfe & carson20 has been updated in recent texts1 to classify and predict the prognosis for resolution of preputial injuries in bulls1 (table 1). additionally, there are variations on anatomical terminology for the prepuce in large animals,6,21,22 and it is paramount that examining veterinarians understand preputial anatomy of bulls, particularly during circumstances of injury where confusion can occur. authors’ recommend mailto:rory.nevard@sydney.edu.au http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10415 2 citation line: clinical theriogenology 2024, 16, 10415, http://dx.doi.org/10.58292/ct.v16.10415 and prefer to use that described by ashdown et.al.6,22 when considering the definitions of portions of the preputial tissue involved in injury so to avoid confusion or inadvertent incorrect anatomical descriptions. further, it is also important to highlight that although the preputial tissue is continuous with that of the penile integument, and may resemble that of a mucous membrane, it is in fact lined with stratified squamous epithelium and the preputial wall contains inner and outer concentric fibrous layers that are organized differently to penile mucosa.6,22 regardless of the technique for medical management, core principles of nonsurgical treatment involve managing edema, improving lymphatic drainage, reducing inflammation, and preventing further injury. this may include topical hydrotherapy with daily cold water hosing in acute stages, bandaging the prolapsed tissue around a placed urinary egress tube, applying topical emolients, placing a sling, and treating with nonsteroidal antiinflammatories and antimicrobials systemically.1,5,9 failing successful medical management, or for cases of chronic prolapse, either surgery or salvage slaughter is generally indicated.4 known surgical repair techniques include either simple laceration repair, preputial resection (i.e. ‘reefing’ procedure) or preputial amputation.4,5,13 due to the advent of farm animal insurance in some countries outside of us, bulls with prolapses that are not amenable to medical management are often culled or claimed for reproductive loss of use. additionally, due to financial limitations and concerns over use of prolonged general anesthesia in ruminants,23 surgical procedures may be limited to certain institutions or simply not elected by owners. this may result in either salvage slaughter, or potentially even the adoption of salvage procedures such as ring amputation that are not completely restorative.24 ultimately, this may dissuade producers from electing for fully corrective surgical procedures in future, and therefore exploring and/or revisiting alternate procedures are of value to bovine practitioners. detailed presurgical (assessment, medical management, restraint, and anesthesia) and surgical descriptions are provided in this report. case presentation an 8 year, ~ 900 kg brahman bull, was presented for surgical management of a grade iii20 prolapsed prepuce of ~ 6 months duration. bull was identified with this condition by the owner after deployment in a large paddock for breeding of ~ 40 heifers. bull had previously sired calves successfully in previous seasons with no evidence of prior injuries. owner pursued treatment and surgery due to amiable and calm temperament of the bull despite the severity of injury. treatment examination bull was initially examined while restrained in a crush to determine the extent of injury. bull was bright, alert and responsive, and in good body condition (bcs 4/5). careful inspection and palpation of the prepuce revealed prolapse severity (figure 1). attempts to exteriorize the penis were not initially successful that was attributed to narrowing of the preputial lumen by the swollen prolapsed tissue and/ or retraction of retractor penis muscles. prolapse was palpated and internal channel of prolapse was also examined to determine if there was evidence of strictures or if penis could be exteriorized. other incidental findings included overgrown claws (possibly due to reduced sexual activity as a result of preputial injury). prolapsed tissue was cleaned, cold water hosed and bandaged around a ~ 40 cm urine egress tube made from plastic garden tubing. a sling using shade-cloth material was also placed to aid in rectifying the remaining edematous tissue prior to surgery (figure 2). treatment with a broad-spectrum antimicrobial (intramuscular oxytetracycline 10 mg/kg once a day, alamycin®, norbrook) and a nonsteroidal antiinflammatory (subcutaneous meloxicam (metacam®, boehringer) 0.5 mg/kg once every 3 days) was initiated. bull was not fed for 36 hours prior to surgery and had water restriction for 12 hours. anesthesia and restraint bull was initially restrained in a tip-table crush. intravenous sedation (via caudal coccygeal vein) consisted of 0.02 mg/kg xylazine hydrochloride (xylazil-100, ilium) and 0.02 mg/kg butorphanol tartare (butorgesic, ilium); caudal coccygeal epidural anesthesia was induced (4.5 ml of 2% lignocaine hydrochloride [lignocaine 20, ilium]). bull was then tipped into right lateral recumbency on the tip table and pudendal nerve block was induced.25,26 ischiorectal fossa region was clipped and surgically prepared on both tail sides. after creating 3-4 ml table 1. preputial injuries categories: description, treatment, and prognosis1 category description treatment and prognosis i simple preputial prolapse with slight to moderate edema without laceration, necrosis, or fibrosis either conservative or surgical treatment with good prognosis ii the prolapsed prepuce has moderate to severe edema, may have superficial lacerations or slight necrosis, but has no evidence of fibrosis surgery is the usual course of therapy with a good to guarded prognosis iii there is severe edema of the prolapsed prepuce with deep lacerations, moderate necrosis, and slight fibrosis surgery is indicated and the prognosis is guarded iv the prolapsed prepuce has been exposed for quite some time and has severe edema, deep lacerations, deep necrosis, fibrosis, and often abscess surgery and salvage by slaughter are the only options, and a guarded to poor prognosis follows surgery http://dx.doi.org/10.58292/ct.v16.10415 citation line: clinical theriogenology 2024, 16, 10415, http://dx.doi.org/10.58292/ct.v16.10415 3 bleb of 2% lignocaine hydrochloride (lignocaine 20, ilium), a spinal needle (18 gauge, 4 inch) was inserted through a hypodermic needle (14 gauge 1.5 inch) that was used as a cannula and guide. point of the spinal needle was directed (while palpating transrectally) toward the lesser sacro-sciatic foramen, 30 ml of 2% lignocaine hydrochloride (lignocaine 20, ilium) was deposited immediately cranial and caudal to the lesser sciatic foramen a cannula (20 gauge, 32 inch) was placed in the left auricular vein (pulse rate, respiratory rate, mucous membrane color, and rectal temperature were monitored during the procedure). immobilization was achieved with 3 mg/kg intravenous ketamine hydrochloride (ketamil, ilium); butorphanol, ketamine, and xylazine boluses were given during the procedure. nasal oxygen (12 liters/minute) was given and bull was blindfolded during recumbency. after pudendal nerve block, 50 ml of lidocaine infiltrated at the proximal prepuce, further sedation allowed good surgical condition. penis was fully extended from prepuce and the entire preputial tissue was examined to plan for surgical incisions (figure 3). sheath skin was clipped, skin and preputial and penile mucosa were scrubbed with dilute chlorhexidine and sterile water routinely. distal portion of the prolapsed preputial tissue was tied with a long gauze for immobilization. a disposable plastic drape was then placed over the surgical field. surgery first, 2 stay sutures were placed on cranial and caudal aspects of prolapsed skin proximal to planned amputation site to ensure correct anatomical tissue apposition after amputation. using a size 22 scalpel blade, a transversely positioned oblique circumferential incision was created on the exposed tissue, ~ 2 cm distal to the junction of the sheath and prepuce (figure 5). using metzenbaum scissors, careful blunt and sharp dissection were alternately used to transect the subcutaneous tissue. large vessels were ligated with usp 2-0 rapidly absorbable monofilament suture (glycomertm631 [biosyntm], covidien) while bleeding caused by transection of minor vessels was contained with a handheld electrocautery unit (kaustolux, dlc). using a scalpel, preputial subcutaneous tissue was transected, and additional stay sutures were placed to ensure that the internal channel of tissue was correctly reattached to the preputial skin in its previous anatomical position. apposition of edges of transected skin was then completed using usp  2-0  rapidly absorbable monofilament suture (glycomertm631[biosyntm], covidien) in a simple continuous pattern interrupted at cranial and caudal aspects of the preputial circumference. while closing, internal (visceral) preputial skin was meticulously included in each bite to ensure correct apposition to parietal preputial tissue. stay sutures were figure 1. close examination of prolapsed prepuce; note edema and sunburnt preputial skin. figure 2. a. edematous prepuce tightly bandaged against a urine egress tube made from rubber tubing; b. placement of sling (rubber esmarch’s tourniquet straps and shade-cloth material). http://dx.doi.org/10.58292/ct.v16.10415 4 citation line: clinical theriogenology 2024, 16, 10415, http://dx.doi.org/10.58292/ct.v16.10415 removed and as prepuce was released, surgical site was immediately retracted inwards. surgical site and entire preputial tissue were covered with 1% silver sulphadiazine (flamazinetm, simth & nephew), a shortened sterilized nasogastric tube was placed into prepuce, and prepuce was then bandaged. bull’s feet were trimmed to correct the overgrown claws. diagrammatic (figure 4 [a-f] and visual representation of surgical events are provided (figure 5 [a-d]). aftercare after recovery from sedation/anesthesia, the shade-cloth material sling was replaced and bull was examined twice daily. three days postoperatively, the bandage was removed, and surgical site was inspected. transrectal palpation of accessory sex glands was also performed to facilitate penile relaxation, but was unsuccessful on first attempt. topical ointment (1% silver sulfadiazine, flamazinetm, smith & nephew) was applied and the tube and bandage were replaced. four days after surgery, the sling, bandage and tube were removed, along with the sling. daily observation for urination and penile extension was employed and it was apparent that the bull was able to protrude the penis beyond the hair line at the external orifice of the prepuce by the 5th day postsurgery. bull’s claws were also trimmed for a second time during hospitalisation. outcome after 10 days of hospitalization, bull was discharged, owner was instructed to give complete sexual rest (at least 2 months) for the bull. no evidence of stricture or tubal contraction was noted, and inspection of apposed tissues revealed no evidence dehiscence or infection. approximately 6 months later, a follow up with the owner revealed that successful intromission had been observed and a number of females were successfully bred, supported by positive pregnancy diagnosis. discussion successful treatment of a severe and chronic preputial prolapse in a mature bos indicus brahman bull using an updated version of the preputial amputation (posthectomy) technique is reported for the first time. lay terminologies for surgical descriptions have been used interchangeably;13,19 however, we prefer to categorize ‘reefing’ with preputial resection and anastomosis, and ‘circumcision’ with preputial amputation (or posthectomy), as indicated.27 to authors’ knowledge, there are no published reports of using a rapidly absorbable suture (such as biosyntm) to close the internal to external preputial integument during a preputial amputation in bulls, despite being recently published in stallions.28 preputial prolapse repair, particularly in north america, are often performed in specialist referral hospitals under controlled theatre conditions and with meticulous hospital aftercare. it is therefore clinically relevant to explore alternate techniques that can be performed in field conditions and reduce postoperative handling. in a case series of 51 surgical repairs5 it was identified that success rates of preputial injuries were higher if a preputial resection could be performed compared to amputation (90 versus 43% success). surgical success rate was also reported to be higher when the procedure was conducted under full general anesthesia in an operating theatre, as expected, compared to injectable or local anesthesia. generally, preputial amputation is reserved for chronic prolapse cases where complete extension of the penis is not possible, and the preputial resection/reefing technique is often considered to yield more favourable outcomes than amputation.19,29 higher incidence of wound contracture stenosis was reported13 for preputial amputation (or posthectomy). however, in a different case series, preputial circumcision (i.e. amputation) indeed resulted in somewhat favourable outcomes, with 76% of bulls reproductively sound for ≥ 1 year after surgery.30 reefing procedure may not always be feasible in scenarios where sterile theatre conditions are not available or replicable, particularly in animals of high anesthetic risk, those with behavioral limitations or for clients with cost constraints. additionally, other factors such as location of injury, ability to exteriorise the penis, and breed/subspecies may further influence decision making on surgical technique.27 for instance, bulls of the british bos taurus subspecies may not have enough preputial skin to facilitate amputation or posthectomy.13,27 nonetheless, it is indeed valuable to reconsider alternate procedures that can provide similar success rates to those reported in texts,13 along with shortening surgical time and minimizing postprocedural handling. although complete penile extension was achieved in this case, it was decided to perform a preputial amputation (circumcision) technique similar to that described earlier31 and later32 using rapidly absorbable suture material for the apposition of the preputial skin on closure. suture material (2-0 biosyntm) used may lose up to 50% of tensile strength at 2-3 weeks and is completely absorbed at ~ 3 months,33 as opposed to other suture materials such as polydioxanone (pds) suture that has minimal absorption within this same time frame.34 previous reports on surgical prolapse have suggested using absorbable suture materials such as polydioxanone,35 and even nonabsorbable sutures such as supramid® and wound staples.13 however, given that the internal visceral layer of skin in the prepuce is delicate, there is a possibility of irritation when using longer absorbing sutures that may result in undesirable sequalae such as further inflammation and wound breakdown. other key aspects of this technique is that the amputation line is oblique, rather than straight/transverse which results in a postamputation orifice that is oval as opposed to circular.31 this may mitigate the need for an additional ‘v’ incision to be incorporated into the closure that has been advocated by some practitioners, but lacks substantial evidence.19,36 additionally, horizontal mattress sutures were not placed proximal to the line of amputation as described,27,31 and may not be necessary for most cases. distal amputated figure 3. fully extended penis after pudendal nerve block; note the extent of preputial skin damage and tissue to be removed. http://dx.doi.org/10.58292/ct.v16.10415 citation line: clinical theriogenology 2024, 16, 10415, http://dx.doi.org/10.58292/ct.v16.10415 5 portion was also removed in its entirety without staged incision and closure in ‘thirds’.13 both these modifications reduced surgical time considerably as closure was performed in halves as described.27 however, substantial hemorrhage was encountered during the procedure. therefore, without appropriate hemostasis, it is possible for incorrect anatomical apposition of tissues and excessive postoperative swelling. nevertheless, minimal postoperative swelling was reported and spontaneous full penile extension was achieved within 1 week postsurgery, eliminating the need to perform daily bandage changes or considerable ongoing intervention. specific steps of the procedure are outlined in figure 5. a key aspect worth mentioning by the surgeons in this case report is that the internal linings of the prolapsed portion of the prepuce were spared by bluntly dissecting distally to the initial incision site (figure 5b) which may have preserved more skin that is less likely to be involved in the inflammatory process and hence avoided removing too much tissue to prevent full penile extension in future.19,27 postoperative complications such as incisional dehiscence, suture abscesses and focal incisional hernias were figure 4. sequence of events during surgery: a. incised and dissected preputial skin down to the internal (visceral lamina); note the bleeding despite attempts at meticulous hemostasis, b. proximal aspect of prepuce after prolapsed section removal; note exposed tissue that had to be joined by placing sutures from internal to external preputial tissue, c. closure of the surgery incision using 2/0 biosyntm absorbable suture material in a simple continuous fashion; semi-circular half of the ovoid orifice was closed at a time, d. finished closure of the surgery site prior to prepuce retraction into sheath, and e and f. application of compressive bandage to assist with postoperative swelling. a b dc e f http://dx.doi.org/10.58292/ct.v16.10415 6 citation line: clinical theriogenology 2024, 16, 10415, http://dx.doi.org/10.58292/ct.v16.10415 not encountered as mentioned.27,30 further, no evidence of wound contracture stenosis was apparent throughout hospitalization and neither was there any impediment to full penile extension, erection, and intromission as visually confirmed by the owner during paddock service. other benefits to performing amputation as opposed to resection (i.e. ‘reefing’) are that it negates the need for full penile extension, and could minimize trauma to penis which needs to be fully exteriorized to perform the latter technique. interestingly, alternatives to resection and anastomosis technique have just been recently described28,36 further suggesting this updated technique as a viable option. this amputation technique has only received limited mention in some salient texts on reproductive surgery,32 despite others reporting reasonable outcomes.27,30 besides minimizing surgical trauma, surgeons’ commitment to key components of halstead’s principles such as aseptic technique, attention to hemostasis, minimization of deadspace, and use of rapidly absorbed small diameter monofilament suture may have contributed to the positive outcome. further, reported sequelae (wound contracture and stenosis) to amputation technique can be overcome by employing the aforementioned techniques. this case served as an example for successful resolution of a grade iii chronic preputial prolapse through a slightly modified approach to the typical preputial amputation techniques.13,20,31,35 learning points • preputial amputation is a reasonable alternative to preputial resection (‘reefing’) for surgical repair of chronic preputial prolapses. • use of rapidly absorbable suture may assist in reducing ongoing irritation and facilitate rapid healing; some steps in previous procedures may be bypassed to reduce surgical time. • modifications to historical techniques for preputial amputation may allow for surgery to be performed figure 5. a. intended incision (dotted line) site and location (cranial and caudal aspect of the section to be removed) of stay sutures, b. circumferential incision (using a scalpel blade) in an angled plane and removal of tissue using metzenbaum scissors and dissected distally to the closure site to allow more skin within the internal channel of the prolapse to be apposed during the closure, c. after amputation, recognize tissues to be reapposed in correct anatomical plane; note it is crucial that any bleeding vessels are dealt with using appropriate haemostatic techniques (electrocautery or ligation), and d. closure is achieved using rapidly absorbable 2-0 glycomertm631 [biosyntm] suture in a continuous fashion semicircularly from the distal stay suture to the cranial stay suture. bites should be taken from the inside of the preputial skin to the outside, so to ‘roll’ the tissue outwardly. after one-half of the closure is completed, the same is done for the other semicircle of the closure. a c b d http://dx.doi.org/10.58292/ct.v16.10415 citation line: clinical theriogenology 2024, 16, 10415, http://dx.doi.org/10.58292/ct.v16.10415 7 without the need for general anesthesia or a sterile surgical suite. • meticulous hemostasis, conservative resection, gentle tissue handling, and accurate anatomical apposition of tissues are key to ensuring surgical success. acknowledgement authors thank dr. cassandra macdonald for surgical illustrations. conflict of interest none to report and no funding is associated. author contributions rn conceived the idea, rn wrote majority of manuscript, ml,  cq and hl reviewed it, and all authors approved submission. references 1. maxwell h: inability to breed due to injury or abnormality of the external genitalia of bulls. in: hopper rm: editor. bovine reproduction. ist edition, hoboken, nj; wiley-blackwell: 2021. p. 155–172. 2. memon m, dawson l, usenik e, et al: preputial injuries in beef bulls: 172 cases (1980-1985). j am vet med assoc 1988;193: 481–485. 3. parkinson tj, mcgowan m: abnormalities affecting reproductive function of 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segmental posthetomy in a four stallions case series. animals 2021;11:1145. doi: 10.3390/ani11041145 29. desrochers a, jean g, anderson d: surgical management of preputial injuries in bulls: 51 cases (1986-1994). can vet j 1995; 36:553–556. 30. baxter gm, allen d, wallace ce: breeding soundness of beef bulls after circumcision: 33 cases (1980-1986). j am vet med assoc 1989;194:948–952. http://dx.doi.org/10.58292/ct.v16.10415 https://doi.org/10.1016/j.cvfa.2008.02.002 https://doi.org/10.1016/j.cvfa.2008.02.002 https://doi.org/10.1016/s0034-5288(18)33846-3 https://doi.org/10.2527/jas1970.306949x https://doi.org/10.2527/jas1970.306949x https://doi.org/10.1017/s1751731118000939 https://doi.org/10.1017/s1751731118000939 https://doi.org/10.1016/j.cvfa.2016.05.009 https://doi.org/10.1111/avj.12900 https://doi.org/10.1111/avj.12900 https://doi.org/10.1136/vr.86.7.192 https://doi.org/10.1136/vr.86.7.192 https://doi.org/10.1136/vr.91.7.165 https://doi.org/10.1136/vr.86.6.161 https://doi.org/10.1111/j.1751-0813.1971.tb09207.x https://doi.org/10.1016/s0749-0739(17)30624-7 https://doi.org/10.1016/s0749-0739(17)30624-7 https://doi.org/10.1079/pavsnnr20061021 https://doi.org/10.1016/j.cvfa.2016.05.002 https://doi.org/10.1016/j.cvfa.2016.01.015 https://doi.org/10.21423/bovine-vol1995no29p13-21 https://doi.org/10.3390/ani11041145 8 citation line: clinical theriogenology 2024, 16, 10415, http://dx.doi.org/10.58292/ct.v16.10415 31. turner as, mcilwraith cw, hull bl: techniques in large animal surgery. 2nd edition, philadelphia, pa; lea & febiger: lippincott williams & wilkins: 1989. 32. wolfe df, moll hd: large animal urogenital surgery. 2nd edition, baltimore; williams & wilkins: 1999. p. 261–263. 33. chu cc: 11 materials for absorbable and nonabsorbable surgical sutures. in: king mw, gupta bs, guidoin r: editors. biotextiles as medical implants. woodhead publishing: 2013. p. 275–334. 34. dart aj, dart cm: 6.636 suture material: conventional and stimuli responsive. in: ducheyne p: editor. comprehensive biomaterials. oxford; elsevier: 2011. p. 573–587. 35. wolfe df: restorative surgery of the prepuce. in: hopper rm: editor. bovine reproduction. 2nd edition, hoboken, nj; wileyblackwell, 2014. p. 142–154. 36. lopes ma, papa fo: acrobustitis-phimosis in bulls: postoplasty technique performed with the animals in a standing position. anim reprod 2023;20:e20230047. doi: 10.1590/1984-3143-ar2023-0047 http://dx.doi.org/10.58292/ct.v16.10415 https://doi.org/10.1590/1984-3143-ar2023-0047 2016: endometrial tissue concentrations of ceftiofur derivatives following intrauterine infusion in both non-infected mares and mares with experimentally induced endometritis endometrial tissue concentrations of ceftiofur derivatives following intrauterine infusion in both non-infected mares and mares with experimentally induced endometritis camilla j. scott,a ghislaine a. dujovne,b bruce w. christensenb awilliam r. pritchard veterinary medical teaching hospital, school of veterinary medicine, university of california-davis; bdepartment of population health and reproduction, school of veterinary medicine, university of california-davis,davis, ca abstract intrauterine infusion of antimicrobials is common practice for the treatment of bacterial endometritis, however evidence based guidelines are lacking. the objective of this study was to determine endometrial concentrations of ceftiofur derivatives following intrauterine infusion in noninfected mares and those with experimentally induced endometritis. it was hypothesized that endometrial tissue concentrations of desfuroylceftiofur-acetamide (dca; active metabolite of ceftiofur) would remain above the minimum inhibitory concentration (mic) for common uterine pathogens for greater than 24 h in both groups of mares. endometrial biopsy samples were collected from six mares during estrus immediately prior to intrauterine infusion of 1g ceftiofur and at 4, 8, 12, 24, 36 and 48 h after infusion. desfuroylceftiofur-acetamide levels in endometrial tissue were measured using liquid chromatographymass spectrometry (lc-ms) at each time point. six further mares were then chosen that had been shown to be susceptible to breeding induced endometritis. once in estrus they were inoculated with 107 colonyforming units (cfu) of streptococcus equi zooepidemicus (s. zooepidemicus). 24 h following inoculation, 1g of ceftiofur was infused into the uterus and endometrial biopsies taken immediately prior to and at 6, 12, 24, 48, 72 and 96 h after infusion to determine tissue concentrations of dca. in non-infected mares, concentrations of dca were above mic90 for s. zooepidemicus and escherichia coli (e. coli) for the 48 h-testing period. in mares with experimentally induced endometritis, concentrations of dca were only above mic90 for e. coli for 6 h and s. zooepidemicus for 24 h. keywords: mare, bacterial endometritis, ceftiofur introduction bacterial endometritis is a leading cause of infertility in the mare and a major cause of economic loss to the equine breeding industry.1 the use of intrauterine antimicrobials to treat endometritis is common practice;2-4 evidence-based guidelines, however, for suitable antibiotic choice, effective dose rates, and treatment lengths are lacking. while in vitro susceptibility patterns can be determined following uterine culture of mares with endometritis5,6 there is limited in vivo research of commonly used antimicrobials. this is especially true at sites of active infection7 as pharmacokinetic studies are typically performed on non-infected mares rather than those with active endometritis. streptococcus equi zooepidemicus (s. zooepidemicus) and escherichia coli (e. coli) are the two most common causes of bacterial endometritis in the mare.5,6,8 ceftiofur is widely used to treat bacterial endometritis caused by these agents, both via systemic administration and intrauterine infusion.3,7 an online survey reported ceftiofur to be the most commonly used antimicrobial for intrauterine infusion.9 ceftiofur is a third generation cephalosporin that has good spectrum of activity against gram-positive and some gram-negative organisms due to increased resistance to gram-negative beta-lactamases,10 it is hydrolysed in the liver to form its most prevalent metabolite; desfuroylceftiofuracetamide (dca).11 ceftiofur is licensed in the united states for treatment of respiratory infections in horses caused by s. zooepidemicus,10,12 but despite its widespread use for treating endometritis, there are limited data on its distribution in the uterus following intrauterine infusion.13,14 the objective of this study was to compare endometrial tissue concentrations of the ceftiofur derivative; dca following intrauterine infusion in both non-infected mares and those with experimentally induced endometritis. it was hypothesized that endometrial tissue concentrations of dca would remain clinical theriogenology • volume 8 number 2 • june 2016113 above target concentrations for greater than 24 h in both non-infected mares and those with experimentally induced endometritis. materials and methods selection of mares two groups of six, non-pregnant mares of mixed breeds, ages (range 5 to 21 yrs.) and weights (range 525 to 578 kg) were selected. the mares were housed at the center for equine health at the university of california, davis on dry lots with ad lib access to hay and water and twice-daily feed of a balanced concentrate. non-infected mares. twelve mares were examined daily by transrectal palpation and ultrasonography for evidence of follicular growth, uterine edema development, intrauterine fluid accumulation, and cervical tone. once deemed to be in estrus, a baseline uterine biopsy was taken with a kevorkian biopsy forcep in an aseptic manor as previously described15 and histopathology evaluation was performed to rule out underlying uterine pathology such as the presence of inflammatory cells, periglandular fibrosis and lymphatic lacunae. the biopsies were graded using the kenney-doig scale.16 endometrial samples were also taken in an aseptic manor for bacterial culture using a double-guarded swab (mofa global, verona, wi) and cytology using a cytobrush (mofa global, verona, wi)17 to confirm the absence of any bacterial infection. any mares showing evidence of uterine pathology (grade iib or iii endometrium due to fibrosis or inflammatory changes or >3 to 4 polymorphonuclear neutrophils (pmn) per high power field) 14 were removed from the study. six mares were selected to obtain the noninfected mare group. endometritis mares. a second group of mares were chosen to be experimentally induced with bacterial endometritis. these mares were older than ten years of age (range 11-22 years), with biopsy scores of iia-iii and were free from bacterial endometritis as determined by uterine culture and cytology. in addition these mares were tested to determine susceptibility to breeding induced endometritis, as described by woodward et al.18 in short, the mares were infused with killed spermatozoa during estrus and evaluated for their ability to clear induced uterine inflammation, with susceptible mares retaining intrauterine fluid 48 h and 96 h following insemination and resistant mares clearing the fluid within 48 h. the aim of selecting susceptible mares was to help ensure that they would not resolve the bacterial induced endometritis prior to the sampling process. of sixteen mares tested, six mares fit the inclusion criteria. experimental design non-infected mares. once the mares (n=6) were determined to be in estrus by transrectal ultrasound examination, i.e. a follicle of ≥30 mm in the presence of uterine edema, a sterile infusion pipette was manually guided through the cervix to facilitate intrauterine infusion of 1 g ceftiofur hydrochloride (naxcel®, zoetis, florham park, nj) diluted in sterile saline to 60 ml total volume, as per standard infusion protocol. endometrial biopsies were taken immediately prior to infusion (time point 0) and then at 4, 8, 12, 24, 36 and 48 h after infusion. the tissue samples were stored in sterile plastic tubes at -20° c until analysis. desfuroylceftiofur-acetamide concentrations in the endometrial tissues were then measured by liquid chromatography–mass spectrometry (lc-ms; see below) at each time point.19 endometritis mares. once the susceptible mares were determined to be in estrus by criteria described above, the perineal area was aseptically prepared and they were inoculated with a total of 107 cfu of s. zooepidemicus in 10 ml of phosphate buffered saline (pbs). twenty four hours following inoculation endometrial culture, cytology and ultrasound examination were performed to confirm inflammation i.e. positive endometrial culture, >3 to 4 pmns per high power field on cytology and intrauterine fluid accumulation on ultrasound examination. endometrial biopsies were then taken immediately prior to the intrauterine infusion of 1g ceftiofur hydrochloride (time point 0) and then again at 6, 12, 24, 48, 72 and 96 h after inoculation to determine tissue concentrations of dca by lc-ms. based on the results of the non-infected group of mares, where endometrial tissue concentrations were clinical theriogenology • volume 8 number 2 • june 2016 114 above target concentrations for the duration of the 48 h testing period, it was decided to extend the duration of sampling in the endometritis mares to 96 h. in order to take the same number of total biopsy samples the 4 and 8 h biopsies were not taken and instead replaced by the 6 h sample. all of the biopsies were performed on the same estrous cycle to reduce the effects of potential variability in subsequent cycles. multiple biopsies have not been shown to be harmful; no significant effect on cytokine expression was associated with repeat biopsies in a study by christoffersen et al.20 the institutional animal care and use committee of the university of california, davis, approved all experimental procedures. bacterial inoculation the s. zooepidemicus isolate used as a model to induce endometritis in the susceptible mares was obtained from a clinical sample submitted to the microbiology laboratory at the university of california, davis. the colonies were streaked on a blood agar plate and incubated for 24 h at 35° c in 5% co2. after incubation colonies were transferred to 10 ml sterile 0.85% saline (108 cfu per ml as determined by nephelometer) and then diluted 1:10 using sterile 0.85% saline to a final concentration of 107 cfu of s. zooepidemicus per 10 ml inoculum. the inocula were kept on ice until immediately prior to transcervical infusion via an infusion pipette aseptically passed through the cervix into the uterus. liquid chromatography tandem-mass spectrometry ceftiofur residues were quantified in endometrial tissue using a method similar to that described by drillich et al.19 the method converts all residues of ceftiofur into dca prior to analysis, as dca is a more stable analyte to measure. the concentration of dca was measured in endometrial tissue by lcms. the technique was optimized to provide a lower limit of quantitation of 0.1 μg/g for dca and a lower limit of detection of 0.03μg/g. statistical analysis descriptive statistical analysis was carried out using statxact 11 (cytel software corporation, cambridge, ma). the distributions of concentrations from the non-infected horses were compared to those with endometritis at each of the separate sampling times using the exact wilcoxon-mann-whitney test. significance was set at p < 0.05. all values are represented as means ± standard error of the mean (sem). results non-infected mares following intrauterine ceftiofur infusion, endometrial tissue concentrations of dca declined from mean concentration of 873.3 μg/g (± 81.1) at 4 h, to 43.4 μg/g (± 23.6) at 48 h (graph 1). at no stage did any mare have evidence of active endometritis either clinically or based on culture results. one mare was excluded from the study following the 24 h biopsy due to minor uterine bleeding. endometritis mares following inoculation with s. zooepidemicus, all six mares in the second experiment had bacteriological evidence of endometritis (positive culture results), as well as clinical evidence of uterine inflammation, including intrauterine fluid accumulation, positive cytology (>3 to 4 pmns per high power field) and excessive uterine edema. endometrial tissue concentrations of dca declined from 173.7 μg/g (± 57.0) at 6 h, to 4.9 μg/g (± 4.1) at 96 h following intrauterine ceftiofur infusion (graph 2). non-infected vs. endometritis mares initial dca endometrial concentrations in the endometritis mares (173.7 μg/g [± 57.0] at 6 h) were lower than the non-infected mare group (873.3 μg/g [± 81.1] at 4 h) and remained so throughout the study. when comparing the same time points across the two groups, endometrial tissue concentrations of ceftiofur were significantly lower at 12 h in mares with experimentally induced endometritis compared to clinical theriogenology • volume 8 number 2 • june 2016115 non-infected mares and were lower at both 24 h (46.8 μg/g [± 37.3] vs. 171.0 μg/g [± 101.3]) and 48 h (20.4 μg/g (± 13.1) vs. 43.4 μg/g (± 23.6)) although not significant. discussion mic90 (0.25 μg/ml)21 of s. zooepidemicus was exceeded in endometrial tissue for the duration of the 48h-testing period in the non-infected mares. this provides evidence that a single-dose ceftiofur infusion will be effective for at least 48 h in this population. in the mares with experimentally induced endometritis, tissue concentrations of dca were above mic90 for s. zooepidemicus in all six mares at 12 and 24 h, supporting the current practice of once daily infusions of 1g ceftiofur.4 however at 48 h tissue concentrations were below mic90 for s. zooepidemicus in one of the infected mares (0.02 μg/g), suggesting that an every-other-day infusion protocol could result in treatment failure in a proportion of clinical cases of s. zooepidemicus endometritis. based on an mic90 of 2.0 μg/ml for e.coli,21 endometrial tissue concentrations of dca were above target concentration in all non-infected mares for the 48 h testing period. in the endometritis group, tissue concentrations were only above mic90 for 6 h in all mares; they were below 2.0 μg/g in one mare at 12 h, three mares at 24 h and four mares at 48 h. published mic90 for e. coli do vary, with older reports quoting 0.5 μg/ml.22 this may not, however, reflect the current situation and unpublished data from e. coli isolates obtained in the microbiology laboratory at university of california, davis suggest an mic90 of 4.0 μg/ml. when deciding if ceftiofur is an appropriate antimicrobial to use for e. coli endometritis, it is advisable to determine the specific mic90 of the isolate in question. to our knowledge this is the first study directly comparing the distribution of ceftiofur following intrauterine infusion in both non-infected and endometritis mares. when comparing the same time points across the two experiments, tissue concentrations of dca were significantly different at 12 h in the endometritis mares than the non-infected mares and lower at both 24 h and 48 h. this suggests that care should be taken when extrapolating data from studies performed on non-infected mares, as they may not be reflective of those with endometritis. ideally mares with experimentally induced endometritis would be more suitable models for future pharmacokinetic studies evaluating the efficacy of antimicrobials for the treatment of bacterial endometritis. it is speculated that mares with experimentally induced endometritis may have had local prostaglandin release or increased blood flow due to inflammation, which may have affected the clearance of dca, and hence explain the differences detected between the two groups. clinical signs of uterine inflammation were seen in all mares experimentally induced with endometritis but histological examination of the endometrium was not performed as in other studies.23 determining the dca levels in uterine luminal secretions was not performed and is a limitation of the study. mares in the non-infected group did not have any evidence of uterine fluid at any stage and hence measuring luminal concentrations was unlikely to be relevant. in the endometritis mares, however, it would have been interesting to determine dca levels in the luminal fluid, since bacteria often reside within the uterine lumen and hence would be targeted in this location. desfuroylceftiofur-acetamide levels in systemic circulation were also not determined following intrauterine infusion. while ceftiofur is absorbed by the mucosa and would therefore be expected to be present in the circulation, there is limited evidence to support this in the case of other antimicrobials including clavulanate and amikacin, hence the decision not to measure systemic levels.23,25 an endometrial biopsy was taken prior to the infusion of the ceftiofur hydrochloride to ensure that endometrial levels of dca were non-detectable at this stage. it is unknown, however, if the minor trauma inflicted at the biopsy site may have affected the endometrial absorption of the antimicrobial. it was felt that the importance of having a time point 0 (baseline result) outweighed this concern. the individual variability seen was interesting and while the repeatability of this could be determined with a larger sample size, it is likely to reflect the spectrum of mares with endometritis that are treated and both their ability to absorb ceftiofur and the potential for uterine evacuation of antimicrobials. while the use of systemic antimicrobials has been discussed to overcome these issues, clinical theriogenology • volume 8 number 2 • june 2016 116 dca concentrations in endometrial tissue following intrauterine infusion were higher in this study in non-infected mares (873.3± 81.1 μg/g) and in endometritis mares (173.7 ± 57.0μg/g) compared to those reported following systemic treatment (1.7 ± 0.5μg/g).26 in addition there have been conflicting results in studies testing the penetration of endometrial tissue concentrations of ceftiofur following systemic administration.26,27 conclusion this study supports the use of once daily intrauterine infusions of 1 g ceftiofur for cases of s. zooepidemicus endometritis. however, when treating bacterial endometritis caused by pathogens with a higher mic90 such as e. coli, ceftiofur may not an appropriate choice for a once daily infusion protocol. further research would be justified to determine steady state endometrial tissue concentrations following repeated intrauterine dosing of ceftiofur and the effect of increasing the initial dose of ceftiofur on endometrial tissue concentrations. acknowlegement this study was funded by the center for equine health, university of california, davis. references 1. riddle wt, leblanc mm, stromberg aj: relationships between uterine culture, cytology and pregnancy rates in a thoroughbred practice. theriogenology 2007;68:395-402. 2. causey rc: uterine therapy for mares with bacterial infections. in: mckinnon, editor. current therapy in equine reproduction. st. louis: w.b. saunders; 2007. p. 105-115. 3. lu kg, morresey pr: reproductive tract infections in horses. vet clin north am equine pract 2006;22:519-552. 4. brinsko sp, blanchard tl, varner dd, et al: endometritis. in: hartman, editor. manual of equine reproduction. 3rd ed. saint louis: mosby; 2011. p. 73-84. 5. davis ha, stanton mb, thungrat k, et al: uterine bacterial isolates from mares and their resistance to antimicrobials: 8,296 cases (2003–2008). j am vet med assoc 2013;242:977-983. 6. albihn a, baverud v, magnusson u: uterine microbiology and antimicrobial susceptibility in isolated bacteria from mares with fertility problems. acta vet scand 2003;44:121-129. 7. leblanc mm: the current status of antibiotic use in equine reproduction. equine vet educ 2009;21:156-167. 8. hurtgen jp: pathogenesis and treatment of endometritis in the mare: a review. theriogenology 2006;66:560-566. 9. dascanio, jj: how and when to treat endometritis with systemic or local antibiotics. proc annu conv am assoc equine pract 2011. p. 24-31. 10. haggett ef, wilson wd: overview of the use of antimicrobials for the treatment of bacterial infections in horses. equine vet educ 2008;20:433-448. 11. brown sa, jaglan ps, banting a: ceftiofur sodium: disposition, protein-binding, metabolism, and residue depletion profile in various species. acta vet scand suppl 1991;87:97-99 12. folz sd, hanson bj, griffin ak, et al: treatment of respiratory infections in horses with ceftiofur sodium. equine vet j 1992;24:300-304. 13. bermudez v, sifontes l, navarro n, et al: effects of intrauterine infusion of sodium ceftiofur on the endometrium of mares. proc am assoc equine pract; 1995. p.261-263. 14. ricketts sw: treatment of equine endometritis with intrauterine irrigations of ceftiofur sodium: a comparison with mares treated in a similar manner with a mixture of sodium benzylpenicillin, neomycin sulphate, polymixin b sulphate and furaltadone hydrochloride. pferdeheilkunde 1997;13:486-489. 15. brinsko sp, blanchard tl, varner dd, et al: breeding soundness examination of the mare. in: hartman, editor. manual of equine reproduction. 3rd ed. st. louis: mosby; 2011. p. 39-53. 16. kenney rm, doig pa: equine endometrial biopsy. in: morrow da, editor. current therapy in theriogenology. 2nd ed. philadelphia: wb saunders; 1989: p. 723-729. 17. cocchia n, paciello o, autetta l, et al: comparison of the cytobrush, cottonswab, and low-volume uterine flush techniques to evaluate endometrial cytology for diagnosisng endometritis in chronically infertile mares. theriogenology 2012;77:89-98. 18. woodward em, christoffersen m, campos j, et al: susceptibility to persistent breeding-induced endoemtritis in the mare: relationship to endometrial biopsy score and age, and variations between seasons. theriogenology 2012;78:495501. 19. drillich m, arlt s, kersting s, et al: ceftiofur derivatives in serum, uterine tissues, cotyledons, and lochia after fetal membrane retention. j dairy sci 2006; 89:3431-3438. 20. christoffersen m, woodward em, bojesen am, et al: effect of immunomodulatory therapy on the endometrial inflammatory response to induced infectious endometritis in susceptible mares. theriogenology 2012;78:991-1004. clinical theriogenology • volume 8 number 2 • june 2016117 21. wayne pa: performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals (secondinformational supplement). clinical and laboratory standards institute; 2013. 22. salmon sa, watts jl, yancey rj, jr: in vitro activity of ceftiofur and its primary metabolite, desfuroylceftiofur, against organisms of veterinary importance. j vet diagn invest 1996;8:332-336. 23. parlevliet jm, paccamonti dl, barker sa: cefquinome concentrations in endometrium after intrauterine treatment of cobactan 4.5% in mares and inflammatory response of the endometrium to this treatment. reprod domest anim 2009;44:189-193. 24. van camp sd, papich mg, whitacre md: administration of ticarcillin in combination with clavulanic acid intravenously and intrauterinely to clinically normal oestrous mares. j vet pharmacol therap 2000;23, 373-378. 25. orsini ja, park mi, spencer pa: tissue and serum concentrations of amikacin after intramuscular and intrauterine administration to mares in estrus. can vet j 1996; 37:157-160. 26. witte ts, bergwerff aa, scherpenisse p, et al: ceftiofur derivates in serum and endometrial tissue after intramuscular administration in healthy mares. theriogenology 2010;74:466-472. 27. cervantes cc, brown mp, gronwall r, et al: pharmacokinetics and concentrations of ceftiofur sodium in body fluids and endometrium after repeated intramuscular injections in mares. am j vet res 1993;54:573-575. graph 1: non-infected mares: endometrial tissue concentrations of dca (desfuroylceftiofur-acetamide) at 8, 12, 24, 36 and 48 h. 8 12 24 36 48 0 200 400 600 800 1000 time (h) d c a c o n c . ( μg /g ) 12 24 48 72 96 0 50 100 150 200 250 time (h) d c a c o n c . ( μg /g ) clinical theriogenology • volume 8 number 2 • june 2016 118 graph 2: mares with experimentally induced endometritis: endometrial tissue concentrations of dca (desfuroylceftiofur-acetamide) at 12, 24, 48, 72 and 96 h. graph 3: endometrial tissue concentrations of dca (desfuroylceftiofur-acetamide) in non-infected mares (black bars and o) and mares with experimentally induced endometritis (grey bars and ☐) at 12, 24 and 48 h post intrauterine infusion of 1 g ceftiofur. an asterisk indicates significant differences in dca tissue concentrations at 12 h. 12 24 48 0 200 400 600 800 1000 time (h) d c a c o n c . 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/cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /converttocmyk /destinationprofilename (u.s. web coated \(swop\) v2) /destinationprofileselector /usename /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1 contact jamie stewart jlstewart13@vt.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11640, http://dx.doi.org/10.58292/ct.v17.11640 research report pregnancy outcomes and calf growth in beef cows given modifiedlive virus vaccination at synchronization for timed artificial insemination jamie stewart,a john currin,a sherrie clark,a tracey redifer,a vitor mercadantea,b adepartment of large animal clinical sciences, virginia-maryland college of veterinary medicine, blacksburg, va, usa bschool of animal sciences, college of agriculture and life sciences, virginia polytechnic institute and state university, blacksburg, va, usa abstract the objective was to compare reproductive outcomes and calf traits after vaccination of suckled beef cows using a commercial modified-live virus (mlv) vaccine that contained bovine herpesvirus 1 and bovine viral diarrhea virus (types 1 and 2) at the initiation of a timed artificial insemination (ai) program. in experiment 1, cows given mlv vaccine in the previous year were enrolled during the fall 2019 and spring 2020 breeding seasons. at initiation of a 7-day co-synch + cidr synchronization protocol (10 days before breeding), cows were given either mlv vaccine or nothing (cont). cows were inseminated 60-66 hours after removal of the cidr insert and subsequently exposed to bulls starting ~ 1 week after ai. all cont cows received a killed virus vaccine at mid-pregnancy. for experiment 2, available cows from fall 2019 were reenrolled and given the same vaccination treatment as in the previous season. treatment with mlv vaccine did not have effects on ai pregnancy rates (p ≥ 0.31), total season pregnancy rates (p ≥ 0.12), or ai pregnancy losses (p ≥ 0.41). there was also no effect of vaccination treatment on calf birth weight (p ≥ 0.27), weaning weight (p ≥ 0.14), or average daily gain (p ≥ 0.14). in summary, cows given mlv vaccinations on day 10 before breeding experienced no deleterious effects on pregnancy outcomes or calf traits. these findings supported the use of mlv vaccination in previously vaccinated cows at synchronization for timed ai. keywords: artificial insemination, cattle, breeding, modified-live virus, synchronization introduction in beef cow-calf herds, exposure to common abortifacients, such as bovine herpesvirus 1 (bohv-1) and bovine viral diarrhea virus (bvdv) produces a serious threat to reproductive efficiency and overall production. in addition to well-documented deleterious effects on the mid-pregnancy fetus and pregnancy maintenance, both viruses also have adverse effects on ovulation, corpus luteum formation, and early embryonic development when exposed early in the breeding season.1–6 fortunately, there are various effective approved multivalent vaccines available for use in cattle. these products, frequently referred to as ‘5-way vaccines,’ protect against bohv-1 and bvd (both types 1 and 2), and common respiratory viruses, parainfluenza 3 and bovine respiratory syncytial virus. variations of these products also commonly include bacterins to protect against other important reproductive pathogens, namely campylobacter fetus ssp. venerealis and several leptospira spp. serovars. among the various commercial 5-way vaccine products for cattle, there are 2 general classes: modified live virus (mlv) vaccine or a chemically inactivated/killed virus (kv) vaccine. the mlv vaccines have multiple advantages over the kv vaccines. authors of a meta-analysis reported that although both vaccine types decreased abortion and fetal infection in cows exposed to bvdv, the relative risk for pregnancy loss was less with the use of mlv vaccines.7 similarly, mlv and kv vaccines against bohv-1 reduced the risk of abortion in cattle by ~ 60%; however, mlv vaccines had a greater duration of protective immunity and stimulated higher neutralizing antibody titers.8 despite its proven efficacy, notable controversies surrounding the use of mlv vaccines involve their effects on conception rates when given near breeding and on fetal losses when given during pregnancy. it is recommended that commercial multivalent vaccines containing modified-live bohv-1 and bvdv components be given a minimum of 30 days before breeding or insemination to avoid adverse effects on fertility.9 mailto:jlstewart13@vt.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11640 2 citation: clinical theriogenology 2025, 17, 11640, http://dx.doi.org/10.58292/ct.v17.11640 on the contrary, manufacturers of commercial kv vaccines typically recommend giving at 2-4 weeks before breeding, and also state that they are safe to use in pregnant cows and heifers, regardless of vaccination history. although kv vaccines are labeled as safe for use in pregnant cows, many mlv vaccine product labels state that they can only be safely given to cows during pregnancy if the cows have been previously vaccinated. a case report calls this claim into question after abortions occurred in a group of 55 heifers vaccinated with a usda-licensed ‘5-way’ vaccine product at 7-8 months of pregnancy.10 the heifers had been vaccinated with the same product on 3 earlier occasions prior to breeding. seven heifers aborted fetuses between 45-55 days after vaccination, of which, bohv-1 infection was diagnosed in the 6 submitted specimens, based on detection of bohv-1 antigen and isolation of bohv-1 from pooled tissue.10 to err on the  side of caution, many practitioners recommend using only  kv vaccinations during pregnancy, and reserve the use of  mlv vaccines only for prebreeding. necrotic oophoritis, abnormal estrous cycles, and decreased pregnancy rates are well-documented deleterious outcomes of mlv vaccination near the onset of estrus in naïve heifers,11–14 which is why the label specifies that vaccination should occur no sooner than 30 days before breeding. however, the reproductive effects of mlv vaccine in previously vaccinated cattle have been inconsistent. in the largest field study to date, our research group reported no deleterious effects of mlv vaccine given on day 10 before timed artificial insemination (ai) compared to cows receiving kv vaccine.15 however, the biggest limitation of our previous study was the lack of a true negative control. authors of a study reported a decrease in ai pregnancy rates when previously vaccinated cattle were given either a mlv or chemically altered/inactivated (ca/ia) vaccine between 27-29 or 30-37 days before breeding (52%), compared to those vaccinated with either product at 46-89 days before breeding (64%).16 there is, therefore, a need to critically evaluate the timing of our prebreeding cowcalf vaccination protocol with a true negative control to better understand its effects on fertility. timed ai is a tool that beef cow-calf producers can use to improve calving distribution, enhance pregnancy rates, and increase subsequent calf value.17 vaccination of cows > 30 days before breeding may not be practical for well-managed farms that wish to implement timed ai and maintain a 365 days calving interval. synchronization of ovulation for ai already requires a minimum of 3 trips through the chute. prebreeding vaccination would add an additional handling event, increasing both cattle stress and producer costs. vaccination at synchronization could reduce stressors on cattle by decreasing the number of trips through the chute. although we demonstrated that mlv vaccination at cidr insertion (10 days before ai) did not have adverse effects on reproductive outcomes or calf traits compared to kv vaccine,15 there is still a need to compare the use of mlv vaccine to a control group receiving no prebreeding vaccine. therefore, the objective was to assess the reproductive outcomes (ai and breeding season pregnancy rates and ai pregnancy losses) and subsequent calf traits (birth and weaning weights) of previously vaccinated suckled beef cows in commercial cow-calf operations vaccinated with mlv vaccine 10 days before breeding. we hypothesized that reproductive outcomes and calf traits do not differ between mlvvaccinated and control cows vaccinated with kv vaccine at mid-pregnancy. materials and methods cows all cows utilized were cared for in accordance with the practices outlined in the guide for the care and use of agricultural animals in agricultural research and teaching.18 since data  collected from routine farm visits were used, animal care and use committee approval was not required, and the study is exempt. suckled angus-cross beef cows (n = 2,912) housed at 11 locations during fall of 2019 (8 locations; n = 1,107), spring of 2020 (5 locations; n = 911), and fall of 2020 (7 locations; n = 894) were enrolled in this study. all cows belonged to commercial cow-calf operations managed by the virginia  department of corrections agribusiness program. replacement heifers in these cow-calf operations are raised internally and vaccinated with an mlv vaccine at weaning and then again before breeding each season. experiment 1 included cows bred in the fall 2019 and spring 2020 seasons. these enrolled cows had all been vaccinated the previous year (fall 2018/spring 2019) with a mlv vaccine (pyramid 10, boehringer ingelheim, duluth, ga, usa) given at cidr insertion (10 days before timed ai). experiment 2 included cows bred fall 2020 that were also used in experiment 1. vaccination, estrus synchronization, and ai for experiment 1 (fall 2019/spring 2020), cows were blocked within herd by breeding status from the previous breeding season (ai versus bull bred) and age (primiparous versus multiparous), then randomly assigned to either of 2 study groups: 1. modified-live virus vaccine (mlv, n = 1,003; pyramid 10, boehringer ingelheim, duluth, ga, usa) given subcutaneously at the labeled dose at initiation of a 7-day co-synch + cidr synchronization protocol19 or 2. unvaccinated (cont, n = 1,015). for experiment 2 (fall 2020), each cow received the same treatment (mlv, n = 406; versus cont, n = 439) that they were assigned to in experiment 1. a total of 145 cows (57 in fall 2019, 39 in spring 2020, and 49 in fall 2019) were excluded from ai due to calving late in the previous breeding season but were still given vaccination and remained in the breeding group for natural service. at treatment (10 days before ai), cows were assigned a body condition score (bcs) and received 100 µg of intramuscular gnrh agonist (factrel, zoetis, parsippany, nj, usa) and an intravaginal insert containing 1.38 g of progesterone (eazi-breed cidr, zoetis). the cidr insert was removed after 7 days, and 25 mg of intramuscular prostaglandin f2α analog (dinoprost tromethamine, lutalyse, zoetis) was given. at cidr removal, cows were also fitted with an estrus-detection patch (estrotect breeding indicator, rockway, inc, spring valley, wi, usa). at 60-66 hours after cidr removal and prostaglandin treatment, cows received intramuscular gnrh agonist and were inseminated by trained ai technicians (12 technicians total) using commercially obtained frozen semen from 16 proven bulls selected for breeding based on phenotype and pedigree. estrus detection patches were observed at ai, and a cow was determined to be in estrus if it had an activated (color change from gray to red), lost, or partially activated (≥ 50% color change) estrus patch. drugs and vaccinations were given by trained veterinarians, technicians, or veterinary students using single-dose syringes with a new needle for each cow. all cows, regardless of treatment, were commingled immediately after vaccination within their assigned breeding groups to be exposed to bulls starting 1 week after ai. intact angus http://dx.doi.org/10.58292/ct.v17.11640 citation: clinical theriogenology 2025, 17, 11640, http://dx.doi.org/10.58292/ct.v17.11640 3 bulls that had successfully passed a breeding soundness examination were turned in with each breeding group at a ratio of 1:20-1:40 for ~ 65 days during each season. cows in the cont group were subsequently vaccinated at 2-5 months pregnancy with a kv vaccine (virashield 6l5, elanco, greenfield, in, usa). pregnancy diagnosis percentage of cows pregnant by ai was determined by transrectal ultrasonography performed by experienced veterinarians (blinded to treatment) between 40-80 days after ai. presence of a fetal heartbeat was used to determine fetal viability and crown-rump length of the fetus or embryo was used to determine age (ai versus bull bred). confirmation of pregnancy status was performed at 1-2 months after initial pregnancy detection and additional pregnancies from natural service were similarly recorded. cows that were diagnosed as pregnant by ai at first pregnancy diagnosis, and were no longer pregnant at second pregnancy diagnosis, had confirmed abortions, or did not calve were designated ai pregnancy losses. pregnancy losses to those bred by the bull were not included in analyses. the total season pregnancy rate for each herd was determined using a combination of pregnancy diagnosis results and final calving data provided by each farm and included both ai and bull-bred cows. calving day was determined for each cow by calculating the difference between the recorded calving date and day 0 of the calving season. day 0 was established to be 283 days after timed ai for ai-bred cows or 283 days after the first day of bull exposure for cows that were only bred by natural service. calf birth weights and weaning weights were also recorded and provided by farm personnel. calves were weaned between 5-9 months, at an average of 230 ± 0.7 days for calves born to fall-bred cows in 2019, 228 ± 0.8 days for calves born to spring-bred cows in 2020, and 236 ± 0.7 days for calves born to fall-bred cows in 2020. average daily gain (adg) at weaning was calculated for each calf by subtracting the birth weight from the weaning weight and dividing the age (in days) at weaning. data analyses cow was the experimental unit in this study as the treatment was applied to each individual cow. all statistical models included farm as a random variable to allow for its use as an error term for interpretation across all farms. days postpartum (dpp), bcs at cidr insertion, percentage of cows detected in estrus, calving day within each herd’s calving season, calf birth weights, calf weaning weights, and calf adg at weaning were assessed by anova in r, with treatment and season included as fixed variables for experiment 1 and just treatment for experiment 2. the model for calf weaning weights and adg also included calf birth weight and calf sex as independent covariates, and the model for calving day included dpp and cow age as independent covariates. the binomial outcomes of ai pregnancy rates, total season pregnancy rates (ai + bull bred pregnancies), and ai pregnancy losses were analyzed using a generalized linear mixed-effects model (glmer procedure) in r with treatment, season, and dpp included as fixed variables. the mixed effect model for ai pregnancy rates also contained the effects of ai technician, bcs at cidr insertion, and estrus expression as independent covariates. the mixed effects model for season pregnancy rates also contained the effects of bcs at cidr insertion and breeding groups as independent covariates. the 145 cows that were excluded from ai were not included in the model for ai pregnancy rates or ai pregnancy losses, but were included in all other analyses. significance was set at p ≤ 0.05, and tendencies were discussed between p = 0.06 and 0.10. results prebreeding variables, bcs at cidr insertion, dpp at ai, and percentage of cows having activated or lost estrus-detection patches at ai for experiments 1 and 2 are summarized (table 1). in experiment 1, bcs, dpp, and estrus expression differed (p ≤ 0.05) between seasons, but did not differ (p ≥ 0.38) between treatment groups. there was also no treatment (p ≥ 0.51) by season interaction for any of these variables. similarly, none of these variables differed (p ≥ 0.56) between treatment groups for cows in experiment 2. the reproductive outcomes in mlv versus cont cows from experiments 1 and 2 are summarized (figure). in experiment 1, there were effects of estrus expression (p < 0.01), bcs (p < 0.01), dpp (p < 0.01), and ai technician (p < 0.01) on ai pregnancy rates, but no treatment (p = 0.31), treatment by season (p = 0.94), or treatment by dpp (p = 0.54) interaction (figure). similarly, there were effects of estrus expression (p < 0.01), bcs (p = 0.04), and dpp (p = 0.04), but no effect of treatment (p = 0.93) or treatment by dpp (p = 0.11) interaction on ai pregnancy rates in experiment 2 (figure). in experiment 1, there was a main effect of bcs (p < 0.01) on total season pregnancy rates, but there were no treatment (p = 0.37), season (p = 0.60), or treatment by season (p = 0.71) interaction (figure). in experiment 2, bcs tended (p = 0.08) to have an effect on total season pregnancy rates, but there was also no effect of treatment (p = 0.12; figure). there were [aq1] table 1. prebreeding variables; body condition score (bcs) at cidr insertion, days postpartum (dpp) and estrus expression at ai; modified-live virus (mlv) group were vaccinated 10 days before timed ai and unvaccinated (cont) group received kv vaccine at mid-pregnancy season bcs dpp estrus expression cont mlv cont mlv cont mlv fall 2019 5.5 ± 0.03 5.5 ± 0.03 78 ± 0.7 78 ± 0.7 57% (303/534) 55% (283/516) spring 2020 5.2 ± 0.04 5.3 ± 0.04 83 ± 0.8 83 ± 0.9 65% (282/431) 65% (289/443) p-value 0.38 0.63 0.59 fall 2020 5.5 ± 0.04 5.5 ± 0.04 74 ± 0.8 73 ± 0.7 59% (259/439) 57% (231/405) p-value 0.69 0.81 0.56 http://dx.doi.org/10.58292/ct.v17.11640 4 citation: clinical theriogenology 2025, 17, 11640, http://dx.doi.org/10.58292/ct.v17.11640 no effects of treatment (p = 0.95), season (p = 0.70), or treatment by season interaction (p = 0.42) on ai pregnancy losses in experiment 1 (figure), nor were there effects of treatment (p = 0.41) on ai pregnancy losses in experiment 2 (figure). calf traits for mlv versus cont cows in both experiments 1 and 2 are summarized (table 2). in experiment 1, there were effects of season (p < 0.01) and dpp (p < 0.01) on the average day within the calving season that cows gave birth, but no treatment (p = 0.59) or treatment by season (p = 0.42) interaction. figure. reproductive outcomes in cows given mlv vaccine 10 days before timed ai compared to unvaccinated cows (cont) given kv vaccine at mid-pregnancy in experiments 1 (a, c, e) and 2 (b, d, f). a. in experiment 1, there were no treatment (p = 0.31), treatment by season (p = 0.94), or treatment by days postpartum (p = 0.54) interactions (a). b. similarly, there were no effects of treatment (p = 0.93) or treatment by dpp (p = 0.11) interaction on ai pregnancy rates in experiment 2. c. there were no effects of treatment (p = 0.95), season (p = 70), or treatment by season (p = 0.42) interaction on total season pregnancy rates in experiment. d. treatment also did not affect total season pregnancy rates in experiment 2 (p = 0.12). e. ai pregnancy losses did not differ by treatment (p = 0.95), season (p = 0.70), or treatment by season (p = 0.42) in experiment 1. f. ai pregnancy losses also did not differ between treatments (p = 0.41) in experiment 2. http://dx.doi.org/10.58292/ct.v17.11640 citation: clinical theriogenology 2025, 17, 11640, http://dx.doi.org/10.58292/ct.v17.11640 5 similarly, there was an effect of dpp (p = 0.01) on calving day in experiment 2, but no treatment effect (p = 0.73). in experiment 1, there was an effect of season (p = 0.02) and calf sex (p < 0.01) on calf birth weight, but no treatment (p = 0.58) or treatment by season (p = 0.90) interaction. in experiment 2, there was an effect of calf sex (p < 0.01) on calf birth weight, but no treatment effect (p = 0.27). in experiment 1, there were main effects of season (p < 0.01), calf birth weight (p < 0.01), and calf sex (p = 0.02) on weaning weight, but no treatment (p  = 0.14) or treatment by season (p = 0.23) interaction. in experiment 2, there were main effects of calf birth weight (p < 0.01) and calf sex (p < 0.01) on weaning weights, but no treatment effect (p = 0.32). similarly, there were main effects of season (p < 0.01), calf birth weight (p < 0.01), and calf sex (p < 0.01) on adg, but no treatment (p = 0.14) or treatment by season (p = 0.17) interaction in experiment 1. there were main effects of calf birth weight (p < 0.01) and calf sex (p < 0.01) on adg in experiment 2, but no treatment effect (p = 0.35). in both experiments, calf weaning ages did not differ between treatments (p ≥ 0.79). there tended to be an effect of season (p = 0.09) on weaning age in experiment 1, but no treatment by season interaction (p = 0.73). discussion the results of the current study demonstrated that vaccination of suckled beef cows with a commercial mlv product at initiation of a 7-day co-synch + cidr synchronization protocol (10 days before ai) did not have adverse effects on reproductive outcomes or subsequent calf growth traits. most notably, ai pregnancy rates were not affected by mlv vaccination on day 10 before breeding in either experiment 1 (fall 2019: 48-51%; spring 2020: 58-60%) or 2 (fall 2020: 59-60%). this finding was in contrast to our previous study, where ai pregnancy rates were higher in cows given mlv versus kv vaccine at cidr insertion the fall breeding season only,15 despite having 2 fall breeding seasons represented in the current study. we previously theorized that since respiratory pathogen exposure is greatest in the cooler fall/winter months in the usa,20 cows given mlv vaccine may have had greater protection against the effects of these pathogens on fertility and early embryonic development.1–6 it is worth noting that since both our previous and the current studies were field trials, exposure to pathogens could not be controlled. as a result, pathogen exposure could have been less in the years of our current study compared to those in our previous study. regardless, the lack of any deleterious effect of prebreeding mlv vaccination on ai or total season pregnancy rates in both of our studies challenged the notion that mlv vaccination at < 30 days before breeding is detrimental to cow fertility. in addition, there were no effects of prebreeding mlv vaccination on estrus expression or ai pregnancy losses, consistent with findings in prior studies.15,16 the timing of prebreeding mlv vaccinations has been a notable health management controversy, due to inconsistent findings among researchers. replacement heifers given a combination mlv vaccine containing bohv-1 and bvdv 9 day before breeding did not differ in estrus expression or pregnancy outcomes (by either ai or natural service) compared to those vaccinated on day 30 before breeding.21 in a similar study, beef heifers were vaccinated with either a mlv or kv (with no bohv-1/bvdv component) vaccine on day 2 after an unsynchronized estrus and then revaccinated with the same product either 10 or 31 days before natural service.22 neither vaccine type nor timing of vaccination had an effect on the duration of interestrus intervals, estrus expression, total pregnancy rates, or pregnancy losses.22 on the contrary, authors of another study reported decreased ai pregnancy rates in spring-calving beef cows and heifers given mlv vaccine (52%) compared to those given a chemically altered/inactivated (ca/ ia) vaccine (60%) on ≥ day 27 before breeding.16 interestingly, cows vaccinated with either a mlv or ca/ia vaccine between days 27-29 or 30-37 before breeding had lower pregnancy rates (52%) compared to those vaccinated using either product on days 46-89 before breeding (64%).16 these findings called into question the use of either product close to breeding, as it was suggested that the nonspecific inflammation from vaccination could negatively impact fertility.16 in contrast, there were no adverse effects on ai or total season pregnancy rates in beef cattle given a ca/ia vaccine (47%; 98%) on days 60 and 30 before breeding compared to saline-treated controls (43%, p = 0.49; 96%, p = 0.14).23 similarly, there was also no reported effect on ai or season pregnancy rates in beef cattle given a mlv vaccine (40%; 95%) on days 60 and 30 before breeding compared to saline-treated controls (43%, p = 0.21; 96%, p = 0.34)23 similar to the findings of our current study. due to the concerns with ca/ia vaccine given close to ai,16 we elected to have a control group in the current study that table 2. summary on calving day, birth weights, weaning weights, weaning age, and average daily gain (adg) of calves born to cows treated with a modified-live virus (mlv) vaccine 10 days before timed ai and unvaccinated cows (cont) that were given kv vaccine at mid-pregnancy season calving day birth weight (kg) weaning weight (kg) weaning age (days) adg (kg/day) cont mlv cont mlv cont mlv cont mlv cont mlv fall 2019 9.9 ± 0.9 9.9 ± 0.9 34.9 ± 0.3 35.1 ± 0.3 244 ± 1.9 248 ± 2.1 230 ± 1.0 229 ± 1.0 0.912 ± 0.008 0.934 ± 0.009 spring 2020 5.2 ± 0.8 6.0 ± 0.9 34.3 ± 0.3 34.5 ± 0.3 256 ± 2.0 256 ± 1.9 228 ± 1.1 228 ± 1.1 0.976 ± 0.008 0.975 ± 0.008 p-value 0.59 0.58 0.18 0.80 0.14 fall 2020 6.4 ± 0.9 5.8 ± 0.9 34.6 ± 0.3 35.1 ± 0.3 250 ± 2.0 253 ± 2.3 236 ± 1.0 236 ± 1.0 0.916 ± 0.008 0.927 ± 0.010 p-value 0.73 0.27 0.32 0.79 0.35 http://dx.doi.org/10.58292/ct.v17.11640 6 citation: clinical theriogenology 2025, 17, 11640, http://dx.doi.org/10.58292/ct.v17.11640 received no prebreeding vaccine, but instead received a kv vaccine at mid-pregnancy. a controlled study evaluated the efficacy of mid-pregnancy (day 118) ca/ia vaccination to heifers that had received 2 mlv vaccinations previously.24 heifers received an initial ca/ia vaccine booster during their first pregnancy. during second pregnancy, beef heifers were exposed to bvdv pi animals (days 95-111 of pregnancy), revaccinated with ca/ia vaccine on day 118, and inoculated with bohv-1 on day 210 of pregnancy.24 the results of that study suggested that this combination mlv/ca/ia vaccine protocol provided adequate protection against fetal infection and loss following controlled exposure to bvd and bohv-1.24 the cows enrolled in experiment 2 herein similarly received kv vaccines mid-pregnancy in 2 subsequent pregnancies after having previously received mlv in the years prior with no issues regarding fetal loss. although these results also supported the use of kv or ca/ia vaccine at mid-pregnancy in multiple pregnancies, it is worth noting that there was no known (or controlled) pathogen exposure in these herds during the current study. also, the kv vaccine used in our study does not guarantee protection against development of persistently infected bvd calves, whereas most mlv vaccines guarantee this protection. an important distinction among many of these studies with contradictory results is that the mlv and kv vaccines used were from different manufacturers. the kv vaccine product used16,24 contained chemically altered strains of bohv-1 and parainfluenza 3 viruses, modified live bovine respiratory syncytial virus, and a liquid, adjuvanted preparation of inactivated bvd viruses (type 1 and 2), which is why we have denoted it as a ca/ia, and is the only ‘kv’ vaccine product known to guarantee protection against the development of persistently infected bvd calves. the kv vaccine product used in our current and previous15 studies contained all inactivated viruses but did not provide protection against the development of persistently infected bvd calves. in addition to different kv vaccine products, mlv vaccines among these studies also came from various manufacturers and contained different adjuvants. an earlier study compared the use of 2 commercial mlv vaccines in postpartum beef cows (n = 807) at 2 intervals before ai (day 30 versus day 10); authors reported no effect of vaccine product or time of treatment on ai (53-56%) or seasonal (85-89%) pregnancy rates.25 data are still lacking on a more recent and thorough comparison of commercial mlv and kv cattle vaccine products at various intervals before breeding. the mean calving day did not differ between cows receiving prebreeding mlv vaccines versus kv vaccination at mid-pregnancy. in our previous study, mlv-vaccinated cows calved, on average, 2 days sooner than kv-vaccinated cows (~ 8.4 versus ~ 10.3 days), regardless of the season.15 there was a difference between seasons in experiment 1, with decreased mean calving day in the spring (5.2-6 days) versus the fall (9.9 days), but no interaction with the vaccine treatment. several factors may have roles in these differences, including cow nutrition, weather, pathogen exposure, and bull genetics. pregnancy length has been reported to be negatively correlated (genetically) with all growth traits except birth weights, suggesting that faster growing fetuses may trigger earlier parturition.26 pearson’s correlation test determined a moderately negative correlation between the mean calving day and weaning weights (r = − 0.30; p < 0.01) of the current study and from data collected in the previous study (r = − 0.35; p < 0.01) (author communication). neither calf birth weights, adg, nor weaning weights were affected by vaccine treatment in the current study or previously.15 therefore, it appeared unlikely that calf growth traits may have previously affected pregnancy length and mean calving day. more likely, the greater fall ai pregnancy rates observed in the previous study could have a role in the observed differences on mean calving day.15 future studies that evaluate the downstream effects of vaccinations on calf growth traits with controlled pathogen exposure are needed to better understand these findings. in conclusion, there were no differences in reproductive outcomes or calf traits between suckled beef cows given mlv vaccine 10 days before ai compared to those given kv at mid-pregnancy. pregnancy rates observed were consistent with industry standards of ~ 45-55% ai pregnancy rates and ~ 90-95% total seasonal pregnancy rates. however, based on contradictory results among several large field trials, there is still a need to more thoroughly investigate the effects of vaccine protocols on pregnancy outcomes based on factors such as manufacturer, season, geographical location, pathogen exposure risks, and farm goals. understanding how these factors impact production systems can help veterinarians provide up-to-date and practical vaccination recommendations to cow-calf producers. acknowledgments authors thank all the farm managers and workers at the virginia department of corrections for their valuable assistance and partnership throughout this study. in addition, we acknowledge clinical-year veterinary students and technicians at the virginia-maryland college of veterinary medicine, who assisted in synchronization, vaccinations, and pregnancy diagnosis. we extend our gratitude to zoetis and estrotect for their donation of synchronization products. conflict of interest authors have no conflict of interest to declare. references 1. miller jm, van der maaten mj: experimentally induced infectious bovine rhinotracheitis virus infection during early pregnancy: effect on the bovine corpus luteum and conceptus. am j vet res 1986;47:223–228. pmid: 2420240 2. miller jm, van der maaten mj: early embryonic death in heifers after inoculation with bovine herpesvirus-1 and reactivation of latent virus in reproductive tissues. am j vet res 1987;48:1555– 1558. pmid: 3434897 3. miller jm, van der maaten mj, whetstone ca: effects of a bovine herpesvirus-1 isolate on reproductive function in heifers: classification as a type-2 (infectious pustular vulvovaginitis) virus by restriction endonuclease analysis of viral dna. am j vet res 1988;49:1653–1656. pmid: 2847601 4. grooms dl, brock k, ward la: detection of bovine viral diarrhea virus in the ovaries of cattle acutely infected with bovine viral diarrhea virus. j vet diagn invest 1998;10:125–129. doi: 10.1177/104063879801000201 5. archbald lf, gibson cd, schultz rh, et al: effects of intrauterine inoculation of bovine viral diarrhea-mucosal disease virus on uterine tubes and uterus of nonpregnant cows. am j vet res 1973;34:1133–1137. http://dx.doi.org/10.58292/ct.v17.11640 https://doi.org/10.1177/104063879801000201 citation: clinical theriogenology 2025, 17, 11640, http://dx.doi.org/10.58292/ct.v17.11640 7 6. archbald lf, fulton rw, seger cl, et al: effect of the bovine viral diarrhea (bvd) virus on preimplantation bovine embryos: a preliminary study. theriogenology 1979;11:81–89. doi: 10.1016/ s0093-691x(79)80020-5 7. newcomer bw, walz ph, daniel givens m, et al: efficacy of bovine viral diarrhea virus 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virus and bovine herpesvirus-1 afforded by annual revaccination with modified-live viral or combination modified-live/killed viral vaccines after primary vaccination with modified-live viral  vaccine. vaccine 2017;35:1046–1054. doi: 10.1016/j. vaccine.2017.01.006 25. whittier wd, baitis hk: timing of vaccinations in estrous synchronization programs. proceedings, applied reproductive strategies in beef cattle 2005; p. 147–156. 26. bourdon rm, brinks js: genetic, environmental and phenotypic relationships among gestation length, birth weight, growth traits and age at first calving in beef cattle. j anim sci 1982;55:543–553. doi: 10.2527/jas1982.553543x http://dx.doi.org/10.58292/ct.v17.11640 https://doi.org/10.1016/s0093-691x(79)80020-5 https://doi.org/10.1016/s0093-691x(79)80020-5 https://doi.org/10.1016/j.theriogenology.2014.09.028 https://doi.org/10.1016/j.prevetmed.2017.01.005 https://doi.org/10.1016/j.prevetmed.2017.01.005 https://doi.org/10.21423/aabppro20015171 https://doi.org/10.2460/javma.241.2.189 https://doi.org/10.1016/0093-691x(90)90071-z https://doi.org/10.2460/ajvr.1990.51.07.969 https://doi.org/10.1016/0378-1135(85)90017-3 https://doi.org/10.1016/j.theriogenology.2012.10.005 https://doi.org/10.1016/j.theriogenology.2023.01.027 https://doi.org/10.1016/j.theriogenology.2023.01.027 https://doi.org/10.21423/bovine-vol52no1p53-58 https://doi.org/10.1016/j.cvfa.2016.01.006 https://doi.org/10.2527/2006.842332x https://doi.org/10.21423/bovine-vol51no2p159-164 https://doi.org/10.1016/s0093-691x(97)00056-3 https://doi.org/10.1016/j.theriogenology.2014.11.015 https://doi.org/10.21423/aabppro20163477 https://doi.org/10.1016/j.vaccine.2017.01.006 https://doi.org/10.1016/j.vaccine.2017.01.006 https://doi.org/10.2527/jas1982.553543x immunohistochemical evidence for kisspeptin signaling in equine gonadotrophs immunohistochemical evidence for kisspeptin signaling in equine gonadotrophs christianne magee,a kelly kirkley,a luke sylvester,b brittany runyan,a colin claya aanimal reproduction and biotechnology laboratory, department of biomedical sciences college of veterinary medicine and biomedical sciences colorado state university, fort collins, co bdepartment of biochemistry, college of natural sciences colorado state university, fort collins, co kisspeptins (kp) are a family of amidated peptides known to initiate and maintain reproductive function in all mammalian species studied to date. our laboratory and others have provided immunohistochemical evidence for a kisspeptin gonadotropin releasing hormone (gnrh) neuronal mechanism in the equine hypothalamus and have demonstrated that iv administration of equine kisspeptin decapeptide (ekp 10) elicits a rise in peripheral luteinizing hormone (lh) concentrations in mares. we hypothesized that kp has a direct effect on equine gonadotrophs to elicit lh synthesis and secretion. both gnrh receptor and kiss1r are gαq/11 coupled receptors and a rise in intracellular calcium concentration is necessary for lh secretion. by quantifying the rise in intracellular calcium after exposure of equine primary pituitary cells to gnrh and/or ekp 10, our laboratory identified 3 distinct populations of equine pituitary cells: 1 population responded to both ekp 10 and gnrh, a second population responded to only ekp 10, and a third population responded only to gnrh. although these findings supported a pituitary mechanism for ekp 10 at the level of the gonadotroph, pre-treatment of diestrus mares with gnrh antagonist (antide 1.0 mg iv) eliminated any measurable change in peripheral lh after 1.0 mg ekp 10 iv. the body of evidence for kisspeptin regulation of pituitary cell function is growing. objective was to establish immunohistochemical evidence for kp signaling at the level of the equine pituitary gland. to confirm kiss1r expression in the equine pituitary and to determine if gonadotrophs express kiss1r, coimmunofluorescence studies against kiss1r (akr 001, alomone labs, israel) and the lh subunit β (518b7, courtesy of jf roser) were performed on frozen diestrus mare hemipituitaries (n = 8). images of immunolabeled tissues were captured using scanning confocal microscopy and analyzed via direct cell counting of at least 4 fields per tissue per mare, using dapi to identify individual cells. based on cell counts, on average 26.7% of all cells within the equine pituitary expressed kiss1r and 21.17% of all pituitary cells were positive for lhβ. additionally, in congruency with previous data, 3 populations were identified including kiss1r only positive cells (18.2%), lhβ only positive cells (13.3%) and cells positive for both kiss1r and lhβ (8.4%). data provided evidence that cells of the equine pituitary, including gonadotropes, were primed to directly respond to kisspeptin signaling and that further exploration to understand the role of kisspeptin in pituitary function is needed. keywords: equine, kisspeptin, pituitary, immunohistochemistry 521 clinical theriogenology • volume 11, number 3 • september 2019 522clinical theriogenology • volume 11, number 3 • september 2019 untitled omniblank: 2014: lameness in the pasture bull lameness in the pasture bull gary d. warner, justin box elgin veterinary hospital, elgin, tx lameness in a herd bull in the pasture can be just as devastating to breeding performance as any penile injury or orchitis event. even minor problems, such as a simple puncture wound inducing a subsolar abscess, can lead to a lack of desire to service females and/or maintain body condition in the pasture. many issues may resolve themselves over a period of time, but at what cost to reproductive efficiency? in order to facilitate accurate diagnosis and treatment of bovine lameness, the practitioner should have adequate devices available to aid in the restraint of an animal. hydraulic tilt chutes or tilt tables offer a great advantage when evaluating the lower limb and will often lead to a more accurate diagnosis when such tools are available. every item available for analyzing lameness in the equine is of value when evaluating bovine lameness. analyzing the lame bull proper evaluation of the lame bull starts with characterization and localization of the lameness. a practitioner must be careful not to let the caretaker’s history persuade him/her into looking at the wrong area. examine the bull and observe what he tells you. is there swelling or inflammation present on or around the coronary band? how does he bear weight on his claw? does he rotate his toes in or out when he moves? does he subluxate his fetlock? does he fully flex his tarsus? hopefully the ability to properly restrain the bull in lateral recumbency is available because standing examination using rope restraint of a compromised limb in a beef animal in a squeeze chute is difficult at best. if a hydraulic tilt chute is utilized, access to the lower limb is excellent; however; access to the upper joints like the shoulder and stifle is limited. as always, the hoof is examined first. if it is overgrown, it should be trimmed and close attention should be paid to any cracks, fissures, or discolored sites. all suspect areas should be pursued with curettage. lower limb lameness bulls with a history of being developed in a feedlot or gain test may be more predisposed to various foot problems like: white line disease and subsequent sub-solar abscess, axial wall separation, and abcessation due to wall injury. previous sub-clinical laminitis is usually the causative agent of most of the hoof lameness presented. the desire for carcass data from ultrasound evaluation encourages producers to push bulls with excessive grain diets in order to improve carcass values. this has led to an increase in laminitis and the problems that arise secondary to a laminitic event. there are varying opinions on how to deal with sub-solar abscesses in the bovine. the preferred method involves elevating the affected claw by applying a block (shaped like the sole) to the unaffected claw. most often, an antiseptic bandage is applied with or without parenteral antibiotic administration. some practitioners prefer to use analgesics as part of a treatment plan. axial wall cracks and overrun/overgrown walls are diagnosed as part of the visual examination and are the result of over-feeding at some point in the animal’s life (usually during the yearling development phase). like sub-solar abscesses, resolution is achieved with debridement of the separated wall and bandaging. care should be taken when performing curettage on a lateral wall crack. cutting too deep into sensitive lamina may cause over-proliferation of granulation tissue and a delay in healing. interdigital fibromas are another common lameness problem in herd sires, particularly in bulls that are extremely heavy and have a smaller hoof. the development of the fibroma occurs when the suspensory ligament of the fat pad “breaks down” allowing the fat pad to prolapse distally into the interdigital space. eventually, hypertrophy of the skin of the interdigital space causes development of the fibroma. these are not usually painful, but if they become large enough to come in contact with the soil surface, the fibroma will get ulcerated and possibly develop a localized infection. sometimes the fibroma becomes so large that it may be pinched between the claws which causes discomfort and lameness. resolution of the lameness problem is relatively simple and straightforward with surgical reduction being 517 clinical theriogenology • volume 6, number 4 • december 2014 the preferred method. it is always recommended that the interdigital fat pat be removed while reducing the fibroma (particularly if the capsule is breached while dissecting the fibroma). the surgical site is left open to granulate and heal by second intention. some surgeons prefer to just wire the toes together while others provide additional bandage support. some just prefer to bandage the foot. usually the surgical site is healed in seven to ten days, and the bull can be placed back in service after an additional seven to ten days of rest. hairy heel warts are another dilemma for the herd sire. most often, they are observed in the younger bull being developed in close confinement (i.e. feedlot or pasture with intensive supplementation). it can become a chronic problem if left unattended. it is particularly contagious in a naïve population of cattle, especially when congregating in areas that have soil with higher moisture content (around water troughs, common pathways through pens, etc.). this problem is easily treated by immersion of the hoof with hoof baths or by a single antibiotic bandage application. parenteral antibiotics do not seem to be effective as a treatment for resolution of the problem. vertical hoof cracks the prevalence of hoof cracks or fissures observed in practice has increased dramatically over the last few years. genetics as well as more intense management probably play a significant role in their development. faster growth, higher concentrate rations, and decreased availability of trace minerals may all play a part in the development of vertical hoof cracks. it is thought that subclinical laminitis following episodes of subclinical rumen acidosis may be the most significant cause of this problem. the push for improved performance numbers as an aid to evaluate and market young herd sires may be the greatest determining factor to continued lameness in these individuals. lameness is thought to occur as weight bearing load is applied to the cracked hoof wall with instability of the wall creating abnormal pressure on the underlying structures of the hoof (figure 1) the cracks may occur as an isolated incident affecting one claw, or they may affect several claws at one time. wall cracks may become infected when soil or other debris collect and are packed within the fissure. this can also induce pressure necrosis of the sensitive lamina. figure 1 vertical hoof crack. the lesion in this picture has the appearance of a rupture which could be the result of mechanical stress and bending. repair can be accomplished by curettage of the hoof crack and treatment of any infection present, which can be accomplished by bandaging with antibiotics/antiseptics and parenteral antibiotics if needed. closure of the defect should not be completed until the surface of the crack is completely dry and hardened. repair can be performed by drilling horizontally across the margin of the fissure and lacing stainless steel wire across the crack followed by application of a pliable acrylic. another method consists of application of para-aramid synthetic cloth (kevlar®, dupont, wilmington, de) intermittently with acrylic compound directly into the fissure followed by stabilizing the hoof wall. stabilization allows 518clinical theriogenology • volume 6, number 4 • december 2014 normal wall growth from the coronary band and usually allows the repair to gradually wear out as the normal wall grows in (figures 2a and 2b figure 2a repair of a vertical hoof fissure with kevlar® figure 2b completed application of kevlar® and acrylic and acrylic. septic arthritis of the coffin joint this condition is a common sequella to chronic infectious processes involving the hoof and/or interdigital space. a common presentation is a diffusely swollen area involving the coronary band and sometimes the entire digit. it is also not uncommon to see a draining lesion in the area of the extensor process or at the heel bulb region. usually, the animal is significantly lame or non-weight bearing, and most of the time production is limited and can involve serious weight loss. radiographs usually reveal active osteomyelitis with lytic areas appearing in both the second and third phalanges and an obliterated joint surface (figure 3). figure 3 osteomyelitis with lytic (black and white arrow) and proliferative bony lesions. note the obliterated joint space with roughened articular surfaces (white arrow head). economical treatment of this condition may be achieved without sacrifice of the entire claw. first of all, a block is applied to the good claw to relieve any weight-bearing on the affected claw. next, 519 clinical theriogenology • volume 6, number 4 • december 2014 after thoroughly cleansing the area, a tourniquet is placed proximal to the fetlock and a local anesthetic is injected into the vasculature of the distal limb. continued preparation involves creating a window to enter the coffin joint for debridement. this can be created at the heel bulb or mid hoof just distal to the coronary band. when draining tracts are involved, it seems better to approach from the heel bulb area. this will allow complete debridement of both the coffin joint and navicular bursa. occasionally the navicular bone and associated deep digital flexor tendon are involved and must be removed. this is facilitated by entry at the rear of the hoof. a one-half inch drill bit is introduced into the area of the coffin joint/navicular area, and the drill is used as a router of sorts to clean the necrotic material from the joint. the goal is to extend the curettage to a point just proximal to the extensor process of the coffin bone. a large curette is used to facilitate removal of debris and to insure all necrotic bone has been removed. it is necessary to completely debride the affected area lest healing will be slowed markedly. aggressive lavage of the joint with a sterile antiseptic solution is recommended and facilitated by use of a squeeze bulb to create positive pressure. a pack is usually placed in the joint for two days and a bandage applied. parenteral antibiotics are administered daily and bandage changes with lavage of the joint are undertaken every two days for two to four times. once purulent drainage ceases, the limb may be cast above the fetlock for five to six weeks. casting supports the toe that has been elevated and aids in prevention of tendon and joint laxity. an alternative to casting is simply wiring or applying acrylic between the toes to immobilize the diseased toe. after five to six weeks, the cast may be removed along with the block, and after a recuperative period, the animal should be nearly clinically sound. most cases are 80% improved in overall lameness, but few are completely asymptomatic. injuries of the proximal limb after a rather lengthy discussion of various problems caused by different etiologies in the lower limb, lameness involving the upper limb is usually the result of one etiology: trauma. trauma can occur to the limb in many forms: punctures, lacerations, blunt force from other animals, putting a limb where it should not be, or even hematogenous spread of bacteria in the case of localized bruising. injuries such as long bone fractures to a metacarpal or metatarsal (these are most common) or tibia or radius fractures (most often the result of bulls fighting) are routinely dealt with in large animal practice. these injuries respond well to external fixation via trans-fixation casting or application of thomas splint/casting. subluxation of the fetlock or pastern can occur for various reasons although most are thought to occur after the limb is caught up in a hole in the ground or a cattle guard and attempts to dislodge cause the injury. limbs trapped in cattle guards will often luxate one or both of these joints during attempts to dislodge. often times, alignment can occur after a significant amount of traction has been applied to the limb. usually, a device such as a “calf jack” is placed on the sternum and attached to the hoof by a wire. holes are drilled in the hoof wall to allow the wires to pass through. this method aids in fatigue of muscle, ligament and tendon associated with the luxation. brute force is then utilized to replace the joint to its original position. when this cannot be accomplished via closed reduction, the joint is opened and joint surfaces debrided so that reduction is accomplished via leverage. the goal is reduction with subsequent arthrodesis of the affected joint. the bull can still function very well whether the affected joint is in the fore or rear limb. once the luxation is reduced, the limb is cast (usually full limb and preferably with trans-fixation pinning). lacerations to the caudal lower limb (particularly below the fetlock) should be considered as serious as a limb fracture because they can be just as detrimental to overall performance. lacerations in this area can easily involve the pastern or coffin joint and will likely include a portion of the deep digital flexor tendon sheath. lacerations to the tendon sheath are rarely cared for properly immediately after the initial insult, and thus require extensive treatment in order to resolve. proper care should involve installation of a drain in the tendon sheath with aggressive lavage for several days. vascular perfusion of the lower limb with appropriate antibiotic therapy as well as parenteral antibiotics and analgesics are necessary. placing a block on the good toe always aids in ambulation and resolution usually occurs 520clinical theriogenology • volume 6, number 4 • december 2014 within two weeks. additionally, placing the lower limb in a light-weight cast does help markedly with recovery after resolution of the infection. injuries of the upper limb upper limb injuries, particularly of the shoulder and stifle, are probably the most common cause of lameness in the breeding bull after problems with the hoof. in forelimb lameness, if the problem is not found in the hoof, the shoulder is the next most likely area of concern. shoulder injuries can occur during dismount of cows or other bulls, slipping on slick surfaces, or because of lateral concussive injuries during a bull fight. most of these injuries are thought to be more soft tissue in nature (i.e. capsular tears or muscle/ligament injury) rather than trauma to the joint surface. once again, this injury can be difficult to diagnose without proper facilities for restraint. although the shoulder can be injected while the bull is standing in a squeeze chute, it is preferred to do the injection with the animal in lateral recumbency with the limb properly restrained. any serious injury such as a proximal fracture of the humeral head or scapula will not show a positive response when the joint is anesthetized. positive response (meaning resolution of lameness or at least 80% of it) to anesthesia injected into the joint can predict a fair outcome for treatment. preference is given to directly medicating the joint utilizing the longer acting corticosteroids such as betamethasone or triamcinolone in combination with hyaluronic acid. restricting activity after medicating is extremely important with a rest period ranging from 30 to as much as 90 days. treatment of most shoulder injuries has been very successful, particularly when given rest after medicating. much like the shoulder is a usual suspect in forelimb lameness, the stifle is the usual cause of lameness in the rear limb if the hoof is not involved. most stifle injuries are due to blunt force trauma initiated by another animal. the blow is usually delivered to a preoccupied bull that is in the act of servicing a female or during a bull fight. most stifle injuries affect the soft tissue structures of the stifle: anterior and posterior cruciates, the collateral ligaments and/or the menisci (primarily medial). a diagnosis may be made by clinical signs alone and the prognosis may be reached by evaluating the degree of lameness experienced by the bull. diagnostics and potential for treatment may be augmented by the use of a tilt table. with the bull in lateral recumbency, the ability to take radiographs and possibly do a joint centesis are markedly improved, thus a more accurate diagnosis is possible. increased effusion is common, particularly in more extensive injuries. effusion is a negative prognostic indicator and radiographs of the stifle are the best tool to reach a proper prognosis, the more caudally displaced the femur in comparison to normal of the tibia, the poorer the prognosis (figure 4). aggressive therapy early after injury will aid in the healing of all but the most severe injuries. the best time to treat is within the first two to three weeks after the insult. figure 4 stifle effusion and caudally displaced femur. the preferred approach to treatment starts with an aggressive lavage of the stifle with saline solution combined with an anti-inflammatory of the practitioner’s choosing. most often, a longer acting 521 clinical theriogenology • volume 6, number 4 • december 2014 anti-inflammatory is administered to the joint before the bull is removed from the table. a second lavage is recommended about two weeks after the first lavage followed by the injection of a long-acting corticosteroid and hyaluronic acid. parenteral anti-inflammatories are also recommended initially and during the rest period which may be as long as six months post-treatment. the bull is confineed to a stall for a period of up to 60 days and then a small paddock or pasture for the balance of the period; it is extremely important that each bull be housed separately. the injured bull should not be housed with any other cattle. those bulls with severe stifle injuries that have the genetic merit to produce frozen semen may benefit from external fixation of the stifle to allow for fibrosis of the damaged structures. although a thomas splint will not aid in resolution of the injury, immobilization of the joint and secondary fibrosis of the structures of the stifle will allow the bull to get around sufficiently to produce viable semen. fractures of the long bones with the salvage value of cattle today passing historical highs, the opportunity to repair limb fractures in all cattle makes economic sense. cattle tolerate external fixation very well with minimal aftercare. most heal nicely within teo to three months (with additional time for rehabilitation of the limb), and the individual can be put back into production. any limb fracture below the carpus or tarsus should be cast; the repair may be augmented by the use of a trans-fixation pin placed perpendicular through the bone immediately proximal to the fractured bone (pin placed thru the distal radius if the bull has a fractured metacarpus). for fractures occurring below the stifle or the elbow (fractures of the radius or tibia), the use of a thomas splint combined with casting of the limb is the best approach. the thomas splint (t-splint) works best for those animals that are more athletic. cattle that are very excitable or obese do not handle splinting well. the reason for this is it takes a lot of effort to arise from recumbency with the affected limb in extension and if the animal does not take care when moving with the splint they can fall on the splinted limb and cannot get up once they are down without assistance. because of this, disposition is an important consideration when evaluating options for repair. transfixation casting having proper equipment (tilt table) and sedation is ideal for this type of repair. a combination of sedatives and analgesics may be used to facilitate getting the animal into lateral recumbency when a table is not available. tourniquet placement proximal to the site of pin placement will allow for vascular perfusion with an anesthetic agent such as lidocaine. this provides local anesthesia/analgesia and facilitates placement of the transfixing pin. although large diameter intramedullary pins (1/4” -3/8”) can be used, the length of time the pin is effective is limited by the lack of solid purchase in the cortex of the bone. threaded pins that are specifically designed to be used as transfixation pins will remain in place longer and therefore improve the effectiveness of the transfixation cast. placement of a threaded pin requires the drilling of a pilot hole and a tap to facilitate placement of the pin through the cortex. several wires are placed through holes drilled in the walls of both claws to aid in manipulation of the limb and allow for proper reduction of the fracture. if need be, traction may be applied using these wires to aid in dis-traction and better alignment of the fracture. once part of the cast has been applied, the wire is incorporated into the cast insuring a good anchor to the distal extent of the limb. this casting technique, along with aggressive medical management has been used to facilitate healing of compound and commuted fractures of the metatarsus and metacarpus. typically, the transfixation pin will have to be removed before the cast is ready to be removed because of osteolysis at the pin site. this usually occurs about six weeks after pin placement. the cast is then removed at eight to ten weeks after application, and if needed, a splinted bandage is applied to support the limb for an additional one to two weeks. on average, it takes about two months for rehabilitation of the limb to occur after cast removal (figure 5). 522clinical theriogenology • volume 6, number 4 • december 2014 figure 5 transfixation cast in place on a charolais bull. thomas splint application in order to have the most effective thomas splint application, it is best to have several different sizes on hand so that the animal can have the most appropriate fit. splint construction allows for length adjustment, which enables the splint to be custom fitted according to the size of the patient (figure 6) usually, the animal is placed in lateral recumbency with the use of a tilt table, rope, casting harness, or by chemical restraint. the splint is applied by placing the limb through the loop and attaching the hoof to the plate at the distal extent of the splint. this is accomplished by drilling holes in the hoof wall and placing wire through the holes and into the foot plate on the splint. traction is applied to the limb before the splint is locked in place in order to facilitate alignment of the displaced bone. if there is diffuse swelling, a robert jones bandage is applied to the limb for seven to 14 days before casting to allow for edema reduction. this is crucial for long-term fracture immobilization. whenever the limb is of sufficient health, a cast is applied to the limb from the hoof to the loop of the splint, incorporating both the cast limb and the splint together. if good alignment of the fracture site has been achieved, some of the tension on the splint may be relieved to better facilitate the animals ability to move about freely. applying the cast with the hock in slight flexion is preferable so that movement and use of the leg is maintained. the splint is usually left on the animal for eight to 12 weeks (figure 7) length of time is determined by the severity of the fracture and how well the animal tolerates the splint. rehabilitation may take longer than if transfixation casting is used because of the traction applied to the limb and the way the animal must use the splint. forelimbs will rehabilitate much faster than rear limbs and exercise is a necessary component to any rehabilitation program. some individuals experience significant pain after removal of these devices and analgesics administered after removal can greatly facilitate an animal’s recovery. 523 clinical theriogenology • volume 6, number 4 • december 2014 figure 6 thomas splint with screws figure 7 thomas splint in place on front limb. note position of the to allow for length adjustment. loop loop caudal to the elbow and cranial to the point of the shoulder. (black and white arrow). foot plate (white arrow head) suggested reading goonewardene la, hand rk: a study of hoof cracks in grazing cows—association with age, weight, and fatness. can j anim sci 1995;75:25-29. greenough pr: bovine laminitis and lameness: a hands-on approach. edinburgh: saunders elsevier; 2007. starke a, heppelmann m, kehler w, et al: septic arthritis of the distal interphalangeal joint in cattle; comparison of digital amputation and joint resection by solar approach. vet surg 2007;36:350-357. steiner a, ossent p, mathis ga: intravenous regional anesthesia and antibiotic therapy applied to the limbs of cattle: indications techniques and complications. schweiz tierheilkd 1990;132: 227-237. stokka gl, lechtenberg k, edwards t, et al: lameness in feedlot cattle. vet clin north am 2001;17:189-207. 524clinical theriogenology • volume 6, number 4 • december 2014 1 contact ramanathan kasimanickam ramkasi@wsu.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 review report laparoscopic artificial insemination in sheep: review and cost benefit analysis andrew myers, ramanathan kasimanickam college of veterinary medicine, washington state university, pullman, wa, usa abstract artificial insemination in sheep has vastly increased in popularity as preserved semen is readily available. laparoscopic artificial insemination (lai), a surgical procedure, involves deposition of semen directly into the uterus using a laparoscope. we have reviewed lai, compared benefits to natural breeding and transcervical artificial insemination, described procurement, and provided a cost benefit analysis. lai is an advanced assisted reproductive technique with several benefits, including higher pregnancy rates requiring fewer sperm per breeding than vaginal or transcervical artificial inseminations. although lai involves a higher start-up cost compared to other techniques, it is an economically feasible option. with the continual development of new equipment, techniques, and semen preservation technologies, lai can enhance herd genetics without investment in expensive genetically superior studs and maintaining them. lai with an appropriate estrus synchronization protocol results in higher numbers of superior quality lambs per ewe for higher profit margin and higher lambing rates for producers, making lai a profitable service for a veterinary practice. keywords: sheep, artificial insemination, laparoscopy, fertility, economic analysis introduction artificial insemination (ai) technologies have dramatically increased the ability of producers to shift the genetics of their herd to meet milk and meat market demands.1 development of ai and semen preservation has allowed producers to access top-tier genetics from around the world.2 in the small ruminant industry, ai is a high-impact assisted reproductive technology. it supports genetic improvement and contributes to conservation of genetic resources. in addition, it is a safe and effective method of reproduction while contributing to animal health by controlling the spread of several infectious diseases. the successful implementation of ai depends on effective animal management, semen evaluation and preservation, estrous cycle control, estrus detection, and appropriate insemination methods. although the use of ai dates to the 1700’s, it was not heavily researched or used until the 1900’s.3 now, ai is a widespread practice and is used throughout the world. many variations and methods of ai have been developed, with species responding differently to each type of approach. laparoscopy artificial insemination (lai) was first reported in the 1980’s.4,5 in ewes, unlike cows, the major limiting factor is the difficulty to pass the insemination pipette through the cervix for intrauterine semen deposition. ewe’s cervix is small, narrow, rigid, and tortuous (figure 1)6 and it is relatively more difficult to navigate the inseminating pipette without transrectal cervical manipulation. for this reason, semen is usually deposited at cervical entrance or in the cervix (intracervical) resulting in poor pregnancy rates. not only cervical navigation is bypassed in lai, it also results in higher pregnancy rates compared to vaginal artificial insemination (vai) and transcervical artificial insemination (tcai).7,8 for this reason, fixed time intrauterine insemination is performed via laparoscopy; commonly used in large-scale breeding programs worldwide. although lai is a minimally invasive procedure it requires veterinary expertise and raises animal welfare concerns and demands specialized equipment and more labor compared to cervical insemination. although several alternative approaches have been proposed to replace laparoscopy, lai technique continues to be the gold standard when higher pregnancy rates are required. in small ruminants, there are 3 types of ai techniques: vaginal (pericervical), cervical (intracervical) or intrauterine (transcervical and laparoscopic technique) semen deposition.7 objectives were to: 1. review lai literature; 2. compare benefits of laparoscopic artificial insemination over natural breeding and tcai; 3. describe the procedure; and 3. perform economic analyses and determine the break-even point for lai service to become profitable. mailto:ramkasi@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11080 2 citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 intravaginal (pericervical) artificial insemination vaginal insemination is performed with fresh or chilled semen. in this method, the semen is deposited in the cranial vagina (fundus) using an infusion rod. the procedure is easy and quick to perform under field conditions and may result in lower but still acceptable pregnancy rates. vaginal ai is best suited to use after estrus detection during the natural breeding season. the ideal timing of ai is before ovulation (i.e. 12-18 hours after estrus onset);9 recommended semen volume is 0.2-0.5 ml and the number of progressively motile sperm is minimum of 300 × 106.7,10 intravaginal ai is effective in ewes with fresh semen compared to extended (chilled) semen. however, conception rates after vaginal insemination are lower with pharmacological estrus synchronization. additionally, pregnancy rates following vaginal insemination with frozen semen are not acceptable. cervical insemination with fresh or liquid semen and intrauterine insemination with frozen semen via laparoscopy require smaller insemination doses. intracervical artificial insemination intracervical ai with fresh or chilled semen is the ideal choice in small ruminants since the external cervical os is located/visualized with illumination. the insemination pipette is passed through the speculum into the cervix, without excessive force, to a depth of 5-12 mm. semen is deposited after positioning the pipette in the cervix. special attention is necessary to minimize semen backflow. unlike intravaginal ai, conception rates achieved with fresh or chilled semen via this method are acceptable after pharmacological estrus synchronization.7,9,11 the ideal time for ai is 48-65 hours after intravaginal progesterone inserts removal or 15-17 hours after the onset of detected estrus.12-14 originally, use of frozen semen was limited because of the low lambing percentages (25-35%) with cervical ai. this was associated with the reduced viability of frozen sperm resulting in lower numbers of viable or undamaged sperm reaching fertilization site. currently, with the development of cervical ai skills and good semen evaluation and processing methods, pregnancy rates are improved.15 insemination volume and the number of motile sperm required are 0.2-0.5 ml and 150-200 × 106, respectively.7,10 since lai is prohibited in most of the north european countries and requiring special and costly equipment and well-qualified technicians, importance is given to cervical ai with frozen semen.16,17 in goats, intracervical ai is the most widely practiced method since caprine cervix is relatively easier to traverse than ovine cervix. intrauterine artificial insemination lack of perfect nonsurgical procedures for ewes severely limits its use. direct transfer of semen into uterine lumen with the laparoscopic technique has resulted in acceptable results, but it has disadvantages. alternatively, transcervical intrauterine ai techniques (e.g. guelph system of transcervical ai) have been developed.10,18-24 transcervical ai procedure require special positioning of the animal, cervical retraction and stabilization, and specially designed instruments for stabilization and passing insemination pipette through the cervix. recently developed methods allow the semen to be deposited atraumatically, even deeper into the uterine horns by the transcervical route.20-22 however, the process of manipulating an ai catheter through the cervix still has been linked to reductions in pregnancy and lambing rates.22 it has been hypothesized that cervical trauma and vaginal/cervical stimulation caused by the catheterization may activate pathways interrupting pregnancy between days 3-14.21,22 intracervical application of hyaluronan 52 hours after sponge removal improved cervical relaxation and increased cervical entry.25 among other methods, the use of drugs (hormones,26,27 chemokines,28 myorelaxants,29 beta-adrenergic blocking agents30,31) and the design of inseminating catheters32,33 associated with manipulation of the cervical canal (i.e. guelph system17-20) have been investigated with some success. the procedure of transcervical ai could be even simpler if it were sufficient to deposit the semen in only 1 uterine horn. there are opinions to the contrary,5 but evidence from lai in sheep indicates that the deposition of semen in a single uterine horn leads to fertilization of ova from both ovaries.34 insemination volume and the number of  motile sperm required are 0.2-0.5 ml and minimum 100-200 × 106, respectively.7,10 trained and experienced inseminators may enter the cervix in 75-85% of ewes.20,21 however, transcervical ai cannot be used with an acceptable success rate in nulliparous ewes. cervical injury, abscesses, infections and poor pregnancy rates are associated with this technique, but they vary by operator, semen dose, ewe condition, and experience.22,24 pregnancy rates are generally lower than those achieved by laparoscopic ai (fresh: 40-70%; frozen: 30-70%). laparoscopic intrauterine ai the novelty of laparoscopic intrauterine insemination of ewes represents the most remarkable development in ovine ai industry. with the laparoscopic technique, decreased amount of semen is deposited into uterine horns lumen. the number of sperm required for each insemination is lower and the volume of semen is proportionally higher.35,36 this allows more suitable dilution rates and, therefore, better preservation and protection of sperm. laparoscope-aided intrauterine ai improves pregnancy rate achieved with cryopreserved semen and facilitates the widespread dissemination of valuable genotypes. when correctly performed, this method has a success rate of > 60%, but this rate will vary depending on the quality of semen, the time of the year, the ewe’s condition, and the skill of the inseminators. conception rates achieved with frozen semen used by this method are figure 1. image of mold of ewe’s cervical canal with cervical folds and rings (courtesy of dr. brian buckrell) a b http://dx.doi.org/10.58292/ct.v17.11080 citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 3 higher than for intracervical insemination, because of better cryopreservation of sperm and deposition of semen in the uterus. the main disadvantages are the need for expensive laparoscopic equipment, invasive surgery (animal welfare issues), and the technical expertise needed to perform the procedure. its cost usually limits this procedure for expensive semen, valuable animals or breeding companies. current techniques recommend depositing half the total dose of semen in the middle region of each uterine horn without regard to ovulation site;9,37 however, deposition in 1 horn had acceptable conception rates.8,37 in estrus-synchronized ewes, ideal timing of ai is 48-65 hours12-14,37 after the removal of progesterone inserts and 12-18.5 hours38 after estrus detection. insemination volume and the number of motile sperm required are 0.25 or 0.5 ml and minimum of 20-40 × 106, respectively.9,15,16,38 an average ejaculate diluted appropriately can be utilized to inseminate 50 ewes. an experienced team in a well-equipped and organized operation can inseminate 250-500 ewes in a day with this technique. factors influencing the reproductive outcome application of ai in sheep production requires appropriate knowledge of animal management, seasonality, breeds involved, and production system of small ruminants. several factors influence ai program success in small ruminants (table 1). sheep breed can influence the length of breeding season; sheep breed should be selected based on operation objective. in accelerated breeding programs (increase the number of lamb crops per ewe; 3 lambing in 2 years and 5 lambing in 3 years) it is ideal to have sheep breeds with a long breeding season. sheep breeds and breeding season length are provided (table 2).71,72 laparoscopic ai animal selection careful selection of animals is vital to achieve good reproductive outcome and to deliver the best monetary results to clients. as mentioned above, there are several factors pertinent to ewe and ram, among others that influence lai results. selection of ram and ewe provides the best improvement in genetics and conception rate. implementing ai allow producers with an opportunity to choose genetically superior semen with desirable traits, based on operation objectives. frozen semen technology provides the ability to obtain semen from genetically top studs throughout the world. however, reproductive performances with fresh, cooled, and frozen semen can differ (table 3). guidelines for animal welfare animal welfare is important in all aspects of animal husbandry and veterinary medicine. animal health and well-being should be considered in breeding protocol and ai. precautions should be taken to prevent undue stress and pain. sheep should be sedated for lai and given preemptive pain medication and prophylactic antibiotics.78 effort should be made to maintain sterility of all instruments entering the abdominal cavity.78 table 1. factors influencing the reproductive outcome factors references female factors: age of the ewe/parity 39-41 genetics 42 ewes’ body condition 43,44 prolificacy 45,46 health management 47 nutritional management 48-51 synchronization and induction of estrus 52-54 stress 55,56 hygiene 57,58 animal management 59,60 male factors: age of the ram 61 genetics 42 scrotal circumference 62,63 superior quality semen 6,41,61,64,65 others: farm 41,66 year 41 season 41,66,67 semen type 68,69 ai technique 66,68 ai technician 41,66 insemination time, method 6,67,70 interval from lambing to ai 41 table 2. length of breeding season and breed of sheep length of breeding season breeds long (6-8 months) dorset finn polypay rambouillet rideau romanov medium (4-6 months) canadian charollais hampshire oxford suffolk short (4 months) leicester north country cheviot scottish blackface shetland texel http://dx.doi.org/10.58292/ct.v17.11080 4 citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 the animal should be in the cradle only for a short duration to prevent adverse effects associated with dorsal recumbency. time spent in the trendelenburg position should be minimal due to pressure on lungs. therefore, animals should only be positioned when everything is ready. if an animal develops breathing complications, it should be returned to a normal position. after lai, sheep should be monitored for postanesthetic complications. when lai is performed correctly, complications are mitigated with lower incidence of surgical complications. a comprehensive list of lai complications is provided.37 complications include rupture or puncture of abdominal viscera, subcutaneous emphysema, hematoma or subcutaneous bleeding, abscess formation, peritonitis, sepsis, intra-abdominal adhesions, and uterine bleeding.37 synchronization of estrus synchronization of estrus is vital for ai program success. ewes are often artificially inseminated at a fixed time. the basis for implementation of synchronization protocols is to hormonally regulate the estrous cycle at the herd level; this facilitates scheduling of the procedure at a fixed time and increases fertility. various synchronization protocols are available for small ruminants; protocol used should be customized with the owner’s and veterinarian’s goals and schedules. there are several pharmaceuticals available in the usa for synchronization protocols. prostaglandin (pgf2a) lyses a corpus luteum in cycling ewes to induce estrus and is given twice (7-12 days apart) for luteal regression and estrus expression (table 4).79-82 estrus detection is required for the 2-dose pgf2α protocol. since estrus expression occurs over a period, timed ai is not possible. it was not successful due to poor fertility and embryonic loss83 compared to synchronization protocols with progestogen, progesterone, and ecg;84 however, there was no impact on fertilization.85 efficiency of pgf2α in combination with the ram effect was explored.86 the most common method of estrus synchronization involves progesterone followed by a single dose of equine chorionic gonadotropin (ecg) or pg600. this requires progesterone, controlled internal drug release (cidr)73 or compounds with progesterone-like activity (progestogen sponges, map, medroxyprogesterone acetate;87,88 or fga, fluorogestone acetate73,89) in intravaginal devices that are used 12-14 days prior to ai. at removal, a single injection of ecg73,90 or pg60091,92 is given subcutaneously to induce ovulation. androgenized teasers can also be introduced to ewes along with ecg treatment to help stimulate estrus.93,94 most ewes will enter estrus 24-36 hours later, peaking at 48 hours.95 laparoscopic intrauterine ai of ewes with semen is normally performed table 4. effect of prostaglandin f2α on estrus response and pregnancy in ewes prostaglandin f2α dose interval (days) season type of breeding er (%) pr (%) reference cloprostenol 120 μg 7 breeding natural 91 73.3 80 9 94 75.9 11.5 94 75.9 cloprostenol 126 μg 9 breeding 100 81 68.25 μg 9 75 38.5 μg 9 87.5 dinoprost tromethamine 10 mg single dose breeding natural 60 82 cloprostenol sodium 150 μg 70 d-cloprosternol 150 μg 70 er: estrus response rate; pr: pregnancy rate table 3. reproductive outcome in laparoscopic intrauterine insemination* semen type insemination dose (x 106 sperm) number of ewes reproductive outcome (%) season references fresh 50 in each horn 300 pr 70.1 breeding 73 n/a 2,508 pr 82.2 breeding and anestrus 69 cooled 20 in each horn 265 lr 75.3 breeding and out of season 74 frozen 50 103 lr 62.1 out of season 75 75 243 pr 50.2 breeding 76 n/a 25,939 pr 70.1 breeding and anestrus 69 80 198 lr 63.6 breeding 77 *synchronization regimens and insemination timing referred were different; pr: pregnancy rate, lr: lambing rate http://dx.doi.org/10.58292/ct.v17.11080 citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 5 48-65 hours after cidr removal (depending on whether fresh or frozen-thawed semen is used), with a minimum of 25 × 106 motile sperm deposited per ewe.9 several types of intravaginal devices resulted in varying reproductive outcomes. cidrs are more accessible, quick to insert, and a more efficient form of progesterone.96 however, protocols must be adapted depending on the progesterone and treatment form. cidrs resulted in an earlier response and ovulation compared to sponges, 55 and 60 hours, respectively.95 the delay in the commencement of ovulation was observed in november and december when most ewes are expected to be in anestrus.95 cidr treatment produced less variation around ovulation timing97 and a greater tendency for higher conception rates, 69 compared to 58.6% with other intravaginal devices. further, during breeding season, cidr had significantly higher pregnancy, fertility, twinning rate and fecundity compared to sponges during breeding and out of season.73,98 another study, conducted during the breeding season, had higher fertility yields in short-term (6-7 days) cidr-based treatments (75%) and the long-term (12-14 days) cidr-based treatment (70%) than in the long-term (12-14 days) sponge-based treatment (45%).99 poor fertility in sponge groups was attributed to change in vaginal ph (~ 8) and vaginal microbiome (a higher incidence of salmonella spp. and staphylococcus aureus).99 conversely, the percentage of ewes lambing and twinning rate are similar between sponges and cidr-treated ewes during breeding and out of season.96,100 fga sponges resulted in higher pregnancy than cidrs in autumn (54.7 versus 67.4%), but not spring (59.2 versus 56.2%), suggesting a seasonal impact on the effectiveness of estrus synchronization.101 however, another study reported no difference between pregnancy rates in march, april, and may inseminations compared to summer months.69 the type of progestogen could also be a reason for the variability. a lower conception rate was observed with map (64.6%) compared to fga (74.7%) or cidr (71.7%).69 during breeding season, ecg treatment in ewes after progesterone removal increased rates of ovulation,102,103 conception and lambing.73,103 treatment with 250 iu (72.9%) or 300 iu (79.1%) ecg doses resulted in higher pregnancy rate compared to 200 iu (62.4%). in nonseasonal ewes, a higher ecg dose (400 iu) at cidr removal normally led to a superovulatory effect or increase in prolificacy, measured as twins and triplets.101 during breeding season, gnrh treatment at estrus onset or before luteinizing hormone (lh) surge or 36 hours after progesterone device removal,104,105 had no improvement in the number of pregnant ewes compared to current protocols97 despite improved synchronization of anestrus ewes.102 gnrh has been used in sexed semen106,107 or in multiple-ovulation embryo transfer programs,102,108 where timing of ovulation is critical for success. estrus response and pregnancy for various combinations of progesterone and gonadotropin are provided (table 5) cidr is the only approved form of progesterone device in the usa. currently, ecg is either unavailable or backordered in the usa and pg600 does not perform as well as ecg. standard doses of pg600, typically intended for pigs, can lead to overstimulation of the ovaries, resulting in reduced pregnancy rates due to issues with ovulation and embryo survival; therefore, a lower dose of pg600 is usually recommended for sheep.91,109 additionally, pg600 in small ruminants is considered extra-label. therefore, other products, such as gnrh agonists (gnrh, hcg94) or fsh,110,111 should also be considered. combinations of various progesterone and gonadotropin formulations to induce estrus for natural or artificial insemination in ewes are provided (table 5). postthaw semen evaluation evaluation of postthaw semen is important to determine the quality. although postthaw motility is the primary criterion, evaluation of membrane integrity, viability and functional tests such as hypo-osmatic swelling and sperm dna and acrosomal integrity are used to objectively evaluate sperm quality. however, in the field, motility and concentration are commonly used indicators to determine semen quality and quantity. briefly, semen straws are removed from the liquid nitrogen storage tank and placed in a 37°c water bath for 1 minute, then dried with a paper towel. the straws are cut with scissors, and the samples are emptied into glass vials, which are placed on a 37°c digital dry block warmer and swirled for 1 minute. a 7.5 µl sample is then pipetted to the isperm (madison, wi, usa) base chip for analysis of total motility and concentration.117 procedure laparoscopy requires insertion of a cannula/trocar through the abdominal wall, distension of the abdominal cavity with sterile air or co2, and visual examination of the abdominal organs with an illuminated telescope. in lai, 2 ports of entry are created in the abdomen: 1 for the laparoscope to visualize the uterus and the other for passing the ai gun for insemination. equipment list for transcervical artificial insemination and laparoscopic insemination in small ruminants is provided.118 the procedure is relatively quick once ewes are ready for ai. the procedure can take < 1 minute to a few minutes per animal, depending on experience. it is advisable to have several working groups: technicians to help sedate ewes, load them into the cradle and prepare ewes for lai and monitor them; technician to thaw and evaluate semen; personnel to perform ai; and a group to monitor recovery. sedation can be accomplished through a single intravenous injection of an anesthetic combination. a mixture of 1,000 mg of ketamine, 100 mg of xylazine (α-2 adrenoceptor agonist, table 6), and 10 mg of butorphanol. a dose of 0.1 ml/20 lbs can sedate a small ruminant long enough to undergo the lai procedure while maintaining minimal side effects and short recovery times.119 intramuscular (0.05-0.3) or intravenous (0.016-0.1) xylazine alone or in combination with butorphanol (intravenous xylazine [0.1-0.2] + intravenous butorphanol [0.01-0.02]) or (intramuscular xylazine [0.02] + intramuscular butorphanol [0.05-0.07] are used.121,122 xylazine and other α-2 agonists have several dose-dependent adverse effects and require careful consideration.123 for instance, small ruminants are particularly sensitive to the pulmonary effects of these drugs. the primary cause of these pulmonary effects is the activation of pulmonary intravascular macrophages (pims). once activated, pims release prostaglandins and other vasoactive substances leading to alveolar edema, increased transpulmonary pressure, reduced pulmonary compliance, and pulmonary congestion. ketamine (1 mg/kg) combined with xylazine (0.05 mg/kg; 20 mg/ml) and butorphanol (0.025 mg/kg) are used to make a stock solution for the lai procedure.120 ewes are given intramuscularly 0.02 ml/kg. after 10 minutes, the sedated females are ready to stand alongside of an artificial insemination table and for rolling into dorsal recumbency. http://dx.doi.org/10.58292/ct.v17.11080 6 citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 ta b le 5 . e ff ec ts o f c o m b in at io n s o f v ar io u s p ro ge st er o n e an d g o n ad o tr o p in fo rm u la ti o n s o n e st ru s re sp o n se a n d p re gn an cy fo ll ow in g n at u ra l o r ar ti fi ci al in se m in at io n in e w es p ro ge st er o n e p 4 tr ea tm en t d u ra ti o n (d ay s) g o n ad o tr o p in s p ro st ag la n d in s se as o n b re ed in g ty p e er ( % ) p r ( % ) r ef er en ce fg a 4 0  m g 14 ec g 2 00 i u a t p 4 w it h d ra w al b re ed in g se as o n n at u ra l 1: 5 ra m :e w e 93 .3 60 .0 89 fg a 4 0  m g 5 ec g 2 00 i u a t p 4 w it h d ra w al + c lo p ro st en o l 1 00 m g at p 4 w it h d ra w al 86 .7 86 .7 fg a 4 0  m g 5 c lo p ro st en o l 1 00 m g o n d ay 0 + e c g 2 00 i u a t p 4 w it h d ra w al 92 .3 92 .3 fg a 4 0  m g 5 c lo p ro st en o l 1 00 m g o n d ay 0 + g n r h 1 00 m g 30 h o u rs af te r p 4 w it h d ra w al 66 .6 33 .3 g n r h 1 00 m g o n d ay 0 + c lo p ro st en o l 1 00 m g o n d ay 5 + 20 0 iu o n d ay 5 91 .7 66 .7 fg a 4 0  m g 5 c lo p ro st en o l 1 00 m g o n d ay 5 a t p 4 w it h d ra w al b re ed in g se as o n ta i (+ 5 2 h o u rs o r + 60 h o u rs a ft er p 4 w it h d ra w al ) 52 h o u rs : 5 0. 0 60 h o u rs : 3 3. 3 89 fg a 4 0  m g 5 c lo p ro st en o l 1 00 m g o n d ay 5 a t p 4 w it h d ra w al + g n r h (1 00 m g gi ve n 3 0 h o u rs a ft er p 4 w it h d ra w al 52 h o u rs : 4 0. 0 60 h o u rs : 2 0. 0 fg a 4 0  m g 5 c lo p ro st en o l 1 00 m g o n d ay 5 + e c g 2 00 i u o n d ay 5 a t p 4 w it h d ra w al + g n r h ( 10 0 m g gi ve n 3 0 h o u rs a ft er p 4 w it h d ra w al 52 h o u rs : 2 0. 0 60 h o u rs : 6 0. 0 c id r 0 .3  g 14 ec g 6 00 i u a t p 4 w it h d ra w al b re ed in g se as o n tl a i 96 .9 77 .8 73 fg a 3 0  m g 14 ec g 6 00 i u a t p 4 w it h d ra w al 95 .7 62 .4 m a p 6 0  m g 12 c o n tr o l o u t o f b re ed in g se as o n n at u ra l 1: 6 ra m :e w e  5 6 88 m a p 6 0  m g 12 ec g 5 00 i u a t p 4 w it h d ra w al  8 8 c id r 0 .3 g 14 ec g 4 00 i u a t p 4 w it h d ra w al o u t o f b re ed in g se as o n tr an sc er vi ca l a i 10 0. 0 75 .0 90 g n r h 4 00 m g at p 4 w it h d ra w al 10 0. 0 75 .0 g n r h 5 00 m g at p 4 w it h d ra w al 10 0. 0 70 .0 g n r h 6 00 m g at p 4 w it h d ra w al 10 0. 0 75 .0 h c g 2 00 i u a t p 4 w it h d ra w al 10 0. 0 75 .0 h c g 3 00 i u a t p 4 w it h d ra w al 10 0. 0 80 .0 h c g 4 00 i u a t p 4 w it h d ra w al 10 0. 0 75 .0 c id r 0 .3 g 9 c lo p ro st en o l 1 20 m g o n d ay 7 + p g 60 0 5 m l b re ed in g se as o n n at u ra l m at in g 3: 16 – r am :e w e 77 .0 91 c lo p ro st en o l 1 20 m g o n d ay 7 + p g 60 0 1. 5 m l 87 .5 c lo p ro st en o l 1 20 m g o n d ay 7 o n ly 37 .5 (c on ti nu ed ) http://dx.doi.org/10.58292/ct.v17.11080 citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 7 ta b le 5 . ( c on ti nu ed ) p ro ge st er o n e p 4 tr ea tm en t d u ra ti o n (d ay s) g o n ad o tr o p in s p ro st ag la n d in s se as o n b re ed in g ty p e er ( % ) p r ( % ) r ef er en ce c id r 0 .3 g 5 3 m l p g 60 0 (2 40 i u e c g , 1 20 i u h c g ) at p 4 w it h d ra w al a n es tr u s n at u ra l m at in g 1: 25 – r am :e w e 79 .2 71 .9 92 c o n tr o l 94 .2 66 .7 c id r 0 .3 g 5 3 m l p g 60 0 (2 40 i u e c g , 1 20 i u h c g ) 1 d ay b ef o re p 4 w it h d ra w al 79 .6 51 .7 c o n tr o l 84 .3 59 .9 fg a 4 5  m g 11 0. 1 m g o f p g f 2α o n d ay + 6 μ g o f in tr am u sc u la r g n r h 3 6 h o u rs af te r sp o n ge w it h d ra w al b re ed in g c er vi ca l a i 70 .0 71 .4 54 11 33 0 iu a n d 0 .1 m g o f p g f 2α d ay 9 83 .3 80 .0 11 33 0 iu a n d 0 .1 m g o f p g f 2α o n d ay 9 + 6 μ g o f in tr am u sc u la r g n r h 3 6 h o u r af te r sp o n ge w it h d ra w al 86 .7 80 .8 13 0. 1 m g o f p g f 2α o n d ay 1 2 + 30 0 iu a t sp o n ge w it h d ra w al 80 .0 75 .0 13 30 0 iu a t sp o n ge w it h d ra w al 76 .7 73 .9 1 m a p 6 0  m g 14 ec g 3 00 i u a t p 4 w it h d ra w al n o n b re ed in g n at u ra l 80 83 .3 11 2 m a p 6 0  m g 14 ec g 6 00 i u a t p 4 w it h d ra w al 80 10 0 c o n tr o l 10 0 m a p 6 0  m g 6 ec g 3 00 i u + 75 μ g d -c lo p ro st en o l, 1 d ay b ef o re p 4 w it h d ra w al b re ed in g n at u ra l 72 .7 45 .5 11 3 9 ec g 3 00 i u + 75 μ g d -c lo p ro st en o l, 1 d ay b ef o re p 4 w it h d ra w al 72 .7 36 .4 12 ec g 3 00 i u + 75 μ g d -c lo p ro st en o l, 1 d ay b ef o re p 4 w it h d ra w al 80 20 fg a 4 0  m g 12 ec g 1 40 i u 1 d ay b ef o re p 4 w it h d ra w al n o n b re ed in g n at u ra l 10 0 70 .0 11 4 fg a 4 0  m g 12 ec g 2 80 i u 1 d ay b ef o re p 4 w it h d ra w al 10 0 73 .7 fg a 4 0  m g 10 c o n b re ed in g n at u ra l 89 62 .5 a 10 3 fg a 4 0  m g 10 ec g 1 00 i u 92 .7 58 .8 a fg a 4 0  m g 10 ec g 2 00 i u 95 .8 93 .7 b fg a 4 0  m g 10 ec g 4 00 i u 91 .7 10 0b fg a 4 0  m g 14 ec g 5 00 i u b re ed in g a i 10 0 90 .6 b c 11 5 fg a 4 0  m g 14 ec g 6 00 i u 10 0 93 .7 b fg a 4 0  m g 14 ec g 7 50 i u 10 0 10 0a c o n tr o l 97 .1 79 .4 c fg a 4 0  m g 12 ec g 3 00 i u b re ed in g n at u ra l 64 36 11 6 ec g 4 50 i u 77 .7 19 .3 ec g 6 00 i u 86 .3 36 ec g 7 50 i u 80 .9 19 c id r : c o n tr o ll ed in te rn al d ru g re le as e; f g a : fl u o ro ge st o n e ac et at e; m a p : m ed ro xy p ro ge st er o n e ac et at e; e r % : e st ru s re sp o n se r at e; p r % : p re gn an cy r at e; a i: a rt ifi ci al in se m in at io n ; t a i: t im ed a rt ifi ci al in se m in at io n ; t la i: t im ed la p ar o sc o p ic a rt ifi ci al in se m in at io n ; t re at m en ts w it h o u t co m m o n s u p er sc ri p ts d if fe re d ( p < 0 .0 5) . http://dx.doi.org/10.58292/ct.v17.11080 8 citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 intramuscular (0.05-0.2 mg/kg) or intravenous (0.02-0.05 mg/kg) acepromazine is the most used agent in this group. compared to xylazine, it has a delayed onset of action. the maximum effect after intravenous treatment may take 15-20 minutes, and its duration of action lasts 4-6 hours. although acepromazine has minimal respiratory effects, it may cause arterial hypotension, especially at higher doses or in hypovolemic animals. furthermore, acepromazine lacks analgesic properties, and its sedative effect is weaker than xylazine. acepromazine is most effective when used in combination with other drugs. benzodiazepines (intravenous diazepam: 0.5-1.0 mg/kg given slowly, and intravenous, subcutaneous, or intramuscular midazolam 0.3-0.5 mg/kg) have minimal effects on the cardiovascular and respiratory systems;124 used for sedation in small ruminants as the sole drug, but they are preferred in combination with other agents.125 once ewes are sedated and loaded onto the cradle on dorsal recumbency, the posterior ventral abdomen (30 × 30 cm area cranial to the udder) is surgically prepared (clipped and scrubbed with chlorhexidineand sterile saline-soaked gauze). once the site is sterilized, injection of local anesthetic (2% lidocaine) is given 5 inches cranial to the teats and 2 inches off the midline on each side with care to avoid large veins. after a few minutes (for the local anesthetic to take effect), the cradle is lifted to a 40 degree angle, with the abdomen at a higher elevation than the head (figure 2). a small incision through the skin (but not the abdominal wall) is made in each of the spots where the local anesthetic was injected. a trocar is carefully passed through the abdominal wall along with cannula and removed to insufflate the abdomen with co2 before passing a second trocar through the second incision. the trocar is removed from the second cannula and a laparoscope is passed through 1 canula while forceps is passed through the other (figure 2). once the uterus is visualized through the laparoscope, it is manipulated into position for insemination with forceps. once the uterus is secured in a desirable position, the forceps is withdrawn and replaced with the insemination gun loaded with semen. the insemination gun is used to puncture and deposit semen in both uterine horns at the midway point between the utero-tubal junction and the base of the horn. once this is accomplished, equipment is withdrawn from the abdomen, abdomen deflated, and incisions are closed with staples, sutures, or tissue glue; however, sutures and staples are avoided.37 ewes should be allowed to recover in a quiet pen while monitored for adverse effects. in general, ewes readily walk away. intravenous yohimbine (0.1-1.0 mg/kg) is the antidote for xylazine to reverse effects. intramuscular or intravenous atipamezole may also be used for reversal (0.1-0.2 mg/kg). diazepam and midazolam are reversed with intramuscular or intravenous flumazenil (0.02 mg/kg).126 estimated input cost for laparoscopic artificial insemination service purchasing the reusable equipment to perform lai is an investment upfront but can quickly turn into profit. to perform lai, it is necessary to have the equipment to enter the abdomen, visualize the uterus and deposit the semen in the table 6. α-2 adrenoceptor agonist and doses120 drug treatment route dose dexmedetomidine (μg/kg) intravenous intramuscular 5-10 10-30 detomidine (μg/kg) intravenous intramuscular 3-20 20-30 romifidine (μg/kg) intravenous intramuscular 3-40 40-80 xylazine intravenous intramuscular 0.016-0.1 0.05-0.3 figure 2. laparoscopic artificial insemination procedure in an ewe (courtesy of dr. salman waqas) http://dx.doi.org/10.58292/ct.v17.11080 citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 9 lumen of the uterus. although 5.0, 6.5, 7.0, 7.5, and 10 mm laparoscopes are available, 5 mm is a popular choice among veterinarians and will be the size of equipment mentioned unless otherwise mentioned. purchasing refurbished or used medical equipment has been a popular choice and its price is used in our cost analysis. necessary reusable equipment includes, laparoscope, trocars, cannulas, a co2 tank, scope, light source, forceps, a laparoscopy cradle, lai gun, and semen processing equipment. laparoscope with light source and forceps: cost of a laparoscope can vary depending on whether it provides digital imaging or a lens, degree of offset, and type of light source; new $7,000 or used for $2,500. a 0-degree scope with a lens is ~ $250. a used halogen light source with a fiber optic cable is ~ $1,000 or a portable handheld endoscope light source (~ $150) may be more suitable for field use. laparoscopic forceps is required to manipulate the uterus or omentum before insemination and can be purchased for ~ $100. trocar and cannulas: available in 3 sizes: 5, 7.5, and 10 mm. it is desirable to have smaller size cannulas as it allows for smaller incisions and smaller rigid 5 mm laparoscopes, require no trocar adaptors for ai guns, carry reduced risks of abdominal perforation and prolonged lifespan due to lesser wear and tear. trocars and cannulas with a port are required to enter and insufflate the abdomen; 2 are needed as 1 will be for the scope and the other for the forceps to manipulate the uterus or insemination pipette. a set of these can be ~ $500 for a new set or $250 for a used set. co2 tank: is required to insufflate the abdomen. insufflating the abdomen allows for more space to manipulate and visualize the uterus. a medical grade insufflation air unit or a generic tank and regulator is used. medical grade unit, co2 tank with regulator, and a commercial vacuum pump can be purchased for $8,750. a used tank and regulator with a hose can be purchased for ~ $300. cradle: is needed to hold the sheep in dorsal recumbency at an incline during the procedure. a new cradle can be purchased for ~ $1,800. used units are not always readily available but can sometimes be purchased for < $1,000. although 2 cradles are ideal, only 1 cradle was considered for economic analysis. an lai gun for use with robertson insemination pipette, sheaths and semen straws (minitube usa, inc, verona, wi, usa) can be purchased as a new set for $400 or conversely a less costly robertson insemination pipette with a syringe can be used for semen deposition. other available options include laparoscopic sheaths (biogenesis, bloomington, il, usa) for universal small ruminant ai gun and ovine transcap guide with aspic sheath for mini straws (imv technologies, 61300 l’aigle, france). semen processing equipment can include a microscope, a slide warmer, semen straw cutters, and a semen thawing unit. a generic microscope can be purchased for ~ $300, or a semen specific microscope for ~ $2,500. a slide warmer can be purchased for ~ $200. semen straw cutters are relatively inexpensive at $15 each. a semen thawing unit can be purchased for ~ $200. cost of new and refurbished laparoscopic ai equipment is provided (table 7). costs associated with training (such as course fee, travel, hotel, etc.) and practicing on smaller flocks to gain proficiency are key factors in calculating breakeven costs. however, these factors are not included in this economic analysis because training costs and the time required to train an individual can vary substantially. in general, a set of refurbished equipment can be purchased for ~ $4,000. fiber optic light sources and scopes with digital image abilities will increase the cost and are often not necessary. buying new equipment or job-specific equipment can also increase the cost but may increase the lifespan of the equipment. many companies sell lai kits (e.g. ‘sheep lai set’ from medit) with fiber optic light source, 0-degree scope, and 2 trocars and cannulas for $2,300. these can be a viable choice for veterinarians who like to upgrade their kit or purchase new equipment guaranteed to be compatible. consumables are also needed to perform lai. consumables can include insemination pipettes and semen. robertson insemination pipettes can be purchased in bundles and the cost is ~ $20 or less per pipette. semen cost is variable, depending on the breed and source. breakeven cost assessment (refurbished equipment procurement with laparoscopic artificial insemination service) breakeven point (table 8) at which lai service would become profitable was determined using current market prices, with the following assumptions. veterinarian made only 1 trip to inseminate required number of ewes over a 3-hour period, ~ 25 ewes/hour. expenses used/refurbished equipment cost $4,000.00 technician $18.00/hour trailer rental $15.00/hour needle/syringe/blade/glue $3.00/ewe sedation $0.50/ewe synchronization drug cost $19.00/ewe (cidr, pg600 and pgf2α) table 7. cost of new and refurbished laparoscopic ai equipment equipment new ($) used/refurbished ($) laparoscopy 7,000 2,500 light source 7,000 1,400 trocar/canula 500 250 video camera and screen 3,500 air insufflation unit 8,500 300 cradle 1,800 1,000 lai gun 500-550 200 semen processing 3,000 715 http://dx.doi.org/10.58292/ct.v17.11080 10 citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 veterinary service revenue lai service charge $80.00/ewe farm call $75.00/trip performing lai in 71 ewes would breakeven the expenses needed to procure the refurbished laparoscopic equipment. economic advantages of laparoscopic ai over natural and transcervical ai economic advantage of lai over tcai and natural service was determined using a crude economic analysis (table 9a, 9b, 9c) with following assumptions. assumptions used for laparoscopic artificial insemination: lai was performed in 10 ewes with 70% pregnancy rate and 1.6 lambs/ewe (show-quality lambs) ram lamb: ewe lamb ratio 50:50 cost for frozen semen $ 100/insemination dose feeding lambs to 90 lbs $ 52.50/lamb technician $ 18.00/hour trailer rental $ 15.00 needle/syringe/blade/glue $ 3.00/ewe sedation $ 0.50/ewe synchronization drug cost $ 19.00/ewe (cidr, pg600 and pgf2α) market price for ram lamb $1,100/lamb market price for ewe lamb $1,000/lamb assumption used for transcervical artificial insemination: tcai was performed in 10 ewes with 25% pregnancy rate and 1.6 lambs/ewe (show quality lambs) ram lamb: ewe lamb ratio 50:50 cost for frozen semen $100/insemination dose feeding lambs to 90 lbs $ 52.50 technician $ 18.00/hour trailer rental $ 15.00 needle/syringe/blade/glue $ 3.00/ewe sedation $ 0.50/ewe synchronization drug cost $ 19.00/ewe (cidr, pg600 and pgf2α) market price for ram lamb $1100 market price for ewe lamb $1000 assumptions for natural breeding: natural breeding was performed in 10 ewes with 66% pregnancy rate and 1.6 lambs/ewe (show quality lambs) ram lamb: ewe lamb ratio 50:50 ram value $7500.00 feeding lambs to 90 lbs $52.50/lamb feeding ram for a year $700.00/ram synchronization drug cost $19.00/ewe (cidr, pg600 and pgf2α) market price for ram lamb $600/lamb market price for ewe lamb $600/lamb breakeven point at which lai services become profitable was determined using current market prices (table 8); analysis assumed that veterinarian made only 1 trip and inseminated 71 sheep in 3 hours. cost to benefit for lai, tcai, and natural breeding from a producer’s standpoint are compared (tables 9a-c). though natural service and transcervical ai breeding programs produced ~ $2,000 profit, laparoscopic ai produced ~ an additional $6,800 monetary benefit. in addition to monetary benefits, lai has several others. although there is a higher overhead cost associated with lai over more conventional ai methods, it is still a table 8. economic analysis of breakeven (expense and profit) cost associated with procurement of refurbished laparoscope and laparoscopic artificial insemination service itemized list price ($) per quantity cost ($) revenue ($) expense: laparoscope 4,000.00 1 4,000.00 technicians 18.00/hour; 3 hours 2 108.00 trailer rental 15.00/hour; 3 hours 1 45.00 consumables/ewe needle/syringe/blade/glue 3.00 71 413.00 sedation (60 kg ewe) 0.50 71 35.50 synchronization (cidr-s, pg600, pgf2α) 19.00 71 1349.00 total expenses: 5,750.50 charges lai 80.00 71 5,680.00 farm call 75.00 1 $75.00 total revenue 5,755.00 http://dx.doi.org/10.58292/ct.v17.11080 citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 11 practical option for many veterinarians. the technical skills and equipment required for lai are more substantial than tcai but the benefits of lai include bypassing the cervix, access to a deeper site of semen deposition, more efficient use of sperm, and higher conception rates translating to added value service delivered to clients. frozen semen dose for lai is 20-25 × 106 sperm (300-400 × 106 and 150-200 × 106 for transvaginal and transcervical ai, respectively). sexed semen option is available for sheep and is worth considering. however, it should be based on the producer’s goals; it is vital to perform a cost benefit analysis to assess the value of sexed semen in each specific scenario. conclusion laparoscopic artificial insemination is a highly effective and proven method of artificial insemination that has several advantages over traditional insemination techniques. one of the primary benefits of lai is its precision and ability to achieve higher success rates that translates into improved reproductive outcomes for livestock producers. although there is an initial investment required to purchase the specialized equipment, the costs are relatively modest when table 9a. economic analysis for profit estimation generated after implementing laparoscopic artificial insemination procedure (expenses and revenues considered, based on the assumptions listed*) itemized list price ($) per quantity revenue ($) cost ($) profit ($) expenses: farm call 75.00 1 trip 75.00 lai service charge 80.00 10 ewes 800.00 cost of frozen semen 100.00 10 doses 1,000.00 synchronization drug 19.00 10 ewes 190.00 needle/syringe/blade/glue 3.00 10 30.00 sedation 0.50 10 5.00 trailer rental 15.00 1 15.00 technician 18.00/hour 1 18.00 feeding lambs to 90 lbs (52.50) 11* 577.50 *total lambs (pregnancy rate 70%; 1.6 lambs/ewe) 11.2 profit: sale of ram lambs 1,100.00 5 5,500.00 sale of ewe lambs 1,000.00 6 6,000.00 total 11,500.00 2,710.50 8,789.50 table 9b. economic analysis for profit estimation generated after implementing transcervical artificial insemination procedure (expenses and revenues considered, based on the assumptions listed*) itemized list price ($) per quantity revenue ($) cost ($) profit ($) expenses: farm call 75.00 1 trip 75.00 tai service charge 60.00 10 ewes 600.00 cost of frozen semen 100.00 10 doses 1,000.00 synchronization drug 19.00 10 ewes 190.00 needle/syringe/blade/glue 3.00 10 30.00 sedation 0.50 10 5.00 trailer rental 15.00 1 15.00 technician 18.00/hour 1 18.00 feeding lambs to 90 lbs (52.50) 4* 210.00 *total lambs (pregnancy rate 25%; 1.6 lambs/ewe) 4 profit: sale of ram lambs 1,100.00 2 5,500.00 sale of ewe lambs 1,000.00 2 6,000.00 total 4,200.00 2,143.00 2,057.00 http://dx.doi.org/10.58292/ct.v17.11080 12 citation: clinical theriogenology 2025, 17, 11080, http://dx.doi.org/10.58292/ct.v17.11080 compared to the long-term advantages. additionally, the overhead costs associated with this service are minimal, making it an economically viable option for veterinary practices. for practitioners who already have a sufficient client base, the equipment expenses can often be recouped within just 1 breeding season, providing a strong return on investment. however, several factors must be carefully considered to ensure the success of artificial insemination in sheep. these include the quality of the semen used, proper storage conditions, animals’ fertility level, effective management practices, and strict adherence to scheduling protocols. additionally, producer compliance with recommended procedures and the timing of insemination relative to the breeding season are all critical to achieving the desired outcomes. it is essential that these factors are monitored and controlled to minimize risks and optimize results. any variation or oversight in these areas can significantly impact the success rates of the procedure. the economic benefits of laparoscopic artificial insemination are substantial, especially to veterinarians who can provide this service to a broader client base. by increasing conception rates and reducing the need for costly repeated matings, lai can ultimately save producers money while also contributing to higher overall productivity. for veterinary practices, adding lai procedures provides a valuable opportunity to differentiate themselves in a competitive market. effectively communicating these benefits to clients is crucial, as it helps to underscore the advantages of the service and fosters trust between practitioners and producers. lai not only enhances the economic viability of a practice but also adds a layer of expertise and specialization, further enriching the practice’s reputation and service provided. conflict of interest none to report. author contribution authors equally contributed to this work. references 1. davis tc, white rr: breeding animals to feed people: the many roles of animal reproduction in ensuring global food security. theriogenology 2020;150:27-33. doi: 10.1016/j.theriogenology. 2020.01.041 2. foote rh: the history of artificial insemination: selected notes and notables. j anim sci 2002;80:1-10. doi: 10.2527/animalsc i2002.80e-suppl_21a 3. ivanoff ei: on the use of artificial insemination for zootechnical purposes in russia. j agric sci 1922;12:244-256. doi: 10.1017/ s002185960000530x 4. spanner ea, de graaf sp, rickard jp: factors affecting the success of laparoscopic artificial insemination in sheep. anim reprod sci 2024;264:107453. doi: 10.1016/j.anireprosci.2024.107453 5. killen id, caffery gj: uterine insemination of ewes with the aid of a laparoscope. aust vet j 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current therapy in large animal theriogenology. 2nd edition, st. louis, mo; saunders: 2007. p. 629-641. table 9c. economic analysis for profit estimation generated after implementing natural service breeding program (expenses and revenues considered, based on the assumptions listed*) itemized list price ($) per quantity revenue ($) cost ($) profit ($) expenses: cost of the ram 7,500.00 1 7,500.00 feeding ram for 1 year 700.00 1 700.00 ram bse 45.00 1 45.00 synchronization drug 19.00 10 ewes 190.00 feeding lambs to 90 lbs (52.50) 11* 577.50 *total lambs (66% pregnancy rate; 1.6 lambs/ewe) 11 profit: ram lambs 600.00 5 3,000.00 ewe lambs 500.00 6 3,000.00 sale price of ram after 1 year 5,000.00 1 5,000.00 total 11,000.00 (9,012.50) 1,987.50 http://dx.doi.org/10.58292/ct.v17.11080 https://doi.org/10.1016/j.theriogenology.2020.01.041 https://doi.org/10.1016/j.theriogenology.2020.01.041 https://doi.org/10.2527/animalsci2002.80e-suppl_21a https://doi.org/10.2527/animalsci2002.80e-suppl_21a 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2017: squamous cell carcinoma of the vulva and perineum in a katahdin ewe   squamous cell carcinoma of the vulva and perineum in a katahdin ewe jessica klabnik-bradford,a* sierra r. guynn,a sherrie clark-deener,a geoffrey saundersb adepartment of large animal clinical sciences; and bdepartment of biomedical sciences and pathobiology, virginia-maryland college of veterinary medicine, blacksburg, va summary an 8 year-old, 12 weeks pregnant, white katahdin ewe was presented for a malodor associated with a vulvar lesion. physical examination was unremarkable with the exception of an ulcerated, necrotic lesion involving the entire vulva and a small part of the surrounding perineum. squamous cell carcinoma was suspected. eight weeks after initial presentation the ewe was again presented for dystocia due to malposition of the first lamb. one deceased and two live lambs were delivered with vaginal assistance. in the four days following the dystocia the dam was lethargic, anorexic, and did not properly care for her lambs. with the visible progression of the lesion and suspected poor prognosis for future reproduction the owner decided to humanely euthanize the ewe. necropsy confirmed squamous cell carcinoma on histopathology. bacterial pneumonia was considered to be the cause of the ewe’s clinical signs at the time of euthanasia based on lung pathology and culture. no squamous cell carcinoma metastases were found. keywords: squamous cell carcinoma, vulva, perineum, ewe background in sheep, squamous cell carcinoma is an uncommon lesion typically found on the ears and muzzle, but is well documented on the vuvla, perineum, and tail. to date, squamous cell carcinoma has only been described in wooled-breeds. predisposing factors include tail docking, mules operation, solar exposure, branding, and papilloma virus infection. rate of metastasis of squamous cell carcinoma appears to be relatively low. very limited information about the reproductive prognosis for vulvar squamous cell carcinoma diagnosed during pregnancy is published. to the authors’ knowledge, this is the first report of squamous cell carcinoma in a haired sheep and the first report regarding pregnancy maintenance after presentation. although this ewe did not have the predisposing management factors described in the literature, she was pink-skinned and kept in a poorly-shaded pasture. case presentation an 8 year-old white katahdin ewe was presented to the production management medicine service at the virginia-maryland college of veterinary medicine in february for a malodor associated with a vulvar lesion. the owner had noticed a mass on her vulva prior to presentation but could not recall when the mass first appeared. six weeks prior to presentation the ewe had been confirmed 40 to 45 days pregnant via abdominal ultrasound and a mass was not noticed at that time. the number of fetuses was not counted. upon presentation, the physical findings were within normal limits and the ewe was approximately 85 days pregnant. the ewe’s famacha score was 2/5 (1= bright pink/ideal; 5 = very pale.) an ulcerated, necrotic lesion was found involving the entire vulva and a small part of the surrounding perineum with thickening of the affected integument (figure 1.) the lesion had a purulent discharge and an associated malodor. the lesion was cleaned with chlorhexidine solution. differential diagnosis the most likely diagnoses for this vulvar/perineal lesion was squamous cell carcinoma with differential diagnoses including hyperkeratosis, papilloma, other integumentary neoplasia, or infected laceration/trauma.                                                              * current address: department of large animal clinical sciences, college of veterinary medicine, university of tennessee-knoxville, knoxville, tn clinical theriogenology • volume 9 number 1 • march 201791   treatment biopsy was recommended to confirm a diagnosis. the ewe was intended as a production animal and it was expected that she would be rejected from an abattoir with such a lesion. therefore, the owner declined the biopsy with the intention of humanely euthanizing the ewe after her lambs were weaned. at discharge (approximately 85 days pregnant), the owner was instructed to clean the lesion at least weekly with chlorhexidine gluconate and monitor the ewe for change in attitude, change in appetite, and abortion. additionally, the owner moved the ewe from the poorly shaded pasture into the barn where she could be monitored more closely. two weeks after presentation (approximately 99 days pregnant), maggots were found throughout the lesion. the recommendation to apply permethrin (catron ® iv, bayer, shawnee mission, ks) topically and was performed daily by the owner for two weeks and then every three days until parturition. due to the progression of the lesion over those two weeks and the value of the pregnancy, the owner requested a second pregnancy ultrasound evaluation. the ewe was reconfirmed pregnant via abdominal ultrasound the following week (three weeks after initial presentation; approximately 106 days pregnant). outcome nearly eight weeks after initial presentation (approximately 141 days gestation), the ewe presented for dystocia. the first lamb was in posterior presentation, dorsal-sacral position, with left hip and right hock flexion. that lamb was dead upon assisted vaginal delivery. the second and third lambs were in a normal presentation, position, and posture and were delivered vaginally with minimal assistance. the second and third lambs were both alive upon delivery. the live lambs were fed 60 ml of the dam’s colostrums by orogastric intubation. the dam was not treated at that time but it was noted that she was not attentive to the lambs. the day after parturition the owner reported the ewe was anorexic and lethargic. the owner administered electrolytes (163.4g, hydra-lyte, agrilabs, st. joseph, mo) and nutritional drench (150g, aas drench mix with extra calcium, advanced agri solutions llc, lancaster county, pa) via an orogastric tube. four-days after parturition the ewe remained lethargic and anorexic. she had not been producing enough milk for two lambs so the lambs were bottle fed milk replacer. the vulvar lesion had progressed to involve more of the perineum and the ventral aspect of the anus (figure 2.) due to grave prognosis for future reproduction and a poor prognosis for being able to raise the lambs on her own, euthanasia was elected (15ml iv, fatal-plus, vortech pharmaceuticals ltd, dearborn, mi) and a necropsy was performed. at necropsy the vulvar and perineal lesion measured roughly 12 cm x 7 cm and was confirmed to be a squamous cell carcinoma by histopathology (figure 3.) the endometrium was grossly ulcerated and on histopathologic examination was found to be covered with a layer of neutrophils; no squamous cell carcinoma was seen in the uterus (figure 4.) local lymph nodes did not contain histopathologic evidence of metastasis. lung pathology included cranioventral consolidation with 40% of the left lung and 20% of the right lung being affected (figure 5.) aerobic culture of the lung lesions resulted in isolation of bibersteinia trehalosi, pasteurella multocida, and trueperella pyogenes. all three bacteria are considered pathologic in sheep and were considered to be the cause of the ewe’s clinical signs at the time of euthanasia. no squamous cell carcinoma metastases were found and pneumonia was confirmed by histopathology. although the ewe was euthanized for a poor productive prognosis, it is unknown if treating the pneumonia would have allowed her to raise the lambs on her own. at two months of age both lambs were thriving. discussion squamous cell carcinoma in sheep is considered an uncommon occurrence, with reported prevalence of 0.5-3%. in certain instances, outbreaks have been reported in which the prevalence increased up to 18%.1 in outbreak situations, it has been hypothesized that photosensitizing agents or carcinogens have contributed to the increased prevalence; however, no evidence has been found to support those theories.2,3 clinical theriogenology • volume 9 number 1 • march 2017 92   the majority of squamous cell carcinoma cases are in merino or merino-cross ewes.2-4 one author reported squamous cell carcinoma in two merino wethers.4 other breeds in which squamous cell tumors have been reported include awassi,5 berrichon du cher,1 and khorasan-kordi.6 all of these breeds are wooled breeds. in this report squamous cell carcinoma was found in a katahdin ewe, a haired breed. increased risk of squamous cell carcinoma occurs with increased age, although it has been confirmed in sheep as young as three years of age.3 solar radiation is known to contribute to the development of epithelial neoplasia in many species. it has been linked to the development of basal cell and squamous cell carcinomas in humans, squamous cell carcinoma in goats, and epitheliomata of cats with white pinnae. pigmented eyelids in cattle result in decreased squamous cell carcinoma incidence. it is very plausible that sunlight contributes to the development of squamous cell carcinoma in sheep, as the most commonly affected areas are those exposed to sunlight.1,2 sheep undergoing mules’ operation appear to be at a greater risk of developing vulvar or perineal squamous cell carcinoma.2,3 the mules’ operation is the surgical removal of wooled skin around the perineum in order to stretch the woolless region to reduce myiasis and fecal matting. shorter tail docking also results in increased sunlight exposure to the perineal area and therefore may affect the incidence of squamous cell carcinoma in that area.2,3 in the current case, the ewe developed a perineal squamous cell carcinoma without having either the mules’ operation or her tail docked. papilloma viruses are well-documented to contribute to squamous cell neoplasias in humans and horses. a possible link has been found between ovis aries papillomavirus 3 and squamous cell carcinoma in sheep.7 in cattle, branding has been associated with papilloma and squamous cell carcinoma formation at the site of branding. heat branding may contribute to squamous cell carcinoma at that site in sheep.5 lesions are most common on the ears and muzzle2,4; although perineal, vulvar and tail lesions are also well documented.2-4 individual case reports have demonstrated lesions on the eyelids1 and on wooled-areas (thoracic2 and lumbar.6) in one study, metastasis was found in 12% of cases. metastatic sites included submandibular lymph nodes, parotid lymph nodes, prescapular lymph nodes, the parotid salivary gland,4 and lungs.2 several treatment options are available for squamous cell carcinomas in humans and sheep are considered an excellent model for human squamous cell carcinoma. chemotherapy studies in sheep have demonstrated reduction in tumor size and growth.8 sheep are a minor food production species and withdrawal times for chemotherapeutic agents have not been established. therefore, if treated, the sheep would have to be permanently removed from the food supply. the ability to accomplish permanent removal of a food animal from the food chain is controversial. learning points:  in sheep, squamous cell carcinoma is typically found on the ears and muzzle, but is well documented on the vuvla, perineum, and tail.  rate of metastasis of squamous cell carcinoma appears to be relatively low.  very limited information about the reproductive prognosis for vulvar squamous cell carcinoma diagnosed during pregnancy is published. references 1. mendez a, perez j, ruiz-villamor e, et al: clinicopathological study of an outbreak, of squamous cell carcinoma in sheep. vet rec 1997;141:597-600. 2. lloyd lc: epithelial tumors of the skin of sheep. br j cancer 1961;15:780-789. 3. vandegraaff r: squamous cell carcinoma of the vulva in merino sheep. aust vet j 1976;52:21-23. 4. ladds pw, entwistle kw: observations on squamous cell carcinomas of sheep in queensland, australia. br j cancer 1988;35:110-114. 5. veruham i, perl s, nyska a: skin tumours in cattle and sheep after freezeor heat-branding. j comp path 1996:114;101-106. 6. najarnezhad v, aslani mr: unusual case of cutaneous squamous cell carcinoma in a ewe. iranian j vet sci technol 2012;4:49-53. 7. alverti a, pirino s, pintore f, et al: ovis aries papillomavirus 3: a prototype of a novel genus in the family papillomaviridae associated with ovine squamous cell carcinoma. virology 2010;407:352-359. clinical theriogenology • volume 9 number 1 • march 201793   8. harker gj, stephens fo. comparison of intra-arterial versus intravenous 5-fluorouracil administration on epidermal squamous cell carcinoma in sheep. eur j cancer 1992;28: 1437-1441. figure 1. on initial presentation, an ulcerated, necrotic lesion involved the entire vulva and a small part of the perineum. figure 2. at the time of euthanasia, the vulvar lesion had progressed to involve more of the perineum and involve the ventral aspect of the anus. clinical theriogenology • volume 9 number 1 • march 2017 94   figure 3. the vulvar lesion was confirmed to be a squamous cell carcinoma by histopathology as seen in this image. figure 4. image of the katahdin ewe’s uterus with an incision made on the dorsal aspect of the right uterine horn and uterine body. on necropsy, the endometrium was found to be grossly ulcerated but no gross lesions associated with squamous cell carcinoma were found. histopathologic examination found the endometrium to be covered with a layer of neutrophils. figure 5. cranioventral consolidation was seen in 40% of the left lung and 20% of the right lung. (editor’s note: the photographs in this manuscript are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 9 number 1 • march 201795 clinical theriogenology • volume 9 number 1 • march 2017 96 omniblank: 2017 in vitro efficacy of anti-protozoal compounds as a novel treatment of tritrichomonas foetus in vitro efficacy of anti-protozoal compounds as a novel treatment of tritrichomonas foetus jennifer h. koziol,a* misty a. edmondson,a sue h. duran,a thomas passler,a julie a. gard,a kellye joiner,b dwight f. wolfea adepartments of clinical sciences and b pathobiology, college of veterinary medicine, auburn university, auburn, al abstract bovine trichomoniasis is a sexually transmitted disease caused by tritrichomonas foetus, an obligate parasite of the reproductive tract of cows and the surfaces of the penis and prepuce of bulls. with the ban of efficacious drugs for treatment of t. foetus such as the nitromidazoles in the united states, veterinarians are currently left without legal means of treatment. oxfendazole, oxibendazole, and ponazuril are anti-protozoal drugs that can be legally used in food animals. in vitro experiments were performed using pure cultures of t. foetus trophozoites in diamond’s medium with the addition of the individual drugs in replicate. cell cultures were evaluated at predetermined time points using neubauer hemocytometers to determine the number of viable trophozoites. cultures were examined for reversibility to the motile trophozoite form or continued replication of organisms following the administration of the drug. in our final study, complete kill of the organism was achieved and all replicates remained negative for reemergence of trophozoites following five days of culture. keywords: tritrichomonas foetus, bull, benzimidazole, ponazuril, treatment introduction tritrichomonas foetus, an extracellular flagellated protozoan, is the organism responsible for the sexually transmitted disease commonly known as bovine trichomoniasis. this organism is an obligate parasite of the female reproductive tract and surface of the male’s prepuce and penis in cattle.1 tritrichomonas foetus is a pleomorphic organism that can exist in both the trophozoite and pseudocyst form both of which can be transmitted during coitus.1-4 the bull, an asymptomatic carrier of the t. foetus organism, transmits the protozoan to the female during coitus with reports of a single mating with an infected bull inducing an infection in 95% of susceptible nulliparous cows.5 infected cows may experience vaginitis, pyometra, embryonic death, and abortion.6 the impact of decreased calf crops and prolonged calving seasons that occur as a sequela to the clinical infection in the cow can be quite substantial as infection rates amongst herds have been reported from 5-50% with variability based upon geographical location.7-10 speer and white speculated that a 5% calf loss due to trichomoniasis in us beef cattle herds would translate into 1,685,250 calves lost based on 1989 calf production as estimated by the usda.11 this equated to a $650 million annual loss to the industry. the usda has estimated the united states calf crop to be approximately 35.1 million in 2016.12 assuming a five percent loss of calves due to t. foetus infection at current market prices, the loss to the industry would approach 1.5 billion dollars. while most cows mount a short term immune response and eventually clear the infection for the year, the bull fails to establish an immune response to the infection allowing for the establishment of the chronic carrier status.13 this fact combined with the severe economic impact due to loss of calves along with the current lack of a legal effective treatment all contribute to the decision that bulls found positive for the disease are culled from the herd as part of disease management. this results in additional losses to the producer in the form of monetary expenditures of replacing the bull, and in some cases the loss of valuable livestock genetics. with the increasing value of cattle, the opportunity to treat bulls infected with t. foetus has once again become a priority for cattle producers as opposed to culling and replacing infected bulls. several treatments for trichomoniasis were used successfully in the past, including: bovoflavin salve® (farbwerke hoechst ag, frankfurt, germany), ipronidazole, and metronidazole.14 bovoflavin * current address: college of veterinary medicine, purdue university, west lafayette, in 539 clinical theriogenology • volume 9 number 4 • december 2017 salve® contained trypaflavine at a concentration of 0.5% and was applied to the penis and prepuce of infected bulls. when the salve was applied to 57 bulls, 55 bulls proved to be cured by examination two to four weeks following treatment.14 bartlett treated eight infected bulls with bovoflavin salve® and found that seven were cured by a single series of treatment.15 nitromidazoles were also reported to have similar successes when applied topically.16 unfortunately, bovoflavin salve® is no longer manufactured and nitromidazoles became illegal for use in food animals.17 the objective of this project was to investigate in vitro the use of novel therapeutic drugs for the treatment of t. foetus. extrapolating from the previous treatment successes of the use of topical treatments such as bovoflavin salve® and nitromidazoles, novel therapeutic dosage formulations were tested in vitro for their ability to kill or reduce the number of t. foetus organisms in culture. various formulations were tested using two types of antiprotozoals, ponazuril (po) and benzimidazoles including oxibendazole (ox) and oxfendazole (oxf). if found to be successful these antiprotozoal drugs would be utilized to form a novel chemotherapeutic that could be applied to the prepuce and penis of infected bulls in similar fashion to the successful bovoflavin salve ®. benzimidazoles were chosen as a potential treatment for t. foetus as they specifically affect the colchicine binding-site of �-tubulin monomers inhibiting microtubule assembly and disassembly.18,19 microtubules play a vital role in the cytoskeleton of the trichomonad. the pelta, flagella, spindle and basal bodies as well as the axostyle, the organelle responsible for cell division, are all formed from these structures.20-22 moreover, benzimidazoles can also act as a lipid-soluble proton ionophore, inducing direct and indirect biochemical changes including inhibition of glucose uptake, glycogen depletion, inhibition of the fumarate reductase system and uncoupling of electron transport-associated phosphorylation.23 the exact mechanism of action the triazine agents such as po is currently unknown. totrazuril sulfone, more commonly known as po, has been found to have clinical application in the treatment of neospora caninum in cattle and may have clinical application for treatment of other protozoal disease in cattle including t. foetus.24 materials and methods experimental design two in vitro experiments were performed using pure cultures of t. foetus trophozoites. in the initial study ox (anthelcide eq® zoetis, florham park, nj) or po (marquis® merial, duluth, ga) paste formulations were utilized. in the second study oxf powder (u.s. pharmacopeial convention rockville, md) was utilized. samples were evaluated at predetermined time points using neubauer hemocytometers to determine the number of viable, motile organisms. live trophozoites were determined as those protozoa exhibiting pear shaped bodies, externalized flagella and displaying motion. non-motile trophozoites were characterized by the presence of the pear shaped bodies with externalized flagella but lack of motion and were not counted. cultures were tested for reversibility to the motile trophozoite form or replication of organisms. parasites the cdtf3 strain of t. foetus, a pure culture cultivated at auburn university, was used in this study. the parasites were cultivated in trypticase–yeast extract-maltose diamond’s medium (dm),25 supplemented with ten percent heat-inactivated fetal bovine serum (atlanta biologics, norcross, ga) at 37�c for 24-48 hours until appropriate numbers of trichomonads were present to perform the required number of replications for the study. following culture, t. foetus organisms were washed in sterile phosphate buffered saline (pbs;vwr, radnor, pa) centrifuged at 4000g for ten minutes at room temperature and re-suspended in 1 ml of pbs and then inoculated into 15 ml sterile conical tubes (bd falcon, franklin lakes, nj) or tissue culture wells (sarstedt, inc., newton, nc) containing dm. statistics data were analyzed using statistical software package stata se v.14.2 (statacorp, college station, tx). significance of treatment group, time, and their interaction were assessed by a mixed 540clinical theriogenology • volume 9 number 4 • december 2017 effects model with treatment group as a fixed effect and replicate as a random effect within time. differences between groups and times were assessed by comparisons of least squares means with bonferroni adjustment for multiple comparisons. statistical significance was set a p<0.05. drug susceptibility and reversibility experiment i trophozoites (3.75 x 104 cells/ml) washed in pbs were inoculated into dm for a total of 10ml of dm in each tube. at time 0, each of the following treatments were added to culture tubes: 1) control: 0.5ml dm; 2) 75 mg po (0.5ml); 3) 50 mg ox (0.5ml). five replicates were completed for each treatment. following incubation at 37�c, samples were taken every two hours for a total of 12 hours, the tubes were vortexed prior to removal of a 20�l sample. from these samples, the surviving organisms were counted utilizing disposable neubauer hemocytometers (c-chip®, nanoentek inc., pleasanton, ca). to evaluate if the antiprotozoal induced the formation of the pseudocyst stage or non-motile trophozoites that could be reversed and the organism would return to a motile trophozoite, at 12 hours following treatment each formulation was centrifuged at 4000g for ten minutes at room temperature. the supernatant fluid was removed and the pellet was re-suspended in 10ml of fresh dm without drug and evaluated microscopically daily for five days for the presence of t. foetus organisms in culture. drug susceptibility and reversibility experiment ii trophozoites (3.75 x 104 cells/ml) were cultured as described above and added to tissue culture wells for a total of 3ml of dm in each well. at time zero, each of the following treatments was added to tissue culture wells: 1) control 4ml dm; 2) 150 mg ox (1ml) for a total of 4ml; 3) 450mg po (1ml) for a total of 4 ml. each treatment was applied to tissue culture wells in duplicate. following growth at 37�c on a rocker plate (hoefer inc., holliston, ma), samples were collected every 4 hours for a total of 24 hours. a 20�l sample was removed from each well, and the surviving organisms counted utilizing disposable neubauer hemocytometers. cultures were microscopically examined every four hours for a total of 24 hours for the presence of t. foetus organisms in culture by counting organisms as described in experiment i. at 24 hours after treatment and following counting of organisms, each formulation was removed from the tissue culture well and centrifuged at 4000g for ten minutes at room temperature. the supernatant fluid was removed and the pellet was re-suspended in 4ml of dm and placed once again in a tissue culture well before being re-incubated at 37�c. this process was repeated every 24 hours for five passages (120 hours) to allow for re-emergence of any organisms. results in experiment i, po and ox significantly inhibited parasite growth at concentrations of 75 mg ml-1 and 50 mg ml-1, respectively. the variables of treatment group, time, and their interaction were statistically significant (p<0.0001). counts for the control treatment were significantly greater than for po and ox treatment groups (p<0.0001) but counts for po and ox groups did not significantly differ (p=0.99). however, following re-culture at 12 hours and examination at 24 hours, reestablishment of motile organisms was observed. (figure). in experiment ii the concentration of benzimidazole was increased with the use of oxf solution at 150 mg ml-1. oxfendazole had substantial inhibitory effects on trichomonad growth in the culture following an incubation period of 4 hours and non-motile trophozoites were maintained for 24 hours and reversibility of organism to motile trophozoites was not demonstrated in continued cultures. once again the variables of treatment group, time, and their interaction were statistically significant (p<0.0001). cultures containing po could not be evaluated as there was limited visibility when viewed microscopically and therefore removed from the study. discussion in the present study, the in vitro vulnerability of bovine t. foetus to po, ox, and oxf was evaluated utilizing susceptibility and subsequent reversibility tests. the efficacy of each anti-protozoal medication was evaluated using a susceptibility technique. the time of sampling varied between the two 541 clinical theriogenology • volume 9 number 4 • december 2017 studies due to the fact that no significant difference was noted at evaluation between two hours and four hours in experiment i and between four and eight hours in experiment ii. in experiment i as hypothesized by the authors, ox and po in the form of approved drug formulations for horses of anthelcide eq® and marquis® respectively demonstrated an antiprotozoal effect on t. foetus, by severely inhibiting the growth of organisms in the culture. susceptibility tests determining growth inhibition and reversibility tests demonstrated statistically similar results between ox and po with ox causing a sharper decline in organisms as compared to po. in an attempt to induce a permanent reduction in organisms in experiment ii, oxf was substituted for ox to increase concentration of drug per culture without dilution in the oil-based suspension formulation. when the oxf solution was added to culture, no motile trophozoites were detected four hours after inoculation. following continued incubation, the culture continued to remain negative for motile trophozoites. as well, tissue culture wells and a rocker plate were utilized in this experiment to ensure that the drug continued to mix within the media and did not settle out of the suspension. in conclusion, the described experiments demonstrated that oxf solution inhibited the growth of the bovine strain cdtf3 of t. foetus in vitro and may offer a new treatment option. limitations of this study include not using video microscopy or transmission electron microscopy to further elucidate the effects of our novel chemotherapeutic on the morphology and ultrastructure of the t. foetus organism. acknowledgement this work was supported by theriogenology foundation; the authors thank them for their generous support. conflict of interest the authors have no conflicts of interest to report. references 1. roberts sj: veterinary obstetrics and gynecology:theriogenology. woodstock: published by author;1986. 2. pereira-neves a, benchimol m: tritrichomonas foetus: budding from multinucleated pseudocysts. protist 2009;160:536-551. 3. pereira-neves a, campero cm, martinez a, et al: identification of tritrichomonas foetus pseudocysts in fresh preputial secretion samples from bulls. vet parasitol 2011;175:1-8. 4. pereira-neves a, nascimento lf, benchimol m: cytotoxic effects exerted by tritrichomonas foetus pseudocysts. protist 2012;163:529-543. 5. parsonson im, clark bl, dufty jh: early pathogenesis and pathology of tritrichomonas foetus infection in virgin heifers. j comp pathol 1976;86:59-66. 6. rae do, crews je: tritrichomonas foetus. vet clin north am food anim pract 2006;22:595-611. 7. bondurant rh, anderson ml, blanchard p, et al: prevalence of trichomoniasis among california beef herds. j am vet med assoc 1990;196:1590-1593. 8. rae do, chenoweth pj, genho pc, et al: prevalence of tritrichomonas foetus in a bull population and effect on production in a large cow-calf enterprise. j am vet med assoc 1999;214:1051-1055. 9. rodning sp, wolfe df, carson rl, et al: prevalence of tritrichomonas foetus in several subpopulations of alabama beef bulls. theriogenology 2008;69:212-217. 10. rae do, crews je, greiner ec, et al: epidemiology of tritrichomonas foetus in beef bull populations in florida. theriogenology 2004;61:605-618. 11. speer ca, white mw: bovine trichomoniasis. better diagnostics and control could save beef industry $650 million annually. large animal veterinarian 1991;46:18-20. 12. service nas. cattle inventory. http://usda.mannlib.cornell.edu/mannusda/viewdocumentinfo.do?documentid=1017; 2017. 13. clark bl, parsonson im, dufty jh: experimental infection of bulls with tritrichomonas foetus. aust vet j 1974;50:189-91. 14. hammond d, fitzgerald p, binns w, et al: the efficacy of bovoflavin salve in the experimental treatment of trichomoniasis in bulls. cornell vet 1953;43:121. 15. bartlett d: further observations on experimental treatments of trichomonas foetus infection in bulls. am j vet res 1948;9:351-359. 542clinical theriogenology • volume 9 number 4 • december 2017 16. gasparini g, vaghi m, tardani a: treatment of bovine trichomoniasis with metronidazole (8823 rp). vet rec 1963;75:940. 17. davis jl, smith gw, baynes re, et al: update on drugs prohibited from extralabel use in food animals. j am vet med assoc 2009;235:528-534. 18. lacey e: the role of the cytoskeletal protein, tubulin, in the mode of action and mechanism of drug resistance to benzimidazoles. int j parasitol 1988;18:885-936. 19. meloni bp, thompson rc, reynoldson ja, et al: albendazole: a more effective antigiardial agent in vitro than metronidazole or tinidazole. trans r soc trop med hyg 1990;84:375-379. 20. benchimol m: new ultrastructural observations on the skeletal matrix of tritrichomonas foetus. parasit res 2005;97:408-416. 21. benchimol m: trichomonads under microscopy. microsc microanal 2004;10:528-550. 22. benchimol m, engelke f: hydrogenosome behavior during the cell cycle in tritrichomonas foetus. biol cell 2003;95:283-293. 23. mccracken ro, stillwell wh: a possible biochemical mode of action for benzimidazole anthelmintics. int j parasitol 1991;21:99-104. 24. dirikolu l, yohn r, garrett ef, et al: detection, quantifications and pharmacokinetics of toltrazuril sulfone (ponazuril®) in cattle. j vet pharmacol ther 2009;32:280-288. 25. diamond ls: the establishment of various trichomonads of animals and man in axenic cultures. j parasitol 1957;43:488-490. figure. averages for each culture were obtained by averaging the 5 replicates at the individual time points. both po and ox alone demonstrated a 98% reduction in motile organisms after two hours of culture. however, following re-culture at 12 hours and examination at 24 hours, reestablishment of motile organisms was observed. 0 5000 10000 15000 20000 25000 30000 2 4 6 8 10 12 24 m o ti le �c el ls /m l hours�after�inoculation� experiment�i control�(dm) po�avg ox�avg 543 clinical theriogenology • volume 9 number 4 • december 2017 2016: the use of artificial insemination gun protective plastic sheath at the time of artificial insemination did not improve fertility of beef cattle the use of artificial insemination gun protective plastic sheath at the time of artificial insemination did not improve fertility of beef cattle ramanathan kasimanickam department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa abstract a strict hygienic artificial insemination (ai) technique is critical to maximize reproductive outcomes. the objective of this study was to evaluate the effect of ai gun protective plastic sheaths use on ai pregnancy rates (ai-pr) in beef cattle. angus cross beef cows (n=321) and angus cross beef heifers (n=430) were randomly assigned to one of two groups: with (trt) or without (con) the use of disposable plastic sheath during the ai procedure. accounting for estrus expression at or prior to ai (p<0.01), the use of protective sheath neither in cows (sheath, 59.4% [95/160] vs. no sheath, 55.9% [90/161]; p>0.1] nor in heifers (sheath, 58.3 [127/218] vs. no sheath, 56.7% [121/212]; p>0.1) improved ai-pr. both cows and heifers that expressed estrus at or prior to ai had greater ai-pr compared to that did not express estrus (cows: estrus, 64.4% [116/180] vs. no estrus, 48.9 [69/141]; heifers: estrus, 64.6 [144/213] vs. no estrus, 50.2 [104/207]; p<0.01). in conclusion, carrying out a hygienic ai technique is critical for ai success; however, the use of protective sheath did not improve reproductive outcomes in beef cattle. keywords: beef cattle, artificial insemination, ai gun, protective sheath, pregnancy rate introduction artificial insemination was the first reproductive biotechnology applied to improve reproduction and genetics of farm animals.1 the utilization of ai has an enormous impact worldwide in many species, mainly among domestic livestock including cattle, sheep, goats, pigs, horses, poultry (turkeys, ducks, fowl) and rabbits. the worldwide acceptance of ai technology provided a thrust for the development of other assisted reproductive technologies, such as pharmacological regulation of estrous cycle, multiple ovulation and embryo transfer, in-vitro embryo production, sexing of sperm and embryo, cryopreservation of gametes and embryo, and somatic cell nuclear transfer and cloning.1,2 in cattle the safest and best method of insemination is the recto-vaginal method which involves traversing the anatomic barriers of the female reproductive tract, the cervix, and deposition of frozen-thawed semen in the body of the uterus under hygienic conditions. the efficiency of insemination depends on the deposition of proper numbers of normal spermatozoa at the appropriate site in the reproductive tract at the precise time relative to estrus. in addition, an adequate and clean ai technique is recommended to improve reproductive outcome.3 improper handling of instruments and unsanitary conditions may lead to lower fertility. cattle fertility is influenced by several factors including herd environment and management practices.4 it should be noted that during the ai procedure, not only semen but also bacteria and debris may be introduced into the lumen of the uterus. studies revealed that dairy cows that developed post-ai subclinical endometritis had lower conception rates.5 this could be due to either uterine reaction to sperm, existing infection, or introduction of pathogens from the vagina into the uterus. in beef cows endometrial cytology did not predict final pregnancy status or day of conception when samples were collected >50 d postpartum, indicating that beef cows were able to clear uterine inflammation after resumption of ovarian cyclicity.6 however, a recent study that followed beef cows for 130 days postpartum concluded that cows with subclinical endometritis had an increase in time to conception and a decrease in the risk of pregnancy.7 use of protective sheaths (double sheath insemination procedure) prevents introduction of vaginal contaminants including pathogenic organisms into the uterus via the ai gun. previous studies reported no improvement in first service conception rates3,8 and improved pregnancies per ai for second or greater clinical theriogenology • volume 8 number 2 • june 2016103 services in dairy cattle9 following the use of ai catheter protective sheaths during ai compared to no protective sheath use. use of protective sheaths incurs additional cost to beef producers whereas failure to use protective sheaths may lower pregnancy rate. this study was designed with an intent to provide a comprehensive recommendation on whether or not to use ai gun protective sheaths while performing ai in beef cattle. the objective of this study was to evaluate the effect of ai gun protective plastic sheaths (ps) use on ai pregnancies in beef cattle. materials and methods animals and breeding management cows. a total of 321 angus cross beef cows from three similarly managed commercial cow-calf operations inseminated during spring of 2014 were included. within locations cows were randomly assigned to ovsynch+ controlled internal drug release (cidr) or co-synch+cidr groups (figure 1a) and were assigned a body condition score (bcs; 1-emaciated; 9-obese) at initiation of synchronization protocol (day 0). cows in ovsynch+cidr group received a cidr (1.3 g of progesterone; per vagina; eazi-breed™ cidr® cattle insert; zoetis animal health, new york, ny) vaginal insert and 100 µg gonadorelin diacetate tetrahydrate (gnrh; 2 ml; im; cystorelin®, merial inc., duluth, ga) on day 0, 25 mg prostaglandin f2alpha (pgf; dinoprost tromethamine sterile solution, 5 ml; im; lutalyse®, zoetis animal health) and cidr removal on day 7, 100 μg gnrh, im, 48 h later on day 9 and insemination on day10, 66 hrs after cidr removal. cows in co-synch+cidr group received treatment similar to cows in ovsynch+cidr group except the second gnrh (100 μg; im) was administered at the time of ai. sires (n=5) were selected and assigned to cows based on sire traits and to avoid inbreeding. inseminators (n=6) included in the study were experienced but differed among locations. cows were maintained in pastoral conditions and during the winter months supplemented with hay. the cows were fed to meet nrc recommendations.10 at the time of ai, cows were randomly assigned to one of the two treatment groups, with (trt, n = 160) or without (con, n = 161) the use of ai catheter protective sheath. the sheath (30 cm in length × 0.7 cm in diameter) was a rigid polyvinyl chloride tube (king et al., 1984). the sheath was funnel shaped at one end, where the insemination catheter was introduced, whereas the other end was sealed with a prescored soft rubber cap that can be easily punctured when pressure was applied. for all services performed in the trt group, the ai catheter protected with a sheath was introduced in to the vagina; at the cranial portion of the vagina adjacent to the cervical os, the sheath was punctured by withdrawing it back, exposing the ai catheter that was advanced through the cervix into the uterine body for semen deposition. in the con group, the ai catheter was introduced to the vagina without the use of sheath and was advanced through the cervix for deposition of the semen in the uterine body. all cows were fitted with a heat detector aids estrus detection aids (kamar® heatmount detector patches; kamar, inc., steamboat springs, co) or estrus alert patches (western point inc., apple valley, mn) or chalk at cidr removal. after cidr removal, the cows were observed twice daily until insemination for estrus and estrus detection aid status (estrus, activated aids or lost aids with mount marks vs. no estrus, intact aids) and estrus status (standing to be mounted) was recorded. a cow was determined to be in estrus if she was observed to stand for mounting or if she had an activated, lost (with mount marks) or partially-activated aid. the timing of cidr insertion, cidr withdrawal, and interval from cidr withdrawal to timed-ai was recorded for each animal. age of the cows was retrieved from the records. two weeks later, intact angus bulls were placed with cows (approximately1:30 to 1:50), across treatments, for the remainder of the 60 to70 d breeding season. cows were examined for pregnancy status approximately 70 days after ai by ultrasonography of the uterus and its contents to differentiate cows bred by ai or natural service.11,12 the criteria considered were the size of the amniotic vesicle, fetus, and placentomes. the ai pregnancy rate was calculated as the number of cows pregnant to ai divided by the total number of cows inseminated. clinical theriogenology • volume 8 number 2 • june 2016 104 heifers. a total of 430 angus cross beef heifers from four similarly managed commercial cowcalf operations inseminated during spring of 2013 were included. the goal of the participating farms was to have heifers calve at two year of age. the heifers were fed to meet nrc recommendations.10 at enrollment, heifers were assigned a bcs (1 to 9; 1, emaciated; 9, obese) and a reproductive tract score (rts, 1 to 5; 1, immature, anestrus; 5, mature, cycling). within the herd, heifers were randomly assigned to long term (lt)-72 (14-d cidr-pgf-gnrh; n=203) or short term (st)-72 (5-d co-synch+cidr; n=227) estrous synchronization protocol groups (figure 1b). briefly, heifers in the lt-72 group received a cidr from days 0 to 14, followed by 25 mg of pgf 16 days later (day 30). in order to inseminate heifers in both lt and st groups at the same time, the synchronization of heifers in st-72 group was initiated on day 25. heifers in the st-72 group received a cidr and 100 µg of gnrh on day 25 followed by 25 mg of pgf at cidr removal on day 30 and a second dose of pgf six hours later (day 30). artificial insemination was performed at 72 h (day 33) after cidr removal. all heifers were given gnrh (100 µg, im) at the time of insemination. the sires (n=7) were selected and assigned to heifers based on sire traits and to avoid inbreeding. inseminators (n=7) were experienced but differed among locations. at the time of ai, heifers were randomly assigned to one of the two treatment groups, with (trt, n=218) or without (con, n=212) the use of protective sheath as described previously. all heifers were fitted with a heat detector aids estrus detection aids (kamar® heatmount detector patches;kamar, inc., steamboat springs, co or estrus alert patches; western point inc., apple valley, mn) or chalk at cidr removal. after cidr removal, the heifers were observed twice daily until insemination for estrus and estrus detection aid status (estrus, activated or lost aids with mount marks vs. no estrus, intact aids) and estrus status (standing to be mounted) was recorded as described previously. the timing of cidr insertion, cidr withdrawal, interval to the second pgf injection and timed-ai was recorded for each animal. age of the heifers was retrieved from the records. two weeks later, intact angus bulls were placed with heifers (approximately1:30 to 1:50), across treatments, for the remainder of the 60 to70 d breeding season. heifers were examined for pregnancy status approximately 70 days after ai by ultrasonography of the uterus and its contents to differentiate heifers bred by ai or natural service sires.11,12 the criteria considered were the size of the amniotic vesicle, fetus, and placentomes. the ai pregnancy rate was calculated as the number of heifers pregnant to ai divided by the total number of heifers inseminated. statistical analyses data were analyzed with a statistical software program (sas version 9.4 for windows, sas institute, cary, nc). mean bcs, age, days postpartum, the mean interval (h) cidr insertion to cidr withdrawal, and mean interval (h) from cidr withdrawal to timed-ai in cows between trt and con groups were analyzed using one-way anova (proc glm of sas). similarly differences in the mean bcs, rts, age, the mean interval (h) from cidr insertion to cidr withdrawal, the mean interval (h) from first to second pgf injection and the mean interval from cidr withdrawal to timed-ai in heifers between trt and con groups were analyzed using one-way anova (proc glm of sas). proc glimmix of sas was used to examine the differences in ai pregnancy rate between trt and con groups. variables included in the model for cows were treatments (sheath vs. no sheath), synchronization treatments, bcs categories (≤ 5 vs > 5), days postpartum categories (≤ 60 vs. > 60 days), age categories (2, 3 to 6 and > 6 yrs), estrus (expression at or prior to ai vs. no expression) and appropriate interaction. variables included in the model for heifers were treatments (sheath vs. no sheath), synchronization treatments, bcs categories (≤ 5 vs > 5), age categories (≤ 15 vs.>15 mo), rts (2 to 5), estrus (expression at or prior to ai vs. no expression) and appropriate interactions. in the model, ai sires, inseminators and locations were used as random effects. all main effects were retained during model reduction in the model. the ‘p’ value at 0.05 was considered significant. results clinical theriogenology • volume 8 number 2 • june 2016105 the mean (± sem) age, mean bcs and mean days postpartum for cows are given in table 1. the mean age, mean body condition scores and mean days postpartum in cows did not differ between trt and con groups (table 1; p>0.1). similarly, the mean (± sem) age, mean bcs and mean rts for heifers are given in table 1. the mean age, mean bcs and mean rts in heifers did not differ between trt and con groups (table 1; p>0.1). the proportion of rts 5, 4 3, and 2 were 51.9 (223), 21.6 (93), 17.2 (74) and 9.3% (40), respectively. the mean time (h) from cidr insertion to cidr withdrawal, and interval from cidr withdrawal to timed-ai in cows did not differ between trt and con groups (p>0.1). similarly, the mean time (h) from cidr insertion to cidr withdrawal, interval from first to second pgf injection and time from cidr withdrawal to timed-ai in heifers did not differ between trt and con groups (p>0.1). accounting for estrus expression at or prior to ai (p<0.01), the use of protective sheath did not improve ai-pr in cows (sheath, 59.4 [95/160] vs. no sheath, 55.9% [90/161]; p>0.1; figure 2). the aipr for synchronization treatments (ovsynch+cidr, 58.8% [100/170] vs.co-synch+cidr, 56.3% [85/151]), bcs categories (≤ 5, 57.4% [58/101] vs. > 5, 57.7% [127/220]), days postpartum categories (≤ 60, 56.7% [148/261] vs. > 60 days, 61.2% [27/60]) and age categories (2, 52.0% [13/25]; 3 to 6, 59.2% [115/192] and > 6 yrs, 54.8% [57/104]), were not significantly different (p>0.1;table 2). cows that expressed estrus at or prior to ai had greater ai-pr compared cows that did not express estrus (estrus, 64.4% [116/180] vs. no estrus, 48.9% [69/141]; p<0.01). accounting for estrus expression at or prior to ai (p<0.01), the use of protective sheath did not improve ai-pr in heifers (sheath, 58.3% [127/218] vs. no sheath, 56.7% [121/212]; p>0.1; figure 2). the ai-pr for synchronization treatments (st-72, 58.6% [133/227] vs.lt-72, 56.3% [115/203]), bcs categories (≤ 5, 57.1% [93/163] vs. > 5, 58.1% [155/267]), and rts categories (5, 60.9% [136/223], 4, 56.5% [52/93], 3, 55.4% [41/74], 2, 47.5% [19/40]) were not significantly different (p>0.1;table 2). age categories had a trend for differences in ai-pr (≤ 15, 55.4% [(56/101]; > 15 mo, 58.4% [192/329]; p<0.1). heifers that expressed estrus at or prior to ai had greater ai-pr compared heifers that did not express estrus (estrus, 64.6% [144/213] vs. no estrus, 50.2% [104/207]; p<0.01). discussion the results of the present study revealed that the use of protective sheath did not improve ai-pr in beef heifers and in beef cows. overall ai-pr was comparable to studies that used similar reproductive protocols.13,14 the purpose of using protective sheath is to minimize contamination of the ai catheter at the time of ai in order to prevent introduction of pathogens into the uterus and to improve reproductive outcome. although bacterial isolation was not performed in this study, a study in dairy cattle showed a greater proportion of recognized potential and opportunistic uterine pathogens isolated from samples taken from the ai gun in cows inseminated without the protective sheath compared with cows inseminated with the protective sheath.9 the recognized uterine pathogens isolated were trueperella pyogenes, prevotella melaninogenica, escherichia coli, fusobacterium necrophorum, and proteus spp., potential uterine pathogens isolated were bacillus spp. and pasteurella spp., and opportunistic uterine contaminants isolated were streptococcus spp., providencia spp., klebsiella spp., and corynebacterium spp. these pathogens have been associated with postpartum uterine diseases and reduced fertility in dairy cattle. even though, the prevalence and severity of uterine disease in beef cattle is not as great as those in dairy cattle (17% vs. 51% prevalence at 7 wk. postpartum, respectively),6,15 it is possible that these pathogens could reside in the vagina of beef cattle. beef females included in this study were synchronized by cidr protocols that required vaginal placement of inserts from 5 to 14 days. the placement of an intravaginal device provides the opportunity for bacterial vaginitis. bacterial culture of swabs of the vagina after a 7-d treatment period revealed moderate growth of coliforms, environmental streptococcus spp. and staphylococcus spp., and other gram-positive, rod-shaped organisms.16 bulman et al sampled close to the cervical os after 14 d of progesterone-releasing intravaginal device (prid) treatment and isolated enterococcus spp., escherichia coli, and proteus spp. from 90% of the treated animals and 40% of animals examined with a speculum.17 clinical theriogenology • volume 8 number 2 • june 2016 106 however, from repeated sampling, the authors reported no bacterial growth in 90% of the cows 7 d after device removal and in 100% of the cows 14 d after device removal.17 it should be noted that application of vaginal insert did not reduce reproductive outcome in beef cattle. in general, beef females are exposed to bulls during the rest of the breeding season after the first ai. this poses a greater risk of beef females acquiring pathogens from beef bulls. female reproductive tract contamination by ureasplasma spp., mycoplasma spp.18 and venereal pathogens such as campylobacter fetus19,20 and tritrichomonas fetus21 poses a greater risk for reduced reproductive outcomes. in lactating dairy cows, a higher proportion of polymorphonuclear neutrophils (pmn) (>15%) immediately before22 and 4 h after ai5 were associated with poor reproductive performance. this indicates that the dairy cows with lowered reproductive performance possibly suffered from poor uterine clearance and poor immunological response due to reduced estradiol concentration around the time of ai.23,24 it should be noted that beef cows are able to clear uterine inflammation after resumption of ovarian cyclicity.6 beef cows that exhibited estrus had significantly greater preovulatory peak estradiol concentrations than cows not exhibiting estrus.20 similarly, preovulatory plasma estradiol concentrations on day of ai for dairy cows diagnosed pregnant was greater compared to cows that are not pregnant.25 results from these studies indicated that preovulatory estradiol concentrations were greater in beef cows compared to dairy cows. this elevated estradiol concentrations in beef females could possibly contributed to the required uterine clearance and immunological response around the time of ai resulted in similar ai pregnancy between trt and con groups. beef females that expressed estrus had greater ai pregnancy compared to those that did not express estrus. preovulatory estradiol is essential for preovulatory luteinizing hormone secretion,26 and preparation of ideal oviduct and uterine environments for proper embryo development.27,28 therefore it is possible that elevated concentrations of estrogen in cows that expressed estrus had effects on follicle development and ovulation, and effects on uterine environment to provide a more conducive environment for fertilization and subsequent embryo development. conclusions in conclusion, a hygienic ai technique is critical for ai success; however, the use of protective sheath did not improve reproductive outcomes in beef cattle. beef females that expressed estrus had greater ai pregnancies likely as a result of differences follicle development and favorable uterine environment for fertilization and subsequent embryo development. acknowledgements the author thanks participating beef cattle producers for the successful completion of this study. the author acknowledges drs. matthew asay and philip firth for their assistance with this project and thank zoetis animal health for donation of eazi-breed® cidr cattle insert, lutalyse®, and factrel® for the heifer experiment. references 1. foote rh: review: dairy cattle reproductive physiology research and management-past progress and future prospects. j dairy sci 1996;79:980-990. 2. vishwanath r: artificial insemination: the state of the art. theriogenology 2003;59:571-584. 3. king gj, bellissimo dj, penner wj: routine use of protective sheaths in cattle inseminations did not improve fertility. can vet j 1984;25:327-328. 4. windig jj, calus mp, veerkamp rf: influence of herd environment on health and fertility and their relationship with milk production. j dairy sci 2005;88:335-347. 5. kaufmann tb, drillich m, tenhagen ba, et al: prevalence of bovine subclinical endometritis 4 h after insemination and its effects on first service conception rate. theriogenology 2009;71:385-391. 6. santos nr, lamb gc, brown dr, et al: postpartum endometrial cytology in beef cows. theriogenology 2009;71:739745. 7. ricci a, gallo s, molinaro f, et al: evaluation of subclinical endometritis and consequences on fertility in piedmontese beef cows. reprod domest anim 2015;50:142-148. clinical theriogenology • volume 8 number 2 • june 2016107 8. richards mw, spitzer jc, newman sk, et al: bovine pregnancy and non-return rates following artificial insemination using a covered sheath. theriogenology 1984;21:949-957. 9. bas s, hoet a, rajala-schultz p, et al: the use of plastic cover sheaths at the time of artificial insemination improved fertility of lactating dairy cows. j dairy sci 2011;94:793-799. 10. nrc: nutrient requirements of beef cattle. seventh edition. washington, dc: natl acad press; 2000. 11. hughes ea, davies dar: practical uses of ultrasound in early pregnancy in cattle. vet rec 1989;124:456-458. 12. ginther oj: ultrasonic imaging and animal reproduction: fundamentals book 1. cross plains(wi): equiservices publishing; 1995. p. 782 and 147-155. 13. kasimanickam r, schroeder s, hall jb, et al: fertility after implementation of longand short-term progesterone-based ovulation synchronization protocols for fixed-time artificial insemination in beef heifers. theriogenology 2015;83:1226-1232. 14. kasimanickam r, hall jb, currin jf, et al: sire effect on the pregnancy outcome in beef cows synchronized with progesterone based ovsynch and co-synch protocols. anim reprod sci 2008;104:1-8. 15. gilbert ro, shin st, frajblat m, et al: the incidence of endometritis and its effect on reproductive performance of dairy cows. in: proc 12th int conf prod dis farm anim, 2004;12:30 [abstract]. 16. walsh rj, leblanc sj, bernooy e, et al: safety of a progesterone-releasing intravaginal device as assessed from vaginal mucosal integrity and indicators of systemic inflammation in postpartum dairy cows. can j vet res 2008;72:43-49. 17. bulman dc, mckibbin pe, appleyard wt, et al: effect of a progesterone releasing intravaginal device on mik progesterone levels, vaginal flora, milk yield and fertility of cyclic and non-cyclic dairy cows. j reprod fertil 1978;53:289-296. 18. mulira gl, saunders jr, barth ad: isolation of ureaplasma diversum and mycoplasmas from genital tracts of beef and dairy cattle in saskatchewan. can vet j 1992;33:46-49. 19. schurig gd, hall ce, burda k, et al: infection patterns in heifers following cervicovaginal or intrauterine instillation of campylobacter (vibrio) fetus veneralis. cornell vet 1974;64:533-548. 20. truyers i, luke t, wilson d, et al: diagnosis and management of venereal campylobacteriosis in beef cattle. bmc vet res 2014;10:280. 21. corbeil lb, hodgson jl, jones dw, et al: adherence of tritrichomonas foetus to bovine vaginal epithelial cells. infect immun 1989;57:2158-2165. 22. williams bl, senger pl, stephens lr, et al: relationships between days post partum, observed estrus and uterine microflora in commercial dairy cows. theriogenology 1988:30:555-561. 23. lacetera n, scalia d, franci o, et al: short communication: effects of nonesterified fatty acids on lymphocyte function in dairy heifers. j. dairy sci 2004;87:1012-1014. 24. leroy jlmr, vanholder t, delanghe jr, et al: metabolic changes in follicular fluid of the dominant follicle in highyielding dairy cows early post partum. theriogenology 2004;62:1131-1143. 25. perry ga, swanson ol, larimore el, et al: relationship of follicle size and concentrations of estradiol among cows exhibiting or not exhibiting estrus during a fixed-time ai protocol. domest anim endocrinol 2014;48:15-20. 26. baratta m,west la, turzillo am, et al: activin modulates differential effects of estradiol on synthesis and secretion of follicle-stimulating hormone in ovine pituitary cells. biol reprod 2001;64:714-719. 27. miller bg, moore nw, murphy l, et al: early pregnancy in the ewe: effects of oestradiol and progesterone on uterine metabolism and on embryo survival. aust j biol sci 1977;30:279-288. 28. geisert rd, zavy mc, biggers bg, et al: characterization of the uterine environment during early conceptus expansion in the bovine. anim reprod sci 1998;16:11-25. clinical theriogenology • volume 8 number 2 • june 2016 108 figure 1: schematic presentation of synchronization protocol figure 1a cidr day 0 gnrh pgf2a, day 7 day 10 gnrh + tai 66 h cidr day 0 gnrh pgf2a day 7 day 10 tai 48 h gnrh day 9 18 h ovsynch + cidr co-synch +cidr clinical theriogenology • volume 8 number 2 • june 2016109 figure 1b note: in order to inseminate heifers in both lt-72 and st-72 groups at the same time, the synchronization of heifers in st-72 group was initiated on day 25 of lt-72 protocol. figure 2: mean (± sem) artificial insemination (ai) pregnancy percentage in beef cattle inseminated with or without ai gun protective sheath. 59.4 55.9 58.3 56.7 0 20 40 60 80 100 sheath no sheath sheath no sheath cows heifers (95/160) (90/161) (127/218) (121/212) p > 0.1 pgf2a cidr pgf2a 6 h ftai @ 72 h from cdir removal st 72 (5-d co-synch+cidr) day 25 day 14 day 30 am day 33 gnrh gnrh + ftai ftai @ 72 h from cdir removal pgf2a cidr day 0 day 14 day 30 day 33 gnrh + ftai lt 72 (14-d cidr – pgf – gnrh) clinical theriogenology • volume 8 number 2 • june 2016 110 table 1.mean ± sem of age, body condition scores, and days postpartum of beef cows belonging to treatment groups in different locations cattle groups variables treatment n mean ± sem cow age (yr) con 161 5.73 ± 0.21 trt 160 5.58 ± 0.21 bcs con 161 6.07± 0.08 trt 160 6.43 ±0.45 dpp con 161 52.8 ± 0.67 trt 160 52.9 ± 0.68 heifers age (mo) con 212 15.3 ±0.43 trt 218 15.6 ± 0.37 bcs con 212 5.93 ± 0.97 trt 218 6.12 ± 1.49 rts con 212 4.09 ± 1,08 trt 218 4.14 ± 1.07 sem, standard error of the mean; con, ai gun with no protective sheath; trt, ai gun with protective sheath; bcs, body condition score (1 to 9; 1, emaciated; 9, obese); dpp, days postpartum; rts, reproductive tract score (1 to 5; 1, immature, anestrus; 5, mature, cycling); table 2. effect of explanatory variables on ai pregnancy rates from beef cows and beef heifers that were inseminated with (trt) and without (con) the use of a protective plastic sheath (ps) beef cattle explanatory variables degrees of freedom f value p value cow protective sheath 1 0.08 0.78 synchronization treatment 1 0.41 0.52 estrus 1 8.07 0.005 age 2 0.71 0.49 body condition score 1 0.10 0.79 days postpartum 1 0.43 0.52 heifer protective sheath 1 0.09 0.82 synchronization treatment 1 0.42 0.52 estrus 1 9.72 0.002 age 1 2.86 0.09 body condition score 1 0.10 0.92 reproductive tract score 3 0.53 0.66 refer to figure 1 for cow and heifer synchronization treatment; estrus expression of estrus at or prior to ai vs no expression; cow age categories (yrs)2, 3 to 6, > 6 years; body condition score (1 to 9; 1, emaciated; 9, obese) categories, ≤ 5 and > 5; dpp, days postpartum categories ≤ 60 and > 60; reproductive tract score (1 to 5; 1, immature, anestrus; 5, mature, cycling), categories 2 to 4; heifer age categories (mo), ≤ 15 and > 15; cow covariance parameter estimates: location, 0.07831; ai sire, 0.06429; inseminators, 0.03344; residual 0.28937; fit statistics bic =1276.06; -2 res log likelihood =1254.51; heifer covariance parameter estimates: location, 0.10586; ai sire, 0.08713; inseminators, 0.08237; residual, 0.22820; fit statistics, bic = 1386.8; -2 res log likelihood = 1371.8; clinical theriogenology • volume 8 number 2 • june 2016111 clinical theriogenology • volume 8 number 2 • june 2016 112 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default 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/useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2012: position statement: welfare of breeding dogs position statement: welfare of breeding dogs the american college of theriogenologists and society for theriogenology promote the breeding of healthy, genetically superior dogs to maintain a diverse canine population that meets the needs of society for companion dogs and working dogs. the college and society support practices to promote optimal health of all breeding dogs. purpose-bred dogs are maintained subject to regulation by institutional and government agencies, while similar guidelines for noninstitutional breeders are lacking. this position statement refers to care and management of breeding of dogs intended for personal ownership. it is the position of the act and sft that: -animals must be provided water, food, proper handling, health care, and environments appropriate to their species and use, and should be cared for in ways that prevent and minimize fear, pain, distress, and suffering. specifically, all breeding animals should be housed in clean, properly sized facilities that permit them to express normal behavior, include environmental enrichment, and are appropriate for stage of life. male and female dogs may be co-housed in social units except for those times when bitches are in estrus. specific attention to individual temperament to avoid inter animal aggression is required. regular observation of and interaction with dogs by handlers must occur. dogs should have access to a balanced diet that is appropriate for their life stage and fed to them in a manner that will permit them to maintain a body condition score (bcs) of 4 or 5 out of 9, excepting certain breeds of dogs such as sight hounds that are naturally lean in body type. fresh water should be available. dogs should have routine health care and disease prophylaxis including regular veterinary examination, vaccination, internal and external parasite control, dental care and coat care when applicable. dogs intended for breeding should be evaluated for hereditary disorders before being bred. owners of breeding dogs should develop a breeding plan with a veterinarian to minimize or eliminate production of puppies with hereditary defects. all dogs intended for breeding should be appropriately tested for canine brucellosis to prevent spread of this disease. at a minimum, both members of a breeding pair should be tested prior to each breeding. all dogs intended for breeding should be regularly tested for canine brucellosis, either at the time of breeding or every six months. -intact male dogs should be regularly evaluated by a veterinarian for prostate and testicular disease. bitches should be regularly evaluated by a veterinarian for pyometra and mammary neoplasia. decisions about when to spay or castrate individual dogs and bitches no longer intended for breeding should be made with the counsel of a veterinarian. bitches should not be bred before they are physically mature and should not be bred on the first estrous cycle without the advice of a veterinarian. bitches may be bred on consecutive estrous cycles if they maintain or regain their breed appropriate body condition and are deemed healthy on the basis of veterinarian examination prior to the onset of the next proestrus. -bitches approaching or exceeding the critical age at which reproductive efficiency for their breed or type declines and pregnancy-associated risks increase should not be bred without the counsel and advice of a veterinarian. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201225 clinical theriogenology • volume 4 number 1 • march 2012 clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 26clinical theriogenology • volume 4 number 1 • march 2012 omniblank: 1 © 2023 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2023, 15, 10521, http://dx.doi.org/10.58292/ct.v15.10521 a note of appreciation editorial team thanks the following scholars for reviewing manuscripts published in volume 15 henry annandale scott bailey orsolya balogh theresa beachler (2) margaret casal john cavalieri *sherrie clark-deener *gail colbern (3) *jill colloton (2) dinesh dadarwal freitas dell’aqua mariana diel de amorim hilary french *allan gunn *vanmathy kasimanickam (3) dale kelley *kara kolster (2) *michelle kutzler (3) *réjean lefebvre (2) catharina lindeforsberg charles love jim morehead szaboles nagy christopher premanandan jo randall *jen roberts (2) *cyril stephen dawna voelki *brian whitlock william whitler (2) *editorial board member numbers within parentheses indicate manuscripts reviewed http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v15.10521 preliminary insemination trial to determine the fertility of stallion sperm stored for 3 and 7 days at 17ºc in a long term liquid storage medium preliminary insemination trial to determine the fertility of stallion sperm stored for 3 and 7 days at 17ºc in a long term liquid storage medium jennifer clulow,a aleona swegen,b john aitken,b zamira gibbb ascone equine group, scone, new south wales, australia buniversity of newcastle, newcastle, new south wales, australia a synthetic medium has been developed which allows stallion sperm to be stored at 17ºc for up to 7 days;1 our objective was to determine fertility of sperm stored in this medium. an ejaculate from 2 pony stallions was collected and stored for 3 and 7 days duration prior to insemination. two thoroughbred, 1 quarter horse and 1 standardbred mare from 5 8 years of age were used. mares were examined using transrectal ultrasonography and ovulation induced by giving 1.25 mg of biorelease deslorelin™ when a follicle > 35 mm and grade 3 uterine edema was identified. a fixed time artificial insemination protocol was used, with mares bred 24 hours after ovulation induction with a minimum of 500 x 106 progressively motile sperm deposited into the uterine body. ovulation was detected the following day in 3 mares, whereas the fourth mare ovulated 48 hours after insemination following semen storage for 8 days (10 days after semen collection). a uterine lavage (1 liter of 0.9 % sterile saline) was done on all mares 12 hours after insemination and 10 iu of oxytocin given. mares were examined 14 days after ovulation for the presence of embryo. all mares conceived on the first cycle, with twins identified in 2 mares. one mare was allowed to maintain her pregnancy to term and delivered a healthy filly at 332 days pregnancy. liquid storage in this synthetic medium for up to 7 days at 17˚c did not affect fertility of stallion sperm. future use of this medium will greatly simplify sperm transport logistics and artificial insemination regimens for equine veterinarians. keywords: stallion, storage, insemination, pregnancy, sperm references 1. gibb, z, clulow, jr, aiken, rj, et al: first publication to describe a protocol for the liquid storage of stallion spermatozoa for 7 days. j equine vet sci 2018;66:37-40 461 clinical theriogenology • volume 11, number 3 • september 2019 462clinical theriogenology • volume 11, number 3 • september 2019 keywords: stallion, storage, insemination, pregnancy, sperm references 1. gibb, z, clulow, jr, aiken, rj, et al: first publication to describe a protocol for the liquid storage of stallion spermatozoa for 7 days. j equine vet sci 2018;66:37-40 omniblank: 2019 comparison of deslorelin and histrelin for induction of ovulation in mares comparison of deslorelin and histrelin for induction of ovulation in mares jennifer morrissey, ryan ferris, patrick mccue department of clinical sciences college of veterinary medicine and biomedical sciences colorado state university, fort collins, co abstract ovulation induction agents are used to synchronize ovulation to optimize reproductive management in breeding programs. the goal of this retrospective study was to compare interval from treatment to ovulation in mares given deslorelin acetate versus histrelin. reproductive records of 134 estrous cycles from 91 privately owned mares managed for insemination with frozen semen were evaluated. deslorelin acetate (1.8 mg) or histrelin (0.5 mg) were given as an intramuscular injection when a dominant follicle was > 35 mm in diameter, in the presence of uterine edema. average interval from deslorelin administration to ovulation (34.8 ± 11.4 hours) tended (p = 0.054) to be shorter than the average interval from histrelin administration to ovulation (38.0 ± 8.1 hours). for both hormones, the highest percentage of ovulations occurred between 37 and 44 hours after treatment. when an ultrasonographic examination was done at or prior to 36 hours post treatment, ovulation was detected in 36.6 and 18.3% of mares given deslorelin and histrelin, respectively. in conclusion, interval from treatment to ovulation and percentage of mares that ovulated within 48 hours were not significantly different between mares receiving 1.8 mg of deslorelin acetate versus 0.5 mg of histrelin. furthermore, sequential scheduled ultrasonographic examinations are recommended after any gnrh agonist treatment, as some mares ovulate earlier than anticipated. keywords: mare, induction, ovulation, deslorelin, histrelin introduction ovulation inducing agents are frequently administered to mares to optimize reproductive management in live-cover and artificial insemination programs and to synchronize ovulation of embryo donors and recipient mares.1-7 common products for inducing ovulation include human chorionic gonadotropin (hcg) and gonadotropin releasing hormone (gnrh) agonists, such as buserelin, deslorelin and histrelin. native gnrh, a 10-amino acid peptide produced in the hypothalamus, stimulates secretion of luteinizing hormone (lh) and follicle stimulating hormone (fsh) from the anterior pituitary.8 agonists of gnrh such as deslorelin and histrelin are synthetic 9 amino acid peptides with enhanced potency compared to native gnrh. in previous studies, 84 100% of mares ovulated within 48 hours after administration of deslorelin acetate4,7,9-11 and 82 100% ovulated within 48 hours after administration of histrelin.12-14 the goal of this retrospective study was to compare interval from treatment to ovulation in mares given deslorelin acetate or histrelin. materials and methods reproductive records of 134 estrous cycles from 91 privately owned mares managed for insemination with frozen semen in the 2015 and 2016 breeding seasons were evaluated. transrectal ultrasonographic examinations were performed on each mare, including evaluation of ovarian follicle development, presence or absence of a corpus luteum, uterine edema score and presence or absence of uterine fluid. diameter of all follicles e 30 mm on each ovary were measured with electronic calipers and recorded. edema score was graded on a scale ranging from 0 (no edema), 0.5 (trace edema), 1 (mild edema), 2 (moderate edema), to 3 (marked edema). in 2015, all mares were given deslorelin acetate, whereas in 2016, all mares were given histrelin. deslorelin acetate (sucromate™, thorn-biosciences, louisville, ky) or histrelin (doc lane’s veterinary 123 clinical theriogenology • volume 11, number 2 • june 2019 pharmacy, lexington, ky) were given as intramuscular injections at a dose of 1.8 mg or 0.5 mg, respectively, when there was a dominant follicle > 35 mm in diameter in the presence of uterine edema. all gnrh agonist treatments were given at 8:00 pm, with the goal of inducing ovulation approximately 40 hours later at 12:00 (noon). ultrasonographic examinations were performed at ~ 6 hour intervals after hormone administration until ovulation was detected. all protocols and procedures used were approved by the university institutional animal care and use committee (16-6876a). a student’s t-test was used to compare, between the two hormones, follicular size, uterine edema score and intervals from treatment to ovulation, whereas a fisher’s exact test was used to compare percentage of mares that ovulated at each time point. for all analyses, p < 0.05 was considered significant. values are reported as mean ± sd. results there was no significant difference between mares given deslorelin versus histrelin for diameter of the dominant follicle at treatment or edema scores (table). although ovulation tended (p = 0.054) to occur earlier in mares given deslorelin versus histrelin (34.8 ± 11.4 versus 38.0 ± 8.1 hours, respectively), there was no difference in the percentage of mares that ovulated at any time interval after treatment (figure). in both groups, the highest percentage of ovulations occurred between 37 and 44 hours after treatment. all mares treated with deslorelin ovulated within 48 hours. however, 6 mares given histrelin failed to ovulate within 48 hours; 4 mares formed hemorrhagic follicles and 2 mares given 2,500 iu of hcg at 48 hours after the initial dose of histrelin ovulated within 2 days. discussion in this study, mares were given a gnrh agonist to induce ovulation to facilitate insemination of a single dose of frozen-thawed semen immediately after detection of ovulation. treatment at 8:00 pm was done in an attempt to induce ovulation approximately 40 hours later, at 12:00 (noon) to optimize availability of trained personnel. mares were examined the morning after gnrh agonist therapy and at ~ 6 hour intervals thereafter until ovulation was detected. mares that did not ovulate within 48 hours after gnrh agonist therapy were subsequently given hcg in a further attempt to induce ovulation. the objective of this retrospective study was to compare efficacy of a commercial deslorelin acetate preparation with that of a compounded histrelin preparation. the deslorelin product was used at the manufacturer’s recommended dose of 1.8 mg. previous work at our facility indicated that doses of histrelin from 0.5 to 1.0 mg had the same efficacy for inducting ovulation;14 therefore, 0.5 mg was given in the present study. all mares were privately owned and had been presented for reproductive management in breeding program using frozen-thawed semen. consequently, it was not possible to have an untreated or placebo-treated control group. the highest percentage of mares ovulated between 37 and 44 hours after administration of either product. interestingly, there was a statistical trend for an earlier ovulation following deslorelin administration. perhaps the higher dosage of deslorelin used (1.8 versus 0.5 mg) elicited an earlier and higher lh response. the average interval from deslorelin to ovulation in this study (34.8 ± 11.4 hours) seemed shorter than the 38.6 hours and 41.4 hours previously reported.4,7 it is also noteworthy that ovulation was detected in 36.6% of mares given deslorelin and 18.3% of mares given histrelin on an ultrasonographic examination performed prior to the 37 44 hour time window. variability in interval to ovulation may have been due to differences between mares in their proximity to spontaneous ovulation at the time of gnrh agonist administration. these ‘early ovulations’ emphasized the need for close monitoring of mares given a gnrh agonist, to facilitate insemination with a single dose of frozen semen in the immediate post-ovulation period. all 41 mares given deslorelin ovulated within 48 hours, whereas six of the 93 mares (6.5%) given histrelin failed to ovulate within 48 hours. presumably sample size had an effect, as previous studies using deslorelin to induce ovulation reported ovulation failure rates ranging from 1.2 to 9.0%.4-6,10 124clinical theriogenology • volume 11, number 2 • june 2019 conclusions interval from hormone administration to ovulation and percentage of mares that ovulated within 48 hours after treatment were not significantly different between mares receiving 1.8 mg of deslorelin acetate versus 0.5 mg of histrelin. in addition, sequential scheduled ultrasonographic examinations are recommended after any gnrh agonist treatment, as some mares ovulate earlier than anticipated. conflict of interest the authors have no conflicts of interest to disclose. references 1. squires e: gonadotropin-releasing hormones. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames: wiley-blackwell; 2011. p. 1820-1824. 2. mckinnon ao, mccue pm: induction of ovulation. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames: wiley-blackwell: 2011. p. 1858-1869. 3. ginther oj: reproductive biology of the mare: basic and applied aspects. 2nd edition, cross plains: equiservices; 1992. p. 279-282. 4. ferris ra, hatzel jn, lindholm arg, et al: efficacy of deslorelin acetate (sucromate) on induction of ovulation in american quarter horse mares. j equine vet sci 2012;32:285-288. 5. hemberg e, lundeheim n, einarsson s: successful timing of ovulation using deslorelin (ovuplant®) is labour-saving in mares aimed for single ai with frozen semen. reprod dom anim 2006;41:535-537. 6. meinert c, silva jfs, kroetz i, et al: advancing the time of ovulation in the mare with a short-term implant releasing gnrh analogue deslorelin. equine vet journal 1993;25:65-68. 7. samper j, jensen s, sergeant j: timing of induction of ovulation in mares treated with ovuplant or chorulon. j equine vet sci 2002 22:320-323. 8. alexander sl, irvine chg: gnrh. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames: wiley-blackwell; 2011. p. 1608-1618. 9. mccue pm, magee c, gee ek: comparison of compounded deslorelin and hcg for induction of ovulation in mares. j equine vet sci 2007;27:58-61. 10. farquhar vj, mccue pm, vanderwall dk, et al: efficacy of the gnrh agonist deslorelin acetate for inducing ovulation in mares relative to age of mare and season. j equine vet sci 2000;20:8-11. 11. lindholm arg, bloemen ehg, brooks rb, et al: comparison of deslorelin and buserelin in mares: lh response and induction of ovulation. animal reprod sci 2010;121s:s68-s70. 12. kiser am, sudderth ak, brinsko sp, et al: comparison of efficacy of two dose rates of histrelin for inducing ovulation in broodmares. j equine vet sci 2013;33:820-822. 13. voge jl, sudderth ak, brinsko sp, et al: comparison of efficacy of two dose rates of histrelin to human chorionic gonadotropin for inducing ovulation in broodmares. j equine vet sci 2012;32:208-210. 14. lindholm, arg, ra ferris, db scofield, et al: comparison of deslorelin and histrelin for induction of ovulation in mares. j equine vet sci 2011;31:312-313. 125 clinical theriogenology • volume 11, number 2 • june 2019 table. reproductive end points in mares given deslorelin versus histrelin to induce ovulation. end point deslorelin histrelin number of cycles 41 93 follicle diameter (mm) 42.0 ± 5.1 42.1 ± 4.8 edema score 1.9 ± 0.6 1.9 ± 0.7 interval to ovulation (hours) 34.8 ± 11.4a 38.0 ± 8.1b a,bwithin a row, values without a common superscript tended to differ (p = 0.054) figure. intervals from treatments (deslorelin or histrelin) to ovulation. 0 10 20 30 40 50 60 70 0-12 13-24 25-36 37-44 44+ fail o vu la tio n ra te (% ) interval to ovulation (hours) histrelin deslorelin 126clinical theriogenology • volume 11, number 2 • june 2019 << /ascii85encodepages false /allowpsxobjects false /allowtransparency false /alwaysembed [ true ] /antialiascolorimages false /antialiasgrayimages false 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tools: genetic testing for genetic defects in various breeds genomic tools: genetic testing for genetic defects in various breeds bob weaber department of animal sciences and industry, kansas state university, manhattan, ks over the last several years the beef seedstock sector has had to deal with a number of recessive genetic defects. the utilization of assisted reproductive technologies including embryo transfer and artificial insemination has allowed breeders to concentrate selection to a relatively small number of animals. while many breeders avoid matings of half-sibs or sires to daughters to reduce the accumulation of inbreeding, it is not unusual for very prominent sires to appear several generations back in pedigrees of both the sire and dam of a particular individual. it is in this case when there is an increased chance for the appearance of a progeny affected by a recessive genetic defect. autosomal recessive genetic defects are inherited congenital abnormalities. these genetic mutations occur on one of the 29 pairs of autosomal (non-sex coding) chromosomes. animals that inherit a single defective or mutated gene and one normal copy of the gene are called heterozygotes and are not affected by the disease but are carriers of the defect passing it on to half of their progeny on average. animals that have two copies of the normal or unmutated gene are called homozygous normal. animals that inherit two copies of the defective gene are homozygous for the recessive forms of the gene and are phenotypically affected by the abnormality. the carriers (heterozygotes) and homozygous normal animals do not exhibit the deleterious condition and are phenotypically indistinguishable. the fact that the normal animals and heterozygotes are indistinguishable makes phenotypic selection to eliminate defect carriers ineffective. to illustrate this difficulty in phenotypic selection to eliminate recessive carriers consider a more common trait like coat color. both homozygous black animals and heterozygous black (red carriers) are phenotypically black and indistinguishable. only when two black animals produce a red calf can we infer the genetic makeup of the color parents as both being heterozygotes (red carriers). all breeds carry some genetic defects. presumably some of the autosomal recessive lethal genetic conditions affect embryos during gestation and cause early embryonic loss. these defects are difficult to detect and may simply be diagnosed as ‘reproductive failure’. a number of defects have been documented in beef breeds, several quite recently. a brief description of several genetic defects are listed below. due to space limitations this is not a complete listing of defects. condition description breeds affected tibial hemimelia (th) affected calves are born with twisted rear legs with fused joints, malformed or missing tibia, large abdominal hernias and/or a skull deformity. dna test available to identify carriers. shorthorn maine-anjou chianina pulmonary hypoplasia with anasarca (pha) underdeveloped heart and lungs, marked increase in calf size caused by fluid retention (anasarca) of fetus. dna test available to identify carriers. shorthorn maine anjou chianina ideopathic epilepsey (ie) a neurological disorder in which affected calves have seizures. dna test available to identify carriers. hereford 551 clinical theriogenology • volume 6, number 4 • december 2014 arthrogryposis -multiplex (am)* many environmentally caused forms appear but one form is inherited as a simple recessive trait. the joints of all four legs are fixed symmetrically and a cleft palate is present. am in angus includes twisted malformation of spine and fixed leg joints. dna test available to identify am carriers. charolais angus* contractual arachnodactyly (ca) inherited as simple recessive, ca is caused by a deletion of genomic dna. calves are abnormal at birth and exhibit arched spine, hyperextension of fetlocks, muscle contracture resulting in inability to straighten the upper limb joints. post-natal improvement of clinical signs. angus dwarfism at least three types of dwarfism documented in cattle and thought to be caused different simply inherited recessive genes. angus hereford brahman dexter hypotrichosis (hairlessness) partial to complete lack of hair. hair grows in and falls out so affected animals may have varying appearance over time. dna test available to identify carriers. hereford protoporphyria light sensitivity causing open sores and scabs. liver function is also affected and animals may suffer from seizures. inherited as simple recessive. dna test available to identify carriers. limousin osteopetrosis (marble bone) long bones are solid and without developed marrow. bones are brittle and break easily . calves are usually born dead 2 to 4 weeks pre-term. dna test available to identify carriers in red angus. angus red angus holstein hydrocephalus -internal * -neuropathic hydrocephalus(nh)** excessive fluid in brain ventricles (internal) or in cranium (external). dna test available to identify nh carriers. hereford* many other breeds angus** 552clinical theriogenology • volume 6, number 4 • december 2014 management of genetic defects in seedstock or commercial beef herds can be quite challenging. in the case of seedstock herds, suspect animals or those known to be the progeny of carriers should be tested when dna diagnostic tools are available and economically practical. carrier animals may be retained in the breeding herd, but breeders should test all progeny to determine carrier status prior to marketing as breeding stock. carrier calves should only be sold to feeders and designated for slaughter only. the table below details the expected results if a homozygous normal sire is mated to a carrier (heterozygote) cow the resulting progeny will include on average one-half defect free and one-half carrier calves with no affected calves. also included in the table is the expected proportion of progeny of various genotypes when a carrier sire is mated to a carrier dam. on average one-quarter of the calves will be free of the defect, one-half will be carriers and one-quarter will be affected. if the animals are affected by a lethal defect, the surviving animals will include one-third defect free calves and two-thirds will be carriers of the defect. if you have a calf born with a suspected congenital defect, photograph the affected calf and contact your breed association immediately to arrange for tissue collection and reporting instructions. in a commercial herd that has had affected calves or is striving to prevent introduction of a known defect, bulls should be dna tested as the primary means of control. pedigree inspection and reduction of matings of closely related animals may provide reductions in the production of affected calves but not as effective as dna marker testing. mating of carrier cows to non-carrier or clean bulls will result in the production of no affected calves. it is frequently not economically practical to cull commercial cows on the basis of their carrier status. if a dna test is available, all new sire purchases or semen used for artificial insemination should be from sires that are not carriers. non-carriers may also be determined by pedigree if both sire and dam have tested free of the defect. if a dna test for a defect is not available, the strategic use of a planned crossbreeding system may eliminate the appearance of affected calves. care should be taken to select a breed that has not had any calves produced in recent generations that are affected by the same defect one is trying to eliminate. at the commercial level, autosomal recessive defects can be effectively managed through careful sire and/or breed selection without extensive culling of the beef cow herd. 553 clinical theriogenology • volume 6, number 4 • december 2014 table . a.) expected calf genotypic frequencies from mating of non-carrier sire to carrier dam for deleterious recessive genetic defect. b.) expected calf genotypic frequencies from mating of carrier sire to carrier dam. a. non-carrier sire genotype (aa) carrier dam genotype (aa) a a a aa aa a aa aa genotypes frequency aa (homo. normal) 50% aa (hetero. carrier) 50% aa (homo. recessive affected) 0% b. carrier sire genotype (aa) carrier dam genotype (aa) a a a aa aa a aa aa genotypes frequency aa (homo. normal) 25% aa (hetero. carrier) 50% aa (homo. recessive affected) 25% phenotypes frequency frequency in surviving calves aa (normal) 25% 33% aa(normal) 50% 67% aa (affected-dead) 25% na a = normal genotype a = defective/mutated genotype 554clinical theriogenology • volume 6, number 4 • december 2014 2014: increase in average scrotal circumference of canadian beef bulls increase in average scrotal circumference of canadian beef bulls albert barth western college of veterinary medicine, university of saskatchewan, saskatoon, sk, canada the basis for scrotal circumference measurement in temperate climates, short growing seasons and long winter-feeding periods impose seasonal cattle management patterns upon producers. efficient and economical herd management depends a great deal on maintaining short, well-defined, calving seasons. the advantages of short breeding seasons are applicable to tropical and subtropical regions as well as in temperate regions.1 short calving periods result in a uniform calf crops that can be managed in groups for vaccination, castration, weaning, weighing for performance records, and for calf sales. it also allows cow herds to be managed in groups for maintaining vaccination programs, observation of breeding activity, synchronization of estrus and artificial insemination, pregnancy testing, culling for non-pregnancy and poor performance, and good nutritional management. in essence, the length of the calving period is dictated by the length of the breeding season. in well-managed herds, it is normal to expect beef cows to produce a calf every 12 months, and for >90% of the herd to be pregnant and due to calve within about a 65-day period. short breeding seasons require highly fertile females. good record keeping, nutritional management, vaccination and pregnancy testing, heifer selection and development, are some of the components of maintaining a highly fertile female population. early puberty in heifers is necessary in order for heifers to cycle in synchrony with the cows. in well-managed herds breeding seasons will begin three weeks after the normal end of the calving period. since heifers are usually selected from last year’s calving period, it is clear that heifers must already be cycling when they are 13 to 15 months old; preferably, heifers would have completed two or three estrous cycles by the beginning of breeding to ensure optimum fertility.2 beef heifers that conceive early and calve as two-year-olds have a greater probability of becoming pregnant as first calf females the following year3 and tend to calve earlier in subsequent years.4 however, the average age of puberty (first estrus) in well-managed, early-maturing breeds and in crossbred heifers is about 12 months of age, i.e., 50% of heifers have cycled once.5-8 by 13-14 months of age, about 90% should have cycled once. in purebred herds with later maturing breeds, such as hereford and limousin,5 having sufficient numbers of cycling replacement heifers at the start of the breeding season may be difficult. selection for increased scrotal circumference (sc) is an essential practice since scrotal circumference of sires is highly correlated with age at onset of puberty in female and male offspring.9-12 in two studies, correlations of 0.66 and 0.97 were found between breed means for scrotal circumference and fertility of female offspring.13,14 for producers that sell purebred bulls and purchase bulls for genetic improvement and for frozen semen sales, early bull puberty is immensely important. in order to capture the genetics of high-priced yearling bulls and return on investment, i.e., to have semen for sale in mid-winter, useful semen must be produced by the time a bull is 12 months old. however, only 30 to 41% of bos taurus bulls produced satisfactory semen at 12 months of age and only 51-75% by 14 months of age(barth ad, unpublished data).15,16 early puberty is also very important in bulls intended for natural service in commercial beef herds. in the past 20 years, it has become common to use yearling bos taurus bulls as primary breeders. the use of bulls at an early age reduces production costs, shortens the generation interval and may increase genetic gains. however, the variability of onset of puberty, among and within breeds, has resulted in great variability in reproductive performance of young bulls among producer herds. poor reproductive performance by yearling bulls may be due partly to inadequacy in mating ability; however, semen quality is a more important factor. age of puberty, and subsequently maturity, is the main factor involved in semen quality of yearling bulls. therefore, the successful use of yearling bulls will depend on production systems that hasten maturity. testis size is highly correlated with age at onset of puberty in male and female progeny.9,17 although the benefits of larger testes include a greater capacity to produce sperm and good quality semen,18-20 the most important reason for selection for larger sc measurement is to promote early puberty 509 clinical theriogenology • volume 6, number 4 • december 2014 in male and female offspring. fortunately, testis size is quite highly heritable with estimates of heritability ranging from 0.44 to 0.69.19 therefore, rapid progress can be made in selection based on sc. keywords: scrotal circumference, puberty, selective breeding, genetic improvement availability of scrotal circumference data it would seem that sc data should be accessible through records of breeding soundness evaluations in veterinary practices and veterinary institutions throughout north america and many other countries of the world; however, in order to be useful sc measurements must be reliable and the age of bulls must be accurately recorded, especially in yearling bulls. in yearling bulls testis size increases a great deal in a short period of time; a few weeks in age makes a big difference in sc. therefore, for yearlings, birth dates must be recorded, or at the very least, bull age must be known to be accurate within two weeks. unfortunately, very few clinicians record birth dates and often do not record age in months. in order to ensure the reliability of sc measurements, those doing the measurements must be well experienced and this may not be the case among all clinicians that evaluate bulls. the use of springtension sc measuring tapes to ensure application of similar tensions among users should improve repeatability of measurements, however, simple tapes without springs are widely used. therefore the procurement of reliable sc data by breed and age is difficult. however, over the years a few researchers have produced useful data. the following table shows sc measurements from several such studies.21 table 1. scrotal circumference data (means, numbers in parenthesis) corrected to 365 ± 14 days of age for bulls from various times and locations. cates 1972-81 sk lunstra 1988 ne barth 1995-98 mb coulter 1981 ab schumann 1990-92 sk arteaga 1998 ab, sk, mb n weighted mean of all bulls mean of research group means simmental 34.8 (376) 33.7 (238) 33.3 (174) 36.0 (401) 33.4 (29) 35.7 (28) 1246 34.7 34.5 br swiss 33.8 (245) 34.1 (15) 260 33.8 33.8 gelbvieh 33.9 (233) 33.7 (13) 32.5 (-) 34.5 (15) 261 33.9 33.6 pinzgauer 33.7 (144) 144 33.7 33.7 charolais 32.0 (576) 31.7 (197) 32.3 (330) 33.1(607) 32.0 (76) 34.7 (101) 1887 32.5 32.6 limousin 29.1 (222) 29.6 (61) 30.3 (276) 30.2 (1) 30.9 (7) 345 29.8 30.0 blonde d’aquit 27.2 (7) 31.9 (8) 15 29.7 29.5 salers 30.2 (40) 29.4 (758) 30.4 (5) 45 29.5 30.0 tarentais 32.0 (14) 14 32.0 32.0 maine anjou 32.1(60) 32.1 (-) 33.7 (4) 64 32.2 32.6 hereford 31.2 (551) 30.4 (256) 32.9 (46) 32.6 (946) 30.5 (24) (33.9) (8) 1567 31.9 31.9 angus 33.1 (703) 32.2 (449) 33.6 (29) 33.9 (260) 32.8 (59) 34.4 (297) 1051 33.2 33.3 shorthorn 31.2 (138) 32.5 (147) 32.8 (20) 167 31.9 32.2 red poll 32.5 (222) 32.9 (28) 250 32.5 32.7 galloway 30.6 (132) 132 30.6 30.6 it would appear from examination of the data in table 1 that the ranking of sc by breed was quite similar between studies and did not change between the 1970’s and the early 1990’s. the latest work shown in table 116 seems to indicate that average sc began to increase in the last half of the 1990’s. a possible explanation for this is that for the decades leading up to 1994, the society for theriogenology system for bull breeding soundness evaluation (bse) was used throughout north america and evidently it did not exert much pressure on breeders to select for sc. in canada, in 1994, a switch was made to the bull bse system of the western canadian association of bovine practitioners (wcabp).21 the wcabp system introduced selection for sc based on age and breed. the back of bse forms provided data on sc and information for producers which encouraged selection for increased sc. furthermore, on the front of bull bse forms, sc was recorded as ‘above average’, ‘average’, ‘below average’ or ‘below 510clinical theriogenology • volume 6, number 4 • december 2014 minimum’. most breeders likely would not want to purchase bulls that were ‘below average’ in sc. perhaps for these reasons, there was a fairly rapid response to selection for increased sc in canada in the later part of the 1990’s and beyond. this supposition would imply that there would not have been a corresponding increase in sc in bulls in the usa. however, there is some sharing of cross-border genetics, particularly in terms of frozen semen sales among purebred breeders and breeders are not bound by bse systems in selection for increased testis size. increase in average testis size of canadian beef bulls in recent years recently (2006-11), sc data were obtained from bulls evaluated for breeding soundness by 41 veterinary practices in western canada and from record of performance test stations from 16 regions of quebec.22 bull age ranged from one to 11 years, however, the majority of bulls were aged one to two years since this is the most common group evaluated for breeding soundness. data were available for 10,968 bulls, 8361 from western canada and 2607 from quebec. twenty-one beef breeds were represented. the mean sc for black and red angus bulls did not differ at any age, thus data were combined under the angus breed. similarly, sc data for polledand horned hereford bulls did not differ at any age and data were combined under the hereford breed. fewer than ten animals were present in each age group for ten breeds including belgian blue, brown swiss, dexter, fleckvieh, maine anjou, piedmontese, red poll, shaver, tarantaise and texas longhorn breeds. therefore, these breeds were not included in any data analysis. scrotal circumference measurements for steel tapes, reliabull tapes and coulter tapes did not differ (p<0.05), but measurements for ‘other’ (homemade tapes) did differ, therefore, data for ‘other’ tapes were not used. scrotal circumference data did not differ between different practices or regions of canada. thus, useful data were available for 10,579 bulls for 11 beef breeds including angus, hereford, simmental, charolais, gelbvieh, limousin, shorthorn, speckle park, blonde d’aquitaine, salers and galloway. testis growth is very rapid from the time of weaning at about seven months of age until maturity at about 15 mo of age. thereafter, testis growth rates decrease quickly and by two years of age about 90% of final testis size is achieved; growth ceases at about four years of age.23 the change in sc per day of age was determined from data reported previously24,25 and from unpublished data (barth). the rate of change in sc does not differ between breeds,26 but rather those breeds that have larger testes at a year of age start out with larger testes at the time of weaning. for example, the sc of limousin bulls averaged 4 cm less at eight months of age than simmental bulls and this difference carried through to 12 months of age.25 the rate of increase in sc (sc/d) between 240 and 365 d of age for seven breeds of beef bulls appeared not to have changed between 1981 (0.0670 cm/day15) and 2000 (0.0647 cm/day25). two studies from 1986-198927,28 showed that the increase in sc between 330 and 430 days was 0.024 to 0.026 cm/day. a subset of data from 2006-2011 study22 for yearling angus bulls, in which age was available in days, indicated that testis growth rate for a similar age group (sc =0.024 cm/day) was in agreement with the earlier data. this gave us some confidence in using the older data for the rate of change in sc in young bulls. table 2 shows the rate of change in sc that was used to correct sc data to 365 days of age (table 3) in yearling bulls for ten beef breeds, angus, hereford, shorthorn, galloway, red poll, charolais, simmental, maine anjou, limousin, and salers.22 511 clinical theriogenology • volume 6, number 4 • december 2014 table 2. estimated increase in scrotal circumference (sc) per month of age for ten breeds of beef bulls. increase in sc age in months daily (cm) monthly (cm) 10-11 0.062 1.86 11-12 0.050 1.50 12-13 0.038 1.14 13-14 0.026 0.78 14-15 0.014 0.42 simmental yearling bulls had a significantly larger average sc than any other breed (table 3). there was no significant difference between angus, charolais and gelbvieh breeds. although, not different than gelbvieh, herefords had a significantly smaller sc than angus and charolais. speckle park, salers, limousin and blonde d’aquitaine had a significantly lower sc than previously mentioned breeds, but were not different from each other. table 3. the mean (± sd) scrotal circumference corrected to 365 days of age for yearling bulls of 10 common beef breeds in canada. breed n mean ± sd simmental 1050 36.06 ± 2.50a angus 3004 34.62 ± 2.21b charolais 1469 34.55 ± 2.82b gelbvieh 190 34.41 ± 2.63bc hereford 272 33.93 ± 2.41cd shorthorn 55 33.03 ± 1.60de speckle park 55 32.04 ± 1.77ef salers 31 31.84 ± 2.84 ef limousin 290 31.52 ± 2.22f blonde d’aquitaine 16 31.24 ± 2.13f a,b different superscripts indicate significant differences (p<0.05). scrotal circumference data for bulls ≥2years of age were not corrected, however, for bulls recorded as two years old, age was known to be accurate within ± 2 months of two years. at two years of age, gelbvieh, charolais and simmental had a significantly larger sc than all other breeds with the exception of herefords (table 4). the mean sc for angus, hereford, galloway and shorthorn did not differ. limousin and speckle park bulls had significantly smaller sc than most breeds except for galloway and shorthorn. 512clinical theriogenology • volume 6, number 4 • december 2014 table 4. the mean (± sd) scrotal circumference for 2-year-old bulls (± 2 months) of 9 common beef breeds in canada. breed n mean ± sd gelbvieh 43 40.17 ± 2.53a charolais 271 39.28 ± 2.83a simmental 223 39.19 ± 2.58a hereford 268 39.15 ± 2.69ab angus 1031 38.55 ± 2.45b galloway 23 38.39 ± 2.97bc shorthorn 40 37.86 ± 2.15bc limousin 80 35.72 ± 2.05c speckle park 21 35.43 ± 2.52c a,b different superscripts indicate significant differences (p<0.05). tables 5 and 6 provide data for three-year-old bulls and for bulls ≥4 years of age. the differences in mean sc among breeds are not very large and therefore, breed rankings based on sc in these tables may be due small sample sizes and other population differences within regions from year to year. table 5. the mean (± sd) scrotal circumference for three-year-old bulls of six common beef breeds in canada. breed n mean ± sd charolais 114 41.09 ± 2.47a gelbvieh 28 40.48 ± 2.72a simmental 193 40.30 ± 3.06a angus 489 39.91 ± 2.67ab hereford 49 39.48 ± 2.55b limousin 56 36.67 ± 2.53c a,bdifferent superscripts indicate significant differences (p<0.05). table 6. the mean (± sd) scrotal circumference for • 4-year-old bulls of six common beef breeds in canada. breed n mean ± sd simmental 249 43.05 ± 2.50a charolais 150 42.29 ± 2.56ab angus 624 41.78 ± 2.68b gelbvieh 44 41.48 ± 2.83b hereford 89 41.12 ± 2.41b a,bdifferent superscripts indicate significant differences (p<0.05). the means for sc for oneand two-year-old bulls from 1972-1998 was determined from published reports21 and compared to data from 2006-201122 in tables 7 and 8. there has been a significant increase in sc over time for all breeds shown in yearling and two-year-old bulls. the largest increase in yearling bulls was seen for the hereford and charolais breeds, and the smallest increase was seen in the shorthorn and gelbvieh breeds. this might be because the gelbvieh breed was uncommon in the earlier decades, but became very common in the last two decades, whereas, the shorthorn breed was common in the early decades and very uncommon in the last two decades. thus differences in sc based on large amounts of data may not be comparable between the earlier decades and the last two decades in these two breeds. the hereford and charolais breeds have been in common use for the past four decades, thus the results of selection pressure for sc would be easily detectable. at two years of age, the increase in average sc was greatest in the limousin breed which has also been in common use for the past four decades. 513 clinical theriogenology • volume 6, number 4 • december 2014 table 7. difference in mean sc in one-year-old bulls of seven beef breeds determined from data of six researchers from 1972 to 199821 and data from 2006-2011.22 *mean from the present study differs from previously reported means (p<0.05). table 8. difference in mean sc in two-year-old bulls of six beef breeds from 1987 data29 and data from 2006-2011.22 *mean from the present study differs from previously reported means (p<0.05). during the period from 1994 to 2013, in the method of bull bse of the wcabp,21 the suggested minimums in sc for a satisfactory classification were established by disqualifying bulls that were below one standard deviation of the mean for their breed and age. in 2012, the wcabp established a committee to study the recent sc data and to make recommendations on minimum sc by age and breed for the future. the data indicated that some of the breed rankings had changed and for some of the breeds, especially the most common breeds used in the past two decades, the minimum sc should be increased. the committee felt that a great deal of progress already had been made in selection for testis size and to continue to disqualify bulls in the bottom standard deviation for sc likely placed more selection pressure than necessary. it was suggested that bulls in the bottom tenth percentile for sc should be disqualified for a satisfactory classification in the future. there were insufficient data to make recommendations for many breeds that are not present in large numbers in canada at the present time. for these breeds the wcabp committee recommended that sc data from studies prior to the year 2000 should likely continue to be used for breeding soundness evaluations. table 9 shows the recommended minimums for sc by age and breed adopted by the wcabp for bull bses going forward. table 9. suggested minimum scrotal circumference (cm) for 2014 and forward. age (mo) simmental, angus, charolais, gelbvieh, pinzgauer, b. swiss/braunvieh hereford, shorthorn, maine anjou, red poll, s. devon, holstein speckle park, salers, tarentais limousin, blonde d’aquitaine, galloway texas longhorn 12 32 31 30 29 28 13 33 32 31 30 29 14 34 33 32 31 30 15 34.5 33.5 32.5 31.5 30.5 16-20 35 34 33 32 31 21-≥30 35 35 34 33 32 some have expressed their belief that the beneficial effect of sc on age at puberty has already been sufficiently achieved for many breeds and fear that continued selection for larger sc for all breeds may lead to semen quality issues in bulls with excessively large testes. these are valid questions and breed mean sc (cm) 1972-1998 mean sc (cm) 2006-2011 difference (cm) angus 33.3 34.6 1.3* charolais 32.6 34.6 2.0* gelbvieh 33.6 34.4 0.8* hereford 31.9 33.9 2.0* limousin 30.0 31.5 1.5* shorthorn 32.2 33.0 0.8* simmental 34.5 36.1 1.6* breed mean sc (cm) 1987 mean sc (cm) 2006-2011 difference angus 37.2 38.6 1.4* charolais 36.3 39.3 3.0* gelbvieh 40.2 hereford 36.1 39.2 3.1* limousin 32.2 35.7 3.5* shorthorn 34.9 37.9 3.0* simmental 38.8 39.2 0.4* 514clinical theriogenology • volume 6, number 4 • december 2014 going forward, research on the effect of testis size on both age of puberty and semen quality should be continued. it would appear that at least for breeds that have smaller testes, e.g., those in the last three columns of table 9, progress in selection for increased testis size is still needed. at the present time, a small amount of data exists in which sc of angus and charolais bulls aged 13.5 to 15 months was grouped as 30-32.9 cm, 33-35.9 cm, and so on, up to 44.9 cm. preliminary analysis showed that fewer bulls had satisfactory semen quality when sc was 30-32.9 cm. there were no differences in the proportions of bulls with satisfactory semen quality in the other sc groups; however, there were only four bulls in the group that had a sc of 42-44.9. references 1. day ml, nogueria gp: management of age at puberty in beef heifers to optimize efficiency of beef production. anim front 2013;3:6-11. 2. byerley dj, staigmiller rb, berardinelli jg, et al: pregnancy rates of beef heifers bred either on puberal or third estrus. j anim sci 1987;65:645-650. 3. burris mj, priode bm:. effect of calving date on subsequent calving performance. j anim sci 1958:17:527-533. 4. lesmeister jl, burfening pj, blackwell rl: date of first calving in beef cows and subsequent calf production. j anim sci 1973;36:1-6. 5. gregory ke, lunstra dd, cundiff lv, et al: breed effects and heterosis in advanced generations of composite populations for puberty and scrotal traits of beef cattle. j anim sci 1991;62: 2795-2807. 6. thallman rm, cundiff lv, gregory ke, et al: germplasm evaluation in beef cattle cycle iv: postweaning growth and puberty of heifers. j anim sci 1999;77:2651-2659. 7. wiltbank jn, gregory ke, swiger la, et al: effects of heterosis on age and weight at puberty in beef heifers. j anim sci 1966;25:744-751. 8. arije gf, wiltbank jn: prediction of age and weight at puberty in beef heifers. j anim sci 1974;38:803-810. 9. lunstra dd, ford jj, echternkamp se: puberty in beef bulls: hormone concentrations, growth, testicular development, sperm production and sexual aggressiveness in bulls of different breeds. j anim sci 1978;46:1054-1062. 10. brinks js, mcinerney mj, chenowith pj: relationship of age of puberty in heifers to reproductive traits in young bulls. proc west sect am soc anim sci 1978;29:28. 11. king rg, kress dd, anderson dc, et al: genetic parameters in herefords for puberty in heifers and scrotal circumference in bulls. proc west sec anim sci 1983;34:11. 12. lunstra dd:. testicular development and onset of puberty in beef bulls. beef res prog rep no 1. clay center (ne): us meat res cent arm-nc-21; 1982. p. 28. 13. gregory ke, lunstra dd, cundiff lv, et al: breed effects and heterosis in advanced generations of composite populations for puberty and scrotal traits of beef cattle. j anim sci 1991;69:2795-2807. 14. toelle vd, robison ow: estimates of genetic correlations between testicular measurements and female reproductive traits in cattle. j anim sci 1985;60: 89-100. 15. cates wf: observations on scrotal circumference and its relationship to classification of bulls. proc annu meet soc therio; 1975. p. 1-18. 16. arteaga aa, baracaldo m, barth ad: the proportion of western canadian beef bulls with mature spermiograms at 11 to 15 months of age. can vet j 2001;42:783-787. 17. moser dw, bertrand jk, benyshek ll, et al: effects of selection for scrotal circumference in limousin bulls on reproductive and growth traits of progeny. j anim sci 1996;74:2052-2057. 18. almquist jo, branas rj, barber ka: postpuberal changes in semen production of charolais bulls ejaculated at high frequency and the relation between testicular measurements and sperm output. j anim sci 1976;42:670-676. 19. coulter gh, foote rh: bovine testicular measurements as indicators of reproductive performance and their relationship to productive traits in cattle: a review. theriogenology 1979;11:297-311. 20. smith mf, morris dl, amoss ms, et al:. relationships among fertility, scrotal circumference, seminal quality, and libido in santa gertrudis bulls. theriogenology 1981;16:379-397. 21. barth ad: bull breeding soundness evaluation. 2nd ed. saskatoon (sk): western canadian association of bovine practitioners; 2000. 22. garcia-guerra a, hendrick s, barth ad: increase in average testis size of canadian beef bulls. can vet j 2013;54:485490. 23. coulter gh: puberty and postpuberal development of beef bulls. in: morrow da, editor. current therapy in theriogenology. 2nd ed. philadelphia: wb saunders; 1986. p. 142-148. 24. cates wf, nicholson hh, crow gh, et al: testicular development in record of performance bulls. proc annu meet soc therio; 1981. p. 16-30. 25. barth ad, ominski kh: the relationship between scrotal circumference at weaning and at one year of age in beef bulls. can vet j 2000;41:1-6. 26. lunstra dd, gregory ke, cundiff lv: heritability estimates and adjustment factors for the effects of bull age and age of dam on yearling testicular size in breeds of bulls. theriogenology 1988;30:127-136. 27. smith ba, brinks js, richardson gv: relationships of sire scrotal circumference to offspring reproduction and growth. j anim sci1989;67:2881-2885. 515 clinical theriogenology • volume 6, number 4 • december 2014 28. bourdon rm, brinks js: scrotal circumference in yearling hereford bulls: adjustment factors, heritabilities and genetic, environmental and phenotypic relationships with growth traits. j anim sci 1986;62:958-967. 29. coulter gh, mapletoft rj, kozub gc, et al: scrotal circumference of two-year-old bulls of several beef breeds. theriogenology 1987;27:485-491. 516clinical theriogenology • volume 6, number 4 • december 2014 2012: preliminary report on scrotal circumference and semen parameters in fertile adult omani rams preliminary report on scrotal circumference and semen parameters in fertile adult omani rams mushtaq a. memon,a musab h. al-busaidi,b osman m. gaafarb adepartment of veterinary clinical sciences, washington state university, pullman, wa; bdepartment of animal and veterinary sciences, sultan qaboos university, al-khoud, muscat, sultanate of oman abstract sheep are one of the important livestock species in the sultanate of oman. there is one major breed of sheep in oman. at present, the breeding rams are selected on the basis of physical appearance. no breeding soundness examination (bse) is performed before animals are used for breeding. the objective of the preliminary study was to evaluate reproductive potential of omani rams utilizing established criteria for bse. eleven rams one to five years old were evaluated for breeding soundness, including physical examination, body weight (bw), scrotal circumference (sc), semen collection and evaluation. scrotal circumference (25.50 ± 1.52 cm) of rams weighing 54.9 0kg (± 1.70) was smaller than reported in other breeds. semen parameters including volume (0.84± 0.12 ml), sperm mass activity (2.64±0.17 of 3.0), sperm motility (76.82 ± 2.05%), sperm concentration (1.91± 0.31 billion/ml), and normal sperm (97.18 ± 0.33%) were within normal range. study of larger number of rams is needed to establish the minimum and maximum bse criteria for omani rams. keywords: omani rams, breeding soundness examination, reproductive evaluation introduction sheep are one of the important livestock species in oman; the second in number (351,066), after goats (15,571,148), followed by cattle (301,588), and camels (117,299) in the sultanate of oman.1 there is one major breed of sheep in oman2. they are predominantly black but some white sheep are observed. they are thin-tailed, small in size; males are mostly horned and females polled.3 evaluation of the male for breeding soundness is an important part of reproductive management of a flock. breeding soundness evaluation also is a first step in semen preservation for a long-term preservation of genetic material and preservation of biodiversity. evaluation of the male for breeding soundness is based upon sc, sperm motility and morphology of semen. it has been shown that highly fertile rams settled ewes earlier in the breeding season resulting in producing more twins. the quality of rams’ semen was significantly correlated with the twinning rate.4 since a large number of high quality spermatozoa is necessary for successful breeding in sheep,5 ewe fertility is closely related to sperm production in the ram. currently, in oman, the breeding rams are selected only on basis of physical appearance. taller and heavier rams are preferred. no bse is performed before rams are used for breeding on the government farms or sold to private farm owners for improvement of their sheep production. the society for theriogenology (sft) has recommended measurable bse criteria, including sc, sperm motility and sperm morphology in the bull and the ram.6,7 based upon sft criteria, bulls are declared satisfactory, questionable and unsatisfactory potential breeders. although limited information is available on various aspects of bse of different sheep breeds, no reports are available on omani rams. the objectives of the current preliminary study were to evaluate reproductive potential of native omani rams utilizing established criteria of bse. to the best of our knowledge, this is the first report on bse of the native omani rams. materials and methods eleven fertile adult rams one to five years old were evaluated for breeding soundness. the rams were a part of the flock used for breeding ewes on two farms. the rams were weighed and the scrotum was examined for presence of two normal testes. scrotal circumference was measured using a standard scrotal metal-tape (nasco, ft. atkinson, wi) at the widest mid-scrotal point. semen was collected using an electroejaculator (ee; standard precision electronics, denver, co) as described earlier.8 semen volume and sperm mass activity, motility and morphology were evaluated by established techniques.8,9 in summary, sperm mass activity was evaluated by placing a drop of undiluted semen on pre-warmed clinical theriogenology • volume 4 number 2 • june 2012159 microscope slide and evaluated for wave motion under a microscope. the mass activity was graded from 0 to 3 (0 – no activity, 1 – slow/irregular, 2 – moderate, 3 – fast wave motion). for sperm motility, a small portion of semen was diluted 1:10 (semen: normal saline). a drop of diluted semen was placed on pre-warmed microscope slide, covered with cover slip and evaluated under microscope as percentage of sperm moving in forward progressive manner. sperm concentration in each ejaculate was calculated by utilizing dilution pipettes (platelett unopette®, b-d #5855, becton, dickinson and company, franklin lake, nj) and a hemocytometer (exodus breeders corporation, york, pa) and the number of sperm per ml was calculated. for sperm morphology, a drop of semen was placed close to an end of a microscope slide and mixed with a drop of eosin-nigrosin stain (society for theriogenology, montgomery, al) and smear was made. the smear was air-dried, and sperm were evaluated under an oil immersion lens of a microscope at 1000x. two hundred sperm were evaluated and abnormalities were classified according to barth and oko.10 the primary abnormalities were considered as sperm head abnormalities, proximal cytoplasmic droplet, abnormal mid-piece, and tightly coiled tails; while the secondary abnormalities included distal cytoplasmic droplet, bent tails, and loose heads. results table 1 summarizes the mean (± se) bw, sc and semen evaluation parameters (semen volume; sperm mass activity, motility, concentration, and normal sperm). the semen parameters represent all semen samples collected by ee. table1. breeding soundness examination parameters of 11 native omani rams bw (kg) sc (cm) s. vol (ml) s. ma (03) s. mot (%) s. conc (bill/ml) n. sperm (%) mean 54.90 25.50 0.84 2.64 76.82 1.91 97.18 se mean 1.70 1.52 0.12 0.17 2.05 0.31 0.33 minimum 47.20 23.50 0.30 1.50 65.00 0.50 95.00 maximum 62.50 29.00 1.60 3.00 90.00 3.60 99.00 sc= scrotal circumference, bw=body weight, s. vol=semen volume, s. ma=sperm mass activity, s. mot=sperm motility, s. conc=sperm concentration, n. sperm=normal discussion the average sc (25.50 ± 1.52 cm) of rams weighing 54.90 ± 1.70 kg were smaller than reported in other breeds,11,12 however the sc shows marked variation in seasonal breeds. differences of up to 30% in testicular volume have been reported between spring and autumn.13 no seasonal effect on reproduction is reported in omani rams. scrotal circumference greater than 33 cm is recommended for adult rams.11 others have recommended sc greater than 36 cm for shearlings and 32 cm for ram lambs.14 however, minimum values for classifying an animal based upon sc are controversial, as they can vary due to age, season, breed and individual variation.15 smaller sc found in this study may be appropriate for omani rams, as they were with flocks of ewes which were pregnant. studies with larger number of rams are needed to establish the minimum and maximum sc in omani rams. semen parameters, including volume (0.84± 0.12 ml), sperm mass activity (2.64±0.17 of 3.0), sperm motility (76.82 ± 2.05%), sperm concentration (1.91± 0.31 billion/ml), and normal sperm (97.18 ± 0.33%) were within normal range as found in previous studies.4,5,7 acknowledgements this research project was conducted as a part of the study entitled “characterization, evaluation and conservation of indigenous animal genetic resources in the sultanate of oman,” supported by the government of the sultanate of oman. the authors appreciate dr. ahmed tibary’s help in data analysis. references 1. anonymous: agriculture census 2004 -2005. ministry of agriculture, sultanate of oman, 2005. clinical theriogenology • volume 4 number 2 • june 2012 160 2. mahgoub o, kadim it, al-dhagab a, et al: an assessment of omani native sheep fiber production and quality characteristics. j agric marine sci 2010;9-14. 3. anonymous: livestock sector review and project identification final report, vol 1. the sector review. a report prepared for oman ministry of agriculture and fisheries by hunting technical services ltd and sudanese investment and consultations co. ltd. muscat, oman. p. 103. 4. hulet cv: prediction of fertility in rams: factors affecting fertility and collection, testing, and evaluation of semen. vet med small anim clin 1977;72:1363-1367. 5. colas g, court m: production of spermatozoa, storage of semen and artificial insemination in the sheep. proc symp management of reproduction in sheep and goats, university of wisconsin, madison, 1977. p. 31-40. 6. elmore rg, bierschwal cj, martin ce, et al: a summary of 1127 breeding soundness examinations in beef bulls. theriogenology. 1975;3:209-218. 7. ott rs, memon ma: breeding soundness examinations of rams and bucks: a review. theriogenology 1980;13:155160. 8. memon ma, bretzlaff kn, ott rs: comparison of semen collection techniques in goats. theriogenology 1986;26:823827. 9. memon ma, mickelsen dm, goyal ho: examination of the male reproductive tract and evaluation of potential breeding. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. philadelphia: wb saunders; 2007. p. 515-518. 10. barth ad, oko r: abnormal morphology of bovine spermatozoa. ames: iowa state university press; 1989. 11. kimberling cn, parsons ga: breeding soundness evaluation and surgical sterilization of the ram. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. philadelphia: wb saunders; 2007. p. 620-628. 12. söderquist l, hultén f. normal values for the scrotal circumference in rams of gotlandic breed. reprod domest anim 2006;1:61-62. 13. rosa hjd, bryant mj: seasonality of reproduction in sheep. small rumin res 2003;48:155-171. 14. scott pr: breeding soundness examination in rams. in: sheep medicine. london: mason publishing ltd; 2007. p. 7476. 15. ridler al, smith sl, west dm: ram and buck management. anim reprod sci 2012;130:180-183. clinical theriogenology • volume 4 number 2 • june 2012161 1 contact jessica klabnik jlk0066@auburn.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12959, http://dx.doi.org/10.58292/ct.v17.12959 review report advances in complementary livestock artificial reproductive techniques jessica klabnik,a julia duboisb adepartment of clinical sciences, auburn university, auburn, al, usa bsilver rose equestrian center, llc. kodak, tn, usa abstract assisted reproductive techniques are beneficial to increasing animal agricultural production and improving veterinary medicine. foundational techniques (e.g. artificial insemination and in vitro embryo production) have paved the way for both complementary and adjunct techniques to arise as technologies and research advance. female/offspring focused technologies include those to increase precision of estrus detection, genomic selection through embryo biopsy, and gene editing via ‘clustered regularly interspaced short palindromic repeats’ (crispr) and crispr associated protein 9 (cas9), whereas male focused technologies include sex sorting after freezing and thawing (‘reverse sex sorted semen’), multisire straws for artificial insemination, and spermatogonial stem cell transplantation. although improvement in efficiency and feasibility is paramount, these recently developed and emerging techniques will likely become important to ensuring that animal products are produced efficiently and humanely. this review highlights these newer advances, with a particular focus on how they may impact mammalian agriculture and veterinary medicine. keywords: assisted reproductive techniques, crispr-cas9, embryo biopsy, estrus detection, reverse sex sorted sperm, spermatogonial stem cell transplant introduction assisted reproductive techniques (arts) include any technique and/or technology that changes the expected outcome of natural service. purposes of arts in agricultural veterinary medicine and/or animal production include increasing reproductive efficiency, preserving genetics, and treating infertility.1,2 incorporation of arts to increase reproductive efficiency includes greater geographic dissemination of genetics,2 promoting earlier conception,3 increasing an individual’s number of offspring,4 and combating negative consequences of heat stress.5 the goal of genetic preservation in animal production is often related to dissemination of genetics in the relatively near future. however, in certain cases, such as heritage breeds, the goal is to maintain or promote genetic diversity in the immediate and more distant future.1 infertility treatment is a less common purpose of arts in production animals compared to companion animals or humans, but may still occur for high-value individuals whose cause of infertility was not identified. heritable conditions that impact fertility and/or performance of offspring may be grounds for classifying an animal as unsatisfactory during routine breeding soundness examinations.6 ultimately, utilization of arts for infertility treatment in production animals is only recommended when the cause of infertility is not heritable, when the fertility of offspring is not important (e.g. terminal-use sires) or in the case of extremely limited genetic diversity. synchronization of estrus and ovulation,7 artificial insemination with or without the use of sex sorted semen,8 multiple ovulation embryo transfer, and in vitro embryo production and transfer4 are currently the most commonly used arts. these techniques, along with somatic-cell nuclear transfer9 have been extensively reviewed. the purpose of this review article is to highlight and expand on arts that have arisen from foundational techniques, with a particular focus on how they may impact mammalian agriculture and veterinary medicine. these arts include: 1. those that increase precision of estrus detection; 2. genomic selection through embryo biopsy; 3. gene editing and ‘clustered regularly interspaced short palindromic repeats’ (crispr) and crispr associated protein 9 (cas9); 4. reverse sex sorted semen; 5. multisire straws for artificial insemination; and 6. spermatogonial stem cell transplantation. mailto:jlk0066@auburn.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12959 2 citation: clinical theriogenology 2025, 17, 12959, http://dx.doi.org/10.58292/ct.v17.12959 increasing precision of estrus detection estrus detection was vital to reproductive efficiency when artificial insemination was first implemented and still is critical in many dairy systems that are not utilizing fixed time insemination. estrus detection is traditionally time consuming, with visual observation of the animal particularly difficult in pasture-based management systems. the simplest technique for improvement of estrus detection is to implement tail-head patches and/or paints that change colors or wear off when the female is repeatedly mounted and are commercially available in a variety of styles. imprecise interpretation of when a patch is ‘triggered’ for artificial insemination can lead to variation of pregnancy outcomes, along with reduced reproductive and economic efficiency. certain patch style designs attempt to combat this ambiguity. for example, heat seekertm patches (beacon automation, muswellbrook, nsw, australia) are curved to the back to watch progression of estrus and have a larger surface area for detection. however, these still require regular visual observation of the animals and reapplication maintenance when patches fall off, either due to poor adhesion or normal wear and tear. heatwatch® (abs, deforest, wisconsin, usa) was the first patented patch technology that radiotransmitted the number of times and duration of mounting to computer software. this technology reduced the need to visually assess animals and minimized ambiguity in detecting estrus, but still required labor to analyze data on a computer. other challenges of this technology were the short transmission radius, patches falling off, and expense, resulting in this technology being no longer commercially available. however, revival and progression of this technology have recently emerged; heatsiecker® (heatsiecker, martell, nebraska, usa) has an expected 1-2 miles radius and sends data in real-time to a cell phone app and utilizes a smaller and more cost-effective patch. initial set up carries an expense due to purchasing the antenna utilized to send the data, but the patches utilized are not of much greater expense to those already on the market. users are reporting that convenience and accuracy are reasons they will continue to use the system. precision livestock farming is the concept of using technology to constantly monitor production animals to better assess health, welfare, production, and calving.10,11 for estrus detection, this includes pedometers/accelerometers as a tool to detect estrus-related increases in activity.12 rumination data have been investigated for estrus detection, based on decreases in feeding and rumination.13,14 activity monitoring can be a challenge due to the variability between animals and among housing systems. regardless, other physiologically based technologies are likely to follow. one potential physiological marker that could be capitalized on includes increases in basal body temperature at and around estrus.15 current temperature assessment tools include rumen boluses,16 subcutaneous rfid biosensor device,17 and tail-head surface temp via wearable sensor18; these currently work well for detection of heat stress or diseases but are not sensitive nor specific enough for practical estrus detection without further calculation and analysis. infrared thermography has been proposed as an estrus detection tool due to its ability to evaluate the whole animal and could be utilized either in a barn setting or pasture setting via infrared thermography cameras that are low maintenance, noninvasive and therefore relatively stress free on the animal, and low in labor input.19 other physiologic parameters investigated, but currently not practical, include vaginal conductivity, pheromones detected via trained dogs, and milk progesterone concentrations.20 the recent development and widespread implementation of artificial intelligence presents a unique opportunity to improve estrus detection utilizing a combination of physiological data types, resulting in a practical report for the producer. ultimately, these combinations of technologies, in conjunction with the development of artificial intelligence, will provide more accurate detection of estrus and increase reproductive efficiency. genomic selection through embryo biopsy embryo biopsies are typically performed on morula or blastocyst stage embryos after either an embryo flush or in vitro production. the goal of a biopsy is to remove enough cells needed for further analyses without damaging the embryo. embryos are then frozen while awaiting results of the desired analyses. viability and pregnancy rates of grade 1 and grade 2 (by international embryo technology society standards) were not affected by embryo biopsy for either in vivo or in vitro produced embryos.21 however, a decline in viability due to embryo biopsy may occur in grade 2 in vivo produced embryos compared to grade 1 embryos.22 method of choice for cell removal depends on the stage of embryo and/or technician experience and training22; acquired cells can be directly used for some single-trait analyses (e.g. sex determination) via polymerase-chain reaction. if the number of cells does not provide enough starting material of dna to be used directly, whole genome amplification is performed prior to genomic analysis.23 genomic analysis is performed utilizing single-nucleotide polymorphisms (snp)-chips of various densities; density refers to the snps directly tested with the specific chip. first, the genotype of each snp is determined; proportion of snps that give a result (a.k.a. call rate) can range from ~ 75-95%.22 next, the rest of the genomic sequence is inferred from reported whole genomes. this technology depends on the fact that nucleotide sequences that exist close together on a chromosome are more likely to be transferred together during crossing-over events than sequences that exist farther apart or are on different chromosomes. therefore, when 1 snp is determined, the sequences that are likely to be next to it (i.e. have been correlated with that snp) can be inferred (i.e. imputation).24 outcomes can then be used to generate breeding value estimations or provide direct information regarding sex, color, poll-status, milk protein variants, or genetic disease status. although embryo biopsy and genomic selection can be individually utilized, the combination of these 2 technologies is particularly powerful. considerable genetic gain, the amount of increase in performance achieved per unit of time, results from increasing selection combined with a reduced generation interval. the generation interval is further reduced when biopsied embryos are generated from prepubertal heifers via  oocyte-pick up and in vitro embryo production.25 intramuscular kisspeptin treatment has been used to manage puberty in livestock by inducing luteinizing hormone surge26 and could be used to promote ovarian function in prepubertal females (5-10 months) resulting in mature oocytes. combining this with round spermatids of immature males for intracytoplasmic sperm injection27 results in multidirectional acceleration of the generational interval. the integration of genomics with such technologies can provide direct information regarding sex, color, poll-status, milk protein variants, or genetic disease status. it can also be used to generate breeding value estimations from equations for a specific trait on progeny’s performance by removing environmental factors.22 the resulting embryos could be transferred to a mature recipient http://dx.doi.org/10.58292/ct.v17.12959 citation: clinical theriogenology 2025, 17, 12959, http://dx.doi.org/10.58292/ct.v17.12959 3 animal, hastening access to desired genetic traits. altogether, this technology allows for genetic progression of valuable donor animals before they are mature by transferring their embryos to mature recipients, which have had successful pregnancies, to act as surrogates.27 as there are several steps in genomic selection through embryo biopsy, errors can occur at many places. technician errors are possible in terms of labeling or damage to the embryo. errors in genotyping can occur when cells are not representative of the whole animal, such as in mixoploidy cells. the most common error that occurs during enzymatic-preamplification is when only 1 allele is amplified. this leads to a loss of heterozygosity and is commonly called allele-drop out.22 if both sire and dam are already genotyped, the imputation step itself can be very accurate (~ 99%). overall accuracy increases with increasing density of the snp-chip28 and when the reference population is larger.29 in general, overall accuracy of the snp call rate is very high (90-> 99%).22,30 the accuracy of estimated breeding values also depends on the heritability of the trait and the number of progeny but may range 0.65-0.85.31 what may be of more practical use at this stage is increased genetic testing and creating databases of dams and sires to improve future offspring. a national or global database can be beneficial for all breeds and species as it can help producers make the best decisions. with the capabilities to ship semen anywhere in the world, the ability to find the best genetics for increased production can be of great benefit. some examples are cattle that are more heat tolerant and genetic disposition for twins or triplets in sheep or swine with certain leptin receptor gene alleles to improve meat quality.32 knowing which sires have those genes can allow for their introduction into the herd or flock without extensive on-farm genetic testing. gene editing and clustered regularly interspaced short palindromic repeats and associated protein 9 a group of technologies that could have a large impact on livestock production and reproduction are those that enable gene editing. although gene editing is unlikely to occur on the farm itself, it could become important in the selection of replacement animals, as some modifications can be inherited. gene editing, genetic modification, genetic engineering and transgenics are all terms to describe changing the genomic sequence of an organism. once the genome is modified, the organism may be called a genetically modified organism (gmo), transgenic organism, and/or a genetically engineered organism. previously, several generations of focused breeding were required to incorporate a new trait and it was often difficult to do without impacting other traits. gene editing can produce livestock with a desired specific trait while minimizing unintended consequences such as decreased resistance to disease.33 when gene editing results in a modification that can then be inherited by the genetically edited animal’s offspring, the procedure may be termed germline editing.34 gene editing technologies act as scissors to cut dna and then either add, delete, or change the sequence at that location. originating gene editing technologies include zinc finger nucleases starting in 1985, followed by transcription activator-like effector nucleases. however, in 2012 the discovery of the crispr/cas9 system for gene editing quickly became the most common method because of its efficiency, ease of use, and lower costs.34–36 the crispr/cas9 system was ‘discovered’ because it is based on the natural system that bacteria and archaea use to protect themselves against viruses; crispr are essentially repeated sequences of genetic code. the first cripsr rna (crrna) recognizes the target site that is ~ 20 nucleotides in size. the second rna, called a trans-activating crispr rna (tracrrna), hybridizes with the crrna. this essentially tells the crispr-cas9 enzyme where to cut in the dna. once complexed with the rnas, the cas9 nuclease then cleaves the dna strands. for the purpose of gene editing, a new sequence can be incorporated if desired when the dna repairs itself. the 2 crispr rnas needed are designed with online tools to minimize potential off-target sites and estimate efficiency.33 gene editing has been proposed for several livestock species with several broad purposes. gene editing could impact complex traits, such as reproductive traits, by helping to select for several genes simultaneously.37 an example of successful gene editing is pigs that are genetically modified to lack the receptor for the porcine reproductive and respiratory syndrome (prrs) virus were completely resistant to the disease,33,38 increasing reproductive efficiency. another example is poll status that has been modified in cattle with gene editing technology to improve animal welfare by removing the need to dehorn.36 another purpose of gene editing is to affect the composition or palatability of the final agricultural product. for example, cattle and goats have been genetically modified to lack beta-lactoglobulin in their milk; this protein is most commonly responsible for milk allergy in humans.33 increased production and increased production efficiency are 2 prominent goals of genetic modification in livestock. this may be through enhanced disease resistance, such as decreasing susceptibility of pigs to coronavirus, african swine fever, and senecavirus a resistance.34 research is ongoing regarding reducing myostatin expression with gene editing. myostatin negatively regulates skeletal muscle growth and is responsible for the ‘double muscling’ of belgian blue and piedmontese cattle. gene editing technology has been used to produce myostatin knockouts of cattle, pigs, sheep, goats, and tilapia.33 although the aforementioned purposes are the current focus of the majority of genetically modifying livestock, another possibility is the use of this technology to treat diseases (called ‘gene therapy’). globally, there are many regulatory agencies with oversight of gmos to ensure safety of the products and the environmental sustainability.35 in the us, regulation of agricultural gmos for agricultural purposes is coordinated through fda, usda, and the epa.39 although this review focuses on agricultural mammals, it is important to note that there are only 3 gmo animal products currently commercially available. the first is the aquadvantage salmon® (aquabounty, harvard, ma, usa) that have increased feed efficiency and grow more quickly than conventional salmon.40 the second is galsafetm pigs (revivicor inc., blacksburg, va, usa), edited to have an inactive ggta1 gene.34,41 by inactivating the ggta1 gene, the meat from these pigs does not contain galactose-alpha-1,3-galactose, commonly called ‘alpha-gal’, responsible for alpha-gal syndrome in humans and contributing to meat consumption allergies. as a result of this technology, people with alpha-gal syndrome are able to consume pork products or receive allergy-free pharmaceuticals and medical implants derived from these pigs.41–43 http://dx.doi.org/10.58292/ct.v17.12959 4 citation: clinical theriogenology 2025, 17, 12959, http://dx.doi.org/10.58292/ct.v17.12959 approval by the fda to use prrs-resistant pigs for these purposes was granted in april 2025.38,44 postthaw sex sorted sperm since becoming commercially available in 2003, sexed semen for cattle has grown in popularity. this is unsurprising as the difference in value between offspring of 1 sex versus the other can be quite substantial in certain industries.45 traditionally, the decision to offer sexed semen needed to be made prior to freezing. however, the ability to sort sperm after thawing allows for delaying the decision about offering sexed semen commercially, therefore increasing the number of sires with sexed semen available. when semen is frozen, thawed, and then sorted in the industry it is called ‘reverse sex sorted (rss) semen’. although semen is typically not washed during cryopreservation and therefore some seminal plasma is frozen with the sperm, it is truly the sperm that are sorted, leading to the ambiguity between the industry term and the more scientifically accurate rss sperm. the process of creating rss semen is similar to traditional sorting that has been reviewed.8 in very brief terms, sperm dna is stained with hoechst 33342 that fluoresces relative to the amount of dna present. utilizing flow cytometry, differences in fluorescent intensity are measured. sperm flow single file through charged plates that direct sperm into 2 populations. the main differences in technology between traditional and rss semen is how the semen is handled prior to and after sorting.8 reverse sex sorted semen must initially be removed of freezing extenders, sorted, and then is not currently refrozen in cattle. a single report after refreezing does exist, with efficiency that is not up to industry standards.46 it has been successfully refrozen in the ram.47 when using the same number of motile sperm (15 x 106), pregnancy rates (p > 0.05) were similar between controlled (i.e. frozen-thawed; 59.5%), sex-sorted frozen-thawed (51.3%), and frozen-thawed, sex-sorted and refrozen-thawed (38.9%) groups. offspring of both sex-sorted groups were > 92% of the desired sex.47 future studies could explore these species-specific differences in order to make refreezing a possibility in cattle production. in the last 20 years, advances in sperm handling and preparation for sorting have increased such that fertility of sex-sorted sperm in cattle approaches that of conventional straws. however, since rss sperm is no longer frozen, application for artificial insemination is limited.8 therefore, rss sperm is currently only used for in vitro production of embryos. new discoveries in semen handling at cooled (5°c) or at room temperature with the addition of extenders up to 6 hours could make sorting prior to freezing more accessible.48 conventionally, 2 frozen straws are used to make 1 dose of rss sperm for in vitro embryo production, but this can vary with sperm number and quality. the blastocyst rate and quality are similar to conventionally sorted when rss sperm used for in vitro embryo production. it is typically processed at ~ 90% purity. (m. hockett, personal communication, august 15, 2022). although rss sperm is currently not refrozen due to extremely low efficiency, a single calf has been generated from refrozen rss sperm (i.e. frozen-thawed-sorted-refrozen sperm).46 advances in sex-sorting technologies, such as sorting with raman spectroscopy,27,49 will likely continue to focus on decreasing sperm damage and improving refreezing capability to ultimately improve reproductive efficiency with such techniques. if a farm is already utilizing artificial insemination, introducing sexed semen based on the needs of the herd can be readily performed and therefore cost effective.50 multisire or heterospermic straws utilizing sperm from multiple sires (heterospermic) into 1 frozen straw for artificial insemination is another technique utilized to help increase the chances of pregnancy. this is not a new concept as the idea has been around since the 1960s, but the market has progressed such that the topic has been revived, primarily for the dairy industry’s recent adaptation of beef-on-dairy.51 utilizing beef cattle semen on a dairy cow creates an economic advantage when the offspring is going to be utilized for beef, but the dam’s value is based in her milk production. one hypothesized reason for increased fertility with this technique is that there is a greater variety of sperm survival rates with multiple ejaculates mixed together that allows for a wider ovulation window.51 multisire straws are currently marketed through stgeneticstm (navasota, texas), labeled as super conventional®. several blends include: 1. beef on dairy that are male offspring focused; 2. blends focused on calving ease for young heifers to create replacement females; and 3. beef blends for mature cows to produce excellent market features in male offspring. conception rate of first, second, and third service is described as 49%. pooled dairy semen does not appear to have a large effect on conception rates compared to beef semen, likely due to the similar breeding ability of dairy males resulting from the practices in place for culling.51 spermatogonial stem cell transplantation although artificial insemination is prominent in some industries, such as dairy production, a large proportion of the livestock in the world are still bred by natural service.52,53 this practice hinders the gain in animal production since reproduction efficiency occurs rapidly when males of superior genetics are able to sire calves through artificial insemination (i.e. a wider dissemination of genetics). in some instances, an animal with the desired genetics cannot or should not be transferred to the desired environment. for example, bos taurus cattle are poorly adapted to tropical environments,54 yet bos taurus x bos indicus animals are economically desirable in these locations.55 one solution that has been proposed is through ‘surrogate sires’ the transfer of male spermatogonial stem cells, such that 1 male can produce sperm that contains another’s genetics. it has been a challenge to ensure that the recipient only produces the donor’s sperm yet maintaining a high level of fertility. recent advances in spermatogonial stem cell transplantation (ssct) have resulted in pregnancies post natural service in the bovine.56 the original research for this technique is decribed.57 there are 2 animals involved in ssct: the donor and the recipient. donor animals contribute testicular tissue that is injected as a single-cell suspension into the rete testis of the recipient. recipient males have intact seminiferous cords and stroma but lack the germ cells needed to produce their own sperm. recipients are currently derived through nanos2 gene knockout via crispr-cas9 technology. in mice, knockout females and nanos2+/males are fertile. if true for all species, the ability to produce recipients through breeding schemes would increase feasibility. research is ongoing to determine how to identify the best recipients for transfer and at what age transfer will have the highest rate of success. it is currently unknown how to identify the ideal donor beyond the desire to disseminate his genetics. nanos2 gene knockout mice, pigs, goats, and cattle have been produced. in mice, ssct has been successful; offspring of the donor’s genetics have been generated through fertile recipients. recipient boars have generated sperm with donor genetics, but not at the number desired for http://dx.doi.org/10.58292/ct.v17.12959 citation: clinical theriogenology 2025, 17, 12959, http://dx.doi.org/10.58292/ct.v17.12959 5 an acceptable level of fertility. sperm of normal morphology and motility were produced from recipient bucks. due to the long generation interval, research with cattle was of the lowest priority. however, a nanos2 knockout bull calf was produced with similar testicular histology to other species.57 recent advancement demonstrates recipient bulls ejaculate donor sperm that is capable of surviving cryopreservation.56 in livestock, ssct techniques could allow for superior donor genetics to be disseminated more rapidly than with artificial insemination alone. for example, a more heat stress-tolerant bull could produce sperm of a less heat stress-tolerant, but higher genetic value bull, resulting in offspring with intermediate heat stress-tolerance when bred to heat-tolerant females. conclusion growing technology in art is a pivotal step to improve livestock management (decrease time or increase efficacy). precision farming technology for estrus detection and reverse sex sorted semen, and multisire straws for artificial insemination are all techniques with the ability to increase reproductive efficiency. in addition to growing use of artificial intelligence, established technology can be paired together to assist with decision making on the farm. genetic focused advances may be achieved through genomic selection through embryo biopsy, gene editing via crispr-cas9, and spermatogonial stem cell 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https://doi.org/10.1016/j.anireprosci.2009.08.007 https://doi.org/10.1016/j.theriogenology.2006.08.005 https://doi.org/10.3390/biology10080806 https://doi.org/10.1016/j.anireprosci.2016.04.008 https://www.beefcentral.com/genetics/sexed-semen-could-be-the-next-drought-mitigation-strategy-for-beef/ https://www.beefcentral.com/genetics/sexed-semen-could-be-the-next-drought-mitigation-strategy-for-beef/ https://www.beefcentral.com/genetics/sexed-semen-could-be-the-next-drought-mitigation-strategy-for-beef/ https://doi.org/10.1017/s175173111800054x https://doi.org/10.3390/ani11123563 https://doi.org/10.3390/ani11123563 https://doi.org/10.1093/jas/skaa015 https://doi.org/10.1111/j.1439-0388.1996.tb00637.x https://doi.org/10.1111/j.1439-0388.1996.tb00637.x https://doi.org/10.1073/pnas.2010102117 2009: neonatal and pediatric ultrasonography-part ii 1 neonatal and pediatric ultrasonography-part ii 2 3 a. p. davidson, t. w. baker 4 department of medicine and epidemiology, school of veterinary medicine, university of 5 california, davis, ca, usa 6 introduction 7 pediatric patients are commonly presented to the veterinarian because of signs 8 referable to the abdominal cavity. presenting signs can be due to congenital anomalies, 9 dietary indiscretion, parasitic infestation and infectious disease. abdominal ultrasound 10 examination is a particularly useful diagnostic tool in the pediatric patient because it is 11 noninvasive and can usually be performed without sedation or anesthesia. ultrasonography 12 provides valuable clinical information about the peritoneal cavity, great vessels, abdominal 13 viscera and lymph nodes and thus greatly facilitates diagnostic differentiation between 14 congenital and acquired disorders.1 ultrasonographic techniques will be described in this 15 paper. 16 keywords: ultrasound, neonatal, congenital, acquired, pediatric 17 disorders of urogenital development 18 in the past, veterinary pediatric ultrasonography has been hampered by the small size of 19 neonatal organs. recent advances in pediatric veterinary ultrasonography are encouraging. 20 abdominal ultrasound can facilitate the diagnosis of congenital urogenital disorders because 21 ectopic, distended ureters and changes in renal architecture are usually readily imaged.2 the 22 presence and location of cryptorchid testes can often be detected with ultrasound.1 23 ultrasonographic examination of the bladder disclosing urolithiasis can provide information 24 155 suggesting congenital hepatic vascular anomalies. ammonium biurate urolithiasis is suggestive 25 of hyperammonemia, common with portosystemic shunt disorders (described below).3,4 26 the most common familial disorders in cats and dogs include renal agenesis, renal dysplasia, 27 polycystic kidneys, renal amyloidosis, basement membrane disorders, and tubular 28 dysfunction (fanconi's syndrome).5 29 renal agenesis 30 congenital renal agenesis resulting in the absence of a kidney can be confirmed with 31 ultrasound. the contralateral kidney typically has normal internal anatomy, but is enlarged as 32 a consequence of obligatory hypertrophy. renal function of the pediatric patient does not 33 equate that of the adult until 4-6 months of age, therefore compensatory renomegaly may not 34 be apparent until that time.3 35 renal dysplasia 36 until reliable genetic markers are marketed and thus available for the various breed 37 specific congenital renal dysplasias(i.e. persian cats) , ultrasound provides the best method 38 of screening young dogs and cats for these likely heritable disorders. early ultrasonographic 39 screening is possible in platycephalic breeds in which morphologic changes are grossly 40 evident (i.e., cairn terriers, german shepherd dogs).3,5 (fig1) 41 ectopic ureter 42 congenital ectopic placement of a distal ureter into the urethra, vestibule or vagina is 43 usually associated with ureteral dilation with or without renal pelvic dilation. dilation of the 44 ureter improves the sensitivity of the ultrasound study; however, the diagnosis can be elusive. 45 visualization of a nonvascular fluid filled structure with a hyperechoic wall passing dorsal to 46 the urinary bladder, or obvious insertion of the structure into the proximal urethra suggest the 47 diagnosis. visualization of the ureteral jets in the bladder suggests normalcy, however some 48 156 ectopic ureters insert initially into the bladder and additionally tunnel distally to terminate in an 49 abnormal site. visualization of the dilated ureter usually occurs near the urinary bladder. (fig 50 2) visualization of the bladder neck and proximal urethra may be obscured by pubic bone, 51 making identification of such termination difficult.1,2 52 hydronephrosis can eventually result from an uncorrected ectopic ureter due to flow 53 impedance at the abnormal site of insertion. (fig 3) urinary tract infection is commonly 54 associated with ectopia, due to accompanying urethral sphincter mechanism anomalies. if 55 not detected and treated, urinary tract infection can progress to pyelonephritis and ureteritis. 56 infection and its associated inflammation in the tract can further alter the ultrasonographic 57 appearance of the kidneys, bladder, ureters and urethra.1 58 contrast enhanced computed tomography is the most sensitive and specific modality 59 for the diagnosis of ectopia, but, like double contrast radiography, requires anesthesia, 60 making initial evaluation with ultrasound desirable when ectopia is suspected clinically. the 61 condition is thought to be heritable (mode not known), and is more commonly symptomatic in 62 females due to the greater strength of the male urethral sphincter and longer length of the 63 urethra.5 64 ureterocele 65 a ureterocele is an uncommon congenital dilation of the ureter near the bladder, 66 appearing as a cystic structure within the bladder lumen or wall. (fig 4) the ureterocele 67 occurs most commonly in association with an ectopic ureter. diagnosis can be made by 68 scanning the urinary bladder in the transverse plane and watching for strong peristalsis of the 69 adjacent ureter.1 70 patent urachus 71 157 the urachus permits the flow of urine from the bladder into the allantoic sac of the 72 fetus, and normally atrophies at birth. a patent urachus in the neonate is characterized 73 clinically by urine dribbling from the umbilicus. the fluid filled urachus can be identified 74 ultrasonographically, extending cranially from the cranioventral bladder wall. if an 75 incompletely patent urachus is present in the neonate, a urachal diverticulum may result, 76 seen as a divot in the apex of the bladder. (fig 5) urachal diverticula can predispose the 77 bladder to recurrent infection because of abnormal bladder flow in the region, surgical 78 excision can be indicated.1 79 cryptorchidism 80 ultrasound identification of cryptorchid testis(es) can confirm cryptorchidism in 81 pediatric patients with bilateral involvement whose neutering status is unknown. 82 ultrasonographic localization of undescended testes can assist the surgeon in planning the 83 approach (i.e., inguinal vs. cranial abdominal). a retained testis can be positioned anywhere 84 between the ipsilateral kidney and the scrotum. a systematic evaluation of the region from 85 the caudal renal pole to the inguinal canal can identify an oval, homogenously echogenic 86 structure with a mildly hyperechoic border representing the parietal and visceral tunics. the 87 epididymis is usually distinctly less echoic than the testicular parenchyma, as in the scrotal 88 testis. the cryptorchid testis will maintain the anatomic structure, the median testes (a 89 hyperechoic slash), and normal testicular echogenicity despite being reduced in size as 90 compared to a scrotal testis.1 (fig 6) 91 ultrasonography is also the procedure of choice to detect undescended testicles in 92 pediatric or adult dogs and cats. clinical evaluation via serum lh concentration or 93 testosterone stimulation tests can increase support the diagnosis. an ultrasound examination 94 158 may also detect nonpalpable scrotal testicular tumors and neoplastic transformation in 95 abdominal testes.1 (fig 7) 96 disorders of digestive system development 97 hernia 98 congenital peritoneopericardial diaphragmatic hernias occur in both the dog and cat; 99 ultrasonography provides an additional modality for their diagnosis. as with other 100 diaphragmatic hernias, careful evaluation for continuity of the echogenic diaphragm 101 differentiates a true hernia from mirror image artifacts. evaluation of the pericardial contents 102 can be made from the subcostal (across the liver) or intercostal (using the heart as an 103 acoustic window) approach. abnormal pericardial contents can include falciform fat, liver, gall 104 bladder and/or intestines. congenital inguinal and scrotal hernias can similarly be confirmed 105 by ultrasonographic identification of intestines in the subcutaneous space of the affected 106 groin. this can be a dynamic finding. mesenteric fat may alternatively be entrapped through 107 the hernia.1,6 (fig 8) 108 congenital hiatal hernias are more difficult to confirm with ultrasound because of the 109 inherent difficulty imaging the gas filled stomach and the intermittent nature of the disorder. 110 stomach wall with characteristic rugal folds can be imaged crossing the diaphragm into the 111 thoracic cavity. fluoroscopic evaluation can be more informative in these cases. 112 a developmental anomaly resulting in extrusion of a portion of the gastrointestinal tract 113 outside of the body wall, occurring within the umbilical canal (omphalocele) or lateral to the 114 umbilical canal (gastroschisis), has been reported in humans and occurs in both dogs and 115 cats. the condition is usually hopeless in small pediatric patients presented to the 116 veterinarian hours after birth; however, a 30-70% survival rate is reported in humans with 117 immediate post partum surgical intervention. diagnosis is made pre partum with abdominal 118 159 ultrasound, based on the recognition of fetal gastric wall (rugal) structures or intestinal 119 contents in an abnormal location. earlier surgical intervention before inevitable septic 120 contamination occurs may improve the prognosis in veterinary patients.1,5 121 enteric anomalies 122 pyloric stenosis secondary to hypertrophic gastritis has been reported in a pediatric 123 dog. focal circumferential thickening of the pylorus primarily involving the muscularis is 124 typical. 125 enteric duplication or agenesis can be confirmed ultrasonographically in pediatric 126 patients. duplication is rare, can occur anywhere in the intestinal tract and the clinical signs 127 may be nonspecific. a fluid filled juxtaintestinal formation with variable peristalsis and 128 contents can be seen. enteric agenesis usually results in severe clinical signs in the neonatal 129 period. ultrasonographic findings usually include marked fluid and gas distention of bowel 130 proximal to the defect.5 131 several breeds of dogs have a reported genetic predilection to small intestinal 132 disease. normally, the small bowel appears sonographically as four distinct layers. the 133 bowel lumen is hyperechoic, as gas and ingesta are compressed. the layer just outside the 134 lumen is the mucosa; it is hypoechoic and normally the thickest appearing section. outside 135 the mucosa is the submucosa, it is hyperechoic to the mucosa and about one third the 136 thickness. the muscularis, the bowel muscle layer, is outside of the submucosa and appears 137 as a very thin hypoechoic black line. (fig 9, 10) 138 an immunoproliferative enteropathy is seen in the basenji breed which is 139 characterized by lymphangectasia, intermittent diarrhea, weight loss, hypoalbuminemia and 140 hyperglobulinemia, and lymphoplasmacytic mucosal infiltrates throughout the gi tract. 141 histopathology is diagnostic, however abdominal ultrasonography can identify bowel in which 142 160 disruption of the normal layering has occurred. chinese sharpei dogs have been identified 143 with a lymphoplasmacytic-eosinophilic infiltrative enteropathy that is characterized by poor 144 weight gain, weight loss, or intermittent diarrhea episodes, with onset of signs typically 145 between 2 to 6 months of age. infiltrative enteropathies can be characterized 146 ultrasonographically as having changes in the normal bowel wall layering.5 147 portosystemic shunt 148 portosystemic shunts (pss) are congenital malformations of the hepatic portal venous 149 drainage system and can have either a familial, i.e. genetic, or random occurrence. 150 congenital pss can be either intrahepatic or extrahepatic; breed predilections for 151 extrahepatic shunts include yorkshire terrier, maltese, poodle, miniature schnauzer, 152 dachshund, lhasa apso, pekingese, pug, and shih tzu, whereas intrahepatic shunts are 153 more commonly identified in large breed dogs such as golden retrievers, german 154 shepherds, irish wolfhounds, irish setters, and samoyeds. pss are uncommon in cats. 155 ultrasonography provides a rapid and noninvasive method for screening patients 156 suspected to have congenital pss. although scintigraphy (transcolonic portal scintigraphy or 157 transplenic portography) is considered the most reliable noninvasive method of documenting 158 a pss, its availability is limited to specialty and university practices, and its use dictates 159 special handling of the radioactive patient for at least 12 hours. mesenteric portography, 160 although more invasive and requiring general anesthesia, is a highly reliable method of 161 confirming and localizing pss.1,7 162 abdominal ultrasonography is a useful diagnostic tool and is routinely done when a 163 pss is suspected. (fig 11,12) it is non-invasive and requires no anesthesia however 164 diagnostic accuracy is highly operator dependent, and the pss will be confirmed in only 165 approximately 60-80% of cases. the liver may be small and difficult to image in patients with 166 161 congenital portosystemic shunts. imaging the liver from the standard ventral approach can be 167 improved in some cases by using the left ventral intercostal and right dorsal intercostal 168 approaches. the presence of ascites can facilitate the study, as can adding fluid to the 169 stomach, and positioning the patient to shift gas away from the scanhead and shift abdominal 170 organs caudally.(fig 13) ultrasound evaluation of portosystemic anomalies can be facilitated 171 by positive pressure ventilation under anesthesia for the same reason. cystic calculi, most 172 commonly ammonium biurate, should increase the clinical suspicion of pss. urinary bladder 173 calculi (radiolucent and radiopaque) produce a strong acoustic shadow when viewed 174 ultrasongraphically.1 (fig 14) 175 post operatively, ultrasound can be used to evaluate portal blood flow following 176 surgical banding or coil embolization. extrahepatic shunts most commonly arise from the 177 portal vein, splenic vein or left gastric vein in the dog, and from the left gastric vein in the cat. 178 (fig 15) identification of a shunting vessel emptying into the caudal vena cava is difficult but 179 confirmatory. intrahepatic shunts can be more difficult to identify because of patient size, 180 bowel gas and liver size. clipping the hair coat intercostally on the right can allow for 181 transverse vessel stacking (of the aorta, vena cava and portal vein) and allow visualization of 182 ductal shunts. there can be right and left shunting of the ductus.1,7 183 references 184 1. baker tw: baker tw: find your friends: location and appearance of normal 185 abdominal viscera, liver, spleen, kidney and urinary bladder. in: what’s that? a 186 beginner’s guide to veterinary abdominal ultrasound. lakewood, co: aaha press, 187 2009. 188 189 162 2. lamb cr: ultrasonography of the ureters. vet clin north am sm anim pract 190 1998;28:823-848. 191 192 3. nyland tg, mattoon js, herrgesell ej, et al: urinary tract. in: nyland tg, mattoon js, 193 editors. small animal diagnostic ultrasound, 2nd ed. philadelphia: wb saunders; 194 2002. p.158-195. 195 196 4. nyland tg, hager da: sonography of the liver, gallbladder, and spleen. vet clin 197 north am sm anim pract 1985;15:1123-1148. 198 199 5. gough a: disease summaries. in: breed predispositions to disease in dogs and cats. 200 gough a, thomas a, editors. oxford: blackwell publishing; 2004. p. 179-229. 201 202 6. suter pf, lord pf: abnormalities of the diaphragm. in: suter pf, editor. thoracic 203 radiography: a text atlas of thoracic disease in the dog and cat. wettswil, switzerland: 204 pf suter; 1984. p. 180-204. 205 206 7. penninck dg: gastrointestinal tract. in: nyland tg, mattoon js, editors. small animal 207 diagnostic ultrasound, 2nd ed. philadelphia: wb saunders; 2002. p. 207-230. 208 163 button24: button25: button26: button27: button28: button29: button30: button31: button32: button33: button34: button35: button36: button37: button38: button39: button40: button41: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false 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/pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2016: hypoglycemia and ketosis in a pregnant bitch hypoglycemia and ketosis in a pregnant bitch nima nakahara, rachael-kate llewellyn, philip george aveston thomas queensland veterinary specialists, stafford heights, brisbane, queensland, australia summary a four-year-old female afghan hound in near-term gestation presented for dystocia. on presentation the patient had dull mentation, a low body condition score (2.5/9) and was unable to ambulate. the patient had delivered two pups before presentation and on physical examination a palpable pup in the vagina. fetal heart rates were measured with transabdominal ultrasound and fetal bradycardia was observed (measured between 155-190 beats per minute). a cesarean section was indicated. clinical pathology showed a marked hypoglycemia (2.1mmol/l), ketonemia and lymphopenia. urinalysis showed hypersthenuria, ketonuria, mild proteinuria and bilirubinuria. dextrose 50% was administered parenterally at 1g/kg and isotonic fluid therapy was commenced. the patient was anaesthetized and a routine cesarean was performed. prior to discharge the patient had normal mentation, was eating, ambulatory and blood glucose was 8.8mmol/l. a catabolic energy state for a bitch in late gestation may result in hypoglycemia, ketonemia and ketonuria. inappropriate energy balance throughout the periparturient period may be detrimental to both the bitch and her pups. keywords: hypoglycemia, ketonemia, ketonuria, pregnancy, cesarean section background hypoglycemia with ketonemia and ketonuria in the preparturient bitch has been poorly described in the literature, hence the pathophysiology and definition of the disorder in the bitch has been difficult to demonstrate.1 pregnancy toxemia, as defined in other domestic species, is a metabolic disorder, commonly occurring in the final third of gestation. it is associated with an inability to maintain glucose homeostasis and a negative energy balance. undernourishment in late gestation and exposure to stressors (environmental and physiological) that can promote glucose consumption has been associated with the disease.2 clinicopathological findings of pregnancy toxemia in species where it is well defined, namely sheep and other small ruminants, include severe hypoglycemia, cortisol-induced hematology changes (neutrophillia, lymphopenia, eosinopenia) and evidence of dehydration.3 ketonemia and ketonuria are also common findings in pregnancy toxemia of small ruminants and occur as a result of the altered metabolism of carbohydrates and fats,4 allowing for the production and accumulation of ketone bodies. hypoglycemia is generally considered an unlikely cause of dystocia in the bitch.5 to the authors’ knowledge, there have been five reported cases of suspected pregnancy toxemia in dogs. irvine6 briefly reported a case series of three patients with hypoglycemia in 1964, and jackson7 reported a case of hypoglycemia-ketonemia in a bitch in 1980, with minimal database and information. another more recent brief communication was published in 2009 detailing preparturient hypoglycemia in a great dane bitch,8 which was the first report in a large breed dog. the aim of the present case report is to contribute to the limited database of cases pertaining to the rare occurrence of severe hypoglycemia and ketosis in the pregnant bitch. case presentation history a four-year-old female afghan hound presented to the theriogenology services at a private specialist hospital for dystocia. she had commenced stage ii labor five hours prior to presentation, and the caregiver had found a deceased pup. a second pup was delivered two hours after the first, which was a male and clinically normal at birth. no further pups were delivered, and the owner had observed no abdominal straining prior to presentation. history at presentation indicated that the patient had been particular about eating; she had initially been fed a dry dog food following insemination, however in the later stage of gestation she had been fed chicken frames and necks. recently her appetite had been reduced and she had subsequently become inappetent. the patient had been inseminated surgically 56 days prior to presentation. clinical examination at time of insemination, including reproductive examination and vaginal cytology, was normal and in accordance with the stage of estrous. three progesterone profiles were obtained prior to surgical insemination. a body condition score (bcs) of five out of nine was given at time of clinical theriogenology • volume 8 number 2 • june 2016127 insemination and was determined as normal. the bitch was inseminated with 140 million frozenthawed sperm. pregnancy examination occurred four weeks after insemination and the bitch was found to be healthy and normal. transabdominal ultrasound identified a minimum of eight conceptuses with heartbeats, which were all of appropriate size for the stage of gestation. uterine and placental anatomy was normal. clinical findings on presentation to the clinic, the patient was unable to right, rise or ambulate on her own and had dull mentation. the patient had a body weight (bw) of 23-kg (50.7lb) and bcs of 2.5/9. cardinal signs were as follows: temperature 37.5°c (99.5°f), heart rate 90 beats per minute (bpm), respiratory rate 16 breaths per miute with appropriate effort, mucous membranes were pink and dry with capillary refill time of two seconds. on reproductive examination the patient had a swollen vulva consistent with parturition, and a soft and dilated vagina. a pup in caudal presentation with normal posture was palpated in the cranial vagina. no ferguson reflex was detected. transabdominal ultrasound revealed a normal uterus and abdomen; multiple pups were visible and fetal heart rates were measured on m-mode. fetal heart rates were measured between 155-190 bpm. laboratory findings biochemistry, performed in-housea on a plasma sample, revealed marked hypoglycemia (2.1mmol/l; reference range 4.11-7.95mmol/l), mild elevation of urea (10 mmol/l; reference range 2.5 to 9.6mmol/l), likely pre-renal (see urinalysis below), with all other values within normal range. marked elevation of ketones in whole blood was found on in-house human point-of-care (poc) analyzerb with the value of 1.9 mmol/l. a blood sample was taken after surgery and was submitted to external pathology laboratory for measurement of b-hydroxybutyrate, which showed mild elevation (1.6mmol/l; normal reference under 0.5mmol/l). resting cortisol level of 158 nmol/l; reference range 15 to 170 nmol/l and low insulin of 3.3 mu/l; reference range 5 to 20 mu/l. progesterone was 3.18 nmol/l (1.0 ng/ml), appropriate with parturition. complete blood count, performed in-house,c revealed a normal hematocrit for late pregnancy (hct, 34.5% reference range 25% to 40%), a normal wbc count (9,400 wbc/μl; reference range 5,050 to 16,760 wbc/μl), and a mild lymphopenia (760 lymphocytes/μl; reference range 1,050 to 5100 lymphocytes/μl). urinalysis performed in-house initially on sample collected via cystocentesis, with results confirmed by an external laboratory revealed hypersthenuria (urine specific gravity of >1.060), indicating adequate urinary concentration, bilirubinuria (large), ketonuria reflecting a catabolic state (over 8 mmol/l), trace glucose (5.5mmol/l), ph of 7.0, and mild proteinuria (1g/l). cytologic interpretation demonstrated few epithelial cells, low cellularity and low number of red blood cells on the background, likely to reflect iatrogenic contamination from cystocentesis. in-house venous blood gas analysisd revealed mild metabolic acidosis with compensatory alkalosis (ph 7.35; reference range 7.38 to 7.44, pco2 31.3mmhg; reference range 40 to 50 mmhg and hco3 16.8mmol/l; reference range 18 to 26 mmol/l) with a standard base excess of -7.8mmol/l, reference range -5 to 1 mmol/l. electrolytes were within normal range (na+ 145 mmol/l; reference range 141 to 156 mmol/l, k+ 4.42mmol/l; reference range 4.0 to 5.6 mmol/l, ionized ca 1.13 mmol/l; reference range 0.98 to 1.45 mmol/l and cl112 mmol/l; reference range 110 to 120 mmol/l). treatment after the initial blood glucose test, 1g/kg (2.2g/lb) of 50% dextrose diluted 1:3 with 0.9% saline was administered slowly intravenously. parenteral isotonic fluids (lactated ringer’s solution) were commenced at 10ml/kg/hr (4.5ml/lb/hr). general anesthesia was induced and a cesarean section was performed soon after initial examination and eleven pups (six females and five males) were delivered to neonatal intensive care for resuscitation. one pup had ruptured fetal membranes when it was isolated in the uterus and was initially resuscitated but died shortly after birth. the deceased pup delivered at home was of unknown sex and health status, with the live pup born at home male and clinically normal. the bw of all pups born alive, via cesarean or at home, ranged from 375g to 515g (0.83 to 1.14lb), with a mean of 434.6g (0.96lb). all pups were assessed and were anatomically within normal limits. pups born via cesarean required moderate resuscitation. single doses of clinical theriogenology • volume 8 number 2 • june 2016 128 oxytocin 10 iu and meloxicam 0.2mg/kg (0.44mg/lb) were given intra-operatively subcutaneously to the bitch. outcome repeat measurements of blood glucose in the bitch with in-house human point-of-care glucometere 30m after dextrose administration was 7.7 mmol/l (initial 2.0 mmol/l), post-operatively 7.8mmol/l and pre-discharge levels (three and half hours after admission) was 8.8mmol/l. blood ketone levels had dropped to 1.6mmol/l post-operatively then to 0.9mmol/l immediately prior to discharge. venous blood gas, which was measured three hours after surgery, demonstrated ph 7.34, pco2 35.5mmhg, hco3 18.5mmol/l with a base excess -6.2 (reference ranges as above). electrolytes were all within normal limits. brief postmortem examination of the deceased pup delivered by cesarean section showed an externally normal female, bw 465g (1.02lb), with a small amount of serous nasal discharge bilaterally. grossly, there was a failure in inflation of the right cranial and right caudal lung lobes. the right middle, and the left cranial and caudal lung lobes were adequately inflated. no other gross abnormalities were detected. the patient was discharged with pups three and one-half hours after initial presentation. the bitch had normal mentation, was ambulatory and comfortable. before discharge, all pups were assisted to nurse twice and the patient ate well. of the 13 pups born, 11 survived and were vocal, suckling and mobile at the time of discharge. no medications were dispensed on discharge. communication from the owner two weeks after surgery showed the patient and all pups doing well. discussion during pregnancy there is a considerable change in the metabolic energy requirements of the dam, with a marked increase in glucose utilization by the fetuses and the placenta. the ability of glucagon, norepinephrine and cortisol to respond to hypoglycemia is blunted in pregnancy.9 in pregnancy toxemia, regulatory mechanisms fail to raise blood glucose levels resulting in a catabolic state and hypoglycemia and mobilization of fat that releases fatty acids and ketones as an alternative energy source. in severe cases, as in the present report, ketones accumulate in circulation resulting in ketonemia and ketonuria alongside the hypoglycemia. significant hypoglycemia occurs in fasted animals (dogs, sheep, rats and guinea pigs) during late gestation and, in the ewe, the severity of hypoglycemia is directly related to the number of fetuses.10 while a causative relationship has not been established, the bitch in the present report had a large litter size, which may have contributed to the development of hypoglycemia and ketosis. the quality and quantity of the diet may also be contributory factor for development of hypoglycemia. starvation in pregnancy has been proven to decrease blood glucose significantly in humans and further prolonged starvation increases plasma fatty acids and β-hydroxybutyrate concentrations.11 this was observed in the present case, where the patient had lost body condition and had been inappetent, resulting in lowered blood glucose and an increase in ketones. there are marked species differences in the condition termed pregnancy toxemia. in the ewe there is severe metabolic disease with high mortality and poor response to treatment.10 in ewes, pregnancy toxemia commonly occurs in the last third of the gestation, is associated with nutrition and the severity is directly proportional to the number of fetuses. in dairy cattle, it is a disease of the postpartum period and occurs at peak lactation, again associated with negative energy balance. in women it is referred to as pre-eclampsia; a multisystemic disease causing multi-organ failure, hypertension, proteinuria, abnormal placentation, premature birth and fetal death. in the canine species it appears to be prepartum disease with moderate to severe clinical signs including that of hypoglycemia; loss of appetite, restlessness, tremors, ataxia, seizures and coma. while it is likely that this patient was at or near the end of gestation at presentation (56 days following insemination), further cases would be useful to determine if hypoglycaemia and ketosis are a cause of pre-term parturition in dogs. in all the reported canine cases, including the present case, response to treatment has been excellent, with full recovery occurring soon after the pups have been born or delivered. the present case, when considered with available literature, suggests that pregnancy toxemia in the bitch is likely associated with poor nutrition during gestation, a negative energy balance in the peripartum period, clinical theriogenology • volume 8 number 2 • june 2016129 poor body condition, litter size and environmental stressors. bitches that have poor body condition in early gestation and in the prepartum period, as well as starvation or reduced appetite in the prepartum period are likely to have an increased risk of pregnancy toxemia. the bitch in the present report was uniparous, further studies would required to determine if this is a contributing risk factor in the development of pregnancy toxemia. in all species, treatment is aimed at rectifying the catabolic state and providing supportive care, which the present case indicates is also appropriate management in the bitch. learning points • hypoglycaemia with ketonemia and ketonuria can occur in bitchs during late gestation as a result of a catabolic energy state. • correction of hypoglycemia and dehydration and a cesarean section may be appropriate treatment. • inappropriate nutrition in the periparturient period may be a precipitating factor of the condition in the pregnant bitch. • with early recognition and treatment, the prognosis for survival may be excellent. a. idexx catalyse in house biochemistry b. point of care analyzer for blood ketone, optium xceed β-ketone c. idexx procyte in house hematology d. blood gas analyzer on co-ox abl80 flex e. point of care analyzer for blood glucose, accu-check performa references 1. johnson ca: glucose homeostasis during canine pregnancy: nsulin resistance, ketosis, and hypoglycemia. theriogenology 2008;70:1418-1423. 2. edmondson ma, pugh dg: pregnancy toxemia in sheep and goats. in: anderson de, rings dm, editors. food animal practice. philadelphia: wb saunders; 2009. p. 144-145. 3. abba y, abdullah ffj, chung elt, et al: biochemical and pathological findings of pregnancy toxemia in saanen doe: a case report. j adv vet anim res 2015;2:236-239. 4. brozos c, mavrogianni vs, fthenakis gc: treatment and control of peri-parturient metabolic diseases: pregnancy toxemia, hypocalcaemia, hypomagnesemia. vet clin north am food anim pract 2011;27;106-107. 5. johnson ca: disorders of pregnancy. vet clin north am small anim pract 1986; 16:477-482. 6. irvine chg: hypoglycaemia in the bitch. nz vet j 1964;12:140-144. 7. jackson rf, bruss ml, growney, et al: hypoglycemia-ketonemia in a pregnant bitch. j am vet med assoc 1980;177:1123-1127. 8. ghaffari ms, najafiyan hr: diagnosis and management of preparturient hypoglycaemia in a great dane bitch. comp clin pathol 2009;18:467-468. 9. canniff km, smith ms, lacy db, et al: glucagon secretion and autonomic signaling during hypoglycemia in late pregnancy. am j physiol regul integr comp physiol 2006;291:788-795. 10. schlumbohm c, harmeyer j: twin-pregnancy increases susceptibility of ewes to hypoglycaemic stress and pregnancy toxemia. res vet sci 2008;84:286-299. 11. metzger be, vileisis ra, ravnkar v, et al: “accelerated starvation” and the skipped breakfast in late normal pregnancy. lancet 1982;588-592. clinical theriogenology • volume 8 number 2 • june 2016 130 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy 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/convertcolors /converttocmyk /destinationprofilename (u.s. web coated \(swop\) v2) /destinationprofileselector /usename /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2014: reproductive emergencies in camelids reproductive emergencies in camelids a.tibary, l.k. pearson department of veterinary clinical sciences, college of veterinary medicine and center for reproductive biology, washington state university, pullman, wa summary theriogenological emergencies represent the overwhelming majority of emergencies in camelid practice. the objective of the present paper is to discuss the diagnosis, treatment and prognosis for the most common male and female camelid reproductive emergencies. male reproductive emergencies are dominated by traumatic injuries to the external genitalia, urolithiasis, and heat stress which severely compromise future reproductive ability. in the non-pregnant female, most emergencies are related to iatrogenic trauma or congenital abnormalities. common emergencies of genital origin in the pregnant female are uterine torsion and vaginal prolapse. non-surgical and surgical approaches to obstetrical emergencies are discussed. management of postpartum emergencies (i.e. uterine prolapse, vaginal prolapse and uterine hemorrhage) is also presented. introduction in theriogenology practice, emergencies are defined not only in terms of concerns for the welfare of the patient but also for its future reproductive life. the challenge often faced when presented with reproductive emergencies is how to preserve the life of the patient and its reproductive ability. in the case of pregnant females, the life and welfare of the neonate are of concern. this paper presents the approach to diagnosis and clinical management of various reproductive emergencies in the male and female camelids. this review is an update on the topic and draws primarily on the clinical experience of the authors as there are very limited controlled studies on clinical reproduction and emergency care in camelids.1 reproductive emergencies in the male many acute disease processes may have some severe repercussions on the reproductive process in the male, however this review is limited to those diseases and accidents which directly affect urogenital system or associated organs. male camelids are often presented for emergency care when there is sudden onset of visible abnormalities in the external genitalia or severe colicky behavior. common visible abnormalities include acute preputial or scrotal swelling, preputial prolapse or paraphimosis, and external genitalia trauma (laceration and hemorrhage). behavioral changes that may be of urogenital origin and requiring immediate care include persistent straining and dysuria, colic, downer male, or complete lack of libido. general approach to examination of the male for reproductive emergency history and initial physical evaluation on the farm should direct the practitioner as to whether the case can be handled on the farm, in the hospital, or referred to a specialty center. accurate weight, body condition score and degree of dehydration are important in the assessment of the patient. in severely compromised animals or when surgical management is the only option, baseline complete blood count (cbc), blood biochemistry and urinalysis should be considered. an intravenous catheter should be placed immediately in depressed animals. ultrasonography of the abdomen and reproductive organs should be part of the physical examination. acute scrotal swelling compromise of testicular thermoregulation should be taken seriously as its negative effect on spermatogenesis may be long lasting or definitive in the male camelid. acute scrotal swelling is generally due to heat stress, trauma or a local or systemic infectious process. testicular torsion and scrotal hernia are commonly considered as a differential in other large animal species but we have not encountered these situation in our in camelids practice. practitioners should be aware that some cases 579 clinical theriogenology • volume 6, number 4 • december 2014 present as sudden onset of scrotal swelling just after shearing. however these cases may actually be chronic and are only noticed at the time of shearing (testicular tumors, lymphomas, etc.). heat stress scrotal and preputial edema and development of severe hydrocele are features of heat stress in south american camelids (sac). factors predisposing to heat stress include prolonged high ambient temperature and humidity, inadequate shade, long fleece, dark coat color, and obesity. the risk for heat stress is exacerbated by stresses such as transportation, exercise, fighting, and breeding.2 hyperthermia results from impaired evaporative cooling mechanism particularly under humid conditions. scrotal edema may be the first clinical sign in the male. the exact pathophysiology of the scrotal and ventral abdominal edema is not well understood. contributing factors may include inability of the pampiniform plexus and testicular artery to cope with the fluid turnover or vascular thermal injury resulting in impaired wall permeability and extravasation of intravascular proteins, electrolytes, and fluid into the interstitium.1,2 spermatogenesis (sperm production and semen quality) is severely impaired when llamas are kept at an ambient temperature of 29°c for four weeks.3,4 these temperatures are common in many regions of world where sac have been introduced. spontaneous recovery may be observed but the male may become infertile for various length of time.5 hyperthermia, increased salivation, anorexia, depression, ataxia, muscular weakness, dehydration, ketosis/hepatic lipidosis, and dyspnea/hyperpnea are seen in advanced cases.2 an inflammatory or stress leukogram is almost always present and anemia may be observed secondary to hemolysis. serum biochemical abnormalities may include hypophosphatemia, hypocalcemia, hypomagnesemia, hyponatremia, hypochloridemia, hypoor hyperkalemia, hyperglycemia, and elevated serum aspartate aminitransferase (ast) and creatine phosphokinase (cpk) concentrations. serum glucose concentration > 300 mg/dl has been associated with a poor prognosis. death may ensue due to severe electrolyte imbalances, damage to the thermoregulatory center in the hypothalamus and multiorgan failure.2 the first step in stabilization of the heat stressed sac is cooling (shearing, spraying the ventral abdomen with cold water, fan), rehydration and correction of metabolic abnormalities with oral or intravenous fluids. intravenous isotonic sodium bicarbonate solution may be required to treat metabolic acidosis. maintenance fluid rates in adults are 30-40 ml/kg/day and crias 80-120 ml/kg/day. pulmonary edema is a serious risk if fluids are administered too fast (>120 ml/kg/hour). other supportive therapies include nasal oxygen insufflation, non-steroidal anti-inflammatories, antioxidants (vitamin e and selenium) and broad-spectrum antibiotics. steroids such as dexamethasone may be indicated in advanced cases. therapeutic diuresis with furosemide is indicated in animals with respiratory distress due to pulmonary edema. heat stress is best prevented by timely shearing, adequate watering (clean, cool water), and providing shade and cooling (fans, sprinklers). obese animals are more predisposed. predisposing factors such transportation, excessive handling and breeding should be avoided during the hot humid days. heat stress index (hsi), expressed as the ambient temperature (°f) + humidity (%), and is considered too high when it reaches or surpasses 160. in camels, scrotal edema may be a sign of acute trypanosomiasis. traumatic injuries traumatic injuries are usually due to fighting amongst males. scrotal traumatic injuries are relatively rare in the wild population of camelids, probably because of the strict social organization. severe traumatic fighting injuries are more common in camels during the rutting season. the extent of the injury may vary from scrotal skin lacerations to severe testicular rupture and hemorrhage. testicular hemorrhage may not show any external signs of laceration and is only identified by ultrasonography of the scrotal content. treatment protocols should be centered on reducing local swelling and preventing 580clinical theriogenology • volume 6, number 4 • december 2014 infectious complications, and a booster with tetanus toxoid. unilateral castration is the treatment method of choice for severe unilateral testicular trauma involving the tunica vaginalis and testes.1,6 orchitis, epididymitis and peri-orchitis inflammatory changes of the testis and associated organs are often noticed as a sudden onset of hind limb lameness, reluctance to breed or visible swelling of the scrotum. infection spread hematogenously (brucella abortus, brucella meletensis and alpaca fever due to s. equi zooepidemicus) or may ascend from scrotal wounds.5,7 treatment with systemic antimicrobials is often unrewarding and orchidectomy is the best option for the welfare of the male and salvage of reproductive ability if the affection is unilateral. acute preputial swelling acute preputial swelling may be due to complications from urolithiasis or traumatic injuries. the etiology of urinary calculi in the camelid is not well understood but is suggested to be similar to that in other domestic ruminants.8,9 clinical signs include persistent straining, odontoprisis, inappetence and ileus, anorexia, dribbling blood tinged urine, and signs of abdominal discomfort.1 more severe clinical signs ensue in cases of complete blockage and rupture of the urethra or bladder. young males (11 to 15 months of age) may present as an emergency because of frequent straining and urine dribbling. this phenomenon is not necessarily due to urolithiasis but could be due to prostatic enlargement during puberty. in camels, preputial swelling is a clinical sign of acute trypanosomiasis. in racing camels, the placement of a preputial ring to prevent masturbation behavior may results in adhesions and obstruction of urine flow. physical examination often reveals tachycardia, tachypnea and elevated rectal temperature. complete blood count may reveal an elevated white cell count and neutrophilia with a left shift, increases in fibrinogen, increased creatinine kinase and aspartate aminotransferase activity, hyperglycemia, hypercreatininemia and increased urea nitrogen. serum electrolyte abnormalities include hyponatremia, hypochloremia and hyperkalemia. fluid obtained by abdominocentesis or from the preputial swelling shows increased creatinine concentration.1 increased serum urea nitrogen and creatinine concentrations suggest presence of uroperitoneum. transcutaneous ultrasonography of the ventral abdomen may show subcutaneous free fluid and tissue edema in the case of urethral rupture and large volumes of free fluid in the abdominal cavity in the case of urinary bladder rupture. in the latter case, the urinary bladder may not be possible to image. transrectal ultrasonography may reveal dilation of the pelvic urethra if the bladder is intact. prognosis is grave in the presence of hydroureter and hydronephrosis. uroliths are often located in the distal penile urethra about 7 to 12 cm from the penile orifice but may occasionally be found immediately proximal to the sigmoid flexure. the urethral recess at the ischial arch makes catheterization of the urinary bladder impossible. surgical management (tube cystotomy, urethrotromy) are a possibility but they carry a poor prognosis particularly for reproduction. preputial/penile trauma and prolapse preputial lacerations are usually a consequence of masturbation behavior (breeding the ground or objects) or complications from foreign objects within the prepuce. hair rings around the penis are commonly found in llamas and suri alpacas. presenting complaints may be similar to those seen with urolithiasis. in some cases the only sign is preputial bloody or purulent discharge. complication resulting in local adhesions and prevention of urination are possible.5,6 the penis and prepuce are evaluated under heavy sedation or general anesthesia (table). early management of preputial and penile injuries should center on providing adequate protection of the traumatized tissue and prevention of infection and complication with urine scalding. replacement of the healthy prolapsed preputial mucosa and its retention with a purse string suture is very helpful in early cases. daily cleaning of the sheath with saline and application of local anti-inflammatory and 581 clinical theriogenology • volume 6, number 4 • december 2014 antimicrobial ointment for three to five days will help reduce the chance of further complications. sutures may be removed after a week to ten days. excessive preputial prolapse with slight necrosis requires circumferential resection and anastomosis of the prepuce. long-standing lesions carry a poor prognosis particularly if there is overt cellulitis and tissue necrosis. tissue necrosis is common problem with these cases and may include the penis due to pressure ischemia. surgical debridement and or phallectomy may be required in these cases. rectal prolapse rectal prolapse is often seen in obese male camels following excessive breeding. the condition is best treated with sexual rest. surgical management may be required for long-standing cases. rectal prolapse may also be the consequence of excessive straining due to urolithiasis. soft palate prolapse in camels the soft palate (dulla) is exteriorized frequently during rutting season in the dromedary.6 impaction of this diverticulum with food or the presence of a foreign body results in entrapment of the tissue under the molars and traumatization during mastication. onset of edema and even abscessation are a common complication resulting in a permanent exteriorization and dysphagia.10 in rare cases, the soft palate is swollen but not exteriorized and blocks the normal respiration which may lead to asphyxiation. surgical management ( i.e. palatectetomy) is the best course of action. excision of the prolapsed dulla is performed under heavy sedation or general anesthesia after ligation of the large vessels. post-surgical management includes nsaids, antimicrobials and tetanus prophylaxis. animals should be on soft food for three to four days after surgery.1 emergencies in the non-pregnant female the most common reproductive emergencies in non-pregnant females are traumatic injuries during breeding or iatrogenic injuries during reproductive examinations. severe trauma to the pelvis and genital organs may occur when heavy males breed younger or lighter females resulting in neurologic syndrome. breeding trauma followed by excessive straining may lead to vaginal and/or even rectal prolapse. this is often seen in the case of congenital abnormalities (persistent hymen, vaginal aplasia) or vaginal/cervical adhesions. rectal or colonic injuries may occur following excessive transrectal palpation or ultrasonography.11,12 the distance between the anus and the peritoneal reflection is very small (4 cm in llamas, 2 to 3 cm in alpacas and 6 to 10 cm in camel). complete rectal tears in camelids are rapidly complicated by peritonitis due fecal contamination. often the only clinical sign is reluctance to stand, lethargy and progressive dehydration a few hours after a reproductive examination. severe toxic shock and death follows within 8 to 24 hours if no medical action is taken.1 patients should be immediately referred to a surgical facility. videoendoscopic evaluation after epidural anesthesia or under general anesthesia may help decide if a transanal repair is possible or if celiotomy should be considered. transanal repair is successful if the laceration is not deep. celiotomy with pubic symphysiotomy is the only option for caudal injuries and in particular for alpacas.12 iatrogenic uterine perforations are due to aggressive placement of foley catheters, infusion pipettes and biopsy forceps. they become an emergency if a major blood vessel is included in the injury or if an irritating substance (iodine) is infused into the abdominal cavity. affected females present with colicky signs consistent with peritonitis or hemoperitoneum. anemia is a feature if there is enough blood loss. supportive therapy includes non-steroidal anti-inflammatory drugs and antimicrobials. blood transfusion and surgical intervention may be indicated if the packed cell volume is < 8%. vaginal perforation with severe bleeding may be controlled by vaginal compression packs. 582clinical theriogenology • volume 6, number 4 • december 2014 emergencies in the pregnant female camelids rely on the corpus luteum for progesterone secretion and maintenance of pregnancy. therefore severe illness associated with an inflammatory or extreme stress response may rapidly lead to luteolysis and abortion with all its complications. pregnant females may present with a variety of emergency clinical syndromes including severe colic or respiratory distress, downer (lateral or sternal continuous recumbency), anorexia, diarrhea, depression, neurologic conditions, excessive straining, profuse diarrhea, severe abdominal wall edema or defects, vaginal discharge, premature lactation or vulvar dilation, and vaginal prolapse. some of these presentations may have a genital origin. the cardinal rule in handling emergencies in the pregnant female is to perform a thorough clinical evaluation of the dam, evaluation of the fetus, and rule in or out genital origin of the presenting complaint after stabilization of the dam. the main emergencies of genital origin in the pregnant female are uterine torsion, vaginal prolapse, impending abortion and uterine rupture. general approach to handling emergencies in the pregnant female clinical evaluation of the dam. any concern on the part of the owner regarding behavior of the midto late-term pregnant camelid should be taken seriously. a detailed history should be obtained, including breeding dates, time and methods used for pregnancy diagnosis, history of previous illness of reproductive disorders, onset and duration of the clinical problem, and recent treatments administered. if the female is in severe distress, blood samples should be taken immediately and the female stabilized before further examination. oxygen insufflation should be initiated and a jugular venous catheter should be placed immediately to allow fluid therapy and emergency anesthesia if needed. choices of drugs and dosage for sedation should take into account their effect on the fetus. butorphanol tartrate (0.05-0.1 mg/kg im) provides good sedation and has minimal effect on the cardiovascular system. however, a mild decrease in systemic vascular resistance has been observed that can be of consequence if the blood flow to the uterus is already restricted.13 the fetus and uteroplacental unit as well as all abdominal viscera and the peritoneal cavity, should be evaluated. abdominocentesis and peritoneal fluid analysis should be considered. a caudal epidural block and infusion of a mixture of lidocaine and lubricant into the rectal cavity help reduce straining and facilitate per rectum evaluation for uterine torsion, presence of pelvic or abdominal masses and imaging of the bladder. vaginal examination may be performed on select cases (presence of abnormal discharge). digital examination of the vagina and cervix does not present any value in the authors’ opinion except when the cervix is open. fetal and uteroplacental evaluation normal fetal heart rate in midto late-pregnancy should be between 1.6 to 1.8 times that of the dam. fetal heart rate tends to range from 80 to 115 bpm in the last trimester of pregnancy and decreases to 80 bpm a few days before parturition. in our emergency cases, fetal stress is evidenced by a heart rate that is constantly above 130 bpm or constantly below 50 bpm. fetal activity is maximal in the first half of pregnancy and is extremely reduced in the last two months of pregnancy.1 similar observations were reported recently in alpacas.14 presence of twins is better confirmed by abdominal radiography in lateterm pregnancy. fetal biometrics may provide an idea on fetal growth and pregnancy stage but most measurements are not very accurate in our experience. fetal fluids are difficult to appreciate because the amount of amniotic and allantoic fluid in this species is low. values for fetal growth in alpacas have been generated recently. birth weight is significantly correlated to size of aorta, kidney and radius.14 the combined uteroplacental thickness (cupt) should be evaluated in the fetal horn (left uterine horn) only, as the placenta may appear thicker in the non-pregnant horn. in the authors’ experience camelids with cupt > 8 mm in the last trimester are more prone to placental detachment and fetal compromise. in a recent study on alpacas, the mean cupt increased from 3.8 ± 0.7 mm at 5 months to 583 clinical theriogenology • volume 6, number 4 • december 2014 6.1 ± 0.7 at term.14 excessive edema of the uterine horn or premature placental detachment are relatively easy to detect and require immediate intervention if the female is at term. ancillary laboratory testing although a stress leukogram is often present in many of the presenting females, neutrophil count, immature band neutrophil count, neutrophil morphology, packed cell volume and fibrinogen concentration are very valuable in evaluating inflammatory and toxic states. anemia may be due to blood loss or onset of other problems such as mycoplasma hemolamae infection. blood biochemistry will help identify electrolyte imbalances and detect risk for hepatic lipidosis which is the major concern in stressed pregnant females that are anorexic. hypoproteinemia is often found in old pregnant females and may be a predisposing factor to metabolic complications. in some cases the serum may be grossly hyperlipemic (white). however, lipemia and ketonemia are not always present in hepatic lipidosis. elevated levels of non-esterified fatty acid (nefa; > 400 µmol/l) and ß-hydroxybutyrate (bhb) concentrations are significant indicators of stress and liver compromise. liver compromise is also indicated by elevated bile acids, gamma-glutamyl transferase and aspartate transaminase, and sorbitol dehydrogenase.8 marginal calcium and magnesium levels or hypocalcemia may be noted in late-term pregnant females and require monitoring during the correction and convalescent period. specific conditions in the pregnant female uterine torsion. uterine torsion is a major differential in colic or depression in the pregnant sac. the condition is possible but not as common in camels.15 clinical signs with uterine torsion range from mild episodes of discomfort to severe colic, diarrhea and anorexia. on physical examination tachypnea and tachycardia are common. complete blood count and blood chemistry changes are consistent with a stress leukogram with various metabolic changes (hepatic lipidosis) depending on the duration and severity of the problem.16 diagnosis is based on transrectal palpation of the broad ligaments as described in other large animal species. palpation of the broad ligament can elicit painful reaction. diagnosis by vaginal palpation has been reported by practitioners but it is not reliable in our experience unless the torsion also includes the cervix which is rare. diagnosis may not be possible until exploratory laparotomy in case of severe colic. alternately, the female could be palpated under general anesthesia which provides more relaxation of the anal sphincter and perineal area.17 although the majority of uterine torsions in camelids are clockwise, a significant proportion (15 to 20%) are counterclockwise.16 uterine torsions can be corrected by rolling, or surgically after celiotomy. both techniques are very efficient. rolling should be considered only if the uterus and its vasculature are not compromised. rolling of alpacas on the farm may be performed without sedation if help is available. sedation or even general anesthesia may be required for llamas. the pain usually disappears immediately after correction of the torsion and females return to normal activity immediately unless they have been anorexic in which case correction of metabolic disorders (hepatic lipidosis) should be part of the follow-up plan. surgical correction may be performed following flank or midline laparotomy. midline laparotomy is the preferred method in later stage of pregnancy.16 complications of uterine torsion include uterine rupture/hemorrhage, endotoxemia and death of the patient. splenic torsion concurrent with uterine torsion has been described in one case with persistent pain following correction.17 progesterone supplementation after correction of the torsion is still a subject of debate. a recent report on 60 cases of uterine torsion in our clinic showed a survival rate of 96.7% for females and 78.3% for fetuses.16 cria survival was significantly (p < 0.05) higher following rolling (100%) than emergency cesarean section (70%). however, females that underwent a cesarean section had a more severe torsion. the most common blood biochemistry abnormalities were toxemia (leukophilia due to neutrophilia, with toxic neutrophils evident on a blood smear; 19.9%), hypocalcemia (<9.0 mg/dl; 5%), toxemia plus hypocalcemia (5%), toxemia plus hepatic lipidosis (3.4%), and elevated creatinine 584clinical theriogenology • volume 6, number 4 • december 2014 kinase (1.7%). all cases of toxemia had concurrent hyperglycemia. the presence of toxemia and blood biochemistry changes tended to be higher with a prolonged interval to presentation.16 uterine rupture. uterine rupture is often due to severe or inadequate clinical management of a uterine torsion. females usually present in advanced stages of shock and in lateral recumbency. abdominocentesis may reveal large volumes of serosanguinous or bloody fluid. severe pain with presence of serosanguinous peritoneal fluid may also be observed in case of splenic torsion.17 the only option is surgical intervention to remove the fetus and salvage the uterus. complete hysterectomy should be considered if there is severe compromise of the uterus. vaginal prolapse. vaginal prolapse is relatively common in all camelid species in the last two months of pregnancy. predisposing factors include age (older females), parity, and body condition (obese and very thin females).1 prolapse of the entire vagina and exteriorization of the cervix is rare but possible. prolonged periods of prolapse lead to increased inflammation of the prolapsed tissue and even severe necrosis of the vaginal mucosa. a complication with ascendant infectious placentitis is possible. in camels, myiasis of the prolapsed tissue is not uncommon. chronic cases are at risk of abortion and/or rectal prolapse because of persistent tenesmus. rectal and vaginal prolapse may be the only sign of uterine torsion, dystocia or abortion. the prognosis for the life of the fetus and dam is relatively good if the condition is treated early. in camels, the vaginal tissue is maintained in place by placing a bühner suture around the vulva. in alpacas and llamas, a shoelace suture pattern is sufficient. sheep vaginal prolapse retainers may help in some cases in alpacas. more advanced cases of prolapsed vagina with increased tenesmus may require epidural anesthesia. other complications of pregnancy. other complications of pregnancy in camelids include ventral abdominal herniation, prepartum downer syndrome, metabolic diseases and premature lactation/placentitis. hydrops of fetal fluids is extremely rare in camelids. ventral herniation during pregnancy is often a complication of previous abdominal surgeries including cesarean section. induction of abortion or parturition may be contemplated to save the female. abortion is easily induced at any stage with an intramuscular administration of cloprostenol (total dose of 250 micrograms in llamas and alpacas and 500 mg in camels). the same dosage is sufficient for induction of parturition with good neonatal survival if pregnancy stage is > 330 days and there is sufficient mammary gland development and colostrum production. abortion or parturition occurs about 18 to 22 hours after administration of prostaglandin.18 in a few situations, a second administration of cloprostenol is necessary. clients should be warned that induction of abortion or parturition in the compromised female may predispose to dystocia. in our practice, fetal heart rate is monitored closely following induction and a cesarean section is performed if there are no signs of parturition at 20 hours and fetal heart rate is < 70 bpm. obstetrical emergencies normal parturition and proper obstetrical techniques have been reviewed by the authors in detail elsewhere.19 it is estimated that about 5% of all camelid births will require some assistance and about 2% will require advanced obstetrical expertise. obstetrical problems are an emergency in camelid because of the relatively explosive and short stages of parturition which are similar to the equine. dystocia of maternal origin include: uterine inertia, uterine rupture, and failure of appropriate dilation of the cervix or vestibulum. uterine torsion and failure of cervical dilation requires delivery by cesarean section. however it is important to confirm that the dam is at term. dystocia of fetal origin occurs most commonly as a result of malpositioning or malposture and to a lesser degree, presence of malformations, twins and large size fetuses. the most common fetal causes of dystocia are carpal or shoulder flexion, head deviations (lateral and ventral) and bilateral hip flexion. transverse presentations are possible and are a common reason for cesarean section. fetal abnormalities causing dystocia include hydrocephalus, schistosomus reflexus, contracted tendons, ankylosis of the hind limbs or neck, and conjoined twins. other anomalies that may complicate delivery include fetal anasarca and emphysematous fetus resulting from the death and production of gas 585 clinical theriogenology • volume 6, number 4 • december 2014 from decomposition. although twining is rare in camelids, a few twin births have been reported. delivery of twins may be complicated by presentation of both fetuses to the birth canal at the same time or in the case of conjoined twins. in our experience all twin dystocias have required cesarean section in order to preserve female reproductive tract integrity. general considerations for obstetrical manipulations three major differences between camelid and ruminants need to be kept in mind when dealing with an obstetrical situation. 1) the pelvic inlet is narrower, 2) the cervix and vaginal are more prone to laceration and severe inflammation often leading to adhesions, 3) risks for neonatal hypoxia and death are increased by the forceful uterine and abdominal contraction and the rapid detachment of the microcotyledonary placenta. most dystocias present to the practitioner during the second stage of parturition. prolongation of the first stage of parturition is primarily due to failure of cervical relaxation or uterine torsion. transrectal palpation and digital examination should allow diagnosis and cesarean section is the best course of action in these cases. in the case of dystocia during the second stage of labor, assessment of cervical dilation and fetal disposition is performed following sedation and/or epidural analgesia. guidelines for assisted vaginal or controlled vaginal (under heavy sedation or general anesthesia) delivery are similar to procedures recommended for the equine and should be kept to no more than 20 minutes. elevation of the hind quarters may facilitate manipulation. the use of clenbuturol when available greatly reduces uterine contractions and facilitates obstetrical manipulation. the use of epinephrine for uterine relaxation has been reported anecdotally but there is no described protocol for this. in our experience, fetotomy is not an option in alpacas and most llamas and camels. cesarean section remains the best approach if controlled vaginal delivery cannot be achieved in less than 20 minutes. analgesia and anesthesia for obstetrical manipulation most simple obstetrical manipulation can be performed without sedation. caudal epidural analgesia is often sufficient. sedation with xylazine has been used by the author particularly in camels. the drug is known to increase uterine artery resistance and increase myometrial contraction in ruminants leading to higher risk of fetal hypoxia. these effects have not been studied in camelids. our group has moved away from induction of anesthesia with ketamine because it has been associated with neonatal depression. we prefer induction with profofol for cesarean section or for surgical management of uterine torsion. because propofol is primarily metabolized by the liver, its use in females with concurrent hepatic lipidosis should be monitored. maintenance of general anesthesia with isoflurane or sevoflurane is ideal, because these inhalation anesthetics are rapidly eliminated. cesarean section techniques for cesarean delivery in camelids have described in detail elsewhere.19,20 we recommend flank approach in camels and any severely compromised sac. left paravertebral (flank) approach. most females will tolerate the operation with a line or inverted “l” block anesthesia (lidocaine diluted to 1% with isotonic bicarbonate or saline, with the total dose not to exceed 4.4 mg/kg bw) following sedation, caudal epidural and physical restraint in the sitting sternal position. after surgical preparation, an oblique incision is made extending from the angle formed by tuber coxae to the level of bottom of the last rib. the incision line should be parallel to the direction of the quadriceps when the animal is sitting in the sternal position. the subcutaneous muscle and fascia and the external oblique muscle are incised while the internal oblique and transverse abdominal muscle may be gridded along the muscle fibers. the peritoneum is incised together with the transverse abdominal muscle. the gravid uterine horn (always the left) is grasped around a fetal limb and gently exteriorized from the incision. an incision is made over the limb at the greater curvature and the fetus is exteriorized. if the placenta is still attached, it should be left in place but peeled off along the uterine incision to avoid 586clinical theriogenology • volume 6, number 4 • december 2014 entrapment during closure of the uterus and dehiscence. because of the type of placentation, mural bleeding is common and hemstasis is provided by oversewing the margins of the uterine incision using a continuous interlocking pattern using absorbable suture material (#1 chromic gut in camels or #0 polydioxanone [pds] in sac). the uterine wall is closed in a cushing, utrecht, or lembert pattern (#0 or l polyglactin 910, #0 polydioxanone or poliglecaprone in alpacas, #1 chromic gut in llamas and camels). some practitioners prefer to administer oxytocin into the uterine wall. the uterine wall and abdominal cavity may be lavaged with a warm sterile saline solution containing antibiotics (one liter isotonic saline solution containing antibiotics (potassium penicillin g 22,000 u/kg body weight [bw], sodium ampicillin 20 mg/kg, or sodium ceftiofur 1 mg/kg), antiinflammatory drugs (flunixin 1 mg/kg), and heparin (20 to 40 units/kg). carboxymethyl cellulose (cmc; 14 ml/kg bw, intraperitoneally [ip]) may be used to prevent postoperative adhesions. the abdominal cavity is closed in three layers using a simple continuous pattern. the suture is anchored every 2 to 3 cm to the transverse muscle to remove any dead space and prevent formation of pockets. the skin is closed using a continuous ford interlocking suture pattern. ventral midline approach. midline celiotomy approach is performed with the patient in dorsal recumbency under general anesthesia and is ideal if the uterus is compromised or need to be completely exteriorized. a midline celiotomy incision (25 to 30 cm in alpaca and 35 to 40 cm in llamas) is made through the skin, subcutaneous fat, cutaneous trunci muscle and linea alba from about 4 cm cranial to the border of the mammary gland extending towards the umbilical scar. the uterus is identified by direct palpation and exteriorized. an incision is made through the uterine wall along the greater curvature. the cria is removed and the umbilicus clamped and transected. the linea alba is closed by appositional pattern with interrupted horizontal mattress, cruciate pattern sutures, or continuous suture pattern (#2 polyglycolic acid or #1 polydioxanone or polyglactin 910). closure of the skin may be done with staples, horizontal mattress suture pattern or preferably with subcuticular suture pattern (#2-0 polyglactin 910 or polyglecaprone). postoperative care, complications and prognosis. postoperative care includes pain management using butorphanol tartrate or flunixin meglumine. antimicrobial prophylaxis should be continued for five to seven days depending on the condition of the uterus and fetus at the time of surgery. fluid therapy may be indicated in some cases. the dam should be monitored for postpartum metritis and toxemia. the placenta is generally expelled within a few hours if the cervix is open or two to four days if it was closed at the time of surgery. oxytocin may be administered (20 iu im in camels and 5 to 10 iu im in sac) every four hours during the first 24 hours after surgery. complications include retained placenta, incisional infection, hernia, peritonitis, intestinal adhesions, and infertility. however, these complications are very minimal when the surgery is performed early in dystocia and sterile technique is used.21,22 the most common complications seen in 63 camel cesarean sections, performed by the author and colleagues in the field, were incisional infections (n=6), peritonitis (n=2), and herniation (n=1) (tibary and anouassi, personal observations). the rebreeding success rate is excellent (>70%) and most females will be rebred at three to four months after surgery.21,22 the earliest successful rebreeding after cesarean section seen by the author is 45 days. we generally recommend least 45 to 60 days sexual rest. neonatal resuscitation following dystocia neonatal monitoring following dystocia is extremely important as obstetrical manipulation is the leading risk factor for hypoxia, failure of passive transfer and sepsis.1,23,24 postpartum emergencies postpartum emergencies are often due to complications of obstetrical situations. in addition to the main genital problems that may alarm the owner (i.e. traumatic injuries, bleeding, uterine prolapse, persistent straining and retained placenta) some of these cases present with ataxia, prolonged recumbency and various degrees of anorexia or depression as the primary complaint. evaluation of the postpartum female should include a complete history and a detailed account of the obstetrical situation including 587 clinical theriogenology • volume 6, number 4 • december 2014 placental delivery. the female should be assessed by complete physical examination, cbc and blood biochemistry, transabdominal and transrectal ultrasonography and vaginal examination. excessive fluid in the abdomen warrants abdominocentesis. recto-vaginal tear due to the small perineal body and the powerful expulsive efforts in camelids, recto-vaginal tears are common following overt obstetrical manipulations. cases seen in our practice are often a complication of fetotomy. recto-vaginal tears may be repaired immediately or a few weeks after second intention healing.5,25 uterine bruising and tears postpartum uterine tears are not as dramatic as in the equine species unless there is involvement of a large vessel or severe contamination of the uterus and peritonitis. uterine involution is very rapid in the camelid and small, dorsal uterine tears may heal quickly with the only sequela of infertility due to peri-uterine adhesions. peritonitis is possible if there is severe contamination associated with placental retention.26 it is not clear how uterine tears occur in camelids and although most are associated with obstetrical manipulation, we have seen cases following normal parturition. if a uterine tear is detected in the early postpartum period by direct vaginal palpation, an attempt could be made to induce uterine prolapse after treatment with epinephrine and epidural anesthesia. alternately, the uterine tears can be repaired after celiotomy. adjunctive therapy for peritonitis is indicated and should include abdominal lavage and systemic broad-spectrum antimicrobial and antiinflammatory therapy, along with fluid therapy for cardiovascular support. postpartum hemorrhage in camelids, the vascular supply to the reproductive tract presents several differences compared to ruminants and equine, characterized by prominent caudal and cranial vaginal arteries and a large arcuate cervical artery.27 most of the postpartum hemorrhage cases diagnosed by our group consist of rupture or laceration of the vaginal uterine artery. excessive manipulation, and in particular fetotomy, may cause sufficient erosion of the mucosa and laceration of the artery. unfortunately, many of these hemorrhages are missed as no outward signs are noted until it is too late. in many females, blood accumulates within the uterus for a few hours followed by cardiovascular collapse. in one case, the female was found dead in her stall two hours after delivery. ruptured vaginal arteries may be sutured if easily accessible. hemorrhage may be managed medically by administration of aminocaproic acid and placement of compresses.28 blood transfusion should be considered in females with a pcv <10%. uterine prolapse partial or total uterine prolapse can occur secondary to dystocia, manual removal of a retained placenta or excessive use of oxytocin (high dosage and frequency). uterine prolapse is more common in camels than sac and is often associated with hypocalcemia, selenium deficiency and retained placenta. dairy camels seem to be more prone to uterine prolapse.29 uterine prolapse occurs generally immediately (the first 30 minutes) after parturition or abortion. techniques for replacement are similar to those reported in cattle and small ruminants under sedation and epidural analgesia. the placenta is often easily peeled off and should be removed if possible before replacement of the uterus. the female is positioned in sternal recumbency with the hind quarters slightly elevated. the uterus should be inspected for any lacerations or hemorrhage. the area of major risk for hemorrhage is located near the cervix where the uterine artery may be exposed. the uterus is cleaned with warm dilute povidone iodine solution before replacement. a bühner suture is used in camels and a shoelace pattern can be used around the vulvar lips in alpacas and llamas. uterine prolapses tend to reoccur if the uterine horns are not fully extended. hysterectomy should be considered if the uterine tissue has sustained severe damage.1 588clinical theriogenology • volume 6, number 4 • december 2014 rectal and vaginal prolapse postpartum rectal or vaginal prolapse is often associated with persistent straining after dystocia and presence of severe necrotic vaginitis or pelvic masses. necrotic vaginitis may be so severe that urination becomes difficult. vaginal prolapse alone can be seen up to three weeks following parturition. these are often due to a ruptured vestibulo-vaginal sphincter. a cerclage suture may help retain the vagina until complete involution occurs. other complications of the postpartum period emergency postpartum complications in camelids include a vast array of conditions which often manifest themselves as lethargy and depression progressing towards a downer female syndrome. the approach to diagnosis of the causes of downer syndrome is similar to that used in the bovine. predisposing factors include septic metritis, necrotic vaginitis, retained placenta, hypocalcemia, dystocia, pelvic injuries, hemorrhage and presence of compressive lesions. milk fever and toxic mastitis have been described in dairy camels but not in sac. severe swelling of the vulva and vagina are painful conditions associated with overt obstetrical manipulation. females experiencing these complications may show persistent straining and abandon their neonate. untreated vaginal and cervical inflammation may lead to complete vaginal adhesions and development of pyometra. females with severe inflammation of the birth canal should be treated with systemic and local anti-inflammatory drugs. daily application of cold compresses and local application of a vaginal tampon covered with ointment (lanolin, antimicrobials) helps prevent adhesions. conclusion reproductive emergencies involve not only saving the health but also the reproductive future of the patient. emergencies in the pregnant female present a peculiar challenge in that the fetus has to be taken into account. one of the main challenges in emergency care in camelids is the lack evidence-based scientific data on treatment and outcome assessment. although extrapolation from other species has been possible, it is important to remember species peculiarities especially with regard to fluid therapy. handling of obstetrical situations is particularly important as many female camelids lose their reproductive ability due to iatrogenic vaginal adhesions and cervical trauma from lengthy manipulation. emergency drugs and protocols (table) should be available in order to reduce the time to address critical care and improve outcome. supportive treatment in cases of emergency should always include nutritional support and stimulation of appetite and gastrointestinal function (vitamin b complex and transfaunation). references 1. tibary a, rodriguez j, sandoval s: reproductive emergencies in camelids. theriogenology 2008;70: 515-534. 2. middleton jr, parish sm: heat stress in a llama (lama glama): a case report and review of the syndrome. j camel practi res 1999;6: 265-269. 3. schwalm a, erhardt g, gerken m, et al: influence of heat stress on thermoregulation and fertility in shorn and unshorn male llamas (lama glama). tieraerztl prax ausg g nutztiere 2008;36: 324-328. 4. schwalm a, gauly m, erhardt g, et al: changes in testicular histology and sperm quality in llamas (lama glama), following exposure to high ambient temperature. theriogenology 2007;67: 1316-1323. 5. tibary a, vaughan j: reproductive physiology and infertility in male south american camelids: a review and clinical observations. small rumin res 2006;61: 283-298. 6. tibary a, anouassi a: male reproductive disorder and surgery. in: camelid theriogenology; 1997. 7. aubry p, swor tm, barrington gm, et al: septic orchitis in an alpaca. can vet j 2000;41:704-706. 8. van saun rj: nutritional diseases of south american camelids. small rumin res 2006;61: 153-164. 9. gutierrez c, corbera ja, faye b: obstructive phosphate urolithiasis in a dromedary camel: a case report. j camel pract res 2008;15: 77-79. 10. gahlot tk, jhirwal sk, bishnoi p, et al: facial paralysis, glossoplegia and injured soft palate in a camel. j camel pract res 2004;11: 157-158. 11. steffen s, grunert e: perforation of the rectum in a llama mare caused by rectal palpation (veterinary expert opinion). dtsch tierarztl wochenschr 1995;102: 330-331. 12. stone wc, lindsay wa, adams gp, et al: rectal and colonic injury in the llama anatomic considerations and surgicalmanagement in 4 llamas. vet surg 1993;22: 62-66. 589 clinical theriogenology • volume 6, number 4 • december 2014 13. garcia-pereira fl, greene sa, keegan rd, et al: effects of intravenous butorphanol on cardiopulmonary function in isoflurane-anesthetized alpacas. vet anaesth analg 2007;34: 269-274. 14. ferrer ms, jones m, anderson de, et al: ultrasonographic parameters of fetal well-being and development in alpacas. theriogenology 2013;79: 1236-1246. 15. bolbol ae, el-eknah mm, aboul-fadle w, et al: caesarean section in female camels. proc second sci cong egypt soc cattle dis; 1993. vol 1. p. 82-89. 16. pearson lk, rodriguez js, tibary a: uterine torsion in late gestation alpacas and llamas: 60 cases (2000-2009). small rumin res 2012;105: 268-272. 17. smith jj, dallap bl: splenic torsion in an alpaca. vet surg 2005;34: 1-4. 18. bravo pw, bazan pj, troedsson mht, et al: induction of parturition in alpacas and subsequent survival of neonates. j am vet med assoc 1996;209:1760-1762. 19. tibary a, rodriguez js, anouassi a, et al: management of dystocia in camelids. j am assoc bovine pract 2008;166176. 20. tibary a: surgery of the reproductive tract in camelids. in: current therapy in large animal theriogenology. youngquist rs, threlfall wr, editors. 2nd ed. st. louis: saunders elsevier; 2006. p 905-918. 21. campbell a, pearson lk, tibary a: cesarean section in alpacas and llamas at a referral center technique, survival, and postoperative fertility: 24 cases (2000-2012). clin theriog 2013;5: 360. 22. miller ba, brounts sh, anderson de,et al: cesarean section in alpacas and llamas: 34 cases (1997-2010). j am vet med assoc 2013;242: 670-674. 23. dolente ba, lindborg s, palmer je, et al: culture-positive sepsis in neonatal camelids: 21 cases. j vet intern med 2007;21: 519-525. 24. tibary a, rodriguez j, walker p, et al: neonatal care and neonatal emergencies in camelids. j am assoc bovine pract 2008;177-184. 25. anwar s, purohit gn: occurrence and surgical repair of third degree perineal lacerations in adult female camels (camelus dromedarius) by one-stage (goetz) technique. open vet j 2013;3: 75-79. 26. bedford sj, hawes m, paradis mr, et al: peritonitis associated with passage of the placenta into the adbominal cavity in a llama. j am vet med assoc 1996;209: 1914-1916. 27. león m e, sato s a, navarrete z m, et al: macroscopic anatomy, irrigation and venous drainage of female reproductive apparatus of llama (lama glama). rev invest vet perú 2011;22: 1-8. 28. pearson lk, tibary a: clinical management of postpartum hemorrhage following failure of cervical dialation in an alpaca. clin therio 2014.(in press) 29. agab h: diseases and causes of mortality in a camel (camelus dromedarius) dairy farm in saudi arabia, j camel prac res 2006;165-169. 590clinical theriogenology • volume 6, number 4 • december 2014 table: formulary for emergency veterinary care of camelids drug llamas and alpacas camels remarks sedatives/anesthetics/analgesics and reversal agents acepromazine 0.02-0.05 mg/kg iv, im, sq 0.05 mg/kg iv, im sedation, use with care in depression animals, hypotensive. avoid in emergency butrophanol 0.05-0.1 mg/kg im, sq 0.05-0.2 mg/kg im ketamine 2-4 mg/kg iv 4 mg-6 mg/kg im 5-8 mg/kg im avoid iv route in pregnant animals ketoprofen 2 mg/kg iv, im, sq 2 mg-3 mg/kg iv, im lidocaine 2% (caudal epidural) 1 ml /100 kg 1 ml/100 kg for local anesthesia use to effect (toxic dose 4 mg/kg) talazoline 2 to 4 mg/kg im or sc iv slow 0.2 mg/kg iv caution cardiac asystole possible yohimbine hcl 0.125 mg/kg iv 0.25 mg/kg iv reversal agent for xylazine neonatal resuscitation aminophylline 2 – 4 mg/kg sq 4-7 mg/kg sq bronchodilator atropine 0.04 mg/kg iv, sq 0.04 mg/kg im, sq use if bradycardia doxapram hcl 5-10 mg/kg iv, im, sl 0.20.3 mg/kg iv, sl use caution as effects do not last very long antimicrobials ampicillin sodium 6-10 mg/kg iv tid 6.6 mg/kg iv tid ceftiofur (solution) 2 -4 mg/kg iv bid 2 mg/kg iv bid ceftiofur (suspension) 2.2 mg/kg sq, im sid 1-2 mg/kg, im sid enrofloxacin 5-10 mg/kg sq sid 2.5 to 5 mg/kg sc or im sid florfenicol 20 mg/kg sq q48 hours 20-40 mg/kg sq single dose gentamicin 5 mg/kg im, sid 4 mg/kg im bid nephrotoxic oxytetracycline la 20 mg/kg sq 10-20 mg/kg im every 2 to 3 days procaine penicillin g 22,000 to 44,000 iu/kg sc or im bid or sid 22,000 iu/kg im bid hormones cloprostenol 250 µg im 500 µg im total dose misoprostol 400-600 µg cervical 1-2 mg topical for cervical dilation oxytocin alpacas; 5-7.5 iu im llama; 5-10 iu im 20 -30 iu im iv drip at a rate of 1 iu per minute hydroxyprogester one caproate 250 mg im 500 mg im every 3 weeks do not use in the last 4 weeks of pregnancy anti-inflammatory drugs flunixin meglumine 1.1 mg/kg iv or sq bid or sid fluid therapy* 591 clinical theriogenology • volume 6, number 4 • december 2014 other useful drugs clembuterol** 300 micrograms as a single dose, im or slow iv obstetrical manipulations diphenhydramine 0.5-1mg/kg im or iv anaphylactic shock epinephrine 1 ml/50 kgs (1:1000 dilution) 0.01 mg/kg anaphylactic shock, uterine myorelaxant vitmanin b complex** 5-10 ml adult 0.02-0.04 ml/ kg buscopan (nbutylscopolammon ium bromide) 4 mg/ ml 1 ml/10 kg spasmolytic, analgesic *fluid therapy, the most common balanced salt solution used are lrs and normosol, bicabonate should be available to correct acidosis, dextrose can be used at 1.5% to 5% depending on degree of hypoglycemia. fluids can be spiked apporiatly to correct hypocalcemia (calcium bromogluconate) or hypokalemia (kcl) or any other metabolic disturbance. **clembutrurol is not available in some countries including the usa, ***each ml contains: b1 (100 mg), b2 (5 mg), b3 (100 mg), b6 (10 mg), b12 (100 mcg) 592clinical theriogenology • volume 6, number 4 • december 2014 2019 news from the society for theriogenology and the american college of theriogenologists dr. david e. bartlett award for lifetime achievement in theriogenology dr. ahmed tibary dr. ahmed tibary is professor of theriogenology at the college of veterinary medicine, washington state university, pullman, wa, usa. dr. tibary graduated in 1980 with a doctorate in veterinary medicine from the institut agronomique et vétérinaire hassan ii (iav-hasson ii), morocco. he earned an ms (1984), doctorate s science (1988) from iav hassan ii and a phd (1989) in theriogenology from the university of minnesota. he was certified as a diplomate of the american college of theriogenologists in 1991. dr tibary started his career in morocco doing clinical practice research on equine and ruminant theriogenology. he worked as a clinical assistant professor of equine theriogenology at the university of minnesota from 19901992. he then accepted a position as the scientific director of the veterinary research center in abu dhabi in the united arab emirates where he helped develop a referral center for infertility and artificial breeding in arabian horses and camels. in 1998, he joined the college of veterinary medicine at washington state where he is now a professor of the comparative theriogenology service providing clinical service and conducting research in equine, ruminant, and camelid reproductive medicine and surgery. dr. tibary devoted most of his career to teaching and conducting applied research on equine, ruminants and camelid theriogenology. he served as veterinary consultant to the royal stables and the national stud farms in morocco and the amiri stable in abu dhabi (uae). he received several awards recognizing his excellence in teaching and clinical practice including the norden distinguished teacher award (2001 and 2005), the faculty member award by the washington state veterinary medical association (2003), the wsu veterinary continuing education award (2002) and the veterinary achievement award by the alpaca research foundation (2007). he was named the 2011 “theriogenologist of the year” by the american college of theriogenologists. dr tibary provided large animal reproductive medicine and surgery continuing education programs for veterinarians around the world. he is the author of several scientific publications, 6 books and over 130 book chapters on camelid, equine and ruminant reproduction. dr. tibary served in several capacities in the act and sft leadership. 2019 proficiency awards the "proficiency in theriogenology award”, sponsored by the society for theriogenology, is a student award to be given annually to one senior at each veterinary school/college in the united states and canada. congratulations to all award recipients (as of 4/16/2019)! julie van scoik (va-md regional college) emily ligon (texas a&m university) mary briggs (midwestern university) brittany shumack (auburn university) ashley davenport (university of georgia) catherine c. thurston (university of pennsylvania) cory anderson (oklahoma state university) austin knapp (ross university) meredith versteeg (atlantic veterinary college, upei) renee saxton (oregon state university) ashley allemand (louisiana state university) kathleen flanagan (kansas state university) nicole sugai (university of illinois) alex hegg (purdue university) matty may (mississippi state university) 91 clinical theriogenology • volume 11, number 2 • june 2019 theriogenologist of the year dr. robert lofstedt dr. rob lofstedt graduated from the university of pretoria in 1973 and spent a couple of years in private practice and industry. with stop-offs at the western college of vet med, iowa state and tufts university, he was awarded an ms in reproductive endocrinology, became board certified in theriogenology and entered academia. his broad interest in reproduction and education has been reflected by memberships in the american society of animal science, society for the study of reproduction, consortium of north american veterinary interactive new concept education (convince), american association of bovine practitioners, society for theriogenology and of course, the american college of theriogenologists. he has served as a consultant for companies, as an expert witness for the avma and as a consultant to the national geographic society. in the last 32 years, he has served as a professor and adjunct professor of theriogenology at the atlantic veterinary college. he has a long-standing association with the act, once an examiner for act board certification and now, as an emeritus member of the act. dr. lofstedt also served on the veterinary council of his home province and as its president from 1997 to 1998. he was recently elected a life member of the peivma. dr. lofstedt is the author of 22 refereed papers, 4 as co-author, nine textbook chapters, nine proceedings and several lay publications. he has been an invited speaker for 14 professional presentations and has conducted many ce meetings for veterinarians on all domestic species. from 1996 to 2006 he served as lecturer and clinical instructor in the uk for the british equine veterinary association. dr. lofstedt’s professional interests include veterinary information technology, virtual reality, digital image manipulation & special illustration techniques. some of this is reflected in the form of lori, an on-line image bank. he has been married for 43 years and has three children (a proven sire). call for nominations-2020 theriogenologist of the year award nomination due date november 15, 2019. the american college of theriogenologists is seeking nominations for “theriogenologist of the year,” an award which recognizes a theriogenologist for recent, outstanding contribution(s) to the discipline of theriogenology. the award is accompanied by a plaque and monetary honorarium of $1,500. 2020 nomination form qualifications include: a. act diplomate in good standing b. has made recent, outstanding contribution(s) to the discipline of theriogenology. 1. in most cases the contribution(s) will have been made within the past 5 year period. 2. outstanding contribution(s) may take many forms and will be judged in part on furthering awareness and understanding of the discipline of theriogenology and theriogenologists, including mentorship and training. 3. note: the award is not intended to recognize career achievement. 92clinical theriogenology • volume 11, number 2 • june 2019 https://www.theriogenology.org/page/toynomination 2020 bartlett award nominations sought nominations are now being sought for the 2020 david e. bartlett award for lifetime achievement in theriogenology, presented annually to a distinguished individual who has made important contributions to the field of theriogenology. the award intended to cement the efforts of the society and college toward common goals in animal reproduction, to reward and inspire excellence, improve the visibility of theriogenology, and to annually recognize the efforts of the founders particularly honors dr. david e. bartlett, first president of the college. among his many accomplishments, dr. bartlett was responsible for deriving the terms “theriogenology” and “theriogenologists,” and was instrumental in gaining hard-won avma recognition for the american college of theriogenologists in 1971. the 2020 david e. bartlett award for lifetime achievement in theriogenology will be presented during the annual therio conference in pittsburgh, pennsylvania. the recipient will be notified in advance, allowing family and friends to be on hand. the honoree will address those in attendance and will have the option of submitting a contribution to the conference proceedings. the honoree will receive $1,000 cash, an engraved statue of nandi, and complimentary registration, hotel accommodations and transportation to the 2020 conference. please submit the accompanying preliminary entry form and nominate an individual to add to the list of preeminent recipients. entry forms must be received no later than january 4, 2020. nominations may also be made online at http://www.therio.org/bartlettawardform. you must be signed in as a sft member to access the form. it is located under the 2020 therio conference and also under the bartlett award recipient list on the sft website. 2020 dr. david e. bartlett award for lifetime achievement in theriogenolo y preliminary entry form sponsored by the theriogenology foundation in cooperation with the society for theriogenology & american college of theriogenologists purpose: to reward a distinguished theriogenologist who has made important contributions to the field of theriogenology. nominee: full address: current employment and position: home phone: business phone: fax: email: provide a short description of nominee’s qualifications for this award. use a separate sheet of paper containing a maximum 350 word narrative bio, highlighting nominee’s key accomplishments. nominated by: full address: home phone: business phone: fax: email: nominations must be received by january 4, 2020 submit online at: http://www.therio.org/bartlettawardform or mail to: theriogenology foundation, bartlett award 761 tiger oak drive pike road, al 36064-3063 93 clinical theriogenology • volume 11, number 2 • june 2019 therio foundation 2019 conference fundraising events tf silent auction 25.0 the passing along of boutique items from colleague-to-colleague through the silent auction has become a signature event of our annual conference. we are limiting the number of items to 25 again this year as it worked out well last year! here are some suggestions for items: *educational items (text books, etc.) *arts and crafts *liquor/wine *sporting events *veterinary equipment once you decide on what your priceless creation or generous gift will be, please e-mail the attached form along with pictures of the item(s) by june 15th, 2019 to: jgfloydjr@gmail.com. get your donation into the spotlight by submitting early…….the final item list will be posted on the website by july 1 so the membership has a month to start saving their dollars to bid! after 25 items have been selected, we will respectfully ask you to hold your gift until 2020! no on-site donations can be accepted this year. thank you for your continued support of the theriogenology foundation silent auction!!! we look forward to seeing you in savannah!! click here for the silent auction donation form. awards dinner and story night this year’s live auction will be held in conjunction with the first annual veterinarian story night. purchase your ticket at: therio.org. sealed bids for live and silent auction items are accepted until july 23. items will be posted online and updated as submitted. please complete the sealed form on the tf website at: http:// www.theriofoundation.org/? page=sealedbidform. craft beer tasting fundraiser for the theriogenology foundation during the opening session reception wednesday evening, july 24 or the awards dinner and story night reception, july 26, attendees will have the chance to experience some locally brewed craft beer and help the theriogenology foundation at the same time. for $40 you will receive four samples of beer. once you have sampled and chosen your favorite, you will get a full serving of that beer. proceeds will benefit the theriogenology foundation. tickets may be used at the event of your choice. 94clinical theriogenology • volume 11, number 2 • june 2019 mailto:jgfloydjr@gmail.com https://www.theriofoundation.org/resource/resmgr/2019_auction/silent_auction-updated_4.12..pdf https://www.therio.org/events/eventdetails.aspx?id=934617 http://www.theriofoundation.org/?page=sealedbidform http://www.theriofoundation.org/?page=sealedbidform http://www.theriofoundation.org/?page=sealedbidform thank you! a huge thank you to the following individuals for all their efforts in planning the 2019 conference! annual conference program chair dr. colin palmer equine track program chair dr. sara lyle production animal track program chair dr. leonardo brito small animal track program chair drs. michelle kutzler and jenny sones small animal symposium chairs drs. michelle kutzler and jenny sones student small animal seminar & lab chairs drs. michelle kutzler and jenny sones educators’ forum chair dr. hilari french silent auction chairs drs. jim floyd and will schultz thank you to our 2019 sponsors! (as of 4/17/19) platinum sponsors idexx lane manufacturing, inc. merck animal health rood & riddle equine hospital and veterinary pharmacy gold sponsors sonosite-a fujifilm company universal imaging, inc. silver sponsors american college of theriogenologists theriogenology foundation bronze sponsors biomerieux multimin usa parsemus foundation thank you to our 2019 exhibitors! (as of 4/17/2019) all vet imaging/microvet diagnostics the american kennel club, inc. aurora pharmaceutical biomerieux botupharma usa, inc. breeder's choice chemometec a/s elsevier, inc. emberequine fertility technology resources idexx imv technologies usa/imv imaging jorgensen laboratories karl storz lane manufacturing, inc. merck animal health multimin usa parsemus foundation puppywarmer sonosite-a fujifilm company universal imaging, inc. veterinary books by success concepts zoetis click on the links below or visit www.therio.org for 2019 therio conference news and registration information! conference registration early bird rates end june 23! hotel registration reduced room rates end june 17! 95 clinical theriogenology • volume 11, number 2 • june 2019 https://www.therio.org/page/2019therioconf https://www.therio.org/events/eventdetails.aspx?id=934617 https://www.marriott.com/event-reservations/reservation-link.mi?id=1549500132008&key=grp&app=resvlink wednesday, july 24 act educators’ forum opening session abstracts opening session reception small animal symposium (additional registration required) opening session abstracts effects of granulocyte colony stimulating factor on the peripheral leukocyte function in postpartum dairy cows dinesh dadarwal doxycycline diffuses to the fetoplacental unit and did not cause apparent complications to the resulting foal fernanda d’el rey dantas , presented by igor canisso comparison of three (3) different intramuscular vehicles with or without progesterone on serum progesterone concentrations in non-cycling mares jamie kaczor validation of an in-clinic immunoassay for measurement of canine progesterone elizabeth schooley virulence factors and antimicrobial resistance of e. coli isolated from canine pyometra and its relationship to biofilm formation tessa fiamengo evaluation of angiogenesis in equine hydrops pouya dini the immune system’s role in placental detachment cristina rosales, presented by christina marth granulosa cell tumor in a double yellow-headed amazon parrot (amazona oratrix) julia bumberry thursday, july 25 bartlett award and address theriogenologist of the year award and address dr. jerry rains memorial abstract competition sft business meeting act business meeting 2019 annual conference agenda 96clinical theriogenology • volume 11, number 2 • june 2019 student case presentations student/mentor reception opening reception in the exhibit hall student small animal seminar & lab (additional registration required) dr. jerry rains memorial abstract competition cryosurvival of epididymal and ejaculated bovine spermatozoa frozen in liposomeand egg yolk-based extenders zachary turner evaluation of commercial semen extenders and crystalloids for short term cooled extension of epididymal spermatozoa in squamates and chelonians sean perry safety and efficacy of intrauterine kerosene infusions to enhance endometrial cup regression in mares giorgia podico analytic approach for endometrial polymorphonuclear cells cytology threshold for the definition of cytological endometritis in primiparous and multiparous dairy cows shaked druker anti-müllerian hormone concentrations and antral follicle count in dairy and beef heifers subjected to a modified cidr + cosynch protocol and the association with pregnancy sarah stanley reproductive hormones of pregnant mares in early aluteal cycles and reproductive parameters of post-treatment cycles chun kuen mak fetal and maternal immune response to ascending placentitis carleigh fedorka genomic testing does not correlate with performance trait phenotype in crossbred calves mackenzie roberts student case presentations idiopathic infertility in a friesian stallion lindsay hilburger subfertility in a stallion caused by a genetic mutation affecting the acrosome reaction katelyn kimble dystocia due to a schistosomus reflexus in a miniature dachshund colleen kutzler a case of testicular torsion in a 1.5 year old intact cane corso nichole whitfill spermatic cord enlargement due to lymphangioma breanthony baker prolonged estrus in a 17 month old intact doberman gabrielle montone 97 clinical theriogenology • volume 11, number 2 • june 2019 friday, july 26 scientific abstract presentations awards dinner reception awards dinner and story night plenary session a primer to genome editing claudia klein epigenetic and transcriptional regulation of early mammalian embryo development pablo ross mixed species abstracts localized uterine body pyometra causing urinary obstruction in an american bully bitch lacey rosenberg uterine perforation secondary to metritis and placenta percreta in a labradoodle bitch lacey rosenberg luteinizing hormone receptor-mediated proliferation of isolated canine t-lymphoma cells camryn flint reproductive remnants, mammary sequelae, and renal agenesis in a domestic short hair cat audrey kelleman metastatic ovarian stromal tumor in a young bitch alyssa helms the hatching artefacts in hermann’s tortoise (testudo hermanni) after artificial incubation mateja stvarnik pregnancy outcome of a mare with renal failure luis de aguiar evaluation of embryo formation via comparisons of oocyte harvesting methods mckayla meyer production animal abstracts a delayed timed artificial insemination 78 hours after cidr removal does not affect pregnancy outcomes in dairy heifers treated with a 4-day cosynch+cidr protocol roberto palomares factors associated with bulls not classified as satisfactory potential breeders at the university of tennessee veterinary medical center nancy conkey telemetric monitoring of calving using a novel calf alert device cyril stephen effect of semen collection method on semen quality in developing bulls jarrett proctor 98clinical theriogenology • volume 11, number 2 • june 2019 effect of injectable trace minerals on mrna expression of toll-like receptors in the testes of bulls with heatinduced testicular degeneration mohammad ibrahim effects of nerve growth factor-β added to extenders for cryopreservation of electro-ejaculated and epididymal harvested bull semen jamie stewart direct effects of nerve growth factor-β, purified from bull seminal plasma, on steroidogenesis and angiogenic markers of the bovine pre-ovulatory follicle jamie stewart nerve growth factor-β increases small luteal cell number and enhances steroidogenic activity in the bovine corpus luteum jamie stewart equine abstracts cidr: another tool for the transitional mare (retrospective findings in 24 mares) ashton broman enrofloxacin crosses the equine placenta in early pregnancy without inducing gross lesions in the fetus robyn ellerbrock cryosurvival of cooled-transported stallion semen frozen in three commercial extenders brittany lister is stallion age related to dystocia or neonatal foal morbidity? james thompson can we preserve immunoglobulin g in frozen colostrum of mares for three years? anne josson-schramme maternal serum and allantoic fluid concentrations of activin a in experimentally-induced equine placentitis harutaka murase a preliminary study comparing pregnancy rates and live-foal rates using individually sheathed or non-sheathed insemination pipettes justin mcnaughten preliminary insemination trial to determine the fertility of stallion spermatozoa stored for 3 and 7 days at 17ºc in a long-term liquid storage medium jennifer clulow case reports prostaglandin facilitated mare foal bonding in a pregnant nurse mare justin mcnaughten delayed sperm production in a presumed sertoli cell only syndrome case in two boars affected by severe combined immunodeficiency malavika adur body pregnancy twin reduction in the mare by colpotomy sabrina hall 99 clinical theriogenology • volume 11, number 2 • june 2019 diagnosis and management of cystic endometrial hyperplasia in a pot-bellied pig julie strachota life-threatening complications of a third-degree perineal laceration in a quarter horse mare viviane gomes equine placentitis caused by stenotrophomonas maltophilia, a multiple-drug-resistant organism viviane gomes inducing parturition in goat with pregnancy toxemia using aglepristone vanessa peixoto de souza clinical assessment and treatment of infertility in a greater one-horned rhinoceros (rhinoceros unicornis) monica stoops abstract poster presentations successful treatment of presumptive cystic ovarian disease in okapi (okapia johnstonii) anneke moresco induction of ovulation in donkeys using gnrh or hcg ava kent immunohistochemical evidence for kisspeptin signaling in equine gonadotropes christianne magee long term reproductive examination of 3 dogs treated postnatally with deslorelin acetate cristina gobello uterine flush fluid proteome of mares in estrus and diestrus: qualitative differences and functional classification firdous khan improvement of canine semen vitrification using sucrose and soy lecithin extender maja zakosek pipan personalized medicine – the future of veterinary medicine? margret casal four healthy litters produced from a sire with semen retrieved from urine after retrograde ejaculation mateja stvarnik microsatellite-based characterization of nigerian indigenous dogs (canis lupus familiaris) in southwest nigeria oluwatoyin ajala metritis in early lactation in dairy cows: energy balance, ovarian function, incidence of subclinical endometritis, and reproductive performance shaked druker small animal track the use of frozen semen in the bitch stuart mason transcervical insemination in the bitch stuart mason 100clinical theriogenology • volume 11, number 2 • june 2019 elective caesarean section in the bitch stuart mason production animal track evolution of synchronization protocols and the use of ftai in beef cattle in south america gabriel bo control of estrus and ovulation in beef heifers and cows david patterson practical considerations for implementation of ai programs for beef cattle sandy johnson equine track rethinking sperm motility stuart meyers antisperm antibodies and immune-mediated infertility. are we missing something? maria soledad ferrer panel discussion stuart meyers, maria soledad ferrer saturday, july 27 so you want to become a diplomate? career paths in theriogenology student case poster presentations small animal track current state of spay/neuter programs in the u.s. and effect on overall animal numbers david haworth how outdated perceptions have reshaped the dog marketplace patti strand joint disorders and cancers sometimes associated with spaying and neutering dogs benjamin hart neoplastic considerations of spaying and neutering dogs evan sones traditional surgical and laparoscopic vasectomy in dogs and cats robert mccarthy growing interest in hormone-sparing dog sterilization and recommendations for standard identification methods linda brent production animal track sexed semen production speaker tbd embryonic stem cells in livestock breeding pablo ross 101 clinical theriogenology • volume 11, number 2 • june 2019 fertility programs to achieve high 21-day pregnancy rates in dairy herds paul fricke economic impacts of reproductive performance in dairy herds julio giordano control of ovarian function for in vivo and in vitro embryo production gabriel bo methods for and implementation of pregnancy diagnosis in cattle paul fricke factors affecting bull sexual development leonardo brito equine track michelle leblanc memorial equine lectures management of twins from 12-120 days; selected advanced caudal reproductive tract surgeries angus mckinnon genetic predisposition for developing equine arteritis virus carrier state: adopting proper biosecurity measures when breeding mares to a persistently infected stallion udeni balasuriya a review of the equine oviduct: pathology, evaluation and current treatments maria schnobrich when and how to perform a transvaginal ovarian biopsy in a mare claire card reproductive effects of equine endocrine disease teresa burns panel discussion claire card, maria schnobrich student case poster presentations management of a uterine prolapse and dehiscence in a queen catarina benavides pyometra in a tennessee walking horse mare blair bennett use of a probang for successful field management in a case of chronic paraphimosis ashley davenport ectasia of the rete testes in an angus bull jordan farrell diagnosis of a metastatic ovarian teratoma in a pregnant bitch darian gordon 102clinical theriogenology • volume 11, number 2 • june 2019 mammary gland fibroma in a quarter horse mare lindsay hilburger clockwise 180° uterine torsion in a brown swiss cow with twins maría juárez a molar pregnancy in a hereford donor cow lauren merritt a case of polioencephalomalacia in a breeding bull madelyn pelletier vulvar squamous cell carcinoma in a german shepherd dog samantha perry recurrent abortion and vaginal papillomatosis in a brangus cow caitlin sanders abnormal behavior in the mare associated with a granulosa cell tumor joshua trumble non-obstetric vaginal laceration in a recently spayed mixed-breed bitch julia zuercher 103 clinical theriogenology • volume 11, number 2 • june 2019 2019 therio conference special events small animal symposium sponsored by idexx act educators’ forum sponsored by theriogenology foundation opening session abstracts student small animal seminar & lab sponsored by sonosite-a fujifilm company dr. david e. bartlett award for lifetime achievement in theriogenology theriogenologist of the year award sponsored by universal imaging, inc. dr. jerry rains memorial abstract competition sponsored by merck animal health sft business meeting act business meeting student case presentations sponsored by idexx exhibit hall opening reception theriogenology foundation craft beer tasting student quiz bowl sponsored by merck animal health student t-shirt contest scoty award sponsored by idexx plenary speakers drs. claudia klein and juan p. ross sponsored by american college of theriogenologists theriogenology foundation silent auction annual awards dinner reception annual awards dinner sponsored by merck animal health dr. john steiner award for practitioner excellence theriogenology foundation veterinarian story night and live auction small animal symposium agenda sponsored by idexx a crash course in veterinary microbiomics candace lyman vaginal microbiome: what not to do candace lyman monitoring reproductive biome health using cytology and histopathology christopher premanandan canine placentation normal gross and histologic structure as well as confounding features during evaluation christopher premanandan the art of measuring progesterone: understanding immunoassays elizabeth schooley canine placental microbiome jenny sones reproductive microbiome alterations: clinical management of pyometra michelle kutzler reproductive microbiome alterations: clinical management of prostatitis michelle kutzler student small animal seminar & lab sponsored by sonosite-a fujifilm company canine reproductive ultrasonography 101 luis de aguiar, viviane gomes lab: reproductive ultrasonography luis de aguiar, viviane gomes, michelle kutzler, jenny sones roundtable lunch: future of companion animal theriogenology lab: progesterone, relaxin, and brucella canis assays luis de aguiar, viviane gomes, michelle kutzler, jenny sones 104clinical theriogenology • volume 11, number 2 • june 2019 https://www.therio.org/page/2019therioconf#small%20animal%20symposium%20agenda 2019 board of directors candidates dr. hilari french dr. hilari french is a third-generation veterinarian, following in her father and grandfather’s footsteps into the profession. she attended the school of veterinary medicine at louisiana state university and received her dvm in 2009 and phd in 2011. she then moved to the university of illinois and completed a large animal theriogenology residency as well as a second residency program for the food animal practice of the american board of veterinary practitioners. she joined the american college of theriogenologists in august 2014 and the american board of veterinary practitioners in october 2015. hilari joined ross university school of veterinary medicine in 2013 as an assistant professor of theriogenology and was promoted to an associate professor in 2017. she is currently serving as the interim department head of clinical sciences and her research focus is on immunocontraceptive use to control feral donkey populations in the caribbean. hilari is married to dr. adam silkworth and they have just welcomed a baby boy, vander, into their family in february 2019. dr. heath king dr. king grew up on a commercial cattle operation that he and his family still operate in eastern mississippi near the town of macon. he graduated veterinary school from mississippi state university in 2007 and stayed there to complete an internship and residency in theriogenology. he and his wife are both employed by mississippi state university college of veterinary medicine as associate clinical professors. together they have two small boys ages 6 and 4. his wife cathleen is a large animal surgeon that works primarily in the equine hospital. dr. king provides theriogenology services for canine, bovine, and equine patients that present to the teaching hospital. his special interests include large animal urogenital surgery and embryo transfer. his current research efforts involve collaborating on projects involving fetal programming and the use of messenger rna to invoke a local immune response for prevention of sexually transmitted diseases. dr. jennifer koziol dr. jennifer koziol is a native of oklahoma. she received her bachelors of science in 2009 and doctor of veterinary medicine in 2012 from oklahoma state university. following graduation, she accepted an internship in comparative theriogenology at iowa state university working with all species. after completing the internship she continued her formal training as a theriogenology resident at auburn university dr. koziol achieved diplomate status in the american college of theriogenologists in 2015 and was awarded a masters of biomedical sciences in 2016. following completion of her formal training dr. koziol joined the faculty at purdue university where she serves as an assistant professor in theriogenology/production medicine and faculty advisor for the sft student chapter. 105 clinical theriogenology • volume 11, number 2 • june 2019 her clinical interests focus on all aspects of large animal reproduction while her research interest focuses on the investigation of tritrichomonas foetus in the bull along with initial descriptions of the microbiome of the urogenital tract in the male bovid. dr. koziol co-authored the 2nd edition of the society for theriogenology bull breeding soundness manual which released in 2018. dr. riley thompson dr. thompson received her bs in agriculture from tennessee tech university in 2012 and graduated with her dvm from the university of tennessee in 2017. during her dvm education, dr. thompson served as the scaaep co-president, theriogenology club secretary, and class of 2017 treasurer. she also was inducted into the phi zeta veterinary honor society and was awarded the utcvm outstanding senior theriogenology student award. she is currently a phd student in a co-appointment between the university of tennessee and smithsonian conservation biology institute (scbi). her research is focused on the endometrium of equids, both domestic and nondomestic horses, including przewalski’s horses and persian onagers. specifically, her research concentrates on how to successfully cryopreserve equine endometrial explants and development of equine endometrial organoids as a novel method for in vitro cell culture. in addition, dr. thompson is also receiving training at scbi in artificial reproductive techniques in several other ungulate species, including scimitar-horned oryx, dama gazelle, eld’s deer, and hartmann’s mountain zebra. dr. thompson’s projected graduation date for her phd is may 2020, and she is planning to continue her research on the equine endometrium as a postdoctoral fellow following graduation. outside of work, dr. thompson enjoys reading, baking, and hiking with her dachshund, sherlock. dr. wendy vaala wendy received her v.m.d. from the university of pennsylvania school of veterinary medicine where she also completed an internship in large animal medicine and surgery followed by a residency in large animal medicine. she remained on staff as assistant professor of medicine until 1996 during which time she helped establish the nicu and high -risk pregnancy program at new bolton center. the next nine years were spent in private equine practice in new jersey where she started medicine referral services and established two nicus and high-risk pregnancy programs. in 2004, wendy joined intervet, inc. (now merck animal health) as a sr. equine technical service veterinarian. her current position is director of life-cycle management for companion animal and equine. her responsibilities include working collaboratively with members of r&d, business development, marketing and professional veterinary services to support and develop existing and new biologics and pharmaceuticals for companion animals and horses. wendy is board-certified in large animal internal medicine. her special interests include equine neonatology and perinatology, parasitology and infectious diseases. she is an invited lecturer at state, national, and international veterinary conferences. she has authored numerous articles for scientific journals and text books and served as guest editor for the wb saunders clinics of north america issue on perinatology and assistant editor for wiley-blackwell’s equine reproduction textbook. wendy and her husband live in western wisconsin on a farm overlooking the mississippi river. 106clinical theriogenology • volume 11, number 2 • june 2019 special events 2019 bartlett-proficiency awards toy award 2019-nom form 2020 bartlett nomination sponsors and exhibitors tf fundraising events 2019 2019 conference agenda-june 2019 sft board noms 2012: the american college of theriogenologists first forty years the american college of theriogenologists – first forty years augustine peter department of veterinary clinical sciences, college of veterinary medicine, purdue university, west lafayette, in abstract the past 40 years of the history of the american college of theriogenologists (act) is summarized. the act became a recognized veterinary specialty organization in 1971. from 28 charter diplomats, currently, it has grown to 441 diplomates. the growth of the act, its contribution to the veterinary profession and society, its future and challenges, and its sister-organizations are described under appropriate headings. regardless of the changing times in the profession and society, technology, and challenges, the future for the act diplomates is very promising in practice, industry, government, and institutions of professional education because of its committed members and their contributions to the veterinary profession, humanity, and the animal kingdom. keywords: theriogenology, american college of theriogenologists, society for theriogenology, theriogenology foundation, clinical theriogenology introduction the american college of theriogenologists became a recognized veterinary specialty organization at the 108th annual meeting of the american veterinary medical association (avma) in detroit, michigan in july of 1971 and was chartered under the laws of the state of illinois. the act came into existence to fill a long-recognized need to unify, identify, and advance that branch of veterinary medicine concerned with the multitudinous phases of reproduction with which veterinarians are involved. it was organized (http://www.theriogenology.org/) to promote the advancement of knowledge; to encourage excellence in undergraduate, graduate, and postgraduate education; and to increase capacities for research and service in theriogenology. the name theriogenology (therio = beast or animal + gen/genesis = beginning, birth, reproduction + ology = study of) was coined by professor herbert howe, department of classics, university of wisconsin and has replaced the phrase veterinary obstetrics, gynecology, and andrology (semenology) from the veterinary vocabulary. dr. david bartlett (american breeders service, deforest, wisconsin) was instrumental in accomplishing this process and gave life to this term by his untiring effort. the subject theriogenology has become an integral part of the veterinary curriculum. as theact celebrates its 40 year anniversary, it is appropriate to reflect on its vibrant life today and consider future issues facing the college under following headings: purpose, progress, and pride. purpose the act was established as a certifying agency to develop a mechanism for veterinarians to become specialists in animal reproduction via residency programs (with or without a graduate component) or extensive clinical experience in theriogenology. other purposes included encouraging research and clinical investigation in theriogenology with publication of findings and developing continuing-education programs for disseminating information and increasing practitioner’s knowledge in theriogenology. founding fathers the act organizing committee consisted of david bartlett (american breeders service), clarence bierschwal (university of missouri), lloyd faulkner (colorado state university), john kendrick (university of california), fayne oberst (michigan state university), stephen roberts (cornell university), and raimunds zemjanis (university of minnesota). the 28 charter diplomates included the organizing committee and drs. bill adams, les ball, cliff barker, bill brown, jim callahan, ed carroll, bill cates, tracy 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ge directly rela of theriogenol omates, unan diplomates. t ology. credentialing mmittee embers) president treasure rience with m study and me mentorship p of the acvsernate route c see figure 1). equire a study effectively dir ove their likel n the college ative training rovided at http committees n the leadersh and committe ip aking a signif honorary mem g (providing n by theriogen gnificant con l meetings, si ated to the fie logy. honora nimously supp the act has t examination committee (8 members) pt r ajor emphasis entorship pro program must -ar. an add candidates to y and mentors rect the prepa lihood of succ e and practitio route must id p://www.ther hip and the va es of act ficant contribu mbers of the a training oppo nology reside ntribution to th ignificantly co eld of therioge ary members ported by the truly been enr exec scienti informat abstract com (8 memb president elect secretary s in theriogen gram. t be pre-appro itional chang sit the certify ship program aration of alter cess. the me oners seeking dentify a men iogenology.o arious commit ution to the fi act. honora ortunities, ser nts, participat he literature), ontributed to enology, or o of the act (s executive boa riched by its h cutive board m co (12 * ific tion/ mmittee bers) t vice directo nology includ oved and inclu e in 1993, rep ying examinat for alternativ rnative route entorship prog certification ntor and subm org/. ttees is provid ield of theriog ary members rvice on gradu tion in journa , attended and o the advancem other unique a see appendix ard and affirm honorary mem d mission ommittee* members) * ad hoc e president ors at large (3) ding successfu ude a diploma placed the qua tion with a stu ve route appli candidates fo gram has also in the act. mit a plan of st ded in figure genology duri have particip uate committe al clubs, parti d/or presented ment of scien and significan x v) were nom med by a majo mbers’ contrib nominating committee (4 members) immedi presi ) executive ul completion ate of either a alifying udy and icants was or the certifyi o served to cre individuals tudy for appro 2. ing their caree pated in ees, teaching cipation on th d at the societ ntific or clinic nt contribution minated by at ority vote of t butions to awards nom committ (7 memb * ad h ate past ident e director n of a act ing eate oval. er he ty for cal n to t least the minating tee* bers) oc clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201269 clinical theriogenology • volume 4 number 1 • march 2012 internationalization and diversity one of the wishes of the founding fathers was to avoid provincialism in the organization.1 thankfully, the act has been and is being greatly enriched by graduates of veterinary schools from other parts of the world. as indicated in table 1, act diplomates serve in many countries. it is worth noting that the act has become a leader in internationalization and diversity as evidenced by its membership as well as its leadership. the act has developed a working relationship with ecar by way of boardmember exchange visiting to participate in each other’s board meetings. it is expected act will continue to develop additional working relationships as other theriogenology programs develop across the globe, particularly on the continents of australia and asia. table 1. distribution of act diplomates by geography and type of employment country active emeritus honorary total united states 281 49 17 347 canada 43 3 1 47 argentina 1 1 australia 14 14 brazil 1 1 colombia 1 1 denmark 1 1 france 1 1 germany 2 2 jordan 3 3 malaysia 1 1 netherlands 1 1 2 new zealand 6 6 south africa 7 7 sweden 1 1 2 switzerland 1 1 united kingdom 2 1 3 venezuela 1 1 totals 366 54 21 441 breakdown by employment type academia 152 private practice 124 government 13 retired 56 industry 24 not noted 58 other 14 sister organizations society for theriogenology the first official meeting of the group was known, at the time, as the "rocky mountain society for the study of breeding soundness of bulls." the organization was founded by a small group of veterinarians for the purpose of collecting and disseminating information related to evaluation of bulls for breeding soundness. during the 1956 meeting, the organization’s official name was changed to "the society for the study of breeding soundness of bulls", as its membership expanded beyond the rocky mountain region. the society was chartered under the laws of colorado in 1961. between 1960 and 1962, the society experienced rapid growth as it wrestled with the challenge of training and certifying its members. the original constitution stated members would undergo a training session followed by a subsequent clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 70clinical theriogenology • volume 4 number 1 • march 2012 evaluation of bulls under the supervision of a member of the board of directors. the society was given a charter under the laws of colorado in 1961. regional groups formed first in texas followed by the southeast group, the montana group, the missouri valley group and the california group. in 1963 the board adopted a new constitution with modifications resolving the issue of requirements for members and establishing dues. during the same meeting the name was again changed to “the american veterinary society for the study of breeding soundness." in 1974, the name was changed one final time to the "society for theriogenology" (sft) and its annual conferences began (appendix vi). the diplomates of the act have regularly contributed to these conferences since their inception. the scope of the sft was broadened to include all species served by veterinary medicine. currently sft (http://www.therio.org) membership includes 1881 veterinarians and 732 veterinary students. student members also belong to their respective student chapters in veterinary schools across the u.s. and canada. each student chapter is supervised by theriogenology faculty at the institution (see appendix vii). growth of the act has been assisted immensely by the sft and many act diplomates are also members of the sft. theriogenology foundation the theriogenology foundation, formed in 2009 as a collaborative effort of act and sft, is working to secure and allocate resources dedicated to advancing the science and practice of animal reproductive medicine. the foundation is committed to providing educational opportunities for students, interns, residents, and new faculty through travel grants and sponsorship of guest lecturers. individual donors can direct their contribution to support research in equine, food/production animal, wildlife/exotic and companion animal reproductive medicine (http://therio.org/displaycommon.cfm?an=1&subarticlenbr=346). official journal in 2009, the journal, clinical theriogenology (http://st.omnibooksonline.com/index.html) was launched to promote scholarship in the clinical aspect of theriogenology. the purpose of the journal is to publish, in a timely manner, peer-reviewed manuscripts relevant to the clinical practice of theriogenology in livestock, companion animals, and wildlife for veterinary practitioners, academic clinicians, animal scientists, and veterinary students. until 2005 the proceedings (presentations, abstracts, and posters) of the annual sft conferences were published by the management office. from 2006 until 2008 they were published in the journal theriogenology. since 2009 the proceedings are published in this newly created official journal of act and sft. the journal is growing under the editorship of act diplomate, robert youngquist, the founding editor. management prior to 1990, the act was administered by volunteer diplomates. since 1990, the act has utilized the services of a professional management company including gmo, inc. (1990-1998), walker associates (1998-2003), and franz management services (2003-present). dr. charles franz, a veterinarian and owner of franz management services, currently serves as the executive director for act and sft. as executive director, dr. franz represents both groups in a number of professional organizations including the american society of veterinary medical association executives (http://vmaexecs.org/index.cfm) and the american board of veterinary specialties (abvs) of the avma as the act’s permanent alternate member. franz management services has been instrumental in helping both organizations create greater visibility for theriogenology. pride the act continuously strives to maintain a standard of excellence among its members through its certification process. candidates must possess a broad knowledge of theriogenology in order to achieve diplomate status. credentialed candidates demonstrate a documentable competency in animal clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201271 clinical theriogenology • volume 4 number 1 • march 2012 reproduction and sit the certifying examination. certification is awarded to those candidates demonstrating a mastery of theriogenology. contributions to the profession having established a culture of excellence, focusing on the reproductive system, the act provides the veterinary profession a unique, multi-species specialty. through their respective curriculum committees, the vast majority of veterinary medical schools and colleges have established separate and identifiable courses devoted to the study of theriogenology. these actions are in large part, based upon a need in the veterinary medical education for a thorough understanding of reproductive behavior and function. through their scholarly endeavors, diplomates of the act have distinguished themselves in educational, investigational, and clinical programs. these efforts include, but have not been limited to, development of educational courses for undergraduate, professional, and graduate-level curricula; continuing-education opportunities for graduate veterinarians and the general public; publication of manuscripts in critically-reviewed scientific journals on all aspects of basic and applied reproductive research; service on editorial advisory boards of veterinary medical journals; and development and/or maintenance of residency and graduate-training programs which focus on reproductive processes. many institutions of higher learning have and continue to utilize the talents of diplomates to serve as administrators and academic leaders. though a relatively small group, the act diplomates are widely involved in international, national, state and regional veterinary conferences. capacity attendance and consistently high approval ratings from participants at these events are testimony the act is meeting a definitive need of the profession. in addition to the annual sft meeting in which a majority of the speakers are diplomates of the act, diplomates are extensively involved with other organizations including species-focused groups (american association of equine practitioners, american association of bovine practitioners, american association of swine practitioners, etc.). a comfortable degree of convergence, interchange of programs and activities between the system-specialty and species-specialty is very important for the profession.1 the diplomates are frequently called on to lecture and teach laboratories at these meetings, as well as to serve in leadership roles in these professional associations. a speaker’s bureau is available on the act website (http://www.theriogenology.org/) with contact information for diplomates willing to speak on various topics in a variety of species. in partnership with the sft, members of the act have worked to establish basic standards for evaluating and maintaining reproductive performance of both male and female animals. this is demonstrated by the development and maintenance of current standards and forms for evaluating male animals for breeding soundness. to hasten technology transfer to practicing veterinarians, the act and sft conceptualized, developed and distributed manuals for breeding soundness examination and fact sheets (1988-1992) detailing assorted topics in animal reproduction. subsequently symposia were developed as a more effective means of disseminating information in greater depth. copies of all symposia proceedings are annually distributed to the entire membership of the act and sft. new information, which often results from research conducted by diplomats is disseminated to professional (veterinary medical) and graduate students, or clinical residents in formal training programs, and to private practitioners. diplomates serve the general public and practicing veterinarians through medical/surgical care and diagnostic support for individual animals with reproductive problems, as well as reproductive assessment/management of healthy animals. diplomates also serve in the federal and state food inspection regulatory bodies and as state veterinarians. the act continually seeks to raise awareness of the college and to encourage veterinary students to aspire to diplomate status after graduation. as part of these goals, act established a student outreach fund to help student organizations cover the costs of inviting diplomates to speak at local events. the fund helps facilitate act diplomate visits and interaction at veterinary schools. it is hoped these visits will stimulate student interest and enthusiasm for the act by broadening students’ awareness of the many opportunities open to our diplomates. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 72clinical theriogenology • volume 4 number 1 • march 2012 educational forums educational forums are conducted at the annual conference with a goal of bringing interested dipolmates together in discussion of ways to improve/strengthen our educational contribution to societal and institutional needs. scholarship consistent with the rise of theriogenology have been numerous technical and scientific advances in knowledge and new technical products applicable to veterinary practice.2,3 more than 7000 peerreviewed publications have been published (pubmed search) by the members of the act. the act has excellent rapport with scientists in other areas of study such as animal science, biology, economics, and other fields of science. this collaboration has resulted in the completion of many research projects with scientists in other disciplines. some diplomates work in non-clinical departments to enable them to fully commit to research. other act diplomates serve as editors of journals and provide assistance to researchers in the developing world in the art of scientific writing. many theriogenology books have been edited (appendix viii) by diplomates over the years with or without chapter contributions from scientists from related areas. many more diplomates have contributed chapters to other related areas in veterinary medicine. in the 1980’s, 16 comprehensive manuals dealing with different aspects of theriogenology in different species were published. subsequently, fact-sheets dealing with specific theriogenology topics were created. since january 1996, a “theriogenology question of the month” has regularly appeared in the journal of the avma. as of today 82 contributions have been made by theriogenologists (appendix ix), most of which originated from theriogenologists and theriogenology residents while in a training program. it has become yet another great source of information for practitioners and veterinary students. as unique means of preserving scholarship, one act diplomate, dr. maarten drost is cataloging digital contributions of visual images in theriogenology in a collection known as, “the drost project visual guides” (http://drostproject.org/drost_information.html), a resource that is helping many. contribution to society the act recognizes the importance of heightening public relations, in order that the public will be more aware of the contributions theriogenology makes to society. the diplomates of the act are uniquely qualified to assist with a host of societal concerns raised by the general public, including the preservation of endangered species, overpopulation of animals and mankind, as well as animal welfare. the favorability of the specialty of theriogenology by the veterinary profession and public remains high. as consultants, diplomates of the act receive scores of telephone calls and referred cases annually from general practitioners. the diplomates of the act function in many capacities within society, ranging from teaching and research in both basic and clinical programs, to employment by governmental agencies, industry, or private practice. many diplomates serve as reproductive consultants to practicing veterinarians or industry. other diplomates are helping plan for and meet the future needs of society by addressing numerous issues facing our world including 1) food production for an everexpanding human population (by increasing reproductive efficiency in food-producing animals), 2) the preservation of endangered species (by studying reproductive physiology of numerous species, preservation of gametes, cloning, and the effect of captivity on reproductive performance), 3) population control of certain wildlife, feral, and companion animals (through development of sterilants, contraceptives, and methods to terminate pregnancy), and 4) studying the impact of environmental toxicologic problems on reproductive systems. the diplomates are also addressing the economic viability of farmers, ranchers, and breeders who depend on animal reproduction for their livelihood. efforts to diagnose treat, and control diseases affecting the reproductive system and the neonate are unending, and require the impact/usefulness of emerging biotechnological advancements on reproductive performance remain a high priority area of study. position statements on a number of issues developed jointly by the act and sft are posted at http://www.theriogenology.org/displaycommon.cfm?an=1&subarticlenbr=53 and these include: clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201273 clinical theriogenology • volume 4 number 1 • march 2012 • position statement on mandatory spay-neuter • basis for position on mandatory spay/neuter in the canine and feline • position statement on the definition of theriogenology in veterinary practice • position statement on the use of ultrasonography imaging to determine reproductive status • position statement on the evaluation for the breeding soundness of animals it is expected that the list will expand based on societal and professional needs. assistance to international veterinarians working in theriogenology the zemjanis outreach fund was established in 1994 to honor the memory of (http://therio.org/displaycommon.cfm?an=1&subarticlenbr=345) raimunds zemjanis, a longtime member of the sft, and a charter diplomate of the act. the fund is supported by donations from members and corporate sponsors and is intended for partial support of veterinarians traveling from developing countries to the united states for educational purposes. future the future of the act is bright. the act’s membership reflects a broad cross-section of the profession including private practice, academia, industry, and government. over the years the act membership and leadership have been quite diverse, signifying health and vigor. twenty one percent of the act diplomates reside and practice outside the usa and graduates of international veterinary schools regularly serve in leadership positions of the act. these trends bode well for the global growth of the act. demonstrating a commitment to quality training for individuals seeking diplomate certification, the act recently established a training and credentialing committee (tcc). in addition to reviewing applicants for credentialing, the tcc is charged with developing and maintaining appropriate standards for credentialing and certification. just as act strives to assure the public and profession through its rigorous and comprehensive certifying examination that its diplomates possess and demonstrate a mastery of theriogenology, act will soon provide assurances of continued mastery among its diplomates. to prove this assurance, in the immediate future, act will initiate two important tasks based, in part, on recommendations from the advisory board on veterinary specialties (abvs) including maintenance of certification and a job-task analysis. maintenance of certification is scheduled to begin for the college by 2015. the act will continue to expand its educational efforts in order to keep the profession wellinformed about theriogenology-related topics. the diplomates of the act will continue to organize, contribute, and participate in symposia and annual scientific meetings in conjunction with the sft. similarly, members of the act will continue to present information in the local, state, national, and international organizations. the progress of the act depends on the ability of its members to provide basic education in theriogenology to undergraduate students while training veterinarians aspiring to attain diplomate status. it is imperative that the act continues to contribute to scholarship by encouraging quality post-graduate programs in theriogenology. in this regard, research (scholarly or scientific investigation or inquiry) and graduate education (the knowledge or skill obtained or developed by a learning process) should be major components of residency programs and residents should be expected to generate new knowledge not just the users of the old.4 a collaborative approach with departments of animal science will immensely help to address the research training needs of residents. further specialized training in specific areas will enable the act members to participate in developing the farm-animal economy of the country. the objectives for training theriogenologists in the farm-animal production have been outlined.5 in two ways this need can be met; the act with support from the sft can find ways to improve and increase diplomates participation in cattle consultation services6 and also plan to provide a dairy theriogenology program as a subspecialty of act. it has to be clearly noted that this subspecialty should follow after clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 74clinical theriogenology • volume 4 number 1 • march 2012 successful certification in comparative theriogenology. a close working relationship with production medicine programs7 and when act grows into a larger college these goals can be easily accomplished. regarding undergraduate training, the legitimate inclusion of comparative theriogenology in the dvm core curriculum and post-graduate training cannot be overemphasized8,9 and the need is felt more than ever. in this regard, a core comparative theriogenology curriculum was created and provided to all avma-accredited colleges of veterinary medicine. this document was intended to act as a guide for the curriculum committees and to support the role of diplomates in the veterinary colleges. in just ten short years, the act will reach its 50 year anniversary and the act leadership will likely have considered implementing some of the proposals listed in these pages. the future for the act diplomates is very promising in practice, industry, government, and institutions of professional education because of its committed members and their contributions to the veterinary profession, humanity, and the animal kingdom. a final point to ponder in general, the discipline of theriogenology has not fared well in some teaching institutions. two common scenarios resulting in declining numbers of theriogenologists positions in academia include; retirement of an act diplomate is followed by either replacement with another specialty individual or not replaced at all or replaced by ‘part-time’ theriogenologists. second, the educational training that ought to be rightfully provided by a theriogenologist to student clinicians is given by specialists from other disciplines due to lack of a critical mass in theriogenology programs.10 considering the scope of benefits provided by act diplomates as described in this article, it is imperative that schools and colleges of veterinary medicine find the value in theriogenologists and continue to make them a part of the resources available at academic institutions. dedication the manuscript is dedicated to two renowned theriogenologists, dr. david bartlett, founding president of the american college of theriogenologists for making theriogenology a reality in the veterinary profession and professor william bosu for his contribution to comparative theriogenology and for nurturing the author. acknowledgments the author enjoys the support and friendship of gary nie, an erudite scholar, whose assistance with this manuscript is appreciated. drs. bob hudson, dale paccamonti, willis parker, and bob youngquist are recognized for their help with this manuscript. thanks to ms. chanda siler for creating the figures. some of the information is borrowed from the five-year reports to the abvs prepared by the act representatives to the abvs; the author thanks these diplomates for their leadership. the author extends special thanks to dr. charles franz and franz management services staff for providing needed information in the compilation of historical and current facts. references 1. bartlett de: theriogenology: from concept to actuality. theriogenology 1985;24:131-145. 2. bartlett de: factors influencing the evolution of theriogenology. j am vet med assoc 1989;195:51-55. 3. kenney rm: thirty seven years working with cattle, horses, and other creatures. what do the next thirty seven hold? proc annu meet soc therio;1991. 4. archbald lf: potpourri and lagniappe from a comparative theriogenologist. proc annu meet soc therio; 2005. 5. lamond dr: theriogenology in veterinary training in relation to animal production. theriogenolgoy 1974;2:161-166 6. rice l: where now, brown cow? proc annu meet soc therio; 2006. 7. whitmore hl: dairy theriogenology: practitioner, professor and producer perspectives. theriogenology 2008;70:262269. 8. root kustritz mv, chenoweth pj, tibary a: efficacy of training in theriogenology as determined by a survey of veterinarians. j am vet med assoc 2006;229:514-521. 9. root kustritz mv, tibary a, chenoweth pj: availability of theriogenology training at north american and caribbean veterinary colleges. j vet med educ 2006;33:140-144. 10. peter at: from the president of the american college of theriogenologists (letter). clin therio 2011;3:9-10. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201275 clinical theriogenology • volume 4 number 1 • march 2012 appendix i: presidents of act 1971-1973: david bartlett 1973-1974: fayne oberst 1974-1975: lloyd faulkner 1975-1976: william adams 1976-1977: barton gledhill 1977-1978: william wagner 1978-1979: clarence bierschwal 1979-1980: edward mather 1980-1981: richard mcfeely 1981-1982: robert hudson 1982-1983: maarten drost 1983-1984: willis parker 1984-1985: robert youngquist 1985-1986: charles martin 1986-1987: brad seguin 1987-1988: howard whitmore 1988-1989: ronnie elmore 1989-1990: shirley johnston 1990-1991: stanley dennis 1991-1992: alfred caudle 1992-1993: theodore lock 1993-1994: patricia olson 1994-1995: chester rawson 1995-1996: walter threlfall 1996-1997: dwight wolfe 1997-1998: michelle leblanc 1998-1999: dickson varner 1999-2000: louis archbald 2000-2001: carla carleton 2001-2002: richard fayrer-hosken 2002-2003: dale paccamonti 2003-2004: douglas freeman 2004-2005: margo macpherson 2005-2006: john steiner 2006-2007: margaret root kustritz 2007-2008: gary nie 2008-2009: ahmed tibary 2009-2010: charles estill 2010-2011: augustine peter appendix ii: bartlett award winners 1984: david bartlett 1985: stephen roberts 1986: elmer woelffer 1987: raimunds zemjanis 1988: lloyd faulkner 1989: clarence bierschwal 1990: les ball 1991: robert kenney 1992: victor shille 1993: robert hudson 1994: donald walker 1995: william wagner 1996: willis parker 1997: a. c. asbury 1998: borje gusstafsson 1999: charles martin 2000: shirley johnston 2001: robert youngquist 2002: patricia olson 2003: robert hillman 2004: maarten drost 2005: louis archbald 2006: lawrence rice 2007: irwin liu 2008: harold whitmore 2009: bradley seguin 2010: robert carson 2011: dwight wolfe appendix iii: theriogenology symposia 1992: equine reproduction symposium 1993: canine theriogenology short course 1994: small ruminant short course 1995: bovine theriogenology short course 1996: mare reproduction symposium swine reproduction symposium 1997: the canine male reproduction symposium reproductive pathology symposium 1998: stallion reproduction symposium canine female reproduction symposium 1999: cryobiology symposium canine reproduction symposium 2000: the periparturient mare & neonate symposium 2001: canine symposium (comment: the annual conference was to start september 12, 2001. the act/sft annual conference was cancelled, but the proceedings were distributed later) 2002: technician/breeder’s symposium (small animal) toxicology symposium 2003: small ruminant symposium (sheep, goats, camelids) bovine symposium (dairy and beef) feline symposium (primarily female) 2004: small animal neonatology symposium equine neonatology wet laboratory clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 76clinical theriogenology • volume 4 number 1 • march 2012 2005: pharmacology symposium small animal symposium equine symposium and wet laboratory 2006: bovine symposium (dairy) small animal symposium (contraception methods) and wet laboratory (early spay/neuter techniques) 2007: no symposia 2008: bovine symposium (beef) small animal symposium and wet laboratory (ultrasonography) 2009: small animal symposium small animal breeders session 2010: reproductive pathology symposium small animal breeders session small animal laparoscopic surgery wet laboratory 2011: small ruminant symposium canine breeders symposium dairy symposium appendix iv: theriogenologist of the year award winners 1999: richard fayrer-hosken 2000: michelle leblanc 2001: robert youngquist 2002: dickson varner 2003: katrin hinrichs 2004: donald schlafer 2005: dirk vanderwall 2006: terry blanchard 2008: ina dobrinski 2009: john kastelic 2010: margaret root kustritz 2011: ahmed tibary appendix v: honorary members of act harold amstutz fuller bazer william bosu patrick concannon c. de bois stig einarsson melvyn fahning borje gustafsson john herrick cleon kimberling clyde kirkbride duane kraemer reuben mapletoft sue mcdonnell richard saacke john simons john spitzer edward squires william thatcher walter zent appendix vi: sft annual meetings 1974 columbia, missouri 1975 cheyenne, wyoming 1976 lexington, kentucky 1977 st. paul, minnesota 1978 oklahoma city, oklahoma 1979 mobile, alabama (hurricane cancelled) 1980 omaha, nebraska 1981 spokane, washington 1982 milwaukee, wisconsin 1983 nashville, tennessee 1984 denver, colorado 1985 sacramento, california 1986 rochester, new york 1987 austin, texas 1988 gainesville, florida 1989 coeur d' alene, idaho 1990 toronto, ontario, canada clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201277 clinical theriogenology • volume 4 number 1 • march 2012 1991 san diego, california 1992 san antonio, texas 1993 jacksonville, florida 1994 kansas city, missouri 1995 san antonio, texas 1996 kansas city, missouri 1997 montreal, quebec, canada 1998 baltimore, maryland 1999 nashville, tennessee 2000 san antonio, texas 2001 vancouver, british columbia (cancelled) 2002 colorado springs, colorado 2003 columbus, ohio 2004 lexington, kentucky 2005 charleston, south carolina 2006 st. paul, minnesota 2007 monterey, california 2008 st. louis, missouri 2009 albuquerque, new mexico 2010 seattle, washington 2011 milwaukee, wisconsin appendix vii: faculty supervisors for sft student chapters aime johnson alabama stuart meyers and wendell cole california patrick meyers colorado audrey kelleman florida richard fayrer-hosken georgia cliff shipley illinois augustine peter indiana bruce christensen iowa robert larson kansas bruce eilts louisiana sandra ayres and carlos gradil massachusetts carla carleton michigan peggy root kustritz minnesota kevin walters mississippi dietrich volkmann missouri sylvia bedford-guaus new york carlos pinto ohio lionel dawson oklahoma tracey chenier ontario michelle kutzleroregon patricia sertich pennsylvania rob lofstedt prince edward island refean lefebvre quebec claire card saskatchewan john dascanio st. kitts tulio prado tennessee charles love texas rick bridges tuskegee beverly purswell virginia ahmed tibary washington harry momont – wisconsin appendix viii: partial list of recent theriogenology texts reproductive biology of the mare, 1st edition, ginther oj, 1979. current therapy in theriogenology, 1st edition, morrow da, 1980. veterinary obstetrics and genital diseases (theriogenology), 3rd edition, roberts sj, 1986. current therapy in theriogenology 2nd edition, morrow da, 1986. reproductive biology of the mare, 2nd edition, ginther oj, 1992. equine reproduction, 1st edition mckinnon ao, voss jl, 1992. ultrasonic imaging and animal reproduction: fundamentals – book 1, ginther oj, 1995. ultrasonic imaging and animal reproduction: horses – book 2, ginther oj, 1995. allen’s fertility and obstetrics in the horse, 2nd edition, england gcw, 1996. current therapy in large animal theriogenology, 1st edition, youngquist rs, 1997. manual of equine reproduction, 1st edition, blanchard tl, varner dd, schumacher j, 1997. ultrasonic imaging and animal reproduction: cattle book 3, ginther oj, 1998 large animal urogenital surgery, wolfe df, moll hd, 1999. equine breeding management and artificial insemination, 1st edition, samper jc, mckinnon ao, pycock j, 1999. canine and feline theriogenology, johnston sd, root kustritz mv, olson pns, 2001. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 78clinical theriogenology • volume 4 number 1 • march 2012 arthur’s veterinary reproduction & obstetrics, 8th edition, noakes de, parkinson tj, england gcw, 2001. sheep & goat medicine, pugh dg, 2002. manual of equine reproduction. 2nd edition blanchard tl, varner dd, schumacher j, love cc, brinsko sp, rigby sl, 2002. small animal theriogenology (practical veterinarian), root kustritz mv, 2003. allen’s fertility and obstetrics in the horse, 3rd edition, england gcw, 2005. the dog breeder's guide to successful breeding and health management, root kustritz mv, 2005. current therapy in equine reproduction, samper jc, mckinnon ao, pycock j, 2006. current therapy in large animal theriogenology, 2nd edition youngquist rs, threlfall wr 2007. ultrasonic imaging and animal reproduction: color-doppler ultrasongraphy book 4, ginther oj, 2007. equine breeding management and artificial insemination, 2nd edition samper jc, 2008. clinical canine and feline reproduction, an evidence-based text, root kustritz mv, 2010. manual of equine reproduction, 3rd edition, brinsko sp, blanchard tl, varner dd, schumacher j love cc, hinrichs k, hartman d, 2010. equine reproduction, 2nd edition, mckinnon ao, squires el, vaala we, varner dd, 2011. blackwell's five-minute veterinary consult clinical companion: equine theriogenology, carleton cl, 2011. appendix xi: theriogenology question of the month globosus amorphus. pearson lk, rodriguez js, tibary a. j am vet med assoc 2011;15;238:1261-1263. trauma-induced paraphimosis. beltaire ka, tanco vm, bedford-guaus sj. j am vet med assoc 2011;238:161-164. pregnancy, pyometra, mucometra, hydrometra, hemorrhage, and urometra in quarter horse-paint mare. tranquillo gg, kelleman aa, sertich pl. j am vet med assoc 2009;235:1161-1164. scoliosis. occhipinti ll, carleton cl, holcombe sj, chaney kp, agnew dw. j am vet med assoc 2009;234:751-753. neonatal ingestion. holmes sp, memon ma, fite cl. j am vet med assoc 2009;234:205-207. unilateral anorchidism. burns jg, petersen nk. j am vet med assoc 2008;233:1553-1554. follicular cysts. schwarze ra, threlfall wr. j am vet med assoc 2008;233:235-237. endometrial polyp. estrada a, ferrer ms, brounts sh, milligan ma, lillich jd, debey b. j am vet med assoc 2008;232:14731475. cause of postestral vulvar discharge. kustritz mv. j am vet med assoc 2008;232:841-843. fetal hydrocephalus. hodder ad, ball ba. j am vet med assoc. 2008;232:211-213. sertoli cell tumor. luby cd, middleton jr, youngquist rs, kim dy, evans at. j am vet med assoc 2007;231:1503-1505. the infected bitch should be euthanized. the other dogs in the kennel should be quarantined. marley ms, rynders pe. j am vet med assoc 2007;231:867-869. bilateral testicular neoplasia. roberson jr, andrews ga. j am vet med assoc 2007;231:531-534. remnants of paramesonephric ducts. kutzler ma, hofmaster il. j am vet med assoc 2007;231:213-215. abscess in the left hemiscrotum, septic urethritis, and inflammation of the right vas deferens. chenier ts, estrada at, koenig jb. j am vet med assoc 2007;230:1469-1472. bilateral testicular neoplasia. roberson jr, andrews ga. j am vet med assoc 2007;230:827-829. neoplasia of the os penis. root kustritz mv, fick jl; j am vet med assoc 2007;230:197-198. hypovolemic shock. scoggin cf, mccue pm. j am vet med assoc 2006;229:1571-1575. unilateral abdominal cryptorchidism. zacharias jr, baird an, hawkins jf. j am vet med assoc 2006;229:937-939. behavior problems in a stallion caused by a nephrolith. dallmeyer md, turner rm, mcdonnell sm, sertich pl, dolente ba, parente ej, diaz om; j am vet med assoc 2006 ;229:511-513. large ossified remnant of the yolk sac. harbo jm, mausling rd, schlafer dh, vanderwall dk. j am vet med assoc. 2006;229:215-217. bleeding varicose veins. christensen bw, troedsson mh, roberts jf, pozor ma, macpherson ml, eichelberger ac. j am vet med assoc 2006;228:1507-1510. sexual immaturity in a prepubertal dog. corrada y, hermo g, gobello c.j am vet med assoc 2006 ;228:855-856. what are the 3 management options for a mare with twin fetuses at this stage of gestation? gray ga, dascanio jj, kolster ka. j am vet med assoc 2006;228:207-209. cystic remnants of the mesonephric (wolffian) ducts (ie, gartner cyst). lefebvre rc, bélanger am. j am vet med assoc 2005;227:1565-1567. hypospadias. hardy rm, kustritz mv. j am vet med assoc 2005;227:887-888. urospermia. dascanio jj, witonsky sg. j am vet med assoc. 2005;227:225-227. seminoma, spermatocele, sustentacular cell tumor.threlfall wr, robertson jt, munsterman as, oglesbee mj, hubbell ja. j am vet med assoc. 2005;226:1649-1650. endometrial cups. willis la, riddle wt. j am vet med assoc 2005;226:877-879. persistence of an intact hymen (imperforate hymen). raggio i, lefebvre r, vaillancourt d. j am vet med assoc 2005;226:205207. acute peritonitis secondary to intra-abdominal semen deposition. slater la, davidson ap, dahlinger j. j am vet med assoc 2004;225:1535-1537. how would you confirm a diagnosis of mycotic infection in an aborted fetus? allison n. j am vet med assoc 2004;225:849851. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201279 clinical theriogenology • volume 4 number 1 • march 2012 theriogenology question of the month. gobello c, baschar h, arias d, tortora m, giordano a. j am vet med assoc 2004;225:207-208. lymphosarcoma infiltrating the uterus, cervix, and vagina. aljarrah ah, gill ms, sod ga. j am vet med assoc 2004;224:1591-1593. acute metritis secondary to retained fetal membranes and a retained nonviable fetus. grundy sa, davidson ap. j am vet med assoc 2004;224:844-847. what is the importance of the fetal bronchopneumonia? strelow l. j am vet med assoc 2003;223:1423-1425. leiomyosarcoma. hinojosa am, holyoak gr, moll d, ritchey jw. j am vet med assoc 2003;223:786-788. in utero infection of the doe by cvv. edwards jf, angulo ab, pannill ec. j am vet med assoc 2003;222:1361-1362 inguinal hernia or hydrocele. bentley va, rashmir-raven a. j am vet med assoc 2002;221:1409-1411. treatment options for erosive seminal vesiculitis caused by acinetobacter calcoaceticus. blanchard tl, woods ja, brinsko sp, varner dd, boothe dm. j am vet med assoc 2002;221:793-795. measurement of serum progresterone concentrations during diestrus. progresterone concentrations during diestrus. pacchiana pd, kustritz mv. j am vet med assoc 2002;220:1465-1467. mineralization of the puppies was suggestive that the bitch would whelp in approximately 11 days. root kustritz mv. j am vet med assoc 2002;220:747-748. histologic examination of tissue sections of the mass. edens ms, heath am. j am vet med assoc 2001;219:1683-1685. torsion of the spermatic cord. pinto cr, paccamonti dl, partington b, mcfadden k. j am vet med assoc 2001;219:13431345. functional ovarian remnant containing estrogen-secreting follicles or ovarian remnant containing an estrogen-secreting neoplasm. kustritz mv, rudolph kd. j am vet med assoc 2001;219:1065-1066. x-chromosome monosomy (xo syndrome). kutzler ma. j am vet med assoc 2001;219:751-752. laparotomy to assist replacement of the uterus. garcia-seco e, gill ms, paccamonti dl.j am vet med assoc 2001;219:443444. differential diagnosis of ovarian tumor, ovarian hematoma and ovarian abscess. peavey cl, trostle ss, bosu wt, cooley aj. j am vet med assoc 2001;218:861-863. endometrial cups. kutzler ma. j am vet med assoc 2001;218:205-206. azoospermia attributable to bilateral epididymal hypoplasia. blanchard tl, woods ja, brinsko sp. j am vet med assoc 2000;217:825-826. an ectopic fetus. nack ra. j am vet med assoc 2000;217:182-184. bacterial placentitis attributable to a gram-positive filamentous branching bacillus organism. wolfsdorf ke, williams nm, donahue jm. j am vet med assoc 2000;216:1915-1916. transmissible venereal tumor (tvt). hasler ah, weber wt. j am vet med assoc 2000;216:1557-1559. erratum in: j am vet med assoc 2000;217:42. pyometra, hydrometra, or mucometra. von reitzenstein m, archbald lf, newell sm. j am vet med assoc 2000;216:12211223. specific aversion to handling for semen collection and to personal approaching the genital area. bedford sj, mcdonnell sm. j am vet med assoc 2000;216:491-493. agarose gel immunodiffusion (agid) serologic testing for brucellosis. kustritz mv. j am vet med assoc 2000;216:181-182. cryptorchid testis. bodri ms. j am vet med assoc 2000;217:1465-1466. scrotal enlargement caused by lymphosarcoma associated with bovine leukemia virus. mccain d, estill ct. j am vet med assoc 1999;215:1777-1779. septic orchitis-periorchitis and epididymitis. kasaback cm, rashmir-raven am, black ss. j am vet med assoc 1999;215:787789. persistent estrus caused by functional granulosa cell tumor of the left ovary. purswell bj, parker na, bailey tl, dascanio jj, sponenberg dp. j am vet med assoc 1999;215:193-195. priapism or paraphimosis. kustritz mv, olson pn. j am vet med assoc 1999;214:1483-1484. malignant teratoma of the ovary. trasti sl, schlafer dh. j am vet med assoc 1999;214:785-786. the goat is pseudopregnant (hydrometra). waldow d. j am vet med assoc 1999;214:195-196. toxicosis associated with fescue grass infected with the endophyte neotyphodium coenophialum (formerly acremonium coenophialum). mirza mh, costa lr, paccamonti d, seahorn tl. j am vet med assoc 1998;213:1405-1406. benign prostatic hypertrophy (bph), prostatitis, and prostatic neoplasia. root kustritz m, merkel l. j am vet med assoc 1998;213:807-809. torsion of the spermatic cord of the right testis. pinto cr, eilts be, paccamonti dl, burba dj.j am vet med assoc 1998;213:205-206. azoospermia associated with 79,xxy chromosome complement (canine klinefelter's syndrome). nie gj, johnston sd, hayden dw, buoen lc, stephens m. j am vet med assoc 1998;212:1545-1547. confirmation of fetal viability via ultrasonography. estill ct. j am vet med assoc 1998;212:817-818. conservative medical management, using cold-water hydrotherapy. sedrish s, seahorn t. j am vet med assoc 1998;212:197198. rupture of the prepubic tendon with additional tearing of the abdominal tunic. mirza mh, paccamonti d, martin gs, ramirez s, pinto c.j am vet med assoc 1997;211:1237-1238. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 80clinical theriogenology • volume 4 number 1 • march 2012 imperforate hymen. buote pl, ragle ca, memon ma. j am vet med assoc 1997;211:703-704. induction of parturition. hawkins jf, sertich p, dallap bl, pozor ma. j am vet med assoc 1997;211:163-164. endometrial polyp in a beagle. schlafer dh, yeager ae, concannon pw. j am vet med assoc 1997;210:759-761. excessive hemorrhaging from ovarian hematomas on both ovaries. sedrish sa, johnson pj. j am vet med assoc 1997;210:179180. scrotal hematocele. blanchard tl, varner dd, brinsko sp. j am vet med assoc 1996;209:2013-2014. cryptorchidism. burba dj, sedrish sa, paccamonti dl. j am vet med assoc 1996;209:1705-1706. transrectal palpation used to diagnose uterine torsion in a horse. perkins nr, hardy j, frazer gs, threlfall wr. j am vet med assoc 1996;209:1395-1396. unilateral scrotal hydrocele. pinto c, paccamonti d, eilts b, angel kl. j am vet med assoc 1996;209:1073-1074. histologic examination of ovarian tissue to confirm the diagnosis of a granulosa cell tumor. sedrish sa, valdes-vazquez ma, oliver j. j am vet med assoc 1996;209:731-732. vaginal constriction, probably a congenital malformation. archbald lf, wolfsdorf k. j am vet med assoc 1996;208:16511652. uterine rupture. waldow d. j am vet med assoc 1996;208:831-832. placental insufficiency, probably the result of twin fetuses. wolfsdorf k. j am vet med assoc 1996;208:201-202. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201281 clinical theriogenology • volume 4 number 1 • march 2012 clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 82clinical theriogenology • volume 4 number 1 • march 2012 omniblank: dystocia due to a schistosomus reflexus in a miniature dachshund dystocia due to a schistosomus reflexus in a miniature dachshund collen kutzler,a margret root-kustritz,a michelle kutzlerb acollege of veterinary medicine, university of minnesota, st. paul, mn bcollege of agricultural sciences, oregon state university, corvallis, or schistosomus reflexus (sr) is a rare type of fetal monstrosity reported primarily in cattle, with a prevalence ranging from 0.01 1.3% of bovine dystocias.1 it has also been reported in sheep,1 goats,1 horses,2 donkeys,3 rhinoceroses,4 and sea turtles5 but not in dogs. a 10 year old intact miniature dachshund was presented with a dystocia following an unplanned mating to her great grandson. bitch was clinically stable on physical examination. small loops of fetal bowel protruded from the vulva. perineal area was cleaned with an antibacterial soap and a digital examination of vestibule and vagina was performed. digital palpation revealed an abnormal fetus in a ventral transverse presentation. sterile lubricant was instilled into vestibule and fetus was delivered vaginally with traction. gross fetal abnormalities included spinal inversion, exposure of abdominal viscera, lung and diaphragm hypoplasia, and limb ankyloses with positioning of the legs adjacent to the skull. three remaining puppies were born without difficulty or abnormalities. embryological mechanism by which sr develops is unknown, but it is reasonable to consider that the origin of this ventral wall closure defect occurred as early as after gastrulation. a genetic origin for sr was suggested in transforming growth factor (tgf) ß2 and tgf ß3 knockout mice.6 in addition, the localization of transacting sirna generating (tas) genes to the x chromosome suggests that sr may be an x linked disorder.7 despite a potential genetic origin, sr occurs infrequently and sporadically in domestic animals. keywords: canine, congenital, deformity, dog, fetus, midline references 1. laughton kw, fisher kr, halina wg, et al: schistosomus reflexus syndrome: a heritable defect in ruminants. anat histol embryol 2005;34:312-318. 2. irwin mr, pulley lt: schistosomus reflexus in an equine fetus. vet med small anim clin 1975;70:44-45. 3. dubbin es, welker fh, veit hp, et al: dystocia attributable to a fetal monster resembling schistosomus reflexus in a donkey. j am vet med assoc 1990;197:605-607. 4. lankton js, vanderhart dj, terrell sp: schistosomus reflexus-like malformation in a southern white rhinoceros (ceratotherium simum simum). j zoo wildl med 2014;45:708-711. 5. bárcenas-ibarra a, rojas-lleonart i, lozano-guzmán ri, et al: schistosomus reflexus syndrome in olive ridley sea turtles (lepidochelys olivacea). vet pathol 2017;54:171-177. 6. dünker n, krieglstein k: tgfß2 / tgfß3 / double knockout mice display severe midline fusion defects and early embryonic lethality. anat embryol (berl) 2002;206:73-83. 7. parvari r, welstein y, ehrlich s, et al: linkage localization of the thoraco‐abdominal syndrome (tas) gene to xq25–26. am j med genet 1994;49:431-434. 507 clinical theriogenology • volume 11, number 3 • september 2019 508clinical theriogenology • volume 11, number 3 • september 2019 omniblank: 1 contact amelia andersson aia30@msstate.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11097, http://dx.doi.org/10.58292/ct.v17.11097 case report abdominal ectopic pregnancy in a mixed-breed dog amelia andersson,a kasey forton,a tom campbell,b jeremy shapero,b jacob shivley,b brittany baughmana adepartment of pathobiology and population medicine, college of veterinary medicine, mississippi state university, starkville, ms, usa bdepartment of clinical sciences, college of veterinary medicine, mississippi state university, starkville, ms, usa abstract an intact, ~ 4-6 year female, mixed-breed dog was presented to an academic spay-and-neuter clinic for a routine ovariohysterectomy and transitioned to an abdominal exploratory after palpation of 2 intraabdominal, firm masses during surgical preparation. intraoperatively, the masses appeared to arise from the small intestine with intimate association to the surrounding mesentery. due to the level of communication between the masses and the small intestine, euthanasia and postmortem examination were elected. at postmortem evaluation, 3 highly vascularized and encapsulated masses were within the small intestinal mesentery; when excised, each contained a well-developed fetus and 2 were in the early stages of mummification. the largest fetus was free within moderate serosanguinous fluid, and had a thick vascular structure wrapped around the left forelimb. histologically, this structure was composed of 2 vessels, consistent with an umbilical vein and artery. the fetal capsule contained moderate number of multinucleated basophilic cells, suspicious for placental trophoblasts. the uterus and ovaries had no gross lesions. although limited in utility due to the postmortem and ex-vivo nature of image acquisition, computed tomography displayed appropriate late-pregnancy development of fetal skeletons and faint, mineral attenuating structures within the maxillary and mandibular alveoli consistent with developing dentition. some changes consistent with fetal death were noted in the smallest fetal skeleton. the diagnosis was a full-term abdominal ectopic pregnancy. this case report describes a canine abdominal ectopic pregnancy diagnosed during postmortem examination, with additional characterization using advanced imaging and histopathology. keywords: dogs, ectopic pregnancy, histopathology, imaging introduction the subject of reproduction is dynamic and although less is understood about pregnancy abnormalities in companion animals, knowledge from human medicine can be extrapolated and applied to veterinary cases to further our understanding. the normal pregnancy length in dogs is ~ 61 days.1 fertilization of 1 or more ova occurs in the uterine tube and ~ 1 week later, the resultant zygote migrates through the uterine horn, with implantation as a blastocyte occurring ~ 2 weeks after fertilization.1 approximately day 22, the embryo is developed and the fetal phase continues from ~ day 35 to parturition.1 failure at any point in this process can result in a failed pregnancy. one such abnormality, termed ectopic pregnancy, occurs when pregnancy develops in an abnormal location, with the type of ectopic pregnancy based on the location of fetal development. although this phenomenon is recognized in veterinary species, it is better described in human medicine where this pathology is more often diagnosed and studied. in animals and humans, ectopic pregnancies are of 2 types, namely, tubal or abdominal.2-4 the former is described as fertilization and fetal development within the uterine tube and the latter is pregnancy in the abdominal cavity.2 abdominal ectopic pregnancies are further subdivided into a primary form, where the fertilized oocyte adheres to the mesentery or viscera in the abdominal cavity, or a secondary form, when there is rupture somewhere along the reproductive tract, such as the uterine tube or uterus, after fetal implantation, with the fetus within the abdominal cavity.3,4 various modalities exist to help diagnose pregnancy in small animals. diagnostic imaging is particularly useful in estimating fetal development as mineralization of the fetal skeleton is apparent radiographically at or beyond 45 days of pregnancy.5 radiographic alignment of the fetal skeleton can also be helpful in evaluating viability, as axial or appendicular skeletal malalignment or collapse of the fetal skull bones may suggest fetal death.5 additionally, overlap or compression of fetal mailto:aia30@msstate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11097 2 citation: clinical theriogenology 2025, 17, 11097, http://dx.doi.org/10.58292/ct.v17.11097 structures into smaller than normal dimensions is suggestive of mummification.5 in veterinary and human medicine, histopathology of the female reproductive tract and fetal structures in cases of ectopic pregnancies is another diagnostic modality contributing to the working knowledge of abnormalities during pregnancy. albeit better reported in human literature, location of blastocoele-lining cells called trophoblasts is a key component in categorizing the type of ectopic pregnancy, as trophoblastic growth and neovascularization are microscopic components of primary abdominal pregnancies.6 secondary abdominal pregnancies follow rupture of the uterus.2,4 tubal abdominal pregnancies are the most common type of ectopic pregnancy in humans, given the female anatomy;6 however, this category is considered extremely rare, if not impossible, in companion animals, as the only reported cases in veterinary literature are in nonhuman primates.2 we describe an abdominal ectopic pregnancy in a shelter dog with an emphasis on postmortem diagnostics including advanced imaging, histopathology of the female reproductive tract and fetal tissue, and immunohistochemical staining of the fetal tissue. case presentation an intact, ~ 4-6 year female mixed-breed dog was presented to the shelter medicine service for a routine ovariohysterectomy. during 4 months prior to presentation, the dog was cared for by another shelter. dog gave birth to a litter of pups 5 months before first adoption. dog’s reproductive cyclicity or breeding history was not known. as per staff, during the 4 months prior to surgery, the dog was timid but otherwise had a normal mentation, ate and drank normally, and maintained normal bodily functions. on arrival, spay/neuter clinic performed a cursory physical examination. dog had a body condition score of 5/9 and weighed 22 kg. temperature, pulse, and respiratory rates were within normal limits, and auscultation of the heart revealed normal heart sounds, rate, and rhythm. palpation of the abdomen revealed 2, firm, midto cranial-abdominal masses. due to financial limitations of the sheltering organization, preoperative bloodwork and imaging were not performed. the attending clinician, in conjunction with the sheltering organization, decided to proceed with ovariohysterectomy, in addition to an abdominal exploratory, to continue investigation of the abdominal masses. case management after surgical preparations, ~ 15 cm skin incision was made on ventral midline extending 5 cm cranially to the umbilicus. the subcutaneous tissues were bluntly dissected using metzenbaum scissors that facilitated entry into the body wall using a stab incision through linea alba and extension with mayo scissors. palpation of the internal organs revealed the masses to be unassociated with the retroperitoneum and instead were intimately associated with the intestinal tract. intestines were gently moved for better visualization of the masses. in so doing, a third mass was identified and additionally, large portions of the jejunum and ileum were firmly adhered to the 3 masses. based on the extent and severity of the adhesions, and particularly the degree of mesenteric vasculature adhesion, resection and anastomosis would be the most effective means to remove the masses, but at great cost to the length of the small intestinal tract. uterus appeared grossly intact upon cursory evaluation. outcome surgical observations were relayed to the shelter coordinator and euthanasia was elected. dog was euthanized intraoperatively using intravenous euthasol with subsequent closure of the body wall and submitted for postmortem evaluation. postmortem findings the body condition score was 4/9 (with 5/9 being ideal) and there was mild autolysis. a ventral midline incision (appropriately opposed) was noted consistent with the aforementioned abdominal surgery with no gross changes. a scant amount of yellow-tinged, clear mucoid material oozed from the vulva. within the thoracic cavity, the lungs were mottled pink to purple to dark red and were wet and heavy on palpation. on cut surface the lungs oozed a scant to moderate amount of serous fluid. a scant amount of white, stable foam was present at the tracheal bifurcation. within the lumen of the right atrium and ventricle were ~ 10-15 long, slender, white adult nematodes morphologically consistent with dirofilaria immitis. abdominal cavity contained ~ 75-100 ml of serosanguineous fluid. liver was moderately enlarged with rounded margins and had an accentuated reticular pattern in addition to multifocal fluid-filled cysts ranging from 1-4 mm in diameter. stomach contained a scant amount of yellow to white mucoid ingesta and there was a locally extensive area of reddened glandular mucosa. within the mesentery, there were 3 large masses (figure 1). the first, and largest mass (figure 2) was red to brown, round, smoothly marginated, soft on palpation and measured ~ 12 x 10 x 8 cm. the second mass was red to brown, multilobulated, hard and measured ~ 11 x 6 x 5 cm. the third, and smallest mass was grossly similar to the medium-sized mass and measured ~ 10 x 7 x 5 cm. the largest mass had a thick, tan, fibrous capsule containing ~ 40-50 ml serosanguineous fluid and a fully developed, haired fetus with grossly normal external features. the fetus was not adhered to the inner capsular wall but appeared to have an umbilicus with an enlarged and congested umbilical artery and vein tightly wrapping around the left forelimb. unfortunately, due to how friable the cord was, upon fetal manipulation, the umbilicus separated from the capsular wall. the second and third masses contained smaller fetuses intimately adhering to the capsular wall and were shrunken and appeared in the early mummification stages. no fetal capsules had any appreciable gross vasculature connecting the capsule to the mesentery; however, multiple jejunal arches were dilated and congested. on cut surface, the uterine lumen was empty. the uterine mucosa was mildly reddened and on palpation a slight depression between the left uterine horn and the uterine body was appreciable although no gross defects were present. the right uterine horn, ovaries, and uterine tubes were grossly normal. no connection between the reproductive tract and the fetal capsules were identified; remainder necropsy findings were within normal limits. tissue from the uterine body was submitted for bacterial culture. http://dx.doi.org/10.58292/ct.v17.11097 citation: clinical theriogenology 2025, 17, 11097, http://dx.doi.org/10.58292/ct.v17.11097 3 bacteriology histopathology tissues collected during postmortem examination were fixed in 10% neutral buffered formalin for 24 hours, then sectioned and prepared for histologic evaluation following standard procedures utilizing hematoxylin and eosin stain. on histopathologic evaluation of the largest fetus, the capsule was composed of thick, mature dense fibrous connective tissue containing many haphazardly arranged, small-caliber vessels. the capsule was lined by simple squamous epithelial cells containing multifocal loose collagen attachments with figure 1. thoracic and abdominal contents (a), note 3 irregularly shaped multilobulated masses (arrows); gastrointestinal tract (b); and jejunal mesentery containing 3 fetal masses (c). a b c specimen type culture type results uterine stump aerobic mild growth of klebsiella pneumoniae ssp. pneumoniae and streptococcus bovis anaerobic mild growth of clostridium perfringens http://dx.doi.org/10.58292/ct.v17.11097 4 citation: clinical theriogenology 2025, 17, 11097, http://dx.doi.org/10.58292/ct.v17.11097 scattered lymphocytes, plasma cells, and hemosiderophages, and had scattered hemorrhage. the thin membrane covering the fetus was multifocally hypercellular (figure 3a). the luminal surface contained moderate numbers of variably sized, deeply basophilic multinucleated cells occasionally having intracytoplasmic tan material (figure 3b). these multinucleated cells lined the luminal surface of multiple vessels and were suspected to be placental trophoblasts (figure 3c). evaluation of the vascular structure wrapped around the fetal forelimb (figure 2b) was histologically hindered by severe autolysis but appeared to be composed of 2 tortuous luminal structures. one vascular structure had a thick smooth muscle wall and the other was lined by endothelium, and thus these structures were interpreted to be the umbilical artery and vein, respectively. in multiple longitudinal and cross sections of the uterine body, the endometrial interstitium was expanded by moderate fibrous connective tissue and clusters of lymphocytes, plasma cells, and hemosiderophages. endometrial glands contained intraluminal eosinophilic material with cellular debris and chronic hemorrhage. occasional clusters of amorphous basophilic material were also present at the luminal surface. no gross lesions were associated with the depression in the uterine body palpated during postmortem evaluation and there were no appreciable lesions consistent with a site of implantation or placentation. immunohistochemical staining on tissue from the fetal capsule was performed to better characterize the cell origin of the multinucleated cells suspected to be trophoblasts. majority of the multinucleated cells had intracytoplasmic immunoreactivity for histiocytic markers iba-1 and cd18. multinucleated cells were not immunoreactive for cytokeratin, an epithelial cell marker. figure 2. largest and most developed ectopic fetus (a) and is exposed (b); note thick vascular structure (arrow) is tightly wrapped around left forelimb; fetus after opened capsule (c); note fetus is covered by thin, friable, tan membrane (asterisk). a b c http://dx.doi.org/10.58292/ct.v17.11097 citation: clinical theriogenology 2025, 17, 11097, http://dx.doi.org/10.58292/ct.v17.11097 5 figure 3. light microscopic images of largest ectopic fetus stained with hematoxylin and eosin: (a) thick fetal capsule and multifocal hypercellular (caret) luminal surface with moderate numbers of variably shaped multinucleated cell (4 x); (b) multinucleated, deeply basophilic cells (asterisk) lining the luminal surface of the fetal capsule (40 x); (c) cross section of a vessel from the fetal membrane with intraluminal multinucleated cells (10 x); (d) locally extensive region of jejunal serosa (outlined in brackets) with numerous naked hair shafts situated within a collagenous stroma (4 x); and (e) hair shafts surrounded by low numbers of multinucleated cells (arrows [20 x]). ^ ^ ^ ^ ^ ** a b d e c http://dx.doi.org/10.58292/ct.v17.11097 6 citation: clinical theriogenology 2025, 17, 11097, http://dx.doi.org/10.58292/ct.v17.11097 within the serosa of the segment of jejunum intimately associated with the largest fetal capsule, was a small focus containing multiple naked hair shafts (figure 3d) surrounded by multinucleated cells and fibroblasts (figure 3e). lamina propria was expanded by lymphocytes and plasma cells and contained dilated and congested vessels. few crypts were expanded by scant cellular debris. one locally extensive focus contained large, prominent lymphoid follicles expanding and distorting overlying mucosa. other notable findings in other nonreproductive organs included neutrophilic and histiocytic bronchopneumonia with numerous intravascular and intraalveolar microfilaria, in addition to severe pulmonary edema and congestion. pulmonary vessels were severely dilated and 1 focal, severely dilated artery was bisected by smooth muscle forming 2 vessels containing intravascular erythrocytes and degenerate neutrophils (neovascularization). microfilaria were also present in multiple vessels, within the interstitium of the heart, and within multifocal renal glomeruli. the spleen had moderate lymphocytolysis and lymphoid depletion, possibly related to stress and chronic antigenic stimulation associated with heartworm disease. diagnostic imaging computed tomography (ct) was performed on the gastrointestinal tract after it was removed during postmortem evaluation. there were 3 fetal skeletons, each partially encircled by a thick, soft tissue to soft tissue-hyperattenuating capsule were noted amongst the small intestines (figure 4). there was moderate amount of gas within the capsules and surrounding the fetuses (might have been entirely secondary to postmortem change and examination). numerous small intestinal loops were closely associated and border effaced with the capsules, consistent with the adhesions noted during laparotomy. the 2 larger fetal skeletons displayed appropriate osseous development for a pregnancy of 42-45 days and all fetuses contained several, small, smoothly marginated, mineral attenuating structures within the maxillary and mandibular alveoli, consistent with developing dentition (figure 5). the smallest fetal skeleton had mild cranial collapse with a step defect between the developing parietal bones and dorsoventral narrowing of the cranial cavity (figure 6). this fetus also had an abnormal, mixed kyphotic and lordotic conformation of its vertebral column. these changes may suggest earlier fetal death than the other, larger fetuses or secondary to mummification. additional information regarding blood supply to the fetuses and capsules could not be obtained due to the postmortem and ex-vivo acquisition of the images, in addition to the limitations in spatial resolution and contrast resolution in noncontract enhanced ct. figure 4. maximum intensity projection computed tomographic image (bone window) of the gastrointestinal tract and ectopic fetuses. the fetuses are surrounded by soft tissue to soft tissue-hyper attenuating (30-90 hounsfield unit) capsules and gas. multiple loops of small intestine approximate and efface with these capsules. figure 5. maximum intensity projection computed tomographic image (bone window) of the largest fetal skeleton. small, smoothly marginated, mineral attenuating structures are noted within the mandibular and maxillary alveoli (yellow arrows) consistent with developing teeth. the cranial structures and vertebral column (not entirely in plane) are normal. http://dx.doi.org/10.58292/ct.v17.11097 citation: clinical theriogenology 2025, 17, 11097, http://dx.doi.org/10.58292/ct.v17.11097 7 discussion there is limited information in veterinary literature outlining etiology and mechanism for canine ectopic pregnancies, with even fewer details characterizing prevalence, histopathology, and advanced imaging. there are no official criteria to classify ectopic pregnancies in animals; however, basic categories based on location of fetal implantation and mechanism of extrusion from the uterus have been developed for research and educational purposes. despite differences in female reproductive anatomy between humans and domestic animals, the literature from both fields of medicine subcategorize ectopic pregnancies into 4 categories: cervical, ovarian, tubal, and abdominal or peritoneal,3,7 with the former 2 predominantly reported in human literature. in humans and veterinary species, tubal ectopic pregnancy refers to pregnancies in which the zygote adheres more cranially to the uterine body, usually in the uterine tube, thus hindering it from descending into the uterine body or implanting in the uterine horns, respectively.3,4,7 abdominal ectopic pregnancies are significantly more uncommon in humans compared to domestic species and occur when the zygote implants outside of the reproductive tract, most commonly as a result of internal trauma, such as a uterine rupture, resulting in pregnancy occurring within the peritoneal cavity.7 depending on the mechanism and location of implantation, abdominal pregnancies are further divided into primary and secondary abdominal ectopic pregnancies. when implantation occurs within the mesentery or abdominal viscera by either a fertilized or unfertilized ovum (later fertilizes in the abdominal cavity), the pregnancy is categorized as a primary abdominal ectopic pregnancy. a true primary abdominal ectopic pregnancy is further characterized with histopathology by trophoblasts and neovascularization between the fetal capsule and supporting tissue structures.6 secondary abdominal ectopic pregnancies occur after implantation when there is rupture of either the uterine tube or uterine body and expulsion of the fetus within the mesentery of peritoneal viscera of the abdomen.3 various outcomes may occur depending on whether the fetal placenta is retained or detached, or if the conceptus is able to maintain a viable blood supply from the tissue in which it is implanted. failure to maintain a viable vascular network for blood flow and fetal nutrient supply can result in mummification or fetal death. studies on ectopic pregnancies in companion animals are limited, likely due to underdiagnosis. in many cases, especially in cases of adoptions, companion animals are obtained already spayed or if intact, have limited to no history regarding the animal’s reproductive history. this is especially important as a history of having had previous litters or a history of trauma increases the likelihood and suspicion for ectopic pregnancy. in the present case, prior to adoption, the dog was on the street with no history other than the information of having 1 previous litter. due to this we speculate this case as a secondary abdominal ectopic pregnancy; however, there was no gross or histological evidence of a uterine horn or uterine body rupture. histopathology of the uterus had signs of chronicity, albeit minimal, based on lymphoplasmacytic inflammation, few hemosiderophages, and fibrosis throughout the endometrial interstitium. although there was no evidence of rupture or trauma within the reproductive tract, previous trauma cannot be ruled out as the regions of fibrosis may indicate a site of past repair. additionally, no infectious agents or neoplastic features were present histologically. bacterial culture of the uterine stump was performed out of caution; however, given the degree of autolysis and low growth of mixed gram positive and negative bacteria, these findings were considered insignificant. a limitation is the largest, most well-developed fetus was the only fetal specimen examined histologically and neither the uterine tube nor the ovaries were evaluated histologically. to authors’ knowledge there are limited current literature reports detailing histopathology of ectopic pregnancies in dogs. dogs have a zonary and endotheliochorial type placentation and the normal histologic layers consist of the placental labyrinth, the junctional zone, and the glandular zone.8 the junctional zone is where trophoblasts reside and, early in pregnancy, they invade the maternal endothelium and endometrial glands.8 in human medicine, a histopathologic diagnosis of tubal ectopic pregnancy had villi, trophoblasts and embryos were within fallopian tubes.3 abdominal ectopic figure 6. transverse computed tomographic image (bone window) of the skull of the smallest fetus (left) and sagittal maximum intensity projection of the same fetus’s vertebral column (right). there is a small step defect between the developing parietal bones (yellow arrow) and dorso-ventral collapse of the cranium (yellow bracket). there is moderate lordosis of the lumbar spine and kyphosis of the thoracic spine (blue arrows). these changes coupled with its smaller size may indicate earlier demise of this fetus. http://dx.doi.org/10.58292/ct.v17.11097 8 citation: clinical theriogenology 2025, 17, 11097, http://dx.doi.org/10.58292/ct.v17.11097 pregnancies are difficult to diagnose as there has to be placentation and/or a mummified fetus with a ruptured uterine tube or uterus.2 this is especially difficult given the rapidly regenerative nature of the myometrium and the reason why rupture sites are not usually identifiable.2 in this case, villi were not appreciable; however, within the fetal membrane there were multifocal clusters of large, multinucleated, deeply basophilic cells.9 differentials for multinucleated cells within fetal tissue included trophoblasts, with syncytiotrophoblasts favored over cytotrophoblasts given the large number of nuclei present within individual cells, versus a multinucleated histiocytic cell. cytotrophoblasts are mononucleated giant cells whereas syncytiotrophoblasts are multinucleated giant cells; they serve as a connection between dam and fetus, based on their location within or around maternal vessels.8,10 immunohistochemical (ihc) staining of the multinucleated cells in this case was largely immunoreactive for iba-1 and cd18 and negative for cytokeratin. the ihc results identified the multinucleated cells to be of histiocytic rather than epithelial cell (i.e. trophoblast) origin, as initially suspected. however, the timeframe and degree of fetal development fits more closely with the cells being syncytiotrophoblasts. other immunohistochemistry stains (positive identifiers of placental trophoblasts), cd31, ck7, ck8/18, and ckae1/ae3 were not performed, thus these cells being true placental trophoblasts cannot be ruled out.10,11 multinucleated histiocytes likely represent an inflammatory response to free fluid, keratin, or squamous epithelium, as similar histologic features were described in a case study involving a pregnant woman who underwent multiple intrauterine procedures during her third trimester, resulting in leakage of amniotic fluid.11 this is less likely in the current case since the degree of inflammation in the fetal tissue was minimal and there was no evidence of fluid leakage. the histiocytic infiltration in combination with the hair shaft granuloma was most likely a response to irritation from fetal hair, keratin, or amniotic fluid, thus, multinucleated cells can be speculated to be a secondary reaction to migration of fetal hair from the ectopic fetus.11 the former was more likely in this case as multifocal cells predominantly contained intracytoplasmic tan material, likely consistent with amniotic fluid, although this pigment can also be a feature of syncytiotrophoblasts.11 no apparent phagocytosis of keratin or squamous epithelial cells was appreciable in this case. trophoblasts may have an indirect role in vascular remodeling in the dam, as a study in mice reported evidence of vascular remodeling leading to spiral artery dilation.12 although the source did not indicate remodeling as a direct result of trophoblast cell invasion into the uterus, the authors did not rule out an indirect association between trophoblasts and changes in the spiral arteries.12 this emphasizes the need to understand the role of vascular remodeling in association with the viability of small animal ectopic pregnancies, and the role of trophoblasts in the growth and development of an ectopic fetus. this dog had vascular remodeling of the pulmonary artery secondary to chronic heartworm disease but there was no evidence connecting these lesions to the ectopic pregnancy. the normal ct features of canine fetuses are poorly described in literature. this is likely because ct incurs a higher dose of ionizing radiation than radiography and is generally contraindicated in pregnant dogs without cause to justify the risk to the developing fetuses.5 in pregnant human patients, ct is generally avoided when other modalities can be used to make  a diagnosis and is considered inappropriate in the diagnosis of ectopic pregnancy.6 however, the dose of ionizing radiation for ct studies of the thorax and head are considered low risk to the developing fetus and guidelines for cross sectional imaging of pregnant people have been developed.2 additionally, ct of human fetuses is used for diagnosis of certain conditions and has been documented to improve outcomes when indicated.1 cross-sectional imaging in ectopic pregnancies have been described in humans; however, there is little published information on advanced imaging in canine ectopic pregnancy.6 a single case report of a presumed secondary abdominal ectopic pregnancy in a dog diagnosed via ct has been published.6 in this report, a heterogeneous rounded mass containing discontinuous, mineral attenuating structures and a discernable vertebral column was noted within the abdomen of an intact female maltese dog. the mass was devoid of any connection or origin of any of the abdominal organs and was associated with the omentum and mesentery when surgically excised. due to the more organized vertebral column, an abdominal ectopic pregnancy was diagnosed.6 in the current case, the ectopic fetuses were also closely associated with the mesentery, yet were far more organized and developed with respect to their skeletal structures. additionally, only 2 had gross signs of mummification, with the largest being free within the capsule and surrounded by fluid. small, developing teeth were noted on ct. when fetal teeth are observed radiographically in the pregnant dog, parturition can be expected within 8 days.5 however, with ct having superior contrast resolution and eliminating superimposition of structures compared to radiography, it is difficult to extrapolate the significance of observable, developing teeth; it may support the diagnosis of a full-term ectopic pregnancy in the current case. additionally, a history of recent pregnancy with or without known trauma during pregnancy, may heighten the index of suspicion for ectopic pregnancy, especially when the mating history is unknown.4 in many human cases, patients diagnosed with abdominal ectopic pregnancies have a good prognosis as long as the mass  or masses do not cause secondary complications such as torsion, altered peristalsis, or inflammation secondary to necrosis from fetal infection or fetal vascular occlusion.3 complications are often accompanied by clinical signs such as  anorexia, abdominal distention, diarrhea, or vomiting, although clinical presentations can vary depending on where the zygote implants and how much viscera is secondarily affected. as a result, surgical removal of the ectopic fetus(es) is considered the treatment of choice. in this case, euthanasia was elected based on financial limitation and the extensive need for resection and anastomosis (might have resulted in prolonged time under anesthesia and postsurgical complications). coinfection with dirofilaria immitis in this dog might also have contributed to the decision to euthanize as higher worm burdens are costand time-intensive to treat and often associated with an overall compromised health status. although clinical signs associated with heartworm infection were not reported in this dog, the number of microfilaria present histologically in the lung, heart, and kidneys would likely have resulted in further anesthetic and lifelong complications, such as cardiopulmonary or vascular compromise, if left untreated. http://dx.doi.org/10.58292/ct.v17.11097 citation: clinical theriogenology 2025, 17, 11097, http://dx.doi.org/10.58292/ct.v17.11097 9 this case report characterized diagnostic findings associated with an ectopic pregnancy in a dog through the examination of advanced imaging, postmortem and histopathological evaluation with the goal to further understand this phenomenon and better develop management techniques when diagnosed. taking into account the history and signalment, an abdominal ectopic pregnancy is a valid, albeit rare, differential diagnosis to consider in any intact female dog presenting with an abdominal mass. learning points • histologic criteria for primary abdominal ectopic pregnancies include trophoblasts and neovascularization  • secondary abdominal ectopic pregnancies follow injury or trauma to the reproductive tract • diagnostic imaging is an incredibly useful modality to diagnose ectopic pregnancies • ectopic pregnancy is a valid differential for a veterinary patient presenting with abdominal masses and an unknown reproductive history acknowledgement authors thank the radiology, necropsy, histology, and microbiology laboratory technicians and support staff at mississippi state university college of veterinary medicine for their contributions. conflict of interest authors have no conflict of interest to declare. references 1. pretzer sd: canine embryonic and fetal development: a review. theriogenology 2008;70:300-303. doi: 10.1016/j.theriogenology. 2008.04.029 2. corpa, jm: ectopic pregnancy in animals and humans. reproduction 2006;131:631-640. doi: 10.1530/rep.1.00606 3. hayashi t, sano k, konishi i: histopathological findings of ectopic pregnancy in contraceptive-wearing woman. j clin med res 2023;15:384-389. doi: 10.14740/jocmr4924 4. hughes k: abdominal ectopic pregnancy and impaired postnatal mammary gland development, consistent with physiologic agalactia, in a wild european rabbit, oryctolagus cuniculus. front vet sci 2019;6:e254. doi: 10.3389/fvets.2019.00254  5. kinns j, nelson n: uterus. in: thrall de: editor. textbook of veterinary diagnostic radiology. 7th edition, st. louis, mo; elsevier: 2018. p. 880-881.  6. myung hw, lee aj, kim jy, et al: secondary abdominal pregnancy with foetal mummification diagnosed using computed tomography in a dog: a case report. vet med 2016;61:51-55. doi: 10.17221/ 8682-vetmed  7. kopelman tr, bogert jn, walters jw, et al: computed tomographic imaging interpretation improves fetal outcomes after maternal trauma. j trauma acute care surg 2016;81:1131-1135. doi: 10.1097/ta.0000000000001210 8. sarli g, castagnetti c, bianco c, et al: canine placenta histological findings and microvascular density: the histological basis of a negative neonatal outcome? animals 2021;11:e1418. doi: 10.3390/ani11051418 9. adamson sl, lu y, whiteley kj, et al: interactions between trophoblast cells and the maternal and fetal circulation in the mouse placenta. dev biol 2002;250:358-373. doi: 10.1006/dbio.2002. 0773 10. mainenti m, wettere aj: trophoblast emboli in the lung of a snowshoe hare (lepus americanus). vet pathol 2022;59:353-357. doi: 10.1177/03009858211071011  11. sheridan t, askin fb, ji h: multinucleated foreign body giant cells in placental membrane. pediatr dev pathol 2005;8:493-496. doi: 10.1007/s10024-005-0027-6 12. chen cm, fergus cv, kaimal a, et al: guidelines for computed tomography and magnetic resonance imaging use during pregnancy and lactation. am j obstet gynecol 2008;112:333-340. doi: 10.1097/aog.0b013e318180a505  http://dx.doi.org/10.58292/ct.v17.11097 https://doi.org/10.1016/j.theriogenology.2008.04.029 https://doi.org/10.1016/j.theriogenology.2008.04.029 https://doi.org/10.1530/rep.1.00606 https://doi.org/10.14740/jocmr4924 https://doi.org/10.3389/fvets.2019.00254  https://doi.org/10.17221/8682-vetmed  https://doi.org/10.17221/8682-vetmed  https://doi.org/10.1097/ta.0000000000001210 https://doi.org/10.3390/ani11051418 https://doi.org/10.1006/dbio.2002.0773 https://doi.org/10.1006/dbio.2002.0773 https://doi.org/10.1177/03009858211071011  https://doi.org/10.1007/s10024-005-0027-6 https://doi.org/10.1097/aog.0b013e318180a505  2014: histopathologic changes in testes three days after administration of zinc gluconate neutralized with arginine as an intratesticular injection for contraception histopathologic changes in testes three days after administration of zinc gluconate neutralized with arginine as an intratesticular injection for contraception margaret v. root kustritz, jennifer brazzell, jonna swanson, graham brayshaw college of veterinary medicine, university of minnesota, st. paul, mn abstract five prepuberal mixed breed dogs were treated with zinc gluconate neutralized with arginine as an intratesticular injection, which is approved by the food and drug administration as a non-surgical sterilant for puppies aged 3-10 months with testicular width between 10 and 27 mm. the product was administered following manufacturer’s recommendations. three days later, all dogs underwent surgical castration and the testes, epididymes, and a portion of the spermatic cord submitted for histopathologic evaluation. both testes from all five dogs showed variable degrees of liquefactive and coagulative necrosis with focal acute neutrophilic orchitis, hemorrhage, and edema. percentage of the testis showing degenerative and necrotic changes averaged approximately 45.5% and ranged from 10-90% among the ten testes examined. unaffected seminiferous tubules were lined by sertoli cells and spermatogonia with rare elongated spermatids; this was compatible with the prepuberal life stage of the animals. neutrophilic and lymphohistiocytic epididymitis with edema and vasculitis was present in six of the ten samples. funiculitis (inflammation of the spermatic cord) was present in nine of the ten samples. this is the first report of testicular and epididymal histologic change within days of administration of the product and the only report documenting changes in the vas deferens at any point after administration of the product. key words: castration, sterilization, non-surgical, zinc arginine, intratesticular introduction chemical castration may be preferred to surgical castration for logistical and societal reasons. compounds used are easy to administer. it has the potential to be a less expensive option that may be suitable for large scale castration operations, especially in countries with feral dog populations that are difficult to control because of lack of availability of veterinarians and cultural or social impediments to culling for population control. reported compounds have a high margin of safety. finally, minimal observation of dogs is required after treatment.1-5 in dogs, reported chemical castration techniques include injection of agents into the vas deferens or epididymis, where formation of fibrous occlusions precludes ejaculation of fluid from the testes and epididymes, and injection of agents into the testicular parenchyma, which may be associated with direct toxic effects on germ cells, inflammation and necrosis of testicular tissue, and eventual testicular atrophy or fibrosis. products reported for injection in the vas deferens or epididymis in the dog include formaldehyde, formalin, chlorhexidine gluconate, chlorhexidine diacetate, methylcyanoacrylate, silver nitrate, ethanol, and potassium permagnate.6-10 products reported for intratesticular injection include zinc gluconate neutralized with arginine and with or without dmso or proprietary carriers, calcium chloride, glycerol, freund’s complete adjuvant, bacillus calmette guerin (bcg), and dexamethasone.1,11-18 the only product that has been approved by the food and drug administration for use in the united states is 0.2 m zinc gluconate neutralized with l-arginine to a ph of 7.0, first approved under the brand name neutersol® and more recently, as zeuterin®. zinc arginine effects contraception directly by inhibiting replication of germ cells and disrupting the sertoli cell barrier and indirectly by inducing local immune and inflammatory responses and by affecting testosterone synthesis and metabolization.1,19-21 presently the product is approved for dogs aged 3-10 months, with testicular width between 10 and 27 mm.22 the product should not be used if the dog is does not have two descended testes, if the testes or epididymes are malformed or diseased, or if there is pre-existing scrotal irritation or dermatitis. manufacturer recommendations for administration are the following. withhold food for 12 hours prior to treatment to decrease risk of vomiting. active dogs should be sedated to ensure they do not move during administration of the product. although administration of the product will cause mild testicular distension, sedation is not required to control pain; the only afferent nerves associated with pain sensation 473 clinical theriogenology • volume 6, number 4 • december 2014 are located in the testicular capsule, not the testicular parenchyma.23,24 place the dog in dorsal recumbency. do not shave or clip the scrotum and clean it with a non-irritating disinfectant. measure each testis separately using the calipers supplied by the manufacturer. use three needles and two syringes for each dog. one needle is inserted into the product container and is left in place to permit drawing the product into the syringes. once the correct volume is drawn up, a 28 gauge 1/2 -5/8” needle is attached to the syringe for administration of the product into the testis. the testis is held securely but without squeezing it. palpate the head of the epididymis and insert the needle next to it, in the dorsal cranial portion of the testis. insert the needle and administer the product slowly. if any resistance is felt, withdraw the needle and do not repeat the injection. the product should flow freely into the testis. once administration is complete, hold the testis and syringe with compressed plunger in place for one minute, and then release everything at once, withdrawing the needle. repeat with the other testis. care must be taken not to introduce the compound into the scrotal skin or scrotal sac. after treatment, the dog must not be permitted to bite or lick at the scrotum and should not be housed on hard or wet surfaces. following injection, mild testicular swelling beginning 24 hours and peaking at about 48 hours after treatment is reported but with minimal pain response or change in behavior exhibited by treated dogs.25 reported decrease in testicular size is variable long-term. rare negative side-effects reported include vomiting, biting or licking at the scrotum, scrotal ulceration or draining tracts, and very rarely, scrotal perforation.1,25,26 efficacy in one study was reported to be 98.7% (221/224 treated dogs) with 76.3% of those dogs having no seminal fluid collected at attempts six months after treatment (aspermia) and 22.3% having no spermatozoa in the ejaculate six months aftertreatment.25 overall, complete azoospermia was reported by three to six months after-treatment.25,27 the manufacturer recommends that treated dogs not be permitted near bitches in estrus for at least 60 days after injection.22 change in serum testosterone is variable but generally is described as being decreased by about 50%; significance of this decline in regards to development of testosterone-dependent disease and exhibition of testosterone-dependent behavior is unknown.25,27 this is a report documenting acute histopathologic changes in the testes, epididymes, and spermatic cords after administration of zinc arginine as a contraceptive agent administered by intratesticular injection in dogs. materials and methods five prepuberal dogs, aged 3-5 months and of mixed breeds, with weights ranging from 7.3 to 19.0 kg, that had been relinquished to a humane organization were treated with zeuterin® as a training exercise for veterinarians and veterinary students to permit them to use this drug for population control. the intent was always that these dogs would undergo surgical castration at the humane society shortly after zeuterin® treatment, as chemical castration is an uncommon procedure in this region and it was perceived that dogs with visible testes were less likely to be adopted despite having been treated with a chemical castration agent. all of the dogs were normal on physical examination and had been treated with dewormer and vaccinated. all were sedated with dexmedetomidine (500 mcg/m2 im) and continuously monitored by a veterinary student while under sedation. the intratesticular injection of zeuterin® was performed according to manufacturer’s directions as described previously. sedation was reversed with atipamezole (equal volume as for dexmeditomidine im) and dogs were treated with carprofen (4.4 mg/kg sq) and once verified to be alert, were returned to the humane society. three days after treatment with zeuterin®, all dogs underwent surgical castration by an experienced veterinarian at that facility. after induction of anesthesia and primary surgical preparation of the pre-scrotal area, a mixture of lidocaine (2%, 0.5 ml/10 kg body weight) and bupivacaine (0.5%, 2.0 ml/10 kg body weight; volume split into two equal aliquots) was injected into the testes. a 22 gauge needle was inserted into the caudal pole of each testis and directed toward the spermatic cord and the fluid infused. testes were surgically removed within five minutes of injection. 474clinical theriogenology • volume 6, number 4 • december 2014 testicles were submitted for histopathologic evaluation. all testes, epididymes, and spermatic cords were evaluated by one pathologist. percentages of degenerative and necrotic change were estimated based on the two-dimensional histology image. results both testes from all five dogs showed variable degrees of liquefactive and coagulative necrosis with focal acute neutrophilic orchitis, hemorrhage, and edema (figures 1-4). percentage of the testis showing degenerative and necrotic changes averaged approximately 45.5% and ranged from 10-90% among the ten testes examined. unaffected seminiferous tubules were lined by sertoli cells and spermatogonia with rare elongated spermatids; this was compatible with the prepuberal life stage of the animals.28 neutrophilic and lymphohistiocytic epididymitis with edema and vasculitis was present in six of the ten samples. funiculitis (inflammation of the spermatic cord) was present in nine of the ten samples. funiculitis was regionally most extensive in the portion of the spermatic cord directly abutting associated tissues and was therefore most likely an extension of inflammation in the associated testis and epididymis. inflammation in the spermatic cord varied between neutrophilic and lymphohistiocytic with varying degrees of necrosis, hemorrhage, and edema. severity was commensurate with that seen in the associated epididymis and testis. percentage of the cord involved could not be estimated as amount of spermatic cord submitted varied among dogs. discussion the dogs in this study had been relinquished to a humane organization and had no permanent owner at the time of castration so a decision was made to perform surgical castration as that was perceived to be associated with greater likelihood of those dogs being readily adopted. all five dogs were adopted from the humane organization within three days of surgery; four of the five were adopted within one day of surgery. chemical castration was demonstrated to be well accepted by owners in two studies, both of which were completed in the galapagos islands. in the first, owners were permitted to choose if they wanted the dogs chemically castrated when pets were microchipped. twenty-three participants were questioned three to seven days after treatment and the majority considered the method to be good or excellent.29 in the second, pet owners were permitted to choose between surgical and chemical castration of their animals. one hundred three of 161 dogs (64%) underwent chemical castration and the technique was reported to be well accepted by the community.4 dogs in this study were treated with an injectable anti-inflammatory drug at the time of treatment, at the recommendation of the manufacturer and the veterinarians leading the training. this therapy is believed to minimize the dog biting and licking at the scrotum after therapy and potentially causing scrotal injury. while it may be perceived that this would preclude desired inflammatory change within the testis, this has not been demonstrated to be true. in one study comparing groups of dogs treated with zinc gluconate by intratesticular injection either with or without simultaneous administration of an antiinflammatory drug, treatment was equally effective at inducing azoospermia in the two groups.19 histologic changes within the testis reported here are appropriate for short-term exposure to the compound. all testes were injected with lidocaine and bupivicaine immediately prior to castration but there was insufficient time for changes specific to exposure to these drugs to be evident in testicular tissue. histology of testes after administration of zinc gluconate with l-arginine previously has been reported only after prolonged exposure of the testicular tissue to the compound. at 60-75 days after treatment, coagulation necrosis, fibrosis, and atrophy of seminiferous tubules was reported.3 at five months, reported changes included decreased number of germinal cells, lack of elongated spermatids in the seminiferous tubules, and seminiferous tubules lined only with vacuolated sertoli cells, and degenerative changes in the testicular parenchyma.30 at one year after treatment, there was increase in connective tissue especially around the rete testis, and decreased diameter of the rete testis.27 by two years after treatment, there was severe atrophy of the testis with decreased thickness of the basement 475 clinical theriogenology • volume 6, number 4 • december 2014 membrane, decreased height of the germinal epithelium, and decreased diameter of the seminiferous tubules.25 histology of the epididymis after intratesticular injection with zinc gluconate with l-arginine is more rarely reported and histology of the vas deferens after this treatment has not been reported. in this study, acute inflammation of the epididymis and spermatic cord was observed. other studies report lack of spermatozoa in the coils of the epididymis, decreased height of columnar epithelium, and increase in fibrous tissue one year after treatment and severe atrophy of the epididymis two years after treatment.25,27 in a survey of veterinarians in nigeria, 67.4% of those surveyed expressed an interest in alternatives to surgical castration but only 9.7% had attempted any alternative.31 the authors are unaware of a similar survey of veterinarians in the united states. publications documenting research in nonsurgical contraceptives have increased in number six-fold since the 1980s.32 it is hoped that information such as that presented here will help veterinarians better understand the use of these products. references 1. alliance for contraception in cats and dogs: contraception and fertility control in dogs and cats. ebook. available at www.acc-d.org/accd%20symposia. pp 50-55. accessed 06-16-14. 2. soerensen b, silva pjs, christhavo fg: [advantages of chemical castration compared with the traditional method of surgical castration]. univ marilia 2007;1:15-20. 3. tepsumethanon v, wilde h, hemachudha t: intratesticular injection of a balanced zinc solution for permanent sterilization of dogs. j med assoc thai 2005;88:686-689. 4. levy jk, crawford pc, appel ld, et al: comparison of intratesticular injection of zinc gluconate versus surgical castration to sterilize male dogs. am j vet res 2008;69:140-143. 5. oliveira ecs, moura mrp, de sa mjc, et al: permanent contraception of dogs induced with intratesticular injection of a zinc gluconate-based solution. theriogenology 2012;77:1056-1063. 6. pineda mh, reimers tj, faulkner lc, et al: azoospermia in dogs induced by injection of sclerosing agents into the caudae of the epididymides. am j vet res 1977;38:831-838. 7. perez meg, ramirez rmp, lacroix ce, et al: [sterilization in the dog by injection of methylcyanoacrylate in the tail of the epididymis]. vet mex 1994;25:261-265. 8. barnett bd: chemical vasectomy of domestic dogs in the galapagos islands. theriogenology 1985;23:499-509. 9. dixit vp, lohiya nk, aryam m, et al: the effects of chemical occlusion of vas deferens on the testicular function of dog: a preliminary study. acta eur fertil 1975;6:348-353. 10. pineda mh, hepler di: chemical vasectomy in dogs. long-term study. theriogenology 1981;16:1-11. 11. soto frm, viana wg, mucciolo gcb, et al: evaluation of efficacy and safety of zinc gluconate associated with dimethyl sulfoxide for sexually mature canine males chemical neutering. reprod domest anim 2009;44:927-931. 12. jana k, samanta pk: sterilization of male stray dogs with a single intratesticular injection of calcium chloride: a dosedependent study. contraception 2007;75:390-400. 13. alliance for contraception in cats and dogs: acc&d statement and recommendations regarding calcium chloride injection for chemical castration. available at www.acc-d.org/accd%20docs/accd-recommcalcchlor2.pdf. accessed 06-17-14. 14. immergart hm, threlfall wr: evaluation of intratesticular injection of glycerol for non-surgical sterilization of dogs. am j vet res 2000;61:544-549. 15. dixit vp: chemical sterilization of male dogs (canis familiaris) after single intratesticular administration of methalibure (ici-33828), dexamethasone, metoprion (su-4885, ciba), niradazole (33644-ba, ciba), alphachlorohydrin (u-5897) and danazol. indian j exp biol 1979;17:937-940. 16. talwar gp, naz rk: immunological control of male fertility. arch androl 1981;7:177-185. 17. naz rk, talwar gp: immunological sterilization of male dogs by bcg. intl j androl 1981;4:111-128. 18. talwar gp, naz rk, das c, et al: a practicable immunological approach to block spermatogenesis without loss of androgens. proc natl acad sci usa 1979;76:5882-5885. 19. oliveira ecs, muller pm, silva flm, et al: oral administration of an anti-inflammatory drug does not compromise the efficacy of intratesticular injection of zinc gluconate as a contraceptive for dogs. anim reprod sci 2012;132:207-212. 20. fahim ms, wang m, sutcu mf, et al: zinc arginine, a 5-alpha-reductase inhibitor, reduces rat ventral prostate weight and dna without affecting testicular functions. andrologia 1993;25:369-375. 21. kellokumpu s, rajaniemi h: effect of zinc on the uptake of human chorionic gonadotropin (hcg) in rat testis and testosterone response in vivo. biol reprod 1981;24:298-305. 22. zeuterin® package insert. available at www.zeuterin.com/images/pdf/zeuterincmcpackageinsertfinal.pdf. accessed 06-16-14. 23. christensen gc: the urogenital apparatus. in: evans he, christensen gc, editors. miller’s anatomy of the dog. philadelphia: wb saunders; 1979. p. 544-601. 476clinical theriogenology • volume 6, number 4 • december 2014 24. davis jr, langford ga, kirby pj: the testicular capsule. in: johnson wr, gomes wr, editors. the testis. new york: academic press inc, 1970. p. 285-293. 25. technology transfer inc. freedom of information summary: neutersol® injectable solution for dogs. available at www.fda.gov/downloads/animalveterinary/products/approvedanimaldrugproducts/foiadrugsummaries/ucm11802 4.pdf. accessed 06-06-14. 26. forzan mj, garde e, perez ge, et al: necrosuppurative orchitis and scrotal necrotizing dermatitis following intratesticular administration of zinc gluconate neutralized with arginine (esterilsol) in 2 mixed-breed dogs. vet pathol 2014;51:820-823. 27. fahim ms, wang m, sutcu mf, et al: sterilization of dogs with intraepididymal injection of zinc arginine. contraception 1993;47:107-122. 28. frame sr, hurtt me, green jw: testicular maturation in prepubertal new zealand white rabbits. vet pathol 1994;31:541-545. 29. soto frm, bittencourt do, neves am: [experiment with using chemical sterilant with microchips for male dogs in the city of redencao da serra-state of sao paulo]. rev cienc vet 2011;7:16-23. 30. oliveira ecs, moura mr, silva va, et al: intratesticular injection of a zinc-based solution as a conceptive for dogs. theriogenology 2007;68:137-145. 31. ayisat at: approaches to canine castration in nigeria [abstract]. clin therio 2013;5:388. 32. massei g, miller la: nonsurgical fertility control for managing free-roaming dog populations: a review of products and criteria for field applications. theriogenology 2013; 80:829-838. figure 1: cross section of a zeuterin™ injected testis with a portion of the epididymis (e). approximately 40% of the seminiferous tubules have undergone coagulative necrosis (*); 0.4x magnification, hematoxylin and eosin. 477 clinical theriogenology • volume 6, number 4 • december 2014 figure 2: cross section of a zeuterin™ injected testis with a portion of the epididymis (e). there is a focus of liquefactive necrosis and hemorrhage occupying approximately 60% of the testicular parenchyma. 0.4x magnification, hematoxylin and eosin. figure 3: longitudinal section of a zeuterin™ injected testis including the epididymis (e) and spermatic cord (s). a linear area of liquefactive necrosis and hemorrhage extends from the testis through the tunica albuginea into the epididymis and spermatic cord. 0.4x magnification, hematoxylin and eosin. 478clinical theriogenology • volume 6, number 4 • december 2014 figure 4: higher magnification of figure 3 showing the area of liquefactive necrosis and hemorrhage extending from the testis between an area of coagulative necrosis (*) and normal seminiferous tubules (t) outward into the interstitium of the spermatic cord and epididymis. normal epididymis is visible on the left. 2x magnification, hematoxylin and eosin. (editor’s note: photographs in this manuscript are available in color in the online edition of clinical theriogenology.) 479 clinical theriogenology • volume 6, number 4 • december 2014 480clinical theriogenology • volume 6, number 4 • december 2014 omniblank: 1© 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11643, http://dx.doi.org/10.58292/ct.v17.11643 foreword it is our pleasure to bring you another special edition of clinical theriogenology. this edition focuses on therapeutics in theriogenology including estrus manipulation and diagnosis and treatment of selective ovarian diseases across species. this issue includes eight review articles contributed by several authors. we thank our colleagues for contributing to this special issue and hope you enjoy this collection of manuscripts. aime johnson, kristina lu, erin runcan guest editors http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11643 2009: applying ultrasound to the individual dairy cow and herd level reproductive management applying ultrasound to the individual dairy cow and herd level reproductive management 1 2 k. mcsweeny 3 2456 courtney dr., loveland, co, usa 4 5 6 modern dairy cattle have been described as subfertile.1 implementing timed breeding 7 synchronization programs (tai) can improve heat detection efficiency, but conception rates can 8 remain low. the majority of synchronization programs are initiated regardless of where the cow 9 is within her estrous cycle, and it has been shown that conception rates vary greatly depending 10 upon when these programs are started in relation to the cow’s estrous cycle.2 most important in 11 predicting the stage of a cow’s estrous cycle is identifying the presence of an active corpus 12 luteum (cl). ultrasound has been found to be far superior to rectal palpation in predicting an 13 active cl and high progesterone levels,3 and thus better predicting when cows fall into the 14 optimum period to initiate or continue synchronization programs. by using ultrasound, cows can 15 be assessed and synchronization programs can be modified when cows fail to respond to the first 16 gnrh injection. 17 ultrasound can also be used as a heat detection aid, especially for dairies that utilize tail 18 chalk. accuracy of heat detection when using tail chalk can vary, and often cows in diestrus or 19 even pregnant are bred. using ultrasound, cows with an active cl can be avoided, and only cows 20 appearing in estrus will be bred. 21 incorporating ultrasound intensively into tai and estrus detection programs can improve 22 pregnancy rates dramatically. individual cow management along with techniques and strategies 23 to apply these protocols on any size dairy will be discussed. 24 25 275 keywords: ultrasound, synchronization, timed ai 26 27 introduction 28 modern dairy cattle have been described as subfertile1 and the expanding dairies of the 29 western u.s. seem to be experiencing some of the greatest reproductive inefficiency. according 30 to dhi-provo data, since 1990, days open have increased by more than 30 days for dairies in the 31 western united states, with conception rates declining almost 25% during that same period. heat 32 detection rates followed the same downturn during the 90’s, but with greater implementation of 33 tai, conception rates have since risen to values approaching 45 to 50%. while the greater use of 34 synchronization programs has improved heat detection efficiency, conception rates have still 35 continued to decline. it is not uncommon to find herds in the western united states with 36 conception rates to timed breeding programs mired in the 20% range. there are multiple 37 contributing factors to this problem, such as those related to abnormal reproductive physiology, 38 poor timing of initiation, and non-compliance to synchronization protocols. 39 materials and methods 40 in order to achieve acceptable reproductive performance additional management tools need 41 to be implemented to overcome this subfertility. timed breeding synchronization programs are 42 one such tool but conception rates with these programs, especially in expanding western dairies 43 under current management conditions, are far from desirable. reproductive ultrasound is another 44 tool available to dairy practitioners to improve reproductive performance. due to practical 45 limitations and the cost of ultrasound equipment, dairy practitioners have been slow to adopt this 46 technology. recent significant improvements and less expensive portable ultrasound units make 47 practicality and cost less of a concern. 48 276 reproductive ultrasound in the dairy industry is most often thought of as a means of early 49 pregnancy diagnosis.2 with significant decreases in conception rates and failure to observe 50 visual estrus, being able to identify open cows as soon as possible and then initiate a timed 51 breeding program can improve pregnancy rates (percent of eligible cows becoming pregnant 52 over a 21-d period) by increasing the heat detection rate. however conception rates to 53 synchronization programs still are below desired levels and keep pregnancy rates low. currently 54 in the western u.s. dairy industry, the majority of cows found not pregnant at pregnancy 55 diagnosis are immediately started on a synchronization program or in some cases the program is 56 initiated a week prior to pregnancy diagnosis as because gnrh has not been shown to affect 57 pregnancy.3 upon being diagnosed not pregnant, cows are then given prostaglandin and 58 continued in the synchronization program. both of these strategies initiate synchronization 59 programs no matter where the cow is within her estrous cycle. 60 it has been shown that conception rates vary greatly depending upon when synchronization 61 programs are started in relation to the cow’s estrous cycle. most studies find that the ovsynch 62 timed ai program results in the greatest conception rates when initiated between d 5 to 12 of the 63 estrous cycle.4-6 research conducted in colorado on commercial dairies found that a large 64 percentage of cows at first service or diagnosed not pregnant are not within this optimal 5 to 12 d 65 window to start synchronization programs. when these cows are allowed to continue through 66 programs, conception rates are extremely low. not only are these non-synchronized cows outside 67 the d 5 to 12 window, but a large percentage are acyclic, have cystic ovaries, contain dead 68 fetuses, or have pyometra, which make them poor candidates for enrollment in a synchronization 69 program.7 70 277 an advantage of ultrasound over rectal palpation is the ability to completely assess ovarian 71 structures and better predict when cows fall into the optimum period to initiate or continue in 72 synchronization programs. most important in predicting the stage of a cow’s estrous cycle is 73 identifying the presence of an active corpus luteum. ultrasound has been found to be far superior 74 to rectal palpation in predicting an active corpus luteum and high progesterone levels.8,9 by 75 using ultrasound, cows can be assessed and synchronization programs can be modified when 76 cows fail to respond to the first gnrh injection. combining ultrasound with a synchronization 77 program can be a powerful management tool to maximize not only heat detection rates, but also 78 conception rates resulting in improved overall pregnancy rates. 79 ultrasound can also be used as a heat detection aid, especially for dairies that utilize tail 80 chalk. accuracy of heat detection when using tail chalk can vary, and often diestrus or even 81 pregnant cattle are bred. by using the ultrasound, cows with an active corpus luteum can be 82 avoided, and only cows appearing in estrus (hyperechoic uterus, ovulatory follicle and no corpus 83 luteum) will be bred. 84 however, western u.s. dairies are also experiencing an increased cow-to-employee ratio, 85 which makes increasing the level of reproductive management a challenge. in order to 86 implement this reproductive management system, dairies must increase the number of times 87 cattle are handled for ultrasound examination, injections, and artificial insemination. only dairies 88 capable of handling large numbers of cattle in a short period of time will be able to incorporate 89 these protocols effectively. currently most large dairies in the western u.s. utilize lock-ups to 90 handle cattle. this system requires that cows actively enter and lock themselves, which is not 91 always ideal. almost 100% of the time this system results in some loose cattle requiring time to 92 find and then restrain. 93 278 management/palpation rails were created and gained popularity with the advent of bovine 94 somatotropin. if designed properly, this system can be very efficient in working large numbers 95 of cattle in a short period of time. currently on one client’s 10,000 cow dairy, our practice 96 incorporates ultrasound exclusively and provides 100% tai. cattle are handled in a roofed 97 double-40 palpation rail. with three ultrasonographers, our practice scans 300-450 cows/hour, 98 and using multiple ai technicians we inseminate approximately 250 cows/hour. by 99 ultrasounding, injecting, and inseminating hundreds of cows in a very short time period, we 100 maintain the time intervals for synchronization and thus maximize conception rates. only 101 producers willing to restructure their reproductive management and build facilities around it will 102 be able to implement these protocols effectively. 103 conclusion 104 cows have changed so much reproductively over the last 20 years that it takes greater 105 attention to detail and a better understanding of reproductive physiology to get cows pregnant. 106 producers are reluctant to truly admit that cows have changed. they still want to manage cows 107 reproductively as if they were in the 1980s. acknowledging that cows are not the same and then 108 implementing intensive management protocols to overcome these obstacles is critical to 109 improving reproduction. everything we thought we knew about the reproduction of lactating 110 dairy cows has to be reconsidered. thinking outside the box and applying new strategies to 111 reproductive management can pay big returns. incorporating ultrasound intensively into tai and 112 estrus detection programs can improve rates dramatically, but requires a different mindset for 113 managing cows. 114 managing reproduction is unlike any other task on the dairy. to succeed, attention to detail 115 has to border on obsessive-compulsive. synchronization programs require attention to detail, 116 279 strict adherence to injection schedules, and proper ai technique. managing high numbers of 117 cows within these programs also requires strict data entry and retrieval within dairy software 118 programs. so, no matter how well the cows are started on these programs, if compliance is 119 inadequate, results will be inferior. failure to strive for perfection in every aspect of reproductive 120 management is doomed to poor results. finding people that have the work ethic and are 121 knowledgeable of reproduction is exceedingly difficult for producers. 122 other tasks within the dairy industry have been contracted over the years, such as crop 123 production and calf/heifer production. by designing a veterinary practice around the 124 management of reproduction and employing highly skilled individuals to conduct all aspects of 125 reproduction from ultrasound, injections, and ai to data entry/retrieval reproduction can be 126 improved. the greatest chance of reproductive success occurs when incorporating ultrasound 127 into both synchronization and heat detection programs on dairies. 128 references 129 1. thatcher ww, bilby tr, bartolome ja, et al: strategies for improving fertility in the 130 modern dairy cow. theriogenology 2006;65:30-44. 131 132 2. kastelic jp, curran s, pierson ra, et al: ultrasonic evaluation of the bovine conceptus. 133 theriogenology 198829:39-54. 134 3. chebel cc, santos jep, cerri rla,et al: effect of resynchronization with gnrh on d 135 21 after artificial insemination on pregnancy rate and pregnancy loss in lactating dairy 136 cows. theriogenology 2003;60:1389-1399. 137 138 280 4. vasconelos jlm, silcox rw, rosa gj, et al: synchronization rate, size of the ovulatory 139 follicle, and pregnancy rate after synchronization of ovulation beginning on different 140 days of the estrous cycle in lactating dairy cows. theriogenology 1999;52:1067-1078. 141 142 5. stevenson js, tiffany sm: resynchronizating estrus and ovulation after not-pregnant 143 diagnosis and various ovarian states including cysts. j. dairy sci 2004;87:3658-3664. 144 145 6. bartolome ja, silvestre ft, kamimura s, et al: resynchronization of ovulation and 146 timed insemination in lactating dairy cows i: use of the ovsynch and heatsynch protocols 147 after non-pregnancy diagnosis by ultrasonography. theriogenology 2005;63:1617-1627. 148 149 7. mcsweeney kd, garry f, olson j, et al: unpublished data. 150 151 8. sprecher dj, nebel rl, whitmanss: the predictive value, sensitivity and specificity of 152 palpation per rectum and transrectal ultrasonography for the determination of bovine 153 luteal status. theriogenology 1989;31:1165-1172. 154 9. bicalho rc, galvão kn, guard cl, et al: optimizing the accuracy of detecting a 155 functional corpus luteum in dairy cows. theriogenology 2008;70:199-207. 156 157 281 << /ascii85encodepages false /allowtransparency false 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perineal laceration was diagnosed on examination. one bitch was treated with primary intention closure, the other with second intention closure. both bitches received prophylactic antibiotics and healed well. the bitch that was treated with second intention healing developed a vestibule stricture. the future breeding potential of both bitches had not been established at this time. keywords: perineal laceration, second degree, treatment, antibiotics background perineal laceration in the dog has not previously been described in the literature and little is currently known about treatment options. while perineal lacerations in the mare are relatively common and first intention repair is broadly accepted, repair in humans by first and second intention is still in contention. case presentation case 1 a three year old, 30 kg, multiparous american staffordshire was referred to the queensland veterinary specialist hospital for evaluation of a perineal laceration. the bitch was seen at the referring veterinarian for an acute laceration of the vulva and vestibule that morning. the bitch had given birth naturally with no assistance to six pups 24 hours previously. the first pup had been passed at 0400 on april 30, 2012 and the sixth pup at 0930 on april 30, 2012. of the six pups, one was stillborn, two suffered maternal birth trauma, and the remaining three were healthy. she had then suckled and mothered the pups well that day and did not appear to be in labor. the owners found the bitch the next morning with another dead pup in her whelping box and noticed the bitch to be bleeding from the vulva. the dead pup was found in two pieces. routine prophylaxis (vaccination, deworming and heartworm prevention) was up to date and she had been historically healthy. she was taken immediately to the referring veterinary hospital. on admission to the referring clinic the bitch was bright and alert. her mucous membranes where pink and capillary refill time was less than two seconds, heart rate was 140 beats per minute and respiratory rate was 16 breaths per minute. the abdomen was swollen. her rectal temperature was 40.4°c. there was a large amount of edema of the vulva and a hematoma present in the vagina. initial treatment was administered at the referring veterinary clinic before referral to queensland veterinary specialist. treatment at the primary care clinic included: placement of an intravenous catheter into the cephalic vein and delivery of intravenous crystalloids at 180 ml/hr (6 ml/kg/hr), enrofloxacin 150 mg (5mg/kg) administered subcutaneously, amoxicillin with clavulonic acid 262 mg (8.7 mg/kg) administered subcutaneously and 300 µg buprenorphine (10 µg/kg) administered subcutaneously. on presentation to the specialist hospital she was bright, alert and responsive with a heart rate of 120 beats per minute and was panting. her mucous membranes were pink and tacky, she had a capillary refill time of two seconds and strong femoral pulses. her estimated body condition was 6/9 and her physical examination did not reveal any other abnormalities. she urinated on presentation with no abnormalities seen. reproductive examination revealed a macerated tear of the dorsal vulval commissure extending rostrally into the perineal body to create a cavernous dead space approximately 7 cm in diameter. there was also a communicating tunnel from the vagina to the skin of the perineal body creating a hole in the dorsal vagina that extended into the perineal body (figure 1a and b). the 57 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 injury was classified as a second-degree perineal laceration as there was tearing of the perineal body including the connective tissue and paravaginal muscle at the level of the vulvovaginal sphincter. blood was taken for packed cell volume and total protein analysis. packed cell volume was 36 percent and total protein was 62 g/l (6.2 g/dl). abdominal radiographs were taken to screen for retained fetuses. no fetuses were visualized within the uterine horns or the birth canal. a brief abdominal ultrasound examination was performed to screen for free abdominal fluid. no free fluid was noted. she was placed back on intravenous fluid therapy of intravenous crystalloid solution (compound sodium lactate; hartman’s solution) at 6 ml/kg/hr overnight. the three pups were healthy (two females and one male) and weighed 375 g, 372 g and 435 g, respectively. pups were temporarily weaned while the bitch was hospitalized. the bitch was re-evaluated the following morning under general anesthesia. anesthesia was induced with alfaxalon1 to effect and maintained with isoflurane; no premedication was given. a delayed primary closure method was planned. treatment consisted of debridement of a small amount of necrotic tissue and continuation of antibiotic treatment. the bitch was maintained on 500 mg amoxicillin with clavulonic acid (16.6 mg/kg) po bid for seven days. instructions were given to the owner to keep the bitch rested and confined to a small inside room with appropriate bedding and to wash the external vulva three times a day with plain warm water until re-examined in 48 hours. further re-checks were performed 8, 23, and 30 (figure 1c) days after initial presentation. healing was evident at all re-checks and primary closure was not performed. the laceration was healed by second intention alone. uterine involution was regular and no abnormalities in defecation or urination were noted. the re-check 30 days after initial presentation revealed a mostly healed vulva, however the vestibule was rolled out on a small area dorsally. a vestibular stricture was present with an internal diameter of 1cm. the vulva was healed well when the patient was examined eight weeks after presentation, however the vestibular stricture remained (figure 1d). case 2 a three year old maltese cross 4.7 kg maiden bitch was presented to a referring practice for a perineal laceration. the bitch had whelped during the night and delivered four live pups with no assistance. the bitch had been feeding the pups and mothering them well after birth. the owner noticed a wound in the perineal region in the morning and presented the bitch to the referring veterinarian. on presentation she had a temperature of 39.0°c, a heart rate of 200 beats per minute and was panting. mucous membranes were pink and moist with a capillary refill time of less than two seconds. the abdomen was enlarged but palpation did not elicit pain. the vulva was flaccid and intact with no hemorrhage or obvious trauma. there was a laceration in the perineum that connected with the vestibule (figure 2a). a rectal examination was performed and no rectovaginal fistula was found. the injury was classified as a second-degree perineal laceration as there was tearing of the perineal body including the connective tissue and paravaginal muscle. immediate closure for primary intention healing was performed. a 22 gauge catheter was placed in the right cephalic. anesthesia was induced with intravenous alfaxalon to effect and maintained with isoflurane; no premedication was given. intravenous fluid therapy of compound sodium lactate at 70 ml/hr (10 ml/kg/hr) was administered. the perineal wound was in the shape of an inverted “u”. the epidermal edges and subcutaneous tissues were torn and had jagged edges. the epidermis and subcutaneous tissues were debrided. the superficial perineal fascia was closed with simple continues sutures using 3-0 monofilament glyconate. the epidermis was closed with simple interrupted sutures of 3-0 polymerized caprolactam (figure 2b). post-operative palpation of the rectum and vagina revealed no stricture or fistula. meloxicam (0.2 mg/kg) administered subcutaneously, amoxicillin with clavulonic acid (12.5 mg/kg) and metoclopramide (0.48 mg/kg) were administered post-operatively. metoclopramide was given to stimulate prolactin release; it acts by blocking dopamine from inhibiting the production of prolactin and therefore has been used to increase milk production.2 the bitch was discharged and the owners were instructed to administer amoxicillin and cluvulonic acid (11 mg/kg q 12 hr for seven days), enrofloxacin (5.4 mg/kg q 24 hr po for three days), and meloxicam (0.1 mg/kg q 24 hr for five to seven days), and to wash the perineum with chlorhexidine once or twice daily. several rechecks of the wound were performed and sutures were removed 14 days after surgery. no impairment of urination or defecation was noted; 58clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 uterine involution appeared to be within normal limits. re-examination 14 days after surgery revealed scarring of the dorsal vaginal wall. no further follow-up was recorded. discussion this report describes the signalment, history and clinical presentation of two cases of perineal laceration that occured at the time of whelping. the author was unable to find other case reports describing perineal laceration in dogs (or other small animals). perineal lacerations are commonly encountered in equine and human medicine and are divided into four categories. first degree lacerations can be described as tearing of the vulvar lips only and second degree lacerations as tearing of the perineal body including the connective tissue and paravaginal muscle at the level of the vulvovaginal sphincter.3 third degree lacerations are described as cloaca formation from a tear extending through the rectovaginal shelf, perineal body and sphincter and vulvar lips. fourth degree lacerations are characterized by communication between rectum and vagina without disruption of the perineal body, anal sphincter, or vulvar lips.3 the two cases described in this report were classified as second degree lacerations using these definitions. two types of repair were used, primary closure was used in case 2 and secondary intention closure was used in case 1. in humans closure of first and second degree spontaneous perineal rupture by suturing (primary) or not suturing (secondary intention healing) was compared.4 these authors found no clinical difference in pain or wound complications between women that received primary closure compared to those that healed by second intention.4 they also noted that analgesia use was higher in women that received primary surgical closure. perineal lacerations are frequently encountered in equine obstetrics. in mares, repair of second degree lacerations by surgical closure of the perineal body and vulvar lips is advised to prevent urine pooling and pneumovagina formation.3 in some cases urethral extension may be required to correct pooling of urine in the cranial vagina.3 delayed primary closure of third degree perineal lacerations is advised and has been well-documented in the horse by the use of a single or two stage technique.5 it is uncertain from these two cases if there are any underlying risk factors that may have contributed to the perineal lacerations. both dogs were multiparous, had multiple pups, unassisted deliveries and were of differing breeds. in humans several predisposing factors have been identified including nuliparity, increased birth weight, assisted delivery and ethnicity. further research in small animals may help identify risk factors for perineal laceration in dogs however parity, birth weight, litter size and breed warrant investigation. both cases were treated with prophylactic antibiotics. case one was treated with a single dose on enrofloxacin (5 mg/kg) and a seven day course of amoxicillin and clavulonic acid (16.6 mg/kg – dosed due to tablet convenience). the second case was treated with amoxicillin and clavulonic acid for seven days and enrofloxacin for three days. prophylaxis for prevention of infection of perineal laceration has been advised in human medicine.4 duggal et al, 2008, compared patients who received a single dose of an intravenous cephalosporin prior to primary closure to those that received a placebo and found that patients who received prophylactic antibiotics had lower rates of wound complications than those who did not.6 future breeding potential of these two bitches is currently unknown as it is too early to rebreed them. follow-up examinations will be necessary to determine if complications that will impede mating and whelping are present. in conclusion, perineal lacerations are an uncommon condition in bitches but can be treated similarly to perineal laceration in humans. there does not seem to be any significant difference between closure by primary intention or secondary intention in the dog. broad spectrum antibiotic use was indicated in both cases and no subsequent infections were encountered. however, a single dose of intravenous cephalosporin may be all that is indicated in cases of primary closure.6 predisposing factors for perineal laceration and future breeding potential of both bitches are uncertain at this stage and warrant further investigation. learning points: • primary intention or secondary intention healing were both successful mechanisms of closure • closure by secondary intention may lead to stricture formation 59 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 • broad spectrum antibiotic use prevented secondary infections in both cases • single dose of intravenous cephalosporins prior to primary closure may be sufficient to prevent infection figire 1a. case 1 at presentation. macerated tear of the dorsal vulval commissure extending rostrally into the perineal body. figure 1b. case 1 at presentation. cavernous dead space present within the perineal body. a. dorsal vulval commissure b. vestibule c. ventral vulval commissure a b c a. dorsal vulval commisure b. perineal body a b 60clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 figure 1c. case 1 at four week re-check. the vestibule was rolled out on a small area dorsally. figure 1d. case 1 at eight week re-check. the vulva and vestibule are healed however a vestibular stricture remained. a. vestibule b. ventral vulval commissure b a a. vestibule a 61 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 figure 2a. case 2 at presentation. laceration of the perineal body that connects with the vestibule. figure 2b. case 2 post-operatively. references 1. murrell j: alfaxalone (alfaxan cd-rtu). compan anim 2009;14:89-90. 2. mccallum r, sowers j, hershman j,et al: metoclopramide stimulates prolactin secretion in man. j clin endocrinol metab 1976:42:1148-1152 3. held j, blackford j: surgical correction of abnormalities of the female reproductive organs. in: youngquist rs, editor. current therapy in large animal theriogenology. philedelphia: wb saunders;1997. p 195-208. 4. elharmeel sm, chaudhary y, tan s, et al: surgical repair of spontaneous perineal tears that occur during childbirth versus no intervention. cochrane database syst rev 2011; doi:10.1002/14651858.cd008534.pub2. 5. o’reilly jl, maclean aa, lowis tc: repair of third degree perineal laceration by a modified goetz technique in 20 mares. equine vet educ 1998;10:2-7. 6. duggal n, mercado c, daniels k, et al: antibiotic prophylaxis for prevention of postpartum perineal wound complications: a randomized controlled trial. obstet gynecol 2008;111:1268-1273. (editor’s note: the photographs in this report appear in color in the online edition of clinical theriogenology.) a. perineal body b. vestibule c. vagina a b c a. dorsal vulval commissure b. ventral vulval commissure b a 62clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 2014: simulations in veterinary education simulations in veterinary education margaret v. root kustritz college of veterinary medicine, university of minnesota, st. paul, mn a medical simulation is defined as “a device or set of conditions that aims to imitate real patients, anatomic regions, or clinical tasks.”1 simulations and models are being used increasingly in veterinary training. possible reasons for this movement include societal pressure to decrease use of live animals for teaching; fewer faculty available to provide training in technical skills; less money available for expensive laboratory training; increasing tertiary caseload at veterinary teaching hospitals, which minimizes student exposure to common presentations and opportunities to practice common technical skills; and lack of required internship/residency training.1,2 another reason may be increasing pressure from society for competency-based education. “while student learning is clearly the goal of education, there is a pressing need to provide evidence that learning or mastery actually occurs.”3 the great value of teaching using simulations, including models, is that there are no consequences to failure, animal use is minimized, opportunities for repeated practice are readily available, and the system can be optimized for each student to ensure progressive acquisition of skills and knowledge.4 in a simulation, students can explore “what if” without consequence, increasing their learning.5 simulations may enhance student abilities, specifically decreasing things like time to complete procedures or increasing their overall skills when working with client-owned animals.6,7 one concern about use of models is lack of a clinical context; students must be reminded that technical skills are not carried out in a vacuum and must learn to complete those technical tasks while also considering communication skills, teamwork, and other aspects of professionalism.8 models also are best used in a progressive manner, for example as a first step to be followed by practice on cadavers, and then on live animals. in one study evaluating simulation training for laparoscopic surgical skills, novices showed gains after training on a simulator but experienced practitioners did not, suggesting that they had already mastered the level of training that was possible using that particular simulator.9 technical expertise requires students to undertake deliberative practice, defined as repeated attempts at a given skill with continual incorporation of feedback.8,10 best use of simulators includes the following features:10-12 students are presented with specific goals and established procedures to meet those goals. students also are provided with examples of what not to do. use of a specific rubric clarifies student expectations and enhances consistency of grading. feedback is provided during the learning experience. learners have the opportunity to repeat the procedure, incorporating feedback to enhance performance. expectations for student responsibility for deliberative practice are articulated. the simulation provides a constant feeling of challenge and may vary in difficulty as the learner’s skills increase. simulations are embedded within a larger curriculum. simulators can be low fidelity or high fidelity, with fidelity defined as approximation of a reallife situation. examples of low fidelity simulators include plastic tubing filled with colloidal oatmeal as a doppler flow phantom for ultrasound training, and gelatin-filled examining gloves to mimic parenchymatous organs and latex tubing to represent hollow organs for surgery training.13,14 examples of high fidelity simulators include dynamic multi-media environments into which students can be immersed while performing tasks with team members, on-line case simulations that require students to demonstrate steps in clinical decision-making, and virtual reality representations of animal models based on 3d medical imaging and computer-based tracking of student performance.15-17 while extremely sophisticated simulators exist, with some incorporating virtual reality elements to make the experience as authentic as possible, this is not always associated with greater learning. in one study comparing training in placement of intravenous catheters using a student driven interactive multimedia system with a virtual reality simulator to an instructor-led demonstration and practice on plastic 607 clinical theriogenology • volume 6, number 4 • december 2014 arms, skill acquisition was the same between the two groups and the students were more satisfied with the latter method because they craved the immediate feedback specific to their situation.4 higher fidelity simulators also are expensive and may require special expertise to use, both as a student and an instructor. surgical skills are difficult for students to acquire because of concerns about use of live animals for teaching and anxiety on the part of the students. many studies have been done evaluating use of models and cadavers as alternatives to surgery training on live, anesthetized animals. a megastudy identified 17 studies in veterinary medicine, human medicine, undergraduate coursework and high school biology courses evaluating dissection skills and surgical skills, and showed that alternative instructional methods in all cases were equivalent or superior to training with live, anesthetized animals. these included computer simulations, plastic models, soft tissue models, and cadavers.18-21 a recent study evaluated use of video games as a method of enhancing student surgical skills; skills in laparoscopy were enhanced by video game training but traditional surgical skills were not.22 similarly, in a study evaluating use of high-fidelity models versus live animals for training in performance of ovariohysterectomy of dogs, those students trained on live animals showed much greater confidence and skill in the abdominal approach than did those students trained using a simulation.20 this highlights need for evaluation of specific microskills being developed within a simulation and promotes the concept of creating a context that is as realistic as possible to ensure student acquisition of skills on models is transferable to live animals. a study evaluating cardiopulmonary resuscitation (cpr) training using hands-on training along with an interactive computer program or watching of videotaped or live demonstrations identified equal ability of students to gather knowledge but better technical skills in the interactive computer group than in the group that watched demonstrations.23 a canine cpr model also has been described and was associated with increased student knowledge and clinical proficiency in cpr.24 human simulators may be modified to produce a high fidelity veterinary simulation. an example is use of a human anesthesia simulator to help students practice anesthetic monitoring and handling of critical events; students trained on the simulator showed increased skills on clinical patients and were overwhelmingly supportive of use of this model for training before working on client-owned animals.6 two models for training in transrectal palpation have been described in the veterinary literature. the haptic cow uses touch feedback and computer-based instructor visualization of student progress within the model to help students learn the tactile skills of transrectal palpation. students report great value in not being pressed for time while using the model the way they would be if they were examining a live animal, and having the instructor know where they are in the tract and what they are feeling, in a way that could not be replicated using a live animal. students expressed concern that the simulation was too low in fidelity, stating, “the real cow pushes your hand out.”25 the breed’n betsy is another model for transrectal palpation. students trained on this model in one study had more difficulty localizing organs and evaluating structures on the ovary than did students trained using live animals.26 the society for theriogenology and american college of theriogenologists identified a core curriculum for dvm training, which includes the technical skills listed below (table). it may behoove those members interested in education to more systematically consider what models might already exist or might be created to ensure progressive acquisition of theriogenology skills in veterinary students. in one study evaluating transrectal palpation of cows, it was determined that students need to perform transrectal palpation on more than 200 cows before they are able to consistently identify and evaluate structures.26 there is great value in finding more ways for our students to practice skills to help them reach the level of competence desired. references 1. scalese rj, issenberg sb: effective use of simulations for the teaching and acquisition of veterinary professional and clinical skills. j vet med educ 2005;32:461-467. 2. smeak dd: teaching surgery to the veterinary novice: the ohio state university experience. j vet med educ 2007;34:620-627. 3. kochevar dt: the critical role of outcomes assessment in veterinary accreditation. j vet med educ 2004;31:116-119. 608clinical theriogenology • volume 6, number 4 • december 2014 4. engum sa, jeffries p, fisher l: intravenous catheter training system: computer-based education versus traditional learning methods. am j surg 2003;186:67-74. 5. williamson kb: instructional technology in medical education. in: distlehorst lh, dunnington gl, folse jr (eds); teaching and learning in medical and surgical education: lessons learned for the 21st century. new york: psychology press; 2009. p.120-124. 6. modell jh, cantwell s, hardcastle j, et al: using the human patient simulator to educate students of veterinary medicine. j vet med educ 2002;29:111-116. 7. abutarbush sm, naylor jm, parchoma g, et al: evaluation of traditional instruction versus a self-learning computer module in teaching veterinary students how to pass a nasogastric tube in a horse. j vet med educ 2006;33:447-454. 8. kneebone r, baillie s: contextualized simulation and procedural skills: a view from medical education. j vet med educ 2008;35:595-598. 9. fransson ba, ragle ca: assessment of laparoscopic skills before and after simulation training with a canine abdominal model. j am vet med assoc 2010;236:1079-1084. 10. ambrose sa, bridges mw, dipietro m, et al: how learning works: 7 research-based principles for smart teaching. san francisco: jossey-bass;2010. p. 145-148. 11. debie mh, lipman lja: the use of digital games and simulators in veterinary education: an overview with examples. j vet med educ 2012;39:13-20. 12. pivec m, kearney p: games for learning and learning from games. informatica 2007;31:419-423. 13. dennison se, delaney fa: an in-house phantom as an alternative to commercially available doppler flow phantoms. vet radiol ulreasound 2010;51:545-547. 14. perez-rivero jj, rendon-franco e: experience of the use of table-top simulators as alternatives in the primary surgical training of veterinary undergraduate students. altern lab anim 2012;41:p10-p11. 15. schlachter jg. virtual veterinary emergency department: a software system that presents dynamic interactive medical scenarios for teaching veterinary medicine. athens ga, 2004. available at https://getd.libs.uga.edu/pdfs/schlachter_jason_g_200408_ms.pdf, accessed february 4, 2014. 16. dhein cr: online small animal case simulations, a.k.a the virtual veterinary clinic. j vet med educ 2005;32:93-102. 17. lee s, lee j, lee a, et al: augmented reality intravenous injection simulator based 3d medical imaging for veterinary medicine. vet j 2013;196:197-202. 18. patronek gj, rauch a: systematic review of comparative studies examining alternatives to the harmful use of animals in biomedical education. j am vet med assoc 2007;230:37-43. 19. carpenter lg, piermattei dl, salman md, et al: a comparison of surgical training with live anesthetized dogs and cadavers. vet surg 1991;20:373-378. 20. greenfield cl, johnson al, schaeffer dj, et al: comparison of surgical skills of veterinary students trained using models or live animals. j am vet med assoc 1995;206:1840-1845. 21. griffon dj, cronin p, kirby b, et al: evaluation of a hemostasis model for teaching ovariohysterectomy in veterinary surgery. vet surg 2000;29:309-316. 22. towle millard ha, millard rp, constable pd, et al: relationships among video gaming proficiency and spatial orientation, laparoscopic, and traditional surgical skills of third-year veterinary students. j am vet med assoc 2014;244:357-362. 23. reder s, cummings p, quan l: comparison of three instructional methods for teaching cardiopulmonary resuscitation and use of an automatic external defibrillator to high school students. resuscitation 2006;69:443-453. 24. fletcher dj, militello r, schoeffler gl, et al: development and evaluation of a high-fidelity canine patient simulator for veterinary clinical training. j vet med educ 2012;39:7-12. 25. baillie s, mellor dj, brewster sa, et al: integrating a bovine rectal palpation simulator into an undergraduate veterinary curriculum. j vet med educ 2005;32:79-85. 26. bossaert p, leterme l, caluwaerts t, et al. teaching transrectal palpation of the internal genital organs in cattle. j vet med educ 2009;36:451-460. 609 clinical theriogenology • volume 6, number 4 • december 2014 table: technical skills by species in core curriculum technique species transrectal reproductive examination bovine, equine ultrasound of the reproductive tract bovine, equine breeding soundness examination / semen collection and evaluation bovine, equine, small animal passage of pipette / biopsy instrument / insemination pipette into uterus bovine, equine neonatal resuscitation bovine, equine, small animal anesthesia bovine, equine, small animal teat surgery bovine caslick’s surgery bovine, equine ovariectomy / ovariohysterectomy bovine, equine, small animal castration (descended and cryptorchid) bovine, equine, small animal cesarean section bovine, equine perineal reconstruction equine 610clinical theriogenology • volume 6, number 4 • december 2014 2009: managing the problem beef embryo donor managing the problem beef embryo donor 1 j.w. shull 2 brazos valley genetics, p.o. box 10345, college station, tx, usa 3 4 abstract 5 6 defining the problem beef embryo donor and the methods of managing each problem 7 can differ with each donor presented. to manage the problems presented to the practitioner 8 the answer may include any one, or a combination of conservative medical or surgical 9 solutions. the donor’s life and physical health are rarely at risk when dealing with embryo 10 production issues. therefore, the treatment protocol should be based on the possibility of 11 increasing her embryo production, and whether the expense involved is justified. 12 keywords: problem donor, management, treatment, expense 13 introduction 14 what is a problem donor? the argument can be made for defining the problem donor 15 as that animal with physical or reproductive problems that deny her any possibility of 16 producing viable embryos on the day of embryo collection or one that just gives fewer 17 embryos than the average. in embryo transfer practice the definition of a problem donor is 18 primarily dependent upon the owner of the cow. one owner’s definition of a problem donor 19 can be another owner’s definition of acceptable. the financial as well as emotional 20 investment is weighed in defining whether or not the individual donor has a problem. if the 21 beef donor is the son’s or daughter’s present or past show heifer, or if she is the dam of the 22 steer that won a major show, then her status as a problem donor may differ when compared 23 to the embryo donor that is used only to fill an embryo purchase quota. emotional 24 283 involvement and potential windfall gain, as compared to economic returns based on embryo 25 production per collection can drastically alter the definition of the problem donor. the 26 difficulty is to arrive at a definition of the problem beef donor that allows the embryo transfer 27 practitioner to make consistent recommendations for managing a problem while at the same 28 time taking into account the variations of expectations that result from each individual case. 29 donor embryo production 30 the average number of viable embryos recovered per collection from stimulated beef 31 donors as reported by the american embryo transfer association (aeta) in 2007 was 32 6.52.1 in 2006 embryo production was 6.501 and review of the past averages reveals there 33 have not been appreciable changes since the reporting began. this gives a baseline for the 34 discussion with the owner, and allows him or her to make treatment decisions based on the 35 animal’s peers. 36 donor management problems 37 the problems that are routinely seen in the beef donor that result in less than expected 38 results can be categorized into problems associated with the owner, the practitioner, and 39 those directly related to the animal. the owner’s problems can be the easiest or the most 40 difficult to solve. many times the owner’s problem is solved through education. helping the 41 owner understand what is and is not a problem is crucial. if he/she has been misinformed by 42 breeders, journals, or other sources about expectations of embryo production a quick review 43 of the national averages1 will resolve the problem. a second owner-related issue that can 44 lead to a donor problem is the use of frozen semen from a sire with poor fertility. the 45 pressure to use the new up-and-coming sire or the bull that sired the high selling lot at the last 46 breed sale may take precedence over possible fertility issues. even if a history of fertility 47 284 problems with the sire in question can be documented the decision to change sire choice to 48 one with good fertility can be a difficult task. 49 the second group of problems that can negatively affect the donor’s production are 50 problems related to the practitioner. the donor problems that can arise from the practitioner 51 range from inexperience, substandard donor protocols, inadequate embryo equipment, 52 handling facilities, weather, and cattle breed and age variations to name a few. 53 the third category of problems is that linked directly to the animal herself. these 54 decreases in production can be related to age, breed, malnutrition, obesity, disease, lameness, 55 genetic or congenital defects, reproductive disease, trauma, or dystocia. 56 solutions and treatments 57 to assess and devise a management plan for the many causes of decreased production 58 in the donor requires addressing all three categories of problems. 59 owner-related problems 60 owner education is the key to an understanding of embryo transfer goals. it is not 61 always easy for the owner to hear that not all donors give “25 number 1 eggs” at every 62 collection, but it is better to give realistic goals to the owner before the expenses and time 63 required for a successful or non-successful superovulation and collection are incurred. 64 embryo production can be extremely high but there are many more donors that give no 65 viable embryos in a collection as opposed to those that produce twenty five or more that all 66 grade as quality score one.2 sire selection is a difficult decision for the owner. in a 67 superovulation program it should be given considerable attention. with embryo production 68 the practitioner’s primary goal, the most consistent sire selection criteria available is to use 69 semen with known collection results. whenever possible obtain frozen semen that you have 70 285 personal experience using in your practice. the same semen from the same cane from the 71 same bull taken from the same ranch nitrogen storage tank is the best way to insure 72 consistent sire results. keep detailed records on every sire and refer to the results when 73 discussing sire assignments with the owner. he will make the final decision, but your 74 knowledge of previous sire success or failure can be a big influence. 75 practitioner-related problems 76 the failure to reach production goals because of mistakes, inexperience, and lack of 77 specific reproductive knowledge by the practitioner is not a minor problem. if the 78 practitioner can not devote a large percentage of their time to embryo work they will not 79 know what equipment is necessary in specific situations, what programming differences are 80 needed related to breed and age, and how to deal with individual animal variations to obtain 81 the maximum production at each collection. the virgin heifer is a good example. she can be 82 a productive embryo producer if managed properly. by monitoring the estrous cycle, 83 decreasing the total amount of fsh, minimizing external stresses, and using specific 84 collection equipment and techniques a practitioner can increase the percentages of success 85 with young heifers. if the virgin heifer is managed as an adult the results will be 86 disappointing. the opposite end of the spectrum is the obese older american breed 87 (brahman influenced) donor. finding the proper stimulation protocols, and having the 88 equipment and ability needed to effectively collect this cow type is quite a challenge. there 89 are 1800 pound beefmaster donors that require less fsh than a virgin heifer, have an 90 extremely large cervix and ovaries that are larger during postpartum anestrous than most 91 bovine superstimulated ovaries. the list of normal variations in the beef donor is lengthy but 92 286 the point is, the practitioner must be knowledgeable of the different skills, techniques, and 93 donor requirements needed to handle the variations as they are presented. 94 animal-related problems 95 many of the donor problems related to the cow can be solved with time. very 96 commonly a young normal heifer is presented as a potential donor and has not yet reached 97 puberty. a post partum cow or a thin nursing cow is presented and is not yet cycling. these 98 are not true problems but normal occurrences in the cow’s life. a group of reproductive 99 problems that can be treated either conservatively or aggressively, depending on the specific 100 problem’s effect on embryo production are urine pooling, pneumovagina, and vulvar tears. 101 my approach to this group of problems is conservative as long as no uterine involvement is 102 detected. i have adopted this approach because of the examination of recipient pregnancy 103 records. notations on the transfer record for each of these three problems were made every 104 time a recipient was presented for transfer and the pregnancy results were examined. the 105 pregnancy rates of those affected recipients with urine pooling, pneumovagina, or vulvar 106 tears, but no indication of uterine involvement, did not differ from the non-affected 107 recipients. the donor with vulvar tears that allow vaginal fecal contamination and the urine 108 poolers that show signs of metritis are surgically repaired. the true problems that affect 109 production are often times problems that cannot be reversed. blockage of the oviduct 110 (whether cystic or caseated), chronic mucometra, and severe uterine/ovarian adhesions are a 111 few examples. these problems can be handled in one of two ways, either sell the donor or 112 refer her to an in-vitro fertilization (ivf) clinic. the options are discussed with the owner as 113 to the cost of the procedure and expected results. if the value of the donor’s offspring warrant 114 the expense then ivf is a good option. 115 287 conclusion 116 i hope i have made you aware that most of the problem donors that are presented can 117 be managed conservatively with success. the beef cow is one of the most reproductively 118 resilient animals veterinarians will ever encounter. the nutritional, physical, infectious, 119 genetic, and iatrogenic stresses that the cow can withstand and still be reproductively prolific 120 is quite remarkeable. 121 references 122 1. stroud b: annual report of the aeta statistics committee for the calendar year 123 2007. a closer look. am embryo transfer assoc fall 2008:23:14. 124 2. stringfellow d, seidel sm: certification and identification of the embryo. manual of 125 the international embryo transfer society, 3rd ed; 1998. p 106-107.126 127 288 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding 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/legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2019 cushioned centrifugation improves recovery rate and viability of canine sperm cushioned centrifugation improves recovery rate and viability of canine sperm christine winslow,a,b candace lyman,a shelby hayden,a,c jessica cohen,a,d jared taylor,a g. reed holyoaka acenter for veterinary health sciences, oklahoma state university, stillwater, ok bcurrent address: animal health institute, midwestern university, glendale, az ccurrent address: kansas center for equine reproduction, lawrence, ks dcurrent address: legacy veterinary hospital, frisco, tx abstract the objective was to compare recovery rate, motility and viability of canine sperm after cushioned and conventional (non-cushioned) centrifugation. our hypothesis was that cushioned centrifugation improves sperm recovery rate relative to conventional, non-cushioned centrifugation without detrimental effects on sperm total motility or viability. five adult dogs of varying breeds were collected weekly for 3 weeks. each sample was processed, divided, and aliquots were centrifuged with and without cushion media. the aliquots were resuspended and analyzed for percent recovery, percent viability, and change in total motility. cushion centrifugation had significant effects on recovery rate, and a numerical yet not statistically significant effect on total motility relative to conventional, non-cushion centrifugation of canine semen. percent recovery was higher (p = 0.041) in cushioned centrifugation samples and a greater loss in viability (p = 0.003) was observed in non-cushioned centrifugation samples. keywords: canine, cushion centrifugation, semen processing, sperm, semen quality introduction centrifugation is commonly done to remove excess canine seminal plasma. however, conventional, non-cushioned centrifugation techniques utilizing higher speeds (e 1620 x g) impose high mechanical forces, significantly decreasing sperm quality.1 lowering the centrifugation speed to d 720 x g helps prevent sperm damage, but decreases sperm recovery.1 since sperm concentration can vary with age and collection method, especially in older dogs, the practitioner’s ability to adequately concentrate semen without damaging sperm becomes a clinical concern. based on laboratory analysis in several studies, density gradient centrifugation of canine semen is effective in concentrating and selectively recovering higher quality sperm and is also capable of removing cellular contaminants.2-4 however, effects of cushioned centrifugation alone on recovery rate and quality, as assessed by viability and motility of canine sperm, have apparently not been reported. cushioned centrifugation of stallion semen effectively mitigated sperm damage and improved recovery rate.5-7 similar results were reported for boar semen.8 perhaps cushioned centrifugation would also be beneficial for dog semen. the objective was to compare sperm recovery, motility, and viability of canine semen after cushioned and non-cushioned centrifugation. our hypothesis was that cushioned centrifugation improves recovery rate without inducing significant detrimental effects on viability and motility of sperm relative to conventional, non-cushioned centrifugation. materials and methods study design five adult dogs, aged 1.8 6.2 years (mean 4.8) were used. the breeds were rhodesian ridgeback, chesapeake bay retriever, saint bernard, great dane, and labrador retriever cross. all dogs were privately owned and remained with their owners except during semen collection. prior to acceptance into the study, each dog was subjected to a full physical examination and semen evaluation, and tested for brucellosis using a rapid benchtop assay (anigen rapid canine brucella ab test kit, bionote, gyeonggi, korea). the study protocol was approved by the institutional animal care and use committee at oklahoma state university. to reduce bias and increase consistency, only 2 individuals collected and processed semen samples, each one responsible for a unique set of tasks. 127 clinical theriogenology • volume 11, number 2 • june 2019 sample collection and processing a cleanout collection was performed 7 days prior to the first day of sample collection and processing in dogs (n = 5). thereafter, 1 ejaculate was collected from each dog every 7 days for 3 weeks (total of 15 samples). to improve statistical power, each collection was regarded as an individual sample. no sexual activity was allowed between collections. neat semen was analyzed for concentration and volume (determined by weight) to determine total sperm in the ejaculate. sperm concentration and viability (live-dead ratio by propidium iodide exclusion) were evaluated using a nucleocounter® sp100™ (chemometec a/s, allerod, denmark), according to manufacturer’s instructions. an aliquot of semen was then diluted to a concentration of 25 x 106 sperm/ml with caninespecific semen extender (canipro™ apx2 chill 5, minitube of america, verona, wi) to evaluate total motile sperm. all sperm motility samples were analyzed at a concentration of 25 x 106 sperm/ml following placement on a covered slide-warmer (37°c) for 10 minutes, using a computer assisted sperm motility analyzer (ivos version 12.0, hamilton-thorne research, hamilton, ma), as described.9 extended semen containing identical amounts of sperm were placed into 2 conical vials (15 ml). in 1 of these 15 ml conical vials, 0.25 ml of cushion media (cushion fluidtm, minit� b, tiefenbach, germany) was layered under the extended semen, as described.5,6 the vial containing cushion media was then centrifuged at 1500 x g for 10 minutes, whereas the vial without cushion media was centrifuged at 750 x g for 10 minutes. the supernatant was removed via vacuum aspiration (cook medical, llc, bloomington, in). for vials containing cushion media, a 7.62 cm spinal needle was inserted to the base of the vial in order to gently aspirate as much of the cushion media as possible without disrupting the sperm pellet layer. the volume of cushion media removed was minimal, totaling < 0.1 ml in each instance. the sperm pellet was suspended with extender containing 20% (v/v) of the subject’s own fresh seminal plasma to 50 x 106 sperm/ml, as described.6 fresh seminal plasma was obtained by collecting and subjecting the third fraction of the ejaculate to centrifugation at 2000 x g for 10 minutes, followed by filtering the supernatant through tandem 5.0 and 1.2 µm nylon filters (cameo 30 n syringe filter, nylon, 30 mm; sigma-aldrich, st. louis, mo) fitted to an all-plastic syringe to remove any remaining sperm. the sample was immediately analyzed for sperm recovery rate, viability, and total motility. statistical analyses analyses were performed for impact of treatment on sperm recovery rate, viability, and total motility. recovery rate was calculated as ([sperm count/ml after treatment] / [sperm count/ml prior to treatment] x 100). reduction in viability was calculated as [percentage of viable sperm prior to treatment – percentage viable sperm after treatment], and reduction in motility was calculated similarly [percentage of motile sperm prior to treatment – percentage of motile sperm after treatment]. a general linear model for repeated measures was done using commercial software (spss 21). treatment (cushioned versus non-cushioned centrifugation) was considered a fixed effect. main effects were considered only when no dog by treatment interaction was present. results percent recovery mean percentage of sperm recovered post centrifugation was 98.7% (range 75.9 -128.7). method of centrifugation affected (p = 0.041) percent recovery, with the cushioned centrifugation approach resulting in a larger percent recovery (103.5 versus 93.8%). there was no significant effect of dog on percent recovery and there was no treatment by dog interaction. percent viability mean percent of viable sperm was 89.4% prior to centrifugation, 89.5% after cushioned centrifugation and 86.2% after non-cushioned centrifugation, with a difference among dogs (p = 0.002) and no treatment by dog interaction. there was a greater loss in viability (p = 0.003) in non-cushioned 128clinical theriogenology • volume 11, number 2 • june 2019 versus cushioned approaches. viability in cushioned samples was essentially unchanged when compared to diluted and non-centrifuged samples. percent motility treatment had a highly variable impact on total motility and varied among samples from individual dogs. changes in motility following centrifugation ranged from a decrease of 16% for a noncushioned sample (90% initial motility down to 74%) to an increase in motility of 9% for 1 cushioned sample (69% initial motility to 78%). mean changes were decreases of 3 and 6% for cushioned and noncushioned centrifugation, respectively. no dog by treatment interaction was detected for change in total motility, and neither dog nor treatment affected motility following centrifugation (p = 0.224 and p = 0.18, respectively). discussion in the present study, cushioned centrifugation improved sperm recovery rate when compared to conventional, non-cushioned centrifugation without negatively affecting viability of canine sperm. results were consistent with those obtained in other domestic species.5,7,8 however, there was no significant advantage in terms of percent loss of total motility. given that the recovery rate of the cushioned samples was high, partial removal of the cushion media did not appear to negatively impact recovery rate in this study. higher recovery rate for the cushioned samples was attributed to an increased sperm sedimentation rate due to a higher centrifugal force, whereas non-cushioned samples were centrifuged at 720 x g to preserve viability.1 in stallions, less conservative non-cushioned centrifugation protocols can yield good recovery rates without significant loss of sperm quality,10 although such data have apparently not been reported for dogs. perhaps canine sperm might remain viable with an acceptable recovery rate when centrifuged with moderate force even without the use of cushion medium (may require increased time). as viability can also be affected by increased duration of centrifugation, additional studies are needed to determine the optimal protocol if cushion centrifugation is to be avoided, for instance due to expense, unfamiliarity with the technique, or practitioner preference. that average recovery rate for cushioned centrifugation samples was 103.5%, a value greater than what was present in the original sample, was unexpected. according to the manufacturer, the nucleocounter® sp-100™ is accurate to within 4% on repeated analyses of the same sample for measurement of concentration, whereas accuracy for viability is repeatable to within 8%. applying margins of error, recovery rate for the cushioned protocol would potentially be as low as 99.5% given the calculated average of 103.5%. however, since the initial (pre-centrifugation) measurements would also be subject to some degree of variation, original values may have been underestimated on average, thereby inflating subsequent readings. all analyses using the nucleocounter® sp-100™ were performed by the same individual in order to mitigate interoperator differences. the degree of change in viability varied among samples compared between dogs, but there was no interaction between a particular treatment and individual dogs. perhaps sperm that responds poorly to conventional centrifugation still undergoes a noticeable loss of viability even with cushioned media. however, use of a cushion protocol reduced loss in viability, blunting a detrimental effect of the standard processing technique. the study population was comprised of dogs with normal initial semen quality based on published values with regard to sperm numbers, motility, viability, and morphology (data not shown).11 dogs with low sperm numbers or poor viability, such as aged or smaller breed dogs, may benefit the most from a cushioned centrifugation protocol to remove excess seminal plasma while minimizing damage to the sperm. a practical observation was that use of cushion fluid made dissolution of the sperm pellet subjectively easier and may have contributed to sustaining semen quality by reducing the vigor and time required to re-suspend sperm in solution. the average effect on motility was a decrease in total motility relative to the initial, uncentrifuged samples. although there was a numerical difference between the cushion and non-cushion protocols (decreases of 3 and 6% respectively), these were not statistically significant. this was consistent 129 clinical theriogenology • volume 11, number 2 • june 2019 with work done in the boar, although stallions have some variation. in that regard, there is not agreement in the literature as to whether or not centrifugation has a detrimental effect on stallion sperm motility.8,9 based on the results of this study, no statement on fertility of the centrifuged canine sperm can be made. however, centrifugation is becoming more common for canine semen processing in practice and no significant detrimental effects would be expected in a dog with good initial semen quality when handled appropriately during processing. in stallions, incorporating small amounts of iodixanol cushion medium into the ejaculate during processing had no appreciable effect on semen quality or fertility.5 the effects of incorporating cushion fluid into the ejaculate and its subsequent effects on fertility in the bitch are unknown. further investigation with a fertility trial comparing non-centrifuged, cushioned centrifuged, and non-cushioned centrifuged semen is required to elucidate this effect. post-thaw motility and viability parameters of cryopreserved semen subjected to cushioned and non-cushioned centrifugation have yet to be compared. if sperm centrifuged without cushion were less likely to survive cryopreservation due to increased membrane damage, standard clinical procedures during semen cryopreservation would need to be altered. in conclusion, cushioned centrifugation improved sperm recovery rate and maintained viability relative to conventional, non-cushioned centrifugation of canine semen. these findings supported the use of a cushion solution when performing centrifugation as a component of semen processing in dogs. funding funding for this project was provided by a grant from the research advisory committee at oklahoma state university and the bullock endowed chair. cassettes for the nucleocounter® sp-100™ (chemometec a/s, allerod, denmark) and the cushion fluidtm (minit� b, tiefenbach, germany) were donated by the manufacturers. acknowledgements the authors thank søren pedersen and chemometec a/s, allerod, denmark for their donation of test cartridges used during the study and for technical support. conflict of interest the authors have no conflicts of interest. references 1. rijsselaere t, van soom a, maes d, et al: effect of centrifugation on in vitro survival of fresh diluted canine spermatozoa. theriogenology 2002;57:1669-1681. 2. dorado j, alcaráz l, duarte n, et al: centrifugation on puresperm® density-gradient improved quality of spermatozoa from frozen-thawed dog semen. theriogenology 2011;76:381-385. 3. phillips tc, dhaliwal g, verstegen jp: separation of viable, motile sperm from red blood cells and dead spermatozoa: a comparison of four density gradient centrifugation media in the dog. theriogenology 2008;70:578-579. 4. hinshinuma m, sekine j: separation of canine epididymal spermatozoa by percoll gradient centrifugation. theriogenology 2004;61:365-372. 5. waite ja, love cc, brinsko sp, et al: factors impacting equine sperm recovery rate and quality following cushioned centrifugation. theriogenology 2008;70:704-714. 6. bliss sb, voge jl, hayden ss, et al: the impact of cushioned centrifugation protocols on semen quality of stallions. theriogenology 2012;77:1232-1239. 7. len ja, beehan dp, lyle sk, et al: cushioned versus noncushioned centrifugation: sperm recovery rate and integrity. theriogenology 2013;80:648-653. 8. matás c, decuadro g, martínez-miró s, et al: evaluation of a cushioned method for centrifugation and processing for freezing boar semen. theriogenology 2007;67:1087-1091. 9. schäfer-somi s, aurich c: use of a new computer-assisted sperm analyzer for the assessment of motility and viability of dog spermatozoa and evaluation of four different semen extenders for predilution. anim reprod sci 2007;102:1-13. 10. len ja, jenkins ja, eilts be, et al: immediate and delayed (after cooling) effects of centrifugation on equine sperm. theriogenology 2010;73:225-231. 11. johnston sd, root kustritz mvr, olson pns: canine and feline theriogenology. new york: wb saunders company; 2001. p. 289. 130clinical theriogenology • volume 11, number 2 • june 2019 abstract introduction materials and methods study design sample collection and processing statistical analyses results percent recovery percent viability percent motility discussion references << /ascii85encodepages false /allowpsxobjects false /allowtransparency false /alwaysembed [ true ] /antialiascolorimages false /antialiasgrayimages false /antialiasmonoimages false /autofiltercolorimages true /autofiltergrayimages true /autopositionepsfiles true /autorotatepages /none /binding /left /calcmykprofile (u.s. web coated \050swop\051 v2) /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /checkcompliance [ /none ] 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] /pdfx1acheck false /pdfx3check false /pdfxbleedboxtotrimboxoffset [ 0 0 0 0 ] /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxoutputcondition () /pdfxoutputconditionidentifier () /pdfxoutputintentprofile () /pdfxregistryname () /pdfxsetbleedboxtomediabox true /pdfxtrapped /false /pdfxtrimboxtomediaboxoffset [ 0 0 0 0 ] /pagelayout /singlepage /parsedsccomments true /parsedsccommentsfordocinfo true /parseiccprofilesincomments true /passthroughjpegimages true /preservecopypage true /preservedicmykvalues true /preserveepsinfo true /preserveflatness false /preservehalftoneinfo false /preserveopicomments true /preserveoverprintsettings true /startpage 1 /subsetfonts true /transferfunctioninfo /apply /ucrandbginfo /preserve /useprologue false /srgbprofile (srgb iec61966-2.1) >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2010: improving neonatal survivability in swine* improving neonatal survivability in swine* c.a. smith american veterinary medical association, schaumburg, il, usa introduction the swine industry in the united states is composed of approximately 6.01 million females that farrow more than 11.8 million times annually, producing 112 million piglets. preweaning loss of piglets is a major problem confronting the swine producer. approximately 15 to 25% of all piglets die during parturition and the first few weeks of life.1 many of these deaths occur during parturition and the first 72 hours of life.2 keywords: neonatal survival, swine, parturition, postpartum care prepartum care of the sow and gilt efforts to improve neonatal survivability should begin with the pregnant sow (gilt). preparturient females should be housed in a well-maintained environment of 18.5° c (65° f). females exposed to environmental temperatures of 37.8° c (100° f) for 17 hours per day during the last week of gestation farrowed more stillborn piglets.3 the use of fans or drip-cooling systems for the sows may be necessary to provide relief from heat-related effects in sub-tropical environments, while heaters may be necessary to provide an adequate thermal environment in colder climates. provision of appropriate nutritional requirements is imperative. in some cases, the feeding of energy-supplemented diets to sows during the latter stages of gestation has improved the survivability of piglets.4 diets supplemented with animal fat during the last five days of gestation decreased the number of stillbirths.4 many modern swine facilities are not designed to use liquid fats in the diet of sows. dry fats are commercially available but may be expensive. the labor requirements of individual feeding of diets to sows during late gestation would not be unreasonable. however, excess energy in the diet can be detrimental. fat sows can have an increase in stillbirths and greater mortality rate for piglets during the first three days after farrowing.5 animals housed to allow exercise have lower stillbirth rates than animals that are tethered or confined in crates.6 modern swine facilities are often not designed to allow exercise of animals during late gestation and lactation. the loss of piglets due to crushing and chilling during exercise periods, as well as the extra labor required to manage these systems, have been deterrents to their use in commercial swine ventures. however, animal welfare concerns may influence the type of sow housing. care at parturition the length of gestation in swine is typically 114 to 116 days. variations may be due to the breed of sow or boar involved. large litter sizes may decrease gestation length.7 physical appearance and behavioral changes signal impending parturition. swelling and increased reddening of the vulvar lips occurs about four days prior to farrowing. the mammary glands will usually become swollen and turgid during the last seven to ten days of gestation. a serous secretion may be present up to 48 hours prepartum, changing to a milky fluid about 24 hours prepartum. an abundance of milk ,which can be easily expressed from the teat, indicates farrowing is imminent and usually will commence within six hours. rectal temperature will usually rise 0.5° c (1° f) during the last 12 hours prior to farrowing. respiratory rates will * originally published by the society for theriogenology and the american college of theriogenologists as publication p-1 (2/91) in the theriogenology handbook (revised 2010). 65 increase substantially during the 12 to 24 hour period prior to parturition but return to a nearnormal rate just prior to the birth of the first piglet. frequency of defecation and urination are often increased during the 12 hour prepartum period. behavioral changes begin during the last 24 hours prepartum. females will exhibit increased restlessness and will lie down and get up frequently. animals will attempt “nestbuilding” by scraping bedding or food into a pile. animals in crates will vigorously paw, root or chew on the bars of the crate. periods of increased activity are interrupted occasionally by short periods of rest. this pattern of behavior continues until shortly (15 to 60 minutes) before birth of the first piglet at which time the sow becomes quiet and settles into lateral recumbency. abdominal straining will begin, often accompanied by drawing the rear legs toward the abdomen. a small amount of viscous fluid, often blood-tinged and many times containing some meconium, will be passed from the vulva during this time, followed by the delivery of the first piglet within 15 to 20 minutes. parturition delivery of the piglets is accompanied by mild abdominal straining, a small amount of fluid and a very noticeable wiggling of the tail. the duration of the second stage of labor is usually one to five hours, but may be considerably longer without adverse effects on the piglets. the normal interval between piglets is about 15 to 20 minutes. the interval between stillborn piglets tends to be greater, as does the interval between the next to last and last piglet of the litter. fetuses are most frequently (60%) born in anterior presentation. posterior presentations usually create no problem and are considered normal. fetuses born in anterior presentation have the snout delivered first, with the front legs positioned along the sides of the piglet, while the rear legs are extended and delivered first in posterior presentations. fetal membranes are usually passed after the completion of farrowing. expulsion may occur anywhere from 20 minutes to ten hours after birth of the last piglet but normally occurs within a few hours. individual fetal membranes may be expelled during parturition in the interval between piglets. retained fetal membranes are rare in the pig; retained fetal membranes may be an indication that piglets are still present in the uterus. deaths during parturition due to stillbirths dystocia is a rare event in porcine parturition, but the losses due to stillbirths are severe and may account for 60% of all preweaning deaths. most stillborn pigs (67%) are intra-partum rather than pre-partum deaths. seventy percent of these intra-partum deaths show signs of viability at the time of their delivery, as evidenced by weak gasping respirations or a slow, faint heartbeat. asphyxia, indicated by the presence of meconium-stained amniotic fluid, results in piglets that are severely depressed at birth due to profound acidosis and hypercapnia.8 the asphyxia may be related to the high incidence of ruptured umbilical cords (94%) observed in these piglets. delayed farrowing may also influence stillbirth rate. highest stillbirth rates are seen in parturitions greater than four hours duration with 82% of all stillborn piglets occurring in the last third of the litter. the interval preceding stillborn piglets, while highly variable, tends to be longer than the interval preceding liveborn piglets. forty percent of females account for almost 100% of stillbirths.9 sows predisposed to stillbirths can usually be predicted prior to farrowing. sows at seventh parity or higher, sows that consistently farrow greater than ten piglets, and sows that have previously farrowed litters containing stillborn piglets are good candidates to produce stillborn piglets at subsequent parturitions.10 66 assistance during parturition and the peripartum period indications for manual examination and intervention during farrowing have historically included the presence of a foul smelling vaginal discharge, cessation of abdominal straining following birth of only one or two piglets, active abdominal straining with an interval of greater than 30 minutes since the birth of the last piglet, and the presence of abdominal straining without the birth of the first piglet. a farrowing house attendant can decrease both the stillbirth rate and the rate of preweaning mortality.11 current implementation includes assisted manual delivery on all sows beginning with the birth of the first piglet. examinations are performed at intervals of five to ten minutes until farrowing is completed to prevent asphyxiated piglets. this is preferable to waiting 30 minutes before intervening and only recovering an expired piglet. immediately following birth, the attendant should remove any fetal membranes and mucus from the head and face of the piglet. vigorous massaging of the piglet or swinging the piglet in a head-down position is helpful in clearing airways and stimulating respiration. it is recommended that the naval cord be dipped in disinfectant as a precaution to infection. ligation of the umbilical cord is not routinely performed, but can be utilized in the occasional piglet in which hemorrhage from the umbilical cord is a problem. the attendant should help the neonates remain warm. first teat contact usually occurs within eight to nine minutes after birth with first successful suckling within 30 minutes after birth. assistance should be provided to enable colostrum intake if the piglet has not done so within 30 minutes after birth. parturition induction commonly used methods of parturition induction for synchronized farrowing will result in greater than 70% of females farrowing during the next working day. however, better utilization of personnel and better results may be obtained by providing continuous assistance 48 hours immediately after induction since greater than 90% of treated females will farrow during this time. the use of prostaglandin followed by administration of five to ten units of oxytocin 20 to 24 hours later results in approximately 50% of females farrowing within three hours after oxytocin administration.12 pregnant females should be at least 111 days or later in gestation when induced to avoid birth of small, lightweight piglets that are more likely to succumb. the ideal use of prostaglandin would be to calculate the length of gestation for each herd from on-farm records and induce parturition one to two days before the anticipated day of delivery. aftercare of the neonate feces should be removed from behind the sow to prevent contamination of the newborn. the newborn piglet should have a warm, dry environment. heat lamps or heating pads are equally effective. ideally, a heat source should be positioned at the rear of the sow and also on the sides of the sow to create the warm microenvironment desired. the heated area on the side of the sow should be maintained at approximately 35° c (95° f) to maintain the body temperature of the newborn piglet. heating devices must be used with caution. excessively high temperatures will actually induce piglets to lie away from heated areas.13 additionally, the heating device should warm only the piglet area; the ambient room temperature should remain at or below 21° c (70° f) to maintain sow comfort. the use of a farrowing crate or stall will prevent excessive movement of the sow and limit crushing of the newborns. the use of heat lamps or heating pads will provide a source of heat to induce piglets to rest in those warmed areas rather than lying in contact with the sow, thus reducing the risk of the sow crushing or injuring piglets. the use of crush bars will prevent the sow from rapidly becoming recumbent at the onset of nursing episodes and trapping piglets beneath her. 67 piglets suffering from splayleg can be treated by taping the rear legs together to provide stability for the piglet when it attempts to stand. cross-fostering of piglets to other sows should occur within the first day after farrowing. conclusions improving neonatal survivability is an achievable goal for swine producers. prepartum care of the sow is necessary, as are personnel trained in assisting farrowing females. pharmacological induction of parturition and familiarity with the signs associated with the onset of parturition will enable timely assistance for the neonate. a warm, dry environment protected from unintentional trauma by the sow is essential. references 1. vailancourt jp, marsh we, dial gd: perinatal mortality in 48 north american swine herds. swine health prod 1994;2:13. 2. nielsen nc, christensen k, bille n, et al: preweaning mortality in pigs. i. herd investigations. nord vet med 1974;26:137-150. 3. omvedt et, nelson re, edwards rl, et al: influence of heat stress during early, mid and late pregnancy of gilts. j anim sci 1971;32:312-317. 4. coffey mt, seerly rw, martin rj, et al: effect of level, source and duration of feeding of supplemental energy in sow diets on metabolic and hormonal traits related to energy utilization in the baby pig. j anim sci 1982;55:329-336. 5. bilkei-papp g, papp gb: perinatal losses-general condition of sows. iii. experiences obtained with prednisolone treatment. magyar allatorvosok lapja 1994;49:680-683. 6. svendsen j, andreasson b: perinatal mortality in pigs: influence of housing. proc 6th int cong pig vet soc 1980. p. 3. 7. bevier gw, dzuik pj: the effect of number of fetuses and their location in the uterus on the incidence of stillbirth. proc 4th int congr pig vet soc 1976. p. d21. 8. randall gcb: perinatal mortality: some problems of adaptation at birth. adv vet sci comp med 1978;22:53-81. 9. blackwell te: predicting stillbirth problems. compend contin educ pract vet 1988;9:f371-374. 10. zaleski hm, hacker rr: variables related to the progress of parturition and probability of stillbirths in swine. can vet j 1993;34:109-113. 11. wendler a: utilizing a farrowing attendant to achieve 95% survival. comp contin educ pract vet 1988;10:241-246. 12. welp c, jochle w, holz w: induction of parturition in swine with a prostaglandin analog and oxytocin: a trial involving dose of oxytocin and parity. theriogenology 1984;22:509-520. 13. xin h, bundy ds, zhou h: comparison of 250 w versus 175 w radiant heat lamps for swine farrowing operations. iowa state univ swine research report, ames, ia, 1996. p. 129-132. additional reading 1. fangman tj, amass sf: postpartum care of the sow and neonates. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: saunders elsevier; 2007. p. 778-788. 2. roberts sj: veterinary obstetrics and genital diseases-theriogenology. 3rd ed. woodstock, vt: published by the author; 1986. p. 245-259. 3. cutler rs, fahy va, cronin gm: preweaning mortality. in straw be, zimmerman jj, d’allaire s, et al, editors. diseases of swine. 9th ed. ames: blackwell scientific publishing; 2006. p. 997-1006. 68 2010: common problems during pregnancy in the bitch common problems during pregnancy in the bitch kara a. kolster springfield veterinary center, glen allen, va introduction achieving pregnancy through proper breeding or artificial insemination at the appropriate time is the first step in producing a healthy litter of puppies. the next hurdle is keeping the dam and conceptuses healthy through full-term gestation. this review will discuss signs, treatment, and prevention of common complications in canine pregnancy for which breeders and clinicians should be aware. keywords: canine, pregnancy, infectious, herpesvirus, nutrition, eclampsia bacterial infection infection is a potential complication during pregnancy and a common concern of breeders. bacterial infection may ascend through the cervix, spread hematogenously, or through oronasal contact. exposure can occur at the time of breeding or at any point during gestation. bacterial infection with brucella canis is a significant concern in all breeding dogs. infection causes abortion in the female, and epididymitis and orchitis leading to sterility in the male. there is no effective treatment for b. canis in the dog. it is highly contagious and is spread through most body fluids, including semen, vaginal secretions, urine, nasal secretions, and saliva.1 most breeders are aware that b. canis is transmitted through sexual contact, however many do not realize it is also spread through artificial insemination and non-reproductive routes. all breeding stock should be tested regularly for b. canis: females prior to each breeding, and males ideally prior to each breeding or at least twice yearly. positive results on a screening test, such as the rapid card agglutination test (rcat), must be confirmed by a more specific test (agar gel immunodiffusion, polymerase chain reaction, or blood culture) as false positives occur.2 if b. canis is identified in a kennel, the recommended treatment is euthanasia of affected dogs, and monthly testing of all individuals until negative results are achieved in the kennel for three consecutive months.2, 3 antibiotic treatment of affected individuals can lead to improvement in clinical signs and negative serologic test results, but does not effectively eradicate the organism. once infected, a dog is a persistent carrier and can revert to shedding of organisms at any time.3 b. canis is transmissible to humans, particularly immunocompromised individuals, and causes recurrent fever and flu-like signs.1,2 bacterial infection with a variety of other organisms can affect pregnancy in the bitch. the most commonly isolated organisms from the cranial vagina are escherichia coli, streptococcus species, pasteurella species, and staphylococcus species.1 any of these normal flora can cause opportunistic infection. campylobacter and salmonella species have also been implicated in canine fetal loss.1 3 elevated progesterone levels during diestrus and pregnancy maintain closure of the cervix and also cause immune system suppression. bacteria introduced during estrus and breeding should be cleared from the uterus prior to cervical closure. if bacteria remain within the uterus or gain entrance through the cervix during pregnancy, infection can occur due to diminished immune reaction. bacteria may attack the placenta and fetus, or cause systemic disease in the dam which leads to fetal loss. necropsy of aborted or stillborn fetuses may show characteristic lesions depending on the causative organism and is often the best diagnostic tool. culture of placental tissue or fetuses may also identify a bacterial cause. tissue should be refrigerated, not frozen, for best results. signs of bacterial infection during pregnancy can include vaginitis, mucopurulent or hemorrhagic vulvar discharge, lethargy, inappetance, abortion or premature labor. diagnostic workup of the bitch with a suspected intrauterine infection should include thorough physical examination, abdominal ultrasound to evaluate fetal viability and abnormal uterine contents, complete blood count, progesterone level, guarded-swab aerobic vaginal culture, vaginal cytology, and mycoplasma culture if indicated. treatment is initially with broad spectrum antibiotics pending culture and sensitivity results. certain antibiotics, such as fluoroquinolones and tetracyclines, are contraindicated during pregnancy due to potential negative effects on fetuses. 509 treatment success, as measured by the ability of the bitch to carry the litter to term and deliver viable puppies, is dependent upon the stage of gestation when infection is first identified, severity of infection, presence of dead or macerated fetuses in the uterus, premature drop in progesterone levels due to local prostaglandin release, and ability to quickly institute appropriate antibiotic therapy. viral infection viral transmission is typically through oronasal exposure. the most significant virus in canine reproduction is herpesvirus. exposure of a naïve bitch during the last three weeks of gestation will cause stillbirth or neonatal loss. exposure to neonates during the first three weeks of life also results in near 100% fetal mortality. herpesvirus causes mild, if any, clinical signs in otherwise healthy adult dogs. transient upper respiratory signs and vesicles on the penis or vagina may be seen.4 herpesvirus complications can be prevented by exposing the bitch prior to breeding, isolating dam and neonates during the susceptible period, and by providing proper heat sources to neonates. maintaining body temperature at 98-100 degrees f will help prevent viral replication. treatment of neonates infected with herpesvirus is generally unrewarding. some individuals may be saved with temperature regulation, prevention of secondary bacterial infection, and supportive care. however, significant neurologic and renal complications are reported in dogs that have survived neonatal herpesvirus. bitches may be exposed to numerous other viral agents that cause pregnancy complications. the best prevention is maintaining strict biosecurity practices: do not expose pregnant bitches to public areas such as dog shows, isolate pregnant bitches and puppies from other dogs that have been outside the home environment, and maintain clean hands, clothes, and shoes for caretakers and visitors when handling pregnant bitches and puppies. premature labor any process in the bitch that causes prostaglandin release and decrease in serum progesterone can lead to premature labor and delivery of puppies. these causes can include infection, trauma, primary hypoluteodism or other hormonal aberrations. accurate gestational aging is necessary, as frequently the lack of precise ovulation timing makes determining due date difficult and the bitch may actually be in labor with full-term puppies. treatment of pre-term labor should be directed at the primary cause, if known. options include the use of tocolytics, progesterone monitoring and supplementation, and uterine contractility monitoring. inadequate nutrition nutrition of the pregnant bitch is an important factor in producing a healthy litter. current recommendations for feeding the pregnant bitch are to provide increased (28-30%) protein, increased fatty acids, and moderate levels of carbohydrates.5,6 inadequate nutrition during pregnancy can result in the birth of small weak pups and increased neonatal mortality. appropriate nutrient balance should be provided from breeding through weaning, however caloric needs do not increase until the last three weeks of gestation when the fetuses are growing significantly in size. prior to this, organogenesis and development are occurring, which do not require increased caloric intake for the dam. increasing calories too soon can lead to obesity, which has a negative impact on whelping ease and increases the risk of dystocia and cesarean section. changing to a higher protein food during pregnancy may require a decrease in total amount fed to maintain steady caloric intake. caloric requirements during late gestation increase to approximately 150-200% of maintenance levels, depending upon breed size and litter size. more frequent smaller meals (e.g. three to four times daily) are useful when the distending uterus pressures the stomach. supplementation with appropriate levels of omega-3 and omega-6 fatty acids can improve litter health and puppy trainability.7 many other supplements may actually be detrimental to an otherwise balanced diet and are therefore not routinely recommended. 510 trauma injury may occur due to everyday accidents and physical limitations of late pregnancy. increased abdominal size and change in weight distribution can make previously routine activities dangerous for the pregnant bitch. blunt trauma to the abdomen, such as a fall or hit-by-car, can cause placental abruption or uterine rupture. consequences of acute trauma to the dam can also cause prostaglandin release and luteolysis. penetrating abdominal wounds can cause direct trauma to the uterus and fetuses. most other types of physical injuries do not directly affect fetal health. however, potential risk to the fetuses should be considered when treating the bitch with anesthetics, antibiotics, or analgesics. hypocalcemia also known as eclampsia or puerperal tetany in the dog, hypocalcemia most commonly occurs during the first few weeks postpartum but may occur prior to whelping.1 small breed dogs with large litters are at increased risk.8 initial signs of restlessness, stiffness, trembling, behavior changes, and facial pruritis progress to tetany and convulsions within minutes to hours. clinical hypocalcemia should be treated as an emergency. puppies typically must be weaned or supplemented with bottle-feeding to reduce strain on the dam after she is stable. dietary imbalance of calcium and phosphorus during pregnancy predisposes a bitch to develop hypocalcemia. a diet with calcium:phosphorus ratio ranging from 1:1 to 1.2 :1 is reportedly suitable for pregnancy.1,8 additional calcium supplements should not be added to the diet during pregnancy, however they may be helpful during heavy lactation in bitches prone to developing hypocalcemia. conclusions while many canine pregnancies are routine and uneventful, the potential for pregnancy complications in the bitch exists from many causes. many problems can be prevented with proper knowledge and husbandry practices. it is important for breeders and clinicians to be aware of risks, warning signs and treatment strategies when complications occur. references 1. johnson sd, kustritz mvr, olson pns: canine and feline theriogenology. philadelphia: saunders; 2001. 2. hollett rb: update on canine brucellosis. theriogenology 2009;1:287-925. 3. pretzer sd: bacterial and protozoal causes of pregnancy loss in the bitch and queen. theriogenology 2008;70:320-326. 4. verstegen j, dhaliwal g, verstegen-onclin k: canine and feline pregnancy loss due to viral and non-infectious causes: a review. theriogenology 2008;70:304-319. 5. kelley rl: nutritional management of the bitch: pre-breeding to whelping. proc canine reprod breed 2001. p. 14-17. 6. rosmos dr, palmer hj, muiruri kl, et al: influence of a low carbohydrate diet on performance of pregnant and lactating dogs. j nutr 1981;111:678-689. 7. kelley rl, lepine aj: improving puppy trainability through nutrition. proc iams canine pediatr symp 2005. p. 30-34. 8. drobatz kj, casey kk: eclampsia in dogs: 31 cases (1995-1998). j am vet med assoc 2000;217:216-219. 511 512 omniblank: 2010: induction of parturition in swine* induction of parturition in swine* craig a. smith american veterinary medical association, shaumburg, il, usa introduction reduction of preweaning losses, decreasing the number of stillborn piglets and improving piglet survivability and productivity are all realistic goals that can be achieved by the swine industry. improved knowledge of parturition, the ability to control its onset and the ability to influence the duration of farrowing are important steps in improving performance in the farrowing house. keywords: induced parturition, swine, preweaning survival, piglet mortality the onset of parturition the gestation length in swine is typically 114-116 days. variations may be due to the breed of sow and boar involved. sows with large litters may have slightly decreased gestation length.1 the initiation of parturition in the pig is apparently controlled by the fetuses,2 with the presence of at least two live fetuses necessary to initiate the hormonal cascade that results in farrowing. the proposed mechanism functions as follows: at the appropriate time at the end of gestation, the fetuses release adrenocorticotrophic hormone (acth) from the fetal pituitary, which stimulates fetal adrenal hypertrophy and production of glucocorticoids (primarily cortisol) by the fetal adrenals. the increased levels of cortisol stimulate prostaglandin f2alpha (pf2a) release from the uterus. the increased concentrations of pgf2a cause luteolysis and a decrease in progesterone concentrations and promote release of relaxin from the corpora lutea and oxytocin from the posterior pituitary. relaxin acts to soften and relax the cervix, while the absence of progesterone allows the oxytocin and pgf2a to increase myometrial activity and uterine contractions that initiate parturition and result in birth of the piglets. induction of parturition pharmacological control of the onset of parturition is paramount to maximizing sow productivity and profitability of the swine producer by allowing scheduling of farrowings. prostaglandin f2alpha and its analogs will induce the onset of parturition. the use of pgf2a or one of its analogs allows more effective utilization of farrowing facilities and farrowing house personnel by minimizing weekend and night farrowings and, when necessary, scheduling additional personnel to supervise farrowings. improved supervision of farrowing sows should improve piglet survivability. improved synchrony of farrowing allows for better cross-fostering of piglets. improved uniformity of piglet age ultimately results in better piglet performance in the farrowing house that ideally is carried over to the nursery. lastly, induction of parturition with prostaglandin reduces the occurrence of the mastitis-metritis-agalactia (mma) syndrome in herds with high incidence.3 parturition-inducing compounds prostaglandin prostaglandin f2alpha is the only prostaglandin approved for use in swine in the united states. prostaglandin analogs are as efficacious as pgf2a but tend to have fewer and less severe * originally published by the society for theriogenology and the american college of theriogenologists as publication p-2 (2/91) in the theriogenology handbook (revised 2010). 61 side effects. these side effects are usually quite mild and short-lived, and may include restlessness, erythema, increased respiratory rates, salivation, defecation and urination. vomiting is a rare occurrence. if side effects appear they will occur within ten minutes of administration and dissipate by 60 minutes postinjection.4 the dose of pgf2a is 10 mg, i.m. the dose of cloprostenol is 175 ug, i.m., but doses from 125 to 250 ug have been used.5 a dose of 5 mg pgf2a or 87.5 ug cloprostenol administered in the vulvar mucosa can reliably induce parturition and the side effects are less severe than for i.m. administration of the higher dose.6 administration of a prostaglandin product will result in females farrowing the first piglet 24 to 32 hours after treatment. hence, most treatments are administered in the morning so the bulk of farrowings will be accomplished during normal hours of the following workday. a shorter treatment to farrowing interval is observed in animals that are close to spontaneous farrowing.7 prostaglandin and progesterone prostaglandins have been used in conjunction with progesterone and progesterone-like compounds to improve the synchrony of the onset of farrowing.8 the use of progestational compounds administered daily on days 109 to 112 during late pregnancy prevented spontaneous farrowings. the subsequent use of prostaglandin at the time of withdrawal of the progesterone compound provided well-synchronized farrowings with no apparent adverse effects on the piglets. prostaglandin and oxytocin improved synchronization and a decreased interval from treatment to parturition can be obtained by using oxytocin. five to 30 units of oxytocin administered 20 to 24 hours after prostaglandin treatment will result in the onset of farrowing within three to six hours in approximately 50% of the females.9 twenty to 30 units of oxytocin will usually result in shorter intervals to birth of the first piglet but may also result in a higher incidence of dystocia due to stimulation of strong, asynchronous uterine contractions. the intravenous injection of two to three units of oxytocin intensifies normal myometrial activity within 20 seconds in sows, while dosages of ten units result in uterine spasms lasting ten to 15 minutes.7 dosages of oxytocin greater than ten units may also interfere with the sow’s ability to release milk early in the postpartum period. therefore, the dose of oxytocin to assist parturition in swine should be five to ten units administered i.m. to the sow no sooner than 20 hours after induction with pgf2a.5 dexamethasone administration of dexamethasone (75 mg/sow/day s.c. for three days) on days 101 to 103 of gestation resulted in a shortened gestation, 112 days versus 114 days.10 the need for repeated treatment, inability to accurately and efficiently control the onset of farrowing and decreased piglet birthweights and viability make this an unacceptable method of induction. furthermore, the addition of dexamethasone to pgf2a-induced farrowing does not suggest a beneficial role for the use of corticosteroids in farrowing induction.11 development of new compounds and regimens the increased use of drugs for parturition induction and synchronous farrowing will depend in large part on governmental approval of compounds. the prostaglandin analogs are prime examples of efficacious compounds that will be increasingly utilized in the future as they become legally available. among the other induction agents is epostane, a competitive inhibitor of the 3-beta hydroxysteroid dehydrogenase enzyme system. this system is responsible for the conversion of metabolic precursors to progesterone. administration of epostane at oral dosages 62 of 5 to 10 mg/kg bodyweight or s.c. injections of 5 mg/kg bodyweight results in a decrease in progesterone concentrations and the initiation of parturition within approximately 32 hours.12 epostane is currently not approved for use in the united states. agents that improve the synchrony of the onset of parturition may also be commercially utilized. relaxin is effective at improving the synchrony of farrowing following prostaglandin induction with more sows farrowing within a 12-hour time span.13 the administration of xylazine (2 mg/kg bodyweight i.m.) 20 hours after prostaglandin treatment resulted in the birth of the first piglet 1.5 hours after xylazine.14 some of the treated sows showed signs of sedation following xylazine administration. neither relaxin nor xylazine are approved for such use in swine. tocolytic compounds the use of tocolytic compounds may enhance our abilities to improve the survivability of neonatal pigs. clenbuterol is a beta 2-sympathomimetic compound shown to have long-lasting uterine relaxant effects.15 clenbuterol stimulates adenyl cyclase in cell membranes and outflow of intracellular calcium from the myometrial cells, making them unresponsive to oxytocin. this results in an interruption of the parturition process, which can occur within five minutes after administration. although the greatest benefits may be the use of cloprostenol during stage i parturition to prevent the onset of stage ii and farrowing during night-time hours, it can interrupt stage ii labor even after the birth of several piglets. resumption of normal parturition occurs in a few hours when the drug’s effects wear off, with no apparent adverse effects on the health or vitality of the piglets. in contrast, injection of pgf2a followed by injection of propanolol, which blocks beta1and beta 2-adrenergic receptors, did not influence time of onset of parturition.16 disadvantages the results achievable with induced parturition are directly attributable to the level of care provided during the peripartum period. the use of prostaglandin to induce farrowing may actually increase the number of piglets which expire at or shortly after birth by causing the premature birth of some piglets or the birth of smaller piglets that are more susceptible to the extra-uterine environment.17 likewise, stillbirth rates will not be improved unless trained personnel are present to assist with the delivery and care of neonates. conclusions the routine use of parturition induction is a valuable tool in modern swine production. correct implementation can improve efficiency of labor and facility utilization, and decrease neonatal death loss, thus increasing profitability. the routine use of parturition induction by itself will not improve piglet survivability without a commitment by the swine producer to: 1) provide personnel to supervise and assist at the time of farrowing; 2) provide a warm, dry environment for the newborn piglet; 3) ensure colostral intake by the newborn piglet; and 4) utilize management strategies (cross-fostering of piglets, all in-all out batch farrowings) made possible by the use of parturition induction. 63 references 1. bevier gw, dziuk pj: the effect of number of fetuses and their location in the uterus on the incidence of stillbirth. proc 4th int pig vet soc congr, 1976. p. d21. 2. first nl, bosc mj: proposed mechanisms controlling parturition and the induction of parturition in swine. j an sci 1979;48:1407-1421. 3. cerne f, jochle w: clinical evaluations of a new prostaglandin analog in pigs. i. control of parturition and of the mma syndrome. theriogenology 1981;16:459-467. 4. dial gd: clinical applications of prostaglandins in swine. j am vet med assoc 1984;185:1523-1528. 5. pressing al: pharmacologic control of swine reproduction. vet clin north am food anim pract 1992;8:707-724. 6. friendship rm, templeton cl, deckert ae: an evaluation of vulvomucosal injections of prostaglandins for induction of parturition in swine. can vet j 1990;31:433-436. 7. dial gd, almond gw, hilley hd, et al: oxytocin precipitation of prostaglandininduced farrowing in swine: determination of the optimal dose of oxytocin and optimal interval between prostaglandin f2a and oxytocin. am j vet res 1987;48:966-970. 8. gooneratne a, hartmann pe, mccauley i, et al: control of parturition in the sow using progesterone and prostaglandin. aust j biol sci 1979;32:587-595. 9. welp c, jochle w, holtz w: induction of parturition in swine with a prostaglandin analog and oxytocin: a trial involving dose of oxytocin and parity. theriogenology 1984;22:509-520. 10. north sa, hauer er, fist nl: induction of parturition on swine and rabbits with the corticosteroid dexamethasone. j anim sci 1973;36:1170-1174. 11. cessar g, kirkwood rn, friendship r, et al: sow and litter performance following farrowing induction with prostaglandin: effect of adjunct treatments with dexamethasone or oxytocin. j swine health prod 2005;13:81-85. 12. martin pa, hammitt dg, strohbehn rs, et al: induction of parturition in swine and epostane, a competitive inhibitor of 3beta-hydroxysteroid dehydrogenase. j anim sci 1987;64:497-506. 13. butler wr, boyd rd: relaxin enhances synchronization of parturition induced with prostaglandin f2alpha in swine. biol reprod 1983;28:1061-1065. 14. ko jch, evans le, hsu wh, et al: farrowing induction with cloprostenol-xylazine combination. theriogenology 1989;31:795-800. 15. zeroin k, kondig h: the control of myometrial function during parturition with a beta-mimetic compound, planipart. theriogenology 1980;14:21-35. 16. kirkwood rn, thacker pa: effect of propanolol on the onset and duration of parturition in sows. can vet j 1995;36:238-239. 17. young im, harvey mja: routine induction of farrowing with dinoprost in a commercial breeding unit over a year. vet rec 1984;115:539-541. additional reading 1. cowart rp: parturition and dystocia in swine. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: saunders elsevier; 2007. p. 778-784. 2. roberts sj: veterinary obstetrics and genital diseases-theriogenology. 3rd ed. woodstock, vt: published by the author; 1986. p. 245-259. 3. almond gw, flowers wl, batista l, et al: diseases of the reproductive system. in straw be, zimmerman jj, d’allaire s, et al, editors. diseases of swine. 9th ed. ames: blackwell publishing; 2006. p. 113-147. 64 1 contact réjean lefebvre rejean.lefebvre@umontreal.ca © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2025, 17, 10988, http://dx.doi.org/10.58292/ct.v17.10988 research report prepartum vaginal microbiota and postpartum uterine microbiota in cows ali bazzazan,a marcio costa,b mariela segura,c réjean lefebvrea adepartment of clinical sciences, faculty of veterinary medicine, university of montréal, saint-hyacinthe, quebec, canada bdepartment of biomedical sciences, faculty of veterinary medicine, university of montréal, saint-hyacinthe, quebec, canada cresearch group on infectious diseases in production animals, and swine and poultry infectious diseases research centre, faculty of veterinary medicine, university of montréal, saint-hyacinthe, quebec, canada abstract postpartum uterus clears bacteria within 6 weeks; however, up to 40% of dairy cows cannot eliminate uterine infection. cultureindependent approaches based on the analysis of the 16s rrna gene indentified reduced richness and diversity in the reproductive tract of cows with endometritis. goal was to characterize the vaginal microbiota before calving and the uterine microbiota after calving in cows that had or not developed endometritis. we compared the results of dna sequencing with conventional culture methods in uterine samples collected 21 days after calving. vaginal samples (via cytobrush) from multiparous cows (n = 61) were obtained 1 week before calving (-1w) and uterine samples 1 week (+1w), 3 weeks (+3w), and 5 weeks (+5w) after calving. samples of healthy cows (n = 11) and cows with endometritis (n = 11) were selected for dna extraction and 16s rrna sequencing. richness and diversity measures in the uterine microbiota were not different between healthy and diseased groups (chao p = 0.17, simpson p = 0.29) at -1w, +1w and +5w. uterine microbiota composition differed (p = 0.03) between groups only at 3 weeks after calving. diseased cows had a lower relative abundance of firmicutes and bacteroidetes and a higher abundance of actinobacteria than healthy cows. bacteria that grew in culture were often represented within the most abundant bacterial genera in the dna sequencing 3 weeks after calving. keywords: microbiota, dairy cows, vagina, uterus introduction approximately 95% of cows have some degree of uterine contamination with bacteria and inflammation after calving, regardless of signs of disease.1,2 loss of anatomical barriers and the negative pressure created uterine contraction and relaxation enhance the vacuum effect and bacterial ascent to the uterus, allowing rapid colonization within minutes.3,4 there is also evidence that bacteria could reach the uterus through blood circulation.5 gram-negative bacteria dominate during the first week after calving, gradually replaced by gram-positive organisms by day 15 postpartum, when 78% of cows still have bacteria in the uterus.6 during normal uterine involution, the uterus clears bacteria at week 6 after calving;7 however, up to 40% of dairy cows cannot eliminate infection.8 persistence of pathogenic bacteria in the uterus is associated with delayed uterine involution, postpartum uterine diseases (puds), and reduced reproductive efficiency.9-11 based on culture-dependent studies, most common bacteria involved in puds are trueperella pyogenes (t. pyogenes), escherichia coli (e. coli), fusobacterium necrophorum, prevotella melaninogenica, and bacteroides spp.12,13 culture-independent approaches based on the analysis of 16s rrna gene (the most conserved ribosomal rna among bacteria and has the most variability between species) have indentified that dairy cows with puds had distinct microbial profiles and  reduced richness and diversity compared to healthy cows.14-18 nonconventional pathogens have also been identified by  dna sequencing, such as species of bacteroides and  helcococcus. in contrast, other bacteria normally associated  with diseases, such as e. coli, mycoplasma and ureaplasma, were abundant in healthy postpartum cows.19 it remains  unclear whether changes in the normal composition of the uterine microbiota (dysbiosis) are associated with puds.20 mailto:rejean.lefebvre@umontreal.ca http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.10988 2 citation line: clinical theriogenology 2025, 17, 10988, http://dx.doi.org/10.58292/ct.v17.10988 most studies investigating puds in dairy cows have focused on the early postpartum period before the completion of uterine involution and the reestablishment of a complete partitioning of the reproductive tract.21 vaginal microbiota is a potential source of uterine contamination after calving and needs to be investigated and understood.22 we hypothesized that prepartum vaginal microbiota and the uterine microbiota in postpartum cows differ between healthy and diseased dairy cows. objective was to use dna sequencing to characterize bacterial communities in the genital tract of healthy dairy cows during the peripartum period and make associations with clinical endometritis. we compared the uterine bacteria obtained by conventional culture methods with the microbiota profile of dna sequencing from samples collected 3 weeks after calving. materials and methods animals and management this research was conducted in compliance with the experimental practices and standards approved by the animal care committee of the university of montréal (#211-03) in compliance with the canadian council of animal care guidelines. three commercial dairy herds in the same region of quebec (canada) were recruited, and multiparous cows (n = 61) were included in the study between june 2016 and february 2017. reproductive and health data were compiled in a databank using health record management software (dsahr, sainthyacinthe, quebec, canada j2s 3a5). rolling herd average milk production for the 3 herds was 9,000 kg. all farms had a tied-stall barn system. cows were milked twice daily and fed a total mixed ration formulated mainly with corn and hay silage to meet the dietary requirements for the various stages of lactation (nrc, 2011). farms were visited weekly by the same veterinarians and cows were vaccinated twice (days 40 and 26 before parturition) with 2 ml intramuscular e. coli (j-vac, merial inc., athens, ga, usa) vaccine and once (days 15-40 after calving) with 2 ml intramuscular bovine viral diarrhea virus, types 1 and 2, infectious bovine rhinotracheitis, pi-3, and bovine respiratory syncytial virus (bovi-shield gold fptm 5 l5, zoetis, parsippany, new jersey 07054, usa) vaccine, and injected with 5 ml selenium (mu-se, intervet canada corp., kirkland, quebec, canada h9h 4m7) 60 days before calving. sampling and experimental design based on the predicted date of calving (dsahr software), cows were examined 1 week before calving (-1w), then 1 (+1w), 3 (+3w), and 5 weeks (+5w) after calving (figure 1). examinations included an assessment of lameness, cyclicity (presence of a corpus luteum) via transrectal and ultrasonographic examinations, body condition, and milk somatic cell count. blood samples were collected for hematologic profile; visual vaginal discharge23 and cervical appearance24 were also performed. before calving, transrectal examination was performed to confirm pregnancy. it involved the assessment of placentomes, fetus viability, and uterine artery (fremitus). in addition, a vaginal (fornix) cytobrush sample was collected 1 week before calving and endometrial cytobrush samples were collected at 1, 3, and 5 weeks after calving. after cleaning cow’s perineal area with soap and water, 70% isopropyl alcohol was sprayed and dried using paper towels. visual cervical assessment was via vaginal speculum examination before a sterile cytobrush rod (covered with 2 sterile sanitary sheaths) was introduced into the vagina and guided through the cervix per rectum.25 once the tip of the rod reached cervical opening and uterine body, first and second sanitary sheaths were pulled back, respectively, and the cytobrush was pushed out of the rod and rotated against the dorsal wall of the uterine body with gentle pressure of the index finger through the rectum. cytobrush was retracted into the rod and each sanitary sheath before removed from the cervix and the vagina, respectively. once outside the genital tract, the cytobrush was gently rolled onto a sterilized microscope slide. the cytobrush was then cut with sterile scissors, placed in a sterile 2 ml cryovial, and stored at −80°c within 5 minutes. for the sampling at +3w, an additional uterine cytobrush sample was collected and transferred into a tube containing an anaerobic transport medium (bbl culture swab, bd, mississauga, on, canada), placed on ice and processed in the bacteriology laboratory within 2 hours after collection. cytology slides were stained using the may-grunwald-giemsa stain, 300 cells (neutrophils and endometrial cells) were counted per slide in multiple fields by one person and the number of polymorphonuclear neutrophils  (pmns) was assessed. cows were not enrolled in a systematic synchronization protocol, and reproductive data from cows were collected for at least 300 days after calving. case definition no antibiotic was used before and during the sampling period. from the 61 cows, diseased cows (n = 11) were identified figure 1. timeline of sampling and genital examination of prepartum and postpartum dairy cows (n = 22) from 3 dairy herds in quebec (fx; fornix, ut; uterine mucus) http://dx.doi.org/10.58292/ct.v17.10988 citation line: clinical theriogenology 2025, 17, 10988, http://dx.doi.org/10.58292/ct.v17.10988 3 based on 2 criteria at 5 weeks after calving: 1. positive clinical endometritis; cows with purulent vaginal discharge (pvd) of grade ≥ 223 and 2. positive subclinical endometritis; cows with > 5% polymorphonuclear neutrophils (pmns) on uterine endometrial cytology (number of pmns/number of total cells.26 control animals (n = 11) were characterized by the absence of all criteria (pvd < 2 and pmns < 5%). dna extraction, 16s rrna gene sequencing cytobrushes were stored at −80 °c after collection until dna extraction. frozen samples were dipped in 1 ml of phosphate-buffered saline (pbs) and dna was extracted from the suspension using the powersoil dna extraction kit (qiagen) according to the manufacturer’s protocol. the v4 region of the 16s rrna gene was amplified by polymerase chain reaction (pcr) using the forward 515f (gtgccagcmgccgcggtaa) and reverse 806r (ggactachvgggtwtctaat) primers and the following conditions: denaturing for 3 minutes at 94°c, followed by 35 cycles of 45 seconds at 94°c, 60 seconds at 50°c, and 90 seconds at 72°c, with a final elongation step of 72°c for 10 minutes. the pcr products were sequenced in an illumina miseq platform using reagents for 2 x 250 cycles at the mcgill university and génome québec innovation centre. the mothur software was used to perform the bioinformatic analysis.27 contigs assembly was made from the original fastq files, excluding sequences longer than 300 bp, containing ambiguities, and having polymers longer than 8 bp. sequences were aligned using the silva 16s rrna reference database and clustered at 97% similarity. taxonomic classification was obtained using the ribosomal database project. richness (total number of species) was estimated by the chao1 index, and diversity was measured by the simpson index (number of species accounting for their evenness). for beta diversity, the bray-curtis dissimilarity index was used to compare community composition among samples. uterine bacterial culture and identification uterine cytobrush samples were collected at +3w for routine bacterial culture (aerobic and anaerobic) using standard methods for bacteriological testing (api system, biomérieux, marcy l’etoile, france). cytobrush samples were stored in a culture tube (bbl culture swab, bd, mississauga, on, canada), placed on ice, and transported to the diagnostic laboratory of the faculty of veterinary medicine within 2 hours. for microbiological analysis, the solution was plated onto sheep blood agar with a sterile disposable plastic eye (soy agar with 5% sheep blood, becton, dickinson and co., sparks, md, usa). plates were incubated for 48 hours at 35°c under aerobic conditions and then examined. when growth was observed, the number of colonies was graded as rare, 0 (1 colony), few, +1 (between 2-5 colonies), +2 (> 5 colonies), and +3 (> 5 colonies after dilution (1 in 2 dilution) whereas colony types were identified based on morphology, pigmentation, and hemolytic patterns. beta hemolytic, catalase-negative minuscule colonies demonstrating coliform gram-positive rods were identified as t. pyogenes. they were isolated using the standard procedure used at the diagnostic laboratory of the faculty of veterinary medicine (pon-bac-019). the samples were plated on brucella agar containing neomycin (100 mg/ml) and incubated anaerobically at 35°c for 5 days for culture of other bacterial species. when gram-negative rods were observed, colonies were examined using the api 20 a gallery system to identify fusobacterium necrophorum and prevotella melaninogenica. for isolation of e. coli, cytobrush samples were plated on blood agar and macconkey agar (oxoid inc., ottawa, on, canada) at 37°c. at the reference laboratory for e. coli (ecl; faculty of veterinary medicine, university of montréal), 5 typical lactose-positive e. coli colonies from the macconkey agar plates were streaked with blood agar for isolation and further identification. isolates were submitted to 3 biochemical tests (indole spot, simon’s citrate and motility) for confirmation of e. coli. isolates of e. coli was stored in tryptic soy broth containing 30% glycerol at −80 °c (becton, dickinson and co., sparks, maryland, usa). collection and analysis of blood blood samples were collected aseptically by coccygeal venipuncture from 3 cows into collection tubes containing edta for hematology, including red blood cell count, hemoglobin concentration, packed cell volume, erythrocyte sedimentation rate, corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total leukocytic count, and differential leukocytic count. data analyses downstream analyses were carried out in rstudio (version 3.6.3; r core team, vienna, austria) using the ggplot2, vegan, factominer, dplyr, tidyverse, and phyloseq packages unless otherwise stated. uterine and vaginal alpha diversity metrics were examined for normal distribution by proc univariate in rstudio. repeated measures analysis of variance was performed on all alpha diversity indices, with fixed effects of the day, status, and their interaction with the cows. differences in uterine bacterial composition were investigated according to disease presence and days after calving, and their outcomes were evaluated using a nonparametric multivariate analysis of variance (permanova) accounting for repeated measures. linear discriminant analysis effect size (lefse) was used to describe the statistical significance and biological relevance among healthy and diseased cows at the genus level. the lefse was performed using the online galaxy interface with uterine health status as the main class, dim at sampling as the subclass, and the cow as the subject, using an alpha of 0.05 and an effect size threshold of 3.5. significance level was set for all statistical tests as p value < 0.05.28 results descriptive statistics the study population consisted of 85 cows, but an initial exclusion resulted in the removal of cows due to culling (n = 5), use of antimicrobials (n = 4), metabolic diseases (n = 5), and missing data (n = 10). a total of 61 cows ended the follow-up period without systemic illness (no dystocia, fetal membranes retention, and mastitis). dna sequencing of vaginal (prepartum) and uterine (postpartum) samples from 22 cows classified as healthy (n = 11; parity 1.7 ± 0.5 [mean ± sd], and bcs 2.8 ± 0.2) and diseased cows (n = 11; parity 1.9 ± 0.5 [mean ± sd], and bcs 2.7 ± 0.3) was performed. one healthy (9%) and 5 diseased (45%) cows had cervicitis grade 2 at 5 weeks after calving and no significant hematological changes and somatic cell counts and cyclicity among cows were measured. http://dx.doi.org/10.58292/ct.v17.10988 4 citation line: clinical theriogenology 2025, 17, 10988, http://dx.doi.org/10.58292/ct.v17.10988 microbial community analyses a total of 9,480,588 paired-end reads were obtained, of which, 7,820,890 passed all quality control after bioinformatic analysis. results from the alpha diversity of microbiota of healthy and diseased cows from 1 week before calving to 5 weeks after calving are provided (figure 2). overall and at each sampling time, alpha diversity of the uterine microbiota was not different (chao’s p value = 0.17, simpson’s p value = 0.29) between healthy and diseased groups. the principal coordinate analysis addressing the microbial composition (beta diversity) revealed distinct microbiota profiles (p = 0.03) between healthy and diseased groups at 3 weeks after calving (figure 3). no difference (p = 0.35) was observed for the other sampling times. taxonomic composition the relative abundance of the most abundant phyla (figure 4a) and genera (figure 4b) are illustrated. according to taxonomical annotation, the most common bacterial phyla in the uterine microbiota in both groups of cows were bacteroidetes, firmicutes, actinobacteria, fusobacteria, proteobacteria, and tenericutes (figure 4). during the postpartum period, cows with endometritis had a lesser relative abundance of firmicutes figure 2. box plot of the distribution of alpha diversity indices of healthy and diseased cows from 1 week before calving to 5 weeks after calving. chao richness estimator (a) and the simpson diversity index (b) of the uterine microbiota postpartum (n = 22). healthy (red, n = 11) and diseased (blue, n = 11) cows were identified retrospectively on the vaginal discharge and the number of neutrophils on endometrial cytobrush. the bar inside the box marks the median. alpha diversity for bacterial genera and phyla were similar between healthy and diseased cows. a repeated measures analysis was performed on all alpha diversity, with fixed effects of the day, status, and their interaction with the cows. bars represent sd (p ≤ 0.05). healthy diseased healthy diseased a b figure 3. beta diversity (principal coordinate analysis (bray–curtis) for bacteria phyla (a) and genera (b) at three weeks after calving a b http://dx.doi.org/10.58292/ct.v17.10988 citation line: clinical theriogenology 2025, 17, 10988, http://dx.doi.org/10.58292/ct.v17.10988 5 and bacteroidetes and a higher abundance of actinobacteria than healthy cows. the number of unclassified bacteria was < 2% in all samplings. as per taxonomic assignment the dominant uterine bacterial genera were bacteroides, trueperella, porphyromonas, fusobacterium, escherichia, streptococcus, and clostridium sensu stricto (figure 5). at the genus level, diseased cows had a higher relative abundance of bacteroides, trueperella, fusobacterium,  and peptoniphilus than healthy cows at 3 weeks after calving. diseased cows also had a higher relative abundance (p = 0.03) of trueperella from 1 week before to 5 weeks after calving than healthy cows (figure 6). linear discriminant analysis effect size diseased cows had discriminately higher abundances (lda sores > 3.6) of helcococcus, peptoniphilus, and trueperella than healthy cows at 3 weeks after calving (figure 6). no significant differences between healthy and diseased cows were observed at the other samplings. bacterial culture gram-positive and gram-negative bacteria were cultured from the cytobrushes of healthy and diseased cows 3 weeks after calving. most of the diseased cows had t. pyogenes in large amounts (9 out of 11, more than 5 colonies) and small amounts of e. coli (3 out of 11, 1 colony), enterococcus (3 out of 11, 1 colony), and streptococcus uberis (2 out of 11, 1 colony). among healthy cows, only 4 out of 11 had a few colonies of staphylococcus spp. (1 colony), corynebacterium (1colony), and bacillus spp. (1 colony). discussion objective of this study was to use dna sequencing to investigate bacterial dynamics in prepartum vagina and in postpartum uterus of cows that remained healthy with those that developed endometritis during the transition period. our results revealed that cows developing endometritis had distinct profiles of the uterine microbiota 3 weeks after calving compared to cows that remained healthy. this suggested that figure 4a. color-coded bar plot showing the relative abundance of predominant bacteria at the phylum levels. relative abundance of the most dominant bacterial phyla in dairy cows (n = 22) in samples collected 1 week before calving and 1 week, 3 weeks, and5 weeks after calving. only the seven most common phyla are represented (98% of the reads). based on their uterine health status in 5th week postpartum, cows were retrospectively selected and classified as healthy and diseased groups. figure 4b. relative abundance of predominant bacteria at the genus level. samples were collected one week before and one, three and five weeks after calving. only the 14 most common genera are represented. based on their uterine health status in the 5th week postpartum, cows were retrospectively selected and classified as healthy and diseased groups. http://dx.doi.org/10.58292/ct.v17.10988 6 citation line: clinical theriogenology 2025, 17, 10988, http://dx.doi.org/10.58292/ct.v17.10988 the uterine microbiota goes through early changes before clinical signs of the disease that could be used to predict cows at higher risk by using key bacterial markers. our data also confirmed that known uterine pathogens are associated with clinical endometritis. similar results were observed29 in the phylum and genera levels in cows with clinical and subclinical endometritis compared to healthy cows in equivalent times (days 10, 21, and 35 postpartum). in the present study, t. pyogenes was the most abundant bacteria in 9 of the 11 diseased cows by culture-dependent technique. other studies observed that t. pyogenes was present in the uterus of most postpartum cows with endometritis using culture-independent methods30 and culture-dependent methods.31 our results from sequencing of the 16s rrna gene supported that cows with endometritis had an increased relative abundance of bacteroidetes and actinobacteria at the phylum level, as well as bacteroides  and  trueperella  spp. at the genus level. interestingly, bacteria that grew in culture often appeared within the most abundant bacteria in the16s rrna gene sequencing. the culture-dependent results have identified several genera (e. coli, enterococcus, streptococcus uberis, staphylococcus spp., corynebacterium, and bacillus spp.) in a very small number (1 colony). these bacteria are among the most common isolated intrauterine bacteria and have been described as potential or opportunistic pathogens18,32,33 streptococci and staphylococci were isolated in postpartum cows with a reduced risk of endometritis.12 postpartum dairy cows with intrauterine a-hemolytic streptococci had improved reproductive performance.34 these bacteria may interact with recognized pathogenic members of the uterine microbiota to favor a healthy or diseased uterine cavity. in the present study, romboutsia and other bacteria unclassified at the genus level were associated with a healthy uterine environment, suggesting that further studies searching for the markers capable of predicting the occurrence of diseases are justified. in addition, truperella spp., peptoniphilus and helcococcus were significantly higher (lda sores > 3.5) in the disease group compared to the healthy group at 3 weeks after calving (figure 6). intrauterine infusion of t. pyogenes in holstein heifers has caused clinical endometritis.35 many factors influence bacterial pathogenicity, including bacterial load, the presence of virulence factors, and positive interactions among species.36 in fact, t. pyogenes has a synergic interaction with f. necrophorum and p. melaninogenica to support their growth and colonization of the uterine cavity around 3 weeks postpartum.36 as helcococus and peptoniphilis belong to the fusobacterium cooccurrence group, they may act synergistically with t. pyogenes and facilitate the uterine dysbiosis observed 3 weeks after calving and increase the risk of clinical endometritis. this synergic interaction between trueperella and the gram-negative anaerobes like fusobacterium has been suggested as a mechanism to overcome uterine defence, culminating with endometritis.9,33,36,37 a strong co-occurrence between obligate anaerobes like peptoniphilus spp. in postpartum and more specifically, around 35 days after calving was also observed.29 in addition to the bacterial load and virulence factors, the sequence at which bacteria grow in the uterus may affect the risk of puds. however, there was no association between t. pyogenes and f. necrophorum in cows with clinical endometritis at 5 weeks after calving38; therefore, larger studies are needed. figure 5. relative abundance of the most dominant bacterial genera in healthy and diseased postpartum dairy cows (n = 22) in samples collected 1 week before, and 1 week after, 3 weeks, and 5 weeks after calving. cows were retrospectively selected based on their uterine health status in the 5th week postpartum and classified as healthy (n = 11), and diseased cows with clinical and subclinical endometritis (n = 11; > 50% purulent vaginal discharge and > 5% endometrial pmn). no differences (p > 0.20) in relative abundance in bacteria genera between healthy and diseased cows. http://dx.doi.org/10.58292/ct.v17.10988 citation line: clinical theriogenology 2025, 17, 10988, http://dx.doi.org/10.58292/ct.v17.10988 7 e. coli is detected in the uterus within a few days of calving before the invasion of other bacteria and is more associated with metritis in the early postpartum than endometritis. using culture-dependent and pcr-based methods, e. coli was isolated between 1 and 3 days after calving.7,14 in the present study, healthy cows had a higher relative abundance of escherichia/shigella than diseased cows 1 week after calving. likewise, rare e. coli in the uterus of dairy cows with metritis was identified by metagenomic sequencing analysis.4 in other studies, e. coli was identified more frequently in cows with metritis than in healthy cows.38 the lack of knowledge on the interaction between e. coli and other bacteria like t. pyogenes and f. necrophorum and the diversity of e. coli strains (virulent factors) may explain the conflicting results. in the present study, virulent factors of e. coli were not assessed. based on the rationale that the ascending colonization of the uterus from the vagina is the most evident pathway for puds, the investigation of the vaginal microbiota before calving may improve our understanding of the sequential events leading to clinical endometritis. frimicutes, bacteroidetes, proteobacteria, and actinobacteria were isoalated in the vagina of cows during the postpartum period.39–42 in the present study, the dominant bacterial genera in the vagina were bacteroides, trueperella, porphyromonas, fusobacterium, escherichia/shigella, streptococcus, and clostridium sensu stricto. vaginal microbiota in dairy cows was associated with the occurrence of reproductive tract diseases.43 however, an important point of the present results is that trueperella and fusobacterium were present in the vagina 1 week before calving, sustaining the importance of these 2 species in the ascending colonization of the uterus after calving and their role in the etiology of endometritis in dairy cows in the postpartum period. these results are in line with the study14 that identified fusobacteria in the vagina of dairy cows 7 days before calving. genital microbiome may vary among species and even individuals of the same species,4 with nutrition, farm management19 and phase of estrous cycle44 influencing the microbiota composition.45,46 nevertheless, breeds under various management can present similarities in their vaginal microbiota41 and no difference between sampling techniques or phase of estrous cyle were observed.47 the 3 farms of the present study were localized close to each other and had similar herd health and feeding management reducing data variation since the geographic origin may be a significant factor of variation of the microbiome.48 all animals were of the same breed (holstein) and had similar body conditions at sampling time. limitations of the present study included small sample size, short sampling interval before calving and short interval of sampling (9 months) during the year. nevertheless, trueperella spp. in the vagina of cows before calving, with its increased relative abundance in the uterus at 3 weeks after calving, suggests that trueperella could be used as a predictor of postpartum clinical endometritis in dairy cows and could justify larger prospective studies to test the ability of this marker to predict endometritis in a broader population and eventually be used as an accurate and rapid test for prognosis of puds. conclusion the microbiota composition of the uterine microbiome was significantly different between healthy and diseased cows before clinical signs of endometritis (3 weeks postpartum). in cows with clinical endometritis, t. pyogenes, peptoniphilus, and helcococcus were discriminately in higher abundance than in healthy cows. t. pyogenes in the vagina of cows before calving and its increased relative abundance in the uterus 3 weeks after calving suggested that bacterial markers before calving could also be used as predictors of postpartum clinical endometritis in dairy cows. larger prospective studies to test the ability of these markers to predict endometritis should be performed. funding theriogenology fund of the faculty of veterinary medicine. conflict of interest none to report. figure 6. linear discriminant analysis (lda) effect size plot showing the differences in uterine microbiota among cows diagnosed in the 5 weeks after calving as healthy (n = 11), and diseased cows (n = 11; > 50% purulent vaginal discharge and > 5% endometrial pmn). histograms show the lda effect size computed for features at the genera levels. enriched features for healthy cows are indicated with positive lda scores (green), and enriched features in diseased cows are indicated with negative lda scores (red). only features with p > 0.05 and an effect size cut-off of 3.5 are plotted. healthydiseased http://dx.doi.org/10.58292/ct.v17.10988 8 citation line: clinical theriogenology 2025, 17, 10988, http://dx.doi.org/10.58292/ct.v17.10988 authors’ contribution ab collected data, analysed and interpreted data, and drafted the mansucript; 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sci rep 2022;12:14790. doi: 10.1038/s41598-022-18971-6 http://dx.doi.org/10.58292/ct.v17.10988 https://doi.org/10.1016/j.theriogenology.2020.01.040 https://doi.org/10.3168/jds.2017-13135. https://doi.org/10.1016/j.theriogenology.2014.08.018 https://doi.org/10.1016/j.theriogenology.2014.08.018 https://doi.org/10.3390/ani13010082 https://doi.org/10.1016/j.theriogenology.2015.09.043 https://doi.org/10.3168/jds.2018-15595 https://doi.org/10.1016/j.theriogenology.2017.08.010 https://doi.org/10.1016/j.theriogenology.2020.10.018 https://doi.org/10.3389/fmicb.2018.02691 https://doi.org/10.1039/c7ra10553c https://doi.org/10.1371/journal.pone.0143294 https://doi.org/10.1371/journal.pone.0143294 https://doi.org/10.1007/s42770-019-00120-3 https://doi.org/10.1016/j.rvsc.2020.03.027 https://doi.org/10.4238/2015.june.12.4 https://doi.org/10.4238/2015.june.12.4 https://doi.org/10.3389/fmicb.2024.​1419914 https://doi.org/10.3389/fmicb.2024.​1419914 https://doi.org/10.3389/fanim.2023.1111636 https://doi.org/10.3390/ani12040460 https://doi.org/10.1016/j.theriogenology.​2023.11.004 https://doi.org/10.1016/j.theriogenology.​2023.11.004 https://doi.org/10.1038/s41598-022-18971-6 2011: effect of body condition at initiation of synchronization on estrus expression, pregnancy rates to ai and breeding season in beef cows effect of body condition at initiation of synchronization on estrus expression, pregnancy rates to ai and breeding season in beef cows ramanathan kasimanickam,a william d whittier,b john f. currin,b john b. hallc adepartment of veterinary clinical sciences, washington state university, pullman, wa; bdepartment of large animal clinical science, virginia polytechnic institute and state university, blacksburg, va; cnancy m. cummings research education and extension center, university of idaho, carmen, id abstract the objective was to examine the influence of body condition score (bcs) at synchronization initiation on estrus and pregnancy rates. data were retrospectively collected from beef cattle breedings that occurred between fall 2003 and spring 2008 on 12 beef farms. the cows (n=5510) included in the analysis were synchronized with progesterone based co-synch or ovsynch protocols. the bcs (1emaciated; 9-obese) of all cows were recorded on day 0 of synchronization at the time of insertion of a controlled internal drug release device (cidr; eazi-breed™ cidr®, pfizer animal health, new york, ny). at the time of cidr removal, pressure sensitive mount detectors (kamar heatmount® detector, kamar products, inc., zionsville, in) were placed on all cows to aid in identification of cows displaying estrus until artificial insemination (ai). cows were inseminated at observed estrus or at a fixed time and bulls (approximately 1:40 bull:cow ratio) were introduced 14 days after ai and maintained for a 45 to 50 d breeding period to impregnate cows that failed to conceive to ai. cows were examined for pregnancy at 55 to 70 days and again at 120 days after ai. the bcs ranged from 3 to 8. the expression of estrus, ai and breeding season pregnancy rates were influenced by bcs (p<0.05). the estrus expression rates were 41.8%, 40.5%, 50.5%, 53.0%, 56.4% and 40.4% for bcs 3 to 8, respectively. the fixed-time ai pregnancy rates were 36.7%, 47.4%, 51.8%, 52.9%, 50.9% and 44.9% for bcs from 3 to 8, respectively. breeding season pregnancy rates were 74.7%, 78.2%, 86.4%, 90.2%, 89.9%, and 87.9% for bcs from 3 to 8, respectively. in conclusion, a minimum bcs of 5 should be achieved prior to the breeding season to ensure acceptable reproductive performance in beef cows managed on forage. keywords: beef cows, body condition score, synchronization, estrus expression, pregnancy introduction in a beef cattle operation, failure of cows to become pregnant during a breeding season of 85 days is the most important factor reducing net calf crop.1 optimum reproduction in beef cows is often limited by prolonged postpartum anestrous intervals. suckling and nutrition are major regulators of the duration of the postpartum anestrous interval. reduced nutrient intake prepartum results in thin cows at calving, a prolonged postpartum anestrous interval, and fewer cows in estrus during the breeding season.2,3 greater postpartum nutrient intake can enhance the secretion of luteinizing hormone (lh) and follicular growth. metabolites and metabolic hormones could mediate the effects of nutrient intake on reproductive function.4,5 effects of nutrition on reproduction may be more pronounced in thin and fat cows than in cows with moderate body condition. sufficient body energy reserve is necessary for acceptable reproductive performance. body condition score has been shown to be a good practical and applicable indicator of body energy reserves when compared to body weight.6,7 body condition score at calving and nutrient supply during the early postpartum period affect the return to ovarian cyclic activity and subsequent pregnancy rates.8,9 the bcs of 5 at calving is critical to ensure acceptable postpartum reproduction in mature cows. however the change in bcs after calving may influence the reproductive outcome. the objective of this study was to determine the influence of body condition score at initiation of synchronization (30 to 90 d postpartum) on estrus expression rate and pregnancy rates for the ai and natural breeding seasons. 29 materials and methods cattle and synchronization protocols data used in this study were retrospectively collected from fall 2003 to spring 2008 beef cattle breedings that occurred on 12 virginia correctional center beef farms. angus crossbred beef cows (n=5510) from 12 locations (six spring and six fall breeding locations) were synchronized with ovsynchcidr or co-synch-cidr protocols (figure 1). cows (n=2176) synchronized with the ovsynch-cidr protocols received 100 g of gonadotropin-releasing hormone (gnrh; cystorelin, merial, athens, ga) and a cidr on day 0, 25 mg prostaglandin f2 (pgf, lutalyse, pfizer animal health, new york, ny) and cidr removal on day 7, 100 g gnrh 48 h after pgf on day 9, and fixed-time ai 16 h after gnrh on day 10. cows (n=3334) synchronized with the co-synch-cidr protocol received 100 g gnrh and a cidr device on day 0, 25 mg pgf and cidr removal on day 5 (5-d co-synch-cidr; n=830) or 7 (7-d co-synch-cidr; n=2504), and 100 g gnrh on day 10 at the time of ai, 72 h (5-d co-synchcidr) or 66 h (7-d co-synch-cidr) after cidr removal. cow bcs were recorded on day 0 of synchronization. estrus detection at the time of cidr removal, all cows received a pressure sensitive mount detector to aid in identification of cows displaying estrus. cows were observed for at least 30 minutes in the morning, at noon and late afternoon on day 8, 9 and 10. a cow was determined to be in estrus if it was observed to stand for mounting or had an activated (color change from white to red), lost (with mount marks) or partially activated pressure sensitive mount detector. bulls (approximately 1:40 bull:cow ratio) were introduced 14 days after ai and maintained for a 45 to 50 d breeding period. pregnancy determination the pregnancy status of each cow was determined 55 to 70 d after fixed time ai either by perrectal palpation or by trans-rectal ultrasonography (sonosite® 180 plus™, sonosite inc., bothell, wa) reproductive parameters estrus expression rate was calculated by dividing the number of cows that expressed estrus by number of cows that received a pressure sensitive mount detector; ai pregnancy rate (pr) was calculated by dividing the number of cows that become pregnant to ai by number of cows inseminated; breeding season pr was calculated by dividing the number of cows pregnant following ai and natural service exposure by total number of cows exposed to breeding. data management cows were excluded from the analysis if they failed to calve during the season or if they received the synchronization treatment within 30 days of calving or 90 days after calving. cows inseminated at observed estrus in 2004-05 were included in the analysis to account for ai pregnancy. five-d co-synch cidr and 7-d co-synch cidr were pooled as co-synch-cidr [5-d co-synch-cidr ai-pr = 53.1% (291/548) vs. 7-d co-synch-cidr ai-pr=50.5% (1406/2786); p=0.26. note that data for two doses of pgf were excluded from 5-d co-synch-cidr data from 2006 fall breeding10]. cows were inseminated by experienced (with previous experience of a minimum of 1000 inseminations) technicians and clinicians. inseminators randomly inseminated cows at each location. since most inseminations occurred on the same day across several locations, different groups of inseminators inseminated cows at different locations, ai technicians were not included in the model. sires were selected to avoid inbreeding and assigned randomly within locations. during a single breeding season, all ai sires were not utilized in all farms. also, the number of cows inseminated was low for some sires; hence, ai sires was offered as a random effect. 30 statistical analyses the data were analyzed using a statistical software program (sas version 9.1 for windows, sas institute, cary, nc). the mixed procedure was used to evaluate differences in estrus expression rates, ai and breeding season pregnancy rates among the body condition scores. the data were managed to provide appropriate contemporaries for comparisons. the independent variables included in the estrus expression rate model were location (12), breeding season-year nested within location, bcs (3 to 8), days post-calving at protocol initiation (31 to 40; 41 to 50; 51 to 60; 61 to 70; 71 to 80; 81 to 90), dam age groups (2, 3 to 6 and ≥7 years) synchronization protocol × bcs, breeding season × bcs and breeding season × dam age groups. the independent variables included in the ai pregnancy rate model were location, breeding season-year nested within location, bcs, days post-calving at protocol initiation, dam age groups, synchronization protocol (ovsynch-cidr and co-synch-cidr), synchronization protocol × bcs, synchronization protocol × age groups, breeding season × bcs and breeding season × dam age groups. the independent variables included in the breeding season pregnancy rate model were location, breeding season-year nested within location, bcs, dam age groups, breeding season × bcs and breeding season × dam age groups. results mean  se values of bcs and age of cows from different locations are shown in table 1. body condition scores ranged from 3 to 8. the estrus expression rate for all cows was 51.8% (2854/5510). the ai pregnancy for all cows was 51.5% (2835/5510) and the breeding season pregnancy for all cows was 87.9% (4843/5510). estrus expression accounting for location, breeding season-year location, age groups and days post-calving, estrus expression was influenced by bcs (table 2; p<0.05). estrus expression rates were 41.8%, 40.5%, 50.5%, 53.0%, 56.4% and 40.4% for bcs 3 to 8, respectively (figure 2). estrus expression rates were ranged from 37.2% to 54.8% for days post-calving (figure 3) and were significantly different among age groups (table 5). decreased number of two-year-old cows expressed estrus compared to older groups. no interactions of breeding season × body condition score and breeding season × dam age groups were observed (p>0.1). ai pregnancy rate the ai pregnancy rate ranged from 48.3 to 56.4%. accounting for location, breeding seasonyear location, synchronization protocol and days post-calving, pregnancy to ai was influenced by bcs (table 3; p<0.05). pregnancy rates to ai were 36.7%, 47.4%, 51.8%, 52.9%, 50.9% and 44.9% for bcs 3 to 8, respectively (figure 2). pregnancy rates to ai ranged from 42.1% to 54.5% for days post-calving (figure 3) but did not differ among age groups (table 5; p>0.1). the ai pregnancy rates were not different between the ovsynch-cidr (52.3%) and co-synch-cidr (50.9%) protocols (table 6; p<0.05). however, ai and breeding season pregnancy rates between the fall breeding season (52.9%) and the spring breeding season (50.1) were significantly different (table 6; p<0.05). in addition, ai pregnancy rates varied among locations (figure 4; p<0.05). no interactions of synchronization protocol × body condition score, breeding season × body condition score and breeding season × dam age groups (p>0.1) were observed. breeding season pregnancy breeding season pregnancy rates ranged from 78.7% to 95.4% and were influenced by bcs (p<0.05; table 5). the breeding season pregnancy rates were 74.7%, 78.2%, 86.4%, 90.2%, 89.9%, and 87.9% for bcs from 3 to 8, respectively. no differences between breeding season pregnancy rates were detected among age groups (table 5). however, breeding season pregnancy rates between the fall breeding season (92.9%) and the spring breeding season (86.8%) were significantly different (table 6; 31 p<0.05). the breeding season pregnancy varied among locations (figure 4; p<0.05). no interactions of breeding season × body condition score were observed (p>0.1). discussion the results of this study indicate that the bcs at breeding influenced estrus expression, and ai and breeding season pregnancy rates. reduced nutrient intake during the prepartum period increases the interval from parturition to first estrus in beef cows.8,11,12 restricted energy suppresses the hypothalamic secretion of luteinizing hormone releasing hormone (lhrh). however the mechanisms through which restricted energy intake suppresses lhrh may be through alterations in the growth hormone (gh), insulin like growth factor (igf)-1, and igf binding protein (igfbp) axis in response to nutritional stress.13 the igf-1 was greater between two and ten weeks post-calving in cows that resumed ovarian cyclicity than in cows that remained anestrus. days post-calving and age of dam significantly affected estrus expression. estrus expression rates increased as the days post-calving increased. decreased number of two-year-old cows expressed estrus compared to older cows. primiparous cows utilize energy for growth after parturition which results in a low lh pulse frequency and longer postpartum anestrous interval, one to four weeks longer than in multiparous cows.14 in the current study, cows with bcs 5, 6 and 7 achieved greater than 50% ai pregnancy rates (figure 2). lake, et al., showed that first service conception rates were 36.1% for cows with bcs 4 and 50.0% for cows with bcs 6.15 the authors of that study suggested that the cows should be managed to achieve a bcs of >4 before parturition to improve reproductive success. they also showed that cows with bcs 4 at parturition are capable of maintaining this body condition score during lactation. dietary energy restriction has a negative impact on reproduction. among cows that lost body condition during the mid-trimester of gestation, those that increased their nutrient intake one to three months before calving had substantially improved pregnancy rates compared to cows that continued to lose body condition until parturition.6 however, cows that maintained body condition from mid-gestation until calving had a greater pregnancy rate than cows that lost and regained body condition. reproductive performance is decreased in primiparous compared to mature cows. the stress of calving, lower intakes of high forage diets and the requirements for growth and lactation impose nutritional demands that are often not met. inadequate nutrient intake before or after calving has a greater detrimental effect on reproduction in heifers than in cows. heifers bred to calve at two years of age resume ovarian cyclicity 20 to 40 days later than mature cows. doornbos, et al., reported that multiparous cows have greater pregnancy rates than heifers.16 kress, et al., evaluated calving rates among young multiparous cows and found that cows  5 years old had a greater calving rate than young cows.17 renquist, et al., demonstrated a quadratic relationship between age and pregnancy rate and concluded that reproductive performance of ten-year-old cows decreased.18 however, they suggested that age was not a significant determinant when bcs was included in the model and concluded that the effect of age is associated with the decreasing bcs of older cows at breeding. in the current study, the ai pregnancy rates were not different among age groups (p>0.1). this may be attributed to a good heifer management and nutritional program in these locations. it is interesting to note that the estrus expression rate was lower for the two-year-old cows compared to older cows. breeding season pregnancy rates were influenced by bcs (p<0.05; figure 4). the breeding season pregnancy rates were 76.4%, 84.7%, 88.0%, 90.2%, 89.2%, and 87.0% for bcs 3 to 8, respectively; and ai and breeding season pregnancy rate between fall and spring were significantly different. more cows were pregnant during the fall compared to the spring season. in virginia the primary grasses during spring are fescue (festuca arundinacea), bluegrass (poa pratensis) and clover (trifolium pratense). fescue toxicity and heat stress may play a role in reduced pregnancy during the spring season compared to fall. in females, the fescue endophyte may affect ovarian gamete maturation, ovulation, gamete transport and fertilization, conceptus transport, and/or embryo attachment and the effects on the postpartum anestrous interval are possibly caused by neurohormonal imbalances that lead to improper ovarian function.19-21 the possible effects endophyte-infected fescue in the male include sperm 32 production, sperm motility, libido, and testicular development.22 these effects may be reflected in this study by reduced ai and breeding season pregnancy rates in spring compared to fall. days post-calving at protocol initiation significantly influenced the ai pregnancy rate. it is interesting to note that the days post-calving also affected estrus expression rates indicating that the late calving cows are still under the influence of postpartum anestrus. however if the cows were in moderate body condition they become pregnant. lents, et al., showed that bcs at calving influenced the size of the dominant follicle at the first postpartum estrus in mature suckled cows and suggested that cows be managed to calve in moderate bcs.23 there were no differences in the ai and breeding season pregnancy between ovsynch-cidr and co-synch-cidr synchronization protocols. no interactions between synchronization protocols by bcs and between synchronization protocols by days post-calving were observed. a progesterone-supplemented protocol is recommended for improving fertility by inducing cyclicity in anestrous postpartum beef cows. if anestrus is the problem, then all cows should benefit from progesterone-supplemented synchronization protocols. the results indicated that cows with bcs 5 to 7 achieved > 50% ai pregnancy. ciccioli, et al., concluded neither bcs at calving nor postpartum nutrition influenced estrous behavior at the first postpartum estrus.24 they suggested that the lack of effect of bcs at calving on reproductive performance in that study could be related to the minimal differences in bcs of the thin and moderate condition cows at calving, or that all cows had less than optimal bcs at calving for adequate performance. spitzer, et al., demonstrated that bcs 4 to 6 at calving influences the duration from parturition to estrus.25 they also found greater pregnancy rates for cows with a bcs of 6 at parturition compared with cows with a bcs of 4 or 5. greater nutrient intake postpartum can have a positive effect26-28 or no effect29-32 on duration of the postpartum anovulatory interval. results of other experiments indicate that postpartum energy intake may influence pregnancy rate at the first postpartum estrus,11,33 however, the effect of energy intake on pregnancy rate was not significant. reduced nutrient intake during estrous cycles did not affect fertilization rate3 but did reduce conception rate.34 lack of consistency among studies may involve the amount of energy intake, duration of the feeding period, bcs at calving, bcs change during postpartum period, and age of cows. fuel sensors, such as glucose, insulin or leptin, are known to be directly involved in the regulation of fertility at each level of the hypothalamuspituitary-gonadal axis. ciccioli, et al., suggested that the concentrations of igf-1, leptin, insulin, glucose, nonesterified fatty acid, or thyroxin in blood may not individually signal the onset of postpartum ovarian function, but they may act in concert with other factors to indicate the adequacy of nutrients.24 so it is plausible that differences in endocrine function or metabolic signals during the postpartum period could influence ovarian activity and fertility. in summary, the bcs of beef cows at the time of initiation of synchronization protocol influenced estrus expression, ai and breeding season pregnancy rates. a minimum bcs of 5 should be achieved prior to the breeding season to ensure acceptable reproductive performance. further research is needed to elucidate the mechanism(s) that control postpartum ovarian activity and estrous behavior to maximize reproductive efficiency in beef cattle. lipid biomarkers play a key role in reproduction. it would be of interest to investigate the impact of different types of lipid biomarkers, perhaps integrated into feed, on ovulation capacity and embryonic development. acknowledgements the authors thank select sires inc, plain city, oh for providing partial financial support. the authors also extend their gratitude to the staff of virginia department of corrections for their support with data collection. references 1. wiltbank jn, warwick ej, vernon eh, et al: factors affecting net calf crop in beef cattle. j anim sci 1961;20:409415. 2. bossis i, wettemann rp, welty sd, et al: nutritionally induced anovulation in beef heifers: ovarian and endocrine function preceding cessation of ovulation. j anim sci 1999;77:1536-1546. 33 3. perry rc, corah lr, cochran rc, et al: influence of dietary energy on follicular development, serum gonadotropins, and first postpartum ovulation in suckled beef cows. j anim sci 1991;69:3762-3773. 4. wettemann rp, bossis i: energy intake regulates ovarian function in beef cattle. proc am soc anim sci 2000. available from: http://www.asas.org/jas/symposia/proceedings/0934.pdf 5. armstrong jd, cohick ws, harvey rw, et al: effect of feed restriction on serum somatotropin, insulin-like growth factor-i-(igf-i) and igf binding proteins in cyclic heifers actively immunized against growth hormone releasing factor. domest anim endocrinol 1993;10:315-324. 6. wagner jj, lusby ks, oltjen jw, et al: carcass composition in mature hereford cows: estimation and effect on daily metabolizable energy requirement during winter. j anim sci 1988;66:603-612. 7. houghton pl, lemenger rp, horstman la, et al: prediction of postpartum beef cows body composition using weight to height ratio and visual body condition score. j anim sci 1990;68:1428-1437. 8. richards mw, spitzer jc, warner mb: effect of varying levels of postpartum nutrition and body condition at calving on subsequent reproductive performance in beef cattle. j anim sci 1986;62:300-306. 9. selk ge, wettemann rp, lusby ks, et al: relationships among weight change, body condition and reproductive performance or range beef cows. j anim sci 1988;66:3153-3159. 10. kasimanickam r, day ml, rudolph js, et al: two doses of prostaglandin improve pregnancy rates to timed-ai in a 5day progesterone-based synchronization protocol in beef cows. theriogenology 2009;15:762-767. 11. richards mw, wettemann rp, schoenemann hm: nutritional anestrus in beef cows: body weight change, body condition, luteinizing hormone in serum and ovarian activity. j anim sci 1989;67:1520-1526. 12. bishop dk, wettemann rp, spicer lj: body energy reserves influence the onset of luteal activity after early weaning of beef cows. j anim sci 1994;72:2703-2708. 13. roberts aj. nugent iii ra, klint j, et al: circulating insulin-like growth factors i, insulin-like growth factor binding protein, growth hormone, and resumption of estrus in postpartum cows subjected to dietary energy restriction. j anim sci 1997;75:1909-1917. 14. yavas y, walton js: postpartum acyclicity in suckled beef cows: a review. theriogenology 2000;54:5-25. 15. lake sl, schooljegerdes ej, atkinson rl, et al: body condition score at parturition and postpartum supplemental fat effects on cow and calf performance. j anim sci 2005;83:2908-2917. 16. doornbos de, bellows ra, burfening pj, et al: effects of dam age, prepartum nutrition, and duration of labor on productivity and postpartum reproduction in beef females. j anim sci 1984; 59:1-10. 17. kress dd, doornbos de, anderson dc: performance of crosses among hereford, angus, and simmental cattle with different levels of simmental breeding: v. calf production, milk production and reproduction of threeto eight-yearold dams. j anim sci 1990;68:1910-1921. 18. renquist bj. oltjen jw, sainz rd, et al: effects of age on body condition and production parameters of mulitparous beef cows. j anim sci 2006;84:1890-1895. 19. paterson j, forcherio c, larson b, et al: the effects of fescue toxicosis on beef cattle productivity. j anim sci 1995;73:889-898. 20. rhodes mt, paterson ja, kerley ms, et al: reduced blood flow to peripheral and core body tissues in sheep and cattle induced by endophyte-infected tall fescue. j anim sci 1991;89:2033-2043. 21. porter jk, thompson fn jr: effects of fescue toxicosis on reproduction in livestock. j anim sci 1992;70:1594-1603. 22. alamer ma, erickson bh. effect of fungus-infested fescue on testicular development and hormonal secretions in the beef bull (abstract). j anim sci 1990;68(suppl 1):40. 23. lents ca, white fj, ciccioli nh, et al: effects of body condition score at parturition and postpartum protein supplementation on estrous behavior and size of the dominant follicle in beef cows. j anim sci 2008;86:2549-2556. 24. ciccioli nh, wettemann rp, spicer lj, et al: influence of body condition at calving and postpartum nutrition on endocrine function and reproductive performance of primiparous beef cows. j anim sci 2003;81:3107-3120. 25. spitzer jc, morrison dg, wettemann rp, et al: reproductive responses and calf birth and weaning weights as affected by body condition at parturition and postpartum weight gain in primiparous beef cows. j anim sci 1995;73:1251-1257. 26. wright ia, rhind sm, whyte tk, et al: effect of body condition at calving and feeding level after calving on lh profiles and duration of the post-partum anoestrous period in beef cows. anim prod 1992;55:41-46. 27. stagg k, diskin mg, sreenan jm, et al: follicular development in long-term anoestrous suckler beef cows fed two levels of energy postpartum. anim reprod sci 1995;38:49-61. 28. vizcarra ja, wettemann rp, spitzer jc, et al: body condition at parturition and postpartum weight gain influence luteal activity and concentrations of glucose, insulin, and nonesterified fatty acids in plasma of primiparous beef cows. j anim sci 1998;76:927-936. 29. wright ia, rhind sm, russel ajf, et al: effects of body condition, food intake and temporary calf separation on the duration of the post-partum anoestrous period and associated lh, fsh, and prolactin concentrations in beef cows. anim prod 1998;45:395-402. 30. whittier jc, clanton dc, deutscher gh: effect of post-partum levels of nutrition on productivity of 2-year-old heifers. anim prod 1988;47:59-64. 31. stagg k, spicer lj, sreenan jm, et al: effect of calf isolation on follicular wave dynamics, gonadotropin and metabolic hormone changes, and interval to first ovulation in beef cows fed either of two energy levels postpartum. biol reprod 1998;59:777-783. 34 32. wiltbank j n, rowden ww, ingalls je, et al: influence of post-partum energy level on reproductive performance of hereford cows restricted in energy intake prior to calving. j anim sci 1964;23:1049-1053. 33. spitzer jc, niswender gd, seidel ge jr, et al: fertilization and blood levels of progesterone and lh in beef heifers on restricted energy diet. j anim sci 1978;46:1071-1077. 34. hill jr, lamond dr jr, henricks dm, et al: the effects of undernutrition on ovarian function and fertility in beef heifers. biol reprod 1970;2:78-84. table 1. mean  se values of body condition score and age of beef cows in different locations. season location body condition score age (y) fall 1 5.27  0.05 4.79  0.11 2 6.31  0.09 5.47  0.38 3 5.53  0.04 5.62  0.13 4 5.43  0.05 4.23  0.06 5 5.64  0.03 4.02  0.08 6 5.55  0.06 4.18  0.16 spring 7 5.54  0.12 5.38  0.25 8 5.28  0.05 5.23  0.25 9 5.79  0.40 4.24  0.62 10 5.21  0.04 4.44  0.34 11 5.65  0.08 4.36  0.29 12 5.17  0.10 5.19  0.22 table 2. the general linear model for the effects on estrus expression rate of angus-cross beef cows (n=5510). source d.f. sum of squares f ratio prob > f location 11 82.4517 4.1268 <0.0001 breeding season-year within location 44 63.4232 6.6214 <0.0001 body condition score* 5 2.2319 4.1246 <0.0338 days post-calving at protocol initiation† 5 5.8309 18.3948 <0.0015 age group‡ 2 2.9817 5.9985 <0.03238 d.f – degrees of freedom; *1-emaciated and 9-obese; †days post-calving groups: 31-40; 41-50; 51-60; 61-70; 71-80 and 81-90; ‡age groups: 2; 3-6 and >6 35 table 3. the general linear model for the effects on ai pregnancy rate of angus-cross beef cows (n=5510). source d.f. sum of squares f ratio prob > f location 11 16.6242 4.1253 <0.0001 breeding season-year within location 44 58.5227 23.2381 <0.0001 body condition score§ 5 2.0121 3.6128 <0.0313 days post-calving at protocol initiation¶ 5 11.1174 3.9213 <0.0001 age group** 2 1.2158 1.5781 <0.3316 synchronization protocol†† 1 5.2459 24.5168 <0.0123 synchronization protocol by body condition score 5 0.0216 0.6921 <0.4571 breeding season by body condition score 11 0.04328 0.7081 <0.6570 synchronization protocol by days postcalving at protocol initiation 5 0.0721 0.7218 <0.8212 d.f – degrees of freedom; §1-emaciated and 9-obese; ¶days post-calving groups: 31-40; 41-50; 51-60; 61-70; 71-80 and 81-90; **age groups: 2; 3-6 and >6; ††refer to figure 1 for treatment; table 4. the general linear model for the effects on breeding season pregnancy rate of angus-cross beef cows (n=5510). source d.f. sum of squares f ratio prob > f location 11 14.4218 22.1246 <0.0001 breeding season-year within location 44 87.2445 7.8351 <0.0001 body condition score‡‡ 5 2.4513 4.7862 <0.0211 age group§§ 2 3.2193 0.5781 <0.2714 breeding season by body condition score 11 1.6739 0.8463 <0.5438 d.f – degrees of freedom; ‡‡1-emaciated and 9-obese; §§age groups: 2; 3-6 and >6; table 5. effect of age of the dam (in years) on the ai and breeding season pregnancy* in angus cross beef cows (n=5510) age groups n estrus expression rate (95% ci) ai-pr (95% ci) breeding season pr (95% ci) 2 1317 47.9 (43.1, 50.9)a 53.5 (48.2-57.6)a 86.8 (81.1, 90.8)a 3 to 6 2920 54.0 (49.7, 57.8)b 52.7 (48.2-56.7)a 87.7 (80.9, 94.1)a > 6 1273 50.8 (47.1, 53.1)b 50.3 (41.4-53.3)a 88.7 (84.3, 91.4)a ab different superscripts within column are statistically significant, p<0.05; ci – confidence interval pr – pregnancy rate 36 table 6. effect of synchronization protocol, breeding season on the ai and breeding season pregnancy* in angus cross beef cows (n=5510). source level n ai-pr (95% ci) breeding season pr (95% ci) synchronization¶¶ ovsynch-cidr 2176 52.3 (48.9, 57.4)a 87.4 (83.4, 91.7)a co-synch-cidr 3334 50.9 (48.2, 53.7)a 88.2 (81.8, 93.6)a breeding season spring 2816 50.1 (48.9, 52.4)a 86.8 (83.8, 89.6)a fall 2694 52.9 (49.6, 55.4)b 89.0 (85.6, 93.3)b ab different superscripts within source and between level are different (p<0.01) ¶¶refer to figure 1 for treatment ci – confidence interval pr – pregnancy rate cidr – controlled internal drug release device 37 ovsynch-cidr day 0 day 7 am day 9 pm day 10 am gnrh pgf2 48 h gnrh 16 h tai 7-d co-synch-cidr day 0 day 7 am day 10 am gnrh pgf2 66 h gnrh+tai 5-d co-synch-cidr day 0 day 5 am day 8 am gnrh pgf2 72 h gnrh+tai figure 1. treatment protocols for estrous synchronization. angus crossbred beef cows (n = 5510) from 12 locations (six spring and six fall breeding locations) were synchronized with ovsynch-cidr, or cosynch-cidr protocols. cows synchronized with ovsynch-cidr protocols received 100 g gonadotropin-releasing hormone (gnrh; cystorelin, merial, athens, ga) + controlled internal drug release device (cidr; eazi-breed cidr, pfizer animal health, new york, ny) on day 0, 25 mg prostaglandin f2 (pgf; lutalyse, pfizer animal health) and cidr removal on day 7, 100 g gnrh 48 h after pgf on day 9, and fixed-time ai 16 h after gnrh on day 10. cows synchronized with cosynch-cidr protocols received 100 g gnrh + cidr device on day 0, 25 mg pgf and cidr device removal on day 5 (5-d co-synch-cidr; n=830) or 7 (7-d co-synch-cidr; n=2504), and 100 g gnrh on day 10 at the time of ai, 72 h (5-d co-synch-cidr) or 66 h (7-d co-synch-cidr) after cidr removal. cow bcs (1-emaciated; 9-obese) were recorded on day 0 of synchronization. five-d cosynch cidr and 7-d co-synch cidr were pooled as co-synch-cidr for the analysis. cidr cidr cidr 38 rabc different superscripts within estrus expression are statistically significant (p<0.05) ab different superscripts within ai pregnancy are statistically significant (p<0.05) ab different superscripts within breeding season pregnancy are statistically significant (p<0.05) figure 2. influence of body condition score on on estrus expression, and ai and breeding season pregnancy (mean percentage ± sem) in cows (n=5510) following porgesterone supplemented synchronization protocol. numbers in paenthesis are number of cows for the corresponding body condition scores. numbers in black are percentage of cows expressed estrus. numbers in white are percentage of cows pregnant to ai. numbers in the box are percentage of cows pregnant for the breeding season pregnancy. 41.8 40.5 50.5 53.0 56.4 40.4 36.7 47.4 51.8 52.9 50.9 43.9 74.7 78.2 86.4 90.2 89.9 87.7 0.0 20.0 40.0 60.0 80.0 100.0 3 4 5 6 7 8 p er ce nt ag e body condition score estrus expression ai pregnancy breeding season pregnancy a bc c c a (79) (348) (1834) (2089) (1103) (57) a b b bbb bb a a abab 39 abc different superscripts within ai pregnancy are statistically significant (p<0.05) abc different superscripts within ai pregnancy are statistically significant (p<0.05) figure 3. effect of days post-calving at protocol initiation on estrus expression and ai pregnancy (mean percentage ± sem) in cows (n=5510) following porgesterone supplemented synchronization protocol. numbers in parenthesis are number of cows for the corresponding days post-calving. numbers in black are percentage of cows expressed estrus. numbers in white are percentage of cows pregnant to ai. numbers in the box are percentage of cows pregnant for the breeding season pregnancy. 37.2 47.9 50.9 54.5 52.8 54.8 42.1 48.8 51.6 54.5 52.3 50.9 0.0 20.0 40.0 60.0 80.0 100.0 31 to 40 41 to 50 51 to 60 61 to 70 71 to 80 81 to 90 p er ce nt ag e days postcalving at protocol initiation estrus expression ai pregnancy (309) (543) (796) (1007) (1959) (896) a b bc bc bcc a b bc ccc 40 ab d if fe re nt s up er sc ri pt s ar e st at is ti ca ll y si gn if ic an t ( p < 0. 05 ) f ig ur e 4. e ff ec t o f lo ca ti on s (n = 12 ) on a i pr eg na nc y (m ea n pe rc en ta ge ± s e m ) in b ee f co w s (n = 55 10 ) sy nc hr on iz ed w it h pr og es te ro ne s up pl em en te d pr ot oc ol s. n um be rs in p ar en th es is a re n um be r of c ow s in se m in at ed f ro m th e co rr es po nd in g lo ca ti on . 48 .3 53 .4 55 .1 48 .3 55 .2 48 .7 56 .4 52 .5 49 .5 50 .6 50 .7 52 .5 81 .2 91 .5 91 .2 79 .8 93 .2 78 .7 95 .4 94 .6 88 .3 87 .6 90 .1 91 .6 0. 0 20 .0 40 .0 60 .0 80 .0 10 0. 0 1 2 3 4 5 6 7 8 9 10 11 12 percentage l oc at io ns a i pr eg na nc y b re ed in g s ea so n p re gn an cy (4 10 ) (2 79 ) (3 34 ) (7 61 ) (8 27 ) (4 21 ) ab c a ab ab ab c bc a ab c ab c ab c c (8 12 ) (3 48 ) (4 35 ) (1 81 ) (3 26 ) (3 26 ) ab c a a a b c bc bc bc bc bc (4 10 ) (2 79 ) (3 34 ) (7 61 ) (8 27 ) (4 21 ) ab c a ab ab ab c bc a ab c ab c ab c c (8 12 ) (3 48 ) (4 35 ) (1 81 ) (3 26 ) (3 26 ) ab c a a a b cd bc bc bc bc bc cd d 41 42 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 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[612.000 792.000] >> setpagedevice 2014: management of common surgical problems of the bull genitalia management of common surgical problems of the bull genitalia dwight f. wolfe food animal section, department of clinical sciences, college of veterinary medicine, auburn university, auburn, al abstract numerous congenital or acquired defects of the bull genitalia prevent normal breeding efficiency. this presentation will review the anatomy and pathophysiology of congenital defects and acquired conditions of the penis and prepuce of bulls that compromise reproductive soundness and that are amenable to surgical repair. medical and surgical options for returning bulls to breeding soundness will be discussed. keywords: bull, penis, prepuce, injury introduction diagnosis and management of defects or injuries of the bull penis and prepuce requires good understanding of the normal anatomy as well as the physiology of erection and coitus. the non-erect penis of the bull is contained within the sheath which is a double invagination of skin along the ventral abdominal wall. the skin of the sheath is covered with hair and terminates distally at the preputial orifice where it joins the non-haired epithelium of the prepuce.1 the prepuce terminates at the free portion of the penis several centimeters proximal to the glans penis at the preputial ring.2 the diameter and length of the prepuce varies considerably among bulls of varying breeds and ages but the prepuce of an adult bull is 35 to 40 cm long and approximately 4 cm in diameter. the prepuce of bos indicus breeds averages 5.5 cm longer than bulls of the bos taurus breeds and they have a more pendulous sheath and larger preputial orifice which is 2 to 4 cm in diameter in bos taurus breeds may exceed 10 cm in some bos indicus bulls.3 multiple interdigitating layers of elastic tissue between the preputial epithelium and the tunica albuginea of the penis allow the penis to glide within the sheath from full retraction to full extension. there is wide variation among bulls such that the penis extends 25 to 60 cm beyond the preputial orifice during full erection therefore full excursion of the glans penis may be greater than 1 meter.3,4 penile and preputial conditions of young bulls the most common anomalies of the penis, prepuce, or sheath of young bulls that prevent breeding soundness are penile fibropapillomas, persistent frenulum, and incomplete separation of the penis and prepuce. penile fibropapillomas fibropapilloma or warts caused by bovine papilloma virus are fairly common in young bulls reared in groups. the virus is believed to enter the penile skin through wounds or abrasion sustained during homosexual activity among young bulls and causes neoplastic growth of fibroblasts which is not locally invasive or metastatic. often several bulls in a group will develop penile fibropapillomas and affected bulls frequently do not have obvious lesions on other parts of the body.5,6 penile fibropapillomas are usually easily removed with the bull restrained on a tilt table or in a squeeze chute. manually extend the penis and following antiseptic preparation of the surgical field infiltrate 2 to 4 ml 2% lidocaine hydrochloride subcutaneously across the dorsum of the penis proximal to the lesion for regional anesthesia of the surgical site. carefully identify the urethra to avoid incising this tissue during excision of the growth. dissect the skin of the penis at the base of the lesion until the growth is completely removed with either sharp dissection with a scalpel or with a co2 laser to assist hemorrhage control. ligate any small vessels and close the skin defect with #0 absorbable suture. bulls treated for penile fibropapilloma should have enforced sexual rest and be examined for healing or regrowth four weeks following surgery before entering breeding service.7 503 clinical theriogenology • volume 6, number 4 • december 2014 persistent frenulum, and incomplete separation of the penis and prepuce as young bulls complete puberty the penis grows in length and diameter and a sigmoid flexure develops. the surface epithelium of the free portion of the penis is firmly attached to the epithelium of the prepuce at birth and these interdigitating tissues begin to separate at approximately four weeks of age and proceeds caudally until complete separation occurs between wight and 11 months of age. the separation rarely occurs prematurely in young bulls, perhaps caused by juvenile attempts at mounting and penile extension, resulting in hematoma formation from hemorrhage of the surface epithelial layers. affected bulls may have swelling along the distal sheath with evidence of hemorrhage on the preputial hairs. following sexual rest complete recovery may be expected unless excessive fibrosis develops between the surface epithelial layers which may permanently prevent penile extension.7 more commonly incomplete separation of the penis and prepuce which prevents complete penile extension is seen in bulls over 11 months of age. manual traction of the free portion of the penis and prepuce may complete the separation but should be avoided if the tissues are tightly adhered such that tearing or hemorrhage will be created. this condition is thought to be associated with later maturity and perhaps may have an undesirable heritable component. during normal separation of the epithelium of the penis and prepuce of young bulls the frenulum, a thin band of collagenous connective tissue on the ventral midline that extends over the basal 80% of the free end of the penis, ruptures allowing complete separation of the glans penis and prepuce. when this band of tissue fails to rupture the penis can extend but the persistent frenulum impairs straightening of the tip of the penis and may prevent intromission.8,9 the persistent frenulum is easily diagnosed as a band of tissue from the median raphe at the posterior of the glans penis to the prepuce. this epithelium covered band may be thin or broad and usually contains one or more blood vessels. persistent frenulum is easily surgically repaired by ligating each end of the frenulum and transecting the tissue to reduce the possibility of hemorrhage. although controversial, the owner should be advised that this condition is considered to be heritable and retaining his sons as sires is not recommended.9 breeding injuries the most common breeding injuries that prevent reproductive soundness are preputial lacerations. bulls with preputial injury may prolapse the preputial epithelium distal to the end of the sheath or develop phimosis wherein the bull is unable to freely extend the penis and prepuce through the end of the sheath. the extent of damage of the surface epithelium and underlying peripenile elastic tissue determines the prognosis and therapeutic approach for returning the bull to breeding soundness. primary preputial prolapse in the bull is usually the sequela to bruising or contusions during breeding or to frostbite or balanoposthitis caused by herpes viral infection (ibr-ipv). secondary preputial prolapse may occur with penile hematoma or urethral rupture. bulls with preputial frostbite may undergo considerable necrosis of the preputial epithelium and heal with mild to severe stenosis of the preputial lumen causing phimosis.10,11 due to their pendulous sheath and longer prepuce bos indicus breeds and their crosses more commonly develop preputial prolapse following preputial laceration. during intromission the prepuce is abruptly forced caudally forming a collar of taut tissue at the preputial orifice which may become forcefully entrapped between the bull’s abdomen and the vulva and pelvis of the cow during the ejaculatory lunge. the subsequent contusion and occasionally laceration or bursting of the skin on the longitudinal axis of the ventral aspect of the prepuce leads to edema which quickly develops in the traumatized skin and underlying elastic tissue leading to prolapse of the prepuce. as the penis is withdrawn into the preputial cavity the longitudinal tear of the surface epithelium assumes a transverse orientation which effectively shortens the ventral aspect of the prepuce and in bos indicus breeds may not be fully retracted into the preputial cavity. as edema accumulates in the damaged tissues the prolapsed prepuce increases in size and the laceration is evident as a transverse wound on the caudal aspect of the prolapsed tissues. the edematous prolapsed prepuce frequently sustains additional trauma with subsequent extreme mutilation, cellulitis, necrosis, fibrosis and risk of frostbite in colder climates.10,12 504clinical theriogenology • volume 6, number 4 • december 2014 laceration of the prepuce of bos taurus breeds usually does not lead to preputial prolapse as the damaged prepuce is typically withdrawn into the sheath where swelling of the sheath may be observable. minor injuries are often unnoticed and heal without complication with only superficial scarring visible on the surface epithelium of the prepuce.10-12 alternatively some bos taurus bulls develop paraphimosis wherein the damaged tissues will not allow retraction of the penis into the sheath. often bulls with moderate to severe laceration retract the penis into the sheath and develop phimosis due to stricture of the injured prepuce. the most critical factors for determining the return to breeding soundness are the length of the preputial skin that is disrupted and the extent of damage to the peripenile elastic tissue. in order to allow sufficient penile and preputial extension for breeding the remaining prepuce must be at least 1.5 times the length of the free portion of the penis following surgical repair. the peripenile elastic tissue must also be sufficiently free of scar tissue to allow complete extension and retraction of the penis and prepuce.11 medical management of preputial prolapse the prolapsed tissues should be cleaned with antiseptic scrub and an emollient antiseptic ointment applied. frequently this symptomatic treatment allows bulls with a minor laceration and prolapse to resume breeding soundness following at least 60 days sexual rest.10-12 for bulls with more extensive tissue damage and edema that prevents returning the prepuce into the preputial cavity compression bandaging or other support is indicated. change the bandage every second or third day or sooner if it becomes loose or extremely soiled. bulls with preputial laceration and prolapse may sustain considerable and possibly deep necrosis and slough of preputial skin and elastic tissues after the first few bandage changes. these healing tissues should be covered by healthy granulation tissue before reversion into the preputial cavity or surgery is considered. when the initial edema subsides in bulls with mild to moderate preputial prolapse the prepuce should be reverted into the preputial cavity and held in place by elastic bandaging with a urinary drainage tube incorporated into the bandage. placing a purse-string suture in the preputial orifice should be avoided due to the risk of abscess formation and subsequent stenosis of the preputial orifice. because they do not usually prolapse the prepuce after injury bulls of bos taurus breeds are more likely to develop phlegmon within the sheath or retropreputial abscess. these bulls have a guarded prognosis for returning to breeding soundness and systemic antibiotics, irrigation of the preputial cavity with mild antiseptic solutions and hydrotherapy may reduce the risk of severe complications. following a minimum of 60 days of forced sexual rest evaluate the prepuce for scar formation that restricts penile extension prior to resuming breeding. phimosis or impaired extension is very likely since the original longitudinal wound healed transversely and may be associated with significant fibrosis of the elastic tissues. surgical management of preputial injury circumcision. resection and anastomosis of the prepuce, frequently called circumcision, provides the best prognosis for return to breeding soundness when scar tissue in the prepuce prevents normal penile extension and coitus. circumcision requires full extension of the penis and prepuce which may require incision of fibrous stenotic areas of the prepuce. the guideline for the maximum amount of prepuce that may be removed is that the remaining prepuce must be at least one and one-half times the length of the free portion of the penis. repeat of the prolapse is likely when the bull resumes breeding if the prepuce is left excessively long. alternatively, if the prepuce is shortened excessively the bull will be unable to completely extend the penis. following aseptic preparation of the surgical field apply a 2.5 cm penrose drain around the penis and prepuce at the preputial orifice as a tourniquet and make circumferential incisions through the preputial epithelium proximal and distal to the area to be removed. with a scalpel or co2 laser make a longitudinal incision through the epithelium to join the circumferential incisions. remove the skin of the injured area by blunt and sharp dissection taking care to only remove fibrotic tissues. ligate or seal all visible elastic tissue vessels by judicious electrocautery then remove the 505 clinical theriogenology • volume 6, number 4 • december 2014 tourniquet and ligate any remaining vessels to ensure complete hemostasis. appose the subcutaneous elastic tissue with #0 chromic gut in a simple interrupted pattern or in a simple continuous pattern tied at each quadrant of the circumference of the prepuce. close the skin of the prepuce in similar fashion ensuring end-to-end apposition of the skin edges and place a 2.5-cm penrose drain over the free portion of the penis to provide urine drainage away from the surgical site. secure the penrose with 3 or 4 absorbable sutures through the latex tubing and the skin of the penis. revert the penis and prepuce into the preputial cavity and place a semi-flexible rubber tube of maximum diameter into the lumen of the prepuce and firmly wrap the distal portion of the haired sheath with elastic tape to serve as a pressure bandage on the incision. remove the pressure bandage and rubber tubing three to fice days postoperatively and leave the penrose drain on the penis until skin sutures are removed in ten days. ensure a minimum of 60 days sexual rest and evaluate the bull prior to resuming breeding.11-13 preputial reconstruction by scar revision. for bulls with insufficient prepuce to allow resection and anastomosis the bull may be returned to breeding soundness with scar revision and preputial reconstruction. with the prepuce prepared for aseptic surgery extend the penis and excise only the superficial transverse scar tissue. return the edges of the preputial incision back to their original longitudinal orientation and loosely place an absorbable bootlace suture in a longitudinal plane such that the free ends are toward the sheath. suture a 2.5-cm penrose drain over the free potion of the penis and revert the penis into the preputial cavity. tighten the previously placed preputial sutures to appose the skin edges. the goal of this procedure is to allow the surgical wound to heal in a longitudinal plane and to maintain normal elastic tissue function. since no elastic tissue or preputial skin is removed first intention healing should reduce the risk of preputial stenosis and allow subsequent penile extension. many bulls begin to masturbate a few weeks following surgery and will stretch contracted tissues without permanent damage. most bulls require 60 to 120 days to regain full penile extension and forceful attempts to extend the penis should be avoided as the potential trauma increases the likelihood of excessive scar formation.11 inability to extend the penis (phimosis) the most common cause of phimosis, the inability to extend the penis, in adult bulls is preputial stenosis resulting from scar tissue. the stenosis is most often a sequelae of either breeding injury laceration or frostbite. either case may be corrected surgically by circumcision if removal of the damaged tissues leaves sufficient healthy prepuce and elastic tissue for normal penile extension. the most limiting factors for the likelihood of returning to breeding soundness for a bull with preputial laceration are the extent of the damage to the peripenile elastic tissue and the length of preputial skin that is lacerated. after reconstructive surgery the remaining prepuce must be at least 1.5 times as long as the free portion of the penis in order for full penile extension.11,12 references 1. ashdown r: functional anatomy of the penis in ruminants. vet anat 1973;14:22-25. 2. ashdown r, rickets, wardley r: the fibrous architecture of the integumentary coverings of the bovine penis. j anat 1968:103;576-578. 3. bellinger cr: a comparison of certain parameters of the penis and prepuce in various breeds of beef cattle. res vet sci 1971;299-304. 4. ashdown r, pearson h: anatomical and experimental studies on eversion of the sheath and protrusion of the penis in the bull. res vet sci 1973;15:13-24. 5. mcentee k: fibropapilloma of the external genitalia of cattle. cornell vet 1950;40:304-312. 6. olson c, robl mg, larson ll: cutaneous and penile bovine fibropappilomatosis and its control. j am vet med assoc 1968;153:1189-1104. 7. wolfe df, rodning sp: diagnosis and management of juvenile anomalies of the penis and prepuce. in: anderson de, rings m, editors. current veterinary therapy, food animal practice. 5th ed. st. louis: saunderselsevier; 2009. p.340341. 8. ashdown r, pearson h: the functional significance of the dorsal apical ligament of the bovine penis. res vet sci 1971;12:183-184. 9. carroll ej, aanes wa, ball l: persistent penile frenulum in bulls. j am vet med assoc. 1964;144:747-749. 506clinical theriogenology • volume 6, number 4 • december 2014 10. wolfe df, hudson rs, walker df: common penile and preputial problems in bulls. compend contin educ pract vet 1983;447-455. 11. wolfe df, beckett sd, carson rl: acquired conditions of the penis and prepuce. large animal urogenital surgery. baltimore: williams and wilkins, 1998; p. 237-272. 12. memon m, dawson l, usenik e, et al: preputial injuries in bulls. j am vet med assoc 1988;193;484-485. 13. cardwell w: the surgical correction of preputial and penile disorders o the bull. southwestern vet 1961;summer:270273. 507 clinical theriogenology • volume 6, number 4 • december 2014 508clinical theriogenology • volume 6, number 4 • december 2014 omniblank: 2014: comparison of three methods for termination of pregnancy in cats using a dopamine agonist and prostaglandin f2alpha comparison of three methods for termination of pregnancy in cats using a dopamine agonist and prostaglandin f2alpha amin ahmadzadeh,a beverly j. purswell,b stephen m. boylec adepartment of animal and veterinary science, center for reproductive biology, university of idaho, moscow, id; bcenter for reproductive excellence using advanced technology and endocrinology, and ccenter for molecular medicine and infectious diseases, virginia-maryland regional college of veterinary medicine, virginia tech, blacksburg, va abstract this study compared the effect and efficacy of a natural prostaglandin f2alpha (pgf) a dopamine agonist (cabergoline; cbr), or the combination of pgf and cbr in pregnancy termination in domestic cats. after pregnancy confirmation by palpation and ultrasonography. treatments on 15 cats (in two trials) were initiated between day 35 41 after first mating. animals were assigned randomly to one of three treatment groups: cbr was given orally (n=10; 50 mcg/cat/day) for five consecutive days (day 0 to 5); pgf (n=10; 2.5 mg/cat/day, s.c.) for five consecutive days; or cbr and pgf (n=9, one cat was eliminated from the study). the queens were monitored by ultrasonography on days three and five during treatments and every day after completion of the treatments for three days to assess pregnancy status and fetal viability. the percentage of cats aborted was different (p < 0.05) among treatments. in the cbr group, six of ten cats aborted within three days after the last dose. three days after the completion of the initial treatment, the only observed side effect was a mild bloody vaginal discharge. in the pgf group, only one of ten cats aborted during the three-day period following the completion of pgf treatment. ultrasonography revealed viable fetuses (presence of heart beat) during and after treatment in the nine cats that did not lose their pregnancy. all nine cats in the cbr + pgf group aborted within three to six days after the initiation of the treatment (five cats after two doses, three cats after three doses, and one cat after five doses). these results indicate that a combination of cbr and pgf is a quick and effective method for pregnancy termination in cats. at 35 to 41 days after mating, treatment with cbr (at the above dose) alone was less effective in pregnancy termination compared with cbr+ pgf. furthermore, pgf treatment alone, at least at this dose, was not effective in pregnancy termination in cats during 35-41 days after mating. keywords: abortion; pregnancy; cat; cabergoline; prostaglandin f2alpha introduction current practical methods to terminate pregnancy after unwanted mating in cats include pgf administration, use of a dopamine agonist such as cbr, or progesterone receptor antagonist.1 prostaglandin f2α has been reported to induce abortion by causing a premature luteolysis and thus cessation of progesterone secretion in dogs.2 high and low doses of pgf successfully induced abortion in bitches when administered between 35-55 days of pregnancy.2 however, using pgf as an abortifacient in cats is controversial and results are highly variable. for instance, administering natural pgf successfully terminated pregnancy in four queens at 33 days of gestation.3 also, it has been shown that pgf (0.5 to 1.0 mg/kg)� injected after day 40 of pregnancy induced abortion in queens.4 however, using a similar treatment regimen, it was reported that pgf (400 mcg/kg bw) induced luteolysis in cats, but without subsequent abortion.5 prolactin is a luteotropic hormone in dogs and cats. inhibition of pituitary prolactin release by the dopamine agonist (cbr) initiated abortion through a marked suppression of plasma progesterone.6 it has been shown that daily administration of cbr during mid-pregnancy was effective in pregnancy termination in 80% of treated queens.3 these results were similar to others who recorded 92.3% abortion in queens treated orally with cbr on day 40 of pregnancy.6 however, in feral cats, it has been shown that oral administration of cbr (5 to 15 mcg/kg/ bw) for four to nine days during estimated 36-41 days of pregnancy, resulted in 100% abortion at 40.5 ± 6.19 days of pregnancy.7 467 clinical theriogenology • volume 6, number 4 • december 2014 the abortion success rate of progesterone receptor antagonist treatment is reported to be only 67%.1 in another study, two cats were treated with cbr (15 µg⁄kg; daily, orally) combined with alfaprostol (10 ug⁄kg; every other day, s.c.) starting on day 45 after mating. this treatment was not successful, although the cats were treated seven and eight times, respectively.8 modification of the above protocols by combining pgf and cbr might provide a more effective approach for termination of pregnancy. for instance, in dogs, the combination of low dose of pgf and oral cbr was shown to induce abortion in 100% of the treated bitches.9,10 moreover, it was reported that the combination of a synthetic pgf and cbr treatment during mid-gestation induced abortion in all five treated queens with minor side effects 11. the results of termination of unwanted pregnancies with dopamine agonist or pgf in cats are inconsistent (0 to 100% success rate) and the research on use of pregnancy with dopamine agonist and pgf combined is limited. moreover, to our knowledge, the effect of dopamine agonist, pgf and their combination on pregnancy termination in cats has not been compared in side by side studies. the objectives of the present study were to simultaneously compare the efficacy and clinical properties of crb, natural pgf, and their combination in termination of pregnancy in queens, and to observe any side effects of these treatments. materials and methods animals the virginia tech institutional animal care and use committee approved all the procedures used in these experiments. fifteen adult female cats (18.1 ± 0.6 months old) with average body weight of 4.2 ± 0.7 kg were used in two trials. cats were group housed, fed once a day with a commercial dry feline diet (feline maintenance,® hill’s pet nutrition, topeka, ks), and given access to water ad libitum. cats were exposed to a lighting schedule of 10 h dark and 14 h light. when estrous behavior was observed, all queens were left with a fertile male cat for five days. estrous behavior and occurrence of mating was monitored three times daily. the date of the first successful mating was recorded. in both trials, all queens were bred by the male during the five-day period. all queens were housed together during pregnancy and after abortion. on day 25-28 after first mating, pregnancy was confirmed by abdominal palpation and a real time ultrasound scanner, equipped with a 5 mhz transducer probe (aloka co., ssd-500, mure, mitaka-shi, tokyo, japan). to estimate the stage of pregnancy, ultrasound images were used and fetal size was measured by an experienced veterinarian to better estimate the age of conceptus.12 thirty days after the completion of the first trial, a second trial was conducted using the same animals and using the same procedures as in trial one. none of the queens in trial two received the same treatment as in trial one. however, in the second trial, one cat was eliminated from the study due to respiratory problems unrelated to the experiment. treatment treatments were initiated between day 35 41 after first recorded mating and ten days after pregnancy was confirmed. animals were assigned randomly to one of three treatment groups. an oral formulation of cbr (50 mcg/ml; galastop®, vetem, centralvet, milan) at a daily dosage of 50 mcg per cat (approximately 12 mcg/kg bw) was used. natural pgf (lutalyse®, zoetis, florham, nj) was administered s.c. at a daily dosage of 2.5 mg per cat (approximately 0.6 mg/kg bw). after detection of pregnancy, and between day 35-41 after mating, cats were randomly divided into three treatment groups. group 1 received oral cbr (50 mcg per cat; n = 10) for five consecutive days or less if abortion had occurred earlier; group 2 received (s.c.) 2.5 mg per cat of pgf for five days or less if abortion had occurred earlier (n =10); group 3 received both oral cbr (50 mcg per cat) and pgf (2.5 mg per cat, s.c.) for five days or less if abortion had occurred earlier (n =9). to examine fetal presence and viability, the queens were monitored by ultrasonography on day 3 and 5 after initiation of treatment during treatments and every day after completion of the treatments for three days. the presence of potential side effects after daily treatment (tachypnea, prostration, vocalization, and emesis) was monitored and recorded. 468clinical theriogenology • volume 6, number 4 • december 2014 statistical analysis data from both trials were analyzed by genmod procedure on sas.13 the full statistical model included the effects of treatment (cbr, pgf or cbr + pgf), trial (trial 1and trial 2) and two-way interactions. statistical significance was defined as p ≤ 0.05. if the treatment was significant, preplanned contrasts were carried out to compare the proportion pregnancy loss means among treatments (cbr + pgf vs. cbr and vs. pgf and cbr vs pgf). results there was no effect of trial or trial by treatment interaction. there was an effect of treatment on percentage of cats which lost their pregnancy (p < 0.05). pregnancy losses were 100% (9/9) and 60% (6/10) for crb + pgf and crb groups, respectively, and were greater (p < 0.05) than the pgf group (10%, 1/10; table ). in the cbr group, oral administration of cbr for five days induced abortion in six of ten cats (60%) within three days after the completion of the treatment. in four of these six cats, abortion involved the observed expulsion of fetuses. in the other two cats, no fetus expulsion was observed. ultrasonography conducted on day five after start of the treatment indicated the presence of non-viable fetuses as no heartbeats were detected. oral cbr did not produce any side effects except hemorrhagic vulvar discharges were noticed in all queens on day four and five of treatment. in the pgf group, subcutaneous administration of pgf failed to induce abortion within eight days after the initial treatment period except in one of ten cats. that one abortion occurred at the end of eighth day in both trials, during the treatment period (five days) and three days after the last injection, ultrasonography revealed normal fetuses that appeared viable as evidenced by heartbeats. side effects occurred in the queens approximately ten minutes after drug administration and included tachypnea, vocalization, and emesis. after the third injection, queens seemed to adapt to the drug and side effects diminished considerably by the fourth and fifth injection. there were no signs of a hemorrhagic vulvar discharge in the cat that lost its pregnancy and other cats in this group. in the cbr + pgf group, a combination of pgf and cbr resulted in a rapid loss of pregnancy in all cats (100%) within 3-6 days after initiation of treatment, which was greater (p ≤ 0.05) the other two treatment groups. five cats aborted by day 3 (after two doses), three cats aborted by day 4 (after three doses), and one cat aborted on day 6 (after five doses) after the start of the treatment. abortion involved observed expulsion of fetuses in all cats. the side effects observed with this treatment group (cbr + pgf) were similar to those observed in cbr and pgf groups, including occasional emesis, prostration, and vocalization. furthermore, hemorrhagic vulvar discharge was observed. because we wanted to terminate pregnancy in the 13 cats that remained pregnant (four cats in the cbr group and nine cats in the pgf group), on day 9 and 10 after treatment initiation, these cats received two doses of cbr + pgf which resulted in abortion in all 13 cats, two days (four cats) or three days (nine cats) after the last dose. abortion involved observed expulsion of fetuses in 12 cats. the side effects were similar to those observed in cbr + pgf treated cats. discussion to our knowledge, this is the first study that simultaneously examined the effect of the dopamine agonist cbr, pgf, and their combination for inducing abortion in cats. it was reported that a combination of cbr and synthetic pgf resulted in termination of pregnancy in all cats (five cats) compared with controls.11 however, in the present study, abortions occurred within 3-6 days after the start of treatment. in contrast, in the previous study,11 abortion occurred within 9 ± 1 days. the differences between the present experiment and the previous report could be attributable to cbr treatment protocol, the type and regimen of pgf used, and (or) the stage of gestation at when treatment was initiated. in the previous study,11 synthetic pgf (cloprostenol) was administered every two days (45 injections) starting on day 30 after mating whereas in the present study, we administered natural pgf (dinoprost tromethamine) daily between day 35-40 after mating. moreover, we administered cbr for 469 clinical theriogenology • volume 6, number 4 • december 2014 only five consecutive days, whereas, in the previous study, cbr was administered every day for a mean of 11 days. five doses of cbr (50 mcg per cat) induced abortion in only six of the ten cats three days after the completion of the treatment. the results are in accord with previous findings7 that recorded about 92% abortion rate in queens treated orally with cbr on day 40 of pregnancy. furthermore, the daily use of cbr injections from day 30 after mating terminated pregnancy in 80% of the treated cats.3 these results, however, are different from others who showed that 4-9 days of cbr treatment at an average of 36-41 days of estimated pregnancy, resulted in 100% abortion at 40.5 ± 6.19 days after mating.7 it should be noted in that study, confirmation of pregnancy and abortion was solely based on changes in behavior, body confirmation, and clinical and social interaction.7 the findings of this experiment combined with others3,6,7 indicate that oral administration of cbr can be effective in inducing abortion in cats; however a longer treatment period (>5 days) may be required in order to reach 100% efficacy.7 the mechanism of action by which crb induced pregnancy loss cannot be derived from this study as no blood hormones were measured. corpora luteal are necessary to maintain pregnancy in the second half of gestation in cats.14 furthermore, prolactin is an essential luteotropic hormone in cats and it appears to act by sustaining corpus luteum lifespan and function after 25 days of gestation in cats.14 there is substantial evidence that cbr is an ergoline derivative with potent, selective and long-lasting inhibitory activity on prolactin and can inhibit prolactin secretion in cats.3,14 therefore, cbr-induced inhibition of prolactin causes a substantial decrease in luteal progesterone and pregnancy loss.3,11,14 in the current study and the previous studies pregnancy loss occurred between seven and ten days after initiation of oral cbr treatments.11,14 it is hypothesized the delay between luteolysis induced by cbr and abortion may be due to slower rate of decline in progesterone (through indirect inhibition of prolactin), as well as existence of small amount of progesterone from the cat’s placenta in second half of pregnancy that is limited in duration. this small amount of progesterone may delay pregnancy loss, but is insufficient to overcome the marked decrease in luteal progesterone caused the luteolytic effect of cabergoline.3,14 in the pgf group, with exception of one queen, subcutaneous administration pgf did not result in abortion within eight days after the first injection. these results were similar to previous research that showed pgf was luteolytic in cats, but did not induce abortion.5 our findings in pgf-treated group are contrary to others,3 which showed that intramuscularly injection of natural pgf successfully (100%) induced abortion in queens (four cats) when administered on day 30 to 33 after mating. the exact reason(s) for the observed difference between the present experiment and the previous study3 is not clear except that pgf was administered intramuscularly during an earlier stage of gestation. whether the route and time (relative to mating) of pgf treatment contributes to the effectiveness of pgf as an abortifacient drug, requires more research. it is apparent that the use of cbr alone during day 35-41 of pregnancy has advantages to that of pgf treatment alone as it can induce abortion without noticeable side effects. however, a five-day treatment was not sufficient to produce 100% pregnancy termination. in contrast, the combination of pgf and cbr was 100% effective in pregnancy termination and required a shorter treatment period (three to five days). it is argued that pgf through its direct luteolytic action combined with cbr by inhibiting prolactin have synergistic effect in decreasing progesterone and result in 100% pregnancy termination compared with cbr or pgf alone.14 these results provide further evidence that a combination of cbr and pgf is a quick and effective method for pregnancy termination in cats during the second half of pregnancy. at 35 to 41 days after mating, treatment with cbr (at the above dose) alone was less effective in pregnancy termination compared with cbr+ pgf. furthermore, pgf treatment alone, at least at the dose used here, was not effective in pregnancy termination in cats during 35-41 days after mating. references 1. georgiev p, bostedt h, goericke‐pesch s, et al: induction of abortion with aglepristone in cats on day 45 and 46 after mating. reprod domest anim 2010;45:e161-e167. 470clinical theriogenology • volume 6, number 4 • december 2014 2. lein dh, concannon pw, hornbuckle we, et al: termination of pregnancy in bitches by administration of prostaglandin f-2 alpha. j reprod fertil suppl 1989;39:231-240. 3. verstegen jp, onclin k, silva ld, et al: abortion induction in the cat using prostaglandin f2 alpha and a new antiprolactinic agent, cabergoline. j reprod fertil suppl 1993;47:411-417. 4. nachreiner rf, marple dn: termination of pregnancy in cats with prostaglandin f2 alpha. prostaglandins 1974;7:303308. 5. wycoff jt, ganjam vr: use of prostaglandin f2α in the cat. proc fed am soc exp biol 1979;38:1189-1193. 6. jöchle w, arbeiter k, post k, et al: effects on pseudopregnancy, pregnancy and interoestrous intervals of pharmacological suppression of prolactin secretion in female dogs and cats. j reprod fertil suppl 1989;39: 199-207. 7. jöchle w, jöchle m: reproduction in a feral cat population and its control with a prolactin inhibitor, cabergoline. j reprodfertil suppl 1993;47:419-424. 8. erünal-maral n, aslan s, findik, m, et al: induction of abortion in queens by administration of cabergoline (galastop™) solely or in combination with the pgf2α analogue alfaprostol (gabbrostim™). theriogenology 2004;61:1471-1475. 9. onclin k, silva ldm, verstegen jp: termination of unwanted pregnancy in dogs with dopamine agonist cabergoline in combination with a synthetic analogue of pgf2α either cloprostenol or alphaprostol. theriogenology 1995; 43:813822. 10. onclin k, verstegen jp: practical use of a combination of a dopamine agonist and a synthetic prostaglandin analogue to terminate unwanted pregnancy in dogs. j small anim pract 1996;37: 211-216. 11. onclin k,verstegen jp: termination of pregnancy in cats using a combination of cabergoline, a new dopamine agonist, and a synthetic pgf2 alpha, cloprostenol. j reprod fertil suppl 1997;51:259-263. 12. zambelli d, catagnetti c, belluzzi s, et al: correlation between the age of the conceptus and various ultrasonographic measurements during the first 30 days of pregnancy in domestic cats (felis catus). theriogenology 2002;57:1981-1987. 13. sas institute inc., sas 9.1.3 cary, nc: sas institute inc.,2000-2004. 14. onclin k, verstegen jp: in vivo investigation of luteal function in dogs: effects of cabergoline, a dopamine agonist, and prolactin on progesterone secretion during mid-pregnancy and-diestrus. domest anim endocrinol;1997;14:25-38. 15. verstegen jp, onclin k, silva ld, et al: regulation of progesterone during pregnancy in the cat: studies on the roles of corpora lutea, placenta and prolactin secretion. j reprod fertil suppl 1993;47:165-173. table: effect of treatment with cabergoline (5 days), prostaglandin f2alpha (5 days), or their combination (2 to 5 days) on efficacy of pregnancy termination starting at days 35 to 41 after mating in queens. treatment dosage no. of dose efficacy pterm/trta time of pregnancy termination pgf (n=10) 2.5 mg (s.c.) 5 1/10 (10%)a 8 days after end of treatment cabergoline (n = 10) 50 mcg (oral) 5 6/10 (60%)b 3 days after end of treatment cabergoline + pgf (n = 9) 50 mcg & 2.5 mg (oral & s.c.) 2-5c 9/9 (100%)c on day 3 of treatment (n=5); on on day 4 of treatment (n=3); a day after end of treatment (n=1) a pterm/trt: number of pregnancy termination/number of queens treated. * a,b,c within a column, values with different superscript differ (p ≤ 0.05) 471 clinical theriogenology • volume 6, number 4 • december 2014 472clinical theriogenology • volume 6, number 4 • december 2014 omniblank: 2010: a pilot study: comparison of pregnancy rates from vitrified jersey embryos utilizing direct transfer methods with pregnancy rates of jersey and holstein embryos frozen in ethylene glycol or glycerol by slow-cooling methods a pilot study: comparison of pregnancy rates from vitrified jersey embryos utilizing direct transfer methods with pregnancy rates of jersey and holstein embryos frozen in ethylene glycol or glycerol by slow-cooling methods richard g. steel evergreen veterinary reproductive services, tillamook, or abstract it has been previously reported that pregnancy rates from frozen jersey embryos are lower than pregnancy rates from other breeds of cattle. this study incorporated 1.5m ethylene glycol or 1.4m glycerol as cryoprotectants and utilized slow-cooling freeze programs. the present study was undertaken to determine if the vitrification of jersey embryos in 0.25 cc straws could result in a more favorable pregnancy rate than previously reported. in addition, an in-straw dilution of cryoprotectants with resulting direct transfer (dt) of embryos was evaluated to determine if this methodology is compatible with vitrification processes. ten superovulations of nine jersey donor cows yielded 50 embryos that were vitrified and subsequently thawed and transferred into suitable recipients using dt methodology. the resulting pregnancy rate (28%) is numerically lower than retrospective pregnancy rates achieved from jersey embryos slow-cooled in 1.4m glycerol or 1.5m ethylene glycol (46.2% and 39.9% respectively). however, two jersey donors achieved reasonable pregnancy rates with their vitrified embryos (50%). this study has shown that average pregnancy rates of vitrified jersey embryos utilizing dt methodology is numerically lower than average pregnancy rates of conventional, slow-cooled embryos. however, due to the wide range of pregnancy rates in this study, individual donor cows may have different cryotolerances to this vitrification method. further modifications of vitrification systems utilizing dt methods may eventually improve overall pregnancy rates of frozen jersey embryos. keywords: vitrification, pregnancy rates, direct transfer, slow-cooling, jersey embryos introduction cryopreservation of bovine embryos has been a routine procedure performed in commercial embryo transfer businesses for many years. pregnancy rates resulting from the transfer of frozen bovine embryos are for the most part very predictable as evidenced by the widespread domestic and international commercial activity of selling frozen embryos. many types of biologic specimens have been successfully cryopreserved; however, there have been some exceptions related to a decreased tolerance to chilling. some notable examples are porcine embryos,1 oocytes,2 in-vitro produced embryos3 and bos indicus embryos.4 several reports in the literature have described lower pregnancy rates of frozen jersey embryos when compared to pregnancy rates of frozen embryos from other dairy breeds.5-7 there has been some speculation about the reasons for this occurrence, especially with the discovery that jersey embryos may have a relatively higher lipid content than embryos from other breeds.7,8 some investigators have observed that embryos with higher lipid concentrations experience depressed frozen embryo pregnancy rates.1,7,9,10 this has lead the industry to test other types of cryoprotectants and procedures that may be more appropriate for jersey embryos. vitrification, although not a new process,11 is a freezing procedure that has been successfully applied to many types of specimens.12-20 recently, studies have compared pregnancy rates from embryos frozen by slow-cool conventional methods with embryos frozen by vitrification.21-30 some of these studies have shown vitrification to be superior to conventional freezing techniques.22,27,31-33 most vitrified specimens undergo a serial dilution of cryoprotectants in a laboratory setting prior to culture and/or transfer. in-straw dilution of cryoprotectants was described a number of years ago34 and has the advantage of bypassing any laboratory environment by allowing dt of an embryo into a recipient animal after thawing and equilibration. however, with the advent of 1.5m ethylene glycol as a cryoprotectant in slow-cooling systems, serial dilution of cryoprotectants is not necessary, making use of this cryoprotectant and dt the method of choice in the embryo transfer industry. ninety percent of all bovine embryos thawed in the us in 2007 utilized the dt method.35 445 to date, there have not been sufficient data generated to determine if vitrification of jersey embryos would increase frozen embryo pregnancy rates. there have also been very few studies involving in-straw dilution of cryoprotectants of vitrified embryos with subsequent dt.26,36,37 thus, the aim of this study was to compare pregnancy rates of vitrified jersey embryos utilizing in-straw dilution of cryoprotectants and direct dt with retrospective pregnancy rates of jersey and holstein embryos frozen in glycerol or ethylene glycol by conventional slow-cooling methods. materials and methods animals ten superovulations were performed on nine lactating jersey donor cows at least two years of age on five different dairies. donors were examined for normal reproductive health before enrollment. one donor cow was superovulated twice. recipient animals were both lactating cows and virgin heifers and included jersey and holstein breeds. superovulation all donors were treated with a progesterone-containing vaginal insert (eazi-breed™ cidr®, pfizer animal health, new york, ny) at random stages of the estrous cycle four days prior to superstimulation. donors also received 100 mcg gonadotropin-releasing hormone (gnrh, cystorelin®, merial, duluth, ga) 60 hours prior to superstimulation. superstimulation was accomplished with a decreasing dose of follicle stimulating hormone (fsh; folltropin®-v, bioniche, bogart, ga) twice daily over four days for a total dose of 300 mg. thirty-seven mg and 25 mg dinoprost tromethamine (lutalyse®, pfizer animal health) was given at the same time as the fifth and sixth fsh injection, respectively. vaginal inserts were removed when the sixth fsh injection was given. donor cows were inseminated twice with one unit of semen at 12 and 24 hours after the onset of estrus. embryos were collected 7.5 days after the onset of estrus. recipient synchronization recipients were synchronized with either one or two injections of 25 mg dinoprost tromethamine or a progesterone-containing vaginal insert. recipients synchronized with a single injection of dinoprost tromethamine were observed for estrus two to five days after treatment. recipients synchronized with two injections were treated at an interval of 12-14 days and observed for estrus two to five days after the second injection. progesterone-containing vaginal inserts were placed at random stages of the estrous cycle and left in place for seven days. twenty-four hours prior to insert removal, recipients were injected with 25 mg dinoprost tromethamine. the animals were observed for estrus for five days after removal of the inserts. only recipients detected in standing estrus and that had a mature corpus luteum on the day of transfer were used. embryos frozen conventionally retrospective data from the last 15 years were compiled to compare pregnancy rates utilizing slow-cooling protocols. grade 1 or 2 jersey and holstein embryos were frozen by conventional slowcooling methods with either 1.4m glycerol (gly) or 1.5m ethylene glycol (eg). all embryos were thawed for six seconds in air, then 15 seconds in 30 °c water. embryos frozen in glycerol were passed through a three step serial dilution in the presence of 0.5m sucrose. after serial dilution, embryos were transferred within one hour of thawing. embryos frozen in ethylene glycol were immediately loaded into a transfer gun after thawing and transferred within 10 minutes. embryos vitrified an aluminum block, partially submerged in a liquid nitrogen bath, was utilized as a vitrification chamber. this device contains holes 123 mm deep with a diameter that allows a 0.25 cc straw to fit snugly against the metal sidewall with as little air interface as possible. grade 1 and 2 embryos were first 446 exposed to vitrification solution 1 (5.0m ethylene glycol) for three minutes then placed in a 15 ul drop of vitrification solution 2 (7.0m ethylene glycol plus 0.5m galactose with 18% ficoll™ [ge healthcare, piscataway, nj]). embryos were immediately loaded into a 0.25 cc straw in the following manner: 173 ul dilution solution (1.0m galactose), a 10 mm air bubble, embryo in a 15 ul micro drop of vitrification solution 2, a 10 mm air bubble, with the remaining space filled with diluent solution. straws were then sealed and embryos were visualized to confirm their exact location prior to plunging. embryos were exposed to vitrification solution 2 for 45 seconds after which the straw was inserted into the vitrification device for 60 seconds. straws were then immediately plunged into liquid nitrogen and stored in goblets until transfer between one hour and 30 days later. embryos were thawed six seconds in air, then 20 seconds in 35 °c water. straws were dried and shaken in a downward manner until all media within the straw was mixed. immediately after thawing, embryos were visually located during a four minute equilibration at 25 °c to ensure they were not in the end of the straw that was to be cut. embryos were then loaded into a transfer gun and transferred into a suitable recipient within ten minutes after equilibration. pregnancy examination all animals were manually examined for pregnancy 35 to 60 days after the calculated date of conception. results a total of 52 grade 1 and 2 embryos were recovered from ten superovulations of nine jersey donor cows for an average of 5.2 embryos per flush (table 1). fifty embryos were vitrified and two embryos were discarded during the vitrifying procedure. this was due to the fact that these two embryos were exposed to vitrification solution 2 longer than 45 seconds due to technical difficulties and were excluded from this study. a total of 50 vitrified jersey embryos were thawed and transferred into suitable recipients resulting in 14 pregnancies (28%). pregnancy rates for embryos frozen from each individual donor cow ranged from 0 to 50% (table 1). pregnancy rates on individual dairies ranged from 0 to 41.7% (table 2). upon ultrasonic examination at 75 days gestation, one recipient on farm 2 was found to have twin female fetuses, the result of spontaneous splitting of a vitrified embryo after thawing. discussion an average pregnancy rate of 28% achieved from the transfer of 50 vitrified jersey embryos is numerically lower that the average pregnancy rates achieved from the transfer of jersey and holstein embryos slow-cooled in 1.4m glycerol or 1.5m ethylene glycol (table 3). pregnancy rates of jersey embryos, regardless of freezing technique, were numerically lower than pregnancy rates of frozen holstein embryos. although data are limited, vitrified embryos from two donors resulted in pregnancy rates of 50.0%. a wide variation of pregnancy rates (0-50.0%) from individual jersey donors is apparent in this study of vitrified embryos, and, in the author’s experience, jersey embryos frozen by conventional slow-cooling methods.* the variation between jersey donor cows that achieve above average pregnancy rates with frozen embryos, and those that underachieve on a consistent basis, indicates there may be differences in freezing tolerances among individual donors within the same breed. this pilot study did not confirm the results of a previous study in which bovine embryos were vitrified in 6.5m glycerol and cryoprotectants diluted after thawing in 1m sucrose prior to dt. utilizing 728 recipients, pregnancy rates from vitrified embryos were similar to those from embryos slow-cooled and frozen in 1.5m glycerol with a three step serial dilution after thawing (45.1% vs. 44.5%, respectively).36 with the methods used in the current study, larger data sets would be necessary to test for significant differences in pregnancy rates between vitrified jersey embryos and jersey embryos frozen with conventional slow-cooling methods. it is important to note that proper placement of the embryo within the vitrifying environment is extremely important when making critical evaluations of this process. prior to this study, 50 unfertilized * r. steele, unpublished observations 447 bovine ova were vitrified and it was noted that two ova were improperly located within the diluent solution after the straws were sealed in the current study, all embryos were visually located prior to freezing to confirm their proper location within the vitrification environment. in addition, upon thawing and mixing of diluent within the straw , embryos were located and tracked during equilibration to ensure they did not drift too close to the sealed end that was to be opened prior to transfer. conclusion vitrification of mammalian embryos has been shown to be successful in a variety of clinical settings. perhaps the greatest challenge in undertaking this study was to incorporate an in-straw dilution and subsequent direct transfer method of vitrified embryos. can acceptable pregnancy rates be achieved with vitrified embryos without the time and expense of passing embryos through a serial dilution of cryoprotectants? can proper vitrification occur within the confines of a 0.25 cc straw where a large sample size (15 ul micro drop) and the insulating effects of the straw may be detrimental to the process? both of these aspects would have great bearing on achieving an ultra-rapid cooling rate, a factor of critical importance in the vitrification process. this study has shown that average pregnancy rates from vitrified jersey embryos are numerically lower than the average pregnancy rates of jersey embryos slow-cooled by conventional methods. however, some individual exceptions were noted as vitrified embryos from two jersey donors achieved a 50% pregnancy rate. donors that achieve high average pregnancy rates often demonstrate the potential of a new methodology. although jersey embryos may possess unique characteristics in their cryotolerance, modification of current vitrification processes utilizing dt methods, combined with further field trials with larger numbers of animals, may help improve overall pregnancy rates with frozen jersey embryos. acknowledgements this study was supported by a grant from the ajcc research foundation of the american jersey cattle association. the author thanks both denise steel and stephanie miranda for technical assistance. table 1. summary table of superovulation and vitrification results. †one embryo discarded due to improper timing of vitrification process. superovulation number total recovered total grade 1 & 2 total grade 3 total degen. total unfert. total vitrified total thawed total preg. preg rate (%) 1 15 3 0 0 12 3 3 0 0.0 2 15 12 1 0 2 11† 11 2 18.2 3 7 5 0 1 1 5 5 2 40.0 4 5 0 0 0 5 0 n/a n/a n/a 5 6 3 0 0 3 2† 2 0 0.0 6 13 10 0 2 1 10 10 5 50.0 7 6 6 0 0 0 6 6 1 16.7 8 22 8 0 0 14 8 8 4 50.0 9 3 0 0 2 1 0 n/a n/a n/a 10 7 5 1 0 1 5 5 0 0.0 totals 99 52 2 5 40 50 50 14 28.0 averages 9.90 5.20 0.20 0.50 4.00 5.00 448 table 2. summary of pregnancies obtained at five different jersey farms. table 3. pregnancy rates resulting from the transfer of fresh and frozen holstein and jersey embryos. holstein embryos jersey embryos embryos no. transferred % pregnant no. transferred % pregnant fresh‡ 3376 65.3 1161 62.8 frozen gly‡ 836 57.7 593 46.2 frozen eg‡ 1111 53.8 193 39.9 vitrified ----50 28.0 ‡historical data5 references 1. berthelot f, martinat-botte f, vajta g: cryopreservation of porcine embryos: state of the art. livest prod sci 2003;83:73-83. 2. otoi t, kamamota k, koyama n, et al: cryopreservation of mature bovine oocytes following centrifugation treatment. cryobiology 1997;34:36-41. 3. massip a, mermillod p, dinnyes a: morphology and biochemistry of in-vitro produced bovine embryos, implications for their cryopreservation. hum reprod;1995;10:3004-3011. 4. visintin ja, martins jfp, bevilacqua ev, et al: cryopreservation of bos tarus vs bos indicus embryos: are they really different? theriogenology 2002;57:345-359. 5. steel r, hasler jf: pregnancy rates resulting from the transfer of fresh and frozen holstein and jersey embryos. reprod fertil dev 2004;16:182-183. 6. kieler m: embryo comparisons for five dairy breeds. proc am embryo transf assoc; 2008. p. 49-50. 7. weathers j: early indications of breed differences for cryopreservation of embryos in cattle [thesis]. lubbock (tx): texas tech university; 2008. 8. pegoraro lm, barros s, sinowatz f, et al: lipidic content in jersey blastocysts compared with holstein and ivp embryos. reprod fertil dev 2004;16:268. 9. george f, daniaux c, genicot g, et al: lipid content, resistance to cryopreservation and sex ratio of in-vitro bovine blastocysts produced in a serum free media. reprod fertil dev 2006;18:188-189 10. abe h, yamashita s, satoh t, et al: accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media. mol reprod dev 2002;61:57-66. 11. vafta g, nagy zp: are programmable freezers still needed in the embryo laboratory? review on vitrification. reprod biomed online 2006;12:779-796. 12. siedel g: new methods of cryopreserving bovine embryos. proc am embryo transf assoc 2007. p. 98-101. 13. ishimore h, saeki k, inai m, et al: vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide. theriogenology 1993;40:427-433. 14. stachecki j, garrisi j, sabino s, et al: a new safe, simple and successful vitrification method for bovine and human blastocysts. reprod biomed online 2008;17:360-367. 15. vajta g: vitrification of the oocytes and embryos of domestic animals. anim reprod sci 2000;60-61:357-364. 16. campos-chillos lf, suh tk, barelo-fimbres m, et al: vitrification of early stage bovine and equine embryos. theriogenology 2009;71:349-354. 17. kuwayama m, vajta g, kato o, et al: highly efficient vitrification method for cryopreservation of human oocytes. reprod biomed online 2005;11:300-308. 18. frers l, hepburn j, mandriaza-munoz j, et al: vitrification of bovine in-vitro produced and in-vivo embryos. reprod fertil dev 2009;21:131-132. 19. vanderzwalmen p, zech n, greindl aj, et al: cryopreservation of human embryos by vitrification. gynecol obstet fertil 2006;34:760-769. farm total pregnancies total transferred pregnancy rate (%) 1 0 5 0.0 2 7 25 28.0 3 5 12 41.7 4 2 5 40.0 5 0 3 0.0 totals 14 50 28.0 449 20. chun-chia h, tsung-hsien l, shee-uan c, et al: successful pregnancy following blastocysts cryopreservation using super-cooling ultra rapid vitrification. hum reprod 2005;20:122-128. 21. da silva fm, metelo r: relation between physical properties of the zona pellucida and viability of bovine embryos after slow-freezing and vitrification. reprod domest anim 2005;40:205-209. 22. stehlik e, stehlik j, datayama kp, et al: vitrification demonstrates significant improvement versus slow freezing of human blastocysts. reprod biomed online 2005;11:53-57. 23. vajta g, kuwayama m: improving cryopreservation systems. theriogenology 2006;65:236-244. 24. dupras r, dupras j, chorfi y: cell integrity of holstein and jersey embryos. comparison of two freezing methods. reprod fertil dev 2008;20:149-150. 25. liebermann j, tucker mj: comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. fertil steril 2006;86:20-26. 26. massip a, vanderzwalmen p, ectors f: recent progress in cryopreservation of cattle embryos. theriogenology 1987;27:69. 27. loutradi, k, kolibianakis em, venetis ca, et al: cryopreservation of human embryos by vitrification or slow freezing: a systematic review and meta-analysis. fertil steril 2008;90:186-193. 28. massip a: cryopreservation of embryos of farm animals. reprod domest anim 2001;36:49-55. 29. liebermann j, dieti j, vanderzwalmen p, et al: recent developments in human oocyte, embryo and blastocyst vitrification: where are we now? reprod biomed online 2003;7:623-633. 30. kolibianakis em, venetis ca, tariatzis, bc: cryopreservation of human embryos by vitrification or slow freezing: which one is better? curr opin obstet gynecol 2009;21:270-274. 31. kuleshova l, lopata a: vitrification can be more favorable than slow cooling. fertil steril 2002;78:449-454. 32. al-hasani s, ozmen b, koutlaki n, et al: three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing? reprod biomed online 2007;14:288-293. 33. urman b: vitrification of human embryos is superior to conventional slow freezing (review). fertility weekly 2008 nov 10. 34. leibo s: a one step method for direct nonsurgical transfer of frozen-thawed bovine embryos. theriogenology 1984;21:767-790. 35. american embryo transfer association: statistical report 2007. champaign (il): aeta. 36. van wagtendonk-de leeuw am, den daas jhg, rall wf: field trial to compare pregnancy rates of bovine embryo cryopreservation methods. theriogenology 1997;48:1071-1084. 37. campos-chillon lf, walker dj, de la torre-sanchez jf, et al: in vitro assessment of a direct transfer vitrification procedure for bovine embryos. theriogenology 2006;65:1200-1214. 450 1 contact camilo hernández-avilés chernandez@cvm.tamu.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10832, http://dx.doi.org/10.58292/ct.v16.10832 review report stallion sperm cryopreservation: concepts in cryopreservation-induced sperm damage, processing, analysis, and utilization in assisted reproductive technologies* camilo hernández-avilés equine fertility laboratory, department of large animal clinical sciences, college of veterinary medicine and biomedical sciences, texas a&m university, college station, tx, usa abstract semen cryopreservation remains the only viable method for long-term stallion sperm preservation. increased demand for frozen/ thawed sperm in assisted reproductive technologies (arts) continues to result in a considerable body of research determining the effects of cryopreservation on stallion sperm and clinical interpretation of sperm quality in frozen/thawed semen. some concepts regarding the impacts of cryopreservation on stallion sperm or methods for sperm cryopreservation might limit the widespread implementation of this technique in private practice settings. clinically relevant data on stallion sperm cryopreservation, various protocols for sperm cryopreservation, concepts on postthaw sperm quality analysis, and utilization of frozen/thawed sperm for arts are summarized. keywords: stallion sperm, cryopreservation, cryoprotectants, semen extender, postthaw quality introduction sperm cryopreservation, to date, is the primary method for long-term sperm preservation in domestic animals, including horses. since the initial reports by russian,1 british,2 japanese,3,4 german,5 and polish6 scientists regarding stallion sperm cryopreservation and the first report of successful insemination of a mare with frozen/thawed (epididymal) sperm,7 there has been an exponential increase in the ‘quest’ for suitable and repeatable methods for sperm cryopreservation that may eventually replace fresh or cool-stored semen utilization for artificial insemination (ai) of mares. multiple benefits of sperm cryopreservation and ai in horses have been described.8 because of these, european, asian (japan, china), and north american workers have dedicated extensive research programs focused on optimizing ai in mares with frozen/thawed semen, trying to emulate dairy breeding industry programs of 1960’s to 1970’s. studies conducted in 1970’s to 1980’s9 were reviewed,8,10 which reported 30-60% per cycle pregnancy rates in *presented at the 2024 society for theriogenology conference, published after peer review. mares  inseminated (daily until end of estrus) with frozen/ thawed sperm. recent retrospective studies11 (> 2,000 mares  bred in 2004) reported 46-48% per cycle pregnancy  rates in mares inseminated twice in their cycle (24  hours apart). currently, similar per cycle pregnancy rates can be obtained by 1 insemination performed immediately after  induced (with a synchronizing agent) ovulation (determined via transrectal palpation and ultrasonography, every 6-8 hours, starting 12-24 hours after  treatment).12-15 variations16 of this protocol ‘simpli fied’ ovulation synchronization, frequency of transrectal examinations, and ai after detected ovulation. overall, it is  apparent that insemination of frozen/thawed stallion sperm can yield widely accepted fertility rates (i.e. per cycle  pregnancy rates of 40-60%) for commercial settings,  provided adequate breeding management, and mare  and stallion intrinsic fertility are met. nevertheless, any of these results are affected by other factors, such as  availability of frozen sperm from a given stallion (often limited by stallion owner/agent), semen processing and resulting postthaw sperm quality, and availability and knowledge of the personnel (i.e. veterinarian) involved in insemination. mailto:chernandez@cvm.tamu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10832 2 citation: clinical theriogenology 2024, 16, 10832, http://dx.doi.org/10.58292/ct.v16.10832 more mares are currently bred with frozen/thawed sperm than before; yet, use of this technology for ai may still be less than fresh or cool-stored semen. such a trend has not changed since mid-1990’s,17 and may be attributed to multiple factors, including specific breed registries regulations (e.g. only until 2000, the american quarter horse association [world’s largest breed registry] allowed foals registration conceived by ai with frozen/ thawed sperm), difficulties in standardization of stallion sperm cryopreservation methods under field conditions,18 lack of consensus regarding ‘minimum’ criteria for postthaw sperm quality,18,19 and the relative ‘low’ relationship between in vitro sperm quality assays and in vivo stallion fertility.20-23 although the aim is not to provide extensive review of what has or has not been done regarding sperm cryopreservation in stallions, author prefers to review some relevant concepts on stallion sperm cryopreservation and discuss specific recent data that originated (author and his coworkers) regarding cryopreservation of stallion sperm, analysis of postthaw quality in frozen/thawed stallion semen, and frozen/thawed stallion sperm use for intracytoplasmic sperm injection (icsi) in mares. cellular and molecular changes due to cryopreservation of stallion sperm: what else can we ask them to survive? author names this section as a tribute to a seminal review paper24 by roy hammerstedt who in conjunction with james graham and john nolan conducted several studies on certain effects of cryopreservation on sperm plasma membranes, particularly in ruminants, using flow cytometry-based assays to study sperm function.25-27 certainly, the title (‘what we ask them to survive’) of the paper24 attested to challenges researchers and clinicians faced then that we still face when devising methods to maximize survival and fertilizing ability of frozen/ thawed stallion sperm. after all, it is surprising that sperm subpopulations are still able to survive and maintain fertilizing competence after sustaining such marked change in their membrane composition and overall function as a result of: 1. temperature-associated metabolic restriction; 2. conformational and functional changes at the organelle level (i.e. plasma membranes, mitochondria) due to exposure to supra (5-0°c) and subzero (< 0°c) temperatures; 3. osmotic changes during cryopreservation and after thawing caused by exposure to high molecular weight penetrating and nonpenetrating cryoprotectants and water freezing; and 4. resulting cellular (i.e. oxidative stress, apoptosis) and molecular (e.g. protein posttranslational modifications) changes that follow osmotic stress (figure 1). some of these changes will be discussed. sperm plasma membrane, similar to somatic cells, is composed of a phospholipid bilayer with extracellular, intracellular, and embedded transmembrane proteins, fatty acids, and a glycocalyx with potential roles during sperm oviduct/oocyte interactions.28-30 more comprehensive reviews31-34 described composition and function of this fascinating organelle, particularly during fertilization. considering sperm plasma membrane intricacy and composition, it is not surprising that multiple changes at the phospholipid or protein conformation level would result in membrane damage and subsequently, sperm death. for this review, consider the multiple steps required to freeze stallion sperm (figure 2). although initial dilution of the ejaculate with a prewarmed skim milk or casein-based extender (generally isosmotic: 300-350 mosm/kg) does not result in considerable changes to plasma membrane, temperature reduction (from 37 to ~ 22-20°c) during centrifugation theoretically would cause changes in sperm metabolic rate35 and semen freezing extender addition would affect plasma membrane organization due to hyperosmolality (semen freezing extenders have an osmolality that ranges 800-1200 mosm/ kg).36 additionally, temperature reduction during the initial prefreezing cooling phase (20-5°c) will induce conformational modifications to plasma membrane architecture due to  the so-called ‘thermotropic phase’ change, whereby the plasma  membrane phospholipids change from a ‘fluid’ (liquid-crystalline) to a ‘gel’ state due to interactions among hydrophobic phospholipid acyl chains (figure 1).37-40 these changes, in conjunction with reduced metabolic rate due to temperature restriction, have induced higher membrane permeability, mainly to ions such as potassium and calcium,41 alterations in membrane potential and permeability, loss of intracellular calcium regulation, and reduced sperm motility and membrane intactness in other mammalian sperm models, such as boars or ruminants (figure 1).24,41 to author’s knowledge, some of these changes have not yet been studied in stallion sperm, but it is generally assumed that similar alterations would occur during cooling and cryopreservation. after dilution of sperm with a freezing extender and exposure to lower temperatures (i.e. 5°c), additional conformational changes occur to plasma membrane due to ‘lyotropic membrane phase transitions,’ wherein water within the plasma membrane begins to freeze due to exposure to temperatures near 0°c.42 furthermore, when temperature reaches below 0°c, extracellular ice formation begins due to extracellular water freezing. such an increase in extracellular milieu osmolality further induces osmotic changes that will cause sperm dehydration (figure 1). although sperm dehydration is pivotal for survival after cryopreservation and thawing, excessive cell dehydration would result in plasma membrane rupture and cell death.43 studies44-46 have reported that freshly ejaculated stallion sperm can withstand specific ranges of ‘anisosmolality,’ mainly when sperm are suspended in a medium at 300 mosm/ kg (isosmolar), rapidly exposed to hyperosmolar conditions (900 mosm/kg) and then exposed again to isosmolar conditions. interestingly, a transmission electron microscopy study47 reported that hypoosmotic conditions (75-100 mosm/kg) are more harmful to plasma membrane and mitochondrial structure of fresh stallion sperm compared to hyperosmotic conditions (900 mosm/kg), particularly when sperm are rapidly removed from hyperosmotic to hypoosmotic media. such data can be interpreted as rapid exposure from hyperosmotic (during freezing) to isoosmotic conditions (after thawing) causes most damage to stallion sperm. nevertheless, serial dilution of cryopreserved stallion sperm into a skim milkbased extender with decreasing concentrations of cryoprotectant (glycerol [gly]) to avoid sudden changes in sperm osmolality did not yield higher motility or plasma membrane intactness than immediate exposure from hyperosmotic to isosmotic conditions.48 hence, the exact mechanism by which exposure to hyperosmotic conditions during cryopreservation (i.e. cryopreservation extender, water freezing, or both) and hypoosmotic conditions during thawing affect stallion sperm quality is not entirely understood. changes to fresh stallion sperm exposed to osmotic imbalances (also known as ‘osmotic excursions’) in plasma membrane and mitochondrial structure and function (i.e. intactness and membrane potential) have been extensively reported.44-47 additionally, it has been described that exposure of fresh stallion sperm to osmotic excursions, particularly iso-to-hyperosmolality, results in changes in sperm cell volume but not dramatic decreases in http://dx.doi.org/10.58292/ct.v16.10832 citation: clinical theriogenology 2024, 16, 10832, http://dx.doi.org/10.58292/ct.v16.10832 3 the percentage of plasma membrane-intact sperm.49 in contrast, exposure from hypo-to-isosmolality resulted in mitochondrial membrane potential collapse.49 such changes in mitochondrial membrane potential have been associated with decreased mitochondrial function, deregulation of reactive oxygen species (ros) production, and occurrence of mitochondrial-mediated apoptosis (figure 1).50-54 relationship between osmotic and oxidative stress (or decreased oxidation-reduction [redox] balance) in fresh stallion sperm was initially demonstrated55; stallion sperm exposed to osmotic excursions display an increased production of superoxide anion, a common byproduct of mitochondrial metabolism that in turn was potentially associated with activation of nicotinamide adenine dinucleotide phosphate oxidase.55 additionally, multiple studies50-54 have elucidated various cellular and molecular changes stallion sperm undergo after a freezing/thawing cycle (figure 1). after freezing and thawing, a subpopulation of plasma membrane-intact sperm had increased concentrations of lipid peroxidation byproduct 4-hydroxynonenal, activated caspases (3 and 7), membrane depolarization, and increased intracellular sodium concentrations and also reduced mitochondrial membrane potential and sperm motility. readers are referred to more extensive reviews regarding the effects of cryopreservation procedures on stallion sperm, particularly those focused on the occurrence of oxidative insults due to redox imbalance.56-58 interestingly, these studies reported the relevance of mitochondrial function in stallion sperm subjected to freezing/ thawing that further emphasizes the pivotal role of mitochondria for normal function of stallion sperm under in vitro conditions.59-62 hence, devising methodologies to maintain the mitochondrial function of stallion sperm has become a common objective for researchers working on stallion sperm cryopreservation. utilization of newer laboratory techniques, such as mass spectrometry-based technologies to study protein (proteomics), lipid (lipidomics), and metabolite (metabolomics) composition of sperm may shed light not only on cryoinjury mechanisms but also on potential targets for improving current protocols for sperm cryopreservation in stallions. in this regard, mass spectrometry-based techniques have been recently employed to determine changes in stallion sperm proteome after a freezing/thawing cycle, whereby a lower abundance of proteins associated with oxidative phosphorylation (the main metabolic pathway utilized by freshly ejaculated stallion sperm to produce atp), and some proteins associated to sperm-oocyte interactions were observed in frozen/thawed compared to freshly ejaculated sperm.63 studies also reported that stallion sperm subjected to a freezing/thawing cycle display a lower abundance of antioxidant figure 1. a graphical representation of some of the cellular changes that stallion sperm undergo during cryopreservation, including a. plasma membrane conformation changes due to reduced temperature; b. metabolic rate reduction due to reduced temperatures; c. osmotic-associated stress due to exposure to hyperosmolality during freezing; and d. oxidative stress associated to osmotic damage to the sperm mitochondria. figure created with biorender (www.biorender.com). ros: reactive oxygen species; hufa’s: highly unsaturated fatty acids http://dx.doi.org/10.58292/ct.v16.10832 http://www.biorender.com 4 citation: clinical theriogenology 2024, 16, 10832, http://dx.doi.org/10.58292/ct.v16.10832 enzyme systems, such as superoxide dismutase that are pivotal for redox balance maintenance.64 moreover, studies from this same group of researchers have also attempted to identify proteins of differential abundance in stallions that display higher postthaw motility (i.e. ‘good freezers’) when compared to ‘poor freezer’ stallions. these proteins include those associated with mitochondrial function, antioxidant enzyme systems, and seminal plasma-associated proteins.65,66 in summary, these new molecular biology technologies are promising tools to elucidate further the ‘pathophysiology’ of cryopreservation-induced damage in stallion sperm, provided a proper validation of some of these ‘biomarkers’ (i.e. proteins, lipids, and metabolites) is performed. in the years to come, we will observe an exponential increase in the utilization of these techniques in equine reproductive medicine. some clinically related concepts regarding stallion sperm cryopreservation after considering the vast array of cellular and molecular changes that stallion sperm undergo during cryopreservation and thawing, it is evident that specific cryopreservation process steps would be of higher interest while designing methods to maximize sperm survival after thawing. to author, it is noteworthy that many current techniques (semen extenders, cooling, freezing, and thawing methods) for stallion sperm cryopreservation were initially adapted from other species (i.e. cattle) and have not changed dramatically during last 2 decades. many commercial and ‘homemade’ preparations of semen extenders employed currently were used and reported in scientific literature; nevertheless, most of these formulations are based on the same components initially devised figure 2. a graphical representation of a typical cryopreservation protocol utilized in the author’s laboratory for stallion sperm. figure created with biorender (www.biorender.com). http://dx.doi.org/10.58292/ct.v16.10832 http://www.biorender.com citation: clinical theriogenology 2024, 16, 10832, http://dx.doi.org/10.58292/ct.v16.10832 5 more than 7 decades ago. surprisingly, such a simple mixture of sugars, phospholipid sources, antibiotics, and penetrating cryoprotectants can prolong fertilizing capacity of sperm subpopulations for an indefinite period! one of the long-standing concepts of stallion sperm cryopreservation has been the use of slow prefreeze cooling rates to maintain adequate postthaw sperm quality, particularly when utilizing gly as a penetrating cryoprotectant. as mentioned above, changes in the plasma membrane phase (liquid-to-gel phase) are caused by noncontrolled temperature reduction; thus, using fast prefreeze cooling rates (> 1°c/minute) would result in the so-called ‘cold shock.’ sperm plasma membrane composition, particularly cholesterol-to-phospholipid ratio, has been suggested as a critical factor that allows sperm to withstand plasma membrane transition phases discussed above.29,67 optimal cooling rate for stallion sperm exposed to temperatures from 20 to 5°c has been reported to range between –0.1 to –0.01°c/minute to minimize plasma membrane phase transitions.68 such a ‘slow’ cooling rate will require ~ 120 minutes before exposure to liquid nitrogen vapors. slow (0.05-0.1°c/minute) cooling (from 20 to 5°c) of stallion sperm diluted in skim milk + 4% egg yolk + gly (inra-82) semen freezing extender yielded higher postthaw motion characteristics compared to rapid (–60°c/minute) prefreeze cooling.69 the study also compared postthaw motility of sperm cryopreserved in inra-82 versus sperm cryopreserved in a 20% egg yolk + gly + lactose-ethylenediaminetetraacetic acid (edta) extender; prefreeze cooling rate did not influence postthaw quality of sperm cryopreserved in lactose-edta, higher postthaw motion characteristics were generally obtained with the inra-82 extender. in contrast, higher postthaw motion characteristics and plasma membrane intactness in stallion sperm cryopreserved in lactose-edta (le®; animal reproduction systems [ars], ontario, ca, usa) than in an inra-82-based extender (mfr5®; ars), independent of prefreeze cooling rate employed, have been reported.70,71 furthermore, stallion sperm processed in le® extender at –5 (5 minutes cooling), –1 (20 minutes cooling), or –0.1°c/minute (120 minutes cooling) yielded similar postthaw sperm quality parameters, whereas sperm processed in the mfr5® extender required a ‘slow’ cooling rate (–0.1°c/minute) to yield an acceptable postthaw sperm motility and membrane intactness values (30-40% motility, 40-50% plasma membrane-intact sperm).71 under field conditions, it is common to use ‘fast’ prefreeze cooling rates when utilizing semen cryopreservation extenders that contain low molecular weight penetrating cryoprotectants (i.e. amides, dimethylformamide [dmf], or methyl formamide [mf]). in general, several authors recommend the use of prefreeze cooling periods (20-5°c) of 20-30 minutes (≥ –1°c/ minute) before exposing sperm to liquid nitrogen vapors.72-74 the rationale behind this concept is that amides have a higher permeability coefficient due to their low molecular weight and thus would penetrate sperm plasma membrane faster. we recently compared the use of semen extenders with (skim milk + egg yolk + 2% gly + 3% mf [cryomax mfr5®; ars; cmmfr5]; 2% egg yolk 3% mf [cryomax le®; ars; cmle]) or without (skim milk-egg yolk-gly [mfr5®]; egg yolk-gly [le®]) amides when exposing stallion sperm to ‘slow’ (–0.1°c/ minute; prefreeze cooling phase of 120 minutes before freezing) or ‘moderate’ (–1°c/minute; prefreeze cooling phase of 20 minutes before freezing) cooling rates.71 although we observed higher postthaw sperm motion characteristics and plasma membrane intactness in the face of lower lipid peroxidation or apoptosis in stallion sperm cryopreserved in cmle® or le® than in mfr5® or cmmfr5® extenders, these results were not associated with precooling rate or penetrating cryoprotectant employed. as such, our studies indicated that prefreeze cooling rate, and thus the period required to cool stallion sperm from 20 to 5°c before exposure to liquid nitrogen vapors, is dictated by egg yolk quantity in freezing extender and milk proteins (presence or absence), and not by the presence of low molecular weight cryoprotectants such as amides. an additional concept regarding stallion sperm cryopreservation has been replacement of high molecular weight cryoprotectant gly (92.09 g/mol) by cryoprotectants of lower molecular weight, including ethylene glycol (62.07 g/mol), dimethyl sulfoxide (dmso; 78.13 g/mol), methyl formamide (mf; 59.06 g/mol), and dmf (73.09 g/mol). studies indicated that exposure and rapid removal of fresh stallion sperm from a medium that contained 1.0 m gly resulted in the highest proportion of sperm with plasma membrane damage compared to stallion sperm subjected to the same conditions (rapid exposure and removal of cryoprotectant) but with 1.0 m ethylene glycol, dmso, or propylene glycol.44 these studies suggested stallion sperm was considerably sensitive to rapid cryoprotectant removal, particularly when exposed to gly. in the early 2000s, various studies in colorado,75 brazil,73,76-78, and france79 indicated that supplementation of semen freezing extenders with dmf yielded similar or higher postthaw sperm quality parameters when compared to gly-containing extenders. other studies did not report any advantage regarding postthaw sperm motility or plasma membrane intactness when cryopreserving stallion sperm with amides.80,81 studies by brazilian72 and colorado/saskatchewan82 researchers reported higher fertility rates for stallion sperm cryopreserved with dmf than with gly, whereas french workers did not observe differences in pregnancy rates.79 currently, the hypothesis regarding benefits of cryopreserving stallion sperm with amide-containing semen extenders is that due to their lower molecular weight, less osmotic stress is caused to stallion sperm, diminishing plasma membrane and mitochondrial damage. nevertheless, conflicting results have been published in this regard. a study83 indicated that stallion sperm cryopreserved in skim milk and egg yolk-based semen freezing extender supplemented with either 4% dmf or a combination of 2.5% dmf and 1.5% gly yielded higher postthaw plasma membrane-intact sperm (4-5% increase) and a lower proportion of sperm with low mitochondrial function (5-10% decrease) than sperm cryopreserved with 4% gly. conversely, mitochondrial oxygen consumption of stallion sperm cryopreserved in an amide-containing semen freezing extender (botucrio®, botupharma usa, phoenix, az, usa) was higher compared to gly-containing extenders (inra-96®, mfr5®, le®) despite similar postthaw motion characteristics, plasma membrane intactness, and ros production were observed in stallion sperm cryopreserved with le® than in botucrio®.85 we recently studied whether cryopreservation of stallion sperm with gly-containing extenders (le® or mfr5®) yielded a higher proportion of sperm with early plasma membrane changes (associated with apoptosis) or higher lipid peroxidation in plasma membrane-intact sperm (associated with mitochondrial dysfunction and redox imbalance) when compared to the same semen extenders containing a combination of gly and mf (cmle® or cmmfr5®).72 we did not detect differences in any of these parameters when comparing le® versus cmle® or mfr5® vs. cmmfr5®; in contrast, a higher proportion of early plasma membrane changes and lipid peroxidation in plasma membrane-intact sperm were observed in stallion sperm cryopreserved in mfr5® or cmmfr5® versus le® or cmle®. we concluded that even when amides have a higher http://dx.doi.org/10.58292/ct.v16.10832 6 citation: clinical theriogenology 2024, 16, 10832, http://dx.doi.org/10.58292/ct.v16.10832 plasma membrane permeability coefficient than gly,46 they do not confer higher protection to the sperm plasma membrane nor mitochondria (based on ros production) than only with gly. an aspect that warrants further research, even though the limitations of performing in vivo fertility trials in horses, is determining whether the fertility of stallions whose sperm has been cryopreserved in amide-containing extenders is higher than the obtained when inseminating mares with sperm cryopreserved in gly-containing extenders. finally, another factor that warrants further consideration are the beneficial effects of increasing the cholesterol concentrations at sperm plasma membrane to enhance sperm survival after cooling and cryopreservation. cholesterol-to-phospholipid molar ratio in plasma membranes of stallion sperm (0.36) has been reported to be lower than that of bull (0.45), rabbit (0.88), or man (0.99) whereas closer to that of rooster (0.30) and boar (0.26) sperm.29,67 this has been suggested as a main factor that affects the ability of stallion sperm to withstand the cooling and cryopreservation processes.67,85 hence, various methods have been devised to incorporate more cholesterol in sperm plasma membrane, with use of cholesterol-loaded cyclodextrins (clcs) the most popular. these cyclodextrins are cyclic oligosaccharides with a hydrophilic external face and a hydrophobic core that can ‘encapsulate’ compounds like cholesterol.85 several studies have indicated that the addition of clcs (generally 1.5 mg clc per 120 x 106 sperm) resulted in a 5-10% increase in postthaw motility and plasma membrane intactness compared to nonclc-supplemented extenders.86-88 although some authors initially suggested that increasing the molar cholesterol concentration by adding clcs to stallion sperm also reduced acrosomal exocytosis and in vivo fertility rates,87 more recent studies have refuted those findings.89 considering the data above, it would be expected then that stallions whose sperm yield higher postthaw sperm quality after cryopreservation also would have a higher cholesterol-to-phospholipids ratio in their sperm plasma membranes, but a preliminary study did not find a linear association between postthaw sperm motility and plasma membrane cholesterol-to-phospholipid ratio of fresh sperm.90 certainly, more research regarding the relationship between sperm plasma membrane composition and tolerance to cooling or cryopreservation in stallions is required. interestingly, recent data indicated that egg yolk replacement in semen cryopreservation extender by clcs also allowed the reduction in the concentration of penetrating cryoprotectant (gly) required to maximize the postthaw motility and plasma membrane intactness of stallion sperm.91 currently, addition of clcs, in conjunction with purified milk proteins (caseins), to semen cooling extenders is common (i.e. botusemen gold®; botupharma usa), and it would be interesting to determine whether egg yolk could be replaced from semen cryopreservation extenders for stallions by adding clcs. perhaps this would lead to developing more ‘chemically defined’ media for long-term storage of stallion sperm. postthaw sperm quality analysis in stallions: beyond the concept of progressive motility one of the long-standing ‘dilemmas’ in theriogenology, particularly in equine reproductive medicine, is the relationship between sperm quality measures and in vivo fertility. the sperm quality assay most employed by practitioners in the field is the estimation of sperm motility, as it is assumed that motile sperm subpopulations would reach the oviduct after insemination and fertilize the oocyte. although this may be partially true, many changes in sperm caused by cryopreservation may be unintentionally ignored by just assessing postthaw motility. many years ago, to create standard values or guidelines for practitioners to ‘judge’ whether sperm quality in a given sample (frozen/thawed sperm) would be consistent with a positive outcome (pregnancy), the equine breeding industry adopted a minimum of 30% (35% for european countries) ‘progressively’ motile sperm, to deem that an ejaculate was commercially acceptable.17,19,92 whereas tracking down the exact publications that suggested such a cutoff value was difficult for the author, it appears that by the mid 1970’s when german,9 polish,93 and (then) czechoslovakian94 scientists began developing commercial programs for stallion sperm cryopreservation, the terms ‘progressively motile’ ‘motile’ and ‘30%’ were already incorporated, and often used interchangeably, in scientific jargon. none of these studies described why such a cutoff value was used nor what the concept of ‘progressivity’ entailed. still, it can be assumed that these values and terminologies were extrapolated from studies conducted in other species. examining more carefully at other publications at that time regarding stallion sperm analysis and fertility potential, the concept of progressive motility was defined as ‘cells that are actively moving forward’95 or ‘cells that are moving across the field of view.’96 neither of these definitions specifically dictated how ‘forward’ or ‘across the field’ should or should not be interpreted, but basically, they suggested that any sperm that are not moving in circles can be deemed as progressive. although this argument may seem philosophical rather than clinical, author considers this debate necessary for multiple reasons: 1. definition of progressive motility is not standard per se, even when computer assisted sperm analysis (casa) systems are employed; progressive motility is a composite measure (average path velocity – vap [µm/s]; straightness – str [%]) that can be altered by modifying the threshold values that a given casa system uses to detect progressive or nonprogressive sperm, resulting in differences on postthaw progressive motility97; 2. stallion sperm tend to display curvilinear rather than linear trajectories, potentially due to the high incidence of abaxial attachment of the sperm midpiece that should not be considered as a morphologic abnormality but rather a distinctive (i.e. normal) stallion sperm feature.98-101 thus, unless a spermatozoon is moving in very tight circles in a ‘rotational’ rather than ‘translational’ fashion, it is difficult to argue that such spermatozoon is not being ‘progressively motile’ (figure 3); 3. using percentual values as an indicator of sperm quality rather than the actual absolute value of sperm displaying a given characteristic (e.g. motile or ‘progressively motile’) leads to misconceptions regarding the fertility potential of a clinical sample (i.e. batch of cryopreserved semen), as doing it so would ignore that sperm motility, or the lack of thereof, is of a compensable nature. even when the percentage of motile (or ‘progressively motile’) sperm is low in a straw of cryopreserved sperm, if more straws are inseminated then the critical number of sperm displaying such characteristic (i.e. motility) required to ‘theoretically’ achieve maximal fertility can be reached; and 4. the relationship between in vivo fertility and postthaw motility (including progressive motility) is generally low,20-23 and a combination of assays may provide a better appraisal of fertility potential of a sample, as long as it is understood that this will not be 100% accurate in predicting fertility. it has recently been brought to author’s attention that the semen standards established by the world breeding federation of sport horses (wbfsh)92 for frozen/thawed stallion sperm are not currently available for consultation and that various european laboratories that process frozen stallion sperm are no longer abiding to the historical threshold value of 35% or more progressive motility to consider an ejaculate http://dx.doi.org/10.58292/ct.v16.10832 citation: clinical theriogenology 2024, 16, 10832, http://dx.doi.org/10.58292/ct.v16.10832 7 as commercially acceptable. yet, many other laboratories, publications (scientific and lay), and textbooks still refer to that cutoff value. introduction of additional sperm quality assays, primarily based on fluorescent dyes, has allowed us not only to study in depth the changes that sperm undergo during the cryopreservation process but also provided us a better picture of other attributes that are necessary to be considered, besides sperm motility, to judge the fertility potential of frozen/thawed stallion sperm. indeed, estimating plasma membrane intactness (also termed ‘viability’) should be considered as important as estimating sperm motility. it is assumed that immotile sperm are ‘dead,’ although these sperm may still possess an intact plasma membrane and may resume motion if their surrounding conditions (i.e. following insemination) are changed. this has been demonstrated for fresh and cool-stored stallion sperm that has been exposed to high levels of seminal plasma (i.e. 50%, volume:volume),102 and that still may yield acceptable fertility following 4 days of cooled storage despite displaying < 5% motile sperm (having > 70% viable sperm).103 certainly, in these studies, the common factor was that the stallions enrolled presented high intrinsic sperm quality (i.e. sperm morphology), which should be considered when interpreting discrepancies between sperm motility and viability. in frozen/thawed stallion sperm, we observed that differences between values of postthaw sperm motility and viability are lower (i.e. the absolute difference between motility and viability is minimal) when the intrinsic sperm quality (i.e. sperm morphology and dna quality) is high. at the same time, such discrepancies increased as the intrinsic sperm quality decreased.104 from a clinical perspective, this indicated that it is highly likely that poor postthaw sperm motility is related to low intrinsic sperm quality from a particular stallion (or ejaculate) and that the analysis of postthaw sperm viability, in conjunction with sperm motility and morphology analyses, would offer additional insight regarding the quality of that batch of frozen/thawed semen. in general, sperm viability is determined by using exclusion dyes, that is, nonpermeable dyes to the plasma membrane. under field conditions, eosin/nigrosin (e/n) stain (also known as hancock’s stain) has been extensively reported for such purpose. although it appears that e/n yields similar results regarding sperm viability in fresh stallion semen,102,105 it does overestimate this parameter when analyzing coolstored103 or frozen/thawed sperm.105 considering the increased utilization of fluorescence-based techniques for sperm quality analysis in clinical settings, practitioners have become more familiar with other dyes, such as propidium iodide (pi) or ethidium homodimer-1 (ethd-1) that are nonpermeable to the plasma membrane and thus will identify nonviable (‘dead’) sperm populations (using pi or ethd-1, sperm will fluoresce red upon excitation with a 488 nm laser). a very popular semiautomated, fluorescence-based cell counter (nucleocounter sp-100™ [nc], chemometec, allerød, figure 3. representative sperm track printouts for sperm motion characteristics analyses in stallion semen using a computerassisted sperm motion analyzer (casa) system (ivos ii®, hamilton thorne, beverly, ma, usa) before (a) and after cryopreservation (b). note that the percentage of progressively motile sperm (pmot), defined by this system as vap ≥ 30 µm/second and str ≥ 50% (light blue tracks), was lower in fresh (total motility [tmot] – 85%; pmot – 36%; curvilinear velocity [vcl] – 232 µm/ second) than in frozen/thawed sperm (tmot – 69%; pmot – 53%; vcl – 114 µm/second). additional images of sperm displaying motile (c and d) or progressively motile (d and f) tracks from other stallion samples are presented. note the similarities among the trajectories of these sperm despite the system classifying them differently. definitions for pmot were vap ≥ 30 µm/ second and str ≥ 50% for images c and d, whereas vap ≥ 50 µm/second and str ≥ 75% for images e and f. percentages of postthaw motion characteristics were tmot – 55% and pmot 41% for c and d, and tmot – 54% and pmot 34% for e and f. http://dx.doi.org/10.58292/ct.v16.10832 8 citation: clinical theriogenology 2024, 16, 10832, http://dx.doi.org/10.58292/ct.v16.10832 denmark) utilizes microfluidic cassettes preloaded with pi to determine sperm concentration and viability.106-108 for stallion sperm, use of nc for estimation of sperm viability has been compared to flow cytometry (fc) when analyzing fresh or cool-stored semen.107 this study indicated that, overall, both methods (nc and fc) yielded similar results in terms of sperm viability. thus, nc could be considered a less expensive and more ‘user friendly’ alternative to fc. we recently compared nc and fc use to estimate sperm viability in frozen/ thawed stallion semen.104 our results indicated that across a broad spectrum of intrinsic stallion sperm quality (i.e. sperm morphology and dna quality) ‘agreement’ (interpreted as the absolute difference between 2 measures or 2 measurement methods) between both methods was consistently high. while writing this portion of the document, the author wonders if soon the equine breeding industry will move away from utilizing just progressive motility as an indicator of postthaw sperm quality and will adopt both ‘more advanced’ measures of sperm quality (e.g. viability, dna quality) in conjunction with more ‘traditional’ sperm quality assays (e.g. sperm morphology and total motility) that are less deceiving and may have a higher relationship with in vivo fertility of stallions. by any means, the author’s intent is not to suggest that sperm motility should not be considered when analyzing postthaw sperm quality, but rather such a parameter should not solely be the measure of postthaw sperm quality utilized to deem whether an ejaculate is ‘freezable’ or not, nor to determine the fertility potential of a batch of frozen/thawed stallion sperm. interestingly, a study conducted with fresh and cool-stored stallion sperm had a significant relationship between measures of sperm total motility and normal morphology with the first-cycle pregnancy rates of 88 stallions that was not evident when analyzing progressive motility.109 furthermore, in a recent work,110 combination of a battery of assays (casa-derived measures of sperm total motility, morphology, viability, and dna integrity determined by flow cytometry) accounted for up to 95% of in vivo fertility variation from a group of 42 stallions utilized to breed a minimum of 25 mares/stallion with frozen/thawed semen.110 although more extensive studies in commercial settings are warranted, it is evident that the concept of progressive motility as a measure of sperm quality should be thoroughly revisited. use of frozen/thawed stallion sperm for in vitro production of equine embryos by icsi: does it only take one? in vitro production (ivp) of equine embryos by icsi has become a routine procedure in equine reproductive medicine.111-113 it would not be surprising that, shortly, more pregnancies will be established by ivp embryos than by conventional embryo transfer (in vivo embryos). use of frozen/thawed semen for ivp in horses is quite advantageous as straws of frozen semen can either be ‘cut’ (8-10 small cuts made from a 0.5 ml straw; icsi cuts) or thawed and refrozen into more dilute straws (1-2 x 106 sperm/ml; icsi doses).114 additionally, frozen semen can be utilized at any time of the year without coordinating semen collection and shipment with the timing required for fertilization of in vitro-matured oocytes, and doses of frozen/ thawed sperm from old or deceased stallions that were frozen many years ago can still be utilized. it is safe to assume that currently, the primary driving force behind icsi use in the equine breeding industry is related to the stallion, as many stallion owners or managers have realized the convenience of utilizing icsi to maximize the offspring available from their genetically valuable stallions. the first study115 to report the use of frozen/thawed stallion sperm for icsi, with resulting pregnancies utilized sperm from a fertile and a subfertile stallion frozen in an egg yolkgly-based extender and produced 3 pregnancies from the fertile stallion and 1 from the subfertile stallion following icsi on in vivo-matured oocytes that were transferred into the oviducts of recipient mares within 4 hours after icsi. first pregnancies and live foals after utilizing frozen/thawed sperm for icsi on in vitro-matured equine oocytes recovered from abattoir-derived ovaries were reported in the uk,116 and first comparisons on the efficiency of oocyte activation and initial embryonic development (i.e. 16 cells) of in vitro matured equine oocytes fertilized by icsi with either fresh or frozen/ thawed sperm were reported in the usa.117 at the 2002 international symposium on equine reproduction (ft. collins, co, usa; https://www.sciencedirect.com/journal/ theriogenology/vol/58/issue/2), most of the works related to icsi in horses reported the use of frozen/thawed stallion sperm for oocyte fertilization.118-121 since then, all publications about ivp in horses have employed frozen/thawed stallion sperm, except for a recent preliminary report122 from a commercial icsi program that used cool-stored sperm, whereby almost 1 fold increase in embryo production (26 versus 13%) and 20% more pregnancies were observed compared to frozen/thawed sperm. more controlled studies, whereby the same stallions and mares are enrolled, are necessary to determine whether cool-stored sperm would yield higher embryo production than frozen/thawed sperm. as described above, one of the advantages of utilizing frozen/ thawed stallion sperm is sperm that was cryopreserved many years ago can be thawed, rediluted at a much lower sperm concentration (1-2 x 106 sperm/ml) and then refrozen. blastocyst production after icsi using stallion sperm frozen once (control) or twice in a skim milk-egg yolk-gly-based extender yielded similar blastocyst development rates (27 versus 23%, respectively).123 however, the cleavage rate (> 8 blastomeres per embryo) was higher in the control group (75 versus 55%).123 these researchers also indicated that refrozen sperm from a subfertile stallion (whose percentage of sperm dna damage was > 25% [considered high124]) yielded a similar proportion of blastocysts by icsi compared to sperm that was frozen once (9 versus 9%, respectively). although the experience with refrozen sperm in the us has been overall satisfactory, a recent retrospective report from a clinical program in europe indicated that the experience with refrozen sperm has yielded poor cleavage and blastocyst rates (21 and 4%, respectively) after icsi.113 during 2023, in our commercial icsi program at the equine fertility laboratory – texas a&m university, we utilized refrozen sperm from 4 stallions (reprocessed between 2010 and 2013, when these stallions were already dead) to fertilize in vitro-matured oocytes recovered by transvaginal oocyte aspiration (tva) from 6 mares (ramírez-agámez, hernández-avilés: unpublished observations; table). overall, the cleavage and blastocyst rates obtained were 52 and 21%, respectively, slightly lower than those obtained in our program during the same period for frozen/thawed sperm that has been cryopreserved once (64 and 29%, respectively) but within values that are currently considered as commercially acceptable. our group recently studied whether some factors during cryopreservation of stallion sperm would impact the outcomes (i.e. cleavage and blastocyst rates) after icsi on in http://dx.doi.org/10.58292/ct.v16.10832 citation: clinical theriogenology 2024, 16, 10832, http://dx.doi.org/10.58292/ct.v16.10832 9 vitro-matured equine oocytes.125 particularly, we were interested in determining whether the amount of egg yolk (le® or cmle® versus mfr5® or cmmfr5®) and the penetrating cryoprotectant combination (le® or mfr5® versus cmle® or cmmfr5®) would yield more or less ivp embryos after icsi. overall, semen frozen in mfr5® yielded almost 3 times more blastocysts than semen frozen in le® (26 versus 10%, respectively) and 5 times more blastocysts than semen frozen in cmle® or cmmfr5® (5 and 5%, respectively). interestingly, in that study, sperm frozen in mfr5® also yielded the lowest postthaw motility (38%) and higher percentage of lipid peroxidation in viable sperm (35%) compared to other treatments. two main conclusions were obtained from this experiment: 1. apparently, extenders formulated with amides and glycerol yield lower blastocysts compared to gly-only containing extenders and 2. higher blastocyst rates were obtained in sperm that was cryopreserved only with gly, and in which higher membrane destabilization (lipid peroxidation) resulted after cryopreservation. to date, it is still unknown why extenders formulated with amides yielded considerably lower rates of blastocyst production after icsi. still, we hypothesized that a potential toxic effect of these cryoprotectants might have had a role in the ability of in vitro matured oocytes to become embryos. current studies in our laboratory focus on determining whether cryopreservation of stallion sperm with amide-only containing semen extenders would also yield a lower blastocyst rate after icsi than gly-only containing semen extenders. interestingly, in that same study, we thawed ejaculated or epididymal sperm from a fertile stallion that was cryopreserved initially in cmle® extender and refrozen in mfr5® extender.125 then, icsi doses from each group were utilized for icsi on in vitro-matured oocytes, and differences in cleavage and blastocyst rates were not observed (57 versus 77% and 17 versus 17%, respectively). our results not only confirmed those previously reported123 regarding blastocyst production by icsi using refrozen stallion sperm but also indicated that sperm that was initially cryopreserved in cmle® (which yielded 5% blastocyst rate after icsi) could be ‘rescued’ by refreezing in mfr5® extender. another point worth discussing is the relationship between postthaw sperm quality parameters and icsi outcomes in horses. in human reproductive medicine, application of icsi was initially intended to overcome infertility due to poor sperm quality or low sperm numbers.126-128 currently, utilization of icsi has outgrown conventional in vitro fertilization (ivf), and it has become the procedure of choice for ivp of human embryos.129-131 studies in human reproductive medicine focused on the relationship between postthaw sperm quality and icsi outcomes (pregnancy rates and live births) have revealed that analysis of sperm dna quality may provide the highest predictive value of positive icsi outcomes.132-135 in horses, some experimental studies have indicated that sperm of poor postthaw motility can still be utilized to produce ivp embryos and pregnancies.121,123 to author’s knowledge, only 1 study conducted in a commercial icsi program has determined the relationship between certain postthaw sperm quality parameters (i.e. normal morphology – e/n staining; viability – e/n staining; plasma membrane function – hypoosmotic swelling test [host]; dna damage – sperm chromatin dispersion assay) blastocyst production, and pregnancy rates after icsi.136 these researchers estimated sperm postthaw quality after sperm selection by swim-up; they reported that percentage of viable sperm and percentage of sperm with functional plasma membrane (host +) were predictors of positive icsi outcomes (blastocyst development and pregnancy on day 28 after transfer). it is important to mention that these researchers estimated such parameters only after sperm selection before icsi that would bias the relationship between initial postthaw sperm quality and icsi outcomes. further studies in this area are warranted to determine whether there is/are a minimum value/s of postthaw quality that should be considered acceptable, even for icsi purposes. concluding remarks although alternative methods for sperm storage in liquid form (i.e. semen extenders for ambient temperature storage)137 or in the frozen state (i.e. sperm vitrification)138,139 have been recently reported and might have promising results regarding sperm quality parameters and fertility potential, production and utilization of frozen/thawed sperm is still, and most likely will be, the main method for long-term preservation of stallion sperm. many studies have been conducted in the last 2 decades regarding cellular and molecular effects of cryopreservation on stallion sperm, that indicated most of the deleterious effects of this procedure are associated with osmotic and oxidative stress and their consequences to various sperm organelles. remarkably, mitochondria of stallion sperm are a sensitive target to cryopreservation-induced damage; thus, current and future studies will focus on preserving adequate mitochondrial function. although various advances in sperm quality estimation have been published in the last 2 decades, estimation of postthaw sperm quality still heavily relies on the table. postthaw sperm motility (determined subjectively; phase-contrast microscopy at 200 x magnification), sperm morphology (assessed by differential interference contrast [dic] microscopy; 1,000 x magnification), number of mares, transvaginal oocyte aspiration (tva) cycles, number of in vitro-matured oocytes fertilized by intracytoplasmic sperm injection (icsi), cleavage and blastocyst rates of 4 stallions whose sperm was frozen between 1995-2005, then thawed and refrozen to prepare icsi doses. all icsi doses were cryopreserved after 2010 using the mfr5® semen freezing extender (animal reproduction systems, ontario, ca). stallion percent motility percent normal sperm mares (n) tva/icsi cycles ivm oocytes fertilized cleaved embryos/icsi session blastocysts/ icsi session a 5% 43% 2 3 16 10 (63%) 3 (19%) b 10% 55% 1 1 12 3 (25%) 2 (17%) c 5% 61% 2 2 12 7 (58%) 4 (33%) d 2% 37% 1 3 8 5 (63%) 1 (13%) cleavage rate was determined on day 5 posticsi, and a cleaved embryo (> 8 blastomeres). blastocyst rate was determined from day 7 to day 10 posticsi. http://dx.doi.org/10.58292/ct.v16.10832 10 citation: clinical theriogenology 2024, 16, 10832, http://dx.doi.org/10.58292/ct.v16.10832 analysis of sperm motility. even though this assay is fundamental, postthaw sperm quality analyses and clinical interpretations should depend not only on this test but also on a combination of assays that can be applied under field conditions. application of assisted reproductive technologies, and particularly icsi, in horses has increased the utilization of frozen/thawed stallion sperm. with the recent report regarding a repeatable method for conventional ivf in horses,140 we will witness an increase in the number of studies that will validate the use of frozen/thawed sperm in ivf. although ivf may not replace icsi in a commercial setting, most research efforts will likely be focused on studying the relationship between postthaw sperm quality and in vitro fertility in stallions. conflicts of interest no conflicts of interest to declare. acknowledgments legends premier stallion season auction, texas a&m university, clinical icsi program at the equine fertility laboratory at texas a&m university, and department of large animal clinical sciences, texas a&m university have funded various studies conducted by the author and his coworkers. author thanks his colleagues charles love, luisa 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braga dapf, provenza rr, et al: oocyte ability to repair sperm dna fragmentation: the impact of maternal age on intracytoplasmic sperm injection outcomes. fertil steril 2021;116:123-129. doi: 10.1016/j.fertnstert.2020.10.045 136. gonzalez-castro ra, carnevale em: association of equine sperm population parameters with outcome of intracytoplasmic sperm injections. theriogenology 2018;119:114-120. doi: 10.1016/j.theriogenology.2018.06.027 137. gibb z, clulow jr, maclellan lj, et al: storing stallion sperm in spermsafe™ at 17°c may improve fertility by reducing mptp formation. j equine vet sci 2023;125:104589. doi: 10.1016/j. jevs.2023.104589 138. consuegra c, crespo f, dorado j, et al: vitrification of stallion sperm using 0.25 ml straws: effects of volume, concentration and carbohydrates (sucrose/trehalose/raffinose). anim reprod sci 2019;206:69-77. doi: 10.1016/j.anireprosci. 2019.05.009 139. diaz-jimenez m, rota a, dorado j, et al: first pregnancies in jennies with vitrified donkey semen using a new warming method. animal 2021;15:100097. doi: 10.1016/j.animal.2020. 100097 140. felix mr, turner rm, dobbie t, et al: successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation. biol reprod 2022;107:1551-1564. doi: 10.1093/biolre/ioac172 http://dx.doi.org/10.58292/ct.v16.10832 https://doi.org/10.1016/s0093-691x(02)00771-9 https://doi.org/10.1016/s0093-691x(02)00777-x https://doi.org/10.1016/s0093-691x(02)00777-x https://doi.org/10.1016/j.jevs.2023.104634 https://doi.org/10.1016/j.jevs.2023.104634 https://doi.org/10.1016/j.theriogenology.2005.04.035 https://doi.org/10.1016/s0093-691x(98)00199-x https://doi.org/10.1016/s0093-691x(98)00199-x https://doi.org/10.1016/j.theriogenology.2022.10.014 https://doi.org/10.1016/j.theriogenology.2022.10.014 https://doi.org/10.1016/0140-6736(92)92425-f https://doi.org/10.1016/s0015-0228(16)57666-3 https://doi.org/10.1016/s0015-0228(16)57666-3 https://doi.org/10.1016/s0015-0282(16)58462-3 https://doi.org/10.1016/s0015-0282(16)58462-3 https://doi.org/10.1001/jama.2014.17985 https://doi.org/10.1001/jama.2014.17985 https://doi.org/10.1016/j.fertnstert.2020.05.032 https://doi.org/10.1016/s0140-6736(21)00535-3 https://doi.org/10.1093/humrep/del326 https://doi.org/10.1093/humrep/del326 https://doi.org/10.1016/j.rbmo.2014.10.018 https://doi.org/10.1038/s41598-023-45091-6 https://doi.org/10.1038/s41598-023-45091-6 https://doi.org/10.1016/j.fertnstert.2020.10.045 https://doi.org/10.1016/j.theriogenology.2018.06.027 https://doi.org/10.1016/j.jevs.2023.104589 https://doi.org/10.1016/j.jevs.2023.104589 https://doi.org/10.1016/j.anireprosci.2019.05.009 https://doi.org/10.1016/j.anireprosci.2019.05.009 https://doi.org/10.1016/j.animal.2020.100097 https://doi.org/10.1016/j.animal.2020.100097 https://doi.org/10.1093/biolre/ioac172 1 contact sara lyle saraklyle3@gmail.com © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 10638, http://dx.doi.org/10.58292/ct.v17.10638 case report infertility caused by oophoritis in a dog resolved by hemiovariectomy amber nebel-karp,a david mahoney,b michael whitacre,c beth bauer,d sara lylec‡ aanimal hospital of east davie, advance, nc, usa bsouth mountain pet care & canine reproduction, sandy, ut, usa cdepartment of clinical sciences, college of veterinary medicine, north carolina state university, raleigh, nc, usa dcarolina ranch animal hospital & resort, garner, nc, usa abstract canine oophoritis is a rare ovarian pathology that has been reported to cause infertility. however, diagnostic work-up, treatment options, and prognosis of return to fertility after treatment for oophoritis were not reported. we report oophoritis in a dog that was diagnosed via histopathology after hemiovariectomy; diagnostic tests, treatment, and outcome of fertility are included and compared to other domestic species and women. additionally, pathophysiology and prognostic outcome of oophoritis in women are discussed in the context of future directions and possible avenues of investigation in canine oophoritis. keywords: canine oophoritis, infertility, ovarian nodule, hemiovariectomy background canine infertility is oftentimes a heartbreaking issue for breeders and veterinarians to deal with as there can be multiple facets to this problem. in certain cases, ‘apparent infertility’ may be due to inappropriate breeding time; it can be addressed by breeding at the most fertile window, 4-6 days after luteinizing hormone (lh) surge, depending on the type of semen utilized, and measuring serial blood progesterone concentrations. most serious breeders utilize a veterinary team for breeding management to determine the optimal day(s) of estrus to breed. if dogs are bred at optimal day(s), the next step in a diagnostic work up for infertility is assessing semen quality. good semen quality is > 70% normal morphology and progressive motility with at least 200 x 106 progressively motile sperm per breeding dose.1 after addressing appropriate breeding day(s) and semen quality, brucellosis should be considered; in negative brucellosis cases, determining the cause of infertility is challenging. a possible structural cause for female dog infertility is endometrial degeneration due to cystic endometrial hyperplasia (ceh) in dogs > 5 years or subclinical endometritis. ovarian and/or uterine pathology was noted in 42/76 dogs presenting for elective ovariohysterectomy that were clinically normal.2 most common uterine pathologies observed were ceh, periglandular fibrosis, and endometritis. most common ovarian pathologies observed were hyperplasia of the rete ovarii, follicular cysts, and oophoritis.2,3 canine oophoritis is a rare ovarian pathology that has been reported to cause infertility. in the past 3 decades, 3 cases4,5 of canine oophoritis have been reported; a prospective study2 investigated the prevalence of ovarian and uterine pathology in clinically normal dogs at ovariohysterectomy. we report the diagnosis and treatment of a dog with left ovarian granulomatous oophoritis. case presentation a 5-year, maiden german shepherd dog, was presented in august 2020. the referring veterinarian determined ovulation via measuring serial serum progesterone concentrations. during breeding management, abnormal estrous cycles and shortened interestrous intervals were observed. abnormalities noted were failure of appropriate progesterone increases above ovulation concentrations and split estrus with an interestrous interval of < 2-3 months. similar to her dam, this dog had estrous cycles lasting for 21 days and cycled every 4 months. the first breeding attempt was in november 2019 and ovulation was determined via serum progesterone concentrations (chemiluminescence, idexx, westbrook, me). the dog ‡current affiliation: veterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la, usa mailto:saraklyle3@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.10638 2 citation: clinical theriogenology 2025, 17, 10638, http://dx.doi.org/10.58292/ct.v17.10638 tested negative for brucellosis (rapid slide agglutination test; zoetis, parsippany, nj). the estimated lh surge (2.3 ng/ml of progesterone) occurred on november 16, 2019. however, progesterone concentrations did not increase and the cycle was assumed to be a split estrus. the dog came into proestrus in late december 2019, ~ 4 weeks from the end of previous cycle. progesterone concentrations never increased > 1.1 ng/ ml in the following 20 days. the dog was prescribed compounded mibolerone (safeway compounding pharmacy, newark, de) to assist in extending her interestrous interval to have a fertile estrous cycle. mibolerone treatment was initiated on january 13, 2020 and discontinued on may 7, 2020 when the drug availability became limited. her next estrous cycle began in july 2020, with proestrus starting on july 9, 2020 and followed via determination of serial progesterone concentrations every 2-4 days until they reached 3.2 ng/ml on august 4, 2020. testosterone concentrations were 0.1 ng/ ml (< 0.1 ng/ml normal for a female dog). vaginal cytology revealed lack of cornification throughout the cycle. since the dog had another slow increase in progesterone concentrations she was referred. complete blood count and serum chemistry findings were normal. breeding management consisted of progesterone assay (tosoh bioscience aia 360, san fransico, ca) and vaginal cytology. progesterone concentrations on initial presentation were 5.91 ng/ml that suggested potential ovulation; however, her vaginal cytology revealed < 10% cornification with low cellularity. disagreement between progesterone concentrations and vaginal cytology could not be immediately explained. transabdominal ultrasonography was performed; the uterus was normal with no cystic endometrial changes. ovaries were heterogeneous and asymmetrical; the right ovary measured 0.8 x 1.9 cm and the left ovary was 1.8 x 2.5 cm. a single anechoic structure on the right ovary measured 0.2 cm and a hypoechoic nodule in the left ovarian parenchyma measured 1.7 cm (figure 1a); the left ovarian nodule was vascularized, as evidenced on doppler ultrasonography. the radiologist labeled the anechoic structure on the right ovary as a potential cyst or cystic functional body. potential differentials for the left ovary nodule were complicated cyst, atypical functioning body, or neoplasia. the decision was for another progesterone concentrations in 1 week to determine if the estrous cycle would continue to progress. a week later, the dog was presented for follow up breeding management; her progesterone concentrations had decreased to 2.73 ng/ml (similar to findings on repeat vaginal cytology). a follow up ultrasonography was performed on september 9, 2020. minimal changes were observed in the left ovary (still enlarged with a 1 x 1.7 cm hypoechoic nodule in the cranial aspect). the nodule appeared more cavitated in the center compared to initial ultrasonography. the right ovary appeared normal (no anechoic structure on reexamination). mild development of mammary tissue was noticed; due to these findings and lack of change in the nodule, among previous differentials, neoplasia (with granulosa-theca cell tumor) appeared more likely. we suspected left ovarian abnormality as the reason for abnormal estrous cycles. submitting a sample for antimüllerian hormone (amh) to uc davis versus conserving the dog’s breeding potential via hemiovariectomy was discussed. owners decided to have hemiovariectomy with histopathology submission via referring veterinarian and not to submit a serum sample for amh. treatment surgery was scheduled via the referring veterinarian; left hemiovariectomy was performed on september 18, 2020. surgery and recovery were uneventful. when the ovary was bisected, a cavitated center was identified and a black mucous material oozed from the cut surface (figure 1b). histopathology revealed that the structure was not neoplastic; diagnosis was granulomatous oophoritis characterized by lymphocytes, macrophages, plasma cells, fibrosis, and hemorrhage (figure 1c and 1d). outcome after left hemiovariectomy, the dog came into estrus in january 2021. she was naturally bred based on progesterone concentrations that measured > 20 ng/ml near the end of estrus; this time there was no abnormalities in cyclicity. the dog was diagnosed pregnant on february 25, 2021, and whelped 6 healthy pups on march 26, 2021. since surgery, the dog whelped and reared 3 healthy litters: 6 (all surviving past weaning), 9 (8 surviving past weaning), and 7 pups (all surviving past weaning) before retiring from the breeding program. discussion it is unclear what caused oophoritis in this case. oophoritis has been reported2 to be extremely rare with prevalence of 6.67.9% in 42/76 clinically healthy dogs with reproductive abnormalities at ovariohysterectomy. in other species (e.g. pigs and cattle) oophoritis has been linked with modified-live vaccinations of pseudorabies,6 infectious bovine rhinotracheitis,7 and noncytopathogenic bovine pestivirus (bovine viral diarrhea virus).8,9 in mares, extremely rare cases of oophoritis or salpingo-oophoritis were reported in repeated transvaginal aspiration of oocytes.10–12 in dogs, oophoritis was reported in a 3.5-year rhodesian ridgeback4 that had prolonged estrus and an abnormal corpus luteal structure with inflammatory invasion leading to luteal insufficiency. oophoritis5 causing primary anestrus in 2 dogs has been reported, but none presented ultrasonographic structure as we have described. in women, oophoritis may be more common, albeit still rare, as there is more information available on lymphoplasmacytic oophoritis in women. the most common presentation and clinical history in women with oophoritis is premature ovarian failure, also known as premature ovarian insufficiency/ dysfunction or premature menopause, affecting 1–2% of all women; autoimmune oophoritis is thought to cause 5% of poi cases.13–18 clinical signs of premature ovarian insufficiency (poi) in nulligravid to multigravid women < 40 years was amenorrhea (at least for 4 months); women commonly had pelvic pain, hot flashes, low libido, vaginal dryness, infertility, and changes in their menses cycle. hormonal profiles of these women were hypergonadotropic hypogonadism characterized by hypoestrogenism (< 50 pg/ml), hypoandrogenism, and elevated (> 40 iu/ml) follicle-stimulating hormone (fsh) concentrations were on 2 occasions, 1 month apart. most women had a medical history of either having autoimmune thyroiditis (hashimoto’s) and adrenal insufficiency (addison’s) known as autoimmune polyendocrine syndrome types i and ii.13–19 studies suggested that 80% of women that had addison’s disease usually developed autoantibodies against cyp21-ɑ-hydroxylase, cyp11, or cyp17-ɑ-hydroxylase in the adrenal cortex and ovary.20–26 this caused a selective http://dx.doi.org/10.58292/ct.v17.10638 citation: clinical theriogenology 2025, 17, 10638, http://dx.doi.org/10.58292/ct.v17.10638 3 theca cell dysfunction in the ovary leading to decreased steroid production (diminished androstenedione and estrone precursors) and hypoestrogenism. not only was steroid production affected due to the autoantibodies, lymphoplasmacytic destruction occurred primarily in secondary and graafian follicles that have theca interna and externa layers, initially sparing primordial and primary follicles. autoantibodies against oocytes ooplasma were identified in surveillance studies in cattle27–29 presenting for infertility and in thymectomized mice studies.30,31 autoimmune oophoritis was definitively diagnosed solely through ovarian biopsy and histopathology (as in the case). research to correlate less invasive testing with oophoritis diagnosis suggested testing for autoantibodies against cyp21ɑ-hydroxylase using enzyme linked immunoassay or indirect immunofluorescence.19 others have diagnosed salpingo-oophoritis via laparoscopy,32 ovarian cytology,33 and ultrasonography. approximately 20% of women with poi and autoimmune oophoritis experience spontaneous ovulations, with 5-10% of these women conceiving naturally and delivering a child.17,19,34 apart from hormonal testing of estrogen, progesterone, testosterone, lh, and fsh, ultrasonography is a key feature in the preliminary diagnostic work-up for oophoritis.35–37 ovarian presentations were enlargement, asymmetry, multiple or singular cystic structures, or lack of follicular development;20 it was theorized that the pathogenesis for women who developed multiple cystic follicles despite low estrogen was due to elevated fsh driving follicular development without estrogen’s negative feedback and producing higher concentrations of inhibin b.20 treatment for autoimmune oophoritis is difficult and most often unsuccessful. most treatment entails assisted reproductive technologies in controlled ovarian hyperstimulation protocols. protocols utilize gonadotropin-releasing hormone to aid in suppressing fsh, thus increasing ovarian sensitivity and human chorionic gonadotropin38 for ovulation induction when follicles reach an inducible size (≥ 18 mm). this has limited success as observed in some studies where only 3/15 women ovulated after treatment38 and might have interfered with spontaneous ovulation in a small subset of women with * ba * * dc * * * * 50 x20 x figure 1. a. ultrasonographic image of left ovary (craniocaudal view), note hypoechoic structure measuring 1 x 1.7 cm (asterisk) that remained consistent in size at reexamination; b. left ovary bisected after hemiovariectomy, note dark pedunculated mass containing debris (asterisks); and c and d. histopathologic (h&e stain) confirmation of granulomatous oophoritis (asterisks). http://dx.doi.org/10.58292/ct.v17.10638 4 citation: clinical theriogenology 2025, 17, 10638, http://dx.doi.org/10.58292/ct.v17.10638 poi.17 other reproductive laboratories added adjunct therapy,39 supplementing women with dehydroepiandrosterone sulfate to improve androgen concentrations and secondary increase in estrogen. this group observed success with 5/6 women having ovarian responses of a single follicle and 3/6 resulting in ovulation. two of the 3 ovulations were spontaneous resulting in pregnancies and delivery of healthy children in both women. although results were substantial, the success of treatment can be limited depending on the definition of success. is the definition of success having follicular development, even of a single follicle leading to ovulation or is it conceiving and delivering a child? these varying definitions of success, if not explicitly stated, can have substantial impacts on a patient’s expectations and compliance during treatment. autoimmune oophoritis has been anecdotally treated successfully using glucocorticoids via immunosuppression in the early stages before complete follicle deprivation occurred. glucocorticoids worked synergistically with controlled ovarian hyperstimulation and increased the success of treatment or return of menses in the short-term.40,41 possible sequelae to oophoritis reported in women are ovarian torsion from large cysts/follicles,40 adrenal insufficiency within 9 years of poi, osteoporosis, ischemic heart disease, increased risk of mortality, and psychological distress.13,15–19 osteoporosis due to hypoestrogenism necessitates estrogen supplementation until menopausal age (50 years) to reduce pathologic fractures.13,17 as mentioned, autoantibodies against adrenal cyp21-ɑ-hydroxylase is the main etiology causing autoimmune oophoritis.19 women with these autoantibodies should be tested for adrenal insufficiency if not already diagnosed to avoid an adrenal crisis during pregnancy. fasted baseline cortisol and acth stimulation tests are poor screening tools, as women in this population tend to have low sensitivity and specificity to these tests.17 appropriate diagnosis and management of adrenal insufficiency have to be referred to an endocrinologist and are not discussed here. there are no reports of these sequelae occurring in dogs; however, they cannot be ruled out as a possible presentation for oophoritis. pathogenesis of oophoritis is multifactorial with various genetic and environmental components. in women, a hereditary link has been theorized as similar familial history was observed in some patients.13–15 other causes of oophoritis are vaccination against human papilloma virus,13 galactosemia,13 chemotherapy,13,16 smoking,12 mumps,42,43 and zika virus.43,44 mumps virus caused gonadal infection in 5% of women and 20% of men, with oophoritis reported more often in women affected before puberty.42 interestingly, mumps caused transplacental infection of fetuses resulting in offsprings experiencing oligomenorrhea and ovarian hypoplasia.42 after the zika virus epidemic in 2015, acute oophoritis and follicular apoptosis occurred in mice after infection with zika-virus via t lymphocyte-mediated destruction.42,43,44 subsequently this was confirmed; however, no difference in oocyte yield, fertilization rate, or blastulation rate were observed between treated and control mice.43 it is unclear in this case how or why this dog developed oophoritis, as no pertinent medical history of hypothyroidism or addison’s was mentioned in the record. furthermore, there is limited information on canine oophoritis to know if a similar pathogenesis occurs as in women. the only familial history mentioned was short interestrous intervals of 4 months. however, since she was a german shepherd dog, short interestrous intervals are fairly common in this breed.45–47 research is needed to determine whether there is a link with oophoritis and other endocrine insufficiencies like autoimmune thyroiditis and addison’s in canine patients. to fully elucidate this, a complete thyroid panel with inclusion of autoantibodies could have been performed in this case. however, the inclination to have this bloodwork without any clinical signs was low at initial presentation. typical addison’s was also ruled out due to normal electrolytes (serum chemistry) and no clinical signs in the history. testing for atypical addison’s disease was not performed in our case. additional serum hormonal testing in this case could have been recommended to describe the dog’s hormonal status that would include fsh, lh, estrogen, testosterone, and amh. this would have provided more information in the disease process and would have allowed comparisons to women. however, other hormonal testing was declined in favor for a definitive diagnosis via histopathology after a hemiovariectomy. although serum estrogen concentrations were not determined, one can suspect hypoestrogenism due to the indirect bioassay obtained via vaginal cytology. throughout each bleeding episode, no cornification was observed from november 2019 to september 2020 on vaginal cytology and it was consistent with anestrus (low cellularity and low cornification). this provided preliminary evidence of hypoestrogenism in this dog as observed in women with poi. progesterone concentrations reported previously were possible because of luteinization of the follicular wall without ovulation in any monitored cycle. despite the shortcomings of limited serum hormonal assays or endocrine tests, we have documented oophoritis as the cause for estrous cycle abnormalities and infertility. to the authors’ knowledge, for the first time, successful treatment and return to fertility in a dog diagnosed with unilateral granulomatous oophoritis is reported. learning points • oophoritis is a cause of infertility in dogs • hemiovariectomy may be curative in unilateral oophoritis • dogs can have a successful breeding career after hemiovariectomy conflict of interest none to report. references 1. hollinshead fk, hanlon dw: factors affecting the reproductive performance of bitches: a prospective cohort study involving 1203 inseminations with fresh and frozen semen. theriogenology 2017;101:62-72. doi: 10.1016/j.theriogenology.2017.06.021 2. maya-pulgarin d, gonzalez-dominguez ms, aranzazu-taborda d, et al: histopathologic findings in uteri and ovaries collected from clinically healthy dogs at elective ovariohysterectomy: a cross-sectional study. j vet sci 2017;18:407-414. doi: 10.4142/jvs.2017.18.3.407 3. sasidharan jk, patra mk, singh lk, et al: ovarian cysts in the bitch: an update. top companion anim med 2021;43:1-7. doi: 10.1016/j.tcam.2021.100511 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and reproduction: sex-linked dysgenesia of the gonad after neonatal thymectomy in mice. science 1969;166:753-755. doi: 10.1126/science.166.3906.753 31. taguchi o, nishizuka y, sakakura t, et al: autoimmune oophoritis in thymectomized mice: detection of circulating antibodies against oocytes. clin exp immunol 1980;40:540-534. 32. ledger wj: laparoscopy in diagnosing and managing patients with suspected salpingo-oophoritis. am j obstet gynecol 1980;138:1012-1016. doi: 10.1016/0002-9378(80)91098-4 33. piseddu e, masserdotti c, milesi c, et al: cytologic features of normal canine ovaries in different stages of estrus with histologic comparison. vet clin pathol 2012;41:396-404. doi: 10.1111/j.1939165x. 2012.00453.x 34. ayala c, celis me: experimental autoimmune oophoritis and ɑ-melanocyte-stimulating hormone. expert rev endocr metab 2010;5:539-547. doi: 10.1586/eem.10.11 35. patrelli t, franchi l, gizzo s, et al: can the impact of pelvic inflammatory disease on fertility be prevented? epidemiology, clinical features and surgical treatment: evolution over 8 years. j reprod med 2013;58:425-433. 36. singh n, tripathi r, mala ym, et al: xanthomatous oophoritis following uterine artery embolization: successful conservative surgical management with favourable outcome. bmj case rep 2013; 2013:1-5. doi: 10.1136/bcr-2013-010184 37. torre a, amado a, joao d, et al: xanthogranulomatous salpingo-oophoritis presenting as an ovarian tumor. bmj case rep 2022;15:1-4. doi: 10.1136/bcr-2021-248197 38. van kasteren y, hoek a, schoemaker j: ovulation induction in premature ovarian failure: a placebo-controlled randomized trial combining pituitary suppression with gonadal stimulation. fertil steril 1995;64:273-278. doi: 10.1016/s0015-0282(16) 57722-x 39. gleicher n, darmon s, molinari e, et al: a form of secondary ovarian insufficiency (soi) due to adrenal hypoandrogenism as http://dx.doi.org/10.58292/ct.v17.10638 https://doi.org/10.1177/030098589703400304 https://doi.org/10.2460/ajvr.1990.51.07.969 https://doi.org/10.1177/104063879801000201 https://doi.org/10.1177/104063879801000201 https://doi.org/10.1016/s0093-691x(02)01136-6 https://doi.org/10.2746/042516403775467243 https://doi.org/10.1111/j.2042-3306.2012.00606.x https://doi.org/10.1111/j.2042-3306.2012.00606.x https://doi.org/10.5114/pm.2017.68592 https://doi.org/10.1016/j.autrev.2014.01.003 https://doi.org/10.1016/b978-012595961-2/50064-0 https://doi.org/10.1016/j.fertnstert.2004.11.059 https://doi.org/10.1196/annals.1429.006 https://doi.org/10.1038/cmi.2014.97 https://doi.org/10.1210/jc.2004-1985 https://doi.org/10.1007/s43032-022-00970-7 https://doi.org/10.1007/s43032-022-00970-7 https://doi.org/10.1095/biolreprod61.3.635 https://doi.org/10.4049/jimmunol.177.3.1988 https://doi.org/10.1016/j.fertnstert.2005.04.060 https://doi.org/10.1111/j.1600-0897.1989.tb00628.x https://doi.org/10.3168/jds.s0022-0302(89)79498-4 https://doi.org/10.1016/s0749-0720(15)30647-2 https://doi.org/10.1111/j.1751-0813.1974.tb06881.x https://doi.org/10.1126/science.166.3906.753 https://doi.org/10.1016/0002-9378(80)91098-4 https://doi.org/10.1111/j.1939-165x.2012.00453.x https://doi.org/10.1111/j.1939-165x.2012.00453.x https://doi.org/10.1586/eem.10.11 https://doi.org/10.1136/bcr-2013-010184 https://doi.org/10.1136/bcr-2021-248197 https://doi.org/10.1016/s0015-0282(16)57722-x 6 citation: clinical theriogenology 2025, 17, 10638, http://dx.doi.org/10.58292/ct.v17.10638 new infertility diagnosis. endocrine 2021;72:260-267. doi: 10.1007/s12020-020-02512-0 40. bradbury r, mclean m, smith h: successful live birth following a short course of glucocorticoid suppression in a patient with autoimmune polyglandular syndrome type 2 and premature ovarian insufficiency: a case report. j obstet gynaecol res 2023;49: 2194-2198. doi: 10.1111/jog.15684 41. kalantaridou sn, braddock dt, patronas nj, et al: treatment of autoimmune premature ovarian failure. hum reprod 1999;14: 1777-1782. doi: 10.1093/humrep/14.7.1777 42. morrison j, givens jr, wiser wl, et al: mumps oophoritis: a cause of premature menopause. fertil steril 1975;26:655-659. doi: 10.1016/s0015-0282(16)41233-1 43. caine ea, scheaffer sm, broughton de, et al: zika virus causes acute infection and inflammation in the ovary of mice without apparent defects in fertility. j infect dis 2019;220:1904-1914. doi: 10.1093/infdis/jiz239 44. broughton de, schaeffer s, halabi j, et al: zika virus causes acute oophoritis mediated by t lymphocytes in a mouse model. fertil steril pcrs abstracts 2018;109:e4-e5. doi: 10.1016/j.fertnstert. 2018.02.019 45. sokolowski jh, stover dg, van ravenswaay of: seasonal incidence of estrus and interestrous interval for bitches of seven breeds. j am vet med assoc 1970;171:271-273. 46. christie dw, bell et: some observations on the seasonal incidence and frequency of oestrus in breeding bitches in britain. j small anim pract 1971;12:159-167. doi: 10.1111/j.1748-5827. 1971.tb06213.x 47. sokolowski jh: reproductive patterns in the bitch. vet clin north am 1977;7:653-666. doi: 10.1016/s0091-0279(77)50076-7 http://dx.doi.org/10.58292/ct.v17.10638 https://doi.org/10.1007/s12020-020-02512-0 https://doi.org/10.1111/jog.15684 https://doi.org/10.1093/humrep/14.7.1777 https://doi.org/10.1016/s0015-0282(16)41233-1 https://doi.org/10.1093/infdis/jiz239 https://doi.org/10.1016/j.fertnstert.2018.02.019 https://doi.org/10.1016/j.fertnstert.2018.02.019 https://doi.org/10.1111/j.1748-5827.1971.tb06213.x https://doi.org/10.1111/j.1748-5827.1971.tb06213.x https://doi.org/10.1016/s0091-0279(77)50076-7 2019 luteinizing hormone receptor is immunoexpressed within the canine thyroid luteinizing hormone receptor is immunoexpressed within the canine thyroid khawla h. zwida, michelle a. kutzler department of animal and rangeland sciences, oregon state university, corvallis, or abstract gonadectomy has been implicated as a risk factor for development of canine hypothyroidism, due to loss of negative feedback to the anterior pituitary, resulting in persistently elevated luteinizing hormone concentrations. receptors for luteinizing hormone have been identified in various canine gonadal and extragonadal tissues, including bladder and skin. the aim was to investigate if luteinizing hormone receptors were also expressed in the canine thyroid. formalin-fixed, paraffin-embedded thyroid tissues from eight dogs of various sexes and breeds were subjected to routine immunohistochemical methods. expression of luteinizing hormone receptors was evident in thyrocytes from all thyroid tissue sections examined; the percentage of positive luteinizing hormone receptors cellular expression was 46.5 ± 23.8% (mean ± standard deviation). immunoexpression of luteinizing hormone receptors was localized to thyroid follicular epithelial cells, with no immunoreactivity in other thyroid constitutive structures or in any negative control tissue sections. this is apparently the first report to demonstrate luteinizing hormone receptors expression in the canine thyroid gland. functional studies are needed to determine whether unregulated hypersecretion of luteinizing hormone after gonadectomy affects thyroid function. keywords: dog, hypothyroidism, immunohistochemistry, luteinizing hormone receptor introduction hypothyroidism is the most common endocrine disorder of dogs, affecting 0.2 0.8% of dogs.1-3 the most common presenting dermatological and metabolic symptoms are alopecia, dry/poor quality coat, seborrhea, lethargy, exercise intolerance, weight gain, skin hyperpigmentation, pyoderma and cold intolerance. these symptoms occur in combination more than 60% of the time.1,3,4 hypothyroidism is more common with advancing age;5 the mean age at diagnosis is 7.2 years (range, 0.5 15 years).3 the risk of developing hypothyroidism is higher in certain breeds, including doberman pinschers, miniature schnauzers, golden retrievers, cocker spaniels, shetland sheepdogs, irish setters and dachshunds.3,4,6,7 in these high-risk breeds, young dogs may also develop hypothyroidism.7 one gene has been implicated in hypothyroidism and lymphocytic thyroiditis in several dog breeds, including the doberman pinscher, rhodesian ridgeback, english setter and giant schnauzer. this gene is the major histocompatibility complex dog leukocyte antigen haplotype or allele associations.8,9 this is in contrast to humans, in which there are several genetic risk factors for development of autoimmune thyroid disease,1 including a polygenic predisposition or autoimmune hypothyroidism resulting from one of seven genes (aire, foxp3, il2ra, itch, lrba, stat1, and stat3).10,11 gonadectomized dogs have a higher risk for developing hypothyroidism, with an overall incidence of 30%, significantly higher than the general dog population.7 it is noteworthy that hypothyroidism also affects 10 15% of postmenopausal women,12 an endocrine state with many hormonal similarities to gonadectomized dogs. following gonadectomy or menopause, circulating luteinizing hormone (lh) concentrations increase significantly and remain persistently elevated, due to a lack of negative hormonal feedback. previous research has demonstrated the presence of lh receptors (lhr) within normal and adenomatous human thyroid glands.13 however, effects of lhr activation in the human thyroid has not been well described, but may involve intracellular signaling similar to thyroid stimulating hormone (tsh) receptor activation (e.g. adenylate cyclase). to our knowledge, the presence of lhr in the canine thyroid gland has not been reported. we hypothesized that lh receptors are present in the canine thyroid and that lhr localizes to cells with tsh receptors. material and methods formalin-fixed, paraffin-embedded archived canine thyroid tissues from 8 dogs were provided by the oregon state university veterinary diagnostic laboratory and used for this investigation (table). in clinical theriogenology • volume 11 number 1 • march 201923 addition, formalin-fixed paraffin-embedded archived skin tissue from another dog was used as a positive control, as canine skin expresses abundant lhr.14 serial 6-μm sections were cut from paraffin blocks to determine lhr and tsh receptor expression on adjacent tissue sections. sections were mounted on polyl-lysine-coated slides, deparaffinized in xylene, rehydrated in a graded ethanol series (100, 75, and 50%, respectively) and subjected to heat-induced epitope retrieval (#s1700, dako, carpinteria, ca). for this, slides were placed in boiling sodium citrate in a nordicware® tender cooker and boiling continued in a microwave for a total of 10 minutes. thereafter, slides were left in solution and allowed to cool to room temperature for ~ 20 minutes. tissue-specific endogenous peroxidase activity was inhibited by incubating slides in 3% hydrogen peroxide and nonspecific binding blocked with 1% horse serum. goat polyclonal anti-human lh receptor (sc-26341, santa cruz biotechnology, dallas, tx) or mouse monoclonal igg1 tsh receptor (sc-53542, santa cruz biotechnology) were applied at dilutions of 1:50 and 1:100, respectively. primary antibodies were incubated for 1 hour at room temperature. negative controls from each tissue were similarly treated, but without primary antibody. slides were then reacted for 30 minutes with biotinylated horse anti-goat igg (sk-5300 vector laboratories, burlingame, ca) and incubated for 30 minutes with avidin-biotin-peroxidase complex (pk6105, abc kit, vector laboratories) followed by a brief incubation with nova red peroxidase substrate (sk4800, vector laboratories,). slides were counter-stained with hematoxylin, dehydrated and mounted. cells positively expressing lh and tsh receptors were those with red staining in the cytoplasm. cells were counted from five randomly selected fields per antibody per dog with a leica dm4000b microscope using bright field microscopy at 400x magnification. the number of cells evaluated per dog in these fields was 281.3 ± 142.3 (mean ± sd). representative images from each dog were digitally captured using a qimaging camera (qicam 12-bit, #qic-f-m-12-c, qimaging, surrey, bc) and qcapturepro image capturing software (qimaging). data were reported as mean ± sd. results immunostaining for lhr was strong in epidermis and hair bulbs (figure 1). however, there was no tsh receptor expressed in skin (data not shown). expression of lhr was evident in all thyroid tissue sections, with a mean ± sd percentage of positive cellular expression of 46.5 ± 23.8%. the pattern of lh receptor immunoexpression was localized to thyroid follicular epithelial cells, with no signs of immunoreactivity in other thyroid constitutive structures (figure 2). some cells had cap-like immunostaining. it was noteworthy that lhr immunostaining in the canine thyroid could not be differentiated from that of tsh receptor (figure 3), whose immunoexpression has been previously reported in cows and rats.15 thyroid stimulating hormone receptor expression was 56.0 ± 13.3% (mean ± sd). there was no positive staining evident in any negative control tissue section (figures 1-3). discussion immunoexpression of lhr has been described in many canine tissues including the skin,14,16 lower urinary tract (bladder and urethra),17,18 and adrenal cortex,19 lymph node,20 and musculoskeletal tissues (ligaments, synovia, subchondral bone).21 however, this was apparently the first report that the canine thyroid gland expressed lhr. using a cut-off of > 25% of cells staining positive for lhr, 16.7% of normal human thyroids were positive for lhr.13 if a cut-off of > 25% of cells staining positive were applied for dogs, 6 out of 8 (75%) thyroids examined in the current study would be positive for lhr. molecular mechanisms underlying regulation of lhr expression in gonadal and non-gonadal tissues have been investigated. in chickens, lh increases ovarian lhr gene expression, whereas activin a prevents lh-induced increases in lhr gene expression.22 expression of lhr is inhibited posttranscriptionally in rat luteal cells by mevalonate kinase.23 estrogen reduced ovine lhr gene expression in several extra-gonadal tissues, including medulla oblongata, hypothalamus, ruminant stomach (except reticulum), intestinal tissues (except colon), pancreas, liver, kidney, and uterus. in contrast, progesterone increased lhr gene expression in various tissues, including the hypophysis, olfactory bulb, rumen, small intestine, kidney, oviduct, and uterus.24 regulation of lhr expression in the thyroid gland needs additional investigation. in prepubertal clinical theriogenology • volume 11 number 1 • march 2019 24 rats with ptu (6-n-propyl-2-thiouracil) induced hypothyroidism, thyroid lh receptor gene expression was not significantly different from controls.25 in humans, there was increased thyroid lhr expression in women with high serum lh concentrations.13 however, thyroid glands from these patients were adenomatous and it is not known if the women were hypothyroid at sample collection. in the current study, serum tetraiodothyronine (t4) and lh concentrations in these dogs were unknown, due to use of archived tissues. in women who have undergone gonadectomy, lh concentrations are three times higher after ovary removal, whereas both triiodothyronine and t4 concentrations are significantly reduced after ovary removal.26 this observation provides evidence that a causal relationship could exist in dogs as well. that gonadectomized dogs have significantly elevated circulating lh concentrations and are at greater risk for developing hypothyroidism7 is an impetus to determine effect of reducing lh concentrations on thyroid hormone secretion. the current study provided evidence that lh receptors may have a role in thyroid hormone secretion. however, more research is needed to determine whether lhr expression is increased in gonadectomized dogs. acknowledgement the authors thank kay fischer and the oregon state university histology laboratory for technical assistance. authors’ contributions zwida contributed to the design of the study, performed all of the immunohistochemistry experiments, and image acquisitions. kutzler conceived and contributed to the design of the study and oversaw immunohistochemistry and image acquisition. both authors drafted the manuscript, critically revised it, gave final approval and agreed to be accountable for all aspects of the work in ensuring that questions relating to the accuracy or integrity of any part of the work are appropriately investigated and resolved. conflict of interest the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the authors thank the ministry of scientific research and higher education in libya for graduate school financial assistance and dr. howard meyer of chippewa kennels for funding research supplies. references 1. mooney ct: canine hypothyroidism: a review of aetiology and diagnosis. n z vet j 2011;59:105-114. 2. johnson mb, hattersley at, flanagan se: monogenic autoimmune diseases of the endocrine system. lancet diabetes endocrinol 2016;4:862-872. 3. panciera dl: hypothyroidism in dogs: 66 cases (1987-1992). j am vet med assoc1994:204:761-767. 4. dixon rm, mooney ct: canine serum thyroglobulin autoantibodies in health, hypothyroidism and nonthyroidal illness. res vet sci 1999;66:243-246. 5. bagchi n, brown tr, parish rf: thyroid dysfunction in adults over age 55 years. a study in an urban us community. jama intern med 1990;150:785-787. 6. nesbitt gh, izzo j, peterson l, et al: canine hypothyroidism: a retrospective study of 108 cases. j am vet med assoc 1980;177:1117-1122. 7. milne kl, hayes hm jr: epidemiologic features of canine hypothyroidism. cornell vet 1981;71:3-14. 8. kennedy lj, huson hj, leonard j, et al: association of hypothyroid disease in doberman pinscher dogs with a rare major histocompatibility complex dla class ii haplotype. tissue antigens 2006;67:53-56. 9. wilbe m, sundberg k, hansen ir, et al: increased genetic risk or protection for canine autoimmune lymphocytic thyroiditis in giant schnauzers depends on dla class ii genotype. tissue antigens 2010;75:712-719. 10. johnson mb, hattersley at, flanagan se: monogenic autoimmune diseases of the endocrine system. lancet diabetes endocrinol 2016;4:862-872. clinical theriogenology • volume 11 number 1 • march 201925 11. sanyal d, raychaudhuri m: hypothyroidism and obesity: an intriguing link. indian j endocrinol metab 2016;20:554557. 12. giri a, edwards tl, legrys va, et al: subclinical hypothyroidism and risk for incident ischemic stroke among postmenopausal women. thyroid 2014;24:1210-1217. 13. liu j, chen g, et al: serum levels of sex hormones and expression of their receptors in thyroid tissue in female patients with various types of thyroid neoplasms. pathol res pract 2014;210:830-835. 14. welle mm, reichler im, barth a, et al: immunohistochemical localization and quantitative assessment of gnrh-, fsh-, and lh-receptor mrna expression in canine skin: a powerful tool to study the pathogenesis of side effects after spaying. histochem cell biol 2006;126:527-535. 15. schmid kw, jasani b, morgan jm, et al: light microscopic immunocytochemical demonstration of thyroid-stimulating hormone (tsh) receptors on normal rat thyroid cells. j histochem cytochem 1988;36:977-982. 16. venencie py, méduri g, pissard s, et al: luteinizing hormone/human chorionic gonadotrophin receptors in various epidermal structures. br j dermatol 1999;141:438-446. 17. ponglowhapan s, church db, scaramuzzi rj, et al: luteinizing hormone and follicle-stimulating hormone receptors and their transcribed genes (mrna) are present in the lower urinary tract of intact male and female dogs. theriogenology 2007;67:353-366. 18. ponglowhapan s, church db, khalid m: differences in the expression of luteinizing hormone and follicle-stimulating hormone receptors in the lower urinary tract between intact and gonadectomised male and female dogs. domest anim endocrinol 2008;34:339-351. 19. galac s, kars vj, klarenbeek s, et al: expression of receptors for luteinizing hormone, gastric-inhibitory polypeptide, and vasopressin in normal adrenal glands and cortisol-secreting adrenocortical tumors in dogs. domest anim endocrinol 2010;39:63-75. 20. ettinger a, zwida k, kutzler m: normal and neoplastic canine lymphocytes express luteinizing hormone receptors. clin theriogenology 2017;428. 21. kiefel c, kutzler m: luteinizing hormone receptor expression in canine anterior cruciate and femoral head ligaments. iscfr viii 2016; p.151. 22. davis aj, brooks cf, johnson pa: activin a and gonadotropin regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger rna in avian granulosa cells. biol reprod 2001;65:1352-1358. 23. nair ak, menon km: regulation of luteinizing hormone receptor expression: evidence of translational suppression in vitro by a hormonally regulated mrna-binding protein and its endogenous association with luteinizing hormone receptor mrna in the ovary. j biol chem 2005;280:42809-42816. 24. wang lh, zhang w, gao qx, wang f: expression of the luteinizing hormone receptor (lhr) gene in ovine nongonadal tissues during estrous cycle. genet mol res 2012;11:3766-3780. 25. rao jn, liang jy, chakraborti p, et al: effect of thyroid hormone on the development and gene expression of hormone receptors in rat testes in vivo. j endocrinol invest 2003;26:435-443. 26. de leo v, d'antona d, lanzetta d: thyrotropin secretion before and after ovariectomy in premenopausal women. gynecol endocrinol 1993;7:279-283. clinical theriogenology • volume 11 number 1 • march 2019 26 table. signalment of the subjects used to determine lh receptor immunoexpression in canine thyroid tissue. the expression of thyrocytes varied considerably among individuals. breed reproductive status age thyroid cells expressing lh receptors labrador retriever spayed female 2 months 5% american pit bull terrier intact male 5 months 51% labrador retriever spayed female 6 months 19% maltese spayed female 2 years 62% rottweiler intact male 6 years 66% irish setter castrated male 10 years 38% cairn terrier spayed female 12 years 60% clinical theriogenology • volume 11 number 1 • march 201927 figure 1. representative image of immunohistochemical localization; arrows indicate immunoexpression for lh receptor in the canine epidermis (scale bar = 10 μm). inset image is negative control for lh receptor immunostaining. figure 2. representative image of immunohistochemical localization; arrows indicate a low level of immunoexpression for lh receptor in the canine thyroid (scale bar = 10 μm). inset image is negative control for lh receptor immunostaining. clinical theriogenology • volume 11 number 1 • march 2019 28 figure 3. representative image of immunohistochemical localization; arrows indicate a high level of immunoexpression for lh receptor in the canine thyroid (scale bar = 10 μm). inset image is negative control for lh receptor immunostaining. figure 4. representative image of immunohistochemical localization; arrows indicate a low level of immunoexpression for tsh receptor in the canine thyroid (scale bar = 10 μm). inset image is negative control for tsh receptor immunostaining. clinical theriogenology • volume 11 number 1 • march 201929 clinical theriogenology • volume 11 number 1 • march 2019 30 2019_vol1_05a 2019_vol1_05b omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.6 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2014: non-surgical methods of contraception and sterilization in select domestic and wildlife species non-surgical methods of contraception and sterilization in select domestic and wildlife species meredith wikler,a lisa k. pearson,a,b alexis j. campbell,a,b a. tibarya,b a comparative theriogenology, department of veterinary clinical sciences, college of veterinary medicine, and bcenter for reproductive biology, washington state university, pullman, wa abstract population control of domestic and wildlife species is an important consideration of veterinarians and wildlife management organizations. despite decreased rates of euthanasia in united states animal shelters, millions of dogs and cats continue to be euthanized each year. additionally, almost half of the united states feral horse population is currently housed at government-sponsored facilities and lands, and other populations, such as white-tailed deer, have expanded to be considered pest species in some areas of the united states. standard surgical sterilization techniques have been used to help regulate small animal populations; however, other, more efficient means of population control are needed, particularly for freeroaming and large animal species. non-surgical contraception and sterilization methods have the potential of fulfilling this need. although the ideal method has yet to be developed, vaccines against gonadotropin-releasing hormone and zona pellucida glycoproteins have shown promise as contraceptive agents, and intra-testicular injections have been used to induce sterility in males. this review will focus on methods of non-surgical contraception and sterilization which have been researched in cats, dogs, horses, and select wildlife species, and discuss current or future research which may result in increased efficacy, prolonged immune response, and fewer adverse effects in the target species. keywords: gnrh vaccine, zona pellucida vaccine, immunocontraceptive, zinc gluconate, infertility introduction regulation of population size in pet and free-roaming populations is an important consideration for veterinarians and wildlife management organizations. the humane society of the united states estimated that although the rate of euthanasia has decreased approximately 77% since the 1970s, 3.4 million dogs and cats were euthanized in united states animal shelters in 2013.1 additionally, a number of wildlife populations have exceeded the available resources of their habitat, which can impact natural ecosystems. in these cases, animals may be removed from the population by round-up and adoption (feral horses), increased issuance of hunting tags (white-tailed deer), or euthanasia. in 2007, approximately 46.4% of the united states feral horse population was housed in government-funded facilities at a cost of $40 million per year.2 animal overpopulation is a serious concern, and although standard surgical sterilization methods have shown effect in small animal shelter populations, there remains a need for regulation of population size in free-roaming animal populations. in the past few decades, non-surgical methods of contraception and sterilization have become a focus of research. currently several products are in use in the united states and other countries, and have shown efficacy where surgical sterilization methods are undesirable or impractical. non-surgical contraception or sterilization provides several advantages over the current surgical sterilization methods of castration and ovariohysterectomy or ovariectomy. administration of an injection to regulate reproductive ability or to induce sterilization is much less invasive compared to reproductive surgery. pet owners that are averse to anesthesia or surgical sterilization may be more accepting of non-surgical methods. these agents have the potential to greatly increase the number of animals that can be rendered infertile or sterile in population settings, including free-roaming dog populations, feral cat colonies, and wildlife populations. non-surgical contraception or sterilization requires less surgical expertise, equipment, and time than traditional surgical sterilization. several methods of non-surgical contraception or sterilization have been researched, with variable results. currently, there are two methods of non-surgical contraception and one method of non-surgical sterilization which are used or show promise to successfully inhibit or reduce reproductive ability, or induce sterility. two methods use the concept of immunocontraception: the gonadotropin-releasing hormone (gnrh) vaccine and the zona pellucida vaccine. the third method uses intratesticular injections clinical theriogenology • volume 6 number 2 • june 201493 to induce sclerosis and arrest of spermatogenesis. the objective of this paper is to review the methods of non-surgical contraception and sterilization which are currently in use, or which have shown promising results in research trials. these methods will then be discussed in the context of their use and success within select domestic and wildlife species, including cats, dogs, horses, and several wildlife species, with a focus on the control of free-roaming populations. challenges in development of non-surgical methods of sterilization the ideal non-surgical contraceptive or sterilization method would need to meet several criteria, and has yet to be determined. these properties include easy administration, single administration in the animal’s reproductive lifetime, 100% efficacy (including both sexes and all ages), and induction of rapid and permanent sterility.3-5 in addition, pertaining to pet cats and dogs, the ideal method would eliminate sex-related behaviors including estrous cyclicity, roaming, urine spraying, and aggression. in wildlife species, where herd social hierarchy relies on maintenance of sex steroid production (such as in feral horse harems), the ideal contraceptive mechanism must not eliminate production of sex steroids yet prohibit fertility.6 finally, the ideal product must be cost-effective, able to be produced in large quantities with little variation in quality control, and result in few side effects. the development of a product with all of these desired properties is challenging. the michelson grant and prize program was developed to provide $75 million in research monies to develop a one-dose product which would result in permanent sterility in male and female dogs and cats, with a $25 million prize allocated for the successful research group.7 currently $14 million has been allocated to research the “ideal” product.7 the major challenge with the use of immunocontraception is that it requires targeting self-antigens – as a result, self-tolerance must be overcome in order to induce an immunologic response.8 variable responses exist between individuals, and measuring this response poses a problem because it is unclear if circulating antibodies are representative of efficacy.9 long-lasting antibody titers against self-antigens are difficult to achieve, often requiring several boosters for a prolonged response.10 side effects due to affected downstream functions of the target hormones, or due to adjuvants required to stimulate a sufficient response, are also a concern.8 in addition, delivery of non-surgical methods of contraception to free-roaming feral or wildlife animals presents its own set of challenges. currently all formulations require injection, which is achieved either by remote darting from vehicles or aircraft or by hand (typically with the animal tranquilized or anesthetized). access to feral dogs and cats usually requires trapping.9 if formulations were developed which could be administered orally, baiting would need to be species-specific and have a wide margin of safety.9 in addition, it would be difficult to identify which animals have already been treated unless animals are permanently identified, which in some small animals has involved tattoo placement in the pinna or inguinal region.11 for pet dogs, cats, and horses, administration of non-surgical contraceptive vaccines could be easily incorporated into yearly wellness examination visits with the veterinarian. the goals of a non-surgical contraception or sterilization program vary greatly when considering individual animals versus a population. in companion animals, the induction of complete sterility is vital in each individual animal, as owners are unlikely to consider non-surgical methods acceptable if efficacy is not comparable to surgical methods. in contrast, it is not as vital that every individual animal is rendered infertile in a population setting. for example, studies have shown that only 70-80% of female cats in a feral cat population, or more than 95% of males which breed in polygamous mating systems, must be made infertile in order to control the population size.12,13 furthermore, regulation of male fertility is only effective in contained or captive populations. in companion animals, a predictable duration of effect is important, but the need for booster vaccines would be more easily managed than in feral or wildlife settings. it must be recognized, however, that failure of owner compliance with booster vaccines would lead to resumption of fertility in many cases. in contrast, long duration of infertility must be induced in population settings since frequent boosters would be a severely limiting factor in terms of practicality.9 in fact, a single dose of an immunocontraceptive vaccine has resulted in infertility of several years duration in female white-tailed deer, feral swine, and horses.14-18 in feral cat colonies, however, animals tend to have shorter lifespans clinical theriogenology • volume 6 number 2 • june 2014 94 and models suggest that a three-year duration of efficacy in females would be sufficient to regulate population size.12 with any population, there are concerns about the presence of individuals which are "nonresponders" to a vaccine; studies of immunocontraceptives have consistently shown that a small percentage of the population fails to produce an antibody response. this may reflect individual animal genetics, and may lead to a population which, after rendering infertile all susceptible animals, is repopulated by “non-responder” animals, a trait which may be heritable, and which may limit effectiveness of immunocontraceptives in future generations.5 the concept of immunocontraception reproductive function is regulated via the hypothalamic-pituitary-gonadal axis. gonadotropin releasing hormone secreted from the hypothalamus stimulates production of luteinizing hormone (lh) and follicle-stimulating hormone (fsh) from the pituitary gland, which act on the ovaries and testes.19 in females, these hormones regulate follicular activity, ovulation, and the production of estrogen and progesterone. in males, these hormones act on the testes to produce testosterone and support spermatogenesis. immunocontraception relies on the stimulation of antibody production against self-antigens which are involved in the regulation of reproductive function in order to inhibit fertility.9 the goal is to initiate an immune response against a specific hormone or a protein which is integral in the hypothalamicpituitary-gonadal axis in order to induce infertility. the target antigen can be any hormone or protein that plays a mandatory role in reproductive cyclicity and fertility. two targets that vaccines have had success at stimulating an effective immune response against to induce infertility are gnrh and zona pellucida glycoproteins. these two methods of immunocontraception will be discussed in further detail. zona pellucida vaccine zona pellucid (zp) vaccination acts by eliciting an immune reaction against the zp glycoproteins, which form a matrix around the oocyte and which are essential for spermatozoa binding, acrosome reaction, and potentiation of fertilization.8 the glycoprotein which is targeted by the vaccine is zp3, which is specifically responsible for spermatozoa binding and induction of the acrosome reaction.20 the antibodies generated in response to vaccination antagonize the spermatozoa receptor sites, preventing fertilization.20 it is also hypothesized that cell-mediated immunity may also play a role in the effects of the zp vaccine by causing atresia of ovarian follicles and ovulation failure.20,21 since the target of the zp vaccine is far downstream in the hypothalamic-pituitary-gonadal axis, there is little effect on reproductive cyclicity – animals continue to cycle and may be breed.10 vaccinated female white-tailed deer and horses were observed to continue normal estrous cyclicity; however, in some cases beyond the normal breeding season.22,23 maintenance of sexual behaviors is advantageous in wildlife species, where these behaviors are important in maintaining social interactions and a social hierarchy. however, in females in which antibody titers waned, births were observed later in the year than in non-vaccinated animals, which may put offspring at risk for non-survival in winter months.24 in companion animal species, retention of sexual behaviors is not desirable, and can be viewed as a disadvantage of this contraception method. in addition, continued production of sex steroids may result in the development of hormone-dependent uterine or mammary diseases.12 furthermore, in contrast to what has been observed in white-tailed deer and horses, it was observed that long-term treatment (i.e. more than five years of annual vaccination) resulted in irreversible changes within the ovary, including cessation of follicular activity, ovulation failure, and undetectable estrogen levels in bitches.21,25 use of zp glycoproteins as a target for immunocontraception has a number of advantages. since these proteins are specific only to the zp, there is no concern of cross-reactivity with other tissues within the body, as demonstrated using porcine zona pellucida (pzp) antibodies and canine and equine somatic tissue samples.26 portions of the amino acid structure of the zona pellucida glycoproteins are highly conserved among related species, allowing one product to be cross-reactive in other species.12 most zp vaccines which have been used in wildlife species are porcine in origin; these proteins have shown crossclinical theriogenology • volume 6 number 2 • june 201495 reactivity and efficacy in many species, including horses, elephants, and deer.6,15,18,22,27 porcine zona pellucida proteins are readily available from slaughterhouses, and therefore inexpensive to obtain. however, pzp proteins have shown a lack of cross-reactivity in more distant species, such as cats and dogs, which will be discussed further in this paper. there are also a number of disadvantages of the zp vaccine. since zp glycoproteins are only associated with oocytes, this method of contraception can only be used in female animals. like most selfantigens, zp proteins are poor immunogens and require a powerful adjuvant, increasing the risk of adverse injection site reactions.10 furthermore, studies have shown that initial vaccination has short-term effects, and booster vaccines are required for prolonged antibody production.10 a booster vaccine two to six weeks after the initial vaccine, followed by an additional booster one to two years later, can induce infertility for several years.10 a newer vaccine (spayvactm, spayvactm-for-wildlife, inc., new jersey) composed of liposome-encapsulated zp antigens, induced multi-year infertility in white-tailed deer with a single injection.15 adverse effects have been studied in research trials involving domestic and captive wildlife species, but would be difficult to monitor in free-roaming populations. reported side effects of the zp vaccine in horses included increased intrauterine edema (identified ultrasonographically) and estradiol levels, which suggested ovarian dysfunction such as anovulation. in dogs and cats, injection site reactions included both granuloma and abscess development, and in cats granulomas were also found in the lung and brain, associated to the type of adjuvant used.28,29 several cats also developed hypercalcemia and renal dysfunction, and one died of vaccine-induced sarcoma.29 some bitches showed prolonged proestrus and estrus with persistently elevated estradiol levels and no rise in progesterone.28 histologic examination demonstrated ovarian follicular cysts.30 immune-mediated oophoritis has been observed in some treated animals, but is largely attributed to the adjuvant used.29,31 a pzp vaccine is commercially available from the science and conservation center at zoo montana in billings, mt. its effects have been evaluated in wild horses, white-tailed deer, bison, african elephants, seals, and a variety of other species (table).32 the cost is $24 per dose and $15 for each of two adjuvants.32 spayvactm is not yet commercially available. research has shown contraceptive ability in grey seals, fallow dear, white-tailed deer, and other mammals.33 cost of administration of vaccines increases depending on the ability to gather and restrain animals to administer the vaccine. gnrh vaccine the goal of the gnrh vaccine is to stimulate the formation of antibodies against this hormone, and therefore inhibit the downstream release of gonadotropins (lh, fsh) from the pituitary gland. by removing the effects of an upstream hormone in the hypothalamic-pituitary-gonadal axis, suppression of fertility can be achieved. this includes effects such as the suppression of reproductive behaviors, gonadal atrophy, spermatogenic arrest in males, and anestrous in females.34-37 gonadotropin releasing hormone is essential for reproduction in both males and females, making it an effective target for immunocontraception in both sexes. the amino acid structure of gnrh is widely conserved between species, suggesting that one immunocontraceptive product has the potential for use in multiple species. because inhibition of gnrh has downstream effects, the production of sex hormones is blocked, resulting in the inhibition of sex-related behaviors. for example, a significant decrease in aggression has been noted in gnrh-vaccinated males of various species, including bulls, llamas, alpacas, boars, and elephants.34,35,38,39 although studies are lacking, it would be expected that the inhibition of sex hormone production would also limit the development of sex hormone-related diseases, such as mammary gland neoplasia, testicular tumors, pyometra, and prostatic disease.40 however, other conditions seem to have a higher prevalence in animals spayed or neutered at a young age, suggesting that the persistence of sex hormone production may be beneficial in decreasing the risk of diseases such as prostatic carcinoma, osteosarcoma, cranial cruciate ligament rupture, hemangiosarcoma, and urinary bladder sphincter incompetence in female bitches.40,41 using gnrh as a target for immunocontraception presents several disadvantages. although the conservation of the gnrh amino acid sequence between species can be advantageous in terms of efficacy across species, this poses a problem if the product is used in bait for wildlife species. for example, an clinical theriogenology • volume 6 number 2 • june 2014 96 oral gnrh vaccine for deer is currently being developed, but presents a challenge since strict species specificity is required.42 otherwise, wildlife species that are not intentionally targeted may become infertile with exposure to the vaccine. in addition, the advantage of eliminating sex-related behaviors in pets and free-roaming dogs and cats is viewed as a disadvantage in wildlife populations, where the behaviors driven by sex hormones are vital to maintaining social organization.10 furthermore, gnrh is a very small peptide, and therefore serves only as a weak immunogen; it must be administered in combination with carrier proteins and adjuvants in order to induce a sufficient and prolonged immune response.12 since gnrh is released from the hypothalamus and contained within the blood brain barrier, the only opportunity for antibodies to act on the hormone is during its transport through the hypophyseal portal system before binding to receptors within the pituitary gland. therefore, these antibodies must be present at high concentrations in order to be effective.8 studies have consistently shown that the gnrh vaccine is less effective in males than females.12 this is thought to be the result of constant pulsatile gnrh secretion in males, which is likely more difficult to counteract than episodic pulsatile secretion that occurs in females. in addition, all studies involving gnrh immunocontraception have shown that a small percentage of the study population fails to respond to the vaccine with significant antibody titers; a cause for this consistent finding is unknown.12 gonacon™ is a gnrh vaccine that was developed by the national wildlife research center. it was approved for use in white-tailed deer in 2009 and feral horses in 2013. the vaccine is listed as a restricted use pesticide and its administration is limited to united states government organizations. it has also shown efficacy in california ground squirrels, prairie dogs, wild horses, elk, and norway rats.42 worldwide, other commercially manufactured gnrh vaccines provide temporary inhibition of reproductive cyclicity and sex hormone-related effects; these vaccines include equity® (zoetis, inc., australia and zoetis, inc., new zealand), which is registered for use in female horses in australia and new zealand, and improvac® (zoetis, inc., australia), which is approved for use in boars in over 60 countries.39 a commercially produced gnrh vaccine (canine gonadotropin releasing factor immunotherapeutic®, zoetis, inc., florham park, new jersey) was licensed in the united states from 2005-2008 for the treatment of benign prostatic hypertrophy in dogs, but is no longer on the market. however, studies have suggested its potential use as an immunocontraceptive. further research is warranted to evaluate its effects on fertility.34,43 intratesticular injections sterilization via intratesticular injections involves injecting chemical irritants into the testes to disrupt spermatogenesis and to initiate a local inflammatory response. this inflammation allows encroachment of the blood-testes barrier and permits the entrance of lymphoid cells, resulting in an immune-mediated reaction against testicular tissues. the outcome of this process is azoospermia and atrophy of the testis and epididymis (due primarily to decreased testosterone and seminiferous tubule diameter).44,45 zinc gluconate has been the most extensively studied chemical used as an intratesticular sterilant injection, and will be the topic of this section. unlike the methods of contraception discussed earlier in this paper, intratesticular injections of zinc gluconate neutralized by arginine resulted in sterility in 99.6% of treated dogs, although long-term follow-up studies have not been performed to substantiate the permanent sterility claim made by the manufacturer.45 importantly, its use is limited to males which can impact the ability to regulate population sizes. however, for contained or captive populations, implementation of this method of sterilization can have significant effects and reduce need for repeated trapping and vaccination.46 anesthesia is not a requirement for intratesticular injections, although sedation is recommended by the manufacturer to reduce accidental extratesticular injection which can lead to irritation, dermatitis, ulceration, or necrosis.45,47 histologically, zinc gluconate injections resulted in testicular degeneration and sloughing of the germinal epithelium which resulted in azoospermia, as well as vacuolization of both sertoli and leydig cells.44 in field trials, a 77% decrease in testicular size, 52% reduction in prostate size, and up to 52% reduction in testosterone levels were observed.47 clinical theriogenology • volume 6 number 2 • june 201497 use of zinc gluconate injections in cats demonstrated suppression of male sex-related behaviors including aggression, roaming, and marking, which was associated to decreased testosterone levels.48 furthermore, it is hypothesized that decreased testosterone may decrease the risk of sex hormone-related diseases, including benign prostatic hypertrophy, prostatitis, and testicular neoplasia, although no formal studies have been performed and it remains to be determined if a threshold level of testosterone is required.45,47 there are no studies which have evaluated the reproductive behavior of animals after treatment; it is not known if males will attempt to breed or can ejaculate. zinc gluconate injections may be used for sterilization of male dogs and cats in shelters, or in countries or regions where surgical castration is not culturally acceptable or economically feasible. the manufacturer currently is targeting animal shelters, with an offer to provide the product at one-fifth the cost of a surgical castration.45 intratesticular injections reduce the risk of complications associated with anesthesia and surgery but do have known complications. in field trials, the most common side effects were pain upon palpation of the scrotum after injection (6.3%), neutrophilia (6.3%), vomiting (4.4%), and anorexia (4.1%).47 twelve of 1738 dogs required veterinary care post-treatment, five of which required surgical castration (0.003%).47 it should also be noted that although histologic disruption of spermatogenesis is observed within days of administration, extragonadal sperm reserves may allow the animal to successfully reproduce; the manufacturer recommends to keep treated males away from intact females for 60 days.45 the use of zinc gluconate as a form of chemical castration in species besides cats and dogs has not yet been described, so this method of non-surgical sterilization is currently limited in its scope of application. non-surgical contraception and sterilization methods used in cats, dogs, horses, and wildlife non-surgical contraception and sterilization in cats as a species that undergoes early sexual maturity, is seasonally polyestrous, and can produce several large litters each year, cats are extremely prolific. in the united states alone, there are estimated to be more than 90 million free-roaming cats.12 free-roaming cat populations may be a nuisance, harbor pathogens that can be transmitted to humans and other animals, and compromise many native species due to predation. trap-neuter-return programs have begun to replace lethal methods of control in order to manage these exponentially growing cat populations. however, the time and resources needed for these programs make them challenging to sustain, making non-surgical contraception or sterilization methods more ideal to control free-roaming cat populations. gonadotropin releasing hormone vaccination, zp vaccination, and intra-testicular injections have each been researched in order to determine their efficacy in cats. the gnrh vaccine is the method that has shown the most promising results during research trials in cats. in a study of gnrh immunocontraception of male cats, six out of nine vaccinated cats developed high gnrh antibody titers, had undetectable serum testosterone, marked testicular atrophy, and the absence of viable sperm.37 however, the onset of these effects was delayed and varied between individuals, and all but one cat became fertile again by three years after treatment.37 the remaining three vaccinated cats had an intermediate response in terms of the same variables described above, but breeding trials were not done to determine if they were still fertile.37 in a study of gnrh immunocontraception of female cats, all 15 vaccinated cats developed a high gnrh antibody titer.49 breeding trials showed that 93% of the cats remained infertile for the first year following vaccination, 73% for two years, 53% for three years, 40% for four years, and 27% were still infertile at five years when the study ended.49 one model found that successful treatment of 60% of the juvenile females in a population with a three-year duration of contraception could be successful in stopping population growth; these results are very encouraging.49 although the potential need for booster vaccines could be a limiting factor, the use of a gnrh vaccine in free-roaming cat populations would be ideal for a number of reasons. gonadotropin releasing hormone vaccines are effective in both males and females, making it a better choice for population control than either of the other methods of non-surgical contraception and sterilization; gnrh vaccination would also result in the elimination of secondary sex behaviors in treated cats. clinical theriogenology • volume 6 number 2 • june 2014 98 evaluation of the efficacy of pzp vaccination in cats demonstrated no effects on ovulation, fertilization, conception, or litter sizes.11 vaccinated cats developed antibody titers; however, antibodies did not have cross-reactivity with feline zp. the zp glycoproteins from other closely-related species (i.e. cows, minks, ferrets, and dogs) were also evaluated as components of a zp vaccine for cats, but each of these vaccines elicited the formation of antibodies that were much more reactive against the zp glycoproteins of the species of origin than against feline zp proteins, and pregnancy rates were unaffected.50 development of feline zona pellucida-derived (fzp) antigenic epitopes resulted in inhibition of both spermatozoa binding (57.3%) and fertilization (41.5%) of feline oocytes in vitro.51 additionally, use of dna vaccines using fzp proteins in plasmid vectors was examined. twelve cats were treated with one of two vaccines (against different zona pelludia epitopes), and of nine which mated, two became pregnant and each delivered two kittens.52 one vaccinate sustained fetal resorption, and three developed pseudopregnancy. all ovaries demonstrated normal histology and follicular dynamics after ovariohysterectomy.52 although newer vaccine formulations using feline-specific antigens appear to have promise for contraceptive use, zp vaccines may be less than ideal for controlling free-roaming cat populations. the trapping of feral cats is indiscriminate, and therefore, much time would be wasted trapping male cats. requirement for booster vaccines remains a concern. as mentioned previously, the zp vaccine would not suppress the estrous cycle, and sexual behaviors would continue even if the female was rendered infertile. current research on immunocontraceptive vaccines in cats is focused on the use of recombinant technology using viral or bacteriophage vectors.8,53,54 researchers are exploring the use of feline herpesvirus-1 (fhv-1) as a vector, with the potential that if an animal’s infection recrudesces, the vaccine would “self-booster.”8 furthermore, fhv-1 is not transmissible to dogs, which would limit potential spread of vaccine virus. the efficacy of intratesticular injections has recently been evaluated in cats. a study using intratesticular zinc gluconate in male cats showed that 83% became azoospermic, and the remaining cats had decreased sperm concentration and motility.48 although there were no significant changes in testosterone levels of these cats, testicular atrophy, absence of penile spines, and a decrease in sexual behaviors were observed.48 use of intratesticular injections in cat populations will likely have a limited effect on controlling population sizes since only males would be infertile, whereas targeting females for contraception has a much greater overall effect.12 furthermore, ability to copulate or ejaculate has not been examined after intratesticular injections of male cats. although intratesticular injections would require sedation and more handling than administration of gnrh vaccination, it holds potential for permanent sterility in treated animals. further research is warranted. non-surgical contraception and sterilization in dogs approximately 75% of the world’s total dog population consists of homeless stray and feral dogs.55 many of these animals, especially in countries with large free-roaming dog populations, are responsible for fatal dog attacks and human rabies deaths, in addition to other zoonotic infections.56,57 dog removal campaigns and euthanasia in shelters provide only short-term relief of these problems, while large-scale surgical sterilization campaigns have shown to dramatically improve these issues.55,56 however, much like trap-neuter-release programs for cats, these campaigns are greatly limited by cost and availability. non-surgical methods of contraception or sterilization could provide similar benefits as surgical sterilization, but in a more efficient and inexpensive manner. unlike the trials performed in cats, gnrh has shown limited efficacy in dogs. a gnrh vaccine (canine gonadotropin releasing factor immunotherapeutic®) was previously available in the united states for the treatment of benign prostatic hyperplasia in dogs. studies of this vaccine demonstrated a decrease in testicular and prostatic volumes, as well as a decrease in testosterone production in treated dogs.43 however, the study was only short-term, and fertility was not directly evaluated. a gnrh vaccine developed by the usda for wildlife species (gonacon™), showed unsatisfactory results in a pilot study in male dogs, with inconsistent effects on fertility and severe injection site reactions.55 another study in male dogs showed that, although gnrh antibodies were produced, the antibodies failed clinical theriogenology • volume 6 number 2 • june 201499 to have an effect on spermatogenesis and rapidly declined despite booster immunizations.58 in contrast, a study evaluating vaccination with gnrh conjugated to a canine distemper protein in male dogs showed encouraging results – the vaccine was highly immunogenic, resulting in testicular degeneration, sertoli and leydig cell atrophy, and spermatogenic arrest.59 however, the treatment group contained only two dogs and the duration of the vaccine’s effects were not evaluated.59 in female dogs, gonacontm was administered in combination with rabies vaccination; development of antibodies to both immunogens was observed.56 however, no reproductive evaluations or breeding trials were performed.56 in another study, female dogs were administered gonacontm with or without concurrent rabies vaccination.60 all animals developed titers to both immunogens. granulomatous myositis at the injection site developed in seven of 14 animals. two animals were pregnant at the time of vaccine administration and delivered normal litters. animals were euthanized and necropsy demonstrated pituitary congestion, edema, and coagulative necrosis.60 ovaries demonstrated follicular atresia. further investigation of the contraceptive effects of gnrh vaccines on female dogs is needed. one reason which dog owners may elect to delay or elect against surgical sterilization of female digs is the risk of development of urinary bladder sphincter dysfunction, a disorder with incidence of 4.9% to 20% and higher incidence in bitches spayed at less than three months of age.40 it has been suggested that elevated lh levels after ovariohysterectomy have effects on development of bladder sphincter dysfunction. recently, it was demonstrated that treatment of affected spayed bitches with gnrh vaccination resulted in resolution of incontinence in four of nine dogs (44.4%), potentially due to a decrease in lh levels.61 more research in this area is warranted. unlike the reversible effects on fertility observed in horses and white-tailed deer, administratin of zp vaccines to dogs not only has been shown to provide contraception, but in some cases irreversible effects on ovarian function. effective contraception was achieved after a series of booster doses in bitches. the pzp proteins were effective at eliciting a cross-reactive antibody response, and treated female dogs failed to conceive in breeding trials.21 however, treated bitches also demonstrated prolonged proestrus and estrus cycles, failure of progesterone to rise during estrus, and development of follicular ovarian cysts, which may represent anovulation.28,30 immune mediated oophoritis was observed in some dogs.31 findings suggested that contraception may be due to not only inhibition of spermatozoa binding, but also to alteration of follicular dynamics and ovulation. most side effects of the zp vaccines were attributed to the adjuvant used. no long-term studies have evaluated the duration of contraception. due to the observed side effects and requirement for booster doses, zp vaccination does not appear to be the best option for control of population sizes of free-roaming dogs. the results of these studies demonstrate that current gnrh and zp vaccine formulations do not provide consistent contraceptive effects in dogs. the most promising research models appear to be recombinant vaccine vectors including canine distemper virus or canine herpesvirus.8,59 continuing research in these areas may lead to development of more effective, longer acting vaccines for control of canine reproductive ability. the use of intratesticular injections of zinc gluconate neutralized by arginine for sterilization of male dogs has been approved for use in the united states and several other countries. from 2003-2005 a product called neutersoltm was available in the united states, which now is manufactured by a different company under the name zeuterintm (ark sciences, irvington, new york). zeuterintm was approved for use in 2014 for use in male dogs aged three to ten months, with normal, descended testes. injection of zinc gluconate results in sclerosis, arrest of spermatogenesis, and testicular degeneration. testosterone levels of treated dogs decreased 41-52%.45 the effects are thought to be permanent; however, no longterm studies have evaluated testicular function in treated animals. side effects and recommendations for use were discussed earlier in this manuscript. use of intratesticular injections may be beneficial to regulate male reproductive function in captive or contained populations; however, it has been estimated that at least 95% of males in a population must be infertile to affect population growth.13 clinical theriogenology • volume 6 number 2 • june 2014 100 non-surgical contraception in horses in the past few decades, the population of feral horses within the united states has increased drastically from approximately 25,000 to 69,000. approximately half of these horses range freely on public land, while the other half are maintained in government facilities at the cost of millions of dollars per year.2 the large number of feral horses compromises the survival of native species and causes damage to natural ecosystems.2,18 the pzp vaccine has been widely used in free-ranging horses in order to control population sizes. studies have shown that a single injection of pzp can provide multiple years of infertility in an individual mare, but the efficacy gradually declines each year.17 application of the pzp vaccine in feral horse population control programs typically includes annual vaccination, with ovulation failure in individuals and 100% population efficacy occurring after five consecutive injections.23 however, 100% efficacy is unnecessary in controlling populations. a study in a herd of wild horses demonstrated that the pzp vaccine could successfully stop population growth within two years and noticeably decrease the herd size within eight years.62 although the target of the pzp vaccine is far downstream in the hypothalamicpituitary-gonadal axis and should have no effect on reproductive cyclicity or behavior, some studies have shown vaccinated mares may display increased and prolonged reproductive behaviors.23 given the social nature of horses, such changes may alter the interactions and structure of the population as a whole. gonadotropin releasing hormone vaccination has been shown to be successful in inhibiting reproduction in horses in several studies, but with a variable duration of induced infertility.6,63 a series of two vaccinations a few weeks apart was sufficient at rendering most individuals infertile for one breeding season.10,64 in one study, a primary injection and booster several weeks later resulted in anestrous in all mares.63 all but four of the mares returned to cyclicity at a mean time of 418 days (range 232–488 days); older mares showed a trend towards earlier return to cyclicity.63 however, it can be hypothesized that anestrous would be maintained with implementation of annual booster vaccination. one potential benefit of gnrh vaccination is its efficacy in both sexes. in addition to inhibiting cyclicity in females, gnrh vaccines have been shown to temporarily suppress testicular function, greatly affecting semen quality and reducing testosterone levels in stallions.36,65 however, since reproductive behaviors are vital to maintaining the complex social structure of feral horse herds, the inhibitory effects of gnrh vaccination on reproductive hormones and behaviors would have a significant detrimental effect on harem and bachelor herd function when used in free-ranging populations.10 non-surgical contraception in wildlife species the use of immunocontraceptives to control the population sizes of wildlife species has gained popularity over the past few decades. the table summarizes published studies on immunocontraception in several wildlife species. all of these studies focused on the efficacy of immunocontraceptive vaccines in females, which showed high success rates. vaccines were administered using sedation and injection by an operator, or remote darting. antibody titers, hormone levels, ultrasonographic examinations, and birthing rates were all used to determine effectiveness of the vaccines. it is important to keep in mind that most of these studies suffer from small sample sizes and were limited to captive or isolated herds. although this type of setting allows tracking of the animals over time in order to determine effects on fertility and adverse effects, it may not be representative of the true efficacy seen in large-scale use within free-roaming populations. in addition, many studies have conflicting results. these differences may be attributed to variation in vaccination components, such as dose, antigen structure, and adjuvant used. conclusion a non-surgical sterilization method that would fit all of the criteria for the “ideal” technique has not yet been developed. however, even an immunocontraceptive that fails to have 100% efficacy may be sufficient for population control, as long as an adequate number of females within the population are rendered infertile.12 one of the main limiting factors of the techniques that have been developed is the duration of efficacy. the need for booster vaccinations limits the practical use in population management, and methods that induce long-term infertility or permanent sterility are needed. in order to clinical theriogenology • volume 6 number 2 • june 2014101 address this, more recent research approaches have focused on developing an immunocontraceptive with a stronger and more prolonged immune response. this may be achieved in the near future by the use of more effective adjuvants, incorporation of antigens into matrices that allow for slow-release, and development of recombinant vaccines with viral or bacteriophage vectors.8,10 non-surgical contraception and sterilization methods may be used in individual companion animals, but would be especially useful in large-scale settings for population control. these techniques may provide a more efficient, less costly, and less invasive method of rendering animals infertile compared to surgical sterilization. references 1. the humane society of the united states: statement on euthanasia. available at http://www.humanesociety.org/about/policy_statements/statement_euthanasia.html#.uz2o24xvq4m. accessed april 1, 2014. 2. the wildlife society: feral horses: get the facts. available at http://joomla.wildlife.org/documents/policy/feral_horses_1.pdf. accessed july 5, 2013. 3. griffin b, levy j: the future is not far away: nonsurgical sterilization. proc north am vet conf 2009. 4. o'rand mg, lea ia: designing an effective immunocontraceptive. j reprod immunol 1997;36:51-59. 5. cooper d, larsen e: immunocontraception of mammalian wildlife: ecological and immunogenetic issues. reproduction 2006;132:821-828. 6. killian g, miller l, diehl nr, j, et al: evaluation of three contraceptive approaches for population control in wild horses. usda national wildlife research center staff publications paper 370. 2004. 7. michelson prize and grants: available at http://www.michelsonprizeandgrants.org/michelson-prize. accessed april 1, 2014. 8. munks m: progess in development of immunocontraceptive vaccines for permanent non-surgical sterilization of cats and dogs. reprod domest anim 2012;47:223-227. 9. purswell b, kolster k: immunocontraception in companion animals. theriogenology 2006;66:510-513. 10. kirkpatrick j, lyda r, frank k: contraceptive vaccines for wildlife: a review. am j reprod immunol 2011;66:40-50. 11. gorman sp, levy jk, hampton al, et al: evaluation of a porcine zona pellucida vaccine for the immunocontraception of domestic kittens (felis catus). theriogenology 2002;58:135-149. 12. levy j: contraceptive vaccines for the humane control of community cat populations. am j reprod immunol 2011;66:63-70. 13. jewgenow k, dehnhard m, hildebrandt tb, et al: contraception for population control in exotic carnivores. theriogenology 2006;66:1525-1529. 14. miller l, gionfriddo j, fagerstone k, et al: the single-shot gnrh immunocontraceptive vaccine (gonacon) in whitetailed deer: comparison of several gnrh preparations. am j reprod immunol 2008;60:214-223. 15. rutberg at, naugle re, turner jw, et al: field testing of single-administration porcine zona pellucida contraceptive vaccines in white-tailed deer (odocoileus virginianus). wildlife res 2013;40:281-288. 16. killian g, miller l, rhyan j, et al: immunocontraception of florida feral swine with a single-dose gnrh vaccine. am j reprod immunol 2006;55:375-384. 17. killian g, thain d, diehl n: four-year contraception rates of mares treated with single-injection porcine zona pellucida and gnrh vaccines and intrauterine devices. wildlife res 2008;35:531-539. 18. gray me, thain ds, cameron ez, et al: multi-year fertility reduction in free-roaming feral horses with single-injection immunocontraceptive formulations. wildlife res 2011;38:475-481. 19. engelking l: metabolic and endocrine physiology. 2nd ed. jackson: teton newmedia; 2006. p. 26-28. 20. kutzler m, wood a: non-surgical methods of sterilization and sterilization. theriogenology 2006;66:514-525. 21. gupta s, srinivasan v, suman p, et al: contraceptive vaccines based on the zona pellucida glycoproteins for dogs and other wildlife population management. am j reprod immunol 2011;66:51-62. 22. miller l, johns b, killian g: long-term effects of pzp immunization on reproduction in white-tailed deer. vaccine 2000;18:568-574. 23. nunez c, adelman j, rubenstein d: immunocontraception in wild horses (equus caballus) extends reproductive cycling beyond the normal breeding season. plos one 2010;5:e13635. 24. miller la, johns be, killian gj: immunocontraception of white-tailed deer using native and recombinant zona pellucida vaccines. anim reprod sci 2000;63:187-195. 25. kirkpatrick j, turner j, liu i: applications of pig zona pellucida immunocontraception to wildlife fertility control. j reprod fertil suppl 1996;50:183-189. 26. barber mr, fayrer-hosken ra: evaluation of somatic and reproductive immunotoxic effects of the porcine zona pellucida vaccination. j exp zool 2000;286:641-646. 27. delsink a, van altena j, grobler d, et al: regulation of a small, discrete african elephant populatino through immunocontraception in the makalali conservancy, limpopo, south africa. s afr j sci 2006;102:403-405. clinical theriogenology • volume 6 number 2 • june 2014 102 28. mahi-brown c, yanagimachi r, hoffman j, et al: fertility control in the bitch by active immunization with porcine zonae pellucidae: use of different adjuvants and patterns of estradiol and progesterone levels in estrous cycles. biol reprod 1985;32:761-772. 29. munson l, harrenstien la, acton ae, et al: immunologic responses and adverse reactions to freund's-adjuvanted porcine zona pellucida immunocontraceptives in domestic cats. vaccine 2005;23:5646-5654. 30. mahi-brown c, yanagimachi r, nelson m, et al: ovarian histopathology of bitches immunized with porcine zonae pellucidae. am j reprod immunol 1988;18:94-103. 31. fayrer-hosken ra, dookwah hd, brandon ci: immunocontrol in dogs. anim reprod sci 2000;60-61:365-373. 32. the science and conservation center: immunocontraceptive reproductive control utilizing porcine zona pellucida (pzp) in federal wild horse populations. available at http://www.sccpzp.org. accessed july 20, 2013. 33. spayvactm-for-wildlife inc: answers to frequently asked questions about wildlife applications for spayvactm. available at http://terramar.bc.ca/faq.html. accessed july 19, 2013. 34. donovan c, grossman j, pattoon k: effects of a commercial canine gonadotropin releasing hormone vaccination on intact male llamas and alpacas. j vaccines 2013; doi: 10.1155/2013/181834. 35. de nys h, bertschinger h, turkstra j, et al: vaccination against gnrh may suppress aggressive behavior and musth in african elephant (loxodonta africana) bulls a pilot study. j s afr vet assoc 2010;81:8-15. 36. turkstra j, van der meer f, knaap j, et al: effects of gnrh immunization in sexually mature pony stallions. anim reprod sci 2005;86:247-259. 37. levy j, miller l, crawford p, et al: gnrh immunocontraception of male cats. theriogenology 2004;62:1116-1130. 38. price e, adams t, huxsoll c, et al: aggressive behavior is reduced in bulls actively immunized against gonadotropinreleasing hormone. j anim sci 2003;81:411-415. 39. improvactm: frequently asked questions. availabe at http://www.improvac.com/sites/improvac/en-nz/pages/faq.aspx. accessed july 20, 2013. 40. root kustritz m: determining the optimal age for gonadectomy of dogs and cats. j am vet med assoc 2007;231:1665-1675. 41. bryan jn, keeler mr, henry cj, et al: a population study of neutering status as a risk factor for canine prostate cancer. prostate 2007;67:1174-1181. 42. usda aphis: gonacon birth control for deer. available at http://www.aphis.usda.gov. accessed july 19, 2013. 43. donovan c, greer m, kutzler m: physiologic responses following gonadotropin-releasing hormone immunization in intact male dogs. reprod domest anim 2012;s6:403-405. 44. oliveira ecs, moura mr, silva jr va, et al: intratesticular injection of a zinc-based solution as a contraceptive for dogs. theriogenology 2007;68:137-145. 45. ark scinces: zeuterintm. available at www.arksciences.com/products.html. accessed june 10, 2013. 46. levy j, crawford p, appel l, et al: comparison of intratesticular injection of zinc gluconate versus surgical castration to sterilize male dogs. am j vet res 2008;69:140-143. 47. weedon j: neutersol: a clinical perspective. proc north am vet conf; 2009. 48. oliveira e, fagundes a, melo c, et al: intratesticular injection of a zinc-based solution for contraception of domestic cats: a randomized clinical trial of efficacy and safety. vet j 2013;197:307-310. 49. levy j, friary j, miller l, et al: long-term fertility control in female cats wtih gonacon, a gnrh immunocontraceptive. theriogenology 2011;76:1517-1525. 50. levy j, mansour m, crawford p, et al: survey of zona pellucida antigens for immunocontraception of cats. theriogenology 2005;63:1334-1341. 51. ringleb j, rohleder m, jewgenow k: impact of feline zona pellucida glycoprotein b-derived synthetic peptides on in vitro fertilization of cat oocytes. reproduction 2004;127:179-186. 52. eade ja, roberston id, james cm: contraceptive potential of porcine and feline zona pellucida a, b and c subunits in domestic cats. reproduction 2009;137:913-922. 53. samoylova ti, cox nr, cochran am, et al: zp-binding peptides identified via phage display stimulate production of sperm antibodies in dogs. anim reprod sci 2010;120:151-157. 54. samoylova ti, cochran am, samoylov am, et al: phage display allows identification of zona pellucida-binding peptides with species-specific properties: novel approach for development of contraceptive vaccines for wildlife. j biotechnol 2012;162:311-318. 55. levy j: non-surgical methods of sterilization: what's new? proc world small anim vet assoc world cong; 2009. 56. bender sc, bergman dl, wenning km, et al: no adverse effects of simultaneous vaccination with the immunocontraceptive gonacon™ and a commercial rabies vaccine on rabies virus neutralizing antibody production in dogs. vaccine 2009;27:7210-7213. 57. wu xf, franka r, svoboda p, et al: development of combined vaccines for rabies and immunocontraception. vaccine 2009;27:7202-7209. 58. ferro v, khan m, mcadam d, et al: efficacy of an anti-fertility vaccine based on a mammalian gonadotrophin releasing hormone (gnrh-i) a histological comparison in male animals. vet immunol immunop 2004;101:73-86. 59. jung m, moon y, cho i, et al: induction of castration by immunization of male dogs with recombinant gonadotropinreleasing hormone (gnrh)-canine distemper virus (cdv) t helper cell epitope p35. j vet sci 2005;61:21-24. clinical theriogenology • volume 6 number 2 • june 2014103 60. vargas-pino f, gutierrez-cedillo v, canales-vargas ej, et al: concomitant administration of gonacon (tm) and rabies vaccine in female dogs (canis familiaris) in mexico. vaccine 2013;31:4442-4447. 61. donovan ce, gordon jm, kutzler ma: gonadotropin-releasing hormone immunization for the treatment of urethral sphincter mechanism incompetence in ovariectomized bitches. theriogenology 2014;81:196-202. 62. kirkpatrick j, turner a: achieving population goals in a long-lived wildlife species (equus caballus) with contraception. wildlife res 2008;35:513-519. 63. schulman m, botha a, muenscher s, et al: reversibility of the effects of gnrh-vaccination used to suppress reproductive function in mares. equine vet j 2013;45:111-113. 64. card c, raz t, leheiget r, et al: gnrh immunization in mares: ovarian function, return to cycling, and fertility. proc annu conv am assoc equine pract; 2007. p. 576-577. 65. malmgren l, andresen o, dalin a: effect of gnrh immunisation on hormonal levels, sexual behavior, semen quality and testicular morphoology in mature stallions. equine vet j 2001;33:75-83. 66. frank k, lyda r, kirkpatrick j: immunocontraception of captive exotic species iv. species differences in response to the porcine zona pellucida vaccine, timing of booster inoculations, and procedural failures. zoo biol 2005;24:349-358. 67. killian g, kreeger t, rhyan j, et al: observations on the use of gonacon in captive female elk (cervus elaphus). j wildlife dis 2009;45:184-188. 68. miller l, rhyan j, drew m: contraception of bison by gnrh vaccine: a possible means of decreasing transmission of brucellosis in bison. j wildlife dis 2004;40:725-730. table. reported immunocontraception efficacy using zona pellucida (zp) and gonadtropin-releasing hormone (gnrh) vaccination in females of selected wildlife species species method used efficacy duration references white-tailed deer zp gnrh 76% 100% multi-year with boosters 24 months 22 14 african elephants zp 100% multi-year with boosters 10, 27 various bear species zp 100% 30 months 66 zebra zp 96.5% 56 months 66 bighorn sheep zp 100% 14 months 66 elk zp gnrh 91% 90-100% unknown duration 3 years 5 67 grey seal zp 92% unknown duration 5 feral swine gnrh 100% 36 weeks 16 bison gnrh 100% 1 year 68 clinical theriogenology • volume 6 number 2 • june 2014 104 2010: effect of attitudes toward study, study behaviors, and use of study aids on successful completion of the certifying examination of the american college of theriogenologists   effect of attitudes toward study, study behaviors, and use of study aids on successful completion of the certifying examination of the american college of theriogenologists margaret v. root kustritz college of veterinary medicine, university of minnesota, st. paul, mn abstract candidates who had taken the certifying examination of the american college of theriogenologists within the previous ten years were surveyed. data were collected regarding attitudes toward study, self-directed study behaviors, and use of study aids including textbooks, journal articles, websites, list-serves, and group study. study attitudes varied slightly between successful and unsuccessful candidates, with successful candidates more likely to strive for perfection on all tasks and unsuccessful candidates more likely to view completion of the examination necessary for future success. successful candidates exhibited more self-directed study behaviors and to a greater extent than did unsuccessful candidates. the most commonly used study aids are described. keywords: education, examination, study aids introduction reproductive endocrinology, obstetrics, gynecology, andrology, neonatology, and advanced reproductive technologies comprise the discipline of theriogenology in veterinary medicine. to be certified as a specialist in theriogenology, a candidate must be a graduate veterinarian and complete either one year of clinical experience followed by a two to three year residency in theriogenology or complete a mentored program of study while in clinical practice. at the culmination of training, candidates must pass a comprehensive examination evaluating all aspects of theriogenology in all domestic animal species. overall pass rate on the certifying examination averages 56.8%, with 80.7% of candidates passing on their first try.1 the examination is offered annually and failure to pass in a given year is associated with monetary expense if the examination is taken subsequently and may be associated with inability of unsuccessful candidates to progress in their career if board certification is a requirement of employment or promotion. statistical analysis of parameters of the certifying examination from 2000 to 2008 revealed no correlation between success on the examination and route of preparation or species focus, suggesting that extent and type of preparation are what determine success on the examination.1 there is evidence in the literature correlating self-regulatory behaviors with higher test scores in high school and college courses.2,3 these behaviors are self-generated and are the thoughts, feelings, and actions necessary for attainment of goals.4 self-regulated students actively participate in their own learning by altering the environment and their behavior to promote cognitive processes.3,5 examples of successful self-regulatory study behaviors include goal setting and planning, keeping records and monitoring progress, rehearsing verbally and in writing, transforming and reorganizing information, and rewarding oneself for completion of tasks.2 self-regulation also includes self-awareness of when to access outside assistance.6 the strongest correlations between test scores and self-regulatory behaviors are for time management and creation of a study environment.7 motivation can be described as a combination of attainment value, the inherent drive a candidate has to succeed; interest value, the enjoyment one gets from working toward the goal; and utility value, the knowledge that achievement of the goal will enhance one’s life or career.8 the hypothesis is that those who successfully complete the certifying examination of the american college of theriogenologists (act) exhibit more self-regulatory behaviors and higher attainment value, interest value, and utility value than those who are not successful. 467   materials and methods all candidates of the certifying examination for the last ten years were offered a survey assessing self-regulatory behaviors and motivation (survey 1), and a separate survey documenting study aids used (survey 2). the survey was available electronically and all results were anonymous. candidates were contacted by email. survey 1 asked candidates to rate attainment value, interest value, and utility value, respectively, by evaluating statements about their desire for perfection, how much they enjoyed studying for the examination, and how much it would benefit them to successfully complete the examination. the scale used was 0 = statements were not at all true, 1 = statements were somewhat true, 2 = statements were true, and 3 = statement were very true. candidates also were asked to rate on a scale from 1 = rarely to 5 = commonly their use of time management, creation of a specific study environment, seeking of outside assistance, goal setting and planning, keeping records and monitoring, rehearsing and memorizing, organizing and transforming, and positive self-consequences. survey 2 asked candidates how they had used various study aids, including textbooks, journal articles, proceedings, websites, and list-serves. finally, candidates were asked how they had changed their study habits if they had taken the examination more than once, and were asked for general comments. results survey requests were sent to 135 candidates. thirty-two responded to survey 1 and 45 respondents completed survey 2. some individuals completed both surveys. survey 1 was completed by 27 candidates who passed the certifying examination and five candidates who did not. of the 27 who passed, 19 passed on their first try, five on the second try, and three on the third try. attainment value was assessed with the statement, “as a person, i strive to complete every task i undertake as perfectly as possible.” successful candidates rated this as true while unsuccessful candidates rated it slightly lower, with mean scores of 2.0 and 1.6, respectively. interest value was assessed within the statement, “i enjoyed preparing for the american college of theriogenologists certifying examination.” successful candidates and unsuccessful candidates both rated this as somewhat true, with mean scores of 1.3 and 1.4, respectively. utility value was assessed with the statement, “i see great value in passing the american college of theriogenologists certifying examination as a means of helping me achieve future career goals.” successful candidates rated this slightly lower than unsuccessful candidates, with mean scores of 2.1 and 2.4, respectively; both groups considered it to be a true statement. successful candidates used all self-regulatory behaviors to a greater extent than unsuccessful candidates and were much more likely to consciously use time management (table 1). for both groups, creation of a dedicated study space was used frequently. other significant behaviors used by successful candidates included time management, goal setting and planning, and organizing and transforming. survey 2 evaluated use of various study aids. textbooks were used by all respondents, with 82.6% reading and taking notes on entire texts or chapters. this was commonly described as useful for creation of study aids that were then used repeatedly over the study period. texts most commonly used were those providing a broad knowledge base (appendix 1). journal articles were used by all respondents, with 91.4% reading and reviewing select articles with written or oral discussion. articles were chosen as support for clinical cases seen, to promote better understanding of material read in textbooks, or as part of a faculty-led journal club. commonly used journals were those specific to reproduction (appendix 2). proceedings were used by all respondents, with 73.9% reading selected articles or abstracts. the proceedings most commonly used were those from the society for theriogenology annual meeting and from species-specific practitioner organizations (appendix 3). websites were used by 80.0% of respondents. a few candidates had taken specific board preparation courses. most used websites to read compiled notes or view compiled images (appendix 4). list-serves were used by 48.5% of respondents. candidates were equally likely to read questions from a list-serve and not respond or to read and respond to a question with subsequent grading. a small 468   percentage (7.9%) of respondents used list-serve questions to promote peer study. list-serves used most commonly were those supported by the act for board preparation and the society for theriogenology for species-specific case discussions. no respondents reported use of ning, facebook, or other participatory sites for sharing of discussion and documents. finally, face to face meetings for small group study were used by 58.7% of respondents, with most participating as residents in training at their own institutions. guided courses of study offered at the institution, including courses in the dvm curriculum and specific graduate courses, were described as valuable. sample test questions were used by some groups, with some timed to better mimic the actual examination. participation in group meetings promoted timely review of materials and ensured discussion of topics and species outside the candidate’s comfort zone. no respondents reported use of skype or other participatory sites for virtual group meetings. specific suggestions from candidates to promote successful completion of the certifying examination included finding a study partner or group with which to study; ensuring dedicated time for study, as much as six to eight weeks just prior to the examination if possible; reading and seeing cases as much as possible; preparation of study notes and guides for ongoing review; reviewing material in multiple formats including reading texts and answering questions; and consideration of taking an intermediate examination, such as the australian college of veterinary scientists membership examination. other suggestions included flunking the examination once so you knew what to expect, prayer, and beer. discussion successful candidates employed all types of self-regulatory study behaviors to a greater extent than did unsuccessful candidates. this is in agreement with previous work, in which it was demonstrated that successful candidates scored higher on all portions of the examination and in all species than did unsuccessful candidates, suggesting the there is no replacement for broad and deep preparation for the certifying examination.1 attainment value did vary somewhat between the two groups, with the successful candidates more likely to agree with the statement that they strive for perfection than those in the unsuccessful group. neither group had great interest value. surprisingly, the unsuccessful candidates agreed more strongly with the statement of utility value; this may be because they are still striving to reach this goal or because the successful candidates have not realized all they had hoped after successfully completing the examination. conclusion the most important things a candidate can do to prepare for the certifying examination of the act are setting aside time specific for study; creating a study environment with minimal distractions; reading and organizing material from relevant texts, journal articles, and proceedings; using websites judiciously for review of images and other materials, and setting specific goals. the act can help candidates by supporting appropriate residency training programs and mentorships, continuing to provide list-serves for board preparation and case-based discussions, and perhaps helping to create study groups for face-to-face or virtual meetings and journal clubs. references 1. root kustritz mv, kastelic j: statistical analysis of the certifying examination of the american college of theriogenologists, www.theriogenology.org/associations/2753/files/statistical%20analysis%20of%20the%20certifying%20examination% 20of%20the%20act%201.20.09%20final.doc, accessed 05-10-10. 2. kitsantas a: test preparation and performance: a self-regulatory analysis. j exptl educ 2002;70:101-113. 3. judd js, bail ft: secondary students’ strategies and achievement. acad exch quarterly 2006;10:116-120. 4. zimmerman bj: attaining self-regulation: a social cognitive perspective. in: bockaerts m, pintrich p, seidner m, editors. self-regulation: theory, research and applications. orlando (fl): academic press; 2002. p. 13-39. 5. bandura a: self-efficacy: the exercise of control. new york: wh freeman; 1997. 6. orange c. using peer modeling to teach self-regulation. j exptl educ 1999;68:21-39. 469   7. kitsantas a, winsler a, huie f: self-regulation and ability predictors of academic success during college: a predictive validity study. j adv acad 2008;20:42-68. 8. eccles js: subjective task value and the eccles et al model of achievement-related choices. in: elliot aj, dweck cs, editors. handbook of competence and motivation. new york: guilford; 2005. p. 105-121. table 1. use of self-regulatory study behaviors by successful and unsuccessful candidates self-regulatory behavior successful candidate mean score * unsuccessful candidate mean score time management = specific setting of time limits devoted to study 4.1 2.2 study environment = creation and maintenance of dedicated study space without distractions 4.0 3.2 seeking info and help = specific requests for assistance from social and academic contacts 3.4 2.4 goal setting and planning = setting strategic goals and planning study activities 3.9 2.8 keeping records and monitoring = maintaining records of study progress 2.7 2.0 rehearsing and memorizing = engaging in overt practice in written or verbal format 3.3 2.6 organizing and transforming = reorganizing material from lectures, texts, or other sources 3.7 2.8 self-consequencing = rewarding oneself for task completion 2.4 1.4 * 1 = never used, 2 = rarely, 3 = occasionally, 4 = regularly, 5 = commonly appendix 1: textbooks most commonly used by candidates taking the act certifying examination 1) current therapy in large animal theriogenology, 2nd edition. youngquist rs, threlfall w, editors. st. louis: wb saunders; 2007. 2) canine and feline theriogenology. johnston sd, root kustritz mv, olson pn. philadelphia: wb saunders; 2001. 3) pathways to pregnancy and parturition. senger pl. pullman (wa): current conceptions; 2005. best used early in preparation to lay groundwork 4) equine reproduction. mckinnon ao, voss jl, editors. ames (ia): wiley-blackwell; 2003. 5) current therapy in equine reproduction. samper jc, pycock j, mckinnon ao, editors. philadelphia: wb saunders; 2006. 6) manual of equine reproduction, 3rd edition. brinsko sp, blanchard tl, varner dd, et al. st. louis: mosby; 2010. 7) color atlas of reproductive pathology. buergelt cd. st. louis: mosby; 1997. 8) veterinary obstetrics and genital diseases – theriogenology, 3rd edition. roberts sj. woodstock (vt): published by the author; 1986. 9) canine and feline endocrinology and reproduction, 3rd edition. feldman ce, nelson rw. philadelphia; wb saunders: 2003. 470   appendix 2: journals most commonly used by candidates taking the act certifying examination 1) theriogenology 2) animal reproduction science 3) veterinary clinics of north america small animal, food animal, and equine practice appendix 3: proceedings most commonly used by candidates taking the act certifying examination 1) society for theriogenology annual meeting 2) american association of equine practitioners 3) american association of bovine practitioners 4) american association of swine practitioners 5) international meetings – european college of animal reproduction (ecar), international congress on animal reproduction (icar), world small animal veterinary association (wsava) appendix 4: websites most commonly used by candidates taking the act certifying examination 1) drost project – www.drostproject.org 2) cornell pathology – w3.vet.cornell.edu/nst/ 3) university websites – colorado state university, louisiana state university, university of california-davis, university of florida, university of minnesota 4) international veterinary information service – www.ivis.org 471 472 omniblank: 1 contact jamie stewart jlstewart13@vt.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10416 research report evaluating the use of ultrasonography with doppler to assess peripubertal development and predict breeding soundness examination classifications in ram lambs jamie stewart,a olivia reiff,a alyssa helms,a sherrie clark,a scott greiner,b kevin pelzera adepartment of large animal clinical sciences, virginia-maryland college of veterinary medicine, virginia polytechnic institute and state university, blacksburg, va, usa bschool of animal and poultry sciences, virginia polytechnic institute and state university, blacksburg, va, usa abstract the objectives were to evaluate changes in testicular characteristics of peripubertal rams and determine their association with breeding soundness examination (bse) classifications. we hypothesized that testicular echotexture and blood flow assessed via ultrasonography with doppler would predict subsequent bse classifications. ram lambs (n = 40) were examined on intake for performance testing in may. in may and june, scrotal circumference (sc) and scrotal skin temperature measurements were obtained. additionally, ultrasonography with doppler was used to calculate total testicular volume (ttv), assess pixel intensity, and measure resistive (ri) and pulsatility (pi) indices of the right testicular artery. rams underwent bse ~ 10 weeks after the initial evaluation and either passed or failed based on sc and sperm quality. sc, ttv, and pixel intensity increased between may and june, whereas ri and pi decreased (p < 0.01). sc and scrotal skin temperature were greater (p = 0.03) for rams with an ‘excellent’ bse classification than those that failed. sc was also positively correlated (r = 0.53; p < 0.01) with percent normal sperm at bse. testicular pixel intensity, ri, and pi did not differ (p ≥ 0.28) based on bse classification. decreased (p = 0.01) heart rates on intake in may were reported in rams with ‘excellent’ bse classifications and associated with greater percent normal sperm (r = 0.42; p = 0.01) at bse. in conclusion, ultrasonography with doppler can be useful for assessing peripubertal development in ram lambs but provided limited capability in predicting bse classification. keywords: ram lambs, testicular volume, testicular pixel intensity, heart rate introduction although the number of sheep operations in us has declined during the past 40 years, size of us sheep industry has recently stabilized with potential for growth in the foreseeable future.1 to improve profitability within the sheep industry, producers will rely heavily on the development of new technologies that allow for effective dissemination of valuable genetics. specifically, there is a need to select for animals that are less prone to developing diseases following exposure to certain pathogens and parasites, due to growing concerns with drug resistance. the virginia cooperative extension reported on the increasingly widespread resistance of parasites to commercial dewormers on virginia sheep farms.2 the barber pole worm, haemonchus contortus, is the most detrimental of these parasites with the ability to cause severe anemia and death in affected small ruminant hosts. the use of terminal sire crossbreeding systems has the potential to not only improve growth performance in lambs, but also promote parasite resistance.3 early selection of reproductively sound rams is the most efficient way to improve genetics within a flock. the virginia tech southwest ag research and extension center ram test was initiated in 2012 to help quantify growth and parasite resistance in rams. although programs such as these have proven to be useful for selecting genetically valuable sires, the submission of peripubertal ram lambs precludes our ability to predict their reproductive soundness until after the test is completed. as a result, producers may submit ram lambs that perform well in growth performance and parasite resistance testing, but still fail the breeding soundness examination. unfortunately, this practice may discourage producers from mailto:jlstewart13@vt.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10416 2 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10416 submitting ram lambs for testing due to the expense and uncertainty as to whether they will actually be useable for breeding programs. there is, therefore, a need to develop technologies that may predict future reproductive soundness in ram lambs before they are subjected to performance testing. evaluation of breeding soundness in rams includes an assessment of overall health, reproductive organs, and semen evaluation, resulting in a satisfactory, questionable, or unsatisfactory classification.4 ram lambs subjected to performance testing are typically peripubertal, making it difficult to assess semen quality since spermatogenesis is just beginning. the length of sperm transit in the ram is ~ 42 days (6 weeks), so semen evaluation after performance testing is usually suitable for predicting reproductive soundness prior to sale. however, evaluation of testicular architecture at spermatogenic onset may predict subsequent semen quality and aid in earlier ram selection. histological evaluation of testicular biopsy samples is one method to evaluate spermatogenic function with no detectable detriment to testicular development in rams.5 however, its usefulness is severely limited by the amount of time and money required to process and analyze samples. scrotal ultrasonography may be a noninvasive alternative to help predict future fertility in rams. testicular echotexture on ultrasonography was significantly correlated with changes in testicular microstructure, including seminiferous tubule diameter and presence of mature germ cells in ram lambs.6 similarly, testicular pixel intensity in bulls was a useful indicator for pubertal and mature status and was associated with sperm production, sperm morphology, and seminiferous tubule area.7 additionally, blood indices via doppler of internal iliac artery that supplies blood to accessory sex glands, predicted sperm motility and vigor in dorper rams.8 the objective was to determine if testicular ultrasonography with doppler evaluation of the testicular artery may provide veterinarians with an effective means for predicting breeding soundness examination classifications in ram lambs submitted for performance testing. materials and methods procedures used in this study were approved by the institutional animal care and use program at virginia tech (protocol # 19-075). enrolled ram lambs (n = 40) were randomly selected from those housed at the shenandoah valley agricultural experiment station in raphine, va for routine performance testing. ram lambs ranging from 2 to  7 months were classified as either fall-born (average age:  5.9  ± 0.3 months) or winter-born (average age: 3.9  ±  0.2  months). represented breeds included suffolks (n  =  19), dorpers (n  =  11), and katahdins (n  =  10). on arrival to the facility in late april, ram lambs received oral doses of fenbendazole (10 mg/kg; panacur, merck animal health, rahway, nj, usa), levamisole (8 mg/kg; prohibit, agri laboratories, st. joseph, mo, usa), and moxidectin (0.2 mg/kg, cydectin, elanco, greenfield, in, usa) anthelmintics and were vaccinated for respiratory pathogens (1 ml intranasal; inforce 3, zoetis, parsippany, nj, usa), clostridium perfringens type  c and d and tetani (2 ml; barvac cd&t ovine, boehringer ingelheim animal health, duluth, ga, usa), and contagious ovine ecthyma (ovine ecthyma vaccine live virus, colorado serum company, denver, co, usa). rams  were examined on intake by a veterinarian to detect any signs of illness or testicular and epididymal abnormalities. evaluation of ram lambs enrolled ram lambs were examined at 1 week after intake (may) and again ~ 7 weeks later (june). at first examination in may, heart and respiratory rates, body condition score (bcs) on a 1-5 scale,9 and famacha score (1-5)10 were determined. in both may and june, scrotal circumference (sc) was measured with a flexible tape by securing it snugly against the greatest horizontal diameter.11 a small strip of hair/wool was shaved on each testis as needed to facilitate scrotal skin temperature measurements and testicular ultrasonography. rectal temperature was measured using a digital thermometer, and scrotal skin temperature was measured at the proximal, middle, and distal end of the scrotum using a handheld infrared thermometer held ~ 2 inches from the scrotum (figure 1a). the difference between the proximal and distal end of the scrotum was calculated and recorded as the scrotal skin thermal gradient, whereas the middle temperature was recorded as the scrotal skin temperature. the mean scrotal skin temperature and scrotal skin thermal gradient between 2 testes was recorded and used for analyses. ultrasonography was used to measure the length (l) and width (w) of testis to calculate the estimated testicular volume (tv = 0.5236 x l x w2) and ratio (ratio = w/l). total testicular volume (ttv) for each ram was calculated by adding the tv for the left and right testis. the average testicular ratio between 2 testes in each ram was calculated for analyses. images obtained via b-mode ultrasonography, where the mediastinum testes in a vertical plane was evident, were saved and used for pixel intensity measurements (figure 1b). computer analysis of each image was performed using image analysis software (image j, us national institutes of health, md, usa), as described.12 briefly, measurements were taken on each image in 4, ~ 10 mm circles within 10 mm of the mediastinum testis, where the parenchyma appeared homogenous. inside the circles, pixel intensity was measured according to a shade on a 1 to 255 gray-scale (1 = black; 255 = white). the average pixel intensity between 2 testes in each ram was calculated for analysis. testicular blood flow was also assessed using ultrasonography equipped with color doppler (exapad mini, imv imaging). pulsed wave spectral doppler was performed on the right testis testicular artery to measure pulsatility index (pi) and resistive index (ri) (figure 1c). ram lambs underwent a routine breeding soundness examination (bse) ~ 10 weeks after the initial evaluation (3 weeks after the second evaluation) in july. in addition to a focused reproductive examination, semen was collected using a hand-held ram ejaculator (lane manufacturing) and evaluated by an experienced veterinarian for sperm motility and morphology. total sperm motility was estimated by immediately evaluating a drop of semen at low power under light microscopy. concentrated samples were diluted with prewarmed saline to facilitate motility assessments. sperm morphology was assessed by staining a small drop of semen with eosin-nigrosin stain and counting 100 sperm under light microscopy with oil immersion at 1,000 x magnification. each sperm was classified as morphologically normal or abnormal, with abnormalities further classified into primary or secondary defects, as described.13,14 based on modified parameters,15 rams were allocated into 4 bse classifications: 1. ‘excellent’rams with a minimum sc of 31 cm (fall-born) or 30 cm (winter-born) with normal sperm morphology ≥ 90% and sperm motility ≥ 50%; 2. ‘satisfactory’ rams with a minimum sc of 31 cm (fall-born) or 30 cm (winter-born) with normal sperm morphology ≥ 70% and sperm motility ≥ 30%; 3. ‘questionable’ rams with a sc http://dx.doi.org/10.58292/ct.v15.9221 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10416 3 of 28-31 cm (fall-born) or 28-30 cm (winter-born) with normal sperm morphology 30-70% and sperm motility ≥ 30%; and 4. ‘fail’ rams with sc < 28 cm with normal sperm morphology < 30% and/or sperm motility < 30%. data analyses analyses were performed using r (https://www.r-project.org/). testicular measurements (sc, ttv, ratio, pixel intensity, ri, pi, scrotal skin temperature, scrotal skin thermal gradient) were analyzed by anova to assess differences based on fixed effects of bse classification and month of evaluation using ram id as a random variable and breed and age category (fall versus winter born) as independent covariates. for scrotal skin temperature and thermal gradient, rectal temperature was also included as an independent covariate. physical examination parameters at first evaluation (may) were also analyzed by anova to assess differences based on the fixed effect of bse classification, using ram id as a random variable and breed and age category as independent covariates. a pearson’s product-moment correlation test was used to identify associations between testicular/physical examination measurements obtained in may or june and percent normal or motile sperm at the final bse in july. a correlation test was also performed to determine associations between testicular size measurements (sc versus ttv) among months. strong correlations were designated as those with an r > 0.40, with moderate correlations at r = 0.3-0.40, weak correlations at r = 0.25-0.30, and no correlation at r < 0.25. significance was declared at p ≤ 0.05 with tendencies discussed between p = 0.06 and 0.09. results testicular size and shape the sc measurements obtained in may, june, and july were all positively and significantly correlated with one another, as were ttv measurements (table 1). not surprisingly, the strongest correlations were between ttv and sc in may (p < 0.01) and between ttv and sc in june (p < 0.01). percent normal sperm in july was moderately and positively correlated with sc in may (p = 0.01) and strongly correlated with ttv in may (p < 0.01; table 2). there was no association between june or july sc or june ttv with percent normal sperm in july. there was also no association between testicular ratio in may or june and percent normal sperm. in addition, percent motile sperm did not have any associations with sc, ttv, or testicular ratio at any month. the effects of bse classification and month on sc, ttv, and testicular ratio are summarized (table 3). there were main effects of age category (p < 0.01), breed (p = 0.03), month (p < 0.01), and bse classification (p = 0.03) on sc measurements, but no bse classification by month interaction (p = 0.65). rams that failed bse in july had smaller (p = 0.03) sc measurements than those that were classified as ‘excellent’. sc measurements were smaller (p < 0.01) in may (25 ± 0.6 cm) than in june (30 ± 0.4 cm), and the older fallborn lambs had greater (p < 0.01) sc (30 ± 0.6 cm) than the younger winter-born lambs (26 ± 0.6 cm). suffolk ram lambs tended to have smaller (p = 0.09) sc measurements (26 ± 0.7 cm) than katahdins (29 ± 1.0 cm); but neither differed (p < 0.01) from the dorpers (28 ± 0.9 cm). it is worth noting that the dorper ram lambs were, on average, older (6 ± 0.3 months) than the katahdins (4.4  ±  0.4 months; p < 0.01) and the suffolks (3.9 ± 0.2 months; p < 0.01); however, the katahdins and suffolks did not differ (p = 0.54) in age from each other. similarly, there were main effects of age category (p < 0.01), month (p < 0.01), and bse classification (p = 0.02) on ttv measurements, but no effect of breed (p = 0.17) or bse classification by month interaction (p  =  0.28). rams classified as ‘excellent’ tended to have greater ttv measurements than those classified as ‘questionable’ (p  =  0.06) or that failed (p  =  0.07). ttv measurements in may (110  ±  10 cm3) were smaller (p < 0.01) than in june (217 ± 11 cm3; p < 0.01), and the older fall-born lambs had greater (p < 0.01) ttv  (204  ±  13 cm3) than the younger winter-born lambs (142 ± 12 cm3). there were main effects of month (p < 0.01) figure 1. a. scrotal skin temperature measurements were acquired using an infrared thermometer, held ~ 2 inches from the scrotum. the middle-most part of the testis was used for the scrotal skin temperature, whereas the difference between measurements acquired at the proximal and distal-most aspects of the testis were used to calculate scrotal skin thermal gradient. b. to determine testicular pixel intensity, 4 ~ 10 mm spots were measured around the mediastinum using imagej software and averaged for each testis. c. to measure testicular blood flow, a linear probe was positioned over the scrotal neck of the right testis using b-mode ultrasonography until the testicular vascular cone was visualized. pulsed wave spectral doppler was then used to measure the resistive index and pulsatility index of the right testicular artery. http://dx.doi.org/10.58292/ct.v15.9221 https://www.r-project.org/ 4 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10416 and age category (p = 0.05) on testicular ratio, but no effect of breed (p = 0.23), bse classification (p = 0.88) or bse classification by month (p = 0.68) interaction. testicular ratio measurements in may (0.70 ± 0.02) were smaller (p < 0.01) than those in june (0.77 ± 0.01). testicular echotexture, blood flow, and temperature testicular pixel intensity in may and june had no association with percent normal or motile sperm at bse in july (table 2). likewise, there was no main effect of bse classification (p  =  0.28) or bse classification by month interaction (p  =  0.42) on pixel intensity in ram lambs (table 3), but there were main effects of month (p < 0.01), breed (p < 0.01), and age category (p < 0.01). testicular pixel intensity was greater (p < 0.01) in june (78 ± 3.1) than in may (59 ± 2.4) and was greater (p = 0.02) in the younger winter-born ram lambs (70 ± 3.1) than older fall-born ram lambs (65 ± 2.7). interestingly, pixel intensity was greater in suffolk ram lambs (76 ± 3.2) than dorpers (60 ± 3.9; p < 0.01) or katahdins (59 ± 3.4; p < 0.01); however, the katahdins and dorpers did not differ (p = 0.93). table 1. correlation coefficients between scrotal circumference measurements of peripubertal ram lambs obtained in may, june, and july and total testicular volume measurements obtained in may and june testicular parameters sc may sc june sc july ttv may ttv june sc may 0.74*** 0.41*** 0.90*** 0.55*** sc june 0.74*** 0.58*** 0.65*** 0.82*** sc july 0.41*** 0.58*** 0.27* 0.64*** ttv may 0.90*** 0.65*** 0.27* 0.44*** ttv june 0.55*** 0.82*** 0.64*** 0.44*** ***p < 0.01, **p = 0.01-0.05, *p > 0.05. table 2. correlation coefficients between testicular parameters of peripubertal ram lambs measured in may or june and percent morphologically normal sperm or percent motile sperm collecting during a routine breeding soundness examination in july testicular parameters month percent normal sperm percent motile sperm scrotal circumference may 0.39** 0.16 june 0.15 0.07 july −0.007 0.12 total testicular volume may 0.53*** 0.21 june 0.04 −0.12 testicular ratio may −0.17 −0.2 june −0.1 −0.008 testicular pixel intensity may 0.07 −0.1 june −0.17 −0.22 resistive index may 0.11 −0.006 june −0.01 −0.09 pulsatility index may −0.009 −0.05 june 0.08 −0.04 scrotal skin temperature may 0.24 0.17 june 0.17 0.25 scrotal skin thermal gradient may 0.06 −0.05 june 0.21 0.1 rectal temperature may 0.02 0.23 june −0.02 −0.12 body condition score may 0.1 0.12 heart rate may −0.42** −0.24 respiratory rate may 0.08 0.08 famacha may −0.20 −0.23 ***p < 0.01, **p = 0.01-0.05. http://dx.doi.org/10.58292/ct.v15.9221 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10416 5 neither ri nor pi in may or june had an association with percent normal or motile sperm at bse in july (table 2). there was a main effect of month on ri (p = 0.03) and pi (p = 0.02), but no effects of breed (p ≥ 0.30), age category (p ≥ 0.50), bse classification (p ≥ 0.69), or bse classification by month interaction (p ≥ 0.75) on either. in may, ri (0.53 ± 0.03) and pi (0.97  ±  0.1) were greater than in june (ri: 0.43  ±  0.03; pi: 0.66 ± 0.08). neither scrotal skin temperature nor scrotal skin thermal gradient in may or june had an association with percent normal or motile sperm at bse in july (table 2). there was a main effect of bse classification (p < 0.01) and a tendency for a bse by month interaction (p = 0.07) on scrotal skin temperature, as well as interactions with rectal temperature (p < 0.01; table 3) and age category (p = 0.03), but not month (p = 0.16) or breed (p = 0.12). ram lambs that were classified as ‘excellent’ had greater scrotal skin temperatures than those classified as ‘questionable’ (p < 0.01) or that failed (p = 0.03). there were no effects of bse classification (p = 0.94), rectal temperature (p = 0.60), breed (p = 0.82), age category (p = 0.58), or bse classification by month (p = 0.60) interactions on scrotal skin temperature gradient, but there was a tendency (p = 0.09) for interaction with month (table 3). physical examination parameters interestingly, there was a moderate and negative correlation (p = 0.01) between the heart rate measured in may and the percent normal sperm at bse in july (table 2). there was no association between heart rate and percent motile sperm. body condition score, rectal temperature, respiratory rate, and famacha score also did not have any association with percent normal or motile sperm at bse in july (table 2). there was a main effect (p = 0.03) of bse classification on heart rates of ram lambs on intake in may, but no interactions with breed (p = 0.26) or age category (p = 0.99). heart rates of rams on intake in may were lower (p = 0.05) in rams classified as ‘excellent’ than those that failed bse (figure 2a). there were no differences in respiratory rate (p  =  0.91), rectal temperature (p = 0.87), or bcs (p = 0.14) measured in may based on bse classification in july (figures 2a and 2b). there were also no effects of breed (p = 0.93) or age category (p = 0.65) on respiratory rates; a main effect of breed (p  =  0.04), but not age category (p = 0.60) on rectal temperature; and a main effect of age category (p < 0.01), but not breed (p  =  0.76) on bcs. there was a main effect of bse classification (p = 0.02) and a tendency for age category (p = 0.07) to affect famacha score, but no interaction of breed (p = 0.85). ram lambs that failed bse had higher (p = 0.02) famacha scores than those classified as ‘questionable’ (figure 2b). discussion the results presented herein demonstrated that ultrasonography with doppler to assess testicular blood flow and echotexture can be used to assess peripubertal development in ram lambs, but is not predictive of sperm motility, morphology, or bse classification. not surprisingly, rams that failed bse in july had smaller sc measurements in may and june compared to those classified as ‘excellent.’ it is worth noting that the suffolk rams were the youngest breed group represented (average 3.9 months), but their sc did not differ in size from the dorper rams (average: 6 months). though the suffolk rams did not differ significantly in age from the katahdin rams (average: 4.4 months), they tended to have smaller sc. these findings highlighted important breed characteristics that need to be considered when measuring sc and determining an ideal minimum size in small ruminants. there were also moderate to strong correlations between both sc and ttv measurements obtained in may and the percent of normal sperm measured at the july bse, but no association between the percent normal sperm and the june sc/ttv measurements. the association with testicular size in may could be indicative of rams entering puberty at an earlier age that would account for those with better sperm morphology. it is possible that a reevaluation of those that did not pass bse in july may have yielded a passing score at a later date as the rams progressed further into puberty. seasonality may also have a role in the minimal differences observed, as the performance test is performed during the summer, when daylength is longest. the breeding season subsequently commences in august as the daylength begins declining. suffolk sheep are a strongly seasonal breed, so it would make sense that their testis size would not be as big in size until the daylength begins decreasing in august compared to katahdin and dorper sheep that are less influenced by photoperiod. an interesting, yet unexpected, finding was that the rams that were classified as ‘excellent’ during their bse in july had, on average, lower heart rates upon intake in may compared to those that failed bse. correspondingly, there was a moderate table 3. a summary of testicular measurements obtained in may or june based on breeding soundness examination (bse) classification assigned to a ram lamb in july month bse classification sc (cm) ttv (cm3) testicular ratio pixel intensity ri pi sst (°c) gradient (°c) may excellent 25.4 ± 0.9a 130 ± 17a 0.70 ± 0.03a 57 ± 4.7a 0.56 ± 0.05a 0.99 ± 0.17a 31.1 ± 0.4a 2.7 ± 0.3 satisfactory 25.6 ± 1.6ab 120 ± 26ab 0.71 ± 0.06a 59 ± 6.5a 0.56 ± 0.09a 1.16 ± 0.37a 30.2 ± 0.7ab 2.1 ± 0.3 questionable 24.6 ± 1.1ab 109 ± 21b 0.69 ± 0.05a 61 ± 4.5a 0.48 ± 0.07a 0.76 ± 0.13a 29.4 ± 0.5b 2.7 ± 0.6 fail 22.9 ± 1.4b 73 ± 17b 0.72 ± 0.04a 59 ± 3.6a 0.50 ± 0.06a 0.99 ± 0.20a 29.7 ± 0.4b 2.2 ± 0.4 june excellent 31.0 ± 0.6c 235 ± 16c 0.76 ± 0.02b 73 ± 5.4b 0.44 ± 0.06b 0.77 ± 0.16b 30.8 ± 0.4a 1.9 ± 0.2 satisfactory 29.6 ± 1.1cd 220 ± 24cd 0.74 ± 0.03b 75 ± 6.9b 0.36 ± 0.06b 0.52 ± 0.11b 30.5 ± 0.5ab 2.4 ± 0.4 questionable 28.9 ± 0.7cd 175 ± 16d 0.80 ± 0.03b 83 ± 8.5b 0.42 ± 0.08b 0.56 ± 0.12b 30.4 ± 0.4b 1.8 ± 0.4 fail 29.4 ± 1.0d 214 ± 28d 0.78 ± 0.01b 85 ± 4.5b 0.45 ± 0.08b 0.66 ± 0.13b 30.5 ± 0.4b 2.0 ± 0.3 a-dwithin a column, means without a common superscript differed (p < 0.05). http://dx.doi.org/10.58292/ct.v15.9221 6 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10416 and negative correlation between the may heart rate measurement and percent of normal sperm observed at the july bse. ram temperament may be one explanation for this finding, as it seems feasible that calmer rams would be more likely to have a decreased heart rate upon intake. there are a limited number of studies, however, that have reported minimal to no association between male temperament and semen quality in bulls16,17 and rams.18 another factor to consider would be individual animal susceptibility to heat stress, as the heart rate increases with elevated ambient temperatures.19 one might expect the opposite effect, however, with rams experiencing elevated heart rate being better able to thermoregulate testicular temperature due to increased efficiency of sensible heat exchange through conduction.20 interestingly, scrotal skin temperature was also greater in rams that classified as ‘excellent’ versus those that classified as ‘questionable’ or failed, and there was a weak and negative correlation (r = −0.28, p = 0.01) between mean scrotal skin temperature and heart rate. these findings may represent a link between heart rate and scrotal skin temperature that results in decreased susceptibility to the negative effects of heat stress on spermatogenesis. despite these findings, there were no notable changes in the rectal temperature or respiratory rate based on the bse classification, nor was there an effect of ram age or breed. further work is needed to better understand the connection between heart rate and scrotal skin temperature and its associated effect on sperm quality. the scrotal skin thermal gradient measured herein (1.852.50°c) was greater than those reported for haired rams previously (0.5-0.9°c),21 but more in line with those measured in bulls (1.6°c)22 and cross-bred wooled rams (2.8°c).23 interestingly, the mean scrotal skin temperature measured in the current study was lesser (29.5-30.7°c) than those reported in haired rams (32-34°c)21 and more similar to those reported in bulls (~ 29.9°c).22 environmental factors may have contributed to these differences as our study was performed during the summer months in southeast us, whereas the previous ram study was performed in the summer months in brazil,21 and the bull study was performed during summer months in canada.22 interestingly, a study in cross-bred wooled rams determined that the scrotal skin temperature measurements differed based on ambient temperatures of 10°c (26.1-28.9°c) versus 25°c (27.6-30.5°c), but the gradient remained the same (2.8°c).23 on the contrary, haired sheep experienced an increase in the temperature gradient in the summer months versus winter.20 based on these findings, there appears to not only be differences in scrotal skin temperature gradients based on ram breed, but also potential interactions between breed and ambient temperature and humidity indices. although we identified no effect of breed in our statistical model for the temperature gradient, we did have a combination of haired and wooled sheep in our study, and the breed interaction with environmental temperature fluctuations warrants further investigation. regardless, the scrotal skin thermal gradient did not differ based on bse classification or have any correlation with sperm quality in the current study. further studies that investigate substructural sperm characteristics, such as plasma membrane integrity, dna fragmentation index, or presence of reactive oxygen species may provide more insight into the relevance of any potential seasonal and breed differences. similar to the changes in testicular size, testicular ratio also increased from may to june, but did not differ based on bse classification. the ratios measured in may were consistent with a long/ovoid shape (testicular ratio of 0.626-0.750) versus those measured in june that were more consistent with an ovoid/spherical shape (testicular ratio of 0.751-0.875).24–26 in contrast to our findings, authors of another study reported that the testes of dorper rams raised in a subtropical climate became longer and less spherical (increased length, decreased depth) once they reached sexual maturity versus peripuberty.27 since we did not assess the rams in the current study past the peripubertal stage, it is still possible that the testicular shape would continue to change throughout the year. interestingly, bulls with longer shaped testes produced more sperm per day than those with more spherical shaped testes, despite the latter having an equal or larger sc.24 further investigations into the relationship between puberty, testicular shape, and semen quality in rams may be of benefit for clinicians and researchers to better understand its potential usefulness for determining or predicting breeding potential. testicular pixel intensity of ram lambs increased between may and june. these findings are consistent with bulls, where testicular echogenicity increased during sexual development.7 specifically, it was noted that echogenicity began to increase at 16-12 weeks before puberty and reached maximum values 4-0 weeks before puberty.7 ram lambs in the current study were 2-7 months of age at entry into the test station, and, on figure 2. a summary of physical examination parameters measured in may based on breeding soundness examination (bse) classification assigned to a ram lamb in july. a. *heart rate was lower (p = 0.03) in rams that were classified as ‘excellent’ in july than those failed. b. *famacha score was greater (p = 0.02) in rams that failed july bse than those that were classified as ‘questionable’. there were no differences in respiratory rate, rectal temperature, or body condition score based on bse classifications. http://dx.doi.org/10.58292/ct.v15.9221 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10416 7 average, will undergo puberty between 5 and 7 months of age.28 consistently, pixel intensity measurements were greater in peripubertal dorper rams compared to postpubertal rams.27 we identified that the younger, winter-born ram lambs had greater pixel intensity overall compared to the older, fall-born ram lambs that may suggest that the greater pixel intensity observed in june may correspond with near peak values at puberty, and future studies with data expanding into the postpubertal stages could likely find similar patterns as those reported previously. we also identified some differences between breeds in the current study, with the less seasonal breeds (dorpers and katahdins) having decreased pixel intensity measurements than the more seasonally influenced suffolks. differences in ages may have influenced these breed differences, with suffolks being significantly younger than the dorpers. still, further studies are needed to better evaluate the  associations between breeds, seasonality, and testicular pixel intensity to better understand their connection to spermatogenesis. it has been reported that pixel intensity is significantly correlated with seminiferous tubule cell density, the presence of more pre and undifferentiated spermatogonia, and the mitotic and postmitotic phases of spermatogenesis in the peripubertal ram lamb.6,29 therefore, we were surprised that testicular pixel intensity did not differ in may or june between rams who passed or failed bse in july; nor was there any correlation with sperm motility or morphology. these findings are consistent with those reported in bulls, where there was no biological correlation between pixel intensity measurements and semen quality.12 interestingly, pixel intensity measurements in bulls were useful for identifying animals suffering from testicular fibrosis.12 there is, therefore, still a need to investigate its use in diagnosing subclinical testis pathologies in rams. a recent study that also utilized testicular ultrasonography to predict fertility in rams similarly identified no correlation between pixel intensity measurements (measured separately as the number of black versus white versus grey pixels) and sperm motility or morphology.30 interestingly, using ultrasonography video clips and a software program called ecotext®, researchers were able to measure the density of tubules, the percentage of the total area occupied by the lumen of the tubules in the parenchyma, and the mean diameter of the lumen of the seminiferous tubules at high resolution.30 the authors of the article reported a positive correlation between tubular density and both progressive motility and normal sperm morphology.30 in contrast to our study, researchers of that study also reported a small, positive correlation between pi and total sperm motility (r = 0.203), but no correlation with ri (r = 0.186). it is worth noting that semen was collected from each ram monthly over the course of a year in that study.30 in another study, it was determined that rams undergoing a ‘standard frequency’ for semen collection (2 consecutive ejaculates per day and 2 collection days per week) had decreased tubular area and diameter and increased pixel intensity (greater number of grey and white pixels, but lesser number of black pixels) and pi than those that were abstained from collection for a month.31 therefore, the contradictory findings in our study can be related to the fact that the rams were not collected as frequently or as a result of us evaluating only peripubertal ram lambs versus sexually mature rams. in peripubertal ram lambs, we identified that blood flow increased from may to june (as evidenced by decreased pi and ri). in a previous study, there were no differences in doppler measurements of the testis artery reported between peripubertal and sexually mature dorper rams.27 therefore, it would be interesting to know if testis blood flow follows a similar pattern as pixel intensity where it increases in the peripubertal stages and reaches maximum values at puberty. it was also reported that the volume of ejaculate, but no other semen characteristics, was inversely related to the pi of the testicular arteries in dorper rams.27 these findings are consistent with ours in that we identified no significant correlation between doppler measurements in may or june and sperm motility or morphology in july. neither semen volume nor concentration were assessed in the current study, as these can fluctuate greatly when collection is performed by electroejaculation. in mature, fat-tailed rams (2-4 years), marked increases in testicular blood flow were observed within the breeding season, with the least blood flow (highest pi and ri values) measured in june/july.32 interestingly, both pi and ri were significantly correlated with sperm concentration and progressive motility (both assessed on the same day) in that study.33 similar testicular blood flow patterns and correlation with sperm quality were reported in polish heath33 and chois breed34 rams. in the current study, neither ri nor pi (measured in may or june) differed based on ram bse classification in july, nor was there any correlation with sperm quality. we elected to measure these classifications several weeks before the bse to evaluate the effects on spermatogenesis (~ 42 days in rams) rather than on the same day as in previous studies. additionally, since testing was performed outside the natural breeding season (when blood flow would be decreased), it would be interesting to know if a correlation existed if these rams were evaluated into the subsequent breeding season. there is, therefore, a need to further assess the relationship between testicular blood flow and sperm quality amongst different age groups and breeds of sheep both within and outside of the natural breeding season. in conclusion, the results of this study supported the use of ultrasonography with doppler for evaluation of peripubertal ram lambs. although the information provided herein is limited in its ability to predict bse classifications of ram lambs, it does provide a baseline for future studies that should account for factors, such as breed, age, ambient temperature, and season. additionally, the use of advanced technologies such as computer automated sperm analyzers and  flow cytometry may be beneficial in demonstrating a deeper connection between testicular blood flow, echotexture, sperm movement patterns, and substructural sperm characteristics. acknowledgement the authors of this paper thank gabe pent and the staff at the steele’s tavern ram test station for allowing us to use rams housed at the test station for this study. additionally, we thank the clinical year veterinary students at the virginia-maryland college of veterinary medicine who provided technical assistance throughout this project. this project was supported using funding generously provided by the virginia agricultural council. conflict of interest none to declare. http://dx.doi.org/10.58292/ct.v15.9221 8 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10416 references 1. lupton cj: impacts of animal science research on united states sheep production and predictions for the future. j anim sci 2008;86:3252-3274. doi: 10.2527/jas.2008-1148 2. o’brien d, schoenian s, whitley n: dewormer resistance on virginia sheep farms 2017. available from: https://vtechworks.lib. vt.edu/handle/10919/5523 [cited 16 october 2023]. 3. weaver ar: evaluation of terminal sire breeds for hair sheep production systems. master of science. virginia polytechnic institute and state university; 2017. 4. ott rs, memon ma: breeding 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maquivar mg, smith sm, busboom jr: reproductive management of rams and ram lambs during the pre-breeding season in  us sheep farms. animals 2021;11:2503. doi: 10.3390/ ani11092503 29. giffin jl, franks se, rodriguez-sosa jr, et al: a study of morphological and haemodynamic determinants of testicular echotexture characteristics in the ram. exp biol med 2009;234:794-801. doi:  10.3181/0812-rm-364/asset/images/large/10.3181_0812 rm-364-fig4.jpeg 30. carvajal-serna m, miguel-jiménez s, pérez-pe r, et al: testicular ultrasound analysis as a predictive tool of ram sperm quality. biology 2022;11:261. doi: 10.3390/biology11020261/s1 31. montes-garrido r, riesco mf, anel-lopez l, et al: application of ultrasound technique to evaluate the testicular function and its http://dx.doi.org/10.58292/ct.v15.9221 https://doi.org/10.2527/jas.2008-1148 https://vtechworks.lib.vt.edu/handle/10919/5523 https://vtechworks.lib.vt.edu/handle/10919/5523 https://doi.org/10.1016/0093-691x(80)90124-7 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https://doi.org/10.1016/s0093-691x(96)00245-2 https://doi.org/10.1016/j.livesci.2019.06.005 https://doi.org/10.1016/j.livesci.2019.06.005 https://doi.org/10.1016/s0093-691x(98)00009-0 https://doi.org/10.1111/rda.13213 https://doi.org/10.1111/rda.13213 https://doi.org/10.3390/ani11092503 https://doi.org/10.3390/ani11092503 https://doi.org/10.3181/0812-rm-364/asset/images/large/10.3181_0812-rm-364-fig4.jpeg https://doi.org/10.3181/0812-rm-364/asset/images/large/10.3181_0812-rm-364-fig4.jpeg https://doi.org/10.3390/biology11020261/s1 citation: clinical theriogenology 2024, 16, 10416, http://dx.doi.org/10.58292/ct.v16.10416 9 correlation to the sperm quality after different collection frequency in rams. front vet sci 2022;9:1035036. doi: 10.3389/ fvets.2022.1035036/bibtex 32. hedia mg, el-belely ms, ismail st, et al: monthly changes in testicular blood flow dynamics and their association with testicular volume, plasma steroid hormones profile and semen characteristics in rams. theriogenology 2019;123:68-73. doi: 10.1016/j. theriogenology.2018.09.032 33. kozłowska n, faundez r, borzyszkowski k, et al: the relationship between the testicular blood flow and the semen parameters of rams during the selected periods of the breeding and non-breeding seasons. animals 2022;12:760. doi: 10.3390/ ani12060760 34. ntemka a, kiossis e, boscos c, et al: effects of testicular hemodynamic and echogenicity changes on ram semen characteristics. reprod domest anim 2018;53:50-55. doi: 10.1111/rda. 13279 http://dx.doi.org/10.58292/ct.v15.9221 https://doi.org/10.3389/fvets.2022.1035036/bibtex https://doi.org/10.3389/fvets.2022.1035036/bibtex https://doi.org/10.1016/j.theriogenology.2018.09.032 https://doi.org/10.1016/j.theriogenology.2018.09.032 https://doi.org/10.3390/ani12060760 https://doi.org/10.3390/ani12060760 https://doi.org/10.1111/rda.13279 _goback _hlk163050477 _hlk147914372 2019 surgical removal of a retained fetus and subsequent ovariohysterectomy in a sow surgical removal of a retained fetus and subsequent ovariohysterectomy in a sow eleas wu, charles estill, william whitler lois bates acheson veterinary hospital, carlson college of veterinary medicine oregon state university, corvallis, or abstract routine sterilization is no longer a procedure reserved only for companion dogs and cats. with the rise in popularity of household pet pigs and farm animal sanctuaries, spaying gilts and sows is becoming a more common hospital procedure. a 2-year-old crossbred sow presented for an elective ovariectomy procedure. with a history of previous litters and recent estrous cycles, the surgery was expected to proceed as routine. however, during surgery, a firm mass and ulcerative lesion on the uterus were detected. the surgical plan was adjusted and a complete ovariohysterectomy performed. evaluation of tissues revealed a retained fetus in the right uterine horn. pain management was implemented for several days after surgery and the patient recovered without complication. keywords: porcine, sow, retained fetus, ovariohysterectomy background fetal mummification in swine is highest amongst domesticated species, ranging from 1.5 6.8%.1,2 this is likely due to large litter sizes reducing uterine space for adequate fetal growth and development in the final trimester. fetal mummification is possible after 35 40 days of gestation when fetal skeletons have formed. mummification occurs when a lack of oxygen and a dry environment kill bacteria, halting decomposition of a dead fetus. over several weeks, the fetus dehydrates and skin develops a characteristic leather-like appearance. risk factors for fetal mummification include poor management practices, improper nutrition, environmental factors, mycotoxins, and various infectious diseases, including porcine parvovirus, porcine reproductive and respiratory syndrome virus (prrsv), porcine enterovirus, and leptospirosis. litter also has a role; sows and gilts with litters > 12 piglets, have a 14.5 times greater risk of fetal mummification versus those with litters < 10 piglets.3 infectious causes of fetal loss can result in early embryonic death (may be clinically inapparent with a return to estrus), reduced litter size and increased fetal mummies per litter. typically, females show no outward clinical signs, although anorexia, fever, cyanosis of the ears, and coughing were reported with prrsv infections.4 sows and gilts with fetal mummies may have irregular interestrus intervals or unaltered cycle length, but become repeat breeders.5 diagnosis of fetal mummification is generally established after the sow or gilt has farrowed. evaluation of fetuses, live, stillborn, and mummified, aid in determining whether physiologic or infectious causes were likely responsible. typical litter sizes with higher than the average number of mummified fetuses implicate an infectious agent, as compared to a typical litter with no mummified fetus or only a single mummified fetus. serology, including fluorescent antibody tests and enzyme-linked immunosorbent assay, through paired samples from the dam or taking blood samples from surviving piglets prior to suckling may be helpful. additional diagnostics may include fluorescent antibody tests and polymerase chain reactions performed on the mummified fetus(es). case presentation a 2-year-old crossbred pet sow presented to the lois bates acheson veterinary hospital’s theriogenology service for an elective spay (ovariectomy) procedure. she was rescued from a hoarding situation with limited previous history. the patient had farrowed at least 2 litters, with complications during both pregnancies. she experienced rectal and/or vaginal prolapses during late gestation followed by difficult farrowings. the date of her last farrowing was unknown. she had been observed in estrus by her current owner based on behavioral changes and vulvar swelling several weeks prior to presentation. exposure to a boar following her last farrowing was unknown. 135 clinical theriogenology • volume 11, number 2 • june 2019 on physical examination, the patient was estimated to be ~ 225 kg with a body condition score of 3/5. her mammary chains palpated without abnormalities and no milk could be expressed from her teats. her vulva was pale, with no evidence of swelling or discharge. all vital signs remained within normal limits throughout hospitalization. differential diagnosis differential diagnoses considered were: pregnancy, macerated or mummified fetus, neoplasia, or a fibrous tissue adhesion from a previous uterine tear. following removal of the uterus and evaluation, the fetus was suspected to have been retained from a previous parturition. treatment patient was sedated with 500 mg ketamine hcl injection (ketaset®, zoetis, florham park, nj), 50 mg butorphanol (torbugesic®; zoetis, florham park, nj) and 500 mg xylazine (anased® la; vet one, boise, id) iv in the right auricular vein using a 20 gauge butterfly catheter set. due to inadequate sedation, an additional 300 mg ketamine hcl injection (ketaset®, zoetis) and 200 mg xylazine (anased® la; vet one) were administered iv in the left auricular vein. she was then transported to the surgical suite and placed in left lateral recumbency for intubation. an 11 mm (internal diameter) endotracheal tube was placed and the patient maintained on isoflurane (flurisotm; vet one). the patient was rolled into dorsal recumbency and the caudal and mid-abdomen was clipped and prepared using alternating 7.5% povidone iodine scrub and 70% isopropyl alcohol. a 20 gauge intravenous catheter was placed in the left auricular vein and 2 liters of normosol-r (hospira, lake forest, il) were administered throughout the procedure. flunixin meglumine (banamine-s®, merck animal health, madison, nj) 275 mg and 1250 mg ceftiofur crystalline free acid (excede® for swine, zoetis) were administered im behind each ear. a 15 cm incision was made on the ventral midline 5 7 cm cranial to the sow’s pelvis. the uterus was identified and a firm, fetus-like mass, ~ 15 cm in length, was palpated through the incision. further evaluation of the of the right uterine horn confirmed the presence of a mass and ulcerative lesion (figure 1) on the uterine serosa. with owner permission, the procedure was continued, adjusting the surgical plan from an ovariectomy to an ovariohysterectomy. the right ovary was exteriorized and both a circumferential and a transfixation ligature were placed around the ovarian pedicle using 0 polydioxanone (pdstm, ethicon, somerville, nj) absorbable suture. the right uterine horn was exteriorized and small segments of the broad ligament were sectioned for ligation, due to increased vascularization of the broad ligament. ligation of the broad ligament vessels was achieved using 0 polydioxanone (pdstm, ethicon) absorbable suture. the left ovary was exteriorized and ligated in the same manner as the right ovary. due to increased vascularization within the broad ligaments, it was deemed advantageous to utilize a ligasuretm (covidien, dublin, republic of ireland) for electrical hemostasis and cutting. the ligasuretm (covidien) was used to cauterize the remainder of the broad ligament on both sides. the uterine artery and veins of the uterine body were ligated using 0 polydioxanone (pdstm, ethicon) absorbable suture with a miller’s knot. the ligasuretm (covidien) was then used to transect and seal the uterine vessels and body. a parker-kerr pattern was placed over the uterine stump using 0 polydioxanone (pdstm, ethicon) absorbable suture. the abdomen was lavaged with sterile saline. the abdominal wall was closed routinely using 2 polydioxanone (pdstm, ethicon) absorbable suture in a simple continuous pattern. the subcutaneous layer was closed using 2-0 polydioxanone (pdstm, ethicon) absorbable suture, also in a simple continuous pattern. an intradermal pattern using 3-0 poliglecaprone 25 (monocryltm, ethicon, somerville, nj) was used to oppose skin edges. uterine content and ovaries were examined (figures 2 and 3). outcome the patient recovered without complication from surgery and anesthesia. she remained in the hospital overnight for observation. she started eating the evening after surgery and multiple defecations occurred prior to discharge. her pain was managed using 75 mg meloxicam (0.25 mg/kg; cipla ltd., 136clinical theriogenology • volume 11, number 2 • june 2019 mumbai, india) orally once a day for 3 days. several weeks after discharge, the patient was adopted by a farm animal sanctuary and exhibited no further signs of estrus. she is housed with several other neutered male and female pet pigs. discussion to the authors’ knowledge, the only reported case of a retained fetus in a sow was published in 1932.6 however, the incidence is undoubtedly higher, as clinical signs of a retained fetus are vague and may only result in infertility or anestrus. commercial sows and gilts with reproductive failure are frequently culled without further diagnostics. rare cases may result in endometritis or metritis, due to fetal maceration with vaginal discharge and systemic disease. in the present case, a non-typical ulcerative lesion on the serosal surface of the uterus located adjacent to the retained fetus was attributed to pressure necrosis. as routine sterilization is becoming an important part of companion swine medicine, this case is a valuable reminder to evaluate the entire reproductive tract before closure. pet pig ovariectomies are gaining favor over ovariohysterectomies, as they are a simpler procedure and require shorter duration general anesthesia.7 swine have a long and tortuous uterus and it is important to thoroughly palpate the uterus for abnormalities, especially retained fetuses. a retrospective analysis of any case often leads to further possible diagnostics and areas to improve case management and patient care. as history was vague in this case, performing a serum progesterone assay may have proved beneficial for confirming stage of the estrous cycle. however, although the ovaries had evidence of suspected recent ovulations, as supported by the presence of corpora hemorrhagica or young corpora lutea (figure 3), a single progesterone sample may not have been diagnostic. sera obtained 10 days apart were likely needed and not practical for this case. further, obtaining a uterine culture may have assisted in antimicrobial treatment options if complications arose following surgery. the case is also a reminder of maternal recognition of pregnancy in swine and the fetuses’ role in inhibiting luteolysis. our patient likely resumed regular estrous cycles, despite the presence of a deceased fetus, as there was no embryonic signal for pregnancy and thus prostaglandins were likely released by the endometrium for effective luteolysis. learning points • ovariectomies and ovariohysterectomies are becoming routine elective procedures for pet pigs. • thorough evaluation of the entire reproductive tract during traditional and laparoscopic ovariectomies is recommended. • the rate of fetal mummification in domestic livestock species is highest among swine, with increased risk due to poor management, improper nutrition, environmental factors, mycotoxins and infectious diseases. references 1. dial gd, marsh we, polson dd, et al: reproductive failure: differential diagnosis. in: leman ad, straw be, mengeling wl, d'allaire s, taylor dj, editors. diseases of swine. 7th edition, ames: iowa state university press;1992. p . 88-137. 2. van der lende t, van rens bt: critical periods for foetal mortality in gilts identified by analysing the length distribution of mummified foetuses and frequency of non-fresh stillborn piglets. anim reprod sci 2003;75:141-150. 3. borges vf, bernardi ml, bortolozzo fp, et al: risk factors for stillbirth and foetal mummification in four brazilian swine herds. prev vet med 2005;70:165-176. 4. sims ld, glastonbury jrw: the pathology of the pig: a diagnostic guide. barton, australia: pig research and development; 1996. 5. lefebvre, rc: fetal mummification in the major domestic species: current perspectives on causes and management. veterinary medicine (auckland, nz) 2015;6:233-244. 6. baumgartner, wj: remains of a fetal pig in the uterus of a sow. transactions of kansas academy of science (1903). 1932;35:81-84. 7. biedrzycki a, brounts sh: a less invasive technique for spaying pet pigs. vet surg 2013;42:346-352. 137 clinical theriogenology • volume 11, number 2 • june 2019 figure 3. ovary with corpora hemorrhagica or early corpora lutea. figure 2. retained fetus after removal from uterine horn. figure 1. uterus prior to removal; note the ulcerated lesion (arrow) and enlarged mass within right uterine horn. 138clinical theriogenology • volume 11, number 2 • june 2019 << /ascii85encodepages false /allowpsxobjects false /allowtransparency false /alwaysembed [ true ] /antialiascolorimages false /antialiasgrayimages false /antialiasmonoimages false 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spontaneous lymphoma affecting the testicles and nasal cavity of a one-year-old bull 1  atypical spontaneous lymphoma affecting the testicles and nasal cavity of a one-year-old bull jennifer h. koziol, esther g. martinez romero, jennifer w. koehler, dwight f. wolfe, herris s. maxwell college of veterinary medicine, auburn university, auburn, al summary a one-year-old bull was presented to the j. t. vaughan large animal center at auburn university with a six-week history of mucopurulent discharge from both nares. the owner reported that the bull had increased stridor of the last several weeks that did not respond to systemic antibiotics. on physical examination the bull had stenotic nares due to bilateral masses reducing the diameter of the nasal cavities. the bull’s scrotum was enlarged due to enlargement of both testes, with a scrotal circumference of 53 centimeters. on palpation the testicles were firm with adhesions within the vaginal cavity. the heads of the epididymides were more firm than normal. due to the poor prognosis for future fertility and the ongoing respiratory distress the bull was euthanized and submitted for necropsy. at necropsy, multiple masses were present in the nasal passages, testes, epididymis, and haired skin. histopathological examination of sections supported a diagnosis of sporadic multicentric bovine lymphoma. keywords: atypical lymphoma, testicular lymphoma, nasal lymphoma, spontaneous lymphoma background bovine sporadic multicentric lymphoma occurs independently of bovine leukemia virus infection. tumor involvement of either the nasal cavity or testicle is rare in cases of sporadic and enzootic bovine lymphoma, and concurrent involvement has never been reported. nonetheless, this case highlights the importance of adding neoplasia due to enzootic bovine lymphoma or sporadic bovine lymphoma to differential lists when examining bulls for scrotal enlargement. case presentation a one-year-old mixed-breed beef bull was presented to the j. t. vaughan large animal teaching hospital at auburn university in march 2015, for a complaint of nasal discharge and increased respiratory effort of six-weeks duration. the owner reported that initially a bilateral serosanguineous discharge was present from both nares. the serosanguineous discharge progressed to a mucopurulent discharge associated with increased respiratory effort over the following two weeks. at that time the owner administered tulathromycin (draxxin® zoetis animal health, florham park, nj) systemically and the bull’s clinical signs appeared to improve. approximately one week prior to presentation clinical signs of mucopurulent discharge and increased respiratory effort returned. on initial examination the bull was depressed but responsive, with a body condition score of 4/9. rectal temperature was 103.1°f. cardiac auscultation was unremarkable, with a regular heart rhythm and rate of 56 beats per minute. the bull had a significant increase in inspiratory effort and was tachypneic with paradoxical abdominal effort with mucopurulent discharge from the nares (fig. 1). there was a marked decrease in lung sounds beyond the seventh intercostal space bilaterally. both nasal passages had visible masses protruding into the nasal canal causing severe stenosis of the passage and were believed to be cause of the respiratory distress. the scrotum was greatly enlarged with a circumference of 53 centimeters and the scrotal contents were abnormal. both testes were firm on palpation and adhesions could be palpated within the vaginal tunic (fig. 2). the heads of the epididymides were also enlarged and more firm than normal on palpation . ultrasonic examination revealed the testes and heads of the epididymides to be enlarged and hyperechoic. no lung pathology was detected on ultrasonic evaluation. multiple small circular masses were also present on the upper and lower right eyelid. 4047534949 clinical theriogenology · volume 8, number 1 · march 2016 2  differential diagnosis due to the involvement of both the nasal passages and the testes, differential diagnoses for the nares included: mycotic rhinitis, nasal granuloma, neoplasia or sinonasal cysts and for the testes orchitis, epididymitis, periorchitis, and neoplasia. outcome severe stenosis of the nasal passages and palpable scrotal pathology resulted in a poor prognosis and the bull was euthanatized and submitted for necropsy. at necropsy, numerous 5 mm to 3.5 cm in diameter, well demarcated, slightly raised masses were present on the upper and lower right eyelid, inguinal region, and haired skin over the right lateral thorax and flank. both testicles were diffusely firm, pale yellow to tan with an imperceptible mediastinum. the head and tail of the epididymides were diffusely enlarged by multifocal to coalescing, white to tan, firm masses. there was bilateral mucopurulent discharge from the nostrils. multiple expansile, individual-to-coalescing, light grey to purple nodules ranging from 1 cm to 4 cm in diameter were on the alar and basal folds of the nasal mucosa and conchae and were partially occluding the passages of the dorsal and middle meatus. on light microscopic examination, the cutaneous masses were poorly circumscribed, densely cellular, and composed of sheets of monomorphic neoplastic round cells that infiltrated the dermis, and separated and surrounded the adnexae, pre-existing collagen bundles, and blood vessels (fig. 3). neoplastic cells were large (three times the diameter of an erythrocyte) with distinct cell borders and scant pale, granular, eosinophilic cytoplasm. nuclei were round to ovoid, with occasional cerebriform convolutions of the nuclear membrane, coarsely stippled chromatin and 1-2 basophilic nucleoli. there were more than 100 mitotic figures in ten 400x fields. the nasal passages, testes, and epididymides contained monomorphic round cells similar to the neoplastic cells in the haired skin (fig. 4). the neoplastic cells invaded the nasal submucosa and surrounded the nasal cartilage, and partially effaced the testicular and epididymal parenchyma. the final diagnosis was multicentric lymphoma, large cell-high grade. histological features were strongly suggestive of a b-cell lymphoma. however, with immunohistochemical staining, the neoplastic cells did not exhibit cytoplasmic immunoreactivity for cd3 or pax5. discussion bovine lymphoma is classified on epidemiological and clinicotopograhical criteria into two types: enzootic bovine leukosis (ebl), and sporadic bovine leukosis (sbl). enzootic bovine leukosis is endemic in the usa. it is the predominant cause of lymphoma in cattle and is associated with infection with bovine leukemia virus (blv). bovine leukemia virus is an exogenous c-type oncovirus of the retroviridae family infecting the blymphocyte cell lineage.1,2 enzootic bovine leukosis usually occurs in animals greater than three years old, with higher incidence in dairy herds compared with beef herds. the virus can be transmitted either horizontally or iatrogenically to susceptible hosts via biological material containing b-lymphocytes such as blood, milk/colostrum or saliva. insect vector transmission may play a role in blv transmission.1 once infected, 80-90% of animals become blv seropositive. of these seropositive animals, approximately 30% will develop a persistent lymphocytosis but less than 5% develop clinical disease or lymphoma.1,3 in contrast, sbl has no known etiology, does not have an association with blv infection, and occurs uncommonly in cattle less than three years of age. sporadic bovine leukosis has a much smaller incidence, affecting 0.5 to 1.2 out of every 100,000 cattle compared to enzootic bovine leukosis.1 sporadic bovine leukosis has four clinical presentations: the cutaneous form, juvenile/calf form, thymic/adolescent form, and the atypical form which is reserved for those cases that do not fit any of the previous three categories.1,4,5 the cutaneous form is the most commonly reported and the thymic form the least commonly reported. the cutaneous form typically affects cattle aged one to hree years and manifests primarily as skin nodules. it has been reported that that cases of the cutaneous form may undergo a brief period of remission in which the nodules regress; however, four to six weeks after regression a generalized lymphoma returns and is always fatal. the calf or juvenile form occurs most 4148545050clinical theriogenology · volume 8, number 1 · march 2016 3  commonly in calves less than six months of age, but is occasionally reported in animals up to two years of age.1,6 the calf form presents as a generalized lymphadenopathy, with weight loss, depression, and weakness followed by death two to eight weeks after the onset of the disease. the thymic or adolescent form is universally fatal, occurs in animals six to 24 months old, and is more common in beef than dairy calves. lymphoma of the thymus presents as a large, firm enlargement extending cranially from the thoracic inlet. the cutaneous form of sbl is associated with a helper/inducer t-cell origin and the thymic form arises mainly from a t-cell origin. the calf form originates from band t-cell lines.2 b-cell lymphoma is also possible with sbl.5 in europe, due to the eradication of blv positive animals from most of the continent, sporadic bovine leukosis is the predominant type of leukosis. only one case of lymphoma invading the testis has been reported and disease in that animal was reportedly due to enzootic bovine leukosis secondary to infection with bovine leukemia virus.3 similarly, a single case with invasion of atypical sbl into the nasal cavity of a 22 month old brown swiss heifer has been reported. to the authors’ knowledge, the case outlined in this report is the first in which there was both testicular and nasal involvement with sbl. while there is no means of prevention or treatment, it is important to remember that lymphoma is the most common neoplasm of young cattle and should be included on differential lists for stenosis of the nasal passage or scrotal enlargement. learning points  lymphoma is the most common neoplasm in young cattle.  sporadic bovine leukosis should be considered in cases of scrotal enlargement in bulls less than three years of age.  enzootic bovine leukosis is the predominant cause of lymphoma in the united states and is associated with bovine leukemia virus. in contrast sporadic bovine leukosis has no known etiology with a low prevalence as compared to the enzootic form. references 1. nasir ks: sporadic juvenile thymic lymphoma in a 6-month-old holstein heifer. can vet j 2005;46:831-833. 2. dubreuil p, lanevschi a, perrone ma, et al: atypical sporadic lymphosarcoma in a 7-month-old holstein heifer. can vet j 1998;39:431-433. 3. mccain d, estill ct: theriogenology question of the month. scrotal enlargement caused by lymphosarcoma associated with bovine leukemia virus. j am vet med assoc 1999;215:1777-1779. 4. klintevall k, berg a, svedlund g, et al: differentiation between enzootic and sporadic bovine leukosis by use of serological and virological methods. vet rec 1993;133:272. 5. braun u, brammertz c, maischberger e, et al: t-cell lymphoma in the nasal cavity of a brown swiss heifer. acta vet scand 2015;57:8. 6. grunberg w, eisenberg sw: atypical form of sporadic bovine leukosis (sbl) in the netherlands. vet rec 2013;173:398. 4249555151 clinical theriogenology · volume 8, number 1 · march 2016 4  figure 1. mucopurulent nasal discharge was present bilaterally. figure 2. the scrotum was greatly enlarged with a circumference of 53 centimeters and the contents were abnormal. 4350565252clinical theriogenology · volume 8, number 1 · march 2016 5    figure 3. haired skin. the tumor is composed of sheets of monomorphic neoplastic lymphocytes that infiltrate the dermis, and separate and surround the pre-existing collagen bundles (h&e). bar = 20 µm figure 4. testicle. the testicular architecture is effaced by neoplastic lymphocytes (h&e). bar = 50 µm. (editor’s note: the photographs in this manuscript are available in color in the online edition of clinical theriogenology.) 4451575353 clinical theriogenology · volume 8, number 1 · march 2016 2014: female pseudohermaphroditism in a dorset sheep: a case report female pseudohermaphroditism in a dorset sheep: a case report bret r. mcnabb,a alan j. conley,a n. james maclachlanb adepartment of population health and reproduction and bdepartment of pathology, microbiology and immunology, school of veterinary medicine, university of californiadavis, davis, ca summary a four-month old dorset lamb was presented to the university of california-davis (ucd) veterinary medical teaching hospital (vmth) for correction of an inguinal hernia and castration. on physical examination, testicles were not found in the scrotum and it was presumed to be a cryptorchid ram with an inguinal hernia. surgical exploration of the abdomen identified an intact uterus, cervix, vagina and bilateral ovaries. the urethra exited the bladder via a functional fibroelastic penis. the lamb was slaughtered at 11 months of age and the reproductive tract was harvested. the lamb had a 54-xx genotype and the gonads were histopathologically confirmed to be ovaries. a fully intact female internal reproductive tract, in conjunction with functional male external genitalia, identified this lamb as a female pseudohermaphrodite. in livestock, freemartinism and male pseudohermaphrodites are more commonly reported, whereas female pseudohermaphroditism is rare. in this case report we describe the first documented case of female pseudohermaphroditism in sheep. keywords: female pseudohermaphrodite, intersex, sheep, sexual differentiation background intersexuality, or abnormal sexual development, is relatively uncommon in livestock, although it has been repeatedly documented in all species. intersexuality is discordance between or among at least two of the three levels of sexual differentiation, specifically: genetic (at the chromosomal level, ie xx and xy), gonadal (presence of testis or ovaries) and somatic (associated with development of the internal genital tract or external genitalia). the presence of both ovarian and testicular tissue within the gonads results in a true hermaphrodite, with varying phenotypic expressions. this may result from mosacism or chimerism, in which multiple cell chromosomal populations exist within one individual that are derived from the same or different zygotes, respectively. where the lack of concordance is between the gonadal sex and the form of the internal or external genitalia, the condition is referred to as pseudohermaphroditism. the most common cause of female pseudohermaphroditism (ovaries accompanied by masculinization of the internal ducts, with either retention of the mesonephric or regression of the paramesonephric ducts, and/or virilization of the external genitalia) in livestock species is freemartinism in cattle, wherein heterosexual twins/triplets become xx/xy blood chimeras and display a variety of phenotypes often accompanied by gonadal dysplasia. in contrast, freemartinism is far less common in sheep and other species.1 although male pseudohermaphroditism has been reported in many species, including sheep,2 we are unaware of documented instances of female pseudohermaphroditism that do not result from freemartinism. in this report we describe a case of female pseudohermaphroditism in sheep. case presentation a four-month old 22.7 kg dorset lamb thought by the owner to be a male was presented to the ucd vmth for evaluation and surgical correction of an inguinal hernia and castration. the lamb was twin to a ewe lamb and clinically healthy, though noticeably smaller than male flock cohorts. the physical appearance was overtly male with a prepuce positioned normally and opening ventrally. the scrotum was relatively small and the animal had teats that appeared larger than expected for a ram. no testicles were palpable but there was evidence of herniation into the right scrotal compartment. ultrasound examination identified loops of intestine in the scrotum without obvious testicles or epididymi. clinical theriogenology • volume 6 number 2 • june 2014105 the sheep was anesthetized and surgical exploration initially involved the removal of the distal third of the scrotal skin in a customary approach for castration. the hernia sac was unremarkable however no testicles or epididymi were found in the scrotum. the sac was briefly explored and its contents appeared to be unremarkable loops of bowel. scrotal ablation was performed in a routine manner. a second incision was made parallel to the right inguinal ring in an attempt to locate testis, but no male reproductive structures were found. however, an intact uterus and bilateral ovaries were identified amongst the herniated loops of bowel that had been within the scrotum. the loops of bowel and reproductive organs were replaced into the abdomen and the inguinal rings and external layers were closed surgically. the lamb remained below the average body weight of its flockmates over the next three months, but was apparently attractive to the ram lambs during which time swelling developed in the perineal area. a blood sample was drawn and serum testosterone concentration was determined to be 54 pg/ml. this was considered to be inconsistent with the presence of functional testicular tissue.3 treatment and outcome the lamb was slaughtered at 11 months of age, providing an opportunity to examine the reproductive organs. the lamb’s physical appearance had not significantly changed since surgical exploration of the abdomen, but the perineal swelling had continued to increase in size (figures 1 and 2). a fully intact, grossly normal bicornuate uterus was present in the abdomen. two ovaries with evidence of follicular development were positioned in the abdomen as would be expected for a normal female. distal to the uterine body and cervix was a blind vagina distended with mucoid fluid. adjacent to the distal end of the vagina was a bi-lobed, amorphous mass resembling paired seminal vesicles, one of which contained purulent exudate. the urinary bladder communicated with the urethra, which ran through a juvenile penis still attached distally to the preputial mucosa (figure 3). histopathology confirmed the presence of bilateral ovaries with active folliculogenesis on each gonad. primary and secondary follicles with unremarkable oocytes were present, as well as a discrete corpus luteum indicative of recent ovulation. while follicular development was ongoing, the ovary itself was hypoplastic and disorganized with a paucity of follicles. there was no evidence of testicular tissue within either ovary. the uterus was histologically unremarkable in both horns. the mass adjacent to the vagina contained glandular tissue resembling an undifferentiated accessory sex gland. the secretory glandular epithelium was transitional with prominent squamous metaplasia. these metaplastic changes are consistent with estrogenic stimulation seen in other species.4 genotyping from a preserved muscle biopsy was performed by the ucd veterinary genetics laboratory. following fibroblast amplification, genotyping for amelogenin only showed presence of the x-variant, indicating this animal was chromosomally xx. polymorphic genetic markers typically used for parentage analysis found no evidence of chimerism. discussion normal sexual differentiation in mammals is directed by the sex chromosome constitution (xx, xy, or variations thereof). specifically, the sry-region of the y chromosome drives normal testicular development in utero. clinically, the sex (female or male) is defined by the presence of ovaries or testis. the stabilization and development of the mesonephric (wolffian) ducts, and concomitant regression of the paramesonephric (mullerian) ducts, is induced by testicular testosterone and anti-mullerian hormone (amh), respectively, synthesized and secreted by the testes if the fetus is male. the external genitalia and accessory sex glands are also induced to develop from embryonic primordia by dihydrotestosterone (dht) synthesized locally within these tissues from circulating testosterone. the ovaries of female fetuses are relatively inactive endocrinologically and in the absence of testosterone and amh the mesonephric ducts regress and the paramesonephric ducts develop into oviducts, uterus and anterior vagina. similarly, in the absence of testosterone and dht, the genital tubercle remains undeveloped and genital folds develop into the lips of the vulva. the fetus in question was a female pseudohermaphrodite with extensive virilization of the external genitalia suggestive of androgen exposure in utero. clinical theriogenology • volume 6 number 2 • june 2014 106 freemartinism is the most common cause of female pseudohermaphroditism in livestock but is an unlikely explanation in this case. freemartinism is common and well-documented in cattle, less so in sheep, and is thought to be more common in breeds with high ovulation rates and multiple lambs.5 in each case, it is associated with placental anastomosis between multiple fetuses that results in blood chimerism. when placental anastomosis involves fetuses of different genetic sex, development of the female fetus is thought to be disrupted by the testosterone and amh circulating from the male co-twin. exposure of testosterone and amh from the male to the female results in varying degrees of gonadal and phenotypic dysgenesis, and subsequent subfertility and infertility of each offspring. the formation of blood chimeras (xx/xy) is the basis for confirmatory karyoptype testing.1 the female in question is unlikely to be a freemartin because she was not co-twin to a male, but rather to a fertile female sibling that later became pregnant and lambed uneventfully. the likelihood of a third male fetus that developed long enough to have undergone sexual differentiation but subsequently died without leaving any evidence of its existence in utero seems remote. true hermaphroditism must be considered in cases like this, and has been reported in sheep with either bilateral ovotestis6,7 or one testicle and one ovary.8 these cases were phenotypically female with a female reproductive tract, and confirmed with histopathology of the gonads to have both ovarian and testicular tissue. we have eliminated this possibility with histopathology. male pseudohermaphroditism (testis with female genitalia) has been reported in the sheep2,9 and may result from decreased androgen production or androgen insensitivity in target tissues. the higher incidence of these cases is reasonable, in that without testosterone, amh and dihydrotestosterone the female tract is by many accounts the default pathway for development. the opposite is quite unusual. female pseudohermaphroditism is extremely rare but has been reported in cattle,10,11 dogs,12 humans,13-15 and bears, including polar bears.16 these phenotypes range from slight clitoral hypertrophy and infertility to fully-developed external male genitalia. while some cases resulted from mosaicism (sry-positive and not), others had only xx chromosomal compositions, suggesting extra-gonadal hormonal influences. investigation into polar bears in norway with a relatively high incidence of female pseudohermaphroditism suggested exposure to chemical environmental pollutants, specifically polychlorinated biphenyls (pbcs), which may disturb the differentiation of organs with gonadal hormone receptors.16 in any instance, there is a finite window during gestation and sexual differentiation when exposures to such factors must occur to have such severe effects on normal fetal development. the etiology of pseudohermaphrodism in this ewe remains unknown, but it is consistent with the effect of exposure to androgens during days 30-60 of gestation which induces a similar degree of virilization in ewe lambs experimentally exposed to testosterone.17,18 the lack of any abnormality in the female co-twin argues strongly that the androgen presumed to induce genital masculinization of affected ewe lamb must have been synthesized by the lamb itself, even if not gonadal. exogenous androgen exposure during a very limited developmental window is plausible when all offspring are affected, but not when affected offspring are accompanied by normal siblings as in this case. in humans, female pseudohermaphroditism is a common sequel to congenital adrenal hyperplasia,13,15,19 an autosomal recessive disorder. female fetuses with a deficiency in the enzymatic pathway necessary for cortisol synthesis (most frequently due to lack of 21-hydroxylase) have increased acth levels due to lack of feedback, leading to over-stimulation of the adrenal zona reticularis and overproduction of adrenal androgens.20 male genitalia will develop despite an xx-karyotype, though these individuals often have concurrent electrolyte imbalances because of adrenal dysfunction. other than humans, this condition has been reported to occur only twice, both instances in domestic cats.21,22 enlargement of the adrenal glands of the affected ewe were not noted at slaughter, nor were there any other symptoms suggestive of adrenocortical deficiency, but no other possible cause seems plausible to the authors based on what is known. sex reversal, as occurs when the genetic sex and gonadal sex are divergent, was excluded based on genetic analysis and gonadal histopathology. while there must have been unusual androgen exposure to this developing lamb, endogenous estrogen and progesterone must have concurrently been secreted. this sheep had bilateral ovaries, a fully clinical theriogenology • volume 6 number 2 • june 2014107 intact female reproductive tract and functional external male genitalia. while adrenal glands were not assessed postmortem, they remain a plausible source for the male phenotype seen here. the undifferentiated accessory sex gland present must have developed under the presence of testosterone and subsequent conversion to dht, and the apparent effect of estrogen on the glandular tissue is to be expected with functional ovaries. similar effects, including epithelial hyperplasia and squamous metaplasia, are seen in the prostates of dogs with estrogen exposure23 that often results from estrogensecreting tumors. it is difficult to adequately explain the unusual presentation of this sheep. the chromosomal sex matches the functional ovarian tissue, however the androgens inducing penile and scrotal development cannot be readily explained. there is no known androgen or chemical exposure in utero, as the female twin to this sheep was bred and maintained a normal pregnancy. the presence of the sry-gene is unlikely in this case, as is underlying gonadal mosaicism, though these cannot be entirely ruled out in this case and thus the true cause in this sheep therefore remains unknown. female pseudohermaphroditism is a rare occurrence in domestic species but should be considered when sexual ambiguity is present. learning points  sexual differentiation in mammalian species is complex, with the potential for error at any stage.  female pseudohermaphroditism is rare in domestic animals, and the underlying cause may remain ambiguous some cases.  all levels of intersexuality should be considered when sexual ambiguity is present. acknowledgements joan palmero dvm, lee v. millon, dana van liew references 1. padula am: the freemartin syndrome: an update. anim reprod sci 2005;87:93-109. 2. bruere an, mcdonald mf, marshall rb: cytogenetical analysis of an ovine male pseudohermaphrodite and the possible role of the y chromosome in cryptorchidism of sheep. cytogenetics 1969;8:148-157. 3. falvo re, buhl ae, reimers tj, et al: diurnal fluctuations of testosterone and lh in the ram: effect of hcg and gonadotrophin-releasing hormone. j reprod fertil 1975;42:503-510. 4. barrack er, berry sj: dna synthesis in the canine prostate: effects of androgen and estrogen treatment. prostate 1987;10:45-56. 5. parkinson tj, smith kc, long se, et al: inter-relationships among gonadotrophins, reproductive steroids and inhibin in freemartin ewes. reproduction 2001;122:397-409. 6. bunch td, callan rj, maciulis a, et al: true hermaphroditism in a wild sheep: a clinical report. theriogenology 1991;36:185-190. 7. fayrer-hosken ra, huber tl, miller dm, et al: infertility in a ewe as a result of ovotestis. j am vet med assoc 1992;200:1528-1529. 8. smith kc, long se, parkinson tj: a case of true hermaphroditism in a sheep. vet rec 1996;138:497-498. 9. bruere an, macnab j: a cytogenetical investigation of six intersex sheep, shown to be freemartins. res vet sci 1968;9:170-180. 10. burton mj, momont hw: pseudohermaphroditism in a holstein heifer. vet rec 1986;119:155-156. 11. takagi m, yamagishi n, oboshi k, et al: a female pseudohermaphrodite holstein heifer with gonadal mosaicism. theriogenology 2005;63:60-71. 12. weaver ad, harvey mj, munro cd, et al: phenotypic intersex (female pseudohermaphroditism) in a dachshund dog. vet rec 1979;105:230-232. 13. jorge jc, echeverri c, medina y, et al: male gender identity in an xx individual with congenital adrenal hyperplasia. j sex med 2008;5:122-131. 14. rajender s, rajani v, gupta nj, et al: sry-negative 46,xx male with normal genitals, complete masculinization and infertility. mol hum reprod 2006;12:341-346. 15. marino ta: embryology and disorders of sexual development. perspect biol med 2010;53:481-490. 16. wiig o, derocher ae, cronin mm, et al: female pseudohermaphrodite polar bears at svalbard. j wildl dis 1998;34:792-796. 17. clarke ij, scaramuzzi rj, short rv: effects of testosterone implants in pregnant ewes on their female offspring. j embryol exp morphol 1976;36:87-99. clinical theriogenology • volume 6 number 2 • june 2014 108 18. roselli ce, estill ct, stadelman hl, et al: separate critical periods exist for testosterone-induced differentiation of the brain and genitals in sheep. endocrinology 2011;152:2409-2415. 19. kirli ea, karnak i, ciftci ao, et al. an unexpected diagnosis in children with male phenotype and bilateral nonpalpable gonad: congenital adrenal hyperplasia with female genotype. pediatr surg int 2013. 20. hanley na, arlt w. the human fetal adrenal cortex and the window of sexual differentiation. trends endocrinol metab 2006;17:391-397. 21. knighton el. congenital adrenal hyperplasia secondary to 11beta-hydroxylase deficiency in a domestic cat. j am vet med assoc 2004;225:238-241, 231. 22. owens sl, downey me, pressler bm, et al. congenital adrenal hyperplasia associated with mutation in an 11beta hydroxylase-like gene in a cat. j vet intern med 2012;26:1221-1226. 23. mahapokai w, van den ingh ts, van mil f, et al. immune response in hormonally-induced prostatic hyperplasia in the dog. vet immunol immunopathol 2001;78:297-303. figure 1. ante-mortem picture of a female pseudohermaphrodite sheep. note the significant perineal swelling and lack of a vulva. this sheep has a prepuce and a functional penis. figure 2. sheep in a flipped position. note the prepuce (a), enlarged teats (b) and perineal swelling (c). there appears to be a remnant where the vagina would have had an exterior opening. clinical theriogenology • volume 6 number 2 • june 2014109 figure 3. gross image of the entire reproductive tract of a sexually ambiguous sheep. note the bilateral ovaries (a), intact uterus (b), cervix (c), vagina (d), urinary bladder (e) and fibroelastic penis (f). (editor’s note: the photographs in this paper are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 6 number 2 • june 2014 110 2010: uterine torsion in the mare* uterine torsion in the mare* t.l. blanchard, t.s. taylor, d.d. varner, s.p. brinsko department of large animal clinical sciences; college of veterinary medicine and biomedical sciences, texas a&m university, college station, tx, usa introduction uterine torsion is less common in mares than in cows, perhaps due to a more stable anatomical suspension of the equine reproductive tract (i.e., sublumbar ovarian attachment, dorsally attached uterine broad ligaments). however, uterine torsion accounts for a significant percentage of the more serious cases of dystocia in mares. exact causes of uterine torsion are unknown. suggested contributing factors include sudden falls or vigorous movement of a large fetus in a relatively small volume of fluid. uterine torsion in the mare is reported to be as likely to occur in the last half of gestation as during labor. at the texas veterinary medical center, mares presented with uterine torsion are more commonly in midto late-gestation. clinical signs of uterine torsion are primarily those of intermittent unresponsive, often severe, colic. impending parturition may be suspected due to the mare’s restlessness, sweating, anorexia, and frequent attempts at urination. severity of clinical signs is related to the degree of uterine rotation and vascular impairment. immediate correction is required to restore circulation and improve chances of fetal survival. while torsions of less than 180 degrees may or may not require correction, rotation is frequently ≥360 degrees. kweywords: uterine torsion, dystocia, mare, parturition diagnosis diagnosis of uterine torsion is confirmed by detection of displacement in either direction, since clockwise and counterclockwise torsions occur. palpation of the uterus per rectum reveals a tense broad ligament spiraling in the direction of uterine rotation. the other broad ligament is usually palpable as it disappears beneath the uterus. occasionally only one broad ligament is palpable, coursing from one side of the twisted uterus dorsally over and down to the other side. identification of the ovaries serves to distinguish broad ligaments from taut mesentery. anterior vaginal constriction spiraling in the direction of torsion is only occasionally evident on vaginal examination, in contrast to its more frequent occurrence in the cow. manual correction of uterine torsion preparation of the mare for standing intervention, with or without sedation, was described in the previous theriogenology handbook fact sheet on correction of dystocia by mutation and traction.1 manual correction of uterine torsion is reserved for term mares in which the cervix is dilated sufficiently to permit the hand to be passed into the uterus to manipulate the fetus. the obstetrician grasps a portion of the fetus ventrolaterally while resting the arm on the pelvic floor. the fetus is gently rocked until enough momentum is gathered to lift it upward and over within the uterus opposite to the direction of the torsion. if successful, the fetus and uterus will rotate into the normal position and the mare should enter second stage labor as the genital tract fully dilates. administration of 10-20 units of oxytocin intravenously may promote cervical dilation and delivery if congestion of the genital tissues has compromised the dilation process. fetal delivery should be assisted. if cervical dilation does not proceed, cesarean section is indicated. * originally published by the society for theriogenology and the american college of theriogenologists as publication e-2 (3/89) in the theriogenology handbook. 123 rolling to correct uterine torsion the rolling technique for correction of uterine torsion is primarily used in preterm mares and other cases in which the cervix is closed. some authors have cautioned against rolling of the mare to correct uterine torsion, primarily because of increased risk of separation of the chorioallantois and uterine rupture. clinicians at the texas veterinary medical center, however, have had good success encountering few such problems when using the technique described by schaffer2 and modified by bowen.3 the mare is cast in lateral recumbency (on the side of the direction of torsion) after being anesthetized with a short-acting anesthetic regimen, and maintained with inhalation anesthesia. the mare is hobbled, ropes are applied to the limbs, and a wooden plank (2” x 12” x 12’) is placed as shown in figure 1. the mare is gently rolled in the direction of the torsion to her other side (figures 2 and 3) while a person kneels in the middle of the plank. one person cradles the mare’s head during the rolling procedure to protect it and prevent trauma to the cervical spine. the genital tract is examined per rectum to determine if the torsion has been corrected or it is determined that rolling is not beneficial. reexamination is performed in the standing mare after recovery from anesthesia to ensure that the uterus has remained in its normal position and that the fetus is viable. in term mares, delivery should be assisted. close monitoring for several days in preterm mares is necessary due to the potential for fetal death, abortion, and other complications such as uterine rupture. figure 1. position of mare and plank prior to rolling figure 2. uterine (fetal) position maintained by weight of plank during rotation of mare. figure 3. position following reduction of 180 degrees of uterine torsion. surgical correction of uterine torsion surgical correction of uterine torsion is generally reserved for cases that are unresponsive to rolling or for the clinician who does not have immediate access to adequate personnel or 124 equipment for general anesthesia and rolling. the reader is referred to reviews on the procedure for surgical correction for discussion of preparation, anesthesia, and surgical approaches.4-6 if a standing laparotomy is performed, uterine torsion is corrected by reaching under the uterus, grasping the legs of the foal and gently rocking the foal and uterus with a pull-and-lift motion until it is returned to a normal position. if room permits, one hand can be used to grasp a fetal bone through the underside of the uterus and pull toward the operator while the other hand is simultaneously used to push against the dorsal aspect of the uterus. following rotation of the uterus, the ovarian ligaments are traced along their course from their dorsal attachments to the tips of the uterine horns to confirm attainment of the normal uterine position. the ventral abdominal approach should be used with suspected uterine tears, emphysematous fetuses or uterine devitalization. in uncomplicated cases of uterine torsion, return of the uterus to its normal position is accomplished by grasping the limbs of the foal and rotating the uterus. cesarean delivery prior to correction of the rotation is sometimes necessary with the previously mentioned abnormalities, or with very large term or twin fetuses. acknowledgement original drawings by mr. donald connor, senior multimedia specialist, university of missouri. references 1. blanchard tl, varner dd, brinsko sp, et al: equine obstetrics: mutation and delivery by traction. clin therio 2010, in press. 2. schaffer w: zur extraabdominalen retorsioin der gebaermutter nach bach. arch tierheilk 1946; 88:44. 3. bowen jm, gadboury c, bousquet d: non-surgical correction of a uterine torsion in a mare. vet rec 1976;99:494-496. 4. taylor t, blanchard tl, varner dd, et al: management of dystocia in the mare: uterine torsion and cesarean section. compend cont educ pract vet 1989;11:1265-1273. 5. vandeplassche m, spincemaille j, bouters r, et al: symposium. (1) some aspects of equine obstetrics. equine vet j 1972;4:105-109. 6. pascoe jr, meagher dm, wheat jd: surgical management of uterine torsion in the mare: a review of 26 cases. j am vet med assoc 1981;179:351-354. additional reading 1. roberts sj: veterinary obstetrics and genital diseases. 3rd ed. woodstock, vt: published by the author; 1986. p. 277-352. 2. walker df, vaughn jt: bovine and equine urogenital surgery. philadelphia: lea and febiger; 1980. p. 229232. 125 126 omniblank: 2010: early embryonic death in mares early embryonic death in mares dale l. paccamontia and elaine carnevaleb adepartment of veterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la; bequine reproduction laboratory, colorado state university, fort collins, co introduction early embryonic death (eed) is defined as loss of the conceptus before organogenesis is complete, generally regarded as before 40 days of gestation in the mare. using ultrasonography, estimates of eed range from 5-23% between 12 to 50 days of gestation.1-4 whether critical periods exist during which the incidence of eed is highest has not been firmly established.1 overall, pregnancy losses are greatest early in gestation, before 35 days or even earlier before the embryo enters the uterus.1,4,5 estimates of the incidence of eed rely on the method used to diagnose pregnancy and the ability to detect eed. keywords: early embryonic death, mare, infertility diagnosis although continued low progesterone is not compatible with pregnancy maintenance, a single low progesterone value does not necessarily indicate impending eed. low progesterone concentration at more than one sampling is required before a diagnosis of eed or non-pregnant can be made.6-8 estrone sulfate is a product of a viable conceptus and, therefore, can be used as an indicator of pregnancy.9 however, estrone sulfate is not a reliable indicator of fetal viability until at least 44 days of gestation,10 and may not be useful as a valid indicator until after 80 to 90 days of gestation. early pregnancy factor, based on the rosette inhibition test, has been proposed in a number of species as a means to diagnose pregnancy and therefore detect eed at a very early stage. although shown to be unreliable for detection of pregnancy and non-pregnancy in cattle11,12 and horses,12 it has been suggested to be useful for monitoring the viability of equine embryos.13 when eed occurred between days 11-15 after ovulation, no ultrasonographic indication of impending eed was noted.14 later in gestation, however, indications of impending eed include an abnormal appearance of the conceptus, continued mobility of the vesicle, fluid surrounding the vesicle, absence of a heartbeat, decreased volume of conceptual fluids, disruption of conceptual membranes or edema of the endometrial folds.14-16 (figures 1 and 2) between days 20 and 45 of gestation, eed was first characterized by a speckled or granular appearance of the conceptus.17 the conceptus then decreased in size and lost clear definition before disappearing completely within four to seven days of the onset of the granular appearance.17 doppler ultrasonography has been suggested as a tool to assess embryo viability or impending death.16,18 in a normal pregnancy, blood flow is greater in the uterine arteries of the gravid horn. a relative decrease in flow in the arteries of the gravid horn compared to the nongravid horn was observed after the administration of exogenous prostaglandin f2a (pgf).18 embryos that are smaller than normal may be lost at an increased rate compared to normal sized embryos, while oversized vesicles are not at risk.19 abnormal embryo development has been associated with the formation of trophoblastic vesicles that can be observed after day 22 of gestation that lack an embryo proper.20 potentially, these structures are formed after embryonic insult and death of the inner cell mass, the cells within the embryo that will form the embryo proper. (figure 3) the trophoblast layer of the embryo continues to grow, resulting in a vesicle without development of the embryo proper. the vesicle may be small compared to a normal vesicle. although the characteristic bulge associated with pregnancy may not be palpable, uterine and cervical tone are usually typical of a pregnant mare. diagnosis of eed by palpation and ultrasound is facilitated when the vesicle collapses and the contents spread out into the uterus. although pregnancy loss can usually be detected by a loss of uterine tone and the palpable bulge, the bulge in the uterus may remain palpable for up to a week after conceptus death.9 cervical patency at the time of embryonic loss indicates that embryos are probably lost by passage through the cervix rather than by resorption.21 if the mare is on supplemental progesterone, the progression of embryo loss may be changed, as the cervix will remain closed. 99 figure 1. embryonic vesicle in the left uterine horn (left image), with fluid within the lumen of the right uterine horn (right image). the fluid within the uterine lumen moved to surround the embryonic vesicle. in most cases, embryonic death will occur. figure 2. aborting fetuses with disruption of membranes and granular debris. heartbeats were not imaged in the thorax, and fetal features are less defined. after being lavaged from the uterus of a mare, the fetus and membranes were intact, but degenerative changes were occurring. 100 figure 3. a 7-day embryo collected from the uterus of a mare. the embryo has a layer of trophoblast cells that will form the placental membranes. the thicker area of cells represents the inner cell mass, the cells responsible for formation of the embryo proper causes fertilization rates are apparently very high in horses,22,23 although differences may exist between normal and subfertile mares.22 one problem in the assessment of fertilization and eed is that the equine embryo remains in the oviduct for approximately 5.5 days before entry into the uterus,24 and ovulatory or oviductal problems may affect early pregnancy rates. when recently ovulated oocytes or early embryos were recovered from the oviducts of mares, significantly fewer recently ovulated oocytes or embryos were recovered from old mares (≥ 20 years) than young mares (two to ten years), suggesting failure of ovulation or oviductal pickup of the oocyte. therefore, fewer oocytes had the potential for fertilization and embryo development in the older mares, although fertilization rates were not different.25 genetic abnormalities play a significant role in pregnancy loss in other species,26 and similar findings can be expected in horses. chromosomally abnormal cells have been identified in morphologically normal equine embryos27 and an increased incidence of eed has been associated with certain family lines28 or stallions.29 the majority of chromosomal abnormalities leading to eed may not be inherited but arise during formation and ageing of gametes. these abnormalities may result in an inability of the resulting zygote to develop into a viable embryo.30 early embryonic death due to aged gametes may be increased when mating is not closely synchronized with ovulation.30-32 although insemination after ovulation can result in pregnancy, the incidence of eed is higher than when insemination occurs before ovulation33 and increases as the interval from ovulation to insemination lengthens.32 observable or unobservable defects in the embryo soon after its formation may alter or affect its metabolic function. differential transport of unfertilized ova and embryos by the oviduct is apparently a function of the embryo rather than the oviduct.23,34 prostaglandin e2 (pge), secreted by the equine embryo, is apparently responsible for instigating embryo transport.35,36 administration of exogenous pge has reportedly hastened embryo transport.34 some degree of synchrony between the embryo and the uterus is critical for successful establishment of pregnancy. therefore, pregnancy might not be established if an embryo was unable to produce sufficient quantities of pge to induce 101 oviductal transport at the required time. similarly, conditions resulting in increased concentrations of pge may hasten transport resulting in early arrival of the embryo. substances produced by the embryo may be important for its continued survival after arrival in the uterus. equine embryos produce estrogen which may be important in maternal recognition and maintenance of pregnancy.37 estrogen plays a role in the increased tone seen in the early pregnant mare which is influential in fixation of the embryo38 and has a synergistic effect with progesterone to stimulate uterine specific proteins39 which contribute to histotrophe. in a retrospective study, embryo morphology was graded before transfer as excellent, good (minor morphological imperfections) or fair/poor (moderate or severe changes in morphology).40 after transfer into recipient mares, pregnancy rates at 16 days were significantly higher for excellent and good embryos than for fair/poor embryos. however, although pregnancies were established at 16 days, the embryo loss rate from days 16 to 50 was higher for good than excellent embryos, and the embryo loss rate was similar for good and fair/poor embryos.40 results of the study demonstrate that relatively minor morphological defects in embryos may indicate reduced potential for normal embryo and fetal development. unrestricted conceptus mobility is essential for pregnancy maintenance and prevention of luteolysis.41 if uterine contractions, the main propulsive force stimulating embryo movement,14 are not sufficient to move the embryo throughout the uterus and inhibit luteolysis, eed may result. uterine contractility is reduced in old versus young mares.42 removal of the endogenous progesterone source resulted in a reduction in uterine contractions and conceptus mobility and a failure of embryonic fixation.21 the embryo was subsequently found more often in the uterine body,21 a condition associated with increased eed.43 it appears that prostaglandins also play a key role in embryonic mobility.44 inhibition of prostaglandins with flunixin meglumine results in a marked decrease in embryonic migration.44 experimental restriction of conceptus mobility has resulted in luteolysis followed by conceptus loss; however, administration of a progestogen was able to rescue the pregnancy.41 pathology of the uterus such as transluminal adhesions or endometrial cysts could restrict conceptus mobility sufficiently to mimic the experimental model resulting in eed. a critical period in the establishment of pregnancy occurs at 14 to 16 days of gestation when the presence of the conceptus is needed to inhibit luteolysis.41 factors of conceptus origin inhibit the ability of the endometrium to release pgf between days 12 to 16 after ovulation and are essential for maintenance of luteal function.45 if function of the corpus luteum is not maintained because the conceptus fails to block pgf release, either from inadequate migration or from retarded development of the conceptus, pregnancy loss will result.41 (figure 4) a twin pregnancy rarely results in the birth of two healthy viable foals. if twins survive until late gestation, abortion, stillbirth or dystocia are likely outcomes. fortunately, a natural reduction mechanism exists, provided both conceptuses are fixed in the same uterine horn, whereby one conceptus of a pair undergoes eed. the mechanism for embryonic reduction is hypothesized to be the result of deprivation of nutrients due to one conceptus preventing contact of the trilaminar omphalopleure of the other conceptus with the endometrium.46 reproductive status of the mare may reflect maternal-embryonic interactions. not surprisingly, pregnancy rates are lower and the incidence of eed higher for infertile mares than for normal mares.47,48 embryos were recovered at a reduced rate from subfertile mares,49-52 although in one study, a majority of subfertile mares contributed a transferable embryo on at least one occasion after repeated embryo collection attempts.53 similar pregnancy rates have been found between subfertile and normal mares at two days after ovulation; however, the pregnancy rate at day 14 was reported to be greatly reduced in subfertile mares.54 in general, subfertile mares tend to be older than fertile mares; therefore, some aspects of subfertility may be directly associated with aging. in the past, fertility was considered to be primarily dependent on uterine environment. pregnancy rates were similar for embryos from normal and subfertile mares when transferred into normal mares.49 moreover, initial pregnancy rates were similar after transfer of embryos to normal and subfertile recipient mares; however, subsequent eed was greater for subfertile recipients.51 recent evidence indicates other factors are involved. transfer of embryos from normal mares to normal and subfertile recipients resulted in similar pregnancy rates at day 12 or 28, indicating that the uterus of a subfertile mare is capable of supporting an embryo from a normal mare.55 embryos collected from the oviducts of normal and subfertile donors four days after ovulation and transferred 102 figure 4. an embryonic vesicle at 11 days (left image) with a regressing cl on the right ovary. unless exogenous progesterone is administered, the mare will return to estrus, and eed will occur. to normal recipients resulted in lower pregnancy rates at day 14 for mares receiving embryos from subfertile donors than for mares receiving embryos from normal donors.22 other researchers have also found a lower pregnancy rate and an increased incidence of eed after transfer of embryos from subfertile mares when compared to those obtained from maiden mares.52 in addition, abnormal embryos have been collected more often from subfertile mares than from maiden mares.56 maternal aging has been shown to affect fertility and embryo loss in the mare. extremes of age, either young or old, negatively affect fertility.57,58 the high rate of eed in yearling mares has been attributed to immaturity, inadequate nutrition or physical stress.57 older mares were found to have a lower pregnancy rate and a higher loss rate compared with younger mares,22,48,58 and a decrease in live foaling rate.2,58-60 in a study of nearly 1400 mares in newmarket, mares three to eight years of age experienced a 5% pregnancy loss between 15 and 35 days of gestation, while mares nine to 13 years of age had a 14% loss and mares 14 years of age or older showed a marked increase to approximately 22% eed.3 in a controlled experiment, using semen from the same stallion and similar breeding management, the embryo loss rate between 12 and 39 days was 11% for young mares (five to seven years) and 62.5% for older mares (≥ 15 years),42 suggesting differences in the ability of mares of different ages to establish and maintain a pregnancy. the effect of advanced age, however, is difficult to separate from acquired subfertility61 and is confounded by the effect of parity since the two are generally correlated.52 in one study, the incidence of embryonic resorption increased with parity.2 the effect of age on fertility, at least in part, originates in the oocyte. when embryos were collected from the oviducts of young mares (two to nine years) and old mares (≥ 20 years) significantly fewer cells and poorer morphology were noted for embryos from old mares.25 in a subsequent study, the researchers used oocyte transfer to compare fertility of oocytes collected from the follicles of young mares (six to ten years) or old mares (20 to 26 years).62 mature oocytes were transferred into the oviducts of young, inseminated recipients, and embryo development rates were determined. more oocytes from young than old mares (11/12, 92% and 8/26, 31%) resulted in embryonic vesicles. results of the study indicate that intrinsic defects occur in the oocytes of old mares, and these defects may affect fertility and early embryo loss. the repeatability of eed may depend on the inciting cause. some authors indicate that mares which experience eed are more likely to again experience loss,63 while others have failed to find 103 such an association.48 oviductal pathology may result in eed or fertilization failure. during post mortem examinations, salpingitis, which might affect embryo transport, oviductal secretions, and embryo support, was not uncommon.64 almost all oviducts were patent, although adhesions involving infundibula were common.64 uterine pathology may affect histotrophe production, which the early embryo relies on, and result in retarded embryonic development. higher pregnancy rates have been reported when recipient mares had normal endometrium compared with mares that had moderate uterine pathology.50 endometrial cysts are common in older mares. most mares over 17 years of age have some cysts, and mares over 11 years of age are four times as likely to have cysts as younger mares. the effect of cysts on fertility is unclear, because studies are often confounded by other factors such as age and parity. a study of nearly 300 thoroughbred mares found that cysts did not affect the ability to establish or maintain pregnancy; however, the time of initial pregnancy examination was not controlled and embryonic losses occurring before pregnancy examinations were not detected.65 the odds ratio of establishing pregnancy was suggestive of a negative effect.65 other studies have corroborated the quantitative effect of cysts on fertility. if endometrial cysts are present in sufficient number or size, a decrease in fertility results; however, the effect on fertility is less than that caused by delayed uterine clearance. although endometrial cysts may interfere with maternal recognition of pregnancy, once pregnancy is established they do not increase the likelihood of pregnancy loss.65 progesterone, normally supplied by the corpus luteum, is essential for the maintenance of pregnancy.63,66,67 evidence pointing to primary luteal insufficiency as a major cause of eed is lacking.5,68 secondary luteal insufficiency due to premature luteal demise as a result of uterine pathology is a more likely cause of eed.8 pregnancy loss after ovariectomy or pgf administration can be prevented by progestogen supplementation.6,7,63,66 progesterone dependent endometrial proteins have been demonstrated in the mare, and the lack of these proteins following luteolysis may be associated with a decrease in the quality of histotrophe, contributing to eed.69 supplemental progesterone, however, has not improved pregnancy rates in normal mares receiving embryos.70 although pgf release from the uterus is normally responsible for lysis of the corpus luteum, other potential sources of pgf exist which can cause either complete luteolysis or decreased luteal function resulting in eed.71 endotoxemia or other febrile conditions can stimulate pgf release sufficient to cause decreased luteal activity precipitating eed.71 mares in early gestation, before endometrial cup formation, are at more risk of pregnancy loss. although it is commonly believed that the developing corpus luteum is unaffected by pgf until five days after ovulation, studies have shown that some mares will respond as early as three days after ovulation.72 furthermore, irvine, et al.73 have shown that repeated low doses of pgf are very effective in inducing luteolysis. these studies would seem to indicate that inflammatory conditions, even soon after ovulation, warrant attention. prostaglandin inhibitors such as flunixin meglumine are effective in blocking pgf release, but must be administered soon after the onset of endotoxemia.71,74 delaying flunixin until two hours after endotoxin administration failed to prevent pregnancy loss.74 exogenous progestogens, however, can maintain pregnancy even after luteal activity is compromised,71,74 but should be continued until placental progestins are capable of maintaining pregnancy.75 human chorionic gonadotropin, although generally regarded as luteotrophic, has been shown to induce luteolysis, resulting in eed when given between 24 and 39 days of gestation.76 endometritis is an important cause of eed in mares48 and usually causes loss before maternal recognition of pregnancy. once pregnancy is established, endometritis is a less frequent cause of eed,4 provided anatomical defects are corrected. embryonic death may result either indirectly as a result of inflammatory mediated prostaglandin release affecting luteal function or embryo migration or from a direct embryotoxic effect of the inciting agent or inflammatory process. for example, altered intrauterine concentrations of pge, which play an important part in oviductal transport of embryos,34 has been associated with endometritis in mares.77 direct embryotoxic effects of pathogenic organisms have been reported,56 as well as luteolysis secondary to induced endometritis.15 an inflammatory reaction to sperm cells in the uterus is part of the normal physiologic response to mating. in normal mares, fluid and debris are easily evacuated from the uterus and the inflammatory response subsides relatively quickly. a delay in uterine clearance results in persistent mating-induced endometritis (pmie) which will result in eed, either directly or through luteolysis.78 treatment of pmie often includes administration of drugs such as oxytocin or cloprostenol, a pgf analog, to 104 augment uterine contractility and improve clearance. oxytocin is generally regarded as safe when administered during the periovulatory period. cloprostenol, on the other hand, has been shown to depress progesterone concentrations during mid-diestrus when given in the immediate post-ovulatory period.79-82 although the literature on the effect of post-ovulatory cloprostenol on pregnancy rates is equivocal,79,81,82 it would seem advisable to err on the side of caution and restrict cloprostenol use to the pre-ovulatory period and use only oxytocin after ovulation. contagious equine metritis, a transmissible venereal disease caused by taylorella equigenitalis, commonly results in eed after initial infection.83 clinical signs are confined to the mare and vary from acute endometritis to an inapparent carrier state.83 the stallion can also maintain the organism as a carrier.83 control has been achieved by identifying carriers, improved hygiene during breeding, use of artificial insemination and surgical ablation of the clitoral sinuses.83 mare reproductive loss syndrome is associated with both eed and abortion. although the exact etiology is still unclear, reproductive losses have been linked to ingestion of eastern tent caterpillars, specifically the setae of the caterpillars.84 administration of west nile virus vaccine to pregnant mares has been shown to be safe and is not associated with eed.85 early embryonic death has been associated with conception occurring too soon after foaling.59,86 other studies have not substantiated these claims and have found that although pregnancy rates may be lower, no greater losses occur after foal heat breeding than after later breedings.48 examining factors associated with an increase in eed, mckinnon et al. reported that pregnancy rates are lower in mares bred on foal heat when intrauterine fluid was observed ultrasonographically.87 a similar relationship was found between the presence of intrauterine fluid at foal heat and a greater risk of eed.88 in a study examining recovery rate and embryo quality in mares bred on foal heat compared with mares bred at a later estrus, the authors concluded that eed was not the reason for lower pregnancy rates in mares bred on foal heat, and hypothesized that sperm transport or oviductal conditions were more likely to blame.89 the effect of lactation on the incidence eed is likewise unclear. lactating mares have been reported to have a lower,59 a higher,86 or the same60 incidence of eed as nonlactating mares. an increased incidence of pregnancy loss has been associated with nutritional stress.90,91 mares suffering from malnutrition experienced an increase in eed, which could be averted by supplemental feeding.91 in a later study, the authors reported an association of poor quality protein in the ration with increased pregnancy losses, which they attributed to inadequate production of progesterone by the corpus luteum.92 restriction of dietary intake was reportedly successful in reducing twin pregnancies to singletons in 60% of treated mares.93 although reportedly beneficial in cows, nutritional supplementation with chelated or inorganic minerals failed to show an effect on conception, pregnancy rate, or eed in mares.94 no adverse effects on reproduction have been observed in obese mares.90,95 when mares at 16 to 22 or 32 to 38 days of gestation were hauled for nine hours without food or water, progesterone and adrenocorticotropic hormone increased, indicating acute stress; however, pregnancy losses were found to be no different than in non-transported mares.96,97 stress due to pain, infectious disease or emotional disturbances such as weaning have been associated with decreased progesterone.98 administration of corticosteroid similarly has resulted in a drastic yet transient decrease in progestogen which is apparently not a result of luteolysis mediated by pgf.98 repeated palpation or ultrasound examinations have not been associated with an increase in eed.99,100 long-term anabolic steroid treatment has untoward effects on subsequent reproductive performance. a trend towards higher eed was observed in mares treated during the previous year with anabolic steroids.47 occasionally, there may be a need to induce eed. choice of the best method depends on the stage of gestation.101 within five days of ovulation, no consistently effective method exists because the developing corpus hemorrhagicum is relatively unresponsive to the effects of pgf and because the embryo, still within the oviduct, is protected from the uterine environment. from five days after ovulation until the formation of the endometrial cups at approximately 35 to 38 days, administration of pgf is the preferred method to terminate pregnancy. other methods such as manual crushing of the vesicle and uterine infusions, though effective, carry the possibility of untoward side effects.101 after the formation of the endometrial cups, repeated injections of pgf are usually needed to induce eed. in the case of twins, manual crushing of one vesicle is the technique usually employed to 105 reduce the pregnancy to a singleton. most other reported methods do not consistently achieve the same success.101 prevention or treatment effective methods to reduce contamination at breeding and treat endometritis should be employed to allow establishment of pregnancy. procedures such as caslick’s surgery are necessary if anatomic defects such as poor perineal conformation are present. maintenance of good general health and nutrition will likely improve pregnancy rates and reduce eed. failure to maintain pregnancy due to premature release of pgf attributable to inflammatory processes has been successfully treated with flunixin meglumine.102 in the same report, repeated eed attributed to a failure of the normal process of maternal recognition of pregnancy to maintain luteal function was successfully prevented with altrenogest despite regression of the primary corpus luteum and low endogenous progesterone.102 although nonsteroidal anti-inflammatory drugs can inhibit pgf release resulting from endotoxin infusion, administration must occur within a very short time of endotoxin release, limiting their usefulness under natural conditions.71,74 conceptus death may not occur until as long as 48 hours after luteolysis,74 and embryos can apparently continue to grow for some time after loss of endogenous progesterone.21 therefore, sufficient time exists to institute progestogen therapy to rescue the pregnancy.21,71,74 pregnancy has been maintained in mares lacking sufficient endogenous progesterone with the daily administration of progesterone in oil or altrenogest, but was subsequently lost if therapy was stopped before day 50 of gestation.66 results of ovariectomy after day 50 are variable with some mares able to maintain pregnancy without exogenous progestogen. to ensure pregnancy maintenance, exogenous progestogen therapy should be continued through the first four months of gestation, until the placenta can provide adequate progestogens for pregnancy support. some workers suggest that if the uterine environment is normal, progesterone therapy is not needed.8 the difficulty lies in determining if the uterine environment is normal from the viewpoint of the conceptus. administration of altrenogest on days six to 20 to supplement endogenous progesterone or buserelin on day ten to bolster endogenous progesterone production did not significantly affect progesterone concentrations or conceptus size.103 if exogenous progestogen therapy is to be instituted, sufficient dosages should be used. minimum dosages to maintain adequate circulating concentrations in plasma include daily administration of 200 to 300 mg progesterone in oil or 22 mg altrenogest.7,104 lower doses or less frequent administration of these products were insufficient to maintain pregnancy.6,7 aqueous forms of progesterone are cleared too rapidly to be of use for pregnancy maintenance.7 a long-acting injectable formulation of progesterone administered on a weekly basis has been shown to be effective in maintaining pregnancy.105 pregnancy was not maintained in ovariectomized mares using a number of synthetic progestogens, including medroxyprogesterone, norgestomet and megestrol acetate; however, altrenogest at 0.044 mg/kg was able to maintain pregnancy.106 although 22 mg altrenogest is reportedly sufficient for maintaining pregnancy,67 other workers suggest that higher doses are needed to provide optimal uterine and cervical tone and are preferred for pregnancy maintenance.67,107 mckinnon, et al.67 propose that if the progesterone concentration at 24 hours after exogenous progestin therapy is 2.5 ng/ml or more, exogenous therapy may be unnecessary. viability of a pregnancy should be reaffirmed when long-term progestogen therapy is practiced. retention of a conceptus after fetal death may result from continued treatment.108 intensive management alone may accomplish the objective of getting a mare to carry a foal to term. in a report of six mares which had lost their pregnancies two to five times in the preceding six years, five had normal term pregnancies without any special treatment other than good management and being mated to a highly fertile stallion.109 acknowledgement. figures courtesy of dr. elaine carnevale. references 1. ball 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caterpillars in pregnant mares. j am vet med assoc 2005;225:717-721. 85. vest dj, cohen nd, berezowski cj, et al: evaluation of administration of west nile virus vaccine to pregnant broodmares. j am vet med assoc 2004;225:1894-1897. 86. merkt h, gunzel ar: a survey of early pregnancy losses in west german thoroughbred mares. equine vet j 1979;11:256-258. 87. mckinnon ao, squires el, harrison la, et al: ultrasonographic studies on the reproductive tract of mares after parturition: effect of involution and uterine fluid on pregnancy rates in mares with normal and delayed first post partum ovulatory cycles. j am vet med assoc 1988;192:350-353. 88. malschitzky e, schilela a, mattos alg, et al: intrauterine fluid accumulation during foal heat increases embryonic death. pferdeheilkunde 2003;19:646-649. 89. huhtinen m, reilas t, katila t: recovery rate and quality of embryos from mares inseminated at the first postpartum estrus. acta vet scand 1996;37:343-350. 90. henneke dr, potter gd, kreider jl: body condition during pregnancy and lactation and reproductive efficiency of mares. theriogenology 1984;21:897-909. 91. van niekerk ch: early embryonic resorption in mares. j s afr vet med assoc 1965;36:61-69. 92. van niekerk fe, van niekerk ch: the effect of dietary protein on reproduction in the mare. vii. embryonic development, early embryonic death, foetal losses and their relationship with serum progestagen. j s afr vet med assoc 1998;69:150-155. 93. merkt h, jungnickel s, klug e: reduction of early twin pregnancy to single pregnancy in the mare by dietetic means. j reprod fertil suppl 1982;32:451-452. 94. ley wb, thatcher cd, swecker ws, et al: chelated mineral supplementation in the barren mare: a preliminary trial. j equine vet sci1990;10:176-181. 95. kubiak jr, evans jw, potter gd, et al: postpartum reproductive performance in the multiparous mare fed to obesity. theriogenology 1989;32:27-36. 96. baucus kl, squires el, ralston sl, et al: the effect of transportation stress on early embryonic death in mares. proc 10th equine nutr physiol symp 1987. p. 657-662. 97. baucus kl, ralston sl, nockels cf, et al: effects of transportation on early embryonic death in mares. j anim sci 1990; 68:345-351. 98. van niekerk ch, morgenthal jc: fetal loss and the effect of stress on plasma progestagen levels in pregnant thoroughbred mares. j reprod fertil suppl 1982;32:453-457. 99. irwin cfp: early pregnancy testing and its relationship to abortion. j reprod fertil suppl 1975;23:485-488. 100. villahoz md, squires el, voss jl, et al: some observations on early embryonic death in mares. theriogenology 1985;23:915-924. 101. paccamonti dl: elective termination of pregnancy in mares. j am vet med assoc 1991;198:683-688. 102. darenius k, fredriksson g, kindahl h: allyl trenbolone and flunixine meglumine treatment of mares with repeated embryonic loss. equine vet j suppl 1989; 8:35-39. 103. stout tae, tremoleda jl, knaap j, et al: effects of treatments to prevent early pregnancy loss in the mare. pferdeheilkunde 2003;19:711. 109 104. allen wr: is your progesterone therapy really necessary? equine vet j 1984;16:496-498. 105. vanderwall dk, williams jl, woods gl: use of a compounded proprietary long-acting progesterone formulation for maintenance of pregnancy in mares. proc annu meet soc therio 2003. p. 8. 106. mckinnon ao, lescun tb, walker jh, et al: the inability of some synthetic progestagens to maintain pregnancy in the mare. equine vet j 2000;32:83-85. 107. hinrichs k, sertich pl, kenney rm: use of altrenogest to prepare ovariectomized mares as embryo transfer recipients. theriogenology 1986;26:455-460. 108. meyers pj, varner dd: abortion of a mummified fetus associated with short uterine body in a mare. j am vet med assoc 1991;198:1768-1770. 109. darenius k, kindahl h, madej a: clinical and endocrine studies in mares with known history of repeated conceptus losses. theriogenology 1988;29:1215-1232. 110 2019 ex utero intrapartum correction of omphalocele in an english bulldog fetus ex utero intrapartum correction of omphalocele in an english bulldog fetus karen a. von dollen,a valery f. scharf,a lysa p. posner,b emilee j. luckring,a ayako oda,b shelby hedgepeth,a katelyn e. ellis,a theresa m. beachler,a c. scott bailey,a sara k. lylea adepartment of clinical sciences, bmolecular and biomedical sciences north carolina state university, raleigh, nc abstract a fetal abdominal wall defect was diagnosed by ultrasonography during routine cesarean section staging of a full-term english bulldog bitch. the omphalocele was reduced and repaired prior to delivery at the time of cesarean section. the neonate recovered without incident. throughout his first month of life, there was no significant difference in weight gain or clinical status compared to his littermates. however, the body wall defect recurred at ~4 weeks of age in the form of an umbilical hernia. at ~6 weeks of age, the patient was presented for perforation of omentum through the skin overlying the hernia and an umbilical herniorrhaphy was performed. keywords: abdominal wall defect, omphalocele, prenatal ultrasonography, neonatal surgery introduction prenatal diagnosis of abdominal wall defects is common in human medicine, with potential to identify defects as early as 12 weeks of gestation.1 a recent prospective study suggests that the rate of detection in women is ~95%, with most diagnoses before 20 weeks of gestation.2 the practice of screening for congenital defects has not been widely described in veterinary literature, but represents an opportunity to prepare for immediate surgical correction at delivery. a similar case of prenatal diagnosis of omphalocele by ultrasonography has been described, which resulted in euthanasia of the neonate at delivery, due to the severity of presentation.3 to the authors’ knowledge, the current case represented the first reported prenatal diagnosis of omphalocele in a dog subsequently corrected at the time of cesarean section (c-section). surgical correction at delivery reduces the risk of abdominal sepsis and compromise of herniated organs, while maintaining fetal oxygenation through the umbilical vasculature. since the fetus had a continued vascular connection to the dam, we expected the fetus to remain anesthetized, eliminating the need for a separate anesthetic event in the neonatal patient. case presentation a 4-year-old, 26-kg multiparous english bulldog bitch was presented to the veterinary teaching hospital at north carolina state university for timed c-section staging at 60 days after lh surge. ovulation timing and insemination had been performed at the same institution, with lh surge estimated from serial serum progesterone concentrations and used as day 0 of gestation. on routine ultrasonographic evaluation of fetuses to determine maturity, a bulge of mixed echodense contents was visualized in the region anterior and superior to the umbilicus in a single fetus (figure 1, a-c). all examinations were done with a 4 to 8 mhz micro-curved array transducer (fujifilm sonosite s9, universal medical, bothell, wa). on the basis of the ultrasonographic findings, a presumptive diagnosis of congenital abdominal wall defect was made. color flow doppler was used to characterize the vascular supply to the herniated tissue, which was determined to be separate from the umbilical vasculature and urachus (figure 1, d). treatment the bitch was staged for c-section through routine diagnostics, including serial serum progesterone concentrations and ultrasound examinations of fetuses to gauge fetal maturity and monitor for indications of fetal stress. the dam was examined daily from days 60 to 65 (day of surgery). the bitch’s serum progesterone concentration at day 64 was 4.8 ng/ml, with fair fetal maturation as evidenced by ultrasonographic appearance of kidneys and gastrointestinal tracts. at day 65, serum progesterone concentration was not measured due to lack of laboratory availability. the decision to proceed with cclinical theriogenology • volume 11 number 1 • march 201967 section was made based on ultrasonographic maturity of the fetuses (significantly improved gastrointestinal peristalsis from the previous day). the presence of milk expressible from all teats in the bitch provided additional support for this decision. the bitch was routinely prepared for surgery, including cephalic intravenous catheter placement and administration of methylprednisolone (250 mg iv; solu-medrol®; pfizer, new york, ny) with the goal of improving fetal lung maturity. following pre-oxygenation by facemask, anesthesia of the bitch was induced with propofol (140 mg iv). the bitch was intubated and anesthesia maintained with desflurane in oxygen. ventilation was continually assisted following intubation until all puppies (six) were delivered. anesthetic monitoring included heart rate, respiratory rate, peripheral oxygen saturation, blood pressure and capnography, all of which were maintained within normal ranges. the bitch received a single dose of intraoperative cefazolin (580 mg iv) before delivery, as well as buprenorphine (0.5 mg iv) immediately after all puppies were delivered. a standard bilateral hysterotomy was performed; one surgeon removed the unaffected neonates from the right uterine horn and uterine body while a second surgeon removed neonates from the cranial aspect of the left uterine horn until the affected neonate was identified in the caudal aspect of the left uterine horn. the amnion was removed from the affected neonate and the umbilical vasculature left intact during the procedure. dynamic herniation of small intestine through the defect was held in reduction with a cotton tip applicator while herniorrhaphy was performed. the hernia sac was excised and the external rectus fascia freshened using tenotomy scissors. the external rectus sheath was closed in a simple continuous pattern using 3-0 polydioxanone suture (pdsii, ethicon, bridgewater, nj). subcutaneous and skin layers were closed in a single layer using 4-0 nylon (ethilon, ethicon) in a simple continuous pattern. both layers were closed leaving an ~5 mm defect at the caudal aspect of the original defect to prevent premature occlusion of umbilical vasculature (figure 2). after closure of the defect, the umbilical cord was clamped and sharply transected ~2 cm distal to the umbilicus. the neonate had spontaneous respiration by completion of the abdominal wall repair and resuscitation was successful using standard techniques, including vigorous manual stimulation with a dry towel and external heat (lamp). following resuscitation, buprenorphine (0.005 mg sc) was administered to the male, 445 g neonate. the goal of the surgery was to enclose and protect abdominal contents until the patient was mature enough to have a permanent umbilical hernia repair. following delivery of the last neonate, the bitch underwent an elective ovariohysterectomy. the external rectus fascia was infiltrated with bupivacaine (45 mg) at closure. the interval from induction of anesthesia to start of ventral celiotomy was ~9 minutes, interval from induction of anesthesia to start of omphalocele correction was ~15 minutes, total surgical time was 58 minutes, and total anesthetic time from induction to extubation was 83 minutes. neonates were united with their dam following her recovery from anesthesia. the affected neonate was observed to nurse without incident, with no appreciable deficits compared to littermates. the litter was evaluated for congenital defects: no further defects were identified. each puppy received 3 ml frozen-thawed canine plasma delivered by orogastric intubation (standard practice at performing hospital). the bitch and puppies were discharged on the day of surgery following confirmation that the bitch had fully recovered from anesthesia and all puppies were observed to nurse. outcome over the following days, communication was maintained with the owner regarding the neonate’s clinical status. according to the owner, he had increased vocalization intermittently during the first 24 hours after discharge, but maintained an appetite and activity level similar to littermates. on day 12 following discharge, the puppy was presented for a re-check examination and suture removal. the puppy was bright, alert, and responsive with vital parameters within normal limits. the incision was clean, dry, and intact with a thin eschar present overlying the surgical site (figure 3). the sutures had migrated from the wound edges and were removed. on day 30, the puppy was presented for recheck evaluation after the owner noted a soft protrusion in the region of the umbilicus. the puppy was otherwise doing well at home. on examination, clinical theriogenology • volume 11 number 1 • march 2019 68 a reducible umbilical hernia ~3 cm in width and 4 cm in length was diagnosed (figure 4). given the age of the puppy and the risk that immediate surgical correction could result in an increased likelihood of herniation as the puppy continued to grow, it was decided to delay surgical correction of the abdominal wall defect until the puppy was more mature. the patient was discharged with instructions to monitor carefully for any change in the character of the hernia or the patient’s clinical status. sixteen days after the initial recurrence was evaluated (at 46 days old, weight 4.4 kg), the patient was presented on emergency for perforation of omentum through the abdominal skin over the site of the umbilical hernia (figure 5). emergency surgery to correct the hernia was elected. the puppy was sedated with fentanyl (13 µg iv) and anesthesia was induced with midazolam (1 mg iv), propofol (4 mg iv), and ketamine (8 mg iv). the puppy was intubated with a 4.5 mm endotracheal tube and anesthesia was maintained with isoflurane in oxygen and fentanyl administered as a constant rate infusion (cri). anesthesia monitoring included heart rate, respiratory rate, blood pressure, capnography, and temperature. blood glucose concentrations were serially evaluated and remained > 150 g/dl. cefazolin (100 mg iv) was administered immediately before surgery. a ventral midline celiotomy was performed and a 2 cm portion of devitalized omentum (observed externally) was excised using ligation and electrocautery. the edges of the abdominal wall defect (5 x 3 cm) were debrided and closed using 2-0 pds in a simple continuous pattern. the subcutaneous tissue was closed in a simple continuous pattern using 3-0 pds and the subcuticular layers closed in a continuous horizontal mattress pattern using 4-0 poliglecaprone suture (monocryl, ethicon), with skin edges reinforced using tissue glue. recovery from anesthesia was uneventful. the fentanyl cri was continued in the postoperative period for ~36 hours (3 µg/kg/hour, decreased to 1.5 µg/kg/hour prior to discontinuation). antibiotic therapy was provided in the form of ampicillin-sulbactam (97 mg iv every 8 hours) while hospitalized. all medications were discontinued at time of discharge after 2 days of postoperative hospitalized monitoring. communication was maintained with the owner and at time of writing this case report, the patient is nearly 10 months old and thriving, with no recurrence of the umbilical hernia (figure 6). discussion abdominal wall defects are commonly encountered in veterinary practice, with varying incidence across species. in mammalian embryology, a physiologic herniation of the bowel through the umbilicus occurs in early gestation. in dogs, this resolves by 40 days of gestation.5 failure of resolution results in omphalocele, as occurred in the present case. the incidence of congenital abdominal wall defects in canines was 0.6% in > 1600 puppies examined from a cohort of pet store puppies.4 given the hereditary nature of many umbilical hernias, this incidence is likely to vary by breed and mate selection and may actually be higher due to euthanasia of affected fetuses soon after delivery. due to the prenatal diagnosis of an abdominal wall defect, this litter was destined for homes as pets, with no intention for puppies to be used as breeding animals. in a litter-bearing species such as the dog, pre-partum transabdominal ultrasound is limited in its ability to singularly scrutinize the entirety of every fetus; therefore, the risk of false negative findings of canine fetal defects exists when using this modality. surgical correction of umbilical herniation in dogs has been described, with favorable prognosis and excellent long-term outcome.6 umbilical hernias are covered by a layer of subcutaneous tissue and skin which provide protection from both evisceration and abdominal sepsis. in contrast, omphaloceles are a subset of umbilical hernias that lack skin coverage and are often significantly larger than umbilical hernias. a transparent membrane of amniotic tissue initially covers the herniated organs but is easily ruptured; thus, euthanasia of neonates with omphaloceles is often recommended at delivery. surgical repair of omphaloceles in a litter of 5-day-old kittens was attempted but failed due to suture pull-out from the fascia of the abdominal wall.7 in humans, the optimal timing of omphalocele repair remains undetermined, with current practice involving immediate correction at birth, stepwise correction throughout the neonatal period, or delayed closure after epithelialization overlying the defect has occurred.8 omphalocele repair at the time of c-section in dogs may allow for survival of canine neonates that would otherwise perish due to abdominal sepsis and/or trauma to abdominal organs. maintenance of clinical theriogenology • volume 11 number 1 • march 201969 oxygenation and anesthesia via the umbilical vessels during herniorrhaphy at the time of c-section obviates both the need for establishing an airway in the neonate and a separate high-risk anesthetic episode. consequently, this case has further implications for salvage of endangered species, with correctable congenital defects, that would otherwise be euthanized at the time of delivery. traditional repair of ventral midline hernias requires tension-free closure of the defect using nonabsorbable or slowly absorbable suture in the external rectus fascia, followed by separate closure of subcutaneous and cutaneous tissues. in the present case, separate closure of the subcutaneous tissue was not feasible, due to the small size of the patient. tension on the repair and subsequent suture pull-through from the fascial layer is a common cause of herniorrhaphy failure. tension can be mitigated by using synthetic materials or tension-relieving sutures. the use of synthetic materials in this patient was not feasible for several reasons, including the patient’s size and growth potential, surgical time and precision required to apply surgical mesh and potential for contamination of the synthetic material, predisposing to infection. a simple continuous pattern was chosen for both layers in an effort to minimize surgical and anesthetic time for both neonate and dam and because precision of suture placement was limited due to movement of the neonate during suture placement. no tension on the herniorrhaphy was noted at the initial surgical repair. during omphalocele correction, the affected neonate displayed spontaneous movement, which made surgical repair challenging. concurrently, the dam was assessed as appropriately anesthetized. since omphalocele correction occurred ~15 minutes after induction, it would be expected that plasma propofol concentrations would be low or nonexistent. therefore, the dam and the neonate should have been anesthetized primarily by desflurane. the most likely cause of neonate movement was pain/surgical stimulation as the neonate moved, primarily in response to surgical stimulation. why the dam appeared properly anesthetized while the neonate appeared not to be was unknown and unexpected. possible explanations for differences in either pain response or pain threshold include: differing inhalant concentrations in the dam versus the neonate (gas anesthetics move via pressure gradient from dam to neonate), an immature pain pathway in the neonate, and/or progesterone in the dam acting as an analgesic. fetal activity as monitored through electrocorticogram and muscle activity involves interruptions in the fetal sleeplike state secondary to a decrease in external (or cutaneous) temperature, but not core body temperature, which would have occurred at the time of exteriorization from the uterus.9 this physiologic response could provide additional explanation for movement during omphalocele repair. furthermore, development of descending inhibition in the spinal cord is not fully developed until after 1 week of age in the neonatal rat, leading to an exaggerated neural response secondary to skin stimulation.10 in the current case, the response of the neonate could be attributed to this lack of inhibition and would explain the difference in reaction between the two patients (dam and neonate). although many dams undergoing c-section receive opioids for pain from the celiotomy, these drugs are often withheld until all puppies have been severed from the maternal blood supply to prevent neonates from having unnecessary adverse side effects (e.g. sedation, hypoventilation and decreased appetite). standard of care at our teaching hospital is to administer opioids after all puppies are delivered. in retrospect, it may have been beneficial to administer a short-acting opioid, e.g. fentanyl, to the dam immediately before surgery on the neonate, or increase the desflurane concentration (or both), to assure adequate anesthesia to the neonate.11 in addition to humane implications, movement made surgical correction more difficult and therefore should prevented. administration of buprenorphine after resuscitation did not alter ability to nurse or ventilate as compared to littermates. the owner’s description of increased vocalization during the first 24 hours of life was likely due to pain from the surgical repair. consideration of persistent pain should be included following this type of repair. although the herniorrhaphy supported the neonate for approximately the first month of life, longterm success of omphalocele repair may be improved with increased analgesia and placement of interrupted, wide-biting mattress sutures in the external rectus fascia. due to the rapid rate of neonatal growth, however, owners should be prepared for the potential need for revision surgery. this was apparently the first report of repair of omphalocele in a dog at the time of c-section. ultrasonographic prenatal diagnosis of a large ventral midline defect allowed for necessary surgical and anesthetic planning clinical theriogenology • volume 11 number 1 • march 2019 70 to maximize likelihood of successful repair. herniorrhaphy of an omphalocele at the time of c-section may improve survival of affected neonates. learning points  prenatal diagnosis of abdominal wall defects is possible with ultrasonography.  knowledge of a congenital defect may allow for surgical repair of body wall defects at the time of c-section.  analgesia of neonate during surgery and following delivery may facilitate herniorrhaphy. conflict of interest the authors have no conflicts of interest or sources of funding to disclose. references 1. ekin a, gezer c, taner ce, et al: fetal abdominal wall defects: six years experience at a tertiary center. clin exp obstet gyn 2015;42:327-330. 2. fleurke-rozema h, van de kamp k, bakker m, et al: prevalence, timing of diagnosis and pregnancy outcome of abdominal wall defects after the introduction of a national prenatal screening program. prenat diagn 2017;37:383-388. 3. assis ar, bairros aa, martins amq, et al: omphalocele of canine fetus detected by ultrasonography. world small animal veterinary association world congress proceedings 2009. 4. ruble rp, hird dw: congenital abnormalities in immature dogs from a pet store: 253 cases (1987-1988). j am vet med assoc 1993;202:633-636. 5. evans he, christensen gc: prenatal development. in: miller’s anatomy of the dog. 3rd edition, philadelphia; wb saunders co; 1993. p. 32-97. 6. fossum tw: small animal surgery. textbook-e-book. 4th edition, st. louis; elsevier health sciences; 2013. p. 364367. 7. howard dr: omphalocele in a litter of kittens. vet med small anim clin 1973;68:879 8. mortellaro ve, st. peter sd, fike fb, et al: review of the evidence on the closure of abdominal wall defects. pediatr surg int 2011;27:391-397. 9. gluckman pd, gunn tr, johnston bm: the effect of cooling on breathing and shivering in unanaesthetized fetal lambs in utero. j physiol 1983;343:495-506. 10. fitzgerald m, koltzenburg m: the functional development of descending inhibitory pathways in the dorsolateral funiculus of the newborn rat spinal cord. brain res 1986;24:261-270. 11. fisk nm, gitau r, teixeira jm, et al: effect of direct fetal opioid analgesia on fetal hormonal and hemodynamic stress response to intrauterine needling. anesthesiology 2001;95:828-835. figure 1 (a-d): transverse ultrasound images of fetal abdominal wall defect obtained during c-section staging. panels a-c demonstrate appearance of protruding contents (arrows). arrow heads indicate body wall margins; distance between arrow heads approximately 2 cm. panel d illustrates color flow doppler to identify umbilical cord vasculature and urachus (asterisk), which were separate from the defect. clinical theriogenology • volume 11 number 1 • march 201971 figure 2. closure of omphalocele at time of delivery. an approximately 5 mm opening was left at the caudal aspect of the original omphalocele to allow continued patency of the umbilical vasculature while repair was performed. figure 3. healing of incision at age 12 days. figure 4. herniation at site of surgical correction, age 30 days. hernia contents were non-painful and reducible on examination. figure 5. protrusion of omentum through umbilical hernia, age 46 days. figure 6. healed incision at age 114 days. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<feff04120438043a043e0440043804410442043e043204430439044204350020044604560020043f043004400430043c043504420440043800200434043b044f0020044104420432043e04400435043d043d044f00200434043e043a0443043c0435043d044204560432002000410064006f006200650020005000440046002c0020044f043a04560020043d04300439043a04400430044904350020043f045604340445043e0434044f0442044c00200434043b044f0020043204380441043e043a043e044f043a04560441043d043e0433043e0020043f0435044004350434043404400443043a043e0432043e0433043e0020043404400443043a0443002e00200020042104420432043e04400435043d045600200434043e043a0443043c0435043d0442043800200050004400460020043c043e0436043d04300020043204560434043a0440043804420438002004430020004100630072006f006200610074002004420430002000410064006f00620065002000520065006100640065007200200035002e0030002004300431043e0020043f04560437043d04560448043e04570020043204350440044104560457002e> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2010: canine neonatal mortality   canine neonatal mortality michelle a. kutzler oregon state university, department of animal science-companion animal industries, corvallis, or abstract neonatal losses within the first few months of life are a common yet unavoidable problem in canine reproduction. it is important to understand the unique physiology of the canine neonate so that the main non-infectious and infectious causes of mortality can be anticipated and prevented. birth weight is the single most important predictor of neonate survival. the neonate’s body weight should be monitored twice daily for the first two weeks and any loss or failure to gain should be investigated. clinical signs preceding neonatal mortality may include low or no weight gain, continuous vocalization, separation from the dam or other littermates, depression, abdominal distension, or loss of suckle reflex. there are numerous causes for neonatal mortality including peripartum hypoxia, growth retardation (intrauterine and postnatal), maternal aggression, neglect or rejection, nutritional deprivation resulting in hypoglycemia or dehydration, hypothermia, and pathogens (bacterial, viral and parasitic). postmortem examinations provide essential information for causes of neonatal mortality and should be pursued aggressively to prevent further losses. therapeutic goals of preventing neonatal mortality include supportive care and identification and eradication of the cause when known. in many cases, a number of simple actions can significantly reduce neonatal mortality. keywords: canine, mortality, neonate, puppy introduction neonatal losses within the first few months of life are a common yet unavoidable problem in canine reproduction. the overall expected mortality rate in young dogs should be less than 12-15% of full-term births by the age of weaning. however, this could range from 5-35% depending upon factors that predispose to neonatal losses.1-9 of those deaths, approximately 50% are lost within the first seven days. puppies are born much less mature than newborns of many other domestic species and thus are more dependent on care during the few weeks of life. it is important to understand the unique physiology of the canine neonate so that the main non-infectious and infectious causes of mortality can be anticipated and prevented. fading puppy syndrome is a term that is used far too frequently to account of ignorance of normal neonatal needs and poor management10-12 the definition of a canine neonate is somewhat nebulous. generally speaking, a neonate is less than six weeks of age (time of weaning) at which point the puppy would be referred to as pediatric. however, newborn puppies are less mature than newborns of other species. the most critical period in neonates is the first three weeks because this is when neurologic and behavioral maturation is completed. after three weeks, the eyelids and ear canals are open, thermoregulation is established and the puppy is capable of eating solid food. the following definitions have been suggested for neonatal puppies: perinatal period (less than one day), neonatal period (one to 21 days), maturation period (21-28 days) and preweaning period (28-42 days)10,13 clinical examination of the canine neonate clinical signs preceding neonatal mortality may include low or no weight gain, continuous vocalization, separation from the dam or other littermates, depression, abdominal distension, or loss of suckle reflex.6 the severity of clinical signs correlates negatively with survival, and death can occur within 18 to 24 hours from onset of clinical signs depending on the cause. a systematic physical examination should be performed on all neonates, especially if illness is suspected. daily weight gain is an excellent indicator of general health as healthy puppies gain weight continuously.14 neonates should be weighed at birth and then twice a day to accurately measure weight 513   gain.15 digital scales are more accurate and gram scales work better for small and toy canine breeds. it is normal for neonates to lose a little bit of weight (mostly water) in the first 24 hours. neonates that lose more than 10% of the birth weight in the first day have a poor prognosis. after that, puppies should gain 5% to 15% of their birth weight daily, doubling their birth weight by ten days of age. this equates to one to three grams per day per pound of anticipated adult body weight. as neurologic maturation is not complete until 21 days, accurate examination of the neurologic system requires understanding of when various reflexes (rooting, suckling, righting) develop and disappear. presence of weakness in any of the reflexes indicates an ill neonate. the predominant posture is flexion for the first three to four days postnatally and then extensor tone will dominate,6 the panniculus or cutaneous trunci reflex is present at birth. flexor (pain or withdrawal) reflexes and a pain response to a noxious stimulus are present shortly after birth. a crossed extension reflex (pinching one foot and having the opposite limb extend) is present up to three weeks of age.16 in the normal neonatal puppy, the rooting and suckling reflexes disappear between 25 to 28 days.6,17 however, these reflexes may persist longer in orphans.10 the oral cavity should be free of congenital defects (e.g. cleft palate). the hydration status should be assessed by checking moisture of the mucous membranes because skin turgor of neonates is not as developed as in adults. if dehydrated, skin on the ventral abdomen and muzzle may appear a deeper red color. the skull should be examined for the presence of open fontanelles. a cranial nerve examination can be conducted in puppies after three weeks of age. although the palpebral and corneal reflexes are present at birth, they are more easily visualized after the eyelids open.18 puppies’ eyes open at nine to 16 days.19 the menace response in puppies may not appear until after three weeks because the retina is not fully developed before this time. visual depth perception develops by approximately 28 days.20 ear canals open in puppies between ten and 14 days of age, but sound orientation does not occur until approximately 21 days.20 evaluation of mental attitude (e.g. depression, hyperexcitability with excessive vocalization) is also important. in the first two weeks, neonates spend most of their time nursing or sleeping.20,21 they may crawl around on the lower part of their thorax for the first two weeks and after that time, they are able to lift their heads and maintain an upright position. vocalizations occur as they seek food or are disturbed, but continuous crying is abnormal. it is abnormal for a healthy neonate to cry for longer than 20 minutes. continuous crying usually indicates a cold or hungry puppy, but it may be a clinical sign of other painful or infectious processes. on thoracic auscultation, the lungs should be clear and free of fluid. in a healthy puppy, breathing is regular and unlabored. the normal respiratory rate in neonatal puppies is 25 to 35 breaths per minute for the first two weeks of life, which decreases to normal adult rates between three to four weeks of age. the work of breathing in neonates is increased as a result of elevated airflow resistance due to the narrow diameter airways and the pliable chest wall (increased compliance). neonates are more susceptible to respiratory fatigue and hypoventilation. for puppies that are delivered following cesarean, spontaneous breathing and vocalizing at birth are positively associated with survival at a week of age. initial respiratory rate of newborn puppies is ten to 18 breaths per minute and they lack the typical pause between breaths that is normal in adult animals.22 heart rate is higher in neonates (>200 beats per minute) whereas blood pressure is lower (60±5 mmhg).23,24 the neonatal heart rhythm should be a regular sinus rhythm that is not associated with breathing patterns because vagal reflexes do not develop until approximately eight weeks of age. normal rectal temperature in the canine neonate is 35.0 to 37-2 c (95 to 99 f), which increases to 36.1 to 37.8 c (97 to 100 f) by 14 days. poikilothermy gives way to autonomic thermoregulation by 28 days.25 the abdomen should be soft and not painful. sick puppies lack normal bowel sounds on abdominal auscultation. the umbilicus should be dry and without any surrounding redness. discoloration on the umbilicus or the ventral abdominal skin is indicative of sepsis and requires aggressive antimicrobial therapy. the presence and patency of the anus should be checked. the ability to urinate (by stimulating the area around the urethral opening) should be verified. color of the urine 514   should be checked as a relative assessment of hydration status. low specific gravity and glucosuria are common normal findings in neonates.26 orphans will need manual stimulation of the orifices with cotton or a soft cloth to elicit this reflex by owner the first two weeks until voluntary control of these functions is developed.27 if the perineum is not stimulated, the bladder will continue to fill, causing abdominal distension. the anogenital reflex persists until three to five weeks of age, at which time postural control develops, which enables the puppy or kitten to assume a proper stance for urination.28 neonates have decreased functional capacity of many organ systems because of incomplete development of these organs at birth. as they age, organ function increases. hematology and serum biochemistry can be helpful to detect abnormalities of these organ systems as long as age appropriate reference ranges are used (table 1). however, whenever possible, it is recommended to use the reference ranges established for each laboratory because of the lack of standardization among veterinary diagnostic laboratories. neonates normally have mild serum phosphorus elevations as well as mild blood urea nitrogen, albumin, globulin, cholesterol, and hematocrit reductions. in addition, oneto three-day-old neonates have alkaline phosphatase concentrations that are 30 times higher than adult values due to colostral alkaline phosphatase.29 similar to alkaline phosphatase, gamma-glutamyltransferase concentrations are markedly increased in the first day after colostrum ingestion, with values being approximately 100 times higher than adult values.29 these differences are short-lived, resolving within the first two weeks, but can be used as a surrogate marker of effective colostrum ingestion in puppies and kittens.30 although an initial diagnostic database including a complete blood count and serum chemistry profile is desirable, obtaining a blood sample volume sufficient for running these tests may not be possible due to small body size of neonatal patients. the estimated blood volume of a canine neonate is 90 ml/kg of body weight,19 such that not more than 10 ml/kg of body weight should be taken.31 small volume (0.6 ml) blood collection tubes are available from becton, dickinson and company (franklin lakes, nj; http://www.bd.com/vacutainer/pdfs/vs7629_productcat.pdf, bd microtainer blood collection tubes, bd microtainer plastic clad micro-hematocrit tubes). in most cases, a minimum database consisting of a hematocrit, total protein, glucose, and bun is sufficient for making a diagnosis. an ear prick blood sample can yield a sufficient sample volume if only evaluating blood glucose concentrations.19 urine samples are generally easy to collect via perineal stimulation. in normal neonates, urine specific gravity is lower than adults, while urine protein and glucose may be higher. causes of canine neonatal mortality there are numerous causes for neonatal mortality, most of which can be prevented. these have been divided into the following general categories: peripartum hypoxia; growth retardation (intrauterine and postnatal); maternal aggression, neglect or rejection; nutritional deprivation resulting in hypoglycemia or dehydration; hypothermia; and pathogens (bacterial, viral and parasitic). of course, in many instances, overlap exists between these categories. not all of the causes of canine neonatal mortality will be reviewed. for a more complete list of causes, as well as treatment regimens and prevention strategies, readers are referred to the text by peterson m, kutzler ma, editors: small animal pediatrics—the first 12 months of life (st. louis: elsevier; 2011). how the neonate is delivered into its postnatal existence is critical for its future survival. if the neonate is delivered naturally and if the mother does not remove the fetal membranes covering its mouth and nose, suffocation can occur. if the neonate is delivered by cesarean section, the amnion should be removed and a bulb syringe can be used to suction out the oronasal passages. stronger methods of suction are not recommended due to potential injury to the airways and vagally-mediated bradycardia and laryngospasm.32 although historically a common method for removing amnionic fluid from the respiratory tract of neonatal dogs and cats, the swinging of newborns is no longer advocated because of potential cerebral hemorrhage from concussion. 515   spontaneous breathing and vocalizing within one minute following delivery is highly correlated with neonatal survival. neonatal resuscitation utilizes methods that assist with oxygen delivery to hypoxic tissues and prevent deterioration of acid-base stability by supporting ventilation and circulation and keeping the newborn warm.22 vigorous rubbing with gentle intermittent stretching stimulates breathing and clears airways of fluid. a tactile respiratory reflex can be stimulated in the genital and umbilical regions for the first three days of life.33 in addition, in the mildly depressed neonate, vigorous tactile stimulation of the lumbar area by rubbing the fur backwards will help to elicit crying and further clear airways.22 sublingual injections of pharmacological respiratory stimulants (e.g. doxapram) are rarely used in resuscitation of human babies34 and their use in veterinary medicine is controversial.22 their effectiveness is greatly decreased if the brain is hypoxic so administration during hypoventilation is unlikely to result in a favorable response.31 the use of the renzhong acupressure point (jenchung, gv26) stimulated with a 25-gauge needle to elicit spontaneous breathing has some effect.35 the needle is inserted into the nasal philtrum at the base of the nostrils and rotated when bone is contacted (figure1). figure 1. a line drawing depicting the renzhong acupressure point (jenchung, gv26) to elicit breathing in apneic canine neonate. a 25-gauge needle would be inserted into the nasal philtrum at the base of the nostrils (arrow) and rotated when bone is contacted. courtesy of dr. robin waldvogel and the chintimini kennel club. if the neonate does not breathe despite these resuscitation efforts, an attempt to expand the lungs should be made. a tight-fitting mask can be applied to the face of the neonate and not more than 20 to 30 cm of water pressure can be applied for approximately three seconds until the chest wall expands.22 the head should be extended to limit the amount of air forced into the stomach. once the lungs have been expanded, artificial respiration can be continued at a rate of 30 breaths/minute, lasting approximately one second each, with no more than 10 cm of water pressure until the neonate is breathing spontaneously.22 ventilatory support is critical for the stressed neonate and high concentration oxygen is typically used. temporary introduction of 30 to 40% oxygen environment may reduce distress if respiratory function continues to be inadequate. because of the short-term nature of the oxygen therapy, the risk of oxygen toxicity is minimal.22 following respiratory resuscitation (in puppies with persistently low heart rates), lateral chest compressions with the thumb and forefinger should be begun at a rate of one to two beats/second, pausing 516   for respiration.22 in very barrel-chested breeds (i.e. bulldogs), sternal compression may be more effective. the decreased heart rate in stressed puppies is likely due to myocardial hypoxia and is not vagally mediated.36,37 there is little effect of atropine on heart rate in puppies before 14 days of age and because the low heart rate is most often due to myocardial hypoxia, myocardial damage could occur due to increased oxygen demand.36,37 therefore, the most important treatment for neonatal bradycardia is to increase respiration and attempt to correct the myocardial hypoxia.33 neonatal survival at seven days of age following a cesarean section delivery is positively correlated when dams are anesthetized with propofol (4 to 6 mg/kg) and isoflurane as opposed to other general anesthetics.38 certain drugs, such as ketamine, thiopental, and xylazine, should be avoided.38,39 a local block with lidocaine or bupivacaine will help reduce the level of isoflurane gas anesthesia.38,39 neonatal survival is higher following an elective cesarean section compared to an emergency cesarean section, with the overall 80% of neonates surviving to seven days of age. for elective cesarean section, a short-acting steroid such as prednisolone sodium succinate (solu-delta-cortef®, pfizer animal health, new york, ny) administered two to 12 hrs prior to surgery at a dose of 1.0 mg/kg has been shown beneficial in neonatal survival. since neonates have relatively small airways, large tongues, and small nostrils, they are susceptible to hypoxia from the presence of fluid or mucus within the airways. in the normal birth event, spontaneous recovery of hypoxia and acid-base stability occurs over two to three hours.40 hypoxia in the newborn is usually due to an inability to completely inflate the lungs, which can be the result of inadequate lung surfactant production.41 before delivery, the presence of surfactant can be indirectly documented using the “foam stability test”.42 this technique was originally used in human obstetric medicine as a rapid test to determine the timing of a cesarean section. one milliliter of amniotic fluid is collected (via ultrasound-guided centesis or laparotomy) and is mixed with one ml of 100% ethanol in a glass tube. the solution is then vigorously shaken for 15 seconds. if surfactant is present, a ring of bubbles will form at the fluid-air interface and remain for at least 15 minutes (figure 2). in human obstetrics, this method was used as a surgery room test to indicate fetal readiness for delivery. results of this test in elective cesarean section delivery of canine fetuses have indicated that adequate surfactant production is not present until 62 days past the onset of the luteinizing hormone (lh) surge for female canine fetuses and 63 days past the onset of the lh surge for male canine fetuses. this sex difference is consistent with observations in human obstetrics, as female fetuses begin surfactant production earlier than males.43 birth weight is an important survival determinant in most mammalian species. low birth weight is associated with higher neonatal mortality because it is accompanied by immature physiological processes that can lead to extrauterine adaptive failure (e.g. inability to suckle and maintain blood glucose concentrations).44 normal birth weight in puppies is from 100 to 200 grams in small breeds, from 250 to 350 grams in medium breeds, from 400 to 500 grams in large breeds, to 700 to 900 grams in giant-breed dogs.45 newborns that are less than 25% of the average birth weight are at a higher risk of developing hypoglycemia, hypothermia, and pneumonia. birth weight is not influenced by gender but is influenced by parental age and state of health, placental sufficiency, litter size, gestational nutrition, infections (bacterial, viral and parasitic), maternal environment (sanitation, stress) and congenital abnormality. purebred puppies are more prone to congenital and hereditary defects that could result in neonatal mortality.6 inbreeding not only affects total puppy mortality, but also puppy mortality attributable to infection.7 neonatal body weight should be monitored daily to ensure normal weight gain. fading (or wasting) is characterized by a progressive clinical decline associated with anorexia, growth failure, depression, dehydration, and increasingly poor response to environmental stimuli.10-12,44 neonates that die within the first 48 hours have most likely succumbed because of starvation rather than infectious disease. inadequate nutrition leads to dehydration and muscular weakness. extrinsic problems that predispose 517   figure 2. before an elective cesarean section, the presence of surfactant can be indirectly determined using the “foam stability test.” one ml of amniotic fluid is mixed with one ml of 100% ethanol in a glass tube and the solution is shaken vigorously for 15 seconds. if surfactant is present, a ring of bubbles will form at the fluid-air interface and remain for at least 15 minutes. (from ruaux c: the respiratory system. in:peterson m, kutzler ma, editors: small animal pediatrics: the first 12 months of life. st. louis: elsevier; 2011). offspring to wasting include: inadequate lactation quality and quantity, excessive handling of the neonate, inexperienced or behaviorally maladjusted dam, inattentive caretakers, exposure to infectious agents. within the first 12 hours, neonates that have lost less than 10% of their birth weight or gained weight were at a lower risk for mortality.46 puppies should gain weight daily at a rate of two to seven grams per day for each kilogram of anticipated adult weight and they should double their birth weight by ten days of age. puppies not gaining weight require supplementation. if one puppy is not gaining weight while the rest of the litter is, a physical examination should be performed. providing the underfed puppy time alone with the mother may be beneficial. the rest of the litter may be pulled away from the dam for five to ten minutes, three or four times per day to give the underweight neonate an opportunity to increase milk intake. if the mother's milk is slow to come in, supplemental feedings are necessary for neonates that are not gaining weight or for orphaned puppies. a neonates’s caloric requirement approximates 230 to 260 kcal/kg body weight daily (given over multiple feedings).10,47 most commercial milk replacers deliver approximately 1.0 kcal/ml.10,47 the total daily feeding should be divided into at least eight feedings per day (approximately every two to three hours) during the first two weeks of life to reduce the risk of stomach overdistension, abdominal discomfort, diarrhea and aspiration. smaller and toy breeds require more frequent feedings to prevent hypoglycemia. the frequency of feedings can slowly be decreased as the amount being fed increases. formulated puppy milk replacer is available commercially (i.e. esbilac, just born, renurse, vetalac). both powder and liquid forms are available. powdered formula lasts longer, since the unused powder can be frozen for six months. once powdered formula has been reconstituted, contents should be used within 48 hours, provided the unused portion is refrigerated in a glass container. liquid milk replacer should be used within 48 hours once the can is opened, provided the unused portion is refrigerated. ingredients and caloric density of the puppy milk replacer can vary with manufacturer. puppies normally get their energy from fat during the first two weeks of life.47 milk replacer is made 518   from bovine milk and is lower in protein, calories, fat, calcium, phosphorus, and carbohydrates. lactose concentrations are low in colostrum and lower in canine milk compared to bovine milk, which can cause diarrhea in puppies fed milk replacer.47 it is advised to dilute milk replacer 25% to 50% with water or a balanced electrolyte solution for the first two days of feeding to minimize the occurrence of diarrhea. because a neonate's stomach has a limited capacity, it is imperative that this ratio be correct to maintain proper hydration and meet the daily caloric requirement without overextending its fluid capacity. when choosing a milk replacer, it is important to review the product's energy content-to-fluid concentration ratio. commercial milk replacer is superior to homemade versions because commercial products generally provide the correct balance of protein, fat, carbohydrates, vitamins, and minerals needed for growing neonates. if commercial milk replacer is temporarily unavailable, an emergency formula may be used. the emergency formula consists of one cup (250 ml) of whole cow's milk, three egg yolks, one tbsp (15 ml) corn oil, 1 drop high-quality oral multiple vitamin solution and a small pinch of salt.10 this formula is strictly for emergencies and should be replaced with a commercial milk formula as soon as possible. puppies that are fed milk replacers should not be expected to maintain growth rates in parallel to those fed with dam’s milk and will take longer to double their birth weight.10 neonatal nutritional supplementation can be provided by either via bottle or tube feeding. when choosing a supplementation method, the strength of the neonate is the primary selection criterion. bottle feeding might not be the best for weak neonates because their suckle reflex may be diminished and there is a possibility of aspiration into the lungs. in this case, tube feeding might be a better alternative. this process is not particularly difficult but needs to be done correctly. however, placement of orogastric tubes can be stressful to neonates, and if long-term feeding will be necessary, it may be more beneficial to place another type of feeding tube. regardless of the supplementation method, prior to the feeding it is extremely important to make sure that the body temperature of the neonate is above 35.5º c (96 f). if the neonate's body temperature is too low, ileus develops and the ingested material will start to ferment instead of being digested, resulting in a bloated and distressed neonate.10 a red rubber catheter feeding tube should be measured and marked to help ensure that the tip of the tube is placed in the stomach. depending on the size of the neonate, the clinician should use a 5french (for puppies less than 200 grams), 8-french (for puppies 200 to 350 grams), 10-french (for puppies 350 to 500 grams) or 14-french (for puppies over 500 grams) [45]. prior to placing the feeding tube, the distance from the tip of the nose to the last rib should be measured. seventy-five percent of that distance should be marked on the tube with a permanent marker (figure 3a). the feeding tube should extend into the puppy’s stomach, but not far enough to cause kinking or looping and return to the esophagus. it is important to remeasure and re-mark weekly, as the patient grows. place the puppy in sternal recumbency with its head elevated and flexed (not extended). the puppy is not strongly or forcibly restrained. excessive restraint can provoke struggling and increase risk for aspiration. insert the tip of the tube along the left side of the roof of the mouth into the esophagus, following the path of least resistance (figure 3b). no force is needed and most neonates will swallow the feeding tube easily. advance to the mark that is measured on the tube. if the catheter is accidentally inserted into the trachea, the neonate might cough as a sign of the incorrect placement.45 another indication of incorrect catheter placement will be that the tube will not be able to be inserted fully to the premeasured mark. correct placement of the feeding tube can also be ensured by the presence of negative pressure using the syringe on the end of the tube. tube placement can also be checked by placing a stethoscope on the stomach, infusing a small amount of air into the tube, and listening for air in the stomach45 if the tube is in the trachea, remove the tube and start over. once the tube is confirmed in the stomach, aspirate a small amount of gastric contents to check for any remaining formula and then attach a syringe containing the milk replacer. administering the feeding is done with minimal restraint. some puppies “prefer” to crawl around on a flat surface while being fed, with only sufficient restraint to keep the head elevated and maintain the tube placement.45 if the stomach is empty, infuse the formula over a two-minute period to prevent overfeeding or feeding too 519   figure 3. passing an orogastric tube is not particularly difficult but needs to be done correctly. first, 75% of the distance from the tip of the nose to the last rib should be marked on the tube (a). the puppy is minimally restrained with the head elevated and flexed and the tube is passed along the left side of the roof of the mouth into the esophagus, following the path of least resistance (b). (from rickard v: birth and the first 24 hours. in peterson m, kutzler ma, editors: small animal pediatrics: the first 12 months of life. st. louis: elsevier; 2011). 520   fast. the maximum stomach capacity for a puppy is approximately 40 ml/kg body weight (0.7 fluid oz (four teaspoons) per lb).45 many clinicians prefer to initiate supportive feeding with meals approximating 50% of calculated volumes, to avoid volume-related aspiration accidents, and to adjust gut flora to the regimen.10 monitor for gastric distention as over-distention can cause delayed gastric emptying, bloat, and diarrhea. regurgitation can occur through the nose. excess air intake while nursing can also cause bloat and make the puppy uncomfortable. after the feeding is complete, the catheter needs to be kinked before pulling it out to avoid milk dripping from the tip and being aspirated into the lungs. a new feeding tube should be used after eight to ten feedings as the tube will become less rigid and more likely to kink.45 feeding tubes should be disinfected and rinsed thoroughly between feedings in the same day. successful hand rearing of puppies can depend on several factors, including appropriate feeding schedule, selection of milk replacer, meeting caloric needs of the neonate, and proper feeding methods. young dogs that are being tube fed should be monitored carefully for nasal discharge, regurgitation, bloating, abdominal discomfort, and diarrhea. these symptoms can indicate undesirable changes in gut flora or impeding medical complications (e.g. sepsis). neonates being supplemented with milk replacers need to be monitored for constipation. warm water enemas can be performed as needed if that condition occurs. after each meal, neonates need to be stimulated to urinate and defecate by rubbing the perineal and preputial areas with a cotton ball that has been soaked in warm water. gastrointestinal motility is reduced in neonates less than 30 days old.48 the formula should be warmed to body temperature to enhance gastrointestinal motility and prevent hypothermia. in addition, it is strongly advised to ensure proper body temperature, as hypothermia can also cause delayed gastric emptying. maternal aggression towards the neonates and cannibalization can be significant causes of neonatal mortality.12 at times, calcium solutions (cal-pho-sol®; 1 ml/10 lb sc; neogen corp., lexington, ky) have been used to help with hypocalcemia-associated aggression. care must be used with other preparations injected subcutaneously to avoid skin irritations. light tranquilization with acepromazine (0.01 to 0.02 mg/kg) might be necessary initially until the problem resolves. appeasing pheromone diffusers or collars may also be helpful in creating a calm, comfortable environment in the whelping room. in the cases of aggression, it might be necessary to place neonates in a small plastic box that has small round openings cut out for ventilation and longitudinal slits in the lid, so the mother can smell and hear her offspring, as well as see them move. neonates are removed from the container for supervised nursing for the first 48 to 72 hrs until the new mothers are fully desensitized. in some cases of maternal neglect or rejection that do not include aggression, placing a few drops of oxytocin topically into the dam’s nostrils will assist in mothering behavior. this author has had success with warming a placenta that was saved and frozen following delivery, rubbing the warmed placenta over the neglected or rejected neonates, and then allowing the dam to consume the remainder of the placenta and clean off the puppies. although not directly associated with maternal neglect, rejection and aggression, problems with the release of milk (galactostasis) or amount of milk present within the mammary gland (hypogalactia or agalactia) will result in neonatal mortality secondary to lack of adequate colostrum ingestion and starvation. excessive noise and activity in the whelping room should be avoided to reduce maternal stress and allow for normal milk letdown. for galactostasis, the dam can be given low doses of oxytocin (0.2 to 2 u im) 15 to 20 minutes before nursing in an attempt to induce milk release. very attentive dams may be reluctant to leave their offspring to feed themselves, which results in decreased milk production. food and water should be readily available to avoid this problem. for medical management of hypogalactia or agalactia, metoclopramide (0.1 to 0.2 mg/kg po tid) or domperidone (2.2 mg/kg po bid) have been used to help increase milk production. as a complementary treatment, acupuncture points li4 and si1 have also been used to promote lactation. water is the most common essential nutrient in which most animals are deficient. neonates are especially susceptible to dehydration as a result of the physiologic immaturity of their kidneys. although their bodies are more than 80% water, their ability to conserve water (concentrate urine) is significantly diminished since kidneys do not fully mature until six to eight weeks of age. because of the immaturity 521   of the autonomic nervous system, canine neonates have an impaired ability to change systemic vascular resistance and cardiac contractility in response to dehydration. water deficits are most frequently caused by prematurity; vomiting, diarrhea or pneumonia; excessive ambient temperature; and reduced intake secondary to inadequate suckling or poor lactation. the daily water requirement for neonates is 130 to 220 ml/kg of body weight per day.47 oral rehydration is preferred if gastrointestinal function is normal and the neonate is not hypothermic.10 since compromised neonates have increased susceptibility to bacterial infections, parenteral routes for rehydration need to be used with a scrupulously sterile technique. subcutaneous or intraperitoneal administration are the routes used most frequently used to correct dehydration uncomplicated by hypoglycemia but are less desirable since absorption rates are slower and less predictable. intravenous (external jugular) or intraosseous (io; femur or humerus) administration is more difficult and stress producing,10 but necessary if dehydration is concurrent with hypoglycemia, which is often the case. dehydration should be corrected with balanced crystalloid fluids (lactated ringer’s solution or normosol-r® with 5% dextrose; hospira, inc., lake forrest, il). in all cases, administration of warmed fluids is necessary. fluid deficits should be incrementally corrected over 12 to 24 hours. an initial rate of 30 ml/kg of body weight should be used to correct dehydration and then adjusted as needed based on response.31 a maintenance fluid dose in neonates is three to six ml/kg of body weight/hour.22 serial weight assessments are the most accurate method for assessing hydration in neonates and for continued monitoring for reoccurrence. careful monitoring is essential because fluid overload is possible due to the diminished kidney function. it is best to use a microdrip administration set or a syringe pump to avoid fluid overload.31 many neonatal deaths result from inadequate nutritional intake or the inability of the neonate to adequately digest and absorb nutrients as a result of their immature digestive system. a healthy mother who is well-nourished should be able to provide complete nutrition for her puppies for their first four weeks. a healthy puppy will nurse vigorously several times a day and gain weight on a daily basis. if insufficient milk is available for consumption or if neonates are unable to nurse, hypoglycemia will result in neonatal mortality within 48 hours. glucose in the blood is closely regulated and normally maintained by three major mechanisms: intestinal absorption, hepatic production, and, to a lesser degree, renal production. glucose levels are lowest immediately after birth and then significantly increase after approximately three days with normal suckling. at birth, the gastrointestinal tract must transition from processing amniotic fluid to digesting milk. the release of hormones and digestive enzymes and the activation of secretion, motility, and absorption are adaptations that begin shortly after birth. these changes are critical to allow the gastrointestinal tract to perform required functions. the intestinal mass of puppies increases in the first 24 hours. intestinal growth is decreased if milk replacer is fed in place of colostrum, possibly caused by missing hormones, fat, or other colostral components. neonates have decreased pancreatic digestive enzymes, which permits intestinal absorption of immunoglobulins from the colostrum. pancreatic lipase production increases over the first three weeks as the amount of milk fat increases, and pancreatic amylase production does not begin until after 21 days of age. however, canine milk contains the digestive enzyme amylase, which helps digest milk sugars in the neonatal gastrointestinal tract. neonatal hepatic glycogen storage is minimal and dependent upon maternal nutrition during pregnancy. hepatic glycogen reserves are depleted within 24 hours during fasting, resulting in hypoglycemia (serum glucose <40 mg/dl).31 maintaining normoglycemia is important for the neonates’ neurologic status because they have a brain-to-body mass carbohydrate requirement two to four times greater than adults. in addition to having reduced potential for glycogenolysis, they have minimal capacity for glucose derivation from gluconeogenic branched chain amino acids. in comparison with adults, neonatal dogs have poor glycemic regulation that is attributed to a relative insensitivity to endogenous insulin and suboptimal counter-regulatory hormone responses (cortisol and epinephrine). postweaning transient juvenile hypoglycemia is of particular concern in miniature and toy breeds of dogs as a result of poor glycemic regulation. puppies with infectious disease (sepsis) or a portosystemic shunt will also develop hypoglycemia from increased glucose utilization. in addition, disease states such as 522   diarrhea, dehydration, or hypothermia may exacerbate hypoglycemia. to prevent hypoglycemia, neonates should not be fasted prior to anesthesia. glucose supplementation is essential for sick neonates, and the treatment is aimed at achieving a normoglycemic state. lactate-containing fluids are preferred when treating symptomatic hypoglycemic puppies because lactate precedes use of alanine or glutamine for gluconeogenesis in puppies and because lactate is preferentially used in the brain. any fluids that are administrated should be warmed to 37 c (100º f) to prevent hypothermia. in the event of a hypoglycemic crisis, neonates should be treated with ten to 25% dextrose at one to two ml/kg of bodyweight iv.31 neonates have an immature metabolic regulatory mechanism, therefore, hyperglycemia needs to be watched for, and dextrose administration needs to be adjusted accordingly. dextrose should not be administered subcutaneously at these concentrations because of the potential of skin sloughing. if the neonate is not too weak, has a good circulation, and is attempting to nurse, then a 50% dextrose solution can be applied to the gums. however, oral dextrose or corn syrup rarely reverses a hypoglycemic crisis. l-carnitine (50 mg/kg po bid) increases the liver’s ability to convert fat to glucose, and it can be given to prevent hypoglycemic episodes from recurring. thermoregulation is another challenge that is exacerbated in immature dogs. puppies have a larger surface area-to-body weight ratio, with high radiation and evaporative heat losses, an inability to shiver and vasoconstrict in response to decreasing temperatures, a small amount of subcutaneous fat and high water composition, and an immature thermoregulatory system.49 hypothermia can decrease neonatal responsiveness to attempts at resuscitation, as it leads to bradycardia (40 to 50 beats/minute), tissue hypoxia, and metabolic acidosis.50 after the puppy is born, its body temperature will gradually decrease from the dam's body temperature to that of a neonate over the first 30 minutes of life.50 normal body temperature of newborn puppies ranges from 35.0 to 37.2 c (95 to 99 f).10,50 temperature guidelines for the prevention of hypothermia in puppies are 29 to 32 c (85 to 90 f) for the first week of age, 27 c (80 f) for the second and third weeks and 21 to 24 c (70 to 75 f) for the fourth and fifth weeks and 21 c (70 f) thereafter.49,51 ambient temperature should be monitored via thermometer and drafts must be prevented. excessive ambient heat is recognized by changes in litter positioning (separated vs normal huddling), character of respiration (hyperthermia results in hyperventilation and open-mouthed breathing), elevated rectal and skin temperature, and distress vocalizations that express discomfort.10 external heat sources most commonly used are heat lamps, heating pads, and warm water bottles. the latter two need to be used with caution since weak neonates might not be able to crawl away from the heat source, resulting in burns and thermal injury. another important factor is humidity, which should be maintained at 50 to 55% to avoid excessive drying of the skin and dehydration. however, humidity in excess of 60% can result in hyperthermia and other complications. hypothermia suppresses appetite (resulting in failure to suckle), possibly as an adaptive response to reduce risk for aspiration. hypothermia also causes intestinal ileus at temperatures below 34.4 c (94 f).10 decreased intestinal motility can cause fermentation of milk in the stomach, resulting in bloat and severe abdominal pain. subsequently, there is increased pressure on the diaphragm, which causes labored breathing and dyspnea. these factors in turn cause the neonate to swallow more air and thus worsen the bloating. severe bloating can result in circulatory collapse and death. if hypothermic neonates are tube fed, the milk replacer is usually regurgitated and aspirated. hypothermic neonates should be slowly warmed over 30 minutes to two hours and not faster than 1º c (2 f) per hour.10 rapid warming increases metabolic demand, with risk of exceeding the delivery capacity of circulatory and pulmonary function; this can cause loss of cardiovascular integrity, secondary hypoxia, cerebral changes, and sepsis. if the body temperature is raised more than 2 c (4 f) per hour, life-threatening organ failure (specifically the heart and kidneys) can result. neonates should be rotated often to ensure even warming, and rectal temperatures should be checked frequently. warm iv or io fluids can also be given to raise the body temperatures, but the temperature of the fluids should not be 523   more than two degrees higher than that of the body. slow warming followed gastrointestinal parenteral fluid support are the primary therapeutic steps. oral alimentation should not be re-established until normothermia is achieved.44 neonates that have been given supportive warming should be monitored frequently recurrence of hypothermia. to prevent hypothermia, environmental temperatures need to be increased because higher temperatures will help neonates maintain their core temperature. if neonatal anesthesia or surgery is necessary, appropriate steps should be taken to prevent hypothermia by limiting heat loss, such as clipping only a minimal amount of hair and avoiding alcohol-containing antiseptics. side effects associated with hypothermia include cardiovascular alterations (bradycardia, hypotension, decreased cardiac output, arrhythmias), prolonged recovery times and drug metabolism, as well as increased infection rate and decreased wound healing.15 infectious diseases are the second most important cause of mortality and account for a large proportion of newborn losses.4 however, infectious causes of neonatal mortality are generally secondary to management problems. predisposition to infection depends on: stress, environment, exposure to pathogens, and decrease in systemic immunity due to inadequate colostrum ingestion. colostrum deprivation following birth hypoxia greatly increases risk for septicemia, especially in low birth weight puppies.52 puppies that do not ingest adequate colostrum are more susceptible to infection until about 35 days of age. in puppies, negligible amounts (<5%) of maternally derived igg are transferred transplacentally.53,54 the remainder of igg is transferred in the colostrum, resulting in an initial spike in globulins immediately after colostral ingestion. canine colostrum has fifty times the concentration of antibodies than canine milk (500 to 2200 mg/dl compared to 10 to 30 mg/dl, respectively). an adequate ingestion of colostrum must occur within the first 24 hours to acquire passive immunity from the mother. gut permeability to immunoglobulins starts to decline eight hours after birth, and no further absorption is possible after 48 to 72 hours.55 the half-life of igg in puppies is ten days. failure of passive transfer is typically diagnosed by measuring igg in serum via radial immunodiffusion (rid) method. however, this test is not readily available and the results can be difficult to interpret. adequate colostrum ingestion can be indirectly assessed in neonatal puppies by measuring serum alkaline phosphatase and/or gamma glutamyl transferase as both of these enzymes are found in colostrum at concentrations 30 times and 100 times adult serum concentrations, respectively.30 for neonates that have not received adequate colostrum, maternal serum can be administered orally to a neonate less than 12 hours old via a feeding tube in the amount of 150 ml/kg of body weight (divided into multiple feedings). if neonates did not receive any colostrum within the first 24 hours, serum can be administered at a dose of 50 ml/kg of body weight sc, three times at six to eight hour intervals. hyperimmune canine serum preparations can be purchased (www.hemopet.org) and may be particularly useful for the treatment of herpes virus infections (see below). hypoxia predisposes neonates to fatal infectious disease. hypoxia can result from respiratory distress syndrome after compression of umbilical circulation,40 obstetric procedures,38 or hypoxic damage,56 hypothermia,57 hypoglycemia,58 dehydration and congenital abnormalities.14 neonatal sepsis occurs in puppies secondary to hypoxia-induced bacterial translocation from the intestinal tract into the bloodstream, resulting in a fatal necrotizing enterocolitis, even in the absence of mucosal lesions.56 however, colostrum-fed puppies did not develop enterocolitis, illustrating the importance of adequate colostrum ingestion.59 bradycardia and hypotension frequently accompany hypoxia.10 infection with bacteria, rather than viruses, is the most common cause of infectious mortality at this young age.4 the most common route of entry for bacterial organisms is through the umbilicus. however, bacteria can also be introduced orally. neonates are born with a sterile gastrointestinal system and develop their own intestinal flora over the first few days. the neonate's digestive system is very fragile and is easily affected by its diet, the environment, or pathogens because the physical defense against infection is reduced in the newborn. since the production of gastric hydrochloric acid has not yet fully developed, stomach acidity is lower in neonates than in adults.60 as a result of this decrease in the 524   acid barrier, the defense against infectious agents is decreased, allowing greater survival of bacteria and increased susceptibility to gastrointestinal infections. the most common manifestation of gastrointestinal upset is diarrhea. the most commonly reported bacterial causes of neonatal sepsis include: escherichia coli, and other gram-negative enteric organisms (e.g., enterobacter sp.), campylobacter sp., clostricium. perfringens type a, -hemolytic streptococcus sp. (e.g., s. canis), staphylococcus sp. (s.aureus, s.intermedius), and salmonella.3,4,61-66 unfortunately, post-mortem cultures are how most neonatal bacterial infections are diagnosed. aspiration of meconium, milk or other substances can result in pneumonia and systemic bacterial infection. aspiration pneumonia may occur following bottle-feeding or with misplacement of a gastric feeding tube. around the time of weaning and the transition to solid food, aspiration pneumonia can also occur secondary to esophageal or pharyngeal dysfunction, congenital megaesophagus, persistent right aortic arch, or cricopharyngeal achalasia. the clinical signs of aspiration pneumonia are essentially the same as infectious pneumonia (e.g., increased respiratory rate, fever, and a productive cough). respiratory sounds may be harsh. crackles may be auscultated over the affected regions of the thorax. wheezing is less common but may be heard if there is significant bronchial involvement. radiographic signs of aspiration pneumonia are variable and may not be present until 12 to 24 hours after aspiration. bacterial pneumonia typically shows a cranioventral distribution of interstitial to alveolar patterns, viral pneumonias tend to show a more diffuse, interstitial pattern. right cranial and medial lung lobes are most commonly affected in animals who aspirate while in sternal recumbency. other lobes may be affected depending on positioning at the time of aspiration. a high percentage of neonatal puppy mortalities have combinations of pulmonary congestion, edema, hemorrhage, and atalectasis.10 treating pneumonia in a neonatal dog is often unrewarding so efforts should be concentrated on prevention. neonates with pneumonia usually require intensive monitoring in a 24-hour care facility. oxygen supplementation via nasal oxygen cannula or use of a humidified oxygen environment cage is needed to prevent hypoxia and bradycardia. ventilation is improved when inflammatory mucus and exudates are cleared from the airways. maintaining airway hydration by saline nebulization and mucolytic agents (e.g., n-acetylcysteine) will help clear exudates from the respiratory tract. cough suppressants are contraindicated. antibacterial therapy is critical in the management of bacterial pneumonia. relatively long-term antibiotic therapy is required for either four weeks or until ten days after radiographic resolution. ideally antibiotic selection is based on the results of culture and sensitivity from lower airway fluids or lung aspirates. however, the collection of these samples may represent a severe anesthetic risk in compromised neonatal patients. awaiting results of these cultures may also unduly delay therapy in critical patients. subsequently, empirical selection of an antibiotic that is safe in neonates is generally used.67 when selecting an antibiotic that is safe in neonates, drugs that require major hepatic metabolism should be avoided.68,69 highly protein-bound drugs will exert a greater clinical effect on neonates compared to adults as a result of the higher quantities of unbound or active drug in the plasma due to the lower plasma albumin concentration of neonates. renal clearance is decreased until the puppy nears six weeks of age.68,69 since elimination is decreased, the half-life of drugs is longer and the interval between doses should be increased. compared to adults, neonates have increased percentage of total body water and decreased percentage of body fat. as a result, the volume of distribution for the water-soluble drugs is increased, so the dose of water-soluble drugs used in neonates should also be increased. in contrast, the volume of distribution for the lipid soluble drugs is decreased. in this case, the dose of lipid soluble drugs in neonates should be decreased.31 treatment with antibiotics prior to sensitivity results could be life-saving. thus empirical use of safe, broad-spectrum bactericidal antibiotics is usually recommended. safe antibiotics in the neonate include cephalosporins, penicillins, clavulanic acid, macrolides, trimethoprim-sulfonamide, and amikacin (if properly hydrated).49 ceftiofur, a third-generation cephalosporin, administered at a dose of 2.5 mg/kg 525   bid sc for five days, is one of the better choices because it has minimal effects on the normal neonatal gastrointestinal flora. combinations of amoxicillin with clavulanic acid or trimethoprim with sulfonamide are also reasonable first-line choices for antibiotic therapy. fluoroquinolones should be avoided because of the potential for cartilage damage in growing animals. common viral causes of neonatal mortality include canine herpesvirus, canine minute virus (parvovirus type i), canine parvovirus type ii (cpv-2), canine distemper virus. puppies infected with canine distemper virus transplacentally can develop neurologic signs within six weeks of birth.70 canine parvovirus type 1, the causative agent of minute virus of canines, also causes birth of weak puppies.71 neonatal infections with canine herpes virus can be acquired in utero, by contact with infectious vaginal fluids during passage through the birth canal, by exposure to infected littermates, from oronasal secretions of the dam, or by fomite transmission. herpes virus is not stable in the environment and must be acquired from a persistently infected carrier. infection with canine herpesvirus causes an acute, rapidly fatal illness associated with hepatic necrosis. puppies may have predisposition as a result of their poor thermoregulation and inability to mount a febrile response. generalized, fatal infections develop in puppies exposed when less than three weeks of age. puppies exposed when older than three weeks are comparatively resistant and often develop mild or inapparent infection and passage of maternal protective antibodies or lymphocytes in colostrum can prevent illness in neonates. clinical signs of herpesvirus infection in puppies under three weeks of age include lethargy, decreased suckling, persistent crying, yellow-green diarrhea, rhinitis, abdominal pain, and incoordination. a distinct feature is the absence of fever. thrombocytopenia, evident by petechial hemorrhages, develops associated with disseminated intravascular coagulation as a result of the vasculitis. diffuse necrotizing vasculitis and spread of virus into parenchymal organs, including the adrenal glands, kidneys, lungs, spleen, and liver, result in multifocal organ necrosis. meningoencephalitis is common, although puppies infected at less than one week of age usually die before neurologic signs develop. survivors that had neurologic signs may have permanent neurologic deficits, most commonly cerebellar vestibular deficiencies. ocular involvement may cause panuveitis, cataracts, keratitis, retinitis, and subsequent blindness. death frequently occurs within 24 to 48 hours after onset of clinical signs. definitive diagnosis is made based on history, clinical signs, pathologic changes, and virus isolation. gross pathologic findings include disseminated multifocal 1to 2-mm hemorrhages and areas of necrosis that are distinctly circumscribed in the liver, kidney, and lungs. herpes viral replication might be inhibited by maintaining body temperature above 38º c (101 f). acyclovir suspension can be attempted at a dose of 20 mg/kg po every six hours for seven days. impeccable hygiene (from whelping through weaning) and strict biosecurity measures can reduce the incidence of canine herpesvirus mortalities. in countries where available, prophylactic canine herpesvirus vaccination at the time of breeding and at pregnancy diagnosis is a successful way to prevent disease outbreaks and neonatal losses. common parasitic causes of neonatal mortality include strongyloides stercoralis, neospora caninum and toxoplasma gondii. strongyloides stercoralis is a zoonotic parasite that is transmitted from dam to pups via milk.72 overcrowding and poor hygiene in a kennel are predisposing factors.73,74 young animals with heavy burdens have diarrhea and weakness, which progresses to emaciation and death, sometimes secondary to c. perfringens overgrowth.65 fenbendazole is the drug of choice in treatment of s. stercoralis infection.72 prenatal or lactationally acquired protozoal infections with neospora and toxoplasma can result in acute severe neonatal illness and death.75,76 systemically infected puppies can appear normal at birth and continue to nurse but insidiously become lethargic, inappetent, and dyspneic and may demonstrate mucopurulent oculonasal discharge and develop progressive neurologic abnormalities with illness, culminating in death.76 dissemination to multiple organs usually involves the liver and leads to jaundice, hepatomegaly, and abdominal effusion.76 multifocal necrotizing hepatitis or cholangiohepatitis causes anterior abdominal pain and peritoneal effusion and usually is associated with vomiting, diarrhea and jaundice. diagnosis is definitively confirmed by finding tachyzoites in histologic or cytologic specimens.76 clindamycin is the drug of choice for treating clinical toxoplasmosis in dogs 526   and also is recommended for pregnant animals.77 oral and parenteral dosing is similar, 10 to 20 mg/kg body weight bid po or im for four weeks. clinical response is usually evident as early as 48 hours. postmortem examinations provide essential information for causes of neonatal mortality and should be pursued aggressively to prevent further losses. if possible, the entire remains should be shipped via next-day delivery to a veterinary diagnostic laboratory for examination. cases from which only tissue samples were sent were significantly less likely to lead to a conclusive diagnosis. the body should be kept in the refrigerator (not frozen) until shipping and shipped with ice packs. conclusion in conclusion, more neonates die from improper husbandry and inadequate nutrition than infectious disease. hypoglycemia, dehydration, hypoxemia and hypothermia are the main mechanisms for neonatal losses. birth weight is the single most important predictor of neonate survival. the neonate’s body weight should be monitored twice daily for the first two weeks and any loss or failure to gain should be investigated. therapeutic goals of preventing neonatal mortality include supportive care and identification and eradication of the cause when known. in many cases, a number of simple actions can significantly reduce neonatal mortality. acknowledgements i thank susan craig for manuscript research and preparation. references 1. anderson ac: puppy production to weaning age. j am vet med assoc 1957;130:151-158. 2. bowden rst, hodgman sfj, hime jj: neonatal mortality in dogs. proc world vet cong 1985; p. 1009-1013. 3. mosier je: canine pediatrics-the neonate. proc am anim hosp assoc 1981; p. 339-347. 4. nielen al, van der gaag i, knol bw, et al: investigation of mortality and pathological changes in 14-month birth cohort of boxer puppies. vet rec 1998;142:602-606. 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62. popischil a: todesursachen bei hunde und katzenwelpen. praktische tierarzt 1993;74:838-847. 63. schafer-somi s, spergser j, breintenfellner j, et al: bacteriological status of canine milk and septicaemia in neonatal puppies. j vet med b inf dis vet public health 2003;50:343-346. 64. askaa j, jacobsen kb, soerensen m: neonatal infections in puppies caused by escherichia coli serogroups 04 and 025. nord vet med 1978;30:486-488. 65. sasaki j, goryo m, asahina m, et al: hemorrhagic enteritis associated with clostridium perfringens type a in a dog. j vet med sci 1999;61:175-177. 66. munnich a: the pathological newborn in small animals: the neonate is not a small adult. vet res commun 2008;32(suppl 1):s81-85. 67. poffenbarger em, ralston sl, olson pm, et al: canine neonatology. part 2: disorders of the neonate. compend contin educ pract vet 1990;13:25-37. 68. berde cb, sethna fn: analgesics for the treatment of pain in children. n engl j med 2002;347:1094-103. 69. boothe dm, bucheler j: drug and blood component therapy and neonatal isoerythrolysis. in: hoskins jd, editor. veterinary pediatrics-dogs and cats from birth to six months. philadelphia: wb saunders co; 2001. p. 35. 70. greene ce, appel mj: canine distemper. in: greene ce, editor. infectious diseases of the dog and cat, 3rd ed. st. louis: elsevier; 2006. p. 25. 71. carmichael le, schlafer dh, hashimoto a: pathogenicity of minute virus of canines (mvc) for the canine fetus. cornell vet 1981;81:151-171. 72. dillard kj, seppo ams, anttila m: case report: strongyloides stercoralis infection in a finnish kennel. acta vet scand 2007;49:37-43. 73. overgaauw pam, van knapen f: dogs and nematode zoonoses. in: macpherson cnl, meslin fx, wandeler ai, editors. dogs, zoonoses and public health. oxton (uk): cabi publishing; 2000. p. 213. 74. hendrix cm, blagburn bl, lindsay cs: whipworms and intestinal threadworms. vet clin north am small anim pract 1987;17:1355-75. 75. dubey jp, lappin mr: toxoplasmosis and neosporosis. in: greene ce, editor. infectious diseases of the dog and cat, 3rd ed. st. louis: elsevier; 2006. p. 754. 76. lavely ja: pediatric neurology of the dog and cat. vet clin north am small anim pract 2006;36:475-502. 77. dubey jp, lappin mr. toxoplasmosis and neosporosis. in: greene ce, editor. infectious diseases of the dog and cat, 2nd ed. philadelphia:wb saunders co; 1998. p. 493. 529   table 1. normal hematology and serum biochemistry reference ranges for canine neonates from birth to 8 weeks of age. birth week 1 week 4 week 8 rbc (× 106/l) 4.7-5.6 3.6-5.9 3.6-4.9 4.5-5.9 hemoglobin (g/dl) 14.0-17.0 10.4-17.5 8.5-10.3 10.3-12.5 pcv (%) 45.0-52.5 33.0-52.0 27.0-33.5 31.0-39.0 nrbc/100 wbc 0-13 0-11 0-4 0-1 reticulocytes (%) 4.5-9.2 3.8-15.2 4.6-6.6 1.0-6.0 total wbc (× 103/l) 6.8-18.4 9.0-23.0 8.5-16.4 12.7-17.3 segmented neutrophils 4.4-15.8 3.8-15.2 3.7-12.8 6.2-11.8 band neutrophils 0-1.5 0-4.8 0-0.3 0-0.3 lymphocytes 0.5-4.2 1.3-9.4 1.0-8.4 3.1-6.9 monocytes 0.2-2.2 0.3-2.5 0.3-1.5 0.4-1.7 eosinophils 0-1.3 0.2-2.8 0-0.7 0-1.2 platelets (× 103/l) 178-465 282-560 130-360 240-435 albumin (g/dl) 1.76-2.75 1.71-2.5 2.17-2.97 2.38-3.22 alp (u/l) 452-6358 195-768 153-490 153-527 alt (u/l) 9.1-42.2 4.1-21.4 4.3-17.4 10.3-24.3 bilirubin (mg/dl) 0.04-0.38 0.01-0.18 0.02-0.15 0.01-0.11 bun (mg/dl) 29.5-118 29.1-66.7 13.1-46.2 16.8-61.4 calcium (mg/dl) 10.4-13.6 11.2-13.2 10.4-13.2 10.8-12.8 cholesterol (mg/dl) 90-234 158-340 177-392 149-347 creatinine (mg/dl) 0.37-1.06 0.28-0.42 0.25-0.83 0.26-0.66 ggt (u/l) 163-3558 — — — ldh (u/l) 1.8-17.0 0.2-17.7 1.2-9.0 1.6-7.3 glucose (mg/dl) 76-155 101-161 121-158 122-159 tp (g/dl) 3.7-5.77 3.26-4.37 3.71-4.81 4.04-5.33 triglycerides (mg/dl) 45-248 52-220 36-149 39-120 phosphorus (mg/dl) 5.26-10.83 8.35-11.14 8.66-11.45 8.35-11.14 rbc, red blood cells; pcv, packed cell volume; nrbc/100 wbc, number of nucleated red blood cells per 100 white blood cells; total wbc, total white blood cell count; alp, alkaline phosphatase; alt, alanine transferase; bun, blood urea nitrogen, ggt, gamma-glutamyltransferase, ldh, lactate dehydrogenase; tp, total protein. adapted from center sa, et al: effect of colostrum ingestion on gamma-glutamyltransferase and alkaline phosphatase activities in neonatal pups. am j vet res 1991;52:499-504; kuhl s, et al: reference values of chemical blood parameters for puppies during the first 8 weeks of life. dtsch tierärztl wschr 2000;107:438-443; harper ej, et al: age-related variations in hematologic and plasma biochemical test results in beagles and labrador retrievers. j am vet med assoc 2003;223:1436-1442; earl fl, melveger ba, wilson rl: the hemogram and bone marrow profile of normal neonatal and weanling beagle dogs. lab anim sci 1973;23:690-695. 530 2009: synchronization of estrus and ovulation: a practitioner's perspective synchronization of estrus and ovulation: a practitioner’s perspective 1 h. maxwell 2 department of clinical sciences, college of veterinary medicine, auburn university, 3 auburn, al, usa 4 abstract 5 artificial insemination as a management tool can be facilitated by synchronization 6 of estrus or ovulation. although estrus detection will remain an important part of 7 successful breeding programs, timed artificial insemination is increasingly used in cattle 8 herds. the practicing theriogenologist must utilize his or her knowledge of the 9 physiology of the bovine estrous cycle and reproductive pharmacology, consider 10 constraints imposed by availability of labor and facilities, and choose among the 11 available protocols the one which is most likely to be successfully implemented. this 12 article reviews the physiology of the bovine estrous cycle, and some of the protocols 13 currently in use. 14 keywords: bovine estrous cycle, estrus synchronization, artificial insemination 15 introduction 16 artificial insemination as a management tool has been extensively utilized by 17 dairy producers for over 50 years, and use in beef cattle has become increasingly 18 common over the last 2 decades. heat detection and insemination based on estrus signs 19 and behavior remain necessary components for most successful artificial insemination 20 programs, but the veterinary practitioner is increasingly called upon to suggest and 21 monitor protocols aimed at increasing efficiency or convenience of artificial insemination 22 programs by synchronizing estrus or ovulation. knowledge of the physiology of the 23 195 estrous cycle, reproductive pharmacology, and management of cattle and people are all 24 necessary components of successful programs. 25 estrous cycle control has developed in distinct phases, as the events of the estrous 26 cycle have been understood and pharmacologic agents have become available. today, 27 the most successful protocols combine control of the length of the luteal phase with 28 control of the emergence of follicular waves, often with control of the time of ovulation 29 and timed artificial insemination. an understanding of the physiology of the estrous 30 cycle, including emergence of follicular waves, development of the dominant follicle, 31 follicular atresia, ovulation, development of the corpus luteum (cl), and luteolysis is a 32 necessary prerequisite to evaluate and implement synchronization strategies. 33 integration of the physiology of the estrous cycle with reproductive 34 pharmacology, the age, breed and lactation status of the targeted group, the animal 35 handling skills and facilities present on the farm or ranch, the availability of sufficient 36 skilled personnel at critical times, effective communication and record keeping are all 37 important to success of protocols. the practicing theriogenologist must take all of these 38 into consideration as he or she consults with their clients. 39 a brief outline of critical points is presented below, and discussion of the some of 40 the more common protocols which have evolved follows. 41 the bovine estrous cycle 42 cattle are reproductively non-seasonal, polyestrous, monovulatory, with an 43 interestrus interval ranging from 18-23 days. observable estrus behavior is detectable 44 even in the absence of the male. 45 196 heat, or estrus, is best demonstrated by standing to be mounted, whether by a bull 46 or by other females in the herd. typical estrus behavior is triggered by rising estradiol in 47 an environment where progesterone concentrations are falling. 48 the rising concentrations of circulating estradiol accompanying estrus induce the 49 leutinizing hormone (lh) surge, and the lh surge induces ovulation. ovulation follows 50 the onset standing heat by 24 to 32 hours. 51 ovulation is followed by development of the cl, a transient endocrine organ that 52 arises on the ovary at the site of ovulation. the cl produces a number of endocrine 53 products, the most important for this discussion being progesterone, which is necessary 54 for maintenance of pregnancy following insemination. should pregnancy not occur, the 55 cl will regress and another ovulatory follicle will develop. this follicle will produce 56 sufficient estradiol to again induce psychic estrus and the lh surge, and ovulation will 57 follow. the cycle will repeat indefinitely in the absence of pregnancy. this very basic 58 outline of the events of the estrous cycle has proven to be sufficient for insemination 59 programs based on heat detection. as we move to more precisely control the estrous 60 cycle, an understanding of other, less obvious events becomes necessary. 61 the cycle is sometimes conveniently divided into the luteal and follicular phases, 62 based on the dominant ovarian structure present. the follicular phase begins with the 63 regression of the cl and encompasses about 20% of the cycle, the period from the onset 64 of luteolysis until ovulation. estradiol from the developing dominant or preovulatory 65 follicle is the predominant sex steroid during the follicular phase. the luteal phase 66 begins at ovulation, and continues until regression of the cl. during this time, the sex 67 steroid dominance shifts to progesterone produced by the cl. the luteal phase includes 68 197 both metestrus, the period of cl development following ovulation, and diestrus, the 69 period spanned by the life of the mature cl. follicles continue to emerge and grow 70 during the luteal phase, and although they produce estradiol, luteal progesterone inhibits 71 lh sufficiently to prevent development to preovulatory size 72 the endocrine signal that results in regression of the cl in the absence of 73 pregnancy has been known to be prostaglandin f2alpha (pgf2ά) for several decades. 74 more recently, the signal responsible for maternal recognition of pregnancy, which 75 effectively blocks luteolysis, has been shown to be interferon tau produced by the fetal 76 trophoblast.1 the signals that result in emergence, growth, and regression of ovarian 77 follicles became evident following the advent of ultrasound as a research tool coupled 78 with endocrine assays in the early 1980’s.2 as mechanisms regulating the ovarian cycle 79 were discovered and pharmacologic agents became available, opportunities to develop 80 protocols to control the events of the estrous cycle developed. 81 follicular dynamics 82 during the 1980’s, ultrasound technology allowed basic research that described 83 waves of follicular growth and regression. these revelations were coupled with endocrine 84 assays to explain the dynamic nature of ovarian follicles. understanding of the processes 85 that initiated follicular emergence, selection, atresia, dominance and ultimately ovulation, 86 coupled with strategic administration of the limited number of available pharmacologic 87 agents have become the basis for programs to manipulate the estrous cycle and in many 88 instances precisely control the time of ovulation. the basic research on follicular 89 dynamics has been reviewed extensively in recent publications,3,4 and the reader is 90 198 referred to these resources for a more detailed description. a brief summary, applicable 91 to clinical practice follows. 92 estrus is characterized by the presence of a large ovarian follicle, producing 93 sufficient estrogen to induce the psychic changes associated with estrus behavior and to 94 induce a surge of lh, which eventually results in ovulation of the follicle and release of 95 the oocyte. following ovulation, the granulosa and thecal cells surrounding the follicular 96 antrum transform into luteal cells (luteinization) and the cl develops. steroid production 97 shifts from estrogen to progesterone and this period of progesterone dominance initiates 98 the luteal phase. 99 at the time of the lh surge which initiates the events of ovulation, both lh and 100 follicle-stimulating hormone (fsh) are released from the pituitary. this release of fsh 101 is followed closely by a second surge of fsh, and these peri-ovulatory surges of fsh are 102 associated with the emergence of a cohort of small follicles from the ovarian follicular 103 pool shortly after ovulation. emergence, defined as the last day the potential dominant 104 follicle was less than 4 mm, is perhaps more easily understood as the time that follicles 105 are easily detectable with commonly used ultrasound equipment. emergence of follicles 106 is generally coincident with the peak of the fsh surge. as growth of these follicles is 107 stimulated by fsh, follicular hormonal activity increases and the antral follicles produce 108 a number of endocrine products, including the protein hormone inhibin, and later 109 estradiol.5 estradiol and inhibin exert negative influence on the anterior pituitary to 110 decrease or inhibit release of fsh from the anterior pituitary, so that following follicular 111 emergence, fsh concentrations decline. fsh has a long half life relative to lh, but 112 concentrations of fsh remain sufficient to support follicular growth only to a diameter of 113 199 8-9 mm,6 although exogenous fsh administration will allow follicular development to 114 continue past this stage.7 115 within the cohort of emerged follicles, a single follicle either has or gains a slight 116 developmental advantage as fsh concentrations approach their nadir. as this 117 developmentally advantaged follicle grows, it acquires additional lh receptors in the 118 granulosa cells surrounding the follicular antrum and oocyte.8 the acquisition of lh 119 receptors is critical for continued development of the follicle, and lh support allows this 120 follicle to continue progressive development as the growth rate of the other follicles in 121 the cohort declines. the time at which the growth rate of the future dominant follicle 122 exceeds the growth rate of the largest dominant follicle is referred to as follicular 123 deviation and the largest follicle is referred to as the dominant follicle. as this selected 124 follicle continues to grow in response to pulsatile release of lh it continues to produce 125 estradiol and inhibin, and fsh support for the remaining follicles is lost. these 126 subordinate follicles, lacking lh receptors in a low fsh environment, begin the process 127 of atresia as the dominant follicle continues to grow. as the dominant follicle grows and 128 produces estradiol, a positive feed back loop with the hypothalamus increases the 129 frequency of gonadotropin-releasing hormone (gnrh) pulses, which in the face of 130 continued inhibition of fsh release, results in increased pulsatile lh, but not fsh, 131 release from the pituitary. 132 coincident with the development of this first post ovulatory follicular wave, and 133 eventual development of a single dominant follicle, is the development of the cl and the 134 associated increase in circulating progesterone concentrations. as noted above, the 135 dominant follicle grows and increasingly produces estrogen which feeds back on the 136 200 hypothalamus to increase gnrh and elicit lh pulses from the pituitary. however, in the 137 high progesterone environment of the luteal phase, gnrh release is limited, and the 138 resultant lh pulsatility is insufficient to support continued development of the dominant 139 follicle to pre-ovulatory size. the dominant follicle, deprived of sufficient lh for 140 continued development, eventually joins the other members of the cohort, and undergoes 141 atresia. atresia of the dominant follicle removes the source of estradiol and inhibin, and 142 shortly after its demise, another surge of fsh from the anterior pituitary stimulates 143 emergence of a second follicular wave.9 the second follicular wave develops in a 144 manner similar to the first, and a new dominant follicle develops. the dominant follicle 145 of the second follicular wave may go on to become the ovulatory follicle, or may undergo 146 atresia and be replaced by a dominant follicle from a third follicular wave which will 147 become the ovulatory follicle. 148 in the non-pregnant animal, the endometrium releases pgf2ά by day 16 -17 post 149 ovulation, and destruction of the cl (luteolysis) follows. as circulating progesterone 150 concentrations decrease following luteolysis, the inhibitory effect of progesterone on 151 gnrh release is removed. lh pulse frequency increases, and the dominant follicle 152 responds with continued growth. this growth is accompanied by increasing estrogen 153 production, and feedback on the hypothalamus further increasing pulsatile release of 154 gnrh, which it turn acts on the pituitary to increase lh pulse frequency. follicular 155 development continues in this low progesterone environment with the production of 156 estrogen eventually reaching the threshold necessary to trigger the lh surge. 157 most cattle exhibit either 2 or 3 follicular waves during an estrous cycle. it is 158 necessary to recognize that follicles which achieve dominance can only reach pre-159 201 ovulatory status in the low progesterone environment that follows luteolysis, and that any 160 of the dominant follicles produced in either 2 or 3 wave cycles can grow to ovulatory size 161 if luteolysis is induced and the inhibitory effect of progesterone removed. figure 1 is a 162 schematic representation of ovarian follicular development during a typical three wave 163 estrous cycle. 164 pharmacologic control of the estrous cycle 165 programs to control the time of estrus and ovulation developed as the events 166 controlling various portions of the estrous cycle were recognized, and as products for 167 pharmacologic manipulation of these events became available. even though the number 168 of pharmacologic agents available is limited, producers and veterinarians are offered an 169 ever increasing number of protocols for control of the estrous cycle, with seemingly 170 endless variations and refinements. veterinarians actively practicing in the field as 171 theriogenologists are asked and expected to evaluate estrus and ovulation synchronization 172 programs, and make recommendations regarding selection and implementation on 173 specific premises. understanding the available hormones and their interactions with the 174 events of the estrous cycle are essential in this task. 175 pharmacologic agents to control the bovine estrous cycle 176 opportunities for manipulation of the estrous cycle in cattle are limited to control 177 the length of the luteal phase, initiation a new follicular wave, and control the time of 178 ovulation. many of the currently available programs rely on control of all three. precise 179 control of ovulation has increasingly led to adoption of timed artificial insemination 180 (tai) protocols. 181 202 drug availability is influenced by legal constraints which prohibit the use of some 182 drugs and classes of drugs. products must be available from commercial sources, and the 183 use of compounded drugs for estrous cycle control is prohibited by the animal medical 184 drug use clarification act (amduca). enforcement of restrictions seems likely to 185 increase, driven by food safety and consumer demands. 186 available products meeting these criteria and commonly utilized in clinical 187 settings are limited to progesterone and progestins, prostaglandin f2ά (pgf2ά) and its 188 analogs, and gnrh agonists. other potentially useful products such as fsh, lh, or 189 human chorionic gonadotropin (hcg), are uncommonly utilized in synchronization 190 protocols. noticeably, and deliberately absent from this list are injectable estrogenic 191 compounds, which although valuable and historically widely used, are not currently 192 approved for estrus control in food animals, and not commercially available in the united 193 states. as mentioned earlier, compounded products, including estrogen, must be 194 avoided. table 1 provides a list of some commercially available products. 195 control of the length of the luteal phase 196 the length of the luteal phase may be extended by administration of exogenous 197 progesterone or progestins, or truncated by the administration of luteolytic doses of 198 prostaglandin. 199 extending the luteal phase with progestins 200 early attempts at synchronizing estrus focused on administration of progesterone 201 or progestins, followed by acute withdrawal. in this manner, the period of progesterone 202 dominance is extended beyond the normal lifespan of the corpus luteum. exogenous 203 progesterone inhibits release of lh, preventing development of ovarian follicles to 204 203 preovulatory size. removal of the exogenous source or progesterone from groups of 205 cattle results in a relatively synchronized estrus followed by ovulation. an additional 206 benefit of progestin therapy is the ability to hasten the onset of estrous cyclicity in 207 animals that may be anestrous at the beginning of the protocol. 208 melengestrol acetate. melengestrol acetate (mga), an orally active 209 progestational steroid, was developed in the 1960s and first marketed to both improve 210 feed efficiency and rate of gain in feedlot heifers and suppress estrus behavior. 211 suppression of ovulation and estrus behavior occurs when consumption was 212 approximately 0.5 mg per head per day.10 213 early efforts at synchronization with mga in cycling cattle relied on feeding 0.5 214 to 1 mg daily for 14 to 18 days, a period sufficient to allow spontaneous luteal regression 215 in all animals within a group. because the luteal phase is extended past the time of 216 luteolyis, follicular development and ovulation is suppressed. following withdrawal of 217 mga from the diet, follicular development resumes and a synchronized estrus follows. 218 a majority of animals treated in this manner exhibit signs of estrus and ovulate 3 to 7 219 days following mga withdrawal. pregnancy rates to this synchronized estrus are 220 variable and generally disappointing. the reduction in fertility, however, is not apparent 221 in subsequent cycles. because the reduction in fertility is confined to the first post 222 treatment estrus, programs have developed to take advantage of the synchrony of the 223 second post treatment cycle. these programs are plagued by decreasing synchrony of the 224 second post treatment estrus due to the inherent variability of estrous cycle length. these 225 protocols require that the synchronization be planned well in advance of the onset of the 226 breeding season. 227 204 the reduced fertility of the first synchronized estrus following mga withdrawal 228 has been attributed to altered development of ovarian follicles. dominant follicles which 229 would either undergo atresia or ovulate during a normal ovarian cycle persist beyond 230 their normal lifespan in the sub-luteal progesterone environment provided by the 231 exogenous mga. following withdrawal of the progestin, these persistent follicles grow, 232 produce sufficient estradiol to induce estrus and the lh surge, and ovulate, but the 233 oocytes associated with these follicles are often developmentally compromised. 234 administrating mga in the feed for shorter intervals partially overcomes the 235 negative effects of persistent follicles, but synchrony of estrus is decreased. 236 incorporating higher doses of mga has not been effective in overcoming problems 237 associated with development of persistent follicles.11 although some reports indicate 238 acute administration of progesterone late in the artificially lengthened luteal phase will 239 induce follicular turnover,12 the lack of fda approval for injectable progesterone 240 products precludes use of this strategy. 241 several variations of the initial mga protocol utilizing pgf2ά have been 242 developed to overcome problems associated with lack of synchrony of the second post 243 treatment estrus, and will be discussed in a later section. 244 intra-vaginal progesterone. intra-vaginal delivery of progesterone as a method of 245 extending or controlling the length of the luteal phase of the cycle has been available in 246 the united states since 2002. currently the only fda approved device is the eazi-breed 247 cidr® (pfizer animal health, new york, ny, usa). the eazi-breed cidr® has 248 been available for many years in other countries prior to introduction to the u.s. and a 249 205 large number of clinical and research trials have demonstrated the efficacy of 250 progesterone delivery by this route. 251 labeled protocols for eazi-breed cidr® specify that the pessary be placed intra-252 vaginally for 7 days, with administration of pgf2ά either one day prior to pessary 253 removal, or on the day of removal. the protocols yield similar results.13 this 254 combination approach allows precise control of the length of the luteal phase, and 255 synchronizes estrus. because of inherent variations in follicular wave emergence, estrus 256 activity following this protocol is not synchronized sufficiently to permit appointment 257 breeding. the range of estrus activity is often much more tightly controlled than in 258 programs which use prostaglandins as the sole agent. 259 progesterone-releasing pessaries are often incorporated into other synchronization 260 programs to improve the degree of control of the luteal phase, and like other progestin 261 based synchronization protocols, may hasten the onset of cyclicity in anovular cattle.14,15 262 truncating the luteal phase with prostaglandin f2ά 263 almost 3 decades ago, pgf2 ά and its analogs were introduced as the first drugs 264 approved to control the estrous cycle in cycling cows. prostaglandin is released from the 265 endometrium after mid-cycle in non-pregnant cattle, and result in regression of the cl, 266 removing the progesterone mediated inhibition of lh pulsatility. although the 267 developing cl is resistant to the effects of prostaglandin, sensitivity increases as the cl 268 matures, and by day 6 post-estrus, the luteal phase can be terminated in virtually all cows 269 administered exogenous pgf2ά. 270 administration of pgf2ά or its analogs after day 5-6 of the estrous cycle results 271 in cl regression and loss of progesterone dominance. when administered to groups of 272 206 cycling cattle, the synchronized end of the luteal phase is followed by a synchronized 273 estrus. 274 following the commercial introduction, pgf2ά protocols rapidly became popular 275 in estrus synchronization protocols both for artificial insemination and in synchronizing 276 estrous cycles for recipients in embryo transfer programs. the two commercially 277 available products, dinoprost (lutalyse®; pfizer animal health, new york, ny, usa) 278 and cloprostenol (estrumate®;intervet/schering–plough animal health, summit, nj, 279 usa), are in similar in their action, and while cloprostenol has a longer duration of 280 action, their effects are clinically comparable.16 281 following a single injection of pgf2ά to randomly cycling groups of cows or 282 heifers, 60 to 80% can be expected to be in estrus within a few days following luteolysis. 283 poor to no response is expected in cattle which have ovulated recently. cattle which have 284 undergone spontaneous luteolysis would typically exhibit estrus coincidentally with their 285 “synchronized” herd mates. single injection protocols can be tailored to a producer’s 286 needs is several ways. 287 traditional estrus detection with artificial insemination for 6 to 7 days, followed 288 by pgf2ά administration to those not previously inseminated increases the percent 289 response by removing those with immature cls from the pool. animals injected are 290 expected to exhibit estrus in 2 to 5 days. drug costs are very low in this protocol, but the 291 number of days during which animals need to be observed and bred is fairly high. these 292 single injection programs are useful and widely practiced in many farm settings, but 293 many management situations exist in which a shorter window of estrus synchronization is 294 207 desired. in these cases, group synchronization utilizing 2 injections of prostaglandin may 295 offer advantages.17 296 in two injection schemes, animals which respond to the first injection and animals 297 in the group which have undergone spontaneous luteolysis just prior to administration of 298 the drug are expected to be in heat in from 2 to 5 days. those animals with an immature 299 cl, (~ day 1-5 of the cycle) are not be expected to respond to the injection of pgf2ά, and 300 luteal development will continue. at the time of the second injection, all animals in the 301 group should be an appropriate stage of the luteal phase and expected to exhibit a 302 synchronized estrus. 303 although label indications for dinoprost suggest a two injection scheme with the 304 injections separated by 10 to 12 days, 14 day intervals are more commonly used and 14 305 day programs are considered equally effective if not superior to the shorter intervals 306 specified on the product label. 307 strategies aimed at identifying cows at the appropriate stage of the cycle to 308 respond to pgf2ά injections eliminate injections in animals that will not respond to 309 exogenous prostaglandin. transrectal palpation for the presence of a cl, determination 310 of milk progesterone concentrations,18 or identification of the cl with ultrasound have 311 been investigated as methods to identify cattle with functional luteal tissue. although 312 these techniques can be effective, routine administration of pgf2ά to non-inseminated 313 animals at random stages of the estrous cycle has remained the most common, and 314 perhaps most economically justifiable, procedure. 315 estrus synchronization protocols which use pgf2ά as the sole agent are 316 characterized by an inherent variability in the time of the onset of estrus, typically 317 208 exhibiting a bell shaped curve with estrus activity beginning 2 days post injection, 318 peaking in 3 to 4 days, and declining rapidly after day 5. while this synchronization of 319 estrus is useful and advantageous compared to observing and handling cattle over an 320 entire estrous cycle, synchrony of ovulation is too variable to allow timed artificial 321 insemination protocols. 322 mga plus prostaglandin f2ά 323 the availability of pgf2ά products led to estrus synchronization protocols which 324 combined feeding mga for various lengths of time with injections of prostaglandin. 325 feeding mga for 14 to 18 days, as described above, results in synchrony of estrus, but 326 poor fertility is associated with the first estrus following prolonged feeding of the 327 progestational agent. because estrous cycles in cattle fed mga are synchronized 328 following withdrawal of the progestin, there is an opportunity to administer pgf2ά to a 329 group of animals with a synchronized luteal phase following mga withdrawal. 330 treatment with prostaglandin late in the luteal phase reduces the variability in the interval 331 from pgf2ά injection to estrus. following treatment with pgf2ά, synchrony of the 332 second estrus following mga withdrawal is better than in mga protocols which do not 333 control the luteal phase length prior to the second synchronized estrus, and fertility to the 334 synchronized estrus is not compromised. initial protocols injected prostaglandins at 17 335 days after withdrawal of mga, and more recently protocols in which prostaglandin 336 injection has been delayed until 19 days after mga withdrawal have shown tighter estrus 337 synchrony.19 detection of estrus and breeding based on the signs of estrus are 338 recommended. these combination programs are quite effective and economical in 339 209 situations where feed intake is adequately controlled. the length of treatment in these 340 mga protocols remains a disadvantage, requiring a long lead time prior to insemination. 341 shorter mga programs combined with prostaglandin injections at the time of mga 342 withdrawal either did not overcome the infertility associated with longer feeding 343 periods,20 or did not show improvement over pgf2ά alone.21 344 synchronization of the emergence of the follicular wave 345 following atresia of the dominant follicle, a new cohort of follicles emerges in 346 response to fsh, and the transition from the dominance of one follicle to emergence of a 347 new cohort of developing ovarian follicles is termed follicular turnover. treatments 348 aimed at ending the period of follicular dominance will induce follicular turnover, 349 allowing more precise control of the events and timing of the estrous cycle. 350 at least three methods to induce synchronous emergence of a new follicular wave 351 are practiced, but only one lends itself to mass synchronization schemes.22 aspiration or 352 ablation a mid-cycle dominant follicle removes the inhibitory effects of estradiol and 353 inhibin on pituitary fsh release, and is followed by wave emergence in 1 to 2 days. 354 administration of an acute dose of exogenous estradiol or a combination of estradiol and 355 progesterone results in emergence of a new follicular wave in approximately 3-4 days. 356 administration of gnrh can induce ovulation or luteinization of a dominant follicle, 357 removing the source of inhibin and estradiol, followed by emergence of a new follicular 358 wave in 1.5-2 days.22,23 359 follicular ablation is not practical in on farm settings involving synchronization 360 of the estrous cycle of groups of cattle. as previously noted, the lack of commercial 361 sources of injectable estrogens and the legal atmosphere surrounding the use of 362 210 compounded pharmaceuticals in food animals effectively removes administration of 363 estrogen or estrogen/progesterone injections as an option for synchronizing follicular 364 wave emergence. 365 although not specifically approved by the food and drug administration for this 366 purpose, gnrh is available in the united states and licensed for use in cattle. 367 administration of gnrh is currently the basis for control of follicular wave emergence 368 and ovulation in many synchronization programs. 369 gonadotropin-releasing hormone 370 gnrh, a decapeptide hormone produced in the hypothalamus and transported to 371 the anterior pituitary, influences the secretion of the hormones fsh and lh. commercial 372 formulations have been available in the united states and worldwide for nearly 3 373 decades, and are approved for treatment of cystic ovarian disease. currently, no meat or 374 milk withdrawal periods are required for use of this product in cattle. 375 endogenous gnrh stimulates release of lh and fsh, with a varying magnitude 376 of response and ratio depending on the ovarian structures and resultant hormonal mix 377 present. during the luteal phase of the cycle, high progesterone concentrations limit the 378 release of gnrh, and subsequently lh pulse frequency. conversely, during the 379 follicular phase, removal of the negative influence of progesterone is followed by 380 continued growth of the dominant follicle, and increasing production of estradiol by the 381 follicle triggers increasing pulsatile release of gnrh from the hypothalamus. this 382 feedback loop culminates in the pre-ovulatory lh surge, which induces ovulation and 383 initiates the onset of another luteal phase. 384 211 exogenous gnrh administered in the luteal phase during the period of follicular 385 dominance results in ovulation or luteinization of dominant follicles, and is followed by 386 emergence of a new follicular wave. administration shortly after follicular wave 387 emergence is less likely to result in initiation of a new follicular wave. administration 388 of exogenous gnrh following spontaneous or induced luteolysis can be used to induce 389 an lh surge and control the time of ovulation. the actions of gnrh have been 390 incorporated into synchronization protocols which control the time of follicular wave 391 emergence and ovulation. 392 ovsynch and related protocols 393 in 1995, a protocol for tai using sequential administration of gnrh, pgf2ά, 394 and gnrh to synchronize follicular wave emergence, luteolysis, and ovulation was 395 introduced.24 this protocol, widely known as ovsynch, was the first allowing 396 appointment breeding to gain widespread acceptance. the basic program with numerous 397 modifications is the basis for many of the successful ovulation synchronization protocols 398 used today. 399 figure 2a illustrates a timeline schematic for the ovsynch protocol. figure 2b 400 outlines an injection scheduling calendar for ovsynch. on day 0, all eligible cattle 401 receive gnrh, followed 7 days later by pgf2ά. forty eight hours later, a second 402 injection of gnrh is administered, and tai, without regard to heat detection, is carried 403 out 16 to 20 hours following the second gnrh. the initial injection of gnrh is intended 404 to synchronize emergence of a new follicular wave following induction of ovulation of a 405 dominant follicle. the corpus luteum formed following this diestrus ovulation will not 406 become susceptible to the luteolytic effects of prostaglandin for several days, and will 407 212 provide a source of progesterone should the cl which developed following the previous 408 ovulation be destroyed. the prostaglandin injection 7 days after initiation of the protocol 409 will initiate luteolysis and allow continued development of the dominant follicle which 410 arose from the induced follicular wave to pre-ovulatory size. the second injection of 411 gnrh will induce an lh surge, with ovulation following in approximately 28 hours.24 412 not all randomly cycling cattle have equivalent ovarian structures. synchrony of 413 ovulation is improved if a dominant follicle is present at the time of the first gnrh 414 injection compared to initiation prior to the time of follicular deviation. the day of the 415 cycle at which ovsynch is initiated influences pregnancy rates to timed artificial 416 insemination. cows in which ovsynch was initiated near mid-cycle had greater rates of 417 synchronous ovulation following the first injection of gnrh, and programs beginning on 418 day 5 to 12 of the estrous cycle resulted in greater pregnancy rates.25,26 419 many modifications to the initial ovsynch protocol have been developed with the 420 goal of increasing rate of synchronous ovulation and pregnancy. the most common have 421 utilized 2 prostaglandin injections, approximately 2 weeks apart, with the last injection 12 422 or 14 days prior to the first gnrh injection of ovsynch. these programs increase the 423 percentage of animals at the optimum stage of the cycle when the first injection of gnrh 424 is administered, and are collectively referred to as pre-synch ovsynch programs, or 425 prostaglandin pre-synch programs. pregnancy rates to tai are improved compared to 426 ovsynch alone.27 a sample schedule for a pre-synch ovsynch protocol is shown in 427 figure 3. the long lead time (initiation of hormone injections 36-38 days prior to 428 insemination) present an obstacle to use of pre-synch, which is minimized in dairy herds 429 213 by initiating the protocol during the voluntary waiting period during which breeding is 430 commonly withheld for 45 to 70 days following parturition. 431 more recently, protocols utilizing both gnrh and prostaglandin to increase the 432 percentage of cows that ovulate following the first gnrh injection have been developed 433 with improvement in pregnancy rate following timed artificial insemination similar to or 434 better than that seen with pre-synch ovsynch programs.28,29 the injection schedule for 435 the g6g protocol described by bello is illustrated in figure 4. the injection of pgf2ά 436 which initiates the protocol induces luteolysis of midand late-cycle cl’s. two days 437 later, an injection of gnrh synchronizes ovulation following luteolysis, and initiates a 438 new follicular wave in animals which have a dominant follicle. an ovsynch protocol is 439 initiated six days later. the goal is to optimize the number of animals with a dominant 440 follicle at the onset of ovsynch, and ultimately synchronization of ovulation at the 441 completion of ovsynch.28 the g6g protocol has a shorter lead time than pre-synch, but 442 has scheduling disadvantages. increased pregnancy rates to timed artificial insemination 443 compared to ovsynch alone are reported. 444 mga plus pgf2ά plus gnrh protocols 445 estrus synchronization protocols incorporating oral administration of mga, 446 followed with administration of pgf2ά have been modified by incorporating gnrh near 447 the time of the first post mga estrus to synchronize the first follicular wave and 448 ovulation.30-32 these programs generally allow for shorter duration of mga feeding, and 449 offer the potential advantage of hastening the onset of cyclicity in the late pre-pubertal 450 period or post partum period. adaptations allowing fixed-time artificial insemination33 451 214 or heat detection are utilized. figure 5 illustrates a treatment schedule for the mga 7-11 452 synch modification with fixed time insemination. 453 synchronization of ovulation and timing of insemination 454 gnrh administration in the low progesterone environment following luteolysis 455 results in an lh surge followed by ovulation in 24 to 32 hours. protocols which used 456 estrogen to induce ovulation enjoyed some popularity,34 but no approved product is 457 available for veterinarians at this time. agents with lh activity, such as hcg, could be a 458 suitable substitute for gnrh, but protocols utilizing this strategy are not common. 459 timing of insemination should include ample opportunity for sperm capacitiation 460 prior to ovulation, and insemination at 16 to 20 hours following the ovulation-inducing 461 dose of gnrh is the most common recommendation.35 earlier insemination, including 462 at the time of administration of gnrh (co-synch protocol), may result in acceptable 463 pregnancy rates, while insemination later than 24 hours after gnrh leads to less 464 favorable outcomes. 465 in the original ovsynch protocol, the second gnrh injection, administered to 466 synchronize ovulation, was given 48 hours following induction of luteolysis with pgf2ά. 467 this timing was convenient for dairies, and resulted in grouping of chores associated with 468 the protocol around times when cows were handled or locked up. recent studies suggest 469 that delaying the administration of the second gnrh injection until 56 hours after 470 induced luteolysis will improve pregnancy rates.36 modification of any protocol should 471 take into consideration farm schedules to insure compliance, particularly if handling or 472 restraint occurs outside the normal daily or weekly routine. 473 choosing and implementing the right protocol 474 215 many estrus synchronization and tai programs are available, and when 475 implemented properly, most are effective. selecting a protocol for a particular herd will 476 depend on the ability of herd management to comply with the time lines inherent in each 477 protocol. a lack of commitment or cavalier attitude concerning the timing of injections 478 and breeding will negatively impact the ultimate success any program. 479 an old saying in the dairy business is “when heat detection is everybody’s job, it 480 usually means it is nobody’s job”. this wisdom is applicable to scheduling and 481 administration of protocols. unless a job is a priority, compliance will suffer. 482 accurate record keeping and proper administration of all hormones are essential. 483 injection schedules can become very complicated when dealing with large herds and 484 when multiple protocols used within a herd. both pcdart and dairycomp 305 485 have routines which will generate action lists to facilitate scheduling of injections. 486 timed artificial insemination programs may overcome some management 487 problems associated with estrus detection, but management problems with nutrition, 488 housing, semen handling, and concurrent disease are not eliminated. 489 drug cost, availability of trained technicians, class of livestock and the facilities 490 available for repeatedly handling cattle will influence the choice of protocols for 491 individual farms. selection of the appropriate protocol for a particular herd or 492 management system means selection of the protocol herd management will be most likely 493 to efficiently and consistently implement, rather than the newest or most popular protocol 494 available. 495 discussion 496 216 today, theriogenologists have an increasing number of options to synchronize 497 estrus and ovulation. these systems developed in concert with an increased 498 understanding of reproductive endocrinology and the proper selection and 499 implementation of any of the available protocols requires an understanding of follicular 500 dynamics, luteolysis and ovulation. programs which allow for the successful utilization 501 of tai require much more precise control of the events of the estrous cycle than those 502 which incorporate estrus detection. although the legal and regulatory environment 503 restricts or forbids access to certain potentially useful pharmacologic agents, successful 504 strategies utilizing available agents have been devised, and offer adequate control of the 505 cycle. 506 references 507 508 1. bazer fw, spencer te, ott tl: interferon tau: a novel pregnancy recognition 509 signal. am j reprod immunol 1997;37:412-420. 510 2. adams gp, matteri rl, kastelic jp, et al: association between surges of follicle-511 stimulating hormone and the emergence of follicular waves in heifers. j reprod 512 fertil 1992;94:177-188. 513 3. lucy mc: the bovine dominant ovarian follicle. j anim sci 2007;85:e89-e99. 514 4. adams gp, jaiswal r, singh j, malhi p. progress in understanding ovarian 515 follicular dynamics in cattle. theriogenology 2008;69:72-80. 516 5. kaneko h, terada t, taya k, et al: ovarian follicular dynamics and 517 concentrations of oestradiol-17 beta, progesterone, luteinizing hormone and 518 217 follicle stimulating hormone during the periovulatory phase of the oestrous cycle 519 in the cow. reprod fertil dev 1991;3:529-535. 520 6. ginther oj, kot k, kulick lj, et al: emergence and deviation of follicles during 521 the development of follicular waves in cattle. theriogenology 1997;48:75-87. 522 7. hampton jh, bader jf, lamberson wr, et al: gonadotropin requirements for 523 dominant follicle selection in gnrh agonist-treated cows. reproduction 524 2004;127:695-703. 525 8. beg ma, bergfelt dr, kot k, et al: follicular-fluid factors and granulosa-cell 526 gene expression associated with follicle deviation in cattle. biol reprod 527 2001;64:432-441. 528 9. adams gp: comparative patterns of follicle development and selection in 529 ruminants. j reprod fertil (suppl) 1999;54:17-32. 530 10. patterson dj, kiracofe gh, stevenson js, et al: control of the bovine estrous 531 cycle with melengestrol acetate (mga): a review. j anim sci 1989;67:1895-532 1906. 533 11. kojima fn, chenault jr, wehrman et al: melengestrol acetate at greater doses 534 than typically used for estrous synchrony in bovine females does not mimic 535 endogenous progesterone in regulation of secretion of luteinizing hormone and 536 17beta-estradiol. biol reprod 1995;52:455-463. 537 12. anderson lh, day ml: acute progesterone administration regresses persistent 538 dominant follicles and improves fertility of cattle in which estrus was 539 synchronized with melengestrol acetate. j anim sci 1994;72:2955-2961. 540 218 13. ambrose dj, emmanuel dg, colazo mg, et al: pregnancy rates to timed 541 artificial insemination in holstein heifers given prostaglandin f2alpha twenty-542 four hours before or concurrent with removal of an intravaginal progesterone-543 releasing insert. j dairy sci 2008;91:2678-2683. 544 14. yavas y, walton js: induction of ovulation in postpartum suckled beef cows: a 545 review. theriogenology 2000;54:1-23. 546 15. chebel rc, santos je, cerri rl, et al: reproduction in dairy cows following 547 progesterone insert presynchronization and resynchronization protocols. j dairy 548 sci 2006;89:4205-4219. 549 16. salverson rr, dejarnette jm, marshall ce, et al: synchronization of estrus in 550 virgin beef heifers using melengestrol acetate and pgf2alpha: an efficacy 551 comparison of cloprostenol and dinoprost tromethamine. theriogenology 552 2002;57:853-858. 553 17. thatcher ww, chenault jr: reproductive physiological responses of cattle to 554 exogenous prostaglandin f2alpha. j dairy sci 1976;59:1366-1375. 555 18. peters ar, riley gm, rahim se, et al: milk progesterone profiles and the double 556 injection of cloprostenol in post partum beef cows. vet rec 1980;107:174-177. 557 19. lamb gc, nix dw, stevenson js, et al: prolonging the mga-prostaglandin 558 f2alpha interval from 17 to 19 days in an estrus synchronization system for 559 heifers. theriogenology 2000;53:691-698. 560 20. beal we, chenault jr, day ml, et al: variation in conception rates following 561 synchronization of estrus with melengestrol acetate and prostaglandin f2alpha. j 562 anim sci 1988;66:599-602. 563 219 21. chenault jr, mcallister jf, kasson cw: synchronization of estrus with 564 melengestrol acetate and prostaglandin f2alpha in beef and dairy heifers. j anim 565 sci 1990;68:296-303. 566 22. martinez mf, adams gp, kastelic jp, et al: induction of follicular wave 567 emergence for estrus synchronization and artificial insemination in heifers. 568 theriogenology 2000;54:757-769. 569 23. roche jf, austin ej, ryan m, et al: regulation of follicle waves to maximize 570 fertility in cattle. j reprod fertil (suppl) 1999;54:61-71. 571 24. pursley jr, mee mo, wiltbank mc: synchronization of ovulation in dairy cows 572 using pgf2alpha and gnrh. theriogenology 1995;44:915-923. 573 25. vasconcelos jl, silcox rw, rosa gj, et al: synchronization rate, size of the 574 ovulatory follicle, and pregnancy rate after synchronization of ovulation 575 beginning on different days of the estrous cycle in lactating dairy cows. 576 theriogenology 1999;52:1067-1078. 577 26. moreira f, de la sota rl, diaz t, et al: effect of day of the estrous cycle at the 578 initiation of a timed artificial insemination protocol on reproductive responses in 579 dairy heifers. j anim sci 2000;78:1568-1576. 580 27. moreira f, orlandi c, risco ca, et al: effects of presynchronization and bovine 581 somatotropin on pregnancy rates to a timed artificial insemination protocol in 582 lactating dairy cows. j dairy sci 2001;84:1646-1659. 583 28. bello nm, steibel jp, pursley jr: optimizing ovulation to first gnrh improved 584 outcomes to each hormonal injection of ovsynch in lactating dairy cows. j dairy 585 sci 2006;89:3413-3424. 586 220 29. souza ah, ayres h, ferreira rm, et al: a new presynchronization system 587 (double-ovsynch) increases fertility at first postpartum timed ai in lactating dairy 588 cows. theriogenology 2008;70:208-215. 589 30. kojima fn, salfen be, bader jf, et al: development of an estrus synchronization 590 protocol for beef cattle with short-term feeding of melengestrol acetate: 7-11 591 synch. j anim sci 2000;78:2186-2191. 592 31. funston rn, ansotegui rp, lipsey rj, et al: synchronization of estrus in beef 593 heifers using either melengesterol acetate (mga)/prostaglandin or mga/select 594 synch. theriogenology 2002;57:1485-1491. 595 32. wood sl, lucy mc, smith mf, et al: improved synchrony of estrus and 596 ovulation with the addition of gnrh to a melengestrol acetate-prostaglandin 597 f2alpha synchronization treatment in beef heifers. j anim sci 2001;79:2210-598 2216. 599 33. bader jf, kojima fn, schafer dj, et al: a comparison of progestin-based 600 protocols to synchronize ovulation and facilitate fixed-time artificial insemination 601 in postpartum beef cows. j anim sci 2005;83:136-143. 602 34. stevenson js, tiffany sm, lucy mc: use of estradiol cypionate as a substitute 603 for gnrh in protocols for synchronizing ovulation in dairy cattle. j dairy sci 604 2004;87:3298-3305. 605 35. pursley jr, silcox rw, wiltbank mc: effect of time of artificial insemination on 606 pregnancy rates, calving rates, pregnancy loss, and gender ratio after 607 synchronization of ovulation in lactating dairy cows. j dairy sci 1998;81:2139-608 2144. 609 221 36. brusveen dj, cunha ap, silva cd, et al: altering the time of the second 610 gonadotropin-releasing hormone injection and artificial insemination (ai) during 611 ovsynch affects pregnancies per ai in lactating dairy cows. j dairy sci 612 2008;91:1044-1052. 613 614 222 figure 1. 615 616 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 bovine estrous cycle corpus hemorrhagicum functional corpus luteum uterus releases pgf2α follicular development in absence of cl estrus 617 618 figure 1: development of successive waves of ovarian follicles during the estrous cycle. 619 shortly after ovulation, a cohort of follicles emerges; a single follicle gains an advantage 620 that allows it to become the dominant follicle, only to undergo atresia in the high 621 progesterone environment of the luteal phase. a second follicular wave emerges around 622 day 8-9 of the cycle, and the dominant follicle that follows meets the same fate. a third 623 wave emerges on day 16-17 following demise of the second wave dominant follicle. 624 prostaglandin-induced luteolysis destroys the source of progesterone, the inhibition of 625 lh pulses is removed, and this third wave dominant follicle develops to preovulatory 626 size. estradiol produced by the dominant follicle increases past the threshold necessary 627 223 to induce the lh surge, and ovulation follows. image courtesy of dr. m. daniel givens, 628 auburn university 629 630 631 224 632 figure 2a. 633 634 ovsynch day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 gnrh pgf2α 5. inseminate 16-20 hours after gnrh injection. gnrh 1. ovulation of dominant follicle 2. new follicular wave 3. luteolysis 4. induce lh surge and ovulation 635 636 figure 2a: ovsynch protocol. an injection of gnrh on day 0 induces ovulation 637 or luteinization of a dominant follicle, and a new follicular wave emerges 2 days later. 638 injection of prostaglandin f2alpha on day 7 induces luteolysis, and injection gnrh on 639 day 9 induces an lh surge, with ovulation 28 hours later. insemination at 16-20 hours 640 after the second gnrh injection (~8 hours ahead of ovulation) allows time for sperm 641 capacitation prior to ovulation. 642 643 225 figure 2b. sunday monday tuesday wednesday thursday friday sat week 1 gnrh week 2 pgf2ά gnrh tai (16-20 hours post gnrh) figure 2b: injection calendar for a pre-synch ovsynch protocol that avoids weekend chores. pgf2ά = prostaglandin f2alpha, gnrh = gonadotropin-releasing hormone, tai = timed artificial insemination. 226 figure 3. sunday monday tuesday wednesday thursday friday sat week 1 pgf2ά week 2 week 3 pgf2ά week 4 week 5 gnrh week 6 pgf2ά gnrh tai (16-20 hours post gnrh) figure 3: injection calendar for a pre-synch ovsynch protocol which utilizes 2 prostaglandin injections, 14 days apart, with the second injection given 14 days prior to the start of ovsynch. this protocol minimizes the number of days per week on which injections are given, avoids weekend chores, and places all prostaglandin injection on the same day of the week. a modification in which the first two prostaglandin injections are given on wednesday of week 1 and 3 could be utilized and may have advantages. pgf2ά = prostaglandin f2alpha, gnrh = gonadotropin-releasing hormone, tai = ttmed artificial insemination. 227 figure 4. sunday monday tuesday wednesday thursday friday sat week 1 pgf2ά gnrh week 2 gnrh week 3 pgf2ά gnrh tai (1620 hours post gnrh) figure 4: injection calendar for a modified g6g protocol. g6g shortens the lead time prior to insemination compared to pre-synch ovsynch, but has the disadvantage of having assigned chores related to the protocol on more days of the week. scheduling to avoid weekend chores may be more complicated with this protocol. pgf2ά = prostaglandin f2alpha, gnrh = gonadotropin-releasing hormone, tai = timed artificial insemination. 228 figure 5. sunday monday tuesday wednesday thursday friday sat week 1 mga (first day) mga week 2 mga mga mga mga mga (last day) pgf2ά week 3 gnrh week 4 pgf2ά gnrh and tai (60 hours after pgf2ά) week 5 week 6 figure 5: treatment calendar for mga 7-11 synch with tai. mga feeding begins on day 0, and is discontinued on day 7. prostaglandin injections are given on day 7, and followed on day 11 with gnrh injections. on day 18, prostaglandin is injected to end the luteal phase, and gnrh is injected and cows are inseminated at 60 hours following the prostaglandin injection. variations eliminating the last gnrh injection utilize heat detection and breeding based on the signs of estrus. 229 mga = melengestrol acetate incorporated into feed, pgf2ά = prostaglandin f2alpha, gnrh = gonadotropin-releasing hormone, tai = timed artificial insemination. 230 table 1. class drug trade name route of adminstration manufacturer progestatational agents melengestrol acetate progesterone mga® eazi-breed cidr® oral intravaginal pessary pfizer pfizer prostaglandin f2ά and analogs dinoprost tromethamine cloprostonol lutalyse® estrumate ® intramuscular injection intramuscular injection pfizer gnrh agonists gonadorelin diacetate tetrhydrate cystorelin® ovacyst® intramuscular injection intramuscular injection merial 231 gonadorelin hydrochloride factrel® intramuscular injection fort dodge animal health table 1: commercially available products used to manipulate the estrous cycle of cattle. 232 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) 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/legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: accuracy of pregnancy specific protein-b test for early pregnancy diagnosis in dairy cattle accuracy of pregnancy specific protein-b test for early pregnancy diagnosis in dairy cattle j. e. romanoa, j. larsonb adepartment of large animal clinical sciences, college of veterinary medicine and biomedical sciences texas a&m university, college station, tx, usa, bdepartment of animal science, college of agricultural, food and environmental sciences, university of minnesota, st paul, mn, usa early pregnancy diagnosis is vital for efficient reproductive management of cattle. ideally, the diagnostic test used should correctly identify both pregnant and non-pregnant females. the objective of this study was to evaluate the accuracy of the pregnancy specific protein b test (psp-b) for early pregnancy diagnosis in dairy cattle. plasma samples from two hundred forty six lactating cows more than 80 days postpartum were collected three times at days 28, 30 and 35 after breeding (estrus= day 0). each plasma sample was analyzed in duplicate using an elisa test for psp-b. test results for psp-b were reported as: pregnant, non-pregnant or uncertain (repeat open or repeat pregnant). at the same time, each female was examined by transrectal ultrasonography (trus) with a linear 5-7.5 mhz transducer. trus was used as a criterion standard test for comparison with psp-b results. a positive pregnancy diagnosis was made when an embryo plus extra embryonic membranes was observed by trus. the agreement between psp-b and trus diagnoses was compared by using kappa values. the prevalence of pregnancy at days 28 determined by trus was 46.3% (114/246). sensitivity of psp-b test at days 28, 30 and 35 was 93.9%, 96.0% and 97.2%, respectively (p>0.05). specificity of psp-b test for the same days was 95.5%, 93.9% and 93.6% (p>0.05). the positive predictive values for days 28, 30 and 35 were 94.7%, 92.2% and 92.0%, respectively (p>0.05). the negative predictive values for the same days were 94.7 %, 96.8% and 97.8% (p>0.05). the accuracy of psp-b at days 28, 30 and 35 was 96.0 %, 95.4% and 97.5%, respectively (p>0.05). however, when compared at the same days with trus differences were detected (p<0.001). the percentage of uncertain samples among the entire number of specimens analyzed was 5.6% (40/721). a significant reduction in the percentage of samples that required repetition from days 28, 30 to 35 post-ai was detected [8.5 % (21/246), 4.8 % (11/229) and 3.3 % (8/246), respectively (p<0.01)]. kappa value at days 28, 30 and 35 was 0.89 (ci 95%: 0.84-.95), 0.89 (0.84-0.95) and 0.90 (0.85-0.96) (p>0.05). it was concluded that the agreement between psp-b and trus was very good. psp-b test was not a 100% accurate test of pregnancy compared with trus at days 28, 30 or 35 after breeding. false negative results were due to low levels of psp-b in pregnant animals. false positive results were due to persistence of pregnant levels of psp-b in females with pregnancy loss. key words: cattle, pregnancy diagnosis, psp-b, transrectal ultrasonography, accuracy 538 2009: treatment strategies in the perinatal mare and foal treatment strategies in the perinatal mare and foal 1 p.r. morresey 2 rood and riddle equine hospital, po box 12070, lexington, ky, usa 3 abstract 4 a large number of conditions are recognized in the peripartum period that have 5 the potential to impact the health and future performance of both the mare and the foal. 6 peripartum hemorrhage can be insidious in onset and result in profound abdominal pain, 7 hypovolemia and in severe cases death of the mare. those mares that survive the initial 8 hemorrhage may succumb some time after the apparent stabilization of the hematoma. 9 treatment is aimed at promoting hemostasis and improving cardiovascular performance 10 while not promoting further hemorrhage. many drugs in common use have been chosen 11 due to anecdotal reports of success or extrapolation from studies on horses or other 12 species. large, multi-center controlled trials of therapeutic agents are lacking, this being 13 expected given the critical nature of the condition. the compromised neonatal foal is a 14 challenge to the attending clinician, who must balance the immediate medical needs of 15 the foal with the financial requirements of the client. following the initial assessment, 16 resuscitation of the neonate must be performed in a timely and efficacious fashion. the 17 attending clinician should establish a coherent and consistent approach to cardiovascular 18 resuscitation of the foal. following this event, stabilization of the neonate and 19 preparation for transport to a referral center if required can occur. a thorough knowledge 20 of the expected neonatal development of the foal will aid in selection of foals requiring 21 specialist intervention. 22 23 451 keywords: mare, peripartum, hemorrhage, foal, neonatal, resuscitation 24 introduction 25 the peripartum period for both the mare and the foal can be a period of great risk 26 to life and future potential. a myriad of problems exist, and it is beyond the scope of this 27 presentation to cover them all. therefore a selected few will be covered in detail: 28 peripartum hemorrhage in the mare, neonatal foal resuscitation, stabilization and 29 preparation for referral. 30 mare 31 considerations in the periparturient mare 32 the periparturient mare does not differ substantially from the non-pregnant mare 33 with respect to general husbandry. routine exercise, a balanced diet and regular 34 preventative health care (teeth, deworming and vaccination) should all continue as 35 before. 36 differences become important when they involve the overall health of the mare 37 and the effects on placental function that systemic illness can have. the goals of therapy 38 should address resolution of the precipitating problem, systemic support for the mare 39 (and therefore fetoplacental unit) and the avoidance of fetal hypoxia. 40 the overall health of the mare will be affected by any disease process resulting in 41 inappetance, fever or proinflammatory mediator production with systemic release. 42 nutritional insult resulting in weight loss during mid-gestation has detrimental effects on 43 placental development which results in reduced fetal growth.1 endocrine function of the 44 foal is also affected.2 45 452 colic is also of greater concern in the periparturient mare, as this condition 46 presents a serious diagnostic challenge to the practitioner through limitations imposed 47 upon examination by the presence of the gravid uterus. accurate differentiation between 48 gastrointestinal conditions and gestational accidents is essential but difficult in many 49 cases. early gastrointestinal problems can appear similar to initial stages of labor. 50 peripartum gastrointestinal conditions include large colon volvulus or displacement, cecal 51 rupture, small intestinal volvulus, enterocolitis and direct trauma to the intestine.3 52 trauma to the gut may result in ischemic necrosis of the affected areas.4-6. 53 compromise to the gastrointestinal tract results in the onset of endotoxemia with 54 profound circulatory dysfunction and proinflammatory stimuli occurring. serious 55 metabolic insults can affect both the mare and concurrently the fetus. treatment of the 56 gastrointestinal disease may involve general anesthesia with abdominal exploration 57 further insulting the fetoplacental unit. 58 hypoglycemia of the mare resulting from inappetance or fasting while colic 59 treatment continues decreases glucose delivery to the fetus. administration of glucose to 60 late-term pregnant mares is recommended to avoid this, as is supplementation of feed 61 material by nasogastric intubation where practical. oxygen therapy is also recommended 62 where placental function is suspected to be compromised.7 63 reproductive problems in the peripartum period with apparent colic as a 64 presenting clinical sign include uterine torsion, uterine rupture, uterine laceration, uterine 65 bruising and hemorrhage of the blood vessels supplying the reproductive tract.3 66 peripartum hemorrhage 67 453 rupture of and subsequent hemorrhage from the uterine artery is the most 68 common cause of death in mares post partum.3 the external iliac artery, utero-ovarian 69 artery, and uterine artery have also been implicated.8 in a review of central kentucky 70 mares, reproductive complications accounted for the majority (57 of 98 cases, 58%) of 71 deaths in peripartum mares.9 of those that died of reproductive complications, rupture of 72 a uterine artery was determined to be the cause of death in 40 cases (70%). the 73 incidence of peripartum hemorrhage in the mare has not been determined by retrospective 74 studies of large numbers of mares, instead reports of clinical cases are found in the 75 literature. hemoperitoneum itself is a significant cause of abdominal discomfort in the 76 horse, with approximately 13% of all cases due to rupture of uterine vessels.10 77 although usually considered a problem of the post partum period, a number of 78 reports exist of cases in the prepartum period.8,11 peripartum hemorrhage has been 79 reported to occur at any age, however older mares are considered to be at greater risk.12 80 age-related degeneration of arterial vessels associated with the reproductive tract is 81 suspected to be the reason for the increased incidence in older mares, coupled with the 82 increased mechanical stresses imposed by the gravid uterus. uterine contractions and 83 obstetrical manipulations further increase stress on the vessel wall.13 copper deficiency 84 was identified as a contributing factor in mares experiencing fatal hemorrhage, whereas 85 non-fatal hemorrhage mares had comparable copper levels to their non-affected cohorts.14 86 peripartum hemorrhage can occur in any of the following forms: hemorrhage into 87 the peritoneal cavity, hemorrhage retained either within the broad ligament of the uterus 88 or within the uterine wall (mural hemorrhage) or hemorrhage into the uterine lumen. 89 combinations of these may occur, necessitating thorough evaluation of mares affected by 90 454 seemingly less serious forms of hemorrhage so as to avoid non detection of life-91 threatening episodes. 92 hemorrhage into the peritoneal cavity can lead to profound hypovolemia, pain 93 and result in peracute death. if confined to the broad ligament or uterine wall, pain can 94 still be significant but prognosis for life is better. these hematomas may be incidental 95 findings during routine reproductive examinations, or may become acutely apparent some 96 time after foaling following the onset of abdominal hemorrhage. hemorrhage within the 97 uterine lumen is usually of less significance due to the relatively small amount of blood 98 lost from the circulating pool in most cases. 99 diagnosis 100 clinical parameters of horses experiencing acute blood loss and hemoperitoneum 101 have been reviewed.10,15 consistent signs included depression, tachypnea, tachycardia, 102 poor pulse quality, pale mucous membranes, prolonged capillary refill time, cool 103 extremities and abdominal discomfort. signs occurring less often included abdominal 104 distention, sweating, ataxia, and a mass in the broad ligament palpated per rectum. 105 clinical pathology findings include anemia, neutrophilia, lymphopenia, 106 thrombocytopenia, hypoproteinemia, hypocalcemia, and azotemia. measurements of 107 hemostasis (prothrombin time, parital thormoboplastin time, template bleeding time) are 108 usually normal. 109 the diagnosis of hemoperitoneum is confirmed by transabdominal 110 ultrasonography and abdominocentesis. lesions at necropsy may include a ruptured 111 miduterine artery or ruptured broad ligament hematoma. on occasion, the source of 112 hemorrhage may be the iliac vessels. 113 455 rectal examination is controversial, with the need to make a diagnosis balanced 114 by the concern of worsening hemorrhage. there is no evidence that rectal examination of 115 mares suffering prepartum hemorrhage adversely affects outcome.12 rectal examination 116 may not aid diagnosis, as the hemorrhage may dissect between tissue planes and not form 117 a discrete, palpable mass in the broad ligament. careful transrectal ultrasonography may 118 greatly aid diagnosis due to the ability to detect non-palpable lesions. 119 treatment 120 due to the sporadic nature of the condition, treatment modalities are derived by 121 extrapolation from studies on humans, laboratory species and anesthesia studies in the 122 horse. a number of treatments have been recommended, with both scientific and 123 anecdotal backing for those in common usage: 124 ε-aminocaproic acid. the effects of ε (epsilon) aminocaproic acid (eaca) on 125 coagulation and fibrinolysis in healthy horses have been reviewed.16,17. partial 126 thromoplasin time (ptt) was found to be significantly decreased and α2-antiplasmin 127 activity was significantly higher. fibrinogen was significantly lower than baseline. 128 bolus dosage is more practical in field situations; however a recent review17 established 129 an efficacious constant rate infusion protocol. 130 the procoagulant eaca is a synthetic anti-fibrinolytic amino acid. similar 131 agents are widely used in human medicine to arrest hemorrhage. the lysine binding sites 132 of plasminogen become saturated with eaca which displaces plasminogen from the 133 fibrin surface stabilizing the hemostatic plug.18 134 naloxone. naloxone is anecdotally reported and widely used in the treatment of 135 postpartum mare hemorrhage.3 experimental evidence shows that endorphins released 136 456 by stress act on opiate receptors to depress cardiovascular function during hemorrhagic 137 shock.19 naloxone acts as a μ-opioid receptor competitive antagonist and also has 138 (lesser) antagonist action at the κand δ-opioid receptors. the hemodynamic effects of 139 blood loss were shown to be ameliorated by intravenous administration of naloxone as 140 evidenced by an increase in arterial pressure, left ventricular function and cardiac 141 output.20,21 regional blood flow differences were noted in the dog, with naloxone 142 improving circulation to the myocardium, intestine, liver and adrenal.22 in the horse, 143 naloxone (0.20 mg/kg iv) immediately following acute hemorrhage was found to counter 144 the increase seen in heart rate.23 however, this would result in a considerably higher 145 dose than that commonly in usage (8 mg) for the hemorrhaging mare. at available 146 concentrations (0.4 mg/ml) a volume of 250ml would have to be infused to achieve the 147 higher dose rate shown to be effective in acute hemorrhage situations. 148 dexamethasone or other corticosteroids. the beneficial effects of 149 dexamethasone administration during hemorrhagic shock has been shown in dogs.24,25. 150 increased mean arterial pressure was noted, as was improved blood flow to the 151 pulmonary, gastrointestinal and renal circulations. furthermore, less cell damage was 152 evident as shown by decreased plasma enzyme elevations referable to damaged tissues. 153 formalin. formalin activation of platelet function during fixation in vitro has 154 been reported.26 the procoagulant properties of formalin (aqueous formaldehyde) in the 155 horse have been critically reviewed.27 in spite of a reported decrease in clotting and 156 bleeding time in goats,28 administration was shown to have no effect on primary or 157 secondary hemostasis in normal or aspirin-treated horses.27 behavioral effects, 158 tachycardia, lacrimation, salivation and muscle fasciculations were seen at higher doses. 159 457 despite no effect on coagulation seen in that study, the usage of intravenous formalin for 160 its purported hemostatic properties is widely practiced in equine medicine. 161 yunnan baiyao. yunnan baiyao (or yunnan paiyao, literally white medicine 162 from yunnan) is a hemostatic powder of largely unknown constituents. purported uses 163 include hemostasis, relief of pain, diminishment of swellings and the improvement of 164 circulation to the tissues. the mechanism of action is unknown. experimentally, 165 template bleeding time of halothane anesthetized ponies was decreased when compared 166 to baseline values following the administration of yunnan baiyao 4 h prior to and 167 immediately preceding induction of anesthesia.29 activated clotting time was not 168 affected in this study. anecdotal reports indicate widespread usage by equine 169 practitioners with reported favorable results. 170 acepromazine. the pharmacokinetics and pharmacodynamics of intravenous 171 acepromazine have been extensively reviewed in the horse.30,31 use in the hemorrhaging 172 mare is controversial as concerns are held for the potential exacerbation of hypovolemia. 173 however, use allows the hypotensive restoration of adequate circulatory blood volume 174 with a diminished chance of dislodging the hemostatic plug. 175 butorphanol. butorphanol tartrate is widely used for control of pain and 176 chemical restraint in the hemorrhaging mare. concurrent judicious use of the α2 177 adrenergic agonists (xylazine, detomidine) further aids in control of anxiety. 178 butorphanol is a partial agonist/antagonist at the μ opioid receptor and an agonist at the κ 179 opioid receptor. therefore, potential for antagonism exists with the concurrent usage of 180 butorphanol and naloxone. 181 458 blood transfusion. whole blood is the fluid of choice in cases of hemorrhagic 182 shock. however, this is often not available; therefore isotonic polyionic solutions are 183 administered to maintain circulating volume, with consideration of their relatively short 184 time within the vascular space. when blood is lost from the intravascular compartment, 185 central venous pressure decreases and blood lactate concentration increases significantly 186 when compared with baseline values in the healthy horse.32,33 surprisingly, heart rate 187 and venous blood gas analysis do not change significantly in the initial period. 188 therefore, blood lactate concentration is a useful measure of hypovolemia in horses in 189 situations of acute loss before other parameters become abnormal. also, it may be useful 190 to indicate the need for blood transfusion and monitor responses of horses when whole 191 blood is administered.32 192 plasma. the administration of plasma is widely practiced for the provision of 193 clotting factors and oncotic support to the hemorrhaging mare. this is useful in 194 situations where anemia is severe once the mare is stabilized and whole blood is not 195 available. in an emergency situation, this is less practical as plasma needs to be 196 administered slowly and the benefit of administration will not be realized in a clinically 197 relevant time frame. volumes in common usage (1 l) are unlikely to measurably affect 198 oncotic pressure and hemodynamic performance. 199 hypertonic saline. in situations of acute blood loss, the restorative fluid used is 200 of lesser importance as long as an appropriate volume is given.34 in human medicine, 201 considerable interest has been shown in the use of hypertonic saline dextran (hsd) in 202 situations where significant hemorrhage has occurred.35 controversy still surrounds the 203 use of hypertonic solutions for rapid restoration of intravascular volume.36 in the 204 459 hemorrhaging mare, use of hypertonic saline is widely practice but similarly controversial 205 due to the possibility of rapid plasma volume expansion causing a deleterious rapid spike 206 in blood pressure. 207 hetastarch. hetastarch, 6% hydroxyethyl starch solution, is an artificial colloid 208 used as a plasma volume expander. it has oncotic activity only and is not a blood or 209 plasma substitute. hetastarch is elimintated over a prolonged period by the kidneys. in 210 situations where rapid plasma volume expansion is needed, hetastarch can be given as a 211 series of rapid bolus doses in contrast to plasma which must be slowly administered. for 212 this reason it offers an attractive way to rapidly ameliorate the effects of acute blood loss. 213 however, one retrospective study suggests that intraoperative use of hetastarch in human 214 cardiac surgery may increase bleeding and subsequent blood transfusion requirements.37 215 polyionic replacement fluids. volume restoration by polyionic fluids is widely 216 practiced in the hemorrhaging mare. these fluids rapidly leave the vascular space (30 217 minutes) and do not provide a long term solution to hypovolemia, but instead provide a 218 rapid transient means to combat blood loss. when used in conjunction with colloids 219 (plasma and hetastarch) or hypertonic saline a more prolonged effect can be expected. 220 care must be taken to avoid overzealous plasma volume expansion to preserve the 221 hemostatic plug. 222 lidocaine infusion. the historical use of lidocaine as a systemically 223 administered analgesic for intractable human pain has been reported.38 analgesic effects 224 in horses have only been relatively recently reported.39 when practical, a constant rate 225 infusion of lidocaine is an excellent analgesic for the hemorrhaging mare, especially if 226 hemoperitoneum is present. an appropriate dosage regimen is an initial lidocaine 227 460 loading dose (1.3 mg/kg iv) as a slow bolus, followed by a constant rate infusion (0.05 228 mg/kg/min iv), preferably using a fluid pump however this is not essential. 229 management 230 although controversy exists as to the utility of various therapeutic agents, 231 ensuring the mare is as calm as possible and not exposed to undue stress is widely agreed 232 upon. care should be taken during restraint to not excessively stress the mare by using a 233 combination of physical and chemical restraint. 234 broad spectrum antimicrobial therapy is indicated to prevent the establishment of 235 bacterial overgrowth in any hematomas or stagnant pools of blood post hemorrhage. 236 anti-inflammatory therapy should be maintained following the initial insult to minimize 237 pain and distress, which may lead to increased blood pressure and restarting of 238 hemorrhage. 239 fluid therapy should be approached with caution and closely monitored. rapid 240 plasma volume expansion can lead to hemodilution, loss of the hemostatic plug and 241 restarting of hemorrhage. this must be weighed against the necessity of restoration of an 242 adequate circulating volume in the hypovolemic mare. the mare will succumb to 243 hypovolemia not anemia in the acute phase of blood loss.34 the signs of hemorrhage and 244 hypovolemia are well known: visible distress or colic signs, muscle fasciculations, 245 sweating along the flanks, flehmen, elevated heart and respiratory rates, and palor of the 246 mucous membranes. should these signs return during fluid restoration, an immediate 247 decrease in the rate of admission should be considered. in the healthy horse, where 248 potential for hemorrhage is not present, one-half of the calculated fluid deficit can be 249 administered rapidly, with the remainder of the deficit given over the ensuing 24 h. 250 461 however, in the mare affected by peripartum hemorrhage, this initial rapid high volume 251 administration is not possible. 252 a representative treatment plan for the author follows. subsequent to the 253 diagnosis of hemorrhage, an intravenous catheter is placed and an initial 5 l bolus of 254 polyionic fluids containing 20 g ε-aminocaproic acid as a hemostatic agent is given over 255 30 min. concurrent with this, acepromazine is administered intramuscularly to allow 256 hypotensive circulating volume restoration and to act as a mild calming agent. unless the 257 mare is showing obvious signs of cardiac compromise, naloxone is not administered. 258 broad spectrum antimicrobial coverage is initiated (k penicillin and gentamicin, 259 alternatively trimethoprim-sulfamethoxazole) for a minimum of five d. analgesia and 260 anti-inflammatory therapy is provided by flunixin meglumine. when this is insufficient, 261 a constant rate infusion of lidocaine is given until 48 h following the last noted abdominal 262 pain. hemostatic therapy is continued for 2 to 3 d, allowing ultrasonographic evidence of 263 the cessation of hemorrhage and stabilization of the hemorrhage site (if visible or 264 palpable) to be noted. the author avoids the use of oxytocin in post partum hemorrhage 265 mares, and is judicious with rectal evaluation. uterine lavage, when attempted after 2 to 266 3 d, involves the establishment of a siphon and avoids distension of the uterus whenever 267 possible. undue stress from any source is avoided. this is especially important with 268 mares protective of the foal, where procedures involving the foal are minimized and 269 absences will be avoided if at all possible. 270 an appropriate fluid plan following stabilization of the hypovolemic mare is to 271 provide a maintenance rate of polyionic intravenous fluids (2 ml/kg/h) until water intake 272 is deemed sufficient. a pcv that is low but stable is acceptable. the author considers a 273 462 pcv of 15% that is stable acceptable, however a blood cross match is initiated at this 274 point. the pcv will begin to slowly rise (1 to 2% daily) once hemorrhage ceases and the 275 bone marrow responds. resorption of peritoneal blood (if present) aids in this increase. 276 should the mare reach a pcv of 12% and continue to decrease, up to 20% of the 277 mare’s circulating volume should be replaced with whole blood over 2 to 3 h by 278 transfusion from a compatible donor. in this case, it should be expected that the pcv 279 will begin to slowly decrease again over a period of 2 to 3 d as the transfused red blood 280 cells are removed from circulation. 281 useful drug dosages for postpartum mares are summarized in table 1. 282 the foal 283 peripartum risk factors 284 peripartum factors affecting the neonatal foal may be divided into three broad 285 categories. 286 maternal health. systemic illness with fever, gastrointestinal compromise 287 (potential for endotoxemia), and surgical manipulation are deleterious to the fetus. 288 nutritional status also affects fetal health.1 289 reproductive conditions of the mare. history of previous neonatal compromise, 290 known placental pathology (infection, thickening, separation), abnormalities of the birth 291 canal, vulvar discharge, and loss of colostrum prepartum alert the clinician to potential 292 neonatal difficulties. 293 parturient events. the neonate can be affected by an abnormal gestation length, 294 prolonged labor, dystocia, premature placental separation, and premature rupture of the 295 umbilical cord. meconium aspiration leads to hypoxic injury and pulmonary disease, 296 463 with presence of meconium in the amniotic fluid or amnion sometimes the only 297 indication of this event. 298 resuscitation of the compromised neonate 299 prior preparation in anticipation of an emergency is paramount to success. a 300 readily available collection of necessary equipment and drugs, kept within an easily 301 accessible and portable container aids in achieving a successful outcome. a list of drug 302 dose rates useful in resuscitation situations should be kept within the drug kit (table 2). 303 preparation of the foal for resuscitation includes drying and generally stimulating 304 the neonate, and clearance of respiratory and oral secretions by suctioning to maximize 305 the airway. it is convenient and safest to position the foal in lateral recumbency with any 306 rib fractures down. 307 abcde of resuscitation 308 establishing a clear and consistent protocol for resuscitation of the neonate is 309 important when seeking to avoid delays in action during emergency situations (table 3). 310 the order of activities can easily be remembered by the abcde protocol. techniques, 311 drug dosages and break points determining changes in action may vary between 312 individual clinicians due to preference and case experience. the aim of establishing a 313 regular and sustainable cardiac and respiratory rhythm however is common to all 314 protocols. 315 indications for referral 316 the normal newborn foal displays a fairly predictable progression from the time 317 of delivery to the onset of appropriate foal behavior and physiology (table 4). 318 464 significant deviation from these benchmarks strongly suggests that referral for advanced 319 care should be considered. 320 evaluation of the at risk foal includes a physical examination and consideration 321 of the gestational history and laboratory values if available (table 5). although the foal 322 may appear normal initially, rapid deterioration is possible and subtle deviations in 323 physical findings and blood values may be the only indication of impending trouble. 324 stabilization of the foal and preparation for referral 325 if the foal is showing signs of distress it may be prudent to transport the foal as 326 soon as possible even if separately from the mare. send colostrum from the mare for 327 later administration and the placenta for examination if available. 328 if hypothermic, ensure the foal remains warm. use blankets, insulated foal 329 covers, or provide external sources of heat such as warmed fluid bags. avoid excessively 330 warming the hypovolemic foal as increasing circulation at the periphery can lead to 331 profound falls in blood pressure. 332 if breathing difficulties are present, place an intranasal oxygen cannula. insert the 333 tip to the level of the eye socket. portable oxygen tanks can be set to provide 5l oxygen 334 flow per minute. 335 fluids should be administered if dehydration or hypovolemia are present. half 336 the calculated deficit (deficit in liters = bodyweight in kg x % dehydration) can be given 337 rapidly prior to referral. maintenance fluid rates for foals are higher than the adult horse, 338 being 5% to 10% of body weight daily, i.e., 2 to 4 ml/kg/hr. when calculating the 339 required maintenance rate, consider all sources of fluid intake for the foal to avoid over 340 hydrating the compromised foal. this is especially important if the foal is recumbent as 341 465 exceeding 10% of bodyweight can promote pulmonary edema formation. glucose 342 supplementation (2.5% or 5% dextrose in polyionic fluids) can be given if blood glucose 343 levels are low, however rapid rehydration of the foal should always use non-glucose 344 containing fluids. care must be exercised with the addition of glucose to fluids as over 345 supplementation results in hyperinsulinemia and subsequent worsening of hypoglycemia. 346 placement of an indwelling feeding tube will aid in administration of colostrum as 347 well as provide a vehicle for continued feeding of the foal (if appropriate) during 348 transport to the referral facility should this be distant from the farm. ensure the foal is 349 fed standing (if able to rise) or only when in sternal recumbency. reflux of gastric 350 content is still possible with a correctly placed nasogastric tube. avoid overfeeding the 351 sick foal: a useful rule of thumb is to feed 10% of the bodyweight of the foal as milk over 352 a 24 h period. divide this amount into 12 equal feeds at two h intervals. should colic or 353 nasogastric reflux occur post feeding, discontinue immediately. 354 antimicrobial therapy should not be delayed in the foal suspected of sepsis (table 355 2). collection of a blood culture using aseptic technique before administration of 356 antimicrobials is desirable to improve chances of yielding the causative infectious agent. 357 this can be shipped with the foal for bacteriologic examination and antimicrobial 358 sensitivity analysis. 359 anti-inflammatory treatments are also indicated in the foal that has sustained 360 physical trauma or is febrile. lipid derived inflammatory mediators are important in the 361 pathophysiology of hypoxic ischemic encephalopathy (hie) suggesting prompt use of 362 non-steroidal anti-inflammatory drugs (nsaids) and anti-oxidant therapies is warranted. 363 the nsaids vary in their potential for gastric mucosal and renal toxicity, especially if 364 466 used in the dehydrated patient; therefore care must be exercised in selection for the 365 compromised neonate. 366 if neurological dysfunction is present, control of cerebral edema and seizure 367 activity (if present) are indicated. cerebral edema results from any traumatic, ischemic 368 or hypoxic insult to the neonate. the onset of signs of neurological dysfunction is 369 usually delayed, becoming apparent after a 24 to 48 h period of apparently normal 370 development. often the only suggestions of impending problems are the gestational 371 history, parturient events, and subtle early neonatal behavioral abnormalities. should 372 seizure activity ensue, control of seizure activity in the first instance is necessary to avoid 373 rapid exhaustion of the foal and secondary injuries predisposing to bacterial sepsis. 374 placement of leg wraps and provision of padding adjacent to the foal will minimize 375 trauma. 376 if the foal is sufficiently medically stabilized and the owner is compliant, 377 transport to a referral facility is possible with the following considerations: 378 timeliness. nothing is worse than a referral too late. increased costs of treatment 379 to the owner coupled with a decreased prognosis result in a loss to us all as a profession. 380 owner financial resources. hospitalization will be expensive. complications are 381 provided at no extra cost. continuous nursing care is expensive but imperative. 382 ensure secure vascular access; if you place an intravenous catheter ensure that it will 383 remain in place with all attachments. this may be replaced with a longer-term catheter in 384 hospital. 385 467 history. where possible, a written account encompassing treatment to the time of 386 arrival at the hospital. if the responsible person is not coming with the foal, encourage 387 the client to bring your billing/record sheets. 388 up to date blood work is imperative. blood collected before referral will likely 389 be repeated, to establish both a baseline and to gauge response to previous treatments. 390 mare compliance. is it necessary for the mare to accompany the foal? if so, will 391 she adjust to a hospital setting? 392 summary 393 peripartum hemorrhage in the mare may become a life-threatening emergency 394 depending on the structures involved and the extent of blood loss. treatment of the mare 395 centers on promoting hemostasis and restoring circulating blood volume in a fashion that 396 does not excessively raise blood pressure and risk restarting hemorrhage. many 397 established treatments are controversial. the neonatal foal may require resuscitation 398 following delivery as the result of gestational or parturient events. although many 399 conditions can be managed on the farm, a thorough clinical exam should be performed to 400 identify those foals in need of specialist intervention. appropriate stabilization and 401 preparation for transport and referral increase the probability of success. 402 references 403 1. wilsher s, allen wr: effects of a streptococcus equi infection--mediated 404 nutritional insult during mid-gestation in primiparous thoroughbred fillies. part 1: 405 placental and fetal development. equine vet j 2006;38:549-557. 406 468 2. ousey jc, fowden al, wilsher s, et al: the effects of maternal health and body 407 condition on the endocrine responses of neonatal foals. equine vet j 2008;40:673-408 679. 409 3. dolente ba: critical peripartum disease in the mare. vet clin north am equine 410 pract 2004;20:151-165. 411 4. platt h: caecal rupture in parturient mares. j comp pathol 1983;93:343-346. 412 5. dart aj, pascoe jr, snyder jr: mesenteric tears of the descending (small) colon as 413 a postpartum complication in two mares. j am vet med.assoc 1991;199:1612-414 1615. 415 6. zamos dt, ford ts, cohen nd, et al: segmental ischemic necrosis of the small 416 intestine in two postparturient mares. j am vet med assoc 1993;202:101-103. 417 7. wilkins p: monitoring the pregnant mare in the icu. clin tech equine pract 418 2003;2:212-219. 419 8. rooney j: internal hemorrhage related to gestation in the mare. cornell vet 420 1964;54:11-17. 421 9. dwyer r, harrison l: post partum deaths of mares. university of kentucky equine 422 disease quarterly 1993;2:5. 423 10. dechant je, nieto je, le jeune ss: hemoperitoneum in horses: 67 cases (1989-424 2004). j amvet med assoc 2006;229:253-258. 425 11. pascoe rr: rupture of the utero-ovarian or middle uterine artery in the mare at or 426 near parturition. vet rec 1979;104:77. 427 428 469 12. arnold ce, payne m, thompson ja, et al: periparturient hemorrhage in mares: 73 429 cases (1998-2005). j amvet med assoc 2008;232:1345-1351. 430 13. gruninger b, schoon ha, schoon d, et al: incidence and morphology of 431 endometrial angiopathies in mares in relationship to age and parity. j comp pathol 432 1998;119:293-309. 433 14. stowe hd: effects of age and impending parturition upon serum copper of 434 thoroughbred mares. j nutr 1968;95:179-183. 435 15. pusterla n, fecteau me, madigan je, et al: acute hemoperitoneum in horses: a 436 review of 19 cases (1992-2003). j vet intern med 2005;19:344-347. 437 16. heidmann p, tornquist sj, qu a, et al: laboratory measures of hemostasis and 438 fibrinolysis after intravenous administration of epsilon-aminocaproic acid in 439 clinically normal horses and ponies. am j vet res 2005;66:313-318. 440 17. ross j, dallap bl, dolente ba, et al: pharmacokinetics and pharmacodynamics of 441 epsilon-aminocaproic acid in horses. am j vet res 2007;68:1016-1021. 442 18. verstraete m. clinical application of inhibitors of fibrinolysis. drugs 1985;29:236-443 261. 444 19. gurll nj, vargish t, reynolds dg, et al: opiate receptors and endorphins in the 445 pathophysiology of hemorrhagic shock. surgery 1981;89:364-369. 446 20. faden ai, holaday jw: opiate antagonists: a role in the treatment of hypovolemic 447 shock. science 1979;205:317-318. 448 21. vargish t, reynolds dg, gurll nj, et al: naloxone reversal of hypovolemic shock 449 in dogs. circ shock 1980;7:31-38. 450 470 22. lechner rb, gurll nj, reynolds dg: effects of naloxone on regional blood flow 451 distribution in canine hemorrhagic shock. proc soc exp biol med 1985;178:227-452 233. 453 23.weld jm, kamerling sg, combie jd, et al: the effects of naloxone on endotoxic and 454 hemorrhagic shock in horses. res commun chem pathol pharmacol 1984;44:227-455 238. 456 24. ferguson jl, roesel of, bottoms gd: dexamethasone treatment during 457 hemorrhagic shock: blood pressure, tissue perfusion, and plasma enzymes. am j 458 vet res 1978;39:817-824. 459 25. hellman a, haggendal e, lundberg d: hemodynamic effects of massive doses of 460 dexamethasone in controlled hypovolemic shock in the dog. acta anaesthesiol 461 scand 1982;26:222-224. 462 26. pfueller sl, hosseinzadeh p, firkin bg, et al: activation of platelet coagulant 463 activities by formalin. thromb haemost 1978;39:546-548. 464 27. taylor el, sellon dc, wardrop kj, et al: effects of intravenous administration of 465 formaldehyde on platelet and coagulation variables in healthy horses. am j vet res 466 2000;61:1191-1196. 467 28. ali m, abdus s: comparative studies on systemic coagulants in ruminants. indian 468 vet j 1973;50:27-31. 469 29. graham l, farnsworth k, cary j: the effect of yunnan baiyao on the template 470 bleeding time and activated clotting time in healthy halothane anesthetized ponies. 471 proc int’l vet emergency crit care soc 2002. p. 790. 472 471 30. marroum pj, webb ai, aeschbacher g, et al: pharmacokinetics and 473 pharmacodynamics of acepromazine in horses. am j vet res 1994;55:1428-1433. 474 31. ballard s, shults t, kownacki aa, et al: the pharmacokinetics, pharmacological 475 responses and behavioral effects of acepromazine in the horse. j vet pharmacol 476 ther 1982;5:21-31. 477 32. magdesian kg, fielding cl, rhodes dm, et al: changes in central venous 478 pressure and blood lactate concentration in response to acute blood loss in horses. j 479 am vet med assoc 2006;229:1458-1462. 480 33. hurcombe sd, mudge mc, hinchcliff kw: clinical and clinicopathologic 481 variables in adult horses receiving blood transfusions: 31 cases (1999-2005). j am 482 vet med assoc 2007;231:267-274. 483 34. nolan j: fluid resuscitation for the trauma patient. resuscitation 2001;48:57-69. 484 35. dubick ma, atkins jl: small-volume fluid resuscitation for the far-forward 485 combat environment: current concepts. j trauma 2003;54:s43-s45. 486 36. dubick ma, bruttig sp, wade ce: issues of concern regarding the use of 487 hypertonic/hyperoncotic fluid resuscitation of hemorrhagic hypotension. shock 488 2006;25:321-328. 489 37. knutson je, deering ja, hall fw, et al: does intraoperative hetastarch 490 administration increase blood loss and transfusion requirements after cardiac 491 surgery? anesthesia analgesia 2000;90:801-807. 492 38. mccleane g: intravenous lidocaine: an outdated or underutilized treatment for 493 pain? j palliat med 2007;10:798-805. 494 472 39. doherty tj, frazier dl: effect of intravenous lidocaine on halothane minimum 495 alveolar concentration in ponies. equine vet j 1998;30:300-303. 496 497 473 table 1: useful drug dosages for the postpartum mare sedation: care should always be exercised in the administration of sedation to the pregnant mare. most agents cause hypotension which will exacerbate the negative effects of hypovolemia. xylazine: 0.25 to 1 mg/kg iv or im. potent hypotensive effects. detomidine: 0.01 to 0.02 mg/kg iv or im. less hypotensive potential than xylazine. butorphanol: 0.01 to 0.02 mg/kg iv or im. give as needed, may be repeated every 8 to 12 h. effects will be attenuated where naloxone is administered concurrently. acepromazine: 0.02 mg/kg im q 12h. potent hypotensive agent, use with caution. analgesia: flunixin: 1 mg/kg iv q 12h lidocaine: load with 1.3 mg/kg slow iv bolus of lidocaine 20% followed by 0.05 mg/kg/min iv as a constant rate infusion. may be conveniently made by adding 450 ml lidocaine 20% to a 3 l fluid bag and administering at 1 ml/kg/h. hemostatic agents: ε-aminocaproic acid: 40 mg/kg iv load then 20 mg/kg iv q 6-8h. administer in 1 l fluids over 15-30 min. five or more doses may be required if hemorrhage ongoing. yunnan baiyao: 8 mg/kg po q 6h. administer 16 x 0.25g capsules in water as paste q 6h. duration of treatment is highly variable, being 2 to 4 d. 474 circulatory support: naloxone: 0.01 to 0.02 mg/kg iv. administered in initial treatment phase, may be repeated. polyionic fluids: 2 ml/kg/h iv (maintenance rate). continued throughout the period when fluid intake by other means deemed inadequate. hypertonic saline: 2 to 4 ml/kg iv. controversial due to potential for a rapid rise in blood pressure. given during the initial fluid resuscitation period. hetastarch: 6 to 10 ml/kg iv. administered during the initial fluid volume restoration period only. 475 table 2: useful drug dosages for the foal. antimicrobials: duration of treatment is governed by clinical response. a minimum of five d should be administered in the absence of adverse reactions. k penicillin: 40000 to 50000 units/kg iv q 6h ceftiofur sodium: 2 mg/kg im q 12h or 5 to 10 mg/kg iv q 6-12h amikacin: 25 mg/kg iv q 24h trimethoprim-sulfamethoxazole: 30 mg/kg po bid seizure control and metabolic support: diazepam 0.05 to 0.4 mg/kg iv. short-acting control following acute onset of seizure activity. may repeat 2 to 3 times as required to establish control. phenobarbital 4 to 10 mg/kg iv. long-acting control useful when initial agent fails. may be repeated at 12 h intervals for continued control, or change to oral phenobarbital at 4 mg/kg po q 12h. thiamine 10 mg/kg iv q 24h. cerebral metabolic support. useful in initial stages of hypoxic insult. control cerebral edema and inflammation: hypertonic saline 7 ml/kg iv as 3% solution. shown to aid in control of cerebral edema. useful in initial period following insult. 476 mannitol 1 mg/kg iv as 20% solution. may repeat at 12 h intervals until appropriate mentation returns, often 2 to 3 d sufficient. flunixin meglumine: 1 mg/kg iv q 12h. continue while clinical signs evident. ketoprofen: 2 mg/kg iv q 24h. as for flunixin. less ulcerogenic potential. control oxidative damage: vitamin c 100 mg/kg iv q 24h. useful in the initial stages following onset of cerebral compromise. three d treatment or more may be required and this is safely administered. vitamin e 20 iu/kg sc q 24h. requires prolonged administration to reach therapeutic levels in central nervous system. dimethyl sulfoxide 1 g/kg iv q 12h as 10% solution. useful during period when ongoing damage is suspected to be occurring, often up to three d post insult. respiratory stimulant caffeine 10 mg/kg po load, then 2.5 mg/kg po q 6h. continue until appropriate respiratory pattern is established. may cause hyperactivity and lower the seizure threshold in some foals. 477 table 3: resuscitation protocol for the compromised neonate. airway intubate by nasotracheal route with largest practical endotracheal tube extend neck and twist as arytenoids reached to ease passing size 8 to 10 mm suitable for average foals pass tube to nares to minimize dead space compress chest and palpate esophagus to ensure correct placement breathing respiratory arrest usually precedes cardiac arrest in the neonatal foal establish rate of 8 to 10 breaths/minute with 1 second inspiration period use 100% oxygen if available, however room air is acceptable the use of a self-inflating resuscitation bag with a pressure limiting valve (ambu), or any other similar delivery device, will aid ventilation and avoid excessive inflation pressure doxapram (controversial) at 0.5 mg/kg iv circulation thoracic compressions if hr less than 60, especially if less than 60 and not increasing minimize interruptions (no longer than 10 seconds) rapid compressions (aim for 100/minute) establish vascular access if response is not immediately favorable 478 drugs usage and rationale based on experience and extrapolation heart rate less than 60 and not increasing continue compressions to distribute drugs epinephrine at 0.02 mg/kg (0.5 to 1 ml of 1:1000 per 50 kg foal). intratracheal dose 5 to 10x this, however absorption is poor repeat every 3 to 5 minutes until response is noted administer 10 ml/kg of a balanced electrolyte solution. a 2 to 4 ml/kg hetastarch bolus may be useful to rapidly expand circulating volume avoid inducing hyperglycemia with dextrose containing solutions as resulting hyperinsulinemia is depressive dobutamine at 3 to 40 μg/kg/min iv is useful to improve pulse pressure and peripheral perfusion everything else monitor foal progress pupillary light responses: dilated pupils indicate lack of cerebral perfusion stop when hr above 60 (pause 10 seconds max) spontaneous breathing (pause 30 seconds) end point determination 479 table 4: times of importance to the neonatal foal. sternal recumbency: the foal should right itself and be able to remain sternal within 5 to 10 minutes of birth. standing: within 60 minutes (range of 15 to 165 minutes). compromised neonates tend to remain recumbent longer, further exposing themselves to pathogens. suckle reflex: usually develops within 20 minutes of birth, although may be much sooner. suckling: the foal should suckle the mare within 2 h (range 35 minutes to 7 h). urination: first urination occurs at 6 h for colts, 10 h for fillies. urine production: approximately 6 ml/kg/hr. decreases may result from decreased fluid intake, increased losses or compromises in renal function. obstruction or disruption due to rupture and uroperitoneum are possible in the compromised neonate, or one which sustained trauma during parturition. defecation: foals display abdominal straining within the first few h after of birth, and pass meconium completely within 24 h. colostrum stimulates gi motility. any interference with gi motility will prolong passage of meconium increasing the likelihood of impaction. 480 table 5: examination of the neonatal foal. • rectal temperature appropriate neonatal range 99 °f to 101.5 ºf. neonatal foals are unable to regulate body temperature to the same degree as older foals. • cardiovascular system appropriate neonatal heart rate (hr) range is between 70 to 120 beats per minute. hr is highly labile, however rate and rhythm is regular. pulses are synchronous with the heart beat and easily palpable. deviations may indicate arrhythmia (electrolyte abnormality, congenital cardiac anomaly). a murmur associated with a pda can occur for the first few days of life. • respiratory system within the first hour of life, the respiratory rate (rr) of a normal foal can rise up to 80 breaths per minute. the rr decreases progressively over the next few days, with a subsequent range of 30 to 40 breaths per minute. increased rr may indicate compromised pulmonary function, pain, excitement or fever. the magnitude of the thoracic excursion is indicative of the respiratory effort. any decrease may indicate fatigue. nostril flaring may be the only indication of increased respiratory effort. • musculoskeletal system 481 joint distension: indicative of sepsis, coagulopathy (hemarthrosis), trauma. • integument decubital ulcers indicate trauma, unseen seizure activity or the occurrence of prolonged recumbency. pitting edema suggests hypoproteinemia or cardiac dysfunction. icterus suggests sepsis, neonatal isoerythrolysis, or hepatic disease. hemorrhage on the mucous membranes suggests sepsis, coagulopathy or direct trauma. • gi function any occurrence of colic has the potential to indicate a life-threatening episode and should be thoroughly investigated. • urinary function acute renal failure may occur as the result of decreased in utero blood supply or be iatrogenic from nephrotoxic drug usage. uroperitoneum results from a ruptured bladder, ruptured urachus or torn ureter. • ocular examination hyperemia of the sclera indicates birth trauma, sepsis, or coagulopathy. entropion results from weight loss or dehydration leading to enophthalmia. corneal opacity is the result of ulceration, a depressed blink response, lack of tear production, or exposure keratitis in the depressed neonate. 482 anterior chamber: can reflect systemic inflammation and sepsis. fibrin deposition (aqueous flare) and hypopyon may result. • mentation and nervous function hypoxic ischemic encephalopathy causes loss of affinity for the mare, generalized depression and a lack of vigorous suckling. low level seizure activity may appear as muscle fasciculations, chewing fits, or unexplained cutaneous trauma. the menace response is absent from the neonate and is not an indication of vision. this reflex will take between 4 to 14 d to develop. hematology • normal neonatal range (birth to 1 week): rbc range from 7.4 to 11.4 million cells/ul. wbc range from 4.9 to 13.6 thousand cells/ul. • neutrophils (n): approximately 5500 cells/μl are present at birth with this increasing to 8000/μl within the first 12 h of life. • lymphocytes (l): may decrease to approximately 1400 cells/μl within a few hours of birth, thereafter they increase to approximately 5000 cells/μl by 3 months of age. a transient decrease to below 1000 cells/μl may occur in some normal foals, but this may also indicate infection or immune compromise. serum chemistry 483 in the absence of established foal parameters in many laboratories, normal adult equine values are often used for assessment of neonatal foal health. • serum electrolyte concentrations are maintained within a narrow range and do not differ substantially from established adult values. • glucose is elevated compared to adults due to frequent suckling. hypoglycemia is cause for concern as this may indicate of sepsis or decreased feeding activity. • plasma protein levels at birth vary considerably between foals. • serum creatinine and bun are unreliable indicators of newborn foal renal performance as the placenta is primarily responsible for elimination of waste products; therefore an increase reflects placental dysfunction. however, bun and creatinine may be initially elevated decreasing to adult levels by 3 to 5 d. • total bilirubin may be elevated as mild icterus is common. consider sepsis, isoerythrolysis or hepatic disease. • creatine kinase is raised by muscle trauma from delivery. • alkaline phophatase is elevated due to rapid growth in the neonate. • ggt elevations may be of colostral origin. • fibrinogen is uniformly low in the healthy neonate (up to 200mg/dl). elevated levels indicate in utero challenge with prenatal response. • igg concentration: there is a strong association between the occurrence of sepsis and an immunoglobulin concentration less than 400mg/dl. catabolism of immunoglobulins may occur in the compromised neonate. 484 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 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/openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2012: case report: ileo-jejunal entrapment in a uterine mesometrium rent of a non-pregnant mare case report: ileo-jejunal entrapment in a uterine mesometrium rent of a non-pregnant mare paula a. schaffer,a jennifer m. sonis,b jacobo s. rodriguez,c sushan hana adepartment of microbiology, immunology and pathology; bdepartment of clinical sciences; cequine reproduction laboratory; college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co summary a ten-year-old, non-pregnant arabian mare presented with acute uncontrollable abdominal pain. a diagnosis of small bowel compromise was made on clinical and trans-abdominal ultrasonographic examination. euthanasia was elected by the owner and the necropsy examination revealed herniation and venous infarction of five meters of distal jejunum and the entire ileum within a 7.5 cm rent in the right mesometrium of the uterus. small bowel entrapment in a mesometrial rent is rare in domestic animals with few case reports in the literature, but interestingly has been relatively well-characterized in primiparous and multiparous women. entrapment in a mesometrial rent may be considered as a differential diagnosis for acute small bowel compromise and colic in non-pregnant and pregnant mares. keywords: broad ligament, entrapment, hernia, horse, small intestine background mesometrial rents have the potential to cause life-threatening small bowel entrapment in both humans and animals. this rare condition is well-documented in humans, but few published reports exist in the veterinary literature, and cases may be under-reported. mesometrial herniation in women can have a promising surgical prognosis if treated early, and similar success has been historically reported in mares.1-3 in general, herniation and venous infarction of the small intestine is a common cause of acute abdominal pain and euthanasia in horses.4,5 small intestinal hernias in horses most frequently occur in association with spontaneous, congenital, or traumatic rents in the abdominal mesentery, cecocolic fold, diaphragm,6 and within natural foramina including the gastroepiploic foramen, the inguinal ring of stallions, and the umbilicus of foals.7-10 small bowel entrapment is rarely documented in rents within the lateral ligaments of the urinary bladder, mesodiverticulum and uterine mesometrium.4,8,9,11-13 the pathogenesis of rent formation in these unusual structures has not yet been determined and documentation of additional cases is necessary in order to improve our ability to make early diagnoses and to instigate successful surgical intervention. case presentation a ten-year-old non-pregnant arabian mare was presented to the colorado state university equine critical care service with a four hour-history of acute abdominal pain. on presentation, the mare was tachycardic (70 beats/minute) and had a normal capillary refill time (< 2 seconds). five liters of bright green watery reflux was obtained upon passing a nasogastric tube. on rectal palpation, loops of small intestine were moderately distended and there was mild gas distension of the large colon. transabdominal ultrasound revealed non-motile, distended small intestine and large amounts of flocculent free abdominal fluid. fluid obtained by abdominocentesis was serosanguinous with an elevated total protein level of 3.8 g/dl. a strangulating small intestinal lesion was suspected and exploratory laparotomy was recommended. the owner declined surgery and euthanasia was elected due to poor prognosis without surgical intervention. outcome the mare was submitted for postmortem examination. at necropsy the abdominal cavity contained approximately ten l of yellow-tinged serosanguinous and fibrinous fluid. approximately five meters of small intestine from the level of the distal jejunum to the ileo-cecal junction was herniated through a 7.5 cm diameter rent in the center of the right mesometrium of the uterus just cranial to the uterine artery (figure 1). the incarcerated small intestine was dilated five cm in diameter. the serosal surface was diffusely mottled dark red and purple, and the wall was thickened one to two cm with transmural clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201255 clinical theriogenology • volume 4 number 1 • march 2012 hemorrha mucus, an mesentery mesentery hemorrha figure 1. i herniated a was light p upper right evalu normal gl there were firm fibro this site. glands, se the media septicemi histop of the gut sloughed expanded segmental herniation fibrosis an mesotheli though a t ge and edema nd sloughed n y had a line of y. incarcerate ge and edema in situ photogr and incarcerate pink, gas disten t quadrant (aste uation of the r istening seros e no lesions in ous adhesion t additional gr evere and diff astinum and o a and shock. pathology of with ischemi and infiltrated by edema, bl l lymphoplasm n and necrosis nd consisted o ial reactivity. traumatic rent a. the intesti necrotic muco f demarcation ed mesentery a which exten raph of the post ed within a rent nded and sharp erisk). right mesomet sal membrane n the uterus a to the adjacen ross findings fuse pulmonar n the epicard incarcerated j ic necrosis ch d by colonies lood and scatt macytic enter s. the margin of normal fibr lack of infla t occurring se inal lumen wa osa admixed w n that sharply was friable, d nded from the t-mortem exam t in the right m ly demarcated trium (figure e, with no evi and ovaries. t nt mesocolon, included hem ry congestion ial surface, al jejunum and haracteristic o s of mixed bac tered inflamm ritis which ma ns of the mes rovascular me ammation or everal years in as filled with with finely gro delineated in discolored da e mesenteric b mination. five mesometrium of from incarcera e 2) revealed t idence of hem the left meso , possibly ind morrhagic line n with petechi ll consistent w ileum confirm of entrapment cteria. the su matory cells. ay have occur ometrial rent esometrial str fibrosis is con n advance wit serosanguino ound feed ma ncarcerated m ark purple, and border to the m meters of dista f the uterus (ar ated bowel. th that the rent w morrhage, infl ometrium had dicative of prio ear streaks in ial and ecchym with endotoxe med segmenta (figure 3). t ubmucosa and small intestin rred secondar lacked inflam roma covered nsistent with th termination ous fluid, clot aterial. the a mesentery from d markedly th mesenteric ro al jejunum and rrow). non-her he right ovary i was smoothly lammation, or d a five x three or inflammati the cortex of motic hemorr emia and sugg al, transmural the mucosa w d tunica musc ne orad to the ry to adjacent mmation, hem d by smooth s a congenitall n of fibrosis a tted blood and associated m adjacent no hickened with oot. d ileum were rniated small b is visible in the y bordered by r fibroplasia, e x three cm f ion or trauma both adrenal rhages throug gestive of l venous infar was extensive cularis were e hernia had t small bowel morrhage, or erosa with m y formed rent and remodelin d ormal h bowel e and focal a at ghout rction ely ild t ng clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 56clinical theriogenology • volume 4 number 1 • march 2012 was also c hemorrha syndrome figure 2. d of the rent and uterine discussio the p be second from vario cystitis an rent forma parturient artery at m possibly r anomaly.1 the te had been b pregnancy this unrem hemorrha mesometr of the left the relatio lacked inf considered. t ge and coagu e). dorsal view of were smooth w e arteries (arrow on pathogenesis o dary to pelvic ous causes of nd peritonitis) ation. hemor t mares.14,15 h mid-gestation resulting in re 1,2,15 en-year-old n bred by live c y with unassis markable heal ge of the mes rial defect and t mesometrium on of this lesio flammation an the zona glom ulative necrosi f the uterus. a with no hemorr ws) were gross of mesometria trauma such f abdominal in ) which may r rrhage from u hematomas ca or parturition ent formation. non-pregnant m cover as a thre sted parturitio lth history, it i sometrium du d permanent r m and mesoco on to the mes nd fibrosis, a merulosa of th is, consistent 7.5 cm rent w rhage or fibros sly normal. al rent format as may be ass nflammation result in tears uterine or ova an form in the n. improper h .15 abdomina mare in this c eeand four-y on and no hist is possible th uring pregnanc rent formation olon suggests ometrial rent congenital de he adrenal cor with endotox was present in th sis. the uterine tion in the ma sociated with (i.e. perforati in the ligame arian vessels i e mesometriu healing may r al rents in gen case report ha year old, and tory of dystoc at the mare m cy and partur n in the right s a prior histor and prior pre efect is also c rtex was exte xic shock (wa he right mesom e body (arrowh are has not be h dystocia, pre ing or necrotiz ent and subse s a well-docu um due to rup result in focal neral may also ad no previous successfully cia or post-pa may have had rition, which m mesometrium ry of at least egnancies is p considered lik ensively disrup aterhouse-fri metrium (asteri head), uterine h een determine evious abdom zing enteritis equent healing umented comp pture of the m l necrosis of t o be a spontan s history of co produced a fo artum complic subclinical p may have resu m. indeed, the focal pelvic s purely specula kely in this ca pted by derichsen isk). the marg horns (open arr ed. defects m minal surgery, , colitis, metr g with perman plication in p iddle uterine the mesometr neous congen olic. the ma foal from each cations. desp elvic trauma ulted in a e gross adhes serositis, thou ative. as the se. gins rows) may or ritis, nent ostrium, nital are h pite or sion ugh rent clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201257 clinical theriogenology • volume 4 number 1 • march 2012 t literature colitis of 3 explorator through w made to th intestine a performed complicat with acute jejunum w intestine w defect wa recovered figure 3. p ischemic n extensive h arterioles ( to the animal sp rare, but w and 4-7% history of occur.17 d tomograph intestine, two case repo published in 36 hours dura ry standing la what was presu he mare’s prio appeared viab d. the mare r tion. in the 1 e colic of six were herniated was successfu s not closed o d from colic su photomicrogra necrosis of the i hemorrhage an (arrow). h&e, e authors’ kno ecies or in co well-described of all interna f uterine surge diagnosis of b hy scan, and surgical resec orts of mares w 1972 and 198 ation. the ma aparotomy.2 a umed to be a or breeding h ble and no res recovered afte 980 report, a hours duratio d in a rent in t ully reduced a or ablated in t urgery and de aph of incarcera intestinal wall. nd edema of the bar 500 µm. owledge, no c ompanion anim d in women, i al hernias.16-22 ery, salpingiti bowel herniat surgical corre ction and ana with small int 80. in the 197 are had sever at surgery, th congenital de history and the ection was re er surgery an four-year-old on.3 a ventral the right mes and resection this case, and elivered a nor ated jejunum. . the mucosa w e submucosa, a comparable re mals. interes in which it ac 2 in women m is, or endome tion in a meso ection can be stomosis if ne testinal hernia 72 report, a th e abdominal p he entire jejun efect in the le e rent could n equired. abla d was subseq d belgian mar l midline celi ometrium. s and anastomo gross feature rmal foal three there was tran was sloughed w and marked ven eports are ava tingly, small ccounts for < mesometrial r triosis, thoug ometrial rent performed by ecessary, and ation in a mes hree-year-old pain on rectal num and most eft mesometriu not be grossly ation of the le quently used f re in the eight iotomy was p imilar to the osis was not n es of the rent e months late nsmural venou with moderate nous dilation (a ailable regard bowel hernia 0.1% of all c rents are assoc gh congenital is based on cl y laparoscopi d ablation of th sometrial rent pony mare p l palpation an t of the ileum um, though n y visualized. t eft broad ligam for breeding w th month of g erformed and first case, the necessary. th were not prov er. us infarction, he inflammation asterisks) and ding this cond ation in a mes auses of intes ciated with m rents are also linical signs a ic reduction o he mesometri t are in the resented for a nd underwent m had herniate no reference w the herniated ment was without gestation pres d five meters o e herniated he mesometri vided. the m emorrhage, and (arrowhead) a congestion of dition in other sometrial rent stinal obstruct multiparity or o thought to and computer of herniated ial defect to acute ed was d ented of ial mare d and r large t is tion a rized clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 58clinical theriogenology • volume 4 number 1 • march 2012 prevent recurrence. ablation is achieved either by suturing the defect closed or by surgically incising the mesometrium to connect the defect with the lateral mesometrial fold. in humans, mesometrium defects are classified into three types based on location in reference to the round ligament.23 small intestinal herniation in a mesometrial rent should be considered as a differential diagnosis in maiden mares and mares that have previously foaled or are currently pregnant which present with signs of acute abdominal pain and small bowel entrapment. the time elapsed between presentation of initial signs and surgical intervention, as well as extent of small bowel incarceration likely determine the outcome, though at least some surgical cases had long-term favorable outcomes.2,3 learning points • herniation of intestine through rents in the mesometrium is an important differential for acute colic in maiden mares and mares that have previously foaled or are currently pregnant. • mesometrial rents may occur congenitally in the mare, and post-parturient trauma or abdominal inflammation may also be a risk factor, as is often the case in women. • meticulous examination of the mesometrium should be considered in post-parturient mares to identify possible mesometrial lesions that could result in future rent formation. • intestinal herniation through a mesometrial rent should be a differential diagnosis for a mare presenting with acute colic even if the previous reproductive history is unremarkable. references 1. arnold ce, payne m, thompson ja, et al: periparturient hemorrhage in mares: 73 cases (1998-2005). j am vet med assoc 2008;232:1345-1351. 2. lamothe p, giguere g, marcoux m: hernia of the broad ligament of the uterus in a mare. mod vet pract 1972;53:49. 3. becht jl, mcilwraith cw: jejunal displacement through the mesometrium in a pregnant mare. j am vet med assoc 1980;177:436. 4. tennant b, wheat, jd, meagher, dm: observations on the causes and incidence of acute intestinal obstruction in the horse. proc annu conv am assoc equine pract 1975. p. 426-438. 5. wheat jd: causes of colic and types requiring surgical intervention. j s afr vet med assoc 1975;46:95-98. 6. santschi em, juzwiak js, moll hd, et al: diaphragmatic hernia repair in three young horses. vet surg 1997;26:242245. 7. archer dc, proudman cj, pinchbeck g, et al: entrapment of the small intestine in the epiploic foramen in horses: a retrospective analysis of 71 cases recorded between 1991 and 2001. vet rec 2004;155:793-797. 8. gayle jm, blikslager at, bowman kf: mesenteric rents as a source of small intestinal strangulation in horses: 15 cases (1990-1997). j am vet med assoc 2000;216:1446-1449. 9. gayle jm, macharg ma, smallwood je: strangulating obstruction caused by intestinal herniation through the proximal aspect of the cecocolic fold in 9 horses. vet surg 2001;30:40-43. 10. voermans m, butler cm, van der velden ma, et al: incarcerated umbilical hernia in the horse: a case with a review of the literature. tijdschr diergeneeskd 2004;129:142-149. 11. freeman de, koch db, boles cl: mesodiverticular bands as a cause of small intestinal strangulation and volvulus in the horse. j am vet med assoc 1979;175:1089-1094. 12. hawkins jf, schumacher js, mcclure sr, et al: small intestinal incarceration through the lateral ligament of the urinary bladder in a horse. j am vet med assoc 1993;202:89-90. 13. jenei tm, garcia-lopez jm, provost pj, et al: surgical management of small intestinal incarceration through the gastrosplenic ligament: 14 cases (1994-2006). j am vet med assoc 2007;231:1221-1224. 14. leblanc mm: common peripartum problems in the mare. equine vet sci 2008;28:709-715. 15. ueno t, nambo y, tajima y, et al: pathology of lethal peripartum broad ligament haematoma in 31 thoroughbred mares. equine vet j 2010;42:529-533. 16. fafet p, souiri m, ould said h, et al: internal hernia of the small intestine through a breach of the broad ligament, apropos of a case. review of the literature. j chir (paris) 1995;132:314-317. 17. guillem p, cordonnier c, bounoua f, et al: small bowel incarceration in a broad ligament defect. surg endosc 2003;17:161-162. 18. haku t, daidouji k, kawamura h, et al: internal herniation through a defect of the broad ligament of the uterus. abdom imaging 2004;29:161-163. 19. hiraiwa k, morozumi k, miyazaki h, et al: strangulated hernia through a defect of the broad ligament and mobile cecum: a case report. world j gastroenterol 2006;12:1479-1480. 20. ishihara h, terahara m, kigawa j, et al: strangulated herniation through a defect of the broad ligament of the uterus. gynecol obstet invest 1993;35:187-189. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201259 clinical theriogenology • volume 4 number 1 • march 2012 21. natalini g, trancanelli v, gerli p, et al: internal hernia through an orifice of the broad ligament of the uterus. minerva chir 1980;35:1359-1362. 22. nozoe t, anai h: incarceration of small bowel herniation through a defect of the broad ligament of the uterus: report of a case. surg today 2002;32:834-835. 23. cilley r, poterack k, lemmer j, et al: defects of the broad ligament of the uterus. am j gastroenterol 1986;81:389391. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 60clinical theriogenology • volume 4 number 1 • march 2012 2010: position statement on the use of megestrol acetate for population control in feral cats position statement on the use of megestrol acetate for population control in feral cats boards of directors of the society for theriogenology and the american college of theriogenologists montgomery, al the diplomates of the american college of theriogenologists (act) and members of the society for theriogenology (sft) support population control of feral cats, but do not endorse the use of synthetic progestogens for contraception in feral cat colonies. products containing megestrol acetate, the progestogen approved for estrus suppression in dogs under the brand name ovaban®, have been suggested as a method for estrus suppression in feral cat colonies. megestrol acetate has not been approved by the fda for use as a contraceptive in cats and no progestational product is approved for this use. progestagens, including megestrol acetate, may be available to veterinarians for treatment of individually owned cats, but only within the strict confines of a veterinarian-client-patient relationship, including a veterinary prescription. progestogen administration in cats can be associated with adverse health issues, including development of diabetes mellitus, mammary hypertrophy, mammary neoplasia, and acromegaly in both female and male cats. inadvertent administration to pregnant female cats may induce teratogenic changes in developing fetuses, and may prevent onset of normal labor. an oral bait route of administration cannot ensure accurate dosing and there is no assurance that animals other than the intended targets would not ingest the medication. the potential population control benefit is outweighed by the known adverse effects of progestogen administration in cats, and it is the position of the society and college that such use should be discouraged. 443 444 omniblank: 2012: breeding soundness examination of native omani bucks breeding soundness examination of native omani bucks mushtaq a. memon,a musab h. al-busaidi,b abdullah r. al-waelyc adepartment of veterinary clinical sciences, washington state university, pullman, wa; bdepartment of animal and veterinary sciences, qaboos university, al-khoud, muscat, sultanate of oman; canimal production research center, ministry of agriculture, wadi quriyat, sultanate of oman abstract goats are the most important livestock in the sultanate of oman. there are three major goat breeds in oman: dhofari, batina, and jabal akhdar. at present, the breeding bucks are selected on basis of physical appearance. taller and heavier bucks are preferred. no breeding soundness examination (bse) is performed before animals are used for breeding. the objectives of the study were to evaluate reproductive potential of omani bucks utilizing established criteria for bse. fifty bucks (19 dhofari, 18 batinah, 13 jabal akhdar), 1-5 years old were evaluated for breeding soundness, including physical examination, body weight (bw), scrotal circumference (sc), semen collection and evaluation. there was a significant difference in bw (p<0.001) and in sc (p<0.01) between dhofari, batinah and jabal akhdar. it is concluded that bse should be performed to evaluate omani bucks intended for breeding, and for semen preservation of genetically superior bucks; to facilitate artificial insemination for genetic improvement; and preservation of biodiversity in omani goats. keywords: omani bucks, breeding soundness examination, reproductive evaluation introduction goats are the most important livestock species in oman, considering the largest number (15,571,148) of the livestock population, followed by sheep (351,066), cattle (301,588), and camels (117,299) in sultanate of oman.1 there are three major distinct breeds of goats in oman: dhofari, batinah, and jabal akhdar which contribute 40, 55, and 5% of the total goat population, respectively.2 evaluation of the male for breeding soundness is an important part of a reproductive management of a herd. breeding soundness evaluation also is a first step in semen preservation for long-term preservation of genetic material and preservation of biodiversity. evaluation of the male for breeding soundness is based upon sc and motility and morphology of sperm. it has been shown that highly fertile rams settled ewes earlier in the breeding season, and resulted in production more twins. the quality of rams’ semen was significantly correlated with the twinning rate.3 since a large number of high quality spermatozoa are necessary for successful breeding in the sheep,4 sheep fertility is closely related to sperm production in the ram. bongso, et al5 reported significant correlation between buck sc with age and bw. the rate of male sexual development seemed to vary with goat breed, birth weight and nutrition. currently, in oman, the breeding bucks are selected only on basis of physical appearance. taller and heavier bucks are preferred. no bse is performed before bucks are used for breeding on the government farms or sold to private farm owners for improvement of their goat production. the society for theriogenology (sft) has recommended measurable bse criteria, including sc, sperm motility and sperm morphology in the bull and the ram.6,7 based upon sft criteria, bulls are declared satisfactory, questionable and unsatisfactory potential breeders. although limited information is available on various aspects of bse of different goat breeds5,8,9 but no reports are available on omani goat breeds the objectives of the current study was to evaluate reproductive potential of native omani bucks utilizing established criteria of bse, and to establish male bse values for breeding recommendations. to the best of our knowledge, this is the first report on buck bse of dhofari, batina, and jabal akhdar, the native omani goat breeds. materials and methods fifty bucks 1-5 years old were evaluated for breeding soundness. the breed distribution was 19 dhofari, 18 batinah, and 13 jabal akhdar. the bucks were weighed and the scrotum was examined for presence of two normal testes. scrotal circumference was measured using a standard scrotal metal-tape clinical theriogenology • volume 4 number 2 • june 2012153 (nasco, ft. atkinson, wi) at the widest mid-scrotal point. semen was collected using artificial vagina (av) or electroejaculator (ee; standard precision electronics, denver, co) as described earlier.10 semen volume, sperm mass activity, individual sperm motility and morphology were evaluated by established techniques.7,11 in summary, sperm mass activity was evaluated by placing a drop of undiluted semen on pre-warmed microscope slide and evaluated for the wave motion under a microscope. the mass activity was graded from 0 to 3 (0 – no activity, 1 – slow/irregular, 2 – moderate, 3 – fast wave motion). for individual sperm motility, a small portion of semen was diluted 1:10 (semen: normal saline). a drop of diluted semen was placed on pre-warmed microscope slide, covered with cover slip and evaluated under microscope as percentage of sperm moving in a forward progressive manner. sperm concentration in each ejaculate was calculated by utilizing dilution pipettes (platelett unopette®, b-d #5855, becton, dickinson and company, franklin lake, nj) and hemocytometer (exodus breeders corporation, york, pa) and the number of sperm/ml was calculated. for sperm morphology, a drop of semen was placed close to an end of a microscope slide and mixed with a drop of eosin-nigrosin stain (society for theriogenology, montgomery, al), and smear was made. the smear was air-dried, and sperm were evaluated under oil immersion lens of a microscope at 1000x. two hundred sperm were evaluated and abnormalities were classified according to barth and oko.12 the primary abnormalities were considered as sperm head abnormalities, proximal cytoplasmic droplet, abnormal mid-piece, and tightly coiled tails; while the secondary abnormalities included distal cytoplasmic droplet, bent tails, and loose heads. statistical analysis the original experimental design was a random 3×2 factorial design with three breeds of goats (dhofari, batinah and jabal akhdar) and two methods of collection (av and ee). data were analyzed using microsoft excel spreadsheets, sas (2000). mean statement was used to generate means, standard deviation, minimum and maximum values. glm procedures on the sas were used for analyses of variance to test for effects of breed, method and their interaction. data were then pooled after finding no effect of method or interaction and analyzed by analyses of variance. microsoft excel was used to plot relationship between bw and sc and polynomial equation and r2 were generated. results table 1 summarizes the mean (± sd) age, body weight, sc and semen evaluation parameters (semen volume; sperm mass activity, motility, concentration, and normal sperm). the semen parameters represent all semen samples collected from three breeds by av or by ee. out of 50 bucks, 16 (dhofari 9, batinah 4, jabal akhdar 3) were collected by av, and the remaining 35 ((dhofari 11, batinah 18, jabal akhdar 13) were collected by ee. the analysis from all three breeds was combined because there was no significant difference between semen parameters between breeds (table 2). there was a significant difference (p<0.001) in bw between the smaller dhofari and the larger batina and jabal akhdar breeds. the sc showed significant (p<0.01) difference between dhofari, and batina and jabal akhdar. the sc difference represents the bw of each breed (figure). among the semen parameters tested, only sperm motility was significantly (p<0.01) different between dhofari, and batina and jabal akhdar (table 2). discussion the positive relationship between bw and sc found in the current study is in agreement with earlier studies.13,14 bucks weighing 55-60 kg had significantly greater testicular size, semen volume and the total number of live sperm.14 in our study, the bw and sc of the bucks ranged from 22 to 77 kg and from 17 to 27 cm, respectively. in comparison with zaraibi breed bucks15 with smaller bw (25-27 kg) had similar sc (25-27cm) as noted in our study. a similar trend in bw (60-75 kg) and sc (24-27cm) was noted in saanen bucks under tropical conditions.16 in the study by ahmad, et al,16 sc in saanen bucks showed seasonal variation, with smallest sc (23.88 cm) during summer and the largest sc (26.54 cm) during autumn. seasonal affects on reproductive performance is also reported in western iran.17 our study was conducted during january in northern oman at 21° latitude.18 the goats raised in areas away clinical theriogenology • volume 4 number 2 • june 2012 154 from equator are more affected by season.19,20 the goat breeds living in latitudes lower than 300 do not present significant differences in reproductive activity as a consequence of seasonal influence.21 the seasonal effect, if any on reproductive characters of omani goats raised at 210 latitude is not known. in conclusion, it is recommended that bse should be utilized to evaluate omani bucks intended for breeding. breeding soundness evaluation would be a desirable first step for screening bucks for semen preservation of genetically superior bucks; to facilitate artificial insemination for genetic improvement; and preservation of biodiversity in omani goats. acknowledgements the study was conducted during first author’s fulbright fellowship at sultan qaboos university (squ), oman. the authors wish to express their gratitude to prof. osman mahgoub for helping in statistical analysis. assistance provided by prof. e. johnson and his staff for semen evaluation is greatly appreciated. valuable assistance provided by staff at squ agriculture experiment station; animal production research center, wade quriyat; squ department of animal & veterinary sciences; ministry of agriculture, oman is gratefully acknowledged. references 1. anonymous: agriculture census 2004 -2005. ministry of agriculture, sultanate of oman, 2005. 2. anonymous: feasibility study for the establishment of goat production project in 1988. final report. oman ministry of agriculture and fisheries and the arab company for livestock development, muscat, 1986. p. 547–607. 3. hulet cv: prediction of fertility in rams: factors affecting fertility and collection, testing, and evaluation of semen. vet med small anim clin 1977; 72: 1363-1367. 4. colas g, court m: production of spermatozoa, storage of semen and artificial insemination in the sheep. proc symp management of reproduction in sheep and goats, university of wisconsin, madison, 1977. p. 31-40. 5. bongso ta, jainudeen mr, zaharah as: relationship of scrotal circumference to age, body weight, and onset of spermatogenesis in goats. theriogenology 1982;18:513-524. 6. elmore rg, bierschwal cj, martin ce, et al: a summary of 1127 breeding soundness examinations in beef bulls. theriogenology. 1975;3:209-218. 7. ott rs, memon ma: breeding soundness examinations of rams and bucks: a review. theriogenology 1980;13:155160. 8. mittal jp, ghosh pk: characteristics of parbastar breed of goats from rajasthan desert. indian j anim sci 1985;55:673-678. 9. ali bh, mustafa ai: semen characteristics of nubian goats in the sudan. anim reprod sci 1986;12:63-68. 10. memon ma, bretzlaff kn, ott rs: comparison of semen collection techniques in goats. theriogenology 1986;26:823827. 11. memon ma, mickelsen dm, goyal ho: examination of the male reproductive tract and evaluation of potential breeding. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. philadelphia: wb saunders; 2007. p. 515-518. 12. barth ad, oko r: abnormal morphology of bovine spermatozoa. ames: iowa state university press; 1989. 13. al-ghalban am, tabbaa mj, kridli rt: factors affecting semen characteristics and scrotal circumference in damascus bucks. small rumin res 2004;53:141-149. 14. zamiri mj, heidari ah: reproductive characteristics of rayini male goats of kerman province in iran. anim reprod sci 2006;96:176-185. 15. barkawi ah, eldayed eh, ashour g, et al: seasonal changes in semen characteristics, hormonal profiles and testicular activity in zaraibi goats. small rumin res 2006;66:209-213. 16. ahmad mmm, makawi sa, gadir aa: reproductive performance of saanen bucks under tropical climate. small rumin res 1997;26:151-155. 17. talebi j, souri m, moghaddam a, et al: characteristics and seasonal variation in the semen of markhoz bucks in western iran. small rumin res 2009;85:8-22. 18. anonymous: oman latitude and longitude. maps of the world. http://www.mapsofworld.com/lat_long/; accessed january 22, 2012. 19. corteel jm: collection, processing and artificial insemination of goat semen. in: gall c, editor. goat production. london: academic press; 1981. p. 171-191. 20. tuli rk, holtz w: the effect of season on seminal characters in boer goat bucks in the northern temperate zone. in: recent advances in goat production. proc papers presented at the 5th int conf on goats, 1992; new delhi, abstract no. 1195. 21. chemineau p: sexual behavior and gonadal activity during the year in the tropical creole meat goat. i. female estrus behavior and ovarian activity. reprod nutr dev 1986;26:441-452. clinical theriogenology • volume 4 number 2 • june 2012155 table 1: accumulative values of breeding soundness examination of 50 omani native breed bucks variable mean ± sd minimum maximum age (days) 712.83±479.21 123 1,724 sc (cm) 22.84±2.21 17 27 bw (kg) 45.25±13.44 21.40 77.00 s. vol (ml) 1.12±0.24 0.50 1.50 s. ma (0-3) 2.31±0.87 0.0 3.0 s. mot (%) 75.60±10.53 50 90 s. conc (bil/ml) 1.62±0.78 0.1 3.7 n. sp (%) 97.26±1.83 90 99 sc= scrotal circumference, bw=body weight, s. vol. =semen volume, s. ma=sperm mass activity, s. mot=sperm motility, s. conc. =sperm concentration, n. sperm = normal sperm table 2: breeding soundness examination parameters (mean ± sd) distributed by breed of 50 native omani breeds bucks variable goat breeds breed effect dhofari batinah jabal akhdar bw (kg) 32.22±2.03 52.34±2.23 58.53±2.62 *** sc (cm) 21.53±0.49 24.01±0.57 23.80±0.66 ** s. vol (ml) 1.13±0.05 1.13±0.06 1.09±0.07 ns s. ma (0-3) 2.03±0.19 2.66±0.25 2.43±0.28 ns s. mot (%) 68.96±2.10 79.35±2.68 79.50±3.08 ** s. conc (bill/ml) 1.58±0.18 1.85±0.23 1.65±0.26 ns n. sp (%) 96.94±0.40 96.76±0.51 97.33±0.59 ns sc= scrotal circumference, bw=body weight, s. vol=semen volume, s. ma=sperm mass activity, s. mot=sperm motility, s. conc=sperm concentration, n. sperm=normal sperm means on the same line which are denoted with no or similar superscripts do not differ significantly (p>0.05). * : p<0.05; **: p<0.01; ***: p<0.001 clinical theriogenology • volume 4 number 2 • june 2012 156 figure: relationship between body weight and scrotal circumference in omani goats of various breeds y = 0.0003x2 + 0.0856x + 18.268 r2 = 0.5139 15 17 19 21 23 25 27 29 10 20 30 40 50 60 70 80 90 body weight (kg) s cr ot al c irc um fe re nc e (c m sc poly. (sc) clinical theriogenology • volume 4 number 2 • june 2012157 2012: survey of owners of dogs with prostate carcinoma for identification of risk factors   survey of owners of dogs with prostate carcinoma for identification of risk factors margaret v. root kustritz university of minnesota college of veterinary medicine, st paul, mn abstract fifty owners of dogs with prostate carcinoma were sent questionnaires to identify risk factors for development of disease. response rate was 68.1%. the population was not shown to be unique in regards to occurrence of neoplasia in related dogs, occurrence of other disease in affected dogs, age at castration, diet, activity level, housing, or exposure to tobacco smoke. keywords: prostate, neoplasia introduction prostatic carcinoma is an uncommon, high morbidity, high mortality tumor in dogs. risk factors other than intact status have not been identified in dogs. prostatic neoplasia in humans occurs as two distinct stages, a hormone-dependent phase followed by a more invasive, androgen-independent phase. in humans, risk factors for development of malignant prostatic neoplasia include age, genetics, and intact status. about one-third of men aged 50 years have some degree of neoplastic change in their prostate and over 90% of men aged 90 years or more have neoplastic change in their prostate.1 up to 10% of prostatic tumors in men are considered to have a hereditary component, with increased incidence in men with an affected father or brother.2,3 in dogs, breed risk was identified in several studies.4-6 however, prostatic neoplasia is not listed as a hereditary disorder in the canine inherited disorders database.7 in men, the risk due to intact status is increased in those who have never had significant androgen exposure, either because of prepuberal castration or congenital hypogonadism, and is decreased in those who have gone through puberty and have normal serum concentrations of testosterone and dihydrotestosterone.8 castration has been identified as a risk factor for prostatic neoplasia in older dogs.4-6,9-11 discussion on this topic at the society for theriogenology annual meeting in 2010 led to a consensus that a survey of owners of dogs with prostatic carcinoma might be one way to help us identify potential risk factors worthy of further study. materials and methods medical records were identified as previously described.12 surveys were sent by surface mail to the owners of 50 dogs having confirmed cases of prostatic neoplasia without accompanying prostatic disease. the owners had given their permission to be contacted questions that were asked are listed in the table. results three surveys were returned by the postal service. of the 47 successfully mailed, 32 were returned for a response rate of 68.1%. dogs with prostatic neoplasia were 10.2 ± 1.9 years of age at the time of admission and 26 breeds were represented. none of the dogs were intact at the time of diagnosis. at the time of the survey, two of the dogs were still alive and were receiving palliative therapy. responses to the questions are listed in the table. general comments about topics not included in the questionnaire were concerns about water source, exposure to flea/tick products, and possible association with dogs being inside for long periods of time without permitting them to urinate. discussion results of this survey suggest that the questions asked have not identified factors which might make these dogs a unique population. limited information was available to owners regarding possible heritability of this condition. because breed predisposition has been identified, veterinarians may wish to clinical theriogenology • volume 4 number 2 • june 2012149   counsel owners against continuing to use fresh or stored semen from males or to breed to bitches that have produced affected offspring. these efforts are hampered by late onset of the disease. the majority of the dogs in this study were castrated at three years of age or younger. this is in agreement with demographic data suggesting most dogs in the united states that are castrated undergo surgery at less than four years of age, with the greatest increase in number castrated between six and 12 months of age.13 in studies documenting castration as a risk factor for development of prostatic neoplasia, age at castration was not specifically correlated with either increased or decreased risk of disease. percentage of dogs that were castrated varied greatly among studies and many dogs underwent castration late in life as one component of treatment for prostate disease, clouding ability to draw firm conclusions. prostatic carcinoma in dogs is of ductal-urothelial origin; these are androgen-independent tissues.11,14,15 it may be that castration and subsequent removal of androgen and possibly non-androgen secretory products of the testes is a component of tumor initiation or progression, either as a promoter or via removal of inhibition. it has been shown that prostatic neoplasms from castrated dogs are more anaplastic than are those from intact dogs; frequency and pattern of metastases may or may not vary between these groups.5,9 at present, it can be stated that castration is not preventative against development of prostatic neoplasia and that lifelong exposure to androgens or non-androgen secretory products of the testes is not required for initiation or progression of prostatic neoplasia. most dogs in this study were fed commercial dry food and were moderately active. again, this agrees with the literature; most dogs in the united states are fed commercial dry food and are considered by their owners to be moderately to very active.16 the owners reported that their dogs were of normal weight. several studies have demonstrated that owners tend to rate their animals as normal weight even though their veterinarian deemed those animals to be overweight, with lack of agreement in up to 44.1% of cases.17,18 body condition scoring data were not available for all dogs in the electronic medical record in this study, and many different clinicians performed the body condition scoring, making it a less consistent measure. since castration is a risk factor for obesity, if the owner perception is correct, this is a population of castrated dogs of normal weight, which is unusual compared to the general population of castrated dogs.19,20 it is not clear how being of normal weight could be a risk factor for neoplasia. the lack of obesity in this population may be due to the owner-reported activity level of this population or to weight loss secondary to prostatic disease. one could argue that castrated dogs live longer than intact dogs so predisposition to neoplasia is only due to increased longevity of these animals. studies do not agree regarding longevity of castrated versus intact dogs, with lack of statistical significance often attributed to wide standard deviation in the two groups.21-23 mean age at time of death or euthanasia of castrated dogs is 9.9-10.8 years, while mean age at time of death or euthanasia of intact dogs is 6.2-10.9 years.21-23 mean age at time of diagnosis of prostatic neoplasia is about 10.0 years.5,10,11,24 finally, studies do not agree as to whether or not intact dogs develop prostatic neoplasia earlier or later in life than do castrated dogs, with one study supporting that hypothesis and other studies refuting it. 5,6,9,11 environmental concerns were not demonstrated to be risk factors in this study. there is little information about exposure to tobacco smoke or other environmental irritants as a cause of neoplasia in dogs.25 there is no information in the literature regarding exposure to substances in water that may be associated with neoplasia. thirty-six surveys were sent to addresses in suburbs of a large metropolitan area, suggesting most of the dogs would be drinking tested city water, not well water. reports of increased incidence of neoplasia due to exposure to flea and tick products are based on historical concerns about increased transitional cell carcinoma after exposure to flea and tick dips and shampoos.25 there are no reports of neoplasia associated with currently marketed flea and tick products.26 recent identification of prostatic neoplasia in a 2200-year-old human mummy suggests that industrial environmental exposures are not a likely sole cause of disease.27 hypotheses for pathogenesis of neoplasia secondary to induced urine retention include increased urine concentration and crystalluria leading to urinary tract infections, or direct induction of inflammation, and persistent exposure to toxins excreted in urine with decrease micturition frequency.25 there are documented associations between inflammation and neoplasia in the human literature.28 clinical theriogenology • volume 4 number 2 • june 2012 150   however, in human medicine, chronic prostatic inflammation is more commonly associated with benign disease than with malignant transformation of tissue.29 conclusion results of this study do not provide guidance as to likely risk factors associated with increased incidence of prostatic neoplasia in dogs. further work should perhaps be directed at identifying changes in prostate histomorphology and function secondary to the endocrine changes and subsequent prostatic atrophy associated with castration in dogs. references 1. garnick mb: the dilemmas of prostate cancer. sci am 1994;270:72-81. 2. isaacs wb, xu j, walsh pc: hereditary prostate cancer. in: chung lwk, issacs wb, simons jw, editors. prostate cancer: biology, genetics, and the new therapeutics. totowa (nj): humana press;2001. p. 13-28. 3. carter bs, beaty th, stewberg gd, et al: mendelian inheritance of familial prostate cancer. proc natl acad sci usa 1992;89:3367-3371. 4. teske e, naan ec, van dijk em, et al: canine prostate carcinoma: epidemiological evidence of an increased risk in castrated dogs. molec cell endocrinol 2002;197:251-255. 5. cornell kk, bostwick dg, cooley dm, et al: clinical and pathologic aspects of spontaneous prostatic carcinoma: a retrospective analysis of 76 cases. prostate 2000;45:173-183. 6. bryan jn, keeler mr, henry cj, et al: a population study of neutering status as a risk factor for canine prostate cancer. prostate 2007;67:1174-1181. 7. canine inherited disorders database, http://ic.upei.ca/cidd/, accessed march 20, 2012. 8. ross rk, reichardt jkv, ingles sa, et al: the genetic epidemiology of prostate cancer. in: chung lwk, issacs wb, simons jw, editors. prostate cancer: biology, genetics, and the new therapeutics. totowa (nj): humana press; 2001. p. 111-121. 9. bell fw, klausner js, hayden dw, et al: clinical and pathologic features of prostatic adenocarcinoma in sexually intact and castrated dogs: 31 cases (1970-1987). j am vet med assoc 1991;199:1623-1630. 10. obradovich j, walshaw r, goullaud e: the influence of castration on the development of prostatic carcinoma in the dog. j vet intern med 1987;1:1830187. 11. sorenmo ku, goldschmidt m, shofer f, et al: immunohistochemical characterization of canine prostatic carcinoma and correlation with castration status and castration time. vet comp oncol 2003;1:48-56. 12. root kustritz mv: value of specific history and physical examination findings and diagnostic test results for differentiation of benign from malignant prostate disease in dogs. clin therio 2012;4:49-54. 13. trevejo r, yang m, lund em: epidemiology of surgical castration of dogs and cats in the united states. j am vet med assoc 2011;238:898-904. 14. mahapokai w, xue y, vangarderen e, et al: cell kinetics and differentiation after hormonal-induced prostatic hyperplasia in the dog. prostate 2000;44:40-48. 15. leav i, schelling kh, adams jy, et al: rose of canine basal cells in prostatic post natal development, induction of hyperplasia, sex hormone-stimulated growth, and the ductal origin of carcinoma. prostate 2001;47:149-163. 16. slater mr, robinson le, zoran dl, et al: diet and exercise patterns in pet dogs. j am vet med assoc 1995;207:186190. 17. courcier ea, mellor dj, thomson rm, et al: a cross-sectional study of the prevalence and risk factors for owner misperception of canine body shape in first opinion practice in glasgow. prev vet med 2011;102:66-74. 18. white ga, hobson-west p, cobb k, et al: canine obesity: is there a difference between veterinarian and owner perception? j small anim prac 2011;52:622-626. 19. sloth c: practical management of obesity in dogs and cats. j small anim prac 1992;33:178-182. 20. crane sw: occurrence and management of obesity in companion animals. j small anim prac 1991;32:275-282. 21. moore ge, burkman kd, carter mn, et al: causes of death or reasons for euthanasia in military working dogs: 927 cases (1993-1996). j am vet med assoc 2001;219:209-214. 22. bronson rt. variation in age at death of dogs of different sexes and breeds. am j vet res 1982;43:2057-2059. 23. michell ar: longevity of british breeds of dogs and its relationships with sex, size, cardiovascular variables and disease. vet rec 1999;145:625-629. 24. krawiec dr, heflin d: study of prostatic disease in dogs: 177 cases (1981-1986). j am vet med assoc 1992;200:1119-1122. 25. kelsey jl, moore as, glickman lt: epidemiologic studies of risk factors for cancer in pet dogs. epidemiol rev 1998;20:204-217. 26. raghavan m, knapp dw, dawson mh, et al: topical flea and tick pesticides and the risk of transitional cell carcinoma of the urinary bladder in scottish terriers. j am vet med assoc 2004;225:389-394. 27. "cancer found in egyptian mummy" http://www.twincities.com/ci_19849430?iadid=search-www.twincities.comwww.twincities.com, accessed january 30, 2012. clinical theriogenology • volume 4 number 2 • june 2012151   28. morrison wb: inflammation and cancer: a comparative view. j vet intern med 2012;26:18-31. 29. karakiewicz pi, benayoun s, begin lr, et al: chronic inflammation is negatively associated with prostate cancer and high-grade prostatic intraepithelial neoplasia on needle biopsy. intl j clin prac 2007;61:425-430. table: responses to owner questionnaire regarding possible risk factors for prostate carcinoma in dogs question responses comments are you aware of any dogs related to [name of dog], male or female, who also suffered from cancer? yes = 3 no or unaware of any = 22 not answered = 7 1 female littermate with cardiac tumor, 1 male littermate with presumed splenic tumor, 1 halfbrother with oral melanoma was [name of dog] being treated for any other health conditions at the time of diagnosis of cancer? yes = 8 no = 19 not answered = 5 1 each with ruptured mitral valve, seizure disorder, hip dysplasia, mast cell tumor on tongue and bladder stones, crystalliuria and chronic uti, benign skin masses, oral osteosarcoma, endocrine disease and dermatitis at what age was your dog castrated? less than one year = 19 1-3 years = 8 3-6 years = 1 6-9 years = 1 more than 9 years = 0 unknown = 2 not answered = 1 exact ages known were: 3 months = 1 4-5 months = 1 6 months = 7 9 months = 1 24 months = 1 what diet was your dog fed for most of his life? commercial dry food = 34 commercial canned food = 3 homemade diet = 0 human food = 3 not answered = 1 fifteen different foods were listed. those most commonly fed were iams (n=8), science diet (n=5), and prescription diets from the veterinarian (n=3) what was your dog’s general body condition during most of this lifetime? underweight = 0 normal weight = 26 overweight = 5 not answered = 1 what was his general activity level? hunting dog = 2 very active house pet = 14 regular walks = 8 inactive house pet = 7 not answered = 1 was he housed with other animals? yes = 23 no = 8 not answered = 1 13 with at least one dog, 5 with at least one cat, 4 with at least one dog and cat, 1 with dogs and a bird where was [name of dog] most commonly housed? indoors = 28 outdoors in covered facility = 1 outdoors in open facility = 0 indoors with outside run = 2 not answered = 1 were tobacco products smoked in the household during his lifetime? yes = 2 no = 28 not sure (recently adopted) = 1 not answered = 1 clinical theriogenology • volume 4 number 2 • june 2012 152 2014: the association between sperm function and field fertility in bulls the association between sperm function and field fertility in bulls jacob c. thundathil, gayathri d. rajamanickam, john p. kastelic department of production animal health, faculty of veterinary medicine, university of calgary, calgary, ab, canada introduction a thorough evaluation of bulls to predict their fertility potential is critical for viable cattle production. the importance of fertility prediction is even more important when the bull is used for single-sire mating or artificial insemination (ai). traditional semen analysis may identify semen or bulls that are grossly abnormal, but cannot identify sub-fertile bulls that are producing apparently normal semen. it has been clearly documented that bulls classified as satisfactory differ in their field fertility, necessitating development of new approaches to precisely document potential variation in the fertility of these bulls. in that regard, various submicroscopic approaches such as computer-assisted sperm analysis (casa) and flow cytometry-based evaluation of sperm characteristics, are widely used. these improvements in semen evaluation have led to several studies correlating various end points to fertility of semen used for ai.1,2 however, the desired level of accuracy in fertility estimations based on in vitro assays has not been achieved, due to the inadequacy in fertility measurements and lack of suitable in vitro tests. number of females used for insemination and their reproductive health, number of sperm in the inseminate, and insufficient variations in fertility of males can all influence associations between in vitro tests and fertility. every sub-compartment of sperm must be intact and functional in order to fertilize an oocyte; furthermore, the association between fertility and almost any fertility assay depends on the efficacy of the assay to measure multiple sperm traits. for example, sperm must have normal motility, morphology and plasma membrane integrity. each ejaculate contains a heterogeneous population of sperm, with variations in the functional attributes needed for fertilization. unfortunately, most laboratory assays give a population mean for each attribute, which confounds the true number of fertile sperm in the population. in addition, assays are often prone to error due to judgement or bias. current methods, including genomics and proteomics, are still confined to the research community because of their sophisticated instrumentation and advanced bioinformatics tools. regardless, it is predicted that these “omics” methods will play a major role in future diagnostics. the present review describes the current laboratory methods available for semen analysis in bulls as an aid for predicting their fertility following ai. traditional breeding soundness evaluation fertility of a bull is predicted by breeding trials (breeding many normal, fertile females and assessing pregnancy rates) or conducting a breeding soundness evaluation (bse). since no single measurement or criterion reliably predicts fertility, several criteria are usually evaluated. the standards of the society for theriogenology (www.therio.org) are intended to assess the likelihood of a bull establishing pregnancy in >25 healthy, cycling females in a 65-70 d breeding season. the bse classifications are based on a physical evaluation and acceptable thresholds for testicular development, sperm motility, and normal sperm morphology. although libido is a major determinant of bull fertility, this aspect of fertility is not routinely evaluated during the bse. in addition, evaluation of bulls for venereal diseases is also not a common practice during bse. a standard bse includes assessment of size, muscling, body condition, and freedom from disease and physical defects (i.e. abnormal angularity of legs, hoof problems, etc.). the scrotum should have a distinct neck, testes should be freely moveable, similar in size, firm and resilient, with normal epididymides and spermatic cords. scrotal circumference (sc) is measured by forcing the testes to the bottom of the scrotum and using a flexible tape to apply moderate tension at the largest circumference. the sc is highly correlated with paired testis weight, which is correlated with daily sperm production and semen quality.3 sperm motility is estimated by examining semen on a clean, warm slide and is reduced by extreme temperatures and environmental contaminants. mass motility (affected by both individual sperm motility and sperm concentration) is detectable at low power, but progressive motility should be assessed 533 clinical theriogenology • volume 6, number 4 • december 2014 under medium power (~400 x); a cover slip is applied and concentrated samples are diluted with warm, fresh saline. sperm morphology should be evaluated under oil immersion. semen is diluted in eosin-nigrosin (smear) or 10% neutral buffered formalin (wet mount) and at least 100 sperm cells (up to 300 if there are multiple abnormalities) are evaluated. in general, fertility will be decreased with >30% morphologically abnormal sperm or >20% head defects.3 a bull that is healthy and sound, with an adequate sc, >70% morphologically normal sperm, and >30% progressive motility, is designated a satisfactory potential breeder.3 furthermore, a bull with temporary conditions (likely to resolve) is designated classification deferred; this typically includes recent puberty, an injury or lameness that is likely to resolve, or temporary testicular degeneration (e.g. due to hot weather). finally, a bull with undesirable heritable defects, small sc, debilitating injury or disease, or permanent testicular degeneration, is designated an unsatisfactory potential breeder. although bse can identify bulls that are producing grossly abnormal semen, this approach cannot identify variations in fertility among bulls that are classified as satisfactory potential breeders. consequently, the use of bulls that meet minimum standards in natural breeding or their semen for ai can result in pregnancy rates that differ by 20-25 %.4 therefore, it is important to develop fertility tests to detect this variation in fertility. the following sections of this manuscript are a review of recent advances in evaluation of submicroscopic characteristics of sperm that are apparently normal. sperm kinematic parameters computer assisted sperm analysis is the preferred system for objectively evaluating sperm motility in a commercial setting. with the advent of casa, it is possible to identify various sperm kinematic characteristics simultaneously that cannot be identified by subjective semen analysis. as sperm progress along a trajectory, the casa system calculates motility, velocity, linearity and lateral displacement of the sperm head (alh). several studies have highlighted the importance of total and progressive motility and its association to field fertility.5,6 farell et al7 also reported high correlation between several casa parameters (linearity, average path velocity, curvilinear velocity, total motility and beat cross frequency[bcf]) and fertility, as determined by their 59d non-return rate to first service. kasimanickam et al8 reported that alh and bcf in conjunction with either straight line velocity (vsl) or average path velocity (vap) were significantly correlated to fertility. we recently reported that alh was significantly higher in low(lf) versus high-fertility (hf) bulls.9 this finding suggested that sperm from hf bulls have higher vigor just after thawing in comparison to those from lf bulls. consistent with these results, significantly higher percentage of hyperactivated sperm were recorded in hf bulls. three types of sperm motility patterns have been described: 1) forward progressive motility; 2) transition phase to hyperactivated motility; and 3) hyperactivated motility. based on these criteria, we inferred that postthaw sperm from hf bulls represent a “transition phase” from forward progressive to hyperactivated motility. sperm from lf bulls at post-thaw had a lower alh and numerically higher linear motility representing the “forward progressive” motility pattern, which drives the sperm in a more-or-less straight line. “transition phase” is a motility pattern usually exhibited by physiologically normal sperm that are in progress to hyperactivation. these differences in motility between hf and lf bulls may reflect submicroscopic differences at the structural (plasma membrane) and/or functional level in the sperm among these bulls. based on these findings, we postulated that frozen-thawed sperm from hf bulls are more efficient in undergoing hyperactivation compared to lf bulls, influencing their ability to cross the barriers of the female reproductive tract and reach the site of fertilization. to test this hypothesis, we subjected post-thaw sperm from both lf and hf bulls to a swim-up procedure that provided a barrier between post-thaw semen and the swim-up medium and evaluated the concentration of post swim-up sperm and their motion characteristics under capacitating conditions. interestingly, based on casa analysis of post-swim-up sperm, alh in lf bulls was significantly lower than that of hf bulls, whereas linearity (lin), straightness (str),and wobble (wob) were significantly higher, suggesting that postswim-up sperm from hf bulls were in transition to hyperactivation. concentration of sperm recovered after swim-up (expressed as a percentage of viable sperm present in the post-thaw sample) and the 534clinical theriogenology • volume 6, number 4 • december 2014 proportion of viable sperm in post-swim-up sperm were significantly different between hf and lf bulls and positively correlated with fertility.9 although a significant correlation of sperm concentration after swim-up and fertility have been reported,10 our experimental approach provided an opportunity to normalize concentration of sperm recovered after swim-up for the concentration of viable sperm used for the swim-up procedure. based on low recovery of sperm in the post-swim-up preparations from lf bulls, we inferred that viable sperm from lf bulls were inherently compromised to pass through sodium hyaluronate, indirect evidence for their reduced ability to pass through the barriers of the reproductive tract and populate the site of fertilization. based on these results and observed differences in sperm kinematic parameters, we postulated that sperm from lf bulls have a reduced ability to undergo hyperactivation under appropriate physiological conditions, which affects their ability to pass through the female reproductive tract, and interact with the oocyte, thereby contributing to reduced fertility.11 sperm plasma membrane integrity and viability the integrity of the sperm plasma membrane is often linked to the viability of sperm; consequently, most sperm viability tests assess the intactness or leakiness of the cell plasma membrane.12 cell viability is often assessed after staining sperm with membrane-impermeable fluorescent dyes with affinity for the dna. viable cells are able to exclude these dyes and prevent the staining of the nucleus. some of the membrane-impermeant probes that have been successfully used to assess sperm viability include hoechst 33528, yopro-1, and propidium iodide (pi).13 another method to assess membrane integrity includes the use of membrane-permeant dna dyes such as sybr-14.14 these probes can enter the living cell by virtue of their amphipathic nature, but are immediately deacylated by intracellular esterases, thereby rendering the dye membrane-impermeable. thus, viable cells will have the entrapped sybr-14 whereas in dead cells, the dye easily leaks out due to the damaged membrane.13 recently, combinations of stains are available, which can simultaneously label the viable and non-viable cells. the live/dead® sperm viability kit (invitrogen, carlsbad, ca) employs both sybr-14 and propidium iodide, which is routinely used for evaluating viability of frozen-thawed semen. this kit is reliable and user friendly, as it does not involve any laborious processing steps that might affect cell viability. another added advantage is that egg-yolk particles in the extender will not interfere with the flow cytometer, as they will remain unlabelled for both the dyes used in the kit.15 sperm viability, evaluated by fluorescent labelling of sperm coupled with flow cytometry, had significant correlations between viability and fertility in the stallion,16 bull,17 and boar.18 we recently demonstrated that the percentage of viable sperm at post-thaw was higher in hf versus lf bulls.9 furthermore, there were a significantly lower percentage of the dead and moribund cells (stained with propidium iodide and sybr-14, indicating compromised viability) in hf vs lf bulls. in addition, the rate of conversion of live to moribund sperm was lower in hf compared to lf bulls. it is noteworthy that the proportion of dead and moribund sperm and also rate of conversion of live to moribund sperm were negatively correlated with field fertility.11 sperm mitochondria several fluorescent dyes such as rhodamine 123 (r123), mitotracker (mito), and jc-1 have been used to evaluate the functional status of sperm mitochondria. both r123 and mito dyes are transported into actively respiring mitochondria, which then fluoresces green. all actively functioning mitochondria stain green with r123 and mito and therefore one cannot distinguish sperm that exhibits different respiratory rates.14,19 more recently, jc-1 has been used to distinguish sperm with poor and highly functional mitochondria.19,20 this fluorescent dye stains green in normally functioning mitochondria but when the respiratory rate increases, the stain forms aggregates that fluoresce orange.20 therefore, jc-1 not only differentiates between functional and non-functional mitochondria, but also permits us to assess various levels of mitochondrial function. studies have highlighted the importance of functional mitochondria to sperm motility, irrespective of whether r12321,22 or jc-119,20 is used to evaluate mitochondrial function. 535 clinical theriogenology • volume 6, number 4 • december 2014 sperm dna damage the quality of the sperm dna transmitted during fertilization is essential to support embryo development and fertility. programmed cell death or apoptosis is a major factor that has been implicated in dna damage in sperm and is negatively associated with fertilization rate, implantation, and successful pregnancy23,24 and also related to the field fertility of bull semen.25 increased percentages of apoptotic sperm decrease bull fertility by decreasing the percentage of viable sperm.26,27 the structure of the mature sperm dna is extremely compact due to the presence of the protamines in the sperm chromatin and can display various abnormalities, such as damage to the dna in the form of singleor doublestranded breaks, protein defects which interfere with histone or protamine conversion and thereby affecting dna compaction, and defective tertiary chromatin configuration. all of these defects could impair nuclear decondensation and jeopardize embryonic development, since the oocyte would not be able to overcome these forms of damage.28 moreover, damaged paternal dna could be incorporated into the genome of the embryo, leading to errors in transcription or translation in embryo development, ultimately having a trans-generational effect.29 due to its importance, evaluation of dna integrity of the semen has increased over the past years. for example, a single-cell dna gel electrophoresis assay (comet) can distinguish between normally condensed sperm nuclei (minimal migration on an agarose gel) and more loosely packed dna (tailing of dna). mice with low expression of protamine 2 demonstrates tailing of dna in this assay, highlighting the dependence of dna condensation on protamines.30 chromomycin a3 (cma3) is a fluoroprobe, which binds to deprotaminated dna (at gc specific regions) in a loosely packed chromatin structure.31 lolis et al reported that low cma3 staining in human sperm correlated with higher in vitro fertilizing rates than samples with high cma3 staining. the second classical method to detect the intactness of the sperm dna is by taking advantage of the metachromatic properties of acridine orange (ao) in the sperm chromatin structure assay (scsa). in that regard, normal intact dna is fully resistant to acid denaturation (green fluorescent in double stranded dna), whereas damaged dna (red fluorescent in single stranded dna) is susceptible to denaturation, with the extent of damage detected using flow cytometry. several studies have reported a high correlation of the scsa tests with fertility in several species, including bull,32,33 human,34 stallion35 sperm; hf males had less chromatin denaturation compared to those with lf sperm samples. furthermore, dna fragmentation can also be assessed by using terminal deoxynucleotidyl transferasemediated dutp nick-end labeling (tunel) assays, which identify single stranded dna areas by labeling free 3-oh termini with modified nucleotide analogs. a combined tunel/pi procedure is now available, which takes advantage of the accuracy of tunel and differentiation of viable and dead sperm populations based on pi intensity.36 sperm acrosome integrity acrosome integrity is traditionally measured during frozen semen evaluation by evaluating a wet film of semen under the microscope, ideally with differential interference contrast (dic)-capabilities, which provides a clear view of the acrosomal ridge. however, fluorescent staining of acrosome coupled with flow cytometry can be used to objectively evaluate acrosomal integrity on a larger number of sperm, thereby improving reliability of this sperm function test. acrosome integrity is usually measured with fluorescently labeled plant lectins. for example, pisum sativum agglutinin (psa), a pea plant agglutinin that binds to α-mannose and α-galactose moieties of the acrosome matrix,37 whereas a lectin from peanut, arachis hypogaea agglutinin (pna) binds to β-galactose moieties of the acrosome.38,39 however, it is believed that pna has less non-specific binding to other areas of the sperm and thereby it is favored over psa.40 in both the cases, the lectins are not inherently fluorescent, but can be labeled with fluoroprobes (e.g. fitc). acrosome-intact sperm emit bright fluorescence throughout the acrosomal region, whereas partial, patchy or focal fluorescence from the acrosomal region indicates partial acrosome reaction. finally, an acrosome-reacted sperm, sperm will be fluorescence-free, except for a fluorescent band at the acrosome-postacrosomal region.41 536clinical theriogenology • volume 6, number 4 • december 2014 membrane fluidity and capacitation status the capacitation status of sperm is usually determined with the fluorescent antibiotic chlortetracycline (ctc), which binds to the plasma membrane in ca2+/mg2+ dependent manner.13 uncomplexed ctc (neutral) binds with free calcium in the intracellular compartments and becomes negatively charged and highly fluorescent. in non-capacitated sperm, an overall staining of the sperm head is apparent (f pattern). more prominent staining near the apical area and decreased staining in the posterior regions is evident in capacitated sperm (b pattern).42 loss of ctc staining from the sperm head indicates that the cell has undergone an acrosome reaction.43 this capacitation-associated b pattern has also been noticed in frozen-thawed sperm (cryo-capacitation like changes).44,45 in ai bulls, the percentage of non-capacitated sperm was significantly related to fertility.46,47 another method to assess capacitation is to stain the sperm with merocyanine 540 (m540). in capacitated sperm, a decrease in the packing order of the phospholipids was reported in the outer leaflet of the plasma membrane, which allowed the dye to intercalate into the hydrophobic core of the membrane.48 under capacitating conditions, membrane fluidity changes detected by m540 staining preceded the calcium influx measured by ctc staining.49 therefore, m540 and ctc could be used to monitor calcium dependent and independent events associated with capacitation. fluorescent phospholipid binding proteins such as annexin-v (binds phosphatidylserine) can also be used to monitor changes in the phospholipid asymmetry of the plasma membrane. in uncapacitated sperm, phosphatidylserine (ps) is confined to the cytoplasmic side of the phospholipid bilayer, whereas in capacitated sperm, it is exteriorized to the outer leaflet (owing to changes in membrane fluidity).50 annexin-v binding is considered more sensitive than other probes in detecting membrane changes associated with cryopreservation, since translocation of ps usually precedes the loss of membrane integrity.51 however, correlation between this assay and the ai bull semen fertility was variable.26,52 in vitro fertilization and related techniques sperm is also tested for its ability to bind to homologous oviductal epithelium in vitro. this binding prolonged their life and kept them in an uncapacitated state,53 thus providing insight into the fertilizing capabilities of a semen sample.54 however, only a marginal correlation was identified between the outcome of these tests and fertility.55 effective binding of the sperm to either homologous or heterologous zona pellucidae (zp) is yet another in vitro test to evaluate the fertilizing ability of sperm. zona binding tests use either whole or hemi-zp. fertility is also evaluated using zona-free hamster oocyte penetration tests; ability of a sperm sample to fertilize oocytes is deduced based on the ability of these sperm to penetrate zona-free oocytes and develop into normal male pronuclei.56 a positive correlation was reported between this test and sire fertility.57,58 it is noteworthy that in vitro fertilization (ivf) is the gold standard test to determine the relative fertility of semen sample across several domestic species, as it is the only method that closely resembles sperm-egg interaction in vivo during fertilization. furthermore, a statistically significant relationship between in vitro and in vivo fertility was documented in several studies.59-61 the ‘omics’ revolution the use of different “omics” approaches such as genomics (genes), transcriptomics (rna) and proteomics (proteins) have widened our knowledge, not only on the regulatory networks that govern gamete function, but also indicated that subtle differences in their expression (genes and proteins) could positively or negatively influence the fertility of the animal. high-throughput technologies such as dna and rna sequencing, dna microarray, and mass spectrometry, along with proper bioinformatics tools, may provide better cues for annotation of genes and proteins in the context of gamete biology. sperm genomics recent advances in the field of genome sequencing have provided great insight into genetic differences among sires. these genetic factors may contribute partly to variations in fertility among sires and hence bull fertility could be improved by genetic selection. for example, druet et al62 reported that 537 clinical theriogenology • volume 6, number 4 • december 2014 moderate (0.15-0.3) and high (close to 0.6) heritabilities exist for semen parameters such as volume and concentration, motility and percentage of abnormal sperm, respectively. the most abundant genomic variation, single nucleotide polymorphisms (snps), have been linked to economically important traits in the horse, pig and cattle63-66 information about specific snps related to fertility could facilitate earlier and accurate prediction of fertility and would speed genetic selection programs. khatib et al67 reported that snps in the fgf2 and stat5a gene are related to male fertility. feugang et al68 reported that polymorphisms in the integrin beta 5 gene (itb5b) may have a role in holstein bull fertility. functional studies on itg5b suggested that it might have a role in sperm-egg interaction. dai et al69 reported that snps in the follicle stimulating hormone beta subunit (fshb) were associated with lower semen quality, poor freezability and ultimately a lower non-return rate. thus, identifying individual genes that are responsible for differences in fertility among sires would give us opportunities to improve bull fertility through selective breeding. ultimately, this snp technology would pave the way for marker-assisted selection in choosing young bulls as sires for the next generation. sperm mrna profiles at the time of fertilization, sperm delivers coding and non-coding rna to the oocyte. studies have highlighted the presence and subcellular localization of a specific population of rnas in mature sperm, yet very little is known about its purpose and function. for example, kumar et al70 localized cmyc rna in the tail mid-piece, whereas there are other reports71 that the mrnas for the transcription factors nuclear factor-kappa b (nfκb), interferon consensus sequence binding protein (icsb), protein kinase c-jun n-terminal kinase 2 (jnk2), growth factor heparin-binding egf-like growth factor (hbegf) and epidermal growth factor receptor (erbb3) are present in the sperm head. it is noteworthy that recent developments in global gene expression analysis have allowed us to evaluate the functional diversity of rnas present in ejaculated sperm. various transcripts profiling platforms have identified a myriad of diverse transcripts.72-74 furthermore, semen quality was associated with diverse transcripts that are differentially expressed between bulls of different fertility status. in a study by kasimanickam et al,75 there was a strong association between the mrna abundance for adenylate kinase (ak) 1, integrin β (ib) 5, tissue inhibitor of metalloproteinases (timp) 2, lactate dehydrogenase c, outer dense fiber 2, phospholipase c zeta (plcζ) 1 and sire conception rates. lalancette et al74 reported that mitogenactivated protein kinase kinase, testis-specific serine/threonine protein kinase 3, lim/homeobox protein, and sh3 protein transcripts (along with several others) were differentially expressed in sperm from hf bulls. thus the heterogeneous rna content of the ejaculated sperm could be used as a fingerprint for the genomic analysis to assess semen quality, in terms of both spermatogenesis quality and fertility outcome.71,76-78 therefore, development of a reliable method for the routine isolation of rna from sperm will serve as an important step to develop novel non-invasive procedures to evaluate bull fertility. sperm proteomics by focusing on analysis of expression profiles, proteomics could lead to novel biomarkers that could be linked to bull fertility. in the past decade, mass spectrometry (ms) approaches have enabled global identification and quantification of proteins. in that regard, 2d-page and 2d-dige techniques coupled with ms have not only facilitated individual protein identification, but also their posttranslational modifications. technologies such as isobaric tags for relative and absolute quantitation (itraq) enable relative quantification of the number of peptides (and thus proteins) between two or more samples. as proteins define a cell phenotype, changes at the proteome level could lead to differences in phenotypes influencing economically important traits. for example, fertility-associated antigen (faa), a heparin binding protein was greater in the sperm membranes of beef bulls with greater fertility potential.79 high fertility bulls also had greater abundance of two seminal plasma proteins, osteopontin and prostaglandin-d-synthetase in their semen sample as compared to lf bulls80,81 another protein, clusterin was also in higher concentration in lf bulls compared to hf bulls.82 in addition, several studies have highlighted the differential expression of proteins in bulls with known fertility records.83-85 five proteins, enolase 1 (eno1), atp synthase, h+ transporting mitochondrial f1 complex 538clinical theriogenology • volume 6, number 4 • december 2014 (atp5b), apoptosis-stimulating of p53 protein 2 (aspp2), alpha-2-hs-glycoprotein (ashg), phospholipid hydroperoxide glutathione peroxidase (gpx4) were highly expressed in the hf bulls, whereas voltage dependent anion channel 2 (vdac 2), ropoporin-1, ubiquinol-cytochrome-c reductase complex core protein 2 (uqcrc2) were more represented in lf bulls.83 in a study by soggiu et al,85 alpha enolase was significantly down-regulated in the estimated relative conception rate (ercr)group, whereas two other proteins, isocitrate dehydrogenase, and triosephosphate isomerase were upregulated in ercr(lf) group in comparison to ercr+ (hf) group. d’amours et al84 reported that t-complex protein 1 subunits (cct5 and cct8), epididymal sperm-binding protein e12 (elspbp1), proteasome subunit α type 6 and binder of sperm 1 (bsp1) were more expressed in lf bulls than in hf bulls. adenylate kinase 1 (ak1) and phosphatidylethanolamine-binding protein (pebp1) were more expressed in the hf group compared to the lf group. a two-fold enrichment was noticed in epidermal growth factor (egf) and platelet-derived growth factor (pgdf) signaling pathways in hf versus lf sperm. in addition, peroxisome proliferated activator receptor (ppar) signaling, interleukin (il)-4 signaling, chemokine signaling, insulin growth factor (igf)-1 signaling was identified only in hf sperm.86 in the same study, two proteins, atp synthase, h+ transporting, mitochondrial f1 complex (atp5b) and cytochrome c oxidase subunit iii (cox3) were higher in the hf group in comparison to the lf group. shojaei et al87 demonstrated that a cohort of sperm proteins was differentially expressed in normal versus pyriform sperm derived from the same bulls. several proteins involved in sperm capacitation, sperm-egg interaction and sperm cytoskeletal structure were decreased in pyriform sperm, whereas proteins, which regulate antioxidant activity, apoptosis and metabolic activity were increased. contents of reactive oxygen species and ubiquitinated proteins were higher in pyriform sperm. we concluded that comparing normal versus morphologically abnormal sperm was a suitable experimental model for identifying important sperm functional proteins, which may serve as novel markers for fertility predictions. epigenetics the epigenome represents specific marks around the chromatin that can influence gene expression.88,89 these epigenomic marks are influenced by environmental conditions (e.g. toxicants, nutrition, temperature, etc.). although these environmental triggers do not alter the dna sequence, they induce chemical modifications to dna and influence dna compaction, which in turn controls gene expression. some of these epigenetics modifications are transient modulators of the chromatin structure, influencing access of transcription machinery to particular stretches of dna. for example, histones can be modified by acetylation, phosphorylation and methylation, thereby modifying the affinity of these proteins to dna. however, changes in methylation patterns (addition of methyl groups to the cytosine residues) of the dna are considered permanent epigenetic marks. therefore, evaluating changes in epigenetic marks and associated changes in phenotypes may have implications for identifying epigenetic contributions to regulation of fertility. summary traditional bse remains as the first-line of approach to identify bulls or their semen that are grossly abnormal. however, bulls classified as satisfactory based on a bse still differ in fertility. identification of these variations in fertility may allow us to eliminate bulls or their semen from breeding programs, or allow ai centers to make necessary adjustments in the semen doses to maximize sale of elite sires while preserving optimal fertility. since fertility is a trait influenced by a multitude of factors, no single fertility test is sufficient to precisely document fertility. therefore, a combination of fertility tests to specifically evaluate ability of sperm to pass through barriers of the female reproductive tract and reach the site of fertilization, undergo successful capacitation, bind with the oocyte, undergo an acrosome reaction, penetrate zona pellucidae, fuse with the oocyte plasma membrane, initiate zona and vitelline blocks, induce resumption of meiosis of the oocyte by releasing sperm factors, undergo dna decondensation and form a male pronucleus, fuse with the female pronucleus, initiate cleavage and effectively contribute to embryo-specific gene expression to support developmental competence of resulting embryos. in addition to existing sperm function assays, emerging genetic approaches may 539 clinical theriogenology • volume 6, number 4 • december 2014 enable us to improve fertility predictions and facilitate genetic screening of bulls for fertility at an early age. such advancements in fertility predictions will substantially improve the economic viability of the livestock industry. references 1. ostermeier gc, sargeant ga, yandell bs,et al: relationship of bull 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capacitation. mol hum reprod 2004;10:535-541. 79. bellin me, oyarzo jn, hawkins he, et al: fertility-associated antigen on bull sperm indicates fertility potential. j anim sci 1998;76:2032-2029. 80. moura aa, chapman da, killian gj: proteins of the accessory sex glands associated with the oocyte-penetrating capacity of cauda epididymal sperm from holstein bulls of documented fertility. mol reprod dev 2007;74:214-222. 81. gerena rl, irikura d, urade y, et al: identification of a fertility-associated protein in bull seminal plasma as lipocalintype prostaglandin d synthase. biol reprod 1998;58:826-833. 82. ibrahim nm, gilbert gr, loseth kj, et al: correlation between clusterin-positive spermatozoa determined by flow cytometry in bull semen and fertility. j androl 2000;21:887-894. 83. park yj, kwon ws, oh sa, et al: fertility-related proteomic profiling bull spermatozoa separated by percoll. j proteome res 2012;11:4162-4168. 84. d'amours o, frenette g, fortier m, et al: proteomic comparison of detergent-extracted sperm proteins from bulls with different fertility indexes. reproduction 2010;139:545-556. 85. soggiu a, piras c, hussein ha, et al: unravelling the bull fertility proteome. mol biosyst 2013;9:1188-1195. 86. peddinti d, nanduri b, kaya a, et al: comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility. bmc syst biol 2008;2:19. 87. shojaei saadi ha, van riemsdijk e, dance al, proteins associated with critical sperm functions and sperm head shape are differentially expressed in morphologically abnormal bovine sperm induced by scrotal insulation. j proteomics 2013;82:64-80. 88. rajender s, avery k, agarwal a: epigenetics, spermatogenesis and male infertility. mutat res 2011;727:62-71. 89. jenkins tg, carrell dt: the sperm epigenome and potential implications for the developing embryo. reproduction 2012;143:727-734. 542clinical theriogenology • volume 6, number 4 • december 2014 2010: population characteristics of 453 bitches undergoing 510 cesarean section procedures between 1999 and 2009-a retrospective study population characteristics of 453 bitches undergoing 510 cesarean section procedures between 1999 and 2009–a retrospective study* stephanie e. ayres,a philip g.a. thomasb apet emergency and specialist centre, 1103 dandenong rd, malvern east, vic, australia; bqueensland veterinary specialists, stafford heights, qld, australia abstract objective: to evaluate the characteristics of a population of bitches undergoing cesarean section and report the associated etiological diagnoses. study design: retrospective case series of 510 cases of cesarean section performed on 453 bitches between 1 july 1999 and 31 june 2009. methods: medical records were reviewed for information related to signalment, reproductive history, gestation, clinical features, ultrasonographic findings and treatment in bitches undergoing cesarean section. the number of pups was reviewed and deaths or fetal abnormalities recorded. an etiological diagnosis was sought for each case based on reported findings. results: median age was four years and brachycephalic breeds were overrepresented. it was common for non-maiden bitches to have previously undergone cesarean (87/152). forty-one bitches (9.1%) had anatomical vaginal abnormalities, 81 (17.9%) had uterine abnormalities and 19 (4.2%) had placental abnormalities. fetal heart rates were detected in 96.6% of 409 cases in which ultrasound was successfully performed. the most common induction agent was alfaxalone. the utrecht suture pattern was used in 418 cases (88.9%). few anesthetic or postoperative complications were reported. mean litter size was 5.6, with a mortality rate of 11.5%. the origin of dystocia was considered to be maternal in 63 cases (13.2%), fetal in 177 cases (37.0%) and related to litter size in 79 cases (16.5%). clinical relevance: these results suggest that brachycephalic and dolichocephalic breeds are more likely to require cesarean section than mesocephalic breeds. risk appears to be reduced in small breeds and increased in giant breeds compared with medium and large breeds. alfaxalone and propofol both confer minimal risk to the bitch when used for induction of anesthesia. the utrecht suture pattern is appropriate for closure of the hysterotomy. keywords: cesarean, canine dystocia, alfaxalone, utrecht suture pattern, fetal ultrasound introduction cesarean section is a commonly performed procedure both in general practice and in emergency centers. it is usually performed in cases of dystocia or fetal distress, when there is a history of difficulty with natural birth, or when difficulty is expected due to anatomic abnormalities, breed or other factors. however, breeders are increasingly electing to proceed with cesarean section in the absence of the aforementioned factors, based on estimation of parturition date using progesterone assays or at the first indication of parturition. the largest study of cases of cesarean section to have been performed to date involved 808 bitches from multiple veterinary hospitals throughout the united states and canada.1 this study investigated bitch signalment, perioperative management and mortality rates but did not assess historical or clinical features of these cases, and did not speculate as to the etiology of dystocia. the two other recent relatively large-scale reports on dystocic bitches included both medically and surgically treated cases.2,3 this study aims to provide a large-scale and up-to-date report on aspects of signalment, parity, reproductive history, gestation, reasons for presentation, clinical features, ultrasonographic findings, treatment methods and mortality rates in bitches undergoing cesarean section in northern brisbane, australia. previous articles in the scientific literature regarding cesarean section also have not addressed the seasonality of this procedure. this study examines the day, month and season of * preliminary data were presented at the acvm college science week in july 2009 and in an abbreviated report at the acvm college science week in july 2010. 451 accession, hypothesizing based on the personal experience of staff that the frequency of accession would be increased on sundays, when primary care veterinarians are often not open for business. the use of alfaxalone as an induction agent in the management of dystocia has been rarely reported or discussed in the literature. similarly, the utrecht suture pattern has previously not been investigated as a means of closing the hysterotomy incision in dogs. this study will provide an insight into the appropriateness of this anesthetic agent and suture pattern and may provide the clinician with further anesthetic and surgical alternatives when performing cesarean section. materials and methods the medical records database at queensland veterinary specialists (qvs) and pet emergency room (pet er) was searched for records of dogs examined between july 1999 and june 2009 for which an invoice item ‘cesarean section’ had been entered. this yielded 453 cases; a further 57 cases were recovered while searching through individual records. bitches were included in the study if they had undergone cesarean section within the specified dates and an adequate history was available. fifteen dogs were excluded because the medical records had not been satisfactorily completed. ultimately, 510 cases of cesarean section performed on 453 bitches were included; 46 bitches had more than one cesarean section performed within the study period. in this paper, when the word ‘cases’ is used then all procedures are considered individually, whereas the word ‘bitches’ denotes that each bitch is counted once only regardless of the number of times she presented. the breed, weight and age of the bitch at the time of cesarean section were recorded for each case. breeds were classified as small, medium, large or giant and as brachycephalic, mesocephalic or dolichocephalic (see appendix for classifications). in each case, parity and history of past cesarean sections (yes or no) were recorded where possible. the date and time of insemination were recorded when available and the interval from insemination to cesarean section was thus calculated. bitches that had presented more than once for cesarean section within the study period were counted only once for the purposes of calculating breed prevalence. to compare the study population to a control population of purebred bitches registered with the australian national kennel council (ankc) between 1999 and 2008, only 404 purebred bitches were included.4 in order to reduce the effect of bias caused by the presentation of multiple animals of the same breed by individual breeders, these calculations were repeated on a population of 307 purebred bitches in which only one bitch of any given breed was included for each breeder. all cases were treated on an individual basis for calculations involving age, reproductive history and gestation since these parameters were variable between presentations occurring on separate occasions. if medical management had been attempted prior to cesarean section, this was noted. the date of presentation was recorded and from this data the day, month and season of presentation were determined. the client’s reason for presentation and any concurrent disease or medications at the time of presentation were recorded. the stage and duration of labor, presence or absence of the ferguson reflex, degree of mammary development, presence or absence of a palpable pup or chorioallantoic sac in the vagina and the lowest measured fetal heart rate were recorded for each bitch. vaginal abnormalities or discharge were noted. for bitches that had presented more than once during the study period, abnormalities that were noted on more than one occasion were counted once only. no bitch had independent abnormalities noted on different occasions. a record was made of the anesthetic agents used, type and rate of intra-operative fluid administration and perioperative medications. surgical complications were noted. the suture pattern and material used to close the hysterotomy were recorded. any abnormalities in the appearance of the uterus and placentae at surgery were noted. the number of pups delivered alive and dead vaginally and by cesarean, number of immediate post-operative deaths, total litter size and fetal abnormalities were recorded. for the purposes of calculating the proportions of males and females, cases were included only if the number of males and females were reported as a proportion of the total number delivered by 452 cesarean. any complications for which bitches re-presented to pet er or qvs that arose as a result of pregnancy, lactation or surgery were recorded. for each case of dystocia, an attempt was made to determine etiology based on the available history. if a recorded fetal or maternal abnormality was considered likely to have caused dystocia, then the dystocia was attributed to that cause. if no physiological or anatomical abnormalities were detected, or if abnormalities were considered unlikely to have contributed to dystocia, then the dystocia was classified as idiopathic and further classified into one of four categories: (1) failure to commence second stage labor; (2) cessation of second stage labor prior to passage of all pups; (3) prolonged unproductive second stage labor or (4) apparently normal progression of labor with detection of fetal distress. data analysis/statistical methods the primary aim of this paper is to report the epidemiological characteristics of the studied population and not to examine the relationships between variables or to compare the population with a control population, which would be difficult to define. as a result, statistical analysis was seldom required. reported statistics were calculated using one of two types of analysis. for comparing observed frequencies with expected frequencies for multiple discrete variables, a chi square calculation was used. for comparing binomial variables in cohorts within the studied population, fisher’s exact test was used. all reported p-values are two-tailed. a value of <0.05 was considered statistically significant. results signalment and history the most over-represented breeds were staffordshire terrier (n = 48 cases, 10.6%), labrador retriever (n = 33, 7.3%), greyhound (n = 32, 7.1%), british bulldog (n = 27, 6.0%) and golden retriever (n = 15, 3.3%). of 404 purebred bitches, 176 (43.6%) were brachycephalic, 160 (39.6%) were mesocephalic and 78 (19.3%) were dolichocephalic. a statistically significant difference was found between the proportions of bitches of different head conformations in the purebred portion of the study population compared with the ankc-registered purebred population (p < 0.0001). brachycephalic (odds ratio [or] = 1.36) and dolichocephalic (or = 1.88) breeds were over-represented, whilst mesocephalic (or = 0.69) dogs were underrepresented. when the study population was altered so that multiple bitches of the same breed were excluded if they were presented by the same breeder, the proportions of bitches of different head conformations remained significantly different than those of the control population (p < 0.0001) and the same trends were noted. in 296 cases, a weight was recorded on the date of cesarean section. median weight was 21 kilograms (median, 23.1; mode, 20; range, 2.1 to 86.8 kg). of 404 purebred bitches, 124 (30.7%) were classified as small breed, 93 (23.0%) were medium, 157 (38.9%) were large and 30 (7.4%) were giant. when the proportions of bitches of different sizes were compared between the study population and the ankc-registered purebred population, a statistically significant difference was noted (p = 0.016). small breeds were under-represented (or = 0.85) whilst giant (or = 1.55) breeds were over-represented. when the study population was altered so that multiple bitches of the same breed were excluded if they were presented by the same breeder, statistical significance remained (p = 0.0439) and the same trends were noted. median age at presentation was 4 years (interquartile range [iqr], 2 to 5; mean, 4; mode, 2; range, 1 to 13 years). figure 1 demonstrates the distribution of ages. of 361 cases for which parity was recorded, 194 (53.7%) were maidens and 167 (46.3%) had past litters. of the 167 nonmaiden cases, the presence or absence of past cesareans was recorded for 152. a previous cesarean section had been performed on 87 (57.2%) of these cases; 65 (42.8%) had not required surgical intervention. methods of insemination for 274 bitches where this was recorded are summarized in table 1. of 154 bitches for which a single date of insemination was recorded, the mean interval from insemination to cesarean was 60.0 days (iqr, 59 to 61; median, 60; mode, 60; range, 49 to 70). the distribution of gestation periods is shown in figure 2. 453 in 53 cases, a temperature drop had been noted on one occasion in the lead-up to parturition. in two cases, two separate temperature drops had been recorded. in total, 55 cases (10.8%) had at least one recorded temperature drop. presentation occurred prior to the onset of second stage labor in 47 of these cases (85.5%). in 46 cases (9.0%), medical treatment had been provided prior to the decision to proceed with cesarean. the treatments provided are summarized in table 2. presentation the emergency service received 256 cases (50.2%), whilst 254 cases (49.8%) presented as clients of the reproduction service. a theriogenologist performed 317 cesareans (62.2%); 152 (29.8%) were performed by a pet er staff member and 41 (8.0%) were performed by a qvs surgical specialist or resident. bitches presented more commonly on a sunday (n = 90, 17.7%) or monday (n = 83, 16.3%) and in autumn (n = 147, 28.8%), but these trends were statistically insignificant (p = 0.1353 and p = 0.1767, respectively). by far the most common reason for the owner presenting their bitch was a perceived abnormality in the progression of labor–this was the situation in 372 cases (73.1%). this abnormality generally involved one of failure to progress to second stage labor (n = 115, 31.9%), prolonged labor without normal delivery of puppies (n = 173, 48.1%) or cessation of active labor (n = 69, 19.2%). in three cases (0.8%) the clinician felt that labor was apparently progressing normally but fetal distress or obstruction was detected. in 12 cases, there was insufficient information to determine the type of abnormality. a check-up prior to or during parturition was the reason for presentation in 69 (13.6%) and 35 cases (6.9%), respectively. in 20 of these cases, the owner had detected a clinical abnormality. elective cesarean had been planned in 29 cases (5.7%). in three cases (0.6%), undelivered puppies were detected during a post-partum check-up. in one case the reason for presentation was not recorded. of 491 cases for which the stage of labor was recorded, 268 (54.6%) were in second stage labor. estimates of duration of labor as reported by the owner were available for 220 cases and are summarized in table 3. of 223 cases in which second stage labor had not begun, 114 (51.1%) were suspected to be in first stage labor based on history and clinical findings. physical examination at least one vaginal abnormality was noted in 46 bitches (10.6%) at the time of physical examination. these abnormalities included stricture, stenosis or scarring of the vagina or vestibule (21 bitches, 4.6%), vaginal or vulvar hypoplasia (10 bitches, 2.2%), vaginal pain or evidence of trauma (6 bitches, 1.3%), a membranous or band anomaly (4 bitches, 0.9%) and vaginal hyperplasia (1 bitch, 0.2%). four bitches had had a vaginal anomaly corrected previously (0.9%). in 124 cases (24.3%), vaginal discharge was described by the owner or observed by the clinician, including green/black (n = 45, 8.8%), watery/clear (n = 41, 8.0%), bloody (n = 9, 1.8%), mucoid (n = 5, 1.0%), yellow (n = 2, 0.4%), brown (n = 2, 0.4%), serosanguineous (n = 1, 0.2%) or purulent/cloudy (n = 1, 0.2%) discharge. lochia were observed in six cases (1.2%). in 11 cases the discharge was not well-described. of 208 cases for which mammary development and milk production was recorded, 185 (88.9%) had good development and colostral milk in all glands, six (2.9%) had development of only some glands, eight (3.8%) had scant colostral milk, seven (3.4%) had no mammary development, one (0.5%) had mammary development but no milk and one (0.5%) was producing non-colostral milk. of the 203 bitches for which relevant information regarding the ferguson reflex was recorded, 27 (13.3%) displayed the reflex. a pup was palpable on digital vaginal examination in 147 cases (28.8%) and chorioallantois was palpable in 38 cases (7.5%). other clinical abnormalities included diarrhea (n = 10 cases); abdominal pain (n = 4); dermatopathies (n = 3); injected (n = 1), cyanotic (n = 2), pale (n = 2) or tacky (n = 2) mucous membranes; icterus (n = 1); pyrexia (n = 2); hematuria (n = 1); signs of hypovolemia (n = 2); dyspnea (n = 2); parasitic anemia (n = 2); cardiac murmur (n = 2) and intratracheal pus or uteroverdin suggesting aspiration (n = 2). dehydration, hypoglycemia, cardiac arrhythmia, 454 splenic mass, hepatopathy, pregnancy diabetes and ataxia each occurred in single cases. five cases had more than one abnormality. vomiting (n = 33, 6.5%) and ventral edema (n = 14, 2.7%) are known to occur in association with late pregnancy or parturition. it was noted that eight of the 14 cases with ventral edema were british bulldogs. no bitch presented on more than one occasion with edema. the increased incidence of ventral edema in british bulldogs compared with other breeds is highly statistically significant (p < 0.0001). it was also noted that the incidence of vomiting was increased in labrador retrievers (6/33 cases), and this association too was statistically significant (p = 0.0144) ultrasonographic examination non-zero fetal heart rates were detected in 395 (96.6%) of 409 cases in which ultrasound was successfully performed. in 306 of these cases (77.5%), the lowest recorded fetal heart rate was 180 or less. the average lowest recorded heart rate was 159.7 beats per minute (bpm; iqr, 148 to 180; median, 160; mode, 160; range, 40 – 300). in 50 cases (12.2%), at least one fetus with a heart rate of zero was detected, and in 101 cases (24.7%), there was at least one fetus with a normal heart rate (200 – 220 bpm). in ten cases (2.4%) both normal and zero heart rates were detected; 268 cases (65.5%) had neither zero nor normal heart rates detected. in 57 of 409 (13.9%) successful ultrasonographic examinations, additional abnormalities were detected. these included reduced (n = 33) or increased (n = 4) chorioallantoic fluid surrounding at least one fetus, fetal anasarca (n = 3), abnormal cardiac ventricular emptying (n = 1), abnormal or indistinct fetal echostructure (n = 3), abdominal free fluid (n = 3), transverse fetal presentation (n = 3), flocculent amniotic material (n = 3), abnormal uterine appearance (n = 2), a mass of tissue or organic matter within the uterus (n = 2), a discrepancy in fetal size (n = 2), excessive (n = 2) or reduced (n = 1) intraluminal uterine fluid, loss of definition of uterine contents (n = 1), inability to visualize the urinary bladder (n = 1) and the presence of partiallyaborted concepti (n = 1). in six cases, multiple ultrasonographic abnormalities were identified, including one case with three concurrent abnormalities. anesthetic the induction agent used was alfaxalone (alfaxan, cd-rtu, jurox, rutherford, nsw aust.) in 396 cases (78.3%), propofol (diprivan, astrazeneca, brussels, belgium) in 102 cases (20.2%), thiopentone (ilium thiopentone, troy laboratories, smithfield, nsw aust.) in five cases (1.0%) and mask isoflurane (isorrane, baxter healthcare pty. ltd., old toongabbie, nsw aust.) in three cases (0.6%). alfaxalone was first used in march 2002. propofol continued to be used frequently until april 2003, after which time it became routine to use alfaxalone. isoflurane was used to maintain anesthesia in 497 cases (98.0%). halothane (fluothane, ici pharmaceuticals, melbourne, vic aust.) was used on ten occasions between 1999 and 2003. few anesthetic complications were encountered. under a diazepam (pamlin, parnell laboratories, alexandria, nsw aust.)/propofol/isoflurane anesthetic one bitch suffered hypotension, tachypnea and tachycardia. under alfaxalone/isoflurane anesthetics one bitch suffered sudden recovery and one vomited post-operatively, but that bitch had also vomited pre-operatively. intravenous crystalloid solution was recorded to have been administered to 497 cases (97.5%) at rates varying from maintenance to shock rates. most commonly, fluids were administered at four times (n = 178, 35.8%) maintenance rates. two cases received glucose in saline. in 11 cases, fluid administration was not recorded. surgery of 470 cases in which suture pattern was recorded, a utrecht pattern was used in 418 (88.5%). the use of other patterns is summarized in table 4. ovariohysterectomy (ohe) was performed in six cases. the most commonly used suture materials were 2/0 or 3/0 polyglyconate (maxon, davis and geck, danbury, ct), accounting for 410 cases (99.0%), including five ohe’s. three cases (0.7%) were sutured using polydioxanone (pds ii, ethicon inc., somerville, nj), and catgut was used for one ohe (0.2%). in 96 cases, the suture material utilized was not recorded. 455 complications at surgery included five bitches with excessive hemorrhage (n = 5), milk contamination of the surgery site (n = 2 [1 bitch]), severe scarring of the linea alba (n = 1) and requirement for concurrent splenectomy (n = 1). four cases required emergency ohe for uterine rupture (n = 2) or middle uterine artery hemorrhage (n = 2). two cases with unilateral uterine ischemia required emergency hemiovariohysterectomy. a further two ohes were performed on an elective basis. twelve bitches received a packed red blood cell transfusion. no bitches died. in 502 cases, peri-operative medications were recorded. the most commonly administered medications were oxytocin (ilium syntocinon, troy laboratories, smithfield, nsw aust.; n = 424, 84.4%), meloxicam (ilium meloxicam, troy laboratories, smithfield, nsw aust.; n = 274, 54.6%), amoxicillin-clavulanic acid (clavulox, pfizer, mt. eden, auckland nz; n = 307, 61.2%), methadone (methone, parnell laboratories, alexandria, nsw aust.; n = 58, 11.6%) and diazepam (n = 52, 10.0%). outcome a total of 81 bitches (17.9%) had at least one uterine abnormality noted at the time of surgery. five of these bitches had abnormalities noted on more than one occasion. two simultaneous independent abnormalities were noted in 17 bitches, and three bitches had three independent abnormalities. the most common observed abnormality was uterine tearing or rupture (n = 21/453 bitches, 4.6%), followed by adhesions (n = 17, 3.8%), ischemia (n = 10, 2.2%), hematomata (n = 9, 2.0%), hyperemia or subserosal hemorrhage (n = 9), atonicity (n = 7, 1.5%), stenosis (n = 7), distension (n = 7), torsion (n = 7), cystic changes (n = 5, 1.1%), friability (n = 5), thinning of the uterine wall (n = 4, 0.9%), myometrial thickening (n = 4), failure to dilate (n = 4), stricture (n = 3, 0.7%), folding of a uterine horn (n = 3), myometrial hypercontraction (n = 2, 0.4%), endometritis (n = 1, 0.2%) and a uterine polyp (n = 1). nineteen (4.2%) bitches had placental abnormalities as follows: excessive ease of placental detachment (n = 6), placentitis (n = 5), remnants of aborted concepti (n = 4), excessive hemorrhage from placental sites (n = 1) and abnormal placentation with absent underlying endometrium and myometrium (n = 1). a total of 2736 pups were born from 489 litters for which relevant information was available. of these, 387 (14.1%) were born vaginally, 71 (18.3%) of which were dead on delivery, and 2350 (85.9%) were delivered by cesarean section, 157 (6.7%) of which were dead on delivery. overall, 8.4% of pups were dead on delivery. a further 86 pups failed to be resuscitated (n = 42, 48.8%) or were euthanatized for fetal deformities (n = 44, 51.2%) in the immediate post-operative period, giving an overall short-term puppy mortality rate of 11.5%. the mean litter size was 5.6 (iqr, 3 to 8; median, 5; mode, 5; range, 1 to 16). particularly small (one or two puppies) or large (eight or more puppies) litters were observed in 76 (14.9%) and 84 cases (16.5%), respectively. the number of males and females as a proportion of the total number delivered by cesarean was recorded for 272 litters. males accounted for 571 (47.7%) and females 626 (52.3%) of the puppies born from these litters. this deviation from the expected 1:1 ratio is not statistically significant (p = 0.1117). a total of 29 bitches gave birth to 38 pups with cleft palate, and 17 bitches produced 41 pups with anasarca. a variety of other fetal abnormalities were observed, including eight fetuses with ventral hernias (born to eight bitches), seven with hydrocephalus (four bitches), five with limb deformities (five bitches), four with tail deformities (two bitches), four with oronasal deformities (two bitches), two with anencephaly (two bitches), two with facial deformities (two bitches), one with polydactyly, one with syndactyly, one with evisceration, one with an ulcerated dermal mass, one with anury and one with hindlimb amelia. seven fetuses had unspecified anatomic abnormalities (six bitches). three pups born to two bitches had no apparent placental attachment and five pups born to one bitch had brown mucoid material around a marginal hematomata in the chorioallantois. five mummified pups were delivered from five bitches. macerated pups were recovered from 23 bitches. fifteen bitches produced puppies with premature passage of meconium. fifteen bitches had immature puppies. 456 post-operative complications were rarely reported. the most common diagnoses were metritis (n = 7) two to 23 days post-operatively, mastitis (n = 9) three days to one month postoperatively and hypocalcaemia (n = 6) one to five days post-operatively. within ten days of surgery, four cases presented for wound dehiscence, two for herniation at the incision site, one for nasal discharge, one for ileus and one for laryngitis. two cases presented for subinvolution of placental sites nine and 11 weeks after surgery. four bitches presented for more than one complication. it should be noted that there are likely to have been other complications for which owners presented to their primary care veterinarian, and in these cases there was no record of these complications having occurred. etiology of the 510 cases, 479 (93.9%) were considered to be in dystocia. of 31 cases that were not, 25 underwent elective cesarean procedures, four were aborting and two underwent cesarean prior to the commencement of parturition–one because abnormal fluid accumulation and an undefined mass were observed on ultrasound and one because of abnormal ‘nasty’ vaginal discharge. dystocia was considered to be of maternal origin in 63 cases (13.2%), of fetal origin in 177 cases (37.4%), and a result of a particularly small (n = 30, 20 of which were singletons) or large (n = 49) litter size in 79 cases (16.7%). in 160 cases (33.0%), the cause of dystocia was not obvious and classification was instead based on the criterion that was used to diagnose dystocia. the specific causes of dystocia are reported in table 5. thirty-one bitches underwent elective cesarean, 29 of which presented with this in mind. reasons for proceeding with surgery included previous dystocia (n = 5, 16.1%), breed risk (n = 5, 16.1%), singleton litter (n = 4, 12.9%), vaginal abnormality (n = 2, 6.5%), passage of a dead puppy (n = 1, 3.2%), a splenic mass requiring laparotomy (n = 1, 3.2%) and nulliparity in an old bitch (n = 1, 3.2%). the reason was not known in 11 bitches, but three of these bitches had large litters, three had previously undergone cesarean section, one had a singleton pregnancy and one was an at-risk breed. three (9.7%) bitches presented for elective cesarean with no obvious predisposition to dystocia or other indication for cesarean section. ten bitches (34.5%) presenting for elective cesarean had reduced fetal heart rates on ultrasound. discussion many studies have attempted to identify the factors influencing the risk of dystocia or requirement for cesarean section, and the associated outcomes. factors considered to be associated with increased risk of dystocia include immaturity, old age, anatomical or physiological abnormalities, small or large litter size, absolute or relative fetal oversize, fetal malposition or malpresentation and fetal anatomical abnormalities. it is to be hoped that these data allow clinicians to gain perspective with regard to the prevalence of various abnormalities in cases of dystocia, and hence be guided in the most important aspects of clinical examination. in particular, it is important to realize when dystocia has been caused by an irreversible maternal abnormality, so that future breeding can be discouraged and/or so that cesarean section can be planned for subsequent pregnancies. in the current study, three of the five commonest breeds–staffordshire terrier, labrador and golden retriever–were also among the five most popular breeds registered with the ankc between 1999 and 2008. greyhounds and british bulldogs were commonly presented by breeders as clients of the reproduction service. three breeders were responsible for presentation of 21 of the 27 british bulldogs, whilst six breeders presented 21 of the 32 greyhounds. this is likely to have contributed substantial bias and makes it difficult to judge the true prevalence of these breeds. retrospective studies of bitches in dystocia have, to date, failed to demonstrate a definitive relationship between breed size and occurrence of dystocia. in one study, mediumsized breeds were over-represented compared with all registered bitches and in another study, incidence of dystocia was increased in toy and miniature breeds.2,3 the comparison between this study population and the population of ankc-registered purebred dogs is not ideal, as it is includes both males and females throughout australia, whereas the study population is 457 exclusively female and located primarily in brisbane. bearing this in mind, it is still interesting to note that small breeds are significantly less prevalent and giant breeds more prevalent in the studied population compared with the 'control' population. this result contrasts with the commonly held belief that small size confers a greater risk of dystocia. part of the discrepancy may be a result of differing methods of classification with regard to size. it is widely accepted that brachycephalia is associated with increased risk of dystocia, and the current results lend support to this theory. more surprising is the finding that dolichocephalic breeds also appear to be significantly more likely to require cesarean section than mesocephalic breeds. these relationships remained statistically significant even after removing bitches of the same breed presented by the same breeder from the population under analysis to reduce bias. the result that 53.7% of bitches were maidens supports previous findings by gaudet2 and funkquist, et al.5 in which the percentages of maidens were reported at 67.0% and 51.0%, respectively, but disagrees with the 28.0% reported by walett darvelid and linde-forsberg.3 of the non-maidens, the percentage that had previously undergone cesarean (57.2%) was increased in the current study compared with previous reports. the apparently high incidence of dystocia in bitches that have previously experienced dystocia, even in idiopathic cases, suggests that there may be a genetic influence on risk of dystocia as has been previously proposed.6 in one study of cases of cesarean section where only 21.7% of non-maiden bitches had previously undergone cesarean, it was reported that radiography was a major diagnostic method, with no mention of ultrasonography.5 many of the bitches in the current study had previous cesareans performed at qvs or pet er. in this setting, the routine use of ultrasonography to detect fetal stress may have led to more frequent early surgical intervention rather than medical management, potentially accounting, at least in part, for the observed discrepancy. the combination of an attitude towards performing early cesarean section and client willingness to proceed with surgery, particularly those presenting to the reproduction service, may also explain the reduced percentage of cases to have received medical treatment prior to cesarean section in the current study. only 9.0% of cases had received prior medical treatment in the current study, compared with previous reports of 42.0% and 74.8%.2,3 in general practice, cost constraints may lead to the decision to try medical treatment before resorting to surgical means. the normal gestation period as measured from breeding date has been previously reported to be 57-72 days.7 all calculated gestation periods in the current study fell within the range of 58-70 days except for one case, in which the bitch was diagnosed with late term abortion. the mean interval from insemination to cesarean of 60.0 days is considerably shorter than the mean of 63.8 days reported by walett darvelid and linde-forsberg.3 these results are skewed by the high percentage of artificially inseminated bitches, as these procedures are generally performed one to several days later than an untimed natural mating. interestingly, though, even when artificially inseminated bitches are excluded the mean gestation period is shorter at 60.47 days. clients and veterinarians should be aware of this high variability in normal gestation length to avoid incorrectly diagnosing prematurity or failure to commence labor. it is worth noting that 54 bitches presented prior to the detectable onset of labor with no clinical abnormalities detected by the owner, and were subsequently determined to require cesarean section. this may prompt clinicians to encourage clients to attend pre-partum check-ups even if no perceived abnormalities are present, to allow early detection of dystocia and hence early intervention if required. for high-risk or valuable pregnancies, there may be a case for onceto twice-daily ultrasound checks or at-home monitoring in the form of tocodynamometry and doppler fetal heart checks in the lead-up to parturition. it is interesting to note that ventral edema occurred in late pregnancy in 29.6% of british bulldogs, a significantly higher proportion than the general study population. it may be worth warning clients of this; similarly, clients should be warned that vomiting is a common feature of first stage labor, possibly more so in labradors. the statistical significance of the increased frequency of vomiting observed in labradors may be a result of type ii error due to small sample size. in 45.4% of all cases, second stage labor had not commenced at the time of presentation and in 31.9% this was the owner’s reason for presentation. in these cases, since the first stage of labor is poorly defined and sometimes lengthy, the clinician without access to ultrasonography 458 may be forced to wait until 24 hours have elapsed or until second stage labor commences before being able to confidently diagnose dystocia and consider cesarean section. this is a concern as the fetuses are at risk of hypoxia and death during this time. with fetal ultrasound, the detection of low heart rates gives an early indication of fetal distress and allows more timely intervention. it has been shown that fetal heart rate is relatively stable during pregnancy at around 200-225 bpm but that there is a significant decrease at the time of parturition and that this is associated with fetal hypoxia.8,9 opinions vary as to how low the heart rate must be to warrant emergency intervention, but it is generally agreed that persistent heart rates <180 bpm indicate fetal distress.10,11 in one study, rapid intervention reduced fetal mortality from 9% to 3%.12 in this study, fetal heart rates of 180 or less were detected in 77.5% of cases where ultrasound was successfully performed. survival rates are higher than those reported by moon, et al. in a study where the varying approaches of a wide range of general practices would dictate inconsistent if not infrequent use of ultrasonography.1 survival rates of 92% immediately post-surgery and 87% at two hours post-surgery in the aforementioned study of 3,410 cesarean-derived puppies compare with respective survival rates of 93.3% and 89.7% in the current study. the difference between two-hour survival rates is statistically significant (p = 0.0022) and the difference between immediate survival rates (p = 0.0662) approaches statistical significance using fisher’s exact test, but the practical significance of either observation is difficult to quantify–even if the observed increase in survival rate is attributable to the use of fetal ultrasound, an increase in survival rate of 1-3% may not be incentive enough to use it routinely. further research is required in order to appreciate the advantage, if any, that fetal ultrasound confers in improving neonatal survival. a controlled study comparing populations evaluated with and without fetal ultrasound would be required to prove an association between survival rate and utilization of fetal ultrasound. of the few anesthetic complications that were encountered, there was no strong evidence to contraindicate any anesthetic protocol. the bitch that suffered poor pulse pressure, tachypnea and tachycardia under an anesthetic protocol of diazepam, propofol and isoflurane was one of 48 bitches in which an identical protocol was followed; none of the other bitches suffered complications. it is known that rapid recoveries can be a complication with either alfaxalone or propofol, particularly without premedication.13,14 it would seem, based on the results of the current study, that both alfaxolone and propofol are good choices of induction agent and that isoflurane is an appropriate agent for maintenance of anesthesia. overall, studies conducted recently have found low bitch mortality following cesarean section but this is the first study to report a zero mortality rate.1,3,15,16 the effect of anesthetic agents and perioperative medications on puppy morbidity and mortality was not examined in the current study. a double-layered closure using a continuous inverting suture pattern is reportedly preferred for closing the hysterotomy but, to the authors’ knowledge, no study has compared the use of different patterns and associated complications.17 in the current study, the utrecht pattern was used in 418 cases. polyglyconate was used in all but one of these cases. whilst this study did not involve direct follow-up of cases subsequent to discharge, medical records were searched for any subsequent visits indicating possible complications of surgery involving dehiscence of the uterine incision site and none were identified. there may be instances in which these complications may have been handled by a primary care veterinarian; however, 229 cases in which the utrecht pattern was used were handled by the reproduction referral service and it would be expected that these cases at least would have returned to qvs for re-evaluation if complications had arisen. no definite conclusions can be drawn without a controlled prospective study involving detailed follow-up, but these results suggest that the use of polyglyconate in a utrecht pattern is a good choice for closing the hysterotomy, and provides a basis for further research in this area. etiology was largely determined based on subjective judgment as to whether recorded abnormalities were likely to have caused dystocia. this method leaves obvious capacity for error. in a clinical setting, however, this information is applicable as the clinician’s judgment is the principal deciding factor in assigning etiology when extensive diagnostic tests are not indicated, practical or available. two studies have examined the etiology of dystocia in bitches with relatively large sample sizes.2,3 in these studies, dystocia was attributed to fetal causes in 25% and 40% of cases. 459 the current data support these statistics, with 37.4% of cases considered to be of fetal origin. most commonly the underlying cause was fetal malorientation in previous studies (15.4% and 15.8% of cases), whereas absolute fetal oversize was more common in the current study. in the remaining 75% and 60% of cases, respectively, dystocia was deemed to be of maternal origin. in both studies, physiological causes of dystocia were categorized using the terms ‘complete primary uterine inertia’ (48.9% and 7.4%), ‘partial primary uterine inertia’ (23.1% and 34.7%) and ‘secondary uterine inertia’ (0.0% and 12.6%). the high variability between the values obtained in these two studies may be at least in part due to the unclear definitions of these terms. to avoid such uncertainty in the current study, if no anatomical or physiological abnormality with reasonable capacity to cause dystocia was directly observed, the dystocia was classified as idiopathic. for this reason, comparisons cannot be made between this and previous studies with regard to overall prevalence of maternal dystocia. the percentage of cases attributed to relative fetal oversize agreed with previous reports, but the percentages attributed to uterine (6.0% compared with 2.1% and 1.1%) and vaginal abnormalities (2.4% compared with 1.1% and 0.5%) were increased. we expect uterine abnormalities to be detected more commonly since all uteri were examined at cesarean section. obviously, this is not the case in studies where some bitches are treated medically. in this study 17.9% of bitches had at least one reported uterine abnormality, compared with previously reported incidences of 4.8%, 1.6%, 12.1% and 16.2%.2,3,16,18 the large discrepancy observed here may demonstrate one advantage to proceeding with cesarean section over medical management, since it would appear that we may be missing uterine disease in cases where the uterus is not visualized. this failure of diagnosis may result in later complications. vaginal abnormalities may have been reported more commonly in the current study since many cases were examined by a reproduction specialist, who may receive referrals of cases with vaginal abnormalities and may also be more adept at detecting subtle abnormalities. furthermore, the common use of surgical insemination means that pregnancy may have been achieved in bitches with vaginal abnormalities capable of precluding natural conception. at least one vaginal abnormality was noted in 10.6% of cases, compared with 0.4% and 0.8% in previous studies.2,3 vaginal abnormalities commonly necessitate surgical intervention, so the lack of medically managed cases in the current study may also have influenced this observed increase in prevalence compared with previous reports. it should be noted as well that vaginal abnormalities were not emphasized as a measurable outcome in these earlier studies and hence some cases may not have been recorded; however, this is still a notable difference. these statistics are higher than one might expect and serve to highlight the importance of a thorough physical examination. there are obvious limitations to the epidemiological data yielded from this study. the use of an emergency and referral based population is unlikely to be completely representative of the populations of bitches presenting to primary care veterinary clinics and may lend itself to inaccuracies, particularly involving breed prevalence. naturally, there are many limitations inherent to any retrospective study including the absence of certain data points in many cases, and the absence of scheduled follow-up. it is difficult to quantify the bias attributable to these factors and, as such, no attempt has been made to do so. it follows then that these results should be interpreted with due caution. conclusion this study aims to describe a population of bitches undergoing cesarean section and hence make observations that may direct clinicians in their recommendations to clients with pregnant bitches and aid in the formulation of an appropriate clinic protocol for managing cases in which cesarean section may be considered to maximize success. it has been demonstrated that risk of requiring cesarean section appears to be increased in brachycephalic, dolichocephalic and giant breeds, and decreased in mesocephalic and small breeds. no concrete conclusions can be drawn from the study without a suitable control population, but it highlights some possible trends that may serve as the basis for future studies. on the basis of these results, a clinician should be able to confidently use alfaxalone or propofol in the anesthetic protocol for cesarean section since both have been shown here to confer minimal risk to the bitch when used for induction of anesthesia. the absence of significant post460 operative complications in these bitches provides compelling evidence that the utrecht pattern is appropriate for hysterotomy closure. one of the most notable differences between this study and that performed by moon, et al. is the routine use of ultrasonography. it is certainly possible that this difference may be at least in part responsible for the observed increase in pup survival in the current study, and it will be interesting to see the results of future studies that investigate this possibility. references 1. moon pf, erb hn, ludders jw, et al: perioperative management and mortality rates of dogs undergoing cesarean section in the united states and canada. j am vet med assoc, 1998;213:365-369. 2. gaudet da: retrospective study of 128 cases of canine dystocia. j am anim hosp assoc 1985;21:813-818. 3. walett darvelid a, linde-forsberg c: dystocia in the bitch: a retrospective study of 182 cases. j small anim pract 1994;35:402-407. 4. australian national kennel council. national animal registration analysis 1986 – 2009. available from: http://www.ankc.org.au/national-registration-statistics.aspx. 5. funkquist pme, nyman gc, lofgren amj et al: use of propofol-isoflurane as an anesthetic regimen for cesarean section in dogs. j am vet med assoc 1997;211:313-317. 6. jones de, joshua jo: reproductive clinical problems in the dog, 2nd ed. london: wright; 1988. p. 80-112. 7. root kustritz mv: small animal theriogenology. st. louis: butterworth heinemann; 2003. 8. verstegen j, silva ldm, onclin k, et al: echocardiographic study of heart rate in dog and cat fetuses in utero. j reprod fertil suppl 1993;47:175-180. 9. monheit ag, stone ml, abitbol mm: fetal heart rate and transcutaneous monitoring during experimentally induced hypoxia in the fetal dog. pediatr res 1998;23:548-552. 10. davidson ap: uterine monitoring during pregnancy. proc annu meet soc therio 1998; p. 123-125 11. smith fo: challenges in small animal parturition–timing elective and emergency cesarean sections. theriogenology 2007;68:348-353. 12. rondano vl, lien dh, concannon pw: radiographic evaluation of prenatal development in the beagle– correlation with time of breeding, lh release and parturition. vet radiol 1984;25:132-141. 13. ferre pj, pasloske k, whittem t et al: plasma pharmacokinetics of alfaxalone in dogs after an intravenous bolus of alfaxan-cd rtu. vet anaesth analg 2003;33:229-236. 14. nolan a, reid j: pharmacokinetics of propofol administered by infusion in dogs undergoing surgery. br j anaesth 1993;70:546-551 15. mitchell b: anaesthesia for cesarean section and factors influencing mortality rates of bitches and puppies. vet rec 1966;79:252-257. 16. navarro ja, friedman jr: a clinical evaluation of xylazine and ketamine hcl for cesarean section in the dog. vet med small anim clin 1975;70:1075-1079. 17. gaudet da, kitchell be: canine dystocia. compend contin educ pract vet 7:1406-1418, 1985. 18. robbins ma, mullen hs: en bloc ovariohysterectomy as a treatment for dystocia in dogs and cats. vet surg 1994;23:48-52 461 appendix classification of breeds by head type: brachycephalic: lowchen maltese terrier staffordshire terrier pug cavalier king charles spaniel bulldog (all varieties) mastiff (all varieties) chow chow rottweiler shih tzu pekingese chihuahua lhasa apso papillon boxer pomeranian bichon frise dogue de bordeaux boston terrier leonberger saint bernard mesocephalic: golden retriever labrador retriever dalmatian corgi west highland white terrier alaskan malamute cocker spaniel samoyed poodle silky terrier fox terrier jack russell terrier border collie cattle dog rhodesian ridgeback kelpie old english sheepdog weimaraner bearded collie schnauzer bull terrier viszla hamiltonstovare lakeland terrier husky scottish terrier shepherd (all varieties) soft wheaten terrier kangal tenterfield terrier miniature pinscher beagle chinese crested irish setter puli schipperke dalmatian basset hound dolichocephalic: greyhound italian greyhound rough coated collie great dane dobermann dachshund whippet afghan classification of breeds by size: small: bichon frise chihuahua chinese crested miniature dachshund italian greyhound pekingese miniature pinscher pomeranian toy poodle pug schipperke miniature schnauzer shih tzu cavalier king charles spaniel australian terrier boston terrier miniature fox terrier jack russell terrier lakeland terrier maltese terrier silky terrier west highland white terrier tenterfield terrier medium: afghan beagle french bulldog australian cattle dog bearded collie border collie corgi kelpie puli samoyed standard schnauzer siberian husky cocker spaniel scottish terrier soft wheaten terrier staffordshire terrier whippet large: alaskan malamute bulldog (all varieties except french) shepherd (all varieties) basset hound boxer chow chow rough coated collie dalmatian dobermann greyhound hamiltonstovare hungarian vizsla irish setter old english sheepdog standard poodle golden retriever labrador retriever rhodesian ridgeback rottweiler giant schnauzer bull terrier weimaraner giant: dogue de bordeaux great dane kangal leonberger mastiff (all varieties) saint bernard 462 figure 1: age distribution of bitches undergoing cesarean section 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 11 12 13 age (years) figure 2: distribution of gestation periods of bitches undergoing cesarean section 0 5 10 15 20 25 30 35 40 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 interval from insemination to cesarean section (days) number of bitches natural transvaginal surgical total 463 table 1: methods of insemination method of insemination n = natural mating 131 (47.8%) transvaginal insemination 83 (30.3%) surgical insemination 54 (19.7%) natural and transvaginal insemination 4 (1.5%) natural and surgical insemination 1 (0.4%) transvaginal and surgical insemination 1 (0.4%) table 2: medical treatments provided prior to surgery type of medical treatment n = oxytocin (once only) 16 oxytocin (more than once) 7 manipulation (successful) 13 manipulation (unsuccessful) 4 manipulation (successful), oxytocin 2 oxytocin, calcium 2 glucose 1 parturition induction agent 1 table 3: duration of labor at time of presentation, as reported by owner duration n = 0-3 hours 66 3-6 hours 82 6-9 hours 38 9-12 hours 11 12-18 hours 15 18-24 hours 1 24-36 hours 3 36-48 hours 3 >48 hours 1 table 4: suture patterns used for closing the hysterotomy pattern n = utrecht single-layer 416 utrecht double-layer 2 cushing 19 cushing/lembert 10 unspecified double-inverting 5 unspecified single-inverting 11 simple interrupted/continuous with inverting oversew 6 appositional 1 464 table 5: etiology of dystocia etiology n = maternal (n=63) vaginal/vestibular abnormalities 12 relative fetal oversize 5 uterine torsion 10 uterine rupture 2 uterine luminal narrowing 9 other uterine abnormalities 10 systemic disease 4 hydrops 3 excessive chorioallantoic fluid 2 premature placental separation 5 hypoglycemia 1 fetal (n=177) absolute fetal oversize 36 fetal malposition 8 fetal malpresentation 18 postural abnormality 3 deformity 9 fetal death 7 fetal dysmaturity 1 fetal obstruction, idiopathic 95 litter size (n=79) small (1 or 2) 30 large (8 or more) 49 idiopathic (n=160) failure to commence stage ii labor 89 premature cessation of stage ii labor 28 prolonged unproductive stage ii labor 35 labor apparently progressing normally 2 progression of labor not recorded 6 465 466 omniblank: 2010: guidelines for using the canine breeding soundness evaluation form* guidelines for using the canine breeding soundness evaluation form* original authors: b.j. purswell,a g.c. althouse,b m.v. root kustritzc avirginia-maryland regional college of veterinary medicine, virginia tech, blacksburg, va, usa; bschool of veterinary medicine, university of pennsylvania, kennett square, pa, usa; ccollege of veterinary medicine, university of minnesota, st. paul, mn, usa revised by: scott pretzer,d cheryl lopatee dabilene animal hospital, abilene, ks, usa; ereproductive revolutions, inc., aurora, or, usa introduction the canine breeding soundness examination (bse) is a minimum data base with which to evaluate the reproductive capability of a dog. interpretation of the results of parameters evaluated remains a challenge to the veterinarian, particularly in the area of infertility. for example, the threshold between fertility and infertility may change for an individual dog when there is an increase in numbers of breedings required over a short period of time depleting the number of normal sperm cells available. an example of a bse form is provided (figure 1). use of this form or one similar to it promotes consistency. the form provides an area for identifying the animal properly to ensure accuracy of medical records. the date of the examination in important in serial examinations of an individual; fertility parameters may change over time. since the spermatogenic cycle in the dog is approximately two months, subsequent examinations, if needed, are usually performed at two month intervals. the animal’s age is of particular importance due to the associated increased incidence of reproductive problems in the aged dog or the transient infertility in the prepubertal dog. breeding history of the dog should be obtained if available. many dogs are presented for examination after fertility problems have been encountered; this should be noted in the record as the reason for the evaluation. it is helpful to know when the last successful breeding took place in order to assess the chronological progression of any problems. brucella canis testing is advisable in all canine reproductive examinations. due to the problem of false positive results in many screening tests, it is helpful to know which test was used.1 pedigree of the animal may be potentially helpful when researching the possibility of hereditary problems or the early loss of fertility as a familial problem. a thorough physical examination should be performed as a part of the examination for breeding soundness in the dog. the physical condition, weight, and chronic health problems may adversely affect a given dog’s reproductive capabilities. obesity, skin disease, and endocrine disorders are just some examples of common conditions found in the dog that could adversely influence the dog’s fertility. the penis and prepuce should be examined for inflammation or abnormality. the spermatic cord and scrotum should be examined for any problems including those that might interfere with the thermoregulation of the testes. the prostate gland is the only accessory sex gland in the dog and is responsible for the volume of the canine ejaculate. because disorders of the canine prostate gland are common (benign hyperplasia with or without overlying infection, with associated signs of hematuria, hemospermia, etc.), digital examination of the * originally published by the society for theriogenology and the american college of theriogenologists as publication sa-c1 (8/92) in the therioigenology handbook. 51 prostate per rectum with concurrent abdominal palpation should be performed for determination of prostate size, symmetry and texture, and for assessment of pain. the epididymides should be palpated and any abnormalities noted. the tail of each epididymis should be located on the caudal pole of each horizontally positioned testis. the body and head of the epididymides can be palpated along the dorsal and anterior aspects of the testes. the testes should be examined and total scrotal width measured with calipers. although testicular size varies widely between breeds, size should be recorded for purposes of comparison at subsequent examinations. softening of the testes may indicate testicular degeneration, hypoplasia, atrophy, or abnormal development. hardness of the testes may indicate testicular fibrosis, neoplasia, inflammation, or the presence of a prosthesis. an appreciation of normal testicular consistency and practice are necessary to pick up subtle changes. semen collection as general rule, five days of sexual rest should be allowed prior to semen collection and evaluation.2 conversely, if an animal has not ejaculated within ten days, collecting a second ejaculate may be necessary to provide a reliable indication of the dog’s spermiogram. the adverse effects of sperm cell ageing may be seen in the first ejaculate obtained following prolonged sexual rest with increased numbers of secondary abnormalities such as detached heads and distal cytoplasmic droplets.3 the number of these abnormalities should be dramatically reduced in a second ejaculate if, indeed, they are due to aging. when presented for semen collection, the animal needs to acquaint himself with the collection area and the personnel involved so as to be as comfortable and relaxed as possible. he should be allowed the opportunity to urinate and defecate prior to the examination. in some dogs, urination immediately prior to collection will cause urine contamination of the ejaculate (urospermia), which may affect semen quality. the area used for semen collection should have good surface traction to prevent slipping. it is best to keep distractions to a minimum to maintain the dog’s concentration. it may even be necessary to exclude the owner; sometimes the owner of the dog can be a detrimental influence on the collection procedure. conversely, the owner’s presence may be beneficial to the collection procedure for certain dogs. dogs’ likes and dislikes should be noted in the record for future reference. the most commonly used method for collection of semen from the dog is digital manipulation. a director cone may or may not be used (figure 2).4,5 latex director cones, thoroughly rinsed with hot water and dried between uses, may be used multiple times. disposable polyethylene director cones also are available. digital manipulation with a director cone has the advantage of preventing loss of any part of the ejaculate during collection (figure 3). the disadvantage is increased contamination of the ejaculate (microbial and particulate) from the penile movements within the director cone. the director cone or collection tube should be vented to prevent a vacuum within the cone. this vacuum may impede semen flow or forcibly dislodge the collection tube during semen collection. any lubrication used in the director cone must not come in contact with the semen due to potential spermicidal activitiy.6 finally, it may be difficult to separate the three fractions if so desired using a director cone. digital manipulation without a director cone has the advantage of requiring the least amount of equipment. it is also a relatively simple procedure and contamination of the semen is held to a minimum. the primary disadvantage of this technique is the potential for losing the sperm-rich fraction due to the vigorous thrusting some dogs exhibit during ejaculation. care also must be taken to prevent damage to the tip of the penis during collection due to any thrusting activity by the dog against the collection tube. the collection tube can be either glass or plastic. in either method, graduated polypropylene plastic conical tubes have the advantage of immediate volume determination (figure 2). semen can be collected from a dog with or without the presence of a bitch. many male dogs can be collected without the presence of a bitch using digital manipulation alone. the 52 ejaculate collected without a bitch present is usually adequate to assess a dog’s breeding soundness. if the presence of a bitch is necessary, it is desirable but not necessary that she be in proestrus or estrus. the benefits of utilizing a bitch during semen collection include better responsiveness of the dog to collection and a higher total number of sperm in the ejaculate.7,8 if an estrual bitch is unavailable, frozen-thawed vaginal swabs from a disease-free estrual bitch can be used through their direct application to the perineal area of a tractable bitch. with either method, the dog should be allowed to sniff the bitch’s genital area before and during semen collection. if no teaser bitch is available, administration of 0.1 mg/kg of prostaglandin f2alpha 15 minutes prior to collection also has been shown to increase total number of spermatozoa in the ejaculate.8 prior to collection, the collector may wish to wipe the preputial orifice and penile tip with a moistened cotton pledget to reduce ejaculate contamination with preputial discharge. grasp the penis and prepuce behind (proximal to) the bulbus glandis (bulbus) in a tourniquet-like manner with a thumb and forefinger maintaining moderate but consistent digital pressure (figure 4). by doing this, the collector is trying to mimic the copulatory lock, or tie. using the other three available fingers, apply pulsating pressure directly on the bulbus. the dog usually responds readily to this manipulation by initiating the erection process and may begin thrusting movements. some dogs my require stimulation of the penis and bulbus through the prepuce prior to this simulated tie. once a partial erection is initiated, retract the prepuce behind the bulbus by placing the free hand at the point of attachment of the prepuce to the abdomen and directing the prepuce caudally to expose the bulbus if so desired (figure 5). a dog may experience pain or give an incomplete ejaculate if erection occurs while within the prepuce. conversely, some dogs maintain erection far longer than necessary if the prepuce is retracted behind the bulbus. either method is acceptable as long as the ejaculation is complete and detumescence and replacement of the penis into the prepuce is ensured before the dog is kenneled. during the thrusting movements, maximal erection is gained and the first of the three fractions of the ejaculate is emitted. the volume of this first fraction (f1) is variable. this fraction may be difficult to separate from the second, sperm-rich, fraction (f2). practically, f1 and f2 are often collected together to prevent losing the sperm-rich fraction. fraction 1 is clear to slightly cloudy. the dog will cease thrusting, almost abruptly, and will usually attempt to “tie” by lifting his leg over the arm of the collector, thus directing his penis backwards between his rear legs; not all dogs will attempt this. after a brief pause, the dog emits f2, which can be identified by its milky-white color. fraction 2 also varies in volume. after emitting f2, the dog begins to emit the third fraction (f3) originating from the prostate gland. fraction 3 can be distinguished from the sperm-rich fraction by the change in appearance from cloudy to clear. pulsations can be felt in the urethra and observed as contractions of the anal sphincter as f3 is ejaculated. the collector can stop collecting f3 once adequate dilution of the sperm-rich fraction has occurred to permit appropriate microscopic analysis. it is advisable to keep the exposed penis protected from damage by restraint of the dog until detumescence has occurred. this can be encouraged by the use of a water-soluble lubricant on the penis itself, which also helps to alleviate any drying of the penile mucosa. special attention should be afforded to long-haired breeds where hair may become entangled around the penis during detumescence and retraction of the penis into the prepuce. semen evaluation the semen must be protected from external insults during and after the collection process. temperature, chemical, and mechanical insults to the sperm cells should be avoided. to prevent temperature insults, all equipment coming into contact with the ejaculate should be at room temperature. attempts should be made not to shock the sperm cells with equipment and temperatures that are either too hot or too cold. chemical damage (latex gloves and director cones, water, detergent residues, bacteriostatic lubricants, etc.) should be avoided.9,10 finally, any 53 aspiration of the ejaculate into syringes or pipettes should be done slowly to avoid mechanical to the cells. normal values for semen evaluation in dogs are described in table 1.7,11-13 color fraction 1 should be clear to slightly cloudy. fraction 2 should be some variant of milkywhite and homogenous. if f2 is clear or only slightly cloudy, this may indicate decreased sperm numbers or complete azoospermia. yellow discoloration of f2 may indicate urine contamination or presence of a purulent exudate. red or brown discoloration of f2 indicates presence of fresh or hemolyzed blood; this may be indicative of either reproductive tract disease or trauma at collection. fraction 3 should be clear. red or brown color of f3 strongly suggests the presence of a disease process such as prostatic disease, trauma or injury, or neoplasia of the reproductive tract. volume measure the volume of the collected ejaculate before removing semen for anything else. this volume will be needed to calculate total number of spermatozoa in the ejaculate. the volume of f1 and f2 can be read directly from the calibrated tube into which it was collected or measured in a syringe after removal from another sort of collection vessel. the volumes vary greatly between individuals and among breeds. the volume of the separate f3 need not be recorded as this is even more variable depending on collection technique and duration, and because this fraction contains no spermatozoa. ph to measure ph, a drop of semen should be applied to the ph paper; do not immerse the paper into the semen sample. normal values range from 6.5-7.0. changes in ph may indicate that prostatic infection is present, and may guide the choice of antibiotic therapy.14 motility motility should be measured as soon as possible after collection. place a drop of semen on a slide and examine at 100-200x magnification. the evaluator may or may not wish to compress the drop with a cover slip; either technique is appropriate as long as the evaluator is consistent. a conventional light microscope can be used with a decreased light setting. assessment also can be performed, with excellent results, on a phase contrast microscope. very concentrated samples may need to be diluted to allow for visualization of individual spermatozoa. diluents used include the dog’s own prostatic fluid (f3), phosphate-buffered saline or 2.9% sodium citrate. motility parameters that should be assessed are total motility (% of spermatozoa that are moving), progressive motility (% of spermatozoa that are moving in a straight line), and speed of motility (slow/medium/fast). commonly accepted values are greater than 70% total motility, greater than 80% progressive motility for the sperm that are motile, and greater than 80% with a speed rating of “fast”. concentration concentration of spermatozoa is measured on the collected ejaculate containing f2. the wbc dilution system (bd unopette™ reservoir #365854, becton-dickinson, franklin lake, nj) may be used. draw semen up into the 20 microliter pipette and dispense it into the 2 ml diluent container according to kit instructions; the diluted solution can be used immediately. dispense the solution into both chambers of a hemacytometer. the number of sperm in the center 1 mm square (the square that fills the field using the 10x objective) equals the number of million sperm per milliliter in the ejaculate. for quality control, count both sides of the hemacytometer. if the two values are not within 10%, reload the hemacytometer and count again. 54 total sperm number of spermatozoa in the ejaculate the total number of sperm in the ejaculate is calculated as the concentration (millions of spermatozoa per ml) times the volume (ml per ejaculate). a commonly accepted normal value is greater than 300 million spermatozoa in the ejaculate. large breed dogs have a higher total number of spermatozoa per ejaculate than do small breed dogs due to the larger testicular mass. morphology morphology is most often assessed by staining the semen sample and observing the cells under 1000x magnification (oil immersion). morphology may be assessed without staining using a phase contrast microscope after fixing a sample with formol-buffered saline at a dilution of 1:9. different sample preparations cause artifacts that will be seen as morphologic abnormalities; percentage of morphologically normal spermatozoa should be fairly consistent regardless of method.15 the two most common stains used are a rapid wright’s giemsa stain (dif-quik™, baxter healthcare, miami, fl) and the eosin-nigrosin stain (morphology stain, lane manufacturing, denver, co) to stain with a rapid wright’s giemsa stain, place one drop of semen on a slide and smear out as for a blood smear. allow to air dry. immerse the slide into each of the three solutions in the same order as used for any type of cytology, allowing the slide to sit in each solution for five minutes. rinse the slide completely and let it air dry before evaluation under oil immersion. to stain with eosin-nigrosin, place one drop of semen and a similar-sized drop of stain on one end of a glass slide. gently mix the two together with a pusher slide, and draw out a thin film similar to a blood smear. allow to air dry as quickly as possible; placing the slide on a warming plate (37 °c) and blowing on the slide will hasten the drying process and help to prevent staining artifacts.16 spermatozoa stained with rapid wright’s giemsa stain appear purple on a clear background. normal spermatozoa stained with eosin-nigrosin stain appear white against a black or violet background. eosin-nigrosin stain is taken up by spermatozoa that have abnormal plasma membranes and so appear pink against a dark background. it might be assumed that such spermatozoa are non-viable but no significant correlation between “live-dead” ratio and fertility has been described in dogs. under oil-immersion, examine and count at least 100 spermatozoa. the number of normal spermatozoa in 100 is the percentage morphologically normal spermatozoa. total normal for the sample is calculated by multiplying the total number of spermatozoa in the ejaculate by percentage morphologically normal. commonly accepted values are greater than 80% morphologically normal spermatozoa and greater than 200 million total normal spermatozoa in an ejaculate. abnormal spermatozoa may be classified as having primary defects (those that occur during spermatogenesis, including defects in head shape, bent midpiece, persistent proximal cytoplasmic droplet, and doubling of any portion of the spermatozoon) or secondary defects (those that occur during epididymal maturation or slide preparation, including detached heads, persistent distal cytoplasmic droplets and bent tails). correlation between specific defects and fertility in dogs is poorly defined. cytology to assess cytology, place a drop of undiluted semen on a glass slide, drawn out as for a blood smear, and allowed to air dry. immerse the slide into the three solutions of a rapid wright’s giemsa stain as for any cytologic preparation. the presence of white blood cells (wbcs), red blood cells (rbcs), epithelial cells, and bacteria is noted with a designation of 0 to 4+ to indicate relative amounts (table 2). fraction 3 is evaluated separately; presence of rbcs or wbcs may indicate prostate disease. to evaluate a fairly large acellular sample, centrifuge the semen and process the pellet as 55 described above. a fair number of epithelial cells may be observed, especially after a period of sexual rest. the presence of occasional wbcs and bacteria is normal. if infection of the reproductive tract is suspected based on history and physical examination findings, culture of seminal fluid should be performed even if there does not appear to be a large number of wbcs in the sample.17 table 1. normal seminal characteristics for the dog.7,11-13 unfactionated ejaculate f1 f2 f3 color opaque to white clear milkywhite clear volume (ml) variable 0.253.0 0.4-3.0 1-30 ph 6.5-7.0 ----6.57.0 concentration (million/ml) 65 (10-200) ------ total number of sperm in ejaculate (million) >300 ------ total motility (%) >70 ------ progressive motility (%) >80 ------ velocity (speed) fast ------ normal morphology (%) >80 ------ table 2. quantification of cells other than spermatozoa in canine semen designation number of cells per high power field (hpf) 0 <1 1+ 1-3 2+ 4-6 3+ 7-10 4+ >10 references 1. johnson c, jacobs j, walker r: diagnosis and control of brucella canis in kennel situations. proc soc for therio 1991; p. 236-239. 2. foote rh: the influence of frequency of semen collection, fractionation of the ejaculate, and dilution rate on the survival of stored dog sperm. cornell vet 1964; 54:89-97. 3. christiansen ij: andrology of the normal male. in: christiansen ij, editor. reproduction in the dog and cat. philadelphia: baillere tindall; 1984. p. 99-107. 4. pineda mh: a simple method for collection of semen from dogs. canine pract 1977;4:14-18. 5. hopkins sm, evans le: artificial insemination. in: mcdonald le, pineda mh, editors. veterinary endocrinology and reproduction, 4th ed., philadelphia: lea and febiger; 1989. p. 366-367. 6. froman dp, amann rp: inhibition of motility of bovine, canine, and equine spermatozoa by artificial vagina lubricants. theriogenology 1983; 20:357-362. 7. boucher jh, foote rh, kirk rw: the evaluation of semen quality in the dog and the effects of frequency of ejaculation upon semen quality, libido, and depletion of sperm numbers. cornell vet 1958;48:67-86. 8. root kustritz mv, hess m: effect of administration of prostaglandin f2alpha or presence of an estrous teaser bitch on characteristics of the canine ejaculate. theriogenology 2007;67:255-258. 9. england gcw, allen e: factors affecting the viability of canine spermatozoa. i. potential influences during processing for artificial insemination. theriogenology 1992;37:363-371. 10. althouse gc, ko jch, hopkins sm, et al: effect of latex examination and vinyl examination gloves on canine spermatozoal motility. j am vet med assoc 1991;199:227-229. 56 11. harrop ae: the physiology of reproduction in the dog and bitch. in: harrop ae, editor. reproduction in the dog. baltimore: williams and wilkins co., 1960. p. 64-77. 12. bartlett dj: studies on dog semen. ii. biochemical characteristics. j reprod fertil 1962;3:190-205. 13. olar tt, amann rp, pickett bw: relationships among testicular size, daily production and output of spermatozoa, and extragonadal sperm reserves in the dog. biol reprod 1983;29:1114-1120. 14. olson pns: disorders of the canine prostate gland. proc soc for therio 1984. p. 46-60. 15. root mv, johnston sd, olson pn, et al: effect of stains and investigators on assessment of morphology of canine spermatozoa. j am anim hosp assoc 1998;34:348-352. 16. shaffer he, almquist jo: vital staining of bovine spermatozoa with a eosin-aniline blue staining mixture. j dairy sci 1948;3:677-678. 17. root kustritz mv, johnston sd, olson pn, et al: relationship between inflammatory cytology of canine seminal fluid and significant aerobic, anaerobic or mycoplasma cultures of seminal fluid: 95 cases (1987-2000). theriogenology 2005;64:1333-1339. figure 1. sample canine bse form. available at the society for theriogenology website. 57 figure 2. equipment used in the manual collection of canine semen: director cone (collecting funnel) and graduated polypropylene conical tubes. figure 3. manual collection of canine semen using a director cone; penis directed backwards. 58 figure 4. manual collection of canine semen without a director cone; bulbus glandis retained within the prepuce; penis directed backwards. figure 5. manual collection of canine semen without a director cone; prepuce retracted caudally to expose the bulbus glandis; penis directed backwards. 59 60 omniblank: 82 contact erin runcan runcan.1@osu.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11647, http://dx.doi.org/10.58292/ct.v17.11647 review report estrous cycle manipulation in dogs gail mcrae,a erin runcan,a aime johnsonb aveterinary clinical sciences, college of veterinary medicine, the ohio state university, columbus, oh, usa bdepartment of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa abstract domestic dogs provide a unique challenge to the veterinary practitioner to manipulate estrous cycle. due to an obligatory 120-day anestrus, dogs cycle much less frequently than other domestic species. this can pose a problem for owners and breeders wishing to produce pups at a certain part of the year or temporarily prevent pregnancy due to competition, health, or convenience. we describe the most common pharmacological methods available to practitioners for induction and suppression of the canine estrous cycle with a brief discussion on the efficacy of anecdotal nonpharmacologic methods. keywords: dogs, estrus suppression, estrus induction introduction the domestic dog estrous cycle has 4 stages: anestrus, proestrus, estrus, and diestrus. unlike other species, dogs cycle infrequently, typically only once or twice a year, which can be problematic for breeders and owners wishing to produce pups during the most fertile years. the reason for this prolonged period between estrous cycles is a prolonged anestrus that is necessary to complete uterine involution. in dogs, this process takes ~ 120 days to ensure a fertile cycle.1 anestrus is defined as the period between estrous cycles and can range from 5-14 months (average 7 months).1 if the dog is not cycling regularly, owners may wish to induce an estrous cycle sooner than what is physiologically normal for that individual. additionally, in certain instances, owners may wish to alter when a dog comes into estrus due to availability of a stud dog or convenience around a show or availability to assist with whelping and raising a litter. in some cases, anestrus may be abnormally prolonged. abnormal anestrus can be primary or secondary; primary anestrus is defined as having no estrus by 24 months of age whereas secondary anestrus is exhibiting no signs of estrus 10-18 months after an estrus.2,3 alternatively, owners may wish to suppress estrous behavior temporarily such as during performance competition or when pregnancy is undesirable. therefore, practitioners need to have knowledge of available protocols. there are many well-written reviews published in the literature that thoroughly outline all mechanisms studied to alter the period of estrus.1,3 to list all protocols is beyond the scope of this article, and instead we aim to illustrate the most clinically useful and relevant techniques currently available to veterinary practitioners. estrus induction to chemically induce an estrous cycle, the closer the manipulation mimics the natural hormonal events prior to proestrus, the better the outcome. largely, this has been attributed to increased frequency and concentrations of gonadotropin releasing hormone (gnrh) pulses, and subsequently luteinizing hormone (lh) and follicle stimulating hormone (fsh).2–5 the most common means to induce a fertile estrus include dopamine agonists, gnrh agonists, gonadotropins, and estrogens.1,3,6 dopamine agonists dopamine agonists inhibit prolactin and directly act on the hypothalamus to provide a gnrh stimulatory effect.3 few dopamine agonists have been studied, with the most clinically applicable being bromocriptine and cabergoline. estrus induction with bromocriptine is successful and relatively inexpensive but has been associated with vomiting when used as a sole medication.1,6,7 the recommended dose for oral bromocriptine is 0.3 mg for 3 days then an increased dose of 0.62.5 mg/kg (depending on dog’s size) continuing for 3-6 days after the onset of estrus. the primary benefit of this altered protocol is to reduce the side effect of vomiting.7 cabergoline works similar to bromocriptine and is less likely to induce mailto:runcan.1@osu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11647 citation: clinical theriogenology 2025, 17, 11647, http://dx.doi.org/10.58292/ct.v17.11647 83 vomiting; however, 23% of dogs that received cabergoline for at least 14 days also had reversible coat color changes.8 although not life-threatening, these coat changes can be problematic for owners who are continuing to show their dogs during treatment and should be communicated to clients as a possible unwanted side effect of the drug. another dopamine agonist, cabergoline, is most commonly used in practice. the protocol for oral cabergoline is 0.005 mg/kg (once daily until 1-8 days after proestrus has started)1,2,9; it is very important not to discontinue this medication too soon since ovulation failure can occur. return to estrus will vary depending on the stage of the cycle in which cabergoline treatment is started. on average, if used in early anestrus the treatment duration is 20 ± 2 days, if in mid anestrus 14 ± 3 days, and if in late anestrus 6 ± 1 days.9,10 in many cases, cabergoline can be expensive, especially when compounded, and therefore the potential for prolonged duration of treatment should be disclosed to clients. another benefit of using cabergoline is that when used in primary and secondary anestrus resulted in better pregnancy and whelping rates on the induced cycle.2 gnrh agonists gnrh agonists, used short term, can increase the amount of fsh and lh and has been used to induce estrus. there are a few published protocols to induce estrus that recommend intravenous or subcutaneous gnrh agonist (0.2-0.4 µg/kg every 90 minutes) to mimic the pulsatile fashion in dogs.1 however, because of the cost and labor involved in intravenous or subcutaneous treatment, using long-acting implants, mini-pups, or long-acting injections have also been studied. although effective, these methods are not currently available. deslorelin implants (ovuplant®, dechra, uk and 4.7 mg or 9.5 mg deslorelin acetate (suprelorin-f®, virbac animal health, westlake, usa) have been used. ovuplant implant is no longer available in the usa but is labeled for use in horses in other countries. suprelorin-f® is available in the usa but is only on label for use in ferrets. its use in other species is strictly prohibited in the usa; however, it is labeled for use in dogs in other countries and can be legally imported in special circumstances. these implants should be placed in the dog somewhere easily accessible (e.g. caudal to umbilical area or medial aspect of the thigh) as they need to be removed after ovulation.10 the primary benefit of the implant is that estrus induction and fertility were high as long as the implant was removed appropriately.4,5 implants removal at lh surge resulted in anovulatory cycles in some dogs11 due to this, it is now recommended to remove the implant after ovulation. most dogs come into estrus between 2-7 days after treatment regardless of the time during anestrus the implant was placed.4,5,11 seven out of 32 dogs failed to ovulate and their implants were removed after 15 days to prevent unintended effect of suppressing estrus.11 gonadotropins porcine fsh has been used to induce estrus; however, these protocols have not been successful, and anaphylaxis has been reported.1,12 other protocols used equine chorionic gonadotropin (ecg) and human chorionic gonadotropin (hcg) with varying degrees of success. used alone, ecg did not always induce spontaneous ovulation; therefore, ecg is often combined with hcg in these protocols.1 it has also been noted that hcg decreased progesterone expression leading to reduced pregnancy and ovulation rates.1,13 an injectable product containing 80 iu ecg and 40 iu hcg per ml is available in the usa and is labeled for swine (p.g. 600®, merck animal health, summit, nj). although highly effective in inducing estrus, this product resulted in a high degree of anovulation and therefore is not recommended due to the availability of more effective protocols.1 unfortunately, a commercial product containing only ecg is not currently available. additionally, a few protocols sequentially (50 iu/kg intramuscular ecg and 500 iu of hcg after 7 days) were used, resulting in 80% pregnancy rate.14 treatment with ecg (20 iu/kg ecg) and hcg (25 iu/kg) after 5 days resulted in pregnancy in 10% of dogs and neither whelped due to embryonic resorption.2 estrogens prior to lh pulses increase there is an increase in estradiol concentrations that occur naturally in dogs. this potentially primes the system for the increased pulses of lh. oral diethylstilbestrol (0.1-5 mg/kg once daily for 6-14 days) was used6 to induce estrus; ovulation and pregnancy rates were 46-100% and 31-100%, respectively. domestic dogs are very sensitive to estrogens; usage has been associated with increasing risk for pyometra, myelotoxicity, fever, petechiation, melena, hematuria, and dyspnea.15 due to these risks, and the availability of other protocols, estrogens are not currently recommended for estrus manipulation. nonpharmacological therapies anecdotally, breeders have tried many other nonpharmaceutical methods to induce estrus. it is widely known that cycling dogs when housed with others will often synchronize estrus in what is known as the ‘dormitory effect.’ it is believed that pheromones have a role in this synchronization that can shorten anestrus by as much as 30 days.6 this option however is not feasible for breeders with 1 dog or those that are actively competing and travelling. the additional stress of these events may also have an inhibitory effect on estrus due to increases in circulating cortisol concentrations. another anecdotal method that has been suggested is the addition of b-complex vitamins to the diet. b-complex vitamins such as biotin, riboflavin, and folic acid are essential for reproduction. in human studies, the addition of vitamin b12, folic acid, and folate have improved fertility, especially in women with polycystic ovary syndrome.16,17 high supplementation (50 mg/kg/day) of vitamin b6 was able to suppress prolactin in pseudopregnant dogs as effectively as cabergoline.18 therefore, it may be possible for vitamin b6 to have a similar effect in estrus induction. unfortunately, as no controlled studies have been performed in dogs, it is unknown if additional supplementation beyond a balanced diet is beneficial to induce cyclicity. increasing daylength and exposure to artificial lighting has also been suggested as a method to improve cyclicity in anestrous dogs. domestic dogs are nonseasonal breeders, as evident by the ability for them to become pregnant at any time of the year; however, it is still perpetuated that daylength has a role in their cycle. in free-roaming dogs, especially in tropical areas (e.g. india), temperature and season appear to have a http://dx.doi.org/10.58292/ct.v17.11647 84 citation: clinical theriogenology 2025, 17, 11647, http://dx.doi.org/10.58292/ct.v17.11647 role in number of offspring produced.19 however, this does not seem to be true when evaluating controlled populations of domestic dogs in kennel environments. there was no evidence in seasonal variation of estrus or litter size in a guide dog population in the uk.19 addition of melatonin implants did not have any effect on the interestrus interval of dogs, suggesting lack of involvement in regulation of the reproductive cycles.20 estrus suppression some pet owners may be reluctant to surgically sterilize their pets but still want to suppress the breeding behavior and the estrous cycle. alternatively, owners or trainers do not want competition dogs to have estrus during the competition season. an ideal estrus suppression method should be effective, safe, and reversible. progestogens synthetic progestogens have been widely used for years to prevent estrus.21 the exact mechanism is unknown, but it may be simulation of a diestrus phase. there are many formulations available, with variable affinity for the progesterone receptor. progestogens include megestrol acetate (ma), medroxyprogesterone acetate (mpa), and proligestone. oral ma can be given daily and is metabolized well. oral ma (2.2 mg/kg, once a day for 8 days) was given to dogs (n = 389) in early proestrus; estrus was suppressed in 92%.22 a dose of 0.55 mg/kg given to anestrous dogs for 32 days resulted in estrus suppression in 98%.22 there was a 0.8% incidence of pyometra but no other side effects were noted.22 oral ma given for 2-4 weeks during anestrus followed by 3-4 months without treatment and another 2-4 weeks of treatment has also been reported to be effective.1 treatment should begin ~ 1 month before the expected estrus. medroxyprogesterone acetate (mpa or depo-provera) is available as a long-acting injection or oral tablet. the injection should be given at a dose of 2-3 mg/kg with a maximum of 60 mg per animal.23 injections may be repeated every 3-5 months. proligestone is labeled for estrus suppression and is given subcutaneously at a dose of 10-30 mg/kg. chlormadinone acetate that is similar to ma is given orally at a dose of 2 mg/kg once weekly.24,25 progestogens listed have a variable return to estrus. for mpa, the average return to estrus is 2-9 months.26 average return to estrus for proligestone is 9-12 months but could be up to 2 years. although progestogen supplementation for the suppression of estrus is common, it has side effects that must be noted. side effects are dependent on the formulation used, dose given, the duration of treatment, and cycle stage of the female at treatment. the most common side effects are weight gain, lethargy, and increased appetite.22,27 hair loss, thinning of skin, or hair color change is possible due to the progesterone effects on the glucocorticoid receptor. mammary development and neoplasia are also reported to have a higher incidence after progestogens treatment.22 a more serious complication is the increased incidence of cystic endometrial hyperplasia resulting from prolonged exposure to progestogens; 45% of dogs treated with mpa for estrus suppression had uterine lesions at spay.28 progestogen treatment is not recommended in dogs that have underlaying uterine pathology, diabetes mellitus, mammary neoplasia, or liver disease.3 the best use of progestogens is during anestrus in young, healthy dogs for as short a duration as possible and at the lowest possible dose. it is recommended that a maximum of 2 estrous cycles be suppressed followed by a natural estrus.3 progestogens should not be used in prepubertal animals. if used during pregnancy, they can cause dystocia and fetal death at whelping by failing to allow cervical relaxation. androgens androgens (e.g. testosterone and mibolerone) are widely used for estrus suppression. androgens negatively feedback on the hypothalamus that downregulate gnrh and therefore fsh and lh; androgens do not have direct effects on the uterus like progestogens. these androgens can cause masculinization (e.g. mounting behavior) of the female, enlargement of the clitoris (figure 1), and increased aggression. vaginal discharge and vaginitis have also been described.29 anabolic steroid use is contraindicated in females with liver disease or breeds (e.g. bedlington terrier) that are prone to liver dysfunction. they should not be used in prepubertal or pregnant dogs. the dose of mibolerone is 16 μg/kg orally once a day. mibolerone should be started at least 30 days before the start of proestrus and should be continued no more than 24 months.30 it is important to note that mibolerone is only available via compounding pharmacies in the usa and is frequently unavailable. return to estrus occurs on average within 70 days but could be as long as 7 months. testosterone can be oral (25-50 mg/kg twice weekly) or injectable (testosterone propionate 110 mg once weekly).1 gnrh agonists suppression of gnrh leads to suppression of both lh and fsh that suppress cyclicity in the dog. gnrh agonists can be used off-label to prevent estrus. deslorelin is the most common gnrh agonist and is available in an implant form. figure 1. clitoral hypertrophy in a racing greyhound treated with testosterone for estrus suppression http://dx.doi.org/10.58292/ct.v17.11647 citation: clinical theriogenology 2025, 17, 11647, http://dx.doi.org/10.58292/ct.v17.11647 85 the constant release of deslorelin cause an initial stimulation followed by inhibitory desensitization of gnrh receptors in the pituitary. the initial stimulation usually leads to estrus in most dogs. however, once over, hormones reach baseline concentrations. the implant should be placed during anestrus to avoid estrogen stimulation in the face of high progesterone of diestrus leading to hormone imbalance and risk of pyometra.31 once the implant is placed, it will induce an initial estrus 5-8 days after placement, followed by prevention of estrus for 2-27 months.31 use in prepubertal dogs resulted in estrus suppression for up to 23 months.32 however, the length of estrus suppression is extremely variable among dogs. a clinically available implant is suprelorin® (4.7 mg deslorelin) authorized for treatment of adrenal disease in ferrets. as previously mentioned, this implant can be difficult to attain and is not available in some countries (e.g. usa). the most important issue with the use of the deslorelin implant is the initial flare-up. treatment with progestogens (megestrol acetate at 2 mg/kg) starting 1 week before the implant suppressed the initial estrus.33 side effects of deslorelin implants include ovarian cysts, prolonged estrus (15%), induced lactation (11%), and 2-6% other effects (e.g. vomiting, cystitis, behavioral changes, and allergic reactions). removal of the implant is implicated in these cases; therefore, most practitioners place it subcutaneously just cranial to the umbilicus or medial to the thigh. gnrh antagonists gnrh antagonists directly suppress gnrh and therefore lh and fsh. because it is a direct suppression, there is no initial flare-up. gnrh antagonists have been widely used in human medicine, but not much has been recently published in dogs. subcutaneous acycline to females in early proestrus caused estrus suppression for 3 weeks; there were no side effects noted.34 conclusion although many methods have been tried, only limited pharmacologic methods are commercially available, safe, and reliable to manipulate dog’s estrous cycle. for estrus induction, dopamine agonists and deslorelin implants are the best methods; however, availability and adverse effects still need to be considered. for estrus suppression, more options have been researched, however availability is often unreliable and dependent on compounding. regardless of the protocol, a thorough discussion with the client is warranted regarding efficacy, side effects, and expectations before choosing any pharmacological method. nonpharmacological methods have fewer adverse effects but without formal research, no concrete recommendations can be made. therefore, these therapies may be unreliable on a large scale. in conclusion, further research is needed to develop the ideal treatment for estrous cycle manipulation in dogs. conflict of interest none to declare. references 1. kutzler ma: estrous cycle manipulation in dogs. vet clin north am small anim pract 2018;48:581-594. doi: 10.1016/j.cvsm.2018.02.006 2. nak d, nak y, simsek g: comparison of the use of cabergoline and gonadotrophin to treat primary and secondary anoestrus in bitches. aus vet j 2012;90:194-196. doi: 10.1111/j.1751-0813.2012.00913.x 3. maenhoudt c, santos nr, fontbonne a: manipulation of the oestrous cycle of the bitch-what works… for now. reprod domest anim 2018;53:44-52. doi: 10.1111/rda.13364 4. kutzler ma, wheeler r, lamb sv, et al: use of a gnrh agonist implant (ovuplant®) for estrus induction. proc intl symp on canine and feline reprod 2008. 5. volkmann dh, kutzler ma, wheeler r, et al: the use of deslorelin implants for the synchronization of estrous in diestrous bitches.  theriogenology 2006;66:1497-1501. doi: 10.1016/j. theriogenology.2006.01.033 6. kutzler ma: induction and synchronization of estrus in dogs.  theriogenology 2005;64: 766-775. doi: 10.1016/j. theriogenology.2005.05.025 7. zöldág l, fekete s, czáky c, et al: fertile estrus induced in bitches  by bromocryptine, a dopamine agonist: a clinical trial. theriogenology 2001;55:1657-1666. doi: 10.1016/s0093-691x (01)00510-6 8. gobello c, castex g, broglia y, et al: coat colour changes associated with cabergoline administration in bitches. j  small  anim pract 2003;44:352-354. doi: 10.1111/j.1748-5827.2003.tb00166.x 9. wiebe vj, howard jp: pharmacologic advances in canine and feline reproduction. top companion anim med 2009;24:71-99. doi: 10.1053/j.tcam.2008.12.004 10. verstegen jp, onclin k, silva ldm, et al: effect of stage of anestrus on the induction of estrus by the dopamine agonist cabergoline in dogs. theriogenology 1999;51:597-611. doi: 10.1016/ s0093-691x(99)00013-8 11. fontaine e, mir f, vannier f, et al: induction of fertile oestrus  in the bitch using deslorelin, a gnrh agonist. theriogenology  2011;76:1561-1566. doi:  10.1016/j.theriogenology. 2011.06.031 12. bouchard g, youngquist rs, clark b, et al: estrus induction in the bitch using a combination diethylstilbestrol and fsh-p. theriogenology 1991;36:51-65. doi: 10.1016/0093-691x(91)90433-e 13. kusuma ps, tainturier d: comparison of induction of oestrus in dogs using metergoline, metergoline plus human chorionic gonadotrophin, or pregnant mares’ serum gonadotrophin. j reprod fertil suppl 1993;47:363-370. 14. stornelli mc, garcia mitacek mc, gimenez f, et al: pharmacokinetics of ecg and induction of fertile estrus in bitches using ecg followed by hcg. theriogenology 2012;78:1056-1064. doi: 10.1016/j.theriogenology.2012.04.012 15. sontas hb, dokuzeylu b, turna o, et al: estrogen-induced myelotoxicity in dogs: a review. can vet j 2009;50:1054-1058. 16. thornburgh s, gaskins aj: b vitamins, polycystic ovary syndrome, and  fertility. curr opin endocrinol  diabetes  obes 2022;29: 554-559. doi: 10.1097/med.0000000000000773 17. cueto ht, jacobsen bh, dam laursen as, et al: dietary folate intake and fecundability in two preconception cohorts. hum reprod 2022;37:828-837. doi: 10.1093/humrep/deac002 18. silva mc, guedes peb, silva fl, et al: use of pyridoxine hydrochloride  in the interruption of lactation in female dogs with pseudopregnancy. anim reprod 2021;18:e20200062. doi: 10.1590/1984-3143-ar2020-0062 19. wigham e, moxon r, england g, et al: seasonality in oestrus and litter size in an assistance dog breeding colony in the united kingdom. vet rec 2017;18:371. doi: 10.1136/vr.104217 http://dx.doi.org/10.58292/ct.v17.11647 https://doi.org/10.1016/j.cvsm.2018.02.006 https://doi.org/10.1111/j.1751-0813.2012.00913.x https://doi.org/10.1111/rda.13364 https://doi.org/10.1016/j.theriogenology.2006.01.033 https://doi.org/10.1016/j.theriogenology.2006.01.033 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10.3390/ani13081316 21. murray g, eden e: progesterone to delay estrum in bitches. vet med 1952;47:467-468. 22. burke tj, reynolds ha, jr.: megestrol acetate for estrus postponement in the bitch. j am vet med assoc 1975;167:285-287. 23. schaefers-okkens a, kooistra hs: use of progestogens. tijdschr diergeneeskd 1996;121:335-337. 24. evans jm, sutton dj: the use of hormones, especially progestagens,  to  control oestrus in bitches. j reprod fertil suppl 1989;39:163-173. 25. sawada t, tamada h, inaba t, et al: prevention of estrus in the bitch with chlormadinone acetate administered orally. j vet med sci 1992;54:595-596. doi: 10.1292/jvms.54.595 26. max a, jurka p, dobrzynski a, et al: non-surgical contraception in female dogs and cats. acta scientiarum polonorum zootechnica 2014;13:3-18. 27. støvring m, moe l, glattre e: a population-based case-control study of canine mammary tumours and clinical use of medroxyprogesterone acetate. apmis 1997;105:590-596. doi: 10.1111/ j.1699-0463.1997.tb05057.x 28. von berky ag, townsend wl: the relationship between the prevalence of uterine lesions and the use of medroxyprogesterone acetate for canine population control. aust vet j 1993;70:249250. doi: 10.1111/j.1751-0813.1993.tb08041.x 29. concannon pw, meyers-wallen vn: current and proposed methods for contraception and termination of pregnancy in dogs and cats. j am vet med assoc 1991;198:1214-1225. doi: 10.2460/ javma.1991.198.07.1214 30. root kustritz mv: managing the reproductive cycle in the bitch. vet clin north am small anim pract 2012;42:423-437. doi: 10.1016/j.cvsm.2012.01.012  31. maenhoudt c, santos nr, fontbonne a: suppression of fertility in adult dogs. reprod domest anim 2014;49 suppl 2:58-63. doi: 10.1111/rda.12306 32. lacoste d, dube d, trudel c, et al: normal gonadal functions and fertility after 23 months of treatment of prepubertal male and female dogs with the gnrh agonist [d-trp6, desglynh210] gnrh ethylamide. j androl 1989;10:456-465. doi: 10.1002/ j.1939-4640.1989.tb00140.x 33. wright pj, verstegen jp, onclin k, et al: suppression of the oestrous responses of bitches to the gnrh analogue deslorelin by progestin. j reprod fertil suppl 2001;57:263-268. 34. valiente c, romero gg, corrada y, et al: estrous cycle interruption with a low and a high dose of the gnrh antagonist, acyline, in bitches. theriogenology 2009;79:408-411. doi: 10.1016/j. theriogenology.2008.08.007 http://dx.doi.org/10.58292/ct.v17.11647 https://doi.org/10.3390/ani13081316 https://doi.org/10.1292/jvms.54.595 https://doi.org/10.1111/j.1699-0463.1997.tb05057.x https://doi.org/10.1111/j.1699-0463.1997.tb05057.x https://doi.org/10.1111/j.1751-0813.1993.tb08041.x https://doi.org/10.2460/javma.1991.198.07.1214 https://doi.org/10.2460/javma.1991.198.07.1214 https://doi.org/10.1016/j.cvsm.2012.01.012  https://doi.org/10.1111/rda.12306 https://doi.org/10.1002/j.1939-4640.1989.tb00140.x https://doi.org/10.1002/j.1939-4640.1989.tb00140.x https://doi.org/10.1016/j.theriogenology.2008.08.007 https://doi.org/10.1016/j.theriogenology.2008.08.007 2014: effect of beef cow temperament at mid-gestation on reproductive performance effect of beef cow temperament at mid-gestation on reproductive performance ramanathan kasimanickam department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa abstract temperament is defined by reaction characteristics in response to human handling. an excitable temperament has proven to have detrimental effects on production and reproduction traits. the objective of this study was to evaluate the effect of temperament at mid-gestation on reproductive performance of beef cows. angus and angus cross beef cows (n=1325) from seven locations were included in this study. cows were grouped with bulls with satisfactory breeding potential and free of venereal disease at bull to cow ratios of 1:25 to 1:30. all cows were given a body condition score (bcs; 1-emaciated; 9-obese) and chute-exit and gait score (1 = slow exit, walk; calm temperament; 2 = jump, trot or run; excitable temperament) at pregnancy diagnosis six months after the beginning of the breeding season. pregnancy status and stage of gestation were determined by per-rectal palpation and/or by ultrasonography. cows that were excited had a lower breeding season pregnancy rate compared to calm cows (88.6% [599/694] vs. 92.7% [585/631]; p<0.0001). cows with excitable temperament took 15 more days to become pregnant in the 85 day breeding season compared to calm cows (median days to pregnancy: 30 (20, 60) vs. 15 (10, 40) days; p<0.0001). in conclusion, the modified 2-point chute exit-gait scoring method can be used to identify cattle with calm temperaments at mid-gestation. even though assessing temperament before breeding season makes sense, the producers still can make culling decisions at mid-gestation. cows with calm temperaments in a beef operation will have a higher pregnancy rate and take less time to become pregnant during the breeding season. reducing the proportion of excited cows could improve reproductive performance of the beef operation. . keywords: beef cows; temperament; facility type; breeding season; pregnancy rate; days to pregnancy; introduction profitability of a beef operation is determined by the amount of calf crop produced in a year. pregnancy achieved early in the breeding season and overall breeding season pregnancy rates are two determining reproductive parameters for the annual calf crop.1,2 many factors, including temperament of cattle, affect annual calf crop production. temperament is defined by reaction characteristics of cattle when exposed to human handling.3 cattle with less than optimal temperaments are more excitable while those with better temperaments are calmer and more docile.3-8 calm cattle are less stressed than excitable cattle, demonstrated by lower circulating blood cortisol, prolactin, and substance-p concentrations.9,10 cattle temperament has been studied extensively for its effect on production parameters such as decreased average daily gain,5,6,11 dry matter intake,12,13 feed efficiency,14 and growth.5,6,10 further, excitable cattle tend to produce poor quality carcasses due to decreased marbling,15 decreased meat tenderness,16,17 and increased percentage of dark cutters.15 reproductive parameters previously studied include temperament’s effect on fixed time artificial insemination success and pregnancy rates.8,10,17-20 temperament scoring techniques used to identify calm or excitable cattle vary from computerized analysis to individual observation.3,4,21,22 these methods are subjective and/or objective. chute exit velocity and gait methods with 5 or 6 point scales have been studied.19,21,22 most studies that utilize the 5 point temperament scoring system categorized scores 1 and 2 as calm cattle and scores 3 to 5 as excited cattle for the analysis.19,22 hence, a modified two point scoring method10,20 utilized in this study. the ideal time to assess cattle temperament is before breeding. this will facilitate decision making on whether or not to include excited cows in the breeding program and for culling. temperament scoring by chute exit and gait method requires individual cow handling. in western states of the usa, 36.6% operations utilize pregnancy diagnosis by palpation.23 in a majority of these operations cattle are handled individually at the time of pregnancy diagnosis. even though timely pregnancy diagnosis is 453 clinical theriogenology • volume 6, number 4 • december 2014 critical to cull open cows early in order to reduce feed cost, often pregnancy diagnosis was performed during mid-gestation, approximately 6 months after the beginning of the breeding season on these farms. the objective of the study was to evaluate the effect of cattle temperament assessed at midgestation at pregnancy diagnosis on the reproductive performance in beef cows. the hypothesis was that beef cows with excitable temperaments at handling will have lower breeding season pregnancy rates and will become pregnant later in the breeding season. materials and methods angus and angus cross beef cows (n=1325) from 2013 spring breeding at seven locations in washington were included in this study. angus bulls were grouped with cows (1:25 to 1:30 bull to cow ratio) for 85 days. only bulls with satisfactory breeding potential and free of tritrichomonas fetus were included. all cows were given a body condition score (bcs; 1-emaciated; 9-obese) and chute exit-gait score (1 = slow exit, walk; calm temperament; 2 = jump, trot or run; excitable temperament) at pregnancy diagnosis six months after the onset of the breeding season. cows were maintained in the pasture, vaccinated against routine respiratory and reproductive diseases, and received ad libitum commercial mineral supplements. cows’ pregnancy status and stage of gestation were determined by per-rectal palpation and/or by ultrasonography (aloka-500, sysmed lab inc., chicago, il). the gestation length was determined by sizes of the placentomes and fetus. outcomes measured to assess reproductive performance were breeding season pregnancy rates (%) and interval from beginning of breeding season to pregnancy. data were analyzed with a statistical software program (sas, version 9.4 for windows, sas institute, cary, nc. mixed model (proc glimmix) was used to determine differences in breeding season pregnancy rates amongst temperament score groups. for the determination of pregnancy rates, temperament (calm vs. excitable), bcs (<5 and ≥ 5) and two-way interactions were included in the model. locations, animal handlers (n=28) and natural service sires (n=51) were considered as random variables. models were built by manual reverse stepwise elimination. the p value was set at <0.05 for inclusion and >0.10 for exclusion. kaplan-meier survival estimates (proc lifetest) were used to determine the differences in the crude median days to become pregnant during breeding season between calm and excitable groups. graphs of cumulative pregnancy risk over time were generated. in kaplan-meier analysis, cows may either experience the “event of interest” (i.e. pregnancy) or ‘censored’ (i.e. non-pregnancy at the end of breeding season). this approach allows cows that are censored to contribute to the days at risk for pregnancy as long as they are in the 85-day study breeding season, without making assumptions about what would have occurred had they remained for a longer period of time. in this study, cows that were identified as non-pregnant at the time of the pregnancy examination were censored on the last day of the 85-day breeding period. the log-rank test was used to compare the overall equality of temperament score survivor functions, and follow-up pair-wise comparisons were conducted using a bonferroni-corrected log-rank test to limit the experiment-wise type-i error rate to 5%. restricted mean survival times were obtained as the area under kaplan-meier survivor curves. univariate analysis, proc gnemod, was used to determine the proportion of excited cows in different locations. p-values of <0.05 were considered statistically significant. results of the study population (n=1325), 52.3% (694) of cows were identified as excited and 47.4% (631) as calm. the percentage of excited cows in a location varied from 13.6%% to 70.3% (p<0.01). the mean bcs of the cows was not significantly different between calm and excited group (5.98±0.11 vs. 6.04±0.09). accounting for bcs categories (p<0.0001), breeding season pregnancy rate was different between calm and excited cows (p<0.0001; table). the pregnancy rate was 92.7 (585/631) vs. 86.3% (599/694) for calm and excited cows, respectively. cows with bcs ≤5 had lower breeding season pregnancy rates compared to cows >5 bcs, 74.5 (187/251) and 91.9% (987/1074) respectively 454clinical theriogenology • volume 6, number 4 • december 2014 (p<0.0001). no significance in bcs by temperament score interaction on the breeding season pregnancy rate was observed (p>0.1). calm cows became pregnant earlier in the breeding season compared to excited cows (log rank test; chi-square -55.7; p<0.0001; figure ). the percentage of cows that remained non-pregnant at the end of the breeding season and median days to pregnancy (25th and 75th percentile) is given in the table presented below the figure. discussion this study observed that beef cows with calm temperament had a significantly higher probability of becoming pregnant early in the breeding season. in this study the temperament was assessed at midgestation. in a normal physiological status, it is reasonable to expect that tw0-thirds of non-pregnant cows should become pregnant in each 21 day estrous cycle. if each cow gets three chances to become pregnant during the breeding season, then 97% cows should become pregnant at the end of the breeding season. in this study, 86% cows with excitable temperament compared to 93% of calm cows became pregnant at the end of the breeding season. furthermore, excitable cows took longer time to become pregnant in this study. increased stress biomarkers, including elevated substance-p, prolactin and cortisol concentrations, in excited cattle suppress gonadotropin releasing hormone and luteinizing hormone.10,21,22,24,26 further, βendorphin is involved in regulating luteinizing hormone secretion in postpartum beef cows.23 these hormonal disturbances plausibly cause delay in resumption of ovarian cyclicity in excited cows after calving.24-26 in addition, it is also possible that excitability affects follicular dynamics resulting in hindrance in estrus expression. further, reduced ovarian steroidogenesis and increased prostaglandin2α in excited cattle may have caused early embryonic death in this group.10 it should be noted that lactation stress could also cause similar untoward physiological changes. however, the nutritional requirement (maintenance + lactation) should be adequate to prevent the lactation stress in most beef cows if not under environmental stress.27 temperament was negatively associated with bcs and nutritional status in growing cattle.5,6 in this study no bcs by temperament interaction effect on breeding season pregnancy rate was observed. voisinet et al observed that cattle that became agitated during handling had 14% lower body weight gain compared to calmer cattle.16 the reasons for reduced productivity in excitable cattle are: increased blood cortisol concentrations and other stress markers stimulate muscle and fat metabolism at a greater rate than calm cattle; and excitable cattle have more frequent meals, but overall decreased intake.5 the excitable cattle had decreased time spent eating, and increased activity looking for “threats” rather than consuming, resulting in body condition loss.5,6 further, it is possible that excited cows also affect the temperament of other calm cows. in this study the temperament was assessed at mid-gestation concurrent with pregnancy diagnosis. it should be noted that temperament scoring at two to four weeks prior to the beginning of the breeding season also associated with the reproductive performance of beef cattle.10,20 culling nonpregnant cows alone will remove an excitable cow 11.4% of the time. however, it would be prudent to exclude excited cows from the breeding program due to their lower reproductive performance. moreover excitable cattle are dangerous to cattle handlers and to other animals and could also cause damage to the facility. causes of cattle temperament include genetics, inappropriate cattle handling and poor facility design. culling excited and non-pregnant cows and utilizing bulls with high docility expected progeny difference score offers options to reduce the number of excitable cows on a beef farm. further acclimation of beef cattle to handling and proper facility design could reduce the excitability.10,19,20,22 the median days to become pregnant between calm and excited cows in the present study were shorter compared to previous study,19 15 and 30 vs 35 and 59 respectively. the observed differences in median days to become pregnant in these studies may plausibly be caused by other management factors. nevertheless, the observed differences in median days to become pregnant between calm and excited cows were significant in these two studies. 455 clinical theriogenology • volume 6, number 4 • december 2014 conclusion the modified 2-point chute exit-gait scoring method can be used to identify cattle with calm temperaments at mid-gestation. even though assessing temperament before breeding season makes sense, producers still can make culling decisions at mid-gestation. cows with calm temperaments in a beef operation will have a higher pregnancy rate and take less time to become pregnant during the breeding season. reducing the proportion of excited cows could improve reproductive performance of the beef operation. acknowledgements the author thanks all participating beef producers. the author also thanks veterinary students stephanie schroeder, katy hanson, marion fisher, shelley gerstner, micall gooderidge, kathleen gutierrez, and vaughn fish, college of veterinary medicine, washington state university for their help. table. explanatory variables*, temperament score, and body condition score (bcs) influencing pregnancy loss in angus cross beef cows (n=1325) effect degrees of freedom f value p value temperament score 1 18.23 <0.001 bcs categories 2 15.80 <0.0001 temperament score – 1, calm slow, walking; 2, excitedjumping, trotting, or running; bcs categories ≤5 and >5; bcsbody condition score 1-emaciated; 9-obese; natural service sires, locations and animal handlers were offered as random variables; *co-variance parameter estimates – natural service sire 0.1977; location 0.3898; animal handlers 0.0946; residual 0.1895; fit statistics bic = 1442.6; -2 res log likelihood =1388.3; 456clinical theriogenology • volume 6, number 4 • december 2014 figure. survival curve for the effect of temperament on the probability of non-pregnancy* during the breeding season in beef cows temperament % non-pregnant cows (censored) median days to become pregnant confidence limits 25th percentile 50th percentile calm (0) 7.3 15 10 40 excited (1) 11.4 30 20 60 * instantaneous relative risk of pregnancy on daily basis references 1. mathis c, sawyer j: beef cow efficiency in the southwest. nm state university extension. available at: http://www.safmm.nmsu.edu/pubs/_b/b-217.pdf. accessed 5/31/13. 2. rae do: assessing performance of cow-calf operations using epidemiology. vet clin. north am food anim pract 2006;22:53-74. 3. fordyce ge, dodt rm, wythes kh, et al: cattle temperaments in extensive beef herds in northern queensland. aust j exp agric 1988;28:683-687. 4. vetters mdd, engle te, ahola jk, et al: comparison of flight speed and exit score as 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2011;89:52-57. 16. voisinet bd, grandin t, o’connor sf, et al: bos indicus-cross feedlot cattle with excitable temperaments and tougher meat and higher incidence of borderline dark cutters. meat sci. 1997;46:367-377. 17. behrends sm, miller rk, rouguette fm, et al: relationship of temperament, growth, carcass characteristics and tenderness in beef steers. meat sci 2009;81:422-438. 18. cooke rf, bohnert dw, meneghetti m, et al: effects of temperament of pregnancy rates to fixed-time ai in bos indicus beef cows. livest sci 2011;142:108-113. 19. cooke rf, arthington jd, austin br, et al: effects of acclimation to handling on performance, reproductive, and physiological responses, of brahman-crossbred heifers. j anim sci 2009;87:3403-3712. 20. kasimanickam r, asay m, schroeder s, et al: calm temperament improves reproductive performance of beef cows. reprod domest anim 2014 (submitted) 21. cooke rf, scarpa a, mueller c, et al: effects of temperament on reproductive and physiological responses of beef cows. 2010 beef research report available at: http://beefcattle.ans.oregonstate.edu/html/ publications/documents/beef038-cowdisposition.pdf. accessed 5/15/13. 22. cooke rf, bohnert dw, cappellozza bi, et al: effects of temperament and acclimation to handling on reproductive performance of bos taurus beef cattle. j anim sci 2012;90:3547-3555. 23. http://www.aphis.usda.gov/animal_health/nahms/beefcowcalf/downloads/beef0708/beef0708_dr_partii.pdf .accessed 5/31/13. 24. dobson ha, ribadu ay, noble km, et al: ultrasonography and hormone profiles of adrenocorticotrophic hormone (acth)-induced persistent ovarian follicles (cysts) in cattle. j reprod fertil 2000;120:405-410. 25. osawa t, nakao t, moriyoshi m, et al: plasma β-endorphin around parturition and its relationship to cortisol level and resumption of pituitary and ovarian function in dairy cows. anim reprod sci 1998;52:27-38. 26. wagner wc, li ps: influence of adrenal corticosteroids on postpartum pituitary and ovarian function. in: karg h, schallenberger e, editors. factors influencing fertility in the postpartum cow. the hague: martinus nijhoff; 1982. p. 123-147. 27. subcommittee on beef cattle nutrition: nutrient requirements: excess and deficiencies. in: nutrient requirements of beef cattle. 6th revised edition.. washington: national academic press; 1984. p. 1-24. 458clinical theriogenology • volume 6, number 4 • december 2014 1 contact eduardo prado eprado@utk.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10997, http://dx.doi.org/10.58292/ct.v16.10997 case report colpotomy in a cow to remove a fetal bone from the uterus eduardo prado, tulio prado, daniel prado, tanner drum, brittany veerasammy, joe smith department of large animal clinical sciences, college of veterinary medicine, university of tennessee, knoxville, tn, usa abstract a 5-year, aberdeen angus cow was presented to assess a hard/foreign structure that was felt in the uterus via transrectal palpation. further diagnostics including transrectal ultrasonography were performed; a fetal bone was in the left uterine horn. removal of the bone through the cervix was unsuccessful. colpotomy was elected; removed bone was half of a fetal mandible, presumably a remnant from fetal maceration. uterus was sutured with a simple continuous pattern; the cow was treated with antimicrobials and antiinflammatory drugs and recovery was uneventful. ovum pick up was performed 5 months after surgery; several oocytes were collected from both ovaries and 6 embryos were obtained via in vitro fertilization. cow was pregnant 6 months after surgery. keywords: beef cow, uterine horn, macerated fetus, fetal bone, colpotomy background there is a risk of pregnancy loss in cattle and depending on the stage the embryo/fetus is resorbed or expelled. embryonic loss results in resorption whereas fetal loss in expulsion or retention.1 fetal ossification starts ~ day 45 of pregnancy in cattle.2 a retained dead fetus can become mummified or macerated. if pregnancy loss occurs after 3 months without proper fetal expulsion, the process of maceration starts in ~ 4 to 5 days.2 a retained nonviable fetus can be removed medically or surgically. hysterotomy via flank or ventral midline approach is preferred; however, if the structure in the uterus is < 30 cm, a hysterotomy via colpotomy is feasible.3 cows that had a macerated fetus have a poor prognosis for breeding and carrying pregnancy to term than cows with mummified fetus; damage by incrusted bones to the endometrium is more severe.3,4 case presentation a 4-year, aberdeen angus, multiparous cow was presented to assess a hard/foreign structure that was felt in the uterus by the referring veterinarian via transrectal palpation. owner stated that the cow was due to calve ~ 3 months prior to presentation. mammary gland development was noticed around the due date but no calf was delivered and a few weeks after the due date, the cow had clinical signs of estrus. cow was bred via artificial insemination during 2 estrous cycles, nonpregnancy was diagnosed and the cow was in estrus ~ 10 days before presentation. cow was bright, alert, and responsive; vital parameters were: heart rate 36 beats per minute, respiratory rate 30 breaths per minute, and temperature 100.1 f. transrectal palpation and ultrasonography were performed; a foreign body, potentially a fetal bone (palpated as a single long piece) was in the left uterine horn. uterine horns felt similar in size (~ 20 to 22 cm); had no fluid and were hyperechogenic. right ovary had a 6 mm follicle and the left ovary had a corpus luteum (cl). based on the history, top differentials were mummified or macerated fetus; latter was more possible since cl does not persist with a macerated fetus.5 treatment cow was initially treated medically; 2 intramuscular prostaglandin (lutalyse®) injections were given 24 hours apart. although the cl was lysed, the foreign object was not expelled and no abnormal discharge or unusual mucus secretion was observed at estrus. removal of fetal structure via manual extraction through the cervix was attempted. cow was placed in a head lock and received an epidural injection (between last sacral and first coccygeal vertebrae) with 4 ml of lidocaine (lidocaine 2%). additionally, 400 mg of synthetic prostaglandin e1 ( misoprostol) mixed with sterile obstetrics lube was applied to cervix for softening and dilation. however, enough dilation of the cervical rings was not possible for manual removal of the foreign body. cow was started on oral meloxicam mailto:eprado@utk.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10997 2 citation: clinical theriogenology 2024, 16, 10997, http://dx.doi.org/10.58292/ct.v16.10997 (1 mg/kg once daily) to prevent any possible swelling due to vaginal and cervix manipulation. hysterotomy via colpotomy was elected and cow was placed in a cattle transporter chute. high and low epidurals were given with 3 ml of mepivacaine hydrochloride (carbocaine®) on each site to block the perineal area, immobilize the tail, and reduce defecation during surgery. cow was sedated with intravenous acepromazine 10 mg (1 ml) via left jugular. cow’s tail was bandaged with roll brown gauze and secured to her body. perineal area was scrubbed with iodine solution and water. a 28 french foley catheter was placed in the urinary bladder to decrease its size and prevent urine contamination during the procedure. vaginal cavity was flushed 3 times with  diluted chlorhexidine and isotonic saline solution. subsequently, a pack of sterile gauze was soaked in lidocaine, and it was attached to the surgeon’s hand with a sterile umbilical tape. the gauze pack was introduced to the vaginal cavity and pressed over the proposed incision site for about 5 minutes to desensitize the area. an incision was made in the vaginal cavity at 10 o’clock position; extended 1 inch horizontally and surgeon’s fingers widened the incision. surgeon’s hand was introduced through the incision and uterus was reached. uterus was carefully handled within the abdominal cavity to prevent uterine wall rupture and potential intrabdominal contamination. thereafter, uterus was exteriorized through the vaginal incision. traction to the uterus resulted in partial rupture of uterine horn wall; opening was widened to ~ 3 inches with # 5 scalpel blade, and the bone (half of fetal mandible;16 x 1.5 x 7 cm) was extracted (figure 1). uterine incision was closed with a simple continuous pattern (figure 2), using absorbable suture (# 0 vicryl polyglactin) and returned to its normal position. vaginal incision was left open to heal by second intention; buhner stitch was applied around the vulva to prevent evisceration. on the day of surgery, cow received clostridium perfringens type c and d, and tetanus booster (bar-vacr cd/t) vaccinations. antibiotics and antiinflammatory drugs were given. cow was started on intramuscular penicillin g procaine (22,000 iu/kg [penone protm, 300,000 iu/ml]) twice daily for 3 consecutive days. cow was also given (at the base of ear) 2 injections (72 hours apart) of ceftiofur crystalline free acid (6.6 mg/kg [excede® 200 mg/ml]) after the procedure for suspected local infection observed during surgery. on the day of surgery, intravenous flunixin meglumine (1 mg/kg [prevailtm 50 mg/ml]) was given and continued for 4 days with oral meloxicam (1 mg/kg once daily) and was reduced to 0.5 mg/kg once daily for 4 more days. figure 2. exteriorized uterus after fetal mandible removal and incision (note incision site [arrow]) closure figure 1. extracted fetal mandible (16 x 1.5 x 7 cm) from the uterine horn http://dx.doi.org/10.58292/ct.v16.10997 citation: clinical theriogenology 2024, 16, 10997, http://dx.doi.org/10.58292/ct.v16.10997 3 outcome cow was hospitalized for a week after surgery. transrectal palpation was performed 4 days after surgery to retract the uterus to prevent adhesion. buhner stitch was removed 5 days after surgery. it was recommended to keep the cow confined alone in a small pasture for ~ 4-6 weeks, with no reproductive activity. cow was examined 4 months after surgery by the university theriogenology department. transrectal palpation and ultrasonography were performed; no abnormalities were detected. reproductive tract was completely mobile; left ovary had a cl and right ovary had a 9 mm follicle. vaginal speculum examination revealed a fully healed colpotomy incision. a few weeks later, ovum pick up was performed and several oocytes were collected and 6 embryos were obtained via in vitro fertilization. cow was pregnant 6 months after surgery. discussion fertility in cows that had a macerated fetus decreased substantially due to fetal bones damage to endometrium.6 however, there has been anecdotal evidence of cows carrying pregnancy after a history of a macerated fetus. in cows that have had a mummified fetus, pregnancy rates a few months after the mummy extraction via hysterotomy was ~ 36%.7 additionally, 1 mare was diagnosed pregnant a few weeks after removal of fetal bones in a case of fetal maceration.8 medical treatment for fetal bones expulsion can be attempted with prostaglandin treatment. however, if it is not effective, a surgical option is necessary. hysterotomy can be performed via colpotomy or caudal flank.3-7 colpotomy is a very common technique used in mares for ovariectomy to eliminate unwanted behavior or diseased ovaries.9 it is also indicated for unilateral ovariectomy in cows for removal of diseased ovaries.10 advantages of colpotomy approach is that it can be performed standing in a squeeze chute; however, it is recommended for moderately enlarged uterus with structures no larger than 30 cm in diameter.3 -11 cow in this case received multiple treatments that are considered extralabel usage in the us. procaine penicillin g dose was extralabel. ceftiofur product was used as an extralabel indication (localized infection to the foreign body); however, this is permissible under the third-generation cephalosporin order of prohibition, as the label route, dose, and frequency were used. antiinflammatories were used extralabelly for postoperative pain and misoprostol was also used in an extralabel fashion. this extralabel usage is permitted in the us within a valid veterinarian-client-patient relationship, and before discharging the case, food animal avoidance databank was consulted for withdrawal recommendations. in cows with high genetic value, assisted reproductive techniques can be applied (e.g. ovum pick up for in vitro fertilization). embryo flushing is also an option to bypass the uterus and obtain offspring from cows that had uterine damage because of pregnancy. fertility may continue to be normal in cows that have experienced any type of fetal abnormalities during pregnancy. learning points • cows that exceed the due date should have reproductive tract evaluation by a veterinarian • hysterotomy via colpotomy is a feasible procedure to perform in cows with retained structures in the uterus • assisted reproductive techniques options are available to produce offspring from cows with a history of uterine damage conflict of interest none to report. references 1. baumgartner w: fetal disease and abortion: diagnosis and causes. in: hopper rm: editor. bovine reproduction. 2nd edition, hoboken; john wiley & sons: 2021. p. 667-716. 2. roberts s: diseases and accidents during the gestation period. in: veterinary obstetrics and genital diseases. 2nd edition, ithaca; published by the author: 1971. p. 107-198. 3. hopper r, hostetler d, smith j, et al: surgical removal of a mummified fetus via colpotomy. bovine pract 2006;40:57-58. doi: 10.21423/bovine-vol40no2p57-58 4. hopper r: surgical correction of abnormalities of genital organs of cows. in: youngquist r, threlfall w: editors. current therapy in large animal theriogenology, 2nd edition, st. louis, missouri; elsevier: 2007. p. 463-472. 5. lefebvre r: fetal mummification in the major domestic species: current perspectives on causes and management. vet med (auckl) 2015;6:233-244. doi: 10.2147/vmrr.s59520. pmid: 30101110; pmcid: pmc6067784. 6. sood p, vasishta nk, singh m: use of a novel surgical approach to manage macerated fetus in a crossbred cow. vet rec 2009;165:347-348. doi: 10.1136/vr.165.12.347. pmid: 19767639. 7. lefebvre rc, saint-hilaire e, morin i, et al: retrospective case study of fetal mummification in cows that did not respond to prostaglandin f2alpha treatment. can vet j 2009;50:71-76. pmid: 19337617; pmcid: pmc2603657. 8. morrissey j, pinsky t, roy mf: fetal maceration and partial retention of fetal bones in 2 mares. can vet j 2024;65:133-137. pmid: 38304478; pmcid: pmc10783584. 9. prado t, schumacher j: how to perform ovariectomy through a colpotomy. equine vet educ 2017;31:209-213. doi: 10.1111/ eve.12801 10. prado t, schumacher j, dawson l: surgical procedures of the genital organs of cows. vet clin north am food anim pract 2016;32:727-752. doi: 10.1016/j.cvfa.2016.05.016 11. irons pc: hysterotomy by a colpotomy approach for treatment of foetal mummification in a cow. j s afr vet assoc 1999;70:127-129. doi: 10.4102/jsava.v70i3.772 http://dx.doi.org/10.58292/ct.v16.10997 https://doi.org/10.21423/bovine-vol40no2p57-58 https://doi.org/10.2147/vmrr.s59520 https://doi.org/10.1136/vr.165.12.347 https://doi.org/10.1111/eve.12801 https://doi.org/10.1111/eve.12801 https://doi.org/10.1016/j.cvfa.2016.05.016 https://doi.org/10.4102/jsava.v70i3.772 2014: reproductive performance of an angus cow after removal of a macerated fetus via caudal flank laparotomy and hysterotomy reproductive performance of an angus cow after removal of a macerated fetus via caudal flank laparotomy and hysterotomy j.a. herrmann,a s.r. satheb acollege of veterinary medicine, university of illinois, urbana, il; bcollege of veterinary medicine, iowa state university, ames, ia summary a seven year-old angus cow in normal body condition was presented with a history of chronic vaginal discharge, failure to cycle and a palpable uterine mass. clinical examination revealed fetal bones in the right uterine horn and a purulent vaginal discharge. a diagnosis of fetal maceration and metritis was presented to the owner, with surgical removal of the macerated fetus as the only treatment option. a poor prognosis for recovery of reproductive function was given. due to the past value of the cow as an embryo donor, the owners elected to proceed with surgical intervention. fetal bones, including one embedded in the endometrium, were removed via caudal flank laparotomy and hysterotomy. aggressive post-surgery medical treatment of the infected and damaged uterus, combined with five months of sexual rest, resulted in a return to normal fertility. keywords: macrated fetus, hysterotomy, metritis, fertility background fetal mummification and maceration have been reported in cattle but the incidence has been described as low and sporadic, with an estimated incidence of mummification between 0.43 and 1.8% of pregnancies.1 in cattle, pregnancy is supported by progesterone produced by the corpus luteum and, after approximately 150 to 200 days of gestation, by the placenta and adrenal glands as well.2,3 bovine fetal mummification most commonly occurs during the fourth through sixth months of gestation with fetal autolysis, resorption of fetal fluid in a sterile environment and without concomitant loss of the corpus luteum or dilation of the cervix.1,3 bovine fetal maceration is also associated with a functional corpus luteum but with further autolysis of fetal soft tissues, resulting in only fetal bones remaining in the uterus.3 fetal maceration is usually accompanied by a partially dilated cervix, fetid vaginal discharge, metritis and often, clinical illness.1,4 many causes of fetal mummification and maceration have been proposed, including most of those that can cause abortion, such as viral and bacterial infections, genetic factors and developmental abnormalities. however, since the mummification and maceration processes occur long after fetal death, the cause is often not determined. diagnosis of either fetal mummification or maceration, through either trans-rectal palpation or ultrasound examination, is not difficult. in cases of fetal mummification, the dam is usually not clinically ill and seldom shows signs of an interruption of gestation. the standard treatment for fetal mummification is the administration of prostaglandin f2alpha, with expulsion of the fetus expected within 96 hours.3,5 for dams that do not respond to initial treatment, prostaglandin f2alpha can be repeated if a corpus luteum is still present. alternatively, the combination of estradiol-17beta and oxytocin has been reported to be effective.7 medical treatment of fetal mummification generally leads to a resumption of normal reproductive function.1,3-5,7,10 for cases that do not respond to medical therapy, surgical removal of mummified fetuses through hysterotomy via either colpotomy or laparotomy has been successful, with an eventual return to normal reproductive function.1,5-9 the treatment of fetal maceration, on the other hand, is often more challenging. fetal maceration is frequently associated with clinical signs of anorexia, pyrexia, elevated pulse and respiration, atrial fibrillation, straining, and subsequent development of metritis or pyometra.1,4,10 in long-standing cases, fetal bones can become embedded in the endometrium and chronic damage to the endometrium can be significant. response to prostaglandin or estrogen therapy is often unrewarding. long term treatment of the infected uterus is necessary and often unsuccessful.1 the prognosis for a return to normal reproductive function is often poor and slaughter is often recommended.1,4 clinical theriogenology • volume 6, number 1 • march 201447 47 case presentation a seven year-old angus cow was presented to the theriogenology service at the veterinary teaching hospital at the university of illinois (uiuc vth) with a history of vaginal discharge, failure to cycle and a uterine mass. the owner reported that the cow had been used as a successful embryo donor and had been bred by artificial insemination 11 months earlier. she was confirmed pregnant via transrectal palpation approximately 60 days after breeding. four months prior to presentation, the animal developed a partial vaginal prolapse when she was approximately 7.5 months pregnant. the owner placed a buhner’s stitch to prevent a further prolapse and called the referring veterinarian a few days later after he noticed a foul smelling vaginal discharge. the referring veterinarian found a hard mass in the right uterine horn on trans-rectal palpation and vaginal examination revealed an abnormal foul smelling discharge coming from a partially open cervix. fetal bones could be felt in the cervical canal extending into the lumen of the uterus and were extracted. however, the entire fetal skeleton could not be extracted due to the small diameter of the cervix. the cow was treated with 25 mg dinoprost tromethamine by intramuscular injection and the owner was instructed to have the cow rechecked by the veterinarian in ten days. instead, the owner administered the same dose of dinoprost tromethamine on several occasions during the three months prior to referral and more fetal bones were reportedly extracted through the cervix of the cow by the owner. more than three months after the referring veterinarian first saw the cow, she was referred to the uiuc vth. the cow was examined by the theriogenology service of the uiuc vth. body temperature, pulse and respiratory rates were found to be within the normal range. a weight of 1885 lbs. (856.3 kgs.) and a body condition score 6/9 on the standard beef cattle scale were recorded. a trans-rectal examination, using both manual palpation and a 5.0 mhz real-time ultrasound probe, was performed to evaluate the reproductive tract. the right uterine horn was found to be enlarged (approximately 22 cm diameter) and contracted around a distinct, hard mass midway between the uterine bifurcation and oviduct. the left uterine horn was palpated and found to be of normal size and consistency. both ovaries had numerous developing follicles, ranging in size from 6 mm to 16 mm diameter. the left ovary had a 32 mm corpus luteum. vaginal examination with a penlight and speculum revealed the cervix open to approximately 35 mm diameter with roughened and indurated mucosa around the external os of the cervix and grey, fetid discharge coming through the cervix. it was possible to pass two fingers inside the cervical canal and small pieces of fetal bones were extracted. the size of the cervical canal precluded a more detailed examination. a diagnosis of fetal maceration was made and surgical removal via hysterotomy was offered as the only reasonable treatment option. a poor prognosis for the recovery of reproductive function was given based on the long-standing nature of the condition and the potential for widespread damage to the uterine mucosa and subsequent fibrosis. the owners, who wanted to retain the animal as a potential embryo donor, elected to proceed with the surgical option. treatment the animal was restrained in a hydraulic chute with a head gate. light sedation was achieved by intravenous administration of 10 mg xylazine and 20 mg of butorphanol tartarate via the left jugular vein. the right paralumbar fossa was clipped and prepared for aseptic surgery. after considering the very dorsal position of the right uterine horn containing the mummified fetus in the abdominal cavity, a diagonal incision was planned, starting slightly cranial and ventral to the right ilium and extending to a distance of about 15 cm (figure 1). the proposed incision site was infiltrated with 90 ml of 2% lidocaine hydrochloride to achieve local analgesia and the right flank was draped after final skin preparation. the skin, external and internal oblique and transversus abdominis muscles were incised with a scalpel and the peritoneum was opened with mayo scissors. the abdominal cavity was entered and the enlarged segment of the right uterine horn was gently lifted and exteriorized through the skin incision. the intra-luminal uterine mass was palpated through the uterine wall and the entrance into the abdominal cavity was packed off with sterile laparotomy towels. clinical theriogenology • volume 6, number 1 • march 2014 48 48 the right uterine horn was incised along its greater curvature directly over the hard palpable mass. fetal skull, along with a segment of the cervical spine, was extracted through the hysterotomy incision. further exploration revealed numerous parts of the fetal skeleton, including scapulae, additional thoracic and lumbar vertebrae, ribs, and metacarpal bones, all of which were subsequently extracted (figure 2). before closure, the uterine lumen was examined through the hysterotomy incision by the surgeons and per vaginum by an assistant to determine if all debris was removed from the uterine lumen. during this exploration and palpation of the uterine lumen, a piece of bone was found to be embedded between the uterine mucosa and the myometrium at the base of the right uterine horn and thus could not be extracted through the original uterine incision. before attempting to remove this embedded piece, the uterus was flushed with 0.05 % povidone iodine solution using a uterine flushing catheter (bivona equine cahteter, partnar animal health, london, on, canada) inserted into the uterine lumen. closure was routine using an inverting utrecht suture pattern and 0 polydioxanone (pds, ethicon inc., somerville, nj). the right uterine horn was then further exteriorized through the abdominal wall incision and the base of the right uterine horn was palpated to localize the embedded piece of bone. an incision was made through the uterine serosa and muscularis directly over the bone fragment and, after some blunt dissection, the bone was loosened from the surrounding fibrous connective tissue and removed. it was observed that the uterine lumen could not be palpated through the serosal surface incision due to proliferation of fibrous connective tissue. the serosal incision was then closed with a lembert pattern followed by a cushing’s pattern in a double inverting layer pattern using 0 polydioxanone suture material, was rinsed with normal saline to wash away any blood clots and placed back inside the abdominal cavity. the abdominal wall was closed in a standard fashion in three layers, using 1 polydioxanone suture material and the skin in a fordinterlocking suture pattern with extra heavy polymerized caprolactam. the cow was administered 25 mg dinoprost tromethamine by intramuscular injection and was also given an injection of long-acting ceftiofur at the rate of 6.6 mg/kg body weight subcutaneously in the middle third of the posterior aspect of the left ear. the cow was released to the owner who was advised to work closely with the referring veterinarian so that postoperative care could be managed effectively. initial recommendations were for the referring veterinarian to recheck the cow ten days after surgery and communicate his findings to the university theriogenologists. outcome the referring veterinarian performed a trans-rectal ultrasound examination cow ten days after surgery and found that the right horn had decreased to approximately 12 cm in diameter and was fluid filled. upon manual palpation, mucus and purulent exudate were expressed through the vulva. palpation and ultrasonography of the ovaries revealed a 35 mm corpus luteum and a 15 mm follicle on the left ovary and no significant structures on the right ovary. there were no alterations in the animal’s temperature, appetite or behavior. the cow was treated with long-acting ceftiofur, long-acting oxytetracycline at the rate of 13.6 mg/lb subcurtaneously and 25 mg dinoprost tromethamine intramuscularly. the incision site showed presence of a purulent discharge from the most ventral aspect, which was flushed and cleaned with dilute povidone iodine. at twenty-two days after surgery the referring veterinarian performed a trans-rectal ultrasound examination and found that the right horn had decreased to approximately 7.5 cm diameter, was fluidfilled and felt fibrotic, with thickening of the uterine wall. examination of the ovaries revealed follicles of various sizes on both ovaries with a mature corpus luteum on the left ovary. the cow was eating, drinking and behaving normally. blood samples were taken for complete blood count (cbc) and chemistry panel and the uterine discarge was cultured. after consultation with the uiuc vth theriogenology service, the cow was again treated with short-acting ceftiofur subcutaneously at the rate of 2 ml/100 lbs. per day for seven days and 25 mg dinoprost tromethamine intramuscularly. in addition, the referring veterinarian administered long-acting oxytetracycline. the incision site had healed and sutures were removed. clinical theriogenology • volume 6, number 1 • march 201449 49 hematology and serum biochemistry values were within normal limits. culture results from the uterine exudate sample revealed a nearly pure culture of arcanobacter (trueperella) pyogenes infection sensitive to ampicillin. at 30 days after surgery, and after consultation with the uiuc theriogenology service, intrauterine therapy with 3 grams of ampicillin sodium diluted in 80 cc of sterile water was performed by the referring veterinarian and repeated every other day for four treatments. the animal was re-examined after ten days. the right uterine horn was smaller, approximately 5.0 cm in diameter and almost equal to the left uterine horn in size. the right ovary had a palpable 40 mm corpus luteum and a few developing follicles while the left ovary was unremarkable. no apparent uterine discharge was noted at the vulvar commissure and no fluid was noted in the uterus on trans-rectal ultrasound examination. the cow was administered 25 mg dinoprost tromethamine and was turned out in the pasture with a few other cows. the owner reported observing several periods of estrous behavior over the next 60 to 70 days, consistent with 20 to 22 day estrous cycles. the cow was given reproductive rest for a total of five months after surgery, was subsequently enrolled in the farm’s superovulation protocol and flushed, yielding viable grade #1 and #2 embryos. the cow was superovulated and flushed once more, again yielding viable grade #1 and #2 embryos, and was subsequently bred by artificial insemination and became pregnant. discussion when cases of bovine fetal mummification or maceration are diagnosed, veterinarians and owners are faced with three options: cull for slaughter, treat medically by inducing abortion or treat surgically by removing the fetal tissue through hysterotomy. it has been suggested that the dam’s fertility after removal of a mummified fetus via medical induction of abortion is generally good. 1,3,4,11-16 however, little has been reported in the literature regarding the reproductive capacity of cows subsequent to surgical removal of mummified fetuses. in any surgical approach to the uterus, access can be difficult. the colpotomy approach leaves little room for manipulation or visual inspection of the fetus and is often impractical for large mummified fetuses. the caudal flank approach is often limited by the location of the uterus dorsal in the caudal abdomen, often partially within the pelvic canal, as well as the thickness of the abdominal wall muscles just anterior and ventral to the ilium. in one case series of fourteen cows that had mummified fetuses, five of 11 cows (45%) that had the mummified fetus removed by laparotomy and hysterotomy and none of the three cows that had mummified fetuses removed per vaginum subsequently became pregnant. however, in that report, all fetuses were mummified, not macerated, and there was no indication of significant uterine pathology.6 medical and surgical treatment of bovine fetal maceration have been dismissed as ineffective and impractical because of the amount and severity of uterine pathology associated with long-standing cases of fetal maceration.1,3,4 adding to that argument, many cows with fetal maceration commonly develop endometritis10 or pyometra, two additional conditions that have been shown to be strongly associated with impaired fertility and increased cull rates.17-19 as additional support for a poor prognosis for cows with fetal maceration, the authors could find only one report of a cow becoming pregnant after the removal of a macerated fetal bone that was embedded in the endometrium. however, there was no documentation of pregnancy status.20 in contrast, this case provides evidence of better than expected surgical and reproductive outcomes, despite significant uterine pathology in a long-standing case of fetal maceration. following surgery and intensive postoperative medical treatment, the patient was superovulated and produced viable embryos twice before she became pregnant, all within nine months of initial referral and surgical treatment. part of the postoperative medical therapy was the extra-label use of aqueous sodium ampicillin as an intrauterine infusion. the authors felt that its use followed the american veterinary medical association’s judicious use principles21 because its selection was based on culture and sensitivity of the organism isolated from the uterus, because the cow was not lactating, was not going to be slaughtered within thirty days of administration and because the antibiotic has a narrow spectrum and was placed in clinical theriogenology • volume 6, number 1 • march 2014 50 50 direct contact with bacteria in the uterine lumen. the authors believe that the follow-up care provided by the referring veterinarian and the diligence of the owners in committing to long-term therapy were keys to the cow’s recovery. the case presented here suggests that, in cases of fetal maceration, clinicians should not necessarily be pessimistic about an individual cow’s chances for recovery of reproductive function, even when complicated by significant uterine damage, metritis and pyometra. surgical removal of macerated fetal tissue, followed by aggressive medical treatment, can be an effective treatment option for high value cows. learning points � cows with retained macerated fetuses can regain reproductive function and fertility. � surgical removal of macerated fetal tissue, even when complicated by the growth of endometrium over retained fetal bones, can be an effective treatment option for high value cows. � patience and long-term postoperative therapy are necessary for recovery of reproductive function. references 1. roberts sj: veterinary obstetrics and genital diseases. 3rd ed. woodstock (vt): published by the author; 1986. p. 213217. 2. wendorf gl, lawyer ms, first nl: role of the adrenals in the maintenance of pregnancy in cows. j reprod fertil 1983;68:281-287. 3. barth ad: induced abortion in cattle. in: morrow da, editor. current therapy in theriogenology. 2nd ed. philadelphia: wb saunders; 1986. p. 205-208. 4. thomas, pga: induced abortion. in: youngquist rs, threllfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st. louis: wb saunders; 2007. p. 309. 5. hirsbrunner g, knutti b, burkhardt hw, et al: surgical and conservative methods for removing mummified fetuses in cattle. schweiz arch tierheilkd 2004;146:515-521. 6. lefebvre rc, saint-hilaire e, morin i, et al: retrospective case study of fetal mummification in cows that did not respond to prostaglandin f2a treatment. can vet j 2009;50:71-76. 7. erb re, morrison ra: effect of mummified fetuses on the prolificacy of holsteins. j dairy sci 1957;40:1030-1035. 8. irons pc: hysterotomy by a colpotomy approach for treatment of foetal mummification in a cow. j s afr vet assoc 1999;70:127-129. 9. wolfe df, riddell mg, mysinger w, et al: a caudal flank approach for the collection of oviductal stage bovine embryos. theriogenology 1990;34:167-174. 10. sheldon im, lewis gs, leblanc sl: defining postpartum uterine disease in cattle. theriogenology 2006:65:15161530. 11. gee rw: the occurrence and treatment of foetal mummification in the bovine. aust vet j 1958;34:256-258. 12. azizunnesa bc, sutradhar bc: a case study on mummified foetus in a heifer. univ j zool rajshahi univ 2010;28:6163. 13. moore aa, richardson gf: uterine torsion and fetal mummification in a cow. can vet j 1995;36:705-706. 14. drost m: complications during gestation in the cow. theriogenology 2007;68:487-491. 15. de araújo aa, brasil af, moura aa, et al: fetal mummification in a cow with twin gestation and retention of the fetuses in the vagina: a case report. rev ciênc agron 2006;37:113-116. 16. yilmaz o, celik ha,yazici e, et al: twin mummified foetuses in a holstein friesian cow: a case report. vet med (praha) 2011;56:573-576. 17. leblanc sj, duffield tf, leslie ke, et al: defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows. j dairy sci 2002;85:2223-2236. 18. gilbert ro, sang ts, guard cl, et al: prevalence of endometritis and its effects on reproductive performance of dairy cows. theriogenology 2005;64:1879-1888. 19. sheldon im, dobson h: postpartum uterine health in cattle. anim reprod sci 2004;82-83:295-306. 20. rezakhani a, oloumi mm, ahmadinia: atrial fibrillation in a cow with fetal maceration. can vet j 1996;37:625-626. 21. american veterinary medical association: judicious therapeutic use of antimicrobials. https://www.avma.org/kb/policies/pages/judicious-therapeutic-use-of-antimicrobials.aspx; accessed december 5, 2013. clinical theriogenology • volume 6, number 1 • march 201451 51 figure 1. surgical site for removal of macerated fetus. figure 2. macerated fetal bones removed from a seven year-old angus cow via hysterotomy. (editor’s note: photographs in this paper are available in color in the online version of clinical theriogenology.) clinical theriogenology • volume 6, number 1 • march 2014 52 52 1 contact theresa beachler beachler@iastate.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11198, http://dx.doi.org/10.58292/ct.v17.11198 case report x chromosomal monosomy with bilateral ovotestes in a labrador retriever dog hayley hunt,a michael yaeger,b amanda fales-williams,b sharon wagner,a eleas wu,c theresa beachlerc aveterinary clinical sciences, college of veterinary medicine, iowa state university, ames, ia, usa bveterinary pathology, college of veterinary medicine, iowa state university, ames, ia, usa cveterinary diagnostic and production animal medicine, college of veterinary medicine, iowa state university, ames, ia, usa abstract a 2 year intact, phenotypically female labrador retriever dog was presented for evaluation of persistent white vaginal discharge after a perceived normal estrous cycle. on repeat evaluations, the dog was determined to be in persistent cytologic estrus ~ for 2 months. abdominal ultrasonography revealed suspected bilateral ovarian cysts and cystic endometrial hyperplasia. after ultrasound-guided cystic aspiration, the dog was treated with a 3 day course of gonadotropin-releasing hormone (gnrh). four months after initial treatment, the dog developed behavioral and physical signs of estrus that lasted ~ for 4 weeks; systemic progesterone concentrations never increased > 1.0 ng/ml. repeat abdominal ultrasonography revealed another cyst-like structure on the right gonad. similar treatment (cyst aspiration and gnrh treatment) was given. paired cystic fluid and blood serum hormone concentrations were analyzed at each aspiration; testosterone concentrations were higher in serum and cystic fluid. ovariohysterectomy and karyotyping diagnoses were bilateral ovotestis and x chromosomal monosomy (77, xo). keywords: dogs, sexual development, ovotestis, x chromosomal monosomy background disorders of sexual development are uncommon findings that contribute to infertility or altered reproductive characteristics and behaviors. sex chromosome aneuploidies are well-known causes of chromosomal sex abnormalities in domestic animals and have been reported in dogs.1–7 dogs have 78 total chromosomes compromised of 76 autosomes and 2 sex chromosomes (xx versus xy). x chromosomal monosomy (77, xo) or a single x chromosome has been diagnosed in multiple breeds, including doberman pinschers, miniature american eskimos, and miniature poodles, and mosaicism of the condition in a limited number of others.1–7 x chromosomal monosomy, also referred as turner’s syndrome in people; common clinical findings were small stature, webbing on the skin of the neck, congenital heart disease, metabolic and endocrine disturbances, and infertility due to hypergonadotropic hypogonadism.8 affected dogs with x chromosomal monosomy were infertile with frequent hypoplastic afollicular gonads, small stature, and excessive skin along the ventrum.1,2 we describe a case of x chromosomal monosomy in an externally phenotypic female dog with bilateral ovotestes. case presentation a 2 year, intact maiden female labrador retriever dog was presented for evaluation of persistent scant white vaginal discharge of ~ 2 months duration, 3 months after an apparently normal estrous cycle (figure 1). this cycle was the second estrous cycle with external signs of vulvar swelling and serosanguinous vulvar discharge lasting ~ for 4 weeks after a cycle that had similar signs for 3 weeks. dog was initially evaluated by the primary care veterinarian and started on a week’s course of twice-daily oral amoxicillin trihydrate/clavulanate potassium (13.75 mg/kg) and once daily oral enrofloxacin (10 mg/ kg) for suspected vaginitis. clinical signs of intermittent vaginal discharge appeared after cessation of treatment. dog weighed 23 kg with a body condition score of 4/9. physical examination revealed minimal to no vulvar edema with no vulvar discharge. mammary glands palpated within normal limits with 5 nipples on the left mammary chain and 4 nipples on the right mammary chain. all other physical examination findings were unremarkable. digital vaginal mailto:beachler@iastate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11198 2 citation: clinical theriogenology 2025, 17, 11198, http://dx.doi.org/10.58292/ct.v17.11198 examination was normal (no abnormalities in the caudal vaginal vault nor evidence of foreign material) and vaginal lavage appeared normal. serial vaginal cytologic examinations over the next 2 months identified cytological estrus (vaginal epithelial cells exhibiting > 80-90% cornification of superficial and anuclear epithelial cells) without evidence of inflammation (figure 2a).9 serum progesterone concentrations were 0.336 ng/ml. abdominal ultrasonography revealed bilateral cystic structures within gonads; right gonad had a 2.1 x 1.6 cm ovoid anechoic cyst and left gonad had an 8 mm cystic structure. uterus was identified from the cervix to the uterine horns and demonstrated diffuse endometrial hyperplasia with multifocal cystic structures throughout the endometrial wall. proximal right uterine horn additionally contained a focal amount of anechoic intraluminal fluid (figure 2b). differential diagnoses initial differential diagnoses primarily for the clinical finding of persistent cytologic estrus included cystic ovarian disease, exposure to exogenous hormones, or ovarian neoplasia. as per owner there was no exogenous hormonal exposure (e.g. topical hormone replacement therapy). diet consisted of a commercial dry adult dog food with no known source of elevated phytoestrogens. abdominal ultrasonography suggested cystic ovarian disease with concurrent endometrial hyperplasia. treatment ultrasound-guided aspiration utilized an extension set attached to a 3 ml syringe and a needle (22 g x 1.5”); 1.5 ml of translucent, gold color fluid was collected from the larger right cystic structure. immediately after aspiration, cystic structure collapsed. dog was treated with intramuscular gonadorelin (cystorelin®, boehringer ingelheim, ridgefield, ct) at 75 µg/kg once a day for 3 days. paired hormonal analyses of aspirated cysts (university of california, davis endocrinology laboratory) had marked increases in estradiol, progesterone, and inhibin b in cystic fluid compared to serum (table 1). serum progesterone remained at basal concentrations (0.75 ng/ml), and serum antimüllerian hormone (amh) was 0.18 ng/ml. dog was closely monitored at home with no reoccurrence in vaginal discharge and returned to vaginal cell populations consistent with cytologic anestrus within a month of initial therapy. abdominal ultrasonography (2 months after initial treatment) revealed gonads with no apparent follicular or cystic activity and marked improvement in endometrial hyperplasia and size of gross intramural cysts. approximately 4 months after initial treatment, dog entered behavioral estrus (vulvar swelling, serosanguinous vulvar discharge, and attraction to and from an intact male dog) within the household. it was elected to pursue breeding management, and the dog was presented for serial vaginal cytology and progesterone assays. during next 20 days, dog was assessed every 3-5 days. she remained within cytological proestrus; vaginal epithelial cell populations never increased figure 1. physical appearance (a) and genital mucoid discharge (b) figure 2. vaginal cytology (a) and transabdominal ultrasonography image (b) at initial cystic aspiration; note cornified vaginal epithelial cells composed of superficial and large intermediate cells (20 x magnification; bar = 200 µm) and large cystic structure in right gonad and intrauterine fluid in right uterine horn. http://dx.doi.org/10.58292/ct.v17.11198 citation: clinical theriogenology 2025, 17, 11198, http://dx.doi.org/10.58292/ct.v17.11198 3 > 50-70% cornification and progesterone concentrations remained < 1.0 ng/ml. after 3 weeks, the owner elected to discontinue breeding management and continued to monitor her closely. although bodily signs of estrus diminished during the following days with reduced vulvar edema and cessation of serosanguinous discharge, dog remained attractive to the intact male dog within the household. repeat examination 2 months later (6 months after initial treatment) revealed > 90% cornification on vaginal cytology, consistent with cytological estrus. focused reproductive ultrasonography findings were similar to earlier one; a large right gonadal cyst (1.4 x 1.0 cm) with no significant cystic changes in the left gonad. right gonadal cyst was again aspirated (ultrasound-guided) and the dog was again treated with intramuscular gonadorelin (cystorelin®). aspirated cystic fluid and serum were submitted for paired hormonal analysis. due to the unusual nature of the case, testosterone was also analyzed in addition to progesterone, estradiol, amh, and inhibin b; there were marked increases in cystic fluid progesterone and estradiol compared to serum with relatively high testosterone concentrations in cystic fluid and serum (table 2). ovariohysterectomy was performed and hormone analysis was not repeated. gonads and surrounding tissues did not have overt cystic lesions (figure 3). histopathology of gonads was consistent with bilateral ovotestes with central area with a solid sheet of interstitial cells that was multifocally interrupted by scattered seminiferous tubules lined by sertoli cells, and occasional primary spermatogonia noted in the left gonad (figure 4a). surrounding the seminiferous tubules, small spindle cells were present, consistent with thecal cells, and the gonads were covered by simple cuboidal epithelium. no follicles or ova were observed in either gonad. left ovotestis was surrounded by a tubular structure, most consistent with an oviduct. right ovotestis was surrounded by several tubules lined by columnar epithelium supported by a moderately cellular fibrovascular stroma, most consistent with epididymis. uterus contained diffuse cystic endometrial hyperplasia with moderate to markedly dilated endometrial glands (figure 4b). karyotyping (texas a&m molecular cytogenetics laboratory) had a total of 77 chromosomes characterized by a single x chromosome consistent with x chromosomal monosomy (77, xo). concurrent pcr testing for the sex determining region y (sry) gene was negative (figure 5). discussion normal sexual development in mammals involves 3 stages; chromosomal or genetic sex as determined at fertilization (xx versus xy), gonadal sex (ovaries versus testes), and the expression of the internal and external phenotypic sex through the internal reproductive tract and external genitalia.3 errors or alterations in any of the 3 components of sexual development cause disorders of sexual development in the dog and result in a wide range of anatomical abnormalities, physiologic behaviors, and infertility. initially, the dog in this case report was believed to have cystic ovarian disease due to the appearance of a large gonadal cystic structure on abdominal ultrasonography and signs of concurrent estrogenization of tubular genitalia, with the development of endometrial hyperplasia and prolonged cytologic estrus on vaginal cytology. ultimately, histopathology and chromosomal analysis were crucial for diagnosis of a disorder of sexual development, with an abnormality in both the chromosomal and gonadal sex. karyotype analysis suggested a pure x chromosomal monosomy within blood-derived cells (n = 30 cells). although a pure x chromosomal monosomy and aneuploidy explained the lack of follicular development, many of the internal and external physical characteristic, ovotestes (histopathology) is uncommon, with negative expression of the sry gene. unilateral or bilateral ovotestes were reported in xx/xy mosaicism or chimerism cases.10–12 in this case, karyotyping was performed on blood-derived leukocytes, not fibroblasts. the ultimate cause of ovotestis formation is unknown in this case and there was no evidence of mosaicism, chimerism, or even a mixoploidy in cell lines. sry-negative xx sex reversal (78, xx) with testes or ovotestes, has also been reported in multiple breeds of dogs, such as the american cocker spaniel, kerry blue terrier, and english cocker spaniel.3 similar findings have not yet been described to authors knowledge in a case of pure x chromosomal monosomy. table 1. hormonal analysis of paired serum and cystic fluid at initial cystic gonadal aspiration hormone serum cystic fluid progesterone (ng/ml) 0.75 15.1 estradiol (pg/ml) 21.8 3,689.7 amh (ng/ml) 0.18 2.8 inhibin b (pg/ml) 6 155,700 table 2. hormonal analysis of paired serum and cystic fluid at second (6 months after initial treatment) aspiration hormone serum cystic fluid progesterone (ng/ml) 1.6 > 30 estradiol (pg/ml) 14.2 9,750.7 amh (ng/ml) 0.84 > 12 inhibin b (pg/ml) < 6 73,640 testosterone (pg/ml) 179.7 3,188.1 figure 3. gross appearance of the gonads at ovariohysterectomy; right gonad (a) and left gonad (b). note thumb and index finger grasping the tip of each respective uterine horn. http://dx.doi.org/10.58292/ct.v17.11198 4 citation: clinical theriogenology 2025, 17, 11198, http://dx.doi.org/10.58292/ct.v17.11198 the initial presenting complaint was persistent scant mucoid vaginal discharge. a 6 month, doberman pinscher pup that had mucopurulent vulvar discharge was diagnosed as x chromosomal monosomy at prophylactic spay.5 unlike this case (had evidence of virilization with an enlarged clitoris) our case did not have enlarged clitoris despite bilateral ovotestes. abnormal estrous cycles have also been noted in other cases of x chromosomal monosomy, similar to this case with signs of hyperestrogenism.1,5,6 in our case, diffuse endometrial hyperplasia was also present and likely related to altered hormonal milieu. however, other findings of systemic hyperestrogenism were not noted on physical examination nor the comprehensive bloodwork prior to ovariohysterectomy. the ultimate origin of observed gonadal cystic structures was not fully determined on histopathological analysis at ovariohysterectomy since there were no gross cystic dilations of the ovary or surrounding structures. we hypothesized that figure 4. light microscopic images of reproductive tract stained with hematoxylin and eosin; scattered seminiferous tubules within stroma of left ovotestis (a [10 x magnification]) and diffuse endometrial hyperplasia and glandular dilation (b [20 x magnification]). http://dx.doi.org/10.58292/ct.v17.11198 citation: clinical theriogenology 2025, 17, 11198, http://dx.doi.org/10.58292/ct.v17.11198 5 dilation of the tubular structures within or surrounding the right ovotestis were most consistent with mesonephric ducts or epididymis, especially due to concurrent high testosterone concentrations. dog’s external characteristics and behavior, including intermittent vulvar edema, serosanguinous vulvar discharge, and attraction to and from intact males, may also be attributable to both the localized and systemic hormonal milieu. serum amh concentrations were correlated to follicular reserve and litter size in 78, xx intact female dogs, and were used for diagnosing testicular tissue in 78, xy intact males.13,14 increases in amh have also recently been examined to identify testicular tissue in cases of canine disorders of sexual development.15 serum amh concentrations obtained at the second cystic aspiration were consistent with the laboratories current reference intervals for both intact females and intact males, whereas serum amh remained within the inconclusive interval for both intact females and males at the first cystic aspiration. the amh concentrations detected in this case most likely due to the duality of the internal gonads and sertoli cell amh production, as no follicles were observed in either gonad. we presented a case of x chromosomal monosomy with bilateral ovotestis in a phenotypic female dog using karyotype analysis and histopathology of gonadal tissue. conflict of interest none to declare. acknowledgement authors thank the uc davis endocrinology laboratory and dr. allen conley for interpreting hormone results. learning points • sex chromosomes disorders are relatively uncommon findings in dogs • karyotyping and histopathology of internal reproductive organs are crucial for final diagnosis in suspected cases of disorders of sexual development • x chromosomal monosomy may be considered as a cause in cases of abnormal estrous cycles or suspected infertility references 1. szczerbal i, malek e, rigillo a, et al: non-mosaic x monosomy (77,x) in a female dog with signs of virilization. j appl genet 2023;64:169-172. doi: 10.1007/s13353-022-00739-3 2. szczerbal i, switonski m: clinical cytogenetics of the dog: a review. animals 2021;11:947. doi: 10.3390/ani11040947 3. poth t, breuer w, walter b, et al: disorders of sex development in the dog-adoption of a new nomenclature and reclassification of reported cases. anim reprod sci 2010;121:197-207. doi: 10.1016/j. anireprosci.2010.04.011 4. noto nt, raudsepp t, kolb e, et al: a rare finding of double barr bodies and x-monosomy/x-trisomy mosaicism in a dog with presumed idiopathic epilepsy. vet clin pathol 2023;52:583-587. doi: 10.1111/vcp.13261 5. smith fw, buoen lc, weber af, et al: x-chromosomal monosomy (77, xo) in a doberman pinscher with gonadal dysgenesis. j  vet  intern med 1989;3:90-95. doi: 10.1111/j.1939-1676.1989. tb03085.x 6. lofstedt rm, buoen lc, weber af, et al: prolonged proestrus in a bitch with x chromosomal monosomy (77, xo). j am vet med assoc 1992;200:1104-1106. doi: 10.2460/javma.1992.200. 08.1104 7. mayenco aguirre am, padilla ja, flores jm: canine gonadal dysgenesis syndrome: a case of mosaicism (77,xo-78xx). vet rec 1999;145:582-584. doi: 10.1136/vr.145.20.582 8. gravholt ch, viuff mh, brun s, et al: turner syndrome: mechanisms and management. nat rev endocrinol 2019;15:601-614. doi: 10.1038/s41574-019-0224-4 9. johnston sd, root kustriz mv, olson pns: vaginal cytology. in: johnston sd, root kustriz mv, olson pns: editors. canine and feline theriogenology. philadelphia, pa; saunders: 2001. p. 32-40. 10. hare wc: intersexuality in the dog. can vet j 1976;17:7-15. pmid: 769934. 11. bosu wt, chick bf, basrur pk: clinical, pathological and cytogenetic observations on two intersex dogs. cornel vet 1978;68: 375-390. 12. wright s, lago-alvarez, y, champion c, et al: sexual development disorder in a dog. clinical theriogenology 2024;16:10439. doi: 10.58292/ctv16.10439 ba figure 5. blood karyotype analysis of 30 cells; representative cytogenetic analysis from 1 depicted cell (a) and molecular analysis of sex chromosomes (b). http://dx.doi.org/10.58292/ct.v17.11198 https://doi.org/10.1007/s13353-022-00739-3 https://doi.org/10.3390/ani11040947 https://doi.org/10.1016/j.anireprosci.2010.04.011 https://doi.org/10.1016/j.anireprosci.2010.04.011 https://doi.org/10.1111/vcp.13261 https://doi.org/10.1111/j.1939-1676.1989.tb03085.x https://doi.org/10.1111/j.1939-1676.1989.tb03085.x https://doi.org/10.2460/javma.1992.200.08.1104 https://doi.org/10.2460/javma.1992.200.08.1104 https://doi.org/10.1136/vr.145.20.582 https://doi.org/10.1038/s41574-019-0224-4 https://doi.org/10.58292/ctv16.10439 6 citation: clinical theriogenology 2025, 17, 11198, http://dx.doi.org/10.58292/ct.v17.11198 13. hollinshead fk, walker c, hanlon dw: determination of the normal reference interval for antimullerian hormone (amh) in bitches and use of amh as a potential predictor of litter size. reprod domest anim 2017;15:35-40. doi: 10.1111/rda.12822 14. prapaiwan n, manee-in s, thanawongnuwech, et al: antimullerian hormone levels in serum and testes of male dogs: relations with neuter status and bilateral abdominal cryptorchidism. theriogenology 2023;208:171-177. doi: 10.1016/j.theriogenology. 2023.06.015 15. walter b, flock u, leykam c, et al: serum anti-mullerian hormone concentrations as a diagnostic tool to identify testicular tissue in canine disorders of sexual development. domest anim endocrinol 2022;78:106654. doi: 10.1016/j.domaniend.2021. 106654 http://dx.doi.org/10.58292/ct.v17.11198 https://doi.org/10.1111/rda.12822 https://doi.org/10.1016/j.theriogenology.2023.06.015 https://doi.org/10.1016/j.theriogenology.2023.06.015 https://doi.org/10.1016/j.domaniend.2021.106654 https://doi.org/10.1016/j.domaniend.2021.106654 2010: equine obstetrics: mutation and delivery by traction* equine obstetrics: mutation and delivery by traction* t. l. blanchard, d.d. varner, s.p. brinsko, t. taylor department of large animal clinical sciences; college of veterinary medicine and biomedical sciences, texas a&m university, college station, tx, usa introduction successful intervention in equine dystocia requires an adequate understanding of normal parturition and a thorough knowledge of the causes of dystocia. the clinician must first accurately diagnose contributing fetal and/or maternal abnormalities in order to properly formulate a therapeutic plan. judgment and experience are paramount in performing obstetrical procedures so that unnecessary injury to the dam and/or fetus is avoided and the mare’s subsequent fertility is maintained. parturition is typically divided into three characteristic stages (1 to 3): stage 1, the preparatory phase, usually lasts 30 minutes to four hours. during this stage the fetus rotates into dorsosacral position and uterine contractions force the foal and surrounding fetal membranes into the dilating cervix. stage 2, the most active stage of parturition, involves passage of the foal through the birth canal and usually requires only 20-30 minutes. this phase begins with fetal passage into the birth canal, is accompanied by vigorous abdominal straining and rupture of the chorioallantoic membrane, and ends with delivery of the foal. stage 3 involves passage of the fetal membranes and uterine involution. if either the first or second stage of parturition is prolonged, dystocia should be suspected. the reported incidence of dystocia in the mare is 1-4%. most equine dystocias are attributed to abnormalities in fetal presentation, position and posture, with postural abnormalities being the major contributor, due to the long fetal extremities. fetal oversize appears to be rare in horses. maternal contribution to equine dystocia, such as defects in the abdominal wall, occasionally occurs. prompt examination of the birth canal and fetus is indicated when dystocia is suspected. the equine fetal membranes separate rapidly from the endometrium during parturition thereby depriving the fetus of oxygen if it is not promptly delivered after the onset of second stage labor. early intervention maximizes the chances of delivery of a viable foal with no injury incurred by the dam. keywords: dystocia, obstetrics, parturition, mare obstetrical equipment high quality obstetrical equipment, kept clean and sterilized between uses, should be readily available for obstetrical intervention. minimal equipment should include materials necessary to cleanse the hindquarters of the mare, obstetrical chains or straps with handles, sleeves, lubricant, and fetatome with saw wire and krey hook. a clean bucket, cotton, tail wrap, and disinfectant are used to clean and prepare the mare’s hindquarters and perineal area for examination. proper lubrication is required to protect the genital tract during manipulative procedures. water-based lubricants, such as lubrivet™ (butler schein animal health, dublin, oh) or other carboxymethylcellulose solutions are generally preferred. good protection is provided for the fetus and genital tract with such lubricants, and large volumes can be pumped into the uterine lumen and around the fetus through a sterile stomach tube when necessary. if fetotomy is necessary, petroleum jelly can be applied to the fetus and birth canal for additional protection. however, if the obstetrician anticipates that a cesarean section will be necessary, petroleum jelly should be avoided since it is not water soluble and, if spilled into the abdominal cavity, can induce peritonitis. examination of the mare the mare should be standing during the initial examination when possible. restraint should be the minimum required to protect both the clinician and mare from injury. caudal epidural anesthesia * originally published by the society for theriogenology and the american college of theriogenologists as publication e-1 (3/89) in the theriogenology handbook. 119 (1.0-1.25 ml 2% lidocaine/100 kg body weight) is occasionally necessary to minimize straining and facilitate examination of the fetus and birth canal. slowly walking the mare during the examination is usually sufficient to control straining. sedation is avoided whenever possible because of associated disturbances in fetal respiratory and cardiac function. if sedation is necessary, intravenous injection of 0.07 mg/kg acepromazine maleate or an initial injection of 0.44 mg/kg xylazine followed in 4-8 minutes by intravenous injection of 0.044 mg/kg butorphanol tarterate will usually reduce straining and provide more pronounced sedation and analgesia. rarely, it is necessary to induce general anesthesia and elevate the mare’s hindquarters to provide room for examination and manipulation. strict sanitation and effective lubrication should always be maintained whenever the genital tract is invaded. the obstetrician’s hands and arms should be scrubbed with a disinfectant soap prior to examination. the fetus and birth canal are thoroughly examined for evidence of previous trauma and the viability, presentation, position, and posture of the fetus are determined. the fetus is also evaluated for deformities, and the amount of room available for manipulation is assessed. obstetrical operations mutation of abnormal presentation, position, and posture of the fetus, followed by delivery with traction, is the most common method of relieving dystocia in mares. in order to permit passage of a fully developed fetus, the extremities must be extended. to accomplish mutation it is helpful to first repel the fetus from the maternal pelvis into the uterus where more space is available for manipulation to correct fetal malposture or malposition. such maneuvers are sometimes facilitated by first pumping 4-8 liters of lubricant into the uterine lumen and around the fetus. an alternate form of delivery should be selected if the uterus is devoid of fetal fluids and contracted tightly around the fetus, since repulsion in such cases is likely to result in uterine rupture. to correct flexion of an extremity, the proximal end should be repelled while the middle portion is rotated laterally. traction can then be applied to the distal end of the extremity, either by hand or by traction on a chain or strap placed around the pastern, until the limb is fully extended. this reduces the longitudinal arc the foot must go through in the birth canal, effectively increasing room for limb extension. extreme care should be taken to protect the genital tract from injury when applying traction on the distal end of a flexed extremity. uterine rupture is commonly associated with this manipulation, particularly when correcting hock flexion in posterior presentation, as more room is required to extend a flexed hindlimb than a flexed forelimb. the operator should always cup the hand over the foal’s hoof as it is brought forward. to correct lateral or ventral head posture, the fetal limbs and/or body are repelled while the muzzle or jaw is grasped and pulled toward the pelvic inlet. it is sometimes easier to correct abnormal head or neck posture with one of the forelimbs flexed at the carpus, giving more space than that offered when the forelimbs are extended. if the fetus is in a transverse ventral position, it is often easier to extend the two hindlimbs and proceed with delivery of a posteriorly presented fetus than to convert the fetus into anterior presentation with the forelimbs and head and neck extended. once the fetus is presented normally (i.e., anterior longitudinal presentation; dorsosacral position; with the extremities, including the head and neck, extended), delivery can proceed. if the fetus is presented posteriorly, delivery can proceed once the hindlimbs are extended. obstetrical chains or straps are placed around the fetlocks, and an additional loop is placed in a half-hitch around the pasterns, with the eye of the straps or chains on the dorsal aspect of the limb. traction devices should not be used on the head of a live fetus, except where necessary to direct the head into the pelvic inlet. extractive force should never be applied to the head as this may result in injury to the bones of the skull or cervical spine. the amount of traction applied to the equine fetus should not exceed the force that can be applied by the pulling of two or three persons. this amount of traction is seldom necessary since relative fetal oversize is uncommon in the equine. traction should only be applied during the dam’s abdominal press. if delivery does not progress rapidly once traction is applied, the obstetrician should examine the birth canal to ensure that a flexed limb is not situated between the fetal body and the maternal pelvis. delivery should be as prompt as possible since the umbilicus may become compressed in the maternal pelvis, 120 reducing blood supply to the fetus. this is particularly important with delivery of the fetus presented posteriorly, as rupture or compression of the umbilicus commonly occurs and quickly leads to fetal anoxia. the reader is referred to various veterinary obstetrics textbooks for indepth study of mutation procedures and delivery by traction. references 1. vandeplassche m, spincemaille j, bouters r, et al: symposium (1): some aspects of equine obstetrics. equine vet j 1972;4:105-109. 2. vandeplassche m: obstetrician’s view of the physiology of equine parturition and dystocia. equine vet j 1989;12:45 49. additional reading 1. roberts sj: veterinary obstetrics and genital diseases. 3rd ed. woodstock, vt: published by the author; 1986. p. 277 352. 2. benesch f, wright jg: veterinary obstetrics. london: bailliere, tindall, and cox; 1950. p. 121-302. 121 122 omniblank: contact jennifer roberts jennifer_3.roberts@boehringer-ingelheim.com © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11644, http://dx.doi.org/10.58292/ct.v17.11644 review report anovular conditions impacting fertility in dairy cattle and considerations for clinical management jennifer roberts,a clare scullyb aboehringer ingelheim animal health, duluth, ga, usa bdepartment of veterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la, usa abstract reproductive performance is a primary driver of herd profitability and reproductive failure or infertility is one of the main causes of culling in usa dairy herds. nearly ⅓ of dairy cows in usa experience anovulation in early lactation leading to increases in days open and reductions in first service conception rates. anestrus and anovulation in dairy cows can be attributed to many causes including parity, postpartum uterine or systemic diseases, nutritional deficiencies, body condition loss, and stress. identification and management of anovular cows, including strategies to reduce the incidence of anovulation, are critical to the overall reproductive performance of the herd. the aim of this review is to highlight causes, recent research on anovulatory conditions, and clinical approaches to management to improve dairy herd reproductive outcomes.  keywords: anovulation, anestrus, dairy cattle, infertility  introduction optimizing profitability is a primary focus of producers and veterinarians in the usa dairy industry. one of the drivers of maintaining profitability within a dairy herd is the ability to achieve optimal reproductive performance and minimize the number of days open after calving. many factors influence the number of days open or the calving to conception interval including, but not limited to, herd management, nutrition, metabolic disease during early lactation, uterine health, and ovarian function. failure of cyclicity in early lactation or anovulation has been a focus of researchers and clinical veterinarians for decades. in cattle, anovulation is broadly defined as lack of ovulation during the normal 21-day estrous cycle. the term anovulation is often used interchangeably with anestrus; however, it is important to distinguish anovulation from anestrus as it is not uncommon for cows in the postpartum period to have a silent estrus at first postpartum ovulation. four types of anovulation have been described in cattle. type i involves follicle growth only to emergence, type ii is characterized by follicle growth to deviation with failure to reach ovulatory size, type iii is follicle growth to ovulatory size or larger and may be referred to as persistent or cystic follicles, and type iv is anovulation as a result of a prolonged luteal phase1,2 in dairy production, postpartum anovulation of any type can delay cows’ ability to conceive and result in economic losses due to increases in the number of days open or culling due to subfertility.  incidence of anovulation in the usa varies by region, farm, and parity; analysis of data across 4 geographical regions identified 28.5% anovulation rate (8.1-49.6% among farms).3 the ranges emphasized the variability of the condition across various management systems. in addition, there is also opportunity for veterinarians to influence herds’ reproductive health and performance by focusing on diagnostics and interventions to reduce the economic impact of anovular conditions. postpartum anovulation uneventful calving followed by normal uterine involution and return to cyclicity are critical milestones in the postpartum period of dairy cows. although this appears simple, in theory, the return to cyclicity depends upon an intricate series of events involving an array of molecular and cellular signaling, with the production and release of hormones throughout the hypothalamic-pituitary-ovarian axis. the modern dairy cow experiences many stressors in the postpartum transition period that can disrupt the delicate balance of this complex mailto:jennifer_3.roberts@boehringer-ingelheim.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11644 citation: clinical theriogenology 2025, 17, 11664, http://dx.doi.org/10.58292/ct.v17.11664 3 biological phenomenon. on average, the first postpartum ovulation occurs around 30 days in milk (dim); however, this can be impacted by nutritional status, uterine health, and presence or absence of concurrent diseases.4-7 during the postpartum period, it is common to observe a voluntary waiting period (vwp) during which, despite cows exhibiting estrous signs, producers elect to delay insemination to allow uterine involution completion to ensure uterine environment readiness for subsequent pregnancy. length of vwp varies depending on farm management but is typically 60-80 days postpartum. as many as 20-30% of cows may be anovular at the end of the vwp, which can decrease conception rates after first artificial insemination (ai) and increase days open.8 postpartum diseases have substantial impact on ovular status of dairy cows. among anovular cows, 63.8% were diagnosed with at least 1 postpartum health problem.7 prevalence of retained fetal membranes, metritis, ketosis, and digestive disease (e.g. displaced abomasum) and lameness were significantly higher among anovular compared to cyclic cows. additionally, cows experiencing 1 health event had 2-fold increase in risk of anovulation whereas cows with 2 or more health events were 3 times more likely to be anovular compared to cows that remained healthy in the postpartum period.7  nutritional status also has an important role in return to cyclicity in the postpartum period. the transition from the dry period to early lactation involves a major shift in energy requirements as milk production commences whereas dry matter intake does not increase as rapidly to keep up with the demand for increased energy. consequently, dairy cows in early lactation experience a state of negative energy balance (neb) that can result in diversion of nutritional resources away from the reproductive tract.2 the mechanism by which neb delays onset of first postpartum ovulation has been attributed to low blood concentrations of glucose, insulin and insulin-like growth factor-i, and inhibition of lh pulse frequency.4 in addition, increases in circulating non-esterified fatty acids (nefa) and ketone bodies that are often associated with neb in cows can have negative effects on oocytes and granulosa cells9 and high nefa concentrations have been associated with higher risk of anovulation around 63 dim.6 a study evaluating blood metabolites and activity data in cows ovulating early or late in the postpartum period, defined as before or after 33 dim, implied that earlier ovulating cows had lower serum concentrations of free fatty acids, beta-hydroxybutyrate, and haptoglobin and spent more time eating and less time resting in the first 3 weeks after calving compared to cows that ovulated later. additionally, early ovulating cows lost less body condition in the first 9 weeks postpartum.5 in a study evaluating 942 holstein primiparous and multiparous cows, those with a lower body condition score (bcs) at 35 dim were more likely to be anovular compared to those with higher bcs.7 the interconnectedness of nutrition, postpartum health, and reproductive performance underscores the importance of focusing on the postpartum period when working up cases of poor reproductive performance in dairy herds.  diagnosis of postpartum anovulation is via transrectal palpation or ultrasonographic examination or measurement of serum or milk progesterone concentrations. the most practical and commonly implemented methods in commercial dairies are transrectal ultrasonography or palpation. absence of corpus luteum at either of 2 ultrasonographic examinations performed at a 14-day interval has been used as criteria for determining an anovular state.7 anovular cows diagnosed with small ovaries via transrectal palpation alone may be referred to as static; however, even in the case of small ovaries, there may still be follicular activity and growth beyond emergence of the deviation stage of development, making ultrasonography a more reliable means to assess the ovarian activity of anovular cows.1 despite the apparent lack of ovarian activity in cows with small ovaries on transrectal palpation or ultrasonography, follicles that have reached the deviation stage may be capable of responding to exogenous gonadotropin releasing hormone (gnrh) and presynchronization protocols using timed injections of gnrh and prostaglandin have been successful in reducing the prevalence of anovular cows in early lactation.1,10-12  resolution of anovular conditions can be achieved using progesterone exposure to reinitiate the hypothalamic responsiveness to estradiol to stimulate an lh surge from the anterior pituitary. in anovular cows, a minimum of 3 days of progesterone exposure improved ovulation.13 from a practical standpoint when designing protocols to be implemented on farm, 7 days of progesterone exposure is more commonly used to reduce labor associated with additional handling of synchronized cows. although exogenous progesterone sources are available for use in lactating dairy cows, a more widely adopted strategy to increase circulating progesterone involves a presynchronization protocol that induces ovulation with subsequent formation of a cl and increase in endogenously produced progesterone. presynch-ovsynch and double ovsynch are presynchronization protocols used in many dairy herds. multiple studies have implied that gnrh and prostaglandin (pgf)-based presynchronization like double ovsynch is more effective for inducing ovulation and decreasing the number of anovular cows prior to the breeding ovsynch portion of the protocol as compared to a pgf-based presynchronization like presynch-ovsynch.14 cows with progesterone concentrations < 1 ng/ml at the final pgf injection of ovsynch are more likely to experience a short luteal phase and significantly lower pregnancy rate per ai (p/ai) than cows with serum progesterone > 1 ng/ml.8 therefore, an important component in management of anovular cows in the postpartum period should be implementation of presynchronization protocols capable of achieving higher serum progesterone concentrations at final pgf injection of the protocol.  uterine health and anovulation uterine involution and clearance of uterine contamination in the postpartum period is another critical factor in return to cyclicity. reproductive challenges of cows experiencing retained fetal membranes, puerperal metritis, and delayed involution have been well documented.3,15-19 effects of postpartum uterine diseases on fertility and cyclicity have been attributed to bacterial uterine contamination leading to systemic inflammation and endotoxins in follicular fluid and systemically. the inflammatory process may lead to a decrease in lh secretion, slower follicular growth and reduced size of the dominant follicle, and reduced steroidogenesis within the follicle, all of which negatively impact return to cyclicity.3 the intimate association of the uterine vein and ovarian artery that allow for countercurrent exchange of prostaglandin from http://dx.doi.org/10.58292/ct.v17.11664 4 citation: clinical theriogenology 2025, 17, 11664, http://dx.doi.org/10.58292/ct.v17.11664 the uterus to the ovary also has the potential to transport endotoxins and other proinflammatory cytokines from an infected postpartum uterus to the ovary. endotoxins reach follicular fluid causing inflammation, oxidative stress, and ultimately, delayed oocyte development and ovulation.20 anovular cows had higher concentrations of endotoxin in follicular fluid and a greater degree of systemic inflammation as measured by serum haptoglobin compared to ovular cows in the early postpartum period.21 however, it is important to note that a robust local inflammatory response within the uterus on the day of calving is positively correlated with return to cyclicity.21 the reported benefits of inflammation on the day of calving are consistent with other research indicating that dysregulation of the inflammatory process in the immediate postpartum period contributed to retained fetal membranes.22 consequently, some degree of inflammation is beneficial to postpartum uterine health whereas increased inflammation associated with increased bacterial contamination of the uterus is detrimental to fertility.  clinical endometritis or subclinical endometritis, identified clinically as cows with purulent vaginal discharge (pvd) or abnormal endometrial cytology after 21 days postpartum, respectively, also result in negative effects on postpartum fertility and cyclicity.3,23 cytological endometritis (ce) is often used as a proxy for subclinical endometritis. cows with subclinical endometritis may go undiagnosed by farm personnel due to lack of visible clinical signs. in contrast, cows with pvd, or clinical endometritis, are easily identified and may be managed more aggressively to address uterine inflammation. clinically, these conditions are often considered less severe compared to puerperal metritis but any postpartum condition that results in prolonged inflammation has the potential to reduce first service conception rates, increase days open and reduce fertility, thereby contributing to increased risk of culling and decrease in productive life of dairy cows.  cows diagnosed with ce experience significantly prolonged postpartum anovulation (or = 1.52) compared to cows that are not diagnosed with ce.6 combined with anovulation, effects of ce are even more substantial. individual and combined effects of anovulation and cytological endometritis in 1,569 cows across 3 states were evaluated.23 cyclicity was determined via serial transrectal ultrasonographic examinations at 35 and 49 dim. cows without a corpus luteum at both time points were diagnosed as anovular. cows that were anovular and were diagnosed with ce had a first service pregnancy per ai (p/ai) of 21.3% compared to healthy cyclic cows that achieved p/ai of 46.7% whereas cows that were healthy but anovular had a p/ai of 37.9%.23 in addition, cows that were anovular, with or without ce, had more days open compared to healthy cows with or without ce. although this study demonstrated that uterine disease combined with anovulation has an additive negative effect on reproductive performance, the reduction in p/ai for healthy, anovular cows in this study reinforced the importance of return to cyclicity and its impact on fertility.  in another study, the combined effect of anovulation and purulent vaginal discharge (pvd) in 10,995 cows in 16 herds across usa were evaluated.3 diagnosis of pvd was made at 28 dim and anovulation was diagnosed via transrectal ultrasonographic examinations at 40 and 54 dim. the mean prevalence of anovulation and pvd was 28.5 and 25.7%, respectively.3 cows diagnosed with both pvd and anovulation had decreased first service conception rates and increased days open compared to healthy cows or cows with either pvd or anovulation alone.  cows that are pregnant at 130 dim are more likely to maintain or gain body condition in the first 30 days of the next lactation, leading to a greater chance of pregnancy and reduced pregnancy losses after insemination.24 this concept, referred to as the high fertility cycle, relies on prompt insemination of cows in early lactation with the goal of maintaining health and fertility in each subsequent lactation. anovulation and uterine disease in the postpartum period have the potential to make this goal unattainable, leading to farm reproductive losses. therefore, management practices should be aimed at optimizing postpartum uterine health and reducing the herd prevalence of metritis and endometritis, as the effects of these conditions combined with anovulation are detrimental to herd reproductive performance. large ovary syndrome and anovulation large ovary syndrome (los) is a collection of conditions that may cause anovulation in the cow and includes ovarian cysts, abscesses, hematomas, and neoplasia. among los cases referred to a veterinary teaching hospital, 60% were granulosa theca cell tumors (gtct).25 although rare, gtct is the most common ovarian neoplasm reported in cattle and leads to anovulation via production of anti-müllerian hormone (amh), inhibin, and estradiol that arrest follicular development on the contralateral ovary through negative feedback to the hypothalamus and pituitary.26 the predominance of gtct cases in referral centers is most likely related to the fact that other causes of los, particularly ovarian cysts, are managed on farm by the clinician and not referred. in a commercial dairy, it is unlikely that gtct cases would be treated as it is more economical to cull these animals from the herd. however, in animals of high value or genetic merit, the clinician may be requested to investigate or treat an animal with suspected gtct. the diagnosis of gtct can be presumptively made via transrectal ultrasonography and confirmed via hormonal assays or histopathology; amh and inhibin may be used to confirm gtct diagnosis versus other causes of los.26,27 unilateral ovariectomy to remove the affected ovary can lead to favorable outcomes including return to fertility in the majority of cases; however, other reproductive comorbidities such as pneumovagina or urovagina may contribute to culling.25 therefore, it is important to consider all factors, including economics and genetic value of the animal, prior to investing in gtct treatment to resolve anovulation in these cases.  cystic ovarian disease cystic ovarian disease (cod), another condition broadly classified within los, represents a significant challenge in dairy cattle management, impacting reproductive efficiency and economic sustainability within the industry. this section aims to elucidate the complexities of cod, from its etiology to its diagnosis and management strategies. cystic ovarian disease is defined as the presence of anovulatory follicles > 17 mm in diameter in the ovaries that interfere with cyclicity and persist for more than 6 days, with low to intermediate concentrations of progesterone http://dx.doi.org/10.58292/ct.v17.11664 citation: clinical theriogenology 2025, 17, 11664, http://dx.doi.org/10.58292/ct.v17.11664 5 indicative of the absence of a corpus luteum.28,29 the fluid-filled structures in the ovaries are classified as follicular or luteal cysts based on steroid production. the disease etiology and progression are not entirely understood; however, environmental and hereditary factors are speculated that affect clinical outcomes. the most consistent hypothesis for occurrence of cod is a dysfunction of the neuroendocrine system, specifically in the hypothalamic-pituitary axis. cystic ovarian disease is caused by a disturbance of the estrogen positive feedback on the hypothalamus that leads to failure of preovulatory lh surge and consequently leads to anovulation.1 the disturbance is associated with an unresponsiveness of the hypothalamus, caused by either decreased estradiol or abnormal progesterone concentrations. another factor that can predispose cows to develop ovarian cysts is increased milk production and associated stress, through the adrenocorticotropic hormone (acth) and cortisol secretion. the secretion of both acth and cortisol suppresses the preovulatory lh surge and increased acth can downregulate the expression of lh receptor mrna in the ovarian follicle.30 additionally, delayed uterine involution, retained fetal membranes, and peripartum hypocalcemia can have a role in the development of cysts.31 the heritability of this disease is speculated to be low but breeds and certain bloodlines selected for higher milk production had increased incidence of ovarian cysts.32,33 prevalence of cod varies; follicular cysts occurred in 6 to 19% of lactating dairy cows1 and was 4.6% in abattoir sourced tracts.34 ovarian cysts develop more commonly in dairy cattle, specifically during the early postpartum period and before the first ovulation. presence and development of these fluid-filled structures affect fertility by increasing the calving intervals, delaying the first postpartum ovulation and estrus, and subsequently increasing the days to first ai.35,36  although through the years transrectal palpation has been the most common method of diagnosing cod, the diagnostic accuracy of palpation alone, as well as the ability to differentiate follicular versus luteal cysts, is relatively poor.37 as a way of improving diagnosis and management of cystic cows, other tests can be performed to better assess ovarian cysts. ultrasonography is the most reliable tool in diagnosing cod since native anatomical ovarian structures can be differentiated from true cysts. these cysts are distinguished via ultrasonography by their wall thickness. follicular cysts have a wall thickness of ≤ 3 mm whereas luteal cysts are ≥ 3 mm thick.38 to further differentiate cystic ovarian structures, color doppler ultrasonography (cdu) was used to measure ovarian vascular function; follicular cysts had notably lower blood flow values than luteal cysts and the diagnostic accuracy of cdu was superior to b-mode ultrasonography.39 the combination of wall thickness measurement and blood flow area measurements increased the sensitivity to differentiate a follicular from a luteal cyst. the evaluation of progesterone concentrations in combination with the ultrasonography analysis may also aid in differentiation of the type of cyst, as progesterone concentration thresholds for follicular and luteal cysts are < 1 ng/ml and ≥ 1 ng/ml, respectively.39 some behavioral abnormalities that might be observed in cattle with cod are related mostly to the neuroendocrine imbalance. one abnormality the cow may present with is  nymphomania, a frequent, irregular, and prolonged estrus. other clinical signs at evaluation include elevation of tail-head, relaxation of pelvic ligaments, and even masculine phenotype in chronic cases.38 although all of these are possible clinical signs of the disease, prolonged anestrus due to failure of ovulation is the most commonly observed manifestation of cod in dairy cattle. it is recommended to perform transrectal palpation or transrectal ultrasonography for evaluation of the cyst wall thickness and plasma progesterone concentrations to have a more accurate diagnosis of cod.  for many years, the treatment of cod was manual rupture of the cysts via transrectal palpation. however, this treatment is no longer recommended due to the potential negative effects including ovarian trauma, hemorrhage, and an increased risk of ovariobursal adhesions.31 fortunately, many studies have reported that approximately 60% of cystic cows undergo spontaneous recovery and cyst regression by the first postpartum ovulation.31 the mechanism by which the cysts regress spontaneously is still unknown. nowadays the most common treatment for cod involves the use of gonadotropin releasing hormone (gnrh) to stimulate an lh surge from the anterior pituitary, resulting in luteinization of the cyst.40 the efficacy of these treatments varies across studies and the significance, or lack thereof, of reported rates of resolution is likely attributed to small numbers of cows in each study. human chorionic gonadotropin hormone (hcg) has also been used in the treatment of cod as it has lh-like activity and luteinizes the cysts, leading to increased progesterone concentrations;31,41 the resulting luteal cysts are then treated with prostaglandin f2α (pgf2α) that cause the structure to regress, resulting in expression of estrus within 2-5 days.  treatment with gnrh and pgf2α in combination is beneficial for luteal and follicular cyst regression and early return to estrus.31 the use of exogenous progesterone with or without the ovsynch protocol are other options for the treatment of cod in cows. exogenous progesterone treatment can reestablish the hypothalamic response to the estrogen positive feedback mechanism and return the animal to normal cyclicity. single controlled internal drug release device (cidr) for 14 days in cows with endocrinologically active cysts (determined via circulating estradiol concentrations) initiated emergence of a new follicular wave within 3 days after cidr insertion.42 however, this treatment was ineffective for cysts that were not estrogen producing. the ovsynch artificial insemination protocol has also been used successfully on cows with cystic ovaries. in a study comparing ovsynch to an exogenous progesterone source for 7 days followed by an injection of pgf2α (with estrus detection as an option) for treatment of cystic cows, the use of the ovsynch protocol increased the percentage of cows inseminated, but the conception rates and pregnancy rates did not differ across groups.35 using the findings of this  study, an economic analysis was conducted showing that treatment of cod with a timed ai protocol had an $11.39 advantage over a cidr-based protocol.43 ultimately, the decision on which protocol to implement for management of cod depends on the farm’s ability to achieve protocol compliance, value of the cow, stage of lactation at diagnosis, and the cost benefit ratio of each treatment option.  cystic ovarian disease remains as a substantial concern in dairy cattle management, demanding ongoing research efforts and collaborative initiatives. preventing cod necessitates a multifaceted approach encompassing genetic http://dx.doi.org/10.58292/ct.v17.11664 6 citation: clinical theriogenology 2025, 17, 11664, http://dx.doi.org/10.58292/ct.v17.11664 selection, nutritional management, prioritization of animal welfare, and evidence-based reproductive protocols. by enhancing our understanding of its etiology, refining diagnostic techniques, and implementing effective management strategies, clinicians can work with dairy clients to minimize the economic burden of cod and optimize reproductive outcomes. technology for identification of anovular cows the availability and affordability of agricultural technologies has been increasing over the past decade. the use of automated activity monitors (aam) on dairy farms has enabled producers to gather more data on health and reproductive parameters than ever before. automated activity monitor systems use sensor technology incorporated into ear tags, neck collars, leg bands, or boluses allowing dairy producers to monitor cow activity and rumination. the associated software determines baseline data for each cow and monitors daily movement facilitating detection of changes in activity patterns that are associated with behavioral estrus. the challenge with increasing amounts of data is determining how to best use the available data to make management decisions to improve production at the herd level. with respect to anovulation, the use of aam in the postpartum period may enable farmers to identify anovular cows and act more promptly to improve reproductive outcomes.  anovular cows in the postpartum period were identified via aam; 35.9% were in the first 30 dim,44 20.8% at 60 dim,45 and 17% at 80 dim.46 although the rates decreased as dim increased, these studies were consistent with previous data indicating that, on average, > 20% of cows remain anestrus at the end of vwp. lack of estrus expression before 60 dim is negatively associated with reproductive performance with cows that did not express estrus had prolonged interval to first ai, reduced estrus duration and intensity, longer days to pregnancy, and reduced first service pregnancy per ai (p/ai) compared to cows that were detected in estrus.45,47 furthermore, cows that had 2 or more estrus events prior to the end of vwp were more likely to be inseminated by 100 dim with a higher first service p/ai, and increased likelihood of pregnancy by 200 dim compared to cows with 0 or 1 estrus event in the same period.45  lack of estrus expression may not always coincide with lack of ovulation. from a clinical perspective, at the herd level, methods to assess the true ovarian status of individual cows are rarely practical. blood progesterone measurements are expensive, require additional labor, and samples must be submitted to an outside laboratory. in-line milk progesterone monitoring is not widely available, and serial ultrasonography of ovarian structures requires additional time, labor, and veterinary cost. however, the use of confirmatory tests is crucial in understanding the validity of the on-farm aam systems that many producers use to identify anovular or anestrous cows.  based on blood progesterone to assess resumption of cyclicity, 9% of cows were truly anovular despite 17% diagnosed as anestrus.46 in an assessment of sensitivity and specificity of aam to detect resumption of cyclicity compared to serum progesterone, aam had a sensitivity and specificity of 34.1 and 84.0%, respectively.44 the authors attributed the low sensitivity for identification of cyclic cows to the silent estrus often observed in early postpartum cows. however, the higher specificity indicated that the aam system was able to correctly identify a majority of the anovular cows. although this means that some cows diagnosed as anovular may be cyclic and could be enrolled into a synchronization protocol aimed at managing anovular cows, there is little risk in doing so. enrollment in a synchronization protocol in early lactation improves p/ai to first service, reduces days open, and improves overall reproductive performance, regardless of ovular status.48 it is noteworthy to consider a presynchronization protocol such as double ovsynch over a basic ovsynch protocol for synchronization of anovular cows as a management strategy. although anovular cows ovulated in response to gnrh injections, conception rates were lower for anovular cows compared to ovular cows whether they are inseminated after detected estrus or ovsynch.48 a combination strategy of aam to detect anovular cows and enrollment in an appropriate synchronization protocol is likely most beneficial in improving reproductive performance and the greatest return on investment of these technologies. targeted reproductive management programs aim to make the best use of aam along with customizing management protocols for groups of cows within a herd, particularly those that are anestrus and anovular. these programs typically involve allocation of cows to various management strategies for first service insemination based on estrus expression identified by automated activity alerts during the vwp and the use of these data as a proxy for cyclicity. the feasibility of implementing these programs is still a subject of research. for example, prioritization of ai after estrus detection based on estrus expression prior to the end of vwp resulted in similar proportion of cows pregnant around 150 dim despite a lower first service p/ai compared to a double ovsynch protocol.49 targeted reproductive management using aam may provide additional options to improve or at least maintain reproductive performance in herds whose goal is to reduce the cost and labor associated with enrolling all cows in a timed ai protocol for first service.  future directions until now, research on anovular conditions focused on the underlying mechanisms, contribution of postpartum diseases, and the use of commercially available formulations of various reproductive hormones to initiate resumption of ovarian activity. there are opportunities to explore hormones for management of anovular conditions that target the hypothalamic-pituitary-ovarian axis via different mechanisms than current commercially available reproductive hormones. synthetic kisspeptin, a peptide hormone, has a role in facilitation of the gnrh surge, and can stimulate ovulation in postpartum cows.50 a single injection of kisspeptin was capable of inducing ovulation in postpartum anovular cows with a dominant follicle > 10 mm in diameter, followed by a normal length estrous cycle. cows receiving kisspeptin had significant increases in fsh and lh compared to controls but the ability of the kisspeptin-induced lh surge to cause ovulation was dependent on follicular size. although this study only enrolled a limited number of cows, there is a potential for an alternate clinical tool in the management of anovulatory cows. future studies need to examine the effects of kisspeptin treatment on days open, first service p/ai, and pregnancy losses to determine if this management tool has positive impact on fertility in anovular postpartum cows. an additional hurdle for http://dx.doi.org/10.58292/ct.v17.11664 citation: clinical theriogenology 2025, 17, 11664, http://dx.doi.org/10.58292/ct.v17.11664 7 kisspeptin use on a larger scale is that there is currently no commercially available product in the usa; however, the importance of reproductive performance to the usa dairy industry and high prevalence of anovulation in postpartum cows could make development of such a product a viable option for future application. conclusion regardless of cause, anovulation has substantial negative effects on reproductive performance and occurs at a relatively high rate in usa dairy cattle. management practices to reduce within-herd rates of anovulation should be focused on maintaining nutritional status and minimizing body condition loss in the postpartum period, reducing risks for uterine and nonuterine diseases by supporting immune function and overall health, and reducing stress in the postpartum period. early identification of anovular cows using activity monitoring systems, ultrasonographic examinations during routine herd health visits, or milk progesterone monitoring may allow for enrollment in targeted reproductive management programs to improve reproductive outcomes in this subgroup of cows. clinical knowledge of the benefits and shortcomings of each management strategy will enable the reproductive practitioner to better assist dairy herd clients achieving their herd reproductive goals.  conflict of interest jennifer roberts is employed by boehringer ingelheim animal health; authors have no other conflicts of interest to declare. acknowledgements authors acknowledge paola n colon guerra’s contribution; assisted with editing and reference verification of the section on cystic ovarian disease. references 1. wiltbank mc, gümen a, sartori r: physiological classification of anovulatory conditions in cattle. theriogenology 2002;57:21-52. doi: 10.1016/s0093-691x(01)00656-2 2. peter at, vos plam, ambrose dj: postpartum anestrus in dairy cattle. theriogenology 2009;71:1333-1342. doi: 10.1016/j. theriogenology.2008.11.012 3. pinedo p, santos jep, galvão kn, et al: combined effect of purulent vaginal discharge and anovulation on pregnancy status in a large multi-state population of holstein cows. jds commun 2023;4:106-110. doi: 10.3168/jdsc.2022-0271 4. butler wr: nutritional interactions with reproductive performance in dairy cattle. anim reprod sci 2000;60-61:449-457. doi: 10.1016/s0378-4320(00)00076-2 5. banuelos s, stevenson js: transition cow metabolites and physical traits influence days to first postpartum ovulation in dairy cows. theriogenology 2021;173:133-143. doi: 10.1016/j. theriogenology.2021.08.002 6. dubuc j, duffield tf, leslie ke, et al: risk factors and effects of postpartum anovulation in dairy cows. j dairy sci 2012;95:18451854. doi: 10.3168/jds.2011-4781 7. monteiro plj, 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https://doi.org/10.3168/jds.s0022-0302(06)72576-0 https://doi.org/10.3168/jdsc.2023-0463 https://doi.org/10.3168/jds.2022-22616 https://doi.org/10.3168/jds.2017-13505 https://doi.org/10.3168/jds.2020-19705 https://doi.org/10.3168/jds.s0022-0302(03)73921-6 https://doi.org/10.3168/jds.2022-22082 https://doi.org/10.1530/raf-21-0046 2009: ecbolic and tocolytic agents in bovine reproduction ecbolic and tocolytic agents in bovine reproduction 1 m. a. edmondson 2 department of clinical sciences, college of veterinary medicine, auburn university, 3 auburn, al, usa 4 abstract 5 the use and efficacy of ecbolic and tocolytic agents to treat and manage 6 reproductive conditions in the bovine have been controversial topics at times. this is in 7 part due to our limited understanding and lack of research with regard to the pregnant and 8 post partum uterus in the cattle. this article reviews the anatomy and physiology of the 9 bovine uterus and research evaluating the use and efficacy of various ecbolic and 10 tocolytic compounds. 11 keywords: bovine uterus, ecbolic, tocolytic 12 introduction 13 ecbolic agents have been administered in an attempt to treat and prevent many 14 reproductive conditions in the periparturient cow which most commonly include retained 15 fetal membranes, post-partum endometritis and metritis. tocolytic agents have been used 16 to inhibit uterine contractility in a variety of clinical situations which include delaying 17 parturition and facilitating obstetrical manipulations. to further understand the uses and 18 possible roles of ecbolics and tocolytics in bovine practice, one must first understand the 19 anatomy and physiology of the uterus and the pharmacology of these agents. 20 anatomy and physiology of the uterus 21 the uterine wall is composed of three layers. the innermost layer, the 22 endometrium, lines the lumen of the organ and consists of columnar epithelium and 23 underlying stromal tissues. the middle layer, the myometrium, which consists 24 predominately of smooth muscle cells, also contains blood and lymph vessels, nerves, 25 309 immune cells, and connective tissue. the outer layer, the serosa, is a thin layer which 26 covers most of the uterus and is composed of mesothelial cells. the individual smooth 27 muscle cells of the myometrium are the physiologic units of uterine contraction. the 28 muscle fibers of the outer longitudinal layer are arranged parallel and those of the inner 29 circular layer are arranged concentrically around the long axis of the uterus. advancing 30 gestation is accompanied with hypertrophy and hyperplasia of the uterine smooth muscle. 31 the increase in size can be up to threeto fivefold by the end of gestation.1,2 32 smooth muscles are innervated by the sympathetic nervous system and have α 33 and β receptors. the α receptors are responsible for muscular contractions while the β 34 receptors are responsible for relaxation. the β1 receptors are confined to the heart, 35 adipose tissue, and small intestine. stimulation of β1 receptors leads to increased cardiac 36 automaticity, positive chronotropic and inotropic effects, and elevated free fatty acids. 37 the β2 receptors are found in the smooth muscle of the uterus (myometrium), vascular 38 smooth muscle, and bronchioles. stimulation of β2 receptors causes relaxation of the 39 uterus, vasodilation, and bronchodilation. activation of these β2 receptors leads to an 40 elevation of in camp, mediated through adenylate cyclase. these increased levels of 41 camp prevent myosin light-chain kinase (mlck) activity through both decreased 42 phosphorylation and inhibition of release of stored intracellular calcium thus inhibiting 43 uterine contraction.1,2 44 uterine contractility 45 the contractile activity of the uterus is directly related to the electrical activity in 46 the smooth muscle cells. this activity is characterized by cyclic depolarization and 47 repolarization of the plasma membrane and action potentials. contraction of the smooth 48 310 muscle cells occurs by the interaction of the myofilaments which are composed of 49 myosin and actin. the sarcoplasmic reticulum also plays a key role as it is the site of 50 calcium storage. in the resting stage, the intracellular calcium in the smooth muscle is 51 low. contraction of the smooth muscle is preceded by an increase in free intracellular 52 calcium levels. calcium ions bind with calmodulin which then activates myosin kinase. 53 myosin kinase in turns phosphorylates a myosin head. the phosphorylated myosin head 54 then binds with an actin filament, thus inducing smooth muscle contraction.2 55 during pregnancy, the uterine smooth muscle is relatively quiescent, displaying 56 weak, localized, and poorly coordinated contractions. in contrast, during parturition, the 57 contractions are forceful, sustained, regular, and well-synchronized. factors responsible 58 for initiating the process of parturition include 1) an increase in the number of gap 59 junctions which allows cell coupling and interactions, 2) decreased production of nitric 60 oxide thereby inhibiting uterine relaxation, and 3) stretch of the myometrium which 61 enhances contractility.2 62 ecbolic agents (oxytocics) 63 ecbolic agents or oxytocics are compounds that hasten uterine evacuation by 64 stimulating uterine contractions of the myometrium. the best known and most widely 65 used ecbolics are oxytocin and prostaglandins (pg). ecbolics have been used for the 66 treatment of retained placentas and for the treatment and prevention of metritis. 67 oxytocin 68 in 1906, sir henry dale reported that an extract of the neurohypophysis had 69 oxytocic effects. the use of this extract to induce labor was first reported as early as 70 1911. the name given to this compound was oxytocin, derived from the greek word 71 311 meaning “swift birth.” oxytocin is a neuropeptide hormone produced by magnocellular 72 cells in the hypothalamus, transported to the posterior pituitary via axons, and stored and 73 secreted by the posterior pituitary. another major function of oxytocin in mammals is 74 stimulation of milk letdown from the mammary glands. in the 1950s du vigneaud and 75 others established the structure and synthesized oxytocin. oxytocin was the first peptide 76 to be synthesized.3-5 77 oxytocin is a potent stimulus for uterine contractions. the action of oxytocin is 78 mediated by binding to specific oxytocin receptors present in the uterus when the uterus 79 is under the influence of estrogen. thus oxytocin is considered to be effective in causing 80 uterine contraction and uterine evacuation if administered within 48 to 72 hours 81 postpartum. the binding of oxytocin to its receptors causes an increase in intracellular 82 calcium which results in contraction of myometrial cells from the uterus. oxytocin has 83 three specific actions on the uterus which include 1) induction of myometrial 84 contractions, 2) release of pgf2α from the endometrium, and 3) release of pge2 from 85 cervical mucosa. most formulations of oxytocin contain 20 united states pharmacopeia 86 (usp) units/ml with package inserts recommending up to 100 usp units. this dosage 87 recommendation seems high considering that 1.0 iu of oxytocin achieves physiologic 88 levels comparable to those seen during milking. thus, an oxytocin dosage of 10 iu is a 89 supra-physiologic dosage.6 90 prostaglandins 91 prostaglandins were discovered in the early 1930s as substances present in human 92 seminal plasma that induced, and sometimes relaxed, uterine muscle activity in vitro. 93 von euler named these substances prostaglandins believing that they came from the 94 312 prostate gland. although, it was later discovered that the seminal vesicles were the major 95 source of prostaglandins; thus, prostaglandins is really a misnomer. in 1957, bergstrom 96 and sjovall reported the first isolation of prostaglandins and determined their structures. 97 by the 1960s, sammuelsson and colleagues began describing the prostaglandin metabolic 98 pathways. in 1971, vane reported that aspirin inhibited pg production and later showed 99 that non-steroidal anti-inflammatory drugs acted via pg inhibition. bergstrom, 100 sammuelsson, and vane were awarded the nobel prize in 1982 for their work with pgs.5 101 when biologically active pgs are released into the blood they are metabolized by 102 enzymes in the liver, kidney, and particularly the lung. after one passage of pgf2α 103 through the lungs, over 90% of the pg is metabolized. this is one reason why most pgs 104 have very short half-lives in blood (<1 min). in addition to the lung, the placenta also has 105 very high concentrations of pg dehydrogenase. thus, it is very unlikely the biologically 106 active pgs can cross the placenta and affect the fetus. prostaglandins are capable of 107 causing muscle relaxation and muscle contraction depending upon the branch of the 108 receptors (relaxant or stimulatory receptors). the relaxant receptors act via adenylate 109 cyclase to elevate levels of intracellular cyclic adenosine monophosphate (camp) and 110 phosphokinase a (pka) activity. this in turns relaxes smooth muscle via elevated 111 camp which induces sequestration of intracellular calcium. the stimulant receptors act 112 via enhancing intracellular phosphokinase c (pkc) activity and intracellular calcium. 113 the activity through this receptor stimulates smooth muscle contraction by elevating 114 intracellular calcium levels. this discussion will focus on the stimulatory effects of pgs, 115 specifically pgf2α, on the uterus.5,6 116 313 during the immediate postpartum period, serum concentrations of pgf2α and its 117 metabolites are elevated. these elevations are thought to facilitate uterine involution. 118 prostaglandin f2α has many actions, including luteolysis, stimulation of myometrium, and 119 constriction of blood vessels. in addition, pgs also have a relaxant effect on the cervix. 120 thus, pgf2α is used to treat a variety of medical conditions. these conditions include 1) 121 induction of parturition, 2) lysis of corpus luteum (cl) for cases of pyometra, 3) lysis of 122 cl for management of the estrous cycle, 4) induction of abortion, and 5) evacuation of 123 the uterus in cases of metritis or endometritis. prostaglandins can be used to induce 124 parturition and abortion by its uterotonic effect and by its luteolytic effect. 125 prostaglandins used in veterinary medicine include cloprostenol sodium, dinoprost 126 tromethamine, fenprostalene, fluprostinol sodium, alfaprostol, and luprostiol.6 127 cloprostenol sodium. cloprostenol (estrumate®, intervet/schering-plough 128 animal health corp., summit, nj, usa) is a powerful luteolytic agent and causes rapid 129 regression of the cl and arrests its secretory activity. cloprostenol is available in a 130 concentration of 250 mcg/ml and is a pg analog that is administered by intramuscular 131 injection for all indications in both beef and dairy cattle. cloprostenol is used in beef or 132 dairy cattle to induce luteolysis. it is recommended by the manufacturer for unobserved 133 or undetected estrus in cows cycling normally, pyometra or chronic endometritis, 134 expulsion of mummified fetus, luteal cysts, induced abortions after mismating and to 135 schedule estrus and ovulation for controlled breeding.7 136 dinoprost tromethamine. dinoprost tromethamine (lutalyse®, pfizer animal 137 health, new york, ny) is the naturally-occurring pgf2α as the tromethamine salt. each 138 ml contains 5mg of dinoprost and is luteolytic in cattle at 25 mg (5 ml) administered 139 314 intramuscularly. dinoprost tromethamine is labeled for estrus synchronization, treatment 140 of unobserved (silent) estrus and pyometra (chronic endometritis) in cattle; and for 141 abortion of feedlot and other non-lactating cattle.7 142 other pgs. alfaprostol and luprostiol are prostaglandin analogs that are used 143 mainly outside of the united states. these drugs have similar effects and uses as the 144 other synthetic prostaglandins. 145 xylazine 146 xylazine produces uterine contraction by stimulation of α2-adrenergic receptors in 147 the uterus. the pregnant bovine uterus appears to be more sensitive to xylazine-induced 148 contractility than the non-pregnant uterus, particularly after 270 days of gestation. this 149 enhanced susceptibility may involve hormone-related changes in α-adrenergic receptor 150 populations in myometrial tissue. in one study, administration of xylazine (10mg 151 intravenously) significantly increased uterine motility during late gestation. thus, the use 152 of xylazine in the last month of gestation is contraindicated in cattle because of the 153 increased tendency for induction of premature parturition. xylazine has no practical uses 154 as an ecbolic due to its sedative and muscle relaxant properties.6,7 155 estrogen 156 estrogen has been used in an attempt to initiate and/or strengthen myometrial 157 contractions. however, the use of estrogen is controversial. because estrogen levels 158 normally decrease dramatically once the calf is expelled, it appears that normal uterine 159 involution can proceed without the influence of estrogen in the normal cow. studies have 160 shown no beneficial effects on the prevention of metritis or reproductive performance, 161 and that the use of estrogen may actually have a negative effect on subsequent fertility.8 162 315 it is believed that contractions induced by estrogen may force septic uterine contents not 163 only through the cervix but also into the uterine tubes which results in severe bilateral 164 salpingitis.9 research has shown that estrogen treatment postpartum has a negative 165 impact on uterine motility where the normal uterine contractions changed to a sustained 166 contraction or spasm.8 in addition, one study demonstrated that the use of oxytocin in an 167 estrogen-primed uterus did not increase the contraction frequency and thus did not 168 enhance the myometrial effect of oxytocin. thus, scientific evidence does not support 169 the use of exogenous estrogens in the postpartum cow. 170 use of ecbolics to treat retained fetal membranes and uterine infections 171 two common problems that are encountered in periparturient dairy cattle and 172 occasionally in beef cattle are retention of fetal membranes and metritis. retained fetal 173 membranes is one of the most important factors that predisposes cattle to uterine 174 infections. cattle that have retained fetal membranes are six times more likely to develop 175 a uterine infection than are cows without retained fetal membranes. primary retention of 176 the fetal membranes results from lack of detachment from the maternal caruncles, 177 whereas secondary retention is related to mechanical difficulty in expelling the already 178 detached fetal membranes. greater than three-fourths of cows expel the placenta by 6 179 hours post-partum with the majority of the remaining cows expelling the placenta before 180 12 hours postpartum. because the incidence of retained fetal membranes and postpartum 181 disease varies with parity, the definition of retained fetal membranes may also be ageor 182 parity-dependent. suggestions have been made to define retained fetal membranes from 183 8 to 48 hours; however, 12 hours is widely used to define retained fetal membranes.9 184 316 detachment of the placenta in the cow involves separation of the cotyledon villi 185 from the caruncular crypts without tearing of either fetal or maternal epithelia. for 186 appropriate separation of the cotyledon villi from the caruncular crypts, proteolytic 187 enzymes (collagenases) act to open the cotyledon thereby releasing the caruncle. 188 collagenase activity of cotyledon villi during delivery is increased in healthy cows and 189 decreased in cows with retained fetal membranes. the cellular sources of collagenase 190 and proteolytic enzymes responsible for placental release in the cow are unknown. 191 however in laboratory animals and humans, myometrial cells, fibroblasts, and leukocytes 192 have been identified as sources of collagenase in the uterus. by day 6 postpartum, the 193 caruncle is disorganized; by day 15, caruncles are completely sloughed as a result of 194 necrosis. retained fetal membranes are detached by caruncle necrosis within 6 to 10 195 days and not later than 17 days postpartum. the surface of the endometrium is covered 196 by new epithelium by day 26 to 30 postpartum. after placental detachment, uterine 197 involution is completed in about 39 days in normal cows and 50 days in cows with 198 retained fetal membranes. lack of uterine motility is not considered a reason for primary 199 retention because uterine motility is normal or above normal in cows with retained fetal 200 membranes.9 201 factors reportedly contributing to the development of retained fetal membranes 202 include periparturient hypocalcemia, dystocia, abortion, twinning, stillbirth, and 203 induction of parturition. there are several management approaches that have been used 204 for cows with retained fetal membranes of which many are controversial and lack 205 scientific evidence to support their use. these options include no intervention, manual 206 removal of the fetal membranes, antimicrobial therapy, and hormone therapy. no 207 317 intervention in an otherwise healthy cow is a common practice that allows the fetal 208 membranes to liquefy and necrose until they are passed. these cows should be 209 monitored closely for any signs of septicemia or toxemia in which case systemic therapy 210 is necessary. manual removal of the fetal membranes was once practiced but has fallen 211 from favor due to the likelihood of causing trauma to the uterine wall, the high incidence 212 of leaving tags of the fetal membranes within the uterus, and iatrogenic contamination of 213 the uterus. intrauterine antibiotic therapy is beyond the scope of this discussion but will 214 be discussed in a subsequent presentation. hormone therapy is still one of the most 215 common methods for managing cows with retained fetal membranes.6,9,10 216 the majority of cattle experience bacterial contamination of the uterus at the time 217 of parturition. in the normal cow, the uterus is cleared of this bacterial contamination by 218 four weeks postpartum. when these bacteria are not cleared by the cow’s defense 219 mechanisms, a uterine infection ensues. numerous bacteria have been isolated from the 220 cow’s postpartum uterus, some of which may be incidental and not cause problems. 221 uterine infections are most commonly due to arcanobacterium pyogenes. the gram 222 negative anaerobes fusobacterium necrophorum and bacteroides melaninogenicus are 223 frequently associated with a. pyogenes. other organisms that may be associated with 224 uterine disease in the cow include pseudomonas aeruginosa, staphylococci, hemolytic 225 streptococci, coliforms, etc. clostridium sp. may occasionally infect the uterus and cause 226 a severe gangrenous metritis or tetanus. uterine infections are associated with retained 227 fetal membranes, dystocia, and delivery of twins. metritis is the result of severe 228 inflammation involving all layers of the uterus – endometrial mucosa and submucosa, 229 muscularis, and serosa. metritis usually develops during the first week after calving and 230 318 is associated with dystocia, retained fetal membranes, and calving trauma. affected 231 cattle may be septic and present with fever, depression, and anorexia and a copious fetid 232 vaginal discharge may also be present. endometritis is characterized by inflammation of 233 the endometrium extending no deeper than the stratum spongiosum. cows with 234 endometritis are usually not systemically ill, and bacteria are usually eliminated after a 235 few estrous cycles. pyometra is a collection of purulent exudate within the uterus with 236 the persistence of the corpus luteum, and suspension of the estrous cycle. pyometra 237 usually develops in cows that have their first postpartum ovulation before bacterial 238 contamination of the uterus has been eliminated. the corpus luteum that is associated 239 with the infection persists because intrauterine fluid prevents luteolysis. thus 240 progesterone persists and suppressed uterine defense mechanisms.9,10 241 several different hormones have been used in an attempt to manage retained fetal 242 membranes and uterine infections with the ecbolic agents oxytocin and pg being the 243 most common. oxytocin appears to stimulate myometrial contraction by 1) direct 244 activation of receptors on myometrial cells and 2) indirect stimulation of contraction 245 through the release of stimulatory pgs from the endometrium. circulating oxytocin 246 binds to myometrial receptors which leads to rapid uterine contraction and an increase in 247 pgf2α levels. it is believed that pgf2α stimulates the release of more oxytocin and also 248 enhances the sensitivity of the myometrium to oxytocin.3 as little as 2.5 iu of oxytocin 249 intravenously will cause the proximal ends of the uterine horns to respond within 30 to 50 250 seconds when progesterone levels are low in a cycling cow, and this increase in 251 myometrial activity persists for up to 80 minutes.11 studies such as this in cycling cows 252 have supported the idea that the myometrium is only responsive to oxytocin when 253 319 estrogen is dominant; whether oxytocin is effective in cows with toxic metritis is 254 unclear.6 one study indicated that as little as 5 units of oxytocin intravenously can 255 initiate a more intense rhythm of contraction in cows with retained fetal membranes.12 256 other studies refute this evidence and suggest that oxytocin was of no benefit to 257 postpartum cows with retained fetal membranes; however, in these two studies a dose of 258 60 to 100 units of oxytocin was administered which causes a spasm of the uterus versus a 259 progressive contraction.13-15 it also appears that the traditional dose of oxytocin (40 260 units) when administered intravenously causes an initial tetanic spasm of the uterus.16 261 most of the studies demonstrating the positive effect of exogenous oxytocin have used 262 the intravenous route of administration instead of the more commonly used intramuscular 263 route of administration. however, one study did show that the myometrial response 264 following administration of 20 to 30 units of oxytocin was similar following 265 administration via intravenous, intramuscular, and subcutaneous routes of 266 administration.16 a day two to three protocol of repeated 20 unit (1.0 ml) oxytocin 267 injections administered at least three hours apart or three doses evenly spaced between 268 milkings, etc has been suggested.6 although the frequent administration with low dose 269 oxytocin appears to be impractical in most situations, it would appear to induce a more 270 physiologic response than current therapeutic protocols which use infrequent 271 administration at supra-physiologic dosages which induce tetanic uterine spasms. the 272 most physiologic uterotonic dose of oxytocin has not been determined. 273 despite much research, the ability of exogenous pgs to have a direct effect on 274 periparturient uterine activity in cattle has been a controversial issue among researchers 275 and clinicians. although a few studies indicate that pgf2α may reduce the incidence of 276 320 retained fetal membranes, subsequent studies have failed to confirm these results and 277 many report that exogenous pg has no effect. many of these studies lack sufficient 278 numbers of animals, lack control animals, and used concurrent medications which make 279 interpretation of the results difficult. it appears that suboptimal uterine contraction is 280 rarely the cause of retained fetal membranes in a nontoxic cow. studies have shown that 281 the presence of retained fetal membranes alone doubles the rate and increases the 282 frequency of uterine contractions. in another study, cows that had evidence of uterine 283 infection (fetid, sanguine-purulent lochia) at up to day 15 post partum had significantly 284 higher concentrations of pgf metabolite (pgfm) than did cows that had a mucopurulent 285 to purulent lochia.17 in addition, studies indicate that a single intramuscular injection of 286 pg before the formation of a functional corpus luteum will have no beneficial effect in 287 the post-partum cow.12,16 even when the pg dose was doubled, there was still no 288 increase in uterine tone. however, luteolytic doses of pgf2α (25 mg) administered by 289 rapid intravenous injection on day 2 postpartum did cause an increase in uterine 290 contractions.16 however, by day four postpartum the stimulatory effect was noticeably 291 decreased. intravenous administration of pg also has significant side effects (dyspnea, 292 salivation, milk ejection, frequent urination) that make it impractical to use particularly in 293 a toxic cow. only when luteal tissue is present on the ovary is it widely accepted that 294 exogenous administration of pg has a beneficial effect on the postpartum cow. 295 intramuscular injection of pgf2α may not be uterotonic because the pgf2α is metabolized 296 almost entirely into pgfm upon a single passage through the lungs. using pg to lyse the 297 cl allows for removal of the immunosuppressive effects of luteal progesterone which 298 may aid in the resolution of chronic postpartum endometritis.6 currently, there is no 299 321 scientific evidence that intramuscular or subcutaneous injections of either natural or 300 synthetic pgf2α aids in the expulsion of retained fetal membranes. in addition, 301 administration of pg during the immediate postpartum period has not been shown to 302 have an effect on the rate of uterine involution.18 303 some studies indicate that the use of pg may improve overall reproductive 304 performance in cows that are not affected by periparturient diseases. in addition, cows 305 affected with dystocia, retained fetal membranes, or both that were treated with pgf2α 306 early post partum followed by a second treatment 14 days later experienced a higher 307 conception rates to first service than non-treated cows.19 308 conclusion 309 although more research is needed on the postpartum cow uterus, there is no 310 proven scientific evidence that supports the routine use of ecbolic agents as a treatment 311 for the pathologic postpartum uterus. based on scientific research, exogenous estrogen 312 and pg at published doses appear unable to stimulate the appropriate rhythmic 313 contractions necessary to empty the pathologic post partum uterus. there is some 314 evidence that supports the use of exogenous oxytocin to stimulate uterine contractions 315 that are similar to those contractions observed during stage ii of labor. however, these 316 studies used intravenous oxytocin rather than the more common intramuscular route of 317 administration. 318 tocolysis 319 tocolysis is derived from greek with “tokos” meaning childbirth and “lysis” 320 meaning capable of dissolving. a tocolytic agent is a compound that is capable of 321 inhibiting uterine contractions. tocolytics were originally designed for use in human 322 322 medicine to interrupt premature labor and have, over time, been used more commonly in 323 veterinary medicine. the use of tocolytics to inhibit uterine contraction has a number of 324 potential clinical applications in cattle. delaying parturition for controlled calving may 325 be useful if parturition were occurring at a time that decreased fetal survival (nocturnal 326 delivery). obstetrical manipulations such as correction of malpresentation and 327 malposition, repulsion and rotation of the fetus, correction of uterine torsion, ease of 328 extraperitoneal lifting of uterus during cesarean section, and replacement of uterine 329 torsion may be aided by the use of tocolytic agents. some believe that these drugs may 330 also be useful in the area of embryo transfer. 331 tocolytic agents 332 a variety of tocolytics have been used for the aforementioned applications to 333 cause uterine relaxation. ethyl alcohol, magnesium sulfate, progesterone, prostaglandin 334 synthetase inhibitors, calcium channel blockers, epinephrine, and β sympathomimetics 335 have all been used to induce uterine quiescence. however, the unpredictable efficacy and 336 adverse side effects make some of these drugs less acceptable than others for the 337 induction of uterine relaxation.20 338 ethanol 339 in the mid 20th century, ethanol was a commonly used tocolytic agent in human 340 patients to halt pre-term labor. it is believed to effectively inhibit the secretion of 341 oxytocin and interfere with prostaglandin synthesis. ethanol was given intravenously in 342 humans at a rate to maintain a blood alcohol level of 0.9 to 1.6 mg/liter. of course, side 343 effects were observed which included nausea, vomiting, depression, intoxication of 344 323 mother and fetus, and acidosis. research has since demonstrated that ethanol is not 345 effective in delaying parturition.21 346 magnesium sulfate 347 magnesium sulfate has also been used as a tocolytic. magnesium sulfate is a 348 central nervous depressant which blocks neuromuscular transmission and lowers 349 acetylcholine. in 1959, the tocolytic properties of magnesium sulfate were first 350 described. the exact mechanism by which magnesium sulfate exerts its tocolytic effects 351 is unknown. however, one possible mechanism may be its ability to block nerve 352 transmission and/or by its actions as a calcium antagonist. magnesium sulfate has been 353 given intravenously as a 10% solution to delay parturition for 24 to 48 hours. most 354 human studies do not indicate a significant ability for magnesium sulfate to prolong 355 pregnancy. the use of magnesium sulfate as a tocolytic is no longer recommended due 356 to its lack of effect at preventing preterm deliveries and because of its association with a 357 higher risk of perinatal death.22 358 progesterone 359 the actions of progesterone on the pregnant myometrium include relaxation of 360 myometrial smooth muscle, blocking the action of oxytocin, and inhibition of gap 361 junctions. progesterone decreases the concentration of myometrial oxytocin receptors 362 which counteracts the effect of estrogens. progesterone also inhibits pg production by 363 the placenta. in human research, progesterone has been found useful for the maintenance 364 of tocolysis to increase gestational age at delivery or as a preventative agent in women 365 with high-risk pregnancies.23 progesterone has been shown to enhance the tocolytic 366 effect of some of the beta sympathomimetics (ritodrine) when used in human patients.24 367 324 progesterone prolongs gestation length when administered during advanced pregnancy 368 and, as a result, chances of dystocia increase due to additional weight gain of the fetus. 369 prostaglandin synthetase inhibitors 370 prostaglandins are known to be important mediators in uterine contractility. at 371 the time of parturition, there are increased concentrations of arachidonic acid, pg e2 and 372 pgf2α. prostaglandins increase intracellular free calcium levels which may increase the 373 frequency of uterine contractions. thus, nonsteroidal anti-inflammatory drugs that 374 inhibit pg synthesis in the uterus have been considered for use as tocolytics. 375 indomethacin, a product used in human medicine, has been studied, although with a small 376 sample size, in human patients and was found to delay parturition for 48 hours. 377 indomethacin is relatively safe as far as the maternal side effects, but crosses the placenta 378 and causes concerns regarding fetal pulmonary hypertension, gastrointestinal 379 inflammation, and hemorrhage.22 flunixin meglumine (banamine®, intervet/schering-380 plough animal health), has been considered for use as a tocolytic agent in cattle based 381 on its ability to block pg synthesis. although the effects of flunixin meglumine have not 382 been evaluated in a controlled study, the label actually warns against use of flunixin 383 meglumine in late gestation as it is “known to have the potential to delay parturition 384 through a tocolytic effect”.7 one study evaluated the effect of flunixin meglumine on 385 uterine contractility on a small group of postpartum cows and concluded that flunixin 386 meglumine inhibited pg production by more than 80% and decreased spontaneous 387 uterine motility.25 the effects of flunixin meglumine on uterine contractility have not 388 been evaluated in the pregnant cow. 389 calcium channel blockers 390 325 calcium channel blockers have been used for their tocolytic effects in human 391 medicine since the 1980’s. calcium channel blockers work to inhibit uterine contraction 392 by blocking the influx of calcium into the cells of the myometrium through disruption of 393 the voltage-operated calcium channels.22 some studies indicate that nifedipine is as 394 effective as magnesium sulfate and beta agonists with fewer side effects. limited 395 research has shown that the calcium channel blocker, nifedipine, is capable of blocking 396 xylazine-induced uterine contractions in goats. one study found that nifedipine at 80 397 mcg/kg given intravenously was able to delay parturition in sheep for six to seven 398 hours.26 side effects associated with the use of calcium channel blockers for tocolysis 399 include fluid retention and decreased cardiac output which can result in pulmonary 400 edema.22 401 epinephrine 402 epinephrine is an adrenergic that has both α and β activity; therefore, it is capable 403 of relaxing smooth muscle. the use of epinephrine in veterinary medicine has been 404 primarily limited to emergency situations to treat anaphylactic shock or cardiac 405 resuscitation and because of its vasoconstrictive properties as an additive to local 406 anesthetics to decrease absorption and prolong effect.7 however, epinephrine has also 407 been administered to cattle at 10 cc per cow of the 1:1000 solution as a slow intravenous 408 infusion to cause uterine relaxation and quiescence.6,20 this use of epinephrine in cattle 409 has been primarily to facilitate obstetrical procedures and conditions such as cesarean 410 section, fetotomy, uterine prolapse, and uterine torsion. uterine relaxation is almost 411 immediate following intravenous administration. side effects which may be seen with 412 326 rapid administration and overdose of epinephrine include severe increases in blood 413 pressure, cardiac arrhythmias, pulmonary edema, and dyspnea.20 414 β sympathomimetics 415 as previously mentioned stimulation of β1 receptors leads to increased cardiac 416 automaticity, positive chronotropic and inotropic effects, and elevated free fatty acids 417 while stimulation of β2 receptors causes relaxation of the uterus, vasodilation, and 418 bronchodilation. although β sympathomimetic tocolytics may be β2 selective, they do 419 retain some β1 activity which accounts for the side effects that may be observed.22 an 420 ideal β tocolytic would be completely β2 selective. however, no such drug exists. 421 therefore, the benefit of uterine relaxation is often accompanied by side effects 422 attributable to β1 activity. maternal side effects with β sympathomimetics are associated 423 with cardiovascular complications which include tachycardia, arrhythmias, and ischemia. 424 in humans, the most common complication is pulmonary edema which occurs in 425 approximately 5% of patients.22 426 clenbuterol and isoxsuprine have been widely used to induce uterine relaxation. 427 isoxsuprine was the first beta sympathomimetic used for tocolysis. one study 428 demonstrated that isoxsuprine was able to induce tocolysis within 10 to 15 minutes of 429 administration with a duration of one to 1.5 hours.27 however, the lack of discrimination 430 between β1 and β2 receptors and the resulting tachycardia have limited its use. 431 clenbuterol is a specific β2-adrenergic agonist and thus has fewer side effects on 432 extrauterine tissues than does isoxsuprine. clenbuterol also has the longest duration of 433 action (eight to 10 hours) of any of the β sympathomimetics.28 terbutaline is another 434 specific β2-adrenergic agonist that has been used as a tocolytic in humans. limited 435 327 research has demonstrated the tocolytic effects of terbutaline in rats, sheep, and buffalo, 436 and preliminary pharmacokinetic data suggests that it may be a useful tocolytic in 437 cattle.29 terbutaline shares many of the side effects associated with other beta 438 sympathomimetics which include tachycardia, cardiac arrhythmias, muscle 439 fasciculations, hypotension, and hyperglycemia of both dam and fetus.29 although the 440 beta sympathomimetic tocolytics do have some side effects, these side effects are 441 considered to be a minor concern, particularly when using the more specific β2-442 adrenergic agonist. due to the more predictable efficacy and minor side effects, the beta 443 sympathomimetics are generally considered to the best choice for tocolysis.20 444 however, it is important to remember that none of these drugs are approved for 445 use in food animals in the united states. high doses of clenbuterol have been used as 446 repartitioning agents to promote protein deposition while lowering fat deposition which 447 improved carcass composition. this use of clenbuterol has been associated with acute 448 poisonings in humans who consumed meat from clenbuterol-fed animals. in 1990 in 449 spain, 135 people had to be hospitalized after consuming tainted veal and liver. in 1994, 450 another 140 people suffered from dizziness, heart palpitations, breathing difficulty, 451 tremors, and headaches. in addition, clenbuterol was banned in the united states, 452 europe, and canada in 1997 due to reports of aplastic anemia due to human intoxication 453 with clenbuterol subsequent to its use as a repartitioning agent in cattle.30 the use of 454 clenbuterol in food-producing animals remains illegal in the united states. however, 455 abuse of clenbuterol in show cattle in united states has been reported.30 456 use of tocolytic agents 457 328 tocolytic agents have been used historically to treat or to assist in the treatment of 458 numerous conditions in cattle. these uses include 1) threatened abortion/preterm labor, 459 2) controlled calving/nocturnal delivery, 3) reduction in neonatal morbidity and mortality 460 associated with dystocia, 4) aid in obstetrical operations such as cesarean section and 461 fetotomy, 5) treatment of uterine prolapse, 6) treatment of uterine torsion, and 7) embryo 462 technologies. research on the use of tocolytics in ruminants has revealed several factors 463 that may affect tocolysis. the parity of animal may be important to consider as heifers 464 were found to respond faster to clenbuterol and have a longer duration of tocolysis when 465 compared to cows.31 the amount of cervical dilation and the position of the fetus may 466 also have an affect on tocolyis. in animals where the cervix was fully dilated or fetal feet 467 were found to be passing into cervical area, clenbuterol was only able to delay labor for a 468 maximum of a few hours.32 another study evaluated the use of clenbuterol for 469 postponing parturition at various stages in cows. this study found that cows treated 470 during the second stage of labor postponed calving by two hours. in addition, cows 471 treated during the first stage of labor calved 5.2 to 9.7 hours later than control animals.33 472 another study evaluated the use of clenbuterol in beef heifers. this study found that 473 administration of clenbuterol during the first stage of labor (cervical dilation of 5 cm) was 474 able to delay parturition by increasing the length of the first stage of labor with no 475 adverse effects on the fetus or the dam.28 one report suggests that the use of clenbuterol 476 will reduce neonatal morbidity and mortality in dystocia, will aid in obstetrical operations 477 such as cesarean section and fetotomy. the authors of this study also reported less 478 requirement of epidural anesthesia when clenbuterol was administered versus controls, 479 easier correction of malpresentation and malposition, correction of uterine torsion and 480 329 uterine prolapse, and no increase in the incidence of retained fetal membranes in bovine 481 dytocias.34 another study evaluated the use of clenbuterol in seventeen cows undergoing 482 cesarean section and concluded that the use of clenbuterol in these animals resulted in 483 decreased uterine tone to the uterus which allowed for easier exteriorization and suturing 484 of the uterus.35,36 485 conflicting reports exist as to the usefulness of tocolytics with regard to embryo 486 technologies. some have speculated that relaxation of the uterus would improve embryo 487 recovery when used in donor animals and increase pregnancy rates in the recipient 488 animals. however, research and subjective evaluation have not shown any significant 489 improvement in recovery rates of embryos or pregnancy rates in recipients. 490 conclusions 491 it is important to remember that the much of the research on the efficacy and side 492 effects of tocolytic drugs have been conducted in the field of human medicine. there are 493 limited studies in ruminants which are burdened by the lack of critical evaluation and 494 subjective interpretation of the studies’ results. among the β-sympathomimetic agents 495 used in reproduction, only clenbuterol and isoxsuprine have been widely used in clinical 496 management of obstetrical disorders apart from embryo biotechnology with encouraging 497 therapeutic results. the efficacy of these drugs is mostly assessed clinically. however 498 successful the beta sympathomimetics may be as tocolytics, their use is still extra-label 499 and the use of clenbuterol is illegal. thus more research regarding new tocolytic agents, 500 efficacy, and the pharmacokinetics of these drugs needs to be studied in detail to ensure 501 wide use with awareness of adverse effects of drug metabolites, if any. 502 503 330 references 504 505 1. dailey ra: female reproductive system, nonhuman mammal. in: knobil e, neill j, 506 editors. encyclopedia of reproduction, vol. 2. st. louis: elsevier; 1998. p. 229-239. 507 2. jain v, saade gr, gartfield re: uterine contraction. in: knobil e, neill j, editors. 508 encyclopedia of reproduction, vol. 4. st. louis: elsevier; 1998. p. 932-942. 509 3. devries ac, carter cs: oxytocin. in: knobil e, neill j, editors. encyclopedia of 510 reproduction, vol. 3. st. louis: elsevier; 1998. p. 630-634. 511 4. irwin jc, giudice lc: decidua. in: knobil e, neill j, editors. encyclopedia 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myometrium during caesarean operation in cattle. aust vet j 1984;61:401-585 403. 586 587 588 335 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true 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/usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2012: recommendations for management of breeding dogs: a review recommendations for management of breeding dogs: a review margaret v. root kustritz college of veterinary medicine, university of minnesota, st paul, mn abstract information on housing, diet, and management of breeding for bitches and stud dogs is reviewed. veterinarians were surveyed and their recommendations and sources from which they derived those recommendations compiled. there is little research published in the veterinary literature supporting recommendations regarding breeding management of animals, requiring veterinarians to extrapolate from relevant related studies that have been published and to rely on their experience. keywords: theriogenology, breeding management, canine, animal welfare introduction small animal theriogenologists increasingly are requested to provide information about husbandry and breeding of dogs for creation of legislation or to act as expert witnesses in legal proceedings. the literature on topics of interest is sparse, making it difficult to argue for suggested guidelines with science as a basis. the literature was reviewed and practitioners surveyed to determine current practices and their scientific basis in the management of breeding dogs, and a comparison made to breeding within wild dog populations. note that this review will not address general veterinary care of breeding dog populations including regular physical examination by a veterinarian, vaccinations, control of internal and external parasites, and dental care. nutrition will be addressed only for pregnant and lactating bitches. literature review general breeding management dogs should not be bred to near relatives. inbreeding is defined as mating between individuals with one or more common ancestor. close inbreeding is defined as mating of siblings or parents to offspring and is associated with an inbreeding coefficient of 0.25. moderate inbreeding is defined as mating between half-siblings, uncle and niece, aunt and nephew, or grandparent and grandchild and is associated with an inbreeding coefficient of 0.125.1 inbreeding is associated with decreased fertility, decreased litter size, and reduced longevity; this is called inbreeding depression.2-6 all personnel breeding dogs are encouraged to work with a veterinarian directly to create a breeding plan for each dog that includes evaluation of pedigrees and assessment of pros and cons of each animal, to permit pairing that builds on each animal’s strengths and offsets their weaknesses, and to reduce inbreeding depression while maintaining breed standards. stud dogs housing. stud dogs may be housed with other males or in mixed gender groups. group housing is preferred to solitary housing as it is associated with decreased animal vocalization and increased resting behavior.7,8 animal housing should be constructed in a manner that decreases constant stimulation, for example by not requiring constant exposure to movement of other animals or humans, and ameliorates noise.9 age at onset of puberty, defined as normal breeding behaviors and semen quality, is positively correlated with body size and occurs anywhere from six to 18 months of age.10 male dogs raised without exposure to other dogs of either gender are less likely to show normal breeding behaviors as adults.11 male dogs must be housed near enough to female dogs to be familiar to those bitches if they are likely to permit them to breed.12 however, it has been well-demonstrated that incidences of intermale aggression increase in groups of male dogs when housed with or near estrous females. therefore, it is recommended that a suitable location be available for housing stud dogs away from bitches in heat as needed.12,13 all dogs in kennel situations benefit from human interaction including petting, grooming, and observing clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201227 clinical theriogenology • volume 4 number 1 • march 2012 health and behavioral conditions.14 human caregivers are encouraged to clearly differentiate behavior associated with distress from those traits the dogs have learned will garner attention.15 pros and cons of castration. there is much discussion regarding the health implications of leaving male dogs intact. this question arises most commonly when a male dog develops testosteronedependent disease, such as benign prostatic hypertrophy, or disease of the reproductive tract, such as testicular neoplasia, or is otherwise determined not to be of value as a breeding animal. there are good published reviews of benefits and detriments of castration in dogs.16,17 benefits of castration include management of the disease conditions described above and decrease in sexually dimorphic behaviors, most notably mounting, roaming, and urine marking.18,19 some reports also suggest that castrated males live longer than intact males.20,21 detriments include increased incidence of prostatic neoplasia,22-26 transitional cell carcinoma,27,28 osteosarcoma,29,30 and hemangiosarcoma,31,32 anterior cruciate ligament injury,33-35 obesity,36-38cognitive decline,39 and possibly diabetes mellitus.40 testing before use as breeding animal. it is strongly recommended that all dogs to be used for breeding be evaluated for hereditary disorders specific to their breed. all national breed clubs in the american kennel club must have a health subcommittee, and information about specific conditions and available testing usually is available through those groups and several breed-associated web sites. brucellosis testing should be performed in all dogs periodically, whether or not they are actively being used at stud. some advocate testing of females only; this has no basis in science. transmission of brucella canis by ingestion or inhalation is at least as common as by sexual contact and infected males may spread the disease through marking with urine containing brucella organisms.41 the screening method most commonly used for testing, the rapid slide agglutination test (rsat), has good negative predictive value.42 it becomes accurate eight to 12 weeks after exposure so there is little wisdom in testing any more often than every three months.43 polymerase chain reaction (pcr) testing is accurate sooner after exposure and has very good negative and positive predictive value,32 however, testing more frequently is not recommended because incidence of canine brucellosis generally is reported as low but is gaining recognition by its occurrence in many states. surveillance for reproductive tract disease. common diseases of intact male dogs are testicular neoplasia and prostate disease. testicular neoplasia most commonly develops in dogs over three years of age and is very common, with a reported incidence of 0.9%.44 benign prostatic hypertrophy is reported to be present histologically in 75 to 80% of dogs aged six years or more.45-47 morbidity of these disorders is low and castration is curative. bitches housing. there is little in the literature regarding appropriate housing for breeding bitches. basic principles are as for the male. bitches housed together will eventually go through estrus about the same time (the dormitory effect).48 breeders and veterinarians may use this event to their advantage when trying to induce estrus in another bitch. it may also be a detriment as many bitches will whelp at or about the same time, increasing the workload for human caregivers. there are some reports suggesting that interfemale aggression may be increased when bitches are in heat.13 estrous bitches should definitely be housed away from intact males except when they are actively being bred on that cycle. human interaction is required as described for the male. there is little information in the literature regarding housing of pregnant and lactating animals. one breeding facility described significantly lower puppy mortality in bitches and litters housed in pens (10 x 20 feet or 13 x 24 feet) compared to those housed in cages (8 x 10 feet or 12 x 12 feet).49 most reports suggest that a specific whelping area or box be provided with absorbent, clean nesting material. one case report documents gastric impaction due to ingestion of enrichment materials in a whelping box, suggesting caution with materials used for construction and strict observation of these animals.50 naïve bitches exposed to canine herpesvirus in the last three weeks of gestation may develop placentitis and give birth to stillborn, macerated or weak pups that die soon after delivery.51 for this reason, many advocate housing pregnant bitches away from the rest of the kennel population. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 28clinical theriogenology • volume 4 number 1 • march 2012 nutrition (pregnant and lactating). there are no specific changes in nutrition required in the first five weeks of gestation in bitches. in the last trimester, energy requirements increase by 1.25 to 1.5 times.52 bitches should be fed an energy-dense commercial diet, such as a growth or performance diet, with a minimum of 22% protein.53 most diets for pregnant and lactating bitches contain about 30% protein and 20% fat on a dry matter basis.52 ideally, bitches are fed several small meals daily late in gestation, recognizing lack of filling ability of the stomach as it is compressed by the gravid uterus. supplementation with calcium is not recommended unless it is required to balance a home-made diet or the bitch is symptomatic for hypocalcemia. vitamin supplementation is not recommended. energy requirement during lactation may be as high as 300% above maintenance.52 lactating bitches should be free-fed the same diet they received in late gestation and have ready access to clean fresh water at all times. energy requirement does not vary by breed but does vary by litter size.54 bitches should be monitored and fed to maintain her body condition. pros and cons of gonadectomy. there is much discussion regarding the health implications of leaving female dogs intact. there are good published reviews of the benefits and detriments of gonadectomy in bitches.16,17,55 current research suggests that there is no significant difference in intraoperative or post-operative complications, or long-term side-effects when comparing ovariectomy to ovariohysterectomy.56-58 the greatest reported benefit of gonadectomy is a substantial decrease in incidence of mammary neoplasia.59 mammary neoplasia is the most common tumor in female dogs, with an incidence of 3.4%,6063 and the most common malignancy of female dogs, with 50.9% of mammary tumors found to be carcinomas.60,64-66 the largest advantage in decreasing its incidence is achieved by spaying before the first heat.59 there is a decreasing benefit over time, with some benefit up to nine years of age.59,67 as bitches age, the benefit may be only in decreasing incidence of benign mammary tumors but because even benign mammary tumors may be associated with lymphangiogenesis and changes in local microscopic anatomy, a benefit still exists.68 a second advantage is in decreasing incidence of reproductive tract disease, most notably pyometra. cystic endometrial hyperplasia-pyometra complex (ceh-p) occurs in 15 to 17% of dogs greater than four to five years of age and in up to 24% of bitches by ten years of age.69-71 it is associated with endotoxemia and significant secondary renal dysfunction potentially leading to renal failure,72-77 anemia,78 passage of bacteria into the abdominal cavity,79 and decreased immune function.80 breeds at increased risk for developing pyometra include airedale terrier, cavalier king charles spaniel, irish terrier, bernese mountain dog, english cocker spaniel, golden retriever, leonberger, miniature schnauzer, rough collie, rottweiler, and st. bernard.71,81 ovariohysterectomy is curative in most cases, but reported mortality rates are 0 to 17%.82 detriments of gonadectomy include increased incidence of transitional cell carcinoma,27,28 osteosarcoma,29,30 hemangiosarcoma,31,32 cutaneous mast cell tumors,83 anterior cruciate ligament injury,3639 obesity,36-38 urethral sphincter mechanism incompetence,57,84-88 heightened reactivity or aggression in some breeds,89-93 and possibly diabetes mellitus.40 finally, intact female dogs may live longer than spayed female dogs, at least when considering death from natural causes.94,95 testing before use as breeding animal. recommendations for testing for hereditary disorders and brucellosis testing are the same as for the male. age and frequency of breeding. three questions often posed to veterinarians regarding breeding management of bitches are the youngest age at which a bitch should be bred, the oldest age at which a bitch should be bred, and the frequency with which she should be bred. bitches enter puberty on average at nine to ten months of age.96 age at onset of puberty, defined as the first visible signs of proestrus and estrus, is positively correlated with body size. it has been demonstrated that concentration of reproductive hormones is lower and periods of sexual receptivity shorter in the first estrous cycle than in the second, when hormone concentrations and durations of sexual receptivity reach average adult values.97 there are no studies evaluating health considerations of breeding bitches when very young. one could consider “maturity” as the earliest age at which a bitch should be bred. sexual maturity is the onset clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201229 clinical theriogenology • volume 4 number 1 • march 2012 of puberty. physical maturity, defined as closure of the growth plates of the major long bones, occurs about the time of puberty in dogs.98 behavioral maturity occurs at an age approximately twice that of sexual maturity.99 considerations for oldest age at which to breed bitches revolve around identified health detriments including decreased conception rate, decreased litter size with coincidental increase in puppy size and risk of dystocia. one author defined a “critical age” as that at which litter size decreased by 15% or more from the average for that breed.53 critical age is negatively correlated with body size, with toy breeds reaching critical age at ten years of age or older, medium breeds at eight to nine years of age, large breeds at five to six years of age, and giant breeds at less than five years of age.53 evaluation of breeding productivity in large commercial kennels usually identifies five years of age or five litters as the cut-off point for breeding in bitches.100,101 frequency of breeding is not addressed with specific studies in the literature. all bitches go through a prolonged diestrus after every estrus, regardless of pregnancy status, and then go into an anestrus period such that most bitches cycle about every seven to eight months.102 the period between estrous cycles may or may not be prolonged by pregnancy and lactation.103-105 estrous cycling is not seasonal in most breeds of domestic dog; exceptions are the basenji and tibetan mastiff.96,98,101,106-110 seasonality of whelping may be due to human interaction and only breeding bitches at times most desirable for raising or selling puppies.107 many are concerned about breeding bitches on consecutive cycles, assuming that the risks of pregnancy and whelping and the metabolic demands of pregnancy and lactation should not be imposed on a bitch sequentially. because all bitches go through a prolonged luteal phase with subsequent decline in progesterone and rise in prolactin, bitches may experience false pregnancy, defined as lactation in the absence of whelping. it has been reported that a history of overt false pregnancy three or more times in a bitch’s life may be associated with increased incidence of mammary neoplasia,67 suggesting that there is no benefit in not breeding a bitch on every cycle in this regard. the other consideration is uterine health. it has been well-demonstrated that ceh-p is most common in nulliparous bitches and that pregnancy is protective against these pathologic changes on a given cycle.69,81,111,112 pregnancy is not protective long-term, suggesting that it is the physiologic and immunologic aspects of pregnancy that are protective, not alterations in development of endometrial pathology.112 the infection component of pyometra is due to invasion of the bitch’s own fecal flora, and not necessarily exposure to bacteria from an animal to whom she is bred.113,114 involution of the uterus after whelping requires anywhere from three to 4.5 months,110, 115,116 which usually falls well within the normal anestrus period in bitches. one review of a population of dogs in europe stated that pyometra was more likely to occur in household pets than in “country dogs” that were permitted to breed freely.117 studies demonstrating increased interestrous interval in bitches that whelp compared to non-bred bitches suggests a natural mechanism to ensure proper duration between pregnancies.104,105 again, this suggests that bitches can be bred sequentially. wild dog populations wild canid populations are unlikely to be inbred and suffer from inbreeding depression unless geographic limitations on dispersal of pack members or other ecological factors prohibit mating of genetically diverse individuals.1,2,118 animals sense relationship through olfaction, most likely by identifying major urinary proteins or proteins of the major histocompatibility complex (mhc), and use that information to select unrelated mates.119 wild dogs are more likely to cycle seasonally, with variation in seasonality associated with latitude.120,121 in packs of wild dogs, the bitches will cycle together and many of the subordinate bitches will not bear young. because they may go through false pregnancy, they participate in raising of offspring from the dominant pack members, to which they most likely are related, giving them an opportunity to pass on their genetics even if they are not bred.122,123 incidence of ceh-p in one population of adult stray dogs was 5.4%,121 much lower than the 9% incidence of ceh-p described for a clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 30clinical theriogenology • volume 4 number 1 • march 2012 similarly aged population of owned bitches.124 average lifespan of wild dogs is much less than that of domestic dogs, as they succumb to predation and disease.123 survey and results a survey was distributed electronically to veterinarians who are members of the american college of theriogenologists or members of the society for theriogenology. a large number of participants received the survey as members of both organizations, making it difficult to know to exactly how many unique individuals the survey was sent. forty-nine people responded to questions about management of breeding animals and where they got the information guiding their decisions about management, and provided demographic information. for the purposes of this survey, “the literature” refers to published materials and information provided through continuing education, “experience” refers to personal experience, and “anecdote” refers to knowledge gained through others’ experience. forty-two of the respondents practiced in the united states and three in canada. others practiced in australia and countries in europe; some respondents practiced in more than one country. those practicing in the united states were from 30 states, representing all of the country except the southwest. some respondents practiced in more than one state and two were not in small animal practice at the time of this survey. when asked about specific laws in their country, state, or other municipality regarding management of breeding animals, 11 of 46 respondents (23.9%) were unaware of any such laws and 15 (32.6%) were sure there were no such laws. laws described revolved around housing (n = 1); limiting or controlling size of facilities or number of animals housed there (n = 7); restricting breeding, usually by mandating maximum number of litters per bitch over a given time period (n = 2); and regulations on veterinary care, including use of surgical artificial insemination (n = 1) and reporting of brucellosis (n = 1). fifteen of 48 veterinary respondents (31.2%) were dog breeders. when asked about brucellosis testing recommendations for bitches and stud dogs, the majority of respondents recommended bitches be tested at time of breeding (27 of 48, 56.3%) and males be tested at the time of breeding (27.1%) or twice yearly (29.2%). some felt that there should be no hard-and-fast regulation but that instead testing should be based on potential exposure, including at entry into a new kennel or after travel of a given dog or its kennel-mates to shows or other outside venues. for male dogs, recommendations also varied with how frequently the dog was used for breeding, perhaps as a reflection of potential exposure, and many people recommended testing at the time of semen collection for immediate use or for storage as chilled or frozen semen (20.8%). finally, some respondents specifically noted that brucellosis testing should be performed for all bitches and stud dogs presented for infertility. few respondents gave information about source for their recommendation; four stated that it was based on the literature and three based it on their own experience. recommendations for housing of intact male dogs was almost uniformly split between individual housing separate from all other dogs (11 of 48, 22.9%) and housing with other males and females unless the females were in heat (25.0%). many noted that it depends on the breed in question and their possible dog-aggressive tendencies (20.8%) and on the individual male dog and his temperament (16.7%). five people noted that they had never been asked to give a recommendation regarding housing of intact male dogs. when questioned about resources guiding recommendations, one person noted use of the literature (2.1%) while 25 (52.1%) noted personal experience and six (12.5%) noted anecdote as their source. youngest age at which bitches should be bred was noted to vary by breed (10 of 48, 20.8%), but most respondents felt that the second heat or two years of age was the earliest a bitch should be bred (56.3%), with most including the proviso that all appropriate testing for hereditary disease be done by this time. other recommendations included at the first heat (8.3%) or at the third heat (6.2%), or at ten to 12 months of age (8.3%), 12 to18 months of age (4.2%), or 18 to 24 months of age (4.2%). one person stated that they follow recommendations of that breed’s national club. oldest age at which bitches should be bred was widely noted to depend on breed, health of the bitch, and number and quality of the pups produced in earlier breedings (16.7%). responses for specific ages included five to six years of age (6.2%), six to seven years (12.5%), seven to eight years (8.3%), and eight to ten years (16.7%). one person referenced the critical age, as described earlier, and two people suggested it should not be based on clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201231 clinical theriogenology • volume 4 number 1 • march 2012 age but on maximum number of litters in a lifetime, with either two or three litters suggested as that maximum. the recommendation for frequency with which bitches should be bred overwhelmingly was every cycle as long as the bitch and pups were healthy and the bitch had regained body condition after lactation (54.2%). other responses included no more than once per year (10.4%), two cycles in a row and then skip a cycle (12.5%), and every other cycle (6.2%). one person noted that veterinarians and breeders may be limited by regulations outside of their control, for example, breed club requirements in some countries permitting only one litter per bitch to be registered annually. sources for all of these recommendations were experience (45.8%), followed by the literature (29.2%), and anecdote (20.8%). diet of pregnant and lactating bitches was strongly recommended to change in the last trimester of confirmed pregnancy and throughout lactation (12 of 47, 25.5%) to a diet higher in calories, protein and fat, such as a puppy, growth, or performance diet (51.1%). six respondents (12.8%) changed diet earlier, at one month of gestation. other diets recommended included any association of american feed control officers (aafco) or balanced diet appropriate for this life stage (14.9%), a high quality adult food (12.8%), a named specific diet (6.4%), and a mixed of dry and canned food (2.1%). two respondents specifically stated that raw diets should not be fed. there was no clear agreement on supplements, with three people each suggesting vitamin or fish oil supplements, and two people stating that no supplements should be used. respondents also noted that diet and feeding recommendations may be based on the bitch’s body condition at the time of breeding and throughout pregnancy and lactation (10.6%), on litter size (6.4%), and on breed (4.3%). diet and feeding recommendations were based largely on the literature (55.3%), followed by experience (27.7%), anecdote (12.8%), and consultation with a nutritionist or food company representative (6.4%). housing of pregnant and lactating bitches was strongly based on disease control concerns, with 25 of 46 respondents (54.3%) recommending that from late pregnancy through lactation bitches be housed separate from other dogs in the facility. most noted that the environment, whether it be a home environment or a kennel, should have appropriate temperature controls, be easily cleaned to minimize spread of disease, and provide minimal stress to the bitch and pups. housing recommendations were largely based on experience (56.5%), followed by the literature (30.4%), and anecdote (13.0%). conclusions there is little literature to support many of the recommendations made by veterinarians regarding management of breeding dogs. this limits our ability to guide legislators uniformly and may result in inappropriate legislation or legal findings. from this literature review and responses from the survey, it is clear that nutrition during pregnancy and lactation is the topic that is best researched and documented. much work remains to be done better defining appropriate housing, management of breeding frequency, and testing for brucellosis. many of the regulations in place govern breeding to try to control “puppy mills,” broadly defined as breeding facilities with large numbers of animals geared toward production without appropriate management of the individual breeding animals and their needs. because of this, many laws limit the number of breedings or litters per year or over a lifetime. it is clear that it may be in a given bitch’s best interest to be bred consecutively and legislation perhaps should be geared more toward maximum number of litters in a lifetime followed by mandated ovariohysterectomy than toward limiting number of litters per year. veterinarians responding to this survey largely were unaware of local laws regarding management of breeding animals despite there being such laws in 25 of the 30 states represented.125 efforts should be made by veterinarians to be aware of and to participate in creation of such laws locally and nationally. the chair of the advisory council on the welfare issues of dog breeding in the united kingdom sums up concerns about indiscriminate dog breeding by stating, “breeding excessive numbers of dogs in conditions that are far from ideal is only profitable because of the ignorance and attitudes of a significant number of potential purchasers.”126 it is essential that veterinarians to educate the public about the dogs they purchase and to assist legislators and court officials in developing and upholding laws that promote clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 32clinical theriogenology • volume 4 number 1 • march 2012 animal welfare without unfairly penalizing conscientious dog breeders. the farm animal welfare council in the united kingdom defines five freedoms, ideal states of maintenance for animals. these are (1) freedom from hunger and thirst, (2) freedom from discomfort, (3) freedom from pain, injury and disease, (4) freedom to express normal behaviors, and (5) freedom from mental distress and fear.127 we may promote these freedoms and animal welfare in regards to housing and breeding management of stud dogs and bitches by resolving the following: • all breeding animals should be housed in a manner that permits them to show normal behaviors and does not promote mental distress, with specific attention paid to individual dog’s temperaments. this includes housing male and female dogs in appropriate social units except for those times when bitches are in estrus, and ensuring clean facilities of appropriate size for the animal’s stage of life. human observation of and interaction with dogs must take place regularly. • dogs should have access to a balanced diet that is appropriate for their life stage and fed to them in such a manner as to readily permit them to maintain body condition. clean fresh water should be available (or easily accessible). • all dogs intended for breeding should be evaluated for hereditary disorders before being bred and should be regularly tested for canine brucellosis, either at the time of breeding or every six months. all breeding dogs should have a breeding plan developed with help of a veterinarian to ensure pairings to promote production of pups that are healthier than the parents. • all dog owners should recognize the pros and cons of elective gonadectomy and work with a veterinarian to make the best decision regarding timing of castration or spay for individual animals. all male dogs left intact should be regularly evaluated for prostate and testicular disease. all bitches should undergo ovariectomy or ovariohysterectomy when they are no longer to be used for breeding. • bitches should not be bred before they are physically mature and should not be bred on the first estrous cycle. • bitches should not produce more than five litters or be bred beyond the critical age for dogs of their breed, defined by body size. • bitches may be bred on consecutive estrous cycles if they maintain or regain body condition after whelping and lactation before onset of the next proestrus. references 1. marshall tc, coltman dw, pemberton jm, et al: estimating the prevalence of inbreeding from incomplete pedigrees. proc royal soc london b 2002;269:1533-1539. 2. spiering pa, syzkman gunther m, somers mj, et al: inbreeding, heterozygosity and fitness in a reintroduced population of endangered african wild dogs (lycaon pictus). conserv genet 2011;12:401-412. 3. lockyear km, waddell wt, 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karayannopoulou m, kaldrymidoy e, et al: the lymph drainage of the neoplastic mammary glands in the bitch: a lymphographic study. anat histol embryol 2006;35:228-234. 69. dow c: the cystic hyperplasia-pyometra complex in the bitch. vet rec 1958;70:1102-1108. 70. fukuda s: incidence of pyometra in colony-raised beagle dogs. exp anim 2001;50:325-329. 71. egenvall a, hagman r, bonnett bn, et al: breed risk of pyometra in insured dogs in sweden. j vet intern med 2001;15:530-538. 72. vanbree h, deschepper j, capiau e: the significance of radiology in the diagnosis of pyometra (endometritis post oestrum) in dogs: an evaluation of the correlation between radiographic and laboratory findings in 131 cases. j vet med a 1988;35:200-206. 73. hardy rm, osborne ca: canine pyometra: pathophysiology, diagnosis and treatment of uterine and extra-uterine lesions. j am anim hosp assoc 1974;10:245-268. 74. stone ea, littman mp, robertson jl, et al: renal dysfunction in dogs with pyometra. j am vet med assoc 1988;193:457-464. 75. okano s, tagawa m, takase k: relationship of the blood endotoxin concentration and prognosis in dogs with pyometra. j vet med sci 1998;60:1265-1267. 76. heiene r, vanvonderen ik, moe l, et al: vasopressin secretion in response to osmotic stimulation and effects of desmopressin on urinary concentrating capacity in dogs with pyometra. am j vet res 2004;65:404-408. 77. heiene r, kristiansen v, tiege j, et al: renal histomorphology in dogs with pyometra and control dogs, and long term clinical outcome with respect to signs of kidney disease. acta vet scand 2007;49:13-21. 78. deschepper j, vanderstock j, capaiu e: anaemia and leucocytosis in one hundred and twelve dogs with pyometra. j small anim prac 1987;28:137-145. 79. vandeplassche m, coryn m, deschepper j: pyometra in the bitch: cytological, bacterial, histological and endocrinological characteristics. vlaams dierg tijd 1991;60:207-211. 80. faldyna m, laznicka a, toman m: immunosuppression in bitches with pyometra. j small anim prac 2001;42:5-10. 81. niskanen m, thrusfield mv: associations between age, parity, hormonal therapy and breed, and pyometra in finnish dogs. vet rec 1998;143:493-498. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201235 clinical theriogenology • volume 4 number 1 • march 2012 82. johnston sd, root kustritz mv, olson pn: disorders of the canine uterus and uterine tubes (oviducts). in: johnston sd, root kustritz mv, olson pn, editors. canine and feline theriogenology. philadelphia: wb saunders; 2001. p. 216. 83. white cr, hohenhaus ae, kelsey j, et al: cutaneous mcts: associations with spay/neuter status, breed, body size, and phylogenetic cluster. j am anim hosp assoc 2011;47:210-216. 84. reichler im, pfeiffer e, piche ca, et al: changes in plasma gonadotropin concentrations and urethral closure pressure in the bitch during the 12 months following ovariectomy. theriogenology 2004;62:1391-1402. 85. stocklin-gautschi nm, hassig m, reichler im, et al: the relationship of urinary incontinence to early spaying in bitches. j reprod fertil 2001;suppl 57:233-236. 86. angioletti a, defrancesco i, vergottini m, et al: urinary incontinence after spaying in the bitch: incidence and oestrogen-therapy. vet res comm 2004;28 suppl 1:153-155. 87. holt pe: urinary incontinence in the male and female dog or does sex matter? available at: www.vin.com. accessed 10/01, 2004. 88. spain cv, scarlett jm, houpt ka: long-term risks and benefits of early-age gonadectomy in dogs. j am vet med assoc 2004;224:380-387. 89. duffy dl, serpell ja: non-reproductive effects of spaying and neutering on behavior in dogs. proc symp nonsurgical contraceptive methods for pet population control, alexandria va, 2006:1-2. 90. kim hh, yeon sc, houpt ka, et al: effects of ovariohysterectomy on reactivity in german shepherd dogs. vet j 2006;172:154-159. 91. o’farrell v, peachey e: behavioural effects of ovariohysterectomy on bitches. j small anim prac 1990;31:595-598. 92. wright jc, nesselrote ms: classification of behavior problems in dogs: distributions of age, breed, sex and reproductive status. appl anim behav sci 1987;19:169-178. 93. reisner ir: dominance-related aggression of english springer spaniels: a review of 53 cases. appl anim behav sci 1993;37:83-84. 94. michell ar: longevity of british breeds of dog and its relationship with sex, size, cardiovascular variables and disease. vet rec 1999;145:625-629. 95. waters dj, kengeri ss, clever b, et al: exploring mechanisms of sex differences in longevity: lifetime ovary exposure and exceptional longevity in dogs. aging cell 2009;8:752-755. 96. rogers al, templeton jw, stewart ap: preliminary observations of estrous cycles in large, colony raised laboratory dogs. lab anim care 1970;20:1133-1136. 97. chakraborty pk, panko wb, fletcher ws: serum hormone concentrations and their relationships to sexual behavior at the first and second estrous cycles of the 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bitches of seven breeds. j am vet med assoc 1977;171:271-273. 104. linde-forsberg c, wallen a: effects of whelping and season of the year on the interoestrus intervals in dogs. j small anim prac 1992;33:67-70. 105. christie dw, bell et: some observations on the seasonal incidence and frequency of oestrus in breeding bitches in britain. j small anim prac 1971;12:159-167. 106. bouchard g, youngquist rs, vaillancourt d, et al: seasonality and variability of the interestrous interval in the bitch. theriogenology 1991;36:41-49. 107. tedor jb, reif js: natal patterns among registered dogs in the united states. j am vet med assoc 1978;172:11791185 108. smith wc, reese wc: characteristics of a beagle colony. i. estrous cycle. lab anim care 1968;18:602-606. 109. sokolowski jh: reproductive features and patterns in the bitch. j am anim hosp assoc 1973;9:71-81. 110 kutzler ma: estrus induction and synchronization in canids and felids. theriogenology 2007;68:354-374. 111. dow c: the cystic 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canine uterus – gross anatomical and histological features. reprod domest anim 2009;44 (suppl 2):152-155. 116. al-bassam ma, thomson rg, o’donnell l: normal postpartum involution of the uterus in the dog. can j comp med 1981;45:217-232. 117. benesch f, pommer a: radiographic diagnosis of pyometra and hydrometra in carnivores. wein tier monat 1930;17:1-12. 118. randall da, pollinger jp, wayne rk, et al: inbreeding is reduced by female-biased dispersal and mating behavior in ethiopian wolves. behav ecol 2007;18:579-589. 119. cheetham sa, thom md, jury f, et al: the genetic basis of individual-recognition signals in the mouse. curr biol 2007;23:1771-1777. 120. totton sc, wandeler ai, gartley cj, et al: assessing reproductive patterns and disorders in free-ranging dogs in jodhpur, india to optimize a population control program. theriogenology 2010;74:1115-1120. 121. ortega-pacheco a, segura-correa jc, jimenez-coello m, et al: reproductive patterns and reproductive pathologies of stray bitches in the tropics. theriogenology 2007;67:382-390. 122. voith vl: functional significance of pseudocyesis. mod vet pract 1980;61:75-77. 123. somers mj, graf ja, szykman m, et al: dynamics of a small re-introduced population of wild dogs over 25 years: allee effects and the implications of sociality for endangered species’ recovery. oecologia 2008;158:239-247. 124. ewald bh: a survey of the cystic hyperplasia-pyometra complex in the bitch. small anim clin 1961;1:383-386. 125. american veterinary medical association: regulation of companion animal breeders and dealers. available at: http://www.avma.org/advocacy/state/issues/breeder_bills-table.pdf. accessed 08/23, 2011. 126. crispin s: tackling the welfare issues of dog breeding. vet rec 2011;168:53-54. 127. farm animal welfare council: five freedoms. available at: www.fawc.org.uk/freedoms.htm. accessed 08/05, 2011. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201237 clinical theriogenology • volume 4 number 1 • march 2012 clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 38clinical theriogenology • volume 4 number 1 • march 2012 omniblank: 2014: infectious diseases in new world camelids infectious diseases in new world camelids christopher cebra college of veterinary medicine, oregon state university, corvallis, or we have been lucky over the years that camelids in north america have had relatively few problems with contagious disease. herd outbreaks of infections were rare. individual infections were more commonly the result of opportunistic invasion of damaged tissues, rather than aggressive, spreadable infections. in recent years, our expanding knowledge and improvement in diagnostic methodologies have enabled us to identify a number of important infectious agents. some of these are of very sporadic importance. others appear to be more common. the amount of press they receive is not always directly proportional to their importance. viruses rotavirus and coronavirus these two viruses are recovered from crias with diarrhea. coronavirus is common and has been associated with outbreaks of diarrhea affecting camelids of all ages. coronavirus appears to be more contagious, and frequently affects the gastrointestinal tract. rotavirus appears to be rare in the u.s, but is occasionally brought to a farm by one infected animal, only to spread over a couple weeks to all animals on the farm. the diarrhea is usually self-limiting, though young crias can require intensive fluid management. diagnosis is achieved by electronmicroscopy of feces. the camelid coronavirus is related to the bovine coronavirus, but it is not known whether bovine diagnostic tests detect it or whether it can cross-infect between species. treatment is mainly supportive. prevention is chiefly through quarantine. bovine viral diarrhea virus bovine viral diarrhea virus (bvdv) has recently gained prominence in discussions of camelid herd health matters. previously, the virus was thought to only transiently affect camelids and only cause mild signs. more recently, the syndrome of persistent infection (pi) has been detected in alpacas. persistent infection is a well known syndrome in cattle and sheep and requires infection of a pregnant dam. if the strain of bvdv (type ib) is right, the dam becomes viremic, and the timing is right, the cria can be born with a lifelong viral infection. infected crias typically grow poorly and are prone to bouts of other infections, but can also appear normal. another important factor is that pi crias continue to shed the virus, and act as the source of infection for the next generation. the timing of infection to create the pi state is not known in camelids. in cattle it lasts most of the first 45% of pregnancy. the original source of infection can be a bovid, a pi cria, or a person (or piece of equipment) who travels from farm to farm. multiple genetic types have been identified in camelids, most likely due to separate cross-over events from another species, presumably a bovid. in camelids with exposure to cattle, unless the disease is eliminated from the cattle, and possibly deer, the risk will continue. in camelids without such exposure, personal hygiene and detection of pi crias are paramount. herd control involves several strategies. to determine if a herd has been exposed, serum antibody titers (red top tube) are determined. vaccination of a herd, even though it might somewhat decrease (not eliminate) the chance of pi crias, ruins the usefulness of herd titers to determine exposure. by this thinking, herds without titers should be considered low risk, because they have not been, or are not currently being, exposed to the virus. if a herd shows antibody evidence of exposure, finding the source is important. if cattle or deer can be ruled out, and a pi camelid is suspected, the polymerase chain reaction (pcr; purple top tube) is the test of choice for screening the herd. a positive pcr suggests the camelid is infected and should be isolated for retesting. a positive retest 3 weeks later suggests pi, though one cria reverted to negative after 8 weeks. because of their risk to the herd, pi crias should not be allowed any contact with pregnant 593 clinical theriogenology • volume 6, number 4 • december 2014 camelids. a negative pcr, except in crias only a few months old, rules out the pi state for the life of the camelid. the pi status can only develop in the gestating crias. adults and crias on the ground can become infected, and may show some mild nasal, oral, or ocular discharge and irritation, but cannot become pi. treatment for camelids with acute bvd is mainly supportive. treatment of pi camelids may not be in the herd’s best interest. prevention is through elimination of all pi animals (ruminant or camelid) in contact with pregnant dams. viral encephalitides west nile virus and eastern encephalitis virus both can affect camelids. only west nile virus is present on the west coast, and it has not reached all areas. both are spread from birds by mosquitoes and thus have strong seasonality. unvaccinated camelids are at the greatest risk, especially when the disease first moves into an area, or when migrating birds arrive from various sources during mosquito season. a three-shot course of the killed virus vaccine, with a booster before mosquito season, appears to offer good protection. the clinical signs involve neurologic dysfunction, including weakness, inability to hold the head upright, dullness, lethargy, and recumbency. diagnosis is achieved by antibody tests. many other species, including man, are susceptible, but camelids are not thought to produce enough virus to be considered infective to other species. treatment is mainly supportive. hyperimmunized plasma and specific antiviral drugs may be helpful. adenovirus adenovirus has been recognized for a long time, but its importance has always been questioned. early research at oregon state university suggested that most camelids were exposed, based on antibody titers, but evidence of clinical disease was rare. putative lesions have been found in the gastrointestinal tract (erosions), liver (zonal hepatitis), and lung (pneumonia). these diseases appear to be uncommon, but can be severe in individual animals, especially crias. detection is by antibody titer or isolation of the virus from diseased tissues. adenoviruses create inclusion bodies that may be seen on microscopic examination of tissues. currently, there are no firm recommendations for prevention. equine herpesvirus equine herpesvirus (ehv)1 gained some notoriety in the 1980s, but appears to be altogether rare. this herpesvirus spreads from equids to camelids and causes acute blindness, seizures, weakness, and other neurologic signs. it is usually fatal within a few days. the reported cases often had contact with zebras, not horses, and the only case the author has seen was in a llama with a confirmed immunodeficiency. currently, there are no firm recommendations for prevention. separation of camelids from equids, especially immunocompromised camelids and exotic equids, should decrease risk. recent work suggests zebras have ehv9, which is not normally carried by domestic horses, but is infectious for a variety of species, but the development of camelid cases with confirmed ehv1 during the recent california outbreak also suggest that this virus is the main culprit. papillomavirus the papillomavirus of camelids was first described in 2003. it induced single or multiple solid growths on the nose, lip, and cheeks. masses occasionally wax and wane in size, and often regrow after excision. although the nature of these appears similar to the equine sarcoid, and it has been suggested that the bovine papilloma virus may play a role, a unique camelid papilloma virus was identified in one case. it is not known how this spreads, but most papillomaviruses invade already damaged tissue. animal to animal transmission is likely, but there may also be an immune component. outbreaks are rare, suggesting that transmission is inefficient, or that the immune defect is rare. diagnosis is by biopsy. some may regress spontaneously, though most appear to persist until excised, and may regrow. other treatments are not required. 594clinical theriogenology • volume 6, number 4 • december 2014 contagious ecthyma/orf/soremouth this parapox virus usually affects sheep and goats, and can spread from those species to camelids. transmission does not require direct contact, because the virus is long-lived and can contaminate the environment. thus, areas inhabited by infected sheep or goats up to a year previously could be sources. once in a herd, orf could presumably become endemic, most severely affecting newborns each year while sparing immunocompetent adults. the most common lesions are ulcerative and proliferative sores on the lips and nose. in other species, foot and udder lesions are also common, but those are not usually mentioned in camelids. in most animals, the lesions spontaneously regress within two months, but some, presumably immunodeficient, animals maintain infections several months longer. the disease causes discomfort on eating, which is most important in crias. food intake or weight gain should be monitored to avoid more serious problems. a vaccine exists for orf, but it can itself cause the disease. its use may offer some advantage in the face of an outbreak, but it should not be used in orf-negative herds. people can get orf from infected animals or the vaccine, so wear appropriate protective gear and exercise caution. rabies rabies is an uncommon disease on the west coast. it is worthy of mention mainly because it can spread to people and because vaccination in other species appears to afford good protection. vaccination of camelids in rabies-prone areas is also recommended. clinical signs of rabies can be very variable. they typically involved a behavior change, either duller or more aggressive, and some degree of swallowing dysfunction. suspect camelids should be isolated at once and outmost caution taken to avoid contact with saliva or other body fluids. bacteria mycoplasma haemolama mycoplasma haemolama is a bacterium that parasitizes the blood. it was formerly known as eperythrozoa. the parasite invades red blood cells and presumably leads to their destruction. clinical signs can include anemia, fever, poor growth, weight loss, and lethargy. most infected camelids show none of those signs, or very low grade anemia at worst. however, camelids with poor immune system function may show severe disease, including packed cell volumes in the low teens to upper single digits. the mechanism of transmission is unknown. because the parasite resides in the blood, the role of biting insects has been suggested. additionally, it is possible that crias born to infected dams are themselves born infected. our latest research suggests the infection is usually life-long. the numbers and clinical significance of the parasite are low when the camelid is healthy, but become more prominent when other diseases or stressors decrease immune system function. the older method of identification, examination of a blood smear, is useful for the clinical disease, but the newer pcr test is required to identify carriers. tetracycline appears to be the most effective treatment, and may be used against the clinical disease. salmonella salmonella is luckily still rare in camelids. this bacterium usually invades through the gastointestinal tract, and can affect the gut, liver, and occasionally other organs. clinical signs can include diarrhea, fever, lethargy, anorexia, weakness, and shock. it is becoming more common in horses and cattle, probably as those species become more intensively managed. large farms, overcrowding, contact with other infected species, and feeding contaminated feedstuffs all increase risk. diagnosing salmonellosis usually involves culturing the organism from infected feces or tissues. a bovine vaccine exists, but the low prevalence of infection in camelids rarely justifies its use. 595 clinical theriogenology • volume 6, number 4 • december 2014 clostridial diseases clostridial diseases including enterotoxemia and type a clostridiosis are among the best known bacterial infections in camelids. in fact, in the u.s., they are not that common. clostridial organisms are as deadly as they are because they make potent toxins. botulism, tetanus, blackleg, and enterotoxemia are examples. tetanus and botulism are both rare in camelids, the former potentially because of vaccination; the latter being associated on rare occasions with infected rodents milled into feeds. the muscle and liver clostridia are likewise rare, often being associated with wounds or liver flukes in other species. enterotoxemia and type a clostridiosis both affect the gastrointestinal tract. in south america, these are associated with the high cria mortality rate, but recent evidence suggests that they require previous gut damage to start their process. coccidia are the most likely cause for this previous damage. in the u.s., the intestinal clostridia occasionally affect or kill camelids. signs are fairly short before the animal dies. colic, low-grade diarrhea, fever, and severe lethargy may be seen. at oregon state, clostridium perfringens type c is the usual isolate, even when type a is suspected. preexisting intestinal damage, again usually due to coccidiosis, is suspected in some cases. all ages are affected. in ruminants, rapid growth and high carbohydrate diets are implicated. in oregon camelids, we have some concern about overingestion of lush spring pasture, but all in all, clostridiosis is not that common. the disease is usually diagnosed by characteristic signs or culture. the culture must be performed antemortem or soon after death, because clostridia and found in the gut of normal camelids and rapidly proliferate postmortem. alpaca fever/streptococcus zooepidemicus alpaca fever is a sporadic but serious condition in this country. it is caused by streptococcus zooepidemicus, a bacterium most commonly associated with respiratory disease in horses. camelids may become infected by inhalation of bacteria, and also possible by ingestion or wound invasion. the bacteria enter the blood stream and infect various organs or body cavities. sometimes the infection remains diffuse and other times it localizes into abscesses. the camelid shows signs based on the location of the infection. common signs are fever, lethargy, weakness, decreased feed intake, increased respiratory effort, and colic. diagnosis is made by culturing the organism from an infected site. presumptive diagnosis can be made in camelid with compatible signs from which a pus-like fluid is recovered from the chest or abdomen. treatment is supportive with the addition of antibiotics and anti-inflammatory drugs to decrease fever and inflammation. s. zooepidemicus is usually sensitive to many common antibiotics including penicillin, ceftiofur, and florfenicol. this disease is considerable more common in south america and is associated with poor management. in north america, it is sometimes linked to exposure with horses. many healthy horses have this bacterium in their nasal passages. camelids usually keep their distance from horses and the risk of transmission appears to be low, but owners of both horses and camelids, or camelid owners with equine neighbors should use common sense to reduce the risk of transmission. leptospirosis leptospira interogans primarily colonizes the urogenital tract. the reservoir populations are most commonly cattle or deer, who both can become infected without necessarily becoming severely sick. transmission can occur through direct contact with infected urine or with water contaminated by infected urine. the organism can cross into the body through most mucous membranes, and travels through the blood to the kidney or reproductive organs. it is primarily known as a cause of fetal loss or late term abortion in camelids. in rare cases it may also damage the kidneys or other organs. in the latter cases, other signs of infection, including fever, abnormal urine, lethargy, anorexia, or other systemic signs, may be present. because the organism is hard to isolate, diagnosis is usually supported by demonstrating rising antibody titers in affected animals. remember that in the case of fetal loss or abortion, the dam usually appears clinically normal. 596clinical theriogenology • volume 6, number 4 • december 2014 caseous lymphadenitis corynebacterium pseudotuberculosis causes lymph node abscessation in small ruminants and occasionally camelids. in sheep, shearing is the major route of transmission and most of the abscesses are external. in goats, ingestion or inhalation are greater problems and many of the abscesses are internal. in camelids, both are possible. in the u.s. camelid population, it is rare, but it can be a serious problem in some herds, affecting multiple animals. presumably, initial entry into the herd comes from contact with infected small ruminants, contaminated material, or contaminated shearing equipment. pus draining from open abscesses is highly infective, and the organism can persist in the environment for some time. antibiotics can be used to try to limit the severity of infection, but it is very hard to eradicate. vaccines may limit severity in infected herds. fungal diseases cryptococcus cryptococcus is associated with bird droppings and certain soil types. it is common around the pacific rim, particularly in the northwest u.s. and british columbia. spores are usually inhaled and can invade the lung or infect other organs via the blood. in oregon, the two most common syndromes are chronic respiratory disease or neurologic diseases. the respiratory form is characterized by a rapid respiratory rate, exercise intolerance, and eventual weight loss and decreased feed intake. death ensues within about two months. the neurologic form is marked by rapid onset of severe dullness, blindness, and lethargy. affected animals usually die within a week. diagnosis can be reached by isolating the organism or potentially demonstrating antibody titers. antifungal treatment may be attempted, but have not historically been successful. man can get this disease. direct transmission from animals to people is extremely rare. coccidiomycosis/valley fever coccidiodes immitis spores lie dormant in dry soils and become airborne when the soil is disturbed. the organism is common in the drier areas of the southwest, though we have seen one case in oregon. it is inhaled, and similar to cryptococcus, can invade the lung or travel through the blood to remote sites. any organ can be infected. skin and bones are common sites. signs are slowly progressive, and in any endemic areas, any camelid with chronic, progressive signs should be considered a suspect. poor-doing is the most common sign, though more specific respiratory signs or thickened skin may also be seen. diagnosis can be reached by isolating the organism or potentially demonstrating antibody titers. antifungal treatment may be attempted, but have not historically been successful. people can get this disease. animals with diffuse diseases can very rarely spread it to people. be cautious around camelids with compatible signs. dermatophytosis/ringworm ringworm is generally uncommon in camelids. when it occurs, it leads to fiber loss and skin crusting on the legs, perineum, and face. winter housing may increase the risk of transmission for one camelid to another. diagnosis is by examining infected hairshafts under a microscope, biopsy, or dermatophyte culture. a variety of treatments exist. most involve clipping the infected area and scrubbing it with antiseptics or specific antifungal agents. vaccines no specific camelid vaccines exist in north america. most bovine and equine vaccines appear to be safe in camelids, but camelids often require more vaccine doses to achieve the same antibody response. degree of protection is a total unknown. 597 clinical theriogenology • volume 6, number 4 • december 2014 some vaccines make practical sense because they work well in other species and prevent horrible diseases. examples of this are the tetanus vaccine and, in some places, rabies vaccine. the enterotoxemia vaccine (type c&d toxoids) fits in a similar category. the muscle and liver clostridia (7way vaccine) are rare and the vaccine is not benign. it should be used with caution in pregnant camelids. other vaccine usages arise as new diseases emerge. west nile virus is an example of this. the bovine rotavirus/coronavirus may afford some protection against neonatal diarrhea. the leptosporosis vaccine may help prevent abortion. of the myriad of vaccines available, most should only be used after determining their usefulness in your area. 598clinical theriogenology • volume 6, number 4 • december 2014 1 contact susan robertson surobertson@csu.edu.au © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12298, http://dx.doi.org/10.58292/ct.v17.12298 case report a case study of dystocia and abortion in angora goats susan robertson, allan gunn, bruce allworth gulbali institute and school of agricultural, environmental and veterinary sciences, charles sturt university, wagga wagga, nsw, australia abstract this observational study investigated the incidence of dystocia over 3 years in a commercial angora goat herd and the occurrence of spontaneous abortion in 1 year. the does (n = 41-127) were grazed extensively and were shedded overnight during late pregnancy for shearing. incidence of doe mortality (≤ 3%) and assisted dystocia delivery (≤ 9%) were lower in 2022 and 2023; in 2024, doe mortality (13.4%) was associated with a high rate of abortion and dystocia (19.7%) in the first week of the predicted kidding period. abortion in very late pregnancy in 2024 increased doe mortality due to their inability to expel dead, decomposing full-term kids. although the cause of abortion was not established, stakeholders should be aware of the high risk of abortion with angora does from noninfectious causes. keywords: dystocia, mortality, abortion, pregnancy, nutrition background high rates of abortion are a potential risk in goat reproduction in many breeds,1 particularly in angora does.2 abortion in goats has been associated with infectious3 and noninfectious causes.4,5 undernutrition is reported to be a key noninfectious cause of abortion, with low blood glucose, low body condition score, and mineral deficiencies being risk factors for high rates of abortion (> 50%).5,6 undernutrition in late pregnancy has also been associated with lack of viability in live-born kids.7 although abortion can occur at any stage of pregnancy, it is more common from days 90 to 120 of pregnancy.2 this is consistent with increased placental sensitivity to high peripheral cortisol concentrations from days 101 to 125 (compared to other periods of pregnancy),wherein high cortisol may result in abortion after 36 hours of restricted nutrition and stress due to transport.8 inadequate nutrition increases fetal cortisol, promoting regression of the corpus luteum (sole source of progestogen in goats) with subsequent abortion.9 failure of corpora lutea to produce progestagens results in loss of pregnancy in does within 2 days, although if > 1 corpus luteum is present, pregnancy may continue for varying periods.10 short-term events such as cold snaps, transport or shearing of angora does therefore create a risk of abortion due to an induced period of fasting.2,11 although the occurrence of abortion in goats is well documented,1,2,4 the occurrence in late pregnancy and implications on fetal death, dystocia, and doe mortality are not. this study reports observations of an abortion event and the associated incidence of dystocia, and contrasts with previous years dystocia incidence. case presentation this study used historical data from a private farm so was exempt under the charles sturt university animal ethics committee (project 2024ea-17). retrospective kidding records were obtained for an angora herd in southern australia, managed under extensive grazing conditions. mixed-age (1.5-8.5 years) does were typically mated in early may for 6 weeks; vaccinated against clostridial diseases annually, drenched as required to manage gastrointestinal worms, and managed to maintain does in a target condition score 3/512 through regular physical assessment. the goat herd did not have any known history of abortion, and there had been no introduction of new goats nor contact with other goat herds since 2022. shearing in each year was conducted in february to april then again in late september during late pregnancy by competent, professional shearers using electric handpieces in a shearing shed. no other procedures were undertaken on does at september shearing. for the september shearing, does were shorn on day 143 from buck introduction in 2022 and 2023 after a single overnight fast (with no access to feed and water for ~ 16.5-20.5 hours). in 2024, does were mated from may 7, mailto:surobertson@csu.edu.au http://dx.doi.org/10.58292/ct.v17.12298 2 citation: clinical theriogenology 2025, 17, 12298, http://dx.doi.org/10.58292/ct.v17.12298 2024, and were due to commence kidding on october 4. does were shedded overnight with no feed or water on 2 occasions prior to shearing; on day 140 from buck introduction (due to the threat of rain) and again on day 144 from buck introduction (immediately prior to shearing). does were shedded for < 20 hours on each occasion and were grazing ample green pasture (estimated 1,500 kg dm/ha) such that nutrition was only limited by fasting while shedded. the pasture in 2024 was primarily lucerne (medicago sativa) and annual grasses (mostly barley grass – hordeum leporinum) with no known toxic plants. grain supplements were not required; mineral supplements were not offered due to no known soil mineral deficiencies in the region. does were inspected daily after shearing and throughout the kidding period, with some kids tagged and weighed at birth. full kidding records were available for the 2 previous years. no samples from aborting goats were collected for laboratory testing due to the owner not initially recognizing elevated rates of dystocia with an abortion storm. the owner had > 40 years of experience in commercial sheep management and assisting dystocia in ewes. outcome after prekidding shearing in 2024, no clinical signs consistent with pregnancy toxaemia were observed and does appeared healthy. elevated rates of doe mortality and dystocia occurred in the first week of the kidding period (6-11 days after shearing) and days 150-155 after buck introduction. out of 127 does, 17 does died and 16 surviving does needed assistance during the 6 weeks of kidding period (table 1). mortality of does was recorded as follows: euthanasia due to inability to deliver kids (6/17), euthanasia due to signs consistent with septicaemia with decomposing fetuses (2/17), euthanasia for gangrenous mastitis (2/17), dead pregnant does without kidding (3/17), and does dying after giving birth to live kids (4/17). a total of 24 does were assisted during kidding, with only 4 of the assisted does giving birth to live kids. commonly, dystocia was not associated with fetal parts evident at the vulva, but a malaligned fetus within the reproductive tract. where the kid could be moved and presentation assessed, anterior presentation with one or both forelegs flexed and deviation of the head (head back, down or crown presenting) were the most common forms of malposture in 2024 (table 2). all fetuses were fully developed and appeared full-term. most dead fetuses delivered in the first week of kidding were in advanced stages of decomposition with signs reported including dehydrated cloudy eyes, generalized edema, sloughing of mohair, brown to blue skin, brown amniotic fluid, and putrid odor. these signs are consistent with death occurring several days before, strongly suggesting abortion.13 dead fetuses appeared to hamper delivery. for assisted does (where kids were delivered), 7 had single, 10 had twins and 1 had triplet. live kids were born unassisted to 15 does in the first 4 days of the kidding period. in comparison, shearing during late pregnancy was not associated with any perceived abortion event in either of the previous 2 years nor with abnormally high dystocia. in those years, the incidence of assisted delivery (≤ 9%) and doe mortality (1-3%) were markedly lower than for 2024. oversize kids were not considered a causative factor in any cases of dystocia in any year, with maximum birthweights of 3.7 kg (table 2). the does were in better body condition (3.5-4/5 score) at kidding in 2024 than in previous years (3.0). discussion late-term abortions have been reported as presenting sometimes live fetuses, without mention of dystocia.11 the table 1. percentage of does (number of does in brackets) assisted at delivery or dead for sequential intervals from the due date of kidding (day 150 after buck introduction) day of kidding 2022 2023 2024 assisted (%) deada (%) assisted (%) deada (%) assisted (%) deada,b (%) -3 to -1 0 0 1.0 (1) 0 0 0 1-7 2.4 (1) 2.4 (1) 4.0 (4) 1.0 (1) 13.4 (17) 9.4 (12) 8-14 2.4 (1) 0 1 (1) 1.0 (1) 0.8 (1) 1.6 (2) 15-21 0 0 0 0 3.1 (4) 2.4 (3) 22-28 0 0 0 0 1.6 (2) 0 29-57 2.4 (1) 0 3.0 (3) 1.0 (1) 0 (0) 0 total assisted or dead 7.3 (3) 2.1 (1) 9.0 (9) 3.0 (3) 19.7 (24) 13.4 (17) number of does in herd 41 100 127 ainclude euthanized at assisted delivery; binclude 2 does euthanized on days 11 and 19 due to gangrenous mastitis or cancer table 2. percentage malalignment of dystocia cases (number of does in brackets) particulars 2022 (%) 2023 (%) 2024 (%) head deviated 33 (1) 67 (4) 33 (6) both kids at once 0 17 (1) 11 (2) one or both forelegs flexed 33 (1) 17 (1) 39 (7) posterior presentation 0 0 6 (1) breech presentation 0 0 11 (2) deformed fetus 33 (1) 0 0 total number 3 6 18 birthweight (kg) 1.5-3.2 2.2-3.3 1.8-3.7 http://dx.doi.org/10.58292/ct.v17.12298 citation: clinical theriogenology 2025, 17, 12298, http://dx.doi.org/10.58292/ct.v17.12298 3 occurrence of decomposing fetuses and their impact on the incidence of dystocia and doe mortality in the present study is therefore noteworthy. etiology of 2024 abortion storm reported here cannot be determined. infectious causes cannot be excluded; however, with the herd history and the predilection for goats to abort after short-term nutritional stress events,2,11 a noninfectious (stressor related) etiology is a more likely explanation. there is clear evidence that nutritional restriction causes substantial abortions;5,14 restriction to 75% of maintenance requirement during late pregnancy caused 52% of does to abort 2 weeks after the change in ration.15 in comparison, angora does fed to maintenance requirements have resulted in low rates of abortion (2%).14 it is unclear why abortion occurred in 2024 but not in previous years if the cause was the fasting/handling event, given does were always shorn during late pregnancy and subjected to fasting immediately prior to shearing. it is possible that the additional shedding event prior to shearing in 2024 may have provided an additive nutritional deprivation stress effect. additionally, the good condition of does in 2024 may have contributed to the occurrence of abortion. if the shearing/fasting event was the cause, this is difficult to avoid during some stage of pregnancy, given angora goats are often shorn at 6-month intervals. since abortions can occur at any stage of pregnancy, management to reduce the risk would appear prudent and simultaneously reducing the risk of weather-induced abortion due to cold weather2 that may be more likely when recently shorn (risk of hypothermia). minimizing the period does are off feed when shedded for shearing and the provision of high-energy supplement immediately prior to shearing may reduce the risk and warrants further investigation. aggressive handling by humans has been reported as being associated with fetal loss in dairy goats.16 although rough handling did not occur at shearing in the present report, the process of shearing and the presence of unfamiliar persons could be perceived by goats as highly aggressive. similarly, a low social status has been associated with abortion.4 a low status may result in reduced access to feed leading to ‘nutritional restriction abortion’. when shedded overnight for shearing, lower status does may be unable to escape from aggressive does, resulting in stress and increased movement for a prolonged period that possibly increases the risk of abortion. regardless of the cause of the abortion storm in 2024, the consequences of late-term fetal death on rates of dystocia and doe mortality were severe. malalignment, particularly malposture, rather than excessive birthweights, caused dystocia in the event reported here. the alignment of fetuses involved in dystocia were consistent with reported patterns in other breeds.17 in extensively grazed herds with minimal monitoring to allow intervention in cases of dystocia, the level of doe mortality could be much higher after an abortion storm or fetal death without expulsion, than that reported here. it is suggested that abortion in very late pregnancy may increase doe mortality due to does being unable to expel decomposing, term lambs that is less probable in mid-pregnancy due to the smaller fetal size. these larger dead fetuses could be more susceptible to malposture during parturition, as the fetus is unable to participate in the required actions to correctly engage in the birth canal resulting in dystocia and likely death of the dam. abortion cases near term may be confused with malalignment and dystocia, overlooking the opportunity to improve welfare and production through preventive management. the prevalence of abortion in does in australia is unknown; nevertheless, veterinarians should be aware of the high risk with angora does and consider both noninfectious and infectious causes as risk factors. strategies to manage dystocia in commercial herds need to focus on prevention. this will vary with the cause, such that in the case of a dystocia storm as reported here, laboratory analyses to diagnose infectious or noninfectious cause may allow appropriate current and future interventions. since nutritional restriction is a potential cause, timing events earlier in late pregnancy may reduce the risk of fetal death causing dystocia along with minimizing the period of restriction. where does must be yarded for several hours or overnight, the provision of an energy supplement such as lupin grain immediately before or during yarding may reduce the risk. in small herds, drenching with 50 ml propylene glycol may be a feasible option. however, this will only be beneficial if there is a nutrient deficit interaction inducing fetal compromise, as hypothesized in this case. daily surveillance of does for 2 weeks after any handling or stress event may enable early detection of abortion or dystocia, and intervention (assistance or euthanasia) for the latter. learning points • high rates of abortion are a potential feature of goat reproduction • rates of dystocia in goats can be increased after an abortion storm during late pregnancy • abortion cases near term may be confused with fetal malalignment and resulting dystocia • abortion storms may be associated with periods of stressors such as undernutrition and handling that could be ameliorated by appropriate surveillance and management • dystocia was associated with malposture rather than oversize kids in all years conflict of interest none to declare. references 1. mellado m, valdéz r, garcía je, et al: factors affecting the reproductive performance of goats under intensive conditions in a hot arid environment. small rum res 2006;63:110-118. doi: 10.1016/j. smallrumres.2005.02.016 2. shelton m, groff j: improving reproductive efficiency in angora goats. san angelo; texas agricultural experiment station: 1984. 3. menzies pi: vaccination programs for reproductive disorders of small ruminants. anim reprod sci 2012;130:162-172. doi: 10.1016/j.anireprosci.2012.01.010 4. engeland iv, waldeland h, andresen ø, et al: foetal loss in dairy goats: an epidemiological study in 515 individual goats. anim reprod sci 1997;49:45-53. 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https://doi.org/10.1007/s11250-012-0090-8 https://doi.org/10.2527/jas1951.102411x https://www.angoras.co.za/article/abortion-in-angora-goats https://www.angoras.co.za/article/abortion-in-angora-goats https://doi.org/10.2460/ajvr.1976.37.04.403 https://doi.org/10.1016/j.smallrumres.2016.10.027 https://doi.org/10.1016/j.smallrumres.2016.10.027 https://doi.org/10.1016/j.physbeh.2016.02.001 _hlk184643381 2012: intrauterine fetal growth restriction, adaptation and programming-a review intrauterine fetal growth restriction, adaptation and programming–a review ramanathan k. kasimanickam,a vanmathy r. kasimancikam,a john p. kastelicb adepartment of veterinary clinical sciences, washington state university, pullman, wa; bagriculture and agri-food canada, lethbridge, ab canada abstract intrauterine fetal growth restriction (iugr) is fetal adaptation in response to a wide range of pathophysiological factors, including an inadequate supply of oxygen, nutrients, or both. in particular, low birth weight has been associated with subsequent disorders in adult life, including cardiovascular, neuro-developmental, metabolic, and reproductive diseases, independent of adult life style. animal models are excellent tools to study the effects of genetic, molecular and cellular events that modulate intrauterine growth and development. various experimental approaches have been used, involving alteration of numerous metabolic and physiologic events, with substantial variations among studies in both methods and species used. intrauterine fetal growth restriction remains a challenging problem in humans and is a major concern in agricultural animal since it affects production parameters. the objective of this manuscript is to review animal models which explore fetal adaptation, including iugr, and evaluate their outcomes, to suggest new opportunities for further research, and the potential for interventions to reduce or eliminate gestational events with deleterious effects on fetal, neonatal, and adult health. keywords: fetus, placenta; animal model; growth restriction introduction intrauterine fetal growth restriction results in unduly high perinatal morbidity and mortality.1-4 intrauterine fetal growth restriction refers to a condition in which a fetus is unable to achieve its genetically determined potential size. the clinical description of iugr is on the basis of birth weight below the tenth percentile, or fetal body weight less than two standard deviations from the mean of study population.5,6 several pathophysiological factors can disturb or restrict fetal growth, including chromosomal abnormalities, gene defects, poor placental function, and maternal stress due to environment or nutrition. the physiological adaptations of the fetus to an adverse intrauterine environment in response to pathophysiological factors, including restrictions on its growth rate, depend on the nature, timing and intensity of extrauterine or intrauterine challenges. adaptation of the fetus to a suboptimal intrauterine environment is of clinical importance in determining long-term, postnatal health.7 the physiological, neuroendocrine or metabolic adaptations that help the fetus to survive a period of intrauterine deprivation result in permanent programming of proliferation and differentiation events within key tissue and organ systems and pathological consequences in adult life.8,9 this is the so called barker's hypothesis, after david j. p. barker, who published the theory in 1997.7 the magnitude of fetal adaptations for both in utero survival and postnatal health outcomes provided the momentum for experimental studies to elucidate the nature and consequences of specific fetal adaptations to a compromised or poor intrauterine environment. this review summarizes animal model studies focused on the range of adaptations the fetus makes in response to a suboptimal uterine environment. underlying mechanisms normal growth has been described as, “the idiom of the genetic potential to grow which is neither abnormally constrained nor promoted by internal or external features”. in this definition, internal features relate to the fetus, whereas external features relate to the placenta, uterus, dam, and environment. however these internal and external characteristics, including the fetus, dam, and utero-placental unit, act collectively; therefore, the problem of interactions among the fetus, placenta and dam must be considered. furthermore, the genetically determined growth course at each stage of gestation has to be understood before growth restriction can be imposed. however, due to inherent interactions among numerous components, it is very difficult to determine the physiologic growth curve of an individual fetus.10 in clinical theriogenology • volume 4 number 2 • june 2012133 addition a fetus can achieve normal fetal growth despite altered internal and/or external features, since the maternal, utero-placental and fetal tissues are capable of adapting to alterations. furthermore, since the placenta and fetus are developing, they are not in a steady state. consequently, the same experimental method may have different effects, depending on the stage of gestation when it is applied, emphasizing the need to carefully consider the timing of experimental alterations. normal intrauterine growth takes place in phases–an embryonic and a fetal phase. the embryonic phase consists of proliferation, organization and differentiation of the embryo, whereas the fetal phase consists of continuing growth and functional maturation of the various tissues and organs. the fetal phase of intrauterine development depends on genetic, placental, and maternal factors. according to time of adverse intrauterine nutrition, a fetal growth restriction is classified as symmetric or asymmetric,11 as shown (table 1). table 1: comparison of symmetric and asymmetric fetal growth symmetric asymmetric occurs early in gestation occurs late in gestation entire fetus is proportionately small fetus is normal length but reduced weight multifactorial genetic, infectious or toxic effects due to placental insufficiency postnatal make-up growth is rare postnatal make-up growth possible adapted from 11 factors that cause fetal growth restriction numerous factors can result in restriction of fetal growth, including: maternal factors  undernutrition  maternal low birth weight  maternal age  chronic hypertension (gestational induced)  pre-eclampsia  diabetes environmental factors  toxicity  high altitude  hyperthermia  nutritional deficiencies placental factors  abnormal placentation  chronic abruption, infarcts, focal lesions  ischemia  chronic inflammatory conditions (e.g. villitis or placentitis) fetal factors  chromosomal anomalies  genetic conditions  congenital malformations  intrauterine infections  multiple fetuses impacts of fetal programming on postnatal life  neurologic, endocrine and cardiac problems  diabetes  delayed puberty clinical theriogenology • volume 4 number 2 • june 2012 134  poor ovarian follicular development  fewer germ, sertoli and leydig cells  poor placentation/fewer uterine carunlces  poor uterine capacity  reduced litter size  poor postnatal growth  poor meat quality treatment modalities  exogenous progesterone or synthetic progestins  exogenous growth hormone  dietary supplementation of energy, protein, or both  antioxidants  manipulations of arginine-nitric oxide/polyamine  sildenafil citrate  aspirin experimental models of restriction of intrauterine fetal growth various experimental approaches have been used to induce iugr in several species, including mice, rats, guinea pig and sheep (it occurs spontaneously in virtually all species). experimental methods to induce iugr include a reduction in utero-placental blood flow by vascular occlusion or ligation, a reduction in materno-fetal contact by cornual ligation or removal, placental embolization, carunclectomy, genetic manipulation of the fetus, fetal infection, a reduction in maternal nutrient intake (energy, protein, or both), maternal overfeeding, and maternal hyperthermia (figure 1).12-40 figure 1: experimental approaches to produce intrauterine fetal growth restriction (iugr) in animals. both nutritional and placental insufficiency models resulted in iugr and postnatal maladies, including endothelial dysfunction, hypertension, insulin resistance, type 2 diabetes, reduced cognitive clinical theriogenology • volume 4 number 2 • june 2012135 development, and cardiac diseases. fetal growth parameters and hormones in the ovine iugr fetus relative to a control are shown (table 2); that the iugr fetus weighed less than the control validated the model. table 2: fetal growth parameters and hormones in the growth-restricted ovine fetus (relative to control) carunclectomy hyperthermia maternal overfeeding embolization uterine artery ligation birth weight ↓ na ↓ ↓ na placental weight ↓ ↓ ↓ ↓ ↓ fetal weight ↓ ↓ ↓ ↓ ↓ relative organ weight ↑/= ↑/= ↑/= ↑/= ↑/= fetal plasma acth ↑ ↑ fetal plasma cortisol ↑ = females; ↑ males = ↑/= ↑ noradrenaline ↑ ↑ ↑ adapted from 40-42 placental vascular development the placenta has a major role in regulation of fetal growth. the sow and mare have a diffuse placenta, with chorionic villi distributed over the entire surface of the chorion, whereas in ruminants the chrioallantois attaches to discrete sites on the uterine wall. placental area of attachment continues to increase as gestation advances43,44 and vascular development of the placenta increases ~200% from mid to late gestation.44 in the cow, cotyledonary growth progressively increases throughout gestation,45,46 whereas in the ewe, growth of the cotyledonary mass is exponential during the first 70–80 days of pregnancy, thereafter slowing markedly until lambing. table 3: comparison of percentage change in capillary vascularity from midto late pregnancy in sheep (days 50 to 140) and cattle (days 125 to 250) placentomal tissue vascularity parameter sheep cattle caruncle cad ↑ 214% ↓30% cnd ↑ 37% ↑151% csd ↑140% ↑32% capillary size ↑45% ↓68% cotyledon cad ↑437 ↑186% cnd ↑1093% ↑80% csd ↑576% ↑172% capillary size ↓ 25% ↑71% cad capillary area density (a flow-related measure); cnd capillary number density (an angiogenesis-related measure); csd capillary surface density (a nutrient exchange-related measure). studies on changes in placental vascularity in response to nutrient-restriction in ewes and cows are very limited, and appear to be largely lacking in sows. seven days of fasting during mid-pregnancy in the ewe decreased vascular endothelial growth factor (vegf) mrna levels and placental weights on day 90; however, placental weights were similar at lambing in nutrient-restricted and control ewes.47 in nutrient-restricted cows (days 30 to 125 of gestation) there was a decrease in total placentome weight, including both the cotyledonary and caruncular portions, on day 125 compared to control cows. this inhibition in total placentome weight persisted, even after realimentation until day 250;46,48 however, only the weight of the cotyledonary tissue remained suppressed at day 250.48 in contrast, in several sheep models of maternal nutrient restriction from early to mid-pregnancy followed by realimentation, there was significant compensatory growth of the entire placentome.46,49 although maternal nutrient delivery clinical theriogenology • volume 4 number 2 • june 2012 136 during pregnancy has been shown to program fetal growth and development, both during pregnancy and later into adult life, it appears that maternal nutrition also programs placental development. in the cow, realimentation after ~90 days of nutrient restriction is the stimulus not only for altering placental vascularity and development, but also placental function.50,51 animal models several researchers used animals to study various aspects of fetal development. those research scenarios could be used as animal models and compared with existing fetal growth restriction models to advance knowledge regarding mechanisms underlying iugr. some scenarios worthy of investigation include: 1. large offspring syndrome in cows 2. uteroplacental hypoxia in sheep 3. embryonic/fetal adaptation in mares 4. low birth weight piglets nutritional status and environmental events can alter important physiological parameters of the embryo and fetus. this resetting is called as fetal or developmental programming and it can continue into childhood and adulthood to trigger disorders. the fetal cellular environment can modify the gene expression pattern in various tissues and organs, with long-term functional consequences in neonatal period and adulthood. fetal programming involves primary alterations and secondary compensations. according to the concept of fetal programming, any detrimental circumstance during the critical period of in-utero development affects the subsequent pattern of growth and development of tissues and organs and may predispose the affected fetus to metabolic disorders later in life.7 the placental angio-architecture is vulnerable to any alterations in the maternal environment. adverse uterine milieu produces an altered placental vascular phenotype and function. in utero, adverse events are critical factors in determining fetal growth, quality of life and overall health. underlying mechanisms of fetal programming caused by various perturbations in the maternal compartment and how they are transmitted across the placenta remain to be completely established. several vascular factors including bfgf, egf, pdgf, angiopoietin 1 and 2, and the vegf family, have been identified as important placental angiogenic regulators. up-regulation of angiogenic factors vegf, bfgf and enos have been documented in iugr pregnancies. differential expression of proand anti-angiogenic factors such as vascular endothelial growth factors (vegf) and placental growth factors (plgf) throughout gestation indicates that both play essential roles in placental angiogenesis.52-54 the imbalance of proand anti-angiogenic mediators during gestation results in impeded fetal growth and other complications. clinical theriogenology • volume 4 number 2 • june 2012137 figure 2: imbalance of proand anti-angiogenic mediators during gestation results in oxidative damage, placental ischemia, impeded fetal growth, and other complications. large offspring syndrome in cattle in vitro production of bovine embryos often affects embryonic gene expression and organogenesis, leading to large offspring syndrome (los). in cattle, the developmental competence of in vitro produced embryos is compromised compared to in vivo embryos. embryos produced in vitro differ from those produced in vivo with regards to morphology, timing of development, embryonic metabolism, intercellular communication, cell number, and gene expression.55-61 a recent study compared three embryo production systems to identify genes affected by in vitro processes and associated with the los; 1) embryos produced in vitro (ivf), 2) embryos fertilized in vivo followed by in vitro culture (in vivo derived, ivd), and 3) embryos generated entirely in vivo by artificial insemination (ai-ivd).47 there were 306, 367, and 200 genes differentially expressed between the ai-ivd, ivf and ivd, and ai and ivf comparisons, respectively. interestingly, 44 differentially expressed genes were identified between the ai embryos and both the in vitro and in vivo embryos, making these genes potential candidates for los. comparing these genes in an iugr model would provide additional insight into fetal programming. in another study, in vitro produced bovine embryos cultured in high concentrations of either serum or bovine serum albumin (bsa) had significantly more cells in day 7 blastocysts, a larger blastocyst on day 12, and differences in relative abundance of transcripts for heat shock protein 70.1, cu/zn-superoxide dismutase (sod), glucose transporters-3 (glut-3), glut-4, bovine-fibroblast growth factor (bfgf), and insulin-like growth factor 1receptor (igf1-r) when compared with in vivo embryos; these differences provided evidence that the culture system had an important role in development.61 furthermore, calves derived from in vitro embryos were significantly heavier than those derived from sheep oviduct culture, superovulation, or ai.62 clinical theriogenology • volume 4 number 2 • june 2012 138 figure 3: factors altering fetal and placental development and fetal programming (adapted from 47-49) cloning by nuclear transfer of somatic cells (scnt) is associated with high rates of embryonic and fetal mortality in various species.63,64 in bovine pregnancies with a fetus produced by scnt, a poorly developed placenta with very few placentomes and limited vascularization has frequently been described during early pregnancy;63,64 that the extra-embryonic tissues fail to develop normally in scnt clones causes abnormalities in implantation and placental development. in placentas of scnt pregnancies examined between 180 and 280 days of gestation, there were indications that placental overgrowth preceded fetal overgrowth,64,65 perhaps due to alterations in placental genes during early development. for the fetus to survive, the placenta must overcome and compensate for developmental defects caused by programming errors; furthermore, in cloned offspring, adaptation to these changes may cause iugr.65 altered placental development due to scnt-induced genomic perturbations would be consistent with such observations. it is apparent that the pre-implantation embryo is extremely sensitive to subtle changes in environment with long-term consequences for the health of the offspring. genes that are implicated in these studies can be studied further using gene silencing or transfection models to determine their pathophysiology in the development of placentation and iugr. uteroplacental hypoxia in sheep intrauterine growth restriction, often associated with functional placental insufficiency, increased perinatal mortality and morbidity. although no animal model can fully simulate human pregnancy, the pregnant sheep has been used extensively to investigate maternal-fetal interactions. embryo stress heat shock culture conditions paternal and/or maternal altered gene expression and imprinting altered gene blastomere function altered placental and fetal development clinical theriogenology • volume 4 number 2 • june 2012139 figure 4: placental oxidative stress and intrauterine growth restriction. placental oxidative stress induced by generation of an abundance of oxidative free radicals cause lipid peroxidation and alters angiogenesis, which reduces nutrient delivery to the fetus, causing fetal growth restriction. development of the placental vascular tree is regulated by several growth factor families. based on differential expression of vascular endothelial growth factors (vegf) and placental growth factors (plgf) throughout gestation, both have essential roles in placental angiogenesis.66-68 the latter is divided into two phases, branching and non-branching.66-68 branching angiogenesis, which occurs during the early phases of gestation under relatively hypoxic conditions, is responsible for expansion of the preexisting vascular bed, and is driven by vegf. non-branching angiogenesis promotes elongation of preexisting capillary loops and occurs later in gestation under the influence of plgf. the balance of these two growth factors throughout gestation is responsible for formation of a normal placenta.67,68 the increasing transplacental oxygen gradient generates changes in angiogenic growth factors which may be associated with the underlying pathophysiology of post-placental hypoxic fetal growth restriction. an altered state of placental oxygenation is associated with aberrant villous morphology.69-71 a fetal growth restricted placenta that develops under maternal hypoxia (e.g. at a high altitude) is characterized by increased branching angiogenesis and reduced vascular impedance.72,73 in contrast, pregnancies with restricted fetal growth are characterized by the absence of end diastolic flow in the umbilical artery and increased vascular impedance, predominantly associated with non-branching angiogenesis. placental oxidative stress induced by ischemic perfusion has been implicated in the pathophysiology of fetal growth restriction. placental tissues from iugr pregnancies have decreased placental anti-oxidant capacity, and lower mrna expression of cu/zn superoxide dismutase (an important enzymatic antioxidant) and non-enzymatic antioxidant vitamin e.74-76 based on several studies, decreased bioavailability of endothelium-derived no, due to oxidative destruction of no by ros, may intermittent perfusion of placental intervillous space fluctuating oxygen concentrations generation of oxygen free radicals (ros/rns) placental oxidative stress direct damaging effect • altered angiogenesis • lipid peroxidation • cytokine synthesis intrauterine growth restriction clinical theriogenology • volume 4 number 2 • june 2012 140 contribute to the impaired endothelium-dependent vasodilatory responses, for which treatment with antioxidant vitamins apparently have a beneficial role.74-76 tocopherols are micronutrients which act as free radical scavengers, i.e. chain-breaking antioxidants. the major tocopherols in mammalian tissue are alpha and gamma. under specific circumstances, gamma tocopherol might scavenge reactive species more effectively than alpha tocopherol.77 in that regard, gamma tocopherol was 35% more effective than alpha tocopherol at inhibiting systemic lipid peroxidation. furthermore, gamma tocopherol is incorporated into the endothelial cells more rapidly than alpha tocopherol, and supplementation with gamma tocopherol was significantly more effective than alpha tocopherol at inhibiting iron-induced lipid peroxidation and occlusive thrombus formation.78 in addition to free radical scavenging properties, since gamma tocopherol has specific activities with key inflammatory enzymes, it may have an added advantage of suppressing inflammation during pregnancy. supplementation of tocopherols may reduce utero-placental hypoxia and promote placental vascular development by reducing hypoxia. the sheep may be an appropriate model to study methods to overcome placental hypoxia, namely, the use of antioxidants to promote vasculogenesis. embryonic/fetal adaptation in mares at the histological level, the exterior surface of the allantochorion in mares consists of highly complex microplacentomes, each packed with fetal and maternal capillaries, which enables materno-fetal exchange of nutritional, gaseous and waste products.79-84 a healthy and functional feto-maternal unit (placenta and uterus) is prerequisite for a successful pregnancy in the mare;84 anything affecting this fetomaternal attachment may result in abortion or weakness in the newborn foal. in that regard, intrauterine development of the fetus and fetal birth weight are closely related to the total area of contact of the allantochorion. in mares, placental compromise is evident in twinning, age-related endometrial degeneration, and maternal size. in twinning, allantochorions of the two conceptuses compete for the limited surface area of endometrium, whereas in endometrial degeneration, the area of placental exchange is reduced. placental compromise related to maternal size has been well characterized by crossing large shire horses with small shetland ponies; the foal from the shetland mare weighed only half that of its half sibling born from the shire mare. importantly, this difference persisted into adult life. tischner created three pairs of sexmatched, full sibling konik pony foals, whereby one of each pair had been transferred as a blastocyst to the uterus of a larger draft-type recipient mare, whereas the sibling was carried to term in its genetic pony mother.85 all three transferred foals were bigger at birth and grew faster while nursing their larger surrogate mothers.86 in other studies, embryos were transferred between thoroughbred (t) mares and pony (p) mares to establish four types of pregnancies: t-in-p, p-in-t, and, as controls, t-in-t and p-in-p. the t-in-t foals were heaviest and the p-in-p foals were lightest at birth.87 the p-in-t foals were heavier than their t-in-p counterparts. further, differences in birth weight were paralleled by differences in placental mean weight, volume, total surface area and density of microcotyledons on the allantochorion and the total microscopic area of fetomaternal contact at the placental interface. thus, intrauterine development of the fetus and the resulting size of the foal are closely related to the total area of contact of the allantochorion. the principal vasculogenic and angiogenic factor vegf, and its two major receptor molecules, flt and kdr, are localized throughout most of gestation on the two principal secretory cell types of the equine placenta, the glandular and lumenal epithelia of the maternal endometrium and the trophoblast of the fetal allantochorion.88 perhaps, vegf, flt and kdr interact in the mare to facilitate development of maternal and fetal vascular networks for exchange of gases, nutrients and waste products throughout gestation. in this regard, mares could be used as model to study fetal adaptation during development. the use of t-p models will address both the restricted (t-p) and over-fed (p-t) scenarios. questions addressed by these studies include: 1. what are differences in the placental vasculogenesis gene expression in restricted and overweight maternal conditions? clinical theriogenology • volume 4 number 2 • june 2012141 2. what are differences in gene expression when there is fetal growth restriction in contrast to fetal adaptation in those conditions? low birth weight in piglets in pigs, birth weight is a major determinant of postnatal survival. since attempts to alleviate the detrimental effects of low birth weight after birth have achieved only limited success, it appears that the in utero environment during a critical stage of gestation can program fetal gene expression and metabolism, leading to low birth weight. the capacity of the placenta to transport nutrients has a direct effect on fetal growth. the placenta supplying the smallest fetus in the uterus is disproportionally lighter than those supplying average-sized fetuses in the same litter.89 furthermore, there is a positive relationship between placental blood flow and fetal weight.90 therefore, there is a generalized reduction in the ability of placentas supplying small fetuses to deliver nutrients. supplementation of l-arginine has been studied extensively studied using pig models. arginine, a conditionally essential amino acid,91 is the nitrogenous precursor of nitric oxide (no), a vasodilator and a cell signaling molecule.92,93 it has been established that no and other products of arginine catabolism, e.g. proline and polyamines, are crucial for cell growth, migration, and proliferation, as well as angiogenesis.94 gilts were supplemented with 0.0, 0.4, or 0.8% l-arginine between 0 and 25 d of gestation.95 dietary supplementation with 0.4 or 0.8% l-arginine enhanced its concentrations in maternal plasma (64 and 98%, respectively) as well as the vascularity of chorionic and allantoic membranes, compared to the control group.95 reproductive performance, including numbers of corpora lutea and fetuses, as well as placental and fetal weights, and embryonic mortality, did not differ between the 0.4% l-arginine and control groups. however, supplementation with 0.8% l-arginine between 0 and 25 d of gestation, increased placental vascularity, but adversely affected reproductive performance of gilts.92 interestingly, giving rats 1.2% l-arginine for 7 d (immediately after breeding) reduced embryonic mortality by 30% .96 similarly, supplementing 0.83% l-arginine to gilts between 30 and 114 d of gestation increased the number of live-born piglets by two per litter.97 the pig may be an appropriate model for experiments dealing with both birth weight and neonatal nutrition. there can be up to a 3-fold difference in body weight among littermates in normally fed sows, thus providing a natural form of intrauterine growth restriction.98,99 in addition, this model allows artificial rearing, which facilitates modulation and control of feed intake during the neonatal period. moreover, comparisons of selection methods based on uterine capacity over treatment modalities could also be evaluated. fetal programming epigenetic refers to the study of changes in gene function that may be mitotically and/or meiotically inherited, but are not related to changes in dna sequence.100 several mechanisms of epigenetics have been reported, including dna methylation, modification of histones and modification of gene expression by non-coding small interfering or long rnas.101 interestingly, mirnas down-regulate or weaken several target genes; and the key mechanisms underlying this process are dna methylation and histone acetylation.100 these molecular mechanisms effectively determine if gene transcription or gene silencing occurs. in brief, histone acetylation and deacetylation are associated with gene transcription and silencing respectively. the histone tails can also be methylated and can activate or silence gene expression. other examples of epigenetic modifications include phosphorylation, ubiquitinization and sumoylation.102 epigenetic mechanisms are important in imprinting where one gene allele derived from one parent is silenced, whereas the allele of the same gene but inherited from the other parent is expressed. embryonic development is characterised by two periods of epigenetic programming, which occur during gametogenesis and the pre-implantation period of pregnancy, respectively. the pre-implantation embryo is extremely sensitive to subtle changes in the environment, with long-term consequences for the health of the offspring. although underlying mechanisms of epigenetic perturbations are unknown, there are indications of altered epigenetic programming of embryo and endometrium, particularly at the clinical theriogenology • volume 4 number 2 • june 2012 142 maternal-fetal interface. suboptimal physiology of the endometrium can subtly or substantially affect embryo development before implantation, with visible and sometimes severe consequences on placentation, fetal development and pregnancy outcome. thus, whereas pregnancy outcome relies on embryo quality, it appears that the developmental course of the embryo is also epigenetically driven by endometrial mechanisms from the very earliest stages of pregnancy.103 in the context of assisted reproductive technologies, developing diagnostic and prognostic tools based on endometrial factors will be valuable, with objectives to estimate the reproductive capacity of the mother, to correct suboptimal endometrial functionality and to assess the developmental potential of the embryo. on a more fundamental basis, an essential issue will be to carefully analyse situations in which embryos can overcome the control exerted by the endometrium.104 in ruminants, a major difficulty in elucidating pregnancy-related gene functions at the materno-fetal interface is the number of genes to be analyzed. to bypass this problem, alternative options have been published. in that regard, lentiviral infection of blastocyst-stage embryos is an effective approach to study trophoblast gene function.105-107 as mentioned above, fetal programming may be achieved through several mechanisms. at a cellular level, programming affects aspects of cell cycle dynamics and apoptosis, whereas at the subcellular level, telomerase shortening and altered function of various enzyme systems have been documented. these are important mechanisms during development and differentiation, allowing various tissues and organs to develop from the same original genotype. the consequence of methylation in nutritional programming has been ably demonstrated in animal models. a sheep model with methionine deficiency (induced by dietary deficiency of vitamin b12 and folate; figure 5) around the time of conception, demonstrated that male offspring were adipose and insulin resistant, and that these changes were associated with methylation of several genes.108,109 these experiments highlighted an important role in nutritional programming. perhaps reductions in dietary methionine and folate during the periconceptional period will cause widespread epigenetic alterations to dna methylation in offspring, and modify adult health-related phenotypes.110 furthermore, genes which increase susceptibility to diabetes also reduce birth weight by influencing insulin-mediated growth, i.e. low birth weight and type 2 diabetes are both phenotypes of the same genotype.109,111 it was observed that the rare mutation in glucokinase gene (gck) reduces insulin secretion in β-cells. if the gck mutation was inherited paternally, birth weight was reduced by 500 gm, provided the mother was normal. however, if the baby inherited the gck mutation maternally, birthweight was normal (since maternal diabetes negated the effect of mutation) or it was 500 g above average if the mutation was not inherited.112 maternal undernutrition and lower socio-economic status remain the most important causes of low birth weight worldwide. numerous animal models of under-nutrition and micronutrient supplementation have confirmed the role of nutritional fetal programming. imprinted genes have diverse functions, notably including regulation of fetal growth.113,114 much attention has been directed to the igf signaling pathway, which has a major influence on fetal size. this pathway contains two components encoded by oppositely imprinted genes, namely, igf2 (a growth promoting factor from the paternal allele) and igf2r (a growth inhibitory factor from the maternal allele). these genes fit the parent-offspring conflict hypothesis for the evolution of genomic imprinting.115,116 it appears that not all components of growth regulatory pathways are encoded by imprinted genes; perhaps within a pathway, the influence of a single gene by each of the parental genomes may be sufficient for the parent-offspring conflict to be enacted. several imprinted genes are known to influence energy homeostasis. in that regard, some, including igf2 and growth factor receptorbound protein 10 (grb10), may coordinate growth via glucose-regulated metabolism.117-119 since perturbation of fetal growth can be associated with metabolic disorders in adulthood, these imprinted genes are considered candidates for involvement in this phenomenon of fetal programming. clinical theriogenology • volume 4 number 2 • june 2012143 figure 5: nutrition and dna methylation. factors during pregnancy influencing development of complex traits in adult life should be expanded to include epigenetic factors, e.g. dna methylation, which occurs in utero. epigenetic factors regulate gene expression and thereby cell differentiation and organogenesis. the process of epigenotype establishment is sensitive to environmental conditions, with nutrition being one of the most important factors. for example, dna methylation depends on availability of several nutrients, including methionine and vitamins b6, b12, and folate. conclusion in summary, poor fetal growth can result from a myriad of fetal, placental, and maternal conditions. based on numerous studies in various populations, there is a clear association between intrauterine growth restriction and the occurrence of obesity, type 2 diabetes mellitus, cardiovascular, neurological disorders, and reproductive disorders later in life. animal studies have confirmed that these associations are 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piglets. cloning stem cells 2007;9:97-106. 117. jaenisch r: dna methylation and imprinting: why bother? trends genet 1997;13:323-329. 118. holliday r, grigg gw: dna methylation and mutation. mutat res 1993;285:61-67. 119. zeisel sh: epigenetic mechanisms for nutrition determinants of later health outcomes. am j clin nutr 2009;89:1488s1493s. clinical theriogenology • volume 4 number 2 • june 2012 148 clinical theriogenology 2022; 14: 259 rho kinases inhibitor effects on postthaw survival of slow-cooled bovine blastocysts abdallah abdelhady, luis henrique de aguiar, rhasaan bovell, yoke lee lee, ziqi guo, mariana diel de amorim, soon hon cheong department of clinical sciences, college of veterinary medicine cornell university, ithaca, ny slow-cooling cryopreservation protocols for direct transfer of bovine blastocysts have allowed convenient thawing and transfer into recipients without additional embryo manipulations. rho-associated coiled-coil-containing protein serine/threonine kinases (rock) inhibition has improved postthaw viability of vitrified bovine embryos produced in vitro; however, it has not been evaluated in slow-cooled bovine embryos. aim was to determine, if treatment with 10 μm y-27632, a rock inhibitor, for 2 hours prior to freezing and/or in the postthaw culture media affects embryo viability. we hypothesized that rock inhibition improves postthaw viability (reexpansion, hatching, and lack of degeneration) in slow-cooled cryopreserved embryos. in vitro produced bovine embryos (n = 448) in the early or expanded blastocyst stage were randomly assigned to 1 of 5 groups. the nonfreeze group (n = 104) was loaded into straws containing freezing media but was not cryopreserved. the control-control group (n = 97) were cryopreserved in cryologic slow-rate freezer and represents the standard cryopreserved embryo. remaining 3 groups were cryopreserved in the same way with rocki exposure prefreezing only (rocki-control) (n = 84), rocki postthaw only (control-rocki) (n = 79), or rocki both prefreezing and postthaw (rocki-rocki) (n = 84). embryos were observed postthaw for 48 hours using time-lapse videography. outcomes measured included: proportions of embryos that reexpanded, hatched, or degenerated. data were analyzed using logistic regression anova, and interval to reexpansion and hatching data were analyzed using cox’s proportional hazard model. all statistical analysis was performed in jmp pro v16. group and replicate were included in all models and significance was set at p < 0.05. reexpansion rates (%) were: 99.0a, 75.3b, 84.5ab, 92.4a, and 94.0a; hatching rates (%) were: 76.9a, 59.8b, 59.5b, 73.4ab, and 80.1a; and degeneration rates (%) were: 8.7a, 26.8b, 17.9b, 8.9a, and 7.1a for nonfreeze, control-control, rocki-control, control-rocki, and rocki-rocki groups, respectively, with different superscripts representing the respective significant differences. risk ratios (representing how quickly embryos reached the outcomes) for reexpansion was highest for nonfreezea, followed by rocki-rockib, control-rockic, rocki-controlcd, then control-controld. risk ratios for hatching was highest for rocki-rockia, followed by nonfreezeab, control-rockibc, rocki-controlcd, then control-controld. exposure abstracts opening session of embryos to rock inhibitor prefreeze and postfreeze resulted in the best postthaw outcome with reexpansion, hatching and degeneration rates similar to uncryopreserved embryos and significantly better than the control-control group without supplementation. exposure to rock inhibitor postthaw appeared to have a greater benefit compared to exposure only prefreezing. these results suggested that rock inhibitors may be beneficial for postthaw survival; however, embryo transfer trials are required. keywords: cattle, embryo, slow-rate freezing, rho kinase inhibition, cryodamage uterine inflammatory response after misoprostol during estrus and diestrus in commercial embryo donor mares gabrielle amorim,a odilon oliveira,a lorenzo segabinazzi,a,b igor canissoa,c adepartment of animal reproduction, são paulo state university (unesp), botucatu, brazil bdepartment of veterinary medicine, university of illinois urbana champaign, il cross university school of veterinary medicine, basseterre, st. kitts, west indies uterine tube obstruction is the leading cause of unexplained subfertility in mares. over the years, numerous techniques were developed to unblock uterine tubes with variable success. in recent years, misoprostol, a synthetic analog of prostaglandin e1 (deep uterine horn treatment with a flexible pipette) has regained popularity in equine practice. however, there have been some concerns regarding rare anaphylactic reactions in some mares and an exacerbated postinfusion uterine inflammatory response to misoprostol; however, this has yet to be critically studied. in addition, debate exists whether mares should receive misoprostol in diestrus or estrus, with the latter offering the advantage of potentially unblocking the mare close to breeding. this study aimed to determine the uterine inflammatory response in mares after infusion with misoprostol during estrus or diestrus. we hypothesized that misoprostol infusion induces a greater inflammatory response than sham infusion; similarly, estrus results in a greater inflammatory response than diestrus. forty-four estrous cycles of light breed mares (n = 11) were sequentially randomly assigned to receive misoprostol or sham infusion during estrus and diestrus. the misoprostol and sham infusions were performed by directing a flexible pipetted (minitube, germany) deep into 1 uterine horn, and then the same pipette was redirected to the opposite side uterine horn. each uterine horn received 200 µg of misoprostol diluted in 3 clinical theriogenology 2022; 14: 260 ml of lactate ringer’s solution (lrs), whereas the sham inoculation consisted of 3 ml of lrs. the cycles assigned for estrus treatment received it when preovulatory follicles ranged from 28 32 mm, and diestrus cycles were treated 8 days postovulation. the day after each infusion, mares had uterine lavage performed with 2 liters of lrs. transrectal ultrasonography of the reproductive tract was performed to quantify uterine edema and intrauterine fluid accumulation immediately before (0), 24, 48, and 72 hours after treatments. at similar time points, uterine cytology samples were harvested to count the number of neutrophils in a high-power field (40 x). once a ≥ 35 mm follicle and edema were first detected, ovulation was hastened with deslorelin (1 mg intramuscular) during estrus or the subsequent estrus after diestrus infusion. mares received 5 mg of intramuscular dinoprost 24 hours after infusion in estrus and diestrus. statistical analyses were performed with graph prisma. neutrophil counts were analyzed with anova repeated measures and tukey’s as post-hoc. edema scores and intrauterine fluid accumulation were analyzed with kruskal-wallis and dunn’s post-hoc. embryo recovery rates were evaluated with fisher’s test. there were effects of time (p = 0.04) but no effects of group (p = 0.96), or stage of the estrous cycle (p = 0.26), or interaction between group and time for edema scores (p = 0.97). similarly, there were effects of time (p = 0.03) but no effects of group (p = 0.32), stage of the estrous cycle (p = 0.86), or interactions between group and time or stage of the estrous cycle for neutrophil counts (p = 0.87). uterine fluid scores did not change over time (p = 0.39), groups (p = 0.22), or stage of the estrous cycle (p = 0.77). embryo recovery was similar for estrus (misoprostol 46 versus sham 46%) versus diestrus (misoprostol 67 versus sham 46%) (p > 0.05). in conclusion, 200 µg of bilateral misoprostol treatment did not exacerbate uterine inflammation in mares. keywords: mares, uterine tube, infertility, prostaglandin e, endometritis effect of sexual rest on cooling and freezing ability of stallions giorgia podico, kianna spencer, humberto magalhaes, igor canisso department of veterinary clinical medicine, university of illinois, urbana, il prolonged sexual rest, particularly during winter, is apparently detrimental to semen quality, yet it is common to collect and ship or freeze semen from sexually rested stallions. when a sexually rested stallion is presented in a hasty situation, practitioners harvest and evaluate the first ejaculate, and based on quality, the semen is extended (1:1) and kept at room temperature while a second collection is attempted in approximately 1 hour, if the stallion’s libido and physical capabilities allow. if the second ejaculate has better quality, practitioners use it for further processing. if both ejaculates have identical marginal quality, either or both ejaculates are processed further. although this approach is routinely applied, there is little evidence to support it. thus, this study aimed to assess the cooling and freezing abilities of sexually rested stallions collected 1 hour apart early in the breeding season. we hypothesized that the second ejaculate of the season has superior cooling and freezing ability compared to the first ejaculate. stallions (n =10) were collected twice, 1 hour apart, on a dummy mount with a teaser mare present. gel-free volume, concentration, total sperm ejaculated, and percentage of normal sperm were determined after collection. gel-free semen was extended at 50 x 106/ml in inra96; part of it was immediately stored, and the remainder was cushion-centrifuged, with the pellet resuspended in inra96 at 50 x 106/ml. extended and resuspended semen were stored in botuflex containers for further evaluations at 24 and 48 hours. after cushion-centrifugation, half of the pellets were resuspended in botucrio at 200 x 106/ml, loaded in 0.5 ml straws, and frozen over liquid nitrogen. total motility (tm) and progressive motility (pm) were evaluated with casa. sperm membrane integrity (smi) and mitochondrial membrane potential (mmp) were assessed with zombie green and mitotracker deep-red via spectral flow cytometry. sperm parameters were assessed at 0, 24, 48 hours of cooling and before and after thawing. a thermal longevity test was conducted on postthaw semen for 4 hours, with motility assessment every 30 minutes. data were analyzed with paired student’s t-test, linear mixed model, and tukey’s post-hoc. significance was set at p < 0.05. gel-free volume was not different (p > 0.05) between first 77.7 ± 10.8 ml and second 64.0 ± 6.7 ml ejaculates. sperm concentration (182.4 ± 35.1 x 106/ml versus 93.1 ± 1 x 106/ml), and total sperm ejaculated (11.4 x 109 versus 6.0 x 109) decreased by half between first and second ejaculates, respectively (p < 0.05). morphologically normal sperm did not vary (p > 0.05) between ejaculates (68.3 ± 6.9 versus 74.0 ± 6.6%). there were no effects (p > 0.05) of time, of ejaculates, or their interaction on tm, pm, and smi. processing semen through cushion-centrifugation was beneficial and increased (p = 0.02) tm, pm, smi compared to noncentrifuged semen. mmp was not affected (p > 0.05) by any factors or interactions. tm, pm, and smi were reduced (p < 0.05) postthaw and during the thermal longevity test; however, there were no differences (p > 0.05) between ejaculates or interactions. in conclusion, semen had adequate cooling and freezing ability despite sexual rest. cushion-centrifugation was a suitable technique to process semen from sexually rested stallions. keywords: stallion, sexual behavior, semen quality, semen cryopreservation a novel sperm filtration system based on microfluidics jesper nielsen,a mette christoffersen,b jacob møllenbachc avetgruppen ansager hestehospital, ansager, denmark breproduction and obstetrics, department of veterinary clinical sciences university of copenhagen, denmark cmotility count aps, valby, denmark objective was to compare equine semen parameters before and after filtration in a novel device based on microfluidics (vetcount™ harvester). hypothesis was that microfluidic-based filtration with the device improves semen parameters related to sperm quality. pilot prospective analyses were performed in stallion semen samples. semen samples evaluated originated from commercially frozen batches from standardbred, arabian, and warmblood stallions. the tested vetcount™ harvester clinical theriogenology 2022; 14: 261 device contains a filter with 10 µm micropores, and the filtration of sperm across the filter is based on fluid dynamics and active movement. the device allows 1.0 ml of frozen thawed semen sample to be filtered into 0.8 ml of skim milk-based extender. samples (n = 55) were all filtered using the device. concentration, percentage of progressive motile sperm (pms), viability using propidium iodide, and percentage of morphological normal sperm were evaluated before and after filtration of the samples. acrosome damage and dna fragmentation analyses were performed on a smaller sample size (n = 20). sperm concentration and viability were evaluated using nucleocounter® sp-100. percentage of pms and morphologically normal sperm, acrosome damage and dna fragmentation were evaluated using casa androvision®. wilcoxon signedrank tests were performed to assess statistical differences for sperm outcomes before and after filtration. data were presented as median (25 and 75% percentiles). significance was set at p < 0.05. median sperm concentration was 259 x 106/ml (176 -368) before filtration and 18.4 x 106/ml (13 29.1) after filtration. following parameters improved significantly after filtration through the vetcount™ harvester device: pms from 30 (12.9 36.6) to 76.0% (69.7 83.1) (p < 0.0001); viability (from 38.1 (30.0 46.0) to 59.0% (47.0 71.0) (p < 0.0001); morphologically normal sperm from 52.0 (45.0 -59.0) to 81% (76.0 86.0) (p < 0.0001); acrosome damaged sperm from 16.6 (11.3 20.2) to 2.5% (1.7 4.2) (p = 0.0005); and sperm with dna fragmentation from 6.38 (5.61 10.6) to 0.9% (0.6 1.42) (p < 0.0001). recovered volume from the device was 0.7 ml. extracted semen samples contained 14.6 % (8.7 22) of pms in the native sperm samples. total number of pms after filtration was 9.9 x 106 (5.8 15.9 x 106). percentage of pmss, sperm viability, sperm morphology, sperm with acrosome damage and sperm with dna fragmentation were substantially improved in a frozen-thawed semen sample using the filtration technique in the microfluidic-based device. in practice, the device does not allow filtration of dead and damaged sperm through the micropores in the filter. this provides an improved semen sample to inseminate the mare without the negative impact of dead and damaged sperm to the uterine environment. vetcount™ harvester device is presented as a new technique that in the future may improve the fertility of frozen equine semen samples for artificial and can improve the quality of a semen sample for insemination or other assisted reproductive techniques (e.g., intracytoplasmic sperm injection). keywords: stallion, semen quality, sperm, microfluidic-based filtration a novel method for oocyte recovery from equine postmortem ovaries jessica lederman,a christian bisiau,a christina divine,a jennifer hatzela adepartment of clinical sciences, college of veterinary medicine and biomedical sciences fort collins, co equine postmortem gamete recovery frequently occurs in the equine industry and is considered an emergency procedure. obtaining postmortem oocytes provides owners with a last chance to further the genetic line of their mare and obtain valuable offspring. currently, the industry standard for oocyte recovery uses varying sizes of bone curettes to scrape the interior follicular walls to manually detach the tightly attached cumulus oocyte complex (coc). this study aimed to evaluate a new method for oocyte recovery from equine postmortem ovaries using tenex technology, a minimally invasive procedure used to perform a percutaneous tenotomy of fasciotomy. we hypothesized that the use of tenex technologies to ultrasonically detach the coc from the follicular wall while maintaining the structure and integrity of the oocyte is an effective method for postmortem equine oocyte recovery. ovaries were obtained through scheduled euthanasia and ovariectomy procedures in mares. ovaries were extracted immediately after euthanasia or obtained shortly after ovariectomy procedures and processed within 24 hours after collection. ovaries were stored in a passive cooling device in an effort to maintain room temperature (~ 22°c) until processing occurred. a single ovary from each mare’s pairs were randomly assigned to either a control or treatment group. both ovaries were trimmed of excessive fascia prior to processing and commercial embryo flush media was utilized. ovarian follicles on control group ovaries were processed traditionally, through manual scraping with bone curettes. curettes were rinsed thoroughly into a 50 ml conical tube filled with embryo flush media. treatment group ovaries were processed with tenex technology to aspirate individual ovarian follicles using a double lumen needle with ultrasonic energy and irrigation capabilities. with built-in suction, the machine allowed for contents to be collected into a 250 ml sterile plastic bottle. the commercial flush media collected from both control and treatment groups were placed into separate petri dishes to facilitate coc identification under dissecting microscopes. data were analyzed using a one-way anova test with significance set at p ≤ .05. the average period spent processing control and treatment ovaries was 37.50 minutes and 34.11 minutes, respectively. treatment ovaries processing were 3.39 minutes shorter than control ovaries, although not significant (p = 0.342). an average of 3.25 cocs were recovered from control ovaries (45.70%) and an average of 2.17 cocs were recovered from treatment ovaries (33.10%) that was significant (p = 0.006). oocyte structure and integrity were not affected by this method of oocyte recovery. this study evaluated an alternative method for equine oocyte collection from postmortem ovaries. although  the oocyte recovery rate did not currently support transitioning to this method in particular, novel methods should continue to be explored in an attempt to improve successful outcomes. keywords: mae, postmortem, ovary, oocyte, gamete gene expression in day 13 postovulation pregnant and nonpregnant mare endometrium michael byron, bradley back, soon hon cheong, mariana diel de amorim department of clinical sciences, college of veterinary medicine cornell university, ithaca, ny equine embryo has maximal mobility between days 11 14 postovulation to deliver signals to the endometrium for pregnancy maintenance. embryonic factor(s) signal to the uterus to turn ‘on‘ or ‘off‘ genes to stop luteolysis in an intricate feto-maternal interaction. insulin-like growth factor binding protein 1 (igclinical theriogenology 2022; 14: 262 fbp1) is a uterine receptivity marker in the cow and sheep, and its expression is increased by prostaglandin e2 and prostacyclin (pgi2). prostaglandin i2 synthase (ptgis) and prostaglandin i2 receptor (ptgir) have been characterized in ruminants and pigs; however, such investigation of prostaglandin-related genes is lacking in the horse during early pregnancy. we hypothesized that the relative expression of prostaglandin-related genes differs in the endometrium of pregnant mares compared to nonpregnant mares at day 13 postovulation. objective was to better understand the role of prostaglandins in the endometrium during early pregnancy. light breed mares (n = 13) were examined daily via transrectal palpation and ultrasonography from estrus to the day of ovulation (d0), and randomly assigned to nonpregnant (np; n = 7) group or pregnant (p; n = 6) group. mares assigned to p were bred using artificial insemination with > 500 x 106 normal and progressively motile sperm from a single fertile stallion. on day 13 postovulation, a uterine lavage was performed to retrieve the conceptus, and an endometrial biopsy was collected, snap frozen in liquid nitrogen, and stored at – 80°c until analysis. total rna was extracted from endometrial biopsies and evaluated for expression of following genes: igfbp1, igfbp2, nr3c1, creb, crtc2, ppar-g, ptges, ptgs2, ptger2, ptger4, ptgfs, ptgfr, mpges2, ptgis, and ptgir using real-time pcr. mean threshold cycle (cq) was determined and then normalized to the reference gene (gapdh and actb) (δct). statistical analyses were performed with graphpad prism 9 using a student’s t-test or mann-whitney test to compare differences between np and p endometrium at p < 0.05. pregnant mare endometrium had higher (p = 0.005) expression of igfbp1 compared to np and ptgis expression was slightly higher (p = 0.09) in p mares. np endometrium had higher (p = 0.03) expression of ptgir compared to p endometrium the remainder of the genes had no significant differences. igfbp1 increases during pregnancy in the horse. this protein can increase the availability of growth factors within the endometrium and the uterine lumen, helping to support endometrial and conceptus growth in preparation for fixation of the conceptus. contrary to our results, in pregnant pig endometrium, ptgir expression increases compared to cycling animals. pgi2 has a role in camp signaling and angiogenesis in pig uterus, with an increase during early pregnancy, suggesting a possible regulation by embryonic factors. further investigation on protein concentrations and functional roles of igfbp1 and ptgir in the equine endometrium and conceptus at various points in early pregnancy is warranted that may help to clarify the differing results observed in horse and pig. keywords: pregnant mare, insulin-like growth factor binding protein, prostaglandins genital mycoplasma prevalence in the healthy and subfertile breeding dog robyn ellerbrock, patricia xavier department of large animal medicine, college of veterinary medicine university of georgia, athens, ga canine reproductive failure can be the result of poor breeding timing, genetic incompatibility, inflammation, or infectious disease. whereas bacteria (e.g., brucella canis) cause reproductive failure, mollicutes (e.g., mycoplasma) have a more controversial role in canine infertility. at least 17 mycoplasma spp. have been isolated from dogs, with m. canis and m. cynos as the most common isolates. m. canis is considered an opportunistic pathogen whereas m. cynos is a proven respiratory pathogen. we hypothesized that a similar pattern would be noted in breeding dogs, with the presence of m. cynos in semen or vaginal samples decreasing fertility, whereas the presence of m. canis not impacting pregnancy rates. objectives were to determine mycoplasma prevalence in the population of dogs presenting for breeding management to the university of georgia veterinary teaching hospital and to determine pregnancy rates in mycoplasma-positive dogs. dogs (n = 13) presented with a history of infertility and dogs (n = 65) presented for routine breeding management were enrolled. for each dog, 100 µl of semen (n = 31), or a vaginal cytology swab (n = 47) were collected during routine breeding management. nucleic acids were extracted using a qiaamp cador pathogen kit (qiagen, hilden, germany) and a qiacube automated nucleic acid extraction system (qiagen) and all samples were analyzed for m. cynos and m. canis using a previously validated multiplex real-time pcr assay. pregnancy rates between positive and negative dogs were compared by student’s t-test. three of 13 dogs presented with a history of infertility were positive for m. cynos, and 3/13 were positive for m. canis. of 65 dogs presented for routine breeding management, m. canis was detected in 11.9% (5/42) of bitches and 12% (3/25) of stud dogs. m. cynos was detected in bitches and 45.8% (11/25) of stud dogs presenting for routine breeding management. when bitches were bred with semen samples with a known mycoplasma status, 50% (5/10) bred with semen positive for m. cynos conceived, 0/2 positive for m. canis conceived, and 89% (8/9) conceived with known negative samples. of mycoplasmanegative bitches that were subsequently bred, 68% (17/25) conceived, whereas 0/6 m. canis-positive bitches conceived, and 0/1 m. cynos positive bitches conceived. collectively, this suggests that both m. cynos and m. canis may contribute to bitch infertility. contrary to our hypothesis, m. cynos was present in almost half of canine ejaculates, and the presence of m. cynos in semen samples decreased conception rates (p = 0.035), but did not prevent pregnancy in the bitch. whereas no bitches positive for mycoplasma spp. conceived, and no bitches bred with m. canis positive semen conceived, there was a low prevalence of mycoplasma-positive bitches in the breeding population, and follow up studies are recommended. in conclusion, both m. canis and m. cynos were detected in genital samples of both the healthy and infertile bitch and stud. whereas conception rates were lower when mycoplasma was detected in either the bitch or the stud, presence of m. cynos in the semen still resulted in 50% conception in the bitch. keywords: dogs, fertility, pregnancy next-generation sequencing correlation with cultured samples soon hon cheong,a rebecca franklin-guild,b yamilka lago-alvarez,a luis henrique de aguiar,a mariana diel de amorim,a craig altierb adepartment of clinical science bdepartment of population medicine and diagnostic sciences college of veterinary medicine, cornell university, ithaca, ny clinical theriogenology 2022; 14: 263 culture is the gold standard for pathogen detection and identification; however, many organisms fail to be cultured. fungal culture may be time-consuming, and overgrowth of bacteria may prevent diagnosis of fungal agents. next-generation sequencing (ngs) is a powerful method for detecting and identifying the presence of microbial dna in samples. studies comparing these 2 methodologies in equine uterine samples are lacking. objective was to determine the correlation between fungal and bacterial culture results with ngs of the same swab used for the cultures. purpose was to test the hypothesis that ngs and cultures are strongly correlated and ngs may provide a broader picture of the uterine microbiome. uterine swabs (n = 63) from mares submitted for fungal culture with or without bacterial culture to the cornell animal health diagnostic center were used. the same swab used for culture was frozen at – 20°c and submitted in a single batch for ngs of the internal transcribed spacer region for fungal and 16s ribosomal rna gene for bacterial organisms through a commercial molecular diagnostic laboratory (microgenvet). for ngs, fungal and bacterial species were reported if the organism comprised at least 2% of the identified organisms. kappa coefficient was calculated using sas v9.4 and interpreted as excellent (> 0.75), fair to good (0.40 0.75) or poor (< 0.40). fungal cultures (n = 63) were assigned to 3 groups: positive, negative, and contaminated (bacterial overgrowth prevented diagnosis of fungal agents). none of the 3 contaminated samples had fungal dna by ngs. of the 16 positive fungal cultures, 5 samples did not identify fungal agents on ngs, 2 samples identified other species of fungal agents, and for 8 (50%) samples, ngs identified the same organism as culture. of the 44 negative samples, 5 samples identified fungal organisms by ngs that may be due to potential higher sensitivity of detection, whereas 39 negative samples (88.6%) had agreeing ngs results. fungal culture and ngs had 78.3% agreement. the kappa coefficient for fungal culture and ngs was 0.41. bacterial culture results (n = 54) were assigned to 3 groups: positive, no growth, and no significant organisms (growth of bacteria classified as nonpathogenic). of the 29 positive bacteria culture samples, ngs identified the same organism in 22 (75.9%) samples and ngs identified different organisms in the remaining 7 samples. ngs detected bacterial presence in 5/14 of no growth samples. of the 11 samples with no significant organisms, 2 samples had bacterial dna from pathogens. the agreement between bacterial culture and ngs was 77.8% with a kappa coefficient of 0.56. in conclusion, culture and ngs had fair to good correlation, and ngs has potential as a valuable diagnostic option for equine endometritis. keywords: mare, endometrium, culture, next-generation sequencing, microbiome antimicrobial activity of nonbiological alternative therapies against important microorganisms causing endometritis in mares mariana mazzuchini,a,b lorenzo segabinazzi,a,c marco alvarenga,a igor canissoa,b adepartment of animal reproduction, são paulo state university (unesp), botucatu, brazil bdepartment of veterinary clinical medicine, university of illinois urbana champaign, il cross university, basseterre, st. kitts, west indies endometritis is the leading cause for antibiotic usage in mares, and their indiscriminate use selects microorganisms resistant to it; thus, the development of alternative therapies is needed. this study aimed to assess the in vitro antimicrobial activity of nontraditional therapies (ntt) against microorganisms isolated from mares with endometritis. we hypothesized that ntt antimicrobial activity varies with the type of agent and microorganism. the ntt evaluated included a commercial anti-septic product (botukiller,® bk, botupharma, brazil); lactated ringer’s solution (lrs as controls), lrs with 10% h202, ozonated lrs (o3, 60 µg/ml of ozone gas for 10 minutes), coca-cola® (cc), and coca-cola-zero-sugar® (cz). the percentage of inhibition (pi) and minimum inhibitory concentrations (mic) of each ntt was performed with the microdilution broth method using 96-well flat-bottom microplates. clinical isolates of streptococcus equi (strep), escherichia coli (e. coli), pseudomonas aeruginosa (pseudo), klebsiella pneumoniae (kleb), staphylococcus aureus (staph), and candida albicans (candida) cultured from mares with clinical endometritis were used in the study. each ntt sample was run 3 times, and within each run, 3 wells were used per microorganism. the first 3 lanes (1 3) of each plate served as the negative controls that consisted of 100 µl ntt and 100 µl of mueller-hinton (mh) (1:1). thereafter, the remaining lanes (4 12) contained serial dilutions of ntt as 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128. row h (lanes 4 12) served as positive controls and contained no ntt. after that, 5 μl (5 x 104 colony-forming unit) of each isolate was added to lanes 4 12. then plates were incubated at 37°c for 24 hours. the optical density of the wells was measured at 570 nm using a spectramax m2 spectrophotometer (molecular devices, san jose, ca). the pi was calculated by the difference between the mean of the triplicates in each group and the mean of the negative control and the positive control. to determine the mic, 10 μl of resazurin solution (0.01%) was added to each well, and the plates were kept at 37°c for 1 hour subsequently, a visual reading was performed to determine microorganism activity; if the well became pink, microbial activity was present, whereas no microbial activity was present if the well remained blue. data were evaluated by anova and tukey’s post hoc test. significance was set at p < 0.05. there was a reduction (p < 0.05) in pi when compared to the positive controls for the different ntt. specifically, h2o2 pi’s were 1/8 e. coli and pseudo, 1/16 staph, 1/32 candida, and 1/64 kleb. the pi results for bk were 1/2 strep, 1/4 staph, 1/8 e.coli, 1/16 pseudo, 1/32 kleb, and 1/64 candida. the o3 results were 1/2 staph, e.coli, and kleb. the pi for cz was 1/4 e.coli. the ntt having mic values were bk 1/2 pseudo; 1/4 staph; kleb; candida; 1/8 e.coli; and h2o2 1/8 e.coli; pseudo, 1/32 kleb and candida and 1/64 staph. cc had no antimicrobial activity. in conclusion, of all ntt tested in the present study, bk and h2o2 had the highest in vitro antimicrobial activity against microorganisms causing endometritis in mares. furthermore, the dose-dependency suggested a different efficacy against the 6 infectious agents tested here. keywords: mare, endometritis, alternative antimicrobials, bacteria, fungus clinical theriogenology 2022; 14: 264 competition session retrograde flushing followed by slicing-floating optimized epididymal sperm recovery in donkeys and horses giorgia podico, igor canissoa department of veterinary clinical medicine, university of illinois, urbana il harvesting epididymal sperm often represents the last opportunity of preserving the genetic material of a valuable sire. it can be accomplished via retrieval by retrograde flushing or by slicing the tail of epididymis and floating sperm into an extender. our clinical experience suggests that starting epididymal flushing with retrograde flushing followed by slicing-floating optimizes sperm recovery during cryopreservation; however, this has not been critically studied. this study aimed to assess the sperm parameters of epididymal sperm harvested by applying both techniques on the same epididymis of horses and donkeys. we hypothesized that the combination of 2 techniques increases the total number of sperm harvested without affecting the semen quality. epididymides from donkeys (n = 16) and horses (n = 20) were harvested after castration or emergency euthanasia. the tails of the epididymis were dissected and weighed. each tail was subjected to retrograde flushing with 5 10 ml of freezing extender (botucrio). after the extender, 20 25 ml of air was used to push the remaining extender and semen into the collection tube. the epididymis was then sliced in 1 2 mm pieces, covered with 5 10 ml of freezing extender, and left in incubation for 15 20 minutes at room temperature. then semen was filtered through a gauze and recovered in a conical tube. recovered sperm from both techniques were evaluated separately for volume, concentration, and total sperm count. thereafter, sperm concentration was adjusted to 200 × 106/ml, loaded in 0.5 ml straws, frozen, and then plunged into liquid nitrogen. thereafter, the samples were thawed at 38oc for 60 seconds. postthaw total motility (tm) and progressive motility (pm) were evaluated with casa. sperm membrane integrity (smi) and mitochondrial membrane potential (mmp) were assessed with zombie green and mitotracker deep red via spectral flow cytometry. data analyzes were carried out via mixed model in r. significance was set at p < 0.05, a tendency at 0.1 < p < 0.05. donkey epididymis weighed 19.8 ± 3.2 grams and the horse 16.2 ± 2.2 grams. the concentration tended to be lower (p = 0.09) in the horse than in the donkey (764 ±5 9 ×106/ml versus 549 ± 68.5 × 106/ml) total sperm harvested was affected by both technique, species, and their interactions (p < 0.05); the slicing technique (4.5 × 109 versus 10.9 × 109) and the horse (5. 5× 109 versus 10.6 × 109) yielded a lower amount of sperm (p < 0.05). use of retrograde flushing followed by slicing-floating resulted in 64 and 43% more sperm per harvest, in the donkey and horse, respectively. tm and pm before freezing were not influenced (p > 0.05) by the type of technique or by the species. after thawing, smi and mmp were not affected (p > 0.05) by the technique or the species. tm and pm were not influenced (p > 0.05) by the technique or the species but by their interaction (p = 0.005); the slicing technique applied to the horse resulted in lower (p < 0.05) tm and pm. results suggested that the combination of both techniques was superior to a single technique, and a suitable method to optimize epididymal semen harvest in donkey and horse. keywords: conservation, epididymal sperm, cryopreservation effects of different treatment approaches on microbial populations in vaginal discharge of cows with clinical metritis jennine lection,a,b emily van syoc,a,b,c asha miles,b,d julia hamilton,e adrian barragan,e erika gandaa,b aintergraduate degree program in integrative & biomedical physiology huck institutes of the life sciences bdepartment of animal science, college of agricultural sciences cintegrative & biomedical physiology and clinical & translational sciences dual-title phd program pennsylvania state university, university park, pa danimal genomics and improvement laboratory, agricultural research service united states department of agriculture, beltsville, md edepartment of veterinary and biomedical sciences, college of agricultural sciences; pennsylvania state university, university park, pa clinical metritis (cm) has many costs to the dairy producer, including decreased milk production, reduced reproductive performance, and treatment costs. the more commonly isolated bacterial species in cm cases are escherichia coli, fusobacterium necrophorum, and prevotella spp. the current treatment strategy involves systemic antibiotics, typically penicillin or a third-generation cephalosporin; however, there is increasing public health concern about judicious use of antibiotics and prevention of drug-resistant pathogens. objective was to evaluate the effects of various treatment approaches, a nonantibiotic treatment (intrauterine dextrose) versus systemic antibiotic treatment (subcutaneous ceftiofur) on the microbiome of the vaginal discharge of dairy cows diagnosed with cm at 7 ± 3 days in milk. we hypothesized that the clinical cure rates are similar between 2 treatment groups, and therefore, their microbial populations are also similar. cows were enrolled from a dairy farm in central pennsylvania and were screened for cm with a clinical theriogenology 2022; 14: 265 metricheck® device at 7 ± 3 days after calving. cows presenting with reddish-brown fetid watery discharge were diagnosed with cm and eligible for enrollment. eligible cows were blocked by parity and randomly allocated to 1 of 2 treatments: i. intrauterine dextrose (dex): cows received 1 liter of an intrauterine 50% dextrose solution for 3 days starting on the day of diagnosis; and ii. systemic ceftiofur (conv): cows received 2 injections of ceftiofur (6.6 mg/kg of bw; excede, zoetis inc.) 72 hours apart, starting on the day of diagnosis. cows were evaluated for clinical cure rate at 7 and 14 days after diagnosis. vaginal discharge samples were collected using a metricheck® device at enrollment day (study day 0, before treatment), day 7, and day 14 for a subset of enrolled cows (dex = 13, conv = 14). vaginal discharge samples were analyzed for 16s rrna gene sequencing to evaluate changes in the microbiome between treatment groups. clinical cure rate between the 2 treatment groups was not different (day 7, p = 0.56; day 14, p = 0.69, glimmix procedure of sas, cary, nc).1 alpha diversity (richness of microbes within each sample) did not differ (welch’s t-test) between 2 treatments at any of the 3 time points or between pre and posttreatment. beta diversity (comparison of microbial communities between cows) based on permutational multivariate anova analysis differed between treatment groups at time of diagnosis (p = 0.024) and again at the second recheck (p = 0.015), but not at the first recheck (p = 0.112). when analyzing differential relative abundance through wald’s test, bacteria of the prevotella and fusobacterium genera were more abundant in the vaginal discharge of the conv group compared to dex group at the first recheck, though at the time of diagnosis there was increased relative abundance of prevotella in the conv cows and fusobacterium in the dex cows. whereas 16s rrna analysis did not provide information on the viability of these bacteria, these changes in relative abundance paired with the similar clinical cure rate of the dex group compared to the conv group provided evidence that dextrose may be a lowcost antibiotic alternative treatment for cm in dairy cows. keywords: antibiotic alternatives, cattle, dextrose, metritis, microbiome, postpartum reference 1. hamilton j, barragan aa, hovingh e, et al: assessment of the effects of intrauterine dextrose infusion on clinical cure rate, daily milk yield, and daily rumination in postpartum dairy cows diagnosed with clinical metritis. j dairy sci 2020;103 (suppl. 1): 164. canine splenic hemangiosarcoma cells express luteinizing hormone receptors in vitro khawla zwida,a lineth truijillo,b michelle kutzlera adepartment of animal and rangeland sciences, oregon state university, corvallis, or bdepartment of integrated biology, oregon state university, corvallis, or hemangiosarcoma is a rapidly growing, highly invasive cancer arising from the lining of blood vessels. more than half of all canine hemangiosarcoma primary tumors arise within the spleen. compared to other breeds, german shepherds, golden retrievers, and labrador retrievers are overrepresented. in addition to breed, gonadectomy (spaying/neutering) increases the likelihood for developing hemangiosarcoma. spayed female dogs have 2 10 times the risk for developing splenic hemangiosarcoma compared to intact female dogs. following gonadectomy, luteinizing hormone (lh) concentrations are significantly and persistently elevated. our laboratory has previously demonstrated that formalin-fixed canine splenic hemangiosarcoma tissues express lh receptors (lhr). we hypothesized that isolated canine splenic hemangiosarcoma cells also express lhr. objective was to use immunocytochemistry to determine the percentage of cells expressing lhr in each cell line. immortalized cell lines isolated from 4 dogs with a primary splenic hemangiosarcoma (dal-4, dhsa, gracehsa, efs; kerafast, inc., boston, ma) were used. cells were fixed onto coverslips and incubated with either a rabbit polyclonal antihuman lhr antibody (#nls1436, novus biologicals, littleton, co) or a universal negative control (#nc498h, biocare medical, pacheco, ca). cells were then incubated with horse antirabbit igg conjugated to fitc (#nb 7159, novus biologicals). the coverslips were then inverted and mounted to slides with medium containing dapi (#h-1500, vectashield® hardsettm antifade mounting medium with dapi, burlingame, ca). three randomly selected fields from each cell line were captured using fluorescence microscopy (leica microsystems, germany) at 400 x magnification. the mean ± sd percentages of lhr positive cells for each cell line was compared by oneway anova (graphpad prism 8.4.3) and significance was defined as p < 0.05. percentages of cells positive for lhr were 6.9 ± 2.5, 8.5 ± 1.0, 11.8 ± 3.1, and 17.2 ± 4.5% in dal-4, dhsa, grace-hsa, and efs, respectively. percentage of lhr positive cells was greater (p = 0.0142) in efs compare dhsa and dal4. this is the first study to report lhr expression in isolated canine hemangiosarcoma cells. our laboratory is currently examining the effect of lhr activation on hemangiosarcoma cell proliferation with increasing concentrations of lhr agonists. since lhr induced splenic hemangiosarcoma cell proliferation, future clinical trials could begin using gnrh agonists to reduce lh concentrations in gonadectomized dogs to prevent the development or recurrence of hemangiosarcoma. keywords: dog, cancer, gonadectomy, immunocytochemistry validating thyroid hormone testing using chemiluminescence in llamas and alpacas kristen bird, tim hazzard, michelle kutzler department of animal and rangeland sciences, oregon state university, corvallis, or hypothyroidism is an endocrine disease in llamas and alpacas resulting from insufficient concentrations of thyroid hormones. hypothyroidism is a cause of infertility in female camelids.¹ elevated thyroid stimulating hormone (tsh) concentrations with normal thyroxine (t4) concentrations are early signs of hypothyroidism.² unfortunately, hypothyroidism is rarely diagnosed in camelids because of a lack of published normal ranges. objectives were to: i. validate a commonly used chemiluminescence assay for determination of camelid tsh and t4 concentrations; and ii. establish normal ranges for tsh and t4 concentrations in male, gelded, and female camelids. it was hypothesized that the chemiluminescence assay accurately measures tsh and t4 clinical theriogenology 2022; 14: 266 concentrations in healthy camelids. to validate the precision of the immulite 1000 (siemens) assay for tsh (#tk9) and t4 (#kt4), 7 pooled samples of varying concentrations from each species were run in octet. to validate the linearity of the tsh assay, pooled samples of varying concentrations were run in triplicate and then spiked with a known concentration and run in duplicate. to validate the linearity of the t4 assay, pooled samples of varying concentrations were run in triplicate and then spiked or diluted with known concentrations and run in quadruplet or triplicate, respectively. data on pooled samples before and after spiking or dilution were compared using linear regression. following validation, tsh and t4 concentrations were measured on archived serum samples from healthy, adult llamas and alpacas (n = 125). coefficients of variation for precision of the assay were < 9%. the correlation coefficient for linearity was high for all pools tested (r² ranged from 0.94 0.99). normal ranges (mean ± 2 standard deviations) of tsh and t4 concentrations were established for alpacas (tshmales (n = 16): 21.9 86.8 pg/ml; geldings (n = 9): 12.3 95.3 pg/ ml; females (n = 12): 20.4 99.1 pg/ml; t4 males (n = 8): 3.59 10.8 µg/dl; geldings (n = 10): 3.65 7.88 µg/dl; females (n = 9): 3.04 7.71 µg/dl) and llamas (tshmales (n = 29): 19.3 118.0 pg/ml; geldings (n = 43): 21.1 106.0 pg/ml; females (n =14): 13.7 158.0 pg/ml; t4 males (n = 9): 3.75 7.73 µg/dl; geldings (n = 9): 5.17 9.94 µg/dl; females (n = 9): 4.22 11.2 µg/dl). this is the first study to validate a method for measuring tsh concentrations in camelids. further research is needed to evaluate what role abnormalities in t4 and tsh concentrations have in camelid subfertility. keywords: camelid, hypothyroidism, precision, thyroid stimulating hormone, thyroxine references 1. hamir an, timm ki: nodular hyperplasia and cysts in thyroid glands of llamas (lama glama). vet rec 2003;152:507-508. 2. almandoz jp, gharib h: hypothyroidism: etiology, diagnosis, and management. med clin north am 2012;96:203-221. endometrial injection of mesenchymal stem cells may revert endometriosis in mares lorenzo segabinazzi,a,b marcio carmo,a carlos fonsecaalves,a fernanda landim,a igor canisso,a,c marco alvarengaa adepartment of animal reproduction, são paulo state university, botucatu, brazil bross university school of veterinary medicine, basseterre, st. kitts, west indies cdepartment of veterinary clinical medicine, university of illinois, urbana, il endometriosis is characterized by degenerative changes of the endometrium leading to poor conception rates and higher pregnancy losses. chemically irritant agents (e.g., kerosene) have been proposed to revert endometriosis; however, animal welfare concerns and questionable results have discouraged many practitioners from using it. conversely, stem cell therapy has emerged as a promising tool in regenerative medicine. therefore, this study aimed to determine the potential regenerative effects of stem cells therapy injected directly into the endometrium of mares with endometriosis. we hypothesized that mesenchymal stem cells (mscs) improve endometrial health of mares with endometriosis. utilizing the kenney-doig scale, light breed mares (n = 15) with endometrial biopsies classified as grade iib or iii were enrolled. ten mares were treated with an endometrial injection of autologous bone marrow mesenchymal stem cells (mscs, 12 ×106 in 6 ml of pbs); 5 mares were sham-injected (6 ml of pbs). injections were guided with an endoscope (series 160 olympus). each treatment consisted of 12 sites (0.5 ml/point) following a horizontal line from the tip of 1 uterine horn to the contralateral uterine horn. uterine biopsies were taken during diestrus at 15 days before (day -15) and 60 days (day 60) after endometrial injections. endometrial score (h&e stain), intensity of fibrosis (masson’s trichrome blue), the extension of collagen type iii, progesterone (mouse monoclonal antiprogesterone, clone pgr 636, dako cytomatuon, carlinteria, ca) and estrogen alpha (mouse monoclonal anti-er-alpha, clone ep1, dako) receptors (immunohistochemistry) were evaluated in endometrial samples before and after treatment. intensity of fibrosis and extension of collagen type iii were assessed in biopsy samples using the software avsoft bio-view spectra 4.0.1. distribution of estrogen and progesterone receptors were evaluated using a semiquantitative scoring system: 0: absence of staining; 1: 1 25% of positive cells; 2: 26 50% of positive cells; 3: 51 75% of positive cells; and 4: > 75% of positive cells. data were analyzed with kruskal-wallis and dunn’s post-hoc. significance was set at p < 0.05. endometrial score improved in 6 mares (3 mares improved to grade i and 3 mares to grade iia) treated with mscs, whereas no changes were observed in the remaining 4 treated mares or 5 controls. morphometric evaluation had a reduction in endometrial fibrosis (p = 0.02; day -15: 541198 ± 750341 pixel2; day 60: 448714 ± 241518 pixel2) and collagen type iii (p = 0.007; day -15: 726869 ± 63200 pixel2; day 60: 130685 ± 7555 pixel2) in mares treated with mscs. additionally, an increase in the distribution of progesterone receptors (p = 0.03; day -15: 3; day 60: 4), immunolabelling for estrogen receptors tended to increase after mscs therapy (p = 0.08; day -15: 1; day 60: 3). in conclusion, endometrial injection of mscs reverted endometriosis in 60% of mares and increased steroid hormone receptors in the endometrium. this technique has the potential to rescue fertility of mares by regenerating endometrium; however, this remains to be confirmed. keywords: barren mares, endometritis, chronic degenerative endometritis, fibrosis, cell therapy does uterine microbial population vary due to uterine sampling techniques and estrous cycle stage in healthy postpartum dairy cows? mohanathas gobikrushanth,a champika fernando,b janet hill,b dinesh dadarwala adepartment of large animal clinical sciences bdepartment of veterinary microbiology western college of veterinary medicine, university of saskatchewan, saskatoon, sk, canada metagenomic sequencing for genital microbial profiling is increasingly used to enhance our understanding of the impact of clinical theriogenology 2022; 14: 267 microbiota on physiological functions, uterine diseases, and hence dairy cows’ fertility. researchers have utilized uterine swab (us), uterine cytobrush (uc), and low-volume uterine lavage (ul) methods to sample the uterus for bacterial population using culture and culture-independent metagenomic sequencing.1 interestingly, variations in uterine microbial profiles that may occur due to inherent differences in sampling methods have not been studied. as these techniques differ in the sample’s depth and representative portion of the uterus sampled and the hormonal milieu differs between estrus and diestrus phases, we hypothesized that uterine microbial profiles differ among uterine sampling techniques and phases of estrous cycle. therefore, we compared uterine microbiome profiles determined using routine bacteriological culture and next-generation sequencing as follows: i. among 3 sampling techniques; and ii. estrus and diestrus phases of estrous cycle. clinically healthy postpartum dairy cows (n = 15) from the rayner dairy research and teaching centre (university of saskatchewan) between 50 and 60 days postpartum were selected. each cow was sampled using 3 uterine sampling techniques during estrus and diestrus phases induced during the presynchronization protocol and confirmed based on transrectal palpation and assessment of ovarian structures via transrectal ultrasonography. for each cow, the us and uc samples were taken from 1 uterine horn (randomly assigned), whereas the ul sample was collected from the opposite uterine horn using sterile techniques. by the end of the experiment, 87/90 expected samples were collected. one-half of each sample was subjected to bacterial culture by incubating aerobically at 37oc for 48 hours on colombia blood agar and resulting colonies with different morphologies were selected for identification by pcr and sanger sequencing of the 16s rrna gene. preliminary results from bacterial culture indicated that the proportion of countable colony-forming units (cfu; defined as 25 250 cfu/plate2) was higher (p = 0.04) for uterine samples collected at diestrus than estrus phase of the estrous cycle (15/45, 33% versus 8/42, 19%, respectively; chi-square = 3.89). however, the proportion of countable cfu did not differ (p = 0.68) among the sampling techniques (us versus uc versus ul: 6/29, 21% versus 7/29, 24% versus 10/29, 34%, respectively; chi-square = 0.78). in summary, the culturable bacterial load varied with the estrous cycle stage but not among uterine sampling techniques. keywords: cattle, endometrial sampling, bacteriome, nextgeneration sequencing references 1. ong ct, turni c, blackall pj, et al: interrogating the bovine reproductive tract metagenomes using culture-independent approaches: a systemic review. anim microbiome 2021;3:41. 2. maturin l, peeler jt: chapter 3. aerobic plate count. in: food and drug administration (fda), bacteriological analytical manual online. 8th edition, berlin; silver spring: 1998. generation of feline oviductal and endometrial organoids riley thompson, mindy meyers, fiona hollinshead department of clinical sciences, colorado state university, fort collins, co organoids are 3d, spherical cell clusters formed in vitro that are capable of regeneration and self-organization with similar function to their tissue of origin. organoids overcome 2 major short-comings of the classic explant and 2d monolayer cell culture approaches by: i. maintaining viability in vitro longer (months); and ii. retaining the ability to physiologically respond to various stimuli similar to their in vivo organ counterpart, respectively. furthermore, organoids provide physiological structures that support intercellular interaction between multiple cell types and, therefore, offer a potentially superior model for exploring disease mechanisms and investigating potential therapeutics. importantly, organoids reduce the need for whole-animal studies, making research more efficient and cost effective while improving animal welfare. to date, equine endometrial organoids are the only reproductive organoids reported in domestic animals.1 therefore, our objectives were to: i. establish endometrial and oviductal organoids derived from feline tissues; and ii. trial various culture media to determine optimal growth conditions. we hypothesized that medium reported for generation of mouse oviductal organoids2 provide the highest growth and viability. three culture media1-3 were trialed with oviductal organoids using previously reported methods1 with minor modifications (scraping the tissue surface to release cells rather than collecting tissue biopsies). dissociated cells were plated at a concentration of 5,000 per well, and oviductal organoids were assessed after 2 weeks in culture for growth and viability using brightfield microscopy in conjunction with orgaquant python script4 and fluorescein diacetate and propidium iodide staining, respectively. data were analyzed using anova with tukey’s post-hoc test, and significance was set at p < 0.05. establishment of feline endometrial and oviductal organoids was successfully achieved for the first time. the organoids appeared as round, cystic structures of epithelial cells, similar to endometrial organoid reports in mares and women.1,3 utilizing orgaquant, the number of established organoids varied (p < 0.005) among 3 media trialed. organoid medium reported for the growth of equine endometrial organoids1 facilitated the greatest growth and cellular viability of feline oviductal organoids. results demonstrated that feline reproductive organoids can be established utilizing existing protocols with minor modifications as a novel 3d cell culture model that may more closely recapitulate in vivo anatomy and physiology long-term that will facilitate reduced reliance on research animals and the associated cost and welfare concerns of live-animal experimentation. keywords: queen, cat, cell culture, in vitro, 3d, fallopian tube references 1. thompson re, johnson ak, dini p, et al: hormone-responsive organoids from domestic mare and endangered przewalski’s horse endometrium. reproduction 2020;160:819-31. 2. xie y, park es, xiang d, et al: long-term organoid culture reveals enrichment of organoid-forming epithelial cells in the fimbrial portion of mouse fallopian tube. stem cell res 2018;32:51-60. 3. turco my, gardner l, hughes j, et al: long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium. nat cell biol 2017;19:568-577. clinical theriogenology 2022; 14: 268 4. kassis t, hernandez-gordillo v, langer r, et al: orgaquant: human intestinal organoid localization and quantification using deep convolutional neural networks. sci rep 2019;9:1-7. cystic ovarian disease in cattle: diagnosis and differentiation via color doppler ultrasonography and hormone assay zachary turner,a fabio lima,b alan conley,b bret mcnabb,ab joan dean-rowe,ab richard pereiraab alivestock herd health and reproduction service bdepartment of population health college of veterinary medicine, university of california, davis, ca most frequently cited definition of cystic ovarian disease (cod) in cattle is an abnormally persistent follicle (> 7 10 days) with a diameter > 20 mm that clearly interferes with normal ovarian cyclicity.1 discrimination between luteal and follicular ovarian cystic structures has traditionally been conducted by measuring the rim of luteal tissue. most common practice in field diagnosis is via transrectal palpation examination with or without the use of a b-mode ultrasonography (though misclassification of ovarian structures is estimated to be 40 50%). color doppler ultrasonography utilizes blood flow area measurements within the ovarian structure that has been proposed as a potential indirect measure for plasma progesterone (p4) concentrations and that apparently increase in luteal cysts. objective was to compare the diagnostic accuracy when differentiating luteal and follicular ovarian cysts using measures collected via b-mode and color doppler transrectal ultrasonography. the consensus definition was used to identify the condition and confirmed by 2 examinations 10 days apart. a 3 mm luteal rim width was used to differentiate follicular and luteal cysts during reproductive examinations. blood flow area measurements were recorded within the rim of cystic structures and calculated using a standard video analysis protocol. multiparous dairy cows (n = 36) were enrolled during routine herd reproductive examination visits, with 22 and 14 having follicular and luteal cysts, respectively. cows were examined using a mini-exapad (easi-scan, imv imaging ltd., rochester, mn) ultrasound with color doppler capabilities. blood samples were collected from each cow to measure p4 serum concentrations, using ≥ 1 ng/ml concentrations as threshold value reference standard for luteal cyst diagnosis. receiver operating characteristic (roc) analyses were generated to compare the accuracy when differentiating follicular and luteal ovarian cysts using measurements collected via b-mode (e.g., luteal rim width) and color doppler (e.g., blood flow area) ultrasonography, using p4 as the gold standard measure. among cystic structure measured parameters, luteal rim width and blood flow area were selected for further analysis because they presented the best roc curves for differentiating cod, with an area under the curve of 0.75 and 0.80, respectively. luteal rim width measurements alone resulted in a sensitivity and specificity of 50 and 86%, but was improved by using color doppler flow area measurements (with values of 79 and 86%, respectively). when combining the use of luteal rim and blood flow area for differentiating cystic ovarian structures, in a parallel approach resulted in sensitivity and specificity of 93 and 73%, and in series approach resulted in sensitivity and specificity of 35 and 100%. in conclusion, discriminating between luteal and follicular ovarian cysts in dairy cattle via doppler ultrasonography had higher diagnostic accuracy compared to b-mode ultrasonography alone; an opportunity for veterinary practitioners to enhance diagnostic ability. keywords: cyst, luteal, follicular, endocrine, progesterone reference 1. vanholder t, opsomer g, de kruif a: aetiology and pathogenesis of cystic ovarian follicles in dairy cattle: a review. reprod nutr dev 2006;46:105-119. clinical theriogenology 2022; 14: 269 mixed animal session homozygous deletion on chromosome 29 leading to disorder of sexual development anne sheerin,a amy leibeckb arood and riddle equine hospital, lexington, ky bgenesee valley equine clinic, scottsville, ny disorders of sexual development (dsd) are not uncommon in the horse, affecting 5.6% of the population. dsds affect sexual differentiation, development and function of the gonads, and may be characterized by atypical chromosomal, phenotypic, or gonadal sex. homozygous deletions in chromosome 29 have been a risk factor for dsds. this report describes the homozygous deletion of 200,000 base pairs in chromone 29 with abnormal genitalia in a 12-month appaloosa filly. horse was presented for an abnormally swollen vulva. upon parting the vulvar lips, a band of pigmented tissue of ~ 1 cm thick in the vestibule, extending 10 cm cranially to the vestibulovaginal fold. the tissue extended from the dorsal to ventral commissure of the vulva. an abnormal urethral opening and enlarged clitoris were observed protruding from the ventral aspect of this septum. filly was sedated and a speculum examination revealed a normal vagina and cervix. due to the abnormal appearance and suspicion of a dsd, a heparinized blood sample was submitted for a chromosomal and pcr analysis. cytogenetic analysis (e.g., karyotyping analysis) performed at texas a&m revealed that the filly had 64 chromosomes with a normal morphology, 2 x chromosomes; however, it was homozygous for a large deletion in chromosome 29. the pcr test for the y-linked sry gene was negative and the pcr test for the x-linked androgen receptor gene was normal. the fertility of this filly is unknown at this time. it has been noted that 8.1% of reproductively or developmentally abnormal horses had a homozygous deletion; however, 79% of horses with a homozygous deletion of chromosome 29 were reproductive or developmentally abnormal.1 the deletion includes the aldo-keto reductase family 1 c (akr1c) gene which is a ketosteroid reductase in steroid hormone development. akr1c is involved in androgen and estrogen metabolism, and all pathways to testosterone and dihydrotestosterone production. these mutations have also been associated with human dsd. altered akr1c has been associated with prostate and breast cancer, endometriosis, and xy dsds in humans. keywords: disorder of sexual development, chromosome, gene deletion reference 1. ghosh s, davis bw, rosengren m, et al: characterization of a homozygous deletion of steroid hormone biosynthesis genes in horse chromosome 29 as a risk factor for disorders of sex development and reproduction. genes 2020;11:251. effect of the sexedultra™ genesis iii technology on the quality and 24 hours longevity of sex-sorted stallion sperm camilo hernández-avilés,a luisa ramírez-agámez,a sheila spacek,a charles love,a pablo ross,b cristina cabrera,b juan moreno,b juan sampera adepartment of large animal clinical sciences college of veterinary medicine and biomedical sciences texas a&m university, college station, tx bst genetics, navasota, tx use of flow cytometry techniques for the sex-sorting of stallion sperm is lower than that reported for food-producing animals. stallion-related factors and sperm susceptibility to the sex-sorting process resulted in reduced sperm quality and longevity over time that accounted for the limited use of this technology in horses. consequently, sex-sorted stallion sperm has been used for insemination of mares closely located to the sorting facility, that limits the commercial applicability of this technology. recently, a proprietary technology (sexedultra™ genesis iii, st genetics, navasota, tx) has become available for sex-sorting of bull sperm. this technology reduced the sorting time and increased the sorting efficiency, quality, and fertility of sex-sorted sperm. a study was designed to determine the effects of this process on stallion sperm. three ejaculates from 4 stallions (n = 12), were diluted with inra-96® and processed by colloid centrifugation. the resulting pellet containing at least 2.5 x 109 sperm was resuspended in a proprietary chemically defined medium (st extender) and stored for ~ 60 minutes at (~ 20°c) before starting the sex-sorting process. sperm quality parameters in fresh semen immediately after colloid centrifugation before staining (fresh), immediately after sex-sorting (x-sorted fresh) and following 24 hours of cooled storage at 6°c (x-sorted cooled) were recorded and analyzed. parameters included: total sperm motility (tmot; %), progressive motility (pmot; %) determined by casa; viability/acrosomal intactness (vai; %), lipid peroxidation in viable sperm (vlpp; %), and sperm dna damage (compα-t), determined by flow cytometry. data were rank-transformed before analysis using the general linear model (jmp pro 16). statistical significance was set at p clinical theriogenology 2022; 14: 270 < 0.05. mean tmot was higher in fresh than in x-sorted fresh or x-sorted cooled semen (88 versus 80 versus 67%; p < 0.05). mean pmot was similar in fresh and x-sorted fresh semen, while higher than in x-sorted cooled (52 versus 59 versus 34%; p < 0.05). mean vai was similar in fresh and x-sorted fresh semen, while higher than in x-sorted cooled semen (84 versus 85 versus 78%, respectively; p < 0.05). mean vlpp was lower in fresh than in x-sorted fresh semen, and higher in x-sorted fresh than in x-sorted cooled semen (8 versus 22 versus 27%, respectively; p < 0.05). mean compα-t was similar in x-sorted fresh and x-sorted cooled semen, whereas lower than in fresh semen (3 versus 4 versus 7%; p < 0.05). results indicated that sex-sorting and subsequent cooled storage induced a reduction in stallion sperm motility and increased sperm lipid peroxidation. other sperm quality parameters (e.g., viability and dna integrity) during the fertilization process were maintained after sex-sorting and 24 hours of cooled storage. sex-sorted stallion sperm processed using the sexedultra™ technology can still maintain acceptable quality for breeding purposes either as fresh sorted or cooled sorted. further studies will determine if fresh and cooled, sex-sorted semen processed with this novel technology will retain its fertilizing potential and could be used for commercial breeding purposes. keywords: stallion sperm, sex-sorting, sexedultra™, cooled storage extending teasing time increases bacterial load and decreases semen longevity in normal libido normospermic stallions giorgia podico, guilherme novello, lorenzo segabinazzi, fabio lima, igor canisso department of veterinary clinical medicine, university of illinois, urbana il normal libido stallions are inadvertently or purposely over teased to increase semen collection. however, the intensity and duration of teasing before semen collection increase the seminal plasma in the ejaculate that is thought to be detrimental to semen quality. this study aimed to determine whether extending the teasing time of normal libido stallions affected semen quality. we hypothesized that extending teasing time reduces semen cooling and freezing abilities and increases bacterial contamination. forty semen collections of 10 stallions were performed at 48 hours intervals. for each stallion, 2 collections were performed using standard teasing (i.e., a successful ejaculate was procured < 5 minutes after penile washing), and 2 collections included extended teasing (i.e., after penile washing, stallions were handheld for 10 minutes before being allowed to mount the dummy mount). immediately after collection, gel-free semen and gel volumes were weighed. sperm concentration was determined with a nucleocounter. the gel-free semen was extended at 50 x 106/ml in inra96 cushion-centrifuged, and half of the pellets were resuspended in inra96 at 50 x 106/ml and stored in equitainer for further evaluations at 24 and 48 hours. the second half of the pellets were resuspended in an egg yolk-based extender (botucrio) at 100 × 106/ml, loaded in 0.5 ml straws, and frozen over nitrogen vapor. total motility (tm) and progressive motility (pm) were evaluated with casa. sperm membrane integrity (smi) and mitochondrial membrane potential (mmp) were assessed with zombie green and mitotracker deepred via spectral flow cytometry. sperm parameters were assessed at 0, 24, 48 hours of cooling before and after thawing. raw and cooled semen were aerobically cultured (48 hours), and maldi-tof identified isolates. data were analyzed with a paired student’s t-test, linear mixed model, fisher’s exact test, and tukey’s posthoc. significance was set at p < 0.05. gel-free and gel semen volumes increased with extended (p < 0.05) teasing time from 45.5 ± 5.0 ml to 76.4 ± 5.5 ml and from 4.7 ± 2.4 ml to 21.3 ± 4.9 ml. sperm concentration reduced (p < 0.05) from 344 ± 56×106/ml to 189 ± 28 x 106/ml when the teasing was extended. isolates included actinobacillus spp., corynebacterium spp., staphylococcus spp., and pseudomonas spp. the extended teasing time increased (p < 0.05) the number of bacterial isolates in the raw semen from 5 to 8. cooled semen at 24 and 48 hours had higher percentages of positive cultures in the extended teasing but not statistically significant (24 hours, standard 25 versus extended 35%; 48 hours, standard 20 versus extended 25%). tm and pm decreased after freezing and overtime after cooling (p < 0.0008) but were not affected by the teasing time (p > 0.05). smi was not affected by group (p > 0.05) or freezing (p = 0.7), but there was an interaction (p = 0.03) between freezing and group. during cooling, smi was affected by group (p = 0.013) but not by time (p = 0.7) or by their interaction (p = 0.27); extended teasing resulted in lower smi at 0, 24, and 48 hours. mmp was not affected by time (p = 0.82) or group (p = 0.2) or their interaction (p = 0.5) during cooling. mmp was affected by freezing (p < 0.001) but not by group (p = 0.4) or interaction (p = 0.3). in conclusion, extended teasing decreased the longevity of the semen upon cooling and increased the bacterial load; however, it did not affect postthaw semen quality. keywords: stallion, seminal plasma, sperm viability, semen freezing luteal tissue area and immunoreactive concentrations of progesterone in plasma of bred and nonbred mares ilaria colombo,ab giorgia podico,a alba rudolf-vegas,ac carmen alminana,c stephan bauersachs,c igor canissoa adepartment of veterinary clinical medicine, university of illinois urbana-champaign, il bdepartment of veterinary medical sciences, university of bologna, italy cinstitute of veterinary anatomy, university of zurich, lindau, switzerland progesterone is a pivotal hormone to maintain pregnancy in the first trimester in mares. low progesterone concentrations (< 4 ng/ml) have been associated with early pregnancy loss. some clinicians collect 1 blood sample from mares 2 10 days postovulation to determine whether the mare needs progestin supplementation. a study demonstrated that mares becoming pregnant have higher progesterone on day 5 postovulation than those failing to become pregnant; however, it remains to be determined if progesterone varies on other days. in some clinical theriogenology 2022; 14: 271 species, seminal plasma has a luteotropic property. therefore, the objective of the present study was to evaluate the immunoreactive progesterone concentrations and the luteal tissue area in non-bred, bred becoming pregnant, and nonpregnant mares. we hypothesized that pregnant mares have higher progesterone concentrations than nonpregnant mares 2 10 days postovulation. light breed mares (n = 14) were monitored via ultrasonography every other day until detection of a preovulatory follicle (diameter ≥ 35 mm in the presence of endometrial edema). then mares received deslorelin acetate (1.8 mg, intramuscularly) to induce ovulation. twenty-four hours later, mares received an ai (~ 2 x 109 progressively motile sperm extended in 50 ml of inra 96) or a sham-ai (50 ml of inra 96). ovulation was confirmed by ultrasonography. the number of corpora lutea and the luteal tissue area were recorded daily until 10 days postovulation. immunoreactive progesterone concentrations were assessed daily from the day of the ovulation to 10 days postovulation. pregnancy diagnosis was carried out at 10 and 13 days postovulation. a total of 52 estrous cycles were completed; 15 were part of the control group (sham-ai), and 37 were part of the bred group, with 17 cycles nonpregnant and 20 pregnant. data were analyzed with a mixed model, tukey test as post-hoc, and pearson’s coefficient of correlation. both immunoreactive progesterone concentration and luteal tissue area varied (p = 0.001) with time postovulation but were not affected (p > 0.05) by group. the number of ovulations caused an increase (p = 0.0001) in the immunoreactive progesterone concentrations and luteal tissue area. there was a weak but positive association between the number of ovulations and immunoreactive progesterone concentrations (r = 0.2; p = 0.0001) and a moderate positive association between the number of ovulations and luteal tissue area (r = 0.54; p = 0.0001). the lack of change in the progesterone concentrations and luteal tissue area between bred and nonbred mares suggested that horse seminal plasma did not affect luteal function in mares. all mares had progesterone above 4 ng/ml after 5 days postovulation. thus, it is possible that if mares with abnormal progesterone concentrations were used, the results could have been different. in conclusion, pregnancy was not associated with higher progesterone concentrations or greater luteal tissue area. keywords: mare, corpus luteum, pregnancy, seminal plasma testicular atrophy following traumatic incident in young german shepherd dog jenica emerson,a alyssa shelby,b bruce christensenb aschool of veterinary medicine, university of california, davis, ca bkokopelli assisted reproductive services, sacramento, ca a 2-year, intact male german shepherd dog, was presented for nonpainful unilateral scrotal swelling of 3 days duration. scrotal swelling can be caused by infectious organisms, neoplasia, autoimmune disease, spermatic cord torsion, inguinal herniation, or direct trauma and can rapidly result in infertility.1 owner wanted to preserve breeding potential and saw no improvement after administering an antibiotic and a nonsteroidal antiinflammatory from their regular veterinarian. left testis was difficult to palpate due to edema and the right testis was smaller than expected. diagnostics included an in-house rsat brucellosis test (negative), semen collection/evaluation with oligospermia and teratospermia (6.66% motility, 12% normal morphology, 11 x 106 total sperm), prostatic fluid cytology (many neutrophils), and prostatic fluid culture (negative). ultrasound evaluation revealed that both testes and prostate parenchyma had normal echotexture, whereas the left testis was small (2.57 x 2.4 x 2.54 cm) compared to the right testis (2.93 x 2.38 x 3.38 cm) and was surrounded by mixed echogenicity fluid. left testis, accompanying tunics, and 6 cm of the left spermatic cord were surgically removed in a closed castration to prevent further inflammation and subsequent degeneration of the right testis. additional diagnostics on the removed testis included a culture (negative) and histopathology (severe testicular atrophy with steatitis) which confirmed the diagnosis of inguinal herniation. eleven days later, the scrotum was enlarged again. ultrasound examination revealed a homogeneously echogenic, normal right testis and left scrotal side filled with hypoechoic fluid and hyperechoic debris. daily antibiotics were continued, and a nonsteroidal antiinflammatory was prescribed along with cold compresses. currently, the client recalled that the patient’s inguinal area got caught on a fence prior to the initial visit. at a recheck ultrasound examination 7 days later, the was limited fluid and fibrin-like tissue present in the left scrotal sac and a normal right testis. semen collection/evaluation 124 days after the previous collection/evaluation had normospermia (85.25% motility, 74% normal morphology, 425.94 x 106 total sperm). it is important to conserve breeding potential in sporting and working breeds with valuable genetics which, with their activities, have an increased risk of traumatic injuries. specifically in this case, traumatic inguinal herniation and the resultant inflammation caused an increase in scrotal temperature resulting in testicular atrophy.2 this case highlighted how successful management of testicular trauma can preserve breeding potential. keywords: dog, orchitis, hemicastration, inguinal hernia references 1. pugh cr, konde lj: sonographic evaluation of canine testicular and scrotal abnormalities: a review of 26 case histories. vet radiol 1991;32:243-250. 2. memon ma: common causes of male dog infertility. theriogenology 2007;68:322-328. diaphragmatic hernia involving the gravid uterus in a dog michela ciccarelli,ab tania pereza aveterinary clinical science, bschool of molecular bioscience college of veterinary medicine washington state university, pullman, wa a pregnant, 4-year german shorthair pointer bitch, was presented for severe progressive tachypnea and depression. the day of pregnancy was unknown, the only information available was that she was bred 52 days prior to the presentation. bitch was quiet and alert with hyperemic mucous membranes. abdomen was soft and appeared nonpainful on manual palpation. several fetuses were palpable in the caudal abdomen. clinical theriogenology 2022; 14: 272 ventrally located lung sounds were muffled on auscultation. radiographs and an ultrasonographic examination revealed a diaphragmatic hernia with intrathoracic displacement of a portion of the uterus. an emergency herniorrhaphy was pursued with the owner requesting to allow the bitch to maintain the pregnancy if possible. preoperative medications included methadone and cefazolin. general anesthesia was induced with propofol to effect. anesthesia was maintained with sevoflurane in 100% oxygen. the bitch was maintained on mechanical ventilation at a respiratory rate of 5 breaths/minute. a ventral midline celiotomy was performed to reduce the herniated abdominal organs and repair the diaphragm. a 7 cm right-sided radial tear extending from the tendinous portion of the diaphragm into the pars sternalis was noted. tip of the left horn of the uterus containing 2 fetuses, the spleen, and right medial and lateral lobes of the liver, pancreas, and a large portion of the greater omentum were in the thoracic cavity. herniorrhaphy was performed using both modified lambert and simple continuous suture patterns. a rotational flap of the internal oblique muscle was used to aid in the closure of the most ventral portion of the hernia due to high tension. porcine submucosa was sutured over it as reinforcement. performance of an intra-operatory transuterine ultrasonographic examination of the fetuses revealed that the placental sites were intact, fetal heartbeats were present in all puppies evaluated, however, low heart rates (80 100 beats/minute) were attributed to uterine circulatory compromise and the effect of anesthesia; however, the decision to allow the pregnancy to continue was made. following surgery, the bitch received supportive medical care for 3 days until its systemic status declined. she was obtunded and had severe ventral edema and swelling of the vulva. blood chemistry and cbc showed hypoalbuminemia, leukocytosis with a left shift neutrophilia, and increased liver enzymes. transabdominal ultrasonography revealed evidence of fetal death with lack of fetal heart rate and increased opacity of the amniotic fluid. an emergency cesarean surgery was performed. six of 11 pups were saved; 1 fetus was underdeveloped and had a very elongated neck, presumably from entrapment within the lumen of the diaphragmatic hernia. to our knowledge, this is the first report that describes a pregnancy with survival of several fetuses following surgery for a diaphragmatic hernia with a gravid uterus displaced to the thorax. keywords: cesarean surgery, ultrasonography, pregnancy, herniorrhaphy survey of golden retriever breeders’ expectation of reproductive services michela ciccarelli,ab kincade audette,a eduardo arroyoa aveterinary clinical science, bschool of molecular bioscience college of veterinary medicine, washington state university, pullman, wa the importance of client communications has long been recognized in veterinary medicine. however, to a much lesser extent has this relationship been explored regarding reproductive service provided to breeders. previous studies reported feelings of disconnect and distrust between the 2 parties. in a recent report,1 dog breeders commented about the apparent lack of training in theriogenology among veterinarians. conversely, veterinarians expressed disinterest in working with breeders who seem arrogant, argumentative, or inflexible.  our goal was to identify possible areas of intervention to improve communications by way of utilizing a survey format to further define the relationship between breeders and veterinarians. a survey link was distributed to the golden retriever club of america list-serv, including ~ 3,900 members, with 127 complete responses used for this study. we designed the survey to have 3 areas of emphasis for analysis: communication, furthering education specific to theriogenology, and most sought-after services to provide in a veterinary clinic. communication techniques included active listening, nonjudgmental language, and trust to create a teamwork mentality and a successful breeder-veterinarian relationship. areas needing increased assistance from veterinarians were breeding soundness examinations, cesarean surgeries, and cleft palates. the main reasons for conflicts between breeders and their veterinarian were inexperience (21.2%), timing of cesarean surgeries (19.2%), lack of availability (15.4%), disagreements on when to spay or neuter (8%), communications barrier (6%) and vaccination recommendations (6%). the primary veterinary services sought by the breeders were interpretation of progesterone concentrations/cycle management (82.7%), ultrasonographic evaluation of the pregnancy (69.3%), prepartum radiographs to determine the number of fetuses present (64.6%), and health clearances (64.6%). a concerning aspect was that 19.2 and 28.8% of the participants never or rarely have had a breeding soundness exam performed before mating their pets. breeders can include a niche clientele of individuals passionate about the health and wellbeing of their animals and the future generations of dogs. the improvement of the continuing veterinary education and the enhancement of veterinary curriculum in veterinary schools are at the base of the betterment of the relationship between breeders and veterinarians who share the common goal of improving the welfare and health of the pets. keywords: theriogenology, dogs, mating, genetic counseling, breeders reference 1.englar re, schettler ka, ostrom sa: survey of communication challenges that impact relationships between veterinarians and dog or cat breeders and proposed solutions for retaining breeders as clients. j am vet med assoc 2021;258:407-415. optimizing centrifugation for cooled canine semen processing: preliminary data nicole sugai, julie cecere, orsolya balogh department of small animal clinical sciences virginia-maryland college of veterinary medicine, blacksburg, va semen processing is critical during preparation for shipped chilled semen or for semen cryopreservation to remove the seminal plasma. there is a deficiency in the knowledge for ideal processing techniques or settings for canine semen. purpose of this study is to provide recommendations for optimizing clinical theriogenology 2022; 14: 273 semen processing to yield improved shipped chilled semen conditions in dogs. our hypothesis was that higher centrifugation speeds and longer processing times will increase sperm recovery rates but reduce sperm viability and motility. our study aimed to determine the optimal combination of time and g force centrifugation allowing the best outcome during cooled storage. healthy, client-owned stud dogs (n = 9) of 2 5.5 years of age, > 15 kg and various breeds were collected using manual technique. only dogs with a negative brucella canis serology and an ejaculate with appropriate concentration, ≥ 70% total motility and ≥ 40% normal morphology, were included. ejaculates were divided into 6 equal volume aliquots, each subjected to 1 of 6 treatments: centrifugation at 400, 720 or 900 g, each for 5 or 10 minutes, followed by extension of the sperm pellet to the original aliquot volume with caniplus chill lt (minitube). samples were then cooled for 24 hours according to standard packaging technique in a minitube canine shipping box. semen evaluations of concentration and viability by nucleocounter® sp-100™ and total and progressive motility (tm, pm) by casa (spermvision™, minitube) were performed before centrifugation (t0), immediately post-centrifugation (t1) and after 24 hours of cooling (t24). sperm recovery rate (rr, %) was calculated as (t1 concentration)/ (t0 concentration)*100 from nucleocounter® sp-100 values. data were analyzed by linear mixed model with significance p < 0.05, and results presented as mean±sd. overall rr was 98.1 ± 9% and similar (p = 0.912) among treatment groups. sperm viability, tm and pm were not affected (p ≥ 0.779) by centrifugation type but all variables decreased (p < 0.001) over time. across all samples, a significant decrease in sperm viability from t0 (84.8 ± 6.8%) was detected by t1 (76.3 ± 6.4%) with further decrease by t24 (69.1 ± 8.4%). tm/pm decreased significantly from t0 (87.6 ± 5.9%/80.5 ± 13.4%) and t1 (84.9 ± 7%/81.8 ± 7.3%) to t24 (80.7 ± 13.3%/73.7 ± 15.9%). in conclusion, our preliminary results suggested that sperm viability was affected by both processing and cooling, whereas significant changes in tm and pm were not observed immediately postcentrifugation. at this time, the specific centrifugation speed or duration used in this study did not significantly affect rr and sperm viability or motility. this study is still ongoing, and we anticipate completion of this project by may 2022. keywords: dog, chilled semen, centrifugation, computer assisted sperm analysis hydrops fetalis diagnosed via transabdominal ultrasonography tiffany hoffman,a morgan agnewb arutgers university, camden, nj bphiladelphia animal hospital, philadelphia, pa a 3-year, golden retriever bitch, was presented to philadelphia animal hospital 59 days after breeding with edema in the hind limbs and a decreased appetite. hydrops was suspected after decreased detail was noted on a single lateral abdominal radiograph performed by the referring veterinarian 1 day prior to presentation. hydrops fetalis and hydrops amnion are congenital disease processes that result in a failure of fetal fluid homeostasis, seen primarily in brachycephalic breeds and golden retrievers.1 more importantly, hydrops fetalis and hydrops amnion increase the risk of fetal and maternal mortality. a transabdominal ultrasonography revealed 2 fetuses in the left uterine horn with heart rates over 180 beats/ minute, excellent gi definition and slightly echogenic amniotic fluid suggestive of meconium contamination. fetus in the right uterine horn had an edematous abdominal wall, pleural, pericardial, and peritoneal effusion with an abdominal diameter of 9 cm. a complete blood count and chemistry revealed mild anemia, but was otherwise unremarkable. dexamethasone was given subcutaneously at a dose of 0.2 mg/kg to accelerate fetal lung maturation.2 cesarean surgery was performed the following day. an excessive amount of fluid was present in the allantois of all fetuses. two viable pups were removed from the left uterine horn, whereas a nonviable puppy with hydrops fetalis and a significant cleft palate was removed from the right uterine horn. biopsies of the uterus and chorioallantois were submitted to ohio state university. there were no marked findings in the uterine biopsy, whereas the fetal membranes presented labyrinthine mineralization of varying severity. no evidence of inflammation or lesions that may have induced dystrophic mineralization were present. this patient was bred the following cycle to a different stud and delivered five normal puppies via planned cesarean surgery. this case illustrated the use of ultrasonography to diagnose fetal abnormalities in late term pregnancy. keywords: hydrops fetalis, golden retriever references 1 hopper bj, richardson jl, lester nv: spontaneous antenatal resolution of canine hydrops fetalis diagnosed by ultrasound. j small anim pract 2004;45:2-8. 2. vannucchi ci, regazzi fm, barbosa mm, et al: cortisol profile and clinical evaluation of canine neonates exposed antenatally to maternal corticosteroid treatment. reprod dom anim 2012;47:s6:173-176. clinical theriogenology 2022; 14: 274 melatonin improves testicular hemodynamics and sperm quality in rams subjected to mild testicular heat stress abdallah shahat,a,b jacob thundathil,a john kastelica adepartment of production animal health, university of calgary, calgary, ab, canada bdepartment of theriogenology, cairo university, giza, egypt melatonin is a potent free-radical scavenger with antiinflammatory, antioxidative, and antiapoptotic effects. our objective was to determine effects of melatonin on testicular blood flow and sperm quality after mild heat stress (hs; scrotal neck insulation) in rams. dorset rams (n = 12) with good semen quality were group-housed indoors (~18°c). once weekly for 2 weeks, doppler indices (resistance index; ri and pulsatility index; ri) were measured in the supratesticular artery and semen was collected (electroejaculation). then, rams were randomly allocated into 2 equal groups: melatonin group (mel), was given subcutaneously 36 mg melatonin in 1 ml corn oil under the ear or a control group with only corn oil (cont). on day 15 after treatment, rams were subjected to mild hs for 96 hours with blood flow measurements and semen collection performed once weekly for the next 6 weeks. sperm motility was assessed with casa (sperm vision®) and morphology using eosin-nigrosin. data were analyzed using repeated measures, with a bonferroni test for post hoc analysis. for total and progressive motility, there were group, week, and group*week interaction effects (p < 0.005) for total and progressive motility and total abnormalities, plus group and week effects for ri and pi (p < 0.005), with no significant difference before treatment. changes in total and progressive motility and sperm abnormalities were evident at week 1 posths, but mel mitigated (p < 0.05) these effects from weeks 2 to 6. furthermore, both pi and ri were reduced (i.e., better flow, p < 0.05) in mel versus cont rams on weeks 1, 3, 4, 5, and 6 after hs. in mel rams, sperm motility and total abnormalities had recovered at weeks 5 and 6, respectively, whereas cont rams had not completely recovered by week 6. in conclusion, melatonin treatment before hs heat stress significantly improved testicular blood flow and protected sperm motility and morphology, with potential for mitigating effects of hs under field conditions. keywords: rams, heat stress, melatonin, sperm quality, doppler indices, supratesticular artery safety of intrauterine infusion of the honey/propolis in normal postpartum dairy cows: a pilot study ali torabi,a willian mori,a réjean lefebvrea adepartment of clinical science, faculty of veterinary medicine university of montreal, saint-hyacinthe, qc, canada endometritis affects dairy cows during the postpartum period (pp) leading to prolongation of voluntary waiting period, infertility, and financial loss. the advocated treatment of the condition is intrauterine infusion of antibiotics. however, in the context of rising antibiotic resistance worldwide, legislative restrictions for antibiotic uses, and increasing numbers of organic farms, there is an urgent need for alternative therapeutic approaches. as an alternative treatment for endometritis, some products of honeybees such as propolis and honey (pro-honey) can substitute conventional treatments. we hypothesized that pro-honey (organic buckwheat honey with 5% propolis) is a good alternative to treat endometritis in dairy cows. however, the safety of the product needs to be assessed first. objective was to determine the acute clinical response of the endometrium to the intrauterine infusion of pro-honey in normal postpartum dairy cows. healthy cows (n = 31) with intact uterus and cycling normally formed 6 groups: group 1 (n = 6), 2 (n = 5), and 3 (n = 5) received an intrauterine infusion of 30 ml of pro-honey, honey alone, and cephapirin (positive control) respectively within 30 50 daysin-milk (dim). in group 4 (n = 3, negative control), cows were not infused. in groups 5 (n = 8) and 6 (n = 4), pro-honey and honey alone were infused into uterus of cows with more than 50 dim, respectively. transrectal ultrasonography, vaginal examination, cytological, and bacteriological evaluations of the uterus via cytobrush sampling at 0 (before infusion), 48, 96, and 192 hours were performed to assess acute response of the uterus. percentage of pmns (polymorphonuclear cells) in the uterus reached the utmost level rapidly at 48 hours before returning to the initial level of 96 hours in all groups with the exception of group 4 (p < 0.001) where no increase was measured. at 48 hours, number of pmns in cows of group 1 (53.8 ± 30) was higher (p < 0.05) compared to groups 2, 3, 4, 5, and 6 with 12.6 ± 7, 16.8 ± 25, 0.3 ± 0.6, 15.9 ± 9, and 10.1 ± 8 pmns respectively. at 48 hours, pro-honey triggered a stronger innate immune response of the uterus in cows in early pp (30 50 dim) than those more advanced in pp. bacterial culture were negative and no changes were observed with ultrasonography. in conclusion, pro-honey triggered a prompt innate immune response of the uterus in normal postpartum cows before returning rapidly to a quiescent status. results demonstrated that intrauterine infusion of the pro-honey in normal postpartum dairy cows is safe and stimulates an innate improduction animal session clinical theriogenology 2022; 14: 275 mune response that could be potentially advantageous in cases of endometritis. keywords: dairy cows, pro-honey, honey, intrauterine infusion, endometritis nonsurgical approach to sterilizing avian species using a domestic chicken model brianna o’malley,a sandra ayres,b jennifer graham,c yamin li,d qiaobing xud advm candidate class of 2024 bdepartment of biomedical sciences, cdepartment of clinical sciences cummings school of veterinary medicine, tufts university, north grafton, ma ddepartment of biomedical engineering, tufts university, medford, ma overpopulation of certain avian species can have detrimental effects on the natural and human environment. zoonotic diseases, nitrogenous pollution, and damage to human property and natural ecosystems are some concerns being raised by wildlife control agencies. current population control efforts include hunting, poisoning, and predator introduction, all of which have substantial impacts on animal welfare of the target species. companion bird species are subject to various diseases related to reproductive dysfunction. these diseases range from behavior manifestations of aggression and feather picking to difficulties in egg production, neoplasia and cloacal prolapse. current therapies can be cost limiting, or in surgical cases, life threatening, creating a need for noninvasive sterilization. effects of injecting domestic chickens with high-dose antibody-guided lipid nanocomplex (lnp) carrying the cytotoxic agent saporin were studied. antibodies for anti-müllerian hormone ii receptors were used to target theca and granulosa cells to stop hormone production and ultimately egg production. a previous study’s use of 100 nmol solution yielded egg laying cessation for 16 days whereas this project increased this dose to 1000 nmol.1 we hypothesized that increasing the dose would extend the period of egg laying cessation. rhode island red laying hens (n = 6) formed a control group and test group of 3 hens each. test group was given lnp intramuscular injections whereas the control group was given an intramuscular injection of saline equal to the lnp volume (1.4 ml). egg laying was monitored for 30 days. hens were euthanized, and necropsied. oviduct and ovaries were removed weighed and photographed. reproductive tracts were placed in formalin and histological sectioning of ovaries were made and stained with hematoxylin and eosin. follicles of each slide were evaluated for theca and granulosa cell layers. follicles between 1 2 mm were counted on each slide and rated on a scale from 1 to 5 based on granulosa cell architecture and organization (1 = healthy, uniform layer; 5 = complete lack of granulosa layer present). hens injected with lnp stopped egg laying for a minimum of 23 days; 1 control hen stopped laying for 26 days. on histology, all chickens had follicles present with a viable granulosa layer. follicle count between 1 2 mm in size varied between 8 17 follicles per slide. all control chickens had at least 2 follicles within this size range with a granulosa rating of 1/5 indicating intact and viable granulosa layer. test group had no follicles within this size range with a viable granulosa layer (1/5 rating). average follicle rating for the control group ranged between 2.5/5 to 2.9/5 (sd 1.28 2.89), and average follicle rating for the test group was 4/5 (sd 0.57 0.72). lack of statistical power was attributed to small sample size. these results reflect that increased lnp dose could potentially lead to long cessation of egg laying. future studies with larger test groups are needed to determine if granulosa cellular architectural changes are significantly different after lnp treatment. chicken monoclonal antibodies should be investigated for increased efficacy over the commercial mammalian sourced antibodies. keywords: gonadal suppression, fertility, avian egg laying, granulosa cell, nanocomplex, saporin-6 reference 1. ayres s, graham j, li,y, et al: a novel method to reduce egg laying in companion avian species using a hen model. clinical theriogenology 2020;12:356. anti-müllerian hormone and inhibin b concentrations in peripubertal beef bulls before and after castration giorgia podico,a edgar garrett,a parker henley,b daniel shike,b alan conley,c igor canissoa adepartment of veterinary clinical medicine, bdepartment of animal sciences university of illinois at urbana-champaign, urbana, il cdepartment of population health & reproduction, university of california, davis, ca anti-müllerian hormone (amh) and inhibin b have roles in the prenatal testicular differentiation and postnatal testicular development leading to puberty. also, the presence of inhibin b has yet to be confirmed in the peripheral blood of bulls. in numerous species, amh has been used as a sertoli cell biomarker to evaluate testicular function without the need of a stimulation test. the present study aimed to assess reproductive hormones of peripubertal beef bulls before and after castration and to determine the relation of them with semen quality parameters. we hypothesized that both amh’s and inhibin b’s peak coincide with puberty and that their level positively correlates with testicular function and semen quality. spring-born angus × simmental bulls (n = 10) were enrolled starting their first fall. bulls had semen and blood collected once a month for 8 times and then they were surgically castrated from 3 days after the last collection. serum was harvested and stored until analyses. semen was collected via electroejaculation; volume, concentration, total sperm, total and progressive motility were assessed to determine the onset of puberty. scrotal circumference was determined. puberty was defined when at least 50 x 106 sperm with at least 10% motility was present in the ejaculate. bulls had amh and inhibin b measured at each timepoint with immunoassays. in addition, inhibin b and amh were measured right before and after castration. data were normalized by the onset of puberty and analyzed with r. a mixed model, pearson’s correlation, and clinical theriogenology 2022; 14: 276 tukey’s as post hoc were used. significance was set at p < 0.05. a strong correlation was defined by r > 0.7, a moderate by 0.3 < r < 0.7, and a weak one by r < 0.3. bulls reached puberty at 11 ± 0.3 months of age. amh and inhibin b concentrations were significantly affected by time. prepubertal amh concentrations were significantly higher than postpubertal concentrations at every timepoint. amh concentrations at puberty were 902.3 ± 484.2 pg/ml. two pre-pubertal peaks of amh concentrations were noted at 5 (3,385 ± 733 pg/ml) and at 3 (2545 ± 603.9 pg/ml) months before puberty; conversely, the lowest amh concentrations were at 6 months after puberty (529.3 ± 720 pg/ml). inhibin b peaked 5 months before puberty (74.1 ± 11.9 ng/ml), and again 3 and 5 months after puberty (79.8 ± 9.7 ng/ml,78.4 ± 11.4 ng/ml). the concentrations of inhibin b decreased after castration (before 49.3 ± 6.9 ng/ml, after 6.7 ± 4.9 ng/ml) (p < 0.05). concentrations of amh were 575.1 ± 42.8 pg/ml before castration and were undetectable after castration. there was a moderate positive correlation between inhibin and total motility (r = 0.41) and progressive motility (r = 0.38). in conclusion, puberty did not coincide with peak of amh nor inhibin b; further studies are needed to examine the value of inhibin b as marker for bull fertility. keywords: endocrinology, testicular function, blood marker effects of ram seminal plasma on corpus luteum function and pregnancy outcomes of ewes jamie stewart,a nicole sugai,a kevin pelzer,a vitor mercadante,ab scott greiner,b alan ealyb adeptartment of large animal clinical science, va-md college of vet med, blacksburg, va bdepartment of animal and poultry sciences, virginia tech, blacksburg, va in us, profitability within the sheep industry relies on improvement of reproductive technologies to allow for dissemination of superior genetics. laparoscopic artificial insemination (lai) with frozen semen is the most efficient way to improve flock genetics; however, there is a need to improve estrus synchronization protocols. role of seminal plasma proteins is of interest in synchronization programs, given that nerve growth factor-beta influences ovulation dynamics in camelids and cattle. objective was to determine if there are factors present in ram seminal plasma that can improve pregnancy outcomes in ewes bred by lai. we hypothesized that systemic treatment of ram seminal plasma at lai improves corpus luteum (cl) function and pregnancy outcomes. semen was collected and pooled from 10 rams, then centrifuged to remove sperm and debris. ampicillin was added to both seminal plasma and pbs injections to equal a final concentration of 41 mg/ewe. crossbred ewes (n = 36) were synchronized using an intravaginal progestin device (cidr) that was removed after 14 days. a 2 ml dose of prostaglandin (lutalyse) was given intramuscularly at the time of cidr removal. at 48 hours after cidr removal, ewes were assigned to 1 of 3 treatment groups: sp (n = 12): ewes that received 2 ml intramuscular injection of seminal plasma at the time of lai; neg (n = 12): ewes that received a 2 ml intramuscular injection of pbs at the time of lai; and pos (n = 12): ewes that were turned out with a ram. ewes undergoing lai received 0.25 ml frozen ram semen deposited laparoscopically in both uterine horns using an aspic. blood was collected before and ~ 4 8 hours after lai or ram breeding for quantification of estradiol, luteinizing hormone, and nerve growth factor-beta. rams were removed from the pos group after 3 days. subsequent blood collections were performed every 4 days until day 32 to assess serum progesterone concentrations (days 4 20) and pregnancy-specific protein b (days 24, 28, and 32). whole blood was flash frozen in trizol on days 16 and 20 to quantify expression of interferon-stimulated genes in peripheral blood leukocytes. transrectal ultrasonography was performed on days 8, 12, and 16 to measure the maximum cl diameter. transabdominal ultrasonography was performed on day 52 to confirm pregnancy status. data were analyzed using anova (cl diameter) and a wald test (pregnancy outcome) in r. blood hormone and gene expression data are pending. the maximum cl diameter did not differ (p = 0.21) amongst treatments nor were there any effects of day (p = 0.24) or treatment × day interactions (p = 0.67). pregnancy rates did not differ (p = 1) between sp (33%; 4/12) and pos (33%; 4/12), but tended to be higher (p = 0.07) than neg ewes (9%; 1/11). these preliminary data suggested that factors in the seminal plasma may be beneficial for improving pregnancy outcomes in ewes undergoing lai; however, further work is needed to determine large-scale implications. keywords: ewes, breeding, ram seminal plasma, synchronization feeding spent hemp biomass does not affect testis weight, seminiferous tubule diameter or dazl immunoexpression in rams melanie meador, niamh nolan, serkan ates, massimo bionaz, michelle kutzler deptartment of animal and rangeland sciences, oregon state university, corvallis, or spent hemp biomass (shb) is a byproduct following cannabidiol extraction of hemp. male mice fed cannabidiol for 34 days had a 30% reduction in fertility rate and a 23% reduction in litter size.1 objective was to determine the effects of feeding shb on fertility in rams. we hypothesized that feeding shb to rams reduces testis mass and seminiferous tubule diameter. additionally, we hypothesized that the expression of the spermatogenesis-associated protein dazl (deleted in azoospermia-like) is reduced following feeding shb. a feeding trial was performed with 6-month polypay rams. rams were randomly assigned to 5 feeding trial groups (7 per group) and fed either alfalfa (control) or shb at 10 or 20% of their total diet for 4 weeks, with 4 weeks withdrawal from shb, or shb at 10 or 20% for 8 weeks. at the conclusion of the feeding trial, rams were euthanized via penetrating captive-bolt. the testes were removed, weighed, and then a 0.5 cm3 from each testis was fixed in 10% buffered formalin and paraffin embedded. serial sections were made from each testis and mounted onto charged slides before routine immunohistochemistry was performed using a rabbit anti-dazl antibody (1:500; bioss, boston, ma) or a universal negative control antibody. an adjacent tissue section was stained with hematoxylin and eosin for histomorphometry. images of all slides were captured using a leica dm4000b microscope and leica dfc295 camclinical theriogenology 2022; 14: 277 era. diameters were measured from 5 representative seminiferous tubule cross-sections per testis. immunoexpression of dazl was determined using quantitative image analysis (nih image j software). a one-way anova was used to compare variables between groups and significance was defined as p < 0.05. compared to controls, feeding shb to rams did not significantly affect total testicular weight, seminiferous tubule diameter, or dazl immunoexpression. based on these results, we concluded that shb can be used as a safe feed source for rams without negatively affecting their fertility. keywords: cannabidiol, fertility, immunohistochemistry, sheep reference 1. carvalho rk, souza mr, santos ml, et al: chronic cannabidiol exposure promotes functional impairment in sexual behavior and fertility of male mice. reprod toxicol 2018;81:34-40. measuring intracellular ice from freezing and ice growth during warming patrick crane,a abdallah abdelhady,a luis henrique de aguiar,a mariana diel de amorim,a robert thorne,b soon hon cheonga adepartment of clinical science, college of veterinary medicine bdepartment of physics, cornell university, ithaca, ny survival of oocyte and embryos postthaw depends on the amount of cryodamage incurred during the freezing and thawing process. intracellular ice formation is a critical cause of cryodamage and its growth is exponentially greater during warming than during cooling, but measuring intracellular ice during thawing is challenging. our objective was to determine if synchrotron x-ray diffraction is able to accurately measure intracellular ice formation during freezing and if time-resolved x-ray diffraction can measure ice growth during thawing. purpose of this proof-of-concept study is to test our hypothesis that properly vitrified oocytes and embryos have little intracellular ice from freezing but some ice growth will occur during thawing even in well-vitrified samples. bovine oocytes and embryos were incubated in standard vitrification media containing 15% ethylene glycol, 15% dimethylsulfoxide, and 0.5 m sucrose or in diluted vitrification media, mounted on specialized cryoloops designed for synchrotron x-ray diffraction, and plunged in liquid nitrogen using the nanuq™ cryopreservation system (mitegen). vitrified samples were placed in a cryostream of liquid nitrogen vapor at t = 100 k and the amount of intracellular ice present was detected by measuring diffracted synchrotron x-rays. to measure ice growth during thawing, the nitrogen cryostream was blocked and simultaneously replaced by a flow of warm room temperature gas directed at the sample. x-ray diffraction 'movies' were acquired with 0.05 s frames starting 1 s before thawing. no ice was observed in vitrified oocytes and embryos mounted on nanuq™ cryoloops, with minimal excess surrounding liquid when standard vitrification media was used. however, when diluted vitrification media was used, ice was first observed at 40% strength of the original concentration. time-resolved x-ray diffraction data showed ice nucleation and growth during warming even when using standard vitrification media, with warming rates of approximately 500 k/s. the maximum ice diffraction intensity and maximum ice grain size, which are likely correlated with degree of cellular damage, increase with decreasing vitrification media concentration and increasing amount of excess liquid around the sample. the nanuq™ cryopreservation system used with cryoloops delivers cooling rates of ~ 600,000°c/min, approximately 20 x faster than the current fastest cooling systems. this ultra-fast cooling rate allows cryoprotectant concentration to potentially be reduced to 50% strength without ice formation on cooling, and reduces ice formation during warming. in conclusion, time-resolved x-ray diffraction is a sensitive and quantitative method for measuring ice growth during warming, when the largest intracellular ice fractions and grain sizes were detected. this method may be used to correlate maximum ice fractions with biologically relevant metrics of cryodamage. keywords: cattle, vitrification, intracellular ice, synchrotron, oocyte, embryo manipulating cumulus-oocyte-complexes and embryos using microfluidics soon hon cheong,a sarah kenney,a julia o’connor,a elizabeth rhoadesb,c adepartment of clinical science, bdepartment of microbiology and immunology college of veterinary medicine, cornell university, ithaca, ny ccornell nanoscale facility, cornell university, ithaca, ny oocytes and embryos experience very different physical conditions in vivo compared to the conventional in vitro production system of embryos. microfluidics is a powerful tool to manipulate cells, including gametes, which involves the manipulation of fluids in micro-channels. the fluidic flow and physical environment in microfluidic devices are much more representative of in vivo conditions and may improve outcomes and allow automation of in vitro fertilization. completing in vitro embryo production in microfluidics requires many steps which introduce challenges in controlling the fluidic flow. objective of this proof-of-concept study is to determine if controlling outflow and inflow can manipulate oocytes and embryos including positioning of the oocytes for intracytoplasmic sperm injection in-a-chip. we hypothesized that regulating outflow channels using valves and inflow channels using syringes and fluidic pumps results in successful production of embryos in a chip. microfluidic channel patterns were created using su-8 negative photoresist on 100 mm silicon wafer exposed to ultraviolet light, then the unexposed portion was removed using su-8 developer. microfluidic devices were produced by pouring polydimethysiloxane (pdms) on the wafer and bonding the pdms device onto glass slides. fluidic ports were created using coring needles. bovine cumulus-oocyte-complexes (cocs) were inserted into the device and the insertion port sealed using pipette tips sealed with pdms. the cocs were moved to a maturation chamber of the device where maturation media was continuously flowed at 20 µl/minute for 22 hours. the cocs were denuded by using holding media with hyaluronidase passed through narrowing clinical theriogenology 2022; 14: 278 channels with side channels to hold the cumulus cells. this process was inefficient and most oocytes were not completely denuded. the denuded oocytes were held in a holding chamber for intracytoplasmic sperm injection (icsi) which was backloaded and the sperm immobilized using xyrcos laser before injection. injection was very inefficient with a steep learning curve. after injection, the putative zygote was moved to a culture chamber with continuous flow of embryo culture media (20 µl/minute). embryos were observed daily for development and proper running of the microfluidic device. embryo development rate was very low with a single blastocyst formation after 200 injections. manipulation of cocs and embryos within the microfluidic device was challenging and there was significant risk of bubble formation and contamination during the prolonged culture. additionally, icsi was challenging due to the oocytes not being held by holding pipettes but rather nested in an injection cradle. future modifications are required to increase optimization of the device and process. keywords: microfluidics, intracytoplasmic-sperm injection, oocyte, embryo clinical theriogenology 2022; 14: 279 identification of candidate proteins associated with impaired acrosomal exocytosis in thoroughbred stallions using data-independent acquisition mass spectrometry camilo hernández-avilés,a susan weintraub,b luisa ramírez-agámez,a charles scoggin,c brian davis,d terje raudsepp,d dickson varner,a charles lovea adepartment of large animal clinical sciences, dveterinary integrative biosciences college of veterinary medicine and biomedical sciences texas a&m university, college station, tx bdepartment of biochemistry and structural biology university of texas health science center, san antonio, tx cleblanc reproduction center, rood & riddle equine hospital, lexington, ky thoroughbred (tb) stallions that carry the linked susceptibility genotypes a/a-a/a in exon 5 of the fkbp6 gene (eca13; equcab 3.0) are uniquely subfertile because they exhibit normal-to-excellent conventional sperm quality parameters. however, when exposed to known inducers of acrosomal exocytosis (ae), sperm from affected stallions undergo ae at a lower rate than sperm from fertile stallions. in affected stallions, a higher cholesterol-to-phospholipid ratio in sperm membranes has been reported compared to sperm membranes from fertile stallions. a clear causation of the fkbp6 exon5 genotypes and impaired acrosomal exocytosis (iae) is still unknown. sperm proteome in frozen/thawed semen from 3 fertile tb stallions (fkbp6 genotypes = g/g-a/a, a/ga/a, and g/g-a/c, respectively; n = 3) and 3 subfertile tb stallions (fkbp6 genotype = a/a-a/a; n = 3) were studied using mass spectrometry. sperm were incubated for up to 6 hours under conditions (reported before) to induce spontaneous ae in viable sperm (ae-viable). at hours 0, 2, 4, and 6, sperm aliquots were analyzed for ae using flow cytometry (fitc-psa and fixable live/dead red stain), and the sperm proteome was analyzed via data-independent acquisition mass spectrometry (dia-ms). student’s t-tests, two-way anova with benjamini-hochberg multiple testing correction (fdr q-value 0.05) were used to determine differences between experimental groups in ae-viable and protein relative abundance. at hours 4 and 6 of incubation, the mean ae-viable was higher (p < 0.05) in fertile than subfertile stallions (hour 4 = 41 versus 14%; hour 6 = 44 versus 16%, respectively;). a total of 2,220 proteins was identified by dia-ms. using strict selection criteria (fdr 1.0%, q-value < 0.05, and log2fold > 0.584 or < -0.584 [e.g., fold change in protein abundance between fertile and subfertile stallions > 1.5]), 140 proteins were differentially abundant in sperm from subfertile stallions compared to fertile stallions (83 less and 57 more abundant). principal component analysis indicated that these proteins explained 65% of the difference in the sperm proteome between stallion groups. analysis using the reactome database and homo sapiens orthologs revealed that the proteins of lower abundance in sperm from the subfertile stallions were mostly overrepresented in the metabolism (32 proteins) and the ‘metabolism of lipids’ (18 proteins) pathways. of interest, 2 proteins were common between these pathways, arsf (arylsulfatase f; fold change 4.02; p < 0.05) and zpbp (zona pellucida-binding protein; fold change 3.36; p < 0.05), are acrosomal enzymes with fundamental roles during sperm-oocyte binding by interacting with proteins in the zona pellucida, including zp3. surprisingly, none of the proteins was differentially abundant between fertile and subfertile stallions appeared to have a relationship with the protein encoded by the fkbp6 gene. these results indicated that dia-ms is a powerful tool to identify candidate proteins that contribute to etiology of iae in tb stallions. these proteins might also be useful to help understand the fertilization process in horses. keywords: stallion sperm, impaired acrosomal exocytosis, proteomics, mass spectrometry, cholesterol relationship between routine dental care and pregnancy loss in thoroughbred broodmares dale kelley,a riley maddux,b anna houp,b brad tannerb adepartment of veterinary clinical sciences, college of veterinary medicine oklahoma state university, stillwater, ok brood & riddle equine hospital, lexington, ky there is a concern among some veterinarians that routine dental care (reducing long cheek teeth) during an equine pregnancy could release bacteria into the bloodstream resulting in pregnancy loss. data regarding dental procedures, in women, has mixed findings; no effect and to beneficial effect on pregnancy outcomes. objective was to determine if a relationship existed between routine dental care (either prior to or during pregnancy) and pregnancy loss in thoroughbred broodmares. records from an equine hospital and 3 well-managed thoroughbred breeding farms located in central kentucky between january 1, 2013 and january 1, 2021 were used. data set contained 253 mares representing 414 routine dental procedures (reduction of long cheek teeth) and pregnancies. information collected from clinical and farm records consisted of mare identification, mare age at dental procedure, farm, date of the dental procedure, date of last breeding, mare’s abortion status, abortion date, and foaling date. dental proceequine session clinical theriogenology 2022; 14: 280 dure time was categorized as having occurred either when the mares were nonpregnant, during 1st trimester (0 114 days), 2nd trimester (115 228 days) or 3rd trimester (> 229 days) of pregnancy. sas (9.4) freq procedure was used to analyze the frequency of time of dental care, abortions by trimester, and by farm. abortion rate was modeled using sas glimmix procedure using a binary distribution with the time of dental procedure occurring, farm, year of dental procedure and age in the model. a random statement was used with mare as the subject to account for repeated observations of mares within the data set. significance was set at p < 0.05. dental care was performed 58 times with nonpregnant mares, 86 times during the 1st trimester, 74 times during the 2nd trimester and 196 times during the 3rd trimester. abortion rate based on the time dental care was performed was 8.6% for nonpregnant mares, 2.3% for during the 1st trimester, 4.1% during the 2nd trimester and 0% during the 3rd trimester. percent abortion rate for each farm was 1.2, 1.0, and 0.2 respectively. as per regression model there was no effect of trimester (p = 0.511), farm (p = 0.623) or year (p = 0.896); however, there was an effect (p = 0.027) of age. an odds ratio for age was calculated as 1.126 (ci: 1.014 1.251), indicating for each 1-year increase in age, mares were 1.126 times more likely to abort. results suggested that routine dental care to broodmares given any time or during pregnancy was a safe procedure at any time and did not increase the risk of pregnancy loss. keywords: mare, pregnancy, float, dentistry, abortion evaluation of corpus luteum function following a single dose of a proprietary slow-release oxytocin formulation in mid-diestrus in the mare holly clement,a dirk vanderwall,a stephen kirschner,b shawn zimmerman,a ian sroufe,a matthew barnesa adepartment of animal, dairy and veterinary sciences school of veterinary medicine, utah state university, logan, ut bwildlife pharmaceuticals, inc, r & d department, windsor, co need for minimally invasive, cost effective and reversible estrus suppression in the mare has a long-standing history in the equine industry. these objectives have been met utilizing oxytocin to prolong corpus luteum (cl) function. in ≥ 70% of treated mares cl function was prolonged with 60 units of intramuscular oxytocin treatment once daily on days 7 14 postovulation. need for 8 daily injections and reproductive examinations during estrus are drawbacks to widespread clinical application of this protocol. to address these disadvantages, a proprietary slow-release oxytocin (sr-ot) formulation was used with the aim to identify a single dose at an optimum day in mid-diestrus to induce prolonged cl function. it has been established1 that 1.0 ml (2,400 iu oxytocin) of sr-ot given intramuscularly on days 7 and 10 postovulation induced prolonged cl function in 75% of treated mares. this represents a 75% reduction in the number of oxytocin treatments required to induce prolonged cl function. current work aimed to further reduce sr-ot to a single treatment, reduce the frequency of transrectal palpation and ultrasonographic examinations to every other day and achieve ≥ 70% prolonged cl function in treated mares. we hypothesized that a single 4,800 iu dose of sr-ot provides a sufficient dose and duration of delivery such that if given on day 8 or 9 postovulation prolonged cl function would be achieved in ≥ 70% of treated mares. mares in estrus were examined via transrectal palpation and ultrasonographic examination every other day to determine the day of ovulation (defined as day 0). jugular blood samples were collected on day 0, monday, wednesday, and friday through day 40 to determine serum progesterone concentrations. mares (n = 16) were evaluated and randomly assigned to 1 of 2 groups, with 8 mares receiving no treatment (control) and 8 mares receiving a single intramuscular dose of sr-ot (4,800 iu oxytocin) on day 8 or 9 (treated). given that mares were examined every other day, treatment day may have been day 9 for some mares. the proportion of mares in each group with prolonged cl function was compared using fisher’s exact test (graphpad software, inc., san diego, ca). a probability of p < 0.05 was considered significant. mares were classified as having prolonged cl function if their serum progesterone concentrations remained > 1.0 ng/ml throughout the sampling period. corpus luteum function was prolonged (p > 0.10) in 1/8 (12.5%) of untreated control mares and 4/8 (50%) of treated mares. in conclusion, a single intramuscular dose of 4,800 iu sr-ot on day 8/9 failed to prolong cl function in ≥ 70% of treated mares. small sample size limited the ability to find a significant difference in the proportion of mares with prolonged cl. keywords: mare, estrus suppression, oxytocin, corpus luteum, prolonged corpus luteum function reference 1. sarnecky ba, vanderwall dk, mason hm et al: evaluation of a proprietary slow-release oxytocin formulation on corpus luteum function in mares. j equine vet sci 2019;77:28-30. seminal microbiome and reproductive outcomes of louisiana thoroughbred stallions fiona herzog,a kalie beckers,a sadie thompson,b tabitha kearns,b jerold murrell,c gary king,b jenny sonesa adepartment of veterinary clinical science, school of veterinary medicine louisiana state university baton rouge, la bdepartment of biological sciences, louisiana state university, baton rouge, la cclear creek stud farm, folsom, la microbiome of semen has yet to be extensively investigated in mammals. meta-analysis of seminal microbiome studies in humans has identified associations with bacterial genera and semen quality. prevotella increased in reduced sperm motility subjects whereas lactobacillus was associated with normal sperm morphology. although this analysis was not able to determine the effects of seminal microbiota on human fertility, in a bull study increases in lawsonella were correlated with reduced fertility. few reports have described the presence of a seminal microbiome in normal stallions in europe; however, reproductive outcomes were not reported. evidence suggested that the microbiome of reproductive body sites can vary depending on environmental and host factors. uterine microbiclinical theriogenology 2022; 14: 281 ome composition in mares, is influenced by the geographical location and estrous cycle stage. our purpose was to describe the seminal microbiome of healthy fertile stallions housed on a commercial stud farm in louisiana. semen was collected in january during a routine preseason breeding soundness examination. we hypothesized that the microbiome composition of stallion semen during their prebreeding soundness examination is correlated with stallion fertility. ejaculates from 5 thoroughbred stallions were collected into an aseptically prepared missouri artificial vagina prior to the 2018 breeding season. microbial dna was extracted from semen and the v4 variable region of 16s rrna gene was used for dna amplification and identification. the selected stallions, mean age 14.4 years (range 10 19), were determined to be in good health and satisfactory breeding potential during an annual breeding soundness examination. no pathogenic microorganisms were identified on aerobic culture of the semen or reproductive tract. parameters of semen quality were determined to be adequate based on total motility: mean 83.75% (range: 70 95), progressive motility: mean 74.38% (range: 60 85), viability: mean 76.23% (range: 65 84.3), morphology: mean 63% normal (range: 53 87), concentration: mean 182.63 x 106/ml (range: 55.6 – 451 x 106/ml), total sperm per ejaculate: mean: 10.55 x 109 (range: 5.24 16.9 x109). stallion fertility for the 2018 breeding season was determined to be sufficient based on seasonal pregnancy rates as determined by day 14 postovulation transrectal ultrasonography: stallion 1, 43/47 (91.49%), stallion 2, 34/38 (89.47%), stallion 3, 50/55 (90.90%), stallion 4, 5/5 (100%), and stallion 5, 5/5 (100%). the semen microbiota had a high diversity (shannon index) and low richness (chao index) and was dominated by 2 bacterial classes, namely bacteroidia and clostridia. in conclusion, our results suggested that equine semen dominated by bacteroidia and clostridia in january could reflect a healthy seminal microbiome for stallions in louisiana. further studies are required to elucidate environmental and host factors that may influence the microbiome of stallion semen, the implications for potential future fertility and clinical value as part of the breeding soundness examination. keywords: stallion, microbiome, fertility, semen effect of freezing extender and cryoprotectant on blastocyst production after intracytoplasmic sperm injection luisa ramírez-agámez,a camilo hernández-avilés,a dale kelley,b charles lovea acollege of veterinary medicine and biomedical sciences texas a&m university, college station, tx bcollege of veterinary medicine, oklahoma state university, stillwater, ok intracytoplasmic sperm injection (icsi) using frozen/thawed sperm is a common procedure to obtain embryos from either fertile or subfertile mares and stallions. stallion-associated factors that impact the efficiency of icsi have been less studied than mare-associated factors. semen extenders for freezing stallion sperm vary in their composition, particularly egg-yolk concentration, presence of milk products, and penetrating cryoprotectant-type. for some stallions, dimethylformamide-containing extenders, irrespective of the egg-yolk concentration or presence of milk products in the base freezing extender, are reported to result in higher postthaw motility and in vivo fertility than glycerol-containing extenders. to date, no studies have determined effects of these extender components on embryonic development after icsi. current experiment determined the effect of 4 commercially available semen extenders containing either glycerol (le or mfr5) or a combination of glycerol and dimethylformamide (cmle or cmmfr5) on the blastocyst rates resulting after icsi. both le and cmle extenders are formulated with 20% egg-yolk (v:v), whereas mfr5 and cmmfr5 extenders are formulated with milk and 4% egg-yolk (v:v). immature oocytes were recovered via transvaginal, ultrasound-guided follicle aspiration and matured in vitro. after maturation, oocytes were injected by piezo-driven icsi using sperm from each 1 of 3 fertile stallions that was frozen using each 1 of 4 freezing extenders. sperm quality parameters: % total motility (tmot), % viability, and sperm % dna damage (compα-t) were determined immediately after thawing (3 ejaculates per stallion, n = 9). after injection, the presumptive zygotes were evaluated for cleavage on day 5 and for blastocyst formation on days 7 10. effects of semen freezing extender, stallion, and their interaction on postthaw semen quality and blastocyst rates after icsi were analyzed using the general linear model and chi-square analysis (jmp pro 16.0). significance was set at p < 0.05. mean tmot was higher (p < 0.05) in cmle and le than in mfr5 or cmmfr5 extenders (52 and 49% versus 39 and 38%, respectively). mean viability and compα-t were not affected (p > 0.05) by the extender (range: 50 56% and 9 14%). an extender-by-stallion interaction for blastocyst rate was not detected (p > 0.05). extender mfr5 resulted in a higher (p < 0.05) blastocyst rate (embryos/injected oocytes) than extenders le, cmmfr5, or cmle (26% [21/82] versus 10% [8/77] versus 5% [4/82] versus 5% [4/80], respectively). extenders with higher egg-yolk content (cmle, le) had higher sperm quality than extenders with lower egg-yolk content and milk (cmmfr5, mfr5). however, extender mfr5 had the highest blastocyst rate after icsi, suggesting an interaction among the freezing extender components (i.e., low egg-yolk quantity, milk proteins, and glycerol) and embryonic development after icsi. results also indicated that the presence of dimethylformamide in the freezing extender, irrespective of egg-yolk concentrations or the presence of milk proteins, negatively affected the in vitro development of equine embryos. keywords: mare, intracytoplasmic sperm injection, frozen semen, extender, blastocyst antimicrobial activity of platelet-rich plasma against common microorganisms causing endometritis in mares mariana mazzuchini,ab lorenzo segabinazzi,ac marco alvarenga,a igor canissob adepartment of animal reproduction, são paulo state university (unesp), botucatu, brazil bdepartment of veterinary clinical medicine, university of illinois urbana champaign, il cross university, basseterre, st. kitts, west indies clinical theriogenology 2022; 14: 282 ever evolving microbial resistance and tightening regulations against the indiscriminate use of antimicrobials warrant the development of alternative therapies to treat endometritis in mares. platelet-rich plasma (prp) has emerged as an alternative therapy to modulate persistent breeding endometritis, due to its antiinflammatory and potential antimicrobial properties. however, limited work has been done to assess the antimicrobial properties of prp against microorganisms causing endometritis in mares. therefore, this study aimed to assess in vitro antimicrobial activity of prp against microorganisms causing endometritis in mares. we hypothesized that prp antimicrobial activity is microorganism-dependent. percentage of inhibition (pi) and minimum inhibitory concentrations (mic) of prp were assessed with the microdilution broth method using 96-well flat-bottom microplates. clinical isolates of streptococcus equi (strep), escherichia coli (e. coli), pseudomonas aeruginosa (pseudo), klebsiella pneumoniae (kleb), staphylococcus aureus (staph), and candida albicans (candida) cultured from mares with clinical endometritis were used. blood samples were collected from 6 mares (3/mare) and double centrifuged to produce prp. each prp sample was run 3 times, and within each run, 3 wells were used per microorganism. first 3 lanes (1 3) of each plate served as the negative controls that consisted of 100 µl prp (1056 ± 198 ×103 platelets/μl) and 100 µl of mueller-hinton (mh) (1:1) added to10 μl calcium chloride 10%. thereafter, the remaining lanes (4 -12) contained serial dilutions of prp as 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128. row h (lanes 4 -12) served as positive controls and contained no prp. then, 5 μl (5 ×104 colony-forming unit) of each isolate was added to lanes 4 12. then plates were incubated at 37°c for 24 hours. optical density of the wells was measured at 570 nm using a spectramax m2 spectrophotometer (molecular devices, san jose, ca). the pi was calculated by the difference between the mean of the triplicates in each group and the mean of the negative control and the positive control. to determine mic, 10 μl of resazurin solution (0.01%) was added to each well, and the plates were kept at 37°c for 1 hour. subsequently, a visual reading was performed to determine microorganism activity; if the well became pink, microbial activity was present, whereas no microbial activity was present if the well remained blue. data were analyzed by anova and tukey’s post hoc test. significance was set at p < 0.05. platelet-rich plasma had a greater inhibition (p < 0.05) compared to the control up to titers of 1:4 (candida); 1:16 (staph); 1:32 (e. coli); and 1:128 (strep; pseudo; and kleb). all prp samples inactivated e. coli in the mic assay at 1:2, whereas pseudo was inactivated in 33% of the samples at 1:2. the staph was inactivated only in 16% of the samples treated with prp. the remaining microorganisms (strep, kleb, and candida) were not inactivated. in conclusion, we demonstrated that prp antimicrobial activity in vitro was doseand microorganism-dependent. the findings hint that greater platelet counts could result in superior clinical efficacy and that not all microorganisms are susceptible to prp. keywords: equine endometritis, antibiotic, platelet-rich plasma, subfertility pentoxifylline reduces postbreeding uterine fluid accumulation in older embryo donor mares and may enhance embryo recovery odilon oliveira,a gabrielle amorim,a lorenzo segabinazzi,a,b igor canissoc adepartment of animal reproduction, são paulo state university (unesp), botucatu, brazil bdepartment of clinical veterinary medicine, university of illinois, urbana, il cross university school of veterinary medicine, basseterre, st. kitts, west indies most donor mares in embryo transfer programs have impaired uterine clearance due to uterine and cervical problems. additionally, repeated breeding cycles, reproductive procedures, and embryo flushing make embryo donors prone to endometritis. anecdotally, pentoxifylline, an antiinflammatory and rheolytic drug, has been used in mares to enhance blood flow in the reproductive tract and reduce uterine inflammation. however, this has not been critically studied. this study assessed the postbreeding inflammatory response and embryo recovery of subfertile donor mares treated with pentoxifylline. we hypothesized that pentoxifylline supplementation mitigates the postbreeding inflammatory response in embryo donor mares. estrous cycles (n = 42) of 7 embryo donor mares (age 16 ± 3.7 years; 13 ± 3 years of donating embryos) were studied. during first 2 cycles, mares received pentoxifylline (17 mg/kg/day) in 50 ml of broodmare-plus® (botupharma). the 2 subsequent cycles served as washout (no supplementation), and then in the last 2 cycles, mares received 50 ml of broodmare®. mares were monitored daily, and then once a preovulatory follicle was detected, ovulation was induced with intramuscular deslorelin (1 mg). all mares were bred with fresh extended semen (1 x 106) from the same stallion. immediately before (0), 24, 48, and 72 hours postbreeding, mares had transrectal ultrasonography performed to determine ovulation, quantify uterine edema, and intrauterine fluid accumulation (iuf). at similar timepoints, uterine cytology sample was collected, stained with diff-quik®, and polymorphous nuclear cells were counted in 10 high power fields. embryo collections were performed 8 days postovulation, and mares received an intramuscular injection of 5 mg of dinoprost (lutalyse®, zoetis, parasipanny, nj) to return to estrus. all mares had at least 1 ovulation confirmed before the onset of the study, and then each mare started in a new group 8 days postovulation. data were analyzed with graph prisma (san diego, ca). neutrophil counts were analyzed with anova repeated measures and tukey’s as post-hoc. edema scores and intrauterine fluid accumulation were analyzed with kruskal-wallis and dunn’s post-hoc. embryo recovery rates were evaluated with fisher’s test. there was an effect (p = 0.032) of time for neutrophil counts in the washout cycle but not (p > 0.05) in the other 2 groups. neutrophil counts did not differ (p > 0.05) across groups. edema scores had effects (p = 0.046) of time but no effects (p > 0.05) of group. similarly, there was an effect (p = 0.038) of time for iuf, and mares receiving pentoxifylline had reduced (p = 0.034) postbreeding iuf compared to washout cycles but not different (p > 0.05) than the group receiving just broodmare®. embryo recovery was not different (p > 0.05) across groups (but remarkably difclinical theriogenology 2022; 14: 283 ferent as follows: washout cycle was 29% (4/14); broodmare® was 36% (5/14), and pentoxifylline (broodmare-plus®) was 57% (8/14). mares in the study were subfertile older mares used as embryo donors for multiple years, thus, justifying the low embryo recovery for the cycles with no pentoxifylline. in conclusion, supplementation with pentoxifylline may be an alternative to improve the postbreeding uterine clearance. it may enhance embryo recovery in mares serving as donors for multiple years; however, this remains to be confirmed with a larger number of mares. keywords: donor mares, endometritis, uterine fluid, inflammation, fertility a retrospective study of the impact of transrectal palpation on teaching mares sheila megehee,a candace lyman,b reed holyoaka adepartment of veterinary clinical sciences, college of veterinary medicine oklahoma state university, stillwater, ok bdepartment of clinical sciences, college of veterinary medicine auburn university, auburn, al transrectal palpation (trpalp) in mares is considered a potentially life-threatening, noxious procedure, particularly when performed by inexperienced individuals. such a risk may prompt teaching institutions to consider replacing live animal laboratories with model simulators. however, removal of live animals from the veterinary curriculum would significantly minimize the amount of real-life experiential education provided to veterinary students. objectives were 3 fold: i. identify the incidence of mare injury associated with trpalp in teaching mares, ii. identify the number of intravenous sedation utilized to circumvent averse behaviors (i.e., behaviors interpreted as ‘dislike’), and iii. identify the duration of individual mares’ careers/cited reasons for retirement. we predicted that the incidence of mare injury from trpalp will be low; additionally, we hypothesized that averse behaviors during trpalp are not a commonly cited reason for mare retirement. transrectal palpation records (2019 2021) of 48 mares (age 4 26 years) were evaluated. analyzed data were collected during years 2 and 3 laboratory courses, senior student clinical rotations, and other student teaching opportunities. documented mare injuries from trpalp included any amount of blood on rectal sleeves and any degree of rectal tearing. individual mare medical records documenting date of entry into the teaching herd, date of retirement, and the reason for retirement were analyzed. per our standing iacuc, mares may be transrectally palpated up to 15 times in a 7-day period. a total of 5,801 trpalp events occurred between 2019 2021, with a mare injury incidence rate of 0.76% (44/5,801); no injury required surgical correction or resulted in death. sedation was utilized to minimize averse behaviors 0.34% of the time (20/5,801) with 11 of 48 mares having received sedation. individual mare careers ranged from 1 22 years with 10 of 48 mares retiring between 2019 2021; no retirement was related to averse behaviors or injury experienced during trpalp. based on these data, the probability of a mare retiring from the teaching herd because of averse behaviors or injury from 2019 2021 was 0; however, being that trpalp can certainly result in injury to a horse, our probability of 0 does not indicate that an injury leading to the retirement or death of a mare is impossible. if trpalp was a noxious procedure, one would expect teaching mares to progressively develop aversion behaviors and require increased use of sedation, although this was not the situation in our herd. whereas it is prudent to perform trpalp with caution and restraint, these data indicate that injury to teaching mares can be kept to an extremely low rate of occurrence. therefore, our impression is that concerns for animal welfare (i.e., averse behaviors and mare injury) should not be cited as a reason to minimize live animal use within the veterinary curriculum. keywords: transrectal palpation, behavior, injury, attrition rate analysis of ph of common intrauterine therapies in mares yamilka lago-alvarez, bradley back, luis henrique de aguiar, mariana diel de amorim, soon hon cheong department of clinical science, college of veterinary medicine, cornell university, ithaca, ny intrauterine therapies are commonly used to treat endometritis in mares and have some advantages over systemic treatments, including being able to achieve higher local concentrations and physically dilute or disrupt biofilm in the uterus. saline (0.9% nacl) (sal) and lactated ringer’s solution (lrs) are 2 commonly used fluids for intrauterine lavage and treatments. sterile saline does not contain buffers and is more acidic compared to lrs. however, some clinicians are concerned that lactate-utilizing bacteria may benefit from lactate in lrs. intrauterine therapies may be irritating to the mare’s uterus due to the drug carrier or due to extreme ph. the ph of commonly used intrauterine therapies is not reported and may be different when combined with sal or lrs. objective of this descriptive study was to report the ph of commonly used intrauterine therapies in mares diluted with sal or lrs. we hypothesized that intrauterine therapies diluted with lrs are closer to neutral compared to sal. the ph of intrauterine therapies was assessed with a hand-held device (laqua twin ph meter, horiba) and a benchtop ph meter (accumet ab15+, fisherbrand). difference in ph by fluid used, treatment, and device were analyzed using linear regression anova in jmp pro v16, with significance set at p < 0.05. the average ph of the following intrauterine therapies were: sal (5.5 ± 0.03), lrs (6.3 ± 0.03); ceragyn flush in sal (3.2 ± 0.03) and lrs (3.2 ± 0.03); vinegar 2% in sal (3.6 ± 0.03) and lrs (4.3 ± 0.03); vinegar 10% in sal (3.6 ± 0.02) and lrs (3.7 ± 0.17); povidone iodine solution 1% in sal (3.2 ± 0.03) and lrs (5.0 ± 0.09); hydrogen peroxide 1% in sal (5.6 ± 0.15) and lrs (6.0 ± 0.02); dimethyl-sulfoxide (dmso) 10% in sal (5.4 ± 0.09) and lrs (6.2 ± 0.02); dmso 30% in sal (5.9 ± 0.02) and lrs (7.1 ± 0.09); n-acetylcysteine 3.3% in sal (5.8 ± 0.07) and in lrs (5.9 ± 0.06); gentamycin 2 grams in sal (6.2 ± 0.02) and lrs (6.2 ± 0.05); ampicillin 1 gram in sal (9.1 ± 0.05) and lrs (9.0 ± 0.08); ampicillin 2 grams in sal (9.3 ± 0.15) and lrs (9.8 ± 0.04); and ceftiofur na 1 gram in sal (6.5 ± 0.08) and lrs (6.6 ± 0.00). there was no significant effect of fluid or device used but a significant effect of the clinical theriogenology 2022; 14: 284 treatment type. there was a strong correlation (r2 = 0.9563) between the handheld and benchtop ph meters. some commonly used infusions are very acidic, including ceragyn infuse (ph = 3.6) that has the same ph as undiluted gentamycin that is known to be irritating to the endometrium, whereas tricide (ph = 8.4), and dmso (ph = 11.8) are alkaline. knowledge on the ph of commonly used intrauterine therapies of mares is valuable to guide decision making, balancing treatment of bacteria that thrive in neutral ph and acidophiles, and treating biofilm without harming the endometrium. keywords: mare, intrauterine therapy, ph, uterine infusion clinical theriogenology 2022; 14: 285 pregnancy and spontaneous partial abortion associated with salmonella in a bitch anum ahmed,a connor ellis,b audrey kellemana adepartment of large animal clinical sciences bdepartment of small animal clinical sciences college of veterinary medicine, university of florida, gainesville, fl an intact, 2-year mixed breed bitch (bernese mountain dog x standard poodle) at 50 days postbreeding was presented for a greenish-brown vulvar discharge of 12 hours duration. dog had a stillbirth 8 hours prior. bitch was not fed a raw diet. due to hyporexia, the owners provided cooked chicken and eggs. physical examination revealed a body condition score of 3/9 (royal canin© scale) and nonfetid brown vulvar discharge. mammary gland secretions were absent. on vaginal examination, neither ferguson reflex, fetuses, nor membranes were palpable. vaginoscopy revealed brown mucoid discharge that obstructed cervical viewing. abdominal ultrasonography revealed premature fetuses, with normal heart rates between 222 300 beats/minute. abdominal radiographs revealed 10 fetuses. serum progesterone concentrations were 1.25 ng/ml; bitch had a mild monocytosis and was hypocalcemic. an in-house serum brucella canis rapid slide agglutination test and herpesvirus titer were negative. a specimen of vaginal discharge was obtained for aerobic culture, and cytology revealed nonseptic neutrophilic inflammation. due to infection and risk of sepsis, ovariohysterectomy was offered but declined by the owner. overnight, the bitch was given intravenous lactated ringer’s solution, terbutaline, fenbendazole, altrenogest, amoxicillin, and clavulanate potassium (clavamox®), and clindamycin, and was discharged with oral medications the following day due to financial constraints. during hospitalization, another stillborn pup was spontaneously delivered; gross necropsy lesions were absent. on physical examination 2 days later, the bitch was clinically stable with vulvar discharge. abdominal ultrasonography revealed live fetuses with slightly increased fetal maturation of kidneys and gastrointestinal tract, but 1 dead fetus. the bitch was not azotemic, but urine specific gravity was isosthenuric at 1.008. preliminary vaginal culture results grew salmonella sp.; based on susceptibility, clindamycin was discontinued and therapy with ciprofloxacin initiated. five days after initial presentation, the bitch was febrile with erythematous pinnae. a marked acute inflammatory leukogram with mild hypoglycemia and hypocalcemia was present. final vaginal culture had salmonella enterica subsp. houtenae serovar iv 50:z4,z23:-. due to this organism, altrenogest and terbutaline were discontinued to allow the bitch to whelp. after the last dose of altrenogest, a stillbirth occurred at 12 hours and whelping began at 60 hours, respectively. two live pups and 1 additional stillborn were delivered at home. upon presentation for dystocia, fetal heart rate was 120 beats/minute for 1, indicative of severe fetal distress, and absent for another. calcium gluconate and oxytocin treatment resulted in birth of 1 live pup. without further contractions, cesarean section resulted in the delivery of 1 live pup and 1 necrotic fetus. uterine aerobic culture resulted in no growth and the bitch remained intact. due to risk of sepsis, the 4 pups were given ceftiofur, trimethoprim sulfa, and subcutaneous 0.9% sodium chloride. one pup was meconium stained; another died at home 2 days later. seven days postpartum, the bitch was stable, and 3 remaining pups were clinically normal. placental histopathology revealed acute suppurative chorioplacentitis, with salmonella as the presumptive etiology. this case demonstrated the significance and challenges of medical management of fetal-placental infections in the bitch. keywords: dog, pregnancy, salmonella, infection, abortion, dystocia unusual disorder of sexual development in a horse: a monorchid 64, xy, sry-positive phenotypic mare brittany middlebrooks,a patrick mccue,a brad nelson,a emily may,a charlie barton,a christina divine,a jerry bouma,b alan conleyc adepartment of clinical sciences, bdepartment of biomedical sciences colorado state university, fort collins, co cdepartment of population health and reproduction, university of california, davis, ca disorders of sexual development (dsd) are uncommon in horses and monorchidism conditions are very rare. this case report outlines the clinical assessment of a mare with a behavioral issue that led to the diagnosis of dsd. a 4-year quarter horse mare was referred with a history of stallion-like behavior and an elevated serum testosterone concentration. differential diagnoses included granulosa theca cell tumor, pregnancy, disorder of sexual development, estrus, and anabolic steroid treatment. oral examination revealed erupting canines and the size were consistent with that of a mare. external genitalia were normal in appearance for a mare. a normal mammary gland with 2 small bilaterally symmetric teats were present. transrectal ultrasonography revealed a structure in the right abdomen consistent with the morphologic appearance of a testis. no gonadal structure was identified on the left side. a vaginal speculum examination revealed a short, ‘blind-ended’ vaginal cavity, with no cervix. endocrine evaluation consisted of serum testosterone concentrations pre(731 pg/ml) and post(2 hours: 762 pg/ml; clinical cases session clinical theriogenology 2022; 14: 286 15 hours: 773 pg/ml) human chorionic gonadotropin (hcg) treatment, anti-müllerian hormone ([amh] 96 ng/ml), estrone sulfate (11 ng/ml), and inhibin b (76 pg/ml). all but inhibin-b concentrations were above the upper limit of the laboratory reference ranges for a nonpregnant mare. cytogenetic analysis revealed a 64, xy karyotype. sex determining region of the y chromosome (sry) was identified by pcr analysis. a standing laparoscopic procedure was performed and a cryptorchid testis was identified on the right side of the abdomen and removed. a similar gonadal structure was absent on the left side. histopathology of the excised right gonad revealed sertoli cells, leydig cells, and spermatogonia within degenerate seminiferous tubules with no evidence of spermatogenesis. additionally, a nest or island of adrenal cortical tissue was noted histologically within the cryptorchid testis and epididymal tissue was noted adjacent to the testis. immunohistochemistry confirmed amh presence in sertoli cells. reevaluation 5 weeks after surgery revealed resolution of the stallion-like behavior and normal endocrine parameters. in summary, a phenotypic mare with stallion-like behavior was diagnosed as a monorchid, 64, xy, sry-positive, disorder of sexual development (dsd). monorchidism was diagnosed by a combination of laparoscopic exploration of the abdomen, identification and removal of 1 gonad, and a subsequent decrease in testosterone and amh concentrations to that consistent with a gelding. presence of ectopic adrenal cortical tissue within the cryptorchid testis was unexpected, but has been reported in gonads of horses and other species. finally, diagnosis of dsd conditions was based on a combination of clinical signs, endocrine evaluation, chromosomal analysis, and histopathology. keywords: mare, monorchidism, disorder of sexual development, karyotype, cryptorchid references 1. bugno-poniewierska, m, raudsep, t: horse clinical cytogenetics: recurrent themes and novel findings. animals (basel) 2021;11:831. 2. marino, g, quartuccio, m, rizzo, s, et al: ectopic adrenal tissue in equine gonads: morphofunctional features. turk j vet anim sci 2012;35:560-565. 3. parks, ah, scott, ea, cox, je, et al: monorchidism in the horse. equine vet j 1989;21:215-217. five-year mare with no history of foaling and early pregnancy loss had retained endometrial cups erin lohbeck, etta bradecamp rood and riddle equine hospital, lexington, ky a 5-year, maiden dutch harness horse mare, was presented for a routine breeding soundness examination prior to undergoing breeding management that would utilize cooled, shipped semen. transrectal ultrasonographic examination revealed normal ovaries and uterus. cervix appeared normal (speculum examination). mare was in early estrus with a dominant follicle on the left ovary and a grade ii edema; samples for uterine culture and cytology were obtained. cytology was normal and the culture had no growth. due to schedule conflicts, mare was not scanned for another 5 days. at that time, mare appeared to have a corpus luteum (cl) on the right ovary the opposite ovary from where the dominant follicle had been documented. mare was examined 6 days later with plans to induce estrus (prostaglandin treatment): however, at this time she had a 40 mm follicle on the left ovary with the cl still present on the right ovary. prostaglandin was not given. four days later, the mare was again evaluated and appeared to have a cl on the left, small follicles on the right and moderate endometrial edema. mare continued to be closely monitored due to erratic cycling with transrectal ultrasonography performed every other day. during owner consultation, owner maintained that the mare was sold as a maiden with no reported reproductive history. over the course of her management, the mare proceeded to form a new cl without a true estrous cycle ~ every 7 days. at this time, blood equine chorionic gonadotropin (ecg) concentrations were determined (bet labs) and were 2.9 iu/ml (normal 0.0 in nonpregnant mares), lending to a diagnosis of retained endometrial cups. further investigation by the owner into the mare’s history revealed that the mare was reportedly bred 2 years prior but never produced a foal. the owner was given the option of endometrial cup removal via laser ablation or chemical curettage; chemical curettage with kerosene was performed. mare became appropriately cyclic immediately after treatment and was bred. mare conceived on the first cycle following treatment and is currently ~ 210 days in foal. endometrial cups produce ecg and form from the chorionic girdle of the fetal membranes in the mare at ~ 35 days and regress naturally at ~ 120 -150 days pregnancy. endometrial cups will regress on their own at 120 150 days as the allantochorion takes over the role of progesterone production. retaining endometrial cups past this point is abnormal but should be considered in mares with erratic cycling, incomplete estrous cycles, or luteinization of partly developed follicles. diagnosis is best confirmed via detection of ecg. although chemical curettage with kerosene is typically ineffective in mares with higher ecg concentrations, in the authors’ experience it can be useful in treatment of mares with lower ecg concentrations. keywords: mare, retained endometrial cups, equine chorionic gonadotropin management of postsurgical complications in a cat diagnosed with feline mammary fibroepithelial hyperplasia jamie douglas, robyn wilborn, aime johnson department of clinical sciences college of veterinary medicine, auburn university, auburn, al a 1-year, intact, american domestic shorthair queen was presented for complications associated with feline mammary fibroepithelial hyperplasia (fmfh). on presentation, the cat had pyrexia, anorexia, dull mentation, and glandular enlargement along the entirety of both mammary chains. mammary tissues were firm, edematous, and erythematous. patient displayed hypersensitivity to palpation of the affected area and exhibited hunched posture. approximately 3-cm, nonhealing ulcer was noted along midline at site of previous abdominal incision. the ulcer was black, malodorous, and diffusely necrotic. a 3-cm, healing incision was identified along the patient’s right flank. following its adoption, the patient was diagnosed as pregnant clinical theriogenology 2022; 14: 287 and pregnancy termination with simultaneous ovariohysterectomy (ohe) was elected. the initial surgical approach occurred via ventral midline incision but was halted due to excess hemorrhage from hyperplastic mammary tissues. forty-eight hours later, the patient underwent ohe via flank laparotomy. patient was presented 1 week following flank ohe due to patient’s continued mammary enlargement, nonhealing abdominal wound, and overall malaise. after examination, swab specimen of the ventral midline ulcer was submitted for bacterial culture and sensitivity. initial intervention included fluid resuscitation to correct dehydration, pain and wound management, and prolactin antagonism treatment. immediate pain management occurred via continuous rate infusion (cri) of hourly ketamine (0.1 0.5 mg/kg/hour) and once every 12 hours oral gabapentin (25 mg). a nonsteroidal antiinflammatory oral drug was incorporated (robenacoxib, 6 mg every 24 hours) as an antipyretic to reduce inflammation associated with the patient’s wound and mammary hyperplasia. a course of dopamine receptor agonist oral cabergoline (25 µg) was given every 24 hours to suppress lactation via reduction of prolactin. given the patient’s febrile status and chronicity of the mammary ulceration, oral marbofloxacin (25 mg) was given every 24 hours while awaiting culture and sensitivity results. patient sedation followed by debridement of devitalized mammary tissue was performed. silver sulfadiazine (ssd) was liberally applied along the length of the debrided wound to stimulate epithelialization and topically treat infection. wound debridement followed by application of ssd was continued daily. bacterial culture results revealed heavy growth of staphylococcus intermedius, resistant to marbofloxacin. antibiotic therapy was then adjusted to oral clindamycin (150 mg), given every 12 hours, effective per susceptibility report. due to extensive tissue injury, hyperbaric oxygen therapy (hbot) was incorporated in the treatment plan to facilitate delivery of oxygen-rich plasma to damaged cells. cat received a total of 3 hbot treatments of hbot at 48-hour intervals. one week after admission, the patient appeared bright, appetent, afebrile, and comfortable. mammary hyperplasia had started to resolve, evidenced by decreased mammary size, and the patient was discharged to the care of the owners. this case provided an account of fmfh, a dysplastic response of mammary tissue to elevated progesterone concentrations. tissue-healing and patient health were compromised in this case of fmfh, often a clinically benign condition, because of complications associated with ventral midline incision. to avoid additional insult of mammary tissues, flank incision for ohe may prove a superior approach in cases of fmfh. collaboration between theriogenology service and pain management fields, and careful wound management proved beneficial for this patient. keywords: cat, mammary fibroepithelial hyperplasia, hyperbaric oxygen therapy, pain, wound pregnancy in a sry-negative xx sex reversal pig after removal of the ovotestis jewel toenges,a michela ciccarelli,ab ahmed tibary,a jon oatleyab acollege of veterinary medicine, bschool of molecular bioscience washington state university, pullman, wa disorders of sexual development have been documented in over 20 species including pigs in which sex reversal of xx sry-negative animals has been reported to occur in 0.1 0.6% of the population. reports of xx sry-negative sex reversal have also been documented in dogs, goats, and horses. in these animals, 1 or both gonads has ovarian and testicular tissue and underdeveloped female reproductive tract is often present. owing to high testosterone concentrations from testicular tissue, pregnancy is unlikely even when normal uterine structure is present. whether removal of testicular tissue can rescue the ability to achieve a pregnancy has not been fully explored for any species with xx sry-negative sex reversal. here, we identified a newborn gilt with a ‘skyhook’ vulva, a common trait associated with sex reversal in pigs. at 5 months of age, the gilt displayed signs of estrus but the behavior was characterized as aggressive and reflected masculinity. artificial insemination was attempted several times with semen from a proven fertile boar but the insemination pipette could only be advanced 10 cm into the vagina (average vaginal length in pigs is 25 cm). gilt never achieved a pregnancy from these breeding attempts. subsequent attempts at estrous cycle synchronization did not result in normal signs of estrus. abdominatl ultrasonographic examination revealed uterus and left ovary appeared normal; however, contralateral gonad was abnormally shaped with a testis-like echotexture. on exploratory laparotomy, right gonad was suspected to be an ovotestis and was removed. on gross examination, the gonad had 2 distinct tissue types with clear demarcation. the presumed testicular portion was smooth and compact, whereas the ovarian portion was smaller and presented a few small follicles and 1 corpus luteum. left ovary appeared normal and had corpora lutea (8 -10). histologic examination confirmed the diagnosis of ovotestis. the tissue consisted of leydig cells and seminiferous tubules but germline was absent. cytogenic evaluation revealed the gilt to be xx, 38; sry-negative. four months after the removal of the ovotestis, the animal presented a normal estrus with complete immobility reflex and was artificially inseminated yielding a pregnancy that produced 6 healthy piglets. this novel case exemplified surgical removal as a treatment to aid in return to fertility in sry-negative xx sex reversal gilts with a remaining fully developed female reproductive tract. keywords: gilt, sex reversal, hermaphroditism, pregnancy small colon rupture in a postfoaling mare patricia egli,a kara lascola,a ji-hang yin,b maninder sandey,b robyn wilborn,a candace lymana adepartment of veterinary clinical sciences, bdepartment of pathobiology college of veterinary medicine, auburn university, auburn, al a 15-year, american quarter horse mare, was presented to a referral hospital for further evaluation and treatment of postpartum colic. night before presentation, the mare experienced a dystocia (anterior presentation, dorsosacral position, right forelimb flexion at the elbow); the mare delivered a foal before farm personnel could correct the foal’s posture. fetal membranes passed normally; shortly thereafter the mare appeared acutely uncomfortable/agitated but remained quiet with sedation and analgesia. following morning, the referring veterinarian diagnosed the mare with a large colon impaction prior to referral. at presentation, the mare was quiet, alert, and reclinical theriogenology 2022; 14: 288 sponsive with a mild tachycardia (56 beats/minute); mucous membranes (mm) were pink and moist with a capillary refill time of < 2 seconds. other vital parameters were within normal limits (wnl). borborygmi were present in all quadrants and bilateral plaques of ventral edema, considered normal for late pregnancy, were present. passage of a nasogastric tube yielded 9 liters of net enterogastric reflux. postpartum hemorrhage (pph) was suspected via transabdominal ultrasonography with moderate amount of free, hypoechoic fluid. to minimize the risk of introducing bacteria into a hemoabdomen, and taking into account the plaques of ventral edema, an abdominocentesis was not performed. amid concern of pph, a complete transrectal palpation was not performed. a vaginal examination with a polanski speculum revealed mild vaginal bruising and a grade i perineal laceration at the dorsal commissure of the vulva. additionally, unusual gas distention of an empty rectum was appreciated. a presumptive diagnosis of pph and resulting hemoabdomen with a large colon impaction was made. initial medical treatment included intravenous fluid therapy and observation; enteral fluid therapy for treatment of colon impaction was not pursued due to reflux. a serum chemistry panel and complete blood count eventually revealed hemoconcentration, severe neutropenia with a degenerative left shift, and marked toxic changes; broad spectrum antibiotics were initiated due to updated concerns of a uterine or intestinal rupture. overnight, the mare developed tachycardia (80 beats/minute) with continued reflux (~ 23 liters every 2 hours), but appeared stable and mm remained wnl. worsening tachycardia was noted the following morning (120 beats/minute) warranting further diagnostics which revealed an increasing packed cell volume (66%) and additional abdominal fluid accumulation. abdominocentesis revealed severe septic inflammation with mixed bacteria and mm acutely appeared dusky. exploratory enterotomy was declined due to sudden, rapid deterioration and the mare was euthanized. a 15.0 cm tear/rupture was identified in the small colon during necropsy examination, located 60 cm cranial to the anus. the affected segment was dark red to purple. the peritoneal cavity contained ~ 3 liters of yellow to red-tinged fluid with flecks of fibrin, blood clots, and fecal material. the serosal surface of the small and large intestine and peritoneum were diffusely dark red. no intestinal impaction, uterine or bladder rupture was identified. the gross findings confirmed a small colon tear/rupture resulting from segmental ischemic necrosis. foaling in the face of malposture was considered as a potential catalyst for the small colon’s segmental ischemic necrosis and rupture. keywords: mare, dystocia, colic, postfoaling complication hamartoma in the reproductive tract of a thoroughbred mare sarah hargrove,a dale brown,a laura kennedy,b maria schnobricha arood & riddle equine hospital, lexington, ky buniversity of kentucky veterinary diagnostic laboratory, lexington, ky vascular hamartomas are rare, even more so in equine patients. hamartomas are benign, noncancerous tumor-like malformations that are made up of an abnormal mixture of cells that are native to the anatomic location. they grow at the same rate as the original tissue and are mostly due to a developmental error. hamartomas have been reported in equine fetal tissues including lung and liver, subcutaneous tissue of a pelvic limb, spinal cord, and an ovary. more specifically vascular hamartomas have been reported in the tongue, dorsal carpal region, and an ovary of adult equine patients. a 6-year, maiden mare, recently retired from racing, was first evaluated in late january to evaluate the reproductive tract prior to breeding. at that time the referring veterinarian noted cervical bruising; the bruising did not resolve after 60 days of observation and the mare was then referred for further examination. transrectal palpation and ultrasonographic examination revealed the mare to be in diestrus (corpus luteum and medium follicles) with both uterine horns having thickened endometrial folds with no edema but multifocal small echogenic flecks within the endometrium were noted. these numerous, multifocal, small hyperechoic regions between 0.5 2 cm were noted in the endometrial wall of the uterine body. cervix appeared edematous and had multifocal to diffuse, small (0.5 cm) hyperechoic flecks. hysteroscopy revealed severe discoloration with purple to black, hemorrhagic tissue in the vagina, throughout the cervix, and entire uterine body. strands of fibrous adhesions were present throughout the endometrium coalescing on pale crater-like lesions between 2 3 cm in diameter distributed throughout the endometrial surface. vaginoscopy revealed purplish-brown discoloration extending from a linear ventral streak on the vaginal floor to encompass the entire cervix. the appearance was consistent with a bruise or trauma. histologically, there were numerous dilated vascular structures lined by bland endothelial cells admixed with normal endometrial structures. neoplastic features were not identified, consistent with a vascular hamartoma. a complete blood cell count and chemistry, and coagulation profile were submitted to due concerns for clotting disorders and other systemic diseases and consequences. the bloodwork revealed a decreased platelet count, with all other blood parameters within normal limits. a poor prognosis was given for the mare’s future breeding potential due to a diffusely abnormal cervix and uterine body. an abnormal presentation of uterine benign tumor that was diagnosed as a uterine hamartoma is described. keywords: hamartoma, equine, uterine hamartoma acute testicular degeneration in a 6-year paso fino stallion stephanie walbornn,a charles loveb arood and riddle equine hospital in wellington, wellington, fl bdepartment of large animal clinical sciences, texas a&m university, college of veterinary medicine, college station, tx azoospermia refers to absence of sperm in an ejaculate. etiology ranges from reversible/transient (i.e., complete blockage of the ampullae) to end-stage testicular degeneration. a 6-year paso fino stallion was presented to rood & riddle equine hospital (rreh) in wellington in october 2021 for a breeding soundness evaluation following recent transfer of ownership and importation into the us just a few months prior. prior to purchase, 2 semen evaluations were performed in columbia (december 2020 and april 2021) and were reported to contain motile, morphologically normal sperm. however, 2 months clinical theriogenology 2022; 14: 289 prior to presentation to rreh, the stallion was diagnosed with azoospermia. stallion’s history included previous production of live foals and treatment provided for piroplasmosis in early 2021. a total of 7 ejaculates were collected on 3 separate days and no sperm were observed in any ejaculate even following semen centrifugation and cytologic evaluation. stallion had signs consistent with a normal ejaculation, including tail flagging and 5 6 ejaculatory jets. alkaline phosphatase concentrations were measured on 5 ejaculates and concentrations ranged from 1158 5770 u/l (normal 6,913 -22,180 u/l). to rule out retrograde ejaculation, the bladder was catheterized after a collection attempt, the urine centrifuged and examined microscopically, but no sperm were identified. ultrasonographic and manual examination of the external (testes/epididymides) and internal genitalia (ampullae) were normal. the total testicular volume was very small, 86 cc (average – 240 cc), suggesting a primary testicular problem. two testis biopsies, 1 from each testis, were collected using a trucut biopsy punch and submitted for histopathologic interpretation. biopsy results indicated complete absence of spermatids and spermatocytes and only a small number of spermatogonia and sertoli cells within the seminiferous tubules, indicating severe testicular degeneration. it is not clear what caused the apparent acute testicular degeneration. due to the severity of the testicular degeneration, the likelihood of regeneration of the seminiferous tubules was extremely low, and retirement from breeding was recommended. one should consider in this case that the testicular degeneration may be associated with piroplasmosis and/or subsequent treatment. equine piroplasmosis is a disease endemic to south america that is caused by infection with intracellular parasites, theileria equi and babesia caballi. the disease is characterized by fever, lethargy, anorexia, peripheral edema, petechiation of mucous membranes, anemia, and thrombocytopenia. treatment consisted of intramuscular injections with imidocarb diproprionate every 72 hours for 2 or 3 treatments. although studies demonstrating direct causation between piroplasmosis and poor semen quality are lacking, infertility has been reported in stallions. any disease process that causes systemic illness and an increase in body temperature may have a negative effect on spermatogenesis. keywords: stallion, testicular degeneration, azoospermia, piroplasmosis clinical theriogenology 2022; 14: 290 bulls voluntarily access shade during hot weather, reducing scrotal subcutaneous temperatures and improving sperm quality abdallah shahat,ab juan castillo,a jacob thundathil,a john kastelica adepartment of production animal health, faculty of veterinary medicine university of calgary, calgary, ab, canada bdepartment of theriogenology, faculty of veterinary medicine, cairo university, giza, egypt our objectives were to establish relationships among bull location (shaded versus nonshaded), intrascrotal and ambient temperatures, and sperm quality. we hypothesized that in hot ambient temperatures, bulls seek shade, reducing intrascrotal temperatures and improving sperm quality in comparison to bulls without shade. adult angus bulls (n = 6) were randomly and equally allocated into 2 groups in adjacent, outdoor paddocks, with 1 containing artificial shade (metal structure ~ 3.5 x 6 meters and 2.5 meters high with a roof and 3 walls) to be accessed as desired (shaded group) and monitored with a motion camera. temperature data loggers surgically implanted in scrotal subcutaneous tissues recorded temperature once each hour. semen was collected by electroejaculation once weekly for 9 weeks. during the 10 hottest days, the percentage of time that a bull accessed shade increased with ambient temperature, ranging from 7.6 to 86.7% for ambient temperatures < 25°c and > 33°c, respectively. over the 10 hottest days, scrotal subcutaneous temperature in the no-shade group was associated with ambient temperature, with the highest scrotal temperature (36.5°c) recorded when the ambient temperature was ~33°c. conversely, bulls with access to shade had lower (p < 0.001) scrotal subcutaneous temperatures during high ambient temperatures, particularly when they accessed shade. during the 4 hottest days, bulls that could access shade did so most of the time from 12.00 to 17.00, coinciding with peak ambient temperatures. for total and progressive forward motility, there were group effects (p < 0.001 and p = 0.023, respectively; better motility in the shade group). furthermore, for total abnormalities and acrosome integrity, there were group effects (p > 0.001 for each), and a time effect (p = 0.009) for acrosome integrity. for sperm kinematics (vap, vcl, vsl, str, and lin) there were group effects (p < 0.005, p = 0.011, p < 0.010, p = 0.020, and p = 0.046, respectively), whereas alh had a time effect (p < 0.05). in summary, during hot weather, bulls voluntarily accessed shade, lowering scrotal subcutaneous temperatures and improving sperm quality. keywords: bulls, heat stress, shade, intrascrotal temperature effect of recovery rate on in vitro embryo production alex wittorff,a candace lyman,b ryan coy,c reed holyoaka adepartment of veterinary clinical sciences, college of veterinary medicine oklahoma state university, stillwater, ok bdepartment of clinical sciences, college of veterinary medicine auburn university, auburn, al croyal vista southwest, purcell, ok with the advent of transvaginal aspiration (tva) of oocytes becoming a clinically relevant procedure in mares for the purposes of in vitro embryo production, a symbiotic relationship between primary veterinarians managing patient cycles and the referral facility offering outpatient tva services has developed. in such circumstances it is common that aspirated oocytes are shipped to yet another location where laboratory personnel perform intracytoplasmic sperm injection (icsi) and embryo culture. unfortunately, primary veterinarians and clients may be forced to rely on opinion provided by wordof-mouth when choosing facilities, if providers of tva and icsi services do not to provide colleagues and owners with in-house results and statistics. additionally, the majority of scientific reports available associated with tva and icsi are associated with benchtop results and typically do not continue beyond blastocyst development to report heartbeat positive pregnancy rates, likely due to a lack of access to the embryo recipient records. as little scientific research is available to provide practitioners and clients with the information necessary to make informed choices when choosing facilities, the objective was to determine the most important step for producing in vitro embryos and successful live foal rates. we hypothesized that a critical component in successful in vitro embryo production is the number of oocytes recovered from any given mare, as compared to other steps in the production process. a retrospective examination of the latest complete annual records (2020) involving our facility in collaboration with an exclusively equine reproduction veterinary practice, focused on 275 tva sessions completed over the course of 12 months. a range of 2 22 oocytes were collected from a total of 46 mares. per tva session, an average of 11 oocytes were recovered (1,381 follicles punctured and 1,111 oocytes collected, 80% recovery rate) and an average of 7 oocytes matured and underwent icsi per tva session (748 oocytes injected, an oocyte maturation rate of 25%). an average of 2 embryos per tva session were obtained. the average pregnancy rate of recipient mares receiving embryos derived from our collaboration was 87% with a live foal rate of 57%. at our institution, follicles as small as < 5 mm are punctured and included when calculating recovery rates. although aspiration of very poster session clinical theriogenology 2022; 14: 291 small and large follicles may have a negative impact on recovery rate, we included every follicle-like structure aspirated in our calculations. for our data set we concluded that the highest embryo maturation and live foal rates were achieved when a higher number of oocytes are recovered, leading to higher numbers for potential embryo development and pregnancy. to be most beneficial, a streamlined process should exist between the selection of mares with higher numbers of appropriately sized follicles and coordination between the equine practitioner, tva specialty practice, icsi lab, and the recipient management. as collaborators in this working partnership, working as a team to offer such services has enhanced the working relationship between practitioners and specialists and provided our clients with a greater degree of success. keywords: mare, transvaginal oocyte aspiration, oocyte recovery deoxynivalenol and deepoxy-deoxynivalenolinduced alterations in theca cell function as a major cause of infertility in dairy cows ali torabi,a fatemeh aminmarashi,a maryam hezaveheib aresearch center for reproduction and fertility, faculty of veterinary medicine montreal university, st-hyacinthe, qc, canada bdepartment of embryology, reproductive biomedicine research center royan institute for reproductive biomedicine, tehran, iran tricothecene mycotoxins such as deoxynivalenol (don) and its metabolite deepoxy-don (dom-1), can alter major intracellular signaling pathways within theca cells that can perturb normal folliculogenesis in the ovary resulting in infertility in dairy cows. they function through the activation of a specific tyrosine kinase receptor that transduces the signal by activating several intracellular signaling pathways. in our experimental study, bovine ovarian theca cells were collected from adult cows during the follicular phase of estrous cycle and were cultured at a density of 500 000 viable cells for 5 days. cells were treated on day 5 of the culture with 1 ng/ml don and dom-1 for 30 minutes and a mass spectrometry approach was used to identify changes in the proteome profile of the cells. we identified approximately 93 peptides that were phosphorylated and 254 peptides that were dephosphorylated in response to don and dom-1 compared to non-treated control cells. gene ontology analysis indicated that the abundance of proteins associated with cell proliferation such as mapk3/1, mapk14, gngt1, edn1 and ywhab were up-regulated in the don and dom-1 compared to the control group. this study reports for the first time that don and dom-1 at sub-toxic level can activate major mitogen-induced proliferative molecules within theca cells that can stimulate tumorigenesis in the ovary. keywords: bovine ovary, deoxynivalenol, deepoxy-deoxynivalenol, proteome, theca cell a case of hemosemen in a merino ram allan gunn,a susan robertsona,b,c aschool of agricultural, environmental and veterinary sciences, and gulbali institute of agriculture, water and environment research bfred morley centre, graham building charles sturt university, wagga wagga, australia aim was to investigate a case of hemosemen in a ram. a 2½ year, merino ram was examined as part of a routine flock breeding soundness examination. ram was from a brucellosis free flock, and had been on the property for at least 12 months with no previous history suggestive of any health nor reproductive abnormalities. ram was bright, alert and responsive, with a body condition score of 3.5/5, a scrotal circumference of 35 cm, and no obvious abnormalities palpable within the scrotum on 22 feb 2021. semen sample collected via electro-ejaculation was red in colour, suggestive of hemosemen (hemospermia). a diffquick stained smear made from a sample of the ejaculate had red blood cells without leukocytes. morphological assessment (1,000 x magnification) with differential interference contrast microscopy on a 10% buffered formol saline preserved sample resulted in a spermiogram with 93% morphologically normal sperm. an assessment on 07 apr 2021 resulted in a hemosemen sample of similar appearance, but with a more pink colouration on gross evaluation. scrotal circumference was 32 cm, and 90% sperm were morphologically normal. there were fewer erythrocytes detected on a diffquick stained smear from an ejaculate sample, but there were obvious (~ 3 per high power field (hpf) x 1000 magnification) neutrophils visible microscopically. a pink coloured ejaculate was collected at a third examination on 07 jun 2021. abnormal ejaculate was confirmed to be from within the urethral process, illustrating the blood cells were not from a superficial lesion. there were 68% morphologically normal sperm, with most (29%) being distal mid-piece reflex abnormalities. a diffquick stained smear revealed fewer erythrocytes than in the original sample, and more than 3 neutrophils per hpf. occasional macrophages were visualised. transrectal ultrasonography of the pelvis revealed an enlarged vesicular gland, ~ 35 mm in diameter, compared to 15-20 mm in a control ram. ram was treated with long acting oxytetracycline every second day for 3 treatments. ejaculate was less pink in color when re-examined on 13 jan 2022. there were more than 5 neutrophils visible per hpf on microscopic examination of a stained smear, and erythrocytes were scant. mild blood contamination with moderate numbers of degenerate and nondegenerate cells were reported from a laboratory submitted microbiological and cytological assessment. coccobacilli and cocci were visualised on cytospin examination, and the presence of crystals. bacteriological culture revealed moderate mixed growth of staphylococcus aureus and histophilus somni. histophilus ovis and actinobacillus seminis are reported as causes of inflammation in the reproductive tract of rams. hemosemen is rarely reported in ruminants, and to our knowledge there are no reports of hemosemen in the ram. this is the first report of hemosemen in a ram with identification of histophilus somni in the ejaculate. this case illustrated a case of hemosemen that is likely to have been caused by histophilus somni infection of the vesicular glands. a clinical theriogenology 2022; 14: 292 6-week out of season, breeding with this ram resulted in 32% pregnancy rate compared to another ram (63%). keywords: hemosemen, hemospermia, ram, ejaculate, histophilus somni seasonal effects of temperature in semen quality: heat-sensitive and heat-tolerant bulls jacob netherton,a allan gunn,b benjamin robinson,a ana villaverde,c kim colyvas,d ced wise,e tylah russo,a amiee dowdell,a rachel ogle,a mark bakera acollege of health and medicine, university of newcastle, callaghan, australia bschool of agricultural, environmental and veterinary sciences, and gulbali institute of agriculture, water and environment research charles sturt university, wagga wagga, australia cindependent researcher, sao paulo, brazil dcollege of engineering, science and environment university of newcastle, callaghan, australia erocky repro, alton downs, australia breeding soundness examination (bse) in bulls is a risk assessment on the likely infertility of a bull. recommendation is for this assessment to be done at least 2 months prior to a producer’s mating start date, reflecting the time taken for spermatogenesis and epididymal maturation time. adverse effects of heat on the production of viable sperm are well documented. if male thermoregulating capacity is affected, the production of viable sperm can be compromised. many cattle breeders in australia aim for spring (august/september) calving, which means breeding typically occurs during the summer months (november-march). as a result, bse is recommended to be carried out from july to december (winter-early summer in australia) to facilitate timely assessment of the likelihood of infertility in bulls, and allow time for remediation, retesting or replacement of those bulls. objective was to assess the likelihood of bse representing the bulls’ spermiogram at the time of the breeding period. ejaculates (n = 1,271) from 79 bulls of 11 breeds that were submitted for bse, were assessed using standard in vitro fertility parameters where poor quality was determined to be fewer than 30% progressively motile sperm and/or fewer than 50% morphologically normal sperm. importantly, each bull was assessed using several ejaculates from throughout the year. from the results, there appeared to be at least 2 bull classifications based on spermatozoa production effects. these were correlated to climatic conditions prevailing at, or prior to, the time of sperm collection. bulls that consistently produced good quality sperm throughout the year were classified as ‘heat tolerant’ bulls. those that produced varying quality ejaculates during the year were classified as ‘heat sensitive’ bulls. for the heat sensitive bulls, an analysis of the correlation between sperm quality and maximum temperature (lagged from 0 to 40 days) showed that the highest correlation (r = 0.42; 95% ci [– 0.5, – 0.34], p < 0.001) was at 17 days prior to the day sperm quality was measured. this supported the notion that there was a delayed effect of temperature on sperm quality. based on a nominal logistic regression of spermiograms for 12 heat sensitive bulls it would be predicted that 63% of spermiograms would have poor quality 17 days after the ambient temperature reached 34˚c, i.e., decreased semen quality and a likelihood of being less fertile (p < 0.001). knowledge of the inability of a bull to tolerate heat stress is unknown at the time of bse. based on this information, a cohort of 17 bulls were subjected to artificial heat stress of 40˚c for 12 hours during winter, and their spermiograms monitored for 12 weeks. data indicated that 3 bulls were heat susceptible with abnormal sperm morphology between 3 and 8 weeks after heat stress, before returning to normal by 10 weeks, illustrating that the classification of heat sensitive and heat tolerant bulls is scientifically sound. work is continuing to define more clearly heat sensitivity, determine the mechanisms of heat tolerance and sensitivity in bulls, and to locate genetic markers that can be used to predict these phenotypic traits. keywords: breeding soundness examination, bull, spermiogram, heat-tolerant, heat-sensitive fungal endometritis in a cavalier king charles spaniel bitch andrea hesser josey ranch pet hospital, carrollton, tx a 3-year, cavalier king charles spaniel bitch presented for infertility testing after multiple attempts of breeding without successful pregnancy. dog had been bred with fresh chilled semen via transcervical insemination in 2 cycles prior without a pregnancy being identified. a 6-month interestrus interval had been observed, cycle progression normal, and semen quality and quantity deemed acceptable for a favorable outcome. on physical examination, the bitch appeared in excellent physical health. based on recent cycle activity, the bitch’s progesterone concentrations were assessed in late diestrus until progesterone was < 1.5 ng/ml. dog was sedated, and cervix visualized using a rigid ureteroscope. a small biopsy forceps was then passed through the cervical os to acquire multiple endometrial biopsies. several endometrial biopsies were submitted for histopathologic assessment that had fibrin mats containing septate fungal hyphae and small oblong or teardrop-shaped yeast forms within the endometrium. mild cystic change was also observed within the endometrium. bitch was treated with ketoconazole 25 mg/kg orally every 24 hours for 90 days. at the onset of next estrus, intrauterine bacterial culture was performed via low volume lavage using a ureteroscope and 4 french catheter that yielded growth of haemophilus haemoglobinophilus. azithromycin was given at 10 mg/kg orally every 24 hours during estrus prior to breeding. ovulation timing followed, and surgical insemination performed with chilled semen of excellent quality and quantity. at insemination, the uterus had a few small adhesions associated with the uterine body and a few small intramural cystic lesions that were expressed during the procedure. bitch presented at 31 days postovulation that revealed no evidence of pregnancy. bitch was culled from the breeding program after this attempt, but other approaches may have been considered to identify and speciate the fungal infiltrates, and repeat biopsy to assess if treatments had been successful. intrauterine treatments may also be considered if oral therapy was not resolving presence of the organisms. though these treatments have not been reclinical theriogenology 2022; 14: 293 ported in dogs, they are well described in equids and some livestock species. keywords: fungal endometritis, endometritis, canine infertility postbreeding inflammatory response in interand intra-species breeding in equids giorgia podico,a kianna spencer,a joão bittar,b igor canissoa acollege of veterinary medicine, university of illinois urbanachampaign, urbana, il bcollege of veterinary medicine, university of florida, gainesville, fl anecdotally, it has been suggested that subfertile mares have better fertility when bred with donkey semen (d-s) than horse semen (h-s), and this could be due to donkey seminal plasma (d-sp). horse seminal plasma (h-sp) and d-sp have immunomodulatory properties on endometrium of females of their own species; however, it is unknown if d-sp also modulates the mare endometrium. this study aimed to: i. compare the inflammatory response of horse mares inseminated with d-s or h-s or infused with d-sp or h-sp; and ii. assess embryo recovery in mares bred with d-s and h-s. we hypothesized that: i. sperm results in a greater inflammatory response than seminal plasma regardless of species; and that ii. d-sp downregulates endometrial inflammatory response compared to h-sp. five cycles of 22 mares were randomly assigned in a crossover design to a uterine infusion with donkey semen (d-s) and seminal plasma (d-sp) and horse semen (h-s) and seminal plasma (h-sp) or saline. mares were examined via transrectal ultrasonography, and ovulation was induced when a dominant follicle (> 35 mm) was detected. semen and seminal plasma were obtained from a jack and a stallion. uterine edema, intrauterine fluid accumulation, number of neutrophils on uterine cytology were assessed at 0, 6, 24, and 48 hours postinfusion or at ai. progesterone concentrations were assessed at 0, 6, 24, 48, 72, 96 hours postinfusion and after day 8 postovulation. embryo flushing was performed on 8 days postovulation. r-studio was used for data analyses. kruskal-wallis rank-sum test was used to compare the scores given to uterine fluid and edema; a mixed linear model was used to compare the other variables. significance was set at p < 0.05 and a statistical tendency a 0.05 < p < 0.1. although there was a transient increase in the amount of intrauterine fluid after infusion at 6 hours, there was no difference (p < 0.05) among groups. embryo recovery and quality were not different (p > 0.05) between groups; however, it was numerically higher in d-s than h-s (59 versus 55%). there were effects of time (p < 0.05) and group (p < 0.05) but no time by group interaction (p > 0.05) on progesterone concentrations. after 8 days postovulation, h-s had higher (p = 0.046) progesterone concentrations than d-sp, tended to have higher (p = 0.084) concentrations than saline), and had similar (p = 0.7) concentrations to h-sp. neutrophil counts changed (p = 0.001) over time and there was an interaction (p < 0.001) between group and time. at 6 hours postinfusion, the number of neutrophils on uterine cytology in d-s tended to be lower (p = 0.074) than h-s. infusion of d-sp had an antiinflammatory effect (p < 0.05) compared to s-s and s-sp. in conclusion, d-s and h-s had similar postinfusion inflammatory responses and embryo recovery. d-sp results in a lower postinfusion inflammatory response than other combinations. seminal plasma might be the reason that subfertile mares appear to have better fertility when bred with donkeys. keywords: donkey, endometritis, horses, seminal plasma intrauterine chlorine dioxide for treatment of fungal endometritis in marwari horses balachandran ramanathan,a etta bradecampb acomprehensive equine services, india brood and riddle equine hospital, lexington, ky two valuable mares of a rare marwari breed of horse were presented with prolonged lengths of infertility (one for 4 years, and the other for 2 years). mares had been treated by multiple veterinarians with various regimens of antibiotics and antiinfective agents for chronic endometritis. mares were checked for a basic breeding soundness examination and the findings were the following. mare 1 (barren for 4 years): ovaries had multiple follicles and a corpus luteum. cervix was closed with pendulous well-toned uterus containing severe amounts of echogenic fluid accumulation in the body. mare 2 (barren for 2 years): one ovary had 45 mm follicle and the other ovary had multiple small follicles. cervix was relaxed and 30% dilated, and the uterus contained a small amount of echogenic fluid in the right horn and body with a fair to poor uterine tone. mare also had mucopurulent whitish vulvar discharge that was also observed on the medial aspect of one hock. endometrial cytology performed on efflux from small volume uterine lavage from both mares revealed fungal and yeast elements along with severe inflammation. both cytologic smears contained neutrophils, mononuclear inflammatory cells, and plasma cells. cellular debris also contained several coccobacilli. due to the chronicity and refractory nature of the endometritis, an experimental trial with chlorine dioxide was suggested. three days of intrauterine lavage was performed followed by infusion of 1 liter saline with 375 ppm, chlorine dioxide. production of chlorine dioxide was carried out as per the patented formulation (sureshot-eq, india), to accomplish a final concentration of 375 ppm. no adverse reaction or pain response was noted and the efflux on subsequent uterine lavage improved. after 3 consecutive days of treatment the mares had a caslick’s performed and were sent home. mares were examined via transrectal palpation and ultrasonography 4 weeks after treatment. mares had active ovaries, appropriate uterine edema and negligible intrauterine fluid. mares were bred and the referring veterinarian was advised to perform a postbreeding lavage with warm saline. mare 1 was in foal at day 45. mare 2 mare was lost to follow-up. chlorine dioxide has been used historically as an active nonantibiotic biocide. we used a patented technology to produce low volume consistent release of chlorine dioxide at the rate of 375 ppm lasting for 4 hours. in this patented system, the end result of the reactions are sodium chloride and water. additionally, chlorine dioxide has been effective in disrupting biofilm. further research is needed to determine the effectiveness of chlorine dioxide for the treatment of endometritis. clinical theriogenology 2022; 14: 294 keywords: mare, fungal endometritis, chlorine dioxide, marwari lactate-induced spontaneous acrosomal exocytosis in stallion sperm camilo hernández-avilés,a luisa ramírez-agámez,a dickson varner,a charles lovea adepartment of large animal clinical sciences college of veterinary medicine and biomedical sciences texas a&m university, college station, tx an in vitro, physiologic model of acrosomal exocytosis (ae) is needed to study sperm function and fertility potential in stallions. currently, the calcium ionophore a23187 is used to measure the ability of the acrosome to react but is recognized as a potent nonphysiologic inducer of ae. as such, a23187 may not diagnose more subtle forms of acrosome dysfunction. incubation of stallion sperm under capacitating-like conditions by using a medium that contains only lactate as an energy source (lac-mw) resulted in a high rate of spontaneous ae in viable sperm (% ae/viable). here, we determined if the lac-mw model could be used as a more physiologic method to study acrosomal function in stallion sperm, taking into consideration other factors such as semen storage methods for the transport of equine sperm. in experiment 1, fresh semen (n = 12 ejaculates) was incubated with a23187, lac-mw, or control (inra-96®) for up to 6 hours and analyzed for ae/ viable using flow cytometry (fitc-psa and fixable live/dead red). in experiment 2, fresh semen (n = 21 ejaculates) was either incubated in lac-mw (as in experiment 1), cool-stored for 24 hours (cooled), or frozen in liquid nitrogen (frozen). after cooled storage or freezing/thawing, sperm were incubated in lac-mw for up to 6 hours and analyzed for ae/viable. in experiment 3, semen from 5 stallions (stallions a, b, c, d, or e; n = 15 ejaculates) were analyzed after 24 hours of cooled storage using a23187 or lac-mw. also, cool-stored semen from these stallions was used to breed 143 mares (range 24 35 mares/stallion). for all experiments, ae/viable was determined after 6 hours of incubation. in experiment 3, the total ae (only for a23187), and the per-cycle pregnancy rate (pc/pr) per stallion were also calculated. for all experiments, rank-transformed data was analyzed using the mixed linear model (sas 9.4). statistical significance was set at p < 0.05. in experiment 1, the mean ae/viable was higher in lac-mw than a23187, but similar in a23187 and control (42, 8 and 8%, respectively; p < 0.05). in experiment 2, mean ae/viable was similar in fresh, cooled, or frozen semen (37, 38, and 46%, respectively; p > 0.05). in experiment 3, whereas differences in total ae (using a23187) were not observed among all stallions (range: 92 94%; p > 0.05), there were differences in mean ae/viable (using lac-mw) (a e: 56, 45, 42, 33, and 24%, respectively; p < 0.05). for these stallions, the pc/ pr were a: 72%, b: 65%, c: 60%, d: 54%, and e: 45%. we concluded that the use of lac-mw to induce spontaneous ae can be considered as a more physiologic in vitro alternative than a23187 to study acrosomal function in stallion sperm, primarily because sperm viability is preserved during ae. this approach appeared to be not affected by the stallion sperm storage method. we did not observe a faster occurrence of spontaneous ae in frozen/thawed semen, suggesting that freezing/thawing did not induce capacitation-like changes in stallion sperm. keywords: stallion sperm, acrosomal exocytosis, acrosome function, lactate, storage, fertility severe cleft palates affected an entire english bulldog litter casey rubin,a morgan agnewb atemple university, philadelphia, pa bphiladelphia animal hospital, philadelphia, pa a 5-year, female english bulldog, was presented on day 4 of estrus for a reproductive examination and determination of serum progesterone concentrations. bitch had 4 previous breedings and 3 previous pregnancies. one pup in a previous litter was euthanized due to a severe cleft palate. bitch had multiple reabsorptions in the previous litter but delivered 4 viable pups. on examination, all parameters were within normal limits. a vaginal cytology revealed predominantly superficial cells with a few bacteria. vaginal mucosa appeared edematous on vaginoscopy. patient’s initial serum progesterone concentrations were 18.13 ng/ml on an in-house minividas machine, corresponding with 4 days post lh surge. due to the previous reabsorptions and history of cleft palates in pups, the owner was giving enrofloxacin, pentoxifylline, and folic acid prescribed by another veterinarian. enrofloxacin treatment was discontinued 4 days postbreeding. the following day, the patient was bred with fresh semen via transcervical insemination with 1.07 x 109 progressively motile sperm. patient was bred again the following day via artificial insemination. transabdominal ultrasonography was performed 24 days postbreeding and revealed 5 viable pups and 2 reabsorptions. one pup in the left uterine horn had a thickened placenta and areas of placental separation. patient was given pentoxifylline at a dose of 10 mg/kg and augmentin at a dose of 10 mg/kg. a recheck ultrasonography was recommended in 5 7 days but declined. bitch was presented for a cesarean surgery on her calculated due date. a complete blood count and chemistry profile were within normal limits. all 6 pups were viable; however, had severe cleft palates, and the owner elected euthanasia. patient’s female offspring has been bred to the same stud dog and is due in march of 2022. after further questioning, it was apparent that the stud had sired 3 previous litters with 1 litter of entirely nonviable anasarca pups. university of california, davis has been contacted to determine if any useful dna samples can be submitted for research purposes. this case study illustrated the importance of obtaining a full breeding history on the stud and bitch prior to breeding and demonstrated why a genetic test for cleft palates in bulldogs would be invaluable to breeders. keywords: cleft palate, bulldog, anasarca clinical theriogenology 2022; 14: 295 granular vulvitis/balanoposthitis in a cattle herd and ureaplasma diversum detection using quantitative real-time pcr and culture dinesh dadarwal,a mallory norfield,a fritz schumann,a navgeet singh,a jarrett ferrier,b megan russnak,b kamal gabadagea adepartment of large animal clinical sciences western college of veterinary medicine, university of saskatchewan, saskatoon, sk, canada biron creek veterinary hospital, sedgewick, ab, canada we aimed to characterize the granular lesions on the mucosae of the vulva/vestibule of females and prepuce/penis of males with the detection of ureaplasma diversum using rt-pcr and culture (gold-standard). a beef cow-calf herd experienced abortions in pregnant heifers last year (cause undetermined). during the current year, 12 heifers returned to estrus within the first 3 weeks of the breeding period (75 days, 2 bulls, 43 heifers). a month into breeding, vaginal and preputial samples from 11 heifers (returned to estrus) and 2 bulls were submitted for ureaplasma rt-pcr (pds, saskatoon). a month after the end of the breeding period, 43 heifers (group 1), 3 second-parity (group 2), and 5 multiparous (group 3) cows were assessed for pregnancy status and granular vulvitis. bulls (n = 5, 1 each for group 1 3 and 2 others) were assessed for granular balanoposthitis. two swabs from the vestibular region of randomly selected heifers (n = 23) and all cows; and prepuce of all bulls were submitted for rt-pcr (pds, saskatoon) and bacteriological culture (ahl, guelph). all 11 heifers and 2 bulls tested positive for ureaplasma rt-pcr on first sampling. on the second examination, 29/43 group 1 heifers, 4/5 group 2 cows and 3/3 group 3 cows had granular vulvitis, whereas 3/5 bulls had balanoposthitis. out of 23 sampled heifers, 18 had granular vulvitis. overall, 28/36 tested animals had lesions, with 61% (17/28) being culture-positive, whereas 63% (5/8) of animals without lesions had a positive culture. all 5 bulls were positive for ureaplasma rt-pcr as well as culture. ureaplasma rt-pcr had an accuracy of 83% compared to culture as a gold standard with sensitivity and specificity of 95% and 64%, respectively. currently, the calving data for different groups at this farm is being gathered. in conclusion, ureaplasma can be cultured in cattle with and without granular lesions. rt-pcr likely overestimated ureaplasma presence.  keywords: ureaplasma, cattle, granular lesions, vulvitis, balanoposthitis developmental competence of immature equine oocytes after in vitro maturation elizabeth metcalf,a david battaglia,a ryan ferris,b thomas o’learya aandrology division; department of obstetrics & gynecology oregon health & science university; portland, or bsummit equine, woodburn, or with increasing demand for in vitro production of equine embryos over the past 10 years, the value of each oocyte recovered has increased. intracytoplasmic sperm injection (icsi) is performed to fertilize oocytes reaching meiosis ii stage of development, determined by microscopic evidence of an extruded polar body (pb), signifying nuclear maturation. cytoplasmic maturation, during which replication of organelles, proteins and rna’s necessary for postfertilization development occurs, is more difficult to evaluate. however, equine oocytes contain large amount of cytoplasmic lipids, and the migration and polarization of lipid granules can be microscopically visualized in mature oocytes. furthermore, the distribution of oocyte lipids may reflect a level of cytoplasmic capacitation (cc) and developmental competence. although the cleavage rate of equine oocytes fertilized following maturation culture does not appear to be significantly different based on lipid polarization,1 this study evaluated the migration and polarization of cytoplasmic lipids (granularity) following in vitro maturation to determine if blastocyst development can be predicted in the absence of pb extrusion. we hypothesized that evidence of cytoplasmic lipid polarization may predict developmental competence of oocytes failing to demonstrate nuclear maturation following maturation culture. cumulus-oocyte-complexes (n = 411) obtained from ovarian follicles by transvaginal aspiration were stored in holding medium overnight at 20oc before undergoing incubation at 38.5oc in 50 µl drops of equilibrated maturation media.2 after 28.5 hours, oocytes were denuded following exposure to 80 iu hyaluronidase, and evaluated for the presence of an extruded pb and lipid reorganization. cytoplasm of immature (mi) oocytes that did not exhibit pb extrusion was graded as a percentage of dark granular appearance. oocytes were fertilized by conventional icsi using icsi-fertile stallions’ semen. fertilized oocytes were cultured2 and examined under inverted 1,000 x light microscopy. culture had 26% (n = 106) immature (mi [without a visible polar body] and 66% (n = 273) mature (m11 [denuded with an intact membrane and an extruded polar body]) oocytes. of the mi oocytes injected, 23% (n = 24) were deemed to have attained some level of cytoplasmic capacitation (cc = 40 70% granularity). a cleavage rate of 25.5% was recorded for injected mi oocytes. of the 24 mi oocytes with some cc, 37% cleaved and continued to develop, with 7 of the embryos developing to the blastocyst stage. of the 82 mi oocytes that did not demonstrate cc, 22% cleaved; however, no blastocysts developed. in conclusion, blastocyst developmental potential was 6.7% per total mi’s injected and 19% per mi oocytes with at least a 40 70% cc. injected oocytes reached a level of cytoplasmic maturation (without nuclear maturation) resulted in blastocyst development. keywords: equine oocyte, maturation, icsi, development, blastocyst references 1. ambruosi, b, lacakandra, gm, iorga, ai, et al: cytoplasmic lipid droplets and mitochondrial distribution in equine oocytes: implications on oocyte maturation, fertilization, and developmental competence after icsi. theriogenology 2009;71:1093-1104. 2. foss, r, ortis, h, hinrichs, k: effect of oocyte transport protocols on blastocyst rated after intracytoplasmic sperm injection in the horse. equine vet j suppl 2013;45:39-43. clinical theriogenology 2022; 14: 296 efficacy of intrauterine infusion of lyophilized interleukin-1 receptor antagonist protein in the prevention of persistent breeding-induced endometritis in the mare emily may,a christina divine,a savannah rocha,b david frisbie,a tawfik aboellail,b patrick mccuea adepartment of clinical sciences, bdepartment of microbiology, immunology and pathology colorado state university, fort collins, co a transient uterine inflammatory response is an inevitable and natural consequence of breeding by either live-cover or by artificial insemination. inflammation is due to an antigenic response to sperm. a majority of young, reproductively normal mares eliminate inflammation within 24 48 hours. unfortunately, the postbreeding inflammatory response may develop into a pathologic condition referred to as persistent breeding-induced endometritis (pbie). this occurs in ~ 15% of broodmares. chronic inflammation is associated with decreased pregnancy rates, premature luteolysis, and a predisposition to infectious endometritis. aim was to compare the efficacy of intrauterine infusion of a lyophilized interleukin-1 receptor antagonist protein (irap) product versus oral firocoxib in the prevention of pbie. we hypothesized that intrauterine infusion of lyophilized irap would improve clinical outcomes associated with pbie. quarter horse mares (n = 8) aged 10 18 years with a biopsy score of ≥ iia were randomly assigned to 1 of 3 treatment groups in a cross-over study design. treatment groups consisted of i. intrauterine infusion of 20 ml of sterile pbs (control); ii. intrauterine infusion of allogenic lyophilized irap reconstituted with 20 ml sterile pbs; and iii. oral administration of 0.3 mg/kg firocoxib. treatments were given 4 hours prior to insemination with 1 x 109 dead sperm. mares received only1 treatment per estrous cycle. transrectal ultrasound examinations and uterine sample collections were performed 4 hours prior to and again at 6, 24, 48, and 72 hours after insemination. at each time point, assessments of intraluminal uterine fluid depth and echogenicity, and endometrial edema were performed. brush cytology samples were collected for uterine cytology and cytokine analysis. cytokines to be evaluated included il1β, il6, cxcl8 (formerly known as il8), tnfα, il10, and il-1ra. additionally, an endometrial biopsy was collected 72 hours postinsemination for histopathologic evaluation of inflammation. regardless of treatment group, mares mounted an inflammatory response at 6 hours postinsemination as indicated by an increase in intrauterine fluid depth and echogenicity, an increase in endometrial edema, and an increase in inflammatory cells on cytology. a progressive decrease in all parameters was noted over the 72-hour observation period. there were no differences among treatment groups relative to intrauterine fluid depth or echogenicity, endometrial edema pattern, or number of inflammatory cells on uterine cytology over the 72-hour period. cytokine analysis and evaluation of endometrial tissue inflammation are pending. in conclusion, neither intrauterine infusion of allogenic lyophilized irap nor oral treatment of firocoxib 4 hours prior to insemination mitigated the uterine inflammatory response to sperm within confines of this endometritis model. keywords: mare, endometritis, breeding, irap, firocoxib effect of nerve growth factor-β given after artificial insemination giorgia podico,a igor canisso,a,b phillip peixoto,a wilson coelho jr.,a lais cunha,a leonardo ribeiro,a shelby crump,a fabio limac adepartment of veterinary clinical medicine bdepartment of comparative biosciences, university of illinois, urbana, il cdepartment of population health and reproduction school of veterinary medicine, university of california, davis, ca objectives were to determine the effects of nerve growth factor-β (ngf), purified from bull’s seminal plasma, given at artificial insemination (ai) on progesterone postai, interferon-stimulated genes (isg), and pregnancy per ai (p/ai) for lactating holstein dairy cows enrolled in a timed ai protocol. we hypothesized that ngf treatment at ai would increase plasma progesterone postai concentrations, upregulate relative abundance of isg, and improve p/ai in lactating dairy cows. holstein cows (n = 557) from a single commercial dairy farm were blocked by parity and randomly assigned to receive an injection containing 296 µg of bovine purified ngf at ai diluted in 2 ml of phosphate-buffered saline (ngf, n = 275), or 2 ml of phosphate-buffered saline (control, n = 282). plasma progesterone concentrations and corpus luteum (cl) size were assessed in a subset of cows (ngf, n = 32; control, n = 36) at days 7, 14, and 19 postai. relative mrna abundance of isg (isg15, mx1, mx2, rtp4) was assessed in peripheral blood leukocytes on day 19 postai. pregnancy diagnosis was performed at 37and 65-days postai. there was an interaction effect between treatment and parity for plasma progesterone, with ngf resulting in greater plasma progesterone postai in primiparous cows, but there were no effects in multiparous cows. however, plasma progesterone and isg did not differ between treatments. there were no effects of ngf for p/ai at 37 days postai (ngf 40.0 versus control 41.6%), 65 days postai (ngf 36.0 versus control 38.1%), or for pregnancy loss (ngf 8.4 versus control 7.7%). current study revealed that effects to ngf in lactating holstein cows were minor and contingent with parity for progesterone concentrations, with no improvement in isg relative abundance or p/ai. keywords: cattle, ngf, progesterone, interferon stimulated gene power doppler as a tool to perform early indirect pregnancy diagnosis in donkeys humberto magalhaes, igor canisso department of veterinary clinical medicine, university of illinois, urbana, il horse embryo induces a differential blood flow in the uterus of pregnant mares; however, in donkeys this is unknown. doppler ultrasonography is becoming increasingly available in portable ultrasound machines. a sensitive, early indirect clinical theriogenology 2022; 14: 297 detection of pregnancy could facilitate breeding decisions and improve reproductive efficiency. this study aimed to assess the uterine blood flow of jennies as an early indirect pregnancy diagnostic test. we hypothesized that uterine vascular hemodynamics increase in pregnant jennies, thus, serving as early indirect pregnancy diagnostic tests. multiparous jennies (n = 44) had daily transrectal ultrasonography until a preovulatory follicle of 30 mm was detected. jennies received deslorelin to hasten ovulation. by 24 hours postdeslorelin, jennies were bred with fresh semen (> 250 x 106 sperm) from 1 jack. ovulation was confirmed by ultrasonography (well. d, medical electronics co, shenzhen, china) performed every other day. transrectal ultrasonography was performed from days 7 10 postovulation to assess the blood flow of each uterine horn with power doppler. vascularization of the endometrium was analyzed using spot meter techniques that measured mean pixel intensity. serum progesterone concentrations were analyzed by radioimmunoassay 7 10 days postovulation. pregnancy diagnosis with b-mode ultrasonography was performed on days 7, 8, 9, 10, and 15 postovulation. data assessed for normality with shapiro-wilk’s test and mixed models. sensitivity, specificity, positive predictive value (ppp), and negative predictive value (npv) for blood flow on days 7 10 postovulation were calculated to identify pregnant animals confirmed 15 days postovulation. significance was set as p ≤ 0.05 and tendency as 0.05 < p < 0.1. out of 44 jennies, 23 (53%) were confirmed pregnant on day 15 postovulation. b-mode ultrasonography identified 17% (4/23) of the embryonic vesicles on day 9 and 39% (9/23) on day 10; no embryonic vesicles were identified on days 7 and 8. pregnant jennies had greater (p = 0.007 uterine vascular blood flow in comparison with nonpregnant jennies (144.8 ± 1.5 versus 139.2 ± 1.3 pixels/ unit). there was no effect of time (p = 0.48) or interaction between group and time (p = 0.70) for the uterine blood flow. progesterone concentrations did not change (p = 0.81) within 7 10 days postovulation and there was no interaction (p = 0.49) between group and time. progesterone concentrations tended to be greater (p = 0.07 in pregnant than nonpregnant jennies. there was no effect of time (p = 0.79), group (p = 0.77), or interaction between time and group (p = 0.92) for uterine horns. sensitivity on days 7 to 10 were 76, 42, 60, and 62%, respectively. whereas specificity from days 7 to 10 were 70, 50, 50, and 75%, respectively. the ppv and npv for days 7 10 were 71 and 15%; 75 and 20%; 75 and 33%; and 80 and 27%, respectively. in conclusion, power-doppler ultrasonography had high sensitivity and specificity for indirect detection of pregnancy in donkeys, particularly, 7 and 10 days postovulation, whereas standard b-mode ultrasonography first detected a small percentage of pregnancies on days 9 and 10. blood flow was not associated with progesterone concentrations. pregnant jennies tended to have higher progesterone than nonpregnant jennies. keywords: donkey, b-mode, power doppler, blood flow, pregnancy outcome impending parturition is characterized by a reduction in progestogens and estradiol concentrations with no correlations with mammary gland secretions in donkeys humberto magalhaes, igor canisso department of veterinary clinical medicine, university of illinois, urbana, il ability to predict parturition allows timely assistance for the dam and neonate; however, limited information is known in the donkey, including the endocrine events upon imminent parturition. this study aimed to describe progestogens and estradiol concentrations in periparturient donkeys and associations with mammary gland secretions. multiparous jennies (n = 37) were monitored daily from 350 355 days of pregnancy to delivery. the ph of mammary gland secretions was assessed daily with a hand-held device (laqua twin, horiba, irvine, ca). aliquots of mammary secretions were frozen daily and then assessed retrospectively for electrolyte concentrations (ca2+, mg2+, k+, and na+) (unicel 800, beckman coulter, switzerland) from 5 days before foaling. serum samples were collected daily from each jenny and frozen at – 20oc until analysed. progestogens and estradiol concentrations were analyzed by radioimmunoassay 5 days prepartum. statistical analyses were performed with graph prisma (version 9.1, san diego, ca). progestogens and estradiol concentrations were analyzed with a mixed model and tukey test. associations between steroid concentrations, pregnancy length, mammary gland secretions, and the progestogens:estradiol ratio (p/e) were determined with pearson’s coefficient of correlation. statistical significance was set as p < 0.05. sensitivity, specificity, negative predictive value (npv), and positive predictive values (ppv) were calculated using a cut-off value for p/e ratio of < 10 (1:10) for parturition in 24 hours. the mean gestation length was 374 ± 8.7 days and ranged from 357 to 390 days. there were effects of time for progestogens and estradiol concentrations (p < 0.0001) and interaction for the concentrations of these hormones and time (p = 0.01). concentrations of progestogens reduced from 18.8 ± 2.7 to 8.8 ± 0.87 ng/ml from 96 to 24 hours before parturition (p = 0.008). estradiol concentrations declined from 96 to 24 hours prior to parturition (140.6 ± 8.3 -115.5 ± 8.4 pg/ml) (p = 0.01). the p/e ratio had 70% sensitivity for parturition in 24 hours, whereas the specificity was 35%. the ppv and npv values were 54% and 52%, respectively. there was a weak (r = – 0.02) and negative correlation between progestogens concentration and gestational length, and a weak (r = 0.17) and positive correlation between estradiol concentrations and pregnancy length. there was a weak correlation (r = 0.17) between ph of the mammary gland secretions and steroid hormones concentration (r = 0.17 progestogens; r = 0.01 estradiol), and a weak and negative correlation with the p/e ratio (r = – 0.07). progestogens presented a weak and negative correlation for ca ( r = – 0.22) and na (r = – 0.07) and weak and positive correlations for k (r = 0.19) and mg (r = 0.03). estradiol was moderately, negatively correlated with ca (r = – 0.39) and k (r = – 0.18) and positively correlated with na (r = 0.31) and mg (r = 0.40). in conclusion, the p/e ratio had moderate sensitivity but low specificity for parturition in 24 hours. there were weak correlations between the p/e ratio, mammary gland ph, and clinical theriogenology 2022; 14: 298 electrolytes. this species demonstrated a reduction in these hormones like horses, but no distinct peak in estradiol as observed in ruminants. keywords: donkey, steroid concentrations, progestogens, estradiol ratio, predict foaling donkeys have a higher incidence of hemorrhagic anovulatory follicles and multiple ovulations in spring humberto magalhaes, igor canisso department of veterinary clinical medicine, university of illinois, urbana, il in horses, hemorrhagic anovulatory follicle (haf) is a type of ovulation failure associated with fall and spring transitions, equine metabolic syndrome, cloprostenol treatment, retained endometrial cups, aging (> 16 years), and use of nonsteroidal antiinflammatory drugs. to date, haf has not been well described in donkeys. thus, this study aimed to assess the associations between season, body condition scores (bcs) of mares with haf, and multiple ovulations. we hypothesized that season and bcs are associated with haf and multiple ovulations in jennies. estrous cycles (n = 190) of 103 jennies kept in a humid subtropical climate were analyzed. jennies had transrectal ultrasonography every other day and when a preovulatory follicle (≥ 30 mm) was detected, they were bred with fresh semen at 48-hour intervals (> 250 x 106 sperm) until ovulation was confirmed via ultrasonography. jennies were bred in the spring, fall, and summer. day of ovulation and first day of detection of haf (i.e., excessive specks floating in the follicle antrum) were designated as day 0. follicle size and bcs (1 5) were recorded during each ultrasonography. statistical analyses were performed with mixed models followed by sidak for parametric data and kruskal-wallis followed by dunn’s test for nonparametric data. significance was set as p ≤ 0.05 and tendency as 0.05 < p <0.1. overall incidence of haf was 9.4% (18/190 cycles). the same jennies displayed 27.7% (5/18) of haf (4 in the spring and 1in summer). there was an effect (p = 0.0009) of the season on haf incidence. spring cycles (19.6%; 12/61) had a greater (p = 0.0009) incidence of haf than summer (6.6%; 3/45), and fall (4.7%; 3/45) cycles with no differences (p > 0.99) between the latter 2 seasons. season did not affect follicular size for regular cycles (spring 38.2 ± 4.17 mm, summer 38.1 ± 4.78 mm, and fall 37.8 ± 2.88 mm) or haf cycles (spring 36.6 ± 3.6 mm, summer 39.1 ± 5.7 mm and fall 35.3± 1.63 mm), and there was no interaction (p > 0.05) between season and follicular size, consistent with a nonseasonal species. follicular sizes at last ultrasonography before day 0 were similar (p = 0.53) between haf (37.1 ± 3.9 mm) and regular cycle (37.6 ± 4.1 mm). there were no effects (p = 0.59) of season or interaction with bcs. there was an interaction (p < 0.0001) between follicle size before day 0 and there was no interaction (p = 0.86) with follicles from regular cycles. higher bcs (5) presented larger (p < 0.05) haf sizes before day 0 (41.5 ± 2.1 mm) in comparison to bcss 2 (36 ± 3.6 mm), 3 (35.5 ± 3.5 mm), and 4 (37 ± 4.3 mm). there was an effect (p = 0.0004) of the season on the incidence of multiple ovulations. spring cycles (21.3%; 13/61) had a greater incidence (p = 0.61) of multiple ovulations than fall (3.6%, 3/84; p=0.003); however, not different than summer (13.3%; 6/45), and the latter was not different (p > 0.99) from each other. there was no apparent association between haf and multiple ovulations. in conclusion, jennies had a higher incidence of haf and multiple ovulations in spring than in summer and fall. size of the preovulatory follicle of cycles representing normal ovulation did not differ from those with haf. season did not alter haf size. keywords: jennies, hemorrhagic follicle, haf, season field study on effects of postbreeding uterine therapy on fertility in donkeys humberto magalhaes, igor canisso department of veterinary clinical medicine, university of illinois, urbana, il postbreeding therapies (e.g., uterine lavage, ecbolic, and antiinflammatory drugs) are routinely applied to promote uterine clearance and enhance fertility in horses. however, the literature is scant in donkeys. additionally, it has been suggested that donkeys are prone to postbreeding inflammation. thus, aim of the study was to assess the effects of postbreeding therapies on pregnancy rates and losses in donkeys. we hypothesized that jennies treated 6 hours postbreeding have higher fertility than untreated jennies. estrous cycles (n = 250) of 132 jennies were included in the study. jennies were examined every other day until a preovulatory follicle (30 mm) was detected via transrectal ultrasonography. then jennies were bred every other day with fresh semen (> 250 x 106 sperm) until ovulation was confirmed. ovulation was not induced. six hours after the first-ai, cycles were randomly allocated into 4 groups: a. uterine lavage (1 liter of lactated ringer’s solution (lrs) with 100 ml of dmso added (lrs-dmso; n = 67 cycles); b. uterine lavage, (1 liter of lrs + 20 iu intravenous oxytocin) (lrs-oxy; n = 65 cycles); c. 20 iu single intravenous oxytocin treatment (oxy; n = 43 cycles); and d. no treatment (control; n = 75 cycles). number of ai/cycle was recorded and compared among groups. pregnancy diagnosis was performed on day 15 postovulation and confirmed on day 45 postovulation; losses between the first and second pregnancy diagnosis were accounted for as pregnancy loss. data were analyzed with graph prisma (version 9.1, san diego, ca). pregnancy rates and losses were analyzed with multiple logistic regression. significance was set at p ≤ 0.05 and tendency as 0.05 < p < 0.10. pregnancy rates were not different (p = 0.54): lrs-dmso 47.7% (32/67-cycles); lrs-oxy 32.3% (21/65-cycles); oxy 32.3% (43/75-cycles); and control 37.3% the pregnancy losses were not different (p = 0.93): lrs-dmso 6.25% (2/32-pregnancies); lrs-oxy 4.7% (1/21-pregnancies); oxy, 2.3% (1/43-pregnancies); and control 1.3% (1/75-pregnancies). there were no differences (p = 0.11) in ai/cycle number among groups: lrs-dmso (1.3 ± 0.4), lrs-oxy (1.1 ± 0.3), oxy (1.3 ± 0.4), and control (1.4 ± 0.5). in conclusion, uterine therapy did not improve pregnancy rates compared to control. keywords: jennies, endometritis, dmso, oxytocin, pregnancy rate clinical theriogenology 2022; 14: 299 response to deslorelin and fertility according to follicle size in donkeys humberto magalhaes, igor canisso department of veterinary clinical medicine, university of illinois, urbana, il follicle size is the primary criterion used to determine when mares or jennies need to be bred. in horses, endometrial edema is also included in the criteria. however, donkeys are notorious for not having as reliable endometrial edema patterns as horses; thus, follicle size is even more relevant. one recent study reported ovulatory response to deslorelin in small donkeys, but not fertility. this study aimed to determine the ovulatory response and fertility according to follicle size at deslorelin administration in large donkeys. we hypothesized that follicle size at induction affects donkeys’ ovulatory response and fertility. estrous cycles (n = 100) of 64 jennies received deslorelin to induce ovulation, and 83 cycles of 51 jennies did not receive deslorelin and were considered controls. the cycles were arbitrarily arranged based on the follicle sizes at induction/control as follows: size-1 (30 33mm [n = 27 induced and n = 26 control]); size-2 (34 -37 mm [n = 40 induced and n = 38 control]); and size-3 (38-41 mm [n = 33 induced and n = 29 control]). ovulation was confirmed via transrectal ultrasonography performed at 12 hour intervals. after detecting predetermined follicle size, jennies were bred every other day until ovulation with fresh semen from 1 jack (> 250 x 106 sperm). pregnancy diagnosis was performed on days 15 and 45 postovulation. data were analyzed with mixed models followed by sidak for parametric data and kruskal-wallis, followed by dunn’s test for nonparametric data. there was an effect of group (sizes 1-3) (p < 0.0001) and induction (p = 0.004) on the ovulatory follicle size. induction of ovulation decreased ovulatory sizes among groups (p < 0.05), size-1 30.6 ± 0.89 mm versus 32 ± 1.17 mm; size-2 35.9 ± 0.84 mm versus 36.8 ± 0.4 mm; and size-3 39.2 ± 0.9 mm versus 39.6 ± 1.2 mm for induced and control cycles, respectively (p > 0.05). percentage ovulatory response arranged by 12 hour intervals were: size-1 (induced versus control) 12 24 hours (3.3 versus 0%); 24 36 hours (7.4 versus 2.4%); and 36 48 hours (15.8 versus 5.2%); size-2 (induced versus control) 12 24 hours (18 versus 9.5%); 24 36 hours (25.6 versus12.7%); and 36 48 hours (19.8 versus 10%); size-3 (induced versus control) 12 24 hours (5 versus 5.4%); 24 36 hours (2.2 versus 1.2%); and 36 48 hours (0 versus 1%). five jennies did not ovulate within 48 hours in the control group, and 3 in the induced group. deslorelin resulted in 97% (97/100) of cycles ovulating in 48 hours, whereas for the control 47.4% (39/83) ovulated in the same interval (p = 0.002). pregnancy rates were similar (p = 0.37) between induced (38%) and control (29%) cycles. pregnancy loss was similar (p = 0.34) between induced 13% (5/38) and control 25% (6/24) cycles. induced cycles had fewer (p < 0.0001) ai/cycle than control. percentage of ai per cycle was (induced versus control): oneai 74 versus 61.4%; two-ai 26 versus 25.3%; and three-ai 0% versus 13.3%. number of ai/cycle (induced versus control were: one-ai/cycle (55 versus 17.8%); two-ai/cycle (45 versus 50%); three-ai/cycle (0 versus 17.7%). in conclusion, deslorelin was 97% efficient at concentrating ovulations in 48 hours and at reducing ovulatory follicle size and number of ai/cycle. pregnancy rates were similar between the deslorelin and control groups. keywords: jennies, deslorelin, ovulation, pregnancy rate effects of season on fertility and losses in donkeys humberto magalhaes, igor canisso department of veterinary clinical medicine, university of illinois, urbana, il donkeys are deemed nonseasonal polyestrous species. they display regular cyclicity throughout the year. other nonseasonal polyestrous species (e.g., cattle and pigs) experience seasonal variations in fertility and pregnancy losses. season does not seem to affect fertility in donkeys kept in tropical weather; however, the seasonal variation in fertility and pregnancy losses has not been assessed in jennies kept under continental weather. this study aimed to evaluate the effects of the season on the fertility, number of ai/cycle, and pregnancy losses in donkeys kept under continental weather. we hypothesized that season affects fertility and losses in jennies. one hundred and forty-four cycles of 82 jennies were evaluated. the cycle distributions were as follows: spring (n = 53), summer (n = 46), and fall (n = 45). transrectal ultrasonography was performed every other day until detection of a preovulatory follicle (> 30 mm). then, jennies were bred every other day with fresh semen from 1 jack (> 250 x 106 sperm) until detection of ovulation. ovulation was confirmed by ultrasonography performed at 24-hour intervals. pregnancy diagnosis was performed on days 15 and 45 postovulation. data were analyzed with mixed models followed by tukey for follicle size, number of ai/cycle, and season. chi-square test was used to assess pregnancy rates and losses. there was no difference (p = 0.42) in follicle size across seasons, consistent with nonseasonal species. follicle sizes before ovulation related to the ai/cycle were: 39 ± 0.4mm, 41 ± 3.3mm in spring; 41.3 ± 4.7mm, 38.4 ± 7.9 mm in summer; and 41.5 ± 5.2mm, 39.3 ± 4mm in fall for 1 and 2 ai/cycle, respectively. pregnancy rates were similar (p = 0.91 [39.6, 32.06, and 31.1% for spring, summer and fall respectively]. there was no difference (p = 0.33) in pregnancy loss across seasons with 4.7, 6.6, and 5.1% for spring, summer, and fall, respectively. there was no effect on ai/cycle (p = 0.41), season (p = 0.95,) or interaction between ai/ cycle and season (p = 0.21). the fertility related to the season and number of ai/cycle were 11/30 versus 10/23; 12/35 versus 3/11; 9/29 versus 3/16 for 1 versus >1 ai/cycle in spring, summer, and fall, respectively. there was no difference (p = 0.85) on the overall ai/cycle and fertility; 1 ai/cycle accounted for 62/94 versus 34/50 for > 1 ai/cycle. in conclusion, season did not affect follicle size prior to ovulation, number of ai/cycle, pregnancy fertility, or pregnancy loss. the number of ai/cycle did not affect fertility. keywords: jennies, follicle size, ai/cycle, fertility clinical theriogenology 2022; 14: 300 effect of different extenders used for selection of equine sperm by the swim-up technique joaquin paredes,a laura favetta,b eduardo ribeiro,c mariana diel amorim,d michael neal,e tracey cheniera adepartment of population medicine, bdepartment biomedical sciences ontario veterinary college, university of guelph, guelph, on, canada cdepartment animal biosciences, ontario agricultural college university of guelph, guelph, on, canada ddepartment of clinical sciences, college of veterinary medicine cornell university, ithaca, ny eone fertility, burlington, on, canada equine sperm are highly susceptible to damage under suboptimal incubation conditions. successful assisted reproductive technologies (art) rely on these functional aspects such as motility which may be impaired by sperm processing methods. we hypothesized that various extenders affect sperm function influencing motility parameters of the recovered samples. frozen-thawed sperm from 7 stallions was diluted (25 x 106 sperm/ml) and evaluated after being incubated with 1 of 4 sperm media extenders including: i. modified human tubal fluid (h-htf); ii. modified chatot-ziomek-bavister (spczb); iii. modified tyrode’s solution (sp-talp); and iv. control (inra 96). samples were maintained at room temperature and motility was evaluated by computer assisted sperm analysis (casa) at 4 time points (0, 2, 6, and 12 hours). statistical analyses were performed using anova for repeated measures. type of media and culture time had a significant interaction on total motility (tm, p = 0.0059) and progressive motility (pm, p = 0.0069). the inra extender had the highest values for tm and pm at all time points and had no significant reduction in values over time. in comparison to inra, h-htf did not differ in tm and pm; however, sp-czb had lower tm (p = < 0.0001) and pm (p = < 0.0001) in comparison to control, with a significant reduction at 12 hours for tm (p = < 0.01) and by 6 hours for pm (p = 0.011). similarly, sp-talp had lower tm (p = < 0.001) and pm (p = < 0.0001), and a reduction at 6 hours for tm (p = 0.0169) and by 2 hours for pm (p = 0.03). additionally, we evaluated the role of the same extenders, except inra, on sperm selection using the swimup method. paired frozen-thawed sperm samples from 6 stallions were incubated under 1 ml of media (h-htf/ sp-czb/ sp-talp) at 37° c at a 45-degree angle, for 2 incubation times (20 and 60 minutes). after incubation, 60% of the upper fraction of the supernatant was recovered and evaluated for motility (casa), morphology (hancock stain), viability (hancock stain), concentration (hemocytometer) and recovery rates. statistical analysis was performed using a two-way anova. extenders and time had no effect on morphology, viability or recovery rates, although both affected tm (p = < 0.01; 0.027) and pm (p = < 0.001; 0.017). the media also had effects on path velocity (vap, p = < 0.001), curvilinear velocity (vcl, p = < 0.001) and linear velocity (vsl, p = < 0.001). when comparing between groups, h-htf media had higher values for tm (p = < 0.001), pm (p = < 0.001), vap (p = < 0.001), vcl (p = < 0.001) and vsl (p = < 0.0001) in comparison to sp-czb, and similarly, higher values for tm (p = 0.01), pm (p = < 0.001) and vsl (p = 0.03) in comparison to sp-talp; the latter had higher values for vap (p = 0.011) only in comparison to spczb. based on these results, it is evident that extenders used in sperm selection influence sperm functions, such as motility, and considering that no changes in recovery were detected, we concluded that the main influence of extender are changes in motility parameters of recovered sperm. this evidence affirmed the importance of culture media selection to optimize sperm recovery to improve equine art outcomes. keywords: stallion sperm, extender, motility, selection, swimup effects of semen cooling extender and sperm prestaining with hoechst 33342 on the longevity of cool-stored, sex-sorted stallion sperm juan samper,a camilo hernández-avilés,a luisa ramírezagámez,a sheila spaceka pablo ross,b cristina cabrera,b juan moreno,b charles lovea acollege of veterinary medicine and biomedical sciences texas a&m university, college station, tx bst genetics, navasota, tx standardizing a protocol that allows stallion sperm to be collected in a remote location, being sent to a semen sex-sorting laboratory, and sent back to a breeding farm would permit wider dissemination of this technology in the equine breeding industry. immediately prior to sex-sorting, sperm is stained with a dna probe (e.g., hoechst 33342). we studied the effect of using 2 commercial semen extenders for shipping semen and the effect of prestaining sperm before being sent to a sex-sorting laboratory, on the quality of cool-stored, sex-sorted stallion sperm. we hypothesized that there would be a significant extender effect and a significant interaction between extender and duration of exposure to the hoechst 33324. three ejaculates from each of 4 stallions (n = 12) were diluted at a ratio of 1: 3 with either inra-96® (inra) or botugold® (bg) semen extenders, either prestained or not with hoechst 33342 (yes or no) and stored for 24 hours at 6°c. after storage, the semen was processed by colloid centrifugation and sent to a sex-sorting laboratory. sperm quality parameters were recorded immediately after sorting at 24 hours, and after cooled storage of sex-sorted sperm at 48 hours. at each time point (24 or 48 hours), sperm total and progressive motility (% tmot and % pmot, respectively) were analyzed using casa, whereas sperm viability (% viab) and dna damage (% compa-t) were determined using a nucleocounter sp-100™ and flow cytometry, respectively. at each time point, comparisons among treatments were made in rank-transformed data using the general linear model (jmp pro 16.0). statistical significance was set at p < 0.05. a stallion-by-treatment interaction was observed in 2/4 stallions (p < 0.05). at 24 hours, inra-no, bg-no, and inra-yes resulted in similar (p > 0.05) mean tmot (73 versus 75 versus 72%, respectively) whereas inra-no and bg-no had higher (p < 0.05) tmot than bgyes (70%). mean pmot was similar (p > 0.05) in inra-no, bg-no, and bg-yes (50 versus 60 versus 52%, respectively), whereas higher (p < 0.05) than in inra-yes (39%). mean viab was higher in bg-no than in inra-yes (85 versus 82%), whereas similar to bg-yes and inra-no (83 and 84%, respectively; p > 0.05). mean compa-t was similar (p > 0.05) among clinical theriogenology 2022; 14: 301 all treatments (range: 4 5%). at 48 hours, mean tmot and pmot were similar (p > 0.05) in inra-no and bg-no (39 and 16 versus 45 and 20%, respectively), whereas higher (p > 0.05) than in inra-yes and bg-yes (24 and 12% versus 26 and 10%, respectively). mean viab and compa-t were not affected (p > 0.05) by any of the treatments (range: 72 77% and 8 10%, respectively. the sperm quality parameters observed in sex-sorted stallion sperm that was prestained before submission, although slightly lower than in the nonstained sperm, were considered acceptable when compared to those observed in sex-sorted stallion sperm that was not prestained. these results indicated that stallion sperm can be processed either in inra-96® or botugold® semen extenders, pre-stained with hoechst 33342, cool-stored for 24 hours, submitted to a sex-sorting laboratory, and then cooled again after sex-sorting for additional 24 hours, opening the possibility of making this technology accessible to a wider range of breeders. keywords: stallion sperm, sex-sorting, cooled storage, hoechst 33343, inra-96®, botugold® clinical observations of bacterial culture results after intrauterine infusion of misoprostol karen von dollen,a kristina lu,a sara lyle,b justine elam,a karen wolfsdorfa ahagyard equine medical institute, lexington, ky bdepartment of clinical sciences, college of veterinary medicine north carolina state university, raleigh, nc deep horn intrauterine deposition of misoprostol has been demonstrated to be an effective method of achieving pregnancies in mares that have otherwise been subfertile.1 as the use of this technique has increased in recent years, sporadic anecdotal reports of profound uterine inflammation have been reported. as part of standard practice, the authors routinely sample the uterus by low volume lavage for culture and cytology on the day following misoprostol infusion. to objectively assess uterine response to misoprostol infusion, the records of 36 mares infused with misoprostol during a 1-year period (august 2020 august 2021) were evaluated. mares were presented for this procedure for failure to have produced a 14-day pregnancy despite appropriate breeding timing and no evidence of bacterial endometritis by industry standard screening methods (endometrial swab). two 200 µg tablets of misoprostol (cytotec®, pfizer) were dissolved in 3 ml sterile saline and 2 aliquots were made, 1 for each uterine horn. aliquot was deposited at the distal end of each uterine horn by standard deep horn insemination technique using a flexible pipette and transrectal guidance. the following day, 1 liter of sterile lactated ringer’s solution (lrs) was instilled into the uterus and immediately collected back into the original bottle. this low volume lavage was submitted for culture and cytology to a single laboratory. bacterial growth was reported as no growth, scant (< 10 colony forming units), light (growth on 1 quadrant of plate only), moderate (growth on 2 quadrants of plate), or heavy (growth on 3 quadrants of plate). of the 36 mares infused with misoprostol, follow-up was available for 32. of these, 7 (22%) had no bacterial growth, 5 (16%) had scant bacterial growth, 6 (19%) had light bacterial growth, 10 (31%) had moderate bacterial growth, and 4 (13%) had heavy bacterial growth. mares were then treated for bacterial endometritis based on these results at the discretion of the attending clinician. use of intrauterine misoprostol has expanded throughout clinical practice since publication of the technique. whereas increased detection of previously undiagnosed bacterial endometritis was not included as a benefit of the procedure, results suggested that this may be an unintended utility. sixty-three percent (20/32) of mares infused with misoprostol responded to the infusion with clinically significant bacterial endometritis (light, moderate, or heavy bacterial growth) that had otherwise gone undiagnosed during previous workup. this phenomenon remains unexplained, but potential hypotheses include stimulation of dormant bacteria through the inflammatory insult of intrauterine infusion of a foreign substance, physical irritation of the endometrium by the deep horn infusion technique, or acute immune system downregulation secondary to misoprostol which allows for bacterial proliferation. despite not yet understanding the underlying mechanism by which this process occurs, results from this group of mares suggested that there is substantial clinical value in culturing mares following misoprostol infusion. keywords: mare, endometritis, misoprostol, barren reference 1. alvarenga ma, segabinazzi lg: application of misoprostol as a treatment of unexplained infertility in mares. j equine vet sci 2018;71:46-50. incidence and effect of low serum progesterone concentrations at day 6 postovulation on pregnancy rates in mares karen wolfsdorf,a elam justine,a marie gantz,b ben espy,c karen von dollen,a kristina lu,a william allend† ahagyard equine medical institute, lexington, ky bgluck equine research center, lexington, ky cben espy pllc, san antonio, tx dthe paul mellon laboratory, newmarket, uk (†deceased) low serum progesterone (p4) concentration is 1 factor that may contribute to early pregnancy failure (epf) in the mare. the threshold value for pregnancy maintenance is 2.5 4 ng/ ml. mares with epf are routinely placed on exogenous p4. this is expensive, poses human health risks, immunosuppressive, and potentially unnecessary. therefore, we hypothesized that measuring serum p4 concentrations on day 6 (d6) postovulation can enhance the efficacy of p4 supplementation on pregnancy rates. progesterone concentrations were measured in 341 thoroughbred mares on d6 postovulation, over 500 bred estrous cycles. the study spanned 2 consecutive breeding seasons. during estrus, daily rectal ultrasonography was performed, +/endometrial culture and human chorionic gonadotropin or deslorelin was given 24 hours prior to mating. ovulation was confirmed 12 24 hours after breeding via transrectal ultrasonography. blood collected on d6 post ovulation (day 1 defined when the preovulatory follicle was replaced by a corpus hemorrhagicum) was cooled within 6 clinical theriogenology 2022; 14: 302 hours after collection and centrifuged for serum collection within 36 hours. an accurate quantitative enzyme linked immuno assay measured serum p4. in year 1, p4 concentrations were determined immediately and < 4 ng/ml initiated altrenogest supplementation. in year 2, samples were stored at – 20°c and subsequently analyzed. pregnancy was diagnosed 14/15 days postovulation via transrectal ultrasonography. mares were assigned to 3 groups based on d6 p4 concentrations (group 1, < 4 ng/ml; group 2, 5 6 ng/ml; group 3, > 6 ng/ml) and pregnancy rates determined. pregnancy rates among 3 groups were compared in each season, using a chisquared test. group 1 pregnancy rates were compared using a chi-squared test between years 1 and 2. in addition, pregnancy rates were compared between years 1 and 2 for mares with p4 concentrations of < 2.5 and > 2.5 4.0 ng/ml. in year 1, 15/149 (10.06%) and year 2, 38/351(10.8%) mares exhibited p4 concentrations of < 4 ng/ml respectively. pregnancy rates for 3 groups during years 1 and 2, respectively were: group 1, 7/15 (47%), 21/38 (55.8%); group 2, 22/40 (55%), 67/104 (64.4%); and group 3, 62/94 (65.9%), 130/207 (62.8%). there was no difference among groups or between years 1 and 2 pregnancy rates with p4 concentrations of < 4 ng/ml or < 2.5, > 2.5 4.0 or > 4 ng/ml, or between < 2.5, > 2.5 4.0, or > 4 ng/ml in year 2. results indicated that primary luteal insufficiency occurred in 10 -11% of estrous cycles. exogenous p4 supplementation did not increase pregnancy rates. there was no difference in pregnancy rates of mares with concentrations of < 2.5 ng/ml or > 2.5 < 4 ng/ml (61.3 versus 50%) when no exogenous p4 was given. although exogenous p4 therapy did not appear beneficial, and numbers in this category were small, earlier measurement of serum p4 concentrations on d6 may reduce overuse of exogenous p4. results further suggested that other factors are more likely to be the cause of epf or poor pregnancy rates in the mare. keywords: mare, progesterone, early pregnancy loss, luteal insufficiency ovulatory response to gnrh-agonist during early and late fall in mares kianna spencer,a giorgia podico,a ameer megahed,b kristi jones,b joão bittar,b igor canissoa adepartment of veterinary clinical medicine, university of illinois urbana-champaign, il bdepartment of large animal clinical sciences, university of florida, gainesville, fl deslorelin acetate (deslorelin) is the most popular drug used to induce ovulation in mares, likely due to reliable response in older mares and consistent availability. response to this gnrh-agonist during spring and summer is reported as ~ 90% with ovulation occuring ~ at 40 hours.1 however, despite its efficacy and popularity, its ability to induce ovulation in the fall is debated. endometrial edema score is an important clinical criterion to determine a follicle’s readiness to respond to induction of ovulation; however, it is suggested that endometrial edema is lower in the fall, hence the presumed poor gnrh-response. objective was to assess the ovulatory response of deslorelin in mares during fall by comparing success in inducing ovulation, interval to ovulation, edema scores, and parameters of the resultant corpus luteum (cl) in early and late fall. we hypothesized that deslorelin has a better ovulatory response during early fall than late fall. mares (n = 22) kept in 40o latitude were checked via transrectal ultrasonography until a preovulatory follicle was detected then ovulation was induced with intramuscular deslorelin (1.8 mg). ovulation was confirmed by ultrasonography performed at 24 and 36 hours postinduction and repeated at 2 hour intervals. serum progesterone concentrations (chemiluminescence) and luteal tissue area (via ultrasound-calipers) were determined daily to assess cl function. a dose of intramuscular pgf2α (7.5 mg) was given 8 days postovulation and each cycle was repeated up to 5 times. associations between local weather and endpoints were analyzed. cycles were grouped as early (n = 55) and late fall (n = 45) based on date of induction. cycles with spontaneous ovulation < 24 hours (n = 6) or failure more than 48 hours (n = 2) from deslorelin were discarded. parametric data was analyzed with mixed models, anova, tukey’s test, and pearson’s correlation. nonparametric data were analyzed with fisher’s test, kruskal-wallis, and dunn’s test. significance was defined as p < 0.05. deslorelin effectively induced ovulation in 90% of cycles. spontaneous, failure, and multiple ovulations were similar (p > 0.05) between early and late fall. the interval from induction to ovulation was identical (p = 0.55) in early (40.6 ± 0.4 hours) and late (41.2 ± 0.5 hours) fall. percentage of mares ovulating between 36 48 hours and 38 44 hours postdeslorelin did not vary (p > 0.05), between early (91 and 62%) and late (95 and 60%) fall respectively. follicle size at induction tended (p = 0.07) to be smaller in early (36.4 ± 0.4 mm) than late (37.4 ± 0.4 mm) fall. edema scores varied (p < 0.001) with time relative to ovulation and were lower (p = 0.01) in late fall. there was a negative correlation (r = – 0.88, p = 0.05) between edema score on interval to ovulation. progesterone concentrations varied (p < 0.001) with time but did not differ (p = 0.73) between early and late fall and correlated weakly with the luteal area (r = 0.13; p = 0.031). lower temperature was associated with smaller follicle size at induction (p = 0.0021) and ovulation (p = 0.009), and lower relative humidity was associated with larger follicle size at ovulation (p = 0.032). in conclusion, the findings here demonstrated for the first time that mares respond satisfactorily to gnrh-agonists in the fall. despite lower endometrial edema scores in late fall, the indistinguishable difference for other parameters between early and late fall suggested poor response to gnrh-agonist in the fall, independent of edema score. keywords: ovulation, deslorelin acetate, progesterone, luteal function reference 1. ferris ra, hatzel jn, lindholm arg, et al: efficacy of deslorelin acetate (sucromate) on induction of ovulation in american quarter horse mares. j equine vet sci 2012; 32:285-288. clinical theriogenology 2022; 14: 303 sperm parameters and fertility of donkey semen cooled using skimmed milk, sodium caseinate, and egg yolk-based extenders lorenzo segabinazzi,a,b mariana gobato,a verônica scheeren,a rafael bandeira,a camila dell´aqua,a josé dell´aqua jr,a marco alvarenga,a frederico papaa afaculty of veterinary medicine and animal science são paulo state university (unesp), botucatu, sp, brazil bross university school of veterinary medicine, basseterre, st. kitts, west indies in brazil and the us, the production of mules is increasingly prized, and the high value of top mules has generated an interest in donkey reproduction. however, donkey semen does not well tolerate cooling with skim milk-based (skm) extenders. therefore, this study aimed to compare semen parameters and fertility of cooled donkey semen extended in 3 different extender bases in the presence and absence of seminal plasma. in experiment 1, 18 ejaculates from 6 mature jacks were harvested and divided into 6 aliquots. three of them were immediately diluted at 50 x 106 sperm/ml in 3 commercially available semen extenders: skm containing cholesterol-loaded cyclodextrin (clc) (botuspecial®); sodium caseinate-based with clc (sc, botugold®); or egg yolk-based (ey, botucrio®) extenders. remaining aliquots (n = 3) were also diluted at 50 x 106 sperm/ml with skm (n = 1) or sc (n = 2) and then subjected to centrifugation at 600 x g/10 minutes. after centrifugation, the supernatant was discarded, and the sperm pellet of 2 out of 3 samples were resuspended with the same semen extender (skm-c and sc-c); 1 sample previously diluted with sc was resuspended in ey (ey-c). after centrifugation, sperm were resuspended at 100 x 106 sperm/ml. all samples were stored in a passive cooling container at 5°c for 48 hours. total motility (tm), progressive motility (pm), and percentage of rapid sperm (rap) were assessed with casa. plasma membrane stability (pms) and mitochondrial membrane potential (mmp) were assessed with the combination of yo-pro® and mitostatusred with flow cytometry. semen was assessed before (0) and 24 and 48 hours after cooling. in experiment 2, 118 estrous cycles of 15 mares were used for fertility assessment during 2 breeding seasons. mares were randomly bred 24 hours postinduction of ovulation with semen from 2 jacks (jack 1, n = 90; jack 2, n = 28) cooled for 24 hours with either of the treatments (skm, sc, ey, skm-c, sc-c, or ey-c). pregnancy diagnosis was performed at day 15 postovulation; mares then received intramuscular dinoprost (5 mg) to return to estrus. data were evaluated by anova and tukey’s post hoc test. pregnancy rates were evaluated by fisher’s exact test. significance was set at p < 0.05. there were no differences (p > 0.05) in tm, pm, rap, pms, and mmp for semen in either group at hour 0. there was a reduction (p < 0.05) in tm, pm, rap, pms, and mmp over time across groups. groups ey, scc, and ey-c had superior (p < 0.05) tm, pm, rap, pms, and mmp than skm. semen in the skm-c and sc groups had intermediate values (p > 0.05). centrifugation positively affected (p < 0.05) sperm parameters in donkey semen extended in skm (skm versus skm-c). mares bred with semen extended in sc (74%, 14/19), sc-c (89%, 17/19), ey (90%, 18/20), or ey-c (79%, 15/19) had a greater (p < 0.05) conception rate than mares bred with skm (30%, 6/20). mares bred with skm-c had intermediate conception rates (62%, 13/21). in conclusion, sc containing clc and ey-based extender appeared more affordable to preserve the sperm characteristics and fertility in cooled donkey semen. additionally, centrifugation positively impacted donkey semen extended in skmbased extenders. keywords: mule, jack, stallion, artificial insemination, cryopreservation feeding spent hemp biomass does not negatively affect ram fertility melanie meador,a niamh nolan,a massimo bionaz,a serkan ates,a michelle kutzlera adepartment of animal and rangeland sciences oregon state university, corvallis, or industrial hemp was recently removed from the controlled substances act and classified as an agricultural product. after cannabidiol (cbd) extraction, several tons of spent hemp biomass (shb) containing a residual amount of cbd is left over. shb is high in crude protein, omega-3 fatty acid, and omega-6 fatty acid, suggesting it could be used in livestock diets. however, use of shb as feed in livestock diets have not been approved by the fda yet because of the paucity of information about cannabinoid residuals and their effects on animal health, production, reproduction, and product quality. chronic exposure to cannabinoids, including cbd, can negatively affect fertility in mice. objective was to determine the reproductive effects of feeding shb to rams. we hypothesized that feeding shb to rams may adversely affect fertility. a feeding trial was performed with 6-month-old polypay rams. rams were randomly assigned to 5 feeding trial groups (7 per group) and fed either alfalfa (control) or shb at 10 or 20% for 4 weeks, with 4 weeks withdrawal from shb, or shb at 10 or 20% for 8 weeks. blood samples were collected from the jugular vein on days 0, 28, 56, and serum was analyzed for testosterone concentrations via chemiluminescence (immulite 1000®, siemens). at the conclusion of the feeding trial, rams were euthanized via penetrating captive-bolt and testes were removed. sperm was collected from the vas deferens and evaluated for motility (total, progressive, speed), morphology, and concentration using routine methods. an analysis of variance was used to analyze the date and significance was defined as p < 0.05. testosterone concentrations increased (p < 0.01) over time in all groups but there was no effect of animal (p = 0.58), treatment (p = 0.30), or time*treatment (p = 0.75). however, rams that were fed either 10 or 20% shb for 8 weeks had higher (p < 0.05) total and progressive sperm motility and higher sperm motility speed compared to controls there was no difference (p > 0.05) in sperm concentration or the percentage of morphologically normal sperm per group based on these results, we concluded that shb does not have a negative reproductive effect on male endocrinology and may improve fertility with respect to sperm motility. keywords: cannabidiol, hemp, motility sperm, testosterone clinical theriogenology 2022; 14: 304 effect of caslick’s surgery as a treatment for behavioral problems in mares mette christoffersen, louise christensen, camilla hughes, hanne pedersen section for reproduction and obstetrics department of veterinary clinical sciences university of copenhagen, denmark referred vaginal pain causes behavioral problems in mares (including decreased rideability), and a direct link between windsucking, pneumovagina, and subsequent chronic irritative vaginitis causing referred vaginal pain syndrome has previously been demonstrated. aim was to retrospectively evaluate the effects of caslick’s surgery in mares with a reported history of behavioral problems. an additional aim was to describe the owner’s perception of the mare’s clinical presentation related to the behavioral problems. a retrospective study using an online questionnaire was performed. participants were recruited on danish equestrian social media platforms. questionnaire included 19 questions and was designed in surveymonkey®. general variables (age, breed, use, and competition participation) and specific variables related to behavioral symptoms, estrous cycle, and age at the time of caslick’s surgery was used in statistical analyses. total population was categorized into 3 groups based on the effect of caslick’s surgery and further divided into groups based on the frequency of occurrence of behavioral symptoms. all comparative analyses were carried out using chi-square tests. statistical significance was set at p < 0.05. in total, 406 mare owners reported that their mare had caslick’s surgery performed due to behavioral problems. ‘full effect’ (disappearance of symptoms) was reported by 129 owners (31.8%), ‘some effect’ (behavioral problems became less apparent) in 152 mares (37.4%), and no effect was reported by 125 owners (30.8%). symptoms were present ‘constantly’ in 53.2% of mares (215/404), ‘in relation to estrus’ in 28.7% (73/404), and with ‘varying pattern’ in 28.7% (116/404). the mares reported to express symptoms ‘constantly’ had higher prevalence of the symptoms ‘increased sensitivity in the flank region’ (p = 0.013), ‘excessive tail swishing’ (p = 0.006), and ‘opposing signals from rider’s legs’ (p = 0.013) compared to mares expressing symptoms in a ‘varying pattern’. additionally, the prevalence of mares ‘refusing to move forward’ was higher (p = 0.005) within the group of mares expressing symptoms ‘constantly’ compared to mares only showing symptoms ‘in relation to estrus’. age ranged from 3 24 years (mean: 10.0 years). the ‘full effect’ group contained significantly more mares aged ≤ 6 years at the time of surgery compared to ‘no effect’ (p = 0.033) and ‘some effect’ (p = 0.006). more mares (p = 0.008) within the age interval ≤ 6 years were present in the group ‘full effect’ compared to ‘some effect’. mares were reported to participate in competitions by 65.7% of the owners. higher prevalence of competing mares was in the groups ‘some effect’ (p = 0.002) and ‘full effect’ (p = 0.0009) compared to ‘no effect’. time to effect was reported to be within 3 months in 83.7% (‘full effect’) and 83.5% (‘some effect’). within the group ‘no effect’, 37.6% (47/125) of the owners reported to have another reason for the mares’ problems. caslick’s surgery resolved or markedly improved behavior in almost 70% of the cases, and in most of these mares, the effect was observed within 3 months postoperatively. despite the bias of the owners’ subjective perceptions/observations, we concluded that caslick’s surgery will solve behavioral problems in mares suffering from referred vaginal pain syndrome. keywords: mares, behavioral problem, caslick’s surgery sperm protein reactive with antisperm antibody expression in domestic and wild horse ovaries mikaela lee, brynley cozzi, michelle kutzler department of animal and rangeland sciences oregon state university, corvallis, or sperm protein reactive with antisperm antibody (sprasa) is a lysozyme-like protein previously identified in domestic and wild horse ovarian follicles.1 objective was to use image analysis software to compare sprasa ovarian immunoexpression in all follicle stages. we hypothesized that sprasa expression does not differ between domestic and wild mares. routine immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections, followed by image analysis using fiji software. heat-induced epitope retrieval with sodium citrate (ph 6.1) was used. anti-sprasa polyclonal antibody (#hpa023633, atlas antibodies) was applied at 1:200 dilution and immunostaining specificity was verified by replacing the primary antibody with negative control rabbit serum on adjacent sections. sections were then reacted with one-step horseradish peroxidase-conjugated polymer anti-rabbit igg (ih-8064-custom-orsu, immunobioscience) followed by a novared peroxidase substrate (#sk-4800, vector labs). representative images of each follicle stage from each ovary were digitally captured using qcapturepro image capture software by a single observer at 200 x magnification (ml). cellular expression of sprasa was then quantified in primordial, primary, secondary, and tertiary follicles using fiji software with rgb stack and manual thresholding to isolate areas of staining. the granulosa cells and theca cells were outlined using the freehand selection tool and mean gray value was measured. results (mean ± sem) were compared between domestic and wild mares using a student’s t-test and significance was defined as p < 0.05. sprasa expression was greater in domestic compared to wild mares in granulosa cells of primordial (p = 0.0022), primary (p < 0.001), secondary (p = 0.025), and tertiary follicles (p = 0.0078), and in theca cells of tertiary follicles (p = 0.022) (figure 1). wild equid population in us has drastically exceeded the carrying capacity of the public lands where they are managed.2 immunization against sprasa may prove to be a permanent, nonsurgical immunologic method for sterilizing horses, because it targets all stages of ovarian follicles. figure 1. sprasa expression in equine follicles clinical theriogenology 2022; 14: 305 keywords: mare, immunocontraceptive, mare, granulosa cell, follicle references 1. cozzi b, kiesler z, kutzler m: sperm acrosome associated 3 protein expression in equine primordial, primary, secondary, and tertiary follicles. clinical theriogenology 2021;13:85-88. 2. bureau of land management. wild horse and burro on-range population estimates. 2019. how to work-up a mare exhibiting stallion-like behavior patrick mccue department of clinical sciences, colorado state university, fort collins, co veterinarians are occasionally asked to evaluate a mare exhibiting stallion-like behavior, which may include mounting or herding other mares, aggression toward other horses or people or other manifestations. an initial approach to the issue would be to formulate a differential diagnosis (ddx) for the abnormal behavior. goals of the ddx are to provide a list of potential causes, guide diagnostic tests and treatment protocols, rule out life threatening or time-critical conditions and provide a pathway to a final diagnosis. differential diagnoses for stallion-like behavior in mares includes an ovarian granulosa-theca cell tumor, pregnancy, estrus, dominant/alpha mare, disorder of sexual development, iatrogenic, idiopathic and inaccurate assessment of behavior by the mare owner or trainer. initial diagnostic plan should include a complete and accurate history, a general physical examination (evidence of increased muscle tone, masculine physical features, ‘cresty’ neck, abnormal perineal region) and transrectal palpation/ultrasound (evaluation of gonads, presence/absence of a uterus, pregnancy status). additional diagnostic tests that may be indicated pending the initial evaluation include hormone analysis (anti-müllerian hormone, inhibin, testosterone, progesterone), observation of behavior, vaginal speculum examination (is the vaginal vault complete and is a cervix present), karyotype (chromosome analysis), behavioral response to hormone therapy (altrenogest), tissue biopsy with histopathology, and other tests. mares in behavioral estrus may exhibit mounting behavior. mounting behavior is uncommon in mares compared to the incidence rate in cows. mares that exhibit mounting behavior have been reported to have higher testosterone concentrations than mares being mounted. a dominant mare may exhibit aggressive behavior toward other (subordinate) mares in the herd or harem. dominance behavior may be more evident with introduction of a new mare into the herd. dominant mares may have elevated testosterone concentrations of ovarian or adrenal origin. a granulosa-theca cell tumor is a benign, unilateral ovarian tumor associated with a small, inactive contralateral ovary. the presence of thecal cells is associated with elevated testosterone concentrations and subsequent expression of aggressive or stallion-like behavior. diagnosis is based on behavioral characteristics, transrectal palpation/ultrasound and hormone analysis. mares in mid to late pregnancy have elevated blood testosterone, which may lead to aggressive or stallion-like behavior. the source of testosterone is the fetal-placental unit; levels peak at ~ 7 months of gestation. mares given an anabolic steroid (e.g., boldenone undecylenate), may exhibit behavior characteristic of stallions, including aggression or fighting, flehmen response, elimination or marking behavior, and mounting and/ or herding of other mares. disorders of sexual development may be associated with stallion-like behavior in a horse with a female phenotype. one example is a 64 xy, sry-positive horse with cryptorchid testes, elevated testosterone and variable differentiation of the perineal region. diagnosis of this condition is chromosomal analysis of a blood or hair sample. in summary, an owner or trainer complaint of aggressive or stallion-like behavior is uncommon, but not rare. acquisition of a complete history and an initial thorough, systematic reproductive evaluation will dictate additional diagnostic tests that will hopefully lead to a definitive diagnosis. keywords: mare, stallion-like behavior, testosterone, karyotype induction of labor in the mare: review of principles, procedures, and pitfalls patrick mccue department of clinical sciences, colorado state university, fort collins, co induction of labor allows for an opportunity to have professional assistance available to assist with foaling and care of the newborn foal from a high-risk pregnancy. clinical indications for a controlled induction of labor in a mare include management of high-risk pregnancies, and monitoring of labor in mares that have had a history of dystocia, stillbirth, premature separation of the placenta, or other foaling complications. induction of labor may also be performed as an emergency procedure on a late-term pregnant mare with a life-threatening medical condition, such as severe acute laminitis, ruptured prepubic tendon, placental hydrops or other issues. elective induction of labor in the mare for nonmedical reasons is generally not recommended. induction of foaling in mares can be predictable and successful with strict adherence to guidelines for assessment of fetal readiness for birth. however, emergency medical situations may over-ride the standard guidelines. to be considered for induction of labor, a mare should have a gestation length of at least 330 days, have an enlarged udder and engorgement of the teats with colostrum and, if measurement is possible, a mammary fluid calcium carbonate level of greater than 200 ppm. hormonal techniques evaluated for induction of labor in the mare have included corticosteroids, prostaglandins and oxytocin. corticosteroids (e.g., dexamethasone) are effective in inducing parturition in ruminants, but generally unreliable in inducing labor in mares. prostaglandins have been used successfully in mares, but interval from administration to rupture of the chorioallantois and subsequent foaling is not consistent or predictable. in author’s opinion, the most reliable technique for induction of labor in mares is treatment with oxytocin. dosage and treatment regimens described for oxytocin include treatment of small (2.5 20 units) oxytocin boluses, intravenously or intramuscularly, at 10 30 minute intervals, treatment of a larger bolus (40 60 units) intramuscularly, and treatment of 75 100 units of clinical theriogenology 2022; 14: 306 oxytocin diluted in 1 liter of saline through an intravenous catheter at a rate of 1 unit/minute or until the fetus is delivered. author prefers giving small intravenous boluses of oxytocin for induction of labor in mares. the interval from first oxytocin treatment (5 units) the first signs of labor (uterine contractions, discomfort, sweating) is 5 10 minutes; a second dose of oxytocin (10 units) is given 15 minutes after the initial dose. rupture of the chorioallantois usually occurs within 5 10 minutes after the second dose and delivery of the fetus usually 10 15 minutes thereafter. in conclusion, controlled induction of labor can allow for a safe, predictable foaling when breeding farm and veterinary medical support personnel and emergency equipment are available. conversely, induction of labor can be disastrous if guidelines for selection of mares are not followed or if an accurate breeding date is not known. induced parturition may also be associated with dystocia, retained fetal membranes, premature placental separation, dysmaturity, and failure of passive transfer. keywords: mare, induction, labor, obstetrics, oxytocin outcome of periparturient peritonitis secondary to uterine perforation in mares peter morresey rood and riddle equine hospital, lexington, ky this study investigated signalment and presenting signs of mares with suspected uterine perforation and their association with duration of hospitalization, survival, and subsequent fertility. it was hypothesized that affected mares differ from clinical and clinicopathological reference ranges of nonaffected mares, and survivors and nonsurvivors also differ in the magnitude of these changes. medical records of mares (n = 46) presented to a large central kentucky referral hospital between 2011 and 2020 with uterine perforation and resultant periparturient peritonitis were reviewed. affected mares ranged between 4 and 22 years of age with mean ± standard deviation (mean ± sd) of 10.5 ± 3.7 years. duration of hospitalization was (mean ± sd) 7 ± 5 days, with surviving mares 3 ± 3.94 days (range 0 10 days) and nonsurviving mares 8 ± 5.03 days (range 4 22 days). most consistent findings on initial examination were tachypnea 76%, apparent abdominal discomfort 63%, and tachycardia 56%. clinicopathological abnormalities commonly detected were hyperlactatemia 80%, bandemia 63%, hemoconcentration 49%, and leukopenia 33%. peritonitis was diagnosed on the basis of abdominocentesis, transabdominal ultrasonography, and exploratory celiotomy. common peritoneal fluid abnormalities included elevated protein concentrations 100% and leukocytosis 94%. all mares suspected of uterine perforation underwent exploratory celiotomy. location of uterine defect was evenly distributed between the uterine body or the horns. most common postsurgical complication was peritonitis 75%. medical treatment consisted of treatment of intravenous fluids, antibacterials, nonsteroidal antiinflammatory drugs, and peritoneal lavage (intraoperative and postoperative). survival to discharge was 82.6% (38/46). of nonsurvivors, 87.5% (7/8) were humanely euthanized based on prognosis 71.4% (5/7), intraoperative findings 14.3% (1/7), and postanesthetic misadventure 14.3% (1/7). factors associated with nonsurvival (odds ratio, 95% confidence interval) included hypercreatininemia (39, 1.86 817.63), leukocytosis (5.16, 0.65 40), absence of fever (3.66, 0.35 38.03), increased peritoneal fluid volume (3.20, 0.33 31.42), and absence of apparent abdominal pain at admission (1.87, 0.22 15.93). sample size limitations are evident in calculation of confidence intervals. for surviving mares not lost to follow up, 80% (24/30) produced another foal with (mean ± sd) 1.48 ± 0.66 years (range 1 3 years) until successful delivery. in summary, survival of mares with uterine perforation leading to peritonitis is good. reproductive potential is favorable. presenting signs and clinicopathological variables available at initial examination differ from healthy mares and these are also differentially associated with nonsurvival. keywords: mare, peritonitis, uterine perforation, foaling, hypercreatininemia, colic a case of paraphimosis of 7 years’ duration in a percheron stallion peyton draheim,a sophie boorman,a reid hanson,a candace lymana adepartment of veterinary clinical sciences college of veterinary medicine auburn university, auburn, al a 15-year, percheron stallion, was presented for evaluation of paraphimosis of 7 years’ duration. available history indicated that the paraphimosis was subsequent to a 2013 kicking injury. vital parameters were within normal limits (wnl). penis and prepuce were markedly firm and swollen, glans penis extended to tarsus level, and chronic penile exteriorization was evident (epithelium was thickened, friable, and cool to the touch). since the stallion had an apparent lack of sensation, as evidenced by the lack of response when the penile skin was pinched, a phallectomy and castration were recommended. prior to induction of general anesthesia, the stallion received prophylactic antibiotics and antiinflammatory drugs; presurgical packed cell volume (pcv) was 32% with total solids (ts) of 8.7 g/dl. following anesthetic induction, the periscrotal area and penis were aseptically prepared and a stallion urinary catheter was placed. to reduce the likelihood of urethral stricture, the williams’ technique was selected. use of electrocautery and an esmarch tourniquet at the base of the penis provided hemorrhage control. a urethrostomy was performed by making 3 cm long incisions, forming a triangular-shaped wedge on the ventrum of the penis. catheter was removed and a stoma was created by securing the urethral mucosa to the epithelium with simple interrupted sutures. penis was amputated 5 cm distal to the urethrostomy site. a simple interrupted suture pattern provided compression and closure of the corpus cavernosum around the urethral lumen. finally, a closed castration was performed with serra emasculators. no intraoperative hemorrhage was noted. recovery from anesthesia was uneventful and the patient experienced mild postoperative hemorrhage from the stoma. physical examination findings were unremarkable. approximately 12 hours postsurgery, the gelding presented with dull mentation, pale mucous membranes, tachycardia (72 beats/minute), and tachypnea (32 breaths/minute). gelding was given infusions clinical theriogenology 2022; 14: 307 of aminocaproic acid, 0.05% formalin, and a whole blood transfusion (7 liters). by the following afternoon, pcv had increased from 18 to 22% and ts increased from 5 to 6.8 g/ dl; vital parameters were wnl. gelding was discharged 5 days postsurgery with normal physical examination findings. medications included oral twice daily phenylbutazone (1.5 gram), trimethoprim sulfamethoxazole (30 mg/kg), and red cell® (30 ml). instructions provided to owners included stall rest, daily hydrotherapy, and short hand walks twice daily to manage postoperative edema. four days after discharge, owners returned the gelding for assessment of dull mentation; vital parameters were wnl except for mild tachycardia (56 beats/ minute). urethral stoma appeared to be healing appropriately; pcv was 16% and ts was 7.4 g/dl at presentation. gelding remained stable and was discharged 2 days later. instructions included continued stall rest and hydrotherapy in addition to application of femycin ointment to penis. this case described resolution of a severely protracted paraphimosis and successful management of common, albeit concerning, postoperative complications. keywords: paraphimosis, stallion, phallectomy, castration evaluating susceptibility of fungal biofilm to mucolytics used to treat mares yamilka lago-alvarez,a theodore kapogiannis,a claire wang,a joyce jin,a shinyee su,a rebecca franklin-guild,b mariana diel de amorim,a craig altier,b soon hon cheonga adepartment of clinical sciences, bdepartment of population medicine and diagnostic sciences college of veterinary medicine, cornell university, ithaca, ny fungal endometritis in mares causes infertility and is difficult to treat. fungal agents form biofilms that impede the immune system and may enhance antifungal resistance. mucolytics are commonly used to disrupt fungal biofilm but the efficacy of these intrauterine treatments is not well known. we aimed to investigate the susceptibility of candida sp. biofilm to commonly used intrauterine treatments. we hypothesized that biofilm from various candida species have distinct susceptibility to mucolytics. biofilm was produced by culturing 5 candida isolates (1 c. albicans, 2 c. parasilopsis, and 2 c. tropicalis) obtained from clinical cases of equine endometritis in rpmi media for 24 hours in 96-well plates. initial biofilm amount was determined by spectrophotometric absorbance at 600 nm. biofilm was exposed to 1 of 15 treatments: lactated ringer’s solution (lrs) as control; ceragyn lavage, ceragyn uterine infuse, dimethylsulfoxide (dmso) at 10, 20, and 30%; hydrogen peroxide at 0.5 and 1 %; povidone iodine at 1 and 1.5%; n-acetylcysteine at 3.3 and 20%; tricide®; and vinegar at 2 and 10%. treatment was applied to the biofilm for 5 minutes, then washed before repeating spectrophotometric reading. each isolate was tested at least 3 times and each treatment was tested in 4 replicates each time. percent reduction in biofilm absorbance was calculated and used as the dependent variable. statistical analysis was performed using linear regression anova with dunnett’s post hoc test in jmp pro v16 with significance set at p < 0.05. independent variables tested were: replicate, treatment, species, isolate, and interactions. there was a significant interaction of treatment by species, indicating various fungal species had varied susceptibility to the mucolytics, and the analysis was repeated stratifying for fungal species. isolate and treatment were still significant, indicating that even within a fungal species, biofilm from various isolates have varied susceptibilities to mucolytics, and all results were reported by isolate. for c. albicans, treatment with dmso at 30% and hydrogen peroxide at 0.5 and 1% had higher percent biofilm reduction compared to lrs. for c. parasilopsis, biofilm from 1 isolate was more susceptible to dmso at 20 and 30%, and hydrogen peroxide at 0.5 and 1%; whereas biofilm of the other isolate was more susceptible to dmso at 10, 20, and 30%, povidone iodine at 1 and 1.5%, n-acetylcysteine at 20%, and tricide compared to lrs, but was not susceptible to hydrogen peroxide. for c. tropicalis, biofilm from 1 isolate was susceptible to dmso at 10, 20, and 30%, hydrogen peroxide at 0.5 and 1%, and n-acetylcysteine at 20%; whereas biofilm of the other isolate was only more susceptible to dmso at 30% compared to lrs. replating fungal biofilm posttreatment for 24 hours for c. albicans resulted in no significant difference in growth compared with lrs, whereas for c. tropicalis and c. parasilopsis treatment with ceragyn lavage and uterine infuse, dmso at 20 and 30%, tricide®, and n-acetylcysteine resulted in higher inhibition of fungal growth. these results indicated a need for more studies to understand fungal biofilm susceptibility. keywords: mare, candida, mucolytic, biofilm efficacy of nano-colloidal silver treatment for postpartum endometritis in dairy cows gamal el-amrawi department of theriogenology, faculty of veterinary medicine, alexandria university, egypt postpartum endometritis due to bacterial infection is 1 of the most common postpartum diseases affecting dairy cows, and antibiotics are widely utilized for treatment. recently, bacterial resistance to antibiotics has become the most important problem. aim was to detect the efficacy of nano-colloidal silver in treatment of postpartum endometritis in dairy cows. we hypothesized that intrauterine treatment of nano-colloidal silver eliminates bacterial infection causing endometritis, due to its wide spectrum of bacteriostatic and bactericidal activity against enormous range of bacterial strains at low concentration. consequently, such recovery can be detected initially by peripheral concentrations of some chemokines and acute phase proteins and subsequently by pregnancy rate. in large private dairy farm, holstein cows (n = 21) were diagnosed with postpartum endometritis at 35 40 days postpartum via clinical and ultrasonographic examinations. cows received 40 ml of intrauterine nano-colloidal silver solution (25 ppm/ml, particle diameter of ~ 15 25 nm) for 5 consecutive days and 1 dose of prostaglandin f2α analogue (cloprestenol) for cows with a corpus luteum. blood samples were drawn just before the treatment and on day 7 after treatment. serum concentrations of ceruloplasmin, crp were assessed (immune-turbidmetry), in addition to serum amyloid –a (saa), haptoglobin, (tnf-α), and (il-6) using elisa kits. after treatment, uterine status was monitored via transrectal ultrasonography. clinical theriogenology 2022; 14: 308 animals were reexamined at 49 54 days postpartum to assess recovery rate. cured animals were artificially inseminated on their observed estrus; pregnancy was diagnosed on day 40 after insemination. data were analyzed by student’s t-test using spss software. treatment with nano-colloidal silver, decreased (p < 0.001) serum concentrations of saa, crp, tnf-α, and il-6. haptoglobin and ceruloplasmin concentrations did not change (p < 0.001) after treatment. uterine secretions decreased or disappeared, and endometrial thickness decreased on day 7 after treatment. at reexamination, 14 of 21 cows (66.6%) had recovered from endometritis and pregnancy rate was 71.4% (10/14). in conclusion, the study indicated the effectiveness of nanocolloidal silver in treatment of postpartum endometritis in dairy cows. so, it could be used instead of antibiotic treatment for antibiotic resistance problems. further studies are needed to investigate the effect of different treatment period and solution concentrations of nano-colloidal silver on treatment of endometritis in dairy cows. keywords: dairy cows, postpartum endometritis, nano-silver transcriptomic analysis reveals the complex regulatory networks in equine chorioallantois during spontaneous term labor hossam el-sheikh ali,a,b barry balla agluck equine research center, department of veterinary science university of kentucky, lexington, ky bcollege of veterinary medicine, mansoura university, egypt equine chorioallantois (ca) undergoes complex physical and biochemical changes during labor. however, the molecular mechanisms controlling these changes are still unclear. therefore, the aim of the study was to characterize the transcriptome of equine ca during spontaneous labor and compare it to normal preterm ca. placental samples were collected postpartum from mares with normal term labor (tl group, n = 4) and from preterm not in labor mares (330 days ga; ptnl group, n = 4). our study identified 4,137 differentially expressed genes (degs) (1,820 upregulated and 2,317 downregulated) in ca during tl as compared to ptnl. tl was associated with the upregulation of several proinflammatory mediators (mhc-i, mhc-ii, nlrp3, cxcl8, and mif). also, tl was associated with the upregulation of matrix metalloproteinase (mmp1, mmp2, mmp3, and mmp9) with subsequent extracellular matrix degradation and apoptosis, as reflected by upregulation of several apoptosis-related genes (atf3, atf4, fas, fos, and birc3). additionally, tl was associated with downregulation of 21 transcripts coding for collagens. the upregulation of proteases, along with the downregulation of collagens, is believed to be implicated in separation and rupture of the ca during tl. furthermore, tl was associated with downregulation of transcripts coding for proteins essential for progestin synthesis (srd5a1 and akr1c1) and angiogenesis (vegfa and rtl1), as well as upregulation of prostaglandin synthesis-related genes (ptgs2 and ptges) that could reflect the physiological switch in placental endocrinology and function during tl. in conclusion, our findings revealed the equine ca gene expression signature in spontaneous labor at term that improves our understanding of the molecular mechanisms triggering labor. keywords: mare, parturition, placenta, pregnancy, chorioallantois, transcriptome clinical theriogenology 2022; 14: 309 evaluation of chemiluminescent assays for canine progesterone and its clinical implications heather gosnell, pamela haney,a melissa singletary,a lucia lazarowski,a robyn wilbornb acanine performance sciences bdepartment of clinical sciences, auburn university, al detection dogs perform a vital role in national security. currently there is a national shortage of domestic detection dogs within us, so it is important to maximize reproductive efficiency. working with auburn’s canine performance sciences program, the goal of this study was to evaluate the relative accuracy of ovulation timing using 2 commonly used methods to determine serum progesterone concentrations and determine whether the differing values would influence clinical decisions. we hypothesized that progesterone concentrations measured at auburn university’s veterinary teaching hospital (auvth) and measured at east alabama medical center (eamc) do not differ enough to alter the treatment plan for the patient. two different chemiluminescent immunoassays were used. auvth uses the immulite 1000 whereas the eamc uses the access clia. the complete estrous cycle of 4 dogs were followed from the start of proestrus to diestrus was detected using vaginal cytology. data were analyzed statistically focusing on values of 1 10 ng/ml since these are the most clinically relevant. a bland-altman analysis and passing bablock regression were used along with a wilcox signed rank test to analyze the data. although progesterone concentrations increased over the course of the estrous cycle, there were significant differences between these 2 methods. for the wilcox signed rank test, the median difference between the 2 methods was compared to zero and a p value of 0 was determined. as the values increased, the difference between them increased. the bland-altman highlighted the proportional systematic bias between the 2 methods. mean difference between 2 methods was 1.29 with a confidence interval of 0.89 1.7. because 0 does not fall in the confidence interval of the mean difference, the bias can be considered significant. the line of regression for the passing bablock has a slope of 1.2726 and y-intercept of 0.4074, which highlights the poor agreement between the two methods. in conclusion, the access clia reported consistently higher values than the immulite 1000. there was poor agreement between the 2 methods of measurement, with the access machine reading an average of 1.29 ng/ml higher than the immulite 1000. however, the access clia has not been validated for use in the dog and should ideally be compared to a radioimmunoassay, where reference ranges for canine progesterone concentrations were determined to compare accuracy. further evaluation is necessary to determine if this difference was substantial enough to influence clinical decisions. findings suggested that it is possible. lastly, currently there is no clinical way to confirm the time of canine ovulation. with the use of vaginal cytology, cytologic diestrus can help confirm the date of ovulation in retrospect and was the most reliable tool available for this study. metagenetic analysis of pregnant mare’s placental microbiome kalie beckers,a viviane gomes,a christopher schulz,b gary childers,b jenny sonesa aveterinary clinical sciences, school of veterinary medicine louisiana state university, baton rouge, la bdepartment of biological sciences, southeastern louisiana university, hammond, la placentitis is the leading cause of infectious abortion in the horse and contributes to roughly 19% of all abortions in us. it continues to cause episodic abortions, in addition to weak and/or growth restricted offspring.  it has been reported that the equine placenta harbors a unique microbiome as collected at term delivery from healthy mares. aim was to identify the core microbial communities in various body sites of the pregnant mare in early pregnancy to describe a core microbiome that may be perturbed in pathologic pregnancies such as in placentitis. we hypothesized that the equine placenta harbors a distinct resident microbiome in early pregnancy when characterized by metagenetics and that there is a disparity in bacterial communities from the oral, vaginal and fecal microbiome. furthermore, we believed metagenetics reveals distinct communities and indicator taxa that characterize healthy and dysbiotic states in the equine placenta. eight pregnant pony mares (bred to the same stallion) were used between 40 120 days of pregnancy. sample swabs were collected from oral cavity, vagina, anus, and the allantoic portion of the allantochorion. the v4 region of the 16s rrna gene was amplified for illumina miseq sequencing to examine core bacterial communities present in various body sites. microbial community composition of the pregnant ponies by body site differed (p = 0.001, permanova with bray-curtis dissimilarity of 16s amplicon sequence variant’s relative abundance). the allantois was different from feces (p = 0.006), oral cavity (p = 0.02), and the vagina (p = 0.016) using a permanova of bray-curtis dissimilarity. using beta dispersion, a calculation based on centroid distance between samples, the allantois grouped most closely together with the vagina. microbial communities within the feces were also similar to some vaginal samples, but the allantois and feces were significantly different from each other. alpha diversity measuring shannon diversity matrix student research sessions clinical theriogenology 2022; 14: 310 was different (p = 0.0002) with the body sites being a compounding variable meaning there was a difference in richness and evenness in the different microbial communities. when using tukey’s multiple comparisons, the allantois was most similar to the oral cavity when using alpha diversity alone. in conclusion, metagenetics revealed distinct community differences in the oral, fecal, vaginal, and allantoic cavities of the horse. equine placenta had some similarities in its microbial communities to the oral cavity (alpha diversity) and the vagina (beta diversity). further research is needed to investigate how bacteria are translocated to the placenta from these other body sites and how they contribute to the development of placentitis. we intend to develop a screening method using next generation sequencing to rapidly identify microbial community dysbiosis to monitor mares that could potentially develop placentitis. keywords: mare, pregnancy, placenta, microbiome effect of corn-based diets on beef bull semen characteristics luke trapp,a manuel chamorro,a laura huber,b richard hopper,a julie schnuelle,a jessica rush,a jessica cowley,a katelyn waters,a yatta boakaric adepartment of clinical sciences, bdepartment of pathobiology college of veterinary medicine, auburn university, auburn, al ctexas a&m college of veterinary medicine, texas a&m university, college station, tx in modern production operations, bull development and maintenance nutrition are often unbalanced leading to detrimental effects on semen quality. there are unpublished reports recognizing a correlation between high corn-based feedstuff consumption and poor results on breeding soundness evaluations (bse) in bulls. we hypothesized that the consumption of high corn-based diets negatively affect sperm by increasing morphologic defects, resulting in bulls having an unsatisfactory classification on a bse. healthy beef bulls were presented to veterinary teaching hospitals for bse’s were enrolled with mean age 42 months and average pounds/day corn-based diet consumed being 15 pounds. all bulls in this study were given free choice trace minerals. angus was the most commonly presented breed (47%), followed by brahman influence (23%), and hereford (17%). a total of 8 bulls (16%) were classified as unsatisfactory and 39 classified as satisfactory with average normal cells being 72% and most common deformity a midpiece abnormality. preliminary data were used to create a statistical model with results of bse (unsatisfactory/satisfactory), age, weight, amount of corn product consumed and sperm abnormalities. computed models were used to analyze and compare characteristics of age, weight, and corn product consumed (pounds/day) between bulls with a satisfactory/unsatisfactory bse result. statistical significance was set at p<0.05. no significant associations were established at this time (p > 0.05). however, with each 1 month increase in age, the odds of satisfactory classification on bse decreased by 98.74% (ci 100 40.05%, p =0.25); for each unit increase in pounds/day of consumption of corn product, the odds of satisfactory classification on bse decreased by 99.42% (ci 100 54.45%, p = 0.27); and for each unit increase in weight, the odds of satisfactory classification on bse decreased by 8.03% (ci 24.54 1.07%, p = 0.25). bulls maintained on a grass diet seemingly had fewer morphological defects analyzed at time of bse. in conclusion, amount of corn-based product, age of bull, and weight had a negative relationship with satisfactory characteristics for a bse. additional data are needed to further assess effects of corn-based diet on semen morphology to make accurate and practical recommendations for producers. keywords: bull, corn, sperm morphology clinical theriogenology 2022; 14: 311 a case of penile denervation in a brangus bull cody davis,a jessica cowley,a yatta boakari,b katelyn waters,a jenna stockler,a herris maxwella, richard hoppera acollege of veterinary medicine, auburn university, auburn, al bcollege of veterinary medicine and biomedical sciences texas a&m university, college station, tx a 3-year, brangus bull, was presented for evaluation of a preputial injury that had been managed medically for several weeks. at the second visit, the owner reported that the patient had a 50% pregnancy rate in the previous breeding season and had been observed having difficulty in achieving intromission. based on the history and the owner’s description of the bull’s courtship and breeding behavior, impotence due to an inability to copulate (impotentia coeundi) was suspected. in order to better define the condition, a test mating was arranged to allow evaluation of the bull’s ability to breed. bull was placed in a 12 x 15 foot stall with a heifer in estrus. bull initiated normal courtship behavior and had interest in the female. an initial mount was not accompanied by erection (false mount) and the bull resumed courtship behavior after dismounting. at the next mounting attempt, the bull achieved full erection and extension of penis. bull was observed making normal searching motions with glans penis but failed to locate the vulva. bull did not achieve intromission, and the erect penis eventually extended along the left side of the heifer before the bull dismounted. on a subsequent breeding attempt, bull mounted the heifer appropriately, initiated penile searching motions, probing the area of the escutcheon with the fully erect penis, but failed to achieve intromission, ultimately placing the erect penis along the right side of the heifer prior to dismounting. observation of active searching with the glans penis accompanied by failure to achieve intromission with the fully erect penis is diagnostic of damage to the dorsal nerve of the penis. supplemental testing of nerve conduction may be used to confirm the diagnosis, although the equipment and expertise required is seldom available to private practitioners. in this case, the owner declined further diagnostics, and chose to salvage the bull at slaughter. an alternative method of determining dorsal penile nerve function is via a bovine artificial vagina after proper stimulation and mounting are allowed.1 however, lack of ejaculate via artificial vagina method could also indicate inadequate stimulation from female exposure, that the artificial vagina does not match the bull’s preference, or musculoskeletal pain during mounting.1 this method may also not be definitive since the more caudal portions of the penis may still be adequately stimulated if the penis is inserted far enough into the artificial vagina. penile denervation should not interfere with erection or production of viable semen.2,3 affected bulls of sufficient value can have semen collected via electroejaculation for cryopreservation.2,3 keywords: penile denervation, preputial prolapse, test mating, brangus references 1. wolfe df, moll hd: examination and special diagnostic procedures of the penis and prepuce. in: wolfe df, moll hd: editors. large animal urogenital surgery. 2nd edition, baltimore: williams & wilkins; 1999. p. 221-231. 2. maxwell h: inability to breed due to injury or abnormality of the external genitalia of bulls. in: hopper rm: editor. bovine reproduction. 2nd edition, hoboken: joe wiley & sons; 2021. p. 155172. 3. maxwell hs, edmondson ma: diagnosis and management of injuries to the penis and prepuce of bulls. in: anderson de, rings dm: editors. food animal practice. 5th edition, saint louis: wb saunders; 2009. p. 345-356. hydroallantois as a result of a longhorn and bison mating hannah stokley, jessica cowley, herris maxwell department of clinical sciences, college of veterinary medicine auburn university, auburn, al a 6-year, longhorn domestic cow (bos taurus), was presented for acutely nonambulatory and recumbent condition. cow had extreme bilateral abdominal distension and had been recumbent for ~ 5 hours. it was determined that cow was in active labor. it was suspected that cow had been bred by a bison (bison bison) bull. delivery was assisted, and an extreme volume of serous fluid was expelled from the uterus. other than the large amount of fluid expelled there were no complications with the delivery and a live and seemingly healthy heifer calf was delivered. a diagnosis of dystocia due to hydroallantois was determined. hydroallantois is a condition that complicates 1:7,500 pregnancies.1 in uncomplicated pregnancies, the volume of allantoic fluid is ~ 15 liters,2 whereas in pregnancies complicated by hydroallantois there can be up to 175 liters of fluid in the allantoic cavity.3 hydorallantois occurs due to ineffective placental membranes.4 supportive care was given including intravenous boluses of hypertonic saline and plasmalyte, intramuscular injections of dexamethasone, and assistance in standing. after 4 days of hospitalization with no student clinical case session clinical theriogenology 2022; 14: 312 improvement she was euthanized and a necropsy performed. relevant necropsy findings were consistent with severe adventitious placentation and a venous thrombus occluding the caudal vena cava. adventitious placentation is characterized by abnormal placentome development, decreased numbers of placentomes, and decreased vascularization of the placenta.1 pregnancies sired by a bison bull with a bos taurus dam, result in abortions in 1:1.5 confirmed pregnancies.5 of the live births, 90% were female.5 abortions occurred in 7% of confirmed pregnancies sired by a bos taurus bull to a bison cow, of the live births there was a 1:1 sex ratio.5 most common cause of abortion in bison and domestic cattle crosses has been hydramnios.6 hydramnios has more viscus fluid (25 liters).7 in uncomplicated pregnancies the amnionic fluid is between 3 5 liters.7 hydramnios is caused by a lack of fetal ingestion of the amnionic fluid usually associated with fetal facial deformities.8,9 there has been no evidence of facial deformities in historical records kept of attempted crosses between a bison bull and bos taurus cow. similar issues with adventitious placentation leading to hydroallantois, as commonly observed in abnormal offspring syndrome, have been associated with a lack of epigenetic processing of genes important to placental growth on the y-chromosome.10,11 failure of hybrids are normally attributed to dissimilarities of the karyotypes of the parent species, the only structural difference between the karyotypes of the domestic cattle and bison are that of the y-chromosome.12 in bison, the y-chromosome is acrocentric whereas in domestic cattle it is metacentric.12 therefore, it has been hypothesized that bison bulls lack a gene on the y-chromosome critical for placentation that domestic cattle possess. it is also hypothesized that the bison cow has a gene that negates the importance of the gene that regulates placental growth on the y-chromosome. keywords: hydroallantois, adventitious placentation, bison cross references 1. maxwell h: dystocia and accidents of gestation. in: bovine reproduction. hopper r: editor. 2nd edition, hoboken; wileyblackwell: 2021. p. 603-604. 2 arthur gh: some notes on the quantities of fetal fluids in ruminants, with special reference to “hydrops amnii.” br vet j 1957;113:17-28. 3 hanna wb, scott wd: an outbreak of placental hydrops in a beefalo herd. can vet j 1989;30:597-598. 4. jackson, pgg: handbook of veterinary obstetrics. london; w.b. saunders company limited: 1980. p. 221. 5. hedrick pw: conservation genetics and north american bison (bison bison). j hered 2009;100:411-420. 6. basrur pk: bovine hybrids. current therapy in theriogenology. 1986;2:433-437. 7. arthur gh: the fetal fluids of domestic animals. j reprod fertil suppl 1969;9:45-52. and limits for therapy of amniotic hydrops in cattlea review. dtsch. tierarztl. wochenschr., 106: 210-212 8. zdunczyk s, grunert e: possibilities and limits for therapy of amniotic hydrops in cattlea review. deutsche tierarztliche wochenschrift 1999;106:210-212. 9. drost m: complications during gestation in the cow. theriogenology 2007;68:487-491. 10. guillomot m, taghouti g, constant f, et al: abnormal expression of the imprinted gene phlda2 in cloned bovine placenta. placenta 2010;31:482-490.  11. wang x, miller dc, harman r et al: paternally expressed genes predominate in the placenta. proc natl acad sci u s a. 2013;110:1070510710. 12. basrur pk, moon ys: chromosomes of cattle, bison, and their hybrid, the cattalo. am j vet res 1967;28:1319-1325. a case of suspected vaginal hyperplasia in a chihuahua jordan farrell, chase turner, aime johnsona, robyn wilborn,a candace lymana adepartment of clinical sciences, college of veterinary medicine auburn university, auburn, al vaginal hyperplasia is often associated with younger intact females.1 a 10-year, recently spayed female chihuahua, was referred for evaluation of suspected vaginal hyperplasia. patient was suspected to have pyometra and was spayed 3 days prior to presentation. prolapsed tissue became evident through the vulva 1 day after surgery. initial differential diagnoses included vaginal prolapse, vaginal hyperplasia, or vaginal neoplasia. although initially suspected by the referring veterinarian, the likelihood of vaginal hyperplasia in this patient was low based on the history. vaginal hyperplasia is more often associated with younger intact females and under the influence of estrogen. suspected pyometra indicated that the patient was likely in diestrus and under the influence of progesterone. patient had a body condition score of 9/9 and a 2 cm in diameter section of tissue was protruding through the vulvar lips. on further evaluation, the tissue appeared well pedunculated and originated from the ventral vaginal wall. prolapsed tissue appeared dry and black in color, suggesting necrosis. due to the tissue originating from the ventral vaginal wall and the urethra identified in a normal location, it became less likely to be a vaginal prolapse or hyperplasia. vaginal neoplasia became the most likely diagnosis. patient was sedated with hydromorphone (0.1 mg/kg) and intravenous dexmedetomidine (375 µg/m2). stalk of the protruding tissue was clamped with crile hemostatic forceps and transected near the base of the mass with electrocautery, taking care to avoid iatrogenic damage to the vaginal epithelium. once the initial mass was removed and the patient relaxed under sedation, a second, smaller mass was identified near the same location. second mass was also transected and both were submitted for histopathology. dexmedetomidine was reversed with atipamezole (375 µg/m2) and the patient recovered uneventfully. two most common vulvovaginal neoplasms include leiomyoma and leiomyosarcoma. ninety percent of cases are benign leiomyoma.2,3 histopathological evaluation of the mass revealed subepithelial fibrous stroma with a densely cellular, nonencapsulated neoplasm. vaginal mucosa was extensively ulcerated with intralesional coccoid bacterial colonies. pathologist confirmed a diagnosis of leiomyoma. leiomyoma is a benign neoplasia affecting the muscle cells within the vulva. it is often a solitary lesion in spayed females, but may be diffuse and hormonally responsive in intact females.4,5 as performed in this case, primary treatment for leiomyoma is local excision clinical theriogenology 2022; 14: 313 and ovariohysterectomy. this case identified the importance of taking a complete and thorough history as the smallest piece of information (e.g., reason for spaying) can be the key to narrowing differentials and for accurate diagnosis. keywords: dog, vaginal neoplasia, leiomyoma, vaginal hyperplasia references 1. saba cf, lawrence ja: tumors of the female reproductive system. in: vail dm, thamm dh, liptak jm: editors. small animal clinical oncology. 6th edition, w.b. saunders: 2019. p. 597-603. 2. brodey rs, roszelj f: neoplasms of the canine uterus, vagina, and vulva: a clinicopathologic survey of 90 cases. j am vet med assoc 1967;151:1294-307. 3. thacher c, bradley rl: vulvar and vaginal tumors in the dog: a retrospective study. j am vet med assoc 1983;183:690-692. 4. herron ma: tumors of the canine genital system. j am anim hosp assoc 1983;19:981-994. 5. jones c, hunt rd, king nw: genital system. in: jones tc: editor. veterinary pathology. 6th edition, baltimore; williams and wilkins co.: 1997. medical treatment of pyometra in young queens linda alvarenga, aime johnson department of clinical sciences auburn university college of veterinary medicine, auburn, al two intact female cats from the same cattery were presented with vaginal discharge. a 10-month, intact siamese female, was presented with purulent vaginal discharge without a history of breeding. abdominal ultrasonography revealed 0.7 cm of fluid within uterus. pyometra was diagnosed and treatment consisted of enrofloxacin (15.6 mg once daily subcutaneously [sq] for 7 days) and dinoprost (lutalyse; 0.3 mg twice daily intramuscularly [im] for 2 days). lutalyse was discontinued on uterine fluid clearance. patient remained alert, responsive, and afebrile throughout treatment. queen recovered adequately and was placed with a male 1 month later. she delivered 7 healthy kittens. a second 1-year queen presented with vaginal discharge after she was with a male. on ultrasonography, multiple fluid filled loops of uterus with 0.5 1 cm flocculated fluid was noted. moderate degenerated neutrophils were noted in vaginal cytology. general physical examination was normal. pyometra was diagnosed and the patient was started on enrofloxacin (9.5 mg im once daily) and lutalyse (0.19 mg sq twice daily for 7 and 2 days). fluid within the uterus decreased daily and completely resolved after 48 hours. enrofloxacin was continued for 14 days. final examination after a week revealed normal sized uterus with no discharge. queen was placed with a male 1 month later, bred successfully, and gave birth to 4 healthy kittens. pyometra is either acute or chronic inflammation of the uterine wall with accumulation of purulent material resulting from cystic endometrial hyperplasia (ceh).1 in queens, incidence of pyometra is lower than in bitches because queens are induced ovulators.2 over time, prolonged secretion of progesterone leads to endometrial hyperplasia, and bacterial contamination leads to pyometra.1 in some reports, female cats housed alone or without an opportunity to copulate experienced pyometra that suggested spontaneous ovulation.1 in the first queen, spontaneous ovulation is suggested as the underlying cause of the patient’s pyometra because there was no male. this queen was able to successfully breed and had 3 more litters after her first litter. abdominal ultrasonography or radiography are indicated to determine uterine size and shape and to rule out pregnancy.1 bacterial culture and measurement of serum progesterone concentrations supported a diagnosis of pyometra.1 escherichia coli is the predominant bacterium present in pyometra; however, more than 1 species may be present and some cultures may be negative.3 for reproducing females, standard treatment of pyometra involves multiple injections of prostaglandin to evacuate purulent material from the uterus, plus antibiotic therapy.1 prognosis for reproductive success following medical treatment depends on the severity of endometrial pathology1 and response to treatment. although treatment may be successful in queens, reoccurrence rate is 70% within 2 years.4 these cases are important because pyometra is commonly observed in older animals that have had exposure to progesterone causing uterine pathology. in these young cats, pyometra was not due to uterine pathology and hence a higher success rate. keywords: cat, pyometra, cystic endometrial hyperplasia, vaginal discharge references 1. johnston s, kustritz m, olson p: disorders of the feline uterus and uterine tubes. canine and feline theriogenoloy. 1st edition, philadelphia; saunders company: 2001.p. 463-469. 2. hollinshead f, krekeler n: pyometra in the queen. j fel med surg 2016;18:21-22. 3. hagman r: pyometra in small animals. vet clin north am small anim pract 2018;48:640-641. 4. wiebe v, howard j: topics in companion animals. pharmacologic advances in canine and feline reproduction 2009;24:78-79. disorder of sexual development in a chinese crested dog with mixed testicular tumors rebecca schwartz,a nicole sugai,b kristin eden,c caitlin castaneda,d matt jevit,d terje raudsepp,d julie cecereb avirginia-maryland regional college of veterinary medicine, blacksburg, va bdepartment of veterinary clinical sciences cdepartment of pathology virginia-maryland regional college of veterinary medicine blacksburg, va ddepartment of veterinary integrative biosciences molecular cytogenic laboratory texas a&m college of veterinary medicine and biomedical sciences parkway college station, tx a 10-year, intact female chinese crested dog, was presented for evaluation and further diagnostics due to persistent symptoms of vulvar swelling and vaginal discharge, with an 8-year clinical theriogenology 2022; 14: 314 history of acyclicity. generalized hyperpigmentation and truncal alopecia were identified, along with no aberrations of the female phenotype. vaginal cytology at multiple previous veterinary visits confirmed the influence of estrogen. positive assay results for anti-müllerian hormone confirmed gonad presence. exploratory abdominal laparotomy was performed and gonadal tissue and associated tubular structures were submitted for histopathology. histopathologically, gonads were identified as abnormal testes containing sertoli and interstitial (leydig) cell tumors. histopathologic diagnosis of testes and concurrent normal external phenotypically female genitalia in the patient led to a diagnosis of a disorder of sexual development (dsd). karyotype evaluation was pursued, and the molecular analysis revealed a mosaic pattern of xx (80%) and xy (20%) cells among the blood lymphocytes, and pcr test was positive for the y-linked sry gene. skin biopsy karyotype (to determine if this is a case of xx/xy leukocyte chimerism or a case of whole body xx/xy mosaicism) is pending. hair follicles are currently examined for sry status. this is the first known case report of a canine with a karyotypic diagnosis of mixed sex chromosomes with completely normal female phenotypic external genitalia. this patient was diagnosed with a dsd at the chromosomal level as a suspected blood chimera or whole body xx/xy mosaicism. the pathophysiology of this dsd is from sry gene expression on the genital ridge during sexual differentiation to begin testicular differentiation. whereas male external genitalia development is hormonally driven, the female phenotypic development is not hormonally driven.1 in this patient, it was suspected that an aberration in the process of testicular development led to an androgen deficiency, allowing the external genitalia to develop as female. clinical signs that led to the patient’s specialty referral were due to the patient’s abdominally retained testes and resultant hormonally active sertoli cell tumors that caused a feminizing paraneoplastic syndrome.2,3 subsequently, a diagnosis of a dsd was also made. after recovery from surgery, the patient’s clinical signs have completely resolved. this case illustrated a distinct presentation for hormonally active sertoli cell tumorigenesis and demonstrated that surgery is curative for feminizing paraneoplastic syndrome. keywords: dog, disorder of sexual development, chimerism, mosaicism, sertoli cell tumor, leydig cell tumor references 1. wilhelm d, palmer s, koopman p: sex determination and gonadal development in mammals. physiol rev 2007;87:1-28. 2. quartuccio m, marino g, garufi g, et al: sertoli cell tumors associated with feminizing syndrome and spermatic cord torsion in two cryptorchid dogs. j vet sci 2012;13:207-209. 3. foster ra: male genital system. in: maxie mg: editor. jubb, kennedy, and palmer’s pathology of domestic animals: volume 3. 6th edition, philadelphia; saunders wb: 2015. p. 465-510. cervical wedge resection treatment in a pyometra mare thomas burke, aime johnson, candace lyman, robyn wilborn, fred caldwell department of clinical sciences auburn university college of veterinary medicine, auburn, al a 15-year, pony mare, with no known previous history was obtained by the current owner. during a routine pregnancy examination (exposed to a stallion), she had intrauterine fluid and purulent vulvar discharge. further treatment was declined, and she returned to normal, nonbreeding activity. approximately 4 months later, she was represented with excessive vaginal discharge. distended uterus (12.5 cm) with purulent fluid in the vaginal canal and throughout the uterine body and horns were detected via transrectal palpation and ultrasonography. digital examination of the cervix revealed excessive transluminal adhesions and lack of patency. adhesions were broken manually and a catheter was passed through the cervix. five gallons of purulent material was drained and a uterine lavage was performed to clear the purulent material from uterus. a gram stain and culture were performed on a sample from the collected material. gram stain had substantial number of gram-positive cocci; however, aerobic culture results were negative. uterine lavages were continued daily with dilute betadine solution (0.05%) until the fluid returned clean. four days after presentation, the mare was placed in a set of stocks and sedated with butorphanol (3 mg) and detomadine (3 mg) and an epidural containing carbocaine (40 mg) and xylazine (50 mg) was placed. cervix was retracted caudally using stay sutures and a triangular section of the dorsal portion of the caudal cervix (~ 2/3 of the cervix) was removed. after surgery, intramuscular ceftiofur crystalline-free acid (3,000 mg) was given. lanolin-based ointment containing dexamethasone and oxytetracycline was applied to cervix to prevent adhesion formation. ointment was applied every 3 4 days for 2 weeks along with manual breakdown of adhesions. a month after surgery, the mare’s cervix had healed adequately. digital examination of cervix revealed that the resection was still open and the cervix was patent. minimal fluid (via transrectal ultrasonography) was present in the uterus. surgery was considered a success and the mare has returned to her athletic function with no clinical symptoms associated with uterine distension since the cervical wedge resection. pyometra is often caused by poor perineal conformation or cervical adhesions preventing drainage of the infected uterus and resulting in purulent fluid accumulation.1-4 traditional treatment of fluid accumulation caused by pyometra involves uterine lavage, intrauterine antibiotics, and continual breakdown of cervical adhesions to maintain patency.1-4 this method is often used in mares planned for breeding. without proper maintenance of a patent cervix, the animal is susceptible to repeat infections or failure of the uterus to adequately drain resulting in recurrent accumulation of fluid.2 a cervical wedge resection performed on mares that did not respond to traditional therapies or are no longer desired for breeding enables the cervix to remain permanently open and any fluid to freely drain, thereby preventing subsequent fluid accumulation.2 this method can be used as an alternative option to ovariohysterectomy as abdominal surgery on mares clinical theriogenology 2022; 14: 315 with pyometra increases risk of contamination within the abdomen that may result in severe complications.2,5 keywords: mare, pyometra, cervical wedge resection, cervix references 1. sharma s, thapak s: chronic pyometra in a thoroughbred mare. vet pract 2010;11:116-117. 2. arnold ce, brinsko sp, varner dd: cervical wedge resection for treatment of pyometra secondary to transluminal cervical adhesions in six mares. j am vet med assoc 2015;246:1354-1357. 3. rais mnb, adzahan nm, mohamad ma et al: equine pyometra: a case report. j ag vet sci 2013;3:61-63. 4. prestes nc, medrado fe, rodriques lt et al: total hysterectomy as treatment for recurrent chronic pyometra in mares with total cervical adhesion-case report. j eq vet sci 2018;63:61-64. 5. freeman de, rötting ak, köllman m et al: ovariohysterectomy in mares: 17 cases (1988 2007). amer assoc eq pract proc 2007;53:370373. clinical theriogenology 2022; 14: 316 in utero diagnosis of bilateral cataracts and hydrops in a mule pregnancy alexandra grillos, catherine renaudin, eduardo santos villanueva, bianca martins, soledad martin-pelaez, lauren huggins, eunju choi, ghislaine dujovne, pouya dini school of veterinary medicine, university of california davis, davis, ca a 17-year, thoroughbred mare, was bred to a donkey jack. a routine monthly transrectal ultrasonography was performed during pregnancy. at 186 days of pregnancy, the fetus was abnormally located deep in the mare’s abdomen. only a large amount of fetal fluid could be imaged transrectally. two weeks later, at 200 days pregnancy, the head was detected transrectally via ultrasonography, having an eye with a hyperechogenic lens, suggestive of congenital cataract. transabdominal ultrasonography revealed several fetal abnormalities, such as small biparietal diameter and small eye volume consistent with intrauterine growth retardation (iugr), and hyperechogenic bowels. fetal heart rate was within the normal limit but on the lower side of the range. as pregnancy advanced, iugr became more pronounced and more abnormalities were observed with an abnormal twisted umbilical cord, hyperechogenic medulla of the right kidney, posterior presentation at 240 days of pregnancy, and an increased maximum fluid depth. additionally, amniotic and chorioallantoic membranes were juxtaposed with a minimal amount of allantoic fluid, suggesting a hydrops amnion condition. fetus was identified dead by the loss of heartbeat at 272 days without clinical signs of impending abortion in the mare. amniotic membrane was punctured with a thoracic trocar, and ~ 20 liters of fluids were slowly evacuated. fetus was manually delivered in a posterior presentation, with extensive umbilical cord twists. fetal membrane had an enlarged avillous area at cervical star and fetus had bilateral congenital fetal cataracts and abnormal right kidney. histologically, the right kidney had a severe diffuse lobulation/malformation, and there was lymphocytic inflammation in the upper esophagus. this is the first report of an in utero diagnosis of fetal congenital cataracts associated with hydrops, highlighting the importance of ultrasonographic assessments of pregnancy and fetal well-being. keywords: mare, congenital cataracts, hydrops condition successful pregnancy in a dog treated for a deep surgical infection with abdominal wall abscess after surgical artificial insemination alexis nelson,a alec sherman,a adrien aertsens,a eleas wu,b theresa beachlerb adepartment of veterinary clinical sciences bdepartment of veterinary diagnostic and production animal medicine college of veterinary medicine, iowa state university, ames, ia dogs are bred via natural mating or artificial insemination (ai). methods of ai include transvaginal, transcervical, and surgical. surgical ai is frequently performed with either frozen or poor-quality semen, in cases of previous infertility, or on an elective basis.1 as in any surgical procedure, there are potential complications after surgical ai, including postsurgical infection, unsuccessful fertilization, and complications associated with anesthesia. a 3-year, labrador retriever bitch, was presented to the emergency service 2 days after surgical insemination for anorexia and lethargy. dog was pyrexic, lethargic, tachycardic, and a diffuse cellulitis with associated pain on palpation of her incisional site were noted. a complete blood count had severe leukocytosis characterized by a mature neutrophilia, eosinophilia, and monocytosis. abdominal radiography and ultrasonography confirmed soft tissue swelling and fluid accumulation around the incisional site. septic exudate was aspirated via ultrasonography guided technique. after initial stabilization with intravenous fluids, hydromorphone and gabapentin for analgesia, and ampicillin/sulbactam as an antimicrobial, surgical exploration was performed and confirmed a deep surgical site infection of subcutaneous tissue and an abdominal wall abscess communicating with the abdominal cavity. surgical debridement with peritoneal lavage was performed and the abdomen was closed after placement of an active jackson-pratt® drain that was removed after 4 days. skin and subcutaneous layers were left open for 3 days for a vacuum-assisted closure to allow secondary wound closure. continued medical support consisted of gabapentin, hydromorphone, carprofen, and intravenous ampicillin/sulbactam. microbial culture was positive for staphylococcus pseudintermedius sensitive to beta-lactamase inhibitors. clinical status improved quickly and the patient started eating well on day 2 after surgery. she was discharged 7 days after admission on oral carprofen and amoxicillin-clavulanic acid. approximately 1 month after discharge, her incision site was healed, and she was diagnosed pregnant (medium to large litter size) via abdominal ultrasonography. at 66 days after estimated luteinizing hormone surge, she successfully whelped and subsequently raised 7 viable pups without complication. student poster session clinical theriogenology 2022; 14: 317 keywords: dog, surgical insemination, abscess, pregnancy reference 1. makloski, cl: clinical techniques of artificial insemination in dogs. vet clin north am small anim pract 2012;42:439-444. postparturient metritis in a primiparous belgian mare allison wallace, kianna spencer, giorgia podico, igor canisso college of veterinary medicine, university of illinois urbanachampaign, urbana il metritis is a common postpartum complication in domesticated species, including horse. although all equine breeds are affected by metritis, it appears that thoroughbred and belgian horses are more severely affected by this disease. if left untreated, metritis can be life-threating and result in laminitis. retained fetal membranes and dystocia are common predisposing factors to metritis, but mares with an otherwise normal parturition and release of fetal membranes can also develop the condition. clinical signs include fever (typically > 102.5°f); depression, tachycardia, purulent vulvar discharge, and secondary laminitis in advanced cases. treatment consists of a combination of broad-spectrum antibiotics, nonsteroidal antiinflammatory drugs, uterine lavage, ecbolics, and in severe cases, fluid therapy. a 4-year, primiparous belgian mare, delivered a filly uneventfully at 340 days of pregnancy. fetal membranes were passed completely 1.5 hours postpartum, and the filly stood and nursed by 2 hours postpartum a dose of flunixin meglumine (1.1 mg/kg) was given intravenously to prevent any foaling induced pain. by 2 hours postpartum, the mare had mild signs of foal rejection (stall walking, pinning her ears at the foal, and not standing to nurse) that was mitigated following treatment with intramuscular cloprostenol (500 µg) and butorphanol (10 mg) treatment. twenty-four hours postfoaling examination was unremarkable. by 48 hours after foaling, the mare had fever (103.7°f; 99 -101.5°f), tachycardia (60 beats/minute; 28 44 beats/minute), tachypnea (30 breaths/minute, 10 24 breaths/minute) and mucoid vulvar discharge. uterine lavage was performed with 0.1% iodine solution and the fluid recovered had abundant strands of fibrin and debris. the mare was given intravenously potassium penicillin g (22,000 units/kg, every 6 hours), gentamicin (6.6 mg/kg, every 24 hours), and oral metronidazole (15 mg/ kg, every 8 hours) for 5 consecutive days. intravenous fluid therapy (lactated ringers, 3 liters/hour) was initiated. a commercial multi-electrolyte solution (cmpk, containing calcium; magnesium, phosphorus and potassium was included at 50 ml/hour) and oxytocin (4 units/hour) were given via constant rate infusion for ecbolic action to aid in uterine clearance. other treatments included flunixin intravenous meglumine (1.1 mg/kg, every 12 hours), oral omeprazole (4 mg/kg every 24 hours) and probios (60 grams loading dose and then 30 g every 24 hours). mare developed a slightly increased digital pulse and temperature that were treated with distal limb cryotherapy for 48 hours. uterine lavage (80 liters/ session) was repeated 3 times/day in the first 72 hours, and then reduced to 2 times a day from 96 to 120 hours; and then once daily from 144 to 168 hours postpartum. clinical signs progressively improved starting 12 to 96 hours postpartum. mare was discharged after the last treatment with sulfadiazine oral trimethoprim (24 mg/kg, every 12 hours) for 7 days and intramuscular oxytocin (20 units, every 12 hours). breeding the mare on foal-heat was strongly discouraged. mare was reexamined by the referring veterinarian at the '30-day heat'. mare was rebred, diagnosed pregnant at 15and 25-days postovulation. prognosis for metritis can be poor for draft mares due to secondary laminitis. in the present case, early diagnosis and aggressive intervention resulted in successful therapy for metritis. persistent müllerian duct syndrome in a german shorthair pointer anna tomlinson,a heath king,a brittany baughman,a alicia olivier,a matt mcgee,b darcie sidelingera amississippi state college of veterinary medicine mississippi state, ms bcorinth animal care center, corinth, ms a 2-year, intact male, german shorthair pointer, was presented for a routine castration. prepuce was located further caudal than normal, and penis was malformed and hypoplastic. scrotum was not present, and testes were not appreciable on palpation. cryptorchidism was suspected. a ventral midline laparotomy was performed to remove suspected retained testes. in the abdomen, there was a structure that resembled a uterus, with uterine horns and a testis at the end of each horn. this structure was removed; histopathology confirmed that the reproductive tract had bilateral testes, both with an epididymis that was attached to a hypoplastic uterus. testes had a pampiniform plexus and were composed of seminiferous tubules that lacked spermatogonia, but had sertoli and leydig cells. each testis was connected to müllerian and wolffian duct systems. uterine tissue was devoid of glands. although karyotyping was not performed to confirm the diagnosis, clinical findings and histopathology were consistent with persistent müllerian duct syndrome (pmds), a form of male pseudohermaphroditism. during normal fetal development, müllerian inhibiting substance (mis) is secreted and causes regression of the müllerian ducts. in male dogs with pmds, there is a genetic defect in mis or its receptor, misrii, so the müllerian ducts persist. müllerian ducts are responsible for development of the uterus, fallopian tubes, and cranial vagina. with pmds, these normally female structures are present in a dog that is phenotypically male.1 persistent müllerian duct syndrome is a heritable, autosomal recessive trait primarily observed in homozygous male miniature schnauzers and is considered rare but has been reported in other breeds.2 a gonadectomy, as performed in this case, is recommended in dogs with pmds to prevent further reproductive disorders such as pyometra, urinary tract infection, or prostate infection.3 keywords: dog, persistent müllerian duct syndrome, pmds, pseudohermaphrodite clinical theriogenology 2022; 14: 318 references 1. wu x, wan s, pujar s, haskins me, et al: a single base pair mutation encoding a premature stop codon in the mis type ii receptor is responsible for canine persistent müllerian duct syndrome. j androl 2009;30:46-56. 2. meyers-wallen vn: genetics of sexual differentiation and anomalies in dogs and cats reprod fertil suppl 1993;47:441-452. pmid: 8229960. 3. cinti f, sainato d, charlesworth t:. a case of persistent mullerian duct syndrome in a dog. j small anim pract 2020;62:311-311. anencephaly in a french bulldog pup blaire arney, peyton draheim, candace lyman department of clinical sciences college of veterinary medicine, auburn, al neural tube defects (ntds) are the second most common neonatal abnormality reported in human medicine. in veterinary medicine, 3 25% of neonatal mortality in livestock and companion species has been associated with developmental anomalies. it is not unusual that french bulldogs are reported to possess ntds in the form of vertebral column abnormalities, including spina bifida. however, to date, there have not been any reported cases of the ntd identified as anencephaly in the french bulldog. an intact, 4-year, female french bulldog, 60 days into pregnancy with her third litter for a prepartum management of an elective cesarean surgery. all previous litters were healthy and fully developed upon delivery. dog’s ovulation date had previously been determined to be october 30, 2021, via serum progesterone concentrations; a transcervical insemination was subsequently performed with chilled semen on day 2 after ovulation. on day 60 of pregnancy, the bitch appeared normal in physical examination and had marked mammary development. a transabdominal ultrasonographic examination of the uterus was performed; multiple fetuses were visualized with heart rates within normal limits (190 220 beats/minute), indicating that the fetuses were not distressed. all visualized fetuses had good cortico-medullary definition of the kidney and detailed intestinal definition, although peristalsis was only seen in 2 fetuses. shortly thereafter, serum progesterone concentrations were 2.0 ng/ml and the patient was prepared for cesarian surgery. one pup was diagnosed with the more common ntd of a cleft palate that extended the entirety of the hard and soft palates. however, the second pup had less common ntd in that it was missing the dorsal aspect of its skull and cerebrum, leaving the entirety of the eye globes exposed along with what little portion of brain matter existed. a diagnosis of anencephaly was made based on macroscopic observation. both pups were subsequently euthanized, leaving 5/7 completely healthy and viable puppies. at the owner’s request, ovariohysterectomy was performed concurrently with the cesarean surgery. recovery from anesthesia was uneventful, with recently nursed pups and bitch were discharged. neonatal abnormalities are undesirable in any litter. however, until corresponding factors have been identified, clients and practitioners need to be aware that developmental abnormalities of varying degrees can occur. cleft palates, umbilical hernias, and atresia ani are common conditions that every veterinarian should routinely inspect neonates for prior to discharge. in humans, it is estimated that 1 in every 4,600 infants is afflicted with the ntd of anencephaly. the specific cause of anencephaly in individual cases are unknown but are thought to include chromosomal abnormalities, environmental factors, folic acid deficiency, or exposure to teratogenic medications. recommendations for pet owners in these cases may also include avoidance of a repeat breeding between the impacted litter’s dam and sire. this case illustrated that clients and practitioners should be prepared for varying degrees of neonatal abnormalities, even if previous reports may not exist. keywords: french bulldog, anencephaly, neural tube defect iatrogenic foreign body in urinary bladder secondary to vaginal cytology procedure connor cashman, erin runcan, marco coutinho da silva college of veterinary medicine, ohio state university, columbus, oh a 2-year, multiparous golden retriever bitch, was presented for breeding management. dog had bloody vaginal discharge for 5 days. during vaginal smear collection the bitch unexpectedly jumped and the tip off of the wooden cotton swab was broken and it was not palpable in the caudal vagina nor was not seen on the floor. a rigid endoscope was used to perform a vaginoscopy, but the missing segment of the cotton swab was not visualized. abdominal ultrasonography revealed a normal, small, hypoechoic bladder with no evidence of foreign material. dog was taken outside to confirm that she was able to urinate. dog was sent home with instructions to monitor for changes in behavior until her next scheduled appointment in 2 days. owners returned with the bitch 2 days later and reported that she was urinating normally the day following the accident but had started to experience discomfort when urinating within 12 hours after presentation. transabdominal ultrasonography was performed again that now revealed a linear hyperechoic structure in the lumen of the bladder. dog was placed under general anesthesia and underwent a cystoscopy. foreign material (broken cotton tip of the swab) located in the trigone of the bladder was removed. there was moderate urinary bladder and urethral trauma from the inadvertent catheterization of the urethra and the splintered wooden shaft of the cotton tipped applicator freely floating within the urinary bladder. dog was sent home with carprofen 2.2 mg/kg and amoxicillin 15 mg/kg to help manage inflammation and prevent infection from the procedures. progesterone concentrations were monitored and had a normal profile. dog was bred via transcervical insemination with frozen semen 5 days after cystoscopy and pregnancy confirmed via transabdominal ultrasonography at 30 days of pregnancy. she then went on to have 8 healthy pups delivered via caesarean surgery. cotton tipped applicator was probably broken off within the urethra during the vaginal cytology procedure and traveled (retrograde) to the urinary bladder. we suspect that the tip was not visualized in the initial ultrasonography performed immediately after it was broken was most likely due to the limitations of visualizing the pelvic urethra. however, the combination of an attentive owner and subsequent ultrasonography enabled prompt removal of the tip of the swab without any detrimental effects on the bitch’s health and fertility. clinical theriogenology 2022; 14: 319 keywords: dog, cystoscopy, vaginoscopy, vaginal cytology, foreign body cryptorchidism and laparoscopic assisted cryptorchidectomy in nigerian dwarf goats dane schwartz, eduardo arroyo, ahmed tibary comparative theriogenology service, department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa cryptorchidism is reportedly uncommon in small ruminants. objective was to describe clinical features and laparoscopic assisted cryptorchidectomy in 18 nigerian dwarf goats presented at the wsu theriogenology services. all goats were companion animals and ranged from 2 months to 1 year. there were 15 (83%) unilateral and 3 (17%) bilateral cryptorchids. two of 3 bilateral goats were purchased as wethers. owners were concerned when they began to display male like behavior and odor at ~ 6 months of age. the remaining bilateral goat was presumably castrated by banding at 1 week of age. of the 15 unilateral, 11 retained right testis and 4 retained left. in this case series there were 3 full brothers (2 bilateral, 1 unilateral) and 5 half-brothers (same sire). retained testis were located intra-abdominally near the bladder, via transcutaneous inguinal ultrasonography with curvilinear 3.5 5 mhz probe. retained testis length and width were 21.8 ± 2.1 mm and 18.3 ± 1.5 mm, respectively (mean ± sd). for laparoscopic assisted cryptorchidectomy, goats were fasted for 18 hours. after sedation with butorphanol, anesthesia was induced with propofol and midazolam and maintained with isoflurane in oxygen. animals were placed in dorsal recumbency, and the scrotum and ventral abdomen prepared aseptically. a 1 cm skin incision was made ~ 4 6 cm lateral to midline, contralateral to the side of the retained testis. abdominal cavity was insufflated with medical grade co2 through a teat cannula prior to insertion of a 6 mm trocar and canula which served as the laparoscope portal. patient was placed in trendelenburg position to visualize testis. a second incision was made parallel to the inguinal ring on the side of the retained testis. babcock forceps were introduced into the second portal and testis was grasped at the level of the gubernaculum. incision length was extended to allow testis exteriorization. spermatic cord was transfixed with 0 pds suture and testis was excised with metzenbaum scissors. incision was sutured in 2 planes with 2-0 pds to close the body wall with a simple continuous pattern and skin with an intradermal pattern. a single cruciate suture was used to close the laparoscope portal incision. scrotal testis was castrated using a closed technique. for bilaterally cryptorchid cases, both testes were removed from the same paramedian instead of inguinal incision. there were no surgical or postsurgical complications. this case series suggested that cryptorchidism, may be more common and possibly hereditary in nigerian dwarfs. cryptorchidism may be missed by owners during castration. in companion goats, cryptorchidism is undesirable because of the male behavior and odor. laparoscopic assisted cryptorchidectomy is minimally invasive and is highly recommended for small sized animals. keywords: goat, laparoscopy, testicular descent, inguinal, ultrasonography uterine hemangioma in an alpaca (vicugna pacos) dane schwartz, eduardo arroyo, ahmed tibary department of veterinary clinical sciences, college of veterinary medicine washington state university, pullman, wa uterine hemangioma has been described in women, but occurs rarely in animals. here we describe the clinical and pathological features of a case of uterine hemangioma in an alpaca. a 10-year, multiparous female, was presented with a complaint of postmating hemorrhage. alpaca had undergone a term cesarean surgery, due to uterine torsion, 8 months prior to presentation. physical examination parameters were within normal limits. on transrectal ultrasonography, the uterine wall appeared thickened and lined with several round echogenic areas 9 13 mm in diameter. a 4 mm cyst was visualized in the middle of the left uterine horn. right ovary was static, whereas left ovary had multiple small follicles. vaginoscopy did not reveal any abnormalities, and cervix was open. alpaca was sedated with intramuscular butorphanol tartrate (0.05 mg/kg) and hysteroscopy was performed using a 9 mm pediatric gastroscope. body of the uterus and uterine horns had several raised congested areas. two biopsies were obtained from the lesions through the working channel of the endoscope. examination was halted because of bleeding following biopsy. alpaca was treated with flunixin meglumine and long acting ceftiofur, then placed under observation overnight. blood was observed dribbling from the vulva but stopped after 4 hours. biopsies taken by hysteroscopy were nondiagnostic. the owner declined further diagnostics and the animal was discharged with instruction not to breed her until another examination was performed. one year after presentation the animal was euthanized because of severe respiratory distress. no necropsy was performed; however, the uterus was submitted for examination. full thickness histological preparations were made of the raised lesions and surrounding tissue. uterine hemangioma was diagnosed based on presence of irregularly shaped cavernous vascular spaces in the endometrium and between myometrial fascicles. clinical and pathological description of uterine hemangiomas is scarce in veterinary medicine. in humans, the condition can be congenital or acquired and is associated with menorrhagia and pregnancy complications. this case may have been acquired. hysteroscopy should be considered to further investigate lesions observed on ultrasonography. keywords: alpaca, neoplasia, infertility, uterus, hysteroscopy clinical theriogenology 2022; 14: 320 sertoli cell tumor in a cryptorchid dog daniella adams, fiona herzog, alissa st. blanc, carlos pinto department of veterinary clinical sciences, school of veterinary medicine louisiana state university, baton rouge, la cryptorchidism is a relatively common condition in male dogs that can predispose the animal to neoplastic development and feminizing paraneoplastic syndrome. a 6-year, male castrated boxer, was referred for evaluation of chronic generalized hair loss and thickening of skin on its chest and abdomen; pet owner also informed that dog had only 1 scrotal testis and was removed at 6 months. initial evaluation (dermatology service) revealed symmetrical alopecia and seborrhea, lichenification of scrotal skin and an edematous, pendulous prepuce. further evaluation (theriogenology service) revealed an enlarged and elongated nipple; cytology of penis/prepuce revealed cornified epithelial cells. a brief focal transabdominal ultrasonography was performed and a 4 5 cm globoid mass with heterogenous echogenicity was imaged on the left abdomen, ventrocranial to the urinary bladder; the prostate length and width measured 3.2 and 2.8 cm, respectively. surgical removal of the abdominal mass was recommended, and the surgery was scheduled for 4 days atter initial diagnosis of cryptorchidism and presumptive sertoli cell tumor. a preoperative comprehensive abdominal ultrasonography further detected the presence of medial iliac lymphadenopathy and splenic nodules. fine needle aspirates were obtained from the enlarged medial iliac lymph node and spleen; the splenic cytology had hyperplasia and low-grade extramedullary hematopoiesis, and the lymph node cytology, though poorly cellular, had no evidence of neoplastic cells. thoracic radiographs revealed no abnormalities. complete blood cell count and serum chemistry had a stress leukogram and hypercholesterolemia, but it was otherwise unremarkable. during surgery, the left cryptorchid testis and a mildly enlarged medial iliac lymph node were removed. histopathology of these tissues confirmed the mass to be a sertoli cell tumor and the lymph node had no evidence of metastasis. serum inhibin b concentrations decreased from 805 pg/ml prior to surgery to < 6 pg/ml 3 weeks after the surgery, and anti-müllerian hormone (amh) decreased from 8435 ng/ml to 56 ng/ml during that period. patient recovered uneventfully but it was not brought in for an abdominal ultrasonography to rule out potential abdominal metastasis at 3 months after surgery. pet owner reported the dermatoses began to subside by 2 months after surgery and only the 2 cranial thoracic nipples remained mildly enlarged. this report highlighted the practical and diagnostic value of penis/prepuce cytology, brief focal ultrasonographic examination and measurement of serum inhibin b and amh concentrations in diagnosing cryptorchidism and a sertoli cell tumor in dogs. keywords: dog, sertoli cell tumor, cryptorchidism, inhibin b, anti-müllerian hormone fetal mummification in a goat  elizabeth whitt,a hope patrick,b pablo jarrin,a tulio pradoa acollege of veterinary medicine, banimal science department university of tennessee, knoxville, tn fetal mummification occurs in most species; however, is not a common finding in goats. not much is published in goats. a 5-year doe, 1 month after her expected day of kidding, was presented with a history of vaginal discharge and fetal membranes hanging from her vulva. she was bright alert and responsive, and had increased heart rate, elevated respiratory rate and effort along with decreased ruminal contractions. transabdominal ultrasonography was nondiagnostic. bloodwork had multiple electrolyte abnormalities with elevated lactate. vulva appeared hypoplastic and urethra felt firm on palpation. reddish-brown mucoid discharge was noted and fetal membranes were palpable. transrectal ultrasonography revealed a fetus with bony structures resembling thorax with no heartbeat. cesarean surgery was performed via a left flank approach and a mummified fetus was extracted from the left uterine horn. keywords: goat, mummification, cesarean surgery reference 1. lefebvre rc: fetal mummification in the major domestic species: current perspectives on causes and management. vet med (auckl) 2015;6:233-244. umbilical torsion abortion in a multiparous quarter horse mare emma hicks, kianna spencer, giorgia podico, humberto magalhaes, igor canisso college of veterinary medicine, university of illinois urbanachampaign, urbana il umbilical cord torsion is the main cause of abortion in the united kingdom; however, in us it accounts for a small fraction of abortions. a 21-year, multiparous quarter horse mare, was presented for evaluation postabortion with an accompanying 8-month fetus. one week prior to abortion, the mare had an apparently unremarkable transrectal ultrasonographic evaluation. the mare’s past breeding history included susceptibility to persistent breeding induced endometritis and enlarged pendulous uterus. she was up to date on vaccinations, deworming, and had no history of disease. initial physical examination was within normal limits. udder was moderately developed, and milk was present. transrectal palpation and ultrasonography revealed a distended uterus with decreased tone and a large cyst in the uterine body. vaginal examination revealed moderate bruising inside the vestibule and a mild amount of straw-colored fluid in the uterine body and cranial vagina. evaluation of aborted fetus suggested that the fetus was slightly small for its fetal age and had no hair but was otherwise developmentally normal. fetal membranes had thin tissue with diffuse pallor of the chorion and there was clinical theriogenology 2022; 14: 321 lack of focal thickening. necrotic villi near the cervical star indicated premature separation at the caudal pole. umbilical cord was increased in length (~ 122 cm) and had several twists and moderately thickened umbilical vessels and urachus near the carcass. collectively, these findings were consistent with fetal death due to umbilical cord torsion. due to the enlarged uterus, the mare was at risk to develop metritis and secondary laminitis; thus, therapy was initiated to prevent these issues. during hospitalization, large volume of uterine lavage was performed 1 or 2 times per day. an ecbolic agent (oxytocin 20 units, every 6 hours) was given intramuscularly and the mare was exercised daily to aid in uterine clearance. the mare was placed on oral trimethoprim sulfa (30 mg/kg, every 12 hours) to treat potential infection, flunixin meglumine (1.1 mg/kg, every 12 hours) was given intramuscularly to manage inflammation. additionally, a temporary caslick’s vulvoplasty was performed using nonabsorbable suture to reduce severe pneumovagina. after 4 days of hospitalization, mare recovered without having any signs of metritis, but developed urovagina/urometra. as urovagina/urometra can be transient in postpartum/postabortion mares, a reevaluation was scheduled in 60 days. owner was instructed to monitor mare’s rectal temperature daily for 7 days and bring the mare back if any fever or depression was noted. power walks were recommended to aid in uterine involution and reduce urine pooling, along with intramuscular oxytocin (20 units, every 12 hours) for 2 weeks. cause of umbilical cord torsion remains unknown; certain thoroughbred blood lines are prone to it. the condition has been rarely detected in quarter horses. the umbilical length was >100 cm that makes this mare 3 times more likely to abort from umbilical torsion. it remains to be determined if the mare’s incredibly pendulous uterus served as a predisposing factor for umbilical cord torsion. although reoccurrence of umbilical cord torsion appears to be rare, the owner was offered the possibility of performing embryo transfer to prevent the mare from carrying her own foals. a case of placental edema and premature lactation in a thoroughbred mare haley white,a peyton draheim,a candace lyman,a robyn wilborna adepartment of clinical sciences, college of veterinary medicine auburn university, auburn, al a 17-year, thoroughbred mare, was presented at 302 days of pregnancy for intensive management of placentitis. mare was confirmed pregnant at 40 days with no abnormalities identified. referring veterinarian made the diagnosis of placentitis based upon a relaxed and elongated vulva, premature lactation with a milk calcium of > 2,000 ppm and an increased combined thickness of the uterus and placenta (ctup) of 19 mm. on presentation, the mare’s physical examination findings were within normal limits. evidence of premature lactation was noted on the hind legs, but no vulvar discharge was present. on transrectal ultrasonography, ctup measured 21 mm with the placenta appearing markedly edematous around the cervical star; fetal movement was appreciated. a vaginal speculum examination was performed, and findings were unremarkable. a fetal thorax was identified on the right side of the mare via transabdominal ultrasonography with no heartbeat; however, fetal movement was detected on subsequent transrectal palpation. based on these findings, ascending placentitis was the most likely differential. medications included potassium intravenous penicillin (22,000 iu/kg, 4 times daily), gentamicin (6.6 mg/kg, once daily), flunixin meglumine (1.1 mg/kg, twice daily), oral altrenogest (0.088 mg/kg, once daily) and pentoxifylline (8.4 mg/kg, 4 times daily). transrectal and transabdominal ultrasonography were performed regularly to monitor progress. improvement in the ctup at the cervical star was noted so treatment was continued. on day 307 of pregnancy, transabdominal ultrasonography revealed a second fetal thorax with a heartbeat of 80 beats/minute on the mare’s left side. this made complications from a twin pregnancy the top differential. it was hypothesized that recent fetal death incited an inflammatory response that resulted in premature lactation and mimicked the signs of placentitis. with this change, the treatment plan was altered from intravenous antibiotics to oral trimethoprim sulfamethoxazole (30 mg/ kg, twice daily). altrenogest was discontinued at a rate of 2 ml/day. the mare spontaneously delivered a live filly and a deceased colt on day 325 of pregnancy. gross evaluation of the twins indicated that they were full term. physical examination of the filly indicated no abnormalities, but it was decided to give intravenous frozen-thawed plasma based on the assumption that the mare had lost most if not all colostrum due to premature lactation. it was noted that the filly was progressively becoming less active. the filly was sedated with intravenous diazepam (0.1 mg/kg) and butorphanol (0.08 mg/ kg) and placed in lateral recumbency for catheter placement, during which time respiration stopped; all efforts to resuscitate were unsuccessful. the filly and fetal membranes were submitted for necropsy; the pathologist identified no signs of sepsis. fetal membranes were dichorionic with the filly’s fetal membrane being unremarkable and the colt was greenish grey in color and diffusely thickened. although this case appeared as a typical case of placentitis, it emphasized the importance of considering twins as a differential. twins should be detected via repeated thorough transabdominal ultrasonographic examinations and should be reduced early in pregnancy. keywords: mare, placentitis, twins ovarian cysts and cystic endometrial hyperplasia in a young breeding bitch hayley hunt,a sharon wagner,a eleas wu,b theresa beachlerb adepartment of veterinary clinical sciences bdepartment of veterinary diagnostic and production animal medicine college of veterinary medicine, iowa state university, ames, ia an intact, 2-year maiden labrador retriever bitch, was presented for evaluation of scant, persistent white vaginal discharge of ~ 3-months duration, immediately following a clinically normal estrus. repeated vaginal cytology revealed cornified vaginal epithelium consistent with persistent cytologic estrus; however, no external physical or behavioral clinical signs were observed during this time. suspicions for this bitch’s persistent cytologic estrus were hormonally clinical theriogenology 2022; 14: 322 active ovarian cysts or exposure to exogenous hormones. however, there was no known exposure to any hormones or phytoestrogens. abdominal ultrasonography was performed and bilateral ovarian cysts (right 2.2 cm; left 0.8 cm) and cystic endometrial hyperplasia were diagnosed. transabdominal ultrasound-guided aspiration of 3.2 ml of fluid from the right ovary was performed, and cyst fluid and serum were submitted for paired hormonal analysis. there were marked elevations of estradiol and inhibin b within the cyst fluid compared to serum (estradiol 3,689.7 pg/ml versus 21.8 pg/ml; inhibin b 155,700.0 pg/ml versus 6.0 pg/ml). progesterone (15.1 ng/ml versus 0.75 ng/ml) and antimullerian hormone (2.2 ng/ml versus 0.18 ng/ml) were additionally elevated within cyst fluid compared to serum. hyperestrogenism is the hallmark clinical appearance of hormonally active follicular cysts.1 no external or behavioral clinical signs associated with hyperestrogenism were seen in this particular bitch, though cystic endometrial changes were likely due to their prolonged hormonal influence. while the reported treatment for ovarian cysts and cystic endometrial hyperplasia has historically been ovariohysterectomy, medical management was elected due to this bitch being young and a breeding prospect.1 cystorelin (75 µg) was given intramuscularly for 3 days in addition to aspiration of the large right ovarian cyst to cause luteinization and allow for uterine remodeling. recheck examination performed 2 months later revealed resolution of vaginal discharge with noncornified vaginal epithelium, and followup ultrasonography showed a marked improvement in uterine pathology with almost complete resolution of all endometrial cysts. keywords: canine, cystic endometrial hyperplasia, ovarian cyst, estrus references 1. sasidharan, jk, patra, mk, kipjen singh, l, et al: ovarian cysts in the bitch: an update. top companion anim med 2021;43:https://doi. org/10.1016/j.tcam.2021.100511. hysteroscopy on a southern tamandua hope patrick,b elizabeth whitt,a pablo jarrin,a lew strickland,a heather schwartz,c margarita woc colburn,c tulio prado a acollege of veterinary medicine, bdepartment of animal science university of tennessee, knoxville, tn cnashville zoo, nashville, tn currently, there is no report available on the use of a flexible scope in tamandua. we used a flexible multipurpose video endoscope (model 60714 nks) with a 7.9 mm probe. a 12year, intact female southern tamandua (tamandua tetradactyla) was presented for a hysteroscopy examination. several cystic endometrial glands were observed during ultrasonography. hysteroscopy was performed under general anesthesia with a flexible scope. approximately 3 to 4 cm of the flexible scope was introduced transvaginally. vestibule, vaginal vault, cervix, and uterus were identified and evaluated. several hyperplastic endometrial cysts were observed, confirming ultrasonographic findings. this examination suggested that a flexible large animal scope can be successfully used in a tamandua to observe uterus for evaluation and diagnosis of uterine pathologies (e.g., cystic endometrial hyperplasia). keywords: tamandua, hysteroscopy, endometrial cysts references 1. bracher v, mathias s, allen wr: videoendoscopic evaluation of the mare’s uterus: ii. findings in subfertile mares. equine vet j 1992;24:279-284. 2. rossi lf, luaces jp, aldana marcos hj, et al: female reproductive tract of the lesser anteater (tamandua tetradactyla, myrmecophagidae, xenarthra). anatomy and histology. j morphol 2011:272:1307-1313. spontaneous asymmetrical mammary gland fibroadenomatous hyperplasia in a bitch kassandra crissman,a viviane gomes,a chun kuen mak,a jacquelline elliott,b fabio del piero,b jenny sones,a carlos pintoa adepartment of veterinary clinical sciences, bdepartment of pathobiological sciences school of veterinary medicine, louisiana state university, baton rouge, la mammary gland fibroadenomatous hyperplasia (mfh) is a nonneoplastic, progesterone-dependent proliferative process of the mammary glands. mfh is commonly observed in pregnant and nonpregnant queens, usually as a sudden, diffuse, and symmetrical enlargement of the mammary glands with no evidence of lactation. in this report, an 11-month, nulliparous intact female maltese dog, was presented with a 7-day history of asymmetrical mammary gland enlargement, mild anorexia, and lethargy. dog had reportedly undergone her first estrous cycle ~ 33 days prior to presentation. there was no history of exogenous exposure to sex steroid hormones. right caudal thoracic mamma presented severe ecchymosis and a 15 x 10 x 8 cm (length x width x depth) subcutaneous mass, extending towards the right cranial thoracic mamma. left caudal thoracic mamma presented mild ecchymosis and a smaller mass, measuring ~ 7.5 x 6 x 3 cm. mammary papillae were juvenile, and no secretion could be expressed. differential diagnoses included mastitis, abscessation, mfh, mammary neoplasia, and paraneoplastic mammary gland enlargement. noncornified vaginal cytology and blood progesterone concentrations (12.24 ng/ml) were consistent with diestrus. there were no remarkable findings on complete blood count and serum chemistry. ultrasonographically, the masses presented architecture characteristic of mammary tissue. there was no free or encapsulated fluid. fine needle aspirates of the right and left caudal thoracic mammae were submitted for cytology and culture. a 48-hour culture and cytology result in no bacterial growth, suggestive of either epithelial hyperplasia or complex neoplasia. as there was no breeding intention, ovariectomy and biopsy of the mammary glands was elected. histopathological examination of the mammary tissue clinical theriogenology 2022; 14: 323 was consistent with mfh: the specimens were comprised of abundant spindle cells expanded by mild to moderate edema, and multifocal to coalescing embedded basophilic apocrine glandular ducts with very narrow lumen. enlarged mammary tissue gradually reduced in size after ovariectomy, with minimal enlargement of the right caudal thoracic mamma after 25 days surgery and complete resolution ~ 60 80 days after surgery. complications (e.g., skin necrosis, ulceration, and mastitis) did not occur in this case. although mfh is a condition commonly observed in young female cats, it has also been reported in older male and female cats exposed to exogenous progestogens, rats, rabbits, goats, and a sea lion. upregulation of progesterone receptors in the mammary tissue has been speculated as a predisposing factor for mfh. this condition is rarely observed in bitches, with asymmetrical mfh reported in 1 bitch after progestogens treatment. to date, this appears to be the first published report of asymmetrical mfh in a young bitch with no history of exposure to exogenous sex steroids. keywords: dog, fibroepithelial hyperplasia, mammary fibroadenoma, fibroadenomatosis double cidr induced anestrus in boer doe kyra broomfield,a joanna kollipali,a matthew cochran,a marco coutinho dasilva,a erin runcana college of veterinary medicine, ohio state university, columbus, oh since its introduction in 1984, cidr usage in parous ruminants has continued to be a mainstay of reproductive synchronization and management for operations of all sizes. many protocols have been described and published over the years, with most indicating a return to estrus ~ 48 hours after removal, and insemination within the window of 48 hours after device removal. does treated with cidr protocols had similar reproductive performance parameters compared to those treated with fga sponges,1 without the reported effects of malodorous discharge as documented in ovine counterparts.2 however, these results are typically associated with removal of the device from the vagina. a 6-year multiparous boer doe was presented after failing to conceive via laparoscopic ai and natural cover by 2 proven bucks. prior to both breedings, cidrs were placed, but when the time came for removal, neither were detected. doe’s general physical examination was unremarkable apart from mild bilateral serous nasal discharge. on genital evaluation, there was mild bloody serous discharge coming from the vagina and the animal appeared painful on external manipulation. utilizing a sonoscape a6v expert e1v with a rectal probe, a transrectal ultrasonography was performed that revealed 2 distinct cylindrical foreign bodies, in sequential linear fashion, with the most proximal observed in the cranial vagina near the cervix, and a fluid filled uterus. vaginal speculum examination revealed a proliferative scar tissue and serosanguinous discharge (submitted for histopathology). on digital palpation of the vagina, the string of the most distal cidr was felt and removed with minimal manipulation. the more proximal cidr required gentle traction and consistent pressure. at original presentation, the full extent of the soft tissue damage could not be assessed due to substantial swelling. doe was started on a course of meloxicam 20 mg/kg every 24 hours and oxytetracycline 200 mg/kg every 48 hours for 3 doses of each. initial follow up was scheduled for after 2 weeks to provide adequate period for the inflammation to decrease. during this visit, a vaginal speculum was easily passed to 23 cm, and there was no gross evidence of cervical or vaginal scarring. doe seemed much more comfortable on digital evaluation, and the owners were advised to attempt breeding on her following cycle. keywords: boer, cidr, induced, anestrus references 1. martinez-ros p, lozano m, hernandez f, et al: intravaginal devicetype and treatment-length for ovine estrus synchronization modify vaginal mucus and microbiota and affect fertility. animals (basel) 2018;8. doi: 10.3390/ani8120226 2. knights m, singh-knights d: use of controlled internal drug releasing (cidr) devices to control reproduction in goats: a review. anim sci j 2016;87:1084-1089. papillomavirus and vaginal vesicular lesions in a dairy herd stephanie bandoski college of veterinary medicine, cornell university, ithaca, ny papillomavirus has been studied extensively.1-5 perianal lesions were observed in a herd of 150 holstein cows in upstate new york. lesions included anal warts, vaginal warts, and vaginal vesicles. days in milk and lactation were noted to be risk factors for the presence of these lesions, but had minimal bioeconomic impact on reproduction and milk production. cross-sectional data was taken across the herd to document the presence or absence of anal warts, vaginal warts, and vaginal vesicles. the vesicles were scored on a scale from 1 to 5 based on severity.  these cross-sectional data were collected at 3 time points over the course of 6 months to gain information on incidence and persistence. tissue biopsies from 3 cows and a vaginal swab from 1 cow were collected and submitted for  in situ hybridization (ish), virus isolation, polymerase chain reaction (pcr), and bacterial culture. bovine papillomavirus (bpv) was identified from a vaginal wart via ish. the histological pattern of the lesion was not consistent with bpv type-1 or -2 and samples were negative for bpv 1 and 2 via pcr. it was suspected that the bpv identified was a novel strain or a strain not previously associated with the lesions seen in this outbreak. this case raises important welfare concerns on dairies  since the etiology and transmission of these lesions remain unknown. it is postulated that it may be caused by iatrogenic transmission from farm personnel involved in pregnancy detection and artificial insemination on the dairy. treatment included surgical removal of 1 anal wart as it was interfering with palpating, breeding, and defecating. improved care to change rectal sleeves between cows and to use more lubricant was implemented.  clinical theriogenology 2022; 14: 324 references 1. bauermann fv, joshi lr, morh ka, et al: a novel bovine papillomavirus type in the genus of dyokappapappilomavirus. arch virol 2017;162:3225-3228. 2. lima, fabio. “vulvitis and vaginitis in large animals reproductive system.” merck veterinary manual, merck veterinary manual, 8 oct. 2021, https://www.merckvetmanual.com/reproductive-system/vulvitisand-vaginitis-in-large-animals/vulvitis-and-vaginitis-in-large-animals. 3. lunardi m, alfieri aa, otonel raa, et al: bovine papillomaviruses — taxonomy and genetic features. current issues in molecular virology. viral genetics and biotechnological applications, victor romanowski, intechopen, doi: 10.5772/56195. available from: https://www.intechopen.com/chapters/44804 4. moeller rb jr, crossley b, adaska jm, et al: parapoxviral vulvovaginitis in holstein cows. j vet diagn invest 2018;30:464-467. 5. yamashita-kawanishi n, haga t: anogenital-associated papillomaviruses in animals: focusing on bos taurus papillomaviruses. pathogens. 2020;9:993. published 2020 nov 27. doi:10.3390/pathogens9120993 dystocia and foal survival in a mare with seven hour long second stage of labor vaiva palunas, eduardo arroyo, cristian patino, michela ciccarelli, ahmed tibary college of veterinary medicine, washington state university, pullman, wa an 8-year, arabian mare, at 328 days of pregnancy was referred for failure of normal foaling progression. owner reported that the mare had her 'water bag' ruptured 30 minutes prior but since had no contractions. mare was comfortable and interested in feed and there appeared a second membrane at the vulva that was confirmed to be the amniotic sac via photograph and consultation with the referring veterinarian. mare arrived ~ at 6 hours after the initial call. upon arrival the mare was bright, alert, and responsive with mildly increased heart rate (48 beats/minute) and respiratory rate (40 beats/minute), and decreased borborygmi. her mucous membranes were pink and moist with a crt of < 2.5 seconds. she was straining on arrival. transabdominal ultrasonography confirmed a live fetus with a heart rate of 64 beats/minute. epidural anesthesia was given (4 ml 2% lidocaine), and on abbreviated vaginal examination, the fetus was partially engaged in the birth canal in cranial presentation, dorsopubic position, with both front legs extended. during the examination the mare increased abdominal effort and attempted to go down. she was immediately anesthetized for controlled vaginal delivery. fetal maldisposition of lateral deviation of head and neck was discovered and corrected within 15 minutes and a live filly was delivered. the filly displayed normal behavior at birth. the mare retained the fetal membranes that was delivered after oxytocin treatment in lactated ringer’s drip (30 iu in 30 minutes) followed by chorioallantoic distention. postpartum evaluation was normal except for a 30-degree cranio-ventral tilt of the vagina and vaginal vestibule. after 4 days, mare and filly were discharged. rupture of the chorioallantois is considered the beginning of the second stage of parturition. in the mare, the second stage of parturition is rapid and lasts ~ 20 minutes 10 30 minutes). foal survival decreases by 10% for each additional 10 minutes beyond 30 minutes. in this case the foal survived ~ 7 hours after initiation of the second stage of foaling. this is extremely rare. although the reason for this delay could not be determined, failure of the foal to rotate and lack of uterine contractions may have delayed placental detachment and prevented hypoxia. this case illustrated an uncommon outcome of a delayed second stage of labor and underscores the importance of proceeding with care and optimism even if the second stage has been abnormally long. keywords: mare, dystocia, chorioallantois, maldisposition 047_74988 st 2022 vol. 14 #3_color 048_74988 st 2022 vol. 14 #3 049_74988 st 2022 vol. 14 #3_color 050_74988 st 2022 vol. 14 #3 051_74988 st 2022 vol. 14 #3_color 052_74988 st 2022 vol. 14 #3 053_74988 st 2022 vol. 14 #3_color 054_74988 st 2022 vol. 14 #3 055_74988 st 2022 vol. 14 #3_color 056_74988 st 2022 vol. 14 #3 057_74988 st 2022 vol. 14 #3_color 058_74988 st 2022 vol. 14 #3 059_74988 st 2022 vol. 14 #3_color 060_74988 st 2022 vol. 14 #3 061_74988 st 2022 vol. 14 #3_color 062_74988 st 2022 vol. 14 #3 063_74988 st 2022 vol. 14 #3_color 064_74988 st 2022 vol. 14 #3 2012: early pregnancy in the mare: information to interpret, concepts to accrue early pregnancy in the mare: information to interpret, concepts to accrue k.j. betteridge,a b.n. lillie,b b.a. quinn,b r.o. waelchli,a j.i. raeside,a m.a. hayesb departments of abiomedical sciences and bpathobiology, ontario veterinary college, university of guelph, guelph, ontario, canada knowledge of how the mare and her conceptus have to interact during their first month together if pregnancy is to be maintained is growing rapidly. that growth is owed to enormous advances in genomic and proteomic technology coupled to the need to combat the economically important problem of early embryonic loss. the first part of this presentation will describe and discuss some of our recent findings on how certain proteins and cytokines change at the interface between the mare and her conceptus; these we have studied in biopsy samples of endometrium, the uterine environment (as reflected in flush fluid), and three components of the conceptus (the capsule and the biand trilaminar yolk-sac wall) before and after conceptus fixation, and in response to experimental treatment of the mare with the prostaglandin (pg) cloprostenol to induce luteolysis and embryonic death. proteomic profiles of flush and yolk-sac fluids and of capsule-associated proteins were established from trypsin digested peptides by lc ms/ms with scaffold-2 interpretation, or by sds-page and immunoblots. transcriptomes for endometrium and trilaminar trophoblast were determined by agilent 44k expression microarrays. prominent amongst the temporal changes during normal pregnancy are limited proteolysis of capsuleassociated -2 microglobulin, the disappearance of secretory phopholipase-a2 (spla2) from the capsule, cessation of the passage of uterocalin into the yolk sac, and diminishing trophoblastic expression of gm2activator protein (probably involved in lipid and/or phospholipid transportation) — all occurring around the time of fixation. comparisons of the same sets of tissues and fluids from normal (control) pregnancies and from pregnancies failing after treatment of mares with pg have revealed important differences subsequent to the luteolysis. in the endometrium and uterine flush fluid, expression and/or concentrations of both uteroglobin and spla2 are increased by pg treatment and spla2 increases in the capsule and yolk-sac fluid. proteins corresponding to serpina14, anxa1, muc6, and b2m were also increased in uterine flush fluids after pg treatment, with serpina14 being simultaneously increased in yolk-sac fluid. by comparison, vnn2, vnn3, gzmb, cd109 and a2m were consistently reduced in uterine flush fluid from treated mares. microarray analyses of endometrial gene expression have revealed that pg treatment results in 10to 100-fold increases in expression of many genes, most notably six proteins involved in signalling, four related to lipid metabolism (including spla2 and uteroglobin), two associated with extracellular matrix modification, and one with lipid transport. conversely, significant >2-fold reductions in endometrial expression of various genes were also associated with pg treatment. in contrast, gene expression in trophoblasts from control and treated mares were more similar; only 14 significant elevations (in the 2to 5-fold range) resulted from treatment. these included increases in serpins b2 and b10. these studies have provided data that complement the findings of other investigators who have made similar comparisons of expression of proteins and cytokines during the estrous cycle and early pregnancy. they have revealed several alterations in the endometrium and conceptus that might be harmful effectors, or biomarkers of adverse uterine conditions, useful for clarifying the mechanisms of pregnancy establishment and pregnancy failure in mares. the second part of the talk will discuss the advantages and limitations of applying ‘cutting-edge’ techniques to investigations of early pregnancy. the advantages include unparalleled opportunities to obtain a ‘global view’ of concomitant events; limitations include the temptations to associate magnitudes of genomic and proteomic changes with biological significance. the third section of the presentation will be a reminder of some of the many deficits in our knowledge of mare-conceptus interactions that remain to be investigated by techniques old and new, of genetic predispositions to early embryo loss, and of behavioral factors that may affect the progress of pregnancy. all must be integrated if we are to understand “… what a tangled web [mares] weave, when first [they] practice to [conceive]” [apologies to sir walter scott]. 561 clinical theriogenology • volume 4, number 4 • december 2012 562clinical theriogenology • volume 4, number 4 • december 2012 omniblank: 2019 embryo grade, but not developmental stage, is related to embryonic sex in superovulated beef cows embryo grade, but not developmental stage, is related to embryonic sex in superovulated beef cows caitlin wiley,a marianna jahnke,a patrick j. gunn,b tyler dohlmana aveterinary diagnostic and production animal medicine, college of veterinary medicine, bdepartment of animal science, iowa state university, ames, ia abstract knowing calf sex before birth can have advantages for management and marketing. the objective was to detect relationships between embryonic sex and developmental stage and/or embryo quality at recovery. mature, non-pregnant, non-lactating angus and simmental cows (n = 29) were superovulated using a 13-d, controlled internal drug release-based protocol following ultrasound-guided dominant follicle aspiration. cows were artificially inseminated with frozen-thawed semen twice on d 6, 36 and 48 hours after controlled internal drug release-removal and pgf2α. embryos were recovered by non-surgical flush 7 days after first insemination, with an average of nine transferrable quality embryos per donor. embryos of quality grade 1-3 (n = 265) were biopsied, using a micromanipulator with a microsurgical blade to excise approximately 15 20% of the inner cell mass of each embryo. embryo sex was determined with polymerase chain reaction on biopsies, including electrophoresis and analysis of gels to detect a male-specific y-chromosome. stage of embryo had no association with sex (p = 0.6). however, grade was associated with sex (p = 0.03), as proportionally more grade-1 embryos were male (51.2%), whereas a higher proportion of grade-2 embryos were females (75.2%). we concluded embryo quality at recovery was a better predictor of sex than developmental stage. keywords: embryo, embryo transfer, sex determination, superovulation introduction knowing sex of offspring prior to parturition can be economically beneficial. for instance, in the dairy industry, there is a strong interest in heifer calves as replacements, within the herd or for sale to other herds.1-3 conversely, in some sectors of the beef industry, bulls are preferred for supplying feedlots with steers, whereas in other operations, heifers are desired for replacements.4 in addition to marketing benefits, fetal sex determination may assist in improving management at calving, as bull calves have higher rates of dystocia and mortality.5,6 in natural mating scenarios, there is a 51% probability of the conception resulting in a bull calf.3 for producers to influence sex of the offspring, sex-sorted semen, with ~90% accuracy, is the only validated approach to modify the sex ratio.3 timing of insemination is a hypothetical method to influence fetal sex, but efficacy has not been consistent.7-9 to determine sex before calving, there are several options, with variable accuracy. transrectal ultrasonography at 55 98 days after fertilization is one of the most common and least invasive methods to determine fetal sex.10,11 other options for sex determination involve micromanipulation at the embryonic stage, including cytoplasmic analysis,12 male-specific antigens,13 and x-linked enzyme activity;14 however, these are not commonly used due to impracticality and inconsistent results. the most common and reliable method for embryonic sex determination is dna analysis via polymerase chain reaction (pcr).15,16 research has attempted to use developmental stage and/or embryo quality to predict embryonic sex. several studies have been conducted with embryos produced by in vitro fertilization to determine if stage of embryo development at time of transfer has any association with fetal sex.7,8,17,18 the main findings were that male embryos developed faster than female embryos, if insemination occurred at the same time.7,17 however, timing of ovulation and fertilization is nearly impossible to predict in vivo, particularly in superovulated cattle that may ovulate over a 24-h interval. the objective was to determine if there is a relationship between embryonic stage and/or quality to embryonic sex from conventional in vivo-derived recoveries, as limited research has evaluated this relationship in conventional embryos. it clinical theriogenology • volume 11 number 1 • march 201953 was hypothesized there would be no significant difference in sex of embryos due to embryo stage or quality grade. materials and methods general this study was conducted at iowa state university lab animal research station, ames, ia and used embryos recovered from a superovulation study conducted at iowa state university zumwalt station, ames, iowa in august 2016.19 all protocols and procedures used were approved by the iowa state university institutional animal care and use committee. embryos were recovered from angus and simmental cows (n = 29) following a timed, 13-d superstimulation controlled internal drug release (eazi-breed™ cidr®, zoetis, kalamazoo, mi)-based protocol initiated 1 day after trans-vaginal ultrasound guided dominant follicle ablation. cows were artificially inseminated (ai) twice (12 hours apart) according to timed-ai schedule, 1.5 days after cidr® removal and second prostaglandin f2α (pgf2α; lutalyse®, zoetis), concurrent with signs of estrus observed in all cows. each insemination utilized one unit of frozen-thawed conventional semen sourced from a single bull collection, known to have high success rates in previous superstimulation research in our lab. non-surgical embryo recovery was performed 7 days after initial timed-ai and embryos were evaluated according to international embryo technology society standards by american embryo transfer association certified personnel. embryo biopsy following evaluation and washing, embryos were placed in micro-drops of splitting medium (vigro™ splitting plus solution; bioniche animal health, athens, ga) used to immobilize embryos for micromanipulation. biopsies were collected using a bioniche animal health twinning system with a micromanipulator and an olympus ckx41 microscope. a microsurgical blade excised the zona pellucida to allow removal of approximately 4 8 cells from the inner cell mass of the intact embryo contained in a micro-drop of splitting medium. each biopsy was washed with vigro™ retrieval supplement medium (bioniche animal health) and immediately transferred to labeled micro centrifuge tube with 8 µl sterile water. each tube was submerged in liquid nitrogen to ‘snap-freeze’ the samples and then were placed in racks at -18⁰c for approximately 1 month, until time allowed for pcr analysis. embryo sexing was performed with a commercial pcr kit using primers specific to the ychromosome determinant according to the manufacturer’s instructions.20 following electrophoresis, gels were placed on a uv trans-illuminator to determine y-chromosome presence or absence. if the sample failed to produce distinguishable bands, results were not included in the dataset. although 203 of the 265 embryos evaluated had distinguishable results, 62 biopsies were removed from the dataset due to inconclusive results. statistical analysis data were analyzed in sas 9.4 (sas institute inc., cary, nc) using the glimmix procedure for multivariate distribution. embryo served as the experimental unit, with embryo grade, stage and flush group as fixed effects. breed was included in the initial model as a covariate and subsequently removed due to a lack of significance (p > 0.10). animal nested in group served as a random effect. statistical significance was acknowledged at p ≤ 0.05. there were 62 embryo biopsies excluded from the dataset due to inconclusive pcr results. results there was no difference in sex due to stage of embryo (p = 0.6; table 1). however, sex differed in relation to quality grade (p = 0.03; table 2). specifically, there was a greater percentage of males from grade-1 versus grade-2 embryos (51.2 vs. 24.8%, respectively; p = 0.01). clinical theriogenology • volume 11 number 1 • march 2019 54 discussion sex determination before parturition is a growing sector of advanced reproductive technologies. as multiple ovulation embryo transfer (moet) technology is widely used, sexing of embryos will continue to be a requested service and pcr has become the most relevant technology for early embryonic sex determination. sexing of ivf derived embryos has advantages of monitoring maturation of oocytes, specific timing of fertilization, and development rates of embryos, all of which have been hypothesized to impact embryo sex.7,8,18,21 several ivf studies reported correlations between embryonic development stage and sex. previous work18 concluded male embryos develop at a faster rate than females. early blastocysts (stage 5) and blastocysts (stage 6) had a greater proportion of males, whereas morulas (stage 4) had a greater proportion of females. previous work17 reported similar proportions of males developing to more advanced stages by d 8 after insemination. the reasoning for increased rate of development of males is unknown; however, it is hypothesized that the increased growth rate in males is associated with their gonadal sex differentiation.7,22 furthermore, when the maturation state of oocytes was considered, early fertilization resulted in a higher ratio of females, whereas delayed fertilization produced a greater proportion of males.8,21 a potential explanation was that oocytes develop a mechanism allowing for improved processing of y-bearing sperm during late maturation of metaphase ii arrested oocytes. furthermore, xand y-bearing sperm have different signaling messages and surface proteins, enabling oocytes early in maturation to process x-bearing sperm easier, whereas more mature oocytes process ybearing sperm more effectively later in estrus.21,23 however, the current study failed to identify a relationship between stage and embryonic sex. with superovulation protocols, a donor is expected to ovulate multiple ova during a 24 36 hour interval, in contrast to a single ovulation in non-stimulated donors.24 as such, the current study implemented two separate timed inseminations (12 hours apart) to have viable sperm at any point during this window of ovulation. therefore, timing of fertilization relative to ovulation should have been evenly spaced and was not expected to alter the sex ratio, although exact timing of fertilization and rate of development were unknown. embryo quality grade is an important component of embryo evaluation, as it can be used to predict viability after fresh transfer or cryopreservation. quality grades take into account percentage of extruded cells, compactness of inner cell mass, shape, and color of cytoplasm.25 as the developmental stage of embryos increases with in vivo derived embryos, quality grades below a 1 are rarely appreciated, especially once embryos develop to at least stage 6 (blastocyst). one reason for this is the blastocoel cavity expands and fills the perivitelline space, making it difficult to appreciate any extruded cells or debris, which eliminates the potential for downgrading embryo quality. results from the current study, with in vivo-derived embryos, reflect these observations, with all stage 6 and 7 embryos given quality grade 1. meanwhile, quality grade 2 embryos in this study were at stages 4 and 5. in a previous work,26 ovine ivf embryos were evaluated for embryonic sex based on rate of development at 207 hours after insemination. within the advanced development stages (hatched, hatching, and expanded blastocysts), quality grades ranged from 1 4. these more advanced development stage with quality grades 1 3, tended to have a higher proportion of males (57.2%, p < 0.08).26 despite advanced developmental stages, there were variations in quality that were associated with embryonic sex. however, quality grading of ivf embryos is not as well defined as conventional embryo grading, and ivf embryos are often graded harder due to increased cellular debris and extruded cells, especially in the more developed stages, increasing variations in quality grades with more advanced embryos.27 our study had few grades 2 and 3 embryos (n = 29 and 19, respectively); therefore, further evaluation incorporating a larger dataset of in vivo derived embryos is warranted. furthermore, due to the lack of variation in quality grade with more developed in vivo derived embryos as observed in this current study, it may be of value to evaluate the embryonic sex of embryo recoveries performed earlier, perhaps at d 6. these embryos would be less developed with potentially more variation in quality grade, thereby enhancing our ability to determine if quality grade is truly associated with sex determination. clinical theriogenology • volume 11 number 1 • march 201955 conclusion there was a relationship between quality grade of in vivo-derived embryos and their genetic make-up, with a greater proportion of quality grade 2 embryos being heifers and a greater proportion of quality grade 1 embryos being bulls. however, there was no significant relationship between developmental stage at time of recovery and embryonic sex. notwithstanding, limited observations of quality grades 2 and 3 embryos emphasized the need for further research to validate and extend the current findings. acknowledgement this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. conflict of interest none of the authors have any conflict of interest to declare. references 1. kaimio i, mikkola m, lindeberg h, et al: embryo production with sex-sorted semen in superovulated dairy heifers and cows. theriogenology 2013;80:950-954. 2. mikkola m, andersson m, taponen j: transfer of cattle embryos produced with sex-sorted semen results in impaired pregnancy rate and increased male calf mortality. theriogenology 2015;84:1118-1122. 3. seidel ge: economics of selecting for sex: the most important genetic trait. theriogenology 2003;59:585-598. 4. dahlen c, larson j, lamb gc: impacts of reproductive technologies on beef production in the united states. in: gc lamb, n dilorenzo, editors. current and future reproductive technologies and world food production. adv exp med biol 752. new york, springer; 2014. p. 97-114. 5. morris ca, bennett gl, baker rl, et al: birth weight, dystocia, and calf mortality in some new zealand beef breeding herds. j anim sci 1986;62:327-343. 6. dematawewa cmb, berger pj: effect of dystocia on yield, fertility, and cow losses and an economic evaluation of dystocia scores for holsteins. j dairy sci 1997;80:754-761. 7. avery b, madison v, greve t: sex and development in bovine in vitro fertilized embryos. theriogenology 1991;35:953-963. 8. gutierrez-adan a, perez-garnelo s, granados j, et al: relationship between sex ratio and time of insemination according to both time of ovulation and maturational state of oocyte. theriogenology 1999;51:397. 9. rorie rw, lester td, lindsey br, et al: effect of timing of artificial insemination on gender ratio in beef cattle. theriogenology 1999;52:1035-1041. 10. ali a: effect of gestational age and fetal position on the possibility and accuracy of ultrasonographic fetal gender determination in dairy cattle. reprod domest anim 2004;39:190-194. 11. curran s, ginther oj: ultrasonic determination of fetal gender in horses and cattle under farm conditions. theriogenology 1991;36:809-814. 12. wintenbergertorres s, popescu pc: transfer of cow blastocysts after sexing. theriogenology 1980;14:309-318. 13. white kl, anderson gb, bondurant rh: expression of a male specific factor on various stages of preimplantation bovine embryos. biol reprod 1987;37:867-873. 14. williams tj: a technique for sexing mouse embryos by a visual colorimetric assay of the x-linked enzyme, glucose-6phosphate-dehydrogenase. theriogenology 1986;25:733-739. 15. thibier m, nibart m: the sexing of bovine embryos in the field. theriogenology 1995;43:71-80. 16. shea bf: determining the sex of bovine embryos using polymerase chain reaction results: a six-year retrospective study. theriogenology 1999;51:841-854. 17. xu kp, yadav br, king wa, et al: sex related differences in developmental rates of bovine embryos produced and cultured in vitro. mol reprod dev 1992;31:249-252. 18. carvalho rv, delcampo mr, palasz at, et al: survival rates and sex ratio of bovine ivf embryos frozen at different developmental stages on day 7. theriogenology 1996;45: 489-498. 19. wiley c.e., dohlman t.m., jahnke m.m., et al: effects of endogenous progesterone during ovarian follicle superstimulation on embryo quality and quantity in beef cows. clinical theriogenology 2018;10:427-434. 20. herr c, steel t, lascelles a, et al: instruction manual: splitting, biopsy and rapid sexing of cattle embryos. edn., pullmann, wa, ab technology, inc., 1995:58. 21. dominko t, first nl: relationship between the maturational state of oocytes at the time of insemination and sex ratio of subsequent early bovine embryos. theriogenology 1997;47:1041-1050. 22. mittwoch u: sex differentiation in mammals and tempo of growth probabilities vs switches. j theor biol 1989;137:445-455. 23. moor rm, osborn jc, cran dg, et al: selective effect of gonadotropins on cell coupling, nuclear maturation and protein synthesis in mammalian oocytes. j embryol exp morphol 1981;61:347-365. clinical theriogenology • volume 11 number 1 • march 2019 56 24. callesen h, greve t, hyttel p: preovulatory endocrinology and oocyte maturation in superovulated cattle. theriogenology 1986;25:71-86. 25. wright rw, ellington j: morphological and physiological differences between in vivo and in vitro produced preimplantation embryos from livestock species. theriogenology 1995;44:1167-1189. 26. bernardi ml, delouis c: sex-related differences in the developmental rate of in-vitro matured in-vitro fertilized ovine embryos. hum reprod 1996;11:621-626. 27. barfield j: evaluation of in vitro-produced bovine embryos. proceedings ceta/acte & aeta joint convention 2015. table 1. sex determination of bovine embryos, by developmental stage.1 development stage 4 (n = 78) 5 (n = 99) 6 (n = 11) 7 (n = 15) p-value female (%) 56.0 57.2 77.7 60.8 0.6 male (%) 44.0 42.8 22.3 39.2 0.6 1stage based on international embryo technology society classification table 2. sex determination of bovine embryos, by embryo grade.1 quality grade 1 (n = 155) 2 (n = 29) 3 (n = 19) p-value2 females (%) 48.8a 75.2b 64.7ab 0.03 males (%) 51.2a 24.8b 35.3ab 0.03 1 quality grade based on international embryo technology society classification (1 = excellent or good, 2 = fair, 3 = poor). 2 p-values of the main effect of grade. a-c within a row, lsmeans without a common superscript differed (p ≤ 0.05). clinical theriogenology • volume 11 number 1 • march 201957 clinical theriogenology • volume 11 number 1 • march 2019 58 omniblank: << /ascii85encodepages false /allowtransparency false 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: bovine richomoniasis: a review bovine richomoniasis: a review 1 l. strickland 2 department of clinical sciences, college of veterinary medicine, auburn 3 university, auburn, al, usa 4 5 abstract 6 trichomoniasis is a bovine venereal disease that causes substantial economic 7 losses. bulls serve as asymptomatic carriers for the protozoan tritrichomonas foetus, 8 whereas infection in females may result in early embryonic death, abortion, pyometra, 9 fetal maceration, or infertility, all of which negatively influence the profitability of a 10 cattle operation. when allowed adequate recovery time following infection most females 11 mount an immune response and return to normal reproductive status. however, the male 12 can remain infected and remain a risk to a producer’s breeding program. currently no 13 legal treatment for this disease exists in the united states therefore veterinarians and 14 cattle producers must focus on preventive management and surveillance measures such as 15 testing, identification and removal of positive animals. understanding the pathogenesis, 16 prevalence, economic impact, and diagnosis of trichomoniasis will assist with 17 implementation of appropriate prevention and control programs. this paper reviews the 18 pathogenesis, prevalence, economic impact, and diagnosis of trichomoniasis in cattle, as 19 well as common guidelines for the prevention and control of trichomoniasis. 20 keywords: trichomoniasis, tritrichomonas foetus, epithelial crypts. 21 22 23 289 introduction 24 the bovine venereal disease trichomoniasis is caused by the protozoan 25 tritrichomonas foetus (t. foetus). bulls serve as asymptomatic carriers when the 26 organism colonizes the epithelium of the penis or prepuce with no clinical signs. 27 following coitus or artificial insemination with contaminated semen females develop 28 uterine infections that may lead to early embryonic death, abortion, fetal maceration, 29 pyometra or infertility. this manuscript reviews the pathophysiology of bovine 30 trichomoniasis as well as common guidelines for diagnosis 31 venereal disease 32 t. foetus is an obligate parasite of the bovine reproductive tract. similar to most 33 venereal diseases, the male is an asymptomatic carrier while the female suffers 34 identifiable consequences of infection. t. foetus in bulls localizes in the smegma 35 (secretions) of the epithelial lining of the penis, prepuce, and distal urethra.1 the 36 organism does not invade the epithelium, and therefore does not invoke an immune 37 response in the bull.2 t .foetus causes no penile or preputial lesions and does not affect 38 libido.3,4 there are no observable changes in semen quality attributable to the organism’s 39 presence, however in a recent study by benchimol, et al, exposure to t. foetus resulted in 40 decreased spermatozoal motility, agglutination of sperm cells, and eventual 41 phagocytosis.5-7 the only clinical sign that may be observed in an infected bull is a mild 42 transient preputial discharge during the first two weeks of an infection.4 43 infection in young bulls (less than 3-4 years of age) is purportedly most often 44 transient, with disease transmission only occurring if sexual contact with a non-infected 45 cow occurs within minutes to days following breeding of an infected cow.8,9 studies by 46 290 morgan8 and clark9 indicate that clearance of the organism in a young bull is possible 47 within 20 minutes following breeding an infected cow. transmission of t. foetus by a 48 young bull is therefore likely to be a passive, mechanical transmission that differs from 49 transmission associated with a chronically infected older bull. 50 t. foetus infection in the cow occurs during coitus with an infected bull. the 51 organism transverses the cervix and colonizes the entire reproductive tract within 1-2 52 weeks,10 and as the organism multiplies in the uterus it can cause death of the embryo or 53 fetus, most commonly between gestational days 15 to 80.11 pyometra and abortion are 54 often the first physical signs of trichomoniasis noticed in a herd, but these signs occur in 55 fewer than 5% of infected animals.12 infertility due to embryonic death is the most 56 economically damaging clinical sign and occurs in a larger percentage of infected cows. 57 an affected cow’s interestrus interval is usually prolonged because the embryonic loss 58 typically occurs after maternal recognition of pregnancy (days 15-17 of gestation).10 59 unlike the bull, the cow typically mounts an effective immune response to t. foetus,4 but 60 the time it takes to clear t. foetus from the cow’s reproductive tract is quite variable. 61 primary infections may be cleared from the reproductive tract in as little as 95 days13 or 62 as long as 22 months.8 subsequent infections are cleared in about 20 days, indicating an 63 anamnestic response.9 immunity does not persist, however, and the anamnestic response 64 is only significant if re-infection occurs within about 15 months of the primary 65 infection.14,15 a cow in a herd with a long breeding season could therefore become 66 pregnant and infected with t. foetus early in the breeding season, lose that embryo, be 67 infertile for several months, clear the initial t. foetus infection, rebreed, conceive, and 68 carry a calf to term as a result of temporary immunity. the result is that more cows will 69 291 calve later in the calving season than desired, and there is a resultant wide variety in 70 weaning weights rather than just a reduced calving percentage. the later-born calves are 71 then marketed at lighter weights, or the cattle producer will incur increased feeding costs 72 to achieve a desired market weight. in either case the cattle producer will sustain 73 substantial economic losses. 74 economic aspects 75 economic losses due to venereal disease result from culling and replacement of 76 infected animals, a decreased percentage of cows calving or calving later than desired 77 with subsequent reduced calf crop and lower weaning weight caused by failure or delay 78 of conception.14 fitzgerald, et al16 estimated in 1958 that each infected bull in a large 79 herd was responsible for an $800 loss per year. wilson, et al17 estimated a $2.5 million 80 annual calf loss in 1979 due to trichomoniasis in oklahoma replacement heifers. in 1986, 81 fitzgerald estimated that the total economic impact in the usa was $65 million 82 annually.18 in a 1991 study, speer, et al estimated that annual losses could reach near 83 $650 million.19 recently the state of louisiana estimates that current losses exceed $100 84 million for that state alone, so the economic loss is likely considerably greater than earlier 85 studies.20 86 during the 1990’s the idaho legislature approved statutes that prescribe 87 regulations for identifying and eliminating t. foetus bulls within the state and for 88 importation of bulls into that state.21 since that time other states have adopted similar 89 legislation and currently the states of nebraska, north dakota, south dakota, montana, 90 wyoming, idaho, washington, oregon and utah require bulls be test negative for 91 trichomoniasis before being transported into the state, sold, or used on public land.22-29 92 292 the texas animal health commission has recently passed similar requirements30. these 93 regulations reflect the growing concern for control of this bovine venereal disease which 94 is so economically important in the united states. 95 diagnosis of bovine trichomoniasis 96 diagnosis of t. foetus has traditionally relied upon microscopic identification of 97 key morphological characteristics in preputial smegma or cervicovaginal mucus (cvm) 98 incubated in various culture media. such characteristics include three anterior flagella, 99 one posterior flagellum, and an undulating membrane resulting in a jerky movement 100 pattern. however, accurate microscopic identification of t. foetus can be complicated by 101 the presence of other trichomonadid protozoa.31-35 contamination of the preputial orifice, 102 prepuce, or penis with fecal material probably explains the presence of these 103 opportunistic trichomonads. several non-pathogenic protozoa are normal inhabitants of 104 the bovine gastrointestinal tract,36-38 and therefore proper cleaning of the preputial orifice 105 and proper sampling techniques are critical to avoid fecal contamination of diagnostic 106 samples. none of the contaminating trichomonads, however, results in reproductive 107 pathology in cows or bulls.39 therefore, research has recently focused on molecular-108 based assays to accurately differentiate t. foetus33,40-42 from other trichomonads. given 109 the lack of legal therapy for bulls infected with t. foetus in the united states the only 110 reasonable course of action is to slaughter an infected bull. it is therefore imperative to 111 correctly identify t. foetus-infected bulls and not misdiagnose based on the presence of 112 non-pathogenic fecal trichomonads. 113 at present, molecular-based assays are most commonly used as confirmatory tests 114 for bovine trichomoniasis because of the relatively low cost of in vitro cultivation 115 293 compared to molecular-based assays. however, molecular-based assays are currently 116 very effective in diagnosing human trichomoniasis caused by trichomonas vaginalis, 117 with a sensitivity of 95% and a specificity of 98%.43 it is therefore very likely that in the 118 future the preferred diagnostic test for bovine trichomoniasis will be a molecular-based 119 assay, and some researchers have already advocated their use as an independent 120 diagnostic test for bovine trichomoniasis.44,45 121 sampling techniques for detection of trichomoniasis in the male 122 several sampling techniques are utilized for obtaining diagnostic specimens in the 123 bull including: 1) a swab technique;46 2) a dry pipette technique;9,47 3) a wet pipette 124 technique;48 and 4) the douche technique.48 fitzgerald, et al compared the swab and 125 pipette techniques and reported that the number of parasites recovered via the swab 126 technique is only 20% of the number of parasites recovered via pipette scraping.49 the 127 swab technique is therefore rarely used in the united states. the dry pipette technique is 128 one of the most common sampling methods in the u sa, while the douche method is the 129 preferred technique in europe.47 schönmann, et al reported that the two methods are not 130 statistically different.47 131 regardless of technique used, it is generally recommended that bulls be sexually 132 rested 1-2 weeks before testing for t. foetus; otherwise, false-negative results are more 133 likely because breeding mechanically removes many of the organisms from a bull’s penis 134 and prepuce. given the sensitivity of t. foetus cultures, false-negative results are also 135 possible even if a bull has been sexually rested. only with three negative tests at weekly 136 intervals (figure 1) can a veterinarian or producer be 99% sure that a bull is t. foetus 137 negative.50 138 294 139 figure 1. sensitivity (in series) of t. foetus cultures.50 140 sampling techniques for diagnosis of trichomoniasis in the female 141 researchers investigating diagnostic sampling methodologies for t. foetus have 142 focused primarily on optimizing sample collection and culture from bulls because of their 143 propensity to develop chronic infections. the technique most commonly used to sample 144 female cattle for t. foetus is a dry pipette technique.48 an infusion pipette is used to 145 aspirate cvm from the vaginal fornix or near the external cervical os. alternatively, in 146 the case of a post-coital pyometra, an infusion pipette can also be used to aspirate some 147 of the content of the uterus. either sample is then examined directly or placed into 148 appropriate culture medium. culturing t. foetus from cvm has a reported sensitivity of 149 58 to 75%.51 samples can also be evaluated with appropriate molecular-based assays. 150 in vitro culture of trichomoniasis foetus 151 direct microscopic examination of specimens for t. foetus may be diagnostic, but 152 a far more sensitive method for the detection of t. foetus is in vitro culture of preputial 153 smegma in a selective nutrient medium for up to a week.51-53 in vitro culture allows the 154 proliferation of t. foetus to more readily detectable levels. all cultures containing 155 result sensitivity (in series) first test negative 80% second test (one week later) negative 96% third test (one week later) negative 99 295 organisms resembling t. foetus should be confirmed with appropriate molecular-based 156 assays to avoid false-positive results due to fecal trichomonad contamination of culture 157 media.31,32,54 alternatively, samples may be submitted directly for molecular-based 158 evaluation. if polymerase chain reaction-based evaluations are not available, a current 159 study by corbeil, et al suggest that immunofluorescent assay may be useful in the 160 diagnosis of t. foetus.55 161 in vitro culture media 162 various culture and transport media systems have been used including 163 kupferberg medium and broth, claussen’s medium, sutherland medium, trypticase-yeast 164 extract-maltose (tym) medium, diamond’s medium, and most recently the inpouch® 165 tf (biomed diagnostics, white city, or, usa) tritrichomonas foetus culture pouch. in 166 vitro cultivation using either diamond’s medium or the inpouch® tf is currently the 167 most common method used to diagnose t. foetus in the united states. both culture 168 systems are fairly equal in sensitivity.47,56-58 however, the inpouch® tf is somewhat 169 more convenient than diamond’s medium.59 the inpouch® tf has a 12-month shelf-life 170 at room temperature, compared to a much shorter refrigerator-life for diamond’s 171 medium. also, the plastic pouch design of the inpouch® tf is less likely to break or leak 172 than tubes containing diamond’s medium. unfortunately, the inpouch® tf is more 173 expensive than diamond’s medium. 174 for many years, cultivation of microorganisms with motility and morphology 175 resembling t. foetus in either the inpouch® tf or diamond’s medium was considered to 176 be 100% specific. however, accurate microscopic identification of t. foetus has since 177 been shown to be complicated by the presence of other contaminating trichomonadid 178 296 protozoa. all cultures containing organisms resembling t. foetus should therefore be 179 confirmed with appropriate molecular-based assays, or samples should be submitted 180 directly to a laboratory for molecular analysis. contact the laboratory prior to sample 181 collection to verify the appropriate transport medium. 182 treatment of cattle infected with tritrichomoniasis foetus 183 one of the complicating factors associated with bovine trichomoniasis is that 184 there are currently no effective treatments with u.s. food and drug administration 185 approval. historically, the most successful treatment for bulls with trichomoniasis 186 involved systemic treatment with nitromidazole derivatives.51,60-62 despite its 187 effectiveness, the use of nitromidazole derivatives is now illegal in food-producing 188 animals in the u.s. because of their mutagenic and carcinogenic properties, and no 189 alternative treatments are available. however, a recent study by carvalho, et al63 found 190 that t. foetus exposed in vitro to mebendazole resulted in internalization of the flagella, 191 disruption of the nucleus, and cytoplasmic vacuolization. these findings suggest new 192 possibilities in the treatment of trichomonasis. still, the lack of effective approved 193 therapies for bovine trichomoniasis emphasizes the need for appropriate preventive and 194 control measures. 195 prevention and control of bovine trichomoniasis 196 preventing the introduction of t. foetus into a cattle herd and controlling 197 trichomoniasis in an infected herd follow many of the same management strategies and to 198 a large extent focus on herd biosecurity. ideally, every cattle operation should focus on 199 preventing the introduction of t. foetus. 200 297 recommended practices to prevent the introduction of t. foetus into a cattle herd 201 include: 202 1) when possible, avoid grazing cattle on public lands where both bulls and cows 203 have a much greater risk of exposure through coitus with other t. foetus-infected 204 animals.64 205 2) utilize artificial insemination when possible. 206 3) cull all open cows and heifers. 207 4) control animal movement into a herd. maintain good fences to prevent t. foetus-208 infected animals from inadvertently entering a herd, or to prevent uninfected 209 animals from temporarily entering a t. foetus-infected herd and then returning 210 with t. foetus to their uninfected herd of origin. 211 5) purchase virgin bulls and heifers as replacements. buying older bulls and cows as 212 replacements greatly increases the chance of purchasing a t. foetus-infected 213 animal. while older bulls are much more likely to become chronically infected 214 with t. foetus than cows, a small percentage of cows will also become chronically 215 infected. 216 6) test bulls for t. foetus at least once before introducing them into a new herd 217 7) the test should be performed after two weeks of sexual rest. ideally, a bull should 218 have three negative cultures at weekly intervals. 219 8) maintain as young a bull battery as possible. older bulls are considered more 220 likely to develop chronic t. foetus infections. however, any bull exposed to t. 221 foetus in a natural breeding situation is capable of becoming chronically infected, 222 regardless of age. 223 298 9) breed purchased cows and heifers in a separate herd, and cull all open animals. 224 ideally, continue to keep the pregnant animals segregated from the rest of the herd 225 through the next breeding season. 226 10) consider immunization against t. foetus in high-risk herds. 227 recommendations for control of trichomoniasis in an infected herd includes: 228 1) test and cull all infected bulls. infected bulls should be sold for slaughter only. 229 2) decrease the number of bulls per breeding unit. single-sire herds offer the lowest 230 exposure potential. however, single-sire units may not always be practical. 231 3) reduce the average age of the bull herd. older bulls are considered more likely to 232 develop chronic t. foetus infections. however, any bull exposed to t. foetus in a 233 natural breeding situation is capable of becoming chronically infected, regardless 234 of age. 235 4) test bulls for t. foetus at least once before introducing them into a new herd. the 236 test should be performed after two weeks of sexual rest. ideally, a bull should 237 have three negative cultures at weekly intervals. 238 5) utilize artificial insemination when possible. 239 6) reduce the breeding season to 60-90 days and cull all open cows and heifers. if 240 there are too many open cows for culling to be economically feasible, then at least 241 these animals should be separated into a high-risk herd. a long breeding season not 242 only allows propagation of t. foetus, but it may also hide production losses due to 243 reduced weaning weights because of delayed conception. 244 7) culture all cases of pyometra diagnosed in cows or heifers during pregnancy 245 examinations. 246 299 8) submit all aborted fetuses and placental tissue to a diagnostic laboratory. 247 immunization against t. foetus is an extremely important management tool for herds 248 infected with t. foetus. research trials clearly demonstrate the benefit of t. foetus 249 vaccination.64-66 trichguard® (fort dodge animal health, fort dodge, ia, usa) and 250 trichguard® v5l (fort dodge animal health) are currently the only t. foetus vaccines 251 available in the united states. the vaccines require an initial subcutaneous dose followed 252 by a booster dose two to four weeks later. the second injection should precede the 253 breeding season by four weeks. annual revaccination four weeks prior to the breeding 254 season is recommended. 255 references 256 1. parsonson im, clark bl, dufty jh: the pathogenesis of tritrichomonas foetus 257 infection in the bull. aust vet j 1974;50:421-3. 258 2. peter d: bovine venereal diseases. in: youngquist rs, editor. current therapy in 259 large animal theriogenology. 1st ed. philadelphia: w.b. saunders; 1997. p. 355-63. 260 3. johnson ae: incidence and diagnosis of trichomoniasis in western beef bulls. j am 261 vet med assoc 1964;145:1007-1010. 262 4. anderson ml, barr bc, conrad pa: protozoal causes of 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bovine trichomoniasis. in: morrow da, ed. current therapy in 355 theriogenology. philadelphia: w.b. saunders company, 1986. p. 275-279. 356 49. fitzgerald pr, hammond dm, miner mc, et al: relative efficacy of various 357 methods of obtaining preputial samples for diagnosis of trichomoniasis in bulls. 358 am j vet res 1952;13:452-457. 359 50. food animal reproduction and herd health services, university of california at 360 davis: bovine trichomonosis: essential facts and testing. 361 www.vetmed.ucdavis.edu/vetext/inf-be_bov-trichomonosis.html. 2006. 362 305 51. skirrow s, bondurant rh: bovine trichomoniasis. vet bull 1988;58:592-603. 363 52. johnson ae the diagnosis of trichomoniasis in the bull. proc of the us livestock 364 sanitary assoc 1965;69:183-189. 365 53. tedesco lf, errico f, baglivi lp: diagnosis of tritrichomonas foetus infection 366 in bulls using two sampling methods and a transport medium. aust vet j 367 1979;55:322324. 368 54. campero cm, rodriguez dubra c, bolondi a, 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wg, hall mr, hanks dr: bovine trichomoniasis. in: howard jl, 399 editor. current veterinary therapy: food animal practice. 4th ed. philadelphia: wb 400 saunders; 1999. p. 420-425. 401 307 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 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/preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1 contact luke duckworth aggievet15@yahoo.com © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12523, http://dx.doi.org/10.58292/ct.v17.12523 technique report pilot study for evaluating a practical method of diluting goat epididymal semen with seminal plasma luke duckworth, stan bychawski southern veterinary services, tulsa, ok, usa abstract the untimely death of a sire often leads to the need for a postmortem semen collection for advanced reproductive techniques. even with careful attention to detail, postmortem semen inherently has low motility.1 postmortem semen is collected from the epididymis and is void of seminal plasma, which is responsible for nurturing sperm and aiding in motility and maturation. however, sperm are not mixed with seminal plasma from the accessory sex glands until ejaculation. this study describes a practical method of adding seminal plasma to epididymal semen to improve semen motility. kiko goats were anesthetized and fresh semen was collected via electroejaculation. goats were then surgically castrated and semen was collected from the epididymis to mimic a postmortem semen collection. seminal plasma supernatant from the fresh collections was used to dilute the epididymal semen; it increased sperm velocity and amplitude of lateral head movement. keywords: postmortem, goat epididymal semen, seminal plasma, motility introduction the need to preserve semen from a sire at the time of death is challenging because postmortem semen is usually of low quality due to poor motility.2 postmortem semen is collected from the epididymis tail where sperm are stored before developing progressive motility.3 these epididymal sperm are motile and have a flagellum, but swim in a unidirectional pattern.4 progressive motility is not initiated until sperm are mixed with seminal plasma5 (sp) from the accessory sex glands during ejaculation. due to its composition of proteins and metabolites, sp is essential for sperm maturation, nutrition, and motility.6 without sp, epididymal semen has lower fertilization rate than ejaculated semen due to inferior motility.7 a practical method to improve the motility of postmortem sperm would be valuable to any animal industry that utilizes advanced reproductive techniques. sperm function, fertilization, and embryo development are all influenced by sp.8 thus, we hypothesized that addition of sp to an epididymal semen sample increases sperm motility (as demonstrated in human semen).4 commercial synthetic sp is not available and thus sp needs to be collected and processed. in postmortem collection, sp from a live sire can be used as a diluent for epididymal semen. alternatively, sp from a previous live collection can be filtered and frozen for use as needed. this can be performed in a field or clinical setting. materials and methods initial experimental design included comparing epididymal semen and seminal plasma diluted epididymal (spde) semen to fresh semen. given the cost of intact male breeding stock, a pilot study was conducted to evaluate the experimental design for a practical method of adding sp to a postmortem semen sample to increase sperm motility. this project was also intended to teach 4th year veterinary students at oklahoma state university how to perform a fresh semen collection, surgical castration, and postmortem semen collection. in accordance with osu iacuc policy, this project was exempt because it utilized tissues collected from animals for a routine breeding soundness examination that occurred for teaching purposes. five kiko goat kids (8 months of age and ~ 45 kg) were brought to oklahoma state university teaching hospital for semen collection followed by castration in october (northern hemisphere). the first kid was used as a demonstration. each of 4 veterinary students then performed semen collection, surgical castration, and epididymal semen collection on the mailto:aggievet15@yahoo.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12523 2 citation: clinical theriogenology 2025, 17, 12523, http://dx.doi.org/10.58292/ct.v17.12523 remaining kids. food and water were withheld for 12 hours in preparation for surgery. kids were manually restrained and the neck was clipped and prepped with a surgical blade and 70% isopropyl alcohol. the jugular furrow was occluded at the left thoracic inlet and 0.05 mg/kg intravenous xylazine (xylamed, vetone, boise, idaho) and 0.25 mg/kg ketamine (ketamine 200, axtell, pilot point, texas) were given in the left jugular vein. once anesthetized, kids were placed in left lateral recumbency. digital pressure was applied to sigmoid flexure and prepuce to exteriorize the penis. penis was grasped, cleaned, and isolated with gauze sponges. rectum was evacuated of fecal pellets before introducing the 2-electrode rectal probe. electrical stimulation was provided via a pulsator v electroejaculator. the electroejaculator was operated in manual mode. a series of electrical impulses in increasing intensity was applied from levels 1-3. semen was collected into a glass tulip flask. fresh semen was diluted 1:1 with triladyl (minitube, tiefenbach, germany) and a 25 µl portion of the sample was transferred to a falcon tube as a control (fresh). the remaining sample was transferred into a 15 ml conical vial and centrifuged for 10 minutes at 895 g. the sp supernatant was filtered through a 0.22 µm syringe filter and saved to mix with the epididymal sample. fresh semen served as an internal control for each subject. after semen collection, kids were placed dorso-ventrally on a surgery gurney. scrotal neck was isolated manually and pressure was applied to tighten the scrotum around testis. scrotum was clipped and aseptically prepped with 4% chlorhexidine and 70% isopropyl alcohol and 60 mg of 2% lidocaine was used to block each spermatic cord. the distal half of the scrotum was removed via excision with a number 22 scalpel blade. parietal vaginal tunic was excised over epididymal tail. care was taken not to excise the epididymis. testes were then exteriorized through the parietal vaginal tunic, leaving the visceral vaginal tunic intact. the fascia surrounding the spermatic cords was stripped and pushed dorsally. a reimer emasculator was applied around the spermatic cord between the head of the epididymis and the scrotal incision. spermatic cord was crimped to aid in hemostasis. after 2 minutes of crimping, the spermatic cord was severed on the distal side of the emasculator. a pair of curved hemostats was secured to the spermatic cord before the emasculators were released and observed for proper hemostasis. spermatic cord was released when no hemorrhage was observed. similar procedure was repeated for the other testis. castrated testes were held firmly in hand, so the tail of the epididymis was accessible. number 22 scalpel blade was used to gently mince epididymis tail repeatedly until the tissue began to disintegrate. care was taken not to contaminate the epididymal semen sample during mincing. scalpel blade was used to scrape and collect the semen sample as the epididymis was sliced. semen was then rinsed off the scalpel blade into a 15-ml conical vial, using triladyl egg yolk extender. these steps were repeated until the epididymal tail was completely sliced. extended semen sample was then centrifuged for 10 minutes at 895 g. the supernatant was removed and 25 µl semen samples were placed in 2 falcon tubes. then, 25 µl of triladyl extender was added to 1 epididymal sample to mimic a postmortem sample (epididymis), whereas 25 ml of sp was added to the other epididymal sample creating a spde sample (50% seminal). each sample was evaluated using computer assisted sperm analysis (casa). data were analyzed using a paired student’s t-test. results velocity velocity average pathway (vap) measures the average velocity of sperm travel in m/s. compared to epididymal samples, mean progressive vap increased in 3 of 50% seminal samples, remained the same in 1, and slightly decreased in 1 sample. velocity straight line (vsl) measures the straight-line distance a sperm travels between 2 scans in m/s. mean progressive vsl increased in 3 of 50% seminal samples, remained the same in 1, and slightly decreased in 1 sample. velocity curvilinear (vcl) measures sperm velocity over the path traveled and indicates cell vigor in m/s. mean progressive vcl increased in 4 of 50% seminal samples and slightly decreased in 1 sample. progressive vap, vsl, and vcl all were higher (p > 0.5) for the 50% seminal samples compared to epididymal samples. motility progressive sperm number was higher (p > 0.05) in 4 of the 50% seminal samples compared to epididymal samples (table 1). head movement amplitude of lateral head displacement (alh) measures the height/width of head movement and beat cross frequency (bcf) measures the frequency of those movements in μm and hz, respectively (table 2). compared to epididymal samples, alh increased in 3 of 50% seminal samples, stayed the same in 1 sample, and slightly decreased in another sample. bcf stayed relatively unchanged in 3 of 50% samples, slightly increased in 1 sample and slightly decreased in another sample. all 50% seminal samples had a lower (p > 0.5) bcf than fresh collections. discussion seminal plasma increased the velocity and amplitude of lateral head movement of epididymal semen when used as a diluent. the original design for this project was to use each goat as its own control. the fresh collection served as an internal control and allowed veterinary students to perform a semen collection on an anesthetized ruminant. the epididymal and spde samples could be compared against the fresh collection. two problems lead the authors to conclude a fresh collection is not necessary in a larger study. first, not every kid gave a usable fresh collection. second, semen parameters for the ejaculates were poor. some fresh semen collections had lower motility metrics than their corresponding spde samples. we theorize kids were entering puberty and that resulted in lower semen quality than would be expected for mature bucks. given the goal to teach castration and semen collection to veterinary students, young cull bucks were the only animals economically affordable. mature breeding bucks were too high value for the project and producers were uncomfortable selling breeding bucks for a project that included castration. due to the problems encountered, a fresh collection will not be part of the methods in further studies. also, buying enough intact mature animals to castrate is not economical. in future studies, testes will be utilized from a harvest facility. furthermore, only an epididymal sample and spde sample will be evaluated. this will also allow a larger sample size. http://dx.doi.org/10.58292/ct.v17.12523 citation: clinical theriogenology 2025, 17, 12523, http://dx.doi.org/10.58292/ct.v17.12523 3 each fresh semen sample collected was centrifuged and the sp supernatant was used to dilute that buck’s epididymal sample. this was problematic for 2 kids that did not give a fresh semen sample. their epididymal samples were mixed with sp from animal 3. the goal of this project was to provide a real-world option of improving postmortem semen quality. seminal plasma will not be available at postmortem collection; therefore, prepared sp from another sire can be used. in a larger study, we intend to perform fresh semen collections in the same species, mix them together, and use a ‘homogenous donor’ sp preparation to dilute the epididymal semen in the experimental group. this would provide a uniform dilution agent, since sp quality differs among sp donors.9 motility of epididymal semen is ph dependent.2 raw epididymal semen is too dense and has to be diluted for casa evaluation; triladyl was used to rinse epididymal semen off the table 1. progressive motility metrics before and after adding caprine seminal plasma to caprine epididymal semen animal id sample type progressive count (x 106) mean progressive vapa (m/s) mean progressive vslb (m/s) mean progressive vclc (m/s) p value 1 fresh 101 119.01 106.96 193.92 — 1 epididymis 321 205.40 191.73 244.46 0.077 1 50% seminal 412 220.86 205.17 266.23 2 fresh n/a n/a n/a n/a — 2 epididymis 266 232.81 215.84 296.11 0.167 2 50% seminal 293 231.33 215.35 301.64 3 fresh 379 120.91 112.83 174.28 — 3 epididymis 649 211.95 196.67 258.29 0.214 3 50% seminal 322 216.36 199.51 272.55 4 fresh n/a n/a n/a n/a — 4 epididymis 239 189.63 176.04 243.15 0.173 4 50% seminal 642 199.99 187.62 254.56 5 fresh 888 146.85 140.36 175.96 — 5 epididymis 584 188.65 177.73 221.12 0.381 5 50% seminal 599 179.94 169.25 215.79 avelocity average pathway bvelocity straight line cvelocity curvilinear table 2. head movement metrics before and after adding seminal plasma to epididymal semen animal id sample type progressive mean alhe (μm) progressive mean bcff (hz) p value 1 initial 6.64 41.13 — 1 epididymis 8.02 25.83 0.382 1 50% seminal 9.68 25.10 2 initial n/a n/a — 2 epididymis 11.21 23.98 0.442 2 50% seminal 12.18 23.31 3 initial 7.78 32.65 — 3 epididymis 9.88 25.82 0.271 3 50% seminal 9.76 27.57 4 initial n/a n/a — 4 epididymis 9.10 27.81 0.472 4 50 seminal 10.98 25.55 5 initial 7.01 28.36 — 5 epididymis 9.10 24.77 0.423 5 50% seminal 8.64 25.53 eamplitude of lateral head displacement fbeat cross frequency http://dx.doi.org/10.58292/ct.v17.12523 4 citation: clinical theriogenology 2025, 17, 12523, http://dx.doi.org/10.58292/ct.v17.12523 scalpel and thus further diluted the epididymal semen. bovine epididymal semen has a ph of 5.8.2 it is assumed that caprine epididymal semen has a similar ph. triladyl has a neutral ph of 6.9 and increases the ph of the epididymal sample. motility increases very rapidly when ph increases.10 therefore, the baseline analysis of the triladyl diluted epididymal semen was likely artificially elevated. sterile saline has an acidic ph similar to epididymal semen and will be used as the dilution agent for the control sample in future studies. this pilot study supported the hypothesis that adding sp to epididymal semen increases sperm velocity. previous studies had an increase in vap and vsl and a decrease in vcl for ejaculated sperm compared to epididymal sperm.3 increase in velocity on an average pathway and in a straight-line favor increases progressive motility. an increase in velocity along a curved line indicated that sp diluted epididymal semen still followed a curved line, but at a higher velocity. we concluded that increased velocity in all measured categories was evidence that adding sp to epididymal semen increased motility. addition of sp to epididymal semen increased alh, and decreased bcf; alh and bcf are important for fertilization and sperm/ovum interaction.11 a higher alh indicates more sperm cell vigor and favors progressive motility, whereas a lower bcf results in less frequent sperm head movement. a lower bcf increases proximity between the sperm and ovum that ultimately increases the chance for fertilization.11 sperm movement can be classified in 2 phases: progressive mode and attack mode.11 progressive mode occurs in the larger tubular areas of the female reproductive tract and is associated with higher velocity and alh of the sperm. attack mode occurs in the smaller tubular area of the female tract (oviduct) and is characterized by a lower bcf to facilitate proximity between the sperm and ovum. therefore, an increase in alh would likely favor progressive motility whereas a decrease in bcf would favor sperm/egg contact time. the trends observed in changes in sperm velocity, alh, and bcf indicate that expanding to a larger sample size is justified. although the p values were not significant, literature supports the increase of sperm motility when sp is added to epididymal semen. a larger sample size should produce a significant outcome in future studies. increasing the quality of postmortem semen samples at death is only relevant for current and future sires. however, the problem with low-quality postmortem semen still remains for sires that have already expired and had their semen frozen after death. the dilution of postmortem semen with sp is a practical approach that could also improve the quality of frozen-thawed semen. authors will continue this study on a larger scale and also apply frozen-filtered sp to postthaw postmortem semen samples to evaluate the impacts on sperm kinetics. acknowledgment authors thank dale kelley of oklahoma state university school of veterinary medicine for assistance with casa. funding provided by luke duckworth and southern veterinary services, tulsa, ok. references 1. monteiro ga, papa fo, zahn fs, et al: cryopreservation and fertility of ejaculated and epididymal stallion sperm. anim reprod sci 2011;127:197-201. doi: 10.1016/j.anireprosci.2011.08.002 2. acott ts, carr dw: inhibition of bovine spermatozoa by caudal epididymal fluid: ii. interaction of ph and a quiescence factor. biol reprod 1984;30:926-935. doi: 10.1095/biolreprod30.4.926 3. goovaerts igf, hoflack gg, van soom a, et al: evaluation of epididymal semen quality using the hamilton–thorne analyser indicates variation between the two caudae epididymides of the same bull. theriogenology 2006;66:323-330. doi: 10.1016/j.theriogenology. 2005.11.018 4. lindholmer ch: the importance of seminal plasma for human sperm motility.  biol reprod 1974;10:533-542. doi: 10.1095/ biolreprod10.5.533 5. carr dw, acott ts: inhibition of bovine spermatozoa by caudal epididymal fluid: i. studies of a sperm motility quiescence factor. biol reprod 1984;30:913-925. doi: 10.1095/biolreprod30.4.913 6. wang f, yang w, ouyang s, et al: the vehicle determines the destination: the significance of seminal plasma factors for male fertility. int j mol sci 2020;21:8499. doi: 10.3390/ijms21228499 7. chaveiro a, cerqueira c, silva j, et al: evaluation of frozen thawed caudal epididymal sperms and in vitro fertilizing potential of bovine sperm collected from the caudal epididymal. iran j vet res 2015;16:188. doi: 10.22099/ijvr.2015.3054 8. juyena ns, stellate c: seminal plasma: an essential attribute to spermatozoa.  j androl 2012;33:536-551. doi: 10.2164/jandrol. 110.012583 9. turunen t, magris m, malinen m, et al: seminal-plasma-mediated effects on sperm performance in humans.  cells 2022;11:2147. doi: 10.3390/cells11142147 10. carr dw, acott ts: intracellular ph regulates bovine sperm motility and protein phosphorylation. biol reprod 1989;41:907-920. doi: 10.1095/biolreprod41.5.907 11. raveshi mr, abdul halim ms, agnihotri sn, et al: curvature in the reproductive tract alters sperm–surface interactions.  nat commun 2021;12:3446. doi: 10.1038/s41467-021-23773-x http://dx.doi.org/10.58292/ct.v17.12523 https://doi.org/10.1016/j.anireprosci.2011.08.002 https://doi.org/10.1095/biolreprod30.4.926 https://doi.org/10.1016/j.theriogenology.​2005.11.018 https://doi.org/10.1016/j.theriogenology.​2005.11.018 https://doi.org/10.1095/biolreprod10.5.533 https://doi.org/10.1095/biolreprod10.5.533 https://doi.org/10.1095/biolreprod30.4.913 https://doi.org/10.3390/ijms21228499 https://doi.org/10.22099/ijvr.2015.3054 https://doi.org/10.2164/jandrol.110.012583 https://doi.org/10.2164/jandrol.110.012583 https://doi.org/10.3390/cells11142147 https://doi.org/10.1095/biolreprod41.5.907 https://doi.org/10.1038/s41467-021-23773-x 2009: new treatment strategies f 1 or chronic endometritis and post mating endometritis new treatment strategies for chronic endometritis and post mating endometritis 1 m. m. leblanc 2 rood and riddle equine hospital, lexington, ky, usa 3 4 abstract 5 traditional treatments for chronic endometritis and post-mating induced 6 endometritis including intra-uterine antibiotics, uterine lavage and ecbolics do not always 7 resolve an infection or clear uterine fluid. treatment failure may be due to continual 8 contamination of the uterus because of anatomical abnormalities in the caudal tract, 9 degradation of antibiotics in uterine exudate, biofilm production by micro-organisms or 10 prolonged uterine inflammation. older, pluriparous mares are most commonly affected 11 as they are unable to physically clear uterine contamination or inflammation after 12 breeding. nulliparous mares may also develop persistent mating induced endometritis or 13 chronic endometritis if they have an incompetent cervix as it will prevent rapid drainage. 14 repeatedly treating chronically infected mares with intra-uterine antibiotics can lead to 15 multi-drug resistant infections while prolonged inflammation in mares with post mating 16 induced endometritis can eventually result in bacterial or yeast endometritis. because 17 traditional treatments are not always successful, a number of agents and treatment 18 strategies have been investigated. these include buffered chelators that potentiate 19 antibiotics (tris-edta), mucolytics (dmso, kerosene, n-acetylcysteine), corticosteroids 20 (prednisolone, dexamethasone) and immunomodulators (cell wall extracts of 21 mycobacterium phlei and propionibacterium acnes). all have shown some degree of 22 success when cases are selected carefully and protocols are followed. 23 24 keywords: mare, endometritis, chelating agents, mucolytics, immunomodulation 25 26 introduction 27 traditional therapy for chronic endometritis includes removal of the offending 28 organism through uterine lavage, judicious use of ecbolics and antimicrobial therapy for 29 three to five days during estrus in addition to repair of anatomical defects.1,2 uterine 30 irrigation and administration of oxytocin or cloprostenol within eight hours of mating 31 433 followed by a second treatment at 24 hours that may or may not include intra-uterine 32 antibiotics, is a recommended protocol for post mating induced endometritis.3,4 however, 33 these protocols are not always successful in clearing uterine fluid or infection. treatment 34 failure may be due to an inability to physically clear uterine fluid quickly after mating, 35 continual production of uterine fluid secondary to inflammation, or an inability of 36 antibiotics to penetrate exudate or biofilm produced by microorganisms. because 37 treatments have failed, intrauterine buffered chelators (tris-edta; ethylene-diamine 38 tetra-acetic acid (3.5 m)-tromethamine 50mm; rood and riddle veterinary pharmacy, 39 lexingon, ky, usa and tricide®; 8mm disodium edta dehydrate and 20 mm 2-40 amino-2-hydroxymethyl-1,2-propanediol; medical molecular therapeutics, llc, 41 athens, ga, usa), mucolytics (dmso, kerosene, n-acetylcysteine), corticosteroids 42 (prednisolone, dexamethasone) and immunomodulators (cell wall extracts of 43 mycobacterium phlei and propionibacterium acnes) have been investigated and have 44 shown potential as effective therapies for endometritis if used appropriately. some of 45 these agents offer alternatives to repeated use of anti-microbial agents, which is often the 46 major instigating factor for antibiotic resistance. clinical studies on large groups of 47 barren mares are lacking though and need to be performed before true efficacy can be 48 determined. 49 new treatment strategies for chronic endometritis 50 the most critical factor in uterine defense against infection is rapid, physical 51 clearance of inflammatory debris from the uterus after mating or post foaling. some 52 mares have difficulty clearing this debris because they have developed anatomical and/or 53 degenerative defects that interfere with uterine drainage. repeated foaling and breeding 54 can cause anatomical defects such as poor perineal conformation, incompetent vagino-55 vestibular sphincter, vaginal stretching, incompetent cervix, a pendulous uterus or 56 degenerative changes such as an abnormal myometrium, periglandular fibrosis, vascular 57 elastosis, lymphangectasia, scarring and atrophy of endometrial folds or damage to the 58 mucociliary apparatus. older nulliparous mares that are not mated until 10 or more years 59 of age and those that have repeated embryo recovery attempts also experience delayed 60 uterine clearance, often because of cervical malfunction.4-8 the uterus responds to 61 prolonged retention of inflammatory debris by increased mucus production by 62 434 epithelium, transudation of serum proteins, and an influx of neutrophils and 63 immunoglobulins into the uterine lumen. if these substances remain in the uterine lumen 64 for more than 24 to 48 hours, endometrial ulceration and secondary bacterial infections 65 may result.1,9-11 bacterial endometritis is most commonly treated with intra-uterine 66 therapies (i.e. uterine lavage, ecbolics and intra-uterine antibiotics). most uterine 67 infections resolve after a three to five day course of antibiotics as long as inflammation is 68 not severe, antibiotics are not rendered ineffective and anatomical defects do not 69 compromise the mare’s ability to physically clear the uterus of bacteria, inflammatory 70 debris and contaminants. however, if uterine degeneration is severe, the cervix is 71 fibrotic, or the offending organism produces a biofilm, treatment with intra-uterine 72 antibiotics can lead to secondary fungal endometritis or infection with multi-drug 73 resistant bacteria. 74 multi-drug resistant bacteria have been isolated from the uterus of mares after 75 repeated intra-uterine antibiotic treatment including methicillin resistant staphylococcus 76 aureus and multi-drug resistant pseudomonas aeruginosa, staphylococcus epidermis, e. 77 coli, and enterobacter cloacae (personal communication, marianne swintosky, 2008). 78 these findings and the wider implications of antibiotic resistance in humans support 79 development and use of novel strategies to combat equine uterine infections. 80 mucolytics 81 mucus plays an important role in protecting and cleansing of mucosal surfaces 82 such as the respiratory and gastrointestinal tract.12 it may have a similar role in the 83 reproductive tract as the equine endometrium contains cilia and is covered by a mucus 84 blanket.13,14 mucus production at the equine endometrial surface has been demonstrated 85 using alcian blue,14,15 mucicarmine16 and periodic acid schiff stains.14,17 excessive 86 mucus production by the equine endometrium, detectable in uterine lavage fluid18 or 87 uterine biopsy specimens,14,17 is now linked to failure to become pregnant. during acute 88 and subacute uterine inflammation there is an increase in mucus production and in the 89 height of epithelial cells.17 90 solvents and mucolytic agents have been added to uterine irrigation fluids in an 91 attempt to clear exudate, mucus or biofilm. agents used include dmso, kerosene and 92 n-acetylcysteine (20% solution; butler corp, columbus, oh, usa). each compound 93 435 appears to have some beneficial effects. barren mares (n = 16) infused with a 30% 94 solution of dmso after breeding tended to have higher pregnancy rates than mares 95 infused with saline.19 intrauterine dmso therapy also resulted in a significant 96 improvement in endometrial biopsy classification in 18 of 27 mares; whereas only 2 of 18 97 barren mares improved following intrauterine saline treatment. in contrast, intrauterine 98 infusion of 50 ml of commercially available kerosene in 26 mares with varying degrees 99 of endometrial pathology induced diffuse moderate to severe endometritis, severe diffuse 100 edema and production of a serum-like exudates.20 half of the mares exhibited mild to 101 severe necrosis of luminal epithelium. mares were subsequently bred on the next cycle 102 and surprisingly, 50% of the mares with category ii or iii biopsy scores carried foals 103 until term. although kerosene was associated with significant inflammatory changes, 104 pregnancy may have been established because mucus and exudate were removed via 105 destruction and necrosis of uterine epithelium. 106 n-acetylcysteine (nac) is a mucolytic agent that disrupts disulphide bonds 107 between mucin polymers, thereby reducing the viscosity of mucus. in addition, nac 108 possesses antioxidant and possibly some antimicrobial properties.21-23 nac has been 109 used to treat respiratory diseases such as pneumonia, the pulmonary component of cystic 110 fibrosis in humans, meconium impactions in both humans24,25 and equine neonates 111 (morresey pr, personal communication, 2008), and meconium aspiration pneumonia in 112 equine neonates.26 multiple studies support its beneficial anti-oxidative properties 113 especially in chronic inflammatory diseases.21-23,27 we have recently evaluated its effect 114 on the endometrium and epithelium.28 endometrial biopsies were obtained from fertile 115 and barren mares before and after infusion of a 3.3% solution of n-acetylcysteine (day 1) 116 and compared to biopsies obtained from mares infused with saline. the uterus of all 117 mares was irrigated with 2 l of lactated ringer’s solution on days 2 and 3 and a second 118 biopsy obtained. endometrial biopsies were given a kenney grade by a board certified 119 veterinary pathologist and changes in epithelial architecture and mucus blanket were 120 measured by image analysis. data indicated that nac was not harmful to the 121 endometrium and that it may counteract the irritating effect of saline, as reflected through 122 increased cell height in control mares. as further evidence that nac does no harm and 123 may be beneficial, 20 barren thoroughbred mares bred 2 to 5 times in 2007 or 2008 and 124 436 with a history of endometritis were mated naturally to commercial stallions in central 125 kentucky in late may and june 2008. mares received a 0.6% solution of nac either the 126 treatment cycle before (n = 10) or in the 48 h before breeding (n = 10) in addition to 127 conventional treatments. infusion before breeding was associated with higher than 128 expected pregnancy rates as 17 of 20 mares (85%) were pregnant as of february, 2009. 129 prior to this study, the rationale for using nac as a uterine infusion had been the removal 130 of inspissated secretions, exudate and biofilm, (i.e. as a mucolytic). however, since 131 increased vaginal mucus viscosity is documented to inhibit sperm forward progression in 132 cows,29 it is also speculated that nac may improve sperm-transport in mares with 133 excessively viscous mucous secretions by breaking the cross-linking disulfide bridges 134 between mucin polymers. 135 bacterial and yeast biofilms 136 antibiotic failure in chronic endometritis may be due to biofilm produced by 137 some gram negative bacteria, yeast and fungi. bacterial biofilms consist of a 138 heterogeneous community of different bacterial species, surrounded by an extracellular 139 matrix, that co-exist in a symbiotic relationship.30 such biofilms are found throughout the 140 human body, e.g. the oral cavity, the skin, the intestines and the vagina. in most cases, 141 the inhabitants of this community are considered as normal flora and serve as a protective 142 mechanism to prevent the colonization of frank and opportunistic pathogens. if the 143 balance of this biofilm community is upset or disrupted, pathogens may colonize, 144 proliferate, and cause disease.30 biofilms confer antibiotic resistance and therefore 145 contribute to treatment failure. a number of theories have been advanced to account for 146 this increased resistance.31-34 one is simply that the antibiotic is unable to penetrate the 147 extracellular matrix of the biofilm. another is that antibiotics are less active on biofilms 148 due to the lower rate of metabolism and growth. a currently popular theory is that there 149 are “persister cells” within the biofilm community. persister cells are defined as a small 150 subpopulation of essentially invulnerable cells that neither grow or die in the presence of 151 bactericidal agents and exhibit multi-drug tolerance or resistance to antibiotics.30 152 pseudomonas aeruginosa is a potent biofilm producer and is often cultured from 153 the uterus of mares with chronic endometritis. other equine pathogens that produce 154 biofilm and can be isolated from the uterus include staphylococcus epidermis, e. coli, 155 437 enterobacter cloacae and a number of yeast and fungi. these organisms more commonly 156 cause endometritis in older, pluriparous barren mares that have anatomical defects than 157 young, fertile mares, although uterine defenses can be broached in the latter resulting in 158 chronic infection. infections by these organisms can be difficult to treat, are often 159 refractory to a 3 to 5 day course of antibiotics, and may result in a population of bacteria 160 colonizing the uterus that is highly resistant to the drug initially used for treatment. work 161 in other species and in the mare have been shown that buffered chelating agents (tris-162 edta) may potentiate the actions of antimicrobials, dissolve exudate, and break up 163 biofilm. 164 buffered chelators such as first generation tris-edta35-41 and third generation 165 tricide® potentiate the actions of antimicrobials.42 they have been shown to enhance 166 the bactericidal effects of antimicrobials in dogs with refractory otitis,35,37,39 pyoderma,39 167 osteomyelitis,36 multiple fistulas,36,43 rhinitis,44 and cystitis.39,45 uterine isolates of 168 pseudomonas collected from mares exposed to tris-edta solution exhibited decreased 169 viability. 46 others have shown that addition of tris-edta to gentamicin in vitro 170 improved killing of pseudomonas aeruginosa by 1000 fold more than treatment with 171 only gentamicin.47 addition of tris-edta to penicillin, ampicillin, oxytetracycline, 172 neomycin, and amikacin has also been shown to be synergistic.42 a recent study showed 173 that tricide® increased in vitro activity of antifungal drugs against common fungal 174 pathogens isolated from eyes of horses with mycotic keratitis.42 the mechanism of 175 action of buffered chelating agents is not completely understood but it is speculated that 176 the chelating agent (edta) chelates calcium and/or magnesium from the outer 177 membrane of bacteria, thereby altering the integrity and permeability of the cell wall. 178 damage to the cell wall interferes with the effectiveness of the bacterial efflux pump and 179 facilitates osmotic collapse. unlike bacteria, fungal cell walls are composed mainly of 180 polysaccharides (beta-glucans and chitin) and protein. it is hypothesized that removal of 181 divalent cations in the cell wall by third generation chelating agents may alter membrane 182 proteins that are important in maintaining the construction and maintenance of the 183 polysaccharides in the wall.42 184 buffered chelators reportedly have minimum adverse effects when used in 185 joints,36 bones,36 the uterus,48 ears,35,37,41 the bladder,39,45 and mammary glands.36 186 438 treatment with tris-edta, a first generation chelating agent, appears not to be harmful 187 as infusion of 250 ml of 3.5 m edta, 0.05 m tris, ph 8, into the uterus induced an 188 inflammatory response that was no greater than saline.48. the benefit of third generation 189 chelating agents such as tricide® over first generation chelating agents is greater 190 antibiotic stability in third generation chelating solutions (b.w. ritchie, personal 191 communication, 2009). there are no clinical studies on the use of third generation 192 chelating agents in the treatment of bacterial or yeast endometritis. 193 buffered chelating agents must come in direct contact with the bacterial cell wall 194 in order to kill the organism so the volume of solution needed for infusion will vary with 195 the size of the uterus. doses ranging from 200 to 500 ml are recommended. the 196 chelating agent binds to the bacteria within minutes resulting in cell death and 197 accumulation of debris so the uterus should be lavaged within 12 hours to remove these 198 by-products (b.w. ritchie, personal communication, 2009). 199 new treatment strategies for post mating induced endometritis 200 fluid may accumulate within the uterine lumen during estrus because it is not 201 physically drained through the cervix, or production is increased secondary to chronic 202 inflammation, bacterial infection or vestibule-vaginal reflux. degenerative uterine 203 changes such as vascular elastosis may also contribute to fluid accumulation. vascular 204 elastosis appears to indirectly reduce fertility through a reduction in endometrial 205 perfusion, and through disturbances in uterine drainage caused by reduced venous return 206 in capillary beds.49-51 for the past 20 years, treatment of post mating induced 207 endometritis has emphasized methods for improving physical drainage. the currently 208 recommended therapy for improving physical clearance of uterine fluid is uterine 209 irrigation followed immediately by administration of either oxytocin (10 to 25 iu i.v. or 210 i.m.) or cloprostenol (250 μg i.m.) at 4 to 8 hrs after breeding.4,52-58 this treatment has 211 increased pregnancy rates in highly susceptible barren mares.59 a long-acting synthetic 212 oxytocin analog, carbetocin, has recently become available in europe, canada and 213 mexico. it was well-tolerated in a group of horses following intravenous administration 214 of 175 μg. the half-life of carbetocin is about 17 minutes, or 2.5 times that of oxytocin.60 215 the drug may be of benefit in mares where more prolonged uterine contractions are 216 needed. no clinical studies comparing its efficacy with oxytocin have been reported. 217 439 in a mare with a cervix which fails to dilate, such as an aged maiden mare, 218 oxytocin may be ineffective in expulsion of uterine fluid. however, similar to its role in 219 promoting lymphatic drainage, cloprostenol may help to expel uterine fluid through a 220 narrow cervix through sustained uterine contractions. in addition, the cervix may be 221 manually dilated to assist fluid drainage. we have used a compounded misoprostol 222 product (2000 μg/3 ml; rood and riddle veterinary pharmacy, lexington, ky, usa), a 223 synthetic prostaglandin e1 analog, that clinically appears to have resulted in cervical 224 relaxation when applied topically to the cervical epithelium 2 to 4 h before breeding. 225 there is a clinical impression that oxytocin does not always effectively clear 226 uterine fluid in old, pluriparous mares so cloprostenol is frequently given in place of 227 oxytocin. however, cloprostenol has been shown to be associated with a decrease in 228 serum progesterone concentrations if given after ovulation.61,62 because of perceived 229 treatment failures, complications with administration of cloprostenol and the fact that 230 retained uterine fluids contain inflammatory by-products that adversely affect embryo 231 viability; modulation of the immune system has been investigated. 232 recent work has shown that steroids or immunomodulators administered 233 judiciously around the time of mating may increase pregnancy rates in mares with fluid 234 accumulation or uterine inflammation.63-69 immunomodulation by either administration 235 of steroids or immunomodulators may help restore homeostatic local inflammatory 236 mechanisms through reducing pro-inflammatory cytokines. this may be especially 237 helpful in older mares that may be suffering from inflamm-aging. inflamm-aging is a 238 low-grade, systemic inflammatory response associated with advanced age in humans and 239 horses that is characterized by increased inflammatory cytokine production.70,71 240 peripheral blood mononuclear cells collected from old horses have been shown to 241 produce more inflammatory cytokines than mononuclear cells from young horses; 242 moreover, fat old horses have even greater frequencies of lymphocytes and monocytes 243 producing inflammatory cytokines than thin old horses. weight loss in old fat mares 244 reduced the percent of ifnγ and tnfα positive lymphocytes and monocytes and serum 245 levels of tnf α protein. when weight and fat increased in these old horses, there was a 246 significant increase in inflammatory cytokine production.70,71 247 440 a single dose dexamethasone administered within one hour of mating and daily 248 prednisolone administration given before and after mating have improved pregnancy 249 rates in mares with uterine fluid. bucca, et al.67 reported that a single injection of 250 dexamethasone administered within one hour of mating (50 mg, iv; approximately 0.1 251 mg/kg) combined with routine post breeding therapies (uterine irrigation, ecbolic drugs 252 and in some cases intra-uterine antibiotics) resulted in increased pregnancy rates in mares 253 with a history of fluid accumulation after ovulation and in mares with cervical 254 incompetence. treated mares exhibited decreased uterine edema, decreased intrauterine 255 fluid and an increase in uterine fluid clarity. although dexamethasone did not increase 256 pregnancy rates in the general population, pregnancy rates were increased in mares that 257 had 3 or more risk factors for susceptibility to endometritis. risk factors included 258 abnormal reproductive history, abnormal perineal conformation, vulvoplasty not repaired 259 after foaling, an incompetent cervix, positive endometrial culture, > 2 cm of endometrial 260 fluid before breeding, endometrial fluid post mating between 1.5 and 2.0 cm, or a fluid 261 volume > 2cm, and endometrial fluid persisting more than 36 hours after mating. 262 increased pregnancy rates were also observed in mares with a history of intra-uterine 263 fluid accumulation following oral administration of acetate 9-alpha-predinisolone (0.1 264 mg/kg) given at 12 h intervals for 4 days beginning 48 hrs before breeding.66 in contrast, 265 administration of dexamethasone (10 or 20 mg, im) 6 to 12 h after insemination did not 266 improve pregnancy rates of warmblood mares with a history of intra-uterine fluid 267 retention (n=783 cycles).69 a plausible cause for the different results is that steroids 268 block both the cyclooxygenase and 5-lipoxygenase pathways of inflammation. the 5269 lipoxygenase pathway includes leukotriene b, a potent neutrophil chemotactic factor 270 found in uterine fluids of susceptible mares after mating.72,73 reducing neutrophil 271 chemotaxis and the number of neutrophils recruited into the uterus post mating may 272 diminish the severity and length of the inflammatory response. candidates for steroid use 273 should be chosen carefully as misuse in mares with bacterial endometritis may exacerbate 274 the infection. 275 immunomodulators may also improve pregnancy rates, although the mechanism 276 of action remains speculative. immunomodulators induce a nonspecific cell-mediated 277 response predominantly by activation of macrophages and release of cytokines that elicit 278 441 a general increase in immune system activity.74 two immunomodulators are currently 279 labeled and marketed for use in horses. one is a cell-wall extract of mycobacterium phlei 280 (mcwe; settle®, bioniche animal health, bogard, ga, usa) that has been approved as 281 an adjunctive treatment in mares with uterine infection caused by streptococcus equi 282 subspecies zooepidemicus. studies have shown that it modulates the immune response of 283 susceptible mares63,64 and that mares with experimentally induced bacterial endometritis 284 cleared inflammation more rapidly after treatment with mcwe compared to untreated 285 mares.65 the second immunomodulator is propionibacterium acnes (eqstim®, neogen 286 corp, lexington, ky, usa). it is used as an adjunct treatment for horses with equine 287 respiratory disease complex. pregnancy and live foal rates were higher in barren mares 288 with a cytologic diagnosis of persistent endometritis treated with both p. acnes and 289 conventional treatments than in mares treated only with conventional treatments. 68 290 conclusion 291 novel treatment strategies for chronic endometritis or persistent mating induced 292 endometritis have been recently evaluated in mares. treatments for chronic endometritis 293 include adding chelating solutions to potentiate antibiotics, irrigating with mucolytics to 294 dissolve excessive mucus or biofilm and adding oxygen radical scavengers to irrigation 295 solutions to reduce inflammation. although improving physical uterine clearance after 296 mating will remain the primary treatment for mares with persistent post mating induced 297 endometritis, administration of immunomodulators around the time of mating has been 298 shown to improve pregnancy rates. 299 300 references 301 1. causey rc: making sense of equine uterine 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pathogens obtained from horses with mycotic keratitis. am j vet res 405 2006;67:562-568. 406 43. bjorling de, wooley re: edta-tromethamine lavage as an adjunct treatment for 407 multiple fistulas in a dog. j am vet med assoc 1982;181:596-597. 408 44. wooley re, berman ap, shotts jr eb: antibiotic-tromethamine-edta lavage 409 for the treatment of bacterial rhinitis in a dog. j am vet med assoc 410 1979;175:817-818. 411 45. wooley re, schall wd, eagon rg, et al: efficacy of edta-tris-lysozyme 412 lavage in the treatment of experimentally in induced pseudomonas aeruginosa 413 cystitis in the dog. am j vet res 1974;35:27-29. 414 46. kirkland kd, fales wh, blanchard tl, et al: the in vitro effects of edta-tris, 415 edta-tris-lysozyme, and antimicrobial agents on equine gential isolates of 416 pseudomonas aeruginosa. theriogenology 1983;20:287-295. 417 47. wooley re, jones ms, shotts jr eb: uptake of antibodies in gram-negative 418 bacteria exposed to edta-tris. vet microbiol 1984;10:57-70. 419 48. youngquist rs, blanchard tl, lapin d, et al: the effects of edta-tris infusion 420 on the equine endometrium. theriogenology 1984;22:593-599. 421 49. schoon d, schoon h-a, klug e: angioses in the equine endometrium-422 pathogenesis and clinical correlations. pferdeheilkunde 1999;15:541-546. 423 50. liu ikm, esteller-vico a, couto s, et al: assessment of equine endometrial and 424 myometrial vascular elastosis: relationship to biopsy grade, age and parity. the 425 havemeyer foundation. the chronically infertile mare 2008; p. 11-12. 426 51. esteller-vico a, liu ikm, steffey ep, et al: effect of vascular degeneration on 427 utero-ovarian blood flow and perfusion in the cyclic mare. theriogenology 428 2007;68:506. 429 446 52. troedsson mh, scott ma, liu ik: comparative treatment of mares susceptible to 430 chronic uterine infection. am j vet res 1995;56:468-472. 431 53. pycock jf, newcombe jr: assessment of the effect of three treatments to remove 432 intrauterine fluid on pregnancy rate in the mare. vet rec 1996;138:320-323. 433 54. brinsko sp, varner dd, blanchard tl, et al: the effect of postbreeding uterine 434 lavage on pregnancy rate in mares. theriogenology 1990;33:465-475. 435 55. leblanc m, neuwirth l, mauragis d, et al: oxytocin enhances clearance of 436 radiocolloid from the uterine lumen of reproductively normal mares and mares 437 susceptible to endometritis. equine vet j 1994;26:279-282. 438 56. combs gb, leblanc mm, neuwirth l, et al: effects of prostaglandin f2 alpha, 439 cloprostenol and fenprostalene on uerine clearance of radiocolloid in the mare. 440 theriogenology 1996;45:1449-1455. 441 57. rasch k, schoon ha, sieme h, et al: histomorphological endometrial status and 442 influence of oxytocin on the uterine drainage and pregnancy rate in mares. equine 443 vet j 1996;28:455-460. 444 58. knutti b, pycock jf, van der weijden gc, et al: the influence of early 445 postbreeding uterine lavage on pregnancy rate in mares with intrauterine fluid 446 accumulations after breeding. equine vet educ 2000;5:346-349. 447 59. troedsson mht: therapeutic considerations for mating-induced endometritis. 448 pferdeheilkunde 1997;13:516-520. 449 60. schramme ar, pinto cr, davis j, et al: pharmacokinetics of carbetocin, a long-450 acting oxytocin analogue, following intravenous administration in horses. equine 451 vet j 2008;40:658-661. 452 61. troedsson mh, ababneh mm, ohlgren af, et al: effect of periovulatory 453 prostaglandin f2alpha on pregnancy rates and luteal function in the mare. 454 theriogenology 2001;55:1891-1899. 455 62. nie gj, johnson ke, wenzel jg, et al: luteal function in mares following 456 administration of oxytocin, cloprostenol or saline on day 0, 1 or 2 post-ovulation. 457 theriogenology 2003;60:1119-1125. 458 63. fumuso e, giguere s, wade j, et al: endometrial il-1, il-6 and tnfα, mrna 459 expression in mares resistant or susceptible to post-breeding endometritis. effects 460 447 of estrous cycle, artificial insemination and immunomodulation. vet immunol 461 immunopathol 2003;96:31-41. 462 64. fumuso ea, aguilar j, giguere s, et al: immune parameters in mares resistant 463 and susceptible to persistent post-breeding endometritis: effects of 464 immunomodulation. vet immunol immunopathol 2007;118:30-39. 465 65. rogan d, fumuso ea, rodriguez e, et al: use of mycobacterial cell wall extract 466 (mcwe) in susceptible mares to clear experimentally induced endometritis with 467 streptococcus zooepidemicus. j equine vet sci 2007;27:112-117. 468 66. papa fo, dell'aqua jr ja, alvarenga ma, et al: use of corticosteroid therapy on 469 the modulation of uterine inflammatory response in mares after artificial 470 insemination with frozen semen. pferdeheilkunde 2008;24:79-82. 471 67. bucca s, carli a, buckley t, et al: the use of dexamethasone administered to 472 mares at breeding time in the modulation of persistent mating induced 473 endometritis. theriogenology 2008;70:1093-1100. 474 68. rohrbach bw, sheerin pc, cantrell ck, et al: effect of adjunctive treatment with 475 intravenously administered propionibacterium acnes on reproductive 476 performance in mares with persistent endometritis. j am vet med assoc 477 2007;231:107-113. 478 69. vandaele h, daels p, piepers s, et al: the effect of post-insemination 479 dexamethasone treatment on pregnancy rates in mares. the havemeyer 480 foundation. the chronically infertile mare 2008;43-44. 481 70. adams aa, breathnach cc, katepalli mp, et al: advanced age in horses affects 482 divisional history of t cells and inflammatory cytokine production. mech ageing 483 dev 2008;129:656-664. 484 71. adams aa, katepalli mp, kohler k, et al: effect of body condition, body weight 485 and adiposity on inflammatory cytokine responses in old horses. vet immunol 486 immunopathol 2009;127:286-294. 487 72. watson ed, stokes cr, bourne fj: effect of exogenous ovarian steroids on the 488 uterine luminal prostaglandins in ovariectomised mares with experimental 489 endometritis. res vet sci 1988;44:361-365. 490 448 73. watson ed, stokes cr, bourne fj: concentrations of immunoreactive 491 leukotriene b4 in uterine lavage fluid from mares with experimentally induced 492 and naturally occurring endometritis. j vet pharmacol ther 1988;11:130-134. 493 74. cox wi: examining the immunologic and hematopoietic properties of an 494 immunostimulant. vet med small anim clin 1988;6:424-428. 495 496 497 449 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy 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print]'] use these settings to create adobe pdf documents for quality printing on desktop printers and proofers. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes false /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: synchronization of follicular waves with progesterone/estrogen combination before superstimulation with pfsh in alpacas synchronization of follicular waves with progesterone/estrogen combination before superstimulation with pfsh in alpacas y. pichaa,b, j. rodrigueza, s. nielsena, s. sandovala, j. sumarb, a. tibarya a college of veterinary medicine, washington state university, pullman, wa bsumac tarpuy association, mega alpaca project, puno, perú ovarian response to superstimulation with pfsh depends on control of follicular waves before initiation of treatment. we hypothesized that a combination of progesterone and estrogen treatment would produce better synchronization of follicular waves in alpacas than progesterone alone. we tested this hypothesis in two experiments. in experiment one; adult females (five per group) were given no treatment (control), daily progesterone injection (25 mg in oil, im) for 10 days (p4 group) or combination of progesterone (25 mg) and estrogen (5 mg) im for 10 days (p/e group). ovarian activity was monitored daily by transrectal ultrasonography (us) (aloka 500, wallingford, ct, usa, with 7.5 mhz linear transducer) until emergence of a dominant follicle (6 mm in diameter). time between the end of treatment and dominant follicle emergence was compared by anova. in experiment two, adult females (10 per group) were given either p/e for 10 days (group a) or gnrh (cystorelin ®, merial, duluth, ga, usa, 50 mcg, im) followed by p/e for six days and p4 alone for four days (group b). after treatment, all females received five days of pfsh bid (20/20 mg, 15/15 mg, 10/10 mg, 10/10 mg and 10/10 mg). follicular growth was monitored every 48 hours and ovulation induced with hcg (chorulon®, intervet/schering-plough animal health, millsboro, de, usa, 1000 iu, iv) when follicles were between 6 to 8 mm in diameter. ovulation was verified by us seven days after hcg treatment. in experiment one, follicular activity continued during the observation period in the control animals and in three alpacas in the p4 group. none of the alpacas in the p/e group had follicles greater than 2 mm in diameter on the 8th day of treatment. the mean interval in days and standard deviation (sd) to emergence of a dominant follicle was 5 (3.8), 4.2 (1.8) and 7 (0.7) for the control, p4 group and p/e group, respectively. in experiment two, all females responded to pfsh. however, group a had a poor ovulation rate and a higher incidence of anovulatory follicles compared to group b. group b had a uniform follicular population one day after the end of the pfsh treatment. the number of follicles was difficult to ascertain in some females and ranged from five to greater than 12 per ovary. these preliminary results indicate that treatment with a combination of p/e may be an option for control of follicular activity in alpacas before initiation of superstimulation treatment with pfsh. administration of gnrh before p/e treatment provides better synchronization of follicular waves probably through the elimination of dominant follicles. studies are underway to determine the reason for ovulation failure in some of the stimulated females. factors being studied include dosage of progesterone, estrogen, pfsh and follicle size at the time of ovulation induction. acknowledgements: dr. picha was partially funded by the sft/act zemjanis outreach fund keywords: superovulation, synchronization, anovulatory follicles, alpaca 535 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 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<< /tilewidth 256 /tileheight 256 /quality 30 >> /jpeg2000grayimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /antialiasmonoimages false /cropmonoimages false /monoimageminresolution 1200 /monoimageminresolutionpolicy /ok /downsamplemonoimages false /monoimagedownsampletype /average /monoimageresolution 300 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages false /monoimagefilter /flateencode /monoimagedict << /k -1 >> /allowpsxobjects false /checkcompliance [ /none ] /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname () /pdfxtrapped /false /createjdffile false /description << /enu ([based on 'no compression'] [based on 'no compression wbleeed'] [based on '[high quality print]'] use these settings to create adobe pdf documents for quality printing on desktop printers and proofers. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes false /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1 contact pururava sharma pururavasharma@gmail.com © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12822, http://dx.doi.org/10.58292/ct.v17.12822 case report fetal abdominoschisis and chondrodysplastic dwarfism in hydroallantoic buffaloes pururava sharma,a pravesh kumar,b pankaj sood,b akshay sharma,b dawa yangka,c depika thakur,d amit sharmab adepartment of animal husbandry, himachal pradesh, india bdepartment of veterinary gynaecology and obstetrics, dgcn covas, palampur, himachal pradesh, india cministry of agriculture and livestock, royal government of bhutan ddepartment of veterinary gynaecology and obstetrics, abhilashi university, himachal pradesh, india abstract we describe 2 rare cases of hydroallantois in buffaloes, each associated with a distinct severely malformed fetus (abdominoschisis and chondrodysplastic dwarfism, respectively). both buffaloes, in advanced pregnancy, were presented with progressive abdominal distension, anorexia, and reduced ruminal activity. transrectal examination revealed excessive allantoic fluid accumulation with no palpable fetal parts. pregnancy was terminated followed by controlled drainage of allantoic fluid and supportive therapy. biochemical analysis indicated compromised fetal metabolism; however, buffaloes recovered fully without postpartum complications. keywords: abdominoschisis, buffalo, chondrodysplastic, fetal monsters, hydorallantois introduction hydroallantois is a pathological condition of pregnancy characterized by excessive and rapid accumulation of 40-160 litres allantoic fluid within the allantoic cavity,1 typically occurring within 5-20 days prior to parturition.2 this condition arises due to fetal membranes functional or structural abnormalities, particularly allantochorion, and leads to marked bilateral abdominal distension of the dam.3 hydroallantois is infrequently diagnosed and is observed across multiple species, with the highest incidence reported in cattle and buffaloes.4 occasional cases have also been documented in mares,5,6 in an ewe,7 and a dog.8 in severe cases, failure to diagnose and treat the condition at an early stage may lead to recumbency with guarded prognosis. although hydroallantois occurs infrequently in cattle, it is often associated with concurrent fetal and fetal membrane abnormalities.9 pathophysiology of hydroallantois is primarily due to reduced placental vascularization, leading to functional impairment of placentomes and fluid accumulation within the allantoic sac. it may also result from fetal anomalies, including hepatic or  renal dysfunction and umbilical cord torsion.10,11 hydroallantois accounts for 85-90% of bovine dropsical conditions, often characterized by non-functional caruncles in one uterine horn and compensatory enlargement of remaining placentomes.3 fetal monstrosities, severely malformed fetuses are attributed to a combination of genetic mutations and environmental insults.12 although, the overall incidence is relatively low (~  0.5% in domestic animals13) significantly higher prevalence of 7.9-12.8% has been documented in river buffaloes.14 we describe the clinical presentation and successful management of 2 cases of hydroallantois in buffaloes, each associated with distinct severely malformed fetuses (abdominoschisis and chondrodysplastic dwarfism, respectively). case presentation both referred buffaloes were in advanced pregnancy (> 240 days) and presented with a history of progressive to sudden bilateral abdominal distension (figures 1 and 2), accompanied by anorexia, reduced water intake, and scanty fecal output. animals had shallow, laboured respiration, and reduced ruminal motility. both animals were artificially bred (semen from the same bull) and had a history of normal previous parturitions. case 1 a 9-year, multiparous murrah buffalo on day 260 of pregnancy was presented with a sudden onset of bilateral abdominal distension noticed over the last 10-12 days. animal had signs of mailto:pururavasharma@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12822 2 citation: clinical theriogenology 2025, 17, 10416, http://dx.doi.org/10.58292/ct.v17.12822 systemic compromise, including  dullness,  dry muzzle,  congested mucous membranes, and shallow respiration with no signs of parturition. transrectal palpation revealed shortened cervix (suggestive of early cervical changes pertaining to parturition) and a severely distended uterus that occupied most of the abdominal and pelvic cavity; fetal parts were not palpable. vaginal examination revealed a closed cervix. case 2 a  6-year, murrah-crossbred buffalo on ~ day 255 of pregnancy was presented with a gradual abdominal distension progressing over 20-25 days. owner reported reduced appetite but no acute signs of distress. transrectal examination confirmed a grossly distended uterus filled with fluid, with no palpable fetal structures. vaginal examination revealed a firm and closed cervix with no indication of impending parturition.  treatment hydroallantois was the presumptive diagnosis in both cases, based on consistent clinical presentations and diagnostic findings. our goal was to preserve dams’ survival. parturition was induced with 32 mg of intramuscular dexamethasone sodium phosphate (dexona®; zenex animal health, ahmedabad, india) and 25 mg of intramuscular dinoprost tromethamine (lutalyse®; zoetis, mumbai, india). in both cases, cervical dilation commenced within 5-6 hours; allantois was punctured to gradually drain allantoic fluid with a 16 french foley catheter. initially ~ 60-80 liters of fluid was drained. to prevent hypovolemic shock, buffaloes received intravenous fluid therapy consisting of 5 liters each of ringer’s lactate and normal saline. supportive medication included 5 grams intramuscular streptopenicillin (dicrysticin®; zenex ah) and fluid therapy. second stage of parturition (visible abdominal and uterine contractions) was observed (during fluid therapy) after ~ 15 and 17 hours of induction in cases 1 and 2, respectively. allanotoic fluid was collected for electrolytes estimation (table). in case 1, ~ 120 liters of allantoic fluid was expelled during an interval of 12-15 hours. a dead female fetus, exhibiting kyphosis at the second last thoracic vertebra and abdominoschisis with externalized viscera (figure 3), was extracted manually. fetal membranes were manually removed after 42 hours of induction of parturition with gentle teasing and avoiding uterine hemorrhage; had numerous small-sized cotyledons and hypoplasia. in case 2, ~ 100 liters of allantoic fluid were drained in a controlled manner. a dead, malformed female fetus identified as chondrodysplastic dwarf (figure 4) was removed manually; fetus had short limbs, broad thorax, flat face with mandibular prognathism, bulging eyes, and fluid-filled abdomen (figure 5). figure 1. bilateral abdominal distension in a buffalo; note: symmetrical enlargement of the flanks (case 1) figure 2. marked abdominal distension observed from left lateral side (case 2) http://dx.doi.org/10.58292/ct.v17.12822 citation: clinical theriogenology 2025, 17, 12822, http://dx.doi.org/10.58292/ct.v17.12822 3 despite differences in progression and fetal pathology (table), both animals responded favorably to parturition induction and supportive management. animals were given intravenous tranexamic acid (texableed®; vet mankind, new delhi, india) at 5 mg/kg bw, 300 ml of intravenous and 150 ml subcutaneous calcium-magnesium borogluconate (mifex®; elanco, thane, india), and slow intravenous oxytocin (100 iu diluted in normal saline). outcome complete recovery occurred after 5 days in both buffaloes, with no postpartum complications for 60 days as reported by the owner. owners were advised for artificial insemination in the next breeding season. discussion hydroallantois is a relatively uncommon pregnancy disorder, more frequently reported in cattle but also observed in buffaloes;15–18 typically manifests during last trimester, particularly between the 8th and 9th months of pregnancy.3 pathophysiology of hydroallantois primarily involves rapid and excessive accumulation of allantoic fluid, leading to severe abdominal distension. in many cases, the condition is associated with abnormal placentation, particularly the formation of adventitious placental structures resulting from the absence or dysfunction of maternal caruncles.3,19 such changes are often secondary to uterine pathology, including fibrosis or inflammatory conditions that impair normal placentome development and function. additionally, fetal renal dysfunction, altered membrane permeability, impaired active sodium transport across chorioallantoic membrane, and hormonal table. summary of clinical, biochemical and therapeutic findings parameter case 1 case 2 age/parity 9 years/multiparous 6 years/multiparous stage of pregnancy at presentation ~ 260 days ~ 255 days onset and duration of abdominal distension sudden; 10-12 days gradual; 20-25 days vital signs temperature: 100°f; heart rate: 114 bpm; respiratory rate: 32/minutes; dehydration: 3% temperature: 102.2°f; heart rate: 100 bpm; respiratory rate: 37/mins; dehydration: 2% transrectal examination markedly distended uterus; no palpable fetal parts markedly distended uterus; no palpable fetal parts cervical findings closed but shortened closed and firm initial fluid drained ~ 60-80 liters ~ 60-70 liters total fluid drained ~ 120 liters ~ 100 liters electrolytes of allantoic fluid electrolytes (reference range) case 1 case 2 sodium (142.85 ± 10.1 mmol/l) 135.4 mmol/l 140.2 mmol/l potassium (4.67 ± 1.21 mmol/l) 4.77 mmol/l 4.52 mmol/l chloride (92.99 ± 6.72 mmol/l) 95.2 mmol/l 93.1 mmol/l biochemicals of dams’ plasma biochemical (reference range) case 1 case 2 total protein (normal: 5.5-8 grams %) 0.11 grams 1.7 grams bilirubin (1-2 mg%) 0.66 mg 1.1 mg bun (12-25 mg%) 29.4 mg 30.0 mg glucose (45-75 mg%) 30.7 mg 20 mg creatinine (1-2 mg%) 1.05 mg 0.8 mg parameter case 1 case 2 fetal diagnosis abdominoschisis with kyphosis; externalized viscera chondrodysplastic dwarf; short limb, flat face and prognathism fetal membranes hypoplastic with small cotyledons normal without appreciable gross abnormality time to cervical dilation ~ 8 hours ~ 12 hours time to puncture allantois for drainage ~ 15 hours ~ 25 hours time of fetal delivery ~ 30 hours ~ 38 hours time of fetal membranes expulsion ~ 42 hours after parturition induction (with gentle teasing) immediately after calving; spontaneous http://dx.doi.org/10.58292/ct.v17.12822 4 citation: clinical theriogenology 2025, 17, 10416, http://dx.doi.org/10.58292/ct.v17.12822 imbalance have also been implicated in the etiopathogenesis of this condition.18 clinically, hydroallantois is characterized by a rapid increase in abdominal size, loss of body condition, recumbency, and eventual death if untreated.1 the condition often results in a compromised fetus, commonly underdeveloped, edematous, afflicted with congenital anomalies such as ascites, or in some cases, apparently normal but nonviable.3,18,20 chloride concentrations remain lower than plasma, whereas sodium and potassium exhibit inverse trends.21 management of hydroallantois depends on the severity and prognosis. in severe cases, termination of pregnancy is warranted and can be achieved pharmacologically using prostaglandin f2α and corticosteroids.21,22 however, caution must be exercised as rapid removal of the fluid may result in hypovolemic shock and collapse.23 in both cases, tranexamic acid was given empirically after fetal delivery, despite lack of overt uterine trauma or hemorrhage. this decision was based on the application of manual traction during assisted delivery that carries a potential risk of subclinical uterine injury and delayed bleeding, especially in cases involving severe uterine distension and manipulation. although not routinely indicated for hydroallantois management, tranexamic acid has been used off-label in bovine obstetrics to minimize haemorrhagic risk in high-intervention scenarios. its empirical use in such contexts has been described in veterinary literature and may be justified as a precautionary measure.24 in some hydroallantois cases, associated fetal anomalies such as abdominal wall defects may be present. abdominoschisis and omphalocele are congenital conditions characterized by herniation of abdominal organs through a defect in the figure 3. manually extracted dead female fetus exhibiting kyphosis and abdominoschisis figure 4. lateral view of chondrodysplastic dwarf monster http://dx.doi.org/10.58292/ct.v17.12822 citation: clinical theriogenology 2025, 17, 12822, http://dx.doi.org/10.58292/ct.v17.12822 5 abdominal wall. these defects are detectable via prenatal ultrasonography in humans, allowing for early diagnosis and safer maternal outcomes.25 additionally, chondrodysplastic anomalies, such as the bulldog calf phenotype, are often genetically inherited and have been well-documented in specific cattle breeds like dexter. these conditions are typically attributed to disruptions in fetal pituitary development and function.26 antepartum ultrasonography was not performed to assess placental or fetal abnormalities due to referral hospital facility and histopathological examination of fetal membranes was not conducted. lack of genetic testing, pedigree analysis, or follow-up on other offspring from the same sire limits interpretation of a heritable cause. conclusion the cases highlighted a rare association of hydroallantois with severely malformed fetuses (abdominoschisis and chondrodysplastic dwarfism) in buffaloes. successful management required timely diagnosis, controlled fluid drainage, and supportive therapy. same breeding bull used in both cases suggested a possible genetic association. importance of vigilant monitoring in advanced pregnancy and the need to consider genetic factors in recurrent fetal anomalies and monsters is emphasized. learning points • hydroallantois, although rare, can be associated with severe fetal monsters such as abdominoschisis and chondrodysplastic dwarfism in buffaloes, indicating a possible shared pathophysiological or genetic basis. • prompt identification of hydroallantois through clinical and transrectal examination, followed by pharmacological induction, gradual fluid drainage, and supportive care, can ensure successful outcomes and full recovery of the dam without postpartum complications. • genetic link from natural service with the same breeding bull is possible. both buffaloes were bred by the same bull via natural service and therefore a heritable component cannot be excluded. however, no genetic testing was performed, and this association remains speculative. conflict of interest authors declare no conflict of interest. authors contribution diagnosis and treatment: ps, pk, and akshay s; after calving management: dy and dt; all authors contributed to manuscript writing. references 1. noakes de, parkinson tj, england gcw: vaginal wall and cervix through the vulva. in: veterinary reproduction and obstetrics. 10th edition, london; elsevier: 2019. p. 191-192. 2. morrow da: current therapy in theriogenology. 2nd edition, philadelphia; w.b. saunders:1986. p. 207. 3. roberts sj: veterinary obstetrics and genital diseases. new delhi; cbs publishers and distributors: 1971. p. 180-183. 4. roberts sj: veterinary obstetrics and genital diseases. 3rd edition, ann arbor; edwards brothers inc: 1986. p. 223-228. 5. govaere jl, de schauwer c, hoogewijs mk, et al: hydrallantois in  the mare—a report of five cases. reprod domest anim 2013;48:1-6. doi: 10.1111/j.1439-0531.2012.02013.x 6. burton k, holyoak r, bassett a, et al: pregnancy termination in a mare with hydroallantois. clinical theriogenology 2025;17:12176. doi: 10.58292/ct.v17.12176 7. milton a, welker b, modransky p: hydrallantois in a ewe. j am  vet  med assoc 1989;195:1385-1386. doi: 10.2460/ javma.1989.195.10.1385 8. sharma a, sharma p, sharma j, et al: clinical approach to a rare case of hydrallantois in bitch. vet rec case rep 2024;12:e937. doi: 10.1002/vrc2.937 9. napolean re, palanisamy m, selvaraju m, et al: successful management of hydroallantois in a jersey crossbred cow. int j livest res 2012;2:213-216. available from: http://krishikosh.egranth. ac.in/handle/1/5810150310 [cited 22 august 2025]. 10. kapadiya ps, parikh ss, chauhan pm, et al: management of hydrallantois in a jaffrabadi buffalo: a case report. j pharmacogn phytochem 2018;11:1534-1536. 11. prabhakaran kp: management of hydrallantois and pathological description of affected fetus in a murrah buffalo: a case report. buffalo bull 2020;39:117-123. 12. purohit gn, kumar p, solanki k, et al: perspective of fetal dystocia in cattle and buffalo: vet sci dev 2012;2:31-42. doi: 10.4081/ vsd.2012.3712 13. craig jf: fleming’s veterinary obstetrics. 4th edition, london; tindall and cox: 1930. 14. singla vk, sharma rd: analysis of 188 cases of dystocia in buffaloes. ind vet j 1992;69:563-564. 15. rangasamy s, rajasundaram rc, sathiamoorthy t, et al: management of hydroallantois in a non-descriptive cow. indian j anim reprod 2013;34:52-53. 16. resum ns, bhavna ka, kour p: hydroallantois in a cross-bred heifer: a case report. indian j anim health 2016;55:91-94. 17. kumar s, sharma u, pandey ak, et al: hydroallantois in buffalo: a case report. buffalo bull 2012;31:67-69. figure 5. postmortem image depicting fluid filled abdomen along with anasarca http://dx.doi.org/10.58292/ct.v17.12822 https://doi.org/10.1111/j.1439‑0531.2012.02013.x https://doi.org/10.58292/ct.v17.12176 https://doi.org/10.2460/javma.1989.195.10.1385 https://doi.org/10.2460/javma.1989.195.10.1385 https://doi.org/10.1002/vrc2.937 http://krishikosh.egranth.ac.in/handle/1/5810150310 http://krishikosh.egranth.ac.in/handle/1/5810150310 https://doi.org/10.4081/vsd.2012.3712 https://doi.org/10.4081/vsd.2012.3712 6 citation: clinical theriogenology 2025, 17, 10416, http://dx.doi.org/10.58292/ct.v17.12822 18. kumar p, sharma a, singh mm, et al: hydrallantois in buffaloes. buffalo bull 2018;37:437-440. 19. drost m: complications during gestation in the cow. theriogenology 2007;68:487-491. doi: 10.1016/j.theriogenology.2007.04.006 20. morin de, hornbuckle t, rowan ll, et al: hydrallantois in a caprine doe. j am vet med assoc 1994;204:108-111. doi: 10.2460/ javma.1994.204.01.108 21. sharma p, ahuja ak, sharma a, et al: hydrallantois with foetal monstrosity in a murrah crossbred buffalo: a case report. ijah 2024;63:148-149. doi: 10.36062/ijah.2024.15623 22. manokaran s, ravikumar k, nepolean re, et al: hydrallantois in a non-descript buffalo: a case report. indian j field vet 2011;7:69. 23. peiro jr, borges as, yanaka r, et al: hydrallantois in an ewe (case report). ars vet jaboticabal 2007;23:116-119. issn 0102-6380 24. papich mg: saunders handbook of veterinary drugs: small and large animals, 4th edition, st. louis; elsevier-wb saunders: 2016. 25. bartos m: congenital abdominal wall defects: a topic review and a case report of a lethal polymalformation. vet med-czech 2020;20:133-137. doi: 10.17221/72/2020 26. charmila sd, muramoto c, silva awo, et al: chondrodysplasia bulldog type in cattle in the state of bahia, brazil. pesquisa veterinária brasileira 2020;13:536-541. doi: 10.24070/ bjvp.1983-0246.v13i2p536-541 [cited 22 august 2025]. http://dx.doi.org/10.58292/ct.v17.12822 https://doi.org/10.1016/j.theriogenology.2007.04.006 https://doi.org/10.2460/javma.1994.204.01.108 https://doi.org/10.2460/javma.1994.204.01.108 https://doi.org/10.36062/ijah.2024.15623 https://doi.org/10.17221/72/2020 https://doi.org/10.24070/bjvp.1983-0246.v13i2p536-541 https://doi.org/10.24070/bjvp.1983-0246.v13i2p536-541 2014: hermaphroditism in a miniature schnauzer hermaphroditism in a miniature schnauzer john m. haller,a*jack d. smith,b michelle h. fabiania atexas gulf coast veterinary specialists, houston, tx; bdepartment of pathobiology and population medicine, college of veterinary medicine, mississippi state university, mississippi state, ms abstract a nine-year-old male miniature schnauzer was examined because of a one month history of progressive abdominal distention and lethargy. the patient presented with gynecomastia and neither testicle was identified within the scrotum. abdominal radiographs and abdominal ultrasonography revealed bilateral gonadal masses and an enlarged and elongated fluid-filled uterus with echogenic contents, consistent with pyometra. gonadohysterectomy and histopathology were consistent with true hermaphroditism, with the abdomen containing one right testicle with an associated sertoli cell tumor and contralaterally a left ovary with a granulosa cell tumor. the dog recovered from surgery without complications. there appears to be no association between true hermaphroditism and persistent mullerian duct syndrome, a common inherited disorder in the miniature schnauzer breed, where the mullerian ducts fail to regress after testicular development. cryptorchidism is an important clinical problem and affected animals and/or carriers of this genetic defect should not be used for breeding. either persistent mullerian duct syndrome or true hermaphroditism should be considered as a cause for cryptorchidism in the miniature schnauzer breed. keywords: hermaphroditism, cryptorchidism, miniature schnauzer clinical report a nine-year-old male miniature schnauzer was referred for examination following a one month history of progressive abdominal distention and concurrent lethargy. findings observed upon physical examination included a distended abdomen with a pendulous appearance and bilateral perineal hernias. testes were not present within the scrotum nor palpable within the area of the external inguinal rings. a large (approximately 5 cm) non-painful mass was palpated in the mid-abdomen. the prepuce and penis appeared to be normally developed. gynecomastia was also evident. the results of a complete blood count and serum biochemistry tests showed a mildly decreased hematocrit (29%; reference range, 3662%), along with a mild non-regenerative anemia (5.01 x 106/ul; reference range, 5.5-8.5 x 106/ul) and hyperglobulinemia (4 g/dl; reference range, 1.6-3.6 g/dl). imaging diagnosis and outcome abdominal radiographs were performed (figure 1). there were multiple round, smoothly marginated soft tissue structures within the ventral abdomen, which caused caudal displacement of the urinary bladder, dorsal displacement of the small bowel, and dorsal displacement of the pylorus of the stomach on the lateral view. on the ventrodorsal view, an elongated soft tissue opacity extended along the left and right lateral abdominal walls. this tubular structure was approximately 6 cm in width on the ventrodorsal view. there was slightly reduced serosal detail within the ventral mid-peritoneal space. the nipples of several mammary glands were prominent. the primary differentials for the two well-defined oval masses within the ventral abdomen included cryptorchidism and testicular neoplasia. based on these findings an ultrasound examination of the abdomen was recommended and performed. on abdominal ultrasonography, there was echogenic swirling fluid contained within a tubular, elongated and thin-walled structure, which extended cranially to the level of the right lateral liver lobe (figure 2). the structure was located superficial to the duodenum, caudate lobe of the liver and right kidney. there was a heterogenous hypoechoic 9 cm long by 7 cm deep mass within the left *current address: department of veterinary medicine and surgery, college of veterinary medicine, university of missouri, columbia, mo 481 clinical theriogenology • volume 6, number 4 • december 2014 hemiabdomen cranial to the head of the spleen. additionally, a hyperechoic distal shadowing 11 cm long mass was noted within the right abdomen. the urinary bladder was caudally displaced. two medial iliac lymph nodes were enlarged and measured 1.9 cm in diameter. on the basis of the collective ultrasound findings, pyometra, hydrometra, or mucometra with concurrent ovarian or testicular neoplasia associated with hermaphroditism was suspected. the enlarged medial iliac lymph nodes were most consistent with lymphoid hyperplasia. an exploratory celiotomy was performed. a grossly enlarged fluid filled uterus was observed upon entering the abdomen. the uterus joined what appeared to be the cervix just prior to entering the prostate gland. the uterus was ligated immediately adjacent to its entry into the prostate gland. the prostate was enlarged, but appeared smooth and symmetrical. two masses were observed and appeared to be the right and left gonads. each mass was also found to be attached and closely associated to the distal aspect of the fluid-filled uterine horn (figure 3). the left gonadal mass was severely enlarged and irregular. the right gonadal mass was much smaller (approximately 4 cm). a gonadohysterectomy was performed and the abdomen was closed routinely. the uterine body was opened and several hundred milliliters of thick, dark green malodorous fluid consistent with a purulent exudate was drained from the uterus. the dog had an uneventful recovery from surgery, was subsequently discharged and was reported to be normal one month following the surgery. each gonadal mass was submitted for histopathological examination. the larger left gonadal mass was identified as an ovary completely effaced by a variably multicystic and solid mass obliterating the normal ovarian architecture. the mass was composed of nests, tubules and cords of triangular to papillary structures supported by fibrous stromal septae that formed and partially delineated cystic spaces. the neoplastic cells had ill-defined borders and vacuolated eosinophilic cytoplasm. the nuclei were oval and had smudged chromatin. the nucleoli were indistinct. mitotic figures were uncommon. the smaller right gonadal mass was identified as testicle, containing an attached epididymis. the testicle was moderately enlarged and effaced by irregularly sized tubules filled with sertoli cells. the sertoli cells were triangular shaped and were often palisading. they had moderate amounts of vacuolated light basophilic cytoplasm. the nuclei were ovoid and mildly pleomorphic. the tubules were subdivided by a fine fibrous to fibrovascular stroma. the diagnosis was true hermaphroditism, with the abdomen containing one right testicle with an associated sertoli cell tumor and contralaterally, a left ovary with a granulosa cell tumor. discussion since the reproductive tracts of males and females develop fundamentally from the same embryonic primordium, incidental fusion defects or the occasional failure of structures to unite results in the idiosyncrasy of both sexes being identified within the same individual.1 in true hermaphroditism, there is both ovarian and testicular tissue recognized as being either united or separated.1 on the other hand, pseudohermaphroditism is a much more common condition in which the chromosomal and gonadal sexes are identical, but the development of the internal or external genitalia is indeterminate or opposed.2 in normal males, the mullerian ducts regress after testicular development, secondary to androgendependent masculinization.2 mullerian inhibiting substance (mis), which is made by sertoli cells, must be generated at a specific period in the development of the embryo to allow for the proper regression of the mullerian ducts in males.2 the mullerian ducts will eventually develop into the fallopian tubes, uterus, cervix and the cranial portion of the vagina. persistent mullerian duct syndrome (pmds) is a syndrome in which these derivatives do not regress in individuals having normal male chromosomal composition (xy karyotype) and bilateral testicles.2 several cases and case series involving pmds in miniature schnauzers have been reported.2-8 these animals are sometimes cryptorchid (either unilaterally or bilaterally) but have no clinical abnormalities. this is due to the fact that testosterone production or action is not modified in any way, and the animal has the visible appearance of being male.8 a recent study has identified that genetic defects in the mis receptor, misrii, are causative in canines.9 mutation of the misrii receptor gene results in the encoded receptor protein being shortened and impaired or dysfunctional.2 in patients with genetic defects in the mis receptor, the mis bioactivity was equivalent 482clinical theriogenology • volume 6, number 4 • december 2014 during regression of the mullerian ducts, when compared with age-matched controls, confirming normal functional levels of mis.9 persistent mullerian duct synrome has been identified as an inherited disorder in both the miniature schnauzer breed and bassett hound breeds in various countries.9,10 there have been other reports in the literature describing this syndrome in other breeds as well.11-15 persistent mullerian duct synrome is a representation of male pseudohermaphroditism, and is inherited as an autosomal recessive trait in the miniature schnauzer breed.16 other abnormalities in addition to neoplasia and pyometra have also been associated with pmds, including urinary tract infections and prostatitis.8 the uterus of the dog described here was severely dilated with several hundred milliliters of greenish colored, malodorous fluid consistent with a purulent exudate. the severely enlarged uterus was causing displacement of the intraabdominal organs, including severe caudal deviation and compression of the urinary bladder, to the point where it was partially intrapelvic in location. in spite of this, the animal had no clinical history of urinary abnormalities or infection involving the urinary tract. given the fact that the cervix appeared to be communicating with the prostate gland, we suspect that the most likely route of infection for the pyometra was via this communication with the prostate gland and prostatic urethra. the miniature schnauzer of the present report was bilaterally cryptorchid, and therefore, sterile. the patient was never recognized as being abnormal by the owners, and no clinical abnormalities were ever displayed by the dog until, approximately 1onemonth prior to presentation when progressive abdominal distention was noticed by the owners. subsequent examination by the referring veterinarian revealed bilateral perineal hernias, an abdominal mass and gynecomastia. one of the two masses described in this dog was a sertoli cell tumor. sertoli cell tumors are a common sequelae to cryptorchidism in pmds. additionally, pyometra has also been noted in older miniature schnauzers with pmds. the historical and physical examination findings of cryptorchidism, in conjunction with gynecomastia, cultivated the belief that at least one of the intraabdominal testes had undergone neoplastic transformation (figure 4). outward physical signs of feminization in male dogs can be the result of exuberant estrogen being produced by the sertoli cells and is frequently associated with testicular neoplasia, particularly sertoli cell tumors.8 attractiveness to other male intact dogs in our patient could not be confirmed as the dog was never around other dogs. preputial cytology could have been used to quickly verify the exposure to estrogen, by visualizing superficial cells.17 with estrogen exposure, keratinization of the preputial mucosa may be present. the preputial mucosa becomes cornified and cytologically will have a similar appearance to that of vaginal cytology from an estral bitch. preputial cytology is highly sensitive and specific for diagnosis of estrogen producing testicular tumors in canines and in particular, dogs with estrogen-induced alopecia,17 although alopecia was not detected in the patient of this report. repeated exposure to estrogen likely caused the cervix to dilate, leading to an ascending infection, and subsequent pyometra. there is no apparent association between pmds and true hermaphroditism. persistent mullerian duct syndrome is associated with pseudohermaphroditism and true hermaphroditism is a separate disorder. affected animals and/or carriers of this genetic defect should not be used for breeding. this is especially important as the condition cannot be confirmed with physical examination alone. additionally, some animals with pmds may only be unilaterally cryptorchid, allowing them to be capable of fertile matings. a molecular diagnostic test has been reported for pmds in the miniature schnauzer breed to diagnose all affected animals, as well as carriers.10 the test involves polymerase chain reaction amplification and gel electrophoresis, which would allow a relatively quick interpretation.10 clearly, the test would only need to be performed in dogs that are not cryptorchid. there is optimism that this test will allow breeders to avoid breeding affected dogs, as well as carriers. the test would serve to promote the well-being of the miniature schnauzer breed, and should allow this syndrome to be eradicated from the breed completely. the lack of genetic testing including chromosomal analysis, and historical pedigree analysis in our patient was unfortunate as it would have added to the academic knowledge of this case and allowed us to more accurately describe the disorder of sexual development. important information can be attained by including genetic testing with chromosomal and endocrinologic analysis. for example, it would have 483 clinical theriogenology • volume 6, number 4 • december 2014 been easy to test for estrogen production by the sertoli cell tumor, as well as for the presence of progesterone, which could have further validated the suspicion of pyometra in this dog. however, the presence of a testicle with sertoli cells indicates the probable presence of a y chromosome and the likelihood of a normal xy karyotype. the clinical signs of feminization of this patient, in conjunction with historical cryptorchidism and abdominal distention indicated the need for diagnostic imaging to visualize potential neoplastic transformation. it should be noted that the physical examination finding of bilateral cryptorchidism in a miniature schnauzer may signify either pmds or true hermaphroditism, as in the present report. in either case, an exploratory celiotomy should be performed. references 1. bartels je: radiology of the genital tract. in: o’brien tr, editor. radiographic diagnosis of abdominal disorders in the dog and cat. philadelphia: wb saunders; 1978. p 659. 2. breshears ma, peters jl: ambiguous genitalia in a fertile, unilaterally cryptorchid male miniature schnauzer dog. vet pathol 2011;4:1038-1040. 3. frey dc, tyler de, ramsey fk: pyometra associated with bilateral cryptorchism and sertoli’s cell tumor in a male pseudohermaphroditic dog. j am vet med assoc 1965;146:723-727. 4. brown tt, burek jd, mcentee k: male pseudohermaphroditism, cryptorchism, and sertoli cell neoplasia in three miniature schnauzers. j am vet med assoc 1976;169:821-825. 5. marshall ls, oehlert bs, haskins me, et al: persistent mullerian duct syndrome in miniature schnauzers. j am vet med assoc 1982;181:798-801. 6. bruinsma dl, ackerman la: male pseudohermaphroditism in a miniature schnauzer. vet med small anim clin 1983;78:1568-1571. 7. matsuu aya, hashizume takuya, nagano masashi, et al. a case of persistent mullerian duct syndrome with sertoli cell tumor and hydrometra in a dog. j vet med sci 2009;71(3): 379-381. 8. vegter ar, kooistra hs, van sluijs fj, et al. persistent mullerian duct syndrome in a miniature schnauzer dog with signs of feminization and a sertoli cell tumour. reprod dom anim 2010;45:447-452. 9. wu x, wan s, pujar s, et al: a single base pair mutation encoding a premature stop codon in the mis type ii receptor is responsible for canine persistent mullerian duct syndrome. j androl 2009;30:46-56. 10. pujar s, meyers-wallen vn: a molecular diagnostic test for persistent mullerian duct syndrome in miniature schnauzer dogs. sex dev 2010;3:326-328. 11. delamo an, deluca j, zufriategui l, et al: male pseudohermaphroditism in a dog: a case report. commun therio 2001;1:1-11. 12. wernham bgj, jerram rm: male pseudohermaphroditism in a labrador retriever, and a review of mammalian sexual differentiation. nz vet j 2006;54:248-252. 13. nowacka-woszuk j, nizanski w, klimowicz m, et al: normal male chromosome complement and a lack of sry and sox9 gene mutations in a male pseudohermaphrodite dog. anim reprod sci 2007;98:371-376. 14. alam mr, cho yg, cho sj, et al: male pseudohermaphroditism in dogs: three case reports. veterinarni medicina 2007;2:74-78. 15. whyte ana, monteagudo luis v, diaz-otero angel, et al: malformations of the epididymis, incomplete regression of the mesonephric tubules and hyperplasia of leydig cells in canine persistence of mullerian duct syndrome. anim reprod sci 2009;115:328-333. 16. meyers-wallen vn, donahoe pk, ueno s, et al: mullerian inhibiting substance is present in testes of dogs with persistent mullerian duct syndrome. biol reprod 1989;41:881-888. 17. dreimanis k, falk t, cigut m, et al: evaluation of preputial cytology in diagnosing oestrogen producing testicular tumours in dogs. j small anim pract 2012;53:536-541. 484clinical theriogenology • volume 6, number 4 • december 2014 figure 1. right lateral (top) and ventrodorsal (bottom) radiographs. multiple enlarged soft tissue opacities are noted in the abdomen, displacing the small intestinal tract dorsally and the stomach craniodorsally on the lateral image (outlines). enlarged tubular soft tissue opacities are noted along the lateral abdominal walls on the ventrodorsal image (arrows). the nipples are prominent (arrowheads). 485 clinical theriogenology • volume 6, number 4 • december 2014 figure 2. longitudinal ultrasound image of the lateral aspect of the left hemiabdomen. the left uterine horn (u) is markedly enlarged and contains echogenic material within the lumen. a large, rounded, heterogenous and predominantly solid ovarian tumor is noted (arrow). figure 3. exteriorization of the reproductive tract intraoperatively. both tumors can clearly be seen, along with the pyometra, as well as their anatomical association. the cervix is also visible (white arrow). the larger ovarian mass is being held by the surgeon’s hand. the smaller testicular mass is indicated by the arrow. 486clinical theriogenology • volume 6, number 4 • december 2014 figure 4. postoperative image demonstrating the gynomastia. (edotor’s note: the photographs in this paper are available in color in the online edition of clinical theriogenology.) 487 clinical theriogenology • volume 6, number 4 • december 2014 488clinical theriogenology • volume 6, number 4 • december 2014 omniblank: 2014: non-surgical management of vaginal prolapse in a late gestation alpaca (lamos pacos) non-surgical management of vaginal prolapse in a late gestation alpaca (lamos pacos) justin w. mcnaughten, malgorzata a. pozor, martha f. mallicote, amy l. stieler, audrey a. kelleman, margo l. macpherson college of veterinary medicine, university of florida, gainesville, fl summary: a multiparous alpaca at day 358 of gestation was presented with a four week history of intermittent vaginal prolapse. the vaginal prolapse was initially managed by the owner. due to the duration of prolapse and increase in its size, combined with pollakiuria and stranguria, the alpaca was presented to the university of florida, large animal hospital. transcutaneous and transabdominal ultrasonography was performed to assess the prolapsed tissue and to confirm that the fetus was viable. the vaginal prolapse was manually reduced and a commercially available plastic retention device for small ruminants was placed in the vagina and secured to a harness for stability. the retention device was well-tolerated and the hembra successfully delivered a healthy cria with the device in place. keywords: camelid, reproductive emergency, fetal assessment, ultrasound, vagina background vaginal prolapse is a condition that may affect lamoids in late gestation, similar to the condition in cattle and sheep.1,2 there are multiple theories regarding the etiology of vaginal prolapse in camelids, but the accepted predisposing factors include age, parity, and obesity.1 while early recognition and treatment usually carries a good prognosis for the dam and fetus, it is speculated that the condition may reoccur during subsequent pregnancies and that there may be a genetic component. after manual reduction of the prolapse, surgical techniques that involve suturing the vulvar tissue are often used to maintain the prolapse in situ.3,4 the technique that is most often reported is placement of truss sutures, at the muco-cutaneous perineal junction, but these sutures must be removed prior to delivery.3,4 plastic intravaginal prolapse retention devices are commonly utilized in sheep with vaginal prolapse.2 the device places pressure on the ventral vagina preventing the prolapse from reoccurring. the benefit of this device is that it is a non-surgical treatment that does not need to be removed prior to parturition. to the authors’ knowledge, there are no published reports of successful use of such a plastic retention device for the management of vaginal prolapse in an alpaca. case presentation a seven-year-old huacaya alpaca hembra at day 358 of gestation was presented to the university of florida, college of veterinary medicine, with a four week history of intermittent vaginal prolapse. the owner reported that the hembra had successfully delivered four crias prior to this pregnancy without incident. at the onset of signs, the tennis ball sized structure would spontaneously reduce. over the next four weeks, the prolapsed tissue was exteriorized for longer durations, grew increasing large, and the owner was required to reduce the prolapse with manual pressure several times per day. the day prior to presentation, the hembra was observed to be intermittently uncomfortable with pollakiuria and was admitted for evaluation. a softball sized mass (approximately 10 cm diameter) was protruding from the vulva at the time of presentation (figure 1). vital parameters and the remainder of the physical examination were within normal limits. the hembra weighed 81 kilograms with a body condition score of 4/5, indicating moderate over conditioning. ultrasonographic evaluation performed transcutaneously though the prolapsed vaginal tissue revealed an invaginating uterine pouch with fetal membranes and anechoic fetal fluids. there was no evidence that the urinary bladder or gastrointestinal tract were incarcerated. transabdominal ultrasonography performed from the right ventral abdomen confirmed a viable fetus, in anterior presentation, with fetal heart rate of 112-133 beats per minute (bpm). the owner elected to hospitalize the alpaca for management of the prolapse and any potential periparturient complications. 489 clinical theriogenology • volume 6, number 4 • december 2014 treatment with the hembra standing, the vaginal prolapse was cleansed with sterile isotonic saline and lathered in a sterile lubricant. the prolapse was gently massaged with manual cranial pressure until it was reduced. when the hembra subsequently cushed, the tissue again prolapsed. a disposable mare vaginal speculum was used to aid with the second attempt to manually reduce the prolapse. the speculum was inserted into the vagina and gently advanced, aiding in reduction of the prolapse. visual examination was performed at this time to assess the vagina and cervix, in order to ensure that there was not excess vaginal stretching or trauma, both of which have been proposed as predisposing factors for vaginal prolapse.4 the cervix was tightly closed and the mucous plug was present. there was no evidence of vaginal trauma, but the vaginal mucosa was hyperemic. a small amount of tan mucopurulent exudate was noted in the caudal vagina and cytology of this exudate showed polymorphonuclear cells, but bacteria were not identified. based upon visual and cytological evidence, vaginitis caused by chronic intermittent prolapse and repeated irritation from manual reduction was evident. following the second reduction of the prolapse, a commercially available plastic prolapse retainer device (ewe savers, pbs animal health, massillon, oh) was aseptically placed in the vagina. a foal fluid therapy surcingle (line management surcingle, mila international, erlanger, ky) was fitted to the hembra and strings attached to the device were secured to the surcingle buckles to stabilize the device (figure 2). the harness was positioned around the alpaca’s thorax and held in position with an elastic chest strap, as designed for use in a foal. the hembra was administered flunixin meglumine (1.1mg/kg, po) to reduce vaginal inflammation. in addition, ceftiofur (2.2mg/kg, sq, q24hr) was administered due to the chronic nature of the prolapse and concerns for potential bacterial contamination of the genitourinary tract. serial transabdominal ultrasonographic examinations were performed to monitor fetal wellbeing and the hembra was closely observed for signs of parturition. outcome the hembra was monitored for the next five days. the retention apparatus was well tolerated, the alpaca could easily stand, cush, urinate, and defecate. the second day that the device was in place, the vaginal prolapse reoccurred (figure 3) due to loosening of the surcingle and subsequent loosening of the tension on the retention device. the apparatus was assessed daily to ensure that the plastic device remained under tension and subsequently the prolapse did not reoccur. on day 363 of gestation, a live cria was spontaneously delivered vaginally without removal of the retention device. in spite of frequent observations, parturition was unattended. the retention device had passed out of the vagina, but was still attached to the harness. the fetal membranes were expelled 30 minutes after delivery of the cria. vaginal prolapse was observed on two occasions a few hours postpartum, but both incidents resolved spontaneously. physical examination revealed a normal 6.8 kg female cria. the hembra and cria were discharged the following day. there were no further reports of vaginal prolapse from the owner and the hembra has not been rebred. discussion vaginal prolapse is most commonly reported in pregnant camelids in late gestation.1 while the condition is known to occur in alpacas, the frequency is not known. the incidence has been reported to be as high as 15% in sheep.5 cervico-vaginal prolapse may occur, but in most cases the prolapse is limited to 3-5 cm of vaginal tissue.3 in cases where the vaginal prolapse resolves spontaneously, no treatment is necessary.4 inflammation and edema may increase the size of the prolapse, thus impairing spontaneous reduction.3 if the tissue is continually exposed, it may become traumatized resulting in necrosis of the vaginal tissue4 and increased risk of ascending infectious placentitis.3 while it has not been reported in alpacas, the urinary bladder can be found inside the prolapsed vaginal tissue of sheep and cattle.2,6 in one report, the urinary bladder was identified in 41% of sheep with vaginal prolapse that were examined via ultrasonosongraphy.7 in this case, ultrasonography was used to confirm the contents of the prolapsed tissue prior to correction. 490clinical theriogenology • volume 6, number 4 • december 2014 some authors recommend the use of epidural anesthesia prior to manual reduction.3,4 in this case, epidural anesthesia was not required for successful replacement. the prolapsed tissue should be thoroughly cleansed and examined for the presence of any necrosis. if necrotic tissue is identified, it should be debrided prior to reduction of the prolapse.4 while no necrotic tissue was visible in this case, inflammatory cells were found on the cytological examination of the vaginal exudate. despite ascending placentitis being a rare sequela to recurrent vaginal prolapse in camelids, the hembra was treated prophylactically with antimicrobials.8 in cases where the prolapse has become inflamed and edematous, suturing the vulva is often required following manual reduction.3,4 most authors suggest the use of the truss shoelace pattern as the method of choice for retention of vaginal prolapse in camelids.3,4 the procedure requires epidural or local anesthesia prior to placement of two or three loops of suture on either side of the vulva at the mucocutaneous junction lateral to the vulvar lip. umbilical tape is threaded through the loops of suture in a shoelace fashion. a disadvantage of this technique is that the umbilical tape must be removed prior to parturition requiring that the dam be closely monitored for signs of the first stage of labor.1,4 in most cases, prolapsed vaginal tissue arises from the ventral vaginal floor.2 the plastic retention device utilized in sheep is based on the principle that the broad spoon-shaped device places pressure on the ventral vagina floor to prevent the prolapse from reoccurring. in this case the authors elected to utilize this same retention device, theorizing that the hembra would be able to deliver the cria with the plastic device in situ, similar to ewes that are managed for the same condition.2 the plastic retention device was secured to a harness for stability. while serial ultrasonographic fetal assessments are not required for management of cases with vaginal prolapse, they were performed in this case. in late gestation, normal fetal heart rate (fhr) is between 80 and 120 bpm in alpacas.9 alterations in the normal fhr occur in response to activity level and external stimulus. periods of tachycardia or bradycardia are expected, but sustained fhr greater than 130 bpm or less than 70 bpm may be associated with fetal stress.3,10 it is important to note that the fhr may decrease to 80 bpm or less prior to parturition.3 at the time of admission the first fhr was 133 bpm which decreased to 112 bpm over a period of several minutes. over the next several days of hospitalization, the average fhr was 100 bpm. on the morning of delivery the initial fhr was 45 bpm. the bradycardia was a cause for concern and the fetus was balloted through the abdominal wall. subsequent measurements of fhr were 67 bpm and 88 bpm, suggestive of adequate fetal wellbeing and impending parturition. after delivery, the cria was found in the stall and the retention device was found hanging still attached to the harness worn by the alpaca. while the delivery was unattended, less than 20 minutes had elapsed between the last monitoring evaluation and when the cria was found. the second stage of labor in camelids is an expulsive event and similar to the mare, any delay in delivery may be life threating to the fetus. the majority of hembras will deliver within eight to twenty four minutes.4 there was no evidence to suggest that delivery was prolonged or that the retention device impeded passage of the cria. the authors speculate that the elastic nature of the harness allowed the hembra to pass the retention device as the cria was pushed into the vaginal vault. in this case, the apparatus did not need to be removed for partition to occur, unlike truss sutures. vaginal prolapse is not uncommon and in the majority of cases, on-farm management is instituted. use of the plastic prolapse retention device was a novel alternative and non-surgical approach to managing this condition in an alpaca. this case report demonstrates that vaginal prolapse in the late gestation alpaca may be managed successfully through parturition using a non-surgical retainer device. learning points • vaginal prolapse in a pregnant alpaca can be successfully maintained in reduction with an intravaginal retention device (ewe savers). • it is important to maintain the device under adequate tension to ensure that the prolapse does not reoccur. • late gestation ultrasonographic monitoring is a valuable method to monitor fetal wellbeing. 491 clinical theriogenology • volume 6, number 4 • december 2014 references 1. tibary a: surgery of the reproductive tract in lamoids. in: youngquist rs, threllfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st louis: wb saunders; 2007. p. 905-918. 2. mobini s, heath am, pugh dg: theriogenology of sheep and goats. in: pugh dg, editor. sheep and goat medicine. philadelphia: wb saunders; 2001. p. 166-167. 3. tibary a, rodriguez j, sandoval s: reproductive emergencies in camelids. theriogenology 2008;70:515-534. 4. fowler me: surgery. in: medicine and surgery of camelids. 3rd ed. ames (ia): wiley-blackwell; 2010. p 154-156. 5. low jc, sutherland hk: a census of the prevalence of vaginal prolapse in sheep flocks in the borders region of scotland. vet rec 1987;120:571-575. 6. hopper rm: surgical correction of abnormalities of genital organs of cows. in: youngquist rs, threllfall wr, editors. current therapy in large animal theriogenology. 2nd ed. st louis: wb saunders; 2007. p. 467-468. 7. scott pr, gessert me: ultrasonographic examination of 12 ovine vaginal prolapses. vet rec 1998;155:323-324 8. tibary a, fite c, annouassi a, sghiri a: infectious casuses of reproductive loss in camelids. theriogenology 2006;66:633-647. 9. anderson de: assessment of fetal well-being in the camelid. the ohio state university college of veterinary medicine meeting on camelid medicine, surgery and reproduction 2000;312-316. 10. jonker fh. fetal death: comparative aspects in large domestic animals. anim reprod sci 2004;82-83:415-430. figure 1. vaginal prolapse in a seven-year-old multiparous pregnant alpaca at 358 days of gestation. 492clinical theriogenology • volume 6, number 4 • december 2014 figure 2. plastic prolapse retention device (ewe savers) in situ secured to harness. vaginal prolapse is held in a reduced state. figure 3. vaginal prolapse recurrence with the retention device in place when adequate tension was not maintained. (editor’s note: the photographs in this manuscript are available in color in the online edition of clinical theriogenology.) 493 clinical theriogenology • volume 6, number 4 • december 2014 494clinical theriogenology • volume 6, number 4 • december 2014 omniblank: 2010: guidelines for using the bull breeding soundness evaluation form* guidelines for using the bull breeding soundness evaluation form* p.j. chenoweth,a f.m. hopkins,b j.c. spitzer,c r.e. larsend aschool of animal and veterinary sciences, charles sturt university, waga waga, nsw, australia; bcollege of veterinary medicine, university of tennessee, knoxville, tn, usa; cspitzer ranch, fair play, sc, usa; dschool of veterinary medicine, st. george’s university, grenada, west indies introduction the breeding soundness evaluation (bse) is a relatively quick and economic procedure for screening bulls prior to sale or use. it is also a useful tool in infertility investigations. proven benefits from its application include direct effects on herd fertility and indirect effects via genetic relationships with other fertility traits in both the male and female. although the bse classifies bulls into categories which generally perform as predicted, the examination is limited in its ability to consistently predict individual bull fertility. reasons for this include: 1. fertility is complex and influenced by both male and female traits as well as by extraneous factors. 2. the bse is a relatively quick and simple screening procedure which does not attempt to comprehensively assess all aspects of male fertility 3. both knowledge and understanding keep increasing and changing. keywords: breeding soundness evaluation, bull, semen quality, physical examination general procedures the primary mission of the bull is to efficiently impregnate all available females as early in the breeding season as possible. for this he needs good eyesight and musculo-skeletal conformation as well as the necessary reproductive equipment and sex drive to produce and deliver sufficient numbers of fertile spermatozoa when and where necessary. the bse consists of the following steps: 1. physical examination 2. reproductive examination (including scrotal circumference measurement) 3. collection and examination of semen in addition, a libido/mating ability test may be included, as may special tests for diseases (e.g., campylobacterosis or trichomoniasis). these procedures may add predictive value to the assessment process and may be specifically indicated in some situations, but they are not generally part of the routine bse. identification and ownership of bulls permanent and unique identification of each bull is important as well as the establishment of ownership. the form gives provision for breed, age, identification numbers (including brands, tattoos and eartags) and previous history. it is good procedure to identify all samples, slides and work-sheets with the bull identification number. this section includes owner information as well as case number and details of any previous tests. body condition and other body measurements body condition is relevant to reproductive assessment. excessively fat or thin bulls can have semen, libido and mating ability problems. poor body condition may also reflect health problems apart * originally published by the society for theriogenology and the american college of theriogenologists as publication b-10 (7/93) in the theriogenology handbook. 43 from poor nutrition. provision is made for bulls to be categorized into general categories (thin, moderate, good, obese) or for scoring systems to be applied as developed for either beef (1-9) or dairy (1-5) cattle. pelvic size in bulls is related to pelvic size in their daughters, an important consideration in dystocia management. pelvic size measures (height, width, area) can be recorded on the form. the height and width are usually measured by manual insertion of a pelvimeter and multiplied together to give the approximate area. while no specific slot was provided for bull weight, this measure can be inserted in any of the available blank areas on the form. structure, conformation and movement the bull should be observed moving free of restraint in order to observe gait and movement. once in the chute, closer attention can be paid to the limbs, joints and claws. the bull should have good, symmetrical musculo-skeletal conformation and he should walk smoothly. asymetrical or overgrown claws as well as swellings over the lower limb joints are common abnormalities. nb: bull examinations should be undertaken with due regard for the safety of both the examiner and the bull. physical and reproductive examination a thorough physical examination is necessary since the bull must be able to see, eat and move about without discomfort to be a functional breeder. a systematic approach should be employed which emphasizes those aspects most important for breeding success. the bull’s eyes should be examined for impaired vision. common problems here include corneal defects, infectious keratoconjuctivitis (“pink eye”), scarring, dermoids and squamous cell carcinoma. examination of the bull’s teeth allows the examiner to verify age and ensure that dentition is adequate for foraging. the bull’s feet are a common source of problems. overgrown claws in need of trimming, malformed claws and interdigital fibromas are commonly identified problems. the sheath of the bull should be inspected and palpated. problems here might include inflammatory exudates, trauma, fibrosis, abscessation and scaring. the penis, palpable within the sheath, should be symmetrical, freely moveable, and have no abnormal masses associated with it. common problems include abscesses, adhesions, fibropapillomas and hematomas. scrotal conformation should be evaluated. significant differences in the size and shape of either testicle should be cause for further evaluation; a disparity of more than 25% in either testicle should be regarded with suspicion. the most common cause of marked scrotal asymmetry is unilateral testicular hypoplasia although a variety of congenital, traumatic or infections conditions may be implicated. testicles should be palpated for hardness and resiliency; a normal testicle should be firm and resilient (similar to a new tennis ball). the epididymides should be palpated. problems here include inflammation, fibrosis, abscessation, hypoplasia or aplasia. to complete the examination of the external genitalia, the penis of the bull should be exteriorized. commonly encountered problems include a persistent penile frenulum, hair rings, fibropapillomas, infections, trauma and scaring. per-rectal examination of the bull is an essential step so that pelvic structures, particularly the ampullae and vesicular glands, can be evaluated. infections, inflammation and adhesions can occur in both areas as can aplasia/hypoplasia. scrotal circumference scrotal circumference (sc) in bulls is related to sperm production, sperm quality and the age at puberty of related females. it is also fairly highly heritable, at least in beef bulls. the measurement of sc is simple and highly repeatable and does not require expensive or highly technical equipment to perform. for these reasons, it is an important component of the bse. 44 to measure sc, a tape is applied snugly around the greatest circumference of both testicles which should be pushed to the bottom of the scrotum. a good procedure is to take two measurements to ensure accuracy, with two consecutive measurements not varying by more than 1.0 cm. scrotal circumference thresholds minimum recommended scrotal circumference semen collection semen can be collected from bulls by a variety of means including per-rectal massage, the use of an artificial vagina and by electroejaculation. the latter method is the one most commonly employed with range-type bulls. electroejaculators and probes commercially available electroejaculators are available with power being provided by ac current, by internal re-chargeable batteries, or by 12 volt automobile batteries. electroejaculation requires stimulation of pelvic nerves controlling not only emission of semen into the penile urethra but also those controlling erection and ejaculation. newer probe designs have full-length longitudinal electrodes which stimulate all functions simultaneously. preparation and stimulation the bull’s rectum should be emptied of feces before the probe is inserted. the lubricated probe is inserted so the anal sphincter closes behind the main body of the unit. it is helpful to determine the lowest current level at which the animal first shows an obvious physical response. this initial response may be subtle, e.g., a twitch of the tail, a tightening of the anal sphincter, or a tensing of the gluetal muscles. this stimulus “threshold” provides a starting point for subsequent stimulations which should be conducted with a smooth routine to which the bull can easily adapt. with bos taurus breeds, a typical approach is to deliver a smooth increase in probe current from zero to the desired level over a duration of one to two seconds, followed by a more rapid reduction to zero current and a rest period of approximately one second before the next stimulation. once the bull is settled into the routine, five to seven stimulations are given at each succeeding voltage step until erection and ejaculation occur. for machines that do not have separate voltage and current controls, the same stimulation pattern is employed except that the single control is used to generate incremental increases in probe voltage and current until ejaculation occurs. during the early stages of stimulation and erection, clear seminal plasma is often passed which is not generally collected. when the ejaculate turns cloudy, the subsequent jets of semen are collected. it is important to continue stimulation until the ejaculate starts age (months) sc (cm) ≤15 30 ≥15 ≤18 31 ≥18 ≤21 32 ≥21 ≤24 33 >24 34 45 to become clear again. failure to proceed to this point can lead to errors of interpretation in the spermiogram as the initial portion of the ejaculate may contain large numbers of degenerating spermatozoa, especially in bulls which have been sexually quiescent for some time. for the same reason, if an ejaculate shows substandard motility in the absence of an obvious physical cause, the collection of a second sample within a short period of time (e.g. five to ten minutes) can often result in improvement. with bos indicus bulls this stimulation routine is often unsuccessful in eliciting both erection and ejaculation. a more gentle stimulation pattern incorporating longer rest periods can sometimes improve ejaculation success; however this still may not be accompanied by erection so that semen is voided through the preputial lumen. the voiding of semen within the prepuce is a problem as it can increase semen contamination with preputial debris. success in exteriorizing the penis during electroejaculation can sometimes be obtained by pushing on the sigmoid flexure with a clenched fist from the rear of the bull; this technique can also be successful with young bulls. inability to exteriorize the penis means that this organ cannot be properly examined. unless the penis and inner prepuce are extended and examined, the bull cannot be properly classified. variations on these stimulus patters occur with different machines and operators. with difficult bulls some experimentation might well be necessary. in all situations, however, the welfare of the animal is paramount and stimulation should be discontinued if either undue stress is being caused or physical injury to the bull might occur. collection devices semen is collected into a prewarmed insulated or jacketed tube through a funnel or cone. all surfaces coming into contact with semen should be warm, dry, and free of spermatoxic agents. because “cold shock” causes irreversible damage to spermatozoa, efforts to maintain semen at 30-35 °c until the “on-site” evaluation procedures are complete is an important consideration for successful semen assessment. semen evaluation initial impressions volume, density, and gross characteristics of the ejaculate are not “front-line” bse assessments because they have not been shown to be related to fertility. space on the score sheet is, however, provided for recording such information. likewise, the assessment of spermatozoa concentration is not a routine part of the bse; measurement of sc provides a better estimate of sperm production in range-type bulls which are subject to infrequent examinations. however, informal recording of such information may help to monitor the success of semen collection and to interpret gross motility estimation. other gross characteristics which may be noted include evidence of contamination, hemorrhage or inflammatory material. if the ejaculate contains sufficient purulent material for this to be obvious to the naked eye, then the bull should not be classified as satisfactory at least until a benign cause is found. debris or contamination from the sheath may be regarded as being less serous unless it represents active infection. motility motility should be assessed microscopically. two methods of assessing sperm motility are traditionally employed: gross motility (or mass activity) and individual motility (or percent progressive motility). it is good procedure to use both methods as they can differ somewhat in interpretation and precision. with all motility estimates, it is important to protect semen against adverse effects (e.g. cold shock) and do the estimation as soon as possible after semen collection. gross motility, or the amount of swirling (or wave motion) present in an undiluted semen sample, is a function of both sperm concentration and individual motility. under field conditions, gross motility is typically assessed by placing a drop of raw semen on a warmed slide and observing it at 100 46 magnifications (10x eyepiece and 10x objective). with the condenser properly adjusted, mass action or “swirl” can be observed in samples which have adequate numbers of motile spermatozoa. the rankings for this estimate are as follows: mass activity (gross motility) rating rapid swirling very good (vg) slower swirling good (g) generalized oscillation fair (f) sporadic oscillation poor (p) the minimum recommended threshold for gross motility is fair (f) individual progressive motility of spermatozoa is assessed under a brightfield or phase contrast microscope preferably equipped with a warm stage or other means of preventing cold shock of spermatozoa. coverslipped specimens are usually examined at a total magnification of 400x. in dense samples (milky or creamy), the sample should be diluted for proper observation of individual spermatozoa. sodium citrate or skim milk based semen extenders are serviceable diluents; physiological saline (pss) may be used although readings should not be delayed when it is used. the percentage of active, progressively motile cells is estimated. this procedure takes more practice than does the gross motility estimation, but is probably more accurate in experienced hands. individual motility ratings are as follows: percent progressive motility rating ≥70% very good (vg) 50-69% good (g) 30-49% fair (f) ≤30% poor (p) the minimum recommended threshold for individual motility is 30% observation of the semen sample at 400x can also help to identify the presence of abnormal numbers of other cells (e.g. squamous epithelial cells, inflammatory cells or spheroids) within the sample. the identification of aberrant cellular material can benefit from the staining of the semen smear with modified wright giemsa stain, new methylene blue, or other differential blood cell stain, while bacteria are best categorized using a gram stain. morphology of spermatozoa morphology of spermatozoa (differential counts of normal and abnormal cells) is assessed either by phase contrast microscopy (by using preparations “fixed” in formol-buffered saline or pssglutaraldehyde) or by using brightfield microscopy of stained smears. common stains used for this purpose include nigrosin-eosin, william’s stain, modified giemsa, and even india ink. the society for theriogenology recommends nigrosin-eosin for its combination of ease and utility. although this stain is a “supra-vital” stain (i.e., sperm which are “alive” at staining will not absorb the stain while those that are “dead” will partially or completely absorb the red eosin color), here it used for its ability to depict sperm morphology only. with nigrosin-eosin staining of spermatozoa, the most common method is to mix a fraction of a drop of semen with a drop of background or “negative” stain and spread the mixture over the surface of a 47 glass slide which is allowed to air-dry. care should be taken during the smearing process to avoid trauma to sperm. it is also helpful to vary the thickness of the smear to provide a variety of background densities to the stain from which an area can be picked for best microscopic examination. brightfield microscopy of stained smears is best done at 1,000x with an oil immersion lens. at least 100 spermatozoa should be observed in different fields and classified for normality or abnormality. normal sperm should be at least 70% of those counted for the bull to pass the bse (see below). in previous bse systems, sperm abnormalities were classified as being either “primary” or “secondary” with the underlying assumptions being that primary abnormals (considered to be caused during spermatogenesis) were more serious than secondaries (caused subsequent to sperm released into the extragonadal system). more recent knowledge has cast doubt upon these assumptions. in the meantime a system of using “major” and “minor” abnormalities was created to more accurately reflect sperm abnormalities for which fertility data were available. it was apparent that the lists of sperm abnormalities in routine use for both systems were essentially indistinguishable (except perhaps for proximal cytoplasmic droplets). thus, as the primary/secondary scheme is widely used at present, this was retained as the reference point in the present system (see appendix 1 for categories of abnormalities). although total abnormalities only are employed as the threshold in the new bse system, “primary” and “secondary” abnormalities can be collated to arrive at this number. the recording of specific abnormalities (or their category as primary or secondary) can also be useful for the monitoring of bulls and their progress. the minimum recommended threshold for sperm morphology is 70% normal spermatozoa evaluation categories satisfactory bulls which equal or surpass the minimum thresholds for sc, sperm motility and sperm morphology, and which do not show genetic, infectious or other problems or faults which could compromise breeding or fertility. unsatisfactory bulls which are below one or more thresholds and which are unlikely to ever improve their status. also, bulls which show genetic faults or irrevocable physical problems (including infectious disease) which would compromise breeding or fertility are included. classification deferred any bull which does not fit into the above categories and which could benefit from a retest. provision is provided for the scheduling of a retest and this is recommended. this category would include bulls with an “immature” semen profile† as well as any bull whose semen is substandard but considered to be capable of improvement. also in this category are bulls from which a satisfactory ejaculate could not be obtained for reasons unknown as well as bulls with treatable problems such as seminal vesiculitis or footrot. in general, if any doubt exists about a bull fitting into either the satisfactory or unsatisfactory categories, he should be considered as a candidate for a retest and placed into the “classification deferred” category. veterinarians are encouraged to work with their clients to accept higher standards for bulls than the “minimum acceptable” standards employed in this bse system † it is not uncommon for yearling bulls to exhibit higher levels of certain types of spermatozoal abnormalities which are associated with immaturity. such bulls will usually require a second examination before being classified as a satisfactory potential breeder. 48 selected references 1. ball l, ott rs, motimer rg, et al: manual for breeding soundness examination of bulls. j soc therio, xii; 1983. 2. barth al, oko rj: abnormal morphology of bovine spermatozoa. ames: iowa state university press, 1989. 3. bartlett de: theriogenology: from concept to reality. proc soc for therio 1984; p. 221-234. 4. bierschwal cj: revised breeding soundness procedures. proc soc for therio1976; p. 128-130. 5. blom e: the ultrastructure of some characteristic sperm defects and a proposal for a new classification of the bull spermiogram. simposio int di zootechnicia; milan; 1972. p. 125-139. 6. carroll ej, ball l, scott ja: breeding soundness in bulls-a summary of 10,940 examinations. j am vet med assoc1963;142;1105-1111. 7. chenoweth pj: examination of bulls for libido and breeding ability. vet clin north america food anim pract 1983;5:59-74. 8. chenoweth pj, ball l: breeding soundness evaluation in bulls. in: morrow da, editor. current therapy in theriogenology; philadelphia: wb saunders; 1980. p. 330-339. 9. lagerlof n: changes in the spermatozoa and in the testes of bulls with impaired or enhanced fertility. acta path microbiol scand 1934;suppl 19;254. 10. larsen re, chenoweth pj: diadem/crater defects in spermatozoa from two related angus bulls. mol repro dev 1990;25:87-96. 11. saacke rg, dejarnette jm, nebel rl, et al: assessing bull fertility. proc soc for therio 1991; p. 56-69. 12. spitzer jc: influences of nutrition on reproduction in beef cattle. in: morrow da, editor, current therapy in theriogenology. 2nd ed. philadelphia: wb saunders; 1986. p. 320-341. 13. spitzer jc, hopkins fm, webster hw, et al: breeding soundness examination in yearling beef bulls. j am vet med assoc 1988;193:1075-1079. 14. volger cj: effects of elevated testicular temperature on viability of cryopreserved semen and morphological characteristics of ejaculated spermatozoa [thesis]. blacksburg (va): virginia polytechnic institute; 1990. 15. wildman ee, jones gm, wagner pe, et al: a dairy body condition scoring system and its relationship to selected reproductive characteristics. j dairy sci 1982;65:495-501. 16. wiltbank jn, parish n: evaluation of bulls for potential fertility. proc soc for therio 1982; p. 141-154. appendix 1 categories of sperm abnormalities primary abnormalities secondary abnormalities underdeveloped small normal heads double forms giant and short broad heads acrosome defects (e.g. knobbed) free normal heads crater-diadem defect detached, folded, loose acrosome membranes pear-shape head abaxial midpiece abnormal head contour distal droplet small and free abnormal heads simple bent tail proximal droplet terminal coiled tail double bent and coiled tail accessory tail 49 appendix 2 bull breeding soundness evaluation form available to members of the society for theriogenology, p.o. box 3007, montgomery, al 36109. 50 2012: causes and cures for repeated early pregnancy failure in the mare causes and cures for repeated early pregnancy failure in the mare twink allen the paul mellon laboratory of equine reproduction, ‘brunswick’, newmarket, suffolk, uk abstract some 14% of thoroughbred mares lose their pregnancy between initial diagnosis at 12-14 days post-ovulation and term and around 60% of these losses occur during the first 42 days of gestation, with a further 25% between days 42 and 150. discounting infective endometritis as a possible cause of this high early stage loss (pathogenic bacteria or fungi would usually kill the embryo well before initial diagnosis of pregnancy at day 12), and from karyotyping studies in other species, notably women and pigs, it may be reasonably assumed that as many as 50-60% of the early losses stem from chromosomal aberrations occurring at fertilization. they are a matter of ‘good riddance to bad rubbish’ at a sufficiently early stage to generate a second normal pregnancy and they are often seen ultrasonographically as an anembryonic conceptus that simply ‘fades away’ between 20 and 30 days after ovulation. repeated failure to conceive in the face of normal ovulation and an absence of uterine/cervical pathology may be caused by accumulations of spirals of inspisated follicular fluid at the ampullary-isthmus constriction in both oviducts which inhibit the downward passage of the embryo to the uterus. the condition is successfully overcome by laparoscopic application of a human preparation of prostaglandin e2 gel to both oviducts but the question remains as to how many mares in the population have one blocked oviduct thereby reducing their fecundity rather than causing full infertility. as demonstrated convincingly by the late cliff irvine in new zealand, progesterone deficiency is not a significant cause of early pregnancy loss in the mare in spite of the huge quantities of altrenogest and other progestagens shovelled into mares during this period. on the contrary, exogenous progestagen administration is likely to downregulate luteal function and, thereby, endogenous progesterone production, especially if given prior to the luteotrophic support provided by the commencing secretion of equine chorionic gonadotropin (ecg) around day 38-40. likewise, an injection of human chorionic gonadotrophin (hcg) given prior to ecg secretion will also induce luteolysis and pregnancy loss. the horse conceptus must imbibe appreciable amounts of endometrial gland secretions (histotroph) if it is to survive during its first 40 days of ‘free living’ in the mare’s uterus. and even after microvillous attachment and allantochorion-endometrium interdigitation commences around day 40, histotroph imbibition via the highly absorptive areolae situated between the microcotyledons on the allantochorion remains a vital source of nutrients for the growing fetus. hence, severe deprivation of histotroph uptake, such as occurs frequently in the misaligned member of unicornuate twin conceptuses, or as a result of significant scarring of, or excessive cyst formation in, the endometrium at the base of the uterine horn where the conceptus will initially become ‘fixed’ and try to implant, can be lethal to it as a consequence of the resultant starvation. the precise role of the considerable quantities of estrogen secreted during early pregnancy, initially by the conceptus itself up to day 40, and by the primary and secondary corpora lutea (cl) thereafter in response to the luteotrophic stimulus of commencing ecg secretion, has not been determined. it is likely to play an important, and perhaps vital, part in stimulating histotroph secretion, especially in those glands in direct contact with the expanding trophoblast, and to act in concert with vascular endothelial growth factor (vegf) to develop the extensive capillary networks required on both the maternal and fetal sides of the placental interface. whether any deficiency in estrogen production may occur during early pregnancy, from the conceptus membranes or the accessory cl, perhaps caused by a lack of steroid precursor availability occasioned by restricted dietary intake, has yet to be determined. finally, it is clearly important that the horse embryo signals its presence correctly to the maternal organism early in gestation to gain immunological acceptance in the uterus. as in women who suffer repeated early pregnancy failure, active immunization of mares that do likewise against lymphocytes extracted from the peripheral blood of the sire of the conceptus results in live births from a significant proportion of the treated animals. the mechanism of action and true value of such therapy is still hotly debated in human gynaecology circles but it is clear that it can protect the immunologically threatened conceptus in some persistently aborting mares. 563 clinical theriogenology • volume 4, number 4 • december 2012 564clinical theriogenology • volume 4, number 4 • december 2012 omniblank: 67 contact lindsey vansandt lindsey.vansandt@cincinnatizoo.org © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11649, http://dx.doi.org/10.58292/ct.v17.11649 review report estrous cycle manipulation in cats julie barnes, lindsey vansandt center for conservation and research of endangered wildlife, cincinnati zoo and botanical garden, cincinnati, oh, usa abstract for decades, interest in reproductive physiology of the domestic cat has largely been driven by its importance as a model for wild felids and human biomedical research. as such, several assisted reproductive technologies have been established in cats. despite the growing need for feline theriogenology, the application of these tools in clinical practice is extremely limited. we discuss: 1. reproductive physiology of the queen and her unique challenges; 2. estrus induction (photoperiod, social interaction, and pharmacologic [gonadotropins, gnrh agonists]); 3. natural cycle monitoring (blood hormones, fecal hormone metabolites, behavior, vaginal cytology, transabdominal ultrasonography) and ovulation induction (manual stimulation, pharmacologic [gonadotropins, gnrh agonists]); 4. estrus suppression (photoperiod, melatonin, gnrh agonists, progestins); and 5. permanent nonsurgical contraception (immunocontraception, gene therapy). this review will summarize published reports on estrous cycle manipulation in felids, both wild and domestic; notable differences between cats and dogs are highlighted and comments based on the authors’ personal experiences and preferences for application are included. keywords: felids, estrous cycle, estrus induction, ovulation induction, natural cycle, estrus suppression, contraception introduction in veterinary medical research, the number of publications featuring dogs outnumbers cats ~ 3:1.1 cats also remain understudied in theriogenology.2 perhaps, in part, because the domestic cat is viewed as an extremely fecund species. when one considers the estimated 80 million unowned, outdoor cats that live in the usa,3 it may appear incongruous to focus on assisted reproduction in cats. however, there is a growing body of literature that demonstrates that infertility is a major issue in cats. early embryonic collections following natural matings in domestic short hairs produced good-quality embryos only from ~ 73% (38/52) of queens;4 the remaining cats either failed to ovulate (~ 8%) or had degenerating embryos (~ 8%), unfertilized oocytes (~ 10%), or no oocytes/embryos (~ 2%).4 most data in purebred cats are derived from case studies5,6 and self-reported questionnaires,7-10 with 15-42% of queens failing to conceive after natural mating. despite an empiric lack of interest in feline theriogenology, the basic reproductive biology of the cat has been well-studied. this was largely driven by its importance as a model for wild cats.11 it is speculated that modern felids originated from a common ancestor ~ 11 million years ago and as such, their reproductive physiology has remained well-conserved across cat species.12 the domestic cat has also proven to be an important model organism for biomedical research. the genomic organization of the cat is highly similar to humans13 and cats possess ~ 250 naturally-occurring genetic disorders with analogous pathologies to human diseases.14 assisted reproductive technologies (arts), such as in vitro fertilization (ivf), embryo transfer (et), and artificial insemination (ai), have therefore been developed in the domestic cat to aid in the conservation of wild felids,15 to propagate naturally occurring genetic disease models,16 and to produce genetically modified animals.17 although many arts are well-established in the cat, these tools are rarely utilized in general practice (especially compared to dogs) and, for most veterinarians, their clinical experience with cat reproduction begins and ends with neutering. with both the number and professionalism of cat breeders rising, there is an increased (and as of yet unmet) demand for the application of domestic cat art in veterinary medicine. this review considers: 1. the reproductive physiology of the queen and her unique challenges; 2. estrus induction; 3. natural cycle ovulation induction; 4. estrus suppression; and 5. permanent nonsurgical sterilization in the queen. mailto:lindsey.vansandt@cincinnatizoo.org http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11649 68 citation: clinical theriogenology 2025, 17, 11649, http://dx.doi.org/10.58292/ct.v17.11649 reproductive physiology of the queen the queen is seasonally polyestrous; the breeding season begins when daylight length increases and anestrus occurs after a reduction in daylight hours.18,19 cats in equatorial zones may breed continuously, whereas cats at the polar circles only cycle for ~ 6 months.20 in the northern hemisphere, the breeding season usually begins around february and ends by september.21 temperature also has a role, as queens in warmer climates may continue to cycle as late as november before entering anestrus.22 furthermore, periods of extreme heat and/ or humidity can increase the interestrus interval.23 the breed also influences photoperiod sensitivity as long-haired breeds tend to have a shorter, more-defined breeding season than short-haired breeds.24 for cats housed exclusively indoors without exposure to natural light, alterations in the daily photoperiod can be utilized to manipulate the queen’s estrous cycle.25-27 different protocols are reviewed in photoperiod. the queen is categorized as an induced ovulator,28 with the act of copulation serving as the canonical stimulus for release of gonadotropin releasing hormone (gnrh) and the subsequent surge of luteinizing hormone (lh) from the anterior pituitary gland. amplitude of the lh surge is directly correlated with the number of copulations; 8-12 copulations over a 4-hour period produced peak concentrations.29 lh concentrations were significantly lower with only 4 copulations over the same interval, and were even lower with a single breeding. in the aforementioned study, 50% of estrual queens ovulated after 1 copulation, whereas every queen ovulated after > 4 copulations. ovulation was determined by the amount of lh released, not by differences in the follicles’ responses to similar lh concentrations.30 furthermore, ovulation appears to be an all-or-none event, as the number of mature follicles present during the preovulatory period correspond to the number of corpora lutea after mating in the queen. because the queen requires an external stimulus to ovulate, one must consider ovulation induction when designing estrus manipulation protocols without natural mating (e.g. ai and et). it should be easy to manipulate a queen’s estrous cycle because, in the absence of mating, the queen would ostensibly lack corpora lutea and not require luteal control protocols. however, spontaneous ovulation is a well-documented phenomenon in cats.31-35 reports vary greatly in the percentage of females that spontaneously ovulate (35-87%). the rate of each queen also varies widely among reports; some females rarely spontaneously ovulate, whereas others may consistently ovulate without copulatory stimuli. interestingly, wild felids also demonstrate a spectrum of ovulation patterns, with some being almost exclusively induced-ovulators and others exhibiting a high rate of spontaneous ovulation.36,37 to date, felids are the only taxon reported to exhibit both spontaneous ovulation in some individuals and exclusively induced ovulation in others.38 in equids and ruminants, prostaglandins are often employed during a luteal phase to regress the mature corpus luteum (cl) and return the female to estrus within a predictable interval.39 unfortunately, the feline cl is refractory to prostaglandin f2α treatment.40,41 gnrh antagonists that induce luteolysis in dogs42 are also ineffective in cats.43 dopamine agonist cabergoline has been successfully used with41,44 or without41,45,46 a prostaglandin to induce abortion in cats. cabergoline regresses the cl via its inhibitory effect on prolactin secretion. the use of cabergoline in estrus induction protocols has not been reported, likely due to its long treatment period (5-15 days) and undefined interval for cl regression. although aglepristone has also been used to successfully induce abortion in queens, it is important to note that the mechanism of action differs; aglepristone is a progesterone receptor antagonist and cannot regress the feline cl.47 estrus induction there are a variety of indications for estrus induction in queens. as a long-day breeder, cats have a period of anestrus during short days and estrus manipulation is required if out of season breeding is desired. cats can experience primary anestrus (delayed puberty) or secondary anestrus (abnormally long interestrus intervals in an adult queen that previously displayed cyclicity).48 management practices (e.g. photoperiod, social interaction) should be reviewed before initiating pharmacological intervention and prepubertal queens should not be induced with exogenous hormones because they are more likely to develop a high number of cystic follicles.48 spontaneous ovulation should be ruled out before diagnosing secondary anestrus. finally, estrus induction is often utilized for cycle synchronization in arts, such as ai, collection of in vivo-matured oocytes for ivf, and recipient-preparation for et. photoperiod as discussed in the reproductive physiology of the queen, the cat is a long-day seasonal breeder and is highly responsive to changes in photoperiod. increasing exposure to light thus represents one of the easiest and most efficient techniques to induce cyclicity. a light:dark cycle of 8:16 hours was sufficient to cease cyclicity immediately;26 plasma estradiol concentrations were dramatically reduced, with concentrations significantly lower than interestrus concentrations in females exposed to 14 hours of light per day. when females were again exposed to 14 hours of light, cyclicity resumed 12-26 days later (mean 16.3 days). a much longer anestrus was observed in a different study27 where queens took an average of 44.6 days for estrus after returning to 14 hours of light exposure. resumption of estrus occurred significantly sooner (mean 15.6 days) by providing 1 hour of light during the dark period. a light:dark cycle of 12:12 hours (similar to equatorial conditions) created the most productive year-round cyclicity (as defined by percent of successful breedings per week). 20 the same study reported that if a shortened and more-defined breeding season is desired, 2 months at 9:15 hours light:dark followed by 14:10 hours light:dark significantly increased the number of litters born 6 months after the change to 14 hours of light. similarly, if a queen has been maintained under constant artificial lighting conditions for years, reducing the light exposure to 8 hours per day for 2-3 months will allow her to experience a period of anestrus and may improve fertility.49 cats exposed to 24 hours of light will still demonstrate cyclicity, but the rate of estrus is decreased (only a mean 0.8 periods of estrus were observed the first month, and ~ 1 estrus/month was observed the following 2 months).26 plasma estradiol concentrations demonstrated prolonged periods of proestrus (up to 7 days), suggesting initial follicular development was temporarily suppressed. thus, it is not the authors’ recommendation to maintain queens under constant light exposure. http://dx.doi.org/10.58292/ct.v17.11649 citation: clinical theriogenology 2025, 17, 11649, http://dx.doi.org/10.58292/ct.v17.11649 69 finally, cats maintained in a home setting are often exposed to both natural and artificial light. because the artificial lighting in a home setting is not constant, it may not result in predictable ovarian cycles. anecdotally, most intact home-housed cats do not cycle during the short daylight period. social interactions interactions with conspecifics have the potential to influence the estrous cycle of the queen. in the aforementioned photoperiod study,27 queens that returned to long-day light conditions (14 hours) after a period of short-day light exposure (8 hours) took an average 44.6 days to exhibit estrus.27 introduction of other queens in estrus at the change of the photoperiod allowed the queens to resume cyclicity significantly sooner (22.3 days), likely through the influence of estradiol and pheromones.50,51 similarly, it is also advised to house prepubertal females with cycling queens to help stimulate the onset of puberty.51 conversely, inter-female aggression has been cited as a cause for females to not show overt signs of estrus, particularly for timid cats that are lower in the social hierarchy.48 exposure to an intact male has also been documented to affect cyclicity in cats. similar to exposure to an estrual female at the onset of long-day light conditions, introduction of a male shortened the interval to cyclicity resumption, with the same number of days (22.3) until the first estrus was observed.27 the presence of a male can help accentuate signs of estrus51 and even the noncopulatory presence of a tom can increase the rate of spontaneous ovulation.35 pharmacologic estrus induction the goal of a pharmacologic estrus induction is to recapitulate the natural cascade of the hypothalamic-pituitary-ovarian axis, so drugs that mimic the actions of gnrh, follicle stimulating hormone (fsh), and/or lh are utilized. initial studies to induce folliculogenesis in the cat focused on serial injections of porcine-derived fsh. five daily injections were successful in initiating follicular development; however, ovarian hyperstimulation was observed52-54 and the authors reported logistical challenges in giving multiple injections, particularly when this protocol was used in wild felids.55-58 subsequent studies have focused on equine chorionic gonadotropin (ecg) which is longer acting and only requires a single injection.59,60 in cats, ecg is primary used for its folliculogenic activity; however, high dosages or serial treatment can induce ovulation.53 more commonly, human chorionic gonadotropin (hcg) is used as the luteotrophic agent to induce ovulation in feline ovarian stimulation protocols.61,62 however, hcg also demonstrated folliculogenic activity in the cat, including the capacity to stimulate growth and maturation of smaller (< 2 mm) antral follicles.60 thus, although ecg is predominantly folliculogenic and hcg luteotrophic, each exhibits duality in the cat and can mimic the other’s principle action. historically, the most common protocol to induce estrus and ovulation prior to a timed art procedure was to give 100 iu intramuscular ecg, followed by 75 iu intramuscular hcg 80-85 hours later.63 the timing of the procedure depends on which art is employed. queens will ovulate ~ 30 hours after hcg treatment,61 so, oocytes are collected for ivf 25-27 hours (i.e. preovulatory) after hcg treatment63 and ais are conducted 31-33 hours (i.e. postovulatory) after hcg treatment.15,59 one drawback with ecg/hcg is that both are large glycoproteins that persist in circulation for 4-5 days in cats and can induce formation of antigonadotropin antibodies, causing the female to become refractory to future treatment.60,64,65 additionally, due to its folliculogenic activity in cats, hcg can promote undesirable secondary follicular growth and ovulations.66 these ancillary follicles and secondary cls disrupt the postovulatory endocrine environment and potentially have a negative impact on embryo survival following ai or et.67 alternatively, plh has a very short half-life and remains in circulation for just hours after injection.68 a protocol using 100 iu intramuscular ecg and 1,000 iu intramuscular plh with an 85 hour interval between treatments was highly effective for inducing ovulation in et recipients without significant formation of secondary ovarian structures,69 and producing high pregnancy percentages with both et and ai procedures in cats.15 to the authors’ knowledge, there are only 2 sources of plh in the usa (novatein biosciences and prospec bio) and the current price per cat dose ($331-$475) make it cost-prohibitive in most applications.70 anesthesia prior to ovulation may have a detrimental effect on ovulation. queens treated with ecg and hcg that were anesthetized immediately before ovulation demonstrated a low rate of ovulation and a reduced pregnancy rate (14%) following intrauterine ai, compared to ecg/hcg-treated females that were anesthetized immediately after ovulation (50% pregnancy rate).61 alternatively, ovulation-induction with hcg on days 2-4 of a natural cycle demonstrated a higher level of success in the queens anesthetized for ai prior to ovulation (56%) versus queens anesthetized after ovulation (21%).71 thus, the compromising effect of anesthesia on ovulation may be limited to queens exogenously treated for estrus induction. for gonadotropin treatment to be maximally effective in cats, a quiescent ovary at the time of treatment is needed.11 the high rate of spontaneous ovulation in cats complicates one’s ability to artificially control the ovaries because high circulating progesterone concentrations can reduce or even prevent the effectiveness of exogenous hormone treatment.11 regressing the cl, although a typical means of regulating the estrous cycle in other mammals, is ineffective in felids.40 to address this issue, 2 strategies may be employed. the first is to confirm that the queen is nonluteal before initiating treatment. this can be accomplished with vaginal cytology (refer to real-time estrous cycle monitoring) and a serum progesterone assay (nonluteal is defined as < 2 ng/ml progesterone). the second strategy is to suppress estrus before initiating the hormone treatment protocol. a nonpregnant luteal phase in the cat lasts ~ 40 days,72 so the goal is to achieve ovarian inhibition over the same time interval. this allows natural regression of any current cls and prevention of any new spontaneous ovulations. the synthetic progestin levonorgestrel has been used to successfully down-regulate the feline ovary.43 six silastic rod implants (36 mg levonorgestrel/rod) were placed for 39 days before gonadotropin stimulation and then removed 2 days prior to placement.33 queens with elevated estradiol at the time of treatment completed a normal surge before returning to baseline. however, levonorgestrel successfully inhibited the initiation of new estradiol surges throughout the treatment period and the ovaries were highly responsive to the gonadotropin treatment after a 2 day withdrawal period. although http://dx.doi.org/10.58292/ct.v17.11649 70 citation: clinical theriogenology 2025, 17, 11649, http://dx.doi.org/10.58292/ct.v17.11649 highly effective, the biggest drawback to this technique is its labor intensiveness, requiring two anesthesia events before the estrus induction can begin. for this reason, the oral progestin altrenogest is more commonly employed. similar to levonorgestrel treatment, females with increased estradiol at treatment initiation completed a normal surge before returning to baseline.73 unlike levonorgestrel, altrenogest-treated queens had new estradiol surges during treatment, albeit at a significantly lower rate, and none ovulated during treatment. given that the domestic cat can have ovarian follicular activity during a luteal phase, it is not surprising to observe some activity during exogenous progestin treatment.72 the authors investigated 3 doses of  altrenogest, and concluded that the middle dose (0.088 mg/kg) was optimal for the cat as it produced normal baseline estrogen and progesterone concentrations and a more uniform return to follicular activity (10-16 days) versus either the low dose treatment (0.044 mg/kg; 2-12 days) or the high dose treatment (0.352 mg/kg; 9-35 days). exogenous gonadotropin treatment 3-5 days after altrenogest withdrawal produced consistent follicular development and synchronous ovulation,74 normal luteal function,75 improvements in embryo development,75 and high (83-86%) ai pregnancy rates.75,76 the latter strategy (ovarian suppression) is more commonly adopted because the former strategy (confirmation of a nonluteal phase) may include queens in the follicular stage of estrus that respond suboptimally to estrus induction protocols compared to queens in interestrus.43 an inactive ovary contains primordial follicles and follicles in a gonadotropin-independent continuous growth phase resulting in a more uniform population of early antral follicles that are highly receptive to gonadotropin stimulation and creates a more uniform ovarian response.43 furthermore, ecg is often used due to its fsh-like activity in cats to stimulate follicular growth. however, ecg also has lh-like activity and can trigger ovulation if given to a cat that is already in estrus.60 typical timed ai protocols administer ecg ~ 5 days before ai.15 in this example, the queen would ovulate several days early, and the oocytes would likely be too senescent at the time of ai to fertilize. finally, there is some evidence that progestin exposure (either through exogenous treatment or endogenous progesterone from a spontaneous ovulation) primes the ovary to be more sensitive to the effects of gonadotropins.43,74 direct stimulation of the pituitary with gnrh agonists has also been investigated. treatment of a gnrh agonist initially causes an acute stimulatory phase that lasts for several days and is accompanied by a large increase in fsh and lh concentrations.77,78 with prolonged exposure, gnrh receptors are down-regulated, fsh and lh production is reduced, and temporary infertility is induced. deslorelin is a gnrh agonist with a biological potency 10-144 times higher than native gnrh.79 deslorelin is commercially available as a slow-release subcutaneous implant (suprelorin®, virbac). it is registered in the european union (eu), australia, and new zealand for long-term suppression of adult male dogs (see estrus suppression) and, as of june 2022, the 4.7 mg implant has been approved in the eu for use in male cats.80,81 in the usa, suprelorin f® is a food and drug administration indexed product to manage adrenal disease in male and female ferrets.80 thus, any use of suprelorin in the queen is considered extra-label. consistent results were reported using the 4.7 mg suprelorin implants to induce estrus in queens.82 the implants were placed in the umbilical area without sedation or general anesthesia. queens were monitored for estrus via daily behavioral observation, daily vaginal cytology, and every other day transabdominal ultrasonography. estrus was detected 5.0 ± 2.2 days after implant placement in 100% of the 13 queens tested. seven females had behavioral estrus and an average of 4.8 ± 1.6 follicles were detected with ultrasonography. once peak estrus was observed (as defined by vaginal cytology with 100% cornified cells on a clear background), 100 iu of intramuscular hcg was given. peak estrus was identified 4-11 days after implant placement. serum progesterone measured 5-6 days after hcg treatment confirmed ovulation in all queens. three of the females were artificially inseminated twice; procedures were performed 24 and 48 hours after hcg treatment with fresh semen deposited transcervically into the uterine horns. the ais were performed under general anesthesia, and the implant was removed during the first ai. all females became pregnant and gave birth to healthy kittens. a separate study only reported successful estrus induction in 10% (2/20) of queens treated with a 4.7 mg deslorelin implant.83 ten females were treated 3 days after estrus began and another 10 females were treated 7 days after the end of estrus. estrus was induced in 1 female from each treatment group. the 9 females that were treated after the end of estrus and failed to respond to estrus induction had serum progesterone concentrations > 1.5 ng/ml, indicating they were luteal at the time of treatment. on the contrary, the study with 100% estrus induction82 only treated females in anestrus/interestrus, and the authors theorized the disparity of responses could be explained by the difference in stages of the estrous cycle during treatment. indeed, it would appear much like the ability of the ovary to respond to gonadotropins, a quiescent ovary is more capable to respond to gnrh agonist treatment. deslorelin implants have also been utilized to down-regulate ovarian activity prior to ecg/hcg stimulation; 10 queens were treated with 4.7 mg deslorelin implants for 90 days.84 following a 10 day withdrawal period after implant removal, cats were treated with ecg/hcg and spayed 3 days later. the authors recovered ovulated oocytes via oviductal flushing and confirmed viability with propidium iodide dye exclusion, but no further assessments were made for oocyte quality or competence. it is of note that on average, females had 13.1 ± 5.5 cls and 8.5 ± 5.5 follicles, indicating that deslorelin pretreatment did not prevent gonadotropin-induced ovarian hyperstimulation. the combined use of ecg and gnrh has been explored as an alternative protocol for et recipient synchronization in the cat. anestrual queens were treated with 100 iu intramuscular ecg followed 80 hours later with 1 or 2 (12-hour treatment interval) subcutaneous injections of 25 µg gnrh agonist gonadorelin.69 only 1 female (out of 5) in each group ovulated. the 2 ovulatory females had significantly higher serum lh concentrations compared to anovulatory cats, suggesting that an insufficient pituitary release of lh was responsible for ovulation failure. finally, there is a single report on the use of serial intramuscular naloxone treatments (0.04 mg/kg daily for 4 days) in conjunction with a single intramuscular hcg (1,000 iu) treatment http://dx.doi.org/10.58292/ct.v17.11649 citation: clinical theriogenology 2025, 17, 11649, http://dx.doi.org/10.58292/ct.v17.11649 71 to induce estrus and ovulation in the cat via antagonization of the hypothalamic gnrh opioid block.85 eight of the 9 treated females ovulated (based on increase in serum progesterone); hcg treatment appeared to be necessary for ovulation induction, as none of the females (n = 4) treated only with naloxone ovulated. natural estrus ovulation induction one of the largest drawbacks to estrus induction is that it often relies on exogenous gonadotropins to stimulate follicular growth, oocyte maturation, and ovulation. in other mammals, gonadotropin treatment can affect the normal follicular, oviductal, or uterine environment leading to poor quality oocytes or reduced implantation rates.86,87 in cats, exogenous gonadotropins can hyperstimulate the ovary and create an abnormal endocrine environment. compared to naturally cyclic females, gonadotropin-treated queens produce a higher number of total follicles (5 in a natural state72 versus > 10 with gonadotropin treatment),60 a higher number of unovulated follicles54 (ovulation is an all or nothing phenomenon in the naturally-mated queen),30 and more follicular cysts.53 additionally, gonadotropin-treatment in felids has been associated with the production of antigonadotropin antibodies, disruption of oviductal embryo transportation, and reduced embryo quality.32,60,64-66,88 timed estrus induction protocols are often paired with a period of ovarian suppression beforehand. although some data suggest progestins may have a positive role in priming the ovary to favorably respond to gonadotropin treatment,43,73 the entirety of effects exogenous progestins can have on the uterine environment is still unknown. progesterone and its receptors in the uterus have a major role in both maintaining pregnancy and in the progression of disease, creating a delicate balance that is not entirely understood, even in human medicine.89 as a species that demonstrates both induced and spontaneous ovulation, it is difficult to conclude whether progesterone presence prior to an estrus phase is advantageous, detrimental, or has no effect on fertility. ovulation induction following a natural estrus represents a viable alternative to estrus induction and has the potential to reduce or eliminate the need for exogenous gonadotropins. in humans, exogenous ovarian stimulation is associated with a higher rate of pregnancy loss before pregnancy can be clinically detected and a reduced implantation rate compared to natural cycle conception rates.90 natural cycles are aimed at achieving physiological concentrations of estradiol and progesterone, and ovulation can be induced by natural mating behavior, manual stimulation, or exogenous hormone therapy. irrespective of the specific technique used for ovulation induction, reliable and accurate detection of estrus is obligatory for success. real-time estrous cycle monitoring estrus is characterized by a rapid increase in estradiol, from a baseline plasma concentrations of ~ 15 pg/ml to > 20 pg/ml as the ovarian follicles grow into distinct, vesicular structures > 2 mm in diameter.72 serum estradiol could be considered for estrus monitoring, but there is a ~ 1-2 week turnaround time in commercial reference laboratories (e.g. 7-16 days at idexx) and, to the best of the authors’ knowledge, there are no commercially available in-house estradiol assays validated in the cat. the long turnaround paired with the inherent difficulty in serial blood sampling feline patients currently precludes the use of serum estradiol as a useful tool to monitor the estrous cycle in real-time. following natural mating, lh surges within minutes, ovulation occurs 24-32 hours later, and progesterone increases 1-2 days after ovulation.72,91 because cats have no preovulatory surge in progesterone, serum concentrations cannot inform the ideal breeding window as it is used in domestic dogs. however, serum progesterone can be useful to confirm ovulation. the authors recommend waiting a minimum of 5 days after the ovulation-inducing event to perform a serum progesterone test. feces are the major route of excretion for both estradiol and progesterone metabolites in domestic cats.36,92 excreted fecal hormone metabolites accurately reflect hormonal patterns in the blood, considering the appropriate time delay (12-24 hours) for metabolite passage from the blood into the feces. unfortunately, the gut transit delay plus an additional ~ 48 hours for sample shipment, processing, and assaying renders this technology unsuitable for real-time natural cycle monitoring. however, fecal hormone analysis remains a valuable noninvasive tool for retrospective longitudinal hormone monitoring in domestic and nondomestic felids. unlike their canine counterparts, felids do not display vulvar swelling or vaginal bleeding during the estrous cycle.21 because of the minimal overt outward changes, behavior has been the mainstay for monitoring felid estrous cycles. stroking of the flanks and perineal region by a handler may be used to elicit treading of the hind feet and lordosis (bent forelegs with hind quarters elevated and lateral tail deviation).93 other behaviors that may be associated with estrus in the queen include rolling, intense vocalization, frequent urination, and increased restlessness. however, there is a great deal of individual variation in what behaviors are expressed. queens that are particularly affectionate can exhibit estrous behaviors, including lordosis, during times of anestrus. additionally, behavioral estrus can lag behind physiologic estrus, with only 8% of cats demonstrating estrous behaviors on day 1 (as defined by > 20 pg/ml plasma estradiol), whereas 80% of cats show such behavior on day 4.93 for these reasons, it is the authors’ recommendation to pair behavioral observation with vaginal cytology. although not as commonly used as in dogs, vaginal cytology can help accurately determine estrus, especially if performed with regular (ideally daily) sampling. to distinguish other similar periods of the reproductive cycle, 2 or 3 consecutive vaginal cytology samples should be assessed for the proportion of basal, parabasal, intermediate, and superficial epithelial cells, as well as assessing for background polymorphonuclear cells, bacteria, and mucus.94-96 up to 1/3 of females may have signs of estrus before cornified cells are noticed on vaginal cytology;48 instead, clearing of the vaginal smear background (absence of cellular debris) is the most sensitive and earliest indicator of follicular activity, and occurs ~ in 1/3 of cats during proestrus.93 vaginal cytology with clearing of the background, a reduction of cellular debris, and a proportion of superficial cells > 80% is indicative of estrus in both domestic cats and african lions.94-96 given that the cat is an induced ovulator, with vaginal stimuli from the tom during coitus being the canonical inducing agent, it is important to note that vaginal cytology examination alone did not increase the risk for ovulation induction.96 http://dx.doi.org/10.58292/ct.v17.11649 72 citation: clinical theriogenology 2025, 17, 11649, http://dx.doi.org/10.58292/ct.v17.11649 ultrasonography is another tool that can aid in estrous cycle monitoring. although transabdominal ovarian ultrasonography has been described to monitor ovarian follicular growth during estrus, it is not commonly employed in cats.97 in the authors’ experience, follicles are routinely identified as round anechoic structures, but corpora lutea (cl) are not easily visualized. in a study performed with queens in a trap-neuter-release program, only 55% (11/20) of cls found on retrospective histopathology were identified via transabdominal ultrasonography.98 the cls that were readily identified were hyperechoic, large, and/or deformed along the ovarian margins. identification was more challenging and/or not possible when they were isoor hypoechoic to the ovary. because timing of a natural cycle would primarily be based on follicular growth, ultrasonography can be beneficial. verification of ovulation with ultrasonography could provide more immediate feedback, but this information can be achieved by other means (e.g. progesterone monitoring) if the cl(s) is/are not readily identified. induction of ovulation manual stimulation during natural mating, there are 2 overarching factors that determine whether a female will ovulate after copulation: number of stimuli and timing relative to day of estrus. single copulations can induce ovulation in a subset of females (2150%), whereas multiple copulations (3-12) during a 4 hour period in a single day of estrus resulted in higher ovulation rates (83-100%).29,30,99,100 although multiple copulations appear to be consistently superior across studies, there are various reports of the relative day of estrus and mating intervals used. in the first paper to describe manual ovulation induction, queens were stimulated with a glass rod during their first signs of estrus and ovulated 9 out of 12 times.101 in more recent feline manual induction protocols, a series of 5 vaginal stimulations at 30 minute intervals during peak estrus or maximum follicular diameter induced ovulation in 72 (8/11) to 75% (9/12) of queens.2,97 peak estrus is variable among breeding ovulation studies, with most indicating on days 3-5 of estrus.4,29,100 breeding before the third day of estrus can reduce lh secretion and increase the chance of ovulation failure,102 comparable to a report97 that detected maximum follicular diameter on 3.8 ± 0.3 days. pharmacologic ovulation induction early studies reported a wide range of hcg doses (50-500 iu) used to induce ovulation during days 1-2 of natural estrus, given either the day before or on the day of ai.52,103 more recently, several studies have been performed using intravenous hcg treatment on days 2-4 of natural estrus, with either 2 100 iu injections given 24 hours apart or a single 250 iu injection.71,104,105 ovulation rate with either protocol was relatively high in all studies (82.4-95.6%), and these protocols were used in conjunction with vaginal or uterine ais at 15, 20, and/or 30 hours after hcg treatment to successfully produce live offspring. subsequently half-life and bioavailability of hcg was determine and that were similar between intramuscular and intravenous treatments60; however, to the authors’ knowledge, no studies have subsequently been performed to assess ovulation rates with intramuscular hcg utilizing the aforementioned treatment protocol. due to its potential to cause neutralizing immunoglobulins and undesirable secondary follicular growth, it is not the authors’ recommendation to use hcg in natural cycle ovulation induction protocols. because of its small size (9 amino acids), gnrh is not detected by the immune system and therefore can be used repeatedly without the development of antignrh antibodies.106 nor is it associated with ovarian hyperstimulation, likely due to its differing mechanism of action, or more specifically, that it targets the pituitary, which creates another level of opportunity for feedback inhibition.107 treatment with a single intramuscular injection of 25 µg gonadorelin (gnrh agonist) resulted in a sharp increase in serum lh for queens in estrus or anestrus and ovulation was observed in 100% (4/4) of estrual queens.108 a single 25 µg intramuscular injection of gonadorelin given on day 2 or 3 of natural estrus produced a comparable number of ovulations (4.1 ± 0.8) to a single intramuscular injection of 250 iu hcg given during the same time period (4.0 ± 0.9).54 subcutaneous treatment of 25 µg gonadorelin with repeated vaginal stimulations resulted in a 100% (7/7) ovulatory rate on day 3 of estrus.96 a more recent study utilized a single intramuscular injection of 50 µg gonadorelin on day 2-4 of behavioral estrus, with successful ovulation occurring in 84% of treated females.106 the authors have used 2 treatments of gonadorelin 12 hours apart on days 3-4 of natural estrus (defined by vaginal cytology and/or behavior), with successful ovulation induction (defined by laparoscopic ovarian examination and/or fecal progesterone metabolite analysis) in the domestic cat (25 µg), ocelot (50 µg), amur leopard (100 µg), and jaguar (100 µg). a single intramuscular injection of gnrh agonist buserelin (~ 50 times more potent than native gnrh79 and not currently available in the usa) on day 4-6 of natural estrus in the asiatic golden cat (3 µg) and lion (20 µg) resulted in ovulation, and in conjunction with ai, produced live offspring.109,110 the llama is an induced ovulator that relies on ovulation-inducing factors (oifs) in the seminal plasma, rather than the physical act of copulation, to trigger ovulation. beta nerve growth factor (beta-ngf) has been identified as the potent oif in llamas, capable of eliciting ovulation either through intrauterine infusion or intramuscular injection.111,112 beta-ngf has since been detected in the seminal plasma of a variety of other induced and spontaneous ovulator species, although cats have not specifically been investigated.113 sixty-seven percent (4/6) of cats treated on day 2 of natural estrus ovulated in response to subcutaneous cat seminal plasma, versus 0% (0/6) of cats treated with intramuscular cat seminal plasma, and 17% (1/6) of cats treated with subcutaneous purified llama beta-ngf.114 this suggests that cat seminal plasma may contain oifs that help support ovulation in the queen. although not currently available for clinical use, the identification and isolation of cat oif molecules could offer a new avenue for ovulation induction in the queen. finally, as reported in the estrus induction section, plh has commonly been employed to stimulate ovulation after ecg-mediated estrus induction during timed art procedures. to the authors’ knowledge, there are no reports that have utilized plh to induce ovulation from a natural cycle, but it could be considered as an alternative to hcg or gnrh treatment. http://dx.doi.org/10.58292/ct.v17.11649 citation: clinical theriogenology 2025, 17, 11649, http://dx.doi.org/10.58292/ct.v17.11649 73 estrus suppression as discussed above, a quiescent ovary is required for estrus induction to be maximally effective. ovarian suppression protocols that are specifically designed for treatment prior to gonadotropin are reviewed in the pharmacologic estrus induction subsection. this section will address the other reasons safe and reliable estrus suppression is needed in cats. breeders often request estrus suppression in queens to temporarily delay breeding without compromising future fertility.115 even in cats not intended for breeding, owners may be reluctant to choose surgical neutering due to concerns about preexisting conditions or surgical complications.116 ovarian remnant syndrome is a well-documented surgical complication in cats, and in some cases, it may be difficult or impossible to identify and remove the residual ovarian tissue, whereas other means of reproductive control need to be considered.117,118 finally, cat overpopulation (i.e. unowned, outdoor ‘community cats’) is a global concern. the surgical model of trap-neuter-return (tnr) has remained the gold standard for humanely reducing community cat populations. although the rise in subsidized spay-neuters has helped to significantly reduce the rate of shelter euthanasia in usa, tnr is limited by access to resources and veterinarians.119 the covid-19 pandemic amplified the shortage, creating a deficit of over 2.7 million spay/neuters for companion animals in usa.120 this shortage is further magnified in developing countries with limited economic resources and some countries do not legally permit surgical neutering. nonsurgical options for long-term (or permanent) contraception could help augment traditional tnr programs. photoperiod queens are highly responsive to changes in photoperiod; a light:dark cycle of 8:16 hours is sufficient to immediately cease cyclity.26 the time it takes to resume cyclicity after a return to long-day light conditions (14:10 light: dark hours) varies by study (12-46 days), and the interval can be shortened by providing 1 hour of light during the dark period, cohabitation with estrual females, or introduction of a tom.26,27 melatonin photoperiod exerts its effect on the queen’s cyclicity via the retino-hypothalamic pathway to the suprachiasmatic nuclei that in turn regulates melatonin synthesis by the pineal gland.121 melatonin is a neuromodulatory substance that inhibits hypothalamic secretion of gnrh.122 in the cat, melatonin synthesis peaks during the night and serum concentration is ~ 15-fold higher than during the day.123 thus, exogenous melatonin treatment is a logical target for estrus suppression. daily oral melatonin (30 mg/cat), given 3 hours before lightsoff, is effective in suppressing estrus.123 serum melatonin concentrations peaked ~ 1 hour after treatment and remained significantly elevated above endogenous night-time concentrations for at least 8 hours. three of the 6 treated females demonstrated a period of ovarian follicular activity early in treatment (based on fecal estradiol metabolite analysis), but all follicular activity stopped after day 25 of treatment. following 35 days of melatonin treatment, females took 21-40 days (33 ± 2.8) to resume cyclicity. the authors also studied the use of melatonin as an ovarian down-regulation strategy prior to gonadotropin stimulation and ai. although 30 days of treatment prior to ai was successful in down-regulating the ovaries and still allowed the queen to respond to ecg/hcg treatment (given either at the end of melatonin treatment or after a 2 day withdrawal period), the authors did not recommend the use of melatonin as a pregonadotropin suppression protocol because it only marginally reduced ancillary follicle development and had no impact on quantity or quality of embryos produced from ai. some cats may be refractory to daily oral melatonin treatment. therefore, long-term release subcutaneous melatonin implants have also been investigated. most of the studies have utilized an 18 mg implant marketed for use in sheep (melovine®, ceva). similar to oral melatonin treatment, complete suppression may require a period of time and estrus can occur soon after treatment. therefore, estrous stage should be considered at the time of implant placement. approximately 80% of queens implanted during estrus will have estrus behavior shortly after implantation.124 conversely, queens implanted in late interestrus demonstrate estrus in ~ 35% of cases122,124-126 and queens implanted during anestrus127 or early interestrus125 did not come into estrus. the estrus that follows a melatonin implant may be fertile, as 1 queen in estrus after treatment was allowed to breed and produced a live litter.122 typical duration of estrus suppression for queens implanted during interestrus is 1-3 months,122,124-126,128 but larger ranges (21-277 days; ~ 0.79.2 months) have been reported.129 an important consideration of the melovine® implant is that the product is designed for sheep, and each pack contains 25 implants to be loaded sequentially to ewes with a single applicator.81 this would not be considered appropriate by feline practitioners and thus, alternative application schemes, such as insertion through a skin incision,122 should be performed. similarly, preservation of sterility for the remaining implants needs to be addressed. gnrh agonists although gnrh agonist treatment initially causes an acute stimulatory phase,77,78 prolonged exposure leads to desensitization of the gnrh receptors, reducing production and/or release of fsh and lh, inducing a state of infertily.130 most studies investigating the use of deslorelin in the queen have been performed with a 4.7 mg deslorelin implant (suprelorlin®), therefore this review will focus on those data. in cats, the implant is typically inserted through a needle subcutaneously into either the subscapular83,84 or umbilical area,82 with the latter being preferred for easy removal of the implant.81 in male dogs treated with the 4.7 mg implant, serum deslorelin concentrations peaked during the first week after treatment and then gradually decreased, reaching undetectable concentrations around day 80.130 no comparable pharmacokinetic studies have been performed in the cat. clinical data from a study82 suggests an initial peak of deslorelin during the first week of treatment likely occurs in the queen as well. the duration of efficacy, however, is much longer and more variable in queens compared to bitches. in one study, duration of efficacy ranged between 483-1,025 days (~ 16-34 months), with 1 female still clinically suppressed at the study’s conclusion (1,102 days, ~ 37 months).83 other studies have reported efficacy to last 4-14 months,129,131 and 18-26 months.132,133 one of the latter mentioned studies treated 14 cats with 9.5 mg http://dx.doi.org/10.58292/ct.v17.11649 74 citation: clinical theriogenology 2025, 17, 11649, http://dx.doi.org/10.58292/ct.v17.11649 deslorelin implants and 1 (7%) had no suppression of ovarian activity.133 it is unknown whether this large variability in effect duration is due to individual variation of susceptibility to deslorelin, individual variation in the desensitization mechanism, degree of vascularization at the insertion site, or other undescribed factors.80 during the acute stimulatory or ‘flare-up phase’, estradiol concentrations surge and can lead to a behavioral estrus within a few days of implant insertion. the rate of estrus induction varies by study protocol and is largely influenced by the queen’s estrous stage, with interestrus females being the most reliable estrus presenters.82-84,133 ovulation may also occur. in one report, 40% (4/10) of treated cats ovulated (determined with weekly blood progesterone analyses) during the flare-up phase.84 it is important to note that these estrus events can be fertile, as high pregnancy rates were achieved using deslorelin to induce estrus for ai.82 in this case, the implants were removed at the time of the procedure. however, pregnancy can be maintained in deslorelin-treated queens that are mated during the flare-up phase134 or ~ 7-9 days before implant placement.81,135 in one case report of a suspected mismating 8-9 days before implant placement, the queen delivered 4 healthy kittens, but had no maternal interest and had inadequate lactation.135 prolactin was not measured, so the specific mechanism of deslorelin-induced hypogalactia could not be identified. following parturition, the queen entered anestrus and did not have another estrus until 498 days after treatment. another curious variability observed with deslorelin treatment is that a subset of females can have a period of estrus that is not connected to the end of the implant’s action, as these episodes will be followed by another prolonged period of anestrus. a study reported that 1 female (n = 20; 5%) had 2 periods of estrual signs, 138 and 155 days after treatment.83 in another study, 1 female (n = 14; 7%) exhibited estrous behavior to her caretaker 3.5 months after beginning treatment, but did not allow a tom to mount her.133 in a study that removed implants at 3, 6, or 9 months after placement, the authors concluded that ~ 3 weeks are needed during increasing photoperiod to resume cyclicity, and this requirement can increase up to 7 weeks if the photoperiod is decreasing.134 the length of implant placement had no effect on the length of time needed to return to estrus. a return to fertility after termination of deslorelin treatment (via surgical removal of implant or cessation of implant effect) has been consistently demonstrated. studies have confirmed the queen’s capacity to ovulate,84 return to normal cyclicity,83 and produce normal litters.83 the most common side effect reported is weight gain that is often reversible after implant removal/failure without dietary intervention.129 other side effects occurred far less frequently and include persistent estrus,129,134 galactorrhea,129,136 and implant site lesions.129,133 data on the effects of long-term treatment are lacking. there is one case report of a queen treated at 1 year of age and then treated repeatedly every time she showed estrus (which occurred ~ every 2 years). when she presented with estrus at 8 years of age, the female was spayed and her reproductive tract was examined histologically. the ovaries were juvenile in appearance, containing numerous primordial and primary follicles. however, the uterus demonstrated marked endometrial hyperplasia, suggesting that repeated deslorelin stimulation and subsequent flair-up stages can have a negative effect on uterine health.137 overall, deslorelin treatment is regarded as relatively safe in cats, with minimal side effects, a quick return to fertility, and a high rate of efficacy. its largest drawback is the wide range in duration of effect. owners should regularly monitor for signs of estrus and consider intermittent vaginal cytology to more precisely predict when an implant’s effect is waning. progestins progestins are synthetic derivatives of progesterone that bind to the progesterone receptor with a greater affinity than endogenous progesterone.138 progestins have the same biological effects as progesterone and have been used for a variety of clinical cases, such as dermatologic and behavioral disorders.139 their main veterinary application remains as control of the estrous cycle.140 the mechanism of action by which progestins facilitate estrus suppression is not fully known. one well-accepted pathway proposal is through negative feedback on the hypothalamus and pituitary, suppressing release of gnrh, fsh, and lh.141,142 progestins may also inhibit sperm transport by thickening cervical mucus and reducing uterine motility, as well as preventing implantation through endometrial alterations.141,143,144 progestins use in queens has been associated with cystic endometrial hyperplasia-pyometra complex, fibroadenomatous mammary hyperplasia, mammary neoplasia, adrenocortical suppression, and diabetes mellitus.141,145-154 these effects were magnified with long-term treatment, higher dose usage, or when the queens were older and/or had preexisting conditions. it is therefore the authors’ recommendation to not consider progestin therapy as a strategy for long-term fertility control in the queen. however, there is a global shortage of access to spay/neuter programs, which was amplified by the covid-19 pandemic.120 this has prompted animal welfare advocates and organization, such as the alliance for contraception in cats & dogs (acc&d), to suggest the strategic use of megestrol acetate (ma) in queens when spay services are available but delayed.155,156 megestrol acetate (6-methyl-6-dehydro-17α-acetoxyprogesterone, ma) is a potent progestin, with activity estimated to be several times higher than endogenous progesterone.138 it is commercially-available as an oral formulation in several european countries.157 ma became commercially-available in the usa in 1975 as an fda-approved veterinary drug for female dogs (ovaban®, intervet schering-plough); off-label use in the cat was not uncommon.139 in 2008, an extra-label formulation of ma was developed by a private veterinarian and marketed to free-roaming cat colony caretakers (feralstat).156 the intention was to serve as an adjunct to tnr programs by preventing pregnancy in queens waiting to be spayed, although some caretakers elected to use this product in lieu of surgical sterilization. the package insert instructed weekly dosing at ~ 0.1-0.2 mg/kg ma, which was significantly lower than dosing regimens previously reported to be effective at pregnancy prevention. a veterinary consultant for acc&d interviewed several feralstat users, who reported satisfactory results (i.e. generally healthy colony and pregnancy prevention).158 however, no prospective studies have been performed to assess the safety and efficacy of ma given in this dosing regimen. http://dx.doi.org/10.58292/ct.v17.11649 citation: clinical theriogenology 2025, 17, 11649, http://dx.doi.org/10.58292/ct.v17.11649 75 efficacy of weekly treatment of 2.5 mg/cat for at least 30 weeks in 244 cats was assessed.150 twenty-one females demonstrated estrus during treatment. two females that were pregnant prior to treatment initiation had abnormal pregnancies; no other pregnancies were reported. an increase in appetite was reported in 33.6% (89/244) of queens and weight gain was noted in 13% (32/244) of queens. one female (0.4%) that received ma for 3 years developed pyometra and mammary adenocarcinoma. assuming a 4 kg body weight, 2.5 mg/cat would equate to 0.625 mg/kg/week, which is considered a low dose in cats.157 this is an important point as higher progestin dosages are correlated with a higher rate and/or increased severity of side effects. it is of note that most of the european-based formulations list 2.5 mg/week for a maximum of 30 weeks as the suggested dosing regimen. it is contraindicated to start ma treatment when a female is luteal, as adding a synthetic progestin to endogenous progesterone could be equivalent to high-dosage treatment, and it is currently recommended to only treat queens in anestrus or interestrus.157,159 the american college of theriogenologists (act) does not support the use of progestins, including ma, for contraception in free-roaming cat populations. in their joint position statement with members of the society for theriogenology, act cites the potential for inaccurate dosing, inadvertent treatment of pregnant females or nontarget species, and the adverse health events associated with progestin use as reasons for discouraging its use in this manner.160 it was stated that, “[progestins], including megestrol acetate, may be available to veterinarians for treatment of individually owned cats, but only within the strict confines of a veterinarian-client-patient relationship, including a veterinary prescription.” acc&d currently supports the use of ma in queens, “that can be individually treated at prescribed times with an accurate dose, and whose health can be monitored over time …as a stopgap measure to prevent pregnancy in female cats at risk of conceiving while awaiting spay surgery.”155 while historically advising against the use of ma in free-roaming cats, acc&d stated that because of the covid-19 pandemic, and the subsequent strain that it has put on spay/neuter programs, they do support the consideration of short-term, low dose ma treatment in situations where surgical spay is not an immediate option. in conclusion, the authors do not recommend progestin-based contraception as a strategy for long-term fertility control. the use of ma may be considered in females where surgical sterilization is planned but delayed. rigorous data on the safety and efficacy of low-dose ma treatment are lacking; careful patient selection and thorough clinical monitoring is warranted. permanent contraception due to the cost and logistical demands of large-scale tnr programs, considerable effort has been placed towards the development of a single-dose, nonsurgical, low-cost alternative for permanent contraception in the cat. early studies focused on the use of immunocontraceptive vaccines that control fertility by stimulating the production of antibodies against proteins that are essential for reproduction. one such approach utilizes porcine zona pellucida (pzp) glycoproteins extracted from pig ovaries. treatment in several mammalian species (e.g. horse,161 rabbit,162 dog,163 elephant,164 white-tailed deer,165 and seal166) resulted in production of antibodies that bind to the surface of the oocyte, which block sperm penetration and subsequent fertilization. a vaccine formulation that incorporates pzp antigens into multilamellar liposomes (spayvac™, immunovaccine technologies inc) was investigated in the domestic cat, due to its ability to induce long-term contraception in other tested species.167 all vaccinated kittens developed high antipzp antibody titers, but the treatment did not prevent cyclicity or pregnancy. ovarian immunohistochemical analyses revealed that the antipzp antibodies produced by spayvac-treated kittens did not recognize feline zp (fzp). these results align with a study that demonstrated cat and pig zonae pellucidae expressed a very small number of shared antigenic determinants.168 a subsequent study screened native soluble-isolated zps (sizps) isolated from 5 mammalian species: cows, cats, ferrets, dogs, and mink.169 treatments from all species resulted in antisizp antibody production. however, the antisizp antibodies had low cross-reactivity to fzp, as evidenced by low antifzp titers and lack of binding to feline ovaries. the next immunocontraceptive investigated was gonacon™, a gnrh vaccine developed by scientists at the united states department of agriculture-animal and plant health inspection service wildlife service’s national wildlife research center (usda-aphis nwrc) for use in wildlife. because gnrh is the ‘master regulator’ of reproduction, antibodies against hypothalamic gnrh prevents the normal hormone cascade required for sex-steroid production and gametogenesis.170 gonacon was originally developed for use in wild horses and white-tailed deer, but has since been applied to a variety of species, including the cat. after a single gonacon injection, 93% of cats were infertile for the first year after vaccination, whereas 73, 53, 40, and 27% remained infertile for 2, 3, 4, and 5 years, respectively.171 since gonacon was initially produced by the nwrc, it has undergone several formulation changes.172 compared to the formulation tested171 in 2011, the gonacon formulation registered with the environmental protection agency (epa) in 2016 consisted of a different antigen-carrier protein and increased antigen concentration. therefore, our laboratory investigated the safety and efficacy of this updated eparegistered formulation.173 all cats (n = 6) developed antignrh antibodies within 30 days after vaccination. the endpoint titer (1:1,024,000) was similar among all cats, and titers remained at that level throughout the duration of the study (4-6 months). because the vaccine was tested on ovariohysterectomized cats, fertility could not be assessed. therefore, a larger follow-up study in intact females was performed.174 sixty percent (12/20) of gonacon treated females became pregnant within 4 months after breeding trial initiation. two additional females became pregnant within 1 year after treatment, for a total of 70% (14/20) of queens that became pregnant following vaccination. the poor contraceptive efficacy was not anticipated, based on the high rate of contraception in queens treated with the earlier gonacon formulation171 and high antignrh antibody titers observed with the current formulation in ovariohysterectomized queens.173 antibody titers were not performed in this study; therefore, batch to batch variation in vaccine production could not be ruled out. individual vaccine response variation as well as differences in study population and design (the former study was performed with laboratory cats under http://dx.doi.org/10.58292/ct.v17.11649 76 citation: clinical theriogenology 2025, 17, 11649, http://dx.doi.org/10.58292/ct.v17.11649 controlled, indoor conditions, whereas the latter was performed with cats adopted from shelters in an ambient-temperature facility with daily outdoor access) should also be considered. irrespective of the cause for treatment failure, the overarching conclusion was that gonacon cannot currently provide contraception for a sufficient proportion of the population to justify its use for control of free-roaming cats. more recently, our laboratory reported a novel approach for long-term contraception in the cat utilizing anti-müllerian hormone (amh) that plays a critical role in ovarian folliculogenesis.175 at high concentrations, amh inhibits the recruitment of primordial follicles into the pool of growing follicles and decreases the fsh-responsiveness of growing follicles. an adeno-associated viral vector, delivered intramuscularly as a single injection, was used to overexpress amh in adult female queens. fecal hormone metabolite analysis was used to monitor progesterone and estrogen concentrations, and 2 breeding trials (4 months duration) were performed 1 and 2 years after treatment. all control cats produced kittens (3/3), but none of the treated cats became pregnant (0/6). treated cats had a reduction in average progesterone concentrations, a reduction in the rate of spontaneous ovulation, and complete inhibition of coitus-induced ovulation. furthermore, the cats’ amh concentrations remained elevated for 5+ years since initial treatment (unpublished data), indicating that gene therapy treatment may be able to provide contraception for the rest of the cats’ lives. further studies, large-scale production facilities, and fda approval will be required before this product can be made commercially available. in summary, a permanent, nonsurgical approach to sterilization would be a powerful tool for the humane control of free-roaming cat populations and could provide owned-cats with an alternative to surgical spay. although considerable research has been conducted in this field, no permanent, nonsurgical sterilization products are commercially available. despite success in many other mammalian species, immunocontraceptive approaches have not been effective in the cat. however, the application of gene therapy provides an exciting proof of concept and suggests the realization of nonsurgical sterilization in the domestic cat may be on the horizon. conclusion many methods of estrus manipulation exist in felids depending on the goal of the treatment, but success rates vary widely among and within estrus induction, ovulation induction, and estrus suppression protocols. external stimuli such as light and social interactions play a major role in feline cyclicity and should be considered before pharmaceutical manipulation. gonadotropins and gnrh agonists have successfully been used in cats for estrus and/or ovulation induction. melatonin, gnrh agonists, and progestins can all suppress feline estrus. however, dose and duration of treatment should be considered on an individual basis. these tools are useful when working with the feline estrous cycle, but understanding each regimen’s limitations is critical before making an appropriate selection. conflict of interest lv has served on the alliance for contraception in cats & dogs ethical review board and is currently on the scientific advisory board for the michelson found animals foundation michelson prize and grants in reproductive biology program. jb has no conflicts of 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vansandt lm, meinsohn m-c, godin p, et al: durable contraception in the female domestic cat using viral-vectored delivery of a feline anti-müllerian hormone transgene. nat commun 2023;14:3140. doi: 10.1038/s41467-023-38721-0 http://dx.doi.org/10.58292/ct.v17.11649 https://doi.org/10.1111/rda.12853 https://doi.org/10.1177/1098612x18758549 https://doi.org/10.1177/1098612x18758549 https://doi.org/10.1038/s41467-023-38721-0 1 review report what has embryo recovery and transfer taught us about equine reproductive physiology?* sandra wilsher, maarten hoogewijs sharjah equine hospital, al atain area, sharjah, uae abstract equine embryo transfer has been the foundation stone on which modern reproductive technologies and novel research in this species has been built. nonsurgical embryo recovery and transfer methods developed from the original surgical methods, and the ease with which these procedures can be undertaken has enabled these techniques to play a continuing role in original research. we document how embryo recovery and/or transfer have increased our knowledge about many of the events involved in equine reproductive physiology. these include, when the equine embryo enters the uterus, the importance and development of the blastocyst capsule, secretory products of early embryos, early maternal-embryo interactions, effects of uterine asynchrony on early embryo development, the role of endometrial cups and maternal immunological tolerance of extraspecies embryo transfers, and the influence of uterine environmental on fetal growth and development. keywords: fetomaternal interactions, nonsurgical embryo transfer, surgical embryo transfer introduction1 our ability to collect an equine embryo and transfer it successfully to a recipient’s uterus is considered a standard modern breeding technology, which was first successfully accomplished 50 years ago.1 the first report detailed a surgical technique (midline laparotomy) for both recovery and transfer of embryos. these first transfers were undertaken solely for experimental purposes (to study the effect of interspecies transfers between donkeys and horses) and it was not uppermost in the researchers’ minds to make this technique a routine veterinary procedure for the commercial production of horses. since this first successful embryo transfer (et), the technique of nonsurgical embryo recovery (er) and transfer have been further developed (figure 1) and can now be undertaken with relative ease, allowing their routine use in modern assisted reproductive techniques (art).2-4 in addition to having a role in art, both er and et have benefited the horse industry in general, even those sectors that do not allow embryo technologies to be used for breeding (e.g. thoroughbred industry). this is because veterinary researchers have been able to utilize the techniques involved in er and * presented at the 2023 society for theriogenology conference, published after peer review. et to gain a clearer understanding of early embryonic development in the mare, maternal and fetal interactions required to establish ongoing pregnancy, and the influence of maternal uterine environment on fetal health and postnatal growth. additionally, information gleaned from such studies will also pay dividends in unforeseen ways in the wider field of reproductive biology. some of the discoveries relating to equine reproductive physiology that would not have been possible without er and/or et are presented below. this is by no means a complete list and the authors have chosen to discuss some of those that are of personal interest. timing of embryo entry into uterus from early attempts at flushing equine embryos or collection of early embryos from the oviduct either at slaughter or surgery,1,5,6 it became apparent that equine embryos were retained in the oviduct until 5 or 6 days after ovulation. however, determination of ovulation was not possible with any great precision as ultrasonography of the reproductive tract was not yet commonplace and it relied on teasing and transrectal palpation of the ovaries. with the advent of ultrasonography of the reproductive tract,7,8 a more accurate determination of ovulation was possible and when combined with nonsurgical er techniques, experiments could be undertaken contact sandra wilsher sandrawilsher@hotmail.co.uk © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10602, http://dx.doi.org/10.58292/ct.v16.10602 mailto:sandrawilsher@hotmail.co.uk http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10602 2 citation line: clinical theriogenology 2024, 16, 10602, http://dx.doi.org/10.58292/ct.v16.10602 to try and pinpoint the precise timing of embryo entry into the uterus.9,10 using hourly ultrasonography of the ovaries to determine the time of ovulation, mares were subsequently flushed once at 144 ± 0.5, 156 ± 0.5, or 168 ± 0.5 hours after ovulation. with this methodology, it was determined that embryos entered the uterus between 144-156 hours after ovulation.9 however, these researchers noted that the interval between ovulation and fertilization appeared to be inconsistent and/or embryonic development rate differed among individual embryos, as there was variation in the size and degree of development in embryos collected at similar time points.9,10 a subsequent study, using a similar protocol with successive uterine flushings at 3 or 6 hours intervals on the same mare until an embryo was obtained, tried to determine a more precise timing for transport of the embryo through the oviduct.11 there was wide variation in the timing of entry of the embryo into the uterus, with some embryos collected from flushes as early as 144 ± 0.5 hours after ovulation and others as late as 150 ± 0.5 hours. a more recent study12 undertook consecutive day flushing when attempting to recover embryos ≤ 300 microns for vitrification, with er initially undertaken 156 ± 12 hours after ovulation for those bred with chilled semen or 168 hours after ovulation for those bred with frozen semen. if an embryo was not recovered at the first flushing, 2 more flushings were tried at 24 hours intervals. in 7 of 35 mares (20%) an embryo was recovered on the third flush (204 ± 12 hours for chilled semen; 216 hours for frozen semen),12 suggesting that the timing of embryo entry into the uterus may have a wider timespan than originally thought. despite the observed biological variation in the duration of oviductal transport, the knowledge that the majority of embryos will be present in the uterus at 156 hours (6.5 days) after ovulation has been instrumental in obtaining small embryos ≤ 300 microns that were suitable for successful freezing13 or vitrification14 in early cryopreservation protocols. importance and development of equine blastocyst capsule recovery of early embryos from the uterus has also allowed reproductive physiologists to discover more about the equine blastocyst capsule (figure 2). this unique acellular layer develops underneath the zona pellucida (zp) and envelops the equine embryo from ~ days 7 to 21 of pregnancy, once the zp is shed15 the capsule is essential for the embryo’s early survival in the uterus as demonstrated in an elegant experiment in figure 1. a diagrammatic representation of how embryo recovery and transfer fit into both clinical assisted reproductive technologies and research. figure 2. a 600-mm expanded blastocyst (a), loose capsule (arrow) around collapsed (via a fine acupuncture needle) blastocyst (b), and (c) capsular vesicles (100-300 mm), recovered from a recipient mare (via uterine flushing) 48 hours after transfer. http://dx.doi.org/10.58292/ct.v16.10602 citation line: clinical theriogenology 2024, 16, 10602, http://dx.doi.org/10.58292/ct.v16.10602 3 which day-7 embryos were flushed from the uterus and their capsules removed before being transferred to recipient mares.16 none of the 15 denuded embryos developed, whereas 4/6 (67%) control embryos, subjected to the same manipulations without removal of the capsule, developed. this clearly demonstrated that the presence of a capsule is essential for embryo survival.16 analysis of capsules from in vivo-derived embryos has revealed they are composed of mucin-like glycoproteins that are produced primarily by the trophoblast17,18 with possible contributions from the endometrium. macroscopic and molecular changes in its development over time have also been noted. for example, the weight of the capsule increased over time as it migrated throughout the uterus and, once the conceptus became immobile at the base of a uterine horn ~ day 16, the sialic acid content of the capsule markedly declined.18 during in vitro embryo production, the capsule failed to form properly, but following their transfer to recipient mares, such embryos formed a capsule within the uterus.6,19 recent work has demonstrated that denuded embryos or trophoblast vesicles (tvs), when transferred to the uterus of a recipient mare, can produce copious amounts of capsular material that forms vesicles, but they fail to encapsulate the embryo and remain free within the uterus. the number of capsular vesicles that form when tvs are transferred is dependent on the stage of the recipient’s cycle, with more prodigious formation between days 6 and 12, when an in vivo embryo would normally be losing the zp and increasing the thickness of the capsule.20,21 interestingly, equine trophoblast transplanted to immuno-deficient mice produced capsular material.22 further efforts are required to elucidate the manner by which the trophoblast synthesizes capsular material in these atypical situations. secretory products of early embryos recovered embryos have also been used in various experiments to determine steroids, receptors, proteins, peptides,  eicosanoids, and various other products of equine metabolism using a variety of techniques, including in vitro culture, immunological, and molecular methodologies. estrogen and androgen production by the early equine conceptus has been demonstrated from as early as the 1970s23-25 with variations in steroid production from endoderm and trophectoderm.26 more recent studies not only identified that embryonic steroid production is a feature of early pregnancy but mrna for progesterone and estrogen receptors were also present on early conceptuses,27 suggesting a likely role for these hormones in the early development of the conceptus. in addition to steroids, eicosanoids are also produced by the early conceptus, with the production of both pge2 and pgf2a. 28-31 it is believed that these 2 prostaglandins have a role in the transuterine migration of the equine blastocyst before days 16-17 of pregnancy32 but may also be involved in the unknown mechanisms that prevent the demise of the corpus luteum during maternal recognition of pregnancy in the mare. from the 1990s, equine embryos have also been used to examine the array of proteins and peptides that they produce or to characterise single ones.33-36 as technology has progressed, gene expression, transcriptome and proteomics studies have also been conducted on early conceptuses.37-46 some of these studies have aimed to find clues towards the as yet unknown maternal recognition of pregnancy (mrp) signal in the mare and although, to date, a specific protein for this role remains elusive, a vast array of proteins that are up or down regulated and/or expressed has been demonstrated. further work will  help to elucidate how these proteins function in many key  events during early pregnancy and embryo-maternal communication. early maternal-embryo interactions and asynchrony the equine embryo takes ~ 6-6.5 days to traverse the oviduct and enter the uterus, suggesting that successful interaction between the embryo and the uterus may only be possible after this time. however, the high survival rate of day-4 embryos flushed from the oviduct of mares and subsequently transferred to recipients that had ovulated within 1 day of the donor proved otherwise.47-48 this was the first chronological evidence that equine embryos can tolerate considerable asynchrony with the maternal uterine environment. asynchronous et has also demonstrated that the equine embryo can withstand a much wider degree of developmental asynchrony (figure 3) with the uterus than the embryos of cattle, sheep, and pigs. for example, this is illustrated by the successful transfer of day-10 embryos to recipient mares that had ovulated between 7 days after to 2 days before the embryo donor.49,50 in addition to identifying considerable tolerance of equine embryos to asynchrony, these experiments also demonstrated the marked dependency of embryonic development on maternal uterine stage. this was best illustrated by the transfer of day-10 embryos to recipients with the greatest asynchrony (i.e. transfer to day-3 recipient mares equating to 7 days asynchrony). only 3 of 8 embryos transferred with this degree of asynchrony survived past the heartbeat stage, but all 3 had a dramatic slowing of development of both the membranes and the embryo itself to match the stage of the negatively asynchronous uterus. this effect of embryonic development matching uterine rather  than its own age was noted in all the asynchronous transfers.49,50 further studies have also demonstrated that, when placed in a negatively asynchronous uterine environment, the horse conceptus adjusts its transcriptome, resulting in many differentially expressed genes being up regulated, whereas the endometrial transcriptome is only modified slightly by the presence of an asynchronous embryo.51,52 despite embryonic development becoming aligned with uterine age, one event in early embryonic development does not appear to be governed entirely by the uterus. namely, intrauterine mobility of the equine embryo up to days 16-17 of pregnancy. in recipient mares carrying asynchronous embryos, fixation of the conceptus at the base of a horn occurred when the conceptus was ~ 17 days of age, regardless of the degree of uterine asynchrony. this finding would appear to suggest that although an increase in uterine tone has also been associated with conceptus fixation,53-55 intraluminal impediment, with or without increased uterine tone, would appear to be the overriding influence on fixation time since mobility of the transferred embryos ceased at the same conceptus age, regardless of the stage of the uterus. http://dx.doi.org/10.58292/ct.v16.10602 4 citation line: clinical theriogenology 2024, 16, 10602, http://dx.doi.org/10.58292/ct.v16.10602 another unexpected finding from the asynchronous transfer of day-10 embryos was the ability of such embryos to prevent luteolysis and survive when transferred to day-12 recipient mares. it had long been assumed that the equine blastocyst must transmit its antiluteolytic mrp signal to the mare before day 10, if it is to suppress the luteolytic cascade and hence, prevent luteolysis. however, the aforementioned experiment clearly demonstrated that day-10 embryos could provide the mrp signal as late as day 12 and suppress luteolysis of the primary corpus luteum. furthermore, it has been widely accepted that the continuing movement of the embryo until day 16 is essential to enable the mrp signal to be distributed throughout the uterine lumen.56-57 however, in some of the asynchronous recipient mares the conceptus ceased to move when the uterus was only 9 days after ovulation, yet luteostasis still occurred. indeed, luteostasis occurred in all the recipients in which an embryonic vesicle was detected after transfer, regardless of its developmental competence, and including both anembryonic or trophoblastic vesicles and normal embryos suffering delayed development of the embryo proper and its membranes.49,50 recent experiments undertaken by one of the authors utilizing et have also shed new light on the role of embryo migration in mrp signalling. transfer of day-11 embryos to recipient mares on days 10 (n = 4), 11 (n = 6), 12 (n = 6), or 13 (n = 6) after ovulation followed by manual reduction of the embryo 24 hours later resulted in luteostasis in 100, 83, 10, and 83% of mares, respectively. furthermore, when day-11 embryos were transferred to day-11 recipients (n = 5) and manually reduced just 12 hours later, all recipient mares became luteostatic. hence, an intact embryo need only be present in the uterus for 12 hours to cause luteostasis.58 an extension of this work demonstrated that when ruptured day-11 embryos were transferred to recipient mares on days 11 or 12 (n = 8/group) significantly more day-12 recipients entered a period of luteostasis compared to day-11 ones (75 versus 12.5%). control mares that received only transfer medium without a ruptured embryo had an incidence of luteostasis of 12.5% in days 11 or 12 (n = 8/group) recipients. hence, this study suggested that the presence of medium containing blastocoele fluid, a ruptured capsule and trophoblast in the uterine lumen prevented luteolysis around the time of mrp (day 12).59 this work led to the question of how long the mrp signal needs to be given to prevent luteolysis, and to what extent embryo mobility is involved in mrp. endometrial cup reaction and maternal immunological tolerance another unusual feature of equine pregnancy revealed by et  was the remarkable immunological tolerance of the mare’s uterus to accept and carry to term a very ‘foreign fetal occupant.’ a series of elegant experiments conducted in the early 1980s transferred przewalski’s horse embryos (equus ferus przewalskii; 2n = 66), domestic horse embryos (e. caballus; 2n = 64), donkey embryos (e. asinus; 2n = 62) and grant’s zebra embryos (e. quagga; 2n = 46) to horse, donkey, and mule (horse♀ x donkey♂, 2n = 63) recipients and obtained live viable offspring (figure 4) from every type of extraspecies transfer.60 before the first equine et was undertaken in the early 1970s, it had been noted that fetal genotype exerted a marked influence on placental development in equids, especially on the width and general development of the chorionic girdle, the progenitor tissue of the endometrial cups, which secrete equine chorionic gonadotrophin (ecg). dramatic differences in the size, productivity, and lifespan of the endometrial cups in mares carrying horse or mule conceptuses versus donkeys carrying donkey or hinny (donkey ♀ x horse ♂; 2n=63) conceptuses were noted. serum ecg concentrations were higher in mares and jenny donkeys carrying fetuses with horse paternity (horse or hinny, respectively) than in donkeys and mares carrying fetuses with donkey paternity (donkey or mule, respectively). this appeared to indicate that, as has been demonstrated in mice, the paternal genome contributed preferentially to the placenta. however, in a subsequent experiment, demi mule embryos (created by bisecting a mule morula) were transferred to horse and donkey recipients and 2 intact mule blastocysts into 2 further donkey recipients. the results were noteworthy; the 3 donkeys carrying mule embryos produced large endometrial cups had very high ecg concentrations, whereas the mare carrying the other demi mule embryo had typical small endometrial cups and figure 3. compared to the embryos of other large domestic animal species the equine embryo has an amazing ability to tolerate a wide range of donor-recipient asynchrony. in most instances an embryo flushed from a donor’s uterus on day 8 would be transferred to a days 6 or 7 recipient mare. however, equine embryos survive in recipients that ovulate up to 5 days after the donor or up to 2 days before the donor. even though such asynchrony is possible, embryos transferred to asynchronous uteruses had developmental and transcriptome changes in keeping with uterine not embryonic age.49,50 http://dx.doi.org/10.58292/ct.v16.10602 citation line: clinical theriogenology 2024, 16, 10602, http://dx.doi.org/10.58292/ct.v16.10602 5 correspondingly low ecg concentrations. thus, contrary to the view that imprinted paternal genes-controlled development of the chorionic girdle, the extraspecies transfer experiment demonstrated clearly that uterine environment exerts an overriding influence on the whole process.60,61 extraspecies ets also revealed the likely importance of the endometrial cup reaction in the development of the equine allantochorionic placenta and maternal immunological tolerance. although the majority of equine extraspecific pregnancies are carried to term without issue, transfer of donkey embryos to horse mares (d-in-h) resulted in complete failure of the endometrial cup reaction and a consequential absence of ecg from maternal blood. interestingly, the reverse occurred following transfer of horse embryos to jenny donkeys (hin-d) in which very large and active endometrial cups developed and secreted very high ecg concentrations into maternal blood. in the d-in-h model, development of the donkey progenitor chorionic girdle was retarded to such an extent in the recipient horse uterus that it completely failed to invade the maternal endometrium on days 36 to 38 in most cases. nevertheless, the conceptus continued to develop normally to ~ 60 days of pregnancy, beyond which a clear division occurred in the outcome of the pregnancy. in approximately 70% of the d-in-h pregnancies the donkey allantochorion failed to interdigitate with recipient horse endometrium and the developing fetus starved to death ~ days 80-90 as the unattached allantochorion degenerated and was actively attacked by maternal immune cells accumulated in the underlying endometrium. in the remaining 30% of d-in-h pregnancies, however, implantation, interdigitation and placentation, albeit delayed and rather irregular, did occur during days 40-100 of pregnancy. thereafter, the pregnancies continued to term with the birth of live foals that ranged from wellgrown and healthy to dysmature, small and barely viable due to varying degrees of inadequacy of the placenta.60,62 it is tempting to conclude that the lack of ecg secretion, and the consequential failure of plasma progesterone and estrogen concentrations to increase after day 40 as a result of the lack of secondary corpora lutea or resurgence of the primary corpus luteum, was responsible for pregnancy loss in those mares that aborted. however, subsequent supplementation of mares carrying donkey fetuses with altrenogest and/or ecg failed to prevent fetal death. notwithstanding, immunological therapy in the form of either passive immunization using serum from mares carrying normal intraspecific pregnancies, or active immunization with donkey lymphocytes, appeared to increase the rate of a successful pregnancy, although it did not prevent fetal demise in all the treated recipient mares. identifying the exact cause of pregnancy loss in this model is difficult, especially since some form of immunological memory or tolerance seemed to be involved. namely, the small percentage of horse mares that carry a transferred donkey fetus to term will normally do so again and again, whereas the mares that abort donkey fetuses do so repeatedly at earlier stages of pregnancy in successive d-in-h pregnancies.60,62 it is now some 40 years since these experiments were undertaken and suggested an important role for the specialized, invasive trophoblast cells of the chorionic girdle in initiating and driving attachment and interdigitation of the noninvasive placenta while at the same time modulating maternal immunological responses to enable survival of the foreign donkey fetus in the horse uterus. revisiting such interspecies et experiments may well prove timely in trying to unravel the persisting mysteries of the possible role(s) of the equine-unique endometrial cup reaction in promoting and maintaining pregnancy in this genus. fetal growth and development the production of genetically identical horse twins either by transferring blastomeres from 4to 8-cell embryos into empty pig zona pellucida and maturing them to blastocysts in the oviduct of a sheep for subsequent transfer to recipient mares,63 or by bisecting a morula to produce demi embryos64-66 for transfer to 2 recipient mares, has demonstrated the role of maternal uterine environment on fetal growth and development. for example, from a phenotypical point of view, such genetically identical twin foals are very alike in general conformation and appearance, but they frequently had quite marked differences in the white markings on their face and legs; similar variability in white markings have been observed in cloned offsprings.67 hence, the degree of spread of melanocytes from the neural crest during fetal development to dictate the positioning of white markings must, to some part, be dictated by the uterine environment. the size of the recipient mare and, correspondingly, uterine size can also influence markedly, the size and conformation of foals carried. this originates from uterine size controlling placental size and function by limiting the area of microscopic contact between fetal trophoblast and maternal endometrial epithelium at the placental interface. in 1938, before et had been undertaken in equids, the effects of maternal size on foal birthweight (by crossing large shire horses with much smaller shetland ponies) was first demonstrated.68 the latter hybrid offspring were much smaller at birth and the size disparity continued into adulthood. subsequently, konik pony embryos were transferred into larger draught-type recipient mares figure 4. extraspecies transfers (a) a grant’s zebra (e. quagga; 2n = 46) carried to term in a domestic horse (e. caballus; 2n = 64) recipient, (b) a donkey (e. asinus; 2n = 62) carried to term in a domestic horse recipient, and (c) a domestic horse carried to term in a donkey recipient. http://dx.doi.org/10.58292/ct.v16.10602 6 citation line: clinical theriogenology 2024, 16, 10602, http://dx.doi.org/10.58292/ct.v16.10602 before remating the konik pony donors to the same konik pony stallion as controls. in all of the 3 sex-matched pairs of full sibling foals produced in this manner, those from the larger recipient mares weighed considerably more and were taller than their full siblings born from the genetic konik pony mothers; as with the shire and shetland crosses, these disparities in body size persisted in later life.69,70 a similar experiment used reciprocal et between large thoroughbred and small welsh pony mares to again highlight the marked influences of maternal uterine size on placental and fetal growth.71 ponies in thoroughbred recipients (p-in-tb) were born ~ 15% bigger (weight and height) than their pony-in-pony (p-in-p) controls, whereas thoroughbred embryos in pony recipients (tb-in-p) were born some 15% smaller than their thoroughbred-in-thoroughbred (tb-in-tb) counterparts. these considerable bodyweight differences correlated closely with differences in the weight and gross area of the respective allantochorions and with the total microscopic area of contact at the feto-maternal interface calculated stereologically. the birth size differences persisted to 3 years of age and although a degree of ‘catch-up growth’ occurred in the undersized tb-in-p foals, they nevertheless remained appreciably smaller than their normal tb-in-tb controls.72 in addition to changes in placental morphology, the function of the feto-placental unit as an endocrine organ was also modified. for example, pony mares carrying thoroughbred foals had much higher maternal plasma concentrations of progestagens during the second half of pregnancy than the other 3 groups, presumably as a result of fetal stress initiating the production of pregnenolone, the precursor for progestagens, from the fetal adrenal glands.73 in addition, both growth-restricted (tb-in-p) and growth-enhanced intrauterine existence (p-in-tb) in the above animals altered cardiovascular function and other metabolic systems in postnatal life.74,75 more recently, the influence of both breed and maternal size on placental and fetal development in an et model was reexamined; possible effects on the epigenome of equine embryo technologies were reviewed.76 conclusion the experiments outlined above clearly demonstrated the great potential value of er, horse-horse et, and intraand extraspecies et in dissecting out the stage-related occurrence and significance of early maternofetal interactions in driving the development and the physiological efficiency of the diffuse, epitheliochorial placenta in the horse and other equids. they have also highlighted the amazing, and apparently unique, mechanism of the equine uterus in its immunological tolerance towards a range of genetically diverse fetuses. further, they have identified the long-lasting influences on the morphological and endocrinological functions of transferred embryos in their preand postnatal lives. the ease with which both er and et can be undertaken in equids has and remains a significant boon to research in equine reproduction and reproductive biology in general. conflict of interest none to report. references 1. allen wr, rowson lea: transfer of ova between horses and donkeys. proc 7th int congr anim reprod ai, munich. 1972. p. 484-486. 2. kraemer dc: a history of equine embryo transfer and related technologies. j equine vet sci 2013;33:305-308. doi: 10.1016/j. jevs.2013.03.007 3. allen wr, wilsher s: historic aspect of equine embryo transfer. j equine vet sci 2020;89:10298. doi: 10.1016/j.jevs.2020.102987 4. stout tae, griffiths h: clinical insights: 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et al: steroid secretion by different cell types of the horse conceptus. j reprod fertil 1987;35:363-369. 27. rambags bpb, van tol hta, van den eng mm, et al: expression of progesterone and oestrogen receptors by early intrauterine equine conceptuses. theriogenology 2008;69:366-375. doi: 10.1016/j. theriogenology.2007.10.011 28. watson ed, sertich pl: prostaglandin production by horse embryos and the effect of co-culture of embryos with endometrium from pregnant mares j reprod fertil 1989;87:331-336. 29. weber ja, woods gl, freeman da, et al: prostaglandin e2 secretion by day 6 to day 9 equine embryos. prostaglandins 1992;43:5559. doi: 10.1016/0090-6980(92)90064-z 30. vanderwall dj, woods gl, weber ja, et al: pge2 secretion by the conceptus and binding by non-pregnant endometrium in the horse. equine vet j 1993;15:24-27. doi: 10.1111/j.2042-3306.1993. tb04818.x 31. stout tae, allen wr: prostaglandin e2 and f2a production by equine conceptuses and concentrations in conceptus fluids and 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incubation medium of early equine conceptuses preliminary results. j equine vet sci 2012;32:400. doi: 10.1016/j.jevs.2012.05.014 37. klein c, troedsson mht: transcriptional profiling of equine conceptuses reveals new aspects of embryo-maternal communication in the horse. biol reprod 2011;84:872-885. doi: 10.1095/ biolreprod.110.088732 38. beckelmann j, budik s, bartel c, et al: evaluation of xist expression in preattachment equine embryos. theriogenology 2012;78:1429-1436. 39. de ruijter-villani m, van boxtel pr, stout ta: fibroblast growth factor-2 expression in the preimplantation equine  conceptus and endometrium of pregnant and cyclic mares. theriogenology 2013;80:979-989. doi: 10.1016/j. theriogenology.2013.07.024 40. de ruijter-villani m, deelen c, stout ta: expression of leukaemia inhibitory factor at the conceptus-maternal interface during preimplantation development and in the endometrium during the oestrous cycle in the mare. reprod fertil dev 2015;28:1642-1651. doi: 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upregulation of placental imprinted genes. placenta 2017;57:175-182. doi: 10.1016/j. placenta.2017.07.007 53. ginther oj: fixation and orientation of the early equine conceptus. theriogenology 1983;19:613-623. 54. griffin pg, ginther oj: uterine morphology and function in postpartum mares. j equine vet sci 1991;11:330-339. doi: 10.1016/ s0737-0806(06)81262-1 55. carnevale em, ginther oj: relationships of age to uterine function and reproductive efficiency in mares. theriogenology 1992;37:1101-1115. doi: 10.1016/0093-691x(92)90108-4 56. mcdowell kj, sharp dc, grubaugh w, et al: restricted conceptus mobility results in failure of pregnancy maintenance in mares. biol reprod 1988;39:340-348. doi: 10.1095/biolreprod39.2.340 57. griffin pg, ginther oj: effects of the embryo on uterine morphology and function in mares. anim reprod sci 1993;31:311-329. doi: 10.1016/0378-4320(93)90015-j 58. wilsher s, newcombe jr, ismer a, et al: the effect of embryo reduction and transfer on luteostasis in the mare. anim reprod sci 2022;242:107002. doi: 10.1016/j.anireprosci.2022.107002 59. wilsher s, ismer a, newcombe j, et al: transfer of day 11 ruptured embryos can prevent luteolysis in recipient mares. equine j vet sci 2022;113:103989. doi: 10.1016/j.jevs.2022.103989 60. allen wr, short rv: interspecific and extraspecific pregnancies in equids: anything goes. j hered 1997;88:384-392. doi: 10.1093/ oxfordjournals.jhered.a023123 61. allen wr, skidmore ja, stewart f, et al: effects of fetal genotype and uterine environment on placental development in equids. j reprod fertil 1993;97:55-60. doi: 10.1530/jrf.0.0980055 62. allen wr: immunological aspects of the equine endometrial cup reaction and the effect of xenogeneic pregnancy in horses and donkeys. j reprod fertil 1982;31:57-94. 63. allen wr, pashen rl: production of monozygotic (identical) horse twins by embryo micromanipulation. j reprod fertil 1984:71:607-613. doi: 10.1530/jrf.0.0710607 64. mckinnon ao, carnevale em, squires el, et al: 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resulted in a full thickness cervicouterine tear secondary to abnormal reproductive tract anatomy. ewe was euthanized and determined to have uterus didelphys via field necropsy. this incidental finding demonstrated that pregnancy can be carried to term despite substantial anatomic abnormality, highlighting the importance of a thorough reproductive tract examination prior to assisted delivery. keywords: sheep, dystocia, congenital anomaly, uterus, cervix background in the embryo, female genital tract arises from paramesonephric (müllerian) ducts to form oviducts, uterus, cervix, and vagina in the absence of müllerian inhibitory substance and testosterone. paramesonephric ducts fuse caudally to form the uterine body, cervix, and cranial vagina, creating a single lumen.1 uterus didelphys (uterus bicorpor bicollis) is a rare congenital abnormality of the female reproductive tract, consisting of a divided uterine body, double cervix, and a single or double vagina.2,3 in uterus didelphys animals, if fertilization is achieved via ovulation from the ovary ipsilateral to cervix, pregnancy in the corresponding uterine horn can be established that can be carried to term but the outcome may not be ideal. a case of pregnant uterus didelphys ewe is presented, highlighting challenges associated with delivery of a fetus. case presentation a 2-year, hampshire ewe was presented for sustained and unprogressive labor. based on recorded natural breeding dates, the ewe was determined to have a full-term pregnancy. the ewe had been actively straining for 1 hour at home after they had observed vaginal discharge. based on history, it was determined that ewe was entering stage ii of labor. on physical examination, the ewe appeared stable, temperature was 38.6°c (38.3-39.8°c), respiratory rate was 92 breaths per minute (15-35 bpm), and heart rate was 152 beats per minute (70-80 bpm). rumen contraction rate was 3 strong contractions in 2 minutes (1-2 per minute). ewe’s famacha score was 2/5 (1: normal and 5: anemic) and body condition score was 2/5. ewe’s udder was fully developed with expressible colostrum. treatment ewe’s perineum was disinfected, followed by vaginal palpation that revealed a small vulvar opening and abnormal anatomy within the vagina. the left side vagina had a small, firm, palpable depression in the vaginal wall (later identified as a tightly closed external cervical os). there was a second opening on cranial vagina’s right side, presumed to be a partially dilated (3 fingers diameter) cervix. the cervical tissue was soft and pliable. based on palpation, a diagnosis of ‘ring womb’ (failure of cervical dilation) was made. the owner opted for vaginal delivery, and so manual dilation was performed. after 10 minutes of gentle manual dilation, the chorioallantoic membrane protruded from the vagina. once this membrane was ruptured, front legs and fetal head were palpable within the cervix. a head snare was carefully applied to the fetus. after manipulation of fetal head and forelimbs through the incompletely relaxed cervix, a dead fetus was delivered. outcome attempts to resuscitate the fetus were unsuccessful. vaginal palpation of the ewe after delivery identified a full-thickness contact tessa marshall tsmarsha@illinois.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12651, http://dx.doi.org/10.58292/ct.v17.12651 mailto:tsmarsha@illinois.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12651 2 citation: clinical theriogenology 2025, 17, 12651, http://dx.doi.org/10.58292/ct.v17.12651 dorsal tear at the cervicouterine junction. ewe was euthanized because of its limited value and poor prognosis for survival. field necropsy was performed, and reproductive tract was exteriorized. a single vestibule and vulva (figure 1), a septum in the cranial vagina was visible as a dorsoventral tissue fold, and 2 cervices leading to individual uterine horns were identified, characterizing uterus didelphys. the necropsy also revealed an extensive full-thickness uterine tear in the right uterine horn (where the fetus was) caudal to the cervix (figure 1). on the left, cervix, cervical rings, mucosa, and nonpregnant uterine horn were intact. however, the right cervix and vaginal mucosa had submucosal hemorrhage and some multifocal superficial tears (figure 2). discussion reproductive efficiency (fertilization) may be compromised in animals with uterus didelphys if bred via artificial insemination because of the possibility of semen being deposited in the contralateral cervix to the ovulated ovary.2,3 however, in natural breeding, because semen is deposited in the cranial vagina, fertilization and establishment of pregnancy is possible. abortion, premature delivery, retained fetal membranes, and infertility are considered more common in cows with uterus didelphys, than in normal cows, because they have a unicornual placentation of the fetus.2 in comparison, there have been reports that the ovine placenta has the ability to adapt and maintain fetal nourishment in utero despite a reduction of the placentome figure 1. postmortem image of the reproductive tract (note the uterine tear in right uterine horn and in nonpregnant left uterine horn, both cervices, and caudal vagina). dvf: dorsoventral fold; lc: left cervix; lh: left uterine horn; rc: right cervix; rh: right uterine horn; ut: uterine tear http://dx.doi.org/10.58292/ct.v17.12651 citation: clinical theriogenology 2025, 17, 12651, http://dx.doi.org/10.58292/ct.v17.12651 3 numbers associated with a unicornual pregnancy. pregnancies have been maintained by compensatory placentome growth.4 uterus didelphys also results in a reduction in available space in the uterine cavity. this space restriction with unicornual placentation may be detrimental to fetal growth in multiple pregnancies (twins and triplets).4 in the present case, naturally bred ewe conceived and maintained a singleton pregnancy to term, despite uterus didelphys. although the outcome was unfavorable for both dam and fetus, it highlighted the potential for pregnancy with such a congenital anomaly. fetus was of normal size for a singleton lamb and would likely have been delivered without assistance in a dam with normal anatomy. in a large sheep abattoir survey (33,506 tracts were examined), the prevalence of congenital abnormalities was 3.3% and uterus didelphys was 0.02%.5 the outcome for this ewe might have improved if a decision to perform cesarean surgery was made after initial vaginal palpation determining failure for full cervical dilation. the decision made at examination is often fraught with complicating factors; nevertheless, in this case, cesarean surgery might have resulted in a live dam and fetus. however, economics (value of the animal and cost) and animal ability to breed back are important factors to make such decisions. furthermore, the feasibility of providing cesarean surgery should be considered. ewe was a primiparous, unproven, grade dam. it is possible that the congenital anatomical abnormality would not have been identified during surgery, in which case, the ewe might have been rebred presenting similar problems at next lambing. in a survey of congenital abnormalities of ewes at abattoirs in england, there was 1 parous sheep with uterus didephlys.5 this highlighted the importance of taking time during cesarean surgery in primiparous animals to assess reproductive anatomy and advise clients on the risks of rebreeding if the dystocia was not a result figure 2. postmortem image of caudal reproductive tract; left cervix, divided cranial vagina with submucosal hemorrhage, caudal vagina, vestibule, and vulva. cav: caudal vagina; crv: cranial vagina; dvf: dorsoventral fold; lc: left cervix; v: vestibule: vu: vulva http://dx.doi.org/10.58292/ct.v17.12651 4 citation: clinical theriogenology 2025, 17, 12651, http://dx.doi.org/10.58292/ct.v17.12651 of fetopelvic mismatch or a malpresentation. certainly, this type of anatomic abnormality compromises the life of ewe and fetus, and it will depend on the obstetrical skills of the clinician to detect and resolve them appropriately. conflict of interest none to report. learning points • reproductive tract should be completely examined in dystocia cases • congenital anomalies of the reproductive tract can support pregnancy to term • during caesarean surgery in primiparous ewes, examine the uterus for normal anatomy references 1. schlafer dh, foster ra: female genital system. in: jubb, kennedy, and palmer’s pathology of the domestic animals: volume 3. 6th edition, st. louis, 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incidence of retained fetal membranes or improve reproductive performance in crossbred zebu cows. oxytocin therapy immediately after parturition does not reduce the incidence of retained fetal membranes or improve reproductive performance in crossbred zebu cows. r.a. palomares a,c, e. sotoa, j.c. gutiérreza, g. portilloa, j.c. boscana, m.a. monteroa, y. lópeza, g.s. frazer b aunidad de investigación en reproducción animal. facultad de ciencias veterinarias. universidad del zulia, maracaibo, venezuela; bdepartment of veterinary preventive medicine. college of veterinary medicine. the ohio state university, columbus oh, usa; cdepartment of pathobiology, college of veterinary medicine, auburn university, auburn, al, usa oxytocin plays a crucial role during parturition and uterine involution. many pharmaceutical companies recommend the administration of oxytocin to prevent retained fetal membranes (rfm) and improve reproductive performance (rp) in cows. however, there are limited reports to support this approach, and the results are contradictory. the objective of this study was to determine the effect of oxytocin therapy after parturition on the rfm incidence and rp in dual purpose cows under tropical conditions. five hundred thirty six multiparous, crossbred zebu cows were randomly assigned to two groups: oxy (n=280): 30 iu of oxytocin (pituifral® c.a. laboratorios asociados, venezuela) injected i.m. immediately after normal parturition, and again 6 hours later and c (n=256): control. expulsion of fetal membranes was evaluated 24 hours after delivery. cows were subsequently inseminated12 hours after they were detected in estrus. data were analyzed using proc logistic and glm (sas®, cary, nc,usa). oxytocin had no effect on the incidence of rfm (4.6 vs 3.1% for oxy and c, respectively, p>0.05). cows in oxy and c had similar conception and overall pregnancy rates (54.0 vs 47.8% and 75.4 vs 73.4%, respectively, p>0.05). there were no differences between oxy and c for calving to first estrus (83.6 ± 3.7 vs 77.2 ± 3.8 days) and calving to conception intervals (113.6 ± 5.0 vs 110.5 ± 5.2 days) anestrus (13.6 vs 13.7%), repeat breeding (21.8 vs 20.7%) and culling rates (15.7 vs 16.4%). oxytocin therapy after parturition did not reduce the incidence of rfm or improve rp in crossbred zebu cows under tropical conditions. keywords: oxytocin, calving, reproductive performance, zebu, cows 536 1 contact erin lohbeck elohbeck@roodandriddle.com © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12589, http://dx.doi.org/10.58292/ct.v17.12589 case report diagnosis, treatment, and outcome of luteoma in a mare erin lohbeck,a etta bradecamp,a brett woodie,a rachel doenges,a mariah slack,a francine calvarusob arood and riddle equine hospital, lexington, ky, usa buniversity of kentucky veterinary diagnostic laboratory, lexington, ky, usa abstract a 5-year, grade mare in a commercial recipient herd was presented for transrectal palpation and ultrasonography; mare had a follicle on the left ovary that continued to grow for ~ 2 months with typical cyclic activity on the right ovary. blood was submitted for granulosa cell tumor (gct) endocrine panel (inhibin b, testosterone, and antimüllerian hormone [amh]). serum testosterone concentrations (49.5 pg/ml; normal range: 20-45 pg/ml) and amh concentrations (> 14 ng/ml; normal range: 0.1-6.9 ng/ml) were higher, and inhibin b concentrations were normal (61.9 pg/ml; normal range: 60.1-100 pg/ml). based on abnormal left ovarian structure and gct panel results, left ovariectomy was performed; left ovarian luteoma was diagnosed histologically. mare resumed cyclicity on the right ovary and returned to the recipient mare herd. increased inhibin b and amh concentrations were previously reported in 2 mares with luteoma (diagnosed after ovariectomy); however, this is the first report documenting increased testosterone concentrations. although luteoma is an uncommon diagnosis, it should be considered as a differential for abnormal ovarian structures, abnormal behavior, and altered ovarian hormone profile. keywords: horse, ovary, luteoma, testosterone, inhibin b, antimüllerian hormone background abnormalities of the equine ovary are overrepresented by granulosa-theca cell tumors and granulosa cell tumors (gct). these subtypes of ovarian sex cord tumors comprise 85% of reported ovarian neoplasms diagnosed histologically.1 other documented ovarian abnormalities that can occur include hemorrhagic anovulatory follicles, abscesses, thecomas, teratomas, sertoli cell tumor of the ovary, leydig cell tumor, androblastoma, arrhenoblastoma, interstitial gland tumor, and lipid cell tumor of the ovary.2 a luteoma is a neoplastic proliferation of luteal cells that are derived from granulosa cells and are mesodermal in origin. to date, 3 cases of luteomas have been described in the mare.3,4 due to the nature of the affected tissues, many of the ovarian sex  cord tumors produced excess concentrations of antimüllerian hormone (amh), testosterone, and inhibin b.1 presumptive diagnosis of ovarian sex cord tumors in a mare is made based on abnormal ovarian structures identified by transrectal palpation and ultrasonography of the ovary, positive results of serum ovarian hormone panel, and stallion-like behavior; however, ultimately histopathology is required to confirm ovarian neoplasia and type.1 a luteoma is a benign neoplastic proliferation of luteal cells that is rarely diagnosed in mammalian species but has been documented in domestic animals and in women.3,5-8 in  women, luteoma is specific to pregnancy and produces excess androgens, specifically testosterone.5 true incidence is unknown due to varying clinical signs and most of them are identified at cesarian surgery; however it is rare.5 increased hormone concentrations cause changes such as hirsutism and virilization during the second half of pregnancy, and 50% of female infants may experience virilization.5 dissimilar to other species, luteomas in women regress spontaneously following parturition.5 in cattle, the incidence is low (2.2%).6 in cattle, luteomas are typically unilateral and produce progesterone, causing the cow to enter a state of prolonged diestrus that is perceived by owners as a period of anestrus.7 however, if the luteoma is minimally hormonally active, the cow may continue to cycle and conceive.7 after removal of the affected ovary, the cow may return to cyclicity but it is not guaranteed.7 in the female dog, the incidence is also reported to be low, with a single case reported that had clinical signs of abdominal distension and signs associated with hyperadrenocorticism.8 ovarian hormone profile has not been described in the dog and the mechanism for which luteoma induced mailto:elohbeck@roodandriddle.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12589 2 citation: clinical theriogenology 2025, 17, 12589, http://dx.doi.org/10.58292/ct.v17.12589 hyperadrenocorticism is unclear. however, following removal of the ovary and treatment for a subsequent hypoadrenal crisis, the dog’s condition was normalized.8 mares diagnosed with a luteoma typically present with aggressive behavior leading to difficulty in management.4 hormone profiles of documented mares indicated 2 mares with increases in inhibin b with varying increases in amh concentrations and no deviation in plasma testosterone, and an additional mare with no deviations in ovarian hormone profile.3,4 our objective is to describe a luteoma case in a mare that subjectively displayed no aggressive behavior, yet had increases in serum testosterone and amh concentrations with no increase in inhibin. case presentation a 5-year, maiden recipient mare used for commercial embryo transfer was noted to have an enlarged left ovary during routine reproductive transrectal ultrasonography. prior to the development of the abnormal structure, the mare’s reproductive cycles appeared normal with appropriate follicular dynamics and ovulations. following a successful embryo transfer with subsequent development of an anembryonic vesicle, the mare was reported to ovulate appropriately over the subsequent estrous cycle. mare was evaluated by transrectal ultrasonography to determine the possibility of prostaglandin f2α treatment; a 35 mm follicle and a corpus luteum were identified on the left ovary, follicles (20 mm) were present on the right ovary, no uterine edema or intraluminal fluid was observed, and cervix was closed and toned. intramuscular prostaglandin f2α (250 µg; estrumate, merck animal health, rahway, nj) was given and mare was evaluated 2 days later. mare had a 52 mm follicle on the left ovary and a 32 mm follicle on the right ovary; however, there was no uterine edema or intraluminal fluid, and the cervix remained closed. mare’s reproductive tract was evaluated daily by transrectal palpation and ultrasonography. over the next 5 days, the follicle on the right ovary continued to enlarge, grew to 48 mm with firm tone, moderate uterine edema developed, and the follicle ovulated appropriately. the follicle on the left ovary continued to grow to 52 mm and remained firm but did not ovulate. embryo transfer was not possible due to lack of in vivo embryo production by the anticipated donor mares. mare was evaluated 9 days later for prostaglandin treatment. mare’s enlarged follicle on the left ovary measured 93 mm. normal follicular dynamics were observed on the right ovary throughout the examination period. approximately 30 days after initial observation, the structure on the left ovary measured 152 mm with hypoechoic contents and a subjectively thickened rim. on initial examinations, transrectal palpation findings of the left ovary were consistent with a normal ovary with a large dominant follicle and ovulation fossa. however, as the abnormal structure continued to grow to its final diameter of 152 mm, palpation of structures such as the ovulation fossa became less defined and more difficult to appreciate due to the gross size of the ovary and difficulty in manipulation. on day 30, serum was submitted (clinical endocrinology laboratory at uc davis) for gct endocrine profile (amh, inhibin, and testosterone). serum testosterone concentrations (49.5 pg/ml; normal range: 20-45 pg/ml) and amh concentrations (> 14 ng/ml; normal range: 0.1-6.9 ng/ml) were higher, and inhibin b concentrations were normal (61.9 pg/ml; normal range: 60.1-100 pg/ml). treatment due to the persistence and continued growth of the structure on the left ovary combined with the results of the gct panel, the decision was made to remove the left ovary. transrectal ultrasonography was conducted before surgery. what was earlier described as an anechoic follicular structure appeared hyperechoic and had hyperechoic strands (figure 1). standing unilateral left ovariectomy was performed with a flank approach. ovary had to be drained (500 ml of fluid was drained via suction) before removal. ovary was large (~ 10 x 5 cm) and had a well demarcated unencapsulated mass (figure 2); there was hemorrhage within its stroma. ovary was submitted for histopathology.9 outcome mare recovered from surgery and was returned to the recipient herd. an incisional infection developed at the site of the flank laparotomy ~ 3 weeks after surgery, with swelling at the most ventral part of the incision site. a focal ultrasonographic examination of the swelling was performed that was figure 1. transrectal ultrasonographic images of the abnormal structure on the left ovary immediately prior to ovariectomy. both images are from the same evaluation of the structure but are different planes of view; note the hyperechoic appearance of the antral fluid in some fields and thin hyperechoic strands in others (only 12 cm depth is depicted, but the structure extended beyond this field) http://dx.doi.org/10.58292/ct.v17.12589 citation: clinical theriogenology 2025, 17, 12589, http://dx.doi.org/10.58292/ct.v17.12589 3 suspicious for abscess formation. the swelling was aspirated, and 23 ml of purulent discharge was recovered and submitted for aerobic culture. the culture recovered 2 bacteria: streptococcus dysgalactiae ssp equisimilis and escherichia coli. this infection was treated (2 treatments 4 days apart) with intramuscular ceftiofur crystalline free acid (3,200 mg; excede, zoetis servies llc, parsippany, nj). since ovariectomy was in september, routine transrectal palpation and ultrasonography were not performed due to low numbers of possible embryo transfers at that time of year. following complete recovery from surgical site infection, prior to winter, mare was evaluated for 1 cycle (culture and cytology were obtained); mare was cycling appropriately with 1 (right) ovary. to date, mare has not yet had an embryo transfer. on histopathologic examination, the mass consisted of polygonal cells with abundant, finely vacuolated to granular eosinophilic cytoplasm. nuclei were round with stippled chromatin and 1-3 nucleoli (figure 3). no mitotic figures were observed; based on histopathologic findings, the mass was identified as luteoma. discussion the presentation and diagnostic findings of this equine luteoma case differed from those reported3,4 with regards to ovarian hormone profiles, ovarian appearance, and clinical signs. although the most classic clinical sign associated with reported luteomas was aggressive behavior, our mare had no history of this type of behavior. this could provide a discrepancy in indicators for this abnormality, or it was possible that tumor was identified early and was removed before behavior changes started to develop. previous assumptions that testosterone is the driving factor for aggression and stallion-like behavior have recently been challenged by ovarian hormone panel results that do not indicate increases in testosterone or other hormones, despite stallion-like aggression being displayed by the mare in question.3 this suggests that there may be other factors driving the behavior that require more research and evaluation to determine the cause. reports have documented that luteomas rarely cause ovarian enlargement with minimal ultrasonographic notable abnormalities.3,4 this mare differed from reported cases in that the figure 2. removed left ovary (a) and cut section (b); note large cavity previously filled with fluid a b figure 3. light microscopic images of the mass at 20 x magnification (a) and 400 x magnification (b) stained with hematoxylin and eosin; note polygonal cells with abundant, finely vacuolated to granular eosinophilic cytoplasm, round nuclei with stippled chromatin and 1-3 nucleoli and no mitotic figures a baaaaaaaaaaaaaaaaaaaaaaaaaaaa bbbbbbbbbbbbbbbbbbbbbba b http://dx.doi.org/10.58292/ct.v17.12589 4 citation: clinical theriogenology 2025, 17, 12589, http://dx.doi.org/10.58292/ct.v17.12589 affected ovary was substantially larger than the other. in a mare, bilateral ovariectomy was performed due to lack of diagnostic evidence to support removal of one ovary over the other.4 in this case, the left ovary was clearly abnormal, with the right ovary demonstrating normal follicular development, ovulation, and luteal formation. because of this, unilateral left ovariectomy was performed. bilateral ovariectomy, as performed in previous cases, aimed to remove the body’s main source of estrogen and decrease or eliminate clinical and behavioral signs associated with estrus.10 however, 35% of mares undergoing bilateral ovariectomy are reported to continue to have mild signs of behavioral estrus.10 it is unclear as to whether another source of estrogen, purely behavioral components, or if the removal of progesterone and its inhibition on expression of estrus contributed to continued low-level estrus behaviors.10 bilateral ovariectomy successfully resolved behavioral concerns in 64-86% of cases, as reported from the perspective of the owners.11 however, in these cases, mares that had no histologic evidence of gct were also reported to have an improvement in behavior.11 it should be noted that this reference was published based on the results of a survey given to mare owners, so false positives due to subjective bias had the potential to affect results. these authors proposed that this may indicate normal concentrations of reproductive hormones in some mares with behavioral concerns (as assessed by owners).11 classification of ovarian sex cord tumors is based upon the predominant cell population compared to the rest of the surrounding tissue. similarly to previously excised luteomas, this tumor featured polygonal cells with finely vacuolated to granular eosinophilic cytoplasm. absence of spindle cells and call-exner bodies further supported the diagnosis of luteoma, as either of these cell types would indicate granulosa cell tumor or thecoma.2 call-exner bodies are identified as eosinophilic proteinaceous deposits that are surrounded by granulosa cells that are commonly, but not always observed in histologic examination of a gct.2 thecomas, although rare, have been documented to be macroscopically yellow due to lipid and microscopically composed of spindle cells with vacuolated cytoplasm containing lipids.12 it is commonly accepted that a serum ovarian hormone profile demonstrating increases in testosterone, amh and/or inhibin b is associated with gct, with amh being more specific for gct than either inhibin b or testosterone.1,13 the sensitivity of increased serum inhibin concentrations in gct was as high as 96%; however in this study, 74% of mares with other ovarian abnormalities also had increased concentrations.13 other studies cited sensitivity of inhibin to be 60,14 73,15 85,16 and 87%.17 in the same study, the sensitivity of an increase in serum amh was 100% sensitive for a gct, with only 4.8% of mares with another ovarian abnormality having an increase.13 a second study determined the sensitivity of amh to be 98%.18 in the case of testosterone, the sensitivity of increased testosterone concentrations in gct was 55%.16 in contrast to the hormone profiles associated with gct, the changes in amh, testosterone, and inhibin b serum concentrations in luteomas remains unclear. in 3 reported cases, serum amh was increased in 2 cases, serum inhibin b increased in 1 case, but no change in testosterone in any cases.3,4 the mare presented in this case had increases in amh and testosterone. the findings in this case demonstrated that the diagnosis of a specific ovarian neoplasm cannot be based solely on diagnostic imaging and amh, testosterone or inhibin concentrations. in the mare, the corpus luteum is comprised of large luteal cells that are almost completely derived from granulosa cells.19 this is in contrast to ruminants in which the luteal cells originate from both the theca and granulosa cells as small and large luteal cells, respectively.19 the luteal cells are responsible for production of progesterone in diestrus and maintenance of pregnancy until placental progesterone is produced ~ day 180 of pregnancy.19 one might suspect that progesterone would also be increased in the case of a luteoma due to its structure; however, this is currently unknown as it was not reported. in this case, progesterone was not tested; however, it is believed that it was not abnormally elevated due to the presence of estrous signs such as uterine edema. in these cases, histopathology is required in order to classify the tumor. however, the varying presentation can suggest that clinical signs with no other explanation of cause may be sufficient to warrant ovariectomy, despite no aberrations in serum ovarian hormone concentrations. following unilateral ovariectomy in the fall, the mare continued to cycle appropriately on the right ovary. routine transrectal palpation and ultrasonography will resume in the spring, and it is uncertain whether the mare will continue or resume cycling following transition and retain fertility. this is the only known published case of luteoma treated with unilateral ovariectomy.3,4 additional case studies would be required to determine future fertility after unilateral ovariectomy in mares diagnosed with luteoma. in theory, the affected ovary could have been left since mare was cycling; however, it is likely that mare would eventually stop cycling due to the negative feedback from hormones produced by the luteoma. this factor had a role in the decision to ovariectomize, due to recovery time and the desire to use the mare as an embryo recipient in the following breeding season. learning points • aberrations in serum ovarian reproductive hormone concentrations (amh, inhibin b, and testosterone) that are consistent with gct may also observed in luteomas • normal estrous cyclicity may be observed in a mare with a unilateral luteoma • absence of behavioral aggression does not rule out the presence of an ovarian sex cord tumor conflict of interest none to declare. references 1. mccue pm, roser jf, munro cj, et al: granulosa cell tumors of the equine ovary. vet clin north am equine pract 2006;22:799-817. doi: 10.1016/j.cveq.2006.08.008 2. agnew dm, maclachlan nj: tumors of domestic animals. hoboken; john wiley & sons: 2017. p. 689-701. 3. renaudin cd, kelleman aa, keel k, et al: equine granulosa cell tumors among other ovarian conditions: diagnostic challenges. equine vet j 2020;53:60-70. doi: 10.1111/evj.13279 4. combs e, beachler t, troy jr, et al: luteoma in two mares treated by ovariectomy. equine vet educ 2021;38:318-322. doi: 10.1111/ eve.13582 http://dx.doi.org/10.58292/ct.v17.12589 https://doi.org/10.1016/j.cveq.2006.08.008 https://doi.org/10.1111/evj.13279 https://doi.org/10.1111/eve.13582 https://doi.org/10.1111/eve.13582 citation: clinical theriogenology 2025, 17, 12589, http://dx.doi.org/10.58292/ct.v17.12589 5 5. korenaga trk, tewari ks: gynecologic cancer in pregnancy. gynecol oncol 2020;157:799-809. doi: 10.1016/j.ygyno.2020.03.015 6. mcentee k: ovarian neoplasms. in: reproductive pathology of domestic mammals. burlington; elsevier science: 1990. p. 69-93. 7. grunert e: ovarielle dysfunktionen [ovarian disfunction]. in: grunert e, berchtold m, de kruif a, et al: editors. fertilitätsstörungen beim weiblichen rind [fertility disorders in female cattle]. 3rd edition, berlin; parey: 1999. p. 111-142. german. 8. yamini b, vandenbrink pl, refsal kr: ovarian steroid cell tumor resembling luteoma associated with hyperadrenocorticism (cushing’s disease) in a dog. vet pathol 1997;34:57-60. doi: 10.1177/030098589703400112 9. mckinnon ao, voss jl, colburn, gt: ovariectomy. in: mckinnon ao, voss jl: editors. equine reproduction. 1st edition, media; williams & wilkins: 1993. p. 452-453. 10. hooper rn, taylor ts, varner dd, et al: effects of bilateral ovariectomy via colpotomy in mares: 23 cases (1984-1990). j am vet med assoc 1993;203:1043-1046. 11. stratico p, hattab j, guerri g, et al: behavioral disorders in mares with ovarian disorders, outcome after laparoscopic ovariectomy: a case series. vet sci 2023;10:1-11. doi: 10.3390/vetsci10080483 12. prestes nc, nogueira de moraes c, maia l, et al: ovarian tumor in a mare—thecoma—case report. j equine vet sci 2013;33:196200. doi: 10.1016/j.jevs.2012.06.007 13. murase h, ball b, tangyuenyong s, et al: serum anti-mullerian hormone concentrations in mares with granulosa-cells tumors versus other ovarian abnormalities. j equine vet sci 2018;60:6-10. doi: 10.1016/j.jevs.2017.10.012 14. sherlock ce, lott-ellis k, bergren a, et al: granulosa cell tumours in the mare: a review of 52 cases. equine vet educ 2016;28:75-82. doi: 10.1111/eve.12449 15. almeida j, ball ba, conley aj, et al: biological and clinical significance of anti-mullerian hormone determination in blood serum of the mare. theriogenology 2011;76:1393-403. doi: 10.1016/j. theriogenology.2011.06.008 16. ball ba, conley aj, almeida j, et al: a retrospective analysis of 2,253 cases submitted for endocrine diagnosis of possible granulosa cell tumors in mares. j equine vet sci 2014;34:307-313. doi: 10.1016/j.jevs.2013.07.005 17. mccue pm: equine granulosa cell tumors. proc am assoc equine pract 1992. p. 587-593. 18. ball ba, almeida j, conley aj: determination of serum anti-mullerian hormone concentrations for the diagnosis of granulosa-cell tumours in mares. equine vet j 2013;45:199-203. doi: 10.1111/j.2042-3306.2012.00594.x 19. niswender d, nett tm: luteal phase. in: mckinnon ao, voss jl: editors. equine reproduction. 1st edition, media; williams & wilkins: 1993. p. 172-175. http://dx.doi.org/10.58292/ct.v17.12589 https://doi.org/10.1016/j.ygyno.2020.03.015 https://doi.org/10.1177/030098589703400112 https://doi.org/10.3390/vetsci10080483 https://doi.org/10.1016/j.jevs.2012.06.007 https://doi.org/10.1016/j.jevs.2017.10.012 https://doi.org/10.1111/eve.12449 https://doi.org/10.1016/j.theriogenology.2011.06.008 https://doi.org/10.1016/j.theriogenology.2011.06.008 https://doi.org/10.1016/j.jevs.2013.07.005 https://doi.org/10.1111/j.2042-3306.2012.00594.x 2010: maximizing litter size in the bitch maximizing litter size in the bitch cheryl lopate reproductive revolutions, inc., aurora, or introduction when we plan to breed a bitch, one of our main goals is to maximize litter size. having large litters provides several functions: 1) ensures that there will be enough stimulus from the fetuses to initiate labor; 2) increases the genetic diversity of the litter; 3) helps controls fetal size; 4) maximizes the choice of puppies for clients and breeders; 5) increases socialization activity among the puppies. in order to increase reproductive efficiency we must understand the factors we can influence and those that are predetermined. by making proper choices, veterinarians and breeders can improve their litter statistics. keywords: dog, litter size, breeding, ovulation, pregnancy breed small breed dogs tend to have smaller litters than large breed dogs, while the giant and large breed dogs tend to have the largest litters. the american kennel club (akc) evaluated litter size from their 15 most popular breed registrations over a three year period and the results are reported in table 1. genetics some bloodlines are more prolific than others. in any given breed, there are lines that tend to produce at the high end of the litter size range while others produce at the low end of the range. this likely has to do with ovulation rate more than uterine capacity, since most bitches of similar breed have similar sized uteri. there is a genetic component to pathology of the bitch’s uterus as well, with some lines and breeds developing inflammation, scarring and related changes at much earlier ages then others. there may be a genetic component to the male’s fertility as well, although the impact on litter size is not clear. one common example of genetics and infertility in the male is immune mediated orchitis and epididymitis which has been shown to be familial in some cases. in these cases, the male typically proves to be fertile at a young age, only to lose their capacity to successfully mate in early – middle age. so the impact on litter size in these cases is likely minimal. the genetic combination of the male and female may have more impact genetically if there is some type of chromosomal incompatibility between the selected sire and dam lines either resulting in fertilization failure or embryo/fetal development. in humans it has been shown that 15% of miscarriages are due to genetic incompatibility. the gene pool for each breed of dog is certainly far smaller than the human gene pool, so there must be some impact on pregnancy rate and litter size associated with genetic incompatibility. the extent of this impact is not known. inbreeding can impact litter size. the inbreeding coefficient measures how inbred an individual is and ranges from 0 (completely unrelated individuals) to 1.0 (completely inbred; i.e., brother/sister mating). as the inbreeding coefficient increases, the litter size decreases and the number of stillborn puppies increases. breeders tend to choose the animal(s) they want to breed on factors other than reproductive performance, such as conformation, athletic ability, temperament, etc. in other litter bearing species, like the rabbit and pig, it has been shown that selectively breeding for litter size quickly increases the average litter size of the line. so if litter size is of the utmost importance, breeders should selectively chose the bitches with the highest ovulation rates and they will likely see coincident increases in their litter size. age very young and very old bitches are less fertile than bitches between 1.5 and 5 years of age. very young bitches tend to ovulate fewer follicles, while older bitches have more chance for pathology in the uterus in the form of acute or chronic inflammation and cystic change which results in an inhospitable environment for the fetuses, a greater incidence of resorption, abortion, and stillbirths. if a bitch is bred from 1.5 to 2 years of age onward, on every cycle, the first litter born will have a lower litter size than the 489 second litter because the uterine wall has never been stretched before and so space is somewhat constrained coupled with decreased ovulation rate. the subsequent three litters tend to increase in size and then from the fourth litter onward litter size will begin to decline again (figure 1). if a bitch is middle aged for her first litter, one cannot expect her maximal litter size to compare to a bitch bred at two years for her first litter, since some aging change has already likely occurred in her uterus, negatively impacting litter size. reproductive performance will decrease at an earlier age in large and giant breed bitches compared to small and medium breeds. the largest litter a bitch will produce will therefore vary with her breed and size. for large and giant breed bitches we consider the height of their reproductive potential to be around five years of age, while for toy and small breeds, they might not reach their reproductive peak until eight to ten years of age (see table 2 – extrapolated from the three year akc study). when deciding when to breed your bitch for the first time, this should be taken into consideration, and breeding instituted well before their peak performance is anticipated in order to maximize litter size during her reproductive lifetime. ovulation timing one of the most important determinants of litter size is breeding the bitch during her fertile period. we know that when bitches have atypical cycles they will stand to be bred at either prior to or after their fertile period. even experienced males will be interested in the female outside their fertile window. thus, the use of receptive behavior +/the instincts of the stud dog alone will result in nonpregnancy in a portion of bitches that stand outside the fertile window. breeding outside the fertile window may still result in pregnancy because of the length of time sperm can survive in the female’s reproductive tract, but litter size will not be optimized. accurate ovulation timing is necessary in order to be sure that viable sperm are delivered to the oviductal reservoir so that they are present as the eggs are maturing. poor breeding management is the number one cause of non-pregnancy or small litter size. determination of the appropriate time to breed a bitch requires accurate ovulation timing including vaginal cytology, speculum examination of the vaginal mucosa, serum progesterone concentrations and sometimes luteinizing hormone (lh) measurement. the more of these techniques that are combined to determine the fertile window the more accurate the assessment will be. in some cases, vaginal cytology will be performed until the bitch appears to be mid-late proestrus and then the addition of progesterone (and lh) measurement may be initiated. the bitch ovulates immature eggs (ova or oocytes) and they require two to three days to mature before fertilization can begin. after the eggs mature, they will remain healthy and fertilizable for about two to three days and then will slowly start to degenerate. we now know that ova may be fertilized for up to 200 hours (eight days) post-ovulation if they can be accessed by spermatozoa. often the limiting factor regarding fertilization is the ability of the sperm to gain access to the uterus. once progesterone begins to rise rapidly the cervix begins to close. the cervix closes within 7 +/1 days after the lh surge. for bitches whose cervices close by day 6 after the lh surge, they will often still be in standing heat, but their closed cervix will block the entry of sperm to the uterus and thereby prevent fertilization from occurring even though the eggs may still be viable. in cases, where bitches are being bred for the first time late in the fertile window, use of an insemination technique that bypasses the cervix (transcervical or surgical insemination) may be a more appropriate choice. type of breeding depending on the type of semen being used, the quality of the semen, the age of the dog and/or bitch, and number of inseminations, a decision must be made on what type of breeding should be performed. the types of breeding that are available include natural breeding, vaginal artificial insemination (vai), transcervical artificial insemination (endoscopic or norwegian catheter technique; tci), or surgical artificial insemination (sai). natural breeding is typically best for younger dogs and bitches with normal fertility and semen quality. novice breeders or aged dogs may have difficulty achieving a normal copulatory lock (inside tie) and sperm numbers delivered to the cervix are 490 significantly reduced during an outside tie. bitches with strictures may also only be able to achieve partial intromission. significant size discrepancies between the dog and bitch may preclude normal mating. fear or anxiety of either the bitch or dog may prevent a successful mating as well. when any of the above situations is presented, if semen quality is compromised or if the bitch has a fertility issue, an alternative form of insemination may be desirable. it has been shown that the percent motility or percent morphologically normal sperm in an ejaculate is not the limiting factor for successful pregnancy, but rather the total number of motile, morphologically normal sperm inseminated. this means that the type of insemination chosen must be such that adequate sperm numbers are delivered to the oviductal reservoirs. vaginal artificial insemination may be used when semen quality and bitch fertility are adequate but a breeding cannot be physically accomplished. vaginal artificial insemination is also commonly used when excellent quality chilled semen with adequate sperm numbers is shipped to the bitch. alternatively, if chilled semen of lesser quality or lower numbers are shipped or if semen quality is somewhat diminished, sperm may be deposited in the uterus via tci. frozen semen is also sometimes inseminated via tci. however, in many cases, if semen quality is poor, if bitch fertility is poor or questionable, or if frozen semen is being used, intrauterine insemination may be more desirable using sai. surgical artificial insemination allows the clinician to get his/her hands on the uterus to assess it for pathology, to manually rupture any cysts that are present, and to equally divide a small or poor dose of semen between both horns closer to the ovaries and oviductal reservoirs. a large scandanavian study evaluated type of insemination performed and showed that uterine insemination, whether by natural mating, tci or sai, and regardless of the type of semen used, resulted in about 20% higher whelping rates than vai. number of inseminations ensuring that the entire fertile window is covered by fresh, viable semen will greatly increase litter size. multiple matings during a single cycle will increase litter size and pregnancy rates. with natural breeding, bitches are ideally bred every other day from one to two days post-ovulation until five to six days post-ovulation. breeding is not recommended on a daily basis as this may result in depletion of sperm from the male’s reproductive tract and thereby decreasing numbers of sperm are present in each subsequent ejaculate. by the time the fertile window is reached, there may not be adequate sperm numbers for conception. allowing 48 hours between matings allows for replenishment of the epididymal tail (the sperm storage site) with adequate numbers of sperm. in cases where the male is young or old (so sperm numbers may be decreased) or sperm numbers are low, it is even more important to allow for this time for replenishment of the sperm numbers. with vai or tci, two inseminations are usually recommended. ideally, the first is done two days after ovulation and the second, two days later. depending on availability of semen, alternate days would be one and three days post-ovulation. when frozen semen is used with tci, two inseminations are performed on each of days three and four post-ovulation. with sai, often a single breeding is performed. with fresh or fresh-chilled semen, a single sai would be performed on either day two, three or four postovulation. with frozen semen, a single sai would be performed on day three or four post-ovulation. if frozen semen quality is diminished, adding a second insemination may increase litter size by expanding the amount of time oocytes are exposed to viable sperm during the fertile window. in these cases, both a tci and a sai may be performed (in no particular order) on days three and four post-ovulation. type of semen historically, breeders felt that conception rates were best with natural breeding, but with good quality semen and a fertile bitch, the use of fresh chilled or frozen semen with good ovulation timing now results in much more comparable pregnancy rates. however, when semen quality is diminished, pregnancy rates and litter size will decrease accordingly. generally speaking, the less manipulation that occurs in semen handling and processing, the better the quality will be. so, fresh semen typically has better success over fresh-chilled, which in turn has better success over frozen semen. when offered the 491 choice of excellent quality frozen semen or moderate to poor quality fresh or fresh-chilled semen, the preference to maximize litter size would be to use the frozen semen. a large scandanavian study evaluated the different types of semen and found that either fresh, fresh-chilled or frozen semen had comparable whelping rates, but they were all about 20% lower compared to natural mating when similar insemination techniques were used. semen quality the higher the quality of the semen, the better the chances of optimizing litter size. when assessing semen quality it is critical to get a recent semen evaluation. semen should be evaluated for motility (both total and progressive), velocity, concentration, morphology and the presence of other cells (white blood cells, red blood cells or immature germ cells). partial semen evaluation or evaluations performed more than two weeks prior to semen collection may not correctly reflect on semen quality at the time of the breeding. particular attention should be paid to semen morphology (shape and size of individual sperm cells) since many times motile sperm may have defects that will prevent them from fertilizing the eggs properly. again, it is the total number of morphologically normal, motile sperm delivered that seems to be the critical factor. in some cases, a dog may have high numbers of abnormal or immotile sperm, but if sperm numbers are high enough this problem may be overcome (compensable). acceptable breeding doses for fresh or fresh chilled semen for vai in medium sized bitches seems to be around 200 to 225 million normal, motile sperm, while acceptable breeding doses for intrauterine insemination of fresh, fresh-chilled or frozen semen seem to be between 100 to 150 million normal, motile sperm. season litter size and pregnancy rates are increased in spring compared to summer in latitudes that have distinct changes in season and daylight hours. nutrition once pregnant, bitches will do everything in their power to provide for the growth and development of their litters including removing their own body mass and nutrients to nurture the growth and development of their puppies. therefore, bitches should be in good body condition prior to breeding. pregnancy diagnosis via ultrasound is recommended to determine first if the bitch is pregnant, and then to estimate fetal numbers. bitches should be fed differently based on litter size. bitches with large litters that are not fed adequately will pull from their own fat and protein stores to provide for fetal growth. if this nutritional deficiency is large enough fetal resorption or even abortion may occur. bitches should be fed a diet that is 30% protein, 20% fat with 20 30% carbohydrates. important supplemental nutrients in the diet include omega 6 and 3 fatty acids which should be at a ratio of 5:1 to 15:1. calcium and phosphorus are the two most important minerals in the diet. feeding of too much calcium or an improper ca:p ratio may result in improper functioning of the bitch’s mechanism for provision of calcium for fetal development, labor and lactation. the ca:p ratio should be between 1.2:1 to 1:1. when choosing a diet the numbers of kcal/cup metabolizable energy is also important. bitches with large litters should be fed diets with at least 450 kcal/cup so that when the bitch approaches term and stomach capacity is decreased due to increased pressure from an ever-enlarging uterus, adequate caloric intake with the proper ratio of all nutrients can be provided. bitches with smaller litters may be fed diets that are at least 350 kcal/cup and still be able to provide adequate calories. breeders should ensure that the diet they chose is manufactured according to association of american feed control officials (aafco) standards. feeding other supplements is generally not required unless specifically recommended by your veterinarian. supplementation of foods like cottage cheese or other dairy products or beef liver will significantly alter calcium, phosphorus and protein content in the diet. so care should be taken when adding supplements to the diet. 492 prior litter size should also be accounted for here. if a bitch has become overly thin after nurturing a large litter and she is bred during the subsequent cycle, her next litter may be smaller because she has not had enough time to recondition herself to feed another large litter. so, her energy stores are still being diverted towards replenishing her own body condition, and are not directed at the fullest potential for ovarian function resulting in decreased ovulation rate and subsequent litter size. summary it is clear that there are many factors involved in determining litter size in the bitch. some are easily controlled, some less so. it is important for the breeder to try to maximize bitch health, promote excellent genetics (including breeding animals with superior reproductive performance), maintain proper nutrition and spend time selecting the dog, type of semen used and form of breeding in order to maximize reproductive efficiency in the kennel. references 1. kelley rl: canine reproductive management: factors influencing litter size. proc annu meet soc therio 2002. 2. root-kustritz mv: decreased litter size in the bitch. proc breeder symp annu meet soc therio 2006. 3. kelley rl: factors influencing canine reproduction and nutritional management of the pregnant bitch. proc tufts anim expo 2001. p. 9-14. 4. linde-forsberg c: what can be learned from 2500 ais in the dog? proc annu wsava congr 2002. 5. mickelson wd, memon ma, anderson pb, et al: the relationship of semen quality to pregnancy rate and litter size following artificial insemination in the bitch. theriogenology 1993;39:553-560. 6. verstegen jp, silva ldm, onclin k: determination of the role of cervical closure in fertility regulation after mating or artificial insemination in beagle bitches. j reprod fertil suppl 2001;57:31-34. 7. tsutsui t, takahashi f, hori t, et al: prolonged duration of fertility of dog ova. proc 6th int symp canine feline reprod and 6th biannual eur vet soc small anim reprod cong 2008. 493 table 1. summary of akc registrations and litter size* breed numbers of litters registered over 3 years litter size (range)† average litter size most commonly reported litter size (% reported of total registrations) labrador retriever 85113 5 – 10 7.6 8 (16.54%) poodle 69755 2 – 5 3.4 3 (25.01%) dachshund 59935 2 – 6 3.9 4 (24.04%) pomeranian 56976 2 – 4 3.0 3 (26.90%) chihuahua 55513 2 – 5 3.3 3 (23.99%) yorkshire terrier 53141 2 – 5 3.3 3 (24.08%) shih tzu 48667 2 – 6 4.1 4 (23.69%) rottweiler 46805 4 – 10 6.6 7 (12.44%) german shepherd 44537 4 – 9 6.6 8 (14.08%) beagle 42823 3 – 7 5.0 5 (17.67%) american cocker spaniel 39019 3 – 7 5.0 5 (18.62%) golden retriever 38233 5 – 10 7.6 8 (16.17%) miniature schnauzer 32235 3 – 6 4.6 5 (21.01%) shetland sheepdog 28421 2 – 6 4.3 4 (20.03%) american boxer 27098 4 – 8 5.8 6 (16.17%) *adapted from kelley rl, proc annu meet soc therio 2002 and root-kustritz mv, proc soc therio breeders symposium 2006. †the number of puppies within +/1 standard deviation of the mean figure 1. effect of litter number on litter size in beagles 5.8 6 6.2 6.4 6.6 6.8 7 7.2 litter number litter size vs number of litters litter #1 litter #2 litter #3 litter #4 litter #5 494 table 2. effect of age on litter size‡ breed age (yrs) at peak litter size§ critical age** labrador retriever 1 3 5 poodle 1 – 5 9 dachshund 1 4 8 pomeranian 1 – 4 10+ chihuahua 1 – 4 10+ yorkshire terrier 1 7 8 shih tzu 1 4 8 rottweiler 1 3 5 german shepherd 1 3 5 beagle 2 3 6 american cocker spaniel 1 3 6 golden retriever 1 3 5 miniature schnauzer 2 3 6 shetland sheepdog 1 4 8 american boxer 1 2 5 ‡adapted from kelley rl, proc annu meet soc therio, 2002 §based on number of puppies reported born ** the year when litter size decreases below 15% of breed average 495 496 omniblank: 2009: legal issues in the pharmaceutical management of bovine reproduction legal issues in the pharmaceutical management of bovine reproduction 1 m. g. riddell, jr. 2 3 american association of bovine practitioners, box 3610, auburn, al, usa 4 5 abstract 6 the ability for the bovine practitioner to implement reproductive management 7 programs is scientifically well-based and clinically proven. however, as is being 8 encountered in many areas of food animal medicine, science is no longer the major 9 determinant of how a veterinarian practices or how a producer manages. extra-label drug 10 use (eldu) allowed under the animal medicinal drug use clarification act was not 11 extended to drugs used for production purposes, such as reproduction. therefore, eldu 12 of drugs for reproductive purposes is not technically allowed in the code of federal 13 regulations (cfr). compounding of drugs from raw pharmaceutical agents, except for a 14 few specifically listed antidotes, is a violation of the cfr. further, the use of illegally 15 compounded products in food animals is ill-advised not only due to the legal and liability 16 issues, but also because of a potential consumer perception issue. a final point to 17 consider is the consumer reaction when current bovine reproductive management 18 programs are presented to the consuming public by anti-agriculture activists. 19 keywords: amduca, compounding, management, reproduction, extra-label 20 21 amduca and eldu in reproductive management protocols 22 the animal medicinal drug use clarification act1 (amduca) of 1994 and the 23 implementing regulations2 were limited to drugs used for therapeutic purposes only. 24 269 numerous comments to the proposed rule supporting the extension of the regulations to 25 drugs used for reproductive management were considered but denied in the final rule. it 26 is important to note that amduca applies only to food and drug administration (fda 27 or the agency) approved drugs. the current situation has resulted in any extra-label use 28 of reproductive drugs being illegal. the u.s. fda has adopted an unofficial position of 29 regulatory discretion. any official position would require the generation and publication 30 of a compliance policy guideline (cpg) and the fda has not indicated that it would 31 consider taking this action. 32 the fda’s reasoning behind placing low regulatory priority on the issue of using 33 drugs approved for reproductive purposes in a manner other than on the label is that these 34 drugs have been proven safe and effective, and the dose, route and species are not altered. 35 for example, the drug most often used in an extra-label manner is gonadotropin releasing 36 hormone (gnrh). gnrh is a decapeptide and poses virtually no risk of residues nor any 37 target animal safety issues. for these reasons, the agency has suggested that they 38 currently will not pursue any actions directed at extra-label use of reproductive 39 hormones. at the same time, the fda has indicated that they would be glad to entertain 40 and possibly facilitate the approval of additional label claims for gnrh products so that 41 the need for eldu would be minimized or eliminated. 42 one final point which should be made regarding eldu of reproductive drugs is 43 that of client informed consent. eldu has been the basis for at least one major lawsuit 44 against a veterinary practice. in today’s litigious society, ensuring that clients are aware 45 of the extra-label nature of any use is appropriate. having an informed and consenting 46 270 client is ever more important when such eldu is not in compliance with the code of 47 federal regulations. 48 compounded drugs 49 compounding medications from unapproved drugs or raw pharmaceutical agents 50 is illegal. references to this can be found in the amduca regulations1 and in an fda 51 cpg.3 there are exceptions in which drugs can be compounded under this cpg, but 52 these are limited to certain specified antidotes only. the most common drug 53 compounded for use in reproductive management protocols is some form of estrogen. 54 estrogens have been proven effective in initiating follicular waves both in embryo 55 transfer programs and in routine breeding management systems. 56 however, in this case science has surpassed the veterinarian’s armamentarium of 57 approved drugs and, while the use of compounded estrogens may be tempting due to 58 effectiveness, the potential for regulatory action and consumer backlash makes the use of 59 compounded estrogens ill-advised. currently the fda lists one estrogen 60 (diethylstilbestrol) on the list of drugs prohibited for extra-label use in food animals. an 61 estrogen (estradiol cypionate) which was registered in 1953 but never approved has been 62 withdrawn from the market by the sponsor based upon an impending request for an 63 official approval application. 64 of all the dugs used in cattle, both label and extra-label, the single drug with the 65 greatest “headline potential” is any form of estrogen. the average consumer’s lack of 66 familiarity with animal husbandry and modern agricultural production practices, make 67 the consideration of consumer acceptance relevant to the discussion of both eldu and 68 the use of compounded drugs in food producing animals. this significant downside to 69 271 the use of a drug compounded from unapproved products (specific antidotes are 70 exceptions) makes such drug use illogical and irresponsible. 71 consumer/public perception 72 animal agriculture has numerous detractors in the form of activists. the issues 73 which are used to malign the animal agriculture industries to the consuming public 74 include, but are not limited to, animal welfare and factory farms. because less than 1% 75 of the population of the u.s. is involved in agriculture, the activist groups find the 76 consuming public easily misled about commonly accepted, well proven, welfare-friendly 77 and accepted management practices. the portrayal of modern agriculture as factory 78 farming or intensive farm animal production is negatively compared to the typical small 79 farming operation of 50 and 60 years ago. with this as background, some of the current 80 reproduction management protocols which utilize multiple injections of various products 81 coupled with timed artificial insemination would represent easy targets for malicious mis-82 representation to the uninformed public. the point of this thread is not to argue that the 83 management protocols are indefensible, rather that the discussion will have to ultimately 84 be directed at a consuming public unfamiliar with any aspect of animal agriculture. the 85 science-based discussions held with colleagues regarding choice or efficacy of protocol 86 will not apply when the production practices are discussed in a public forum. the 87 impacts that these programs have on animal productivity, operational sustainability and 88 the production of a safe, abundant and affordable food supply will be the key 89 communication points. 90 summary 91 272 extra-label use of drugs in reproductive management programs is not allowed 92 under amduca. therefore, eldu of drugs for reproductive purposes is not technically 93 allowed in the cfr. compounding of drugs from raw pharmaceutical agents, except for 94 a few specifically listed antidotes, is a violation of the cfr. in addition, the use of 95 illegally compounded products in food animals is ill-advised not only to the legal and 96 liability issues, but also because of a potential consumer perception issue. a final point 97 to consider is the consumer reaction when current bovine reproductive management 98 programs are presented to the consuming public by anti-agriculture activists. 99 references 100 1. animal medicinal drug use clarification act of 1994, pub. l. no. 103-396 101 (october 22, 1994). 102 2. extralabel drug use in animals, 21 c.f.r. sect. 530 (1996). 103 3. compliance policy guide (cpg) section 608.400 entitled “compounding of 104 drugs for use in animals” 105 (http://www.fda.gov/ora/compliance_ref/cpg/cpgvet/cpg608-400.html) 106 273 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left 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true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname () /pdfxtrapped /false /createjdffile false /description << /enu ([based on 'no compression'] [based on 'no compression wbleeed'] [based on '[high quality print]'] use these settings to create adobe pdf documents for quality printing on desktop printers and proofers. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes false /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: antimicrobial therapy in bovine reproduction antimicrobial therapy in bovine reproduction 1 m.a. edmondson 2 department of clinical sciences, college of veterinary medicine, auburn university, 3 auburn, al, usa 4 abstract 5 the use of antimicrobials, both systemically and locally, has been a hallmark of 6 therapy in the treatment of uterine infections in cattle. however, the use of 7 antimicrobials has not been without controversy regarding their efficacy, effects on future 8 fertility, risk for bacterial resistance and potential residues. this article reviews the 9 immunology and pathophysiology of postpartum uterine infections in cattle and evaluates 10 research regarding the use and efficacy of local and systemic antimicrobial therapies. 11 keywords: bovine uterus, metritis, endometritis, antibiotic therapy 12 introduction 13 the use of antimicrobials has been a conventional therapy in treatment of uterine 14 infections in cattle although the use of antibiotics has not been without controversy. 15 debate continues regarding antimicrobial efficacy, effects on future fertility, risk for 16 bacterial resistance and residues. the proper use of antimicrobials to treat uterine 17 infections must first begin with an appropriate diagnosis and thorough understanding of 18 the immunology of the uterus, the pathophysiology of uterine infections, and the 19 properties of the various antimicrobial agents that may be used therapeutically. 20 normal uterine involution 21 understanding normal uterine involution is vital to understanding and defining 22 postpartum disease in cattle. lochia are normally present until 14 to 23 days 23 233 postpartum.1 after placental detachment, uterine involution is complete in an average of 24 39 days in normal cows. by day 6 postpartum, caruncle septa are disorganized, and by 25 day 15 caruncles are completely sloughed due to necrosis. by day 26 to 30 postpartum, 26 the surface of the endometrium is covered by new endometrium.2 cervical involution is 27 slower than uterine involution and by day 15 postpartum, the diameter of the cervix 28 normally exceeds that of the uterine horns. reported times for gross involution of the 29 uterus and cervix vary from 25 to 47 days. complete histologic uterine involution takes 30 longer than palpable involution and occurs at 42 to 50 days.1 31 infection of the bovine uterus 32 the majority of cattle experience bacterial contamination of the uterus at the time 33 of parturition. in the normal cow, the uterus is cleared of this bacterial contamination by 34 four weeks postpartum and when these bacteria are not cleared by the cow’s defense 35 mechanisms, a uterine infection ensues. numerous bacteria have been isolated from the 36 cow’s postpartum uterus, some of which may be incidental and not cause problems. 37 uterine infections are most commonly due to arcanobacterium pyogenes. the gram 38 negative anaerobes fusobacterium necrophorum and bacteroides melaninogenicus are 39 frequently associated with a. pyogenes. other organisms that may be associated with 40 uterine disease in the cow include pseudomonas aeruginosa, staphylococci, hemolytic 41 streptococci, coliforms, etc. clostridium sp. may occasionally infect the uterus and cause 42 a severe gangrenous metritis or tetanus. uterine infections in the cow are associated with 43 retained fetal membranes, dystocia, and delivery of twins.3 44 metritis is the result of severe inflammation involving all layers of the uterus – 45 endometrial mucosa and submucosa, muscularis, and serosa. metritis usually develops 46 234 during the first week after calving and is associated with dystocia, retained fetal 47 membranes, and calving trauma. these cattle may be septic and present with fever, 48 depression, and anorexia. a copious amount of fetid vaginal discharge may also be 49 present. endometritis is characterized by inflammation of the endometrium extending no 50 deeper than the stratum spongiosum. cows with endometritis are usually not 51 systemically ill, and bacteria are usually eliminated after a few estrous cycles. 52 endometritis is characterized by a mucopurulent or purulent uterine discharge associated 53 with a chronic uterine infection, usually later than 3 weeks postpartum.1 54 diagnosing uterine infections 55 uterine infections are most commonly diagnosed at routine examination of 56 postpartum cows, at breeding, or upon examination of a sick cow. a diagnosis of metritis 57 or endometritis may be made by a variety of methods which include: clinical signs, rectal 58 palpation, vaginoscopy, ultrasonography, uterine culture, uterine cytology, and uterine 59 biopsy. clinical signs of uterine infection vary with the virulence of the causative 60 organism and the severity of predisposing conditions of the cow. uterine discharge may 61 vary considerably in color; however, discharges are not usually considered abnormal 62 unless the uterine fluid is fetid or if the cow has clinical signs of sepsis. palpation per 63 rectum is the most commonly used technique for evaluating the degree of uterine 64 involution prior to breeding and is used to diagnose endometritis through evaluation of 65 the size and consistency of the uterus and cervix and the presence of fluid within the 66 lumen of the uterus. if involution is normal, fluid should not be palpable within the 67 uterine lumen by 14 to 18 days postpartum. in cases of severe metritis, the uterus will be 68 enlarged and friable with occasional adhesions between the uterus and other organs or the 69 235 body wall. although rectal palpation is commonly used for diagnosis of endometritis, it 70 is neither sensitive nor specific. the evaluation of purulent exudate with the aid of a 71 vaginal speculum may also be a useful tool for diagnosing endometritis. 72 ultrasonography has also been used to evaluate and characterize intralumenal fluid 73 within the uterus as well as the thickness of the uterine wall. uterine infections are 74 associated with intrauterine fluid with echogenic particles and a variably thickened 75 uterine wall. bacterial culture is rarely used as treatment must be initiated before culture 76 results are available. culture is more commonly used in cases where cows fail to respond 77 to treatment. endometrial biopsy is not used as commonly in cattle as it is in mares and 78 is reported to have a detrimental effect on future fertility.3,4 79 acute-phase proteins have been evaluated as markers for endometritis in the 80 postpartum cow. these acute-phase proteins include peripheral blood haptoglobin and 81 α1-acid glycoprotein. haptoglobin is synthesized in the liver in response to tissue 82 damage and binds free hemoglobin to protect from the oxidative activity of hemoglobin. 83 serum haptoglobin concentrations increase in dairy cows with acute metritis, but do not 84 increase significantly in cases of chronic metritis.5,6 another study showed that cows 85 with acute postpartum metritis generally have low concentrations of plasma haptoglobin 86 while cows with severe metritis had consistently higher levels.7 a more recent study 87 indicated that cows with ≥1 g/l of haptoglobin on day 3 postpartum are 6.7 times more 88 likely to develop severe or mild metritis. this study also suggests that an acute phase 89 inflammatory response precedes clinical metritis and that haptoglobin screening may 90 assist in the early detection of metritis.8 the α1-acid glycoprotein has been evaluated as 91 well, but the results were less diagnostic than those for haptoglobin.7 the significance of 92 236 these acute phase proteins in the diagnosis of endometritis or metritis is not fully 93 understood. intrauterine oxygen reductase potential (eh) and ph have also been explored 94 as a means to assess the level of bacterial contamination within the lumen of the uterus. 95 the eh values fell in the presence of infection which created an anaerobic environment 96 within the uterus. it is thought that the drop in eh is due to either bacterial metabolism or 97 increased oxygen consumption by polymorphonuclear (pmn) cells.9 the ph of uterine 98 discharge collected from cases of endometritis varied from 6.9 to 7.3 which favors the 99 growth of a. pyogenes.10,11 100 immunology of the bovine uterus 101 the uterine defense mechanisms against contaminating bacteria are maintained in 102 many ways which include 1) anatomically by the simple or pseudostratified columnar 103 epithelium covering the endometrium, 2) chemically by the mucoid secretions from the 104 endometrial glands, and 3) immunologically by pmns and humoral antibodies.10 105 disruption of these natural defense mechanisms allows for invasion and colonization of 106 the endometrium by opportunistic pathogens. inflammation of the bovine endometrium 107 can occur following coitus, artificial insemination, or more commonly postpartum. 108 uterine cellular immunity 109 in the uterus, the cellular defense against bacterial invaders is provided by uterine 110 leukocytes. the pmn population within the uterine lumen increased after 111 experimentally-induced uterine infections.12,13 at approximately 48 hours postpartum in 112 unassisted calvings, leukocytes begin to accumulate in the uterine lumen along with 113 bacterial contaminants.14 this is the beginning of the normal process of uterine 114 involution. in cases of metritis there is an initial decrease in the phagocytic activity of 115 237 uterine pmns.10,15 two to three weeks later when clinical recovery has occurred, the 116 phagocytic activity increases which also coincides with lower numbers of bacteria in the 117 uterine lumen.10,16 118 the cellular immune response of the uterus may be negatively affected by some 119 treatments commonly used to treatment postpartum disorders in the cows.10,17 it has been 120 found that manual removal of fetal membranes, intrauterine antiseptics and disinfectants, 121 and intrauterine antibiotics may inhibit or suppress uterine leukocyte phagocytic activity 122 for several days.10,18 it has also been noted that lugol’s iodine and polyvinyl-pyrrolidone 123 both cause necrosis of the endometrial epithelium and stimulate uterine defense 124 mechanisms and release of prostaglandin f2α.19-22 although these agents stimulate 125 uterine defense mechanisms, they also cause endometrial fibrosis and thus should not be 126 used as an intrauterine therapy.10,19,23 127 elevated blood progesterone concentrations have been found to inhibit both 128 uterine and peripheral blood neutrophil phagocytic activities. the numbers of peripheral 129 blood neutrophils increase slowly from about six weeks prior to parturition and reach a 130 peak on the day of calving.16,24,25 however, maternal and fetal cortisol at the time of 131 calving may suppress neutrophil function.26-28 immediately postpartum, the phagocytic 132 activity of blood neutrophils declines within the uterine lumen.10,29 the cellular defense 133 mechanisms are preserved by an increase in the number of pmn cells.10,24 during the 134 first three weeks postpartum, the number of peripheral blood pmns declines and is likely 135 due to the migration of these cells into the mammary gland and uterine lumen.24,30,31 in 136 addition, the phagocytic activity of the pmns declines which is more marked in older 137 versus younger cows.10,30,31 there is some disagreement as to whether there is a decrease 138 238 in the phagocytic activity of neutrophils in the uterine lumen versus the peripheral 139 blood.32-34 other leukocytes are present in the endometrium of all animals. lymphocytes 140 are found within the endometrial epithelium in both cycling and non-cycling ewes and 141 heifers with little variation in numbers at different stages of the estrous cycle.35,36 142 uterine humoral immunity 143 protective immunoglobulins have been found in the bovine uterine secretions.37-41 144 immunoglobulin a (iga) is produced locally from the mucosa of the bovine uterus 145 whereas igg is produced from two sites. a portion of igg1 is produced locally in the 146 endometrium while the remaining igg1 and all of igg2 is obtained from peripheral 147 circulation.10,37,42 experimental uterine infections with pathogenic bacteria have 148 demonstrated immunoglobulins in cervical and vaginal secretions that appear in the order 149 igm, iga, and igg and disappear in the order igm, igg, and iga.10,43 the concentrations 150 of each immunoglobulin depends upon the site of sampling with igg predominately 151 found in uterine lumen and iga in the vagina.10,13,43,44 both igg and igm concentrations 152 in lochia from healthy cows fall after calving.45 in cows with postpartum disease, both 153 iga and igg concentrations in uterine fluids increase quite rapidly as endometritis 154 develops. however, igm remains low in cattle with endometritis.46 155 intrauterine therapy 156 a variety of antibiotics and antiseptics have been infused into the uterus of cows 157 to treat postpartum infections. intrauterine antimicrobials are used in order to achieve 158 high concentrations at the site of infection but are usually unable to penetrate any deeper 159 than the endometrium.1 the intrauterine use of antimicrobial agents is controversial as 160 some have found intrauterine treatment to be beneficial while others have found these 161 239 agents to have no effect or a detrimental effect. the bovine uterus is an anaerobic 162 environment. thus, antibiotics that are chosen for intrauterine infusion must be active in 163 the absence of oxygen. additionally, most antibiotics depress the activity of uterine 164 neutrophils and interfere with uterine defense mechanisms.3 thus, one must carefully 165 evaluate the evidence regarding intrauterine antimicrobial use and carefully consider both 166 the advantages and disadvantages associated with therapy. 167 historically, intrauterine use of antimicrobials has been a common therapy for 168 treatment of uterine infections. antimicrobials reportedly used for uterine infections 169 include tetracycline, penicillin, cephapirin, chloramphenicol, lugol’s iodine, gentamycin, 170 spectinomycin, sulfonamides, nitrofurasone, povidone iodine solution, urea, and 171 chlorhexidine.1 most of these compounds are not approved for intrauterine use and have 172 no published withdrawal times. there are also reports that intrauterine infusion of 173 antibiotics cause drug residues in milk.47,48 additionally, regulatory guidelines must be 174 adhered to for extralabel use of antimicrobials in food animals. intrauterine therapy is 175 considered an extralabel use, and thus may be prohibited for many antibiotics, 176 particularly in the united states. 177 the organisms that cause most postpartum infections are usually sensitive to 178 penicillin. however, bacterial contaminants present within the uterus during the first 179 several weeks postpartum produce penicillinase which makes penicillin useless if used 180 locally in the early (less than 30 days) postpartum period. by 30 days postpartum, the 181 contaminating bacteria are usually eliminated and intrauterine treatment with penicillin is 182 more likely to be effective.3 other factors may also affect the efficacy of intrauterine 183 240 antibiotic therapy. uterine lochia present during uterine infections contain organic fluids 184 and debris that can render certain antibiotics, such as sulfonamides, ineffective. 185 more recently, oxytetracycline has been the antimicrobial commonly used for 186 intrauterine therapy.3 however, one study indicated that most isolates of a. pyogenes are 187 resistant to oxytetracycline. this study also showed that large doses of intrauterine 188 oxytetracycline did not affect the frequency of isolation of a. pyogenes.3,49 in addition, 189 oxytetracycline and lugol’s iodine are quite irritating and are reported to cause 190 coagulation necrosis of the endometrium.3,50 although some studies indicate improved 191 reproductive performance with the use of intrauterine oxytetracycline, it has been 192 speculated that this improvement may be due to local prostaglandin production due to 193 chemical irritation of the endometrium.51 194 in general, intrauterine infusion of antimicrobials has failed to show any increase 195 in reproductive performance.1 two large field studies evaluated the use of cephapirin 196 benzoate in cows with clinical endometritis and demonstrated an improvement in 197 reproductive performance.52-54 but other studies have shown no improvement in 198 reproductive performance when evaluating intrauterine administration of cephapirin 199 benzoate.53 the appropriate use of intrauterine antibiotics to treat uterine infections still 200 remains controversial as only a limited number of studies indicate the efficacious use of 201 intrauterine antibiotics. 202 numerous antiseptics have been used to lavage the postpartum bovine uterus with 203 iodine and chlorhexidine solutions being most commonly used. many of these solutions 204 are quite irritating to the endometrium and are thought to stimulate endogenous 205 prostaglandin release. one study showed that the incidence of retained fetal membranes 206 241 and endometritis was reduced in cows that received 500 ml of 2% lugol’s iodine 207 immediately after calving and again 6 hours later. however, this study did not evaluate 208 the future reproductive performance of these treated cows.3,55 another study evaluated 209 the use of 50 to 100 ml of 2% povidone iodine solution in the uterus one month 210 postpartum and found that the reproductive performance of normal cows was not 211 improved and that the treatment was detrimental to the fertility of cows with 212 endometritis.3,23 213 systemic antibiotic therapy 214 cattle with metritis often suffer moderate to severe illness. these cattle are often 215 septic with fever, depression, and anorexia. a variety of antibiotics have been 216 recommended for parenteral use in cattle suffering from uterine infections. penicillin or 217 one of the synthetic penicillin analogs and ceftiofur are among the more common 218 antibiotics used systemically in cattle suffering from metritis. systemic use of 219 oxytetracyline may not be efficacious because of the difficulty in achieving the minimal 220 inhibitory concentration (mic) required for a. pyogenes in the uterine lumen.3 however, 221 one study found clinical improvement of cattle suffering from metritis with the use of 222 tetracycline at 10mg/kg.56 223 ceftiofur is a third-generation cephalosporin that has broad-spectrum activity 224 against gram negative and gram positive bacteria.57 ceftiofur is reported to reach all 225 layers of the uterus without causing violative residues in milk. ceftiofur is approved in 226 the united states for systemic administration to lactating cows affected with metritis.3 a 227 subcutaneous dose of ceftiofur at 1mg/kg in postpartum cows results in a concentration of 228 ceftiofur and its active metabolites in plasma, uterine tissues, and lochia higher than the 229 242 mic for most of the common pathogens involved in metritis.58 one study demonstrated 230 that ceftiofur administered at 2.2mg/kg daily for five days was effective in treating cows 231 with metritis.57 another study supported these findings and showed ceftiofur 232 administered at 2.2 mg/kg once daily for five days is as effective for treating metritis as 233 procaine penicillin g or procaine penicillin g with intrauterine infusion of 234 oxytetracycline.59 235 because of the reported lack of efficacy and potential detrimental effects of future 236 fertility, intrauterine infusion of antibiotics is not a favored treatment for most cases of 237 metritis. certain systemic antibiotics have demonstrated their effectiveness at treating 238 uterine infections in cattle. thus, most cases of metritis, especially cows that are toxic, 239 should be treated with systemic antibiotics such as penicillin or ceftiofur. 240 conclusion 241 there are no antibiotics currently approved for intrauterine administration in the 242 united states. intrauterine infusion of antibiotics leads to contamination in milk and 243 tissues for which appropriate withdrawal times have not been ascertained. in addition, 244 the assays used on farms to detect antibiotics in milk may not be accurate. although 245 some studies indicate a positive response to therapy with the use of intrauterine 246 antimicrobials, most studies do not show an improvement in reproductive performance or 247 clinical signs of disease when comparing intrauterine antimicrobial therapy and systemic 248 antibiotic therapy. this information, in conjunction with concerns regarding uterine or 249 endometrial damage and withdrawal times following the use intrauterine antimicrobials, 250 suggests systemic antibiotic therapy as the best treatment for many cases of cows with 251 uterine infections. 252 243 references 253 1. leblanc sj: postpartum uterine disease and dairy herd reproductive performance: a 254 review. vet j 2008;176:102-114. 255 2. eiler h, fecteau ka: retained placenta. in: youngquist rs, threlfall wr, editors. 256 current therapy in large animal theriogenology. 2nd ed. st. louis: saunders; 257 2007. p. 345-354. 258 3. risco ca, youngquist rs, shore md: postpartum uterine infections. in: 259 youngquist rs, threlfall wr, editors. current therapy in large animal 260 theriogenology. 2nd ed. st. louis: saunders; 2007 p. 339-344. 261 4. etherington wg, martin sw, bonnett bn, et al: reproductive performance of dairy 262 cows following treatment with cloprostenol 26 and/or 40 days postpartum: a 263 field trial. theriogenology 1988;29:565-575. 264 5. skinner jg, brown ra, roberts l: bovine haptoglobin response in clinically 265 defined field conditions. vet rec 1991;128:147-149. 266 6. smith bi, donovan ga, risco ca, et al. serum haptoglobin concentration in 267 holstein dairy cattle with toxic puerperal metritis. vet rec 1998;142:83-85. 268 7. hirvonen j, huszenicza g, kulcsar m: acute-phase response in dairy cows with 269 acute postpartum metritis. theriogenology 1999;51:1071-1083. 270 8. huzzey jm, duffield tf, leblanc sj, et al: short communication: haptoglobin as 271 an early indicator of metritis. j dairy sci 2009;92:621-625. 272 244 9. el-azab ma, whitmore hl, kakoma i, et al: evaluation of the uterine environment 273 in experimental and spontaneous bovine metritis. theriogenology 274 1988;29:1327-1334. 275 10. dhaliwal gs, murray rd, woldehiwet z: some aspects of immunology of the 276 bovine uterus related to treatments for endometritis. anim reprod sci 277 2001;67:135-152. 278 11. may tw: investigations relating to intrauterine ph and postpartum endometritis in 279 dairy cattle, with particular reference to actinomyces pyogenes. 280 [dissertation], liverpool: university of liverpool; 1996. 281 12. butt bm, senger pl, widders pr: neutrophil migration into the bovine uterine 282 lumen following intrauterine inoculation with killed haemophilus somnus. 283 j reprod fertil 1991;93:341-345. 284 13. watson ed, diehl nk, evans jf; antibody response in the bovine genital tract to 285 intrauterine infusion of actinomyces pyogenes. res vet sci 1990;48:70-75. 286 14. morrow da: postpartum ovarian activity and involution of the uterus and cervix in 287 dairy cattle. vet scope 1969;xiv:2-13. 288 15. vandeplassche m, bouters r: peurperal metritis in the bovine.in: proceedings of 289 the 4th international congress on animal reproduction and ai; 1976 july 290 12-16; krakow, poland. p. 660-665. 291 245 16. vandeplassche m: new comparitive aspects of involution and puerpal metritis in 292 mare, cow and sow. vet med 1981;36:804-807. 293 17. bouters r, vandeplassche m: postpartum infection in cattle. diagnosis and 294 preventive and curative treatment. j south afr vet med assoc 1977;48:237-295 239. 296 18. vandeplassche m, bouters r: the impact of gynaecological and obstetrical 297 problems resulting out of pregnancy and parturition. in: karg h, 298 schallenberger e, editors. factors influencing fertility in the post-partum 299 cow. 1981 september 30-october 2; freisling, germany. the hague: 300 martinus nijhoff; 1982. 301 19. carleton cl, threlfall wr: determination of changes in the rate of absorption and 302 histologic changes of endometrium following intrauterine lugol's infusion 303 of the postpartum cow (abstract). in: proceedings of the 10th international 304 congress on animal reproduction and ai; 1984 june 10-14; urbana-305 champaign, il. p. 412. 306 20. gupta rc, simha ak, krishnaswamy a; studies in the efficacy of some post-307 service intrauterine infusions on the conception rate in repeat breeding 308 cattle. theriogenology 1983;20:559-564. 309 21. gustafsson bk: therapeutic strategies involving anti-microbial treatment of the 310 uterus of large animals. j am vet med assoc 1984;185:1194-1198. 311 246 22. seguin be, morrow da, lewis tm, et al: luteolysis, luteostasis and the effects of 312 prostaglandin f2α on cows after endometrial irritation. am j vet res 313 1974;35:57-61. 314 23. nakao t, moriyoshi m, kawata k, et al: effect of postpartum intrauterine treatment 315 with 2% polyvinyl-pyrrolidone-iodine solution on reprodcutive efficiency in 316 cows. theriogenology 1988;30:1033-1043. 317 24. guidry aj, paapa mj, pearson re, et al: effects of parturition and lactation on 318 blood and milk concentrations, corticosteroids, and neutrophil phagocytosis 319 in the cow. am j vet res 1976;37:1195-1200. 320 25. vandeplassche m. stimuation and inhibition of phagocytosis in domestic animals. 321 in: proceedings of the 10th international congress on animal reproduction 322 and ai; 1984 june 10-14; urbana-champaign, il. p. 475-477. 323 26. roth ja, kaeberle ml: in vivo effect of ascorbic acid on neutrophil function in 324 healthy and dexamethasone-treated cattle. am j vet res 1985;46:2434-325 2436. 326 27. roth ja, kaeberle ml: effect of in vivo dexamethasone administration on in vitro 327 bovine polymorphonuclear leukocyte function and lymphocyte 328 blastogenesis. am j vet res 1981;43:412-416. 329 28. whitmore hl, bernoco m, liu ikm, et al: effect of parturition on phagocytosis 330 and chemotactic function in peripheral blood polymorphonuclear leukocytes 331 247 in cattle.1988; in: proceedings of the 11th international congress on animal 332 reproduction and ai; 1988 june 26-30; dublin, ireland. p. 528-530. 333 29. newbould fh: phagocytic activity of bovine leukocytes during pregnancy. can j 334 comp med 1976;401:111-116. 335 30. gilbert ro, grohn yt, miller pm, et al: the effect of parity on periparturient 336 neutrophil function in dairy cows. vet immunol immunopathol 1993;36:75-337 82. 338 31. kehrli me jr, nonneke bj, roth ja: alteration in bovine neutrophil function 339 during the periparturient period. am j vet res 1989;50:207-214. 340 32. anderson kl, hamida na, frank a, et al: collection and phagocytic evaluation of 341 uterine neutrophil leukocytes. theriogenology 1985;24:305-315. 342 33. chacin mfl, hansen pj, drost m: effects of stage of the estrous cycle and steroid 343 treatment on uterine immunoglobulin content and polymorphonuclear 344 leukocytes in cattle. theriogenology 1990;34:1169-1184. 345 34. zerbe h, schuberth hj, hoedemaker m, et al: a new model system for 346 endometritis: basic concepts and characterisation of phenotypic and 347 functional properties of bovine uterine neutrophils. theriogenology 348 1996;46:1339-1356. 349 248 35. lee cs, meeusen e, gogolin-ewens k, et al: quantitative and qualitative changes 350 in the intraepithelial lymphocyte population in the uterus of non-pregnant 351 and pregnant sheep. am j reprod immunol 1992;28:90-96. 352 36. vander weilen al, king gj: intraepithelial lymphocytes in the bovine uterus 353 during the oestrous cycle and early gestation. j reprod fertil 1984;70:457-354 462. 355 37. curtain cc, clark bl, dufty jh: the origins of the immunoglobulins in the mucus 356 of cattle. clin exp immunol 1971;8:335-344. 357 38. dhaliwal gs, murray rd, dobson h, et al: presence of antigen and antibodies in 358 serum and genital discharges of heifers after intrauterine inoculation of 359 leptospira interrogans serovar hardjo. res vet sci 1996;60:157-162. 360 39. dhaliwal gs, murray rd, dobson h, et al: presence of antigen and antibodies in 361 serum and genital discharges of cows from dairy herds naturally infected 362 with leptospira interrogans serovar hardjo. res vet sci 1996;60:163-167. 363 40. duncan jr, wilkie bn, hiestand f, et al: the serum and secretory 364 immunoglobulins of cattle: characterisation and quanitation. j immunol 365 1972;108:965-976. 366 41. whitmore hl, archbald lf: demonstration and quantitation of immunoglobulins 367 in bovine serum, follicular fluid, and uterine and vaginal secretions with 368 reference to bovine viral diarrhea and infectious bovine rhinotracheitis. am 369 j vet res 1977;38:455-457. 370 249 42. butt bm, bessre te, senger, pl: specific antibody to haemophilus somnus in the 371 bovine uterus following intramuscular immunization. infect immunol 372 1993;61:2558-2562. 373 43. corbeil lb, schurig jr, duncan, jr: immunoglobulin classes and biological 374 function of campylobacter (vibrio) fetus antibodies in serum and 375 cervicovaginal mucus. infect immunol 1974;10:422-429. 376 44. corbeil lb, hall ce, lein d, et al: immunoglobulin classes in genital secretions of 377 mycoplasma-infected and normal heifers. infect immunol 1976;13:1595-378 1600. 379 45. georgiv s: study of the general immunologic response and microbial content of the 380 uterus of cows in the puerperal period. vet sci 1978;15:69-77. 381 46. aknazarov bk: study of humoral factors of local protective function in cows. 382 veterinariya 1988;8:41-43. 383 47. dinsmore rp, stevens rd, cattell mb, et al: oxytetracycline residues in milk after 384 treatment of cows with retained fetal membranes. j am vet med assoc 385 1996;209:1753-1755. 386 48. kaneene jb, coe ph, smith jh, et al: drug residues in milk after intrauterine 387 injection of oxytetracycline, lincomycin-spectinomycin, and povidone-388 iodine in cows with metritis. am j vet res 1986;47:1363-1365. 389 250 49. cohen ro, bernstein m, ziv g: isolation and antimicrobial susceptibility of 390 actinomyces pyogenes recovered from the uterus of dairy cows with 391 retained fetal membranes and postparturient endometritis. theriogenology 392 1995;43:1389-1397. 393 50. gilbert ro, schwark ws: pharmacologic considerations in the management of 394 peripartum conditions in the cow. vet clin north am food anim pract 395 1992;8:29-56. 396 51. goshen t, shpigel ny: evaluation of intrauterine antibiotic treatment of clinical 397 metritis and retained fetal membranes in dairy cows. theriogenology 398 2006;66:2210-2218. 399 52. leblanc sj, duffield tf, leslie ke, et al: defining and diagnosing postpartum 400 clinical endometritis, and its impact on reproductive performance in dairy 401 cows. j dairy sci 2002;85:2223-2236. 402 53. leblanc sj, duffield tf, leslie ke, et al: the effect of treatment of clinical 403 endometritis on reproductive performance in dairy cows. j dairy sci 404 2002;85:2237-2249. 405 54. mcdougall s: effect of intrauterine antibiotic treatment on reproductive 406 performance of dairy cows following periparturient disease. n z vet j 407 2001;49:150-158. 408 251 55. putro pp: effects of intrauterine dilute iodine solution infusion on the incidence if 409 retained placenta and endometritis in dairy cows. acta vet scand 1988;83 410 suppl:58-65: 411 56. schmitt ej, boucher jf, van den eede ca, et al; comparison of clinical efficacy of 412 ceftiofur and oxytetracycline for treatment of acute puerperal metritis in 413 dairy cows. proc annu conv am assoc bovine pract 2001; p. 199. 414 57. chenault jr, mcallister jf, chester st, et al: efficacy of ceftiofur hydrochloride 415 sterile suspension administered parenterally for the treatment of acute 416 postpartum metritis. j am vet med assoc 2004;224:1634-1639. 417 58. schmitt ej, bergwerff aa: concentration of potentially active ceftiofur residues in 418 plasma, uterine tissues and uterine secretions after post-partum 419 administration of ceftiofur hydrochloride in lactating dairy cows. pharmacia 420 animal health, 2000. 421 59. smith bi, donovan ga, risco ca: comparison of various antibiotic treatments for 422 cows diagnosed with toxic puerperal metritis. j dairy sci 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true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2014: camelid parasitology camelid parasitology christopher k. cebra college of veterinary medicine, oregon state university, corvallis, or introduction like any other animal, camelids are faced with exposure to internal and external parasites. these can be relatively benign or cause severe disease. where each camelid ends up on this spectrum of severity depends on a number of factors, including type of parasite, number of parasites, and host immunity. exposure to parasites is a fact of life. except in very rare cases, we cannot eliminate parasites. therefore, our primary jobs are to promote immunity and prevent large-scale exposure. we attempt to manage parasitism to reduce or eliminate health effects. gastrointestinal tract protozoa new world camelids in appear to be susceptible to five or more species of emeria. these include small coccidia (e. punoensis, e. alpacae, and e. lamae), and large coccidia (e. macusaniensis and e. ivitaensis). these appear to be specific to new world camelids, but transferable among those species. the prepatent period for small eimeria in camelids appears to be about 15 to 21 days. over that time, there are two rounds of schizontogeny (asexual reproduction), the first in the lower small intestine and the second in the large intestine. gametogeny (sexual reproduction) then occurs primarily in the large intestine with destruction of the gut epithelial cells. clinical signs usually appear with gametogeny, though diarrhea may precede fecal oocyst shedding by a few days. because of its predilection for large intestinal damage, eimeria are one of the diseases more commonly associated with diarrhea in camelids. small coccidia also follow the general pattern described for other herbivores, in that older camelids are generally resistant to disease. therefore, higher egg counts should be expected in camelids less than one year of age, and up to a few hundred eggs per gram can be considered normal. counts much higher than 200 eggs per gram are unusual in adults, unless the camelid is otherwise debilitated or in an overcrowded environment. e. macusaniensis is the largest and slowest maturing of the coccidia. the prepatent period is over one month, and it is common for camelids to show clinical signs of disease (colic, weight loss or poor growth, possibly diarrhea) before they develop patent infections. diarrhea is less common than other clinical signs, especially in camelids over one year of age. it affects camelids of all ages more so than small coccidia, which infect but rarely cause clinical disease in camelids older than one year. in crias in contaminated environments, prepatent e. macusaniensis may be responsible for signs mistakenly attributed to the faster developing small coccidia, because the latter are found in the feces. large coccidia also appear to be important in creating an environment conducive to cl. perfringens-related disease. eimeria ivitaensis is a more recently recognized large coccidial parasite. while not as common or apparently as damaging as e. macusaniensis, it has been associated with severe disease in both young and old camelids. cryptosporidium is much smaller coccidian parasite, which can infect many species, including man. in addition to the intestinal epithelium, cryptosporidium has been found in the biliary, renal, and respiratory epithelium of some immunocompromised patients of other species. both c. parvum and c. ubiquitum have been isolated from camelids. recently, cryptosporidium has been implicated in several herd outbreaks of diarrhea in crias around the u.s. adult camelids are less commonly clinically affected, but were found to positive on fecal examination, and hence a possible source of infections. cryptosporidium usually affects younger crias < 21 days old, but has also been found in older crias with diarrhea, and in adults during outbreaks. larger herds with a large number of births close together are most likely to have outbreaks of cryptosporidiosis. 599 clinical theriogenology • volume 6, number 4 • december 2014 giardia giardia is a flagellate parasite. giardia cysts can be found in the feces of many normal animals, including camelids, thus causing many to doubt its pathogenicity. it appears to be able to affect many different hosts, and introduction into a camelid herd is usually thought to occur through infected wildlife. contaminated water courses are considered the usual route, though in moist areas, it is likely that the organism can persist on damp pasture. ingested giardia cysts release trophozoites, which attach to small intestinal mucosal cells. although cell function is impaired, giardia do not reproduce in the cells like eimeria. number of ingested cysts and hence number of affected host cells is likely to be a predictor of the clinical importance of giardia in a specific situation. the incubation period is approximately five days, but has lasted as long as 21 days in humans. with longer incubation periods, the disease can be seen seven to ten days before patency. the more cysts found in the feces, the more likely that giardia is the cause of diarrhea. giardia is primarily a problem in crias up to about seven months of age. due to its predilection for the small intestine, diarrhea is not always found in camelids with severe giardiasis. balantidum coli is an amoeba. this has been described in camels and anecdotally in camelids. in the middle east, camels are postulated to be the reservoir population for human infections. in alpacas, balantidum is thought to cause diarrhea without weight loss in adults, and watery diarrhea in crias. identification coccidia may be identified by fecal flotation and microscopic examination. the average length and width increase from e. punoensis (around 20 x 6μ), e. alpacae (24 x 20μ), e. lamae (36 x 25μ), to e. ivitaensis (88 x 52μ), and e. macusaniensis (94 x 67μ). small coccidia float well in most solutions, and appear to float better in saturated saline than sheather’s solution, and are decreased by centrifugation procedures. thus, a simple suspension or flotation appears best. large coccidia require a denser flotation solution, such as sheather’s solution, and because low numbers may be important at the beginning of the patent period, concentration techniques such as centrifugation-flotation procedures are often helpful. on dead animals, impression smears or histopathology may be helpful. it is important to recognize that the gut may appear grossly normal even with severe coccidiosis, or may have white mucosal punctae, hemorrhagic or fibrinonecrotic areas, or rarely edema and cobblestoning of the serosal surface. the distal jejunum and ileum are the areas of highest parasite concentration. in live animals, diagnosis of prepatent disease is difficult or presumptive. laboratory abnormalities are likewise non-specific, with evidence of shock and fat mobilization more common than electrolyte loss. hypoproteinemia is common, but is also ubiquitous with many other camelid disorders. anemia is rarer. abdominocentesis usually yields a transudate. abdominal imaging is also inconclusive: colicky camelids may have ileus and fluid-distended intestine, though usually to a lesser degree than camelids with gastrointestinal obstruction. thickened bowel walls are rare (<10% of cases). polymerase chain reaction may be helpful in diagnosing prepatent e. macusaniensis infection. cryptosporidium and giardia may be seen on routine parasitologic mounts, but use of immunofluorescence for either or acid-fast staining for cryptosporidium improves visibility. some immunofluorescence kits have a tendency to yield false positives, so performance should be investigated before the kit is put into regular diagnostic use. giardia or balantidium trophozoites may be seen on wet mount of very fresh feces, and cysts can be found using centrifugation in a tube filled with 1.18 sg zinc sulfate solution. other flotation solutions tend to distort cysts. treatment and control if the camelid has either a confirmed or suspected infestation, treatment may be warranted. the most commonly used anticoccidial medications for small ruminants and camelids in north america are amprolium and sulfa antibiotics. both are more effective against the immature forms of the parasite, and should not be expected to immediately reduce fecal shedding. oral sulfa antibiotics are unlikely to be absorbed or effective after the juvenile period. decoquinate may be added to feed to reduce coccidia. 600clinical theriogenology • volume 6, number 4 • december 2014 treatment during the prepatent period does appear to reduce subsequent shedding. in countries where they are available, benzeacetonitrile (-azuril) compounds may be an effective alternative, especially as they kill more stages of the organism, and more rapidly reduce shedding. many affected camelids also require plasma transfusions and antibiotic coverage. in cases of cryptosporidium infection, there is anecdotal information about some agents, but in general, anticoccidials are not known to have a beneficial effect. giardia may respond to high doses of metronidazole or fenbendazole. metronidazole may also be effective against balantidium. herd control is a tricky issue. disease in younger animals should be treated similarly to other forms of parasitism. some degree of shedding may be normal or clinically insignificant prior to the development of immunity. in older animals, the amount of shedding should be lower, but complete lack cannot be expected. signs of disease, protein loss, diarrhea, or weakness support treatment in herds with known or suspected parasitic issues support the argument for treatment. other management changes to reduce stress and overcrowding, and clean up the environment are necessary. herd outbreaks are not common, emphasizing the role of immunocompromise in severe infestations, but have been seen when groups of naïve camelids have been brought to farms with endemic problems. spot fecals may be helpful of assessing the level of herd shedding, but remember that prepatent disease is common and intermittent shedding may occur. for herd treatments, less aggressive medical treatment is necessary, as the goal is to reduce, but not eliminate the parasite. strongyles camelids are susceptible to infection by many of the same nematodes that affect domestic ruminants. the major stomach (c3) worms include haemonchus, ostertagia, teladorsagia, trichostrongylus, camelostrongylus, and marshallagia. the major small intestinal worms include cooperia, nematodirus, trichostrongylus, lamanema. the major large intestinal worm is oesophagostomum, which is found infrequently. of these lamanema is reported to be the most pathogenic in south america. the gastric “h.o.t.” complex (initials of the major worm types) worms and the small intestinal n. battus appear to be more important in north america. as most of these affect the gastrointestinal tract above the colon, they appear to be more important as causes of weight loss, low protein, anemia, and ill-thrift, versus diarrhea. strongyles cause two types of disease. the first type results from grazing heavily contaminated pasture and rapid increase in worm burden. maturing larvae damage the gastrointestinal wall, leading to a decrease in nutrient absorption and an increase in protein or blood loss. if this build-up occurs rapidly, the disease may occur during larval development, and hence few or no eggs may be present in the feces (prepatent disease). the second type of disease occurs in the late winter or spring. larvae ingested towards the end of the previous grazing season arrest their development within the gastrointestinal wall. this is called hypobiosis. towards the beginning of the next grazing season, something triggers these larvae to continue their development. thus, there can be an intense period of larval development, leading to severe gut damage and clinical disease. eggs may not be passed until late in the disease, thus it may also be considered prepatent. haemonchus contortus, or the “barber pole” worm deserves special mention. it is far more damaging than similar worms, in part because it feeds on the host’s blood. blood feeding by l4 larvae and adults may remove 0.05ml blood per worm per day. a typical alpaca blood volume = 0.07 x 50 kg = 3.5l. therefore, each worm removes 0.0014% of blood./day; 1000 worms remove 1.4% of blood/day. the “barber pole” name comes from the double helix of the red, blood-filled intestine and the white, eggfilled ovaries of the adult female worm. adults live in the acidic part of the third gastric compartment. females release eggs which hatch out on pasture. larvae undergo two molts until they become infective l3 larvae. these are susceptible to extreme temperatures, starvation, and desiccation, so they usually gather in water droplets on blades of grass or leafy forage. if eaten, the larvae molt again to l4s and start to feed about 11 days after ingestion on blood from the third compartment wall. it is important to realize 601 clinical theriogenology • volume 6, number 4 • december 2014 that larvae start the blood feeding. these immature worms do not produce eggs, so it is possible, when large numbers of larvae are ingested over a short period of time, for severe disease to occur several weeks prior to the passage of large numbers of eggs. eventually the larvae mature to blood-eating adults and the cycle continues. clinical signs of severe haemonchosis include the common signs of weakness and ill-thrift, but may specifically include severe anemia. camelids with single digit packed cell volumes at our clinic frequently are found to have haemonchosis. most other parasites lead to protein and weight loss, but with only modest red blood cell loss. because of the anemia, qualitative tests examining oral membrane color may be more useful for diagnosing haemonchosis than they are for other parasitic infections. remember again that disease caused by larvae may not be accompanied immediately by high fecal egg counts. the importance of all worm infections appears to be growing with the greater frequency of resistance to common deworming medications. this is a serious problem, especially in the southeastern u.s. resistant worm populations may be crossing over from ruminants, but also may be arising in camelid herds, especially those using frequent dewormings to prevent diseases such as parelaphostrongylus infection. drug resistance can be detected in a variety of ways, such as the fecal egg count reduction test (comparing premedication egg counts to those 10 to 14 days later), or the larval development assay. a variety of suggestions have come forth to combat drug resistance, but most have not been evaluated scientifically. these include use of higher doses, use of novel agents, use or alternate methods (such as parasite-eating fungi, copper wires, and toxic plants), breeding for resistance, and pasture management. all have some potential. regardless of the method chosen, continued surveillance is necessary to check for continued efficacy. as with medications, alternate control strategies have the potential to have diminishing value over time. other worms strongyloides, capillaria (aonchotheca), trichuris, and tapeworms (moniezia) are relatively infrequent findings. trichuris particularly appears to be clinically important. capillaria is known to be an intermittent shedder. low egg counts can be found in heavily parasitized camelids. trichuris (whipworm) eggs can be hard to detect because they do not float very well in saturated salt solution. both trichuris and capillaria have lengthy prepatent periods in ruminants, and may cause prepatent disease in camelids. trichuris affect the colon, whereas most of the other worms affect the third compartment or small intestine. by affecting the colon, trichuris are more likely to cause colic, diarrhea, and straining than the other worms. liver liver flukes camelids are susceptible to several species of flukes, including fasciola hepatica, fascioloides magna,and dicrocoelium dendriticum. f. hepatica appears to be the most important in the u.s., particularly in the pacific northwest, and in england, whereas d. dendriticum is the most important fluke in central europe. like other internal parasites, they tend to cause an ill-thrift syndrome, with the major difference being that clinical pathology tests point more towards hepatic disease, particularly through increases in gamma-glutamyl transpeptidase activity and bilirubin. flukes may also contribute to other disorders including clostridial infections and endocarditis. the primary disease is due to migrating larvae. thus, egg-shedding may not occur until two to five weeks after the onset of abnormalities. worms lamanema chavezi is a gastrointestinal worm that has a phase of larval migration through the liver. it causes severe disease in south america, but appears to be rare here. this liver disease may be fatal and prepatent. 602clinical theriogenology • volume 6, number 4 • december 2014 respiratory tract lungworms lungworm infestations of new world camelids have not been reported in north america, but could potentially occur in areas where camelids are close to infected ruminants or deer. coughing and respiratory difficulty would be the expected signs. nasal bots two species of fly, oestrus ovis and cephenomyia hominivorax, deposit larvae on the nose of camelids. the usual hosts for these flies are sheep and deer, respectively. the larvae mature in the nasal passages, and can cause occlusion and irritation. the common signs are respiratory difficulty, snorting, and nasal discharge, sometimes blood-tinged. the maggots can be seen by endoscopy, or suggested by radiographs. muscle sarcocystis cruzi sarcocysts are protozoal invaders of muscle. the definitive hosts are dogs and wild canids. in order to become infected, camelids must ingest something contaminated with infected dog feces. canine infections are relatively rare in north america, but some imported camelids appear to have arrived with infections. infected camelids pose no risk to other camelids, and can only spread the disease through their meat. the organisms can cause inflammation of muscle and fascia, which can affect gait or function of specific muscles (extraocular in one case), or potentially be linked to muscle disorders such as megaesophagus. although camelids are unlikely to receive heavy infections here, stresses may lead to clinical worsening of preexisting infections. the disease is diagnosed through identification of the organism on muscle biopsy. nervous system parelaphostrongylus tenuis we are lucky not to have this parasite in the west. the brain worm of white-tailed deer infects snails as its intermediate hosts. snails transport the larvae and are subsequently eaten by grazing animals. in aberrant hosts, such as camelids, larvae migrate through the brain and spinal cord inducing neurologic disease. camelids rarely develop patent infections because the long prepatent period (around 90 days) is considerably longer than the average time until development of neurologic signs or death (around 30 to 60 days post-infection). reported signs include incoordination, hind-end weakness, recumbency, curvature to the neck, blindness, seizures, respiratory paralysis, and death. in many parts of the country, deworming strategies are directed against killing p. tenuis larvae before they enter the central nervous system, leading to every 14day dosing during snail season. because of the difficulty in getting eggs, diagnosis is frequently based on compatible signs and geographic location, and the finding of eosinophils within the cerebrospinal fluid. because of the relatively long period between infection and disease, this disease can be found in camelids transported out of the endemic areas. screening for parasites alpacas are not uniform in their parasite loads. the old tenet is that 10% of the animals have 90% of the parasites. these animals usually reflect those that have lower immunity (the old, young, and infirm), or have higher exposure (outcasts, dirt-eaters, crias). if the burden is high enough, these animals 603 clinical theriogenology • volume 6, number 4 • december 2014 will show characteristic signs and should be screened for parasites. if the burden is more moderate, it is harder to identify these animals. we recommend regular parasite screenings. how often they occur depends on the specifics of the herd. based on the natural history of most parasites, problems are most likely to occur 1) when there are a large number of younger animals present 2) shortly after mild, wet weather. considering that most parasites take at least three weeks to develop patent infections, checking feces three+ weeks after the above two events makes sense. in some areas, the condition number two above may be constant, nearly constant, or seasonal. generally, if no animals are showing clinical disease, examining feces from around 20% of the animals should provide reasonable surveillance. this may miss the intermittent heavy shedder, but all animals with a previous history of heavy shedding should be added to or included in the 20%. with most parasites, we try to quantify the fecal egg count, an estimate of the concentration of worm eggs in the feces. in doing so, we hope that these egg counts correlate well to the number of worms or protozoa in the intestine, and hence the risk of disease. unfortunately, this is not always the case. certain parasites cause disease before they shed large numbers of eggs or larvae (e. macusaniensis, t. tenuis, some strongyles, liver flukes, lung worms). which test to run? which test to run depends heavily on the parasite we wish to detect. unfortunately, no one test optimally finds all parasites. many tests completely miss certain parasites. 1) direct smear the simplest test, smearing feces with saline on a slide. this is best for motile protozoa such as giardia and balantidium trophozoites, and decent for most types of worm eggs at high concentrations. it is not particularly good at finding parasites where we worry about low numbers of eggs. 2) saline flotation relies on the difference in specific gravity between saturated (1.18) or more dilute saline and the worm eggs. saline will not float t. tenuis, e. macusaniensis, or liver fluke eggs. many other salts, such as 33% zinc sulfate solution have a similar specific gravity to saline. saline distorts giardia rapidly. 3) sucrose flotation sheather’s solution has a higher specific gravity than saline (1.27), and thus will float all but liver fluke eggs. the high viscosity of this solution may inhibit flotation of small coccidia or nematodirus, but may be better than saline for cryptosporidium and giardia. 4) mcmaster counting chamber. rather than floating the eggs in a tube onto a coverslip, this involves putting a slurry into a counting chamber. the flotation properties become less important. most parasites can be detected this way, but it is not particularly sensitive for low levels of infection. 5) double centrifuge technique feces are dissolved in water, preferably overnight, then centrifuged. the pellet is resuspended in a flotation solution, which then gives it the properties of that flotation technique. the advantages to this technique are the longer dissolving period enables more eggs to be released from fecal matter, and also the pelleting concentrates eggs. this technique appears to be the best way to find small numbers of eggs. we have found that extending the flotation time beyond the recommended ten to 15 minutes out to one to four hours further improves the yield. 604clinical theriogenology • volume 6, number 4 • december 2014 6) sedimentation feces are dissolved in water and either left to settle or pelleted in a centrifuge. a drop or two of the sediment is placed on a slide for microscopic examination. this technique is used most for fluke eggs, which do not float in most solutions. the flukefinder is a specialized system for sedimenting fluke eggs. 7) baermann test this is most commonly done on fresh feces to identify lungworms. if the sample is allowed to sit a few days, strongyloides and strongyle larvae may also hatch out of their eggs and confound the diagnosis. this test involves suspending a fecal sample in cheesecloth within a funnel or cone-shaped vessel. the vessel is filled with warm water and left to sit for at least eight hours. free-living larvae will concentrate at the bottom of the cone and can be pipetted out for examination under a microscope. 8) immunologic methods fluorescent antibody and enzyme immunosorbent tests are available to aid in the detection of cryptosporidium and giardia. a systemic (blood) antibody response to flukes occurs within about 30 days of exposure and may be useful in detecting prepatent disease. table. prepatent periods for important camelid parasites prepatent patent eimeria punoensis 10 days illness rare e. lamae 15-16 days illness with patency e. alpacae 16-18 days illness rare e. macusaniensis 32-43 days 20-40 days illness possible 2-3 weeks before patency cryptosporidium spp. 3-7 days 4-5 days illness with patency giardia duodenalis 5-21 days months illness possible 7-10 days before patency balantidium coli strongyle worms 17-36 days nematodirus battus 14-21 days trichuris tenuis 60 days? capillaria spp. 42 days? moniezia 40 days? dictyocaulus spp. 22-25 days? prepatent disease possible fasciola hepatica 70-84 days? dicrocoelium dendriticum 49-79 days? 605 clinical theriogenology • volume 6, number 4 • december 2014 606clinical theriogenology • volume 6, number 4 • december 2014 omniblank: 2010: case report: breeding soundness examination of 12 native omani bulls case report: breeding soundness examination of 12 native omani bulls mushtaq a. memon,a musab h. al-busaidi,b hamood al-khanjaric and rashid s. al-habsic adepartment of veterinary clinical sciences, washington state university, pullman, wa, usa; bdepartment of animal and veterinary sciences, sultan qaboos university, al-khoud, muscat, sultanate of oman; canimal production research center, ministry of agriculture, rumais, sultanate of oman summary breeding bulls strongly affect the reproductive efficiency of cattle herds, irrespective of whether they are used for natural breeding or artificial insemination. the purpose of this case was to evaluate the reproductive potential of native omani bulls using criteria established by the society for theriogenology for breeding soundness examination (bse). twelve bulls, 2-4 years old were evaluated. the bulls were weighed and assigned a body condition score (bcs) on a scale ranging from 1 through 9. scrotal circumference (sc) was measured and semen was collected with an elctroejaculator. semen was evaluated for sperm mass activity, individual sperm motility, sperm concentration and morphology. two bulls were declared unsatisfactory owing to azoospermia (ejaculate devoid of sperm) in one and a scrotal hernia in the second. key words: omani bull, breeding soundness examination, reproductive evaluation background the sultanate of oman is located in southwest asia on the southeast coast of the arabian peninsula. it borders the united arab emirates on the northwest, saudi arabia on the west and yemen on the southwest. oman is a leading livestock producer in the region with goats being the most numerous (15,571,148), followed by sheep (351,066), cattle (301,588), and camels (117,299).1 the majority of cattle (173,892) are found south of the dhofar mountains. because of the influence of the monsoon this area has the only natural pasture in oman.1 the coast-facing mountain slopes behind the coastal plain are traditional cattle-breeding areas of the jabali tribesmen. until recently, the jabali reared cattle only for their milk. currently with good road communications to northern oman where demand for beef is increasing, the jabali are being encouraged to export their cattle to the north. in the north, the native omani cattle are raised primarily for beef. the native cattle breed has some phenotypic similarity to other zebu cattle found in other countries in the area (fig 1). at present, breeding bulls in oman are selected only by physical appearance. no bse is performed before animals are used for breeding purposes on the government farms or sold to private farm owners for improvement of their livestock. bulls strongly affect the reproductive efficiency of breeding herds, irrespective of whether they are used for natural breeding or artificial insemination. thus, impairment of bull fertility results in great economic loss. therefore, it is essential that bulls be examined both before and during their use in breeding programs in order to identify and remove any individuals with potentially low fertility.2 case presentation twelve omani bulls, 2-4 years old from the northern region of the country were evaluated at the animal production research center, ministry of agriculture, rumais, sultanate of oman following the guidelines established by the society for theriogenology.3 each bull was weighed, scored for body 25 fig. 1. native breed bull from oman. the phenotypic characters of dark brown color, a small hump, medium size dewlap, and wide forehead appear to have some influence from other zebu breeds found in countries in the area, e.g. red sindhi in pakistan. condition (1-9)4 and the scrotum was examined for presence of two normal testes with firm consistency. scrotal circumference was measured using a scrotal tape (nasco, ft. atkinson, wi) at the widest midscrotal point. the accessory sex glands were examined by palpation per rectum. semen samples were collected using an elctroejaculator (standard precision electronics, denver, co) as described by chenoweth and osborne.5 sperm mass activity, motility and sperm morphology were evaluated.6 sperm mass activity was evaluated by placing a drop of undiluted semen on a pre-warmed microscope slide and wave motion was evaluated at 100x magnification. the mass activity was graded from 0 to 3 (0=no activity, 1=slow/irregular wave motion, 2=moderate wave motion, and 3=fast wave motion). for determination of sperm motility, an aliquot of semen was diluted 1:10 with normal saline and a drop of diluted semen was placed on pre-warmed microscope slide, covered with a coverslip and assessed by estimating the percentage of sperm moving forward in a progressive manner. sperm concentration in each ejaculate was measured by diluting the semen 1:100 (bd unopette™ reservoir #365854, bectondickinson, franklin lake, nj) and sperm were counted using a neubauer chamber (bright line™ hemacytometer, exodus breeders corporation, york, pa). to determine sperm morphology, a drop of semen was placed close to one end of a microscope slide and mixed with a drop of eosin-nigrosin stain (society for theriogenology, montgomery, al) and a smear was made. the smear was air-dried, and sperm were evaluated under oil immersion (1000x). two hundred sperm were evaluated and abnormalities were classified as described by barth and oko.7 primary abnormalities included sperm head abnormalities, proximal cytoplasmic droplets, abnormal midpieces, and coiled tails, while secondary abnormalities included distal cytoplasmic droplets, bent tails, and loose heads. outcome all bulls responded adequately to electroejaculation. the ejaculate of one bull was devoid of sperm. semen collection from this bull was attempted twice at an interval of several days with the same results; therefore the bull was declared to be azoospermic (fig 2) and data from this bull were removed. another bull was found to have a scrotal hernia (fig 3). both bulls were declared unsatisfactory potential 26 breeders and removal from the breeding program was recommended. the average body weight, sc, semen volume, sperm mass activity, sperm motility, number of sperm per ejaculate and percent of normal sperm are shown in table 1. discussion the bulls examined were from northern oman, however, the majority of omani cattle, primarily the dhofari breed, are found south of the dhofar mountains.1 scrotal circumference, as a measure of testis size, has been positively associated with sperm production in beef bulls with correlations estimated to be as high as 0.81(see review by rusk8). the mean sc in the bulls examined for this report was greater than that found by others who examined bos indicus (nelore) or canchim bulls (bos indicus x bos taurus crossbred).9 the sperm motility and percentage of normal sperm were also higher in these bulls than in those examined by brito, et al.9 the bull with a scrotal hernia was declared unsatisfactory for breeding due to the possible heritable nature of the condition.10 the bull with azoospermia was unsatisfactory for breeding due to lack of sperm in his ejaculate. it was our purpose to demonstrate that bse of omani bulls before breeding or purchase would enable the veterinarian to remove bulls with inadequate semen quality, poor bcs or physical characteristics that interfere with satisfactory breeding performance. learning points • bse of omani bulls before breeding or purchase will enable the veterinarian to remove bulls with marginal or poor semen quality or physical conditions which compromise breeding performance. • bulls with scrotal hernias and other potentially hereditary conditions can be detected by bse. • infertile or sterile bulls with azoospermia or oligospermia may be detected by bse and removed from the breeding herd. acknowledgements the study was conducted during the first author’s fulbright fellowship at sultan qaboos university (squ), oman. the authors are grateful for valuable support provided by prof. osman mahgoub, prof. e. johnson and their staff; squ agriculture experiment station; the department of animal and veterinary sciences; the ministry of agriculture, oman; and the staff at the animal production research center, rumais, oman. references 1. anonymous: agriculture census 2004-2005. ministry of agriculture, sultanate of oman. 2. carroll e, ball l, scott j: breeding soundness in bulls, a summary of 10,940 examinations. j am vet med assoc 1963;142:1105-1111. 3. chenoweth jp, spitzer j, hopkins f: a new breeding soundness evaluation form. proc soc for therio 1992; p. 63-70. 4. parish ja, rhinehart d: body condition scoring beef cattle. mississippi state university publication 2508, p 1-8. http://msucares.com/pubs/publications/p2508.pdf 2008. accessed november 29, 2009. 5. chenoweth pj, osborne hg: breed differences in the response of young beef bulls to electroejaculation. aust vet j 1978;54:323-366. 6. nelson dd: bull selection and breeding soundness evaluation for the beef producer. http://cru.cahe.wsu.edu/cepublications/eb1601/eb1601.pdf 1997. accessed november 29, 2009. 7. barth ad, oko r: abnormal morphology of bovine spermatozoa. ames: iowa state university press; 1989. p. 89-271. 8. rusk cp, king me, mortimer rg, et al: case study: relationships of scrotal circumference and scrotal volume to growth and semen traits in beef bulls. prof anim sci, march 2002. http://findarticles.com/p/articles/mi_qa4035/is_200203/ai_n9046823/?tag=content;col1 accessed november 29, 2009. 9. brito lfc, silva aedf, unanian mm, et al: sexual development in earlyand late-maturing bos indicus and bos indicus x bos taurus crossbred bulls in brazil. theriogenology 2004; 62:1198-1217. 10. zhao x, du zq, vukasinovic n, et al: association of hoxa10, zfpm2, and mmp2 genes with scrotal hernias evaluated via biological candidate gene analyses in pigs. am j vet res 2009;70:1006-1012. 27 table 1. mean (± s. d.) of reproductive parameters of eleven 2-4 year old omani native bulls reproductive parameter mean ± s.d. body weight (kg) 360.2 ± 41.4 scrotal circumference (cm) 28.2 ± 1.6 semen volume (ml) 4.8 ± 2.7 sperm mass activity (0-3 grade) 1.8 ± 1.1 sperm motility (%) 55.5 ± 13.5 total sperm number (109 ) 869.8 ± 39.4 normal sperm (%) 89.1 ± 13.8 fig. 2. ejaculates from two native omani breed bulls. ejaculate with no sperm (l) and normal milky white color semen (r). fig 3. scrotum with normal testes (l), scrotal hernia (r) of native omani breed bulls. 28 2014: surface architectural anatomy of the penile and preputial epithelium of bulls* surface architectural anatomy of the penile and preputial epithelium of bulls* lew strickland,a† misty edmondson,a herris maxwell,a joseph newton,b james wright,b soren rodning,c robert carson,a dwight wolfea adepartment of clinical sciences and bdepartment of pathobiology, college of veterinary medicine; cdepartment of animal sciences, college of agriculture, auburn university, auburn, al abstract microscopic examinations of epithelial tissues are valuable methods to evaluate surface and histologic architecture. the objectives of this study were to determine: 1) the changes in the thickness of the surface epithelium of the penis and prepuce, 2) the size of epithelial folds, 3) the location and distribution of epithelial folds in the surface epithelium, and 4) examination for the presence of crypts on the penis and prepuce of two groups of beef bulls (bos taurus and bos indicus). bulls two years of age and bulls >5 years of age were selected. neither the area encompassed by epithelial folds (p < 0.05) nor the number of epithelial folds (p < 0.05) differed between age groups based upon image j analysis of penile and preputial epithelium. keywords: penile epithelium, preputial epithelium, crypt, epithelial fold, beef bulls introduction it has long been assumed that as bulls age the folds or crypts on the surface of the penis and prepuce become deeper. the presence of deeper crypts purportedly facilitates the chronic carrier state of bovine venereal disease by providing a protected environment suitable for long term maintenance of infection. no published reports were found to support this assumption and this study was conducted to characterize the surface architectural anatomy of the epithelium and epithelial crypts in younger and older bulls. the gross anatomy of the bovine penis and prepuce has been described and the intricate mechanisms leading to erection and ejaculation defined.1 compounded wrinkles and folds of the preputial epithelium and creases on the surface of the glans penis are present and thought to allow for the mechanics of penile extension during erection.‡ the surface architecture of the genital epithelium has been speculatively discussed for decades but quantitative studies of the microscopic anatomy and the effects of ageing are lacking. it has been suggested that that the epithelial microstructure of the penis and prepuce undergoes significant age-associated changes and that these changes are important in establishment of persistent infection with tritrichomonas foetus (t. foetus) or campylobacter fetus subsp. venerealis (c. fetus).2 persistence of infection with these organisms has been speculated to be related to deeper folds or crypts in the preputial epithelium of mature bulls. a crypt is defined as a blind pit or tube-like structure on a free surface.3 specific structures such as the crypts of liberkühn are well-defined as a lumen of intestinal glands on the surface of the intestinal mucus membrane that serve to secrete or are absorptive in nature.4 however, epithelial crypts are poorly defined on the penis and prepuce of the bull. penile anatomy of the bull the surface of the penis and the prepuce within the preputial cavity are among the very few nonhaired areas of skin present on the bovine. the free portion of the non-erect penis distal to the attachment of the prepuce is approximately 12 cm long and lies within the caudal portion of the preputial cavity when the penis is non-erect. the free portion is capped by a small cushion of asymmetrical, ventrally directed, slightly spiraled tissue which comprises the glans penis.5 * portions of these data were presented at the 2011theriogenology annual conference. † current address: department of animal science, university of tennessee, knoxville, tn. ‡ personal communication, dr. david bartlett. 445 clinical theriogenology • volume 6, number 4 • december 2014 the prepuce of the bull is 35 to 40 cm long and approximately 4 cm in diameter with wide variations among breeds.6 the prepuce is composed of an external, parietal, and visceral layer. the parietal and visceral layers of the prepuce are covered with stratified squamous epithelium. the free portion of the penis is covered with stratified squamous epithelium which is very tightly adhered over the apex. this epithelium transitions caudally to become more loosely attached at the junction of the free portion and prepuce, allowing the epithelium to change its orientation when the penis is extended or withdrawn.5 the distal portion of the penis is encapsulated by the smooth cap-like glans penis, while the remainder of the glans has a rough uneven epithelial surface. the epithelium of this area is characterized by a network of fine epithelial folds which vary from slightly irregular depressions to relatively deep, closely-arranged crevices that produce a papillate appearance.7 it has been alleged that epithelial folds or crypts become deeper as a bull matures and that folding of the epithelium provides a protected environment suitable for the development of a chronic carrier state of venereal diseases.8 materials and methods animals healthy sexually mature bulls were placed into two groups of six (table). group 1 (n=6) consisted of bulls two years of age (± 3 mo.). group 2 (n=6) consisted of bulls five years old or older (five to 12 yrs.). these age groups were selected because most bulls enter the breeding herd at two years of age and most are removed from the herd at five years of age. it was hypothesized that age-related penile and preputial epithelial differences would be apparent by five years of age. all tissue samples were collected from bulls presented to the john thomas vaughan large animal teaching hospital, college of veterinary medicine, auburn university for routine breeding soundness examinations. written consent was obtained from owners prior to tissue collection. each bull was sampled for presence of t. foetus and c. fetus. the auburn university institutional animal care and use committee approved all protocols. biopsy technique three locations were chosen for epithelial biopsy: 1) the distal penis 1 cm proximal to the glans, 2) the proximal portion of the free end of the penis, 1 cm distal to the attachment of the prepuce, and 3) the proximal prepuce 6 cm distal to the attachment of the prepuce and the sheath. each bull was restrained in a livestock chute and the penis was manually extended, held by sterile surgical gauze, and cleaned with water prior to aseptic surgical preparation. the dorsal penile nerves were blocked with 2% lidocaine hydrochloride (5 ml) and a #22 scalpel blade was used to excise a 1cm diameter sample of surface tissue from each target area. each excision area was closed with 0 chromic gut in a cruciate pattern. collection of all samples was conducted by the same investigator to minimize differences of collection methods. tissue samples were pressed onto sections of tongue depressors to reduce artifact contracture and folding during fixation. each tissue sample was immersion fixed and stored in a mixture of 4% paraformalin, 1% glutaraldehyde (electron microscopy services, hatfield, pa), and 150 mm phosphate buffered saline (sigma chemical, st louis, mo) prior to processing. light microscopy all steps of tissue preparation were performed under a ventilated hood in compliance with osha guidelines. tissues for light microscopy were prepared by rinsing in phosphate buffered saline (pbs) in three separate rinses of 20 min each to remove fixative agents. fixative-free tissues were dehydrated with ethyl alcohol (etoh) in graded strengths to displace water from the tissues. fixation was performed in the following manner: 1) each sample was placed in 30% etoh and agitated at room temperature for 30 min, 2) the samples were placed in a series of ascending concentrations of etoh (50%, 70%, 85%, 100%) for 30 min each, 3) samples were washed two times in hexamethyldisilazane (hmds; electron microscopy services) for 30 min each to clear residual etoh, and 4) tissue samples were placed under a fume hood overnight to allow remaining chemicals to be removed. 446clinical theriogenology • volume 6, number 4 • december 2014 tissue samples were placed in molds for embedding in paraffin. samples were processed in a tissue-tek vip e300™ (ames co., inc., elkhart, in) for 2-3 hrs. each sample was cut into 7 micrometer sections using a reichert-jung 2040 autocut microtometm (germany). the sections were mounted on glass microscope slides with resin and allowed to dry for 20 minutes, then stained with hematoxylin and eosin (sigma chemical). each biopsy sample was examined at 40 x magnification to evaluate the morphology of the penile and preputial surface epithelium. evaluation was performed by a veterinary pathologist blinded as to the group and location from which the biopsy specimen was collected. image j software (nih.gov) was used to determine epithelial surface area, the area of epithelial folds, and the number of epithelial folds per section. measurements of each parameter from each location were compared within and between groups. scanning electron microscopy tissues for scanning electron microscopy (sem) were collected and stored in a mixture of 4% paraformalin, 1% glutaraldehyde (electron microscopy service), and phosphate buffered saline (pbs; sigma chemical) at 4°c. the tissues were rinsed in pbs for 20 min with agitation. tissues were fixed in 4% osmium tetroxide/pbs (electron microscopy services) for two hours, rinsed with pbs for three changes of 20 min each, and rinsed with distilled water for 20 min. tissues were dehydrated as previously described for light microscopy. all samples were stored under a vacuum to ensure complete dehydration. the samples were mounted on an aluminum specimen stud (electron microscopy services) and sputter coated with colloidal gold (electron microscopy services) prior to viewing with an evo 50 sem™ (zeiss xvp, usa). images were captured with the evo 50™ (zeiss xvp, usa) computer and stored on removable file. similar to light microscopy samples, surface epithelium of each sample was evaluated by a veterinary pathologist blinded to group and location from which biopsy specimens were collected. differences in the surface epithelium at each anatomical location were compared within and between groups by subjective evaluation. biopsy analysis the following parameters were measured for each biopsy (3.256 mm): 1) area of the epithelium between the basement membrane and the luminal surface, 2) area of epithelial folds under a line drawn from the pinnacles of the surface epithelium at the edges of the epithelial fold, 3) the number of epithelial folds, and 4) presence of crypts.3,4,9 an epithelial fold was defined as a deviation encompassing an area greater than 200 square microns, the area that would accommodate a tritrichomonas foetus organism (20x10 microns). these parameters could be consistently measured with image j software. area of epithelium each sample of epithelium was labeled according to the anatomical location from which it was collected. from each specimen color images of 4080 x 3072 pixel resolution (40x magnification) were acquired with a light microscope (olympus ® bh-2) and digital camera (olympus® dp-71). epithelial borders were traced and epithelial areas were determined using image j analysis software. area of epithelial folds for each sample of epithelium, the borders of the epithelial folds were outlined, the area of the epithelial fold determined and the number of folds recorded using image j software. examination for crypts each biopsy was examined with scanning electron microscopy for presence of structures that met the criteria for crypts as described.3,4,9 the surface epithelium of each biopsy from each bull was evaluated and compared within and between age groups. 447 clinical theriogenology • volume 6, number 4 • december 2014 statistical analysis area of epithelium, area of epithelial folds, and total number of epithelial folds per biopsy were compared within and between age groups by anova using the sas analytical system (sas software, sas institute, cary, nc). a value of p <0.05 was considered significant. results area of epithelium the area of the epithelium did not differ within (p = 0.35) or between (p =0.77) groups (figure 1). the mean ± sd epithelial areas were: group 1: distal 0.8 x 106 ± 0.3 x 106 µm2, middle 0.7 x 106 ± 0.2 x 106 µm2, and proximal 1.0 x 106 ± 0.3 x 106 µm2; group 2: distal 0.8 x 106 ± 0.3 x 106 µm2 , middle 0.7 x 106 ± 0.3 x 106 µm2, and proximal 0.8 x106 ± 0.3 x 106 µm2. area within epithelial folds the area of epithelial folds did not differ within (p =0.07) and between groups (p = 0.15) (figure 2). the areas of epithelial folds (mean ± sd) for group 1 were: distal 0.1 x 106 ± 0.1 x 106µm2, middle 0.1 x 106 ± 0.03 x 106 µm2, and proximal 0.2 x 106 ± 0.1 x 106 µm2. the areas of epithelial folds of group 2 were: distal 0.1 x 106 ± 0.09 x 106 µm2, middle 0.07 x 106 ± 0.05 x 106 µm2, and proximal 0.1 x 106 ± 0.1 x 106 µm2, (figure 2). number of folds the number of epithelial folds did not differ between biopsy sites within (p = 0.56) and between (p=0.41) age groups. the number of epithelial folds (mean ± sd) per site was: group 1 distal 12.3 ± 3.8, middle 9.2 ± 3.4, and proximal 12.7 ± 5.8; group 2 distal 12.3 ± 6.7, middle 10.6 ± 5.3, and proximal 14.1 ± 11.4 (figure 3). electron microscopic evaluation tissues were examined by electron microscopy for presence of epithelial crypts at 31–170x magnification. folds of epithelium were present on all sections examined but no structures comparable to intestinal crypts were identified.9 discussion in 1941, shortly after the initial description of t. foetus, abelein noted that there were numerous infoldings present on the surface of the penis. the author suggested that t. foetus established long-term infection in these crevices.10 it has been stated that presence of deeper crypts in older bulls allows the development of chronic infection with c. fetus subsp. venerealis.2 for decades older bulls have been assumed to have deeper crypts than younger bulls, but little documentation exists to support this conclusion. this study was undertaken to evaluate the differences in the penile and preputial epithelium between different aged bulls at three different anatomical locations. the first objective of this study was to determine the thickness of the epithelial layer of the penis and prepuce as the bull matures. the area of the surface of the epithelium did not differ within or between groups. a second objective of this study was to determine the area encompassed by the epithelial folds and to compare differences within and between groups. area within the epithelial folds did not differ within or between groups. this was unexpected and contrary to the widely held belief that older bulls would have increased areas of the epithelial folds. in each age group the area within the epithelial folds was greater at the proximal and distal sites than the middle. the final objective was to determine the total number of epithelial folds in each age groups. the total number of folds did not differ within or between the groups. also, based upon examination by sem, there is an absence of structures that can be classified as crypts. due to the absence of the crypt or cryptlike structures, the authors propose that epithelial folds be used as the preferred descriptive term. 448clinical theriogenology • volume 6, number 4 • december 2014 architectural characterization and electron microscopic evaluation of penile and preputial epithelium may enhance future studies of the pathophysiology and immunology of venereal infections in bulls. acknowledgement the authors acknowledge the auburn university college of veterinary medicine working group for venereal diseases in animals for their support of this project. references 1. hafez ese: anatomy of male reproduction. in: reproduction in farm animals. 6th ed. philadelphia: lea & febiger; 1993. p. 3-19. 2. bondurant rh: venereal diseaes of cattle: natural history, diagnosis, and the role of vaccines in their control. vet clin north am food anim pract 2005;21:383-408. 3. blood dc, studdert vp: baillière’s comprehensive veterinary medical dictionary. london: wb saunders; 1995. p. 240. 4. samuelson da: textbook of veterinary histology. st. louis: wb saunders; 2007. p. 339. 5. dyce km, sack wo, wensing cjg: textbook of veterinary anatomy. 4th ed. st. louis: wb saunders; 2010. p. 717. 6. bellenger cr: a comparsion of certain parameters of the penis and prepuce in various breeds of beef cattle. res vet sci 1971;12:299-304. 7. hammond dm, bartlett de: the distribution of tritrichomonas foetus in the preputial cavity of infected bulls. am j vet res 1943;4:143-149. 8. bondurant rh: pathogenesis, diagnosis and management of trichomoniasis in cattle. vet clin north am food anim pract 1997;13:345-361 9. skrzypek t, valverde piedra jl, skrzypek h, et al: intestinal villi structure during the development of pig and wild boar crossbreed neonates. livest sci 2007;109:38-41. 10. abelein r: trichomonadenseuche beim bullen und ihre behandlung. berl munich tier wchnschr, july 25, 1941:357362. group 1 group 2 angus 25 mo. angus 26 mo. angus 25 mo. brahman 24 mo. angus 27 mo. angus 26 mo. angus 6 yrs. angus 5 yrs. angus 12 yrs. angus 5 yrs. angus 7 yrs. angus 6 yrs. table. age and breed of bulls. 449 clinical theriogenology • volume 6, number 4 • december 2014 figure 1. mean epithelial area and standard deviation (a-f) per site. area between groups is not different (p= 0.77) a) sd ± 0.3 x 106 b) sd ± 0.3 x 106 , c) sd ± 0.2 x 106 , d) sd ± 0.3 x 106 , e) sd ± 0.3 x 106 , f) sd ± 0.3 x 106. figure 2. mean area of infoldings and standard deviations (a-f). area of infoldings between age groups is not different (p = 0.15) a) sd ± 0.1 x 106 b) sd ± 0.09 x 106 , c) sd ± 0.03 x 106 , d) sd ± 0.05 x 106 , e) sd ± 0.1 x 106 , f) sd ± 0.1 x 106 a) mean area of infoldings within each group was greater in proximal sites than in middle sites (p = 0.025). b) mean area of infoldings within each group was greater in distal sites than in middle sites (p = 0.028). 0 0.2 0.4 0.6 0.8 1 1.2 a b c d e f distal middle proximal ar ea (10 6µ m 2 ) anatomical location mean epithelial area 2 yr old ≥ 5 yr old 0 0.05 0.1 0.15 0.2 0.25 0.3 a b c d e f distal middle proximal ar ea (1 06 µm 2 ) anatomical location mean area epithelial folds 2 yr old ≥ 5 yr old b b a,b a,b a a 450clinical theriogenology • volume 6, number 4 • december 2014 figure 3. total number of folds/3256 µm and standard deviations (a-f). a) mean 12.3 sd ± 3.8, b) mean 12.3 sd ± 6.7, c) mean 9.2 sd ± 3.4, d) mean 10.6 sd ± 5.3, e) mean 12.6 sd ± 5.8 f) mean 14.2 sd ± 11.4. 74 55 76 69 64 85 d i s t a l m i d d l e p r o x i m a l # fo ld s / 3 25 6 m ic ro n s anatomical location total # of infoldings 2 yr old ≥ 5 yr old f a b c d e 451 clinical theriogenology • volume 6, number 4 • december 2014 452clinical theriogenology • volume 6, number 4 • december 2014 omniblank: 2009: streptococcus equi subspecies zooepidemicus resides deep in the chronically infected endometrium of mares streptococcus equi subspecies zooepidemicus resides deep in the chronically infected endometrium of mares m. r. petersena, j.m. nielsenb, h. lehn-jensena, a.m. bojesenc aveterinary reproduction and obstetrics, department of large animal sciences, faculty of life sciences, university of copenhagen, dyrlaegevej 68, dk-1870 frederiksberg c, denmark bansager dyrehospital (ansager large animal hospital), gartnerhaven 5, dk-6823 ansager, denmark cdepartment of veterinary pathobiology, faculty of life science, university of copenhagen, stigbøjlen 4, dk-1870 frederiksberg c, denmark abstract bacterial endometritis is considered a leading cause of infertility in the mare. detection of the causative agent has traditionally relied on the use of the “guarded swab method”. recently, however, attention has been directed towards alternative detection methods after nielsen6 found that a high proportion of infertile mares that were negative for streptococcus equi subspecies zooepidemicus (s. zooepidemicus) based on culture from swabs (21/84=25%), were positive based on culture from endometrial biopsies (61/84=73 %) (p<0,0001). using fluorescence in situ hybridization (fish), bacteria may be visualized within infected tissue, and their spatial distribution can be used to provide information about bacterial pathogenesis. the aim of the present study was therefore to develop a fish procedure to visualize s. zooepidemicus in order to be able to detect the bacteria in the endometrium of experimentally infected mares and in broodmares diagnosed with clinical endometritis. using fish, s. zooepidemicus could be visualized in the endometrium in a majority of the infected mares studied. in the young experimentally infected mares, s. zooepidemicus was 393 located superficially in the uterus, and could not be visualized 48 h after inoculation. however, in the chronic endometritis cases, s. zooepidemicus was found in distinct foci within the endometrium, often just below the luminal epithelia, but also further down, in the stratum compactum, 300 to 500 µm from the endometrial lumen. no s. zooepidemicus could be visualized directly on the luminal surface of the endometrium, but in some instances bacteria were present in the endometrial crypts. keywords: endometritis; streptococcus equi subspecies zooepidemicus, localization, chronic infections, equine, fluorescence in situ hybridization introduction for more than 80 years endometritis has been considered a leading cause of infertility in the mare. 1, 2 streptococcus equi subspecies zooepidemicus (s. zooepidemicus ) has historically been considered as the main pathogen in equine endometritis. 3-6 forty years ago, hughes and loy7 demonstrated that a population of mares could be described as either susceptible or resistant to endometritis based on their ability to clear an infection following experimental inoculation of s. zooepidemicus. since then, substantial efforts have been devoted to identify factors that determine whether a mare is susceptible or resistant to endometritis. compromised uterine clearance and myometrial contractility were demonstrated in susceptible mares8, 9 and have been identified as important components of the pathophysiology of endometritis. 10-12 the diagnosis of endometritis in the mare has routinely relied on using a guarded swab followed by bacterial culture and cytological evaluation of exfoliated endometrial material.13,14 recently, attention has been directed towards alternative detection methods, after it was shown by nielsen6 that the swabbing method was inadequate for detection of uterine pathogens, especially s. zooepidemicus. s. zooepidemicus were isolated from sixty-three of 84 culture positive mares (75%) based on endometrial biopsy whereas only 23 of the same 84 mares (27%) cultured s. zooepidemicus using the swabbing method (p<0,0001). 394 the higher proportion of positive mares using endometrial biopsy as compared to swabs, indicates that s. zooepidemicus may reside below the epithelial lining, in the deeper layers of the endometrium, and is therefore less likely to be detected by the non-invasive swabbing method. information regarding the localization of s. zooepidemicus within the endometrium will affect our perception of s. zooepidemicus induced endometritis and potentially affect the choice of treatment regimen. consequently, we wanted to investigate where in the endometrium s. zooepidemicus was located in mares suffering from endometritis. together with immunohistochemistry and polymerase chain reaction (pcr), fluorescence in situ hybridization (fish) offers an alternative to traditional detection methods based on bacterial culture.15 the fish technique relies on the use of hybridization between bacterial 16s ribosomal-rna and fluorescent labeled complementary oligonucleotides. after hybridization, detection of individual bacterial cells can be made by fluorescence microscopy.16 using fish, bacteria can be identified in their natural environment, and since spatial distribution within the infected tissue can be evaluated, information regarding bacterial pathogenesis can be generated.17 compared to standard epifluorescence microscopy, confocal laser scanning microscopy (clsm) has the advantage of a pinhole to exclude fluorescence that originates outside the focal point of the objective lens, thus increasing the resolution of fine structures.18 as 3d data can be acquired using clsm, detailed visualization of small structures like s. zooepidemicus (0,5-2 µm) within the tissue of interest can be obtained. the primary aim of this work was therefore to develop a fish procedure that allowed visualization of s. zooepidemicus in the endometrium of the mare, and to demonstrate the localization of the bacteria in the endometrium of experimentally infected mares as well as in broodmares diagnosed with clinical endometritis. materials and methods 395 endometrial biopsies were recovered from 4 mares experimentally infected with s. zooepidemicus and from 6 broodmares with symptoms of clinical endometritis. except otherwise stated, all plastic wares were purchased from nunc (vwr international, albertslund, denmark), and all reagents were from sigma-aldrich (vallensbæk, denmark). the media were prepared with freshly produced milli-q water (millipore, hedehusene, denmark). endometrial biopsies from mares experimentally infected with s. zooepidemicus four reproductively normal standardbred mares aged five to ten years and belonging to the teaching herd at the faculty of life science, department of animal science, were used for the experimental infections. the experimental mares were evaluated for cyclicity and clinical signs of endometritis (transrectal palpation/ultrasound and speculum examination of the vagina) every two to three days for a minimum of 14 days before bacterial inoculation. when mares were in estrus, they were inoculated with a s. zooepidemicus isolate cultured from a mare with clinical endometritis (109 colony forming units in 10 ml of brain-heart infusion broth). endometrial biopsies were sampled from the left and right uterine horn before inoculation (0 h) and again 3h, 6h, 24h, 48h and 96h after inoculation. biopsies were collected as described by nielsen.6 in brief, the mare was placed in a set of stocks, the tail was wrapped, the perineal region washed, and a sterile biopsy speculum was introduced through the vagina into the cervical canal. biopsies were recovered using an alligator forceps directed to the base of the left and right uterine horn. the biopsy instrument was not guided by rectal palpation. following collection of a biopsy, a sterile pair of pincers was used to smear the biopsy onto blood agar (5% fetal calf blood, toxin free). blood agar plates were incubated at 37 °c for 48 h and colonies of s. zooepidemicus were identified by the β-hemolysis, colony morphology (mucoid and non-pigmented) and gram stain. 396 experimental mares were re-evaluated by transrectal palpation and ultrasography, a speculum examination of the vagina, and a bacterial culture from endometrial biopsy 18 to 21 days after inoculation. endometrial biopsies from broodmares in clinical practice infected with s. zooepidemicus endometrial samples were collected from broodmares if clinical signs of endometritis were detected, e.g. >2 cm free fluid in the uterus during estrus before and/or after breeding, mucupurulent vaginal discharge, or repeated breeding without establishment of a pregnancy. endometrial biopsies were collected and bacterial culture performed as described by nielsen.6 no information was available concerning the mares or the breeding management including pregnancy status. to increase the chance of visualizing s. zooepidemicus only endometrial biopsies from which β-haemolytic streptococci had been cultured were evaluated by fish. five biopsies were evaluated. fluorescence in situ hybridization (fish) oligonucleotide probes. the streptococcus specific oligonucleotide probe, strept (5`cy3-cactctccccttctgcac-3`), used to target 16s rrna from s. zooepidemicus in this experiment was developed by trebesius et al.19 as a positive control, the general bacteria probe eub338 (5`cy5-gctgcctcccgtaggagt-3`) was used. 20 as a negative control, the reverse and complementary strept probe called non-strept (5`cy3-gtgcagaaggggagagtg-3`) was used. all probes were purchased from tag copenhagen, denmark. in situ hybridization of bacterial cells. prior to hybridization, fixed bacterial cells were bound to poly-l-lysine (sigma, st. louis, usa). teflon coated slides (novakemi ab, enskede, sweden) and dehydrated by sequential washes in 70 and 96% ethanol (3 min. each). ten µl of hybridization buffer (40% formamide, 20 mm tris, ph 7.0, 0.9 m nacl, 0.1% sds) and 5 ng of probe were applied to each slide followed by hybridization in a humidified moist chamber at 46 397 oc. the duration of the hybridization time was at least 1 h. slides were washed in hybridization buffer (46 oc) for 10 m and subsequently transferred to a washing buffer (46 oc) for 15 m. the slides were finally rinsed in milliq-water (46 oc) and air-dried in the dark. the hybridization signal intensity was evaluated with the probes strept, non-strept, and eub338. preparation of tissue sections and in situ hybridization. after smearing onto blood agar, the biopsies were transferred into 10% phosphate-buffered formaldehyde for fixation, and the following day transferred to 70% ethanol and placed at 5 °c until slide processing. the biopsies were embedded in paraffin, cut into 5 µm thick sections, which were mounted on adhesive superfrost/plus slides (menzel-gläser, braunschweig, germany). from each biopsy 10 tissue sections were made, of which four would be hybridized with the strept and four with the eub probe respectively, while the remaining two slides would be labeled with the non-strept probe. prior to hybridization, the slides were deparaffinized in xylene for 3 m and allowed to air dry. the tissues samples were then dehydrated by sequential washing in 70% and 90% ethanol (3 min each), and allowed to air dry. lysozyme (5mg/ml in 100mm tris (ph 8.0), 50 mm edta,) was then added and incubated for 20 min at 37 oc and finally washed in mili-q water. hybridization was performed using 100 μl hybridization buffer (40% formamide; 20 mm trishcl, ph=7.0; 0.9 m nacl; 0.1% sodium dedocyl sulphate [sds]) containing a total of 200 ng of probe per tissue section. only one probe was used for each tissue section. hybridization was conducted in a cmt-hybridization chamber (corning inc., corning, ny, usa) at 46 oc overnight. the slides were then washed in prewarmed hybridization buffer at 46 oc for 15 m and transferred to a washing buffer containing 20% (vol/vol) formamide for 10 m at 46 oc. finally the slides were washed in mili-q water, allowed to air dry before being mounted with a coverslip for microscopy. confocal laser scanning microscopy 398 imaging was performed on a leica confocal microscope (leica tcs sp2; leica microsystems, bensheim, germany). the objective used were leica microsystems hc pl apo ×10/0.40 or hcx pl apo ×63/1.32 (oil). to allow visualization of the endometrial tissue, autofluorescence was excited by the 458 nm laser and the emitted light was detected at the interval from 460 to 529 nm, whereas cy3 and cy5 were excited by the 543 nm and 633 nm lasers, respectively, and emitted light detected at the interval from 540 to 600 nm and 650 to 750 nm, respectively. either single images or a stack of images were used to construct three dimensional visualization of the tissue. image visualization and three-dimensional analysis were performed with the leica tcs sp2 software. results verification of probe specificity fish on pure cultures of the s. zooepidemicus strain used for experimental infections demonstrated that the bacteria were labeled following probing with the streptand eub338probe, respectively. bacterial morphology and number could be determined with both probes, whereas no signal was obtained when the nonstrept probe was applied. endometrial biopsies from mares experimentally infected with s. zooepidemicus results following experimental infections with s. zooepidemicus including clinical signs, bacterial culture, and fish are outlined in table 1. table 1: clinical signs, results of bacterial culture and visualization of s. zooepidemicus by fish at fixed time intervals following uterine inoculation with s. zooepidemicus (0 to 96 h). time from inoculation (h) clinical signs bacterial culture fish 399 0 no abnormalities noted no growth from three mares. two s. zooepidemicus colonies isolated from one mare. no bacteria visualized 3 and 6 increasing amount of fluid (echogenic on transrectal ultrasound evaluation) in the uterus. no discharge or small amount of discharge noted on the speculum examination. large (>40) number of s. zooepidemicus isolated from biopsies from each mare large number of bacteria visualized in the uterine lumen and/or aligned close to the luminal epithelium. bacteria visualized in all biopsies recovered. 24 echogenic fluid in the uterine lumen, mucupurulent exudates from the cervix large number of s. zooepidemicus isolated s. zooepidemicus organized in clusters in distinct foci on the luminal epithelium. very few if any bacteria visualized in the uterine lumen. 400 48 echogenic fluid in the uterine lumen, mucupurulent exudates from the cervix – reduced amounts in three of the four mares compared to 24 h. 10 to 20 colonies of s. zooepidemicus isolated from all mares. few and small clusters of s. zooepidemicus visualized on the luminal epithelium. bacteria visualized in biopsies from two of the four mares. 96 echogenic fluid in the uterine lumen identified in one of the four mares. vaginal discharge noted from one of the four mares. none or max 3 s. zooepidemicus colonies could be identified from three of the mares. a total of 14 s. zooepidemicus colonies were isolated from one mare (mare with clinical symptoms, left + right horn). no bacteria visualized in biopsies from the four mares. mares experimentally infected with s. zooepidemicus presented clinical signs as expected (table 1). at 24 h after inoculation mucupurulent discharge originating from the uterus was noted in all four mares. the amount of fluid in the uterus and vaginal discharge had decreased by 48 h in three of the four mares. by transrectal ultrasound, a medium amount (2 cm) of slightly echogenic intrauterine fluid could be identified in one of the four mares at 96 h after inoculation, and s. zooepidemicus was cultured from the biopsies from this mare. three of the mares cleared the 401 infection within 96 h, whereas one mare did not. s. zooepidemicus was cultured from this mare when she returned to estrus 19 d after inoculation. the mare was then treated on three consecutive days with penicillin (5 million iu), uterine lavage (1-3 l of ringer’s lactate) and oxytocin (10 iu, im q. 8 h, intervet®, intervet denmark as). bacterial culture from a uterine biopsy performed 2 d after the last treatment was negative. autofluorescence from the endometrial tissue, following excitation from the 458 nm laser, was adequate to outline the endometrial structures including luminal and glandular epithelium as well as uterine glands and vessels within the stratum compactum. differentiation of cells in general, e.g. identification of inflammatory cells, was not possible with the described techniques. in most situations, a standard epifluorescence microscope equipped with the correct filters is adequate to evaluate tissue processed for fish, and preferred over clsm because of price and ease of use. endometrial tissue autofluorescence was helpful in determining endometrial architecture, but autofluorescence prevented discrimination between artifacts and probe signals when a standard epifluorescence microscope was used, even though specific filters were applied. this problem was overcome when clsm was used. using the described fish protocol, s. zooepidemicus could be visualized in tissue sections originating from endometrial biopsies. when biopsies were collected 3 to 6 h after inoculation, s. zooepidemicus was localized in large numbers in the uterine lumen and /or in close association with the luminal epithelium. the number of bacteria decreased substantially, especially in the lumen of the uterus when biopsies were collected 24 h after inoculation. at this time, s. zooepidemicus appeared in small clusters on the luminal epithelium. due to the high resolution of the confocal microscope, the characteristic round shape of the s. zooepidemicus could be discerned. in biopsies collected 48 h after inoculation, few and very small clusters of s. zooepidemicus could be visualized on the luminal epithelia, but only in biopsies from two of the four mares, even though 402 bacteria were demonstrated upon culture from all recovered biopsies. no s. zooepidemicus could be visualized in biopsies recovered 96 h after inoculation, although s. zooepidemicus was cultured from one of the four mares. endometrial biopsies from broodmares in clinical practice infected with s. zooepidemicus biopsies collected from five broodmares from a clinical practice from which s. zooepidemicus had been isolated were processed as described above. using fish, s. zooepidemicus could be visualized in the endometrium from three of the five mares. in these likely chronic endometritis cases, s. zooepidemicus were localized primarily in distinct foci just below the luminal epithelium, but also deeper in the stratum compactum 300 to 500 µm from the endometrial lumen. no s. zooepidemicus could be visualized directly on the luminal surface of the luminal endometrium, but in some instances bacteria were present in the endometrial crypts. tissue remodeling with increased amounts of fibrotic tissue was, in some instances, observed in areas where bacteria were visualized; but in most cases presence of bacteria did not seem to entail changes in tissue morphology. examination of endometrial tissue labeled with the eub338 probe (positive control) revealed a similar bacteria localization pattern as observed using the strept probe. control hybridizations without any probe added (negative control) did not result in any specific hybridization signal. discussion using fish, we were able to show a rapid decline in the number of s. zooepidemicus during the first four days following experimental infection of young mares in estrus. however, in endometrial samples from clinical cases, presumably representing a more chronic infection, s. zooepidemicus was demonstrated to reside deeply within the endometrium. to our knowledge this has not been demonstrated before. 403 in our initial visualization experiments it was possible to visualize s. zooepidemicus in biopsies recovered 3 and 6 hours after inoculation, but not in biopsies recovered 24 hours after inoculation, despite the presence of a large number of bacteria in cultures of smears from the same biopsies. it is known that the success of fish is dependent on optimization of tissue fixation and permeabilization to allow the probe to access the bacteria.17, 21 in an attempt to increase the permeation of the 16s rrna probes, the tissue sections were treated with either lysozyme or protein kinase k .21 a pilot study detected no difference between samples treated with either of the two permeabilizing agents. increased permeabilization made it possible to visualize s. zooepidemicus in the endometrium, independent of time from inoculation. why permeabilization was necessary to allow visualization in samples recovered 24 h after inoculation, but not after 3 or 6 h, is difficult to determine. the explanation could be the presence of a bacterial capsule 24 h after inoculation, since s. zooepidemicus capsule synthesis is usually lost following primary in vitro culture. 22 a high degree of autofluorescence originated from the endometrial tissue, especially using epifluorescence microscopy when excitation was induced with a mercury lamp. this was particularly true when dilated uterine glands were present in the endometrium, likely due to mineralized deposits within these dilated glands 23 and perhaps autofluorescence originating from leucocytes, as shown in humans. 24 autofluorescence was reduced when clsm was employed, probably because excitation was induced with only one specific wavelength at a time.18 the reduced autofluorescence, combined with the capacity to freely select emission wavelength, whereby probe signal could be differentiated from background, made specific visualization of s. zooepidemicus in the endometrium possible. localization of s. zooepidemicus deep in the endometrium of chronically infected mares explains why nielsen6 found a higher number of mares were diagnosed with endometritis by culture from a biopsy than from a swab. a recent study using low volume lavage to diagnose 404 endometritis indicated that e. coli in some situations is more frequently isolated (42.2%) than βhemolytic streptococcus (37.6%). 25 other studies using a swab to diagnose endometritis, also conducted in kentucky, found β-hemolytic streptococci to be the pathogen most commonly isolated.5 the difference with respect to pathogens isolated clearly relates to the diagnostic method used and warrants further investigation. the fish technique might be useful in the future to illuminate differences in the pathogenesis of e. coli and s. zooepidemicus induced endometritis in the mare. recently, restriction fragment length polymorphism (rflp) was used to identify the genetic profile of s. zooepidemicus clones present in mares with endometritis.26 genetic profile or rflp pattern were evaluated in 12 mares before and after treatment with antibiotics. following treatment rflp patterns were identical to those before treatment in 11 of the 12 mares, indicating treatment failure. the authors concluded that s. zooepidemicus persisted in the endometrium despite treatment. different systemic or intrauterine treatments are likely to affect streptococci residing in the deeper layers of the endometrium differently. as described by schlafer, 27 the use of molecular based techniques such as rflp, amplified fragment length polymorphism,28 pcr, fish, etc. are likely to impact our knowledge of the pathogenesis of endometritis and facilitate optimal treatment protocols in the years to come. the impact of s. zooepidemicus residing deep in the endometrium of chronically infected mares on endometrial function is difficult to predict. recently, it has been suggested that persistent active inflammation in the endometrium can lead to increased mucus production. 29 riddle, et al.5 demonstrated the lowest pregnancy rate in mares with the highest degree of endometrial inflammation and correlated a high level of inflammation to isolation of β-hemolytic streptococci. chronic infections with s. zooepidemicus deep in the endometrium might induce inflammation and increase mucus production potentially reducing pregnancy rates. we foresee that 405 by using the approach described here we will eventually arrive at a better understanding of streptococcusinduced endometritis. acknowledgements the authors would like to thank parvin faghan for excellent work in the laboratory. references 1. schiebel g: untersuchungen über bakterienflora im uterus steriler stuten.[dissertation] tierarzl hochscule: hanover; 1920. 2. dimmockww, edwards pr: the pathology and bacteriology of the reproductive organs of mares in relation to sterility. bull ky agric exp sta 286; 1928. 3. wingfield digby n, ricketts s: results of concurrent bacteriological and cytological examinations of the endometrium of mares in routine stud farm practice 1978-1981. j reprod fertil suppl 1982;32:181-185. 4. shin sj, lein dh, aronson a, et al: the bacteriological culture of equine uterine contents, in-vitro sensitivity of organisms isolated and interpretation. j reprod fertil suppl 1979;27:307-315. 5. riddle wt, leblanc mm, stromberg aj: relationships between uterine culture, cytology and pregnancy rates in a thoroughbred practice. theriogenology 2007;68:395-402. 6. nielsen jm: endometritis in the mare: a diagnostic study comparing cultures from swab and biopsy. theriogenology 2005;64:510-518. 7. hughes j, loy r: investigations on the effect on intrauterine inoculation of streptococcus zooepidemicus in the mare. proc annu conv am assoc equine pract 1969; p. 289-292. 406 8. troedsson mht, liu ikm, ing m, et al: multiple site electromyography recordings of uterine activity following an intrauterine bacterial challenge in mares susceptible and resistant to chronic uterine infection. j reprod fertil 1993;99, 307-313. 9. troedsson m, liu i: uterine clearance of non-antigenic markers (51cr) in response to a bacterial challenge in mares potentially susceptible and resistant to chronic uterine infections. j reprod fertil suppl 1991;44:283-288. 10. troedsson mht: uterine clearance and resistance to persistent endometritis in the mare. theriogenology 1999;52:461-471. 11 leblanc mm, neuwirth l, asbury a, et al: scintigraphic measurement of uterine clearance in normal mares and mares with recurrent endometritis. equine vet j 1994; 26:109-113. 12. causey rc: making sense of equine uterine infections: the many faces of physical clearance. vet j 2006;172:405-421. 13. card c: post-breeding inflammation and endometrial cytology in mares. theriogenology 2005;64;580-588. 14. liu ikm, troedsson mht: the diagnosis and treatment of endometritis in the mare: yesterday and today. theriogenology 2008;70;415-420. 15. tegtmeier c, angen ï, ahrens p: comparison of bacterial cultivation, pcr, in situ hybridization and immunohistochemistry as tools for diagnosis of haemophilus somnus pneumonia in cattle. vet microbiol 2000;76:385-394. 407 16. delong ef, wickham gs, pace nr: phylogenetic stains: ribosomal rna-based probes for the identification of single cells. science 1989;243;1360-1363. 17. moter a, göbel ub.: fluorescence in situ hybridization (fish) for direct visualization of microorganisms. j microbiol meth 2000;41:85-112. 18. pawley j: handbook of biological confocal microscopy. new york: springer science and business media; 2006. 19. trebesius k, leitritz l, adler k, et al: culture independent and rapid identification of bacterial pathogens in necrotising fasciitis and streptococcal toxic shock syndrome by fluorescence in situ hybridisation. med microbiol immunol 2000;188:169-175. 20. amann ri, ludwig w, schleifer kh: phylogenetic identification and in-situ detection of individual microbial cells without cultivation. microbiol rev 1995;59:143-169. 21. carr el, eales k, soddell j, et al: improved permeabilization protocols for fluorescence in situ hybridization (fish) of mycolic-acid-containing bacteria found in foams. j microbiol meth 2005;61:47-54. 22. timoney jf: the pathogenic equine streptococci. vet res 2004;35:397-409. 23. walter i, helmreich m, handler j, et al: mineralised deposits in the uterine glands of mares with chronic endometrial degeneration. vet rec 2003;153:708-710. 24. monici m, pratesi r, bernabe pa, et al: natural fluorescence of white blood cells: spectroscopic and imaging study. j photochem photobiol b 1995;30:29-37. 408 25. leblanc mm, magsig j, stromberg aj: use of a low-volume uterine flush for diagnosing endometritis in chronically infertile mares. theriogenology 2007;68;403-412. 26. wittenbrink mm, hoelzle k, hoezle le: what´s new in bacteriology of the mare´s gentital tract. pherdeheilkunde 2008; 24:53-55. 27. schlafer dh: equine endometrial biopsy: enhancement of clinical value by more extensive histopathology and application of new diagnostic techniques? theriogenology 2007;68:413-422. 28. bindslev mm, villumsen, mh, petersen mm, et al: genetic diversity of s. equi ssp. zooepidemicus and e. coli isolated from the reproductive tract of the mare [abstract]. reprod dom anim 2008;43:110. 29. causey rc, miletello t, o'donnell l, et al: pathologic effects of clinical uterine inflammation on the equine endometrial mucosa [abstract]. proc annu conv am assoc equine pract 2008; p. 276-277. 409 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 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/openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1 contact júlia marlière julia.marliere@ufv.br © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 13214, http://dx.doi.org/10.58292/ct.v17.13214 review report effects of equine seminal plasma on fertility and impacts on reproductive biotechnologies: a comprehensive review tayná da silva, shara silva, júlia marlière, bruna waddington veterinary department, federal university of viçosa, viçosa, mg, brazil abstract stallion fertility is crucial for the equine industry, yet selection of stallions focuses on phenotypical, temperamental, and performance traits, with limited attention to reproductive performance. seminal plasma is composed of fluids from the testes, epididymis, and accessory glands; it has a central role in sperm survival and preservation by providing energy, antioxidants, and proteins. biochemical composition of seminal plasma, including specific proteins and enzymes, can affect sperm quality and cryopreservation success. literature presents conflicting views on the effects of maintaining, removing, or adding seminal plasma after cryopreservation. individual variations in seminal plasma composition can have substantial impact on stallion fertility and predict the success of these biotechnologies. keywords: stallion, sperm, ejaculate, cryopreservation, fertility introduction stallion fertility is a crucial factor within the equine industry. however, unlike other production species, stallions are primarily selected according to their phenotypical conformation, athletic aptitude, temperament, genetic potential, and financial value, with limited emphasis on reproductive performance.1 several factors can affect fertility, stemming from genetics, climate change, behavioral and environmental characteristics, breeding management, exercise regimen and competition, and pathologies, requiring clinical and pathological diagnoses to determine the reproductive prognosis.2-7 before a new stallion is introduced into reproductive activities, it should have a thorough examination in which features such as pedigree and behavioral characteristics, overall health and reproductive soundness (both anatomical and functional) should be assessed.8 semen biotechnologies are widely employed in the equine industry, offering greater flexibility for artificial insemination that has grown in popularity, particularly when the primary goal is to accelerate genetic improvement through the production of superior offspring.9,10 seminal quality is decisive for reproductive success in biotechnology application. processes such as semen cooling and, in particular, freezing, extend seminal viability but can cause damage to sperm due to irreversible plasma membrane lesions, reducing fertilization potential.11,12 stallion ejaculate consists of a liquid and a cellular fraction, expelled, though not uniformly, in a variable number of squirts. in general, according to sperm concentration, the ejaculate can be divided into 3 consecutive fractions: presperm, spermrich, and postsperm.13 seminal plasma constitutes the liquid portion of the ejaculate and comprises fluids produced and secreted by testes, epididymides, and accessory sexual glands.14 its components have a substantial role in sperm survival by providing energy substrate to sperm, as well as amino acids, lipids, proteins, steroid hormones, enzymatic and nonenzymatic antioxidants, and micronutrients like selenium, zinc, copper, and iron, among others.15-21 there is disagreement regarding the addition or removal of seminal plasma during cryopreservation. therefore, the objective of this review is to compile current available data about the seminal plasma profile and its impacts on reproductive biotechnologies and fertility. seminal plasma as in other mammalian species, stallion sperm are not promptly capable of fertilization once they leave testes.22 posttesticular maturation occurs in 2 steps: maturation and capacitation/acrosome reaction. maturation occurs during sperm transit in the epididymis and capacitation and mailto:julia.marliere@ufv.br http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.13214 2 citation: clinical theriogenology 2025, 17, 13214, http://dx.doi.org/10.58292/ct.v17.13214 acrosome reactions occur in mare’s reproductive tract. both of these events comprise a series of structural and functional changes to sperm so that they become fertilization-competent.19,23 some of the critical changes include acquisition of progressive motility and ability to recognize and bind to zona pellucida, chromatin condensation, cholesterol depletion with increase in membrane fluidity, increase of cytoplasmatic ph, camp, and calcium concentrations, calcium influx through the acrosomal membrane, and exocytosis.24-26 during ejaculation, the prespermatic fraction is the first to be released and removes impurities from the urethra, being mainly composed of fluids originating from the bulbourethral glands.13 spermrich fraction is then released, characterized by its milky appearance due to high sperm concentration (containing up to 80% of the total sperm) and its composition derives from secretions of the epididymis, ampulla, and deferens ducts.27 although the last fraction does not contain substantial sperm, it is responsible for transporting residual sperm from stallion’s urethra through vesicular glands’ secretions.13 the volume of the ejaculate fractions can be altered by excessive teasing or sexual preparation of the stallion and false mounts (increases the volume of seminal plasma) and consequently, the total volume of semen.28 biochemical composition of the seminal plasma proteins and enzymes proteins are the main component of the seminal plasma, and epididymis is the site where ~ 70% of seminal proteins are secreted.29 although it is the most abundant in the seminal plasma, stallion seminal plasma protein concentration is lower than other mammalian species, with a mean concentration of 10 mg/ml. this is of particular interest when evaluating the role of seminal plasma components due to their participation in crucial processes before fertilization.30 exact information regarding fertility-associated proteins remains unclear. although much research has been carried out on the topic, current information about those proteins is still insufficient to establish diagnostic protocols for stallion fertility based on seminal plasma composition.31 some of the major proteins present in the stallion seminal plasma are horse seminal plasma proteins (hsp), a group of 8 recognized low molecular weight proteins.32 with the exception of hsp-4, all hsps are able to bind to the sperm surface at ejaculation;30 2 of these proteins, hsp-1 and hsp-2, account for up to 80% of the total seminal protein content. they belong, although not exclusively, to 3 structurally varying seminal protein groups: hsp-1 and hsp-2 belong to the fibronectin type ii proteins, hsp-3 belongs to the cysteine-rich secretory proteins (crisps), and hsp-7 belongs to the spermadhesins.32,33 hsp-1 and -2 are capable of binding to sperm surface through choline phospholipids present in the gamete membrane, leading to cholesterol efflux, an important event for sperm capacitation.26,34 however, the influence of the concentration of these proteins in seminal plasma on fertility was discussed and is questionable. some reports observed high concentrations of hsp-1 and -2 in the ejaculates of stallions with poor fertility.17,35,36 a possible explanation for this finding is the ability of these proteins to increase the permeability of the sperm membrane, compromising its integrity.37 crisp proteins are divided into 3 main classes, characterized by their structural composition of 16 cysteine residues: crisp1, crisp-2, and crisp-3 (hsp-3).32,33 they are the product of secretions from the epididymis, ampulla and seminal vesicle, and throughout the reproductive tract, crisp-3 is in higher concentration compared to others.38 its presence has effects not only on sperm but also on the reproductive tract of the mare after breeding. in fresh semen, high concentration of crisp-3/hsp-3 in the seminal plasma was associated with increased sperm motility and seminal quality index.39 it also has an immunomodulatory effect on the mare reproductive tract during the physiological uterine postbreeding inflammatory response, suppressing the binding between sperm and polymorphonuclear neutrophils and increasing fertility.40,41 hsp-4 and hsp-5 are proteins with little or no clear functions documented at this time. hsp-4 is presumed to be related to a calcitonin-like product, impacting sperm motility in men, whereas hsp-5 has not yet been associated with any specific protein.32,42 in stallions, the hsp-7 is the only identified member of the sperm adhesins. it is homologous to boar spermadhesin (awn) protein and its participation during sperm binding to the zona pellucida.30,43 hsp-6 and hsp-8 are isoforms of the same protein, belonging to the kallikrein family, but their function is not well established.32 immunoglobulins are also present in the seminal plasma. izumo sperm-egg fusion protein 4 (izumo4) was isolated from equine seminal plasma,44 a protein belonging to the immunoglobulin group. it was suggested that its function is correlated to prefertilization stages, such as capacitation.44 the izumo multiprotein family is composed of 4 proteins; izumo 1, izumo 2, izumo 3, and izumo 4 and has already been described as a sperm membrane protein in mice and humans.45,46 in these species, the izumo immunoglobulins are expressed on the cellular membrane surface, being essential for gamete fusion during fertilization. some enzymes have been isolated and described in equine seminal plasma. alkaline phosphatases are a group of isoenzymes widely distributed through various horse tissues, such as intestinal mucosa, liver, bones, renal tubule, lungs, and placenta.47-50 its sources include testicular and epididymal fluids, and for this reason, it can be used as an ejaculation completion marker in normal stallions. low alkaline phosphatase concentration in the seminal plasma may indicate ejaculatory failure and/or ampullary blockage, being an important clinical tool to differentiate these conditions from azoospermia.51 matrix metalloproteinases 2 and 9 (mmp-2 and mmp-9) are gelatinases responsible for degrading extracellular matrix components during tissue restructuring, in physiological and pathological conditions. they are secreted in latent forms and are present in all fractions of the stallion ejaculate. however, there still no evidence to support the use of metalloproteinases expression as a marker for stallion semen quality.52 enzymatic and nonenzymatic antioxidants the large group of enzymes identified in the stallion seminal plasma is related to the oxidative balance. as aerobic cells, sperm face the oxidative paradox: oxygen is needed for energy production, but its metabolites can cause significant, and http://dx.doi.org/10.58292/ct.v17.13214 citation: clinical theriogenology 2025, 17, 13214, http://dx.doi.org/10.58292/ct.v17.13214 3 sometimes irreversible, cellular damage.53 reactive oxygen species (ros) act as mediators for the transfer of electrons in several normal biochemical reactions. all aerobic organisms are continually subject to the oxidizing effects of these metabolites, as ros is the result of aerobic metabolism.54 stallion sperm, unlike human sperm, predominantly use oxidative phosphorylation to produce energy.53 the increased energy production through phosphorylation results in a rapid production of ros. under normal conditions, ros production is balanced by the action of antioxidant systems, both enzymatic and nonenzymatic. moreover, ros are necessary mediators for physiological sperm processes, such as tyrosine phosphorylation and capacitation.55 however, the accumulation of ros leads to the oxidation of biomolecules, including peroxidation of lipid membranes, protein damage, carbohydrate oxidation, and destruction of nuclear and mitochondrial dna and rna.56 mammalian sperm, due to the high concentration of polyunsaturated fatty acids in the plasma membrane, are particularly sensitive to oxidative damage, especially lipid peroxidation.57 catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase are antioxidant enzymes that act as ros scavengers and have been identified in the stallion seminal plasma.58,59 superoxide dismutase activity has been associated with a function other than ros neutralization. in association with lactoferrin, an iron-binding protein identified in stallion epididymal secretion, superoxide dismutase-3 forms a complex that binds itself to nonviable sperm, facilitating their elimination by phagocytosis in mare’s reproductive tract.18,60 previously mentioned, hsp-1 and hsp-2 have also been suggested to act as chaperones against oxidative damage and may be able to prevent ros production.61 minerals can contribute, alone or in combination with enzymes, to sperm quality. calcium, selenium, zinc, copper, and molybdenum are positively associated with sperm morphology, motility, and fertility, whereas higher iron and chrome concentrations were associated with lower fertility in arabian stallions.21,62,63 copper, zinc, and manganese are important cofactors for superoxide dismutase activity.64 other seminal components the carbohydrate concentration in the stallion seminal plasma is substantially lower compared to bull and ram ejaculates, and it is suggested that the gel secreted by the seminal vesicles is the main source of carbohydrates. inositol and glucose are in stallion semen, whereas fructose is only in insignificant concentrations.65,66 the lipid content of seminal plasma is low, consisting mainly of phospholipids and cholesterol. the phospholipid-to-cholesterol ratio in seminal plasma is crucial for maintaining membrane integrity and controlling the rate of sperm capacitation through the acrosomal reaction. infertile stallions with normal spermiograms have a phospholipid:cholesterol ratio 2.5 times > fertile stallions (figure).67 a considerable number of hormones can be identified in the seminal plasma. leptin is directly correlated with the percentage of abnormal sperm in arabian stallions.68 sperm motility and acrosomal integrity can be positively associated with seminal cortisol, progesterone and insulin concentrations.69 prostaglandin e2 acts on mare’s reproductive tract, increasing the oviduct lumen diameter, thereby facilitating sperm transportation. seminal prostaglandin f2α concentrations are higher in stallions than in bulls and can affect uterine mechanical clearance during physiological postbreeding endometritis.70,71 impacts on biotechnologies: cooling and freezing reproductive technologies are an essential part of the equine industry and the possibility of selling cooled or frozen semen from genetically valuable stallions is one of its pillars.10,72 use of these biotechnologies enables long-term semen storage and preservation, safe transportation over long distances without compromising quality, and reduced risk of spreading reproductive diseases.73 the equine species is considered challenging for cryopreservation, and stallions can be classified as ‘good’ or ‘poor’ freezers based on postthawing seminal evaluations. it is estimated that 20-50% of stallions are poor freezers. poor freezers have low tolerance to the cryopreservation process, resulting in low progressive sperm motility (< 35%) and a high percentage of dead and immotile sperm.74 sperm quality can be assessed in various ways; common methods use total sperm motility, progressive sperm motility, membrane and acrosomal integrity, and chromatin condensation.75 individual differences between seminal plasma composition and the freezability of stallions is important to consider, as some individuals may not have satisfactory results despite having adequate seminal parameters prefreezing or cooling. for this reason, studies have focused on proteomics and specific biochemical profiling of seminal plasma figure. stallion seminal plasma components whose production and secretion sites have been identified. a: testicle; b: epididymis; c: ampulla of vas deferens; d: vesicular gland; e: prostate gland; f: bulbourethral gland; g: bladder. crisp: cysteine-rich secretory protein; hsp: horse seminal plasma protein; mmp: matrix metalloproteinase http://dx.doi.org/10.58292/ct.v17.13214 4 citation: clinical theriogenology 2025, 17, 13214, http://dx.doi.org/10.58292/ct.v17.13214 with the goal of identifying differences between good and poor freezers.69,74,76,77 reduced semen fertility after cooling or freezing processes is correlated with drastic temperature changes, osmotic pressure, ph changes, early capacitation, and oxidative stress, all of which affect cellular architecture and biochemical composition.78-81 at the final stages of spermatogenesis, sperm become incapable of repairing biomolecular damage, and their metabolism becomes simplified, making the damage caused by these processes irreversible.82 in the literature, there is still no consensus among authors on the maintenance, removal, or postthaw addition of seminal plasma to improve sperm parameters. some authors report improved postthaw sperm mobility, increased membrane stability and reduced capacitation rates with the addition of seminal plasma.72,81,83,84 others reported no effect on sperm quality.85-88 there was no significant difference in lipid peroxidation after the postthaw addition of seminal plasma.81 dna integrity was preserved at 24-48 hours by prior removal of seminal plasma, whereas damage was observed with its addition.89 addition of only autologous seminal plasma after 48 hours of cool semen storage was detrimental to total and progressive sperm motility but there was a reported increase in kinematic parameters with the combination of commercial extenders and 2 concentrations of seminal plasma.90 these contradictory results can be attributed to the many differences in methodology present in each study, such as sperm selection, the type of extender used, the use of homologous or heterologous seminal plasma and its concentration when present, the timing of seminal plasma addition (before or postthaw/cooling), and individual variation in seminal plasma composition. individual variations in the composition of the seminal plasma are often observed between good and poor freezers. lower concentrations of vitamin e were in the seminal plasma of good freezers, but the importance of this finding is yet unclear.76 in addition, 6 proteins as discriminant variables were used for identifying good freezer stallions; 1 was mitochondrial nadh dehydrogenase (ubiquinone) a component of the mitochondrial respiratory chain that in its reduced form acts as an antioxidant.74,91 differences in phospholipid profiles, evaluated with lipidomics, were also identified between good and poor freezers.77 good freezer stallions had higher concentrations of cortisol, estradiol, insulin, triiodothyronine, and thyroxine in their seminal plasma.69 conclusion a comprehensive understanding of seminal plasma composition and function in stallions is key to improving the use of reproductive technologies and, consequently, fertility in general. although substantial progress has been made in identification of seminal plasma proteins, enzymes, and antioxidants of fertile stallions, gaps remain in the practical application of this knowledge, particularly in semen cryopreservation. individual variations among stallions highlights the need for personalized approaches to optimize cryopreservation results and ensure sperm fertility. therefore, additional research is needed to develop techniques that account for individual variabilities, aiming to maximize reproductive potential. conflict of interest none to report. references 1. varner dd, gibb z, aitken rj: stallion fertility: a focus on the spermatozoon. equine vet j 2014;47:16-24. doi: 10.1111/evj. 12308 2. giesecke k, sieme h, distl o: infertility and candidate gene markers for fertility in stallions. vet j 2010;185:265-271. doi: 10.1016/j. tvjl.2009.07.024 3. shakeel m, yoon m: heat stress and stallion fertility. j anim sci 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2007;99:65-71. doi: 10.1016/j.anireprosci.2006.03.018 68. el-maaty am, el sisy gm, shaker mh, et al: age-related rump fat, fat percentage, body fat mass, androgens and semen parameters or arab stallions. asian pac j reprod 2014;3:184-191. doi: 10.1016/s2305-0500(14)60024-5 69. el-brady dam: relationship between some seminal plasma hormones and quality of fresh, chilled and frozen-thawed semen in arabian horses. j egypt med assoc 2015;73:23. 70. claus r, dimmick ma, gimenez t, et al: estrogens and prostaglandin f2alpha in the semen and blood plasma of stallions. theriogenology 1992;38:687-693. doi: 10.1016/0093-691x(92) 90031-l 71. troedsson mht, desvousges a, alghamdi as, et al: components in seminal plasma regulating sperm transport and elimination. anim reprod sci 2005;89:171-186. doi: 10.1016/j.anireprosci. 2005.07.005 72. neuhauser s, gösele p, handler j: post-thaw addition of autologous seminal plasma improves sperm motion characteristics in fair and poor freezer stallions. j equine vet sci 2019;72:117-123. doi: 10.1016/j.jevs.2018.10.028 73. alvarenga ma, papa fo, ozanam f, et al: advances in stallion semen cryopreservation. vet clin north am equine pract 2016;32:521-530. doi: 10.1016/j.cveq.2016.08.003 74. gaitskell-phillips g, martín-cano fe, ortiz-rodríguez jm, et al: differences in the proteome of stallion spermatozoa explain stallion-to-stallion variability in sperm quality post-thaw. biol reprod 2021;104:1097-1113. doi: 10.1093/biolre/ioab003 75. al-kass z, morell jm: freezing stallion semen – what do we need to focus on the future? vet sci 2024;11:65. doi: 10.3390/ vetsci11020065 76. mráčková m, zavadilová m, sedlinská m: assessment of the effect of selected components of equine seminal plasma on semen freezability. mac vet rev 2015;38:91-96. doi: 10.14432/j. macvetrev.2015.01.037 77. ramires neto c, freitas-dell’acqua cp, souza ff, et al: identification of phospholipids associated with semen cooling ability of mangalarga marchador stallions. j equine vet sci 2023;125:104613. doi: 10.1016/j.jevs.2023.104613 78. bailey jl, bilodeau jf, cormier n: semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. j androl 2000;21:1-7. pmid: 10670514. 79. loomis pr: advanced methods for handling and preparation of stallion semen. vet clin north am equine pract 2006;22: 663-676. doi: 10.1016/j.cveq.2006.07.002 http://dx.doi.org/10.58292/ct.v17.13214 https://doi.org/10.1111/j.1939-165x.1997.tb00731.x https://doi.org/10.22456/1679-9216.99755 https://doi.org/10.3390/ani12070861 https://doi.org/10.1016/s0093-691x(02)00956-1 https://doi.org/10.1016/j.theriogenology.2021.01.020 https://doi.org/10.1095/biolreprod.114.118539 https://doi.org/10.1111/j.1471-4159.1992.tb10990.x https://doi.org/10.1111/j.1471-4159.1992.tb10990.x https://doi.org/10.1016/s0093-691x(03)00144-4 https://doi.org/10.1016/s0093-691x(03)00144-4 https://doi.org/10.1016/0002-9343(91)90281-2 https://doi.org/10.1071/rd9950659 https://doi.org/10.1071/rd9950659 https://doi.org/10.1016/j.theriogenology.2019.08.032 https://doi.org/10.1016/j.theriogenology.2019.08.032 https://doi.org/10.3390/life11111238 https://doi.org/10.3390/life11111238 https://doi.org/10.3390/life11111238 https://doi.org/10.3390/life11111238 https://doi.org/10.1016/j.bbrc.2016.04.015  https://doi.org/10.1016/j.bbrc.2016.04.015  https://doi.org/10.1071/rd10032 https://doi.org/10.14202/vetworld.2017.214-220 https://doi.org/10.3390/antiox12091675 https://doi.org/10.1530/jrf.0.0240303 https://doi.org/10.2527/jas1976.433626x https://doi.org/10.2527/jas1976.433626x https://doi.org/10.1016/j.anireprosci.2006.03.018 https://doi.org/10.1016/s2305-0500(14)60024-5 https://doi.org/10.1016/0093-691x(92)90031-l https://doi.org/10.1016/0093-691x(92)90031-l https://doi.org/10.1016/j.anireprosci.2005.07.005 https://doi.org/10.1016/j.anireprosci.2005.07.005 https://doi.org/10.1016/j.jevs.2018.10.028 https://doi.org/10.1016/j.cveq.2016.08.003 https://doi.org/10.1093/biolre/ioab003 https://doi.org/10.3390/vetsci11020065 https://doi.org/10.3390/vetsci11020065 https://doi.org/10.14432/j.macvetrev.2015.01.037 https://doi.org/10.14432/j.macvetrev.2015.01.037 https://doi.org/10.1016/j.jevs.2023.104613 https://doi.org/10.1016/j.cveq.2006.07.002 citation: clinical theriogenology 2025, 17, 13214, http://dx.doi.org/10.58292/ct.v17.13214 7 80. morris, gj, acton e, murray bj, et al: freezing injury: the special case of the sperm cell. cryobiology 2012;64:71-80. doi: 10.1016/j. cryobiol.2011.12.002 81. de andrade afc, zaffalon fg, celeghini ecc, et al: post-thaw addition of seminal plasma reduces tyrosine phosphorylation on the surface of cryopreserved equine sperm, but does not reduce lipid peroxidation. theriogenology 2012;77:1866-1872. doi: 10.1016/j.theriogenology.2012.01.003 82. hammerstedt rh: maintenance of bioenergetic balance in sperm and prevention of lipid peroxidation: a review of the effect on design of storage preservation systems. reprod fertil dev 1993;5:675-690. doi: 10.1071/rd9930675 83. aurich je, kühne a, hoppe h, et al: seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation. theriogenology 1996;46:791-797. doi: 10.1016/ s0093-691x(96)00237-3 84. de andrade afc, zaffalon fg, celeghini ecc, et al: addition of seminal plasma to post-thawing equine semen: what is the effect on sperm cell viability? reprod dom anim 2011;48:682-686. doi: 10.1111/j.1439-0531.2010.01729.x 85. moore ai, squires el, graham jk: effect of seminal plasma on the cryopreservation of equine spermatozoa. theriogenology 2005;63:2372-2381. doi: 10.1016/j.theriogenology. 2004.05.032 86. al-essawe e, johannisson a, wulf m, et al: improved cryosurvival of stallion spermatozoa after colloid centrifugation is independent of the addition of seminal plasma. cryobiology 2018;81: 145-152. doi: 10.1016/j.cryobiol.2018.01.009 87. al-essawe e, johannisson a, wulf m, et al: addition of seminal plasma to thawed stallion spermatozoa did not repair cryoinjuries. anim reprod sci 2018;196:48-58. doi: 10.1016/j.anireprosci. 2018.06.018 88. bubeníčková f, šichtař j, dvořáková b, et al: effect of seminal plasma and two types of freezing extenders on characteristics of frozen-thawed stallion epididymal spermatozoa. in: proceedings of the 22nd annual conference of the european society for domestic animal reproduction (esdar), cordoba, spain, 27–29 september 2018. reprod domest anim 2018;53:74–215. doi: 10.1111/rda.13270 89. love cc, brinsko sp, rigby sl, et al: relationship of seminal plasma level and extender type to sperm motility and dna integrity. theriogenology 2005;63:1584-1591. doi: 10.1016/ j.theriogenology.2004.05.030 90. nerhauser s, säcker j, handler j: effects of the addition of autologous seminal plasma to highly concentrated stallion semen after 48 hours of cooled storage. j equine vet sci 2017;49:25-30. doi: 10.1016/j.jevs.2016.09.008 91. pobezhimova tp, voinikov vk: biochemical and physiological aspects of ubiquinone function. membr cell biol 2000;13: 595-602. pmid: 10987383. http://dx.doi.org/10.58292/ct.v17.13214 https://doi.org/10.1016/j.cryobiol.2011.12.002 https://doi.org/10.1016/j.cryobiol.2011.12.002 https://doi.org/10.1016/j.theriogenology.2012.01.003 https://doi.org/10.1071/rd9930675 https://doi.org/10.1016/s0093-691x(96)00237-3 https://doi.org/10.1016/s0093-691x(96)00237-3 https://doi.org/10.1111/j.1439-0531.2010.01729.x https://doi.org/10.1016/j.theriogenology.2004.05.032 https://doi.org/10.1016/j.theriogenology.2004.05.032 https://doi.org/10.1016/j.cryobiol.2018.01.009 https://doi.org/10.1016/j.anireprosci.2018.06.018 http://doi.org/10.1111/rda.13270 https://doi.org/10.1016/j.theriogenology.2004.05.030 https://doi.org/10.1016/j.theriogenology.2004.05.030 https://doi.org/10.1016/j.jevs.2016.09.008 1 contact younis khan younis.khan@vetmeduni.ac.at © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommerical 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12157, http://dx.doi.org/10.58292/ct.v17.12157 case report early pregnancy loss in a mare due to possible acute uterine bacterial infection younis khan,a amr el-shalofy,a,b camille gautier,a‡ christine auricha aclinical center for reproduction, department for small animals and horses, university of veterinary medicine, vienna, austria btheriogenology department, faculty of veterinary medicine, cairo university, giza, egypt ‡current affiliation: unilasalle, collège agrosciences, beauvais, france abstract this case involves an 11-year haflinger mare with breeding induced endometritis. after breeding soundness examination, mare was inseminated twice during 1 estrous cycle. on day 1 (day 0 = day of ovulation), mare was negative for endometrial cytology and culture. on day 10, an embryonic vesicle was visualized via transrectal ultrasonography; progesterone concentrations were 15.8 ng/ml. on day 11, the embryonic vesicle had increased to a diameter of 0.8 cm with clearly visible endometrial edema; progesterone concentrations were 14.4 ng/ml. on day 12, embryonic vesicle was no longer visible; progesterone concentrations were 4.7 ng/ml (possibility of premature luteolysis). bacteriological culture of the uterus with a guarded endometrial swab had a pure culture of streptococcus equi subspecies zooepidemicus (s. zooepidemicus). mare underwent treatment (uterine lavage and systemic penicillin for 5 days) and became pregnant at the next estrus. this case highlighted the possibility of s. zooepidemicus subclinical endometrial infection, not diagnosed by routine endometrial cytology and culture, becoming dormant. keywords: mare, endometritis, acute bacterial infection, early embryonic loss, treatment background early embryonic loss in mares is attributed to a range of factors of both infectious and noninfectious origins.1,2 in horses, early embryonic loss typically refers to pregnancy loss from fertilization to day 42 of pregancy.3 the widespread use of transrectal ultrasonography4–6 has become a must for early pregnancy diagnosis and follow up examinations. endometrial infection is a major cause of impaired fertility in mares.7 routine endometrial culture and cytology obtained before breeding might help to predict the likelihood of conceptus growth and development. subclinical bacterial uterine infection can result in early embryonic death.8,9 streptococcus equi subspecies zooepidemicus (s. zooepidemicus) and escherichia coli (e. coli) are commonly isolated in uterine bacterial infections whereas pseudomonas spp., klebsiella, and leptospira spp., have been less frequently identified.7,10 certain reproductive strains of s. zooepidemicus have adapted horses as their host.11,12 these bacteria can hide within the endometrial tissue and therefore are not easily detectable via standard bacterial culture techniques (e.g. guarded endometrial swab).13–15 furthermore, breeding or uterine instillation of a bacterial growth medium13 may activate these ‘dormant’ streptococci resulting in acute infectious endometritis.13 we describe a case of early embryonic death in a mare assumed to  be associated with subclinical bacterial infection by s. zooepidemicus. case presentation an 11-year haflinger mare, weighing 535 kg with a body condition score 7/9, was enrolled (after breeding soundness examination) in a study approved by the austrian federal ministry for science and research (experimentation license number 2020-0.547.78).16 mare was a maiden and has been bred multiple times with positive pregnancy outcomes while used as an embryo donor. mare had no history of spontaneous resorption and had never carried a foal to term. mare underwent daily transrectal ultrasonography examinations (dp-6600vet; mindray, shenzhen, china). at estrus detection (preovulatory follicle of 3.9 cm in diameter accompanied by mailto:younis.khan@vetmeduni.ac.at http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12157 2 citation: clinical theriogenology 2025, 17, 12157, http://dx.doi.org/10.58292/ct.v17.12157 marked endometrial edema grade > 2/3), mare was inseminated with raw semen from a fertile stallion containing 500 x 106 progressively motile sperm. artificial insemination was repeated 48 hours after the first insemination. on day 1 (day 0 = day of ovulation), an endometrial culture and cytology were collected using a double-guarded uterine culture swab (minitube, tiefenbach, germany) and a cytology brush (minitube). blood samples were collected from the jugular vein into vacuum tubes containing edta (vacuette 9ml k3edta, greiner bio one, kremsmünster, austria). immediately after collection, blood samples were transferred into iced water and subsequently centrifuged at  5°c (1,200 x g, 10 minutes). plasma was aliquoted and  stored at –20°c until progesterone analysis. plasma progesterone concentrations were analyzed by an enzymelinked immuno-sorbent assay (enzo progesterone elisa; enzo life sciences.17 the intraassay coefficient of variation was 4.9%, the interassay coefficient of variation was 7.9%, and the minimal detectable concentrations were 11.1 pg/ ml.16 uterus was examined once daily for intrauterine free fluid via transrectal ultrasonography from insemination day until fluid was no longer detectable. the amount of intrauterine fluid was determined by measuring the area of  fluid at the largest diameter in both uterine horns and  the  uterine body using the electronic calipers of the ultrasound machine and then calculating the total maximal area.18 from day 10 onwards, the uterus was scanned daily for embryonic vesicle and if detected, its diameter was determined in 2 dimensions at a 90° angle with the electronic calipers. the mean of the 2 measurements was calculated. on day 10, transrectal ultrasonography revealed an embryonic vesicle along with slight endometrial edema (figure 1a). the next day, the vesicle had increased in size (figure 1b), but on day 12, the vesicle was no longer visible in transrectal ultrasonography. instead, a considerable amount of hyperechoic intrauterine fluid was present (figures 1c-e). on day 14, endometrial culture (with antibiogram) and cytology (cytobrush) were collected. the endometrial cytology was negative (0% polymorphonuclear neutrophils [pmn]) but the endometrial culture resulted in moderate growth of s. zooepidemicus (result made available on day 16). progesterone concentrations had dropped considerably from day 11 to day 12. detailed description of clinical and laboratory findings is provided (table). based on these findings, an acute endometrial bacterial endometritis followed by premature luteolysis and early embryonic death were diagnosed. beginning on day 14, treatment included were uterine lavage with 0.9% nacl solution and stimulation of mechanical uterine clearance with 20 iu intravenous oxytocin (oxytocin, vana, vienna, austria) given every 4 hours. from day 16, figure 1. transrectal ultrasonographic images of uterus: a. day 10 embryonic vesicle (white arrow) of 0.4 cm and grade 1 endometrial edema; b. day 11 embryonic vesicle (white arrow) of 0.8 cm and grade 2 endometrial edema; c. day 12 embryonic vesicle; d and e. days 13 and 14 embryonic vesicle (note hyperechoic free fluid visible in the uterine lumen* and uterine mucosa edema**) http://dx.doi.org/10.58292/ct.v17.12157 citation: clinical theriogenology 2025, 17, 12157, http://dx.doi.org/10.58292/ct.v17.12157 3 tr an sr ec ta l u lt ra so n o gr ap h y in se m in at io n , e n d o m et ri al sa m p le s, a n d l ab o ra to ry r es u lt s p ro ge st er o n e co n ce n tr at io n s (n g/ m l) tr ea tm en t c yc le # d ay en d o m et ri al ed em a in tr a u te ri n e fl u id ( cm 2 ) em b ry o n ic ve si cl e (c m ) c yt o lo gy (% p m n ) c u lt u re /i n se m in at io n u te ri n e la va ge (0 .9 % n ac l so lu ti o n ) su b cu ta n eo u s o xy to ci n (2 0 iu ) a n ti b io ti c tr ea tm en t -3 3 0 n o t ap p li ca b le in se m in at io n -2 2 8. 2 n o t ap p li ca b le -1 2 4 n o t ap p li ca b le in se m in at io n 0 1 1. 6 n o t ap p li ca b le 1 1 0 0 n o t ap p li ca b le 2. 5 cu lt u re 3 re su lt : n eg at iv e 10 1 0 0. 44 15 .8 11 2 0 0. 8 14 .4 12 3 6. 7 0. 7 4. 7 13 3 7. 1 n o t d et ec ta b le 3. 00 14 3 4. 1 n o t d et ec ta b le 0 cu lt u re 2. 8 o n ce d ai ly ev er y 4 h o u rs 15 2 4. 5 n o t d et ec ta b le o n ce d ai ly ev er y 4 h o u rs 16 3 4. 2 n o t d et ec ta b le re su lt : m o d er at e gr ow th o f  s. z oo ep id em ic us o n ce d ai ly ev er y 4 h o u rs o n ce d ai ly p en ic il li n (2 0, 00 0 iu ) 17 3 5 n o t d et ec ta b le ev er y 4 h o u rs o n ce d ai ly p en ic il li n (2 0, 00 0 iu ) 18 1 1. 9 n o t d et ec ta b le 0 ev er y 4 h o u rs o n ce d ai ly p en ic il li n (2 0, 00 0 iu ) ta bl e. c li n ic al , l ab o ra to ry fi n d in gs ( en d o m et ri al e d em a: 0 : n o n e, 1 : s li gh t, 2 : m o d er at e, 3 : s ev er e) , a n d t re at m en t http://dx.doi.org/10.58292/ct.v17.12157 4 citation: clinical theriogenology 2025, 17, 12157, http://dx.doi.org/10.58292/ct.v17.12157 tr an sr ec ta l u lt ra so n o gr ap h y in se m in at io n , e n d o m et ri al sa m p le s, a n d l ab o ra to ry r es u lt s p ro ge st er o n e co n ce n tr at io n s (n g/ m l) tr ea tm en t c yc le # d ay en d o m et ri al ed em a in tr a u te ri n e fl u id ( cm 2 ) em b ry o n ic ve si cl e (c m ) c yt o lo gy (% p m n ) c u lt u re /i n se m in at io n u te ri n e la va ge (0 .9 % n ac l so lu ti o n ) su b cu ta n eo u s o xy to ci n (2 0 iu ) a n ti b io ti c tr ea tm en t 2 0 0 1. 6 n o t ap p li ca b le ev er y 4 h o u rs o n ce d ai ly p en ic il li n (2 0, 00 0 iu ) 1 0 1. 1 n o t ap p li ca b le ev er y 4 h o u rs o n ce d ai ly p en ic il li n (2 0, 00 0 iu ) 2 0 0 n o t ap p li ca b le ev er y 4 h o u rs 11 0 0 n o t ap p li ca b le 0 cu lt u re 13 0 0 n o t ap p li ca b le re su lt : n eg at iv e 17 2 0 n o t ap p li ca b le in se m in at io n 18 2 6. 3 n o t ap p li ca b le ev er y 6 h o u rs 19 2 7. 9 n o t ap p li ca b le in se m in at io n ev er y 6 h o u rs 20 1 6. 8 n o t ap p li ca b le 3 0 1 0. 8 n o t ap p li ca b le ev er y 6 h o u rs 1 0 1. 82 n o t ap p li ca b le 0 cu lt u re ev er y 6 h o u rs 2 0 1. 14 n o t ap p li ca b le re su lt : n eg at iv e ev er y 6 h o u rs 3 0 0 n o t ap p li ca b le 10 0 0 0. 28 23 .9 11 0 0 0. 47 30 .6 12 0 0 0. 83 27 .1 13 0 0 1. 2 30 .1 14 0 0 1. 71 24 .6 ta bl e. ( c o n ti n u ed ) http://dx.doi.org/10.58292/ct.v17.12157 citation: clinical theriogenology 2025, 17, 12157, http://dx.doi.org/10.58292/ct.v17.12157 5 intramuscular penicillin (vanapen, vana, vienna, austria) was given (20,000 iu/kg bodyweight once daily) and continued for 5 days. outcome mare ovulated on day 19 of cycle 1 (corresponding to day 1 of cycle 2), underwent diagnostic assessments, including endometrial culture and cytology on day 11 of cycle 2; results were negative. mare was inseminated on days 17 and 19 of cycle 2 with raw semen collected from the same stallion as in cycle 1. on days 18-20, a considerable amount of intrauterine fluid accumulation was detected. mare ovulated on day 21 of cycle 2 (i.e. day 1 of cycle 3). an embryonic vesicle was detected via transrectal ultrasonography on day 10. the vesicle continued to develop until day 14; no signs of endometritis or pregnancy loss were detected. mare was treated with intramuscular 250 µg prostaglandin f2α (estrumate, msd, vienna, austria) for termination of pregnancy and induction of estrus according to the experimental protocol of the research study. further examinations during 3 consecutive cycles did not reveal any signs of acute endometritis. discussion this case emphasizes some critical points regarding the diagnosis of activated dormant bacteria, management, and treatment of endometritis in mares, particularly acute bacterial endometritis. endometritis in horses occur either clinically or subclinically. in either case, laboratory tests (e.g. endometrial cytology and culture) are crucial for diagnosis and for identifying the pathogens involved and their susceptibility to antibiotics.4,19 in this case, breeding soundness examination of the mare did not reveal any clinical signs of endometritis, and the endometrial culture performed with a double-guarded swab was negative. endometrial cytology, however, revealed 2.5% pmn on day 1 after ovulation and the mare had not been bred during the previous estrous cycle. this finding might have been considered suspicious because subclinical endometritis in mares without clinical signs was assumed when > 2% of pmn were in an endometrial cytology sample.20 other researchers, however, suggested that a pmn count < 5% is nonindicative for endometrial inflammation.21,22 in this context, the stage of the estrous cycle should be considered because it may influence endometrial cytology. although healthy mares outside estrus have an inflammatory cell count close to zero because of reduced pmn migration into the endometrium during progesterone dominance,20 a small resident population of neutrophils (< 5%) may exist in the endometrium of mares from 24-96 hours after ovulation (i.e. when progesterone concentrations gradually increase).21 based on these suggestions, we interpreted the initial endometrial cytology (2.5% pmn) together with a negative endometrial culture collected on day 1 after ovulation as physiological. progression of the case supports the suggestion to include endometrial histology in breeding soundness examination of mares. this approach considerably improved the likelihood of detecting increased numbers of pmn in the endometrium.23 interestingly, more recent investigations demonstrated that specific bacteria influence endometrial cytology; not only e. coli infections, but similarly dormant streptococci spp. may reduce pmn in endometrial cytology.11,13,24,25 this may be considered as an explanation for the small number of pmn detected in the initial endometrial cytology of the present case. s. zooepidemicus was only determined by endometrial culture once the mare developed acute endometritis during early pregnancy. this might suggest that dormant bacteria were activated during estrus or postovulatory phase. dormant s. zooepidemicus are likely undetectable in a routine endometrial culture obtained by double guarded swab but require activation. for diagnostic purposes this is possible by intrauterine instillation of the bacterial growth medium bactivate.13 in addition, intrauterine misoprostol infusion provoked clinically significant bacterial endometritis in 63% of mares that had previously tested negative for endometritis, highlighting its potential utility in uncovering undiagnosed infections.26 furthermore, culture of an endometrial biopsy was often successful.27 in the present case, repeated intrauterine manipulation associated with insemination and endometrial sample collection or even more intrauterine fluid accumulation indicating delayed uterine clearance after insemination of the mare may have resulted in activation of dormant streptococci. under experimental conditions, intrauterine instillation of bactivate in early estrus resulted in positive endometrial cytology and culture already after 48 hours.13 in the present study, mild signs of endometritis can be suspected by the detection of a mild endometrial edema together with the conceptus on day 10. this is considerably later but unfortunately, no information on transrectal ultrasonographic findings after treatment is provided.13 alternative to activation of dormant streptococci, infection with s. zooepidemicus due to contamination at insemination or culture collection must be considered in the present case. this scenario is, however, relatively unlikely because the stallion used for semen collection was restricted to breeding by artificial insemination limiting transfer of bacteria from another mare. also, any intrauterine manipulation was performed under strict hygienic conditions to minimize the likelihood of bacterial contamination in an experiment where uterine health was a key requirement.16 this approach was highly successful as none of the 7 mares kept together with this mare discussed had any positive culture or any clinical signs of endometritis during the entire experimental period which included a total of 7 closely monitored estrous cycles per mare. although embryonic vesicle size was within the physiological range indicating normal conceptus development on days 10 and 11,28 growth had ceased on day 12 demonstrating a switch to abnormal embryo development often indicative of early pregnancy loss.29 at this period, also progesterone concentrations had considerably dropped which paralleled by increasing endometrial edema indicating return of the mare to estrus.30 in addition, the sudden appearance of free intrauterine fluid suggested an endometrial inflammatory condition. in agreement with good practice of antimicrobial stewardship, antibiotic treatment of the mare was not initiated before the infectious agent, s. zooepidemicus was isolated via endometrial culture.7 nonantimicrobial treatment, however, was already started as soon as embryonic loss and return to estrus were diagnosed to take advantage of the beneficial effects of removal of debris and inflammatory material from the uterus.4,9,28,30 two day delay in initiating antibiotic treatment had no negative impact. identification of bacteria in mares with mild subclinical endometritis may be difficult. streptococci are among the most frequent bacteria causing endometritis in horses, and because some strains may enter a dormant state, their detection is diagnostically challenging. observation of pmn in endometrial cytology samples must therefore be considered cautiously. http://dx.doi.org/10.58292/ct.v17.12157 6 citation: clinical theriogenology 2025, 17, 12157, http://dx.doi.org/10.58292/ct.v17.12157 additional diagnostic approaches may help to identify bacteria that have developed strategies to escape the host immune response.31 learning points • s. zooepidemicus endometrial infections may be dormant in horses • routine endometrial cytology and culture may not detect s. zooepidemicus • advanced diagnostic methods are necessary • endometrial infections are treatable • proper treatment can help mares conceive successfully conflict of interest none of the authors has any financial or personal relationships that could inappropriately influence or bias the content. acknowledgement authors are grateful to julia maderner for expert assistance with progesterone analysis. references 1. tibary a, pearson lk: pregnancy loss in mares. spermova 2015;5:168–188. doi: 10.18548/aspe/0002.37 2. wilsher s: the uterus and early pregnancy failure in the mare. equine vet educ 2019;31:214-224. doi: 10.1111/eve.12844 3. newcombe jr, cuervo-arango j: the relationship between the positive identification of the embryo proper in equine pregnancies aged 18–28 days and its future viability: a field study. j equine vet sci 2012;32:257–261. doi: 10.1016/j.jevs.2011.09.068 4. pascottini ob, aurich c, england g, et al: general and comparative aspects of endometritis in domestic species: a review. reprod dom anim 2023;58:49–71. doi: 10.1111/rda.14390 5. morris lh, mccue pm, aurich c: equine endometritis: a review of challenges and new approaches. reproduction 2020;160: r95–r110. doi: 10.1530/rep-19-0478 6. morris lh, allen wr: reproductive efficiency of intensively managed thoroughbred mares in newmarket. equine vet j 2002;34: 51–60. doi: 10.2746/042516402776181222 7. nocera fp, d’eletto e, ambrosio m, et al: occurrence and antimicrobial susceptibility profiles of streptococcus equi subsp. zooepidemicus strains isolated from mares with fertility problems. antibiotics 2021;11:25. doi: 10.3390/antibiotics11010025 8. cerny kl, little tv, scoggin cf, et al: presence of bacteria on the external genitalia of healthy stallions and its transmission to the mare at the time of breeding by live cover. j equine vet sci 2014;34:369–374. doi: 10.1016/j.jevs.2013.07.008 9. leblanc mm, causey rc: clinical and subclinical endometritis in the mare: both threats to fertility. reprod dom anim 2009;44: 10–22. doi: 10.1111/j.1439-0531.2009.01485.x 10. köhne m, hegger a, tönissen a, et al: frequency of potentially pathogenic bacterial and fungal isolates among 28,887 endometrial samples from mares, with an emphasis on multi-drug resistant bacteria in germany (2018–2022). j equine vet sci 2024;133:105008. doi: 10.1016/j.jevs.2024.105008 11. christoffersen m, söderlind m, rudefalk sr, et al: risk factors associated with uterine fluid after breeding caused by streptococcus zooepidemicus. theriogenology 2015;84:1283–1290. doi: 10.1016/ j.theriogenology.2015.07.007 12. rasmussen cd, haugaard mm, petersen mr, et al: streptococcus equi subsp. zooepidemicus isolates from equine infectious endometritis belong to a distinct genetic group. vet res 2013;44:1-8. doi: 10.1186/1297–9716-44-26 13. petersen mr, skive b, christoffersen m, et al: activation of persistent streptococcus equi subspecies zooepidemicus in mares with subclinical endometritis. vet microbiol 2015;179:119–125. doi: 10.1016/j.vetmic.2015.06.006 14. skive b, rohde m, molinari g, et al: streptococcus equi subsp. zooepidemicus invades and survives in epithelial cells. front cell infect microbiol 2017;7:465. doi: 10.3389/fcimb.2017.00465 15. petersen mr, møller nielsen j, lehn-jensen h, et al: streptococcus equi subspecies zooepidemicus resides deep in the chronically infected endometrium of mares. clinical theriogenology 2009;1:393–409 16. khan y, el-shalofy a, kaps m, et al: in mares resistant to endometrial infection, periovulatory treatment with ecbolic drugs does not influence uterine clearance or luteal development. anim reprod sci 2024;267:107548. doi: 10.1016/j.anireprosci.2024.107548 17. beyer t, rink be, scarlet d, et al: early luteal phase progestin concentration influences endometrial function in pregnant mares. theriogenology 2019;125:236–241. doi: 10.1016/j.theriogenology.2018.11.018 18. freccero f, mislei b, bucci d, et al: effects of intra-uterine fluid accumulation after artificial insemination on luteal function in mares. animals 2022;13:67. doi: 10.3390/ani13010067 19. scott cj, de mestre am, verheyen kl, et al: bayesian accuracy estimates and fit for purpose thresholds of cytology and culture of endometrial swab samples for detecting endometritis in mares. prev vet med 2022;209:105783. doi: 10.1016/j.prevetmed.2022.105783 20. riddle wt, leblanc mm, stromberg aj: relationships between uterine culture, cytology and pregnancy rates in a thoroughbred practice. theriogenology 2007;68:395–402. doi: 10.1016/j.theriogenology. 2007.05.050 21. card c: post-breeding inflammation and endometrial cytology in mares. theriogenology 2005;64:580–588. doi: 10.1016/j. theriogenology.2005.05.041 22. rink be, beyer t, french hm, et al: the fate of autologous endometrial mesenchymal stromal cells after application in the healthy equine uterus. stem cells dev 2018;27:1046–1052. doi: 10.1089/ scd.2018.0056 23. nielsen jm, nielsen fh, petersen mr, et al: diagnosis of equine endometritis–microbiology, cytology and histology of endometrial biopsies and the correlation to fertility. pferdeheilkunde 2012;28:8–13. 24. leblanc mm: advances in the diagnosis and treatment of chronic infectious and post-mating-induced endometritis in the mare. reprod dom anim 2010;45:21–27. doi: 10.1111/j.1439-0531.2010.01634.x 25. christoffersen m, brandis l, samuelsson j, et al: diagnostic double-guarded low-volume uterine lavage in mares. theriogenology 2015;83:222–227. doi: 10.1016/j.theriogenology. 2014.09.008 http://dx.doi.org/10.58292/ct.v17.12157 https://doi.org/10.18548/aspe/0002.37 https://doi.org/10.1111/eve.12844 https://doi.org/10.1016/j.jevs.2011.09.068 https://doi.org/10.1111/rda.14390 https://doi.org/10.1530/rep-19-0478 https://doi.org/10.2746/042516402776181222 https://doi.org/10.3390/antibiotics11010025 https://doi.org/10.1016/j.jevs.2013.07.008 https://doi.org/10.1111/j.1439-0531.2009.01485.x https://doi.org/10.1016/j.jevs.2024.105008 https://doi.org/10.1016/ j.theriogenology.2015.07.007 https://doi.org/10.1016/ j.theriogenology.2015.07.007 https://doi.org/10.1186/1297–9716-44-26 https://doi.org/10.1016/j.vetmic.2015.06.006 https://doi.org/10.3389/fcimb.2017.00465 https://doi.org/10.1016/j.anireprosci.2024.107548 https://doi.org/10.1016/j.theriogenology.2018.11.018 https://doi.org/10.3390/ani13010067 https://doi.org/10.1016/j.prevetmed.2022.105783 https://doi.org/10.1016/j.theriogenology.​2007.05.050 https://doi.org/10.1016/j.theriogenology.​2007.05.050 https://doi.org/10.1016/j.theriogenology.2005.05.041 https://doi.org/10.1016/j.theriogenology.2005.05.041 https://doi.org/10.1089/scd.2018.0056 https://doi.org/10.1089/scd.2018.0056 https://doi.org/10.1111/j.1439-0531.2010.01634.x https://doi.org/10.1016/j.theriogenology.​2014.09.008 https://doi.org/10.1016/j.theriogenology.​2014.09.008 citation: clinical theriogenology 2025, 17, 12157, http://dx.doi.org/10.58292/ct.v17.12157 7 26. von dollen k, lu k, lyle s, et al: clinical observations of bacterial culture results after intrauterine infusion of misoprostol. clinical theriogenology 2022;14:301. 27. nielsen jm: endometritis in the mare: a diagnostic study comparing cultures from swab and biopsy. theriogenology 2005;64:510–518. doi: 10.1016/j.theriogenology.2005. 01.008 28. aurich c, budik s: season does not influence embryo recovery rate and conceptus size until day 14 after ovulation in the horse. reprod dom anim 2015;50:299–303. doi: 10.1111/ rda.12490 29. vanderwall dk, squires el, brinsko sp, et al: diagnosis and management of abnormal embryonic development characterized by formation of an embryonic vesicle without an embryo in mares. j am vet med assoc 2000;217:58–63. doi: 10.2460/javma.2000.217.58 30. mccue pm: ultrasound examination of the pregnant reproductive tract. in: descanio j, mccue p: editors. equine reproductive procedures. 2nd edition, amess; wiley blackwell: 2021. p. 257–261. 31. ferris r: biofilm and latent bacteria in equine uterus. clinical theriogenology 2022;14:229-232. doi: 10.58292/ct.v14.9680 http://dx.doi.org/10.58292/ct.v17.12157 https://doi.org/10.1016/j.theriogenology.2005.​01.008 https://doi.org/10.1111/rda.12490 https://doi.org/10.1111/rda.12490 https://doi.org/10.2460/javma.2000.217.58 https://doi.org/10.58292/ct.v14.9680 _hlk152841185 _hlk193368379 1 contact jenna stockler jew0027@auburn.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12245, http://dx.doi.org/10.58292/ct.v17.12245 case report unilateral scrotal enlargement and cryptorchidism in two boars dane schwartz,a jenna stockler,a miguel saucedo,a oscar huertas,a robert cole,a thomas passler,a katelyn waters,a ricardo stockler,a alyona michael,b adepartment of clinical science, bdepartment of pathobiology, college of veterinary medicine, auburn university, auburn, al, usa abstract two ~ 10-year, intact male vietnamese pot-bellied pig crosses were presented for evaluation of unilateral scrotal enlargement of 1 year’s duration and historic diagnosis of cryptorchidism. ultrasonographic examination of the scrotum was performed; tissue within the scrotum had no defining features of a normal testis. based on physical examination, history, and ultrasonographic findings, testicular neoplasia was the primary differential diagnosis for both boars. due to financial constraints and animals’ age, humane euthanasia was elected. in both boars, histopathologic examination of enlarged testes revealed trabeculae and packets of polygonal cells with eosinophilic to microvacuolated cytoplasm, consistent with leydig (interstitial) cell tumor. not largely reported in swine, neoplasia must be considered a differential for any intact male. this case report demonstrated the importance of early castration for males not intended for breeding. keywords: vietnamese pot-bellied pig, neoplasia, cryptorchidism, leydig cell tumor background cryptorchidism, the inguinal or intraabdominal retention of 1 or both testes, is more common in swine than cattle or sheep,1 although testicular neoplasia is rarely reported.2 we describe left-sided cryptorchidism and neoplastic transformation of the descended testis in 2 cohoused potbellied boars. case presentation two ~ 10-year, intact male, vietnamese pot-bellied pig crosses, weighing 91 and 95 kg, were presented in september 2024 for evaluation of unilateral scrotal enlargement. both were companion animals that had been raised on the same farm with unknown genetic relation. as piglets, both had been diagnosed with left unilateral cryptorchidism and no significant prior medical history was otherwise reported. both boars had developed unilateral scrotal enlargement 1 year prior to presentation, with rapid growth noted over the last 5 months. no behavioral changes were appreciated by the owner. three months prior to referral, a local veterinarian had confirmed that both boars were serologically negative for brucella suis and suid herpesvirus 1 (pseudorabies virus). the referring veterinarian had performed a fine needle aspirate that failed to establish a definitive diagnosis. case management on presentation, boars were bright, alert, and responsive, with normal respiratory rates. due to lack of patient cooperation, the remainder of the physical examination could not be completed. the scrotum of both boars was notably asymmetrically distended with taut skin, prominent vasculature, and firm adherence to an immobile underlying mass that had no discernible testicular or epididymal structures on transcutaneous palpation (figures 1 and 2). retained left testes were not palpable within the inguinal regions. to facilitate ultrasonographic examination of the scrotums using a macro convex variable frequency (3-5 mhz) transducer on a portable ultrasound machine (sonoscape x5, sonoscape medical corp, centennial, co), boars were sedated with intramuscular xylazine (0.3 mg/kg). although the drug was extra label, it was used with owner’s consent in compliance with provision of the animal medicinal drug use clarification act of 1994 and 21 cfr 530. additional restraint was provided with the use of sorting panels to create an enclosed area and then boars were placed in dorsal recumbency. intrascrotal structures measured ~ 19 cm in diameter, exhibited a heterogeneous and mixed echogenic pattern with minimal cavitation, and had no defining mailto:jew0027@auburn.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12245 2 citation: clinical theriogenology 2025, 17, 12245, http://dx.doi.org/10.58292/ct.v17.12245 figure 1. boar 1: a. caudal view of unilateral scrotal enlargement; b. close up view of enlarged scrotum with taut skin and prominent vasculature; and c. profile view figure 2. boar 2: a. close up caudal view and b. profile view http://dx.doi.org/10.58292/ct.v17.12245 citation: clinical theriogenology 2025, 17, 12245, http://dx.doi.org/10.58292/ct.v17.12245 3 features of a normal testis (figure 3). in both boars the left  testis was retained and could not be located ultrasonographically. differential diagnoses for porcine scrotal enlargement include infectious causes of orchitis or epididymitis (e.g. brucella suis),3 and noninfectious conditions such as inguinal hernia, testicular torsion, trauma,4 sperm granuloma, spermatocele, hydrocele, scrotal abscesses, hematoma, and neoplasia. based on history, physical examination, and ultrasonographic findings, testicular neoplasia was the primary differential diagnosis. outcome as a therapeutic option, the owner was offered computed tomography-assisted localization of the retained left testis in both boars followed by bilateral surgical castration under general anesthesia. however, due to financial constraints and quality of life concerns without intervention, humane euthanasia was elected. postmortem findings following euthanasia, the retained and enlarged testis from each boar was removed and submitted for gross examination and histopathology (figure 4). the retained left testes of both boars (4.5 and 7.5 cm in length, respectively) were hypoplastic with severe atrophy and obsolescence of the seminiferous tubules histologically (figure 4). in contrast, the enlarged right testis of each boar measured > 34 cm in length and 19 cm in diameter and weighed 4.2 and 4.5 kg, respectively, was friable, tan, and had a necrotic core comprising 80-90% of the testicular mass, surrounded by subcapsular red-brown viable tissue. histologically, native testicular architecture was effaced by sheets, trabeculae, and packets of neoplastic cells separated by fine fibrovascular stroma. polygonal neoplastic cells consistent with interstitial cells had abundant eosinophilic to finely vacuolated cytoplasm, distinct cell borders, and round nuclei with finely stippled chromatin and 1 or 2 small nucleoli. scattered cytoplasmic invagination, moderate anisocytosis and anisokaryosis with karyomegaly and binucleation were  noted. mitotic activity was mild to moderate, with figure 3. boar 1: a. sagittal plane ultrasonogram of scrotal mass; note heterogeneous complex echo pattern without obvious characteristics of normal testicular tissue and minimal cavitations (~ 19 cm in diameter). unrelated animal: b. sagittal plane ultrasound image of normal testicular parenchyma; note homogenous parenchyma with hyperechoic mediastinum (rete testes) (scale bar on left is in cm) figure 4. boar 1: a. gross image of testis (inset: gross image of retained testis and associated epididymal structure). b. cross section of testis; note areas of necrosis and loss of normal testicular parenchyma (atrophied area is identified [2.54 cm bars]) http://dx.doi.org/10.58292/ct.v17.12245 4 citation: clinical theriogenology 2025, 17, 12245, http://dx.doi.org/10.58292/ct.v17.12245 1 and 8 mitotic figures per 2.37 mm2 in respective masses. necrotic  cores were infarcted with vascular thrombosis, coagulative necrosis of neoplastic cells, hemorrhage, fibrinous to fibrinosuppurative exudation, and mineralization (figure 5). gross pathological examination did not provide evidence of  metastasis; other histologically examined organs were unremarkable. discussion the presentation of synchronous testicular enlargement in multiple animals would typically prioritize infectious, toxic, or traumatic differential diagnoses.4 based on physical examination, history, ultrasonography, and histopathology, unusual concurrent development of right testicular leydig (interstitial) cell tumor was diagnosed in both boars. vietnamese pot-bellied pigs reach puberty as early as 3 months of age, and early castration of nonbreeding boars is recommended to avoid development of aggression, territorial urine marking, sexual behavior, and buildup of malodorous secretions in the preputial diverticulum.5 the growing popularity and increasing life span of companion pigs is associated with rising frequency of porcine neoplastic disease presentation to veterinarians, with over-representation of gastrointestinal and uterine smooth muscle transformation.6 miniature pigs had uterine neoplastic lesions in 20/32 (62.5%) of which smooth muscle tumors were identified in animals > 5 years,7 demonstrating the impact of continued hormonal influence. further research is needed to determine the direct impacts of estrogen and progesterone on neoplastic changes within tissue.7 testicular neoplasia in boars is uncommonly reported; however, pure interstitial cell tumors are rare in this species.8–12 in a recent review of testicular neoplasia of commercial boars, seminomas were documented as the predominant primary testicular tumor, followed by mixed germ cell stromal tumors, with no mention of pure interstitial cell tumors.2 cryptorchidism is the retention of 1 or both testes; surgical removal by laparoscopically assisted, paramedian or inguinal approach cryptorchidectomy has been described in multiple species.13,14 majority of retained testes were located in the abdomen (94%), and the left testis was most commonly retained (57%).13 these findings are consistent with the cases presented here. in general, cryptorchid testes are predisposed to neoplastic transformation. in stallions, development of interstitial cell tumors is predominantly associated with cryptorchidism.14 interestingly, neoplastic transformation in boars described herein was limited to the descended testis, with no evidence of malignancy in the retained gonads. given the unusual contemporaneous development of rightsided interstitial cell tumors in 2 cohoused boars with leftsided cryptorchidism, it is unfortunate that the genetic relation of these animals is unknown. although environmental exposure to a variety of toxicants has been associated with testicular neoplasia in humans15,16 and modeled in rodents,17,18 analogous epidemiological or experimental studies are lacking in pigs. as such, the relative contributions of genetics, environment, and serendipity to this unusual clinical presentation remain undetermined. neoplasia is documented in swine, although not commonly reported in the male reproductive tract. increasing popularity of pet pigs necessitates that neoplasia must be considered a differential in any intact male. additionally, this report demonstrated the importance of client education and the risks with figure 5. boar 1. a. light microscopic image of right testis; note interstitial cell tumor featuring trabeculae and nests of polygonal cells (inset: neoplastic cells with abundant eosinophilic to microvaculated cytoplasm (hematoxylin and eosin stain; 400 x). b.  light microscopic image of normal interstitial cells in a swine testicle unrelated to these boars (hematoxylin and eosin stain; 400 x) http://dx.doi.org/10.58292/ct.v17.12245 citation: clinical theriogenology 2025, 17, 12245, http://dx.doi.org/10.58292/ct.v17.12245 5 maintaining intact males when breeding is not the intended purpose. learning points • neoplasia is a differential for any change in testicular size • leydig cell tumors are not commonly reported in swine • early castration of companion swine is recommended to prevent development of neoplasia • sedation in swine can assist in diagnostic procedures acknowledgement authors thank kaitlyn locke, primary fourth year veterinary student, for exceptional patient care and silas zee for editing images. conflict of interest none to declare. references 1. amann rp, veeramachaneni dnr: cryptorchidism in common eutherian mammals. reproduction 2007;133:541-561. doi: 10.1530/rep-06-0272 2. ausejo r, falceto mv, mitjana o, garrido am, badiola jj, moreno b: testicular tumors in commercial boars with infertility: a gross, histologic, and immunohistochemical study. vet pathol 2025;62(1):20-30. doi: 10.1177/03009858241269836 3. olsen sc, boggiatto p, nol p, et al: brucellosis. in: zimmerman jj, karriker la, ramirez a, et al: editors. diseases of swine. 11th edition, hoboken; wiley-blackwell: 2019. p. 779-786. 4. schwarz l, dürlinger s, martin v, et al: a single, episodic event of unilateral/bilateral scrotal swelling in a group of adult boars at an austrian boar stud. porcine health manag 2023;9:17 doi: 10.1186/ s40813-023-00313-1 5. østevik l, elmas c, rubio-martinez, lm: castration of the vietnamese pot-bellied boar: 8 cases. can vet j 2012;53:943-948. pmid: 23450857 6. newman sj, rohrbach b: pot-bellied pig neoplasia: a retrospective case series (2004-2011). j vet diagn invest 2012;24:1008-1013. doi: 10.1177/1040638712452725 7. ilha mr, newman sj, van amstel s, et al: uterine lesions in 32 female miniature pet pigs. vet pathol 2010;47:1071-1075. doi: 10.1177/0300985810382522 8. roberson jr, andrews ga: theriogenology question of the month. j am vet med assoc 2007;230(6):827-829. 9. duenzel g, wemheuer wm, jaeger k, et al: diffuse seminoma in a breeding boar. dtsch tierarztl wochenschr 2009;116:33-37. available from: https://publications.goettingen-research-online. de/handle/2/17795 [cited 27 may 2025]. 10. mabara s, hashimoto n, kadota k: malignant sertoli and leydig cell tumour in a boar. j comparative pathol 1990;103:369-378. doi: 10.1016/s0021-9975(08)80025-4 11. weaver dm, tyler jw, mille ma, et al: bilateral testicular interstitial cell tumour in an aged boar. vet rec 2000;146:224. doi: 10.1136/vr.146.8.224 12. schwartz d, waqas, ms, arroyo e, et al: laparoscopic-assisted cryptorchidectomy in south american camelids and nigerian dwarf goats. clinical theriogenology 2023;15:98-102. doi: 10.58292/ct.v15.9638 13. skelton ja, baird an, hawkins jf, et al: cryptorchidectomy with a paramedian or inguinal approach in domestic pigs: 47 cases (2000-2018). j am vet med assoc 2021;258:10. doi: 10.2460/ javma.258.10.1130 14. foster r: male genital system. in: maxie g: editor. jubb, kennedy & palmer’s pathology of domestic animals. volume 3, 6th edition, st. louis, missouri; elsevier: 2016. p. 493-494. 15. meeks jj, sheinfeld j, eggener se: environmental toxicology of testicular cancer. urol oncol 2012;30:212-215. doi: 10.1016/j. urolonc.2011.09.009 16. purdue mp, rhee j, denic-roberts h, et al: a nested case–control study of serum perand polyfluoroalkyl substances and testicular germ cell tumors among u.s. air force servicemen. environ health perspect 2023;131:e077007. doi: 10.1289/ ehp12603 17. creasy d, bube a, de rijk e, et al: proliferative and nonproliferative lesions of the rat and mouse male reproductive s ystem. toxicol pathol 2012;40:40s-121s. doi: 10.1177/0192623312 454337 18. newbold rr, hanson rb, jefferson wn, et al: proliferative lesions and reproductive tract tumors in male descendants of mice exposed developmentally to diethylstilbestrol. carcinogenesis 2000;21:1355-1363. doi: 10.1093/carcin/21. 7.1355 http://dx.doi.org/10.58292/ct.v17.12245 https://doi.org/10.1530/rep-06-0272 https://doi.org/10.1177/03009858241269836 https://doi.org/10.1186/s40813-023-00313-1 https://doi.org/10.1186/s40813-023-00313-1 https://doi.org/10.1177/1040638712452725 https://doi.org/10.1177/0300985810382522 https://publications.goettingen-research-online.de/handle/2/17795 https://publications.goettingen-research-online.de/handle/2/17795 https://doi.org/10.1016/s0021-9975(08)80025-4 https://doi.org/10.1136/vr.146.8.224 https://doi.org/10.58292/ct.v15.9638 https://doi.org/10.2460/javma.258.10.1130 https://doi.org/10.2460/javma.258.10.1130 https://doi.org/10.1016/j.urolonc.2011.09.009 https://doi.org/10.1016/j.urolonc.2011.09.009 https://doi.org/10.1289/ehp12603 https://doi.org/10.1289/ehp12603 https://doi.org/10.1177/0192623312454337 https://doi.org/10.1177/0192623312454337 https://doi.org/10.1093/carcin/21.7.1355 https://doi.org/10.1093/carcin/21.7.1355 1 contact alyssa shelby a.thomas2017@gmail.com © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11199, http://dx.doi.org/10.58292/ct.v17.11199 case report scrotal swelling with suppurative and necrotizing epididymitis, and funiculitis in a dog alyssa shelby,a dalen agnew,b bruce christensena akokopelli veterinary center, sacramento, ca, usa bmichigan state university veterinary diagnostic laboratory, lansing, mi, usa abstract an 8-year, intact male dog, was presented with a swollen and painful scrotum. the dog was deemed a valuable part of the client’s breeding program and hemicastration was performed in order to preserve fertility. hemicastration is a viable option and should be considered for preserving fertility in males deemed valuable for breeding. hemicastration can be performed by general practitioners and the affected testis should be submitted for testing (along with serology from the patient) to rule out brucella canis and to determine the cause for testicular/epididymal pathology. keywords: canine, epididymis, funiculitis, enlarged, painful background epididymitis and orchitis may occur separately, jointly (orchiepididymitis), or with extension to the vaginal tunic and adhesions to the scrotum (periorchiepididymitis).1 those conditions typically occur in young dogs (2-3 years of age) but can range from 11 months to 10 years of age. there is no breed disposition reported; dogs may have acute or chronic disease, with the acute disease process associated with pain, lethargy, scrotal edema, and purulent preputial discharge. dogs with chronic orchitis/epididymitis have nonpainful enlargement of scrotal contents that can be associated with atrophy of the unaffected testis.2 both epididymitis and orchitis can occur independent of an infectious process or secondary to an infectious process.1 case presentation an 8-year, 21 kg (48 lb) male australian shepherd dog, was examined because of an enlarged scrotum that was extremely painful to the touch. the owner noticed dog’s difficulty (straining) to defecate, lethargic appearance and decreased appetite. the dog was used for natural breeding 1 week prior to noticing the clinical signs that were originally attributed to back pain until the owner noticed the swollen scrotum. the patient had been demonstrating clinical signs for ~ 7 days before presentation. on physical examination the dog appeared in good general health, vitals were within reference limits and had a body condition score of 4 (on a scale of 1-9). the dog was bright, alert, and afebrile. digital transrectal examination revealed a bilaterally enlarged prostate that was not painful on palpation, smooth and symmetrical. this was considered an unremarkable finding as the prostatic enlargement was consistent with benign prostatic hyperplasia in an older, intact male dog. palpation of the scrotum revealed an enlarged, painful scrotum with firm swelling that appeared to originate in the left testis and extended up to the length of the spermatic cord. there were no notable penetrating wounds or injury appreciated to the scrotum on physical examination. the patient was nonpainful on spinal manipulation. all other physical examination findings were within normal limits. the noted clinical signs of straining to defecate and back pain noted by the owner appeared to be related to the enlarged scrotum. diagnosis and treatment ultrasonography of the left scrotum and testis revealed marked swelling of the spermatic cord (2 cm diameter) and epididymis. evaluation of the right scrotum and testis did not indicate remarkable changes nor findings related to those affecting the left scrotum/testis. left testis measured 2.4 × 2.9 cm and right testis measured 2.2 × 3.1 cm. echogenicity of each testis was homogeneous. rapid slide agglutination test (canine brucellosis antibody test kit d-tech; synbiotics corporation, kansas city, mo) for canine brucellosis was negative. mailto:a.thomas2017@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11199 2 citation: clinical theriogenology 2025, 17, 11199, http://dx.doi.org/10.58292/ct.v17.11199 the owner elected for hemicastration. as the right testis did not appear to be affected the owner requested this testis remain in hopes of preserving the patient’s future fertility. patient’s pain level prevented us to perform a full physical examination or ultrasonography without sedation and a semen collection/analysis. dog was sedated with intravenous butorphanol (10 mg/ml [0.2 mg/kg] 0.44 ml) and dexmedetomidine (0.5 mg/ml [0.005 mg/kg] 0.22 ml) for ultrasonography. preoperative complete blood count revealed a stress leukogram. all other laboratory findings were within normal parameters. dog was transferred to surgery and induced with intravenous propofol (10 mg/ml [6 mg/kg] 12 ml) and maintained on isoflurane and oxygen throughout the procedure. at surgery, torsion of the left testis was not appreciated as the epididymis was in proper orientation, but the left testis vein was grossly distended and there was evidence of pinpoint abscesses in the vas deferens and pampiniform plexus. adhesions to the tunic had to be broken down before the testis could be exteriorized. the tunic was thickened and irregular. numerous firm adhesions were noted surrounding the left testis and cord and a rent in the vaginal tunic surrounding the left cord was noted. small tan to yellow nodules containing liquid contents were scattered within and on the surface of the cord (figure 1) clinically compatible with small abscesses. bacterial culture of the cord was obtained through the rent. open castration was performed and all abnormal tissue was removed as completely as possible. the cord was double ligated with 2 gut suture material. the incision was closed with 2-0 monocryl subcutaneous and surgical glue. immediately after surgery, the surgical site was treated with cold compresses for 10 minutes and the dog was placed on nsaids (meloxidyl) for pain management for 3-5 days duration. the culture swab obtained during surgery yielded an abundant and pure growth of escherichia coli (e. coli), resistant to amoxicillin/ clavulanic acid but susceptible to marbofloxacin, ciprofloxacin, and enrofloxacin. amoxicillin/clavulanic acid treatment that was stopped (initiated after surgery due to abscesses) and the dog was placed on oral enrofloxacin (136 mg [6 mg/kg], 1 tablet twice a day for 30 days). the owner was counseled to have a semen culture 1-2 weeks after completion of the antibiotics and a semen evaluation 3 months later. figure 1. affected testis after surgical removal; note marked congestion/dilation of spermatic cord with dilated epididymis and pyogranulomatous inflammation of epididymal head. figure 2. light microscopic images of epididymis and testis stained with hematoxylin and eosin: a. epididymal ducts are effaced by necrosis, neutrophils, fibrin, and hemorrhage and are surrounded by fibrosis. other less affected ducts are dilated and contain cellular debris (bar = 500 µm); b. seminiferous tubules are variably affected, with some loss of spermatocytes and increased numbers of degenerating spermatocytes including multinucleated giant cells within the lumen (bar = 50 µm). http://dx.doi.org/10.58292/ct.v17.11199 citation: clinical theriogenology 2025, 17, 11199, http://dx.doi.org/10.58292/ct.v17.11199 3 histological evaluation of epididymis and spermatic cord revealed extensive necrosis, hemorrhage, pools of neutrophils and fibrin, and loss of epididymal anatomic detail (figure 2). most cross-sections of the head of the epididymis were obliterated by suppurative inflammation, and the surrounding soft tissue had edema, neutrophils, and hemorrhage. pools of free sperm were also present in the regions of necrosis. cross-sections near the epididymis tail were less affected. vasculature in the vaginal tunic was dilated and congested. a few seminiferous tubules appeared within normal limits with abundant spermatogenesis. the vas deferens in the spermatic cord and the adjoining smooth muscle and connective tissue were also largely obliterated by neutrophils and necrosis. the pampiniform plexus was markedly congested and vessels were dilated. the inflammation extended to the proximal margin of the cord. the final diagnosis was severe suppurative and necrotizing epididymitis and funiculitis. the primary pathogen (e.  coli) was confirmed. the vasodilation in this case suggested that the patient additionally suffered from a spermatic cord torsion of the affected testis. outcome the patient completed followup evaluations with a local veterinarian. although the recommended repeat culture was not performed, a semen analysis was performed 3 months after surgery. dog had limited sperm production but with subjectively low numbers and low motility. five months after surgery the dog was used for natural breeding resulting in a litter of 6 healthy pups. at 9 months after surgery, semen collection and evaluation were performed at another facility, and the dog had an ejaculate containing 650 x 106 total sperm with 98% total motility and 93% normal morphology. discussion differential diagnoses for scrotal swelling include epididymitis, orchitis, neoplasia, and testicular torsion. all dogs presenting with an enlarged scrotum should be tested for canine brucellosis, regardless of age or breeding status. treatment for those disease processes requires removal of affected testes; antibiotic therapy alone is rarely curative.2 the pathogenesis of orchitis/epididymitis involves inflammation of the scrotal contents due to infection or autoimmune destruction.3 infection can take place either by retrograde movement of organisms from the prostate or lower urinary tract, hematogenous spread, or via direct entry from a bite/penetrating wound.1 in the case of retrograde infection orchitis/epididymitis the testis and epididymis will swell and form numerous, tiny, intraluminal abscesses. the chronicity of the infection will frequently cause the epididymis to become fibrotic and the testis to degenerate and scar.2,5 organisms commonly associated with orchitis/epididymitis are e. coli (the most common), brucella canis, proteus vulgaris, staphylococcus spp., streptococcus spp., mycoplasma canis, blastomyces dermatitidis, and canine distemper virus.2-5 as the preputial cavity and urethra are not sterile, it should be noted many of the above-mentioned bacteria are normal flora in the male reproductive tract.1,3,5 bacterial prostatitis is one of the most prevalent diseases in intact male dogs, secondary to benign prostatic hyperplasia/ hypertrophy.6,7 many bacteria mentioned above are common in cases of bacterial prostatitis and this particular case stands to reason that the e. coli cultured in this case might have been  an ascending infection from the prostate/urinary tract system, spreading to the testis via hematogenous spread.7 ultrasonography of the prostate might have been very beneficial in this case to determine if abscessation was apparent in the prostate despite the digital rectal examination findings within normal limits. in cases of suspected orchitis/epididymitis, ultrasonography can be useful for diagnosing acute versus chronic conditions. acute cases of orchitis on ultrasonography may have irregular, poorly defined anechoic areas with possible diffuse hypoechoic patterns and focal abscessations. there is typically enlargement of the testis and epididymis with fluid between the visceral and parietal tunics within the scrotum.8 in cases of chronic orchitis there may be less obvious features noted on ultrasonography with hyperechoic or mixed echogenic parenchyma and reduced testis size. abscesses were observed with chronic orchitis, featured as irregular, hyperechoic walls filled with anechoic/hypoechoic contents.8 testicular torsion, more correctly termed torsion of the spermatic cord, varies from a loose 360º torsion to several tight revolutions. dogs with cryptorchid testicular torsion usually have signs of acute onset of abdominal pain, vomiting, abdominal distension, lethargy, anorexia, stiff gait, dysuria, hematuria, and pyrexia.7,9,10 if testicular torsion involves a scrotal testis, swelling of the scrotum may or may not be painful. torsion is also more commonly reported in cryptorchid testes than with scrotal testes.7,9,10 intraabdominal testes are thought to be more prone to torsion due to greater mobility of the testis within the abdominal cavity.7,9,10 histologic descriptions of nonneoplastic testicular torsion have ischemic necrosis, intratesticular hemorrhage, and epididymal edema.9,10 regardless of etiology, valuable breeding dogs with only 1 affected testis are good candidates to undergo a unilateral orchiectomy as soon as possible to prevent atrophy of the contralateral testis due to heat, compression, extension of an infection, or hormonal suppression from certain tumors. semen quality should return to normal 62 days after therapy.1,2 compensatory hypertrophy of the remaining testis may be evident as early as 3 months after surgery.2 prognosis is better if neither atrophy nor fibrosis is noted in the remaining testis.2 it is noted in stallions that orchidopexy of the remaining scrotal testis is recommended at surgery.9 in dogs, torsion is more common in the undescended (cryptorchid) testis and often both descended and undescended testes are removed.9 orchidopexy is not routinely performed in dogs nor in this case. learning points • testing for brucellosis in all breeding dogs is strongly recommended, especially if they have possible clinical signs for the disease • hemicastration is a viable  option (can be performed by general practitioners) to preserve breeding potential for clients valuable breeding dogs • it is important to perform semen analysis at 2-3 months after hemicastration to determine successful preservation of fertility  • submitting samples for histopathology is ideal for ruling out infectious disease processes that may require systemic treatment http://dx.doi.org/10.58292/ct.v17.11199 4 citation: clinical theriogenology 2025, 17, 11199, http://dx.doi.org/10.58292/ct.v17.11199 acknowledgments authors thank drs. mike johnson and emily o’connell for their collaboration and support with this clinical case report. conflict of interest authors have nothing to declare. references 1. fuchs  km,  de sousa  gc,  denadai  r,  et al:  unilateral orchitis-epididymitis and diffuse periorchitis in a dog due to β-haemolytic streptococcal infection.  vet rec case rep 2023;11:e655. doi: 10.1002/vrc2.655 2. johnston sd, root kustritz mv, olson pn: disorders of the canine testes and epididymes. in: canine and feline theriogenology. philadelphia, pa; saunders: 2001. p. 312-332. 3. pérez c, whitacre md, thakur s, et al: unilateral epididymitis associated with salmonella bacteremia in a dog. clin theriogenol 2012;4:61-65. doi: 10.58292/ct.v4.11253 4. camargo-castañeda am, stranahan lw, edwards jf, et al: characterization of epididymal and testicular histologic lesions and use of immunohistochemistry and pcr on formalin-fixed tissues to detect brucella canis  in male dogs.  j vet diagn invest 2021;33:352-356. doi: 10.1177/1040638720986883 5. root kustritz mv, johnston sd, olson pn, et al: relationship between inflammatory cytology of canine seminal fluid and significant aerobic bacterial, anaerobic bacterial or mycoplasma cultures of canine seminal fluid: 95 cases (1987– 2000). theriogenology 2005;64:1333-1339. doi: 10.1016/j. theriogenology.2005.03.003 6. weese js, blondeau j, boothe d, et al: international society for companion animal infectious diseases (iscaid) guidelines for the diagnosis and management of bacterial urinary tract infections in dogs and cats. vet j 2019;247:8-25. doi: 10.1016/j. tvjl.2019.02.008 7. foster ra: common lesions in the male reproductive tract of cats and dogs. vet clin north am small anim pract 2012:42;527-545. doi: 10.1016/j.cvsm.2012.01.007 8. bracco c, gloria a, contri a: ultrasound-based technologies for the evaluation of testicles in the dog: keystones and breakthroughs. vet sci 2023;10:683. doi: 10.3390/vetsci10120683. 9. raisi a, davoodi f: testicular torsions in veterinary medicine. vet res commun 2022;46:303-313. doi: 10.1007/s11259-021-09873-5 10. whitfill n, johnson a, wilborn r, et al: a case of testicular torsion in an intact cane corso.  clin theriogenol 2029;11:505. doi: 10.58292/ct.v11.10430 http://dx.doi.org/10.58292/ct.v17.11199 https://doi.org/10.1002/vrc2.655 https://doi.org/10.58292/ct.v4.11253 https://doi.org/10.1177/1040638720986883 https://doi.org/10.1016/j.theriogenology.2005.03.003 https://doi.org/10.1016/j.theriogenology.2005.03.003 https://doi.org/10.1016/j.tvjl.2019.02.008 https://doi.org/10.1016/j.tvjl.2019.02.008 https://doi.org/10.1016/j.cvsm.2012.01.007 https://doi.org/10.3390/vetsci10120683. https://doi.org/10.1007/s11259-021-09873-5 https://doi.org/10.58292/ct.v11.10430 2014: equine semen quality following sperm exposure to seminal plasma stored under different conditions equine semen quality following sperm exposure to seminal plasma stored under different conditions a.r. whigham,a t.l. blanchard,a c.c. love,a s.r. teague,a s.p. brinsko,a t.h. welsh jr,b k. hinrichs,c d.d. varnera adepartment of large animal clinical sciences, college of veterinary medicine and biomedical sciences, bdepartment of animal science, college of agriculture and life sciences, and cdepartment of veterinary physiology and pharmacology, college of veterinary medicine and biomedical sciences, texas a&m university, college station, tx abstract this study addressed equine sperm quality following exposure to seminal plasma stored under different conditions. objectives were to compare fresh versus snap-frozen homologous seminal plasma; to compare homologous versus allologous frozen seminal plasma; and to determine the optimal processing/freezing method for long-term preservation of seminal plasma. for the latter objective, seminal plasma was subjected to the following storage conditions: immediate storage of seminal plasma at -20, -80, or -196oc (groups 20, 80 and 196, respectively); storage of seminal plasma at 4oc for 24 h prior to freezing at -20, -80, or -196oc (groups 4-20, 4-80, and 4-196, respectively); or storage of raw semen at 4oc for 24 h prior to isolating seminal plasma and freezing at -20, -80, or -196oc (groups rw20, rw-80, and rw-196, respectively). seven ejaculates were collected from each of three fertile stallions that served as sperm donors and seminal-plasma donors. seminal plasma was also obtained from seven other stallions by centrifugation and filtration of raw semen. following exposure of sperm to seminal plasma treatments and cooled storage of extended semen for 24 h, sperm motion characteristics (total motility [tmot, %], progressive motility [pmot, %], curvilinear velocity [vcl, µm/s]), plasma membrane intactness (pmi, %), acrosomal membrane intactness (ai, %), and sperm dna quality (comp, %) were evaluated. comparison of fresh versus frozen homologous seminal plasma revealed no effect of treatment on any experimental endpoint (p > 0.05). progressive motility was higher in semen exposed to frozen allologous seminal plasma, as compared to frozen homologous seminal plasma, for one of three stallions (p < 0.05). storage method for seminal plasma did not impact pmi or comp; however, group rw-20 yielded lower tmot than group 4-80 (p < 0.05). group rw-20 yielded lower vcl than all other treatments. highest vcl was detected in groups 80, 196, 4-80, and 4-196. a treatment x stallion interaction was detected for pmot. no difference was observed for two stallions (p > 0.05); however, group 20 yielded higher pmot than groups 80 or 196 for the remaining stallion (p < 0.05). our findings suggest that: 1) seminal plasma can be frozen for later use, obviating the need to process fresh semen to supply seminal plasma, 2) allologous seminal plasma may be beneficial for selected stallions, and 3) semen should not be stored in the raw form for an extended period prior to processing seminal plasma for frozen storage. keywords: equine, seminal plasma, sperm, semen quality, storage introduction the effects of seminal plasma on sperm function are an active area of investigation in the horse, in areas as broad as the effects of seminal plasma on post-breeding endometritis;1 effects on quality and fertility of frozen semen;2,3 and effects on sperm quality in cool-stored semen.4-14 both inhibitory and stimulating effects of seminal plasma on sperm capacitation have also been investigated in other species,15-17 and applications to stallion sperm have been proposed.18 raw semen can be subjected a centrifugation/filtration process as a means of harvesting sperm-free seminal plasma for future use; however, information is also unavailable regarding optimal methods for processing and storage of the seminal plasma. another area that has not been well-researched is individual stallion variation in seminal plasma effects on sperm. clinically, there are reports of stallions whose seminal plasma exerts a depressing effect on sperm quality; yet, when seminal plasma from another stallion is substituted, sperm quality improves when the semen is subjected to cooling4 or cryopreservation.2 despite the increasingly 459 clinical theriogenology • volume 6, number 4 • december 2014 widespread use of seminal plasma addition/replacement clinically, data are sparse regarding the effect of adding allologous seminal plasma to semen when incorporated soon after ejaculation. the objectives of this study were to: compare fresh versus snap-frozen homologous seminal plasma and homologous versus allologous frozen seminal plasma on semen quality following cooled storage; and determine the optimal freezing method for long-term preservation of seminal plasma. materials and methods animals sexually-active mature light-breed stallions (n=10) with good sperm quality and no known fertility problems were used in this study. all animals were fed a pelleted diet with access to fresh water and roughage and all were in good body condition. stallions were kept in stalls with occasional turn out in paddocks. two to three daily ejaculates were collected from each stallion to reduce extragonadal sperm reserves prior to collection of semen and/or seminal plasma for the experimental procedures. three stallions (11 to 24 years of age) were used as donors of both seminal plasma and sperm. seven different stallions (9 to 20 years of age) were used as donors only of seminal plasma. semen collection semen was collected using a lubricated missouri-model artificial vagina (nasco, ft. atkinson, wi) fitted with a semen receptacle (animal reproduction systems, chino, ca) containing a nylon mesh in-line filter (animal reproduction systems) to separate gel-free and gel-containing fractions of the ejaculate. stallions were exposed to an ovariectomized mare to stimulate penile erection. the erect penis was rinsed with warm water and then patted dry with clean, disposable towels. after penile cleaning, stallions were again sexually stimulated and allowed to mount a breeding dummy for semen collection. semen processing the gel-free semen volume was measured by weight (model tp600s precision plus top-loading balance, ohaus corporation, florham park, nj) and recorded in ml (1 g ≈ 1 ml), and sperm concentration was determined using a fluorescence-based instrument (nucleocounter® sp-100™, chemometec, allerød, denmark). the gel-free semen was diluted with extender (inra 96; imv, maple grove, mn) and then subjected to cushioned centrifugation, using 40-ml capacity glass nipple-bottom centrifuge tubes.18 to prepare glass nipple tubes for centrifugation, 30 µl of cushion fluid (minitube of america, inc., verona, wi) was pipetted into the bottom of the nipple underneath 1 ml of inra 96 extender. following extension of semen to a concentration of 30 x 106sperm/ml in inra 96, approximately 1 x 109 sperm were carefully layered on top of the inra-96 in the nipple tubes. loaded nipple tubes were centrifuged at 400 x g for 20 min at room temperature. the supernatant was aspirated and the resulting sperm pellet was resuspended in inra 96 extender, then transferred to a 50-ml conicalbottom tube and further diluted with extender and seminal plasma (30-32 ml) to obtain a final concentration of approximately 30 x 106 sperm/ml. extended semen was mixed with seminal plasma (20%, v/v) based on seminal-plasma treatments dictated by experimental protocols. prepared vials of extended semen were then packaged in a commercial semen-transport container (equitainer® ii; hamilton research, inc., south hamilton, ma) for 24 h of cooled storage. following cooled storage, aliquots of extended semen were packaged in capped 0.6-ml polypropylene tubes (fisherbrand™ snap-cap flat-top graduated microcentrifuge tubes, fisher scientific, pittsburgh, pa) and then immediately frozen (-80°c) until analyzed for sperm dna integrity, using the sperm chromatin structure assay (scsa). the remaining contents were warmed for 15 min at 37oc and then evaluated for sperm motion characteristics, as well as plasma-membrane and acrosomal-membrane intactness. seminal plasma processing and storage techniques gel-free raw semen was centrifuged at 2000 x g for 10 min at room temperature using 15-ml plastic conical-bottom tubes (vwr international, llc, radnor, pa). the seminal plasma was decanted 460clinical theriogenology • volume 6, number 4 • december 2014 and filtered through tandem nylon syringe filters (5.0and 1.2-µm pore diameters, spectrum chemical manufacturing corp., new brunswick, nj) to remove any residual sperm. aliquots (1.0to 1.8-ml) of filtered seminal plasma were packaged in capped polypropylene tubes (cryogenic vials [2.0-ml]; corning life sciences, lowell, ma) and stored according to experimental specifications. experimental treatments fresh versus frozen-thawed seminal plasma. seven ejaculates from each of three stallions were subjected to centrifugation and sperm resuspension in extender, as described above. semen was diluted to a final concentration 30 x 106 sperm/ml in extender containing 20% (v/v) freshly prepared (unfrozen) seminal plasma or seminal plasma that was flash-frozen (-196oc) immediately prior to thawing and use. volume of extended semen ranged from 30-32 ml. homologous versus allologous seminal plasma. seven gel-free ejaculates from each of three stallions were subjected to centrifugation and sperm resuspension in extender, as described above. semen was diluted to a final concentration 30 x 106 sperm/ml in extender containing 20% (v/v) frozen (196oc)/thawed seminal plasma from the same stallion (homologous) or from each of seven other seminalplasma donor stallions (allologous). seminal plasma frozen-storage conditions. seven gel-free ejaculates from each of three stallions were subjected to centrifugation and sperm resuspension in extender, as described above. semen was diluted to a final concentration 30 x 106 sperm/ml in extender containing 20% (v/v) allologous seminal plasma (from seven different stallions) that had been processed and stored in various manners. treatment groups consisted of the following: group 20 (seminal plasma processed immediately following collection and frozen at -20°c); group 80 (seminal plasma processed immediately following collection and frozen at -80°c); group 196 (seminal plasma processed immediately following collection and frozen at -196°c); group 4-20 (seminal plasma processed immediately following collection, stored at 4°c for 24 h and then frozen at -20°c); group 4-80 (seminal plasma processed immediately following collection, stored at 4°c for 24 h and then frozen at -80°c); group 4-196 (seminal plasma processed immediately following collection, stored at 4°c for 24 h and then frozen at -196°c); group rw-20 (raw semen stored at 4°c for 24 h, then processed for seminal plasma and frozen at -20°c); group rw-80 (raw semen stored at 4°c for 24 h, then processed for seminal plasma and frozen at -80°c); and group rw-196 (raw semen stored at 4°c for 24 h, then processed for seminal plasma and frozen at -196°c). computer-assisted sperm motion analysis (casma). sperm motion characteristics were analyzed in a manner similar to that previously described.19 warmed (37oc) analysis chambers (fixed height of 20 μm) affixed to microscope slides (leja standard count 2 chamber slides; leja products, b.v., nieuw-vennep, the netherlands) were slowly loaded with a 6-μl volume of extended semen. the slides were then placed on a stage (37oc) and inserted into the casma instrument (ivos version 12.0, hamilton-thorne research) for evaluation. a total of 10 microscopic fields and a minimum of 500 sperm were examined per sample. preset values for the ivos system consisted of the following: frames acquired–45; frame rate–60 hz; minimum contrast–70; minimum cell size–4 pixels; minimum static contrast–30; straightness (str) threshold for progressive motility–50; average-path velocity (vap) threshold for progressive motility–30; vap threshold for static cells–15; cell intensity–106; static head size–0.60 to 2.00; static head intensity–0.20 to 2.01; static elongation–40 to 85; led illumination intensity–2200. experimental endpoints included: 1) percentage of motile sperm (tmot); percentage of progressively motile sperm (pmot); and curvilinear velocity (vcl; μm/s). sperm acrosomal-membrane and plasma-membrane integrity. the intactness (integrity) of sperm acrosomal and plasma membranes was evaluated using a procedure described previously.20 fifty μl of extended semen were added to 133 μl of dulbecco’s phosphate buffered saline (pbs; invitrogen gibco,® carlsbad, ca). three μl of propidium iodide (invitrogen molecular probes, eugene, or; 2.4 mm working solution) and 10 μl pisum sativum agglutinin (psa)-fitc conjugate (sigma-aldrich, st. louis, mo; 0.05 mg/ml working solution) were added to the semen-buffer solution. the samples were incubated at room temperature (approximately 25oc) in the dark for 10 min. fifty μl of semen were then mixed with 1 ml pbs and processed immediately on a flow cytometer (facscan; becton dickinson, 461 clinical theriogenology • volume 6, number 4 • december 2014 mountain view, ca). the sample was allowed to pass through the tubing for 30 sec before evaluation of cells. a cell flow rate of approximately 300 cells/s was used and a total of 5000 events were evaluated per sample. the voltage settings on the flow cytometer were as follows: ssc 240, fl1 798, fl2 657, and fl3 150. the compensation was set at fl1 1.9% of fl2, and fl2 18.8% of fl1. data were acquired using a log scale and analyzed by winlist™ software (verity software house, topsham, me), with scatterplots divided into quadrants: minimal green and red fluorescence (representing sperm with intact plasma membranes and intact acrosomal membranes); minimal green and enhanced red fluorescence (representing spermatozoa with damaged plasma membranes and intact acrosomal membranes; minimal red and enhanced green fluorescence (representing spermatozoa with intact plasma membranes and damaged acrosomal membranes); and enhanced red and green fluorescence (representing spermatozoa with damaged plasma membranes and damaged acrosomal membranes). data were sorted by sperm with intact plasma membranes, regardless of acrosomal status (pmi; %) and sperm with intact acrosomal membranes, regardless of plasma membrane status (ai; %). sperm chromatin structure assay (scsa). the scsa protocol was conducted as previously described.21 all stock solutions (buffer solution [tne; ph 7.4; 0.19 g disodium edta, 0.79 g tris-hcl, 4.380 g nacl in 500 ml deionized water], triton-x [2.19 g nacl, 1.0 ml of 2n hci solution, 0.25-ml triton-x, qs. 250 ml with deionized water], and acridine orange [ph 6.0; 3.8869 g citric acid monohydrate, 8.9429 g na2hpo4, 4.3850 g nacl, 0.17 g disodium edta, 4.0 μg/ml acridine orange stock solution (1.0 mg/ml), qs. 500 ml water]) were kept on ice throughout the duration of the procedure. immediately prior to analysis, semen samples were thawed in a 37°c water bath. nine-µl of frozen-thawed semen were aliquoted into a 5-ml falcon tube and diluted to 200 µl with the tne solution, then 400 µl triton-x solution was added and the mixture was placed on ice for 30 s. a 1.2ml aliquot of acridine orange stain was then pipetted into the tube. the mixture was analyzed via flow cytometry using the following settings: mean green fluorescence at 500 channels (fl-1: 500) and mean red fluorescence at 150 channels (fl-3: 150). all samples underwent an equilibrium period of 30 seconds prior to analysis. five-thousand (5000) events were recorded at a rate of at least 200 events/s. quantification of dna denaturation in each cell was determined by the term alpha-t (αt), which is defined as the ratio of red/(red + green fluorescence). the alpha-t (αt) designation is used to describe the relationship between the amounts of green (double-stranded dna) and red (single-stranded dna) fluorescence. data were acquired in a list-mode and values were calculated using winlist™ software (verity software house). cells outside the main population (comp) was the endpoint measured and represented the percentage of sperm outside the main population. statistical analysis percentage data were arc sine-root transformed for normalization prior to statistical analysis using sas® (sas institute inc., cary, nc). statistical tests were conducted on transformed data. analysis-ofvariance (anova) procedures were used for data analysis, with the tukey test used for mean separation when treatment f ratios were significant (p < 0.05). level of significance was set at p < 0.05. untransformed data are presented in the results section for clarity of interpretation. results comparison of fresh versus frozen homologous seminal plasma no significant treatment differences were detected for experimental endpoints (p > 0.05; table 1), and stallion-by-treatment interactions were not significant (p > 0.05). 462clinical theriogenology • volume 6, number 4 • december 2014 table 1. main effect of fresh versus frozen/thawed homologous seminal plasma on measures of sperm quality (mean ± sem) for three stallions following 24 h of cooled storage (n = 21 ejaculates). laboratory parameter* treatment fresh seminal plasma frozen/thawed seminal plasma tmot (%) 80 ± 1.8 79 ± 2.0 pmot (%) 49 ± 1.7 48 ± 1.5 vcl (µm/s) 183 ± 6.8 187 ± 6.9 pmi (%) 85 ± 0.6 84 ± 0.7 ai (%) 88 ± 1.0 88 ± 1.1 comp (%) 10 ± 0.8 9 ± 0.9 *tmot = total sperm motility (%); pmot = progressive sperm motility (%); vcl = curvilinear velocity (µm/s); pmi = sperm with intact plasma membrane (%); ai = sperm with intact acrosomal membrane (%); comp = percentage of sperm with αt value outside the main population (%). percentage data (tmot, pmot, pmi, ai, and comp) were arc sine-root transformed for normalization prior to statistical analysis. untransformed values for mean and sem are presented in table to ease interpretation but statistical tests were conducted on transformed data. for each dependent variable, treatment differences were not detected (p > 0.05). comparison of frozen-thawed homologous versus allologous seminal plasma table 2 illustrates the main effects of frozen homologous versus allologous seminal plasma on experimental endpoints. no significant differences were detected between homologous and allologous treatment groups for variables tmot, pmi, ai and comp (p > 0.05). allologous seminal plasma yielded a lower vcl when compared to homologous seminal plasma (p < 0.05). for variable pmot, allologous seminal plasma yielded significantly higher values than did homologous seminal plasma (p < 0.05). stallion-by-treatment interactions were detected for pmot, where mean pmot was lower for homologous seminal plasma than allologous seminal plasma for one of three stallions (p < 0.05). treatment differences were not detected for the remaining two stallions (p > 0.05). the source of the allologous seminal plasma which yielded the highest pmot differed with sperm from each of the three donor stallions. table 2. main effect of frozen/thawed homologous versus allologous seminal plasma on measures of sperm quality (mean ± sem) for three stallions following 24 h of cooled storage (n = 21 ejaculates). laboratory parameter* treatment homologous allologous tmot (%) 79 ± 2.0a 82 ± 1.7a pmot (%) 48 ± 1.5b 54 ± 1.9a vcl (µm/s) 187 ± 6.9a 176 ± 7.8b pmi (%) 84 ± 0.7a 85 ± 1.2a ai (%) 88 ± 1.1 a 88 ± 1.1a comp (%) 9 ± 0.9a 8 ± 0.5a *tmot = total sperm motility (%); pmot = progressive sperm motility (%); vcl = curvilinear velocity (µm/s); pmi = sperm with intact plasma membrane (%); ai = sperm with intact acrosomal membrane (%); comp = percentage of sperm with αt value outside the main population (%). percentage data (tmot, pmot, pmi, ai, and comp) were arc sine-root transformed for normalization prior to statistical analysis. untransformed values for mean and sem are presented in table to ease interpretation but statistical tests were conducted on transformed data a,b within row, means with different superscripts differ (p < 0.05). storage methods for frozen preservation of allologous seminal plasma data regarding the effect frozen storage methods for allologous seminal plasma on semen quality are provided in table 3. a main effect of treatment was detected (p < 0.05) for all sperm motion 463 clinical theriogenology • volume 6, number 4 • december 2014 variables. variable tmot was higher for group 4-80 as compared to group rw-20 (p < 0.05), but tmot for both treatment groups was similar to that of the remaining treatments groups (p>0.05). group 20 exhibited higher pmot than rw-80 and rw-196 (p < 0.05), but pmot for these two treatment groups was similar to the remaining treatment groups (p > 0.05). mean vcl was higher in groups 80 and 4-80 than in groups 20, 4-20, rw80 and rw-196 (p < 0.05), and was lower for group rw-20 than that of all other treatment groups (p < 0.05). main effects of treatment were not detected for variables pmi, ai, and comp (p > 0.05). a treatment-by-stallion interaction was detected for pmot (p < 0.05). mean pmot was similar among treatment groups for two of three stallions (p > 0.05). for the remaining stallion, pmot was higher in group 20 than in groups 4-20, 80, 4-80, 196, rw-196 and rw-196. table 3. main effect of frozen-thawed seminal plasma that had previously been processed in various manners and stored for nine months at various freezing temperatures on measures of sperm quality (mean ± sem) in three stallions following 24 h of cooled storage with allologous sperm (n = 21 ejaculates). laboratory parameter* treatment‡ 20 80 196 4-20 4-80 4-196 rw-20 rw-80 rw196 tmot (%) 81 ± 2ab 82 ± 2ab 82 ± 2ab 82 ± 2ab 83 ± 2a 83 ± 2ab 80 ± 2b 82 ± 2ab 80 ± 2ab pmot (%) 58 ± 2.3a 55 ± 2.1ab 54 ± 1.9ab 57 ± 1.8ab 55 ± 1.9ab 57 ± 2.3ab 54 ± 2.1ab 53 ± 2.4b 53 ± 2.1b vcl (µm/s) 166 ± 7.4c 177 ± 7.5a 176 ± 7.8ab 166 ± 7.6c 178 ± 8.3a 177 ± 7.7ab 155 ± 7.4d 168 ± 8.5bc 164 ± 7.6c pmi (%) 85 ± 0.7a 85 ± 0.8a 85 ± 1.2a 84 ± 1.4a 86 ± 0.7a 86 ± 0.8a 86 ± 0.9a 87 ± 0.7a 86 ± 0.6a ai (%) 89 ± 0.9a 89 ± 0.8a 88 ± 1.1a 89 ± 0.9a 89 ± 0.8a 89 ± 0.8a 89 ± 0.9a 89 ± 0.8a 89 ± 0.8a comp (%) 10 ± 0.8a 9 ± 0.7a 8 ± 0.5a 9 ± 0.5a 11 ± 0.7a 10 ± 0.6a 10 ± 0.6a 10 ± 0.6a 9 ± 0.5a *tmot = total sperm motility (%); pmot = progressive sperm motility (%); vcl = curvilinear velocity (µm/s); pmi = sperm with intact plasma membrane (%); ai = sperm with intact acrosomal membrane (%); comp = percentage of sperm with αt value outside the main population (%). percentage data (tmot, pmot, pmi, ai, and comp) were arc sine-root transformed for normalization prior to statistical analysis. untransformed values for mean and sem are presented in table to ease interpretation but statistical tests were conducted on transformed data. †20 = seminal plasma stored only at -20 °c; 80 = seminal plasma stored only at -80°c; 196 = seminal plasma stored only at 196°c; 4-20 = seminal plasma held at 4°c for 24 h and then stored at -20°c; 4-80 = seminal plasma held at 4°c for 24 h and then stored at -80°c; 4-196 = seminal plasma held at 4°c for 24h and then stored at -196°c; rw-20 = raw semen sample stored for 24 h at 4°c and then processed for seminal plasma which was then stored at -20°c; rw-80 = raw semen sample stored for 24 h at 4°c and then processed for seminal plasma which was then stored at -80°c; rw-196 = raw semen sample stored for 24 h at 4°c and then processed for seminal plasma which was then stored at -196°c. a-d within row, means with different superscripts differ (p < 0.05). discussion this study evaluated the effect of seminal plasma processing and storage methods on resulting sperm quality following cooled storage. our results indicate that seminal plasma can be processed and stored under different conditions without adversely affecting sperm quality when using clinically normal stallions. there was no difference between fresh and snap-frozen/thawed seminal plasma on sperm quality, which suggests that seminal plasma can be processed for immediate use, or it can be stored frozen for later use. this permits the clinician more flexibility in the processing of seminal plasma for use with fresh, cooled, or cryopreserved semen. data from a previous study suggested that freezing of seminal plasma prior to mixing with semen could negatively impact sperm motility.22 the current findings are not supportive of this notion. the source of the frozen-thawed seminal plasma could have impacted 464clinical theriogenology • volume 6, number 4 • december 2014 experimental outcomes in these two studies. we evaluated the effects of homologous versus allologous seminal plasma on sperm semen quality to determine if the origin of seminal plasma would affect sperm quality. clinically, there are reports of stallions whose seminal plasma exerts a suppressive effect on sperm quality; yet when seminal plasma from another stallion is substituted, sperm quality improves when the semen is subjected to cooling4 or cryopreservation.2 in the present study, allologous seminal plasma was generally similar to homologous seminal plasma for maintaining sperm motion characteristics, membrane integrity and dna integrity after 24 h of cooled storage; however, we did note a stallion-by-treatment interaction, whereby sperm from one stallion exhibited improved progressive motility following cooled storage when mixed with allologous seminal plasma, as compared to homologous seminal plasma. this finding supports the report of varner et al. where incorporation of allologous seminal plasma improved the sperm velocity in a subfertile stallion.4 it also supports the findings of aurich et al. where the seminal plasma from stallions with good post-thaw sperm quality improved sperm quality of stallions with poor post-thaw sperm quality.2 the fact that sperm from one stallion in our study did show a preference for allologous seminal plasma for pmot suggests that it may be appropriate to test sperm from problematic stallions in the clinical setting to determine whether allologous seminal plasma may be more appropriate than homologous seminal plasma when processing semen for cooled storage. our data suggest that it may be important to test various sources of allologous seminal plasma when performing this procedure, as no single source of allologous seminal plasma yielded consistently high values for sperm quality when added to the semen of the three sperm donors. others have reported inconsistencies in use of homologous versus heterologous (implying allologous) seminal plasma among stallions, but that study involved overnight storage of extended semen containing homologous seminal plasma for transport to the laboratory prior to conducting the experiments.23 this may have confounded experimental results, as components of homologous seminal plasma likely incorporated into sperm membranes during this time.23 the term, heterologous, was inappropriately used by morrell and coworkers, as the term would indicate that the seminal plasma was derived from a different species from the recipient. we evaluated various processing methods and frozen-storage temperatures for seminal plasma in an effort to determine the technique(s) for accomplishing this task that might optimize resulting semen quality following cooled storage. the findings generally supported our hypothesis that sperm quality would not be affected by seminal plasma storage temperature (-20 oc, -80oc or -196oc). we also hypothesized that cooled storage of raw semen for 24 h prior to processing of seminal plasma would be detrimental. while this treatment condition was not dramatically different than the other methods used for processing seminal plasma, the data certainly suggest that some resulting sperm-motility values could be suppressed when this method is used. the seminal-plasma samples in this experiment were stored for nine months prior to use. based on the findings of this study, we consider it feasible for a veterinary practice to store seminal plasma using a conventional freezer (-20oc), especially if liquid nitrogen, dry ice, or a -80oc-freezer is not readily accessible; however, we did not test freezers with automatic defrost (frost-free) systems. it is possible that defrost cycles of such freezers would be deleterious to some components of seminal plasma. in summary, fresh and frozen seminal plasma yielded similar results for sperm quality, regardless of freezing temperature for storage. as such, we contend that seminal plasma can be banked for future use, eliminating the need for processing a fresh sample when seminal plasma is needed. semen from certain stallions may benefit from using allologous seminal plasma, as compared to homologous seminal plasma. differences detected among processing/freezing methods for seminal plasma were slight, suggesting that considerable flexibility may be permitted in techniques for storage. acknowledgment funding for this project was provided by the legends premier stallion season auction, texas a&m university. 465 clinical theriogenology • volume 6, number 4 • december 2014 references 1. troedsson mh, lee cs, franklin rd, et al: the role of seminal plasma in post-breeding uterine inflammation. j reprod fertil suppl 2000;56:341-349. 2. aurich je, kühne a, hoppe h, et al: seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation.theriogenology 1996;46:791-797. 3. moore ai, squires el, graham jk: effect of seminal plasma on the cryopreservation of equine spermatozoa. theriogenology 2005;63:2372-2381. 4. varner dd, love cc, brinsko sp, et al: semen processing for the subfertile stallion. j equine vet sci 2008;28:677685. 5. rigby sl, brinsko sp, cochran m, et al: advances in cooled semen technologies: seminal plasma and semen extender. anim reprod sci 2001:68:171-180. 6. pickett bw, sullivan jj, beyers ww, et al: effect of centrifugation and seminal plasma on motility and fertility of stallion and bull spermatozoa. fertil steril 1975;26:167-174. 7. varner dd, blanchard tl, love cc, et al: effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters. theriogenology 1987;28:709-718. 8. jasko dj, moran dm, farlin me, et al: effect of seminal plasma dilution or removal on spermatozoal motion characteristics of cooled stallion semen. theriogenology 991;35:1059-1067. 9. jasko dj, hathaway ja, schaltenbrand vl, et al: effect of seminal plasma and egg golk on motion characteristics of cooled stallion spermatozoa. theriogenology 1992;37:1241-152. 10. brinsko sp, crockett ec, squires el: effect of centrifugation and partial removal of seminal plasma on equine spermatozoal motility after cooling and storage. theriogenology 2000;54:129-136. 11. love cc, brinsko sp, rigby sl, et al: relationship of seminal plasma level and extender type to sperm motility and dna integrity. theriogenology 2005;63:1584-1591. 12. akcay e, reilas t, andersson m, et al: effect of seminal plasma fractions on stallion sperm survival after cooled storage. j vet med a physiol pathol clin med 2006;53:481-485. 13. foster ml, varner dd, hinrichs k, et al: agreement between measures of total motility and membrane integrity in stallion sperm. theriogenology 2011;75:1499-1505. 14. morrell jm, pihl j, dalin am, et al: restoration of seminal plasma to stallion spermatozoa selected by colloid centrifugation increases sperm progressive motility but is detrimental to chromatin integrity. theriogenology 2012;78:345-352. 15. bedford jm, chang mc: removal of depacitation factor from seminal plasma by high-speed centrifugation. am j physiol 1962;202:179-187 16. manjunath p, therien i: role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation. j reprod immunol 2002;53:109-119. 17. leahy t, badella bm: sperm surface changes and physiological consequences induced by sperm handling and storage. reproduction 2011;142:759-778. 18. töpfer-petersen e, ekhlasi-hundrieser m, kirchhoff c, et al: the role of stallion seminal proteins in fertilization. anim reprod sci 2005;89:159-170. 19. waite ja, love cc, brinsko sp, et al: factors impacting equine sperm recovery rate and quality following cushioned centrifugation. theriogenology 2008;70:704-714. 20. salazar jl, teague sr, love cc, et al: effect of cryopreservation protocol on post-thaw characteristics of stallion sperm. theriogenology 2011;76:409-418. 21. love cc, kenney rm: the relationship of increased susceptibility of sperm dna to denaturation and fertility in the stallion. theriogenology 1998;50:955-972. 22. foster ml, varner dd, hinrichs k, et al: agreement between measures of total motility and membrane integrity in stallion sperm. theriogenology 2010;75:1499-1505. 23. morrell jm, georgakas a, lundeheim n, et al: effect of heterologous and homologous seminal plasma on stallion sperm. theriogenology 2014 apr 1. pii: s0093-691x(14)00161-7. doi: 10.1016/j.theriogenology.2014.03.020. [epub ahead of print]. 466clinical theriogenology • volume 6, number 4 • december 2014 2016: adrenal rest tissue in domestic cats: a case series adrenal rest tissue in domestic cats: a case series margaret v. root kustritz, jennifer l. brazzell, kim culbertson college of veterinary medicine, university of minnesota, st. paul, mn summary paraovarian masses were identified in cats presented to a high-quality, high-volume spay and neuter clinic. the masses were defined histologically as ectopic adrenal tissue. keywords: adrenal, paraovarian mass background the adrenal gland develops embryologically as two separate structures, with the adrenal medulla arising from the neural crest and the adrenal cortex arising from the urogenital ridge along with the differentiating gonad. masses of tissue separate from the main adrenal gland, called adrenal rest tissue, are commonly identified in humans. adrenal rest tissue most often is found near the adrenal gland or associated with the ovaries or testes and contains no medullary tissue, suggesting that these remnants are formed from cells misplaced during embryologic migration.1 adrenal rest tissue is usually of no clinical significance in humans. they are identified in as many as 50% of neonates but are only reported in 1% of adult humans.1 ectopic adrenal tissue near the ovaries is common in mares. it is uncommon in cats and rare in other species.a this is a case series describing adrenal rest tissue identified in six domestic cats. case presentation a two-year-old female domestic longhair cat (dlh) weighing 6.7 pounds was presented to a high-quality, low-cost spay and neuter service by its owner for ovariohysterectomy (ohe). all surgeries described were performed by one of the authors (kc). the cat had no history of health concerns and was not noted by the owner to be in estrus at the time of presentation. information about estrous cycles was not available. the cat was verified to be in good health based on a complete physical examination. she received hydromorphone pre-operatively and was anesthetized with a combination of medetomidine, ketamine, and butorphanol. anesthesia was monitored by assessment of vital signs, pulse oximetry, and capnography. ovariohysterectomy was routine but the cat was noted to have masses near both ovaries (figure 1). in this cat and in all other cats described the masses were readily visible. they were oval, and tan, and were 1-2 mm in diameter. all were located in the suspensory ligament, about 5-10 mm away from the ovary, and did not appear to be encapsulated. no abnormalities of the ipsilateral ovary or uterine horn were noted. the masses were excised, placed in formalin, and submitted for histopathology. the ipsilateral ovary also was submitted, with each tissue submitted in its own jar of formalin. removal of this tissue did not require use of additional suture for control of hemorrhage or otherwise complicate surgery. the linea alba and subcutaneous tissues were closed in a simple continuous pattern with 3/0 or 4/0 polydioxanone suture. subcuticular closure of skin was performed using the same suture. two feral cats, one a female domestic medium hair (dmh) cat estimated to be six to eight months of age and weighing 3.8 pounds and the other a female domestic short hair (dsh) cat estimated to be ten weeks of age and weighing 2.7 pounds were presented for ohe to the same facility. information about estrous cycles was not available. health of the cats was assessed by visual inspection. anesthetic management was as previously described. ovariohysterectomy was routine but both cats were noted to have one paraovarian mass, which was removed, placed in formalin, and submitted for histopathology. closure was as previously described. three cats were presented from rescue organizations for ohe at the same facility. one was a female dmh estimated to be three months of age and weighing 3.0 pounds. the second was a female domestic dsh estimated to be four months of age and weighing 4.6 pounds. the third was a female dsh estimated to be two years of age and weighing 8.7 pounds. the cats were verified to be in good health based on a complete physical examination. the two year old cat was palpably pregnant. anesthetic clinical theriogenology • volume 8 number 2 • june 2016131 management was as previously described; the pregnant queen also received subcutaneous lactated ringer’s solution during surgical preparation. ovariohysterectomy was routine but all cats were noted to have one paraovarian mass, which was removed, placed in formalin and submitted for histopathology. closure was as previously described; 2/0 polydioxanone suture was used for the pregnant cat. differential diagnosis the most likely cause of a discrete, small mass near the ovary is accessory or ectopic adrenal tissue. accessory ovarian tissue is a theoretical possibility. all of the masses were adrenocortical tissue within fibrovascular tissue (mesovarium, mesosalpinx, or mesometrium) (figure 2). outcome all of the cats recovered from anesthesia with no complications and were discharged the same day after they were ambulatory and were judged to be capable of returning to their home environment based on assessment of mentation and normal appearance of the incision and surrounding skin. longterm outcome was not known. discussion the equivalent of adrenal rest tissue has been identified in cats. masses 2-5 mm in diameter and located in the broad ligament near the ovary were removed from 17 cats and submitted for histopathologic evaluation. all of those masses contained adrenal cortical tissue with no adrenal medulla.2 prevalence of adrenal rest tissue in that study was cited as 2.2%.2 while overall uncommon in cats, ectopic adrenal tissue in cats is a finding still more common than in any other species except the mare.a clinical significance, if any, is unknown. the zona reticularis of the adrenal cortex produces and secretes a variety of androgens including dihydroepiandrosterone (dhea), which is a precursor of naturally produced estrogens. there are individual case reports of cats with multiple ovaries,3 but this is rare. it is hypothetically possible, therefore, that either accessory adrenal tissue or additional ovarian tissue could be a cause of estrus signs after ovariectomy or ovariohysterectomy, as is seen in ovarian remnant syndrome. ovarian remnant syndrome occurs more commonly in cats than in dogs.4 affected spayed cats will show signs of estrus anywhere from 17 days to nine years after ohe, with lordosis (behavioral posturing of estrus) the most common sign.5-7 presence of estrogen secretion can be verified by vaginal cytology, using cornification of vaginal cells as a bioassay for serum estrogen concentrations. ovulation can be induced with gonadotropin releasing hormone (gnrh; 25 mcg/cat, im) and blood drawn two to three weeks later to verify elevation in serum progesterone concentration. other diagnostic methods include ovulation induction with human chorionic gonadotropin (hcg), assay of serum anti-mullerian hormone (amh) or luteinizing hormone (lh) concentrations, abdominal ultrasonography, and abdominal exploratory.5,6,8-11 because there are no safe products available for long-term estrus suppression in cats, surgery to remove ovarian remnants is recommended. surgery is best performed when there are identifiable structures (either follicles or corpora lutea) on the remnant tissue. remnants are occasionally found in the abdomen, often wrapped in omentum, but are most commonly are found at one or both ovarian pedicles and should be submitted for histopathologic evaluation, as some will be neoplastic.4,6,12-14 while it appears clear that neither adrenal rest tissue nor accessory ovarian tissue is the usual cause of ovarian remnant syndrome, the true cause of behavioral estrus after ovariectomy or ohe often goes unidentified. ovarian cortical tissue readily revascularizes in the abdomen and it has been suggested that because the feline ovary is not in a bursa, cortical cells are more easily knocked away or caught in a clamp and left behind.8,15 ovarian remnant syndrome is not associated with difficulty of the surgery, experience of the veterinarian, or age or breed of the cat.4 use of a left flank approach for ohe has been hypothesized as a cause of retention of ovarian tissue at the right pedicle.16 surgeon error is a reported cause.5 removal of adrenal rest tissue in these cats was not associated with increased surgical time or increased complications of surgery. it is, therefore, probably not detrimental to cats for it to be removed clinical theriogenology • volume 8 number 2 • june 2016 132 during routine ohe. however, since there are no reported cases of ovarian remnant syndrome in cats due to anything but retained pieces of ovary, it probably is of greatest value for surgeons noting such tissue to be aware that it is adrenal in origin and need not be removed. learning points • paraovarian masses noted at the time of routine ohe in domestic cats are most likely adrenal rest tissue. • clinical significance of adrenal rest tissue in cats is unknown. a.personal communication, rob foster, bvsc, phd, manzvcs, diplomate acvp references 1. barwick td, malhotra a, webb jaw, et al: embryology of the adrenal glands and its relevance to diagnostic imaging. clin radiol 2005;60:953-959. 2. altera kp, miller ln: recognition of parovarian nodules as ectopic adrenocortical tissue. j am vet med assoc 1986;189:71-72. 3. mcentee k: reproductive pathology of domestic mammals. new york: academic press;1990. p. 36-38. 4. miller dm: ovarian remnant syndrome in dogs and cats: 46 cases (1988-1992). j vet diagn invest 1995;7:572-574. 5. england gcw: confirmation of ovarian remnant syndrome in the queen using hcg administration. vet rec 1997;141:309-310. 6. wallace ms: the ovarian remnant syndrome in the bitch and queen. vet clin na 1991;21:501-507. 7. naiman jh, mayhew pd, steffey ma, et al: laparoscopic treatment of ovarian remnant syndrome in dogs and cats: 7 cases (2010-2013). j am vet med assoc 2014;245:1251-1257. 8. denardo ga, becker k. brown no, dobbins s: ovarian remnant syndrome: revascularization of free-floating ovarian tissue in the feline abdominal cavity. j am anim hosp assoc 2001;37:290-296. 9. place nj, hansen bs, cheraskin j-l, et al: measurement of serum anti-mullerian hormone concentration in female dogs and cats before and after ovariohysterectomy. j vet diagn invest 2011;23:524-527. 10. scebra lr, griffin b: evaluation of a commercially available luteinizing hormone test to distinguish between ovariectomized and sexually intact queens. acvim forum, charlotte nc, 2003 [abstract]. 11. rohlertz m, strom holst b, axner e: comparison of the gnrh-stimulation test and a semiquantitative quick test for lh to diagnose presence of ovaries in the female domestic cat. theriogenology 2012;78:1901-1906. 12. ball rl, birchard sj, may lr, et al: ovarian remnant syndrome in dogs and cats: 21 cases (2000-2007). j am vet med assoc 2010;236:548-553. 13. root kustritz mv, rudolph kd: neoplastic ovarian remnant in a cat. j am vet med assoc 2001;219:1065-1066. 14. heffelfinger dj: ovarian remnant in a 2-year-old queen. can vet j 2006;47:165-167. 15. shemwell re, weed jc: ovarian remnant syndrome. obstet gynecol 1970;36:299-303. 16. demirel ma, acar db: ovarian remnant syndrome and uterine stump pyometra in three queens. j feline med surg 2012;14:913-918. clinical theriogenology • volume 8 number 2 • june 2016133 figure 1. normal ovary (large arrow) and extraovarian mass (small arrow). figure 2. histology of adrenal rest tissue composed of adrenocortical cells within the fibrovascular tissue of the mesovarium. 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/documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2010: pathology of male reproductive organs pathology of male reproductive organs robert a. foster department of pathobiology, ontario veterinary college, university of guelph guelph, ontario, canada general introduction diseases and conditions of the male reproductive system are traditionally approached from the perspective of anatomical location, beginning with the scrotal contents, at the site of production of spermatozoa, and ending with the penis and prepuce. this parallels a problems based approach as diseases tend to involve the scrotum and contents, the accessory genital glands and/or the penis and prepuce. if you are interested in one particular species only, the information for other species is still relevant because a disease in one is usually identical to that in another! the veterinary reproductive pathology website (www.uoguelph.ca/~rfoster/repropath/repro.htm) provides additional information for the dog and cat, as do recently updated pathology textbooks.1,2 the nomina anatomica veterinaria3 (nav), which establishes the names in anatomy, lists names of reproductive structures. some differ from commonly used terms. the latest texts in reproductive pathology use the nav terminology and this overview will use the english version of the latin name. keywords: male, reproductive, pathology scrotal contents introduction the testes are often considered the center of the ‘male reproductive universe’. not every disease of the scrotal contents involves the testes, however. orchitis is a common clinical diagnosis, but this term can be misleading because inflammation of the testes is a rare event. the inflammation is usually of the vaginal tunics and epididymis. often times the reaction is to spermatozoa that have leaked into the tissues. the ability of spermatozoa to stimulate a florid inflammatory reaction is overlooked, but is the most important factor when considering prognosis. the testis is recognized as having immune privilege. the development of germ cells occurs long after immune tolerance develops and a constitutive production of anti-inflammatory cytokines in the testis is now recognized. these are directed by local androgens to inhibit proinflammatory cytokines.4 an alteration of this balance results in immune and inflammatory reactions in the testis. spermatic granuloma the inflammatory and immunologic response to spermatozoa is of critical importance in many diseases and conditions of the male reproductive tract. any condition that causes leakage of spermatozoa or spermatozoal antigens into the extratubular compartment is potentially complicated by this reaction. spermatozoa incite a granulomatous and pyogranulomatous reaction known as a spermatic granuloma. macroscopically, these have an appearance similar to pus. spermatozoa have a cell wall similar in composition to keratin as they have many sulfur bonds. spermatozoa produce a foreign body type reaction and the large number of them and their keratin-like composition means that degradation is a slow process. in addition, immune responses are florid with many immunoglobulin producing cells and cd4 and cd8-expressing lymphocytes appear. upregulation of major compatibility complex (mhc) i in epithelial cells also occurs.5 the reaction leads to chronic inflammation and fibrosis, and continued disruption and obstruction of tubes and tubules. spermiostasis, spermatocele and/or further spermatic granulomas are the consequence. diseases of the vaginal tunics primary diseases of the vaginal tunics are rare. secondary involvement after epididymitis (see below) is a much more frequent finding. in domesticated species the most well known primary cause of inflammation of the vaginal tunics is feline infectious peritonitis (fip). scrotal swelling in the cat is a 531 presenting sign that should alert the practitioner to consider fip.6 periorchitis is also seen in traumatic events, such as fight wounds in cats, which allow introduction of bacterial pathogens into the vaginal recess. this recess is continuous with the peritoneum; any disease affecting the peritoneum can and will affect this area. hydrocele or non-inflammatory fluid within the cavity of the vaginal tunics occurs in ascites in all species and in bulls in conditions of suboptimal nutrition.7 disease of the testis and epididymis the testes produce spermatozoa and hormones such as testosterone and inhibin. while early research focused on the hypothalamic–pituitary–testicular axis, there is an enormous body of work on local regulation and cellular crosstalk in the testis. perhaps more complex is the regulation of testicular development and descent as it involves many different hormones, growth factors (including insulin-like growth factors [igf]) and the interaction of systemic and local factors and their receptors. not all are well understood. the regulation of the blood-testis barrier and the effects of inflammation, focusing on the molecular aspects was reviewed recently.8-10 germ cell, sertoli cell and interstitial endocrine cell function is closely interrelated and there is considerable crosstalk among them.11 control of programmed cell death and apoptosis is now identified as a critical process in testicular physiology and pathophysiology.12 it is a normal process that limits potential spermatogenesis by about 25 to 75%. gonadotrophins and intratesticular androgens act as survival factors, whereas external events such as elevated temperature increase apoptosis of spermatocytes. small testes. small testes mean reduced production of spermatozoa because testicular weight and volume (to name a few) are correlated with daily sperm output. scrotal circumference in the bull is highly correlated with daily sperm output, but there are few good measures used in the other species. scrotal circumference can be used in the ram and buck, but the lack of accessible testes for this measurement in the other species means that we must rely on comparing the testes with objects of the same shape and known volume—orchidometers. testicular size is seldom a selection criterion in nonruminants. it is almost impossible to differentiate the cause of small testes in older animals unless the testes have been examined previously. primary testicular hypoplasia and testicular atrophy cannot be accurately differentiated, in part, because hypoplastic testes undergo degeneration. when animals are examined at puberty, one’s confidence in diagnosing hypoplasia is much higher. in general, a hypoplastic testis has a small hypoplastic epididymis attached. the epididymis is usually disproportionately larger in testicular atrophy. the two main processes resulting in small testes are therefore a failure to develop and a reduction in size. failure to develop is a ‘disorder of sexual development’ (dsd)and therefore hypoplasia. a reduction in size is atrophy. disorders of sexual development. testicular hypoplasia exists when the testis remains smaller than expected after puberty. it occurs as an ‘uncomplicated’ or primary condition in normally descended testes, in cryptorchidism, and in conjunction with other disorders of sexual development such as chromosomal anomalies (detailed below). hypoplasia. the failure of one or both testes to grow to a normal size results because of a reduction in the amount of the seminiferous epithelium (lining).13 there is little written about the molecular mechanisms involved in hypoplasia in domestic animals. it is known to be a genetic disease in many species. primary hypoplasia can be either unilateral or bilateral. an affected testis is macroscopically similar to normal testis in almost every way—except size. hypoplasia is the potential end-result of a large number of different abnormalities that may operate at a systemic or local level (in the case of unilateral disease). we are seldom able to identify a cause. it is surprising how little is written 532 about testicular hypoplasia except in the context of cryptorchidism! this is despite a large percentage of bulls (up to 56% of belgian blue bulls) with a scrotal circumference that is too small,14 and many dogs have histological evidence of hypoplasia.15 the reduction in the area of seminiferous epithelium is the result of either some small tubules in the presence of normal ones, or all tubules are small. theoretically there could be a reduction in the number of tubules, the length of tubules, or the diameter of the tubules, or one or more combinations of these. germ cells may be absent or present but fail to produce enough spermatozoa. germ cells may have failed to migrate to the genital ridge in utero, failed to migrate in sufficient numbers, failed to survive, have arrested development, undergo excessive apoptosis or undergo degeneration at some stage of spermatogenesis. hypoplastic testes with a total lack of germ cells are very small and fail to enlarge from their size at birth. although we are aware of these possibilities, we often lack the tools and finances to determine the molecular mechanisms involved. a deficiency of gonadotrophins is associated with hypoplasia (hypogonadotrophic hypogonadism) in humans and mice, but studies in domesticated mammals indicate a normal13 or increased serum concentration of follicle stimulating hormone and luteinizing hormone. testosterone concentration was lower than normal in some bulls with hypoplasia or degeneration while in sheep, testosterone and inhibin concentrations may be normal. kay, et al.16 reported concurrent branching of the testicular artery on the same side as a hypoplastic testis, regardless of side affected. this abnormal blood supply could be responsible in some cases. this should result in uniform change in the testis—it does not explain those cases where there is a mixture of hypoplastic and normal tubules. this phenomenon should be investigated further. recently borel, et al.17 identified a bull they considered to suffer from testicular hypoplasia and demonstrated bvd antigen in germ cells, sertoli cells and blood vessel walls, but not in interstitial endocrine cells. the photographs demonstrated testicular atrophy and degeneration, but the history of ‘hypogonadism’ suggests this case may be one of hypoplasia with secondary atrophy. a cause and effect was not established. the involvement of bvd in testicular hypoplasia should also be investigated. at the genetic level, hypoplasia is known or suspected to be hereditary in the dog, bull, ram, and buck. the exact genetic abnormality is often not established. cryptorchidism. there is much written about cryptorchidism because of the human disease and because it is so common in our domestic animals. it is now classified as a dsd (xy sry+ with testis). there is much discussion about the stages of testicular descent, but there appears to be a consensus emerging (reviewed by amann and veeramachaneni18 and hughes and acerini19). there are three phases of testicular descent: abdominal translocation, transinguinal migration and inguinoscrotal migration. the first phase involves anchoring of the testis to the inguinal ring by the gubernaculum and relaxation of the cranial suspensory ligament, and this may be controlled by insulin-like peptide 3 (insl3) and testosterone. growth and therefore enlargement of the fetus, results in caudal migration. the second phase involves opening of the inguinal ring by an enlarged gubernaculum and intraabdominal pressure. the third phase takes the testis from the subcutaneous location into the scrotum, and is an interaction of androgen, calcitonin gene related protein and the genitofemoral nerve. the exact mechanisms are yet to be elucidated, but they will no doubt be complex and involve an interrelationship of systemic and local factors. the usual locations of the retained testis are adjacent to the inguinal ring, within the inguinal ring, and beneath the inguinal ring in a subcutaneous location, representing failure of one of the phases of migration. cryptorchidism is the potential end result of genetic, hormonal, structural or other abnormalities. for the genetic causes, a polygenic recessive-hereditary basis has been established or suggested in all species. recent molecular techniques have identified a mutation in the insl3 receptor rxfp2 that is strongly associated with the disease in humans.20 exposure to endocrine disruptors interfering with hormonal actions, are blamed for some cases. structural abnormalities such as gonadal splenic fusion are recognized causes. flock outbreaks in sheep suggest environmental causes can operate as well. diagnosis and therapeutic intervention to modify or control testicular descent, based on 533 correcting the exact abnormality, is currently a theoretical possibility only. because of prolonged exposure to increased temperature, the cryptorchid testis is hypoplastic and will eventually degenerate. chromosomal disorders of sexual development. animals with chromosomal abnormalities that affect sexual development result in dysgenesis, hypoplasia and or cryptorchidism. there are many recent reviews of disorders affecting sexual development, with the most recent being from meyers wallen.21 the terminology of dsd includes hermaphrodism, intersex, and ambiguous development. this overview uses the recommendations of hughes22 in naming dsd. briefly the dsd are categorized according to the genotype, presence of the sry gene, the gonadal type and phenotype. for example, at the chromosomal level, xxy sry positive testicular dsd (klinefelter’s-like syndrome) is recognized. affected cats, bulls, dogs, pigs, horses and sheep have an xxy genotype or a mosaicism with xxy chromosomes. the most famous of these is the male tortoiseshell or calico cat. xx sry negative testicular dsd occurs in goats of the saanen breed with the gene for polledness and in dogs. a variety of other dsd have been identified as being associated with hypoplasia and or cryptorchidism including the persistent müllerian duct syndrome (xy sry positive testicular dsd) in schnauzers and bassets. testicular atrophy and degeneration. testicular atrophy is defined as the macroscopic reduction in size of the testis after it has attained its post-pubertal size. degeneration is the histological term. the germinal epithelium reduces in amount and eventually all spermatogenesis stops. there is often spermiostasis, and mineralization of the tubular content. spermatic granulomas may develop. testicular degeneration usually occurs because of external influences (cf. hypoplasia) that interfere with any of the three main cell types of the testis—sertoli cells, interstitial endocrine cells and germ cells. there is a seemingly endless list of potential causes (table 1). the close interrelationship of the sertoli cells, interstitial endocrine cells and germ cells means that insults to any one or several of them eventually affect them all. the manifestation in the testis is not always bilateral or uniform and in some species has a distinct pattern. the testes of bulls frequently degenerate from the ventrum, and rams from the dorsum. mineralization, a common sign of degeneration, may involve whole or part of one seminiferous tubule, or whole regions. testicular biopsies could miss affected areas. maintenance of normal testicular function requires a normal hypothalamic pituitary gonadal axis, and maintenance of the normal paracrine and autocrine functions of the testis.8 any factor that interrupts the endocrine control (such as endocrine disruptors), or interferes with the cytokine milieu will alter spermatogenesis and or induce apoptosis. at the cellular level, the basic mechanisms of degeneration are similar to that of other cells and tissues. the more we learn about the mechanisms involved in other tissues, the more will be identified in the testis.23 for example, a recent review highlighted the effect of oxidative stress on cells of the testis.24 neoplasms. testicular neoplasms are most commonly found in the dog. there is no satisfactory explanation for this, but intact dogs are allowed to live as long as they are able, and they tend to be watched closely. male cats tend to be castrated early and fewer of those that are intact live to old age. neoplasms arise in all species sporadically and, with the exception of the dog, it is possible to predict the histologic type of neoplasm based on species and age. seminoma is the common tumor of the aged, and teratoma is more common in young horses. cats, rams and bucks get neoplasms rarely, and both seminoma and sertoli cell tumor are reported. bulls are more likely to have interstitial cell tumors, but sertoli cell tumors are also reported. three main testicular neoplasms of dogs are the sertoli cell tumor, the interstitial cell tumor and the seminoma. these are reviewed in detail at www.uoguelph.ca/~rfoster/repropath/repro.htm. multiple types of neoplasia may be found in one testis. most primary testicular neoplasms in dogs are benign. exceptions are extremely rare. identification of metastasis is the only way to determine that the neoplasm is malignant; there are no good cytological or histologic markers. recently, an unusual presentation of testicular tumors was reported.25 these occurred in dogs that were previously castrated, and the tumors 534 developed in the spermatic cord and incision site. sertoli cell tumors were most common in dogs and interstitial cell tumors in cats. implantation of testicular tissue during surgery is believed to be the cause. most neoplasms cause enlargement of the testis. in general, seminomas are white, soft and usually bulge on cut section. sertoli cell tumors tend to contain a lot of fibrous tissue so they are white in color and are firm. the interstitial cell tumor is soft, tan or yellow in color, and often contains areas of hemorrhage. sertoli cell tumors, but rarely interstitial cell tumors, can produce a hyperestrogenism-like syndrome and feminization. this is usually manifested by attractiveness to other male dogs, gynecomastia, and alopecia. some develop bone marrow aplasia. affected animals return to normal after removal of the neoplasm. the signs are not always associated with estrogen production and not all dogs will have increased serum estrogen concentrations. in these instances inhibin secretion by the neoplastic sertoli cells inhibits the secretion of follicle stimulating hormone and luteinizing hormone by the pituitary, which in turn, inhibits testosterone production. the imbalance between testosterone and estrogen is responsible for the relative increase in estrogen and feminization. extratesticular signs are much more common when the neoplasm is larger, and therefore is more common in cryptorchid dogs. it is also in these dogs that an unfortunate sequel of testicular torsion can occur. testicular torsion..with the exception of stallions, torsion of the testis is very rare unless there is incomplete descent. a testicular neoplasm is often also present to provide sufficient weight to maintain the torsion. the usual clinical presentation is acute abdominal pain, and the offending mass is blackened due to venous infarction and is sometimes indistinguishable as testis. spontaneous torsion of the cryptorchid testis of boars is seen commonly at slaughter. there are many publications about testicular torsion in humans and rodent models. torsion results in testicular ischemia, and reperfusion results in the release of proinflammatory cytokines by testicular macrophages, sertoli cells, interstitial endocrine cells, and germ cells and they bind to receptors on the same cell types thus having autocrine and paracrine effects.26 oxidative injury also occurs24,27 and is very important. orchitis. apart from bulls in areas endemic for brucella abortus, orchitis is a rare and sporadic disease in domesticated animals. the vast majority of cases in domestic species diagnosed clinically as orchitis are really epididymitis. foci of lymphocytes are occasionally seen in the testes of most species and suggest a subclinical disease. orchitis as the primary manifestation occurs sporadically and is reported in cats with fip, rams and bucks with corynebacterium pseudotuberculosis, pigs with brucella suis or burkholderia pseudomallei and stallions with migrating larvae of strongylus spp. nematodes. sporadic infection with other bacteria will no doubt occur from time to time. the isolated position of the testis suggests that infection with the various agents is hematogenously derived, or occurs by direct traumatic penetration. the growth of bacteria is no doubt aided by the immune privilege of the testis. experimental studies particularly of autoimmune orchitis are numerous and have helped to unravel the molecular mechanisms. the cytokines that are important in the normal regulation of the testis are also involved in immune and inflammatory reactions. as well as the direct effects of effector cells on the testicular cells, there is upregulation of chemokines such as ccl2, 3 and 4, toll-like receptors (tlr) and release of proinflammatory cytokines ifnγ, il-6 and tnfα result in germ cell apoptosis and disrupt spermatogenesis.8 developmental epididymal disease. there are many different developmental diseases that affect the epididymis, but only two that we will deal with specifically here. they are segmental aplasia and spermatic granuloma of the epididymal head. the first of these, segmental aplasia, occurs when a portion is missing. usually it is the tail of the epididymis. as can be expected, the affected side is sterile. although this occurs in all species, it is known to have a hereditary basis in bulls. 535 the second disease is called spermatic granuloma of the epididymal head. in this condition not all efferent ductules attach to the epididymal tube—some end blindly. in the blind ending ductules, spermiostasis develops and subsequently becomes a spermatic granuloma. this disease is recognized as the granulomas only occur in the region of the epididymal head. every case of azoospermia with normal testicular production of spermatozoa, based on biopsy or unilateral castration that i have investigated, had this condition. i now regard the diagnosis of testicular sperm resorption as a cause of azoospermia with great suspicion. infectious epididymitis. bacteria cause most infections of the epididymis. viruses, such as equine arteritis virus and others are reported to induce epididymitis. primary infection with brucella spp. in each species results in epididymitis. b. ovis, b. canis, b. melitensis and b. suis are especially virulent for the epididymis. it is assumed that the infection is systemic and the bacterium localizes in the epididymis. direct infection of the epididymis by penetrating injury is a rare event. secondary infection from periorchitis, or peritonitis is an occasional possibility. almost all species develop infection of the epididymis by the ascending route. this has been studied in the ram where actinobacillus seminus and histophilus somni are common isolates. while the exact mechanism is not known, the work of jansen28 in south africa during the 1980’s indicated that preputial organisms migrate to the accessory genital glands (agg) and infect the epididymis by retrograde movement. the privileged environment of the lumen of the epididymal duct allows the organisms to infect the organ and incite damage. the formation of spermatic granulomas means that the reproductive potential of the affected side is lost. complete return to normal is rare. in the dog, the sequel of self-trauma of the scrotum and systemic affects of infection with endotoxin producing bacteria such as escherichia coli further complicates epididymitis by causing systemic illness. the epididymis relies on innate immune mechanisms to prevent infection. the isolation of the epididymal duct and its narrow and long course must provide a challenge to ascending organisms. the one-way flow of spermatozoa and strong muscular contractions are also detrimental to bacterial passage. antimicrobial molecules such as tlrs are found in the rat epididymis29 and granulocyte chemotactic protein (gcp-2/cxcl6) is present in human epididymis,30 thus microbial pattern recognition molecules are present to prevent infection. the epididymis has no natural local immune system of antigen receptors, recirculation of immunocytes and no local plasma cell population. some aggregates of lymphocytes are occasionally seen in otherwise normal animals. after infection, the epididymis must develop a local immune system, but alas, the damage is usually so extensive, and the sequelae so severe that immunity is too late. even so, the epithelial cells have the ability to express mhc i and ii and lymphocytes and plasma cells can be recruited after challenge.5 spermatic cord the spermatic cord is composed of the deferent duct (ductus deferens), pampiniform plexus, muscle and nerve. there are many diseases that affect this area. the principal ones we will deal with are varicocele and inguinal hernia. when palpating the region of the spermatic cord and the inguinal ring, all of the structures of this area can usually be identified. the deferent duct may have indications of a previous vasectomy, the lymph node may be enlarged with lymphoma, and a large inguinal hernia will be palpated as a sack-like structure that may contain intestines. it is particularly important in the horse to palpate the scrotum of animals that have colic. varicocele is a disease that particularly affects old rams. it can be a cause of subfertility of both testes because of the effects of reduced blood flow that is involved with the formation of varicocele. the bilateral nature of the effects suggests that a failure to thermoregulate is probably simplistic. varicocele is a common disease in humans31 where up to 15% percent of individuals are affected. most are on the left side because the left testicular vein empties into the left renal vein at a right angle, rather than obliquely into the caudal vena cava as occurs in the right testicular vein. the higher pressure in the left 536 testicular vein combined with less effective venous valves results in dilation of the vein and varicocele. except in the ram, where varicocele is more common, most cases of varicocele in animals occur secondary to scaring or other obstruction of the testicular veins. varicocele is usually only recognized in domestic animals when thrombosis develops. there is considerable discussion about the effects of varicocele on fertility. in humans, varicocele can cause failure of testicular development and if the condition develops after puberty, infertility. infertility is thought to be the result of increased scrotal temperature, greater oxidative stress and reduced blood flow resulting in greater fas ligand activity32 which is involved in apoptosis of germ cells. the semen of men with varicocele contains higher levels of oxidants and reduced levels of antioxidants compared to that of unaffected individuals.33 intersititial cell dysfunction from interstitial fibrosis may also be involved. accessory genital glands the agg include the ampullae, vesicular glands (seminal vesicles), prostate, and bulbourethral glands. they are frequently overlooked during necropsy examination, unless there are specific indicators of disease. diseases of the agg are sporadic in most of the species and tend to be incidental findings. disease of the prostate of the dog, and vesicular glands (seminal vesicles) of bulls, are common and important. prostatic disease in the dog the prostate is the only genital gland of the dog and it is prone to diseases that can be difficult to differentiate. the gland is relatively inaccessible and signs of prostatic disease are not always specific to that organ. prostatic hyperplasia. the prostate gland has been the focus of much research, especially in the area of prostatic hyperplasia. canine prostates progressively enlarge with age. a prepubertal dog has a very small prostate, and with puberty, it increases in size to 'normal'. the size tends to reflect body size although the scottish terrier is reported to have a prostate that is about four times the size of dogs of a similar size.34 about 63% of dogs develop progressive enlargement of the prostate with age after puberty.34 the gland can become large enough to cause clinical signs or fecal obstruction rather than urinary obstruction (as occurs in humans). the enlarged prostate hangs over the pelvic brim when the dog is in its normal quadrapedal position, but when the position for defecation is assumed, the prostate is pulled by gravity into the pelvic inlet and with an increase in intraabdominal pressure, forms a 'ball valve' and compresses the colon. the increase in size is reported to be the result of increased interstitial tissue with the overall amount of epithelium not increasing in amount. the lumen of glands, however, increases in diameter with age.35,36 shain and boesel37 examined the androgen receptor content of canine prostates and found that hyperplastic glands had a higher receptor content than normal, but there was no difference in the receptor content per cell and there was no increase in testosterone. moore,et al.38 found that estradiol enhanced androgen effects on the prostate by enhancing an androgen binding protein in the cytoplasm. castration of juveniles results in a failure of prostatic development and reduces the number of prostatic diseases to one–carcinoma (see below). squamous metaplasia will cause a prostate to be larger. it occurs after administering estrogens and in sertoli cell tumors. in all animals, paracrine signals from the stroma define the epithelial type that will develop. it will also define the arrangement of the epithelium, including whether the epithelium will form a gland or a cavity. this whole interaction involves epithelial feedback and regulation (reviewed by cunha, et al.39). steroid hormones regulate this interaction and in the normal prostate, ar is an important factor. in estrogen-induced squamous metaplasia, estrogen stimulates estrogen receptor (er)α, which is especially important in the stroma (the main er of the epithelium is erβ). in er knockout mice, both stromal erα and epithelial erα must be present and stimulated to induce both proliferation and metaplasia. the stratified squamous cells acquire cyclooxygenase 2 activity as a result of estrogen stimulation. 537 prostatitis. prostatitis is a common finding, even in asymptomatic dogs.40 it is reported to occur in canine brucellosis.41 many dogs have foci of inflammatory cells in the interstitial tissues,35 suggesting that subclinical infection may be common. ten percent of dogs had these at six months but 45% had them at seven years of age. sequential studies of prostatitis are lacking, so much is assumed from studies in other animals. it is assumed that prostatitis occurs mostly by ascending infection—organisms travel from the penis and prepuce via the urethra to the prostate. the bacteria isolated from cases of prostatitis include escherichia coli, proteus, klebsiella, staphylococcus, and streptococcus sp., but there is little information on prevalence. hematogenous spread and localization in the prostate is probably the way that infection with brucella canis reaches the prostate, but infection from epididymitis is also possible. there is also the theoretical possibility of infection of the prostate from the bladder and urine. once bacteria infect the prostate, they proliferate within the lumen of the glands, and from there either elicit an inflammatory response or invade. thus there is an acute intraacinar or glandular phase and later an interstitial phase. neutrophils and the contents of their granules contribute to the chemotactic factors of the bacteria to incite an inflammatory response. bacteria are usually gram negative and therefore release endotoxin. with time, the interstitial response will become more obvious, and lymphocytes and plasma cells predominate. this interstitial phase will also be accompanied with fibrosis. abscessation of the prostate is also an outcome of prostatitis. paraprostatic cysts and pseudocysts. the most common 'cyst' attached and external to the prostate is the paraprostatic pseudocyst. some can be 30 cm in diameter or larger and form a spaceoccupying mass in the abdomen.42 it is not known how or from what they arise. some cysts, particularly the smaller ones, are lined by epithelium and they may communicate with prostatic cysts and acini. many prostates have projections of glands from the prostate into and through the capsule—they extend between bundles of the discontinuous smooth muscle of the capsule. many paraprostatic cysts are therefore an extension of cysts in prostatic hyperplasia. other possible origins for paraprostatic cysts are the serosal inclusion cyst and hematomas. those that are in the dorsal midline region of the prostate are probably derived from a cystic uterus masculinus, a remnant of the paramesonephric duct. the structure of the cysts make determining their origin impossible as the inner lining is seldom lined by epithelium. the term pseudocyst is more precise in this instance. the lining is often either fibrous or granulation tissue with an appearance similar to a 'seroma'. the wall is usually of compressed fibrous tissue and the outer lining is mesothelium (serosa). it is not unusual for the pseudocysts to have mineralization of the wall, although the literature is scant.43,44 prostatic neoplasia. prostatic carcinoma is a term with several meanings. the convention is that carcinomas of the prostate are actually adenocarcinomas (from the prostate glandular tissue), and although this is reasonable in humans, it is not necessarily the case in dogs. there are several types of carcinoma in dogs, including adenocarcinoma (presumably from the glands), transitional cell carcinoma (from the prostatic ducts), mixed carcinomas and squamous cell carcinomas. there is disagreement as to which is the most common and this is because subclassifying carcinomas is subjective. prognostically, there is little difference as virtually every dog develops metastasis.45 prostatic carcinomas occur in sexually intact and neutered dogs.46,47 carcinomas in castrated dogs tend to have pulmonary metastasis when diagnosed and transitional cell differentiation is more common than adenocarcinoma. intact dogs have an equal prevalence of transitional cell and glandular differentiation.34,48 prostatic carcinomas occur in sexually intact and neutered dogs.46,47 tumors in castrated dogs tend to have pulmonary metastasis when diagnosed. those carcinomas with transitional cell differentiation tend to occur more commonly in castrated dogs whereas neutered dogs are evenly split between those with and those without transitional differentiation.34,48 the phenotype of carcinomas in the prostate was debated for many years. attempts to separate them morphologically,49 using immunohistochemistry for cytokeratin 7 and arginine esterase,50 and 538 protein expression profiling51 were fruitless as prostatic glandular epithelium and normal bladder epithelium are identical in their expression. it seems reasonable to call all carcinomas of the prostate the generic term 'carcinoma of the prostate' or 'prostatic carcinoma'. a diagnosis of prostatic carcinoma in a dog carries an extremely poor prognosis. metastasis to the lung is most common but about 14% have metastasis to bone, especially the lumbar vertebrae and pelvis.45,48,52 there are no known causes, although there is a slightly increased risk in castrated dogs.47 as already indicated, castration is not protective so the systemic hormonal environment does not appear to be important. lai, et al.53 found that androgen receptors were less common in neoplastic prostatic tissue than in normal prostate. positive staining was in the cytoplasm rather than the nucleus and there were no detectable mutations to dna coding for the ar. there are numerous studies outlining the distribution of ers in canine prostates, and the intranuclear receptors are found in stromal cells and in the epithelial cells. hyperplastic prostates have a similar distribution. grieco, et al.54,55 found a loss of ers in the stromal of all prostates including those with carcinoma. the main er in the epithelium is erβ. in the more 'differentiated' tumors, er was present but with less intense staining. staining was reduced with the less differentiated tumors. the significance of this is yet to be established. gallardo, et al.56 examined ar, er α and β, and progesterone receptor expression in prostatic specimens from dogs with prostatic hyperplasia, prostatitis and neoplasia. expression of these was less common in disease except for progesterone receptor, where more animals had expression in disease conditions. it is widely believed that precursor lesions, such as low grade prostatic intraepithelial neoplasia, do not occur in dogs, although matsuzaki, et al.57 believe they do. high grade prostatic intraepithelial neoplasia however is reported in clinically normal dogs58 and in dogs with carcinoma.59,60 some prostatic carcinomas have cytogenetic abnormalities and of particular interest is one with an abnormality similar to human prostatic carcinoma. the carcinoma studied had trisomy of chromosome 13, which is similar to chromosome 8 of humans and which also has cytogenetic anomalies in some human prostatic carcinomas.61,62 vesicular adenitis (seminal vesiculitis) in bulls vesicular adenitis occurs commonly as a subclinical disease in ruminant species. in bulls it is recognized as an important clinical disease and a cause of infertility and poor freezibility of semen. it is a disease of young bulls predominantly—those less than two years of age. the pathogenesis has not been proven, but it is commonly believed that hematogenous infection is important. this would certainly be the case with brucella abortus or mycoplasma bovis infection. abnormalities of the seminal colliculus are implicated also. ascending infection in a similar mechanism to the pathogenesis of epididymitis in rams28 seems to have been overlooked by many. arcanobacterium pyogenes and histophilus somni are common isolates in north america63 but many bacteria and some mycoplasmas were recovered from clinical cases.64-66 two forms of vesicular adenitis in the bull are recognized, an acute fibrinopurulent form where there are the typical signs of acute inflammation—swelling, pain on palpation and neutrophils in the semen. some bulls may have systemic signs. a chronic interstitial form is the second type and there is a considerable increase in size, excessive fibrosis, firm consistency and loss of lobulation.64-66 penis and prepuce diseases of the penis and prepuce are common in all species. many of the congenital or developmental diseases are part of the dsd. congenitally short penis, lack of sigmoid flexure in ruminants, hypospadias, deviations and other obvious and less obvious anomalies may be seen from time to time. the epithelium of the penis and prepuce are fused until puberty, when there is separation of the epithelium. failure of complete separation will result in a persistent membrane (called a persistent preputial band) or frenulum that may cause deviation of the penis when it is erect. trauma and related diseases67 occur in all species but eversion of the preputial mucosa is very important and common in bulls of the bos indicus and in polled breeds of the bos taurus species. 539 eversion is normal but inadequate preputial muscles and injuries to the everted epithelium are the cause. trauma and desiccation lead to edema, inflammation, and persistent preputial prolapse. mating injuries occur in dogs and horses especially, and the constricting effects of hair (and ‘hair ring’) occur in cats, sheep and goats. forced deviation of the penis (hematoma of the penis, broken penis) is a disease of bulls mostly. deviation of the penis during coitus causes the extremely high pressure generated by the coital thrust to result in rupture of the penis at the level of the insertion of the retractor penis muscle. a hematoma develops and it may be large enough to cause hypovolemic shock. most are not so immediately lifethreatening and will heal with scarring and phimosis.68 paraphimosis is a complication of tranquilizing horses, especially if phenothiazines are used. it also occurs in debilitated horses.69 affected horses are unable to retract the penis, and engorgement and trauma result. dogs frequently develop paraphimosis as a ‘spontaneous’ lesion. most species, but especially cats and ruminants, develop urolithiasis and the stones lodge in the penile urethra. penile necrosis, and ‘water belly’ with bladder and/or urethral rupture, occurs. inflammation and or infection of the prepuce (posthitis) mostly occur as a nonspecific event. inflammation of the penis and prepuce is called balanoposthitis but should more accurately be called phalloposthitis except in the dogs where the head of the penis is the portion of the penis from the bulb to the tip; balanoposthitis is the appropriate term. as with any external site, the prepuce has a normal flora that contains potential pathogens. sexually transmitted organisms are usually found in the prepuce and may not be a cause of disease in males. organisms of importance include the herpesviruses, mycoplasmas and ureaplasmas of most species. specific agents include tritrichomonas foetus, campylobacter fetus, corynebacterium renale and ureaplasma diversum in bulls, and eubacterium suis in pigs. a nonspecific preputial discharge is seen frequently in male dogs. geldings tend to extrude their penis less often than stallions, and they urinate in their sheath. the buildup of smegma and or the effect of urine allow secondary organisms to flourish. outbreaks of posthitis are recorded in wethers, and rarely in rams. there is a combination of factors in this disease. the animals are usually on a high protein diet thus producing abundant urea. the causative agent is corynebacterium renale, a urease-producing organism that breaks down urea to ammonia. it is believed that the ammonia causes ulceration adjacent to the preputial orifice. further damage and infection results in a severe posthitis then preputial obstruction, scarring and eventually death.70,71 boars develop preputial diverticulitis with ulcers and abundant necrotic debris. canine transmissible venereal tumor (ctvt) in endemic areas is common, and diagnosis of masses around the prepuce can usually be made on either fine needle aspiration or incision or excisional biopsy. canine transmissible venereal tumor was the first naturally occurring neoplasm that is spread by the transferring of cells from an affected dog to another. it happens to be venereally transmitted. the second neoplasm to be spread in a similar fashion is the oral facial tumor of tasmanian devils, called devil facial tumor disease. they are therefore allographs. there are several reviews written about the ctvt.72-74 the cells of ctvt have a chromosomal number of 57, 58 or 59. murgia, et al.75 used molecular techniques to demonstrate that neoplasms from different continents and collected decades apart are clonal, and, while there are two subtypes, they have a common origin. the dna of the ctvt is closely related to dna of wolves and east asian dog breeds. the cells of a ctvt are able to avoid detection by the immune cells. they downregulate class i molecules and there is no class ii activity because they secrete inhibitory cytokines (tgfβ1 and il 6).75,76 in the initial proliferative phase of ctvt, they express little mhc class i or ii. after about 12 weeks in an experimental model, lymphocytes stimulated mhc expression and subsequent regression of the tumors.77 most transmissible venereal tumors are exquisitely sensitive to vincristine. squamous cell carcinoma of the penis of the horse is a relatively common condition of older animals. the head of the penis is mostly affected, and about 10% metastasize to the superficial inguinal lymph nodes.78,79 the cause is not known and smegma is no longer considered a likely candidate. 540 young bulls commonly develop fibropapillomas. the papilloma virus, bovine papilloma virus type 2, causes these exophytic lesions. they often occur during the first mating season, and they are fleshy lesions that become ulcerated and cause hemorrhage and pain.80 table 1. some of the known causes of testicular degeneration in mammals, including rodents. advancing age chlorinated naphthalenes epididymitis chemicals chemotherapy halogenated compounds including hexachlorophene nitrogen containing compounds including benzimidazoles nitrofurans heat hormones dexamethasone estrogen testosterone zeranolone metal compound toxicity neoplasia pituitary tumors sertoli cell tumors nutritional disorders negative energy balance fatty 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(http:// creativecommons.org/licenses/by-nc/4.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 10399, http://dx.doi.org/10.58292/ct.v16.10399 review report genomic selection in dairy cattle: impact and contribution to the improvement of bovine fertility* kent weigel, agustin chasco, hendyel pacheco, anil sigdel, fiona guinan, megan lauber, paul fricke, francisco peñagaricano department of animal & dairy sciences, university of wisconsin, madison, wi, usa abstract genomic selection has revolutionized the dairy cattle breeding industry, with ripple effects that have greatly impacted dairy herd management. rate of genetic progress has increased markedly, especially in holstein and jersey breeds, for production, health, and fertility traits. genomic testing of young bulls and heifers provides greater accuracy of selection decisions involving traditional fertility traits, such as daughter pregnancy rate, while creating the opportunity to improve novel traits, such as fetal loss. cameras, wearable sensors, and other precision livestock farming technologies will allow selection for traits such as estrus duration and intensity that require high frequency phenotyping. at the same time, synergies between genomic testing and advanced reproductive technologies have led to rapid and widespread adoption of sexed semen, coupled with mating of females whose offspring are not needed as herd replacements to beef sires. this strategy produces added-value crossbred calves for the beef supply chain, while allowing genetically inferior mature cows that are still producing at a high level to remain in the herd for additional lactations. keywords: fertility, genomic testing, selection impact of genomic selection on dairy cattle improvement programs1 genomic selection using tens of thousands of single nucleotide polymorphism markers dispersed across the bovine genome has transformed dairy cattle breeding programs over the past 15 years, due to commercial availability and widespread adoption of inexpensive genomic testing using dna microarrays. this technology was first used by artificial insemination (ai) companies to assess the genetic merit of young bulls and potential embryo transfer or in vitro fertilization donors, as well as by pedigree breeders who wished to sell calves, heifers, and cows with high breeding values for production and conformation traits at a premium price. these efforts were highly successful in increasing the reliability of genetic predictions for young bulls and heifers, relative to pregenomic era, because the council on dairy cattle breeding (cdcb; bowie, md) very rapidly incorporated genomic data from commercial testing laboratories into routine national genetic evaluations for dairy cattle. the immediate impact was a decrease in generation interval, particularly in the ‘sires of bulls’ and ‘dams of bulls’ pathways, where generation * presented at the 2023 society for theriogenology conference, published after peer review. intervals were reduced from ~ 7.5 and 4.5 years, respectively, to 2.5 and 2.0 years (figure 1). average generation interval in the ‘sires of cows’ pathway also declined, albeit at a slower rate, from ~ 7.0 years to 4.5 years (figure 1). because generation interval is in the denominator of the ‘breeders equation’ for calculating rate of annual genetic progress that features genetic variation, accuracy, and selection intensity in the numerator, average genetic gain per year in key traits such as protein yield accelerated with the switch to genomic selection (figure 2). annual genetic change in female fertility, as measured by daughter pregnancy rate (a function of days open), declined from 1975 to 2000. this downward trend stalled between 2000 and 2010 and began to reverse after genomic selection began in 2009 (figure 2), indicating genomic selection can yield an increase in the rate of genetic improvement for female fertility and augment phenotypic gains achieved through improved reproductive management practices. increased genetic progress by genotyping potentially elite young bulls and heifers represented an early win for the applications of genomic technologies in dairy cattle improvement programs. fifteen years prior to the advent of genomic selection, dairy cattle breeders realized that selection for increased revenue mailto:kent.weigel@wisc.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10399 2 citation line: clinical theriogenology 2024, 16, 10399, http://dx.doi.org/10.58292/ct.v16.10399 figure 1. average generation intervals by year of birth for us holstein bulls and cows from 1975 to present; dashed vertical line indicates when genomic selection was implemented, adapted from guinan et al: 2023.1 figure 2. average predicted breeding value (pbv) by year of birth for us holstein bulls for protein yield and daughter pregnancy rate from 1975 to present; dashed vertical line indicates when genomic selection was implemented, adapted from guinan et al: 2023.1 figure 3. relative economic weights for production and fitness traits in the predicted difference (pd$) and net merit (nm$) indices used by us dairy farmers from 1971 to present. fitness traits include productive life (pl), somatic cell score (scs), body size/weight composition (bsc/bwc), udder composite (udc), foot and leg composite (flc), daughter pregnancy rate (dpr), sire calving ease (sce), daughter calving ease (dce), calving ability (ca$), heifer conception rate (hcr), cow conception rate (ccr), livability (liv), producer-reported health disorders (hth$), residual feed intake (rfi), early first calving (efc), and heifer livability (hliv). negative values indicate that lower values of a given trait are preferable, adapted from vanraden et al: 2021.2 http://dx.doi.org/10.58292/ct.v16.10399 citation line: clinical theriogenology 2024, 16, 10399, http://dx.doi.org/10.58292/ct.v16.10399 3 per cow, in the form of higher production traits such as milk, fat, and protein yields, was not the optimal way to improve farm profitability. genetic evaluations of physical conformation, such as udder composite and foot and leg composite, had been available for decades, but no direct measures of fitness were part of the breeding goal until 1994 (figure 3). in that year, national genetic evaluations for somatic cell score and length of productive life were provided to us dairy farmers, and the latter allowed indirect selection for improved reproductive performance through its favorable genetic correlations with traits such as conception rate and days open. a direct measure of female fertility, namely daughter pregnancy rate, was added to the breeding goal in 2003, as was calving ability that included direct and maternal aspects of calving ease and stillbirth rate. two more direct measures of female fertility, cow conception rate and heifer conception rate, were added into the breeding objective in 2014. it is important to note that some other countries, particularly those in scandinavia that had national veterinary recording systems, implemented national genetic improvement programs for traits such as fertility, calving ability, and mastitis resistance long before these traits received substantial attention in north america. although genetic evaluations for many of the fitness traits were introduced after genomic testing became available, most of them could have been implemented without genomics. that is not the case for traits such as feed utilization efficiency or enteric methane emissions, which require expensive measurement equipment and are too difficult and expensive to record routinely on commercial farms. data from research farms can be combined with genomic testing information from the same animals, allowing implementation of national genomic evaluations for novel traits that cannot be measured on commercial farms. this strategy of enlisting research farms or contract herds with specific data capture technologies can also be used for novel measures of specific aspects of female fertility. impact of genomic testing on dairy herd management initially, genomic testing was used only for potentially elite animals that were expected to be superior based on their pedigrees, performance, or progeny. the ai companies tested their current and prospective bulls immediately, as well as most current and prospective donor dams, but the impact of genomics on commercial dairy farms was limited to indirect gains conferred by increased merit of the bulls represented in their semen tanks. that changed between 2015 and 2020, when farmers began aggressively using semen from beef bulls to mate excess females, which needed a pregnancy to initiate the next lactation but were genetically too inferior to be used as dams of the next generation of replacement heifers. this practice allowed mature cows whose time had passed, genetically speaking, to stay in the herd for additional lactations if they were still producing milk at a high level. in addition, the resulting crossbred calves brought added value upon entering the beef supply chain, relative to the purebred calves that would have been created otherwise, especially in the jersey breed.3 by 2021 (figure 4), majority of first and second inseminations of yearling heifers involved sexed holstein semen and, although many first and second inseminations of young cows still relied on conventional holstein semen, a rapid shift toward beef semen was observed in the repeat inseminations of older cows. market for conventional dairy semen has become relatively small, as the proportions of inseminations to sexed dairy semen and conventional beef semen continue to increase. potential advances in improvement of female fertility although selection for improved female fertility using national genetic evaluations for daughter pregnancy rate, heifer conception rate, and cow conception rate can help stem the decline in reproductive performance observed in previous decades, these traits can be influenced heavily by environmental and management factors, such as length of the voluntary waiting period, presence or absence of heat abatement systems, and hormonal synchronization protocols. at the same time, marked phenotypic improvements in reproductive performance have been achieved through advances in reproductive management practices, specifically hormonal synchronization of estrus and ovulation, followed by timed ai.5 many commercial dairy farms today are achieving levels of reproductive success that would have been unimaginable 20 years ago, but these programs bear short-term costs of additional labor and supplies, and they present a long-term risk of consumer backlash. one aspect of female fertility that has eluded direct genetic selection thus far is pregnancy loss. pregnancy losses during the first 42 days of pregnancy are typically referred to as embryonic loss, whereas pregnancy losses after 42 days of pregnancy are commonly known as fetal loss. rates of embryonic loss are typically higher, around 25 to 40%, than rates of fetal loss that usually range from 8 to 14%.6 however, because fetal losses occur later in pregnancy, their economic impact is much greater, and fetal loss is a heritable trait that could be considered in selection programs. recently, genetic parameters were estimated7 for fetal loss in us holstein cattle by applying linear and threshold models to data from nulliparous heifers and primiparous and multiparous lactating cows that were confirmed pregnant on day 42 after an insemination event (figure 5). fetal loss phenotypes were considered as binary (0 = pregnancy maintained, 1 = pregnancy lost after day 42) or ordinal (0 = pregnancy maintained, 1 = pregnant on day 42 but nonpregnant on day 150, 2 = pregnancy lost after day 150). heritability estimates were low in nulliparous heifers, but threshold model estimates for the binary phenotypes were 8 and 16%, respectively, in primiparous and multiparous cows, indicating the potential for improvement through genetic selection. opportunities may also exist to select for estrous behavior, duration, or intensity, which could in turn improve insemination and conception rates. it is known that high-producing dairy cows tend to have shorter estruses than low-producing cows or yearling heifers. for example, it has been reported8 that cows producing 46.4 ± 0.4 kg milk/day had estruses of shorter duration (6.2 ± 0.4 hours) than cows producing 33.5 ± 0.3 kg milk/day (10.9 ± 0.7 hours), with fewer standing events (6.3 ± 0.4 versus 8.8 ± 0.6, respectively). highproducing cows also had lower intensity of estrus, as measured by the number of standing events per hour, than low-producing cows, such that 53.4% of high-producing cows that estruses characterized as short duration / low intensity (< 8.7 hours and < 2.7 standing events per hour), 23.3% had estruses characterized as long duration / low intensity (≥ 8.7 hours and < 2.7 standing events per hour), and 15.8% had estruses characterized as short duration / high intensity (< 8.7 hours and ≥ 2.7 standing events per hour). estruses that are http://dx.doi.org/10.58292/ct.v16.10399 4 citation line: clinical theriogenology 2024, 16, 10399, http://dx.doi.org/10.58292/ct.v16.10399 short in duration or low in intensity make visual heat detection very challenging, and they can lead to missed estrus events when using pedometers or accelerometers for automated estrus detection. this can, in turn, lead to greater reliance on hormonal synchronization and timed ai programs, which are highly effective but may not be viewed favorably by consumers of dairy products. portfolio of tools available for reproductive management of lactating dairy cows have been reviewed5,9 with specific focus on the use of data from wearable sensors, noting that these systems provide the opportunity to study associations estrus duration, estrus intensity, and pregnancy outcomes. data from a large commercial farm with an accelerometer system were used10 to quantify estrus duration, as well as activity strength (intensity of the increase in activity) and rumination strength (intensity of the decrease in rumination) during estrus, with the latter phenotypes measured relative to a cow’s baseline activity and rumination levels prior to estrus. increases in probability of pregnancy by lactation number and semen type, according to activity strength at the time of insemination are described (figure 6). in every scenario, cows with greater activity strength had higher conception rates, and similar results (not shown) were observed for estrus duration and rumination strength, indicating the possibility of enhancing the duration and intensity of estrus through genetic selection that could lead to less reliance on hormonal synchronization programs. while this article focuses largely on novel female fertility traits under study by our research group at the university of figure 4. relative frequencies of beef, conventional holstein, sexed holstein, and other dairy semen inseminations of yearling holstein heifers and first parity, second parity, or third and later parity holstein cows in the us from 2019 to 2021, by service number, adapted from lauber et al: 2023.4 http://dx.doi.org/10.58292/ct.v16.10399 citation line: clinical theriogenology 2024, 16, 10399, http://dx.doi.org/10.58292/ct.v16.10399 5 wisconsin-madison, other research groups have pursued different traits that may also be strong candidates for improvement through genomic selection. for example, heritability estimates of 0.31 and 0.25 for antral follicle count in lactating holstein cows and nulliparous holstein heifers, respectively, have been reported11 indicating the potential for increasing the number of healthy follicles and oocytes through selection. substantial relationship between anogenital distance and pregnancy status in a seasonal, pasture-based system, has been reported12 suggesting it may have potential as an indicator trait in genetic selection programs. positive associations between plasma concentrations of antimüllerian hormone (amh) and maintenance of pregnancy or pregnancy rate have been reported13; and candidate genes associated with amh concentration and embryo development in holstein cattle have been reported.14 thus, genomic selection can be applied to a broad array of novel fertility traits if a reference population of animals with genotypes and phenotypes can be established. potential advances in improvement of male fertility although selection programs have focused primarily on improving female fertility, significant variability also exists in the male’s contribution to pregnancy. preselection of ai bulls occurs routinely, based on ai company benchmarks for semen quantity and quality,15 so the observed differences between bulls in on-farm conception rates reflect the remaining variation in sperm quality after internal control measures have been applied. for the past 15 years, cdcb has provided sire conception rate evaluations that are phenotypic (rather than genetic) predictions of fertility of semen from individual bulls after accounting for accounting for known environmental and management factors.16 farmers can use this information when purchasing semen, and ai companies can monitor these data to identify or confirm challenges with the semen of individual bulls at a given time. furthermore, it has been reported17 that sires with low versus high evaluations for sire conception rate differ in blastocyst development rate, as well as percentages of unfertilized oocytes and degenerated embryos, in an in vitro experiment. widespread genomic testing of us dairy cattle has facilitated rapid and highly effective investigation of inherited conditions affecting embryonic or fetal loss that can be subsequently managed using the genomic data of ai bulls. in some cases, the functional mutation can be identified, and its mode of inheritance can be deduced, whereas in other cases, only the association between a specific haplotype (haploid stretch of figure 5. heritability estimates for fetal loss in nulliparous, primiparous, and multiparous holstein cattle, using linear or threshold models and considering fetal loss phenotypes as binary or ordinal, adapted from sigdel et al: 2022.7 figure 6. predicted probability of pregnancy for cows in lactation 1, 2, or 3+ when inseminated with sexed, conventional, or beef semen, according to accelerometer-based measures of the increase in activity during estrus relative to the cow’s preestrus baseline activity, adapted from chasco et al: 2023.10 http://dx.doi.org/10.58292/ct.v16.10399 6 citation line: clinical theriogenology 2024, 16, 10399, http://dx.doi.org/10.58292/ct.v16.10399 dna carried by the sperm or egg) and a reproductive outcome can be identified. a novel approach was proposed18 to detecting genomic regions associated with impaired fertility, in which haplotypes that appear frequently in the population in heterozygous form, but for which no living homozygotes have been reported, are targeted for further investigation. with additional research, many of these putative genetic defects can been confirmed and attributed to underlying functional mutations. breed associations maintain lists of genetic variants that are known to affect fertility, coat color, polled status, and many other conditions, and these can be managed at the farm level using computerized mating programs that incorporate genomic data. research aimed at understanding the underlying genetic basis of observed differences in male and female fertility continues at a rapid pace. recently, genomes of 1,102 italian brown swiss bulls using high-density genotypes (454,556 single nucleotide markers per bull) were scanned19 and it was detected that regions on chromosomes 6 and 26 that were strongly associated with sire conception rates and seemed to indicate nonadditive inheritance. bulls that were homozygous for the unfavorable allele at each location had significantly (p  < 0.05) poorer male fertility than their contemporaries (figure  7). widespread genomic testing in cattle and other farm animals, coupled with advances in statistical models and machine learning algorithms, will allow continued advances in our understanding of the roles of specific genetic and epigenetic factors associated with male and female fertility in the future. conclusion genomic selection of dairy cattle has led to monumental changes in dairy cattle breeding programs, especially when coupled with advanced reproductive technologies. widespread genomic testing and advanced reproductive technologies have led to more rapid genetic progress for most economically important traits, and new opportunities have arisen regarding selection for novel phenotypes that are too difficult to measure on commercial farms, as well as those that can be captured electronically using cameras, accelerometers, and other sensor technologies. farmers have shifted quickly to coupling sexed dairy semen with beef semen for generating high-merit replacement heifers for their dairy operations and added-value crossbred calves for the beef supply chain. together, genome-based selection tools and precision livestock farming technologies will continue to aid in the improvement of reproductive performance on dairy farms and the sustainability of our dairy industry. conflict of interest authors declare no conflict of interest. references 1. guinan fl, wiggans gr, norman hd, et al: changes in genetic trends in us dairy cattle since the implementation of genomic selection. j dairy sci 2023;106:1110-1129. doi: 10.3168/ jds.2022-22205 2. vanraden pm, cole jb, neupane m, et al: net merit as a measure of lifetime profit: 2021 revision. aip research report nm$8 (05–21). available from: https://www.ars.usda.gov/arsuserfiles/80420530/ publications/arr/nmcalc-2021arr-nm8.pdf [cited 16 august 2023]. 3. berry dp: invited review: beef-on-dairy—the generation of crossbred beef × dairy cattle. j dairy sci 2021;104:3789-3819. doi: 10.3168/jds.2020-19519 4. lauber mr, peñagaricano f, fourdraine rh, et al: characterization of semen type prevalence and allocation in holstein and jersey females in the united states. j dairy sci 2023;106:3748-3760. doi: 10.3168/jds.2022-22494 5. giordano jo, sitko em, rial c, et al: symposium review: use of multiple biological, management, and performance data for the design of targeted reproductive management strategies for dairy cows. j dairy sci 2022;105:4669-4678. doi: 10.3168/jds.2021-21476 6. wiltbank mc, baez gm, garcia-guerra a, et al: pivotal periods for pregnancy loss during the first trimester of gestation in lactating dairy cows. theriogenology 2016;86:239–253. doi: 10.1016/j. theriogenology.2016.04.037 7. sigdel a, bisinotto rs, peñagaricano f: genetic analysis of fetal loss in holstein cattle. j dairy sci 2022;105:9012-9020. doi: 10.3168/jds.2022-22000 8. lopez h, satter ld, wiltbank mc: 2004. relationship between level of milk production and estrous behavior of lactating dairy cows. anim reprod sci 2004;81:209-223. doi: 10.1016/j. anireprosci.2003.10.009 figure 7. sire conception rates of italian brown swiss bulls carrying 1 or 2 copies of the undesirable allele at single nucleotide polymorphism loci on chromosomes 6 (left panel) and 26 (right panel), adapted from pacheco et al: 2022.19 http://dx.doi.org/10.58292/ct.v16.10399 https://doi.org/10.3168/jds.2022-22205 https://doi.org/10.3168/jds.2022-22205 https://www.ars.usda.gov/arsuserfiles/80420530/publications/arr/nmcalc-2021arr-nm8.pdf https://www.ars.usda.gov/arsuserfiles/80420530/publications/arr/nmcalc-2021arr-nm8.pdf https://doi.org/10.3168/jds.2020-19519 https://doi.org/10.3168/jds.2022-22494 https://doi.org/10.3168/jds.2021-21476 https://doi.org/10.1016/j.theriogenology.2016.04.037 https://doi.org/10.1016/j.theriogenology.2016.04.037 https://doi.org/10.3168/jds.2022-22000 https://doi.org/10.1016/j.anireprosci.2003.10.009 https://doi.org/10.1016/j.anireprosci.2003.10.009 citation line: clinical theriogenology 2024, 16, 10399, http://dx.doi.org/10.58292/ct.v16.10399 7 9. cerri rla, burnett ta, madureira aml, et al: symposium review: linking activity-sensor data and physiology to improve dairy cow  fertility. j dairy sci 2021;104:1220-1231. doi: 10.3168/ jds.2019-17893 10. chasco ja, chebel rc, weigel ka, et al: estrus expression in dairy cows: phenotyping, genetic variability, and association with reproductive performance. j dairy sci 2023;106 (suppl. 1):53. 11. walsh sw, mossa f, butler st, et al: 2014. heritability and impact of environmental effects during pregnancy on antral follicle count  in cattle. j dairy sci 2014;97:4503-4511. doi: 10.3168/ jds.2013-7758 12. grala tm, price md, kuhn-sherlock b, et al: investigating anogenital distance and antral follicle count as novel markers of fertility within a herd of cows with positive or negative genetic merit for fertility traits. j dairy sci 2021;104:12939-12952. doi: 10.3168/ jds.2020-19948 13. ribeiro es, bisinotto rs, lima fs, et al: plasma anti-müllerian hormone in adult dairy cows and associations with fertility. j dairy sci 2014;97:6888-6900. doi: 10.3168/jds.2014-7908 14. gobikrushanth m, purfield dc, colazo mg, et al: the relationship between serum anti-müllerian hormone concentrations and fertility, and genome-wide associations for anti-müllerian hormone in holstein cows. j dairy sci 2018;101:7563-7574. doi: 10.3168/jds.2017-13940 15. harstine b, utt m, dejarnette jm: review: integrating a semen quality control program and sire fertility at a large artificial insemination organization. animal 2018;12:s63-s74. doi: 10.1017/ s1751731118000319 16. kuhn mt, hutchison jl: prediction of dairy bull fertility from field data: use of multiple services and identification and utilization of factors affecting bull fertility. j dairy sci 2008;91:24812492. doi: 10.3168/jds.2007-0743 17. ortega ms, moraes jgn, patterson dj, et al: influences of sire conception rate on pregnancy establishment in dairy cattle. biol reprod 2018;99:1244-1254. doi: 10.1093%2fbiolre%2fioy141 18. vanraden pm, olson km, null dj, et al: harmful recessive effects on fertility detected by absence of homozygous haplotypes. j dairy sci 2011;94:6153-6161. doi: 10.3168/ jds.2011-4624 19. pacheco ha, rossoni a, cecchinato a, et al: deciphering the genetic basis of male fertility in italian brown swiss dairy cattle. sci rep 2022;12:10575. doi: 10.1038/s41598-022-14889-1 http://dx.doi.org/10.58292/ct.v16.10399 https://doi.org/10.3168/jds.2019-17893 https://doi.org/10.3168/jds.2019-17893 https://doi.org/10.3168/jds.2013-7758 https://doi.org/10.3168/jds.2013-7758 https://doi.org/10.3168/jds.2020-19948 https://doi.org/10.3168/jds.2020-19948 https://doi.org/10.3168/jds.2014-7908 https://doi.org/10.3168/jds.2017-13940 https://doi.org/10.1017/s1751731118000319 https://doi.org/10.1017/s1751731118000319 https://doi.org/10.3168/jds.2007-0743 https://doi.org/10.1093%2fbiolre%2fioy141 https://doi.org/10.3168/jds.2011-4624 https://doi.org/10.3168/jds.2011-4624 https://doi.org/10.1038/s41598-022-14889-1 2009: investigation of ovulation induction in alpacas with acupuncture investigation of ovulation induction in alpacas with acupuncture m.a. memona,b, c.m. liua, j. rodrigueza, s. sandovala, a. tibarya,b adepartment of veterinary clinical sciences, college of veterinary medicine bcenter for reproductive biology, washington state university, pullman, wa, usa acupuncture has been used to induce ovulation in several animal species, including induced ovulators such as the rabbit. there is no published information on induction of ovulation in alpacas using acupuncture. a pilot study was conducted to test the hypothesis that acupuncture induces ovulation in alpacas. four alpacas 8-14 years old were used in a cross-over design to compare two treatments, gnrh and acupuncture, for ovulation induction during 2 follicular wave cycles. treatment with gnrh (cystorelin® 50μg/ml im, merial, duluth, ga, usa) or acupuncture was performed when the alpacas had uterine edema and an ovarian follicle 8-10mm in diameter as detected by ultrasonography (us). ovulation was confirmed using us to detect the presence of a corpus luteum on the ovary. all alpacas received cloprostenol (estrumate®, 250 μg, intervet/schering-plough animal health, kenilworth, nj, usa) intramuscularly 7 days after induction of ovulation and were subjected to the second treatment during the next follicular wave (1 to 2 weeks later). acupuncture consisted stimulating the acupoints by aquapuncture; injecting 3 ml vitamin b12 (cyanocoblamin, 1000 μg/ml, vedco, inc, st. joseph, mo, usa) at the bladder (bl) and stomach (st) acupoints (bl 22, bl 51, bl 23, bl 52, st 25), and yan-chi. the acupuncture points used in this study are utilized to treat various causes of infertility. blood samples were collected by jugular venipuncture from each animal before treatment and 7 days after treatment. sera were stored at -20°c until assayed for progesterone concentration (p4). p4 >2ng/ml was considered as evidence of ovulation and development of a corpus luteum. ovulation was induced by gnrh in all alpacas, as evidenced by detection of a corpus luteum with us and a concomitant increase in p4. none of the alpacas ovulated following acupuncture treatment. various reasons are possible for the failure of ovulation induction using acupuncture in this study. contrary to other reflex ovulators such as the rabbit where acupuncture was found to be useful for induction of ovulation, camelids do not have a neuroreflex mechanism for induction of the lh surge. an ovulation-induction factor present in seminal plasma stimulates an lh surge similar to that induced by gnrh. as expected, gnrh induced ovulation in all cases selected based on follicular size and uterine edema. the lack of response to acupuncture in the present study could be due to other factors. acupuncture was shown to cause a significant increase in ß-endorphin levels that lasts for up to 24 hours. the role of neuropeptides, including ß-endorphin, in the regulation of gnrh secretion has been reported. other acupuncture approaches need to be investigated in order to determine the usefulness of this technique in camelids. keywords: acupuncture, ovulation induction, alpaca 534 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false 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true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2019 a novel non-surgical method to reduce fertility using the rat as a model a novel non-surgical method to reduce fertility using the rat as a model kristy l. meadows,a yamin li,b qiaobing xu,b sandra ayresa acummings school of veterinary medicine at tufts university, north grafton, ma btufts university, medford, ma abstract overpopulation of pets, livestock, and some species of wildlife is a world-wide problem that could be addressed by reducing or eliminating fertility. although surgical sterilization, immunocontraceptive vaccines, and chemical castrations are available, their limitations include cost, training, equipment, efficacy, and morbidity. therefore, there is a need for a novel non-surgical method of sterilization for control of animal overpopulation. the goal of the present study was to develop a novel non-surgical sterilization technique employing a lipid-based nanocomplex carrying a cytotoxin, guided to specific gonadal cells by an antibody against anti-mullerian hormone receptor type ii. an intravenous injection of the antibody-guided lipid-nanocomplex negatively impacted aspects of reproduction in rats, including sperm production, estrous cyclicity and testicular and ovarian morphology, without affecting overall health within a 4-week interval after treatment. these results are promising initial steps toward developing a novel non-surgical method of sterilization that may change control of pet and wildlife overpopulation. keywords: non-surgical sterilization, anti-mullerian hormone, nanocomplex, nanoparticle introduction overpopulation of pets, livestock, and some species of wildlife is a world-wide problem and contributes to the spread of disease in humans, pets, and other domestic species. for example, the spread of rabies,1,2 brucellosis,3,4 leptospirosis,4,5 neospora caninum,6 toxoplasmosis,6,7 and tick-borne diseases such as lyme disease, ehrlichiosis, anaplasmosis, and babesiosis,8 have all been attributed to animal overpopulation. further, decimation of native environments and indigenous wildlife, as well as destruction of local flora and fauna can occur as a result of feral cats,9 deer,10 feral horses,11 and feral goats.12 hunting,12,13 euthanasia,14 birth control,15,16 and sterilization17,18 are currently used to control animal overpopulation. while hunting and euthanasia can be effective, preventing animals from reproducing is a better, more socially and ethically acceptable option. although there are contraceptive methods available, such as the zona pellucida vaccine,19 effects are temporary and vaccines need to be given every 2 3 years. in contrast, sterilization can permanently reduce fertility. currently, the most effective method of sterilization involves surgical removal of ovaries and testes which is expensive, requires surgical equipment and trained personnel, and has inherent risks of morbidity and mortality. therefore, there is a need for a novel non-surgical method of sterilization to control animal overpopulation. the goal of the current study was to develop a non-surgical sterilization technique employing a nanocomplex. the non-surgical technique that we have developed consists of an antibody-guided, lipid-based nanocomplex that carries a cytotoxin.20,21 this nanocomplex is guided to specific cells in the gonads by an antibody against the anti-mullerian hormone receptor type ii (amhrii). the amhrii is expressed in the adrenal glands, pancreas, and spleen (among other tissues),22 but is almost exclusively expressed within the gonads, specifically by granulosa and theca cells in the ovary, and sertoli and leydig cells in the testes. the antibody-guided nanocomplex is engineered to carry the cytotoxin saporin,23 which induces apoptosis in the above-listed gonadal cells; these “nurse cells” are essential for oocytes and sperm to develop, undergo meiosis and survive.24-33 they also produce the hormones estrogen and testosterone, which promote reproductive behavior. we hypothesize that the above described gonadal “nurse cells” are destroyed after administration of the nanocomplex, and that this cellular destruction will cause disruption in ovarian follicular development in females and spermatogonia development in males, ultimately causing infertility. clinical theriogenology • volume 11 number 1 • march 201931 preliminary data in rats collected 24 hours after injection of the nanocomplex indicated that apoptosis was occurring in these gonadal cells, particularly in males.34 the goal of the current study was to expand upon the results of our previous pilot work to determine the extent of damage in the gonads during a 4 week interval after nanocomplex injection. materials and methods experimental animals eight male and eight female, 7-week-old sprague-dawley rats were obtained from charles river laboratory (shrewsbury, ma). for 1 week prior to injections, all rats were acclimated to the animal colony to reduce non-specific stress. all rats were pair-housed in light (12:12 hour lighting schedule with lights on during working hours) and temperature (21 24°c) controlled rooms, in shoe-box type cages with corncob bedding, and enviro-dri® (biological associates, melbourne, australia) nest material for enrichment. food (2018 teklad global 18% protein rodent diet, envigo, cambridge, uk) and water were provided ad libitum. males and females were kept in the same room within the animal colony. all animals were maintained in accordance with the national research council (nrc) guide for the care and use of laboratory animals and all animal procedures were approved by the institutional animals care and use committee of tufts university (protocol numbers g2018-158 and g2018-101). nanocomplex production cationic lipidoids, cholesterol, and dioleyl phosphatidylethanolamine (dope) were mixed in a chloroform solution. after evaporating the organic solvent, the thin film formed was rehydrated with sterilized pbs and dispersed by probe sonication. phospholipids like dope are used to provide support to the lipid bilayer and to facilitate endosomal escape during cell internalization. cholesterol is relatively more hydrophobic and is able to stabilize the supramolecular structure and promote the membrane fusion process, which could enhance delivery efficiciency.35 ultrasonication was used to facilitate dispersion of the lipid mixture in aqueous solution to produce lipidoid nanoparticles, which in most cases are liposomes (vesicular structures with lipid bilayers and an aqueous interior). to the rehydrated formations, protein (saporin cytotoxin, sigma aldrich, st. louis, mo) was added in pre-optimized ratios and was complexed with lipoid nanoparticles through supramolecular interactions (hydrogen bonding, electrostatic interactions and hydrophobic interactions).20 lipidoid-protein nanocomplexes were vortexed for another 60 minutes at room temperature and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[amino (polyethylene glycol) 2000]-anti-anti-mullerian hormone receptor type ii (dspe-peg 2000-antiamhrii) was mixed and incorporated into the lipid/saporin nanocomplex for targeted protein delivery. the amhrii antibody was first covalently conjugated to the macromolecular lipid, dspe-peg 2000, through the cyanur-amine conjugation reaction, a widely used technique for protein/antibody labelling.36,37 due to its amphiphilic nature, the dspe-peg 2000-amhrii can easily incorporate into cholesterol/dope/cationic lipidoid nanoparticles, primarily through a hydrophobic interaction between the hydrophobic domain of the nanoparticle and the dioleoyl-tail of dspe-peg 2000-amhrii. the antibody was covalently conjugated onto the nanoparticles and saporin was loaded through supramolecular interactions; therefore, labeling/binding was expected to be stable, as free saporin cannot be efficiently internalized by cells. furthermore, we have used similar methods for complexation and delivery of other proteins, both in vitro and in vivo.38 nanocomplex injections eight-week-old animals were randomly allocated to one of two treatment groups: injection with either sterile saline (male n = 4, female n = 4) or nanocomplex (male n = 4, female n = 4). all injections were administered iv into the lateral tail vein, using a 24-gauge over-the-needle catheter. all test rats received 8.2 nmol of the nanocomplex in 0.1 ml of sterile saline, followed by a 0.1 ml sterile saline flush to ensure that nanocomplexes were delivered. all control rats received a 0.1 ml dose of sterile saline, followed by a 0.1 ml sterile saline flush. rats were weighed twice weekly after injections and were clinical theriogenology • volume 11 number 1 • march 2019 32 maintained in the animal colony for 4 weeks. estrous cycle beginning 2 weeks after injection, female animals were evaluated once daily (between 0900 and 1100). vaginal lavage was performed by manually restraining the rats and placing a small pipette containing 0.25 ml (maximum volume) of sterile saline into the caudal vagina. the saline was gently introduced into the vagina and immediately aspirated. each aspirate was examined using an olympus bx50 microscope (olympus corporation of the americas headquarters, center valley, pa) equipped with a photometrics coolsnap hq2 video camera (photometrics, tucson, az) interfaced with metamorph® (nashville, tn) software. tissue collection and processing rats were euthanized by co2 asphyxiation as recommended by the avma guidelines for the euthanasia of animals: 2013 edition (https://www.avma.org/kb/policies/documents/euthanasia.pdf). gonads were excised and after removing epididymides, were preserved in 10% formaldehyde for histological processing. in all males, the tail of the epididymis was removed along with approximately 6 mm of ductus deferens and placed in a 1.5 ml microcentrifuge tube containing normal saline. these samples remained at room temperature for approximately 6 hours. then,100 µl of fluid was aspirated from the tube, ~50 µl placed onto a slide and examined using an olympus bx50 microscope (olympus corporation of the americas headquarters) equipped with a photometrics coolsnap hq2 video camera (photometrics, tuscon, az) interfaced with metamorph® (nashville, tn) software. quantification of sperm cell count was performed using imagej software (nih, bethesda, md) multipoint cell counter with roi manager. two images (10 x objective + 10 x eyepiece = 100 x magnification) per subject were quantified per high-power field and mean counts used for analyses. hematoxylin and eosin staining formalin-fixed paraffin embedded gonadal tissue was sliced from the central axis of each ovary and testis (six sections from the right and left gonad of each animal were sampled), at five µm per section and mounted onto microscope slides. slides were stained with hematoxylin (gill 3 hematoxylin, statlab, mckinney, tx) and eosin (eosin y, statlab) in an automated stainer (tissue-tek, sakura finetek inc., torrance, ca) using a standard protocol. tissue preparation and staining was performed by the histopathology section at the cummings veterinary school. image production and analysis bright field images were obtained of the ovaries at 2 x magnification and the testes at 20 x magnification using an olympus bx41 microscope, with an attached olympus q-color 5 camera (olympus corporation of the americas headquarters) interfaced with qcapture (qimaging, surrey, bc, canada) software. quantification of spermatogonia and primary spermatocyte counts was performed using imagej software (nih, bethesda, md) multipoint cell counter with roi manager. twenty images per subject were quantified and mean counts were used for analysis. statistical analysis all data were analyzed using an unpaired student’s t-test. significance was defined as p < 0.05. results body weight and wellbeing all animals gained weight and appeared healthy throughout the study. as expected, male rats consistently weighed more and gained weight at a slightly faster rate than female rats.39 there were no significant differences in weight gain as a measure of overall health between nanocomplex-treated and saline-treated controls. clinical theriogenology • volume 11 number 1 • march 201933 estrous cycle nanocomplex-treated female rats had vaginal cytologies with many cells and mixed cell types (including cornified, intermediate, parabasal and white blood cells), making it difficult to determine stages of the estrous cycle (figure 1). all but one saline-treated control had vaginal cytology that indicated normal cyclicity,40,41 whereas the remaining one had constant diestrus, consistent with pseudopregnancy.42 epididymal sperm count nanocomplex-treated males had lower epididymal sperm count (figure 2) compared to salinetreated controls (unpaired t-test, t =3.062, df =14, p < 0.010) and appeared to have fewer live (motile) sperm than controls (figure 3). spermatogonia and primary spermatocyte counts spermatogonia (panel a; unpaired t-test, t =26.9, df = 158, p < 0.01) and primary spermatocytes (panel b; unpaired t-test; t =21.92, df =158, p < 0.01) counts were lower (figure 4) in nanocomplextreated rats compared to saline-treated control rats. under bright-field microscopy, spermatogonia were small, located close to the basement membrane of seminiferous tubules and often flattened on the side facing the basement membrane. primary spermatocytes were large, round cells in a layer next to spermatogonia. testis histology seminiferous tubules of nanocomplex-treated males appeared smaller than saline-treated controls (figure 5). in addition, there appeared to be atypical luminal contents (no sperm or undeveloped sperm, acellular material and immature germ cells), decreased interstitial cells, and disruption of general cohesion of cells in nanocomplex-treated males. ovary histology ovarian stroma of nanocomplex-treated females had notable hyperplasia and appeared larger than saline-treated controls, with some evidence of increased vasculature (figure 6). in nanocomplex-treated females, stromal architecture appeared mildly edematous with decreased, incohesive interstitial tissue and there appeared to be increased numbers of corpora lutea and tertiary follicles. discussion in the current study, an iv injection of an antibody-guided (amhrii antibody) lipidnanocomplex carrying the cytotoxin, saporin, negatively impacted aspects of reproduction in both male and female rats, without affecting general health and wellbeing, for 4 weeks after treatment. there were no significant effects of treatment (compared to saline-treated controls) on body weight gain in either males or females during the 4-week duration of monitoring. in contrast, in studies comparing sham-operated and surgically gonadectomized rats,39 there were differences between the two groups in weight gain. castrated males lost more weight than sham-operated males and never regained that difference in weight.39 furthermore, females had opposite results, with greater weight gain in ovariectomized rats than sham-operated controls, particularly during the first 30 days after surgery.39 an intriguing possibility is that, unlike surgical gonadectomy, the nanocomplex has not removed all support cells from the gonads at 4 weeks after treatment. this could leave behind cells still producing scant amounts of hormones, possibly accounting for the lack of differences in weight gain. overall, at this dose, the nanocomplex procedure did not appear to impact general health of animals, at least within the first 4 weeks after treatment, as indicated by similar weight gains in both groups. vaginal cytologies from nanocomplex-treated females were difficult to evaluate, as there were often many cell types in each aspirate, with no clear dominant cell type. in contrast, three of four saline treated-control females had normal estrous cycles that could be easily identified by evaluating the cell clinical theriogenology • volume 11 number 1 • march 2019 34 types on vaginal cytology.40,41 reproductively senescent, aged rats commonly have irregular estrous cycles as demonstrated by similar vaginal cytologies with many numbers of mixed cell types, presumably due to age-related dysregulation of the hypothalamic-pituitary-gonadal axis.43,44 vaginal cytologies in the current study may have been due to the nanocomplex destroying granulosa cells sufficient to interfere with normal hormone production, particularly of estrogen and anti-mullerian hormone, effectively beginning reproductive senescence. there were significantly fewer sperm in samples from nanocomplex-treated males compared to saline-treated controls. in addition, there appeared to be more motile, or live, sperm per sample in salinetreated control males. because sertoli cells are essential for sperm development and survival, 28,30-33 this significant decrease in sperm numbers was attributed to destruction of sertoli cells by the nanocomplex as hypothesized. it takes approximately 8 weeks for sperm to be produced and travel to the tail of the epididymis; therefore, some sperm may still be in the seminiferous tubules and epididymis of treated rats after 4 weeks, albeit in much lower numbers than in controls. additionally, there were significantly fewer spermatogonia and primary spermatocytes in the seminiferous tubules of nanocomplex-treated males compared to saline-treated controls. furthermore, based on histological examination, architecture was disrupted in the testes of nanocomplex-treated males. tubules appeared smaller compared to saline-treated control males, and there appeared to be less cohesion between the cells within the seminiferous tubules, with elongated spermatids mingling with spermatogonia or primary spermatocytes, rather than appearing in well-organized rings at the lumen of the tubule. based on these changes, we inferred that sertoli cells had been negatively impacted or eliminated by the nanocomplex, as these cells form “channels” in which sperm develop in an ordered fashion, progressing from spermatogonia at the base of the seminiferous tubule, to elongated spermatids or sperm at the lumen.28,31 moreover, abnormal seminiferous tubule formation with decreased numbers of spermatogonia, were also present in a rat model of testicular dysgenesis syndrome,45 suggesting that fertility was likely decreased in the current study. ovaries of treated rats also had irregular morphology, including notable hyperplasia, with decreased, incohesive interstitial tissue that appeared mildly edematous. ovarian hyperplasia with minimal interstitial tissue has been demonstrated as part of a progression toward ovarian atrophy in a rodent model of early reproductive senescence.46 additionally, the ovary-intact rodent model of menopause has similar morphologic changes to those in the current study, including retention of ovarian tissue prior to the progression to complete ovarian atrophy.43 thus, morphologic changes in the current study were consistent with onset of reproductive senescence following treatment with the nanocomplex. in conclusion, the current study demonstrated that 4 weeks after injection with amhrii antibody-guided lipid nanocomplex, fertility in rats was likely reduced, as evidenced by decreased sperm production, decreased spermatogonia and primary spermatocyte counts in males, disruptions in estrous cyclicity in females, as well as changes in both testicular and ovarian architecture. future studies are needed to extend the duration of treatment monitoring to determine impacts on fertility, especially in the male, where the testes could produce sperm from residual germ cells for up to 60 days after injection. in addition, hormonal assays are essential for a more comprehensive understanding of the effects of the nanocomplex on fertility. lastly, breeding experiments will be necessary to determine if the nanocomplex treatment has definitively reduced or eliminated fertility. results of the current study are promising initial steps toward developing a novel non-surgical method of sterilization. this technique could be refined for use as a novel rodent model of menopause and may change our approach to controlling pet and wildlife overpopulation. funding the project described was partially supported by the national center for advancing translational sciences, national institutes of health, award number ul1tr001064. the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih. clinical theriogenology • volume 11 number 1 • march 201935 acknowledgements we thank janine stuczko for her administrative oversight, frances brown for her histopathology support and drs. perry bain and nicholas robinson for their assistance with histological analyses. conflict of interest the authors have no conflicts of interest. references 1. windiyaningsih c, wilde h, meslin f, et al: the rabies epidemic on flores island, indonesia (1998–2003). j med assoc thai 2004;89:1389-1393. 2. rosatte rc, donovan d, allan m, et al: the control of raccoon rabies in ontario canada: proactive and reactive tactics 1994–2007. j wildl dis 2009;45:772-784. 3. mor sm, wiethoelter ak, lee a, et al: emergence of brucella suis in dogs in new south wales, australia: clinical findings and implications for zoonotic transmission. bmc vet res 2016;12:199-208. 4. ridoutt c, lee a, moloney b, et al: detection of brucellosis and leptospirosis in feral pigs in new south wales. aust vet j 2014;92:343-347. 5. gay n, soupé-gilbert m-e, goarant c: though not reservoirs, dogs might transmit leptospira in new caledonia. int j environ res public health 2014;11:4316-4325. 6. dubey jp, jenkins mc, kwok och, et al: seroprevalence of neospora caninum and 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hormone receptor ii protein in the postnatal rat testis from birth to sexual maturity. histol histopathol 2006;21:125-130. 25. baarends wm, hoogerbrugge jw, post m, et al: anti-müllerian hormone and anti-müllerian hormone type ii receptor messenger ribonucleic acid expression during postnatal testis development and in the adult testis of the rat. endocrinology 1995;136:5614-5622. 26. baarends wm, uilenbroek jt, kramer p, et al: anti-müllerian hormone and anti-müllerian hormone type ii receptor messenger ribonucleic acid expression in rat ovaries during postnatal development, the estrous cycle, and gonadotropin-induced follicle growth. endocrinology 1995;136:4951-4962. 27. durlinger a, visser j, themmen a: regulation of ovarian function: the role of anti-mullerian hormone. reproduction clinical theriogenology • volume 11 number 1 • march 2019 36 2002;124:601-609. 28. griswold md: the central role of sertoli cells in spermatogenesis. semin cell dev biol 1998;9:411-416. 29. hirobe s, he ww, gustafson ml, et al: müllerian inhibiting substance gene expression in the cycling rat ovary correlates with recruited or graafian follicle selection. biol reprod 1994;50:1238-1243. 30. josso n, di clemente n, gouedard l: anti-mullerian hormone and its receptors. mol cell endocrinol 2001;179:25-32. 31. hai y, hou j, liu y, et al: the roles and regulation of sertoli cells in fate determinations of spermatogonial stem cells and spermatogenesis. semin cell dev biol 2014;29:66-75. 32. dumesic da, meldrum dr, katz-jaffe mg, et al: oocyte environment: follicular fluid and cumulus cells are critical for oocyte health. fertil steril 2015;103:303-316. 33. young jm, mcneilly as: theca: the forgotten cell of the ovarian follicle. reproduction 2010;140:489-504. 34. meadows k, xu q, ayres s: a rodent model for non-surgical sterilization using an antibody-guided nanoparticle cytotoxin delivery system. clinical theriogenology 2016;8:309. 35. kauffman kj, dorkin jr, yang jh, et al: optimization of lipid nanoparticle formulations for mrna delivery in vivo with fractional factorial and definitive screening designs. nano lett 2015;15:7300-7306. 36. bendas g, krause a, bakowsky u, et al: targetability of novel immunoliposomes prepared by a new antibody conjugation technique. int j pharm 1999;181:79-93. 37. bakowsky h, richter t, kneuer c, et al: adhesion characteristics and stability assessment of lectin-modified liposomes for site-specific drug delivery. biochim biophys acta 2008;1778:242-249. 38. li y, yang t, yu y, et al: combinatorial library of chalcogen-containing lipidoids for intracellular delivery of genome-editing proteins. biomaterials 2018;178:652-662. 39. kakolewski jw, cox vc, valenstein es: sex differences in body-weight change following gonadectomy of rats. psychol rep 1968;22:547-554. 40. marcondes f, bianchi f, tanno a: determination of the estrous cycle phases of rats: some helpful consideration. braz j biol 2002;62:609-614. 41. cora mc, kooistra l, travlos g: vaginal cytology of the laboratory rat and mouse. toxicol pathol 2015;43:776793. 42. frye ca, erskine ms: influence of time of mating and paced copulation on induction of pseudopregnancy in cyclic female rats. j reprod fertil 1990;90:375-385. 43. koebele sv, bimonte-nelson ha: modeling menopause: the utility of rodents in translational behavioral endocrinology research. maturitas 2016;87:5-17. 44. finch ce: the menopause and aging, a comparative perspective. j steroid biochem mol biol 2014;142:132-141. 45. fisher js: human 'testicular dysgenesis syndrome': a possible model using in-utero exposure of the rat to dibutyl phthalate. hum reprod 2003;18:1383-1394. 46. mujoomdar ml, hogan lm, parlow af, et al: pcsk6 mutant mice exhibit progressive loss of ovarian function, altered gene expression, and formation of ovarian pathology. reproduction 2011;141:343-355. clinical theriogenology • volume 11 number 1 • march 201937 figure 1. representative image (40 x objective) of the effect of intravenous nanoparticle administration on estrous cyclicity (panel a). vaginal smears of nanoparticle injected rats displayed many cells and mixed cell types, including white blood cells (a), cornified cells (b), parabasal cells (c), and intermediate cells (d), making it difficult to determine stages of the estrous cycle definitively. panel b displays representative images of normal direct cytology for comparison. figure 2. effect of intravenous nanoparticle administration on epididymal sperm count when compared to saline injected controls (unpaired t-test, t = 3.062, df = 14, *p < 0.01). values are presented as mean +/sem. the x-axis denotes nanoparticle injected animals vs saline injected controls, and the y-axis denotes cell count. clinical theriogenology • volume 11 number 1 • march 2019 38 figure 3. representative images (10 x objective) displaying the effect of intravenous nanoparticle administration on epididymal sperm numbers when compared to saline injected controls. figure 4. effect of intravenous nanoparticle administration on spermatogonia (panel a; unpaired t-test, t = 26.9, df = 158, *p < 0.01) and primary spermatocyte (panel b; unpaired t-test; t = 21.92, df = 158, *p < 0.01) cell counts when compared to saline injected controls. values are presented as mean +/sem. the x-axis denotes nanoparticle injected animals versus saline injected controls, and the y-axis denotes cell count. clinical theriogenology • volume 11 number 1 • march 201939 figure 5. hematoxylin and eosin stained sections of testes (20 x objective) displaying the effect of intravenous nanoparticle administration on seminiferous tubule morphology when compared to saline injected controls. clinical theriogenology • volume 11 number 1 • march 2019 40 figure 6. hematoxylin and eosin stained sections of ovary (2 x objective) displaying the effect of intravenous nanoparticle administration on ovary morphology when compared to saline injected controls. clinical theriogenology • volume 11 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1 research report fate of cystic ovarian follicles, clinical responses, and pregnancy in dairy cows subjected to ovsynch and timed artificial insemination, with or without an intravaginal progesterone device divakar ambrose,a marcos colazo,a,b mohanathas gobikrushanthc adepartment of agricultural food and nutritional science, university of alberta, edmonton, ab, canada bleduc farm animal hospital, leduc, ab, canada ccollege of public health, medical and veterinary sciences, james cook university, townsville, qld, australia abstract our objectives were to determine the fate of cystic ovarian follicles (cof), clinical responses, and pregnancy to timed artificial insemination (tai) in lactating holstein cows subjected to an ovsynch protocol, with or without insertion of a progesterone releasing intravaginal device (prid). cows diagnosed with at least 1 cof (≥ 25 mm) were given 100 µg of gonadorelin acetate (gnrh; day –10 and day –1), 500 µg cloprostenol (prostaglandin f2α; day –3), and tai (day 0). cows randomly received prid® (n = 49) for 7 days or no-prid (n = 62). transrectal ultrasonography was conducted on days –10, –3, 0, 1, and 11 to monitor ovarian responses and on day 32 for pregnancy diagnosis. cows were also categorized as low progesterone (lp; n = 50) or high progesterone (hp; n = 61) based on plasma progesterone concentrations (< or ≥ 1.0 ng/ml) or luteal status (presence or absence of luteal tissue) on day –10. mean (± sem) diameter of cof was 33 ± 0.6 mm. although ovarian responses did not differ by prid treatment, a higher (p = 0.05) proportion of cof in hp compared to lp cows underwent complete luteinization (21 versus 8%) after 1st gnrh treatment. in contrast, the proportion of cows ovulating an existing follicle after 1st gnrh treatment was higher (p = 0.03) in lp than in hp cows (72 versus 54%). no interactions existed between treatment (prid or no-prid) and progesterone categories (lp, hp) in any of the ovarian or endocrine responses. furthermore, 10% of cof ovulated and 62% of other preexisting follicles ovulated after 1st gnrh treatment. a new follicle was detected (day –3) in 95% of cases and 84% ovulated after 2nd gnrh. pregnancy per ai did not differ (p = 0.46) between prid and no-prid cows (37 versus 45%). plasma progesterone at tai was elevated (p = 0.02) in lp than hp cows (0.7 versus 0.2 ng/ml) and pregnancy tended to be lower (p = 0.13) in lp than in hp cows (34 versus 48%). in summary, cows with cof responded normally to ovsynch protocol; although incorporating prid into the protocol was not beneficial, cows with hp status at the start of the protocol tended to have higher pregnancy per ai. keywords: cattle, ovary, cyst, luteinization, ovulation, treatment introduction cystic ovarian follicles (cof) are widely prevalent in lactating dairy cows, occurring usually during the early postpartum period, with incidence from < 20%1 up to 50% in individual herd situations.2 because of cof, dairy industry had substantial economic losses,3 mainly by increased calving interval3,4 and the likelihood of cows being culled.4 although estimated losses of up to $160 per cow per lactation was reported in the 1980’s,5 a recent economic simulation study estimated the annual loss to the global dairy industry due to cof at $4 billion.6 the conventional definition of cof is a large (≥ 25 mm diameter) anechoic follicle-like structure persisting for 10 days or longer in the absence of a corpus luteum (cl), although later definitions have included smaller follicular structures of 17-20 mm in diameter.7 the coexistence of cl with cof has been reported8,9 and it has been opined that cof coexisting with cl are benign persistent structures that exert no negative influence on ovarian follicular growth or pregnancy establishment.10 although managing cof in cattle has been an active topic of research for nearly a century,11,12 treating cows diagnosed with cof continues to be a vexing problem for practitioners and dairy producers. contact divakar ambrose dambrose@ualberta.ca © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12189, http://dx.doi.org/10.58292/ct.v17.12189 mailto:dambrose@ualberta.ca http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12189 2 citation: clinical theriogenology 2025, 17, 12189, http://dx.doi.org/10.58292/ct.v17.12189 exogenous gonadotropin releasing hormone (gnrh), luteinizing hormone (lh), human chorionic gonadotropin (hcg), progesterone, and corticosteroids have been used with varying degrees of success to treat follicular cysts, whereas prostaglandin f2α (pgf2α) is the preferred treatment for luteinized follicular cysts3,4,13,14 that often results in induced estrus within a few days after treatment. for over 2 decades, the protocol used for synchronization of ovulation in cattle, widely known as ovsynch,15 with timed artificial insemination (tai) has been used in cows with cof, achieving reasonable pregnancy per artificial insemination (p/ai).10,16–20 treatment with exogenous progesterone (progesterone-releasing intravaginal device; prid) decreased mean lh and lh pulse frequency, regressed cof, and initiated new follicular growth.21 moreover, prid as a standalone treatment22,23 or progesterone given as controlled internal drug release (cidr) device, with pgf2α and gnrh,17 or incorporating cidr into an ovsynch protocol,19 have been effective in treating bovine cof. it has been emphasized that strategies for therapeutic management of cof should include induction of follicular turnover and an increase in progesterone concentrations that can be achieved by the ovsynch protocol, when used in combination with an exogenous progesterone insert such as cidr.17 although the efficacy of ovsynch/tai treatment and that of cidr, prid, and other forms of progesterone treatments on cows diagnosed with cof have been tested adequately, only a few studies have concurrently compared the efficacy of ovsynch/tai and ovsynch/tai including a progesterone device in treating cows with cof. furthermore, the fate of cof, including that of persistent cysts that linger in coexistence with a cl and their response to treatment, have not been adequately documented. our objectives, therefore, were to determine the fate of cof, clinical responses to treatments, and p/ai in dairy cows subjected to tai following ovsynch, with or without exogenous progesterone treatment in the form of an intravaginal progesterone device. exogenous progesterone treatment can reduce lh pulse frequency resulting in regression of cof and initiation of new follicular growth.4,21 moreover, an elevated progesterone environment at the initiation of ovsynch improved p/ai in cyclic and acyclic dairy cows,24,25 and in cows with cof.19 therefore, we hypothesized that incorporating a prid during the initial 7 days of ovsynch/tai protocol improves p/ ai in cows diagnosed with cof, particularly in cows with low progesterone status at diagnosis compared to those assigned to ovsynch/tai without prid. materials and methods animals and housing this randomized clinical trial conducted over 4 years included 111 lactating holstein cows diagnosed with cof. cows were of mixed parities (mean ± sd, 2.9 ± 1.5; range 1-7) from 9 dairy farms in alberta, including 1 university farm, with a majority (75%) from 2 farms and the remainder from the other 7 farms (figure 1). mean (± sd) days in milk at cof diagnosis was 103 ± 54 (range 54-265) and the mean 305 days milk yield was 10,144 ± 1657 kg (range 7,077-12,921 kg). cows had a mean (± sd) body condition score of 2.90 ± 0.30 using the common 1 (emaciated) to 5 (obese) scale, with scores assigned in 0.25 increments.26 animal use and treatment protocols were approved by the university of alberta animal care and use committee for livestock (aup no. 052/10/09) and cows were cared for in accordance with the requirements of canadian council on animal care. housing types included both tie-stall and free-stall barns and herd sizes ranged 50-450 cows. cows were fed total mixed rations formulated according to national research council guidelines27 with primary ingredients of barley or alfalfa silage, grass or alfalfa hay, and concentrates. cows had unrestricted access to potable water and were milked either twice (8 herds) or thrice daily (1 herd). of the 111 cases, 83 were from 2 farms (1 university, 1 commercial) often used in research and the other 28 cases were directed to the researchers by collaborating dairy practitioners. ovaries were examined via transrectal ultrasonography on day –10, and cows that had at least 1 large (≥ 25 mm in diameter) anechoic structure, regardless of the presence or absence of other ovarian structures,28 were considered to have a cof and randomly assigned to either no-prid or prid treatment group. although persistent cysts existing alongside a cl may not fit the conventional definition of cof, such cases were also included to fulfil our objective of determining their fate and response to treatment. treatments and transrectal ultrasonography cows received 2 treatments of intramuscular gonadorelin acetate (100 μg; fertiline® [gnrh]; vetoquinol inc. lavaltrie, qc, canada) 9 days apart (on day –10 and day –1), 1 treatment of intramuscular cloprostenol (500 μg [pgf2α]; estrumate®; merck animal health, kirkland, qc, canada) on day –3, and were subjected to tai on day 0, ~ 16-20 hours after 2nd gnrh treatment (figure 2). cows (n = 49) received prid® (1.55 gram of progesterone; vetoquinol inc.) during the initial 7 days of ovsynch and 62 cows did not receive prid (no-prid). transrectal ultrasonography was performed on day –10 (at 1st gnrh), day –3 (at pgf2α), and day 0 (at tai) of the ovsynch protocol and on days 1, 11, and 32 after tai. experimental events (figure 1) and the purpose of each event (table 1) are provided. pregnancy was diagnosed (visualization of a viable embryo) via transrectal ultrasonography on day 32 after tai. observations made at ultrasonography including size and location of major ovarian structures and ovarian responses to treatments were manually recorded on paper as ovarian maps and data were later entered into an excel spreadsheet for analyses. if the thickness of the cof wall exceeded 3 mm, it was considered a luteinized follicular cyst. if the antrum of the cof filled completely with presumed luteal tissue of uneven echogenicity on day 7 after 1st gnrh treatment, and this cl-like figure 1. distribution of cows with cystic ovarian follicles 50 33 7 5 4 4 3 3 2 0 10 20 30 40 50 60 a b c d e f g h i nu m be r o f c ow s farm id http://dx.doi.org/10.58292/ct.v17.12189 citation: clinical theriogenology 2025, 17, 12189, http://dx.doi.org/10.58292/ct.v17.12189 3 structure lacked the characteristic homogenous appearance of a cl, the cof was considered to have undergone complete luteinization. disappearance of a cof or another follicle that existed on day –10 in response to 1st gnrh treatment, and appearance of a distinct cl at the same location on day –3, confirmed 1st ovulation. likewise, disappearance, on day 1, of a new follicle detected on days –3 and 0, and the presence of a new cl on day 11 at the same location, confirmed 2nd ovulation in response to 2nd gnrh treatment. blood sampling and quantification of plasma progesterone and estradiol concentrations blood samples were collected by coccygeal venipuncture into evacuated tubes containing sodium heparin (vacutainer, beckton dickinson and co. franklin lakes, nj) in a subset of 77 cows on days –10, –3, 0, and 11 to determine plasma progesterone concentrations, and on days –10 and –3 for plasma estradiol concentrations. samples were placed on ice after collection and centrifuged at 1,500 g for 20 minutes at 4°c, plasma was harvested and frozen at –20°c until assayed. plasma progesterone concentrations were quantified using a solid-phase radioimmunoassay (coat-a-count; diagnostic products corp. los angeles, ca) with a minimum detection limit of 0.1 ng/ml, in 2 separate assays. mean intraand interassay coefficients of variations for low (1.9 ng/ml), medium (3.1 ng/ml) and high (15.0 ng/ml) progesterone reference samples were 14.8, 3.9, and 5.9% and 13.8, 6.5, and 6.2%, respectively. estradiol was quantified using a commercial assay (immuchem double antibody 17β-estradiol 125i ria kit, mp biomedicals, santa ana, ca) with 0.55 pg/ml lowest detection limit. mean intraand interassay coefficients of variations for low (3.6 pg/ml) and high (41.9 pg/ml) estradiol reference samples were 16.9 and 13.1%, and 24.8 and 14.0%, respectively. categorization of cows by progesterone status on day –10 and by type of cystic ovarian follicle cows were retrospectively categorized as having had cof under a low progesterone (lp; n = 50) or high progesterone (hp; n = 61) environment, based on either plasma progesterone concentrations (< 1.0 or ≥ 1.0 ng/ml)29 or absence figure 2. timeline for treatments and procedures 11stst gnrhgnrh pgfpgf2α2α 22ndnd gnrhgnrh taitai pregnancy pregnancy diagnosisdiagnosis day 11day 11day–3 day–3 day 0day 0day–1 day–1 day–10 day–10 day 32day 32 transrectal transrectal ultrasonographyultrasonography blood sampleblood sample transrectal transrectal ultrasonographyultrasonography transrectal transrectal ultrasonographyultrasonography blood sample blood sample transrectal transrectal ultrasonographyultrasonography blood sample blood sample progesterone progesterone blood sample blood sample progesterone progesterone prid (7 days) prid (7 days) or or nono--pridprid day 1day 1 transrectal transrectal ultrasonographyultrasonography blood sampleblood sample table 1. schedule and description of experimental events day activity description or purpose –10 transrectal ultrasonography, blood sample, gnrh + prid transrectal ultrasonography to document characteristics of cof and the presence of other major ovarian structures > 9 mm; blood sample for progesterone and estradiol; 1st gnrh; prid inserted or no-prid inserted. –3 transrectal ultrasonography, blood, pgf2α –prid transrectal ultrasonography to document ovarian response (luteinization of cyst or ovulation of cof or an existing follicle; presence of a new dominant follicle); document major ovarian structures; blood sample drawn to quantify progesterone and estradiol; pgf2α to induce luteal regression; prid removed. –1 gnrh 2nd gnrh to synchronize ovulation; no transrectal ultrasonography and no blood sample. 0 transrectal ultrasonography, blood sample, tai transrectal ultrasonography to measure preovulatory follicle diameter; document ovarian structures including cof; blood sample to quantify progesterone; tai 16-20 hours after 2nd gnrh. 1 transrectal ultrasonography transrectal ultrasonography to confirm ovulation of new preovulatory follicle 11 transrectal ultrasonography, blood sample transrectal ultrasonography to confirm presence of new cl; blood sample to quantify progesterone and determine luteal status. 32 transrectal ultrasonography transrectal ultrasonography primarily to determine insemination outcome (pregnant or not pregnant) and secondarily to determine persistence of cof until pregnancy diagnosis. http://dx.doi.org/10.58292/ct.v17.12189 4 citation: clinical theriogenology 2025, 17, 12189, http://dx.doi.org/10.58292/ct.v17.12189 (lp) or presence (hp) of a cl on day -10. presence of a luteinized follicular cyst was evident by ultrasonography (follicular walls > 3 mm thickness)23,29 but if plasma progesterone data were unavailable, the cow was assigned to hp category. if luteinization was not evident (absence of cl) and progesterone concentration at day –10 was not available, the cow was assigned to lp category. additionally, cows with at least 1 large (≥ 25 mm) follicular structure and no detectable signs of luteinization or cl, and low progesterone concentrations (< 1 ng/ml) at enrollment (day –10) were categorized, retrospectively, as having had ‘true follicular cyst.’ cows with at least 1 large follicular structure (≥ 25 mm) and visible signs of luteinization but no detectable cl or those with progesterone concentrations ≥ 1 ng/ml in the absence of a detectable cl were categorized as having had ‘luteinized follicular cyst’. cows with at least 1 large follicular structure (≥ 25 mm) and a distinct cl on either ovary, with progesterone ≥ 1 ng/ml or without progesterone data were categorized as having had ‘persistent follicular cyst’. we chose the above categorization merely as an alternate approach to understand the clinical responses to ovsynch/tai treatment, based on structurally distinct types of cysts. although the aforesaid categories were used retrospectively to describe the types of cysts and determine insemination outcomes (pregnant versus open) by cyst category, because the effect of prid treatment was not significant, all 3 types of cysts (true follicular, luteinized follicular, and persistent follicular) were considered under the common category of cof during analyses to determine main effects of treatment (no-prid versus prid) and progesterone category (lp versus hp). terms clinical responses and ovarian responses are used synonymously to indicate ovarian changes in response to gnrh, pgf2α or prid treatment and endocrine responses indicate changes in hormone (progesterone and estradiol) concentrations. data analyses data were analysed using sas version 9.4 (sas institute inc., cary, nc) or ibm spss statistics (version 29.0). univariate procedure of sas was used to determine the descriptive statistics such as mean (± standard error of mean; sem), minimum and maximum diameters of cof. freq procedure of sas was used to determine the following outcomes: incidence of cof on right ovary, left ovary, and both ovaries, proportion of cows with > 1 cof, proportion of cows in which the cof grew (that is, became larger in diameter) after starting ovsynch, proportion of cows that ovulated or highly luteinized the cof in response to 1st gnrh, and proportion of cows that developed a new cof during the ovsynch-tai protocol. differences in continuous dependent outcomes such as plasma progesterone and estradiol concentrations by prid treatment (no-prid versus prid), progesterone status at enrolment (lp versus hp) and their interactions were evaluated by anova using mixed procedure of sas, with the effect of farm considered as random. differences in continuous dependent outcomes such as mean diameter of cof at 1st gnrh and preovulatory follicle at 2nd gnrh and binomial dependent outcomes such as the proportion of cows that ovulated or luteinized a cof after 1st gnrh; in which the cof grew in size between 1st gnrh and pgf2α; ovulated an existing follicle after 1st gnrh; developed a new dominant follicle at pgf2α, ovulated a new follicle after 2nd gnrh, and ovulated > 1 follicle in response to 2nd gnrh of ovsynch/tai; were pregnant on day 32 after ai were determined by 2 x 2 factorial analyses between prid treatment (no-prid versus prid) and progesterone status at enrolment (lp versus hp) and their interactions using generalized linear mixed model in spss. in addition, differences in mean bcs, dim, and 305 days my were determined by 2 x 2 factorial analyses between prid treatment (no-prid versus prid) and progesterone status at enrolment (lp versus hp) and their interactions using generalized linear mixed model in spss. differences were declared significant if p ≤ 0.05 and considered trends if p > 0.05 and ≤ 0.15. results mean (± sem), minimum, and maximum diameter of the cof were 33 (± 0.6), 25, and 49 mm; in 72% of the cows cof diameter ranged 25-36 mm, 19% ranged 37-42 mm, and 9% of the cof were > 43 mm diameter (figure 3). prevalence of cof was greatest on the right ovary (58%), followed by the left ovary (26%), and in 16% of the cases cof were present on both ovaries. more than 1 cof was detected in 25% of cases, present either on the same ovary or both ovaries. none of the clinical and endocrine responses or p/ai differed between cows subjected to ovsynch without (no-prid) or with prid insertion during the 1st 7 days of the protocol (table 2). thus, when all cows were considered together, ignoring the prid treatment, the cof either ovulated (11/111) or completely luteinized (17/111) in response to 1st gnrh in 25.2% of cases. cystic ovarian follicles that completely luteinized presented a cl-like structure with nonhomogenous echogenicity at ultrasonography on day –3 at pgf2α treatment. ovulation of an existing follicle other than the cof occurred in 69 instances (62.2% of cases). in response to pgf2α given on day –3, luteal regression occurred in 94.7% of cows. cystic ovarian follicle grew in 28 (25.2%) cases, determined by ultrasonography as an increase in its diameter between 1st gnrh (day –10) and pgf2α (day –3) treatments. development of a new cof (≥ 25 mm) was recorded in 9 (8.1%) cows of lp category; none of the hp category cows developed a new cyst during treatment period of 10 days. in response to 1st gnrh treatment of the ovsynch protocol, a new dominant follicle (mean diameter 17.4 mm) developed in figure 3. distribution of cows (n = 111) based on the diameter (mm) of cof; mean ± sem (33 ± 0.6), minimum (25) and maximum (49) 37 43 21 10 0 5 10 15 20 25 30 35 40 45 50 25-30 31-36 37-42 43-49 nu m be r o f c ow s cys�c ovarian follicle diameter (mm) http://dx.doi.org/10.58292/ct.v17.12189 citation: clinical theriogenology 2025, 17, 12189, http://dx.doi.org/10.58292/ct.v17.12189 5 105 (94.6%; table 2) cases and 93 (88.6%) of those follicles or 83.8% of all cases ovulated in response to 2nd gnrh treatment given on day –1. incidence of ≥ 2 ovulations (defined as ‘double ovulation’) was recorded in 18 cases (16.2%; table 2). mean p/ai determined by transrectal ultrasonography on day 32 after tai was 41.4% (46/111 cows pregnant) when no-prid and prid groups were considered together. information on persistence of cof until pregnancy determination was available in 37 cows. among those, the cof persisted for the entire 42 days (day 10 treatment + day 32 until pregnancy determination) in 14 cows (37.8%) of which 4 were pregnant. thus, based on the subset of cows from whom data were available on persistence (or not) of cof until pregnancy diagnosis, 28.5% of the pregnant cows had a persistent follicular cyst on day 32 after tai when pregnancy was diagnosed. proportions of cows that were categorized as having true follicular cyst, luteinized follicular cyst, and persistent follicular cyst (either ipsilateral or contralateral) were 45 (50/111), 20 (22/111), and 35% (39/111), respectively, and percentages of cows determined pregnant from the above categories were 33.8, 58.5, and 41.3, respectively (p = 0.16). ovulation of a cyst in response to 1st gnrh treatment occurred in 18 (9/50), 0 (0/22), and 5% (2/39); a new cyst developed during the treatment period in 18 (9/50), 0 (0/22) and 0% (0/39); and double ovulation after 2nd gnrh occurred in 28 (14/50), 14 (3/22) and 3% (1/39) of cows categorized as true follicular cyst, luteinized follicular cyst, and persistent follicular cyst, respectively. ovarian and endocrine responses, and p/ai of cows with cof categorized by treatment (no-prid or prid), progesterone category (lp or hp, based on progesterone concentrations or presence of a cl on day –10) and their interactions are presented (table 2). treatment did not affect ovarian (clinical) responses and pregnancy per ai, whereas some ovarian responses differed or tended to differ by progesterone category. mean diameter of cof on day –10 in lp table 2. ovarian responses and pregnancy per ai in cows diagnosed with cof subjected to the ovsynch/tai protocol without (no-prid) or with (prid) an intravaginal progesterone releasing device treatment (trt) for 7 days and categorized as having either low (lp) or high progesterone (hp) at the initiation of the ovsynch/tai protocol observations, ovarian responses and outcomes no-prid prid trt progesterone category trt x progesterone category interaction lp hp lp hp p p p number of cows 26 36 24 25 mean (± sem) diameter of cyst in mm 32.6 ± 1.1 33.9 ± 1.1 31.2 ± 1.1 35.2 ± 1.1 0.95 0.22 0.24 ovulation or luteinization1 of cyst after 1st gnrh, n (%) 8 (30.8) 9 (25.0) 5 (20.8) 6 (24.0) 0.53 0.92 0.32 ovulation of cyst after 1st gnrh, n (%) 5 (19.2) 2 (5.5) 4 (16.7) 0 (0.0) 0.27 0.11 0.11 luteinization1 of cyst after 1st gnrh, n (%) 3 (11.5) 7 (19.5) 1 (4.2) 6 (24.0) 0.88 0.05 0.76 ovulation of existing follicle after 1st gnrh, n (%) 18 (69.2) 23 (63.9) 18 (75.0) 10 (40.0) 0.38 0.03 0.12 cyst grew between 1st gnrh and pgf2α, n (%) 10 (38.4) 6 (16.7) 6 (25.0) 6 (24.0) 0.82 0.18 0.22 new cyst developed during treatment, n (%) 5 (19.2) 0 (0.0) 4 (16.7) 0 (0.0) 1.00 nd nd cows with new dominant follicle at pgf2α, n (%) 24 (92.3) 34 (94.4) 22 (91.7) 25 (100) 0.99 0.99 0.99 mean (± sem) diameter of new dominant follicle in mm 17.4 ± 0.6 16.9 ± 0.5 18.4 ± 0.6 16.9 ± 0.6 0.41 0.11 0.50 ovulation of new follicle after 2nd gnrh, n (%) 22 (84.6) 31 (86.1) 19 (79.1) 21 (84.0) 0.60 0.66 0.83 double ovulation after 2nd gnrh, n (%) 8 (36.6) 4 (12.9) 6 (31.5) 0 (0.0) 0.99 0.99 0.99 cows pregnant on day 32 after ai (%) 10 (38.4) 18 (50.0) 7 (29.1) 11 (45.8) 0.46 0.13 0.76 nd: not determinable 1cyst completely luteinized presenting a cl-like appearance http://dx.doi.org/10.58292/ct.v17.12189 6 citation: clinical theriogenology 2025, 17, 12189, http://dx.doi.org/10.58292/ct.v17.12189 versus hp cows was 31.9 versus 34.6 mm (p = 0.22). proportion of cof that ovulated from lp (18.0%) versus hp cows (3.3%) tended to be higher (p = 0.11), with a mean diameter of 28.7 ± 1.1 (min, max; 25, 34) mm. on the contrary, the proportion of cof that underwent complete luteinization was higher (p = 0.05) in hp than in lp category cows (21.3 versus 8.0%). the mean diameter of cof that completely luteinized was 34.0 ± 1.3 (minimum, maximum; 25, 42) mm. moreover, ovulation of an existing follicle after 1st gnrh was higher (72 versus 54%; p = 0.03) in lp than in hp cows (table 2). although neither the proportions of cows that developed a new dominant follicle in response to 1st gnrh treatment nor their ovulation responses to 2nd gnrh treatment differed between lp and hp cows (table 2), the putative preovulatory follicle tended (p = 0.11) to be larger by 1 mm in lp cows. despite the lack of differences (p = 0.66) in the proportions of lp and hp cows that ovulated a new follicle after 2nd gnrh of ovsynch, cows that started the protocol in a lp environment tended (p = 0.13) to have lower p/ai on day 32 after ai than  cows that started the protocol in a hp environment (34.0 versus 47.5%; table 2). mean body condition score, days in milk, and 305 days milk yield did not differ by treatment, by progesterone category, or their interactions (table 3). progesterone profiles (subset of 77 cows) of days –10, –3, 0, and 11, corresponding to 1st gnrh, pgf2α, tai, and 11 days after tai, assigned to no-prid versus prid as well as from cows classified as lp versus hp are presented (figure  4). progesterone concentrations did not differ (p > 0.30) at any of the days mentioned above between no-prid and prid cows. however, mean progesterone concentrations (ng/ml) at 1st gnrh (0.4 versus 4.2) and at pgf2α (3.4 versus 5.1) were lower (p ≤ 0.02) in lp than in hp cows. on the contrary, plasma progesterone on day 0 (tai) was higher (p = 0.02) in lp cows than in hp (0.7 versus 0.2 ng/ml). plasma estradiol concentrations (pg/ml) of day –10 (1st gnrh) and day –3 (pgf2α) did not differ between no-prid and prid cows. although mean estradiol concentrations were higher in lp than in hp cows on day –10 (2.6 ± 0.3 versus 1.3 ± 0.3; p = 0.01) they did not differ (p = 0.66) on day –3 (1.2 ± 0.2 versus 1.3 ± 0.2). discussion cows diagnosed with cof responded adequately to ovsynch treatment and irrespective of the type of cof (follicular, luteinized follicular or persistent follicular cyst) present at  diagnosis, acceptable p/ai were obtained after tai. nevertheless, our hypothesis that incorporating prid during the initial 7 days of ovsynch/tai improves p/ai in cows diagnosed with cof was not supported. in fact, the prid treatment may have worsened p/ai, as it was numerically lower by 8.5-percentage points than no-prid treatment. besides, none of the ovarian and endocrine responses measured was changed by including a prid into the ovsynch protocol. treating cows diagnosed with cof with gnrh triggered an lh release within minutes,30 peaking at 2 hours, and led to a measurable increase in serum progesterone concentrations in < 20 minutes after gnrh treatment in some cows. moreover, a sustained increase in serum progesterone concentrations occurred up to 14 days, presenting a progesterone profile akin to that of a normal estrous cycle.30 therefore, 1st gnrh treatment of the ovsynch protocol in the present study would have induced an acute lh release that either induced ovulation of lh-receptive cof and dominant follicle(s) or caused luteinization of the cof. although it is impossible to rule out that an endogenous lh surge may have occurred prior to 2nd gnrh treatment in cows that received no prid, leading to the development of a spontaneous cl, it is unlikely. we anticipated cof to ovulate in response to 1st gnrh treatment only in rare instances; however, ovulation of the cof occurred in ~ 10% of the cases, and complete luteinization resulting in the development of a cl-like structure occurred in ~ 15% of cases, indicating the luteotropic effects of lh released in response to 1st gnrh treatment. among cows that underwent complete luteinization of cof, 76% were categorized as hp and 24% as lp. differences in the quantity of luteal tissue present in cof and their lh receptivity may determine how they respond to gnrh treatment30; therefore, variations in progesterone concentrations on day –3 (at pgf2α) and differences in progesterone concentrations between lp and hp cows may be attributed to the above notion. although reports of either complete or partial luteinization of the cof after gnrh treatment are not uncommon, ovulation of cof, either spontaneously or in response to gnrh treatment, is less commonly reported. in a previous study10 with only 18 cows, none of the cof ovulated in response to 1st gnrh although in 2 (~ 11%) cases the cof table 3. differences in body condition score (bcs), days in milk (dim), and 305 days milk yield (my) in kg in a subset of 71 cows diagnosed with cof subjected to ovsynch/tai protocol without (no-prid) or with (prid) an intravaginal progesterone releasing device treatment (trt) for 7 days and categorized as having either lp or hp at ovsynch/tai protocol initiation no-prid prid trt progesterone category trt x progesterone category interaction lp hp lp hp p p p number of cows 17 27 14 13 bcs (mean ± sem) 3.0 ± 0.05 3.0 ± 0.05 3.0 ± 0.05 3.0 ± 0.05 0.84 0.41 0.84 dim (mean ± sem) 104 ± 14 109 ± 11 139 ± 15 102 ± 16 0.32 0.25 0.14 my (mean ± sem) 10,283 ± 485 10,579 ± 396 9,911 ± 551 11,136 ± 536 0.85 0.13 0.35 http://dx.doi.org/10.58292/ct.v17.12189 citation: clinical theriogenology 2025, 17, 12189, http://dx.doi.org/10.58292/ct.v17.12189 7 underwent complete luteinization presenting cl-like structures 7 days after treatment. instances of spontaneous ovulation of cof after persisting for 21 days,31 and induced ovulation of cof in response to gnrh treatment during an ovsynch protocol19 have been reported in isolated cases. ovulation of low-dose-progesterone-induced follicular cysts after persisting for more than 30 days was also reported in one study.32 of the 11 cows that ovulated a cof in response to 1st gnrh treatment, 9 were in the lp category and 2 cows in which progesterone concentrations were not quantified on day –10, were assigned to the hp category based on luteal tissue detected during ultrasonography on day –10. on further examination of data, it was evident that the 9 lp cows that ovulated a cof also belonged to the ‘true follicular cyst’ category and the 2 hp cows belonged to the ‘persistent follicular cyst’ category. although induced ovulation after gnrh treatment occurred more readily when circulating progesterone concentrations were lower than elevated,33 it is likely that the 2 cof categorized as ‘persistent follicular cyst’ were responsive to the gnrh-induced lh surge. furthermore, the mean diameter of the cof that ovulated in response to 1st gnrh was smaller (28.7 mm) compared to that of cof that underwent luteinization (34.0 mm) suggesting that the former may have been in an active growing phase presenting a milieu conducive to induced ovulation. three of the 9 cows (33.3%) that developed a new cyst were diagnosed pregnant at day 32, indicating that even cows that developed a new cof responded positively to the ovsynch/ tai protocol resulting in an acceptable p/ai. overall incidence of double ovulation in the present study (16.2%) was higher than what was reported for cyclic cows of mixed parities (14.1%) and cows with cof (3.6%).16 that the incidence of double ovulation was higher 14/18 (78%) in lp cows than in the hp category 4/18 (22%) was not surprising; because, as previously discussed, induced ovulation after gnrh treatment occurred more readily when circulating progesterone concentrations were lower33 and cof is a known risk factor for double ovulation.34 in addition to the 11 cows that ovulated a cof in response to 1st gnrh treatment, over 60% of cows with cof responded to that gnrh by ovulating an existing follicle and developed a cl on day –3, as determined by ovarian ultrasonography prior to pgf2α treatment. more importantly, 95% of cases developed a new dominant follicle in response to 1st gnrh treatment and 89% of the aforesaid new follicles (or 84% of all cows) ovulated in response to 2nd gnrh treatment, providing evidence that the ovsynch protocol works in cows with cof as well as it does in normal cyclic cows. new dominant follicle development in response to 1st gnrh treatment in 100% of cof cases has been reported10,19 and synchronized ovulation rates in response to 2nd gnrh treatment were 73,16 83,10 and 100%.19 although ovulation response to 2nd gnrh treatment did not differ between lp and hp categories, cows in which the ovsynch protocol was initiated in lp status tended to have reduced p/ai compared to cows in hp status in the present study. it is known that starting the ovsynch protocol during the luteal phase (hp status) results in higher p/ai in cows because adequate exposure to progesterone during ovulatory follicle development increased the fertility of dairy cows subjected to tai programs24,35 and cows that start the protocol in hp status have higher odds of conceiving and sustaining the pregnancy to term through reduced embryo losses. cows categorized as lp in the current study presented no evidence of luteal tissue at ultrasonography and/or plasma progesterone concentrations were < 1 ng/ml at the initiation of the ovsynch figure 4: plasma progesterone profiles at 1st gnrh treatment (day –10), at pgf2α treatment (day –3), at tai (day 0) and 11 days after tai (day +11) in cows that received no-prid (a) versus prid (b) or categorized as lp (c; < 1 ng/ml) versus hp (d; ≥ 1 ng/ ml). standard errors of the means (common to charts a, b, c, and d) on days –10, –3 and +11 were ± 0.3, 0.4, and 0.4, respectively. standard errors of the means on day 0 for charts a, b, c, and d were 0.5, 0.4, and 0.1, 0.1, respectively. 2.62.6 4.64.6 0.60.6 5.45.4 0.00.0 1.01.0 2.02.0 3.03.0 4.04.0 5.05.0 6.06.0 day -10day -10 day -3day -3 day 0day 0 day +11day +11 pr og es te ro ne (n g/ m l) pr og es te ro ne (n g/ m l) nono--prid (n = 35)prid (n = 35) aa 2.22.2 4.14.1 0.40.4 5.35.3 0.00.0 1.01.0 2.02.0 3.03.0 4.04.0 5.05.0 6.06.0 day -10day -10 day -3day -3 day 0day 0 day +11day +11 pr og es te ro ne (n g/ m l) pr og es te ro ne (n g/ m l) prid (n = 42)prid (n = 42) bb 0.40.4 3.43.4 0.70.7 5.55.5 0.00.0 1.01.0 2.02.0 3.03.0 4.04.0 5.05.0 6.06.0 day -10day -10 day -3day -3 day 0day 0 day +11day +11 pr og es te ro ne (n g/ m l) pr og es te ro ne (n g/ m l) low progesterone (n = 36)low progesterone (n = 36) cc 4.24.2 5.25.2 0.20.2 5.35.3 0.00.0 1.01.0 2.02.0 3.03.0 4.04.0 5.05.0 6.06.0 day -10day -10 day -3day -3 day 0day 0 day +11day +11 pr og es te ro ne (n g/ m l) pr og es te ro ne (n g/ m l) high progesterone (n = 41)high progesterone (n = 41) dd http://dx.doi.org/10.58292/ct.v17.12189 8 citation: clinical theriogenology 2025, 17, 12189, http://dx.doi.org/10.58292/ct.v17.12189 protocol. thus, cows in the lp category (no cl, progesterone < 1 ng/ml) may be considered as having ‘true cof’ with little progesterone exposure and at a higher risk of not conceiving. indeed, overall p/ai in lp cows, irrespective of prid treatment, tended to be lower than in hp cows by 13.5-percentage points (table 2). however, when lp cows that received no-prid treatment, hence expected to have the lowest p/ai, were compared against lp cows that received prid, mean p/ai was numerically higher in lp cows by 9.3-percentage points (table 2), suggesting that lp cows with no-prid appeared to have a slight advantage. however, no conclusions may be drawn based on these observations because we did not have an adequate sample size for these tests, a limitation of the study. mean progesterone concentrations at 1st gnrh and at pgf2α were lower in lp than in hp cows, attributable to the lack of prior progesterone exposure. in contrast, mean plasma progesterone concentrations on d 0 (tai), albeit < 1 ng/ml, was higher in lp cows than in hp (0.7 versus 0.2 ng/ml). suprabasal concentrations of progesterone on the day of tai are detrimental to pregnancy establishment35,36 and this may be the main reason why lp cows tended to have reduced p/ai. although the use of ovsynch/tai in the reproductive management of cystic cows has been widely reported,10,16–18,20,37 p/ai has varied 3-41% in the cited studies. one study19 tested the effectiveness of ovsynch/tai + cidr against a protocol involving ai at detected estrus and reported that p/ai in cows with cof subjected to ovsynch/tai + cidr greatly improved (52% > 27%; p < 0.05) compared to control group. nonetheless, few studies have directly compared treatment outcomes to ovsynch and ovsynch + progesterone within the same trial. one such study38 compared p/ai after ovsynch and ovsynch + cidr and reported that p/ai in the ovsynch + cidr group of cows with cof improved (37.5 > 16.2%; p < 0.05) whereas in another study39 that made a direct comparison between ovsynch and ovsynch + cidr in cows with true follicular cysts, the addition of a cidr did not improve p/ai although both ovsynch/tai and ovsynch/ tai + cidr yielded above average p/ai (mean 44.7%). although past studies10,21,23 have demonstrated the positive impact of exogenous progesterone on managing ovarian cysts in dairy cattle, our findings and that of another39 do not support the blanket use of a progesterone device in all cof cases. some studies had significant improvements in p/ai when a progesterone device was incorporated into the ovsynch protocol to manage either cof cows38 or normal cyclic cows;40 however, given the increased input cost associated with prid or cidr, selective use of a progesterone device only where its inclusion would be most beneficial could be justified. in this regard, a study41 that compared the economic benefits of tai versus prid as therapeutic options for managing cof in dairy cows, using a modelling approach, concluded that the overall benefit of treatment by tai was $11.39 higher than by prid. thus, based on our findings and because of a calculated cost advantage to using tai over prid for managing dairy cows with cof,41 addition of exogenous progesterone to the ovsynch/tai protocol cannot be recommended. in summary, ovsynch/tai protocol was effective in dairy cows diagnosed with cof, regardless of the type of cyst present. first gnrh treatment induced ovulation of the cof in ~ 10% of cases and caused complete luteinization in ~ 15% of cases. presence of cof did not hinder the ovulation of an existing follicle or the development of a new follicle. moreover, cof did not interfere with conception or have any apparent harmful effects on early embryonic development at least until day 32 after tai. our findings reiterated previous reports that the application of ovsynch/tai is an effective approach to manage cows with cof to attain normal p/ai. cows with cof that started on the ovsynch program with hp status tended to attain higher pregnancy to tai. indiscriminate inclusion of a prid in the ovsynch/tai protocol, in cows with all types of cof, was not beneficial. acknowledgments research supported by alberta milk, alberta livestock and meat agency, agri-food council, and alberta agriculture and forestry. product donations by schering-plough (estrumate®) and vetoquinol (fertiline® and prid®) are gratefully acknowledged. authors thank leduc farm animal hospital, leduc, alberta, wetaskiwin animal hospital, wetaskiwin, alberta and the dairy producers for their collaboration, and ms. jamie kratchkowski for technical assistance. conflict of interest none to report. references 1. garverick ha: ovarian 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http://dx.doi.org/10.58292/ct.v17.12189 https://doi.org/10.3168/jds.s0022-0302(97)76025-9 http://msdvetmanual.com https://www.msdvetmanual.com/reproductive-system/cystic-ovary-disease/cystic-ovary-disease-herd-problem?query=statham https://www.msdvetmanual.com/reproductive-system/cystic-ovary-disease/cystic-ovary-disease-herd-problem?query=statham https://www.msdvetmanual.com/reproductive-system/cystic-ovary-disease/cystic-ovary-disease-herd-problem?query=statham https://doi.org/10.1046/j.0936-6768.2003.00466.x https://doi.org/10.1016/0167-5877(86)90004-8 https://doi.org/10.3168/jds.2023-24626 https://doi.org/10.3168/jds.2023-24626 https://doi.org/10.1292/jvms.20-0381 https://doi.org/10.1262/jrd1955.21.147 https://doi.org/10.1136/vr.101.16.320 citation: clinical theriogenology 2025, 17, 12189, http://dx.doi.org/10.58292/ct.v17.12189 9 12. cassida le, mcshan wh, meyer rk: effects of an unfractionated pituitary extract upon cystic ovaries and nymphomania in cows. j anim sci 1944;3:273-282. doi: 10.2527/jas1944.33273x 13. elmore rg, bierschwal cj, youngquist rs, et al: clinical responses of dairy cows with ovarian cysts after treatment with 10,000 i.u. hcg or 100 mcg gnrh. vet clin north am large anim pract 1975;70:1346-1349. 14. ijaz a, fahning ml, zemjanis r: treatment and control of cystic ovarian disease in dairy cattle: a review. br vet j 1987;143:226237. doi: 10.1016/0007-1935(87)90085-6 15. pursley jr, mee mo, wiltbank mc: synchronization of ovulation in dairy cows using pgf2α and gnrh. theriogenology 1995;44: 915-923. doi: 10.1016/0093-691x(95)00279-h 16. fricke pm, wiltbank mc: effect of milk production on the incidence of double ovulation in dairy cows. theriogenology 1999;52:1133-1143. doi: 10.1016/s0093-691x(99)00205-8 17. bartolome ja, thatcher ww, melendez p, et al: strategies for the diagnosis and treatment of ovarian cysts in dairy cattle. j am vet med assoc 2005;222:1409-1414. doi: 10.2460/javma.2005.227.1409 18. crane mb, melendez p, bartolome j, et al: association between milk production and treatment response of ovarian cysts in lactating dairy cows using the ovsynch protocol. theriogenology 2006;66:1243-1248. doi: 10.1016/j.theriogenology.2006.03.035 19. kim ih, suh gh, kim uh, et al: a cidr-based timed ai protocol can be effectively used for dairy cows with follicular cysts. anim reprod sci 2006;95:206-213. doi: 10.1016/j.anireprosci.2005.10.003 20. gundling n, drews s, hoedemaker m: comparison of two different programmes of ovulation synchronization in the treatment of ovarian cysts in dairy cows. reprod domest anim 2015;50:893-900. doi: 10.1111/j.1439-0531.2009.01342.x 21. calder md, salfen be, bao b, et al: administration of progesterone to cows with ovarian follicular cysts results in a reduction in mean lh and lh pulse frequency and initiates ovulatory follicular growth. j anim sci 1999;77:3037-3042. doi: 10.2527/1999. 77113037x 22. douthwaite r, dobson h: comparison of different methods of diagnosis of cystic ovarian disease in cattle and an assessment of its treatment with a progesterone-releasing intravaginal device. vet rec 2000;147:355-359. doi: 10.1136/vr.147.13.355 23. zulu vc, nakao t, yamada k, et al: clinical response of ovarian  cysts in dairy cows after prid treatment. j vet med sci 2003;65:57-62. doi: 10.1292/jvms.65.57 24. colazo mg, dourey a, rajamahendran, r et al: progesterone supplementation before timed ai increased ovulation synchrony and pregnancy per ai, and supplementation after timed ai reduced pregnancy losses in lactating dairy cows. theriogenology 2013;79:833-841. doi: 10.1016/j.theriogenology.2012.12.011 25. bisinotto rs, lean ij, thatcher ww, et al: meta-analysis of progesterone supplementation during timed artificial insemination programs in dairy cows. j dairy sci 2015;98:2472-2487. doi: 10.3168/ jds.2014-8954 26. edmonson aj, lean ij, weaver ld, et al: a body condition scoring chart for holstein dairy cows. j dairy sci 1989;72:68-78. doi: 10.3168/jds.s0022-0302(89)79081-0 27. national research council: nutrient requirements of dairy cattle. seventh revised edition, washington, dc; the national academies press: 2001. 28. farin pw, youngquist rs, parfet jr, et al: diagnosis of luteal and follicular cysts by palpation per rectum and linear-array ultrasonography in dairy cows. j am vet med assoc 1992;200:1085-1089. doi: 10.2460/javma.1992.200.08.1085 29. turner zb, lima fs, conley aj, et al: cystic ovarian disease in dairy cattle: diagnostic accuracy when using b-mode and color doppler ultrasound. j dairy sci 2023;106:3411-3420. doi: 10.3168/jds.2022-22498 30. kittok rj, britt jh, convey em: endocrine response after gnrh in luteal phase cows and cows with ovarian follicular cysts. j anim sci 1973;37:985-989. doi: 10.2527/jas1973.374985x 31. sakaguchi m, sasamoto y, suzuki t, et al: fate of cystic ovarian follicles and the subsequent fertility of early postpartum dairy cows. vet rec 2006;139:197-201. doi: 10.1136/vr.159.7.197 32. noble km, tebble je, harvey d, et al: ultrasonography and hormone profiles of persistent ovarian follicles (cysts) induced with low doses of progesterone in cattle. j reprod fertil 2000;120:361366. doi: 10.1530/reprod/120.2.361 33. colazo mg, kastelic jp, davis h, et al: effects of plasma progesterone concentrations on lh release and ovulation in beef cattle given gnrh. domest anim endocrinol 2008;34:109-117. doi: 10.1016/j.domaniend.2006.11.004 34. macmillan k, kastelic jp, colazo mg: update on multiple ovulations in dairy cattle. animals 2018;8:62. doi: 10.3390/ani8050062 35. bruinjé tc, gobikrushanth m, colazo mg, et al: dynamics of preand post-insemination progesterone profiles and insemination outcomes determined by an in-line milk analysis system in primiparous and multiparous canadian holstein cows. theriogenology 2017;102:147-153. doi: 10.1016/j.theriogenology. 2017.05.024 36. colazo mg, lópez helguera, behrouzi a, et al: relationship between circulating progesterone at timed-ai and fertility in dairy cows subjected to gnrh-based protocols. theriogenology 2017;94:15-20. doi: 10.1016/j.theriogenology.2017.02.004 37. lópez-gatius f, lópez-béjar m: reproductive performance of dairy cows with ovarian cysts after different gnrh and cloprostenol treatments. theriogenology 2002;58:1337-1348. doi: 10.1016/ s0093-691x(02)00952-4 38. bartolome ja, sozzi a, mchale j, et al: resynchronization of ovulation and timed insemination in lactating dairy cows. iii. administration of gnrh 23 days post ai and ultrasonography for nonpregnancy diagnosis on day 30. theriogenology 2005;63:16431658. doi: 10.1016/j.theriogenology.2004.07.018 39. kawate n, watanabe k, uenaka k, et al: comparison of plasma concentrations of estradiol-17β and progesterone, and conception in dairy cows with cystic ovarian diseases between ovsynch and ovsynch plus cidr timed ai protocols. j reprod dev 2011;57:267-272. doi: 10.1262/jrd.10-066t 40. hölper m, bretzinger l, randi f, et al: effect of a progesterone-releasing intravaginal device (prid) for 8 days during a modified ovsynch protocol on pregnancy outcomes in lactating holstein  cows. jds commun 2023;4:303-307. doi: 10.3168/ jdsc. 2022-0314 41. de vries a, crane mb, bartolome ja, et al: economic comparison of timed artificial insemination and exogenous progesterone as treatments for ovarian cysts. j dairy sci 2006;89:3028-3037. doi: 10.3168/jds.s0022-0302(06)72576-0 http://dx.doi.org/10.58292/ct.v17.12189 https://doi.org/10.2527/jas1944.33273x https://doi.org/10.1016/0007-1935(87)90085-6 https://doi.org/10.1016/0093-691x(95)00279-h https://doi.org/10.1016/s0093-691x(99)00205-8 https://doi.org/10.2460/javma.2005.227.1409 https://doi.org/10.1016/j.theriogenology.2006.03.035 https://doi.org/10.1016/j.anireprosci.2005.10.003 https://doi.org/10.1111/j.1439-0531.2009.01342.x https://doi.org/10.2527/1999.77113037x https://doi.org/10.2527/1999.77113037x https://doi.org/10.1136/vr.147.13.355 https://doi.org/10.1292/jvms.65.57 https://doi.org/10.1016/j.theriogenology.2012.12.011 https://doi.org/10.3168/jds.2014-8954 https://doi.org/10.3168/jds.2014-8954 https://doi.org/10.3168/jds.s0022-0302(89)79081-0 https://doi.org/10.2460/javma.1992.200.08.1085 https://doi.org/10.3168/jds.2022-22498 https://doi.org/10.2527/jas1973.374985x https://doi.org/10.1136/vr.159.7.197 https://doi.org/10.1530/reprod/120.2.361 https://doi.org/10.1016/j.domaniend.2006.11.004 https://doi.org/10.3390/ani8050062 https://doi.org/10.1016/j.theriogenology.2017.05.024 https://doi.org/10.1016/j.theriogenology.2017.05.024 https://doi.org/10.1016/j.theriogenology.2017.02.004 https://doi.org/10.1016/s0093-691x(02)00952-4 https://doi.org/10.1016/s0093-691x(02)00952-4 https://doi.org/10.1016/j.theriogenology.2004.07.018 https://doi.org/10.1262/jrd.10-066t https://doi.org/10.3168/jdsc.2022-0314 https://doi.org/10.3168/jdsc.2022-0314 https://doi.org/10.3168/jds.s0022-0302(06)72576-0 1 © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 11053, http://dx.doi.org/10.58292/ct.v16.11053 editorial editor’s note dear theriogenology friends, clinical theriogenology is making good progress with strong support from the leadership and membership of our organizations (society for theriogenology, american college of theriogenologists, theriogenology foundation) and hannah street consulting, inc. in addition to publishing the bartlett address (vol. 15 (2023): conference proceedings | clinical theriogenology), we also published a review (after peer review) that was built on the bartlett address (vol. 16 (2024): issue in progress | clinical theriogenology); thank you dr. stuart meyers. as decided, we also published manuscripts (after peer review) created by presenters at the 2023 conference (vol. 16 (2024): issue in progress | clinical theriogenology). we sincerely appreciate the willingness of conference speakers to create full manuscripts, and working with us (editorial and production teams). we also acknowledge the time and effort invested by reviewers of these manuscripts; thank you all. it has been a personal privilege for me to format, edit, and copyedit (initial) 76 abstracts for the 2024 conference that are published in this issue. we express our gratitude to the american college of theriogenologists scientific information/abstract committee (drs. roberto palomares [chair] orsolya balogh, misty edmondson, robyn ellerbrock, jessica klabnik, and karen wolfsdorf) and to drs. jimmy alexander and david christiansen (student abstracts) for their dedication and due diligence. we are impressed with the broad range of topic, techniques, and application of ‘cutting edge’ science; student abstracts reflect the enthusiasm students have for theriogenology and the dedication of our mentoring colleagues (thank you all) in our discipline. abstracts are truly an outcome of enormous amount of time commitment and effort and resources invested by the investigators and their institutions. there is always a possibility of creating manuscripts from abstracts. although some abstracts will need further work before a manuscript can be created, many have sufficient information to become either a case or research report. please consider expanding your abstract into a manuscript for clinical theriogenology. if you decide to publish elsewhere, please acknowledge that an abstract was presented at the society for theriogenology conference and indicate that the abstract was published in clinical theriogenology. we express our profound appreciation to copy editor, dr. john kastelic and production editor, ms. emma csemiczky. dr. michelle kutzler (edtiorial board member) joined us this year to observe the process of copyediting; thanks michelle for your insights and ‘welcome aboard.’ in addition, i recognize the enthusiastic efforts of our ‘point person’ for conference abstracts in our management office (hannah street consulting, inc.), ms. beth gibson; thank you beth, we appreciate all your help. regards, augustine http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.11053 https://clinicaltheriogenology.net/index.php/ct/issue/view/579 https://clinicaltheriogenology.net/index.php/ct/issue/view/579 https://clinicaltheriogenology.net/index.php/ct/issue/view/584 https://clinicaltheriogenology.net/index.php/ct/issue/view/584 https://clinicaltheriogenology.net/index.php/ct/issue/view/584 https://clinicaltheriogenology.net/index.php/ct/issue/view/584 2012: use of an impact score to guide client decision-making about timing of spay-castration of dogs and cats use of an impact score to guide client decision-making about timing of spay-castration of dogs and cats margaret v. root kustritz college of veterinary medicine, university of minnesota, st. paul, mn abstract veterinarians were solicited for opinions regarding morbidity of health disorders associated with gonadectomy of dogs and cats. morbidity score was assigned using a standard scale. average morbidity score was multiplied by incidence to yield an impact score, with positive impact scores associated with better health after gonadectomy and negative impact scores associated with poorer health after gonadectomy. for both genders and species, obesity was associated with a fairly large negative impact score. for female dogs and cats, benefits of ovariohysterectomy (ohe) outweighed detriments and prepuberal ohe is recommended. for male dogs, benefits of castration did not outweigh detriments until the animal was likely to develop age-related, benign diseases of the reproductive tract, or about 2.5 years of age. keywords: ovariohysterectomy, castration, gonadectomy, dog, cat introduction benefits of ovariohysterectomy (ohe) and castration have been well reported in the literature and include societal benefits, mostly regarding population control, and health benefits. there is an increasing body of information identifying increased predisposition to disorders secondary to gonadectomy; reviews of these pros and cons have been published.1-4 the difficulty for the pet owner and practitioner lies in determining what is the greatest good for the animal when balancing association with gonadectomy of detrimental disorders that are common but of low morbidity with disorders that are uncommon but of high morbidity. creation of a single score that incorporates morbidity and incidence would help guide discussion by better permitting the veterinarian to educate the owner without providing either too little information to assist in decision-making or so much information as to be overwhelming. material and methods veterinarians who are members of the society for theriogenology were solicited for their opinions regarding morbidity of disorders associated with gonadectomy in dogs and cats. the morbidity rating scale used was 0 = no morbidity, 3 = morbidity present but does not require treatment, 5 = morbidity requires non-invasive or acute treatment, 7 = morbidity requires invasive or chronic treatment, and 10 = mortality likely despite treatment. the mean morbidity score was multiplied by incidence to create an impact score for each health disorder, with a negative impact score denoting risk to the animal from gonadectomy and a positive impact score denoting benefit to the animal from gonadectomy. disorders included in the survey were those reported to be associated with gonadectomy that are most common or most deleterious to the animal, and that are well described in the literature. results and discussion fifty people responded to the anonymous survey. mean number of years in practice was 23.4 with a range of 3 to 42 years (n = 45). thirty-one respondents (64.6%) were in a small animal exclusive practice, 10 (20.8%) in a mixed animal practice, 5 (10.4%) in a small animal/exotics/avian practice, and 2 (4.2%) in some other type of practice. thirty-one participants completed morbidity ratings in the survey. morbidity ratings and impact scores are shown in the table. female dogs benefits of gonadectomy are decreased incidence of mammary neoplasia when aged and decreased incidence of pyometra. detriments are complications during and after surgery; increased 481 clinical theriogenology • volume 4, number 4 • december 2012 incidence of osteosarcoma, hemangiosarcoma, and transitional cell carcinoma; increased incidence of urethral sphincter mechanism incompetence; increased incidence of rupture of the cranial cruciate ligament (ccl); and obesity. incidence of mammary neoplasia is 3.4% and mean morbidity rating was 7.1. it has been well demonstrated that ohe prior to the first heat cycle greatly reduces incidence of mammary neoplasia later in life, with a decreasing benefit as the bitch goes through successive heat cycles.5-8 pyometra occurs in 15.2% of dogs by four years of age and 23 to 24% by ten years of age.9,10 mean morbidity rating was 6.6. surgery is curative of most cases of pyometra at the time of diagnosis, with reported mortality rate of 0 to 17%.11 medical therapy is not curative, as underlying cystic endometrial hyperplasia persists, leaving the bitch susceptible to redevelopment of pyometra at her next estrous cycle. surgical complications are reported to occur in 6.1% of dogs after gonadectomy, with the vast majority of these complications so mild as to not require veterinary intervention.12 mean morbidity rating was 3.3. risk of some forms of neoplasia is increased with gonadectomy; cause-and-effect of this association is not reported. incidence of osteosarcoma, hemangiosarcoma, and transitional cell carcinoma are 0.2%, 0.2%, and less than 1.0%,13-15 and mean morbidity rating were 9.3, 8.6, and 8.2, respectively. urethral sphincter mechanism incompetence, also called estrogen-responsive urinary incontinence, is very common with reported incidence of 4.9 to20.0%.16-18 mean morbidity rating was 5.3. increased incidence of rupture of the ccl has been reported after gonadectomy, independent of increase in body weight.19-21 incidence of rupture of the ccl is 1.8% and mean morbidity rating was 5.9. obesity is the most common nutrition disorder of dogs, with reported incidence of 2.8%.22 gonadectomy is a known risk factor, perhaps due to changes in metabolic rate or satiety. mean morbidity rating was 4.9. female cats the primary benefit of gonadectomy of female cats is decreased incidence of mammary neoplasia when aged. detriments are complications during and after surgery, and obesity. mammary neoplasia in female cats is common, with reported incidence of 2.5%. tumors virtually always are adenocarcinoma with local invasion and metastasis to distant sites.5,23 mean morbidity rating was 7.7. surgical complications are reported to occur in 2.6% of cats after gonadectomy, with the vast majority of these complications so mild as to not require veterinary intervention.12 mean morbidity rating was 2.8. obesity is the most common nutrition disorder of cats, with reported incidence of 6.4%.24 gonadectomy is a known risk factor, due to changes in metabolic rate.25 mean morbidity rating was 4.4. male dogs benefits of gonadectomy in male dogs are decreased incidence of testicular neoplasia and benign prostatic hypertrophy (bph). detriments are complications during and after surgery; increased incidence of prostatic neoplasia, osteosarcoma, hemangiosarcoma, and transitional cell carcinoma; increased incidence of rupture of the ccl; and obesity. testicular neoplasia is common in middle-aged to older dogs with a reported incidence of 0.9%.26 mean morbidity rating was 5.4. bph is a normal aging change of the prostate in dogs, occurring in 75 to 80% of dogs by 6 years of age.27-29 mean morbidity rating was 4.9. castration at the time of diagnosis is curative of either testicular neoplasia or bph. surgical complications are reported to occur in 6.1% of dogs after gonadectomy, with the vast majority of these complications so mild as to not require veterinary intervention.12 mean morbidity rating was 2.7. risk of some forms of neoplasia is increased with gonadectomy; cause-and-effect of this association is not reported. incidence of prostatic neoplasia, osteosarcoma, hemangiosarcoma, and transitional cell carcinoma are 0.2 to 0.6%, 0.2%, 0.2%, and less than 1%,13-15,30-32 and mean morbidity ratings were 7.7, 9.3, 8.4, and 8.2, respectively. increased incidence of rupture of the ccl has been reported after gonadectomy, independent of increase in body weight.19-21 incidence of rupture of the ccl is 1.8% and mean morbidity rating was 5.9. 482clinical theriogenology • volume 4, number 4 • december 2012 obesity is the most common nutrition disorder of dogs, with reported incidence of 2.8%.22 gonadectomy is a known risk factor, perhaps due to changes in metabolic rate or satiety. mean morbidity rating was 4.6. male cats there are no medical benefits reported after castration of male cats. all benefits are behavioral (decreasing sexual arousal, spraying of foul-smelling urine, aggression) or societal (decreased indiscriminate breeding). detriments are complications during and after surgery and obesity, as in the female cat. mean morbidity ratings for surgical complications and obesity in male cats were 1.2 and 4.1, respectively. conclusion this is the first description of impact factor analysis as a means of guiding client decision-making regarding timing of gonadectomy surgery. the author chose those disorders she believed to be best supported in the veterinary literature and of greatest significance to individual animals. the group of veterinarians solicited for opinions regarding morbidity were members of the society for theriogenology and a moderate number responded. it may be that results would be different if other disorders were investigated and if a different or larger group of veterinarians had been surveyed. for all genders and species, obesity is a significant detriment to gonadectomy. of the disorders listed, this is the one that is most readily controlled by the animal’s owner. most pet owners do not wish their animals to be sexually active because of concerns about undesirable reproductive behaviors in males and females and physical changes associated with heat in females. use of the impact factor helps clients more readily see the effects of gonadectomy and opens the door for valuable communications with their veterinarian regarding proper diet and exercise for their animal. disregarding obesity, it is clear that benefits outweigh detriments for ohe of bitches and queens. because it has been well established that ohe prior to the first heat has the greatest benefit in decreasing incidence of mammary neoplasia later in life in both dogs and cats,33,34 it is recommended that bitches and queens undergo ohe before puberty. for male dogs, the benefit of castration in decreasing incidence of bph is very large because it is such a very common disorder. however, castration at the time of diagnosis is curative and few dogs develop clinical manifestations of bph prior to 2.4 years of age, suggesting that in the absence of any other compounding factors, castration in dogs safely could be put off until that age.27-29 there is little information to permit use of impact factors to guide decision-making regarding castration of male cats. their aggressive natural breeding behaviors, ready sexual arousal, and spraying of foul-smelling urine all are sexually dimorphic behaviors that are readily controlled by castration.35 castration while the cat is prepuberal is recommended for this reason for any male cat intended to be a house pet. 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1998;39:559-566. 19. whitehair jg, vasseur pb, willits nh: epidemiology of cranial cruciate ligament rupture in dogs. j am vet med assoc 1993;203:1016-1019. 20. slauterbeck jr, pankratz k, xu kt, et al: canine ovariohysterectomy and orchiectomy increases the prevalence of acl injury. clin orthop relat res 2004;429:301-305. 21. duval jm, budsberg sc, flo gl, et al: breed, sex, and body weight as risk factors for rupture of the cranial cruciate ligament in young dogs. j am vet med assoc 1999;215:811-814. 22. mason e: obesity in pet dogs. vet rec 1970;86:612-616. 23. verstegen j, onclin k:mammary tumors in the queen. proc soc therio 2003; p. 239-245. 24. verbrugghe a, hesta m, daminet s, et al: nutritional modulation of insulin resistance in the true carnivorous cat: a review. crit rev food science nutr 2012;52:172-182. 25. root mv, johnston sd, olson pn: effect of prepuberal and postpuberal gonadectomy on heat production measured by indirect calorimetry in male and female domestic cats. am j vet res 1996;57:371-4. 26. hahn ka, von der haar ma, teclaw rf: an epidemiologic evaluation of 1202 dogs with testicular neoplasia [abstract]. j vet intern med 1992;6:121. 27. lowseth la, gerlach rf, gillett na, et al: age-related changes in the prostate and testes of the beagle dog. vet pathol 1990;27:347-353. 28. zirkin br, strandberg jd: quantitative changes in the morphology of the aging canine prostate. anat rec 1984;208:207-214. 29. berry sj, strandberg jd, saunders wj, et al: development of canine benign prostatic hyperplasia with age. prostate 1986;9:363-373. 30. bell fw, klausner js, hayden dw, et al: clinical and pathologic features of prostatic adenocarcinoma in sexually intact and castrated dogs: 31 cases (1970-1987). j am vet med assoc 1991;199:1623-1630. 31. weaver ad: fifteen cases of prostatic carcinoma in the dog. vet rec 1981;109:71-75. 32. teske e, naan ec, van dijk em, et al: canine prostate carcinoma: epidemiological evidence of an increased risk in castrated dogs. mol cell endocrinol 2002;197:251-255. 33. schneider r, dorn cr, taylor do: factors influencing canine mammary cancer development and postsurgical survival. j natl cancer inst 1969;43:1249-1261. 34. overley b, shofer fs, goldschmidt mh, et al: association between ovariohysterectomy and feline mammary carcinoma. j vet intern med 2005;19:560-563. 35. hart bl, barrett re: effects of castration on fighting, roaming, and urine spraying in adult male cats. j am vet med assoc 1973;163:290-292. 484clinical theriogenology • volume 4, number 4 • december 2012 table: impact* on health of male and female dogs after gonadectomy disorder female dog male dog female cat male cat mammary neoplasia +24 +19 pyometra§ +100 surgical complications -20 -16 -7 -3 osteosarcoma -2 -2 hemangiosarcoma -2 -2 transitional cell carcinoma§§ -7 -7 prostate neoplasia§§§ -3 testicular neoplasia +5 urethral sphincter mechanism incompetence§§§ -66 benign prostatic hypertrophy§§§ +368 rupture of the cranial cruciate ligament -11 -11 obesity -14 -13 -28 -26 *positive impact score = benefit from gonadectomy, negative impact score = detriment from gonadectomy  § incidence used for calculation of impact score was 15.2%. §§ incidence used for calculation of impact score was 0.9%. §§§ incidence used for calculation of impact score was the average of those reported. 485 clinical theriogenology • volume 4, number 4 • december 2012 486clinical theriogenology • volume 4, number 4 • december 2012 omniblank: 1 case report recurrent preputial sarcoids in a miniature donkey josefina ghersa,a alannah friedlund,a keri thomas,a bruce wobeser,b claire carda adepartment of large animal clinical sciences bdepartment of veterinary pathology, western college of veterinary medicine, university of saskatchewan, saskatoon, sk, canada abstract sarcoids are common dermal neoplasms that have predilection for paragenital tissues in jacks. sarcoids have variable appearance, do not metastasize but may be locally invasive and certain forms may grow rapidly. various treatment options are available for preputial sarcoids, including surgical excision for large masses. we describe a case of mixed sarcoid affecting the preputial tissues of a miniature donkey gelding that was excised, regrew, and required a second surgical procedure involving preputial ablation. keywords: donkey, gelding, sarcoid, preputial lesions background sarcoids are common type of dermal neoplasm that affect domestic equids. compared to horses, donkeys have been overrepresented in diagnostic lab submissions of sarcoids.1 sarcoids do not metastasize and vary in appearance. they may be classified into occult, verrucus, nodular, mixed, fibroblastic, and malevolent types. compared to jennies, jacks are at increased risk for sarcoid formation; paragenital area is the most common location reported for sarcoids in jacks.2 younger animals are more commonly affected; furthermore, there may be a genetic predisposition for sarcoids. the underlying etiology of most sarcoids is a cross-species infection with bovine papilloma type 1 or type 2 virus.3 bovine papilloma viruses have been recovered from many donkey sarcoids, and the type varied by region.1,2 these viruses are very persistent in the environment.3 other cutaneous tumors such as squamous cell carcinoma and melanoma may mimic some features of sarcoids. case presentation a 12-year, grey miniature donkey gelding, weighing 140 kg, was presented for removal of a possible recurrent sarcoid mass in preputial tissues and penile sheath. donkey was gelded at 1 year; for over 9 years had been housed in the same facilities with alpacas, sheep, pigs, and horses. in 2017, the gelding had undergone surgery for excision of 2 (4 cm) ulcerated nodular masses on the external penile sheath and 2 (1 cm) masses on the internal penile sheath. histopathology confirmed mixed sarcoid with incomplete surgical excision. in 2023, the gelding was examined for dropping some feed. a  dental examination and corresponding treatment were performed and subsequently clinical signs of dropping feed resolved. on physical examination, gelding was bright alert and responsive, temperature was 36.9°c, heart rate was 48 beats per minute, and respiratory rate was 24 breaths per minute; cardiopulmonary auscultation was unremarkable. gelding had a body condition score of 7.5/9 and a cresty neck score of 5/5. firm, smooth masses were noted in relationship to the external preputial lamina: a large mass (8 x 4 cm) was located on the ventral aspect, multiple small nodules (2 x 2 cm) were present on the dorsal preputial aspect, and ~ 4 cm pedunculated mass was on the left ventral preputial side (figure 1). the left superficial inguinal lymph node was palpable whereas the right was not palpable. the differential diagnosis from the gross appearance of the masses included recurrent mixed sarcoid, squamous cell carcinoma, and melanoma. a month later, the gelding was evaluated and surgical removal of the preputial masses was recommended. masses observed earlier had coalesced into 1 large ulcerated caudo-ventral mass (12 x 9 cm) with firm and semi-firm areas. there was also an associated pedunculated ulcerated mass (2.7 x 3.3 cm) on the left side of the external preputial lamina. the gross appearance of the tissue (figures 2a and 2b) was compatible with a mixed nodular fibroblastic type sarcoid. the urethra, glans penis, and penile shaft were not compromised by the masses. surgical procedure three months later, gelding was prepared for surgery; feed was withheld for 8 hours, a jugular vein was catheterized, and 3 million iu of intravenous sodium penicillin (fresenius kabi canada ltd., toronto, on, canada) diluted in 0.9% contact josefina ghersa josefina.ghersa@usask.ca © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12221, http://dx.doi.org/10.58292/ct.v17.12221 mailto:josefina.ghersa@usask.ca http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11199 2 citation: clinical theriogenology 2025, 17, 12221, http://dx.doi.org/10.58292/ct.v17.12221 saline solution along with 4.4 mg/kg of phenylbutazone (rafter 8 products inc., calgary, ab, canada) were given. gelding was given 1.25 mg/kg intravenous xylazine (rompun®, elanco canada ltd., mississauga, on, canada) as a premedication and general anesthesia was induced with ketamine 2.2 mg/kg (narketan®, vetoquinol, lavaltrie, qc, canada) and diazepam 0.1 mg/kg (sandoz canada inc, boucherville, qc, canada), and was intubated and maintained with isoflurane (aerrane, baxter corporation, mississauga, on, canada) anesthetic gas. gelding was placed in dorsal recumbency on the surgical table. penis was extended by traction on gauze looped around the collum glandis; ventral abdomen including the prepuce was clipped and aseptically prepared. penis was catheterized and gelding was moved into the operating room and draped for sheath ablation. an elliptical incision was made using a size 22 blade around the sheath from the preputial opening to the caudal extent of the mass. dissection of the mass down to normal tissue was performed using mayo scissors (figure  2c). electrocautery was used as needed to control hemorrhage from smaller vessels. large vessels were ligated using circumferential ligatures of 2-0 poliglecaprone 25 (monocryl™, ethicon) and then transected. penis was palpated in its position underneath the mass during surgery in order to guide dissection. preputial tissue and a margin of adjacent skin were excised with the mass. cranial penile skin was closed using 1  and 0 polydioxanone suture (pds™, ethicon) using a combination of tension relieving and simple interrupted sutures. remaining preputial tissue was sutured circumferentially to the scrotal tissue using 1 polydioxanone suture (pds™, ethicon) in a simple interrupted pattern. dorsal penile skin was closed intradermally with 3-0 poliglecaprone 25 (monocryl™, ethicon). five tension releasing incisions were made on each side of the suture line and 2 incisions were made on either side of the scrotum for drainage (figure 2d). gelding was moved to the recovery room and placed on a mat; recovered uneventfully from anesthesia. postoperative management postoperatively swelling was noted at the surgery site (figure 2e). every 6 hours, a hot pack was placed for 5 minutes in the swollen area. penicillin at 22,000 iu/kg (fresenius kabi, canada) was continued every 6 hours for 3 days. antimicrobial treatment was continued with oral trimethoprim sulfamethoxazole 30 mg/kg (sulfatrim ds, aa pharma inc., toronto, on, canada) twice daily for 5 days. for pain management, phenylbutazone at 4.4 mg/kg (dominion veterinary laboratories ltd., winnipeg, mb, canada) was continued once daily for 4 more days. gelding was also hand-walked every 6 hours and fed hay 4 times daily at a total of 2% of body weight. gross and histopathologic evaluation combined weight of the excised masses was 800 g (figure 2d). masses were fixed in 10% buffered formalin. specimens were obtained from various sections of the masses. fixed tissues were embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin using an automated slide stainer at prairie diagnostic services, saskatoon, sk, canada. tissues had well-demarcated unencapsulated, moderately cellular focally infiltrative neoplastic mass. epithelium was multifocally ulcerated and associated with long, thin rete peg-like skin extensions. cells within the mass were arranged in short streams and arcs within a fibrovascular stroma. mitotic figures were very rare (< 1/10 in 400 x magnification). there were occasional aggregates of inflammatory cells scattered through the mass. surgical margins and complete excision of the mass  could not be accurately assessed due to the size (figure 3). the diagnosis was a mixed sarcoid. outcome end of the month gelding had a follow-up examination; surgical wound cranial to the prepuce had partially dehisced and had secondary bacterial infection. surgical wound was washed with clorhexidine gluconate 4% (stanhexidine®, omega laboratories ltd., montréal, qc, canada) and water and dried. silver sulphadiazine 1% cream (flamazine®, smith & nephew, mississauga, on, canada) was applied over the infected site once daily for 4 days, after washing and drying of the site. systemic antimicrobial treatment was prescribed with oral trimethoprim sulfamethoxazole 30 mg/kg (sulfatrim ds, aa pharma inc.) twice daily for 7 days. gelding was also stabled until complete healing of the infected wound. a week later, second examination was performed: surgical site has made favorable progress with no signs of continuing infection and closed suture wound. three months later, owners reported complete healing of the suture wound and no resurgence of new masses. no urinary complications have been noted to that day. a follow-up examination 6 months later indicated that masses were recurring in the prepuce. figure 1. regrowth of the sarcoid masses following surgical resection in 2017: a. gross appearance of sarcoid masses (white arrow heads and yellow arrow) in preputial area; b. 4 cm pedunculated sarcoid mass (yellow arrow); cr: cranial and ca: caudal side of gelding http://dx.doi.org/10.58292/ct.v17.12221 citation: clinical theriogenology 2025, 17, 12221, http://dx.doi.org/10.58292/ct.v17.12221 3 discussion equine sarcoids represent 72% of all neoplasms in donkeys, and 82% of cutaneous neoplasms.4 the inguinal region is the second most common place of incidence of sarcoids in donkeys, and the prepuce is the most common location in jacks. unlike horses, other cutaneous tumors such as squamous cell carcinoma, melanomas and lymphosarcomas are rare. equine sarcoids are usually diagnosed by clinical appearance. a diagnostic protocol as a tool for the clinical workup and diagnosis of equine sarcoid has been developed.5 the gold standard is biopsy and histopathologic figure 2. mixed preputial sarcoid in 2023: a. preoperative preputial region with a white piece of gauze wrapped around glans penis; b. urinary catheter in penile urethra with preputial area prepared for surgery; c. large circular area surgically resected cranial and caudal to preputial orifice; d. postoperative surgical site after resection of sarcoid masses; e. resected preputial sarcoid tissue; f. postoperative lateral view of the surgical site with penis out of preputial cavity. in all figures, black arrows point glans penis (in b, c, d with urinary catheter in place); yellow arrows refer to pedunculated mass; white arrowheads point to large ulcerated preputial mass; cr: cranial and ca: caudal side of gelding figure 3. light microscopic images of preputial sarcoid: a. superficial epithelial crusting and elongated, thin rete pegs (black arrows) with adjacent neoplastic cells (200 x magnification); b. and c. higher magnification (400 x) of a rete peg showing a picket fence–like arrangement of neoplastic cells along the dermal-epidermal border (black arrow) http://dx.doi.org/10.58292/ct.v17.12221 4 citation: clinical theriogenology 2025, 17, 12221, http://dx.doi.org/10.58292/ct.v17.12221 evaluation. however, biopsy-induced trauma or irritation might induce proliferation and aggravate the mass type and size.6 consequently, other noninvasive diagnostic tools are studied. fine needle aspiration (fna), and subsequent polymerase chain reaction to detect bovine papilloma virus (bpv), has promising results for sampling ulcerated and nonulcerated masses.7 fna creates less damage to the tissue than a biopsy sample and has less false positive results from latent bpv in superficial keratinocytes. other complementary diagnostic tests such as microrna biomarkers have been studied; however, they are not yet as sensitive.8 to the best of the authors’ knowledge, 5 preputial sarcoid cases have been reported in donkeys. a verrucus ulcerated mass on the lateral preputial area was associated with permanent paraphimosis.9 in egypt, 2 preputial sarcoids were reported in donkeys.10 treatment for all cases involved surgical excision of the masses, with no adjunctive therapy. conventional surgical excision alone has been successful in 30-50% of the cases, and unsuccessful cases had recurrence within 6 months.6,10 one jack in egypt had recurrence of sarcoid masses 3 months after first excision.10 on the contrary, successful surgical removal of a sarcoid from a teat has also been reported in a jenny, and 2 years after surgery the jenny has not had any relapses.11 in the present case, the exact time for recurrence of the masses from was unknown, as the owner only noted the masses when they had reached a considerable size. nevertheless, the first excision did not have clear surgical margins and thus recurrence was expected. the second surgery was not successful either in obtaining clear surgical margins and resulted in recurrence of sarcoid masses after 6 months. various treatments for equine sarcoid have been described, with variable results. sarcoid treatments that had regression rates > 90% were brachytherapy, radiation, cryotherapy, intralesional cisplatin, or electrochemotherapy.12 however, there is insufficient evidence to determine the efficacy of one above the other. surgical debulking is often needed prior to forementioned therapies to reduce tumor volume and increase success rate. other therapies that need further investigation are immunotherapy with intratumoral injections of bacillus calmette and guerin, topical application of imiquimod cream, and therapeutic vaccines.6,12 treatment selection primarily depends on resources available, economic factors, owners’ capability and predisposition, masses location in the body, and size and type of sarcoid. in the present case, mass size was the predominant factor taken into consideration to choose surgical removal. adjunctive chemotherapy with biodegradable cisplatin beads was a therapeutic option; however, this was not performed due to cost. moreover, the extent to which the tumor invaded adjacent tissues and compromised parts of  the penis was not known prior to surgical procedure. miniature donkey’s wellness, including the possibility to urinate without complications, was a major consideration for surgery. masses were removed attempting to have as much margin as possible resulting in loss of considerable amount of abdominal skin, external sheath and part of internal sheath. consequently, closure of the surgical wound was challenging. edema and dehiscence were expected due to surgical wound location and surrounding skin tightness. in western canada, donkeys are at higher risk than horses for developing sarcoids.1 bpv has also been widely associated with equine sarcoids. bpv type 2 was the predominant virus in sarcoids.1 papillomavirus infections are mostly species-specific, and the natural host for bpv is cattle. however, bpv may also cause cross-species infection, most commonly in equids resulting in sarcoid lesions. in the present case, for the past 9 years the miniature donkey has not been in contact with any cattle but with other ruminants. bpv may persist in the environment. there was no history to the previous housing situation. it is noteworthy that bpv has been reported to produce wart-like lesions in  sheep and that viral transmission can occur through fomites that contact open skin lesions.13,14 in conclusion, preputial ablation was used for treatment of recurrent sarcoid mass. alternative therapies (e.g. injection of biodegradable cisplatin beads) should be pursued to treat smaller recurring masses. learning points • sarcoids may affect paragenital tissues in donkeys • sarcoids do not metastasize but are locally invasive and may grow quickly • large preputial sarcoid may involve surgical removal including preputial ablation and postoperative monitoring to evaluate regrowth of the sarcoid conflict of interest authors declare no conflict of interest. authors’ contribution all authors contributed to the writing of the manuscript; al and kt performed surgery; bw performed histopathologic evaluation. references 1. wobeser bk, davies jl, hill je, et al: epidemiology of equine sarcoids in horses in western canada. can vet j 2010;51:1103-1108. pmcid: pmc2942047 2. reid s, gettinby g, fowler j, et al: epidemiological observations on sarcoids in a population of donkeys (equus asinus). vet rec 1994;134:207-211. doi: 10.1136/vr.134.9.207 3. knottenbelt d, edwards s, daniel e: diagnosis and treatment of the equine sarcoid. practice 1995;17:123-129,199. doi: 10.1136/ inpract.17.3.123 4. davis cr, valentine ba, gordon e, et al: neoplasia in 125 donkeys (equus asinus): literature review and a survey of five veterinary schools in the united states and canada. j vet diagn invest 2016;28:662-670. doi: 10.1177/1040638716665659 5. haspeslagh m, gerber v, knottenbelt dc, et al: the clinical diagnosis of equine sarcoids-part 2: assessment of case features typical of equine sarcoids and validation of a diagnostic protocol to guide equine clinicians in the diagnosis of equine sarcoids. vet j 2018;240:14-18. doi: 10.1016/j.tvjl.2018.08.010 6. taylor s, haldorson g: a review of equine sarcoid. eq vet ed 2013;25:210-216. doi: 10.1111/j.2042-3292.2012.00411.x 7. gysens l, martens a, haspeslagh m: cross-sectional comparison of superficial swab and fine-needle aspiration: improving the http://dx.doi.org/10.58292/ct.v17.12221 https://doi.org/10.1136/vr.134.9.207 https://doi.org/10.1136/inpract.17.3.123 https://doi.org/10.1136/inpract.17.3.123 https://doi.org/10.1177/1040638716665659 https://doi.org/10.1016/j.tvjl.2018.08.010 https://doi.org/10.1111/j.2042-3292.2012.00411.x citation: clinical theriogenology 2025, 17, 12221, http://dx.doi.org/10.58292/ct.v17.12221 5 diagnostic workup of horses with sarcoids. vet j 2022;289:105916. doi: 10.1016/j.tvjl.2022.105916 8. unger l, abril c, gerber v, et al: diagnostic potential of three serum micrornas as biomarkers for equine sarcoid disease in horses and donkeys. j vet intern med 2021;35:610-619. doi: 10.1111/jvim.16027 9. oliveira m, moreira ar, melo-franco, et al: surgical resolution of preputial sarcoid in a donkey. equine vet j 2013;33:809-812. doi: 10.1016/j.jevs.2012.12.009 10. rizk a, mosbah e, karrouf g, et al: surgical management of penile and preputial neoplasms in equine with special reference to partial phallectomy. j vet med 2013;891413. doi: 10.1155/2013/891413 11. rizk a: unilateral amputation of the teat for treatment of equine sarcoid in a she-donkey. j eq vet sci 2016;46:15-17. doi: 10.1016/j. jevs.2016.07.010 12. offer ks, dixon ce, sutton dgm: treatment of equine sarcoids: a systematic review. equine vet j 2024;56:12-25. doi: 10.1111/evj.13935 13. mazzuchelli-de-souza j, de carvalho rf, módolo dg, et al: first detection of bovine papillomavirus type 2 in cutaneous wart lesions from ovines. transbound emerg dis 2018;65:9. doi: 10.1111/tbed.12892 14. savini f, gallina l, prosperi a, et al: bovine papillomavirus 1 gets out of the flock: detection in an ovine wart in sicily. pathogens 2020;9:429. doi: 10.3390/pathogens9060429 http://dx.doi.org/10.58292/ct.v17.12221 https://doi.org/10.1016/j.tvjl.2022.105916 https://doi.org/10.1111/jvim.16027 https://doi.org/10.1016/j.jevs.2012.12.009 https://doi.org/10.1155/2013/891413 https://doi.org/10.1016/j.jevs.2016.07.010 https://doi.org/10.1016/j.jevs.2016.07.010 https://doi.org/10.1111/evj.13935 https://doi.org/10.1111/tbed.12892 https://doi.org/10.3390/pathogens9060429 1 a note of appreciation editorial team thanks the following scholars for reviewing manuscripts published in volume 16 *editorial board member numbers within parentheses indicate manuscripts reviewed anum ahmed *divakar ambrose (3) chance armstrong christine aurich *scott bailey *orsolya balogh james barrett fernando campos-chillon claire card elaine carnevale michelle caissie jorge chacon tracey chenier *sherrie clark-deener *gail colebern *jill colloton dinesh dadarwal lionel dawson daniela demetrio callum donnelly peyton draheim *ivan filho jacques fuselier edgar garrett mohanthas gobikrushanth *allan gunn camilo hernandez-aviles (2) milan hess mohmmad ibrahim peter irons claudia jimenez-escobar *khalid el allali *vanmathy kasimanickam (2) john kastelic dale kelley jessica klabnik *kara kolster jennifer koziol chun kuen mak *michelle kutzler *réjean lefebvre rasa levstein paul loomis patrick mccue celeste morris amber nebel-karp scott norman malgorzata pozor jo randall jessica rush maria schnobrich claire scully harald sieme brittany southern edward squires *cyril stephen (2) nicole sugai *masa tanuguchi (2) dan tracy stephanie walbornn *brian whitlock 2016: 2015 theriogenologist of the year 2015 theriogenologist of the year r.o. gilbert department of clinical sciences, college of veterinary medicine, cornell university, ithaca, ny i am deeply honored to be recognized in this way, especially by colleagues for whom i have enormous respect. this award was completely unexpected. when first contacted by the act i thought it was a request for a letter of support for someone else. i was completely surprised to learn i was actually to be the recipient. thank you very, very much. i am especially grateful to those who invested time and effort to nominate me or support the nomination, all without breathing a word to me. i hope i am not leaving people out, but these include augustine peter, barry ball, gregg adams, don schlafer and soon hon cheong. many, many thanks to all. i am very proud to be a veterinarian, and very proud to be a theriogenologist. to be elected theriogenologist of the year is an enormous honor and extremely humbling. my thanks to you all. this is where it started. the veterinary college of the university of pretoria, situated at onderstepoort, some 15 miles north of the main campus in central pretoria. the inset shows graduation day in 1977. like all of you, i have been profoundly influenced by several people in my professional life and i would like to mention a few. as a high school pupil, and throughout my veterinary education, i saw practice with dr. margaret hearn. more than anyone else, she probably influenced my decision to become a veterinarian. she was an amazing person, graduating from the royal veterinary college in london in 1935. there were very few women in veterinary colleges at that time. three years later, in 1938, the first loveday report would recommend strongly against use of public funds to educate women in veterinary schools. luckily, professor frederick hobday, then the principal, disagreed and forged on in defiance of prevailing opinion and the later formal report. in 1936 dr. hearn emigrated to south africa with her husband and went into private practice with dr. jack boswell. this was no trivial endeavor. this was the first private practice in south africa. until this time, essentially all veterinarians were in the employ of national or provincial governments. dr. hearn came to be regarded as a specialist surgeon, although there were no formally recognized specialists at that time. so margaret hearn was an indomitable woman. the first time i watched her do surgery her instructions to me were: “you may feel queasy. if you do, fall that way.” she was nearing the end of her remarkable career when i interacted with her but she influenced me greatly. many of you will know bill jenkins, the best teacher i had from grade school to grad school. he taught me physiology and pharmacology at veterinary school. later he moved to the usa, becoming first a professor at texas a&m, then dean at lsu. he went on to become provost, president and ultimately chancellor of the louisiana system. not only was bill jenkins an exceptional teacher, he was a wise mentor who took a keen interest in his students. by the time i graduated i knew i wanted to be a theriogenologist, and that i wanted to work in academia. my theriogenology professor, against the policy of the time, offered me a position upon graduation. when i spoke to dr. jenkins he told me that there would be more opportunities at the university. i should go out into practice. without private practice experience i would be of less value to my own students in the future. i came to respect this advice later and was grateful for it. those of us in academia are often inclined to think we are at the center of the profession and it all revolves around us. not so. the backbone of our great profession consists of the men and women who take care of animals and their owners every day, in the front lines of veterinary practice. i have never forgotten this. so, off to practice i went. my first job was in a town called ladysmith, in the foothills of the drakensberg. this was a lovely area, and the practice was very varied, with dairy and beef cattle, sheep, swine, poultry, racing pigeons, horses including thoroughbred broodmares, polo ponies and pleasure horses as well as all kinds of companion animals. the practice area was large and we spent too much time driving, devoting 80 % of our time to farm calls and making 80 % of our income from companion animals. 7317575 clinical theriogenology · volume 8, number 1 · march 2016 my employer had been in solo practice for seven years, without missing a single day of work. the evening i arrived, he announced he would be leaving on vacation in the morning. so there was a baptism by fire, which i more or less survived. i started in the practice on december 12, 1977. people observing me at that time would not have expected me to be standing before you as an honoree today. on christmas day i had a call for a bovine dystocia, which the farmer had already worked on for some time. i expected it would require a cesarean section, so i packed my equipment into my practice car and drove the 50 or so miles to the farm, taking with me my wife of a little over a month. when we arrived, the cow was in a small pen. neatly arranged around her were tiers of straw bales. the farmer’s entire extended family was visiting him and i realized that watching the c-section was to be the afternoon entertainment. i forgot to mention that i had managed to graduate from veterinary school without even seeing a bovine cesarean; this was to be the first one i ever saw or performed. things went well as i examined the cow, confirmed that the section was indicated, blocked her and prepared her. things even went well as i located and exteriorized the uterus, incised it and delivered the calf. unfortunately, the calf did not survive. at this point i asked my assistant, my new wife, to pass me the suture material, and soon i realized a problem was looming. nobody in veterinary school had told us how much suture material it took to close up a cow. i thought i had thrown in plenty, and a few packs to spare. however, by the time i had closed the uterus, i had used almost all the absorbable suture material i had, and closure of the abdominal wall took all the rest of the sterile suture material – absorbable and non-absorbable. the skin was still gaping open, and my appreciative audience was unaware of my plight. i summoned the farmer and had a brief conversation. he quickly fetched his fishing equipment and a bowl of alcohol. we soaked fishing line in alcohol, and used it to close the incision. cows being resilient patients, she made a good recovery. it did start a trend though, and i think i did bovine cesarean sections on three of the next four christmas days – albeit with the necessary supplies. i obviously needed a lot of help, and i received it. i have been shaped as a veterinarian and particularly as a theriogenologist by a large number of extremely talented people, and i would like to pay tribute to some of them. a few were formal mentors and these included professors brough coubrough and henk bertschinger in south africa and bill bosu in wisconsin. several other distinguished theriogenolgists “adopted” me and treated me as if i were a formal mentee. in this way i received generous advice and help from ollie ginther in wisconsin and although i was not formally a member of his group he allowed me to participate in his group during a very exciting period of development of reproductive ultrasonography in horses and cattle. at cornell, steve roberts and ken mcentee, both retired, gave me regular advice and many of their teaching materials. bob kenney was equally generous with time and advice although he was obviously in pennsylvania, and bob hillman was and remains a source of much information and insight. apart from formal and informal mentoring i have learned a great deal from my interactions with the many extremely talented people who have been my colleagues in pretoria, wisconsin and at cornell, some of whom are listed here, and many of whom have achieved great distinction. not only have i learned from extraordinary theriogenologists and reproductive scientists, but many colleagues in other disciplines have taught me much about my own discipline. so i am grateful to sheila mcguirk, tom divers and ken hinchcliff for much that they have taught me about medical matters, to susie fubini and rick hackett for making me a better reproductive surgeon and to barry cooper and dave slauson for many insightful conversations about pathogenesis of disease in general and inflammation in particular. in the same vein i could never have made progress in research without wonderful collaborators, including ron butler, chuck guard, rodrigo bicalho, stephen leblanc and martin sheldon. teaching is a two way process and i am sure i have learned as much from students and trainees as they have from me. we are extremely privileged in veterinary academia to work with some of the most talented, intelligent, dedicated young people that there are. their curiosity, questions and sometimes even 7427676clinical theriogenology · volume 8, number 1 · march 2016 disagreements lead us continually to new discoveries. i am grateful to all who have challenged me or prodded me to explore new questions. the list of people i have learned from continues. animal owners, animal caretakers and many others have impressed me with their powers of observation and insightful comments, even if they do not always have the right explanations for their observations. perhaps my early career in ladysmith with a menagerie of species initiated my ongoing passion for comparative veterinary medicine and for comparative theriogenology. i am well aware of the demands for species-oriented practitioners in private practice settings. however, i believe that initial veterinary training, and initial specialist training in theriogenology, benefit from a comparative approach. the things we know in some species help us develop new knowledge in other species, sometimes by extrapolation, sometimes by similarities, other times by marked differences. sometimes the fact that we know something in one species is useful even if only because it makes it obvious that we lack knowledge of the same process in another species and provides an incentive to explore or discover the missing information. a broad species knowledge also allows a platform for development of knowledge in wild or exotic species. in some instances, domestic animals make good models for reproductive processes or disorders in humans, and this also represents an arm of comparative theriogenology that we should develop more fully. my career has spanned an extremely exciting period in theriogenology. i graduated in time to be one of the first embryo transfer practitioners in cattle, sheep and pigs in south africa, and now we see cloning, transgenesis, ivf, icsi, gamete and embryo cryopreservation routinely in many species. likewise, our repertoire of diagnoses and treatments of reproductive disorders continues to increase rapidly, with considerable benefits to our patients, our clients, and to society in general. at the same time, there is a great deal of knowledge that resides in each of you, and which never reaches the profession as a whole. i urge you to share this information, formally or informally. i applaud those whose efforts have ensured that we have meetings like this one to exchange ideas and information. similarly, the advent of list serves like the ones that serve diplomates and others with an interest in reproduction, is a great boon to us all. even though i sometimes wonder if my old compatriot rob lofstedt does nothing but wait eyes on the monitor and fingers on the keyboard to respond to any request, i genuinely respect his willingness to contribute freely to debate and greatly appreciate his generosity in sharing his meticulously prepared photographs depicting reproductive processes and disorders. i tend to be a “lurker” on this list serve, and benefit more from the wisdom of others than i contribute. i suspect the same is true for many of you and i urge you to be more forthcoming with your opinions, knowledge, experience and insights. this fuels progress in our discipline faster than ever before. please participate. i have mentioned my respect for private practice. a lot of knowledge is generated in practice and is not readily disseminated to the wider profession. do not be afraid to disagree and to enter into respectful, even if vigorous, debate. without it, our continued advance is not possible. although we have made great progress and continue to do so, and although i have said how much i appreciate informal avenues for exchange of knowledge and opinion, much of our practice in theriogenology is still based on information that would be considered the least reliable of medical evidence. many routine practices lack evidence that they are efficacious, even some that are very common. i urge you all to be skeptical and questioning, and where possible to put habits and theories to the test in a rigorous way, and then to disseminate the results in a way that contributes to improved practice in animal reproduction to the benefit of all. theriogenology is not only fun. it is a critical discipline for our profession and our world. reproduction is fundamental to all animal production systems and improved reproduction is often key to enhancing sustainability and reducing the environmental footprint of such production systems. it is also of cardinal importance in companion and performance animal practice. reproduction is critical to preserving environmental health and species diversity, whether by improving reproductive success or by creative means of contraception. in these ways, our discipline is of enormous importance to the future of 7537777 clinical theriogenology · volume 8, number 1 · march 2016 our profession, and to the future of our society and even our planet. my hope is that we will all enjoy the practice of our discipline and feel some pride that it contributes to the welfare of animals, people and our environment in important ways. i have derived enormous pleasure and pride from being a theriogenologist. this pride and pleasure is only increased by the enormous honor you have bestowed on me today. i am deeply grateful and thank each and every one of you very much indeed. finally, i am grateful to my family. my wife and children have supported my every professional endeavor, and have sacrificed much to allow me to practice the profession i love. to them, my gratitude and love forever. thank you. 47647878clinical theriogenology · volume 8, number 1 · march 2016 2012: incidence, severity and factors associated with intrauterine fluid accumulation in mares after insemination with cooled or frozen semen incidence, severity and factors associated with intrauterine fluid accumulation in mares after insemination with cooled or frozen semen dirk k. vanderwall,a andrew r. schmidt,b raymond c. bostona adepartment of clinical studies, new bolton center, school of veterinary medicine, university of pennsylvania, kennett square, pa; bwisconsin equine clinic and hospital, oconomowoc, wi introduction under commercial breeding farm conditions the incidence of post-breeding intrauterine fluid accumulation in thoroughbred mares bred with natural service was approximately 15% and the incidence and severity of intrauterine fluid accumulation increased significantly with mare age.1 in intensively managed mares inseminated with frozen-thawed semen the incidence of post-breeding intrauterine fluid accumulation varied from 16%2 to 25%,3 and mare age was again found to be a significant factor, since the incidence of intrauterine fluid accumulation in mares aged 3 to 9 years, 10 to 16 years and greater than 16 years was 17%, 28% and 68%, respectively.3 to our knowledge, the incidence of intrauterine fluid accumulation following insemination with cooled semen has not been reported. the objective of this study was to determine the incidence, severity and factors associated with intrauterine fluid accumulation in commercial broodmares inseminated with cooled or frozen semen. keywords: equine, mare, insemination, cooled/frozen semen, intrauterine fluid materials and methods this study was performed by retrospectively examining the reproductive records of 156 client-owned mares (1 to 7 cycles/mare) that were managed for breeding with cooled or frozen semen during the 2011 breeding season. the following information was obtained from each breeding record: mare age, type of semen (cooled or frozen), presence (if any) of intrauterine fluid prior to insemination and presence (if any) of intrauterine fluid after insemination. fluid accumulation after insemination was recorded as none/minimal if no fluid was identified or the amount was so slight that no treatment was deemed necessary; mild when the fluid accumulation was treated with an ecbolic agent only; or moderate/severe when the fluid accumulation was treated with an ecbolic agent and uterine lavage. mare age, type of semen (cooled or frozen) and presence of intrauterine fluid prior to insemination were examined for associations with uterine fluid accumulation after insemination with multinomial logistic regression analysis using stata 12.1 software (statacorp, college station, tx). for analyses involving mare age, mares were categorized into three groups, 3 to 9 years, 10 to 15 years and >15 years. results the proportion of mare cycles with moderate/severe fluid accumulation after insemination with cooled and frozen semen was 17.7% and 28.6%, respectively (p<0.01). the proportion of cycles in mares aged 3 to 9, 10 to 15 and >15 years with moderate/severe fluid accumulation after insemination with cooled or frozen semen was 14.0%, 26.7% and 34.6%, respectively (p<0.05). the proportion of mare cycles with moderate/severe fluid accumulation after insemination with cooled or frozen semen was 18.5% for mares that did not have uterine fluid prior to insemination and 39.7% for mares that did have uterine fluid prior to insemination (p<0.001). discussion the findings from this study that approximately 18% of mare cycles in which cooled semen was inseminated and 28% of mare cycles in which frozen semen was inseminated resulted in moderate/severe intrauterine fluid accumulation after insemination are consistent with previous reports that approximately 15 to 25% of mares develop intrauterine fluid accumulation after insemination. similarly, these results agree with previous reports that aged mares are more likely to develop intrauterine fluid accumulation after insemination compared to younger mares. an important clinically-relevant finding of this study is that the presence of any intrauterine fluid prior to insemination was associated with a greater likelihood of 565 clinical theriogenology • volume 4, number 4 • december 2012 moderate/severe fluid accumulation after insemination. it has been reported that the presence of more than a 2 cm height of uterine fluid during estrus prior to breeding was a predictor of susceptibility to post-breeding endometritis;4 however, the results of this study suggest the presence of any ultrasonographically-detectable intrauterine fluid prior to insemination may be indicative of susceptibility to fluid accumulation after breeding. references 1. zent ww, troedsson mht, xue j-l. postbreeding uterine fluid accumulation in a normal population of thoroughbred mares: a field study. proc annu conv am assoc equine pract 1998; p. 64-65. 2. watson ed, barbacini s, berrocal b, et al: effect of insemination time of frozen semen on incidence of uterine fluid in mares. theriogenology 2001;56:123-131. 3. barbacini s, neechi d, zavaglia g, et al: retrospective study on the incidence of postinsemination uterine fluid in mares inseminated with frozen/thawed semen. j equine vet sci 2003;23:493-496. 4. brinsko sp, rigby sl, varner dd, et al: a practical method for recognizing mares susceptible to post-breeding endometritis. proc annu conv am assoc equine pract 2003; p. 363-365. 566clinical theriogenology • volume 4, number 4 • december 2012 2014: reproductive disorders of male camelids reproductive disorders of male camelids l.k. pearson, a. tibary comparative theriogenology, department of veterinary clinical sciences, washington state university college of veterinary medicine, pullman, wa introduction unlike the cattle and sheep industries, there are no standards for male breeding soundness examination classifications in camelids. some guidelines have been cited in the literature as to minimum requirements for testicular size and semen quality; however, many males, outside of south america, are bred to few females, and may produce offspring despite any observed abnormalities in testes size or semen quality. maximal reproductive performance is often not required of these males. furthermore, males are often selected as a herd sire based on pedigree, fiber quality, conformation, or show performance, and not on reproductive parameters. the most common reasons for which male camelids are presented to the veterinarian for reproductive evaluation include: 1) clinically normal males for breeding soundness examination (bse; including prepubertal and adult males); 2) males with reproductive problems (including infertility and abnormal breeding behavior); and 3) reproductive emergencies (primarily scrotal/testicular or penile injuries). retrospective analysis of cases at the authors’ clinic demonstrated that of 164 males which had been presented for evaluation, infertility was most commonly due to testicular hypoplasia and testicular degeneration (45%) and testicular or epididymal cysts (17%).1 in proven males, the most commonly diagnosed cause of infertility was testicular degeneration secondary to heat stress.1 this paper will discuss the clinical examination of the male camelid for reproductive evaluation, noting specific findings associated to subfertility or infertility. reproductive emergencies will be covered in a subsequent paper in these proceedings. selection of the herd sire selection of a herd sire begins at birth. the male should have two descended testes and no other congenital defects. any significant cardiovascular, musculoskeletal, or visceral defects should remove the animal from breeding consideration due to the unknown heritability of most defects in these species.2,3 the male should be examined by the veterinarian starting at six months of age. testicular measurements as well as ultrasonography should be performed to ascertain the presence of rete testis cysts and record the size and location, if present. presence of cysts will not remove a male from breeding consideration unless they obliterate the entire testicular parenchyma. the initial examination of a male will serve as a baseline as he grows and matures and can be referred to in the case of any changes in the yearly bse or fertility. the male alpaca or llama reaches sexual maturity at 4-5 years of age although many are used for breeding starting at two or three years of age.4 as a general rule all prospective herd sires should undergo a complete veterinary breeding soundness examination prior to use around two years of age. this examination aims to determine the ability of the male to produce and deliver spermatozoa (normal spermatogenesis, sperm transport, and mating behavior). some young males may show signs of immaturity (poor sperm quality and quantity). a final decision on the breeding ability of the male is usually achieved at three years of age. history any male camelid which presents to the veterinarian for reproductive examination should be accompanied by a detailed health and reproductive history, including all previous examinations of testicular size, ultrasonography, and semen. unfortunately, in many cases, the first examination of a male by a veterinarian is upon identification of a problem, whether physically visible (such as penile or scrotal injury) or observed after review of the breeding records (such as increased number of open females or number of breedings or cycles per pregnancy). 571 clinical theriogenology • volume 6, number 4 • december 2014 breeding records are extremely valuable, with the inclusion of the name or identification number of the female which was bred, date and duration of breeding, and outcome (establishment of pregnancy). increased observation of interrupted breeding should be noted. breeding management scheme is also important: breeding in-hand, breeding in a small pen, or pasture breeding. travel history should be included, particularly to shows or other breeding farms where contact with sick animals may have occurred. general health history should include episodes of fever, skin lesions, lameness, colic, or injuries. nutritional history should include the type of feed (hay, concentrates, pasture access), and any hay or trace mineral analysis, if previously performed. for specific complaints regarding the reproductive tract, the duration and severity should be noted. for example, acute onset of scrotal enlargement should be differentiated from chronic. for penile injuries due to masturbation, the frequency, duration, and objects bred should be recorded. for decreased libido or difficulty completing the breeding, a time-frame and associated other events (travel, injury) should be noted. in many cases, males are sold and purchased without a reproductive examination. conscientious owners may request a bse as part of a pre-purchase agreement. examination in these cases may prevent loss of money, time, and labor if a male is unable to perform to expectations. clinical examination physical examination clinical examination, after review of the history and breeding records, begins with general inspection of the animal. any systemic disorders should be investigated (enlarged lymph nodes, heart murmur, etc.). reproductive examination begins with examination of the scrotum. the testes should be present in the scrotum at birth. the scrotum is non-pendulous but may appear so in older males or animals which have experienced testicular degeneration.5 the testes are nearly equal in size and may be slightly off-set. the head of the epididymis may be palpated as a small firm structure. the skin of the scrotum should be examined for punctures, lacerations, or dermatitis. the testes are measured for height and length (table 1),6 and ultrasonography is used to evaluate the parenchyma for presence of rete testis cysts or hyperechoic areas suggestive of fibrosis. the penis may be examined under sedation or anesthesia. the glans is inspected for any lesions, including pustules, lacerations, hair-rings, or abrasions. the preputial attachment should be freed by two to three years of age.4 in a recent study in the authors’ laboratory, 85% of males had observation of preputial detachment by 18 months of age.7 inability to exteriorize the penis in an adult male should raise suspicion of adhesions or persistent frenulum. some males may present dysuria or stranguira. urolithiasis is a common finding. in pubertal animals the prostate may enlarge under testosterone influence resulting in transient stranguria. examination of the internal genitalia may be required, usually as preparation for electroejaculation. the prostate and bulbourethral glands are examined for size and presence of any lesions. the distance from the prostate to the anus is measured to assist in proper placement of the electrodes during semen collection. a table of reproductive lesions which have been diagnosed in male camelids at washington state university is provided in table 2.6 semen collection and analysis semen collection in camelids is difficult due to the nature of ejaculation: semen is dribbled continuously throughout the mating period. there is no discrete ejaculate volume as observed in other species. semen may be collected by one of several methods in camelids. first, the male may be mated to a receptive female and semen aspirated from the female tract using a sterile insemination pipette. this method of semen collection will allow for observation of the libido of the male, and his physical ability to 572clinical theriogenology • volume 6, number 4 • december 2014 mate. however, aspirated semen is typically contaminated with erythrocytes and/or leukocytes from the female tract. an uncontaminated sample may be obtained by electroejaculation (ee). this technique is most successful if the male does not have a full bladder. the animal is placed under general anesthesia in lateral recumbency. an electroejaculation probe with linear, non-circumferential electrodes is placed over the prostate, at the distance measured ultrasonographically earlier in the examination. the penis is exteriorized and held manually with a collection tube. electrical stimulation is applied starting at very low voltage. despite general anesthesia, electrical stimulation will result in hind limb movement and muscle contraction. in a series of studies performed in the authors’ laboratory, ee was shown not to incite a higher stress response than anesthesia alone.8 furthermore, ee resulted in collection of samples adequate for evaluation, ranging in volume from 0.25 to 1.75 ml.9 some males may be trained to serve an artificial vagina (av). however, facilities must have a suitable mounting dummy, often with integrated circulating warm water to maintain ejaculate temperature over the mating period, and the av must be carefully constructed to simulate the intrauterine breeding of these species.10 this technique is not feasible for semen collection in most males. examination of the semen includes motility, volume, color, and viscosity. the motility is variable and dependent on the viscosity of the sample – the authors include an “activity” parameter in viscous samples rather than progressive motility in some cases. morphology is variable – some studies found greater than 70% normal morphology,11 however, 50% normal morphology is not unusual in sexually rested males.6 cytology should not demonstrate leukocytes or erythrocytes in ejaculates collected by ee or av. advanced diagnostic techniques for infertility or subfertility include testicular biopsy, trace mineral assay, endocrinology, and cytogenetics.12 testicular biopsy is useful in cases of azoospermia, testicular hypoplasia, and neoplasia.13 the technique can help distinguish azoospermia of testicular (spermatogenic arrest) or non-testicular (occlusion of the epididymal ducts, ductus deferens, etc.) origin. trace mineral assay can help to identify deficiencies in zinc, copper, and selenium, all minerals which impact sperm development and function. endocrinology testing can determine if poor libido is due to low testosterone or to diagnose cryptorchidism/ectopic testis. cytogenetics can identify chromosomal abnormalities in males which have poor semen quality, infertility, or pregnancy loss in bred females. evaluation of specific complaints poor libido poor libido in male camelids is not well understood as far as the eitiopathogenesis or predisposing factors. in young males, lack of sexual interest may be related to inexperience, shyness, or previous reprimands by handlers.6 in these cases, initiating of breeding in a familiar environment (i.e., on the farm, and not at the veterinary clinic), or observation of a mature male completing a breeding may increase the libido of the young male. alternatively, hormone imbalance or systemic disease may affect sexual performance.6 serum endocrinology can evaluate testosterone levels, and a complete veterinary examination should be performed. young males may require some patience to develop or encourage training of normal sexual behavior. in the adult male, or proven herd sire, any decrease in libido should be immediately investigated. the most common causes of decreased libido are systemic infections, musculoskeletal disorders including arthritis, or poor body condition.6 additionally, heat stress or group housing with females may result in decreased sexual interest over time. abnormal erection male camelids breed intrauterine within the female reproductive tract. therefore, to complete a breeding, the penis must free of its preputial attachments, of sufficient length, of normal shape to penetrate the cervix, and free of lesions. as mentioned previously, preputial detachment of the penis should occur in all males by two to three years of age.4 inability to exteriorize the penis may indicate 573 clinical theriogenology • volume 6, number 4 • december 2014 persistent frenulum or development of preputial-penile adhesions. congenital short penis has been observed in some cases of infertility. in these cases, the male is unable to complete the intrauterine breeding, which can result in infertility both by failure of intrauterine deposition of semen and failure to induce ovulation. penile lesions may include lacerations, hair rings, ulcerations, warts, or other lesions which may inhibit mating ability due to pain. last, one of the authors (at) has seen cases of erection failure due to neurologic disease caused by meningeal worm infection. azoospermia and oligozoospermia many cases of infertility in camelids are due to azoospermia or oligozoospermia, the presence of no or very few spermatozoa in the ejaculate, respectively. azoospermia may be of congenital or acquired pathogenesis. congenital azoospermia is most often due to inherent defects in spermatogenesis or outflow obstruction via segmental aplasia along the epididymal ducts or ductus deferens. testicular hypoplasia affects up to 10% of male camelids.14 the authors have observed a male in which spermatogenesis did not progress through meiosis. in animals with outflow obstruction, spermatogenesis may progress normally (as verified by testicular biopsy). however, ejaculation results only in emission of accessory sex gland secretions, with no testicular component. acquired azoospermia is usually of testicular origin. the most common inciting cause is testicular degeneration, typically due to heat stress, but may be reflective of other systemic processes.9 azoospermia may be suspected based on infertility, failure to aspirate spermatozoa from a mated female, or failure to identify spermatozoa in a collection via ee. confirmation is achieved by testicular biopsy, which may demonstrate varying degrees of testicular degeneration and/or fibrosis.5,13 as the spermatogenic cycle in camelids is thought to be approximately 60 days, sexual rest for at least two months may demonstrate resumption of spermatogenesis if the insult was transient; however, in many cases the effects are permanent. the authors have treated several males with experimental hormone therapy of follicle stimulating hormone (fsh) and luteinizing hormone (lh) to try and increase spermatogenesis, which demonstrated variable efficacy and a high rate of development of savage behavior in treated males.12 azoospermia may also be observed in males with large rete testis cysts which obliterate the entire normal testicular parenchyma.15 the authors have recently shown that measurement of seminal plasma alkaline phosphatase is not a marker of ejaculation in alpacas, compared to in dogs and horses.9 therefore, testicular biopsy remains the gold standard for diagnostic evaluation of azoospermia.13 teratozoospermia high proportion of morphologically abnormal spermatozoa in the ejaculate is classified as teratozoospermia and may reflect inherent defects in spermatogenesis or epididymal maturation.16 in other species, a high percentage of morphologically normal spermatozoa are required for a male to be classified as a satisfactory potential breeder (i.e., 70% in bulls). however, male camelids may demonstrate high percentages of abnormal morphology. studies have demonstrated successful herd sires which only demonstrated at times 50% morphologically normal spermatozoa.16,17 many other nonbreeding males which were submitted to the authors’ practice had less than 30% normal morphology.9 several other studies in both alpacas and llamas have demonstrated highly variable normal morphology.10,11,17,18 as the bse is not standardized for camelid species, and because the proportion of males submitted for bse is low (compared to the number of breeding males in use), much research is needed to determine the parameters of an ejaculate which are required for maximal fertility. males outside of south america are rarely taxed to their reproductive capabilities, and so reduced numbers of normal sperm may not result in reduced numbers of pregnancies. evaluation of teratozoospermia may require use of several staining methodologies.19 the traditional eosin-nigrosin stain for morphology may help to identify the specific morphologic abnormalities which are present. specialized stains such as spermac® are used to identify acrosome integrity. in one case, the authors utilized electron microscopy to diagnose abnormalities of the spermatozoa ultrastructure at the level of the mitochondrial sheath.19 574clinical theriogenology • volume 6, number 4 • december 2014 for diagnosed cases of teratozoospermia, the prognosis is guarded. a thorough evaluation of the nutrition, trace minerals, and general health status of the animal is warranted.12 testicular ultrasonography and biopsy may provide diagnostic information. in some cases, despite at least 60 days sexual rest and correction of all identified management issues, the etiopathogenesis of the condition remains undiagnosed. other causes of poor fertility rete testis cysts. observations of rete testis cysts within the testes of male alpacas have been well documented but the etiopathogenesis and prevalence of these lesions remains poorly defined. ultrasonographic examination was performed on 173 male alpacas presented for castration.20 rete testis cysts were identified in 18.5% of animals; 40.6% of cases were bilateral. cysts ranged in size from 4-45 mm (mean ± sem; 13.3 ± 1.3) in length and 2-28 mm (6.5±0.8) in width. after castration, cysts were aspirated; 44.4% of cysts contained immature spermatozoa. all cystic testes had evidence of spermatogenesis; however, disruption was observed in testes with large cysts. examination of the epididymis of affected testes demonstrated that 20% were completely devoid of spermatozoa. rete testis cysts can be a cause of subfertility or infertility and ultrasonographic examination of the testes should be included in the male breeding soundness examination as they are not palpable externally.21 testicular degeneration. one of the most common causes of acquired subfertility or infertility in male camelids is testicular degeneration. testicular degeneration is hallmarked by disrupted spermatogenesis, decreased sperm output (oligozoospermia), increased abnormal morphology, or azoospermia. histologically, seminiferous tubules may become smaller in diameter, with vacuolization and sloughing of the germinal epithelium. inciting causes may include heat stress, high fever, trauma or inflammation of the scrotum or testes directly, chronic systemic illness, toxic insult, nutritional or hormone imbalance, or advanced age.12 on clinical examination, the testes are smaller than expected for the age of the male, and excess scrotal skin may be apparent, with an increased appearance of pendulous testes. ultrasonographically, the testes may have small hyperechoic areas which suggest fibrosis.22 in many cases, once the inciting damage has occurred, it is irreversible. however, in cases where azoospermia has not yet developed, it may be possible to mate males on a restricted schedule to fertile females and achieve a pregnancy. prognosis the prognosis for the life and breeding potential of an animal is highly dependent on the diagnosis. severe penile injuries may require euthanasia, especially if preputial adhesions have developed. testicular injury, degeneration, or neoplasia may require hemilateral or bilateral castration. azoospermia due to spermatogenic arrest may improve with time and/or with experimental hormonal treatments; however, in many animals re-initiation of spermatogenesis does not occur. in some cases, as long as the male is producing some viable and morphologically normal spermatozoa, restricted breeding schedules may result in establishment of pregnancy in healthy, normal females. conclusion loss of breeding function of a herd sire can have severe effects on a herd, not only in terms of crias produced but also time, labor, and finances. owners of male camelids would be well-served to present the animal at least one or two times annually to the veterinarian for reproductive examination. furthermore, any lesions or abnormalities should be immediately addressed upon identification. males which are known to fight with other males or breed the ground or objects should be inspected daily for injuries and placed in suitable housing (away from females/other males and with appropriate fencing). performance of a reproductive examination prior to purchase of a herd sire is recommended to screen for reproductive abnormalities. 575 clinical theriogenology • volume 6, number 4 • december 2014 references 1. tibary a, rodriguez js, pearson lk: reproductive disorders in alpacas and llamas in a referral center. reprod domest anim 2011;45:51. 2. tibary a, pearson lk, picha y: congenital anomalies in crias. proc north am vet conf; 2011. p. 327-329. 3. pearson lk, tibary a: congenital defects of camelids. proc uc davis camelid symp; 2012. 4. tibary a, vaughan j: reproductive physiology and infertility in male south american camelids: a review and clinical observations. small rumin res 2006;61:283-298. 5. pearson lk, tibary a: infertility of camelids. proc uc davis camelid symp; 2012. 6. tibary a, pearson lk: male fertility attributes. proc univ minnesota camelid health conf; 2012. 7. pearson lk, tibary a: research update on the male breeding soundness examination. proc int camelid health conf vet; the ohio state univ; 2014. 8. picha y, sandoval s, pearson lk, et al: evaluation of stress response to electroejaculation in anesthetized alpacas (vicugna pacos). clin therio 2011;3:380. 9. pearson lk, campbell aj, sandoval s, et al: effects of vasectomy on seminal plasma alkaline phosphatase in male alpacas (vicugna pacos). reprod domest anim 2013;48:995-1000. 10. bravo pw, flores u, garnica j, et al: collection of semen and artificial insemination of alpacas. theriogenology 1997;47:619-626. 11. bravo pw, flores d, ordoñez c: effect of repeated collection on semen characteristics of alpacas. biol reprod 1997;57:520-524. 12. pearson lk, rodriguez js, tibary a: infertility and subfertility in the male. in: cebra c, anderson d, tibary a, et al., eds. llama and alpaca care: medicine, surgery, reproduction, nutrition, and herd health. philadelphia: wb saunders; 2014. p. 194-216. 13. tibary a: testicular biopsy. proc north am vet conf; 2004. p. 297. 14. tibary a, pearson lk, picha y: congenital and possibly hereditary cause of infertility in camelids. proc north am vet conf; 2011. p. 324-326. 15. tibary a, campbell aj, pearson lk: reproductive disorders in the male camelid. proc int camelid health conf, oregon state univ; 2013. p. 154-157. 16. tibary a, pearson lk, campbell aj: reproductive disorders in the male llama and alpaca. proc uc davis camelid symp; 2013. 17. flores p, garcia-huidobro j, munoz c, et al: alpaca semen characteristics previous to a mating period. anim reprod sci 2002;72:259-266. 17. lichtenwalner ab, woods gl, weber ja: seminal collection, seminal characteristics and pattern of ejaculation in llamas. theriogenology 1996;46:293-305. 19. tibary a, pearson lk, anouassi a: camelid andrology. in: chenoweth pj, editor. animal andrology: theories and application. wallingford (uk):centre for agricultural bioscience international. in press. 20. bott i, pearson lk, rodriguez js, et al: incidence and pathologic features of rete testis cysts in alpacas (vicugna pacos). clin therio 2010;2:395. 21. tibary a, picha y, pearson lk: diagnosis of testicular cysts and their significance in infertility in camelids. proc north am vet conf; 2011. p. 332-333. 22. tibary a: testicular ultrasonography and biopsy in small ruminants and lamas. proc annu conf soc therio; 2001. p. 379-385. table 1: recommended testicular size at specific ages in alpacas and llamas alpacas llamas age (months) length (cm) width (cm) length (cm width (cm) 6 1 0.4 2.4 1.4 12 (1 yr) 2.3 1.5 3.4 2.3 18 2.8 1.9 3.5 2.6 24 (2 yr) 3.3 2.2 3.9 2.3 30 3.6 2.4 4.4 2.5 36 (3 yr) 3.6 2.4 4.5 2.7 sires 3.7 2.5 5.4 3.3 576clinical theriogenology • volume 6, number 4 • december 2014 table 2: documented diseases of the reproductive organs in male camelids at the wsu theriogenology service prepuce penis testis and epididymis accessory sex glands preputial edema (heat stress) obstruction laceration prolapse necrosis inflammation (posthitis) warts phimosis prolapse paraphimosis inflammation ulcerations abrasions hair ring penile warts urethral rupture urethritis urolithiasis cryptorchidism ectopic testicles hydrocele testicular degeneration testicular hypoplasia testicular cyst orchitis epididymitis epididymal segmental aplasia prostate hypertrophy prostate abscess 577 clinical theriogenology • volume 6, number 4 • december 2014 578clinical theriogenology • volume 6, number 4 • december 2014 omniblank: 2009: equine metabolic syndrome and cushing's disease: possible role in infertility in the horse equine metabolic syndrome and cushing’s disease: possible role in infertility in the horse m. e. cadarioa, m. m. leblancb againesville, fl, usa; brood and riddle equine hospital, po box 12090, lexington, ky, usa abstract over the last several years, there has been a clear increase in the geriatric horse population. some of those mares are expected to become donors for embryo transfer or to become pregnant, forgetting the consequences of a long life expectancy. for example; illnesses and problems that commonly affect the aged horse such as cushing’s disease (cd) and equine metabolic syndrome (ms) have become more prevalent. these metabolic conditions share traits, e.g., insulin resistance (ir), often in conjunction with obesity, which have been associated with the development of abnormal reproductive function and laminitis in the horse. a large number of obese and ir mares continue to cycle during winter, have longer inter-estrus intervals, prolonged luteal phases and a higher incidence of anovulatory follicles. diagnosis of ms or cd requires a good clinical assessment in combination with the proper laboratory testing. once identified, ms should be managed with a diet poor in carbohydrates and an exercise program that stresses a moderate body condition. treatment for cd comprises appropriate health care and the administration of pergolide, cyproheptadine or a combination. even though a link between infertility and these metabolic conditions has been established, there is a clear need for further investigation. keywords: cushing’s disease, metabolic syndrome, insulin resistance, infertility, mare 485 introduction several endocrine and metabolic conditions had been empirically linked to infertility in the mare including hypothyroidism, metabolic syndrome (ms-insulin resistance) and cushing’s disease (cd) or pituitary pars intermedia disease (ppid). the mechanisms by which these conditions lead to infertility are not completely understood. these metabolic aberrations are gaining attention by veterinary scientists and equine nutritionists as the population of geriatric, obese horses in the usa is increasing and owners are requesting additional care for affected horses. this review discusses the pathophysiology of ms and cd, how they may adversely affect fertility, diagnostics and recommended therapies. during the fall months and in preparation for winter, the horse begins to store energy in the form of body fat. the process is controlled by the hypothalamic-pituitary axis as it induces an increased secretion of pro-opiomelanocortin peptides1 and results in an increase in appetite and adipogenesis in conjunction with the development of a thick hair coat. this “survival mechanism”, disappears by the end of winter in healthy horses but not in horses with ms or cd. metabolic synrrome and, to a certain degree cd, are associated with the acquisition of excessive fat and, more importantly, the persistence of body fat and its negative consequences on the mare’s general heath and reproduction.2 clinical findings that should make one suspicious of ms or cd include obesity in older horses that do not shed their winter coats completely or do so much later in the spring than the remainder of the herd, abnormal estrous cycles, anovulatory follicles and repeated bouts of endometritis. a number of tests are available for diagnosing ms or cd. however, there is no single “gold standard”. 486 equine metabolic syndrome equine ms is closely associated with two disorders: obesity and insulin resistance (ir or glucose intolerance). horses affected with ms also have a predisposition to developing laminitis and mares may exhibit abnormal estrous cycles.3 the obesity is evident as subcutaneous regional adiposity (“cresty neck”, tailhead, shoulders and prepuce in geldings) and affected horses tend to be genetically predisposed requiring fewer calories to keep normal body weight (“easy keepers”). not all obese horses have ir even though obesity increases the horse’s risk of developing the disease. some breeds appear to be more commonly affected than others, including ponies, quarter horses, morgans, arabians and saddlebreds. there is no gender bias.4 pathophysiology: when foods rich in carbohydrates are ingested, they are broken down into glucose. this is followed by a physiological hyperglycemia which triggers the release of insulin from the pancreas. insulin promotes glucose uptake by cells. once in the cell, glucose is either used immediately or it is stored as glycogen or fat. the amount of insulin required for inducing glucose uptake into cells is tightly regulated and is referred to as insulin sensitivity. if a horse develops ir, there is a reduction in the insulin driven uptake of glucose by the skeletal and adipose tissue creating hyperglycemia.2,4 this, in turn, generates hyperinsulinemia in an effort by the pancreas to compensate for the insensitivity. little is known about the factors predisposing to ir; however, obesity appears to be the recurrent trait in horses. it has been suggested that certain adipocites behave as endocrine tissue releasing cortisol, pro-inflammatory cytokines and leptin, which may interfere with glucose uptake by the cells.2 age and diet appear to be important contributing factors to the development of ir. ageing is believed to decrease sensitivity to insulin. 487 obesity in horses and especially in ponies appears to be a risk factor for mild laminitis. the simplest explanation is excessive weight at the hoof–lamellar interface.4 the fact that ponies are at higher risk even though they bear less weight, suggests the genetic predisposition of this breed to ms and ir.2,4,5 glucose is essential for the health and strength of the hoof-lamellar interface. ir creates cell-glucose starvation which may increase the risk of laminitis more than obesity per se. diagnosis characteristics that are common in horses with ms or ir include increased age (8 to 20 years old), obesity, distinctive distribution of body fat (neck, shoulders, rump), “easy keepers” that have difficulty losing weight, a history, presence or predisposition for laminitis, abnormal estrous cycles and decreased pregnancy rates. resting levels of insulin are good indicators of ir since compensatory hyperinsulinemia is a common finding. plasma samples should be taken early in the morning after an overnight fast. horses should be removed from pasture and only fed course roughage overnight. roughage should be available as some horses will become increasingly agitated if no food is available. resting insulin concentrations > 20 µu/ml are suggestive of insulin resistance and > 30 µu/ml are considered diagnostic.4,6 even though resting levels of insulin are good markers, they are not helpful in mild or early ir where the rise of insulin is too small to reach or exceed the reference value. reference values differ among laboratories and should be taken into consideration when interpreting results (insulin >30 µu/ml at university of tennessee vs. >43 µu/ml at michigan state university).6 if one is presented a horse with physical findings consistent with ms or cd that has a normal resting plasma insulin level, a combined glucose-insulin test should be performed.6 this test consists of collecting a baseline glucose blood sample before infusing 150 mg/kg of 50% 488 dextrose solution. immediately after the dextrose infusion, insulin is administered intravenously at 0.10 u/kg. serum samples are collected at 1, 5, 25, 35, 45, 60, 75, 90, 105, 120, 135 and 150 min. however for practical purposes, the test can be limited to 60 minutes. a horse is considered to have insulin resistance if blood glucose levels remain above baseline for more than 45 min.6 stress should be avoided during the test since it can cause transient ir. horses should be kept quiet, allowed to graze and the jugular catheter should be placed the night before for blood collection. metabolic syndrome and cd share a number of clinical signs including abnormal distribution of fat, hyperinsulinemia and insulin resistance, predisposition to laminitis and infertility. cushing’s disease can be differentiated from ms by performing a dexamethasone suppression test (dst; see ppid), as this test will be positive only for ppid. indeed, ms has been described as the presence of insulin resistance without detectable presence of ppid.7 treatment treatment is directed at lowering body fat and consequently insulin levels to <30 µu/ml. horses should be placed on a low carbohydrate diet to reduce caloric intake and partake in a consistent exercise program to increase energy outflow and glucose uptake. grain should be removed from the diet (alone or mixed with molasses) and access to pasture should be limited. medical treatment with levothyroxine sodium (thyro l; vet-a-mix, shenandoah, ia, usa) is warranted if weight loss occurs too slowly or hyperinsulinemia persists after reaching the desirable body weight.4 levothyroxine sodium is administered at a dose of 48 mg/day po for 3 to 6 months to accelerate weight loss in horses, especially those which cannot exercise due to laminitis. persistent hyperinsulinemia/hyperglycemia is treated with a combination of levothyroxine sodium (24 mg/day po) and a biguanide (metformin; 15 mg/kg po bid). the 489 mechanism of action is not known but it appears to inhibit gluconeogenesis and increases glucose uptake.4 its safety in the horse has not been established. in addition, administration of chromium at a dose of 5 to 25 mg/day may improve insulin efficacy in transporting glucose into the cells.4,7 cushing’s disease or pituitary pars intermedia dysfunction cushing’s disease or ppid is a relatively prevalent condition in horses due to an increase in the geriatric population.8,9 onset of clinical signs usually occurs around 18 to 23 years of age but horses as young as 7 years have been affected. there is no sex predisposition and all breeds can be affected. ponies and morgan horses appear to be at a higher risk.10 first described by pallaske 75 years ago, this syndrome is characterized by adrenocortical hyperplasia and excessive glucocorticoid, mineralocorticoid and androgenic steroid secretion in response to a surplus release of adrenocorticotrophic hormone (acth).11 in the horse, there is a hypothalamic or pituitary-dependant hyperadrenocorticism. the presence of a possible benign adenoma in the pituitary gland or the degeneration of dopaminergic neurons at the hypothalamic level had been held responsible.6,9,12 pathophysiology. the hypothalamus controls secretion of acth by the anterior pituitary through the release of corticotrophin releasing hormone (crh). neurons secreting crh are located in the anterior portion of the paraventricular nuclei and their axons terminate in the pituitary median eminence. corticotrophin releasing hormone stimulates secretion of acth, beta-lipotropin (ß-lph) and ß-endorphins. all three originate from the same precursor called pro-opiomelanocortin (pomc),9,11 and all have the same secretory dynamics, that is release is increased in response to stress and hypoglycemia and decreased in response to glucocorticoids. 490 negative feedback inhibition of acth by glucocorticoids is absent in ppid. therefore, acth and pomc related hormones persist despite cortisol elevation.12 chronic exposure to excessive glucocorticoids results in a variety of physical and clinical abnormalities. the most characteristic clinical sign is hirsutism (47 to 100% of affected horses) and the inability to shed the winter coat. it is suggested that hirsutism results from increased melanocyte-stimulating hormone (msh) released from the pars intermedia or increased production of androgens by the adrenal cortex. in women, hirsutism has been associated with clinical hyper-androgenism.13 in addition there are changes in weight, unusual fat accumulation at the neck, tail head and over the croup (29 to 67%). obesity is believed to be the underlying factor for horses with ms to develop ppid at a younger age than average. chronic obesity is thought to result from a pro-inflammatory and pro-oxidative condition that may accelerate degeneration of dopaminergic neurons.7 other characteristics are muscle atrophy (protein catabolism), immunosuppression (and consequent predisposition to retarded wound healing and infections such as endometritis and sole abscesses), polydipsia and polyuria (25%), lethargy, tachypnea, hyperhidrosis (65%) and occasionally, neurologic impairment.6,14 laminitis occurs in 50% of advanced cases. one explanation is that excess of corticoids promote laminar vasoconstriction. the presence of ir seems to exacerbate the condition. broodmares may show persistent lactation (hyperprolactinemia), low grade endometritis (immunosuppression) and anovulatory follicles during the cyclic season.7,9 in humans, 23% of cushing’s patients with persistent lactation and amenorrhea (due to gnrh inhibition by prolactin) have increased prolactin concentrations. the increase in prolactin is due to excessive production and secretion by corticotroph adenomas or an alteration of the regulation at the dopaminergic neurons.6,9,12 491 the most common biochemical abnormality in ppid is hyperglycemia and hyperinsulinemia/ir. other findings include increased hepatic enzymes, cholesterol and triglycerides. horses can also have mild anemia, neutrophilia and lymphopenia. diagnosis presence of hirsutism had greater diagnostic accuracy than any endocrinological test alone or in combination.10 affected horses are usually aged (8 to 20/30 years old), overweight, with the characteristic distribution of body fat (neck, shoulders,), laminitis, and abnormal estrous cycles.7,8,9,10 dynamic endocrinologic diagnostic tests the dynamic endocrinologic diagnostic tests consist of the overnight dexamethasone suppression test (dst), thyrotropin-releasing hormone (trh) stimulation test and the cortisol rhythm assay. dst. in normal horses, administration of dexamethasone (20 mg im/500 kg) decreases the acth release from the pituitary, resulting in a cortisol serum concentration of < 1 µg or 10 ng/dl approximately 19 hours after injection. conversely, in horses with ppid, serum cortisol concentration does not decrease after dexamethasone administration due to continuous acth release. unfortunately, this gold-standard test loses accuracy (sensitivity and specificity) early in the condition when there is still some feedback from the hypothalamic-pituitary axis, as it appears that loss of suppression by dexamethasone is a late event in the pathogenesis.10 the dst results are also affected by the season of the year since the pituitary pars intermedia produces more hormones during the fall (“survival mechanism”). one study showed that falsepositive overnight dst results were common when horses or ponies were tested in the fall.1 492 trh stimulation test. horses with ppid show an increase (30 to 50% from baseline) in serum cortisol concentration 30 to 90 min after trh administration (0.5 to 1 mg, iv) whereas normal horses do not.15 normally, trh produced by the hypothalamus stimulates tsh release from the pituitary. however, in ppid, exogenous trh stimulates acth and hence, cortisol release. it appears to be due to an up-regulation and expression of trh receptors in hyperplastic/adenomatous corticotropes in the pituitary pars distalis and intermedia.10,14 this test is safe to perform in laminitic horses. it has not been critically evaluated in horses early in the disease process. (diurnal) cortisol rhythm assay. this assay is based on the observation that horses with ppid have a loss of the diurnal cortisol rhythm (normally high in morning, low at midnight).10,16 when performing this test, plasma cortisol concentration is measured at 8 am and again at 4 pm. a difference of less than 30% between the morning and afternoon cortisol concentration is considered to be suggestive of ppid. however, this test has not been validated and several factors such as fasting, changes in stabling or laminitis can increase plasma cortisol concentration in the horse. combined dst and trh stimulation test. administration of dexamethasone before trh should suppress acth release from the pituitary par distalis therefore, any increase in cortisol after trh administration is attributed to pars intermedia corticotropes. in performing this test, a baseline plasma cortisol concentration is obtained followed by administration of dexamethasone (20 mg, im/500 kg). three h after the dexamethasone is given, trh (0.5 to 1 mg, iv) is administered. cortisol should be suppressed by the trh. plasma cortisol concentrations are measured 30 min and 21 h after administration of trh (24 h after dexamethasone administration). a plasma cortisol concentration > 1 µg/dl 24 h after administration of 493 dexamethasone or a > 66% increase in cortisol 30 min after trh administration is considered diagnostic for ppid.[8,10 the test is expensive and may be impractical for the ambulatory clinician. accuracy of each test when compared with histological findings is dst/trh 81%; dst 71%; trh 71%. accuracy of hirsutism alone is 86%. single sample endocrinologic tests two single sample endocrinological tests are available and include measurement of endogenous release of plasma adrenocorticotrophic hormone (acth) concentration and insulin concentration. acth concentration test. measuring a single sample of endogenous plasma acth, which is supposedly higher in horses with ppid, is not more accurate than the dst test, does not detect early cases, and it undergoes daily and seasonal variations. a reference value of > 50 pg/ml is considered elevated.10 sample handling is intricate. blood must be collected in cold plastic tubes containing edta, as the acth can be absorbed onto glass and degraded by proteolytic enzymes in blood or serum. it needs to be centrifuged as soon as possible (within 30 to 60 min) and the plasma transferred immediately to a plastic tube for freezing and shipping (should arrive to the laboratory below 60 0f). this test is recommended when one is trying to avoid administration of dexamethasone to laminitic animals. insulin concentration. insulin resistance/hyperinsulinemia has been recognized in horses with ppid. affected horses have increased insulin blood levels attributed to excessive circulating concentrations of cortisol since they have antagonistic metabolic effects. excessive insulin concentrations vary between laboratories and reference values must always be consulted when interpreting the test. cutoff point for normal insulin concentration varies from 25 µu/ml to 57 494 µu/ml7,10 however, > 30 µu/ml is considered diagnostic of hyperinsulinemia. the accuracy of this test compared to histological findings is 92%. this test has the advantage of a single sample for the diagnosis, however, ms should be ruled out since hyperinsulinemia is also present in this condition. douglas has proposed that testing for suspected cases of ppid and ms should start by simply collecting two serum samples 8 to 10 h apart.7 no grain should be given 4 h before both samples and stress should be avoided since both parameters can result in false positives. serum samples are collected for measurement of cortisol, insulin and thyroxine (tt4). the absolute values of cortisol are 20 to 90 ng/ml, however, the test specifically looks at a difference of less than 30% between the two samples since that indicates a loss in the diurnal cortisol rhythm which is considered suggestive of ppid. a consistent insulin value > 25 to 30 µu/ml will be indicative of ir. a typical endocrine profile in a horse with ir and ppid sample time tt4 normal=12.0 ng/ml insulin normal=<25 μu/ml cortisol normal 20-90 ng/ml cortisol normal>30 % difference 8 to 10 am 11.9 ng/ml 35 μu/ml 58 ng/ml 11 % 4 to 6 pm 12.2 ng/ml 55 μu/ml 65 ng/ml from: douglas rh. endocrine assessment and management of insulin resistance and ppid.7 495 horses suspected of having early signs of ppid (“pre-cushingoid”) that have normal values from earlier, appropriate diagnostic tests, should be re-tested at 4 to 6 month intervals avoiding the fall months. the evaluation should include a physical examination, cbc, serum chemistry, acth, insulin concentrations and dst as a baseline profile.10 treatment management of ppid is essential to assure appropriate health care of older animals: body clipping, regular hoof and dental care, enhanced nutrition, and intermittent or long term antibiotics may be required. the dopamine agonist, pergolide mesylate (1 to 5 mg/po/q 24 h/500 kg horse) is most commonly administered since the disease appears to be associated with a loss of hypothalamic dopaminergic innervations. pergolide should be discontinued in pregnant mares approximately 3 weeks before their anticipated foaling date to avoid agalactia (prl suppression). the most common adverse effect of pergolide administration is a mild decrease in appetite. serotonin antagonists (cyproheptadine 0.25 mg/kg/po/q 12 or 24 h, or 200 to 400 mg/500 kg/po/q 24 h) are also used alone or in conjunction with pergolide. this therapy is based on the secretagogue effect of serotonin on acth in rat pars intermedia.10,17 the use of cyproheptadine is controversial since results appear to be similar to the clinical improvement obtained with good management alone. it has been suggested, that a combination of 1 mg of pergolide and 200 mg of cyproheptadine/day accelerates the clinical response to medication, increasing the chances of successfully breeding a mare with history of development of anovulatory follicles or chronic endometritis.6 recent studies have examined the use of trilostane (0.4 to 1.0 mg/kg/q 24 h in feed) in horses with ppid.10,18 this drug is a competitive inhibitor of 3-ß hydroxysteroid dehydrogenase, 496 an adrenocortical enzyme needed for cortisol production. even though some treated horses exhibited a decrease in clinical signs, there is reservation toward using this treatment because less than 20% of horses with ppid have adrenocortical hyperplasia. aging, obesity, insulin resistance and infertility recent studies associate obesity and insulin resistance in horses with development of abnormal reproductive function and laminitis.19 equine ms and cd or ppid share traits that provide plausible explanations for the clinically perceived increased incidence of fertility problems in horses. a) the shared clinical manifestations of both diseases are: aged horse, obesity, regional subcutaneous adiposity (neck, tail head, prepuce), a history, presence or predisposition for laminitis and/or infertility. b) the shared metabolic alterations include: increased insulin and or insulin resistance, +/_ increased glucose (and in some cases increased leptin and “leptin resistance”, cortisol and fatty acid). c) the shared presence of excessive fat, in the form of obesity or regional adiposity, creates at the cellular-molecular level an increase in “adipocytokines” such as tumor necrosis factor-α (tnf-α). therefore, ppid and ms may also have an increased level of tnf-α, a pro-inflammatory cytokine which inhibits the proper function of the insulin receptor. increased fatty acids impair glucose transport through the membrane. increased 11ßhydroxysteroid dehydrogenase-1 (11ß-hsd1) converts cortisone to cortisol generating more adipogenesis and ir.2 497 similar to humans, old horses have been identified as developing a condition referred to as “inflamm-aging”. this condition is a persistent low grade, systemic, chronic, inflammation caused by an increased production of serum levels of il-6 and tnf-α.20 increased body fat, associated with obesity, may be a contributing factor since adipose tissue leads to increased production of inflammatory cytokines.20,21 adams et al, showed that fat, old, horses between 20 and 28 years old, had greater mrna expression of tnf-α and inf-γ from lymphocytes and monocytes and had higher serum concentrations of tnf-α protein than thin old horses.21 the impact of this increase in pro-inflammatory cytokines and infertility is unknown. in humans, elevated tnf-α plays a direct role in the development of obesity-associated insulin resistance and it appears that inflammation may be a key link between obesity and ir in horses as well.19 in women, chronic insulin resistance and hyperinsulinemia are associated with increased duration of the follicular phase of the menstrual cycle. polycystic ovarian syndrome (pos) in women is often associated with obesity and characterized by several clinical signs, neuroendocrine and metabolic alterations. clinical signs that are diagnostic in women are: ovulatory dysfunction, clinical hyperandrogenism (hirsutism, alopecia, acne) and ir/hyperinsulinemia.13 neuroendocrine disturbances associated with pos include an increase in gnrh pulses that selectively stimulate lh release but not fsh. lh stimulates the expression of the p450c17 cytochrome in the theca cells, increasing the production of 17α-hydroxyprogesterone and testosterone. this hormonal environment (↑lh/↓fsh) impairs follicular maturation, decreases the number of granulosa cells and aromatization (↓e2), and inhibits normal ovulation.13 insulin resistance/hyperinsulinemia may have some role since administration of metformin decreases the plasma levels of insulin, lh and androgens in women.13,22 the increased level of insulin and 498 insulin like growth factor-1 (igf-1) directly target the theca cells acting in synergism with lh, stimulating p450c17 cytochrome production and hyperandrogenism. they also decrease the hepatic secretion of the sexual hormone binding globulins (shbg) and the ifg binding proteins (igfbp-1) producing hyperandrogenism by increasing the free testosterone fraction and the igf1 bioactivity.13 there are limited studies in the mare on the endocrine effects of ir, ms, or ppid on reproductive function. one study showed that a great proportion of obese mares continued to cycle during the winter compared to lean mares.23,24 it was speculated that metabolic alterations in the form of excessive energy imbalance may lead to continuous cycling during winter.23,25 later studies found that obese mares had aberrations in the duration of the estrous cycle: longer inter-estrus intervals (36.72+4.77 vs. 26.00+0.54 days), prolonged luteal phases (30.00+5.55 vs. 17.67+0.18 days) and a higher incidence of anovulatory follicles compared to lean mares.23,26 progesterone remained elevated for periods of 37 to 78 days in 83% of the obese mares while the longest time progesterone was elevated in lean mares was 22 days.24 the persistence of elevated serum levels of progesterone in obese mares was thought to be associated with a persistent corpus luteum or luteinization of an anovulatory follicle.27 ultrasonic examination of the reproductive tract of these mares showed large, persistent anovulatory follicles during periods of elevated progesterone suggesting that one or more mechanisms involved in ovulation may be suppressed or inhibited.28 obese mares exhibited lower levels of serum concentrations of thyroxine and leptin and higher levels of insulin and insulin insensitivity compared to lean mares. the mechanism of how hyperinsulinemia and ir may disrupt normal reproductive activity is unknown but the following data suggest a direct effect on the ovary rather than the 499 hypothalamic-pituitary-ovary axis. in domestic species, insulin in combination with igf, promotes follicular development and modulates ovarian steroids secretion as seen in women with pos.13 sessions et al., showed that induction of transient insulin resistance in the mare lengthens the interovulatory interval (mean duration 19.57+2.66 vs. 15.57+ 0.97 d), creates higher serum peak concentrations of progesterone (12.87+4.04 vs. 8.78+2.23 ng/ml) but does not affect serum lh concentrations (3.9+0.34 vs. 3.29+0.13 ng/ml) when compared to control mares.29] in women, hyperinsulinemia affects the menstrual cycle by alterations of the gnrh-mediated lh release and has a direct effect on the ovary by increasing steroid secretions.30,31 this phenomenon is well documented in women with pos where there is an arrest of follicular development. apparently, small follicles that just attained lh receptors, in the presence of insulin and high lh, produce estradiol levels that are similar to levels produced by mature follicles, thereby inhibiting growth and arresting follicles in the immature stage. a similar phenomenon may be occurring in the mare with insulin resistance; however it appears to be due to a direct effect of insulin on the ovary since the hypothalamic-pituitary axis was not affected during an induced short term hyperinsulinemia. peak concentrations of progesterone were higher in mares experiencing transient ir compared to control mares suggesting an increased steroid secretion by the ovary. it has been proposed that insulin has a stimulatory effect on progesterone production by luteal tissue and this effect is mediated by changes in the developing follicle. in studying the repeatability of hemorrhagic anovulatory follicles (haf) in ponies, ginther also deflected the focus from the hypothalamic-pituitary-ovary axis.32 he showed that follicular diameter, levels of lh and progesterone were similar in ovulating and haf mares before and at the day of ovulation. the impact of his findings on obese mares remains unknown. 500 the longer interovulatory interval in obese mares appears to be due to luteinization without ovulation of the dominant follicle (failure to ovulate) extending the luteal phase. one plausible explanation is what happens in women with pos; insulin directly targets the theca cell stimulating p450c17 cytochrome production and hyperandrogenism. increased androgen inhibits fsh secretion and this hormonal environment (↑androgens/↓fsh) impairs follicular maturation, decreases the number of granulosa cells and aromatization (↓e2), and inhibits normal ovulation. further studies need to be performed to confirm this theory by measuring androgens and fsh in mares with persistent ir. another explanation, which does not exclude the prior, is that elevated insulin affects follicle remodeling mechanisms necessary for ovulation. normal follicular growth, ovulation and atresia require remodeling events which are carried out by matrix metalloproteinases (mmp) and tissue inhibition of metalloproteinases (timp). recent studies indicate that exposure to elevated insulin affects the in vitro expression of mmp and timp in preovulatory and atretic follicles. moreover, it specifically disrupts the expression of timp-2 modifying the ratio of mmp/timp expression by equine granulosa cells of large follicles. this alteration may be responsible for the inability of the follicular wall to remodel for development, migration and ovulation.26,33 conclusion although the pathophysiology of metabolic diseases such as ir, ms or cd is not completely known, there is increasing evidence that these conditions may adversely impact fertility of the mare. diagnostic testing for these conditions should be considered in old, obese mares that repeatedly form anovulatory follicles, experience bouts of laminitis or endometritis. while accuracy of diagnostic tests for detecting disease may not be as high as one would like, a combination of tests along with clinical assessment will identify the vast majority of mares. a 501 strict exercise regimen plus removal of feed stuffs rich in carbohydrates from the diet and supplementation with thyroid hormone and/or pergolide should improve fertility in affected mares. references 1. donaldson mt, mcdonell sm, schanbaucher bj, et al: variation in plasma adrenococorticotropic hormone concentration and dexametasone suppression tests results with season, age, and sex in healthy ponies and horses. j vet int med 2005;19:217-222. 2. johnson pj, ganjam sk, turk jr, et al: obesity paradigm: an introduction to the emerging discipline of adipobiology. proc annu conv am assoc equine pract 2006;4150. 3. equine metabolic syndrome. j eq vet sci 2003;23:373-374. 4. frank n: therapeutics in practice: managing equine metabolic syndrome. compend equine 2008;3:348-355. 5. treiber kh, kronfeld ds, hess tm, et al: evaluation of genetic and metabolic predisposition and nutritional risk factors for pasture associated laminitis in ponies. j am vet med assoc 2006; 228: 538-1545. 6. frank n: insulin resistance in the horse. proc annu conv am assoc equine pract 2006;51-54. 7. douglas rh: endocrine assessment and management of insulin resistance and ppid. proc primer congreso argentino de reproduccion equina 2009;81-83. 502 8. mcfarlane d: diagnosing pituitary pars intermedia dysfunction. compend equine 2007;2:208-213. 9. mcfarlane d: role of equine hypothalamic-pituitary pars intermedia axis in health and disease. proc annu conv am assoc equine pract 2006;55-59. 10. schott hc: pituitary pars intermedia dysfunction: challenges of diagnosis and treatment. proc annu conv am assoc equine pract 2006;60-73. 11. pallaske g: sur kasuistik seltnere geschwülste bei den haustieren. z krebsforsch 1932; 36:342–353. 12. feldman ec, nelson rw: hyperadrenocorticism. in: canine and feline endocrinology and reproduction. philadelphia:wb saunders; 1987. p. 137-186. 13. castro acuña v, martinez-martinez l, cravioto m:. resistencia a la insulin en el syndrome de ovaries poliquisticos. rev inves clin 2004;56:763-772. 14. mcfarlane d, dybdal n, donaldson mt, et al: nitration and increase alpha-synuclein expression associated with dopaminergic neurodegeneration in equine pituitary pars intermedia dysfunction. j neuroendocrinol 2005;17;73-80. 15. beech j, garcia m: hormonal response to thyrotropin-releasing hormone in healthy horses and in horses with pituitary adenomas. am j vet res 1985;46:1941-1943. 16. dybdal no, hargreaves km, madigan je, et al: diagnostic testing for pituitary pars intermedia dysfunction in the horse. j am vet med assoc 1994;204:627-632. 17. kreiger dt, amorosa l, linick f: cyproheptadine-induced remission of cushing’s disease. n engl j med 1975;293:893-896. 503 18. mcgowan cm, neiger r: efficacy of trilostane for the treatment of equine cushing’s syndrome. equine vet j 2003;35:414-418. 19. vick mm, adams aa, murphy ba, et al: relationship among inflammatory cytokines, obesity, and insulin sensitivity in the horse. j anim sci 2007;85:1144-1155. 20. horohov dw: immunosenescence and inflamm-aging in the aged horse. in: the chronically infertile mare (abstract book). the havemeyer foundation 2008; page 21. 21. adams aa, katepalli mp, kohler k, et al: effect of body condition, body weight and adiposity on inflammatory cytokines responses in old horses. in: the chronically infertile mare (abstract book). the havemeyer foundation 2008; page 22. 22. sattar n, hopkinson ze, greer ia. insulin sensitizing agents in polycystic ovary syndrome. lancet 1998;351:305-306. 23. vick mm, sessions dr, murphy ba, et al: obesity is associated with altered metabolic and reproductive activity in the mare: effect of metformin on insulin sensitivity and reproductive cyclicity. reprod fertil dev 2006;18:609-617. 24. fitzgerald bp, reedy se, sessions dr, et al: potential signals mediating the maintenance of reproductive activity during the non-breeding season of the mare. reprod suppl 2002;59:115-129. 25. gentry lr, thompson dl, gentry gt, et al: the relationship between body condition, leptin, and reproductive and hormonal characteristics of mares during during the seasonal anovulatory period. j anim sci 2002;80:2695-2703. 26. fitzgerald bp, sessions dr, vick mm, et al: obesity and reproduction in the mare. in: the chronically infertile mare (abstract book). the havemeyer foundation 2008; page 23. 504 27. mccue pm, squires el. persistent anovulatory follicles in the mare. theriogenology 2002;58:541-543. 28. mccue pm. diagnosis of ovarian abnormalities. in: ball ba, editor. recent advances in equine reproduction. international veterinary information service; 2000 available from: http://www.ivis.org. 29. session dr, reedy, vick mm, et al: development of a model for inducing transient insulin resistance in the mare: preliminary implications regarding the estrous cycle. j anim sci 2004;82:2321-2328. 30. diamanti-kandarakis e, bergiele a.: the influence of obesity on hyperadrenogenism and infertility in the female. obese rev 2001;2:231-238. 31. nestler je. obesity, insulin, sex steroids and ovulation. int j obes relat metab disord 2000;24:s71-s73. 32. ginther oj, gastal el, gastal mo, et al: conversion of a viable preovulatory follicle into a hemorrhagic anovulatory follicle in mares. anim reprod 2006;3:29-40. 33. session dr, vick mm, fitzgerald bp. expression of mmp-2 and timp in equine granulosa cells and the impact of elevated insulin in vitro. biol reprod 2007;77:156. 505 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false 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contact maria gallelli mgallelli@fvet.uba.ar © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 13213, http://dx.doi.org/10.58292/ct.v17.13213 technique report successful pregnancy using stallion semen stored at 17ºc for 6 days maria munhoz,a márcio nunes,b maria gallelli,c gustavo carneiro,d marcelo miragayac amk arabians, ajman, uae bgerman standard group, dubai, uae cuniversidad de buenos aires, facultad de cs. veterinarias, argentina and econicet dfederal rural university of pernambuco, recife, pe, brazil abstract aim of this study was to assess the effectiveness of beyond® in maintaining sperm motility at 17ºc with ‘poor cooler’ stallion semen and achieving pregnancy after artificial insemination. an ejaculate was diluted in equiplus (minitube) and divided into 2 aliquots. one was kept at 5ºc (control sample), whereas the other was centrifuged, further diluted in beyond® and kept at 17°c in a semen cooler (minitube) for 10 days. total motility (tm) and membrane integrity were assessed daily. for the control sample, tm was 65 and 35% after 24 hours and 48 hours, respectively, whereas the sample diluted with beyond® still had a tm of 40% after 10 days. six days after storage at 17°c, a mare was inseminated; a healthy colt was born 344 days after ovulation. we are reporting the first successful pregnancy after storing semen from a poor cooler stallion for 6 days at 17°c. keywords: dilution, extender, stallion, semen, pregnancy introduction artificial insemination with cooled semen is a common practice in equine reproduction.1,2 current commercial extenders normally allow semen doses to be stored at 5°c for up to 48 hours after collection but storage at 15°c is not often practiced. this is due to detrimental side effects, mainly related to bacterial growth at this temperature, and increases in reactive oxygen species production in the sample, in response to higher sperm metabolism.3,4 semen survival may vary according to the dilution rate, extender characteristics, and the stallion semen preference for specific formulas.1,5,6 recently, a new extender containing antioxidants, antibiotics, and antifungals that allows stallion semen to survive up to 7 days at 17°c after colloid centrifugation, has been described.7 field trials using this new type of extender after simple centrifugation with a milk-based extender had good sperm progressive motility.8 furthermore, fixed time insemination of frozen-thawed semen after microfluidic selection and storage at 17ºc with this extender for 24 hours, produced embryo recovery rates of ~ 50%.9 cooled semen storage, usually performed at 5°c, is a useful strategy in equine breeding management for stallions with restrictions on frequent collection or those that produce a small number of sperm per ejaculation, allowing the storage of new ejaculates until an insemination dose is reached.10 semen storage also avoids the need for daily ejaculate splitting in reproduction centers to produce fresh, cooled, and frozen doses. this way, a complete ejaculate can be used to produce cooled doses available throughout the week, allowing subsequent collections to be exclusively used for freezing. furthermore, semen storage at 15-17°c would be particularly advantageous for stallions that are poor coolers, as their more sensitive membranes are less stable under colder refrigeration.6,11 fresh semen doses for artificial insemination should preferably be available within a 24-hours window before the expected ovulation time, as fertility can be lower in protocols that delay ovulation versus those that induce ovulation.12,13 therefore, availability of fresh semen doses is required within a short interval after a preovulatory follicle is identified. if the mare ovulates earlier or if the courier transporting the semen doses is delayed, the ovulation can be missed. consequently, breeding management could be improved by ensuring the continuous availability of semen doses. beyond® (minitüb gmbh, tiefenbach, germany) is the first commercial extender of its kind that allows storage of semen doses for up to 14 days at 17°c, maintaining its quality, according to the manufacturer. therefore, the use of this mailto:mgallelli@fvet.uba.ar http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.13213 2 citation: clinical theriogenology 2025, 17, 13213, http://dx.doi.org/10.58292/ct.v17.13213 extender could minimize the partial disposal of ejaculates when all doses are not required within 48 hours after semen collection, reduce the frequency of semen collection, and decrease missed ovulations. aim of this study was to assess the effectiveness of beyond® in maintaining the sperm motility of semen kept beyond normal interval from a poor cooler stallion and achieving pregnancy. materials and methods a 5-year arabian stallion (part of the herd at mk arabians stud in ajman, united arab emirates) was used; stallions on the farm are kept according to national and international regulations for the husbandry and care of equids. based on its reproductive history, the stallion was considered a poor cooler due to its inability to maintain good sperm motility when samples were stored at 5ºc for up to 48 hours14,15 during the previous 2 breeding seasons, with notably worse performance when 15ºc storage was attempted. following the farm’s routine during the breeding season, after properly washing the penis with plain water, the ejaculate of this trained stallion was usually collected on a dummy up to 3  times a week using a properly heated artificial vagina (missouri model; minitube, germany) without lubricant. artificial vagina was equipped with a disposable plastic inner liner attached to a semen bottle containing a disposable semen filter to separate the gel fraction. these ejaculates were frequently used for inseminations with fresh semen or stored at 5°c for up to 24 hours, and 1 of them was used for this trial. the sperm total motility (tm) of raw samples was assessed using optical microscopy by an experienced veterinarian, and the concentration was evaluated with a neubauer chamber. based on previous cooling tests, a milk-based extender (equiplus combi – minitube, germany) was chosen for this ejaculate`s dilution. thus, an aliquot of the sample was diluted in equiplus in 1:1 volume ratio to be further centrifuged (treatment sample), whereas another aliquot was diluted with the same extender to a concentration of 50 x 106 sperm/ml (control sample) and kept into an identified tube. after 30 minutes of incubation at 37ºc, tm and membrane integrity (eosin-nigrosin test) were assessed.16 treatment sample was centrifuged at 600 g for 20 minutes and the supernatant was discarded. the beyond® extender, prewarmed to 37ºc, was added to the sperm pellet to a concentration of 200 x 106 cells/ml, and tm and membrane integrity were evaluated. the ready-to-use semen dose was kept at 17ºc in an adjustable semen cooler (minitube, germany) for 10 days, whereas the control sample was kept at 5ºc. an aliquot from each group was taken daily and warmed to 37ºc in a water bath before motility and membrane integrity assessment (table). after 6 days of cooled storage with daily monitoring, an insemination dose of 18 ml with 70% tm and 50% of membrane integrity, comprising 420 x 106 motile sperm (600 x 106 total sperm), was used for a postovulatory deep intrauterine insemination in a 13-year fertile arabian mare. the criteria used to select the mare for the insemination trial, in addition to a good fertility history, included the presence of an active dominant follicle > 35 mm at 32-38 hours after intravenous hcg (1,500 iu; ovusyn®, syntex, vaca cua, corrientes, argentina) treatment, good uterine edema evaluation (assessed by ultrasonography), and absence of a corpus luteum.17 transrectal ultrasonography (mindray vetus eq 7) was performed to monitor the estrous cycle, and then on days 1, 14, and 23 after insemination; was also performed monthly, to assess pregnancy. six days after semen collection, a mare with a history of normal previous cycle fertility, from the breeding herd that had been monitored for reproductive purposes was used. mare was induced to ovulate and was designated for this stallion. mare was inseminated 38 hours after intravenous hcg treatment; a flexible pipette was introduced through the cervix and guided transrectally to the tip of the uterine horn near the ovulation site. transrectal ultrasonographic examination on day 1 after insemination revealed no possible excessive uterine reaction due to new extender or semen dose and no evidence of persistent inflammatory process.18 results fourteen days after insemination another transrectal examination and ultrasonography were performed; a 22 mm mobile, embryonic vesicle was observed, consistent with the expected size of a 14-day embryo (figure).19 on day 23 of pregnancy, embryo’s heartbeat was identified; amniotic sac was attached at left uterine horn base (figure). fetal sex was diagnosed on day 60 of pregnancy (genital tubercle was close figure. embryonic vesicle (a) and embryo (b) at 14 and 60 days after insemination, respectively; healthy colt was recently born (c, d) http://dx.doi.org/10.58292/ct.v17.13213 citation: clinical theriogenology 2025, 17, 13213, http://dx.doi.org/10.58292/ct.v17.13213 3 to umbilical cord20), progesterone was not supplemented during pregnancy. parturition occurred on day 344 after ovulation; a healthy colt was born (figure). fetal membranes were examined immediately; there were no grossly visible abnormalities. discussion semen quality and fertility maintenance are affected by the extender composition and its interactions with sperm. furthermore, temperature, bacterial contamination, and seminal plasma can also influence these parameters.21 cooled semen samples have a limited storage period, maintaining good motility and pregnancy rate that normally does not exceed 72 hours.22,23 a tm range of 30-50% after 72 hours storage at 4ºc, using 2 skimmed milk-based extenders (inra96 and botusemen gold) was reported.24 in the present study, the tm of sperm diluted in beyond®, an extender without milk compounds in its formula, also decreased over time but maintained acceptable motility values for up to 10 days of storage.25 six days after semen collection, when this sample was used for deep artificial insemination, the tm after warming to 37ºc was 70%. besides providing an adequate environment and cellular nutrition,26 a longer sperm storage period might be achieved avoiding oxidation and reducing the membrane damage caused by fluidity changes when the temperature is reduced below 15ºc.6 however, decreasing temperature is a strategy to slow sperm metabolism, controlling reactive oxygen species production and bacterial growth. therefore, using an extender focused on antioxidation and microbiological control, would allow maintaining semen above 15ºc and so reduce sperm damage.23,27 in the present study, the use of beyond® allowed semen storage at 17ºc, maintaining sperm quality for a longer interval than other protocols that require sample`s cooling at 4-5ºc.24,28 possibly, other sperm parameters might have been benefited using this extender that remain to be evaluated. in conclusion, we report the first pregnancy achieved after storing semen for > 48 hours of a poor cooler stallion. semen can be stored in a new extender (beyond®) for up to 6 days at 17ºc while maintaining sperm fertilization capacity; this opens new possibilities in equine breeding routines, especially for stallions with semen storage difficulties. author contributions and agreement authors contributions; mn designed, mn conducted, mg analyzed data and wrote the manuscript, and gc and mm involved in manuscript preparation. authors have read and approved the final version of the manuscript and have agreed to its submission. conflict of interest none to declare. references 1. aurich c: recent advances in cooled-semen technology. anim reprod sci 2008;107:268-275. doi: 10.1016/j.anireprosci.2008.04.015 2. kowalczyk a, czerniawska-piątkowska e, kuczaj m: factors influencing the popularity of artificial insemination of mares in europe. animals 2019;9:460. doi: 10.3390/ani9070460 3. price s, aurich j, davies-morel m, et al: effects of oxygen exposure and gentamicin on stallion semen stored at 5 and 15  degrees. reprod domest anim 2008;43:261-266. doi: 10.1111/j.1439-0531.2007.00888.x 4. trentin jm, rodrigues mf, pessoa ga, et al: viability of pony stallion semen in different temperature and dilution. acta sci vet 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theriogenology 2000;54:129-136. doi: 10.1016/s0093-691x(00)00331-9 15. segabinazzi l, carmo mt, freitas-dell’aqua cp, et al: post-cooling semen processing and sperm re-suspension as an alternative method to circumvent poor semen cooling in stallions. equine vet j 2024;56:697-710. doi: 10.1111/evj.14085 16. cecere jt: eosin-nigrosin staining in the evaluation of sperm. in: dascanio j, mccue p: editors. equine reproductive procedures.  2nd edition, ames; wiley: 2021. p. 373-376. doi: 10.1002/9781119556015.ch132 17. samper jc, plough t: techniques for the insemination of low doses of stallion sperm. reprod domest anim 2010;45:35-39. doi: 10.1016/j.theriogenology.2005.05.012 18. troedsson mht: uterine clearance and resistance to persistent endometritis in the mare. theriogenology 1999;52:461-471. doi: 10.1016/s0093-691x(99)00143-0 19. mccue p: ultrasound examination of the pregnant mare. in: dascanio j, mccue p: editors. equine reproductive procedures. 2nd edition, ames; wiley: 2021. p. 257-262. 20. curran s: fetal sex determination in cattle and horses by ultrasonography. theriogenology 1992;37:17-21. doi: 10.1016/ 0093-691x(92)90244-l 21. lefrapper l, walston bs, whisnant cs: comparison of various extenders for storage of cooled stallion spermatozoa for 72  hours.  j equine vet sci 2010;35:200-204. doi: 10.1111/ j.1439-0531.2006.00828.x 22. novello g, podico g, segabinazzi lgtm, et al: stallion semen cooling using native phosphocaseinate-based extender and sodium caseinate cholesterol-loaded cyclodextrin-based extender. j equine vet sci 2020;92:103104. doi: 10.1016/j.jevs.2020.103104 23. papin j, stuhtmann g, martinsson g, et al: stored stallion sperm quality depends on sperm preparation method in inra82 or inra96. j. equine vet sci 2021;98:103367. doi: 10.1016/j.jevs.2020.103367 24. rečková z, filipčík r, soušková k, et al: the efficiency of different types of extenders for semen cooling in stallions. anim biosci 2022;35:670-676. doi: 10.5713/ab.21.0300 25. prell mj, mccue pm, moffett pd, et al: motility and fertility evaluation of thawed frozen stallion semen after 24 hours of cooled storage. j equine vet sci 2020;90:102983. doi: 10.1016/j.jevs.2020.102983 26. gibb z, aitken rj: the impact of sperm metabolism during in vitro storage: the stallion as a model. biomed res int 2016;2016:9380609. doi: 10.1155/2016/9380609 27. graham jk: principles of cooled semen. in: mckinnon ao, squires el, vaala et al: editors. equine reproduction. 2nd edition, ames; wiley: 2011. p. 1308-1315. 28. cuervo-arango j, nivola k, vaihkonen l, et al: the effect of storage temperature of stallion semen on pregnancy rates. j equine vet sci 2015;35:611-616. doi: 10.1016/j.jevs.2015.06.003 http://dx.doi.org/10.58292/ct.v17.13213 https://doi.org/10.1053/j.ctep.2007.09.003 https://doi.org/10.1111/j.1439-0531.2010.01728.x https://doi.org/10.1016/s0093-691x(00)00331-9 https://doi.org/10.1111/evj.14085 https://doi.org/10.1002/9781119556015.ch132 https://doi.org/10.1016/j.theriogenology.2005.05.012 https://doi.org/10.1016/s0093-691x(99)00143-0 https://doi.org/10.1016/0093-691x(92)90244-l https://doi.org/10.1016/0093-691x(92)90244-l https://doi.org/10.1111/j.1439-0531.2006.00828.x https://doi.org/10.1111/j.1439-0531.2006.00828.x https://doi.org/10.1016/j.jevs.2020.103104 https://doi.org/10.1016/j.jevs.2020.103367 https://doi.org/10.5713/ab.21.0300 https://doi.org/10.1016/j.jevs.2020.102983 https://doi.org/10.1155/2016/9380609 https://doi.org/10.1016/j.jevs.2015.06.003 _hlk207653505 2019 a comparison of pregnancy diagnosis methods in commercial sheep using lambing as a gold standard   a comparison of pregnancy diagnosis methods in commercial sheep using lambing as a gold standard jennifer roberts,a katherine may,b olumide ajani,c john kaneene d adepartment of large animal clinical sciences, college of veterinary medicine, michigan state university east lansing, mi, bantel biosystems, lansing, mi cdepartment of theriogenology, university of ibadan, ibadan, nigeria ddepartment of large animal clinical sciences and center for comparative epidemiology college of veterinary medicine, michigan state university, east lansing, mi abstract several methods are used for pregnancy diagnosis in sheep, including ultrasonography and detection of pregnancy associated glycoproteins (pag) in blood, using either an idexx rapid visual pregnancy test (rvpt) kit or idexx bovine pregnancy test (bpt). the objective was to compare transabdominal ultrasonography, rvpt and bpt for pregnancy diagnosis in sheep, with lambing results as a gold standard. crossbred ewes (n = 196) from 3 commercial flocks were exposed to a ram for a 60day breeding period. transabdominal ultrasonography was performed 30 days after ram removal and a blood sample was concurrently collected. results of transabdominal ultrasonography, rvpt, and bpt were compared to lambing results. for ultrasonography, rvpt and bpt, sensitivities were 98.6, 97.2, and 95.1%, respectively (95% ci were 95 99.8, 93 99.2 and 90.1 98,) and specificities were 100, 94.3 and 98.1% (95% ci were 93.2 100, 84.3 98.8 and 89.9 99.9). seven pregnant ewes were misclassified as not pregnant on the bpt, whereas 4 of these ewes were also misclassified as not pregnant on the rvpt. we concluded that rvpt, bpt, and transabdominal ultrasonography had similar sensitivity and specificity when compared to lambing results. keywords: pregnancy-associated glycoproteins, sheep, pregnancy diagnosis, transabdominal ultrasonography introduction global sheep production continues to rise to meet consumer demand for sheep meat.1 therefore, it is imperative to improve reproductive efficiency in sheep production. early and accurate diagnosis of pregnancy enables producers to implement management interventions to attain pregnancy in non-pregnant females and aid culling decisions. common methods of ovine pregnancy diagnosis include transrectal or transabdominal ultrasonography and assays for pregnancy associated glycoproteins (pag). ultrasonography is a standard method for pregnancy diagnosis in sheep. one study indicated accuracy of 100% for transrectal ultrasonographic pregnancy diagnosis performed at least 20 days after mating when compared to a second ultrasound examination.2 in another study, specificity, and accuracy of transabdominal ultrasonography in sheep was 100% at 39 days compared to necropsy examinations or lambing.3 despite the high diagnostic accuracy of ultrasonography for pregnancy diagnosis in ewes, limitations include handling facilities, ewe positioning for examination, equipment availability, portability and cost, as well as transducer type and frequency. pregnancy-associated glycoproteins are inactive aspartic proteinases produced by monoand binucleate giant cells in the trophoblast layer of the ruminant placenta.4,5 these proteins cross into maternal circulation via passive diffusion and have been used to detect pregnancy in ruminants, including sheep.6-8 pregnancy-associated glycoproteins is detected in pregnant ewes as early as 20 days after insemination.6 107 clinical theriogenology • volume 11, number 2 • june 2019   although commercially available elisas use bovine anti-pag antibodies for detection of maternal pag, structural similarities between bovine and ovine pag enable detection of ovine pag using bovine assays.9,10 the idexx rapid visual pregnancy test (rvpt) and idexx bovine pregnancy test (bpt) (idexx laboratories, inc., westbrook, me) detect pag in blood of pregnant ruminants. sensitivity and specificity of bpt for pregnancy diagnosis in sheep was 93.5 and 98.9%, respectively, when compared to ultrasonographic pregnancy diagnosis.9 similarly, rvpt had 97.56% sensitivity and 93.75% specificity for pregnancy diagnosis in sheep when using transabdominal ultrasonography as the gold standard at known pregnancy timepoints.11 veterinarians advising clients on methods for pregnancy diagnosis for their flock may consider factors such as flock size, available facilities or equipment, cost and timeliness of results. whereas results of ultrasonography are immediately available, the bpt requires submission to a laboratory for analysis and a subsequent delay in obtaining results. the rvpt is a sandwich elisa that detects pag in maternal samples of whole blood or serum, providing more flexibility in sample collection and processing. the rvpt relies on visual readout of results, can be performed in veterinary clinics by trained personnel, without expensive laboratory equipment, with results available in < 1 hour. the ease of performing the test in the veterinary clinic is appealing for veterinarians in rural areas working with small-holder livestock farms and aiming to provide value-added services. blood pag concentrations vary throughout pregnancy, with unique profiles expressed by various assays.12 one study indicated decreased sensitivity of rvpt (compared to transabdominal ultrasonography) at the end of the first trimester of pregnancy in ewes.13 many factors affect pag concentrations in maternal circulation, including maternal nutrition and placental mass, breed, sex of the fetus, stage of pregnancy and fetal number.12, 14-16 due to wide variations in flock management, breeds and stages of pregnancy in reproductive management of sheep, evaluation of available methods of pregnancy diagnosis compared to lambing results is warranted. the objective was to evaluate diagnostic sensitivity and specificity of transabdominal ultrasonography, rvpt, and bpt in commercial sheep at varying stages of pregnancy, based on a single ultrasonographic examination and concurrently collected blood sample, using lambing as the gold standard. to the authors’ knowledge, this was the first study to evaluate all 3 methods of pregnancy diagnosis under conditions encountered by veterinarians in the field where gestational days were variable but estimated, using lambing results at the gold standard. materials and methods all animals used in this study, conducted from november 2016 to july 2017, were breeding age ewes (1 7 years) from two privately owned flocks and michigan state university teaching and research center, with dorper, shropshire, hampshire, and crossbred (dorset x polypay) sheep breeds. all ewes were managed on grazing pastures with supplemental feeds. all procedures performed during this study were approved by the institutional animal care and use committee at michigan state university (auf #07/16-107-00). transabdominal ultrasonography using an ovi-scan scanner with variable 3.5 5.0 mhz, 170° axial scan transducer (bcf technology, rochester, mn) was performed on all ewes at least 30 days after ram exposure. gestational age was estimated based on fetal measurements and fetal count was obtained during examination.17 concurrently, blood samples were collected by jugular venipunture into an 8.5 ml serum separator tube (bd diagnostics, franklin lakes, nj). samples were transported on ice to the 108clinical theriogenology • volume 11, number 2 • june 2019   laboratory and centrifuged at 400 x g for 5 minutes. serum was pipetted into 1.5 ml polypropylene microcentrifuge tubes and stored for 2 weeks at -20°c until analysis. serum samples were analyzed using the idexx rapid visual pregnancy test (idexx laboratories, inc., westbrook, me) according to the manufacturer’s instructions. briefly, 100 µl of sheep serum samples and assay controls were pipetted into anti-pag antibody coated wells with detector solution and incubated for 7 minutes at room temperature (18 26°c). following incubation, wells were washed 3 times with distilled water. after washing, conjugate solution was added to each well and incubated for 7 minutes at room temperature. wells were washed 3 times with distilled water, followed by addition of substrate solution. after 7 minutes of incubation at room temperature, stop solution was added to each well and color in each well was compared to the positive and negative controls. color development deemed greater than the negative control was considered a positive result, whereas color development equal to or less than the negative control was considered a negative result. only a dichotomous result was reported for rvpt (none of the samples were classified inconclusive). the reader of the rvpt results was blinded to the results of the ultrasound and bpt. the idexx bovine pregnancy test was performed as described,12 using a commercial antigen capture enzyme-linked immunosorbent assay (idexx laboratories, inc., westbrook, me).1 briefly, 100 µl of ovine serum and assay controls were pipetted into anti-pag antibody coated plates with 25 µl sample diluent. plates were sealed and incubated at 37°c for 60 minutes, then washed four times using 350 µl wash solution. detector solution (100 µl) was added to each well, covered and incubated at room temperature (18 26°c) for 30 minutes, followed by four washes. then, 100 µl of conjugate solution was added, the plate covered and incubated at room temperature for 30 minutes, followed by four washes. the elisa was developed by adding 100 µl of substrate and incubating covered for 15 minutes at room temperature. development was stopped with addition of 100 µl stop solution and absorbance recorded at 450 nm using a microtiter plate spectrophotometer. sample od (optical density) values were reported as absorbance of serum samples, minus absorbance of negative controls. samples with od ≥ 0.3 were considered pregnant, whereas values ≤ 0.3 were reported as not pregnant. intra-plate and inter-plate coefficients of variation (cv) for positive controls were 2.7 and 6.3%, respectively. lambing records were maintained for all ewes by producers or flock managers. ewes were included in the final analysis if all data for ultrasonography, rvpt result, bpt result, and lambing date or confirmation of no lambs delivered were recorded. sensitivity, specificity, positive and negative predictive values, and accuracy of each method of pregnancy diagnosis were calculated using lambing as the gold standard. accuracy (acc) was calculated using the formula acc = tp+tn/(tp+fp+fn+tn) where tp = true positive, fp = false positive, tn = true negative, and fn = false negative. kappa analyses were conducted to determine agreement between lambing and each method of pregnancy diagnosis. a mcnemar’s test was computed to test the hypothesis of no significant difference between results of each method of pregnancy diagnosis and lambing. a simple regression analysis was performed for comparison of bpt elisa od values and estimated gestational days. results all ewes (n = 196) examined were either classified as pregnant (n = 141) or not pregnant (n = 55) following transabdominal ultrasound examination. pregnant females ranged from 35 to 92 days estimated gestational age, based on fetal measurements obtained via ultrasonography. for ultrasonography, rvpt and bpt, sensitivity was 98.6, 97.2, and 95.1%, respectively, whereas specificity was 100, 98.11, and 94.3%, when compared to lambing results (table 1). for all 3 109 clinical theriogenology • volume 11, number 2 • june 2019   methods, kappa analyses indicated significant agreements with lambing (table 2). for mcnemar’s analysis, there were no significant differences between either ultrasonography or rvpt and lambing results, but there was a difference (p = 0.0339) between results of bpt and lambing results. the elisa od values from the bpt are shown (figure). seven pregnant ewes were misclassified as not pregnant on the bpt, whereas 4 of these ewes were also misclassified as not pregnant on the rvpt. discussion based on results, we concluded that a single blood sample obtained from ewes at least 30 days after breeding and analyzed using the rvpt can provide timely and valuable information for management of reproduction in sheep flocks. the most useful aspect of this test may be the ability to easily diagnose pregnancy in sheep raised on small-holder livestock farms or in developing countries where access to veterinary care and ultrasonography equipment is limited, but reliable information on pregnancy status of production animals can impact the producer’s livelihood. in addition to ultrasonography or laboratory elisa, veterinarians have the option to expand their billable services by providing pregnancy diagnosis in sheep via in-house pag testing using the rvpt. in the current study, sensitivity of transabdominal ultrasonography was 98.6%, whereas sensitivity and specificity of transabdominal ultrasonography to detect pregnancy in sheep 30 days after insemination was 83 and 100%, respectively, with sensitivity reaching 100% at 39 days after insemination.3 sensitivity of the rvpt and bpt were 97.2 and 95.1%, respectively, comparable to ultrasonography. perhaps some ewes were too early in pregnancy for transabdominal ultrasonography, since the ram was removed only 30 days prior to the examination. however, ultrasonography can provide valuable information about fetal number and viability. furthermore, pag can persist in maternal circulation after pregnancy loss in ruminants and for up to 10 weeks postpartum in sheep, which may result in false positives if the test is performed too soon after pregnancy loss or lambing.12,13,18 false positives obtained during interpretation of the rvpt were likely due to the visual readout and interpretation error of negative wells located adjacent to strongly positive wells or a slight color change in the well due to residual pag following pregnancy loss. in a previous study, specificity of the rvpt increased from 85 to 93% when read using a microtiter plate reader rather than solely relying on visual interpretation.19 the cost of a microtiter plate reader ranges from $4,000 to $5,000 and may be too costly of an investment for a private practice if visual readout results are acceptable. although a few ewes may be misclassified using only visual readout, in the present study, rvpt test closely agreed with lambing results and has potential to provide valuable management information to sheep producers and veterinarians. seven ewes in this study were diagnosed pregnant based on ultrasonography and subsequently lambed but were classified not pregnant with od values ≤ 0.3 on the bpt (figure 1). of these 7 ewes, 4 were also classified as not pregnant using the rvpt and were between 35 and 55 days, based on ultrasonographic fetal measurements. in cows, the profile of pag secretion in early pregnancy declined before increasing again and continuing to rise until calving.20 perhaps pag secretion in sheep has a similar phenomenon that may account for the misclassifications of the 7 ewes in early pregnancy. in this regard, decreased sensitivity of the rvpt at 49 days in ewes was reported.13 future studies are needed to fully characterize the pattern of secretion of pag throughout pregnancy in sheep and evaluate effects on test performance when diagnosing pregnancy in sheep using an assay targeted at bovine pag. there was strong agreement with lambing for 3 common methods of pregnancy diagnosis in sheep. this information provides veterinary practitioners with the evidence needed to counsel clients who are trying to select the optimal method of pregnancy diagnosis for reproductive management of their 110clinical theriogenology • volume 11, number 2 • june 2019   flock, based on available resources and the timeframe for desired results. it is important for veterinarians to recognize the limitations of reading results of the rvpt and potential for misclassification of open or pregnant animals when using pag assays such as the bpt due to changes in pag secretion throughout pregnancy or persistence of pag in maternal circulation after pregnancy loss. misclassifications during pregnancy diagnosis may impact the economic value of the test due to housing a non-pregnant female until the next breeding season; however, implementation of pregnancy diagnosis via any available method is a critical component of sound reproductive management of the flock. while the chosen method of pregnancy diagnosis will depend on many factors including cost, accessibility of services, technical expertise, and flock management, veterinarians can be confident that, based on this study, transabdominal ultrasonography, rvpt, or bpt at varying stages of pregnancy yield results in close agreement with pregnancy outcomes. conflict of interest none. funding funding for this study was provided through the united states department of agriculture – foreign agricultural service (usda-fas) faculty exchange program [grant number fe-cr-16-005]. acknowledgements the authors thank the staff at the michigan state university sheep teaching and research center for their assistance. we also thank ms. nicole keener for administrative support throughout the faculty exchange program. references 1. colby l, 2015. world sheep meat market to 2025. http://beefandlamb.ahdb.org.uk/wpcontent/uploads/2016/01/world-sheep-meat-market-to-2025.pdf (accessed 2/20/2018) 2. romano je and christians cj: early pregnancy diagnosis by transrectal ultrasonography in ewes. sm rumin res 2008;77:51-57. 3. jones ak, gately re, mcfadden kk, et al: transabdominal ultrasound for detection of pregnancy, fetal and placental landmarks, and fetal age before day 45 of gestation in the sheep. theriogenology 2016;85:939-945. 4. sasser rg, ruder ca, ivani ka, et al: detection of pregnancy by radioimmunoassay of a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation. biol reprod 1986;35:936-942. 5. xie sc, low bg, nagel rj, et al: identification of the major pregnancy-specific antigens of cattle and sheep as inactive members of the aspartic proteinase family. proc natl acad sci usa 1991;88:10247-10251. 6. karen a, beckers jf, sulon j, et al: early pregnancy diagnosis in sheep by progesterone and pregnancy-associated glycoprotein tests. theriogenology 2003;59:1941-1948. 7. green ja, parks te, avalle mp, et al: the establishment of an elisa for the detection of pregnancy-associated glycoproteins (pags) in the serum of pregnant cows and heifers. theriogenology 2005;63:1481-503. 8. redden r, passavant c: efficacy of pregnancy-specific protein b assay to detect pregnancy and lambing rates in sheep. sheep and goat research journal 2013;28:21-24. 9. rovani mt, cezar as, rigo ml, et al: evaluation of a bovine pregnancy-associated glycoprotein enzyme-linked immunosorbent assay kit for serological diagnosis of pregnancy in sheep. ciencia rural 2016;46:362-367. 10. xie s, green j, bixby jb, et al: the diversity and evolutionary relationships of the pregnancy-associated glycoproteins, an aspartic proteinase subfamily consisting of many trophoblast-expressed genes. proc natl acad sci usa 1997;94:12809-12816. 11. chaves c, dias da costa rl, duarte kmr, et al: visual elisa for detection of pregnancy-associated glycoproteins (pags) in ewe serum. theriogenology 2017;97:78-82. 12. roberts jn, may kj, veiga-lopez a: time-dependent changes in pregnancy-associated glycoproteins and progesterone in commercial crossbred sheep. theriogenology 2017;89:271-279. 111 clinical theriogenology • volume 11, number 2 • june 2019   13. steckeler p, weber f, zerbe h, et al: evaluation of a bovine visual pregnancy test for the detection of pregnancyassociated glycoproteins in sheep. reprod dom anim 2019;54:280-288. 14. wallace jm, da silva p, aitken rp, et al: maternal endocrine status in relation to pregnancy outcome in rapidly growing adolescent sheep. j endocrinol 1997;155:359-368. 15. ranilla mj, sulon j, carro md, et al: plasmatic profiles of pregnancy-associated glycoprotein and progesterone levels during gestation in churra and merino sheep. theriogenology 1994;42:537-545. 16. patel ov, sulon j, beckers jf, et al: plasma bovine pregnancy-associated glycoprotein concentrations throughout gestation in relationship to fetal number in the cow. eur j endocrinol 1997;137:423-428. 17. vinoles-gil c, gonzalez-bulnes a, martin gb, et al: sheep and goats. in: descoteaux l, colloton j, gnemmi g, editors. practical atlas of ruminant and camelid ultrasonography. ames: wiley-blackwell; 2010. p. 195. 18. giordano jo, guenther jn, lopes g, et al: changes in serum pregnancy-associated glycoprotein, pregnancy-specific protein b, and progesterone concentrations before and after induction of pregnancy loss in lactating dairy cows. j dairy sci 2012;95:683-697. 19. mayo lm, moore sg, poock se, et al: technical note: validation of a chemical pregnancy test in dairy cows that uses whole blood, shortened incubation times, and visual readout. j dairy sci 2016;99:7634-7641. ricci a, carvalho pd, amundson mc, et al: factors associated with pregnancy-associated glycoprotein (pag) levels in plasma and milk of holstein cows during early pregnancy and their effect on the accuracy of pregnancy diagnosis. j dairy sci 2015;98:2502-2514. table 1. sensitivity (se), specificity (sp), positive predictive value (ppv), negative predictive value (npv), and accuracy with 95% confidence interval (ci) of the idexx rapid visual pregnancy test (rvpt), idexx bovine pregnancy test (bpt) and transabdominal ultrasonography (us) compared to lambing in commercial ewes (n = 196). us rvpt bpt se (%) 98.6 97.20 95.1 95% ci 95-99.8 93-99.2 90.1-98 sp (%) 100 94.34 98.11 95% ci 93.2-100 84.3-98.8 89.9-99.9 ppv (%) 100 97.89 99.27 95% ci 93.9-99.2 95.1-99.8 npv (%) 96.36 92.59 88.14 95% ci 87-99 82.6-97 78.2-93.8 accuracy (%) 98.98 96.43 95.92 95% ci 96.3 – 99.8 92.7-98.5 92.1-98.2 112clinical theriogenology • volume 11, number 2 • june 2019   table 2. kappa analysis of transabdominal ultrasonography (us), idexx rapid visual pregnancy test (rvpt) and idexx bovine pregnancy test (bpt) versus lambing for detection of pregnancy in 196 commercial ewes. figure. elisa optical density (od) values for samples tested using idexx bovine pregnancy test (bpt) (n = 196) by estimated gestational days. horizontal dashed line indicates the od cut off value of 0.3 for classification of pregnant or not pregnant on test interpretation. seven ewes diagnosed pregnant by ultrasonography were reported as not pregnant, whereas 4 of these ewes were diagnosed as not pregnant on the rvpt. r² = 0.5988 0 0.5 1 1.5 2 2.5 3 0 10 20 30 40 50 60 70 80 90 100 id ex x b ov in e pr eg na nc y te st o d estimated gestational days us rvpt bpt degree of agreement 0.974 0.900 0.910 95% ci 0.939 1 0.832-0.967 0.844-0.975 p-value < 0.0001 < 0.0001 < 0.0001 113 clinical theriogenology • volume 11, number 2 • june 2019 blank page << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) 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/preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] /pagelayout /singlepage >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: testosterone decreases to basal values within 24 hours following castration in alpacas testosterone decreases to basal values within 24 hours following castration in alpacas m. kutzlera , t. fiamengoa , s. lambb a department of clinical sciences, college of veterinary medicine, oregon state university, corvallis, or, usa bendocrinology laboratory, animal health diagnostic center, cornell university, ithaca, ny, usa the objective of this study was to compare serum testosterone concentrations ([t]) in adult male alpacas (vicugna pacos) following administration of human chorionic gonadotropin (hcg) before and after castration. this information is of diagnostic significance for the determination of retained testicular tissue following castration or in the case of cryptorchidism. jugular blood samples were collected from nine adult alpacas prior to intravenous administration of hcg (2500 iu; chorulon®, intervet, millsboro, de, usa) as well as 2, 4 and 6 post-hcg. alpacas were castrated using routine surgical techniques 24 h post-hcg. testes were removed from the vaginal tunica, the epididimydes were removed, and the testes were weighed. a postcastration hcg response test was performed 24 h after castration (48 h from the previous hcg response test). serum [t] was measured using a double antibody radioimmunoassay (diagnostic products corporation, los angeles, ca, usa). the assay sensitivity was 0.04 ng/ml and the intra-assay and inter assay cvs were <10%. all of the samples were tested within one assay. the mean [t] at each treatment time were compared using an analysis of variance. in addition, [t] were compared to testicular weight using linear regression. significance was defined as p<0.05. the results for individual animals are summarized in the table below. the [t] was significantly increased from pre-hcg values by 2 h post-hcg. the highest mean [t] occurred 6 h post hcg, however [t] did not differ significantly between 2, 4 and 6 h post hcg. at 24 h post-castration (48 h post-hcg), the [t] was below the limits of detection. there was no significant correlation between [t] at any time point and testicular weight. in summary, serum [t] is not correlated with testicular weight and falls to undetectable values within 24 h following castration in alpacas. serum testosterone concentrations (ng/ml) id testicular weight (g) prehcg 2 h posthcg 4 h posthcg 6 h posthcg 48 h post-hcg (24 h post-castration) 1 23.49 3.23 10.32 15.53 20.37 <0.04 2 22.62 7.72 14.30 14.21 18.58 <0.04 3 21.99 3.62 5.98 10.43 8.53 <0.04 4 28.80 4.22 9.89 9.74 13.25 <0.04 5 28.09 4.44 7.15 6.33 6.96 <0.04 6 17.94 7.14 10.65 16.21 19.23 <0.04 7 14.69 0.67 3.81 6.26 4.71 <0.04 8 28.91 9.12 17.66 20.89 24.91 <0.04 9 36.16 5.60 18.48 19.83 24.94 <0.04 24.74 5.08 10.92 13.27 15.72 sd 6.48 2.60 5.06 5.40 7.64 keywords: castration, hcg, alpaca, testes, testosterone x 533 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy 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0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname () /pdfxtrapped /false /createjdffile false /description << /enu ([based on 'no compression'] [based on 'no compression wbleeed'] [based on '[high quality print]'] use these settings to create adobe pdf documents for quality printing on desktop printers and proofers. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes false /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: neoplasia of the reproductive tract of the male dog neoplasia of the reproductive tract of the male dog 1 m. v. root kustritz 2 college of veterinary medicine, university of minnesota, st. paul, mn, usa 3 abstract 4 a brief review of tumors of the male reproductive tract is provided. diagnosis is 5 straightforward in most cases, with resolution of disease easily achieved. the exception 6 to the latter is prostatic neoplasia, a high morbidity, high mortality form of cancer to 7 which we may predispose dogs by castration. 8 keywords: neoplasia, penis, prostate, testes, transmissible venereal tumor 9 introduction 10 neoplasia of the male canine reproductive tract is uncommon and often easily 11 diagnosed. the most common reproductive tract tumor of male dogs worldwide is 12 testicular neoplasia, with transmissible venereal tumor also very common. the most 13 devastating cancer of the reproductive tract of male dogs is prostatic neoplasia. less 14 common tumors include those of the soft tissues of the penis and prepuce, those of the os 15 penis, and those of the scrotal skin and mammary tissues. excellent published reviews of 16 this topic exist.1,2 this manuscript will provide brief reviews of cancer biology and 17 updated information. 18 testicular tumors 19 the three most common tumor types are seminomas, leydig (interstitial) cell 20 tumors, and sertoli cell tumors. in humans, testicular neoplasia is common in young men 21 and is aggressively malignant. in dogs, testicular neoplasia occurs in older individuals 22 93 and is malignant in 10% of cases at most. it is not uncommon for there to be more than 23 one tumor type in a given testes or between testes concurrently.3,4 24 incidence of testicular neoplasia is higher in retained than in descended testes. 25 there is no research documenting increased risk of neoplasia in the descended testis of 26 unilateral cryptorchids.5 controversy exists regarding whether or not the predisposition 27 to neoplasia in retained testes remains if the cryptorchid testis is surgically pulled down 28 and tacked into the scrotum. in human medicine, studies suggest increased incidence in 29 tumor incidence if orchiopexy is delayed until puberty or beyond.5,6 this may suggest 30 that medical therapies to induce testicular descent are beneficial in prepubertal dogs, both 31 by potentially decreasing tumor incidence and making castration of those dogs easier. 32 dogs with testicular neoplasia may present with enlargement of one or both 33 testes. if neoplasia is unilateral, oftentimes there will be atrophy of the contralateral testis 34 due to increased intrascrotal temperature and possibly to hormone secretion by the 35 affected testis. components of an associated paraneoplastic syndrome, most commonly 36 associated with estrogen secretion, include gynecomastia, attraction of male dogs, and 37 bilaterally symmetrical alopecia. malignant tumors mestastasize locally and to the 38 regional lymph nodes and lungs, with occasional reports of hypertrophic 39 osteoarthropathy.7 a fair proportion of dogs (28.3% in one study) have inapparent 40 testicular neoplasia; these dogs may present for testicular atrophy, poor semen quality, or 41 infertility.3 42 diagnosis is by inspection. for dogs with no overt change in testicular size or 43 consistency, testicular ultrasound is the preferred diagnostic technique. fine-needle 44 aspirate (fna) of the testes also may be used to identify abnormal cells. the dog is 45 94 sedated and the scrotal skin cleaned. a 20 ga needle is attached to a 12 cc or larger 46 syringe. the needle is introduced on or just lateral to the midline and redirected several 47 times, with suction at each location. negative pressure is released and the needle 48 withdrawn. the sample is expelled onto a glass slide and submitted to a cytologist for 49 interpretation.8-10 50 treatment is surgical. bilateral castration is recommended. caution during 51 surgery is recommended; there are reports of neoplastic tissue (n = 11 sertoli cell tumors, 52 n = 1 leydig cell tumor) arising in the spermatic cord, at the incision site, on the scrotal 53 skin, and in the inguinal canal, presumably due to transplantation of cells during 54 castration.11 in valuable breeding animals, one may consider removing only the affected 55 testis but the owners must be cautioned that spermatogenesis may be altered by 56 inapparent changes in the remaining testis. 57 in humans, incidence of testicular neoplasia is increasing. this same trend 58 appears to be true in veterinary medicine, with incidence of testicular tumors increased in 59 one study from that described in 1962.12 the primary theory explaining this in human 60 medicine is increase in exposure to environmental toxins. 61 transmissible venereal tumor 62 transmissible venereal tumor (tvt) is a cellular transplant containing an average 63 of 59 chromosomes, rather than a transformed canine cell, which would contain 78 64 chromosomes. analysis of tvts from five continents showed that all are genetically 65 similar, suggesting that all arose from a single mutation. that mutation is believed to 66 have arisen 250 to 2500 years ago in a wolf or east-asian breed of dog.13 67 95 incidence of tvt in one study was 5.4%; this incidence varies greatly by region.4 68 tvt is most common in young, sexually intact, free-roaming dogs, and so is primarily a 69 problem in sub-tropical and tropical areas of the world. 70 most tvts invade only locally and so usually are not considered malignant. the 71 tumor is a meaty and irregular, and is easily ulcerated. lesions may be noted on the 72 mouth and nose; this is from transplantation of cells by licking of the genitalia. 73 occasionally tumor will spread to regional lymph nodes and the internal organs.14 74 metastasis is more common in very young animals and immunosuppressed animals.15 75 diagnosis is by cytology. tvt is easily identified as round cells with abundant 76 cytoplasm and an eccentrically placed round to oval nucleus. 77 chemotherapy with vincristine is the recommended treatment. surgical debulking 78 may be required in some cases. vincristine treatment has been demonstrated to cause 79 resolution of clinical signs with minimal side-effects within 4 treatments in most dogs, 80 and to decrease semen quality in breeding dogs for only up to 15 days after treatment is 81 completed.16,17 treatment failure is more likely if tumors are large, the affected animal is 82 old or immunosuppressed, and if the treatment is performed during hot or rainy months.18 83 prostatic neoplasia 84 the most common prostatic neoplasm reported is adenocarcinoma. it is 85 considered to have metastasized by the time of diagnosis in the majority of cases. it is a 86 disorder of older male dogs and may occur in either intact or castrated dogs. it is the only 87 prostatic disorder commonly seen in castrated dogs. overall incidence of prostatic 88 neoplasia is reported as 0.4 to 0.7%.19,20 it is a high mortality disorder; in one survey of 89 96 72 affected dogs, 58 were euthanized at the time of diagnosis and mean survival for those 90 who survived more than one week from diagnosis was 30 days.21 91 in humans, prostatic neoplasia is hormone-dependent. that is not true in dogs. in 92 fact, castration, and subsequent removal of testosterone, appears to predispose dogs to 93 prostatic neoplasia, with increased risk of 2.8 to 4.3 times reported.22,23 castrated dogs 94 tend to develop more poorly differentiated tumors than intact dogs, suggesting that 95 testosterone has a protective effect.24 number of androgen receptors is decreased in the 96 prostates of dogs with neoplasia compared to dogs with normal prostates or benign 97 prostatic hypertrophy, again suggesting loss of a protective effect of testosterone.25 in 98 one survey of 56 affected dogs, time from castration to onset was variable and there was 99 no difference in mean age at time of diagnosis between intact and castrated dogs, 100 suggesting that castration does not favor tumor initiation but may favor tumor 101 progression.20 102 it has been shown in dogs that castration leads to an increase in number of 103 receptors for endothelin on the prostate; endothelin increases mitogenic responses and 104 uncontrolled cell growth.26 endothelin also supports osteoblastic function and stimulates 105 bony growth at areas of metastasis from prostatic neoplasia in dogs.27 106 dogs may present with signs of prostate disease, such as dripping of bloody fluid 107 from the penis unassociated with urination, hematuria, and passage of ribbon-shaped 108 stools. commonly, dogs also present with stranguria and signs referable to sites of 109 metastasis including stiff gait or ataxia, coughing, and cachexia. 110 because prognosis is grave, definitive diagnostics are required. definitive 111 diagnosis requires collection of a sample directly from the prostate, either as fna or 112 97 biopsy. ultrasound guidance is recommended with either technique. for fna, sedation 113 usually is not required. a 20 ga needle is attached to a 12 cc syringe. the needle is passed 114 into the prostatic parenchyma and negative pressure applied. pressure is released and the 115 needle withdrawn. the sample is expelled onto a glass slide and submitted to a cytologist 116 for interpretation. for biopsy, sedation is required. prepare the area over the prostate as 117 for sterile surgery and place a sterile sleeve over the ultrasound probe. viewing the 118 prostate by ultrasound, trigger the biopsy instrument to see it pass within the prostatic 119 parenchyma. withdraw the biopsy instrument and retrieve the sample; make sure you 120 have an adequate sample before reversing sedation. samples should be submitted to a 121 pathologist for interpretation. a diagnostic sample is more commonly retrieved by biopsy 122 than by fna. side-effects include hematuria and hemospermia.28,29 in humans, seeding 123 of the abdomen with tumor cells and subsequent growth of transplanted cells is reported 124 to occur in 0.009% of cases after these techniques.30 this is less of a concern in dogs, 125 who rarely live long enough after diagnosis for significant secondary tumor development 126 to occur. 127 treatment is palliative. most dogs are treated with anti-inflammatory medications 128 (meloxicam, piroxicam) and antibiotics to control secondary infection. prostatectomy 129 rarely is performed because it cannot cure disease and often causes urinary or fecal 130 incontinence. there are reports of transurethral resection of the prostate using 131 electrocautery, with or without associated radiation and chemotherapy,31 and subcapsular 132 prostatectomy using a laser.32 some of the dogs in these studies showed a resolution of 133 clinical signs and had longer mean survival times than is commonly reported but other 134 dogs in those studies did not respond to treatment or died from complications of therapy. 135 98 that, coupled with the technical skill required to perform this work, makes them 136 unsuitable alternatives for most veterinarians and their patients. 137 tumors of the penis, prepuce, scrotum, and mammary tissue 138 tumors of the soft tissue of the penis and of the os penis are described in the 139 literature. tumors of the soft tissue of the penis often are associated with preputial 140 extension and, along with tumors of the scrotum, are the common skin tumors seen 141 elsewhere including papillomas, squamous cell carcinoma, lymphosarcoma, and mast cell 142 tumors.33,34 tumors of the os penis are less common and may be either benign or 143 malignant. stranguria is a common presenting complaint with tumors of the os penis, 144 which may be confused with fracture of the os penis.34-38 mammary neoplasia rarely is 145 reported in male dogs; one institution reported that male dogs at that institution were 62 146 times less likely to develop mammary neoplasia than female dogs in that population.39 147 mammary tumors usually are benign and easily surgically removed. 148 references 149 1. johnston sd, root kustritz mv, olson pn: canine and feline theriogenology. 150 philadelphia: wb saunders; 2001. 151 2. ogilvie gk, moore as: managing the veterinary cancer patient. trenton nj: 152 veterinary learning systems; 1995. 153 3. peters maj, de rooij dg, teerds kj, et al: spermatogenesis and testicular 154 tumors in ageing dogs. j reprod fertil 2000;120:443-452. 155 4. ortega-pacheco a, rodriguez-buenfil jc, segura-correa jc, et al: pathological 156 conditions of the reproductive organs of male stray dogs in the tropics: 157 99 prevalence, risk factors, morphological findings and testosterone concentrations. 158 reprod dom anim 2006;41:429-437. 159 5. wood hm, elder js: cryptorchidism and testicular cancer: separating fact from 160 fiction. j urol 2009;181:452-461. 161 6. walsh tj, dall’era ma, croughan ms, et al: orchiopexy for cryptorchidism 162 may be associated with lower risk of testicular cancer. j urol 2007;178:1440-163 1446. 164 7. barrand kr, scudamore cl: canine hypertrophic osteoarthropathy associated 165 with a malignant sertoli cell tumor. j sm anim pract 2001;42:143-145. 166 8. dahlbom m, makinen a, suominen j: testicular fine needle aspiration cytology 167 as a diagnostic tool in dog infertility. j sm anim pract 1997;38:506-512. 168 9. romagnoli s, bonaccini p, stelletta c, et al: clinical use of testicular fine needle 169 aspiration (fna) cytology in oligozoospermic and azoospermic dogs. 170 proceedings of the international symposium on canine and feline reproduction, 171 vienna, austria, 2008. 172 10. masserdotti c, bonfanti u, de lorenzi d, et al: cytologic features of testicular 173 tumours in dogs. j vet med a 2005;52:339-346. 174 11. doxsee al, yager ja, best sj, et al: extratesticular interstitial and sertoli cell 175 tumors in previously neutered dogs and cats: a report of 17 cases. can vet j 176 2006;47:763-766. 177 12. grieco v, riccardi e, greppi gf, et al: canine testicular tumours: a study on 232 178 dogs. j comp path 2008;138:86-89. 179 100 13. murgia c, pritchard jk, kim sy, et al: clonal origin and evolution of a 180 transmissible cancer. cell 2006;126:477-487. 181 14. park m-s, kim y, kang m-s, et al: disseminated transmissible venereal tumor 182 in a dog. j vet diagn invest 2006;18:130-133. 183 15. mukaratirwa s, gruys e: canine transmissible venereal tumor: cytogenetic 184 origin, immunophenotype, and immunobiology. a review. vet q 2003;25:101-185 111. 186 16. saratsis ph, ypsilantis p, tselkas k. semen quality during vincristine treatment 187 in dogs with transmissible venereal tumor. theriogenology 2000;53:1185-1192. 188 17. nak d, nak y, cangul it, et al:. a clinicopathological study on the effect of 189 vincristine on transmissible venereal tumour in dogs. j vet med a 2005;52:366-190 370. 191 18. scarpelli kc, valladao ml, metze k: predictive factors for the regression of 192 canine transmissible venereal tumor during vincristine therapy. vet j, in press, 193 corrected proof, available online 23 december 2008. 194 19. merlo df, rossi l, pellegrino c, et al: cancer incidence in pet dogs: findings of 195 the animal tumor registry of genoa, italy. j vet intern med 2008;22:976-984 196 20. teske e, naan ec, van dijk em, et al: canine prostate carcinoma: 197 epidemiological evidence of an increased risk in castrated dogs. molec cell 198 endocrinol 2002;197:251-255. 199 21. mann fa, barrett rj, henderson ra: use of a retained urethral catheter in three 200 dogs with prostatic neoplasia. vet surg 1992;21:342-347. 201 101 22. sorenmo ku, goldschmidt m, shofer f, et al: immunohistochemical 202 characterization of canine prostatic carcinoma and correlation with castration 203 status and castration time. vet comp oncol 2003;1:48-56. 204 23. bryan jn, keeler mr, henry cj, et al: a population study of neutering status as 205 a risk factor for canine prostate cancer. prostate 2007;67:1174-1181. 206 24. lai c-l, van den ham r, van leenders g, et al. histopathological and 207 immunohistochemical characterization of canine prostate cancer. prostate 208 2008;68:477-488. 209 25. gallardo f, mogas t, baro t, et al: expression of androgen, oestrogen alpha and 210 beta, and progesterone receptors in the canine prostate: differences between 211 normal, inflamed, hyperplastic and neoplastic glands. j comp path 2007;136:1-8. 212 26. padley rj, dixon db, wu-wong jr: effect of castration on endothelin receptors. 213 clin sci 2002;103(suppl 48):442s-445s. 214 27. leroy be, sellers rs, rosol tj: canine prostate stimulates osteoblast function 215 using the endothelin receptors. prostate 2004;59:148-156. 216 28. root kustritz mv: collection of tissue and culture samples from the canine 217 reproductive tract. theriogenology 2006;66:567-574. 218 29. powe jr, canfield pj, martin pa: evaluation of the cytologic diagnosis of 219 canine prostatic disorders. vet clin path 2004;33:150-154. 220 30. nyland tg, wallack st, wisner er: needle-tract implantation following 221 ultrasound-guided fine-needle aspiration biopsy of transitional cell carcinoma of 222 the bladder, urethra and prostate. vet radiol ultrasound 2002;43:50-53. 223 102 31. liptak jm, brutscher sp, monnet e, et al: transurethral resection in the 224 management of urethral and prostatic neoplasia in 6 dogs. vet surg 2004;33:506-225 516. 226 32. l’eplattenier hf, van nimwegen sa, van sluijs fj, et al: partial prostatectomy 227 using nd:yag laser for management of canine prostate carcinoma. vet surg 228 2006;35:406-411. 229 33. cerundolo r, rest j, persechino a: what is your diagnosis? cutaneous 230 lymphosarcoma of the scrotum. j sm anim pract 2002;43:1-2. 231 34. root kustritz mv, fick jl. theriogenology question of the month: benign 232 mesenchyoma of the os penis. j am vet med assoc 2007;230:197-198. 233 35. mirkovic tk, shmon cl, allen al: urinary obstruction secondary to an 234 ossifying fibroma of the os penis in a dog. j am anim hosp assoc 2004;40:152-235 156. 236 36. michels gm, knapp dw, david m, et al: penile prolapse and urethral obstruction 237 secondary to lymphosarcoma of the penis in a dog. j am anim hosp assoc 238 2001;37:474-477. 239 37. bleier t, lewitschek hp, reinacher m: canine osteosarcoma of the penile bone. 240 j vet med a 2003;50:397-398. 241 38. patnaik ak, matthiesen dt, zawie da: two cases of canine penile 242 neoplasms: squamous cell carcinoma and mesenchymal chondrosarcoma. j am 243 anim hosp assoc 1988;24:403-406. 244 39. saba cf, rogert ks, newman sj, et al: mammary gland tumors in male dogs. j 245 vet intern med 2007;21:1056-1059. 246 103 << 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/honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1 contact eduardo arroyo eduardoarroyo@ufl.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10820, http://dx.doi.org/10.58292/ct.v16.10820 research report effects of a semen extender treated with coenzyme q10-ubiquinol on the quality of thawed ram sperm eduardo arroyo,a‡ warwick bayly,a swarnita chakraborty,b desmond leadon,c ahmed tibarya adepartment of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa, usa bdepartment of mathematics and statistics, college of arts and sciences, washington state university, pullman, wa, usa cswordlestown little stud, naas county, kildare, ireland abstract objective was to determine whether addition of coenzyme q10 ubiquinol to glycerol extender improved cryopreserved ram sperm quality. semen was collected (electroejaculation) from mature suffolk rams (n = 6) 6 times during the breeding season. pooled semen from each session was processed and cryopreserved in extenders with 150 µmol/liter of ubiquinol (treated) and without (control). thawed cryopreserved semen was evaluated using computer-assisted sperm analysis for motility and kinetic parameters. sperm viability was determined via plasma membrane integrity. after thawing, measurements were obtained at 10, 20, 60, and 120 minutes. in treated samples, there were increases in progressive motility (p < 0.0001), curvilinear velocity (p = 0.002), straight line velocity (p = 0.003) and time average velocity (p = 0.001), and sperm tended (p = 0.07) to have more viability. furthermore, there were time-related decreases in all variables in treated control and samples. ubiquinol improved cryopreserved ram sperm quality. keywords: antioxidants, reactive oxygen species, sperm, small ruminants introduction artificial insemination has been beneficial to the livestock industry, as it allows dissemination of valuable genetics and hastens improvement in genetic makeup of production animals.1 semen processing, storage, and transport should meet high quality standards to maximize conception rates.2 manipulation and storage of semen impairs sperm function. mammalian sperm are highly susceptible to oxidative stress due to high concentrations of polyunsaturated fatty acids in cell membranes and lack of cytoplasmic antioxidants. oxidative stress and accumulation of radical oxygen species (ros) are major factors contributing to poor sperm function and low conception rates.3 during mitochondrial redox reactions via oxidative phosphorylation, ~ 5% oxygen is transformed into ros.4 freezing, thawing, and procedures associated with semen storage induce oxidative stress damage to sperm. these may be factors contributing to cryodamage and alteration of sperm function and structure. oxidative stress is partially responsible for reductions in ‡current affiliation: department of large animal clinical sciences, college of veterinary medicine, university of florida, gainesville, fl, usa postthaw sperm motility, viability, membrane and acrosome integrity, mitochondrial function, dna integrity, and finally, fertility in stallions.5 several approaches have been used to limit ros formation in semen, including systemic supplementation with antioxidants or their addition to semen extenders.6 in men, reduced sperm motility was attributed to a relative deficiency of coenzyme q10 (coq10).4 additionally, energy use by sperm depends on coq10 availability. 7 coq10 exists in 2 forms: ubiquinone and ubiquinol. ubiquinol is the reduced, electron-rich bioactive form of coq10 and a potent nonenzymatic antioxidant. most coq10 in mammalian body (> 90%) exists as ubiquinol. it protects lipids, proteins, and dna by minimizing first stages of lipid peroxidation and participates in recycling of secondary antioxidants like α-tocopherol.8 these reactions result in temporary oxidation of ubiquinol to ubiquinone that serves as an electron carrier in mitochondrial respiratory chain. this function is associated with ros production and reduction of ubiquinone back to ubiquinol.9 coq10 is naturally present in every body cell, including sperm midpiece.10 coq10 has enhanced postthaw mailto:eduardoarroyo@ufl.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10820 2 citation: clinical theriogenology 2024, 16, 10820, http://dx.doi.org/10.58292/ct.v16.10820 sperm quality in men,11 stallions,12 bulls,11 goat bucks,13 roosters,14 and rams.15 several ubiquinone preparations are commercially available but have poor water solubility and limited bioavailability. coq10 is absorbed from the intestine as ubiquinol. intestinal absorption is variable and affected mainly by coq10 formulation type.16 recently, a water-dispersible preparation of ubiquinol was developed and provided better bioavailability compared to conventional coq10 preparations.17 our objective was to determine whether adding water-dispersi ble ubiquinol preparation to a ruminant semen extender improved motion parameters and viability of frozen-thawed ram sperm. we hypothesized that addition of water-dispersi ble ubiquinol to freezing extender improves sperm motility and viability compared to similar extender without ubiquinol. materials and methods in accordance with wsu iacuc policy # 21, this research is exempt because it utilized tissues collected from animas for a routine breeding soundness examination that occurred for clinical purposes and no animals were manipulated for the purpose of this research. animals and study location six mature suffolk rams aged 1-4 years, in good physical condition and with proven fertility were used. rams were raised in pacific northwest of us (46° 44’ 32.4708”, 117° 1’ 3.234), housed separately from ewes, and received a daily diet consisting of alfalfa hay, grass hay, barley, commercial sheep concentrate, canola meal, and ground corn. diet contained, on a dry matter basis, 2.97 megacalorie metabolizable energy/kg, 12.6% crude protein, 0.8% calcium, 0.31% phosphorus, 0.19% magnesium, 1.71% potassium, and 0.92% sodium.18 rams had access to mineral blocks and fresh water. experimental design semen was collected via electroejaculation on 6 days during the september-october breeding season. the low-voltage setting of a ram ejaculator (lane manufacturing, denver, co, usa) with a probe length of 6 inches was utilized. each ejaculate was evaluated for volume and concentration. only ejaculates of > 1 ml with a concentration of > 2 x 109 sperm/ml, total motility > 50%, and abnormal sperm < 10%, determined with a computer-assisted sperm analyzer, were used. ejaculates from each collection day were pooled and divided into 2 equal aliquots and then diluted to achieve the final cryopreservation concentration of 200 x 106/ml. dilutions were made with a commercial semen extender containing 20% egg yolk. either 0 µmol/liter (control) or 150 µmol/liter of a 30% solution (treated) of water-dispersible ubiquinol (petroeuroasia, shizuoka, japan) was added to extender and homogenized for 30 minutes before use. for cryopreservation, semen samples plus triladyl® were transferred into a warm water bath (30°c) and then cooled to 5°c at an approximated rate of 0.27°c/minute. semen was equilibrated for 4 hours at 5°c then loaded into 0.5 ml straws. after equilibration, the straws were placed 4 cm above the liquid nitrogen surface for 20 minutes and then plunged into liquid nitrogen. frozen semen straws were stored for 30 days in liquid nitrogen until postthaw evaluation. semen evaluation all ejaculates fulfilled inclusion criteria. semen evaluations were replicated 6 times on each stored sample. cryopreserved straws were thawed at 37°c for 30 seconds. samples were kept at 37°c and evaluated for sperm motility, kinetics, and viability at 10, 20, 60, and 120 minutes after thawing. sperm motion parameters were determined using computerized assisted semen analysis (casa spermvision®, mofa®, verona, wi, usa) equipped with a monochrome video camera (imi tech, imb-12 ft). for analysis, 3 μl of sample was placed on a prewarmed (37°c) calibrated slide (leja®, imv technologies, maple grove, mn, usa). seven fields were randomly evaluated under a phase contrast microscope 20 x positive low objective (axio 10, carl zeiss) with a warmed stage. the acquisition frame rate was set at 75 frames/second. output criteria of casa included the proportion of motile sperm and the proportion of sperm with progressive motility (pm). sperm motion characteristics were analyzed as follows: if the average velocity was below 10 µm/second, sperm were regarded as immotile, whereas velocity minimums for slow, medium and rapid sperm were 10, 25 and 50 µm/second, respectively. sperm motion velocity was measured in 3 ways: a) straight line velocity (vsl) is the time-average velocity of a sperm head along the straight line between its first and last detected positions; b) curvilinear velocity (vcl) is the average velocity measured over the actual point-to-point track followed by the sperm; and c) time-average velocity (vap) measures sperm head along its spatial average trajectory (i.e. smoothed version of vcl). linearity (lin) is the linearity of the curvilinear trajectory calculated as vsl/vcl × 100. straightness (str) is the lin of the sperm average path calculated as vsl/vap × 100. minimal lin was 50%, and minimal str for progressive fast sperm was 70%. amplitude of lateral head displacement (alh) is the maximum lateral displacement of a sperm head about its spatial average trajectory (i.e. track width). sperm viability was assessed using the sybr14/pi stain (minitube, germany) to test sperm membrane integrity, following manufacturer’s instructions. briefly, 100 μl of semen was mixed with 10 μl of warm sybr14/pi fluorescent stain and incubated for 10 minutes at 37°c. after incubation, 5 µl droplet of the stained sample was placed over a slide, covered with a coverslip, and analyzed under epifluorescence microscopy (400 x). red-stained sperm head was considered to have a nonintact membrane (nonviable, pi positive), and green-stained sperm were regarded as having intact membranes (viable, sybr14 positive). two hundred sperm were evaluated per sample for frozen-thawed samples. data analyses as data were normally distributed (shapiro-wilk test), parametric tests were used. effects of time of incubation, diluent, and their interactions on casa measurements of sperm motility kinetics (pm, vsl, vcl, vap, lin, str, alh) and sperm viability were analyzed by a general linear model randomized complete block design with repeated measures analysis of variance. if significant treatment x time interactions were identified, pair-wise tests for the interaction effects http://dx.doi.org/10.58292/ct.v16.10820 citation: clinical theriogenology 2024, 16, 10820, http://dx.doi.org/10.58292/ct.v16.10820 3 were conducted with the tukey-kramer adjustment to identify significant pairs of interactions. analyses were performed using the sas 9.4 studio online platform (sas 2022 institute, cary, nc, usa) to test treatment effects of extender with ubiquinol on sperm parameters; p < 0.05 was considered significant. values are expressed as mean ± standard deviation. results mean values of sperm motility and kinetics are reported (table 1). postthaw progressive motility was higher (p < 0.0001) in extender containing ubiquinol compared to control. vsl (p = 0.003), vcl (p = 0.002), and vap (p = 0.001) were higher in treated samples. there was an effect of treatment with ubiquinol on alh (p = 0.029) but not on lin (p = 0.67) or str (p = 0.68). the values declined over time for control and treated samples (table 1). values for viability (plasma membrane integrity) at each time point are reported (table 2). there was no significant extender treatment effect on the percentage of sperm with intact membranes, although the difference approached significance (p = 0.073). discussion addition of 150 µmol/liter of ubiquinol to extender improved pm and motion kinetics parameters (vap, vcl, vsl, alh) after thawing. viability of samples exposed to extender plus ubiquinol tended to be higher; however, the improvement (p = 0.073) did not meet the p < 0.05 criterion for significance. concentration of ubiquinol (150 µmol/liter) used was based on a stallion semen report wherein 75 µmol/liter of coq10-ubiquinone had positive effects on certain parameters of sperm motion.19 we tested a higher concentration of ubiquinol because of the possibility that egg yolk in the extender could bind with ubiquinol and make it less available to sperm.20 although causes of subfertility and infertility in male reproduction are multifactorial, oxidative stress has an important role.21 use of exogenous antioxidants improved the quality and lifespan of poor-quality sperm such as in conditions associated with asthenozoospermia and teratozoospermia.22 addition of coq10 and other antioxidants to diet and semen extenders had positive effects on sperm quality.22–24 ubiquinol is the antioxidant form of coq10 and has been widely used to enhance reproductive health and prevent neurodegenerative diseases and musculoskeletal disorders in people.24 several coq10 preparations are available, differing mainly in their redox state and degree of bioavailability, due primarily to their relative insolubility in water. we used a synthesized water dispersible formulation of ubiquinol preparation that has high availability and stability once in solution.17 ubiquinol-treated samples had a higher pm at each time point. these results agree in part with another study on ram semen that had a higher progressive motility after storage for 5 days at room temperature in samples to which 50 µmol/liter ubiquinol was added.15 better pm associated with using ubiquinol-treated extender was likely due to its protective antioxidant effect on sperm plasma membrane. however, an effect on the mitochondrial respiratory chain and the synthesis of atp might have also contributed. higher postthaw pm observed in samples containing ubiquinol was also similar to a study on goat semen; addition of 1 µm of coq10 significantly improved motility of ta b le 1 . p o st th aw r am s p er m m o ti o n c h ar ac te ri st ic s o f se m en f ro ze n in t ri la d yl e xt en d er , w it h o u t an d w it h u b iq u in o l tr il ad yl tr il ad yl + u b iq u in o l 10 m in u te s 20 m in u te s 60 m in u te s 12 0 m in u te s 10 m in u te s 20 m in u te s 60 m in u te s 12 0 m in u te s p m 30 .8 3 ± 7. 62 aa 32 .5 4 ± 8. 62 b b 25 .6 3 ± 5. 24 cc 24 .1 4 ± 7. 38 d d 46 .0 5 ± 1. 79 b a 44 .9 8 ± 1. 77 cb 40 .0 1 ± 2. 36 d c 31 .2 7 ± 4. 08 ed v c l µ m / se co n d 96 .5 3 ± 4. 3aa 87 .0 6 ± 6. 32 b b 87 .0 6 ± 4. 02 cc 87 .4 7 ± 6. 52 ee 10 0. 39 ± 3 .4 5b a 94 .1 0 ± 5. 1cb 92 .6 4 ± 4. 24 d c 94 .9 7 ± 4. 69 ed va p µ m / se co n d 30 .8 4 ± 7. 63 aa 32 .5 5 ± 8. 69 b b 25 .6 3 ± 5. 24 cc 24 .1 5 ± 7. 38 ee 46 .0 5 ± 1. 80 b a 44 .9 8 ± 1. 77 cb 40 .0 2 ± 2. 37 d c 31 .2 8 ± 4. 08 ed v sl µ m / se co n d 44 .8 2 ± 2. 34 aa 41 .4 1 ± 2. 39 b b 39 .7 1 ± 1. 79 cc 39 .7 4 ± 2. 19 ee 46 .4 0 ± 1. 85 b a 44 ± 1 .6 6cb 42 .2 5 ± 1. 17 d c 43 .2 7 ± 1. 98 e d li n ( % ) 0. 46 ± 0 .0 1aa 0. 46 ± 0 .0 1ab 0. 45 ± 0 .0 1ac 0. 45 ± 0 .0 1ad 0. 46 ± 0 .0 1aa 0. 46 ± 0 .0 1ab 0. 45 ± 0 .0 2ac 0. 45 ± 0 .0 1ad st r ( % ) 0. 78 ± 0 .0 2aa 0. 78 ± 0 .0 2ab 0. 77 ± 0 .0 1ac 0. 77 ± 0 .0 2ad 0. 78 ± 0 .0 1aa 0. 78 ± 0 .0 1ab 0. 77 ± 0 .0 2ac 0. 78 ± 0 .0 1ad a lh ( µ m ) 4. 73 ± 0 .4 1aa 4. 63 ± 0 .4 7ab 4. 53 ± 0 .4 7ac 4. 57 ± 0 .3 1ad 5. 08 ± 0 .3 2aa 4. 86 ± 0 .3 9ab 4. 94 ± 0 .4 6ac 4. 7 ± 0. 37 ad ae m ea n s w it h o u t a co m m o n lo w er ca se s u p er sc ri p t in a r ow d if fe re d ( p < 0 .0 5) b et w ee n t re at m en ts . a -e m ea n s w it h o u t a co m m o n u p p er ca se s u p er sc ri p t in a r ow d if fe re d ( p < 0 .0 5) b et w ee n t im es o f tr ea tm en t. http://dx.doi.org/10.58292/ct.v16.10820 4 citation: clinical theriogenology 2024, 16, 10820, http://dx.doi.org/10.58292/ct.v16.10820 frozen-thawed samples. however, no details were given regarding the method of addition of coq10 to extender. in addition, studies on cattle and buffalo bulls,25 stallions,26 boars,27 and rams28 also reported that coq10-supplemented extenders improved sperm motility supporting current results. some decrease in pm of frozen-thawed samples overtime was expected, regardless of whether control or treated semen was used. for the treated extender, reduction in pm may be due to the progressive utilization of bioactive ubiquinol, as the greatest decrease in pm occurred between 60 and 120 minutes. this decrease in pm after 60 minutes, despite ubiquinol presence, may not be practically relevant, because frozen-thawed semen have to be used within 10 minutes to achieve good fertilization rates.29 addition of ubiquinol to semen extender increased vsl, vcl, vap, and alh; vcl and vap are positively correlated with sperm quality. there are no standardized values for farm animals’ sperm kinetics. nonetheless, ram vcl is higher than bulls or men.30 frozen-thawed ram sperm undergo high oxidative stress during incubation as evident by reduction in kinetics parameters over time.31 in our study, ubiquinol-treated frozen-thawed samples had higher values for pm and most motion kinetics parameters at specific time points compared to controls. this may suggest that the addition of water dispersible ubiquinol had a mitigating effect on the oxidative stress imposed on ram semen by freeze-thaw process. addition of ubiquinol to semen extender did not increase (p = 0.073) plasma membrane integrity. there was a high variance in results for this parameter might explain lack of significant treatment effect. nonetheless, mean percentage of sperm with intact membranes was numerically higher at each analysis time following addition of ubiquinol to extender. maintenance of plasma membrane integrity was expected with ubiquinol addition. coq10 regenerated α-tocopherol and increased its action against ros damage to plasma membrane.9 freezing and thawing of semen impose substantial stress on sperm plasma membrane integrity and functionality. increases in ros exacerbate oxidative stress that leads to irreversible cell damage.32 dna damage induced by high concentrations of ros may hasten sperm apoptosis leading to reduced numbers of live sperm.33 addition of coq10 to semen extenders improved sperm plasma membrane integrity and viability in bulls,25 bucks,13 men,11 and roosters.34 although there was no significant difference between treatment and control groups, percentage of viable cells tended to remain higher in ubiquinol-treated samples at each time point. due to lack of direct measurement of free radicals, ros, and total antioxidant capacity,15 we can only speculate that the maintenance of plasma membrane integrity indirectly reflected a reduction in lipid peroxidation and dna fragmentation due to ubiquinol addition, as those parameters were also not directly measured. conclusion there were positive effects of water-dispersible ubiquinol’s addition to semen extender on cryopreserved ram sperm. sperm pm and most kinetics parameters were higher in ubiquinol-treated extender. there was also a trend towards better viability. addition of water-dispersible form of ubiquinol to extenders may improve frozen/thawed stored sperm quality. lack of measurements of lipid peroxidation, dna fragmentation, total antioxidant capacity, and content of ros are major limitations of the study; further research is warranted. conflict of interest none to report. references 1. gibbons ae, fernandez j, bruno-galarraga mm, et al: technical recommendations for artificial insemination in sheep. anim reprod 2019;16:803-809. doi: 10.21451/1984-3143-ar2018-0129 2. salamon s, maxwell wm: storage of ram semen. anim reprod sci 2000;62:77-111. doi: 10.1016/s0378-4320(00)00155-x 3. aitken rj, baker ma, de iuliis gn, et al: new insights into sperm physiology and pathology. in: habenicht uf., aitken rj. (editors). fertility control. handbook of experimental pharmacology, vol 198. berlin, springer: 2010; 198:99–115. doi: 10.1007/978-3-642-02062-9_7 4. amidi f, pazhohan a, shabani nashtaei m, et al: the role of antioxidants in sperm freezing: a review. cell tissue bank 2016;17:745756. doi: 10.1007/s10561-016-9566-5 5. aitken rj, lambourne s, gibb z: the john hughes memorial lecture: aspects of sperm physiology – oxidative stress and the functionality of stallion spermatozoa. j equine vet sci 2014;34:1727. doi: 10.1016/j.jevs.2013.10.120 6. gvozdjáková a, kucharská j, dubravicky j, et al: coenzyme q10, α-tocopherol, and oxidative stress could be important metabolic biomarkers of male infertility. dis markers 2015;2015:827941. doi: 10.1155/2015/827941 7. bailey jl, bilodeau jf, cormier n: semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. j androl 2000;21:1-7. doi: 10.1002/j.1939-4640.2000.tb03268.x 8. ernster l, dallner g: biochemical, physiological and medical aspects of ubiquinone function. bba-mol basis dis 1995;1271:195204. doi: 10.1016/0925-4439(95)00028-3 9. turunen m, olsson j, dallner g: metabolism and function of coenzyme q. biochim biophys acta 2004;1660:171-199. doi: 10.1016/j.bbamem.2003.11.012 table 2. postthaw viability of ram sperm frozen in triladyl extender, without and with ubiquinol triladyl triladyl + ubiquinol 10 minutes 20 minutes 60 minutes 120 minutes 10 minutes 20 minutes 60 minutes 120 minutes viability (%) 60.31 ± 2.65a 50.05 ± 3.16b 28.02 ± 3.23c 31.89 ± 2.06d 80.19 ± 3.70e 68.97 ± 2.22b 49.66 ± 2.53f 35.76 ± 2.51d a-fmeans without a common uppercase superscript differed (p < 0.05) between times of treatment. http://dx.doi.org/10.58292/ct.v16.10820 https://doi.org/10.21451/1984-3143-ar2018-0129 https://doi.org/10.1016/s0378-4320(00)00155-x https://doi.org/10.1007/978-3-642-02062-9_7 https://doi.org/10.1007/s10561-016-9566-5 https://doi.org/10.1016/j.jevs.2013.10.120 https://doi.org/10.1155/2015/827941 https://doi.org/10.1002/j.1939-4640.2000.tb03268.x https://doi.org/10.1016/0925-4439(95)00028-3 https://doi.org/10.1016/j.bbamem.2003.11.012 citation: clinical theriogenology 2024, 16, 10820, http://dx.doi.org/10.58292/ct.v16.10820 5 10. lançoni r, celeghini ecc, giuli v, et al: coenzyme q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm. anim reprod 2021;18:e20200218. doi: 10.1590/1984-3143-ar2020-0218 11. gualtieri r, barbato v, fiorentino i, et al: treatment with zinc, d-aspartate, and coenzyme q10 protects bull sperm against damage and improves their ability to support embryo development. theriogenology 2014;82:592-598. doi: 10.1016/j.theriogenology. 2014.05.028 12. nogueira bg, sampaio bf, souza mi, et al: coenzyme q10 and alpha-tocopherol prevent the lipid peroxidation of cooled equine semen. reprod domest anim 2015;50:1003-1010. doi: 10.1111/ rda.12627 13. yousefian i, emamverdi m, karamzadeh-dehaghani a, et al: attenuation of cryopreservation-induced oxidative stress by antioxidant: impact of coenzyme q(10) on the quality of post-thawed buck spermatozoa. cryobiology 2018;81:88-93. doi: 10.1016/j. cryobiol.2018.02.005 14. raouf sm, taha at: effects of coq10 with vitamin e supplementation on semen quality and seminal plasma parameters of broiler breeder males. iraqi j vet sci 2021;35:65-70. doi: 10.33899/ ijvs.2020.126287.1289 15. wang y, kang y, zhang l, et al: coenzyme q(10) improves the quality of sheep sperm stored at room temperature by mitigating oxidative stress. anim sci j 2022;93:e13708. doi: 10.1111/asj.13708 16. pastor-maldonado cj, suarez-rivero jm, povea-cabello s, et al: coenzyme q(10): novel formulations and medical trends. int j mol sci 2020;21;8432. doi: 10.3390/ijms21228432 17. uchida y, wakimoto k, takahashi h, et al: the bioavailability of reduced coenzyme q10 water-dispersive powder after single oral administration. j altern complement med 2014;11:103-105. doi: 10.1625/jcam.11.103 18. salah n, sauvant d, archimède h: nutritional requirements of sheep, goats and cattle in warm climates: a meta-analysis. animal 2014;8:1439-1447. doi: 10.1017/s1751731114001153 19. carneiro jam, canisso if, bandeira rs, et al: effects of coenzyme q10 on semen cryopreservation of stallions classified as having good or bad semen freezing ability. anim reprod sci 2018;192:107118. doi: 10.1016/j.anireprosci.2018.02.020 20. khalifa ta, lymberopoulos ag, el-saidy be: testing usability of butylated hydroxytoluene in conservation of goat semen. reprod domest anim 2008;43:525-530. doi: 10.1111/j.1439-0531. 2007.00947.x 21. kumaresan a, das gupta m, datta tk, et al: sperm dna integrity and male fertility in farm animals: a review. front vet sci 2020;7:321. doi: 10.3389/fvets.2020.00321 22. balercia g, mosca f, mantero f, et al: coenzyme q(10) supplementation in infertile men with idiopathic asthenozoospermia: an open, uncontrolled pilot study. fertil steril 2004;81:93-98. doi: 10.1016/j.fertnstert.2003.05.009 23. masoudi r, sharafi m, shahneh az: effects of coq10 on the quality of ram sperm during cryopreservation in plant and animal based extenders. anim reprod sci 2019;208:106103. doi: 10.1016/j.anireprosci.2019.06.015 24. hernandez-camacho jd, bernier m, lopez-lluch g, et al: coenzyme q(10) supplementation in aging and disease. front physiol 2018;9:44. doi: 10.3389/fphys.2018.00044 25. saeed am, el-nagar ha, wafa wm, et al: effect of coenzyme q10 as an antioxidant added to semen extender during cryopreservation of buffalo and cattle semen. j anim poult prod 2016;7:403408. doi: 10.21608/jappmu.2016.48748 26. yousefian i, zare-shahneh a, zhandi m: the effect of coenzyme q10 and α-tocopherol in skim milk–based extender for preservation of caspian stallion semen in cool condition. j equine vet sci 2014;34:949-954. doi: 10.1016/j.jevs.2014.04.002 27. pindaru l: effects of coenzyme q10 on sperm viability durign storage of boar semen at 17°c. sci works 2016;61:32-36. 28. daghigh kia h, bolooki z, vaseghi dodran h, et al: effect of adding coenzyme q10 and ellagic acid during cryopreservation on postthaw quality of ram semen. iran j appl anim sci 2017;7:445-451. 29. gillan l, evans g, maxwell wm: capacitation status and fertility of fresh and frozen-thawed ram spermatozoa. reprod fertil dev 1997;9:481-487. doi: 10.1071/r96046 30. castellini c, dal bosco a, ruggeri s, et al: what is the best frame rate for evaluation of sperm motility in different species by computer-assisted sperm analysis? fertil steril 2011;96:24-27. doi: 10.1016/j.fertnstert.2011.04.096 31. balasuriya a, serhal p, doshi a, et al: processes involved in assisted reproduction technologies significantly increase sperm dna fragmentation and phosphatidylserine translocation. andrologia 2014;46:86-97. doi: 10.1111/and.12052 32. srivastava n, pande m: mitochondrion: features, functions and comparative analysis of specific probes in detecting sperm cell damages. asian pac j reprod 2016;5:445-452. doi: 10.1016/j.apjr.2016.10.008 33. aitken rj: reactive oxygen species as mediators of sperm capacitation and pathological damage. mol reprod dev 2017;84:10391052. doi: 10.1002/mrd.22871 34. masoudi r, sharafi m, pourazadi l: improvement of rooster semen quality using coenzyme q10 during cooling storage in the lake extender. cryobiology 2019;88:87-91. doi: 10.1016/j. cryobiol.2019.03.003 http://dx.doi.org/10.58292/ct.v16.10820 https://doi.org/10.1590/1984-3143-ar2020-0218 https://doi.org/10.1016/j.theriogenology.2014.05.028 https://doi.org/10.1016/j.theriogenology.2014.05.028 https://doi.org/10.1111/rda.12627 https://doi.org/10.1111/rda.12627 https://doi.org/10.1016/j.cryobiol.2018.02.005 https://doi.org/10.1016/j.cryobiol.2018.02.005 https://doi.org/10.33899/ijvs.2020.126287.1289 https://doi.org/10.33899/ijvs.2020.126287.1289 https://doi.org/10.1111/asj.13708 https://doi.org/10.3390/ijms21228432 https://doi.org/10.1625/jcam.11.103 https://doi.org/10.1017/s1751731114001153 https://doi.org/10.1016/j.anireprosci.2018.02.020 https://doi.org/10.1111/j.1439-0531.2007.00947.x https://doi.org/10.3389/fvets.2020.00321 https://doi.org/10.1016/j.fertnstert.2003.05.009 https://doi.org/10.1016/j.anireprosci.2019.06.015 https://doi.org/10.3389/fphys.2018.00044 https://doi.org/10.21608/jappmu.2016.48748 https://doi.org/10.1016/j.jevs.2014.04.002 https://doi.org/10.1071/r96046 https://doi.org/10.1016/j.fertnstert.2011.04.096 https://doi.org/10.1111/and.12052 https://doi.org/10.1016/j.apjr.2016.10.008 https://doi.org/10.1002/mrd.22871 https://doi.org/10.1016/j.cryobiol.2019.03.003 https://doi.org/10.1016/j.cryobiol.2019.03.003 2014: clinical management of postpartum hemorrhage following failure of cervical dilation in an alpaca clinical management of postpartum hemorrhage following failure of cervical dilation in an alpaca lisa k. pearson, ahmed tibary comparative theriogenology, department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa summary a 9 year old female huacaya alpaca was maintained on exogenous progesterone (hydroxyprogesterone caproate) during pregnancy due to previous pregnancy loss. dystocia due to partial failure of cervical dilation at 345 days of gestation was complicated by rupture of the vaginal artery. severe hemorrhage was treated with application of pressure to the vaginal artery by use of a vaginal tampon, and administration of aminocaproic acid, supplemental oxygen, and fluid therapy. after stabilization, the female was treated for retained placenta and anemia. unique features of this case include treatment of postpartum hemorrhage with aminocaproic acid, documentation of luteal insufficiency, and failure of cervical dilation after use of exogenous progesterone during pregnancy. keywords: camelid, aminocaproic acid, dystocia, endoscopy, hydroxyprogesterone caproate, progesterone background this case of periparturient hemorrhage in an alpaca is unique in several aspects. first, we documented the use of aminocaproic acid as a component of treatment of postpartum hemorrhage in an alpaca. we also documented luteal insufficiency during pregnancy, a condition long-suspected in many females but rarely documented in alpacas. lastly, this female experienced partial failure of cervical dilation subsequent to exogenous progesterone administration, a complication which has been anecdotally reported in alpacas. this case provides data to the field of camelid theriogenology regarding these conditions. case presentation the breeding management of a 9 year old huacaya alpaca had been overseen by the theriogenology service at washington state university (wsu) for several years. two years previously the alpaca had experienced dystocia at 325 days of gestation due to bilateral carpal flexure of the fetus which the owner resolved. the fetus was non-viable. the female was presented to wsu after the dystocia and hysteroscopy demonstrated no cervical tears. subsequently, the female had been bred several times without establishment of a pregnancy. reproductive evaluation at that time included serial ultrasonography, endometrial cytology, endometrial culture, endometrial biopsy, and hysteroscopy. in summary, she had normal follicular waves and ovulation. the uterus developed edema with small amounts of fluid during estrus, and cytology showed few neutrophils. culture was negative for pathogenic bacteria. the biopsy was classified as category 1a, with few histologic changes. hysteroscopy demonstrated normal uterine appearance and cervical function. a pregnancy was finally established after use of a modified minimum-contamination breeding technique. this entailed the use of systemic antibiotics for one day prior to and two days after breeding (excenel®, 2.2 mg/kg sq, zoetis animal health, florham park, nj), as well as induction of ovulation of a dominant follicle using a gonadotropin releasing hormone (gnrh) analogue (cystorelin®, 50 mcg im, merial, duluth, ga) and use of a systemic anti-inflammatory (flunixin meglumine, 1.1 mg/kg sq). the pregnancy was maintained with exogenous progesterone (hydroxyprogesterone caproate, 250 mg im, unique pharmaceuticals, temple, tx) which was administered every three weeks after ultrasonographic determination of a viable fetus. the exogenous progesterone was administered from 7 to 300 days of gestation. due to the value of the pregnancy and history of infertility, empiric progestagen therapy was elected. however, to retrospectively analyze the alpaca’s endogenous progesterone production during pregnancy, serum was collected and frozen at -20°c throughout pregnancy and analyzed postpartum clinical theriogenology • volume 6 number 2 • june 2014111 (table 1) using a commercial radioimmunoassay (siemens healthcare diagnostics, los angeles, ca). all samples were analyzed in one assay; the intra-assay cv was 5.8%. the female was hospitalized at 324 days of gestation to monitor for parturition. she received oral domperidone therapy (equidone®, 220 mg po, dechra veterinary products, overland park, ks) from days 326 to 332 of gestation due to insufficient mammary development. serial transabdominal ultrasonography using a curvilinear 3.5 mhz transducer (aloka 500, hitachi aloka, tokyo, japan) demonstrated normal fetal heart rate (80 to 96 beats/minute) and movement during hospitalization. at 345 days of gestation, rupture of the chorioallantois occurred and fetal limbs were observed protruding from the vulva despite the lack of obvious abdominal contractions. reproductive examination demonstrated partial failure of cervical dilation, at 6 to 8 cm diameter, which allowed protrusion of the fetal limbs but not the head. cesarean section was considered; however, given the extent of partial cervical dilation, manual dilation and prompt fetal delivery was the treatment option selected. the cervix was manually dilated over 10-15 minutes and a viable male cria was born via assisted vaginal delivery. the cria weighed 7.73 kg, a moderate size for the breed. subsequent to delivery of the cria, profuse arterial hemorrhage from the vulva was evident. treatment at the onset of vaginal hemorrhage, an intravenous jugular catheter was placed and initial blood values obtained. the packed cell volume (pcv) was 31% and serum total protein (tp) was 6.0 g/dl. aminocaproic acid (hospira inc., lake forest, il) was administered at a loading dose of 5 g in 500 ml of lactated ringer’s solution (lrs) iv over 15 minutes. several large blood clots were passed from the vulva. the female collapsed in the stocks and began open-mouthed breathing. supplemental oxygen therapy was initiated. a vaginal tampon was fashioned from brown gauze over roll cotton, covered with sterile lubricant, and inserted into the vagina to apply pressure to the site of hemorrhage. visual inspection ensured the tampon remained in place. fluid therapy was initiated and 3l of lrs were administered over 1 hour, after which pcv was 23% and tp was 4.1 g/dl. the animal’s heart rate increased to 120-152 beats per minute with irregular rate and rhythm due to hypovolemic shock. the vaginal tampon was removed after 1 hour and vaginal palpation demonstrated a tear of several centimeters involving, and just cranial to, the cervix at the 4 o’clock position, and a firmly attached placenta. at that time, the animal had stabilized and was able to walk to a stall. the female was administered oxytocin at a dose of 20 iu im every 4 hours; flunixin meglumine 1.1 mg/kg sq once for pain; ceftiofur (excenel®) 2.2 mg/kg sq every 12 hours for 7 days; and aminocaproic acid 2.5 g in 500 ml lrs iv every 6 hours for a total of 3 doses. packed cell volume and tp were monitored every 4 hours (table 2). routine neonatal cria care was implemented including umbilical treatment and monitoring of adequate nursing. twelve hours after the hemorrhage event, transabdominal unltrasonography did not demonstrate any free fluid in the abdomen, suggesting that the uterine and cervicovaginal tears were not full-thickness. the detached portion of the retained placenta was manually removed. ultrasonographic examination after placental removal demonstrated normal uterine contractions and initiation of involution. twenty hours after the hemorrhage event, endoscopic examination of the reproductive tract was performed. two tears were identified: one in the cranial vagina, involving the first cervical ring, and one in the uterine body, unassociated with placental retention. both tears appeared less than 2 cm in size. no active hemorrhage was noted. by that time cardiac auscultation demonstrated normal rhythm although tachycardia persisted (100 beats per minute). thirty-four hours after the hemorrhage event, the remainder of the placenta was passed and oxytocin therapy was discontinued. the alpaca was administered a b-complex vitamin injection of 5 ml sq and started on red cell® iron and mineral supplementation (farnam companies, phoenix, az) at a dose of 20 ml po, once daily, for 3 weeks to treat anemia. complete blood count (cbc) and serum biochemistry analysis were performed at 10, 82, and 154 hours after the hemorrhage event, as well as at 21 days (table 3). the alpaca displayed leukocytosis due to neutrophilia at 2 of the 4 sampling times with minor derangements in electrolytes and glucose. at both clinical theriogenology • volume 6 number 2 • june 2014 112 82 hours and twenty-one days postpartum, the cbc demonstrated a large number of eosinophils (1.2 x103 and 2.4 x 103, respectively). a fecal analysis was performed 32 days postpartum and was negative for parasites. the inciting cause of the eosinophilia was not identified. twenty-one days after the hemorrhage event, transrectal ultrasonography, using a 7.5 mhz linear transducer mounted on an extension rod, did not demonstrate free fluid in the uterus and involution was proceeding normally. endoscopic examination of the vagina demonstrated that the vaginal tear was granulating normally with no evidence of recent hemorrhage. the developing scar tissue appeared to extend to the first cervical ring although the extent of cervical involvement or function could not be ascertained at that time. endoscopy of the uterus was not attempted. outcome in a follow-up discussion with the owner nine months later, it was noted that the female was comfortable at the farm and had raised the cria to weaning. she had not shown any ill effects although the extent of cervical competency and functionality of the reproductive tract was unknown. the owner did not desire to breed her again. after two years the owner reported that the animal was still in good health. discussion postpartum hemorrhage in alpacas is rare and most often is subsequent to obstetrical manipulations or fetotomy (table 4). this is one major reason why the authors do not advocate fetotomy in alpacas and are more likely to recommend cesarean section in cases of severe dystocia than to spend excessive time attempting vaginal delivery. in the authors’ practice, all obstetrical manipulations are limited to 20 minutes, after which cesarean section is performed. in contrast to mares, the affected artery is typically the vaginal artery, a large vessel easily palpated in the wall of the vagina during obstetrical manipulations.1,2 due to this anatomical location, the site of hemorrhage is typically the uterus or vagina, whereas in mares, hemorrhage into the broad ligament or abdomen is commonly identified. observed clinical signs of acute volume depletion may include cardiovascular shock, tachycardia, pale mucous membranes, muscle fasciculation, sweating, recumbency, and death.3 in alpacas, if the hemorrhage is not noted externally (i.e., expulsion of blood from the vulva), the female is often found dead several hours after parturition.2 if the veterinarian is presented with an alpaca with clinical signs of cardiovascular shock after parturition or is presented with hemorrhage after obstetrical manipulations, the application of pressure to the site of hemorrhage is paramount. this treatment approach is unique to camelids, as the site of arterial rupture is typically the vaginal artery. a large vaginal tampon is easily constructed from common medical supplies and is lubricated with sterile lubricant or antibiotic-impregnated lanolin. one of the authors (at) has treated a postpartum arterial rupture by placing a ligature around the vessel. this was achieved under epidural anesthesia and after caudal retraction of the cervix using long-handled forceps. intravenous catheter placement is recommended for fluid replacement therapy; camelids in general are very susceptible to fluid overload and pulmonary edema, and the use of hypertonic saline and large volumes of crystalline fluids are contraindicated.4 in this case, the female was administered 3l of lrs over one hour after aminocaproic acid in lrs administration (500ml volume over 15 minutes). additional doses of aminocaproic acid (2.5 g in 500ml lrs) were given at 6 hours interval. the maintenance dose of fluids for adult alpacas is 4 to 6ml/kg/hour.5 for this particular 86 kg female, the dose of iv fluids at the time of hemorrhage was approximately 40ml/kg which provided cardiovascular stabilization without the onset of pulmonary edema. this dose is twice that of recommended dosing for volume-depleted alpacas,6 but is reflective of the volume of blood loss during the hemorrhage event. whole blood transfusion has also been described when pcv < 10%.2 there are no studies of aminocaproic acid use in alpacas to determine the optimal dosing and volume of fluid administration, and the treatment options selected in this animal were clinically based. retained fetal membranes are often a component of dystocia and hemorrhage events. the vaginal tampon will need to be removed for passage of fetal membranes. the practitioner may elect to clinical theriogenology • volume 6 number 2 • june 2014113 treat the female with oxytocin intramuscularly after cessation of hemorrhage to assist with placental passage. as opposed to mares, fetal membranes are considered retained in alpacas after six hours and often do not result in systemic illness. presence of retained fetal membranes may act to place pressure on a uterine or vaginal site of hemorrhage; passage of retained fetal membranes may release this pressure or disrupt clot formation, and animals should be monitored for resumption of hemorrhage.3 the vaginal tampon should not be left in place for long periods of time due to the risk of vaginal mucosal irritation and likelihood of straining by the female. uterine lavage as a treatment for retained fetal membranes is often contraindicated, especially in cases of hemorrhage. additional risks of uterine lavage in postpartum females include delay of uterine involution and, in the presence of full-thickness uterine tears, peritonitis. as hemorrhage events in postpartum alpacas are rare, extrapolation of treatment protocols in mares were used by the authors, including the use of aminocaproic acid. aminocaproic acid is a synthetic derivative of the amino acid lysine which has been commonly used in human medicine. although the mechanism of action is unknown, aminocaproic acid acts by inhibiting plasminogen activation which alters the dynamics of clot breakdown, resulting in stabilization and prolongation of a clot without disrupting its formation.7 the pharmacokinetics and pharmacodynamics of this drug have been examined in horses which demonstrated no detrimental effects on healthy horses.7 several dosing protocols have been suggested, including a loading dose followed by intravenous continuous rate infusion to achieve therapeutic plasma concentrations.7 more commonly in horses, a loading dose is followed by a lesser dose at 4 to 6 hour intervals.8-10 in humans, thrombotic complications have been reported, as well as an increase in plasma levels of the drug in patients with renal disease.7 the drug is contradicted for use in patients with disseminated intravascular coagulation. to the authors’ knowledge, this is the first reported use of aminocaproic acid in an alpaca although we have referred to this case previously.3,11 the dosage was extrapolated from the dosing for the equine which is recommended as a loading dose of 20 g per horse in iv fluids, with subsequent doses of 10g per horse in iv fluids every 4 to 6 hours.9,10 as the alpaca in this report was approximately one-fourth the size of an average horse, the dose was administered at that ratio (5g loading dose with subsequent doses of 2.5g). as postpartum hemorrhage events in alpacas are rare, and most of what is known about the disease pertains to broodmares, a review of the literature was performed to compare the disease in the two species (table 4). periparturient hemorrhage events represent a significant cause of death in the broodmare.9,12 in a study of mares which presented to a referral hospital postpartum, not only did mares with postpartum hemorrhage present significantly sooner after foaling, but represented significantly older mares (average age 13 years, range 8 to 21) compared to other postpartum complications such as metritis or large colon volvulus.13 likely this is due to the rapid onset of clinical signs, and represents the degenerative changes within the intima of the affected vessel. the average time from foaling to presentation in that study was 96 hours (range 3 to 720h).13 the wide range reflects the fact that mares tend to hemorrhage into the broad ligament, a self-limiting space which applies pressure to the affected vessel. in contrast, alpacas tend to hemorrhage into the uterus or vagina, which in an infinite space, and which is why most affected animals are found dead within several hours of parturition. data regarding postpartum hemorrhage in alpacas are lacking. the incidence is not known and almost certainly under-reported. complicating diagnosis and characterization of this disease is the lack of standardized anatomical description of the vasculature of the camelid reproductive tract.16,17 the cases which have been described or treated by the authors involved rupture of the vaginal artery. recent studies on the pathology of periparturient hemorrhage in mares have demonstrated that the most common affected vessel is the middle uterine artery. in one study of 31 hemorrhage events, the uterine artery ruptured in 24 cases (77.4%), with most occurring on the left side.14 the majority of these (n=18) ruptured proximally within 15 cm of branching from the external iliac artery. other rupture sites included the internal pudendal artery (n=5) and the internal iliac artery (n=1). similarly, another study demonstrated that 54 of 71 mares which died had rupture of the uterine artery; 33 of these were on the left side.15 this site is hypothesized to be most often affected because the sharp curve which the artery takes at this location may predispose it to rupture when under the high blood pressures associated with clinical theriogenology • volume 6 number 2 • june 2014 114 parturition, with increased risk in mares with atrophy of the tunica media smooth muscle and fibrosis, common in older mares. in addition to the treatments described above in reference to the alpaca of this report, other treatments used in horses for acute events of hemorrhage include: formalin, naloxone, the chinese herb yunnan baiyao, conjugated estrogens, hetastarch, morphine caudal epidural administration, and corticosteroids.8-10,12 to the authors’ knowledge, none of these treatments have been used in alpacas with hemorrhage events. controversy exists regarding the use of acepromazine as a tranquilizer in mares due to its hypotensive effects, but this drug is not a routine part of camelid practice and butorphanol tartrate remains the drug of choice in camelid species for sedation and analgesia. during the examination of mares, there are often concerns about performing transrectal palpation and the types of physical restraint implemented. in alpacas, the goals are to keep the female calm by use of a chute system or small pen. physical restraint may be required but is not as likely to elevate blood pressure as the restraint of an uncomfortable horse (i.e., lip twitch or chain) which may disrupt clot formation. in one study of hemorrhage events in broodmares, only tachycardia was associated with clinical outcome.9 there are not enough cases of hemorrhage events in alpacas to make epidemiological inferences. other major concerns in mares which survive periparturient hemorrhage events are not common features in alpacas and include endotoxemia and laminitis. the female alpaca of this report sustained partial failure of cervical dilation after treatment with exogenous progesterone every three weeks up to 300 days of gestation. previous reports of failure of cervical dilation have been made in alpacas.1,3,18 however, not all females which maintain pregnancy due to exogenous progesterone administration have experienced failure of cervical dilation, and not all females which experience failure of cervical dilation have been treated with exogenous progesterone. furthermore, the number of animals which are administered exogenous progesterone which actually have luteal insufficiency is not known. owners of animals receiving progestagen supplementation should be educated to monitor for signs of labor and dystocia. in the case of the animal of this report, the risk of complication is what led the owner to hospitalize the animal at 324 days of gestation. other causes of failure of cervical dilation include fetal maldisposition (does not engage the cervix) and uterine inertia. in this case, the female alpaca was not demonstrating strong abdominal contractions despite appropriate presentation of the cria. serum calcium levels ten hours postpartum were slightly below reference ranges (7.5 mg/dl; reference range 8.4-10.4 mg/dl) but were not low enough to be considered deficient (< 7.0 mg/dl).19 retrospective analysis of serum progesterone concentrations from day seven of gestation to the day of parturition in the animal of this report supports a diagnosis of luteal insufficiency, considered at progesterone concentrations < 1.0 ng/ml (table 1).2 luteal insufficiency has been commonly suspected in females with unexplained pregnancy loss and no abnormalities as identified by breeding soundness examination and advanced reproductive evaluation such as uterine biopsy and hysteroscopy. as mentioned previously, complete reproductive evaluation did not demonstrate a cause for infertility in this animal. furthermore, the body weight at breeding was high (96 kg). she was placed on dietary modifications throughout pregnancy and after parturition weighed 86 kg. serum progesterone levels were > 2.5 ng/ml until 71 days of gestation. at 95 days of gestation, progesterone was 1.28 ng/ml. the lowest measured serum levels were 0.6 ng/ml at 179 days of gestation, although levels increased slightly in late gestation. fluctuations in progesterone concentrations may be reflective of body weight, hydration status, liver metabolism, and lactation as seen in dairy cattle, but remain to be examined in alpacas. differences in laboratory protocols may also account for some variation, although in our case all samples were run in the same assay.20 this case demonstrates that animals with luteal insufficiency can be successfully maintained to parturition with exogenous progesterone and produce a viable cria. however, as mentioned previously, there are risks associated with this treatment and the owner and practitioner should be prepared for dystocia. last, the alpaca of this report was treated with an iron-mineral supplement which has been demonstrated to increase the risk of copper toxicity in alpacas. its use must be carefully monitored to ensure that toxicity does not occur. clinical theriogenology • volume 6 number 2 • june 2014115 in conclusion, management of high risk pregnancies in alpacas can be challenging. maintenance of proper body condition, use of a minimum contamination breeding technique, and exogenous progesterone use all resulted in a live cria from this female. however, the end result was not without lifethreatening complications. further studies are needed of these types of cases to improve welfare of pregnant animals during gestation, parturition, and the postpartum period. learning points  aminocaproic acid was administered, in conjunction with other therapies, to an alpaca experiencing postpartum hemorrhage, with a successful outcome.  luteal insufficiency was documented and treated by administration of exogenous progesterone up to 300 days of gestation.  failure of cervical dilation occurred subsequent to exogenous progesterone administration and resulted in dystocia and postpartum hemorrhage.  treatment of hypovolemic shock in alpacas should be performed carefully as to not result in fluid overload and pulmonary edema in affected animals. references 1. tibary a, pearson lk, rodriguez js, et al: obstetrical emergencies in camelids. proc int camelid health conf vet; 2010. 2. tibary a, rodriguez j, sandoval s: reproductive emergencies in camelids. theriogenology 2008;70:515-534. 3. pearson lk, rodriguez js, tibary a: postpartum disorders. in: cebra c, anderson d, tibary a, et al., eds. llama and alpaca care: medicine, surgery, reproduction, nutrition, and herd health. st. louis: saunders; 2014. p. 297-309. 4. cebra c: diseases of the respiratory tract. in: cebra c, anderson d, tibary a, et al., eds. llama and alpaca care: medicine, surgery, reproduction, nutrition, and herd health. st. louis: saunders; 2014. p. 421-436. 5. walker pg: fluid therapy in camelids. proc int camelid health conf vet; 2010. 6. bedenice d: approach to the critically ill camelid. vet clin north am food anim pract 2009;25:407-421. 7. ross j, dallap bl, dolente ba, et al: pharmacokinetics and pharmacodynamics of epsilon-aminocaproic acid in horses. am j vet res 2007;68:1016-1021. 8. leblanc mm: common peripartum problems in the mare. j eq vet sci 2008;28:709-715. 9. arnold ce, payne m, thompson ja, et al: periparturient hemorrhage in mares: 73 cases (1998-2005). j am vet med assoc 2008;232:1345-1351. 10. scoggin cf, mccue p: how to assess and stabilize a mare suspected of periparturient hemorrhage in the field. proc annu conv am assoc equine pract 2007. p. 342-348. 11. tibary a, pearson lk, picha y: management of postpartum emergencies in camelids. proc north am vet conf; 2011. p. 320-323. 12. frazer gs: post partum complications in the mare. part 1: conditions affecting the uterus. equine vet educ 2003;15:36-44. 13. dolente ba, sullivan ek, boston r, et al: mares admitted to a referral hospital for postpartum emergencies: 163 cases (1992-2002). j vet emerg crit care 2005;15:193-200. 14. ueno t, nambo y, tajima y, et al: pathology of lethal peripartum broad ligament haematoma in 31 thoroughbred mares. equine vet j 2010;42:529-533. 15. williams nm, bryant uk: periparturient arterial rupture in mares: a postmortem study. j equie vet sci 2012;32:281284. 16. smuts mms, bezuidenhout aj: anatomy of the dromedary. oxford: clarendon press; 1987. 17. leon e, sato a, navarrete m, et al: anatomia macroscopica, irrigacion y drenaje venoso del aparato reproductor femenino de la llama (lama glama). rev inv vet peru 2011;22:1-8. 18. saltet j, dart aj, dart cm, et al: ventral midline caesarean section for dystocia secondary to failure to dilate the cervix in three alpacas. aust vet j 2000;78:326-328. 19. van saun rj, herdt, t: nutritional assessment. in: cebra c, anderson d, tibary a, et al., eds. llama and alpaca care: medicine, surgery, reproduction, nutrition, and herd health. st. louis: saunders; 2014. p.100-123. 20. bott ni, rodriguez js, nielson s, et al: pharmacokinetics of a single injection of progesterone in oil, and factors affecting serum analysis in alpacas. clin therio 2009;1:240. clinical theriogenology • volume 6 number 2 • june 2014 116 table 1. endogenous serum progesterone levels of an alpaca with luteal insufficiency which was maintained on exogenous progesterone from days 7 to 300 of gestation. days of gestation progesterone (ng/ml) 7 3.64 28 2.66 71 2.50 95 1.28 116 1.20 179 0.60 326 1.60 344 0.09 345 parturition table 2. packed cell volume (pcv) and total protein (tp) of an alpaca during and after an event of postpartum hemorrhage. hours postpartum 0 4 8 10 18 24 82 154 21 days pcv (%) 31 23 22 18 14 13 15 14 21 tp (g/dl) 6.0 4.1 4.6 4.5 4.4 4.4 5.5 5.7 6.3 table 3. complete blood count and serum biochemistry analysis of an alpaca after an event of postpartum hemorrhage, select values. abnormalities are highlighted in bold. hours postpartum 10 82 154 21 days total white blood cells (x103/μl) 17.0 9.7 19.5 10.1 neutrophils (x103/μl) (%) 15.0 (88%) 5.0 (52%) 18.3 (94%) 6.8 (67%) eosinophils (x103/μl) (%) 0.3 (2%) 1.2 (12%) 0.6 (3%) 2.4 (24%) bun (mg/dl) 19 20 20 18 creatinine (mg/dl) 1.7 1.4 1.1 1.4 glucose (mg/dl) 326 125 97 122 calcium (mg/dl) 7.5 8.0 8.3 9.0 phosphorus (mg/dl) 7.1 2.8 3.7 7.8 sodium (meq/l) 154 156 156 161 chloride (meq/l) 116 120 117 117 potassium (meq/l) 4.6 4.7 4.8 4.4 table 4. common features of periparturient hemorrhage in mares versus alpacas. mares alpacas incidence most common emergency in postpartum mares12 rare site of hemorrhage uterine artery vaginal artery pathogenesis degeneration of intima of arteries in older or multiparous mares; site of vascular curving or bending with high hemostatic pressures usually secondary to obstetrical manipulations location of hemorrhage broad ligament, abdomen uterus, vagina clinical signs colic, cardiovascular shock acute death survival 84-88%8,12 unknown future fertility 50%8 unknown clinical theriogenology • volume 6 number 2 • june 2014117 2010: equine abortions equine abortions fabio del piero new bolton center, school of veterinary medicine, university of pennsylvania, kennett square, pa numerous and frequent are the etiologic agents able to cause abortion, stillbirth in horses. equine herpesvirus (ehv)-1 causes epidemic abortion, stillbirth, respiratory disease and myeloencephalopathy with paralysis in equidae and camelidae, whereas ehv-4 induces sporadic abortion and mainly respiratory disease. considering the value of some foals, it is clear that both sporadic and epidemic abortions are of great economic importance to the horse industry. rapid diagnosis is paramount. often the mares abort in the last few months of pregnancy with no particular premonitory signs. the fetus is often contained within the fetal membranes with the red chorion on the external part. the fetus may show the following suggestive, but non-specific, features and lesions: meconium staining on the body surfaces, mucosal petechiae, straw colored fluid in the serosal cavities, edematous atelectatic lungs with rib impressions and thymic necrosis. unfortunately, the pathognomonic macroscopic gray foci representing hepatic necrosis are not always present. histologically, if lung, liver and thymus are collected, is always possible to observe the suggestive multifocal coagulative necrosis associated with the intranuclear acidophilic pathognomonic viral inclusions. it is also possible to examine frozen sections and detect lesions shortly after postmortem examination. equine herpesvirus-1 antigen (ehv-1 ag) can be detected in fixed tissues using peroxidase immunohistochemistry, which allows the simultaneous evaluation of lesions and ehv-1 ag localization. equine herpesvirus-1 ag can also be abundant in areas where lesions are mild or absent. the antigen is intranuclear and intracytoplasmic and can be mainly detected in endothelium, epithelium and monocyte-macrophages, but also in myocytes and pericytes. target organs are mainly lung, liver, reticular cells of lymphoid tissue and cortices of adrenal gland, but also intestine, kidney, pancreas and fetal membranes. the virus grows well in rk-13 cells where it produces a cytopathic effect, generally within 24 hours. equine herpesvirus-4 is a slow growing herpesvirus, which replicates in a few days on equine lung cells. virus identification can also be obtained with immunohistochemistry using fluorescent antibodies. considering the latency of herpesviruses, polymerase chain reaction (pcr) techniques may be able to identify the infection, but without the contemporary evaluation of the presence of the lesions it is not possible to differentiate between latency, productive infection and disease. fetal and maternal serology may be of benefit. however, vaccinated dams may not have a representative humoral immune response and fetal death can be very rapid, with insignificant fetal serological response, even in immunocompetent fetuses. equine arteritis virus (eav) is an arterivirus, which is also able to induce respiratory disease, stillbirth, foal mortality and abortion. the aborted fetus can be fresh or moderately autolyzed and pathognomonic lesions are not observed. occasionally there is mild interstitial pneumonia and vasculitis. although with immunoperoxidase histochemistry it may be possible to identify eav ag in fetuses and the allantochorion, serological evaluation of the dam and virus isolation are, at the moment, the most powerful tools used to diagnose eav abortion. on the other hand, lesions are highly suggestive in fatal cases involving adults and foals: interstitial pneumonia, systemic vasculitis, and, in terminal phases of the disease, tubular necrosis. getah virus and equine influenza are also potential causes of abortion. perhaps a vesivirus is able to cause equine abortion, but additional studies are needed to verify this. other rather common infectious etiologic agents of abortion in horses are bacteria and fungi. streptococcus equi var. zooepidemicus induces severe progressive cervical chorionic necrosis and thickening with neovascularization and allantoic hyperplasia lesions progress from the cervical area toward the placental body inducing progressive placental insufficiency. sometimes this is not followed by fetal septicemia. similar gross and histologic findings are observed in cases of aspergillus spp. mitotic placentitis. gram-negative bacteria like escherichia coli, klebsiella spp and others are often able to induce fetal septicemia with dissemination of bacterial emboli in various organs. leptospira interrogans serogroup pomona, serovar kennewicki and gryppotyphosa have been identified as a cause of abortion and neonatal sepsis in horses in kentucky. in this case, placentitis does not ascend from the cervical area, but is most likely hematogenic in origin. examination of the placenta may reveal edema 571 and necrosis of the chorion, enlarged liver and swollen kidneys with pale streaks. histologically there is placental thrombosis, vasculitis and villitis with necrosis followed by hepatocellular dissociation, giant cell hepatopathy, nephritis, pneumonia and myocarditis. it is possible to identify the spiraliform bacteria using steiner and whartin starry silver stains and or immunoperoxidase histochemistry, which allow to simultaneous evaluation of the lesions and bacterial antigens. this technique has made the use of fluorescent antibodies to detect leptospira in the placenta obsolete, where the specificity of the staining is difficult to interpret. higher bacteria may cause equine placentitis and other bacteria have been identified as sporadic causes of abortions in mares. neorickettsia risticii, the etiologic agent of potomac horse fever induces enteritis, splenitis and hepatitis in the fetus. in addition to the relatively common aspergillus, other causes of mycotic abortion include mucor spp., histoplasma capsulatum, candida spp., whereas allescheria boydii and coccidioides immitis are rare. amongst the noninfectious causes of abortion there are umbilical cord abnormalities, which may induce fetal hypoxia and death. umbilical cord torsion is predisposed by an abnormal increase in length. acute and recurrent chronic torsions have been observed. the first is associated with severe segmental to diffuse edema and hemorrhage of the cord, whereas the latter may be characterized by urinary bladder distention, urachal dilatations, chorionic vessel thrombosis and intraluminal mineralization. twinning often ends in abortion. fetal loss appears to be caused by a combination of factors including inadequate nutrition, interfetal incompatibility and immunologic reactions. a sharp line of demarcation can readily identify the placentae from twins where the chorioallantois of the two fetuses comes in contact with each other. other non-infectious causes of abortion include: early placenta separation, placenta edema, contracted foal syndrome, congenital anomalies, atrophy of placental villi, body pregnancy, and fetal diarrhea syndrome. fescue toxicosis is an important cause of perinatal death of foals. it is associated with the endophyte neotyphodium (previously acremonium spp.). in these cases placenta membranes are congested and very edematous. it is proposed that the mare reproductive loss syndrome observed in kentucky, usa, which includes early fetal loss, late fetal loss, uveitis, pericarditis, and encephalitis, is associated with tissue penetration by septic barbed setal fragments (septic penetrating setae) from eastern tent caterpillars (malacosoma americanum). once ingested, these barbed setal fragments migrate through moving tissues, followed by rapid hematogenous spread of bacteria, bacterial emboli, and/or septic fragments of setae (septic penetrating setal emboli). there are other suggested but unproven causes of equine abortion. the dissection of the aborted fetus and placenta and the following ancillary procedures including histopathology, immunohistochemistry, virology, bacteriology, and serology will allow us to obtain a rapid diagnosis and to prevent other fetal and neonatal losses. several control measures are required for the management of an epizootic situation, but of primary importance is the proper diagnosis of the disease involved. once the offending agent is identified, control measure must be implemented. control measures include isolation of affected animals, isolation and examination of any newly introduced animals, maintaining separate implements for sick and healthy animals and separating farm workers into those working with sick animals and those working with healthy animals. depending on the infectious agent, vaccination in the face of an outbreak may be warranted, particularly in previously unvaccinated animals considered at risk. transport of any animals to and from the farm should be prevented during the disease outbreak and for several weeks after the last affected animals are normal. keywords: abortion, equine herpesvirus, equine arteritis virus, placentitis, twinning, mare reproductive loss syndrome 572 2012: safe handling of reproductive hormones routinely used in equine practice safe handling of reproductive hormones routinely used in equine practice dirk k. vanderwall department of clinical studies, new bolton center, school of veterinary medicine, university of pennsylvania, kennett square, pa introduction the use of exogenous reproductive hormones and/or hormone analogs is a mainstay of both clinical practice and research activities in equine reproduction. the national institute for occupational safety and health (niosh) defines hazardous drugs as those that are known or suspected to cause adverse health effects in people from exposure in the workplace, and reproductive hormones such as those routinely used in equine practice are considered hazardous.1 therefore, these agents can have inadvertent deleterious effects on personnel (discussed later), particularly women, who handle and administer them if appropriate precautions are not taken. there are multiple steps during which veterinarians and other veterinary healthcare workers such as veterinary students, technicians and support staff may be exposed to hazardous reproductive drugs including preparing a drug for administration; administering a drug to an animal; and/or during disposal or cleaning of materials used during preparation or administration of a drug (e.g., iv syringes, dose syringes, etc.).2 veterinary workers are most likely to be exposed to hazardous drugs via skin contact/absorption, ingestion or inhalation, though needle stick or sharps injuries also pose a risk for inadvertent drug exposure.2 the objective of this article is to provide an overview of the various formulations of exogenous hormones currently available in the united states for equine reproductive management and discuss personal safety considerations when handling and administering these agents. keywords: exogenous hormone, equine, mare, stallion, personal safety exogenous hormone preparations with the exception of equine follicle stimulating hormone (efsh) all of the major hormones secreted by the hypothalmic-pituitary-gonadal axis are available as exogenous formulations that are used to regulate physiological and/or behavioral reproductive phenomena in horses (table 1). although native gonadotropin releasing hormone (gnrh) is commercially available in aqueous injectable formulations (e.g., cystorelin®, merial ltd., duluth, ga; factrel®, fort dodge animal health, fort dodge, ia), their short half-life limits the usefulness of these products in mares; therefore, the potent gnrh analog deslorelin is widely used instead.3 deslorelin is primarily used to induce ovulation in mares that are in estrus with a growing dominant follicle (≥30 mm).4 administration of deslorelin stimulates the release of luteinizing hormone (lh) from the anterior pituitary gland, which initiates follicular/oocyte maturation and subsequent ovulation approximately 36 to 48 hours after administration. in 2011, a new fdaapproved deslorelin product (sucromate™ equine, thorn bioscience llc, louisville, ky) became commercially available, which supplanted the use of compounded deslorelin products that had been used previously. human chorionic gonadotropin (hcg) is routinely used to induce ovulation in mares because of its lh-like activity.5 like deslorelin, hcg is typically administered to mares that are in estrus with a growing dominant follicle (≥35 mm). the lh-activity of hcg induces follicular/oocyte maturation resulting in ovulation approximately 36 to 48 hours after treatment. because hcg is a large foreign (nonequine) protein, repeated administration to mares will induce antibody formation against the hcg molecule. although the development of anti-hcg antibodies has not been documented to interfere with the efficacy of hcg, it has been demonstrated that aged mares (>15 years of age) tend to be less responsive to hcg.4,5 for mares in which hcg has not reliably induced ovulation, or there is simply concern that a mare will not respond reliably to hcg (i.e., aged mares), the use of deslorelin is generally warranted. in addition to its use in mares, because of its lh-like activity, hcg is also used in male horses as a diagnostic test for the presence of testicular tissue in suspected cryptorchid horses.6 if testicular tissue is present in these animals, administration of hcg will stimulate synthesis and secretion of testosterone from leydig cells within the testicular tissue. a typical hcg-stimulation protocol begins clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201239 clinical theriogenology • volume 4 number 1 • march 2012 with the collection of an initial blood sample for assessment of the baseline testosterone level that is immediately followed by administration of 6,000 to 12,000 units of hcg iv. post-treatment blood samples are collected between one and three hours after hcg treatment and again 24 hours after treatment in order to detect a resultant rise in testosterone. horses with testicular tissue generally have a >four-fold rise in testosterone in one or both post-treatment samples.6 although follicle stimulating hormone (fsh) products are used extensively for superovulation prior to embryo transfer in cattle and other livestock species, the fsh products that are used (e.g., folltropin®-v, bioniche animal health canada inc., belleville, ontario, canada; pluset®, minitube of america inc., verona, wi) are of porcine origin and have not been reliably efficacious for superovulating mares.7 for a period of time, an efsh preparation was commercially available (efsh, bioniche animal health inc., athens, ga) for superovulating mares;8 however, that product was subsequently withdrawn from the market. as an alternative, a recombinant equine fsh (refsh) product is currently being evaluated for superovulating mares, but it is not commercially available at this time.9 exogenous formulations of the ovarian steroid hormones estrogen and progesterone are used extensively, both alone and in combination, to regulate and/or treat numerous physiological and pathological reproductive conditions in mares (table 1).10,11 estrogen is administered to ovariectomized mares to induce estrous behavior for breeding shed activities; to spring “transitional” mares prior to administration of dopamine antagonists such as sulpiride to hasten the onset of ovulation; and to noncycling (or ovariectomized) oocyte or embryo recipient mares to enhance the responsiveness of the uterus to subsequent progesterone treatment. in addition, estrogen may be used as an adjunctive therapy in highrisk pregnant mares, such as those with placentitis. estrogen in combination with progesterone is routinely used for synchronizing estrus/ovulation in cycling mares and it can be used in postpartum mares to delay estrus/ovulation in an effort to enhance the fertility of foal-heat breeding.10,11 progesterone and the synthetic progestin altrenogest are widely used for reproductive management in both nonpregnant and pregnant mares.11 these hormones are used to suppress estrous behavior in nonpregnant performance horses and in spring “transitional” mares to hasten the onset of ovulatory activity. in pregnant mares exogenous progestins are used extensively (whether warranted or not) to augment endogenous progesterone levels in an effort to enhance pregnancy maintenance during early gestation (<100 days). exogenous progestins are also used for maintenance of pregnancy in nonovulating (or ovariectomized) oocyte or embryo recipient mares that lack an endogenous source of progesterone (until fetal-placental production begins) and as an adjunctive therapy in high-risk pregnant mares, such as those with placentitis or severe systemic disease. as noted above for estrogen products, progesterone in combination with estrogen is routinely used for synchronizing estrus/ovulation in cycling mares and it can be used in postpartum mares to delay estrus/ovulation in an effort to enhance the fertility of foal-heat breeding.10,11 in addition to its use in mares, altrenogest has also been administered to stallions to temporarily suppress sexual and aggressive behavior during training and related performance activities. oral administration of twice the labeled dose (0.088 mg/kg) of altrenogest significantly suppressed reproductive behavior in stallions between two and 18 years of age.12,13 in contrast, oral altrenogest treatment at the labeled dose (0.044 mg/kg) had only a slight effect on reproductive behavior in mature stallions greater than five years of age.14 it should be noted however, that in the later study the altrenogest was administered for only 30 days,14 whereas it was administered for 5713 and 24012 days in the two previously cited studies. additionally, it is important to note that there are limited data on the long-term effects of this treatment on future fertility in stallions (particularly when used in young stallions); therefore, the use of exogenous altrenogest in stallions that will subsequently be used for breeding should be pursued very cautiously. exogenous progesterone has also been used empirically to treat selfmutilation behavior in stallions and geldings and anecdotally it has been useful in some of these cases.15 it is important to diagnose the underlying cause of the behavior in these horses before initiating treatment, because some root causes will not respond to progesterone therapy (e.g., pain-induced self-mutilation).15 although exogenous testosterone and related androgens are not widely used in equine reproduction they are occasionally utilized for behavior/libido modification in stallions with sub-optimal clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 40clinical theriogenology • volume 4 number 1 • march 2012 endogenous androgen levels (table 1).16 compounded injectable preparations of testosterone or longacting testosterone conjugates have been used for this purpose, and more recently transdermal delivery has been used by applying testosterone in oil to hairless skin in the perineum or directly on the shaft of the penis.16 when exogenous testosterone is used in stallions it is important to monitor preand posttreatment androgen levels in order to avoid an excessive negative feedback effect of the exogenous testosterone and a subsequent down-regulation of the hypothalamic-pituitary-testicular axis. prostaglandin f2α (pgf2α) has applications in nonpregnant and pregnant mares.17 two formulations of pgf2α are currently available, native pgf2α (dinoprost tromethamine) or the synthetic analog cloprostenol (table 1). the most common use of pgf2α in nonpregnant mares is to induce luteolysis in order to terminate corpus luteum (cl) function and bring mares into heat. more specifically, pgf2α can be used to abbreviate the normal luteal phase to “short-cycle” mares or it can be used to interrupt an abnormally prolonged luteal phase in order to restore normal cyclical ovarian activity. although administration of two doses of pgf2α 14 days apart has been used to synchronize estrus/ovulation, it is not a very reliable way of achieving that goal in mares, so more efficacious methods of estrus/ovulation synchronization are used (e.g., treatment with progesterone and estradiol-17β), which incorporate a single pgf2α treatment to eliminate endogenous progesterone secretion from a cl. prostaglandin f2α is also used in nonpregnant mares for its ecbolic effect to stimulate uterine contractile activity for the treatment of uterine fluid accumulation associated with pathological processes such as persistent mating-induced endometritis. exogenous pgf2α will stimulate uterine contractile activity for a duration of four to five hours, with cloprostenol inducing a more consistent response than native pgf2α, so it is the agent of choice for this application.17 it is important to note, that although historically the cl has been considered to be resistant to the luteolytic effects of pgf2α until day five after ovulation, it has been demonstrated that cl function is altered following administration of pgf2α as early as day two after ovulation.17 therefore, when using exogenous pgf2α to treat mares for fluid accumulation during estrus, treatment is generally not continued beyond the day of ovulation. administration of pgf2α is the preferred method of terminating pregnancy during early gestation in mares.17 prior to approximately 30 to 35 days of gestation, administration of a single dose of pgf2α is reliably abortifacient through its luteolytic effect causing endogenous progesterone levels to fall below the threshold necessary for continued maintenance of pregnancy. after 35 days of gestation, coincident with formation of the endometrial cups and secretion of equine chorionic gonadotropin (ecg) that induces supplemental cl formation, multiple pgf2α treatments may be necessary to induce abortion. after fetal-placental production of progesterone/progestins becomes sufficient to maintain pregnancy without an ovarian/cl source of progesterone (>100 days of gestation), pgf2α can still be used to induce abortion in mares, however multiple treatments will be required over the course of several days. although they are not used nearly as extensively as pgf2α, prostaglandins e1 (pge1) and e2 (pge2) do have therapeutic applications in nonpregnant and pregnant mares (table 1).17 topical application of 1.0 mg pge1 cream to the caudal cervical os and lumen has been used to induce cervical relaxation to aid uterine fluid evacuation after breeding in mares in which the cervix fails to relax during estrus.18 similarly, local application of 2.0 to 2.5 mg pge2 into the cervical canal has been performed to aid cervical relaxation (i.e., “cervical ripening”) prior to induction of parturition.19 cervical ripening with pge2 appeared to favor shorter delivery times and foal vigor, although the number of mares in the study was small and most of the end points that were compared between control and pge2-treated mares were not significantly different. intrauterine deposition of pge2 prior to insemination has been performed in an attempt to enhance pregnancy rates by increasing colonization of the oviduct by fertile spermatozoa. the primary interest in this treatment modality is for situations involving “low-dose” insemination with a sub-optimal number of spermatozoa. in an initial report,20 intrauterine deposition of 0.25 mg pge2 two hours prior to insemination improved pregnancy rates in mares inseminated with a suboptimal number of spermatozoa from a stallion with good semen quality, but the same treatment did not improve pregnancy rates in mares inseminated with a suboptimal number of spermatozoa from a stallion with poor semen quality; therefore, further studies are needed to more fully assess the benefit (if any) of intrauterine administration of pge2 prior to insemination with a suboptimal number of spermatozoa. there is clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201241 clinical theriogenology • volume 4 number 1 • march 2012 increasing interest in the use of pge2 to reestablish oviductal patency in infertile mares, which is based on work by allen et al21 who described laparoscopic delivery of pge2 onto the oviducts of 15 chronically infertile mares. the mares were ten to 21 years old and all of them had failed to become pregnant over a period of one to four years during which they were bred to fertile stallions. none of the mares had any identifiable reproductive pathology that would explain their infertility. laparoscopic application of 0.2 mg of pge2 gel onto the surface of the oviducts was performed, and after treatment 14 of the 15 mares (93%) became pregnant either the same or the following year. the authors hypothesized that over time the mares developed blocked oviducts due to the accumulation of naturally occurring collagenous oviductal masses and that topical application of the pge2 induced changes in circular and longitudinal oviductal smooth muscle contractility that resulted in the cathartic transport of the masses out of the oviduct into the uterine lumen restoring patency. although the results are encouraging, the absence of a group of control mares and the lack of pretreatment evidence that oviductal blockage was the underlying cause of infertility in these mares are limitations of the study; therefore, further work is needed to more fully understand the incidence of infertility caused by pathological oviductal blockage in mares and the suitability of laparoscopic application of pge2 for its treatment. the use of exogenous oxytocin has applications in nonpregnant, pregnant and postpartum mares (table 1).22 in nonpregnant mares oxytocin has revolutionized the clinical management of endometritis, both persistent mating-induced endometritis and infectious endometritis, because of its ability to stimulate uterine contractile activity and enhance clearance of intraluminal fluid. depending upon the clinical circumstances, oxytocin can be used alone for its ecbolic effect or it can be combined with large volume uterine lavage to facilitate evacuation of uterine fluid. oxytocin stimulates uterine contractile activity for approximately 30 to 45 minutes, so repetitive doses are often given during the day in an effort to maximize uterine clearance.22 a newly described use of exogenous oxytocin in nonpregnant mares is administration during diestrus to disrupt luteolysis to prolong cl function as a means of estrus suppression in performance mares. in the initial report on the use of oxytocin for this purpose, administration of 60 units of oxytocin im on days seven to 14 after ovulation induced prolonged cl function through day 30 after ovulation in all six treated mares whereas six saline-treated control mares all underwent luteolysis by day 16 after ovulation.23 in a subsequent report, once daily administration of oxytocin was found to be as effective as twice daily administration for prolonging cl function, which further simplifies the treatment protocol.24 the primary use of oxytocin in pregnant mares is for induction of parturition and numerous protocols for this purpose have been described.22 in postpartum mares oxytocin is routinely used as an aid in the treatment of retained fetal membranes. treatment is generally initiated if the fetal membranes have not been passed within three hours after parturition, and typically consists of administration of ten to 40 units of oxytocin given iv or im, which may be repeated every 30 minutes to four hours or alternatively given as a slow iv drip diluted in physiological saline. for refractory cases, administration of one liter of lactated ringer’s solution containing 100 to 150 ml of 23% calcium gluconate followed by 20 units of oxytocin iv is often successful.22 personal safety given the extensive selection of exogenous hormones that are available and their numerous applications in both mares and stallions it is clear these products are indispensable for the reproductive management of horses. therefore, it seems inevitable that veterinarians and other veterinary healthcare workers will handle and administer many, if not all, of these agents on a regular basis if they are involved in equine reproductive activities. in addition, veterinarians will likely dispense or provide prescriptions for many of these agents to horse owners, managers or other lay personnel further increasing the number of individuals handling and administering these products. most, but not all, commercially-available hormone preparations provide specific human safety warnings and guidelines for safe handling and administration (table 2). in contrast, compounded preparations are generally not accompanied with this type of human safety information (table 2). most of the safety warnings that accompany hormone products address particular concerns for women, specifically pregnant women and women of childbearing age. for example, inadvertent exposure of some drugs (e.g., gnrh analogs and clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 42clinical theriogenology • volume 4 number 1 • march 2012 progesterone/progestins) may lead to disruption of the menstrual cycle; abdominal cramping; increased or decreased uterine bleeding; prolongation of pregnancy; or headaches; while other drugs (e.g., pgf2α, pge1, pge2 and their analogs) may cause abortion (table 2). other safety warnings are more general and are relevant regardless of gender. for example, individuals with asthma or other respiratory conditions, regardless of gender, are urged to use caution when handling drugs such as pgf2α and its analogs, because they can cause bronchiospasms (table 2). a primary emphasis in most of the manufacturer-recommended safety warnings is to avoid direct skin contact since many of the hormones can be readily absorbed through unbroken skin. because of that concern, most products list specific procedures to follow to avoid direct skin contact such as wearing impervious gloves during drug handling and appropriate hand washing immediately afterwards, particularly if exposure is known to occur (table 2). in the case of accidental exposure through other routes, most manufacturers recommend seeking medical attention or consulting a physician immediately (table 2). although safety warnings are not usually provided with compounded hormone products, it would seem prudent to avoid direct contact with skin by wearing impervious gloves and if contact occurs to cleanse the affected area appropriately, and for other routes of exposure to seek medical attention or consult a physician immediately. additional information about preventing occupational exposure to hazardous drugs is available through niosh.1,2 in summary, the use of exogenous reproductive hormones and/or hormone analogs is a mainstay of both clinical practice and research activities in equine reproduction. although these agents are used to regulate reproductive events in horses, they can have adverse effects on people, particularly women, who handle and administer them if appropriate precautions are not taken. for hormone products that are provided with them, manufacturer-recommended safety warnings should be noted and handling guidelines followed. when specific warnings and guidelines are not provided with a product, such as compounded preparations, heeding safety warnings and handling guidelines such as those that are provided with fda-approved products and/or following niosh guidelines1,2 would be a judicious course of action. references 1. niosh alert: preventing occupational exposures to antineoplastic and other hazardous drugs in healthcare settings. u.s. department of health and human services, centers for disease control and prevention, national institute for occupational safety and health, dhhs (niosh) publication no. 2004-165, cincinnati, oh: 2004. available on-line at: http://www.cdc.gov/niosh/docs/2004-165/pdfs/2004-165.pdf. 2. niosh workplace solutions: safe handling of hazardous drugs for veterinary healthcare workers. u.s. department of health and human services, centers for disease control and prevention, national institute for occupational safety and health, dhhs (niosh) publication no. 2010-150, cincinnati, oh: 2010. available on-line at: http://www.cdc.gov/niosh/docs/wp-solutions/2010-150/pdfs/2010-150.pdf. 3. squires el: gonadotropin-releasing hormones. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. oxford: wiley-blackwell; 2011. p. 1820-1824. 4. mckinnon ao, mccue pm: induction of ovulation. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. oxford: wiley-blackwell; 2011. p. 1858-1869. 5. newcombe jr: human chorionic gonadotropin. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. oxford: wiley-blackwell; 2011. p. 1804-1810. 6. arighi m: testicular descent. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. oxford: wiley-blackwell; 2011. p. 1099-1106. 7. squires el, mccue pm: superovulation. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. oxford: wiley-blackwell; 2011. p. 1836-1844. 8. mccue pm, leblanc mm, squires el: efsh in clinical equine practice. theriogenology 2007;68:429-433. 9. meyers-brown ga, mccue pm, niswender kd, et al: superovulation in mares using recombinant equine follicle stimulating hormone: ovulation rates, embryo retrieval, and hormone profiles. j equine vet sci 2010;30:560-568. 10. tibary a: estrogen therapy. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. oxford: wiley-blackwell; 2011. p. 1825-1829. 11. pinto crf: progestagens and progesterone. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. oxford: wiley-blackwell; 2011. p. 1811-1819. 12. squires el, badzinski sl, amann rp, et al: effects of altrenogest on total scrotal width, seminal characteristics, concentrations of lh and testosterone and sexual behavior of stallions. theriogenology 1997;48:313-328. 13. brady ha, johnson nn, whisnant cs, et al: effects of oral altrenogest on testicular parameters, steroidal profiles, and clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201243 clinical theriogenology • volume 4 number 1 • march 2012 seminal characteristics in young stallions. proc annu conv am assoc equine pract 1997; p. 195-196. 14. miller cd, varner dd, blanchard tl, et al: effects of altrenogest on behavior and reproductive function of stallions. proc annu conv am assoc equine pract 1997; p. 197-198. 15. mcdonnell sm: practical review of self-mutilation in horses. anim reprod sci 2008;107:219-228. 16. mcdonnell sm: pharmacological manipulation of stallion behavior. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. oxford: wiley-blackwell; 2011. p. 1415-1418. 17. staempfli sa: prostaglandins. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. oxford: wiley-blackwell; 2011. p. 1797-1803. 18. nie gj, barnes aj: use of prostaglandin e1 to induce cervical relaxation in a maiden mare with post breeding endometritis. equine vet educ 2003;5:221-224. 19. rigby s, love c, carpenter k, et al: use of prostaglandin e2 to ripen the cervix of the mare prior to induction of parturition. theriogenology 1998;50:897-904. 20. woods j, rigby s, brinsko s, et al: effect of intrauterine treatment with prostaglandin e2 prior to insemination of mares in the uterine horn or body. theriogenology 2000;53:1827-1836. 21. allen wr, wilsher s, morris l, et al: laparoscopic application of pge2 to re-establish oviducal patency and fertility in infertile mares: a preliminary study. equine vet j 2006;38:454-459. 22. leblanc mm: oxytocin. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. 2nd ed. oxford: wiley-blackwell; 2011. p. 1830-1835. 23. vanderwall dk, rasmussen dm, woods gl: effect of repeated administration of oxytocin during diestrus on duration of function of corpora lutea in mares. j am vet med assoc 2007;231:1864-1867. 24. vanderwall dk, rasmussen dm, carnahan kg, et al: effect of administration of oxytocin during diestrus on corpus luteum function and endometrial oxytocin receptor concentration in cycling mares. j equine vet sci 2012 (in press). clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 44clinical theriogenology • volume 4 number 1 • march 2012 table 1. exogenous hormone preparations available for equine reproductive management. hormone product (supplier) dose and route(s) of administration to horses primary use(s) in horses deslorelin acetate (gnrh analog) sucromate™ equine (thorn bioscience llc, louisville, ky) 1.8 mg im induction of ovulation. human chorionic gonadotropin (lh activity) chorulon® (intervet/ schering-plough animal health, summit, nj) mares: 3 to 5 units/kg iv or im stallions: 6,000 to 12,000 units iv mares: induction of ovulation. stallions: diagnostic evaluation for testicular tissue (i.e., cryptorchid stallions). equine follicle stimulating hormone (efsh) commercial product not currently available na when a product was available it was primarily used for superovulation in embryo donor mares. estradiol-17β and conjugated estrogens (e.g., estradiol cypionate and estradiol benzoate) compounded formulations see reference #10 1) induction of estrus behavior in ovariectomized mares. 2) pretreatment of non-ovulating (or ovariectomized) oocyte or embryo recipient mares. 3) pretreatment of spring “transitional” mares prior to administration of dopamine antagonists (e.g., sulpiride) to hasten onset of ovulation. 4) high-risk pregnant mares (e.g., placentitis). progesterone compounded formulations 100 to 300 mg in oil once daily im or 1.5 to 2.0 grams once every 7 to 10 days for long-acting formulations 1) hasten onset of ovulation in spring “transition” mares. 2) estrus suppression. 3) supplementation of endogenous progesterone in pregnant mares. 4) maintenance of pregnancy in non-ovulating (or ovariectomized) oocyte or embryo recipient mares. 5) high-risk pregnant mares (e.g., placentitis) or mares with severe systemic illness (e.g., colic/endotoxemia, etc.). altrenogest (synthetic progestin) regumate® solution 0.22% (intervet/ schering-plough animal health, summit, nj) 0.044 mg/kg orally (0.088 mg/kg in high-risk pregnant mares or those with severe systemic disease) 1) hasten onset of ovulation in spring “transition” mares. 2) estrus suppression. 3) supplementation of endogenous progesterone in pregnant mares. 4) maintenance of pregnancy in non-ovulating (or ovariectomized) oocyte or embryo recipient mares. 5) high-risk pregnant mares (e.g., placentitis) or mares with severe systemic illness (e.g., colic/endotoxemia, etc.). 6) suppression of sexual and aggressive behavior in stallions. 7) treatment for self-mutilation in stallions and geldings. progesterone and estrogen combination compounded formulations 150 mg progesterone and 10 mg estradiol17β/day im 1) synchronization of estrus/ovulation. 2) delaying postpartum estrus/ovulation. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201245 clinical theriogenology • volume 4 number 1 • march 2012 testosterone compounded formulations 80 to 100 mg testosterone in oil every-other-day im or once or twice daily trans-dermal application enhancement of libido in stallions. prostaglandin f2α (dinoprost tromethamine) lutalyse® (pfizer animal health, new york, ny) 0.01 to 0.02 mg/kg im 1) induction of luteolysis. 2) termination of pregnancy. cloprostenol (pgf2α analog) estrumate® (intervet/ schering-plough animal health, summit, nj) 0.5 μg/kg im 1) induction of luteolysis. 2) termination of pregnancy. 3) ecbolic for uterine fluid evacuation and/or expulsion of retained fetal membranes. misoprostol (pge1 analog) cytotec® (pfizer inc., new york, ny) 1.0 mg topically to cervix cervical relaxation in nonpregnant mares. prostaglandin e2 (dinoprostone) compounded formulations 2.0 to 2.5 mg topically to cervix or 0.25 mg intrauterine 1) cervical “ripening” prior to induction of parturition. 2) enhanced transport of spermatozoa into the oviduct after insemination. prostaglandin e2 (dinoprostone) prepidil® gel (pfizer inc., new york, ny) 0.2 mg topically onto oviduct restoration of oviductal patency. oxytocin many generic formulations see reference #22 1) ecbolic for uterine fluid evacuation. 2) disruption of luteolysis to prolong cl function. 3) induction of parturition. 4) treatment of retained fetal membranes. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 46clinical theriogenology • volume 4 number 1 • march 2012 table 2. exogenous hormone preparations available for equine reproductive management and their associated safety warnings regarding inadvertent human exposure. hormone product (supplier) safety warning on product label and/or package insert* deslorelin acetate (gnrh analog) sucromate™ equine (thorn bioscience llc, louisville, ky) pregnant women and women of childbearing age should exercise caution when handling this product. accidental administration may lead to disruption of the menstrual cycle. direct contact with skin should be avoided. insoluble in water: if direct contact with skin occurs, first wash with alcohol, then soap and water. in case of accidental human injection, consult a physician immediately. human chorionic gonadotropin (lh activity) chorulon® (intervet/ schering-plough animal health, summit, nj) no specific safety warning regarding human exposure provided with product.** equine follicle stimulating hormone (efsh) commercial product not currently available na estrogen (estradiol 17β or conjugated estrogens such as estradiol cypionate) compounded formulations no specific safety warning regarding human exposure provided with product.** progesterone compounded formulations no specific safety warning regarding human exposure provided with product.** altrenogest (synthetic progestin) regumate® solution 0.22% (intervet/ schering-plough animal health, summit, nj) women of childbearing age should exercise extreme caution when handling this product. regumate is readily absorbed through unbroken skin. impervious rubber or vinyl gloves must be worn when handling regumate. accidental absorption could lead to disruption of the menstrual cycle, abdominal cramping, increased or decreased uterine bleeding, prolongation of pregnancy or headaches. if direct contact with skin occurs wash immediately with soap and water. for eye exposure flush with plenty of water for 15 minutes and get medical attention. if swallowed do not induce vomiting, call a physician. combinations of progesterone and estrogen compounded formulations no specific safety warning regarding human exposure provided with product.** testosterone compounded formulations no specific safety warning regarding human exposure provided with product.** prostaglandin f2α (dinoprost tromethamine) lutalyse® (pfizer animal health, new york, ny) women of childbearing age, asthmatics, and persons with bronchial or other respiratory problems should exercise extreme caution when handling this product. lutalyse is readily absorbed through the skin and may cause abortion. if direct contact with skin occurs wash with soap and water. [note: because of its known side effects, it would seem prudent that pregnant women use extreme caution and/or avoid handling this product for veterinary use]. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201247 clinical theriogenology • volume 4 number 1 • march 2012 cloprostenol (pgf2α analog) estrumate® (intervet/ schering-plough animal health, summit, nj) women of childbearing age, asthmatics, and persons with bronchial or other respiratory problems should exercise extreme caution when handling this product. estrumate is readily absorbed through the skin and may cause abortion and/or bronchiospasms. if direct contact with skin occurs wash with soap and water. [note: because of its known side effects, it would seem prudent that pregnant women use extreme caution and/or avoid handling this product for veterinary use]. misoprostol (pge1 analog) cytotec® (pfizer inc., new york, ny) may cause abortion, pre-term labor or birth defects. no specific handling recommendations provided with product.** [note: because of its known side effects, it would seem prudent that pregnant women use extreme caution and/or avoid handling this product for veterinary use]. prostaglandin e2 (dinoprostone) compounded formulations no specific safety warning regarding human exposure provided with product.** [see note below for prepidil® gel formulation of pge2]. prostaglandin e2 (dinoprostone) prepidil® gel (pfizer inc., new york, ny) use caution when handling this product to prevent contact with skin. wash hands thoroughly with soap and water after use. [note: because this product is used to induce cervical “ripening” in pregnant women, it would seem prudent that pregnant women use extreme caution and/or avoid handling this product for veterinary use]. oxytocin many generic formulations no specific safety warning regarding human exposure provided with product.** * see product label and/or package insert for complete details. ** although no specific safety warning is provided with this product, it would seem prudent to avoid direct contact with skin and if contact occurs to cleanse the affected area, and for other routes of exposure to seek medical attention or consult a physician. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 48clinical theriogenology • volume 4 number 1 • march 2012 2010: advances in sex ratio management in cattle advances in sex ratio management in cattle e. curry, s.e. davis, j.r. gibbons department of animal and veterinary sciences, clemson university, clemson, sc, usa abstract manipulation of the sex ratio of cattle offspring can be documented as far back as 1891 when russell analyzed the relationship between the sex of a calf and the time during estrus at which copulation occurred.1 interestingly, or perhaps embarrassingly, researchers have made little progress in the advancement of sex ratio manipulation, although there have been copious attempts in the last century. most methods of manipulating embryo sex attempt to exploit the differences between xand ybearing sperm. mass and surface proteins have been predicted to differ between xand ybearing sperm and may provide an approach for separation through techniques such as flow cytometry, sperm surface antigen binding, and albumin separation. other studies have discovered correlations between pharmacological influence, semen incubation, the timing of insemination relative to ovulation, and maternal physiological influence on the sex of offspring. still other observations included the effects of acute stressors, drugs, environment, and paternal occupation on the sex ratio of offspring. the purpose of this review is to provide an update on existing sex selection technologies as well as present novel sex selection data in beef cattle. keywords: cattle, sex ratio, sperm introduction swaying the sex ratio is beneficial in agriculture and human fertility medicine. in the dairy industry, female calves are significantly more valuable because of their ability to produce milk, whereas in beef production, male calves are more desirable due to their rapid growth and efficient meat production. in the swine industry, males are undesirable due to boar taint, an objectionable flavor and odor associated with the meat of intact males. further, gilts reach market weight quicker than males thereby decreasing overall feed costs. niche markets, such as endangered species, pets, or laboratory animals may also prove to be useful applications for sex selection.2 to date, there is no effective, practical, inexpensive method for manipulating the sex of offspring. sex determination in mammals, somatic sex of the offspring is determined by the presence or absence of the y chromosome. an xx genotype results in a female while an xy genotype produces a male.2 the haploid oocyte from the female always contains an xchromosome, whereas males produce both xand y bearing haploid sperm, governing which sex will result upon fertilization of the oocyte. it is a common estimation that in most mammalian species, males produce xand ybearing sperm in a 1:1 ratio, resulting in male or female embryos of the same proportion. sry gene the sex-determining region of the ychromosome, or sry gene, was discovered in 1990 and is located near the tip of the chromosome’s short arm.4 it is an intronless region that encodes for a nuclear factor-like protein with a central high mobility group box.5,6 referred to as the testis determining factor (tdf), this protein is responsible for differentiation of the testes, which secrete anti-müllerian hormone (amh) from sertoli cells and testosterone from leydig cells. together, these hormones stimulate masculinization of the fetus. if the sry gene is absent, the gonad differentiates into an ovary. other genes play a role in sexual differentiation, such as sox9,3,7 an autosomal gene that aids in testis formation and is potentially upregulated by sry; steroidogenic factor 1, a transcription factor which may also be regulated by sry to increase levels of amh;8 and wnt4, a growth factor involved in müllerian duct regression and sex-specific cell migration.9 in the bovine, expression of the sry gene can be detected as 29 early as the four to eight cell stage embryo by reverse transcription polymerase chain reaction;10 however, expression of the gene has not been strongly linked to the presence of the tdf protein, perhaps due to the inability to accurately measure tdf or the timing of protein production relative to sry expression.11 history and various techniques of altering the sex ratio flow cytometry flow cytometry has many cell sorting applications and can be used to sort individual sperm cells by mass. moruzzi suggested that differences in dna mass could provide basis for separating xand ybearing sperm.12 because of the x-chromosome’s larger size relative to the y-chromosome, x-bearing sperm have approximately 3.7-4.1% more dna in their genome, depending upon species.13 this subtle difference in dna mass can be exploited by the flow cytometry process. in order to distinguish between x and y chromosomes, cellular dna is stained with a fluorescent dye and a single stream of cells is forced through the flow cell and subjected to a light source. the fluorescence of each cell can be excited into scattering light at a lower frequency than the light source. the scattered light is detected and analyzed, allowing various parameters of the cell to be deduced, such as cellular mass, mass of dna, proteins, ph, pigments, and enzymatic activity. as sperm cells pass through the flow cell, they can be selectively charged as positive or negative based on chromosomal content, and can then be deflected into separate paths as the sperm cells are attracted to either the positive or negative plate depending on which charge was applied. the current sperm sorting system permits the separation of six million x-bearing sperm and six million y-bearing sperm per hour, at a purity of up to 90% depending upon species and differences among individual males.2,14 after a semen sample is sorted via flow cytometry, the desired sperm cells are collected and used for artificial insemination or frozen for later use. although the accuracy of flow cytometry has been demonstrated using bull sperm, the process generally requires fresh semen and results in a reduction of sperm integrity. another point of controversy is the bis-benzimidazole dye, hoechst 33342, used to stain cells prior to flow cytometry. this stain targets dna at adenine-thymine-rich regions of the minor groove and although chromosomal abnormalities have been suggested,15,16 recent advances in the flow sorting technology have not only increased efficiencies and decreased costs,17 but have also facilitated a very low incidence of genotoxicity in vivo in swine.18 depending upon semen quality, there may be bull to bull variation and sexed semen is only available from selected bulls, although the list of available bulls is growing sharply. the flow cytometer equipment costs approximately $250,00019 and can only sort a limited number of cells per day; however, there are a growing number of sorting facilities available with several machines on site. the cost of a straw of sexed semen ranges from approximately thirty to fifty dollars and up to two-hundred dollars for dairy bulls, while the price is quite variable for beef bulls.20 as efficiencies increase (currently around 90% accurate) and fertility approaches close to that of non-sexed semen, the costs are expected to continue to decrease to an economically feasible point for most dairy producers and a growing number of niche beef producers.17 in addition, there may be variations in the flow cytometry accuracy process or resulting fertility among bulls of the same species or breed. although the system is gaining momentum and there has been a substantial increase in the number of bulls from which sexed semen is available, the research applications and opportunities perhaps outweigh commercial utility.14 surface proteins another suggested difference between xand y-bearing sperm is sperm head surface proteins. it is theorized that selectively binding proteins to xor y-bearing sperm will aid in their separation. the hy antigen has been studied to determine whether it is preferentially expressed on y-bearing sperm and whether it could aid in sperm separation; results have been conflicting.21, 22 the h-y antigen was first described by eichwald and silmser in 1955 following the observation that male-to-female skin grafts in mice were rejected while grafts within the same sex, as well as femaleto-male grafts, usually succeeded.23 in an attempt to exploit this difference in cell surface antigens, sills 30 et al. treated human sperm with monoclonal immunoglobulin m (igm) antibodies against the h-y antigen and incubated the mixture with igm antibodies appended to paramagnetic beads.24 the preparation was sorted through exposure to a magnetic field, with anticipated results that the reactive group would be positive for the h-y antigen and the non-reactive group would be negative. fluorescent in situ hybridization (fish) showed that 49% of ybearing sperm did not express the h-y antigen; therefore, the usefulness of this approach has been limited. studies using mice have shown that developing xand y-bearing sperm express different genes; however, these products are shared among gametes due to the intercellular bridges formed during spermatogenesis.25 braun et al. used transgenic hemizygous mice with mouse protamine 1 (mp1) transcriptional regulatory sequences fused to the human growth hormone (hgh) gene to demonstrate that rna and protein can pass through the intercellular bridges among spermatids.26 the hgh gene was transmitted to 50% of a developing sperm population, but subsequent immunocytochemical analysis showed that 90% of sperm contained hgh. the level of sharing may actually be greater; of the homozygous controls in the same study, only 92% were positive for hgh. caldwell and handel also demonstrated that post-meiotic spermatids share mp1 gene products through intercellular bridges.27 although sex-specific antigens have been identified on mammalian spermatozoa, exploiting this specificity has proven to be difficult and unrepeatable, therefore limiting the clinical application of this process.25,28 albumin separation albumin separation is a technique that utilizes the progressive forward motility of sperm and possibly results in the isolation of y-bearing sperm.29 semen is washed and diluted before being layered over columns of bovine serum albumin. using this method, ericsson et al. reported the isolation of 85% human y-bearing sperm with up to 98% progressive forward motility.29 two years later, ross et al. attempted to repeat the same experiment, but results failed to support ericsson’s findings.30 another study attempted albumin separation using bull semen with samples previously analyzed using flow cytometry. results indicated that the ratios of xand y-bearing sperm were the same for the treatment group and the controls.31 ten years later, ericcson co-authored a second study which found that 71% male offspring were produced out of 1,407 human births in 65 fertility clinics in the us and abroad through an albumin separation technique followed by intrauterine insemination on the presumed day of ovulation.32 in contrast, an experiment with rabbits found that the sex ratio was unaltered by the separation of sperm through albumin gradient.33 it was previously believed that this separation process resulted in a sperm sample with a higher representation of y-bearing sperm.34 a group in hong kong determined through fish that human sperm separation did not increase the concentration of y-bearing sperm, rather, they postulated that serum albumin inactivated x-bearing sperm.35 because the mode of action of albumin separation is unknown and results are contradictory and unrepeatable, the utility of this approach has been limited. semen incubation a study of in vitro produced bovine embryos showed that a 24 hour semen incubation period resulted in significantly more female hatched blastocysts when compared with zero or six hour semen incubation periods.36 watkins et al. found that human x-bearing sperm had significantly higher percentages of motility, rapid progression, faster curvilinear velocities, and hyperactivation after a 24 hour incubation when compared to y-bearing sperm.37 because no differences in xor y-bearing sperm morphology or metabolism have been found, the effects of sperm cell incubation and associated mechanism(s) are unknown. commercially available products post-thaw semen treatment products named heiferplustm and bullplustm (emlab genetics, arcola, il) are currently available which are claimed to “speed up” xor y-bearing sperm, respectively, to shift the sex ratio in favor of the desired sex. due to the proprietary nature of the products, the 31 ingredients and mode of action are undisclosed. the manufacturer published internet resources supporting its claim in both hyperstimulated and naturally ovulating cattle, although no control groups were reported.38 recently, an independent trial investigated the effects of heiferplus™ treatment on semen used to breed both single ovulating cows and hyperstimulated cows compared to control groups. results indicated that heiferplus™ did not effectively skew the sex ratio in favor of the female in either hyperstimulated or single-ovulating cows.38 no independent research investigating the efficacy of bullplustm has been reported. timing of insemination another factor that may influence embryo sex is the timing of insemination relative to ovulation. mammalian sperm can reach the oocyte quickly after insemination but may not be competent to fertilize the oocyte. they acquire competence as they move towards the ampulla of the oviduct, but will become unable to fertilize if they remain in the oviduct for an extended period of time.40 it has been observed that uncapacitated sperm temporarily bind to the membranes of the oviducts in the isthmus and are released upon capacitation.41 this event slows capacitation and lengthens the life span of the sperm, maximizing the probability of sperm being present in the ampulla to fertilize the egg even if insemination does not occur at a time coincident with ovulation. ovsynch42,43 is an ovulation synchronization protocol in which 100 µg gonadotropin releasing hormone (gnrh) is administered on day 1, 25 mg prostaglandinf2α (pgf) is administered seven days later, followed by a second injection of 100 µg gnrh on day 9. pursley et al. synchronized ovulation with ovsynch and examined the timing of insemination relative to ovulation.44 cows were inseminated at 0, 8, 16, 24, or 32 hours after the second injection of gnrh. cows inseminated at 0 and 32 hrs had the highest percentage of female offspring (61.0% and 65.0%, respectively), with the 0 hr group having the lowest pregnancy loss (between day 35 and parturition) and the 32 hr group having the highest. this protocol has become widely used in cattle and more recently, sheep.45,46 martinez et al. examined the timing of insemination relative to the onset of estrus in cattle and found that significantly more female calves (73.1%) resulted from inseminations performed from eight to 18 hours following first observed estrus.47 a study in sheep revealed that more females resulted from inseminations five hours prior to ovulation and more males resulted from inseminations occurring five hours after ovulation.48 guerrero evaluated 1,318 human conception cycles from both natural conception and intrauterine insemination to determine if basal body temperature at insemination impacted the sex ratio of human births.49 in natural conception, male births were most common (68.0%) when insemination occurred six days prior to the temperature shift, decreasing to 44.0% on the day of the shift. in artificial insemination, the trend was the opposite.49 in more precise human studies examining ovulation specifically, wilcox et al.50 showed that the timing of sexual intercourse relative to ovulation had no influence on the sex of the child, whereas gray51 reported that conception occurring close to ovulation resulted in a significantly lower proportion of male births.50,51 other studies have found no effects of the timing of insemination on sex ratio. rorie et al. found no difference in sex ratio of calves resulting from inseminations either 20 hr or 10 hr prior to the expected time of ovulation.52 in sows that had recently farrowed and weaned, soede et al. demonstrated that the sex ratio was unaffected in sows inseminated at four hour intervals from the onset of estrus until ovulation.53 in a recent study, angus and angus-cross cows were subjected to ovarian hyperstimulation using follicle-stimulating hormone (fsh).39 on day 3 of fsh administration, two doses of 25 mg pgf were given 12 hours apart. cattle were examined for signs of estrus every six hours and administered 100 µg of gnrh at estrus. cattle were bred with either frozen-thawed semen that had been incubated for 20 minutes at 37 °c (control; n=12) or frozen-thawed semen that had been treated with heiferplus™35 and incubated at 37 °c for 20 minutes, as per manufacturer’s protocol (n=13). chi square analysis revealed that the control group produced a significantly higher (p<0.005) proportion of female embryos (65/104, 65.0%) than the heiferplus™ group (71/167, 43.0%). 32 in two subsequent hyperstimulation studies with beef cattle, similar results were achieved.54 cows (n=8) and heifers (n=14), were synchronized, hyperstimulated, and examined for estrus 36 hours after the initial injection of pgf and every four hours thereafter until estrus was observed. cattle were administered 100 µg gnrh at estrus and inseminated with two doses of frozen-thawed semen from a single ejaculate of one bull 12 hours later. the treatment groups for these studies were subjected to transrectal ultrasound every four hours from 36 to 76 hours after the initial injection of pgf while the controls were not subjected to ultrasound. although there were no significant treatment effects, when grade 1-3 embryos (experiments 1 and 2; n=118) were combined the percentage of female embryos was higher (p<0.05) than the expected ratio of 50:50. this further supports the notion that synchronization of insemination and induced ovulation impacts the bovine embryo sex ratio. frequent rectal palpation/ultrasound ideta et al. found no difference in the sex ratio when hyperstimulated holstein heifers were inseminated at either 48 or 56 hours after pgf with frozen-thawed semen from multiple bulls; however, in a second experiment, they reported that heifers which produced a high percentage of female embryos (>50%) had a shorter duration of estrus (13.2±3.9 hours vs. 24.9±6.5 hours), fewer standing mounts (27.8±5.9 vs. 75.0±18.5 mounts) and lower superovulatory response (8.4±1.0 vs. 19.7±3.1 ova collected) than heifers with a low percentage of female embryos (<50%), respectively.55 ideta et al. observed that heifers which had been subjected to ultrasonic evaluation of their reproductive tracts to examine ovulation patterns tended to produce a higher percentage of female embryos (66.7%).56 these findings led to an additional study to determine the effects of frequent rectal palpation and ultrasonic evaluation on sex ratio.57 estrus synchronization was initiated by insertion of a progesterone-releasing pessary for nine days. a pgf analog was administered two days before the pessaries were removed and heifers were injected with equine chorionic gonadotrophin (500 iu, im) concurrently with pessary removal. hyperstimulation treatment began mid-cycle following estrus with eight decreasing doses of fsh. prostaglandin was administered concurrently with the seventh and eighth fsh injections. the treatment group was rectally palpated and subjected to trans-rectal ultrasonography every four hours from 36 to 76 hours beginning with the initial pgf injection (concomitant with the seventh fsh injection) while controls were not subjected to ultrasound. all cows were artificially inseminated 56 and 72 hours after the initial injection of pgf with frozen-thawed semen from a single bull. embryos were recovered on day 7 after artificial insemination by means of uterine lavage. sex of embryos was determined using loop-mediated isothermal amplification.58 the percentage of female embryos (grades 1-3) was significantly (p<0.05) increased in the treatment group (67.8%) from the expected ratio of 50:50. the authors proposed that acute stress caused by frequent rectal palpation and ultrasound around the time of ovulation skewed the sex ratio towards the female. in contrast, two studies by davis et al. found that frequent ultrasound around time of ovulation did not significantly skew the sex ratio toward the female relative to the controls, which were not subjected to frequent ultrasound, although the controls had an unexpectedly high percentage of female embryos.54 in experiment 1, multiparous cattle were synchronized by ultrasound-guided follicular ablation, a progesterone-releasing pessary was inserted and cattle were administered 25 mg pgf. superovulation was initiated 48 hours following follicular ablation with eight decreasing doses of fsh (once every 12 hours for four days). pessaries were removed 48 hours after initiation of fsh and observations for estrus began 36 hours later and occurred every four hours for 40 hours. gonadotropinreleasing hormone (100µg) was administered at observed estrus and cows were inseminated with two doses of frozen-thawed semen 12 hours later. treated cattle (n=4) were subjected to trans-rectal ultrasound at each estrus detection period while controls (n=4) were not subjected to ultrasound but were processed through the chute. embryos (control n=25; treatment n=23) were collected seven days after insemination, graded based on the international embryo transfer society scale and sex was determined on grade 1-3 embryos (control, n=23; treatment, n=14) using duplex polymerase chain reaction (pcr) 33 and dot blotting.59,60 the ratios of female to male embryos (grade 1-3) in the treatment and control groups were 57.1% (8/14) and 56.5% (13/23), respectively. the sex ratio did not differ significantly between groups or from the expected 50:50 ratio. in experiment 2, cattle were synchronized and hyperstimulated following a protocol similar to that used in experiment 1. multiparous cattle were synchronized by ultrasound-guided follicular ablation, a progesterone-releasing pessary was inserted and cattle were administered 25 mg pgf. superovulation was initiated 48 hours after follicular ablation with eight decreasing doses of fsh (once every 12 hours for four days). pessaries were removed 48 hours after initiation of fsh treatment and observations for estrus began 36 hours later and occurred every four hours for 40 hours. additionally, since cattle were at random stages of the estrous cycle at the beginning of treatment, both groups were administered additional doses of pgf (25 mg) concomitant with the fifth and sixth fsh injection. the treatment group (n=8) was subjected to ultrasound every four hours in conjunction with the estrus detection periods. the control group (n=7) was neither subjected to ultrasound nor processed through the chute. day 7 embryos (control, n=51; treatment, n=49) were collected and sex was determined on grade 1-3 embryos (control, n=41; treatment, n=40) using duplex pcr and dot blotting. the percentage of females for the treatment and control groups was 65.0% (26/ 40) and 73.2% (30/ 41), respectively. these data suggest that the timing of insemination relative to induced lh surge/ovulation (induced by gnrh) may have outweighed either the effects of ultrasound or the acute stressors (processing through the chute) associated with this experiment. other physiological factors oviductal proteins other studies have shown that an equal ratio of xto y-bearing sperm in a semen sample can result in a skewed sex ratio of offspring. recently, oviductal proteins have been shown to interact and possibly influence gamete and embryo development. oviduct-specific glycoproteins and osteopontin have been shown to affect sperm capacitation, gamete binding, fertilization, and embryo development.61 it is possible that other secretions of the oviduct could influence the sex ratio through preferential binding of one sperm type or another to the oocyte or through a mechanism operating on or within the embryo prior to implantation. catt et al. showed that of the total number of ovine embryos produced in vivo, a higher percentage of male embryos were represented, yet the sex ratio at birth was still 50:50 suggesting that more male embryos are lost perhaps during the peri-implantation period.62 a study using in vitro produced bovine embryos found that culture systems using synthetic oviduct fluid medium with fetal calf serum resulted in significantly higher rates of male blastocyst survival when compared to other culture systems, although the mechanism is not understood.63 cervical mucus martin found that sex ratio in human births was altered depending on the length of the follicular phase in women, with longer follicular phases resulting in more females.64 the author suggested that properties of cervical mucus may differ depending on the length of the follicular phase, and that these differences may preferentially select one sperm type over another. in cows, wehner et al. utilized an intravaginal probe to determine the conductivity of cervical mucus as a gauge for insemination times and their effect on sex ratio.65 these authors reported that significantly more heifer calves resulted from inseminations that occurred when impedance values were low (35-45; ~20 hrs prior to expected ovulation) and significantly more bull calves resulted when impedance values were high (50-70; ~10 hrs prior to ovulation). follicular environment at a local level within the follicle, an elevated concentration of testosterone can possibly alter fertilization events by producing an oocyte with an apparent preference for fertilization by a y-bearing sperm cell. recently, a higher proportion of in vitro produced male embryos resulted from oocytes 34 bathed in elevated follicular testosterone concentrations.66 these data suggest that there are differences in the fertilization capabilities of oocytes related to the steroid environment, but not necessarily sex selection at the level of the sperm cell. drugs, environment, and occupation exposure to certain drugs and toxins has been shown to alter the sex ratio. dibromochloropropane (dbcp), a toxic nematocide used extensively until the 1960’s, has been shown to induce testicular dysfunction in workers subjected to high levels of exposure. pregnancies conceived during exposure to dbcp resulted in 35% males whereas those same couples produced 53% males during the pre-exposure period.67 clomiphene citrate is a non-steroidal estrogen antagonist used to treat infertility by inhibiting estrogen binding by receptors in the anterior pituitary. this leads to increased production of fsh followed by lh and results in higher rates of ovulation. the use of clomiphene citrate also resulted in significantly fewer males at birth. sampson et al. found that of 89 clomiphene citrateinduced ovulations followed by intrauterine insemination, 53.9% resulted in females.68 silverman et al. used clomiphene citrate in conjunction with albumin separation prior to intrauterine insemination and reported 51.9% females compared to the control group which had 48.6% females.69 dioxin is a chemical produced as a byproduct of the manufacture of agent orange (2,4dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid), an herbicide used extensively during the vietnam war to defoliate plants and trees. michalek et al. reported that dioxin exposure led to significantly more female offspring.70 the air force health study examined offspring of veterans of operation ranch hand, the unit that handled agent orange during the vietnam war. men who conceived children (n=181) less than one month post-service produced 56.9% females. after an industrial accident in seveso, italy, nine families in which both parents were exposed to dioxin resulted in 100 percent female children (n=12).71 data on the health of the resulting children were not reported. the national toxicology program classified dioxin as a human carcinogen in its second annual report on carcinogens (1981).72 davis et al. concluded that the reduction in male births should be viewed as a sentinel health event that could be linked to environmental factors such as pollution or dioxin exposure in industrialized countries.73,74 retrospective studies have investigated the relationship between certain occupations and the sex ratio of offspring. lyster examined the offspring sex ratio of australian abalone divers.75 of the 58 participants in the study, there were 85 daughters (65.4%) and 45 sons (34.6%), all conceived after their fathers had become divers. other studies have investigated the offspring sex ratio of pilots and astronauts exposed to high g forces. pilots exposed to higher g forces had 60% female children, while those considered to work at low g forces showed no statistical difference in their offspring’s sex ratio.76 after administering a comprehensive questionnaire to 1,000 german pilots, goerres and gerbert of the german air force institute of aviation medicine concluded that the offspring sex ratio of helicopter pilots significantly shifted in favor of females after the one-thousandth flying hour.77 sex determination in non-mammalian species current methods of manipulating the sex ratio in mammals may not be applicable to other organisms. in avian species, the male is homogametic, designated zz, while the female is heterogametic, or zw. in drosophila, the y-chromosome does not produce a male; a male is generated by the lack of two xxs. in other insects, such as bees, fertilized eggs become females while unfertilized eggs develop into males.78 in most reptiles, sex is dependent upon the incubation temperature of the eggs, not by sex chromosomes deposited into the egg by a male. in species that display temperature-dependent sex determination, males and females have identical karyotypes.79 turtle eggs incubated at lower temperatures (26 °c) yield males, while those incubated at higher temperatures (33 °c) yield females. alligators are the opposite, with lower temperatures producing females and increased temperatures resulting in males. differentiation of gonads into ovaries or testes depends upon the incubation 35 temperature of the eggs during a period of development in which the embryo is thermosensitive. it is believed that temperature affects the undifferentiated gonads, since they are the site of estrogen synthesis and aromatase activity.80 selecting for sex at the embryo level following semen sexing by flow cytometry, a single sperm cell of the desired sex can be injected directly into the oocyte in vitro through intracytoplasmic sperm injection. this technique assures an embryo of a chosen sex, but is invasive, inefficient, and can be damaging to the oocyte. in vitro fertilization (ivf) is a process in which the gametes are collected and fertilization takes place in culture. the resulting embryos can then be transferred into a recipient or frozen for later use. studies describe a higher number of male offspring resulting from ivf, possibly due to the rapid early division and growth of male embryos relative to female embryos. embryos at a more advanced stage in development are more likely to be selected for transfer rather than frozen.63 kochhar et al. theorized that because the ychromosome may contain more genes that function as transcription factors, development would be accelerated, whereas the x-chromosome contains genes that code for rate limiting steps required in embryonic developmental pathways.81 comprehensive understanding of the molecular events that control testes development, regulation of tdf, sry, sox9, and wnt4 and to the extent to which these events control sex determination of subsequent offspring remain elusive.3,82 the sex of embryos can be determined prior to embryo transfer by pcr. this technique is useful because it requires only one or two blastomeres, leaving a viable embryo to be transferred into a recipient or frozen for later use. to determine the sex of a mammalian embryo, a dna sequence specific to the ychromosome can be amplified and the product visualized using agarose gel electrophoresis. if a visible band is produced by the primer pair, the specific region of dna is present and the embryo can be identified as a male. lack of that band would signify lack of a y-chromosome, indicating a female embryo. polymerase chain reaction was first used for genetic diagnosis in bovine blastocysts in 1988 and is now a widely used method of sexing bovine embryos.5, 83-87 although it is sensitive and specific, pcr often yields inconsistent results when assessing samples with a limited template, especially when copies of target genes are unequal. almodin et al. reported successful sex determination in 82.0% of embryos, using bovine embryos as models for human preimplantation genetic diagnosis.88 a retrospective study conducted by shea examined the use of pcr for sexing 4,183 embryos in a commercial bovine embryo transfer program.89 results showed that sex could successfully be determined in 90.0% of samples; failures at sexing were attributed to malfunctions of specific pcr reagents, contamination, and insufficient dna template. because pcr assays for embryo sexing have been optimized in order to amplify samples with small amounts of dna, they are also sensitive enough to amplify contamination by a single dna molecule. ethical considerations while the purpose of altering the sex ratio in livestock is to reach financial goals, in human assisted reproduction, couples may want a child of a certain sex in order to avoid sex-linked genetic disorders, such as hemophilia, duchenne muscular dystrophy, and fragile x syndrome, which are inherited through one of the sex chromosomes. because males have only one copy of the x chromosome, any gene present on the xchromosome will be expressed, even if recessive. reasons to desire a child of a certain sex may be for family balancing or to avoid sex-linked diseases; however, in india and china, there are strong politically-driven preferences towards males. a recent census in india showed that the male:female ratio of children under six years of age was 1000:927. in haryana state, the ratio was smaller, with 1000:820.90 in areas of china where the “one child” policy is mandated, the sex ratio is 117:100, but in rural areas of the country where multiple children are permitted, the sex ratio is balanced.91,92 in most other countries, including the us, canada, and the uk, census data show that no sex preference exists.93 36 summary currently, there is no effective, reliable, inexpensive, and safe method for commercially swaying the sex ratio. although flow cytometry is the most effective technique to date, it is expensive, may cause damage to sperm cells, and sexed semen is only available from a limited selection of bulls. a rapid increase in the flow-sorting technology efficiencies in the last few years has resulted in an accuracy >90% of the desired sex. pregnancy rates using frozen, sexed sperm are within 70-90% of conventionally processed unsexed sperm, although the concentration of sperm cells per insemination is 10-20 fold less.94 an extension of this technology has recently resulted in pregnancies from thawed sex-sorted semen which has been refrozen; however, as expected, pregnancy rates have been low and embryo wastage high.95 to date no sperm cell surface proteins have been isolated that are specific to either xor y-bearing sperm and results of albumin separation studies have been conflicting. although certain toxins and environmental factors appear to have an effect on the sex ratio, usually favoring females, the mechanisms are not well understood and the risks of exposure inarguably outweigh the benefits. intracytoplasmic sperm injection, ivf, and embryo sexing require expensive equipment and supplies and are technically challenging, thereby limiting them mainly to human fertility treatments. further, some aggressive techniques may have species-specific efficiencies. because it is difficult to exploit the only known difference between xand y-bearing sperm (mass), perhaps the sex of the embryo can be manipulated by the maternal side through oviductal or ovarian influences. a better understanding of the relationship of the timing of insemination relative to spontaneous ovulation, first observed estrus, or pharmacologically-induced ovulation in both hyperstimulated and single-ovulating cows warrants a more thorough investigation, as promising studies have been reported in cattle, sheep, and human studies.45,47-49 in addition, these techniques could potentially be implemented at the farm level and without expensive equipment. further research is necessary to determine and experimentally or 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antibodies to separated x and y chromosome-bearing porcine and bovine sperm. mol reprod dev 1993;35:189-196. 22. veerhuis r, hendriksen pj, hengst am, et al: the production of anti-h-y monoclonal antibodies: their potential use in a sex test for bovine embryos. vet immunol immunopathol 1994;42:317-30. 23. eichwald ej, silmser cr: untitled communication. transplantation bulletin 1955;2:148-149. 24. sills es, kirman i, colombero lt, et al: h-y antigen expression patterns in human xand y-chromosome-bearing spermatozoa. am j reprod immunol 1998;40:43-47. 25. hendrickson pjm: do x and y spermatozoa differ in proteins? theriogenology 1999;52:1295-1307. 26. braun r e, behringer r, peschon jj, et al: genetically haploid spermatids are phenotypically diploid. nature 1989;337:373-376. 27. caldwell ka, handel ma: protamine transcript sharing among postmeiotic spermatids. proc natl acad sci 1991;88:2407–2411. 28. howes ea, miller ng, dolby c, et al: a search for sex-specific antigens on bovine spermatozoa using immunological and biochemical techniques to compare the protein profiles of x and y chromosome-bearing sperm populations separated by fluorescence-activated cell sorting. j reprod fertil 1997;110:195-204. 29. ericsson rj, langevin cn, nishino m: letters to nature. nature 1973;246:421-424. 30. ross a, robinson ja, evans hj: failure to confirm separation of xand y-bearing human sperm using bsa gradients. nature 1975;253:354-355. 31. beal we, white lm, garner dl: sex ratio after insemination of bovine spermatozoa isolated using a bovine serum albumin gradient. j anim sci 1984;58:1432-1436. 32. beernink fj, dmowski wp, ericsson rj: sex preselection through albumin separation of sperm. fertil steril 1993;59:382-386. 33. zavos pm: sperm separation attempts via the use of albumin gradients in rabbits. theriogenology 1985;23:875-879. 34. hafez ese: reproduction in farm animals. 5th ed. philadelphia: lea and febiger; 1987. 35. rose ga, wong a: experiences in hong kong with the theory and practice of the albumin column method of sperm separation for sex selection. hum reprod 1998;13:146-149 36. lechniak d, strabel t, bousquet d, et al: sperm pre-incubation prior to insemination affects the sex ratio of bovine embryos produced in vitro. reprod domest anim 2003;38:224-227. 37. watkins am, chan pj, patton wc, et al: sperm kinetics and morphology before and after fractionation on discontinuous percoll gradient for sex preselection: computerized analyses. arch androl 1996;37:1-5. 38. williams t: the use of heiferplus in superovulated heifers. 2007; www.emlabgenetics.com/heiferplusdata. 39. curry e, pratt sl, lapin dr, et al: efficacy of a commercially available post-thaw bovine semen sexing kit in both single-ovulating and hyperstimulated cows. anim reprod sci 2009; 116:376-380. 40. eisenbach m: sperm changes enabling fertilization in mammals. curr opin endocrinol diabetes obes 1995;2:468-475. 41. smith tt: the modulation of sperm function by the oviductal epithelium. biol reprod 1998;58:1102-1104. 42. pursley jr, mee mo, wiltbank mc: synchronization of ovulation in dairy cows using pgf2α and gnrh. theriogenology 1995;44:915-923. 43. stevenson js, kobayashi y, thompson ke: reproductive performance of dairy cows in various programmed breeding systems including ovsynch and combinations of gonadotropin-releasing hormone and prostaglandin f2α. j dairy sci 1999;82:506-515 44. pursley jr, silcox rw, wiltbank mc: effect of time of artificial insemination on pregnancy rates, calving rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows. j dairy sci 1998;81:2139 2144. 45. deligiannis c, valasi i, goulas p, et al: synchronization of ovulation and fixed time intrauterine insemination in ewes. reprod domestic anim 2005;40:6-10. 46. gibbons j, busbee la: ovulation synchronization in sheep. biol reprod 2009;81:586. 47. martinez f, kaabi m, martinez-pastor f, et al: effect of the interval between estrus onset and artificial insemination on sex ratio and fertility in cattle: a field study. theriogenology 2004;62:1264-1270. 48. gutiérrez-adán a, pérez-garnelo, granados j, et al: relationship between sex ratio and time of insemination according to both time of ovulation and maturational state of oocyte. zygote 1999;7:37-43. 49. guerrero r: association of the type and time of insemination within the menstrual cycle with the human sex ratio at birth. n engl j med 1974;291:1056-1059. 50. wilcox aj, weinburg cr, baird dd: timing of sexual intercourse in relation to ovulation. effects on the probability of conception, survival of the pregnancy, and sex of the baby. n engl j med 1995;333:1517-1521. 51. gray rh: natural family planning and sex selection: fact or fiction? am j obstet gynecol 1991;165:1982-1984. 52. rorie rw, lester td, lindsey br, et al: effect of timing of artificial insemination on gender ratio in beef cattle. theriogenology 1999;52:1035-1041. 38 53. soede nm, nissen ak, kemp b: timing of insemination relative to ovulation in pigs: effects on sex ratio of offspring. theriogenology 2000;53:1003-1011. 54. davis s, hesser m, curry e, et al: sex ratio of embryos from hyperstimulated beef cattle. biol reprod 2009;81:(suppl. 1);abstract 650. 55. ideta a, hayama k, urakawa m, et al: relationships among estrous behavior, superovulatory response and grade 1 embryo sex ratio in superovulated holstein heifers. j reprod dev 2007;53:1015-1021. 56. ideta a, hayama k, urakawa m, et al: frequent rectal palpations following superovulatory treatment affect sex ratio of embryos recovered from holstein heifers. reprod fertil dev 2008;20:228-228. 57. ideta a, hayama k, kawashima c, et al: subjecting holstein heifers to stress during the follicular phase following superovulatory treatment may increase the female sex ratio of embryos. j reprod dev 2009; doi:10.1262/jrd.20209 58. hirayama h, kageyama s, moriyasu s, et al: rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification. theriogenology 2004;62:887-896. 59. stringfellow da, seidel s: manual of the international embryo transfer society. 3rd ed. savoy, il: international embryo transfer society;1999. p. 170. 60. curry e, pratt sl, kelley de, et al: use of combined duplex pcr/dot blot assay for more sensitive genetic characterization of bovine embryos. biochemistry insights 2008;1:35-39. 61. killian gj: evidence for the role of oviduct secretions in sperm function, fertilization and embryo development. anim reprod sci 2004;82-83:141-153. 62. catt sl, o’brien jk, maxwell wm, et al: effects of rates of development of in vitro produced ovine embryos on sex ratio and in vivo survival after embryo transfer. theriogenology 1997;48:1369-1478. 63. gutiérrez-adán a, lonergan p, rizos d, et al: effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos. theriogenology 2001;55:1117-1126. 64. martin jf: length of the follicular phase, time of insemination, coital rate and the sex of offspring. hum reprod 1997;12:611-616. 65. wehner gr, wood c, tague a, et al: efficiency of the ovatec unit for estrus detection and calf sex control in beef cows. anim reprod sci 1997;46:27-34. 66. grant vj, irwin rj, standley nt, et al: sex of bovine embryos may be related to mothers' preovulatory follicular testosterone. biol reprod 2008;78:812-815. 67. potashnik g, porath a: dibromochloropropane (dbcp): a 17-year reassessment of testicular function and reproductive performance. j endocrinol 1995;37:1287-1292. 68. sampson jh, alexander nj, fulgham dl, et al: gender after artificial induction of ovulation and artificial insemination. fertil steril 1983;40:481-484. 69. silverman ay, stephens sr, drouin mt, et al: female sex selection using clomiphene citrate and albumin separation of human sperm. hum reprod 2002;17:1254-1256. 70. michalek je, rahe aj, boyle ca: paternal dioxin and the sex of children fathered by veterans of operation ranch hand. epidemiology 1998;4:474-475. 71. mocarelli p, brambilla p, gerthoux p, et al: change in sex ratio with exposure to dioxin. lancet 1996;384:409. 72. national toxicology program: second annual report on carcinogens. 2,3,7,8tetrachlorodibenzo-p-dioxin (tcdd); “dioxin”. 1981;cas no. 1746-0106. 73. davis dl, gottlieb mg, stampnitzky jr: reduced ratio of male to female births in several industrial countries: a sentinel health indicator? jama 1998;279:1018-1023. 74. mocarelli p, gerthoux pm, ferrari e, et al: paternal concentrations of dioxin and sex ratio of offspring. lancet 2000;355:1858-1863. 75. lyster wr: altered sex ratio in children of divers. lancet 1982;320:152. 76. little bb, rigsby ch, little lr: pilot and astronaut offpsring: possible g-force effects on human sex ratio. aviat space environ med 1987;july:707-709. 77. goerres hp, gerbert k: sex ratio in offspring of pilots: a contribution to stress research. aviat space environ med 1975;august:889-892. 78. manolakou p, lavranos g, angelopoulou r: molecular patterns of sex determination in the animal kingdom: a comparative study of the biology of reproduction. reprod biol endocrinol 2006;4:59. 79. modi ws, crews d: sex chromosomes and sex determination in reptiles. curr opin genetics dev 2005;15:660-665. 80. pieau c, dorizzi m: oestrogens and temperature-dependent sex determination in reptiles: all is in the gonads. j endocrinol 2004;181:367-377. 81. kochhar hps, peippo j, king wa: sex related embryo development. theriogenology 2001;55:3-14. 82. harley vr, clarkson mj, argentaro a: the molecular action and regulation of the testis-determining factors, sry (sex-determining region on the y chromosome) and sox9 [sry-related high-mobility group (hmg) box 9]. endocr rev 2003;24:466-487. 83. king d, wall rj: identification of specific gene sequences in preimplantation embryos by genomic amplification: detection of a transgene. mol reprod devel 1988;1:57-62. 84. bredbacka p, kankaanpää a, peippo j: pcr-sexing of bovine embryos: a simplified protocol. theriogenology 1995;44:167-176. 39 85. weikard r, kuhn c, brunner rm, et al: sex determination in cattle based on simultaneous amplification of a new male-specific dna sequence and an autosomal locus using the same primers. mol reprod dev 2001;60:13-19. 86. park jh, lee jh, choi km: rapid sexing of preimplantation bovine embryo using consecutive and multiplex polymerase chain reaction (pcr) with biopsied single blastomere. theriogenology 2001;55:1843-1853. 87. alves bca, hossepian de lima vfm, teixeira cm, et al: use of primers derived from a new sequence of the bovine y chromosome for sexing bos taurus and bos indicus embryos. theriogenology 2002;59;1415-1419. 88. almodin cg, moron af, kulay l jr, et al: a bovine protocol for training professionals in preimplantation genetic diagnosis using polymerase chain reaction. fertil steril 2005;84:895-899. 89. shea bf: determining the sex of bovine embryos using polymerase chain reaction results: a six-year retrospective study. theriogenology 1999;51:841-854. 90. registrar general: provisional population totals, census of india. office of the registrar general, new delhi; 2001. 91. plafker t: sex selection in china sees 117 boys born for every 100 girls. br med j 2002;324(7348):1233. 92. institute of philosophy, chinese academy of social sciences: action recommendations on correcting the birth sex ratio imbalance. chinese academy of social sciences; 2004. 93. royal commission on new reproductive technologies: proceed with care: final report of the royal commission. minister of government services, ottawa. 1993;889-890. 94. schenk jl, cran dg, everett rw, et al: pregnancy rates in heifers and cows with cryopreserved sexed sperm: effect of sperm numbers per inseminate, sorting pressure and sperm storage before sorting. theriogenology 2009;71:717-728. 95. underwood sl, bathgate r, ebsworth m, et al: pregnancy loss in heifers after artificial insemination with frozen thawed, sex-sorted, re-frozen-thawed dairy bull sperm. anim reprod sci 2009;doi:10.1016/j.anireprosci.2009.06.004. 40 2009: orchitis and epididymitis orchitis and epididymitis 1 r. b. hollett 2 department of large animal medicine and deans’ office, college of veterinary 3 medicine, the university of georgia, athens ga, usa 4 abstract 5 inflammation of the male testis and/or epididymis produces canine and feline 6 subfertility and infertility. causative agents include bacteria, trauma, non-infectious 7 immune-mediated response, prostatitis, urinary tract infection, or an accompanying 8 endocrine disorder. each possible etiology should be evaluated to both determine the 9 reason for the abnormal reproductive function and to justify its treatment. 10 keywords: orchitis, epididymitis, dog, cat, brucella 11 introduction 12 in the male, loss of reproductive performance is sometimes first observed as 13 failure of conception in the female to whom the male had been bred, or as a painful 14 attempt during a natural breeding. this change in behavior may be the most apparent 15 sign in an otherwise asymptomatic male. a male who historically exhibited normal 16 breeding behavior may become unwilling to mount and gain intromission. an affected 17 male may start scrotal licking to the extent of hair loss and scrotal dermatitis. multiple 18 etiological agents are possible for such behavior. only after obtaining a good history, 19 performing a thorough physical examination, and choosing the appropriate diagnostic 20 tests can the underlying reason for a behavioral change, pregnancy failure in bred 21 females, or purulent preputial discharge be determined. subsequent therapy can then 22 begin which may or may not maintain the male’s potential to be a satisfactory breeder. 23 59 orchitis and epididymitis can occur separately or in concert since each organ has close 24 anatomical positioning and connection to a common excurrent duct system. 25 inflammation of one structure can lead to inflammatory response of the other and thus the 26 term orchiepididymitis or epididymo-orchitis is used in veterinary literature.1-4 this 27 condition is rarely found or perhaps diagnosed among tom cats. 28 anatomy 29 body weight and breed dimensions should correlate with testicular weight, 30 volume, width and, therefore, quantity of ejaculate. each testicle is positioned obliquely 31 within the scrotum of the stud dog and tom cat. the head of the epididymis is attached to 32 the cranial aspect of the testis, the body lying on the dorsolateral surface, and the tail 33 fixed to the caudal end by the ligament of the tail of the epididymis or former 34 gubernaculum testis.5 the epididymis continues as the ductus deferens within the 35 spermatic cord, which also encloses the testicular artery and vein, pampiniform plexus, 36 cremaster muscle, and lymphatics. the cord is wrapped on the outside by the visceral 37 vaginal tunic and overlaid by the parietal vaginal tunic as it passes through the inguinal 38 canal. deep to these two tunics, the testis is also covered by a thick, white, fibrous, 39 tightly adherent capsule called the tunica albuginea. 40 the body of the testis or testicular parenchyma is subdivided into compartments 41 of seminiferous tubules by connective tissue septae. spermatogenesis occurs within these 42 seminiferous tubules. the sections of tubules feed sperm into a collection of spaces and 43 ducts called the rete testis. a band of connective tissue called the mediastinum testis 44 splits the testicle longitudinally and serves as the entry and exit point for testicular blood 45 60 vessels and lymphatics. from the rete testis, sperm cells then move into and through the 46 head, body and tail of the epididymis where spermatozoal maturation and storage occur. 47 descent of the testicles into the scrotum occurs in a similar pattern for the dog and 48 tom cat. as the gubernaculum shortens, each testis passes through its respective inguinal 49 canal and ring. when this passage occurs, the testes is already covered by the visceral 50 tunic and then overlaid with the outer or superficial parietal tunic. completion of this 51 process usually occurs prior to birth in the normal male. however, male cats have been 52 noted to have testicular movement back and forth through the inguinal canal after birth 53 and prior to puberty.6 54 each testis is partitioned into three sections. the interstitial portion contains the 55 leydig or interstitial cells, blood vessels and some support tissue. this section functions 56 to provide hormones and nutrition to its respective area. the second or basal 57 compartment is comprised of sertoli cells and undifferentiated germ cells called 58 spermatogonia. the third and innermost adluminal compartment houses the developing 59 stages of spermatozoa and is separated from the basal section by the immunologically 60 sensitive blood-testis barrier. 61 etiology 62 infectious 63 bacteria enter the testis or epididymis through several routes: from an ascending 64 source through the penile urethra, from descending flora within the adjacent bladder or 65 prostate, and hematogenously. the primary bacterium associated with canine infertility is 66 brucella canis,7 however other brucella spp. (eg. brucella abortus) have been cultured 67 from the epididymis in the canine.8 additional bacteria are hemolytic e. coli, proteus 68 61 vulgaris, staphylococcus spp. as a natural skin contaminant, and mycoplasma spp.3-9 69 also the canine distemper virus10 with its cytoplasmic and intranuclear inclusions, 70 granulomatous epididymitis from mycotic rhodotorula glutinis,11 blastomyces,12 human 71 tuberculosis,3 rocky mountain spotted fever,4 and canine ehrlichiosis (lyme disease) 72 have been reported as potential causes. the distemper virus is spread venereally to 73 susceptible bitches which acquire an endometritis.3 mycoplasma canis is a natural 74 component of urogenital and respiratory mucosal surfaces in the dog and cat13 and can 75 cause purulent prostatitis and epididymitis. a bacterial urinary tract infection can incite 76 orchiepididymitis since there is potential for retrograde or reverse urethral pressure 77 through the ductus deferens. bacterial infections can occur following injuries from 78 pelvic fracture and surgical resection of ileum.3 unusual reports occur when certain stud 79 dogs still sire litters despite heavy bacterial growth of klebsiella pneumoniae and 80 streptococcus after antibiotic treatment for a preliminary diagnosis of epididymitis.14 81 a stray cat that was positive for feline immunodeficiency virus (fiv) and feline 82 leukemia virus (felv) had chronic necrotic and fibrinous orchitis.15 tuberculosis was 83 reported as the cause for a reproductive infection of tom cats in foreign countries.16 84 brucella spp.7,17,18 and periorchitis from feline infectious peritonitis (fip) were noted as 85 well. cats are resistant to infection with brucella canis.19 86 testicular injury from a bite wound or puncture results in injection of bacteria or 87 viral particles into the animal, followed by entry into the venous blood and initiation of 88 an inflammatory response. the cellular influx causes swelling and edema of the 89 immediate area. white blood cells are attracted to the site and begin phagocytosis. the 90 increased blood supply soon spreads a local infection systemically. if the animal 91 62 becomes septicemic with bacteria such as hemolytic e. coli, effects of endotoxemia on 92 the kidney impair glomerular filtration rates. as endotoxemia ensues, the animal 93 becomes febrile. a local thermal effect on the ipsilateral testis diminishes 94 spermatogenesis and promotes agglutination of sperm which turns into oligospermia, 95 anesthospermia, or azoospermia. as the condition becomes chronic, testicular atrophy 96 results. bacterial ascension from proximal prostatic secretions or a urinary tract infection 97 can initiate orchitis and epididymitis. 98 non-infectious 99 non-infectious, atraumatic orchitis or epididimytis occurs with the entry of sterile 100 urine through the ductus deferens into the testicle. blunt force injury to the abdomen, 101 such as happens from an animal being hit by a car, can cause urine to abnormally flow 102 from a full bladder to the testicles.3 103 in humans, even temporary duct blockage secondary to prostatitis can lead to 104 immune-mediated infertility.20 the blood-testis barrier was described in detail by 105 amann in 1989.21 the blood-testis barrier is comprised of tight junctions between 106 adjacent sertoli cells resting on the basement membrane. any breakdown of this barrier, 107 from trauma, infection or an inflammatory response, breaches the immunologically 108 protected status of the testis. this affront allows the immune system to react to the 109 antigenic insult. sometimes the reaction produces a sperm granuloma at the puncture 110 point. the attraction of plasma cells and lymphocytes into the testicle does produce a 111 residue of immunoglobulins within the seminiferous tubules. the loss of testicular 112 function by the tubules, leydig cells and impaired vascular flow results in reduced sperm 113 development. clinically, immune-mediated infertility will be manifested by 114 63 oligospermia or azoospermia. immune-mediated orchitis can be temporary or 115 permanent.20 116 similar to that observed in humans, immune-mediated disease has been associated 117 with infertility in the dog. the incidence of lymphocytic orchitis in dogs is correlated 118 with lymphocytic thyroiditis, a heritable trait in beagles.22,23 males with this 119 autoimmune disorder had lesions of tubular degeneration, atrophy and lymphocytic 120 orchitis. therefore, a history of reproductive failure in a stud dog that has been 121 diagnosed with other endocrine diseases should prompt further analysis. 122 pathophysiology 123 the blood supply to the testes and epididymides comes from the testicular artery 124 and the artery of the ductus deferens, respectively. both structures are influenced by 125 hormones, both play an important role in spermatogenesis, and both tissues are altered by 126 inflammation and antibody formation. existing infections within the bladder or prostate 127 gain entry into the ductus deferens by retrograde route. inflammation from regional 128 lymph nodes reaches the testis or epididymis by lymphatic drainage and then into the 129 blood stream. epididymal occlusion may appear secondary to an infection. the more 130 proximal the blockage to the adjacent testis, the worse the damage to that testicle.24 131 viral agents such as feline coronavirus are engulfed by and replicate in 132 macrophages. these macrophages then travel to target organs and mix with lymphocytes 133 to form a fibrin layer on the tunica albuginea (i.e., periorchitis), a finding consistent with 134 fip.15 since the parietal and vaginal tunics are continuations of the peritoneal cavity, 135 seeding from a contaminated surgical site or traumatic injury can extend infections into 136 these reproductive organs. small abscesses within the lumen or testicular parenchyma 137 64 increase in size and extend fistulous tracts through the scrotal wall. adhesions develop 138 and block tubular patency. gangrenous inflammation occurs within the unyielding 139 tunics, which inhibit testicular mobility and increase scrotal temperature. inflammation 140 of the spermatic cord results in vascular compromise, tissue necrosis and eventually 141 testicular atrophy.3 142 the interstitial compartment houses immune cells that maintain the testis in an 143 immunologically secure location. spermatogonia within this compartment are protected 144 from an autoimmune attack. in addition to the blood-testis barrier, a multitude of factors 145 establish this privileged immune status. androgens influence the inhibition of 146 proinflammatory cytokines.25 proand anti-inflammatory cytokines regulate testicular 147 function relative to both spermatogenesis and steroidogenesis.26 pro-inflammatory 148 cytokines such as interleukin and anti-inflammatory cytokines are linked to testicular 149 development.27 therefore, an insult from infection or trauma induces production of these 150 respective regulatory proteins and likewise disrupts testicular function. timed 151 progression of focal or diffuse inflammation in testicular tissue eventually results in loss 152 of seminiferous tubules by replacement with connective tissue fibrosis. the clinical 153 outcome of this sequence is manifested as infertility. 154 another source of immune protection in the testis is the resident population of 155 macrophages. macrophages assert cytotoxic and phagocytic activity against infection 156 through the cellular inflammatory response.28 for example, brucella canis embeds itself 157 within macrophages that ultimately seek steroid-dependent organs such as the testis and 158 epididymis.7 these immune and inflammatory components combat and modify normal 159 65 internal testicular functions that correspond to owners’ complaints of nonpregnant 160 matings, scrotal swelling or pain. 161 clinical signs 162 historical information provided from the breeder client often supplies the first 163 clue to the diagnosis of orchiepididimytis. breeder clients are conscientious and notice 164 conception failures. owners or handlers observe stud dogs not wanting to ejaculate, or an 165 enlarged scrotum while grooming for a show or field trial. the client may report 166 previous urinary or reproductive tract infections, an increased licking of the scrotum, an 167 obscure hindleg lameness, stiff or altered gait, purulent preputial discharge, or palpable 168 unilateral testicular atrophy. all prior medical problems, treatments, hormonal adjuvants 169 and products should be chronologically recorded for review.29 as one source 170 commented, a client’s conclusion may be ‘infertility in an otherwise asymptomatic dog’.2 171 additional signs include lethargy, anorexia, fever of unknown origin, inappetance, and 172 vomition. physical findings indicate a swollen scrotum and testicle or epididymis, 173 primarily unilateral enlargement, but it can be bilateral. the testis is asymmetrical; the 174 scrotum is reddened or hyperemic and hyperthermic. the dog licks the area causing a 175 ‘lick’ granuloma or scrotal dermatitis and exhibits a level of discomfort and pain during 176 palpation of the testicles. if the dog resists or is reluctant for digital examination, 177 tranquilization might be necessary. the expression of pain can be acute, episodic and 178 increase with intensity during a natural cover or manual collection. a decrease or loss of 179 libido is evident. one older male had a progressive bilateral alopecia and feminization 180 syndrome associated with acute and chronic epididymitis.30 181 66 digital palpation may distinguish swelling of the head, body and/or tail of the 182 epididymis and testes. the swollen area often has a soft, doughy texture in acute cases, 183 and a firm, fibrotic consistency in chronic cases. a small puncture wound may be 184 discovered with or without draining purulent exudate or concurrent orchitis. a 185 spermatocoele, sperm granuloma, or hematoma within the tunic linings or in the vaginal 186 cavity may be found. a serosanguineous or purulent discharge from the dog’s preputial 187 orifice may be present. 188 in summary, the typical clinical signs exhibited with prostatitis include a blood-189 tinged ejaculate, hematuria, and difficulty in defecation. massage of the prostate per 190 rectum may aid in detection of a purulent discharge from the urethra.3 however, like 191 prostatitis, orchioepididymitis can present with similar signs: altered hindlimb gait, 192 lethargy or pain. to help differentiate prostatitis from orchioepididymitis, digital 193 examination would reveal symmetrical or asymmetrical distension of the inflamed 194 prostate. 195 diagnosis 196 a thorough anamnesis should be recorded. historical information should include 197 details of prior reproductive attempts, onset and duration of signs and subtle changes in 198 behavior or clinical signs. a chronologically ordered medical history is beneficial. 199 notations are made of diet, supplements, deworming program, schedule of vaccinations 200 and past medications or surgical therapy. potentially confounding effects of age, 201 temperament and conformation on behavior or locomotion need to be noted. a physical 202 examination, beginning with a tpr, should conclude with a digital evaluation per rectum 203 of the prostate for size, symmetry, consistency, and pain response. palpation of each 204 67 testis for size, shape and consistency can often discern a soft, acute onset condition from 205 a firm, nodular chronic problem. likewise, palpation of the epididimydes can aid in the 206 identification of unilateral or bilateral testicular atrophy based upon the relative 207 prominence of epididymal structures. if severe, acute pain is shown during examination, 208 the primary differential diagnosis is torsion of the spermatic cord. if the scrotum itself is 209 swollen, the examiner then must determine whether it is an intratesticular enlargement or 210 an extratesticular disorder. ultrasonography would assist in differentiation of the 211 location and tissues involved.31 a swollen scrotum should be distinguished from swollen 212 testes.32 symmetrical scrotal enlargement occurs in orchitis, hydrocele, and torsion of the 213 spermatic cord. asymmetrical scrotal shape results from neoplasia, varicocele, 214 epididymitis, abscess, or hematoma. an ultrasound-guided cystocentesis can be 215 performed and urine submitted for cytology and culture. 216 in all cases of orchioepididymitis, a screening test for brucella canis should be 217 submitted with other samples for clinical pathology. a caution should be issued at that 218 time to client and clinic regarding minimization of potential for human exposure to this 219 zoonotic disease until the results are confirmed. 220 for the breeding soundness evaluation, testicular measurements with a caliper 221 document any change in size or shape. the semen collection should be fractionated into 222 separate samples for determination of sperm motility, morphology, concentration, 223 cytology, culture and sensitivity, and ph. sperm can also be placed in extender for 224 computerized analysis. in azoospermic samples, an alkaline phosphatase value from 225 seminal plasma of the second fraction would confirm epididymal patency or blockage. in 226 cases of oligospermia or azoospermia, retrograde ejaculation of sperm into the urinary 227 68 bladder can be diagnosed by the presence of spermatozoa in urine obtained post-228 collection.9,23 a stained cytology slide of the third fraction containing ample bacteria 229 and greater than three to five neutrophils per high power field (100x) is diagnostic for 230 prostatitis. further imaging or follow-up evaluation is indicated. the semen evaluation 231 yields a decreased number of sperm and an increased amount of abnormal morphology 232 from increased agglutination. bacterial epididymitis causes sperm acrosomal 233 degeneration with subsequent loss of the plasma membrane. affected sperm cells cannot 234 penetrate the zona pellucida of the oocyte. infertility results.34 235 except during the active process of ejaculation and urination, seminal fluid 236 normally moves from the ductus deferens into the bladder.21 the prostatic fluid travels 237 cranially into the bladder by normal urethra pressure.35 therefore, bacteria from the 238 ductus can produce an ascending prostatic or urinary tract infection (uti). culture of a 239 urine sample obtained by cystocentesis would identify the causative bacteria, including 240 the opportunist, mycoplasma canis, if the specimen is cultured on appropriate media.13 241 careful manipulation of the scrotum and its contents is useful in the identification 242 of a puncture wound or laceration, a draining tract, a change in skin thickness, altered 243 sensitivity, differentiation of intratesticular or extratesticular origin, or the soft core of an 244 abscess as one sequela of orchioepididymitis. fibrosis and scrotal adhesions restrict 245 testicular mobility, and with degeneration, the consistency changes from soft, edematous 246 tissue to a firm, hard, smaller and fibrotic testis. 247 ultrasonography (us) provides a rapid, non-invasive imaging tool beneficial for 248 diagnosis, prognosis and a therapeutic plan.31,36 with us, abnormal testicular 249 architecture can be differentiated from the diffuse, hypoechoic pattern imaged in normal 250 69 testicular parenchyma.37 fluid in testicular cysts, a spermatocele, or an abscess can also 251 be detected.37 in humans, the most common diagnosis for a swollen, painful scrotum is 252 epididimytis, and it is most commonly diagnosed on ultrasound examination when 253 viewed as increased vascularity in a testis of otherwise normal architecture.38 when a 254 dog presents with an acute onset of scrotal pain and swelling, color flow or power 255 doppler us imaging can distinguish between the lack of arterial and venous perfusion 256 caused by torsion of the spermatic cord and the hyperemic blood flow from an infectious 257 agent.39,40 following an incident of testicular torsion, doppler can visualize disrupted 258 blood flow within the cord and altered echogenicity from testicular infarction. doppler 259 us is helpful in the localization of a lesion in either the tail of the epididymis, the 260 spermatic cord or tunics, or the testis proper. neoplasia, either leydig or sertoli cell 261 tumors, may have hypoechoic and hyperechoic areas that are usually well-defined.4 262 testicular tumors have an irregular, lobulated contour versus orchitis with a moderately 263 enlarged, oval shaped, smooth textured testicle.4,39 tumors of the epididymis are rare.41 264 however, documentation in human cases is more widely reported. 265 one author suggested using fine-needle aspiration (fna) from the caudae 266 epididymides to collect diagnostic samples under sterile conditions from infertile males 267 for cytology, histopathology, culture (aerobic, anaerobic, and mycoplasma).42 fine-268 needle aspiration has been used to confirm obstructive lesion or maturation arrest for 269 oligospermia and azoospermia.43 ultrasonography can select which patient would 270 benefit from this procedure and avoid inherent risk or immunological consequences.36 271 the accuracy and placement of the needle can be improved through ultrasound guidance 272 as well. while a fna sample for a cytology smear is presumptive for diseases such as 273 70 lymphocytic orchitis, a testicular slice biopsy will confirm the diagnosis. it may be 274 advisable to weigh the short-term benefit versus the long-term risk since adverse effects 275 may develop later. focal hemorrhage, interstitial fibrosis and tubular atrophy can occur 276 at a biopsy site, but adjacent tissue and semen quality may remain within normal 277 limits.44,45 with either method, there is risk for sperm granuloma formation or antisperm 278 antibody production as a result of ‘foreign’ protein leaking from the point of surgical 279 extraction through tunics, cavity, parenchyma and blood-testis barrier. one would 280 obviously not perform a testicular biopsy or risk anesthesia in the presence of 281 inflammation.2 282 prostatic disease can be verified by anamnesis, digital examination, culture and 283 sensitivity, cytology of the third fraction or prostatic wash, ultrasonography, and 284 ultrasound-guided aspirate or biopsy. 285 if an orchidectomy of a diseased testis is performed, samples obtained at surgery 286 should be cultured for aerobic bacteria, mycoplasma spp. and fungi, and tissues should be 287 sent for histopathology. 288 pathology 289 epididymitis is one of the more common inflammatory diseases of the 290 reproductive tract. the usual route of entry for pathogens into the epididymis is via an 291 ascending or retrograde mechanism. trauma to the scrotum combined with endotoxin-292 producing bacteria such e. coli results in septicemia. the epididymis lacks a natural 293 local immune system. lymphocytes or plasma cells are recruited after the initial insult 294 from infection occurs. edema progresses to an abscess or sperm granuloma formation. 295 fibrous bands appear between the tunics, which are extensions of the peritoneal cavity,3 296 71 and the epididymis. in time, these adhesions lead to testicular atrophy and firm, nodular 297 ducts. scrotal swelling is caused by the accumulation of fibrinopurulent exudate in the 298 tunic cavity. this cellular response includes lymphocytes, neutrophils and macrophages. 299 extensive luminal fibrosis and spermatocele can occlude the ducts even after successful 300 treatment and decreased signs of inflammation. disruption of the blood-testis barrier 301 initiates an immune-mediated reaction and results in lymphocytic infiltration. 302 inflammation of the seminiferous tubules causes degeneration. the inflammatory 303 response within the testicular parenchyma is suppurative, with abscessation and possible 304 fistula formation to the exterior scrotum.17 lesions in a cat with orchitis were consistent 305 with vasculitis, increased fibrinogen and infarction from simultaneous viral infections.15 306 multifocal necrotic granulomatous inflammation of the pleura, lung, eye and lip were 307 found. 308 treatment 309 similar treatment protocols are suggested for both orchitis and/or epididymitis. 310 once the cause is known, an appropriate regimen can be instituted. antimicrobial 311 therapy is started based upon results of the culture and sensitivity. a minimum duration 312 of treatment is three to four weeks. concurrent sexual rest is implied. another culture 313 should be obtained one to two weeks after the antibiotic has been discontinued. 314 resolution of epididymitis requires the correct antimicrobial choice, dosage and length of 315 administration. too often, failure of client compliance alters the outcome when treatment 316 is stopped prematurely. within days, owners may supplement with their choice of 317 medication in addition to or in place of the one prescribed. immunosuppressive drugs 318 have been used on a short-term basis to treat immune-mediated disease. however, 319 72 continued use of immuosuppressive agents may adversely affect spermatogenesis and 320 lead to infertility. 321 owners should be apprised of possible outcomes and expense. serial monitoring 322 can document changes in semen parameters and allow for alteration of therapy if 323 necessary. brucellosis management requires immediate quarantine and testing of dogs 324 having had contact with the confirmed case. positive dogs are removed from the 325 premises and are either euthanized or retested until two negative results have been 326 obtained. antibiotics will decrease bacteremic signs and neutering does eliminate the 327 target organs, but the chance for relapse is probable. since brucellosis is zoonotic, 328 exposure to pregnant women, children and immunosuppressed people, in particular, 329 should be avoided. treatment for unilateral epididymitis offers a better recovery rate; 330 bilateral infection has a poorer prognosis for a return to satisfactory reproductive 331 performance.23 332 the treatment of choice may be surgical excision of the infected tissue. relief is 333 immediate and the potential for fertility may be salvaged. hemicastration or 334 orchidectomy of the diseased testis not only removes the source of inflammation or 335 benign neoplasia but also decreases the potential for thermal insult on the unaffected, 336 contralateral testis. following unilateral orchidectomy, an immediate reduction in sperm 337 output was followed by increased spermatogenesis and compensatory hypertrophy by the 338 remaining testis.46 if the preliminary histopathology confirms malignancy or an 339 irreversible condition, complete castration may be recommended for the animal’s 340 welfare. if the cause can be reversed, then the male may recoup fertility. if the primary 341 problem is torsion of the spermatic cord, an infectious disease or immune-mediated 342 73 disorder which resulted in secondary damage, any resolution of pre-existing 343 orchioepididymitis may be irrelevant. the progressive fibrosis within the tubular 344 compartments would gradually prevent spermatogenesis and steroidogenesis. an 345 epididymotomy has been suggested as a treatment for bacterial epididymitis in men.47 346 prevention 347 to control exposure and transmission of disease, screen new arrivals to a kennel, 348 test animals at least annually for brucella canis, disinfect facilities regularly especially 349 the whelping environment, reduce fomite contamination during breeding, and employ 350 artificial insemination when possible. judicious use of antibiotics in the female during 351 her estrous cycle and breeding is important to avoid bacterial resistance and birth defects. 352 good management includes periodic monitoring for urinary tract infections and early 353 detection of subfertility or infertility, followed by an accurate diagnosis and prompt 354 treatment. 355 references 356 1. barsanti ja: genitourinary infections. in: greene ce, editor. infectious diseases 357 of the dog and cat. 3rd ed. philadelphia: wb saunders; 2006. p. 954-955. 358 2 feldman ec, nelson rw: disorders of the testes and epididymides. in: feldman 359 ec, nelson rw, editors. canine and feline endocrinology and reproduction. 3rd 360 edition. philadelphia: wb saunders; 2004. p. 971-974. 361 3. lein dh: canine orchitis. in: kirk rw, editor. current veterinary therapy vi. 362 philadelphia: wb saunders philadelphia; 1977. p. 1255-1259. 363 4. ober cp, spaulding k, breitschwerdt eb, et al: orchitis in two dogs with rocky 364 mountain spotted fever. vet radiol ultrasound 2004;45:458-465. 365 74 5. evans he, christensen gc: the reproductive organs. in: evans he, editor. 366 miller’s anatomy of the dog. philadelphia: wb saunders; 1993. p. 504-514. 367 6. soderberg sf: infertility in the male dog. in: morrow da, editor. current therapy 368 in theriogenology. philadelphia: wb saunders; 1986. p. 544-548. 369 7. hollett rb: canine brucellosis: outbreak and compliance. theriogenology 370 2006;66:575-587. 371 8. love r, hemphill j jr, cooper ms, et al: epididymitis in a dog caused by 372 brucella abortus, and treatment with aureomycin. cornell vet 1952;42:37-41. 373 9. olson pn: clinical approach for evaluating dogs with azoospermia or aspermia. 374 vet clin north am small anim pract 1991;21:591-608. 375 10. dahlbom m, andersson m: long term effects of canine distemper. advances in 376 reproduction in dogs, cats, and exotic carnivores: proceedings of the fourth 377 international symposium on canine and feline reproduction. cambridge: journal 378 of reproduction and fertility; 2001. p. 94. 379 11. kadota k, uchida k, nagatomo t, et al: granulomatous epididymitis related to 380 rhodotorula glutinis infection in a dog. vet pathol 1995;32:716-718. 381 12. pyle rl: canine blastomycosis. compend contin educ pract vet 1981;3:963-382 974. 383 13. l’abee-lund tm, heiene r, fruiis nf, et al: mycoplasma canis and urogenital 384 disease in dogs in norway. vet rec 2003;153:231-235. 385 14. johnstone i: breeding difficulties with a stud dog. aust vet j 1985;62:65. 386 15. foster ra, caswell jl, rinkardt n: chronic fibrinous and necrotic orchitis in a 387 cat. can vet j 1996;37:681-682. 388 75 16. johnston sd, root kustritz mv, olsen pns. canine and feline theriogenology. 389 philadelphia: wb saunders philadelphia; 2001. p. 319, 532. 390 17. jubb kvf, kennedy pc, palmer n: male genital system. in: pathology of 391 domestic animals, 3rd ed. new york: academic press; 1985. p. 565-588. 392 18. stein bs: the genital system. in: catlott ej, editor. feline medicine and surgery, 393 2nd ed. santa barbara: american veterinary publications, inc; 1975. p. 303-354. 394 19. memon ma, sirinarumitr k: semen evaluation, canine male infertility, and 395 common disorders of the male. in: ettinger sj, feldman ec, editors. textbook of 396 veterinary internal medicine, 6th ed. philadelphia: wb saunders elsevier; 2005. 397 p. 1693, 1704. 398 20. comhaire fh, de kretser d, farley tmm, et al. towards more objectivity in 399 diagnosis and management of male infertility. world health organization task 400 force on the diagnosis and treatment of infertility. int j androl (suppl) 401 1987;7:1-53. 402 21. amann rp: structure and function of the normal testis and epididymis. j am coll 403 toxicol 1989;8:457-471. 404 22. fritz te, lombard ls, tyler sa, et al: pathology and familial incidence of 405 orchitis and its relation to thyroiditis in a closed beagle colony. exp mol pathol 406 1976;24:142-158. 407 23. meyers-wallen vn: clinical approach to infertile male dogs with sperm in the 408 ejaculate. vet clin north am small anim pract 1991;21:609-633. 409 76 24. johnson ca: disorders of the canine testicles and epididymides. in: morrow da, 410 editor. current therapy in theriogenology. philadelphia: wb saunders; 1986. p. 411 551-552. 412 25. fijak m, meinhardt a: the testis in immune privilege. immunol rev 413 2006;213:66-81. 414 26. schuppe hc, meinhardt a: immune privilege and inflammation of the testis. 415 chem immunol allergy 2005;88:1-14. 416 27. hedger mp, meinhardt a: cytokines and the immune-testicular axis. j reprod 417 immunol 2003;58:1-26. 418 28. hedger mp: macrophages and the immune responsiveness of the testis. j reprod 419 immunol 2002;57:19-34. 420 29. larsen re: evaluation of fertility problems in the male dog. vet clin north am 421 small anim pract 1977;7:735-745. 422 30. kravis em, lorber jh: feminization syndrome associated with epididymitis in a 423 dog. j am vet med assoc 1962;140:803-806. 424 31. johnston gr, feeney da, rivers b, et al: diagnostic imaging of the male canine 425 reproductive organs.vet clin north am small anim pract1991;21:591-608. 426 32. pinto crf, paccamonti dl, partington b: theriogneology question of the month. 427 j am vet med assoc 2001;219:1343-1345. 428 33. root mv, johnston sd, olson pn: concurrent retrograde ejaculation and 429 hypothyroidism in a dog: case report. theriogenology 1994;41:593-600. 430 34. ellington j, meyers-wallen v, suess r, et al: unilateral bacterial epididymitis in 431 the dog. j am anim hosp assoc 1993;29:315-319. 432 77 35. barsanti ja, finco dr: canine prostatic diseases. in: morrow da, editor. 433 current therapy in theriogenology. philadelphia: wb saunders; 1986. p. 553-560. 434 36. pugh cr, konde lj: sonographic evaluation of canine testicular and scrotal 435 abnormalities: a review of 26 case histories. vet radiol 1991;32:243-250. 436 37. rifkin md, kurtz ab: ultrasound of the prostate. in: sander rc, hill m, editors. 437 ultrasound annual 1983. new york: raven press; 1983. p. 85. 438 38. oyen rh. scrotal ultrasound. eur radiol 2002;12:19-34. 439 39. subramanyam br, horii sc, hilton s: diffuse testicular disease: sonographic 440 features and significance. am j roentgenol 1985;145:1221-1224. 441 40. ringdahl e, teague l: testicular torsion. am fam physician 2006;74;1739-442 1743. 443 41. rifkin md, kurtz ab, goldberg bb: epididymis examined by ultrasound. 444 radiology 1984; 151:187-190. 445 42. dahlbom m, makinen a, suominen j: testicular fine needle aspiration cytology 446 as a diagnostic tool in dog infertility. j sm anim pract 1997;38:506-512. 447 43. romagnoli s, bonaccini p, stelleta c, et al: clinical use of testicular fine needle 448 aspiration (fna) cytology in oligozoospermic and azoospermic dogs, 449 proceedings of the 6th international symposium n canine and feline 450 reproduciton; 2008. 451 44. james rw, heywood r, fowler dj: serial percutaneous testicular biopsy in the 452 beagle dog. j sm anim pract 1979;20:219-228. 453 45. buergelt cd: diseases of the male reproductive tract. in: color atlas of 454 reproductive pathology of domestic animals. st. louis: mosby; 1997. p. 40-41. 455 78 46. gunzel-apel a-r, heilkenbrinker t, heilkenbrinker m, et al: reproductive status 456 of the dog after unilateral orchidectomy or unilateral abdominal testis reposition. j 457 reprod fertil (suppl) 1989;39:328-329. 458 47. witherington r, harper wm: the surgical management of acute bacterial 459 epididymitis with emphasis on epididymotomy. j urol 1982;128:722-725. 460 79 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy 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/colorimageminresolution 300 /colorimageminresolutionpolicy /ok /downsamplecolorimages false /colorimagedownsampletype /average /colorimageresolution 300 /colorimagedepth -1 /colorimagemindownsampledepth 1 /colorimagedownsamplethreshold 1.50000 /encodecolorimages false /colorimagefilter /dctencode /autofiltercolorimages true /colorimageautofilterstrategy /jpeg /coloracsimagedict << /qfactor 0.76 /hsamples [2 1 1 2] /vsamples [2 1 1 2] >> /colorimagedict << /qfactor 0.15 /hsamples [1 1 1 1] /vsamples [1 1 1 1] >> /jpeg2000coloracsimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /jpeg2000colorimagedict << /tilewidth 256 /tileheight 256 /quality 30 >> /antialiasgrayimages false /cropgrayimages false /grayimageminresolution 300 /grayimageminresolutionpolicy /ok /downsamplegrayimages false /grayimagedownsampletype /average /grayimageresolution 300 /grayimagedepth -1 /grayimagemindownsampledepth 2 /grayimagedownsamplethreshold 1.50000 /encodegrayimages false /grayimagefilter 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0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname () /pdfxtrapped /false /createjdffile false /description << /enu ([based on 'no compression'] [based on 'no compression wbleeed'] [based on '[high quality print]'] use these settings to create adobe pdf documents for quality printing on desktop printers and proofers. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes false /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: influence of delivery method on neonatal canine viability parameters influence of delivery method on neonatal canine viability parameters b.b. beall, m. l. casal school of veterinary medicine, university of pennsylvania, philadelphia, pa, usa while many important clinical parameters are used to asses viability, clinical outcome and pathology have been well-established in human neonates, foals, calves, and piglets; they are virtually unknown during the immediate postpartum period in the puppy. the aim of this study was to develop a database on neonatal puppies delivered via vaginal delivery, elective cesarean section, and emergency cesarean section and correlate the values with subjective assessment of viability. preliminary studies involved 9 puppies from three litters that were born by natural vaginal delivery (group a) and 3 puppies from one litter that were delivered by emergency cesarean section (group b). hematology samples were collected via venipuncture of the jugular vein. all samples were collected within 26 minutes of birth. significant differences were noted between group a and b in serum glucose concentrations (111±38 vs 54±17 mg/dl), blood urea nitrogen (22.8±5.1 vs 8.0±7.1 mg/dl), and po2 (34.1±12.0 vs 53.7±18.2 mmhg). however, all values were within normal adult ranges. there were no significant differences between the rest of the measured values but the following trends were observed: apgar scores and doppler blood pressures were higher in group a than b, while pco2 and so2 were higher in group b than a. the ranges in serum glucose, sodium, chloride, lactate, hco3, base excess, and so2 were much wider in group a than b while blood pressure ranges were wider in group b. lactate concentrations were higher, while ph and hco3 levels were lower, in all puppies when compared to normal adults, indicating lactic acidosis and tissue hypoxia. there was no difference in the degree of lactic acidosis between groups a and b and base excess was lower in all puppies. interestingly, there was no correlation between lower apgar scores and the degree of acidosis, blood glucose levels or any other parameter measured. in conclusion, significant differences in serum glucose, blood urea nitrogen and po2 exist between puppies delivered via natural vaginal delivery and via emergency cesarean section. furthermore, these results suggest that significant lactic acidosis is present in puppies immediately after birth, but that there is no significant difference in degree of acidosis 531 between puppies delivered via natural vaginal delivery and emergency cesarean section. however, the presence of acidosis in all puppies did not appear to impact viability as all puppies thrived past the neonatal period. further studies will aim to expand the number of puppies assessed in groups a and b, as well as include puppies from another group, planned cesarean section, and examine how long after birth lactic acidosis is present. keywords: pediatrics, neonatal, dog, hypoxia, cesarean section 532 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true 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>> /antialiasmonoimages false /cropmonoimages false /monoimageminresolution 1200 /monoimageminresolutionpolicy /ok /downsamplemonoimages false /monoimagedownsampletype /average /monoimageresolution 300 /monoimagedepth -1 /monoimagedownsamplethreshold 1.50000 /encodemonoimages false /monoimagefilter /flateencode /monoimagedict << /k -1 >> /allowpsxobjects false /checkcompliance [ /none ] /pdfx1acheck false /pdfx3check false /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxtrimboxtomediaboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxsetbleedboxtomediabox true /pdfxbleedboxtotrimboxoffset [ 0.00000 0.00000 0.00000 0.00000 ] /pdfxoutputintentprofile () /pdfxoutputconditionidentifier () /pdfxoutputcondition () /pdfxregistryname () /pdfxtrapped /false /createjdffile false /description << /enu ([based on 'no compression'] [based on 'no compression wbleeed'] [based on '[high quality print]'] use these settings to create adobe pdf documents for quality printing on desktop printers and proofers. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes false /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: cystic ovarian disease in dairy cattle cystic ovarian disease in dairy cattle 1 j. d. smith 2 department of pathobiology and population medicine, college of veterinary medicine, 3 mississippi state university, mississippi state, ms, usa 4 5 abstract 6 practitioners involved in the delivery of veterinary care and who provide 7 consultation to dairy cattle owners must be able to effectively diagnose and treat common 8 conditions which impact the economic stability of the dairy enterprise. this manuscript 9 provides the practitioner with the information to understand the pathophysiology of 10 cystic ovarian disease, choose an appropriate treatment and suggest possible management 11 changes to minimize the economic impact of this disorder. 12 keywords: cystic ovarian disease; anestrus; infertility; gnrh; cattle 13 introduction 14 cystic ovarian disease (cod) has been recognized as a frequent cause of 15 subfertility and poor reproductive efficiency in cattle for almost 100 years and is still 16 considered to be one of the most economically important reproductive conditions 17 affecting dairy cattle worldwide. the major causes of economic loss are due to increased 18 days open in the postpartum period which extends the calving interval, costs associated 19 with treatment and higher culling rates in affected animals.1,2 cystic ovarian disease has 20 also been shown to decrease the pregnancy rate to subsequent ai which leads to an 21 increase in services per conception resulting in increased semen costs.3 22 terms which have been used to describe the condition of persistent anovulation of 23 preovulatory follicles include: cystic ovarian disease, cystic ovarian follicles, cystic 24 ovarian degeneration and cystic ovaries. the most common term used in the literature to 25 describe this condition is cystic ovarian disease. as we understand more about this 26 253 condition and management practices have changed we should revisit the use of the term 27 cystic ovarian disease. the most recent term used to describe this ovarian dysfunction is 28 cystic ovarian follicles (cof).1,3 since “cysts” are often diagnosed in the absence of any 29 obvious clinical signs the term “disease” should likely be replaced by “follicles” as it 30 more accurately describes the condition.3 31 definition 32 the classical definition of cod in cattle is the presence of an anovulatory 33 structure on the ovary which is > 2.5cm in diameter and has persisted for at least 10 days 34 in the absence of a corpus luteum (cl).4 as more knowledge is gained regarding cod 35 the previous definition requires refinement. the size limit of 2.5cm is arbitrary and 36 would exclude cystic follicles which are smaller than 2.5cm. the dominant follicle of 37 dairy cattle typically ovulates on average at a size of 1.6-1.9cm.3 the classic definition 38 that requires presence for 10 days should also be questioned due to the fact that cystic 39 ovarian follicles have been shown to be dynamic structures which change over the course 40 of time. also cows diagnosed with cod are generally not palpated again in 10 days to 41 totally fulfill the classical definition. the necessity for the absence of a cl is also not 42 universally fulfilled. cysts which are non-steroidogenic and thus hormonally inactive 43 may not influence the estrous cycle and thus could be found in the presence of a cl. 44 most cows today however are diagnosed with cod on the basis of a single rectal 45 palpation or ultrasound examination and no attempt is made to assure the structure has 46 been present for 10 days in the absence of a cl. 47 a recent term and definition put forth in the literature to more accurately describe 48 this condition is cystic ovarian follicles. cystic ovarian follicles are defined as follicles 49 254 with a diameter of at least 2 cm that are present on one or both ovaries in the absence of 50 any active luteal tissue and that clearly interferes with normal ovarian cyclicity.3 this 51 definition more clearly defines the condition in relation to our current understanding and 52 its impact on reproduction. 53 cysts are further classified as being follicular cysts or luteal cysts depending on 54 the degree of lutenization and the level of progesterone secretion. both are considered to 55 be different forms of the same condition with luteal cysts being a follicular cyst which 56 has undergone some lutenization.2 follicular cysts do not secrete progesterone whereas 57 luteal cysts secrete varying amounts of progesterone however an absolute threshold has 58 not been determined.3 the ability to accurately classify each cyst is subject to personal 59 interpretation based on clinical as well as laboratory findings. 60 ultrasound can be a very useful tool with which to gather information regarding 61 the classification of cysts. follicular cysts typically have a thin wall (≤ 3 mm) whereas 62 luteal cysts typically have a thicker wall (≥ 3 mm). the follicular fluid is often 63 hypoechoic in follicular cysts whereas in luteal cysts it may contain echogenic strands 64 creating a web-like appearance.5 65 incidence 66 the incidence of cod in dairy cattle varies amongst several studies but is 67 typically between 5-19% with mean of 10-12%.2 the incidence of cod could likely be 68 even higher based on the findings that as many as 60% of cows that develop cod 69 recover spontaneously prior to their first postpartum ovulation and could easily remain 70 undiagnosed. the majority of cod is diagnosed by routine rectal palpations during the 71 first 60 days postpartum at which time cows are being examined prior to breeding. cases 72 255 are also commonly diagnosed between 120-210 days postpartum. these cases are 73 typically diagnosed in cows which have been presented for examination after extended 74 periods of anestrus. 75 there is a genetic predisposition for cod in dairy cattle, however the heritability 76 is low at 0.07 to 0.12.6,7 cystic ovarian disease seems to occur more often in certain cow 77 families. genetic selection attempting to remove sires who produced daughters that 78 developed cod from the breeding pool has been shown to significantly reduce the 79 incidence of cod in swedish herds.3 reduction in the incidence from 10% to 3% was 80 achieved by selection against sires that produced daughters with cod.8 genetic 81 selection as a prevention for cod will be a lengthy endeavor due to the low heritability 82 but can be effective. 83 with routine use of synchronization programs (i.e., pre-sync, ovsync) during the 84 voluntary waiting period to synchronize the first postpartum ai the incidence of cod as 85 diagnosed by rectal palpation could likely be lower than previous studies have identified. 86 however, as the modern dairy cow is under tremendous dietary and production stressors 87 which can predispose her to cod the apparent affect of these programs on clinical 88 incidence may be modulated. 89 clinical signs 90 behavioral signs seen in cows with cod are variable but can generally be 91 classified into two groups, anestrus and nymphomania. the most common clinical sign 92 observed in cows with cod is anestrus, this is especially evident in the early postpartum 93 period. approximately 80% of cows that develop cod early in the postpartum period 94 exhibited anestrus.4 these cows are often presented for examination after failure of the 95 256 herdsman to detect normal postpartum cycling activity. nymphomania is yet another 96 clinical sign which can be seen in cows with cod. these cows often attempt to ride 97 other cows but generally will not stand for mating themselves.4 approximately 10% of 98 cows affected with cod show signs of nymphomania. it appears that as the number of 99 days following calving at which cod is diagnosed increases the likelihood of 100 nymphomania being a clinical observation also increases.4 irregular estrous cycles can 101 also frequently occur in cows with cod which often leads to inappropriate breeding of 102 these cows based on poor or weak signs of estrus. 103 pathogenesis 104 a dysfunction in the normal hypothalamic-pituitary-gonadal axis leading to 105 ovulation failure is the most common accepted mechanism of cod.1,2,9-11 the precise 106 mechanisms leading to the aforementioned dysfunction have yet to be fully elucidated. it 107 is believed that there is a multi-factorial cause with genetic, phenotypic, environmental 108 and management factors involved.3 109 the most widely accepted hypothesis involves the altered release of luteinizing 110 hormone (lh) from the pituitary gland. the pre-ovulatory surge of lh is either absent, 111 insufficient in magnitude or is improperly timed whereas the dominant follicle does not 112 ovulate leading to cyst formation.1-3 there does not appear to be a reduction in gnrh 113 content in the hypothalamus or a reduction in gnrh receptors in the pituitary.3 114 luteinizing hormone content in the pituitary also does not appear to be reduced in cows 115 with cod.3,12 normally pre-ovulatory follicles secrete estrogen which has a positive 116 feedback on the hypothalamic-pituitary axis causing release of lh which is responsible 117 for the subsequent ovulation. there appears to be a lack of responsiveness of the 118 257 hypothalamus to the positive feedback mechanism of estrogen leading to the altered 119 release of gnrh and/or subsequently lh causing the anovulatory state of cod. 120 predisposing factors 121 numerous factors have been associated with the development of cod in cows. 122 this condition appears to more commonly affect high producing dairy cows in their 123 second through fifth lactation. early in lactation when the cow is often in a negative 124 energy balance metabolic disturbances are more common and are often followed by 125 cod. there appears to be a higher incidence of cod during winter months, however 126 photoperiod does not appear to have an effect on the hypothalamic-pituitary-ovarian 127 (hpo) axis. other factors which have been associated with depression of gnrh/lh 128 release and subsequent cyst formation include uterine infections, retained fetal 129 membranes, lameness and stress.3,11 postpartum uterine infections are thought to 130 stimulate cortisol secretion which can suppress the pre-ovulatory surge of lh leading to 131 anovulation and subsequent cyst formation.1 the associated endotoxins and 132 inflammatory mediators can disrupt the normal hormonal pathways that ultimately 133 control ovarian function including ovulation.13 the role of stress in cod is believed to 134 be related to the release of cortisol which appears to block the estrogen induced lh 135 surge.1,10,14 136 diagnosis 137 the diagnosis of cod has historically been made based on the finding during 138 rectal examination of the cow along with her reproductive history. however the 139 collective findings of a rectal examination, an ultrasonographic evaluation of the 140 reproductive tract including the ovaries, progesterone concentrations in blood or milk and 141 258 behavioral abnormalities will allow for a more accurate diagnosis. the accuracy with 142 which a skilled palpator can identify the type of cysts based on palpation alone is 143 relatively poor.15 the dynamic nature of both cysts and developing corpora lutea can 144 complicate the diagnosis when palpation alone is used. a study by farin et al., showed 145 10% of cows that were diagnosed as having cysts based on rectal examination were found 146 to have a structure consistent with a normal corpus luteum by transrectal ultrasound 147 examination.16 in one study evaluating the use of ultrasound the accuracy of a correct 148 diagnosis being made was 74% of follicular cysts and almost 90% of luteal cysts.17 149 progesterone concentrations have been shown to correlate very well with cyst wall 150 thickness with 3 mm being the threshold between follicular and luteal cysts.1,18 when 151 one combines progesterone concentrations in addition to rectal examination and 152 ultrasound findings the accuracy of diagnosis of the cyst type approaches 100% however 153 this is rarely done outside of research settings. although using progesterone testing to 154 accurately determine the cyst type would aid in treatment decisions it is rarely used in 155 practice situations due to the economic considerations. 156 treatment options 157 probably the oldest treatment of cod in cattle is manual rupture of the cyst via 158 rectal palpation. with the advances in our understanding of cod and the availability of 159 effective medical options this treatment can no longer be recommended. the possibility 160 of oviductal or ovarian bursal adhesions arising secondary to the trauma associated with 161 manual rupture and their affects on subsequent fertility are too great to ignore.4 162 hormone therapy aimed at either causing (gnrh) or mimicking (human 163 chorionic gonadotropin; hcg) an lh surge can be used to treat follicular cysts. of these 164 259 two, gnrh is generally chosen first due to its small molecular size which reduces the 165 likelihood of an immune reaction.1,10 after an injection of gnrh a surge of lh from the 166 pituitary occurs within 2 hours.19 this lh surge can cause lutenization of follicular 167 cysts which will undergo spontaneous luteolysis in about 18 days at which time a normal 168 estrous cycle begins. another possibility following gnrh treatment in cows with 169 follicular cysts is ovulation of a dominant follicle followed by a subsequent normal luteal 170 phase. since cystic cows continue to have follicular waves, the response to gnrh is 171 likely due to ovulation of a dominant follicle present with recruitment of a new cohort of 172 follicles rather than lutenization or regression of the cysts.20 the subsequent increase in 173 progesterone concentrations causes the re-setting of the normal hpo axis and resumption 174 of normal cyclicity in most cows. in one study that evaluated the effectiveness of a 175 single injection of gnrh for treatment of cows with ovarian cysts, 72% of cows resumed 176 normal cycling within 20 days of treatment compared to 16% of control cows.19 177 however other studies have not borne out the same results. a study that evaluated the 178 effectiveness of gnrh as a sole treatment for follicular cysts showed no difference in 179 treated animals versus controls. the lack of agreement between numerous studies 180 evaluating the efficacy of gnrh is likely due to the lack of control animals and the 181 number of cows which recover spontaneously. there was no difference in the period of 182 time between treatment with gnrh and resolution of the cyst or in the period of time 183 until a cl was evident.21 this study brings to light the high incidence of spontaneous 184 recovery and somewhat brings into question the effectiveness of gnrh alone as a sole 185 treatment for cows with cod. timing of treatment in the postpartum period does not 186 appear to affect treatment response. in one study, cystic cows were treated with gnrh 187 260 either before or after 60 days following calving. there was no difference in treatment 188 response in the two groups however there were no control animals with which to 189 compare.22 in accurately diagnosed cases a relatively large percentage of cows return to 190 cyclicity following an injection of gnrh, however some of this response could be 191 attributed to spontaneous resolution. 192 a recent pharmacokinetic study attempted to find the optimal dose of gnrh for 193 treatment of cows with cod. the dose of gnrh which was found to guarantee 194 production of a critical maximum plasma lh concentration of 5 ng/ml was 74 ug of 195 gnrh.23 therefore the standard 100 ug dose of gnrh used to treat cows with cod was 196 found to generate a lh concentration of 5.86 ng/ml and should be adequate in most cases 197 of cod. 23 198 human chorionic gonadotropin has been used successfully to treat refractory 199 follicular cysts that fail to respond to gnrh. it has lh-like properties and causes the 200 cyst to lutenize and begin producing progesterone. once the cyst has lutenized it can 201 then be treated with prostaglandin to restore the normal cyclical pattern. its use is often 202 relegated to cases in which gnrh has failed to render a cure. its use has occasionally 203 been noted to stimulate an immune reaction however the importance of this reaction is 204 poorly understood.1 205 prostaglandin is the treatment of choice for luteal cysts and cysts that have 206 undergone lutenization after being treated with gnrh or hcg.1,2,10,20 prostaglandin has 207 no effect on follicular cysts so it is important to accurately diagnose the type of cyst 208 before using prostaglandin alone. after prostaglandin administration luteal cysts regress 209 with estrus occurring in 90% of cows by day 8 post treatment. prostglandin is commonly 210 261 used in the treatment of cysts after a previous injection of gnrh as part of an ovsync 211 protocol. 212 protocols involving a series of hormonal injections aimed at treating the cysts and 213 restoring the cow to normal cyclicity have been proposed.1,11,24-27 the classical ovsync 214 protocol has been employed as a treatment for cysts irrespective of their type. the 215 rational in using an ovsync protocol is to both treat the cyst and eliminate estrus 216 detection and breed the cows with timed ai.11,24-27 progesterone levels in cows with 217 cod which are treated with gnrh are elevated five days after treatment and therefore 218 could be treated with traditional ovsync with good results.24 when cows with cod are 219 subjected to the ovsync protocol pregnancy rates to subsequent timed insemination has 220 range from 17-25%.25,28 221 use of progesterone as a treatment for cows with cod has been proposed for 222 over 40 years.29 now that progesterone impregnated vaginal pessaries have been 223 approved for use in lactating dairy cattle in the united states recent emphasis has been 224 placed on their use in the treatment of cod. progesterone administration has been 225 shown to re-establish the normal feedback mechanisms involving the hpo axis and allow 226 cows with cod to resume normal cyclicity. the duration of progesterone treatment 227 which is sufficient to re-establish normal hypothalamic responsiveness to estradiol 228 appears to be as short as three days.30 there was no difference in the pregnancy rates of 229 dairy cattle with cod when treated with either the ovsync protocol or use of a 230 progesterone-releasing pessary for seven days with prostaglandin administration at the 231 time of pessary removal followed by breeding after heat detection suggesting that use of 232 intravaginal progesterone as a treatment for cod can be effective.27 use of 233 262 progesterone-releasing pessaries in combination with the ovsync protocol has been 234 studied as a treatment for cysts as well. results showed an increase in pregnancy rates in 235 cystic cows treated with ovsync plus progesterone (37.5% pregnancy rate) compared to 236 ovsync alone (16.7% pregnancy rate).26 237 in a recent study, the effectiveness of the opioid antagonist naloxone as a 238 treatment for cows with cod was examined.31 it has been shown that stress may be a 239 contributor to the pathogenesis of cod in cattle. endogenous opioid peptides are 240 involved in many responses to stress including the regulation of various endocrine 241 systems.31 endogenous opioid peptides are believed to block the release of gnrh from 242 the hypothalamus as well as the estrogen-induced lh surge.14 it has been shown that 243 administration of the opioid antagonist naloxone results in elevated lh release in cattle 244 under various physiological states.32 cows diagnosed with cod were treated with 245 naloxone as well as the gnrh agonist buserelin. in this study 77.5% of treated cows had 246 begun cystic regression as viewed with ultrasonography or had begun cycling normally 247 within two weeks post-treatment.31 further investigation of the use of naloxone is 248 warranted and specifically its use in the absence of gnrh products which confound the 249 interpretation of this particular study. 250 conclusion 251 cystic ovarian disease remains an important postpartum condition affecting dairy 252 cattle with a substantial economic impact on the modern dairy farm. although much 253 effort and research has been placed on elucidating the precise mechanism(s) leading to 254 cod its exact cause remains unclear. currently the most effective treatments for cod 255 appear to be those which are capable of resetting the hpo axis thereby re-establishing 256 263 normal feedback mechanisms which results in normal cyclical patterns. both the ovsync 257 protocol, the use of intravaginal progesterone, or the combination of the two appear to be 258 the most practical and effective treatments for most modern dairy operations. due to the 259 fact of a high incidence of spontaneous recovery in cows with cod it has made it 260 difficult to interpret apparent response to various therapies. further research into the 261 cellular and molecular events that are occurring in cows with cod and the interactions of 262 various stressors will hopefully provide us more answers to this very common condition. 263 references 264 1. peter at: an update on cystic ovarian degeneration in cattle. reprod dom anim 265 2004;39:1-7. 266 2. garverick ha: ovarian follicular cysts in dairy cattle. j dairy sci 1997;80:995-267 1004. 268 3. vanholder t, opsomer g, kruif a: aetiology and pathogenesis of cystic ovarian 269 follicles in dairy cattle: a review. reprod nutr dev 2006;46:105-119. 270 4. roberts sj: veterinary obstetrics and genital diseases (theriogenology), 2nd ed. 271 ithaca, ny: published by the author; 1971. p. 421-432. 272 5. jeffcoate ia, ayliffe tr: an ultrasonographic study of bovine cystic ovarian 273 disease and its treatment. vet rec 1995;136:406-410. 274 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naloxone in high production dairy cows. j vet pharmocol ther 2008;31:184-347 186. 348 32. byerley dj, kiser te, bedrand jk, et al: release of lutenizing hormone after 349 administration of naloxone in preand peripubertal heifers. j anim sci 350 1992;70:2794-2800. 351 352 353 268 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage 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reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes false /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1 contact jenny sones jenny.sones@colostate.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10795, http://dx.doi.org/10.58292/ct.v16.10795 review report pregnancy monitoring and parturition in dogs patricio razquin, jenny sones department of clinical sciences, college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co, usa abstract management of term pregnant dogs is a major and frequent clinical situation in small animal reproductive veterinary practices, and can be challenging. whether a pregnancy is planned or unplanned, these cases require careful monitoring to prevent maternal distress and neonatal loss. reproductive system evaluation of female dogs is useful for determination of ovulation during breeding management, pregnancy diagnosis, and for timing of cesarean surgery, among other clinical scenarios. basic understanding of female dog’s reproductive physiology is mandatory to interpret relevant hormonal assays and other complementary diagnostics such as vaginal cytological evaluation and imaging (ultrasonography and radiography) for clinical decisions. this review covers physiology, diagnostics, and clinical outcomes relevant for pregnancy monitoring and parturition. keywords: canine pregnancy, whelping, cesarean surgery, dystocia, neonate introduction* canine reproduction management is commonly practiced by many theriogenologists and general practitioners. knowledge of female dog’s estrous cycle and pregnancy is important for clinical decisions regarding timing of parturition and plans for caesarean surgery. additionally, canine pregnancy has a well-defined length, and neonates cannot survive if delivered 2 days before or after due date. thus, attending veterinarians making decision for elective caesarean surgery must be confident that fetuses are mature enough to support life outside the uterus. to best determine when a dog is ready to whelp, veterinarians must acquire as much history as possible regarding the dog’s estrous cycle and breeding management. the following are important questions: 1. was stage of estrus determined via vaginal cytology or peripheral hormone (i.e. progesterone) concentrations? 2. what was the type (i.e. live cover, artificial insemination) of breeding? and 3. was pregnancy diagnosed prior to presentation? prediction of parturition has the most potential for inaccuracies based on breeding dates alone. objective of this review is to provide basic reproductive physiology and various diagnostics available to make best decisions for veterinarians practicing *presented at the 2023 society for theriogenology conference, published after peer review. female dog reproductive management and plan for caesarean surgery. reproductive physiology age of sexual maturity is 6-15 months.1 dogs cycle regularly in intervals between estrus that ranges from 5-13 months (average 7 months for most breeds).2 estrous cycle stages include: proestrus, estrus, diestrus, and anestrus. estrous cycle proestrus encompasses the period before luteinizing hormone (lh) surge that lasts for 1-3 weeks (average 9 days). during proestrus, gonadotropin releasing hormone pulses from hypothalamus stimulate follicle stimulating hormone (fsh) release that leads to ovarian follicular development. once follicles reach an appropriate size, they produce estrogen, and classical signs of proestrus are observed. these include vulvar swelling, diapedesis of endometrial red blood cells and consequent serosanguineous vulvar discharge, edema of vaginal epithelium, pheromonal secretion causing increased attraction of male dogs, and progressive cornification of vaginal epithelium.2 follicular luteinization begins before ovulation with increasing peripheral progesterone concentrations as estrogen concentrations start to decrease.3 this shift in hormonal mailto:jenny.sones@colostate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10795 2 citation: clinical theriogenology 2024, 16, 10795, http://dx.doi.org/10.58292/ct.v16.10795 concentrations induces receptivity to male dogs and the period is considered estrus. estrus begins 0-1 days after lh surge and receptivity lasts ~ for 7 days.2 preovulatory lh surge coincides with doubling in progesterone concentrations (from < 1 ng/ml during anestrus and proestrus to 1-3 ng/ml at surge) and lasts 1-5 days in dogs4,5 resulting in ovulation of matured follicles. since dogs are polytocous, ovulation occurs over a period of 24-36 hours, starting 48-72 hours after lh surge, when peripheral progesterone concentrations are 4-10 ng/ml.4 clinically, lack of receptivity to male dogs is end of estrus and vaginal cytology is no longer predominantly composed of cornified epithelial cells. cytological diestrus begins when an abrupt decrease of superficial or cornified cells from 80-100% to < 20% is detected in vaginal cytology.6 dogs are unique; ovulating primary oocytes that mature in the oviduct for 48-72 hours before they are capable of fertilization. this occurs once they become secondary oocytes that have completed first stage of meiosis with first polar body extrusion.7,8 secondary oocytes are fertilizable until 60-108 hours after ovulation.8 canine sperm have prolonged longevity in the female reproductive tract, particularly after natural mating, in part because of their retention in uterine crypts and uterotubal junction, especially if fresh and unprocessed semen is used for insemination.9 after fertilization, embryos enter the uterus between 10-12 days after lh surge. they are nourished from uterine lactotroph until implantation at ~ days 17-18 of embryonic life.10 pregnancy pregnancy lasts for 65 ± 1 days from lh surge (or 63 ± 1 days from ovulation).11 canine pregnancy is maintained solely by progesterone produced from corpora lutea (cl), due to lack of placental steroid synthesis.12,13 although maternal recognition of pregnancy is unknown in dogs, important embryo-maternal communication exists to signal decidualization and eventually parturition cascade.10 after implantation, prolactin and lh secretions assume luteotropic roles, and support progesterone production from the cl. plasma prolactin concentrations increase during second half of pregnancy, peak on the day of parturition, decrease for the next 24-48 hours, and then increase again to support lactation. during lactation, there are high plasma prolactin concentrations.14,15 relaxin can be detected in pregnant dogs ~ days 20-25, peaks 2-3 weeks before whelping and remains elevated until term (figure 1). relaxin is involved in pregnancy related remodeling of connective tissue, acting on pubic ligament, pregnant uterus and cervix, and is the endocrine marker of pregnancy in dogs. intraluteal relaxin is also involved in regulating canine cl.16 progesterone is produced by cl thecal cells throughout pregnancy and is essential for pregnancy maintenance. it increases once luteinization starts and continues after ovulation for ~ 2 weeks until it plateaus for 1-2 weeks prior to a steady decrease during remainder of pregnancy. at end of pregnancy, circulating progesterone concentrations decrease below 2 ng/ml. estrogen is produced throughout pregnancy at low concentrations and may contribute to uterine progesterone receptors maintenance. nonpregnant and pregnant dogs’ cl express prostaglandin f2 alpha (pgf2α) receptors. in dogs, no endogenous luteolytic agents are produced by nonpregnant uterus. regression of cl result in steady decrease in progesterone concentrations until they reach < 1 ng/ml (may be longer in nonpregnant dogs).11 in pregnant dogs, this continuous decrease in progesterone concentrations is hastened by luteolysis due to endogenous production of pgf2α from fetoplacental unit. this steep decrease in progesterone concentrations is observed ~ at day 60 of pregnancy. pgf2α is also responsible for cervical relaxation and uterine contractions during parturition.15 parturition circulating progesterone concentrations decrease to baseline 1-2 days before whelping. maternal decidual cells express progesterone receptors and have an important role in signaling prepartum luteolysis.16 additionally, decrease in circulating progesterone concentrations also appear to alter vascular functions, apoptosis and modulates proinflammatory response resulting in parturition.17 furthermore, this is accompanied by decrease in body temperature, typically 1°c 12-24 hours before the onset of parturition. it is important to mention that this is not observed in every dog. in a large retrospective study, only 33% of pregnant dogs had decreases in basal body temperature prior to parturition.18 therefore, a more complex mechanism involving multiple factors apart from progesterone may be involved.15 maturation of fetal pituitary-adrenal axis resulting in production of cortisol, triggering pgf2α release and initiating parturition cascade has been suggested for many years; however studies failed to prove this theory and corticosteroids concentrations at parturition had wide inter-individual variation.15,19 role of exogenous corticosteroid treatment in late canine pregnancy is discussed later in this review. behavioral signs of parturition can precede hormonal and temperature changes with extreme nesting behavior observed 6-24 hours before whelping. although these can give the clinician valuable information, not every dog will exhibit these signs consistently, making it difficult to rely only on clinical observations. stage i of parturition begins with uterine contractions that may not be appreciated externally and ends when the cervix is fully dilated. length of the canine vagina does not allow for cervical palpation. stage ii becomes evident with passage of pups through birth canal. voluntary abdominal straining and a ferguson’s reflex should be present. neonates may present in cranial or caudal position during normal parturition. stage iii begins after fetal expulsion and ends with expulsion of fetal membranes. stage ii and iii often occur simultaneously.20 pregnancy diagnosis duration of pregnancy is shorter in dogs compared to other species. therefore, canine fetuses are born in an immature state and final development of most organ systems must be completed after birth. consequently, substantial development occurs during the last days of canine pregnancy, ~ 61-63 days after ovulation. failure of this last fetal developmental phase has poor prognosis for pups’ survival. once a fetus exceeds its due date by > 2 days, its demand for nutritional support is more than placenta is able to provide resulting in intrauterine fetal distress and eventually death.11 there are several reasons why determination of accurate pregnancy age is important. this is beneficial for planning prenatal care of pregnant dogs as well as organizing veterinary assistance in cases of natural whelping that may require assistance. also, dogs that may require a planned caesarean http://dx.doi.org/10.58292/ct.v16.10795 citation: clinical theriogenology 2024, 16, 10795, http://dx.doi.org/10.58292/ct.v16.10795 3 surgery, such as in cases of singletons, previous history of dystocia, small litters with large pups or very large litters, and high-risk pregnancies that may compromise dog’s health. one of the most accurate methods to determine pregnancy age is by having a thorough breeding management and knowing the day of lh surge or ovulation. on average, canine pregnancy lasts 65 ± 2 days from lh surge, 63 ± 2 days from beginning of ovulation11,21 or 57 ± 3 days from the beginning of cytological diestrus. breeding date is not accurate in determining time of whelping, and parturition can happen anywhere from 58 to 72 days after breeding. breed variation exists regarding pregnancy duration; german shepherds, hound dogs and cavalier king charles spaniels have shorter pregnancy duration and west highland white terriers have longer pregnancy.22-24 we recommend researching literature while working with dog breeds that are not commonly presented in clinical practices. hormonal evaluation a common approach to assess the likely day of lh surge, ovulation, and optimal fertile period is by measuring peripheral progesterone concentrations. this is often performed every other day, in combination with vaginal cytology, once serosanguineous vulvar discharge is detected. in a large study that evaluated 1,300 dogs, in 1,420 estrous cycles the mean (± standard deviation) progesterone concentrations increased from 1.7 ± 0.7 to 2.7 ± 0.6 ng/ml at lh surge and at estimated day of ovulation concentrations were from 4.8 ± 0.9 to 7.2 ± 1.3 ng/ml.25 ovulation occurred when progesterone concentrations were 4-10 ng/ml.26 more than focusing on individual progesterone concentrations (since variable among dogs), a trend in increasing concentrations of progesterone should be identified to estimate the day of lh surge, ovulation, and fertile period. assay and sample handling may affect progesterone measurement, so consistency is recommended for serial monitoring; i.e. sample collection time of day and collecting blood from a fasted or nonfasted patient. a thorough review on assay variability was recently published.27 detection of lh surge by measuring peripheral concentrations is sometimes utilized in canine breeding management.28 radioimmunoassay has long been considered as gold standard due to its accuracy and repeatability, but due to radiation concerns, expertise required, and time to receive result makes this impractical. however, a semiquantitative enzyme linked immunosorbent assay (witness® lh, zoetis, usa) was developed for in-clinic use. detection of lh surge requires daily sampling as the duration can be variable that limits this assay’s utility in many clinical situations. radiological evaluation radiography is valuable to assess pregnancy age since fetal skeleton mineralizes progressively throughout pregnancy. proper radiographic technique and patient restraint is important to obtain good quality images and commonly, figure. graphic representation of canine estrous cycle, pregnancy hormones and timing of methods to determine pregnancy age; x-axis represents days from lh surge. http://dx.doi.org/10.58292/ct.v16.10795 4 citation: clinical theriogenology 2024, 16, 10795, http://dx.doi.org/10.58292/ct.v16.10795 lateral view is usually sufficient for good diagnosis. ventrodorsal view is indicated when fetomaternal mismatch is suspected; however, caution is required when placing lateterm pregnant dogs in this position. radiographs are the best method to obtain fetal count when performed between days 57-65 (reduces risk of missing a fetus) when mineralization is advanced. mineralization of fetal skull is first visible between days 43-46 of pregnancy. scapula, humerus, and femur are visible between days 46-51. radius, ulna, and tibia are visible between 50 and 53 days. pelvis and ribs are visible between days 53 and 59. coccygeal vertebrae and distal extremities are visible between days 55-64. teeth are the last part to mineralize and are usually visible between days 58-63.29 although radiographs are useful for determining fetal numbers prior to whelping and a rough estimate of pregnancy age, they have limited use as indicators of fetal maturation and readiness for birth. ultrasonographic evaluation ultrasonography has been utilized to monitor canine pregnancy and determine pregnancy age for many years. most veterinary clinics have an ultrasound machine available and although expertise is required to become proficient in diagnosis, the noninvasiveness of this technique makes it valuable for practitioners. repeated ultrasonography examinations of up to 3 times per day have been utilized to determine the time of ovulation.30-32 this particular approach requires training and frequent evaluations, making it impractical for most practitioners. amniotic vesicles can be detected as early as day 19 as 1-2 mm rounded anechoic structures in the uterine lumen. embryos can be first detected via ultrasonography on day 22 and heartbeat on day 24.24 between days 19 and 37 of pregnancy, measurement of amniotic sac diameter (inner and outer chorionic cavities) can be easily obtained, and mathematical formulas are available to determine pregnancy age.24,33 these early pregnancy biometric indices are often more accurate than those obtained in late pregnancy. at days 27-28, embryos move away from the endometrial wall and are suspended by fetal membranes. zonary appearance of the placenta is obvious by days 29-31 and the edges curl inward by days 32-34.24 after day 37, several fetal morphometrics can be utilized to determine pregnancy age; e.g. biparietal diameter (bpd), body diameter (bd), deep portion of the fetal diencephalon-telencephalic vesicle (dptv) and femur, humerus, tibia, and kidney lengths. bpd and bd remain the most accurate of all formulas available across breeds. however, practitioners should take into account the breed and dog size when using these formulas.24 fetal organ development can also be used to calculate pregnancy age. serial examinations and measurements from multiple fetuses provide more accurate information. urinary bladder is first visible between 35-39 days, stomach between 36-39 days, kidneys between 39-47 days, and intestine between 57-63 days.24 fetal kidney length has been utilized to determine pregnancy age and it is most accurate between 48-52 days of pregnancy.34 renal cortical thickness (ct), medullary thickness (mt) and ratio of ct/mt can also be used.35 intestines can be visualized from days 39-43 of pregnancy; however, distinct visualization of various layers is not possible until days 48-54 and these layers become increasingly prominent until term.24,36,37 peristalsis becomes easily visible between 62-64 days, and it may be easier to observe in smaller breeds.36 panting may cause difficulties when observing peristalsis so care must be taken.24 once all intestinal layers are visible and peristalsis is consistently prominent on serial examinations, fetal survival ex utero is optimal; however, veterinarians must recognize that variation among individuals exist and this parameter should not be used as a sole parameter when making decisions for cesarean surgery.24,36,37 a thorough review on pregnancy aging based on ultrasonography was published.24 evaluation of fetal maturity changes in fetal heart rate, intestinal layer development and peristalsis, and progesterone monitoring are important to make a well-informed decision (when dogs are ready to whelp or a caesarean surgery can be scheduled). fetal heart rate usually starts to decrease 12-36 hours prior to onset of labor as fetuses begin to meet or exceed full demands of placental function. transient fetal bradycardia may be observed during uterine contractions. starting to monitor dogs 2-6 days ahead of the expected parturition date usually allows for the most accurate assessment of final fetal maturation and readiness for birth and will allow steroid treatment 24 hours prior to delivery to facilitate fetal pulmonary function and thus neonatal resuscitation.38 medical management before caesarean surgery preanesthetic considerations for term pregnant dogs are controversial. preoxygenation and balanced hemodynamics are often performed for all presurgical veterinary patients. however, pregnant dogs have other considerations accounting for neonatal survival. systematic review from human medical literature suggested that antenatal use of corticosteroids in term planned caesarean surgery may reduce admission to neonatal icu due to respiratory distress syndrome; however, more evidence is needed in this area and it still remains controversial.38,39 betamethasone improved pulmonary capacity in canine neonates; antenatal maternal corticoid therapy in dogs improved areas of gas exchange in lungs and aided in the metabolism of pulmonary fluids rather than stimulated surfactant production.40 prenatal treatment with betamethasone can be adopted as clinical lung maturation protocol for pregnancies at risk in order to prevent morbidity/mortality and to increase neonatal survival.41 conclusion determining parturition time in dogs requires a multimodal approach. relying on a single parameter may lead to inaccurate timing of fetal maturity and readiness for delivery. before proceeding with surgical management of dogs, a thorough history, physical examination with a vaginal digital examination, imaging, bloodwork, and determination of peripheral progesterone concentrations are helpful. if findings are indicative of fetal distress and/or fetal readiness (i.e. progesterone < 2 ng/ml), cesarean surgery may be the best option. surgical intervention may also be the best option if dogs have signs of systemic illness, anatomical abnormalities precluding vaginal delivery or an obstructive dystocia that cannot be resolved with gentle manipulation. planned caesarean surgery can carry an excellent prognosis if proper timing and planning is done in advance. conflict of interest none to report http://dx.doi.org/10.58292/ct.v16.10795 citation: clinical theriogenology 2024, 16, 10795, http://dx.doi.org/10.58292/ct.v16.10795 5 references 1. da costa rep, kinsman rh, owczarczak-garstecka sc, et al: age of sexual maturity and factors associated with neutering dogs in the uk and the republic of ireland. vet rec 2022;191:1–11. doi: 10.1002/vetr.1265 2. concannon pw, castracane vd, temple m, et al: endocrine control of ovarian function in dogs and other carnivores. anim reprod 2009;6:172-193. 3. kowalewski mp: regulation of corpus luteum function in the domestic dog (canis familiaris) and comparative aspects of 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https://www.researchgate.net/profile/patrick-concannon-2/publication/237260118_canine_pregnancy_predicting_partu rition_and_timing_events_of_gestation/links/00b4952f6456c8be7e00 0000/canine-pregnancy-predicting-parturition-and-timing-events-of-gestation.p https://www.researchgate.net/profile/patrick-concannon-2/publication/237260118_canine_pregnancy_predicting_partu rition_and_timing_events_of_gestation/links/00b4952f6456c8be7e00 0000/canine-pregnancy-predicting-parturition-and-timing-events-of-gestation.p https://www.researchgate.net/profile/patrick-concannon-2/publication/237260118_canine_pregnancy_predicting_partu rition_and_timing_events_of_gestation/links/00b4952f6456c8be7e00 0000/canine-pregnancy-predicting-parturition-and-timing-events-of-gestation.p https://www.researchgate.net/profile/patrick-concannon-2/publication/237260118_canine_pregnancy_predicting_partu rition_and_timing_events_of_gestation/links/00b4952f6456c8be7e00 0000/canine-pregnancy-predicting-parturition-and-timing-events-of-gestation.p https://doi.org/10.1016/j.theriogenology.2004.11.007 https://doi.org/10.1080/01652176.1993.9694397 https://doi.org/10.1111/rda.14446 https://doi.org/10.1016/j.theriogenology.2018.10.018 https://doi.org/10.1016/j.theriogenology.2018.10.018 https://doi.org/10.1111/j.1748-5827.1990.tb00824.x https://doi.org/10.1111/j.1748-5827.1990.tb00824.x https://doi.org/10.1016/j.cvsm.2023.05.007 https://doi.org/10.1016/s0195-5616(01)50201-6 https://doi.org/10.1016/s0195-5616(01)50201-6 https://doi.org/10.1016/j.cvsm.2018.02.008 6 citation: clinical theriogenology 2024, 16, 10795, http://dx.doi.org/10.58292/ct.v16.10795 follicles by ultrasonographic diagnosis system in dogs. j vet med sci 2000;62:243-248. doi: 10.1292/jvms.62.243 31. bergeron lh, nykamp sg, brisson ba, et al: an evaluation of b-mode and color doppler ultrasonography for detecting periovulatory events in the bitch. theriogenology 2013;79:274-283. doi: 10.1016/j.theriogenology.2012.08.016 32. tsuchida m, komura n, yoshihara t, et al: ultrasonographic observation in combination with progesterone monitoring for detection of ovulation in labrador retrievers. reprod domest anim 2022;57:149-156. doi: 10.1111/rda.14035 33. socha p, janowski t, bancerz-kisiel a: ultrasonographic fetometry formulas of inner chorionic cavity diameter and biparietal diameter for medium-sized dogs can be used in giant breeds.  theriogenology 2015;84:779-783. doi: 10.1016/j. theriogenology.2015.05.012 34. gil emu, garcia daa, giannico at, et al: early results on canine fetal kidney development: ultrasonographic evaluation and value in prediction of delivery time. theriogenology 2018;107:180-187. doi: 10.1016/j.theriogenology.2017.11.015 35. siena g, di nardo f, romagnoli s, et al: relationship between days before parturition and fetal kidney length, cortical thickness, medullary thickness and their ratio in dogs. theriogenology 2022;194:58-63. doi: 10.1016/j.theriogenology.2022. 09.021 36. siena g, romagnoli s, drigo m, et al: ultrasonographic changes in fetal gastrointestinal motility during the last ten days before parturition in dogs. front vet sci 2022;9:1–9. doi: 10.3389/ fvets.2022.1000975 37. gil emu, garcia daa, froes tr: in utero development of the fetal intestine: sonographic evaluation and correlation with gestational age and fetal maturity in dogs. theriogenology 2015;84:681-686. doi: 10.1016/j.theriogenology.2015.04.030 38. sotiriadis a, mcgoldrick e, makrydimas g, et al: antenatal corticosteroids prior to planned caesarean at term for improving neonatal outcomes (review). cochrane database syst rev 2021; 12(12):cd006614. doi: 10.1002/14651858.cd006614.pub4 39. norman j, shennan a, jacobsson b, et al: figo good practice recommendations on the use of prenatal corticosteroids to improve outcomes and minimize harm in babies born preterm. int j gynecol obstet 2021;155:26-30. doi: 10.1002/ijgo.13836 40. regazzi fm, silva lcg, lúcio cf, et al: morphometric and functional pulmonary changes of premature neonatal puppies after antenatal corticoid therapy. theriogenology 2020;153:19-26. doi: 10.1016/j.theriogenology.2020.05.005 41. regazzi fm, silva lcg, lúcio cf, et al: influence of prenatal maternal corticosteroid therapy on clinical and metabolic features and pulmonary function of preterm newborn puppies. theriogenology 2017;97:179-185. doi: 10.1016/j.theriogenology.2017.04.039 http://dx.doi.org/10.58292/ct.v16.10795 https://doi.org/10.1292/jvms.62.243 https://doi.org/10.1016/j.theriogenology.2012.08.016 https://doi.org/10.1111/rda.14035 https://doi.org/10.1016/j.theriogenology.2015.05.012 https://doi.org/10.1016/j.theriogenology.2015.05.012 https://doi.org/10.1016/j.theriogenology.2017.11.015 https://doi.org/10.1016/j.theriogenology.2022.09.021 https://doi.org/10.1016/j.theriogenology.2022.09.021 https://doi.org/10.3389/fvets.2022.1000975 https://doi.org/10.3389/fvets.2022.1000975 https://doi.org/10.1016/j.theriogenology.2015.04.030 https://doi.org/10.1002/14651858.cd006614.pub4 https://doi.org/10.1002/ijgo.13836 https://doi.org/10.1016/j.theriogenology.2020.05.005 https://doi.org/10.1016/j.theriogenology.2017.04.039 1 contact karen moran morankaren89@gmail.com © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12711, http://dx.doi.org/10.58292/ct.v17.12711 case report testicular degeneration, fibrosis, and mineralization in limangus bulls karen moran,a,b raúl picco,c eleonora morrell,d andrea verna,d luis zapata,b laura moiraghi,b guillermina bilbao,a,b julián bartoloméb acomisión nacional de investigaciones científicas y técnicas, buenos aires, argentina bfacultad de ciencias veterinarias, universidad nacional de la pampa, general pico, argentina cactividad privada, villa huidobro, argentina dinstituto nacional de tecnología agropecuaria, balcarce, argentina abstract a group of limangus heifers had a high pregnancy rate after timed artificial insemination but low final pregnancy rate after 75 days of natural service with 2 limangus bulls. bulls (a and b) suspected of subfertility and 2 other bulls (c and d) with the same genetic background were examined. bull a was clinically normal but had no sperm in the ejaculate. bulls b and c had abnormal testicular consistency resembling fibrosis on palpation and ultrasonography; however, semen was normal. bull d was clinically normal. bulls were negative for brucellosis and venereal diseases. bulls a, b, and c were culled and slaughtered. bull a had a normal genital tract; however, nonstructured 5b gene was detected by reverse transcription polymerase chain reaction, suggesting an infection with bovine viral diarrhea virus-1 (pestivirus a). bulls b and c had macroscopic and microscopic signs of testicular degeneration, fibrosis, and mineralization. although the specific cause for testicular lesions in bulls b and c was not identified, these lesions were probably responsible for the low pregnancy rate. keywords: bulls, infertility, testes, degeneration, fibrosis, mineralization background a common reproductive strategy in beef herds is to use timed artificial insemination (tai) at the beginning of the breeding season and 15 days after tai, use natural service (exposing cows or heifers to bulls for 60 days).1 various factors affect bull fertility; breeding soundness examination (bse) is of great value to recognize the fertility of bulls and to maximize the reproductive efficiency of cow-calf operations.2 a standard bse identifies bulls with substantial deficits in fertility but does not consistently identify subfertile bulls.3 routine bse includes physical examination, estimation of sperm production based on scrotal circumference (sc), evaluation of semen quality, and occasionally, libido or copulation ability is assessed.4 ultrasonography is a complementary tool to identify testicular lesions that are not detected by palpation.5 in addition, laboratory investigation of venereal diseases (tritrichomonas foetus and campylobacter fetus venerealis) should be included, since bulls maybe asymptomatic but infected cows will have signs of infertility. other infectious diseases may affect fertility of beef herds; therefore, core vaccination is recommended for bovine viral diarrhea (bvd), infectious bovine rhinotracheitis, leptospirosis, and clostridial diseases.4 we evaluated 4 limangus bulls during the investigation of infertility in a cow-calf operation located in the south of cordoba province, argentina. in the fall of 2018, limangus heifers (n = 62) were subjected to tai and then exposed to natural service 15 days later with 2 limangus bulls. during natural service, more heifers (n = 37) were added to the group. pregnancy per tai was 74% (46/62); however, pregnancy for 75 days of natural service was only 69.8% (37/53). in the winter of 2019, another group of limangus heifers (n = 92) were bred and then exposed to natural service 15 days later with the same 2 bulls. pregnancy per tai was 50% (46/92); however, pregnancy for 75 days of natural service was only 63% (29/46). the referring veterinarian suspected infertility after detecting low sc and abnormal testicular consistency, since heifers were in good body condition and cycling. two months later, a complete bse (including ultrasonography of testis) mailto:morankaren89@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12711 2 citation: clinical theriogenology 2025, 17, 12711, http://dx.doi.org/10.58292/ct.v17.12711 was conducted and preputial swabs for venereal diseases and blood samples were collected in these 2 bulls (a and b) and 2 other bulls (c and d) that were recently included. bulls a and b were 3 years and bulls c, and d were 2 years (confirmed by second and first pair of incisors, respectively). breeding soundness examination and complementary studies bulls underwent bse according to criteria;4 accessory sex glands were assessed by transrectal palpation. external reproductive organs, scrotal temperature, and painful responses were evaluated by palpation. sc was measured by pushing the testis to scrotal bottom by placing the thumb and fingers laterally on the side of scrotal neck and then pushing ventrally. a metal tape was applied over the scrotum and its contents; sc (in cm) was taken at the greatest diameter of the scrotal contents. genital tract ultrasonography was conducted with a b-mode ultrasound scanner (honda hs 1600) with a 7.5 mhz linear array transducer using described methodology.6 high-quality ultrasonographic gel was used to generate clear ultrasonograms. one testis was pushed upwards (out of the transducer’s ultrasound wave) while the other testis was pushed downwards for examination, thus scrotal wall was stretched improving the contact between transducer and scrotum. transducer was applied longitudinally on the scrotal skin to evaluate the testis halfway between the head and epididymis tail.6 semen samples were collected by electroejaculation and evaluated for sperm motility and morphology. clean, prewarmed microscope slides, and coverslips were prepared. sperm motility was evaluated subjectively under brightfield microscopy (400 x); morphology was evaluated using semen smears stained with eosin and nigrosin (e/n) and observed under oil immersion with brightfield microscopy (1,000 x). sperm (n = 200) were evaluated for head abnormalities in each smear.4 in addition, semen was diluted in 10% neutral buffered formalin (wet mount) and sperm (n = 100) were evaluated for tail defects, abnormal acrosomes, detached sperm heads, and proximal droplets using differential interface contrast microscopy (1,000 x).7 serologic testing for brucella abortus was performed using buffered acidified plate antigen assay and preputial swab was obtained for testing sexually transmitted diseases (tritrichomonas foetus and campylobacter fetus) using a polymerase chain reaction (pcr) test. a month later, bulls that failed bse (a, b, and c) were culled and slaughtered; genital tracts were examined. samples of testis (dorsal, mid, and ventral regions) were frozen at –20°c in sterile vials until processed for bvd virus.8 in addition, 2 mm2 samples were taken for histopathological evaluation: 1. dorsal, middle, and ventral regions of testis; 2. head, body, and tail epididymis; 3. vas deferens; 4. vesicular glands; and 5. lesions. samples were fixed in bouin’s solution for 6 hours, then placed in 10% formaldehyde solution (with 3 previous washes) and submitted for histology. fixed tissues were trimmed and subjected to routine paraffin processing. tissue sections were stained with e/n and examined by brightfield microscopy. bse and necropsy findings bull a bse: scrotum had a distinct neck; testes were symmetrical with normal tone and were freely moveable. epididymis and spermatic cords were within normal limits. sc was 35.5 cm, and the scrotal temperature was normal without painful responses. internal reproductive organs, prepuce, and penis were within normal limits. ultrasonography revealed small focal hyperechoic areas (figure 1a) affecting < 1 % of the area examined. semen (5 ml) collected via electroejaculation was without sperm. necropsy: samples were collected from the left and right epididymal tails. sperm from the left had fair gross motility; individual motility was 70% and in e/n-stained slides 98.5% sperm were morphologically normal with ~ 3% head defects. morphologically normal sperm on the wet mount was 93% (1% proximal droplet, 2% detached heads, 2% midpiece defects, and 2% principal piece defects). sperm from the right had good gross motility; individual motility was 70% and in e/n-stained slides 99% sperm were morphologically normal with ~ 2% head defects). morphologically normal sperm on the wet mount was 96% (4% had principal piece defects). small focal white streaks of < 3 mm2 (figure 1b) were observed in both testicular histology. epididymis, vas deferens, ampullae, and vesicular glands were within normal limits. stromal fibrosis (figure 1c) was evident in left testis. ampullae and vesicular glands had mild fibrosis. in addition, nested multiplex reverse transcription polymerase chain reaction (rt-pcr) identified nonstructured 5b (ns5b) gene of bvd virus (bvdv-1 [pestivirus a]). bull b bse: scrotum had a distinct neck; testes were symmetrical and were freely moveable. right testis consistency was abnormal and had palpable intratesticular firm nodules. epididymis and spermatic cords were within normal limits. sc was 35.5 cm, and the scrotal temperature was normal without painful figure 1. bull a: a. ultrasonogram of left testis; note small hyperechoic areas (black arrows). b. cut section of testes; note small < 3 mm2 in both testes (black arrows). c. light microscopic image (1000 x) of left testis; note stromal fibrosis (black arrow) http://dx.doi.org/10.58292/ct.v17.12711 citation: clinical theriogenology 2025, 17, 12711, http://dx.doi.org/10.58292/ct.v17.12711 3 responses. internal reproductive organs, prepuce, and penis were within normal limits. ultrasonography revealed diffuse hyperechoic areas and were more pronounced in the right testis (figure 2a) affecting 20% of the examined area. collection of semen by electroejaculation resulted in 5 ml of ejaculate that had poor gross motility and 65% individual motility. e/n-stained slides had 98.5% morphologically normal sperm (2% with head defects and 1% with midpiece defects). wet mount had 93% morphologically normal sperm (4% with sperm head defects, 1% with midpiece defects, and 2% with principal piece defects). necropsy: left testis had focal white streaks of 5 mm2 in the parenchyma and small areas of hard consistency and granulomatous appearance. right testis had a large lesion of hard consistency and granulomatous appearance from the mediastinum toward the periphery involving 80% of testis (figure 2b). testicular parenchyma had abnormal appearance and atypical color. epididymis, vas deferens, ampullae, and vesicular glands were within normal limits. in histology, intraluminal dystrophic calcification and peritubular fibrosis of the epididymis, fibrous tissue and calcification with mononuclear and polymorphonuclear cells (figure 2c) were observed. the diagnosis was severe stromal and intratubular fibrosis. nested multiplex rt-pcr was negative for ns5b gene of bvd virus identified as bvdv-1 (pestivirus a). bull c bse: scrotum had a distinct neck; testes were symmetrical and were not freely moveable. the tone was abnormal with palpable intratesticular firm swelling nodules in right testis. left epididymis tail was adhered to the testis. right epididymis and spermatic cords were within normal limits. sc was 33.5 cm, and the scrotal temperature was normal without painful responses. internal reproductive organs, prepuce, and penis were within normal limits. ultrasonography revealed large diffuse and focal hyperechoic areas (figure 3a) in testes affecting ~ 30% of examined areas. collection of semen by electroejaculation resulted in 5 ml of ejaculate that had very good sperm gross motility and 65% individual motility. e/n-stained slides had 93.5% morphologically normal sperm (13% sperm head defects). the percentage of morphologically normal sperm in wet mount was 92% (3% defects of sperm head, 1% with proximal droplets, and 4% with principal piece defects). necropsy: there were adhesions between the parietal and visceral vaginal tunics. testes had large white focal and diffuse streaks from the mediastinum toward the periphery of  the parenchyma with lack of bulging (figure 3b). epididymides had adhesions to the vaginal tunic; vas deferens, ampullae, and vesicular glands were within normal limits. testes histopathology revealed 70% mineralization of the seminiferous tubules with active fibroblasts and inflammatory cells (lymphocytes and macrophages) in the interstitial compartment. seminiferous tubules had abnormal epithelium and intratubular concentric laminated calcification. there was a reduction in germinal cells; germinal cells were missing entirely in some tubules (figure 3c). the diagnosis was severe testicular degeneration with moderate interstitial fibroplasia including areas with necrosis. nested multiplex rt-pcr was negative for ns5b gene of bvd virus identified as bvdv-1 (pestivirus a). figure 2. bull b: a. ultrasonogram of right testis; note large hyperechoic areas (black arrows). b. cut section of testes; note the hard consistency and granulomatous appearance of parenchyma (compatible with fibrosis) with severe lesions in right testis (black arrows). c. light microscopic image (400 x) of testis; note mineralized seminiferous tubules with reduction on germinal cells (black arrows) http://dx.doi.org/10.58292/ct.v17.12711 4 citation: clinical theriogenology 2025, 17, 12711, http://dx.doi.org/10.58292/ct.v17.12711 bull d bse: scrotum had a distinct neck; testes were symmetrical with normal tone, and they were freely moveable. epididymis and spermatic cords were within normal limits. sc was 38.0 cm, and the scrotal temperature was normal without painful responses. internal reproductive organs, prepuce, and penis were within normal limits. ultrasonography of testes revealed small focal hyperechoic areas affecting < 1% of the examined area. collection of semen by electroejaculation resulted in 9 ml of ejaculate with very good gross motility and 90% individual motility. e/n-stained slides had 99% morphologically normal sperm (1% defects of sperm head). the percentage of morphologically normal sperm cells in wet mount was 97% (2% of detached normal sperm heads, 1% simple bent tail). discussion there were severe testicular lesions in these 3 bulls. bull a had stromal fibrosis in the left testis and ns5b gene was detected by rt-pcr suggesting an infection with bvdv-1 (pestivirus a). bulls b and c were suspected of testicular fibrosis on palpation and ultrasonography revealed abnormal hyperechoic areas. they also had low sc; macroscopic signs of degeneration, fibrosis, and mineralization that were confirmed by histopathology. testicular degeneration, fibrosis, and mineralization have been described in bulls with reduced fertility; causes suggested were biopsy, trauma, heat, cold, radiation, nutritional disorders, toxic agents, blind efferent ductules, vascular lesions, and aging.9 testicular biopsy could result in hemorrhage, infarction, and degeneration9; however, in this case, no signs of previous biopsy were observed, and the lesions were in the mediastinum and toward the periphery. testicular trauma is rare in bulls,9 and in these cases, classical hematomas and necrotic lesions were not observed. blind efferent ductules or obstruction at the level of epididymis were suspected, especially in bull a that did not respond to electroejaculation. lesions on the hemato-testicular barrier with spermiostasis often cause testicular degeneration, fibrosis, and mineralization.9 however, duct permeability was normal in that bull and no macroscopic and microscopic signs of obstructive lesions were observed in any bulls. infectious agents could cause inflammation of the arterioles and capillaries in the testes inducing necrosis and fibrosis.10,11 in fact, there is evidence that bovine respiratory syncytial virus could be involved in the etiology of testicular fibrosis.11 bvd virus can be detected in the testes for a long duration after acute infection.12 mass motility, sperm concentration, and herd fertility were significantly lower in serologically bvd-positive bulls.13 however, although bvd virus was detected in a testicular sample of bull a, the role of the virus on infertility cannot be determined. testicular degeneration is an acquired condition that results in testes softer than normal on palpation, low concentration of sperm, with a high percentage of morphological defects.4 in the present case, the specific cause of testicular degeneration, figure 3. bull c: a. ultrasonogram of testis; note large hyperechoic areas (black arrows). b. cut section of testes; note severe fibrosis from the rete toward the peripheral parenchyma (black arrows) with lack of bulging (white arrows). c. light microscopic image (400 x) of testis; note seminiferous tubules with abnormal epithelium and intratubular concentric laminated calcification (black arrows) http://dx.doi.org/10.58292/ct.v17.12711 citation: clinical theriogenology 2025, 17, 12711, http://dx.doi.org/10.58292/ct.v17.12711 5 fibrosis, and mineralization could not be confirmed; however, it could have been the cause of low pregnancy rate in this group of heifers. testicular degeneration can decrease sc and it may be temporary if the underlying cause is removed.11 the sc provides a reliable estimate of testicular mass and is useful in estimating daily sperm production and semen quality.4 severe testicular degeneration was diagnosed in bull c and the sc was below the normal range. however, semen parameters were within normal range for all 3 bulls. probably, there was enough healthy testicular tissue to produce a fair amount of normal semen. the lack of sperm at electroejaculation in bull a was probably due to lack of response to the technique. occasionally, it is difficult to collect from bulls that have been active with cows in the last 24 hours or nervous, irritable, or hard to manage.14 although macroscopic lesions in the testes detected by ultrasonography have been associated with low semen quality and an increase in sperm abnormalities,15 others reported that the degree of testis fibrosis was not associated with semen quality,11 and mild lesions would not affect testicular function or sperm quality.16 in the present case, the extension and severity of testicular degeneration, fibrosis, and mineralization probably reduced the amount of testicular tissue producing sperm and consequently reduced fertility. learning points • breeding soundness examination of bulls before breeding season is reaffirmed • ultrasonography of testes is of great value • limangus breeders should be alerted to detect early testicular problems conflict of interest none to declare. references 1. bó ga, baruselli ps: synchronization of ovulation and fixed-time artificial insemination in beef cattle. animal. 2014;8:144-50. doi: 10.1017/s1751731114000822 2. campero cm, bartolomé ja: examen clínico reproductivo. in: campero cm, bartolomé ja, campero lm: editors. toros para carne, evaluación sanitaria y productiva. 1st edition, argentina; hemisferio sur: 2021. p. 105-121. 3. kastelic jp, 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overuse and sexually transmitted infections on seminal parameters of extensively managed bulls. animals 2021;11:827. doi: 10.3390/ani11030827 14. palmer cw: evaluation of breeding soundness: the spermiogram. in: hopper rm: editor. bovine reproduction. 2nd edition, hoboken; john wiley & sons: 2021. p. 102-119. doi: 10.1002/ 9781119602484.ch9 15. arteaga aa, barth ad, brito lfc: relationship between semen quality and pixel–intensity of testicular ultrasonograms after scrotal insulation in beef bulls. theriogenology 2005;64:408-415. doi: 10.1016/j.theriogenology.2004.12.008 16. coulter gh, bailey drc: effects of ultrasonography on the bovine testis and semen quality. theriogenology 1988;30:743-749. doi: 10.1016/0093-691x(88)90309-3 http://dx.doi.org/10.58292/ct.v17.12711 https://doi.org/10.1017/s1751731114000822. https://doi.org/10.1111/j.1439-0531.2008.01186.x https://doi.org/10.1016/j.theriogenology.2013.09.001 https://doi.org/10.1016/j.theriogenology.2013.09.001 https://doi.org/10.1016/j.cvfa.2009.07.006 https://doi.org/10.1016/j.cvfa.2009.07.006 https://doi.org/10.1111/j.1439-0531.2006.00675.x https://doi.org/10.1128/jcm.37.6.2020-2023.1999 https://doi.org/10.1128/jcm.37.6.2020-2023.1999 https://doi.org/10.1016/b978-0-12-483375-3.50020-2 https://doi.org/10.1016/j.anireprosci.2007.05.002 https://doi.org/10.1016/j.vetmic.2009.04.029 https://doi.org/10.3390/ani11030827 https://doi.org/10.1002/9781119602484.ch9 https://doi.org/10.1002/9781119602484.ch9 https://doi.org/10.1016/j.theriogenology.2004.12.008 https://doi.org/10.1016/0093-691x(88)90309-3 2019 unilateral epididymo-orchitis with coagulative and liquefactive necrosis of the spermatic cord, epididymis, and testis of a friesian stallion caused by salmonella spp. serotype hartford unilateral epididymo-orchitis with coagulative and liquefactive necrosis of the spermatic cord, epididymis, and testis of a friesian stallion caused by salmonella spp. serotype hartford sabrina hall, page mauk, jim schumacher, jessica klabnik-bradford, tulio prado department of large animal clinical sciences, university of tennessee, knoxville, tn abstract a 5-year-old friesian stallion was presented for evaluation due to increasing discomfort, scrotal edema and right testicular enlargement, first observed 8 days prior to presentation. hyperechoic areas (consistent with necrosis) within the right testis, anechoic fluid between the visceral and parietal tunics and scrotal edema were observed during transcutaneous ultrasonographic examination. absence of blood flow to the distal 3 cm of the spermatic cord was detected by doppler ultrasonography. testicular necrosis caused by spermatic torsion was suspected and the right testis and distal aspect of the spermatic cord were excised under general anesthesia. during surgery, it was apparent that the right testis and distal portion of the spermatic cord were necrotic, but there was no evidence of torsion of spermatic cord. histopathology findings included severe diffuse necro-suppurative orchitis, epididymitis and cellulitis, with intralesional bacterial rods. on bacterial culture, there were > 500 colonies of salmonella spp.  serotype hartford. keywords: stallion, necrosis, epididymitis, testis, salmonella background a swollen right testis and lack of blood flow within the distal portion of spermatic cord was present in a stallion. spermatic cord torsion leading to necrosis was suspected. based on intraoperative inspection and subsequent histopathology, epididymo-orchitis and not torsion of the spermatic cord caused necrosis. epididymo-orchitis with coagulative and liquefactive necrosis of the spermatic cord, epididymis, and testis due to salmonella spp. have been reported in humans and rams, but apparently not in stallions. this report describes epididymo-orchitis of a stallion caused by a salmonella spp. case presentation a 5-year-old friesian stallion was presented with anorexia, fever, and enlargement of the right testis. the owner, a veterinarian and equine practitioner, had detected an enlarged, firm testis 8 days prior to presentation. two days later, the horse became anorexic and febrile (40.4°c) and was treated for 6 days with sulfadiazine/trimethoprim (22 mg/kg, po, q12h) and phenylbutazone (2.2 mg/kg, po, q24h). six months before, the stallion had a fever of unknown origin for ~ 1 week, accompanied by elevations in white blood cell count and serum fibrinogen concentrations and was positive for equine herpesvirus 2 (ehv-2). the stallion was otherwise healthy and regularly used for breeding. upon presentation, the stallion was bright, alert, and responsive. temperature was 38.5°c and pulse and respiratory rate were within normal limits. no signs of lameness or gait stiffness were present. the scrotum was edematous, right testis was moderately enlarged, warmer than the left testis and abnormally firm, but the stallion did not exhibit signs of discomfort when this testis was palpated. the diameter of the right testis (7.1 cm), as measured with ultrasonography, was moderately larger than that of the left (size of left testis not recorded). ultrasonographically, the parenchyma of the right testis appeared hyperechoic, indicative of necrosis and there was excessive anechoic fluid between the visceral and parietal tunics. doppler ultrasonographic examination revealed a lack of blood flow in vessels in the distal aspect of right spermatic cord. testicular necrosis caused by spermatic cord torsion was suspected. differential diagnosis although the most likely cause of testicular necrosis was a sequela to torsion of the spermatic cord, other differential diagnoses considered included traumatically induced epididymo-orchitis, inguinal herniation, testicular neoplasia, and hydrocele.1,2 causes of scrotal enlargement, other than torsion of the spermatic cord and epididymo-orchitis, were eliminated during ultrasonographic examination of the 139 clinical theriogenology • volume 11, number 2 • june 2019 scrotum and its contents. surgical removal of the right testis and distal portion of its spermatic cord was recommended, as necrosis was suspected. treatment stallion was treated with sulfadiazine/trimethoprim (22 mg/kg, po) and flunixin meglumine (1.1 mg/kg, iv) and sedated with xylazine hcl (1.1 mg/kg, iv). anesthesia was induced with ketamine hcl (2 mg/kg, iv) and midazolam (0.05 mg/kg, iv), and maintained with a combination of isoflurane, ketamine, and xylazine. a balanced electrolyte solution (5 ml/kg/hour, plasma-lyte, baxter healthcare ltd., deerfield, il) was given during surgery. stallion was positioned in dorsal recumbency, with scrotum and inguinal regions prepared for aseptic surgery. a 10 cm, longitudinal, elliptical skin incision was made through the edematous scrotum over the right testis and extended through thickened scrotal fascia to the parietal tunic. right testis and a large portion of its spermatic cord, each encased within the parietal tunic, were separated from surrounding fascia (figure 1). spermatic cord was ligated with 2 encircling sutures of 1-0 polydioxanone suture (pdstm, ethicon, somerville, nj) close to the superficial inguinal ring and far proximal to a line of demarcation between healthy and necrotic tissue, and the spermatic cord was transected distal to the ligatures with an emasculator. incision in the scrotal fascia was closed with 2-0 pds (pdstm, ethicon) placed in a simple-continuous pattern and scrotal skin sutured using 2-0 barbed sutures composed of glycolide, diaxanone, and trimethylene carbonate (v-loc 90, medtronics, louisville, ky) placed intradermally, using a simple-continuous pattern. testis and its parietal tunic were submitted for histolopathology and bacteriology. one day after surgery, the stallion’s temperature was 38.1oc and it had a normal appetite. owner was instructed to walk the stallion in hand at least twice daily, allow the horse to resume regular exercise at 3 weeks after surgery and continue to administer trimethoprim-sulfa (22 mg/kg, po, q12h) for 7 days. owner was also instructed to have stallion’s semen evaluated at ~ 30 and 60 days after surgery. outcome excised testis and the distal 3 cm of the spermatic cord were necrotic and adhered to a 6 7 mm thick parietal tunic (figure 2). torsion of the spermatic cord was not apparent. there were large, glossy colonies of bacteria on columbia blood agar and > 500 lactose-negative bacterial colonies observed on macconkey agar. no bacteria grew on cna agar or on anaerobic cultures. infection of the testis and epididymis was determined to be caused by a salmonella spp., based on bacterial cultures (figure 3). extensive necrosis and hemorrhage were apparent on histopathology. bacterial rods colonized the seminiferous tubules, which were separated and expanded by neutrophils (figures 4 6). based on the findings, the diagnosis was a diffuse and necrosuppurative, bacterial orchitis and epididymitis. despite the isolation of salmonella spp. serotype hartford from the stallion’s semen 30 days after surgery, the owner inseminated 4 mares with the stallion’s semen collected 60 days after surgery. the uterus of these mares was lavaged once, 8 hours after breeding with 1 liter of isotonic saline solution containing 1 gram cefazolin (cefazolin, west-ward pharmaceutical corp., eatontown, nj). all mares became pregnant, but one aborted at 60 days of pregnancy; the owner attributed the abortion to a low serum progesterone concentration. fifty colonies of salmonella spp. serotype hartford were cultured from stallion’s semen 4 months after surgery; however, no salmonella spp. were cultured from stallion’s semen 5 months after surgery. the stallion bred 10 mares within 5 years after surgery and all 10 foaled. discussion epididymo-orchitis is commonly reported in humans, most often as a complication of enteric salmonellosis.3-5 salmonella enteritidis caused orchitis in a 10 week old boy who developed swelling of right testis, thought initially to be caused by torsion of the ipsilateral spermatic cord.3 in one report, salmonella enteritidis was cultured from the urine and blood of a patient with epididymo-orchitis who also suffered from systemic lupus erythematosus.4 in the same report, another patient suffering from diabetes type ii developed a testicular abscess from which salmonella enteritidis was cultured. adult 140clinical theriogenology • volume 11, number 2 • june 2019 human patients have developed suppurative epididymo-orchitis after gastroenteritis, with immunocompromised adults potentially at highest risk.5 epididymo-orchitis caused by salmonella in a ram was reported.6 the testis of this ram was infected with salmonella enterica subspecies diarizonae serovar 61:k:1.5, which has been associated with gastroenteritis and abortion in sheep.6 this ram was subfertile and had decreased semen motility and scrotal enlargement due to thickening of the right epididymis and testis, which was painful on palpation. gross and histolopathological abnormalities of the testis and epididymis were similar to those of the testis and epididymis of the stallion herein. to our knowledge, this is the first report of a pure growth of salmonella spp. isolation from necrotizing epididymo-orchitis in a stallion. infection of the epididymis of mature rams, resulting in orchitis, is nearly always caused by brucella ovis, whereas the most common bacterial species causing epididymo-orchitis in immature rams are actinobacillus seminis and histophilus ovis.7 the present report indicates the importance of considering salmonella infection as a cause of an enlarged testis, particularly if the affected patient is immunocompromised. six months prior to presentation, this stallion had developed an infection caused by ehv-2. there are apparently no reports associating infection caused by ehv-2 with bacterial orchitis of horses; however, ehv-2 causes immunosuppression and increases susceptibility to infection by other viral or bacterial agents.8 coincidentally, foals immunized against ehv-2 were more resistant to pneumonia caused by rhodococcus equi than non-immunized controls.9 learning points: ● coagulative and liquefactive epididymo-orchitis caused by salmonella spp. is reported in a stallion, apparently for the first time. ● epididymo-orchitis caused by salmonella spp. may not reduce a stallion’s fertility, if infection is resolved. ● salmonella infection should be considered as a possible cause of testicular enlargement in stallions. ● infection with ehv-2 may be a predisposing factor in development of epididymo-orchitis caused by salmonella. references 1. lopate c, le blanc m, knottenbelt dc: the stallion. in: knottenbelt dc, le blanc m, lopate c, et al: editors. equine stud farm medicine and surgery. 1st edition; philadelphia: elsevier saunders; 2003. p. 79-112. 2. brinsko sp, blanchard tl, varner dd, et al: surgery of the stallion reproductive tract. in: manual of equine reproduction. 3rd edition; maryland heights: elsevier mosby; 2010. p. 242-275. 3. berner r, schumacher r, zimmerhackl l, et al: salmonella enteritidis orchitis in a 10–week-old boy. acta paediatrica 1994;83:992-993. 4. svenungsson b: suppurative epididymo-orchitis due to salmonella enteritidis. j infect 1984;8:64-66. 5. al-obeid k, al khalifan nn, jamal w, et al: epididymo-orchitis and testicular abscess caused by salmonella enteritidis in immunocompromised patients in kuwait. med princ pract 2006;15:305-308. 6. ferreras mdel c, muñioz m, pérez v, et al: unilateral orchitis and epididymitis caused by salmonella enterica subspecies diarizonae infection in a ram. j vet diagn invest 2007;19:194-197. 7. burgess gw: ovine contagious epididymitis: a review. vet microbiol 1982;7:551-575. 8. nordengrahn a, rusvai m, merza m et al: equine herpesvirus type 2 (ehv-2) as a predisposing factor for rhodococcus equi pneumonia in foals: prevention of the bifactorial disease with ehv-2 immunostimulating complexes. vet microbiol 1996;51:55-68. 9. murray mj, eichorn es, dubovi ej, et al: equine herpesvirus type 2: prevalence and seroepidemiology in foals. equine vet j 1996;28:432-436. 141 clinical theriogenology • volume 11, number 2 • june 2019         figure 1. right testis and distal 3 cm of the spermatic cord excised using closed-technique orchiectomy. figure 3. greater than 500 colonies of salmonella were present on a macconkey agar plate. figure 4. extensive necrotic lesions bordered by hemorrhage. figure 5. epididymis with necrosis and scattered bacterial colonies filling the lumen; note bacterial colony (circle) and necrosis (arrow). figure 2. fibrotic, edematous, and partially necrotic testis. figure 6. seminiferous tubules of the affected testis with separation and expansion of interstitial tissue due to infiltration of neutrophils; note bacterial colony in a seminiferous tubule (circle); st, seminiferous tubule; is, interstitial space. 142clinical 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<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> >> /magnification /fitpage /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false 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/preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] /pagelayout /singlepage >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: ramblings of an itinerant theriogenologist 2009 theriogenologist of the year ramblings of an itinerant theriogenologist j. p. kastelic agriculture and agri-food canada, lethbridge research centre, lethbridge, ab, canada introduction the invitation to prepare a written composition for this award creates a novel opportunity for a ‘bully pulpit’ from which to share my story. in that regard, i have chosen to briefly review my background, education, and activities, and as well to share the philosophical principles on which i operate. it is an incredible honor and privilege to be designated as theriogenologist of the year! i am profoundly grateful to the selection committee, and to my colleagues who nominated and supported me. furthermore, i am deeply indebted to all those who have helped me in ways big and small; it is no exaggeration to say that i accept this award on behalf of numerous persons who have been part of my world. early years and family life is a journey; what an incredible ride it has been for me! i had a very humble start on a small dairy farm near edmonton, alberta, canada. i am the youngest of four children and am eternally grateful to my parents, john and marge kastelic, who taught me the value of hard work, honesty, and the pursuit of excellence. although neither of my parents had the opportunity to complete high school, they have incredible wisdom and life experience. they strongly believed in the value of a good education, and they take enormous pride in the accomplishments of their children, grandchildren, and great39 grandchildren. furthermore, as they approach their 65th wedding anniversary, they are incredible role models of fidelity and long-term commitment. my wife rose and i have six children. angela is a nurse, currently working on a master’s degree and nurse practitioner certification. rachel has training as a child care worker, is married, and lives in new zealand. our next three sons are professional musicians; all are classically trained violinists. john ‘migrated’ to the viola, and is majoring in music theory and composition. tony also switched to the viola and is majoring in viola performance and music theory. gabe continues to play violin and will start his post-secondary studies in the fall. david is still in high school and is interested in the outdoors and farming. rose chose to stay home to raise our children, and for sixteen years, has supervised the education of our children, including home schooling and cyberschooling (computer-based schooling). as the demands on rose’s time for educating our children have diminished, she is now pursuing a long-term goal of learning to play cello and will attend university part-time to pursue language studies. as a teenager, i recall reading the advice that “if you prepare yourself appropriately, your way in the world will become very clear to you.” although i initially lacked the wisdom to understand the implications of that statement, i never forgot it. i certainly never imagined the opportunities and experiences that have come my way. i keep ‘morphing’ into the next phase of my life, from a student, to a veterinarian, scientist, editor, and writing consultant. remarkably, my previous experiences prepare me for the next phase. i am unsure what lies ahead, except that i am incredibly excited, as i have every anticipation that ‘the best is yet to come.’ 40 education i became a veterinarian (university of saskatchewan, 1982) and spent two years in private veterinary practice (primarily cattle). i was interested in specializing in dairy practice and i assured my wife that i would return to my alma mater to ‘spend a couple of years and then get a real job again.’ that plan was soon abandoned when i recognized that i needed training to the phd level for long-term opportunities and job security. when i mentioned this to prof. reuben mapletoft, he suggested that i go to the university of wisconsin-madison. my first reaction was to dismiss this as sheer folly, but the next day, with my wife’s blessings and reuben’s help, i was on the phone with prof. o.j. ginther. i was soon accepted into his laboratory, and arrived in madison in late june, 1985. in collaboration with gregg adams (we were classmates in both veterinary and graduate school), i worked on mares for one breeding season, but thereafter worked exclusively on cattle. the training i received at madison has created life-long opportunities. in 1990, i completed my studies and started working as a research scientist for the canadian federal department of agriculture (similar to usda). professional activities i passed the qualifying examination of the act in 1994; there were seven successful candidates that year, though i was the only one with a y chromosome! i am enormously grateful to be a member of the act. although my background and ongoing activities and contributions are different than most other diplomates, this is a very diverse group, and i have always felt very well accepted. an invitation to present a lecture on ultrasonography in cattle at the brazilian embryo transfer society in 1995 was followed by a ten-month sabbatical (with my 41 family) to brazil in 1997, and subsequently several other visits to south america. i have also been able to make several trips to europe, primarily hungary. in 2007, i was deeply honoured to receive a professor of honorary cause from the faculty of veterinary science, szent istván university, budapest, hungary. remarkably, this institution started in 1787, making it one of the oldest veterinary schools in the world. from 2001 to 2008, i served on the act examination committee. this was an incredible experience, which has fostered cherished friendships. furthermore, at breakfast during an exam planning meeting in nashville, i casually mentioned to mats troedsson that ‘being editor of theriogenology would be my dream job’; this ultimately resulted in a gracious invitation from the late vic shille to serve as his replacement. i travelled to gainesville in early january 2003, and spent a few days with vic as i assumed the position of co-editor-in-chief. in this capacity, i get manuscripts from much of the planet (except europe and africa; those are handled by my colleague co-editor-in-chief, prof. fulvio gandolfi, in milan, italy). at present, my duties include receiving an average of six submitted manuscripts each week, deciding which ones will be sent to review, finding reviewers, interacting with reviewers and authors, and making decisions regarding the suitability of manuscripts for publication. i also do final edits on an average of at least three papers each week, including submitted manuscripts which i accepted, and proceedings papers which were guided through the review and revision by a guest editor. from 2005-2008, the proceedings of the annual sft conference were published in theriogenology. for each of those four years, i had personal responsibility to do the final edits on the proceedings articles prior to publication. i really appreciated the efforts of the authors, reviewers, and guest editors for their involvement in this process. in addition, i 42 was profoundly grateful for the many compliments that i received from members of the society and college regarding the professionalism and high standard that was established with publication of these proceedings in the journal. i have every confidence that this will continue, now that the proceedings will be published under the auspices of the new journal being launched by the society. science and scientific writing my journal appointment provides experience, credibility, and funds to support my other passion; giving lectures and workshops on science and science writing, primarily to those for whom english is not their first language. we expect scientists to conduct and communicate science, but few graduate programs have formal training in scientific writing; i was profoundly lucky to be mentored by prof. ginther, an incredibly skilled and prolific writer. furthermore, to get international recognition, it’s almost mandatory to publish in english. writing in your native language is difficult, whereas writing in a nonnative language is profoundly challenging. remarkably, despite the great need for training in scientific writing, especially for those for whom english is not their native language, there is a paucity of assistance available. i have a substantial publication record (author or co-author of >100 peer-reviewed papers and >200 abstracts, proceedings, reports, etc.), but i have always felt that i am a better editor than a writer. i gave my first presentation on scientific writing at the veterinary school in botucatu, sp, brazil in 1997, followed by a presentation in budapest in 2004. i have subsequently given presentations on scientific writing in these two countries, as well as seven others, in north and south america, europe, and asia. on several occasions, i have done these in collaboration with my wife rose. although 43 english is her native language, rose spoke primarily german before she attended school, has an undergraduate degree in french, spent one year attending university in france, and also has a masters in political science. when we do writing workshops together, rose covers the principles of writing in english, including sentence structure, punctuation, grammar, paraphrasing, and preparing outlines, whereas i cover the principles of conducting science and preparing manuscripts. we genuinely enjoy travelling and working together; most of the time, we are able to include some sightseeing into our trips. philosophy my formal training is in veterinary medicine and reproductive biology, with only a few humanities options, and certainly no formal training in philosophy. in the paragraphs that follow, i have summarized some of my basic philosophies and guiding principles. it is noteworthy that these notes are derived in part from a brief presentation on ethics and philosophy that i usually include in a scientific writing workshop (when time permits). the primary purpose of sharing these ideas is not so that they are universally accepted and adopted, but rather to challenge others to contemplate these issues and formulate their own approaches. education, knowledge, and wisdom although education, knowledge and wisdom are clearly related, i view them as distinctly different. veterinarians spend many years in school, learning a myriad of facts. whereas knowledge is specific information, i really like the definition that education is what you remember after you have forgotten the details. furthermore, i regard wisdom as the ability to apply knowledge. we all know many persons that are highly educated, but 44 lack wisdom. conversely, there are many persons with a paucity of formal education that are extremely wise and have much to contribute. the people factor although much of veterinary medicine is focussed on work with animals, most veterinarians spend considerable time interacting with people. consequently, personal rapport and communication skills are very important, both for oral, as well as written communications. although clinicians and scientists historically often worked as independent professionals, it is becoming much more common to collaborate with others. for a successful collaboration, communication is critically important, along with a clear understanding of responsibilities, expectations, and benefits. in general, long-term collaborations are usually successful only if all persons involved contribute and derive a tangible benefit. personal vision although most companies and organizations have a mission statement, in many cases, it’s debatable whether this mission statement has relevance in day-to-day activities. regardless, i strongly encourage everyone to develop a personal mission statement, and to use it to guide your activities and decisions. my own mission statement is the essence of simplicity; my mission is to help people. thus, i spend much of my day interacting directly with family, colleagues, coworkers, students, and members of society, and indirectly with people from around the world. i recall the late otto radostits saying that if you want to get something done, you ask the busiest person that you know; they will usually agree to help, and will usually do 45 so. conversely, the person who has truly little to do will typically give you an extended litany regarding how busy they are, and they are highly unlikely to assist you. it’s critically important to establish goals; everyone should have short-, medium and long-term goals. these goals should cover a broad range, from short-term practical ones, to others which are fanciful. regardless of the nature of the goal, in the absence of a plan to achieve it, you have a fantasy, and not a dream. furthermore, it is wise to be careful what you wish for, as it may come to pass! problem solving numerous times each day, all of us encounter problems, challenges, and requests for assistance. although it’s easy to feel completely overwhelmed, the key is to keep things in perspective. to cope with these demands, i employ a two-step approach. i first determine if this is something that i can address. although there are many things that i dislike or disagree with, by recognizing that i have no ability to change them, i put them aside, and avoid considerable personal angst and frustration. for things that i can address, on the basis of urgency, importance, and my own schedule, i determine an appropriate time frame for my response. although this approach may seem simplistic, it has enabled me to face a seemingly overwhelming barrage, put aside that which i cannot influence or control, and develop a manageable schedule of response for things which i can affect. it’s mostly attitude it’s been said that your life is approximately 10% what happens to you and 90% how you respond to it. in that regard, happiness is mostly a choice; you will be as happy or unhappy as you choose to be! bad things happen to good people on a daily basis, and life is frequently not fair. there is a well defined series of reactions in response to 46 tragedy; shock, disbelief, denial, anger, and eventually acceptance, the resolve to move ahead, and restoration of normalcy. there are two key issues regarding this series of reactions. firstly, trying to avoid the steps, for example, trying to move ahead without grieving, are usually not successful. secondly, although some events are so profound and tragic that you many never fully recover, prolonged angst and perpetually feeling that you are a victim can be very debilitating. all of us know persons that are consistently upbeat and cheerful, often despite many challenges and difficulties. how do they do it? for myself, i make a conscious effort to adopt a positive outlook as my default mode. furthermore, when confronted with adversity, i try to keep calm and maintain perspective; most things which initially seemed like mountains, are truly speed bumps when given sober second thought. dare to be different it’s been said that it takes ‘different’ people to make a difference. furthermore, i recall one of my mentors telling me that “it’s not conventional, but that never stopped us in the past!” thus, i frequently take an unorthodox approach to many of my tasks and opportunities. furthermore, i frequently encourage others to pursue novel approaches. professionalism veterinarians are highly trained medical personnel, with an expectation of being regarded by society as professional persons. as veterinary professionals, we are obligated to maintain a high level of knowledge and skill, and to provide services that are in the best interests of our patients and clients. however, in my view, professionalism is not merely conferred by accomplishment or education, but rather it is something that we earn in accordance with our conduct, including how we treat our patients, and arguably, more 47 importantly, how we treat others, including clients, staff, and colleagues. i firmly believe, that as professionals, we are held to a higher standard of conduct, in all aspects of our lives, both in our work and our personal lives. in that regard, unscrupulous behaviour in our personal conduct results in a substantial loss of credibility in our work. work-life balance as the world becomes more interconnected and complicated, the overall pace of our lives seems to increase. many working people have challenges maintaining a healthy work-life balance; veterinarians are no exception, and for many, emergency calls may make it even more difficult, particularly for those who have few or no associates who share emergency duty. although there are many articles and pundits who claim that ‘you can do it all,’ in my view, that is not realistic. i firmly believe that everything has consequences, both good and bad. for example, a speaking engagement in a foreign country provides me an opportunity to help others, to meet new persons, and to see a new place. however, it also means time away from my responsibilities and activities, both personal and professional. although communications make it possible to stay in contact, for many aspects, they are not a satisfactory substitute. thus, it is critical to honestly consider the true ‘net benefit’ of any action or decision. furthermore, it is foolhardy to endlessly postpone commitments or actions, as they may never happen. we all have to take personal responsibility for our own health and wellbeing, both mental and physical, including development of appropriate leisure activities. the consequences of failing to address these needs can be truly tragic. 48 21st century the world has changed dramatically in the last few decades, and if anything, the pace of change is accelerating. in its current iteration, the world is an exciting, but complicated place! communication and travel are ‘shrinking’ the world; we have unprecedented opportunities to share ideas, knowledge, and experience with others, both electronically, or within a few hours of travel, we can be virtually anywhere on the planet. thus, there are literally endless possibilities for all of us, in particular for youth and young adults. concurrently, the challenge is to carefully sift through all the ‘noise’ and ‘fluff’, and to capture the real ‘nuggets’, and ultimately make good choices and decisions, based on correct and reliable information. in step with the rise of the ‘information age’, there has been an increased emphasis on marketing. i recall reading the liner notes on a music cd, where the artist acknowledged her ‘marketing team.’ thus, her incredible commercial success was due not only to her substantial talent, but concurrently to a clever group of people with detailed knowledge of marketing. in my view, many other entities (not just musical groups, but the world in general) have very little apparent talent, substance or inherent value, yet they are incredible commercial successes, due primarily to the role of marketing. i think there is an important lesson here for all of us. we live in a highly competitive world and we need to learn to market ourselves, including our knowledge, skills, and our opportunity to provide a service, product, or somehow to add value. i recommend a two-step approach. first, identify your inherent skills, ability, and knowledge, and then work very hard to increase and improve those; to truly achieve to your potential. secondly, market yourself, your skills, and abilities. we have all done 49 this; for example, we had to get good grades and experience to gain entry to veterinary school. as information becomes more readily available and the overall levels of competition continue to rise, we all need to pro-actively enhance our resumes, our knowledge, skill sets, and abilities, so that we can remain competitive. furthermore, in my view, it’s not necessary to put others down to gain a competitive advantage; we need to ‘put ourselves up’, through ongoing evaluation and renewal. conclusion i reiterate my profound gratitude to the selection committee for choosing me to receive this award, to all of those who have helped me along the way, and especially to my family, who provide ongoing love, support, and indeed, a reason to live. it seems appropriate to end with a quote from winston churchill: “we make a living by what we get, but we make a life by what we give.” 50 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true 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documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes false /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2012: an update on vaginal and uterine eversions in cattle an update on vaginal and uterine eversions in cattle lionel j. dawson,a augustine t. peterb adepartment of veterinary clinical sciences, oklahoma state university, stillwater, ok; bdepartment of veterinary clinical sciences, purdue university, west lafayette, in introduction eversion of the vagina or both uterine horns from the vulva are commonly referred to as vaginal and uterine prolapses (from ‘prolabi’ = to fall out) respectively. it is not unusual to encounter these cases in cattle practice, particularly in the developing world. vaginal eversions usually occur before calving, and uterine eversions after calving. there is no relationship between the occurrences of these two conditions. a recent review provided an excellent description on the management of these conditions from the perspective of replacing, repairing, and, if need be, amputating the uterus.1 noteworthy is a detailed description on manual eversion of the uterus to correct a uterine tear, a technique that involves the use of different pharmaceuticals.2,3 the purpose of this review is threefold. primarily, to address these maladies and provide remedies with representative images, both real and created. secondarily, to provide a summary of information reported in the global literature, including practical solutions provided by practitioners. finally, a discussion will follow to address these clinical situations from a productive, fertility, and most importantly, from an animal welfare perspective. keywords: eversion, prolapse, vagina, uterus vaginal eversion eversion of the vagina (figure 1) is a relatively common occurrence, particularly, in cattle compared to other species.4 there appears to be a genetic component to its occurrence in beef cows compared to dairy cows. among the beef breeds, hereford,5 heavier breeds such as charolais and limousin,6 and shorthorns are more affected than others. it has been known that bos indicus breeds are predisposed to this condition along with cervical eversion. many factors have been suggested to contribute to this condition.7-10 the list includes increased intra-abdominal pressure in late pregnancy, extreme cold weather, excess peri-vaginal fat, prior injury to the peri-vaginal tissues, intake of large volumes of poorly digestible roughage, poor vaginal conformation, persistence of the medial walls of the müllerian ducts,11 and changes in hormonal secretions, particularly, increased estrogen as observed in late pregnancy and in estrus. it has been suggested that the higher concentrations of estrogens found during pregnancy can contribute to excessive relaxation and edema of pelvic ligaments that support the vagina. besides the above, incompetence of the constrictor vestibule and constrictor vulvae muscles may have a role7 in the etiology. the condition is observed in embryo donor cows that are exposed to hormonal stimulation of the ovaries. imbalance in calcium and phosphorus, and hypocalcemia has been considered to be a contributing factor by some workers.12 it should be pointed out that the association with hypocalcemia was suggested merely based on the response to calcium therapy than on the clinical signs of hypocalcemia. however, as eloquently suggested,12 it can be tentatively postulated that subclinical hypocalcemia could be derogatory to the internal environment of the physiologically stressed female because of pregnancy and parturition. the majority of vaginal eversions occur in the last few weeks of pregnancy, however, it may occur several months before calving. in some cows, it may occur during estrus. vaginal eversions are classified into four grades according to the severity of eversion and the extent of injury. grade 1 eversion a small intermittent protrusion of the vaginal mucosa through the vulval lips is noticed (figure 1) when the animal lies down. often this tissue becomes dehydrated and traumatized and leads to a grade 2. a grade 1 eversion is readily repaired by applying any one of the retention sutures described later. clinical theriogenology • volume 4 number 2 • june 2012115 figure 1: a grade 1 eversion, vaginal eversion occurs intermittently, particularly, when the animal lies down, reproduced with permission. grade 2 eversion vaginal mucosa protrudes through the vulval lips continuously (figure 2) with the possibility of the urinary bladder getting trapped in the everted organ. if left untreated, grade 2 quickly progresses to a grade 3 eversion. figure 2: a grade 2 eversion, floor of the vagina everts and stays everted. grade 3 eversion the entire vaginal mucosa and cervix protrude continuously through the vulval lips (figure 3) with a trapped urinary bladder and an exposed cervical mucus plug. the cervical seal may liquefy allowing bacterial contamination of the uterus leading to placentitis and fetal death. often these fetuses become emphysematous and cervical dilation is insufficient for a vaginal delivery. this condition is often referred to as a cervical eversion but more correctly should be referred to as a cervico-vaginal eversion. this distinguishes it from the eversion of the cervix alone as sometimes observed in bos indicus breeds. in the latter case, only the cervix everts as a pedunculated mass through the vulva. figure 3: a grade 3 eversion, floor and cervix evert and they stay everted. clinical theriogenology • volume 4 number 2 • june 2012 116 grade 4 eversion a grade 4 is of such a duration that the entire vaginal mucosa appears necrotic and fibrosed (figure 4). infection is so extensive that the urinary bladder may become involved in it and peritonitis is a possible result. figure 4: a grade 4 eversion, either a grade 2 or 3 that has become necrotic and/or fibrotic, reproduced with permission. the objective of treatment in the case of pregnant animals is to replace and retain the vagina within the pelvic canal and to deliver and wean a live offspring. many management and treatment options are described for vaginal and cervical eversions.1,4,7, 9-11,13-33 these can be placed in two general categories: those aimed at permanent reduction while others that afford a temporary solution. it should be mentioned that one approach may not fit all grades. the severity of the eversion, the time to expected delivery, the veterinarian's preference, and the owner's ability or willingness to manage the patient after treatment will dictate the treatment option. the elected initial treatment option may need to be changed after the response of the patient has been assessed. this is particularly relevant, if tenesmus becomes an issue. if the patient is close to parturition, induction is recommended to prevent recurrence prior to parturition. short-term treatment options 1. caslick’s suture 2. buhner stitch 3. bootlace technique 4. horizontal mattress (halstead) retention technique 5. deep vertical mattress technique caslick’s suture: this technique (figures 5 and 6) should be reserved only for a non-irritated grade 1 vaginal eversion that occurs close to parturition and when tenesmus is not expected to be a concern. unfortunately, a simple caslick’s suture in many cases is insufficient. as the intra-abdominal pressure increases, tenesmus begins and the suture is ripped out or the vagina begins to evert below the sutures. further, the vulval softening and stretching that occur closer to parturition contributes to this phenomenon. caslick’s suture may suffice in patients that are not pregnant and have a grade 1 eversion during estrus. this is often the case with embryo donor animals whose ovaries are frequently superstimulated with hormones for the sole purpose of increasing the number of ovulations. clinical theriogenology • volume 4 number 2 • june 2012117 figure 5. a caslick’s suture being applied to a non-pregnant cow with grade 1 vaginal eversion. figure 6: a diagrammatic representation of the caslick’s suture procedure. buhner stitch: this is a very effective treatment for more advanced grades and that are chronic in nature.17,18 to have a successful outcome the stab incisions have to be placed deeply such that the retention line is as cranial to the vulval lips as possible (figure 7). the disadvantage is that it is unforgiving and if assistance is not available when the patient begins to calve, severe trauma to the vulva may occur or the calf is unable to be delivered and dies in utero or the patient may die as a result of uterine rupture and fatal hemorrhage. following epidural anesthesia, the vulva is thoroughly washed with detergent and a 1 cm stab incision on the midline below the vulva and as far forward as practical is made. a second incision is made midway between the anus and dorsal commissure of the vulva (figure 7). the buhner needle is then passed deeply from the ventral incision up one side of the vulva and out through the dorsal incision. the buhner tape (non-wicking) is passed through the eye of the needle and the needle retracted through the ventral incision along with the tape. the procedure is repeated on the opposite vulval lip and the two ends tied leaving about a twofinger opening in the ventral vulva for urination. the preference is to tie a bow knot and then tie the two loops of the bow in a single square knot. this allows the knot to be loosened but to retain the suture so that if the patient should try to evert again it can be replaced and the knot re-tightened. this can also be done in the evenings, if observation of the patient for parturition is not going to occur overnight. clinical theriogenology • volume 4 number 2 • june 2012 118 figure 7: diagrammatic representations of buhner stich procedure. note the locations of dorsal and ventral retention lines. if the procedure is performed properly, the tape should not be visible at the dorsal incision and this helps to prevent feces tracking down the needle paths. it should be stressed to the caretakers that their presence at the onset of calving is crucial for a successful outcome. if observation is a problem, medium sized catgut can be doubled and used as a suture material. it helps in patients that are within ten days of calving as the gut will break during parturition. bootlace technique: this is not a highly preferred technique as the vulval lips are inverted and the suture placement is tedious. however, it is stronger than a caslick’s suture. following an epidural anesthetic procedure and disinfecting of the vulva and surrounding area, 4-5 small eyelets are made with umbilical tape or hog nose rings11 on either side of the vulva in a dorsal to ventral line at the hair to hairless junction on the vulval lips. after placing the eyelets, umbilical or buhner tape is then used to "lace up" the vulva much like a boot (figure 8). as the pattern is tightened, the vulval lips invert. again, the bootlace must be loosened prior to calving to avoid serious trauma to the vagina. clinical theriogenology • volume 4 number 2 • june 2012119 figure 8. diagrammatic representation of a bootlace technique. horizontal mattress (halstead) technique: in this technique, a needle is passed through the vulval lip on the right beginning deep at the junction of the labia with the skin of the rump. the needle is continued across the vulval opening and through the left vulval lip at the same depth as the right. the needle is then passed back from left to right at the same plane as the first passage but beginning approximately 2-3 cm ventral to the first passage. this procedure can be done using a buhner or large cutting needle. place as many sutures as needed to close the vulva down to about two fingers wide to allow for urination. the disadvantage of the technique is that even if the sutures are not pulled really tight, vulval edema frequently develops and can be quite severe. this can be lessened if the suture tension is dispersed by "stents". half inch polyvinyl chloride tubing can be cut the length of the vulva and holes drilled 2-3 cm apart; similarly wooden dowels,24 or latex tubing can be utilized. a stent is placed either side of the vulva and the horizontal mattress suture passed through the holes and tied (figure 9). this approach has good deep retention and more evenly disperses the pressure. figure 9: an example of a horizontal mattress technique, note the location of the stents in the illustration for reduction of vulval edema and for dispersion of pressure. deep vertical mattress technique: this is similar to the horizontal mattress pattern described above. the difference is that a vertical mattress suture pattern is used and stents are recommended to disperse tension on the vulval lips (figure 10). this replacement method can also have serious consequences if clinical theriogenology • volume 4 number 2 • june 2012 120 assistance is not available at the onset of parturition. figure 10 an example of a deep vertical mattress technique applied and buttons are used as stents. permanent treatment options: 1. minchev vaginopexy 2. modified minchev vaginopexy 3. winkler cervicopexy minchev vaginopexy: there are two minchev techniques described that differ only in location of needle insertion. in the original minchev technique the needle is passed through the lesser sciatic foramen. the technique is an excellent prepartum method that aims to retain the vagina in the correct pelvic location by adhesion formation between the sub-mucosa of the vagina and surrounding fascia. an epidural is given and the gluteal area shaved and disinfected. in the original minchev technique, a large s-shaped needle is threaded with ⅜inch umbilical tape and a gauze bandage or large plastic or metal plate is attached at the end of the tape, the needle is then taken in vaginally and the lesser sciatic foramen is located on the dorso-lateral wall of the vagina. the needle is then pushed through the dorsal area of the foramen in order to avoid the pudendal nerve. the needle is pushed through the skin in the gluteal area and the suture pulled up snug (do not tie too tightly) and another button, plate or gauze is tied into the suture. this is repeated on the other side. modified minchev vaginopexy: this technique is similar except the stay sutures are placed anterior to the lesser sciatic foramen 5 cm lateral to the midline and just posterior to the shaft of the ilium, providing more cranial fixation of the vaginal wall (figure 11). care must be taken to avoid the sciatic nerve, pudendal artery and rectum when placing the needle. these structures can be identified cranial and slightly dorsal to the lesser sciatic foramen. the needle is passed through the sacrosciatic ligament approximately 45 cm lateral to the sacrum and just posterior to the shaft of the ileum. there are advantages to this technique, including the fact that cows rarely show tenesmus when the vagina is replaced and the cow can calve unassisted. it is however recommended that the sutures are removed in two to four weeks. a commercial prolapsed repair kit is available (figure 12; pro-button prolapse, jorgensen laboratories, loveland, co). assembled kit is soaked in a disinfectant. the stainless steel pin with the six inch plastic trocar passed through the three inch plastic button is carried vaginally. the trocar is pushed dorsal or anterior to the lesser sciatic foramen pointing dorso-laterally to avoid the rectum, and then through the sacrosciatic ligament and the biceps femoris to the skin outside. the stainless steel pin is removed, and the 2.5 inch plastic button is placed on the protruding trocar. pushing down on the 2.5 inch button, enables the three inch button to be held firmly between the vaginal wall and the sacrosciatic ligament. the cotter pin is inserted through the hole in the protruding trocar. the plastic trocar is cut about an inch above the cotter pin. in severe cases, it may be necessary to place buttons on both sides of the anterior vagina. clinical theriogenology • volume 4 number 2 • june 2012121 however, it should be recognized that recurrence of eversion is possible since only the dorsum of the vagina is held. further, when the internal placement is too far caudal, a partial eversion may occur eliciting straining resulting in total eversion. figure 11: an example of a modified michev technique (top) with a schematic illustration. figure 12: trocar and plastic buttons used for a modified minchev technique. clinical theriogenology • volume 4 number 2 • june 2012 122 winkler technique cervicopexy: this technique33 is a very effective method of vaginal retention because it fixes the cervix in place making it difficult for the vagina to prolapse. the basic procedure is aimed at suturing the cervix to the prepubic tendon. there are numerous descriptions of placing the suture in the cervix and how to suture through the tendon. the two approaches to suture placement are either through a left-paralumbar approach26 or through the vagina. the former is more time-consuming but is safer while the vaginal approach is quicker but can result in broken needles if one is not careful. the vaginal approach could be done in two ways by either including the lower half of the cervix or by just including the external os of the cervix on one side (figure 13). figure 13: schematic representation of a winkler technique for cervicopexy, redrawn with permission. before placing the suture vaginally, an epidural is given and a mild antiseptic douching of the vagina performed. the prepubic tendon is then palpated at its attachment to the pelvic brim. as one moves their fingers laterally about 5 cm on both sides of midline, a small triangular shaped area free of tendon is felt. this is formed by a short band of tendon extending posterolateral to the iliopectineal eminence of the pelvis. a large curved cutting needle is bent into a "u”-shape and 60 inches of non-absorbable suture material is attached. the needle is then carried into the vagina and passed from the inner lumen of the external cervical ring out peripherally. the needle is grasped again and passed down in a medial to lateral direction through the prepubic tendon and carefully curved around to come up through the triangular area. the needle is grasped again, passed through the external cervical ring from the periphery to the center and bought on out of the vagina. some clinicians prefer to pass the “u”-shaped needle (figure 14) through the vaginal wall initially, then pass the needle through the prepubic tendon medial to lateral direction, then pull the needle back through towards the intravaginal part of the cervix, then the needle is directed across the lower half of the cervix and brought out side to tie the knot. this latter procedure may prevent breaking of the “u”-shaped needle. a catheter is then passed through the urethra so that as the suture is tied the urethra and not trapped in the suture. clinical theriogenology • volume 4 number 2 • june 2012123 figure 14: a special needle used for winkler technique for cervicopexy. to tie the suture, the knots are started on the outside of the vulva and slid forward. the suture is tied tight enough to prevent the posterior movement of the cervix. to assist in placing the cervical sutures, knowles' cervical forceps can be used to either fix the cervix cranially making passing of the needle through the cervix easier or in some cows the cervix can be retracted sufficiently to be presented at the vulva again making placing of the cervical sutures easier. other descriptions of this procedure do not advocate passing the needle through the cervical opening but rather through the side of the cervix without penetrating the lumen. this approach is greatly aided by fixation of the cervix with forceps.3,26 the advantages of this approach are that the cow can calve on its own, post-repair tenesmus is rare and the cow can still be examined per vaginum. occasionally the suture in the cervix can break resulting in recurrence of eversion. this procedure is recommended for chronic cases which the owner wishes to keep, especially embryo donor cows in which the problem is likely hormonally induced rather then there being any genetic predisposition. however, if the condition is likely to occur again or if it is probably of hereditary in nature, the affected animal should be culled. uterine eversion uterine eversion occurs mostly within 24 hours after fetal delivery with the majority of cases occurring shortly after parturition. while relatively uncommon (less than 1% of parturitions34), it is nevertheless one of the true emergency situations one encounters in practice. in many instances the everted organ is readily contaminated (figure 15), traumatized physically or by extreme weather (heat/cold), becomes edematous, may hemorrhage and evert severely enough to rupture the uterine vessels leading to rapid shock and death. figure 15: an everted uterus with evidence of contamination. clinical theriogenology • volume 4 number 2 • june 2012 124 owners should be instructed not to transport the animal and to put the everted organ inside a plastic bag to reduce contamination. fortunately, many of these cows are unable to get up and run due to hypocalcaemia, partial paralysis or exhaustion. however, on occasions, the cow is capable of getting up and may become fractious as she attempts to outrun the uterus or knocks the uterus between her hocks (figure 16). since these cases often result in rupture of the uterine vessels and rapid death due to internal hemorrhage, one should approach them very cautiously. figure 16: uterus is further traumatized by knocking between the hocks. many factors8,35 are believed to contribute to uterine eversion including reduced uterine contractility due to decreased uterine activity following primary or secondary uterine inertia. hypocalcemia and reduced prostaglandin and oxytocin receptors and myometrial defects due to overstretching contribute to primary uterine inertia. among others, mere physical exhaustion and dystocia are responsible for secondary uterine inertia. apart from uterine inertia, situations wherein continuing tenesmus as noted in dystocia, manual extraction of calf, retained fetal membrane, delayed cervical involution, and laceration of the reproductive tract can lead to eversions. prolonged recumbency that follows paralysis or hypocalcemia can lead to increases in intra-abdominal pressure, a potential factor for eversion. hypocalcemia is also believed to make the uterus atonic delaying the involution of cervix, a predisposing factor for uterine eversion.36 however, it has been suggested that hypocalcemia can be the result of an uterine eversion rather than a cause of it.37 the role of parity38 and hypocalcemia still remain an enigma in the etiology of uterine eversions.39,40 over-conditioned animals may be more prone to increased intra-abdominal pressure due to increased fat reserves in the abdomen. other non-animal factors can contribute to this condition. for example, outbreaks can occasionally occur due to extreme weather conditions and pasture composition. further it has been suggested that alterations in the availability of dietary calcium, phosphorus, and magnesium may have a role in creating uterine inertia and predisposing animals to this condition. the uterine eversion outbreak noticed in animals kept on a cool-season grass pasture supports this hypothesis. it has also been suggested that high estrogen concentrations in pasture may play a role. the foregoing undoubtedly attests to the multi-factorial nature of this condition. many methods of reduction and practical tips have been described.1,35,36,41-59 replacing the uterus can be done in either the standing or recumbent position depending on circumstances and clinician preference. the animal may have to be cast, rolled, and pushed to lie on its abdomen to facilitate pulling hind legs straight behind to facilitate reduction.35 obviously, if the cow is in the pasture with partial paralysis, the recumbent position is dictated. replacing the uterus in the recumbent position is greatly facilitated by positioning the cow in the sternal position with both hind legs extended out behind (figure 17) with the head end facing downhill (“okie position”). this position tilts the pelvis forward and allows the clinician to get behind the cow and cradle the uterus in their lap as they feed it back into the cow. clinical theriogenology • volume 4 number 2 • june 2012125 figure 17: “okie position”, note the extension and elevation of hind legs. others prefer the standing position (figure 18) if the cow can be adequately restrained and help is available to place a towel or bedsheet (or something similar) under the uterus and lift it up as the clinician replaces the uterus (figure 19) it back into the cow. figure 18: cow is placed in a chute for the standing reduction, a squeeze chute is preferred. figure 19: elevation of the uterus by a sheet helps in reduction. replacement guidelines  give epidural anesthesia  remove as much fetal membrane as possible clinical theriogenology • volume 4 number 2 • june 2012 126  clean the endometrium  apply chlorexidine ointment over the entire uterus and massage  apply a pressure bandage for 10-15 minutes tightened from the tip of the horn. sugar may be applied sparingly. however, care needs to be taken, because sugar will dry the endometrial layer quickly causing the tissue to become friable. this may lead to tears when applying pressure to push in the horns.  depending on dryness the operator may have to apply an ointment that has lubricant and emollient properties  identify the non-gravid horn (strong intercorunal ligament prevents its eversion) by observing for the presence of an oval or slit-like opening bordered by a ring of small cotyledons and reduce it first  begin applying steady pressure at the cervix/uterine body area using both hands with a clenched fist to reduce the everted uterus into the vagina. once the uterine body is reduced into the vagina, the uterine horns are gradually massaged into the birth canal or vagina.  fully invert the horns. if the operator’s arms are not long enough, the rounded bottom of a one liter soda bottle or probang can be used to completely invert the uterus  give non-steroidal anti-inflammatory drugs and parenteral antibiotics  oxytocin should not be given until the uterus has been reduced or replaced  apply a retention suture a probang (figure 20) is commonly used to make sure that the tip of the uterine horn is fully inverted. the rounded edges of probang prevent possible tears that may occur to the friable uterine wall during the procedure. figure 20. a probang that is used to fully invert the uterine horns, note that the ends are rounded. failure to fully straighten the horns can result in re-eversion or ischemic necrosis of a portion of the uterine horn. placement of a suture in the vulva after uterine replacement is controversial. if the uterus responds to oxytocin and the cow is able to stand, then re-eversion is highly unlikely and a vulval stitch is not needed. on the other hand, if the cow is going to be unable to rise and tenesmus recommences, a vulval stitch may be indicated for 24-48 hrs. after reduction, 20 to 30 iu oxytocin should be injected and repeated two more times at 30 minute intervals and calcium therapy instituted if necessary. intra-uterine antibiotics are probably warranted in the case of a severely injured and contaminated uterus but are controversial in the case of a relatively fresh and cleanly reduced eversion. uterine amputation there are two approaches to uterine amputation, open and closed.1,2,60,61 the open approach involves incising the uterine body and location and ligation of major uterine vessels in the stretched broad ligament prior to amputation of the uterus. the advantage of this approach is that any viscera contained within the prolapse can be returned to the abdomen before the uterus is actually removed. with the surgical clinical theriogenology • volume 4 number 2 • june 2012127 approach, the wall of the uterine body is opened from the cranial cervix to the bifurcation of the uterine horns to expose the contents within the everted uterus and the major vessels in the broad ligament (mesometrium). the latter are identified by "fanning out" the mesometrium and the vessels double ligated, preferably when they are not under tension. the vessels are severed between ligatures and the mesometrium incised and allowed to retract into the abdominal cavity. a series of adjacent slightly overlapping mattress sutures are then placed across the uterine body just proximal to the cervix. the uterus is then amputated 2-3 cm below this line of sutures. the stump may then be sutured with a simple continuous pattern. postoperative treatment should include administration of tetanus toxoid, non-steroidal anti-inflammatory drugs, and appropriate antibiotics. in the closed approach (figure 21) the entire organ is ligated and left to slough in 10-14 days. this is a salvage operation applied in cases of severe ischemic necrosis and or lacerations. with this approach, viscera could be unintentionally trapped in the everted uterus with fatal consequences, ligatures for this latter approach can be made of surgical tubing, bungee cord or the bloodless castrating unit used for castrating large bulls works well. figure 20. a closed approach of uterine amputation, note the location of the ligation. discussion vaginal eversion prepartum vaginal and cervical eversions can be perplexing and disturbing problems both to the producer and veterinarian. the expense and effort can be costly at times. these everted structures should be promptly replaced within the vaginal vault and secured such that contamination, laceration, sepsis, fibrosis or necrosis does not occur. this approach will prevent compromise of cervical and vaginal relaxation at the time of parturition and most importantly will enhance the animal well-being by preventing unnecessary injury at the time of parturition.62 some attempts made by producers to correct the situation can have many unintended consequences and can inflict unnecessary pain and injury to the animal. in this regard, client education is paramount. although there may be temporary relief the animal may evert again at the next parturition if the primary cause for an eversion is not corrected.11 it is very important to realize that satisfactory reduction and retention alone are not sufficient since the recurrence rate is high if the primary or inciting cause is not removed. this not only forces subsequent repair but more importantly, in some instances, can result in mutilation of vulval and perivulval tissue, and become an important animalwelfare issue. many techniques have been developed and stood the test of time. each one is unique and the technique chosen depends on the condition and expertise of the clinician. what has been said a few decades ago still stand true, better techniques for handling chronic vaginal eversions are needed.28 new knowledge in this area by developing alternate techniques and improving current techniques will keep our discipline vibrant. not many studies have characterized the future fertility of affected animals. however, we ought to consider the possibility of future occurrence of the condition in light of animal well-being rather than fertility. in this regard, it is pertinent to consider some of the treatments administered. these are aimed in clinical theriogenology • volume 4 number 2 • june 2012 128 preventing recurrence by narrowing the birth canal6,21 by creating adhesions between retroperitoneal surfaces of the vaginal and adjacent or subjacent structures. these methods can prevent recurrence but may result in injury to the animal at the time of parturition. further, should these animals conceive, subsequent pregnancies are frequently complicated by recurrence. hence, consideration should be given to culling affected animals after their calves are weaned.9 animal health advisors have an important role to play to help the producers to make a decision to eliminate these animals from the breeding herd. considering the injury the animal may endure at the present reduction and subsequent reductions, it behooves us to strongly suggest removing such animals from the herd.62 uterine eversion the appearance of a heavy mass of tissue outside the body of an animal can be intimidating and at times the process of reduction can be overwhelming. truly the prospect of performing a successful reduction and the thought of recurrence can place one in mental anguish. we always look for ease of the operation and reduced physical effort on our part to reduce the eversion. in an attempt to facilitate the process of reduction mechanical aids have been used successfully.48,50,56,57 however, raising the rear end of a live cow may appear inhumane.46 next, the application of agents such as sugar45,58 or salt52 to the everted tissue to reduce the edema spectacularly before reduction is not supported uniformly. although the authors prefer a careful application as supported by others in practice,45 it remains controversial8 since it can amplify endometrial trauma. this technique may have certain academic skepticism despite its benefits. the chance of immediate recurrence can be eliminated if the reposition is done completely without allowing invagination of the uterine horn to occur at the ovarian pole. overall, the mortality rate ranges from 18-20 %.8,36 the prognosis for survival and future reproductive performance depends on several factors, but time from the occurrence to treatment is critical for a positive outcome. despite the extensive trauma that may occur before replacement, those that are replaced correctly and survive, have an acceptable prognosis for future fertility.36,63 occasional bladder eversion and intestinal entrapment that follow into the everted uterus may result in a poor prognosis. timely intervention by a professional can minimize animal discomfort and economic loss to the producer. treatment given by the uninitiated nonprofessional has to be discouraged since severe damage can be inflicted to the animal.44 a unique retention technique developed to prevent recurrence59 should be considered to obviate situations such as necrosis, gangrene, severe trauma, necessitating amputation. the chance of recurrence at the next parturition has been discussed 2,64 and the heritability or additive susceptibility with subsequent pregnancies for this condition is not apparent.1 since no known heritable component is recorded there is no increased probability of occurrence at subsequent parturition. therefore one should not be too hasty in recommending salvage of affected cows. the chance of recurrence is minimal and this has to be discussed with the producer. it should be remembered that in the developing world, cattle are an economic asset and the cost of therapy must be balanced against the value of the animal. besides the value of the animal, decisions to keep the animals in the herd is based on their future fertility. the major factor that can influence in this decision is the degree of trauma caused to the uterus and the time it has taken to repair.36 future fertility of cows is reduced and there is a slight increase in risk at the subsequent calving, but the increased risk is not sufficient to justify culling.2 as the profession is emphasizing more and more into animal health and production than restorative therapy, it is incumbent on us to prevent the occurrence of these conditions by careful selection of breeding stock, observing quality animal husbandry methods, providing adequate care to periparturient animals, and finally eliminating potential recurring cases. all the above will enhance the animal well-being and place the profession as animal welfare contributors. acknowledgements the authors thank their mentors (listed in alphabetical order) for providing them an opportunity to learn the art and science of bovine reproduction: drs. c. bierschwal, w. bosu, r. elmore, l. faulkner, v. naidu, k. narasimhan, j. rajasekaran, l. rice, and r. youngquist. dr. g. morgan’s help in providing the information on classifying vaginal eversion is acknowledged. the illustrations are created by dr. t. clinical theriogenology • volume 4 number 2 • june 2012129 barkley of woodward, oklahoma and her help is much appreciated. figure 1 and 4 are reprinted from the color atlas of diseases and disorders of cattle by r. blowey and a. weaver with permission from elsevier. figure 13 is redrawn from the techniques in large animal surgery by d. hendrickson with permission from john wiley and sons. references 1. miesner md, anderson de: management of uterine and vaginal prolapse in the bovine. vet clin north am food anim prac 2008;24:409-419. 2. youngquist rs: surgical correction of abnormalities of genital organs of cows: uterine prolapse. in: youngquist rs, editor. current therapy in large animal theriogenology. philadelphia: wb saunders company; 1997. p. 433-434. 3. hopper rm: surgical correction of abnormalities of genital organs of cows: management of uterine prolapse. in: youngquist rs, threlfall wr, editors. current therapy in large animal theriogenology. 2nd ed. philadelphia: wb saunders; 2004. p. 382-390. 4. roberts sj: vaginal prolapse. in: veterinary obstetrics and genital diseases, 2nd ed. published by the author, ithaca, new york, 1971. p. 189-196. 5. woodward rr, quesenberry jr: a study of vaginal and uterine prolapse in hereford cattle. j anim sci 1956;15:119124. 6. coulthard h: treatment of bovine vaginal prolapse. vet rec 1991;129:151. 7. hudson rs: genital surgery of the cow. in: morrow da, editor. current therapy in theriogenology. 2nd ed. philadelphia: wb saunders company; 1986. p. 346-350. 8. roberts sj: uterine prolapse. in: veterinary obstetrics and genital diseases. 2nd ed. ithica (ny): published by the author, 1971.p.308-313. 9. youngquist rs: surgical correction of abnormalities of genital organs of cows: vaginal prolapse. in: youngquist rs, editor. current therapy in large animal theriogenology. philadelphia: wb saunders company; 1997. p. 436-437 10. wolfe df, carson rl: surgery of the vestibule, vagina, and cervix. in: wolfe df, moll hd, editors. large animal urogenital surgery. 2nd ed. baltimore: williams and wilkins; 1998. p. 398-410. 11. johansen rd: repair of prolapsed vagina in the cow. vet med small anim clin 1968;63:252-256. 12. dhanotiya rs, srivastava rk, pandit rk: a note on postpartum utero vaginal prolapse in gir cows: estimation of serum calcium, phosphorus, proteins, and cholesterol. arch exper vet med 1989;43:79-80. 13. arthur gh: recent advances in bovine obstetrics. vet rec 1966;79:638-639. 14. baker gj: advances in large animal surgery – part 2. vet rec 1967;81: p ii. 15. minchev p: the use of a new surgical method in eversion and prolapse of vagina in animals. veterinariya, 1956;33:58-60 (abstracted in j am vet med assoc 1957;130:344). 16. bouckaret jh, oyaert w, wijverkens h, et al: prolapse vaginae bij het rund, vlaams diergeneesk. tijdschr 1956;25:119132 (abstracted in j am vet med assoc 1957;130:344). 17. buhner f: simple surgical treatment of uterine and vaginal prolapse (translated from german) tierdraztl umsch 1958;13:183-188. 18. bierschwal cj, debois chw: the buhner method for control of chronic vaginal prolapse in the cow (review and evaluation). vet med small anim clin 1971;66:230-236. 19. dhillon ks, singh bb, kumar n, et al: treatment of vaginal prolapse in cows and buffaloes. vet rec 2006;158:312. 20. habel re: prevention of vaginal prolapse in the cow. j am vet med assoc 1957;130:344-345. 21. hattangady sr, deshphande ks: case reports on “through and through” stay suture technique for retention of vaginal prolapse. indian vet j 1967;44:528-530. 22. hentschl af: the button technique for correction of prolapse of the vagina in cattle. j am vet med assoc 1961;139:1319-1320. 23. kerz pd: correction of vaginal prolapse in the bovine. vet med small anim clin 1966;61:888-889. 24. lamp jh, lamp tm: a method for correcting vaginal prolapse in a cow. vet med small anim clin 1981;76:395-396. 25. milne fj: efocaine – an aid in the treatment of bovine vaginal prolapse. j am vet med assoc 1954;124:108-111. 26. noordsy jl: cervopexy as a treatment for chronic vaginal prolapse in the cow. vet med small anim clin 1981;76:383385. 27. norton es: external fixation of the bovine vagina after reduction of a prolapse. j am vet med assoc 1969;154:11791181. 28. pierson re: a review of surgical procedures for correction of vaginal prolapses in cattle. j am vet med assoc 1961;139:352-356. 29. sah sk, nakao t: some characteristics of vaginal prolapse in nepali buffaloes. j vet med sci 2003;65:1213-1215. 30. pittman t: practice tips. can vet j 2010;51:1347-1348. 31. rautenbach gh: the retention of vaginal prolapse in the cow using a purse-string suture. j s afr vet assoc 1984;55:203204. 32. narasimhan ks, quayam sa, gera kl: a method of retention of recurrent prolapse of the vagina in cows. indian vet j 1975;52:311-313. 33. winkler jk: repair of bovine vaginal prolapse by cervical fixation. j am vet med assoc 1966;149:768-771. clinical theriogenology • volume 4 number 2 • june 2012 130 34. roine k, saloniemi h: incidence of some diseases in connection with parturition in dairy cows. acta vet scand 1978;19:341-353. 35. plenderleith b: prolapse of the uterus in the cow. in pract 1986;1:14-15. 36. odegaard sa: uterine prolapse in dairy cows. a clinical study with special reference to incidence, recovery and subsequent fertility. acta vet scand (suppl)1977;63:1-124. 37. richardson gf, klemmer ad, knudsen db: observations on uterine prolapse in beef cattle. can vet j 1981;22:189-191. 38. markusfeld o: periparturient traits in seven high dairy herds, incidence rates, association with parity, and interrelationships among traits. j dairy sci 1987;70:158-166. 39. risco ca, reynolds jp, hird d: uterine prolapse and hypocalcemia in dairy cows. j am vet med assoc 1984;185:15171519. 40. patterson dj, bellows ra, burfening pj: effects of caesarean section, retained placenta and vaginal or uterine prolapse on subsequent fertility in beef cattle. j anim sci 1981;53:916-921. 41. caldwell hs: eversion of uterus in a maiden heifer. vet rec 1933;29:688-689. 42. baxter k: replacing the prolapsed bovine uterus. vet rec 2004;155:344. 43. biggs a, osborne r: uterine prolapse and mid-pregnancy uterine torsion in cows. vet rec 2003;152:91-92. 44. bullard jf: a case of uterine prolapse in a hereford with unusual lay treatment j am vet med assoc 1946;109:462. 45. douch j: uterine prolapse in the cow. vet rec 1986;118:310. 46. parker cd: uterine prolapse in the cow. vet rec 1986;118:310. 47. formston c: uterine prolapse in the cow. vet rec 1986;118:310. 48. foster sj: a pneumatic appliance for the replacement of the prolapsed uterus of the cow. vet rec 1972;91:418. 49. hibberd rc: replacing the prolapsed bovine uterus. vet rec 2004;155:96. 50. johnston rw: uterine prolapse in the cow. vet rec 1986;118:252. 51. levine hd: partial uterine prolapse associated with uterine foreign body in a cow. j am vet med assoc 1990;197:759760. 52. lyons ar: uterine prolapse in the cow. vet rec 1986;43:79-80. 53. garcia-saco e, gill ms, paccamonti dl: theriogenology question of the month: laparoscopy to assist replacement of the uterus. j am vet med assoc 2001;219:443-444. 54. abdullahi us, kumi-diaka j: prolapse of the nongravid horn in a cow with a seven-month pregnancy: a case report. theriogenology 1986;26:353-356. 55. gardner ia, reynolds jp, risco ca, et al: patterns of uterine prolapse in dairy cows and prognosis after treatment. j am vet med assoc 1990;197:1021-1024. 56. white a: uterine prolapse in the cow. uk vet 2007;12:1-3. 57. wilson pj: a pneumatic appliance for the replacement of the prolapsed uterus of the cow. vet rec 1972;90:729-730. 58. munro ib: replacing the prolapsed bovine uterus. vet rec 2004;155:344. 59. narasimhan ks, thangaraj tm, subramanyam r: a method of retention of recurrent prolapse of the uterus in bovines. indian vet j 1967;44:67-73. 60. wenzel jg, baird an, wolfe df, et al: in: wolfe df, moll hd, editors. large animal urogenital surgery. 2nd ed. baltimore: williams and wilkins; 1998. p. 423 61. roberts sj: amputation of the prolapsed uterus. cornell vet 1949;39:438-439. 62. wolfe df: theriogenology to enhance animal well-being. clin therio 2011;3:180-184. 63. jubb tf, malmo j, brightling p, et al: survival and fertility after uterine prolapse in dairy cows. aust vet j 1990;67:2224. 64. murphy am, dobson h: predisposition, subsequent fertility, and mortality of cows with uterine prolapse. vet rec 2022;151:733-735. (editor’s note: many of the photographs in this manuscript are available in color in the online edition of clinical theriogenology.) clinical theriogenology • volume 4 number 2 • june 2012131 2012: value of specific history and physical examination findings and diagnostic test results for differentiation of benign from malignant prostate disease in dogs value of specific history and physical examination findings and diagnostic test results for differentiation of benign from malignant prostate disease in dogs margaret v. root kustritz college of veterinary medicine, university of minnesota, st. paul, mn abstract electronic medical records with a final diagnosis of prostate disease were retrieved from the electronic medical record system of a veterinary academic referral hospital. data gathered included signalment, age at time of admission, clinical signs including gross hematuria, stranguria or dysuria, dripping of bloody fluid from the prepuce unassociated with urination, urinary incontinence and inappropriate urination, rectal tenesmus, and passage of ribbon-shaped stools, and signs of systemic disease. presence of red blood cells (rbcs) and epithelial cells in urine sediment and of mineralization and regional lymph node enlargement on transabdominal ultrasound also were recorded. ninety-nine cases were reviewed. the history and diagnostic findings determined to be significantly associated with benign prostatic hyperplasia/hypertrophy (bph) rather than with prostatic neoplasia were being sexually intact (p > 0.0001) and dripping bloody fluid from the prepuce unassociated with urination (p = 0.007). the history and diagnostic findings determined to be significantly associated with prostatic neoplasia rather than with bph were being castrated (p < 0.0001), stranguria or dysuria (p < 0.0001), rectal tenesmus (p = 0.0009), systemic signs of disease (p = 0.004), and mineralization of the prostate and regional lymph node enlargement visible with transabdominal ultrasound (p < 0.0001 and p = 0.0002, respectively). keywords: prostate, neoplasia, benign prostatic hypertrophy, prostatitis, diagnosis introduction prostate disease is common in men and in dogs. many pet owners are aware of diagnostic and treatment options for human prostate disease and expect their veterinarians to be aware of how those diagnostic tests and therapies work in dogs. the canine prostate encircles the neck of the urinary bladder and is bounded dorsally by the rectum and ventrally by the symphysis pubis and ventral abdominal wall.1 histologically, it consists of two lobes separated by a median raphe; the lobes are separated into lobules made up of secretory epithelium and supportive tissue.1 blood supply is via the prostatic artery, with the dorsal and periurethral areas better vascularized than the ventral portion of the gland.2 there is relatively little smooth muscle compared to the human prostate gland, with distinct differences in distribution and type of muscle at the bladder neck, and there is relatively more glandular tissue.3,4 the canine prostate gland is surrounded by a well defined capsule; histologic studies disagree as to whether a true capsule surrounds the human prostate.1,2,5 secretory function and increase in size of the prostate are androgen-mediated. a significant difference between human male and canine male populations in the united states in regards to prostate disease is intact status; very few men are castrated or have gonadal dysfunction such that they secrete no androgens, while 31.8% of dogs are castrated in the united states.6 benign prostatic hypertrophy/hyperplasia is very common in men; more than 40% of men over 60 years of age have lower urinary tract signs associated with bph.7 benign prostatic hyperplasia/hypertrophy is the most commonly reported disorder of the prostate in dogs.8-10 it is well documented that 50% of intact male dogs have histologic evidence of bph by five years of age.11 benign prostatic hyperplasia/hypertrophy has not been reported in castrated dogs. the primary metabolite of testosterone, dihydrotestosterone, is the androgen that mediates prostate hypertrophy and hyperplasia.12,13 in dogs, the prostate gradually increases in size until four to six years of age, plateaus, and then increases in size again until it begins to atrophy in very aged dogs.11,14,15 the increase in size associated with age is due to a decrease in androgen secretion in the face of continuing estrogen secretion.14 as the prostate increases in size, it may pull the urinary bladder forward into the abdominal cavity, making it difficult or clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201249 clinical theriogenology • volume 4 number 1 • march 2012 impossible to assess on digital rectal examination. the primary clinical sign of bph in men is difficulty in completely emptying the urinary bladder. this clinical sign is less likely to develop in dogs because of the relative lack of activation of smooth muscle cells in the prostate compared to that in men, and subsequent lack of urethral constriction.16 benign prostatic hyperpertrophy/hyperplasia in dogs more commonly is associated with increased size and secretory function of the gland and so has been reported to be evidenced clinically by dripping of bloody prostatic fluid from the urethra unassociated with urination, hematuria, rectal tenesmus, and passage of ribbon-shaped feces.17-20 prostatic neoplasia is the second leading cause of cancer-related death in men.21 there is histologic evidence of prostatic neoplasia in about one-third of men over 50 years of age and in 90% of men over 90 years of age.21 overall incidence of prostatic neoplasia in dogs is reported as 0.7%.22 prostatic neoplasia has been reported in intact and in castrated dogs; castration increases risk of development of prostatic carcinoma by a factor of two to four times.8,23-26 prostatic neoplasia has a different biology in men than in dogs. men develop an androgen-responsive, fairly benign form of prostatic cancer early, followed by a non-androgen-responsive, more aggressive form later in life. dogs develop only non-androgen-responsive, aggressive prostatic neoplasia.27 growth pattern of prostatic neoplasia differs between castrated and intact dogs.28 the association between castration and development of prostatic neoplasia in dogs has not been explained. clinical signs reported in dogs with prostatic neoplasia include stranguria and dysuria, hematuria, rectal tenesmus, anorexia and weight loss, rear limb lameness, and polyuria and polydipsia.18-20,25,29-33 prostatitis is infection of the prostate gland and commonly occurs secondary to either bph or neoplasia as those alterations in anatomy and function of the gland overcome mechanisms that prevent movement of normal bacterial flora from the distal urethra into the prostate. prostatitis may be diffuse or may be localized as an abscess. prostatic cysts also are reported, again varying from diffuse small cysts scattered throughout the gland, often associated with bph, to large, localized cysts that may be within or outside the gland parenchyma. in humans, evaluation for prostate disease by a physician is recommended for all men between the ages of 50 and 75 who have a life expectancy of at least ten years.34 the recommended diagnostic tests are a complete history; digital rectal examination to evaluate prostate size, shape, and consistency, and to assess for pain; urinalysis; and assessment for serum concentration of prostate specific antigen (psa).35 this is recommended for all men, whether or not they are symptomatic, and further diagnostic testing, such as ultrasound and aspirate or biopsy of the prostate, are recommended only in men with symptoms of prostate disease or elevated psa concentrations. in dogs, evaluation of the prostate beyond digital rectal examination is not usually performed in asymptomatic animals. diagnosis of prostatic disease includes a complete history; digital rectal examination to evaluate prostate size, shape, and consistency, and to assess for pain, remembering that the very enlarged prostate may be abdominal and difficult or impossible to assess by rectal examination; ultrasonography to evaluate internal architecture of the prostate and to guide collection of samples for cytology; and if infection is suspected, culture of prostatic fluid, collected by ejaculation or prostatic massage, or aspirates of the gland. because prostatic fluid is secreted constitutively, with much running into the urinary bladder, urinalysis on samples collected by cystocentesis may provide information about the prostate. prostate specific antigen is not used for diagnostic screening in dogs as it is not routinely identified in serum.36 a related but distinct protein, canine prostate specific esterase, has been identified in the dog but concentrations in serum and seminal fluid have not been demonstrated to differ significantly between normal dogs and dogs with various prostate diseases.36,37 the author has received many anecdotal reports from colleagues of clients arguing for unnecessary tests for prostate disease in their dogs based on information from their experience or the human medical literature. this retrospective study was an attempt to determine which factors gleaned from history, physical examination findings, and common diagnostic tests could be used to differentiate likely causes of disease and streamline the diagnostic process. the intent was not to circumvent cytologic or histopathologic assessment of the prostate, which is the gold standard for differentiation of disease types in the dog. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 50clinical theriogenology • volume 4 number 1 • march 2012 materials and methods medical records were retrieved electronically using the system’s embedded search mechanism. the search included male dogs of all breeds, intact or castrated, for any diagnosis containing all or portions of the word “prostate.” search terms included were abscess-prostate, adenocarcinoma-prostate, carcinoma-prostate, cyst-prostate, hypertrophy-prostate, prostatitis, and prostatomegaly. data gathered included diagnosis, breed, and intact status and age at time of admission. clinical signs noted by the owner or by a veterinarian during physical examination that were specifically recorded included gross hematuria, stranguria or dysuria, dripping of serosanguinous or hemorrhagic fluid from the prepuce unassociated with urination, urinary incontinence or loss of housetraining as evidenced by inappropriate urination, rectal tenesmus, and passage of ribbon-shaped stools. also recorded were signs of systemic disease, such as lethargy, anorexia, or lameness. because it was not always clear how referring veterinarians had gathered data sent along with the patient, data were included from urinalysis and transabdominal ultrasound only if those tests were completed at the author’s institution, to minimize variability. recorded from urinalyses were number of rbcs and epithelial cells in the urine sediment. recorded from transabdominal ultrasound reports were presence or absence of prostatic mineralization and regional lymph node enlargement. cytologic diagnoses were included only if performed at the author’s institution, again to minimize variability. categorical data were compared between groups using the χ2 test or fisher’s exact two-tailed test, as appropriate. non-categorical data were compared using anova. significance was set at p< 0.05. results the initial search retrieved 183 medical records from 2002 to the present. eighty-four cases were removed after initial evaluation. twenty-three had been completed incorrectly by a clinician or had been coded incorrectly and did not pertain to prostate disease in dogs. twenty-eight were removed due to an incomplete work-up, usually consisting only of a history and physical examination with any further diagnostics declined by the owner. fifteen were cases with prostate disease secondary to a significant primary condition that would confound source of clinical signs or results of diagnostic tests; examples of these conditions were pyelonephritis, neoplasia of other organs or systems, immune-mediated thrombocytopenia or hemolytic anemia, infectious diseases including blastomycosis and tick-borne diseases, urolithiasis, and paraplegia with urinary and fecal incontinence. ten cases had putative prostatic carcinoma and eight cases had putative bph, with no cytologic confirmation. diagnosis was based on parameters to be evaluated by this study so these cases were removed. ninety-nine cases were evaluated in detail. diagnoses for all cases were verified by evaluation of cytology specimens collected by fine-needle aspirate or by histopathology after necropsy. complete signalment, history and physical examination findings were recorded for all cases. urinalysis was performed in 56 cases. all urine samples were collected by cystocentesis. transabdominal ultrasound was performed in 90 cases. dogs of 44 breeds were identified. breed size varied from toy to giant. breeds most commonly represented were the labrador retriever (n=18), english springer spaniel (n=9), shetland sheepdog (n=8), golden retriever (n=6), and german shepherd dog (n=5). this reflects popularity of these breeds in the area and the clientele of the clinicians at the author’s institution. fifty-seven dogs were diagnosed with prostatic carcinoma; differentiation was not consistently made between adenocarcinoma and transitional cell carcinoma. twenty-nine dogs were diagnosed with bph. of the remaining dogs, eight were diagnosed with bph with overlying prostatitis, two with carcinoma with overlying prostatitis, and one each with bph with an intraprostatic cyst bph with an ossified intraprostatic abscess, and a periprostatic cyst. statistical comparisons were made between the dogs diagnosed with carcinoma and bph. difference in intact status as an aid in differentiation of disease type is significant, with p < 0.0001. benign prostatic hyperplasia/hypertrophy was more common in intact than in castrated dogs; all 29 dogs diagnosed with bph were intact. prostatic carcinoma was more common in castrated than in clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 201251 clinical theriogenology • volume 4 number 1 • march 2012 intact dogs; 54 of 57 dogs with carcinoma were castrated (94.7%). mean age at diagnosis did not vary significantly between dogs diagnosed with bph (7.9 ± 2.9 years [mean +/sd]) and those diagnosed with carcinoma (10.2 ± 1.9 years). hematuria was defined grossly, either by owner description or as evidenced during urine collection, or by noting of rbcs in the urine sediment. hematuria was noted as a clinical sign in 12 of 29 dogs with bph (41.3%) and in 18 of 57 dogs with carcinoma (31.6%). number of rbcs in urine sediment in dogs with bph was on average much lower than that in dogs with carcinoma (3.3 ± 8.0 in 18 dogs versus 32.8 ± 28.8 in 28 dogs). presence or absence of hematuria did not vary significantly between groups by either definition. stranguria (painful urination) and dysuria (difficult or painful urination) were considered synonymous. stranguria/dysuria was significantly more common in dogs with carcinoma (40.4% of 57 dogs) than in dogs with bph (0 of 29 dogs; p< 0.0001). dripping of serosanguinous or hemorrhagic fluid from the prepuce unassociated with urination was significantly more common in dogs with bph (41.4% of 29 dogs) than in dogs with carcinoma (14.0% of 57 dogs; p = 0.007). urinary incontinence and inappropriate urination did not differ significantly between the two groups; this clinical sign was reported in 10.3% of the dogs with bph and in 15.8% of the dogs with carcinoma. number of epithelial cells in urine sediment was higher in dogs with carcinoma (4.14 +/3.6, n=28) than in dogs with bph (0.5 ± 0.8, n = 18) but this difference was not significant. rectal tenesmus is straining to pass stool, which may be of varying consistency. in this population two of 29 dogs with bph (6.9%) were reported to have tenesmus while 24 of 57 dogs with carcinoma (42.1%) were reported to have tenesmus. this difference is significant (p = 0.0009). associated with this is passage of ribbon-shaped stools. none of the dogs with bph and seven of the dogs with carcinoma (12.3%) were reported to pass soft, ribbon-shaped stools. this difference is not significant (p = 0.09). general systemic signs of disease that were reported included anorexia and weight loss, lethargy, hindlimb weakness and ataxia, and abdominal pain. these signs were significantly more common in dogs with carcinoma (35.1%) than in dogs with bph (6.9%; p=0.004). mineralization of the prostatic parenchyma and regional lymph node enlargement were assessed using transabdominal ultrasound. mineralization was noted in 32 of 49 dogs with carcinoma (65.3%) and in none of the 29 dogs with bph; this difference is significant (p<0.0001). regional lymph node enlargement was noted in 24 of the dogs with carcinoma (49.0%) and in three of the dogs with bph (10.3%); this difference also is significant (p = 0.0002). discussion the primary limitations to this study were the study population and limitations in retrieving data based on varying use of the electronic medical record. the study was conducted at a veterinary teaching hospital. dogs with bph are less likely to be referred for diagnosis or care than are dogs with less common or higher morbidity prostate disorders, including prostatic neoplasia. it is possible that the results of this study reflect increased severity of disease in this specific population of dogs. the electronic medical record is a standard software product (universal veterinary information system [uvis]) and despite the fact that all faculty and house officers are trained to use the medical record in a specific way, historical data and laboratory results were saved in varying sites within the record. it is possible that not all data were found by the author, although a considerable amount of effort was put into completely evaluating all material available for each case. benign prostatic disease has not been reported in castrated dogs and was not identified in this study.20,38 any dog that has been castrated at least three months prior to presentation should have prostatic atrophy and no clinical signs suggestive of prostatic disease.39,40 any castrated male dog with prostatomegaly should be considered to have neoplasia. similarly, dogs with stranguria or dysuria as a component of prostate disease should be considered to have neoplasia. smooth muscle is not activated in dogs with prostate disease so any urethral obstruction is due to invasion of the urethra by neoplastic tissue.16 because many owners will elect euthanasia at the time of diagnosis of prostatic neoplasia, definitive diagnosis by collection of samples for cytology or histopathology is strongly recommended. techniques for collection of fine-needle aspirate cytology specimens and biopsy specimens for clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 52clinical theriogenology • volume 4 number 1 • march 2012 histopathology have been described.41 fine-needle aspirate is less invasive and correlates with histopathology diagnosis in 80% of cases.42 the other parameters listed were significant in this study but were present in dogs with either condition. some, for example mineralization of the prostatic parenchyma, also may be associated with prostatitis, which was not evaluated in this study, or with undiagnosed concurrent disease.39,43-45 intact status of the dog and presence or absence of stranguria/dysuria are strong predictors permitting initial differentiation of bph from prostatic neoplasia in dogs. the history and diagnostic findings determined to be significantly associated with bph rather than with prostatic neoplasia in dogs in this study were being sexually intact and dripping bloody fluid from the prepuce unassociated with urination. the history and diagnostic findings determined to be significantly associated with prostatic neoplasia rather than with bph were being castrated, stranguria or dysuria, rectal tenesmus, systemic signs of disease, and mineralization of the prostate and regional lymph node enlargement visible with transabdominal ultrasound. knowledge of these parameters guides further diagnostic testing and therapy (figure). figure. diagnostic key for dogs with prostatic enlargement and history of clinical signs suggestive of prostatic disease: 1. the dog is intact. go to step 2. 1’. the dog was castrated ≥ 3 months ago. go to step 3. 2. the dog has any of the following clinical signs: stranguria/dysuria, rectal tenesmus, systemic signs of disease, mineralization of the prostate, regional lymph node enlargement. go to step 3. 2’. the dog does not have any of the above clinical signs. go to step 4. 3. the dog most likely has prostatic neoplasia. overlying prostatitis may be present. cytology or biopsy specimens should be collected for definitive diagnosis. therapy is palliative.46 4. the dog most likely has bph. cytology or biopsy specimens should be collected for definitive diagnosis. overlying prostatitis may be present and should be assessed by culture of prostatic fluid or tissue. castration is the most effective therapy for bph. medical therapy with finasteride is described.46 references 1. evans he, christensen gc: the urogenital system. in: evans he, ed. miller’s anatomy of the dog. philadelphia: wb saunders: 1993. p. 514-515. 2. stefanov m, martin-alguacil n, martin-orti r: distinct vascular zones in the canine 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northrup n: prostate cancer in dogs: comparative and clinical aspects. vet j 2009;180:149-162. 28. lai c-l, van den ham r, van leenders g, et al: histopathological and immunohistochemical characterization of canine prostate cancer. prostate 2008;68:477-488. 29. durham sk, dietze ae: prostatic adenocarcinoma with and without metastasis to bone in dogs. j am vet med assoc 1986;188:1432-1436. 30. leav i, ling gv: adenocarcinoma of the canine prostate. cancer 1968;22:1329-1345. 31. lee-parritz de, lamb cr: prostatic adenocarcinoma with osseous metastases in a dog. j am vet med assoc 1988;192:1569-1572. 32. hargis am, miller lm: prostatic carcinoma in dogs. compend contin educ pract vet 1983;5:647-653. 33. basinger rr, rawlings ca, barsanti ja, et al: urodynamic alterations associated with clinical prostatic diseases and prostatic surgery in 23 dogs. j am anim hosp assoc 1989;25:385-392. 34. united states preventive services task force: screening for prostate cancer. available at: http://www.uspreventiveservicestaskforce.org/uspstf08/prostate/prostaters.htm, accessed july 25, 2011. 35. roehrborn cg, bartsch g, kirby r, et al: guidelines for the diagnosis and treatment of benign prostatic hyperplasia: a comparative, international overview. urology 2001;58:642-650. 36. bell fw, klausner js, hayden dw, et al: evaluation of serum and seminal plasma markers in the diagnosis of canine prostatic disorders. j vet intern med 1995;9:149-153. 37. dube jy, lazure c, tremblay rr: dog prostate arginine esterase is related to human prostate specific antigen. clin invest med 1986;9:51-54. 38. krawiec dr: canine prostate disease. j am vet med assoc 1994;204:1561-1564. 39. bradbury ca, westropp jl, pollard re: relationship between prostatomegaly, prostatic mineralization, and cytologic diagnosis. vet radiol ultrasound 2009;51:167-171. 40. o’shea jd: studies on the canine prostate gland. i. factors influencing its size and weight. j comp pathol 1962;72:321-331. 41. root kustritz mv: collection of tissue and culture samples from the canine reproductive tract. theriogenology 2006;66:567-574. 42. powe jr, canfield pj, martin pa: evaluation of the cytologic diagnosis of canine prostatic disorders. vet clin pathol 2004;33:150-154. 43. feeney da, johnston gr, klausner js, et al: canine prostatic disease–comparison of ultrasonographic appearance with morphologic and microbiologic findings: 30 cases (1981-1985). j am vet med assoc 1987;190:1027-1034. 44. feeney da, johnston gr, klausner js, et al: canine prostatic disease–comparison of radiographic appearance with morphologic and microbiologic findings: 30 cases (1981-1985). j am vet med assoc 1987;190:1018-1026. 45. vignoli m, russo m, catone g, et al: assessment of vascular perfusion kinetics using contrast-enhanced ultrasound for the diagnosis of prostatic disease in dogs. reprod domest anim 2011;46:209-213. 46. smith j: canine prostatic disease: a review of anatomy, pathology, diagnosis, and treatment. theriogenology 2008;70:375-383. clinical theriogenology • volume 3 number 4 • december 2011clinical theriogenology • volume 4 number 1 • march 2012 54clinical theriogenology • volume 4 number 1 • march 2012 49 contact christina moss csmoore2014@gmail.com © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11651, http://dx.doi.org/10.58292/ct.v17.11651 review report hormonal manipulation of the estrous cycle in mares christina moss,a kristina lub apiedmont equine practice, the plains, va, usa bhaygard equine medical institute, lexington, ky, usa abstract hormonal manipulation of the estrous cycle is a mainstay of clinical equine reproduction practice. purposes of hormonal manipulation include advancement of the breeding season, induction of estrus from anestrus or vernal transition, ovulation timing, estrus synchronization, and estrus suppression. this review summarizes methods and supporting research of hormonal manipulation of the estrous cycle utilized in mares in clinical equine reproduction practice. keywords: mare, cyclicity, ovulation induction, synchronization, estrus suppression introduction mares are seasonally polyestrous with transitional periods occurring at the beginning and end of the period of normal cyclicity. generally, the period of behavioral estrus can last up to a week and diestrus lasts ~ 14-17 days after ovulation. the high monetary value associated with equids and related activities has driven humans to investigate mechanisms for manipulating the normal cycle. for instance, racing industry birth date standards have prompted practitioners to find ways to induce earlier cyclicity in mares and the desire to continue training and competing with valuable mares has led to investigations into mechanisms for suppressing estrus and synchronizing ovulation for embryo transfer. this review focuses on the various hormonal manipulations studied, aiming to demonstrate that although reliable methods for achieving desired results exist, they are not without variability and dependence on multiple factors for success. induction of cyclicity photoperiod manipulation although most mares experience a fall transition into winter anestrus, a proportion of mares continue to exhibit estrous cycles and ovulate throughout the year.1,2 the transition to winter anestrus and subsequent onset of the spring transitional period are primarily dependent on photoperiod or day length. in mammals, the eye is the primary receptor of light for determination of photoperiod. establishment of photoperiod does not occur through use of rod or cone receptors, but rather through photosensitive opsin proteins within retinal ganglion cells, which receive light signals and project these to the ventrolateral part of the suprachiasmatic nucleus (retinohypothalamic tract).3 changes in photoperiod affect circadian ‘clock’ genes within the suprachiasmatic nucleus that serve to maintain the homeostatic state appropriate for the current photoperiod.4 signals are relayed from the suprachiasmatic nucleus to the pineal gland, where neural signaling is then translated to endocrine signaling in the form of increased or decreased melatonin secretion in response to the photoperiod information received initially by the eyes.5 melatonin secretion increases with decreased day length and decreases with increased day length. these changes in melatonin concentrations affect reproductive cyclicity through gonadotropin-releasing hormone (gnrh) within the hypothalamus. hypothalamic content of gnrh tended to be higher in the summer than during the winter in the northern hemisphere; mares treated with melatonin implants during the summer had lower content of gnrh than untreated mares.6 these results are supportive of a relationship between gnrh and melatonin concentrations in the onset of the transitional period. gnrh secretion stimulates gonadotropins secretion from the anterior pituitary and is the first step in a cascade that results in follicular growth and eventually, ovulation. based on our understanding of the impact of photoperiod on the onset of cyclicity in the mare, photoperiod manipulation emerged as a popular method of attempting to initiate earlier cyclicity in mares, often in response to industry-imposed, standard birthdates. as the transition period takes weeks to months, the goal of introducing artificial lighting is not to shorten the transition period but mailto:csmoore2014@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11651 50 citation: clinical theriogenology 2025, 17, 11651, http://dx.doi.org/10.58292/ct.v17.11651 rather to hasten its onset and completion, resulting in resumption of cyclicity. generally, lighting programs are instituted in the late fall (around december 1 in the northern hemisphere). there was no beneficial effect of starting lighting programs on november 1 versus december 1; however, there was an advantage of starting december 1 versus january 1.7 in general, a 100-200 w lightbulb in a 3.5 x 3.5 m box stall should provide sufficient supplementary light;8 however, an array of lighting systems exists such as paddock lighting and light masks.9 low-level blue light (peak wavelength of 468 nm), directed at a single eye via a light mask inhibited melatonin release in thoroughbred mares.10 several lighting schedules have been employed with success, though the classic model is ~ 14.5-16 hours of consistent light exposure, followed by 8-9.5 hours of darkness.11 adding only an additional 2.5 hours of light at sunset was successful at advancing onset of cyclicity and adding another few hours of light near sunrise did not have an additional significant beneficial effect.12 exposure of anestrous mares to a 1-hour pulse of artificial light 18.5 hours after dawn was beneficial.11 this method was proposed to work by interrupting the normal nighttime secretion of melatonin, modulating the inhibitory effect of melatonin on gnrh.13 despite differences in timing, both mechanisms attempt to alter the equine circadian rhythm such that it resembles that of the longer days of the traditional breeding season. gonadotropin releasing hormone and agonists gnrh is released from 2 regions within the female hypothalamus in differing patterns. the tonic center (ventromedial and arcuate nuclei) releases gnrh in a low amplitude, episodic manner, whereas the surge center (preoptic and suprachiasmatic nuclei and anterior hypothalamic area) releases gnrh with higher frequency and amplitude, resulting in a luteinizing hormone (lh) surge. low, episodic secretion of gnrh appears to favor follicle stimulating hormone (fsh) secretion, whereas high amplitude pulses of gnrh favor lh secretion.14 initially during transition, lh secretion is limited due to low pituitary stores, and the amount of lh within the pituitary increases with increasing daylength.15 increases in gnrh during transition stimulate fsh release that results in follicular waves of steroidogenically incompetent follicles that will not ovulate but may produce enough estrogen to cause erratic estrous behavior.16 eventually these follicles gain the ability to produce enough estrogen to provide positive feedback on lh secretion, resulting in an lh surge and ovulation. studies attempting to use gnrh to advance ovulation and seasonal cyclicity have aimed to determine the route and frequency that provide the earliest onset of estrus. one of the first studies of the use of gnrh to induce ovulation in transitional mares used native gnrh twice daily during the transitional period at 10 day intervals for 3-5 injections, in combination with exogenous progesterone to simulate the luteal phase. the protocol was intended to simulate the normal hormonal secretion of cyclic mares and all 5 mares ovulated in response to treatment.17 however, it must be considered with such a long treatment period (30-50 days) in transitional mares, whether the transitional period was completed before the end of the study and mares had naturally regained normal cyclicity. hourly infusions of native gnrh to mares that had been kept under short-day length resulted in ovulation.18,19 similarly, deep anestrus mares treated with a continuous infusion of 0, 50, or 100 ng gnrh/kg/hour for 28 days via osmotic minipumps showed dose-dependent effects on ovulation rates, with the highest percentage of mares (70%) ovulating in the 100 ng gnrh/kg/hour treatment group. mares in shallow anestrus that were treated with the 100 ng gnrh infusion ovulated at 18.6 days versus 41.9 days in untreated controls.20 pulsatile treatment of native gnrh was more effective in inducing lh peak and ovulation. thrice daily injections stimulated follicular growth; however, injection frequency needed to be increased to hourly to stimulate ovulation. continuous release implants, in comparison, reduced labor and provided acceptable, increased ovulation rates compared to untreated control mares.21 anestrous pony and thoroughbred mares were implanted with either 1 or 2 slow-release polymer implants impregnated with either 0.9 or 1.8 mg of the gnrh agonist, ici 118 630/goserelin.22 implants delivered either 30 or 60 µg gnrh analogue, respectively, for 28 days; 76% of pony mares and 88% of thoroughbred mares ovulated within 3-18 days after treatment. seasonally anestrous mares (n = 45) were assigned to 3 groups: 1. control; 2. 40 µg of the intramuscular gnrh analogue, buserelin, twice daily for 28 days or until ovulation; and 3. subcutaneous implants that released 100 µg buserelin per day for 28 days.23 none of the controls, 7 of 15 mares receiving 40 µg intramuscular buserelin, and 9 of 15 mares with subcutaneous implants ovulated within 30 days. implants were more effective in inducing ovulation when implanted during late transition versus earlier in the year,24 possibly due to larger follicles.25 although, there was an increase in follicle development after fitting anestrous mares with subcutaneous implants containing varying dosages of the gnrh analogue, goserelin, the largest follicular diameter achieved appeared to be more dependent on the largest follicular diameter that was present prior to treatment.25 authors also observed only a 30-35% increase in ovulation rate for the groups implanted with either a 3.6 or 5.4 mg gnrh implant. ovulation rate was dependent on dosage, as lower-dose implants had significantly lower ovulation rates.25 as alluded to above, the effects of gnrh treatment on cyclicity and fertility appear to be dependent on the ovarian status and time of year during which treatment is initiated. ovulation was induced in anestrous mares with twice-daily injections of a gnrh analogue starting either on december 23 or january 15.26 percentage of mares ovulating increased when treatment was started on january 15 versus december 23 and the percentage of mares ovulating also increased with the size of the largest follicle at the initiation of treatment, as observed earlier (100% ovulation rate when follicular diameter was > 25 mm at the initiation of treatment).25 ovulation was also induced in mares with inactive ovaries (follicles < 15 mm diameter) with 200 µg of a gnrh analog given twice daily; however, circulating progesterone and lh concentrations were lower and early embryo loss rates were much higher in treated mares versus controls. though the authors induced ovulation and obtained fertilization, the previous anestrous state of the mares seemed to suppress luteal activity such that pregnancies were lost prior to development of the endometrial cups.27 two schemes (increasing and constant dose of deslorelin) of gnrh treatment to anestrous and transitional mares were studied.28 both schemes increased ovulation rates over control, untreated mares, with no differences in ovulation rates between treatment groups. systemic lh concentrations were similar between groups for the first 12 days and higher than controls, but after day 12, the constant-dosage group had http://dx.doi.org/10.58292/ct.v17.11651 citation: clinical theriogenology 2025, 17, 11651, http://dx.doi.org/10.58292/ct.v17.11651 51 higher lh concentrations than the increasing-dosage group, potentially due to decreasing pituitary sensitivity to gnrh with increasing gnrh dosage.28 overall, the depth of anestrus and the gnrh preparation may explain differential responses to treatment with gnrh agonists for the purpose of inducing cyclicity29 and mares induced to cycle out of deep anestrus may even return to anestrus after their first ovulation.18,24-26 the corpus luteum (cl) that forms after gnrh treatment in anestrous or transitional mares is generally identical to that formed during the normal breeding season.18,19,22,25 however, there are examples of failure to form a cl or failure of a cl to function correctly following gnrh treatment.17,20,21,28 equine pituitary extract and gonadotropins stepping down the hypothalamic-pituitary-gonadal axis, the use of pituitary extracts (native and recombinant gonadotropins) aims to directly stimulate follicular growth and ovulation at the gonadal level. gonadotropins are released from the anterior pituitary in response to gonadotropin releasing hormone (gnrh) and act directly on the gonads. the 2 main gonadotropins in the horse are fsh and lh, and these may be purified from pituitary extracts into different ratios. however, depending on the availability of equine pituitary tissue from slaughterhouses, pituitary extract and native gonadotropins may not be available in certain areas of the world. thus, recombinant fsh, both equine and human preparations, would be useful where purification of natural gonadotropins is either not possible, too labor intensive, or cost prohibitive.30,31 equine pituitary fractions were used to induce ovulation in seasonally anovulatory pony mares with follicles < 10 mm on each ovary. in this study, 87% of mares treated with 2 weeks of twice daily injections of pituitary extract ovulated by the end of the treatment period. of the mares that ovulated, 58% had multiple ovulations per estrus.32 results were better in anestrous mares (follicles < 10 mm) treated with equine pituitary extract twice daily for 14 days; treated mares but none of the salinetreated controls ovulated.33 purified pituitary extract from equine pituitary glands obtained from slaughterhouses was used34; extract was injected into seasonally anovulatory mares when at least 1 follicle reached 25 mm. the extract was able to induce ovulation in 95% of mares and 64% of ovulations were multiple, as observed earlier.32 as is the case with other agents discussed thus far (e.g. gnrh) and despite the apparent success of earlier studies, success of induction of ovulation was dependent on follicular size, as the pituitary extract used was less likely to induce ovulation when follicles were < 25 mm.34 deep anestrous (follicles < 15 mm) and transitional (at least 1 follicle > 25 mm) mares were treated with equine pituitary extract for a maximum of 15 days35; 22% of mares in deep anestrus ovulated within the treatment period in response to human chorionic gonadotropin (hcg) given once a follicle reached 35 mm. in comparison, 90% of mares in transition treated with equine pituitary extract ovulated within the treatment period in response to hcg once a follicle reached 35 mm. the mean interval from start of treatment to ovulation was 11.8 days.35 growing follicles were supplemented with equine lh (elh) from pituitary extracts during early transition to stimulate development of steroidogenically active dominant follicles with the ability to respond to an ovulatory stimulus.36 mares received intravenous elh until a follicle reached 32 mm; they then received 3,000 iu of intravenous hcg to stimulate ovulation. although elh did stimulate follicle growth, it failed to generate steroidogenically active follicles responsive to hcg; elh did not hasten the onset of the ovulatory season because the treated mares that did ovulate returned to an anovulatory transitional state.36 equine fsh (efsh) was given to transitional mares; group 1 (n = 10) served as untreated controls, and group 2 (n = 10) were given 12.5 mg of purified intramuscular efsh (bioniche animal health athens, ga, usa) twice daily for a maximum of 15 consecutive days. once 1 or more follicles > 35 mm were detected, efsh treatment was discontinued and  intravenous hcg  was given. in 8 of 10 mares treated with efsh, follicles developed, and ovulation occurred during the 15-day observation period, compared to 0 of 10 control mares. interval from onset of treatment to ovulation was 7.6 ± 2.4 days for these 8 mares that were treated for an average of 5.2 ± 1.3 days with efsh. thus, the efsh treatment was effective in advancing the first ovulation of the year in transitional mares.37 however, decreased dosage and frequency of efsh treatment produced lower ovulation rates than twice daily 12.5 mg efsh treatment.38 similar success was achieved in treating donor mares for embryo transfer during the transitional period with efsh.39 mares were housed under ambient conditions and had transrectal ultrasonography performed starting on january 30. once a follicle reached 25 mm, twice daily efsh treatment was initiated until a follicle reached 35 mm. at that point, efsh treatment was discontinued and hcg was given after 36 hours. mares were then inseminated every 48 hours until ovulation occurred; mean interval from initiation of treatment to ovulation was 7.9 days. treated mares were noted to have more preovulatory follicles and more ovulations than mares in untreated cycles; however, embryo recovery rates were similar and embryos from the treated mares were noted to have more morphologic abnormalities. additionally, approximately half of the treated mares were noted to have a prolonged inter-ovulatory interval between the first (efsh treated) and second (untreated) cycles of the season.39 twice daily efsh were compared to twice daily injections of 63 µg deslorelin acetate for hastening first ovulation of the year in transitional mares.40 interval from onset of treatment to first ovulation was similar; however, the numbers of ovulations and embryos recovered were higher in the efsh-treated mares than in the deslorelin-treated mares. additionally, no differences in embryo quality were noted between treatment groups. more recently, mares in deep anestrus under natural photoperiod were treated with recombinant fsh (refsh) or a combination of refsh/relh and compared to mares maintained under artificial photoperiod. despite lacking the effects of photoperiod manipulation, mares given refsh and refsh/ relh developed preovulatory follicles significantly faster than control mares and all treated mares ovulated within 10 days after beginning treatment. importantly, treated mares that conceived and had pregnancies terminated with prostaglandin at 25 days continued to cycle normally for the season after pregnancy termination.41 however, refsh is not currently commercially available. its arrival to market is much anticipated as it appears to be a viable option for induction of cyclicity, even without photoperiod manipulation. progestin supplementation for induction of cyclicity, natural progesterone or synthetic progestins are used to suppress estrus for a period, allowing synchronization of estrus when the progestin is withdrawn. http://dx.doi.org/10.58292/ct.v17.11651 52 citation: clinical theriogenology 2025, 17, 11651, http://dx.doi.org/10.58292/ct.v17.11651 altrenogest given by mouth or mixed in feed at a dose of 0.044 mg/kg may be used for this purpose. additionally, both shortand long-acting preparations of injectable progesterone exist. short-acting intramuscular progesterone is given at a dose of 150 mg per day, whereas long-acting progesterone is given at a dose of 1.5 g every 7-10 days.42 follicular activity is not fully suppressed with the use of progesterone or synthetic progestins alone. for example, although lh secretion is generally reduced with the use of altrenogest, ovulation sometimes still occurred.43,44 for best results using progestins to induce cyclicity, mares must be in mid to late transition with follicles of at least 20-25 mm in each ovary.45-47 typically, mares are treated for 12-14 days and then the exogenous progestin is withdrawn. on the contrary, other studies refute this finding with results that neither progesterone in oil nor altrenogest advanced first ovulation in mares, regardless of depth of anestrus/transition and follicular size during treatment.15,48 progestin therapy, specifically with altrenogest, was effective at inducing estrus and first ovulation when combined with photoperiod manipulation.45 researchers exposed mares to a 16-hour daily photoperiod for 2 months prior to daily altrenogest treatment for 12 days. more progestin-treated mares had signs of estrus and ovulated within 12 days after treatment than control mares. other forms of progesterone that have been studied include estrogen/progesterone combinations and long-acting progesterone formulations. in anovulatory mares, long-acting progesterone formulations designed to elevate progesterone for 10-12 days were successful in elevating progesterone for 10 days. additionally, noncycling mares that had not responded to photoperiod manipulation were treated with the long-acting progesterone formulation, la300 (bet pharmacy, lexington, kentucky, usa), and 57% of these mares ovulated within 4 weeks. this was in comparison to a control group of mares in which only 7% of mares ovulated.49 long-acting progesterone was utilized to program mares and advance ovulation; however, it was only significant in late transitional mares (follicles 20-25 mm).50 treatment had no effect on the early transition mares but resulted in ovulation in 10-24 days in 10 of 12 mares treated in late transition versus only 3 of 12 control mares during the same period.50 the combination of injectable progesterone and estrogen provides more effective follicular activity suppression than when using a progestin alone; whereas both methods may be useful for inducing cyclicity in late-transition mares, interval to estrus and ovulation was longer for combined progesterone/estrogen therapy than for altrenogest alone.51 the additional follicular activity suppression in the progesterone/estrogen combination stems from estrogen’s suppressive effects on fsh secretion, as fsh drives follicular growth and development. such a progesterone/estrogen protocol was used; 150 mg intramuscular progesterone and 10 mg estradiol-17β were given daily for 15 days, with 10 mg of pgf2α given on the last day of steroid treatment. in 2 trials within this study, mares ovulated 8-14 and 9-16 days after finishing treatment with conception rates of 77 and 62%, respectively. authors concluded that this combination provided satisfactory control of ovulation in mares early in the breeding season, with no adverse effects on fertility.52 other sources of exogenous progesterone include intravaginal devices such as controlled internal drug release (cidr), used extensively to synchronize ruminant reproductive cycles. mares were inserted with cidr-b device (1.9 g of progesterone) for 12 days; when a follicle reached 35 mm, the device was removed and a deslorelin implant was placed.53 follicular diameter at the end of progesterone treatment was larger in mares treated with cidr compared to untreated control mares, with 77% of treated mares having follicles > 35 mm compared to 8% of untreated controls. in treated mares, 80% with follicles > 35 mm ovulated within 24-48 hours in response to implantation of gnrh device.53 similar, positive results have been demonstrated in multiple studies using both transitional mares and truly anestrous mares.54-56 however, vaginitis of varying degrees was noted in mares and thus may make this method of induction of cyclicity less desirable, despite the ease of use.54,55,57 transvaginal aspiration of follicles in a similar vein to the ovarian ‘reset’ provided by progesterone and estrogen combination therapies, transvaginal aspiration (tva) may be used to remove all follicles, such that a synchronized follicular wave emerges following aspiration. in transitional mares, hcg was given when 1 follicle reached 30 mm and tva (follicles > 10 mm) was applied to 8 mares (treatment group). mares (control and treatment) received prostaglandin on day 7 after hcg; 3 of 8 (38%) mares that had tva formed luteal tissue with systemic progesterone concentrations > 1 ng/ml. these mares continued to cycle normally; however, only 1 mare (1/6; 16%) in the control group ovulated in response to hcg and this mare did not continue to cycle after prostaglandin treatment. although the ovulation rate obtained in this experiment was not very high, treated mares that ovulated formed functional corpora lutea and continued to cycle normally after prostaglandin treatment.58 in transitional mares, follicles > 8 mm were aspirated at 3-4 days intervals for 4 total aspirations. these mares ovulated, on average, 9.8 days after the last transitional stage aspiration. however, there were no control mares for comparison in this study nor were any deep anestrous mares included.59 similarly, luteal tissue formed in 9 of 11 mares 8 days after tva was performed;60 however, researchers in this study were only aspirating the dominant follicle in late transitional mares. the remaining 2 mares in the treatment group in this study formed luteal tissue following a second aspiration procedure. again, deep anestrous mares were not studied. overall, tva appears to be an effective means of inducing ovulation in late transitional mares. however, there are inherent risks to tva. development of adhesions, internal hemorrhage, and ovarian abscesses have infrequently been reported,61,62 and transient rectal bleeding in 16% of tva sessions was also reported.63 the equipment, skill, and risks associated with the procedure, when the success rates of other induction methods used in transitional mares are considered, may make this option less desirable. dopamine antagonists/prolactin manipulation dopamine has a role in regulating reproduction through synapses that are present between dopaminergic and gnrh neurons in the median eminence. inhibition of dopamine d2 receptors increased lh secretion during anestrus, suggesting an inhibitory role of dopamine on http://dx.doi.org/10.58292/ct.v17.11651 citation: clinical theriogenology 2025, 17, 11651, http://dx.doi.org/10.58292/ct.v17.11651 53 cyclicity.64 additionally, dopamine inhibits prolactin secretion. although dopamine concentrations in cerebrospinal fluid of mares were higher during the anovulatory period,65 plasma concentrations of prolactin were higher during the breeding season,66 suggesting an inverse relationship. prolactin treatment to anestrous mares stimulated ovarian activity and induced ovulation in seasonally anovulatory mares. recombinant porcine prolactin given to mares (starting mid-january) resulted in ovulation, an average of 5 weeks earlier than placebo-treated mares (february 6 versus march 14).67 dopamine d2 receptors are not only present within the median eminence, but also in theca cells, granulosa cells, luteal tissue, and ovarian cortex tissue,68,69 suggesting an influence on follicular growth. this may be why the use of dopamine d2 antagonists (e.g. sulpiride and domperidone), have a stimulatory effect on follicular development in anestrous mares.70 domperidone is a dopamine d2 and d3 receptor antagonist, whereas sulpiride is a selective dopamine d2 receptor antagonist; sulpiride can cross blood brain barrier but domperidone cannot. both have been used to attempt to shorten the transitional period and hasten the first ovulation of the year. intramuscular sulpiride (1 mg/kg) given daily (starting in late january) and continued until ovulation71 or 200 mg given daily (starting early february) and continued until ovulation or for a maximum of 58 days72 advanced the first ovulation of the year by 21 days and 33 days, respectively. these studies were performed in seasonally anestrous mares maintained under natural photoperiod. similar results were observed in mares exposed to an artificial photoperiod (starting in january, 2 weeks prior to onset of sulpiride treatment)73 and in transitional mares (follicular diameter > 25 mm).74 however, attempts at beginning the sulpiride protocol earlier in january without photoperiod manipulation failed to affect ovarian activity or ovulation, despite increased circulating prolactin concentrations.75 oral domperidone (1.1 mg/kg) treatment to anestrous mares (starting on january 15) resulted in follicular development within 14 days and advanced the average date of ovulation by 78 days; 6 out of 8 mares continued to cycle after the first ovulation, whereas the remaining 2 experienced prolonged intervals to second ovulation (mean = 67  days). domperidone treatment increased prolactin secretion and concentrations of lh and estrogen conjugates; fsh was not affected as with sulpiride treatment.76 in contrast, there was no positive effect on shortening the transitional period in anestrous mares (natural photoperiod) treated with domperidone for 60 days, or transitional mares (artificial photoperiod) treated for 30 days.77 authors proposed that differences in ambient temperatures and climates between the 2 study locations could have had a role in the opposing results.77 similarly, sulpiride was effective in advancing ovulation in a majority (80%) of the mares studied within 38 days of beginning treatment, whereas domperidone was only effective in advancing ovulation in a small subset (20%) of mares over control mares.78 one potential explanation for the failure of dopamine antagonists to stimulate follicular activity is a lack of estrogen in seasonally anestrous mares. estrogen exposure increased pituitary prolactin storage,79 and thus studies have been performed that have added estradiol priming to their dopamine antagonist protocols. seasonally anestrous mares received 10 injections of intramuscular estradiol benzoate (11 mg once every 2 days) with subcutaneous sulpiride (250 mg once daily) beginning on day 11 (day 0 = beginning of estradiol treatment);80 treatment was continued until ovulation or until 35 days of sulpiride treatment. the control group was given only sulpiride. mares that received estradiol benzoate prior to sulpiride ovulated 45 days earlier than mares receiving sulpiride alone. pretreatment with estrogen enhanced the prolactin response to daily treatment with sulpiride, causing both increased concentrations and increased duration of elevated secretion compared to controls. pretreatment with estradiol also significantly increased lh concentrations to concentrations comparable to the preovulatory lh surge. in another experiment (performed in cyclic mares during the summer) by these authors, a similar hormonal response was observed with 1 injection of domperidone microparticles (3 g) after estradiol benzoate treatment.80 a few years later, the combination of domperidone microparticles (3 g) and estradiol (estradiol cypionate [ecp]) was compared to combinations of these 2 medications with long-acting progesterone in anestrous mares; mares receiving estradiol cypionate and domperidone ovulated within the study period of 35 days unlike those not receiving treatment with the combination of these 2 medications. addition of progesterone to ecp and domperidone did not affect the date of first ovulation over mares just receiving ecp and domperidone. an additional experiment within this study showed a positive effect of ecp on advancing ovulation in domperidone-treated mares, regardless of the dose of ecp or domperidone used.81 treatment with domperidone (1.5 g microparticles) beginning 1 day after ecp has also been compared with treatment with either 0.75 g or 1.5 g sulpiride 1, 6, and 11 days after treatment with ecp. using this experimental protocol, 7 of 9 mares treated with 1.5 g sulpiride ovulated earlier than controls. mares receiving domperidone or 0.75 g sulpiride did not ovulate earlier than controls.82 previous studies seem to suggest that sulpiride may be more efficacious than domperidone in inducing cyclicity, though adding estradiol to protocols for either dopamine antagonist has additional positive effects. other dopamine antagonists (receptor blockers), such as fluphenazine and perphenazine, have also been studied. intramuscular fluphenazine (178.6 µg/kg daily for 3 weeks) treatment to anestrous mares resulted in follicular growth but did not advance first ovulation.83 perphenazine, however, advanced follicular growth and first ovulation of the year by ~ 30 days but required ~ 4 weeks of daily treatment.84 prostaglandin therapy intercavernous sinuses (pituitary venous outflow) of late anestrous or transitional mares were cannulated to determine gnrh, lh, and fsh concentrations in response to systemic prostaglandin analogue (luprostiol) treatment.85 an increase in gnrh was noted 20-25 minutes after luprostiol treatment; surprisingly, lh and fsh increased prior to gnrh. pulses of all 3 hormones were noted to be longer in duration but lower in amplitude than those in normally cyclic mares. authors also reported the results of a field study in which mares were treated with alfaprostol, dinoprost, or cloprostenol; 35% of treated mares came into estrus and 68% of mares ovulated within 10 days after prostaglandin treatment.85 however, the authors cited 2 earlier http://dx.doi.org/10.58292/ct.v17.11651 54 citation: clinical theriogenology 2025, 17, 11651, http://dx.doi.org/10.58292/ct.v17.11651 studies in their discussion in which alfaprostol failed to produce estrus and ovulation in transitional and anestrous mares.86,87 as reviewed,38 prostaglandins have been used to initiate cyclicity; however, the duration of anestrus experienced prior to treatment and/or depth of anestrus were unclear from these studies, so exactly how and when prostaglandin treatment induces cyclicity was not well-defined. overall, although prostaglandins may have some ability to stimulate gonadotropin release and ovulation, they have not emerged as a popular choice for induction of cyclicity and first ovulation of the year. manipulation of the cycling mare prostaglandin f2α – luteolysis, antiluteogenesis, and estrus synchronization prostaglandins are lipid compounds (eicosanoids) with diverse effects throughout the body. these compounds are produced from arachidonic acid via the cyclooxygenase pathway. arachidonic acid is converted to endoperoxide pgh2, a precursor of all prostaglandins, by the enzyme prostaglandin-endoperoxide synthase 2 (ptgs2). from there, prostaglandin synthases are responsible for producing the various specific prostaglandins (e.g. prostaglandin f2α [pgf2α]). pgf2α’s effect on the estrous cycle was first described in 1972;88 authors reported that pgf2α caused lysis of the cl, with return to estrus 3-4 days after treatment.88 the main source of pgf2α was suggested to be the uterus, as a study performed the prior year89 had demonstrated that hysterectomized mares experienced prolonged cl function. mare’s cl is highly sensitive to the effects of prostaglandin; it responds to a minimally effective dose (9 μg/kg) of 1 injection of free acid pgf2α, significantly lower than the luteolytic dose in cows.90,91 additionally, pgf2α metabolism is primarily in the lungs and occurs more slowly in mares compared to cows.92 the combination of increased sensitivity of the mare cl to pgf2α and decreased rate of metabolism is necessary, as the mare lacks the utero-ovarian countercurrent exchange mechanism present in other species. thus, pgf2α produced by the uterus must first circulate systemically prior to reaching the ovary, making the concentration that reaches the ovary much lower than in species with a countercurrent exchange mechanism. since the initial report of its effectiveness in shortening diestrus, pgf2α has become a key tool for veterinarians seeking to manipulate estrous cycle lengths, synchronize mares, and terminate pregnancies, among other uses. the most used prostaglandins are naturally occurring pgf2α, dinoprost tromethamine (lutalyse®, zoetis, parsipanny, nj, usa), and the synthetic prostaglandin analogue, cloprostenol (estrumate®, merck and co, inc, rahway, nj, usa).93 recommended dosage for dinoprost tromethamine is 0.01-0.02 mg/kg (5-10 mg or 1-2 ml per 500 kg horse) and for cloprostenol is 0.55 μg/kg (~ 250 mg or 1 ml per 500 kg horse).93 pgf2α can be used in a variety of ways to produce luteolysis;94,95 however, intramuscular treatment is generally preferred due to ease and decreased severity of side effects. side effects include increased gastrointestinal motility, abdominal discomfort, hyperthermia, sweating, and potentially diarrhea, labored breathing, and ataxia. these side effects appear to be dose dependent.96 as some horses may be perceived as being distressed by the side effects occurring at standard doses of pgf2α for luteolysis, researchers have attempted to determine if lower doses of prostaglandin may be used. intramuscular cloprostenol (25  µg; 10th of the recommended dose) given to a standard-sized horse, effectively induced luteolysis.97 similarly, studies using intramuscular dinoprost tromethamine have had success using only 1 dose of 1.25 mg or 2 doses of 0.5 mg given 24 hours apart.98-100 the lower dosages used in these studies were associated with no visible side effects when these were recorded as part of the study protocol.97-99 the cl has been noted to be refractory to lysis until day 5 after ovulation. however, a subset of mares may respond to luteolytic agents earlier than this.91,101 cl regressed with 10 mg dinoprost given on day 3 after ovulation; however, functional resurgence of the cl, evidenced by increased blood progesterone concentrations, was detected in 75% of mares 3 days after treatment and initial regression. on average, the mares in the day 3 prostaglandin group ovulated significantly earlier than mares injected with prostaglandin on day 10.102 other researchers have also investigated the effects of prostaglandin given prior to day 5 after ovulation; however, those studies utilized multi-day protocols with various doses (e.g. 2.5 versus 10 mg dinoprost). although some regression/resurgence of corpora lutea was observed, most mares experienced luteal regression and ovulated as early as 7-9 days after treatment.103,104 branching off these earlier studies, specific research into antiluteogenesis, or the complete prevention of cl formation, has been performed. in addition to dinoprost (10 mg) given on days 0, 1, and 2 of the estrous cycle (ovulation = day 0), 2 additional doses of 10 mg pgf2α were given on days 3 and 4 after the initial 3 days of twice-daily treatment; there was lack of cl formation in 10 of 10 mares treated.105 they also observed a 5 day shorter inter-ovulatory interval in the mares treated with the antiluteogenic protocol and these mares had equal pregnancy rates to controls when bred on the next cycle.105 protocols involving early (days 0-2) prostaglandin treatment have had various results with regards to effects on same-cycle pregnancy rates, with some studies having decreased pregnancy rates with these protocols106,107 and with no effect on pregnancy rates.108 the potential for reduced same-cycle pregnancy rates is important to consider when using prostaglandins for their ecbolic effects postbreeding. however, prostaglandins may be preferred over oxytocin due to their ability to produce lower amplitude, longer lasting contractions of the myometrium and assist in lymphatic flow.109,110 indeed, the use of prostaglandins in the periovulatory period may be beneficial in mares predisposed to developing mating-induced endometritis (due to cervical issues, poor conformation, bred using frozen semen, etc.) or in mares with suspected issues with lymphatic drainages (i.e. mares with endometrial cysts). timing of ovulation is 2-15 days (average 8-10 days) after 1 prostaglandin treatment.88,91,111,112 interval from prostaglandin to ovulation depends on the follicular size and dynamics at treatment. specifically, ovulation may occur within 2-3 days if there is a large, growing follicle on 1 of the ovaries (near the end of the growth phase of a follicular wave) at treatment. conversely, if treated during atresia of previously growing follicles or when the follicles on the ovary are quite small, interval to ovulation will be longer.112,113 endometrial edema may aid in determining whether a large follicle (> 30 mm) present at prostaglandin treatment is http://dx.doi.org/10.58292/ct.v17.11651 citation: clinical theriogenology 2025, 17, 11651, http://dx.doi.org/10.58292/ct.v17.11651 55 destined to ovulate or become atretic. large follicles that will ovulate may be preceded by edema that forms within 24 hours after treatment, whereas treatment when a large atretic follicle is present will not result in endometrial edema for 3-5 days.114 however, some mares with large follicles may not exhibit signs of estrus and may never develop signs associated with rising estrogen (endometrial edema, cervical softening) prior to ovulation.112,115 care must be taken to closely observe other indicators of estrous cycle status (endometrial edema, cervical softening, etc.) when using pgf2α to ‘short cycle’ mares so that timing of breeding coincides with observed or expected ovulation. the variability in interval from treatment to ovulation makes the sole use of prostaglandin for such purposes as estrus synchronization for embryo transfer less than ideal, even when 2 injections (14 days apart) are used. as described above, depending on the follicular status at prostaglandin treatment, ovulation may potentially occur anywhere in a wide range of days after 2 injections of prostaglandin.116 thus it was concluded that, with this variability, at least 10 recipient mares would be needed to have an 80% chance that at least 1 would ovulate within 24 hours of the donor mare using this technique.117 the use of transrectal ultrasonography may improve success; however, the application of additional hormonal manipulations, as will be described in upcoming sections, will also improve success and reduce the number of recipients needed per donor. human chorionic gonadotropin induction of ovulation human chorionic gonadotropin is a glycoprotein synthesized by the chorionic villi of the human placenta beginning very early in pregnancy and excreted in the urine. its secretion starts only a few days after conception and continues throughout the first part of human pregnancy. human chorionic gonadotrophin supports the cl, which is responsible for maintaining pregnancy during this time. secretion declines once the placenta has developed enough to take over maintenance of pregnancy, a scenario that is very similar to the secretion of equine chorionic gonadotropin (ecg) during the first 40 to at least 100 days of pregnancy in the mare. use of hcg to induce ovulation was first suggested in 1939.118 human chorionic gonadotropin has luteinizing-hormone activity in the mare; thus, it promotes cl formation and progesterone production.119 it has become widely used by equine practitioners for this purpose. broodmares bred by live cover often have prescheduled appointments with specific stallions, making more precise timing and control of ovulation necessary. this is especially true during the busiest part of the breeding season, when a mare may not be allowed multiple visits to the stallion in a single estrous cycle. additionally, practitioners breeding mares via artificial insemination must have some control of timing of ovulation as cooled semen, and especially frozen semen, has a limited lifespan. practitioners should aim to inseminate mares with cooled, transported semen within 24 hours before ovulation and within 6-8 hours before or after ovulation for frozen semen. human chorionic gonadotrophin is given to advance ovulation of a follicle or follicles destined for this fate. generally, these follicles are identified using a combination of palpable and ultrasonographic characteristics, in addition to potential known history about the timing of previous cycles. some large follicles may be destined for atresia and thus will not respond to hcg with ovulation. this is the challenge confronting practitioners, especially when close monitoring of the cycle has not been performed up to that point. multiple doses and routes of treatment have been described for hcg, with variable efficacy. differences in response to hcg treatment may be observed across breeds, as some breeds (e.g. friesians) tend to ovulate larger follicles and thus a follicle of a diameter appropriate for ovulation induction in one breed may not have achieved dominance in another breed. in general, dosages ranging from < 1,000 up to 6,000 iu have been used with success across various treatment routes. follicles induced to ovulate must be steroidogenically competent in addition to achieving a larger diameter. during the transitional period, large follicles may appear that are not competent to ovulate and may, as a result, regress. these follicles may not respond to hcg treatment; however, some researchers have had success in treating transitional follicles and causing ovulation earlier than in untreated mares,120 especially when progesterone priming was used.121 human chorionic gonadotrophin has been more effective than gnrh agonists in transitional mares in some studies122 but less effective in others.123 during normal cyclicity, hcg causes ovulation within 48 hours in most mares. intravenous hcg (3,000 iu) treatment to mares with follicles > 30 mm resulted in ovulation (~ 45 hours after treatment).124 a 2,500 iu hcg dose resulted in ovulations occurring earlier than in the previous study (36 hours); however, both studies documented that ovulation resulting from hcg treatment occurred earlier than those resulting from deslorelin.124,125 though hcg may cause ovulation earlier than deslorelin, it has been suggested that it is less reliable, as only 83.3% of hcg-treated mares ovulated within 48 hours versus all mares receiving subcutaneous deslorelin acetate (ovuplant™) in one study.126 ovulations occurred 25-48 hours in 75% of mares after 2,000 iu of hcg treatment.127 various doses of hcg have been used by practitioners and researchers, prompting investigation of potential response differences among doses. ovulation and pregnancy rates among mares treated with 1,500, 3,000, or 6,000 iu doses of hcg were not different.128 in addition to accelerating ovulation, hcg has also been observed to increase the synchronicity of multiple ovulations.129 increased synchronicity results in ovulations occurring < 12 hours apart, as opposed to up to 2-3 days apart.130 this may increase chances of pregnancy or multiple pregnancies; however, this effect was not observed in control mares, despite an increase in synchronicity of multiple ovulations.130 it is important to note that response to hcg, even during the peak breeding season and not during the transitional period, may be more unpredictable in older mares.127,131 one of the suggested reasons for this observation is antibody formation as a result of treatment with hcg. some studies suggest that mares must receive 4 or 5 treatments with hcg during the breeding season before loss of efficacy due to antibody formation,132,133 whereas others noticed reductions in efficacy as early as the second or third treatment.134,135 loss of efficacy is evident as failure of hcg to advance ovulation and failure of hcg to induce predictable ovulations. in contrast, loss of ovulatory efficacy was not observed despite the development of hcg antibodies with half-lives from 1 to multiple months.136 more recently, the same pattern of decreased 48-hour ovulation rates was observed by the third treatment with hcg, but http://dx.doi.org/10.58292/ct.v17.11651 56 citation: clinical theriogenology 2025, 17, 11651, http://dx.doi.org/10.58292/ct.v17.11651 also noted an increase in ovulatory failures in mares > 15 years of age versus mares < 15 years of age.137 similarly, follicular dynamics and oocyte maturation in mares positive for hcg antibodies was investigated.138 antibody-positive mares had less blood flow within the follicular wall and fewer mature oocytes on aspiration at 30 hours after hcg, suggesting an effect of hcg antibodies on oocyte maturation and ovulation.138 it is important to note that both intramuscular and intravenous routes of treatment have been investigated in the aforementioned studies. although hcg has been proven efficacious in advancing ovulation, the risk of antibody formation and diminished or unpredictable response has prompted investigation139 and use of other compounds that may be more reliable across multiple cycles. gonadotropin releasing hormone and analogues – induction of ovulation gnrh is a small peptide released from the hypothalamus in specific patterns throughout the estrous cycle, as described previously in this review. its release stimulates gonadotropins (fsh and lh) release from the anterior pituitary, affecting the gonads (ovaries). the use of gnrh and its analogues in inducing cyclicity has been described above. gnrh analogues, such as deslorelin or buserelin, are widely used among practitioners. past and current formulations of deslorelin include a 2.1 mg, slow-release, subcutaneous implant called ovuplant™, and sucromate® (dechra veterinary products, overland park, ks, usa), an fda-approved intramuscular injection containing 1.8 mg deslorelin acetate. short-term subcutaneous implants of deslorelin acetate to mares when a follicle reached 40 mm resulted in 93% of mares ovulating within 48 hours compared to only 7% in controls.140 additionally, 63% of ovulations occurred between 36-48 hours, which was a tighter window of ovulation when compared to various doses of hcg.140 additional studies have corroborated these findings using the same short-term release implant.141,142 buserelin has also been described to elicit high ovulation rates, with one study citing that 100% of draft mares ovulated in response to 40 μg of intramuscular buserelin given once a follicle had reached 45 mm and endometrial edema was present; average interval to ovulation was 29 hours in treated mares versus 59 hours in controls.143 though draft mares do tend to ovulate larger follicles, it must be considered whether, with follicles that large, ovulation was imminent regardless of treatment with buserelin as an ovulation-inducing agent. buserelin has been compared to hcg for its effectiveness in inducing ovulation in multiple studies. results were variable, with some studies having a similar efficacy144,145 and some had reduced efficacy compared to hcg.146 in comparison to injectable forms of gnrh analogues, the slow-release device ovuplant™ (2.1 mg deslorelin) was developed as a means of releasing a gnrh analogue over multiple days with a single implant. various doses of deslorelin in implant form have been evaluated and although all doses investigated were successful in inducing ovulation earlier than controls, only the 2.2 mg-containing implant was as successful as a 3,000 iu intravenous dose of hcg.124 as has been previously mentioned with injectable deslorelin, ovuplant™ has a tight window of ovulation, with 94% of mares ovulating between 38-42 hours after implantation.147 due to the success in causing ovulation within a narrow time frame, the authors suggested that overnight checks, when employing ovuplant™ for the purpose of frozen semen breeding, were unnecessary.147 unfortunately, no control group was included in this study and ovuplant™ was implanted once a follicle reached 42 mm; therefore, it must be considered whether control mares would have naturally ovulated within a similar time frame with this size follicle. just as age and seasonal effects have been reported for hcg, effects of these 2 factors on ovulation rates in response to ovuplant™ were studied.148 higher ovulation rate was observed in mares aged 10-14 years than in other age groups, with the lowest ovulation rate being in mares aged > 20 years. additionally, ovuplant™ was more effective in inducing ovulation in july-october than in march and april. although implanted deslorelin was demonstrated to have clear benefits, a prolonged inter-ovulatory interval was noted in mares receiving ovuplant™ implants.149-152 investigation into this issue resulted in the discovery that the implant depressed early fsh secretion after ovulation, resulting in delayed emergence of the next follicular wave during early diestrus.150-152 these effects are not observed, in comparison, when hcg is used to induce ovulation.153 it was then demonstrated that removal of the implant 1-2 days after ovulation prevented fsh depression and prolonged inter-ovulatory intervals.152,154 thus, it was recommended to implant ovuplant™ into the lips of the vulva and remove the device 1-2 days after ovulation, if possible.155 efficacy of deslorelin acetate (sucromate®) on induction of ovulation was evaluated.156 deslorelin acetate (138 mares; 168 estrous cycles) was compared to hcg (118 mares; 136  estrous cycles); treatment was given when mares were  in estrus with a ≥ 35 mm follicle. deslorelin acetate  yielded an 89.9% ovulation rate compared to an 82.8% ovulation rate for hcg within 48 hours after treatment.156 additionally, unlike with ovuplant™ implants, prolonged return to estrus was not observed with deslorelin acetate. recombinant luteinizing hormone – induction of ovulation recombinant equine luteinizing hormone (relh) has been studied and is a reliable option for induction of ovulation in mares. once a follicle reached 35-39 mm, mares (3-20 years) were treated with either 1 of 4 doses (0.3, 0.6, 0.75, 0.9 mg) of intravenous relh or hcg (2,500 iu). increasing doses of relh had increasing effectiveness at inducing ovulation within 48 hours after treatment. similar 48-hour ovulation rates were obtained using the 2 highest doses of relh (0.75 and 0.9 mg) compared to hcg; 90 and 80% ovulated within 48 hours in the 0.75 mg and 0.9 mg relh groups, respectively, compared to 85.7% in the hcg group. both hormonal profiles and inter-ovulatory intervals appeared to be similar between control and relh-treated cycles.157 in addition to induction of ovulation, relh increased the number of ovulations, while reducing the number of anovulatory follicles, when combined with refsh for the purpose of embryo recovery in superovulated mares.158 progesterone and progestins +/estradiol – use in estrus synchronization among their many other applications, progesterone and progestins, of which the most popular is altrenogest, can be used to http://dx.doi.org/10.58292/ct.v17.11651 citation: clinical theriogenology 2025, 17, 11651, http://dx.doi.org/10.58292/ct.v17.11651 57 synchronize estrus in mares. this may be desired when there is limited availability of a particular stallion, when it is desired to concentrate breeding times and thus foaling times, or in embryo transfer programs in which recipient mares’ cycles and ovulation must closely match a donor mare. commonly employed protocols involve 10-14 days of oral altrenogest (0.044 mg/kg) or 10 days of intramuscular progesterone (150 mg) and estradiol (10 mg).159 the addition of estradiol, as discussed in previous sections, provides inhibition of fsh, causing more complete ovarian suppression than is achieved with natural progesterone or synthetic progestins alone. both treatment strategies are followed on the last day with an injection of pgf2α. depending on the size of follicle present when progestin therapy is discontinued, interval to ovulation may be anywhere from 3 to 11 days. with the addition of estradiol, which allows for a more uniform population of follicles once therapy is discontinued, most mares will ovulate within 18-23 days after the beginning of steroid treatment.159 in addition to oral and short-term injectable forms, certain sustained release formulations of altrenogest may be useful in estrus synchronization.160 in this study, 225 and 450 mg slow-release altrenogest (biorelease la 150, bet pharm llc, lexington, kentucky, usa) groups had short-term suppression of estrus. however, although ovulation was delayed compared to controls, when individuals in these groups did ovulate, they tended to ovulate within a tight time frame without the use of an ovulation-inducing agent, which may prove useful for purposes of estrus synchronization.160 another sustained-release formulation, the intravaginal device, cidr® was investigated.161 mares received an intravaginal device containing 1.9 gram progesterone (cidr® b, zoetis south africa, sandton, south africa) during various points in their cycles. devices were removed after 12 days and mares were treated with pgf2α. mares were implanted with a short-term deslorelin implant once a follicle reached at least 40 mm in diameter. onset of estrus was synchronized following removal of the cidr®-b devices; however, ovulation was not, due to a lack of synchronization of follicular development during the period of estrus suppression. silent ovulations were noted to occur in 20% of mares while the devices were in place. additionally, as previously noted with intravaginal devices, vaginitis of some degree was noted in all treated mares.161 however, there was no difference in pregnancy rates in mares treated with cidr versus controls despite development of mild vaginitis.162 although progesterone alone can synchronize the onset of estrus, additional agents must be utilized to obtain the tight synchronization of ovulation that is necessary between donor and recipient mares. transvaginal aspiration of follicles – use in estrus synchronization an alternative to steroid-induced (i.e. progesterone and estrogen) synchronization of ovulation is tva of follicles.163,164 follicles > 10 mm were aspirated on day 0 and pgf2α was given 4 days later and hcg was used to induce ovulation, either 6 days after that or when a follicle reached at least 30 mm. this protocol was 96% successful in inducing ovulation within 48 hours after hcg treatment.163 this protocol was then compared to traditional progesterone and estrogen suppression of ovarian activity. ovulation synchronization was similar between the 2 groups, but tva of follicles resulted in a shorter interval from both the start of the study and prostaglandin treatment to ovulation.164 although tva of follicles may not be practical in all settings and requires specialized training, it remains a viable alternative to daily steroid injections for suppression of estrus in the interest of synchronizing ovulation in donor and recipient mares. suppression of estrus suppression of estrous behavior and contraception in the mare are related, but not equivalent concepts. estrus may be suppressed without disrupting a mare’s ability to conceive and removing a mare’s ability to conceive may not remove ‘mareish’ behaviors if, in fact, they are not due to reproductive hormonal cycling. reviews on suppression of estrus were published in this journal,165,166 so the methods discussed in those reviews will not be repeated here. however, new additions to the arsenal of estrus suppression are presented below. the key point to remember, as discussed in the above-mentioned review papers, is that estrous behaviors arise from low or lack of progesterone, hence mares in anestrus and transition with no functional corpora lutea will have variable signs of estrus despite their inability to ovulate during these time frames. therefore, a majority of estrus suppression techniques supplement progestins or aim to prolong luteal function. prolonging the luteal phase – slow-release oxytocin use of specifically timed (days 7-14 after ovulation) oxytocin treatment to prolong the luteal phase was reviewed.165 it was noted that this method requires daily ultrasonography prior to ovulation to determine the exact day of ovulation. it also requires daily injections, albeit intramuscular, making it a potentially cumbersome treatment option. intramuscular oxytocin (60 iu) given once daily from days 7-14 after ovulation (day 0) prolonged the luteal phase for 2 months in 67% of treated mares.167 recently, the use of a proprietary slow-release oxytocin formulation in the suppression of estrus in mares was evaluated.168 initially, 1 ml of intramuscular slow-release oxytocin (2,400 iu) given on days 7 and 10 after ovulation prolonged luteal function in 75% of treated mares. the group then evaluated a single injection of slow-release oxytocin (4,800 iu) given on either day 8 or 9 after ovulation. this single, higher dose did prolong luteal function over the 40-day progesterone-sampling period in 4/8 treated mares; however, they did not achieve the 70% estrus suppression rate that was expected. the authors noted that the small sample size used in the study may have limited their ability to find statistical significance in cl lifespan prolongation between treated and untreated mares.168 intrauterine devices – iupods® the iupod® (pearl pod llc, amherst, ma, usa) intrauterine device system is comprised of 3, 12 x 26 mm, elliptical, magnetic devices that are inserted independently. once free of the insertion tube, the 3 pods self-assemble into a low-energy ring or tripod formation. like the intrauterine marble, the device aims to mimic pregnancy by providing mechanical stimulation to the endometrium, similar to embryo (prior to fixation on days 16-17). when inserted during estrus, average length of diestrus was 73.4 days. in a second experiment, the iupods® were inserted at any stage of the estrous cycle, producing an average prolonged diestrus length of 51.3 days. endometrial scores (obtained before and after device insertion from mares in the first experiment) were not affected by http://dx.doi.org/10.58292/ct.v17.11651 58 citation: clinical theriogenology 2025, 17, 11651, http://dx.doi.org/10.58292/ct.v17.11651 presence of the iupods®. eight mares were randomly selected for breeding in the season after devices removal and 100% of these mares became pregnant.169 results were similar in another study that used a device with the same configuration but larger size (16 x 38 and 16 x 40 mm).170 devices were implanted in 6 mares regardless of estrous cycle stage and mares were placed in a paddock with a known-fertile stallion. none of the mares became pregnant during the 3-month study period and all mares retained the devices. authors did note that fluid was observed on 77% of ultrasonography examinations during the study period, suggesting that the combination of the device and breeding had caused a mating induced endometritis. prolongation of the luteal phase (37 and 91 days) was observed in 2 mares, but for the other mares the average length of diestrus was shortened at 7.8 days, potentially due to uterine inflammation and related premature release of prostaglandin.170 a similar study was conducted in feral equids, wherein devices were implanted in 8 mares in various stages of the estrous cycle. these mares were housed with a fertile stallion for 120 days after being fitted with the device. none of the mares became pregnant during the study period, and 2 of 4 mares conceived within 30 days after device removal; 2 mares were noted to have abundant biofilm on the devices at removal.171 although the above-mentioned intrauterine devices appear to be effective at preventing pregnancy, their presence within the uterus may be associated with intrauterine inflammation and biofilm formation in a subset of mares. conclusion great advances have been made in our ability to manipulate the mare’s estrous cycle, however more research is needed in multiple fields (e.g. estrus synchronization). recombinant hormone products have promise for the  future and as their availability increases, we will surely  know more about how successful they can be compared to traditional hormonal manipulations. in the  meantime, progestins, gnrh analogues, hcg, and prostaglandins have received attention across the decades  that have 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transfer. consequently, methods for vitrification and long-term storage of equine expanded blastocysts were developed. the most successful of these methods require micromanipulator-facilitated blastocoele collapse and subsequent dehydration/rehydration of embryos during vitrification and warming. however, difficulty and expense of these techniques limit blastocyst vitrification/warming to tertiary referral centers staffed by highly trained individuals. a simplified method that would make the process accessible to field practitioners is highly desirable. in conversations with practitioners, we learned that, in the absence of a suitable recipient mare, some field veterinarians attempt manual blastocoele collapse using a small gauge hypodermic needle prior to embryo vitrification with a commercial ‘kit’. pregnancy outcomes following warming and transfer of these embryos are not known. we duplicated this field technique in a controlled setting and determined subsequent pregnancy rates. to further mimic the scenario that might be encountered in general practice, all embryo manipulations were performed by veterinarians familiar with routine handling of fresh embryos, but with little to no experience with embryo collapse or vitrification. twelve grade i, large (> 290 µm) blastocysts were recovered from donor mares following routine embryo flush done 8 days after ovulation. embryos were manually collapsed with a 25 gauge hypodermic needle and vitrified using a commercial kit. six embryos were lost or damaged during collapse, vitrification or warming. the remaining 6 embryos were warmed and transferred to recipient mares by a veterinarian highly experienced with transcervical embryo transfer. no pregnancies resulted; therefore, we suggest that this simplified technique, at least when performed by inexperienced operators, is not suitable for clinical use. keywords: equine, embryo, blastocyst, vitrification introduction embryo transfer (et) is an assisted reproductive technique used when performance or health issues prevent a donor mare from carrying or maintaining a pregnancy. the technique involves breeding a donor mare and recovering the embryo by flushing the donor mare’s uterus on days 7 or 8 after ovulation, when it has typically reached the blastocyst stage and is ~ 300 µm or more in diameter. the embryo is then transferred transcervically into a recipient mare whose estrous cycle has been synchronized with that of the donor. pregnancy rates (prs) following transcervical transfer of fresh embryos range from 74 81%.1,2 expense and labor to maintain a sufficient number of fertile, estrous cycle-synchronized, recipient mares are major limiting factors to clinical et. this is particularly relevant to private practitioners who may not have access to large recipient herds. when multiple donor mares ovulate on the same day and/or when a donor mare ovulates more than one follicle on a cycle, there may be an insufficient number of estrous cycle-synchronized recipient mares to accommodate fresh transfer of each embryo. in this regard, the ability to successfully preserve equine embryos would greatly facilitate equine et by allowing embryos to be stored indefinitely until a suitable recipient is available. preservation of smaller equine embryos (250 400 µm) by vitrification can be very successful, with some reporting up to a 75% per transfer pr following warming.3-9 in particular, pregnancies were reported when a simple, closed vitrification technique was performed on smaller embryos (morula or early blastocyst stage and < 250 µm).10 this method is now available in a commercial ‘kit’ that allows for vitrification with minimal laboratory equipment and minimal training. however, to obtain these smaller 115 clinical theriogenology • volume 11, number 2 • june 2019 embryos, the embryo flush must be carefully timed and performed on day 6 to 6.5 after ovulation or 8 days after properly timed administration of human chorionic gonadotropin (hcg). when a flush is scheduled based on day of ovulation, embryo recovery rates can be significantly lower (23 66%) than those reported for days 7 or 8 flushes (49 82%).10,11 because of the careful timing required for these early flushes, because the embryo is smaller and more difficult to identify, and because of the potential for lower recovery rates, in clinical practice, the vast majority of uterine flushes for embryo recovery are performed on days 7 or 8 after ovulation. however, vitrification and warming of intact, expanded blastocysts (> 400 µm) commonly obtained on days 7 or 8 flushes results in poor success following embryo transfer.4,12 until per transfer prs following vitrification and warming of days 7 or 8 expanded blastocysts approach those achieved following fresh embryo transfer, embryo vitrification will continue to be of limited use in equine clinical practice. it was suggested that the embryonic capsule, together with the large size of the blastocoele cavity, may limit penetration of cryoprotectants into expanded equine blastocysts and therefore may contribute to the low survival of these embryos following vitrification and warming.4,13,14 in this regard, per transfer prs ranging from 46 86% were reported when expanded blastocysts are punctured and/or collapsed prior to vitrification.13,15,16 highest prs were achieved when micromanipulator-facilitated puncture and collapse of the blastocoele was combined with stepwise dehydration in increasing concentrations of ethylene glycol and galactose or sucrose, either by immersion or by direct microinjection of the solution into the collapsed blastocoele, and subsequent vitrification using an open vitrification system with microloader tips.13,15 warming was also performed in an open system and included stepwise rehydration in decreasing concentrations of galactose or sucrose, with or without subsequent embryo culture.13,15 however, technical difficulties and costs involved with these techniques have limited their use to specialty institutions with access to advanced laboratory equipment and individuals with considerable expertise in the use of this equipment. the objective was to develop an alternative method for blastocoele collapse and vitrification that did not require highly trained personnel or the use of a micromanipulator. we combined a manual method of blastocoele collapse with a simple, closed, commercial equine embryo vitrification and warming system. after warming, each embryo was rehydrated using a stepwise system that had previously been reported to result in pregnancies following transfer of collapsed, vitrified expanded blastocysts.13 additionally, all advanced embryo manipulations (embryo collapse, vitrification and warming) were performed by veterinarians who commonly worked with fresh embryos, but who had no prior experience with embryo collapse or vitrification, similar to the situations of many private practitioners working in equine theriogenology. in simplifying these techniques, and in having them performed by individuals with no prior experience with more advanced embryo manipulations, our goal was to develop an approach to blastocyst vitrification that could readily be adapted to clinical practice and so render et more costeffective for the client and more accessible to the equine practitioner. materials and methods animal care and oversight all procedures conducted on live animals were approved by the university of pennsylvania’s institutional animal care and use committee. animals were maintained on pasture with supplemental hay as needed. embryo donor mares six light breed mares, ages 7 15 years, were used for artificial insemination and subsequent embryo recovery. all mares selected as embryo donors were known fertile mares that had been bred with fresh semen and maintained a pregnancy through 14 or 15 days during previous cycles during the same breeding season. these pregnancies were electively terminated by 15 days of gestation. mares had no ultrasonographic evidence of uterine or ovarian pathology throughout this study. 116clinical theriogenology • volume 11, number 2 • june 2019 embryo recipient mares five pony mares, ages 2 7 years, and one light horse mare (age 11 years) were used as embryo recipients. recipient mares were either young maiden mares or known fertile mares that were confirmed pregnant within the 12 months prior to this study. all pregnancies were terminated routinely during early gestation as part of another project and no intrauterine procedures were performed since that time. mares had no ultrasonographic evidence of uterine or ovarian pathology throughout this study. breeding management and embryo recovery donor mares were presented daily to a stallion until behavioral estrus was identified. beginning on the first day of estrus, the reproductive tract of each mare was examined between 7 and 9 am daily by transrectal palpation and ultrasonography to monitor ovarian follicular development. once the mare developed an ovarian follicle > 35 mm in diameter, with associated uterine edema and a relaxed cervix, the mare received hcg (2500 iu, iv; intervet, millsboro, de). the morning following hcg administration, mares were bred via artificial insemination of fresh extended semen collected from one of two resident stallions of proven fertility. semen was extended 1:1 (v:v) with inra 96 (imv technologies, maple grove, mn) and immediately used for breeding. each mare was inseminated with a minimum of 1 x 109 progressively motile spermatozoa. daily transrectal palpations and ultrasonographic examinations were continued until ovulation was detected. most ovulations occurred within 24 hours after insemination and all occurred within 48 hours after insemination. day of ovulation was considered day 0. embryo recovery was performed as described,11 with several modifications. day 8 after ovulation embryos were recovered using a y-junction tubing system (harvet/veterinary concepts, spring valley, wi). the common port of the y tubing was attached to a 34 french silicone catheter with a 60 cc balloon (harvet/veterinary concepts). one end of the y tubing was attached to a 65 µm embryo filter (mai genesis, spring valley, wi) and the other end was connected to a 2 liter bag of embryo flush medium (vigro™ complete flush media, bioniche, belleville, on, canada). the tubing was flushed and filled with medium prior to passage through the mare’s cervix into the uterus. the mare’s perineum was scrubbed three times with non-residue soap (ivory®) and dried. using sterile technique, the silicone catheter was passed through the cervix and the cuff inflated with 60 cc of air. one to 2 liters of embryo flush medium was used for each of three uterine lavages. for each lavage, transrectal ultrasonography was used to confirm that both uterine horns were fully distended with media prior to recovery of the fluid through the in-line embryo filter. cloprostenol (250 µg, im; estrumate®, merck animal health, summitt, nj) was administered to each donor mare following the embryo recovery attempt. the embryo filter was searched under a dissecting microscope. recovered embryos were transferred from the filter dish using a 0.5 cc straw and washed four times in embryo holding media (vigro™ holding media, bioniche). embryo collapse and vitrification embryos were placed in a small amount (~ 0.25 0.5 ml) of holding media in a standard petri dish under a dissecting microscope. the diameter of each embryo was measured before and after blastocoele puncture/collapse using the ‘measurement’ function of the standalone system (sal) for an olympus dp21 camera (tokyo, japan) attached to the dissecting microscope (figure 1a-c). briefly, the ‘scale settings’ on the sal were set to coincide with the monitor adapter and microscope objective magnifications. the sal measurements were obtained using a simplified function, based on the ccd pixels delineated by placement of an electronic cursor. the embryo was held in place using very gentle aspiration applied to a tuberculin syringe attached to a 25 gauge needle (sigma-aldrich, saint-louis, mo). the blastocoele was punctured manually using a second 25 gauge needle attached to a tuberculin syringe. because of the large size of the needle, the puncture was performed using a quick stabbing motion with an attempt to introduce only the tip of the beveled needle into the embryo. rather than applying suction to the syringe used for puncture, blastocoele fluid was allowed to passively diffuse out of the hole created by the needle (figure 1c). 117 clinical theriogenology • volume 11, number 2 • june 2019 following blastocoele collapse, embryos were vitrified according to the manufacturer’s recommendations, using a commercially available embryo vitrification kit (equipro vit-kit™, mofa global, verona, wi) adapted from a glycerol/ethylene glycol-based vitrification method previously described.10 embryos were stored in 0.25 cc straws in liquid nitrogen until warming and transfer the following breeding season. preparation of embryo recipients estrus was synchronized and/or hastened in recipient mares using either a compounded combination of 150 mg progesterone and 10 mg estradiol im once daily for 10 days (hagyard pharmacy, lexington, ky) and 250 µg of cloprostenol im on day 10 (n = 2 pony mares) or a single injection of 250 µg of cloprostenol im (n = 3 pony mares and 1 light horse mare). once uterine edema was identified in association with a > 30 mm follicle, the reproductive tract of each mare was examined daily or every other day by transrectal palpation and ultrasonography to monitor ovarian follicular development. once the mare developed a follicle > 35 mm, with associated uterine edema and a relaxed cervix, the mare received hcg (2500 iu, iv). transrectal palpation and ultrasonographic examinations were continued, daily or every other day, until ovulation was detected. embryo warming and transfer five or 6 days after ovulation was documented in the recipient mare, a single vitrified embryo was warmed and transferred transcervically. embryos were warmed in the closed 0.25 cc straw in a 37ºc water bath for 60 seconds. after warming, the straw was flicked to mix the contents and contents expelled into an empty well of a 4 well plate at 37ºc. using a 0.5 cc straw attached to a 1 ml syringe, the embryo was then transferred through a series of embryo holding media and sucrose solutions, as follows: 0.3 m sucrose for 1 minute, 0.15 m sucrose for 5 minutes, and holding media alone for 5 minutes (figure 2a-d; modified from the “eg/s” method described13). warmed embryos in holding medium were then aspirated into a 0.5 cc straw. the straw containing the embryo was loaded into an embryo transfer gun (bioniche) and the gun was covered with a sterile sheath with side delivery ports for 0.5 cc straws (bioniche). transcervical transfer was performed routinely as previously described.11 after passage of the transfer gun through the cervix, placement of the tip of the gun at the base of a uterine horn was confirmed by palpation per rectum prior to deposition of the embryo into the uterus. the tip of the sheath was examined under a dissecting microscope following transfer to confirm that the embryo had been expelled. unlike advanced embryo manipulations, all embryo transfers were performed by one highly experienced clinician. pregnancy examination pregnancy diagnosis was performed via transrectal ultrasonographic examination of the uterus at least twice between 7 and 30 days after transfer. results embryo recovery, collapse, and vitrification six donor mares were bred via artificial insemination over 17 estrous cycles. fifteen embryos were recovered (88% per cycle embryo recovery rate). only embryos measuring > 290 µm and classified as grade i were used for manual blastocoele collapse and subsequent vitrification (n = 12). the remaining three embryos were either classified as grades ii or iii. embryos used for blastocoele collapse and vitrification ranged in size from 290 725 µm. all 12 embryos were readily held in place and punctured manually in < 5 minutes. once the needle was removed the resulting hole in the trophoblast was large enough to allow > 50% of the blastocoele fluid to diffuse passively out of the embryo over several minutes, without applying suction to the syringe (figure 1). vitrification was done by one of three veterinarians in residency training. these individuals had prior experience with fresh embryo transfer, but had no experience with embryo collapse or vitrification. 118clinical theriogenology • volume 11, number 2 • june 2019 embryo warming and transfer two straws containing vitrified embryos were broken when retrieved from the liquid nitrogen storage tank. an additional 2 straws fractured during warming in the water bath. embryos were not recovered from any of these 4 straws. after warming, 1 embryo was lost during transfer among sucrose solutions. finally, after passage through the sucrose solutions, 1 embryo had the appearance of an empty embryonic capsule containing no trophoblast or inner cell mass. this structure was not transferred. problems associated with these 6 straws/embryos resulted in a 50% embryo loss rate prior to transfer. six embryos were successfully passed through the sucrose gradient and transferred into holding media prior to routine transcervical transfer. none of the transferred embryos resulted in pregnancies at 7 30 days after transfer. data are summarized in table. discussion the objective was to develop a simplified method for vitrification and warming of expanded equine blastocysts that could be used by practitioners with no prior experience in embryo collapse, vitrification or warming and that would result in pregnancies following embryo transfer. the method chosen was designed to mimic a method described by several field practioners. the technique was simple and did not require expensive laboratory equipment; however, it was associated with a high percentage of embryo loss during storage, warming and processing (50% embryo loss) and resulted in no pregnancies following embryo transfer. others have described adequate prs following embryo collapse, vitrification and warming.13,15,16 however, in an attempt to adapt this process for more general use, our chosen methodology differed in one or more ways from each of these more successful methods. differences included the experience of the individuals manipulating embryos and the methods used for embryo puncture and collapse, vitrification and embryo warming and rehydration. to mimic the situation most often encountered in practice, all advanced embryo manipulation procedures were performed by individuals with experience manipulating fresh embryos, but with no experience performing embryo collapse, vitrification, warming or rehydration. after reviewing the vitrification protocol, all were able to complete the collapse and vitrification process readily on the first attempt without assistance, attesting to the ease of this approach. however, 4 straws were broken either upon removal from the liquid nitrogen storage tank or during warming, suggesting that these straws may not have been properly loaded or sealed. additionally, moving the collapsed/vitrified/warmed embryos through the sucrose gradient proved very challenging for individuals unfamiliar with manipulating embryos in hyperosmotic solutions. unlike commercial embryo flush and holding media in which embryos readily sink to the floor of the petri dish, hyperosmotic rehydration gradients cause the embryo to float in suspension for a time until reaching equilibrium and subsequently falling to the floor of the dish. because of these differences, and in spite of the relative simplicity of this technique, operator inexperience still likely contributed to the high percentage of vitrification and embryo loss after vitrification. we inferred that veterinarians experienced only with commercial, fresh embryo transfer may initially find even this simplified technique difficult to execute. to insure that the embryo transfer technique was controlled and would not contribute to a reduction in subsequent pregnancy rates, all embryo transfers were performed by a clinician highly experienced with commercial embryo transfer and with a history or achieving a fresh embryo transfer pr > 85%. similarly, donor/recipient asynchrony was very good and therefore also was unlikely to contribute to a reduction in pregnancy rates. by examining recipient mares daily or every other day, we were able to ensure that embryo transfer was performed on days 5 or 6 after recipient mare ovulation. pregnancy rates are optimal and not significantly different when day 8 embryos are transferred on either days 5 or 6 after the recipient mare’s ovulation. similarly, pregnancy rates are optimal and not significantly different when day 8 embryos are transferred to recipients ovulating 2 or 3 days after the donor.1,17 since embryos were obtained and vitrified on day 8 after donor mare ovulation, our recipient mares ovulated the equivalent of 2 or 3 days after donors. 119 clinical theriogenology • volume 11, number 2 • june 2019 to eliminate the need for micromanipulation equipment, we used small-gauge hypodermic needles attached to tuberculin syringes both to hold the embryo in place and to manually puncture and collapse the blastocyst. even though the holding needle was not purposefully blunted, we observed no apparent damage to the embryo when gentle suction was applied to stabilize the embryo on the tip of the holding needle. however, slight movement of the embryo was noted during subsequent blastocoele puncture, suggesting that this approach did not immobilize the embryo as effectively as a micro holding pipette. after puncture of the embryonic capsule and trophoblast with the hypodermic needle, the resulting hole was large enough to allow for passive diffusion of > 50% of the blastocoele fluid, without suction. however, in previous studies on large equine embryos in which the blastocoele was collapsed more extensively using micromanipulation, pr > 83% were reported following vitrification, rehydration and subsequent embryo transfer.11 similarly, removal of as much as 87 100% of the blastocoele fluid prior to vitrification of human embryos has resulted in the highest embryo survival rates.18–20 it is therefore possible that our passive diffusion method contributed to pregnancy failure by inadequately collapsing the blastocoele. another factor that potentially contributed to the failure of our approach was the size of the needle used for blastocyst puncture. the outer diameter of a 25 gauge needle (~ 510 µm) is > 25 times the size of the outer diameter of most glass microinjection needles (~ 20 µm , figure 3) and the use of even the tip of the bevel resulted in the creation of a large hole in the embryonic capsule and trophoblast (figure 1). in this regard, one embryo appeared as an empty embryonic capsule after warming, suggesting that the trophoblast and inner cell mass may have been lost through the punctured capsule. the embryonic capsule protects the embryo during the motility stage of intrauterine development and damage to the capsule will reduce embryo survival following transfer.21 although more expensive and more fragile than a standard injection needle, glass microinjection needles are available commercially and could be used to manually puncture an equine blastocyst without the need for a micromanipulator. the limitations imposed by the purchase and storage of microinjection needles may be outweighed by their smaller size and the potential reduction in damage to the embryo during puncture. however, the size of the 25 gauge needle is not likely to be the sole cause for pregnancy failure, since a 46.7% pr (7 of 15) was reported following transcervical transfer of vitrified embryos manually punctured with a 25 gauge needle and vitrified using a low-volume, open vitrification system.16 the method of vitrification/warming likely has an important role in embryo survival. we used a vitrification/warming system modified from a previously described ethylene glycol/sucrose (eg/s) method.10,13 by combining micromanipulator-assisted blastocoele collapse with the eg/s vitrification/warming solutions and a low-volume, open vitrification/warming system performed in microloader tips, prs increased to 86% after embryo transfer.13 in an attempt to simplify vitrification, we substituted a closed system in 0.25 ml straws. embryos then were warmed in the closed straws before being expelled to permit stepwise rehydration of the collapsed blastocoele in an open system. although similar closed vitrification/warming systems have resulted in acceptable prs when used on small, intact blastocysts or morulae, this study and others have reported no pregnancies using expanded equine blastocysts (> 400 µm) with or without collapse of the blastocoele.10,12-14,16 all reports to date describing good prs following collapse and vitrification of expanded equine blastocysts used an open vitrification/warming system, combined with a fine-diameter microloader pipette tip.13,15,16 small volume (< 1 µl), open systems facilitate faster and more even cooling and warming rates and have been speculated to help minimize ice crystal formation.5,14,22 given the success rates reported to date, open vitrification systems combined with microloader tips and stepwise rehydration of the collapsed embryo appear to be critical to embryo viability after warming. conclusions our attempt to simplify the methods for collapse and vitrification of expanded equine blastocysts was unsuccessful in that it resulted in no pregnancies following embryo transfer. additional work needs to be done to develop simplified methods of collapse that could be combined with straightforward methods of vitrification and warming to permit preservation of expanded equine blastocysts in a clinical setting. it is 120clinical theriogenology • volume 11, number 2 • june 2019 possible that both the use of the 25 gauge needle for blastocoele collapse and the use of a closed, relatively large-volume system for vitrification/warming contributed to the absence of pregnancies. based on our results, the technique as we described, cannot be recommended for clinical use. acknowledgements the authors thank emily brenner, elle elliman and danielle guilfoyle for their assistance with embryo recoveries and transfers. conflict of interest authors have no conflicts of interest to declare. source of funding supported by a grant from the equine research endowment fund, university of pennsylvania school of veterinary medicine. references 1. mccue pm, ferris ra, stokes j, et al: pregnancy rate and pregnancy loss after transfer of in vivo or in vitro derived equine embryos. j equine vet sci 2016;41:70. 2. cuervo-arango j, claes an, ruijter-villani m, et al: likelihood of pregnancy after embryo transfer is reduced in recipient mares with a short preceding oestrus. equine vet j 2018;50:386-390. 3. squires el, mccue pm: cryopreservation of equine embryos. j equine vet sci 2016;41:7-12. 4. carnevale e: vitrification of equine embryos. vet clin na equine pract 2006;22:831-841. 5. grizelj j, duchamp g, guignot f, et al: ultra rapid open pulled straw vitrification is a perspective for freezing horse embryos. veterinarski arhiv 2009;79:105-117. 6. hochi s, fujimoto t, bruan j, et al: pregnancies following transfer of equine embryos cryopreserved by vitrification. theriogenology 1994;42:483-488. 7. slade np, takeda t, squires el, et al: a new procedure for the cryopreservation of equine embryos. theriogenology 1985;24:45-57. 8. maclellan lj, carnevale em, coutinho da silva ma, et al: cryopreservation of small and large equine embryos pretreated with cytochalasin-b and/or trypsin. theriogenology 2002;58:717-720. 9. hudson j, mccue pm, carnevale em, et al: the effects of cooling and vitrification of embryos from mares treated with equine follicle stimulating hormone on pregnancy rates after nonsurgical transfer. j equine vet sci 2006;26:51-54. 10. eldridge-panuska wd, caracciolo di brienza v, seidel ge, et al: establishment of pregnancies after serial dilution or direct transfer of vitrified equine embryos. theriogenology 2005;63:1308-1319. 11. vanderwall dk: current equine embryo transfer techniques. in: ball ba, editor. recent advances in equine theriogenology: international veterinary information service; 2000. www.ivis.org. 12. campos-chillon lf, cox tj, seidel jr ge, et al: vitrification in vivo of large equine embryos after vitrification or culture. reprod fertil dev 2006;18:151. 13. choi yh, velez ic, riera fl, et al: successful cryopreservation of expanded equine blastocysts. theriogenology 2011;76:143-152. 14. sanchez r, blanco m, weiss j, et al: influence of embryonic size and manipulation on pregnancy rates of mares after transfer of cryopreserved equine embryos. j equine vet sci 2017;49:54-59. 15. diaz f, bondiolli k, paccamonti d, et al: cryopreservation of day 8 equine embryos after blastocyst micromanipulation and vitrification. theriogenology 2016;85:894-903. 16. ferris ra, mccue pm, trundell da, et al: vitrification of large equine embryos following manual or micromanipulator-assisted blastocoele collapse. j equine vet sci 2016;41:64. 17. jacob jc, haag kt, santos go, et al: effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program. theriogenology 2012;77:1159-1166. 18. kader a, choi a, orief y, et al: factors affecting the outcome of human blastocyst vitrification. reprod biol endocrinol 2009;7:99 (doi.org/10.1186/1477-7827-7-99). 19. vanderzwalmen p, bertin g, debauche c, et al: births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. hum reprod 2002;17:744-751. 20. mukaida t, oka c, goto t, et al: artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. hum reprod 2006;21:3246-3252. 21. ginther oj: dynamic physical interactions between the equine embryo and uterus. equine vet j 1985;(s3):41-47. 22. kasai m: advances in the cryopreservation of mammalian oocytes and embryos: development of ultrarapid vitrification. reprod med and biol 2002;1:1-9 121 clinical theriogenology • volume 11, number 2 • june 2019 http://www.ivis.org/ figure 1. appearance of day 8, grade i blastocyst prior to puncture (a), 1 minute after puncture (b) and 5 minutes after puncture (c). note collapse of the blastocoele within the embryonic capsule in panels b and c. the hole in the trophoblast was most apparent in panel c (marked by an arrow). measurements were obtained using the ‘measurement’ function of the standalone system (sal) for an olympus dp21 camera. figure 2. appearance of a collapsed embryo immediately after warming and after mixing of the contents of the warmed straw (a), after transfer into the 0.3 m sucrose solution (b), after transfer into the 0.15 m sucrose solution (c), and after transfer into holding media prior to transfer (d). the bar in all four panels is 600 µm. prior to collapse and vitrification, this embryo was measured as a 725 µm grade i blastocyst. figure 3. comparison of the outer diameter of a 25 gauge hypodermic needle (open arrow, top) and a 20 µm microinjection needle (closed arrow, bottom). table. equine embryo production, recovery and transfer. estrous cycles embryos recovered embryos collapsed and vitrified embryos transferred pregnancies 17 15 (88%) 12 (71%) 6 (35%) 0 (0%) 122clinical theriogenology • volume 11, number 2 • june 2019 morgan agnew, tamara dobbie, jennifer linton, regina turner keywords: equine, embryo, blastocyst, vitrification materials and methods animal care and oversight all procedures conducted on live animals were approved by the university of pennsylvania’s institutional animal care and use committee. animals were maintained on pasture with supplemental hay as needed. embryo donor mares embryo recipient mares five pony mares, ages 2 7 years, and one light horse mare (age 11 years) were used as embryo recipients. recipient mares were either young maiden mares or known fertile mares that were confirmed pregnant within the 12 months prior to this study. all... breeding management and embryo recovery embryo collapse and vitrification preparation of embryo recipients pregnancy examination results embryo recovery, collapse, and vitrification six donor mares were bred via artificial insemination over 17 estrous cycles. fifteen embryos were recovered (88% per cycle embryo recovery rate). only embryos measuring > 290 µm and classified as grade i were used for manual blastocoele collapse and ... all 12 embryos were readily held in place and punctured manually in < 5 minutes. once the needle was removed the resulting hole in the trophoblast was large enough to allow > 50% of the blastocoele fluid to diffuse passively out of the embryo over sev... embryo warming and transfer two straws containing vitrified embryos were broken when retrieved from the liquid nitrogen storage tank. an additional 2 straws fractured during warming in the water bath. embryos were not recovered from any of these 4 straws. after warming, 1 embryo... acknowledgements << /ascii85encodepages false /allowpsxobjects false /allowtransparency false /alwaysembed [ true ] /antialiascolorimages false /antialiasgrayimages false /antialiasmonoimages false /autofiltercolorimages true /autofiltergrayimages true /autopositionepsfiles true /autorotatepages /none /binding /left /calcmykprofile (u.s. web coated \050swop\051 v2) /calgrayprofile (dot gain 20%) /calrgbprofile 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] /pdfx1acheck false /pdfx3check false /pdfxbleedboxtotrimboxoffset [ 0 0 0 0 ] /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxoutputcondition () /pdfxoutputconditionidentifier () /pdfxoutputintentprofile () /pdfxregistryname () /pdfxsetbleedboxtomediabox true /pdfxtrapped /false /pdfxtrimboxtomediaboxoffset [ 0 0 0 0 ] /pagelayout /singlepage /parsedsccomments true /parsedsccommentsfordocinfo true /parseiccprofilesincomments true /passthroughjpegimages true /preservecopypage true /preservedicmykvalues true /preserveepsinfo true /preserveflatness false /preservehalftoneinfo false /preserveopicomments true /preserveoverprintsettings true /startpage 1 /subsetfonts true /transferfunctioninfo /apply /ucrandbginfo /preserve /useprologue false /srgbprofile (srgb iec61966-2.1) >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: antibiotics and other additives for semen extenders to enhance fertility antibiotics and other additives for semen extenders to enhance fertility 1 s. p. brinsko 2 department of large animal clinical sciences, college of veterinary medicine, texas 3 a&m university, college station, tx, usa 4 abstract 5 semen extender formulations have been evolving for over 70 years and this 6 evolution continues. although milk was found to be suitable as a major component of 7 equine semen extenders over 60 years ago, preparation of the extenders remained tedious 8 and time consuming in part, due to the need to heat the milk before use. with the 9 publication of the formulation for a nonfat, dried skim milk–glucose (nfdsm-gluc) in 10 the 1970s, a simple, convenient semen extender became available, prompting a dramatic 11 expansion in the use of equine artificial insemination. this basic formulation of the 12 nfdsm-gluc extender is still in use today, with various modifications. more recent 13 work has further identified the beneficial components of milk, which has led to more 14 defined extender formulations. this paper briefly reviews the development of equine 15 semen extenders and discusses the effect of including various additives such as 16 antibiotics, cryoprotectants, and antioxidants on equine sperm survival and fertility. 17 18 keywords: stallion, semen extender, additives 19 20 369 introduction 21 the search for an optimum semen extender formulation has been an ongoing 22 quest almost from the inception of artificial insemination. despite the fact that artificial 23 insemination of mares is reported to have occurred much earlier than in cows and much 24 of the early work in the field was done in the horse, the development of effective 25 extenders for stallion semen was outpaced by the development of bull semen extenders.1,2 26 this was due in part to the lack of demand for storage of stallion semen, the belief that 27 stallion sperm lacked “the innate resistant factor necessary for storage” and the restrictive 28 attitude of many breed registries toward artificial insemination.1,3 while a thorough 29 review of the development of equine semen extenders is beyond the scope of this 30 manuscript, a brief review of early formulations should help set the stage for where we 31 are today. 32 over the years, numerous extender formulations have been employed in attempts 33 to improve the survivability of stallion semen. included in these formulations are various 34 combinations and levels of: egg-yolk, sugars, buffers, citric acid, electrolytes, gelatin, 35 glycerin, honey, milk products and even blood serum and follicular fluid.1,2,3 the milk 36 products have included mare’s milk, sheep and goat milk, cow’s milk, cream, skim milk, 37 buttermilk, and nonfat dried milk solids (nfdsm).1,2,3 one of the first extenders for 38 stallion semen was a glucose-sulphate-peptone formulation,4 which was later modified to 39 a tartaric-glucose-peptone formulation.5 peptone is a water-soluble mixture of amino 40 acids and peptides derived from the partial hydrolysis of protein. although the source of 41 the peptone used in those early experiments is not stated, it is interesting to note that the 42 370 peptone that can currently be obtained from sigma-aldrich (st. louis, mo, usa) is 43 derived from the enzymatic digestion of the milk protein, casein. 44 for many years, equine semen extender formulations were either the same or very 45 similar to those used for bull semen. results were mixed, sometimes being favorable and 46 oftentimes less than satisfactory. after the discovery that adding egg yolk to a buffer 47 improved the survival of bull sperm6 and lessened its susceptibility to temperature 48 shock,7 a number of investigators incorporated egg yolk into equine semen extenders.8,9,10 49 in 1949, buĭko-rogalevič reported that sperm motility was preserved for 8 to 13 days 50 when stallion semen was diluted in an egg yolk-glucose extender compared to 2.5 days 51 when diluted with glucose and that an 85.5% pregnancy rate was achieved with semen 52 stored for 12 to 42 hours in this egg yolk-glucose extender.9 in a series of experiments, 53 kühr obtained similar results with sperm survival increasing from 8.2 hours in undiluted 54 semen to 100.8 hours in 7% glucose and to 290 hours when semen was diluted in 7% 55 glucose + 5% egg yolk.11 other extender formulations based on successful bull semen 56 extenders were far less satisfactory for stallion semen.2] pace and sullivan reported that 57 the fertilizing capacity of equine semen was depressed almost immediately after mixing 58 with hydrogen ion extenders.12 investigators from several laboratories found that even 59 though various extender formulations could maintain sperm motility, the fertility of 60 semen diluted in these extenders was poor.3,11-14 the inferiority of these extenders is 61 best exemplified by the fact that pregnancy rates were higher when similar numbers of 62 sperm were inseminated using raw semen than with extended semen.12,14,15 as a result, 63 even up through the mid 1970s, it was recommended by some to use raw semen for 64 equine artificial insemination unless the semen was to be stored or unless antibiotics 65 371 needed to be added to the semen because the stallion was shedding pathogenic 66 bacteria.3,14,16 when one examines the composition of the extenders used in many of 67 those studies, it is likely that the glycerol they contained contributed to the poor fertility 68 observed. 69 milk-based extenders 70 milk was used as an extender for stallion semen as early as the 1940s and boiled 71 mare’s milk was reported to yield more favorable results than sheep, goat and even cow’s 72 milk.17 one of the major drawbacks of using fresh milk in semen extenders is the need to 73 heat the milk to 92 to 95 °c for 10 minutes in order to inactivate lactenin, which is toxic 74 to sperm.18-20 because of the heating and pasteurization involved in their manufacture, 75 use of reconstituted dried milk products is thought to alleviate the need for heating when 76 used in semen extenders. in the late 1950s and early 1960s, the chinese established a 77 very successful equine artificial insemination program involving 40 stallions and 78 thousands of mares, using semen diluted in a powdered milk-based extender.21 in 79 comparative studies, cheng reported that both maintenance of sperm motility and 80 pregnancy rates were higher using the powdered milk extender when compared to fresh 81 mare’s milk or sugar-based (glucose or sucrose) extenders.21,22 following up on 82 favorable results with bull semen in the late 1950s, workers at texas a&m evaluated 83 reconstituted buttermilk with glucose added (bmg) as an equine semen extender.1 84 although fertility was not examined, this bmg extender was found to be far superior to 85 mare’s milk, cow’s milk and egg yolk-glucose extenders for preserving sperm motility 86 for up to four days. the dried buttermilk was an “extra grade” product prepared by a 87 company in wisconsin and it may be that limited availability of this product precluded its 88 372 widespread use in semen extenders. however, non-fat dried skim milk had been readily 89 available for years and once kenney and co-workers23 published the recipe for a non-fat 90 dried milk solids-glucose extender (nfdms-gluc), this ‘kenney extender’ as it is 91 known, revolutionized equine artificial insemination in the western world. with the 92 availability of a convenient, reliable semen extender, the use of artificial insemination in 93 horses increased worldwide and the basic formula for this extender has remained virtually 94 unchanged since its publication in 1975. kenney-type extenders are available from a 95 number of commercial sources, differing primarily in the type and level of antibiotic(s) 96 added to the basic formulation. 97 antibiotics 98 inclusion of antibiotics in semen extenders is meant to reduce or eliminate 99 bacterial growth in semen, especially when it is stored, and to help control post breeding 100 endometritis. as with many other extender components, the incorporation of antibiotics 101 was based on satisfactory methods employed with bull semen. however, it was found 102 that the levels of antibiotics commonly used for bull semen extenders were toxic to 103 stallion sperm.1 berry and gazder reported that inclusion of 400 i.u./ml of penicillin and 104 1 mg/ml of streptomycin in their bmg extender was effective in controlling bacterial 105 growth without adversely affecting sperm motility. the original kenney extender 106 contained either 1,500 i.u. of crystalline penicillin/ml and 1.5 mg of crystalline 107 streptomycin/ml or 1 mg/ml of reagent grade gentamicin. antibiotics commonly 108 included alone or in combination in equine semen extenders today are: penicillin, 109 streptomycin, polymixin-b, ticarcillin, timentin, gentamicin, and amikacin. although less 110 commonly used, ceftiofur24 and pipericillin,25 have also been shown to be safe and 111 373 effective antibiotics to include in equine semen extenders. for some very acidic 112 antibiotics, eg. gentamicin and amikacin, buffers also need to be added to adjust ph and it 113 is important to use reagent grade rather than injectable products because the 114 preservatives in the latter can be toxic to sperm. 115 while sperm motility and fertility of stored stallion semen can generally be 116 maintained or improved by extenders containing any of the antibiotics listed above, the 117 choice of which antibiotic to include in the extender may be determined based on specific 118 needs or circumstances. for some normal stallions, certain antibiotics appear to be more 119 favorable than others for maintaining sperm motility in stored semen. certainly, for 120 stallions that are shedding specific pathogens into their semen, the choice of antibiotic to 121 include in the extender should be based on the sensitivity pattern of the offending 122 organism(s). 123 in the 1980s, colorado state university entered into a licensing agreement with a 124 commercial company to market a nfdsm-glu extender. the formulation was 125 essentially the same as the kenney extender except that 1000 iu/ml of polymixin b 126 sulfate replaced gentamicin sulfate as the antibiotic.26 for a number of years, this 127 extender (ez –mixin® original formula, animal reproduction systems, chino, ca, 128 usa) was used extensively in the industry for both fresh and cooled-stored equine 129 semen. later, colorado workers examined the effects of different antibiotics on motion 130 characteristics in stored semen.27 reagent grade amikacin sulfate, ticarcillin disodium, 131 gentamicin sulfate and polymixin b sulfate were added to a nonfat, dried, skim milk 132 glucose seminal extender at concentrations of 1000 or 2000 μg or iu/ml. they found that 133 overall the addition of antibiotics to extender did not significantly improve motion 134 374 characteristics of sperm over control samples but that levels of gentamicin sulfate greater 135 than 1000 μg /ml and polymixin b sulfate equal to or greater than 1000 iu/ml 136 significantly reduced sperm motility. these workers concluded that genatmicin and 137 polymixin b greater than or equal to these levels should be avoided in seminal extenders 138 used for cooled semen. texas a&m workers performed a similar series of experiments, 139 but also evaluated the control of bacterial growth.24 results of this study demonstrated 140 that semen stored in extender containing 1000 iu/ml of polymixin b sulfate resulted in 141 the greatest reduction in sperm motion characteristics and the poorest control of bacterial 142 growth. these workers determined that a nfdms-gluc extender containing potassium 143 penicillin g (1000 iu/ml) and amikacin sulfate (1000 μg/ml) yielded the best 144 combination of motility maintenance and control of bacterial growth. individual stallion 145 effects were also noted. 146 while not an antibiotic, the inclusion of the sugar mannose into semen extenders 147 has been proposed by illinois workers as an alternative to antibiotics for reducing post 148 breeding bacterial endometritis.28 previous work from this laboratory has indicated that 149 this sterioisomer of glucose was able to reduce the adherence of certain bacteria to 150 endometrial tissue.29-31 replacing up to 37 mg/ml of glucose with mannose in nfdsm-151 gluc semen extender did not affect the fertilizing capacity of sperm when immediate 152 insemination was performed on reproductively healthy mares.28 however, whether the 153 inclusion of mannose in semen extenders can control bacterial growth in semen or 154 maintain acceptable pregnancy rates with cooled-transported semen or in susceptible 155 mares requires further study. 156 375 variations on basic components 157 texas a&m workers also developed another variation of the kenney extender. 158 this formulation not only contained the penicillin-amikacin combination but also reduced 159 the level of glucose from 4.9 mg/ml to 2.65 mg/ml with the addition of sucrose at 4.0 160 mg/ml. this tamu extender has proven to be an excellent extender for use in fresh, 161 cool-stored breeding programs and also as a base extender for frozen semen after the 162 addition of egg yolk and glycerol. 163 french workers developed a successful milk-based extender that has been widely 164 used for frozen semen. in addition to sterilized skim milk, glucose and antibiotics, the 165 base inra 82 extender also contains lactose, raffinose, sodium citrate and potassium 166 citrate to which egg yolk and glycerol are added prior to freezing.32 more recently, 167 studies which evaluated the effects of different milk fractions on sperm survival resulted 168 in the development of a defined milk protein extender (inra 96) for use with fresh and 169 cooled semen.33 in this extender, skim milk is replaced with the specific milk 170 component; native phosphocaseinate (nppc) in a hank’s salts solution supplemented 171 with hepes, glucose, lactose (hgll) and bsa. while no difference was detected in 172 sperm motility after 24 h storage of semen in either inra 82 or inra 96, fertility was 173 higher for the semen stored in inra 96.33 this extender was also shown to be as efficient 174 at preserving sperm motility and fertility when semen was stored at 15 ºc as when stored 175 at 4 ºc.34 this extender can also be used for freezing stallion semen. a fertility trial was 176 conducted comparing inra 82 and inra 96 supplemented with egg yolk and glycerol. 177 although motility parameters were significantly higher in inra 82 than in inra 96, the 178 376 inra96 extender significantly improved per-cycle pregnancy rates compared with 179 inra82 (71% versus 40%) in a total of 84 mare cycles.35 180 japanese workers reported that the addition of 2% casein and 5% egg-yolk to a 181 boar semen extender (modena) resulted in superior sperm viability in cooled stored 182 semen compared to kenney extender.36 semen stored in this extender at 5 ºc resulted in 183 14 of 22 mares becoming pregnant within 72 h of storage and 3 of 4 mares becoming 184 pregnant with semen stored within 96 to 120 hours. the problem with adding egg yolk to 185 extenders is that it compromises the ability to accurately assess sperm motion 186 characteristics if the extender is not clarified. 187 workers in austria, evaluated another defined milk protein extender (equipro®, 188 minitüb, tiefenbach, germany) containing caseinate, selected whey proteins, a range of 189 different sugars and glycine.37 interestingly, casein and glycine were components of 190 early extenders such as the cgh-27 extender described by nishikawa in 1975.13 after 191 48 and 72 hours of storage at 5 ºc semen stored in equipro® extender reportedly had 192 significantly higher sperm motility than that stored in a kenney extender. they also 193 reported that centrifugation and removal of 90% of the seminal plasma, which is replaced 194 by the defined milk protein extender, increased the longevity of sperm during storage. 195 seminal plasma 196 the adverse effects of seminal plasma on the survival of equine sperm were 197 recognized as early as the 1930s in the investigations of semen storage.5,38,39 many early 198 investigations not only examined various extender formulations, but also optimal dilution 199 ratios of semen in extender. more recently, colorado workers demonstrated that when 200 using milk-based extenders, complete removal of seminal plasma resulted in significant 201 377 reductions in the sperm motion characteristics of cooled equine semen whereas 202 suspension of equine sperm in extenders containing 5 to 20% seminal plasma maintained 203 motion characteristics for over 72 hours of cooled storage.40,41 subsequently, it has been 204 widely recommended that dilution ratios of at least three to four parts extender to one part 205 semen be used for cooled equine semen, so that the level of seminal plasma does not 206 exceed 20 to 25% by volume and the sperm concentration remains between 25 x 106 and 207 50 x 106/ml. for some stallions, whose sperm do not tolerate the rigors of cooling and 208 storage using simple dilution, centrifugation and partial removal of the seminal plasma to 209 achieve even lower levels (≤ 10 to 12%, v:v) may be necessary to optimize sperm 210 survival.4 however there are other stallions whose seminal plasma is so toxic to their 211 sperm that complete removal is necessary to avoid a rapid reduction in longevity. when 212 complete removal of seminal plasma is required, alternatives to typical milk-based 213 extenders must be employed. 214 padilla and foote43 demonstrated that after centrifugation and complete removal 215 of seminal plasma, the motility of cooled-stored equine sperm was greatly improved 216 when resuspended in a kenney’s nfdsm-gluc extender supplemented with a high-217 potassium modified tyrode’s medium (kmt). however when kmt extender was used 218 in the presence of seminal plasma, motility was reduced, indicating an interaction 219 between seminal plasma and the extender composition. workers at texas a&m 220 confirmed these results, and went on further to demonstrate that fertility was maintained 221 with 13 of 17 mares becoming pregnant using semen stored for 48 h in the kmt 222 extender.44 other work from this laboratory demonstrated that both motility and dna 223 integrity were maintained in sperm from which seminal plasma was removed, followed 224 378 by resuspension in either kenney extender or modified kenney tyrodes-type extender 225 [45]. other investigators have shown that motion characteristics and acrosomal integrity 226 of sperm are maintained when stored 48 hours after seminal plasma removal and 227 resuspension in a kenney extender supplemented with commercially available phosphate 228 buffered saline containing glucose and pyruvate.46 investigators from this laboratory 229 also reported pregnancy rates of 75% (3/4) and 88% (22/25), when this extender was 230 used with semen from two poor cooling stallions in a commercial cooled-transported 231 semen program.47 232 the effects of seminal plasma are not always deleterious and appear to be stallion 233 dependent. when semen from stallions that exhibited low post-thaw sperm motility 234 (<20%) was supplemented with seminal plasma from stallions that produce semen with 235 high post-thaw motility, greater numbers of spermatozoa survived cryopreservation.48 236 cryoprotectants 237 the discovery in the 1930s that the addition of egg yolk to suitable buffers 238 significantly increased the fertilizing capacity of stored sperm from a number of species 239 resulted in the widespread use of artificial insemination in dairy cows.6,49,50 most equine 240 freezing extenders consist of milk, egg yolk, glycerol, various sugars, and electrolytes. 241 while chicken eggs are the most common source of yolk used in semen extenders, yolk 242 from other species has been substituted with favorable results. one study demonstrated 243 that sperm motility parameters were improved when stallion semen was frozen in lactose 244 edta extender supplemented with duck egg yolk rather than chicken egg yolk .51 245 379 glycerol has been one of the most widely used cryoprotectants for frozen semen. 246 however, while a higher level of glycerol often yields better post-thaw sperm motility, 247 higher glycerol levels are also contraceptive in the mare.12,52,53 levels of glycerol in 248 early studies ranged from as low a 1% to as high as 10%. in fact, the first reported 249 pregnancy using frozen-thawed epididymal stallion sperm was obtained using an 250 extender containing 10% glycerol (glycerin).54 many equine semen freezing extenders 251 currently contain approximately 4% glycerol, but some european studies suggest that a 252 final glycerol concentration of 2 to 3.5% may be most appropriate for cryopreservation of 253 equine semen.55,56 however, inra 96 with 6% glycerol was recently reported to 254 improve survivability of cryopreserved equine sperm while not adversely affecting 255 fertility.57 256 because of the tremendous variability observed in the post-thaw motility and 257 fertility of stallion semen frozen in conventional extenders, alternative cryoprotectants to 258 glycerol have been investigated. in one study, the presence of glutamine at 50 mm was 259 not sufficient to offset the need to use glycerol.58 however, it was found that 50 mm 260 glutamine added to a 2.5% glycerol medium significantly improved sperm motility 261 compared to classical freezing medium containing 2.5% glycerol. these workers 262 concluded that glutamine has a synergistic cryoprotective effect with glycerol on 263 cryopreservation of stallion sperm, and suggested that glutamine acts at the extra-cellular 264 level, independently of glycerol. 265 recent studies have demonstrated that both methyl formamide and dimethyl 266 formamide could protect stallion sperm from cryodamage as effectively as glycerol, and 267 it was suggested that these cryoprotectants might provide an alternative for stallions that 268 380 have poor post-thaw sperm motility when frozen in glycerol.59 a new freezing extender 269 botu-crio® (biotech botucatu, botucatu,sao paulo, brazil) has recently been made 270 commercially available. the main difference in botu-crio® compared to other freezing 271 extenders, is the combination of glycerol and methyl formamide as the cryoprotectant. 272 fertility was assessed for good and poor freezing stallions in a retrospective analysis of 273 355 cycles of mares bred with semen frozen in a glucose–edta–lactose extender 274 containing glycerol and on 98 mare cycles for semen frozen in botu-crio®.60 while 275 there was no difference in fertility in the good freezing group between extenders, fertility 276 of the poor freezing group was significantly better for semen frozen in botu-crio®. it 277 was concluded that the botu-crio® extender appears to improve the post-thaw quality 278 and fertility of stallions with semen that is considered to have poor freezability. 279 antioxidants 280 oxidative damage to sperm during storage is thought to be a potential cause of the 281 decline in motility and fertility. endogenous lipase activity in seminal plasma was 282 suggested to be a contributing factor in the adverse effects of seminal plasma on cooled 283 stallion sperm.61 numerous antioxidants have been added to semen extenders, with 284 varying results, in an effort to prevent damage to equine sperm by lipid peroxidation.62-68 285 as with many other extender additives, much of the work with antioxidants has examined 286 in vitro sperm characteristics rather than fertility. 287 the addition of taurine to several different extenders was reported to consistently 288 result in better sperm motility after storage than non-taurine containing extenders.62 289 addition of ascorbic acid was found to increase the percentage of membrane intact sperm 290 stored in a skim milk extender compared to controls.65 in contrast, another study found 291 381 that the addition of the enzymatic scavenger catalase, or a variety of water-soluble or 292 lipid-soluble antioxidants did not significantly improve the maintenance of sperm 293 motility in semen stored at 5 °c in a nfdsm-gluc extender.64 the addition of 2 mm 294 pyruvate to a skim milk extender was beneficial in maintaining sperm motility for semen 295 stored for 48 hours, and based on embryo recovery rates, also tended to improve 296 fertility.69 although lactate dehydrogenase activity was found to be correlated with 297 sperm motility,70 neither pyruvate nor lactate could protect sperm from a h2o2 challenge, 298 and it was suggested that beneficial effects exerted by the addition of pyruvate or lactate 299 to semen extenders were probably resulting from them acting as an energy source rather 300 than as antioxidants.67 quercetin was recently reported to protect sperm from 301 peroxidation after challenge with xanthine-xanthine-oxidase.66 these authors also 302 suggested that addition of quercetin to nfdsm-gluc extender could reduce lipid 303 peroxidation of sperm and thereby prevent premature capacitation of sperm while still 304 allowing the sperm to capacitate and acrosome react after insemination.68 however, this 305 latter conclusion was drawn from the ability of sperm in quercetin treated semen extender 306 to acrosome react after challenge with a23187, which is not very physiologic, and the 307 authors rightly suggested that fertility trials should be performed to determine the 308 effectiveness of quercetin on sperm storage. 309 the value of including additional antioxidants to semen extenders has been 310 challenged by results of more recent experiments, which indicate that there is not a 311 substantial increase in lipid peroxidation during semen storage and that peroxidative 312 damage to sperm membranes is not the predominant cause of reduced semen quality.71,72 313 workers from this laboratory report that the inherent antioxidative activity in stallion 314 382 semen appears to prevent the formation of reactive oxygen species (ros) and that the 315 simple addition of extender increases this activity further.71,72 other workers have also 316 suggested that although equine seminal plasma contains high superoxide dismutase-like 317 activity, sperm themselves have limited glutathione peroxidase and superoxide 318 dismutase-like activity.73 they also suggest that the enzymatic antioxidant activity in 319 equine sperm appears to be predominantly derived from seminal plasma adsorbed onto 320 the sperm plasma membrane and that removal of seminal plasma during semen 321 processing may increase oxidative stress in equine sperm. brazilian investigators reported 322 that lipid oxidation in the seminal plasma appeared to be a general indicator for sperm 323 damage and suggested that both lipid and protein oxidation may aid in the identification 324 of subfertile stallions, but only during the non-breeding season.74 they also reported that 325 ros production levels did not appear to result in compromised sperm dna integrity, 326 which indicated to them that either the measurements were within physiological levels 327 and/or that there is an efficient antioxidant activity in stallion sperm cells.74 328 conclusions 329 the nfdsm-gluc formulation, with slight variations on the basic components 330 ranging from antibiotics to sugars, remains the mainstay of equine semen extenders. 331 inclusion of a variety of other components such as cryoprotectants and antioxidants has 332 also been attempted, with mixed results. differences in laboratory techniques and sample 333 populations of stallions likely contributed to this disparity. use of defined milk proteins 334 has been the most recent major evolutionary step in the development of universally 335 acceptable equine semen extenders and variations on this theme will continue to fuel 336 further research. 337 383 references 338 1. berry gazdeer pj: the viability of spermatozoa as influenced by storage media and 339 by antibiotics. 1960;13:217-220. 340 2. bowen jm: artificial insemination in the horse. equine vet j 1969;1:98-110. 341 3. pickett bw, voss jl, demick ds: stallion seminal extenders. proc annu conv 342 am assoc equine pract 1974; p.155-174. 343 4. milovanov vk. selivanova oa: diluters for the sperm of livestock. probl zhivotn 344 1932;2:75-86. 345 5. gonzaga ac, valenzuela a: preliminary studies on the preservation of the semen 346 of the stallion. philip j anim ind 1934;1:317-377. 347 6. phillips ph, lardy ha: a yolk buffer pablem for the preservation of bull semen. j 348 dairy sci 1940;23:379-403. 349 7. lasley jf, lasley gt, bogart r: some factors influencing the resistance of bull 350 sperm to unfavorable environmental conditions. j anim sci 1942;1:79 (abstr) 351 8. berliner vr: dilutors for stallion and jack semen. j anim sci 1942;1:314-319. 352 9. buĭko-rogalevič an: storage of stallion semen for a long period. konevodstvo 353 1949;5:27-34. 354 10. hejzlar z: the artificial insemination of horses. sborn čsl akad zemĕd vĕd, živoč 355 vyr 1957, 2:597-614. anim breed absrt 1958;26:21. 356 384 11. kühr j; the characteristics of stallion semen and methods of diluting it. sborn čsl 357 akad zemĕd vĕd, živoč vyr 1957;2:557-574. 358 12. pace mm, sullivan jj: effect of timing of insemination, numbers of spermatozoa 359 and extender components on the pregnancy rate in mares inseminated with frozen 360 stallion semen. j reprod fertil 1975;(suppl 23):115-121. 361 13. nishikawa y: studies on the preservation of raw and frozen horse semen. j reprod 362 fertil 1975;(suppl 23):99-104. 363 14. pickett bw, burwash ld, voss jl, et al: effect of seminal extenders on equine 364 fertility. j anim sci 1975;40:1136-1143. 365 15. pickett bw, voss jl: reproductive management of the stallion. proc annu conv 366 am assoc equine pract 1972; p.501-530. 367 16. allen wr, bowen jm, frank cj, et al: the current position of ai in horse breeding. 368 equine vet j 1976; 8:72-74. 369 17. mihailov nn: milk as a diluent for the semen of animals. konevodstvo 1949;6:14-370 16. 371 18. thacker dl, almquist jo: diluters for bovine semen. i. fertility and motility of 372 bovine spermatozoa in boiled milk. j dairy sci 1953;36:173-180. 373 19. flipse rj, patton s, almquist jo: diluters for bovine semen. iii. effect of lactenin 374 and of lactoperoxidase upon spermatozoan livability. j dairy sci 1954;37:1205-375 1211. 376 385 20. pickett bw, squires el, mckinnon ao: procedures for collection, evaluation and 377 utilization of stallion semen for artificial insemination. colorado state univ anim 378 reprod lab bull no 3, fort collins, 1987. 379 21. cheng pl, sheu ck, chung sf, et al: the present situation of artificial 380 insemination of horse in china and some investigations on increasing conception 381 rate of mare and breeding efficiency of stallion. acta vet.zootech.sin 1962;5:18-382 19. 383 22. steele dg. artificial insemination research. the blood horse 1961; p1302-1304. 384 23. kenney rm, bergman rv, cooper wl, et al: minimal contamination techniques 385 of breeding mares. technique and preliminary findings. proc annu conv am assoc 386 equine pract 1975; p. 327-336. 387 24. varner dd, scanlan cm, thompson ja, et al: bacteriology of preserved stallion 388 semen and antibiotics in semen extenders. theriogenology 1998;50:559-573. 389 25. dietz jp, sertich pl, boston rc, et al: comparison of ticarcillin and piperacillin in 390 kenney's semen extender. theriogenology 2007;68:848-852. 391 26. francl at, amann rp, squires el, et al: motility and fertility of equine 392 spermatozoa in a milk extender after 12 or 24 hours at 20 ºc. theriogenology 393 1987;27:517-525. 394 27. jasko dj, bedford sj, cook nl, et al: effect of antibiotics on motion 395 characteristics of cooled stallion spermatozoa.. theriogenology 1993;40:885-893. 396 386 28. king ss, speiser sa, jones kl, et al: equine spermatozoal motility and fertility 397 associated with the incorporation of d-(+)-mannose into semen extender. 398 theriogenology 2006;65:1171-1179. 399 29. king ss, carnevales em, nequine ls, et al: inhibition of bacterial endometritis 400 with mannose. j equine vet sci 1998;18:332-334. 401 30. king ss, young da, nequin lg, et al: inhibition of bacterial adhesion to equine 402 endometrium using specific sugars. am j vet res 2000;61:446-449. 403 31. christiansen ml, johnson se, nequin lg, et al: preventing or treating endometrial 404 streptococcus zooepidemicus infection with d-(+)-mannose in vivo. in: proc 17th 405 equine nutr physiol soc;2001. p73-74 (abstr). 406 32. palmer e: factors affecting stallion semen survival and fertility. poc 10th int cong 407 anim reprod ai 1984;377 (abstr). 408 33. batellier f, magistrini m, fauquant j, et al: effect of milk fractions on survival of 409 equine spermatozoa. theriogenology 1997;48:391-410. 410 34. batellier f, vidament m, fauquant j, et al: advances in cooled semen technology. 411 anim reprod sci 2001;68:181-190. 412 35. pillet e, batellier f, duchamp g, et al: freezing stallion semen in inra96®.-413 based extender improves fertility rates in comparison with inra82. dairy sci tech 414 2008; 88:257-265. 415 36. masuda h, nanasaki s, chiba y. a new extender for preservation of equine 416 spermatozoa at 5 ºc. j equine sci 2004;15:1-5. 417 387 37. pagl r, aurich je, müller-schlösser, et al: comparison of an extender containing 418 defined milk protein fractions with a skim milk-based extender for storage of 419 equine semen at 5 ºc. theriogenology 2006;66:1115-1122. 420 38. milovanov vk: artificial insemination of farm animals. selchozgiz, 3rd ed. 1936. 421 39. walton a: preservation of the fertilizing capacity of horse semen. rec proc am soc 422 anim prod. 1938; p 238-241. 423 40. jasko dj, moran dm, farlin me, et al: effect of seminal plasma dilution or 424 removal on spermatozoal motion characteristics of cooled stallion semen. 425 theriogenology 1991;35:1059-1067. 426 41. jasko dj, hathaway ja, schaltenbrand vl, et al: effect of seminal plasma and egg 427 yolk on motion characteristics of cooled stallion spermatozoa. theriogenology 428 1992;37:1241-1252. 429 42. brinsko sp, crockett ec, squires el. effect of centrifugation and partial removal 430 of seminal plasma on equine spermatozoal motility after cooling and storage. 431 theriogenology 2000;54:129-136. 432 43. padilla aw, foote rh. extender and centrifugation effects on the motility patterns 433 of slow-cooled stallion spermatozoa. j anim sci 1991;69:3308-3313. 434 44. rigby sl, brinsko sp, cochran m, et al: advances in cooled semen technologies: 435 seminal plasma and semen extender. anim reprod sci 2001;68:171-180.. 436 388 45. love cc, brinsko sp, rigby sl, et al: relationship of seminal plasma level and 437 extender type to sperm motility and dna integrity. theriogenology 2005;63:1584-438 1591. 439 46. dawson, gr, webb gw, pruitt ja, et al: effect of different processing techniques 440 on motility and acrosomal integrity of cold-stored stallion spermatozoa. j equine 441 vet sci 2000;20:191-194. 442 47. webb gw, arns mj. use of modified phosphate-buffered saline as a component of 443 semen extenders allows the use of transported semen from two stallions. prof anim 444 sci. 2004;20:278-281. 445 48. aurich je, kühne a, hoppe h, et al: seminal plasma affects membrane integrity 446 and motility of equine spermatozoa after cryopreservation. theriogenology 447 1996;46:791–797. 448 49. phillips ph: preservation of bull semen. j biol chem 1939;130:415. 449 50. lardy ha, phillips ph: preservation of spermatozoa. proc am soc an prod 1939; 450 p 219-221. 451 51. clulow, j r, maxwell, w m c, evans g, et al: a comparison of duck and chicken 452 egg yolk for cryopreservation of stallion sperm. aust vet j 2007;85:232-235 453 52. demick dl, voss jl, pickett bw: effects of cooling, storage, glycerolization, and 454 spermatozoal number on equine fertility. j anim sci 1976;43:633–637. 455 389 53. bedford sj, jasko dj, graham jk,et al: effect of seminal extenders containing egg 456 yolk and glycerol on motion characteristics and fertility of stallion spermatozoa. 457 theriogenology 1995;43:955–967. 458 54. barker cav, gander jcc: pregnancy in a mare resulting from frozen epididymal 459 spermatozoa. can j comp med 1957; 231:47-51. 460 55. sieme h, schafer t, stout ta, et al: the effects of different insemination regimes 461 on fertility in mares. theriogenology 2003;60;1153-1164. 462 56. vidament m: french field results (1985-2005) on factors affecting fertility of frozen 463 stallion semen. anim reprod sci 2005;89:115-136. 464 57. fayer-hosken r, abreu-barbosa c, heusner g,et al: cryopreservation of stallion 465 spermatozoa with inra 96 and glycerol. j equine vet sci 2008;11:672-676. 466 58. khlifaoui m, battut i, bruyas jf, et al: effects of glutamine on post-thaw motility 467 of stallion spermatozoa: an approach of the mechanism of action at spermatozoa 468 level. theriogenology. 2005;63:138-149. 469 59. squires el, keith sl, graham jk: evaluation of alternative cryoprotectants for 470 preserving stallion spermatozoa. theriogenolgy 204;62:1056-1065. 471 60. samper jc, garcia a: post-thaw characteristics and fertility of stallion semen 472 frozen in extenders with different cryoprotectants. anim reprod sci 2008;107:348-473 349. 474 61. carver da, ball ba: lipase activity in stallion seminal plasma and the effect of 475 lipase on stallion spermatozoa during storage at 5 ºc. theriogenology 476 2002;58:1587-1595. 477 390 62. ijaz a, ducharme r. effect of various extenders and taurine on survival of stallion 478 sperm cooled to 5 ºc. theriogenology 1995; 44:1039-1050. 479 64. ball ba, medina v, gravance cg, et al: effect of antioxidants on preservation of 480 motility, viability and acrosomal integrity of equine spermatozoa during storage at 5 481 ºc. theriogenology 2001; 56:577-589. 482 65. aurich je, schonherr u, hoppe h, et al: effects of antioxidants on motility and 483 membrane integrity of chilled-stored stallion semen. theriogenology 1997;48:185-484 192. 485 66. mcniven ma, richardson gf: chilled storage of stallion semen using 486 perfluorochemicals and antioxidants. cell pres tech 2003;1:165-174. 487 67. webb gw, arns mj: effect of pyruvate and lactate on motility of cold stored 488 stallion spermatozoa challenged by hydrogen peroxide. j equine vet sci 489 2006;26:406-411 490 68. mcniven ma, richardson gf. effect of quercetin on capacitation status and lipid 491 peroxidation of stallion spermatozoa. cell pres tech 2006;4:169-177. 492 69. bruemmer je, coy rc, squires el, et al: effect of pyruvate on the function of 493 stallion spermatozoa stored for up to 48 hours. j anim sci 2002;80:12-18. 494 70. pesch s, bergmann m, bostedt h: determination of some enzymes and macroand 495 microelements in stallion seminal plasma and their correlations to semen quality. 496 theriogenology. 2006;66:307-313. 497 391 71. kankofer m, kolm g, aurich j, et al: activity of glutathione peroxidase, 498 superoxide dismutase and catalase and lipid peroxidation intensity in stallion semen 499 during storage at 5 ºc. theriogenology 2005;63:1354-1365. 500 72. pagl r, aurich c, kankofer m: anti-oxidative status and semen quality during 501 cooled storage in stallions. j vet med series a 2006;53:486-489. 502 73. baumber j, ball ba. determination of glutathione peroxidase and superoxide 503 dismutase-like activities in equine spermatozoa, seminal plasma, and reproductive 504 tissues. am j vet res 2005;66:1415-1419. 505 74. morte mi, rodrigues am, soares d, et al: the quantification of lipid and protein 506 oxidation in stallion spermatozoa and seminal plasma: seasonal distinctions and 507 correlations with dna strand breaks, classical seminal parameters and stallion 508 fertility. anim reprod sci 2008;106:36-47. 509 392 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true 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/leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2010: alternative assessment tools   alternative assessment tools margaret v. root kustritz university of minnesota, college of veterinary medicine, st. paul, mn introduction assessment is an essential component of teaching. assessment may be formative, helping students identify gaps in their understanding and building competence, or may be summative, permitting a final evaluation of competence for assignment of a grade or to permit students to advance in a program or career. assessment methods must accurately reflect the learning objectives and activities that preceded their implementation, must be a true test of student learning rather than an exercise in test-taking skills or ability to ascertain what the instructor considers most important, and must permit the instructor to provide consistent and timely feedback. keywords: veterinary education, assessment, multiple choice questions multiple choice questions (mcqs) are written with a stem and several possible answers, or foils. the following are basic rules of mcq construction:1-6  put the central idea in the stem and do not repeat it in the foils  avoid negative construction in the stem (“which of the following is not correct…”)  use proper grammar and ensure the correct foil is not obvious because of grammar clues  do not use the terms “always” or “never” in foils  place foils in logical or numerical order  make sure foils are parallel with respect to grammatical structure, length, and complexity  use “none of the above” with caution; do not use “all of the above” as a foil  three foils is the best number, based on research evaluating number of plausible foils  to write plausible foils, consider most common student misconceptions or create a short answer question one year and use student responses to create foils for the next year  do not worry excessively about distribution of answers; research suggests that students pay no attention to pattern of answers as a test-taking technique  use humor sparingly in the mid-20th century, bloom and colleagues developed a hierarchy of educational objectives in the cognitive domain of teaching and learning that has come to be known as bloom’s taxonomy. at the most basic level is knowledge, ability to remember previously learned material. in increasing order of complexity are comprehension, ability to grasp the meaning of material; application, ability to use information in new situations; analysis, ability to break material down into its component parts; synthesis, ability to re-order material in new ways; and evaluation, ability to compare value of material with other criteria. in veterinary medicine, some material clearly is best assessed at the knowledge level but most information must be assessed at the higher levels of analysis, synthesis, and evaluation. traditionally, people have used mcqs, true-false questions, and matching questions to test lower level learning and have used short answer and essay questions to assess higher level learning. in one study evaluating use of mcqs for assessment of learning across bloom’s taxonomy, of 2913 questions from a test databank 47.3% were written to test knowledge, 24.8% comprehension, 21.8% application, and only 6.5% analysis.1 multiple choice questions are easy to grade and a large databank can be created from which different questions can be drawn with each iteration of the examination to limit potential cheating. in one study mcq testing was superior to assessment by peers, by working through cases or by assessment of a student portfolio, perhaps because it required students to put in significant effort committing the information to longer-term memory.7 the primary concern expressed about mcqs is the perception that instructors purposefully write “trick” questions.8 examples of improper mcq 473   construction that may be perceived as “trick” questions by students include introduction of trivial content, too little distinction between foils, multiple correct answers, and highly ambiguous foils.9 questions must be carefully worded to permit evaluation of higher order learning and student anxiety may prevent their showing their best performance with mcq testing compared to more open-ended question types.10,11 multiple choice questions can be written in a manner that addresses higher level learning. to create mcqs assessing comprehension, students can be asked to interpret information. knowledge of the material is assumed and required for correct interpretation. to assess application, students are expected to know and understand background material and are asked to solve a problem or demonstrate a correct method or procedure. analysis is assessed by requiring recognition of facts versus inferences or faults in reasoning and requires proof that the student understands and can work with the underlying structure of the material provided. synthesis is difficult to assess in a multiple choice question because it requires the student to demonstrate creative ability. to assess the highest level of learning, evaluation, students are asked to judge the value or validity of material presented.12 enhanced multiple choice questions are variations on traditional mcqs that more readily permit assessment of higher level learning. enhanced multiple choice question types include content-dependent mcqs, demonstrated-reasoning mcqs, and confidence-level mcqs.2,13-16 many of these variations break the rules described above for multiple choice testing and are more difficult to grade quickly and consistently. however, they do permit assessment of higher level learning and assessment of other parameters, such as ability of the student to work through ambiguity and demonstrate confidence in their knowledge and skills. content-dependent mcqs are constructed with a body of information provided from which one or more mcqs are drawn. the information provided may be a case study, a chart or diagram, an image of a lesion, or any other information that students can analyze or evaluate.2 be cautious of unconsciously creating questions that are interdependent. demonstrated-reasoning mcqs are standard mcqs associated with an opportunity for students to demonstrate the reason behind their answer(s).14-16 students may be given points for explaining why their chosen answer is correct and why the other foils are not correct. one example of this is giving a given number of points for identifying the correct foil and fewer points for explanation by the student of the correct answer; this may permit instructors to give students credit for their knowledge and reasoning even if they do not choose the correct foil. a final variant of this type of question is conscious use of more than one correct foil with students getting credit for identifying all possible correct answers and getting credit for explaining the rationale for their answers. confidence-level mcqs are the most complex variant.13 six to eight foils are presented for each question, with several clear possibilities, several possibilities student may choose if unsure, and one option consciously entitled “i don’t know.” (figure 1). the goal is to identify not just student learning but also how well informed that student felt in making their decision. any student answering “b” ,” c” or “e” obviously is confident and uninformed. any student answering “d” is confident and misinformed as they clearly do not understand that the answer must be a single whole number. any student answering “f” is not confident and not well informed. this provides interesting information for the instructor regarding student competence and confidence. one concern is that students must know they may get some credit for answering “i don’t know” or they will never choose that answer. for this reason, this type of question is best used for formative, not summative, assessment. script concordance questions are a type of mcq requiring students to demonstrate higher level learning. information is provided and hypotheses generated. students are offered diagnostic test results or other information and asked whether those results make the hypothesis more or less likely (figure 2).1719 in human medicine, script concordance testing results have been shown to correlate well with performance on clinics and with board examination testing when used with medical students, residents, and practicing physicians.20,21 it is recommended that students be permitted to explain the rationale behind their answer. more than one alternative may be considered correct, recognizing lack of confidence by students in absolutely ruling in or ruling out any alternative and variation in student 474   backgrounds and the information those students used to create the “scripts” they have stored in long-term memory about those clinical situations. the use of these alternative testing techniques requires explanation of varying technique to the student so they can demonstrate their knowledge and skills without anxiety about the testing methodology. students may benefit from use of the assessment technique in a formative manner before it is used for summative assessment, to make it clear to the instructor how well students understand the testing technique and to help the students understand how the questions will be scored. for example, when the author first used script concordance testing, she presented the students weekly with a question of this type with the correct answer available to them after their initial attempt. this gave them a comfort level with this type of question before the summative assessment late in the semester and had the added benefit of forcing them to routinely review the material well before the examination. learning objectives are the foundation of any course and should guide content provided, activities and assignments used in the course, and assessments. student preference should not prevent instructors from using inventive assessment techniques to help them best capture assessment of their chosen learning objectives. figure 1. confidence-level multiple choice question the correct answer for the mathematical equation 1+2 is: a. 3 b. 5 c. 256 d. a or c e. a and b f. i don’t know figure 2. script concordance question this type of examination is called a script concordance test. it evaluates not just your knowledge but your ability to use that knowledge to make clinical decisions. read the scenario, think about likely hypotheses and then, for each hypothesis and finding presented, choose likelihood using the scale below. -2 = they hypothesis is virtually eliminated from consideration -1 = the hypothesis becomes less likely 0 = the information has no effect on the hypothesis +1 = the hypothesis is becoming more probable +2 = it can only be this hypothesis scenario: you are presented with a 6-day-old west highland white terrier pup for the complaint of persistent crying and inability to nurse. if you were thinking of… and then you find… the hypothesis becomes… canine herpesvirus decreased red blood cell number -2 -1 0 +1 +2 dehydration pale pink muzzle and groin -2 -1 0 +1 +2 septicemia decreased blood glucose -2 -1 0 +1 +2 475   references 1. masters jc, hulsmeyer bs, pike me, et al: assessment of multiple-choice questions in selected test banks accompanying textbooks used in nursing education. j nurs educ 2001;40:25-32. 2. haladyna tm, downing sm, rodriguez mc: a review of multiple-choice item-writing guidelines for classroom assessment. appl meas educ 2002;15:309-334. 3. briggs dc, alonzo ac, schwab c, et al:: diagnostic assessment with ordered multiple-choice items. educ assess 2006;11:33-63. 4. taylor ak: violating conventional wisdom in multiple choice test construction. coll stud j 2005;39:141-148. 5. mckeachie wj: teaching tips: strategies, research, and theory for college and university teachers. lexington (ma): dc heath and co.; 1994. p. 78. 6. mcmillan jh: classroom assessment: principles and practices for effective instruction. boston: allyn and bacon; 2001. p. 150. 7. struyven k, dochy f, janssens s, et al: the overall effects of end-of-course assessment on student performance: a comparison between multiple choice testing, peer assessment, case-based assessment and portfolio assessment. stud educ eval 2006;32:202-222. 8. dale vhm, wieland b, prikelbauer b, et al: value and benefits of open-book examinations as assessment for deep learning in a post-graduate animal health course. j vet med educ 2009;36:403-410. 9. roberts dm: an empirical study on the nature of trick test questions. j educ meas 1993;30:331-344. 10. turner h, williams rl: vocabulary development and performance on multiple-choice exams in large entry-level courses. j coll read learn 2007;37:64-81. 11. birenbaum m, feldman ra: relationships between learning patterns and attitudes towards two assessment formats. educ res 1998;40:90-98. 12. http://web.uct.ac.za/projects/cbe/mcqman/mcqappc.html#c2, accessed 02-28-10. 13. swartz sm: acceptance and accuracy of multiple choice, confidence-level, and essay question formats for graduate students. j educ business 2006;81:215-220. 14. draper sw: catalytic assessment: understanding how mcqs and evs can foster deep learning. brit j educ tech 2009;40:285-293. 15. ennever f: more than multiple-choice. science teacher 2006;73:54-56. 16. pomplun m, omar mh. multiple-mark items: an alternative objective item format? educ psych meas 1997;57:949 962. 17. smith cs: a developmental approach to evaluating competence in clinical reasoning. j vet med educ 2008;35:375 381. 18. charlin b, tardif j, boshuizen hpa: scripts and medical diagnostic knowledge: theory and application for clinical reasoning instruction and research. acad med 2000;75:182-190. 19. charlin b, van der vleuten c: standardized assessment of reasoning in contexts of uncertainty: the script concordance approach. eval health prof 2004;27:304-319. 20. norcini jj, shea ja, day sc: the use of aggregate scoring for a recertification examination. eval health prof 1990;13:241-251. 21. silbert l, darmoni sj, dahamna b, et al: on line clinical reasoning assessment with script concordance test in urology: results of a french pilot study. bmc med educ 2006;6:45-53. 476 2012: genetics based selection pressure to breed better, healthier dogs genetics based selection pressure to breed better, healthier dogs jerold s bell tufts cummings school of veterinary medicine, north grafton, ma the ability to apply selective pressure to breeding dogs to improve the genetic health of offspring begins with an understanding of the selection tools that we have available to us, as well as the ethical responsibilities to use these tools in assisting with breeding decisions. ethical responsibilities regarding genetic disease as veterinary medical professionals, we are at the front lines of improving the genetic health; of our patients, of breeds, and of all dogs and cats. for most genetic diseases, we know how to either prevent their occurrence, or at least lessen the possibility of producing offspring with genetic disease. this can occur through the genotypic testing of the parents (identification of parents carrying liability genes for genetic disease), phenotypic testing of the parents (identification of parents affected with genetic disease), or pedigree analysis (identification of carrier risk based on the knowledge of carrier or affected relatives). the genetic improvement of dogs and cats will only occur through selective breeding. however, the responsibility for this improvement lies not just with the breeder; but also with the veterinarian, the breed organizations, and the general public. inherent in these responsibilities is the acknowledgement that breeding without genetic testing is irresponsible, and unethical. genetic testing is health quality control. it is no longer acceptable for a breeder to choose two individuals and breed them together without regard to genetic disease control. there is an active debate and inquiry into the genetic health of dog breeds. several reports offer similar recommendations of; avoidance of selection for extreme phenotypes, the use of genetic testing to select for healthy breeding animals, and modification of breeding practices. the kennel club in the uk has initiated a program entitled, “fit for function, fit for life”. at the heart of the program is the belief that all dogs should be able to see, walk, and breathe freely. extreme phenotypes that the kennel club is addressing include the brachycephalic syndrome, excessive skin or eye conformation predisposing to disease, extreme conformation predisposing to lameness, and unstable temperament. the kennel club at their 2012 crufts dog show had six best of breed winners (bulldog, pekingese, clumber spaniel, mastiff, neopolitan mastiff and basset hound) be disqualified from moving on to the group competition due to their failing a veterinary inspection. the kennel club also has an accredited breeder scheme to identify breeders that do health testing and adhere to specific breeding standards. some breed organizations and agencies have embraced the belief that close breeding is the cause of impaired breed health, and have adopted protocols and programs that restrict close breeding. the kennel club in the uk recently adopted a “mate select” program that lists health test results, but also seeks to find a mate that is the least related through pedigree analysis; i.e., outbreeding to produce the lowest inbreeding coefficient and the most heterozygosity. mars veterinary has a optimal selection® panel that selects prospective mates based on genotypic heterozygosity. this process is akin to a species survival plan (ssp) that is utilized when attempting to “rescue” an endangered species. it is using the tool of the inbreeding coefficient as the goal of breeding. the vast majority of dog and cat breeds do not show evidence of genetic depletion as seen in endangered species, such as; low reproductive success, and increased stillborn and neonatal mortality. recommendations to outbreed (only breed to those least related) homogenizes breeds and erases the genetic difference between individuals. it is a self-limiting process. it requires that matings be done between individuals who are different from each other. however, eventually there will be no more “lines” with differences. everyone will be in the center, and no one at the periphery. if a genetic disease or deleterious trait comes up, there will be no “other line” to breed and get away from it. breed gene pool diversity requires distinct lines in order to create selective pressure. prudent breeding practices allow some linebreeding, some outbreeding, and even occasional inbreeding; with different breeders maintaining breeding lines or crossing lines as they see fit. it is the 527 clinical theriogenology • volume 4, number 4 • december 2012 different opinion and breeding actions of breeders that maintain breed diversity. this is not something that can be legislated or regulated. when breeds have issues with genetic diseases, the only way to improve their gene pool is through selection against the specific diseases and their associated liability genes. the types of matings (linebreeding or outbreeding) have no bearing on controlling deleterious genes, or the genetic health of breeds. pure and pedigreed breeds are only endangered if breeders ignore selection for healthy breeding stock. genetic registries in the united states, several genetic registries have been established to assist breeders with genetic disease control. the canine eye registry foundation or cerf (http://www.vmdb.org/cerf.html) is a closed database showing only normal eye examination results by acvo boarded veterinarians. the not-for profit orthopedic foundation for animals (ofa: www.offa.org) has semi-open registries for hip dysplasia, elbow dysplasia, autoimmune thyroiditis, congenital cardiac disease, patella luxation, and other genetic diseases. from the ofa web portal you can look up individual dogs, and their health testing status. this is facebook for dogs; each with their own web pages and information. the canine health information center or chic (www.caninehealthinfo.org) is an open health database that has been established by the akc canine health foundation and the orthopedic foundation for animals. national parent clubs decide to enroll in the chic program, and determine the testable genetic disorders for their breed. (for example, hip evaluation, cerf examination, and thyroid testing.) owners, breeders, and prospective owners can search online for dogs in the ofa/chic database, and view their test results. if a dog completes the recommended testing panel, it receives a chic number regardless of whether it passes all of the tests. chic is about health consciousness, not health perfection. as more testable disorders are identified, few dogs will be normal for all tests. the kennel club in the uk has similar health breeding schemes and a searchable health tests results finder to look up individual dogs. a similar listing of tests is not currently available for cats, however breed related diseases are found on the feline advisory bureau (fab-uk) website: www.fabcats.org/breeders/inherited_disorders. responsibilities of veterinarians with each hereditary disorder, we as a veterinary profession are being called upon to determine what is “normal”, what is “abnormal”, and what screening tests can be performed to allow selection away from disease causing phenotypes. care must be taken, so that selective pressures are not so severe that they limit genetic diversity in the breed gene pool. when a client makes an initial puppy or kitten appointment, we should examine all of the paperwork provided by the breeder or pet store. this includes not only the prior medical care, but the registration paperwork that lists the sire and dam. on receiving the paperwork, the health test requirements for the breed can be identified, and the health test results of the parents searched. if test results are not available on the web-based registries, ask the owner if the breeder provided them with verification of each of the required genetic test results on the parents; i.e., a copy of the official test results from the testing agencies. if no verified test results are available, then the puppy or kitten was not bred by a health conscious breeder. there is no expectation of genetic health in your patient, and you and the owner can only hope for good health. when a client is planning on breeding a dog, you can look up the pre-breeding health test requirements. you can provide many of the tests yourself (radiographs, thyroid profile, or cheek swab or blood samples for genetic tests). for eye examinations or heart examinations by a cardiologist, you can assist your client by providing information on local health screening clinics. you must emphasize the ethical responsibility of pre-breeding genetic testing, or a decision to not breed their animal. genetic testing is a requirement, not a choice. if a client is looking to purchase a purebred or designer-bred dog, you should counsel them on the behavioral and genetic expectations for the selected breeds. provide them with the genetic health test 528clinical theriogenology • volume 4, number 4 • december 2012 requirements. ensure they understand that they should only purchase a pet from parents that have verified results of their breed-specific required health tests. responsibilities of breeders it is the ethical responsibility and obligation of all breeders to perform the available required prebreeding genetic health tests on prospective breeding stock. a breeder is anyone that plans a mating between two animals. these include matings between two members of the same breed, or crosses between two members of different breeds (designer matings). the most common genetic diseases of; canine hip and elbow dysplasia, valvular heart disease, patella luxation, eye disease, and hypothyroidism occur at similar frequencies in mixed-breed versus pure-bred populations. if two animals are purposely bred, then the breed-specific genetic testing for each parent is required. most genetic tests only need to be done once in the prospective breeding animal’s lifetime. others (eye examinations, phenotypic heart examinations, thyroid profile, etc.) should be repeated, depending on the breed specific age of onset of the disorder, and age requirement for diagnosis. if a breeder is not willing or able to have the prescribed pre-breeding genetic tests performed, then they should find a different hobby or profession. cats and dogs are living beings. it is not ethical to forgo the obligation of genetic testing. everyone loves their breed, and their own breeding stock. the more genetic tests that are developed, the greater chance there is of identifying an undesirable gene in an animal. conscientious breeders understand that negative test results limit their breeding options. with direct gene tests, they can use carriers when bred to normal testing mates. for disorders without direct gene tests, they may have to choose a normal relative, as opposed to one they were planning on using in the next generation. matings should be planned that prevent or minimize the risk of producing genetic disease, but do not limit the genetic diversity of the breed. when prospective breeding stock has a carrier or affected test result, you should counsel your client to release this information to the listing health registry. if negative test results are not made available, then other breeders will not be able to ascertain the disease risk of their own breeding stock to make informed breeding decisions. as opposed to the stigma that used to be attached to the appearance of genetic disease, the stigma now rests on those that hide the occurrence of genetic disease. dealing with genetic disorders is a community effort. when making breeding decisions, breeders can search the health registry websites for genetic test results on prospective mates. if test results are not available on animals that have already been bred, then it must be assumed that they are affected or carriers. otherwise the results would be available. when selling a kitten or puppy, breeders should provide new owners with full documentation of the health test results (copies of official test results) on the parents. if early direct genetic testing was done on the puppies, these results should be provided. it is not enough to say that the testing was done. if testing was done then the breeder has the paperwork, and it should be provided. it must be impressed upon the public that health consciousness is one of the most important considerations when getting a puppy or kitten. health guarantees that provide for replacement of puppies or kittens with genetic defects are not a replacement for health testing. such a guarantee is of little value, as no one wants to part with their family member once the emotional bonds have been made. a puppy or kitten is not a toaster. responsibilities of breed organizations/parent clubs it is the responsibility of the breed club to conduct regular breed health surveys to monitor the health of the breed. if breed-related disease is present, it is up to the breed club to promote and fund research to identify phenotypic and genotypic tests that can be used by breeders to improve the genetic health of the breed. for dog breeds, the parent club should work with chic, the kennel club, or other national agencies to select the required and recommended genetic testing that should be performed before dogs are bred. breeders should be counseled to perform pre-breeding health testing. 529 clinical theriogenology • volume 4, number 4 • december 2012 parent clubs should review their breed standards and select against morphological changes that promote disease, morbidity, or mortality. they should counsel their breeders against breeding to extreme standards that can promote disease, and should educate judges to select against morphology that promotes disease. the parent club should also scientifically monitor if significant health issues are being caused by a lack of genetic diversity. if so, they should be open to scientific measures that can increase genetic diversity, including opening of the stud book, or even controlled crossbreeding programs. responsibilities of the general public when a consumer gets a purposely-bred kitten or puppy, the emotional aspect of adding a new member to the family often overwhelms the rational aspect of this important decision. acquiring a new pet should not be an impulse decision. the new pet will hopefully be with the family for the next 10 to 15 years. the public should spend as much time researching this decision as they do when purchasing a new car or a refrigerator. prospective owners need to research whether a specific breed is suitable for them and their home. they also need to research the breed-specific health testing requirements for the selected breed. whether purchasing from a private breeder, one found on the internet, or a pet store, the parental health testing results for both parents should be available. if they are not available, then just walk away – regardless of how cute the puppy or kitten. statements of testing by the breeder, or on a breeder website are not sufficient to document health test status. if the testing has been done, the breeder will be happy to provide the official documentation that they are a health conscious breeder. health guarantees that provide replacement for pets with genetic disease do not eliminate the need for genetic testing. if a breeder states that they do not have the health test documentation, but offer a guarantee of genetic health, the prospective owner should walk away. the breeder has not fulfilled their ethical responsibility and obligation of health testing. the general public is the engine that drives the pet breeding industry. if the general public demands puppies from health tested breeding stock, then the market will change to favor health conscious breeders. if people can easily sell pets to the public on a website without any health tests being done, then there is no market force to change the situation to improve the genetic health of cats and dogs. it is the public’s choice of where they get a puppy or kitten. it is the general public’s obligation to document genetic health testing from breeders. all genetic disease is not preventable. however, the frequency of genetic diseases can be significantly decreased, if not eliminated by valid testing and breeding selection in purposely bred dogs and cats. it is time to put an end to the excuse of ignorance of the breeder, veterinarian, or general public in their roles and responsibilities to improve the genetic health of dogs and cats. it is up to all of us to educate each other about producing genetically healthy cats and dogs, and call for the documentation of health testing of all breeding stock. genetic tests genetic tests vary on what they are able to identify, and therefore how they can be used in managing genetic disease. to understand how we can use genetic tests, we have to understand the types of tests that are available, what they can tell us, and their limitations. phenotypic tests some tests measure the phenotype, or what can be seen in the animal. this may not directly relate to the genotype, or the genes regulating the defect that you are trying to manage. screening for cataracts, ausculting for heart murmurs, hip and elbow radiographs, thyroid profiles, urinalysis for crystals or metabolites, skin biopsy for sebaceous adenitis, and observations on behavioral traits are all tests of the phenotype. most tests of the phenotype only identify affected individuals, and not carriers of disease liability genes. 530clinical theriogenology • volume 4, number 4 • december 2012 linked-marker based tests some defective genes can be linked to a genetic marker, which could be tested for. linkedmarker based tests do not identify the defective gene, but a marker that lies close on the chromosome. if a crossover occurs between the marker and the defective gene during reproduction, the marker will no longer be linked to the defective gene. false positive and false negative results will occur. due to this phenomenon, linkage test results must be compared with results from other family members to determine whether they correlate with the known genotype of relatives. linked marker tests include those for cerebellar ataxia in italian spinone and primary hyperparathyroidism in keeshond. direct mutation based tests direct gene tests are specific for mutations and are a direct measurement of the genotype. they can identify affected, carrier, and normal individuals. these can be run at any age, regardless of the age of onset of the disorder. most direct gene tests identify a mutation that is causative for a genetic disorder. these genes are 100% penetrant, and an affected genetic test result is 100% correlated with clinical disease. however, some direct genetic tests identify a mutation that causes an increased susceptibility for genetic disease. these susceptibility alleles can be part of polygenic/complexly inherited traits, or the cause of incomplete penetrance of (assumed) simple mendelian traits. degenerative myelopathy is considered a complexly (polygenic) inherited disease. an autosomal recessive susceptibility gene has been identified that is homozygous (two abnormal copies) in all dm affected dogs. however, a large proportion of individuals in these breeds are homozygous for this gene and do not become affected. they are considered “at risk”, but not genetically affected. in the wire fox terrier, there is a 91% allele frequency in the breed; however no wire fox terrier has even been diagnosed with degenerative myelopathy. in the boxer, less than 0.5% of dogs develop the disease. testing boxers for the dm susceptibility gene shows 39% testing carrier, and 43% testing homozygous “at risk” for the susceptibility gene. this is an example of a genetic test with low penetrance; indicating that the homozygous state is poorly predictive of clinical disease. there are additional (unidentified) genes that must also be present to produce clinical dm. this test is useful in ruling out a diagnosis of dm in homozygous normal and carrier dogs. however, selecting against 82% (“at risk” and carrier dogs) of the boxer gene pool when making breeding decisions when the vast majority will not produce the disorder is detrimental to the genetic diversity of the breed. similar situations occur in other breeds susceptible to dm. in these breeds, breeding dogs should not be selected against or have their mating choices altered due to carrier or homozygous “at risk” status of dm unless there is knowledge of close (first or second degree) relatives diagnosed with clinical degenerative myelopathy. some breeders feel that any carrier or “at risk” dogs should only be breed to homozygous normal testing dogs. however, requiring that all mating be performed with dogs from only 18% of the population (following the boxer example) would tremendously skew the breed’s gene pool and restrict genetic diversity. this is unnecessary for an extremely low prevalence disease. with genetic tests for lowly penetrant defective genes, selection should only be considered for dogs with families that contain clinically affected individuals. this recommendation should significantly reduce the frequency of clinical disease, as well as the frequencies of other contributory alleles. other examples of low prevalence but high allele frequency diseases are cord1 pra in english springer spaniels (42% “at risk”, 38% carrier, ~1-2% disease prevalence) and miniature dachshunds, and rcd4 pra in gordon setters and irish setters. with rcd4 pra, the average age of clinical diagnosis of the disease is around 10 years of age. however, this is the average age that clinically affected dogs are recognized; not the average age when all homozygous dogs become affected. the actual average “age of onset” of this late-onset disease may be in the teens; when many dogs will already be deceased. now that gordon setters are being tested worldwide, many dogs who are homozygous for the defective gene with normal vision are being identified. with an approximately 30% carrier rate in the small gordon setter gene pool, selection against the gene must necessarily be gradual so as to not restrict the breed’s genetic diversity. 531 clinical theriogenology • volume 4, number 4 • december 2012 other susceptibility genes are found to occur at a greater frequency in affected animals, but are not present in all affected animals. an example is the susceptibility gene for perianal fistula/anal furunculosis in german shepherd dogs. dogs with the susceptibility haplotype (specific sequence of 3 dla genes) have a 5.0x odds ratio for the disease versus those without the haplotype. this risk factor occurs whether the susceptibility haplotype is heterozygous or homozygous; though homozygous dogs develop the disease at an earlier age. another example is the genetic test for pug dog encephalitis, a painful, fatal disease affecting 1-2% of pugs. dogs homozygous for a susceptibility haplotype have a 15.6x odds ratio for developing the disease, but dogs heterozygous for the susceptibility haplotype have no greater risk. many owners and breeders ask what tests should be done in their cats and dogs. the answer depends on whether the cat or dog is going to be a pet, or be used for breeding. for a pet, it is only important to know that it is not going to be affected by a health related disorder. for breeding animals, it is important to know if they carry disease liability genes that they can pass on to their offspring. genetic counseling for pet animals the vast majority of our patients are not breeding animals, but they still require genetic counseling for inherited disorders. we counsel owners of large-breed puppies to feed lower calorie foods to provide for a more uniform growth rate and better joint development. we understand the nutritional counseling needs for fus cats and obese “pre-diabetic” cats. the hallmark of genetic disease is our ability to predict its occurrence before its onset, allowing us to alter its morbidity or mortality. we need to be knowledgeable about what genetic tests are available, and in what patients they should be run. patients from breeds with an incidence of von willibrand’s disease should be tested early in life, so that measures can be taken to prevent excessive hemorrhage during surgery or injury. patients at risk of carrying the mdr-1 mutation should be tested early in life, before drug treatment. in high risk breeds, individual animals should be genetic tested (or verified results documented on parents) before purchase. these include maine coon cats for the autosomal dominant hypertrophic cardiomyopathy gene, and persian and himalayan cats for autosomal dominant polycystic kidney disease. we need to understand the temporal periods when genetic testing will be most accurate, and allow for intervention. puppy hips should be palpated with a gentle ortolani procedure at each vaccine visit, and again at spaying or neutering under anesthesia. juvenile interventional surgery will only benefit those with significant subluxability prior to major growth (for pubic symphysiodesis) or the development of osteoarthritic changes (for triple pelvic osteotomy). genetic testing for hypothyroidism is based on the presence of thyroglobulin autoantibodies. a dog with normal tgaa levels on two tests at least two years apart between two and six years of age is phenotypically normal. however, tgaa levels should not be measured within two to three months postvaccination, as a transient iatrogenic rise can occur during this period. genetic counseling for breeders the goal of genetic counseling is to effectively manage the spread of defective genes, while preserving the health and genetic diversity of the population. genetic counseling recommendations are geared toward preventing the production of affected animals, and reducing the production of carriers. at the same time, recommendations should allow the continuation of breeding lines, to preserve the genetic diversity of the population. historically, genetic counseling has ranged from recommendations to not repeat a mating and outbreed, to recommendations to eliminate all relatives of affected animals from the breeding pool. neither of these two extremes serves the best long-term interest of breeds. outbreeding can prevent the production of affected animals, but it will propagate and further disperse detrimental recessive genes. recommendations for breeding should be based on genetic test results or knowledge of carrier or affected relatives if genetic tests are not available. the testable disorders for each breed should be 532clinical theriogenology • volume 4, number 4 • december 2012 discussed with your clients. for dogs, breed-specific genetic test recommendations are available at the chic website (www.caninehealthinfo.org). there are no breeding recommendations that will fit every situation. protocols for genetic counseling and breeding management of genetic disorders can be based on the known (or unknown) mode of inheritance, and the availability and type of genetic tests. genetic tests should be used to increase the breeder’s options for breeding, and not limit them. in the case of a simple autosomal recessive disorder for which a direct genetic test for carriers is available, the recommendation is to test breeding-quality stock, and breed quality carriers to normaltesting individuals. this prevents affected offspring from being produced. the aim is to replace the carrier breeding-animal with a normal-testing offspring that equals or exceeds it in quality. as each breeder tests and replaces carrier animals with normal-testing animals, the problem for the breed as a whole diminishes. with a genetic test, breeders can positively determine if an individual is a carrier of a defective gene. the typical response of a breeder on finding that their animal is a carrier is to remove it from a breeding program. if a majority of breeders do this, it puts the breed’s gene pool through a genetic bottleneck that can significantly limit the diversity of the breed. the goal of genetic testing is to allow the superior genes of a breeding individual to be propagated, even if the animal is a carrier. one defective gene that can be identified through a genetic test out of tens of thousands of genes, is not a reason to stop breeding. if an owner would breed an individual if it tested normal for a genetic disease, then a carrier result should not change that decision. a direct genetic test does not alter who gets bred, only who they get bred to. we know that most individuals carry some unfavorable recessive genes. the more genetic tests that are developed, the greater chance there is of identifying an undesirable gene in your patient. history has shown that breeders can be successful in reducing breed-wide genetic disease through testing and making informed breeding choices. however, there are also examples of breeds that have actually experienced more problems as a result of unwarranted culling and restriction of their gene pools. these problems include: reducing the incidence of one disease and increasing the incidence of another by repeated use of males known to be clear of the gene that causes the first condition; creating bottlenecks and diminishing diversity by eliminating all carriers of a gene from the breeding pool, instead of breeding and replacing them; and concentrating on the presence or absence of a single gene and not the quality of the whole animal. genetic test results should be used to benefit the overall health of breeds, not to limit it. for autosomal recessive disorders without genetic tests, breeders can still reduce the carrier risk in their matings. a quality individual that is found to be a carrier can be retired from breeding and replaced with a quality, lower-risk offspring. in this way, the carrier risk can be cut in half. by repeating this vertical mating scheme (breeding once to a low-risk mate and replacing with an offspring), you are maintaining the good genes of the line, reducing the carrier risk with each generation, and replacing, not adding to the overall carrier risk in the breeding population. the problem with recessive disorders without carrier tests is the propagation and dissemination of unapparent carriers in the gene pool. multiple offspring should not be placed in breeding homes. an open health registry that is supported by the parent club makes it easier for breeders to objectively assess the carrier risk of prospective breeding animals. by determining the average carrier-risk for the breeding population, breeders can select matings that have a projected risk that is lower than the breed average. using relative-risk assessment as a tool, breeders should replace higher-risk breeding animals with lower-risk offspring that are equal to or better than their parents in quality. a negative aspect of pedigree analysis is that it selects against families, regardless of an individual’s normal or carrier status. on the other hand, it allows for the objective risk assessment and continuation of lines that might otherwise be abandoned due to high carrier-risk. an example of an open health database and relative risk analysis program is cerebellar abiotrophy in the scottish terrier (www.stca.biz/grandcentral/cacentral-ca.asp). autosomal dominant genetic disorders are usually easy to manage. each affected animal has at least one affected parent, but it can be expected that half of the offspring of an affected animal will be free 533 clinical theriogenology • volume 4, number 4 • december 2012 of the defective gene. with disorders that cause death or discomfort, the recommendation is to not breed affected animals. to produce the next generation of a line, a normal full-sibling of an affected animal can be used, or the parent that is normal can be used. a problem with some autosomal dominant disorders is incomplete penetrance, where some individuals with the defective gene may not show the disorder. roughly half their offspring, however, may be affected. if a genetic test is available, this is not a problem. otherwise, relative-risk assessment can identify which individuals are at risk of carrying incompletely penetrant dominant genes. for sex-linked (also known as x-linked) recessive defective genes, selecting a normal male for breeding loses the defective gene in one generation, regardless of his relationship to affected and carrier relatives. carrier, affected, or high risk females should not be used, due to the high risk of producing affected male offspring. if a male is affected, he would have received the defective gene from his carrier mother. all of his daughters will be carriers, but none of his sons. without a test for carriers, you can use relative-risk assessment to breed him to a female that is at low risk of being a carrier. this prevents affected offspring, and a quality son can be selected for replacement. rare sex-linked dominant disorders are managed the same way as autosomal dominant disorders. the difference is that affected males will always produce all affected daughters. most complexly (polygenic) inherited disorders have no tests for carriers, but do have phenotypic tests that can identify affected individuals. these disorders require knowledge of the affected or normal status of full-sibs to prospective breeding animals. open health genetic registries like the orthopedic foundation for animals database provide such information (www.offa.org). individuals whose siblings are normal and whose parents’ sibs are normal have the greatest chance of carrying a low genetic load for the condition. this breadth of pedigree analysis is more important than normalcy in the depth of pedigree (parents and grandparents only.) affected individuals can be replaced with a normal sib or parent, and bred to a low-liability mate. breeders can replace the higher risk parent with a quality, lower risk offspring, and repeat the process. for disorders without a known mode of inheritance or carrier test, breeders should be counseled to use the same control methods as with polygenic disorders. it is distressing to breeders when we confirm a genetic disorder. as veterinary professionals, we can offer positive and practical genetic counseling recommendations to maintain breed lines and genetic diversity, and improve the overall health of breeds. the total elimination of defective genes will probably be impossible for most breeds. the use of these guidelines can assist breeders in making objective breeding decisions for genetic disease management, while continuing their breeding lines. the individual breeder can use genetic tests to; 1) identify carriers, 2) work to breed away from the defective gene(s), and 3) ensure (through testing) that the defective gene(s) is not reintroduced in future matings. each breeder will have their own rate of progress, depending on the frequency of the defective gene(s) in their own breeding animals, and which desirable individuals are carriers. 534clinical theriogenology • volume 4, number 4 • december 2012 1 contact michelle kutzler michelle.kutzler@oregonstate.edu © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommerical 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2024, 16, 10926, http://dx.doi.org/10.58292/ct.v16.10926 research report serum testosterone concentrations in alpacas and llamas after human chorionic gonadotropin stimulation before and after castration reagan haislip,a anna mcallister,b michelle kutzlerc adepartment of fisheries, wildlife, and conservation sciences, college of agricultural sciences bcarlson college of veterinary medicine cdepartment of animal rangeland sciences, college of agricultural sciences, oregon state university, corvallis, or, usa abstract veterinarians perform testosterone stimulation tests in animals suspected of bilateral cryptorchidism and/or incomplete castration. period for circulating testosterone to become undetectable after castration varies among species. to our knowledge, clearance of circulating testosterone in camelids after castration has not been examined. testosterone stimulation with human chorionic gonadotropin (hcg) has been described in several species, including alpacas. however, there is limited information regarding responses to hcg in male llamas. purpose was to measure serum testosterone concentrations [t] in llamas and alpacas after hcg stimulation before and after castration. we hypothesized that [t] significantly increases after hcg treatment and decreases rapidly after castration. testosterone stimulation test (using hcg) was performed in mature male alpacas (n = 9) and llamas (n = 7) 1 day prior to and 1 day after castration. blood samples were collected; 1 sample before and 3 samples (2, 4, and 8 hours) after hcg treatment. in llamas and alpacas [t] increased (p < 0.05) > 2 fold (from prehcg values) within 2 hours after hcg treatment. prehcg [t] were higher (p < 0.05) in alpacas than llamas but did not differ significantly between them after hcg stimulation; 24 hours after castration [t] were < 0.05 ng/ml. keywords: luteinizing hormone, radioimmunoassay, testes, testicular weight introduction veterinarians measure baseline testosterone concentrations and perform testosterone stimulation tests in male animals suspected of bilateral cryptorchidism and/or incomplete castration.1 the interval for circulating testosterone to decrease to undetectable concentrations varies among species after castration. in horses, testosterone half-life is 1 hour; concentrations are similar to geldings at 24 hours after castration.2 in cats, serum testosterone concentrations [t] decreased to 3-12% of precastration concentrations within 1 hour after castration; similar to castrated cats at 24 hours.3 in rats, [t] decreased significantly at 12 hours after castration; however, were detectable until 72 hours after castration.4 to our knowledge, in camelids, clearance of circulating testosterone after castration has not been reported. measuring baseline concentrations and changes to [t] after exogenous gonadotropin releasing hormone (gnrh) or a gnrh analogue (e.g. fertirelin acetate) treatment (induces secretion of endogenous luteinizing hormone [lh])5 is diagnostically useful. binding of lh to receptors in testicular interstitial (leydig) cells stimulates endogenous testosterone secretion.6 another method is human chorionic gonadotropin (hcg) treatment and hcg binds directly to testicular lh receptors. testicular interstitial cells respond to intravenous hcg treatment with immediate cyclic adenosine monophosphate (camp) production and rapid increase in testosterone secretion.7 testosterone stimulation with hcg has been described in several species (table 1). however, information (response to testosterone stimulation with hcg) in llamas is limited to small sample size (intact: n =1; cryptorchid: n = 1; castrated: n = 1).8 purpose was to measure [t] in llamas and alpacas after hcg stimulation before and after castration. we hypothesized that [t] significantly increases after hcg treatment and is undetectable within 24 hours after castration, and has diagnostic value mailto:michelle.kutzler@oregonstate.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.10926 2 citation: clinical theriogenology 2024, 16, 10926, http://dx.doi.org/10.58292/ct.v16.10926 for detecting retained testicular tissue after castration or cryptorchidism. materials and methods mature (6.1 ± 2.9 years; range: 2-10 years) intact male alpacas (n  =  9) and llamas (n  =  7) were used. animal experiments were approved by oregon state university institutional animal care and use committee (protocol #3293). one day prior to and 1 day after castration, testosterone stimulation test (using hcg) was performed. jugular blood samples were collected prior to intravenous treatment of hcg (2,500 iu [alpaca], 5,000 iu [llama], chorulon®, intervet, inc./merck animal health, rahway, nj) and 2, 4, and 8 hours after hcg treatment. llama hcg dose was based on a dose used for testosterone stimulation test in a cryptorchid llama.9 alpaca hcg dose was half of llama dose (alpaca body weight [70.8 ± 12.0 kg; range: 50.0-88.6 kg] was about half of llama body weight [152.6 ± 30.4 kg; range: 104.5-195.4 kg]) and also was similar to a dose used.10 next day, males were castrated under intravenous general anesthesia (telazol®, zoetis, parsippany, nj) using closed castration technique. scrotal incision was made over each testis and fascia surrounding testis was stripped; spermatic cord was crushed using a modified white’s emasculator. vaginal tunic and epididymides were removed (figure 1) and testes were weighed. skin incisions were allowed to heal by second intention. cornell university animal health and diagnostic center was used to measure [t] utilizing a double antibody radioimmunoassay (diagnostic products corporation, los angeles, ca). samples were tested within 1 assay and intra-assay coefficients of variability were < 10%. assay sensitivity was 0.1 ng/ml and the lowest detectable concentration was 0.05 ng/ml. changes in mean ± sem [t] after hcg treatment were analyzed using repeated measures analysis of variance and dunnett’s post hoc analysis (graphpad prism, san diego, ca) and compared between species using student’s t-test (microsoft excel, redmond, wa). simple linear regression was used to compare [t] to testicular weight (microsoft excel; graphpad prism). data are presented as mean ± sem. significance was defined as p < 0.05. results testosterone responses to hcg treatment are summarized (table 2) and illustrated (figure 2). in llamas and alpacas, [t] increased (p < 0.05) > 2 fold from prehcg values within 2 hours after hcg treatment and remained elevated until 8 hours. there were no significant differences in [t] among 2, 4, and 8 hours after hcg treatment. prehcg [t] were higher (p < 0.05) in alpacas than llamas but did not differ significantly after hcg stimulation; 24 hours after castration [t] was  <  0.05 ng/ml. stimulation with hcg also resulted in table 1. review of testosterone stimulation results after hcg treatment in mammals species dose results alpaca 3,000 iu [t] increased 2-4 fold 2 hours after iv hcg injection10 cat 500 iu [t] increased 7-5 fold in 0.5 and 2 hours after iv hcg treatment, respectively18 cow 10,000 iu [t] increased 4-5 fold in 5 days after im hcg treatment19 dog 67.56 iu/kg [t] increased 2-3 fold in 0.5 and 2 hours after sc hcg treatment, respectively20 dog 35 iu/kg [t] increased 2 fold in 6 hours after im hcg treatment, respectively21 dog 150 iu [t] increased 2 fold in 3 hours after im hcg treatment22 dog 25 iu/kg [t] increased 2 fold in 1 hour after hcg sc treatment23 horse 5,000 iu [t] increased 2-5 fold in 1 and 12 hours after iv hcg treatment, respectively2 horse 10,000 iu [t] increased 2-4 fold in 1 and 24 hours after iv hcg treatment, respectively1 pig 3,000 iu [t] increased 10-20 fold in 1 and 28 hours after iv hcg treatment, respectively24 rat 25 iu/kg [t] increased 7 fold in 1 hour after hcg treatment23 sheep 1,000 iu [t] increased 2-8 fold in 0.5 and 3 hours after iv hcg treatment, respectively25 iu: international units; im: intramuscular; iv: intravenous; sc: subcutaneous; [t]: serum testosterone concentrations figure 1. pair of alpaca testes (after vaginal tunic and epididymides removal) prepared for weighing http://dx.doi.org/10.58292/ct.v16.10926 citation: clinical theriogenology 2024, 16, 10926, http://dx.doi.org/10.58292/ct.v16.10926 3 [t] < 0.05 ng/ml at 2, 4, and 8 hours in llamas and alpacas after castration (table 3). prehcg [t] were higher (p < 0.05) in alpacas than llamas but did not differ significantly after hcg stimulation; 24 hours after castration [t] was < 0.05 ng/ml. relationships between testicular weight and [t] are provided (figure 3). there was moderate relationship (r2 = 0.69, p = 0.02) between testicular weight and [t] prior to hcg treatment in llamas, but not in alpacas (r2 = 0.13, p = 0.35). there was a weak relationship between testicular weight and [t] 2 hours after hcg treatment in alpacas (r2 = 0.46, p = 0.04) but not in llamas (r2  =  0.33, p  =  0.17). there was no relationship between testicular weight and [t] at 4 hours (llamas: r2  =  0.12, p = 0.44; alpacas: r2 = 0.19, p = 0.24) and 8 hours (llamas: r2 = 0.12, p = 0.45; alpacas: r2 = 0.25, p = 0.17) after hcg treatment. discussion we demonstrated that testosterone stimulation test is a diagnostic tool to detect testicular tissue in llamas and alpacas and documented that within 24 hours after testicular tissue removal, testosterone concentrations were undetectable; similar to horses and cats.2,3 however, anecdotal evidence (https://open.lib.umn.edu/largeanimalsurgery/chapter/ camelid-castration/) in noncamelid species suggested that males may be fertile and exhibit reproductive behaviors up to 6 weeks after castration; these parameters were not evaluated in the present study. endogenous testosterone secretion can be stimulated by gnrh or hcg; gnrh stimulates endogenous luteinizing hormone release from anterior pituitary that stimulates testosterone secretion from testicular leydig cells, whereas hcg directly stimulates testosterone release from leydig cells. for this reason, hcg is recommended over gnrh for evaluating testicular function.11 in horses, [t] increased nearly 5 fold within 1 hour after hcg treatment.2 in alpacas [t] increased 2-4 fold at 2 hours after hcg treatment.10 similar increases in [t] were not observed in our study in llamas and alpacas at 2, 4, and 8 hours after hcg treatment. however, after castration, hcg treatment did not increase [t] in llamas or alpacas, as in humans.12 increased testicular weight may be correlated with increased steroidogenic cells (e.g. leydig cells); larger mass of steroidogenic tissue increased leydig cell sensitivity and resulted in higher [t].13 this was supported by a study10 in alpacas that demonstrated association (r2 = 0.72; p < 0.001) between testicular weight and [t] 2 hours after hcg treatment. however, in stallions, there was no relationship between testicular weight and [t] before or after hcg treatment.14 in our study, there was only moderate (significant) relationship (r2 = 0.69; p = 0.02) between testicular weight and [t] prior to hcg treatment in llamas, but not in alpacas (r2 = 0.13). there was also moderate (significant) relationship (r2  =  0.46; p  =  0.04) table 2. testicular weights (tw) and serum testosterone concentrations ([t]) relative to the timing of human chorionic gonadotropin (hcg) treatment in alpacas and llamas, minimum (in green font) and maximum (in red font) values are identified in each column for alpacas and llamas; *differed (p < 0.05) from prehcg value alpaca tw prehcg [t] 2 hours posthcg [t] 4 hours posthcg [t] 8 hours posthcg [t] 1 23.5 grams 3.23 ng/ml 10.32 ng/ml 15.53 ng/ml 20.37 ng/ml 2 22.6 grams 7.72 ng/ml 14.30 ng/ml 14.21 ng/ml 18.58 ng/ml 3 22.0 grams 3.62 ng/ml 5.98 ng/ml 10.43 ng/ml 8.53 ng/ml 4 28.8 grams 4.22 ng/ml 9.89 ng/ml 9.74 ng/ml 13.25 ng/ml 5 28.1 grams 4.44 ng/ml 7.15 ng/ml 6.33 ng/ml 6.96 ng/ml 6 17.9 grams 7.14 ng/ml 10.65 ng/ml 16.21 ng/ml 19.23 ng/ml 7 14.7 grams 0.67 ng/ml 3.81 ng/ml 6.26 ng/ml 4.71 ng/ml 8 28.9 grams 9.12 ng/ml 17.66 ng/ml 20.89 ng/ml 24.91 ng/ml 9 36.2 grams 5.60 ng/ml 18.48 ng/ml 19.83 ng/ml 24.94 ng/ml mean ± sem 24.7 ± 1.3 grams 5.1 ± 0.9 ng/ml 10.9 ± 1.7 ng/ml* 13.3 ± 1.8 ng/ml* 14.6 ± 2.5 ng/ml llama tw prehcg [t] 2 hours posthcg [t] 4 hours posthcg [t] 8 hours posthcg [t] 10 35.9 grams 1.5 ng/ml 5.0 ng/ml 5.3 ng/ml 5.71 ng/ml 11 30.1 grams 0.9 ng/ml 6.9 ng/ml 8.5 ng/ml 9.73 ng/ml 12 38.5 grams 2.1 ng/ml 10.1 ng/ml 12.3 ng/ml 15.66 ng/ml 13 32.7 grams 0.6 ng/ml 5.1 ng/ml 6.1 ng/ml 7.51 ng/ml 14 22.0 grams 0.5 ng/ml 6.1 ng/ml 9.4 ng/ml 10.42 ng/ml 15 42.0 grams 3.0 ng/ml 5.5 ng/ml 5.6 ng/ml 5.62 ng/ml 16 9.9 grams 0.2 ng/ml 2.7 ng/ml 3.3 ng/ml 3.58 ng/ml mean ± sem 30.2 ± 1.8 grams 1.3 ± 0.4 ng/ml 5.9 ± 0.8 ng/ml* 7.2 ± 1.1 ng/ml* 8.7 ± 1.5 ng/ml http://dx.doi.org/10.58292/ct.v16.10926 https://open.lib.umn.edu/largeanimalsurgery/chapter/camelid-castration/ https://open.lib.umn.edu/largeanimalsurgery/chapter/camelid-castration/ 4 citation: clinical theriogenology 2024, 16, 10926, http://dx.doi.org/10.58292/ct.v16.10926 table 3. serum testosterone concentrations ([t]) relative to human chorionic gonadotropin (hcg) treatment in alpacas and llamas 1 day after castration; lowest limit of detection for the testosterone assay was 0.05 ng/ml alpaca prehcg [t] 2 hours posthcg [t] 4 hours posthcg [t] 8 hours posthcg [t] 1 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 2 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 3 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 4 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 5 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 6 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 7 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 8 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 9 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml llama prehcg [t] 2 hours posthcg [t] 4 hours posthcg [t] 8 hours posthcg [t] 10 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 11 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 12 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 13 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 14 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 15 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml 16 < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml < 0.05 ng/ml figure 2. serum testosterone concentrations in alpacas (a) and llamas (b) before and 2, 4, and 8 hours after human chorionic gonadotropin (hcg) treatment http://dx.doi.org/10.58292/ct.v16.10926 citation: clinical theriogenology 2024, 16, 10926, http://dx.doi.org/10.58292/ct.v16.10926 5 between testicular weight and [t] 2 hours after hcg in alpacas but not in llamas (r2 = 0.33; p = 0.17). there was no significant relationship between testicular weight and [t] at 4 and 8 hours after hcg treatment in llamas and alpacas (r2 = 0.120.25). these findings suggested that leydig cell responsiveness to hcg is not related to testicular weight. a potential limitation of the current study was that antimüllerian hormone (amh) concentrations were not measured. a recent paper15 stressed the importance of examining amh concentrations in male alpacas. in addition to measuring testosterone concentration after hcg stimulation, amh concentrations can provide evidence for cryptorchid testis. however, testosterone stimulation with hcg remains the gold standard for determining potential cases of cryptorchidism.16,17 conclusion in summary, hcg treatment in mature intact male llamas and alpacas increased [t] by 2 fold within 2 hours and remained figure 3. relationships between serum testicular concentrations and testicular weight in alpacas (a) and llamas (b) before and 2, 4, and 8 hours after human chorionic gonadotropin (hcg) treatment. only relationships before hcg treatment in llamas and 2 hours after hcg in alpacas were different (p < 0.05) http://dx.doi.org/10.58292/ct.v16.10926 6 citation: clinical theriogenology 2024, 16, 10926, http://dx.doi.org/10.58292/ct.v16.10926 elevated for at least 8 hours. in addition, [t] was below detectable limits within 24 hours after castration. acknowledgements authors thank bernadette stang and dr. tessa fiamengo for their technical support. funding morris animal foundation and willamette valley llama foundation. conflict of interest none to report. author contributions mk conceived the idea, garnered 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and castration on serum anti-müllerian hormone concentration in male alpacas. theriogenology 2018;105:174-177. doi: 10.1016/j. theriogenology.2017.09.032 16. marzok m, moustafa a, el-habashi n, et al: evaluation of modified in situ pinhole technique for castration in horses. kafrelsheikh vet med j 2016;14:163-179. doi: 10.21608/kvmj.2016.108687 17. mceachern r, houle am, garel l, et al: lost and found testes: the importance of the hcg stimulation test and other testicular markers to confirm a surgical declaration of anorchia. horm res 2004;62:124-128. doi: 10.1159/000080018 18. memon ma, ganjam vk, pavletic mm, et al: use of human chorionic gonadotropin stimulation test to do detect a retained testis in a cat. j am vet med assoc 1992;201:1602. doi: 10.2460/javma. 1992.201.10.1602 19. murase t, okuda k, sato k: assessment of bull fertility using a mucus penetration test and a human chorionic gonadotrophin stimulation test. theriogenology 1990;34:801-812. doi: 10.1016/ 0093-691x(90)90552-5 20. santana m, batista m, alamo d, et al: influence of sexual stimulation and the administration of human chorionic gonadotrophin on plasma testosterone levels in dogs. reprod domest anim 2012;47:43-46. doi: 10.1111/j.1439-0531.2011.01923.x 21. mialot jp, thibier m, toublanc je, et al: plasma concentration of luteinizing hormone, testosterone, dehydroepiandrosterone, androstenedione between birth and one year in the male dog: longitudinal study and hcg stimulation. andrologia 1987;20: 145-154. doi: 10.1111/j.1439-0272.1988.tb00678.x 22. sundby a, ulstein t: plasma concentrations of testosterone in the male dog and plasma testosterone profile following single intramuscular injection of hcg. acta vet scand 1981;22:409-416. doi: 10.1186/bf03548666 23. tremblay y, belanger a: changes in plasma steroid levels after single administration of hcg or lnrh agonist analogue in dog and rat. j steroid biochem 1985;22:315-320. doi: 10.1016/0022-4731 (85)90432-7 24. andresen o: 5alpha-androstenone in peripheral plasma of pigs, diurnal variation in boars, effects of intravenous hcg administration and castration. acta endocrinol (copenh) 1975;78:385-391. doi: 10.1530/acta.0.0780385 25. rhodes rc, fleming mw: unilateral orchidectomy of ram lambs: acute, chronic and hcg-stimulated androgen levels. comp biochem physiol 1987;87:627-630. doi: 10.1016/0300-9629(87)90372-0 http://dx.doi.org/10.58292/ct.v16.10926 https://doi.org/10.1016/s0737-0806(89)80112-1 https://doi.org/10.1016/j.anireprosci.2020.106670 https://doi.org/10.1016/0378-4320(84)90021-6 https://doi.org/10.1530/acta.0.0680614 https://doi.org/10.1292/jvms.18-0151 https://doi.org/10.1016/s0021-9258(18)33517-8 https://doi.org/10.1016/s0021-9258(18)33517-8 https://doi.org/10.1111/j.1749-6632.1982.tb23171.x https://doi.org/10.1016/s0749-0720(15)30560-0 https://doi.org/10.1111/j.1751-0813.1996.tb13773.x https://doi.org/10.1111/j.1751-0813.1996.tb13773.x https://doi.org/10.3389/fvets.2020.595856 https://doi.org/10.1093/biolreprod/52.monograph_series1.661 https://doi.org/10.1093/biolreprod/52.monograph_series1.661 https://doi.org/10.1159/000280809 https://doi.org/10.2527/jas1982.5551139x https://doi.org/10.1016/j.theriogenology.2017.09.032 https://doi.org/10.1016/j.theriogenology.2017.09.032 https://doi.org/10.21608/kvmj.2016.108687 https://doi.org/10.1159/000080018 https://doi.org/10.2460/javma.1992.201.10.1602 https://doi.org/10.2460/javma.1992.201.10.1602 https://doi.org/10.1016/0093-691x(90)90552-5 https://doi.org/10.1016/0093-691x(90)90552-5 https://doi.org/10.1111/j.1439-0531.2011.01923.x https://doi.org/10.1111/j.1439-0272.1988.tb00678.x https://doi.org/10.1186/bf03548666 https://doi.org/10.1016/0022-4731(85)90432-7 https://doi.org/10.1016/0022-4731(85)90432-7 https://doi.org/10.1530/acta.0.0780385 https://doi.org/10.1016/0300-9629(87)90372-0 1 dr. jerry rains memorial abstracts antimüllerian hormone concentrations and antral follicles in hormonally treated mares hayley moore,a candace lyman,a peyton draheim,b jessica klabnika acollege of veterinary medicine, auburn university, auburn, al, usa bhagyard equine medical institute, lexington, ky, usa antimüllerian hormone (amh) concentrations are correlated with antral follicle count (afc) in the mare. because of this relationship, potential lies in the ability of amh to act as a prognostic tool for assessing a mare’s overall fertility or, more specifically, predicting a mare’s potential as an oocyte donor for the production of in vitro-derived embryos. it is unknown if amh concentrations remain correlated when mares are undergoing hormone therapy protocols aimed at increasing afc to promote oocyte aspiration efficiency. our aim was to determine if afc is correlated to amh concentrations in mares undergoing cycle manipulation with only follicle ablation (controls) or undergoing hormone therapy targeted to increase afc with either progesterone and estradiol (p&e) or with a proprietary drug that supports follicular growth. we hypothesized that amh concentrations are positively correlated with afc in mares undergoing cycle manipulation. data from 2 previous experiments were analyzed. in the first experiment, 10 mares underwent a crossover design in which mares did (p&e group) or did not (control group) receive a dose of a slow-release formulation of p&e (1.5 g and 50 mg, respectively) on day 0. on day 0, all mares underwent transvaginal follicle ablation. on days 0, 3, 6, and 10, afc was determined via transrectal ultrasonography and serum was stored for amh enzyme-linked immunosorbent assay (elisa). in the second experiment, a group of 6 mares (proprietary drug group) underwent transvaginal follicle ablation (day -10) followed by prostaglandin (intramuscular dinoprost tromethamine [5 mg], day -4) to lyse existing luteal tissue and act as a means of estrous cycle synchronization. proprietary drug was given on days 0 through 4. on day 0 through 4 and day 7, afc was determined via transrectal ultrasonography and serum samples were stored for amh elisa. data were analyzed for normality via shapirowilk and correlation was determined by kendall rank correlation coefficient. day 0 amh and afc data were removed prior to analysis, as this was the first day in which estrous cycle manipulation (i.e. drug treatment or ablation) occurred in most mares. significance was set at p < 0.05 for all analyses and performed in r version 4.3.1. there was no correlation (r = 0.17; p = 0.19) between amh and afc in the control group (n = 10), a moderately positive correlation (r = 0.51; p = 0.00011) in the p&e group (n = 10), and a weak positive correlation (r  =  0.36; p  =  0.0062) in the proprietary drug group (n  =  6). this study demonstrated that there was a significant positive correlation between amh concentrations and afc in mares undergoing 2 treatments that support follicular growth. future studies are needed to determine if amh concentrations can be a tool used to help assess oocyte aspiration potential and fertility in the mare. keywords: mare, antimüllerian hormone, antral follicle count l-carnitine and pyruvate increased longevity of ram sperm stephanie ferguson,a jennifer clulow,a erin klein,b zamira gibb,b allan gunna aschool of agricultural, environmental and veterinary sciences and gulbali institute, charles sturt university, wagga wagga, nsw, australia bschool of environmental and life sciences, university of newcastle, callaghan, nsw, australia cooled and cryopreserved ram sperm have poor longevity; additionally, ewe’s cervix is convoluted and complex, necessitating insemination via invasive laparoscopic techniques. we hypothesized that sperm longevity improves by avoiding cooling during storage as demonstrated in other species. this would enable utilization of less invasive artificial insemination techniques and, increase the use of sex-sorted sperm. our aim was to determine if supplementation with l-carnitine and pyruvate increases the lifespan of ambient temperature liquid stored ram sperm. following semen collection (12 ejaculates from 4 rams via artificial vagina), ejaculates were immediately extended to 4 ml total volume with biggers, whitter, and whittingham (bww) solution prior to sperm isolation using a 90% percoll density gradient. sperm pellets were resuspended into 4 treatment groups: 1. bww; 2. bww + pyruvate (5 mm); 3. bww + l-carnitine (100 mm); and 4. bww + pyruvate (5 mm) + l-carnitine (100 mm). media were osmotically balanced to 315 mosm, and 1 mm of d-penicillamine was added to reduce cell to cell agglutination during storage. total and progressive motility were assessed via computer assisted sperm analysis at 7 time points between 0 and 168 hours. sperm were assessed for morphology by a single blinded assessor, whereas another single assessor assigned subjective agglutination scores. total and progressive motility decreased across all samples to 120 hours. pyruvate, l-carnitine and combination treatments had greater (p < 0.05) total motility (13.11% ≤ 12.30, 15.41% ≤ 2.22 and 12.931% ≤ 1.97) compared to control (6.37% ≤ 2.19). additionally, there was improvement (p < 0.05) in progressive motility at 168 hours in the pyruvate, l-carnitine, and combination treatments (11.27% ≤ 1.89, 13.64% ≤ 1.96, 11.28% ≤ 1.68) compared to control (5.5% ≤ 1.85). furthermore, at 168 hours, the l-carnitine supplemented samples had greater (p  <  0.005) © 2024 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v16.11054 2 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 total and progressive motility than other treatments. cell to cell sperm agglutination occurred from 24 hours of storage, despite the addition of d-penicillamine. in conclusion, despite relatively low motility rates, l-carnitine and pyruvate significantly enhanced the longevity of ambient temperature-stored ram sperm after 7 days. further research is required to explore optimized additive concentrations, methods to minimize agglutination and manage contamination. keywords: ram, sperm, storage, ambient temperature, pyruvate, l-carnitine amniotic membrane transcriptome analysis identifies genes and mechanisms associated with inflammatory response during ascending placentitis sophia marchio,a matthew scott,b hossam el-sheikh ali,c claudia fernandes,d kirsten scoggin,c yatta boakaria aschool of veterinary medicine and biomedical sciences, texas a&m university, college station, tx, usa bveterinary education, research, and outreach center, texas a&m university, canyon, tx, usa cmaxwell h. gluck equine research center, department of veterinary science, university of kentucky, lexington, ky, usa ddepartment of animal reproduction and veterinary radiology, school of veterinary medicine and animal science, são paulo university, são paulo, sp, brazil ascending placentitis (ap) in mares is a leading cause of lateterm pregnancy loss, premature birth, and delivery of weak foals. although ap causes substantial economic losses to equine industry, its pathophysiology is not fully understood. recently, it has been elucidated that ap is associated with alteration of amniotic fluid proteomics. however, the molecular mechanisms underlying these changes remain unexplored. we hypothesized that ap is associated with critical transcriptional alterations in amniotic membrane. aim was to characterize the amniotic membrane transcriptome in mares with ap (n = 6) compared to gestationally matched negative controls (n = 6). at 280 days of pregnancy, mares assigned to the placentitis group underwent an intracervical inoculation of streptococcus equi ssp. zooepidemicus (1 x 106 cfu) using a semiflexible artificial insemination pipette, to induce placentitis. after 8 days, mares had an increase (p < 0.01) in combined thickness of uterus and placenta. mares in the control group did not receive any treatment. eight days after inoculation, mares were euthanized. samples of the amniotic membrane were collected and stored in rnalater (#am7021; invitrogen) until rna isolation. paired samples were also fixed in 10% formaldehyde for 24 hours and then transferred to 100% methanol for further histopathological determination of the inflammation score. following rna extraction via commercial kits, next-generation rna sequencing of the samples was performed (novaseq 6000; 150bp, paired-end). following quality assessment, trimming, and gene-level count matrix construction via an hisat2/stringtie2 workflow, raw gene counts were imported to r 4.1.2. data were filtered for zero-counts, normalized with the trimmed mean of m-values method (tmm), and then converted into log2-countsper-million values (log2cpm). weighted gene coexpression network analysis was used for expression network and module construction. according to signed kendall correlation coefficients, the 32,004 genes were constructed into coexpression modules; each module was set to have a minimum of 30 genes, and an intermodular correlation above 0.25 was the threshold to merge the modules. each module was assigned to have unique color identification. module-trait kendall’s tau correlation coefficients were considered significant at |r| > 0.4 and p < 0.05. panther classification system v18.0 was utilized for overrepresentation analysis to functionally annotate genes based on gene ontology (fdr < 0.05). one module (‘brown’) was highly positively correlated (r = 0.81, p < 0.001) with the inflammation score trait. this module enriched for toll-like receptor (tlrs) signaling that includes a series of genes (tlr2, tlr8, ly96) described as upregulated in the chorioallantois and endometrium of mares with placentitis; this mechanism has been determined as a of the critical regulator eliciting inflammation during equine placentitis. tlrs are crucial in recognizing pathogenic bacterial infections, triggering inflammatory cascade; however, activation of the inflammatory response at the fetomaternal interface level must be suppressed to maintain pregnancy, as its early activation can lead to preterm delivery. interestingly, intra-amniotic treatments using tlr antagonists contributed to downregulating the placental inflammatory response in primates. therefore, future research leveraging these tlr mechanisms and associated gene products may result in a potential treatment for placentitis in mares, delaying infection-associated premature birth or abortions. keywords: mare, placentitis, transcriptome, inflammation, toll-like receptor signaling investigating canine infertility through evaluation of full thickness uterine biopsies gail mcrae,a marco coutinho da silva,a erin runcan,a julie stephens,b christopher premanandanc adepartment of veterinary clinical sciences, bcenter for biostatistics, college of medicine, cdepartment of veterinary biosciences, the ohio state university, columbus, oh, usa uterine biopsies have been used routinely in some species (predominantly in mares) to evaluate endometrial health. evaluation of endometrial tissue has the potential to assist with breeding soundness in dogs. however, there is limited information regarding histopathological lesions of the endometrium of dogs with infertility compared to fertile dogs. objective of this study was to compare histopathological differences of uterine biopsies between fertile and subfertile female dogs. we hypothesized that subfertile dogs have a higher incidence and severity of endometrial inflammation, periglandular fibrosis, and endometrial cysts compared to fertile dogs. full thickness uterine biopsies were collected at  hysterotomy site during cesarean surgery from fertile dogs (control; n = 103) or dogs with known history of infertility (experimental; n  =  263). samples were processed for histopathology. paraffin embedded samples were sectioned, placed on a glass slide, and stained with hematoxylin and  eosin. slides were randomized, blinded, and evaluated independently by 2 experienced examiners. samples were assessed for the presence and severity of periglandular fibrosis (number of glands affected and layers), cysts (number), and inflammation. inflammation was further categorized by cell type (neutrophilic, eosinophilic, lymphoplasmacytic, and http://dx.doi.org/10.58292/ct.v16.11054 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 3 histiocytic) and severity (number of inflammatory cells per 400 x field). chi-square tests were used to compare the categorical variables between controls and experimental samples. wilcoxon rank sum test was used to compare age between groups. statistical significance was set at p <0.05. dogs in the control group were younger (p = 0.022) than the experimental group (median 3.5 versus 4.0 years). cellular inflammation was observed more often (p = 0.004) in experimental samples (76%) compared to controls (61%). percentage of samples having neutrophilic inflammation between control and experimental groups was not different (p= 0.714; 45.6 versus 43.5%,). in those with inflammation, chronic inflammation was observed more often (p < 0.001) in experimental group than control group (70 versus 43%). in addition, there was no statistical difference between groups regarding periglandular fibrosis and cyst formation. contrary to our hypothesis, endometrial cysts, periglandular fibrosis, and neutrophilic inflammation were observed at similar frequency in both groups. therefore, these endometrial changes may not be necessarily indicative of fertility issues in dogs. however, subfertile dogs had a higher incidence of chronic inflammation than controls. in mares, periglandular fibrosis is associated with a reduced ability to carry pregnancy to term. based on our results, this may not be the case in dogs and it potentially relates to differences in placentation between species. in conclusion, histopathology is an important tool to evaluate potential fertility in dogs. however, findings should be interpreted carefully and considered species-specific. keywords: dog, uterine biopsy, infertility, endometrium comparison of commercial semen extenders for long-term storage of stallion semen at various temperatures patricio razquin, brittany middlebrooks, patrick mccue colorado state university, fort collins, co, usa use of short-term cooled-stored semen is routine in the equine breeding industry. in most instances stallion semen is stored at 5 to 8°c for 24 to 48 hours prior to insemination. goal was to evaluate the effect on sperm progressive motility of prolonged storage (7 to 15 days) of stallion semen diluted in each of 3 commercial extenders and maintained at 5° or 17°c. our hypothesis was that progressive sperm motility is maintained at a higher percentage in the beyond® extender (minitube) after prolonged storage at 5° or 17°c than either inra96 (imv technologies) or botusemen gold (botupharma usa) extenders. semen was collected from 10 light horse stallions and evaluated for volume (graduated cylinder), sperm concentration (nucleocounter® 200, chemometic) and progressive motility percentage (spermvision®, minitube). two aliquots of semen from each ejaculate were prepared for each of the 3 extenders by diluting raw semen to a concentration of 25 x 106 progressively motile sperm per ml and stored in a whirl-pak® bag. one aliquot of extended semen (x 3 extenders) from each ejaculate was stored at 5°c within a styrofoamtm box inside a refrigerator. the second aliquot of each extended sample for each stallion was stored in a commercial thermoelectric box (minitube) at 17°c. a small (0.5 ml) volume of extended semen was removed each day from each of the 6 aliquots, warmed to 37°c for 10 minutes and evaluated for progressive motility by computer assisted semen analysis. samples stored at 5 and 17°c were evaluated daily for 15 and 7 days, respectively. data were evaluated by one-way analysis of variance and tukey hsd. for aliquots stored at 5°c, there was no difference (p > 0.05) in progressive sperm motility among extenders during the first 10 days of cooled storage. from days 11 to 15, the progressive motility of sperm diluted in the beyond® extender was greater (p < 0.05) than other 2 extenders. for aliquots stored at 17°c, there was no difference (p > 0.05) in progressive sperm motility among 3 extenders during the first 5 days of storage. on days 6 and 7 of storage, progressive motility of sperm diluted in the beyond® extender was greater (p < 0.05) than that of sperm diluted in inra96 or botusemen gold extenders. in summary, there was no difference in progressive motility among 3 extenders during short-term storage at 5 or 17°c. in contrast, the progressive motility of stallion sperm was higher in the beyond® extender during long-term liquid (nonfrozen) storage (≥ 11 days at 5°c or ≥ 6 days at 17°c). fertility trials evaluating storage time and temperatures and microbial culture studies are indicated before long-term storage of liquid stallion semen can be recommended. keywords: stallion, semen, extender, progressive motility, storage gallium nitrate: a potential alternative to traditional antimicrobial treatment of equine endometritis allison deaton,a kaushalya jayathilake,a samantha frontz,b marie montelongo,b jennifer rudd,b dale kelleya adepartment of veterinary clinical sciences, bdepartment of veterinary pathobiology, college of veterinary medicine, oklahoma state university, stillwater, ok, usa bacterial endometritis commonly contributes to infertility in mares. as rates of antimicrobial resistance rise in equine uterine pathogens, there is a need to identify alternative antimicrobial compounds such as gallium. bacteria confuse gallium and iron due to their similar chemical properties, causing disruption of iron-dependent processes and subsequently bacterial cell death. our goal was to assess in vitro effects of gallium on common bacterial pathogens associated with equine endometritis. we hypothesized that gallium nitrate, a highly soluble crystalline form of gallium, inhibits common equine uterine pathogens growth resulting in a concentration-dependent reduction of bacteria. twelve unique bacterial isolates collected via uterine lavage from 10 mares were used to determine the minimum inhibitory concentration (mic) of gallium nitrate for escherichia coli, pseudomonas aeruginosa, and klebsiella pneumoniae (n = 4 isolates each). identification of each clinical isolate was confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (maldi-tof). isolated colonies were then added to sterile water until a 0.5 mcfarland turbidity standard (1.5 x 108 cfu/ml) was achieved using a premade standard and a nephelometer. each isolate was further diluted in a cation-balanced irondepleted muller-hinton (id-camh) broth to a final concentration of 105 cfu/ml. equal molar amounts of gallium nitrate (0.20 g) and tri-sodium citrate (0.23 g) were combined and added to deionized water to create a 100 ml stock solution of gallium nitrate at 2 mg/ml (2,000 µl/ml). the ph of the gallium nitrate solution was measured by a benchtop ph meter (aquasearcher) and adjusted to 7.2 using 0.1m sodium bicarbonate. in a 96-well http://dx.doi.org/10.58292/ct.v16.11054 4 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 plate, 50 µl of sterile water was added to each well to facilitate 2-fold serial dilutions of gallium nitrate, starting at a concentration of 2,000 µl/ml. in replicates of 3, 50 µl of each bacterial suspension was added to each of their respective wells for a total volume of 100 µl per well. plates were then sealed and incubated for 24 hours at 37ºc. growth was visually assessed, and turbidity was quantified using optical density at 600 nm. results were then processed through a statistical analysis system to determine the mean mic of gallium nitrate for each pathogen. mean mic for escherichia coli, pseudomonas aeruginosa, and klebsiella pneumoniae was 1,437.5 ± 864.8 µg/ml, 7.2 ± 5.6 µg/ml, and 2,000 ± 0.0 µg/ml (mean ± sd), respectively. there was inhibition (p < 0.001) for each isolate with pseudomonas aeruginosa. there was also wide variability among individual escherichia coli isolates as demonstrated by a large standard deviation that is thought to be attributed to the variability of the pathogen itself. these data suggested that gallium inhibited the growth of common equine uterine pathogens and maybe an alternative to traditional antimicrobial treatment. additionally, these results allow for future studies testing the safety and efficiency of the solution as an antimicrobial compound for treatment of equine endometritis. keywords: gallium, antimicrobial resistance, endometritis, isolate, pathogen prognostic indicators for survival in goats treated for toxic mastitis hannah maxwell, erin harris, lisa johnson, thomas passler, jessica rush, manuel chamorro department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa severe clinical mastitis, also referred to as toxic or gangrenous mastitis, is a relatively uncommon but life-threatening disease of lactating does. currently, scientific reports of prognostic indicators for survival of goats with toxic mastitis are unavailable. our goal was to evaluate records of goats presented to a veterinary teaching hospital for toxic mastitis in order to identify prognostic indicators for survival to discharge. we hypothesized that the physical examination findings of heart rate, respiratory rate, and rectal temperature upon presentation to the hospital differ between survival and nonsurvival groups. additionally, we hypothesized that medical management factors such as length of hospitalization, treatment with intramammary antibiotics, and treatment with dexamethasone are positively associated with survival. a retrospective study was conducted to evaluate medical records from 2014 to 2024 of goats treated for toxic mastitis. adult does with severe clinical mastitis and signs of systemic compromise (e.g. abnormal body temperature, dehydration, anorexia, and others) were included in the study. a total of 26 medical records met the inclusion criteria, and the association between independent variables and outcome (discharge from the hospital versus nondischarge) was assessed by chi square and fisher’s exact tests. of the 26 goats included, 76.9% (20/26) survived to discharge and 23.1% (6/26) died or were euthanized. initial physical examination findings of heart rate, respiratory rate, and rectal temperature were not associated (p > 0.05) with the outcome. in contrast, hospitalization length ≥ 10 days (p  =  0.006), treatment with intramammary antibiotics (p  =  0.01), and treatment with dexamethasone (p  =  0.04) were independently associated with survival to discharge. treatment of goats with toxic mastitis at this institution carried a fair to good prognosis to hospital discharge. inclusion of intramammary antibiotics and steroids in the treatment regimen may improve survival in some cases of toxic mastitis. keywords: goat, toxic, mastitis, survival, intramammary, dexamethasone effect of inactivated parapoxvirus ovis intramuscular injection on endometrial gene expression in chronic infectious endometritis in mares alanoud alshami,a,b mohammad ababneha afaculty of veterinary medicine, jordan university of science and technology, irbid, jordan bcollege of veterinary medicine and biomedical sciences, texas a&m university, college station, tx, usa chronic infectious endometritis (cie) is a major cause of equine subfertility.1 persistence of cie is partially related to impaired uterine immune response and clearance.2 inactivated parapoxvirus ovis (zylexis®) is an immunomodulator used in the treatment of respiratory disease in horses3; nevertheless, it has not been used for endometritis in the mare. preliminary data indicated that pregnancy rates increased in postpartum mares after zylexis® treatment.4 we studied the effect of zylexis® treatment on endometrial gene expression in mares with cie. we hypothesized that zylexis® affects endometrial genes associated with innate immunity (tumor necrosis factor [tnf]α, interleukin [il]-1β, il-6, il-8, il-10, il-1 receptor antagonist [ra], interferon [ifn]-γ) and mediators of uterine contractility (cytosolic phospholipase a2 [cpla2], secretory pla2 [spla2], and prostaglandin-endoperoxide synthase-2 [ptgs2]) in mares with cie. eight cycling mares (12-15 years) diagnosed with cie due to escherichia coli infection and positive cytology (1-5 pmns/hpf) that were refractory to treatment, were enrolled in this experiment. endometrial biopsies were collected at 0, 4, 8, 24, and 48 hours, starting on day 3 of estrus when mares had a ≥ 35 mm follicle and endometrial edema. after 4 hours, all mares were treated with 2 ml of zylexis® intramuscularly. relative gene expression of tnf-α, il-1β, il-6, il-8, il-10, il-1ra, ifn-γ, cpla2, spla2, and ptgs2 in the endometrial tissue were measured using rt-qpcr. glyceraldehyde 3-phosphate dehydrogenase was selected as a housekeeping gene. results were expressed as fold changes calculated using delta-delta (∆∆) ct method. data were tested for normality using shapiro-wilk test, and fold changes for all genes were analyzed using repeated measures anova or friedman test, followed by lsd or wilcoxon signed-rank test, respectively. significance was set at (p  <  0.05). expression of ifn-γ and ptgs2 differed (p < 0.05) among time points. compared to 4 hours: expression of ifn-γ increased 129-fold at 8 hours, and decreased to 20-fold by 24 hours; and expression of ptgs2 decreased 2-fold at 24 hours. compared to 4 hours: tnf-α and cpla2 tended to decrease (p = 0.07) 2and 1.7-fold at 8 hours, respectively. there was no effect (p > 0.05) of treatment on il-1β, il-1ra, il-6, il-8, il-10, and spla2. in this study, ifn‐γ and tnf-α (innate immune http://dx.doi.org/10.58292/ct.v16.11054 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 5 response genes and proinflammatory cytokines5) and (cpla2 and ptgs2 (mediators of uterine contractility and key enzymes in prostaglandin production6) were affected by zylexis® treatment. upregulation of ifn-γ as a proinflammatory cytokine after zylexis® treatment may have a therapeutic role in the impaired immune response in the mare endometrium. following the spike of ifn-γ at 8 hours, the declining phase of the upregulated ifnγ coupled with decreased ptgs2 expression at 24 hours may indicate the end of inflammatory phase and the initiation of endometritis resolution. further studies are needed to determine the effectiveness of zylexis® in treatment of cie. keywords: mare, inactivated parapoxvirus ovis, chronic infectious endometritis, gene expression references 1. katila t, ferreira-dias g: evolution of the concepts of endometrosis, post breeding endometritis, and susceptibility of mares. animals 2022;12:779. doi: 10.3390/ani12060779 2. leblanc mm: advances in the diagnosis and treatment of chronic infectious and post-mating-induced endometritis in the mare. reprod domest anim 2010;45:21-27. doi: 10.1111/j.1439-0531. 2010.01634.x 3. horohov dw, breathnach cc, sturgill tl et al: in vitro and in vivo modulation of the equine immune response by parapoxvirus ovis. equine vet j 2008;40:468-472. doi: 10.2746/042516408x 322111 4. khalifa t, lymberopoulos a: insights into contribution of non-specific immunostimulants (nis) to reproductive efficiency of lactating mares during vernal transition. reprod domest anim 2008;43s5:86. 5. woodward em, troedsson mht: equine breeding-induced endometritis: a review. j equine vet sci 2013;33:673-682. doi: 10.1016/j. jevs.2012.11.010 6. ababneh mm, troedsson mht: ovarian steroid regulation of endometrial phospholipase a2 isoforms in horses. reprod domest anim 2013;48:311-316. doi: 10.1111/j.1439-0531.2012. 02151.x http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.3390/ani12060779 https://doi.org/10.1111/j.1439-0531.2010.01634.x https://doi.org/10.1111/j.1439-0531.2010.01634.x https://doi.org/10.2746/042516408x322111 https://doi.org/10.2746/042516408x322111 https://doi.org/10.1016/j.jevs.2012.11.010 https://doi.org/10.1016/j.jevs.2012.11.010 https://doi.org/10.1111/j.1439-0531.2012.02151.x https://doi.org/10.1111/j.1439-0531.2012.02151.x 6 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 competitive case reports leiomyoma of tunica albuginea affected breeding performance in a quarter horse stallion alexandra grillos,a erin runcan,a ryan jennings,b jonathan miller,b kayla kownurko,a marco coutinho da silvaa adepartment of veterinary clinical sciences, bdepartment of veterinary biosciences, the ohio state university college of veterinary medicine, columbus, oh, usa a 9-year, quarter horse stallion, was presented in october 2023 for potential oligospermia and a history of reluctance to mount mares during live-cover. oligospermia was noted in a prior semen evaluation. owners also noted that stallion was unwilling to mount and breed mares during last breeding season. at presentation, stallion was hesitant to mount a phantom. semen was collected twice with a missouri model artificial vagina, 1 hour apart, utilizing a hormone-treated, ovariectomized tease mare. during both collections, stallion displayed reluctance to mount the phantom with no signs of obvious lameness that would explain this behavior. first ejaculate had 16.08 x 109 sperm with a total and progressive motility of 64 and 49%, respectively, whereas second ejaculate had 1.18 x 109 sperm with a total and progressive motility of 57 and 31%. approximately 55% of sperm were normal in both ejaculates. we assumed that the second ejaculate was only partial (low sperm number) because stallion had discomfort during collection. therefore, daily sperm output was calculated based on testicular measurements. daily sperm output was estimated to be 6.8 x 109 sperm per day. ultrasonographic examination of the accessory sex glands revealed a 1 cm cyst in caudal left ampulla. testicular ultrasonography revealed a mass (1.65 x 1.53 cm) associated with right epididymis head and related dilation of right pampiniform plexus vasculature. mass was firm on palpation and stallion elicited severe pain when pressed. hemicastration (right testis) was performed and the mass was submitted for histopathology, which revealed that the mass arose from tunica albuginea. there were no substantial microscopic abnormalities of epididymis, ductus deferens, pampiniform plexus, and spermatic cord fascia or testis. mass was identified as benign mesenchymal neoplasm, most likely, of smooth muscle origin. pathologists classified the mass as a leiomyoma arising from tunica albuginea, and excision of the mass was complete. stallion fully recovered from surgery and was discharged the following day with antibiotics and nonsteroidal antiinflammatories. most commonly reported tumors associated with equine testis are seminomas, interstitial cell tumors, teratomas, sertoli cell tumors, and teratocarcinomas; leiomyomas are uncommon. testicular leiomyomas have been described in stallions.1-3 two of these were associated with tunica albuginea, and the third was associated with testicular parenchyma. this is a unique case description of a paratesticular neoplasm that caused discomfort and affected this stallion’s breeding performance. keywords: equine, leiomyoma, tunica albuginea, stallion references 1. johnson rc, steinberg h: leiomyoma of the tunica albuginea in a horse. j comp pathol 1989:100;465-468. doi: 10.1016/00219975 (89)90014-5 2. stoll al, tucker r, witte th, et al: bilateral leiomyoma in the testes of a horse. equine vet educ 2014:27;113-115. doi: 10.1111/ eve.12267 3. weiermayer p, richter b: simultaneous presence of a seminoma and a leiomyoma in the testes of a horse. equine vet educ 2009: 21;172-176. doi: 10.2746/095777309x400306 double type 3 jejunal atresia in a neonatal pup michelle kutzler department of animal and rangeland sciences, oregon state university, corvallis, or, usa a 1-day-old female dachshund pup was presented with weight loss and decreased suckle response. pup weighed 208 g at birth, which decreased to 199 g the next day. at presentation, pup was depressed and had a subnormal body temperature (35.6˚c) with no evidence of meconium on the rectal thermometer. pup also appeared to have mild abdominal distension but did not act painful during abdominal palpation. a urine sample was collected by perineal stimulation. the urine parameters were all within normal limits for a neonatal dog (usg: 1.017 [range 1.006-1.017]; trace protein; trace glucose).1 abdominal ultrasonography revealed some gas-filled bowel with no peristalsis. initial diagnosis was hypothermia with secondary ileus. pup was given prewarmed lactated ringer’s solution subcutaneously (5 ml), glucose (karo syrup) on the oral mucosa, and placed in a humidified incubator (35.5˚c, 50% humidity). within 4 hours, pup’s rectal temperature had increased to 36.7˚c and milk (1 ml) provided by the breeder was given via an orogastric tube. however, within the next hour, pup became agonal, and the decision was made to euthanize. at necropsy, there was small amount of milk in the stomach. serosa of the duodenum and proximal jejunum was dark red, and the lumen was distended along its length to a blind end. after a 1-cm gap, jejunum resumed for 6-cm and then there was a second blind end with a 1-cm gap. distal to the second blind end, the intestinal tract continued to a patent anus. diameter of the jejunum distal to the first blind end was about half that of the diameter proximal to the blind end. on histological examination, the jejunum proximal to the first blind end http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1016/0021-9975(89)90014-5 https://doi.org/10.1016/0021-9975(89)90014-5 https://doi.org/10.1111/eve.12267 https://doi.org/10.1111/eve.12267 https://doi.org/10.2746/095777309x400306 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 7 had transmural congestion and serosal lymphatics were dilated. all other organs were grossly and histologically normal. congenital intestinal atresia can vary from complete blockage by a membrane (type 1) to sections joined by an imperforate cord (type 2) to blind-end atresia in which a segment of intestine is missing (type 3).2 pup in the current report had a double type 3 jejunal atresia. type 3 intestinal stenosis has been reported in 1 foal, 9 calves, 2 lambs, 3 piglets, 1 pup, and 3 kittens.2 there are several hypothetical causes of congenital intestinal atresia, but mesenteric ischemia has been experimentally shown to cause intestinal atresia in fetal dogs.3 mortality can occur in neonatal pups for multiple reasons.1 performing necropsies allows veterinarians to make definitive diagnoses in cases of congenital defects. keywords: canine, congenital defect, neonatal mortality references 1. münnich a, küchenmeister u: causes, diagnosis and therapy of common diseases in neonatal puppies in the first days of life: cornerstones of practical approach. reprod domest anim 2014;49:64-74. doi: 10.1111/rda.12329 2. van der gaag i, tibboel d: intestinal atresia and stenosis in animals: a report of 34 cases. vet pathos 1980;17:565-574. doi: 10.1177/030098588001700505 3. koga y, hayashida y, ikeda k, et al: intestinal atresia in fetal dogs produced by localized ligation of mesenteric vessels. j pediatr surg 1975;10:949-953. doi: 10.1016/s0022-3468(75)80101-1 transient addisonian-like crisis in a late pregnant dog gabriela sousa,a alyssa helms,a nicole sugai,a teresa southard,b bobbi conner,a julie cecere,a orsolya balogha adepartment of small animal clinical sciences, bdepartment of biomedical sciences and pathobiology, virginiamaryland college of veterinary medicine, blacksburg, va, usa a 3-year, pregnant golden retriever dog, was presented 52 days after ovulation for nausea, hyporexia, regurgitation, polyuria, and polydipsia. combined blood count had abnormalities consistent with late-term pregnancy; blood chemistry revealed mild metabolic acidosis, hypocalcemia, hyponatremia, hyperkalemia, and hyperphosphatemia; and there were traces of ketones in urine without glucose. reproductive ultrasonography revealed several viable fetuses. dog was treated with fluids, calcium, and antinausea drugs. two days later, dog was hospitalized because of hyporexia, vomiting, abdominal distention, and acute onset of bleeding from the rectum. a nasogastric tube was placed, and dog received fluids, antiemetics, antinausea, antibiotics, and calcium. despite treatment, dog deteriorated, became tachypneic, and tachycardic, had progressive regurgitation, generalized weakness, and was transferred to an o2 cage. combined blood count was compatible with sepsis/infection and blood chemistry revealed metabolic acidosis, hyponatremia, hyperkalemia, a low na: k ratio (24:1), hypochloremia, and hypocalcemia, raising the suspicion of an addisonian-like crisis. baseline serum cortisol concentrations on the next day were 7.89 ug/dl (reference range 1.007.13 ug/dl). aiming to improve fetal maturation, dexamethasone was started on day 2 of hospitalization and cesarean surgery was performed 2 days later (57 days of pregnancy) because of dog’s continued deterioration despite aggressive medical therapies. evidence of a moderate to a marked increase in allantoic fluid and hiatal hernia were noted at surgery. all 11 pups were delivered alive and had apgar scores of 8-9/10 at discharge on the same day. dog remained hospitalized and progressively improved after aggressive medical management. an acth stimulation test was performed as a differential diagnosis for pregnancy-associated addisonian-crisis and the result was within the reference range. dog was discharged 4 days after surgery and continued to be treated with prednisone for 45 days and recovered well in the following days. she had a follow-up acth stimulation test after discontinuation of prednisone that was within the reference range. pups progressively faded and died a few days after birth. one pup survived and is doing well at the time of this report. necropsy with histopathology on 6 pups that died on various days after birth had lung immaturity with mild edema; conclusion was respiratory failure due to pulmonary immaturity. based on the clinical and laboratory findings and improvement after intensive management of the dog, we hypothesized that the dam had a transient addisonian-like crisis. similar findings were described1,2 despite cortisol concentrations were within the normal range. corticosteroid treatment to improve fetal maturity appeared successful as reported.3 however, according to necropsy and histopathology reports, pups’ lungs stopped maturing after birth, suggesting that glucocorticoids were insufficient to have a persistent influence on lung maturation to prevent respiratory distress syndrome after delivery.4 literature is limited, and based on our knowledge, only a few case reports described similar findings.¹,² this case highlighted that an addisonianlike crisis can be a differential for late pregnant dogs presenting with nausea, vomiting, and hyporexia in addition to conditions such as pregnancy toxemia and hypocalcemia. keywords: pregnancy, canine, hyponatremia, hypochloremia, glucocorticoids references 1. schaer m, halling kb, collins ke, et al: combined hyponatremia and hyperkalemia mimicking acute hypoadrenocorticism in three pregnant dogs. j am vet med assoc 2001;218:897-899. doi: 10.2460/javma.2001.218.897 2. kang jh, kim mj, cho mh, et al: hypoadrenocortical crisis-like transient hyponatremia and hyperkalemia in a near-term pregnant dog. j vet clin 2005;22:450-452. 3. regazzi fm, silva lcg, lucio cf, et al: influence of prenatal maternal corticosteroid therapy on clinical and metabolic features and pulmonary function of preterm newborn puppies. theriogenology 2017:97;179-185. doi: 10.1016/j.theriogenology. 2017.04.039 4. vanucchi ci, silva lcg, lucio cf, et al: prenatal and neonatal adaptations with a focus on the respiratory system. reprod dom anim 2012;47:177-181. doi: 10.1111/rda.12078 http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1111/rda.12329 https://doi.org/10.1177/030098588001700505 https://doi.org/10.1016/s0022-3468(75)80101-1 https://doi.org/10.2460/javma.2001.218.897 https://doi.org/10.1016/j.theriogenology.2017.04.039 https://doi.org/10.1016/j.theriogenology.2017.04.039 https://doi.org/10.1111/rda.12078 8 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 mastitis-induced laminitis suspected in a maiden fell pony mare stephanie walbornn, jose bras, hugo almonte, raul bras rood and riddle equine hospital in wellington, wellington, fl, usa mastitis is described as inflammation and infection of mammary tissue and is less commonly observed in mares. this is due to the smaller size and hidden nature of mammary gland in mares compared to other domestic species.1 mares afflicted with this disease are typically lactating mares that have recently been weaned. mares present systemically ill with clinical signs such as fever, anorexia, swollen mammary gland, and abnormal mammary secretions.1 sequelae to mastitis include fibrosis of the mammary gland, transmission of infectious agent to foal, foal malnutrition, and abortion. we describe a case of suspected mastitis-induced laminitis in a 6-year, fell pony maiden mare that was diagnosed with unilateral mastitis and laminitis. mare was presented for bilateral forelimb lameness. mare had been treated for mastitis for 2 weeks with nonsteroidal antiinflammatories and an antibiotic. at presentation, mare was lame and reluctant to bear weight on her forelimbs and swelling was observed in the left mammary gland. complete blood count and blood chemistry were normal. mammary gland was swollen, painful to the touch, and purulent discharge was milked from the left teat and submitted for aerobic culture and cytology. culture results revealed growth of methicillin-resistant staphylococcus aureus. metabolic testing was performed due to mare’s breed predisposition to metabolic disorders; results were within normal limits. ultrasonography of the udder revealed an abnormal area deep to the skin; due to the small size and location, drainage was not performed. radiographs of all feet were obtained and rotation of front coffin bones was confirmed. ice therapy, broad-spectrum antibiotics, nonsteroidal antiinflammatories, pentoxifylline, and probiotics were implemented as part of mare’s treatment plan, along with frequent milking of the affected udder and cold hosing. a venogram was performed of the front feet that revealed bilateral compromise to the circumflex artery with dorsal flip into the lamellar wedge area and the distal corium was shut down during initial weight bearing views. mare was placed in nanric ultimate cuff shoes with acs padding on the front feet and was scheduled with our veterinary podiatrist for re-evaluation in 30 days. our veterinary podiatrist and surgeon worked closely to manage the mare and keep her comfortable; treatments over the course of several months included therapeutic shoeing, a hoof wall resection, and bilateral deep digital flexor tenotomies. three months after the initial insult, udder was swollen, painful, and hot and an abscess was observed on ultrasonography. abscess was lanced, drained, and flushed over several days and antibiotic therapy was reinstated. mare has had intermittent swelling of the left mammary gland, with and without drainage. courses of various antibiotics have been utilized unsuccessfully to resolve the underlying infection. ultimately, a unilateral mastectomy was performed and tissue was submitted for histopathology. diagnosis was moderate, multifocal, chronic, nodular, pyogranulomatous mastitis with severe fibrosis. mare recovered well from the surgery and minimal swelling was present at the last recheck. keywords: mastitis, laminitis, methicillin-resistant staphylococcus aureus reference 1. canisso i, podico g, ellerbrock re: diagnosis and treatment of mastitis in mares. equine vet educ 2019;33:320-326. doi: 10.1111/ eve.13228 management of persistent overgrowth on routine contagious equine metritis cultures in a warmblood mare peyton draheim hagyard equine medical institute, lexington, ky, usa a 4-year, maiden warmblood mare, was presented for routine taylorella equigenitalis (t. equigenitalis), organism responsible for contagious equine metritis (cem) cultures after imported to united states from netherlands. as per united states department of agriculture (usda) requirements, cultures have to be obtained a total of 3 times, 3 days apart, prior to beginning 5 consecutive days of clitoral washing with chlorhexidine and packing with nitrofurazone. sites to be cultured include clitoral fossa, clitoral sinuses, and endometrium in nonpregnant mares. initial culture samples were obtained from the clitoral fossa and sinuses and submitted to the university of kentucky veterinary diagnostic laboratory. all cultures were unable to be assessed for growth of t. equigenitalis due to overgrowth of other bacteria. after discussion of results and options with the state veterinarian, it was decided to repeat cultures after rinsing clitoris with distilled white vinegar. these cultures again were overgrown with bacteria and were not useful for cem evaluation. a routine aerobic clitoral culture sample was then submitted to the laboratory at hagyard equine medical institute to identify and determine the sensitivity of organism(s) contributing to the overgrowth to guide a targeted treatment plan, with state veterinarian’s approval. culture revealed heavy growth of pseudomonas aeruginosa (p. aeruginosa), sensitive to polymyxin b. clitoris was then treated topically for 5 consecutive days with cleansing with dilute betadine scrub and application of polymyxin b ointment compounded at the hagyard pharmacy. as per usda requirements, 21 days elapsed between the final topical treatment and restarting culture procurement. once again, these clitoral cultures had overgrowth of bacteria. after further discussion with state veterinarian and client, a more robust diagnostic approach was pursued. samples were obtained from endometrium, vagina, and clitoris for routine culture and sensitivity. endometrial culture was obtained via low-volume sterile lavage 1 day after instillation of n-acetylcysteine. endometrial sample yielded scant beta streptococcus species, the vaginal sample grew scant staphylococcus aureus and scant p. aeruginosa, and clitoral sample grew moderate p. aeruginosa. mare was then started on an oral probiotic to promote systemic microflora homeostasis. local treatment was initiated as follows for 5 consecutive days: sterile uterine lavage and infusion of 2 grams of ampicillin, application of medihoney® to the vaginal vault, and application of compounded polymyxin b ointment to the clitoris. systemic treatment with ciprofloxacin and probenecid was initiated concurrently for a total of 14 days. all antimicrobial selection was based on sensitivity results. as per usda guidelines, cem cultures can be procured 21 days after the final topical treatment or 7 days after cessation of systemic antimicrobial treatment. first cem culture sample obtained after this treatment period was again overgrown. after further discussion with state veterinarian, another culture was performed the following day after a http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1111/eve.13228 https://doi.org/10.1111/eve.13228 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 9 more vigorous application of distilled white vinegar and sterile water to clitoris. all subsequent cultures were negative for overgrowth and for t. equigenitalis and after 5 days of clitoral washing and packing, the mare was released from quarantine. keywords: mare, contagious equine metritis, culture, overgrowth transcervical approach for treatment of pyometra in a snow leopard (panthera uncia) julie barnes,a lindsey vansandt,a jennifer niederlander,b bon mazurek,b kurt volleb acenter for conservation and research of endangered wildlife, cincinnati zoo & botanical garden, cincinnati, oh, usa bbuffalo zoo, buffalo, ny, usa pyometra is a serious condition characterized by accumulation of purulent material in the uterus, secondary to bacterial infection. although pyometra has been described in big cats, medical management has not been widely reported. an 8-year, nulliparous snow leopard, underwent exogenous hormonal stimulation and laparoscopic oviductal artificial insemination (ai). on day 60 postai, moderate amount of purulent serosanguineous vaginal discharge was observed, and clavamox and enrofloxacin were initiated. oral misoprostol treatment (200 µg sid) was given on day 65 postai. snow leopard underwent immobilization for a comprehensive reproductive health examination on day 68 postai. transabdominal ultrasonography identified small pockets of hypoechoic fluid in the uterine body and right uterine horn. transvaginal endoscopic examination with a 43 cm rigid 9.5-13.5fr hysteroscope revealed mild purulent discharge within the vaginal lumen, making visualization difficult. a 5-fr cannula was passed through the cervix, and purulent discharge was released from the uterus. ten ml of sterile saline was flushed into the uterus and then aspirated to recover as much fluid as possible; this was repeated 2 more times. aspirated volume initially appeared as a light pink, purulent fluid that transitioned to a mild cloudy consistency by the third series. intramuscular dinoprost (3.4 mg) was given and transcervical cannula remained in place for 28 minutes postinjection. peak back flow occurred 17-21 minutes postinjection. after completing the examination, a 200-µg misoprostol tablet was dissolved in 1 ml sterile saline and given intravaginally. subcutaneous aglepristone was given during the examination (16.8 mg). however, retrospective serum progesterone concentrations (0.4 ng/ml) revealed that the snow leopard was not in luteal phase at examination. a second dose of dinoprost (3.4 mg) was voluntarily given the next day, and a partial dose (due to patient compliance) was given the following day. misoprostol, enrofloxacin, and clavamox were continued 3 days after dinoprost treatment. two-weeks after the initial evaluation, snow leopard was reexamined and was in estrus. uterus and uterine horns (from body to tip) were examined via ultrasonography and no hypoechoic pockets of fluid were noted. a small amount of serosanguineous purulent discharge was present in her vulvar area. during vaginal endoscopy, vestibule appeared to be the source with a minimal amount of discharge, whereas vaginal canal revealed normal pink mucosa with a clear sheen. due to narrowing of the vaginal lumen (reported in domestic cats during estrus), the scope could not be advanced to cervix level. vestibule sample was cultured and treated per the sensitivity results with an additional 10 days of marbofloxacin, after which, the patient recovered uneventfully. due to the disadvantages associated with ovariohysterectomy in large cats, medical management should be considered when treating pyometra. outcome of this procedure provided evidence that combination of antibiotics, prostaglandins, and transcervical therapy can be used to manage the reproductive health of female snow leopards in a zoo-based setting. furthermore, it also increases the chance of identifying animals with pathology linked to genetics. keywords: pyometra treatment, transcervical endoscope, snow leopard mycobacterium avium subsp. hominissuis induced abortion in a mare babiche heil,a,b angela begg,c wendy perriam,a kristen todhunterd agundy veterinary services, scone, nsw, australia bdepartment of veterinary clinical sciences, washington state university, pullman, wa, usa cdiagnostic laboratories pty ltd, new lambton, nsw, australia dlaverty vetnostics laboratory, macquarie park, nsw, australia equine bacterial abortion can be caused by opportunistic organisms ascending from vagina, hematogenous invasion or unknown as in cases of nocardioform placentitis. the result is placental infection, and potentially fetal death. this is the first report of abortion due to placental and fetal infection with mycobacterium avium subsp. hominissuis (mah) in australia. although rare, abortion caused by nontuberculous mycobacterium spp. has been reported in japan (mah), usa (m. avium complex and m. avium runyon group iv), canada (m. avium complex), and australia (m. avium subsp. terrae).1 furthermore, a role for mycobacterium spp. in cases of nocardioform placentitis has been suggested.2 a multiparous, 7-year, thoroughbred mare, was exported for livecover from australia to hokkaido island, japan and returned on day 108 of pregnancy. mare developed premature mammary gland development on day 134 with unremarkable changes to fetoplacental unit (via transrectal and transabdominal ultrasonography). treatment with intramuscular altrenogest (0.3 mg/kg, once every 7 days) and oral pentoxifylline (8.5 mg/kg, twice daily) was initiated and fetoplacental health was regularly monitored via transrectal and transabdominal ultrasonography. treatment was discontinued when the clinical signs subsided. on day 229 of pregnancy, mild placental edema at the cervical star was noted. given the mare’s history, treatment was started with intramuscular altrenogest (0.3 mg/kg, once every 7 days), oral pentoxifylline (8.5 mg/kg twice daily), oral phenylbutazone (2 grams, once daily), and oral sulfadimidine/trimethoprim (25 mg sulfadimidine/5 mg trimethoprim/kg, twice daily) that normalized all parameters after 5 days of therapy. pentoxyfylline, phenylbutazone, and sulfadimidine/ trimethoprim were discontinued but altrenogest treatment http://dx.doi.org/10.58292/ct.v16.11054 10 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 continued. on day 243, mare aborted an emaciated fetus with fetal membranes. grossly, cervical star region of the chorioallantois was edematous with well circumscribed area of caseous exudate. umbilical cord (58 cm) was thickened and there was prominent perivascular edema of the amnion at the point of umbilical cord attachment. pinpoint white foci were noted in fetal liver and white nodules within the caudal lung lobes. final diagnosis based on histopathologic examination was necrotizing granulomatous hepatitis, splenitis and fetal pneumonia associated with a mild multifocal granulomatous amnionitis, funisitis, and placentitis with intralesional modified ziehl-neelsen positive bacilli; mah was identified using 16s rrna pcr and genome sequencing. endometrial biopsy collected 6 months after abortion indicated a severe chronic active lymphoplasmacytic neutrophilic, granulomatous endometritis with moderately extensive early fibrosis. a ziehl-neelsen and fite stain failed to reveal any acid-fast bacteria within the endometrium. low volume lavage prior to breeding revealed gram negative bacteria and neutrophils on cytology and heavy growth of klebsiella oxytoca and proteus mirabilis was isolated from the fluid. extended culture for mycobacterium species remained negative. based on sensitivity testing, the mare was treated with ceftiofur and tris-edta. mare subsequently conceived and carried to term. origin of mah in this case, is uncertain, but the mare could have acquired the organism in japan during her residence for live-cover, where mah has previously been described as a cause of equine abortion. the bacteria could have remained dormant until unknown favourable conditions allowed mah to become opportunistic within the fetal membranes, with subsequent inflammation and fetal death. keywords: equine, abortion, placentitis, fetus, mycoplasma avium subsp. hominissuis references 1. sano y, matsuda k, osaki k, et al: systemic mycobacteriosis in an aborted thoroughbred fetus in japan. j vet med sci 2014;76:16171621. doi: 10.1292/jvms.14-0276 2. van heule m, el-sheikh ali h, schlesener c, et al: the placental microbiome during nocardioform placentitis. j equine vet sci 2023;125:219. doi: 10.1016/j.jevs.2023.104789 stranguria due to cervical sarcoma in a nubian doe ahmed mosallam, ilaria colombo, michela ciccarelli, babiche heil, muhammad waqas department of veterinary clinical science, college of veterinary medicine, washington state university, pullman, wa, usa female reproductive tract tumors in animals may include vaginal leiomyosarcoma, vaginal fibrosarcoma, uterine lymphosarcoma, adenocarcinoma, and leiomyosarcoma. literature on reproductive tract tumors in goats is limited, with sporadic reports of uterine and cervical leiomyomas. approximately 10-50% of genital tumors originate from smooth muscle, of which only 10% are considered malignant. a 7-year, 55 kg maiden nubian doe, was referred for evaluation of a vaginal mass and dysuria. before presentation, owner noticed that the doe had a mucoid, slightly hemorrhagic vulvar discharge, and weight loss. at presentation, the goat’s vitals were within normal limits. however, she had stranguria and abdominal tenderness. abdominal palpation indicated a mass in the caudal abdomen that was confirmed via transrectal and transabdominal ultrasonography. on transabdominal ultrasonography, a 10 cm mass extending from cervix to uterus, compressing urinary bladder neck, was observed. vaginal endoscopy revealed a multinodular, firm and pink-blueish mass extending from caudal vagina to cervix. cervix could not be visualized. vagina was hyperemic and a milky mucoid discharge was present. computed tomography (ct) was performed to determine the lesion’s extent and whether surgery was an option. ct scan revealed a multilobular, heterogeneously enhancing mass with punctate foci of mineralization in the intrapelvic vagina and uterine body, measuring 16.3 (length) x 11.4 (width) x 11.0 cm (height). large vessels branching from the uterine arteries were observed entering mass center from its cranial aspect. there was no evidence of metastasis in ct. however, there was evidence of parasitic cysts or abscessation in the lungs. considering the complex vascularization of the mass, size and location, risks associated with anesthesia, poor to grave prognosis and the invasiveness of surgery, owner opted for humane euthanasia. histopathology of the cervical mass revealed cervical sarcoma, cystic endometrial hyperplasia, and endometritis. presumptive diagnosis was cervical leiomyosarcoma based on tumor location and tissue of origin. leiomyosarcomas are rare malignant neoplasia that develop in smooth muscle of the reproductive tract. they usually exhibit a low-grade malignancy growth pattern, slow invasion, and rarely metastasize.1 diagnosing genital tract tumors in goats is important in usa, where many people own goats as companion animals. although tumors of the female reproductive tract in small ruminants are rare, they can have substantial health implications. this case demonstrated the complex nature of these tumors and highlighted the challenges in diagnosis and treatment. this case also underlined the importance of using vaginoscopy in the diagnosis of cases that present with abnormal vaginal discharge. it allowed direct visual examination of the vaginal and cervix and facilitated the detection of abnormalities that might otherwise be missed. keywords: goat, leiomyosarcoma, neoplasia, stranguria, vagina, cervix, reference 1. whitney km, valentine ba, schlafer dh: caprine genital leiomyosarcoma. vet pathol 2000;37:89-94. doi: 10.1354/vp.37-1-89 http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1292/jvms.14-0276 https://doi.org/10.1016/j.jevs.2023.104789 https://doi.org/10.1354/vp.37-1-89 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 11 competitive poster abstracts sperm chromatin structure assay use in diagnosis of canine infertility tiffany hoffman,a morgan agnewb amasters of comparative biomedical sciences, college of veterinary medicine, university of georgia, athens, ga, usa bagnew veterinary reproductive services, philadelphia, pa, usa a 2-year, maiden pembroke welsh corgi dog, was examined prior to planned artificial insemination. physical examination was unremarkable. serial progesterone measurements were used to estimate the day of lh surge. dog was bred on days 4 and 6 postlh surge. transabdominal ultrasonography (30 days postlh surge) revealed 6 anembryonic vesicles. owner elected to medically induce abortion with aglepristone injections, while empirically treating with enrofloxacin (8 mg/kg, once daily). follow up ultrasonography (by referring veterinarian) confirmed successful evacuation. at next estrus, dog was presented for repeated breeding management; same stud dog was used. thirty days later, dog was presented with pu/pd. transabdominal ultrasonography revealed 3 anembryonic vesicles, 1 reabsorption, and 2 fetuses had visible heart beats. complete blood count, chemistry, and urinalysis unremarkable. cesarean surgery (planned) was performed; submitted uterine biopsy tissue was normal. singleton pup died 3 days postdelivery from an unknown cause. due to the high number of anembryonic vesicles, a genetic cause for the infertility was suspected. an embark panel was previously submitted for the dog that was clear, except for a coefficient of inbreeding (coi) of 42%, with the breed average being 22%. this was considered a possible contribution to the early embryonic loss, and ultimately the dog was retired from breeding. stud dog (proven 11-year, pembroke welsh corgi) had a coi of 14%. a fresh semen sample was flash frozen and submitted to scsa diagnostics, brookings sd (sperm chromatin structure assay [scsa] measures dna fragmentation index [dfi], percentage of sperm containing measurable dna damage along with a high dna stainability [hds], and percentage of sperm with immature, abnormal tertiary chromatin structure). the dfi for this dog was 26.42 and 27.80% (mean 27.1) with an hds of 5.20 and 5.50% (mean 5.4). although the threshold for the dfi of fertile versus infertile dogs has not yet been determined, the mean value is 5.9% compared to subfertile dogs (13.2%).1 with a higher dfi, there is an increased risk of lethal early embryonic defects. the high dfi in this dog likely contributed to the reproductive failure observed. this case demonstrated the use of scsa to aid in diagnosing male canine infertility. keywords: pembroke welsh corgi, sperm chromatin structure assay, dna fragmentation reference 1. ali hassan h, domain g, luvoni gc, et al: canine and feline epididymal semen-a plentiful source of gametes. animals 2021;11:2961. doi: 10.3390/ani11102961 vulvar hemorrhage in a nulliparous aged arabian mare patricia crigler,a lauren paschb aatlantic equine clinic, egg harbor, nj, usa brhinebeck equine llp, rhinebeck, ny, usa a 24-year, nulliparous arabian mare (493.2 kg [1,085 lb]), was presented to rhinebeck equine facility for evaluation of acute onset of hemorrhagic vulvar discharge. mare was bright, alert, and responsive with vital parameters within normal limits. perineal blood staining was evident and the mare frequently passed small volumes of serosanguinous discharge from the vulva, independent of urination. point of care blood work was within normal limits. transrectal palpation revealed no detectable abnormalities within the urinary bladder or the uterus; transrectal ultrasonography confirmed a moderately full urinary bladder and a diestrous uterus, with a corpus luteum on left ovary, no uterine edema, and multiple small follicles on both ovaries. there was a mild echogenic fluid (3-5 cm) collection within the uterine lumen and an irregular spheroid structure (~ 5 x 8 cm) was identified in the mid-uterine body. vaginal speculum examination revealed frank blood in the vagina that appeared to originate from cranial to the cervix. uterus was distended with 2 liters of sterile lactated ringer’s solution. repeat transrectal ultrasonography confirmed that the mass was pendulous and appeared to communicate with the endometrium (cranial to uterine body) via a short stalk. at this time the differential diagnoses included hematoma, abscess, or neoplasia. hysteroscopic examination was performed with a 1.5-meter endoscope, confirming the presence of a hemorrhagic and heterogeneous mass within the cranial uterine body close to the base of the right uterine horn and in direct communication with the endometrium via a vascular stalk. sampling of the mass via endoscope biopsy channel was unsuccessful due to its markedly friable nature. cloprostenol sodium [estrumate, merck animal health] was given to induce luteolysis and facilitate cervical relaxation in anticipation of transcervical mass removal. next day, mare was sedated (intravenous detomidine [0.01 mg/kg] and intravenous butorphanol [0.01 mg/kg]), the perineum was cleaned, and a sterile sleeved hand was passed into the mare’s vagina. gentle digital manipulations were used to dilate the cervix; however, complete dilation was unsuccessful. a caudal epidural was performed with 2% lidocaine (3 mg/ kg) and detomidine (0.02 mg/kg) and a 1.5-meter endoscope was again passed transcervically into the uterus. after insufflating the uterus, a disposable electrosurgical snare [snaremaster 20 mm, olympus medical systems corporations] was passed through the scope’s biopsy channel and tightened around the base of the stalk; electrocautery was used to ligate and provide hemostasis. having freed the mass from its endometrial attachment, gentle transrectal manipulations were used to break off portions of the mass to allow transcervical removal, followed by copious uterine lavage with sterile lactated ringer’s solution. mass fragments were submitted for histopathology that revealed adenocarcinoma. although a specific diagnosis was not possible, this report is valuable to practitioners due to its http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.3390/ani11102961 12 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 thorough approach to management of an intrauterine mass, and most importantly, identification of the condition based on clinical signs. removal method adopted possibly failed to achieve complete margins; however, it provided adequate resolution of mare’s clinical signs. keywords: mare, vulvar hemorrhage, uterine mass, electrocautery, adenocarcinoma. incidental masses during a routine transvaginal reproductive examination in a grizzly bear (ursus arctos horribilis) julie barnes,a zoli gyimesi,b erin currya acenter for conservation and research of endangered wildlife, cincinnati zoo & botanical garden, cincinnati, oh, usa blouisville zoo, louisville, ky, usa an 18.5-year, wild-caught multiparous brown bear, underwent routine reproductive examination prior to shipment. at last year’s examination, a multinodular (cauliflower-like) soft tissue mass (2.5 x 1.9 x 0.6 cm) was observed on the right side of the ventral vulvar mucosa and an incisional biopsy was submitted for histopathology. results revealed benign collagenous expansile lesion suggestive of a polyp. the goal of this year’s procedure was to perform a reproductive examination to characterize and document reproductive pathology among ursidae. vulva measured 76 x 32 x 61 mm (length x width x protrusion). vulvar growth appeared unchanged on the ventral side and measured 2.4 x 1.7 cm. a cotton swab moistened in pbs was passed through a short (~ 10 cm) speculum to sample vaginal epithelial cells. swabs were rolled on glass slides, fixed and stained to differentiate cell types using a papanicolaou stain kit. cytology analysis revealed 4.75% parabasal cells, 43% intermediate cells, 24.75% superficial cells, and 27.5% anuclear superficial cells. aerobic and anaerobic cultures were obtained from perivulvar region, and caudal and deep vagina. all 3 areas cultured klebsiella sp and pseudomonas aeruginosa with no anaerobic growth. perivulvar region also grew aeromonas punctata and enterococcus faecalis, whereas caudal and deep vaginal cultures grew proteus hauseri and staphylococcus schleiferi. vaginal endoscopy with a 43 cm rigid 9.5-13.5fr transcervical insemination (tci) endoscope (karl storz, tuttlingen, germany) with manual insufflation was performed. scope was advanced through the vestibule until the vestibulovaginal junction demarcation. normal light pink mucosa was noted in the vestibule with minimal horizontal striation. a growth protruding from the ventral right vaginal wall was noted adjacent to the urethral opening. it was estimated to be 3-5 mm and was similar in appearance to the vulvar mass. no samples of the mass were taken. vaginal mucosa appeared slightly darker pink with horizontal folds. scope was advanced through the vaginal canal until cervix was visualized. cervix appeared as a normal dorsal out pocketing with pink mucosa at the cranial end of the vaginal canal, and no excessive mucus or abnormalities were noted. distance from the vulva to vestibulovaginal junction was 17 cm and from the vulva to cervical os was 24 cm. bear recovered uneventfully and with no clinical issues related to the masses at this time. although brown bears are not bred for genetic conservation in zoos, they offer valuable insights for comparison with threatened species like polar bears, sloth bears, and andean bears. reproductive examination to collect baseline data on reproductive anatomy and pathology may help assess factors influencing reproductive success or failure. crucial for wildlife conservation, these examinations offer insights into overall health, reproductive trends, and potential threats faced by bear populations. this knowledge aids in developing targeted conservation strategies and contributes to maintaining genetic diversity and sustainable management of bear species, even if not bred in captivity. keywords: transcervical endoscope, reproductive exams, brown bears, vaginal mass bilateral cryptorchidism in a miniature horse and a mustang ilaria colombo, ahmed mosallam, jorge sanclemente, babiche heil, muhammed waqas, michela ciccarelli department of veterinary clinical science, college of veterinary medicine, washington state university, pullman, wa, usa cryptorchidism is the most common disorder of sex development in males. in horses, the prevalence ranges from 2 to 12%, with unilateral cryptorchidism more common (85.795.2%) than bilateral (4.8-14.3%).1,2 this condition is easily diagnosed in unilateral cryptorchid stallions and horses with a good history. however, it becomes challenging in presumed geldings presented with stallion-like behavior. this report describes 2 cases of bilateral cryptorchid stallions, a miniature horse and a mustang, purchased as geldings but later exhibited stallion-like behavior, and were referred for localization of testes and cryptorchidectomy. endocrine evaluation was performed by the referring veterinarian. miniature horse’ serum concentrations of estrone sulfate and testosterone were 25.94 ng/ml (geldings < 0.1 ng/ml; cryptorchid 35-60 ng/ml; intact stallions 140-200 ng/ml) and < 10 pg/ml (geldings < 50 pg/ml; cryptorchid 100-500 pg/ml; intact stallions 800-2000 pg/ml) respectively. mustang, testosterone concentrations were 987.6 pg/ ml (after hcg stimulation). in the miniature horse, transcutaneous inguinal ultrasonography revealed a 3 cm soft tissue structure, consistent with testis and located in the right abdomen dorsal to the vaginal ring. left testis was not identified and transrectal ultrasonography was not performed due to horse’s size. in the mustang, both transrectal and transabdominal ultrasonography revealed 2 soft tissue structures (3 cm), consistent with testes, located in the abdomen dorsal to the respective vaginal rings. before surgery, a blood sample was collected from horses for antimüllerian hormone (amh). serum amh concentrations were 12.2 ng/ml in the miniature horse and 10 ng/ml in the mustang (geldings  <  0.15 ng/ml; cryptorchid and intact stallions > 0.15 ng/ml). laparoscopic cryptorchidectomy was performed. miniature horse underwent general anesthesia and a laparoscopically assisted technique in dorsal recumbency in trendelenburg position. the mustang’s surgery was performed standing using a flank laparoscopic approach, and testes were removed via morcellator.3 testes were successfully visualized above the vaginal rings and removed. histopathology had the following: no evidence of spermatogenesis in seminiferous tubules with enlarged lumen, and vacuolization (consistent with testicular http://dx.doi.org/10.58292/ct.v16.11054 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 13 degeneration due to abnormal thermoregulation). these cases highlighted the importance of endocrine testing to confirm cryptorchidism in presumed geldings exhibiting stallion-like behavior. testosterone estimation may not be reliable (sensitivity 85% and specificity 91%) due to diurnal fluctuations, seasonal effects, and age-related variations, making stimulation by hcg or gnrh necessary to improve diagnostic accuracy. estrone sulfate sensitivity is 88%, and specificity is 84%, and it is not accurate in horses < 3 years of age. amh sensitivity and specificity have not been determined; however, it is considered the best biomarker in cryptorchid horses because only 1 blood sample is required, and the serum concentration is not affected by hourly or seasonal variation. furthermore, these cases suggested that bilateral cryptorchidism is likely underdiagnosed in horses sold as geldings and not exhibiting stallion-like behavior, placing them at risk of developing retained testicular tumors later in life. keywords: stallion, gelding, cryptorchid, antimüllerian hormone, laparoscopy references 1. hayes hm: epidemiological features of 5009 cases of equine cryptorchism. equine vet j. 1986;18:467-471. doi: 10.1111/j.20423306.1986.tb03692.x 2. arroyo e, tibary a: cryptorchidism in stallions. clinical theriogenology 2023;15:31-51. doi: 10.58292/ct.v15.9648 3. sassot ln, ragle ca, farnsworth kd, et al: morcellation for testes extraction in horses undergoing standing laparoscopic cryptorchidectomy. can vet j 2017;58:1215-1220. vaginal polyp prolapse in a queen lily lewis,a hannah maxwell,a scarlett sumner,a rachel neto,b robyn wilborna adepartment of clinical sciences, adepartment of pathobiology, college of veterinary medicine, auburn university, auburn, al, usa a 9-year, domestic shorthair queen, was presented for evaluation of tissue acutely protruding from the vulva and lethargy. her last estrous cycle was 3-4 months prior, and she was reportedly primiparous. on presentation, the queen was systemically stable. intake diagnostics included pcv/ts (42% and 7.0 g/dl, respectively), blood glucose (304 mg/dl), and blood lactate (2.9 mmol/l). these results supported the clinical findings of mild dehydration, in-hospital stress and local urogenital tissue hypoxia. physical examination revealed a spherical, turgid protrusion, of hyperemic tissue measuring ~ 6 x 4 cm appearing from the vulva. interestingly, the tissue appeared to have a lumen, and a small circular region of ischemia was identified at the caudal-most aspect of the protruding tissue. quick assessment via ultrasonography noted no free fluid within the prolapsed structure and an intact urinary bladder in situ. comprehensive bloodwork and abdominal imaging were performed. complete blood count revealed mild thrombocytopenia (deemed artifact), lymphopenia, and monocytosis (stress leukogram). chemistry panel revealed mild hypoalbuminemia (2.67 g/dl), elevated creatinine kinase, and mild electrolyte derangements and no evidence of urinary obstruction (e.g. hyperkalemia or azotemia). diagnostic imaging included transabdominal ultrasonography and abdominal radiographs. right uterine horn was minimally dilated with anechoic fluid, beginning at the bifurcation. a cystic structure (~ 1 cm in diameter) associated with the cervix was identified near the pelvic inlet, dorsal to the urethra. urinary bladder and urethra were noted to be moderately distended, and echogenically stippled urine was appreciated within the bladder lumen. due to the inability to reduce the prolapsed and necrotic tissue, the queen underwent ovariohysterectomy and vaginal tissue resection. uterus, ovaries, and prolapsed vaginal tissue were submitted for histological evaluation. maturing oocytes and stroma were identified in the ovaries that were otherwise unremarkable. cystic endometrial hyperplasia was identified in uterine horns. vaginal tissue was hyperplastic in nature, and an edematous fibrovascular polyp was identified. there were no complications postoperatively and the patient was able to urinate and defecate normally after the procedure. fibrovascular vaginal polyps are uncommon, benign neoplastic masses that are more often reported in middle-aged to older dogs, with limited case reports published in the queen.1,2 often incidental findings, these can go undetected or be managed conservatively. prolapsed hyperplastic vaginal tissue is more common, and a sequela to estrogen dominance during estrus or prepartum. in this case, the presence of follicular tissue may have contributed to the hyperplastic vaginal tissue identified, resulting in prolapse of the polyp. another consideration is that the polyp reached a certain threshold in size, leading to discomfort and increased abdominal press. surgical excision in an attempt to preserve normal anatomy and fertility could be considered in the event that these are identified at an early stage, prior to prolapse and tissue necrosis. thus, though less common, a vaginal polyp is an important differential to consider when presented with prolapsed tissue from the vulva in a dog or cat. keywords: prolapse, polyp, hyperplasia references 1. saitoh y, aikawa t, miyazaki y: complete vaginectomy via a ventral approach with pelvic osteotomy in a cat. can vet j 2022;63:695-700. 2. ortega-pacheco a, gutierrez-blanco e, et al: common lesions in the female reproductive tract of dogs and cats. vet clin north am small anim pract 2012;42:547-559. doi: 10.1016/j. cvsm.2012.01.011 http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1111/j.2042-3306.1986.tb03692.x https://doi.org/10.1111/j.2042-3306.1986.tb03692.x https://doi.org/10.58292/ct.v15.9648 https://doi.org/10.1016/j.cvsm.2012.01.011 https://doi.org/10.1016/j.cvsm.2012.01.011 14 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 use of reproductive tract extracellular vesicles to capacitate bull sperm in vitro brandi dunn,a mindy meyers,b marcie jung,a james graham,a fiona hollinsheadb adepartment of biomedical sciences, bdepartment of clinical sciences, college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co, usa sperm cryopreservation induces plasma membrane damage and premature acrosome exocytosis resulting in reduced sperm longevity, viability, and fertilizing potential (in vivo and in vitro [ivf]). extracellular vesicles (evs) have a physiological role in sperm function through posttesticular modification of sperm, yet that role is not completely understood. objective of this study was to determine if evs from the reproductive tract can affect sperm physiology and fertility. we collected evs from oviducts and accessory sex glands and from in vitro grown 3-d organoid cultures that were stimulated with steroid hormones to mimic in vivo estrus conditions. we hypothesized that evs collected from seminal plasma (sp) and in vivo-derived diestrous oviductal cells (do) would delay sperm capacitation, whereas evs collected from in vivo-derived estrous oviductal cells (eo), and in vitro oviductal organoids (oo) would enhance capacitation. frozenthawed sperm from 6 bulls with proven fertility with ivf were treated with: 1. no evs (control); 2. sp; 3. do; 4. eo; 5. oo; or 6. dilauroylphosphatidylcholine (pc12; + control) after thawing. samples were incubated at 38ºc and assessed at 0.5, 1, 2, and 4 hours posttreatment for motility using microscopy/computer-assisted sperm analysis system (casa), viability (pi), acrosome integrity (fitc-pna), and tyrosine phosphorylation (py20 antibody) using flow cytometry. sperm fertilization potential was determined using ivf. data were analyzed using anova. addition of oo and eo resulted in higher percentages of live-acrosome reacted sperm at 4 hours (8 ± 1 and 6 ± 1, respectively), than sperm treated with sp (2 ± 1; p  <  0.05). additionally, a higher percentage of sperm exhibited capacitation changes at 4 hours (assessed by tyrosine phosphorylation) when treated with oo or eo (67% ± 3 and 55% ± 3, respectively) than control sperm (51% ± 3, p < 0.05). sperm treated with oo tended to fertilize more oocytes in vitro (76%) than control sperm (67%; p = 0.13). this study indicated that oviductal evs enhanced sperm capacitation, and sp evs improved frozen-thawed sperm longevity. these preliminary findings have positive implications to improve fertility rates in commercial bovine assisted reproductive technology and artificial insemination breeding programs. keywords: sperm, cryopreservation, extracellular vesicles, organoids http://dx.doi.org/10.58292/ct.v16.11054 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 15 equine abstracts novel management of chronic pyometra in the mare georgiana crisci, lauren pasch rhinebeck equine, rhinebeck, ny, usa a 24-year, standardbred mare, with an unknown breeding history was presented on farm with mild colic signs and purulent vulvar discharge. transrectal palpation and ultrasonography performed by the referring veterinarian revealed an enlarged uterus filled with fluid, and the mare was referred for further management. on presentation, the mare was bright and alert with normal vital parameters and point of care blood work within reference ranges. purulent discharge was evident on the mare’s perineum, tail and hindlegs. transrectal palpation and ultrasonography revealed a distended uterus with > 15 cm echogenic fluid. intrauterine catheter was passed through the cervix into the uterus; the resulting effluent was thick and diluted as needed with lactated ringer’s solution to facilitate drainage. approximately 50 liters of thick off-white effluent was obtained. mare was treated daily with uterine lavage followed by intrauterine infusion with ceftiofur sodium (ceftiflex, aspen veterinary resources, loveland, co) and intracervical topical steroids (animax, dechra, overland park, ks) to reduce adhesion formation. purulent effluent became more hemorrhagic as treatments continued; hysteroscopy was performed and the endometrium was diffusely inflamed, ulcerated, and hyperemic. mare was discharged on oral sulfadiazine/trimethoprim (equisul, aurora pharmaceutical, northfield, mn) and flunixin meglumine (covetrus, portland, me) and was scheduled to return in 3 4 weeks for further treatment after allowing the endometrium to heal. due to noncompliance, 11 months passed before the mare was presented for reevaluation. mare’s tail was clean and no vulvar discharge was appreciated. transrectal palpation and ultrasonography confirmed a distended uterus with > 10 cm of moderately echogenic fluid. manual cervical examination confirmed the presence of transcervical adhesions that had effectively sealed the cervix closed. careful manual dissection through adhesions and scar tissue was performed until a finger could pass through external os. once again, uterine evacuation, culture, lavage, and antimicrobial infusion were performed serially for 5 treatments. in order to maintain cervical patency and prevent ongoing adhesion formation, an intracervical stent was made with a short, 14 mm internal diameter silicone tracheostomy tube (bivona, gary, in) placed through the cervix, with the cuff inflated at the internal os and the tracheostomy tube flange at the external os. mare was examined at gradually increasing intervals to ensure that the cuff remained inflated and the stent in place. after 3 months, the stent was expelled. manual examination confirmed healing around the stent, and ultrasonography confirmed the absence of intrauterine fluid. without an inflammatory nidus passing through the cervix, it has remained patent for 10 months thus far. chronic pyometra in the mare is an uncommon but potentially significant occurrence, typically secondary to outflow (i.e. cervical) obstruction. this condition may be seen following dystocia without appropriate after care. several options have been described for the management and treatment of pyometra, including bilateral ovariectomy to induce an anestrous state in which the cervix may be moderately relaxed so as to allow drainage, cervical wedge resection1 or a cervicectomy using a fetotome.2 in this case, the mare’s financial value, owner compliance, and cervical anatomy made her a poor candidate for surgical intervention. keywords: mare, pyometra, cervix, adhesions references 1. arnold ce, brinsko sp, varner dd: cervical wedge resection for treatment of pyometra secondary to transluminal cervical adhesions in six mares. j am vet med assoc 2015;246:1354-1357. doi: 10.2460/javma.246.12.1354 2. brinsko sp: amputation of the mare’s cervix for the treatment of pyometra. clinical theriogenology 2023;15:27-30. doi: 10.58292/ ct.v15.9625 incubation of frozen/thawed stallion sperm in a lactate-modified whitten’s medium induced molecular changes associated with the acquisition of fertilizing capacity camilo hernández-avilés, charles love, luisa ramírez-agámez college of veterinary medicine and biomedical sciences, texas a&m university, college station, tx, usa molecular events associated with the acquisition of fertilizing competence (capacitation) include protein tyrosine phosphorylation (pty), acrosomal exocytosis (ae), and relocation of spermoocyte interaction proteins. fresh stallion sperm undergo spontaneous ae while retaining viability (ae/ viable),1,2 when incubated under presumed capacitating conditions (presence of calcium, bicarbonate, and albumin) in a medium with lactate as the only energy substrate (lactate-modified whitten’s medium [lac-mw]). following incubation in lac-mw, a time-dependent change in the occurrence of pty has been reported in fresh stallion sperm.2 in addition, the percentage of ae/viable sperm in fresh and frozen/thawed semen has been reported to be similar.2 whether the occurrence of ae/viable sperm in frozen/thawed stallion sperm is temporally related to changes such as pty or relocation of the sperm-oolemma fusion protein izumo1 is unknown. we processed frozen/thawed semen from 5 fertile stallions by density gradient centrifugation (40% silica particle solution), diluted to 30 x 106 sperm/ml in lac-mw, and incubated at 38.2°c in 5% co2 for up to 6 hours. at 2, 4, and 6 hours of incubation, aliquots were analyzed for ae/viable sperm via flow cytometry, whereas pty and izumo1 expression were http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.2460/javma.246.12.1354 https://doi.org/10.58292/ct.v15.9625 https://doi.org/10.58292/ct.v15.9625 16 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 examined by immunofluorescence. data were analyzed using the mixed linear model. statistical significance was set at p  <  0.05. the mean percent ae/viable sperm was lower (p < 0.05) at 2 hours than at 4 or 6 hours (17, 31, and 44%, respectively). during incubation in lac-mw, the immunofluorescence signal locations generally changed from a caudal position on the sperm to acrosome for both pty and izumo1 and were specifically characterized as follows: pty transitioned from the equator, midpiece and principal piece (location i [l1]) to the acrosome, equator, and midpiece (location ii [lii]); and izumo1 transitioned from the equator (location a [la]) to acrosome region (location b [lb]). at 2 hours, the mean percent of sperm displaying pty-li was higher (p < 0.05) than lii (43 versus 12%), whereas by 4 and 6 hours, the percent of sperm displaying lii was higher (p < 0.05) than li (28 and 22%, respectively). at 2 hours, the mean percent of sperm displaying izumo1-la was higher (p < 0.05) than for lb (32 versus 11%). by 4 and 6 hours of incubation, the mean percentages of izumo1-lb were 34 and 41%, respectively. these values were higher (p < 0.05) than those observed at 2 hours. overall, the sequence of molecular events in frozen/thawed stallion sperm incubated in lac-mw was characterized by the movement of pty from the principal piece to the acrosome, followed by an increase in ae/viable sperm, and then by izumo1 relocation to the acrosome. these molecular changes resembled to similar sequence of events described for capacitation in other species.3 current studies are focused on determining whether incubation in lac-mw could be used to prepare frozen/thawed stallion sperm for conventional in vitro fertilization of in vitro-matured equine oocytes. keywords: stallion sperm, frozen/thawed semen, acrosomal exocytosis, tyrosine phosphorylation, izumo1 references 1. ramírez-agámez l, hernández-avilés c, ortíz i, et al: lactate as the sole energy substrate induces spontaneous acrosomal reaction in viable stallion spermatozoa. andrology 2024;12:459-471. doi: 10.1111/andr.13479 2. hernández-avilés c, ramírez-agámez l, varner dd, et al: lactateinduced spontaneous acrosomal exocytosis as a method to study acrosome function in stallion sperm. theriogenology 2023;210:169-181. doi: 10.1016/j.theriogenology.2023.07.024 3. okabe m. sperm-egg interaction and fertilization: past, present, and future. biol reprod 2018;107:1551-1564. doi: 10.1093/biolre/ioy028 blastocyst development of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection using nonsorted or sex-sorted frozen/ thawed stallion sperm luisa ramírez-agámez,a camilo hernández-avilés,a juan samper,a,b charles lovea adepartment of large animal clinical sciences, school of veterinary medicine & biomedical sciences, texas a&m university, college station, tx, usa bst genetics, navasota, tx, usa intracytoplasmic sperm injection (icsi) using frozen/thawed (f/t) stallion sperm is a common procedure in equine breeding industry. historically, sex-sorted (ss) f/t stallion sperm has yielded lower cleavage (< 30%)1 and blastocyst rates (< 5%)2 after icsi compared to nonsorted (ns) f/t sperm. a new technology for sperm sex-sorting (genesis iii; cytonome®) has been validated by a commercial company (st genetics), in which bovine sperm quality and in vivo fertility (62 versus 64%, respectively) are comparable to ns-f/t sperm.3 our objective was to determine the efficiency of icsi on in vitro-matured equine oocytes fertilized using ss-f/t stallion sperm processed through genesis iii technology. we hypothesized that in vitro-matured equine oocytes fertilized by icsi with ss-f/t sperm have similar cleavage and blastocyst rates to ns-f/t sperm. immature oocytes were collected via transvaginal oocyte aspiration from 8 mares during 65 sessions. a total of 324 in vitro-matured oocytes were fertilized by piezo-driven icsi using nsf/t (n = 160 oocytes) or ss-f/t (n = 164 oocytes) from a fertile stallion. after icsi, injected oocytes in both groups were cultured for 5 days, cleavage rate was assessed (i.e. > 8-16 blastomeres/embryo), and only cleaved embryos were further cultured. on days 7 to 10 posticsi, blastocyst development was assessed. cleavage and blastocyst rates were analyzed by fisher’s exact test, whereas postthaw sperm quality data were analyzed by one-way anova with ls means-tukey’s adjustment test. significance was set at p < 0.05. overall, higher (p < 0.05) cleavage (ns-f/t: 113/160 [71%] versus ss-f/t: 79/164 [48%]) and blastocyst rates (ns-f/t: 42/160 [26%] versus ss-f/t: 23/164 [15%]) were observed in ns-f/t than in ss-f/t sperm. blastocyst rate per cleaved oocytes (ns-f/t: 42/113 [37%] versus ss-f/t: 23/79 [32%]) was not different (p > 0.05). percentage of blastocysts developing on days 7 (33 versus 26%), 8 (19 versus 35%), 9 (38 versus 22%), or 10 (10 versus 17%) was similar (p > 0.05). number of embryos/session was numerically, but not statistically higher (p > 0.05) between ns-f/t (1.4) versus ss-f/t (0.68) sperm. considering these results, in a separate experiment, ns-f/t and ssf/t sperm from 3 fertile stallions were analyzed for postthaw sperm motility, viability, and dna damage. postthaw sperm motility was higher (p  <  0.05) in ns-f/t than in ss-f/t sperm (41 versus 12%), whereas sperm viability was similar (p > 0.05) between groups (54 versus 57%). interestingly, percentage of sperm with damaged dna was considerably higher (p < 0.05) in ssf/t than in ns-f/t sperm (80 versus 5%). compared to earlier reports,1,2 although an improvement in blastocyst development after icsi with ss-f/t sperm was observed, efficiency of the genesis. iii technology for in vitro equine embryo production was lower than ns-f/t sperm. lower cleavage rates observed may be associated with higher percentage of dna damage in ss-f/t sperm, current studies are focused on determining the effect of ss-f/t on initial steps of embryonic development posticsi. keywords: equine embryo, sex-sorted sperm, cleavage, blastocyst, sperm dna references 1. carnevale em, graham jk, suh tk, et al: foals produced after icsi using frozen, sexsorted, refrozen sperm. reprod fertil dev 2008;21:228. doi: 10.1071/rdv21n1ab261 2. colleoni s, spinaci m, duchi r, et al: icsi of equine oocytes with sex-sorted frozen-thawed semen results in low cleavage rate but normal embryo development and pregnancies. reprod fertil dev 2008;1:228-229. doi: 10.1071/rdv21n1ab262 http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1111/andr.13479 https://doi.org/10.1016/j.theriogenology.2023.07.024 https://doi.org/10.1093/biolre/ioy028 https://doi.org/10.1071/rdv21n1ab261 https://doi.org/10.1071/rdv21n1ab262 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 17 3. crites br, vishwanath r, arnett am, et al: conception risk of beef cattle after fixed-time artificial insemination using either sexedultra™ 4m sex-sorted semen or conventional semen. theriogenology 2018;118:126-129. doi: 10.1016/j.theriogenology.2018.05.003 effect of lactobacillus application on mare clitoral microbiome jenny sones,a fiona herzog,b kassie crissman,b kalie beckers,b guoli zhou,c marta cittone,a chin-chi liub adepartment of clinical sciences, college of veterinary medicine and biomedical sciences, colorado state university fort collins, co, usa bdepartment of veterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la, usa cclinical and translational sciences institute, michigan state university, east lansing, mi, usa mare’s lower reproductive tract (lrt) harbors a community of commensal organisms. dysbiosis of lrt may have a role in clinical diseases, including endometritis, metritis, and placentitis. metagenomic/metagenetic analysis of bacterial dna can identify organisms that are not readily cultured and thus may go undetected. recent metagenetic studies have indicated that uterus, placenta and vagina of the mare accommodate microorganisms in varying diversity, abundance and richness. clitoral microbiome has yet to be described in the mare. we tested the following hypotheses: 1. clitoris of estrous mares harbors a unique resident microbiome; 2. topical lactobacillus (lb)-containing probiotic alters clitoral microbiome; and 3. mare conception rates after clitoral lb application will not differ significantly from industry standards. mares (n  =  12) with normal breeding soundness evaluation were utilized in the same breeding season. sterile swabs of the clitoral fossa were collected (0) prior to daily topical lb (1 gram) for 4 days. pharmaceutical grade lb was a silicone-based gel containing lactobacillus pentosus, lactobacillus plantarum and lactobacillus rhamnosus in equal parts totaling 109 -1010 cfu per gram. a second (12 hours) and third swab (48 hours) of the clitoral fossa was collected 12 and 48 hours, respectively, following final lb application. swabs were frozen and stored at –80ºc until dna extraction. on the next estrus, mares were bred by artificial insemination with 1 x 109 motile sperm from a single fertile stallion. conception rates were calculated based on reproductive ultrasonography 14-16 days after ovulation. genomic dna was extracted from frozen swab using qiagen dneasy powersoil extraction kits, screened by gel electrophoresis followed by pcr amplification of the 16s rrna gene prior to multiplex sequencing of the v4 region using an illumina miseq platform. the top 3 most abundant phylum resident in the equine clitoral fossa (0) were firmicutes, bacteroidota, and fusobacteriota; contradictory to equine uterine and vaginal microbiome, with proteobacteria in the top 3 phylum. abundance was evaluated via friedman test with pairwise dunn’s post hoc comparisons. statistical significance was set at p < 0.05. compared to time 0, desulfobacterota decreased at 12 and 48 hours, whereas synergistota only at 48 hours, and fusobacteriota increased only at 12 hours (p < 0.05). at the genus level, lactobacillus sp. was increased in the clitoral fossa at 12 hours but returned (p < 0.05) to 0 levels by 48 hours corynebacterium sp. increased at 12 and 48 hours compared to 0, whereas actinobacillus sp. increased (p < 0.05) in a time-dependent manner. furthermore, mobiluncus sp. and christensenellacea_r-7_group decreased (p < 0.05) at 12 and 48 hours compared to 0. lb had an effect (p<0.05) on beta but not alpha diversity at both 12 and 48 hours. finally, lb treated mareshad an 80% pregnancy rate. our results indicated that mare clitoral fossa microbiome is unique to the vagina and uterus measured by metagenetics, lb application dynamically alters the resident clitoral microbiome, but does not interfere with fertility of normal mares. future investigations are needed to understand the role of the lrt microbiome and probiotics in equine breeding. comparison of commercial vitrification kits for vitrifying equine embryos sandra wilsher, ann ismer, sofia kovacsy, agustina grippo, maarten hoogewijs sharjah equine hospital, sharjah, uae several commercial vitrification kits have been used for vitrifying equine embryos. however, the optimum types and concentrations of intraand extra-cellular cryoprotectants for equine embryos have not been investigated. our aim was to compare the pregnancy rate of embryos vitrified using 2 commercial human vitrification kits (kitazato; www.kitazato. co.jp or vitkitfreeze; www.irvinesci.com). each kit contained an equilibration solution (es) and vitrification solution (vs). the kitazato es comprised 7.5% dimethyl sulfoxide (dmso) and 7.5% ethylene glycol (eg) in medium with hepes, gentamycin, and 0.06 mg/ml hydroxypropyl cellulose (hpc). the vitkit-freeze es varied only in that it contained 20% dextran serum supplement (dss) instead of hpc. the vs of both kits contained 15% dmso and 15% eg in their respective base media, but with the addition of 0.5m trelahose or sucrose for the kitazato and vitkit-freeze, respectively. our hypothesis was trelahose and hpc versus sucrose and dss in the vs improve embryo survival after vitrification. sixty-eight, unpunctured, embryos ≤ 500 mm were vitrified using kitazato (n = 35) or vitkit-freeze (n = 33). embryos were placed in kitazato or vitkit-freeze es for 15 minutes at room temperature (rt), then in kitazato or vitkit-freeeze vs for £ 90 seconds at rt, loaded onto a cryolock and plunged into ln2. before transfer to a day 6 recipient mare, vitrified embryos were warmed in thawing solution (1 mol/l sucrose) at 42oc for 1 minute, then warming solution (0.5 mol/l sucrose) for 4 minutes at rt, before transfer to a holding medium (vigro hold) for 4 minutes. conceptus development in pregnant recipients was checked until day 25 of pregnancy, or day 34 in 2 recipients per vitrification method and size group (g1: ≤ 300 µm; g2: > 300-400 µm; g3: > 400-500 µm). percentages of pregnant mares at day 25 overall, and per size group, were compared between vitrification kits using a z-test. differences in mean embryo size between g1-3 for each vitrification kit were assessed using t-test. significance was set at p < 0.05. no difference existed (p = 0.14) in the overall pregnancy rate between the kitazato or vitkitfreeze vitrification kits (28/35 [80%] versus 20/33 [60.6%], respectively). the mean (± sem) diameter of embryos in g1, g2, and g3 did not vary between kits (g1: 230.8 ± 9.2 versus 253.0± 15.0 mm [p = 0.21]; g2: 349.1 ± 7.6 versus 344.5 ± 9.4 mm [p = 0.71]; g3: 444.2 ± 8.6 versus 451.9 ± 8.7 mm [p  =  0.53]; kitazato versus vitkit-freeze, respectively). pregnancy rates for g1 and g2 were not different between http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1016/j.theriogenology.2018.05.003 18 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 kits (g1:10/12 [83.3%] versus 8/10 [80%], p = 0.72; g2: 9/11 [81.8%] versus 9/10 [90%], p = 0.93; kitazato versus vitkit freeze, respectively). however, for g3, the pregnancy rate was higher (p  =  0.03) with kitazato versus vitkit-freeze (9/12 [75%] versus 3/13 [23.1%]). three recipients lost their pregnancies before day 20, all 3 embryos (225, 350, and 460 mm) were vitrified with the kitazato kit. in conclusion, kitazato improved pregnancy rate over vitkit-freeze for embryos > 400 500 µm. trelahose and hpc have been reported to support a higher glass transition temperature and membrane stability than sucrose, and these properties may have aided the vitrification of larger embryos. higher numbers of kitazato vitrified embryos are required to ensure pregnancy loss is not a risk factor of this methodology. effect of novel water-dispersive ubiquinol supplementation in extender on postthaw stallion sperm quality muhammad-salman waqas, ahmed mosallam, warwick bayly, ahmed tibary comparative theriogenology, department of veterinary clinical sciences, college of veterinary medicine, wsu, pullman, usa supplementation of various preparations of ubiquinone oxidized, ubiquinol reduced coenzyme q10 (coq19) in semen extenders has an antioxidant effect, resulting in sperm cryoprotection. coq10 supplementation can also improve mitochondrial function in sperm resulting in improved postthaw sperm viability and motility. however, the effects of extenders supplemented with coq10 on postthaw semen quality have been variable. extender supplementation with powdered coq10 did not improve postthaw stallion sperm quality.1 coq10 is not water soluble and may precipitate at the bottom of the supplemented extender resulting in decreased coq10 bioavailability in semen extenders. thus, our aim was to test the hypothesis that postthaw coq10 supplementation, using a novel water-dispersive ubiquinol commercial preparation (shiroqe®), improves postthaw sperm quality and longevity. dispersion was composed of 13% ubiquinol, 13.4% modified starch, 6.5% sodium ascorbate, 3% dextrin, 3.5% arabic gum, and 60.6% water. a total of 15 replicates (straws) from 3 stallions frozen in e-z freezing ‘le’® (ez) were evaluated for sperm progressive motility (pm), total motility (tm) and motion kinetics (vcl, str, lin, alh) using computer-assisted sperm analysis (casa). after thawing, the semen was diluted with 4 treatments: ez, inra96® (i), inra96® plus 300 micromolar ubiquinol (i300), and inra96® plus 150 micromolar ubiquinol (i150). the casa evaluations were performed at 10, 60, and 120 minutes postthawing. data were analyzed using 2-way anova under repeated measures. the means were compared using tukey’s test and presented as mean ± sem. effect of time and treatment interaction was not significant for any casa parameters. effects of time and treatment were significant for pm, tm and vcl but not for str, lin and alh. the mean sperm pm, tm, and vcl did not differ among various treatments at 10 minutes and 60 minutes postthawing. at 120 minutes postthawing, the mean sperm pm, tm and vcl were significantly higher for i (21.80 ± 2.62, 28.31 ± 2.66 and 96.60 ± 3.82, respectively) than for i300 (8.405 ± 1.86, 14.71 ± 2.38, 74.03 ± 4.61, respectively). at 120 minutes postthawing, the mean sperm pm, tm, and vcl did not significantly differ among ez versus i, ez versus i300, ez versus i150, i versus i150, and i300 versus i150. sperm tm, pm, and vcl at 60 and 120 minutes were nonsignificantly higher for i than for ez extender. this confirmed that diluting semen, postthaw, in inra96 did not negatively affect sperm quality and longevity. this can be used for insemination under field conditions. in conclusion, adding water-dispersive ubiquinol preparation did not improve postthaw quality of stallion sperm. viscosity of the dispersion can negatively affect sperm quality. however, a study2 suggested that coq10 supplementation of semen extenders enhanced postthaw sperm. keywords: coenzyme q10, stallion, semen, postthaw references 1. ruiz a, waqas s, bayly w, et al: effects of coenzyme q10 (coq10) on equine semen quality after cryopreservation. clinical theriogenology 2018;10:311. 2. masoudi r, sharafi m, shahneh az: effects of coq10 on the quality of ram sperm during cryopreservation in plant and animal based extenders. anim reprod sci 2019;208:106103. doi: 10.1016/j.anireprosci.2019.06.015 http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1016/j.anireprosci.2019.06.015 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 19 small animal abstracts relationship between sire size and breeding dose in dogs daniela cortes,a maria ferrer,a patricia xavier,a claudia escobar,b harvey marquez,b robyn ellerbrocka adepartment of large animal medicine, college of veterinary medicine, university of georgia, athens, ga, usa banimal reproduction clinic, college of veterinary medicine, national university of colombia, bogota, colombia minimum dose for artificial insemination (ai) in dogs has been widely disputed. multiple recent studies proposed that the accepted canine breeding dose is 150-200 x 109 total sperm. however, pregnancies have been reported via surgical insemination with as little as 20 x 106 total fresh sperm. we hypothesized that the minimum total progressive morphologically normal sperm (tpmns) necessary to achieve pregnancy depends on sire size. aim of this retrospective study was to determine the minimum sperm dose likely to establish a pregnancy by either vaginal insemination or transcervical insemination, and to establish recommendations for insemination based on sire size. medical records were evaluated from sires presented for semen collection and ai at 2 veterinary clinics specialized in animal reproduction between 2021-2024. information gathered included age, breed, weight, semen evaluation (volume, color, concentration, and morphology), pregnancy rate, and litter size. sires were categorized into 2 groups: 1. preg: pregnant female and 2. np: nonpregnant female. within each group, sires were classified as: large (> 56 lb), medium (35-55 lb), small (10-34 lb), and toy (< 10 lb). sperm dose was determined by calculating total volume, total sperm (ts), total progressive motile sperm (tpms), and tpmns. for dogs that were bred multiple times during estrus, total semen dose was calculated by combining individual ejaculates. semen volume, ts, tpms, and tpmns were compared by sire size and pregnancy status using a one-way anova, and significance was set at p  <  0.05. twenty-one breeds were included, and sires had a mean age of 3.9 years (range: 1-9 years). for the preg group (n = 46), mean volume, ts, tpms, and tpmns were 5.9 ml, 2.41, 2.01, and 1.74 x 109, respectively, compared to np group (n = 16) of 7.6 ml, 1.06 x 109, 914, and 758 x 106. semen volume was significantly less in the toy group (n = 6) than medium (n = 22, p = 0.03) or large (n = 20, p = 0.01), but no difference was observed in volume between preg and np groups. although no difference was observed in ts by sire size, ts was different (p = 0.01) in np group than preg group. similarly, no difference was observed in tpms or tpmns by sire size; however, tpms and tpmns was different (p  =  0.02) in np group than preg group. pregnancy rate was significantly worse when dogs were bred with < 200 x 106 tpmns or > 5 x 109 tpmns, with 1/7 dogs conceiving when bred with < 200 x 106 tpmns, 19/22 when bred with 200-1200 x 106 tpmns, 26/29 when bred with 1.21 to 4.9 x 106 tpmns, and 0/4 when bred with > 5 x 109 tpmns. in conclusion, there appeared to be minimal and maximal recommended tmpns doses for dogs, and even toy breeds benefitted from a minimum breeding dose of at least 200 x 106 tmpns to achieve pregnancy. keywords: dogs, sperm, artificial insemination, pregnancy rate dag-like sperm defect in a dog suspected of reproductive infection audrey kelleman, jonathan cowart department of large animal clinical sciences, college of veterinary medicine, university of florida, gainesville, fl, usa a 7-year, 50 kg rhodesian ridgeback dog, was evaluated for chronic poor semen quality and prostatitis. dog was not used for breeding nor his semen was used for inseminations. two years prior, 50% of sperm had coiled tails. four months prior, the dog had 80% immotile sperm with 95% coiled tails, and semen culture identified escherichia coli, proteus, and an enterococcus species. therapy with enrofloxacin and later doxycycline did not improve spermiogram and dog was referred. on initial examination, dog appeared clinically healthy. two scrotal testes were present, with normal orientation, symmetry, and turgidity on palpation and with normal appearance of all scrotal content on ultrasonographic examination. epididymides and spermatic cords were symmetrical and normal on palpation. total scrotal width (tsw) by caliper was 43 mm. prostate was nonpainful on transrectal palpation and measured 35-40 mm in width, with left lobe slightly larger (mild mixed echogenicity on ultrasonographic examination). two semen collections had 416 and 700 x 106 total sperm, respectively, with asthenozoospermia (< 10% total motility) and teratozoospermia (3-11% normal morphology). cytological evaluation identified rare neutrophils and apparent sperm accumulation plugs with possible bacteria present. initial new york state veterinary diagnostic laboratory (nysvdl) semen culture confirmed proteus mirabilis and enterococcus faecalis, and identified mycoplasma. in-house serum brucella canis rapid slide agglutination test was negative. basal serum thyroid and testosterone concentrations were normal. therapy, based upon susceptibility testing, was completed with oral ciprofloxacin (10 mg/kg, twice daily, for 8 weeks and concomitant oral amoxicillin (10 mg/kg, twice daily, for 2 weeks). after completion of antibiotic course, proteus and enterococcus were cleared, but mycoplasma canis was identified and susceptibility testing was performed through the nysvdl. oral azithromycin therapy (~ 12 mg/ kg, once a day for 3 days, then every other day for 2 months) was initiated that cleared mycoplasma from the ejaculate. at 9 months after initial presentation, tsw had declined to 35 mm. multiple ejaculates were evaluated during the course of care described. due to continued prominent morphological defects, scanning and transmission electron microscopy were performed. majority of sperm exhibited dag-like defects http://dx.doi.org/10.58292/ct.v16.11054 20 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 in which the flagella were intensely coiled and enclosed within a common plasma membrane. sperm exhibiting daglike defects had coiling of all components of the flagellum including midpiece, principal, and end pieces. transverse sections of the flagella displayed alterations, disarrangements, and/or absence of outer dense fibers and microtubules. other histochemical evaluations indicated that sperm from the dog had normal chromatin integrity; however, only 32% had intact acrosome. there was no improvement during 9-month period and owner later elected castration. testes were not provided for further examination. keywords: dogs, infertility, teratozoospermia, asthenozoospermia, sperm morphology role of prostaglandin-endoperoxide synthases in parturition and dystocia in dogs orsolya balogh,a samantha mccarter,a gabriela sousa,a bruce christensen,b alyssa shelby,b aykut gramc adepartment of small animal clinical sciences, virginiamaryland college of veterinary medicine, blacksburg, va, usa bkokopelli assisted reproductive services, sacramento, ca, usa cdepartment of histology and embryology, faculty of veterinary medicine, erciyes university, kayseri,turkey prostaglandins have key roles in parturition as they contribute to luteolysis, uterine contractions, and cervical dilation. prostaglandin-endoperoxide synthase 2 (ptgs2, also known as cox 2) is the inducible key enzyme for prostaglandin biosynthesis, although historically, ptgs1 is regarded as the constitutively expressed isoform. in dogs, uteroplacental ptgs2 expression significantly increased at prepartum luteolysis compared to midpregnancy,1 and uterine ptgs2 expression did not differ between dystocia types.2 we hypothesized that ptgs2 and ptgs1 are important uterine and placental signals for initiation of parturition in dogs, and their altered expression contributes to primary uterine inertia (pui). our aim was to determine and compare gene expression of ptgs1 and ptgs2 in uterus and placenta of dogs at various stages of parturition or dystocia. uterine biopsies and placental tissues were collected during cesarean surgery in dogs at term pregnancy that are not in labor (tnl, n = 7), in stage 1 labor (tl, n = 6), diagnosed with pui (n = 6), or with obstructive dystocia having strong contractions (od, n  =  5). relative gene expression for ptgs1 and ptgs2 was determined by qpcr. blood was collected before surgery and serum progesterone (p4) concentrations were determined by chemiluminescence. one-way anova, kruskal-wallis test, and paired student’s t-test were used for data analysis, and significance was set at p  <  0.05. serum progesterone concentrations were significantly higher in tnl (range: 3.4-5.3 ng/ml) than in other groups (0.3-1.3 ng/ml). in uterus, ptgs1 expression was significantly higher in tnl than in od, and ptgs2 was significantly lower in tnl compared to other groups. placental gene expression was similar among groups. comparing gene expression by tissue types, ptgs1 was significantly higher in uterus than in placenta in all parturition groups. conversely, ptgs2 expression was higher in placenta than in uterus in tnl and tl, but not in pui and od. ratio of uterus/placenta ptgs1 gene expression was significantly higher in tnl and tl compared to od. ratio of placenta/uterus ptgs2 expression was significantly higher in tnl than in the pui and od groups. in conclusion, these preliminary results using gene expression data indicated that ptgs1 may have an important role in initiating uterine prostaglandin synthesis as labor approaches, with decreasing role during active labor, when ptgs2 becomes more important. in contrast to ptgs2, ptgs1 appeared to be a negligible contributor to placental prostaglandin production in dogs. ptgs1 and ptgs2 expression could not be pinpointed as contributors to pui. keywords: dogs, parturition, prostaglandins, uterus, placenta references 1. kowalewski mp, beceriklisoy hb, pfarrer c, et al: canine placenta: a source of prepartal prostaglandins during normal and antiprogestin-induced parturition. reproduction 2010;139:655-664. doi: 10.1530/rep-09-0140 2. rempel lm, lillevang kta, straten at, et al: do uterine ptgs2, pgfs, and ptgfr expression play a role in canine uterine inertia? cell tissue res 2021;385:251-264. doi: 10.1007/ s00441-021-03427-6 chronic cardiac thrombosis in canine neonates hannah cochran,a morgan agnewb aschool of veterinary medicine, university of pennsylvania, philadelphia, pa, usa bphiladelphia animal hospital, philadelphia, pa, usa a 2-year, maiden cavalier king charles spaniel, was presented for initial progesterone timing prior to breeding. the kennel had previous issues with mycoplasma canis causing reproductive failure, and the dog was given oral doxycycline (10 mg/kg, once a day). physical examination was unremarkable and serum progesterone concentrations were consistent for day 3 postlh surge. dog was bred artificially on days 4 and 6 postlh surge and doxycycline treatment was discontinued on day 10 postlh surge. transabdominal ultrasonography was performed on day 37 postlh surge for pregnancy diagnosis and 5 fetuses with heartbeats were identified. owner elected for planned cesarean surgery on day 61 postlh surge. transabdominal ultrasonography revealed a hyperechoic cystic structure in the ventricle of 2 fetuses. both fetuses had normal heart rates and serum progesterone concentrations were 6.48 ng/ml. following day, cesarean surgery was performed after confirming a drop in serum progesterone concentrations (1.43 ng/ml). eight live pups were delivered and all had harsh bronchovesicular sounds with no cardiac arrhythmias. pups were empirically treated with 2.5 mg/kg of ceftiofur, and 6 out of the 8 pups received dextrose orally during resuscitation. three days after delivery, 2 pups died suddenly and necropsy was performed within 24 hours. postmortem examination revealed an abnormal cystic masslike structure in the heart of both neonates and entire organ was submitted for pathology (pennvet diagnostic laboratories). biopsy report described a focally extensive chronic thrombus adhered to the endocardial surface effacing endothelium at the base of the septal aspect of right ventricle composed of neutrophils, macrophages, erythrocytes, and fragmented to granular material. there was no evidence http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1530/rep-09-0140 https://doi.org/10.1007/s00441-021-03427-6 https://doi.org/10.1007/s00441-021-03427-6 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 21 of bacteria, infectious organisms, or neoplasia. lesion was noted to be unusual with an unknown etiology. it was hypothesized that an in utero infectious or inflammatory insult spread hematogenously from dam to pup; however, other tissue samples were not submitted. there is limited research on diagnosis, etiology, and management of chronic cardiac thrombi in neonatal dogs, however causation and treatment for neonatal thrombosis has been studied in human medicine. it is reported that 2-4% of human infants diagnosed with thrombosis die as direct result of the pathology;1 however, this information is not known in canine population. in humans, prenatal fetal thrombosis is rarely identified via ultrasonography; however, when diagnosed the cause was typically multifactorial.2 treatment of human neonatal thrombi can be managed with observation, through medication (thrombolytics and anticoagulants) or via a thrombectomy, as the definitive treatment depended on patient’s risk of embolization and the underlying cause.2 this case demonstrated the need for additional research on the etiology of antenatal canine fetal cardiac thrombosis that can aid in management or prevention of pathology. this case also demonstrated the use of ultrasonography to identify potential fetal abnormalities to provide a detailed assessment of fetal viability to breeders. keywords: dogs, neonatal, cardiac thrombosis references 1. robinson v, achey ma, nag up et al: thrombosis in infants in the neonatal intensive care unit: analysis of a large national database. j thromb haemost 2021;19:400-407. doi: 10.1111/jth.15144 2. ascensão t, pereira n, branco m: prenatal diagnosis of inferior vena cava thrombosis. acta med port 2022;35:492-493. doi: 10.20344/amp.16201 treatment of male cats with adeno-associated gonadotropin-releasing hormone to achieve nonsurgical sterilization chloe hume, anniston dodson, arthur zimmerman, douglas martin, aime johnson department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa overpopulation of stray cats poses substantial challenges to animal welfare and public health. although traditional surgical sterilization methods are effective, they are resourceintensive, stressful to animals, and require a veterinarian. a nonsurgical technique would eliminate these barriers, remove physical and psychological stressors associated with surgery, and reduce the number of animals euthanized. we explored the potential of adenoassociated gonadotropin-releasing hormone (aav-gnrh) treatment as a nonsurgical alternative for cat sterilization. adeno-associated virus (aav) is a nonenveloped virus that can be modified to target cells and deliver dna. gonadotropin-releasing hormone is produced by the hypothalamus and acts on anterior pituitary gland to produce folliclestimulation hormone and luteinizing hormone. these hormones act on gonads to stimulate the release of testosterone in male and estrogen in female. overproduction of gnrh should lead to downregulation of gnrh through constitutive activation of the receptor. we hypothesized that intravenous treatment of aav containing gnrh to male kittens at 6 weeks induces a reduction in testosterone concentrations and impair sperm characteristics, resulting in infertility. with 4 treated and 4 control (untreated) kittens, the study employed monthly testicular ultrasonography starting at 4 months to calculate total testicular volume (ttv), testicular mass, and percent body weight. once cats reached 6 months, they underwent monthly electroejaculation (eej) to analyze sperm number and characteristics. preliminary results suggested a decrease in sperm production and quality, elevation of testosterone concentrations, and decreased total testicular volume. ongoing research will refine and validate the efficacy of this innovative, nonsurgical approach, offering a step towards addressing the challenges posed by feline overpopulation with ethical and practical considerations. keywords: feline contraception, adeno-associated virus, electroejaculation, gnrh prostaglandin assisted semen collection and cryopreservation of semen in an aged dog with testicular disease luis de aguiar, joy russollilo, audrey kelleman, eduardo arroyo department of large animal clinical sciences, university of florida, college of veterinary medicine, gainesville, fl, usa an 8-year, intact male golden retriever weighing 27.2 kg, was presented for semen collection and cryopreservation. dog sired 1 litter in the past and semen was collected successfully once. prior to visit, a hypoechoic, cyst-like structure of ~ 1 x 1 cm within right testis and multiple smaller cysts within left testis were identified via ultrasonography. hemicastration was scheduled to remove right testis but was postponed for preservation of genetic material. on presentation, dog was bright, alert and responsive and no abnormalities were noticed on physical examination. first 2 collections, 30 minutes apart, using vaginal swabs of estrous females, were unsuccessful and only partial erection was obtained. three days later, after several attempts in the presence of an estrous female and vaginal swabs, only a few drops of first fraction of the ejaculate were obtained. dog’s libido was low and did not posture or thrust during semen collection procedures. four days later, intramuscular dinoprost tromethamine (0.1 mg/kg) was given 20 minutes before semen collection. dog displayed moderate libido and partial ejaculation was obtained containing 31.2 x 106 sperm with 65/40% (total/progressive) motility and 71% morphologically normal sperm. cryopreservation was not performed due to limited sperm count. subsequently, semen was collected 4 times (every 4 days) aided by dinoprost treatment and cryopreserved with caniplus freeze extender with 20% egg yolk (minitube, verona, wi, usa). results were as follows (mean ± sd): total sperm count of 419.3 ± 131.2 x 106 (nucleocounter, chemometec, denmark) with 75.8 ± 3.8% total and 65.5 ± 10.5% progressive motility and 53 ± 6.1% morphologically normal sperm per collection. predominant defects were coiled and kinked tails and abnormal midpieces. post thaw motility was 48 ± 19.9% total and 38.3 ± 17.0% progressive. after castration, testes were measured, and http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1111/jth.15144 https://doi.org/10.20344/amp.16201 22 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 average size was ~ 6.5 x 3.5 x 1.5 cm and cystic structures were not appreciated. on histopathological examination of right testis, a leydig cell tumor, most likely benign due to the low mitotic index and lack of other tissue involvement, was identified. in left testis, a sertoli cell tumor was present, most likely benign due its well-demarcated area, mild pleomorphism and low mitotic count. a possible concern was the transmission of genes associated with these types of neoplasias to next generations; however, there is not enough evidence of their heritability and castration is the treatment approach. in this case, the genetic material of the stud dog was successfully collected through medically assisted ejaculation and subsequently cryopreserved. keywords: chemical-assisted ejaculation, dog, prostaglandin, testicular neoplasia http://dx.doi.org/10.58292/ct.v16.11054 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 23 affected in some primary morphological defects, probably related to impairment during spermiogenesis, since the gene is upregulated in testes as observed in a transcriptomic study.4 these findings supported the hypothesis that izumo1 expression is affected by testicular degeneration, potentially compromising sperm-egg fusion and affecting fertility. moreover, this research opens avenues for further investigations to detect subfertily or infertility in livestock populations. keywords: bulls, infertility, testicular degeneration, sperm, physiology references 1. ferrer ms, palomares ra, hurley dj, et al: changes in serum testosterone and anti-müllerian hormone concentration in bulls undergoing scrotal insulation. dom anim endocrinol 2022;78:106685. doi: 10.1016/j.domaniend.2021.106685 2. ferrer ms, klabnik-bradford j, anderson de, et al: sperm-bound antisperm antibodies prevent capacitation of bovine spermatozoa. theriogenology 2017;89:58-67. doi: 10.1016/j.theriogenology. 2016.10.012 3. hamze jg, sánchez jm, o’callaghan e, et al: juno protein coated beads: a potential tool to predict bovine sperm fertilizing ability. theriogenology 2020;155:168-175. doi: 10.1016/j.theriogenology. 2020.05.025 4. xavier p, moran, k, ellerbrock, re, et al: expression of genes associated with sperm-oocyte interaction in the stallion reproductive tract. j equine vet sci 2023;125:104626. doi: 10.1016/j.jevs.2023.104626 effect of delayed maturation on cloned bovine embryo production luis de aguiar,a,b lee lee,a prasanthi koganti,a abdallah abdelhady,a vimal selvaraj,a soon hon cheonga acollege of veterinary medicine, cornell university, ithaca, ny, usa bcurrent address: large animal clinical sciences, college of veterinary medicine, university of florida, gainesville, fl, usa cloning by somatic cell nuclear transfer is a powerful technology for producing transgenic animals and for genetic preservation of high value and endangered animals. however, the technology is very time consuming, limiting the number of clones that can be produced in a day, making it less efficient. as in equine oocytes, holding a batch of bovine slaughterhouse oocytes could allow a second opportunity to produce cloned embryos. our aim was to determine if holding oocytes overnight has detrimental effects on cloning efficiency. we hypothesized that holding bovine oocytes overnight at room temperature before maturation allows a second cloning replicate the following day with similar embryo development rates production animal abstracts evaluation of bull semen viability before, during, and after scrotal insulation via izumo1 expression and sperm-bound antisperm antibodies patricia xavier, robyn ellerbrock, roberto palomares, daniel gomes, daniela cortes-beltran, maria ferrer department of large animal medicine, college of veterinary medicine, university of georgia, athens, ga, usa most common cause of poor semen quality in bulls is testicular degeneration that can be transient. in a study in our laboratory, bulls recovered preinsulation semen parameters 48 days after scrotal insulation. however, there was persistence of antisperm antibodies (asa), low mitochondrial membrane potential and altered concentrations of antimüllerian hormone and testosterone, in spite of these bulls being classified as satisfactory breeders during routine breeding soundness evaluation.1 bovine sperm-bound asas impaired acrosome reaction and binding to zona pellucida.2 persistence of asas, dysfunctional mitochondria and altered hormonal concentrations beyond regaining normospermia reflect a long-term effect of scrotal insulation on testicular and sperm function that is not detected via routine semen evaluation. these undetected changes could be responsible for long-term infertility in bulls recovering from testicular degeneration and with apparently normal microscopic sperm parameters. it is therefore important to understand how long-lasting asas affect fertilization to make recommendations for testing and management of bulls after undergoing a scrotal insult. in addition, izumo1, a protein involved in sperm-oocyte interaction during fertilization, essential for gamete fusion and for blocking polyspermy, was used to predict bull fertility.3 we hypothesized that testicular degeneration leads to dysregulation of the izumo1 expression, the izumo1 expression is associated with semen microscopic parameters over time and izumo1 expression is not associated with asas. to test this hypothesis, 10 anguscross bulls were collected (aup a2016 01-005-y3a0) via an eletroejaculator twice a week for 3 months. an insulated bag was placed around the scrotum for 8 days. semen sample was evaluated for multiple parameters and semen was cryopreserved using a routine semen freezing protocol. straws were stored in liquid nitrogen until flow cytometry analysis for evaluation of asa binding and izumo1 expression. timepoints analyzed were preinsulation (day 7 [during scrotal insulation]) and days 30 and 60 after insulation. these timepoints were selected to represent negative control, normospermic samples (t0), time when changes were observed on sperm exposed to heat within epididymis (t7), time with most pronounced changes in semen quality (t30), and time when ‘normospermia’ was recovered in the face of persistent asas and endocrine disruption (t60).1,2 positive correlation was observed between izumo1 stained sperm with progressive motility, normal morphology, sperm dna integrity, and live sperm. negative correlation was observed between izumo1 positive and abnormalities in head shape, midpiece, coiled tails, and failure of the bull to pass the breeding soundness evaluation. izumo1 expression was negatively http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1016/j.domaniend.2021.106685 https://doi.org/10.1016/j.theriogenology.2016.10.012 https://doi.org/10.1016/j.theriogenology.2016.10.012 https://doi.org/10.1016/j.theriogenology.2020.05.025 https://doi.org/10.1016/j.theriogenology.2020.05.025 https://doi.org/10.1016/j.jevs.2023.104626 24 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 compared to conventional maturation. bovine oocytes from slaughterhouse ovaries were collected and assigned to 2 groups: conventional maturation (conv) where oocytes were matured for 20 hours in maturation media (ivf biosciences) or delayed maturation (hold) where oocytes were held for 24 hours in holding media (vigro holding plus, vetoquinol, usa) at room temperature (23ºc) before in vitro maturation. after maturation, oocytes were stripped from cumulus cells and matured oocytes with visible polar body were treated with protease to remove zona pellucida. oocytes were then manually bisected and the hemi-cytoplasts obtained were scanned under uv light after staining with bisbenzimide; halves containing the metaphase plate were removed and used as controls. two enucleated halves were reconstructed with a single neonatal fibroblast and fused with 1 single electric pulse. two hours after fusion, embryos were activated with ionomycin and 6dymethylaminopurine. embryos were cultured for 7 days in commercial culture media (ivf biosciences) and evaluated for cleavage and blastocyst rates. regular in vitro fertilized (ivf) oocytes and parthenogenetically activated oocytes were used as controls. differences among groups were assessed using logistic regression anova in jmp pro v17. after 6 replicates, a total of 692 and 680 oocytes were placed in maturation for conv and hold groups, respectively and the maturation rates were similar (conv: 70.2 ± 3.2 versus hold: 67.2 ± 4.0). cleavage (88.3 ± 10.4 versus 86.0 ± 8.1) and blastocyst (21.9 ± 12.6 versus 14.7 ± 8.9) rates for somatic cell nuclear transfer were not different (p > 0.05) between conv and hold groups. holding oocytes overnight did not affect (p > 0.05) development rates for conventional ivf, with similar cleavage (82.8 ± 2.9 versus 79.0 ± 3.5) and blastocyst (31.8 ± 5.5 versus 27.6 ± 5.6) rates for conv and hold groups. parthenogenetically activated, zonafree controls followed a similar trend where cleavage (91.6 ± 4.6 versus 97 ± 1.3) and blastocyst (38.6 ± 8.4 versus 40.5 ± 3.6) rates were not different between groups. in conclusion, oocytes held for 24 hours prematuration supported development of cloned, ivf and parthenogenetically activated bovine embryos. keywords: bovine oocytes, maturation, cleavage, blastocyst, somatic cell nuclear transfer microfluidic and density gradient techniques improved frozen-thawed epididymal bighorn rams’ sperm eduardo arroyo,a‡ jesper nielsen,b jacob møllenbach,c ahmed tibarya acollege of veterinary medicine, washington state university, pullman, wa, usa bvetgruppen ansager hestehospital, ansager, denmark cmotilitycount aps, valby, denmark ‡current affiliation: large animal clinical sciences, college of veterinary medicine, university of florida, gainesville, fl, usa gamete preservation of wild animals is a priority in conservation programs. field techniques to recover sperm from deceased animals may impair its final quality. the variable degree of blood  and tissue contamination may impact cryopreservation. to improve postthaw quality, postmortem-harvested and cryopreserved semen may need to be sorted before use. sperm sorting techniques based on centrifugation can increase the risk of damaging sperm. in contrast, microfluidics uses hydrostatic pressure and capillary forces, avoiding the need for costly equipment and minimizing cell damage. this study aimed to compare sperm motion parameters, morphology, and acrosome integrity of cryopreserved-thawed epididymal bighorn ram sperm before and after sorting. two commercial sorting techniques, vetcount harvester® device (mfd) and bovipure® density gradient centrifugation (dcg), were used. our hypothesis was that semen motion parameters improve equally using either technique. sperm parameters associated with quality were: progressive motile sperm (pms), straight velocity (vsl), average path velocity (vap), curvilinear velocity (vcl), linearity (lin), straightness (str), beat cross frequency (bcf), amplitude of lateral head deviation (alh,) and plasma membrane integrity (pmi). computer assisted sperm analyzer was used for the semen analysis. eosin/nigrosin and spermac® stains were used to assess morphology and acrosome integrity, respectively. total number of morphological normal sperm with intact acrosome was counted by 2 veterinarians. data were normally distributed based on kolmogorov-smirnov and analyzed using a one-way anova. mean sperm concentration was higher (p < 0.001) before filtration (129.56 ± 53.9 x 106/ml) than after dcg (68.42 ± 29.18 x 106/ml) or mfd (52.94 ± 28.76 x 106/ml). pms was higher (p < 0.001) after selection using the dgc (79.70 ± 11.11) or mfd (81.23 ± 5.64) compared to unsorted controls (45.36 ±14.67). sperm motion parameters were higher (p < 0.001) for dgc treatment compared to mcd except alh was higher (p = 0.001) for mcd. sperm morphology and acrosome integrity were higher (p < 0.001) for dgc or mcd. plasma membrane integrity was higher (p < 0.001) for dgc or mfd. in conclusion, microfluidics and single-layer centrifugation improved sperm motion parameters, viability, morphology, and intact acrosome of epididymal frozen-thawed bighorn sperm. microfluidic device was comparable to density gradient centrifugation. therefore, the use of vetcount harvester® microfluidic device has potential as a sperm sorting technique when dgc is not an option. microfluidics system does not require special laboratory equipment and may be advantageous for use in the field. further research is needed to explore the effect of sperm sorted using this technique on artificial insemination, recryopreservation, and assisted reproductive technology outcomes. keywords: microfluidic device, bighorn ram, sperm motion parameters investigation of an outbreak of papillomavirus and vaginal vesicular lesions in a dairy herd soon hon cheong,a stephanie bandoski,a blake nguyen,b toby pinn-woodcock,c leonardo caserta,c mariana diel de amorim,a gerald duhamel,d diego dielc adepartment of clinical sciences, bdepartment of public and ecosystem health, cdepartment of population medicine and diagnostic sciences, ddepartment of biomedical sciences, college of veterinary medicine, cornell university, ithaca, ny, usa a holstein dairy herd milking 150 cows in new york state was investigated for an outbreak of perianal warts (33% morbidity), vaginal warts (11% morbidity), and vaginal vesicles (78% morbidity); 11% of cows had no lesions. cutaneous perianal warts surrounded the anus, perineum, or base of the tail and presented in 2 forms: papular and filiform. mucosal vaginal warts affected the caudal vagina and also presented as papular or filiform. one cow had a large fibropapilloma on the clitoris that http://dx.doi.org/10.58292/ct.v16.11054 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 25 was firm and pedunculated. vaginal vesicles were 1 to 2 mm in diameter and filled with clear fluid with varying degrees of mucosal erythema. no lesions were observed elsewhere on the body, including head or back of affected cows. an initial clinical differential list included: bovine papillomavirus, bovine herpesvirus, parapoxvirus, ureaplasma, and mycoplasma. samples from 3 affected cows were collected: 1. vaginal swab for ureaplasma and mycoplasma; 2. aerobic bacteriological culture; 3. biopsy of vaginal vesicles and warts, and cutaneous biopsy of filiform and papular perianal warts for pcr assays for bovine herpesvirus, pan-herpes, parapoxvirus, and bovine papillomavirus; 4. virus isolation; 5. in situ hybridization (ish) for bovine papillomavirus; and 6. viral sequencing. all cultures and pcr tests were negative except the bovine papillomavirus ish with positive signal restricted to dermal fibroblast-like cells. virus sequencing identified 29.7 and 0.7% of nonhost reads with 99.9% identity with bovine papillomavirus 3 in 2 samples, and 17.4% of nonhost reads shared 99.5% identity with bovine papillomavirus 8. these results indicated the outbreak to be of bovine papillomavirus 3 and 8. a subset of affected cows was reexamined for clinical signs twice during 6-month period, where the proportion of unaffected cows remained 12 and 17%, respectively. risk factors assessment using fisher’s exact text identified multiparous cows with risk ratio (rr) = 2.8 for perianal warts, rr = 1.8 for vaginal warts, and rr = 0.9 for vaginal vesicles compared to primiparous cows, and stage of lactation with cows > 200 days-in-milk with rr = 1.4 for perianal warts and rr = 1.9 for vaginal warts. bioeconomic impact on affected cows at first examination revealed no significant differences in milk production; however, affected cows with vaginal vesicles tended to have higher (p  =  0.05) milk production. fertility tended to be higher (p = 0.07), in cows with perianal warts, whereas 21-day pregnancy rate, pregnancy per ai, and number of inseminations were not different (p > 0.10). between affected and nonaffected cows. presence of lesions was not associated (p > 0.05) with risk of leaving the herd within 8-months after the outbreak this investigation of an atypical outbreak of bovine papillomavirus 3 and 8 in a dairy herd revealed a high prevalence of cows with cutaneous and vaginal lesions without a major impact on economic outcomes. keywords: bovine papillomavirus, genital warts, genital vesicles, outbreak adverse effects of timed exposure to phytoestrogens on beef cattle conception rate jessica wyse,a,b rory nevard,b,c clayton smith,a jeffrey mccormick,a,b saliya gurusinghe,b leslie weston,b cyril stephena,b aschool of agricultural, environmental and veterinary science, bgulbali institute for agriculture, water and the environment, charles sturt university, wagga wagga, nsw, australia csydney school of veterinary science, the university of sydney, camden, nsw, australia phytoestrogenic legume pastures in australia have been observed to affect early conception rates in and early embryonic development in cattle. early embryonic loss (up to 29%) has been observed in cattle grazing legume pastures. we investigated the effect of timed exposure to phytoestrogens resulting from grazing legume pasture on day 35 conception rate in multiparous cattle. a 10-week grazing trial was conducted using 68 multiparous angus cows on mixed lucerne:fescue and ryegrass pastures, with timed grazing treatments applied. cows aged between 3-9 years were allocated randomly to 1 of 4 experimental groups; preestablished annual ryegrass (control) and preestablished lucerne:fescue (60:40) treatment pasture. cows were introduced to control (n = 17) and treatment (t1, n = 17) pasture, at grazing trial start. another group of 17 cows were introduced at 2 weeks after start of grazing trial (t2, n = 17), and a final group were introduced at 4 weeks after start of grazing trial (t3, n = 17). cows were subjected to a progesterone-based estrus synchronization program using a controlled internal drug release intravaginal device (eazi-breed cidr®), estradiol benzoate (bomeroltm 1 mg/ml) and gonadotropin releasing hormone (gnrh, cattlematetm 100 µg/ml). on day 25 after commencement of grazing trial, the cidrs were inserted and cows were given 2 mg of intramuscular estradiol benzoate. on day 34, cidrs were removed and cows were given 500 µg of intramuscular cloprostenol (estrumatetm 250 µg/ml). intramuscular gonadorelin (100) was given 24 hours after cidr removal. cows were subjected to timed artificial insemination (tai) 16 hours after gonadorelin injection, using frozen semen. pregnancy diagnosis was performed using transrectal ultrasonography on day 35 posttai. blood and pasture samples were collected biweekly, starting on day 0 of the trial and continued for 10 weeks. samples were stored at –20 °c before extraction and analysis was performed using ultra high performance liquid chromatography quadropole time of flight mass spectrometry (uhplcms-qtof) and high-performance liquid chromatography triple quadropole mass spectrometry (hplc-ms-qqq). extraction and metabolomic profiling of pasture and blood plasma samples was performed using a preestablished method. numerous phytoestrogens, including coumestrol, were present in plasma and pasture samples. flavonoid apigenin was present in higher relative abundance compared to isoflavones and coumestans identified. relative abundance of coumestrol increased over weeks 2 4, during introduction of t2 and tai. initial results suggested that phytoestrogens present in the treatment pastures may have contributed to a reduction in day 35 conception rates (cr). cr were different (p < 0.01) between timed treatments and were highest for t1 (47%) and lowest for t2 (29%). cattle (7 and 4 years) had cr > 50%; however, there were no differences (p > 0.05) in cr depending on cattle age. this study demonstrated that grazing mixed lucerne pastures, with high abundance of phytoestrogens at least 22 days prior to ai, may significantly reduce day 35 cr > 70%. development of a model to predict which phytoestrogens may be commonly associated with negative impacts on cattle reproduction is also underway. keywords: phytoestrogens, beef cattle, conception rates, lucerne, fescue, coumestrol comparison of breeding soundness evaluation outcome using society of theriogenology and breed-specific standards in yearling wagyu bulls muhammad waqas, ahmed tibary comparative theriogenology, department of veterinary clinical sciences, college of veterinary medicine, wsu, pullman, usa bull breeding soundness examination (bse) is highly recommended for cow-calf operations, 2 to 3 months before http://dx.doi.org/10.58292/ct.v16.11054 26 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 the breeding season. for a bull to be classified as a potential satisfactory breeder, a minimum age-specific scrotal circumference (sc), sperm motility and sperm morphology set by the society of theriogenology (sft) must be met. approximately 15% of tested bulls fail bse because of smaller sc than sft standards. the sft sc standards are not breed-specific. american wagyu bulls have a lighter body weight and reach puberty earlier than continental breeds; therefore, a different breed-specific sc standard has been suggested1 for this breed. our objective was to retrospectively analyze bse results and compare the percentages of bulls failing bse based on sft and breed-specific sc standard for wagyu bulls. we hypothesized that more yearling wagyu bulls fail on the sft standard than on the breed-specific standard. records of bse forms from 93 yearling (12-24 months) wagyu bulls were analyzed using descriptive statistics and the chi-square test. bulls raised at the washington state university beef center were evaluated for bse from 2005 2023. overall, a higher (p < 0.0001) percentage of bulls failed sft standards (61.3%; 57/93) than breed-specific standards (22.6%; 21/93). percentage of bulls failing due to small sc alone was higher (p = 0.02) if sft sc standard was used (91.22%; 52/57) compared to breed-specific sc standard (61.9%; 13/21). substandard sc and poor sperm morphology were the only reasons for bulls to fail in this study, whereas no bull failed bse because of health or any abnormality on physical examination. a higher (p < 0.0001) percentage of bulls (55.9%; 52/93) did not meet sft sc standard, whereas only 8.6% (8/93) of bulls failed breed-specific sc standard. most (69.2%; 36/52) bulls failing sft sc standard met the minimum required sperm motility and morphology standard. among bulls failing based on breed-specific sc standard, a lower percentage of bulls (12.5%; 1/8) passed (p < 0.01) sperm motility and morphology standard. percentage of bulls that failed bse due to poor sperm morphology was similar (sft 14.5 versus breed-specific 16.5, p > 0.05). results stressed the importance of using breed-specific sc standard while evaluating wagyu bulls for breeding soundness. previous reports demonstrated that most bulls failed bse because of poor sperm morphology. this was not the case for wagyu bulls if sft sc standard was used. however, when breed-specific sc standard was used, sperm morphology was the major reason for classifying bulls as unsatisfactory. breedspecific standards for sc could be an important aspect of establishing guidelines for bull bse, particularly in yearlings. keywords: wagyu bulls, scrotal circumference, breeding soundness reference 1. sosa jm, senger pl, reeves jj: evaluation of american wagyu sires for scrotal circumference by age and body weight. anim sci j 2002;80:19-22. doi: 10.2527/2002.80119x http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.2527/2002.80119x citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 27 noncompetitive posters effect of prematuration treatment on nuclear maturation in domestic cat oocytes victor medina,a sabrina cousseau,a carlos pintob adepartment of veterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la, usa bdepartment of ambulatory medicine and theriogenology, cummings school of veterinary medicine at tufts university, woodstock, ct, usa we evaluated whether keeping freshly harvested immature cat oocytes at room temperature for 18 hours before in vitro incubation would affect nuclear maturation. specific objective was to record and compare oocyte nuclear maturation in treated and control oocytes. ovaries were collected from cats undergoing surgical sterilization at a local veterinary clinic. immediately after surgical removal, ovaries were transported to the laboratory in 0.9% saline solution. ovaries were dissected from adjacent tissue and placed in a petri dish containing a heparinized oocyte handling media (opu medium, ivf bioscience, uk). only cumulus oocyte complexes (cocs) with a dark homogeneous cytoplasm and at least 3 layers of cumulus cells were selected. shortly after collection, cocs were placed either into oocyte in vitro maturation (ivm) media (control) or into  a  serum-free hepes-buffered media (eq-hold, ivf bioscience, uk) and kept for 18 hours at room temperature before incubation for in vitro maturation (treatment). after  24 hours, cocs were removed from incubator, denuded by repeated pipetting, and fixed in 2% paraformaldehyde. oocytes were washed 3 times in 75 µl drops of phosphate buffered saline with 20% bovine serum albumin  and mounted on a glass slide with 4’,6-diamidino-2-phenylindole (dapi, invitrogen, thermo fisher scientific) to stain oocyte dna. confocal laser scanning microscopy was used to evaluate chromatin configuration. differences in nuclear maturation between control and treated groups were analyzed by chi-square test, with significance set at p ≤ 0.05. proportion of oocytes undergoing nuclear maturation after incubation did not differ (p > 0.1) between control and treated oocytes, (24 versus 28% and 30 versus 35%, respectively). a subset of oocytes held at room temperature for 18 hours were also analyzed by confocal microscopy, indicating that most remained at the germinal vesicle stage (37/80; 46.25%). holding immature domestic cat oocytes for 18 hours at room temperature in a simple media did not affect their ability to undergo in vitro maturation once incubated. domestic cat oocytes could potentially be kept at room temperature for 18 hours before arriving at an assisted reproduction laboratory for in vitro fertilization procedures. premature placental separation due to urine pooling in a late-term pregnant mare eduardo arroyo‡ ahmed tibary college of veterinary medicine, washington state university, pullman, wa, usa ‡current affiliation: large animal clinical sciences, college of veterinary medicine, university of florida, gainesville, fl, usa an 18-year, multiparous quarter horse mare, 319 days pregnant, was referred to the veterinary teaching hospital, due to worsening of laminitis. on arrival, the mare was lame 5/5, tachycardic, normothermic, euhydrated and with excessive ventral edema. despite daily treatment of phenylbutazone at home, mare’s pain remained uncontrolled. evaluation of mammary gland secretions was challenging due to marked ventral edema and discomfort during manual stimulation. radiological examination revealed a 10° rotation of third phalanx in both front feet. blood work indicated elevated blood urea nitrogen (bun 58 mg/dl) and creatine (6.0 mg/dl) concentrations (normal: 12-27 and 0.9-1.9 mg/dl, respectively). urine specific gravity of 1.018 revealed isosthenuria. transabdominal ultrasonography revealed a live fetus with a heart rate of 82 beats/minute. combined thickness of the uterus and placenta (ctup) was increased at the uterine body and the cranial aspect gravid uterine (17.7 and 16.0 mm, respectively). on transrectal ultrasonography, the ctup at the cervical star region was 9.2 mm, with no evidence of placental separation. initial treatment consisted of intravenous fluids (lactated ringer’s solution at 3 liters/hour, twice maintenance rate), to increase urine output. pain management included acetaminophen (20 mg/kg, po, twice daily) and gabapentin (30 mg/kg, po, twice daily). oral omeprazole was given to protect gastrointestinal mucosa. altrenogest (0.044 mg/kg, po, once daily) and pentoxifylline (7.5 mg/ kg, po, twice daily) were given to support the pregnancy. mare did not receive any antibiotics as there was no evidence of infectious placentitis. mare received grass hay and a commercial low-protein diet (12.5%) in several meals per day. urine production increased in response to treatment, and on day 7 after arrival (day 326 of pregnancy), urine dribbling was noticed. vaginal examination revealed urine pooling with urine sediment. on transrectal ultrasonography, a 14-cm separation of placenta was evident at cervical region, with fluid accumulation between allantochorion and endometrium. transcervical drainage of the fluid with a foley catheter was performed for 3 consecutive days. fluid was confirmed to be urine. creatine and bun concentrations decreased slightly over time, so on day 10, fluid therapy was discontinued. on day 11, the amount of fluid between allantochorion and endometrium was markedly reduced. on day 332 of pregnancy, mare entered labor and had premature placental separation (‘red bag’). a healthy colt was delivered. due to economic constraints, the mare was discharged with elevated creatinine and bun concentrations. medical reevaluation and follow up were declined. a year later, the owner reported that the mare was apparently healthy. this case illustrated the importance of intervention by a http://dx.doi.org/10.58292/ct.v16.11054 28 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 theriogenologist in the clinical management of a late-term pregnant laminitis mare. a vaginal examination and sampling of the fluid present are important in cases of placental separation and premature cervical relaxation. premature placental separation increases the risk of ‘red bag’ delivery. regular evaluation and monitoring are crucial for high-risk pregnant mares. keywords: creatinine, blood urea nitrogen, fluid therapy, premature placental separation, laminitis cystic endometrial hyperplasia in a jack russell terrier marcelo de oliveira,a alex wittorff,a sunil moré,b adriana villaseñor,b lara sypniewski,a reed holyoaka adepartment of veterinary clinical sciences, college of veterinary medicine, boklahoma animal disease diagnostic laboratory, oklahoma state university, stillwater, ok, usa a 16-year, intact female jack russell terrier presented for lethargy, loss of appetite, increased coughing, and head tilt. medical history included chronic steroid-responsive tracheal collapse and bronchitis. physical examination revealed left-sided head tilt, hyperreflexia in hindlimbs, a wide base stance in the forelimbs, respiratory dyspnea, and malodorous vaginal discharge. vaginal cytology had large numbers of neutrophils with a mixed population of bacteria and a few superficial epithelial cells. treatment with fluid therapy, antibiotics (intravenous ampicillin-sulbactam 30 mg/kg, every 8 hours, and enrofloxacin 10 mg/kg, daily), maropitant (1 mg/kg, daily), and methadone (0.1 mg/kg, every 4-8 hours) was initiated. bloodwork and urinalysis results were unremarkable on presentation but severe neutrophilic leukocytosis with a left shift developed by day 3. diagnostic imaging confirmed severe pulmonary disease and a fluid-filled uterus. transabdominal ultrasonography findings included moderate to severe fluid distention of the uterus consistent with pyometra, multiple small cysts on both ovaries, and a focal, slightly bilobed, oblong centrally located luminal structure (6 x 3 cm) characterized by a thin wall and fluid-filled lumen in the right uterine horn. ovariohysterectomy was recommended due to clinical deterioration, but the risk of anesthetic complications secondary to severe pulmonary disease prompted the owners to choose humane euthanasia and necropsy. postmortem examination revealed mild to moderate hydrocephalus of the lateral ventricles bilaterally, explaining the recent head tilt, as well as severe bronchiolar inflammation consistent with clinical findings. cervix, uterine body, and uterine horns were multifocally expanded by numerous fluid-filled cysts ranging from 0.3-6 cm in diameter. on cut section, the cystic structures exuded variable amounts of clear watery fluid. ovaries contained multiple cysts ranging from 1-2 cm diameter. further histological examination of the uterine horn revealed hyperplasia of endometrial glands that were moderately to severely dilated and contained small amounts of eosinophilic proteinaceous fluid and low numbers of neutrophils. all findings were consistent with advanced stage cystic endometrial hyperplasia. cystic endometrial hyperplasia (ceh) has long been associated as a causative condition for pyometra in dogs due to degenerative endometrial changes and opportunistic bacterial overgrowth within the endometrium. this case presented findings both consistent with normal ceh-pyometra complex findings, but also several extraordinary circumstances. the presence of endometrial cysts throughout the endometrium is to be expected for an intact dog of that age. however, the patient’s advanced age with still functioning ovaries is unexpected, as ovarian senescence can be observed in dogs as young as 7 years of age. size of the largest cyst was also unexpected, as well as no previous history of pyometra in a dog that had not been bred for years, indicating that ceh can be present for years without causing clinical signs of disease. keywords: pyometra, cystic endometrial hyperplasia, ovarian senescence cystic endometrial hyperplasia with segmental mucometra in a geriatric queen michelle kutzler department of animal and rangeland sciences, oregon state university, corvallis, or, usa a 13-year, domestic shorthair nulliparous queen (4.5 kg), was presented for a routine ovariohysterectomy. despite her advanced age, physical examination findings were within normal limits except for the presence of nonpainful, tubular masses in her abdomen. complete blood count, serum chemistry, thyroid hormone concentrations, and urinalysis results were within normal limits. transabdominal ultrasonography revealed an enlarged uterine body (2 x 1.5 cm) and horns (1.5 x 0.75 cm) with numerous anechoic mural cysts (0.2 cm). right uterine horn was also focally dilated (0.5 cm) with fluid of mixed echogenicity. two extra-uterine anechoic cysts (1 cm) were visible along the left uterine horn. both ovaries appeared normal with numerous 2-3 mm follicles. cat was sedated with oral buprenorphine (0.02 mg/kg) and intramuscular dexmedetomidine (0.01 mg/kg). intravenous catheter was placed, intravenous maropitant citrate (1 mg/kg; cerenia®) was given, and cat was maintained on intravenous isotonic fluids (14 ml/hour; phylyte®). anesthesia was induced with intravenous propofol (3 mg/kg) and maintained on oxygen (1 liter/minute) with 1% isoflurane gas. hair on the ventral abdomen was clipped and the skin was prepped with chlorhexidine scrub and alcohol. a ventral midline laparotomy incision was made. right ovarian suspensory ligament was manually broken down, and the ovarian pedicle was ligated using a 2-clamp technique and transected. the process was repeated for the left ovary. broad ligaments on both uterine horns were broken down, however, most of the uterine body was adhered to the colon and bladder. efforts were made to gently breakdown these adhesions, but it was not possible. uterine body was too thick to allow proper clamping, so a self-crushing ligature was used on both sides to include each uterine artery individually. cranial uterine body, with both uterine horns and ovaries, were then excised. a routine 3-layer abdominal closure was performed, and cat recovered from anesthesia without complication. intramuscular atipamezole (0.1 mg/kg; antisedan®) was given to reverse dexmedetomidine and topical buprenorphine (20 mg; zorbium®) was given for analgesia. subcutaneous meloxicam (0.08 mg/kg) was given during recovery and dispensed for additional analgesia oral meloxicam (0.04 mg/kg; once daily for 4 days). a diagnosis of cystic endometrial hyperplasia (ceh) with segmental mucometra http://dx.doi.org/10.58292/ct.v16.11054 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 29 with cystic mesonephric duct remnants was made based on gross examination and confirmed with histology and fluid cytologic evaluation. ceh is a common uterine problem in queens with an 88% incidence reported in cats > 5 years old.1 although ceh and mucometra can predispose to the development of pyometra, the current case illustrated that subclinical ceh can exist for several years in older queens and present as an incidental finding at the time of ovariohysterectomy. keywords: cat, cystic endometrial hyperplasia, cyst, mucometra, uterus reference 1. perez jf, conley aj, dieter ja, et al: studies on the origin of ovarian interstitial tissue and the incidence of endometrial hyperplasia in domestic and feral cats. gen comp endocrinol 1999;116:10-20. doi: 10.1006/gcen.1999.7331 use of anaerobic culture in an aborted mare with necrotic intrauterine tissue peyton draheim, karen wolfsdorf hagyard equine medical institute, lexington, ky, usa a 14-year, multiparous thoroughbred mare, was presented for hysteroscopy after aborting at ~ 7 months of pregnancy. mare was treated during 2023 breeding season for endometritis and during pregnancy for placentitis. during hysteroscopy, a small amount of uterine fluid with specks of debris was observed and sterilely aspirated for submission for culture and cytology. towards right uterine horn tip, a discrete white-tan nodule was identified protruding from the endometrium that was ~ 1-2 cm in diameter. endoscopic forceps were used to obtain samples of the nodule and ultimately to debride the entirety of the mass. on removal of fragments, the mass bled, suggesting vascularization and an intimate association with the underlying endometrium. collected malodorous tissue fragments were submitted for culture to hagyard equine medical institute laboratory and to university of kentucky veterinary diagnostic laboratory for histopathology. aerobic culture revealed no bacterial growth on neither the uterine fluid nor the tissue. due to odor associated with the tissue, an anaerobic culture was requested for the fluid and the tissue. uterine fluid grew moderate bacteroides species, whereas the tissue grew heavy bacteroides fragilis on anaerobic culture; the suggested treatment was systemic metronidazole. on histopathology, the diagnosis was neutrophilic inflammation and tissue necrosis; no evidence of a fungal component was appreciated. it is unknown what exactly this tissue originated from, whether placental or endometrial tissue, or another alternative. location of the mass makes endometrial cup remnants unlikely, as those form at uterine horn base rather than the tip. in addition to systemic, oral metronidazole (15 mg/kg, 4 times daily) was given. mare was treated for 5 consecutive days with deep uterine horn infusion (2 grams of ampicillin suspended in 120 ml of tris-edta). mare is scheduled for an additional hysteroscopy to evaluate current endometrial status and will be bred again during the 2024 season. this case highlighted the importance of considering all diagnostic options, even those that may be less commonly utilized such as anaerobic culture of intra-uterine samples, particularly when those samples appear necrotic and/or are malodorous. given the lack of microbial growth on routine aerobic culture in this case, the mare likely would not have received appropriate targeted treatment for the underlying organism that was revealed only on anaerobic culture. keywords: mare, hysteroscopy, anaerobic, bacteroides, endometrium management of pyometra in an aged warmblood mare peyton draheim, nimet browne, dwayne rodgerson hagyard equine medical institute, lexington, ky, usa a 21-year, warmblood mare, was presented for colic. relevant history included a cervix that did not relax during estrus, as well as beta streptococcus species endometritis that had been treated several months prior with sterile uterine lavage and infusion of 2 grams of ampicillin, in addition to oral equisul® (24 mg/kg, twice daily). mare had foaled once more than 15 years prior. the theriogenology department was consulted when it was discovered that a large, fluid-filled viscus was the uterus rather than part of the gastrointestinal system. on transrectal ultrasonography, the uterus was diffusely dilated with over 10.5 cm of echogenic fluid. vulva was washed with dilute betadine scrub and rinsed with warm water. a sample was then sterilely aspirated from the uterus via insemination pipette and submitted for aerobic culture and sensitivity. mare treated with oral sulfamethoxazole-trimethoprim (30 mg/kg, twice daily for 14 days) and intravenous gentamicin (6 mg/kg for 7 days) rather than intrauterine antibiotic infusion in an effort to prevent instillation of additional fluid into a uterus that was ineffective at accomplishing clearance. a flexible largebore sterile tube was introduced through the cervix and 12 liters of purulent nonodorous fluid was spontaneously drained from the uterus. fibrotic cervix would only permit 1 finger through the canal alongside the tube. after evacuation of fluid, a large-volume nonsterile lavage was performed. it was elected not to treat with an ecbolic as uterine wall integrity was unknown after being stretched to accommodate the expanding fluid volume. further, with an abnormal cervix, promoting uterine contractility was unlikely to substantially aid in fluid clearance. transrectal ultrasonography and large-volume uterine lavage was repeated daily/every other day as indicated by intrauterine fluid accumulation, and culture revealed growth of beta streptococcus species. mare remained comfortable and bright after the initial fluid evacuation. options discussed with the owner included the conservative approach of monitoring the mare at home and draining the uterus as needed, placement of a stent, or cervical wedge resection. it was decided to first attempt cervical stent placement and if that was unsuccessful a wedge resection would be performed. cervical stent was placed as described1 and the mare returned to the farm. three days after placement, mare was examined via transrectal ultrasonography. approximately 2 cm fluid was present at the uterine bifurcation, and the cervical stent had fallen out of the mare the previous day. stent was replaced twice over the following week due to repeated loss. after the third loss, it was decided to pursue cervical wedge resection. surgery was successful and the mare was treated daily with topical application of antibiotic ointment to http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1006/gcen.1999.7331 30 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 the cervix to prevent closure of the created defect. mare did not accumulate > 2 cm of anechoic fluid in the uterus at any time since initial hospitalization. keywords: mare, pyometra, cervix, stent, colic, wedge resection reference 1. krohn j, ennen s, hospes r, et al: use of a cervical stent for long-term treatment of pyometra in the mare: a report of three cases. reprod domest anim 2019;54:1115-1159. doi: 10.1111/ rda.13480 endometrial mineralization in a barren thoroughbred mare karen von dollen,a alan loynachan,b karen wolfsdorfa ahagyard equine medical institute, lexington, ky, usa bdepartment of veterinary science, university of kentucky, lexington, ky, usa an 11-year, multiparous thoroughbred mare, barren from the previous season, was presented for hysteroscopy to evaluate hyperechoic irregularities observed via uterine transrectal ultrasonography. six nearly circular hyperechoic structures ~ 5 mm in diameter were visible. these structures were distributed at the base of each horn and cranial uterine body. mare had been treated with intrauterine infusion of kerosene on the presumptive diagnosis that these structures may have represented retained endometrial cups. no change in ultrasonographic appearance of these structures was appreciated after kerosene infusion. on hysteroscopy, the structures were directly visualized as tan, smooth, bulbous protrusions into the uterine lumen, with no appreciable disruption of the overlying endometrium. standard uterine biopsy instrument was introduced into the uterine lumen alongside the endoscope; removed structures were submitted for culture, cytology, and histopathology. culture and cytology results were unremarkable, with no evidence of inflammation or etiologic agents. on reevaluation ultrasonography (12 days after hysteroscopy), hyperechoic structures were again visible, and  had proliferated in number (~ 14). hysteroscopy and debulking of the structures in the above manner was repeated, and removed tissue was submitted for histopathology. grossly, the interior of the removed tissue contained tan granular material. histopathology of the nodules revealed 2 types of nodules; one had amorphous basophilic granular material (mineral) and a second type resembled endometrial polyps with no mineral component. deposition of mineral was predominantly in the interstitium of the stratum spongiosum. no etiologic agents were identified. mare became pregnant on second breeding attempt after hysteroscopy (~ 2 months from initial hysteroscopy) and maintained pregnancy through 7 months before being lost to follow-up. histopathologic findings in this case most closely resembled reports of benign endometrial calcification in women, an uncommon finding associated with infertility and abnormal menstruation.1 the current case bears some similarity to a case reported of calcified structures incidentally identified during cesarean surgery of a recipient sow that produced 5 cloned piglets with no gross apparent abnormalities.2 to authors’ knowledge, this is the first report of endometrial calcification in a mare. keywords: mare, mineralization, hysteroscopy references 1. abdullgaffar b, almulla a: endometrial calcifications. intl j surg path 2020;6:590-599. doi: 10.1177/1066896920909425 2. kim ga, jin jx, taweechaipaisankul a, et al: mineralized deposits in the uterus of a pig without pregnancy loss. j vet sci 2017;4:563565. doi: 10.4142/jvs.2017.18.4.563 application of reproductive sciences for conservation of america’s last wild ocelots ashley reeves,a william swansonb aeast foundation, san antonio, tx, usa bcenter for conservation and research of endangered wildlife (crew), cincinnati zoo & botanical garden, cincinnati, oh, usa historically, wild ocelots thrived across the southwestern us but because of habitat loss and fragmentation, overexploitation for fur and pet trade, and unregulated hunting, became listed as an endangered species in the us in 1982. currently, ocelots only occupy a small region along the texas coast in 2 known breeding populations within laguna atascosa national wildlife refuge and private ranches in kenedy and willacy counties. the viability of these small populations is threatened by their proximity to low-lying coastlines with the risk of catastrophic weather-related disasters and their lack of interconnectivity promoting inbreeding and reduced genetic diversity. proposed recovery actions include establishing new ocelot populations within historical ranges in texas, introducing novel genetics into existing populations, and investigating the use of assisted reproductive technologies, such as semen cryopreservation and artificial insemination (ai), to establish a genetic resource bank and mitigate declining genetic variation. our objectives were to compare the effectiveness of semen collection and cryopreservation methods in wild ocelots, evaluate the feasibility of laparoscopic oviductal artificial insemination (lo-ai) using frozen semen sourced from wild texas ocelots to produce pregnancies in zoo-based females, and develop a reintroduction and monitoring plan for creation of a founding wild ocelot population in south texas. live-trapping efforts with wild ocelots were conducted each year from november through april. individuals were anesthetized with a combination of intramuscular ketamine (200 mg/ ml; 4-8 mg/kg) and medetomidine (10 mg/ml; 0.05-0.07 mg/kg). semen samples were collected using either a urethral catheterization method and/or a controlled voltage electroejaculation procedure. semen samples were cryopreserved using an ultrarapid freezing technique and/or standardized straw freezing methods and stored in liquid nitrogen until use. postcollection, anesthesia was partially reversed with a single intramuscular atipamezole (25 mg/ml; 1 mg/5 mg of medetomidine) treatment. for ai, female ocelots in north american zoos were fed oral progesterone daily for 30 consecutive days to suppress ovarian activity. after 7 days of progesterone withdrawal, animals were injected with intramuscular equine chorionic http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1111/rda.13480 https://doi.org/10.1111/rda.13480 https://doi.org/10.1177/1066896920909425 https://doi.org/10.4142/jvs.2017.18.4.563 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 31 gonadotropin (ecg), followed by (82 hours later) porcine luteinizing hormone (plh) or human chorionic gonadotropin (hcg) to stimulate follicular development and ovulation. timed lo-ai procedures were conducted ~ 39 hours after plh/hcg injection. fecal samples were collected for 85 days postai to allow analysis of fecal progesterone concentrations and determination of pregnancy status. from 2019 to 2024, semen was successfully collected from 11 wild ocelots. for collection and cryopreservation of semen samples, best results were obtained using a combination of electroejaculation with straw freezing. for ovarian synchronization of zoo-housed females, the majority (13/18, 72%) of attempts resulted in ovulation of 1 or more follicles followed by apparently normal luteal phases. however, no offspring were produced from lo-ai, likely due in part to suboptimal postthaw semen parameters. our findings suggested that further refinement will be necessary for lo-ai with frozen semen to become a viable approach for promoting gene flow between in situ and ex situ ocelot populations and assisting with the recovery of extirpated wild texas ocelots within their natural range. keywords: ocelot, reproduction, semen banking, artificial insemination, conservation transcriptome analysis reveals gene expression changes in the equine myometrium during pregnancy yatta boakari, a* hossam el-sheikh ali,b* claudia fernandes,c kirsten scoggin,b leticia meloc adepartment of large animal clinical sciences, school of veterinary medicine & biomedical sciences, texas a&m university, college station, tx, usa bdepartment of veterinary science, gluck equine research center, university of kentucky, lexington, ky, usa cdepartment of animal reproduction, school of veterinary medicine and animal science, university of são paulo, são paulo, sp, brazil *corresponding authors; authors contributed equally in eutherian mammals, myometrial quiescence (relaxation) is maintained by the action of progestins through progesterone receptors (nuclear receptors; pgr and membrane receptors pgrmc), with consequent suppression of genes associated with the inflammatory cascade and uterine contraction. during late pregnancy, mares typically exhibit uterine contractions several nights before parturition, and these contractions increase in amplitude and frequency as parturition approaches. however, little is known about the molecular mechanisms triggering myometrial activation during the prepartum and partum periods in mares. we hypothesized that the myometrial transcriptomic profile during normal pregnancy exhibit different regulation of genes in a dynamic profile throughout pregnancy. therefore, using rna sequencing, we sought to characterize the myometrial transcriptome of mares at various points in normal pregnancy. myometrial samples were collected after euthanasia from mares at 4, 6, 9, 10, and 11 months of pregnancy (n = 4 mares/group), and rna sequencing was conducted with the novaseq 6000 platform. reads were mapped to the latest reference genome (equcab3.0) using hisat2 v2.0.5. a comparative analysis of gene expression was performed at 2 pregnancy lengths using the edger r package. the p value was corrected using the benjamini and hochberg method. corrected p value < 0.05 and absolute foldchange (fc) of 2 were set to determine differentially expressed genes (degs). database for annotation, visualization, and integrated discovery was used to functionally annotate degs based on gene ontology using the biological process. the highest number of degs was obtained in 6 versus 10 months (3937 degs) and 9 versus 10 months (3925 degs), suggesting that early changes occur in the 10-months myometrium in preparation for labor. designation of the prepartum group (11 months) was based on a fixed pregnancy (330 days). because normal equine pregnancy length varies widely (320-355 days) and many endocrine and physiologic changes occur immediately peripartum, this group might not capture the immediate peripartum changes in the equine myometrium. nonetheless, in comparison to the myometrium collected from normal pregnant mares at 10 months of pregnancy, we identified 2990 degs (1411 upregulated and 1579 downregulated). functional annotation of degs identified that these genes are involved in relevant biological processes such as prostaglandin biosynthetic process (cd74, edn1, ptgis, mif, ptgds, ptges), muscle contraction (ace2, rgs2, kcnq1) apoptosis (casp3, casp4), inflammatory response (ccl13, cxcl6, ptger1, cxcl2, cx3cl1, ccl2, nlrp1), and the maternal process involved in parturition (oxtr, ldoc1, kalrn), among others. moreover, the 11-months myometrium was associated with functional withdrawal of progesterone that was indicated by the downregulation of pgr and pgrmc1 in comparison to 4 and 6 months, respectively. this functional withdrawal might explain the activation of an inflammatory cascade in the 11-months myometrium. in conclusion, for the first time, equine myometrial transcriptome throughout pregnancy was characterized. we provided a comprehensive database of the key upstream regulators, hub genes, transcription factors, and gene coexpression networks associated with equine myometrial activation. this transcriptomic work improved our understanding of equine myometrial activation and provided a foundation for constructing new hypotheses. keywords: mare, pregnancy, uterus, parturition, rna sequencing antioxidants expression increased in ejaculates obtained after standard teasing in stallions giorgia podico, guilherme novello, lorenzo segabinazzi, igor canisso department of veterinary clinical medicine, college veterinary medicine, university of illinois, urbana, il, usa abundant presence of certain proteins like caseins, annexina2, and crisp-3 in stallion semen proved beneficial for cooling and cryopreservation. amount of seminal plasma affects their presence and expression; in a clinical setting, semen collection protocol dramatically affects the amount of seminal plasma present in each ejaculate. our aim was to determine whether duration of teasing time affects the proteome of sperm and seminal plasma; we hypothesized that teasing time affects proteomic profile of stallion sperm and seminal plasma. forty semen collections were performed from 10 stallions at 48 hours intervals. for each stallion, 2 http://dx.doi.org/10.58292/ct.v16.11054 32 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 collections were performed using standard teasing (i.e. a successful ejaculate was procured < 5 minutes after penile washing), and 2 collections included extended teasing (i.e. after penile washing, stallions were handheld for 10 minutes before allowing them to mount the dummy). ejaculate was submitted to serial centrifugations (1,000 ´ g for 20 minutes) until seminal plasma was separated from sperm; an aliquot of both seminal plasma and sperm pellet was flash-frozen in liquid nitrogen and stored at –80°c until analyses. proteomic analyses were performed by lc/ms-ms and then the data were analyzed with maxquant and perseus. protein groups were filtered, transformed (log2 and ztransformation), and normalized; expression of each protein group was compared among experimental groups with multiple-sample test, fisher exact test, and principal component analysis. we identified 1,237 proteins within samples; protein database was restricted to include only proteins in at least 70% of the samples in each experimental group. protein signature did not differ in sperm and seminal plasma in stallions after a standard and extended teasing. one protein (f6ujz8) in the seminal plasma and 2 other proteins (a0a9lorwe3, a0a5fpli4) in the sperm proteome had an increased (p < 0.05) expression in the seminal plasma of stallions collected after standard teasing compared to the extended teasing. based on the uniprot e. caballus database, the 3 accession numbers correspond to prolycarboxypeptidase, copper transport protein atox1 and aspartylglucosaminidase; the 3 proteins were inferred by homology (f6ujz8, a0a5fpli4) or had evidence at the transcript level (a0a9lorwe3). those proteins were reported to be involved in ion transportation and regulation of oxygen radicals. oxygen radicals are common byproducts of the oxphos metabolism typical of stallion sperm; presence of a higher abundance of antioxidants may translate into positive effects on sperm viability and longevity of the semen processed by stallions after a standard teasing time compared to an extended one. keywords: stallion, teasing, proteomic morphodynamics of in vitro produced equine embryos: frozen versus cooled semen fernando campos-chillon,a,b madelyn bisson,b moises barcelo-fimbres,c joy altermatta aveterinary reproduction innovations, san luis obispo, ca, usa bcalifornia polytechnic state university, san luis obispo, ca, usa cmid valley large animal service, turlock, ca, usa in vitro embryo production (ivp) by intracytoplasmic sperm injection (icsi) in horses has become a more common procedure. however, despite modest advancements in technology and efficiency, there is large variability in embryo production, pregnancy, and early embryo loss rates. some of the variability is due to mare’s estrous cycle, stallion effects and even differences among cooled and frozen semen, and ivp culture conditions. we reported an increase in overall ivp efficiency and pregnancy rates when using extended cooled versus frozen semen.1 to elucidate the differences, aim of this study was to evaluate the morphodynamics of embryo zygote first cellular division up to the blastocyst stage and determine the differences in embryo development time points obtained with frozen or extended cooled semen. we hypothesized that embryos obtained from cooled semen have a faster morphodynamic development rate. embryos (n  =  150) were obtained by icsi using frozen (n = 86) or cooled semen (n = 64) of 40 and 23 different stallions, respectively, of 7 breeds. injected oocytes were placed in a time-lapse incubator and cultured for 9 days. embryo morphodynamics were annotated at the first cytoplasmic extrusion (1), 2, 4, 8, morula (m), compact morula (cm), blastocyst (b) and expanded blastocyst (eb) stages using phototune imaging software. furthermore, the first cleavage division was characterized as normal or abnormal (direct, explosive, or indirect) and blastocyst size was measured. data were analyzed by anova and wilcoxon test for stage time, size and first cleavage normality, respectively, using sas. results indicated no differences (p > 0.05) between frozen and cooled semen for: developing times (hours) at 1 (19.8 ± 1.5 and 20.7 ± 1.8), 2 (28.3 ± 1.6 and 30.2 ± 1.8), 4 (41.3 ± 1.6 and 43.2 ± 1.8), 8 (63.1 ± 1.3 and 63.4 ± 1.6), cm (126.7 ± 1.4 and 127.5 ± 2), b (145.4 ± 1.4 and 146.3 ± 2.1) and eb (156.7 ± 2 and 156.5 ± 2.4) stages; normality of cleavage; or blastocyst size (156.5 ± 1.4 and 158 ± 1.6 µm). however, m formation occurred earlier (p < 0.05) in embryos produced with cooled (100.3 ± 1 hour) versus frozen semen (106.8 ± 1.3). in conclusion, morphodynamics of embryos obtained by frozen or extended cooled semen were very similar except at the m stage, the early formation of which has been reported to be a key for embryo developmental competence and most importantly, the fate of the inner cell mass and the trophectoderm cell lines. further studies are warranted, including sperm decondensation parameters and plcz content, and embryo assessment of mitotic aneuploidy and blastomere multinucleation, controlling for stallion and mare effects of cooled and frozen semen in the model. keywords: mare, embryo, intracytoplasmic sperm injection, time-lapse reference 1. campos-chillon lf, altermatt jl: effect of cooled vs frozen stallion semen on the efficiency of equine in vitro embryo production. j equine vet sci 2023;125:104634. doi: 10.1016/j. jevs.2023.104634 chronic placentitis and abortion caused by aspergillus fumigatus in a mare eduardo arroyo, maria cadario college of veterinary medicine, university of florida, gainesville, fl, usa placentitis in pregnant mares commonly results in abortion, premature birth or compromised neonatal foal health if not addressed on time. although the most common causes of placentitis are of bacterial origin, fungal placentitis can occur sporadically. a 12-year, warmblood mare that had previously produced live foals became pregnant after embryo transfer. following the farm’s preventive health program, mare was vaccinated against equine herpesvirus type-1 (ehv1/4-pneumabort-k) on day 150 of pregnancy. routine reproductive examinations were performed throughout pregnancy, fetal cardiac activity, combined thickness of the uterus and placenta (ctup) and cloudiness of fetal fluids. no abnormalities were noted on transrectal ultrasonography. mare aborted on day 160 of pregnancy. no premonitory clinical signs of placentitis, such as premature mammary gland development or vaginal discharge, were observed. abortion occurred http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1016/j.jevs.2023.104634 https://doi.org/10.1016/j.jevs.2023.104634 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 33 overnight and was unattended. fetus and fetal membranes were kept for veterinarian evaluation. uterine lavage was performed due to fetal membranes retention in nonfetal horn. remaining fetal membranes were removed, and mare was treated with sulfadiazine/trimethoprim (30 mg/kg). uterine samples were submitted for culture and cytology. uterine cytology reveled polymorphonuclear neutrophils, some rod-shaped bacteria and rare yeast forms. klebsiella pneumoniae was isolated. no fungal colonies grew after 120 hours in culture. fetal membranes and fetus were submitted for postmortem examination and pcr. fetal membranes weighed 3.3 kg. approximately 90% of the chorionic surface from the fetal horn was thickened (up to 1.0 cm). avillous areas around the lesions were overlaid by a thick milky grey caseous exudate. there was an invagination from the allantois to the chorionic side of the placenta, surrounded by a chronic inflammatory reaction and encapsulated. on histopathology, the lesions were characterized by chronic, fibrosing, necrotizing allanto-chorionitis with squamous metaplasia of the chorionic surface and adenomatous hyperplasia of the allantois. there were intralesional fungal hyphae with mixed bacteria. hairless female fetus was covered with meconium. no internal organ abnormalities were observed. pcr revealed aspergillus fumigatus as the main pathogen. on the third day postabortion to ‘challenge’ or stimulate an acute inflammatory response, mare was given intrauterine 400 µg of misoprostol. the induction of inflammation was aimed to increase the likelihood of pathogens isolation (authors’ experience). uterine lavage was performed 24 hours later and the effluent submitted for bacterial and fungal culture. klebsiella pneumoniae and mycobacterium spp. were isolated displaying a heavy colonies development. no fungus grew in culture after 120 hours of incubation. mare was treated based on the laboratory sensitivity results. this case represented the need for a thorough pregnancy examination at any time in a broodmare. bacterial and fungal culture with typification in cases of abortion should be routinely performed. keywords: mare, chronic placentitis, fungal abortion equine endometrial organoids as an in vitro model to trial novel therapeutics for endometritis riley thompson,a,b mindy meyers,a,b ahmed gad,b,c gerrit bouma,d christianne magee,b,c budhan pukazhenthi,e fiona hollinsheada,b adepartment of clinical sciences, banimal reproduction and biotechnology laboratory, cdepartment of biomedical sciences, colorado state university, fort collins, co, usa ddepartment of biomedical sciences, western michigan university, kalamazoo, mi, usa ecenter for species survival, smithsonian national zoo and conservation biology institute, front royal, va, usa a healthy uterine environment is essential for equine fertility. approximately 20% of thoroughbred mares demonstrate persistent breeding-induced endometritis (pbie), creating a uterine environment that is unfavorable for pregnancy. despite extensive in vivo studies, the exact mechanisms and best strategies for resolving pbie remains elusive. a potential therapeutic for pbie is extracellular vesicles (evs), which are a heterogeneous population of membrane-bound nanoparticles containing proteins and nucleic acids, secreted by equine embryo-derived mesenchymal stem cells (edmscs). we hypothesized that evs secreted by edmscs contain antiinflammatory mirnas that prevent pbie in susceptible mares. using organoids as physiologically-relevant 3d cell cultures, our objectives were to assess: 1. presence of specific antiinflammatory mirnas in edmsc-derived evs; and 2. mirna and mrna of endometrial organoids (n = 6 mares; kenneydoig score iib) pretreated with or without 40 x 106/ml evs 24 hours prior to inflammatory stimulation with frozen-killed sperm (fks), lipopolysaccharide (lps), or pbs (control) for 3 and 12 hours. data were analyzed using anova with tukey’s posthoc test with organoids not pretreated with evs and not stimulated with inflammatory reagents serving as control. significance was set at p  <  0.05. results demonstrated the presence of specific antiinflammatory mirnas (mir-10a, mir-21, mir-24, mir-145, and mir-146a) in evs secreted by edmsc cell lines. however, these antiinflammatory mirnas did not increase in organoids after ev treatment (p > 0.05). treatment of organoids with fks did not result in inflammatory gene expression differences (p > 0.05) compared to control. however, organoids pretreated with evs then stimulated with lps exhibited increased (p  <  0.05) expression of proinflammatory il1 at 3 hours and ptgs2 at 12 hours after lps exposure compared to control organoids. furthermore, organoids, with or without ev pretreatment, stimulated with lps had increased expression of proinflammatory il6 (3 and 12 hours) and antiinflammatory il1rn (12 hours) compared to control organoids. these gene expression changes mirror reports describing inflammation of the equine endometrium associated with breeding in vivo.1,2 in conclusion, these data indicated that: 1. edmsc evs contain antiinflammatory mirnas; 2. pretreatment of organoids with evs failed to alter mirna expression in the recipient organoids under the conditions evaluated; and 3. lps stimulated an inflammatory response in endometrial organoids similar to in vivo, demonstrating their utility as an in vitro equine endometritis model. future studies will investigate the effect of reproductive steroid hormones on inflammatory cytokine protein and gene expression in this organoid model of equine endometritis. keywords: mares, 3d culture, organoid, endometritis, inflammation references 1. woodward em, christoffersen m, campos j, et al: endometrial inflammatory markers of the early immune response in mares susceptible or resistant to persistent breeding-induced endometritis. reproduction 2013;145:289-296. doi: 10.1530/rep-12-0452 2. palm f, walter i, budik s, et al: influence of different semen extenders and seminal plasma on pmn migration and on expression of il-1b, il-6, tnf-a and cox-2 mrna in the equine endometrium. theriogenology 2008;70:843-851. doi: 10.1016/j. theriogenology.2008.04.054 http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1530/rep-12-0452 https://doi.org/10.1016/j.theriogenology.2008.04.054 https://doi.org/10.1016/j.theriogenology.2008.04.054 34 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 use of prostatic extracellular vesicles to improve viability and longevity of frozen-thawed canine sperm alexandra hafey,a mindy meyers,a james graham,b fiona hollinshead,a adepartment of clinical sciences, bdepartment of biomedical sciences, college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co, usa reproductive success after artificial insemination (ai) with frozen-thawed dog sperm is highly variable. even the most effective freezing methods results in compromised sperm function which negatively impacts fertility. cryopreservation can damage sperm membranes, including plasma membrane (resulting in cell death or reduced longevity) and acrosomal membranes (that can cause premature acrosomal exocytosis). adding prostatic fluid (pf) to frozen-thawed canine sperm benefits sperm motility, improves conception rates and results in larger litter sizes.1 extracellular vesicles (evs) are nanoparticles secreted by all cells that mediate cell communication via their cargo. we hypothesized that evs produced by prostatic cells have a physiological role in canine sperm function by modulating cryo-damaged sperm membranes, leading to enhanced sperm longevity, viability, and function. study was conducted to determine if evs, from pf, could ameliorate cryodamage to canine sperm. evs were isolated from pf obtained from 6 young dogs (< 2 years) and were used to treat frozen-thawed sperm from semen collected from 3 young and 3 old dogs (> 6 years). after thawing, sperm were diluted in a capacitating media and treated with: 1. no evs; 2. pf evs; or 3. dilauroylphosphatidylcholine (pc12; + control). sperm were incubated, at 37°c, and subsamples assessed for motility (subjectively and computer aided sperm analysis) and sperm viability, acrosome integrity, and membrane fluidity, using flow cytometry, over 24 hours. in addition, the ability of sperm to bind the zona pellucida was evaluated using a perivitelline membrane (pv) binding assay. sperm from young and old dogs responded similarly to treatments; therefore, data were combined. canine sperm treated with pc12 had higher percentages (p < 0.05) of live acrosome reacted sperm (> 6%) than control sperm (3%) and more sperm binding to pv membranes (10% higher binding). sperm treated with pf evs exhibited similar percentages (p > 0.05) of motile sperm, sperm with fluid membranes and live acrosome reacted sperm throughout incubation, but pf evs did increase sperm binding to pv membranes (11% higher binding). in conclusion, the addition of pf derived evs to frozen-thawed canine sperm may improve some sperm functions and help to improve reproductive performance of cryopreserved canine sperm. keywords: extracellular vesicles, dog, frozen-thawed semen, fertility reference 1. nöthling jo, shuttleworth r, de haas k et al: homologous prostatic fluid added to frozenthawed dog spermatozoa prior to intravaginal insemination of bitches resulted in better fertility than albumin-free talp. theriogenology 2005;64:975-991. doi: 10.1016/j.theriogenology.2005.01.002 http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1016/j.theriogenology.2005.01.002 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 35 student abstracts dr. jimmy alexander student case reports chronic endometritis caused by uterine marble in a mare cindy artola, kianna spencer, giorgia podico department of veterinary clinical medicine, college of veterinary medicine, university of illinois urbana-champaign, urbana, il, usa uterine marbles are used in mares to suppress estrus; effectiveness of this device varies in studies from 11 to 41.3%.1 unfortunately, use of marbles has also been linked to chronic endometritis, uterine fibrosis, adhesions, and subfertility; those risks are exacerbated when they are left in the uterus for a prolonged period.2 a 14-year, retired racing standardbred mare, was presented for breeding management and subfertility workup. history reported that the mare failed to become pregnant during 2 years, despite multiple breeding attempts. initial reproductive examination revealed signs of estrus and poor perineal conformation. uterine culture and cytology were performed that had heavy growth of staphylococcus aureus and severe inflammation. transvaginal procedures revealed cervical adhesions. treatment for endometritis included daily uterine lavages, infusions, antiinflammatory medication (dexamethasone), and oral antibiotic (sulfadiazine trimethoprim 24 mg/ kg, every 12 hours). during one treatment, a hard foreign body was felt within the uterus, and it was removed and identified as a glass marble. during next estrous cycle, mare was inseminated with cooled semen. after ai, mare developed severe postbreeding induced endometritis that was managed with uterine lavages, ecbolics, and oral antiinflammatory medication (firocoxib 0.1 mg/kg, once a day); a double synchronous ovulation was confirmed after 24 hours. a caslick’s vulvoplasty was placed. mare was confirmed pregnant 14 days later, with a singleton pregnancy visualized at the base of left horn. progesterone concentrations were determined and were low to support pregnancy; exogenous progestin was recommended. this case highlighted the importance of a breeding soundness examination prior to breeding and the risks in using uterine marbles leading to subfertility and endometritis. keywords: mare, subfertility, uterine marble, estrus suppression references 1. nie gj, johnson ke, braden td, et al: use of an intra-uterine glass ball protocol to extend luteal function in mares. j equine vet sci 2003;23:266-273. doi: 10.1053/jevs.2003.75 2. de amorim md, chenier t, nairn d, et al: complications associated with intrauterine glass marbles in five mares. j am vet med assoc 2016;15;249:1196-1201. doi: 10.2460/ javma.249.10.1196 fungal placentitis in a mare jenny buchanan, giorgia podico, kianna spencer department of veterinary clinical medicine, college of veterinary medicine, university of illinois urbana-champaign, urbana, il, usa placentitis causes 60% of pregnancy loss, periparturient disease and neonatal morbidity in horses.1 clinical signs include premature udder development, vulvar discharge and spontaneous abortion.2 a 11-year, quarter horse mare was presented for premature udder development on day 290 of pregnancy, raising suspicion of placentitis. external examination revealed no signs of vulvar discharge with intact caslick’s vulvoplasty. moderate mammary development with expressible yellow and cloudy secretions were observed. ultrasonographic examination revealed increased combined thickness of the uterus and placenta (2.3 cm). empirical therapies included intramuscular estradiol cypionate (10 mg, every 72 hours), oral sulfamethoxazole-trimethoprim (30 mg/kg, every 12 hours), oral pentoxifylline (8.5 mg/kg, every 12 hours), and oral firocoxib (57 mg, every 24 hours). due to high risk of dystocia and other complications, mare was hospitalized for foaling, monitoring and assistance. parturition occurred on day 327 of pregnancy with no complications. examination of fetal membranes revealed a 20 x 20 cm avillous area of the chorionic surface surrounded by mildly raised edges with tan mucoid material at the base of gravid horn. fetal membranes were submitted for histopathology and evaluation. histologic analysis revealed severe, locally extensive necrosuppurative placentitis infiltration of the chorion by candida albicans. fungal treatment was performed during estrus (day 30 after parturition). intrauterine infusion of 1% hydrogen peroxide (120 ml), followed with a large-volume uterine lavage, intrauterine fluconazole and nystatin (100 mg/0.5 mu, once a day for 3 days), and oral fluconazole (4 mg/kg, once a day for 21 days). it was hypothesized that nocardioform or ascending placentitis was resolved by long-term antibiotic treatment, allowing growth of opportunistic fungus. this case underscored the importance of thorough diagnostic evaluations and targeted treatment in cases of placentitis. keywords: mare, placental disease, high-risk pregnancy references 1. giles rc, donahue jm, hong cb, et al: causes of abortion, stillbirth, and perinatal death in horses: 3,527 cases (1986–1991). j am vet med assoc 1993;203:1170-1175. doi: 10.2460/ javma.1993.203.08.1170 2. canisso if, ball ba, erol e, et al: comprehensive review on equine placentitis. proc am assoc equine pract 2015;61:490-509. http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1053/jevs.2003.75 https://doi.org/10.2460/javma.249.10.1196 https://doi.org/10.2460/javma.249.10.1196 https://doi.org/10.2460/javma.1993.203.08.1170 https://doi.org/10.2460/javma.1993.203.08.1170 36 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 hemospermia in a stallion caused by a urethral rent jack richison iii, charles love, camilo hernandez-aviles, juan samper department of large animal clinical sciences, school of veterinary medicine, texas a&m university, college station, tx, usa hemospermia in the stallion ranges from small to large (overt) amounts of blood that can result from penile trauma, infectious conditions, penile neoplasias, urethral rents, seminal vesiculitis/ ampullitis, or urethritis.1 a 10-year, american quarter horse stallion was presented in 2023 for acute hemospermia. during the 2022 breeding season, stallion bred ~ 150 mares with normal fertility. on presentation, stallion had a normal tpr with no visible lesions on the penis. a blood (overt) contaminated ejaculate, containing 10 x109 sperm was collected. through a urethral endoscopy an 8-10 mm urethral rent was observed cranial to the bulbourethral gland openings and caudal to colliculus seminalis.2 rent was treated by laser therapy to stimulate healing in combination with a perineal corpus spongiotomy to relieve urethral pressure.3,4 following surgery, a large scrotal swelling developed. urethroscopy was repeated 2 weeks later, and it was determined that the rent had reduced in size but had not healed completely, and a second laser treatment was performed targeting the rent lumen in an effort to stimulate healing from the inside. a third urethroscopy was performed to confirm rent healing. subsequently, 2 ejaculates containing no blood and 2.88 and 0.87 x109 total sperm; 9 and 20% normal sperm and 44 and 22% abnormal dna were collected. based on his total testes volume of 271 ml3 his predicted total sperm of ~ 5.5 x109 sperm was lower than expected, perhaps due to the scrotal swelling and it is yet to be determined if sperm quality improves for the 2024 breeding season. overt hemospermia in this stallion was resolved using a combination of laser therapy and surgical intervention. successful treatment of overt hemospermia is essential to maintain normal fertility5 provided his semen quality returns to normal values. keywords: stallion, hemospermia, urethral rent references 1. pearson, lk, campbell aj, et al: how to diagnose and treat hemospermia: a review and case series. proc am assoc equine pract 2013;59:40-50. 2. amann rp: a review of anatomy and physiology of the stallion. j equine vet sci 1981;1:83-105. doi: 10.1016/ s0737-0806(81)80022-6 3. madron m, schleining j, caston s, et al: laser treatment of urethral defect in geldings and stallions used as the primary treatment or in conjunction with a temporary subischial incision: eight cases (2003-2011). equine vet j 2013;25:368-373. doi: 10.1111/eve.12050 4. schumacher j, varner dd, schmitz dg, et al: urethral defects in geldings with hematuria and stallions with hemospermia. vet surg 1995;24:250-254. doi: 10.1111/j.1532-950x.1995.tb01326.x 5. turner ce, walbornn sr, blanchard tl, et al: the effect of two levels of hemospermia on stallion fertility. theriogenology 2016; 86:1399-1402. doi: 10.1016/j.theriogenology.2016.04.084 unusual case of hemospermia and poor sperm quality in a stallion john deramus, charles love, camilo hernandez-aviles, juan samper department of large animal clinical sciences, school of veterinary medicine, texas a&m university, college station, tx, usa a 12-year, quarter horse stallion, was examined in mid-september 2023, on-farm, due to a history of a sudden reduction in sperm numbers and quality. stallion had normal libido, large testes, and bilateral distention of the cauda epididymides. a presumptive diagnosis of plugged ampullae1 was made and a recommendation of per rectum ampullar massage followed by daily semen collection was suggested. several days later the referring veterinarian reported a brown discolored ejaculate. stallion was referred for further evaluation. complete breeding soundness examination was performed. relevant findings included testicular volume of 410 ml, 1.5 x 109 sperm/ejaculate, 25% total motile and morphologically normal sperm and a thick, brown colored ejaculate. culture of the discolored semen yielded no bacterial growth and no cytological evidence of inflammation or intact red blood cells. ultrasonographic evaluation of the ampullae identified unusual hyperechoic areas in the glandular region. semen collected in an openended artificial vagina localized the brown-tinged semen to the sperm-rich fraction of the ejaculate that originated from ampullae, ductus deferens and cauda epididymis. potential differentials for hemospermia include seminal vesiculitis/ ampullitis,2 habronemiasis, or urethral rent3 and were ruled out based on the ejaculate color (red versus brown) and negative bacterial culture and cytology, and ultrasonographic findings. in retrospect, the initial history (low sperm numbers and poor sperm quality) was likely a result of the prolonged elevated ambient temperature experienced in the state of texas in the summer of 2023,4 rather than sperm accumulation. in this case, ampullar massage probably resulted in an unexpected ampullary hemorrhage event manifested as brown hemolyzed blood. during next 3 months regular ejaculation resulted in elimination of blood and improvement in overall semen quality. keywords: heat stress, sperm accumulation, hemospermia references 1. love cc, riera fl, oristaglio r, et al: sperm occluded (plugged) ampullae in the stallion. proc soc theriogenol 1992;17:127. 2. blanchard tl, varner dd, hurtgen jp, et al: bilateral seminal vesiculitis and ampullitis in a stallion. j am vet med assoc 1988;192:525-526. 3. pearson l k, campbell a j, et al: how to diagnose and treat hemospermia: a review and case series. proc am assoc equine pract 2013;59:40-50. 4. freidman r, scott m, heath se, et al: the effects of increased testicular temperature on spermatogenesis in the stallion. j reprod fertil 1991;suppl 44:127-134. http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1016/s0737-0806(81)80022-6 https://doi.org/10.1016/s0737-0806(81)80022-6 https://doi.org/10.1111/eve.12050 https://doi.org/10.1111/j.1532-950x.1995.tb01326.x https://doi.org/10.1016/j.theriogenology.2016.04.084 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 37 successful treatment of fungal and bacterial endometritis in an embryo donor mare megan murphy, kianna spencer, humberto magalhaes, giorgia podico department of veterinary clinical medicine, college of veterinary medicine, university of illinois urbana-champaign, il, usa fungal endometritis is a rare but potentially devastating cause of infertility in mares. infection can result from a variety of causes and often presents together with bacterial endometritis; thus, treatment can be complex and often carries a poor prognosis for future reproductive performance. a case of a 13-year, multiparous mare, presented for artificial insemination and embryo transfer after pregnancy loss is described. culture and cytology indicated severe inflammation and infection with candida spp.; treatments included daily intrauterine antifungals (fluconazole 100  mg, nystatin 0.5 mu), uterine lavages (saline with iodine solution 0.05%, dmso 10%) and oral fluconazole (10 mg/kg followed by 5 mg/kg, once daily). during the following estrous cycle, further tests confirmed the presence of a concomitant bacterial endometritis due to streptococcus zooepidemicus. treatments included daily uterine lavages and infusions (ceftiofur 1 gram, nystatin 0.5 mu), intramuscular ecbolics (oxytocin 20 ui), an oral antiinflammatory (firocoxib 0.1 mg/kg), an oral hemorheological (pentoxifylline 8 mg/kg, twice a day), and an oral antifungal agent. mare was bred with cooled semen, and a double ovulation was confirmed within 24 hours. two goodquality blastocysts were recovered 8 days after, thoroughly washed, and transferred into 2 recipient mares. with the concern of transmitting yeasts, recipient mares were also treated prophylactically with an antifungal (fluconazole 10 mg/kg followed by 5 mg/kg, once daily). unlike other equine pathogens such as equine viral arteritis that is known to be highly transmissible through embryos, yeasts are not mentioned in the list of diseases and pathological agents by the oie in the terrestrial animal health code 4.8.14.¹ recipient mares recently delivered healthy foals, proving that the treatment protocol, in this case, was effective in controlling not only the endometritis of the donor mare but also in preventing the spread of infection to the recipient mares. keywords: embryo transfer, pregnancy loss, fugal endometritis reference 1. oie: terrestrial animal health code. chapter 4.8, article 4.8.14. 2019. available from: https://www.woah.org/en/what-we-do/ standards/codes-and-manuals/terrestrial-code-online-access/?id=169&l=1&htmfile=chapitre_coll_embryo_equid.htm [cited 4 february 2024]. pyometra and cystic endometrial hyperplasia secondary to cervical neoplasia in a doe evie rider, shannon reed, mariah pearson, sophia marchio, yatta boakari department of large animal clinical sciences, school of veterinary medicine & biomedical sciences, texas a&m university, college station, tx, usa an 11-year, nigerian dwarf cross-doe, was presented with a 3-day history of straining to urinate. doe had never been bred but presented with an enlarged udder that had been present for ~ 3 years. physical examination was unremarkable. on abdominal radiographs, a soft tissue structure filled with fluid was observed in the expected location of urinary bladder. an enlarged uterus with thickened walls and heterogeneous fluid was observed on transabdominal ultrasonography. based on presenting complaint and examination findings, a uterine mass and pyometra were suspected, and ovariohysterectomy was scheduled. after surgery, uterus and ovaries were submitted for histopathology. a grossly pale tan-to-white mass measuring 3 x 2.5 x 1 cm attached to uterus and cervix and cystic endometrial hyperplasia were observed. histology was performed, and findings were consistent with a leiomyoma with mild, multifocal, chronic lymphoplasmacytic endometritis with cystic endometrial hyperplasia. when doe returned for reexamination, there was minimal edema around incision site, and ultrasonography revealed no fluid in the uterine stump. leiomyomas have been reported in small ruminants,1,2 but it is not a common disease. this is likely due to does not living long enough to develop neoplasia. however, with the increased popularity of pet goats, we are noticing more cases because they are living longer. presence of leiomyoma in the cervical area is likely the cause of the other clinical signs such as cystic endometrial hyperplasia, pyometra, and straining to urinate noticed by the owner. cystic endometrial hyperplasia in ruminants is associated with hyperestrogenism,3 that could be due to the increased steroid receptors present in leiomyomas.4 pyometra is commonly caused by contamination from bacteria in the vagina ascending into the uterus. the mass prevented the physiological closing of the cervix in the nonpregnant state and allowed an ascending infection that resulted in the pyometra. keywords: does, cervical neoplasia, pyometra, cystic endometrial hyperplasia references 1. hananeh wm, ismail zb, daradka mh: tumors of the reproductive tract of sheep and goats: a review of the current literature and a report of vaginal fibroma in an awassi ewe. vet world 2019;12:778-782. doi: 10.14202/vetworld.2019.778-782 2. linton jk, heller mc, bender sj, et al. neoplasia of the tubular genital tract in 42 goats. j am vet med assoc 2020;256:808813. doi: 10.2460/javma.256.7.808 3. jubb kvf, kennedy pc, palmer n: the female genital system. in: maxie mg: editor. pathology of domestic animals. 5th edition, st louis; saunders ltd; 2015, p. 460-466. 4. millán y, gordon a, de los monteros ae, et al: steroid receptors in canine and human female genital tract tumours with smooth muscle differentiation. j comp pathol 2007;136:197-201. doi: 10.1016/j.jcpa.2007.01.004 http://dx.doi.org/10.58292/ct.v16.11054 https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/?id=169&l=1&htmfile=chapitre_coll_embryo_equid.htm https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/?id=169&l=1&htmfile=chapitre_coll_embryo_equid.htm https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/?id=169&l=1&htmfile=chapitre_coll_embryo_equid.htm https://doi.org/10.14202/vetworld.2019.778-782 https://doi.org/10.2460/javma.256.7.808 https://doi.org/10.1016/j.jcpa.2007.01.004 38 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 dystocia associated with fetal monster in a mare rachael willis, darcie sidelinger, heath king, gabriel gonzalez, chris stokes, nicholas wheeler, julie white college of veterinary medicine, starkville, ms, usa an 11-year, quarter horse, mare presented with dystocia of ~ 8 hours. estimated age of fetus was 268 days. fetus was in posterior presentation, dorsosacral position with bilateral hock flexion. fetus was retropulsed into uterus to allow repositioning of the hocks and placement of obstetrical chains for assisted vaginal delivery of the nonviable fetus. fetus had numerous congenital abnormalities of the skull as well as umbilical torsion. after parturition, ~ 6 cm grade 2 vaginal tear was noted ~ 3 cm into the vagina on the right side. uterus was lavaged with 7 gallons of isotonic, 0.05% betadine solution. mare received intravenous lactated ringer’s solution with calcium, magnesium, phosphorous, and potassium at constant rate infusion of 1 liter/hour for the first 24 hours postpartum, antibiotics, antiinflammatory, oxytocin, and animax ointment. uterus was lavaged twice daily for 4 days, then once daily for 2 additional days. mare exhibited signs of mild colic 3 days postpartum that was medically managed with xylazine and buscopan. mare expelled fetal membranes on the same day. mare was discharged after 8 days of hospitalization. necropsy of fetus revealed following congenital abnormalities: crania bifida, temporalis bone with aplasia cutis, palatoschisis, maxillary brachygnathism, anophthalmia right eye with incomplete cranial orbit, hydrophthalmia left eye, and campylorrhinus lateralis. there were no substantial internal abnormalities outside of the skull. dystocia in mares is considered uncommon, but a recent retrospective study1 reported the incidence of dystocia in healthy mares and mares that had previously experienced dystocia. dystocia frequency was 9.7%;17.5% mares that had previously experienced dystocia compared to 6.7% in mares without a history of dystocia. foal mortality was 1.9%, whereas maternal mortality was 0.5%. keywords: mare, abortion, dystocia, umbilical torsion reference 1. ellerbrock m, krohn j, büttner k, et al: dystocia frequency and causes in horses with pregnancy disorders or a history of dystocia: a prospective study. reprod domest anim 2024;59:e14541. doi: 10.1111/rda.14541 dystocia and uterine rupture after cloprostenol treatment in a pregnant llama ammasie allred, muhammad waqas, ahmed tibary department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa, usa an adult female llama was presented to theriogenology service for retained fetal membranes and emaciation. llama was rescued 1-week before alongside an intact male. a veterinarian was consulted and diagnosed the llama to be open but still gave an unknown dose of cloprostenol to terminate any potential early pregnancy. next day, owners noticed fetal membranes hanging from vulva. lama’s health deteriorated during the following few days and lama was presented. llama was dull and anxious, with normal vital parameters. a foul vaginal discharge was noted. transrectal palpation and ultrasonography, transabdominal ultrasonography and vaginal examination revealed a retained fetus and a tight vestibular sphincter. a baseline cbc yielded left shift with toxic neutrophils and normal blood chemistry. manual manipulation was elected as cesarean surgery was not an option. vestibular sphincter was progressively dilated manually and cervix was fully dilated. approximately 8month fetus (anterior longitudinal presentation, dorsosacral position, head and both forelegs fully extended) was manually delivered. subsequent uterine examination revealed a fullthickness dorsal uterine tear without fresh bleeding. humane euthanasia was elected, and llama and fetus were submitted for necropsy. necropsy confirmed a 10 cm full-thickness tear on the dorsal uterine body and lochia in the abdominal cavity. uterine rupture possibly resulted from uterine contractions with a blocked birth canal due to nondilated vestibular sphincter, leading to subsequent peritonitis, left shift with toxic neutrophils, and clinical signs. this case highlighted the repercussions of misdiagnosis in theriogenology practice on animal welfare and the importance of theriogenology training in the dvm curriculum. although camelid dystocia rates are low, failure of vestibular dilation has been reported to be a maternal cause of dystocia.1 dystocia is an emergency in camelids, and prolonged time to seek treatment contributes to poor prognosis. keywords: camelids, dystocia, vestibular sphincter, abortion reference 1. tibary a, rodriguez j, sandoval s: reproductive emergencies in camelids. theriogenology 2008;70:515-534. doi: 10.1016/j. theriogenology.2008.04.024 http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1111/rda.14541 https://doi.org/10.1016/j.theriogenology.2008.04.024 https://doi.org/10.1016/j.theriogenology.2008.04.024 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 39 dr. jimmy alexander student research posters effects of intrauterine ceftiofur on equine uterine microbiome kalie beckers,a chin liu,a christopher schulz,b gary childers,b jenny sonesc aveterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la, usa bdepartment of biological sciences, southeastern louisiana university, hammond, la, usa cdepartment of clinical sciences, college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co, usa overuse of antibiotics is becoming overwhelming in veterinary medicine resulting in antibiotic resistance and dysbiosis of natural microflora. antimicrobial therapy, such as ceftiofur, a thirdgeneration cephalosporin, is a mainstay for treating reproductive diseases, including endometritis. we hypothesized that an infusion of ceftiofur disrupts healthy mare’s uterine resident microbial community. university-owned mares (n = 8) underwent transrectal palpation and ultrasonography to determine the stage of estrous cycle. a double guarded endometrial swab (mofa®) was inserted transcervically into uterus for characterizing estrual uterus after 3 days of sham (saline) and then subsequently 1 gram of ceftiofur (naxcel®) intrauterine infusion. at third estrus, mares were bred to the same stallion via artificial insemination. the v4 region of the 16s rrna gene was amplified for illumina miseq sequencing to examine core bacterial communities present before and after ceftiofur. microbial community composition of sham and ceftiofur treated mares were not different (p  =  0.321; [permanova with bray-curtis dissimilarity of 16s amplicon sequence variant’s relative abundance]). alpha diversity was not significantly different after ceftiofur, meaning there were no differences in richness and evenness in the microbial communities after treatment. only notable difference was the abundance of christensenellaceae_r-7_ group was lower (p = 0.0428) after ceftiofur (0.14 ± 1.05% versus 2.89 ± 1.07% control) treatment. finally, mares bred by artificial insemination achieved pregnancy. in conclusion, 3-day treatment of ceftiofur did not acutely change the microbial communities within mare’s uterus when sampled directly after treatment. a limitation of this study was time of sampling. ceftiofur may have a long-term effect on the uterine microbiome that may require sampling several weeks after treatment. in conclusion, ceftiofur did not change the healthy uterine microbiome during estrus and did not affect pregnancy rates; however, should be used judiciously because it may have long-term microbial effects. keywords: mare, pregnancy, microbiome, antibiotics probable and actual degrees of homozygosity as a predictor of sperm quality in the domestic cat kristyn burton,a leslie lyons,b dale kelley,c aime johnsond acollege of veterinary medicine, oklahoma state university, stillwater, ok, usa bdepartment of veterinary medicine & surgery, college of veterinary medicine, university of missouri, columbia, mo, usa cdepartment of veterinary clinical sciences, college of veterinary medicine, oklahoma state university, stillwater, ok, usa dscott-ritchey research center, college of veterinary medicine, auburn university, auburn, al, usa important populations of purpose-bred cats which include endangered nondomestics and closed research colonies are bred selectively, representing reduced gene pools and high inbreeding. parameters of sperm quantity and quality are widely regarded as relevant to an individual’s breeding soundness, a relationship well established in many species but largely unexplored in domestic cats. the hypothesis is that poor sperm quality and increased homozygosity are correlated. this study compared semen characteristics with 2 measures of inbreeding, a pedigree-based coefficient of inbreeding (coi, a probable value indicating likelihood of homozygosity) and a genetic analysis (a measure of actual homozygosity), to determine their predictive power regarding ejaculate characteristics in the male domestic cat (felis catis). healthy adult male domestic cats (n = 18) were collected via electroejaculation under anesthesia (ketamine 10 mg/kg, dexmedetomidine 30 µg/kg) 1week after an initial collection. semen characteristics were recorded: ejaculate volume, concentration, total sperm number, total and progressive motilities, and morphology. inbreeding coefficients, calculated for each cat based on 12+ generations of archival records, ranged from 0 to 32.8%. dna samples were then collected via buccal swab and submitted to uc davis’s veterinary genetics laboratory for genotyping by sequencing using the international society of animal genetics standardized panel of single nucleotide variant markers for the cat on their ion genestudio s5 platform. correlations (p < 0.05) were identified between coi and progressive motility, total motility, and percentage of sperm with coiled tails. differences (p < 0.05) in both motilities and percent morphologically normal sperm in individuals with a coi below 10% when compared to those above 10% were also identified. genetic analysis results are pending. understanding correlations between genetic diversity and ejaculate characteristics will provide insight into the effects of inbreeding in felid populations, as well as a possible model for evaluation and selection of potential breeding stock. keywords: domestic cat, inbreeding, fertility, sperm, coefficient of inbreeding http://dx.doi.org/10.58292/ct.v16.11054 40 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 comparison of domestic feline sperm vitality in tes-tris egg yolk, felineoptimized culture medium, and tre-tris-citrate fructose monica lee,a amy miller,b lindsey vansandt,b xiaojuan zhu,c julie barnesb acollege of veterinary medicine, university of tennessee, knoxville, tn, usa bcenter for conservation and research of endangered wildlife, cincinnati zoo and botanical garden, cincinnati, oh, usa cuniversity of tennessee office of information technology, knoxville, tn, usa appropriate sperm handling medium is crucial to maintain sperm viability and ensure fertilization competence for downstream assisted reproduction procedures. however, little is done to optimize a sperm handling medium for the domestic cat. objective was to compare the effects of 3 holding media (testris egg yolk [tey], feline-optimized culture media [focm], and tre-tris-citrate fructose [ttcf]) on sperm morphology, total motility (tm), and progressive motility (pm). adult male domestic cats (n  =  4) were anesthetized and subjected to 1-2 rounds of electroejaculation for sperm collection. semen was immediately split into 3 aliquots and diluted to a concentration range of 15.76-49.04 x 106 total sperm/ml. computer assisted sperm analysis (casa) system-ivos ii (hamilton thorne, beverly, ma) was utilized for assessments of morphology, tm, and pm at the 0, 2, 4, 6, 24, and 48 hours timepoints. data were analyzed with a mixed effect anova model and reported as mean ± sd. statistical significance was set at p < 0.05. across all timepoints, tey had a higher percent normal morphology (89.48 ± 7.25) than focm (79.27 ± 6.74); no other differences were observed (ttcf: 83.53 ± 5.29). at 0 hour, pm was lower in focm (34.2 ± 2.2) than tey (53.3 ± 14.08) and ttcf (57.98 ± 5.95). at 2 and 4 hours, pm was lower in focm (2 hours: 31.28 ± 3.88; 4 hours: 24.95 ± 1.98) than ttcf (2 hours: 52.88 ± 1.72; 4 hours: 41.4 ± 2.68). however, at 24 hours, focm exhibited higher pm (20.15 ± 7.35) than tey (3.8 ± 4.96) and ttcf (2.7 ± 5). similarly, focm also demonstrated higher tm (56.15 ± 15.48) than tey (13.88 ± 17.33) and ttcf (6.93 ± 11.3) at 24 hours. no other differences between treatments were observed for tm. in conclusion, although our findings revealed an initial reduction in pm and tm with focm, it demonstrated sustained pm and tm over a 24-hour period, surpassing the performance of tey and ttcf. these insights contributed valuable information for optimizing sperm handling media in the domestic cat. keywords: domestic cat, sperm handling media, motility, morphology multigenerational toxicity in progeny of zebrafish with developmental trichloroethylene exposure taylor yenrick, katharine horzmann department of pathobiology, college of veterinary medicine, auburn university, auburn, al, usa trichloroethylene (tce) is a legacy environmental toxicant used heavily up through the 1970’s as metal degreaser and an industrial solvent. due to a long half-life and persistence in soil and ground water, tce toxicity is still a concern today. tce is a known carcinogen and has been linked to spontaneous abortion, decreased birth weight, and developmental neurotoxicity. tce is also suspected to cause epigenetic toxicity. this study used zebrafish biomedical model (danio rerio) to test the hypothesis that developmental tce exposure causes toxicity in the exposed group and their offspring. wild-type zebrafish (f0) were developmentally (0-5 days after fertilization) exposed in groups to 0, 5, 50, and 500 parts per billion (ppb; µg/l) tce. reproductive toxicity in f0 zebrafish was assessed through success and fecundancy of dose-matched paired breedings. developmental toxicity in the f1 progeny was assessed via video recorded and computer analyzed embryonic photomotor response and larval visual motor response behavior assays, heart rate analysis, and morphologic assessments. f0 reproductive assays demonstrated no significant alterations in embryo production or embryonic survival. in the f1 progeny, the 5-ppb embryos were hyperactive in the embryonic photomotor response test. on day 5 after fertilization, larval zebrafish had decreased movement and activity and altered turning behavior in all exposure groups. all exposure groups had altered morphologic parameters including body length, head length and width, eye diameter, and jaw length. finally, the 500-ppb progeny demonstrated an increased heart rate (108.7 ± 17.0 bpm) as compared to the controls (100.9 ± 15.8 bpm). despite no toxicity observed in the f0 parental generation, f1 progeny had significant behavioral, physiologic, and morphologic changes. developmental tce exposure in parental generations resulted in toxic effects in the offspring. given the widespread contamination, tce may pose a risk to people and animals living around contaminated sites. keywords: trichloroethylene exposure, zebra fish, toxicity, developmental changes http://dx.doi.org/10.58292/ct.v16.11054 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 41 noncompetitive student posters malignant seminoma in a stallion julia slocum-coffey,a jarrod troy,a tyler harm,b kevin kersh,a theresa beachlerc adepartment of veterinary clinical sciences, bdepartment of veterinary pathology, cdepartment of veterinary diagnostic and production animal medicine, college of veterinary medicine, iowa state university, ames, ia, usa a 22-year, icelandic stallion, was presented with 1-year history of scrotal enlargement. left testis was diffusely enlarged and soft on palpation compared to right testis. scrotal ultrasonography identified a heterogenous multilocular, mass-like effect distorting entire left testis with minimal normal-appearing testicular parenchyma. closed, left hemicastration was performed to conserve stallion’s breeding potential. left testis, on cut surface, contained only a thin rim of normal appearing testicular parenchyma surrounding an extensive multilocular, pale glistening mass. histopathology identified diffuse sheets of round to polygonal cells effacing and replacing normal testicular parenchyma with marked anisocytosis and anisokaryosis with several multinucleated cells. microscopic features were most consistent with seminoma. aggregates of neoplastic cells were identified within the lumen of vasculature indicating lymphovascular invasion and potential metastasis. owners declined additional diagnostics for identification of metastasis. three weeks after surgery stallion was examined for unilateral, left-sided scrotal swelling, and was diagnosed with a subcutaneous incisional infection. ceftiofur crystalline free acid and phenylbutazone were given and continued for 7 days. incision was opened without necessitating suture removal, drained, and the site was copiously lavaged with lactated ringer’s solution under standing sedation. hospital discharge recommendations were to increase movement with forced exercise. seven months after castration, stallion was reported to be doing well with no known concerns. seminomas are the most common equine testicular neoplasm arising from germ cells of spermatic epithelium and have been suggested to have increased metastatic prevalence compared to other domestic species (e.g. dogs). future follow-up assessment of this stallion would allow assessment of right testis, local lymph nodes, and semen quality. keywords: stallion, malignant seminoma, ultrasonography, histopathology, hemicastration presumed prolonged retention of fetal membranes in a mare deborah clark,a robyn wilborn,a jessica cowley,b rachel neto,c jessica klabnika adepartment of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa bdepartment of large animal clinical sciences, virginiamaryland college of veterinary medicine, virginia polytechnic institute and state university, blacksburg va, usa cdepartment of pathobiology, college of veterinary medicine, auburn university, auburn, al, usa ensuring complete expulsion of fetal membranes is important to prevent complications including laminitis and metritis. a 17-year, quarter horse mare, was presented for a breeding soundness examination. mare aborted twins at 6-7 months of pregnancy, 2 years prior and failed to become pregnant during subsequent season. on presentation, mare had multiple small right ovarian follicles, no uterine edema, and poor tone. endometrial biopsy was grade iia and no growth resulted from uterine culture. findings were consistent with mare’s age and seasonal transition; breeding was recommended. mare was presented again 20 days later, presumably just after ovulation. owners gave pgf2α after 14 days. four days later during artificial insemination, a soft tissue mass (~ 6 x 4 x 3 cm) was palpated within the uterine body. mass was retrieved after misoprostol-assisted manual cervical dilation. grossly, it was nonodorous and putty-like in consistency. histopathology identified amorphous dense aggregates of erythrocytes, amorphous to fibrillar beaded eosinophilic material, and rare pyknotic poorly preserved cells. tissue was presumed to be retained fetal membranes, having observed no other gross pathology and inconclusive histopathology. after mass removal, mare’s uterine body was artificially inseminated and lavaged after 4 hours. mare was inseminated with a second breeding dose at ovulation after 2 days. mare was diagnosed pregnant on day 14 day and a heartbeat was confirmed on day 34. mare was infertile possibly due to the uterine mass, presumed to be fetal membranes retained from the mid-term abortion 2 years before. reproductive prognosis for future fertility was considered good. during breeding soundness examinations in subfertile or infertile mares, evaluation of entire uterus is critical not to miss potential foreign objects or material. furthermore, complete fetal membranes’ examination is necessary, especially of both uterine horn tips, after foaling or abortion, and reduces the chance of undetected, retained fetal membranes. keywords: mare, retained fetal membranes, artificial insemination, breeding soundness examination http://dx.doi.org/10.58292/ct.v16.11054 42 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 schistosomas reflexus and uterine tear in a heifer shelby mcallister, kevin walters college of veterinary medicine, mississippi state university, starkville, ms, usa a 2-year, hereford heifer was referred for dystocia after a failed attempt by referring veterinarian. on physical examination, heifer was visibly uncomfortable and 2 of fetus’ feet were protruding from vulva. heifer was confined in a hospital stall and was restrained in a head gate for further examination. as heifer’s vulva and perineum were cleaned and prepped for manual vaginal examination, heifer collapsed into sternal recumbency. halter was placed on heifer’s head to release from head gate while maintaining adequate restraint. halter was tied to the lowest pipe on the head gate and heifer was assisted into left lateral recumbency. vaginal palpation was reinitiated and it was identified that fetus was an anatomical anomaly known as a schistosomus reflexus; uncommon congenital malformation, occurring in 0.01-1.3% of all dystocia cases that is primarily characterized by exposed abdominal and thoracic viscera and extreme spinal inversion producing prominent ventral convex curve. fetotomy was elected; caudal fetal portion was removed and during further removal of fetal parts, entire uterus prolapsed. once reduced, it was noted that there was roughly 300º tear of uterus at cervix level with only a small attachment at the ventral aspect that allowed uterus to protrude through and out of vulva. due to tear’s size and prognosis, owner was consulted and it was decided to humanely euthanize heifer via captive bolt and intravenous kcl. keywords: uterus, dystocia, heifer, schistosomas reflexus, fetotomy dystocia as a consequence of bicornuate pregnancy in a mare haylee hutchins, daniela cortes, patricia xavier, maria ferrer, robyn ellerbrock department of large animal medicine, college of veterinary medicine, university of georgia, athens, ga, usa an 8-year, friesian mare, was presented for dystocia of unknown duration, and with an unknown reproductive history. mare was in the field and determined to be in stage 2 labor. on physical examination, mare was recumbent, mildly hypothermic, tachycardic, and tachypneic. foal’s right front hoof was protruding from vulva; vaginal examination identified that fetus was in transverse presentation, dorso-ileac position, with left forelimb and neck extending into left uterine horn. mare was referred to the veterinary teaching hospital. initial diagnostics on presentation revealed hypocalcemia, hyperglycemia, hyperlactatemia, decreased oxygen saturation, elevated total solids, and respiratory acidosis. transabdominal ultrasonography confirmed absence of fetal heartbeat and fetal death. controlled vaginal delivery under general anesthesia was attempted. first fetotomy cut at left carpus level was achieved; however, it was not possible to reach neck for a second fetotomy cut. ultimately, decision was made to perform cesarean surgery, confirming a bicornuate pregnancy, where fetus was present in both uterine horns. fetus and fetal membranes were successfully removed through hysterotomy, and mare had an uneventful recovery. mare was initially treated with intramuscular procaine penicillin g (22,000 iu/kg, twice daily) and intravenous gentamicin (6.6 mg/kg, once a day), before transitioning to intramuscular ceftiofur (6.6 mg/kg) at discharge. fetal membranes evaluation confirmed a ‘t shape’ form with 2 gravid horns and a type ii vascular pattern. bicornuate pregnancy is rare, with transverse presentation resulting in dystocia and increased fetal mortality due to a reduced placental perfusion and rapid placental separation. this case highlighted the importance of reproductive ultrasonography throughout pregnancy to detect abnormal positions that would benefit from in hospital monitoring and attended parturitions, enabling early interventions when dystocia occurs. keywords: mare, bicornuate pregnancy, dystocia, fetus, pregnancy premature lactation in a recipient mare hailee bornmann, hannah maxwell, chance armstrong, aime johnson department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa a recipient mare, carrying an embryo transfer pregnancy (7 months), was presented for premature udder development. mare had slight vaginal discharge and an increased placental thickness on transrectal ultrasonography. an area of placenta around cervix was detached and fetus could not be palpated. a diagnosis of placentitis was made. placentitis is defined as inflammation or infection of placenta. within equine breeding industry, it has become the leading cause of reproductive loss, with either abortion or delivery of a small, ill-thrift foal. in mare’s placenta, amnion surrounds fetus and chorioallantois attaches to endometrium. these structures protect fetus and provide nutrients and gas exchange for growth. most commonly affected site with placentitis is chorioallantois, leading to a compromise of the attachment site between placenta and endometrium, or thickening of placenta due to inflammation, both of which cause harm to fetus. in this case, ascending infection was the most likely cause, as evidenced by placental separation at cervix. this prevents adequate nutrient and gas exchange in that specific area, leading to fetal death and thus, abortion. septicemia, placental insufficiency, or prostaglandin release from inflammation leading to uterine contractions can cause an abortion.1,2 mare was initially treated with intravenous flunixin meglumine (1.1 mg/kg, twice daily), oral trimethoprim sulfa (30 mg/kg, twice a day) and oral altrenogest (0.088 mg/kg, once daily). fetal heartbeat was not present and no fetal movements were appreciated after multiple attempts. fetus was determined to be dead and medications were discontinued to allow mare to abort on her own. this case depicted the devastating outcome (dead fetus) of a mare with placentitis. keywords: mare, vaginal discharge, placentitis, udder development http://dx.doi.org/10.58292/ct.v16.11054 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 43 references 1. donahue jm, williams nm: emergent causes of placentitis and abortion. vet clin north am equine pract 200;18:443-456. doi: 10.1016/s0749-0739(17)30088-3 2. ruby re, janes, jg: infectious causes of equine placentitis and abortion. vet clin north am equine pract 2023;39:73-88. doi: 10.1016/j.cveq.2022.11.001 successful treatment of 28-hour retained fetal membranes in a mare using umbilical vessel water infusion kayleigh moore, ilaria colombo, ahmed mosallam, babiche heil, muhammed waqas, michela ciccarelli department of veterinary clinical science, college of veterinary medicine, washington state university, pullman, wa, usa expulsion of fetal membranes in mares should occur within 2 hours after parturition, after which, considered retained. complication ranges from asymptomatic to life threatening conditions in as little as 8 hours. several methods are described to treat retained fetal membranes, including oxytocin treatment, manual removal, and water infusion into allantoic cavity and umbilical vessels. umbilical vessel water infusion quickly and gently separates chorioallantois from endometrium by interstitial swelling of membranes.1 an 8-year, clydesdale mare, in dystocia for 4 hours with a dead foal at 307 days pregnancy, was referred. foal was delivered via assisted vaginal delivery and mare retained fetal membranes. mare was started on oral trimethoprim/sulfamethoxazole (30  mg/kg, twice daily), intravenous flunixin meglumine (0.5  mg/kg, 4 times a day) and intramuscular oxytocin (20 iu, 4 times a day). due to broken water line in the hospital, umbilical vessel water infusion could not be performed, and uterus was lavaged twice a day with dilute betadine solution. after 4 lavages, fetal membranes in nonpregnant uterine horn remained tightly attached to endometrium. biochemistry and cbc revealed leukopenia, elevated fibrinogen, decreased segmented neutrophils, and hyperglycemia, placing the mare at high risk of toxic metritis and septicemia. water line was restored 28 hours after foal delivery and fetal membranes were successfully removed intact using umbilical vessel water infusion alternating between 2 available umbilical vessels. mare was discharged next day morning and recovered uneventfully. this case highlighted that even in prolonged retention (> 12-24 hours) of fetal membranes, umbilical vessel water infusion can still be used successfully and it is worth attempting to avoid damage to endometrium and mitigate risks (hemorrhage, impaired uterine involution, tearing of fetal membranes and damage to endometrium) associated with other manual methods to remove retained fetal membranes. keywords: mare, retained fetal membranes, umbilical vessel water infusion reference 1. meijer m, macpherson ml, dijkman r: how to use umbilical vessel water infusion to treat retained fetal membranes in mares. proc annu conv am assoc equine pract 2015;61:478-484. pharmacologically induced ejaculation in an aged stallion kristyn burton,a dale kelleyb acollege of veterinary medicine, oklahoma state university, stillwater, ok, usa bdepartment of veterinary clinical sciences, college of veterinary medicine, oklahoma state university, stillwater, ok, usa a 13-year, quarter horse stallion, was presented for chemical ejaculation due to musculoskeletal limitations that hindered phantom collection. there are 2 protocols for chemical ejaculation that target an α-adrenergic response: combinations of oral imipramine (1.3 mg/kg) and intravenous xylazine (0.3 mg/kg) or oral imipramine, intravenous oxytocin (20 iu), and intravenous detomidine (0.02 mg/ml, imipramine-oxy-det). imipramine was given 1.5 hours prior to other agents, and additional stimulation was provided via estrous mare urine used ~ 15 minutes before ejaculation. chemical ejaculation was first attempted in this stallion on april 11th using imipramine-xylazine; collection was unsuccessful. next day, imipramine-oxy-det yielded 32.0 ml semen with 5.02 x 109 sperm. computer assisted sperm analysis revealed 34% total motility (tm), 12% progressive motility (pm), and 44% normal morphology. stallion was collected again on may 30th, and the same protocol produced 18.6 ml of semen with 5.01 x 109 sperm, 16.4% tm, and 7.9% pm. collection with imipramine-oxy-det failed once on may 31st, then twice on june 16th, with urination in second attempt. six attempts from june 17-20th were also unsuccessful: 5 attempts with increasing doses of detomidine (0.2-0.8 ml) and 1 with xylazine-imipramine reattempt on 20th. on june 21st, stallion was given imipramine-oxy-det with 0.8 ml detomidine and produced 88.4 ml of semen with 15.40 x 109 sperm, 14.1% tm, and 7.7% pm. on august 6th, stallion was collected for the final time using imipramine-oxy-det with 0.8 ml detomidine that yielded an ejaculate volume of 30.3 ml with 14.14 x 109 sperm, and total and progressive motilities of 20.5 and 13.7%. semen was cushion centrifuged and 3 mares were bred during 4 estrous cycles via deep uterine horn artificial insemination, with first and third collections resulting in pregnancies. one mare conceived on first estrous cycle, 1 mare on its second, and 1 mare was not pregnant. ultimately, although challenging due to poor reliability and decreased motility, chemical ejaculation presents a viable option for stallions that are unable to be collected through traditional means. in combination with other semen processing techniques, it is possible to have offspring from stallions with poor semen quality. http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1016/s0749-0739(17)30088-3 https://doi.org/10.1016/j.cveq.2022.11.001 44 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 keywords: stallion, chemical ejaculation, imipramine, oxytocin, sperm motility iatrogenic pneumoperitoneum following vaginoscopic investigation of chronic vaginitis matthew rafferty, mikkel turley, ahmed mosallam, babiche heil, muhammad waqas, michela ciccarelli department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa, usa a 2-year, spayed pitbull mix dog, was referred for evaluation of chronic vaginitis that was diagnosed 1 month prior by the primary care veterinarian via vaginal cytology and culture. primary veterinarian performed vaginal douche with gentamicin and prescribed systemic antibiotics. following appointment, hemorrhagic vaginal discharge and abdominal pain were observed and a second veterinarian was consulted. purulent vulvar discharge was noted, and dog was referred to theriogenology service for further diagnostics and treatment. on presentation, dog appeared depressed, lethargic, and pyrexic. abdominal ultrasonography results were unremarkable. vaginoscopy, using a transcervical insemination scope, was performed to investigate the possibility of stump pyometra and evaluate vaginal mucosa. vaginal mucosa was hyperemic, and a tract was observed on the vaginal dorsal aspect, seemingly terminating in fascia. during the procedure, it was difficult to maintain sufficient vaginal insufflation. immediately after vaginoscopy, dog became tachycardic, vomited several times, and gradually abdomen increased in size. dog was transferred to hospital’s emergency and critical care service; pneumoperitoneum was diagnosed via radiography. dog was stabilized by removal of 500 ml of air from the abdomen and supportive therapy prior to emergency exploratory celiotomy. a 1 cm necrotic perforation of the cranial vagina, communicating with the abdominal cavity was diagnosed and accounted for pneumoperitoneum after vaginoscopy. uterine stump and cervix were removed, and a cranial vaginectomy was performed, utilizing vaginoscopy (transcervical insemination scope) it was ensured that the vaginal necrotic perforation was completely removed; dog recovered uneventfully. this case highlighted the importance of vaginoscopy in the evaluation of dog presented with vaginitis. although rare, clinicians should consider that vaginal cytology, douching, manipulation, and endoscopy can lead to vaginal perforations. keywords: vagina, pneumoperitoneum, vaginoscopy, vaginitis, vaginal perforation left ventrolateral cesarean surgery in a heifer carrying schistosomus reflexus tanya weber, babiche heil, michela ciccarelli, ahmed mosallam, muhammad waqas department of veterinary clinical science, college of veterinary medicine, washington state university, pullman, wa, usa a 2-year, aberdeen angus heifer, was presented with 10-hour history of dystocia. owner reported (via phone) that manual manipulation was attempted and viscera were protruding from vulva. euthanasia was discussed, suspecting maternal evisceration after uterine rupture with poor prognosis. however, photos from the owner confirmed protrusion of dark pink tubular tissues were fetal viscera; schistosomus reflexus was suspected. transrectal and transvaginal palpation revealed an abnormal fetus in uterus and dilated cervix with engaged fetal viscera. vaginal delivery was unsuccessful and fetotomy was not indicated due to unknown extent of fetal malformation. left ventrolateral cesarean surgery was chosen, as heifer went recumbent. a dead, malformed fetal monster of undefined sex, consistent with schistosomus reflexus, characterized by visceral exposure and spinal inversion, was successfully extracted under intramuscular sedation with acepromazine and butorphanol, followed by intravenous xylazine every 15 minutes. uterus, abdominal musculature, and skin were closed by utrecht, simple continuous, and simple interrupted pattern, respectively. heifer was given subcutaneous ceftiofur crystalline free acid and intravenous flunixin meglumine. heifer was clinically normal at discharge after 2-day hospitalization. ten days later, heifer returned for treatment of surgical site infection and dehiscence of several skin sutures. intravenous flunixin meglumine, subcutaneous ceftiofur crystalline free acid, and intramuscular procaine penicillin were given for 3 days. incision was lavaged and allowed to heal by second intention during 17-day hospitalization. heifer recovered uneventfully and was discharged. this case illustrated an important differential for viscera protruding from vulva after attempted manipulation, importance of photos from owners, an approach to cesarean surgery in a recumbent cow, and a known disadvantage of this approach (an increased risk of surgical site infection and wound dehiscence). keywords: schistosomus reflexus, dystocia, cattle, cesarean surgery severe preputial hemorrhage in a neutered male dog natalie buonaiuto, robyn wilborn, hayley moore, elizabeth benton-levith department of clinical sciences, college of veterinary medicine, auburn university, auburn, al, usa substantial, frank hemorrhage from canine prepuce is considered an emergent situation that warrants further evaluation. a 10-year, neutered jack russell terrier, was presented at the emergency service of the hospital with severe hemorrhage from prepuce. approximately 2 months prior, patient was presented to the primary veterinarian with similar signs. a urinary tract infection and/or prostatitis was suspected, and patient was treated with amoxicillin/clavulanic acid and carprofen that appeared to resolve the problem. physical examination was normal except for intermittent hemorrhage from prepuce. dog was sedated to allow for a more detailed examination of penis and prepuce; exteriorized penis was normal. however, a pedunculated mass was located on preputial mucosal surface and was confirmed to be the source of hemorrhage. under sedation, prepuce was manually everted allowing access to base of the mass. mass was 1 x 1.5 cm and was http://dx.doi.org/10.58292/ct.v16.11054 citation line: clinical theriogenology 2024, 16, 11054, http://dx.doi.org/10.58292/ct.v16.11054 45 removed with suture ligation and subsequent excision with no complications. dog recovered uneventfully and was discharged with gabapentin, codeine sulfate, and carprofen. tissue was submitted for histopathology that identified the mass to be an extramedullary plasmacytoma. tumors occurring on canine penis and prepuce are uncommon and are most likely due to venereal disease conditions (e.g. transmissible venereal tumors).1 extramedullary plasmacytomas are normally observed in dogs aged 5-11 years and the growth is usually 1-2 cm in diameter.1–3 most common location for this neoplasm was skin, with nearly ⅓ occurring in mucous membrane sites in oral cavity or rectum.1 extramedullary plasmacytomas are reported in locations such as uterus, third eyelid, and penis.1 surgical excision achieving complete margins is generally curative for these tumors and the prognosis is excellent. keywords: extramedullary plasmacytoma, preputial mass references 1. wypij jm, de lorimier lp: surgery and radiation therapy for extramedullary plasmacytoma of the penile mucosa in a dog. can vet j 2012;53:992-994. 2. kim ms, kim dh, choi us. penile extramedullary plasmacytoma in a dog. reprod domest anim 2010;45:454-457. doi: 10.1111/ j.1439-0531.2009.01555.x 3. rakich pm, latimer ks, weiss r, et al: mucocutaneous plasmacytomas in dogs: 75 cases (1980-1987). j am vet med assoc 1989;194:803-810. http://dx.doi.org/10.58292/ct.v16.11054 https://doi.org/10.1111/j.1439-0531.2009.01555.x https://doi.org/10.1111/j.1439-0531.2009.01555.x 46 index abdallah abdelhady, 23 adriana villaseñor, 28 agustina grippo, 17 ahmed gad, 33 ahmed mosallam, 10, 12, 18, 43, 44 ahmed tibary, 18, 24, 25, 27, 38 aime johnson, 21, 39, 42 amy miller, 40 alan loynachan, 30 alanoud alshami, 4 alex wittorff, 28 alexandra grillos, 6 alexandra hafey, 34 allan gunn, 1 allison deaton, 3 alyssa helms, 7 alyssa shelby, 20 ammasie allred, 38 angela begg, 9 ann ismer, 17 anniston dodson, 21 arthur zimmerman, 21 ashley reeves, 30 audrey kelleman, 19, 21 aykut gram, 20 babiche heil, 9, 10, 12, 43, 44 blake nguyen, 24 bobbi conner, 7 bon mazurek, 9 brandi dunn, 14 bruce christensen, 20 budhan pukazhenthi, 33 camilo hernández-avilés, 15, 16, 36 candace lyman, 1 carlos pinto, 27 chance armstrong, 42 charles love, 15, 16, 36 chin liu, 39 chin-chi liu, 17 chloe hume, 21 chris stokes, 38 christianne magee, 33 christopher premanandan, 2 christopher schulz, 39 cindy artola, 35 claudia escobar, 19 claudia fernandes, 2, 31 clayton smith, 25 cyril stephen, 25 dale kelley, 3, 39, 43 daniel gomes, 23 daniela cortes-beltran, 23 daniela cortes, 19, 42 darcie sidelinger, 38 deborah clark, 41 diego diel, 24 douglas martin, 21 dwayne rodgerson, 29 eduardo arroyo, 21, 24 , 27, 32 elizabeth benton-levith, 44 erin curry, 12 erin harris, 4 erin klein, 1 erin runcan, 2, 6 evie rider, 37 fernando campos-chillon, 32 fiona herzog, 17 fiona hollinshead, 14, 33, 34 gabriel gonzalez, 38 gabriela sousa, 7, 20 gail mcrae, 2 gary childers, 39 georgiana crisci, 15 gerald duhamel, 24 gerrit bouma, 33 giorgia podico, 31, 35, 37 guilherme novello, 31 guoli zhou, 17 hailee bornmann, 42 hannah cochran, 20 hannah maxwell, 4, 13, 42 harvey marquez, 19 haylee hutchins, 42 hayley moore, 1, 44 heath king, 38 hossam el-sheikh ali, 2, 31 hugo almonte, 8 humberto magalhaes, 37 igor canisso, 31 ilaria colombo, 10, 12, 43 jack richison iii, 36 jacob møllenbach, 24 james graham, 14, 34 jarrod troy, 41 jeffrey mccormick, 25 jennifer clulow, 1 jennifer niederlander, 9 jennifer rudd, 3 jenny buchanan, 35 jenny sones, 17, 39 jesper nielsen, 24 jessica cowley, 41 jessica klabnik, 1, 41 jessica rush, 4 jessica wyse, 25 john deramus, 36 jonathan cowart, 19 jonathan miller, 6 jorge sanclemente, 12 jose bras, 8 joy altermatt, 32 joy russollilo, 21 47 juan samper, 16, 36 julia slocum-coffey, 41 julie barnes, 9, 12, 40 julie cecere, 7 julie stephens, 2 julie white, 38 kalie beckers, 17, 39 karen von dollen, 30 karen wolfsdorf, 29, 30 kassie crissman, 17 katharine horzmann, 40 kaushalya jayathilake, 3 kayla kownurko, 6 kayleigh moore, 43 kevin kersh, 41 kevin walters, 42 kianna spencer, 35, 37 kirsten scoggin, 2, 31 kristen todhunter, 9 kristyn burton, 39, 43 kurt volle, 9 lara sypniewski, 28 lauren pasch, 11, 15 lee lee, 23 leonardo caserta, 24 leslie lyons, 39 leslie weston, 25 leticia melo, 31 lily lewis, 13 lindsey vansandt, 9, 40 lisa johnson, 4 lorenzo segabinazzi, 31 luis de aguiar, 21, 23 luisa ramírez-agámez, 15, 16 maarten hoogewijs, 17 madelyn bisson, 32 manuel chamorro, 4 marcelo de oliveira, 28 marcie jung, 14 marco coutinho da silva, 2, 6 maria ferrer, 19, 23, 42 mariah pearson, 37 mariana diel de amorim, 24 marie montelongo, 3 marta cittone, 17 matthew rafferty, 44 matthew scott, 2 megan murphy, 37 michela ciccarelli, 10, 12, 43, 44 michelle kutzler, 6, 28 mikkel turley, 44 mindy meyers, 14, 33, 34 mohammad ababneh, 4 moises barcelo-fimbres, 32 monica lee, 40 morgan agnew, 11, 20 muhammad-salman waqas, 18 muhammad waqas, 10, 25, 38, 44 muhammed waqas, 12, 43 natalie buonaiuto, 44 nicholas wheeler, 38 nicole sugai, 7 nimet browne, 29 orsolya balogh, 7, 20 patricia crigler, 11 patricia xavier, 19, 23, 42 peyton draheim, 1, 8, 29 prasanthi koganti, 23 rachael willis, 38 rachel neto, 13, 41 raul bras, 8 reed holyoak, 28 riley thompson, 33 roberto palomares, 23 robyn ellerbrock, 19, 23, 42 robyn wilborn, 13, 41, 44 rory nevard, 25 ryan jennings, 6 sabrina cousseau, 27 saliya gurusinghe, 25 samantha frontz, 3 samantha mccarter, 20 sandra wilsher, 17 scarlett sumner, 13 shannon reed, 37 shelby mcallister, 42 sofia kovacsy, 17 soon hon cheong, 23, 24 sophia marchio, 2, 37 stephanie bandoski, 24 stephanie ferguson, 1 stephanie walbornn, 8 sunil moré, 28 tanya weber, 44 taylor yenrick, 40 teresa southard, 7 theresa beachler, 41 thomas passler, 4 tiffany hoffman, 11 toby pinn-woodcock, 24 tyler harm, 41 victor medina, 27 vimal selvaraj, 23 warwick bayly, 18 wendy perriam, 9 william swanson, 30 xiaojuan zhu, 40 yatta boakari, 2, 31, 37 zamira gibb, 1 zoli gyimesi, 12 2016: a unique phenotypical presentation of a male pseudohermaphrodite in a dairy bull calf a unique phenotypical presentation of a male pseudohermaphrodite in a dairy bull calf randall bond,a christopher premanandanb adepartment of veterinary preventative medicine, bdepartment of veterinary biosciences, college of veterinary medicine, the ohio state university, columbus oh summary a six month old brown swiss bull calf was referred for surgical correction of a scrotal hernia. palpation and ultrasoography of the scrotum revealed a freely movable luminal structure in the right side of the scrotum and one descended testicle in the left portion of the scrotum. the clinical management plan included surgical correction of the small intestinal inguinal herniation followed by bilateral castration. the calf was anesthetized and taken to surgery. while performing the surgical procedure, it was discovered that intestines were not the structures involved in the hernia but rather were structures of the müllerian duct system; the cervix and uterine horns were found within the scrotum. additionally, the right testicle was attached to the right uterine horn and retained within the right inguinal canal. the uterus, cervix, and bilateral testes were ligated and surgically removed. the inguinal ring was surgically closed in order prevent further herniation. the calf recovered without complications and was returned to the owner. background indirect congenital inguinal herniation involving intestinal structures has been repeatedly reported in calves1 and is by far the most common finding and the primary differential. the classification of indirect versus direct herniation is based on whether the abdominal viscera exits the abdomen through a normal body opening. left sided herniation occurs most commonly.2 the weight of the rumen is thought to predispose to left sided inguinal herniation. bulls which have undergone significant weight loss with large inguinal rings are more predisposed to inguinal herniation. the depletion of the inguinal fat pad occurs with weight loss allowing for structures, such as intestines, to herniate through the inguinal ring into the scrotum. the findings in this case, albeit very uncommon, point to the need to include structures of the müllerian duct system, vestigial or developed, in the differential diagnosis of structures which can herniate through the inguinal ring. hence, in cases of male pseudohermaphroditism it must always be a consideration when evaluating an indirect inguinal hernia in a bull calf. case presentation a six month old brown swiss bull calf was referred to the ohio state university large animal services (osulas) for evaluation and repair of an inguinal hernia and bilateral castration. the calf was initially evaluated by a fair veterinarian. during the weight and health screening, the veterinarian discovered the inguinal hernia and recommended that the hernia be surgically corrected. the calf was otherwise healthy and had been purchased from a neighboring farm. upon presentation to osulas, a physical examination was performed. temperature, heart rate, and respiratory rate were all within normal limits. the animal weighed approximately 200 kilograms. upon palpation of the scrotum, only the left testicle could be palpated along with a freely movable tubular structure within the scrotum on the right side. the descended (left) testicle was of normal size, shape, and firmness for a calf of this age. the other testicle (right) could not be palpated within the scrotum or the adjacent inguinal ring. the calf had a fully formed scrotum and no external female genitalia (figure 1a). an ultrasound examination (easi-scan®; 5.0 mhz linear probe; bcf technologies ltd., bellshill, uk) of the scrotum revealed a right testicle with multiple loops of a fluid-filled luminal structure resembling intestines. the ultrasonic image of the intestine is normally characterized by a single circular hyperechoic muscle layer surrounding hypoechoic to hyperechoic bowel contents. multiple loops of bowel are generally seen and can vary in diameter. movement of the bowel contents and intestinal wall is generally noted with indirect inguinal hernias. in this case, peristalsis was not observed. ultrasonography of the descended testicle revealed homogenous echotexture of the seminiferous tubules with a hyperechoic 4653595555 clinical theriogenology · volume 8, number 1 · march 2016 mediastinum testis. these findings were consistent with a normal ultrasonic image. the remainder of the physical findings were unremarkable. differential diagnosis the primary diagnosis following physical and ultrasonographic examination was unilateral right sided cryptorchidism with small intestinal herniation. less likely differentials included severe right sided testicular hypoplasia and or neoplasia, pseudohermaphroditism, and true hermaphrodism. treatment an intravenous catheter was placed in the right jugular vein and the patient was given 6.6mg/kg ketamine (ketaset®, zoetis inc., florham park, nj) intravenously and 0.22mg/kg xylazine (anased®, akorn inc., decatur, il) intramuscularly. the patient was then intubated and general anesthesia was maintained with sevoflurane (sevoflo®, abbott laboratories, north chicago, il) for the remainder of the surgery. the patient was placed in dorsal recumbency and the surgical site was prepared for sterile surgery in a routine fashion. lactated ringer’s solution (lactated ringer’s injection usp, baxter healthcare corporation, deerfield, il) was administered intravenously at a maintenance rate (2.5ml/kg/hr) for the duration of the surgery. a single 8 cm incision was made through the skin and subcutaneous tissues above the right inguinal ring. the testicle within the scrotum was identified and exteriorized through this incision. there was no attachment of the testis to the visceral vaginal tunic of the scrotum. the tubular structure that was thought to be intestine was firmly attached to the left testicle. this structure was then traced to the opening of the right inguinal canal. as this was done, the right testicle which was attached to the other end of the tubular structure but still within the abdomen, was pulled through the inguinal ring. a fully formed uterus, cervix, and bilateral testes (located in position of normal ovaries) were identified (figure 1b). the structure was then palpated intra-abdominally and appeared to have originated near the pelvic inlet. the uterine horns, cervix, and testes were then ligated with size three chromic gut (chromic gut, ethicon, cincinnati, oh) and excised near the inguinal ring. the inguinal ring, subcutaneous tissue, and skin were closed in routine fashion using size one polyglactin 910 (vicryl®, ethicon, cincinnati, oh) for closure of the inguinal ring and subcutaneous tissues, and size one synthetic suture material (braunamid®, aesculap, center valley, pa) for closure of the skin. recovery was uneventful. the patient received 500 mg flunixin meglumine intravenously (prevailtm, mwi, boise, id) during surgery and given 2,000 mg ceftiofur crystalline free acid (excede®, zoetis inc., florham park, nj) subcutaneously in the middle third of the posterior aspect of the ear where it attaches to the head, for treatment of a mild respiratory infection associated with anesthesia, and dismissed with 1 mg/kg of meloxicam tablets (meloxicam®, unichem pharmaceuticals inc., rochelle park, nj) to be given every other day, orally, for three treatments. the surgically excised tissue was submitted to the applied pathology service at the ohio state university college of veterinary medicine for evaluation. grossly, the specimen consisted of a nearly complete tubular female reproductive tract (cervix, uterine body and uterine horns) (figure 2a). the gonadal tissue was approximately 4.5 cm x 2.5 cm x 2.5 cm. the surface capsular tissue was thick and contained prominent vasculature reminiscent of the tunica albuginea. tubular tissue was present adjacent to the gonadal tissue. the orientation and gross appearance of this tissue resembled an epididymis (figure 2b). the tissue was fixed in 10% neutral buffered formalin and processed in a routine manner for histopathology. histologic examination of the uterus demonstrated normal bovine endometrium, with aglandular segments of endometrium present, which resembled caruncles (figure 3a). a tubular structure was present in the myometrium that was lined by columnar to stratified columnar ciliated epithelium interpreted as a rudimentary ductus deferens (mesonephric duct remnants; figure 3b). sections of the distal tubular reproductive tract demonstrated cross sections of uterine tube, including infundibulum, isthmus and ampulla as well as cross sections of aggregated, highly coiled tubules lined by ciliated columnar epithelium interpreted as an epididymis (figure 3c and d). histologically, the gonadal tissue consisted of numerous structures resembling seminiferous tubules that were lined by polygonal cells interpreted as sertoli cells. these tubules were small and separated by a polygonal to spindle cell 4754605656clinical theriogenology · volume 8, number 1 · march 2016 population, interpreted as leydig cells (figure 3e). a lesser population of round cells typically unattached to the tubular basement membrane were present. immunohistochemical staining directed at gata-4 (santa cruz biotechnology, santa cruz, ca) was performed to identify the sertoli cell population present and to distinguish them from potential germ cells. gata-4 is a transcription factor expressed in multiple tissues that regulate differentiation and cell proliferation. in swine, gata-4 protein expression is observed in sertoli cells, leydig cells and peritubular cells. in addition, gata-4 may play are role in the regulation of expression of muellarian inhibitory substance.22 the gata-4 immunohistochemistry showed prominent nuclear staining of sertoli cells, leydig cells and interstitial fibroblasts; however, a sporadic population of intratubular cells with round morphology and a relatively low nuclear to cytoplasmic ratio did not exhibit gata-4 staining (figure 3f). this population of cells was interpreted as germ cells. a whole blood sample was collected via venipuncture from the patient. the sample was sent to the university of california davis veterinary genetics laboratory for karyotyping. a conventional staining technique was utilized where peripheral blood lymphocytes were isolated from the whole blood sample. the lymphocytes were then cultured for several days under conditions that stimulate them to divide and then be harvested. nuclei of the cells were treated to expose chromosomes which are then stained and photographed. the chromosomes were then examined for number and structure. the calf was determined to have a normal lymphocytic karyotype of a typical male bovid (60 xy). additionally, dna was also extracted from the sample and tested for sex linked traits. there was no evidence of hematopoietic chimerism found and all results were consistent of a normal male bovid. outcome the owners reported that the calf recovered without complications. the calf was meant to be utilized as a show animal but was disqualified due to lack of appropriate weight gain. discussion normal mammalian sex is dependent on the completion of three major stages that are under genetic and hormonal control.3 within any of these stages, errors can occur which significantly influence the subsequent outcome of sexual development of the individual. the first stage is the establishment of chromosomal sex (xx or xy) that occurs at fertilization. based on the chromosomal sex, the genital ridges of the developing indifferent embryo form the sex cords, in which germs cells migrate in order to develop into testes or ovaries.4 determination of gonadal differentiation is dependent on the sry (sex determining region y) gene expression of the y chromosome as well as other factors some of which all are not fully known.5 if differentiation into the male gonadal phenotype takes place, production of testosterone (t) by testicular leydig cells and anti-müllerian hormone (amh) by sertoli cells results.6 these two products promote the differentiation of the mesonephric (wolffian) duct into the male tubular genitalia and regression of the paramesonephric (müllerian) ducts, respectively. in the absence of t and amh, there is a promotion and differentiation of the paramesonephric (müllerian) ducts and regression of the mesonephric (wolffian) ducts to form the female gonadal phenotype. the external genitalia and accessory sex glands are also induced to develop from embryonic primordia by dihydrotestosterone (dht) made within these tissues from circulating testosterone.6 hermaphroditism and pseudohermaphroditism are examples of errors that can occur during this development. errors that occur during this time are considered a condition of intersex. intersex incorporates three conditions: true hermaphroditism, freemartinism, and pseudohermaphroditism. true hermaphroditism is characterized as a combination of ovaries, testes, and/or ovotestes and mosaicism may occur commonly.7 for example, true hermaphrodites may have a testicle on one side, and an ovary on the contralateral side. ovotestes exhibit the presence of testicular and ovarian tissue in the same gonad. a freemartin is the most common form of intersexuality found in cattle.8 it occurs when the developing heterosexual twin fetuses share a vascular placental anastomoses. this connection then consequentially masculinizes the developing female reproductive tract, due primarily to the influence of anti-müllarian hormone.8 typically, the females are sterile and have absence of tubular genitalia.4 4855615757 clinical theriogenology · volume 8, number 1 · march 2016 finally, a pseudohermaphrodite (ph) occurs when there is a difference between the external genitalia and the true phenotypic gonadal sex of the animal.9 a female ph is a genetic and gonadal female with partial masculinization of the external genitalia. in food producing animals, female ph is much less common than male ph.9 this is because female gonadal differentiation is the default without the presence of testosterone, amh, and dihydrotestosterone. some cases of female ph have resulted from mosaicism, while others had xx chromosomal compositions suggesting that extra-gonadal hormonal influences played a role. in one study, a high incidence of female pseudohermaphroditism in polar bears was suggested to be due to polychlorinate biphenyls (pbcs), which may disturb the differentiation of organs.10 conversely, a male ph has testes and female external genitalia.11 male ph can have undescended testes and varying combinations of male and female structures and they may have either xy or xx chromosomal constitution. in male ph, there may be a failure of normal production of androgens by the testicles or androgen insensitivity from the respective tissues. if either of these occur, the outcome resorts to the default, which is female, and the mesonephric system fails to develop.12 in this case, it is speculated that there was androgen insensitivity as the testicles were histologically normal and at least one was present in the correct location. cases of male pseudohermaphroditism have been reported in most domestic species including: dogs,3 cats,13 cattle,12 pigs,14 sheep,15,16 horses,17 white-tailed deer,18 and captive arabian oryx.19 the rate at which this abnormal development occurs is variable within the species.20 in humans, as well as in canines, intersex conditions have been reclassified as disorders of sexual development (dsd). disorders of sexual development classification often takes in account the genotypic abnormalities as well as phenotypic abnormalities observed clinically. bovine intersex conditions have not been reclassified in such a manner; however, utilizing the classification for canines, this case may be referred to as 60 xy persistent müllerian duct disorder of sexual development.21 this particular case demonstrates two intriguing clinical implications. the first is that a male ph can present clinically with the gonads in a scrotal location. this suggests that the condition can be a clinical differential for cryptorchism and testicular hypoplasia/aplasia. in terms of long-term prognosis, the patient had limited reproductive and financial potential. however, it is interesting to speculate on the manner in which the gonads would have continued to develop if surgery was not performed. since germ cells were still present in these testes as shown by the gata-4 immunohistochemical stain, it is possible that spermatogenesis may have taken place at puberty given the intrascrotal location of at least one testis. in conclusion, this case report describes an unusual presentation of a bovine male ph in which male gonadal tissue was present in the scrotum as well as portions of the aberrant female reproductive tract. to the authors’ knowledge, this case is unique in that there have been no other published reports describing this specific type of phenotypic presentation of ph. learning points  in clinical situations of presumptive inguinal herniation, male pseudohermaphrodite should be listed as a possible differential diagnosis along with cryptorchidism, testicular hypoplasia or testicular aplasia.  pseudohermaphroditism is a condition of intersex. it is characterized by having external genitalia that is different from the true gonadal sex of the animal. acknowledgement we gratefully acknowledge lee v. millon and the uc davis veterinary genetics laboratory for the karyotype results presented in this manuscript. references 1. wolfe d, rodning s: diagnosis and management of inguinal hernia in bulls. in: anderson d, rings m, editors. food animal practice. 5th ed. st. louis: saunders elsevier; 2009. p. 356-359. 2. turner as, mcilwraith cw: techniques in large animal surgery. philadelphia: lea and febiger; 1989. p. 313. 3. bigliardi e, parma p, peressotti p, et al: clinical, genetic, and pathological features of male pseudohermaphroditism in dog. reprod biol endocrinol 2011;9:1-7. 4956625858clinical theriogenology · volume 8, number 1 · march 2016 4. ladds pw: congenital abnormalities of the genitalia of cattle, sheep, goats, and pigs. vet clin north am food anim pract 1993;9:127-144. 5. takagi m, yamagishi n, oboshi k, et al: a female pseudohermaphrodite holstein heifer with gonadal mosaicism. theriogenology 2005;63:60-71. 6. mcnabb b, conley a, maclachlan, nj: female pseudohermaphroditism in a dorset sheep: a case report. clin therio 2014;6:105-110. 7. hadjiathanasiou cg, brauner r, lortat-jacob s, et al: true hermaphroditism: genetic variants and clinical management. j pediatr 1994;125:738-744. 8. padula am: the freemartin syndrome: an update. anim reprod sci 2005;87:93-109. 9. jainudeen mr, hafez ese: genetics and reproductive failure. in: hafez, ese, editor: reproduction in farm animals. 5th ed. philadelphia: lea and febiger; 1987. p. 436-451. 10. wiig o, derocher ae, cronin mm, et al: female pseudohermaphrodite polar bears at svalbard. j wildl dis 1998;34:792-796. 11. lott d, benirschke k, mcdonald j, et al: physical and behavioral findings in a pseudohermaphrodite american bison. j wildl dis 1993;29:360-363. 12. moriyama c, tani m, nibe k, et al: two cases of bovine male pseudohermaphrodites with different endocrinological and pathological findings. j vet med sci 2010;72:507-510 13. vallefuoco r, alleaume c, jardel n, et al: type ii atresia ani associated with rectovaginal fistula in a male pseudohermaphrodite kitten. can vet j 2013;54:475-478. 14. halina we, barrales dw, partlow gd, et al: intersexes in swine: a problem in descriptive anatomy. can j comp med 1984;48:313-321. 15. bruere an, mcdonald mf, marshall rb: cytogenetical analysis of an ovine male pseudohermaphrodite and the possible role of the y chromosome in cryptorchidism of sheep. cytogenetics 1969;8:148-157. 16. bruere an, macnab j: a cytogenetical investigation of six intersex sheep, shown to be freemartins. res vet sci 1968;9:170-180. 17. bouters r, vandeplassche m, moor a: an intersex (male pseudohermaphrodite) horse with 64 xx/65 xxy mosaicism. equine vet j 1972;4:150-153. 18. scanlon p, urbson d, sullivan j: a male pseudohermaphrodite white-tailed deer resembling an antlered doe. j wildl dis 1975;11:237-240. 19. padilla l, dutton c, bauman j, et al: xy male pseudohermaphroditism in a captive arabian oryx (oryx leucoryx). j zoo wildl med 2005;36:498-503. 20. payan-carreira r, pires m, quaresma m, et al: a complex intersex condition in a holstein calf. anim reprod sci 2008;103:154-163. 21. poth t, breuer w, walter b, et al: disorders of sex development in the dog-adoption of a new nomenclature and reclassification of reported cases. anim reprod sci 2010;121:197-207. 22. lavoie ha. the role of gata in mammalian reproduction. exp biol med 2003;228:1282-1290. figure 1. left: view of the caudal aspect of the calf showing a fully formed scrotum and no external female genitalia. right:. surgical image of the uterus, cervix, uterine horns, and bilateral testes originating through the right inguinal canal. 5057635959 clinical theriogenology · volume 8, number 1 · march 2016 figure 2. gross images of the surgically excised reproductive tract. a. the entire tubular female reproductive tract is demonstrated in this image in addition to gonads at the distal aspect of each uterine horn. b. gonadal tissue present at the distal aspect of the right uterine horn. note the tubular structure resembling epididymis adjacent to the gonad and the prominent vasculature associated with the tunica albuginea typical of the external surface of the testicle. 5158646060clinical theriogenology · volume 8, number 1 · march 2016 figure 3. a. photomicrograph demonstrating endometrium and myometrium from the tubular reproductive tract. b. photomicrograph of a tubular structure present in the myometrium interpreted as mesonephric duct remnant. c. multiple cross sections of tissue resembling infundibulum of the uterine tube (arrowheads) and epididymal tubules (arrows). d. multiple cross sections of epididymal tubules at higher magnification. e. photomicrograph of gonadal parenchyma containing seminiferous tubules lined by sertoli cells. note the round cells present in the lumen of several tubules that were interpreted as germ cells. f. photomicrograph of gata-4 immunohistochemical stain of gonadal parenchyma. note the strong positive nuclear staining of sertoli cells and the absence of nuclear staining of gonocytes (arrowheads). (editor’s note: the photographs in this manuscript are available in color in the online edition of clinical theriogenology.) 5259656161 clinical theriogenology · volume 8, number 1 · march 2016 2012: advances in ovulation timing in the bitch   advances in ovulation timing in the bitch cheryl lopate reproductive revolutions, aurora, or in order to understand how ovulation timing (ovt) works, it is important to understand the basic physiology of the estrous cycle. ovulation timing consists of vaginal cytology, vaginal speculum examination, progesterone and luteinizing hormone (lh) concentrations. once an understanding of basic ovt is obtained, this knowledge can be used to assist with breeding management decision making. puberty and the estrous cycle puberty occurs on average at nine months of age, with a range of six to 14 months. smaller breed bitches tend to attain puberty earlier than do large breed bitches. the bitch typically has one or two cycles/year; however there is significant individual variation within breeds and between breeds. certain breeds have more frequent cycles (akitas, german shepherd dogs, cocker spaniels and labrador retrievers may have a normal four month interestrous interval) and others less frequent (basenjis and tibetan mastiffs tend to have only one cycle/year). in general, a minimum interestrous interval of 4.5 – 5 months is considered necessary for normal fertility. the estrous cycles consists of 4 phases: proestrus, estrus, diestrus and anestrus. proestrus is when follicle development occurs. estrogen concentrations begin to rise significantly early in this stage and peak near its end, when the follicles are mature. luteinizing hormone is initially produced in small surges which result in thickening of the walls of small follicles present on the ovaries, resulting in a slow, but steady rise in progesterone. at the end of proestrus, a large pre-ovulatory surge in lh occurs, which results in ovulation of any mature follicles, which subsequently causes a rapid rise in progesterone concentrations. on average, proestrus lasts seven to nine days. early in proestrus, the vulva begins to swell and bloody vulvar discharge begins, which is initially bright red in color. this discharge comes from the uterus as a result of leakage of red blood cells across capillary walls following exposure to estrogen. as proestrus continues the discharge lessens and becomes lighter and more straw-colored. most intact males show minimal interest in the bitch early in proestrus but become more and more interested as this stage proceeds. the bitch is typically not receptive during early – mid proestrus but begins flagging and standing for the male towards the end of proestrus or in early estrus. estrus is the stage of the cycle associated with the fertile period (time when eggs are completing maturation or are awaiting fertilization) and sexual receptivity in the bitch. receptive behavior results from the rapid change in the estrogen to progesterone ratio. ovulation occurs over several hours (4 – 30) depending on the number of eggs ovulated. canine eggs are ovulated in an immature state and require two days to mature before fertilization may occur. once mature, the eggs are capable of being fertilized for four to days, provided sperm can gain access to them (see next section). thus, the fertile period lasts six to eight days, beginning with ovulation. estrus generally lasts seven to nine days. during estrus, the vulva softens but remains swollen. there is usually minimal to no vulvar discharge during this stage; although some bitches will have bloody vulvar discharge through estrus and on into diestrus. in most cases, intact male dogs are intensely interested in the bitch during this stage of the cycle. most bitches flag readily and stand to be mated. diestrus is the stage of the cycle associated with progesterone production and pregnancy, if the bitch is successfully mated. it lasts 57 days in the pregnant bitch. if no fertilization occurs or the pregnancy fails, progesterone production can last anywhere from 45 – 72 days. progesterone rises for seven to 21 days after ovulation. it then remains at a plateau for about 10 – 20 days after which is slowly begins to decline until the end of diestrus. receptive behavior ends at the end of estrus or early diestrus and males quickly lose interest in the female in early diestrus. during diestrus when the bitch is not pregnant, vulvar swelling and discharge decreases and returns to its pre-proestrus condition. in the pregnant bitch, vulvar swelling initially decreases but then begins again in late pregnancy, along with relaxation of the pelvic ligaments and perivulvar area in preparation for delivery. in some bitches, there 543 clinical theriogenology • volume 4, number 4 • december 2012   will also be a small amount of clear – mucoid vulvar discharge during diestrus and pregnancy. this discharge normally has no odor or color. anestrus is the stage of the cycle associated with relative ovarian inactivity. intact males are not interested in the bitch nor does the bitch display receptive behavior. there is no vulvar swelling or discharge. vaginal cytology and speculum examination epithelial cell types present on vaginal cytology (from deepest to most superficial) include basal, parabasal, intermediate, nucleated superficial (non-cornified) and anucleated superficial cells (cornified;table 1). other cell types include white blood cells (wbc), red blood cells (rbc), foam cells (clean up cells) and metestrual cells (epithelial cells eating a white blood cell). bacteria, mucus and background debris are also seen at different stages of the cycle. in early proestrus, the deeper cell layers predominate because the vaginal wall is thin. white blood cells, rbcs, bacteria, mucus and background debris are present in larger amounts. as proestrus progresses, the vaginal walls thicken as a result of increasing estrogen concentrations. superficial cells become more predominant, initially with nuclei and eventually becoming more and more anucleated. as proestrus continues, wbcs disappear and rbcs reduce in numbers. bacterial numbers, mucus and background debris diminish to negligible amounts by the end of proestrus. the vaginal mucosa is pink and swollen (edematous) or billowy in appearance during early proestrus. as proestrus continues, it becomes more dehydrated and paler (whiter) in color. this dehydration is termed crenulation and it is most pronounced when anucleated superficial cells predominate at the end of proestrus and continues to be visible throughout estrus. in estrus, the predominant cell type is the anucleated superficial cell. typically there are 70 90% anucleated superficial cells during estrus. the vaginal mucosa is white in color and there is marked crenulation or wrinkling present throughout this stage of the cycle. ovulation cannot be determined using cytology and speculum examination alone. there is an abrupt transition in cell types between estrus and diestrus. on day 1 of diestrus, the number of nucleated epithelial cells increases dramatically. this occurs as a result of the sloughing of the thick layer of cornified cells and exposure of the deeper layers of the wall of the vagina. intermediate, parabasal cells and wbcs return during the first few days of diestrus. the wbcs help with removal of dead sperm and bacteria from the vagina following breeding. two new cell types become evident during early diestrus: metestrual cells and foam cells. the amount of background debris and mucus increases during diestrus. the vaginal mucosa attains a mottled appearance (blotchy pink and red) and becomes flatter. during anestrus, the vaginal wall is thin, so the deep layers of cells begin to predominate again. mostly basal and parabasal cells are present. there are few wbcs present and no foam or metestrual cells. moderate to heavy mucus and background debris are present throughout anestrus. the vaginal mucosa is a solid dark red color and smooth. sample handling samples for progesterone should be obtained on the fasted bitch (at least six to eight hours) to prevent fat in the bloodstream from interfering with results. atraumatic blood draws (using the jugular veins) and care with placing the blood sample directly into the tube, rather than using the vacuum pressure in the tube to draw the blood in, will prevent red blood cell rupture from occurring which also may affect hormone concentrations. use of plain glass red top tubes are recommended. blood should be allowed to clot for 15 30 minutes at room temperature and then the tube should be centrifuged for 5 10 minutes. the serum should be removed promptly and placed in another plain glass red top tube. if the blood will be tested within 48 hours it should be refrigerated. if it will be longer, then it should be frozen. serum separator tubes should be avoided; serum should not be left sitting on top of the red blood cells after centrifugation for more than 10 – 15 minutes; nor should clotted blood be submitted without 544clinical theriogenology • volume 4, number 4 • december 2012   centrifugation and removal of serum. blood obtained for lh samples should be handled similarly but should be frozen until assayed. tests available progesterone concentrations may be evaluated numerically (quantitatively) or in broad ranges (qualitatively with in-house kits). using the same laboratory and testing procedure is recommended during a single cycle. switching between laboratories or types of tests can result in small to large differences in hormone concentrations which may affect interpretation of results especially around the time of ovulation. most veterinary reference laboratories offer once or twice daily progesterone testing, providing quick turnaround for analysis. three in-house progesterone test kits are currently available in the us. these are all subjective tests. results from these test kits are more variable than traditional quantitative testing. current tests available are: target® by biometallics, corp, princeton, nj; ovucheck premate® by biovet, st. anthony, mn; and k9 proges-check® by endocrine technologies, newark, ca. accuracy of these tests varies, with the target® kit displaying the most comparable and consistent results compared to labratory values. these tests give ranges of progesterone based on colormetric changes – for example < 1 ng/ml; 1 – 2.5 ng/ml; 2.5 – 5 ng/ml; or > 5 ng/ml for the target® kit. subjective assessment may result in different observers recording different results, so consistency is critical if these kits will be used for breeding management. numerical testing may be performed using radioimmunoassay (ria), chemiluminescence testing (clia), enzyme-linked fluorescent testing (elfa), or enzyme-linked immunosorbent assay (elisa). most reference laboratories are using clia testing at this time. reproductive practices that have inhouse numerical testing machines are usually clia, elfa, or elisa. as long as there is good internal quality control and consistency, these in-house numerical tests are very accurate. again, using the same testing procedure for all the samples is critical to accurate interpretation of results. most lh testing is done using a qualitative in-house assay called witness lh® (synbiotics corp, kansas city, mo). it provides a positive or negative test value (either >1 ng/ml or < 1 ng/ml) with a turnaround time of 20 minutes. luteinizing hormone testing at the laboratory often has unacceptable turnaround times (2+ days) and most laboratories use the same in-house kit as described above. when using this test on a routine basis, clinics become good at interpreting the difference between weak and strong positive tests, which is important since the lh surge can be < 24 hours in length making the presence of a clear strong positive not possible in some cases. ovulation timing generally speaking, the bitch should be examined for the first time between days 5 and 8 of the cycle. if the onset of the cycle is accurately noted and the bitch has a normal cycle, this will be early enough to catch the lh surge if needed. it is important to remember that in some cases, the early signs of the estrous cycle may be missed so the bitch may be much further along in her cycle than believed. if the bitch will be bred with frozen semen it is more important to start earlier in the cycle than if she is being bred naturally, since determination of the lh surge is significantly more important when using frozen semen. bitches are typically examined every one to three days until ovulation is confirmed. if one is comfortable reading vaginal cytology slides and performing speculum examinations, the need to begin submitting progesterone samples can be delayed until mid-late proestrus is reached. bitches that have slides indicative of early – mid proestrus may have blood samples drawn and the serum frozen and stored in order to be able to backtrack to evaluate progesterone or lh concentrations if future tests indicate. vaginal cytology is typically performed every other day. progesterone concentrations should be performed regularly once vaginal cytology indicates 50% superficial cells and 20% anucleated superficial cells. as the bitch approaches ovulation, every other day to daily sampling may be required depending on the type of breeding being performed and access to laboratory services. it is good practice to always obtain at least one vaginal cytology slide, on the day of presentation, when bitches are presented for ovulation timing in case the initial progesterone concentration is very elevated – in which 545 clinical theriogenology • volume 4, number 4 • december 2012   case the cytology is necessary to determine if the bitch is still within her fertile window or if she has already entered diestrus. regular vaginal cytology and speculum examination can decrease the number of progesterone tests needed and will also alert the veterinarian to abnormalities of the cycle, days to weeks before progesterone will become suspicious. vaginal cytology and speculum examination are indicators of estrogen concentrations and really do not give any clue to what progesterone concentrations may be and vice versa. so while cytology and speculum examination may seem unnecessary when progesterone testing is being performed, it is actually quite helpful. if assessing only progesterone concentrations, every other day sampling is adequate. if lh concentrations are being determined as well, daily sampling is required because the lh surge is short (average – 18 24 hours) and is easily missed with every other day testing. in some practices, only lh testing is performed, and inseminations planned based on the lh surge. this is inadequate for bitches that do not ovulate or have an abbreviated lh surge, so use of lh and progesterone or progesterone alone is more likely to provide accurate breeding management than lh alone. when bitches have normal cycles, progesterone concentrations are correlated to key hormonal events (table 2). ovulation begins after the bitch’s progesterone reaches at least 4 ng/ml (between 4 – 10 ng/ml). ovulation is confirmed by a rise in progesterone of at least 3 ng/ml in 24 hours. some bitches rise 3 ng/ml in 24 hours while others jump by 10 or more ng/ml in 24 hours. the amount of the rise has to do with number of follicles ovulated, the size of the follicles and the amount of progesteroneproducing tissue (luteal tissue) present. increases of less than 3 ng/ml/day are not confirmatory for ovulation and additional samples should be obtained in 1 – 2 days. failing to confirm ovulation is the most common mistake in ovulation timing. following a bitch to the 4 – 5 ng/ml stage and then not obtaining another sample to confirm ovulation may result in conception failure, small litter size and makes determination of an accurate due date more difficult. ovulation timing and type of insemination planned expected spermatozoal longevity fresh semen can be expected to survive at least 48 hours in the bitch and may live significantly longer depending on the dog (up to 11 – 14 days in some cases). fresh, chilled semen can be expected to survive at least 24 hours in the bitch and may live significantly longer depending on the dog. frozen semen has a relatively short lifespan after thawing, on average 8 12 hours. type of hormone analysis recommended natural breeding. use of qualitative progesterone testing alone is typically adequate with natural breeding, although use of numerical progesterone assays are ideal. testing every 2 – 3 days once the cytology is predominantly superficial cells is usually adequate. if using numerical progesterone testing, ovulation should be confirmed by repeating the test 1 – 2 days after reaching 4 – 5 ng/ml. vaginal artificial insemination (vai). progesterone testing (without lh testing) is usually adequate for vai with either fresh or chilled semen. when using shipped, chilled semen, numerical progesterone testing is strongly recommended while when using fresh semen, either numerical or in house tests may be used. ovulation should be confirmed when using numerical testing by repeating the test 1 – 2 days after reaching 4 – 5 ng/ml. transcervical and surgical insemination (tci/sai). progesterone testing alone is sufficient for fresh or fresh, chilled semen, while when using frozen semen the addition of lh testing will help pinpoint the optimal day(s) for breeding. ovulation should always be confirmed prior to inseminating with frozen semen and always prior to anesthetizing the bitch for sai. type of insemination recommended natural breeding is recommended for fertile bitches and dogs with normal semen quality. vaginal ai can be performed using fresh or fresh chilled semen and is recommended for breeders who do not want to risk injury or transmission of venereal disease to the bitch or dog during natural 546clinical theriogenology • volume 4, number 4 • december 2012   breeding, for inexperienced or nervous dogs or bitches when natural breeding is not successful, for bitches with vaginal anatomic defects (strictures or septums), for size discrepancies between dogs and bitches and for shipped, chilled semen that is of adequate quantity and quality that it can be used vaginally. transcervical insemination can be performed using fresh, fresh chilled or frozen semen and is recommended for fresh semen when semen quality is poor or sperm numbers are low in order to introduce an acceptable breeding dose into the uterus. it is recommended if the bitch owner only wants to do one insemination of chilled semen and anesthesia is not desired, although two breedings are usually recommended. it is recommended for large and giant breed bitches to ensure an adequate breeding dose reaches the uterus. when using frozen semen, two breeding doses inseminated 24 hours apart is ideal. surgical insemination can be performed using fresh, fresh chilled or frozen semen and is recommended for all the same reasons as tci (except that anesthesia is required). it is often performed with frozen semen when there is only a single breeding dose available or if the frozen semen is of average or less than average quality. surgical ai is also preferable for bitches with uterine pathology (subacute endometritis and/or cystic endometrial hyperplasia). there is less contamination with sai than any other type of insemination and it also affords the ability to evaluate the uterus and manually rupture any cysts present in the uterus prior to insemination. breeding management natural breeding breeding should commence one or two days after the bitch ovulates. the bitch should be bred every other day for up to three matings or until she is no longer receptive. vaginal insemination the first insemination is performed two days after ovulation. the insemination is repeated in two days. transcervical insemination if using fresh or fresh chilled semen with two inseminations planned, inseminations are ideally performed two and four days after ovulation. if one insemination is planned, insemination on day 2, 3 or 4 after ovulation is equally ideal. when using frozen semen, two inseminations are preferable to one. breedings should be planned on days 5 and/or 6 after the lh surge or days 3 and/or 4 after ovulation. surgical insemination when using fresh or fresh chilled semen, surgical ai is can be performed on day 2, 3 or 4 after ovulation. if semen longevity is poor, waiting to day 3 or 4 after ovulation is preferable. when using frozen semen, sai on day 3 or 4 after ovulation (days 5 or 6 after lh surge) is recommended. the art of ovulation timing when the bitches read the textbook, breeding management is fairly straight forward. it is in the cases where the bitches prove to us that they cannot read, that ovulation timing becomes more art than science. common situations where ovulation timing does not go according to expectations short proestrus. in these situations the vaginal cytology and speculum examination lag behind hormonal changes resulting in progesterone concentrations being significantly higher than expected. bitches are commonly not receptive during the fertile period when this occurs resulting in poor pregnancy rates when bred based on behavior alone. 547 clinical theriogenology • volume 4, number 4 • december 2012   long proestrus. some bitches have an extended proestrus making progesterone testing quite prolonged. owners may become frustrated with the number of tests required due to high costs. bitches progressing slowly through proestrus may only rise at a rate of 0.1 – 0.2 ng/ml per day. bitches are typically not receptive during their fertile period when this occurs resulting in poor pregnancy rates when breeding is based on behavior alone. split heat. bitches may progress through proestrus and enter early estrus, but never rise above an ovulatory threshold. eventually they drop back to baseline and vaginal cytology returns to proestrus or anestrus. after 7 – 21 days, they enter proestrus again. the second cycle is typically fertile and bitches should be bred after ovulation is confirmed. the use of vaginal cytology will help determine when progesterone samples should be obtained and can help determine that a split heat is occurring. anovulatory cycles there are cases where the bitch never ovulates and may be due ovarian cysts, abnormal follicular development, abnormal hormone production of endogenous lh, or in bitches with chromosome defects. bitches that do not test positive on in-house testing. occasionally when using in-house kits, bitches do not have normal colormetric changes associated with the rise in progesterone. these bitches will seemingly remain at baseline while vaginal cytology and speculum examinations progress normally. in bitches where colormetric change is not occurring as expected, numerical progesterone should be submitted to ascertain if this is the situation. stalling. some bitches have progesterone rise to a certain level (2 – 5 ng/ml, or higher) and then fail to progress for several days. if one bases breeding management on a ‘magic number’ (usually 5 ng/ml) then ovulation may be missed by several days. confirming ovulation has occurred is critical to good breeding management. backtracking. some bitches will rise to a certain level and then drop significantly lower for a day or two. care should be taken that this is not a case of laboratory error (ask for validation when values are not as expected) or poor sample handling. rapid rise. once bitches reach mid – late proestrus, they tend to rise at a rate of 0.5 – 2 ng/ml per day. once ovulation occurs, progesterone typically rises at a rate of 3 – 4 ng/ml/day. in some cases, bitches may rise at rates of more than 5 10 ng/ml per day. this rapid rise is normal and does not change the successful window for fertilization. accurate use of ovulation timing allows precise breeding management and increases pregnancy rates and litter size. the most common cause of infertility in the canine is poor breeding management. breeders often depend on their intact males to determine when to breed bitches but this can result in bitches being bred at an inappropriate time. if pregnancy rates are low with appropriate breeding management, other causes of infertility should be evaluated. interpretation of vaginal cytology, speculum examination, progesterone and lh concentrations takes considerable experience. whelping dates can be used to help determine if the day that ovulation was estimated at the time of breeding was accurate. ovulation timing can be fine tuned by use of retrospective analysis. references 1. johnston sd, olson pn and root-kustritz mv: canine and feline theriogenology. philadelphia: wb saunders co; 2001. 2. england g, concannon pw: determination of the optimal breeding time in the bitch: basic considerations. www.ivis.org. 2002. 3. hegstad-davies rl: a review of sample handling considerations for reproductive and thyroid hormone measurement in serum or plasma. theriogenology 2006; 66:592-598. 4. concannon pw, mccann jp, temple m: biology and endocrinology of ovulation, pregnancy and parturition in the dog. j reprod fertil 1989; 39(suppl): 3-25. 548clinical theriogenology • volume 4, number 4 • december 2012   table 1. cell types present on vaginal cytology slides during the estrous cycle of the bitch basal cells parabasal cells intermediate cells superficial cells anucleated superficial cells rbc wbc foam and metestrual cells mucus and background debris early proestrus ++ ++ ++ +/+++ + ++ mid proestrus +/+ + +/++ + late proestrus +/++ ++ + +/ estrus +/+++ +/ diestrus + ++ ++ +/+/+ + + anestrus ++ + +/+/+ table 2. hormonal concentrations associated with key events anestrus or early proestrus 0 – 1.0 ng/ml 0 – 3.14 nmol/l lh surge (2 days prior to ovulation) 1.1 – 1.9 ng/ml 4.08 – 5.97 nmol/l progesterone surge (1 day prior to ovulation) 2.0 – 3.9 ng/ml 6.28 – 12.25 nmol/l ovulation 4.0 – 10 ng/ml 12.56 – 31.4 nmol/l oocytes mature and available for fertilization 10.0 – 34.0+ ng/ml 31.4 106.76+ nmol/l conversion to si units 1 ng/ml = 3.14 nmol/l   549 clinical theriogenology • volume 4, number 4 • december 2012 550clinical theriogenology • volume 4, number 4 • december 2012 omniblank: 2016: effect of flunixin meglumine on prostaglandin metabolites and progesterone in lactating dairy cows effect of flunixin meglumine on prostaglandin metabolites and progesterone in lactating dairy cows j. a. spencer,a k. g. carnahan,a b. shafii,b s. read,a a. ahmadzadeha adepartment of animal and veterinary sciences; bstatistical programs, college of agriculture and life sciences, university of idaho, moscow, id abstract the objectives of this study were to determine the effects of flunixin meglumine (fm), a nonsteroidal anti-inflammatory drug on progtaglandin f2alpha (pgf) secretion, by characterizing plasma prostaglandin metabolites (pgfm), and luteal function by characterizing plasma progesterone (p4) concentrations in lactating holstein dairy cows during the luteal phase of the estrous cycle. on day -35, estrous cycles were synchronized using a presynch-ovsynch protocol. transrectal ultrasonography was performed on days -9, 0, 3, 7, and 15 to confirm ovulation and formation of a corpus luteum (cl). on day 15, cows were stratified by parity and randomly assigned to either treatment or control groups. the treatment group (n = 9) received a total of 2.0 mg/kg body weight of fm (iv), whereas the control group (n = 8) received saline. jugular blood samples were collected at 30 and 0 minutes before treatment for pgfm concentrations. following treatment, blood samples were collected at 30 minutes and every hour for additional 7 hours. blood samples were also collected daily from days 15 to 22 to characterize p4 concentrations. plasma pgfm concentrations did not differ between groups before treatment. following treatment, plasma pgfm remained unchanged in the control group, whereas the mean pgfm concentrations decreased in fm-treated cows (p < 0.05). mean pgfm concentration decreased within 60 minutes following treatment and remained low through the experimental period. mean p4 concentrations before treatment (day 15) did not differ between the control and treatment groups. mean p4 concentrations decreased between days 16 to 22. however, the rate of p4 decline over time tended to be smaller for fm-treated cows compared with controls (p = 0.09). these results suggest that fm treatment decreases plasma pgfm and therefore, may inhibit pgf secretion in lactating dairy cows during the luteal phase of the estrous cycle. moreover, fm may help to extend luteal function by reducing the rate of p4 decline. keywords: flunixin meglumine, prostaglandin, luteal phase, dairy cows introduction reproductive efficiency is an important contributor to dairy farm profitability. many factors can cause reproductive inefficiencies and decrease pregnancy rates (pr). embryonic loss is one factor that contributes to dairy cattle reproductive efficiency and negatively impacts pr.1,2 in dairy cattle, fertilization rates are similar between lactating and nonlactating cows, and it has been reported to be as high as 88% to 98%. others have reported that fertilization rates in dairy cows are approximately 90%.3-5 conception rates (cr) at day 27 to 31 after artificial insemination (ai) are approximately 35 to 45%, indicating that the majority of cows that conceive do not maintain pregnancy following fertilization.3 the majority of embryonic mortality (70 to 80% of the total loss) occurs between days 8 and 16 following fertilization.5 a specific sequence of events must occur in order for embryos to survive and for pregnancy to be maintained. in cattle, these events are in part dependent upon sufficient p4 secretion from the cl and timely secretion of interferon-tau (ifnτ) from the embryo. interferon-tau prevents synthesis and secretion of pgf and stops the luteolytic mechanism.6-8 any disruption in the previously mentioned events may lead to early embryonic death. therefore, it is critical to investigate methods to regulate luteolysis and assist in the maintenance of early pregnancy in dairy cattle. high concentrations of p4 prior to fertilization are necessary to prime the uterus for embryo development and inhibit secretion of pgf9 however, if there are insufficient concentrations of p4, embryonic mortality increases because of an unsuitable uterine environment for embryonic development.10 another possible scenario that may lead to early embryonic loss is if the embryo is 3239454141 clinical theriogenology · volume 8, number 1 · march 2016 incapable of producing sufficient ifnτ, or if secretion of ifnτ is delayed. this may result in untimely maternal recognition, leading to secretion of pgf, luteolysis and embryonic loss.11 several in vivo studies have shown the negative effects of premature secretion of pgf on embryonic survival in beef cows.12-14 given that the majority of embryonic loss occurs between days 8 and 16, and maternal recognition of ifnτ by the embryo occurs between days 14 and 16, embryonic loss may occur due to the inability of the embryo to prevent pgf secretion. any strategy to inhibit or reduce pgf secretion near the time of maternal recognition may help to reduce embryonic loss and improve reproductive performance in lactating dairy cows. non-steroidal anti-inflammatory drugs (nsaid), such as fm, are known inhibitors of prostaglandin synthesis and are used therapeutically for alleviation of pain, fever, and inflammation.15,16 this specific nsaid has been studied to improve pr under various circumstances in beef and dairy cattle. in dairy heifers, the use of fm before luteolysis improved the overall pr.17 a study by schrick and collegues18 showed that when fm was administered during embryo transfer (et) in beef cows, the overall pr was significantly improved. another study by pfeifer et al19 also demonstrated an improvement of pr on day 30 (37% fm vs. 17% control) following ai in lactating dairy cows administered fm 12 hours apart on day 15 and 16 of the estrous cycle. these authors suggested that the observed results were due to the attenuation of pgf2α secretion, therefore delaying luteolysis.17,19 nevertheless, pgf nor its metabolites (pgfm) were measured by these investigators. although some studies have demonstrated a decrease in pgfm and/or an increase in pr/ai, other studies in lactating beef and dairy cows, as well as beef and dairy heifers have shown no difference or a decline in pr/ai when fm was administered following ai. for example, von kruger and heuwieser20 and rabaglino et al,21 observed no differences in pr when fm was administered twice 12 or 24 hour apart on day 15 and 16 after ai in dairy heifers. in lactating beef cows and beef heifers, administration of fm 13 days following ai had either negative effects or showed no improvements on pr/ai.22 once again, pgf nor its metabolites were measured in any of these studies. to our knowledge there are only four studies in which pgfm was measured following fm administration. one study examined fm effects in cyclic dairy heifers and oophorectomized cows, and another used only cyclic dairy heifers. in these studies, a limited number of animals was used (n = 2 to 6), and fm was administered orally 2-4 times daily or intravenously (iv) four times daily for either 7 to 9 days beginning on day 15 or 16 of the estrous cycle.23,24 in both of these studies it was concluded that administration of fm 4 times daily orally or iv decreased pgfm concentrations in cows and heifers and prolonged the cl lifespan in cyclic dairy heifers. an additional study that measured blood pgfm after an intense treatment of fm for 10 days (4 x daily) following et, also demonstrated a decrease in pgfm concentrations following fm treatment.25 lastly, a study in nonlactating beef cows demonstrated that when fm was administered 13 days after ai, pgfm concentrations decreased; however no improvements on pr were observed.26 an intense regiment of fm administration was used in most of these studies, which makes it impractical to implement in a production operation. the results on the effects of fm to improve pr have been inconsistent. moreover, the effects of fm on pgf secretion have not been studied in lactating dairy cows during the luteal phase of the estrous cycle. to our knowledge, there is a lack of evidence on fm effects on pgf secretion and p4 secretion during the maternal recognition period in lactating holstein dairy cows. our hypotheses were that fm would decrease pgfm concentrations and affect p4 secretions following treatment. the objectives of this investigation were to examine the effects of fm administration on pgf secretion by characterizing plasma pgfm, as well as peripheral p4 secretion during the luteal phase of the estrous cycle, around time of maternal recognition. materials and methods this study was conducted at the university of idaho dairy research and education center located in moscow, idaho. the university of idaho animal care and use committee approved all procedures used in this experiment. milk from fm-treated cows was discarded for a minimum of 36 3340464242clinical theriogenology · volume 8, number 1 · march 2016 hours following treatment, based on milk withdrawal guidelines established by the us department of agriculture, food and drug administration (usda-fda). experimental design on day -35, seventeen lactating holstein dairy cows (7 primiparous and 10 multiparous), were presynchronized using two intramuscular (im) injections of pgf (25 mg; lutalyse®; zoetis, florham, nj) 14 days apart (fig. 1). on day -9 (12 days after second pgf) after detection of a cl by transrectal ultrasonography (sonovet 600®, 5 mhz probe; universal ultrasound, bedford hills, ny) all cows were enrolled into an ovsynch protocol and administered gonadotropin-releasing hormone (gnrh; 100 μg, im; cystorelin®; merial, athens, ga; fig. 1). seven days later (day -2), all cows received a pgf injection (25 mg; im) to regress the existing cl previously confirmed on day -9 (fig.1). approximately 48 hours later (day 0) all cows received the final gnrh (100 μg; im; fig. 1). all cows were visually observed (3 to 4 times daily) for estrus behavior from days 15 to 22 of experimental protocol. ovarian examination all cows were subjected to transrectal ultrasonography (sonovet 600®, 5 mhz probe) on days -9, 0, 3, 7, and 15, in order to assess follicular dynamics and to determine ovulation of the dominant follicle and the formation of a cl in the same location (fig.1). ovulation was defined as the disappearance of a follicle ≥ 10 mm diameter and the formation of a cl in the same location. the presence of cl was also confirmed with blood p4 concentrations > 1 ng/ml.27 treatment on day 15, six hours prior to serial blood collection, cows were weighed, assigned a body condition scor (bcs), and the ovaries were examined by transrectal ultrasonography. following detection of a cl, cows were stratified by parity and randomly assigned to either treatment or control groups. the treatment group (n = 9; 5 multiparous and 4 primiparous) received 2.0 mg/kg body weight (bw) of fm (banamine®, schering-plough animal health, union, nj) via the jugular vein (iv) administration, and the control group (n = 8; 5 multiparous and 3 primiparous) received the same volume of saline also via the jugular vein (fig. 1). the average days in milk (dim) for all cow at the time of treatment was 59 ± 4 days. blood collection and processing on the day of experiment (day 15), 6 hours before treatment, cows were fitted with jugular catheters. jugular blood samples were collected at 30 and 0 minutes before treatment, and at 30 minutes and every hour after for 7 hours following treatment to measure plasma pgfm concentrations (fig. 1). additional blood samples were obtained on day 16 to quantify pgfm concentrations 24 hours after treatment. plasma pgfm concentrations are the stable metabolites of pgf in circulation and are known to be correlated to uterine-ovarian pgf in both ovine and bovine species.28-32 daily coccygeal blood samples were also collected from days 15 to 22 for quantification of p4 concentrations (fig. 1). blood samples were collected using chilled collection tubes containing 30 iu heparin and immediately placed on ice. blood tubes were then centrifuged at 4°c for 30 minutes at 2750 × g. plasma was harvested and stored at -20°c until assayed for pgfm and p4 concentrations. hormone assays serial blood samples from day 15 were assessed for pgfm concentrations using an elisa assay (cayman chemical kit 13,14-dihydro 15-keto pgf2α) as previously described.33 samples were diluted 1:1 with an assay buffer, and the standard curve ranged from 2.3 to 5,000 pg/ml. all samples and standard curves were run in duplicates, and the intraand inter-assay coefficient of variance (cv) were 8.8 and 6.8%, respectively. the sensitivity of this assay was 15 pg/ml. daily plasma blood samples from days 15 to 22 were analyzed for p4 concentrations using a solid-phase radioactive-immunoassay (ria; siemens, los angeles, ca). all samples were assayed in duplicates and the standard curve ranged from 3441474343 clinical theriogenology · volume 8, number 1 · march 2016 0.1 to 40 ng/ml. this assay was conducted under equilibrium conditions and the intra-assay cv was 7.8%, with an assay sensitivity of 0.1 ng/ml. statistical analysis differences between treatment groups for bw, bcs, dim, and milk yield were analyzed using the general linear model (glm) in sas 9.2 (sas inst. inc., cary, nc). the model included treatment, parity, and treatment by parity effects. analysis of repeated measures using the mixed model procedure, autoregressive moving average (arma 1,1) in sas 9.2 was utilized to analyze plasma pgfm and p4 concentrations between groups.34 the model included treatment effects, the repeated measure of time, and the interactions between treatment and time. the random effect was cow within treatment, and pre-treatment hormone concentrations were used as a covariate in the model. statistical significance was declared at a p < 0.05 and a tendency at p ≤ 0.1. a linear regression analysis, using glm procedures of sas, were also carried out to analyze the effect of treatments on the p4 concentration over time (day 15 to 22). the fitted model for each treatment took the form of y = β0 + β1 x + ε1 where y was the p4 concentrations, x represented time, β0 was the intercept, β1 was the rate of decline for p4 concentration over time, and ε1 represented the random error under classical regression assumptions. results mean bw, dim, bcs, and milk yield were not different between treatment groups (table). there was no effect of parity or parity by treatment interaction on dim and bcs. as expected, milk yield tended to be different between primiparous and multiparous cows (28.4 ± 3.2 vs. 35.9 ± 2.7 kg, p = 0.08), but there was no effect of parity by treatment interaction on milk yield. in addition, bw differed between primiparous and multiparous cows (552.5 ± 20.0 vs 650.4 ± 16.6 kg; p < 0.05), but there was no effect of parity by treatment interaction on bw. ovarian structures were mapped and recorded using transrectal ultrasonography on days -9, 0, 3, 7, and 15 of experimental protocol (fig. 1). all cows included in this data did not express estrus prior to day 0, the last gnrh injection in the ovsynch protocol. the presence of a dominant follicle, and subsequent formation of a cl in the same location of the dominant follicle were observed in all cows by day 7 and 15 of experiment. all cows had a cl before treatment and on the day of treatment (day 15). also, all cows had plasma p4 concentrations > 1.0 ng/ml on day 15. mean p4 concentrations before treatments were not different between the groups (6.3 vs. 7.2 ± 0.5 ng/ml, for fm and control, respectively). there was an effect of treatment (p < 0.05) and treatment by time interaction (p < 0.01) on mean pgfm concentrations. the mean plasma pgfm concentrations 30 and 0 minutes before treatment did not differ between fm-treated and control cows (129 ± 39 pg/ml for control vs 152 ± 41 pg/ml for fm; fig. 2). flunixin meglumine decreased pgfm secretion (p < 0.05) following treatment and the mean pgfm concentrations were lesser (p < 0.05) in fm-treated cows by 60 minutes after treatment administration and remained lower (p < 0.05) for the fm-treated cows throughout the remainder of the sampling period compared with the control group (57.5 ± 30 pg/ml; fig. 2). plasma pgfm remained unchanged in the control group throughout the sampling period (176 ± 40 pg/ml; fig. 2). also, mean pgfm concentrations on day 16 (22-24 hours following treatment) tended (p = 0.09) to be lower for fm than for control cows (51.4 ± 35 vs. 125.6 ± 33 pg/ml). progesterone concentrations decreased (p < 0.01) over time for both control and treatment groups. there was an effect of treatment by day interaction on plasma p4 concentrations (p < 0.08), which indicated that the rate of p4 decline over time was not similar between the groups. in fact, the slope or the rate of p4 decline over time tended (p = 0.09) to be smaller for fm-treated cows compared with control cows (fig. 3). based on serum p4 concentrations during day 15 to 22, two cows did not exhibit luteolysis (p4 < 0.5 ng/ml) by day 22 in the control group. in contrast, four cows did not exhibit 3542484444clinical theriogenology · volume 8, number 1 · march 2016 luteolysis in fm-treated group. during the seven days after treatment, more cows were detected in estrus in the control group compared with the fm group. in the control group 6 cows exhibited estrus, whereas in the fm group only 4 cows were detected in estrus. discussion in this experiment, fm treatment on day 15 of the estrous cycle decreased plasma pgfm concentrations within 60 minutes following treatment (fig. 2). this indicates that fm has an inhibitory effect on pgf secretion, as plasma pgfm concentrations are known to be correlated to uterine-ovarian pgf concentrations in ovine and bovine species.28-32 therefore, it can be speculated that fm may have inhibited pgf secretion of the uterine-ovarian origin. progesterone concentrations were not different between treatment and control groups on day 15 before fm administration. after treatments, the pattern of blood p4 decline was inconsistent between the fm-treated and the control group. flunixin meglumine treatment tended to delay the rate of decline in p4 concentrations following treatment between days 16 and 22 (fig. 3), indicating that perhaps fm delayed the process of luteolysis. these results suggest that fm may inhibit pgf secretion in lactating dairy cows during the luteal phase of the estrous cycle and affect p4 secretion during the time of maternal recognition. our results are similar to other studies in cyclic dairy heifers, oophorectomized dairy cows and non-lactating beef cows in which fm treatment was able to decrease pgfm concentrations.23,24,26 pfeifer et al19 demonstrated that administration of two doses of fm, 12 hours apart, on day 15 and 16 of the estrous cycle significantly improved pr on day 30 following ai in lactating dairy cows (37% fm vs. 17% control). although the exact mechanism for the improved pr cannot be determined, the authors suggested that the improvement in pr may be due to the attenuation of pgf secretion during the late luteal phase. the findings of the current study support this hypothesis. in the current study, although we did not measure pr/ai, our results indicate that fm treatment may improve pr/ai on day 15, the time in which maternal recognition occurs in cattle by decreasing pgf secretion. flunixin meglumine may be a beneficial reproductive tool in preventing secretion of pgf during the time of maternal recognition, which may cause early embryonic loss. since some studies have indicated an increase in pr/ai in beef cows18 and lactating dairy cows,19 treatment of fm may prove more beneficial when early embryonic loss is more prevalent such as heat stress situations. future research is needed in order to determine the effects of fm during heat stress situations, as well as potential effects on reducing the occurrence of early embryonic loss. references 1. lamming ge, darwash ao, back hl: corpus luteum function in dairy cows and embryo mortality. j reprod fertil 1989;37:245-252. 2. chebel rc, santos jep, reynolds jp, et al: factors affecting conception rate after insemination and pregnancy loss in lactating dairy cows. anim reprod sci 2004;84:239-255. 3. santos jep, thatcher ww, chebel rc, et al: the effect of embryonic death rates in cattle on the efficacy of estrus synchronization programs. anim reprod sci 2004;82:513-535. 4. thatcher ww, staples cr, danet-desnoyers g, et al: embryo health and mortality in sheep and cattle. j anim sci 1994;72:16-30. 5. sreenan jm, diskin mg, morris dg. embryo survival rate in dairy cattle: a major limitation to the achievement of high fertility. in: proceedings of the fertility in the high-producing dairy cow. british society of animal science occasional publication 2001;93–104. 6. anthony rv, helmer sd, sharif sf, et al: synthesis and processing of ovine trophoblast protein-1 and bovine trophoblast protein-1, conceptus secretory proteins involved in the maternal recognition of pregnancy. endocrinology 1988;123:1224-1280. 7. farin ce, imakawa k, hansen tr, et al: expression of trophoblastic interferon genes in sheep and cattle. biol reprod 1990;43:210-218. 8. thatcher ww, guzeloglu a, mattos r, et al: uterine-conceptus interactions and reproductive failure in cattle. theriogenology 2001;56:1435-1450. 9. spencer te, bazer fw: conceptus signals for establishment and maintenance of pregnancy. reprod biol endocrinol 2004;2(49):1-15. 10. thatcher ww, driancourt ma, terqui m, et al: dynamics of ovarian follicular development in cattle following hysterectomy and during early pregnancy. domest anim endocrinol 1991;8:223. 3643494545 clinical theriogenology · volume 8, number 1 · march 2016 11. bazer fw: mediators of maternal recognition of pregnancy in mammals. proc soc exp bio med 1992; p. 373-384. 12. schrick fn, inskeep ek, butcher rl: pregnancy rates for embryo transferred from early postpartum beef cows into recipients with normal estrous cycles. biol reprod 1993;49:617-621. 13. seals rc, lemaster jw, hopkins fm, et al: effects of elevated concentrations of prostaglandin f2α on pregnancy rates in progestogen supplemented cattle. prostaglandins other lipid mediat 1998;56:377-389. 14. hockett me, rohrbach nr, schrick fn: alterations in embryo development in progestogen-supplemented cows administered prostaglandin f2. prostaglandins other lipid mediat 2004;73:227-236. 15. campbell nb, blikslanger at: the role of cyclooxygenase inhibitors in repair of ischaemic-injured jejunal mucosa in the horse. equine vet j 2000;32:59-64. 16. agrawal p, gupta a: nsaids. retrieved from http://en.engormix.com/ma-dairy-cattle/dairy-industry/articles/nsaidst1498/p0.htm. 2010. 17. guzeloglu a, erdem h, salibay mk, et al: effect of the administration of flunixin meglumine on pregnancy rates in holstein heifers. vet rec 2007;160:404-406. 18. schrick fn, hockett me, towns tm, et al: administration of a prostaglandin inhibitor immediately prior to embryo transfer improves pregnancy rates in cattle. theriogenology 2001;55:370. 19. pfeifer lfm, vasconcelos jlm, schneider a, et al: effect of flunixin meglumine at days 15 and 15 after tai on pregnancy rates in lactating holstein cows. j anim sci 2007;85(suppl 1):326. 20. von krueger x, heuwieser w: effect of flunixin meglumine and carprofen on pregnancy rates in dairy cattle. j dairy sci 2010;93:5140-5146. 21. rabaglino mb, risco ca, thatcher mj, et al: use of a five-day progesterone-based timed ai protocol to determine if flunixin meglumine improves pregnancy per timed ai in dairy heifers. theriogenology 2010;73:1311-1318. 22. geary tw, ansotegui rp, macneil md, et al: effects of flunixin meglumine on pregnancy establishment in beef cattle. j anim sci 2010;88(3):943-9. 23. aiumlamai s, odensvik k, stabenfeldt g, et al: regulation of prostaglandin biosynthesis with flunixin meglumine in bovine species. j vet med res 1990;37:16-22. 24. odensvik k, gustafsson h, kindahl h: the effect on luteolysis by intensive oral administration of flunixin granules in heifers. anim reprod sci 1998;50:35-44. 25. odensvik k, gustafsson h: effect of flunixin during asynchronous embryo transfer in the heifer. anim reprod sci 1994;36:13-24. 26. geary tw: effects of adrenocorticotropic hormones and flunixin meglumine on pregnancy retention in beef cows. j anim sci 2012;90:207-211. 27. sellars cb, dalton jc, manzo r, et al: time and incidence of ovulation and conception rates after incorporating estradiol cypionate into a timed artificial insemination protocol. j dairy sci 2006;89:620-626. 28. mitchell md, flint apf, turnbull ac: plasma concentrations of 13,14-dihydro-15-keto-prostaglandin f2α during pregnancy in sheep. prostaglandins 1976;11:319-329. 29. kindahl h, edqvist le, bane a, et al: blood levels of progesterone and 15-keto-13,14-dihydro-prostaglandin f2α during the normal estrous cycle and early pregnancy in heifers. acta endocrinol 1976;82:134-139. 30. kindahl h, lindell jo, edqvist le: release of prostaglandin f2α during the oestrous cycle. acta vet scand suppl 1981;77:143-158. 31. thatcher ww, wolfenson d, curl js, et al: prostaglandin dynamics associated with development of the bovine conceptus. anim reprod sci 1984;7:149-176. 32. zollers jr wg, garverick ha, smith mf, et al: concentrations of progesterone and oxytocin receptors in endometrium of postpartum cows expected to have a short or normal oestrous cycle. j reprod fertil 1993;97:329-337. 33. del vecchio rp, maxey km, lewis gs: a quantitative solid-phase enzyme immunoassay for 13,14-dihydro-15-ketoprostaglandin f2α in plasma. prostaglandins 1992;43:321-330. 34. littell rc, henry pr, ammerman cb: statistical analysis of repeated measures data using sas procedures. j anim sci 1998;76:1216-1231. 3744504646clinical theriogenology · volume 8, number 1 · march 2016 figure 1. experimental protocol. holstein dairy cows were synchronized using a pre-synch-ovsynch protocol. ultrasonography was conducted on days -9, 0, 3, 7, and 15 in order to determine ovulation of a dominant follicle and formation of a corpus luteum in the same location. on day 15, cows received 2.0 mg/kg body weight of flunixin meglumine or the same volume of saline. jugular blood samples were collected at 30 and 0 minutes before treatment, and at 30 minutes and every hour after for 7 hours following treatment to measure plasma pgfm concentrations. from days 16 to 22, daily coccygeal blood sampling was obtained to examine progesterone concentrations. table. descriptive statistics. mean ± standard error (se) for body weight (bw), days in milk (dim), body condition score (bcs), and milk yield in holstein cows treated with flunixin meglumine (fm) or control. treatment bw (kg) dim1 bcs2 milk yield (kg)3 flunixin (n = 9) 4 612.9 ± 23.8 61 ± 4 2.8 ± 0.1 33.3 ± 2.9 control (n = 8) 4 606.7 ± 25.3 58 ± 4 2.7 ± 0.1 32.6 ± 3.1 1 dim on day -8 of experimental protocol. 2 bcs on scale of 1 to 5 in 0.25 increments (1 = emaciated; 5 = over conditioned). 3 milk yield based on closest test date to day of treatments. 4 flunixin = 5 multiparous and 4 primiparous; control = 5 multiparous and 3 primiparous. d -2 d 0 d 7 d 15 d 22 d 9 -30 0 30 1 2 3 4 5 6 7 minutes hours fm or saline pgf2α gnrh gnrh ultrasound d 3 daily blood samples d 21 d 35 pgf2α pgf2α 3845514747 clinical theriogenology · volume 8, number 1 · march 2016 figure 2. prostaglandin metabolite (pgfm) concentrations, between flunixin meglumine (n = 9) and control (saline; n = 8) groups of lactating holstein dairy cows during the luteal phase of the estrous cycle. time -30, and 0 minutes are considered to be jugular blood samples collected prior to treatment. following treatment, blood samples were obtained at 30 minutes, 60 minutes, and every hour for 6 consecutive hours following treatment. * significant difference between treatment groups (p < 0.05). figure 3. linear regression analysis of progesterone between days 16 and 22 for flunixin meglumine and saline (control). the rate of decline (or slope) tended (p = 0.09) to be smaller for the fm-treated cows when compared to the control group. * * * * * * 0.0 50.0 100.0 150.0 200.0 250.0 -30 0 30 60 120 180 240 300 360 p g f m ( p g/ m l ) minutes prostaglandin metabolites flunixin salinetreatment 0 1 2 3 4 5 6 7 16 17 18 19 20 21 22 p ro ge st er on e (n g/ m l ) days after treatment progesterone flunixin saline linear (flunixin) linear (saline) 3946524848clinical theriogenology · volume 8, number 1 · march 2016 2009: advances in canine semen evaluation techniques advances in canine semen evaluation techniques 1 c. lopate 2 reproductive revolutions, inc, 1000 wilsonville rd #55, newberg, or, usa 3 abstract 4 veterinarians are frequently asked to evaluate dog semen for a variety of reasons, 5 including but not limited to: breeding soundness examination, shipment of fresh, cooled semen, 6 cryopreservation; after a conception failure; after an illness suspect to affect fertility; or after 7 testicular neoplasia or prostatic disease is diagnosed. ultimately, the goal of any semen 8 evaluation is to predict how likely it is for a male to be successful when used in a breeding 9 situation. standard semen evaluation in the dog includes determination of semen volume, 10 spermatozoal motility (both total and progressive), velocity, concentration, total 11 spermatozoa/ejaculate, and morphology. semen morphology during a typical breeding 12 soundness examination is typically performed using one of two stains: wright-giemsa or eosin-13 nigrosin. in the dog, standards for normal semen parameters include a semen concentration of 14 > 10 million spermatozoa/kg bodyweight; >70% progressively motile sperm, and >70% 15 morphologically normal spermatozoa. many times, semen quality will exceed these parameters 16 yet fertility of the dog may still be suboptimal. if infection and prostatic disease can be ruled out 17 as causes of the subfertility, the clinician is left with attempting to further evaluate the ejaculate 18 to determine the cause of the infertility. evaluation of the sperm’s function is the next logical 19 step. 20 male factor infertility is said to account for up to 50% of the failed pregnancy attempts in 21 humans.1 no estimates have been made for the canine, but clinical experience would tell us 22 that male factor infertility accounts for a significant portion of either non-pregnancy, early 23 embryonic death, or small litter size. when a standard semen evaluation fails to elicit a clear 24 diagnosis of infertility, additional testing of the ejaculate is desirable. this testing may include 25 additional bright field spermatozoal staining techniques which differentiate different parts of the 26 169 sperm cell; alternate microscopic evaluation of sperm morphology using differential interference 27 contrast or phase contrast morphology; electron microscopy (em), both scanning and 28 transmission; acrosomal testing; hypo-osmotic swelling testing (host); sperm chromatin 29 structure analysis (scsa); computer assisted spermatozoal analysis (casa/asma); fluorescent 30 antibody staining techniques using flow cytometric analysis; anti-sperm antibody (asa) testing; 31 assays for reactive oxygen species (ros); chromosomal studies; and sperm function testing 32 including zona binding assays, sperm penetration assays. 33 while there is some information on the canine in this regard, much of the information 34 presented will be from human, bovine, and other domestic species research, where this topic 35 has been far more extensively evaluated. the data from other species can be extrapolated to 36 the canine although more research is needed to determine if its use is appropriate in the dog as 37 a predictor of fertility. this paper describes the use of these additional diagnostic methods of 38 spermatozoal analysis for infertility assessment. casa will only be covered briefly as another 39 paper in this symposium is dedicated to its use. 40 keywords: canine, semen evaluation, male infertility 41 42 introduction 43 in the human andrology laboratory setting strict criteria have been developed and 44 accepted by the world health organization (who) for the evaluation of sperm morphology.1-8 a 45 normal human sperm has a specified size and shape, with a smooth outline, an acrosome that 46 comprises between 40 and 70% of the sperm head, has no neck, midpiece or tail defects, and 47 has no droplets more than ½ the size of the sperm’ s head. use of these strict criteria makes 48 morphologic examination of semen more uniform and allows for easier comparison of research 49 studies. thus far, no such criteria have been adapted to the domestic species of animals, 50 making semen evaluation less uniform and comparison of infertility studies and application of 51 treatments more difficult. 52 170 bright field microscopy 53 bright field microscopy is one of the simplest forms of semen evaluation since every 54 veterinary clinic possesses the equipment to perform the evaluation. there are many different 55 stains which are available for morphologic assessment of spermatozoa. commonly used stains 56 include eosin-nigrosin (en), modified wright-giemsa, feulgen, india ink, and spermac. each 57 stain has advantages and disadvantages and will be discussed individually. 58 en stain has been a conventional stain used for semen morphology in domestic and 59 non-domestic animals for many years.8,9 slides stained with en have a dark background and a 60 white or pink staining spermatozoon. the equatorial ridge can be clearly seen and some 61 defects in the acrosomal cap and sperm head may be identified including knobbed or ruffled 62 acrosomes, diadem defects, and nuclear vacuoles. the midpiece can be differentiated from the 63 tail-piece or flagellum based on the change in thickness between the two structures. midpiece 64 defects such as thickening, roughening, proximal and distal cytoplasmic droplets, distal 65 midpiece reflexes, coiling and bending may be determined. tail defects including bending and 66 coiling as well as duplicity may be diagnosed. en is also a vital stain. sperm that have intact 67 membranes and normal chromatin will not allow eosin to be drawn into the cell and so stain 68 white, while sperm with damaged membranes and damaged chromatin will stain pink due to 69 eosin uptake. en staining overestimates the number of intact membranes when compared to 70 fluorescent assays (see flow cytometry section).10 use of live:dead information requires that 71 proper staining procedure is used including prompt staining after collection, prevention of cold 72 shock to the sperm, allowing adequate incubation time before smearing the slide, and rapid 73 drying to prevent cracking or cell death. round cells cannot be differentiated with this stain. the 74 stain is hypotonic and this may result in stain artifact, such as coiled tails, if they are not dried 75 properly. cracks in the stain may also be noted if the smear is too thick. 76 modified wright-giemsa stain (diff-quik®; siemens healthcare diagnostics, deerfield, 77 il, usa) is a simple, inexpensive, and rapid method of staining sperm.9 alterations in head 78 171 staining may correlate with dna defects. the sperm head stains deeply basophilic from the 79 equatorial region downward and more lightly basophilic in the area of the acrosomal cap. the 80 midpiece and tail stain eosinophilic. details of the acrosome are not visible with this stain, so 81 only basic alterations in head size and shape are detectable. midpiece and tail defects that can 82 be visualized include cytoplasmic droplets, distal midpiece reflexes, bent and coiled midpieces 83 and tails. this stain has the added benefit of allowing differentiation of round cells in the 84 ejaculate, making identification of wbc vs. germ cells possible. 85 feulgen staining is a time consuming procedure that allows for better visualization of 86 sperm nuclear and head abnormalities.9 the dna in the sperm head stains magenta allowing 87 good nuclear detail and making defects of the nucleus (vacuoles, diadem defects, etc) clearly 88 visible. the acrosome, midpiece and tail do not stain. the stain must be made fresh daily or 89 changes in the ph will affect the intensity of the stain. slides may be evaluated with either 90 bright field or phase contrast microscopy. the use of phase contrast microscopy results in 91 greater color contrast. feulgen staining identified more abnormalities of dna pattern and head 92 shape in the bull sperm nucleus than standard en stain.11 93 india ink is a one-step stain that is simple and inexpensive to use. it provides a black 94 background and a white or clear sperm outline. it does not allow for good visualization of the 95 acrosome or equatorial region. it does however allow for differentiation of alterations in head 96 size and shape, major defects of the midpiece including cytoplasmic droplets, distal midpiece 97 reflexes, bent or coiled midpieces, and bent or coiled tails. 98 spermac® (conception technologies, san diego, ca, usa) is expensive and 99 moderately time-consuming but is technically a simple staining technique.12 it is important that 100 thin smears of semen are made and are air dried for no more than 10 minutes prior to fixing. 101 once fixed, the remaining steps in the staining procedure may be delayed indefinitely. the 102 nucleus of the cell stains dark red while the acrosome stains light green, and the midpiece and 103 tail stain dark green. this is an excellent stain for acrosomal evaluation and also allows for 104 172 good midpiece and tail piece evaluation. assessment of the nucleus is not as accurate, but 105 head size and shape can be evaluated. round cells can be visualized but they cannot be 106 differentiated. 107 papanicolaou stain is commonly used in human andrology labs, but is not commonly 108 used in veterinary applications due to the complexity of the procedure.8,13 109 phase contrast microscopy is a form of microscopy that allows small phase shifts of light 110 through a transparent cell which are converted into contrast and amplitude changes in the 111 image.14 two light rays are focused exactly inside the opening of the condenser annular ring. 112 the two rays are refracted so that they exit the condenser in parallel. the light is minimally 113 refracted on passing through the specimen and it travels in parallel into the objective where it 114 enters the back focal plane of the objective. a phase plate is positioned in the back focal plane 115 to line up with the condenser annulus. 116 differential interference contrast (dic) microscopy is a system whereby a polarized light 117 source enters a prism and is diverted into two beams at 90 degree angles to each other.14 118 these rays pass through a condenser and then the sample. the beams enter and pass through 119 the sample about 0.2μm apart from each other. since the beams pass through different parts of 120 the cell they follow different optical paths. the beams then enter the objective lens of the 121 microscope and passes through a second prism which recombines the rays into one polarized 122 beam. this recombination leads to interference (since they are on different optical paths) which 123 either brightens or darkens the image. the resultant image appears to be three-dimensional. 124 dic microscopy provides excellent resolution and clarity of cellular structure with minimal 125 artifacts. it does require that the sperm sample is in a media of similar refractive index to the 126 cells themselves which means that evaluation of semen in skim milk or yolk extenders is 127 difficult. 128 no staining of the cell is required for either phase or dic microscopy, but for morphology 129 assessment, the sperm are generally fixed in formol buffered saline and viewed at high power 130 173 (40 – 100x). both of these forms of microscopy provide for more detailed visualization of all 131 parts of the sperm cell including the acrosome, nucleus, midpiece and tail piece than bright field 132 microscopy alone. 133 sperm morphologic assessment plays an integral role in predicting success with iui, ivf 134 and art techniques.3-8 determination of teratozoospermia prior to attempting advanced 135 insemination techniques will help with management choices. for example it has been shown in 136 many human studies that by increasing sperm concentration at the time of insemination, 137 patients with teratozoospermia will have a greater success rate with iui and ivf procedures.3-5 138 in humans it has also been repeatedly demonstrated that once the number of normal sperm 139 drops below 14% infertility is a consistent result; with individuals with 0-4% normal forms have 140 the lowest success rates (45%), 5 – 14% normal forms have moderate success rates (75%), 141 while > 14% normal forms have good success rates (85%) when advanced reproductive 142 techniques like ivf and icsi are applied.3-6 143 acrosome stains 144 giemsa stain makes the acrosome appear dark purple.9 it provides good detail of the 145 acrosome but needs to be made fresh for each use and does not allow for evaluation of the 146 sperm nucleus, midpiece or tail. 147 a sperm triple stain of trypan blue stains the spermatozoa blue, bismarck brown stained 148 the post-acrosome region light brown and rose bengal stained the acrosome light red.15 sperm 149 can be differentiated into four groups with this stain: dead sperm with fully or partially inactivated 150 acrosomes, dead spermatozoa with missing or degenerated acrosomes, live spermatozoa with 151 reacted acrosomes, and live spermatozoa with active or normal acrosomes. this staining 152 technique has been used for humans, mouse, bull, horse, goat and boar semen. 153 a one step stain including fast green fcf, rose bengal and ethyl alcohol has been used 154 to stain the acrosome of cat spermatozoa.16 this stain allows differentiation of acrosome intact, 155 174 acrosome reacted or damaged sperm, and acrosome non-intact sperm. the slide is examined 156 using bright field microscopy at 1000x. 157 coomassie blue stain has been used to assess acrosomal integrity in bulls, boars, and 158 stallions.17,18 this staining procedure is relatively simple and results in intense blue staining of 159 intact acrosome and lack of stain uptake in acrosome reacted sperm. the results of coomassie 160 blue staining correlate well with fluorescent staining (see below), dic and bright field 161 microscopy techniques following incubation with calcium ionophore to induce the acrosome 162 reaction.17 163 fluorescent stains, like acridine orange, can be used to evaluate sperm that are 164 extenders in opaque extender, like skim milk.9 acrosomal integrity can be evaluated using 165 fluorescent microscopy, phase-contrast or dic microscopy. 166 acrosome staining can be performed using bis-benzimide dye hoescht 33 258 and a 167 fitc-pisum sativum agglutinin (fitc-psa) after induction of the acrosome reaction by calcium 168 ionophore.19 sperm are incubated in talp media and then calcium ionophore a23 187 is 169 added to induce the acrosome reaction. then the sperm suspension is permealized in 170 methanol and incubated with a lectin in order to bind the fitc-psa probe. the hoescht 33 258 171 is then used to stain the sperm and fluorescent microscopy is used to differentiate acrosome 172 reacted versus non-acrosome reacted sperm. it is a simple, quick technique to assess 173 acrosome status, but it does not allow for morphologic assessment of the cell itself. samples in 174 egg yolk based extenders do not hinder using this technique. 175 trypan blue or congo red stain can be precipitated by neutral red and then stained with 176 giemsa to stain bull, boar and rabbit sperm, but not stallion sperm. this is a simple and reliable 177 staining procedure that results in the stained sperm being classified as live or dead with intact 178 acrosomes, loose or damaged acrosomes, detached acrosome and detached acrosome with no 179 post acrosomal ring.20 180 hypo-osmotic swelling test 181 175 this test is based on the concept that the normal sperm tail membrane will allow fluid to 182 pass into the cell freely under hypo-osmotic conditions. as the fluid flows into the cell, the tail 183 swells. membrane integrity is important in sperm metabolism and changes in membrane 184 properties must occur for capacitation and the acrosome reaction to occur normally.8,21 host 185 not only assesses the morphologic integrity of the plasmalemma but it also assesses its function 186 and biochemical activity.8,10 in humans and bulls, there is strong correlation between the host, 187 the sperm penetration assay, and there is a good interrelationship between host and motility 188 and morphology.8, 10, 22 in bulls and humans, host was a good predictor of success with ivf.10 189 the host is simple, fast and inexpensive.8, 21 one tenth of a milliliter of spermatozoa is 190 incubated in one milliliter of 60 mosmol fructose solution at 37 °c for 45 minutes. then 1– 2 191 drops of this mixture is examined using phase contrast microscopy at 200 x and 400 x. two 192 hundred sperm are counted and the percentage of sperm with curled or swollen tails is 193 determined. host is positively correlated to motility (r = 0.94). the premise being motile sperm 194 have normal membranes and will coil or swell when incubated in a hypo-osmotic solution. 195 as sperm are cooled for increasing amounts of time, decreasing numbers of sperm will 196 be hos+ indicating damage to the sperm membrane with prolonged cooling.21 a similar 197 phenomenon is noted after cryopreservation due to sperm membrane damage. prolonged 198 heating also damages the sperm membrane resulting in few hos+ cells. use of host on 199 rewarmed chilled or post-thaw frozen semen may be predictive of the highest quality samples to 200 be used for insemination by selecting for samples with the highest number of hos+ cells. the 201 host may be a beneficial addition to the semen evaluation in dogs with poor fertility but a 202 normal spermiogram. 203 acrosome assays, acrosome reaction testing and capacitation testing 204 to evaluate acrosome status, sperm must be removed from seminal plasma via 205 centrifugation and then are resuspended in capacitation mediim.23 hyperactivation can be 206 determined through the use of casa. the clinical relevance of hyperactivation has yet to be 207 176 determined. the acrosome reaction can be evaluated through the use of dyes, fluorescent 208 antibodies or lectins. induction of the acrosome reaction is most readily induced following 209 incubation of the sperm first in capacitation media and then calcium ionophore (a23187) is 210 added.8,23 it can be added in high concentration and a short incubation period used (30 – 60 211 minutes) or at low concentration and a long incubation period used (3 hours). the samples are 212 then washed and re-suspended in protein free media and the cells are smeared on slides that 213 are air dried. the slides are then fixed in alcohol and are stained with peanut agglutinin (pna), 214 pisum sativum agglutinin (psa) or fluorescent-labelled lectins and then evaluated with 215 fluorescent microscopy. the number of acrosome reacted cells are then counted and a 216 percentage of all cells is determined.23,24 samples that have high numbers of prematurely 217 reacted cells or which do not respond to incubation with calcium ionophore not likely to be able 218 to complete fertilization.6,7,23,25,26 in humans, there is a high predictive power of induced 219 acrosome reaction and successful ivf outcome.26 220 acrosome reaction can also be detected using staining with fluorescein-conjugated 221 lectins, like psa or pna, plus fluoresceinisothiocyanate (fitc).8,25 this combination of stains 222 evaluates damage to the acrosome while at the same time differentiating acrosome reacted 223 from acrosome intact sperm. psa binds to the acrosomal contents while pna binds to the outer 224 acrosomal membrane.27 acrosomal integrity of canine sperm has been successfully assessed 225 using flow cytometry and staining with fitc conjugated psa and pi.28 capacitation status of 226 chilled and frozen thawed canine sperm has been assessed with a chlortetracycline (ctc) 227 assay and casa for evidence of hyperactivation.29 dog semen has been evaluated for its 228 cryopreservability by first inducing the acrosome reaction with calcium ionophore and then 229 staining the sperm with fitc-pna along with the membrane impermeable dna supravital stain 230 ethidium homodimer1 (ethd-1).30 samples were evaluated with fluorescence microscopy and 231 flow cytometry. the number of cells that underwent the acrosome reaction via ionophore was 232 well correlated with a similar percentage of cells that had acrosome damage post 233 177 cryopreservation. furthermore, the amount of damage to cells caused by acrosome reaction 234 from calcium ionophore was strongly negatively correlated with the number of motile sperm 235 present after freezing. 236 bovine sperm have been incubated with calcium ionophore to stimulate the acrosome 237 reaction and then fixed in formaldehyde.31 afterwards they are stained with naphthol yellow s 238 plus erythrosin b or with naphthol yellow s plus aniline blue. this is a permanent fixative and 239 the use of dic microscopy is required to evaluate acrosomal status. alternatively, bovine sperm 240 may be treated with fluoresceinated psa to assess the acrosome reaction similar to that 241 described previously.27 242 triple staining techniques for acrosomal evaluation have also been described but are 243 more time consuming than the above mentioned techniques and so are not routinely used in the 244 clinical or research setting.8 245 sperm penetration assays 246 these assays asses the ability of the sperm to undergo capacitation, the acrosome 247 reaction, membrane fusion and chromatin decondensation in the presence of an oocyte.4,6-8,23,32 248 sperm must be prepared for the assay by incubating overnight in a capacitation medium or 249 storing in a tes-tris buffer with egg yolk for 24 – 48 hours and then applying thermal shock. 250 after this processing step, the sperm are divided into microdrops and zona-free hamster eggs 251 are added. they incubate for 3 hours and the number of eggs penetrated and the number of 252 sperm/egg are counted. the count is performed by looking for swollen heads within the 253 unstained eggs using phase-contrast or phase-interference microscopy or after staining with 254 acridine orange (ao) and using fluorescence microscopy. use of the tes-tris buffer procedure 255 was more highly correlated with fertility and successful outcomes with ivf in humans in some 256 studies4,6,7,23 while in others it’s predictive power was questionable.26 257 hemizona assays 258 178 this test assesses the availability of the proper molecules on the sperm’s head for it to 259 bind to the zona pellucida and initiate interaction with the oocyte.8,23 a bisected zona pellucida 260 from a normal oocyte is used. each half of the zona is incubated with sperm for 4 hours and 261 then the number of bound cells is counted. zona pellucida binding assays have been used to 262 evaluate the fertilizing capacity of chilled and frozen-thawed canine spermatozoa.33 the test 263 allows an estimation of the damage caused by manipulation of semen on the fertilizing ability of 264 sperm. this test also demonstrates the critical interaction between the zona pellucida and the 265 sperm cell during fertilization and tests multiple sperm functions, including capacitation and 266 ligand-induced acrosome reaction.4,6-8,34,35 of the classic sperm parameters, morphology was 267 the best predictor of the ability of sperm to bind to the zona pellucida.3,4,6,32,34 in conventional 268 ivf studies in humans, defective sperm-zona binding and zona penetration are common causes 269 of failure of fertilization.7,35 there is a high predictive power of sperm-zona pellucida binding 270 and successful outcome with ivf in humans.26,35 271 electron microscopy – transmission (tem) 272 the sperm rich fraction is mixed 1:2 with cacodylate-buffered 6% glutaraldehyde.8,36 273 this mixture is centrifuged, the supernatant removed and the pellet resuspended in 0.1 m 274 sodium cacodylate buffer. this sample is washed a second time and the supernatant removed. 275 the pellet is fixed in a solution of 1% osmium tetroxide in 0.1 m cacodylate buffer and is 276 centrifuged. the osmicated pellet is dehydrated through a graded series of ethanol, is then 277 rinsed in propylene oxide and is then embedded in poly/bed 812 or araldite. sections are cut at 278 80 nm thickness and are then mounted on 300-mesh nickel grids and stained with uranyl 279 acetate and lead citrate for tem. 280 tem may identify lesions of the plasma membrane, acrosome, mitochondria, and 281 nuclear chromatin.8,36 quantification of morphologic defects is not possible but a detailed 282 description of the defects is provided. tem may also help identify and characterize other cells in 283 179 the ejaculate including germ cells, wbc and infectious organisms. dna fragmentation caused 284 by oxidative stress or exposure to toxins may be identified. 285 tem may identify defects of the tail in patients with motility issues that are not apparent 286 with light microscopy.8,37 if a single defect is present in at least 20 – 30 sections it is considered 287 a ciliary dyskinetic condition. total or partial dyein arm deficiency occurs in 3% of human 288 patients with abnormal motility. fragmentation of the plasma membrane and necrosis of the 289 microtubules is typical of necrospermia and is found in 23% of human patients with 290 asthenozoospermia.38 multiple fine ultrastructural defects are noted in another 23% of human 291 patients with this condition. missing outer microtubules, disorganized axonemes, missing 292 central microtubules, additional microtubules above and beyond 9 + 2, thickened and/or 293 disorganized fibrous sheaths, absent radial spokes and translocated tubules are other common 294 defects noted in this group. some of these defects are noted alone and others in combination. 295 in patients whose total motility is > 30% and at least some normal tails were evident, 296 pregnancies using assisted reproductive techniques (art) may be successfully obtained.37 297 a microtubular mass defect was noted on examination of spermatozoa from seven 298 stallions with three of these stallions descending from a single sire.38 there was subfertility in 299 four of these stallions, although it appeared to be at least partially compensable. detailed 300 description of the ultrastructure of the bovine sperm head and midpiece are available.39,40 301 electron microscopy is currently available for clinical cases at the veterinary colleges of 302 auburn university, texas a & m university, and university of saskatchewan. 303 antisperm antibody assay 304 in humans, antisperm antibody production is a common cause of male factor 305 infertility.8,23 there are two commercial assays (spermmar®, conception technologies, and 306 immunobead test®, irvine scientific, santa ana, ca, usa) available for human antisperm 307 antibody assay. these tests provide semiquantitative results regarding the degree of antibody 308 binding present and detect the presence of iga and igg antisperm antibodies. if ≥ 20% of the 309 180 sperm bind to the beads, a sample is considered positive for the presence of antisperm 310 antibodies.23 serum may be assayed for antibodies using a tray agglutination test. at this time, 311 the importance of antisperm antibodies in domestic animals is unclear but may be a useful test 312 for dogs with autoimmune orchitis/epididymitis. 313 flow cytometry for dna and morphology measurement 314 binding of fluorescent dyes to sperm chromatin permits the identification of sperm dna 315 abnormalities and can be measured using a flow cytometer.8,41 sperm are stained using 316 fluorescent assay and then run through the flow cytometer to differentiate cells with normal dna 317 integrity from abnormal. sperm are typically oriented to be in the same plane before they are 318 excited by a laser beam to induce fluorescence and then flow past a fluorescence detector 319 which monitors exactly how much fluorescence each cell has. sperm that have uniform head 320 size and shape display a uniform degree of fluorescence while cells with abnormal size and 321 shape have amounts of fluorescence outside the normal ranges.41 in addition to being able to 322 differentiate morphologically normal from abnormal sperm, flow cytometry can also differentiate 323 sperm with normal motility from those with decreased motility.42 one cause of decreased 324 motility in humans is a result of a break in the dna strands of the sperm nucleus and their 325 mitochondria. flow cytometry and tunel (terminal deoxy-nucleotidyl transferase-mediated 326 deoxyuridine triphosphate-nick end labeling) testing both confirm the presence of these strand 327 breaks in this population of asthenozoospermic men. 328 one of the greatest assets of using flow cytometry for sperm evaluation is the sheer 329 number of sperm that can be evaluated in a short period of time. routine microscopic 330 assessment of sperm involves counting either 100 or preferably 200 cells. but with the use of 331 flow cytometry, thousands of cells can be assessed in a matter of minutes.41 another 332 assessment that can be performed using flow cytometry is that of sperm viability. a dual 333 staining technique using carboxifluorescein diacetate (cfda) and propidium iodide (pi) was 334 used to validate the ability to differentiate live canine sperm from dead sperm.43 plasma 335 181 membrane integrity was also validated in stallion sperm using either cfda and pi or sybr-14 336 stain and pi.25 in these tests, live sperm fluoresce green (from cfda or sybr-14), dead sperm 337 fluoresce red (pi), while dying sperm fluoresce both colors.25,32 338 tests of mitochondrial activity 339 rhodamine 123 (rh123) is a mitochondrial probe which is combined with the viability 340 stains of pi and carboxydimethylfluorescein diacetate (cdmfda) to allow for the determination 341 of sperm with intact membranes (cdmfda+) and functional mitochondria (rh123+) from dead 342 sperm (pi+).25 stallion sperm with functional membranes and mitochondria correlate well with 343 sperm viability and motility.25 344 cytochemical tests for sperm chromatin integrity 345 the integrity of nuclear chromatin results from a combination of factors including sperm 346 maturation processes, damage due to oxidative stress and other endogenous factors.8,44 347 chromatin maturation depends on proper replacement of histone with transition proteins and 348 finally basic protamines. this transition results in compact packaging of the dna and is 349 enforced by cross-linking with protamine-disulfide bonds. in some abnormal sperm, histones 350 may be partially or completely replaced by protamines resulting in loosely packed chromatin. 351 detection of this loose packing may be found with the aniline blue (ab) test. dna is more 352 predisposed to denaturation by heat and low ph when chromatin is packed loosely than when it 353 is tightly packed.44 chromatin proteins in sperm with abnormal dna are more susceptible to 354 staining with acidic dyes like ab, acridine orange and toluidine blue.44 all three of these staining 355 protocols provide a good estimation of the number of sperm with normal vs loosely packed 356 chromatin structure. if more than 30% of sperm have loosely packed chromatin structure an 357 association with increased infertility is noted which correlates with results of scsa (see below). 358 sperm chromatin analysis 359 sperm morphology is well correlated with semen quality.4,7,8,45,46 the head of the sperm 360 consists primarily of nuclear chromatin, so subtle changes in sperm head morphology may be 361 182 related to abnormalities of dna content. measurement of a set of parameters regarding the 362 sperm head (size and shape) and midpiece can align sperm into certain populations of sperm, 363 such that the chromatin content in each sperm head can be determined to be normal or 364 abnormal using automated sperm morphometric analysis (asma). non-compensable defects 365 (those that cannot be overcome by increasing the number of sperm in a breeding dose) are 366 typically related to sperm with abnormal head morphology.4,7,45,46 sperm head shape has been 367 correlated with fertility and resistance to cryopreservation. sperm nuclear dna fragmentation is 368 positively correlated with lower ivf fertilization rates, impaired implantation, increased risk of 369 abortion, and increased risk of disease in offspring, including pediatric cancer.7,8,31,47-49,51 fertile 370 sperm have stable dna which is able to decondense at the appropriate time during the 371 fertilization process such that the oocyte has access to this dna for combination with its own 372 dna complement.48,49,52 373 sperm dna damage may occur on several different levels.4,7,8,31,4850,52 mitochondrial 374 dna damage can occur and be manifest as deletions, point mutations and polymorphism and is 375 associated with decreased semen quality, asthenozoospermia and male infertility. nuclear dna 376 damage may occur as a result of oxidative stress, sperm chromatin packaging and apoptosis. 377 dna damage may occur as a result of environmental factors, pollutants, infection, inflammation, 378 or the presence of ros.48,49 379 for scsa, sperm are treated with an acidic solution (ph1.2) in order to denature their 380 dna in situ.8,46,48 sperm with normal chromatin will not denature under these conditions while 381 abnormal dna will denature. the sperm are then stained with acridine orange. this is a 382 metachromatic dna stain. chromatin which has been denatured into single stranded dna will 383 fluoresce red, while dna which does not denature (remains double stranded) will fluoresce 384 green. the percentage of cells with denatured dna is determined and is called %comp. the 385 percentage of sperm with non-detectable vs detectable dna fragmentation is called the dna 386 fragmentation index (dfi). the percentage of sperm with immature chromatin is called high 387 183 dna stainability (hds). combining the use of scsa and asma may assist in evaluating dogs 388 with normal spermiograms and poor fertility.46,48 389 dna fragmentation may also be evaluated using alkaline single-cell gel electrophoresis 390 testing, tunel assay; comet assay, in situ nick translation, and dna breakage detection-391 fluorescent in situ hybridization assay (dbd-fish). these assays use fluorescence 392 microscopy.7,48-50,52 staining techniques using aniline blue, toluidine blue and chromomycin a3 393 also may be used to identify chromatin packaging defects.52 at this time, few of these assays 394 beyond scsa are used in clinical practice as it remains to be determined what the clinical 395 relevance of negative outcomes means to fertility. there also still remains significant variability 396 in techniques between labs resulting in disparate results. certainly in veterinary medicine, data 397 regarding most of these tests in clinical practice is lacking, although they are slowly being 398 introduced.32 399 on the other hand, scsa, has been accepted as an important tool in the diagnosis of 400 infertility and in prognosticating human couple’s success rates with art.48,49 in humans, dfi is 401 a strong indicator of successful pregnancy outcome.4,7,48-50 prediction of successful outcome 402 with intrauterine insemination is well correlated with the degree of dna fragmentation.48,49 the 403 number of sperm that have dna strand breaks is negatively correlated with their ability to 404 fertilize an oocyte during ivf.48-50 sperm dfi was shown to be negatively correlated with sperm 405 concentration, motility and normal morphology.50 fertilization failure of sperm with dna 406 fragmentation may be able to be overcome by application of art procedures, such as 407 intracytoplasmic sperm injection (icsi), however embryo development may be affected with 408 resultant early embryonic death (eed) or abortion.4,7,32,48-52 in humans, a dna fragmentation 409 rates of > 30% seem to impede fertility, and in couples with high dfi, the use of icsi will 410 improve pregnancy rates over the use of ivf.48,49,52 411 in stallions, scsa has been evaluated and shown to be an indicator of some forms of 412 subfertility or infertility.25,53 subfertile stallions had higher %comp levels than did stallions with 413 184 normal fertility and there was a negative correlation between seasonal pregnancy rate and 414 %comp, % morphologically normal sperm, and % motile sperm. in boars, there is an inverse 415 relationship between farrowing rate and numbers of pigs/litter compared to %dfi.48 pregnancy 416 rates in bulls with high %dfi were lower than for bulls with low %dfi.48 it appears that the 417 threshold for fertility in bulls (10 – 20%) and boars (8%) is much lower than for humans).48 a 418 threshold level is not yet available for dogs. 419 scsa is currently offered for clinical cases at scsa inc (brookings, sd; www.scsa.com) 420 and at texas a & m university college of veterinary medicine. 421 assays for reactive oxygen species 422 ros are very important during the sperm capacitation process in order for tyrosine 423 phosphorylation events to occur normally.7,8 they are normally produced at low levels, 424 however, in some cases of infertility they are produced in much higher amounts. ros interrupt 425 sperm function by causing peroxidative damage to the plasma membrane and thereby impairing 426 motility, the acrosome reaction (exocytosis), and disrupting sperm-oocyte fusion. oxidative 427 stress may also cause mitochondrial dna and nuclear genome damage.7 there are 428 chemluminescent assays using redox-sensitive probes (lucigenin and luminol) for human 429 spermatozoa.7,8 high levels of chemluminesence affect the fertilizing capacity of sperm both in 430 vivo and vitro. the presence of wbcs in the ejaculate will greatly increase the amount of ros 431 present, therefore wbcs must be removed from the samples prior to testing.7 in the presence 432 of seminal plasma, protection from ros produced by wbc is afforded, while for art 433 technologies, these ros will likely have a much more significant role. 434 fluorescence in situ hybridization (fish) 435 this procedure allows for the analysis of chromosome numbers in individual sperm. 436 individuals with oligoasthenoteratozoospermia are at increased risk of having chromosomal 437 abnormalities such as aneuploidy, double aneuploidy and diploidy.1,54 sperm are fixed in a 438 methanol:acetic acid solution and then the dna is decondensed in an acidic salt solution. 439 185 sperm probes for specific chromosomes each fluoresce in specific color ranges are applied and 440 in situ hybridization is performed. in humans, teratozoospermia in the form of macrocephalic, 441 multi-tailed sperm have an increased incidence of aneuploidy.54 other morphologic 442 abnormalities may also be associated with specific chromosomal abnormalities and this area 443 bears the need for further investigation.54 444 computer assisted sperm analysis and automated sperm morphometric analysis 445 casa is a technique employing a computerized system that tracks mean percentage of 446 motile sperm, mean percentage of progressively motile sperm, mean curvilinear velocity and 447 mean straight line velocity per microscopic field.8 the ejaculate is diluted to a specified 448 concentration and the system uses a special gridded microscope slide that accepts a constant 449 volume of semen thus providing a consistent number of sperm to be evaluated each time. the 450 pre-warmed and loaded slide is placed into a thermostatically controlled chamber for analysis. 451 the computer takes video images of the sperm and stores then for analysis. the system 452 recognizes motile from non-motile sperm and other organic debris by comparing luminosity 453 (gray-scale intensity) and size of the object. there are also preset user-defined thresholds for 454 size and luminosity that help prevent mistaking other cells and debris for non-motile sperm. 455 computerized systems have been shown to be more accurate than subjective assessment of 456 sperm motility in human, equine, bovine and canine studies.6,8,32,55-57 casa provides a more 457 discriminating estimation of motility than subjective evaluation with greater repeatability. 458 asma is now also available and provides a more accurate and repeatable evaluation of 459 general sperm morphology.4,6,25,32,46,58,59 with this automated process, as with casa, sperm are 460 diluted in physiologic media to a specified concentration and then a fixed drop placed on a slide 461 and air dried. the slides are then stained and coverslips permanently affixed to the slide before 462 processing in the analyzer. in this way a consistent number of sperm/field may be analyzed. 463 staining method is also important. papanicolaou stain and giemsa stain have been used 464 successfully for morphometric analysis in humans, stallions and dogs. the machine obtains a 465 186 variety of head measurements including length, width, area, perimeter and width/length. a 466 specified gray scale is required and allows differentiation of sperm heads from other cells and 467 debris on the slide. midpiece width and area, distance between the major axes of the head and 468 midpiece, angle of divergence of the midpiece from the head axis can also be assessed. 469 abnormal sperm head, midpiece and tail morphology can be detected 95% of the time with 470 these measurements.45,46,58 471 in the normal dog, a significant variation in head area, length, width and roundness 472 exists, but the analyzer still provides accurate differentiation of teratozoospermic samples.45,46,59 473 ovalness was the least variable factor obtained while length and width had more variation 474 between dogs. within dogs, there was less variation of any measurement. similar variability in 475 sperm head shape and size is noted with scsa analysis of the same dogs, indicating that 476 asma may be a valuable tool when assessing sperm for teratozoospermia.46 477 in vitro fertilization 478 this is the ultimate test of sperm function.8,23 in human medicine, the end point of ivf is 479 what percent of mii oocytes are fertilized and develop to the 8 cell stage by day 3 post 480 insemination.4,32 481 sperm function tests 482 table 1. sperm functions and the sperm function tests that assess them23 483 motility casa light microscopy morphology asma light microscopy em flow cytometry capacitation ivf spa acrosome reaction ivf spa acrosome reaction tests acrosin assays 187 zona pellucida binding ivf hza zona pellucida penetration ivf oocyte-sperm fusion ivf spa casa – computer assisted sperm analysis; asma – automated sperm morphometric 484 analysis; em – electron microscopy; spa – sperm penetration assay; ivf – in vitro fertilization; 485 hza – hemi-zona assay 486 summary 487 it is clear that there is much more to be evaluated regarding male fertility than the basic 488 semen evaluation. the concept that simply providing a specified number of normal appearing, 489 motile sperm at the proper time in relation to breeding will result in acceptable pregnancy rates 490 is clearly a misconception. there are many aspects of sperm function that may affect the 491 functional competence of the sperm cell, beyond its basic size, shape and motility.7 when faced 492 with a dog that has subfertility, the clinician must first rule out the most obvious causes for the 493 problem and them move on to more advanced semen diagnostic testing to exhaust all possible 494 diagnoses. in the process of making the diagnosis, the clinician may discover a method of 495 treating or correcting for the problem. unfortunately, in some cases, even with exhaustive 496 testing, a diagnosis may remain elusive. research in all areas discussed in this paper is needed 497 for all domestic animals including the canine. 498 references 499 1. dursun-durakbasi hg, zamani ag, kutlu r, et al: a new approach to chromosomal 500 abnormalities in sperm from patients with oligoasthenoteratozoospermia: detection of 501 double aneuploidy in addition to single aneuploidy and diploidy by five-color 502 fluorescence in situ hybridization using one probe set. fertil steril 2008;891709–1717. 503 188 2. kruger tf, van der merwe f, van waart j: the tygerberg strict criteria: what are the 504 clinical thresholds for in vitro fertilization, intrauterine insemination, and in vivo 505 fertilization. in: the atlas of human sperm morphology evaluation. new york:informa 506 healthcare; 2009. p. 13–18. 507 3. oehninger s, kruger t: the diagnosis of male infertility by semen quality: clinical 508 significance of sperm morphology assessment. human reprod 1995;10:1037-1038. 509 4. lewis sem: focus on determinants of male fertility: is sperm evaluation useful in 510 predicting human fertility? reproduction 2007;134:31–40. 511 5. coetzee k, kruger tf, lombard cj: predictive value of normal sperm morphology: a 512 structured literature review hum reprod update 1998; 4:73–82. 513 6. bar-chama n, lamb dj: evaluation of sperm function: what is available in the modern 514 andrology laboratory. urol clin north am 1994;21:433–446. 515 7. aitken rj: sperm function tests and fertility. int j androl 2006; 29:69-75. 516 8. mortimer d: practical laboratory andrology. new york:oxford university press; 1994. 517 9. barth ad, oko rj: abnormal morphology of bovine spermatozoa. ames:iowa state 518 university press; 1989. p. 8-18. 519 10. brito lfc, barth ad, bilodeau-goeseels s, et al: comparison of methods to evaluate the 520 plasmalemma of bovine sperm and their relationship with in vitro fertilization rate. 521 theriogenology 2003;60:1539–1551. 522 11. sprecher dj, coe ph: differences in bull spermiograms using eosin-nigrosin stain, 523 feulgen stain, and phase contrast microscopy methods. theriogenology 1996;45:757-524 764. 525 12. oettle ee: using a new acrosome stain to evaluate sperm morphology. vet med 526 1986;81:263. 527 13. kruger tf, franken dr. in: atlas of human sperm morphology evaluation. new 528 york:informa healthcare; 2009. p. 76. 529 189 14. www.wikipedia.com 530 15. vazquez jm, martinez e, roca j, et al: use of triple stain technique for simultaneous 531 assessment of vitality and acrosomal status in boar spermatozoa theriogenology 532 1992;38:843-852. 533 16. pope ce, zhang yz, dresser bl: a simple staining method for quantifying the 534 acrosomal status of cat spermatozoa. theriogenology 1991;35:257. 535 17. larson jl, miller dj: simple histochemical stain for acrosomes on sperm from several 536 species. mol reprod dev 1999;52:445-449. 537 18. personal communication, hannah galatino-homer, 2004. 538 19. jankovicova j, simon m, antalikova, et al: acrosomal integrity status of bovine 539 spermatozoa evaluated by two staining methods. acta vet hung, 2008; 56:133-137. 540 20. kovacs a, foote rh: viability and acrosome staining of bull, boar and rabbit 541 spermatozoa. biotech histochem 1992;67:119–124. 542 21. kumi-diaka j: subjecting canine semen to the hypo-osmotic test. theriogenology 543 1993;39:1279–1289. 544 22. mordel n, dano i, epstein-eldan m, et al: novel parameters of human sperm 545 hypoosmotic swelling test and their correlation to standard spermatogram, total motile 546 sperm fraction, and sperm penetration assay. fertil steril 1993;59:1276–1279. 547 23. ohl da, menge ac: assessment of sperm function and clinical aspects of impaired 548 sperm function. front biosci 1996;1:e96-108. 549 24. varner dd, ward cr, storey bt, et al: induction and characterization of acrosome 550 reaction in equine spermatozoa. am j vet res 1987;48:1383-1389. 551 25. magistrini m, guitton e, levern y: new staining methods for sperm evaluation estimated 552 by microscopy and flow cytometry. theriogenology 1997;48:1229-1235. 553 190 26. oehninger s, franken dr, sayed e, et al: sperm function assays and their predictive 554 value for fertilization outcome in ivf therapy: a meta-analysis. hum reprod update 555 2000;6:160 -168. 556 27. cross nl, watson sk: assessing acrosomal status of bovine sperm using 557 fluoresceinated lectins. theriogenology 1994;42:89-98. 558 28. pena ai, quintela la, heradon pg: flow cytometric assessment of acrosomal status 559 and viability of dog spermatozoa. reprod dom anim 1999;34:495-502. 560 29. rota a, pena ai, linde-forsberg c, et al: in vitro capacitation of fresh, chilled and 561 frozen-thawed dog spermatozoa assessed by chlortetracycline assay and changes in 562 motility patterns. anim repro sci 1999;57:199–215. 563 30 szasz f; sirivaidyapong s, cheng fp, et al: detection of calcium ionophore induced 564 membrane changes in dog sperm as a simple method to predict the cryopreservability of 565 dog semen. mol reprod dev 2000; 55:289-298. 566 31. christensen p, whitfield ch, parkinson tj: the use of bright-field microscopy in 567 evaluating bovine acrosome reaction. theriogenology 1994;42:655–662. 568 32. rodriguez-martinez h: state of the art in farm animal sperm evaluation. reprod fertil 569 dev 2007;19:91-101. 570 33. holst b, larsson b, linde-forsberg c, et al: evaluation of chilled and frozen-thawed 571 canine spermatozoa using a zona pellucida binding assay. j reprod fertil 572 2000;119:201-206. 573 34. oehninger sc: relationship between sperm morphology and binding capacity to the 574 zona pellucida: a critical step leading to fertilization. in: atlas of human sperm 575 morphology evaluation. new york:informa healthcare; 2009 p. 27–34. 576 35. franken dr: new aspects of sperm-zona pellucida binding. andrologia 1998;30:263-577 268. 578 191 36. veeramachaneni dnr, moeller cl, sawyer hr: sperm morphology in stallions: 579 ultrastructure as a functional and diagnostic tool. vet clin north am equine pract 580 2006;22:683-692. 581 37. ryder ta, mobberley ma, hughes l, et al: a survey of the ultrastructural defects 582 associated with absent or impaired human sperm motility. fertil steril 1990;53:556-560. 583 38. heath e, aire t, fujiwara k: microtubular mass defect of spermatozoa in the stallion. 584 am j vet res 1985;46:1121–1125. 585 39. blom e, birch-anderson a: the ultrastructure of the bull sperm. i. the middle piece. 586 nord vet med 1960;12:261–279. 587 40. blom e, birch-anderson a: the ultrastructure of the bull sperm. ii. the sperm head. 588 nord vet med 1965;17:193–210. 589 41. pinkel d, dean p, lake s, et al: flow cytometry of mammalian sperm progress in dna 590 and morphology measurement. j histochem cytochem 1979;27:353-358. 591 42. piasecka m, gaczarzewicz d, laszczynska m, et al: flow cytometry application in the 592 assessment of sperm dna integrity of men with asthenozoospermia, folia histochem 593 cytobiol 2007;45(suppl 1):127–136. 594 43. pena ai, quintela la, herradon pg: viability assessment of dog spermatozoa using flow 595 cytometry. theriogenology 1998;50:1211-1220. 596 44. erenpreiss j, bars j, lipatnikova v, et al: comparative study of cytochemical tests for 597 sperm chromatin integrity. j androl 2001;22:45–53. 598 45. nunez-martinez, moran jm, pena fj: identification of sperm morphometric 599 subpopulations in the canine ejaculate: do they reflect different subpopulations in sperm 600 chromatin integrity. zygote 2007;15:257–266. 601 46. nunez-martinez i, moran jm, pena fj: do computer-assisted, morphometric–derived 602 sperm characteristics reflect dna status in canine spermatozoa? reprod dom anim 603 2005;40:537-543. 604 192 47. lewis se, agbaje i, alvarez j: sperm dna tests as useful adjuncts to semen analysis. 605 syst biol reprod med 2008;54:111-125. 606 48. everson dp, wixon r: clinical aspects of sperm dna fragmentation detection and male 607 infertility. theriogenology 2006;65:979–991. 608 49. erenpreiss j, spano 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/honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1 contact ramanathan kasimanickam ramkasi@wsu.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 review report sperm deoxyribonucleic acid damage and repair ramanathan kasimanickam department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa, usa abstract a greater proportion of inseminated sperm population with fragmented deoxyribonucleic acid (dna), in either natural service or assisted reproductive technology, has been associated with adverse reproductive outcomes, including abnormal embryonic development and failure of implantation, increasing pregnancy loss and reducing pregnancy rates. however, attempts to establish a causal relationship between sperm dna damage and pregnancy success have often resulted in conflicting findings. practical issues include accurate measurement of sperm dna damage and the necessity to reduce impacts of iatrogenic damage. sperm dna repair mechanisms in oocytes and embryos are likely important in addressing sperm dna damage, promoting normal embryo development, and enhancing reproductive outcomes. however, poor reproductive outcomes are likely caused by unrepaired sperm dna damage exceeding a critical threshold and adversely affecting embryo development. this review reports sperm dna damage and repair mechanisms. keywords: sperm, oocyte, embryo, dna damage, dna repair introduction breeding soundness evaluation of bulls is an economically essential element of reproductive management. the prevalence of bulls considered unfit in breeding programs has been investigated and 1 of every 5 bulls in a general population had inadequate semen quality, physical soundness, or both.1,2 in humans, ~ 17.5% of the adult population, roughly 1 in 6 worldwide, experience infertility.3 males are solely responsible for 20-30% of infertility and contribute to 50% of overall cases.4 in clinical settings, sperm fertilization potential is commonly predicted by microscopic assessments of sperm motility and morphology. these regular tests are essential to provide the underlying information on sperm quality under field conditions. however, conventional sperm analysis results are highly variable due to their inherent subjectivity.5 further, it is not always possible to achieve an accurate decision regarding male fertility by routine semen evaluations. in bulls, percentage of progressively motile sperm (threshold of ≥ 30% sperm with forward motion) is determined by sperm motility, and percentage of normal sperm (threshold of ≥ 70% normal sperm) by morphological evaluation.5 though bulls with ≥ 70% morphologically normal sperm sired more calves and bulls with < 50% normal sperm sired fewer calves, a subpopulation of bulls with ≥ 70% normal sperm failed to sire a calf.6 efforts to standardize assessment of these sperm parameters promoted development of more objective computer-assisted sperm analysis (casa) and introduction of new technologies enabled investigation of sperm dna integrity.7 sperm heads contain a condensed haploid nucleus; the deoxyribonucleic acid (dna) is extremely tightly packed, so that its volume is reduced, facilitating sperm transport.8 sperm dna integrity has emerged as one of the most interesting topics in reproductive medicine. the integrity of a sperm genome is a fundamental factor in development of healthy offspring and can be an effective diagnostic tool for male reproductive potential.9 intact dna is defined as complete absence of nicks and breaks, either single-stranded (ssb) or double-stranded (dsb), or any chemical causing alterations in its structure.10 sperm dna fragmentation (sdf) is the accumulation of ssbs or dsbs, measured by the dna fragmentation index (dfi).11 dfi is calculated by the number of single-stranded dna in a sample, divided by the total number of sperm with intact dna, and expressed as a percentage.11 in many studies, elevated sperm dna fragmentation was associated with adverse reproductive outcomes.12 clinical data demonstrating greater sperm dna damage is positively associated with sperm morphological defects (figure 1).13 the negative mailto:ramkasi@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12235 2 citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 impact of high levels of sperm dna damage in natural breeding13 (figure 2) and in assisted reproductive techniques (art) have also been demonstrated.14,15 therefore, assessment of sperm dna integrity could be an independent marker of fertility.16 further, there are strong associations between damage to the paternal genome and embryo development, including effects on newborns and subsequent generations.17 the increasing use of ovum pick-up (opu)/in vitro fertilization in cattle18 and opu/intracytoplasmic sperm insemination in horses19 has raised concerns about unknown parental genome modifications affecting embryo development and future diseases in adulthood stemming from these gamete/ embryo origins.20 subsequently, assessment of dna damage in the male germ line and influence on reproductive outcome has received attention. an understanding of zygote/blastocyst capacity to repair damage would assist reproduction practitioners, and may help to produce healthy, viable embryos. this review summarizes sources of dna damage and repair mechanisms in gametes and embryos, plus in silico analysis of dna repair genes. dna damage in sperm damaged dna has been detected in testicular, epididymal, and ejaculated sperm. elements and assembly of sperm chromatin differ from somatic cells. during spermiogenesis, protamines replace most histones, encompassing chromatin into toroid (a supercoiled structure). as sperm traverse the epididymis, protamines are cross-linked by disulphide bonds, reducing the chromatin to one-sixth the volume in somatic cell nuclei. this dense compaction gives protection against exogenous assaults to sperm dna.21-23 infertile males have relatively high basal sperm dna damage16,17 and their sperm are more susceptible to postejaculation damage.14,15 basis sperm dna damage can arise from exogenous or endogenous sources (table 1) and is due to: 1. faulty chromatin condensation during spermiogenesis, related to an unfitting protamination and inadequate chromatin packaging;24,25 2. the frequency of abortive apoptotic processes, (apoptosis cannot be completed due to nuclear and mitochondrial presence in various compartments in mature sperm);26 and 3. oxidative stress attributable to an imbalance between reactive oxygen species production and antioxidant capacity.27-30 protamination and dna damage during the final phases of sperm transformation ~ 80% of histones are replaced by protamines to enhance sperm nucleus compactness.96 although beneficial for sperm, protamination can have damaging effects on sperm quality, as errors in the replacement process can result in assembly of sperm with dna damage.97,98 while histones are substituted by protamines, temporal breaks occur in the dna due to topoisomerase ii activity that relaxes the dna structure.99 if table 1. causes of sperm dna fragmentation cause references intrinsic causes: deficiencies in recombination during spermatogenesis 31-33 abnormal spermatid maturation 34-36 protamine 1 and 2 ratios 37-41 abortive apoptosis 42-46 oxidative stress 47-64 extrinsic causes: interval after ejaculation 65-67 collection method, extender, postejaculation treatment 68-70 sperm preparation for art 71,72 sex-sorting 73-75 posttesticular oxidative stress 76,77 varicocele 78,79 age 80-82 bacterial infection 83,84 abstinence 85,86 testicular thermoregulation 87-89 reactions to clinical procedures, medications or vaccines 90-92 environmental chemicals 93-95 figure 1. association between percentage of sperm with abnormal morphology and sperm deoxyribonucleic acid fragmentation index (dfi) in bulls (adapted borges et al.12) y = 0.5326x + 3.1242 r = 0.79 10 15 20 25 30 35 20 25 30 35 40 45 50 a b n o rm al s p er m ( % ) sperm dfi (%) figure 2. association between sperm deoxyribonucleic acid fragmentation index (dfi) and pregnancy rate in bulls (adapted borges et al.12) y = -1.3632x + 95.313 r = 0.41 20 25 30 35 40 45 50 40 42 44 46 48 50 s p er m d f i (% ) pregnancy (%) http://dx.doi.org/10.58292/ct.v17.12235 citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 3 these temporal breaks are not repaired properly before the end of spermiogenesis, they will subsequently appear in mature sperm as fragmented dna.100 alternatively, both the quantity of histones that are replaced by protamines and the proportion of protamines (prm)1/prm2 added are typically consistent for each species; therefore, if the proportion is changed, the dna is likely to be poorly packaged, more susceptible to exogenous agents, and more likely to cause infertility.101 the ratio between prm1 and prm2 is ~ 1 in human, and changes in this ratio may cause male infertility.102,103 as prm2 contains fewer cysteine residues than prm1, it produces fewer disulfide bridges, leaving the dna slightly more exposed to adverse effects of external agents.104,105 in addition, abnormal protamine ratios are related to male infertility through aberrant genomic imprinting in infertile humans and bulls.106,107 abortive apoptosis and dna damage apoptosis is genetically controlled, programmed cell death and essential for spermatogenesis. both pro and antiapoptotic molecules involved in various stages of sperm differentiation are summarized (table 2).42 during normal spermatogenesis in mammals, apoptosis results in the loss of up to 75% of prospective sperm,43 as it limits the germ cell population and optimizes the germ-sertoli cell ratio.44 it is a physiological mechanism to eliminate surplus cells during proliferation, removal of derived hormone-dependent cells and elimination of potential harmful cells. during spermatogenesis, sertoli cells select germ cells to pass from mitosis to meiosis; thereafter, ~ 60% of germ cells are marked to be eliminated via apoptosis. however, varying percentages of these marked cells escape apoptotic processes called ‘abortive apoptosis’, in which their dna is partially fragmented, but they maintain capacity to differentiate into mature and even functional sperm45 and will appear in the ejaculate as normal sperm with elevated levels of fragmented dna.46 oxidative stress and sperm dna damage an optimal concentration of reactive oxygen species (ros) in semen is essential for key sperm physiological events, including sperm capacitation, acrosome reaction and sperm-oocyte fusion.47,48 generation of ros in sperm occurs either in the plasma membrane (by nicotinamide adenine dinucleotide phosphate [nadph] oxidase) or in the inner mitochondrial membrane (in the presence of a nicotinamide adenine dinucleotide-dependent oxidoreductase),41,47,49 with the latter being the main source of ros in sperm. sperm midpiece is rich in mitochondria as they need energy to support motility. sperm are also affected by ros in seminal plasma, of either endogenous (leukocytes50 and immature sperm50,51) or exogenous (radiation52,53 and toxins53) origin. normally, ros in seminal plasma is counteracted by antioxidants. however, this balance can be unsettled, and oxidative stress causes lipid peroxidation, which reduces membrane fluidity and damages dna.14,54,55 excessive ros in seminal plasma directly and/or indirectly cause sperm dna damage, abnormal semen parameters, impaired sperm function, and infertility.14,56 the sperm dna may be damaged due to oxidative stress or from reactions between hydroxyl radicals and involve all components of dna, including pyrimidine and purine bases and deoxyribose backbone. reactive oxygen species can further generate dna damage by production of basefree sites, frame shifts, dna-protein cross-linkages, deletions, and chromosomal rearrangements.57 in addition, ros may stimulate protein kinase and poly(adp-ribosyl)ation pathways; therefore, oxidative stress can affect signal transduction pathways and modify expression of genes.58 these collectively may impair sperm functions. however, dna damage, caused by free radicals, may be repaired by specific and nonspecific repair mechanisms, although there is the potential for ill-repair of dna that may contribute to infertility. reactive oxygen species concentration in seminal plasma is maintained in a physiological range by numerous biological antioxidant mechanisms, including enzymatic antioxidants59 (e.g. catalase, superoxide dismutase, and glutathione peroxidase), plus nonenzymatic compounds60 such as vitamins c and e, carotenoids, lactoferrin or coenzyme q10. prostasomes in seminal plasma decrease superoxide radical release by leucocytes, thus reducing oxidative stress.61,62 this antioxidant activity occurs through inhibition of nadph oxidase activity in polymorphonuclear cells (pmns). this arises by rigidification of the pmn plasma membrane by lipid transfer after fusion with a prostasome.63,64 fragmented dna in mature sperm may result from a combination of these processes. for example, apoptosis can occur due to ros-induced sperm damage. furthermore, synergistic effects among damaging effectors are likely to contribute to dna damage and cause sperm death. single-stranded versus double-stranded breaks in normal, healthy sperm, the chromatin is exemplified by a linear arrangement of nucleotides along each dna strand and a lack of ssb and dsb, nucleotide modifications, or base loss.108 sperm chromatin has abundant alkali-labile sites, mainly localized in repetitive dna sequences, prone to dna torsion during chromatin packing. chromatin damage is a comprehensive term that accounts for any defects in dna structure, including: 1. ssb or dsb; 2. base deletion or modification; 3. interstrand or intrastrand dna cross-linkage; and 4. protamine deficiency and/or mispackaging via defective dna-protein cross-links (reviewed109). it may occur during spermatogenesis, spermiogenesis, epididymal transit or postejaculation. sdf relates to breaks at dna strands that are termed ssbs or dsbs. single-strand breaks table 2. pro and antiapoptotic proteins involved in various stages of sperm differentiation stages of sperm differentiation proapoptotic protein antiapoptotic protein primordial germ cells bax, bad, bim, bak bcl-x spermatogonia bax, p53 c-kit spermatocyte bax, fas/fasl bcl-xl, bcl-xs round spermatid fas/fasl bcl-xl, bcl-xs elongated spermatid fas/fasl bcl-x sperm fas/fasl bcl-x http://dx.doi.org/10.58292/ct.v17.12235 4 citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 give rise to free 5’-3’ ends, affecting only 1 dna strand, whereas its template remains undamaged. by contrast, dsbs are characterized by blunt 5’-3’ ends affecting both dna strands. tests currently available to evaluate sdf include: sperm chromatin structure assay (scsa)14-16 terminal deoxynucleotidyl transferase-mediated dutp biotin nick end labelling (tunel) and in situ nick translation (isnt)110-112 assays breakage detection-fluorescence in situ hybridization (dbd-fish) test113 comet assay114,115 single-cell pulsed-field gel electrophoresis technique116 sperm chromatin dispersion test (scdt)117,118 these tests can be grouped in methods that use: 1. enzymatic reactions to label dna breaks: either using a terminal transferase (e.g. tunel) or specific enzymes such as the klenow fragment (e.g. in situ nick translation assay; isnt) to label free 3-oh ends of the nucleotide at the dna break. in the latter, a 5’3’ polymerase activity is combined with a 3’-5’ exonuclease activity for elimination of precoding nucleotides and proofreading. both tunel and isnt assays detect ssbs and dsbs indistinctively. 2. controlled dna denaturation combined with protein depletion as intermediates to reveal dna breaks: these tests apply dna denaturation and/or controlled protein depletion. the scsa relies on controlled dna denaturation to target preexisting dna breaks, whereas the alkaline comet assay and the scdt are based on dna denaturation and controlled protein depletion. like tunel, these assays determine the global sdf without discriminating between sperm with ssb versus dsb. by contrast, the neutral comet assay only uses a controlled protein depletion to exclusively detect dsb whereas the 2-dimensional comet assay uses 2 electrophoretic runs, one in a neutral buffer and another in an alkaline buffer to simultaneously map ssbs and dsbs.119 3. dyes that bind to relaxed guaning cytocine (gc)-rich motifs: includes tests that spot abnormal chromatin packaging by means of the fluorescent antibiotic chromomycin a3 staining, given its preference to bind relaxed dna gc-rich motifs, toluidine blue staining, acridine orange test and aniline blue staining.120 notably, given that histone-complexed dna (stained by acridine orange) fluoresces twice as brightly as protamine-complexed dna,121 this high dna stainability sperm fraction, which represents sperm with excess nuclear histones and faulty chromatin condensation, can also be detected by the scsa. hence, this test also provides information about chromatin compaction. the scsa, comet, scdt, and tunel provide reliable information about sperm dna integrity in subfertility.122 however, optimal thresholds are still to be defined and are vital to understand how each test reports results. the sdf tests can detect sperm with evident dna fragmentation, but sperm with undetectable damage may remain hidden or ambiguous within that population. the latter might not have yet fully expressed sdf at the time of analysis, representing sperm with a dna fragmentation predisposition termed ‘iceberg effect’.65-67,109,123 all of these are valuable tools to acquire information on the state of the sperm dna, but mechanisms of restitution in the oocyte and embryo may provide a confusing view of the real impact of dna damage on pregnancy, contributing to discrepancies about impacts of sdf on pregnancy rates. dna repair mechanisms an intricate balance of genome stability and instability is necessary, controlled by interactions of many dna repair mechanisms. in germ cells, there are some levels of defenses that avoid the production and persistence of dna damage, such as base mismatches, ssbs and dsbs, bulky adducts (disturb dna transcription and replication, and prompt mutations), etc. male and female gametes, and embryos have an array of dna repair pathways including: 1. nucleotide excision repair (ner): corrects a wide variety of helix-distorting dna lesions and crosslinks, primarily caused by environmental agents such us ultraviolet light.123,124 2. mismatch repair (mmr): eliminates dna mismatches produced from recombination between imperfectly matched sequences or from errors during dna replication.125 mismatch repair can also repair oxidative  damage and provide maintenance of repeated sequences.123,126,127 3. base excision repair (ber): corrects small dna alterations that only affect 1 dna strand and that do not distort the structure of the dna helix such as the incorporation of uracil or oxidized bases induced by reactive oxygen species or the presence of ssbs123,128-130 4. homologous recombination (hr); a process that occurs during meiosis but is also used to repair dsbs as well as inter-strand dna crosslinks (mainly due to ionizing radiation).123,131 5. nonhomologous end joining (nhej): repairs dsbs without using the homologous sequence as a template and thus can cause insertions and deletions.123,132,133 the dna direct repair mechanisms, photolyase, alkyltransferase, and dioxygenase-mediated repair processes, provide cells with simple yet efficient solutions to reverse covalent dna adducts.134 these dna repair mechanisms are active in practically all somatic cell types, as well as germ cells,135 but mature sperm and oocytes have differential organization of their repair mechanisms. dna repair mechanisms in sperm spermatogenesis is a complex process that produces sperm, which are unique in structure and function. spermatogenesis can be divided into 3 sequential steps: 1. mitotic proliferation: produces large numbers of sperm over the reproductive lifetime; dna mismatches produced during this step are repaired via the mmr pathway.136 2. meiotic recombination: meiotic recombination and chromosome segregation produce genetically diverse haploid gametes.137,138 regarding recombination, hr and nhej seem to divide their tasks based on the phase of the http://dx.doi.org/10.58292/ct.v17.12235 citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 5 cell cycle and the landscape of the dna break.139 homologous recombination mainly works during s phase and on replication-derived 1-ended dsbs to accurately resolve the damage, whereas the more error-prone nhej process functions primarily during g1 assembly of 2-ended dsbs.139-141 perhaps programmed dna dsbs during meiosis is mainly repaired by hr with high reliability. repair of these breaks is tightly controlled to favor hr (the only repair pathway that can form crossovers). 3. cytodifferentiation of spermatids: a complex remodeling of the haploid genome that involves replacing the majority of histones with protamines.100 dna compaction, an outcome of this process, is achieved by the transient formation of ssbs and dsbs in sperm dna.100,142 spermatids resolve exogenous and programmed dsbs using the alternative nhej pathway143,144 due to their haploid character and the absence of the main components of the classical nhej pathway. it is critical that these transition strand breaks are fixed during this step because persistence of dna breaks in mature sperm can increase sperm dna fragmentation.145,146 in general, mature sperm were deemed incapable of dna damage restoration due to maximum compaction of their dna and reduced transcriptional capacity.147,148 however, human sperm retain a truncated but functional ber pathway containing only the 8-oxoguanine dna glycosylase-1 protein.147,148 the presence of this enzyme is adequate for sperm to detect and remove oxidized base adducts, specially 8-hydroxy-2’-deoxyguanosine (8-ohdg) residues, a prevalent product of oxidative stress.149,150 due to the rest of the pathway being shortened, the abasic (apurinic/apyrimidinic) site produced after excision of 8-ohdg must be then repaired by the oocyte after fertilization and prior to the first round of cell division during early embryo development.149,150 at the end of spermiogenesis, disulfide cross-links are created between protamines, while spermatids pass through the epididymis. this process may be considered an inherent screening mechanism directed at eliminating genetically defective sperm, as the greater the dna damage, the lower disulfide cross-linking is established, resulting in lower-quality sperm with a reduced capacity to fertilize an ovum or produce a good embryo.151,152 dna repair mechanisms in the oocyte oogenesis can be divided into 3 phases: 1. multiplication phase: primordial germ cells initiate differentiation into female germ cells (oogonia) in the early postimplantation embryo 2. growth phase: oogonia divide through mitosis and enter meiosis i until they stop developing at the diplotene stage, in prophase i 3. maturation phase: oocytes complete the first division of meiosis i during ovulation the integrity of the oocyte genome is affected mainly by 2 processes: 1. meiotic recombination during the fetal period 2. the long postnatal period of meiotic arrest (dictyate stage) before meiotic division physiological dsbs are fabricated in association with meiotic recombination during the fetal period; however, this damage is generally repaired at the end of the meiotic prophase i by the oocyte through hr.153,154 failure to repair dna damage caused by recombination directs meiotic checkpoints and activates apoptosis.155,156 regarding the prolonged postnatal interval of meiotic arrest prior to meiotic division, oocyte dna is subjected to a wide range of potential damage that can reduce fertility.157,158 several studies provided strong evidence that oocytes, from the primordial follicle stage in metaphase ii (mii), have the capacity to repair damaged dna and maintain genome integrity.159,160 during oogenesis, genes related to dna repair are expressed at high levels and their mrnas and proteins are accumulated inside oocyte cytoplasm.161 transcripts from all dna repair pathways including direct lesion reversal, ber, mmr, ner, hr, and nhej are represented in mouse, monkey, and human mii oocytes.157,158 these transcripts and proteins have roles during fertilization to address changes in chromatin remodeling and maintain chromatin integrity, and are also used in the zygote until the embryo genome becomes active and can transcribe its own dna repair genes.162,163 metaphase ii stage oocytes have potential to dna repair via nhej in mice.164 however, another study suggested that there may be species differences in the ability of gv (germinal vesical stage) and mii oocytes to undertake dna repair.165 in this study, the overall expression patterns of genes involved in the repair of dna dsbs differed between primates and mice. therefore, it was proposed that rodent oocytes have better dna repair potential than primates.165 dna repair efficiency in the oocyte also decreases with maternal age due to a reduction in mrna levels for dna repair genes.166,167 sperm with dna damage is mostly functional; hence, the damage is transmitted to the oocyte after fertilization.161,164,168 this damage must be rapidly repaired to ensure that genetic information is transmitted reliably and to avoid paternal transmission of chromosomal structural abnormalities and/ or mutations.168 as mature sperm have limited dna repair capabilities, oocytes are initially responsible for repair and remodeling of both paternal and maternal genomes. as demonstrated in mouse models, women with deficiencies in dna repair mechanisms produce embryos with more frequent chromosome/chromatid aberrations and microsatellite instabilities.157,169,170 after fertilization, dna damage repair transcripts and proteins produced by the oocyte are used by the zygote until the embryonic genome is activated and can begin transcribing its own dna repair genes.157,162 this maternal-zygotic transition is species specific; in humans, it typically occurs at the 4-8 cell stage.157,171 in due course, exhaustion of stored maternal mrnas is compensated by zygotic transcription, leading to activation of cell cycle checkpoints and expression of dna repair proteins.157,168 however, impaired oocytes, especially those from older women, may not be able to adequately compensate for paternally derived dna damage. further, as oocytes age, their dna repair efficiency diminishes due to lower expression of key dna repair genes, such as brca1, mre11, rad51, and atm.172 this results in more dsb, chromosomal abnormalities, and increased sensitivity to oxidative stress.157 these findings led to the hypothesis that lower-quality human oocytes may be unable to repair paternally derived dna damage adequately, accounting for poor reproductive outcomes. highly expressed dna repair genes in mii oocytes are provided (table 3). http://dx.doi.org/10.58292/ct.v17.12235 6 citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 prediction and analysis of differentially expressed dna repair genes in mii oocytes the mirnet (http://www.mirnet.ca/, accessed on 4 august 2024) platform was used to perform interaction analysis174 and to select the top 10 dna repair genes in mii oocytes with high degree (number of connections a node has to other nodes) and betweenness (number of connections occurring upon a node) centrality. betweenness centrality measures the extent to which a mirna/gene lies on paths between other mirnas/genes. micrornas/genes with high betweenness may have substantial influence within a regulatory network by virtue of their control over passing information between others.175 genes with a high degree centrality are important for diagnosis of disease, whereas proteins with a high betweenness are important for drug discovery.175 thirty-four dna repair genes in mii oocytes interacted with 45 transcription factors and 2050 mirna (figure  3).  the top 10 dna repair genes in mii oocytes with high degree and betweenness centrality are illustrated (table  4)  and their tissue and single-cell expressions, associated genes, and functions are listed (table 5). dna repair mechanisms in the zygote fertilization induces a cascade of critical events including fusion of paternal and maternal dna and development of the zygote. during this stage, dna has 3 main processes: 1. parental chromosomes initially exist separately as distinct maternal and paternal pronuclei, 2. remodeling of chromatin assembly with active demethylation of paternal dna versus passive demethylation of maternal dna176 3. reparation of ssbs and dsbs in paternal dna177,178 the dna repair in the zygote is considered a maternal trait because until embryonic genome activation occurs (4-cell stage in humans),179 zygote development is supported by maternal transcripts and proteins. as mature sperm have reduced dna repair capacity, a few dna lesions will inevitably remain in sperm dna and will need to be reduced/ eliminated when gametes are enlisted in the zygote. following fertilization, the effects of sperm dna damage depend on the balance between the amount and/or type of dna damage present and the capacity of the fertilized oocyte to fix the damaged sperm dna. it has been suggested that an oocyte has the capacity to repair 8% sperm dna damage180; higher levels of sperm dna damage are associated with a failure to reach the blastocyst stage180 and embryonic loss between embryonic genome activation and blastocyst stages,181 known as a ‘late effect’ from paternal dna damage.182,183 an early paternal effect can compromise reproductive outcomes without increased sperm dna fragmentation. further, sperm dna fragmentation evaluation is useful to detect late paternal effect, which is not associated with morphological abnormalities at the zygote and early cleavage stages.183 sperm dna fragmentation repair in embryos during/after implantation unrepaired dna damage beyond a critical threshold in embryos with normal karyotype generated in vivo and in vitro may cause failure in embryo development after embryo implantation. this type of damage is expressed during/after implantation and has been characterized as late paternal effect.183 there are also indicators of high levels of dna damage in a sperm sample with failure to obtain blastocysts162 and it is believed that some loss of preimplantation embryos occurs between postembryonic genome activation and the blastocyst stage.184-186 short g1 and g2 phases in rapidly dividing blastomeres support the assumption that hr is the dominant dsbr mechanism in the blastocyst. the first step in hr involves rad52, which competes with ku to direct dsbr toward hr rather than nhej.164,165 many animals lacking dna repair enzymes are not viable, with preimplantation death being a common result; however, much of the embryonic loss occurs around implantation, indicating the necessity for dna repair ability when embryonic cells proliferate rapidly and differentiate.166 many dna repair genes involved in dna damage response pathways seem to be expressed in postimplantation mammalian embryos, especially mid-gestational stage onwards. at this stage, spatial table 3. highly expressed dna repair genes in metaphase ii stage (mii) oocytes dna repair genes in mii oocytes references abh2 161,162 apex1 161,162,173 blm 162 ccnh 161,162 cdk7 161,162 chaf1a 161,162 dclre1a 161,162 dut 161,162 fancl 162 fen1 161,162 gtf2h2 161,162 gtf2h5 161,162 h2afx 161,162 kiaa1794 162 lig1 161,162 mbd4 161,162 mgmt 161,162 msh2 161,162 msh6 161,162 pcna 161,162 polb 161,162 rad17 161,162 rad50 161,162 rad51 161,162 rad51c 161,162 rad52 161,162 rbbp8 162 revl1 162 rpa2 161,162 tdp1 161,162 ube2a 161,162 ung 161,162 xpa 161,162 xrcc4 161,162 xrcc6 162 http://dx.doi.org/10.58292/ct.v17.12235 http://www.mirnet.ca/ citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 7 figure 3. gene-mirna-transcription factor interaction network of dna repair genes in mii oocytes; 34 genes interacted with 2050 mirnas and 45 transcription factors (p < 0.05). yellow circles denote genes; green circles denote transcription factors; and size indicates significance. blue squares denote mirnas table 4. top 10 dna repair genes in metaphase ii stage (mii) oocytes with high degree and betweenness centrality high degree centrality high betweenness centrality # id degree betweenness # id degree betweenness 1 msh6 424 352737.6 1 rad51 375 403488.3 2 xrcc6 382 290840.3 2 msh6 424 352737.6 3 rad51 375 403488.3 3 xrcc6 382 290840.3 4 pcna 296 226486.4 4 pcna 296 226486.4 5 apex1 283 199468.7 5 rad50 275 216615.7 6 rad50 275 216615.7 6 apex1 283 199468.7 7 mbd4 229 167614.4 7 h2afx 192 173755.7 8 gtf2h5 226 172415.7 8 gtf2h5 226 172415.7 9 msh2 221 100570.1 9 mbd4 229 167614.4 10 fen1 208 121183 10 fen1 208 121183 http://dx.doi.org/10.58292/ct.v17.12235 8 citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 table 5. top 10 genes (in metaphase ii stage (mii) oocytes with a high degree and betweenness centrality) and their characteristics gene tissue expression single-cell normalized expression (ntpm) associated genes functions apex1 (ber) ovary testis oocyte 95.6 spermatocytes 11.7 spermatogonia 55.0 anp32a fen1 lig1 polb xrcc1 vital role in dna repair and redox regulation fen1 ovary testis oocyte 27.9 spermatocytes 98.9 spermatogonia 67.2 apex1 pcna pold1 lig1 wrn dna replication and repair msh6 (mmr) ovary testis oocyte 27.9 spermatocytes 44.7 spermatogonia 66.0 pcna msh2 mlh1 msh3 pms2 dna mismatch repair rad50 (hr) ovary testis oocyte 8.8 spermatocytes 8.5 spermatogonia 6.6 brad1 brca1 mre11 nbn rbbp8 vital role in dsb repair, dna recombination, maintenance of telomere integrity and meiosis xrcc6 (nhej) ovary testis oocyte 233.1 spermatocytes 584.0 spermatogonia 303.1 aplf xrcc4 xrcc5 lig4 prkdc repair of nonhomologous dna ends, e.g. as required for dsb repair, transposition, and v(d)j recombination. rad51 (hr) ovary testis oocyte 67.6 spermatocytes 65.6 spermatogonia 28.7 xrcc3 blm brca2 rad52 abcd3 homologous strand exchange, a key step in dna repair through homologous recombination mbd4 (ber) ovary testis oocyte 116.2 spermatocytes 79.6 spermatogonia 93.8 dnmt dnmt3a dnmt3b mlh1 tdg dna repair gtf2h5 (ner) ovary testis oocyte 241.2 spermatocytes 336.5 spermatogonia 162.9 ercc2 ercc3 gtf2h1 gtf2h2 gtf2h4 general and transcription-coupled ner of damaged dna. in ner, it opens dna around lesion to allow excision of the damaged oligonucleotide and its replacement by a new dna fragment. essential role in transcription initiation. msh2 (mmr) ovary testis oocyte 19.9 spermatocytes 80.0 spermatogonia 59.0 atm, mlh1 msh3 msh6 pms2 dna mismatch repair system; 2 formed heterodimers bind to dna mismatches to initiate repair. (continued) http://dx.doi.org/10.58292/ct.v17.12235 citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 9 table 6. highly expressed dna repair genes in embryos dna repair genes in embryo references alkbh2 162 apex1 161,162,173 aptx 161,162 blm 162 cdk7 161,162 chaf1a 161,162 dut 161,162 fn1 162 gtf2h5 161,162 h2afx 161,162 mlh1 161,162 mms19l 161,162 msh2 161,162 msh6 161,162 nudt1 161,162 parp1 161,162 parp2 161,162 pcna 161,162 polb 161,162 rad17 161,162 rad23b 161,162 rad51 161,162 rad51c 161,162 rad52 161,162 rpa2 161,162 shfm1 161,162 tdg 161,162 ube2a 161,162 ube2b 161,162 ube2v2 161,162 ung 162 xpa 161,162 xrcc5 161,162 xrcc6 161,162 162 patterns of expression in the embryo become apparent for a few dna repair genes.187 the capacity of the mammalian embryo to repair damaged dna and its selective sensitivity to specific lesions is not well understood. many gaps exist in our current knowledge concerning the roles and expression timings of several dna repair genes in the early stages of embryonic development. the observed developmental stage-specific variations in dna repair gene expression transcripts and proteins highlight complexity of the regulation of these pathways during development. highly expressed genes in embryos are given (table 6). prediction and analysis of differentially expressed dna repair genes in embryo the mirnet (http://www.mirnet.ca/, accessed on 4 august 2024) platform was used to perform interaction analysis174 and to select the top 10 dna repair genes with high degree and betweenness centrality in blastocysts. thirty-four dna repair genes in blastocysts interacted with 249 transcription factors and 2334 mirnas (figure 4). the top 10 dna repair genes in embryo with high degree and betweenness centrality and their tissue and single-cell expressions, associated genes, and functions are in tables 7 and 8, respectively. the dna repair genes expressed in mii oocytes and embryos and their involvement in the repair pathways are listed (table 9). the gene pcna is expressed in both mii oocytes and embryos, involved in 3 pathways (ber, mmr, and nmr). similarly, rad51 is involved in the hr pathway and is expressed in both mii oocytes and embryos. the xrcc6 gene is expressed in mii oocytes and embryos and is involved in the nehj pathway. spatiotemporal expression of these common genes could be diagnostic tools to determine the extent of dna repair by oocytes and embryos. conclusion excellent quality oocytes and embryos have better dna repair capabilities, enabling them to compensate for sperm dna damage and to produce excellent quality embryos that are more likely to develop normally. however, the exact mechanism(s) remain under investigation. additionally, it is unknown what type and/or degree of sperm dna damage is too much for an oocyte and/or embryo to overcome. table 5. (continued) gene tissue expression single-cell normalized expression (ntpm) associated genes functions pcna (ber, ner and mmr) ovary testis oocyte 365.4 spermatocytes 218.6 spermatogonia 119.5 fen1 lig1 polh rfc2 rfc3 increase processivity of leading strand synthesis during dna replication; involved in rad6-dependent dna repair pathway http://dx.doi.org/10.58292/ct.v17.12235 http://www.mirnet.ca/ 10 citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 table 7. top 10 dna repair genes in blastocysts with high degree and betweenness centrality high degree centrality high betweenness centrality # id degree betweenness # id degree betweenness 1 parp1 472 468024.5 1 parp1 472 468024.5 2 fn1 461 306674 2 rad51 374 385735 3 msh6 424 355667.7 3 msh6 424 355667.7 4 rad23b 399 264386 4 parp2 325 343441.9 5 xrcc6 393 319979.2 5 xrcc6 393 319979.2 6 rad51 374 385735 6 fn1 461 306674 7 xrcc5 371 243838.4 7 rad23b 399 264386 8 ube2v2 364 254464.1 8 ube2v2 364 254464.1 9 parp2 325 343441.9 9 xrcc5 371 243838.4 10 pcna 293 237321.9 10 pcna 293 237321.9 figure 4. gene–mirna-transcription factor interaction network of dna repair genes in embryos. thirty-four 34 genes interacted with 2334 mirnas and 249 transcription factors (p < 0.05). yellow circles denote genes, green circles denote transcription factors and size indicates significance. blue squares denote mirnas http://dx.doi.org/10.58292/ct.v17.12235 citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 11 table 8. top 10 genes (in blastocysts with a high degree and betweenness centrality) and their characteristics gene tissue expression single-cell normalized expression (ntpm) associated genes functions parp1 ovary testis oocyte 152.2 spermatocytes 111.5 spermatogonia 226.1 casp3, xrcc5, xrcc6, tim1, hpf1 recovery of cell from dna damage; regulation of differentiation, proliferation, and transformation fn1 ovary testis oocyte 28.7 spermatocytes 2.6 spermatogonia 15.2 itgav, itgb3, itgb1, itga5, yes1 cell adhesion and migration processes including embryogenesis msh6 ovary testis oocyte 27.9 spermatocytes 44.7 spermatogonia 66.0 pcna, msh2, mlh1, msh3, pms2 dna mismatch repair rad23b (ner) ovary testis oocyte 80.1 spermatocytes 51.2 spermatogonia 101.7 ngly1, psmd4, psmd2, ubc, xpc nucleotide excision repair xrcc6 (nhej) ovary testis oocyte 233.1 spermatocytes 584.0 spermatogonia 303.1 aplf, xrcc4, xrcc5, lig4, prkdc repair of nonhomologous dna ends, e.g. as required for dsb repair, transposition, and v(d)j recombination. rad51 (hr) ovary testis oocyte 67.6 spermatocytes 65.6 spermatogonia 28.7 xrcc3, blm, brca2, rad52, abcd3 important role in homologous strand exchange, a key step in dna repair through homologous recombination xrcc5 (nhej) ovary testis oocyte 43.4 spermatocytes 262.4 spermatogonia 169.7 aplf, nhej1, prkdc, lig4, xrcc6 dsb repair and v(d)j recombination ube2v2 (error-free postreplication repair) ovary testis oocyte 65.4 spermatocytes 196.3 spermatogonia 96.0 rps27a, ubc, rad18, ubb, ube2n control of progress through the cell cycle and differentiation, role in error-free dna repair pathway and contributes to the survival of cells after dna damage. (continued) http://dx.doi.org/10.58292/ct.v17.12235 12 citation: clinical theriogenology 2025, 17, 12235, http://dx.doi.org/10.58292/ct.v17.12235 further studies are required to elucidate dna repair capabilities of oocytes and embryos and the extent to which excellent quality oocytes and embryos can overcome high sdf levels. in addition, currently available dna fragmentation tests cannot provide information regarding ‘reparability’ of sperm dna damage. hence, there is a need for tests that predict the reparability of sdf. conflict of interest none to report. references 1. carroll ej, ball l, scott ja: breeding soundness in bulls-a summary of 10,940 examinations. j am vet med assoc 1963;142:1105-1111 2. elmore r, bierschwal cj, martin ce, et al: a summary of 1127 breeding soundness examinations in beef bulls. theriogenology 1975:3:209-218. doi: 10.1016/0093-691x(75)90139-9 3. cox cm, thoma me, tchangalova n, et al: infertility prevalence and the methods of estimation from 1990 to 2021: a systematic review and meta-analysis. hum reprod open 2022;2022:hoac051. doi: 10.1093/hropen/hoac051 4. agarwal a, mulgund a, hamada a, et al: a unique view on male infertility around the globe. reprod biol endocrinol 2015;13:37. doi: 10.1186/s12958-015-0032-1 5. palmer cw, persson y, söderquist l: classification of the potential breeding ability of range beef bulls based on semen quality parameters in samples collected by transrectal massage  a comparison of the swedish and canadian systems. anim reprod sci 2013;140:124-130. doi: 10.1016/j.anireprosci.2013. 06.001 6. fitzpatrick la, fordyce g, mcgowan mr, et al: bull selection and use in northern australia. part 2. semen traits. anim reprod sci 2002;71:39-49. doi: 10.1016/s0378-4320(02)00024-6 7. kutchy na, menezes esb, ugur mr, et al: sperm cellular and nuclear dynamics associated with bull fertility. anim reprod sci 2019;211:106203. doi: 10.1016/j.anireprosci.2019.106203 8. alberts b, johnson a, lewis j, et al: molecular biology of the cell. 4th edition, new york; garland science: 2002. sperm. available from: https://www.ncbi.nlm.nih.gov/books/nbk26914/ [cited 06 august 2024]. 9. shamsi mb, venkatesh s, tanwar m, et al: dna integrity and semen quality in men with low seminal antioxidant levels. mutat res 2009;665:29-36. doi: 10.1016/j.mrfmmm.2009.02.017 10. agarwal a, majzoub a, baskaran s, et al: sperm dna fragmentation: a new guideline for clinicians. world j mens health 2020;38:412-471. doi: 10.5534/wjmh.200128 11. kasimanickam r, nebel rl, peeler id, et al: breed differences in competitive indices of holstein and jersey bulls and their association with sperm dna fragmentation index and plasma table 8. 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al: ku80: product of the xrcc5 gene and its role in dna repair and v(d)j recombination. science 1994;265:1442-1445. doi: 10.1126/science.8073286 200. bohrer rc, duggavathi r, bordignon v: inhibition of histone deacetylases enhances dna damage repair in scnt embryos. cell cycle 2014;13:2138-2148. doi: 10.4161/cc.29215 http://dx.doi.org/10.58292/ct.v17.12235 https://doi.org/10.1073/pnas.0705257104 https://doi.org/10.1073/pnas.0705257104 https://doi.org/10.1016/j.mam.2013.01.003 https://doi.org/10.1016/j.mam.2013.01.003 https://doi.org/10.1126/scitranslmed.3004925 https://doi.org/10.1126/scitranslmed.3004925 https://doi.org/10.1093/molehr/gam038 https://doi.org/10.1093/molehr/gam038 https://doi.org/10.1093/nar/gkaa467 https://doi.org/10.1093/nar/gkaa467 https://doi.org/10.1016/b978-0-12-387038-4.00009-4 https://doi.org/10.3390/cells12141874 https://doi.org/10.1262/jrd.20234 https://doi.org/10.3390/ijms21113882 https://doi.org/10.3390/ijms21113882 https://doi.org/10.1038/332459a0 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https://doi.org/10.1590/1984-3143-ar2023-0021 https://doi.org/10.1126/science.8073286 https://doi.org/10.4161/cc.29215 2019 plasma altrenogest concentrations in mares after intrarectal or intravaginal administration plasma altrenogest concentrations in mares after intrarectal or intravaginal administration k. hornberger, c. c. lyman, j. m. baldrighi, g.r. holyoak center for veterinary health sciences oklahoma state university, still water, ok abstract altrenogest (regu-mate®) is labelled for daily oral administration and is used in mares to suppress estrus behavior, maintain pregnancy and synchronize estrous cycles. it is a very potent progestin, with potential risks associated with transdermal absorption for persons handling or administering this compound. the objective was to determine plasma concentrations of altrenogest following rectal or vaginal administration in mares. altrenogest (0.044 mg/kg) was administered once daily for 5 days; orally, vaginally, and transrectally in 7, 5 and 5 mares, respectively. transrectal palpation and ultrasound examinations were performed on days 1, 3, and 5 to assess cervical tone, endometrial edema, and ovarian structures. venous blood was drawn at time 0 (prior to drug administration) and at 30 and 60 minutes after drug administration. to determine altrenogest concentrations, plasma from each sample was analyzed via ultra-performance liquid chromatography coupled to a triple quadruple mass spectrometer. although absorption of altrenogest occurred after vaginal or oral administration, plasma altrenogest concentrations were less (p < 0.05) than those of orally treated mares. additional studies are needed to determine if plasma altrenogest concentrations after rectal or vaginal administration are sufficient to suppress estrus behavior or maintain pregnancy. keywords: estrus suppression, rectal, vaginal, progestin, progestogen, altrenogest introduction progestins are used in equine reproductive medicine to prevent expression of unwanted estrus behavior, aid in estrus synchronization, hasten onset of cyclicity in late transition mares, aid in maintenance of pregnancy, or treat placentitis.1-9 oral altrenogest (regu-mate®, intervet/merck animal health, madison, nj) treatment effectively binds to the progesterone receptor in the mare at the label dose (0.044 mg/kg), is safe and efficacious for extended intervals in mares and does not affect fertility.4,6,10 oral administration of altrenogest resulted in prompt suppression of estrus behavior that continued until cessation of administration, with estrus occurring within 10 days.11 orally administered altrenogest (0.044 mg/kg) reached maximum plasma concentrations within 15 to 30 minutes after administration in 70% of horses, within 90 minutes in another 20% of horses and within 180 minutes in the remaining 10%.12 mean peak plasma altrenogest concentration was 35 ng/ml (range, 23 75).12 although oral is the primary route of administering altrenogest, this poses some risk to persons administering it, as it is readily absorbed, through intact skin. human risks associated with cutaneous exposure to altrenogest include an altered menstrual cycle or prolonged pregnancy. people with conditions such as thromboembolic disorders, cerebral-vascular or coronary artery disease, known or suspected breast carcinoma, estrogen-dependent neoplasia, undiagnosed vaginal bleeding, oral contraceptive-induced tumors, or liver disease are advised not to handle this product. in addition to potential health implications for persons administering the drug, oral administration can be difficult for owners. the requirement for daily oral administration, often for extended intervals, becomes difficult if a horse develops avoidance behaviors such as tossing their head to resist treatment or spitting out the drug. regarding the latter, the administrator attempts to prevent drug loss by holding the horse’s mouth shut and raising its head, making them vulnerable to drug exposure, even if they are wearing rubber gloves. another option for administration is adding the oil-based drug to the horse's grain; however, loss of drug in the feed bucket, resulting in inadequate dosing, is a practical concern. lastly, there are instances when oral administration to a hospitalized pregnant mare is contraindicated, i.e. refluxing anterior enteritis, intestinal obstruction or ileus, esophageal disease, or facial bone fractures.13 in clinical cases such as these, injectable progestogen formulations are ideal, although access may be delayed if the product is not readily available. clinical theriogenology • volume 11 number 1 • march 201959 alternative drug administration routes have yielded favorable results in various species; an added benefit was increasing duration of drug exposure to mucosal surfaces.14-17 for example, metronidazole was rapidly absorbed after rectal administration in horses. although not superior to oral or intravenous administration, it is a viable, alternative route, as the bitter taste reduces feed intake, even if flavor is added.14 acetylsalicylic acid, used for treatment and prevention of arterial thrombosis in horses, has better bioavailability after rectal versus intragastric administration.15 diazepam, frequently used for seizures, was rapidly absorbed and active after rectal administration in dogs.16 levetiracetum, also used for seizures in dogs, exceeded minimum target concentrations within 10 minutes after rectal administration and sustained them.17 regarding reproductive drugs used in both veterinary and human medicine, not only is intravaginal administration of progesterone or prostaglandins (e.g. pge1, misoprostol and pge2, dinoprostone) a major method of drug delivery, the efficacy of the drug is also enhanced.18-21 intravaginal drug delivery is routinely used for estrous cycle management in ruminants, e.g. controlled internal drugrelease (cidr) devices, progesterone-releasing intravaginal devices (prids), and vaginal sponges. drug administration via rectal or vaginal routes prolongs drug exposure to mucosal surfaces. intravaginal or intrarectal administration routes is expected to be advantageous for administration of altrenogest in horses. these should be viable options for horse owners who are unable to use injectable progestogens due to needle-shyness, for horses that develop injection site reactions, and/or horses averse to oral treatments. the objective was to determine plasma altrenogest concentrations after rectal or vaginal administration. materials and methods animals and structure seventeen healthy mares (14 quarter horse, 3 thoroughbred), from 4 24 years were used. mares were housed at a university research farm, kept on pasture and fed concentrate twice daily with ad libitum access to hay and water. all mares were weighed on day 1 (d1) to ensure accurate drug dosing. horses were handled in accordance with the us department of agriculture guide for care and use of agricultural animals in research (acup vm-15-28). experimental groups assignment of mares to 3 groups was at random. treatments were administered at the same time each day. five mares received intrarectally (0.044 mg/kg12 altrenogest, every 24 hours for 5 consecutive days [rectal group, rg]). five mares received intravaginally (0.044 mg/kg altrenogest, every 24 hours for 5 consecutive days [vaginal group, vg]). a third group, which initially consisted of 2 mares that served as oral-administered controls, received altrenogest orally (0.044 mg/kg) every 24 hours for 5 consecutive days (oral group, og); this group later expanded to a total of 7 mares after addition of 5 mares to serve as og controls. expansion of the oral group occurred 5 months after the initial work. routes of administration for the og group, altrenogest was deposited into the mare’s oral buccal pouch with an oral dose syringe. mares were closely observed for 1 to 2 minutes thereafter and drug loss/oral expulsion noted. mare’s heads were tied to a fence rail to prevent grazing. any drug loss during the next 60 minutes was recorded. for the rg group, manure was not evacuated from the rectum before treatment, to simulate treatment by non-veterinary personnel. a 15 cm pipette was inserted ~5 cm anterior to the anal sphincter and a pre-loaded syringe attached to inject the drug; back flow or resistance during pipette insertion/drug injection was recorded. if a mare defecated within 1 hour after rectal administration, the time was recorded. prior to daily vaginal administration of altrenogest, the vulvar mucosa was examined for signs of irritation and the vulvar commissure was cleaned once using ivory liquid soap and water. vaginal drug clinical theriogenology • volume 11 number 1 • march 2019 60 administration was accomplished by wearing a sterile sleeve and manually guiding a 15 cm pipette through the vulva and vestibule and into the caudal vagina. any back flow or resistance was recorded. blood sampling each day, jugular venipuncture was performed 3 times12 on each mare after administration of altrenogest; once prior to administration, 30 minutes after administration, and 60 minutes after administration. approximately 3 to 5 ml of blood was collected via a vacutainer into blood tubes containing edta. all blood samples were centrifuged at 2000 x g for 10 minutes and plasma was decanted and frozen at -80°c until assayed. reproductive examination transrectal ultrasonographic (sonosite m-turbo®, fujifilm, 7.5 mhz, linear-array transducer) examinations were performed on days 1, 3, and 5. cervical tone was graded on an a to c scale with a indicating a relaxed, open cervix and c indicating a tight, closed cervix. edema was graded from 0 to 3 with a score of 0 indicating no edema and a score of 3 indicating heavy edema. the presence of fluid was noted and when present, echodensity, location and largest pocket size were noted. ovarian follicle size and the presence of a corpus luteum (cl) were recorded. mares were classified as being in estrus (presence of endometrial edema, a/open cervix) or diestrus (lack of endometrial edema, c/closed cervix, presence of a cl). at the conclusion of the 5-day treatment regimen, a speculum examination was performed rectally or vaginally, as dictated by treatment group, to detect evidence of mucosal irritation as a result of administering an oily compound (neobee® m-5, regu-mate® vehicle). hormone assay plasma altrenogest concentrations were determined by ultra-performance liquid chromatography coupled to a triple quadruple mass spectrometer (uplc-ms). the uplc-ms system consisted of an agilent 1290 system coupled with an agilent 6460 triple quad mass spectrometer (agilent technologies, santa clara, ca) equipped with an electrospray ionization (esi) interface. nitrogen gas was used as the drying (10 l/min at 350°c), nebulizing (45 psi) and collision gas. the mass spectrometer was operated in the positive mode and mass transitions were monitored using multiple reaction monitoring (mrm). the standard curve, ranging from 3.9 to 1000 ng/ml, was generated by fortifying mare plasma with known amounts of altrenogest reference standard and trenbolone internal standard (sigma-aldrich, st. louis, mo) that were prepared in 50:50 (v/v) acetonitrile/distillated water and acetonitrile respectively. standard curves were accepted if coefficient of determination (r2) was at least 0.999 and predicted concentrations were within ± 20% of actual concentrations.22 the uplc-ms assay was validated for mare plasma, with the following criteria: linear correlation coefficient (r2) for altrenogest was 0.999; limit of detection was 3.91 ng/ml; lower limit of quantification was 3.91 ng/ml; and upper limit of quantification was 1000 ng/ml. statistical analyses analysis of variance was conducted to compare the plasma altrenogest concentrations. a repeated measures model was used and means compared with protected pairwise comparisons. results altrenogest was absorbed after all 3 routes of administration. mean plasma altrenogest concentrations at 0, 30, and 60 minutes over the 5-day treatment period are shown (figure 1). over the entire treatment period, plasma altrenogest concentrations, were similar in rg and vg mares but lower (p < 0.05) that in og mares (figure 2). clinical theriogenology • volume 11 number 1 • march 201961 control mares in og mares, mean maximal plasma altrenogest concentration was 117.32 ng/ml (range, 31.0 366.5). overall, 35 doses of altrenogest were administered to og mares; on 12 occasions, plasma concentrations peaked at 30 minutes after administration and on 23 occasions, they peaked at 60 minutes. rectal administration mares for rg mares, mean maximal plasma concentration was 35 ng/ml (range, 5.8 100.5). during the treatment period, 25 doses of altrenogest were administered to og mares. following the administration, on 11 occasions plasma concentrations peaked at 30 minutes and on 14 occasions peaked at 60 minutes. vaginal administration mares for vg mares, mean maximal plasma concentration was 18.0 ng/ml (range, 5.4 61.3). all mares always had maximum concentrations at 60 minutes after administration. a recurring challenge in 3 vg mares was moderate difficulty to guide the 15 cm pipette through the vestibule-vaginal sphincter. consequently, drug was deposited within the vestibule to mimic actions of non-veterinary personnel. subsequently, altrenogest was expelled from the vulva shortly thereafter, a problem made worse by clitoral eversion that occurred soon after vaginal drug administration. thus, in these 3 mares, vestibular deposition and clitoral eversion resulted in decreased contact time and drug loss. adverse effects each administration was accompanied by assessment for adverse effects, including monitoring for increased respiration rate, unease, vaginal and/or rectal discomfort, and vulvar/anal redness and discharge were monitored for. no adverse effects were noted following administration of altrenogest by any route. transrectal ultrasonography all mares were cyclic during the 5-day treatment period. of the vaginally treated mares, 4 of the 5 mares were in diestrus and 1 was in estrus. of the rectally treated mares, 2 were in estrus and 3 in diestrus. of the orally treated mares, 2 were in diestrus, 4 were in estrus, and 1 ovulated between days 1 and 3 of the study. discussion oral absorption of altrenogest was variable among mares. mean maximal plasma altrenogest concentration (117.3 ng/ml), after oral administration, was higher than reported (35 and 16 ng/ml)12,13 despite a lower dose (0.044 versus 0.088 mg/kg13). the range for peak concentrations in og mares, 30.2 366.5 ng/ml, was also higher than reported (23 75 ng/ml).12 all peak plasma sample concentrations in og mares were at or above maximal plasma concentrations reported.12 no drug loss via oral expulsion was noted in any og mares. perhaps preventing access to feed and water for 60 minutes after treatment increased concentrations. altrenogest was absorbed via the rectal mucosa into systemic circulation. plasma altrenogest concentrations were higher in the rg versus the vg, but both were less than those in the og. there was individual mare variation (5.8 100.51 ng/ml) in peak plasma altrenogest concentrations following rectal administration. mean maximum plasma concentrations in rectally administered mares was 35 ng/ml, similar to a reported for geldings,12 but much higher than reported for mares (2.54 ng/ml).13 altrenogest was absorbed via the vaginal mucosa into systemic circulation. however, systemic plasma concentrations of vaginally administered altrenogest were vastly lower when compared to orally administered mares, due to suboptimal drug placement, rapid loss by expulsion, and perhaps inadequate absorption. mean maximum plasma altrenogest concentrations after vaginal administration was 18 ng/ml (range, 5.4 61.3), lower than maximum concentrations (23 -75 ng/ml) reported following oral administration.1 clinical theriogenology • volume 11 number 1 • march 2019 62 rectal and vaginal administrations resulted in detectable systemic concentrations of plasma altrenogest; however, concentrations were not as high as oral administration of altrenogest. incomplete and variable absorption of altrenogest may have been due to a number of factors, including drug placement, external elimination, drug dosing, absorption barriers, and inadequate time for absorption. the location of drug placement can affect drug absorption. administration of altrenogest with a 15 cm pipette allowed for the entire dose to be deposited in the caudal rectum, allowing absorption to occur via the pudendal vein into the caudal vena cava, thus bypassing the liver and first-pass metabolism. defecation is expected to reduce drug absorption; an obvious drawback of rectal administration of drugs in a horse is inability to delay defecation. despite this, rectal administration of the drug without manure evacuation was performed to simulate an owner administering the drug at home, as we would not recommend owners doing transrectal palpation, due to risk of injury to the horse and owner. three of the 5 mares defecated within 30 minutes after drug administration on day 2 of the study which likely resulted in a reduction of plasma altrenogest concentrations for that day, since only one of those 3 mares achieved a concentration above the previously reported 23 75 ng/ml concentration after oral administration.12 the vestibule of the mare is ~10 cm in length, whereas the vagina is ~8 cm in length. using a 15 cm pipette, altrenogest was deposited within the posterior vagina. vaginal administration via pipette was more difficult in some mares than rectal administration and should not be recommended for owners to attempt. the presence of a tight vestibule-vaginal sphincter in some mares made it more difficult to reach the vagina; altrenogest was subsequently deposited in the vestibule and was expelled from the vulva via clitoral eversion. whereas drug loss appeared to occur in 3 of the 5 mares that were visually recorded to lose the drug via the vulva, the remaining 2 mares still failed to achieve statistically significant altrenogest plasma concentrations when compared to orally dosed mares. the established oral dose of altrenogest is 0.044 mg/kg,23 but minimum target plasma concentrations of altrenogest are not determined for suppression of estrus behavior or maintenance of pregnancy. a dose of 0.022 mg/kg, was adequate for maintaining pregnancy, at least up until day 40 of gestation.3 even though we demonstrated altrenogest was absorbed in both alternative routes, it is not known whether concentrations of altrenogest absorbed transrectally or transvaginally are adequate for the desired clinical effects. absorption barriers are present in both the rectum and the vagina. despite the presence of feces, plasma concentrations in the rg group were higher than reported wherein a higher dose of altrenogest (0.088 mg/kg) was used.13 absorption may have also been impacted by the endocrine status in the vg mares, since thickness of vaginal mucus or vaginal epithelium (fluctuating by the stage of the estrous cycle) could be a barrier to absorption. the vagina is lined with stratified squamous epithelium, with its degree of secretory activity and thickness fluctuating with endocrine status; progesterone and estrogen can influence the structure and function of vaginal epithelium, since receptors for these hormones are present in the vagina.24 in the estrogen-dominated phase, vaginal cornification ensues and results in an increased number of existing cell layers; vaginal mucus is more voluminous but less viscous.25 during diestrus, mucus viscosity increases, however, volume decreases.25 among the 5 mares treated vaginally, 4 were in diestrus and 1 was in estrus, making it less likely that decreased absorption was due to a thickened vaginal epithelium. interestingly, it was difficult to pass the pipette in the mare in estrus and drug loss was noticed after clitoral eversion, but perhaps this was simply due to individual anatomical variation. conclusions in mares, rectal or vaginal administration of altrenogest are not recommended as alternate methods to oral administration, as plasma altrenogest concentrations 60 minutes after administration were significantly lower compared to mares that had oral administration. further studies are needed to determine if plasma altrenogest concentrations achieved after rectal or vaginal administration suppress estrus behavior or maintain pregnancy. clinical theriogenology • volume 11 number 1 • march 201963 conflict of interest the authors have no conflicts of interest. source of funding oklahoma state university and merck animal health. references 1. hinrichs k, sertich pl, kenney rm: use of altrenogest to prepare ovariectomized mares as embryo transfer recipients. theriogenology 1986;26:455-460. 2. canisso if, beltaire ka, bedford-guaus sj: premature luteal regression in a pregnant mare and subsequent pregnancy maintenance with the use of oral altrenogest. equine vet j 2013;45:97-100. 3. mckinnon ao, squires el, carnevale em, et al: ovariectomized steroid-treated mares as embryo transfer recipients and as a model to study the role of progestins in pregnancy maintenance. theriogenology 1988;29:1055-1063. 4. squires el, stevens wb, mcglothlin de, et al: effect of an oral progestin on the estrous cycle and fertility of mares. j anim sci 1979;49:729-735. 5. wiepz gj, squires el, chapman pl: effects of norgestomet, altrenogest, and/or estradiol on follicular and hormonal characteristics of late transitional mares. theriogenology 1988;30:181-193. 6. hodgson d, howe s, jeffcott l, et al: effect of prolonged use of altrenogest on behaviour in mares. vet j 2005;169:321-325. 7. squires el, heesemann cp, webel sk, et al: relationship of altrenogest to ovarian activity, hormone concentrations and fertility of mares. j anim sci 1983;56:901-910. 8. squires el: use of progestins in open and pregnant mares. anim reprod sci 1993;33:183-193. 9. bailey cs, macpherson ml, pozor ma, et al: treatment efficacy of trimethoprim sulfamethoxazole, pentoxifylline and altrenogest in experimentally induced equine placentitis. theriogenology 2010;74:402-412. 10. shideler rk, voss jl, aufderheide wm, et al: the effect of altrenogest, an oral progestin, on hematologic and biochemical parameters in mares. vet hum toxicol 1983;25:250-252. 11. webel sk, squires el: control of the oestrous cycle in mares with altrenogest. j reprod fertil suppl. 1982;32:193198. 12. machnik m, hegger i, kietzmann m, et al: pharmacokinetics of altrenogest in horses. j vet pharmacol ther 2007;30:86-90. 13. ellis ke, council-trouch rm, von dollen k, et al: pharmacokinetics of intra-rectal altrenogest in horses. j equine vet science 2019;72:41-46. 14. steinman a, gips m, lavy e, et al: pharmacokinetics of metronidazole in horses after intravenous, rectal and oral administration. j vet pharmacol ther 2000;23:353-357. 15. broome ta, brown mp, gronwall rr, et al: pharmacokinetics and plasma concentrations of acetylsalicylic acid after intravenous, rectal, and intragastric administration to horses. can j vet res 2003;67:297-302. 16. papich mg, alcorn j: absorption of diazepam after its rectal administration in dogs. am j vet res 1995;56:16291636. 17. peters rk, schubert t, clemmons r, et al: levetiracetam rectal administration in healthy dogs. j vet intern med 2014;28:504-509. 18. hershko ka, samara n, weintraub a, et al: intramuscular versus vaginal progesterone administration in medicated frozen embryo transfer cycles: a randomized clinical trial assessing sub-endometrial contractions. gynecol obstet invest 2018;83:40-44. 19. malbrue ra, stout rw, pinto cr: pharmacokinetics of oral micronized progesterone and intravaginal progesterone administration in the bitch. clinical theriogenology 2017;9:415. 20. coste mp, catalan c, eyraud jl, et al: cervical ripening after previous cesarean section with dinoprostone vaginal insert. gynecol obstet fertil senol 2017;45:77-82. 21. khan ru, el-refaey h, sharma s, et al: oral, rectal, and vaginal pharmacokinetics of misoprostol. obstet gynecol 2004;103:866-70. 22. lampinen-salomonsson m, beckman e, bondesson u, et al: detection of altrenogest and its metabolites in post administration horse urine using liquid chromatography tandem mass spectrometry increased sensitivity by chemical derivatisation of the glucuronic acid conjugate. j chromatogr b analyt technol biomed life sci 2006;833:245-256. 23. webel sk: estrus control in horses with a progestin. j anim sci 1975;41:385. 24. re g, badino p, novelli a, et al: distribution of cytosolic estrogen and progesterone receptors in the genital tract of the mare. res vet sci 1995;59:214-218. 25. ginther oj: reproductive biology of the mare: basic and applied aspects 2nd edition. cross plains: equiservices;1992. p. 209-215. clinical theriogenology • volume 11 number 1 • march 2019 64 figure 1. mean plasma altrenogest concentrations (over the 5-day treatment period) at 0, 30, and 60 minutes after administration (oral, rectal or vaginal). figure 2. mean plasma altrenogest concentrations, reported by group, for the entire treatment period. a,bmeans without a common superscript differed (p < 0.0001). 1 65 106 0 32 35 0 11 18 0 20 40 60 80 100 120 0 30 60 m ea n pl as m a al tr en og es t (n g/ m l) time (minutes) oral rectal vaginal 91.2a 33.0b 14.4b 0 10 20 30 40 50 60 70 80 90 100 oral rectal vaginal m ea n pl as m a al tr en og es t (n g/ m l) group clinical theriogenology • volume 11 number 1 • march 201965 clinical theriogenology • volume 11 number 1 • march 2019 66 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2019 sedation in combination with local anesthesia was as effective as general anesthesia for canine castrations sedation in combination with local anesthesia was as effective as general anesthesia for canine castrations erika silva cuzmar,a john schumacher,b thomas passler,b william r. ravisc ainstituto de ciencias clinicas veterinarias, facultad de ciencias clinicas veterinarias, universidad austral de chile, valdivia, chile bdepartment of clinical sciences, college of veterinary medicine, auburn university, al cdepartment of drug discovery and development, auburn university, al abstract the object of the study was to compare evidence of pain in dogs castrated while anesthetized with that of dogs castrated while sedated and after desensitizing the testes and site of incision with local anesthetic solution. all dogs were sedated with xylazine (1 mg/kg, intramuscularly). for the local anesthesia treatment, dogs were given 2% lidocaine subcutaneously in the pre-scrotal area and intratesticularly; total doses ranged from 4.2 to 14.9 mg/kg (10.7 mg/kg ± 2.4 mg/kg, mean ± sd). for the general anesthesia treatment, once dogs were sedated, they were given propofol (4.4 mg/kg, intravenously) as a bolus, with an additional bolus of propofol (2.2 mg/kg, intravenously) given if signs of insufficient anesthetic depth were evident during surgery. the interval from sedation to incision and overall duration of castration surgery were similar for both treatments. movement scores during surgery did not differ significantly between treatments. respiratory rates were not significantly different between treatments or over time. however, in both treatments, there was a significant reduction in heart rate during surgery as compared to the preoperative heart rate. based on objective assessments, measured indicators of pain were not different for canine castrations performed under local versus general anesthesia. keywords: dog, castration, sedation, lidocaine, xylazine, propofol introduction male dogs are routinely castrated for population control and to decrease their impulse to roam.1 in many developing countries, free-roaming dogs transmit zoonotic diseases, most importantly rabies. furthermore, roaming dogs are likely to suffer from malnutrition, starvation, fight wounds, abuse, and sexually transmitted diseases.1 access to low-cost neuter programs substantially promotes spay/neuter procedures.2 dogs are typically castrated while under general anesthesia. however, if dogs could be castrated using only sedation and local anesthesia, there is potential for lower anesthetic risk and expense, which could increase the number of dogs castrated. the object of the study was to compare evidence of pain in dogs castrated while anesthetized with that of dogs castrated while sedated and after desensitizing the testes and site of incision with local anesthetic solution. materials and methods dogs owners of 50 various breed male dogs presented for castration signed a consent form allowing participation of their dogs in the study, which was approved by the universidad austral de chile bioethics committee for the use of animals in biomedical research (# 231-2015). dogs were allocated to the following two treatments as follows: after assigning the first dog presented to be castrated under the influence of sedation and local anesthesia (la), the next dog presented was assigned to the alternate treatment to undergo castration under general anesthesia (ga). physical examinations were performed before sedation. dogs were excluded from the study if the heart rate (hr), rectal temperature, or respiratory rate (rr) were outside normal reference ranges, or if a clinical theriogenology • volume 11 number 1 • march 201943 testicular abnormality, e.g. cryptorchidism, was detected. dogs were fasted overnight but allowed access to water until they were sedated with xylazine hcl (xilacina 2% centrovet, santiago, chile; 1.0 mg/kg im). once recumbent, dogs were placed on a surgical table in lateral recumbency. dogs in each treatment were prepared for surgery while sedated and laterally recumbent with the upper pelvic limb flexed and elevated. following surgical preparation, either propofol (ga) or lidocaine (la) were administered. movement scores, heart and respiratory rates were determined before sedation, 3 min after sedation and immediately prior to the skin incision. data collected immediately before the skin incision were used as baseline. heart and respiratory rates were determined by counting heartbeats for 15 seconds with a stethoscope and counting chest movements for 15 seconds, respectively. for dogs in the la treatment, 2% lidocaine hcl (lidocalm® 2%, drag pharma, santiago, chile) was administered by the first author into each testis, until a slight swelling of the testis was detected, and subcutaneously, on the midline, cranial to the scrotum. testicular injections were made by inserting a 21gauge, 16-mm needle on the median raphe and then directing the needle, from its subcutaneous location into the parenchyma of each testis. the volume of lidocaine administered subcutaneously cranial to the scrotum depended on the estimated length of the incision necessary to remove the testes and was generally equal to the dose injected into each testis. for dogs in the la treatment, propofol was to be administered if pain during castration became evident as indicated by vocalization, a head raise, an attempt to stand or purposeful movement. the interval from administration of the sedative to incision, duration of surgery from incision to placement of the last skin suture, and interval from completion of surgery to standing recovery were recorded. the interval from intratesticular injection of lidocaine to the beginning of surgery was retrospectively estimated. after preparing the dog for surgery, anesthesia for dogs in the ga treatment was induced with a bolus of propofol (propofol® 10 mg/ml, b. braun melsungen ag, germany; 4.4 mg/kg iv administered via a hypodermic needle in the cephalic vein). these dogs were not intubated nor given supplemental oxygen. another iv bolus of propofol (2.2 mg/kg) was given if the dog had purposeful movement during surgery (i.e., grades 3 or 4; see grading system below). castrations were performed with the dog in lateral recumbency and its upper pelvic limb elevated in a flexed position. surgery was performed by the first author using a previously described open technique for castration of cats where the ductus deferens is separated from the spermatic cord and tied with the cord vasculature to form a knot.3 parameters assessed during surgery dogs in the ga treatment were maintained at a surgical plane of anesthesia, as indicated by ventromedial eye position, relaxed jaw tone, slow or sluggish palpebral reflex, and lack of movement in response to the surgical incision. heart and respiratory rates and movement during the surgery period (surg) were determined for dogs in both treatments at 3, 5, 10, and 15 minutes after the skin was incised. movement of dogs during the procedure was subjectively graded on a scale of 0-4 where: 0 = no movement; 1 = slight, non-purposeful movement that did not interfere with surgical manipulations; 2 = moderate, non-purposeful movement that interfered slightly with surgery; 3 = purposeful movement that interfered substantially with surgery; and 4 = purposeful movement that made continuation of surgery impossible. no focused attempt was made to associate movement with a surgical maneuver such as skin incision, traction on the testis, or placement of suture. a veterinary anesthesiologist, not blinded to the procedures being performed, determined heart and respiratory rates and graded the movement of dogs during all 48 surgeries. signs that were considered to be indicative of pain included avoidance movement, vocalization, head raise, and attempts to stand. the last four dogs in the la treatment were subjected to a toe pinch before the skin was incised and immediately after the last skin suture was placed, to detect a purposeful response to this noxious stimulus. if the dogs became aroused by the toe pinch, they were allowed to return to a state of sedation before proceeding with the castration. all dogs were given a single dose of ketoprofen (1 mg/kg im) and procaine penicillin g (20,000 iu/kg im) at completion of surgery. after placement of the last suture, dogs were placed on a pad on the surgery room floor in lateral recumbency and interval to standing was recorded. dogs were undisturbed during recovery. sutures were removed after 10 to 14 days, either by the owner or hospital personnel. clinical theriogenology • volume 11 number 1 • march 2019 44 owners were contacted by telephone 4 or 5 days after the surgical procedure to determine if there were post-operative complications and the owner’s evaluation of post-operative pain or abnormal behavior. data analyses statistical analyses were performed using commercial software (jmp® 11.0.1, sas institute inc. cary, nc, usa). data were assessed for normality by visual inspection of frequency distributions and by the shapiro-wilk test. descriptive statistics, including mean, median, standard deviation, minimum, and maximum, were calculated. age, hr, rr, and movement were not normally distributed and were transformed logarithmically prior to statistical analysis. statistical tests were performed to test the null hypothesis that means of each outcome variable were not different between la and ga treatments. outcome variables collected once (age, bodyweight, duration of surgery, time from sedation to incision, and time to stand) were compared using a student’s t-test. for outcome variables assessed at sequential time points during surgery (movement, hr, and rr), average rates determined during baseline assessment and following the skin incision were estimated from the area under the curve for percentage change in the rate from the baseline rate versus time, with average rate calculated as the area under the curve, divided by the duration of observation. movement scores, rr differences, and hr differences were compared between treatments and over time using a mixed model repeated measures analysis, as implemented in the full-factorial design (mixed model) add-on of jmp. for all analyses, p < 0.05 was considered significant. graphs were generated in jmp graph builder, using untransformed data. results two dogs presented for castration were cryptorchid and were excluded from the study; thus, 48 dogs completed the study (24 per group). there was no difference between treatments for dog age (p = 0.77), but dogs in the la treatment were heavier (p = 0.023; table 1). doses of lidocaine administered to dogs in the la treatment ranged from 4.2 to 14.9 mg/kg (mean ± sd of 10.7 mg/kg ± 2.4 mg/kg), with 17 of 24 dogs given > 10 mg/kg. muscle tremors, an early sign of lidocaine intoxication,4 were not observed. one dog had a vocal reaction to intratesticular injection of lidocaine that was interpreted as an expression of pain. two dogs in ga treatment developed purposeful movement during surgery and required additional administration of propofol (2.2 mg/kg, iv), each at 6 minutes after incising the skin. surgical times for both treatments are summarized in table 1. the interval from intratesticular injection of lidocaine to the beginning of surgery was estimated to be ~ 2 minutes. however, the dog that reacted vocally to intratesticular administration of lidocaine was allowed ~5 minutes to return to a state of sedation before beginning surgery. following surgery, dogs in treatment ga took longer to stand (p = 0.017). average percentage changes in hr from baseline values did not differ between treatments (ptreatment = 0.289; figure 1). over time, there were reductions in hr in both treatments, ga (pperiod = 0.008) and la (pperiod = 0.007), at 3, 5, and 15 min (surg) compared to baseline. change in pre rr did not differ between treatments (ptreatment = 0.147; figure 2). however, during surg, percentage decreases in rr from baseline were greater in la versus ga (ptreatment = 0.005), with average percentage changes of -48.9 ± 42.3 versus -13.7 ± 39.7%. the rr were similar (pperiod= 0.977) for pre and surg periods for ga dogs, but for la dogs, decreases in rr were greater (pperiod = 0.008) during surg as compared to pre. movement occurring between assessment times was never greater than that recorded at predetermined time points. movement scores (figure 3) decreased over time in both treatments during surg (ptime < 0.001), with no difference between treatments (ptreatment = 0.496). after an initial decrease (ptreatment = 0.018) in movement for the ga treatment at 3 minutes, movement scores were not different between treatments. average movement during surg did not differ between treatments (ptreatment = 0.202) nine dogs in the la treatment and 16 dogs in the ga treatment did not move during castration. it was noteworthy that movement of dogs in the la treatment did not interfere with surgery and subjectively appeared to be random rather than associated with pain perception. two dogs receiving the lowest doses of lidocaine (4.2 and 5 mg/kg) had movement scores of 1 and 3 at three minutes, but had no movement at clinical theriogenology • volume 11 number 1 • march 201945 five, 10 or 15 minutes. four dogs in la treatment that were subjected to a toe pinch before incising the skin and immediately after placing the last suture responded to this test with signs of pain [i.e., limb withdrawal, purposeful movement, head raise, and standing (one dog)] despite showing no signs of avoidance movement or head raise during orchiectomy. owners were contacted by telephone at 4 or 5 days after their dog’s castration. none of the owners reported signs of post-operative pain or any complications, other than slight swelling and redness of the pre-scrotal incision site. discussion based on objective assessments, indicators of pain did not differ between treatments. furthermore, there were no significant differences in movement, or heart or respiratory rates. although measures of pain were assessed at pre-determined time points rather than specific surgical manipulations that would likely elicit pain, the surgeon and anesthesiologist recorded obvious signs of apparent pain such as vocalization or avoidance movement (such as in one dog during injection). however, no additional signs that might be interpreted as pain were noted. therefore, we attributed any movement of dogs in the la treatment during surgery to insufficient sedation and reaction to environmental stimuli (e.g. noise and touch) rather than pain. castration of standing, sedated stallions is commonly performed without behavioral evidence of pain, after infiltrating the testes or spermatic cord and scrotum with local anesthetic.5,6 almost all testicular surgical procedures performed on men, sedated or unsedated, can be performed using local anesthesia.7-11 in one report, 360 men underwent painless inguinal, scrotal, or testicular surgery (which was an orchiectomy in 40 cases) as unsedated outpatients after the spermatic cord of the testis being operated on and the surgical site were injected with lidocaine.9 in a study that evaluated the analgesic effect of intratesticular and incision line infiltration of local anesthetic (0.5% ropivacaine), 5 of 11 dogs sedated with medetomidine were castrated without movement, whereas two dogs required further sedation to prevent movement during castration.12 intraoperative data collected from one dog in that study was excluded from evaluation due to repeated movement in the absence of a surgical stimulus, despite repeated administration of medetomidine. because of movement, four dogs in that study were given general anesthesia to complete the castration; however, the authors did not offer an opinion as to whether movement appeared to be in response to pain or possibly other environmental stimuli. based on that study, we inferred that sedated dogs can be castrated after anesthesia of the testes and site of incision, as long as restraint is adequate. a limitation of this study was failure to monitor blood pressure. obvious effects of noxious stimuli are tachycardia and hypertension.13 because nociceptive impulses entering the central nervous system induce a sympathetic response affecting hr and blood pressure, these cardiovascular variables are often used in studies involving various species as objective data to indicate nociception.13-15 hemodynamic responses to nociception commonly occur even when anesthetic concentrations are sufficient to prevent motor and ventilatory responses.13 a comparison of blood pressures between the two treatments may have provided evidence of the anti-nociceptive effects of intratesticular lidocaine. physiological reflexes that control these autonomic responses to noxious stimuli are modulated in the cardio-respiratory centers of the medulla oblongata and do not necessarily reach cognitive centers of the brain; therefore, changes in cardiovascular variables are not necessarily evidence of pain perception.15 however, while tachycardia and hypertension in response to surgical manipulation may not necessarily indicate nociception in dogs under general anesthesia, documentation of suppression of hypertension in dogs undergoing castration with intratesticular lidocaine would have provided additional evidence that these dogs did not perceive pain. in a study that evaluated indicators of nociception in stallions undergoing castration, mean arterial blood pressure was a more sensitive indicator of nociception than was hr.14 it was speculated that genital surgery may increase vagal tone, resulting in intra-operative bradycardia. similar findings were reported in dogs undergoing ovariohysterectomy.16 in that study, there was no change in heart rate during the ovariohysterectomy, but blood pressure increased during removal of ovaries, probably concurrent with maximum noxious stimulation. clinical theriogenology • volume 11 number 1 • march 2019 46 we chose to inject the testes rather than the spermatic cord, because the former is simpler. in a study using radiolabeled lidocaine to determine distribution of intratesticularly administered lidocaine in stallions undergoing castration, lidocaine was quickly transported proximally and distributed diffusely throughout the spermatic cord, even though distribution of radiolabeled lidocaine within the testicular parenchyma was poor.17 in men undergoing testicular biopsy, 0.5 to 1 ml of 1% lidocaine injected into a testis adjacent to the tunica albuginea allowed painless biopsy immediately (within 15 seconds) after injection.18 therefore, there appears to be rapid onset of anesthesia of the testis and spermatic cord after intratesticular injection of lidocaine. the interval from intratesticular injection of lidocaine to castration in our study was very short, consistent with rapid desensitization of testis and spermatic cord. we observed no obvious pain response to surgical manipulations from any dog in the la treatment, even though the onset of surgery, for most dogs, was estimated to be ~2 minutes after lidocaine was administered intratesticularly. although 17 of the dogs in the la group received more than the recommended maximum therapeutic dose of lidocaine (10 mg/kg),4 no signs of toxicity, such as seizure, muscle tremor, bradycardia, nausea, and vomiting.4,19 were observed in any la dog. signs of lidocaine toxicity may not have been observed because of a masking effect of xylazine or because much of the lidocaine was removed along with testes and a portion of the spermatic cord, thus decreasing the amount of lidocaine reaching the general circulation. serum lidocaine concentration in dogs peaks ~30 minutes after subcutaneous or intramuscular injection;19 however, in our study, testes were removed much sooner. a much smaller dose of lidocaine (2 mg/kg) was given intratesticularly in a study examining analgesic effects of lidocaine administered into the testes of dogs castrated while anesthetized with an inhalant anesthetic agent (isoflurane).20 this dose of lidocaine significantly decreased the hemodynamic effects of castration compared to a saline control indicating that this dose provided substantial analgesia during castration. that two of the dogs receiving the lowest doses of lidocaine had no movement after the 3minute assessment indicates that administration of lidocaine far below the maximum recommend dose may provide sufficient anesthesia for castration of sedated dogs. in the present study, we used lidocaine due to its broad availability and low cost. however, other local anesthetic agents, e.g., mepivacaine, bupivacaine, or ropivacaine, may be similarly effective. the anesthetic action of each of these local anesthetic agents is longer than that of lidocaine, and therefore, the use of any one of these local anesthetic agents might provide longer post-operative pain relief. one dog displayed signs of pain during intratesticular injection of lidocaine; perhaps this was caused by rapid distention of the testis or a burning sensation related to the low ph of lidocaine (~621). adding sodium bicarbonate to a solution of lidocaine (e.g., 1 ml of 8.4% sodium bicarbonate solution per 9 or 10 ml lidocaine) may decrease pain during injection by increasing ph.21 xylazine was chosen as a sedative because one of the aims of this study was to demonstrate an economical method of dog castration, and due to limited availability of other alpha2-adrenoreceptor agonists in some countries. because xylazine is more likely to cause cardiac arrhythmia than medetomidine or dexmedetomidine, some anesthesiologists consider the use of xylazine in small animal practice unjustified.22 the hemodynamic effects of xylazine can be reduced by administering it intramuscularly rather than intravenously.23 deaths associated with use of xylazine in dogs occur primarily when xylazine is used in combination with a general anesthetic agent.22 xylazine should be administered with caution or not at all to dogs that are debilitated or that have cardiovascular, respiratory, or renal dysfunction.24 it could be argued that general anesthesia in addition to local anesthesia is a better option for castration of dogs than is sedation plus local anesthesia. while this may be true for many circumstances, this study demonstrated that dogs can be castrated using only sedation and local anesthesia without displaying obvious evidence of pain. this technique may be useful for castration of dogs in underdeveloped regions of the world where economic factors make canine population control difficult. limitations of this study include failure to monitor mean arterial blood pressures during castration, as this would have provided better evidence of pain response than heart or respiratory rates. in addition, we relied on arbitrary, pre-determined time intervals to detect evidence of pain rather than trying to correlate components of the surgery most likely to elicit pain with the criteria we chose to evaluate for clinical theriogenology • volume 11 number 1 • march 201947 evidence of pain. a more accurate determination of the minimal time that must elapse between intratesticular injection of local anesthetic and castration would allow surgeons to accurately determine when the procedure can safely begin. finally, the study was not blinded, which may have biased some results. other than observing a purposeful response of one dog to intratesticular administration of lidocaine, no problems were encountered using sedation and local anesthesia to castrate 24 dogs. we concluded that im xylazine and local anesthesia of the testes and surgical site provided a practical option for castration of dogs. given these findings, this protocol warrants further study for castration of dogs when general anesthesia is inappropriate. conflict of interest none of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper. references 1. jackman j, rowan a: free-roaming dogs in developing countries: the benefits of capture, neuter, and return programs. in: salem dj, rowan an, editors. the state of the animals iv. portland, or: humane society press, book news; 2007. p. 55-78. 2. frank jm, carlisle-frank pl: analysis of programs to reduce overpopulation of companion animals: do adoption and low-cost spay/neuter programs merely cause substitution of sources? ecological economics 2007;62:740-746. doi:10.1016/j.ecolecon.2006.09.011 3. hedlund cs: surgery of the reproductive and genital systems. in: fossum tw, editor. small animal surgery 3rd edition, st. louis: mosby elsevier; 2007. p. 702-774. 4. lemo n, vnuk d, radisic b, et al: determination of the toxic dose of lidocaine in dogs and its corresponding serum concentration. vet rec 2007;160:374-375. 5. joyce j, henrickson da: comparison of intraoperative pain responses following intratesticular or mesorchial injection of lidocaine in standing horses undergoing laparoscopic cryptorchidectomy. j am vet med assoc 2006;229:17791783. 6. schumacher j: testis. in: auer ja, stick ja, editors. equine surgery, 4th edition, st. louis: elsevier sanders; 2012. p. 804-840. 7. ather mh, mushtaq a, sulaiman mn: urological surgical procedures under local anesthesia. in: saadatniaki a, editor, clinical use of local anesthetics. rijeka, croatia: intech; 2012. p. 39-58. doi: 10.5772/31828 8. desmond ad, arnold aj, hastie kj: subcapsular orchidectomy under local anaesthesia. br j urol 1987;61:143-145. 9. magoha gao: local infiltration and spermatic cord block for inguinal, scrotal and testicular surgery. east afr med j 1998;75:575-577. 10. riba lwn: subcapsular castration for carcinoma of prostate. j urol 1942;48:384-387. 11. rud o, peter j, kheyri r, et al: subcapsular orchiectomy in the primary therapy of patients with bone metastasis in advanced prostate cancer: an anachronistic intervention? adv urol 2012. doi:10.1155/2012/190624 12. kushnir y, toledano n, cohen l, et al: intratesticular and incisional line infiltration with ropivacaine for castration in medetomidine-butorphanol-midazolam sedated dogs. vet anaesth analg 2017;44:346-355. 13. ilkiw j: balanced anesthetic techniques in dogs and cats. clin tech small anim pract 1999;14:27-37. 14. haga ha, dolvik ni: electroencephalographic and cardiovascular variables as nociceptive indicators in isofluraneanaesthetized horses. vet anaesth analg 2005;32:128-135. 15. johnson c: research tools for the measurement of pain and nociception. animals; 2016. doi:10.3390/ani6110071 16. höglund ov, lövebrant j, olsson u, et al: blood pressure and heart rate during ovariohysterectomy in pyometra and control dogs: a preliminary investigation. acta vet scand 2016;58:80. doi 10.1186/s13028-016-0263-y 17. haga ha, lykkjen s, revold t, et al: effect of intratesticular injection of lidocaine on cardiovascular responses to castration in isoflurane-anesthetized stallions. am j vet res 2006;67:403-408. 18. kamal k, zini a, jarvi k: testicular block using intra-testicular lidocaine: a new anaesthetic technique for percutaneous testis biopsy. can j urol 2002;9:1568-1570. 19. hall lw, clarke kw, trim cm: general principles of local analgesia. in: hall lw, clarke kw, trim cm, editors. veterinary anaesthesia. 10th edition, london: wb saunders; 2000. p. 225-245. 20. huuskonen v, hughes jm, bañon ee, et al: intratesticular lidocaine reduces the response to surgical castration in dogs. vet anaesth analg 2013;40:74-82.2000. p. 225-245. 21. frank sg, lalonde dh: how acidic is the lidocaine we are injecting, and how much bicarbonate should we add? can j plast surg 2012;20:71-74. 22. flaherty d: alpha2-adrenoceptor agonists in small animal practice 1. why they do what they do. in pract 2013;35: 524-530. 23. klide am, calderwood hw, soma lr: cardiopulmonary effects of xylazine in dogs. am j vet res 1975;36:931-935. clinical theriogenology • volume 11 number 1 • march 2019 48 24. paddleford rr, harvey rc: alpha2 agonists and antagonists. vet clin north am small anim pract 1999;29:737-745. table 1. age, bodyweight, and surgical times of interest are summarized for la (sedation and local anesthesia) and ga (general anesthesia) dogs. ________________________________________________________________________________________________________ treatment mean median standard deviation minimum maximum age (months) la 18.9 28.4 6 108 ga 16.5 31.3 4 120 bodyweight (kg)* la 18.6 8.9 5.5 38 ga 13.3 6.6 4.3 26 lidocaine dose (mg/kg) la 10.7 2.4 4.2 14.9 ga n/a n/a n/a n/a duration of surgery (min) la 11.1 2.5 8.0 16.0 ga 10.0 2.5 6.0 19.0 interval from sedation to incision (min) la 21.6 4.8 14.0 33.0 ga 21.8 5.0 11.0 31.0 interval from placement of last suture to standing (min)* la 14.5 10.6 2.0 42.0 ga 23.2 13.4 3.0 58.0 *difference between treatments (p < 0.05); n/a = not administered. treatment means are displayed for normally distributed data whereas treatment medians are displayed for non-normal data. clinical theriogenology • volume 11 number 1 • march 201949 figure 1. average difference in heart rate of each treatment during surgery compared to the average heart rate measured immediately before the skin incision; la (sedation and local anesthesia) and ga (general anesthesia) dogs. figure 2. average difference in respiratory rate of each treatment during surgery compared to the average respiratory rate measured immediately before the skin incision (first point on graph); la (sedation and local anesthesia) and ga (general anesthesia) dogs. -60 -50 -40 -30 -20 -10 0 0 3 5 10 15 duration of surgery (min) c h an g e in h ea rt r at e (% ) la ga -70 -60 -50 -40 -30 -20 -10 0 10 0 3 5 10 15 duration of surgery (min) c h an g e in r es p ir at io n r at e ( % ) la ga clinical theriogenology • volume 11 number 1 • march 2019 50 figure 3. average movement scores during surgery for two groups of dogs. the first point on the graph indicates movement immediately before the skin incision; la (sedation and local anesthesia) and ga (general anesthesia) dogs. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 3 5 10 15 duration of surgery (min) m o ve m en t s co re la ga clinical theriogenology • volume 11 number 1 • march 201951 clinical theriogenology • volume 11 number 1 • march 2019 52 omniblank: << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2019 thyroid dysfunction and iodine deficiency in a miniature jenny associated with neonatal congenital hypothyroidism dysmaturity syndrome thyroid dysfunction and iodine deficiency in a miniature jenny associated with neonatal congenital hypothyroidism dysmaturity syndrome maria lópez-rodríguez,a mike rhodes,b mariana diel de amorim,c gregory starrak,b claire carda adeptartments of large animal clinical sciences and bsmall animal clinical sciences western college of veterinary medicine, saskatoon, sk bnanton veterinary clinic, nanton, alberta, canada cdepartment of clinical sciences, cornell university, ithaca, ny abstract a 7-year-old miniature donkey jenny with a history of stillborn foals in 2012 and 2013 delivered a miniature jack foal with congenital hypothyroidism dysmaturity syndrome in 2014. thyroid dysfunction in the jenny was suspected, therefore, a thyrotropin releasing hormone stimulation test and mineral panel assessment were performed, which revealed an inadequate tetraiodothyronine response (10 nmol/l; reference range: 19.8 48.3 nmol/l) with low serum iodine (0.009 ppm; reference range: 0.02 0.049 ppm) and hair selenium concentration (0.42 ppm; reference range: 1.0 3.0 ppm). these findings were compatible with primary hypothyroidism due to iodine deficiency. the jenny was grazed on a pasture containing mustard plants (flixweed: descurainia sophia; stinkweed: thlaspi arvense; and wild mustard: brassica keber1), which contain glucosinolates; their major metabolites can interfere with iodine uptake or organification, causing thyroid dysfunction. the jenny’s hypothyroidism and iodine deficiency were identified as the underlying cause of congenital hypothyroidism dysmaturity syndrome in the miniature jack foal. background congenital hypothyroidism dysmaturity syndrome (chds) in foals was first described in western canada in 1980.2 affected foals had multiple musculoskeletal abnormalities, a grossly normal or slightly visibly enlarged thyroid gland and histologic evidence of thyroid hyperplasia, small follicles and minimal colloid.2 the syndrome includes: prolonged gestation, hypothermia, severe musculoskeletal abnormalities (mandibular prognathism, contracted tendons, rupture of the common digital extensor tendons, and carpal and tarsal bone dysgenesis) and functional and histologic thyroid abnormalities (including hyperplasia).3 severe cases of chds are usually euthanized due to an inability to stand at birth, sepsis and developmental orthopedic conditions, e.g. tarsal crush syndrome.4 risk factors for chds in equids include inadequate mineral intake, exposure to nitrates and consumption of wild mustard plants such as flixweed (descurainia sophia), stinkweed (thlaspi arvense), wild mustard (brassica keber) or more commercial types of mustard plants known as brassica juncea (brown and oriental mustard) or sinapis alba (yellow mustard), the most common mustard plants in western canada.1,5,6 however, exact etiopathogenesis of chds remains unknown, hampering management to prevent the condition. thyroid dysfunction was reported in donkeys including a report describing 10 adult donkeys with hypothermia.7 seven of these 10 were euthanized due to poor prognosis; 4 of 5 had histologic thyroid abnormalities similar to thyroid abnormalities of foals with chds.7 however, none of the donkeys underwent thyroid function testing through assessment of triiodothyronine (t3) and tetraiodothyronine (t4) concentrations or thyrotropin releasing hormone (trh) stimulation tests.7 serum thyroid hormone concentrations were higher in donkeys than horses.8,9 case presentation a 7-year-old miniature donkey jenny with a history of stillborn foals in 2012 and 2013 of unknown cause was maintained on a pasture containing mustard plants from spring through fall. she was fed grass hay and oats in winter and had access to blue (cobalt iodized: iodine 100 ppm and cobalt 120 ppm) and brown (trace minerals: zinc 4000 ppm, iron 1600 ppm, manganese 1200 ppm, copper 350 ppm, iodine 100 ppm and cobalt 50 ppm) salt blocks. she was bred on pasture and was considered a few weeks overdue for foaling according to the owner’s observations. she foaled on december 9, 2014 with no complications. the jack foal was examined by a veterinarian shortly after birth and a moderate bilateral 143 clinical theriogenology • volume 11, number 2 • june 2019 carpal contracture diagnosed. the foal was able to stand and nurse within a normal time frame. in addition, due to apparent hypothermia, the foal was placed under heat lamps for a few hours after birth. two weeks after birth, flexural limb deformities of the forelimbs were evaluated and a persistent bilateral contracture and carpal valgus diagnosed (figure 1). both front legs were splinted and it was recommended that carpal and tarsal radiographs be done to evaluate the angular limb deformity. at 1 month of age, the foal was presented for physical examination and carpal and tarsal radiographic evaluation. on presentation, the foal was hypothermic and noted to have moderate bilateral carpal contracture with no rupture of the common digital extensor tendons, bilateral carpal valgus deformity and mild tendon laxity of both hindlimbs (figure 2). a dorso-palmar image of the left carpus and latero-medial image of the left tarsus (figure 3) were obtained. major findings were: moderate carpal valgus deformity of the left carpus with approximately 25° degree of angulation (mild: 5 10°; moderate: 15 25° and severe: >25°) with concurrent grade ii/iv incomplete ossification of the left carpal and distal tarsal bones. there was evidence of compression and/or crushing of carpal and tarsal bones due to inadequate load distribution across tarsal and carpal joints. a complete blood count and chemistry panel were done; the most remarkable findings were: neutrophilia with 10.98 x 109/l (reference values: 2.50 6.90 x 109/l), elevated aspartate aminotransferase (ast) at 866 u/l (reference values: 0 228 u/l) and increased gamma-glutamyl transpeptidase (ggt) at 101 u/l (reference values: 0 71 u/l). six weeks later, the foal developed severe respiratory signs compatible with pneumonia and was euthanized. due to the miniature jenny’s reproductive history of 2 previously stillborn foals and clinical signs of her most recent foal compatible with chds, a series of tests were performed to characterize her thyroid function and to determine if dietary risk factors for chds were present (i.e. low trace mineral status, exposure to nitrates or glucosinolates [gsl]). a trh stimulation test10 was performed on the jenny 1.5 months after foaling; serum t3 and t4 concentrations were determined (siemens, immulite assay, endocrine laboratory, prairie diagnostic services, saskatoon, sk, canada) before and after 0.5 mg trh (sigma chemical corp., st. louis, mo) was given iv. serum samples were submitted for evaluation of vitamin e, trace minerals11 (toxicology laboratory, prairie diagnostic services), and serum iodine (animal health laboratory, guelph, on, canada). a mane hair sample was submitted for selenium concentration (colorado state university veterinary diagnostic laboratory, fort collins, co). serum cobalt (0.928 ppm) and selenium (0.108 ppm) concentrations were within normal limits (table). hair selenium (0.42 ppm) and serum iodine (0.009 ppm) concentrations were deficient. serum t3 concentrations were 1.83 and 4.05 nmol/l before and after trh treatment, respectively. serum t4 concentrations were 10 nmol/l both before and after trh treatment. outcome the miniature jenny was moved to another pasture containing no mustard plants and the owner provided additional mineral supplementation. she was bred in 2015 and foaled a normal foal the following year. discussion in primary hypothyroidism, the thyroid is incapable of responding to stimulation of tsh or tsh with elevated concentrations of tsh. in secondary hypothyroidism, secretion of trh or tsh is inadequate and is characterized by lower tsh concentrations. ingestion of goitrogenic plants and deficiency of iodine or its excess are the main causes of primary hypothyroidism.10,12 although primary hypothyroidism in adult horses is rare, in foals it occurs in a specific geographic region in western canada and recognized as congenital hypothyroidism dysmaturity syndrome (chds).2,3 it is characterized by thyroid hyperplasia and severe musculoskeletal abnormalities, mandibular prognathism, moderate to severe angular and flexural limb deformities and incomplete ossification of carpal/tarsal bones.2,3 normally, tarsal and carpal bones ossify in the last 2 to 3 months of pregnancy and the process of endochondral ossification starts centrally within the tarsal/carpal bone and continues to the periphery.13 144clinical theriogenology • volume 11, number 2 • june 2019 there is a grading system, based on radiographic evidence of ossification: grade i: no evidence of ossification of majority of cuboidal bones of the carpus and tarsus; grade ii: all cuboidal bones have some evidence of ossification with rounded shape, however, the proximal physis of the third metacarpus/metatarsus is open; grade iii: all cuboidal bones are ossified with rounded edges and small in size with increased joint space, but the proximal metacarpal/metatarsal physis is closed and grade iv refers to all cuboidal bones ossified with a cuboidal shape and wider joint spaces.13 in this case, incomplete ossification of the carpal cuboidal bones (grade ii/iv) and rounded edges of the incompletely ossified distal tarsal bones were evident (figure 2). unrestricted exercise with angular limb deformities and/or incomplete cuboidal ossification increased probabilities of compression of tarsal and carpal bones, a possibility in this case. compression and/or crushing of the carpal and tarsal bones in this case were attributed to inadequate distribution of weight across tarsal and carpal joints. increased ast without an elevation in creatinine kinase may suggest hepatocellular leakage, whereas ggt in association with elevated ast may be linked to inflammation or hyperplasia of biliary ducts. perhaps hepatobiliary damage and gsl increased these enzymes, although we do not have clear evidence to support this finding. regardless, prolonged gestation, hypothermia, contracted flexor tendons, angular limb deformity, and carpal and tarsal osseous dysgenesis meet the case definition for chds. a main risk factor for chds is ingestion of endocrine disrupters during pregnancy, e.g. gsl, which are common in the brassicae (mustard) family. their major metabolites, e.g. isothiocyanates, thiocyanates and oxazolidinethione, can interfere with the uptake and or organification of iodine and subsequently interfere with synthesis of thyroid hormones.14 therefore, even if iodine intake is adequate, chronic gsl intake may still produce iodine deficiency or exacerbate an existing iodine deficiency.14 higher intake of gsl cause functional iodine deficiency, increase tsh concentrations and the thyroid gland fails to respond to exogenous tsh or trh.14 donkeys are reported to have higher basal serum concentrations of t3 (equine reference value: 1.13 ± 0.1 nmol/l) and t4 (equine reference value: 36.7 ± 2.8 nmol/l) compared to horses.8 thus, reference ranges for horses should not be directly applied to donkeys. postpartum and lactation status can influence circulating thyroid hormones. higher basal serum t3 and t4 concentrations were reported in lactating mares during the first month postpartum compared to nonlactating mares, suggesting higher metabolic rate due to increased energy consumption.15 in addition, serum and milk tsh concentrations significantly increase after foaling, decrease over time and reach nadir concentrations at ~ 60 days postpartum in mares,16 which coincides with increased thyroid hormone secretion.15 likewise, serum and milk leptin concentrations increased after foaling and declined at 60 days post-partum.15 in rodents, leptin directly stimulated thyroid hormone synthesis and enhance iodine transfer through the milk during early lactation.17 failure of t4 to increase following trh injection was compatible with thyroid dysfunction. lower concentrations of selenium and cobalt suggested inadequate dietary mineral intake. the owner reported the presence of mustard plants in the field where the jenny grazed and that she had been observed consuming them, consistent with lower basal t4. mean serum iodine concentrations were lower in this miniature jenny (0.009 ppm) compared to the reported mean level of 0.214 ppm.9 selenium is also involved in thyroid function, as it is necessary for formation of important selenoproteins such as glutathione peroxidase and tissue deiodinases,18 which regulate conversion of t4 into the more metabolically active thyroid hormone, t3 in tissues unable to capture circulating t3. thus, severe selenium deficiency can decrease t3 availability to peripheral tissues, despite a normal hypothalamicpituitary-thyroid axis.19 normal function of the maternal hypothalamic-pituitary-thyroid axis and adequate concentrations of trace minerals (e.g. iodine and selenium) during pregnancy are essential for normal fetal development, especially growth and maturation of the musculoskeletal system and brain. in humans, effects of severe thyroid hormone deficiency in newborns, secondary to an inadequate supply of iodine during pregnancy, can be irreversible and include: cognitive and motor defects, hearing and speech defects and growth retardation (termed neurological cretinism).20 furthermore, depending on the mammalian species and type of placentation, placental transfer of maternal thyroid hormones has an important role in concentrations of 145 clinical theriogenology • volume 11, number 2 • june 2019 t3 and t4 in fetal circulation in early pregnancy. in the second trimester, development of the fetal thyroid gland is complete, which coincides with functional development of the fetal hypothalamo-pituitarythyroid axis; at this time, the fetus is capable of secreting thyroid hormones into fetal circulation. in addition, placental iodide transfer from maternal to fetal circulation is of great importance for thyroid hormone synthesis; therefore, normal production of thyroid hormones by the fetal thyroid depends on maternal iodine uptake and transport through the placenta.21 hence, iodine deficiency caused by inadequate dietary intake or environmental endocrine disruptors (gsl compounds) affecting the thyroid gland and iodine uptake can lead to maternal thyroid dysfunction affecting fetal growth and maturation. we concluded the underlying cause of the chds in the jenny’s foal was iodine deficiency; consumption of mustard plants likely affected the jenny’s ability to store iodine, synthesize thyroid hormone and supply adequate amounts to her fetus. learning points • primary hypothyroidism is present in some adult donkeys. • iodine deficiency can be present in jennies. • equid dams delivering chds foals should be evaluated to determine if maternal iodine and selenium deficiencies are present. • gsl present in the diet can disrupt thyroid function. • changing mineral supplementation practices and removing or minimizing exposure to endocrine disruptors, e.g. gsl, may improve reproductive performance. references: 1. bubar cj, mccoll sj, hall lm. weeds of the prairies. alberta agriculture, food and rural development. alberta, canada. 2000. p 108, 144, 182. 2. allen al. congenital hypothyroidism in horse: looking back and looking ahead. equine vet ed 2014;26:190-193. 3. allen al, townsend hg, doig ce, et al: a case-control study of the congenital hypothyroidism dysmaturity syndrome of foals. can vet j 1996;37:349-351. 4. fretz pb. congenital hypothyroidism dysmaturity syndrome in western canadian foals: clinical manifestations, management, treatment, prognosis and follow-up. proc ann conv am assoc equine pract 1997,40:63. 5. koikkalainen k, knuuttila a, karikoski n, et al: congenital hypothyroidism and dysmaturity syndrome in foals: first reported cases in europe. equine vet educ 2014;26:181-189. 6. hines mt, gay c, talcott c: congenital hypothyroidism and dysmaturity syndrome in foals: diagnosis and possible risk factors. proceedings of the 15th am coll vet int med 1997:363-364. 7. stephen jo, baptiste k, townsend hg: clinical and pathological findings in donkeys with hypothermia: 10 cases (19881998). j am vet med assoc 2000;216:725-729. 8. mendoza fj, perez-ecija ra, toribio re, et al: thyroid hormone concentrations differ between donkeys and horses. equine vet j 2013;45:214-218. 9. elmansury yha, seri hi, elbashir hm, et al: primary data on serum thyroid hormones (thyroxine t4, tiiodothyronine t3) and thyroid stimulating hormone (tsh) of sudanese donkeys (equus asinus): normal level. assiut vet med j 2007;53:113117. 10. breuhaus b. disorders of the equine thyroid. vet clin north am equine pract 2011; 27:115-128. 11. fantuz f, ferraro s, todini l, et al: essential trace elements in milk and blood serum of lactating donkeys as affected by lactation stage and supplementation with trace minerals. animal 2016;7:1893-1899. 12. reed ms, bayly wm, sellon dl. equine internal medicine. 2nd edition. philadelphia: saunders; 2004, p.1340-1342. 13. levine dg. the normal and abnormal equine neonatal musculoskeletal system. vet clin equine 2015;31:601-613. 14. tripathi mk, mishra as. glucosinolates in animal nutrition: a review. anim feed sci and tech 2007;132:1-27. 15. fazio e, medica p, cravana c, et al: seasonal thyroid and lipid profiles in thoroughbred pregnant and non pregnant mares (equus caballus). theriogenology 2016;85:1582-1589. 16. berg el, mcnamara dl, keisler dh. endocrine profiles of periparturient mares and their foals. j anim sci 2007;85:16601668. 17. arbocco fcv, sasso cv, nasif dl, et al: effect of hypothyoidism on the expression of nuclear receptors and their coregulators in mammary gland during lactation in the rat. molec cell endocrinol 2015;412:26-35. 18. stuss m, michaalska-kasiczak m, sewerynek e: the role of selenium in thyroid gland pathophysiology. endokrinologia polska 2017;68:440-454. 19. zarczynska k, sobiech p, radwinska j, et al: effects on selenium on animal health. j elem 2013:329-340. 20. moog kn, entringer s, heim c, et al: influence of maternal thyroid hormones during gestation on fetal brain development. neuroscience 2017;342:98-100. 21. forhead ja, fowden al. thyroid hormones in fetal growth and prepartum maturation. j endocrinol 2014;221:87-103. 146clinical theriogenology • volume 11, number 2 • june 2019 figure 3. dorsopalmar view of the left carpus (a) and lateral view of the tarsus (b) of miniature jack foal at 1 month of age. note incomplete ossification of the carpal cuboidal bones (grade ii/iv) and rounded edges of the incompletely ossified distal tarsal bone. a b figure 1. 23 day old miniature jack foal with bilateral carpal contracture. figure 2. miniature jack foal at 1 month of age. note bilateral carpal contracture and angular limb deformity in the image on left and bilateral tendon laxity and hyperextension of the hind digits in the image on right. 147 clinical theriogenology • volume 11, number 2 • june 2019 table. concentrations of trace minerals, vitamin e and basal thyroid hormones in serum, and selenium in hair of miniature donkey jenny. trace minerals result units reference range magnesium 22.6 ppm 18 – 35 manganese 0.006 ppm 0.006 – 0.07 iron 1.98 ppm 0.084 – 2.57 cobalt 0.928 ppm 0.90 – 15.0 copper 1.25 ppm 0.5 – 2.0 zinc 0.723 ppm 0.47 – 1.70 selenium 0.236 ppm 0.14 – 0.25 molybdenum 0.010 ppm < 0.1 iodine 0.009 ppm 0.02 – 0.049 selenium (hair) 0.42 ppm 1.0 – 3.0 (> 7.0 toxic) vitamin e 2.90 ppm >2 (>4 high) thyroid hormones triiodothyronine (t3) 1.83 nmol/l 1.13 ± 0. 1 (0.76 – 2.0) tetraiodothyronine (t4) 10 nmol/l 36.7 ± 2.8 (19.8 – 48.3) 148clinical theriogenology • volume 11, number 2 • june 2019 << /ascii85encodepages false /allowpsxobjects false /allowtransparency false /alwaysembed [ true ] /antialiascolorimages false /antialiasgrayimages false /antialiasmonoimages false /autofiltercolorimages true /autofiltergrayimages true /autopositionepsfiles true /autorotatepages /none /binding /left /calcmykprofile (u.s. web coated \050swop\051 v2) /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /checkcompliance [ /none ] /coloracsimagedict << /hsamples [ 1 1 1 1 ] /qfactor 0.15000 /vsamples [ 1 1 1 1 ] >> /colorconversionstrategy /usedeviceindependentcolor /colorimageautofilterstrategy /jpeg /colorimagedepth -1 /colorimagedict << /hsamples [ 1 1 1 1 ] 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/treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] /pdfx1acheck false /pdfx3check false /pdfxbleedboxtotrimboxoffset [ 0 0 0 0 ] /pdfxcompliantpdfonly false /pdfxnotrimboxerror true /pdfxoutputcondition () /pdfxoutputconditionidentifier () /pdfxoutputintentprofile () /pdfxregistryname () /pdfxsetbleedboxtomediabox true /pdfxtrapped /false /pdfxtrimboxtomediaboxoffset [ 0 0 0 0 ] /pagelayout /singlepage /parsedsccomments true /parsedsccommentsfordocinfo true /parseiccprofilesincomments true /passthroughjpegimages true /preservecopypage true /preservedicmykvalues true /preserveepsinfo true /preserveflatness false /preservehalftoneinfo false /preserveopicomments true /preserveoverprintsettings true /startpage 1 /subsetfonts true /transferfunctioninfo /apply /ucrandbginfo /preserve /useprologue false /srgbprofile (srgb iec61966-2.1) >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2010: curriculum integration within the context of veterinary education curriculum integration within the context of veterinary education* john cavalieri school of veterinary and biomedical sciences, james cook university, townsville, australia abstract curriculum integration has been applied across a range of educational levels as a means of attempting to improve learning outcomes, but there is a paucity of information that has defined and explored curriculum integration as it relates to the teaching of veterinary science. a definition of curriculum integration in the teaching of veterinary science is suggested, which places the student as life-long learners at the centre of the educational model. learning activities are designed to help students see connections among disciplines and topics thus reinforcing their relevance and helping them to apply their knowledge to different contexts. curriculum integration at james cook university in the first three years of the program has been promoted through the design of assessment, use of case studies, imaging, palpable anatomy, learning of clinical skills, a research project, animal handling, and a personal professional development program. advantages and disadvantages of curriculum integration are discussed. further work is needed to determine if this mode of curriculum design is able to produce veterinary graduates who are better able to meet the challenges of a changing workplace in the 21st century. key words: curriculum integration, education, interdisciplinary studies, life-long learning. introduction changes within the national and international community are likely to affect the way that veterinary science is practiced in the 21st century.1,2 some of these changes include rapid advances in knowledge and technology,3 globalization and internationalization of the profession, changing social, trade and economic constraints, changes in consumer expectations, and greater availability of technicians to perform routine procedures.4,5 if educational institutions are to avoid any disparity between the knowledge and skills required by veterinarians and what they learn, students will need to be equipped with the knowledge and skills that are relevant to a constantly changing workplace. they will need to be lifelong learners who can adapt to a changing knowledge base and be able to search and construct knowledge from a variety of sources. as practices expand and deliver a greater range of services to the public, students will also need to develop social and communication skills, and work effectively in teams.6,7 these evolving educational needs will present new challenges to the development of curricula for education in veterinary science which may require a different approach to traditional educational practice. the use of curriculum integration in veterinary education represents one approach to delivering a learning experience that aims to mimic more closely the way information and problems are encountered in the workplace, and to deliver an authentic learning experience which attempts to meet the educational needs of veterinarians in the 21st century. there, however, appear to be few reports addressing the topic of holistic curriculum integration within veterinary science education programs although a number of reports detail experiences with elements of integration, for example integration of communication and problem solving skills,8,9 specific disciplines10-12 and technologies.13 the aims of this report are to develop a definition and a holistic model of integration that could apply to veterinary education, as well as to examine some of the advantages and disadvantages of curriculum integration. some of the methods that are being used to promote integration within a veterinary science degree program at james cook university are also outlined. historical development of curriculum integration the concept of curriculum integration is not new. as early as the 1880’s separation of knowledge into discrete subjects was criticized as being unnatural and responsible for causing a fragmentation of knowledge.14 in the early 1900’s a number of various forms of curriculum integration were practiced and by 1930 teaching of school subjects in an integrated manner, based on student’s needs was advocated.14 over the years there has been a fluctuating emphasis on integration * reprinted with permission from the journal of veterinary medical education 2009; 36:388-396. 477 within primary and secondary education. more recently there has been a renewed effort to incorporate elements of integration into all levels of education.15,16 this has been encouraged by an exponential increase in knowledge, the need for individuals to solve complex problems which rely on a broad knowledge base, and pedogical reform linked to greater appreciation of the educational value of a more integrated mode of teaching and learning.15 renewed efforts to incorporate integration within medical curricula are also apparent. for example, the latest recommendations of the general medical council which oversees the standards and assessment of undergraduate medical education in the united kingdom are that, “the clinical and basic sciences should be taught in an integrated way throughout the curriculum”. 7 examples, however, of more holistic attempts to include curriculum integration within veterinary education are lacking. integration in relation to veterinary education “a variety of perspectives on curriculum integration have been suggested. these frequently incorporate concepts of students building connections between different topics and disciplines, and the application of knowledge to real world scenarios and problems.15,18 within the context of veterinary education, instead of veterinary science being divided into a series of subjects and disciplines, with an integrated curriculum, students are immersed in a transdisciplinary learning environment that attempts to reflect the complexities of life.19 ingram20 refers to integration as a way of organizing knowledge that counteracts the tendency for knowledge to become fragmented and irrelevant and which assists students to develop a coherent view of life. in explaining the differences in emphasis placed within an integrated compared with a discipline-based curriculum it has been suggested that the “application of knowledge takes precedence over acquisition and mastery of facts alone, activating a dynamic process of question posing, problem posing and solving, decision making, and higher-order critical thinking”.15 the primary philosophical justification for an integrated curriculum comes from the fact that problems are rarely encountered in professional practice that are exclusively presented and solved within the context of a single discipline. more often problems present in the context of requiring knowledge and skills from a range of disciplines. for example, a case of pyometra in a dog requires knowledge of anatomy, physiology, pathology, imaging, medicine, and surgery to solve, while managing the case requires the application of communication and business skills which are all overseen by a code of professional practice and ethics. an integrated curriculum may help students see connections between disciplines, the relevance of material being studied and may enable them to connect and apply their knowledge to a variety of applications. harden21 outlines different degrees of integration in medical teaching. for example, in courses with the least amount of integration these courses are taught in isolation and instructors are largely unaware of what is being taught in other courses. with increasing integration teachers become more aware of what is being taught in other courses (“awareness”), and communicate with other disciplines to ensure that curriculum objectives are achieved (“harmonization”). common themes may be linked between courses (“nesting”), or topics taught in different subjects are aligned in time with similar subjects taught in other subjects (“temporal integration”). further integration occurs when teaching is jointly planned and delivered by different disciplines (“sharing”). for example, teaching sessions on lameness could involve planned delivery from the disciplines of anatomy, imaging, medicine and surgery. integration sessions may be run to highlight areas which are shared in common among subjects which are studied separately (“correlation”) or greater emphasis may be placed on themes which can be explored concurrently with subject-based teaching (“complimentary program”). alternatively, themes can be developed and explored through the disciplines (“multidisciplinary”), for example the use of a body systems-based approach to teaching. high levels of integration operate when courses are taught in an environment of collective planning and teaching where teachers from a number of disciplines collaborate to plan and deliver courses. individual subject content can be combined into a new course which lacks divisions according to disciplines, and where subject content may be delivered according to appropriate themes (“inter-disciplinary”). further integration can be achieved by immersing students in a learning environment that enables them to construct knowledge as part of a real-world, authentic learning experience (“transdisciplinary integration”). given this graduated approach to curriculum integration, it is apparent that elements of 478 integration are included in many undergraduate veterinary degree programs although high level transdisciplinary integration appears to be less common. in attempting to contextualize a definition for the teaching of veterinary science, the author suggests that integration of a veterinary science curriculum involves:  organizing of student learning in an authentic way that is linked to what veterinarians do.  designing the curriculum to reflect how problems are encountered in real life.  promoting linkages between disciplines and topics allowing students to connect and construct their learning across fields, thereby promoting deeper learning and reinforcing greater relevance.  encouraging the development of skills to assist students to be able to locate, assess and use a variety of sources of information. this may help to equip students to be lifelong learners.  enhancing communication, self-management and interpersonal skills, recognizing their critical importance in professional life and the application of knowledge and skills.  acknowledging and embracing the contribution of academic disciplines but limiting their contribution to what is needed in the application of knowledge and sees their place in the context of a more holistic view of knowledge.  interrelating of different subjects and topics, both horizontally between parallel disciplines such as anatomy, physiology and biochemistry, and vertically between different disciplines traditionally taught in the early or latter years of the curriculum, for example, physiology and medicine. in this model, the development of skills is viewed as a tool to reinforce knowledge learnt across disciplines, and is an important component linking knowledge with application. learning activities are designed to help students see connections among disciplines and topics, thus reinforcing their relevance and helping them to apply their knowledge to different contexts. skills learnt within an integrated context may also help students to build new knowledge when problems are encountered where immediate solutions are not known. advantages and disadvantages of an integrated curriculum a number of advantages and disadvantages of curriculum integration are summarized in table 1. advantages associated with an integrated curriculum a) improvements in learning outcomes. a wide body of knowledge supports the value of curriculum integration for improving learning outcomes for students. 22-25 vars23 in summarizing results from over 100 studies spanning 60 years of research concluded that students participating in integrative programs have performed as well or better on standardized achievement tests compared with students enrolled in the more traditional, subject-based disciplines. evidence also exists that integrated learning improves critical thinking, problem solving, retention of knowledge, and relationships with peers and teachers. it also is suggested to help improve motivation, and fosters more positive attitudes to learning.23,24 a number of reports also suggest that graduates are better prepared for their chosen vocations.9,26-29 it is argued that integrative learning requires higher cognitive function through the application of knowledge from a range of disciplines and problem solving, rather than simply knowledge recall.22 dahle et al.24 concluded that vertical integration between clinical medicine and basic sciences within medicine “motivated the students, and stimulated profound rather than superficial learning, a better understanding of important biological principles and better retention of knowledge.” in contrast to an integrated curriculum, traditional, discipline-based curricula, artificially divide and fragment knowledge, often leaving the students to make the necessary connections on their own.30 separation of areas of knowledge may restrict students’ thinking, and it represents an artifice of life that does not reflect true life experiences. lane9 compared problem based learning with a traditional approach to teaching in veterinary and medical science, and cited evidence to suggest that a 479 traditional approach is more likely to result in superficial rather than deep learning, poor recall of knowledge, and frequent student dissatisfaction with their learning experience. b) use of authentic learning benefits of integration are attributed to presenting information and problems that mimic how they are encountered in the real world, and presenting facts in relevant, meaningful31 and connected ways.32 use of authentic learning, connecting knowledge to application in the world beyond the classroom, can stimulate student’s interests, and promote student participation, engagement and satisfaction.33 it can also be argued that learning of basic facts is far easier when those facts are connected with a clinical case, application, or clinical skill. contextualization of knowledge into clinical presentations and problems with integrated learning may be one way that it assists with knowledge retention compared with the learning of knowledge in isolation. c) learning outcomes are focused on specific graduate attributes and competencies veterinary and medical education accreditation bodies have placed increasing emphasis on competencies and essential skills for veterinary practice.34-35 in order to achieve accreditation veterinary educational schools need to demonstrate that students achieve the core knowledge and skills and attributes that are required to achieve the required standards. this has lead to the development of an outcomes-based model of educational practice in some veterinary and medical schools. with this model of education curriculum development is focused on what students should be able to do at the end of their course.36 curriculum integration should facilitate the achievement of an outcomes-based model of educational development by focusing content on what graduates need and accreditation bodies require. it should also enable greater streamlining of course content to what is most relevant for graduates. irrelevant or redundant material can be removed from the curriculum, making learning outcomes clearer to both teachers and students. this should also enable the flexible development of the curriculum as the needs of graduates change enabling the strategic development of subject content that is better aligned with students’ interests and needs, and helps prevent curriculum crowding or overload as knowledge grows. this dynamic approach to curriculum development with changing graduate needs should be better placed to adapt to changes associated with tracking, limited licensure, and increased emphasis on specialization, compared to a more traditional discipline-based curriculum. ingram20 suggests that there is an innate resistance for curriculum to change while knowledge continues to expand. an integrated approach to curriculum delivery is less constrained by narrowly organized subjects; it is more dynamic and can more rapidly assimilate changes in the workplace and society. with a discipline-based educational model, maintaining too tight a focus on disciplines may lead to a tendency for the self perpetuation of subject disciplines, rather than educating students for the world that they will face. integration should provide a framework to adapt our educational practice to a rapidly changing world and facilitate the implementation of outcome-based education. d) promotion of interpersonal and communication skills an appreciation for the relevance of communication, self management, team skills, and ethical reasoning in professional practice has naturally lead to the inclusion of the teaching of personal and professional development within a number of veterinary schools, including james cook university.8 within an integrated curriculum, opportunities can be provided for illustrating and developing personal and professional skills through the solving of problems, management of various scenarios and collaborative exercises. some authors have suggested that benefits of integrating elements of the curriculum within veterinary science include the development of problem solving skills, communication, and team skills, social competence, and life-long learning skills.9,10 e) improvement in student motivation building connections and establishing relevance may promote greater student interest, motivation, and improve student retention, particularly in the early years of a veterinary science course.24,37 rigorous training in foundational sciences without connection to application may result in a dwindling of initial enthusiasm and motivation early in a course, concerns about relevance, and self doubt as to whether students may want to pursue a career in veterinary science.9 setting knowledge, 480 skills and attitudes within the context of how it is applied as veterinarians may provide a degree of emotional appeal to students and encourage motivation. for example, embryological developments can be linked to functional or surgical problems. aspects of anatomy can be explored through discussions concerning surgical approaches to particular anatomical regions or specific physical conditions, highlighting anatomy during physical examination of live animals and by the use of various imaging modalities. physiological processes can be explored using case studies, patient monitors, clinical pathology and pharmacological alteration of function. an advantage of improving student motivation include improved learning outcomes by enhancing deeper learning.24 venville et al.33 argue that some of the main benefits of integrating curricula in relation to secondary school students included improvement in student motivation and interest. the critical importance of motivating students for successful learning outcomes38,39 would suggest that curriculum integration can play an important educational role for students. disadvantages associated with an integrated curriculum a number of potential disadvantages associated with developing and delivering an integrated curriculum are outlined. many operate at the organizational level and so with appropriate leadership, support and a shared vision for change potential problems could be minimized. some of the disadvantages include: a) challenges for staff a number of organizational, teaching and personal challenges confront institutions and staff that are involved in developing an integrated curriculum. delivering an integrated curriculum requires teachers to collaborate and work closely together to design and deliver course content and assessment tasks. resourcefulness and creativity is required by staff as they strategically select which topics are most relevant and how they can be integrated. delivering of learning sessions and assessing integrated content may also require a number of staff from a variety of disciplines to attend simultaneously, which can make scheduling difficult and place more demands on staff time. a high level of cooperation and time to plan and deliver the integrated content is therefore required. greater time demands associated with delivering integrated learning modules has been previously been identified as a potential disadvantage associated with curriculum integration.40 staffing formulas may also need to be revised which could impact on the cost of veterinary education. if staffing issues are not fully met, it could negatively impact on staff morale and research output. teachers may feel less comfortable to deliver integrated teaching.22 educators planning and delivering an integrated curriculum may need to contribute to areas with which they are relatively unfamiliar and to change from the more familiar discipline-based system.22 they may need to revaluate their discipline areas and remove material that may be redundant or irrelevant within the context of an integrated curriculum. it is also conceptually more difficult to prepare integrated teaching materials and it may require staff to downsize individual discipline content. downsizing of content and shift to a different, more collaborative approach to teaching could create a degree of insecurity and anxiety for staff. this may require additional investment in support via training to more smoothly transition the curricula and pedagogical change. curricula drift towards a more familiar discipline-based program could occur as staff are unable to make the transition to an integrated curriculum.33 b) challenges for students students unfamiliar with an integrated learning program need to adjust to the changes in their learning environment. assessment is designed in an integrated manner and students must be fully prepared when undertaking each assessment task and draw upon their entire knowledge base. reference points for solving problems or answering questions are blurred, not constrained by the boundaries of disciplines as students draw upon information learnt across disciplines to answer questions. this may increase the difficulty of assessment tasks which could result in lower academic scores, higher failure rates, and a perceived reduction in student performance if student scores are compared among institutions. greater emphasis on problem solving and application of knowledge and skills requires a higher level of cognitive function than simply factual recall. the need for appropriate standard setting, evaluation, and revising of learning and assessment tasks will be needed 481 to ensure that the desired learning outcomes are being achieved and that students are not overwhelmed by the greater challenges presented with an integrated model of learning and assessment. c) omission of important topics potential trivialization of some areas of knowledge with a failure to engage key aspects of specific disciplines30 and a deficiency of knowledge of the basic sciences have been criticisms of moves to integrate curricula.9 careful monitoring and mapping of the course content, feedback from graduates and employers should, however, help to ensure that essential knowledge and skills are taught and not overlooked. implementation of curriculum integration within a veterinary teaching program at james cook university, a new bachelor of veterinary science degree program was commenced in 2006. an attempt is being made to model the 5-year curriculum on an integrated learning program. traditional science based courses at the university involve completing four, 3-unit subjects per semester. each subject consists of a maximum of approximately 80 to 100 student contact hours over a 13-week study period. accumulation of set numbers of unit points is used to qualify for the earning of various degrees. in the first year of the veterinary science program, students undertake eight equally-weighted, three-unit subjects: chemistry, cell biology and biochemistry, biological principles for agriculture and veterinary science, plant biology, animal biology, physiology and pharmacology, and veterinary professional life 1 and 2.41 the latter two subjects include an introduction to animal industries, animal behavior, ethics, and animal handling, and it is within these two subjects that students are introduced to elements of integration. the second and third years of the program involve a fully integrated curriculum where students study only one, 12-unit subject in the first and second study periods of each year. each12-unit subject consists of about 320 to 380 student contact hours, taught over a 13 to 14 week study period.41 each 12-unit, integrated subject taught in the second and third year of the program consists of a number of themes that are outlined in table 2. themes within the integrated curriculum represent an interdisciplinary concept that provides an organizational framework for developing learning activities that enhance integration between disciplines.42 use of themes offers the advantage of promoting greater connections between disciplines that might not occur when providing instruction in disciplines separately.42 for example, anatomy and physiology are both taught within the theme entitled, structure and function as both anatomical structure and physiological function are intimately connected. the fourth and fifth years of the program are still under development. in the fourth year, integration is being focused within individual species and it will follow a two-tiered, problem-based approach to veterinary science. problems are first presented within species-contexts in which they are normally encountered, for example, companion animals, intensive livestock industries, small and large ruminants, exotic animals and equine. secondly, within these species contexts, problems will be presented in a way which reflects their usual presentation, for example, problems with respiratory symptoms, problems causing reduced production or sudden death, musculoskeletal problems or problems associated with different body systems. these problem themes may be further divided into subthemes. the emphasis remains, however, on the context in which problems are most commonly encountered. in the fifth and final year of the program, students will be able to apply their knowledge to solving clinical problems and scenarios within the fields of veterinary practice, teaching, and government service provision. in addition, they will continue to develop their technical and interpersonal skills. learning activities designed to promote integration activities that are designed to promote integration within the first three years of the program have included the use of case studies, imaging, palpable anatomy, learning of clinical skills, a research project, animal handling, and a personal professional development program. the curriculum is designed in a way to promote both horizontal and vertical integration between topics and disciplines. design of the curriculum around a single,12-unit subject in each study period reduces boundaries between disciplines, reinforces connections, and supports the multidimensional way that problems are encountered in society. assessment includes a range of on-course and supervised assessment tasks. examinations include questions that relate to the entire subject content and examine knowledge, 482 skills, and the application of knowledge. use of scenarios enables questions relating to a number of disciplines to be asked within the same question. item recognition questions, simulated consultations, multiple station assessment tasks, and objective structured clinical examinations (osces) will feature in various parts of the course and be used as instruments to foster integration by including questions directed towards a number of disciplines. multi-disciplinary case studies are used throughout the course to assist students to see how knowledge, skills, and attitudes that they are developing at each stage of the course can be applied in the real world. for example, first year students working in small groups may examine an outbreak of an exotic disease, plant poisoning, or a terminally ill patient. during such an exercise, students learn research and writing skills, learn how to work in teams, and improve their knowledge of the foundational sciences such as biology and chemistry as they relate to the case. in addition, students can be directed to discuss a range of ethical, financial, and communication issues. in this way, students can begin to see how their current knowledge can be applied, but also the wider context in which knowledge and skills are applied in practice. cases can also be used as framework for introducing new knowledge and skills enabling a degree of scaffolding. clinical skills are introduced early in the course to help students see how knowledge can be applied, to enable skills to be progressively developed throughout the course, and also to improve student motivation. for example, while students in their second year of the course are learning aspects associated with the anatomy and physiology of the nervous system, students participate in a practical class that involves assessment of the function of peripheral nerves of dogs and horses and also modify and assess nerve function by performing an inverted-l nerve block on a cow.37 while gross anatomy, histology, and physiology associated with cardiac muscle are taught, exercises in cardiac auscultation or examination of the clinical pathologic findings associated with cardiac injury can be conducted. imaging and palpable anatomy are used to complement didactic lectures, dissections, and prosections in anatomy, but also to link knowledge to application and also to foster the development of clinical skills associated with imaging and physical examination of animals. competence in handling a range of animal species is an essential skill for veterinary graduates.34,43 animal-handling classes also provide an opportunity for integration. topics that are integrated into the animal-handling course include occupational, health and safety, animal welfare, dosage calculations, meat safety, zoonoses, anthelmintic and antiparasitic treatments, regional anatomy, knowledge of the animal industries, and physical examination. similarly, when clinical skills are taught with animals they also reinforce animal handling skills. students within the third year of their program undertake a research project within teams. members of each team are awarded the same mark for the assessment pieces they complete as a team. integration with biometrics, epidemiology, ethics, and welfare is also an advantage of this project. it also enables them to build upon their team, writing, reviewing, and research skills, but also introduces some new knowledge as they research their topics, consider experimental design and management, and write experimental proposals and applications for ethics approval. personal and professional development personal and professional development (ppd) is a theme that is embedded in all stages of the course. emotional intelligence is viewed as one of the key determinants of longevity and success in the workplace,44 so inclusion of learning activities to promote social skills and emotional intelligence is seen as integral to the objective of producing competent veterinarians. the aim of the ppd program is to foster the development of skills that are needed for managing self, improving performance in teams, enhancing communication skills across a range of areas including written and oral communication, and communication in groups, including to the public, clients, and staff. it also aims to promote skills that will enable students to be life-long learners. this includes skills in analysis, reflection, and researching for information. components of the ppd program are therefore integrated with many of the activities that students undertake: oral and written reports; time, self, and group management; and communication across a range of circumstances. an integrated learning model an integrated learning model is illustrated in figure 1. at the center of the model are lifelong learners that are able to adapt to the needs of an evolving workplace. knowledge, skills, and 483 attitudes transcend the limitations of specific disciplines as students make connections within a transdisciplinary environment. during the course, knowledge, skills, and attitudes are continually developed through learning promoted from the foundational, paraclinical, and clinical sciences. a range of skills are developed including clinical, analytical, and communication skills, information literacy, problem solving, and reflective practice. development of emotional intelligence and social competence as well as an understanding of ethics are viewed as being important for successful functioning within the community,44,45 and are therefore an integral component of the educational model. the learning environment is also influenced by a number of learning communities that operate within the educational institution or workplace as well as on national and international scales. professional associations, conferences, literature, and virtual forums and websites all provide important links to the ongoing educational development of the veterinary learner. this illustration conveys the broad educational environment in which an integrated curriculum aims to embed veterinary students. conclusion curriculum integration in a veterinary context refers to the design of the curriculum in such a way that a) enables students to make connections among topics and disciplines, b) reduces boundaries among disciplines, c) is guided by what veterinarians do and are required to do, and d) reflects how problems are encountered in the real world. advantages and disadvantages of this form of curriculum design are apparent, and further work is needed to determine if an integrated curriculum results in improved workplace performance and longevity. curriculum integration at james cook university has been promoted by directed curricular design, methods of student assessment, a personal professional development program, and use of multi-disciplinary case studies, imaging, palpable anatomy, clinical skills, a research project, and animal handling opportunities. acknowledgements i thank k martinez and l westcott for constructive criticism of the manuscript. references 1. heath t: the more things change the more they should stay the same. j vet med educ 2006;33:149-154. 2. willis ng, monroe fa, potworowski ja, et al: envisioning the future of veterinary medical education: the association of american veterinary medical colleges foresight project, final report. j vet med educ 2007;34:1– 41. 3. bushby pa: tackling the knowledge explosion without overlapping the student. aust vet j 1994;71:372–374. 4. brown c, carbajal i, wagner g: preparing the veterinary profession for corporate and trade issues in the americas: proceedings of a conference on synergism and globalization. j vet med educ 2001;28:56-61. 5. gage ed: the globalization of veterinary medical education. j vet med educ 2002;29:201-204. 6. rubin p, franchi-christopher d: new edition of tomorrow’s doctors. med teach 2002;24:368-369. 7. martin ea: managing client communication for effective practice: what skills should veterinary graduates have acquired for success? j vet med educ 2006;33:45-49. 8. mills jn, irwin p, baguley j,et al: development of veterinary communication skills at murdoch university and in other australian veterinary schools. j vet med educ 2006;33:93-99. 9. lane la: problem-based learning in veterinary education. j vet med educ 2008;35:631-636. 10 eysker m: the utrecht model of teaching veterinary medicine and the role of veterinary parasitology. vet parasitol 2002;108:273-281. 11. krockenberge mb, bosward kl, canfield pj: integrated case-based applied pathology (icap): adiagnostic approach model for the learning and teaching of veterinary pathology. j vet med educ 2007;34:396-408. 12. patterson js, stickle je, thomas js, et al: an integrative and case-based approach to the teaching of general and systemic pathology. j vet med educ 2007;34:409-415. 13. baillie s, mellor dj, brewster sa, et al: integrating a bovine rectal palpation simulator into an undergraduate veterinary curriculum. j vet med educ 2005;32:79-85. 14. mccomas wf, wang ha: blended science: the rewards and challenges of integrating the science disciplines for instruction. sch sci math 1998;98:340-348. 15. thompson klein j: integrative learning and interdisciplinary studies. peer rev 2005;7:8-10. 16. huber mt, hutchings p: integrative learning. mapping the terrain http://www.carnegiefoundation.org/dynamic/publications/elibrary_pdf_636.pdf accessed 09/24/09. association of american colleges and universities and the carnegie foundation for the advancement of teaching, stanford, ca, 2004. 17. general medical council: tommorrow’s doctors. <http://www.gmcuk.org/education/undergraduate/gmc_tomorrows_doctors.pdf> accessed 07/25/09. general medical council, london, 2003. 484 18. killen r: programming and assessment for quality in teaching and learning. southbank (in): thomson social science press; 2005. p. 89-94. 19. bhattacharya m, jorgensen l: integrated approach to learning environment design for secondary science teachers. j interact learn res 2007;18:123-133. 20. ingram jb: curriculum integration and lifelong education. a contribution to the improvement of school curricula. advances in lifelong education. volume 6. oxford (in): pergamon press; 1979. 21. harden rm: the integration ladder: a tool for curriculum planning and evaluation. med educ 2000;34:551-557. 22. harden rm, sowden s, dunn wr: educational strategies in curriculum development: the spices model. med educ 1984;18:284-297. 23. vars gf: effects of integrative curriculum and instruction. in: irvin jl, editor. what current research says to the middle school practitioner. <http://www.eric.ed.gov/ericdocs/data/ericdocs2sql/content_storage_01/0000019b/80/17/5e/eb.pdf.> accessed 07/25/09. national middle school association, columbus ohio. 1997, p178-186. 24. dahle lo, brynhildsen j, behrbohm fallsberg m, et al: pros and cons of vertical integration between clinical medicine and basic science within a problem-based undergraduate medical curriculum: examples and experiences from linköping, sweden. med teach 2002;24:280-285. 25. dowden t: relevant, challenging, integrative and exploratory curriculum design: perspectives from theory and practice for middle level schooling in australia. aust educ res 2007;34:51-72. 26. worley p, silagy c, prideaux d, et al: the parallel rural community curriculum: an integrated clinical curriculum based in rural general practice. med educ 2000;34:558-565. 27. katajavuori n, lindblom-ylänne s, hirvonen j: the significance of practical training in linking theoretical studies with practice. high educ 2006;51:439-464. 28. williams le, nettifee-osborne ja, johnson jl: a model for improving student confidence and experience in diagnostic sample collection and interpretation. j vet med educ 2006;33:132-139. 29. abrahamson s: how we learn: concepts, insights, and rationale for integration j vet med educ 2007;34:213-219. 30. hatch t: the differences in theory that matter in the practice of school improvement. am educ res j 1998;35:3-31. 31. smith sr: toward an integrated medical curriculum. med health r i 2005;88:258-261. 32. lake k. integrated curriculum. close-up #16. school improvement research series. northwest regional education laboratory, 1994. <http://www.nwrel.org/scpd/sirs/8/c016.html>. accessed 01/01/ 2008. 33. venville gj, wallace j, rennie lj, et al: curriculum integrations: eroding the high ground of science as a school subject. stud sci educ 2002;37:43-83. 34. collins gh, taylor rm: attributes of australasian veterinary graduates: report of a workshop held at the veterinary conference centre, faculty of veterinary science, university of sydney, january 28-19, 2002. j vet med educ 2002;29:71-72. 35. royal college of veterinary surgeons: essential competencies required of the veterinary surgeon. 2009 examination guidance – annex 1. http://www.rcvs.org.uk/shared_asp_files/uploadedfiles/rcvs/e7d55b38-0ae9-497a-a28a 20f82ed6d019_day_one_comps.pdf accessed 07/25/09. royal college of veterinary surgeons, 2009. 36. davis mh: outcome-based education. j vet med educ 2003;30:227-232. 37. cavalieri j: veterinary student attitudes toward curriculum integration at james cook university. j vet med educ 2009;36:305-316. 38. race p: the lecturer’s toolkit. 2nd ed. abingdon (uk): routledge falmer, 2001. 39. krause k: engaging and promoting student engagement in university learning communities. <http://www.cshe.unimelb.edu.au/pdfs/stud_eng.pdf>. accessed 06/19/09. james cook university, townsville, 2005. 40. grant p, paige k: curriculum integration: a trial. aust j teach educ. 2007;32:1-12. 41. school of veterinary and biomedical sciences: self evaluation report for the veterinary schools accreditation advisory committee. <http://www.jcu.edu.au/vbms/idc/groups/public/documents/staff_publications/jcuprd_047052.pdf > accessed 07/07/09. james cook university, townsville, 2009. 42. lonning ra, defranco tc, weinland tp: development of theme-based, interdisciplinary, integrated curriculum: a theoretical model. sch sci math 1998;98:312-318. 43. veterinary schools accreditation advisory committee (vsaac): policies, procedures and standards 2006. <http://www.avbc.asn.au/documents/pp&p+cvr6-12%5b1%5d.pdf>. accessed 12/07/07. australasian veterinary boards council inc. melbourne, 2006. 44. abraham a: the need for the integration of emotional intelligence skills in business education. bus renaissance quart 2006;1:65-79. 45. rollin be: an introduction to veterinary medical ethics. theory and cases. 2nd ed. ames (ia): blackwell publishing; 2006. 485 figure 1. diagrammatic representation of an integrated learning environment which is emulated within an integrated curriculum in which veterinary students are embedded. 486 table 1. advantages and disadvantages of curriculum integration. advantages of curriculum integration disadvantages of curriculum integration  integrative learning improves learning outcomes, promotes deeper learning and a broader perspective on topics  greater difficulty for students. students must be prepared to be examined on all the learning material  authentic learning connects knowledge to application in the world beyond the classroom potentially improving student’s motivation, participation, engagement, retention and satisfaction  problem and application focus requires a higher level of cognitive function as students solve problems and apply knowledge rather than simply recalling facts  contextualization of knowledge into clinical presentations and problems may assist with knowledge retention compared with the learning of knowledge in isolation  cooperation among staff from different disciplines is required as they plan, implement and assess learning activities  provides emotional appeal, motivating students, encouraging engagement and deeper learning  collaborative, planning, preparation, delivery and assessment can be time consuming. staffing formulas will need to be revised  an emphasis on outcome-based learning focuses learning on achieving specific knowledge, skills and attitudes, and helps remove irrelevant or redundant material from the curriculum  comparisons of student performance with less integrated courses may not be valid  curriculum can be more readily adapted to the changing needs of students and the profession  a deficiency of knowledge of the basic sciences or overlooking of important topics can more easily occur  forces staff to critically evaluate and revise their content. relevance takes precedence over coverage  staff insecurity and anxiety could occur associated with preparation of integrated material, and downsizing of individual discipline content  collaborative learning promotes team and interpersonal skills.  staff training and support required in association with curricula and pedagogical change and the preparation of integrated learning activities  collaborative teaching promotes greater communication and cooperation among staff across disciplines  curricula drift towards a disciplinebased program may occur in the face of difficulties associated with a revised curriculum. staff may default to a more familiar approach  integration of personal and professional development promotes life-long learning, moral and ethical reasoning, team, professional and communication skills  a shared vision is required by staff and students to facilitate the change to an integrated curriculum. appropriate leadership is critical for both inspiration and guidance  draws from, but is not bounded by the constraints of traditional disciplines  some staff may not model integrative thinking with their teaching, denying students visible models of integrated learning 487 table 2. thematic structure of subjects undertaken by students in years 2 and 3 of the veterinary science degree program at james cook university.37,41 theme focus structure and function diversity of living organisms structure and function of plants and animals techniques that are available to study plants and animals disease, defence and chemical agents molecular and cellular basis of disease processes, organisms and bodily defence diversity of organisms that cause disease pharmacology animal production, management and behaviour animal production, welfare and behaviour veterinary services epidemiology and public health veterinary practice knowledge, skills and attitudes required for veterinary practice professional life self understanding, management and personal development team skills communication skills conflict resolution information literacy and lifelong learning skills practice management/business skills 488 2012: non-ovulatory follicles: characteristics, causes, and solutions?     non-ovulatory follicles: characteristics, causes, and solutions? dale paccamonti department of veterinary clinical sciences, school of veterinary medicine, louisiana state university, baton rouge, la ovulatory failure during the breeding season is a frustrating development. non-ovulatory follicles have been called hemorrhagic anovulatory follicles (haf), anovulatory hemorrhagic follicles, hemorrhagic follicles or persistent anovulatory follicles. although most workers agree that anovulatory follicles are more common early or late in the season, they do sometimes occur during the normal breeding season, especially in older mares.1 one investigator examined 737 cycles in 47 mares that were treated repeatedly with cloprostenol (clo) and human chorionic gonadotropin (hcg) over a three year period.2. the failure of ovulation was more common in the autumn (september/october 22%, november 59%), and approximately 50% of the mares developed an anovulatory follicle during at least one cycle, and 26% experienced them during at least two cycles. a tendency for an anovulatory follicle to recur in a mare from one breeding season to the next was noted. others have reported no effect of season3 or the highest incidence from may to august,4 but again found a tendency for some mares to have repeated ovulation failure. while the occurrence of these anovulatory follicles is well recognized, the ability to predict their occurrence has eluded us. using grey-scale b-mode ultrasonography, there are no premonitory signs that can be used to predict that a follicle will fail to ovulate. typically, a haf is recognized by the sudden appearance of hyperechoic floating specks in the antrum of the follicle. over the next few days, hyperechoic strands are observed to develop in the antrum and the wall of the follicle thickens as apparent luteal tissue develops. using doppler ultrasound, comparing follicles the day before ovulation with those at a similar stage of development that would fail to ovulate; increased vascularity in the apical area of the follicle (site of impending ovulation) was noted in follicles destined to fail to ovulate. this difference was attributed to minimal hemorrhage during ovulation vs. considerable hemorrhage during haf formation.1 differentiating a haf from a corpus hemorrhagicum (ch) can be difficult if an examination is not performed shortly after formation. if examined daily, early after formation a haf will have a thinner luteal border, usually less than 3 mm, while a ch will have a border > 5 mm.5 in addition, the haf will have the same or greater diameter than the pre-ovulatory follicle, while the ch will be smaller than the preovulatory follicle diameter.5 with less frequent examinations, as the contents of the structures clot and become organized, the same criteria can be used; however there is overlap. the luteal border of the haf is usually, but not always, thinner than the border of the ch, and the diameter in relation to the maximum preovulatory follicle diameter is usually, but not always, greater in the haf than the ch.5 the progesterone profile after observation of the formation of an haf has been described, and in most cases (88%) when an haf was observed, progesterone was increased. in one report, progesterone rose slowly and progressively to >8 ng/ml in 51% of the cases, while in 13% it rose rapidly to >10 ng/ml. in 25% of the hafs, progesterone reached a plateau of 1 to 4 ng/ml, and in 11% it did not rise above 1 ng/ml.2 others have reported no significant difference in the concentrations of progesterone or luteinizing hormone (lh) between mares that developed an haf and control mares that ovulated, and only a slightly greater concentration of estrogen in the mares with an haf was observed three days before formation.1 other studies have similarly found that concentrations of progesterone in mares with an haf are elevated to >1 ng/ml.6,7 however in mares which formed a haf in response to flunixin meglumine administration, the concentration of progesterone on days 5 and 9 after haf formation was lower than in control mares on corresponding days after ovulation.7 consequently, most mares with an haf will respond to a luteolytic dose of prostaglandin (pg) administered at the appropriate interval after formation of the haf, with a return to estrus and normal ovulation.2 however, as discussed below, treating an haf with a luteolytic dose of clo may in some cases not be the best course of action, especially in mares prone to forming hafs. 567 clinical theriogenology • volume 4, number 4 • december 2012     similar to the inability to reliably predict the occurrence of an haf, the etiology remains elusive. persistent endometrial cups can lead to haf formation,8-10 and may repeat in the same mare.9 the presence of even low levels of equine chorionic gonadotropin (ecg), with its lh-like activity, can disrupt the normal ovulatory process resulting in non-ovulatory luteinized follicles. recent work has revealed a relative lack of angiogenic activity in hafs compared to corpora lutea formed after ovulation.6 other reports have linked the use of clo to induce luteolysis and a return to estrus with an increased risk of formation of hafs.2-4,11 in one study, the histories of two mares that both had an unusually high incidence of hafs were examined.11 in these two mares, the use of clo to induce estrus was associated with a high incidence of ovulation failure. other studies, by the same authors (and including the same mares), examined 207 and 7 mares, respectively, over longer periods of time, with a variable number of estrous cycles included for each mare.3,4 these authors hypothesized that the administration of clo results in a rapid drop in progesterone, which allows lh to rise. if this rise in lh occurs during the development of an immature follicle, before follicular deviation, it could interfere with the intrafollicular metabolism of substances necessary for ovulation and follicular collapse. similarly, in a model using follicle ablation to induce follicular waves and estrus, the mares had an earlier rise in lh and the incidence of hafs was increased.12 another report suggested that the use of hcg, when used in combination with other drugs, could contribute to an increased incidence of hafs.3 alternatively, administration of a prostaglandin inhibitor such as flunixin meglumine can interrupt the ovulatory process, resulting in an increased incidence of hafs. this approach has been used to induce haf formation for experimental purposes.2,13-15 in an attempt to reverse the effect of flunixin, clo was administered around the time that the follicle produces pg during the ovulatory process.12 in the same report, clo was administered to mares after they began to show signs of haf formation to determine if it might be an effective treatment once the initial signs were recognized.15 neither approach was successful. it would appear that once the ovulatory process is disrupted, that it is difficult to get it back on track. certain mares appear to be prone to repeated occurrences of hafs and some researchers feel that conditions such as laminitis may contribute to the occurrence of an haf,1,3,11 which is plausible given the reports linking the use of clo to haf formation. from a retrospective study of breeding records, it has been proposed that the dose of clo affects the chance of haf development, with higher doses more likely to result in haf formation.4 a factor which may play a role but was not described in these reports linking clo usage and haf formation is what stage of follicular development was present at the time of clo administration. the status of follicular activity at the time of clo administration affects the interval to onset of the subsequent estrus and interval to ovulation. it is possible that follicular status at the time of clo administration may also have a role in the formation of an haf. in a mare that is prone to haf development, or when clo administration to a mare with an haf is deemed necessary, it may be prudent to use the lowest effective dose possible. alternatively, it may be wise to forego administration of pg and allow the mare to return to estrus on her own, without hormonal intervention. in some cases, waiting a few days longer for a natural estrus may actually result in getting a mare in foal sooner than with hormonal intervention. references 1. ginther oj, gastal el, gastal mo, et al: incidence, endocrinology, vascularity, and morphology of hemorrhagic anovulatory follicles in mares. j eq vet sci 2007;27:130-139. 2. lefranc ac, allen wr: incidence and morphology of anovulatory haemorrhagic follicles in the mare. pferdeheilkunde 2003;19:611-612. 3. cuervo-arango j, newcombe jr: the effect of hormone treatments (hcg and cloprostenol) and season on the incidence of hemorrhagic anovulatory follicles in the mare: a field study. theriogenology 2009;72:1262-1267. 4. cuervo-arango j, newcombe jr: risk factors for the development of haemorrhagic anovulatory follicles in the mare. reprod domest anim 2010;45:473-480. 5. cuervo-arango j, newcombe jr: ultrasound confirmation of ovulation in mares: a normal corpus luteum or a haemorrhagic anovulatory follicle? reprod domest anim 2012; in press. 6. ellenberger c, muller k, schoon ha, et al: histological and immunohistochemical characterization of equine anovulatory haemorrhagic follicles (ahfs). reprod domest anim 2009;44:395-405. 568clinical theriogenology • volume 4, number 4 • december 2012     7. cuervo a: the effect of treatment with flunixin meglumine at different times relative to hcg administration on ovulation failure and luteal function in mares. anim reprod sci 2011;127:84-90. 8. crabtree jr, chang y, de mestre am: clinical presentation, treatment and possible causes of persistent endometrial cups illustrated by two cases. eq vet educ 2012;24:251-259. 9. allen wr, wilsher s: persistent endometrial cups in the same mare in two successive pregnancies. eq vet educ 2012;24:247-250. 10 willis la, riddle wt: theriogenology question of the month. j am vet med assoc 2005;226:877-879. 11. cuervo-arango j, newcombe jr: the effect of cloprostenol on the incidence of multiple ovulation and anovulatory hemorrhagic follicles in two mares: a case report. j eq vet sci 2009;29:533-539. 12. ginther oj, jacob jc, gastal mo, et al: follicle and systemic hormone interrelationships during spontaneous and ablation-induced ovulatory waves in mares. anim reprod sci 2008;106:181-187. 13. cuervo-arango j, domingo-ortiz r: systemic treatment with high dose of flunixin-meglumine is able to block ovulation in mares by inducing hemorrhage and luteinisation of follicles. theriogenology 2011;75:707-714. 14. ginther oj, cuervo-arango j, beg ma: disruption of periovulatory fsh and lh surges during induced anovulation by an inhibitor of prostaglandin synthesis in mares. anim reprod sci 2011;126:91-95. 15. cuervo-arango j. the effect of systemic administration of cloprostenol on ovulation in mares treated with a prostaglandin synthetase inhibitor. reprod domest anim 2012;47:32-38. 569 clinical theriogenology • volume 4, number 4 • december 2012 570clinical theriogenology • volume 4, number 4 • december 2012 omniblank: 9 contact ramanathan kasimanickam ramkasi@wsu.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 review report estrus and ovulation synchronization strategies in beef cattle ramanathan kasimanickam,a quinlan harting,a rachel hanson,b randa bolera aveterinary clinical sciences, washington state university, pullman, wa, usa banimal health clinic, blackfoot, id, usa abstract various technologies in bioveterinary medicine offer beef producers unique opportunities to improve overall herd genetics. research and technology have greatly enhanced our understanding of cattle reproductive physiology, facilitating induction and synchronization of estrus and/or ovulation in replacement heifers and postpartum cows. these improvements assist beef producers to increase the use of artificial insemination (ai) and facilitate mass breeding at predetermined times. in addition to improving genetics, this helps to increase uniformity (genetics and body weight) at weaning. pregnancy rates following implementation of these approaches are acceptable and generally comparable to breeding after detecting estrus. this review explains dynamics of synchronization, treatment regimens for various protocols, and factors that need to be considered while implementing protocols to achieve greater success. keywords: beef cattle, estrus, ovulation, synchronization, artificial insemination introduction sustainable beef production targets long-term health of the environment, maintains economic viability of the beef enterprise, and addresses consumer concerns. the economic advantage of a cow-calf operation is realized by a sensible and achievable production goal, 1 calf/cow/year, with a mean calving interval of 365 days. approximately 285 days of pregnancy allows only 80 days for the cow to become pregnant again. during this interval, the cow must recover from calving, resume cyclicity, and have 2 or 3 opportunities to become pregnant. synchronization of estrus and/or ovulation is a reproductive management tool to increase efficiency and profitability in beef production. although it is to shorten breeding and calving intervals, other benefits include optimizing labor and time and facilitating ai. in the usa, > 66% of dairy cows are ai bred; however, only 7.9% beef operations use estrus synchronization and 7.6% of operations use ai.1,2 time and labor are the primary reasons producers indicate as barriers to using ai in commercial beef cows and heifers.1,2 there are many options with various treatment regimens for  synchronization of estrus and/or ovulation in beef cattle.  before selecting an approach for mass breeding, it is imperative to assess key elements, including status of cattle intended for synchronization. consideration of key traits such as body weight/condition, pubertal status, pelvic size, and temperament of heifers; and in cows, body condition, days postpartum and temperament, will help improve success. evaluation of resources, including facilities, availability of labor, prior experience, and budget will help to select an appropriate synchronization protocol. in addition, duration of the protocol, number of animal handlings, ability to successfully provide treatments, and proper ai techniques (compliance) are other determining factors for a successful outcome. these key elements are discussed. calf crop and estrus synchronization percentage annual calf crop = × # of calves weaned # of females exposed 100 . although 90-95% of calf crop is achievable in a year, economic benefits are determined by pregnancy rate and pregnancy early in the breeding season which translates to calving rate and calving early in the calving season.1-3 this goal may be attained more easily with estrus synchronization and ai programs. realistically, > 50% of eligible beef females become pregnant to 1 ai after implementation of effective estrus/ ovulation synchronization and ai programs.3-5 estrus synchronization optimizes labor and time and facilitates ai;3 the latter allows access to superior genetics, hastens genetic http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11650 10 citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 improvement within a herd, and is frequently cheaper than natural service (ns).3-5 synchronized females: 1. express estrus at a controlled time; 2. intensify calf uniformity; 3. calve earlier in the season; and 4. wean calves that are older and heavier, all of which can increase economic return. economic return is calculated as: weight of calves at sale (lbs) × calf crop = lbs of beef produced/cow exposed. the following ai and ns combinations (table 1) were used in a trial (n = 1,249) involving 12 cow-calf operations and clearly indicated benefits of using ai in beef operations. there was an increase in pregnancy rate by 5-9% across the 12 locations, fewer assisted births (1.3 versus 2.9%), lower death loss (3.5 versus 5.5%) and overall, 10% more calves when dams and daughters were bred by ai/ai combination compared to ns/ns combination (personal communication, william whittier, december 23, 2024). increased age at weaning, improved pregnancy rates, and potential for increased growth due to improved genetics resulted in reported weaning weight increases (20-40 lbs) for the entire calf crop.6 estrous cycle estrous cycle consists of follicular and luteal phases. the follicular phase includes the interval from corpus luteum (cl) regression to ovulation, including proestrus and estrus stages of the cycle. during the follicular phase, the dominant ovarian structure is a mature and estrogenic follicle that releases estrogen, the predominant hormone. physiological events duing the follicular phase include sexual receptivity, preparation of the dominant follicle for ovulation, gonadotropin release from the anterior pituitary, and ovulation. the luteal phase includes the interval from ovulation (cl formation) to cl regression, including metestrus and diestrus. during the luteal phase, the dominant ovarian structure is a cl that produces progesterone, the dominant hormone. physiological events during the luteal phase include formation of a cl, progesterone production by the cl, uterine quiscence, release of prostaglandin f2α (pgf2α) from the endometrium, and lysis of the cl. control of follicular and lutal phases to synchronize estrus and/or ovulation in all eligible cows, the follicular phase is controlled by initiating emergence of a new follicular wave, whereas the luteal phase is controlled by initiating and/or prolonging the lifespan of the cl by gonadotropin releasing hormone (gnrh) treatment to induce ovulation and cl formation, or by supplementing exogenous progesterone (with or without a cl). liming factors, principles and options to control follicular and luteal phases in beef females with varying physiological status are illustrated in table 2. drugs and dosages prostaglandin f2α intramuscular dinoprost tromethamine : 25 mg intramuscular cloprostenol : 500 µg intramuscular gnrh : 100 µg progesterone controlled internal drug release (cidr) inravaginal insert : 1.38/1.55/1.9 gram for 5-14 days oral melengestrol acetate (mga) : 0.5 mg/head/day mixed in feed oral supplementation of mga is approved for estrus suppression in heifers only (federal register, 1997). use of mga as part of any estrus synchronization protocol in beef cows constitutes an extra-label use of medicated feed that is prohibited by the animal medicinal drug use and clarification act and regulation 21 cfr 530.11(b). food and drug administration (fda) approved pharmacuticals used for synchronization in cattle are given in table 3. estrus synchronization treatment regimens synchronization of estrus in beef females for mass breeding involves: 1. shortening the luteal phase by inducing premature luteolysis or 2. prolonging the luteal phase by maintaining table 1. ai and ns combinations in beef cattle dam bred by daughter bred by ai ai ai ns ns ai ns ns table 2. principles, liming factors and options to control follicular and luteal phases criteria follicular phase luteal phase how could you control? initiating a new follicular wave shortening (or) prolonging cl lifespan what is the limiting factor? presence of a dominant follicle shortening presence of cl and cyclicity are needed prolonging presence of cl and cyclicity do not matter how do you do this? removing dominant follicle shortening lyse cl prolonging supplement progesterone what methods could be employed? giving gnrh/lh/hcg), estradiol (varies with country) remove follicle with ultrasound guided aspiration shortening – give pgf2α – need a responsive/active cl prolonging – induce cl and/or give intravaginal or oral progesterone http://dx.doi.org/10.58292/ct.v17.11650 citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 11 circulating progesterone concentrations via supplementation (daily oral, slow-release injectable (ear implant), or vaginal inserts). the injectable method is uncommon in cattle in north america. shortening luteal phase luteolytic dose of pgf2α or its analog given to eligible beef females (with a mature active cl) results in luteolysis, and estrus7,8 occurs within 2-6 days (figure 1),9 depending on follicle diameter and phase of the follicular wave. when pgf2α is given during growing and static phases, estrus occurs soon (figure 1a), whereas estrus occurs later if pgf2α is given during the regressing phase (figure 1b) as it takes time for a new dominant follicle to emerge and reach preovulatory size. pgf2α causes luteolysis, with subsequent surges in estrogen and lh, with ovulation occuring ~ 24 hours after the lh surge. pgf2α programs pgf2α is generally inexpensive; single, double, or biweekly programs are effective only in cycling females when a cl is table 3. available fda-approved drugs to control and synchronize estrous cycles in cattle (refer animal drugs @ fda for specific information about each drug) regimen drug name (active ingredient) application number and manufacturer sequential use with another drug gonadorelinprostaglandin factrel® injection (gonadorelin injection) nada 139-237 zoetis inc. lutalyse® or lutalyse® highcon (dinoprost tromethamine) fertagyl® (gonadorelin) anada 200-134 intervet, inc. estrumate® (cloprostenol sodium) gonabreed® (gonadorelin acetate) anada 200-541 parnell technologies pty. ltd. cloprostenol sodium cystorelin® (gonadorelin) nada 098-379 boehringer ingelheim animal health, inc. cloprostenol sodium progestin only eazi-breed™ cidr® (progesterone intravaginal insert) nada 141-200 zoetis inc. progestinprostaglandin eazi-breed™ cidr® (progesterone intravaginal insert) nada 141-200 zoetis inc. lutalyse® or lutalyse® highcon prostaglandin only lutalyse® or lutalyse® highcon injection nada 108-901 & nada 141-442 zoetis inc. estrumate® nada 113-645 intervet, inc. prostamate™ (dinoprost tromethamine) anada 200-253 bimeda animal health ltd. estroplan® (cloprostenol sodium) anada 200-310 parnell technologies pty. ltd. synchsure (cloprostenol sodium) anada 200-310 boehringer ingelheim animal health nada: new animal drug applications; anada: abbreviated new animal drug applications figure 1. schematic presentation of interval from pgf2α treatment and estrus expression based on follicle size and phase of the follicular wave in cattle pgf figure a figure b 2α pgf2α pgf2α http://dx.doi.org/10.58292/ct.v17.11650 12 citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 mature/responsive.10 cl becomes responsive to pgf2α ~ 7 days after ovulation. there are several pgf2α protocols, according to management needs (figures 2-4). however, implementation of a successful estrus detection program is essential to achieve good results with pgf2α programs. the ai is performed following the am-pm rule (i.e. a cow should have ai 12 hours after first being observed in estrus).11,12 if a cow is noticed in standing estrus in the am, ai is performed that pm, whereas cows observed in standing estrus in the pm should ai is performed the following am. the am-pm rule requires twice daily ai. however, recent studies recommended that ai should be performed 6-18 hours after first observation of standing estrus.13 conception rate to ai following implementation of pgf2α program is generally similar to ai following spontaneous estrus. weekly monday morning program on monday morning, all eligible cows receive an injection of pgf2α, followed by estrus detection for the remainder of the week and cows detected in estrus have ai following the am-pm rule (figure 2). approximately 65% of cows are expected to exhibit estrus. cows not detected in estrus receive pgf2α the following monday morning along with new group of eligible cows and the same procedure is followed every week. cows given weekly doses of prostaglandin had a 30% higher pregnancy rate than those receiving prostaglandin based on transrectal palpation of a cl.14 biweekly monday morning or double pgf2α program on monday morning, all eligible cows receive an injection of pgf2α, with6 or without ai for cows exhibiting estrus after pgf2α treatment. two weeks later (monday), cows that did not have ai15 or all cows, receive another dose of pgf2α, followed by detection of estrus for the remainder of the week, with ai based on the am-pm rule (figure 3). approximately 85-90% of cows should exhibit estrus. this program requires weekly or biweekly estrus detection. there are several pgf2α programs, including pgf2α treatment for nonpregnant cows with an active cl at pregnancy diagnosis or repeated biweekly pgf2α treatment as a postpartum reproductive management tool16 at 25-32, 39-46, and 53-60 days, with first insemination following the last injection. cows not inseminated after pgf2α injection between 53-60 days are given pgf2α 14 days later. pgf2α programs are popular in dairy operations. however, some beef operations implement pgf2α program before/ after exposing cows to bulls (figure 4). this apporach generally resulted in more pregnancies and more calves compared to no pgf2α program before or after bull introduction. inducing or prolonging luteal phase luteal phase can be achieved by induction of ovulation with gnrh and subsequent cl formation and/or by withdrawal of progesterone following supplementation for 5 or 7 days and a concommitant luteolytic dose of pgf2α will result in a decline in progesterone concentration to basal values and estrus. select synch programs several select synch protocols are available for use in ai or ns breeding programs, including gnrh + pgf2α, cidr + pgf2α, and mga + pgf2α. 17-19 gnrh and pgf2α gnrh treatment on day 0 (random stages of the estrous cycle) is to control follicular wave emergence and/or to induce cl formation; pgf2α is given on days 6 or 7, observe for estrus days 2-6 after pgf2α, and ai cows that express estrus (am-pm rule) (figure 5). conception rate is similar to ai following spontaneous estrus and pgf2α-induced estrus. these programs are ideal for smaller beef operations. figure 2. schematic presentation of weekly monday morning program in cattle monday pgf2α to all eligible cows estrus detection & ai day 2 day 6day 0 figure 3. schematic presentation of biweekly monday morning program: in cattle 61yad41yad0yad monday pgf2α to all eligible cows estrus detection & ai monday, 2 wks. later pgf2α to all cows day 22 figure 4. pgf2α program before/after exposing beef females to bulls http://dx.doi.org/10.58292/ct.v17.11650 citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 13 cidr + pgf2α or select synch + cidr cows receive a cidr (1.3 g progesterone) insert for 7 days and an injection of pgf2α the day before (day 6) (figure 6a) or at cidr removal (day 7) (figure 6b), with improved estrus synchrony and conception rates. when gnrh was given 6 or 7 days prior to pgf2α, 70-83% of cows were in estrus within 4 days.20 it is advantageous to include the cidr when more cows are anestrus and/or when estrus detection before pgf2α treatment is not feasible. with select synch, 5-20% of cattle may exhibit estrus 2 days before pgf2α treatment. the best strategy is to apply both protocols to the same group of cows, placing cidrs in young, thin, and/or late-calving cows. in heifers, progesterone supplentation (oral or intravaginal) promotes cyclicity. mga mga is an orally active progestin; fed at 0.5 mg/day per animal, estrus is suppressed and ovulation is prevented.18,21 feeding mga is specifically approved for estrus suppression only in heifers. level of feeding and consumption of mga are critical to success. in this program, beef heifers are fed mga for 14 days, followed by a pgf2α injection 19 days later on day 33.18,21 this will have heifers in the late luteal stage of the estrous cycle at pgf2α injection, and will maximize conception rate (figure 7a). alternately, if there is a concern about consistent delivery of mga, producers/clinicians can use a cidr insert in place of mga (figure 7b). both mga and cidr control the estrous cycle.22-24 furthermore, a 7-11 modified mga synchroinzation protocol reduces the protocol from 37 to 26 days (figure 8). the 7-11 program25,26 utilizes a 7-day progestin supplementation (mga; fed from days 0 to 7) with pgf2α given at the conclusion of the mga feeding (day 7). gnrh is figure 5. schematic presentation of select synch estrus synchronization program figure 6a. schematic presentation of select-synch + cidr estrus synchronization protocol with pgf2α treatment the day before cidr removal figure 6b. schematic presentation of select-synch + cidr estrus synchronization protocol figure 7a. schematic presentation of 14 d mga + pgf2α estrous synchronization protocol in beef heifers figure 7b. schematic presentation of 14 d cidr + pgf2α estrous synchronization protocol in beef heifers figure 8. schematic presentation of 7-11 synch program http://dx.doi.org/10.58292/ct.v17.11650 14 citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 given on day 11, pgf2α on day 18, estrus detected from days 20-24, with ai using am-pm rule. mga programs are used for ns and ai. field trials involving heifers where mga was used in conjunction with ns, or with pgf2α prior to ai at oberved estrus.27 the pregnancy rate for synchronized estrus was 9 percentage points greater for ns over ai, substantiating the flexibility in implementing precise synchronization protocols with this particular management system.27 further, the mga + select protocol involves an injection of gnrh on day 26 in a 14-day mga + pgf2α program.28,29 although pregnancy rate to synchronized estrus in 2-4 year old cows were similar to both mga-select and mga + pgf2α protocols (62 versus 69%, respectively), the mga + select program is advantageous, as more cows < 5 years of age became pregnant compared to an mga +pgf2α program (71 versus 46%).28,29 substituting cidr inserts for mga in the mga + select protocol in beef heifers were evluated. although pregnancy per ai was greater in cidr versus mga-treated heifers (63 versus 47%; p < 0.01), final pregnancy rate did not differ (p > 0.10) between treatments.25 pregnancy rates to ai were similar following implementation of 14-day mga + pgf2α and 7-11 synch protocols.30 ai success with various protocols is illustreated (table 4). ovulation synchronization ovulation synchronization is aimed at synchronizing ovulation and allowing tai rather than relying on estrus detection to determine when to breed. ovulation synchronization is based on forcing ‘turnover’ of a dominant follicle as it is the main limiting factor to synchronize emergence of a new follicular wave in all eligible beef females at random stages of the estrous cycle at protocol initiation. in addition, the ovulation synchronization program promotes oocyte viability. as illustrated (figure 9), 3 principles are involved and should be achieved in a timely manner to promote success of ovulation synchronization programs. intiation of a new follicular wave and synchronized follcular wave growth can be achieved by gnrh, estrogen (licensed use varies with country), or follicle ablation. gnrh gnrh induces ovulation/luteinization of follicles > 10 mm in 85% of cows and 55% of heifers when injected at random times of the estrous cycle. further, treatment of gnrh at random stages of the estrous cycle inititates new follicular wave emergence in ~ 3-4 days.20,41 estrogen estrogen causes regression of fsh-dependant follicles and luteinization/ovulation of lh dependent/estrogenic follicles via both negative and positive feedback. new follicular wave emergence occurs ~ 4 days later, depending on the type of estradiol used. follicular wave emergence occurs 3.6, 4.1, and 4.4 days after estadiol 17β, estradiol benzoate (eb), and estradiol cypionate, respectively.42-44 it should be noted that when table 4. conception per ai following implementation of estrous synchronization protocols in beef heifers and cows protocol cr/ai% (total female) age group select synch + cidr* 53.8 (323/600) cows31 select synch + cidr* (ss) 65.0 (160/246) cows32 select synch + cidr* (cs) 66.7 (164/267) select synch + ns 56.9 (249/438) heifers33 select synch + cidr* 46.9 (160/341) cows34 14-day cidr + pgf2α 61.7 (267/433) heifers35 5-day select synch + cidr 64.8 (287/443) select synch + cidr* 55.5 (394/710) cows36 2 pgf2α (14 days) 52.3 (376/723) heifers37 select synch 60.2 (100/166) cidr + pgf2α 59.1 (528/894) 5 day select synch + cidr 72 (33/46) cows38 7 day select synch + cidr 72 (36/50) cidr+ pgf2α 61 (197/325) cows39 select synch 70 (217/309) select synch + cidr 67 (230/345) mga/select synch 46.0 (185/402) heifers30 mga/ pgf2α 47.0 (185/394) select synch 47.0 (21/45) cows25 7-11 synch 68.0 (30/44) select synch 65.7 (115/175) cows40 select synch + p4 59.1 (123/208) 2 pgf2α (14 days) 60.6 (86/142) http://dx.doi.org/10.58292/ct.v17.11650 citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 15 using estrogen for follicular suppression, treatment of estrogen and progesterone is advisable (addition of progesterone is to suppress estrogen induced lh surge), whereas estrogen alone is sufficient in combination with a cidr. limitations of synchronizing follicular waves the simplistic explanation is the average length of estrous cycle is 21 days, with a range from 16 to 24 days. a typical bovine estrous cycle has 2 or 3 waves of follicle development, and the dominant follicle of the last wave ovulates. the follicular wave consists of 3 phases (growing, static, and regression) with 3 points (recruitment, selection, and dominance). it should be noted that an 18-21 day range is associated in a 2 wave cycle and a 21-24 day range is associated with a 3 wave cycle.45 two-wave cycles are common in cattle and cycles with 1 or 4 waves cycle occur incidentally. wave length in prepubertal heifers are 8 days. the interovulatory intervals of 3 wave cycles differed from 2 wave cycle in: 1. earlier emergence of the dominant follicles; 2. longer duration; and 3. shorter interval from emergence to ovulation. the reason for 2 or 3 wave cycles is unclear. poor nutrition and stress (3 wave), lifespan of cl (shorter in 3 wave) and slowly growing dominant follicles (2 wave) are associated with the difference.45 differences in number of follicular waves and range of estrous cycle and follicular wave lengths are illustrated (table 5). it depicts variations in follicular dynamics when implementing estrus or ovulation synchronization protocols in a group of eligible beef females at random stages of the estrous cycle. follicular wave length could vary 6-12 days. while implementing synchronization, not only these variations, but prepubertal and peripubertal status in heifers and postpartum anestrus in cows and ovarian disorders such as cystic ovarian degenration and uterine disorders such as clinical and subclinical endometritis should also be taken in to account. ovulation synchronization treatment regimens with combinations of gnrh and pgf2α figure 10 shows the basic ovulation synchronization protocols that utilize the combination of gnrh and pgf2α. both ovsynch and presynch-ovsynch are commonly used in dairy operations. ovsynch includes a first dose of gnrh given on a random day of the estrous cycle (day 0). seven days later, pgf2α (day 7) is given and 48 hours later, a second dose of gnrh is given, with tai 16 hours later,46 requiring 4 handlings. presynchronization, the initial portion of the protocol that precedes the ovsynch portion, is achieved with 2 pgf2α treatments, with a 2 week interval between the first and second pgf2α treatment, in the weeks leading up to the initiation of ovsynch. however, interval between the second pgf2α figure 9. illustration of 3 principles that are involved in the success of ovulation synchronization programs table 5. differences in range of estrous cycle, number and length of follicular waves estrous cycle length 2 wave cycle 3 wave cycle short range – 18 day cycle 9 days per wave 6 days per wave long range – 24 day cycle 12 days per wave 8 days per wave average – 21 day cycle 10.5 days per wave 7 days per wave the underline highlights the difference in the wave’s length between a longand short-range cycle and between a twoand three-wave cycle. figure 10. ovulation synchronization protocols with the combination of gnrh and pgf2α http://dx.doi.org/10.58292/ct.v17.11650 16 citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 treatment and first gnrh of ovsynch protocol is more variable. usually, the selected interval is 10-14 days. the general objective of presynchronization is to increase the percentage of cows that are on days 5-8 of their estrous cycle, with intermediate circulating progesterone concentrations and a healthy, dominant follicle that can ovulate in response to first gnrh of ovsynch protocol treatment. co-synch the co-synch protocol is one of the most commonly used protocols across the industry for tai of beef females. the co-synch protocol, is similar to an ovsynch protocol, except the second gnrh is given at tai, with only 3 handlings. the co-synch protocol is implemented with or without cidr. the co-synch + cidr protocol was reasonably effective among pre/peri pubertal heifers and anestrous cows and it is a good option when a minimal number of animal handlings is desired. co-synch without cidr heifers are injected with gnrh on day 0 and pgf2α is given on day 7. heifers are inseminated 60 ± 4 hours after pgf2α injection and a second dose of gnrh is given at tai (figure 11a). cows are injected with gnrh on day 0, an injection of pgf2α is given on day 7, with tai (and a concommittant injection of gnrh) between 66 and 72 hours after pgf2α injection. co-synch + cidr the co-synch + cidr program is similar to co-synch; in addition, a cidr insert is placed and removed after 7 days (from gnrh to pgf2α treatment). heifers are inseminated at 60 ± 4 hours after pgf2α injection and a second gnrh is given at tai (figure 12a). in cows, insemination is done between 66 and 72 hours after pgf2α, with a second gnrh at tai (figure 12b). pregnany per ai following utilization of various co-synch protocols, with or without cidr, in cows and heifers that received insemination at a various fixed times following cidr removal is given in table 6. 5 day co-synch + cidr this protocol was developed based on the premise that reducing the length of cidr treatment 7-5 days in the co-synch + cidr protocol would increase secretion of estradiol by the preovulatory follicle, decrease incidence of induced ovulation of follicles with reduced estrogenic activity, and potentially improve tai pregnancy rates, based on the assumption that day-4 dominant follicles have higher intrafollicular estradiol-17β.49-51 this protocol is similar to the 7 day co-synch + cidr but involves a shorter interval (5 days) of cidr treatment.49,52 however, it requires giving 2 doses of pgf2α approximately 6 to 8 hours apart (additional handling of cows and cost of second pgf2α). a larger field trial (n = 1817) had a small improvement in pregnancy rates to ai following a 5 day co-synch + cidr protocol compared to the 7 day co-synch + cidr figure 11a. schematic presentation of co-synch ovulation synchroniazation protocol in heifers figure 11b. schematic presentation of co-synch ovulation synchroniazation protocol in cows figure 12. schematic presentation of co-synch + cidr ovulation synchroniazation protocol table 6. pregnancy per ai (p/ai) following implementation of estrous synchronization protocols, with or without progesterone supplementation in beef heifers and cows protocol p/ai (total females) age group co-synch + cidr (72 hours) 65.0% (5470) heifers47 co-synch (72 hours) 55.3% (5099) co-synch + cidr (60-66 hours) 54% (2868) cows48 co-synch (60-66 hours) 52% (871) http://dx.doi.org/10.58292/ct.v17.11650 citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 17 protocol (58.1 versus 55.1%; p = 0.04) in cows,53 whereas in heifers (n = 289), a 5 day co-synch + cidr protocol tended to be greater (63.8 versus 53%; p = 0.07).54 split-time ai pregnancy rates/ai can be optimized with a split-timed ai (stai) approach. the rationale is that pregnancy percentages are greater in beef females that express estrus before insemination. the stai involves not conducting ai in females that have not expressed estrus at the time of tai. however, with heifers that have not expressed estrus at the time of tai, there is an additional interval for behavioral estrus expression to occur before ai, with all heifers that have not expressed estrus by tai being inseminated at the second tai, with gnrh treatment either at first or at second tai to heifers not detected in estrus. in a large field trial in beef heifers (n = 3166), p/ai percentages were 58.9,63.4, 56.5, and 56.5% for 14 days/ stai, 5 days/stai, 14 days/tai and 5 days/tai, respectively. the study concluded the 5 day cidr regimen with 64 + 84 hours split-time ai combination acheived > p/ ai.35 in beef cows (n = 1062), p/ai for cows in the 65 + 85 hours treatment combination was > at 36 days than for cows in the 55 + 75 hours treatment combination (61.0 versus 51.4%), respectively.55 another trial in beef cows (n = 695) was conducted to compare pregnancy percentages per embryo transfer (p/ et) following twice daily compared to split-time (64/84hours) estrus detection in a cidr + select treatment regimen.34 percentage p/et for cows in the split-time and twice daily estrus detection groups did not differ (49.2 [174/354] verus 46.9 [160/341]; p > 0.1). further, percentage conception/et for cows in the split-time and twice daily estrus detection groups were 60.0% (174/290) and 56.3% (160/284), respectively (p > 0.1), whereas conception rates for et at 64 and 84 hours were 61.5% (150/244) and 52.2% (24/46). presynchronization in beef heifers exogenous progesterone hastened cyclicity in pre and peripubertal beef heifers and also increased pregnancy per ai. progesterone (cyclic) status or progesterone supplementation at onset of synchronization protocols are critical for favorable pregnancy outcomes. presynchronization is synchronization of the estrus cycle prior to synchronization for tai. there are various presynchronization methods, including 1 dose of pgf2α (10 days before initiation of protocol) or 2 doses of pgf2α, 10-14 days apart, with the second dose 10-14 days before protocol initiation,56 gnrh alone57 or combined with pgf2α, 57,58 or a cidr for 5, 7, 9, 14 or 18 days53,59,60 before protocol initiation. beef herds with a high percentage of prepubertal or peripubertal heifers at the start of the breeding season may benefit from presynchronization. treatments such as cidr and/or gnrh before initiating a tai program may hasten puberty.38,47,61-63 dominant follicles are present in prepubertal and peripubertal heifers and may be induced to ovulate with exogenous gnrh, depending on follicle size and maturity. however, smaller follicles (< 11 mm) induced to ovulate were less likely to result in pregnancy than ovulation of larger follicles (11-16 mm).19,64-66 using a cidr promotes ovulation in prepubertal and peripubertal heifers47 and anestrus postpartum cows. further, ovarian responses in angus-cross beef heifers presynchronized with cidr-gnrh before a co-synch protocol had more heifers with a cl at pgf2α and increased preovulatory follicle diameter at ai compared to a cidr only before a co-synch protocol.67 recently, 7 & 7 synch protocol (cidr insert and pgf2α on day 0; gnrh on day 7, cidr insert removal and pgf2α on day 14 and gnrh + tai, 66 hours after cidr removal) was compared to 7 days co-synch + cidr protocol in beef cows.68,69 improved pregnancy was observed following implementation of 7 & 7 synch protocol compared to 7-days co-synch + cidr protocol. in beef cattle, 5 days co-synch + cidr protocol resulted in 10% greater pregnancy compared to 7 days co-synch + cidr protocol in cows49 and similar pregnancy rate compared to 14 days cidr protocol in heifers.24 since 5 days co-synch + cidr protocol resulted in greater pregnancy in beef heifers, it would be interesting to compare pregnancy rates following 7 & 5 and 5 & 5 treatment regimens in heifers. preliminary and unpublished results from our trials in beef heifers had greater pregnancy for 7 & 5 protocol when pgf2α (60.1%) was replaced with gnrh (52.8%) at cidr insertion. utilization of sexed semen in a synchronization programs ai with sexed semen should be used only on any cow or heifer that are observed in estrus70-72 following synchronization with any protocol. to improve pregnancy success, it is recommended to use sexed semen on beef females that have exhibited estrus before insemination and use conventional semen on females that have not exhibited estrus, with concurrent gnrh treatment. for best results with sexed semen, ai is performed 16-28 hours after detecting estrus.73,74 variations among bulls exist in pregnancy success following ai with sexed semen due to variation in sperm dna longevity.75 field studies and reports could be used to identify bulls with sufficient number of inseminations and to identify bulls with true differences in fertility. once identified, these bulls could be used in research to assess fertility in sex-sorted semen. sperm dna integrity is an important component of fertility not routinely evaluated by a standard semen analysis. further, extent of sperm dna fragmentation (sdf) correlated with siring capacity.76 reduced cleavage rate and developmental arrest for sexed sperm-derived embryos compared to conventional embryos has also been reported.75 increased amount of sdf in sex-sorted sperm was detected when samples were incubated for 48 hours.75-78 duration of dna integrity in vitro of sexed sperm varied among bulls. bulls with higher fertility following sexed semen ai had sperm dna integrity for longer duration (up to 72 hours), whereas dna longevity was shorter, ≤ 24 hours, for bulls with low fertility.78 however, ai with sexed semen that occurred close to ovulation (28 hours after first standing estrus) eliminated variations in bull fertility. utilization of natural service sire in a synchronization programs ns programs are dependent on bulls for success, and synchronization will result in a larger proportion of females in estrus http://dx.doi.org/10.58292/ct.v17.11650 18 citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 for a shorter interval. any bull used for breeding should have a bull breeding soundness examination done by a veterinarian prior to turnout. as well as evaluating semen quality and scrotal circumference, the veterinarian will also assess the bull’s overall condition and physical structure. individual bulls vary widely in their ability to cover cows. if using synchronization, it is advisable to be on the conservative side with respect to the bull-to-cow ratio. most recommendations are to stock mature bulls at a rate of 1 bull to no more than 25 cows. use of young, inexperienced sires after synchronization is discouraged due to the concentrated breeding window, and bull-to-cow ratios should be reduced if young bulls are used (e.g. 12 cows for a yearling bull and 18 cows for an 18-month-old bull). singlesire breeding pastures also inherently involve more risk. periodic observation of breeding groups is recommended to ensure mating occurs and females do not continue to return to estrus throughout the breeding season. a trial33 was conducted in beef heifers (n = 1,744) to compare estrous response and first service and breeding season pregnancy rates in angus-cross beef heifers that received progesterone-based estrus-synchronization treatment regimens for timed artificial insemination (tai), with or without short-term ns. progesterone-based co-synch tai with a short-term ns treatment regimen resulted in proportionately more pregnancies (60.3%) than without a short-term (54.2%) treatment regimen. in addition, 64/84 hour splittimed ai ([sti] 59.3%) or ns following select-synch (57.3%) treatment regimen could  be  implemented as an alternative, as these treatment regimens resulted in similar pregnancy rate as progesterone-based co-synch tai with short-term ns (60.3%) treatment regimen. synchronization strategies in bos indicus cattle physiological differences between bos indicus and bos taurus include a reduced capacity for lh secretion, greater sensitivity to exogenous gonadotrophins, an earlier lh surge and ovulation with shorter and less overt estrus expression (mainly at night), smaller cl, and lower blood progesterone concentrations in bos indicus cattle.79 this makes it difficult to implement ai programs using estrus syncronization protocols. in bos indicus cattle, estrus response following pgf2α program was ~ 30% less than percentages reported for bos taurus cattle under the same conditions.80,81 the combination of low and variable estrus response and the high incidence of anestrus in animals grazing tropical grasses result in wide variability in estrus response and pregnancy rates. pregnancy rates following implementation of gnrh + pgf2α based ovysnch and co-synch protocols have often been lower than rates reported in bos taurus, with low conception rates in anestrus cows.82 the most useful alternative to increase the number of females that are inseminated are protocols that enable ai without the need for estrus detection, usually called tai; tai protocols using progestin devices, estradiol and equine chorionic gonadotrophin (ecg) have resulted in consistent pregnancy rates in bos indicus and bos indicus crossbred cows. in additon, pregnancy in successive cycles and breeding season pregnancy rates are improved with progestin devices used at the beginning of the breeding season. exogenous control of luteal and follicular development has facilitated application of assisted reproductive technologies in bos indicus-influenced cattle, without necessity of estrus detection and should provide opportunities to improve reproductive performance of beef cattle in tropical climates. estradiol and progestin treatments have been increasingly used over the past several years in estrus synchronization programs in cattle. for example, giving 2 mg of intramuscular eb at insertion of the cidr (day 0); on days 7 or 8 the device is removed and intramuscular pgf2α is given, and 24 hours later, 1 mg of intramuscular eb is given,81 with tai between 52 and 56 hours after device removal. results from 13,510 inseminations in bos taurus and bos indicus crossbred cattle, resulted in an average pregnancy rate of 52.7% (27.8-75.0%).81 factors that influenced pregnancy success were body condition score and cyclicity. treatment of 200-400 iu ecg before ovulation in bos indicus cattle improved ovarian follicular development before ovulation and increased progesterone concentrations during early pregnancy.83 this glycoprotein ecg has fsh and lh like activity in ruminants, with both hormones required for periovulatory follicle maturation. in addition, ecg half-life was estimated to be 45 hours in the bloodstream of cows,84 providing sustained gonadotropin support before ovulation. this effect of ecg is especially important in postpartum anestrus cows where lh pulses are frequently deficient.85 addition of ecg to a progesterone and estradiol-based treatment for tai improves ovulation rate and luteal function in anestrous cows. consequently, ecg has been previously used in conventional progesterone-based treatments.85,86 cyclicity at the initiation of protocol did not affect the pregnancy between cows treated (56.3%) or not treated with ecg (56.5%); however, addition of ecg yielded pregnancy rates close to 50% in cows with a bcs of 2.81 there were greater (p < 0.05) percentages of insemination and pregnancy in a 4 day breeding season in cows treated with cidr + pgf + tw (temporary weaning) + ecg (50.9 and 29.4%) than in cows treated only with cidr + pgf + tw (39.4 and 23.7%).87 clearly, progestin-releasing devices, estradiol and ecg advance resumption of cyclicity in anestrus cows and facilitate tai in suckled bos indicus cows. factors influncing success of synchronization compliance farm personnel should be working closely with veterinarians when they consider which synchronization protocol best fits their goals. additionally, it is crucial to ensure protocols are followed. implementation of a synchronization protocol includes: finding the right cow, using the correct reproductive hormone at the correct dose and route, giving each injection at the correct time on the correct day, and adhering to each step, from the first injection to ai. clear and accurate animal identification, employee training, safe and efficient animal handling, maintaining a record-keeping system and updating it regularly, maintaining product labels, and providing staff with needles, syringes, gloves, etc, are all important in achieving protocol compliance. tracking submission for ai and pregnancy rates are necessary to determine if compliance is being met. it should be noted that missing 5% of cows (or injections) results in 86% compliance across a 3 handling protocol. transportation of beef females after ai transporting cows/heifers after ai should be avoided. embryos are vulnerable to stress-associated changes in circulating hormones and uterine environment during blastocyst formation, hatching, maternal recognition of pregnancy, and attachment to the uterus. it is essential to try to minimize stress and/or major changes after insemination to maximize http://dx.doi.org/10.58292/ct.v17.11650 citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 19 pregnancy success. transportation is recommended 1-4 days after ai or later (day 42) after ai.88-90 although reports suggest that transporting cows 5-42 days after ai can cause 10% decrease in pregnancy rates, in a small trial91 acth treatment increased serum cortisol concentrations but did not increase serum concentrations of prostaglandin f metabolites or cause pregnancy loss during early pregnancy in cows. reproductive tract score in heifers the reproductive tract score (rts) is a 5 point scoring system (1 anestrus/underdeveloped or infantile genitalia; 5 – cycling, mature) based on the size (development) of tubular reproductive tract and ovarian structures are given below (table 7).92 the rts should be used before breeding as a replacement heifer selection criterion. this can be categorized as: prepubertal – score 1; peripubertal – scores 2 and 3; and pubertal – scores 4 & 5. the rts of 1 corresponds to the point in time at which the pattern of lh release is characterized by low-frequency pulses, as the hypothalamic-pituitary axis is highly responsive to estrogen negative feedback. reproductive tract scores of 2 and 3 are associated with the peripubertal phase, at which responsiveness to estradiol negative feedback decreases, causing increases in lh pulse frequency, follicle growth, and estradiol secretion. the decline in estradiol negative feedback and increase in lh secretion promote ovarian follicular growth, and elevated concentrations of estradiol sufficient to induce estrus and the preovulatory lh surge. reproductive tract scores of 4 and 5 are assigned to heifers that have reached puberty, but differ in stage of the estrous cycle at the prebreeding exam (follicular phase = 4; luteal phase = 5). heifers with higher rts achieve higher ai and breeding season pregnancy rates and become pregnant earlier in the breeding season compared to heifers with lower rts.93 temperament score in cows and heifers temperament is a reaction characteristic of cattle to human handling. cattle that remain calm perform better than those that are excitable during handling. in general, excitable temperament has detrimental effects on growth, carcass quality and health of beef cattle. the hypothalamic-pituitary-adrenal axis is exquisitely sensitive to physiological and psychological insults. secretion of glucocorticoids is the classic endocrine response to stress. however, broad endocrine changes occur in response to stress. within seconds to minutes after the onset of stress were increases in catecholamines, cortisol releasing hormones and acth and decreases in gnrh, gonadotropins, prolactin and glucogan secretion.94-96 in addition over hours to days, gonadal steroid hormones decline; these changes inhibit reproductive physiology and behavior, and decrease feeding and appetite. various scoring systems (1-5 or 6 point, or 2 point) have been developed to measure temperament.97-99 current techniques include chute score, flight speed and exit score. calm cows100 and heifers99 had higher ai and breeding season pregnancy rates and become pregnant earlier in the breeding season compared to cattle with excitable temperament. cattle facility design influenced temperament and thus affected reproductive performance.99 cattle handling facility design by temperament group interactions significantly influenced progesterone, cortisol, prolactin and substance-p concentrations.99 inter and intrarater agreements for 2 point temperament scoring were moderate and good. the predictive value for calm and pregnant to ai was 0.87, and excited and nonpregnant to ai was 0.76.99 body condition score in beef cows and heifers the body condition score (bcs) is a 1-9 point (1 – emaciate and 9 – obese) scoring system101 based on visual observation of muscle and fat cover in the area of ribs/thorax, transverse processes, back and gluteal regions and tail head and perineal regions. the bcs is an indirect measure of nutitional status of cows. the bcs can be categorized as thin < 5, moderate – 5, good – 6 and 7, and obese > 7. it is preferrable to feed cattle to have a bcs of 5 at calving and maintained at bcs 5 until breeding.102 it should be noted that bcs has better correlation in beef cows than beef heifers. cows with thin bcs took longer to resume cyclicity after calving due to lower concentrations of igf, estrogen and lh.103,104 adiponectin and leptin are also lower in thin cows.105 thin and obese cows had lesser estrus expression, and lower ai and breeding season pregnancy rates compared to cows with moderate to good bcs.106 cows that maintained or gained body condition after breeding, during first 2 months of pregnancy, had increased pregnancy rates compared to cows that lost body condition.107 conclusion estrus/ovulation synchronization and an ai program are excellent tools that should be planned in collaboration with a veterinarian well-experienced in reproductive management and implemented by experienced personnel who can adhere to the protocol and breeders who can breed many cattle concurrently. the investment of time in selecting ai and ns bulls and carefully following protocols should reduce the calving interval and produce more uniform calves with genetic advantages, making them more marketable and improving profitability. however, sycnhronization will not solve all breeding or management problems. so, the key for success is consideration of all factors and options and making evidence-based decisions. acknowledgments authors acknowledge college of veterinary medicine, washington state university, pullman, wa, usa for the support and thank dr. john kastelic (university of calgary) for reviewing the manuscript. conflict of interest authors declare that they have no conflict of interest. table 7. reproductive tract scoring system based on the size of uterus and ovarian structures tract score uterine horns ovarian structures 1 immature, < 20 mm diameter, no tone no palpable structures 2 20-25 mm diameter, no tone 8 mm follicles 3 20-25 mm diameter, slight tone 8-10 mm follicles 4 30 mm diameter, good tone > 10 mm follicles, possibly cl 5 > 30 mm diameter cl http://dx.doi.org/10.58292/ct.v17.11650 20 citation: clinical theriogenology 2025, 17, 11650, http://dx.doi.org/10.58292/ct.v17.11650 references 1. nahms. part iii: reference of 1997 beef cow-calf production management and disease control. 1998. p. 9-11. available from: https://www.aphis.usda.gov/animal_health/nahms/beefcowcalf/downloads/beef97/beef97_dr_partiii.pdf [cited 22 september 2023]. 2. nahms. part ii: reference of beef cow-calf management 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theriogenology 2011;3:29-41. 106. whitman rh. weight changes, body condition and beef-cow reproduction. phd thesis. colorado state university, fort collins, co, usa, 1975. 107. ciccioli nh, wettemann rp, spicer lj, et al: influence of body condition at calving and postpartum nutrition on endocrine function and reproductive performance of primiparous beef cows. j anim sci 2003;81:3107-3120. doi: 10.2527/ 2003.81123107x http://dx.doi.org/10.58292/ct.v17.11650 https://doi.org/10.1016/0378-4320(92)90123-u https://doi.org/10.1016/0378-4320(93)90104-y https://doi.org/10.1016/j.anireprosci.2009.12.011 https://doi.org/10.2527/jas.2006-587 https://extension.sdstate.edu/sites/default/files/2020-10/p-00189.pdf https://extension.sdstate.edu/sites/default/files/2020-10/p-00189.pdf https://doi.org/10.2527/jas.2010-3564 https://doi.org/10.1016/j.theriogenology.2014.01.008 https://doi.org/10.1016/j.theriogenology.2014.01.008 https://doi.org/10.1016/s0739-7240(02)00146-7 https://doi.org/10.1016/s0739-7240(02)00146-7 https://doi.org/10.1210/edrv.21.1.0389 https://doi.org/10.2527/1997.751249x https://doi.org/10.2527/1997.754892x https://doi.org/10.1111/rda.12368 https://doi.org/10.1111/rda.12436 https://doi.org/10.2527/2006.8441038x https://doi.org/10.2527/1990.683799x https://doi.org/10.2527/1990.683799x https://doi.org/10.1016/j.livsci.2014.10.008 https://doi.org/10.1016/j.livsci.2014.10.008 https://doi.org/10.2527/2003.81123107x 2016: case report: focal squamous cell carcinoma, extensive squamous dysplasia and balanitis of the penis in a stallion case report: focal squamous cell carcinoma, extensive squamous dysplasia and balanitis of the penis in a stallion kristen d. loncar, patrick m. mccue, dean a. hendrickson, ryan a. ferris department of clinical sciences, college of veterinary medicine and biomedical sciences, colorado state university, fort collins, co summary an 18 year old quarter horse stallion was presented with a copious amount of dried purulent debris adhered to his penis. removal of the surface debris revealed a focal squamous cell carcinoma lesion on the glans penis and extensive squamous dysplasia along the shaft of the penis. the focal lesion was removed surgically. additional therapeutic options were discussed. keywords: stallion, penis, squamous cell carcinoma, tumor background an 18 year old american quarter horse stallion had been turned out with a band of mares for three months in 2013 in a pasture breeding program. the owner noticed at the time of turn-out that the horse had a significant amount of debris and areas of discoloration on the shaft of the penis. the stallion was presented to the equine reproduction laboratory at colorado state university prior to the onset of the 2014 breeding season for evaluation of his penis and a concern about the ability of the horse to continue to live cover mares. no difficulties in breeding were observed by the owner during the previous (2013) breeding season. pregnancy rate for the 2013 breeding was not available. case presentation the stallion was in good body condition and weighed 577 kg at presentation. he showed good libido when teased to a mare in estrus. he dropped his penis and gained an erection which allowed for examination of the suspected penile lesions. a layer of dried purulent material was present along the shaft of the penis (figure 1). the exudate was gently rinsed off with warm water and multiple areas of depigmentation and thickening were noted scattered along the penis distal to the preputial ring (figure 2). in addition, a proliferative, raised red mass approximately 1 cm in diameter was visible on the dorsal aspect of the glans penis (figure 3). several unsuccessful attempts were made to collect semen from the stallion using a colorado model artificial vagina. however, the stallion would mount the jump mare, thrust into the artificial vagina several times, but dismount without ejaculation during each collection attempt. pharmacologically induced ex copula ejaculation (commonly called‘chemical ejaculation’) was subsequently performed. the stallion was administered a dose of imipramine hydrochloride orally (3 mg/kg) followed two hours later by intravenous administration of xylazine hydrochloride (0.26 mg/kg).1 the stallion ejaculated into a collection bottle approximately 12 minutes after administration of the xylazine. semen analysis revealed a volume of 53 ml and a spermatozoal concentration of 163 million/ml, for a total of 8.64 billion spermatozoa. sperm motility was analyzed using a computer-assisted sperm analysis (casa) system (spermvision®; minitube usa, delavan, wi) and was determined to consist of 74% total motility and 63% progressive motility. sperm morphology was evaluated using an eosinnigrosin stain.2 a total of 100 spermatozoa were evaluated on each of two stained slides and 73% of the spermatozoa were noted to be morphologically normal. the owner declined further diagnostic tests to evaluate the lesions on the penis. the stallion was initially turned out on pasture with four mares in the month of april and then turned out with a different group of 14 mares in early may and removed from pasture after approximately 90 days. 5461676363 clinical theriogenology · volume 8, number 1 · march 2016 pregnancy evaluation transrectal ultrasonic pregnancy examinations were performed at the ranch on the group of 14 mares three weeks after the stallion was removed from the pasture. nine of the 14 mares were determined to be pregnant. three of the five non-pregnant mares were two years old. the owner reported that all four other mares were confirmed by ultrasonography to be pregnant by another veterinarian. overall, the stallion achieved pregnancies in 13 of the 18 (72.2%) mares he was turned out with in 2014. further diagnostic tests the stallion was returned to the clinic in early january 2015 for additional diagnostic tests on the penile lesions. he was sedated with a combination of 5 mg detomidine hydrochloride and 5 mg butorphanol tartrate, administered intravenously, and placed in examination stocks. culture swabs were collected from the shaft of the penis and urethral fossa and submitted for microbial culture. the penis was washed with a non-residual soap (ivory® liquid; procter and gamble, cincinnati, oh) and rinsed with warm water. lidocaine hydrochloride was infused subcutaneously and biopsy samples were collected using a scalpel blade from the raised red lesion on the glans penis and three sites along the shaft of the penis in areas that appeared thickened and depigmented. all biopsy sites were subsequently closed with 3-0 monofilament absorbable suture on a taper needle (biosyntm, coviden, dublin, ireland). samples were submitted to the colorado state university veterinary diagnostic laboratory for histopathologic evaluation. the stallion was administered a single 6.6mg/kg dose of ceftiofur crystalline free acid (ccfa) (excede®; zoetis, florham park, nj), intramuscularly and 500 mg flunixin meglumine, intravenously, after the biopsy procedures. microbial culture of samples collected from the penile shaft and urethral fossa yielded moderate growth of beta hemolytic streptococcus equi subspecies zooepidemicus and heavy growth of trueperella pyogenes (formerly known as corynebacterium pyogenes and actinomyces pyogenes). histopathologic evaluation of the three penile shaft biopsies indicated moderate to severe lymphoplasmacytic and eosinophilic balanitis with marked acanthosis and dysplasia which were interpreted as pre-neoplastic changes. the biopsy of the glans penis lesion revealed epithelial cells that formed clumps, masses and areas of squamous differentiation with formation of squamous pearls (figures 4 and 5). nuclei were mildly pleomorphic, round and had prominent nucleoli. mitoses were noted in two to three cells per high-powered field (figure 6). the histopathologic diagnosis for the glans penis lesion was squamous cell carcinoma (scc). treatment the first step in the treatment plan was to reduce the chronic infectious and inflammatory processes within the sheath and on the surface of the penis and the second step was to remove the tumor on the glans penis. the stallion was administered trimethoprim-sulfamethoxazole at a dose of 30 mg/kg orally twice daily for ten days. he was also administered prednisolone, starting with a dose of 1.0 mg/kg orally once daily for four days, which was subsequently tapered to 0.5 mg/kg for four days and then 0.25 mg/kg for two additional days. the horse was also administered a dose of ceftiofur crystalline free acid (6.6 mg/kg, intramuscularly) followed by an additional dose four days later. the stallion was sedated with xylazine hydrochloride (0.3 mg/kg) every second day and his penis was gently washed with warm water, rinsed with hypertonic saline and then coated with a layer of silver sulfadiazine cream. after ten days of therapy, the stallion was sedated with a combination of 5 mg detomidine hydrochloride and 5 mg butorphanol tartrate administered intravenously, and restrained in examination stocks. a ring block was performed using a 22 gauge 1.5 inch needle inserted at six separate sites to infuse a total volume of 20 mls of lidocaine subcutaneously in a circumferential ring around the penile shaft midway between the preputial ring and the glans penis (figure 7). three mls of lidocaine 5562686464clinical theriogenology · volume 8, number 1 · march 2016   hydrochloride was infused around and beneath the lesion on the glans penis. the lesion was removed using an elliptical incision approximately 5 mm from the margin of the mass and sufficiently deep to remove the entire mass (figure 8). the incision was closed in two layers using 3-0 monofilament absorbable suture (figure 9). the horse was subsequently administered 500 mg flunixin meglumine and maintained on 1 gram of phenylbutazone orally for an additional two days. outcome two days after the initial procedure, the stallion was again sedated with xylazine hydrochloride to evaluate the surgical area. there was no evidence of swelling, hemorrhage or dehiscence, and the stallion was sent home with instructions to remain on stall rest for two weeks and to remain isolated away from mares to minimize sexual stimulation and development of an erection in order to prevent complications at the surgery site. histopathology of the glans penis mass confirmed the initial diagnosis of squamous cell carcinoma. it was also noted that the tissue margins were comprised of normal tissue, indicating that the tumor had been excised completely. additional therapeutic options were discussed for management of the multiple and extensive dysplastic skin lesions on the shaft of the penis. practical options included topical administration of a chemotherapeutic agent, such as 5-fluorouracil, and long-term oral administration of a cyclooxygenase-2 (cox-2) inhibitor, such as peroxicam or firocoxib. the owner elected to not treat the penile shaft lesions at the current time. the primary factors that led to the decision were that the lesions had been determined histologically to be dysplastic and had not as yet progressed to actual squamous cell carcinoma (even though dysplasia is considered to be a precursor stage to scc), and the stallion was housed on a large pasture and daily administration of an oral medication would be challenging to accomplish. ultimately the owner decided to use the stallion in a pasture breeding program again this year and return him to the clinic for re-examination at the end of the breeding season with the possibility of starting additional therapies at that time. discussion the most commonly reported neoplasms of the penis and prepuce are sarcoids, squamous cell papillomas, carcinomas and melanomas.3 squamous cell carcinoma is a tumor arising from squamous epithelial cells. the penis, prepuce and urethral process are considered to be predisposed sites for development of squamous cell carcinoma.3-5 more specifically, the glans penis has been reported to be involved in 53 to 84% of cases of equine penile and preputial squamous cell carcinoma.6-8 penile scc lesions in horses less than eight years of age tend to be aggressive and commonly metastasize to regional lymph nodes.9,10 factors that predispose horses to development of scc of the penis and prepuce include lack of pigmentation on the genitalia, exposure to the ultraviolet component of solar radiation, infection with equus caballus papillomavirus 2 (ecpv-2),11-13 smegma,14 and poor hygiene which leads to chronic irritation and balanoposthitis.15 infection with ecpv-2 is currently thought to be the main cause for development of scc of the penis and prepuce in the horse.16,17 the condition primarily affects adult horses,6,15,18 with an average age of 18.9 years (range 13 to 30) noted in one report.3 other commonly affected areas include the periorbital and perineal regions. squamous cell carcinoma lesions are often locally invasive and multicentric, metastasize slowly to regional lymph nodes and occasionally metastasize to distant sites such as lung, liver and thoracic lymph nodes.3,7 metastasis to the inguinal lymph nodes for cases of scc of the equine genitalia has been reported to range from 12.5 % to 16.9 %.6,19 clinical signs of scc range from hemospermia to depigmented plaques or non-healing erosive lesions to solid masses with a cauliflower-like appearance.19 diagnosis may be based on physical examination, visual inspection, ultrasonography to determine extent and invasiveness of the tumor, fine needle aspiration or, preferably, a full thickness biopsy. ideally, the biopsy should include the lesion and 5663696565 clinical theriogenology · volume 8, number 1 · march 2016   interface with normal appearing skin. transrectal palpation and possibly thoracic ultrasonography and radiography are also recommended in malignant cases to ascertain metastasis.9 differential diagnoses for scc lesions include sarcoid, papilloma, mast cell tumor, exuberant granulation tissue (‘proud flesh’), habronemiasis, phycomycosis, cutaneous lymphoma, adenocarcinoma/adenoma, neurofibroma, posthitis/balanoposthitis, foreign body reactions and melanoma.9,10 treatment options include surgical procedures, non-surgical interventions or a combination of surgical debulking or debridement followed by a non-surgical therapy. non-surgical interventions include cryotherapy, radiofrequency hyperthermia, radiation therapy, photodynamic therapy, topical or intratumoral chemotherapy, immunomodulators, and other treatments. local or intralesional therapy may be beneficial in reducing systemic side effects of treatments. surgical options are often dictated by the size, anatomic location, invasiveness and local or regional extent of the tumor. a surgical margin of at least 0.5 to 1.0 cm between the tumor and normal tissue is recommended.10 small tumors may only require local resection, but there is a risk of recurrence with local excision.18 larger or more invasive tumors, such as a tumor that has invaded the urethra, tunica albuginea, corpus cavernosum or corpus spongiosum of the penis may require segmental posthioplasty (‘reefing’), partial phallectomy or en bloc penile and preputial resection with penile retroversion.8,20 mair and colleagues6 reported that four of 24 horses that underwent penile amputation for lesions on the glans penis were ultimately euthanized due to recurrence of the tumor. an overall success rate of 55.7 % was reported for stallions with penile and preputial scc.8 small scc lesions can be eliminated by laser ablation3 with a neodymium:yttrium-aluminum garnet, gallium-aluminum-arsenide diode or carbon dioxide laser. lasers vaporize cells along the incision site as well as cause coagulation of small blood vessels, lymphatic vessels and nerves21,22 which leads to less hemorrhage and post-operative pain. mccauley23 reported the use of a carbon dioxide laser in nine horses with squamous cell carcinoma, of which two cases resulted in recurrence of the tumor. cryotherapy involves direct application of liquid nitrogen (-196°c) by spray or probe to the tumor to cool the cells to tumoricidal temperatures of -20 to -40°c for a period of one minute.24,25 repeated cycles of quick freezing and slow warming leads to cellular injury, vascular damage and eventually cell death. in human medicine, a cure rate of 97.3% has been reported following the use of cryotherapy if the tumor is small.26 cryotherapy can be used alone or after surgical debulking of the tumor. radiofrequency hyperthermia involves use of a probe that emits radio waves which create heat inserted directly into a tumor. grier et al27 reported that 80% of bovine and equine ocular squamous cell carcinomas had complete regression and 16% had partial regression of the tumor. the authors concluded that radiofrequency hyperthermia was easy to administer, but not as effective for large tumors as compared with small tumors. hyperthermia can be used as an adjunctive therapy after surgical debulking a scc tumor. radiation therapy has been used extensively in cancer therapy in humans and animals. brachytherapy involves the use of a sealed radiation source such as iridium-192, iodine-125 or strontium90 enclosed in a protective capsule, beads, seeds, pins or wires implanted directly within the target tissue to delivery continuous radiation to the site.28,29 ionizing radiation from the source only effects cells in the local surrounding tissue. brachytherapy can be used alone for small scc lesions or as an adjunctive therapy following surgical debulking of a tumor. wyn-jones30 reported a 100% success rate when using iridium pins to treat a variety of tumors in the horse. mosunic and colleagues31 noted that adjuvant radiation therapy along with the primary treatment was associated will less recurrence of ocular and adnexal scc than primary treatments alone. in contrast, teletherapy or external beam radiotherapy refers to the use of an external radiation source, such as strontium 90, cobalt 60 or other source, to deliver a controlled amount of radiation from a remote source to the affected tissue for a selected period of time.28 radiation has been used in the horse to treat many types of neoplasia, including squamous cell carcinoma, fibrous connective tissue tumors, 5764706666clinical theriogenology · volume 8, number 1 · march 2016   sarcoids, nerve cell tumors, and solitary lymphomas.32 side effects noted with various types of radiation therapy in the horse have included alopecia, delayed healing and infection. photodynamic therapy involves the activation of a photosensitizer drug (a porphyrin compound) with light which results in the formation of reactive and cytotoxic singlet oxygen.33 photosensitizers are preferentially absorbed by proliferating tumor cells, which can then be destroyed when light is delivered to a highly specific area.10 photodynamic therapy is advantageous in that it is non-invasive and effective when treating a large surface area. giuliano and colleagues34 reported that horses with periocular squamous cell carcinoma treated with excision and photodynamic therapy did not exhibit recurrence of the tumor, while 78.5% of horses treated with cryotherapy after excision exhibited recurrence of the tumor. cisplatin (cis-diammine dichloroplatinum) is a broad spectrum chemotherapeutic drug that works by crosslinking dna and interfering with mitotic cell division. damaged cells undergo apoptosis once repair mechanisms fail.35 cisplatin can be mixed with purified medical grade sesame oil to a concentration of 3.3 mg/ml and injected directly into the tumor once every two weeks for a total of four treatments.36 alternatively, biodegradable beads containing cisplatin has also been used in the treatment of equine scc as a slow-release delivery system.37 results often depend on initial size of the tumor and larger tumors (i.e. > 1.5 cm) should be debulked prior to cisplatin treatment. cisplatin can be injected or implanted along the margins of the excision site after debulking or tumor removal. théon and others38 reported a success rate of 88% for horses with squamous cell carcinoma lesions treated with one course of intralesional cisplatin. hewes and sullins37 recommended three applications of cisplatin beads at one month intervals for equine scc. 5-fluorouracil (5-fu) is a fluoropyrimidine that works by incorporation of its metabolites into dna and rna, which leads to improper replication and inhibition of the nucleotide synthetic enzyme thymidylate synthase39 disruption of dna synthesis causes rapidly dividing cancer cells to undergo cell death, whereas normal epithelium adjacent to the tumor remains intact with minimal inflammation. 5-fu is available as a topical cream and a sterile injectable solution and is an alternative to other forms of local chemotherapy.10 paterson40 reported that two horses with facial scc remained in remission after a 30day course of topical 5-fu, while a third horse was controlled by topical administration of 5-fu for seven days every six weeks. fortier and macharg41 described the use of 5-fu cream topically as a treatment for non-metastasized sccs. penile lesions were treated every 14 days with a mean number of five treatments (range two to seven) and treatment was noted to result in complete regression of the lesions. debulking or laser surgery is recommended on large scc tumors prior to 5-fu treatment. mitomycin c is an antimicrobial agent with anti-tumor properties that inhibits dna synthesis and replication.42,43 it has the potential to selectively attack the hypoxic cell component of tumors, which can be advantageous against larger tumors.44 it has been used topically to treat ocular squamous cell carcinoma in the horse, with curative rates reported to be 75% when used alone45 or 90% when used in conjunction with carbon dioxide laser ablation.43 bleomycin is a glycopeptide antitumor agent produced by the bacterium streptomyces verticillus. the mechanism of action is breakage of dna strands. bleomycin has been evaluated in the treatment of periocular scc in horses, but was not as effective when compared to cisplatin.46 piroxicam is a non-steroidal anti-inflammatory drug (nsaid) which suppresses prostaglandin production by inhibiting the cyclooxygenase (cox) pathway. most equine scc tumors express cox-247 and it has been suggested that cox-2 derived prostaglandins may be responsible for tumor growth, angiogenesis and metastasis.48,49 consequently, administration of piroxicam or other cox-2 inhibitors may be beneficial in management of tumors through induction of apoptosis in tumor cells, inhibition of angiogenesis or inhibition of prostaglandin e2 production.47,50-51 piroxicam has been used in the management of squamous cell carcinomas of the lip, face and third eyelid of horses.50-52 immunotherapy involves stimulation of the immune system to reject or destroy cancer cells. bacillus calmette-guerin (bcg), a cell wall extract developed from mycobaterium bovis, has been the most commonly used immunotherapy agent in large animal veterinary medicine. in the horse, bcg has been used in the treatment of sarcoids11 and periocular squamous cell carcinoma in a pony.53 bacillus 5865716767 clinical theriogenology · volume 8, number 1 · march 2016   calmette-guerin has also been used in the treatment of bovine ocular scc, with complete regression of the primary tumor noted in 70% of cases treated with bcg cell wall and 60% regression in cases treated with a live bcg vaccine.54 chemotherapeutic drugs have largely replaced bcg therapy in the horse for the treatment of scc.55 tumor location, size, extent of lymph node involvement, presence of metastases, degree of invasiveness and histopathologic appearance combine to dictate grade and stage of an equine scc. prognosis for a case of scc in a horse is based on the aforementioned features and recurrence of the tumor if it had been treated. a majority of tumors that recur do so within a year and the overall rate of recurrence ranges from 11 to 30%.17 overall, the prognosis is guarded in all cases other than small lesions detected early. in general, penile scc lesions are usually managed by a combination of surgical excision, debulking or amputation potentially in combination with cryotherapy or topical application of 5-fu. a majority of the other treatments described have been used in other anatomical sites (i.e. periocular region) or have not been reported for management of penile scc. in the present case, surgical excision was chosen for treatment because of the location and small size of the tumor. overall a guarded to poor prognosis was given for long-term health of the stallion. it is anticipated that additional squamous cell carcinoma lesions will be identified in this horse in the next few years due to the extensive pre-cancerous squamous dysplasia lesions identified on the penile shaft. the immediate goal of the stallion manager was to be able to utilize the stallion in a pasture breeding program during the upcoming summer months. the future goal is to collect semen for cryopreservation in the event that the stallion is not able to live cover mares. summary an aged stallion was presented with a focal squamous cell carcinoma lesion glans penis, multiple squamous dysplasia lesions on the penile shaft and a secondary balanitis associated with a mixed infection of streptococcus equi subspecies zooepidemicus and trueperella pyogenes. the infection was treated with a combination of local cleansing, topical antiseptics and systemic antibiotics. the scc lesion on the glans penis was completely surgically excised. other options for continued future therapy are discussed. learning points  squamous cell carcinoma is one of the most common neoplasms affecting the penis and prepuce of the adult horse.  tumors are usually locally invasive and metastasize slowly to the inguinal lymph nodes  medical management options include cryotherapy, radiofrequency hyperthermia, radiation therapy, photodynamic therapy, topical or intratumoral chemotherapy, and immunomodulators  tumor recurrence ranges from 11-30% and prognosis is usually guarded unless lesion is small and detected early references 1. mcdonnell sm: oral imipramine and intravenous xylazine for pharmacologically-induced ex copula ejaculation in stallions. anim reprod sci 2001;68:153-159. 2. cecere jt: eosin-nigrosin staining in the evaluation of sperm. in: dascanio j, mccue p, editors. equine reproductive procedures. ames (ia): wiley blackwell; 2014. p. 373-376. 3. edwards jf: neoplasia of the reproductive tract. in: mckinnon ao, squires el, vaala we, et al, editors. equine reproduction. ames (ia):blackwell publishing ltd, 2011. p. 1166-1171. 4. schaffer pa, wobeser b, martin ler, et al: cutaneous neoplastic lesions of 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instrumentation and techniques for carbon dioxide laser in equine surgery. vet clin north am equine pract 1996;12:397-414. 23. mccauley ct, hawkins jf, adams sb, et al: use of a carbon dioxide laser for surgical management of cutaneous masses in horses: 32 cases (1993-2000). j am vet med assoc 2002;220:1192-1197. 24. alam m, ratner d: cutaneous squamous cell carcinoma. ne j med 2001;344:983. 25. theodorescu d: cancer cryotherapy: evolution and biology. rev urol 2004;6(suppl 4):s9-s19. 26. graham g, clark l: statistical analysis in cryosurgery of skin cancer. clin dermatol 1990;8:101-107. 27. grier rl, brewer wg, paul sr, et al: treatment of bovine and equine ocular squamous cell carcinoma by radiofrequency hyperthermia. j am vet med assoc 1980;177:55-61. 28. henson fmd, dobson jm: use of radiation therapy in the treatment of equine neoplasia. equine vet educ 2004;16:315-318. 29. théon ap, pascoe jr: iridium-192 interstitial brachytherapy for equine periocular tumours; treatment results and prognostic factors in 115 horses. equine vet j 1995;27:117–121. 30. wyn-jones g: treatment of equine cutaneous neoplasia by radiotherapy using iridium 192 linear sources. equine vet j 1983;15:361-5. 31. mosunic cb, moore pa, carmicheal kp, et al: effects of treatment with and without adjuvant radiation therapy on recurrence of ocular and adnexal squamous cell carcinoma in horses: 157 cases (1985–2002). j am vet med assoc 2004;225:1733-1738. 32. blackwood l, dobson jm: radiotherapy in the horse. equine vet educ 1994;6:95-99. 33. ericson mb, wennberg a, larko o: review of photodynamic therapy in actinic keratosis and basal cell carcinoma. ther clin risk manag 2008;4:1-9. 34. guiliano ea, johnson pj, delgado c, et al: local photodynamic therapy delays recurrence of equine periocular squamous cell carcinoma compared to cryotherapy. vet ophthal 2014;17(suppl 1):37-45. 35. eastman a: the mechanism of action of cisplatin: from adducts to apoptosis. in: lippert b, editor. cisplatin: chemistry and biochemistry of a leading anticancer drug. wiley online library; 1999. chapter 4. 36. théon ap, pascoe jr, carlson gp, et al: intratumoral chemotherapy with cisplatin in oily emulsion in horses. j am vet med assoc 1993;202:261-267. 37. hewes ca, sullins ke: use of cisplatin-containing biodegradable beads for treatment of cutaneous neoplasia in equidae: 59 cases (2000–2004). j am vet med assoc 2006;229:1617-1622. 38. théon ap, wilson wd, magdesian kg, et al: long-term outcome associated with intratumoral chemotherapy with cisplatin for cutaneous tumors in equidae: 573 cases (1995-2004). j am vet med assoc 2007;230:1506-1513. 39. longley db, harkin dp, johnston pg: 5-fluorouracil: mechanisms of action and clinical strategies. nat rev cancer 2003;3:330-338. 40. paterson s: treatment of superficial ulcerative squamous cell carcinoma in three horses with topical 5-fluorouracil. vet rec 1997;141:626-628. 6067736969 clinical theriogenology · volume 8, number 1 · march 2016   41. fortier la, macharg ma: topical use of 5-fluorouracil for treatment of squamous cell carcinoma of the external genitalia of horses: 11 cases (1988-1992). j am vet med assoc 1995;205:1183-1185. 42. nakata y, nakata k, sakamoto y: on the action mechanism of mitomycin c. biochem biophys res commun 1961;6:339-343. 43. rayner sg, van zyl n: the use of mitomycin c as an adjunctive treatment for equine ocular squamous cell carcinoma. aust vet j 2006;84:43-46. 44. kennedy ka, rockwell s, sartorelli ac: preferential activation of mitomycin c to cytotoxic metabolites by hypoxic tumor cells. cancer res 1980;40:2356-2360. 45. malalana f, knottenbelt d, mckane s: mitomycin c, with or without surgery, for the treatment of ocular squamous cell carcinoma in horses. vet rec 2010;167:373-376. 46. théon ap, pascoe jr, madigan je, et al: comparison of intratumoral administration of cisplatin versus bleomycin for treatment of periocular squamous cell carcinomas in horses. am j vet res 1997;58:431-436. 47. thamm dh, ehrhart ej, charles jb et al: cyclooxygenase-2 expression in equine tumors. vet pathol 2008;45:825-828. 48. leahy km, ornbert rl, wang y et al: cyclooxygenase-2 inhibition by celecoxib reduces proliferation and induces apoptosis in angiogenic endothelial cells in vivo. cancer res 2002;62:625-631. 49. masferrer jl, leahy mk, koki at et al: antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors. cancer res 2000;60:1306-1311. 50. moore as, beam sl, rassnicks km, et al: long-term control of mucocutaneous squamous cell carcinoma and metastases in a horse using piroxicam. equine vet j 2003;35:715-718. 51. iwabe s, ramírez-lópez l, juárez-sánchez, m: the use of piroxicam as an adjuntive treatment for squamous cell carcinoma in the third eyelid of a horse. vet méx 2009;40:389-395. 52. dória rgs, laskoski lm, carvalho mb, et al: use of chemotherapy as treatment of squamous cell carcinoma with metastasis in the face of a horse case report. rev bras med vet 2012;34:336-340. 53. mccalla tl, moore cp, collier ll: immunotherapy of periocular squamous cell carcinoma with metastasis in a pony. j am vet med assoc 1992;200:1678-1681. 54. klein wr, ruitenberg j, steerenberg pa, et al: immunotherapy by intralesional injection of bcg cell walls or live bcg in bovine ocular squamous cell carcinoma: a preliminary report. j natl cancer inst 1982;69:1095-1103. 55. hendrix dvh: equine ocular squamous cell carcinoma. clin tech equine pract 2005;4:87-94. figure 1. shaft of the penis prior to washing. 6168747070clinical theriogenology · volume 8, number 1 · march 2016 figure 2. shaft of the penis after washing showing with multiple areas of depigmentation. figure 3. raised proliferative lesion on glans penis (arrow) and areas of thickened and discolored epithelium (arrowhead). 6269757171 clinical theriogenology · volume 8, number 1 · march 2016   figure 4. histology of the squamous cell carcinoma lesion on the glans penis showing characteristic nesting of tumor cells and invasion into the submucosa. figure 5. squamous pearl. 6370767272clinical theriogenology · volume 8, number 1 · march 2016   figure 6. cell undergoing mitosis in the squamous cell carcinoma. figure 7. placing a ring block around distal end of penis. 6471777373 clinical theriogenology · volume 8, number 1 · march 2016   figure 8. surgical excision of the tumor mass on the glans penis. figure 9. suturing the defect in the glans penis after removal of the tumor mass. 6572787474clinical theriogenology · volume 8, number 1 · march 2016 1 contact marcilio nichi mnichi@usp.br © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12690, http://dx.doi.org/10.58292/ct.v17.12690 research report effects of nonenzymatic and enzymatic antioxidants in cryopreserved canine semen extended with docosahexaenoic acid roberto rosa filho*, nívea vieira*, maíra brito, joão losano, giulia kawai, camilla mendes, bruno rui, ken nagai, álvaro alves, henrique frias, raphaela sousa, fábio sanches, mayra assumpção, marcilio nichi department of animal reproduction, school of veterinary medicine and animal science, university of são paulo, são paulo, sp, brazil abstract polyunsaturated fatty acids (pufas), such as docosahexaenoic acid (dha), could be an alternative to improve cryopreserved canine sperm by increasing membrane fluidity. however, pufas also increase sperm susceptibility to lipid peroxidation. supplementation of the extender with dha and antioxidants could be an alternative. we evaluated supplementation of antioxidants (vitamin e and catalase) of cryopreserved canine semen extended with dha. in experiment 1, ejaculates from 8 dogs were divided into 4 aliquots, with increasing concentrations of dha in the extender (0, 1, 5, and 10 µm) and cryopreserved. sperm motility increased in 5 µm dha and it was used for experiment 2; ejaculates from 8 dogs were divided into 4 groups: control (5 µm dha), vitamin e + dha, catalase + dha, and vitamin e + catalase + dha. samples were evaluated for susceptibility to lipid peroxidation, sperm kinetics, mitochondrial function, dna integrity, and plasma and acrosomal membrane integrity. vitamin e + dha group had beneficial effects on the characteristics of sperm kinetics whereas the catalase + dha group was harmful to sperm mitochondria. vitamin e + catalase + dha group decreased oxidative stress and damage to the plasma membrane and had low mitochondrial membrane activity. addition of dha + vitamin e in canine semen extender improved sperm quality after cryopreservation. keywords: lipid peroxidation, oxidative stress, dog, sperm kinetic, polyunsaturated fatty acid introduction growing interest in increasing reproductive efficiency and societal value of dogs has led to a greater search for reproductive biotechnologies. thus, male gamete preservation is a valuable tool since semen cryopreservation enables and facilitates its transport, storage for prolonged periods, and cost reduction.1 during spermatogenesis, sperm plasma membrane and other specific structures are prepared to react adequately to female genital tract and oocyte.2 sperm membrane is composed of a bilayer of lipids, mainly polyunsaturated fatty acids (pufas), especially docosahexaenoic acid (dha) that are of great importance to sperm, as they provide necessary fluidity characteristics for events associated with sperm permeability and fertilization,3 such as motility and acrosomal reaction.4 plasma membrane fluidity depends on many factors, such as temperature, cholesterol content, and the degree of saturation of the carbon bonds.2 saturated fatty acids guarantee greater rigidity whereas unsaturated ones provide greater fluidity to the membrane due to double bonds,5 an essential characteristic for reproductive events. in addition, lipid composition of sperm membrane in several species influences resistance to cold shock during refrigeration and cryopreservation.6 therefore, polyunsaturated fatty acids are also directly involved in protecting the membrane during cryopreservation.7,8 an alternative would be to supplement the samples with pufas during cryopreservation, protecting sperm against cryoinjury.9 however, the high percentage of pufas makes sperm susceptible to oxidizing agents, leading to lipid peroxidation (due to their large amount of unsaturation, they are more easily oxidized).5 additionally, for canine semen preservation, it is necessary to remove seminal plasma, and with that important antioxidants are removed.10 when reactive oxygen species (ros) act on the unsaturated fatty acids’ double bonds, a lipid peroxidation chain reaction begins and, if not interrupted, leads to loss of membrane permeability and, consequently, sperm function.11 based on these assertions, sperm *cofirst authors mailto:mnichi@usp.br http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12690 2 citation: clinical theriogenology 2025, 17, 12690, http://dx.doi.org/10.58292/ct.v17.12690 treatment with pufas during cryopreservation could provide ambiguous results, but if pufas are incorporated into the sperm membrane, this would make it even more susceptible to ros attack. thus, a plausible alternative would be the association between pufas and antioxidants during sperm cryopreservation, aiming to prevent the enhancement of oxidative events.12 enzymatic and nonenzymatic antioxidants have specificity for certain oxidizing agents; therefore, prior to antioxidant treatment, it is essential to detect which ros the sperm is most susceptible to, in order to perform a specific antioxidant treatment to eliminate this ros.13 also, hydrogen peroxide and hydroxyl radical were the most harmful ros for canine sperm.13 therefore, for an efficient antioxidant treatment, an association between enzymatic and nonenzymatic antioxidants would quell the aforementioned ros.13 catalase and vitamin e are examples of antioxidants that are extremely effective in neutralizing hydrogen peroxide and hydroxyl radical, respectively.14–16 as the largest lipid-soluble antioxidant of sperm, vitamin e (α-tocopherol) acts on the plasma membrane;17 it protects the double bond of unsaturated fatty acids against chain oxidation, donating electrons to a hydroxyl radical.18 it is the most efficient antioxidant to break this chain, reacting with the peroxyl radical, thereby preventing the reaction of this radical with another unsaturated fatty acid.19 it can also act as a membrane stabilizing agent, forming complexes with its phospholipids.20 because of its lipophilic and antioxidant properties, it has a vital role in protecting biological membranes from peroxidative damage.19 catalase is part of an enzyme system that degrades hydrogen peroxide into oxygen and water.21 hydrogen peroxide is an extremely toxic ros due to its ability to cross the membrane freely and to inhibit the enzymatic activities of cellular functions, thus decreasing the antioxidant defenses of sperm.22,23 objective of this study was to determine the ideal concentration of dha, test it in combination in the extender containing 2 antioxidants (vitamin e and catalase) in cryopreserved canine semen, and to verify a possible synergistic effect of these substances in sperm oxidative homeostasis and functionality. material and methods study was approved by the bioethics committee of the school of veterinary medicine and animal science, university of são paulo (protocol number 7693020215). reagents were purchased from sigma–aldrich (st. louis, mo). animals eight dogs (7 belgian malinois and 1 labrador retriever) of proven fertility with a body weight of 28-32 kg and 2-6 years were used. dogs belonged to canine reproduction and distribution center of the 2nd army police battalion (osasco, sao paulo brazil). dogs were periodically collected for the assisted reproductive program. dogs were fed royal canin max adult® twice a day. experiment 1: effect of supplementation with polyunsaturated fatty acid (docosahexaenoic acid [dha]) on canine semen extender eight ejaculates were collected and diluted in a cryopreservation medium to arrive at a final concentration of 100 x 106 sperm per ml. each ejaculate was divided into 4 aliquots and allocated to control group (0 µm dha) and 1 µm dha, 5 µm dha, and 10 µm dha groups. after dilution, samples underwent one-step sperm cryopreservation using an automated machine. experiment 2: effect of vitamin e and catalase supplementation in cryopreserved canine semen in extender containing dha eight ejaculates were collected and diluted in a cryopreservation medium for dogs to arrive at a final concentration of 100 x 106 sperm per ml. each ejaculate was divided into 4 aliquots and  allocated to control group (without antioxidant) and antioxidant groups (0.6 mm vitamin e, 300 u/ml catalase, and 0.6 mm vitamin e plus 300 u catalase/ml1). after dilution, samples underwent one-step sperm cryopreservation using an automated machine. semen collection, cryopreservation, and thawing sperm-rich fraction was collected using the digital manipulation method. each ejaculate was assessed for motility, vigor, and concentration patterns under light microscopy (nikon, eclipse e200, tokyo, japan) and for volume via a graduated tube. sperm concentration (106 sperm/ml) was determined in the neubauer chamber. samples contaminated with urine or blood or with motility below 70% were excluded. seminal plasma was removed by centrifuging semen at 500 x g for 5 minutes (centribio®). pellets were divided into 4 aliquots and suspended at a final concentration of 100 x 106 sperm/ml with 263 mm tris-yolk-fructose-citric acid (tris-hydroxymethyl-aminomethane [trizma-base; t-6791 sigma]), 84.7 mm citric acid monohydrate (t-1909), 63 mm d-fructose (f-2543), egg yolk 20%, gentamicin 10 mg/ml (g-1264), and glycerol 5% and used for each treatment mentioned in the respective experiments. after dilution, seminal aliquots were packed in 0.5 ml straws (imv technologies, france) and allocated in an automated cryopreservation machine tk3000® compacta (tk tecnologia em congelação ltda; uberaba, mg, brazil). subsequently, the samples were refrigerated to 5ºc (0.25°c/minute); straws were then positioned 3 cm above liquid nitrogen (–20ºc) and immersed in liquid nitrogen. samples were stored in liquid nitrogen cylinders and packed in racks. one week after cryopreservation and storage, samples were thawed at 37°c for 30 seconds and subjected to sperm evaluation. sperm evaluation sperm were evaluated for kinetics, plasma and acrosomal membrane integrity, mitochondrial function (activity and mitochondrial membrane potential), dna integrity, and oxidative status. computerized analysis of sperm kinetic patterns kinetic patterns were assessed using computerized sperm analysis (casa; ivos 12.3, hamilton-thorne®, beverly, ma) with equipment settings for dogs (table in appendix). for the analysis, 6 µl of the sample was pipetted in a glass slide with a http://dx.doi.org/10.58292/ct.v17.12690 citation: clinical theriogenology 2025, 17, 12690, http://dx.doi.org/10.58292/ct.v17.12690 3 coverslip heated to 37ºc and ~ 1,000 sperm were evaluated. following variables were considered: motility (%), progressive motility (%), vap (velocity average pathway, µm/second), vsl (velocity straight line, µm/second), vcl (curvilinear velocity, µm/second), alh (amplitude of lateral head displacement, µm), bcf (beat cross-frequency, hz), str (straightness %) and lin (linearity %). sperm speed was classified into 4 groups: fast (%), medium (%), slow (%) and static (%).24 functional sperm tests flow cytometry (guava easycytetm mini system, guava® technologies, hayward, ca) was used to test sperm function with the concentration fixed at 25 x 106 sperm cells/ml after dilution with tris egg yolk. gate details are provided (figure in appendix). a total of 10,000 events per sample were analyzed, and data corresponding to yellow (pm1 photodetector: 583 nm), red (pm2 photodetector: 680 nm), and green fluorescent signals (pm3 photodetector: 525 nm) were recorded after a logarithmic amplification. data were analyzed by flowjo® v10.2 software (flow cytometry analysis software, tree star inc, ashland, or).25 plasma and acrosomal membranes were evaluated with propidium iodide (pi) and fitc probes conjugated to pisum sativum agglutinin (fitc-psa), respectively. this association of fluorophores allocates the sperm population into 4 groups: intact membrane and intact acrosome (imia), intact membrane and damaged acrosome (imda), damaged membrane and intact acrosome (dmia) and damaged membrane and damaged acrosome (dmda). samples were stained with 0.5 mg/ml pi in nacl 0.9% and 100 mg/ml fitc-psa (fitcpsa l-0770) in a 10% sodium azide solution in dpbs. samples were analyzed by flow cytometry after 10 minutes, excited at 488 nm and detected at 630-650 nm (pi) and 515530 nm (fitc). results were expressed as percentage. mitochondrial membrane potential was assessed by flow cytometry. evaluation was made using jc-1 probe (5,5’,6,6’ tetrachloro-1,1’,3,3’ 201-tetraethyl-benzimidazolylcarbocyanine chloride; invitrogen, eugene, or). for the assay, 0.5 μl of the fluorescent probe jc-1 (76.5 mm) was added to the samples and incubated at 37°c for 5 minutes before analysis on the flow cytometer, where there was excitation at 488 nm and detection at 583 nm. samples were classified into percentages of sperm with high (jc-1 high), intermediate (jc-1 intermediate), and low (jc-1 low) mitochondrial membrane potential. mitochondrial activity of 100 sperm were determined using the dab technique (3’3 diaminobenzidine) in phase contrast microscopy at 1,000 x magnification26 and classified into 4  classes, according to the percentage of stained midpiece: completely stained, indicating high mitochondrial activity (dab i); more than 50% colored intermediate piece, indicating average activity (dab ii); more than 50% unstained intermediate piece, indicating low activity (dab iii); and intermediate piece completely unstained, indicating absence of mitochondrial activity (dab iv). results were expressed as a percentage (%) and these classes were used to provide an index of mitochondrial activity. chromatin stability was tested by sperm chromatin structure assay (scsa),27 modified28 for dogs. samples were incubated with 50 µl of tne buffer (0.01 m tris-hcl, 0.15 m nacl, 1 mm edta and distilled water, ph 7.4) and 100 µl acid detergent (0.08 m hcl, 0.15 m nacl, triton x-100 0.1% in distilled water, ph 1.2). after 30 seconds, acridine orange (6 µg/ml stock solution) was added, and each sample was analyzed on flow cytometry after 5 minutes of incubation at 37ºc, excited at 488 nm and detected at 630-650 nm (yellow) and 515-530 nm (green). debris were excluded using a green fluorescence/direct dispersion filter; then the percentage of sperm with dna alteration (ao+) were selected in an αt histogram. to determine the ao positive (ao+) population, a positive control was incubated with hydrochloric acid (1.2 m in acid detergent, ph 0.1) for 5 minutes to induce dna fragmentation in almost 100% of sperm.25 evaluation of sperm resistance to oxidative stress susceptibility to lipid peroxidation was assessed by the thiobarbituric acid [tba] reactive substance (tbars) test that was adapted.29 samples were incubated with iron sulfate (4 mm) and vitamin c (20 mm) at 37ºc for 90 minutes. after these inductions, 10% trichloroacetic acid (600 ml) was added. samples were centrifuged at 20,800 x g for 15 minutes at 5ºc to isolate proteins and debris. subsequently, 800 µl of the supernatant was removed and transferred to cryotubes. then, 1% tba (800 µl) was added to the tubes, which were incubated at 95ºc in a water bath for 15 minutes. in this reaction, malondialdehyde (mda; the primary product of lipid peroxidation) and tba produce a pink-colored complex. this staining was quantified on the spectrophotometer (ultrospec 3300 pro® amersham biosciences, woburn, ma) at a wavelength of 532 nm. results were expressed in nanograms of tbars/106 sperm. to detect ros, a penetrating fluorescent probe was used, which, when oxidized by intracellular free radicals, binds to dna and emits a higher intensity of green fluorescence (cellrox® green, molecular probes, eugene, or). samples were stained with cellrox® green (final concentration of 5 µm) for 30 minutes at 37ºc, and pi was added (final concentration, 6 µm) in the last 10 minutes. samples were analyzed by flow cytometry, excited at 488 nm, and detected at 630650 nm (pi) and 515-530 nm (cellrox® green). sperm data were analyzed for negative oxidative stress and intact plasma membrane (noim), negative oxidative stress and damaged plasma membrane (nodm), positive oxidative stress and intact plasma membrane (poim), and positive oxidative stress and damaged plasma membrane (podm), and expressed as a percentage. data analysis data were analyzed by the sas system for windows (sas, institute inc., cary, nc, 2000). treatment effects were tested for normality of residues (gaussian distribution) and homogeneity of variances using the guided data analysis application. means were compared using the anova method applying the least significant difference test for multiple comparisons. results were presented as means with their respective standard errors of the mean; significance was set at p < 0.05. results experiment 1 groups treated with 5 or 10 µm dha had higher percentage of sperm with progressive motility (figure 1a) and rapid http://dx.doi.org/10.58292/ct.v17.12690 4 citation: clinical theriogenology 2025, 17, 12690, http://dx.doi.org/10.58292/ct.v17.12690 speed (figure 1b) compared to 1 µm group but were similar to control group. the group treated with 10 µm dha had lower percentage of sperm with slow movement (figure 1c) and was not different from control. there was a lower percentage of static sperm in 5 µm dha (figure 1d). other sperm kinetic (table 1) and function (table 2) variables were not different. there was no difference among groups in lipid peroxidation rates from tbars and oxidative stress from cellrox® (table 3). thus, based on the results, 5 µm dha was selected for experiment 2 to test the effect of antioxidants vitamin e and catalase in samples of cryopreserved canine semen in an extender containing dha. experiment 2 the group treated with vitamin e had higher velocity average pathway (vap figure 2a), velocity straight line (vsl figure 2b), total sperm motility (figure 2c), progressive sperm motility (figure 2d), and rapid sperm movement speed (figure 2e) compared to control group. additionally, the group treated with vitamin e did not differ from other treatments for these variables, except for progressive sperm motility, where the percentage of sperm in the group treated with catalase was significantly lower (figure 2d). other kinetic variables were not different among groups (table 4). there was no difference in mitochondrial function between control and vitamin e group in any variable (figures 3a-3d). however, catalase and vitamin e + catalase groups had lower percentage of high mitochondrial activity (dabi) compared to control and vitamin e groups (figure 3a) and a higher percentage of low mitochondrial activity (dabiii) compared to control group (figure 3c). catalase group also had a higher percentage of mitochondrial inactivity (dabiv) compared to control group (figure 3d). control group had higher mitochondrial activity index than vitamin e + catalase and catalase group, vitamin e had higher index than catalase group (figure 3e). no other sperm functional test had a difference among groups (table 5). the vitamin e + catalase group had higher percentage of sperm with negative oxidative stress and intact plasma membrane (noim) compared to control group (figure 4a). figure 1. effect of various concentrations of docosahexaenoic acid (dha) on sperm kinetics variables a,bwithin an end point, columns without a common superscript differed (p ≤ 0.05) table 1. effect of various concentrations of docosahexaenoic acid (dha) on sperm kinetics variables variable control dha 1 µm dha 5 µm dha 10 µm vap (µm/second) 80.07 ± 2.66 76.74 ± 1,96 80.16 ± 3.03 78.25 ± 2.38 vsl (µm/second) 69.04 ± 2.42 65.56 ± 2.00 69.35 ± 3.00 66.79 ± 2.37 vcl (µm/second) 127.52 ± 5.35 124.82 ± 5.27 128.11 ± 4.59 126.30 ± 4.33 alh (µm) 7.66 ± 0.32 7.06 ± 0.34 7.25 ± 0.29 7.55 ± 0.30 bcf (hz) 26.59 ± 1.27 25.92 ± 1.04 26.96 ± 0.90 27.06 ± 0.38 str (%) 85.25 ± 0.92 84.50 ± 1.59 85.50 ± 1.12 84.50 ± 1.07 lin (%) 55.50 ± 1.61 54.25 ± 2.45 55.75 ± 1.73 54.75 ± 1.85 motility (%) 27.12 ± 4.00 18.50 ± 2.54 23.25 ± 3.13 26.00 ± 3.51 medium (%) 19.12 ± 2.65 14.62 ± 1.93 15.87 ± 1.77 18.75 ± 2.42 http://dx.doi.org/10.58292/ct.v17.12690 citation: clinical theriogenology 2025, 17, 12690, http://dx.doi.org/10.58292/ct.v17.12690 5 positive oxidative stress and damaged plasma membrane (podm figure 4b), positive oxidative stress and intact plasma membrane (poim figure 4c), negative oxidative stress and damaged membrane (nodm figure 4d) and lipid peroxidation (tbars figure 4e) were not different among groups. discussion synergistic effects of pufas and enzymatic and nonenzymatic antioxidants added to the canine semen extender were evaluated to prevent exacerbated lipid peroxidation caused by the addition of these fatty acids.30 in experiment 1, 5 µm of dha table 2. effect of various concentrations of docosahexaenoic acid (dha) on functional tests of mitochondrial activity, mitochondrial membrane potential and integrity of sperm plasma, and acrosomal membranes variable (%) control dha 1 µm dha 5 µm dha 10 µm dabi 90.00 ± 1.58 90.00 ± 1.70 88.12 ± 1.53 89.62 ± 1.21 dabii 6.37 ± 1.35 5.00 ± 0.68 6.37 ± 0.68 6.25 ± 0.56 dabiii 2.75 ± 0.67 3.25 ± 1.23 4.12 ± 0.72 3.12 ± 0.91 dabiv 0.87 ± 0.29 1.75 ± 0.56 1.37 ± 0.50 1.00 ± 0.27 jc1high 5.82 ± 3.56 4.30 ± 1.53 7.56 ± 3.45 7.26 ± 2.99 jc1low 73.14 ± 3.85 78.93 ± 3.02 72.22 ± 6.37 71.62 ± 4.16 jc1intermediate 21.02 ± 3.70 16.76 ± 2.82 20.26 ± 3.98 21.13 ± 3.22 scsa 6.94 ± 1.01 5.10 ± 1.18 5.80 ± 1.67 13.34 ± 7.27 imda 2.48 ± 0.94 2.07 ± 0.63 2.16 ± 0.71 2.52 ± 0.95 dmda 64.20 ± 4.93 65.12 ± 3.50 64.15 ± 3.80 58.17 ± 11.11 dmia 21.25 ± 3.40 22.20 ± 2.90 22.45 ± 3.89 24.53 ± 5.13 imam 12.04 ± 3.58 10.60 ± 1.05 11.26 ± 2.51 14.80 ± 5.48 table 3. effect of various concentrations of docosahexaenoic acid (dha) in response to oxidative stress features variable control dha 1 µm dha 5 µm dha 10 µm poim (%) 3.96 ± 1.22 4.00 ± 0.72 6.69 ± 2.08 3.51 ± 0.53 podm (%) 25.80 ± 8.72 33.95 ± 4.90 31.13 ± 5.63 31.22 ± 8.04 nodm (%) 66.32 ± 8.45 58.67 ± 4.92 58.08 ± 6.22 62.13 ± 8.10 noim (%) 3.92 ± 0.82 3.38 ± 0.49 4.06 ± 0.66 3.14 ± 0.51 tbars (ng/106sperm) 234.31 ± 26.73 254.35 ± 32.27 246.56 ± 22.64 205.50 ± 16,81 figure 2. effect of vitamin e and catalase and their combination on sperm kinetics variables in an extender containing dha a,bwithin an end point, columns without a common superscript differed (p ≤ 0.05) http://dx.doi.org/10.58292/ct.v17.12690 6 citation: clinical theriogenology 2025, 17, 12690, http://dx.doi.org/10.58292/ct.v17.12690 had the lowest percentage of static sperm, with same progressive and rapid motility as control group. motility parameters analyzed by casa have high potential in clinical applications since there were significant relationships between aspects of sperm movement and cervical mucus penetration, fertilization in vitro and in vivo.31 also, sperm with fast and linear movements had greater resistance to cryopreservation and are more likely to overcome female reproductive tract barriers to enable fertilization.32 epididymal bull sperm response to dha addition was good9,33 and dha has a role in sperm plasma membrane stabilization in many domestic species.34 human semen incubated with various concentrations of dha (7.2, 14.4, 28.8, 57.6, or 115.2 µm) table 4. effect of vitamin e and catalase and their combination on sperm kinetics variables of in an extender containing dha variable control vitamin e catalase vit e + cat vcl (µm/s) 118.87 ± 4.84 126.51 ± 3.38 124.76 ± 3.27 124.64 ± 6.02 alh (µm/s) 7.6 ± 0.26 7.14 ± 0.23 7.34 ± 0.27 7.32 ± 0.4 bcf (hz) 27.6 ± 0.79 26.8 ± 0.81 25.56 ± 0.77 26.07 ± 1.16 str (%) 83.29 ± 1.36 84.71 ± 0.78 85.5 ± 0.87 84.62 ± 0.5 lin (%) 54.14 ± 2.21 57.57 ± 1.39 57.75 ± 1.54 57.5 ± 1.55 medium (%) 20.71 ± 2.39 22.43 ± 2.57 22.25 ± 3.02 22.75 ± 3.37 slow (%) 63.29 ± 3.75 54.14 ± 6.16 50.62 ± 5.6 51.75 ± 3.38 static (%) 8.14 ± 1.22 9.0 ± 1.98 16.0 ± 4.3 12.37 ± 5.45 figure 3. effect of vitamin e and catalase and their combination on sperm mitochondrial function in an extender containing dha a-cwithin an end point, columns without a common superscript differed (p ≤ 0.05) table 5. effect of vitamin e and catalase and their combination to functional tests of sperm mitochondrial membrane potential and plasma and acrosomal membrane integrity in an extender containing dha variable (%) control vitamine e catalase vit e + cat jc1high 36.80 ± 8.99 38.66 ± 8.49 32.43 ± 5.44 28.09 ± 3.96 jc1low 35.74 ± 2.82 38.26 ± 3.61 44.94 ± 3.91 42.26 ± 2.32 jc1intermidiate 27.46 ± 7.54 32.03 ± 7.22 22.66 ± 3.88 29.66 ± 3.98 scsa 1.36 ± 0.25 1.14 ± 0.32 1.56 ± 0.37 2.01 ± 0.81 dmda 33.67 ± 2.56 30.78 ± 2.48 35.74 ± 3.26 37.12 ± 6.63 imda 2.86 ± 0.32 2.71 ± 0.12 3.44 ± 0.42 3.16 ± 0.39 dmia 17.81 ± 2.06 18.94 ± 3.33 18.54 ± 2.61 20.25 ± 2.92 imia 45.68 ± 3.28 50.40 ± 3.09 42.26 ± 4.40 44.21 ± 3.62 http://dx.doi.org/10.58292/ct.v17.12690 citation: clinical theriogenology 2025, 17, 12690, http://dx.doi.org/10.58292/ct.v17.12690 7 had no significant impact on sperm viability, with the exception of the highest concentration tested (115.2 µm) that was cytotoxic.35 as in the present study, dha supplement to semen extender also increased progressive motility of boar sperm.36 in experiment 2, sperm kinetics were significantly different; the vitamin e group had superior results compared to control group. vitamin e helps to maintain the structural integrity of sperm architecture and prevents mitochondrial dysfunction by inhibiting oxidative stress, preventing reproductive dysfunctions;37 it is an effective antioxidant and has a very important role in protecting the integrity of sperm membrane structure and the structural integrity of mitochondria and tails.37 oral supplementation of vitamin e improved semen quality in dogs with low seminal plasma superoxide dismutase.38 vitamin e addition to extender in cattle semen at a concentration of 1 mg/ml provided protection to sperm plasma membrane from bulls with good quality semen, preserved metabolic activity and cell viability.39 similarly, in boars, a protective effect on post thaw sperm motility and viability was observed when vitamin e was added to the diluter (concentrations of 200 µm1-1) to the extender.40 in dogs, higher values for total motility, rapid steady forward and viability were obtained when vitamin e was added at a concentration of 0.3 mm.1 treatment groups had lower levels of oxidative stress compared to control group, but catalase was most harmful to canine sperm. a reductive stress effect on mitochondria caused by excess of antioxidants may increase ros and induce mitochondrial malfunction,41 and it may be the cause for lower levels of progressive motility and mitochondrial activity index in catalase group and higher percentage of high mitochondrial activity and low percentage of low mitochondrial activity in vitamin e + catalase group. both oxidative stress and reductive stress conditions have detrimental effects on male fertility, resulting in alteration of semen parameters such as concentration, motility, or normal sperm morphology.42 in a recent study, the same authors demonstrated that the establishment of a reductive environment due to high antioxidant concentration has a deleterious impact on sperm motility, vitality, and mitochondrial activity.42 addition of 20 iu catalase/ml to bull sperm extender improved sperm quality; higher sperm motility, lower sperm dna fragmentation and ros production, a higher mitochondrial membrane potential, and higher percentage of sperm with intact acrosome and plasma membranes,43 suggesting that the concentration used in this study was too high. in fact, lower concentrations of antioxidants improved human sperm parameters and chromatin quality when used at a concentration of 100 iu/ml.44 enzymatic and nonenzymatic antioxidants are efficient and have improved post thaw semen quality. however, for many antioxidants, their function or mechanism of action at the cellular level are not clearly known.45 for this reason, the antioxidant treatment of semen is still a field that needs to be studied. we identified the beneficial effects of vitamin e and the deleterious effects of high concentrations of catalase. in conclusion, the addition of 5 µm dha and 0.6 mm vitamin e to canine semen extender improved semen quality after cryopreservation. conflict of interest none to declare. references 1. michael a, alexopoulos c, pontiki e, et al: effect of antioxidant supplementation on semen quality and reactive oxygen species of frozen-thawed canine spermatozoa. theriogenology 2007;68:204212. doi: 10.1016/j.theriogenology.2007.04.053 2. flesch fm, gadella bm: dynamics of the mammalian sperm plasma membrane in the process of fertilization. biochim biophys acta 2000;1469:197-235. doi: 10.1016/s0304-4157(00)00018-6 3. mandal r, badyakar d, chakrabarty j: role of membrane lipid fatty acids in sperm cryopreservation. adv androl 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2016;77:1711-1714. doi: 10.1292/jvms.15-0294 39. beconi mt, francia cr, mora ng, et al: effect of natural antioxidants on frozen bovine semen preservation. theriogenology 1993;40:841-851. doi: 10.1016/0093-691x(93)90219-u 40. pena fj, johannisson a, wallgren m, et al: antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate. anim reprod sci 2003;78:85-98. doi: 10.1016/s0378-4320(03)00049-6 41. angrimani dsr, nichi m, brito mm, et al: the use of reduced glutathione (gsh) as antioxidant for cryopreserved sperm in dogs. arq bras med vet zootec 2018;70:419-428. doi: 10.1590/ 1678-4162-9684  42. panner selvam mk, agarwal a, henkel r, et al: the effect of oxidative and reductive stress on semen parameters and functions of physiologically normal human spermatozoa. free radic biol med 2020;152:375-385. doi: 10.1016/j. freeradbiomed.2020.03.008 43. arslan ho, herrera c, malama e, et al: effect of the addition of different catalase concentrations to a tris-egg yolk extender on quality and in vitro fertilization rate of frozen-thawed bull sperm. cryobiology 2019;91:40-52. doi: 10.1016/j.cryobiol.2019.10.200 44. mohammadzadeh m, ramazani v, khalili ma, et al: medium containing different concentrations of catalase as a strategy for optimising sperm parameters and chromatin in normospermic persons. andrologia 2019;51:1-6. doi: 10.1111/and.13231 45. kumar a, prasad jk, srivastava n, et al: strategies to minimize various stress-related freeze–thaw damages during conventional cryopreservation of mammalian spermatozoa. biopreserv biobank 2019;17:603-612. doi: 10.1089/bio.2019.0037 http://dx.doi.org/10.58292/ct.v17.12690 https://doi.org/10.1111/j.1439-0531.2008.01239.x https://doi.org/10.1016/j.ecoenv.2012.05.014 https://doi.org/10.1292/jvms.15-0294 https://doi.org/10.1016/0093-691x(93)90219-u https://doi.org/10.1016/s0378-4320(03)00049-6 https://doi.org/10.1590/1678-4162-9684  https://doi.org/10.1590/1678-4162-9684  https://doi.org/10.1016/j.freeradbiomed.2020.03.008 https://doi.org/10.1016/j.freeradbiomed.2020.03.008 https://doi.org/10.1016/j.cryobiol.2019.10.200 https://doi.org/10.1111/and.13231 https://doi.org/10.1089/bio.2019.0037 10 citation: clinical theriogenology 2025, 17, 12690, http://dx.doi.org/10.58292/ct.v17.12690 appendix table. settings utilized to assess dog sperm kinetics (casa; hamilton-thorne®, ivos 12.3, usa) settings image capture frames per second (hz) 60 number of frames 30   cell detection minimum contrast 75 minimum cell size (pix) 6   progressive cells path velocity (vap μm/s) 100 straightness (str %) 75   defaults (if < 5 motile cells) cell size (pix) 8 cell intensity 80   slow cells vap cutoff (μm/s) 9.9 vsl cutoff (μm/s) 20   static intensity ts minimum 0.49 maximum 1.68   static size gates minimum 0.8 maximum 4.93   static elongation gates minimum 22 maximum 84 figure. a gate referred to as ‘spitz’ was defined based on green fluorescence intensity and particle size, using a logarithmic scale, to encompass the main sperm population (a), when both green and red fluorescence signals were simultaneously applied to the same population 4 distinct subpopulations were defined (b); iaim (intact acrosome/intact plasma membrane), iadm (intact acrosome/damaged plasma membrane), daim (damaged acrosome/intact plasma membrane), and dadm (damaged acrosome/ damaged plasma membrane). http://dx.doi.org/10.58292/ct.v17.12690 2012: age related and geographical factors affecting fertility and pregnancy in the mare age related and geographical factors affecting fertility and pregnancy in the mare twink allen paul mellon laboratory of equine reproduction,‘brunswick’, woodditton road, newmarket, suffolk, uk abstract forty years ago a failure of significant numbers of maiden, barren and lactating mares to be covered advantageously due to their unwillingness to cycle and ovulate normally during the confines of the arbitrary and out-of-phase thoroughbred ‘covering season’ (mid-feb to mid-june), combined with later stage abortion of undiagnosed twin pregnancies, were probably the major factors acting to reduce fertility in this breed. subsequent development and judicious studfarm veterinary application of drugs like prostaglandin, altrenogest and other progestagen formulations, gonadotrophin-releasing hormone (gnrh) analoges, domperidone and long-acting depot formulations of steroid hormones, together with diagnostic techniques such as the rapid elisa assay kit for progesterone and transrectal ultrasonography for the accurate monitoring of follicular growth, early diagnosis of singleton and twin pregnancy and detection of accumulated fluid and other uterine pathology, have virtually eliminated these original ‘fertility blockers’ in thoroughbred mares and in non-thoroughbred competition horses. the improvements have helped greatly to lift present day ‘diagnosable’ pregnancy rates (i.e. >day 12 after ovulation) to >90% per season. yet some 15% of mares still lose one or more pregnancies each season and around 25% of these losses occur during the second half of gestation. why? and why are so many foals born maladjusted, dysmature or disfigured to the extent that they never perform successfully as athletes? the rates of both early and late pregnancy loss climb steeply with increasing mare age in which, as described originally by the late professor bob kenny and confirmed subsequently by verena bracher of switzerland and heinz schoon and his colleagues in leipzig, degenerative changes occur in both the morphology and secretory functions of the ageing endometrium, including fibrous deposition within the stroma and around the endometrial glands. the former obstructs fluid drainage from the endometrium via the lymph channels and the latter leads to non-functional ‘gland-nest’ formation, thereby reducing both the production of nutrient histotroph and the ability of the endometrium to re-model itself to accommodate the attachment and interdigitation needs of the developing epitheliochorial placenta. just as in twin pregnancy the fetus suffers varying degrees of nutritional deprivation giving rise to dysmature, poorly muscled and long coated foals born well after term or, in the worst case scenario, aborted in later gestation. along similar lines, and as mooted recently by sandra wilsher, failure of the allantochorion to expand the uterus sufficiently, perhaps due to inadequate fluid transport within and through the placenta, may underlie the common occurrence of flexural limb deformities (‘contracted tendons’) in thoroughbred neonates. excessive twisting, leading to vascular occlusion, of the umbilical cord, is perhaps the principal cause of non-infectious midto late-stage abortion in thoroughbred mares in the uk but, curiously, not in america. the question of what induces the fetus to rotate so vigorously in its bath of fluids earlier in gestation remains unsolved. conversely, placentitis of bacterial or fungal cause, occurs more commonly in america than britain. ascending placentitis appears relatively straightforward and it has its origin usually in some degree of cervical incompetence. so-called nocardioform placentitis, on the other hand, which is patchy in its distribution on the placenta and is clearly unrelated to the cervix, is much more intriguing. its apparently limited occurrence to eastern usa and north-eastern australia where the setae-covered, respectively, eastern tent and processionary caterpillars flourish, begs the question as to whether bacteriacarrying setae shed from ingested caterpillars passing through the walls of the intestine and closely adjacent uterus, as demonstrated to occur by judy cawdell-smith and co-workers in australia, may be the crucial etiological factor in the nocardioform placentitis phenomenon. perhaps the terrible mrls abortion storm of 2001/2002 in eastern kentucky is not over yet and the occurrence of abortion versus focal placentitis is merely a matter of the challenge dose of caterpillar setae. in summary, although technological advances in recent years have enabled great strides in achieving conception in thoroughbred and other types of competition mares it is probably true to say that, with the notable exception of twins, the rates of pregnancy loss, both early and late, have continued unchanged, or may even have increased slightly, during the same period. clearly, more research needs to be done to lessen this continuing problem in equine stud management. 571 clinical theriogenology • volume 4, number 4 • december 2012 572clinical theriogenology • volume 4, number 4 • december 2012 omniblank: 2019 uterine rupture with connection to an ectopic fetus in a doe uterine rupture with connection to an ectopic fetus in a doe william whitler, clare scully, charles estill lois bates acheson veterinary hospital, carlson college of veterinary medicine oregon state university, corvallis, or abstract ten weeks after delivery of 2 viable kids, a necrotic, headless fetus was removed surgically from the abdominal mass of a nigerian dwarf doe goat. the outcome was good, although prognosis for future fertility was considered poor. keywords: goat, doe, postpartum, ectopic fetus case presentation a 4-year-old, 25 kg, nigerian dwarf doe goat was presented for an enlarged right ventral abdomen. this doe had delivered 2 live kids without assistance ~ 10 weeks prior to presentation. the swelling was first noted ~ 4 weeks previously, during which time the owner observed weight loss. the owner reported that the mass had doubled in size over the last 10 days. the referring veterinarian had performed an ultrasound directed examination of the mass 10 days prior to admission and visualized suspected fetal remains. a fine needle aspirate at that time recovered purulent material with hair. appetite, water consumption, defecation, and urination were described as normal, and the kids were nursing and growing as expected. on presentation, the doe was bright and alert, her rectal temperature, pulse, respiratory rate, and rumen motility were within normal limits. ocular mucous membrane color was pink, with a famacha score of 1/5. a soft, slightly fluctuant swelling ~ 20 cm in diameter was present along the lateral ventral right abdominal wall, extending cranially from the mammary gland. a soft irregular (~ 10 x 15 cm) mass was palpable within the swelling. ultrasonographic examination of the mass revealed multiple irregular echogenic areas with many highly echogenic foci, each ~ 0.5 1 cm in diameter. plain radiography and computed tomography scan procedures were suggested, but declined by the owner for economic reasons. treatment anesthesia was induced iv with diazepam, (0.1 mg/kg; hospira, lake forest, il), butorphanol, (0.022 mg/kg; torbugesic®, zoetis, kalamazoo, mi) and ketamine (2 mg/kg; ketaset®, zoetis, kalamazoo, mi) and maintained with isoflurane (fluriso® vetone, mwi, boise, id) in oxygen via an oral endotracheal tube. the patient was placed in dorsal recumbency and an area from the xiphoid to the mammary glands was clipped and scrubbed with povidone iodine surgical scrub, then rinsed with isopropyl alcohol. after sterile draping, a 15 cm elliptical incision was made into the mass, oriented in a cranio-caudal direction. approximately 1 liter of purulent material was expressed and a decomposing headless fetus removed (figure 1). a sample of the fluid was collected in an aseptic fashion and submitted for aerobic culture and sensitivity. the surgical site was copiously lavaged. the layer of tissue incised over the mass was < 1 cm thick; it was not clear as to whether the wall of the mass was only cutaneous tissue or a combination of skin and tightly adhered uterine wall. there were 2 tracts along the abdominal wall side of the cavity (figure 2). a 2 cm sample of the incised wall of the mass was submitted for histopathology. two half-inch penrose drains were placed into the surgical site, 1 exiting axially and the other abaxially to the incision at the most dependent location. subcuticular tissues were apposed with 2-0 polydioxanone suture (pds®, ethicon us, llc cincinnati, oh) in a continuous pattern and skin closed with 1-0 polyamide (braunamid, jorgensen labs, loveland, co) in a ford interlocking pattern. the doe was given iv flunixin (1 mg/kg; prevail®, mwi) and ceftiofur suspension sc (6.6 mg/kg; excede®, zoetis), prior to recovery (which was uneventful). the following day, right lateral radiographs in a standing position were obtained (figure 3). the head visible in the radiograph was delivered per vagina. the vagina was lavaged with sterile saline, which 131 clinical theriogenology • volume 11, number 2 • june 2019 subsequently flowed out around the penrose drains through the ventral abdominal skin, confirming the suspicion that 1 tract leading out of the pocket extended towards the cervix. the doe was hospitalized for 6 days after surgery for wound care. initially meloxicam (1 mg/kg; unichem pharmaceuticals, hasbrouck heights, nj) was given orally once daily. however, on day 3 the doe seemed more painful and morphine (0.1 mg/kg; hospira, lake forest, il) was given im every 4 hours for 24 hours. subsequently, a fentanyl patch delivering 25 µg/hour (apotex corp, weston, fl) was applied to the skin of her foreleg after clipping hair. the penrose drains were removed on day 5 and she was discharged from the hospital with instructions to monitor her incision and overall condition. outcome the biopsy of the mass wall suggested dense fibrovascular tissue, with evidence of chronic presumed suppurative inflammation. no specific uterine tissue or body wall musculature was identified. bacterial culture had heavy growth of pasturella sp., in addition to a mixture of contaminants. two weeks post operatively, the owner reported that the doe was doing well with a good appetite and that the surviving kids were growing normally. an area of the incision had dehisced but was granulating well. approximately 2 years after the initial events, the owner reported the swelling in the right inguinal area returned and the referring veterinarian felt it was an enlargement of the original surgical site based on palpation. no ultrasonographic evaluation was done and the patient remained bright, alert with a good appetite and apparently normal urine and fecal production. approximately 5 months later, the doe was found dead in the barn. no necropsy was performed and cause of death was unknown. discussion while ectopic pregnancies and fetus have been reported,1-5 this is not a case of ectopic pregnancy. in ectopic pregnancy the fetus develops outside of the uterus.1 the present case was regarded as unique and not apparently reported in goats. two possible etiologies are suggested: 1) rupture of uterine and abdominal walls, leading to a subcutaneous fistula, gravid uterus herniating and vascular compromise; or 2) decomposing fetus that caused necrosis of the uterine wall and replacement with fibrovasular tissue. although histopathology of the tissue overlying the fetus did not identify uterine tissue, communication with the vagina suggested that vagina and/or uterus communicated with the subcutaneous tissue. location and size of the head in the pelvic canal indicated that the fetus was at least partially within the uterus until close to or up to parturition. the head was retained in the vagina cranial to the urethral orifice. the time of decapitation of the fetal triplet remains unknown. presumably, the normally delivered kids were in 1 uterine horn and this fetus was in the contralateral (right) horn. prepartum herniation of the uterus has been reported in goats.6 although this is a possibility, it was considered unlikely, as the swelling was not noticed until postpartum. it is likely that the rupture occurred intrapartum following delivery of the first 2 kids. while the triplet was retained in the birth canal, straining or some external trauma ruptured the abdominal wall (and possibly the uterus) and the uterine horn and/or body of the fetus exited the abdomen. at some point, the head disarticulated from the body and as the uterine horn and abdominal wall constricted around the neck, the body became necrotic and septic. learning points • consider the presence of a fetus within a subcutaneous mass. • always rule out retained fetuses in a postpartum animal, even if they are acting normally. conflict of interest the authors have no conflicts of interest or sources of funding to disclose. 132clinical theriogenology • volume 11, number 2 • june 2019 references 1. corpa jm: ectopic pregnancy in animals and humans. reproduction 2006;131:631-640. 2. rosset e, galet g, buff s: a case report of an ectopic fetus in a cat. j feline med surg 2011;13:610-613. 3. mitchell kw: ectopic pregnancy in a ewe. vet rec 1989;124:498. 4. sontas bh, toydemir fts, erdogan o, et al: inguinal herniation with hydrometra/mucometra in a poodle bitch. can vet j 2013;54:840-844. 5. noakes de, parkinson tj, england gcw: arthur’s veterinary reproduction and obstetrics, 8th edition, philadelphia: w.b. saunders; 2001. p. 238-241. 6. smith mc, sherman dm: goat medicine, ist edition, philadelphia: lea and febiger; 1994. p. 57. figure 2. incised cavity that had the headless caprine fetus. two tracts (black arrows) appeared to lead into the abdominal cavity. figure 1. headless necrotic caprine fetus removed from an abdominal mass. figure 3. radiograph of abdomen of doe on day after surgery; note the fetal head (white arrow) in the pelvic vagina. 133 clinical theriogenology • volume 11, number 2 • june 2019 conflict of interest the authors have no conflicts of interest or sources of funding to disclose. omniblank: << /ascii85encodepages false /allowpsxobjects false /allowtransparency false /alwaysembed [ true ] /antialiascolorimages false /antialiasgrayimages false /antialiasmonoimages false 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undergraduate curriculum in veterinary reproduction at james cook university an undergraduate curriculum in veterinary reproduction at james cook university john cavalieri school of veterinary and biomedical sciences, james cook university, townsville, qld, australia abstract the curriculum developed to teach a new, five-year undergraduate veterinary science degree at james cook university is outlined and discussed. the curriculum in veterinary reproduction involves delivering 87 h of lectures and 133 h of laboratory and practical classes which comprises 6% of the total number of hours involved in delivering the veterinary degree course. an integrated model of delivering curriculum is utilized where theriogenology is integrated within all five years of the course rather than being delivered as a separate subject. tracking is not possible and all students are exposed to the entire reproductive curriculum although the opportunity is given for final year students interested in theriogenology to utilize electives and externships to further improve their knowledge and skills. a strong emphasis on the development of clinical skills is fostered with horizontal and vertical integration of theriogenology throughout the curriculum and the creation of an extensive set of laboratory classes. stand alone theriogenology rotations are not provided in the final year of the course but clinical aspects of theriogenology are included within other clinical rotations and electives. the curriculum will be continually developed with feedback from staff, students, employers, peer review, and development of further learning aids. keywords: veterinary education, curriculum, theriogenology curriculum introduction the bachelor of veterinary science (bvsc) degree course commenced at james cook university (jcu) in 2006. it is a five-year, undergraduate degree program based on an integrated curriculum. knowledge and skills related to veterinary reproduction are progressively delivered throughout the course. structure and function are emphasized in year 2; pregnancy diagnosis, an introduction to dystocia and infectious agents in year 3; clinical management of reproduction and disorders of the reproductive system in year 4; and clinical application of reproduction in year 5. in year 5 structured learning activities are conducted in the areas of canine, equine and bovine reproduction. additional opportunities are provided through in-practice rotations, electives and extramural experiences. this report provides a brief overview of the bvsc course at jcu and details and discusses the veterinary reproduction curriculum. overview of the veterinary science curriculum the academic year at jcu during years 1 to 4 of the course is divided into two, 13 to 14 week study periods. in year 5 students participate in a 33-week program over the course of the year. in the first year of the course students undertake four, three-unit subjects in study period (sp) 1 and again in sp 2. these introductory subjects cover areas such as animal, plant and cell biology; chemistry; biochemistry; anatomy; an introduction to the animal industries; careers in veterinary science; animal behavior, welfare, and handling; ethics; case studies; and personal professional development. in the second to fourth years of the degree program students are enrolled in each sp in a single 12-unit subject (table 1). these subjects are organized around several interdisciplinary themes which are outlined in table 2. themes within the integrated curriculum provide an organizational framework for developing learning activities that enhance integration between disciplines.1,2 for example, anatomy and physiology are both taught within the theme entitled ‘‘structure and function’’ as both anatomical structure and physiological function are intimately connected. the curriculum in year 5 is aimed at providing students with in-practice experience through a series of structured, “lockstep” rotations, and eight weeks of electives. rotations are conducted through facilities located at james cook university and contracted practices and facilities. on campus these include the school of veterinary and biomedical sciences and the jcu veterinary emergency centre and hospital. off campus rotations are conducted at the jcu townsville veterinary clinic equine clinical teaching centre, aachilpa veterinary hospital, jcu veterinary teaching resource centre in cooperation with the tableland veterinary service and an export 135 clinical theriogenology • volume 3 number 2 • june 2011 abattoir located on the outskirts of townsville. a list of rotations that students complete during the 5year degree program is shown in table 3. extramural practice requirements the extramural practice requirements which students must complete during scheduled university vacation periods are outlined in table 4. placements are structured to provide students with a broad range of experiences across a number of animal industries and clinical veterinary sciences. veterinary reproduction curriculum aspects related to veterinary reproduction are embedded throughout years 1 to 3 of the curriculum in keeping with the integrated model of curriculum delivery. an overview of the animal industries and an introduction to the husbandry associated with a range of species are provided in year 1. reproductive anatomy and physiology are emphasized in year 2, and pregnancy diagnosis, semen evaluation, and infectious agents affecting the reproductive system are covered in year 3. in year 4 the emphasis is placed on clinical reproduction as it relates to individual animal species. where appropriate comparative aspects are emphasized but the focus remains on the individual species as this is the context in which reproductive problems normally present in veterinary practice. clinical issues relevant to reproduction in rabbits, guinea pigs, ferrets, rats, mice, and reptiles are dealt with in a week-long intensive that students undertake examining clinical conditions associated with these species in year 4 while relevant husbandry associated with these species is introduced within years 1 and 2. avian and aquatic animal reproduction is dealt with in lectures which address the biology, physiology and clinical management of these species. lecture topics and practicals relevant to veterinary reproduction in years 2 to 5 are listed in tables 5 to 11. the total number of hours of training, excluding extramural studies and elective rotations encompasses 3755 hours of lectures, practicals, supervised work, clinical work and other learning activities.4 time devoted to training in reproduction comprises a minimum of 6% (220/3755) of the time devoted to intramural studies between years 1 and 5. this includes 87 h devoted to didactic lectures and 133 h devoted to practical and laboratory exercises. the number of boarded or board eligible theriogenology staff involved in the teaching of this discipline is one, although teaching is supported by other staff from the disciplines of anatomy, physiology, and clinical sciences. private practitioners are also used to support teaching. assessment a range of assessment methods and instruments are used to evaluate student learning which have been recently outlined.4 the predominant methods used to summatively assess students are listed in table 12. in addition, presentations, case studies, clinical skill report writing and for some students research proposals and literature reviews may be completed which relate to topics in veterinary reproduction. during the course year 3 and 5 students are summatively assessed in relation to their pregnancy diagnosis skills in cattle and the method for performing rectal and vaginal examination of horses. during year 5 students are formally assessed in regards to their pregnancy diagnostic skills and are expected to accurately perform manual pregnancy diagnosis in cattle over nine weeks of gestation with at least 80% accuracy or remedial instruction is provided. peer review a recent review of the reproduction curriculum by professor bruce eilts, of louisiana state university suggested that the major areas of reproduction were covered during the course. “the reproduction course is well conceived, presented, and adequately covers most aspects of a student’s need for knowledge in reproduction.” a small number of practitioners who were interviewed indicated that they thought that students were well prepared for their clinical experience. interviews with students indicated their understanding of the importance of reproduction in veterinary medicine and felt that they were getting good training in reproduction. suggestions raised in the review included reducing some of the information provided to students and separating information into essential from non-essential information. reducing practicals which involve a single topic, such as pregnancy diagnosis from three hours to one hour, 136clinical theriogenology • volume 3 number 2 • june 2011 while simultaneously reducing the number of students from, for example 30 to 12, may facilitate more active student engagement and provide students with more contact time with animals. increasing staffing levels, reduction in teaching loads in favor of more time for research and/or clinical activities and exposure to clinical cases in some species were highlighted as areas that may assist with staff development, satisfaction and retention in the future. discussion the newly developed curriculum in reproduction at jcu provides an extensive overview of reproductive issues that confront practicing veterinarians. knowledge, skills and attitudes in relation to veterinary reproduction are addressed through didactic lectures, practical classes, case studies, assignments and in-practice training. unlike curricula in many north american or european veterinary schools, the curriculum at jcu is aimed at providing every student with reproductive training in all of the major domestic animals species. opportunities to undertake electives which encompass additional and perhaps more advanced reproductive techniques allow students with a particular interest in reproduction to further develop their knowledge and skills. the number of hours devoted to lectures and laboratory sessions within the curriculum at jcu is greater than what has been reported in one survey of veterinary colleges in north american and the caribbean.5 a maximum of 60 h of lectures and 42 h of laboratory sessions in theriogenology were reported in the survey. a total of 67 h of didactic lectures are included at louisiana state university (eilts b, personal communication) and also at iowa state university (christensen bw, personal communication). the greater number of hours devoted to training in reproduction at jcu could reflect differences in content and emphasis compared to some other veterinary colleges and that training in all of the major domestic species is required for all students. time involved in the teaching of anatomy, physiology, and surgery related to reproductive structure, function and clinical applications was included in the data derived from jcu. it is unclear if this data were included by all survey participants when documenting theriogenology training in north america.5 the undergraduate curriculum at jcu may also require more time to be devoted to the teaching of the basic sciences compared with places where veterinary science is studied as a postgraduate degree. the greater number of hours devoted to the teaching of reproduction at jcu compared to some north american colleges suggests that there is some scope to reduce the amount of time devoted to the teaching of reproduction at jcu. this however, must be balanced with staffing levels, the need to achieve day 1 competencies, a reported concern that there is a need to increase practical exposure to theriogenology in veterinary education and that many reproductive procedures are still regarded as being valuable by practicing veterinarians.6 revisions to the curriculum, in particular shortening many practicals while at the same time reducing student numbers attending individual practicals will in part reduce the total number of student contact hours while at same time should improve teaching quality. further attempts to reduce contact hours will involve investigating the use of blending guided-independent and on-line study with didactic lectures, removing non-essential content and transferring some content to electives. there are at present no stand alone rotations in theriogenology in the final year of the program at jcu. in north america 72.7% (16/22) of schools responding to a survey required some clinical experience in theriogenology.5 the provision of up to eight-week of electives in the final year of the program and the integration of some aspects of clinical reproduction into core and elective rotations does provide students with some exposure to reproduction training in the final year and also enables those interested in reproduction to pursue further training during externships. it is envisaged that clinical training in theriogenology will continue to be integrated into core and elective rotations in the final year of the program. the number of boarded or board eligible faculty at jcu (n=1) is currently less than the mean (± sd) number of 3.3 ± 2.2 reported in north america.5 the current case load, staffing levels, timing of core rotations and the occurrence of peak reproductive activities for some species occurring outside of the academic year will contribute to the ongoing difficulty of offering stand alone theriogenology rotations or electives for final year students at jcu. classes in pregnancy diagnosis commence in the third year of the course, although students are first introduced to rectal palpation in year 2 as part of physical examination exercises in cattle. the philosophy behind introducing rectal palpation in year 3 is that it coincides with when students have completed the relevant anatomy and physiology and it is prior to when students will commence 137 clinical theriogenology • volume 3 number 2 • june 2011 extramural practice with veterinarians. introducing them to the technique in horses and cattle provides students with an opportunity to continue to practice these techniques when they commence their extramural practice. students are first exposed to abattoir specimens, then non-pregnant animals and then in the case of cows they are exposed to pregnancies under five months and then subsequently to pregnancies over five months. access to pregnant mares is limited so the emphasis in horses remains on preparation of the mare for examination, safe examination of the non-pregnant uterus, assessment of vulval conformation and vaginal examination. by the end of year 5 all students should have participated in at least 14 and 32 hours of formal classes involving rectal palpation in mares and cows, respectively. feedback from graduates and employers in the future will be helpful to determine if graduates meet employers’ expectations in regards to pregnancy diagnostic skills and whether any further emphasis on developing pregnancy diagnostic skills are needed within the curriculum. an introduction to obstetrics is taught in year 3 with students being introduced to equipment, normal delivery of the fetus in cranial and caudal presentation, and diagnostic traction. the aim again here is to provide students with basic skills for delivery of a fetus by traction so that they may benefit more from their extramural veterinary practice. in year 4 diagnosis and correction of malpresentations, percutaneous and subcutaneous fetotomy are taught in separate classes. practical classes focusing on male animals include the conduct of breeding soundness examinations (bse) in the dog, bull and stallion. semen evaluation is addressed beginning in year 2, and covered again in year 4 and 5. in year 5 semen processing is introduced. the guidelines issued by the society for theriogenology are used for the evaluation of dogs and stallions while the guidelines issued by the australian cattle veterinarians are used for the evaluation of bulls.7 at jcu the curriculum has been structured to foster integration between different disciplines and to present information and problems in ways veterinarians are likely to encounter them in the workplace.3 veterinary reproductive anatomy and physiology are taught in close association during year 2 of the course while students are also introduced to techniques that are used to prevent reproduction, bse of the bull and semen evaluation. the emphasis here is an attempt to place their knowledge of anatomy and physiology into an applied context. clinical techniques are related back to the relevant anatomy and physiology. for example, the structure and function of the testes are reinforced when students are shown the surgical technique for castration of a colt and a dog, with the relevant anatomy being outlined as the surgeon progresses through the castrations. anatomy of the reproductive tract is highlighted as students practice ultrasound examination of pregnant and nonpregnant tracts in water and the willis spay technique.8 the physiology of the estrous cycle and spermatogenesis are revisited as students use literature and their knowledge of the cycle to determine ways of preventing reproduction. aspects of reproduction can also be introduced in case studies in the early years of the course and ethical problems can be introduced, for example, the ethics associated with sterilization techniques or artificial breeding of dogs. in year 4 the teaching of reproduction is centered mainly within individual species and as such the reproductive course may be less comparative than in some teaching institutions. since reproductive problems usually present within the context of a given species we have elected to teach the majority of the clinical reproduction in the context of individual species. the use of a range of assessment modes provides an opportunity to provide reliable and valid assessment of students as well as provide a degree of diversity which may assist some students who may be disadvantaged by a particular form of assessment.9 time will tell if the standard requirement set for all students in regards to pregnancy diagnosis is too high for the majority of students to attain before completing the final year of the course. feedback from practitioners would however, suggest that achieving such a standard amongst our graduates would be desirable. vertical and horizontal integration of the reproduction components of the course provide an opportunity to scaffold and reinforce the development of knowledge and skills in reproduction throughout the course. vertical integration of clinical skills into the early years of professional degree programs appears to have a positive benefit on student motivation.3 further improvements in the quality of education could perhaps be obtained by the development of interactive cases, exposure to additional clinical cases and expansion of staff numbers with advanced training in reproduction. reviewing of the content of the curriculum will be required as more feedback is obtained from practitioners and external auditors such as the veterinary schools accreditation advisory committee. 138clinical theriogenology • volume 3 number 2 • june 2011 conclusion a detailed curriculum in veterinary reproduction has been developed for the new undergraduate degree program at jcu. highlights include a detailed overview of most topics relevant to practicing veterinarians, an extensive series of practical exercises and where possible vertical and horizontal integration of the educational material throughout the course. positive feedback on the content and effectiveness of the training from an independent reviewer and favorable feedback from students and practitioners have been obtained. further improvement will continue as teaching materials are continually redesigned, and feedback is obtained from students, staff and external reviewers. the quality of training will also likely be improved with development of additional learning aids, recruitment of new staff and the development of more time for research and clinical duties. acknowlegements the influence of dr. mike whitacre and dr. steven van camp in developing the theriogenology curriculum is gratefully acknowledged. the author is grateful for helpful discussions with professor bruce eilts on ways to improve the curriculum and the author acknowledges the usefulness of his on-line theriogenology curriculum at http://www.vetmed.lsu.edu/eiltslotus/theriogenology-5361/ which was frequently consulted when developing this curriculum. references 1. lonning ra, defranco tc, weinland tp: development of theme-based, interdisciplinary, integrated curriculum: a theoretical model. sch sci math 1998;98:312-318. 2. cavalieri j: curriculum integration within the context of veterinary education. j vet med educ 2009;36:388-397. 3. cavalieri j: veterinary student attitudes toward curriculum integration at james cook university. j vet med educ 2009;36:305-316. 4. james cook university school of veterinary and biomedical sciences: self evaluation report for the veterinary schools accreditation and advisory committee june 2010. http://www.jcu.edu.au/vbms/idc/groups/public/documents/staff_publications/jcuprd_056688.pdf. cited 2010 may 11. 5. root kustritz mv, tibary a, chenoweth pj: availability of theriogenology training at north american and caribbean veterinary colleges. j vet med educ 2006;33:140-144. 6. root kustritz mv, chenoweth pj, tibary a: efficacy of training in theriogenology as determined by a survey of veterinarians. j am vet med assoc 2006;229:514-521. 7. entwistle k, fordyce g: evaluating and reporting bull fertility: indooroopilly (qld): australian association of cattle veterinarians; 2003. 8. jubb tf, fordyce g, bolam mj, et al: trial introduction of the willis dropped ovary technique for spaying cattle in northern australia. aust vet j 2003;81:66-70. 9. race p: the lecturer’s toolkit, 2nd ed. abingdon (uk): routledge falmer; 2001. 139 clinical theriogenology • volume 3 number 2 • june 2011 table 1. names of subjects and units of study allocated to individual subjects which are undertaken by students during the bachelor of veterinary science course at james cook university. year study period subjects units 1 1 veterinary professional life 1 3 chemistry: an enabling science 3 cell biology and biochemistry for agricultural and veterinary science 3 biological principles for agricultural and veterinary science. 3 1 2 veterinary professional life 2 3 plant biology for agricultural and veterinary science 3 animal biology for agricultural and veterinary science 3 physiology and pharmacology for veterinary science 3 2 1 integrated animal structure and function 1 12 2 2 integrated animal structure and function 2 12 3 1 transitions from health to disease: identification and management 1 12 3 2 transitions from health to disease: identification and management 2 12 4 1 veterinary clinical sciences 1 12 4 2 veterinary clinical sciences 2 12 5 1-3, 6 veterinary clinical and professional practices 24 table 2. thematic structure of subjects undertaken by students in years 2 and 3 of the veterinary science degree program at james cook university.3,4 theme focus structure and function diversity of living organisms structure and function of plants and animals techniques that are available to study plants and animals disease, defence and chemical agents molecular and cellular basis of disease processes, organisms and bodily defence diversity of organisms that cause disease pharmacology animal production, management and behavior animal production, welfare and behavior veterinary services epidemiology and public health veterinary practice knowledge, skills and attitudes required for veterinary practice professional life self understanding, management and personal development team skills communication skills conflict resolution information literacy and lifelong learning skills practice management/business skills 140clinical theriogenology • volume 3 number 2 • june 2011 table 3. year 5 rotations.4 first half of the year duration (weeks) second half of the year duration (weeks) small animal emergency practice 2.0 equine practice or small animal practice (first accession) — whichever not completed in the lockstep phase 2.0 small animal practice (first accession) 2.0 small animal emergency practice 2.0 small animal medicine and imaging 2.0 dairy cattle and mixed practice (malanda 2) 2.0 surgery and anesthesia 2.0 abattoir 0.8 equine practice 2.0 electives 8.0 dairy cattle practice (malanda 1) 2.0 beef cattle and small ruminants 2.0 composite 1 (epidemiology, veterinary public health, intensive animal production and aquaculture) 2.0 composite 2 (behavior, canine theriogenology, clinical microbiology, toxicology, parasitology, immunology, virology and wildlife, avian, reptiles and exotics) 2.0 table 4. extramural practice requirements.4 nature of work minimum period (weeks) year ‘seeing practice’ – five consecutive days of extramural work experience in an approved veterinary practice prior to entering year 2 1 1 farm/animal industry – 12 weeks of farm/animal industry extramural work experience following completion of the year 1 and prior to the completion of year 3 12 2 to 3 clinical placements – 12 weeks of extramural work experience in approved veterinary premises, working alongside registered veterinarians following completion of year 3 and prior to the commencement of year 5 12 4 141 clinical theriogenology • volume 3 number 2 • june 2011 table 5. reproduction curriculum – year 2. lecture topic hours practical title hours sex determination; hormones 2 reproductive anatomy and histology 3 female reproductive tract and function 1 reproductive biology – hyperactivation of sperm and examination of embryos 3 folliculogenesis 1 prevention of reproduction 4 estrous cycles; ovulation; corpus luteum 1 semen evaluation 4 male reproductive system 1 calf castration, dehorning, branding and bull bse 2 spermatogenesis 1 regulation of gonadal function 1 maturation of the hypothalamic-pituitarygonadal axis and puberty 1 action of steroids on adults 1 sperm transport and fertilization 1 embryo development and placentation 1 maternal recognition of pregnancy of pregnancy 2 parturition 1 olfaction and reproduction 1 reproduction in birds 1 development of the reproductive system 1 table 6. reproduction curriculum – year 3. lecture topic hours practical title hours pregnancy diagnosis in the cow 2 pregnancy diagnosis – examination of abattoir specimens 3 pregnancy diagnosis in the mare 1 examination of the reproductive tract of the mare 11 introduction to obstetrics 2 pregnancy diagnosis in cattle – examination of non-pregnant cows 6 pregnancy diagnosis in the bitch, queen, sow, ewe and doe 1 pregnancy diagnosis in cattle examination of pregnant cows 6 to 16 weeks 5 introduction to obstetrics 3 pregnancy diagnosis in cattle – examination of pregnant cows >20 weeks 3 sheep week – industry visitation including pregnancy diagnosis, husbandry, flock reproductive performance and genetic selection of rams 6 142clinical theriogenology • volume 3 number 2 • june 2011 table 7. equine reproduction curriculum – year 4. lecture topic hours practical title hours castration 1 bse mare (includes synchronization of estrus, culture, cytology & biopsy) 3 equine estrous cycle, anatomy, and estrous behavior 1 diagnosis and treatment of endometritis (includes caslick’s), interpret lab results from mare bse practical, uterine treatment and lavage 3 manipulation of the estrous cycle of mares 1 bse stallion 3 artificial insemination and embryo transfer in mares 1 non-infectious causes of infertility 1 infectious causes of infertility – endometritis 1 problems during pregnancy 1– early embryonic loss, non-infectious causes of pregnancy loss 1 problems during pregnancy 2– infectious causes of pregnancy loss 1 periparturient and postpartum complications in the mare 1 equine urogenital surgery 2 stallion reproductive physiology and bse 1 reproductive problems of stallions 2 assessment of reproductive performance of stallions 1 surgery of the male reproductive tract – stallion 1 table 8. canine and feline reproduction curriculum – year 4. lecture topic hours practical title hours estrous cycle review, estrous behavior, vaginal cytology 1 vaginal cytology 2 breeding management 1 breeding management case studies 2 estrous cycle control, induction of estrus, prevention and termination of pregnancy 1 bse in the male 2 disorders of pregnancy and abortion 1 disorders of the reproductive system 2 problems during pregnancy, periparturient and postpartum disorders 1 bse and reproductive disorders of the male 1 male and female reproductive surgery 2 surgical management of pyometra and cesarean section 1 143 clinical theriogenology • volume 3 number 2 • june 2011 table 9. porcine reproduction curriculum – year 4. lecture topic hours practical title hours the breeding herd 1 pig husbandry procedures 3 the farrowing herd 1 reproductive failure 1 table 10. livestock reproduction curriculum – year 4. lecture topic hours practical title hours overview of bovine estrous cycle and pregnancy 1 obstetrics – correction of malpresentations 3 detection of estrus in cattle 1 obstetrics – percutaneous fetotomy 3 synchronization of estrus 2 obstetrics – subcutaneous fetotomy 3 artificial insemination in cattle 1 artificial insemination (all species) 3 non-infectious causes of reduced reproductive performance in cattle 4 synchronization of estrus and detection of estrus 3 problems during pregnancy in cattle. infectious and toxic causes of reproductive loss 2 bse and semen morphology 2 problems during pregnancy in the cow – physical and metabolic problems 1 evaluation of reproductive records (dairy and beef) 3 induction of parturition and abortion in cattle, sheep, goats and pigs 1 pregnancy diagnosis revision 3 periparturient and postpartum disorders 1 cesarean section in ruminants 1 surgery of female reproductive disorders 1 obstetrics 3 evaluation of reproductive records cattle (dairy, beef) 1 cryopreservation of semen (multiple species) 1 embryo transfer in cattle 1 assisted reproductive technologies 1 bull reproductive anatomy, physiology and bse 1 disorders of the reproductive system of bulls 2 surgical correction of abnormalities of the male reproductive system and preparation of teaser animals 1 veterinary services to the sheep industry general principles of flock and herd investigation 1 investigating low lambing percentage 3 lamb mortality 1 camelid reproduction 1 144clinical theriogenology • volume 3 number 2 • june 2011 table 11. reproduction curriculum – year 5. topic hours canine vaginal cytology and breeding management – case studies 3 canine bse and semen processing 3 stallion bse and semen processing 3 mare breeding management, palpation and case studies 3 bovine pregnancy diagnosis 12 bull bse 6 beef cattle case studies 3 reproductive records analysis 3 elective cesarean section 3 table 12. examples of the major methods used for summative assessment. assessment description multiple choice/extended match questions brief knowledge or problem focused questions with multiple options and a single correct answer. designed to test objective knowledge item recognition questions are associated with images, sounds, videos are posed with 90 seconds allowed to answer questions that are posed scenario-based, short answer questions are presented in relation to case scenarios or problems. questions test objective knowledge, quantitative skills, problem solving and analytical skills multiple station assessment tasks clinical, analytical, professional and practical skills are assessed as students rotate through a series of 10-minute assessment stations viva voce interviews ten minute interviews are conducted with an external expert with the aim of exploring the depth of student knowledge and understanding procedures log list of clinical procedures linked to various species are signed upon satisfactory completion in year 5 145 clinical theriogenology • volume 3 number 2 • june 2011 2012: nutrition for optimum reproductive performance nutrition for optimum reproductive performance kara a. kolster springfield veterinary center, glen allen, va reproductive success has been defined as “the generation and weaning of an adequate sized litter of healthy viable puppies reflecting the genetic potential of both the dam and sire”. many factors work together to determine reproductive success: age and health status of sire and dam, inherent fertility of the breed and of individuals, type and timing of mating, and nutritional status of the dam during breeding, pregnancy and lactation. this is the most nutritionally challenging period in a bitch’s life and providing optimum nutrition will allow her to maintain condition while producing the healthiest possible puppies. the american association of feed control officials (aafco) publishes minimum standards for nutrient levels in animal foods. all commercial diets must meet these standards through feed analysis or feeding trials and specify compliance on the label. these standards are specific to life stage of the animal: adult maintenance, growth and pregnancy/lactation, or all life stages (defined as weaning through adulthood). adherence to aafco standards guarantees that an animal fed that diet is receiving an adequate balanced ration for its life stage. however, an important but undefined distinction exists between adequate and optimum nutrition. this difference can become evident during the physiologic stress of pregnancy and lactation. an adequate but suboptimal diet is unlikely to inhibit reproduction but can limit peak performance. concerns specific to the pregnant bitch include: adolescent bitches that are still growing themselves and thereby competing with fetuses for nutrition; bitches re-bred before recovering from a previous litter and are in a state of nutritional deficit at the onset of next pregnancy; and bitches with placental insufficiency on a diet that is adequate for the dam’s needs but resulting in decreased nutrient transfer to developing fetuses. when evaluating a diet for reproduction, it is important to remember that excess nutrients can cause as much harm as deficiencies. early work in canine nutrition showed that maintenance diets were inadequate for reproduction and that selective supplementation enhanced reproductive performance. bitches fed a maintenance diet showed no outward signs of disease or nutritional deficiency but experienced a higher incidence of perinatal mortality as compared to bitches fed a supplemented diet. this was most evident early in neonatal life, which suggests a prenatal cause. there was also a significant increase in puppy weight gain in supplemented bitches. nutrient deficiencies in the maternal diet have the greatest impact on fetal development during the pre-implantation period and the period of rapid placental development, both of which occur in the first third of pregnancy. deficiencies in maternal nutrition have also been associated with disease in adult progeny. the phenomenon known as “fetal programming” occurs when alterations in the fetal environment result in permanent changes to an individual’s structure, physiology and metabolism that can predispose them to disease in adult life. numerous epidemiologic studies have linked deficient maternal nutrition and intrauterine growth retardation (iugr) to cardiovascular disease in adult offspring. iugr in dogs is known to cause effects such as reduced weight gain in puppies post-weaning. glucose glucose is the primary fuel for almost all cells in the body. an animal’s blood glucose level is a reflection of glucose absorbed from food and glucose produced within the body. these systems work together to create homeostasis, a state of balance within the body. pregnant bitches have altered glucose homeostasis which, if nutritional stress and environmental factors combine, can affect reproductive performance. pregnancy affects blood glucose levels by 1) causing insulin resistance, which leads to a 557 clinical theriogenology • volume 4, number 4 • december 2012 decreased ability to absorb glucose from food, and 2) limiting the body’s ability to produce its own glucose in response to hypoglycemia (low blood sugar). for these reasons, hypoglycemia is common in late-term pregnancy. complicating factors such as decreased appetite, change in type of diet, or other external stress can turn this “normal” condition into a significant problem. seemingly minor changes in the quantity or quality of a bitch’s diet may affect litter size, fetal development, and birth weight. protein suboptimal levels of dietary protein cause reduced maternal weight gain during pregnancy, smaller litter size, iugr, and reduced birth weight of puppies. it is believed that it also causes permanent changes in the puppies’ physiology, as reduced weight gain continues post-weaning. however, excess dietary protein can decrease function of the placenta due to oxidative damage. the aafco minimum standard for protein in a gestation/lactation diet is 22%. more recent studies show that 29-32% protein is ideal. dogs may not be able to effectively utilize greater than 32% protein in their diet. the type and quality of protein is equally as important as the amount. the quality of a protein is related to the amount and balance of amino acids it contains. animal-based protein sources provide the most appropriate balance of amino acids. the exception is an animal protein source that contains primarily connective tissue, which is high in non-essential amino acids. biologic value (bv) is a measure of protein quality based on a food’s balance of essential amino acids. generally, the higher the bv of a protein, the lower the amount that needs to be fed in order to meet an animal’s amino acid requirements. the bv of an egg is set at 100 and all other protein sources are evaluated relative to this standard. mixing protein sources is a way of providing a complete and balanced complement of amino acids without feeding excessive amounts of protein. fat and docosahexaenoic acid essential fatty acids (efa) are important components of the nervous system, especially the brain, eyes, and blood vessels. they accumulate in the brain and retina during fetal and perinatal development. essential fatty acids are transferred from dam to offspring through the placenta and milk. docosahexaenoic acid (dha) is an omega-3 fatty acid. it can be formed in the adult liver but production in young animals is not sufficient to support optimal brain and eye development, so availability of dha in the dam's diet is crucial. maternal efa levels are naturally decreased during pregnancy and lactation, which substantiates the benefit of dietary supplementation during these times. animals that were deficient in dha early in life can experience developmental abnormalities with life-long effects. offspring of dams fed dha-enhanced diets showed improvements in neurologic development, learning and memory ability, and response to stress. both the amount and type of fat in the diet are important. litter size was larger in bitches fed diets with a higher percentage of fat and an optimal omega-6 to omega-3 fatty acid ratio. cold water fish, eggs, and organ meats are all sources of dha. for optimal reproductive success, an 18% fat diet with a 5:1 to 10:1 ratio is recommended. carbohydrates dogs do not generally require carbohydrates in their diet as long as the protein level is high enough to allow production of glucose within the body. however, glucose producing ability is compromised in pregnancy while nutritional demand is increased, setting the stage for significant deficiency if diet is not appropriate. an early study showed many negative effects of feeding a carbohydrate-free diet to pregnant bitches, including: hypoglycemia in dams, increased stillbirth and 558clinical theriogenology • volume 4, number 4 • december 2012 perinatal mortality, decreased mothering behavior, decreased milk quality, and decreased length of lactation. based on available information, the current recommendation is that 20-30% of a diet's calories should be derived from carbohydrates. it is important to remember that this information is not stated on the pet food label but can be calculated. higher levels of carbohydrates are not necessarily harmful, but may indicate that a diet is compromised on protein and other essential nutrients. vitamins and minerals the majority of information available regarding recommended vitamin and mineral levels for gestation/lactation diets is based on aafco standards, which specify minimum adequate but not necessarily optimum levels. most commercial foods contain approximately 250% of the aafco minimum standard, which is expected to be sufficient for all life stages including reproduction. increased intake of a balanced diet late in gestation accounts for increased nutrient needs; it is not necessary to change the diet profile for a pregnant bitch. the only valid reason to utilize dietary supplements is when the diet fails to supply the needed amount of a given nutrient. vitamins and minerals must be consumed in proper balance to maintain health and nothing is supplemented in pure form. for example, a 1.2:1 ratio of calcium to phosphorus is recommended, but a sufficient level of vitamin d is necessary to aid absorption. commonly supplemented meats provide extra protein but also contain much higher levels of phosphorus than calcium. cottage cheese also contains more phosphorus than calcium. it follows that adding unbalanced supplements to a balanced diet will result in unhealthy excesses or deficencies of nutrients. it is generally accepted that vitamins and minerals derived from whole food sources are preferred over inorganic supplements. ideally, animal-based protein and other whole foods (vegetables, grains, etc) should be high on the ingredient list of a pet food. nutriceuticals and supplements docosahexaenoic acid is the best studied dietary supplement in dogs. as discussed previously, it is beneficial at many points during reproduction, and is now commonly added to commercial growth diets. folic acid, also known as vitamin b9, plays a role in dna synthesis, neurotransmitter activity in the brain, and midline closure of the palate during intrauterine development. cleft palate results if fusion between left and right halves of the palate does not occur around day 33 of gestation. this is usually due to either a genetic cause or environmental exposure during pregnancy, such as excess alcohol, nicotine, vitamin a, or corticosteroids. cleft palates have been caused experimentally in puppies by administering a drug that blocks folic acid to the dam during days 25-28 of gestation. administering folic acid to women during the first trimester of pregnancy significantly decreases the risk that their babies will be born with a cleft lip or palate. similar results are seen in dogs where studies show that folic acid supplementation from 15 days prior to breeding through whelping decreased the incidence of cleft palates in boston terriers and french bulldogs. other supplements that may be beneficial to a dam’s health include probiotics and glucosamine. they may or may not provide any advantage to fetuses but are not expected to cause any harm. many commercial foods now include these supplements. there are many quality diets available to meet the nutritional needs of a bitch during gestation and lactation, and no single feeding recommendation is appropriate for all dogs. when selecting a food, consider the overall nutrient levels, the source and quality of ingredients, as well as supplements already 559 clinical theriogenology • volume 4, number 4 • december 2012 included in the diet. a complete and balanced diet does not generally require additional supplements, and adding them may result in an unbalanced diet. reproductive success requires a combination of health, fertility, pedigree, and proper nutrition. each of these factors should be optimized in order to reach peak reproductive performance. references and recommended reading donoghue s: nutrition column. akc gazette 2007-2010. greco ds: nutritional supplements for pregnant and lactating bitches. topics in companion animal medicine 2009;24:46-48. guilloteau a, servet e, biourge v, et al: folic acid and cleft palate in brachycephalic dogs. waltham focus 2006;16:30-33. heinemann km, bauer je: docosahexaenoic acid and neurologic development in animals. j am vet med assoc 2006;228:700705. hoffman l, kelley rl, waltz d: for smarter more trainable puppies: effect of docosahexaenoic acid on puppy trainability. iams company, lewisburg, oh. johnson ca: glucose homeostasis during canine pregnancy: insulin resistance, ketosis, and hypoglycemia. theriogenology 2008;70:1418-1423. kelley rl: canine reproductive management: factors influencing litter size. iams company, lewisburg, oh. langley-evans sc: fetal origins of adult disease. br j nutr 1999;81:5-6. langley-evans sc, sherman rc, welham sj, et al: intrauterine programming of hypertension. biochem soc trans 1999;27:8893. ontko ja, phillips ph: reproduction and lactation studies with bitches fed semipurified diets. j nutr 1958;65:211-218. platt bs, stewart rj: effects of protein-calorie deficiency on dogs: reproduction, growth and behavior. dev med child neurol 1968;10:3-24. reinhart g: fatty-acid balance influences dog litter size. iams company breeder symposium 2003. long beach, ca. romsos dr, palmer hj, muiruri kl, et al: influence of a low carbohydrate diet on performance of pregnant and lactating dogs. j nutr 1981;111:678-689. wu g, bazer fw, cudd ta, et al: maternal nutrition and fetal development. j nutr 2004;134:2169-2172. 560clinical theriogenology • volume 4, number 4 • december 2012 2016: effect of oocyte source and transport time on rates of equine oocyte maturation and cleavage after fertilization by icsi, with a note on the validation of equine embryo morphological classification effect of oocyte source and transport time on rates of equine oocyte maturation and cleavage after fertilization by icsi, with a note on the validation of equine embryo morphological classification n. lewis, k. hinrichs, k. schnauffer, m. morganti, c. mcg. argo school of veterinary medicine, faculty of health and medical sciences, university of surrey, uk abstract production of equine embryos using intracytoplasmic sperm injection (icsi) is rapidly gaining interest in the horse industry. due to increasing client demand, equine practitioners with limited experience in embryology are attempting to set up clinical icsi programs, with little success. we report here studies performed with the purpose of establishing an equine in vitro maturation (ivm)/icsi program. we addressed three objectives: to determine (1) the effect of oocyte source (transvaginal follicle aspiration [tva] vs. abattoir) on maturation, cleavage and blastocyst rates; (2) the impact of time of oocyte recovery (soon after death vs. delayed [median time 7.5 h]) on these parameters in abattoir-derived ovaries; and (3) the correlation of post-icsi embryo morphology with histologically-confirmed nuclear status. maturation rates were greater for tva-derived than for abattoir-derived oocytes (67% vs. 32%, respectively; p <0.01). duration of ovary transport did not affect oocyte maturation (34-48%) or cleavage rates (70-73%). chromatin staining revealed that light-microscopic evaluation was accurate in determining oocyte maturation to metaphase ii (14/15, 93%), but was not accurate in classification of either normal embryo cleavage (8/31 cleaved embryos possessed normal nuclei) or blastocyst formation (7/15 embryos classified as blastocysts were verified on staining). early embryo and blastocyst viability was confirmed by transfer of some embryos into recipient mares. these findings indicate that oocyte source (tva vs. abattoir) can affect results in an equine ivm/icsi system, and suggest that new laboratories should use a systematic approach of comparison of nuclear chromatin staining with morphological classification to validate embryo development after icsi. keywords: horse, icsi, blastocyst, oocyte, embryo introduction the ability to produce foals using icsi has the potential to improve our understanding of early embryo development and has clinical applications in the salvage of valuable equine genetics.1 in mares, this is either following post-mortem oocyte recovery or by collecting oocytes from living sub-fertile mares that cannot provide an embryo for transfer2,3 and in stallions, by offering a mechanism to produce embryos and foals when only limited reserves of semen are available. the in vitro production of viable blastocysts and subsequent pregnancies, encompasses many complex and interdependent procedures, as well as requiring knowledge of cell culture and embryological techniques, such that the development of a successful equine icsi program has been described as “practically unattainable in most situations”.4 interest in the use of equine icsi in both commercial and research applications has rapidly increased worldwide, and client demand has resulted in an increasing number of equine practitioners and equine laboratories with limited experience in in vitro embryo production embarking on the establishment of commercial equine icsi laboratories. given the challenges, successes have been limited. groups that report blastocyst development after equine icsi typically achieve a < 10% blastocyst rate per injected in vitro-matured (ivm) oocyte.5-8 although it is generally understood that equine icsi is feasible, in reality, only a few laboratories have reported high blastocyst rates (>20%) using ivm oocytes9-12 and only two of these have reported >20% blastocyst rates for this procedure more than once (~25-43%).3,11-15 the rate of oocyte maturation is another major determinant of commercial success. if blastocyst rates are low (<10%), production of one blastocyst would take over ten mature (metaphase ii [mii]) oocytes. for many laboratories, oocyte recovery and in vitro maturation rates currently conspire against this. rates of in vitro maturation are variable (44 to 61%), particularly when collecting oocytes post-mortem, as in genetic salvage cases.11,16 clinically, in the case of genetic salvage, a mare often dies or is euthanized at a location distant from the icsi facility. under these circumstances ovaries/oocytes must be optimally retrieved and transported to the icsi facility. despite anecdotal reports, currently, only a single report has 1623292525 clinical theriogenology · volume 8, number 1 · march 2016 detailed the clinical production of foals by icsi under these conditions. in that report, pregnancies were obtained following ovary removal at the location of death and transport of both whole ovaries or recovered oocytes.2 another aspect affecting development of an icsi program is the ability of the practitioner to appropriately identify viable embryos. while some laboratories have validated their classification of embryos as blastocysts by chromatin staining for nuclear status or by achieving high pregnancy rates (>50%) after transfer, some laboratories have assessed blastocyst development based only on morphological appearance.3,6,12,17,18 the morphology of in vitro produced (ivp) equine blastocysts differs from both the morphology of ivp blastocysts in other species and from that of equine in vivoderived blastocysts, thus identification of viable embryos may be problematic for the inexperienced eye. however, to the best of our knowledge, other than early reports from the laboratory in texas which reported both morphological cleavage and cleavage with normal nuclei (41% to 88% of morphologically cleaved embryos had normal cleavage on nuclear evaluation),19-21 essentially no information is available on the relationship of light-microscopic morphological classification to actual embryo status as shown by evaluation after nuclear staining. in this study, we performed three evaluations to establish basic parameters for the development of an equine icsi program. we determined (1) the effect of oocyte source (tva from live mares vs. abattoir-derived) on maturation, cleavage and blastocyst rates after icsi; (2) the relative impact of oocyte recovery soon after death as opposed to a delay following the transport of entire ovaries and (3) the correlation of post-icsi embryo morphology on light microscopy with histologicallyconfirmed nuclear status. materials and methods all procedures were conducted in agreement with the principals of the university of liverpool veterinary ethics committee and with the approval of the royal college of veterinary surgeons. oocyte recovery from abattoir-derived ovaries ovaries were obtained from mares, of unknown age and breed, slaughtered at a uk abattoir for purposes unrelated to the study. slaughter was conducted in accordance with eu legislations ec 852/2004, 853/2004 and 854/2004. ovaries were obtained within 15 min post-mortem, placed in a polythene bag within a polystyrene foam container and maintained at ambient temperature (18-20°c) until all tissues had been processed. ovaries were either transported to the icsi laboratory (transport time 4 hr) or to a local facility as dictated by the study protocols detailed below. on arrival, all visible follicles were aspirated using a 14 g needle and vacuum pump (rocket medical plc., watford, herts, uk) set at approximately 200 mmhg. all plastics (mila international, erlanger, kentucky) were sterilized in ethylene oxide with a minimum gas-off time of 4 wk. a syringe and needle were used to flush each follicle one to two times with flush medium (m199 with hanks salts, 0.4% fetal bovine serum (fbs) (life technologies ltd, paisley, uk), 25 μg/ml gentamicin, 8 iu/ml heparin (sigma-aldrich ltd, gillingham, dorset, uk). ovaries were then sliced at 5-10 mm thickness and additional visible follicles were aspirated and scraped using the 14 g needle with vacuum. aspirated fluid was collected into sterile 500-ml bottles and passed through an embryo filter (emcare icpbio reproduction, auckland, nz). the filter was rinsed with flush medium and cumulus-oocyte complexes (cocs) were located in the recovered tissue under a dissection microscope at 60-120x. identified cocs were moved to manipulation medium (m199 with hanks salts, 10% fbs, 25 μg/ml gentamicin). all manipulations were conducted at ambient temperature (approximately 18-20°c). oocyte collection from live mares oocytes were recovered by tva from six sport-horse type mares aged 7-14 years. the tva procedures were conducted as previously described.10 all visible follicles > 0.5 cm were aspirated; each follicle was flushed up to six times with flush medium. the aspirated fluid was processed within 1-2 hr of aspiration, as described above for fluid aspirated from follicles in abattoir-derived ovaries. 1724302626clinical theriogenology · volume 8, number 1 · march 2016 oocyte holding recovered cocs were divided into groups of eight to 14 and placed in individual 1-ml glass vials (thermoscientific inc., waltham, ma) in eh medium (40% m199 with hanks salts, 40% m199 with earle’s salts, 20% fbs and 25 μg/ml gentamicin).22 vials were sealed and held overnight (12-18 hr) at room temperature (20°c) protected from light. oocyte maturation maturation dishes (pre-prepared with medium and oil) were pre-equilibrated for 12 hr before use. cumulus-oocyte complexes were removed from the eh holding vial, washed in maturation medium (m199 with earle’s salts, 10% fbs, 25 μg/ml gentamicin with 5 mu/ml fsh [sioux biochemical inc., sioux center, ia]) and incubated in groups of 9-12 at a ratio of 10 μl medium per coc in a 4-well dish (thermoscientific), under mineral oil (vitrolife ltd., warwick, uk) for 30 hr at 38.3°c in 5% co2 in air. oocytes were then denuded of cumulus cells by repeated pipetting in manipulation medium containing 80 iu/ml hyaluronidase (vitrolife). oocytes were evaluated by light microscopy using a dissection microscope at 500x. those with a visible polar body were classified as metaphase ii (mii), oocytes with an intact oolemma without a polar body were classified as intact and those with an irregular oolemma or shrunken cytoplasm were classed as degenerating (deg). confirmation of correlation of oocyte morphological classification with chromatin status was performed in experiment 3. icsi and embryo culture frozen-thawed sperm from one fertile stallion was used for icsi. for each icsi session, a single 0.5-ml straw of semen was thawed at 37°c for 30 sec. to perform swim-up, 200 μl of thawed semen was placed under 3 ml of frozen-thawed sp-talp23 and incubated at 37°c for 30 min. sperm suspension from the top of the sp-talp layer was used for icsi. conventional icsi was performed using an integra micromanipulator (research instruments, falmouth, uk) by an embryologist (ks) with extensive experience in human clinical icsi. a standard sharpened icsi needle (5 μm inner diameter; research instruments) was used. denuded mii oocytes were placed in individual 10-μl droplets of manipulation medium and 1-2 μl of sperm suspension was placed in a separate 5-μl droplet of a commercial 9% pvp solution (icsitm; vitrolife). motile spermatozoa were immobilized by crushing of the flagellum with the icsi needle until kinking was observed and aspirated into the icsi needle flagellum first. the oocyte was positioned with the polar body at 6 o’clock and the icsi needle was advanced through the zona pellucida and into the oocyte cytoplasm, essentially to the opposite side of the oocyte. puncture of the oolema was confirmed by visualization of cytoplasmic contents in the icsi needle after suction and the spermatozoon was then deposited in the cytoplasm with a minimum of medium. micromanipulator platform temperature was controlled in relation to ambient temperature in an attempt to perform all icsi manipulations at 37°c. injected oocytes were washed twice in pre-equilibrated culture medium (dmem-f12 [sigma aldrich] with 10% fbs and 25 μg/ml gentamicin)12 to remove manipulation medium and were placed in 20-μl droplets of culture medium under oil (four to ten injected oocytes/droplet, i.e. 2-5μl per injected oocyte) in a 4-well dish, (thermoscientific) at 38.3°c in 5% co2, 5% o2 and 90% n2. except when noted below, embryos were assessed for cleavage on day 3 (day 0 = day of icsi), at which point uncleaved oocytes were moved to a different droplet within the same dish. medium in the original droplet was refreshed by adding 20 μl of fresh culture medium and then removing 20 μl. from day 7 to day 11, embryos were assessed for blastocyst development daily by visualization under an inverted microscope at 200x. embryos were classified as blastocysts morphologically based on an increase in diameter from the previous day and a multicellular appearance with presence of an apparent uniform outer cell layer. transcervical embryo transfer embryos were loaded into 0.25 ml straws using pre-equilibrated culture medium, warmed to 38.3°c. a stainless steel gun with a disposable sheath (both imv technologies, france) was used for the transfer. altrenogest (regumate, msd animal health, buckinghamshire, uk; 0.044 mg/kg bwt po q 24 hr) was administered to each recipient mare beginning on the day of transfer. pregnancy 1825312727 clinical theriogenology · volume 8, number 1 · march 2016 diagnosis was first performed via transrectal ultrasonography on day 14 (day 0= day of icsi). if an embryonic vesicle was not present, examinations were repeated every 2-3 days until day 20 at which point the mare was declared nonpregnant and altrenogest treatment was discontinued. if an embryonic vesicle was visualized on one of these examinations, examination was repeated on days 21, 28, 45, 60 and 90. altrenogest treatment was continued until day 120. oviductal embryo transfer transfer of embryos to the oviduct was performed by flank laparotomy under standing sedation as described previously,24 with the following major modifications: 1) a 15-cm oblique incision was made in the paralumbar fossa contralateral to the site of ovulation, beginning just ventral to the cranioventral aspect of the tuber coxae and extending cranially and ventrally perpendicular to the last rib; 2) injected oocytes/embryos were loaded into a modified 6 f dog urinary catheter attached to a 1 ml all-plastic syringe with < 0.5 ml of culture medium for transfer. evaluation of oocytes and embryos for chromatin status oocytes or presumptive embryos were briefly fixed in buffered formal saline and mounted on a slide with 7 μl mounting medium (9:1 glycerol:pbs) containing 5 μg/ml hoechst 33258 and examined by fluorescent microscopy (range 350-461nm) at 400x. oocytes were classified as mii, mi, gv (having an intact nucleus) or degenerating (no chromatin or abnormal chromatin). zygotes were evaluated for presence of two pronuclei and two polar bodies. cleaved embryos were evaluated to determine the number and status of nuclei. only nuclei that appeared to be normal were included in the number of nuclei recorded; nuclei with signs of degeneration (vacuolization, condensation or fragmentation) were disregarded. embryos were confirmed to be blastocysts if they contained more than 64 normal nuclei and showed arrangement of an outer rim of nuclei in a presumptive trophoblast layer. preliminary study. to establish oocyte collection and maturation procedures, ovaries were obtained from the abattoir and transported to the main laboratory for processing. after eh holding, oocytes were placed in culture in maturation medium as described above, then evaluated morphologically for presence of a polar body after 30 hr culture. experiment one: the effect of oocyte source (abattoir vs. in vivo) on maturation, cleavage and blastocyst rates after icsi. oocyte maturation rates recorded in the preliminary study were low. this study was conducted to determine whether the oocyte source affected maturation rates. oocytes were obtained from abattoir-derived ovaries as described above, or from live mares via tva. after ivm, the proportion of oocytes having polar bodies was recorded for each treatment, and these oocytes were then subjected to icsi. injected oocytes were cultured, and evaluated for blastocyst development from day 7 to day 11. embryos classified morphologically as having developed to blastocyst were either transferred transcervically to the uterus of a recipient mare, or were stained for evaluation of chromatin status. experiment two: effect of ovary transport time on maturation, cleavage and blastocyst rate after icsi. the maturation rate was higher with in vivo-derived oocytes in experiment 1. it was hypothesized that the low maturation rates found in abattoir-derived oocytes in experiments 1 and 2 might be related to the delay during transport of the ovaries to the laboratory. to test this hypothesis, abattoir-derived ovaries were divided into two treatments. group 1: local facility (loc) ovaries collected from mares slaughtered from 7 am to 1 pm were processed at a facility near the abattoir. recovered oocytes were placed into eh medium as described for overnight holding above, then transported to the laboratory. group 2: lab, ovaries from mares slaughtered from 1-5 pm were collected and transported to the main laboratory for processing. recovered oocytes were placed into eh medium for overnight holding. after ivm and icsi, injected oocytes were examined from day 7 to 11 of culture for development to blastocyst, at which time all presumptive embryos were stained for evaluation of nuclear status. in one replicate, for the purpose of evaluating embryo viability, at 66 hr after icsi, the embryos showing the greatest degree of development (uniform morphological cleavage and > six 1926322828clinical theriogenology · volume 8, number 1 · march 2016 cells) as assessed under light microscopy were transferred to the oviduct of a recipient mare on day 1 after ovulation. experiment three: correlation of embryo morphology with nuclear status. staining of embryos at days 7 to 11 in experiments 2 and 3 suggested that morphological classification was not accurately reflecting the nuclear status of embryos. to improve our understanding of the association between equine embryo morphology and nuclear status, a systematic, sequential embryo staining study was performed. oocytes recovered from abattoir-derived ovaries and processed at the main laboratory were matured in vitro, then subjected to icsi and embryo culture as described above, with the modifications that 0.27mm pyruvate was added to the oocyte injection medium and the sperm preparation method was changed to direct swim up.10 staining and assessment of chromatin status was performed at the following times: a) immediately after ivm in oocytes classified as mii on the basis of presence of a polar body; b) at 20 hours post-icsi to evaluate pronuclear status; c) at day 2, 3, 4, or 7 after icsi to determine nucleus number and correlation of morphological cleavage classification with nucleus number; and d) after seven to 11 days of culture. in group d, embryos classified morphologically as blastocysts were stained and evaluated on the day they were identified, then all remaining presumptive embryos were stained and evaluated on day 11 of culture. two day-9 presumptive blastocysts from this group were not stained but were transferred to the uterus of a recipient mare five days after ovulation to evaluate viability. statistical analyses differences in rates of oocyte maturation, cleavage, and blastocyst development were compared among groups by chi-square analysis, with fisher’s exact test used between treatments when a value of less than ten was anticipated for any parameter. results preliminary study five replicates were performed, and 49 oocytes were recovered and subjected to ivm. the rate of maturation to mii was 39%. experiment one: effect of oocyte source (abattoir vs. in vivo) on maturation, cleavage and blastocyst rates after icsi fifteen tva procedures were performed, and 99 oocytes were recovered (table 1). three replicates were performed with abattoir-derived ovaries. overall maturation rates were greater for oocytes obtained by tva than for oocytes from abattoir-derived ovaries (67 % vs. 32%, respectively; p <0.01). there was no significant difference between groups in morphological cleavage rate after icsi (50 -53 %). on day 8 after icsi, on visual appraisal, based on increased diameter and the apparent presence of a uniform outer cell layer, four embryos (two tva and two abattoir), were considered to have developed to the blastocyst stage. three of these were transferred transcervically (one single [abattoir]; one double [tva]) to the uteri of two recipient mares six days after ovulation. no pregnancies resulted. the fourth presumed blastocyst was stained with hoechst 33258; on evaluation, this proved to be a degenerated embryo. experiment two: effect of ovary transport time on maturation, cleavage and blastocyst rates after icsi three replicates were performed (264 oocytes). the time from mare death to placement of oocytes in eh media for the loc treatment ranged from 0.54 hr (median 2.25 hr); in the lab treatment this ranged from 5-10 hr (median 7.5 hr). three metaphase ii oocytes were lost during manipulation and not injected. there was no significant difference in oocyte maturation or cleavage rates between loc and lab treatments (table 2). transfer of the four most advanced embryos at 66 hr culture in one replicate resulted in two embryonic vesicles (4 mm and 5 mm in diameter) detected by transrectal ultrasonography of the recipient mare on day 17 after icsi. these vesicles were lost by day 24. none of the 61 cleaved 2027332929 clinical theriogenology · volume 8, number 1 · march 2016 embryos cultured to day 11 were classified as blastocysts morphologically. however, on evaluation after staining, one blastocyst was identified in this group based on the presence of > 64 cells and an organized outer layer of nuclei. experiment three: correlation of embryo morphology and nuclear status staining and evaluation of chromatin status was performed at sequential steps to validate morphological classification of oocyte and embryo status. in one replicate, 18 oocytes presumed to be in mii based on presence of a polar body were stained to confirm meiotic status; three were lost during manipulation and 14/15 (93%) were confirmed in mii (fig. 1a). a total of 109 oocytes were subjected to icsi. over four replicates, the rate of pronuclear (pn) formation at 20 hr after icsi was 43% (10/23 oocytes; fig. 1b); a further eight injected oocytes were lost during processing. over six replicates, 46 presumptive embryos were stained on day 2, 3, 4, or 7 after icsi. of these, 31 (64%) demonstrated apparent morphological cleavage, but only eight of these (17%) had two or more normal nuclei on staining. in 3/46 embryos (6.5%), the number of nuclei matched the number of visible blastomeres and were appropriate for age (fig. 2a); five embryos possessed two to six normal nuclei, but nucleus number was lower than expected for day.16 the other 23 stained embryos that appeared cleaved morphologically possessed only degenerated nuclei or were completely anuclear (fig. 2b). in five replicates, presumptive embryos were cultured to determine blastocyst development (table 3). seven metaphase ii oocytes were used in other studies and not injected. after culture for 7 to 11 days, 17 presumptive embryos were classified as having developed to the blastocyst stage based on morphological criteria. of these, 15 were stained for nuclear evaluation and two were transferred to the uterus of a recipient mare (results below). evaluation of the 15 presumptive blastocysts after staining revealed seven confirmed blastocysts (e.g. fig. 3a1-3, fig, 4) and eight degenerating embryos (e.g. fig, 3c1-2). in these replicates, at day 11 all remaining structures (not classified as blastocysts) were stained, and one of these was also revealed to be a blastocyst (fig, 3d1-2). notably, as uncleaved oocytes were placed in a separate droplet at day 4 but were kept in culture, we could evaluate changes in these oocytes over time. several known uncleaved oocytes increased in diameter on day 9 (e.g. fig. 3b1), which, on simple morphological evaluation, could have led to mistaken classification of these structures as blastocysts (fig. 3b2-3). transcervical transfer of two day-9 presumptive blastocysts in the final replicate, to the uterus of a recipient mare five days after ovulation resulted in two embryonic vesicles (3 mm and 10 mm in diameter) detected by transrectal ultrasonography on day 14 after icsi. the smaller vesicle was manually reduced at day 16 and the remaining vesicle developed normally. a healthy colt foal was born at 345 days gestation (fig. 5). overall, in these five replicates, including the two transferred embryos, the rate of “blastocyst” development based on morphological assessment was 17/138 (12.3%); however, the confirmed blastocyst development per injected oocyte was 10/138 (7.2%). discussion this study highlights factors that may affect the development of an effective equine icsi program. we found an increased rate of meiotic maturation in oocytes derived from live mares by tva compared to oocytes recovered post-mortem. the maturation rate of post-mortem derived oocytes was not affected by ovary transport time (up to 10 hr). notably, when assessing embryo development in the first two experiments, we found discrepancies between our morphological classification of the embryos on light microscopy and the findings on staining for nuclear evaluation. this led us to evaluate the accuracy of our morphological classifications via step-wise oocyte and embryo staining in exp. 3. the rate of nuclear maturation in our preliminary study with abattoir-derived oocytes was low (39%). to determine if this was related to oocyte source, or to our untested ivm system, we compared maturation rates of tva vs. abattoir-derived oocytes. this showed that with tva oocytes the ivm system produced acceptable rates of maturation to mii (67%) whereas maturation remained low for oocytes from abattoir-derived oocytes, serving to validate the ivm system. we then evaluated whether the long transport time was associated with the low maturation rates in abattoirderived oocytes; however, maturation remained low for oocytes collected after a median time of 2.25 hr or 7.5 hr. it is possible that changes in oocyte meiotic competence had already occurred within our 2128343030clinical theriogenology · volume 8, number 1 · march 2016 shortest handling duration (2.25 hr in the loc group), thus lowering the maturation rates. a study by guignot et al. also found no difference in maturation rates across transport times (1.54 hr vs. 6-8 hr);26 while hinrichs et al. did find a higher maturation rate in oocytes collected < 1 hr of slaughter than in oocytes collected 5-9 hr after slaughter. due to abattoir location and available processing facilities, the loc group represented the most rapid processing time possible in this study. of course, transport time is only one factor affecting rates of meiotic maturation and developmental competence in abattoir-derived ovaries, others including temperature of transport, mare age and health, and exposure of the ovaries to toxic compounds at the abattoir. several studies have investigated optimal transport temperatures for equine ovaries and it was suggested that for periods < 1 hr, ovaries should be kept at 30-37°c but for longer periods, room temperature (22-24°c; as in this study) was more appropriate and resulted in the highest developmental competence.27-29 the final blastocyst rate (number of confirmed blastocysts per injected oocyte) achieved was 7.2%, which is similar to that reported by some established icsi laboratories6,8 but lower than optimal (20-40%).3,10,30 all embryos in this study were produced by conventional icsi. historically, the first equine icsi pregnancy was produced using conventional injection and surgical transfer of early embryos.31 some laboratories applied chemical oocyte activation due to low cleavage rates after icsi,1 although soon after, workers employing piezo drill-assisted icsi indicated that chemical activation was unnecessary.19 on the basis of successes with the piezo drill,12,21,33 it has been hypothesized that this technique is an important aid to equine icsi. in the only study in which piezoassisted and conventional (laser-assisted) icsi were directly compared, no significant difference was recorded, however, blastocyst rates per injected oocyte were low for both methods (4.8% and. 5.1%, respectively).6 the same authors have reported a live foal produced following conventional icsi.34 that study, and ours report blastocyst development at a much lower rate (7.4 % and 7.2% respectively) than do those that use piezo-assisted icsi (> 25%),10,11,14 but clearly there are a myriad of other potential differences among these laboratories. there is a need for a systematic, prospective study within the same laboratory to establish whether piezo-assisted icsi offers a significant advantage over conventional icsi. this is important, as the inclusion of the piezo drill represents a significant additional investment for new laboratories and introduces an additional requirement for technical expertise. difficulties were experienced in correctly interpreting the morphology of cleaved embryos. initially, we used a human grading system, which was shown not to be appropriate for application to equine embryos. the systematic staining of cleaved embryos revealed that only three of 31 embryos, empirically classified as morphologically cleaved, actually possessed the number of nuclei appropriate for age, and we found that many embryos contained anuclear blastomeres.25 the significance of this latter finding is unclear. a recent study in humans reported that presence of anuclear blastomeres had no significant effect on birth rate.35 blastocyst identification was also problematic. even when interpretation was based on images and information from laboratories working in equine icsi,10,36 we still encountered discrepancies between morphological blastocyst appearance and nuclear status (figure 3). it was noted that several known uncleaved oocytes increased in diameter between days 7 and 9 after icsi, and these were shown to have no nuclear chromatin upon staining (figure 3b1-3). this phenomenon has not to the best of our knowledge been presented previously. certain time-dependent cell-cycle and development stages can occur independently of nuclear status,37 therefore it could be hypothesized that there are cytoplasmic developmental clock mechanisms driving oocyte expansion in the absence of cleavage. our findings reinforced the importance of removing uncleaved oocytes to limit uncertainty in later assessments of blastocyst development, and of staining embryos for dna to definitively establish blastocyst development. in practical terms, chromatin integrity cannot be evaluated in presumed blastocysts designated for transfer. here, the benefits gained from coupling morphological assessments with definitive evaluations of nuclear status were invaluable. morphology can be used to reliably select true blastocysts, as shown by the high initial pregnancy rate of ivp blastocysts after transfer reported by experienced laboratories (82/101, 82%;30; 9/13, 69%;3). in our case, morphological selection of two blastocysts in the final replicate of the study resulted in two embryonic vesicles. to the best of our knowledge, this is the first report to document the disparity between morphological and histological assessment in equine ivp embryos. when setting up an icsi 2229353131 clinical theriogenology · volume 8, number 1 · march 2016 laboratory, it is tempting, as we did in experiments 1 and 2, to transfer structures presumed to be embryos to recipient mares in an attempt to establish pregnancies and thus validate the program. however, in retrospect, this was counter-productive as evaluating morphology alone proved to be inaccurate and misleading. our data and the related figures from exp. 3 highlight the need for objective verification of embryo status. we hope that by sharing our experience of identifying discrepancies between morphological classification and actual embryo developmental stage, we have highlighted that morphology must be confirmed by a method such as nuclear staining when laboratories are initially evaluating their rate of blastocyst production. figure 1: photomicrographs of oocytes evaluated after ivm to validate mii status (a) and after icsi to validate fertilization status (b). a1, b1: bright field microscopy 300x; a2, b2: after staining with hoechst 33258, under fluorescence 400x. a1: mii oocyte with polar body (arrow); a2: metaphase plate (arrow) and polar body (closed arrow); b1: injected oocyte 22 hours after icsi b2: fertilization confirmed by visualization of two pronuclei (staining incomplete; arrows) and two polar bodies (closed arrows). 2330363232clinical theriogenology · volume 8, number 1 · march 2016 figure 2: photomicrographs of two embryos 96 hours after icsi. a1, b1: bright field 300x; a2, b2 after staining with hoechst 33258 400x. a1: apparent 10-cell embryo; a2: 9 normal nuclei and one anuclear blastomere (arrow); b1: apparent 8-cell embryo; b2: no normal nuclei. 2431373333 clinical theriogenology · volume 8, number 1 · march 2016 figure 3: photomicrographs of presumptive blastocysts at day 9 after icsi, showing discrepancies between morphological classification and nuclear status. a1 and b1: bright field images (60x) of presumptive embryos shown in panels a2-a3 and b2-b3, respectively, showing relative increase in diameter (arrows). b1-3; structure arising from uncleaved oocyte. left: bright field (300x); right, after hoechst 33258 staining (400x). a2: presumed blastocyst shown in a1, with presumptive trophoblast layer (outer layer of organized cells) and thinning zona pellucida (zp). a3: status as blastocyst (presence of >64 cells with organized outer layer of nuclei) confirmed after staining. b2: structure pictured in b1, having similar morphology to blastocyst; b3: structure confirmed as a degenerating uncleaved oocyte, with no visible chromatin. c1: presumed blastocyst (from cleaved embryo) due to increase in size and thinning of zp. c2: staining revealed structure to be degenerating. d1: presumed degenerating embryo (from cleaved embryo) due to irregular cytoplasmic outline and lack of size increase. d2: staining revealed structure to be blastocyst (penetration of the stain is incomplete). 2532383434clinical theriogenology · volume 8, number 1 · march 2016 figure 4: photomicrographs (bright field 300x) showing sequential development of a blastocyst on days 7 through 9 (a-c) after icsi (blastocyst status was subsequently confirmed by staining). an increase in diameter and in organization of the presumptive trophoblast layer can be seen. figure 5: photograph of icsi foal at 5 days old. editor’s note: the photographs in this manuscript are available in color in the online edition of clinical theriogenology. 2633393535 clinical theriogenology · volume 8, number 1 · march 2016 acknowledgement this project was supported by a technology strategy board, knowledge transfer partnership with r. matson and son of twemlows stud farm, shropshire uk and a bbsrc icase phd studentship. references 1. li x, morris lh, allen wr: influence of co-culture during maturation on the developmental potential of equine oocytes fertilized by intracytoplasmic sperm injection (icsi). reproduction 2001;121:925-932. 2. hinrichs k, choi y, norris jd, et al: evaluation of foal production following intracytoplasmic sperm injection and blastocyst culture of oocytes from ovaries collected immediately before euthanasia or after death of mares under field conditions. j am vet med assoc 2012;241:8-12. 3. galli c, colleoni s, duchi r et al: developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and icsi to embryo culture, cryopreservation and somatic cell nuclear transfer. anim reprod sci 2007;98:39-55. 4. foss r: application of equine oocyte recovery and assisted reproduction techiques to clinical practice. clin therio 2014;6:293-296. 5. alonso a, baca castex c, ferrante a, et al: in vitro equine embryo production using air-dried spermatozoa, with different activation protocols and culture systems. andrologia 2015;47:387-394. 6. smits k, govaere j, hoogewijs m, et al: a pilot comparison of laser-assisted vs piezo drill icsi for the in vitro production of horse embryos. reprod domest anim 2012;47:e1-3. 7. zaniboni a, merlo b, zannoni a, et al: expression of fluorescent reporter protein in equine embryos produced through intracytoplasmic sperm injection mediated gene transfer (icsi-mgt). anim reprod sci 2013;137:53-61. 8. galli c, duchi r, colleoni s, et al: ovum pick up, intracytoplasmic sperm injection and somatic cell nuclear transfer in cattle, buffalo and horses: from the research laboratory to clinical practice. theriogenology 2014;81:138-151. 9. matsukawa k, akagi s, adachi n, et al: in vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection. j reprod dev 2007;53:877-885. 10. foss r, ortis h, hinrichs k: effect of potential oocyte transport protocols on blastocyst rates after intracytoplasmic sperm injection in the horse. equine vet j 2013;45:39-43. 11. galli c, colleoni s, turini p et al: holding equine oocytes at room temperature for 18 hours prior to in vitro maturation maintains their developmental competence. j equine vet sci 2014;34:174-175. 12. hinrichs k, choi yh, love lb, et al: chromatin configuration within the germinal vesicle of horse oocytes: changes post mortem and relationship to meiotic and developmental competence. biol reprod 2005;72:1142-50. 13. colleoni s, lagutina i, lazzari g, et al: new methods for selecting stallion spermatozoa for assisted reproduction. j equine vet sci 2011;31:536-541. 14. jacobson cc, choi y-h, hayden ss, et al: recovery of mare oocytes on a fixed biweekly schedule, and resulting blastocyst formation after intracytoplasmic sperm injection. theriogenology 2010;73:1116-1126. 15. choi yh, love lb, varner dd, et al: effect of holding technique and culture drop size in individual or group culture on blastocyst development after icsi of equine oocytes with low meiotic competence. anim reprod sci 2007;102:38-47. 16. dell’aquila me, masterson m, maritato f. et al: influence of oocyte collection technique on initial chromatin configuration, meiotic competence, and male pronucleus formation after intracytoplasmic sperm injection (icsi) of equine oocytes. mol reprod dev 2001;60:79-88. 17. choi yh, varner dd, hartman dl, et al: blastocyst production from equine oocytes fertilized by intracytoplasmic injection of lyophilized sperm. anim reprod sci 2006;94:307-308. 18. choi yh, varner dd, love cc, et al: production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse. reproduction 2011;142:529-538. 19. choi yh, love cc, love lb, et al: developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa. reproduction 2002;123:455-465. 20. choi y-h, love cc, varner dd, et al: effects of gas conditions, time of medium change, and ratio of medium to embryo on in vitro development of horse oocytes fertilized by intracytoplasmic sperm injection. theriogenology 2003;59:1219-1229. 21. choi yh, love lb, varner dd, et al: factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection. reproduction 2004;127:187-194. 22. choi yh, love cc, varner dd, et al: equine blastocyst development after intracytoplasmic injection of sperm subjected to two freeze-thaw cycles. theriogenology 2006;65:808-819. 23. parrish jj, susko-parrish j, winer ma, et al: capacitation of bovine sperm by heparin. biol rev camb philos soc 1988;35:1171-1180. 24. hinrichs k, matthews gl, freeman da, et al: oocyte transfer in mares. j am vet med assoc 1998;212:982-986. 25. choi yh, harding hd, hartman dl, et al: the uterine environment modulates trophectodermal pou5f1 levels in equine blastocysts. reproduction 2009;138:589-599. 26. guignot f, bezard j, palmer e: effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation. theriogenology 1999;52:757-766. 27. ribeiro bi, love lb, choi yh, et al: transport of equine ovaries for assisted reproduction. anim reprod sci 2008;108:171-179. 2734403636clinical theriogenology · volume 8, number 1 · march 2016 28. love lb, choi yh, love cc, et al: effect of ovary storage and oocyte transport method on maturation rate of horse oocytes. theriogenology 2003;59:765-774. 29. preis k, carnevale em, coutinho da silva m, et al: in vitro maturation and transfer of equine oocytes after transport of ovaries at 12 or 22 degrees c. theriogenology 2004;61:1215-1223. 30. hinrichs k, choi yh, love cc, et al: use of in vitro maturation of oocytes, intracytoplasmic sperm injection and in vitro culture to the blastocyst stage in a commercial equine assisted reproduction program. j equine vet sci 2014;34:176. 31. squires el, wislon jm, kato h: a pregnancy after intracytoplasmic sperm injection. theriogenology 1996;45:306. 32. mohammadi-sangcheshmeh a, held e, ghanem n, et al: g6pdh-activity in equine oocytes correlates with morphology, expression of candidate genes for viability, and preimplantative in vitro development. theriogenology 2011;76:1215-1226. 33. lazzari g, crotti g, turini p, et al: equine embryos at the compacted morula and blastocyst stage can be obtained by intracytoplasmic sperm injection ( icsi ) of in vitro matured oocytes with frozen ± thawed spermatozoa from semen of different fertilities. theriogenology 2002;58:709-712. 34. smits k, govaere j: birth of the first icsi foal in the benelux. vlaams diergeneeskd tijdschr 2010;12:134-138. 35. fauque p, audureau e, leandri r et al: is the nuclear status of an embryo an independent factor to predict its ability to develop to term? fertil steril 2013;99:1299-1304.e3. 36. choi yh, roasa lm, love cc, et al: blastocyst formation rates in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection. biol reprod 2004;70:1231-1238. 37. day m, johnson m, cook d: a cytoplasmic cell cycle controls the activity of a k+ channel in pre-implantation mouse embryos. embo j 1998;17:1952-1960. 2835413737 clinical theriogenology · volume 8, number 1 · march 2016 ta b le  1 : t h e  ef fe ct  o f  o o cy te  s o u rc e  (a b at to ir  d er iv ed  v s.  t v a )  o n  m at u ra ti o n , c le av ag e  an d  b la st o cy st  r at es  a ft er  ic si .   tv a =  tr an sv ag in al  a sp ir at io n  f ro m  li ve  m ar es .  * =  p re su m e d  s ta tu s  b as ed  o n  m o rp h o lo gy . d ay  0 =  d ay  o f  ic si . m ar es  w er e  e xa m in ed  f o r  p re gn an cy  f ro m  d ay s  1 4 ‐2 0 . r ep lic at es   n   o va ri es    n   o o cy te s    n   m ii   n   in je ct ed   n   c le av ed *  n   b la st o cy st s*   n   em b ry o s  tr an sf er re d   (n , t im e  p o st  ic si )  p re gn an ci es   n   a b at to ir  d er iv ed   3   2 4   5 6   1 8  ( 3 2 % )a   1 8   9  ( 5 0 % )  2   1 , d ay  8   0   tv a   1 5   5 2   (2 6  m ar es )  9 9   6 6  ( 6 7 % )b   6 6   3 5  ( 5 3 % )  2   2 , d ay  8   0   2936 42 38 38clinical theriogenology · volume 8, number 1 · march 2016 ta b le  2 :   ef fe ct  o f  o va ry  t ra n sp o rt  t im e  o n  m at u ra ti o n , c le av ag e  an d  b la st o cy st  r at e  af te r  ic si .   *  =  p re su m ed  s ta tu s  b as ed  o n  m o rp h o lo gy .   b la st o cy st  r at es  b as ed  o n   co n fi rm at io n  b y  d n a  s ta in in g  w it h  h o ec h st  3 3 2 5 8  o r  p re gn an cy .   m ed ia n  t im e  d ea th ‐  eh  m ed ia   (h r)   o va ri es   n   o o cy te s  n   m ii   n   in je ct ed   n    c le av ed *  n   b la st o cy st s  n   em b ry o s  tr an sf er re d    (n , t im e  p o st  ic si )  p re gn an ci es   n   lo ca l f ac ili ty   (l o c )  2 .2 5   1 1 4   1 6 6   5 7 (3 4 % )  5 7   4 0  ( 7 0 % )  0   4  ( 6 6 h r)   2   la b o ra to ry   (l a b )  7 .5   8 6   9 8   4 1  ( 4 8 % )  3 8   2 5  ( 6 6 % )  1   0   0   ta b le  3 : r es u lt s  o f  d ay  1 1  s ta in in g  in  e xp er im en t  3 .   b la st o cy st  r at es  b as e d  o n  c o n fi rm at io n  b y  d n a  s ta in in g  w it h  h o ec h st  3 3 2 58  o r  p re gn an cy .   o va ri e s  n   o o cy te s  n   m ii   n   in je ct ed   n   c le av ag e  n   m o ru la e   n   p re su m ed   b la st o cy st s  n   c o n fi rm ed   b la st o cy st s  n   em b ry o s  tr an sf er re d   n   d ay  1 4   p re gn an ci es   n   2 1 4   3 2 6   1 4 5  ( 4 4 % )  1 3 8   8 2  ( 5 9 % )  3   1 7   1 0  ( 7 .2 % )  2   2   3037 43 39 39 clinical theriogenology · volume 8, number 1 · march 2016 2012: topics in canine neonatology and pediatrics topics in canine neonatology and pediatrics cynthia o’connor slade veterinary hospital, framingham, ma neonates puppy mortality can occur in utero, during whelping, after birth, in the first weeks of life, or after weaning and has been reported to be as high as 26%.1 however, about half of all puppies that fail to survive to weaning are stillborn or die within the first three days of life. factors implicated in these perinatal deaths include prematurity, in utero infection with viruses such as canine distemper and canine parvovirus, anatomic birth defects, birth trauma during difficult birth (dystocia), inadequate nutrition, maternal neglect, and environmental stresses. as a result, predisposing factors of perinatal death include factors such as difficult birth (dystocia), malformations from inherited disorders or congenital defects, low birth weight (15% or more lower than the average in the litter), maternal illness and lack of preventative health care (vaccination, parasite control, etc.), unfavorable environmental conditions, and infectious agents. a main focus point of intervention is prevention of predisposing factors as well as early identification and treatment of at risk neonates.2 prevention focuses on pre-breeding health and genetic screening of sire and dam, optimal husbandry, managing labor and delivery to reduce stillbirths, controlling parasitism, reducing infectious disease, preventing injury and environmental exposure, and optimizing nutrition of the dam and neonates.3,4 identifying at risk neonates table 1. an apgar scoring system for routine assessment of newborn puppy viability and short-term survival prognosis5 parameter 0 points 1 point 2 points activity, muscular tone flaccid moderate tone (flexion) in the extremities acive movement, flexion pulse, cardiac rhythm absent to under 110/min 110-220/min >220/min grimace, irritability reflex absent moderate movement responsive, easily irritable, vocalization appearance, mucous membrane color pallor, cyanosis moderate cyanosis pink respiration, respiratory pattern absent weak, irregular rhythm >15/min, regular rhythmic table 2. interpretation points stamina, vitality 0-3 weak 4-6 average, moderate 7-10 normal prompt identification of signs of neonatal distress can be extremely important for providing rapid treatment to less viable newborns. time is certainly a critical factor and the earlier intervention is applied, the more effective and successful that intervention will be. the recent addition of a validated apgar score system (above) for neonatal puppies aids in the identification of at risk neonates so that early intervention can be applied in an effort to reduce neonatal loses. the apgar score system was originally devised in 1952 by dr. virginia apgar as a simple and repeatable method to quickly assess the health of newborn children immediately after birth. it evaluates five criteria which are summarized using the acronym, apgar (appearance, pulse, grimace, activity, and respiration).5 551 clinical theriogenology • volume 4, number 4 • december 2012 it is also important to perform a complete physical examination on all newborns. evaluation should include the oral cavity and mucous membranes which should be moist and bright pink to red/hyperemic from fetal hemoglobin. there should be no evidence of cleft palate with a strong suckle reflex present. the haircoat should be full and clean as missing hair on toes and muzzle may indicate prematurity and meconium staining indicates in utero distress. evaluate the limbs, umbilicus, and urogenital structures for signs of congenital defects (i.e., patent urachus indicated by urine produced out of umbilicus).4,6 a normal neonate will squirm and vocalize when examined and then nurse and sleep quietly when returned to the dam. in addition to the above, a low birth weight is a significant risk factor for neonatal puppy death. birth weights of puppies are breed dependent. a puppy that is less than 15% of the average birth weight of littermates is at high risk. puppies should gain about 5-10% of their birth weight per day so that they double their birth weight by approximately ten days. puppies should also be approximately ten times their birth weight by six weeks of age. the cause of low birth weight or failure to thrive with poor growth is usually difficult to determine and may include factors such as prematurity, inborn errors of metabolism, congenital birth defects, infections, nutritional deficiencies, environmental stressors, and maternal neglect.2,7,8 neonatal and pediatric puppies are not little adults due to continued development following birth, there are significant differences in the physical findings of neonatal and pediatric puppies compared to adults. it is important to recognize these differences in both identifying normal from at risk puppies as well as in the plan for intervention, if needed.9 respiratory rate in neonates is lower and more irregular than adults with the average of 10-18 breaths per minute. a respiratory reflex is present in the genital and umbilical area resulting in stimulation of respiration for the first three days after birth.6 in addition, respiratory control mechanisms in neonates are immature and surfactant production in the lungs is produced just prior to birth. this creates a high susceptibility to hypoxia (low oxygen) in the newborn, particularly those that are premature. there are also significant differences with the cardiovascular system of the neonate. neonatal bradycardia (low heart rate) is most commonly associated with hypothermia (low body temperature) and hypoxia (low oxygen often from poor respiration).4,8 this is a key factor to understand with intervention, making sure the neonate’s body temperature and ventilation are addressed prior to administering medications to elevate heart rate. the cardiovascular system in the neonate is a lower pressure and volume system compared to adults. they have less peripheral vascular resistance and so are less able to react to environmental stressors. they also have a higher heart rate (160-200 beats/min) and cardiac output compared to adults. this elevated heart rate decreases as the puppy develops increased parasympathetic influences (vagal tone) at about four weeks of age. as a result, certain drugs such as atropine, isoproterenol, and dopamine have minimal effect on cardiac function until four to five weeks of age.10 dehydration is always a major concern as newborns are extremely susceptible since they are composed of higher percentage (75%-80%) of water with a large body surface area compared to adults. in addition, immature kidneys have a limited ability to concentrate urine. as a result, water turnover is two to three times that of an adult. unlike an adult, hydration status cannot be accurately assessed using skin turgor or tenting.11 estimates of the degree of dehydration must be determined by weight loss, dryness of the oral mucus membranes and eyes, as well urine specific gravity measurements (a rule of thumb that neonatal urine is a very pale yellow and the darker yellow it becomes, the more of a concern for dehydration). monitoring weight gain is a good indicator of health status. a mentioned above, on average, nursing puppies should double their weight in an average of ten days. in the first 24 hours following birth, it is not uncommon for puppies to not gain as rapidly or even maintain their birth weight while adjusting to life outside of the uterus. however, following the first 24 hours after birth, a normal puppy 552clinical theriogenology • volume 4, number 4 • december 2012 should gain 5-10% of their body weight per day or the puppy should gain two grams per kilogram (kg) of the expected adult weight per day.12 puppies that do not gain after 24 hours of birth or lose weight at any time should be identified as neonates at risk since weight loss is generally one of the first signs of dehydration. thermoregulation is problematic in the neonate and a common cause for death. the shivering reflex and peripheral vasoconstriction responses are not fully developed until at least two to four weeks of age. their relatively large body surface area and lack of insulating fat promotes rapid heat loss. they are very vulnerable to environmental stressors and rely on the ambient temperature, the dam's mothering instincts, and/or littermates for warmth. environmental temperature exposure can be critical as a healthy newborn can only maintain a body temperature 12°f greater than that of the surrounding environment, and so the environmental temperature must be high enough to facilitate maintenance of a body temperature of at least 96-98°f.13 hypothermia negatively impacts immunity, nursing, and digestion.14 the neonatal intestine slows in motility creating a dangerous ileus when body temperatures are below 96°f.15 do not feed a cold puppy! an accurate rectal temperature in neonates requires a digital thermometer that reads to 95°f or lower. humidity is also vital to prevent dehydration, especially at these high environmental temperatures. generally, 55-65% humidity helps to prevent dehydration. exogenous heat should be supplied as needed to prevent hypothermia. a note of caution with heating pads which run the risk of burning neonates incapable of moving away from excessively hot surfaces as well as variable heat, particularly with electric heating pads. below is a chart of the recommended temperature adjacent to neonates.10 these are main considerations for orphaned pups. if the dam is present and has great maternal behavior be careful not to make the dam’s environmental temperature too hot which may result in her discomfort or even neglect of the puppies thereby creating a serious problem. please keep in mind that the recommended temperatures adjacent to neonates are often accomplished with a draft free whelping box in a comfortably warm room with a very attentive dam. table 3. recommended environmental temperatures for neonates weeks of age normal rectal temperature of the newborn recommended environmental temperature immediately adjacent to neonate-major consideration if orphaned or hospitalized first week 96-98°f 85-90°f second week 97-99°f 80°f third week low normal adult 75°f fourth week low normal adult 70-75°f fifth week low normal adult 70°f ninety to 95% of the neonate's passive maternal immunity is derived from colostrum ingestion within the first 24 hours of nursing.16 incompletely developed immune systems during the first ten days of life make neonates vulnerable to systemic infection (most commonly bacterial and viral). failure of passive transfer is a high risk for neonatal mortality. after 12-24 hours, the immunoglobulins are no longer absorbed through the intestinal tract. if failure of passive transfer is suspected, plasma or serum should be given, paying close attention to method of treatment as it should not be given by mouth 1224hours after birth and alternative route of administration must be utilized.17 the normal neonate will sleep quietly and suckle every two to four hours. while it has been shown that a healthy fasting puppy can potentially maintain adequate blood glucose for 12-24 hours through physiologic mechanisms (such as glycogenolysis and gluconeogenesis), this is often not the case with an at-risk neonate who has fewer reserves and is at high risk of hypoglycemia (low blood sugar) when they do not nurse for even short time frames (three to four hours). in addition, it is not recommended to fast pediatric puppies for long periods of time (more than six to eight hours) as their 553 clinical theriogenology • volume 4, number 4 • december 2012 physiologic mechanisms for glucose regulation are not completely matured compared to adults. below is a chart with daily requirements for nutrition as well as fluids.6 neonates should be able to take their daily requirements in five to six meals during the first weeks of life equating to feedings every two to four hours. during the first week of life, puppies can ingest 10-15% of their body weight in milk necessitating the potential for smaller more frequent feedings during this time frame. after the first week of life, they are able in ingest 20-25% of their body weight in milk allowing for a gradual increase in volume and time between feedings.4 table 4. daily nutritional and fluid requirements weeks of age canine neonatal caloric requirements fluids in ml/100 gram body weight per day first week 133 kcal/kg/day 18 mls second week 155 kcal/kg/day 13-22 mls third week 175-198 kcal/kg/day 13-22 mls fourth week 220 kcal/kg/day 13-22 mls the gastrointestinal tract is sterile at birth but is rapidly colonized by variety of bacteria from the dam and the environment. neonates have altered intestinal permeability and higher gastric ph compared to adults. this creates different metabolism for oral therapies in neonates compared to adults. the soft, yellow brown meconium is passed within the first 48 hours. then the normal neonatal feces are pasty yellowish to brownish tan, but they are usually not observed with a fastidious bitch. the stools should become formed at weaning. overfeeding often produces a green or yellow watery stool; and white stools may be observed with lactose intolerance. foamy bright yellow stools may be present with illness such as viral or bacterial diseases. blood tinged stools may be seen in sepsis or with severe parasitism.7 diagnostic testing can be performed on neonatal fecal samples after stimulation to defecate and will aid in the decision for the potential need for treatment. liver function is significantly decreased for the first four weeks which is related to the underdeveloped liver enzyme systems that are generally not equivalent to adult liver function until approximately six months of age. due to immature liver metabolic functions, caution is advised in selection of drugs metabolized and/or excreted via the liver.6 while the numbers of functional nephrons in the kidneys are present at birth, physiological development, as measured by glomerular filtration rate and the ability to concentrate urine, is very immature until at least three weeks of age and not equaling adults until four to six months of age. in the neonate, the ano-genital reflex can be used to stimulate urination for collection and evaluation of urine. the normal specific gravity ranges from 1.006-1.017 (dilute and often very pale yellow in color) and is used to estimate hydration status and response to fluid therapy. mild glucosuria and proteinuria is considered normal for up to six weeks of age.4 the central nervous system development is slow in the neonate. they sleep at least 90% of the time. until four weeks of age, the majority of the sleep is deep, active sleep. active sleep tones and strengthens those muscles necessary to allow the neonate to stand at approximately 14 days then walk by day 21. active learning begins at three weeks followed by important socialization periods. reflexes can be difficult to assess in the neonate because of variations in development and the technical difficulty associated with their small size. puppies are born with a righting, withdrawal, and tactile anal and urinary reflexes. from birth to day three they demonstrate total flexor dominance but by day four extensor dominance predominates until three weeks of age. the suckling reflex is present until three weeks of age. the rooting reflex is present at birth. cross extensor reflexes may be normally present at birth but should not be present after 14 days. crossed extensor reflex after this time period is indicative of upper motor neuron pathology.10,18 neonates cannot voluntarily urinate or defecate. these functions are initially controlled by an ano-genital reflex associated with vigorous stimulation of the perineal area by the dam’s tongue or a wet 554clinical theriogenology • volume 4, number 4 • december 2012 swab. although this reflex is present for up to 28 days in puppies, stimulation is only required for the first 18-21days, thereafter the puppy is able to urinate and defecate on its own. generally the eyelids open at ten to 14 days revealing a cloudy cornea which clears within 24 hours. for the first three weeks vision is poor. the corneal reflex is present as soon as the eyes open however the menace reflex is poor to absent for the first four to 12 weeks. tear production begins when the eyelids open, which is an important consideration if there is a premature opening of the eyelids where lubrication will be needed to prevent eye trauma from dry eye and exposure. the true iris color is generally complete by approximately week eight (some breed variation noted). while the puppy can hear at birth, the external ear canals do not open until ten to 14 days at which time the startle response begins.19 the brain stem auditory response (baer) is the best method of evaluating a puppy’s hearing. routine testing using this evaluation of specific breeds at risk for deafness is generally performed after six weeks of age. identification of those at risk and treatment considerations the clinical approach to identifying disease in neonatal and pediatric puppies includes the need for collaborative efforts by the breeder, primary care veterinarian, and at times veterinary specialists. as with adults, the foundation is a thorough history and physical examination keeping in mind the physical and physiological differences of neonatal and pediatric puppies. additional queries regarding littermates, relatives, and other animals in the environment will assist with the collection of genetic disease, infectious disease, toxin, nutritional, and other important data to be considered in the investigation of disease presentations. diagnostic testing is generally required to further investigate the cause of illness in a diseased animal, and while there are some limitations as well as prioritization that may be needed due to the small size as well as physical differences in neonatal and pediatric puppies, many diagnostics can still be performed and are very beneficial in diagnosis of disease.20 for example, routine tests such as a complete blood cell count, chemistry screen, and urinalysis may suggest specific hematologic or metabolic disorders, and they may help to rule out many acquired disorders. a general rule of thumb is to not exceed 1 ml of blood loss per 100 grams of body weight per week in neonates and pediatric patients. radiology and other imaging techniques may reveal skeletal malformations, echocardiogram may reveal cardiac anomalies, and ophthalmologic examination may further define an inherited eye disease. based on these findings, additional clinical function testing or evaluations for inborn errors of metabolism may be required for diagnosis.21,22 in the case of severe and life threatening illness, histopathology of a tissue biopsy or in some cases a necropsy evaluation from a deceased puppy may be required for a definitive diagnosis. the latter is particularly important when faced with a fading neonatal puppy as this may give information vital to surviving littermates as well as future planned mattings; however this important diagnostic tool is all too often underutilized.  drug therapy, including antibiotic therapy, should be used cautiously in neonatal and pediatric patients, taking into consideration the age, the drug properties, and the possible toxicities. for example, fluroquinolones, such as enrofloxacin and ciprofloxacin, can result in cartilage defects in large breed, growing, puppies who are weight bearing. in consideration of drug properties, water soluble or lipid soluble properties of the drug are significant in the decision making process as neonatal and pediatric puppies have a higher body water composition and lower fat. as discussed above, neonatal and pediatric puppies also have reduced renal and hepatic clearance and lower total protein resulting in more available drug.4 a common indication for antibiotic therapy is in the prevention of sepsis from hypoxia-induced bacterial translocation, and so antibiotics are often one of the interventions considered in at-risk neonates. interventions, particularly drug therapies, should only be performed at the instruction of the primary care veterinarian or veterinary specialist, as neonatal and pediatric puppies are more susceptible to side effects of these medications than are adults. summary neonatal and pediatric puppies are not "little" adults and the recognition of these differences is vital in addressing the identification, diagnosis, and management of at risk neonatal and pediatric puppies. early identification and intervention is critical in an effort to reduce puppy mortality. intervention 555 clinical theriogenology • volume 4, number 4 • december 2012 includes identifying at risk animals with the utilization of apgar scoring mechanisms and physical examination, keeping in mind the important physical and physiological differences between neonatal and pediatric puppies compared with adults. prevention focuses on pre-breeding health, optimal husbandry, managing labor and delivery, reducing infectious disease, and optimizing nutrition of both the dam and puppies. diagnosis and treatment also needs to account for the physical and physiological differences present in neonatal and pediatric patients. the ultimate goal and secret for success is for breeders and veterinarians to work together in an effort to reduce these factors that contribute to neonatal and pediatric losses. references 1. concannon pw: physiology and clinical parameters of pregnancy in dogs. proc world small anim vet cong; 2002. available at: www.wsava.org. 2. kelley rl: canine reproduction: factors influencing litter size. proc annu meet soc therio; 2002. 3. johnston sd, root kustritz mv, olson pns: the neonate from birth to weaning.in: canine and feline theriogenology. philadelphia: wb saunders; 2001. p. 146-167. 4. peterson me, kutzler m: small animal pediatrics. philadelphia: elsevier; 2010. 5. veronesi mc, panzani s, faustini m, et al: apgar scoring system for routine assessment of newborn puppy viability and short-term survival prognosis. theriogenology 2009;72:401-407. 6. hoskins j: veterinary pediatrics. 3rd ed, philadelphia: elsevier; 2001. 7. davidson ap: approaches to reducing neonatal mortality in dogs. in: concannon pw, england g, verstegen j, et al, editors. recent advances in small animal reproduction. ithaca (ny): international veterinary information service; 2003. available at: www.ivis.org. 8. moon pf, erb hn, ludders jw, et al: perioperative risk factors for puppies delivered by cesarean section in the united states and canada. j am anim hosp assoc 2000;36:359-368. 9. münnich a: the pathological newborn in small animals: the neonate is not a small adult. vet res commun 2008;32:s81-s5. 10. poffenbarger em, ralston sl, chandler ml, et al: canine neonatology, part i, physiologic differences between puppies and adults. compen contin educ pract vet 1990;12:1601-1609. 11. davidson ap: topics in pediatrics. proc annu meet swedish soc small animal reprod; 2005. 12. davidson ap: nursing care of neonatal puppies and kittens. in: pratt p, editor. principles and practice of veterinary technology. santa barbara: mosby-year book: 1997. 13. carmichael l: neonatal viral infections of pups: canine herpesvirus and minute virus of canines (canine parvovirus-1). in: carmichael l, editor. recent advances in canine infectious diseases. ithaca (ny): international veterinary information service; 1999. available at: www.ivis.org. 14. davidson ap, grundy sa, foley je: successful medical management of neonatal canine herpesvirus: a case report. commun therio 2003;3:1-5. available at: http://ctheriogenology.lsu.edu/ct/. 15. lawler df, colby ed, editors: pediatrics. vet clin north am small anim pract 1987;17. 16. center sa, randolph jf, manwarren t, et al: effect of colostrum ingestion of gamma-glutamylaminotransferase and alkaline phosphatase activity in neonatal pups. am j vet res 1991;52:499-504. 17. levy jk, crawford pc, collante wr, et al. use of adult cat serum to correct failure of passive transfer in kittens. j am vet med assoc 2001;219:1401-1405. 18. poffenbarger em, olson pn, ralston sl, et al: canine neonatology, part ii, disorders of the neonate. compend contin educ pract vet 1991;13:25-37. 19. mosier je: canine pediatrics: the neonate. proc am anim hosp assoc 1981. p. 339-347. 20. giger u: clinical genetics. in: ettinger s, feldman e, editors textbook of veterinary internal medicine. 6th ed. philadelphia: w.b. saunders: 2005. 21. giger u: new insights into hereditary diseases and genetic predisposition to disease in dogs. proc world small anim vet cong; 2009. available at: www.wsava.org. 22. giger u: peculiarities of feline hereditary disorders. proc world small anim vet cong; 2009. available at: www.wsava.org.  556clinical theriogenology • volume 4, number 4 • december 2012 2012: ethical considerations in the selective breeding of animals ethical considerations in the selective breeding of animals john rossi drexel university school of public health, philadelphia, pa introduction it has been known for some time in veterinary medicine that certain breeds of animals show predilections for particular diseases, and furthermore that particular diseases may be confined entirely to one breed or a small group of breeds. most veterinary textbooks discuss breed predilections for disease as relates to their specific subject matter, and at least one textbook is devoted entirely to the subject of breedassociated disease.1 breed-associated disease can arise in a few ways. first, morphological breed standards can directly cause disease, or can otherwise exacerbate disease. second, because “purebreeding” is essentially inbreeding,* the creation and maintenance of particular breeds decreases genetic heterozygosity as compared to an outbred population, thus increasing the probability that a given individual will possess two copies of rare, recessive disease-causing alleles. third, disease-causing alleles may be inherited though their genetic linkage to loci corresponding to traits that are being selected for.1-5 until very recently the ethical dimensions of animal breeding and its relationship to disease were seldom discussed. this all changed rather dramatically in 2008, when the british broadcasting corporation (bbc) aired the documentary pedigree dogs exposed, which discussed at length health problems in pedigree dogs, and which thrust the issue of breed-associated disease into the public spotlight in a new way. this documentary prompted three separate inquiries into the issue,2,4,6 as well as a handful of academic journal articles.3,5,7 in addition, the companion animal welfare council (cawc) published a report about the health and welfare of selectively bred animals in 2006.8 these reports and articles typically discuss the genetic mechanisms of disease and animal welfare problems associated with selective breeding, and also offer numerous recommendations intended to improve the welfare of selectively bred animals, which variously address breeders, breeding societies, research scientists, veterinarians, regulatory authorities, and consumers. in many cases these recommendations are farreaching. for example, patrick bateson recommends that “all pre-mating tests for inherited disease appropriate to the breed or breeds [be] undertaken on both parents. no mating [should take] place if the tests indicate that it would be inadvisable in the sense that it is likely to produce welfare problems in the offspring and/or is inadvisable in the context of a relevant breeding strategy.”4 however, existing reports and articles concerning selective breeding tend not to include explicit discussion of the ethical principles relevant to the issue; hence recommendations for changes to breeding practices are often unsupported. even where a reader agrees with these recommendations, s/he may wish to gain an understanding of how they might be justified. someone might also think that such recommendations go too far or not far enough. in addition, reports and articles on breed-associated disease may or may not define the concept of “animal welfare,” and where this concept is defined it may require some additional philosophical qualification. hence there is a need to support and contextualize practical discussions about selective breeding by joining them with more philosophical considerations. attention to these philosophical considerations can help to uncover latent assumptions and value judgments, and can also help us to develop an organized framework for thinking through our obligations to animals as concerns breed-associated diseases. the remainder of this article will discuss key philosophical and ethical issues in the selective breeding of animals. while this discussion focuses primarily on the selective breeding of companion animals, many of the following points also apply to selective breeding more broadly, and furthermore the concepts and principles discussed here should be applicable to many issues in veterinary medical ethics. definitions and distinctions before proceeding further, it will be helpful to clarify a few key terms and concepts. as used here, “selective breeding” means breeding to effect, maintain or enhance certain morphological, behavioral, and/or physiological characteristics. this definition covers the breeding of companion 503 clinical theriogenology • volume 4, number 4 • december 2012 animals for appearance, temperament, or function (e.g., herding or guarding, if distinguished from temperament); it also covers the breeding of farm animals for production purposes (e.g., higher milk or meat yield) and the breeding of laboratory animals for purposes conducive to scientific experimentation (e.g., nude mice). however, “selective breeding” as discussed here excludes two key practices, and thus two key ethical questions. first, it excludes the domestication of wild animals, and thus the question of whether this is ethically defensible. second, it excludes the human-managed reproduction of alreadydomesticated animals per se. to argue that selective breeding for certain appearances, behaviors or other characteristics is morally justified is to presuppose that it is morally permissible for humans to breed domesticated animals at all. however, the converse is not true: a person could argue that it is morally permissible for humans to live with domesticated animals and to manage their reproduction, but that it is morally impermissible for humans to selectively breed animals for particular ends in light of the welfare costs that this entails to the animals; or that such selective breeding is only permissible under certain specified circumstances. the question of whether humans ought to live with domesticated animals per se is one that usually arises only on strong animal rights views, and even here all scholars who adopt this kind of view do not hold that it precludes humans’ living with domesticated animals.† for present purposes it is assumed that the continued reproduction of already domesticated animals per se is morally permissible, with the focus being on the selective breeding of such animals for appearance, behavior, or other characteristics. selective breeding can both compromise and enhance animal welfare. for example, we could use genetic tests to screen individuals for disease-causing alleles and remove them from the breeding pool in the hope of reducing the prevalence of particular disease. we could also preferentially breed animals that score very highly on the evaluation of particular healthor welfare-associated traits. both of these strategies involve the use of selective breeding to improve animal welfare. however, for purposes of ethical analysis it makes sense to focus initially on the ways in which selective breeding compromises animal welfare. this is for two reasons. first, selective breeding may be used to ameliorate animal welfare problems that were created by selective breeding in the first place. in these situations, justifying obligations to change breeding practices to improve animal welfare will likely be contingent upon assessing the ethical status of selective breeding when it leads to such welfare problems. using selective breeding techniques to improve animal welfare may impose financial costs upon breeders in the form of screening tests, the removal of valuable animals from the breeding pool, decreases in animal production efficiency, etc. given these costs, a person may reasonably ask why they are obligated to honor such restrictions. answering this question likely requires that we first assess whether it is permissible to selectively breed animals even when it leads to welfare problems. if so, then it is hard to see how breeders or other stakeholders can be morally required to change breeding practices to improve animal health. if not, then an obligation to use selective breeding to improve animal health might be subsumed under a general responsibility to minimize harm or right past wrongs. the second reason why ethical analysis of selective breeding should start with welfare problems is that specific proposals to improve the health of purebred animals may beg important questions‡ about what is ethically permissible. for example, it has been recommended that strategies to improve breed health avoid simply removing all individuals from the breeding pool when such individuals test positive for a particular disorder during screening tests, since removing all such individuals may introduce genetic bottlenecks by shrinking already small effective breeding populations.4 this recommendation presumes that it is ethically defensible to continue selectively breeding animals even when doing so may be associated with the perpetuation of some deleterious characteristics. this recommendation may be sound, but the point certainly requires argument and cannot be assumed. the anatomy of an ethical judgment arguably the best way to approach the ethical analysis of selective breeding is to first ask what kinds of considerations are generally relevant to moral judgments. specific moral judgments are the product of both non-moral facts and moral evaluations.§ moral evaluations tell us what is good, bad, desirable, undesirable, right, wrong, etc. non-moral facts inform us about the specific circumstances we 504clinical theriogenology • volume 4, number 4 • december 2012 are in, or the circumstances that would obtain were we to act a certain way. an example of a non-moral fact relevant to moral judgment is whether a particular procedure causes pain to an animal. we need to know both the relevant non-moral facts and moral evaluations in order to know how we ought to act in a given situation. without moral evaluations, we do not know why certain non-moral facts matter. for example, knowing that a procedure causes pain to an animal is not sufficient to know whether we ought not to perform the procedure. what is needed in addition to this is an understanding of what our moral obligations are when it comes to harming animals. just the same, without knowing the relevant nonmoral facts, we cannot apply moral evaluations to the present circumstances. for example, we cannot apply the principle “do not cause unnecessary pain to animals” if we do not know whether a particular set of circumstances causes pain to an animal. furthermore, judgments about our ethical obligations usually presuppose some idea of what is good or bad for an individual’s welfare. in the preceding example, an implicit judgment is that pain detracts from an individual’s welfare. though moral disagreement in specific circumstances is often thought to be about moral evaluations per se, at times non-moral facts can be of significant importance to moral disagreement. for example, debates about human healthcare policy may hinge not on ethical principles, but rather on different views about the likely economic effects of a particular policy choice. in the context of selective breeding, relevant non-moral facts relate to the physical and behavioral consequences of selective breeding, both in general and as concerns individual matings. relevant moral judgments concern the way in which we define “animal welfare,” our evaluations of the severity of various breed-associated diseases or conditions, and our obligations to avoid imposing risk or harm upon animals (such obligations falling under the umbrella of what philosophers call “normative ethics”). i consider each of these issues below. it should be emphasized that the concept of “animal welfare,” while informed by non-moral facts, is an evaluative concept: it captures ideas about what makes an animal’s life go well or poorly. this may easily be overlooked in situations where the relevant evaluations are non-controversial (e.g., the judgment that being in pain is bad for an animal’s welfare, although it should be noted that not all commentators agree even about this!), in which case it might seem that the judgment that something sets back an animal’s welfare is purely a non-moral judgment. however, moral evaluations will always be in the background, and it is important to be aware of them since they may be controversial. furthermore, our conceptions of animal welfare will determine which non-moral facts are deemed relevant. for example, if animal welfare is defined as the absence of unpleasant sensations (pain, fear, distress, etc.), then the relevant non-moral facts will be those that pertain to whether an animal is experiencing such unpleasant states. but if animal welfare is defined differently, for example by including reference to positive mental states, then additional non-moral facts may be at issue, such as whether an animal is experiencing pleasurable feelings or is able to satisfy preferences. (as an aside, it should also be emphasized that relevant non-moral facts, e.g., scientific facts, often involve value judgments themselves—something that philosophers of science have long studied. hence non-moral facts cannot always be taken as a noncontroversial starting point or “value-free” realm when debating moral issues.) when thinking about the ethics of selective breeding, it is very important to keep judgments about animal welfare and normative ethics distinct. this is for three reasons. first, it’s helpful to sort things out conceptually when addressing ethical questions. discussions of animal welfare frequently conflate prudential value theory and normative ethics—that is, the question of what is good or bad for animals (animal welfare, prudential value theory) and the question of how we should treat animals (normative ethics). if these various concepts are not adequately distinguished, then ethical discussion will be confused, and discussants may talk past each other. second, establishing that something is harmful or beneficial for an animal’s welfare does not entail anything about our moral obligations or lack thereof. most if not all reports on selective breeding discuss its possible detrimental effects on animal welfare, and then proceed directly to a set of recommendations for mitigating these adverse welfare effects. however, this skips over a step that is both necessary and important: defining what obligations we have to protect or promote animal welfare. from a political standpoint, if all parties to a debate happen to agree about our obligations, then omitting a discussion of the basis for our ethical obligations to animals may go unnoticed. but if we are interested in the philosophical justification of various positions, and/or if there is 505 clinical theriogenology • volume 4, number 4 • december 2012 some degree of political disagreement about the nature and extent of our obligations, then we need to go beyond a discussion of the welfare impacts of selective breeding and consider these obligations explicitly. third, it is important to keep the notions of what is good or bad for animal welfare and our moral obligations to animals separate because we need to be careful not to let our normative commitments bias us when defining or assessing animal welfare. scholars who study animal welfare have noted that the definition of this concept may sometimes be inappropriately influenced by political concerns. for example, the discipline of animal welfare science has historical roots in animal research and animal agriculture. both of these activities entail the death of animals. therefore the concept of “humane care and use” came to be defined in such a way that death was not viewed as a harm to animals,9 since otherwise, it would be impossible to conduct these activities in a “humane” way—a conclusion that defenders of these practices wished to avoid. whether death harms sentient animals is a philosophical issue that can be debated, and which furthermore has been debated by philosophers (though the best answer seems to be “yes”). however, we should hope that any conclusion that death does not harm animals will be reached via unbiased, rigorous argument, and not because such a conclusion happens to be politically expedient to the interests of a particular group. it is presently unclear whether the issue of political bias in defining animal welfare will impact the discussion about selective breeding. however, this should at least be noted as a reasonable possibility, given the fact that it is an issue in other areas of animal ethics. for example, a person might argue that an animal only experiences poor welfare if it is afraid, distressed or in significant pain. even on this definition of animal welfare, selective breeding will sometimes adversely affect animal welfare. however, this definition of animal welfare will probably result in a less critical analysis of selective breeding than alternative definitions, since it excludes concerns such as length of life, overall quality of life, mobility and the ease with which animals can satisfy preferences, etc. the point here is not to argue for any particular definition of animal welfare, or for any particular conception of our moral obligations to animals (or lack thereof), but rather just to note that these two determinations are independent of each other. non-moral facts at issue in selective breeding we know with a fair degree of confidence that selective breeding is positively associated with disease as compared to random matings or outcrosses. for example, recent reviews identified a total of 396 inherited disorders in the top 50 breeds. each of the top 50 breeds has at least one conformational aspect predisposing it to a disorder, and though the number of non-conformational inherited disorders varies by breed, the number is usually not less than a few and may be as high as 50 in a given breed.3,5 as compared to an outcrossed population, selective breeding as presently practiced results in greater conformational and non-conformational disease. as discussed below, in order to apply moral principles and evaluations to actual breeding practices, we are going to want to be able to assess for a given mating the risk that offspring will have a particular disease, disorder, or other welfare-relevant phenotype. in the ideal, (at least part of) this risk assessment would be quantitative, in the sense that we would be able to assign with a high degree of confidence the probability that a particular genotype or phenotype would manifest in the offspring of a particular mating. however, making quantitative risk assessments is not presently possible in a large percentage of situations. as concerns non-conformational disease, relatively few genetic tests are currently available, and many traits of interest are polygenic, meaning that for these traits developing predictive genetic tests will not be simple. in the absence of information from genetic tests, we might use phenotypic screens (e.g., hip screening), pedigree analysis, estimated breeding values (ebvs), prevalence information, or simply personal experience with a family line in order to assess risk. in many cases these methods of assessing risk will not yield an exact numerical estimate of probability (although pedigree analysis can sometimes provide this), but while an exact probability is desirable, it may not be necessary from a moral standpoint. rather, what seems necessary is for us to be able to say that we have good reason to believe that a particular mating will have particular effects. 506clinical theriogenology • volume 4, number 4 • december 2012 where for some reason a genuine state of uncertainty exists as to whether a particular mating will produce particular effects, we can broaden our frame of assessment to include the breed at large. there presumably will be situations where we cannot know with a high degree of confidence what the outcome of a specific mating will be, but where over time certain breeding practices (e.g., limiting the number of times an animal may stud, only breeding animals above a certain cut-off point in terms of health or welfare assessment scales) can help us to lower the extent of breed-associated disease, and the risks that an individual animal will face. the preceding comments apply to non-conformational disease. as concerns conformation-related disease (e.g., brachycephalic airway obstruction syndrome), it is assumed here that the effects of specific matings will be somewhat easier to assess. hence, while many reports on the issue of selective breeding have emphasized the need for more information relating to disease prevalence, and the development of more genetic tests2,4,6, such data gaps need not prevent us from applying relevant ethical principles at the present time. yet another non-moral fact of considerable importance to ethical analysis is whether we can selectively breed animals for appearance or function without imposing some additional risk of disease as compared to an outcrossed population. this can be considered an open question at present; however, since selective breeding typically involves decreasing genetic heterozygosity as compared to an outcrossed population, there is reason to suspect that the answer is “no.” defining and assessing animal welfare the term “animal welfare” may be used ambiguously in veterinary medicine, where it could refer to a general concern for animal well-being; those things that make an animal’s life go well or poorly; or a discussion of our obligations to animals. it is the second of these meanings for which the term is used here: those things that make an animal’s life go well or poorly. this use of the term “animal welfare” is most consistent with the way “welfare” tends to be used in the philosophical literature; “welfare” in this sense is also consistent with what philosophers call prudential value. there is much that can be and has been written about animal welfare, even when restricted to its present usage. here i will concentrate on a few points i believe are most important both in discussions of selective breeding and veterinary medical ethics more generally. first, while it is often claimed in both veterinary medicine and animal welfare science that “animal welfare is a science-based concept,” this is not strictly speaking true. rather, welfare is an evaluative concept at bottom: judgments about what is good or bad for an animal, what makes that animal’s life go well or poorly, are by definition evaluative judgments. another way of appreciating the evaluative nature of “animal welfare” is to observe that no amount of scientific data by itself will answer questions about animal welfare. such data can always be met with the question “why is that bad for the animal’s welfare?” for example, data that supports the conclusion that an animal is in pain do not in themselves “prove” that the animal is experiencing poor welfare unless joined to the evaluative judgment that pain (distress, fear, etc.) is bad. so, too, for judgments that an animal cannot express natural behaviors, that it is not “coping” with its environment, and so on. it is important to recognize the evaluative nature of the concept of animal welfare, since the value judgments at play in the concept may be contested between parties. if animal welfare is an evaluative concept at bottom, then why is it so often claimed to be “science-based”? one reason is that scientific data, though not definitive of animal welfare, can be useful and sometimes crucial in assessing an animal’s welfare. for example, it may not be obvious whether animals prefer one housing system over another, and scientific studies designed to assess animals’ preferences can help us to answer such questions. there are also philosophical and sociological reasons why a “science-based” notion of animal welfare gained traction amongst veterinarians and scientists. for some time in the earlyto mid20th century, science was dominated by a philosophy known as logical empiricism (or logical positivism), which held that something could only be called a fact if it was empirically verifiable (or falsifiable). logical empiricism went hand in hand with psychological behaviorism, which held that mental states either could not be fruitfully studied or did not even exist; hence talk of animal mind was replaced by talk of animal physiology and behavior. though behaviorism has long since fallen in philosophical circles as well as human psychology (having been replaced by 507 clinical theriogenology • volume 4, number 4 • december 2012 cognitive psychology), it has left a strong legacy in discussions of animal welfare, and is still evident in some recently published papers.10 a third reason is political: animal welfare science as it is presently institutionalized initially grew out of biomedical research and animal agriculture, and this is reflected somewhat in the tendency to define welfare in scientific terms. this tendency might be explained in part by scientists’ comparatively greater comfort with scientific language and concepts, as opposed to moral language and concepts. however, it should also be noted that a “science-based” conception of the concept of welfare bestows expertise upon scientists while denying expertise to non-scientists, and given the controversy often associated with animal use expertise is a valuable political commodity.** there are a number of practical upshots to recognizing the evaluative nature of animal welfare. first, different considerations deemed relevant to animal welfare, such as behavior and physiology, may at times conflict when examining animal welfare in a specific context (e.g., sows in gestation crates). if these considerations are taken as definitive of animal welfare, then there is an irreducible conflict, whereas if they are taken as evidential considerations for the assessment of animal welfare otherwise defined (e.g., by preference satisfaction, pleasurable sensations, etc.), then the conflict may be merely illusory. it is unclear at present whether this issue will arise when considering the welfare impact of selective breeding, but again, since it is an important issue in animal welfare generally it is best to keep it in mind. second, multiple discussions of animal welfare in the context of selective breeding highlight the need for more “objective” measures; thus severity scales for ranking the welfare impact of various breedassociated diseases have been proposed.11 “objectivity” is a term that can take on multiple meanings, which may not be sufficiently disambiguated when the term is used. one such meaning of “objective” is “true, independently of what anyone happens to think”; another meaning is “quantifiable.” though the first sense of objectivity can apply to moral and scientific judgments alike, in practice these two meanings of “objective” may be run together in discussions of animal welfare, such that it is thought that the only way judgments about animal welfare can be “true, independently of what anyone happens to think” is to attach a quantified measurement to such judgments. however, attaching a number to a concept does not make it a truer representation of that concept unless some rational standard can be identified to determine how numbers should be assigned. severity indices can be useful insofar as they allow for comparisons between different persons’ judgments, and they allow persons to refine intuitive judgments about animal welfare by explicitly considering a number of factors. however, the numbers are still generated by intuitive judgment and stand as proxies to it, so it is important not to overestimate the degree to which such numbers represent “accurate and true” welfare measurements. third, as discussed above the view of welfare as “science-based” tends to place all expertise about animal welfare in the hands of scientists; this purported expertise can be and has been used as a political weapon when different parties disagree about welfare-related issues,†† since it disenfranchises all nonscientists of relevant knowledge. veterinarians, ethologists and animal welfare scientists certainly do possess knowledge and expertise relevant to animal welfare; however, they are not the sole repositories of such expertise, so it is important not to be led astray by “science-based” ideology here. aside from critically interrogating “science-based” conceptions of animal welfare, it is also important to broadly assess the welfare impact of selective breeding. reports or journal articles on selective breeding may or may not define “animal welfare.” in either case, however, it is important to make sure that relevant considerations are not left out of the analysis. one set of concerns relates to how we define welfare. welfare may be defined in terms of pleasure and pain (hedonism), preference satisfaction, the expression of natural behaviors, certain physiological measures, coping, the absence of disease, and perhaps other criteria. the animal welfare science literature is filled with debate about which of these criteria, or which combination of criteria, constitute the best definitions of “welfare.” it is not uncommon for authors to acknowledge that all such criteria are equally important, and that “good welfare” must take each into account. while defining welfare—either human or nonhuman—admits of some philosophical complications that may preclude tidy answers, this kind of response is more an evasion than an answer. here i only emphasize that plausible views of welfare will likely have to make reference to subjectivity; that is, to what an animal experiences or does not experience. even with this 508clinical theriogenology • volume 4, number 4 • december 2012 restriction in place there is still room for debate about how we should define welfare, but the restriction helps to show why some considerations are relevant and why some aren’t. for example, an animal’s ability to fulfill natural behaviors may be relevant to welfare because the fulfillment of such behaviors correlates to the satisfaction of preferences, so it is really preference satisfaction that is definitive of welfare on this view. similarly, definitions of welfare in physiologic terms or in terms of “coping” are suspicious unless it can be shown that such things impact the quality of an animal’s life from the perspective of the animal. otherwise, what would it mean to say that certain things promote or thwart an animal’s well being? even when taking a certain definition of animal welfare for granted, it is important to think broadly about how selective breeding may impact welfare. for example, the cawc restricts usage of the term “welfare” to situations where “only…the quality of consciously, subjectively, experienced feelings (e.g. pain, fear, warmth, pleasure) is at issue in subjective terms.” they focus their discussion of the impact of selective breeding on the ways in which “heritable diseases or characteristics… have the potential to cause, depending in their nature, severe pain, discomfort, anxiety or other unpleasant feelings”.8 however, this ignores the ways in which selective breeding can adversely affect animal welfare by depriving animals of pleasurable experiences. for example, inbreeding (which is by definition part of selective breeding) is associated with shorter lifespan.4 even if animals do not experience “pain, discomfort, anxiety or other unpleasant feelings” while they are alive, we can plausibly say that inbreeding adversely affects their welfare by shortening their lifespan and allowing them fewer opportunities to satisfy interests. similarly, an animal affected by brachycephalic airway obstruction syndrome whose exercise is restricted to prevent discomfort may still be harmed by the condition, since the exercise restriction necessary to ensure its comfort precludes it from running or playing vigorously. problematic arguments for the permissibility of selective breeding in conversation and in print, some problematic arguments may be advanced for why it is morally permissible for us to breed animals, even when doing so harms the animals. before we can get to an appropriate moral framework for exploring selective breeding, we need to dispatch with such arguments. here i briefly review three of the most popular of such arguments; much longer critical analyses are possible. dominion the dominant moral tradition in the west is the judeo-christian religious tradition. it is not uncommon in conversation to hear persons justify various kinds of animal treatment on the grounds that god gave us dominion or stewardship over all of creation. there are two major problems with this kind of argument. first, even if a person accepts dominion in general terms, it is an open question what kinds of specific animal treatment are morally licensed by dominion. for example, matthew scully accepts the concept of dominion, but argues against industrial animal agriculture (“factory farming”) because in his view, dominion requires respectful treatment of animals not provided by industrial farming.12 the theologian andrew linzey argues that the christian tradition requires nonviolence and moral vegetarianism, for example.13 the point here is not that either of these authors’ arguments is correct (they may or may not be), but rather that the concepts of dominion or stewardship cannot be unproblematically invoked to justify specific kinds of animal treatment. since the concept of dominion is open to interpretation, additional argument is needed. the second major problem with religious arguments about animal ethics (or ethics in general) is that they are subject to a number of well-known controversies. such arguments must show at the least that the concept of god is intelligible, that we have good reason to believe that god exists, that there is some connection between god and morality (either because morality consists in doing god’s will, or because it consists in discovering values or purposes that god built into nature), and that specific moral conclusions or principles can be deduced from all of this. this is a significant burden of proof, and one which philosophers have long questioned our ability to meet. at the very least, arguments raised against theological conceptions of ethics need to be addressed head-on, and not skirted. social etiquette often regards religion as an essentially private matter, and one 509 clinical theriogenology • volume 4, number 4 • december 2012 that is beyond appropriate or tasteful criticism. perhaps for this reason, religious views are often invoked in moral debate as “discussion enders”—a way for someone to defend their moral view and place it beyond criticism at the same time. but religious views of morality may be rationally critiqued just like any other moral view. furthermore, morality is by definition a public matter in some respects. it concerns not just our ideas about what makes our own lives meaningful, but how we treat our children, our fellow citizens, animals, and the environment. because our conduct affects others’ interests and not just our own, proponents of views tying morality to religion have a responsibility to defend their position to others, and not just invoke religious views as “discussion enders.” the argument from nonidentity it is sometimes argued that certain kinds of animals, including farm animals and perhaps also certain breeds of companion animal, would not exist were it not for the human purpose for which they were brought into existence (food, breed appreciation, etc.), and that this fact justifies bringing them into existence even if this existence subjects them to some harms, since the harms (e.g., breed-associated disease) are a necessary condition for them being alive in the first place. there are a number of problems with this argument. first, it assumes that being brought into existence is a benefit, and therefore that an animal’s life is worth living on balance. this is sometimes (if not often) questionable in the case of farm and laboratory animals. however, it is probably true that many companion animals have lives worth living on balance. second, it is unclear why the argument justifies bringing animals into existence in conditions that wind up harming them. even if bringing an individual into existence is viewed as a benefit to that individual, the fact that we benefit someone does not give us a right to harm them afterwards. (i cannot justify assaulting my neighbor because i once mowed his lawn for him.) similarly, the fact that we bring someone into existence does not give us ownership over them. we cannot mistreat our children, or treat them well for a period of time before imposing significant harm upon them, just because they would not exist were it not for us. children are not property. third, and more generally, we have no obligation to bring animals or persons into existence—recognizing such an obligation would lead to very strange conclusions—and no harm is committed by not bringing them into existence. however, once they are brought into existence they now have interests, and we have obligations to respect those interests. if bringing animals into existence justifies our harming them, but bringing persons into existence does not justify harming them, then the argument really at play is that animals have lesser moral standing than humans—that is, that animals deserve lesser moral protection than do humans—not that bringing a being into existence justifies harming it. the contract argument a third problematic argument, which is related to the argument from nonidentity, is a version of the contract argument (in the case of farm animals, sometimes called “the ancient contract”). this argument holds that it is acceptable for humans to harm animals in some circumstances because doing so is part of a “fair deal” that benefits the animals on balance. this argument may collapse into the nonidentity argument just considered, since it may be held that the “fair deal” is that the animals have lives that are worth living on balance, or that they receive protection they would not receive in the wild. however, this kind of argument is often represented by contractual language, so it is worth considering the contract metaphor on its own terms. one problem with this argument is that it may presuppose that animals receiving human protection are the same animals that would otherwise be in the wild, but this of course is not true; here we are talking about bringing individual members of domesticated breeds into existence. hence we cannot strictly say that individual animals are better off than they would be in the wild. a second problem is that species or breeds don’t make contracts; individuals do. hence the idea of a contract between humankind and, say, the golden retriever breed just doesn’t make sense. a third problem is that in both moral and legal terms, valid contracts are usually understood as those that were knowingly entered into by rational parties in possession of all the relevant information. in fact, in our legal system individuals usually have to be of adult age in order to sign legally binding contracts. animals are not rational agents; they are not capable of understanding the terms of a contract and acting 510clinical theriogenology • volume 4, number 4 • december 2012 according to rational self-interest in the ways humans can. hence the relevant worry is simply that whatever contract we would invoke would be exploitative, just as we would worry that contracts signed by six year-old children would not ultimately be in their interest. one possible response is to say that whatever contract is under consideration is the “best possible deal” for animals, even if they cannot understand it. but then we run into a familiar problem, which is that it doesn’t make sense for individuals to make agreements to be brought into existence. furthermore, the “best possible deal” for animals would presumably be one where they are brought into existence and not harmed. finally, assuming that an individual is going to be brought into existence, modern proponents of social contract theories of morality often hold that just contracts are those that would be made behind a “veil of ignorance,” whereby parties to the contract do not know anything about their race, gender, socioeconomic status, intelligence, etc. the underlying idea here is fairness: this prevents the formation of exploitative contracts that benefit some parties at the expense of others, and we can easily add species to the list of conditions that would be veiled. if species does not belong on the list, then again we find ourselves at the point where it is really a difference in humans and animals’ moral standing that is motivating the argument. a framework for the ethical analysis of selective breeding knowing that selective breeding can adversely affect animal welfare does not tell us whether or when selective breeding is ethically permissible; in order to arrive at such conclusions we need to appeal to additional ethical principles and considerations. these additional considerations include 1) a general theory of ethical decision-making, and 2) an understanding of humans’ and animals’ comparative moral standing within such a theory. these two things are often conflated, but it is important to keep them separate. one way of seeing the distinction is to exclude animals from the discussion for a moment and note that while most persons accept a thesis of equality between different humans, accepting such equality does not tell us what our obligations to other humans are; it only tells us that when thinking about such obligations, we cannot discount the interests of other persons just because they are not like us (e.g., because of different gender, ethnicity, etc.). so we need additional ethical principles to help explain and derive our obligations. in the long history of western philosophy a large number of prominent philosophers have elaborated moral theories that are intended to explain the origin and authority of morality, and also to guide our conduct by elaborating various principles. famous moral theories include various virtue theories, kantianism, natural law theory, utilitarianism, social contract theory, modern rights theory, and others. rather than ground the present discussion in a particular ethical theory—which would risk making it too parochial—i will proceed by focusing on a principle of nonmaleficence, which holds that we should not harm others. there are two reasons for focusing on this principle. first, essentially all plausible ethical theories recognize a principle of nonmaleficence; this principle is also strongly embedded in our common morality. second, the ethical concern most at issue with selective breeding is that fact that it causes welfare problems for the animals in question. selective breeding is also associated with some benefits to humans; however, the best way to bring these benefits into the context of ethical discussion is to ask the question of when, if ever, it is ethically permissible to bring about harm to others—or to impose risk upon others—because doing so benefits us. the reason for couching the discussion this way is that as concerns humans, a principle of nonmaleficence tends to be recognized as very strict. there may be exceptional circumstances in which it is permissible for us to harm other humans, such as in self-defense or in the cause of a just war. however, outside of these exceptional circumstances it is generally held by both philosophers and non-philosophers alike that we ought not to harm others, particularly when harms are nonconsensual and nontrivial. the next question, then, is whether a principle of nonmaleficence applies to nonhuman animals and, if so, how strongly. here we get into the question of animals’ moral considerability. to say that animals are morally considerable, or that they are owed moral consideration, is to say that animals’ interests matter directly in moral decision-making. there are roughly three options as concerns animals’ moral considerability. first, we might adopt the “no moral consideration view,” which holds that animals do not matter morally in any direct way; that their interests‡‡ do not count directly in any moral calculus. 511 clinical theriogenology • volume 4, number 4 • december 2012 second, we might adopt the “equal moral consideration view,” which holds that animals’ interests deserve equal moral weight or protection as do humans’ interests. third, we might adopt an “unequal moral consideration view,” which holds that animals’ interests matter morally, but to a lesser degree than humans’ interests. the philosophical literature on animal ethics is filled with detailed discussions about the various merits and demerits of these particular positions.14,15 because of space limitations, it is not my intention to argue here for a particular conclusion as regards animals’ moral standing. instead, i will simply trace out what various views imply for selective breeding practices. readers are encouraged to think about which of these positions they find most plausible, and what their favored position implies as to the ethics of selective breeding. no moral consideration views it might seem that on a “no moral consideration view,” we have absolutely no moral obligations to animals, but this is not true. even though animals do not count directly in moral decision-making, they may still count indirectly, since humans take an interest in animals, and since humans’ interests still count on this view. hence “no moral consideration” views are also sometimes called indirect duty views. for example, even on a no moral consideration view it would be morally impermissible for us to poison our neighbor’s dog, since the dog is his property, and since he takes a sentimental interest (we are assuming) in the dog. hence we would harm our neighbor by killing his dog, and since the dog is his rightful property, this harm would be impermissible (just as it would be impermissible for us to vandalize his car). in addition, the way that we treat animals might matter on this indirect duty view since being cruel to animals might make us more likely to be cruel to humans. however, it is hard to see how either of these considerations would be relevant to selective breeding. even though selective breeding may detract from animals’ welfare, harm is not being deliberately inflicted upon animals for sadistic purposes. therefore it is hard to see how the selective breeding of animals would make us more callous to human welfare in the way that deliberate cruelty would (though perhaps this is possible). similarly, when discussing selective breeding we are not typically talking about harming our neighbor’s property, but about what we may do to animals in our own care or remit. therefore, i tentatively conclude that a “no moral consideration view” would not place any meaningful restrictions on the selective breeding of animals, even when this causes disease or other welfare problems. perhaps the most that this view would require in the way of reform is better transparency in breeding and selling practices, so that consumers can make more informed decisions about the breeds of animal they are purchasing and any welfare problems or disease risks associated with the breed. equal moral consideration views an “equal moral consideration” (ec) view, on the other hand, would place extensive restrictions on selective breeding. on this view, animals’ interests deserve the same level of moral protection that we afford to humans’ comparable interests, and since a principle of nonmaleficence applies strongly to humans, it would apply strongly to nonhuman animals as well. in many cases, the welfare impacts of selective breeding are probabilistic—that is, we are talking about imposing a probability of harm (risk) upon animals, rather than a certainty of harm. does this make a difference? probably not. moral considerations around risk are sometimes different than moral considerations around harm, but this usually concerns situations where risk imposition is unavoidable, or where imposing risk is necessary if we are to pursue some basic life interest. neither of these conditions applies to selective breeding, and outside of such conditions we may extend a principle of nonmaleficence from harm to risk without much problem philosophically. on an equal moral consideration view, this would mean that we have stringent obligations not to impose harm or risk upon animals outside of the exceptional circumstances discussed above. hence any time selective breeding imposes harm or risk upon animals over and above what would be seen in the “baseline” of an outcrossed population, it would appear to be impermissible on an equal moral consideration view. in fact, it is unclear whether any selective breeding (other than explicitly for health considerations) would be morally permissible on this view, since it is unclear whether it is possible 512clinical theriogenology • volume 4, number 4 • december 2012 to selectively breed for appearance, productivity, etc. without inbreeding and thus increasing the probability of genetic disease over and above an outcrossed population. that an equal moral consideration view would place extensive restrictions on breeding practices is not surprising— philosophers who endorse this kind of view also frequently argue that we ought not to kill animals for food or fiber, and that most invasive animal research is morally impermissible. and while it is not my present intention to argue for or against a particular view of animals’ moral standing, one brief point is in order here: equal moral consideration views are often dismissed out of hand in veterinary medicine as being radical, extreme, or obviously mistaken. and they are radical in one sense: accepting them would entail very significant changes to the status quo of how humans treat animals. but the fact that a moral view would require significant social change does not in itself invalidate the moral view. many leading scholars in animal ethics have in fact endorsed an ec view16,17, and while this view may or may not ultimately be correct, it should not be dismissed out of hand. unequal moral consideration views it is somewhat more difficult to trace the implications of an unequal moral consideration view as concerns selective breeding. “unequal moral consideration” (uc) does not really describe one view as much as it describes a range of views. we could hold that animals are due less moral consideration than humans, but still maintain that animals are due a very high level of moral consideration that is just slightly less than what we grant to humans; we could also hold that animals are due some moral consideration but very little. both of these are uc views; however, they would entail radically different practices as concerns human obligations to animals. in addition, uc views may accord different degrees of moral standing to different species, depending on their cognitive complexity. some of these complications must be set aside at present. for present purposes, there are two determinations that seem particularly important when assessing selective breeding from a uc perspective, and on which i focus here. first, in light of the possible complexity of uc views, it is helpful to try and identify a lower limit for serious moral consideration of animals’ interests. in my experience, most persons seem to hold some kind of uc view about animals’ moral standing. when attempting to formulate broadly acceptable social policy, then, it will be helpful to try and identify the minimum that we commit ourselves to if we accept the notion that animals have some moral standing. this lower limit can serve as a starting point for us to say “uc views require at least this much in the way of obligations to animals.” from this minimal starting point, we can then gradually increase the level of moral consideration owed to animals and see how such changes impact our moral obligations. we can also continue to have debates about animals’ moral standing, but we should not let points of disagreement obscure points of agreement. i propose here that a reasonable lower limit of serious moral consideration is as follows: “humans should not impose significant harms or risks upon animals for trivial purposes.” the reason why this may be a lower limit for serious moral consideration is that if it were any lower, then animals’ significant interests could be overridden for pretty much any reason whatsoever—but such a view is not consistent with taking animals’ interests seriously from a moral standpoint. this principle sounds similar to the oft-expressed view that “humans should not harm animals unnecessarily.” however, the concept of “necessary harm” is too vague to be useful; “necessary” really means “necessary in order to achieve a particular end,” so we have to evaluate the importance of the particular end for which animals are harmed. other things being equal, harms to animals that are imposed in the process of serving a very important human interest (e.g., curing hiv or cancer, providing necessary food without which we will starve) will be more permissible than harms imposed to animals for less urgent purposes, such as entertainment, luxury, non-essential food, etc. since selective breeding may serve a number of human interests whose significance may vary, its overall ethical defensibility will vary, even on a uc view. therefore the principle “humans should not impose significant harms or risks upon animals for trivial purposes” seems a better candidate for tracing the lower bounds of serious moral consideration, since it helps to clarify the relative importance of interests at stake. 513 clinical theriogenology • volume 4, number 4 • december 2012 second, and given the above principle, we need to ask whether selective breeding imposes significant harms or risks upon animals, and whether these harms are imposed for an important or trivial human purpose. the judgment that a harm or risk is significant is of course evaluative, and reasonable persons may sometimes disagree about whether particular harms or risks should be deemed “significant.” nonetheless, a good case can be made that at least sometimes, selective breeding results in significant harms and risks to animals, since breed-associated diseases or disorders can result in significant pain and distress, shortened lifespan, and difficulty for animals in satisfying some preferences (e.g., by damaging an animal’s mobility, sight, hearing, etc.). for illustrative purposes, examples of breed-associated diseases that severely set back an animal’s interests would (in my view) include, but not be limited to: intervertebral disc disease, hip dysplasia, severe atopy, progressive retinal atrophy, brachycephalic airway obstruction syndrome, elbow dysplasia, and syringomyelia. next, we should assess the importance of the human interests at stake in selective breeding. this is likely to be the most controversial aspect of the ethical discussion, but it is also the most important. reports and articles on the pedigree breeding of dogs and cats typically recommend some reforms to breeding practices to improve animal health and welfare. however, they also tend to take for granted the fact that it is ethically permissible for humans to breed companion animals for desired appearances or functions, without much if any reflection on the importance of the ends for which we are breeding. given the degree to which veterinarians, breeders, and breed enthusiasts may be invested in selective breeding (for economic purposes, social purposes, or because they are attached to a particular breed), we can expect that questioning the importance of selective breeding will be controversial and perhaps unwelcome, particularly if the ethical perspective from which we are questioning this is one that all parties to the debate claim to agree on (and most persons seem to agree that animals have at least some moral standing). nonetheless, if we are to perform an honest ethical analysis we need to assess whether the harms that result to animals from selective breeding can be justified, and this requires looking at the significance of the human interest(s) at stake. as concerns pedigree dog and cat breeding, a number of considerations militate against the view that the human interest in selective breeding is significant. first, it is important to distinguish the human interest in selective breeding per se from other human interests that are related to animals, such as the human interest in enjoying a relationship with a dog or cat. relationships with animals can valuably enrich human existence, but this is a distinct issue from whether breeding animals for particular appearances or functions serves a significant human interest. second, when considering what makes for a good human life, we can (at least roughly) rank various interests according to their importance. most fundamental are our interests in such things as adequate food, shelter, freedom from significant physical and emotional harm, and basic medial care. without these interests being satisfied, it is very difficult for a person’s life to go well for them. also very important are interests in companionship, some degree of control over one’s life, and the ability to exercise one’s intellectual and creative capacities. however, selectively breeding animals for appearance (which comprises most cases of selective breeding of companion animals) serves an aesthetic or recreational interest. this kind of interest is less fundamental and important than the other kinds of interests listed above. furthermore, the aesthetic interest in an animal’s appearance is not on par with, say, our aesthetic interest in enjoying fine works of art. fine works of art represent significant human cultural achievements, whose appreciation can enrich our understanding of ourselves and the human condition. as concerns breeding for appearance, the term “aesthetic” refers to something different, more along the lines of “taste” or “preference.” moreover, while it can be argued that recreation is an important part of a valuable human existence (even if less fundamental than the other things mentioned here), humans can satisfy an interest in recreation or aesthetic enjoyment in myriad number of ways; pedigree breeding is not the sole means by which humans can recreate. when considering alternatives to current breeding practices it is also important to remember that the ethical debate about selective breeding is not just a matter of whether we selectively breed animals, but also how. where the question at issue is whether a particular breed standard becomes modified somewhat, as opposed to whether a particular breed should be done away with entirely, the significance of the human interest at stake becomes even lesser, since breed enthusiasts 514clinical theriogenology • volume 4, number 4 • december 2012 would not have to give up the breeds in which they take an interest. however, even where we are talking about the elimination of particular breeds altogether, it is difficult to argue that such elimination would set back a significant interest for breed enthusiasts. clearly breed enthusiasts would be unhappy about the elimination of a breed of dog or cat to which they are attached. if such a person had devoted a large portion of their time, money and/or energy to breeding or associated activities, it might be argued that eliminating the breed would set back a significant interest of theirs, since such breeding and breed enthusiasm were a large part of their life. on one sense of the term “significant,” such a person’s interest in a particular breed is significant: they care about it a lot. however, not everything that we care about a lot necessarily deserves to be termed “significant” from a moral perspective. otherwise, any of animals’ interests could be overridden for a human purpose, as long as the purpose in question is one that a person cares about enough. but this means that animals’ interests can, in principle, be overridden for any reason whatsoever, and this seems inconsistent with giving animals serious moral consideration. when assessing the significance of an interest from a moral perspective, part of what is being assessed is the overall importance of that interest as relates to an individual living a valuable or fulfilling life. were certain persons who are “breed fanciers” prevented from appreciating a particular breed of animal (because its conformation or temperament was modified, or because we stopped perpetuating a particular breed entirely), they could still enjoy a very fulfilling life, their disappointment at this loss of breed appreciation notwithstanding. furthermore, the kind of loss in question is one where a person first develops an appreciation for a particular breed, and then has this specific form of appreciation taken away from them. were breeding practices changed with respect to appearance, then many persons in the future would never develop such breed appreciation in the first place. nonetheless it is difficult to imagine their lives going more poorly for them because of this, or even their taking notice of it in the first place. this observation is important as we consider policy moving forward. the selective breeding of companion animals (or support services for this) may also be performed for economic and functional (e.g., herding, hunting) purposes. what of the significance of these? while a person’s ability to earn a living and support themselves and their family is clearly important, again we must think about alternatives, and there is any number of alternative ways for a person to derive income. hence it is difficult to say that a person’s economic interest in selective breeding is significant unless there is no alternative way for them to derive income, and in particular unless their ability to derive income depends on their breeding a particular breed of animal, or breeding for extreme conformation within a breed. it is hard to think of examples where this would be the case. furthermore, while consumers may be preferential to some conformations (e.g., pugs with very brachycephalic faces), in my experience they are often ignorant of the adverse health consequences sometimes associated with selective breeding, and often would rather have a healthier example of the breed with a less extreme conformation once these health consequences are explained to them. hence with aggressive client education about health and welfare, consumer demand for some conformations may dry up. as concerns breeding for function, the importance of selective breeding must be assessed against the importance of the activity for which animals are bred. examples of functions include herding, hunting, or higher production from farm animals. some reports on selective breeding seem to presume that breeding for functions like this is less likely to result in adverse welfare consequences than breeding for appearance4, but this seems doubtful in at least some cases (e.g., farm animals). let’s assume for the sake of argument that breeding for herding, hunting, increased productivity, or other functions imposed significant harm or risk upon the animals. what is the significance of the human interest in these functions? since most hunting is carried out for recreational purposes and not to acquire necessary food (even if the animals killed are eaten), this does not seem to be a significant interest. the same can be said for animal agriculture in most (though not all) cases, since persons in the developed world do not need to consume animal products to be healthy; in fact emerging evidence suggests that plant-based diets may be nutritionally advantageous in some respects. furthermore, in the case of breeding for increased production, we are often dealing with a situation where the interest in question is an interest in less expensive animal products, not a situation where consumers are facing a choice between consuming animal products or not consuming them, further lessening the significance of the human interests at stake. 515 clinical theriogenology • volume 4, number 4 • december 2012 in some cases, breeding laboratory animals to model particular diseases might be viewed as representing a significant human interest (assuming fidelity of the model and importance of the disease). as concerns companion animals, perhaps the best candidate for a significant human interest in selective breeding for function would be service dogs, e.g., seeing eye dogs. i conclude here that outside of a few notable exceptions (e.g., seeing eye dogs), the selective breeding of companion animals for appearance or function generally does not serve a significant human interest. therefore, even on a view according only minimal moral consideration to animals, we are led to the conclusion that the selective breeding of companion animals is (generally) impermissible when it results in significant harm to the animals. this conclusion does not prohibit selective breeding for appearance or function entirely. however, it does support the kinds of policy recommendations typically laid out in reports on the issue. for example, this argument supports the modification of extreme breed conformational standards so as to lessen severe disease impacts (e.g., brachycephalic airway obstruction syndrome). it also supports the modification of breeding practices when such practices impose significant risk upon animals, for example by perpetuating genes causative of significant disease where the genetics of disease are not well enough understood to avoid production of diseased animals.§§ even on this view one might argue that certain breeds of animal should not be perpetuated, because it is impossible to maintain a particular breed without imposing significant harm or risk upon the animals. for example, intervertebral disk disease is arguably a significant risk for dachshunds, and given the etiology of this disease it is questionable whether the breed can be maintained as we know it without imposing this risk upon the animals. however, such conclusions would apply at the level of a specific breed, and not to selective breeding for appearance in general. more stringent restrictions of selective breeding practices in a broad way will likely require that we accord animals more than minimal moral standing. for example, the principle outlined here—that we should not impose significant harms or risks upon animals for trivial human purposes—does not rule out the imposition of small or moderate harms or risks for trivial human purposes. however, it should be emphasized that the discussion here has been aimed at identifying the bare minimum that we owe animals if we say that they have moral standing, and what this implies for selective breeding practices. the point of identifying this “bare minimum” is to motivate political agreement, not to identify what we reasonably owe animals. there is still ample room to endorse more stringent restrictions on breeding practices with respect to animal welfare while still endorsing some form of unequal consideration. this conclusion rests on the assumption that the selective breeding of companion animals generally does not serve a significant human interest. there may be exceptional cases in which the human interest served is more significant, in which case restrictions on breeding would have to be arrived at through more stringent principles. in addition, it is always open to defenders of selective breeding to argue that even in routine cases, the selective breeding of companion animals serves a significant human interest—though this conclusion seems dubious. what seems most important for present purposes as concerns unequal moral consideration views is to emphasize the form of argument that these views must take, and to show how they are different than the approach often espoused in veterinary medicine. the american veterinary medical association’s “animal welfare principles” start by listing a number of appropriate human “uses” of animals (e.g., for food, fiber, research, etc.), and while “humane” or “respectful” treatment of animals is advocated, human obligations to protect or promote animal welfare are recognized only insofar as they are consistent with these uses.18 this approach does not provide much guidance in specifying which practices within broad categories of animal use (e.g., agriculture) are permissible or impermissible, for example by delineating which kinds of breeding, housing, or handling practices are consistent with humane or respectful animal agriculture. nor does this approach advance arguments to support its assertions about the permissibility of certain kinds of animal use in general. these are both serious philosophical defects. while there are many details of unequal moral consideration views that remain to be worked out, the general framework of moral considerability outlined here is in my view the best way for us to frame our obligations to animals, and to build upon in future work. as concerns unequal moral consideration views, the advantage of this approach over the “welfarist” approach characteristic of the avma principles is that it forces us to 516clinical theriogenology • volume 4, number 4 • december 2012 honestly assess the significance of the human interests at stake in various kinds of animal use, and whether those interests justify harm to animals. since various kinds of animal use presuppose certain tradeoffs of animals’ interests to serve humans’ interests, assuming the moral defensibility of such activities without providing a supporting argument begs important ethical questions. similarly, the fact that some forms of animal use (e.g., agriculture, or pedigree breeding of dogs and cats for appearance) will be permissible in broad sketch on unequal moral consideration views in broad sketch does not mean that all possible variations of these activities will be. conclusion: animals’ moral considerability and selective breeding the preceding discussion shows that significant changes to current breeding practices of companion animals can be supported just by acknowledging that animals have nontrivial moral status. this is because selective breeding may at times impose significant harm or risk upon animals, and because the human interest in the selective breeding of companion animals seems insignificant in most cases. at a minimal level of moral consideration, however, selective breeding may still be ethically defensible if it imposes small harms or risks upon animals. at any higher level of moral consideration it is questionable whether the selective breeding of companion animals for appearance or function can be justified when it imposes harm or risk. there is also the larger question of whether we should be selectively breeding companion animals for appearance or function at all. in part this depends on an empirical question: is it possible to selectively breed animals for appearance or function without imposing additional harm or risk as compared to an outbred population? it also depends on moral questions: if selective breeding does impose harm or risk by necessity, how much moral consideration is owed to animals, and how significant are the human interests at stake? finally, if animals are accorded no direct moral standing at all, then there are few if any restrictions on breeding practices that can be justified. veterinary responses to ethical issues in selective breeding thus far the discussion has been focused on fundamental moral arguments around the selective breeding of animals for appearance (most commonly) and function. we have not talked about veterinarians’ role in improving breed health. this is purposeful: we cannot know how veterinarians, either individually or as a profession, should respond to this issue until we have some sense of whether current breeding practices are morally defensible. unless we deny that animals have direct moral standing, it would appear that current breeding practices (on the main) are not morally defensible. however, it seems unrealistic to expect the veterinary profession as a whole to renounce selective breeding for appearance in the immediate future, or even to radically change breeding practices to curb deleterious welfare effects—though, to be clear, if the moral arguments indicate that either of these responses is obligatory, then this is how the profession should respond. but in the spirit of moving us forward, i offer a few practical recommendations that we as a profession should be able to implement in the immediate future. most reports on selective breeding and animal welfare emphasize the emerging need for veterinarians to keep abreast of developing tools to screen for breed-associated disease, and to actively counsel clients about the possible welfare effects of breed conformation and breed-associated disease. i agree with these conclusions, but think that the profession needs to go further. first, and most importantly, the veterinary profession needs to engage fundamental arguments around animals’ moral standing and the importance of the human interests that are served by various kinds of animal use or treatment. discussions about veterinary ethics, if they occur at all, tend to take for granted various kinds of animal use and to advocate for “humane” or “respectful” treatment in very general terms. but we have already seen that this is a problem. it is a problem because endorsements of various kinds of animal use in the abstract provide little guidance when thinking about whether specific decisions associated with that use are morally permissible. knowing that is morally permissible to raise and kill animals for food, to use animals in research experiments, or to breed animals for certain human ends does not tell us what 517 clinical theriogenology • volume 4, number 4 • december 2012 kinds of moral restrictions apply to these practices. to say that our treatment of animals should be “respectful” or “humane” does not really help, since these terms are vague. furthermore, whether these activities are morally permissible is itself something that requires argument and cannot be assumed. to date, the veterinary profession has tended to dismiss views calling for substantial changes to the status quo of animal treatment as “radical,” “irrational,” and the product of “strong animal rights views.” but none of these responses is adequate. substantial revision to the status quo of animal treatment does not rest on equal moral consideration or strong animal rights views alone. rather, unequal consideration views are also capable of motivating significant changes to current practices, once it is recognized that harms to animals actually have to be justified by reference to the significance of the human interests at stake. furthermore, the absolute dismissal of any view as “radical” or “irrational” without considering the view on its merits and refuting it is itself an irrational response. the only way that we can know whether a particular moral viewpoint is correct is to assess the reasons for and against the view, and to do so in as rigorous and unbiased a way as possible. without doing this, veterinarians cannot claim the moral or rational high ground when rejecting a particular viewpoint as incorrect, or advancing their own viewpoint as correct. moreover, a large amount of philosophy about animals’ moral standing has been written over the past forty years, much of which questions traditional views about animals’ radically inferior moral standing to humans. in addition, public views about the appropriate treatment of animals are changing, as concerns farm animals, research animals, and companion animals. in many cases the public’s views seem to be more progressive and more protective of animal welfare than the views espoused by mainstream veterinary medicine. from a political standpoint, veterinary medicine can only avoid a genuine engagement with the moral issues for so long before it loses social credibility. second, as a corollary to the preceding point, veterinarians should be receptive to legislative proposals to require veterinary health screens or other services as part of ensuring breed health. such proposals are good for animal welfare and the profession, but historically the veterinary medical profession has sometimes resisted legislative proposals that would empower it (e.g., as concerns laboratory animal protection) because such proposals were viewed as being motivated by “radical” or “extremist” social views.19 third, if veterinarians are also breeders, then whether and how they breed animals is something that is directly under their control. hence moral arguments about what kinds of breeding practices are defensible can be applied directly by these persons; no additional considerations appear to be required. fourth, since veterinarians provide support services for breeders and animal owners, we must think about how veterinarians should respond when clients request services or make decisions that are not in-line with a veterinarian’s values. it is often noted that the veterinarian-client-patient relationship (vcpr) is triangular in nature, and that the veterinarian must serve the interests of both animals and their owners. however, when the interests of animals and owners conflict, the profession has historically tended to side with owners except in egregious situations where neglect or abuse is occurring. though veterinarians ultimately lack control over the decisions that clients make, they do have control over the degree to which they advocate for what they believe is the right decision, as well as their involvement in ethically problematic decisions or practices. veterinarians may need to more aggressively advocate for the health of companion animals, for example by stating outright to a client considering purchase of a particular breed that the veterinarian believes certain breeding practices to be unethical, as opposed to saying something like “well, if it were me, i wouldn’t purchase breed ‘x’, but it’s ultimately your call.” in addition, veterinarians may need to consider conscientious objection in cases where support services are requested by breeders known to be involved in unethical practices, and where education and advocacy by the veterinarian have failed. notes * though some persons distinguish between “inbreeding” and “linebreeding,” i do not—the difference between these two terms is only one of degree and not one of kind. even where two animals of the same breed not known to share a particular ancestor are 518clinical theriogenology • volume 4, number 4 • december 2012 mated, the mating is between two individuals more genetically similar than two individuals of different breeds, or two hybrid individuals. † the reason that some proponents of strong animal rights views view the domestication of animals as wrong is that, in their view, the human relationship with such animals is necessarily exploitative. however, even if someone accepts the core premises of a strong animal rights view, including the premises that animals’ interests deserve equal moral consideration to humans’ interests and that both humans and nonhuman animals have strong negative rights against being harmed, it does not follow logically that humans’ relationship to such animals is exploitative. in fact i think that, apart from special circumstances, this exploitation thesis is mistaken, though i won’t defend this argument here. ‡ the term “beg the question,” as used in philosophy, is a term of logic. it means that a person is assuming the truth of something that requires demonstration by argument. this is distinct from the ordinary-language use of “begs the question” as synonymous with “raises the question.” § these terms are often bundled together in the so-called “fact-value distinction” or “fact-value dichotomy.” unfortunately, these terms have a number of meanings in philosophy, some of which are more controversial than others. i’ll avoid getting into these issues here and simply talk about “non-moral facts” and “moral evaluations,” which should be sufficient to get the job done without attaching any philosophical baggage. the reader should not draw from my use of this distinction the conclusion that moral judgments cannot be true or false, or that science practice (which generates many non-moral facts) does not involve any kinds of value judgments. ** for excellent reviews of these issues, see haynes9 and rollin.20 †† for example, in a recent interview with the humane society veterinary medical association, michael blackwell, stated that “hsvma must take the lead as an affiliate of hsus to help define animal welfare from the perspective of trained veterinary professionals. the veterinary profession cannot do this by riding on the back of the bus while laypersons are driving the bus. rather, we need to take control of the steering wheel to ensure that there are balanced, reasonable and educated voices speaking for the needs of animals.” humane society veterinary medical association monthly news, june 14, 2012. available at: http://action.humanesociety.org/site/messageviewer?em_id=41441.0&dlv_id=44022 ‡‡ by “interest” i mean those things that are pertinent to an animal’s well being; the things that matter to it in the sense of making the animal’s life go well or poorly. an animal’s “welfare” represents the collective set of interests that it has. §§ at the present time, screening tests for genetic disease are relatively few, and we may ask the question of how breeders should proceed in cases where screening tests and prevalence data are not robustly available. in such cases, it may be unknown whether a particular mating is likely to impose a significant risk of genetic disease upon offspring. here ancillary sources of data, such as expected breeding values, may be of use. it may also be necessary to institute certain rules (e.g., rules limiting the number of times an animal may sire offspring) based on the general knowledge that particular kinds of breeding practices are particularly risky when it comes to perpetuating genetic disease. references 1. ackerman l: the genetic connection: a guide to health problems in purebred dogs. lakewood (co): american animal hospital association press; 1999. 2. rooney n, sargan d: pedigree dog breeding in the uk: a major welfare concern? west sussex (uk): royal society for the prevention of cruelty to animals; 2009. 3. asher l., diesel g, summers jf, et al: inherited defects in pedigree dogs, part 1: disorders related to breed standards. vet j 2009;182:402-411. 4. bateson p: independent inquiry into dog breeding. privately published; 2010. available at: http://www.dogbreedinginquiry.com 5. summers jf, diesel g, asher l, et al: inherited defects in pedigree dogs, part 2: disorders that are not related to breed standards. vet j 2010;183;39-45. 6. associate parliamentary group for animal welfare: a healthier future for pedigree dogs. london: apgaw; 2009. available at: http://www.apgaw.org/ 7. rooney n: the welfare of pedigree dogs: cause for concern. j vet behav 2009;4:180-186. 8. companion animal welfare council: breeding and welfare in companion animals. devon (uk): companion animal welfare council, 2006. available at: http://www.cawc.org.uk/reports 9. haynes r: animal welfare: competing conceptions and their ethical implications. new york: springer publishing; 2008. 10. korte sm, olivier b, koolhaas jp: a new animal welfare concept based on allostasis. physiol behav 2007;92:422-428. 11. collins lm, asher l, summers j, et al: getting priorities straight: risk assessment and decision-making in the improvement of inherited disorders in pedigree dogs. vet j 2011;189:147-154. 12. scully m: dominion: the power of man, the suffering of animals, and the call to mercy. new york: macmillan publishing; 2003. 13. linzey a:christianity and the rights of animals. chestnut ridge (ny): crossroad publishing; 1987. 14. degrazia d: animal rights: a very short introduction. new york: oxford university press; 2002. 15. garner r:. animal ethics. malden (ma): polity press: 2005. 16. degrazia d: taking animals seriously: mental life and moral status. cambridge: cambridge university press, 1996. 519 clinical theriogenology • volume 4, number 4 • december 2012 17. regan t: the case for animal rights, revised ed. los angeles: university of california press, 2004. 18 american veterinary medical association (avma). “animal welfare principles.” november, 2006. available at: https://www.avma.org/kb/policies/pages/avma-animal-welfare-principles.aspx 19. rollin b: science and ethics. new york: cambridge university press, 2006, pp. 110-111. 20. rollin b: the unheeded cry: animal consciousness, animal pain and science. new york: oxford university press; 1990. 520clinical theriogenology • volume 4, number 4 • december 2012 2009: endometritis in dogs current knowledge and future considerations endometritis in dogs – current knowledge and future considerations 1 s.k. lyle 2 department of veterinary clinical sciences school of veterinary medicine, 3 louisiana state university, baton rouge, la 4 abstract 5 canine endometritis, as an entity separate from cystic endometrial hyperplasia, is 6 a poorly understood process, yet may be responsible for infertility. inflammatory 7 infiltrates of the endometrium in the absence of proliferative changes are the hallmark of 8 this disease. the ascension of bacteria from the vagina through the cervix is theorized 9 to lead to a chronic, low-grade endometritis, which does not produce overt clinical 10 disease, such as that seen with pyometra. escherichia coli is the organism most 11 commonly implicated in pyometra, and is likely the most common organism involved 12 with endometritis. various uropathogenic virulence factors (uvfs) have been identified 13 from uterine isolates, many of which share common uvfs from isolates producing 14 urinary tract disease in dogs and humans. of these, p fimbria is thought to be crucial for 15 initial bacterial adherence to the luminal epithelium. reduction in mucin-1 expression 16 and immunolocalization in endometrial epithelial cells in early diestrus may be involved 17 with producing a permissive state for bacterial adhesion, allowing colonization. samples 18 that may aid in the diagnosis of endometritis include vaginal or uterine cultures, uterine 19 biopsy, and possibly endometrial cytology and ultrasonography. treatment of 20 endometritis is focused on elimination of infection through the use of antimicrobial 21 agents, and physical clearance of the uterus through the use of prostaglandins, 22 dopamine agonists, and progesterone-receptor antagonists. 23 105 24 keywords: dog, infertility, endometritis, diagnosis, treatment 25 introduction 26 although many clinicians might empirically agree that inflammation of the 27 endometrium may contribute to a reduction in fertility, direct evidence of a causal effect 28 of endometritis on reducing fertility is lacking. subfertility and infertility due to cystic 29 endometrial hyperplasia is more generally agreed upon; however, whether the reduction 30 in fertility is due to the proliferative response of the endometrium or the inflammatory 31 component of the process is unknown. 32 histopathology of the canine endometrium 33 endometrial hyperplasia, with or without cystic changes is considered a post-34 estrual luteal phase disease. age and nulliparity are considered risk factors for 35 development of ceh.1,2 two main hypotheses exist on the pathogenesis of ceh and 36 subsequent development of pyometra. the classical description involves the 37 development of endometrial hyperplasia which subsequently leads to an inflammatory 38 reaction. the accumulation of secretions from the hyperplastic endometrium supports 39 the growth and proliferation of bacteria which ascend through the cervix. establishment 40 of bacterial infection leads to the accumulation of exudate (pyometra). an alternative 41 hypothesis centers on the premise that chronic low-grade uterine infection drives the 42 endometrial proliferative response either by bacterial toxins or inflammatory mediators.3 43 it has also been suggested that endometritis-pyometra can occur independent of 44 endometrial hyperplasia; unfortunately the age distribution of these cases were not 45 defined.4 a variety of stimuli have been used to experimentally reproduce endometrial 46 106 proliferation. these have ranged from china balls,5 suture,6-8 and bacteria,9-13 among 47 others. the proliferative changes observed have been well-characterized, and are not 48 the focus of this discussion. for detailed information on ceh, the reader is directed to 49 several excellent reviews on the topic.1,3,4,14 50 endometritis is also a common finding, and was recently determined to be the 51 most common diagnosis (94 cases) in a survey of 366 canine endometrial biopsies. 52 hyperplasia was the second most common diagnosis (86 cases).15 it is most common 53 to find a plasmacytic infiltrate with subclinical endometritis14 or with ceh with 54 plasmacytic infiltration and little intraluminal fluid accumulation,1 progressing to 55 neutrophilic plasmacytic infiltrates in cases of ceh and significant fluid accumulation 56 (pyometra).1 57 microflora of the reproductive tract 58 debate exists on whether vaginal cultures are useful for determining the 59 presence of intrauterine infection. although most prepuberal and postpuberal bitches 60 were found to have positive vaginal cultures, the majority of uterine cultures were found 61 to be negative.16 similarly, all uterine swabs collected from late diestrus, progestin-62 supplemented bitches were sterile,17 although concurrent sampling of the vagina was 63 not performed. the predominate isolate from infected uteri is escherichia coli,1,18-22 with 64 reported incidences of 73%,1 79.4%,21 and 85%.22 although e. coli in pure culture was 65 the most common isolate from bitches with pyometra, mixed cultures were the most 66 common finding in ‘infertile’ bitches, leading to the conclusion that vaginal cultures have 67 low diagnostic value.20 conversely, other studies found the uterus not to be a sterile 68 environment,23,24 and that vaginal isolates reflected those of the uterus.23 bacteria were 69 107 consistently recovered from the uterus during proestrus and estrus, and post-mortem 70 uterine isolates always reflected those of the cervix and vagina.24 the most common 71 uterine isolates were e. coli, haemophilus spp., α-hemolytic streptococci, 72 corynebacterium spp., streptococcus canis, alcaligenes faecalis, bacteroides spp., 73 pasteurella spp., and proteus mirabilis.24 a method to transcervically collect uterine 74 secretions that is guarded from vaginal secretions would enhance the ability of clinicians 75 to accurately diagnose the presence of bacteria in the uterus. 76 several authors have investigated virulence factors of e. coli isolates from cases 77 of pyometra. early characterizations focused on the presence of the o-18 and k-78 antigen21,22 uropathogenic e. coli strains, which are responsible for urinary tract 79 infections in dogs and cats, may originate from the intestinal tract, and possess a cluster 80 of virulence-related genes encoding for specific o-antigens, type 1 fimbriae, p fimbriae, 81 s fimbriae, α-hemolysin, cytotoxic necrotizing factor 1, and aerobactin (iron-82 sequestering system).25-27 these strains are not canine-specific, and it has been 83 suggested that the dog may serve as a source of uropathogenic e. coli for human 84 urinary tract infections (uti).28,29 biochemical fingerprinting of e. coli isolates from 85 pyometra and uti suggest that these isolates originate from the fecal flora, and the 86 same clone of e. coli is present in cases with concurrent uti and pyometra.30 87 similarly, dna-profiles of e. coli isolates from the urinary bladder and uterus of bitches 88 affected simultaneously with uti and pyometra were 100% identical, and that all 89 colonies from a site were identical, despite macroscopic morphologic differences.31 the 90 papgiii allele, the most frequent allele encoding for p fimbriae in canine and human 91 uropathogenic e. coli isolates,29 had a significantly higher prevalence in e. coli isolates 92 108 from pyometra, and the proportion of strains from pyometra possessing more than three 93 uropathogenic factors was greater than that of fecal strains.32 the presence of p 94 fimbriae is thought to be crucial for bacterial adherence to epithelial cells of the urinary 95 tract. the papgiii adhesion binds to galα1-4 galβ-containing glycolipid receptor and its 96 coreceptor tlr4 present on urinary epithelial cells.33 the presence of these receptors 97 has been confirmed for canine urinary epithelium,34 but has not been investigated in 98 canine endometrium. other virulence genes that have been associated with 99 uropathogenicity and were present in high proportions of isolates from pyometra 100 isolates include fim (type i fimbriae) and sfa (s fimbriae), although the differences were 101 not statistically significant.32 although type i fimbriae are present on many isolates 102 from human utis, the correlation with pathogenicity is considered low.35 escherichia 103 coli bearing s fimbriae bind to human renal proximal tubular cells,36 but only 27.4% of 104 strains were positive for sfa compared to 97.5% of strains carrying fimh (type i 105 fimbriae).37 106 these findings have led researchers to the conclusion that the pathogenesis of 107 both uti and pyometra involve ascension of intestinal strains of e. coli into the lower 108 urinary tract, cranial vagina, and uterus.28,30,32 only a few investigators have attempted 109 to induce infection by inoculation with e. coli. in a series of investigations using an e. 110 coli isolate from a clinical case of pyometra, nomura et al. inoculated the uterus of dogs 111 in either pro-estrus/estrus, diestrus, post-partum, or anestrus with or without cervical 112 ligation.9,11,12 when examined 12 d post-inoculation with cervical ligation, the incidence 113 of pyometra in proestrus/estrus, diestrus, post-partum, and anestrus was 100%, 100%, 114 80%, and 28%, respectively.9 when examined 12 d post-inoculation without cervical 115 109 ligation, the incidence of pyometra in pro-estrus/estrus, diestrus, post-partum, and 116 anestrus was 25%, 89.9%, 70.6%, and 50.6%, respectively. more recently, 5×107 cfu 117 of an e. coli (o2:h:k) isolated from a clinical case of pyometra was inoculated in the 118 uterus of intact bitches at either post-lh day 1-10, 11-20, 21,30, 31-40, 41-50, or 51-60; 119 the incidence of pyometra induced was 16.7%, 90.9%, 78.9%, 62.5%, 40.0%, and 0%, 120 respectively. bitches with induced pyometra were either treated with dinoprost 121 tromethamine and enrofloxacin, or were allowed to spontaneously recover. there was 122 no difference in pregnancy rates between treated and non-treated bitches on the 123 subsequent estrus, and recurrence of pyometra did not occur.13 while this model did 124 induce pyometra in the strict sense of the definition (the presence of pus in the uterus 125 during the luteal phase), the subsequent fertility and lack of recurrence do not fit the 126 typical clinical scenario of bitches with spontaneous pyometra. this model appears to 127 more closely approximate endometritis than pyometra. subsequently, inoculation of the 128 uterus with an e. coli strain possessing five uvfs induced ceh/pyometra in diestrus-129 simulated ovariectomized bitches, while inoculation into the vagina failed to establish 130 uterine infection or endometrial changes.10 differences in these two studies lie in the 131 status of the bitch and potential difference in the pathogenicity of the e. coli strain. 132 host-pathogen interactions 133 limited investigation on the host-response to intrauterine infections exists, but a 134 few noteworthy studies shed some light on mechanisms by which bacterial are able to 135 colonize the endometrium. the proliferative response of peripheral blood monocytes 136 (pbmcs) to a clone of e. coli isolated from the uterus from a dog with pyometra was 137 significantly decreased at day 10 of diestrus compared to proestrus, estrus, day 30 of 138 110 diestrus, or anestrus.38 similarly, the addition of progesterone or 5α-139 dihydroprogesterone to pbmcs collected from anestrous bitches significantly reduced 140 the response to e. coli compared to pbmcs supplemented with estradiol 17-β, 17α-141 hydroxyprogesterone, or pregnenolone; and progesterone reduced the expression of 142 ifnγ by pbmcs compared to estradiol.38 143 recently lactoferrin, an antimicrobial and immunomodulator member of the 144 transferrin gene family which is expressed by epithelial cells and neutrophil granules, 145 has been identified in the equine and canine endometrium.39,40 lactoferrin’s 146 antibacterial property lies within its ability to sequester free iron, thereby inhibiting 147 bacterial growth. in the mare, lactoferrin expression was upregulated during early 148 estrus, protein staining was uninfluenced by cycle and was most intense in the 149 glandular epithelium, and expression of lactoferrin was only increased in mares with 150 delayed physical clearance during early estrus,39 which might represent a response to 151 inflammation. the pattern of lactoferrin expression has also been described in the bitch, 152 where expression increased from proestrus to estrus, then significantly decreased from 153 estrus to day 10 of diestrus, remaining low at day 35 of diestrus and anestrus; a similar 154 pattern was observed with immunohistochemical staining for lactoferrin,40 leading the 155 investigators to conclude that estrogen was involved with the regulation of lactoferrin 156 expression. although reduced lactoferrin expression during diestrus would be a 157 plausible explanation of reduced microbial defenses and increased susceptibility to 158 infection, lactoferrin expression was increased in bitches with pyometra,40 similar to 159 what was observed in mares with delayed uterine clearance and post-mating induced 160 endometritis.39 increased lactoferrin expression in both instances may due to an influx 161 111 of neutrophils.41 while intriguing from a perspective of host-pathogen interactions, 162 unless diminished response in lactoferrin expression and production were observed in 163 bitches suffering from pyometra, reduced lactoferrin activity is unlikely to be responsible 164 for increased susceptibility to infection. 165 mucin-1 (muc1) is an important component of the epithelial cell glycocalyx, 166 functioning as an anti-adhesive molecule; loss of muc1 expression is considered an 167 integral step in allowing adhesion between the trophoblast and the luminal epithelium.42 168 in normal, cyclic bitches, muc1 expression and localization was significantly decreased 169 at day 10 of diestrus and in bitches with pyometra compared to proestrus, estrus, day 170 35, or anestrus. additionally, muc1 expression and adherence of e. coli to endometrium 171 was inversely correlated.43 clearly, further research is needed in the area of host-172 pathogen interactions of the canine uterus. 173 diagnosis of endometritis 174 if bacteria such as e. coli can serve as a stimulus for endometrial proliferation, 175 and hence cystic endometrial hyperplasia; then early diagnosis and appropriate therapy 176 might lead to prolongation of the fertile lifespan of some bitches. for additional detail on 177 specifics for collecting reproductive tract tissues, the reader is directed to an excellent 178 previous review on this topic.44 179 cultures – the most common method used clinically is guarded culture of the anterior 180 vagina during proestrus. as discussed previously, these may or may not reflect a 181 potential uterine pathogen, but in cases in which significant intrauterine infection is 182 suspected, it is the opinion of this author and others,44 that this method should yield a 183 satisfactory sample. it is also possible to collect intrauterine secretions following 184 112 transcervical catheterization with a 4 to 7 fr catheter through a rigid cystoscope or 185 endoscope in the standing bitch.45 unfortunately this sample would also suffer from 186 contamination by vaginal fluids. hysteroscopy in the anesthetized bitch may lessen the 187 contamination, but caused petechia or ecchymoses in 50% of cases, and poor 188 visualization in 37.5% of cases.45 the method providing the most accurate sample of 189 uterine secretions would be that obtained during hysterotomy. in most instances this 190 sample would be obtained concurrent to uterine biopsy.46 191 cytology – endometrial cytology is a commonly used diagnostic tool for the diagnosis of 192 equine endometritis; however, it is not routinely used for evaluation of canine cases. 193 watts et al.47 described the endometrial cytology of the normal bitch in samples 194 collected by transcervical catheterization or at post-mortem.45 endometrial cells were 195 present at all stages, and exhibited degenerative changes during late diestrus, anestrus, 196 and postpartum. during proestrus and estrus, healthy endometrial cells, neutrophils and 197 bacteria were commonly observed. during diestrus and early pregnancy, healthy 198 endometrial cells and neutrophils were most common. during late diestrus and 199 anestrus, evidence of endometrial cell degeneration was observed and lymphocytes 200 and macrophages were the most common leukocyte present. to the authors knowledge 201 there are no published reports regarding changes in endometrial cytology with uterine 202 pathology such as endometritis or cystic endometrial hyperplasia. 203 uterine biopsy – biopsy of the endometrium is maximally invasive, yet provides the 204 most accurate sample for the diagnosis and prognosis for endometritis or other uterine 205 pathology. samples can be obtained by either laparotomy,46 or by a transcervical 206 113 approach;48 however, the later technique only provided a diagnostic sample 31% of the 207 time and was associated with hematomucometra in 44% of cases. 208 ultrasonography – ultrasonography is commonly used to diagnose cystic endometrial 209 hyperplasia with or without pyometra. its usefulness for the diagnosis of endometritis 210 has not been correlated with other diagnostic techniques. 211 treatment of endometritis 212 from the preceding discussion, it is apparent that arriving at an accurate 213 diagnosis of endometritis may be difficult. no controlled studies have been done on 214 therapeutic regimes for the treatment of endometritis. therefore, treatment options are 215 based on what has been recommended for medical management of pyometra, which is 216 focused on eliminating bacterial infection, if present, and stimulating physical clearance 217 of the uterus. the former is achieved by appropriate antimicrobial agents, the latter by 218 terminating the luteal phase through the use of prostaglandins, dopamine agonists, or 219 progesterone-receptor antagonists, and by stimulation of myometrial contractions 220 through the use of prostaglandins. prior to initiating medical therapy, detailed owner 221 counseling regarding the intended use and breeding value of the bitch should occur. 222 ovariohysterectomy should be recommended for bitches without significant 223 reproductive value. 224 antimicrobial agents – when at all possible, antimicrobial agents should be chosen 225 based on results of culture identification and sensitivity patterns. the injudicious use of 226 antibiotics has the potential to select for strains of bacteria with greater antibiotic 227 resistance patterns. to the author’s knowledge multi-resistant staphylococcus aureus 228 (mrsa) has not been isolated from a canine case of endometritis or pyometra. this 229 114 would be a dire situation indeed. recently, mrsa has been recovered from the uterus 230 of mares with extensive history of intrauterine antibiotic therapy.49 systemic 231 administration in the bitch is the most commonly used route of delivery. although 232 transcervical delivery of antibiotics into the lumen is possible, it is doubtful that uniform 233 distribution of drug to the entire endometrial surface would occur. additionally, daily 234 treatment would be required; repeated catheterization adding considerably to the cost of 235 treatment. systemic administration allows for longer treatment regimes (10-14 d), with 236 good penetration of the endometrium. 237 prostaglandin – prostaglandin has been used for many years for the treatment of 238 pyometra.50,51 typical doses of pgf2α (dinoprost tromethamine) range from 100 to 500 239 µg/kg, sq, one to three times daily, and are considered luteolytic. lower doses (10 to 240 50 µg/kg, up to three to five times daily) can also be used to achieve uterine 241 evacuation.52 the use of prostaglandin in the dog is extra-label; therefore, informed 242 client consent is recommended. transient (20 to 30 min) side effects of salivation, 243 defecation, urination, and emesis are not uncommon at the higher doses, but are 244 drastically reduced when lower doses are used. 245 dopamine agonists – inhibitors of prolactin will aid in the rapid reduction in 246 progesterone concentrations, and are frequently combined with low-dose prostaglandin 247 protocols for the treatment of induced abortion.53,54 bromocriptine (25 µg/kg, po, q8 to 248 12 h) or cabergoline (5 µg/kg, po, q24 h), in combination with prostaglandins, can lead 249 to luteolysis with 24 h. 250 progesterone-receptor antagonists – although not available in the united states, 251 aglepristone has been used for pregnancy termination,55 and for treatment of pyometra 252 115 alone56 or combination with cloprostenol for pyometra.57 in a recent study the overall 253 success rate for treatment of pyometra (35 open, 17 closed) with cloprostenol (1 µg/kg) 254 and agelpristone (10 mg/kg, daily) was 84.4% compared to 60% for aglepristone alone; 255 the recurrence rate at 12 and 24 mo was 13% and 19% respectively.58 256 discussion 257 it would seem that there is rather compelling evidence to suggest that subclinical 258 endometritis may precede the development of clinically evident ceh and pyometra. the 259 difficulty lies in the ability to render a diagnosis prior to development of proliferative 260 changes in the endometrium. clearly further research is needed to prove a causal 261 relationship between these two entities. comparison of e. coli strains possessing or 262 lacking uvfs in a model would be a useful first step in that process. further 263 investigation of host-defense mechanisms, such as the presence of toll-like receptors 264 on the endometrium, cytokine signaling involved with endometritis and proliferative 265 changes of the endometrium would also be informative. biofilms have been described in 266 a variety of mucosal systems, and provide a mechanism for pathogen evasion of host 267 recognition and protection from certain treatment modalities. uropathogenic e. coli 268 producing biofilms are implicated in chronic urinary tract infections in people,59 and it 269 has been suggested that some cases of e. coli endometritis produce biofilm.60 from a 270 diagnostic standpoint, a guarded system for transcervical collection of uterine secretions 271 in the standing bitch would benefit not only research endeavors, but collection of 272 diagnostic samples from clinical cases. such information is needed to render an 273 accurate diagnosis, and to progress towards effective treatment strategies. 274 references 275 116 1. dow c: the cystic hyperplasia-pyometra complex in the bitch. vet rec 276 1958;70:1102-1108. 277 2. egenvall a, hagman r, bonnett bn, et al: breed risk of pyometra in insured dogs 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zentralbl veterinarmed a 1992;39:43-56. 321 20. bjurstrom l: aerobic bacteria occurring in the vagina of bitches with reproductive 322 disorders. acta vet scand 1993;34:29-34. 323 21. dhaliwal gk, wray c, noakes de: uterine bacterial flora and uterine lesions in 324 bitches with cystic endometrial hyperplasia (pyometra). vet rec 1998;143:659-325 661. 326 22. sandholm m, vasenius h, kivisto ak. pathogenesis of canine pyometra. j am 327 vet med assoc 1975;167:1006-1010. 328 23. baba e, hata h, fukata t, et al: vaginal and uterine microflora of adult dogs. 329 am j vet res 1983;44:606-609. 330 24. watts jr, wright pj, whithear kc: uterine, cervical and vaginal microflora of the 331 normal bitch throughout the reproductive cycle. j small anim pract 1996;37:54-332 60. 333 25. yuri k, nakata k, katae h, et al: distribution of uropathogenic virulence factors 334 among escherichia coli strains isolated from dogs and cats. j vet med sci 335 1998;60:287-290. 336 26. senior df, deman p, svanborg c: serotype, hemolysin production, and 337 adherence characteristics of strains of escherichia coli causing urinary tract 338 infection in dogs. am j vet res 1992;53:494-498. 339 27. feria c, machado j, correia jd, et al: virulence genes and p fimbriae papa 340 subunit diversity in canine and feline uropathogenic escherichia coli. vet 341 microbiol 2001;82:81-89. 342 119 28. johnson jr, stell al, delavari p, et al: phylogenetic and pathotypic similarities 343 between escherichia coli isolates from urinary tract infections in dogs and 344 extraintestinal infections in humans. j infect dis 2001;183:897-906. 345 29. johnson jr, o'bryan tt, low da, et al: evidence of commonality between 346 canine and human extraintestinal pathogenic escherichia coli strains that 347 express papg allele iii. infect immun 2000;68:3327-3336. 348 30. wadås b, kühn i, lagerstedt as, et al: biochemical phenotypes of escherichia 349 coli in dogs: comparison of isolates isolated from bitches suffering from pyometra 350 and urinary tract infection with isolates from faeces of healthy dogs. vet microbiol 351 1996;52:293-300. 352 31. hagman r, kindahl h, fransson ba, et al: differentiation between pyometra and 353 cystic endometrial hyperplasia/mucometra in bitches by prostaglandin f2-alpha 354 metabolite analysis. theriogenology 2006;66:198-206. 355 32. chen ymm, wright pj, lee cs, et al: uropathogenic virulence factors in isolates 356 of escherichia coli from clinical cases of canine pyometra and feces of healthy 357 bitches. vet microbiol 2003;94:57-69. 358 33. wullt b, bergsten g, samuelsson m, et al: the role of p fimbriae for escherichia 359 coli establishment and mucosal inflammation in the human urinary tract. int j 360 antimicrob agents 2002;19:522-538. 361 34. stromberg n, marklund bi, lund b, et al: host-specificity of uropathogenic 362 escherichia coli depends on differences in binding specificity to gal alpha 1-363 4gal-containing isoreceptors. embo j 1990;9:2001-2010. 364 120 35. hagberg l, jodal u, korhonen tk, et al: adhesion, hemagglutination, and 365 virulence of escherichia coli causing urinary tract infections. infect immun 366 1981;31:564-570. 367 36. kreft b, placzek m, doehn c, et al: s fimbriae of uropathogenic escherichia coli 368 bind to primary human renal proximal tubular epithelial cells but do not induce 369 expression of intercellular adhesion molecule 1. infect immun 1995;63:3235-370 3238. 371 37. tiba mr, yano t, leite dds: genotypic characterization of virulence factors in 372 escherichia coli strains from patients with cystitis. revista do instituto de 373 medicina tropical de sao paulo 2008;50:255-260. 374 38. sugiura k, nishikawa m, ishiguro k, et al: effect of ovarian hormones on 375 periodical changes in immune resistance associated with estrous cycle in the 376 beagle bitch. immunobiology 2004;209:619-627. 377 39. kolm g, klein d, knapp e, et al: lactoferrin expression in the horse 378 endometrium: relevance in persisting mating-induced endometritis. vet immunol 379 immunopathol 2006;114:159-167. 380 40. kida k, baba e, torii r, et al: lactoferrin expression in the canine uterus during 381 the estrous cycle and with pyometra. theriogenology 2006;66:1325-1333. 382 41. bentwood bj, henson pm: the sequential release of granule constituents from 383 human neutrophils. j immunol 1980;124:855-862. 384 42. spencer te, johnson ga, bazer fw, et al: implantation mechanisms: insights 385 from the sheep. reproduction 2004;128:657-668. 386 121 43. ishiguro k, baba e, torii r, et al: reduction of mucin-1 gene expression 387 associated with increased escherichia coli adherence in the canine uterus in the 388 early stage of dioestrus. vet j 2007;173:325-332. 389 44. root kustritz mv: collection of tissue and culture samples from the canine 390 reproductive tract. theriogenology 2006;66:567-574. 391 45. watts jr, wright pj, lee cs, et al: new techniques using transcervical uterine 392 cannulation for the diagnosis of uterine disorders in bitches. j reprod fert 393 1997;51(suppl):283-293. 394 46. downs m, miller-liebl d, fayrer-hosken r, et al: obtaining a useful uterine 395 biopsy specimen in dogs. vet med 1994;89:1055-1059. 396 47. watts jr, wright pj, lee cs: endometrial cytology of the normal bitch 397 throughout the reproductive cycle. j small anim pract 1998;39:2-9. 398 48. gunzel-apel ar, wilke m, aupperle h, et al: development of a technique for 399 transcervical collection of uterine tissue in bitches. j reprod fertil suppl 400 2001;57:61-65. 401 49. leblanc mm: 2009. personal communication. 402 50. meyers-wallen vn, goldschmidt mh, flickinger gl: prostaglandin f2 alpha 403 treatment of canine pyometra. j am vet med assoc 1986;189:1557-1561. 404 51. gilbert ro, nothling jo, oettle ee: a retrospective study of 40 cases of canine 405 pyometra-metritis treated with prostaglandin f-2 alpha and broad-spectrum 406 antibacterial drugs. j reprod fertil suppl 1989;39:225-229. 407 122 52. verstegen j, dhaliwal g, verstegen-onclin k: mucometra, cystic endometrial 408 hyperplasia, and pyometra in the bitch: advances in treatment and assessment of 409 future reproductive success. theriogenology 2008;70:364-374. 410 53. post k, evans le, jöchle w: effects of prolactin suppression with cabergoline on 411 the pregnancy of the bitch. theriogenology 1988;29:1233-1243. 412 54. onclin k, verstegen jp: comparisons of different combinations of analogues of 413 pgf2α and dopamine agonists for the termination of pregnancy in dogs. vet rec 414 1999;144:416-419. 415 55. galac s, kooistra hs, butinar j, et al: termination of mid-gestation pregnancy in 416 bitches with aglepristone, a progesterone receptor antagonist. theriogenology 417 2000;53:941-950. 418 56. breitkopf m, hoffmann b, bostedt h: treatment of pyometra (cystic endometrial 419 hyperplasia) in bitches with an antiprogestin. j reprod fertil suppl 1997;51:327-420 331. 421 57. gobello c, castex g, klima l, et al: a study of two protocols combining 422 aglepristone and cloprostenol to treat open cervix pyometra in the bitch. 423 theriogenology 2003;60:901-908. 424 58. fieni f: clinical evaluation of the use of aglepristone, with or without 425 cloprostenol, to treat cystic endometrial hyperplasia-pyometra complex in 426 bitches. theriogenology 2006;66:1550-1556. 427 59. soto sm, smithson a, horcajada jp, et al: implication of biofilm formation in the 428 persistence of urinary tract infection caused by uropathogenic escherichia coli. 429 clin microbiol infect 2006;12:1034-1036. 430 123 60. leblanc mm, magsig j, stromberg aj: use of a low-volume uterine flush for 431 diagnosing endometritis in chronically infertile mares. theriogenology 432 2007;68:403-412. 433 124 table 1 function, classification, and distribution of virulence factors of escherichia coli isolates from various species. factor serogroup or genes site species o antigens o1, o2, o4, o6, o25 o1, o2, o4, o6, o7, o8, o22, o23, o25, o32, o45, o75, o83, o88 o147 urinary tract [28] uterus [18,21] dog [26,28] human [28] dog [18,21] thermostabile; agglutinating; immunogenic o-specific polysaccharide of the cell wall lipopolysaccharide; possessed by all smooth forms of e. coli type i fimbriae fim (pil) urinary bladder [33] feces [25, 32] uterus [32] dog [25, 32] mouse [33] present on most e. coli; bind cell-bound and secreted mannosylated glycoproteins, tamm horsfall protein, and uroplakins of bladder epitheliam; presumed to be able to bind to endometrium p fimbriae papgiii urinary epithelial cells [25,28,33] feces [25,32] uterus [32] human [28] dog [25,28,32] mediate attachment to galα1→4galβ-containing glycolipid receptor and coreceptor tlr4 s fimbriae sfa urinary tract [25,28]] uterus[25,32] feces [25,32] dog [25,28,32] humans [25,28] bind eukaryotic glycoproteins with a terminal α-sialic acid; bind laminin and plasminogen; may play a role in penetration of e. coli across basement membrane α-hemolysin hlya urinary tract [25,28] uterus [32] feces [25,32 dog [25,28,32] human [25,28] common exotoxin; toxic to a wide variety of mammalian cells cytotoxic necrotizing factor 1 cnf1 urinary tract [25,28] uterus [32] feces [25,32] dog [25,28,32] human [25,28] belongs to a group of bacterial necrotic substances; associated with outer membrane vesicles; activates rho gtpases of host cell leading to macropinocytosis by epithelial cells; may function as a means of entry and survival in epithelial cells aerobactin and other iron-sequestering systems iuc or aer, fyua, iuta, iron urinary tract [25,28] uterus [32] feces [25,32] dog [25,28,32] human [2528] bacterial siderophores (low molecular weight fe(iii)-chelator) 125 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning 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/destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: estrus-ai interval and fixed-time ai pregnancy rates in beef cows inseminated with fresh extended or frozen thawed semen estrus-ai interval and fixed-time ai pregnancy rates in beef cows inseminated with fresh extended or frozen thawed semen t. wisemana, r. kasimanickama, j. m. dejarnetteb, w. d. whittiera adepartment of large animal clinical sciences, virginia-maryland regional college of veterinary medicine, blacksburg, virginia 24061, usa bselect sires inc., plain city, oh 43064, usa we hypothesize that fresh extended semen will improve the ai pregnancy rate compared to frozen thawed semen due to its increased longevity in female reproductive tract. the objective of this trial was to determine the effect of semen type on fixed-time ai pregnancy rate in relation to estrus-ai interval in beef cows synchronized with progesterone based fixed-time artificial insemination (ai) protocols. angus cross beef cows (n=180) were synchronized with cosynch-cidr protocols for a fixed-time ai. briefly, cows were synchronized with 100 μg of gonadotropin releasing hormone (gnrh) + controlled internal drug release insert (cidr) on day 0, 25 mg of pgf2α and cidr removal on day 7 and 100 μg gnrh on day 10 (67 h from cidr removal) at ai (co-synch-cidr). a subset of cows (n=110) received a heatwatch pressure sensor at cidr removal to determine the time of estrus. cows were divided into two groups, inseminated at 47h (early) and 67h (late) from cidr removal with either 3 million cells of fresh extended or 20 million cells of frozen thawed semen. results indicated that cows inseminated at 67h had numerically higher fixed time ai pregnancy compared to cows inseminated at 47h [44.4% (40/90) vs. 33.3% (30/90); p=0.13]. cows inseminated with frozen thawed semen had similar fixed time ai pregnancy compared to fresh extended semen [40.8% (31/76) vs. 37.5% (39/104); p=0.66]. ai-estrus interval was divided into three groups < 0 h (ai occurred before estrus), 0 to 16 h and > 16 h (ai occurred 16 h after estrus). the ai pregnancy outcome for fresh semen for the 3 estrus-ai intervals was similar to frozen semen (figure 1). in conclusion, the fresh semen did not improve the pregnancy rate compared to frozen semen in relation to estrus-ai interval. keywords: beef cows, semen type, fresh semen, synchronization, pregnancy rate 537 2010: treatment of infectious infertility treatment of infectious infertility s.p. brinsko department of large animal clinical sciences, texas a&m university, college station, tx introduction infections infertility has repeatedly been identified as major source of economic loss to the equine breeding industry for over 50 years. despite the development and use of a variety of antimicrobial agents, apparently little progress has been made in reducing the percentage of mares that remain barren due to infectious etiologies which casts some doubt as to the role of bacteria as primary causative agents.1,2 reasons for this are not completely understood, likely because infectious infertility is a multifactorial phenomenon involving breeding management, diagnostic and treatment modalities and inherent resistance to infection. far too often the veterinarian’s antimicrobial armamentarium is relied upon to overcome less than optimal breeding hygiene and management. this practice will not improve the overall fertility on a breeding farm. instead, the veterinarian should play an active role in the breeding program by accurately assessing the condition of the mare’s reproductive tract during estrus and scheduling breedings as close as possible to ovulation. limiting the number of breedings required per cycle and instituting the use of minimum contamination techniques reduces the potential for uterine contamination whether natural cover or artificial insemination is utilized. by being integrally involved with the breeding program, the veterinarian is better able to correctly identify problem mares and initiate therapy when needed. an important aspect of addressing infertility is correctly identifying and treating mares that are truly infected. unfortunately, many breeding farms are under the erroneous assumption that unless mare has a negative bacteriologic uterine culture, she is unfit to breed. the primary role of bacteriologic culture of the mare’s reproductive tract is not to determine whether or not a mare is infected, but rather to identify causative organisms and obtain antimicrobial sensitivity patterns so that appropriate therapy can be instituted. it cannot be over-emphasized that a positive bacteriologic culture in the absence of concurrent signs of inflammation does not indicate infection. ignoring this fact results in treatment of noninfected mares and more importantly, other factors which could be contributing to their infertility may be overlooked. likewise, a negative aerobic culture in the presence of clinical, cytologic, or histopathologic signs of inflammation suggest the culture should be repeated and that other causes of inflammation should be investigated. although anaerobes, viruses and mycoplasma may be isolated from the endometrium, their clinical significance is largely unknown. uteri of mares with accumulations of purulent material may yield no bacterial growth when cultured. apparently, the nature of such fluids may somehow inhibit the recovery of bacteria. pneumovagina and urovagina can cause endometritis without a concurrent bacterial contribution to the inflammation. treatment of infectious infertility begins with correcting predisposing causes. mares with intact uterine defense mechanisms do not become infected even when challenged with massive bacterial inocula, whether naturally or experimentally introduced.3,4 apparently, the initial uterine response to the inoculum is the same for resistant and susceptible mares. however, susceptible mares are unable to maintain an effective response or to mechanically clear the uterine lumen of contaminants so as to prevent infection. in order for a mare’s uterus to become infected there must be a breakdown in her humoral, cellular or mechanical uterine defense mechanisms.5-7 defects in the mechanical barriers probably occur most commonly and are the most easily corrected. the vulvar labia, vestibulovaginal seal, and the cervix are the three mechanical barriers to ascending infection in the mare. disruption of any one of these barriers, as is common in older mares due to age-related changes, multiple foalings or trauma, predisposes the uterus to repeated contamination which can overwhelm the uterine defense mechanisms. surgery to correct pneumovagina and/or urovagina, improving breeding techniques to enhance hygiene and elimination of excessive breedings are the most efficient and beneficial means by 111 which therapy for infections infertility is initiated. many mares spontaneously overcome endometritis once the source of repeated uterine contamination is eliminated. a caslick’s operation (vulvoplasty) can be one of the most effective means of managing infectious infertility. keywords: mare, infertility, endometritis therapeutic strategies over the years intra-uterine infusion with a variety of antibiotic and antiseptic solutions has been utilized in an attempt to eliminate infectious endometritis in mares. these substances have ranged from agents such as kerosene, floor cleaners, chlorine and iodine solutions to potent antibiotics. unfortunately, many of these treatments were instituted without knowledge of their efficacy or potential detrimental effects. as a result, numerous mares have been rendered permanently infertile due to irreparable damage caused by therapies which were more damaging than the original condition being treated. the use of chlorhexidine solution for intra-uterine infusion is strongly discouraged, since it can have devastating effects on the mare’s reproductive tract.8 veterinarians must adhere to the basic tenet of “do no harm” when treating genital infections just as with any other organ system. therefore, the drug and its vehicle should be relatively nonirritating. almost any substance instilled in the mare’s uterus, including physiological saline, will induce some degree of an inflammatory response. however, it is the intensity and duration of this response which is of primary concern with regard to fertility, especially when treatment is performed close to breeding and/or ovulation. inflammation persisting beyond five days post-ovulation can adversely affect fertility by causing early embryonic death either directly or by inducing premature luteolysis. antibiotic therapy the traditional approach to antibiotic therapy for the treatment of infectious endometritis has been via intrauterine infusion; however, some practitioners report satisfactory results using systemic administration of various antibiotics.2,9 while eliminating the potential for repeated genital tract contamination during treatment by the systemic approach is advantageous, lower antibiotic concentrations are generally achieved in the endometrium and uterine lumen depending on the antibiotic used.10-12 while the the effectiveness of systemic treatment of endometritis has not been determined by controlled studies, the fluoroquinolones appear to show promise for systemic treatment of endometritis in mares. therapeutic endometrial levels of enrofloxacin (baytril®, bayer animal health, shawnee, ks) can be achieved when the drug is given orally or parenterally. unfortunately, this drug has poor activity against streptococcus sp., which is the most common bacterial cause of equine endometritis. although ampicillin can achieve high concentrations in the endometrium when given parenterally, administration at four to eight hour intervals is required to maintain adequate levels.10 systemic administration of antibiotics for the treatment of endometritis would therefore necessitate larger doses given more frequently, thereby increasing the cost of treatment. in contrast, when ampicillin (3 grams) is administered via intrauterine infusion, endometrial concentrations of the drug still exceeded minimal inhibitory concentrations for streptococcus sp. 24 hours after treatment.13 since the vast majority of genital infections in the mare are limited to the endometrium, they are likely to respond most favourably to local therapy. however, more severe infections of the uterus and other genital tissues such as postpartum metritis would warrant systemic use of antibiotics. topical treatment of the endometrium by infusion of antibiotics through the cervix constitutes the vast majority of treatments for endometritis. when this approach is utilized, scrupulous attention must be paid to aseptic technique. uterine infusion should be continued for a minimum of three to five days. the volume of the drug and its vehicle should be sufficient to distribute the drug throughout the uterus and provide contact with the entire endometrial surface area. volumes of 200-250 ml are commonly used but often result in a portion of the drug exiting 112 through the cervix, especially when the mare is in estrus. smaller volumes of 60 to 120 ml are adequate to attain the desired result in most mares when combined with gentle uterine massage per rectum. a list of drugs commonly used for the treatment of bacterial endometritis is provided in table 1. antibiotics should be selected based on in vitro culture and sensitivity patterns so that they are as specific as possible for the organisms involved. even though in vitro and in vivo sensitivities may differ, the temptation to routinely use broad spectrum antimicrobial agents must be discouraged since indiscriminate use can lead to the development of resistant strains and/or overgrowth of more pathogenic bacteria, yeasts or fungi. another consideration regarding the antibiotic selection is antibiotic-neutrophil interaction. several antibiotics (e.g. gentamicin, amikacin, tetracyclines, and polymixin b) have been reported to interfere with neutrophil function.7 often times, temporal constraints imposed by the limited breeding season in the equine do not permit the luxury of waiting for culture and sensitivity results before instituting therapy. in these situations, therapeutic agents should be chosen based upon knowledge of the most common causative organisms. the majority (70-80%) of aerobic bacteria confirmed to be associated with endometritis in mares are, in descending order of frequency, streptococcus zooepidemicus, escherichia coli, pseudomonas aeruginosa, and klebsiella pneumoniae.2,6 with this in mind, an antibiotic such as ampicillin which is generally effective against strep. zooepidemicus and many strains of e. coli would be an appropriate choice while awaiting laboratory confirmation. unfortunately, veterinary sources of ampicillin are now limited and the cost of the drug has increased dramatically in recent years. in addition, some practitioners have encountered increased resistance of e. coli to ampicillin. timentin (ticarcillin/clavulanate) and gentamicin are usually effective against most of the causative organisms, but for the reasons discussed above, they should not be used indiscriminately. enrofloxacin is very effective against e. coli as well as a number of other endometrial pathogens. however, intrauterine use of enrofloxacin is not recommended in the mare because its high ph results in an inflammatory response that can take up to two weeks to resolve.14 another therapeutic approach that can be utilized while awaiting culture and sensitivity results is uterine lavage. uterine lavage is especially advantageous if the mare has been unsuccessfully treated with antibiotics previously and where yeast or fungal infections are suspected. uterine lavage uterine lavage alone or in conjunction with antibiotic therapy appears to be beneficial in the treatment of infectious endometritis.15 the rationale for using uterine lavage in the treatment of uterine infections is based upon: 1) reduction of bacterial numbers and removal of exudates from the uterine lumen, 2) enhanced physical clearance of uterine contents by stimulation of uterine contractions, and 3) recruitment of “fresh” circulating neutrophils and possibly opsonins by inducing transient irritation of the endometrium.5 uterine lavage should improve the efficacy of intrauterine antibiotic treatment by reducing foreign material within the uterine lumen. this is especially true for antibiotics of the aminoglycoside and polymixin groups which become highly bound to purulent material.16,17 uterine lavage is typically performed by repeated instillation of one to 1.5 liters of a warmed (42-45 oc) solution by gravity-flow through a large-bore (8 mm inner diameter) ballooncuffed catheter. nulliparous mares may only be able to accommodate 750 ml of lavage fluid. once the desired volume is infused, the catheter can be clamped and the uterus massaged per rectum to distribute the solution throughout the uterine lumen. the effluent is then collected in a clear receptacle to assess volume and clarity. this process is repeated sequentially, several times for several days depending on the character of the effluent. an appropriate antibiotic is infused daily once the effluent is relatively clear. daily lavages can usually be discontinued when the 113 effluent from the first lavage of the day returns with a similar appearance to that which was instilled into the uterus. preferred media for uterine lavage are generally isotonic saline or other balanced salt solutions such as lactated ringer’s solution (lrs), with or without dilute povidone-iodine added. care must be taken when using povidone-iodine solutions since concentrations ≥2% have been shown to induce severe inflammatory responses in the equine endometrium.18,19 stock povidoneiodine solutions are typically 5% or 10% povidone-iodine, equivalent to 0.5% or 1% available iodine, respectively. inflammatory changes were not detectable in equine endometrial biopsy samples six days after treatment with a 0.05% povidone-iodine solution (5 ml of 10% stock solution in one liter of saline.20 bactericidal activity of povidone-iodine in vitro is maintained at concentrations as low as 0.01 to 0.005%.21,22 postbreeding uterine lavage appears to be a viable treatment alternative for mares susceptible to uterine infection, particularly for those mares that suffer from chronic postbreeding endometritis despite the use of minimal contamination techniques. a primary concern is the minimum time interval between breeding and uterine lavage which will not adversely affect fertility. initial reports of successful postbreeding therapy combining uterine lavage followed by plasma infusion delayed treatment until ovulation had occurred. performing uterine lavage as soon as possible after breeding however should minimize the time available for microbial contaminants to multiply and become established. uterine lavage performed within two hours of insemination reduces pregnancy rates in normal mares. however, uterine lavage using either physiologic saline or a 0.05% povidone-iodine solution at four hours post-insemination has no detrimental effect on fertility.23 the ability to perform uterine lavage soon after breeding facilitates removal of bacteria and debris so that uterine contamination is less likely to progress to uterine infection. this technique should be especially advantageous for treating susceptible mares which, due to breed restrictions, must be bred by natural cover and thus are at risk of a higher level of uterine contamination. for mares that have significant luminal fluid while in estrus, uterine lavage with lrs can also be performed immediately prior to breeding without adversely affecting fertility.24 plasma infusion in the early 1980’s, intrauterine infusion of autologous plasma became a popular treatment for infectious infertility; particularly in mares with chronic postbreeding endometritis. both serum and plasma provide neutrophil-enhancing proteins involved in the opsonization of bacteria within the mare’s uterus.5,7 although autologous plasma is recommended for this treatment, heterologous plasma has been used. the possibilities of hypersensitization and transmission of infectious agents should be considered if heterologous plasma is used. the techniques for harvesting and handling plasma for intrauterine infusion have been described.5 successful use of intrauterine plasma infusion has been reported in clinical settings; however, controlled studies present conflicting evidence regarding the beneficial effects of this treatment for correcting infectious infertility. in recent years, this treatment modality has largely gone out of vogue. bacteria-free filtrate intrauterine infusion with bacteria-free filtrate of streptococcus zooepidemicus may improve fertility of some barren mares.25 the rationale for this treatment modality is based upon the filtrate’s ability to chemically stimulate an acute inflammatory response within the uterus, thus recruiting “fresh” neutrophils and opsonins. the uterus is infused once during estrus with 50-80 ml of filtrate which results in an intense inflammatory response that peaks six to ten hours after inoculation. since non-functional neutrophils and other debris within the uteri of chronically infected mares may interfere with the function of the newly recruited neutrophils, it is likely that removing this material by performing uterine lavage prior to filtrate inoculation would enhance the desired response. 114 immunomodulators over the years, a number of products have been used empirically to overcome endometritis by stimulating the mare’s immune system. these include levamisole, mebendazole and nonviable propionibacterium acnes (eqstim®, neogen corporation, lexington, ky). controlled studies evaluating the efficacy of these products for the treatment of equine endometritis are lacking. recently, the usda approved an emulsified product derived from the cell wall of mycobacterium phlei (settle®, bioniche, athens, ga) for the treatment of endometritis caused by streptococcus zooepidemicus. this product is reported to modulate the endometrial immune response, reduce bacterial contamination of the uterus and improve pregnancy rates when compared to placebo treated mares. routes of administration are intravenous or intrauterine and the product can be used as a stand alone therapy or in conjunction with other therapeutic modalities such as antibiotics or uterine lavage. hormonal therapy during estrus, the uterus of the mare is dominated by estrogen and is more capable of eliminating uterine infections than is the progesterone dominated diestrual uterus. factors which may contribute to the estrous mare’s resistance to infection include decreased microbial binding to the endometrium, enhanced neutrophil function and improved physical clearance mechanisms. mares with acute endometritis often have shortened estrous cycles. this results from the inflamed endometrium releasing endogenous prostaglandin, causing premature luteolysis. exogenous prostaglandin administration five to six days post-ovulation can be used to mimic this response in chronically infected mares, thereby increasing the percentage of time that these mares are in physiologic estrus. exogenous administration of estrogens to cyclic mares, on the other hand, had not been demonstrated to be beneficial in the treatment of endometritis. most commercially-available estrogens are esterified or otherwise altered to increase their potency and duration of action, making the mare’s response to such preparations unpredictable. ecbolics such as oxytocin and prostaglandin, are commonly used to treat endometritis by aiding in the evacuation of uterine contents with or without concurrent uterine lavage. however, it is the author’s opinion that if the endometritis has an infectious etiology, ecbolics alone will be less effective than combining the treatment with uterine lavage or antimicrobial therapy. oxytocin (20 units iv or 20 to 40 units im) is the most common ecbolic used and it can be administered during estrus and after ovulation with out detrimental effects, provided that it is not administered within four hours of insemination. prostaglandins can also be used, but they should be used more judiciously because of their longer duration of action and colic-like side effects. when administered at a dose of 250 µg im, the prostaglandin analog, cloprostenol (estrumate®, schering-plough animal health, summit, nj) is a very effective ecbolic with fewer side effects than dinoprost tromethamine (lutalyse®, pfizer animal health, new york, ny). unlike oxytocin, prostaglandins only should be used with caution on or after the day of ovulation on the cycle in which a mare is bred because progesterone levels are adversely affected and pregnancy rates can be reduced.26,27 when older, maiden mares are bred, it is common for them to suffer from persistent postbreeding endometritis. this is often due to failure of the cervix to relax, resulting in poor uterine clearance and fluid accumulation. the use of a prostaglandin e1 analog, misoprostol (cytotec®, g.d. searle & co., chicago, il) enhances cervical softening and dilation. a thick paste is made by mixing crushed tablets with a small amount sterile lubricating jelly and applying the paste in and on the cervix. doses ranging from 200 µg to 2000 µg have been used. lower doses may require four to six hours for maximal effect, while higher doses reportedly are faster acting. in humans, this drug can induce strong uterine contractions, but this has not been 115 observed in mares. the naturally occurring prostaglandin e2, dinoprostone, available from a number of manufacturers in suppository and gel forms for cervical ripening in humans, has also been used in mares. misoprostol appears to be as efficacious as dinoprostone however, dinoprostone is much more expensive.28 mycotic endometritis fungal and yeast infections of the equine uterus occur most commonly in older mares which have undergone intensive intrauterine antibiotic therapy or repeated uterine manipulations such as embryo transfer. these mares usually have poor perineal conformation and/or other disruptions of their uterine defense mechanisms resulting in chronic uterine contamination and therefore, repeated treatment. it is not uncommon for mares with mycotic endometritis to be concurrently infected with resistant strains of pseudomonas or klebsiella.29,30 fungi and yeasts are common environmental and external genital contaminants31,32 which can be introduced at coitus, during gynecologic procedures, or as a result of pneumovagina. the most frequently isolated organisms appear to be species of candida and aspergillus although many other yeasts and molds have been reported.30,32 therapy of mycotic endometritis, as with bacterial endometritis, should be based upon culture and sensitivity results. many yeast infections (candida spp.) can be treated with nystatin, whereas amphotericin b is recommended for fungal infections (aspergillus spp.), although it is also effective against candida. most therapeutic regimens employ human vaginal preparations. common therapeutic drugs for fungal and yeast infections are presented in table 2. it is important to treat the vaginal and vestibular vaults as well as the clitoral area to help ensure elimination of the organisms. saline or sterile water can be used as a vehicle, but sterile water is the recommended diluent for amphotericin b, since a precipitate forms when saline is combined with this drug. good clinical results can be achieved when using povidone-iodine solutions to treat intrauterine mycotic infections however, great care must be taken with this approach. higher concentrations of povidone-iodine are generally required to eliminate mycotic infections than are required to treat bacterial infections. in the author’s opinion, concentrations exceeding 2% povidone-iodine should not be used to treat genital tract infections in mares. some mares are most sensitive than others to the effects of povidone-iodine and must be closely monitored for signs of hypersensitivity. inflammatory responses severe enough to result in transluminal adhesions are possible. recently, the use of lufenuron (program® suspension for cats, novartis animal health greensboro, nc) in conjunction with uterine lavage has reported to be successful for treatment of yeast and fungal endometritis in the mare.33 lufenuron is an insect development inhibitor that works by interfering with chitin formation. since fungal and yeast organisms are surrounded by chitin-rich cell walls it is postulated that the development of these organisms should also be disrupted by treatment. fungal and yeast infections are notoriously difficult to resolve and as with other therapeutic modalities, anecdotal reports of lufenuron therapy have been mixed, however a number of the reports are encouraging. uterine lavage prior to infusion of any antimycotic agent would likely be a valuable adjunct to therapy for the same reasons described for bacterial infections. after the therapeutic course of antimycotics has been completed, correction of conformational defects which contribute to uterine contamination must be performed to prevent re-infection. summary therapy of infectious infertility begins with correcting predisposing factors. therapeutic agents should be selected based on efficacy, specificity and safety. uterine lavage used in conjunction with other therapeutic regimens is likely to improve response to therapy. simply treating uterine infections without eliminating the cause is an exercise in futility. repeated 116 intrauterine infusion of antimicrobials is no substitute for sound reproductive management and could prove to be detrimental. references 1. davis le, abbitt b: clinical pharmacology of antibacterial drugs in the uterus of the mare. j am vet med assoc 1977;170: 204-207. 2. asbury ac: bacterial endometritis. in: robinson ne, editor. current therapy in equine medicine. philadelphia: wb saunders; 1983. p. 410-414. 3. hughes jp, loy rg: investigations of the effect of intrauterine inoculation of streptococcus zooepidemicus in the mare. proc annu conv am assoc equine pract 1969. p. 289-292. 4. peterson fb, mcfeeley ra, david jse: studies on the pathogenesis of endometritis in the mare. proc annu conv am assoc equine pract 1969. p. 279-287. 5. asbury ac: infectious and immunologic considerations in mare fertility. compend cont educ pract vet 1987;9:585-592. 6. neely dp: evaluation and therapy of genital disease in the mare. in: neely dp, liu ikm, hillman rb, editors. equine reproduction. trenton: veterinary learning systems; 1983. p. 40-56. 7. varner dd, blanchard tl: an update on uterine defense mechanisms in the mare. j equine vet sci 1990;10:169-175. 8. blanchard tl, varner dd, schumacher j, et al: endometritis. in: manual of equine reproduction, 2nd ed. st. louis: mosby; 2003. p. 59-68. 9. bennett dg: therapy of endometritis in mare. j am vet med assoc 1986; 188:1390-1392. 10. arbieter kv, awad-maselhem m, kopschitz d, et al.: estimation of antibiotic levels in uterine tissue and blood plasma of the mare following parenteral administration of penicillin and ampicillin. wien tierartzl monaschr 1976;63:298-304. 11. lock tf: distribution of antibiotics in the mare reproductive tract after various routes of administration. j reprod fertil 1982; suppl 32:640-641. 12. pedersoli wm, fazeli mh, haddad ns, et al: endometrial and serum gentamicin concentrations in pony mares. am j vet res 1985;46:1025-1028. 13. love cc, strzemienski pj, kenney rm: endometrial concentrations of ampicillin in mares after intrauterine infusion of the drug. am j vet res 1990;51:197-199. 14. fumoso e, checura c, losinno l, et al: endometrial tissue concentrations of enrofloxacin after intrauterine administration to mares. vet res commun 2002;26:371-380. 15. knutti b, pycock jf, van der weijden gc, et al: the influence of early postbreeding uterine lavage on pregnancy rate in mares with intrauterine fluid accumulations after breeding. equine vet educ 2000;12:267-270. 16. kunin dm: binding of antibiotics to tissue homogenates. j infect dis 1970;121:55-64. 17. shin sj, lien dh, aronson al, et al: the bacteriological culture of equine uterine contents, in-vitro sensitivity of organisms isolated and interpretation. j reprod fertil 1979; suppl 27:307-315. 18. bennett dg, poland hj, kaneps aj, et al: histologic effects of infusion solutions on the equine endometrium. equine pract 1981;3:37-44. 19. van dyk e, lange al: the detrimental effect of the use of iodine as an intrauterine instillation in mares. j s african vet assoc 1986;57:205-210. 20. brinsko sp, varner dd, blanchard tl, et al: the effect of postbreeding uterine lavage on pregnancy rate in mares. theriogenology 1990;33:465-475. 21. berkelman rl, holland bw, anderson rl: increased bactericidal activity of dilute preparations of dilute povidone-iodine solution. j clin microbiol 1982;15:635-639. 22. van den broek pj, buys lfm, van furth r: interaction of povidone-iodine compounds, phagocytic cells and microorganisms. antimicrob agents chemother 1982;22:593-597. 23. brinsko sp, varner dd, blanchard tl: the effect of uterine lavage performed four hours post insemination on pregnancy rate in mares. theriogenology 1991;35:1111-1119. 24. vanderwall dk, woods gw: effect on fertility of uterine lavage performed immediately prior to insemination in mares. j am vet med assoc 2003;222:1108-1110. 25. couto ma, hughes jp: intrauterine inoculation of a bacteria-free filtrate of streptococcus zooepidemicus in clinically normal and infected mares. equine vet sci 1985;5:81-86. 26. brendemuehl j: effect of oxytocin and pgf2α on luteal formation, function, and pregnancy rates in mares. proc annu conv am assoc equine pract 2001. p. 239-241. 27. nie gj, johnson ke, wenzel jgw, et al: effect of administering oxytocin or cloprostenol in the periovulatory period on pregnancy outcome and luteal function in mares. theriogenology 2003; 60:11111118. 28. lokugamage au, forsyth sf, sullivan kr, et al: dinoprostone versus misoprostol: a randomized study of nulliparous women undergoing induction of labor. acta obstet gynecol scand 2003;82:133-137. 117 29. blue mg: fungal endometritis. in: robinson ne, editor. current therapy in equine medicine, 2nd ed. philadelphia: wb saunders; 1987. p. 511-513. 30. pugh dg, bowen jm, kloppe lh, et al: fungal endometritis in mares. compend cont educ pract vet 1986; 8:s173-s181. 31. ball ba, shim sj, patten vh, et al: use of a low-volume uterine flush for microbiologic and cytologic examination of the mare’s endometrium. theriogenology 1988;29:1269-1283. 32. dascanio jj, schweizer c, ley wb: equine fungal endometritis. equine vet educ 2001;13:324-329. 33. hess mb, parker na, purswell bj, et al: use of lufenuron as a treatment for fungal endometritis in four mares. j am vet med assoc 2002;221:266-267. additional reading varner dd, conboy hs, hughes jp, et al: equine reproduction roundtable: treatment of endometritis (parts 1-3). equine pract 1989;11(2):16-23, (3):16-26, (4):17-29. asbury ac, lyle sk: infectious causes of infertility. in: mckinnon ao, voss jl, editors. equine reproduction. philadelphia; lea & febiger; 1993. p. 381-391. lyle sk: endometrial culture and antimicrobial therapy. in: robinson ne, editor. current therapy in equine medicine. 5th ed. philadelphia: wb saunders; 2003. p. 229-231. leblanc mm. new strategies for chronic endometritis and post mating endometritis. clin therio 2010;1:177-183. table 1. suggested intrauterine antibiotics drug dose comments amikacin sulfate 2 grams gram-negative spectrum; buffer with equal volume of 7.5% naco3 ampicillin 1-3 grams streptococci and some e. coli ceftiofur sodium (naxcel®) 1 gram broad spectrum gentamicin sulfate 1-2 grams broad spectrum; buffer with equal volume of 7.5% naco3 kanamycin sulfate 1-2 grams e. coli: toxic to sperm penicillin (k+ or na+ salt) 5 million units economical & very effective against streptococci polymixin b 1 million units pseudomonas; inactivated by organic debris ticarcillin/clavulanate (timentin®) 3-6 grams broad spectrum adapted from 8. table 2. suggested intrauterine antimycotic agents drug dose comments amphotericin b 200-250 mg fungi & yeast; dilute in 100-250 ml of sterile water, makes a relatively insoluble suspension clotrimazole 500-700 mg yeast; crush tablets and mix with 40-60 ml of sterile water lufenuron (program® suspension for cats) 540 mg fungi & yeast; dilute in 60 ml saline; treat once after uterine lavage miconozole 200 mg yeast & fungi; dilute in 60 ml saline & infuse daily up to 10 days fluconozole 100 mg fungi & yeast; adjust ph to 7 & infuse daily for 5-10 days povidone-iodine 1-2% (v:v) yeast & fungi; note strength of stock solution; lavage daily 5-10 days vinegar 2% (v:v) yeast; 20 ml/l saline, lavage daily 5-10 days adapted from 8. 118 1 case report retrospective review of dystocia in south american camelids julianne nussbaum, grace malla, caroline griffin, sarah templeton, andrea lear, eduardo prado, tulio prado, joe smith department of large animal clinical sciences, university of tennessee, college of veterinary medicine, knoxville, tn, usa abstract we describe case management of 30 south american camelids (7 llamas and 23 alpacas) presented to our tertiary care hospital for dystocia. assisted vaginal delivery was successful in 80% of llamas and 50% of alpacas. due to unique camelid anatomy, equid dystocia practices were modified. for llamas and alpacas, neonatal survival rates were 71 and 69%, respectively and nearly all dams (100% [7/7] and 96% [22/23]) were discharged from the hospital. furthermore, 90% (18/20) of cria survived for at least 1 year. it is important to advise owners of potential complications and the need for rapid and appropriate veterinary intervention (including surgical) if a favorable outcome is desired (for both dam and cria). keywords: dystocia, llama, alpaca, assisted vaginal delivery, cesarian delivery introduction dystocia in south american camelids (sacs), as with equid species, is a medical emergency for dam and fetus; however, the unique anatomy (narrow pelvic inlet, vaginal and cervical size) and short parturient window of sacs make handling difficult.1,2 furthermore, forceful uterine contractions lead to premature detachment of fetal membranes and subsequent neonatal hypoxia.3 understanding techniques and outcomes associated with medical intervention (e.g. vagina and cervix are prone to adhesions) are helpful for appropriate case management.3 incidence of dystocia is low (1-5% of births) in camelids, with 1% requiring major medical intervention.2,4 in comparison, dystocia rates are 8.9% in old-world camelids, 2-7 and 8.5% in dairy and beef cattle respectively and 2-13% in horses.4–7 in camelids, dystocia should be suspected if stage 1 is > 6 hours or stage ii does not progress normally 10 minutes after amniotic sac rupture.1,2 dystocia has maternal and fetal causes. the former include uterine torsion, failure of cervical dilation, uterine inertia, and less commonly, fetomaternal disproportion,1 whereas the latter are due to malposition (i.e. neck flexion, front and hind limbs flexure) and/or fetal abnormalities.1  based on a review of medical records, our objectives were to: 1. quantify the types of dystocia in sacs and their clinical workup and treatment options provided within a tertiary referral hospital; 2. present a general overview of dystocia procedures and case handling (adapted from equid and ruminant practices); and 3. determine outcomes (short and long-term dam and cria mortality and dam’s subsequent reproductive success). medical records and data analyses medical records of llamas and alpacas admitted to our teaching hospital from 2006 to 2024 for dystocia as the primary complaint were examined. data on species, age, diagnostics performed, workup prior to presentation, fetal position, medical intervention, days hospitalized, and outcome were collected. as per the criteria adopted, records of 30 animals (7 llamas and 23 alpacas) were identified. for llamas and alpacas, median age was 8.5 years (8.5 ± 3.6, range 4-15 years and 6.9 years (6.9 ± 3.8, range 3-13 years), respectively, and median weight was 162.7 kg (162.7 ± 13.2, range 152.3-177 kg) and 71.5 kg (71.5 ± 17, range 50-91 kg). descriptive statistics and fischer’s exact test (to compare groups) were done with commercial software (graphpad prism version 10.0.0 for windows, graphpad software, boston, ma). contact joe smith jsmit604@utk.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12629, http://dx.doi.org/10.58292/ct.v17.12629 mailto:jsmit604@utk.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12629 2 citation: clinical theriogenology 2025, 17, 12629, http://dx.doi.org/10.58292/ct.v17.12629 observations dystocia type veterinarians referred 3/7 of llamas and 8/23 alpacas after initial assessments; others were presented due to owners’ concerns (4 alpacas were evaluated vaginally by owners) about prolonged parturition.  main reasons for referral were fetal malpresentation and malposition, malposition of fetal extremities (table 1), and uterine torsion (2 llamas and 3 alpacas). analgesia local anesthetic was used for analgesia and to facilitate manual vaginal manipulation. an epidural was used in 1 llama and 8 alpacas (2 patients received only sacrococcygeal epidurals, 1 patient received only lumbosacral epidural, and 1 patient received both sacrococcygeal and lumbosacral epidural; lidocaine 2% (vetone; boise, id)  and carbocaine-v (zoetis; kalamazoo, mi) were used. outcome in llamas, assisted vaginal delivery (table 2) was most frequently used (5/7) as a method of delivery, whereas emergency cesarian delivery was necessary in 2/7. in alpacas, assisted vaginal delivery was used in 10/20 and cesarian delivery in 8/20 (table 2). one alpaca required 3 separate cesarian deliveries (at 3, 5, and 11 years of age). complications included vaginal and uterine prolapse (n = 3); uterine and vaginal laceration (n = 3); vaginal bleeding (n = 1), body wall hernia (n = 2); and seizures with subsequent death (n = 1). therapy ceftiofur was the most common antibiotic (18 cases), including ceftiofur sodium (zoetis) (78.9%, 15/19) and ceftiofur crystalline free acid (zoetis) (21.0%, 4/19). florfenicol (merck; rahway, nj) was used in 4 cases and tulathromycin (zoetis) was used in 2 cases. regarding antiinflammatory agent, flunixin meglumine (vetone) was most commonly used (24  cases) whereas meloxicam (boehringer ingelheim, duluth, ga) was commonly used for patients transitioned to an oral antiinflammatory or for those that did not have an intravenous catheter. additional supportive medications included oxytocin (bimeda; le sueur, mn) (n = 18), domperidone (dechra veterinary products; overland park, ks) (n = 9), and a gastroprotectant (n = 5) or parasiticide (n = 5).  cria survival of the llama cria, 71.4% (5/7) were delivered alive whereas 69% (16/23) of alpaca cria were delivered alive (table 3). for cria that survived delivery, subsequent complications table 1. fetal presentations, positions, and position of fetal extremities in sacs breech (n = 4) anterior presentation, dorsosacral position with limb or neck malposition (n = 8) anterior presentation, ventrosacral position, hind limbs flexed (n = 1) posterior presentation, dorsosacral positioning, hind limb flexed (n = 1) posterior presentation, dorsoilial position, hind limb flexed (n = 1) total (n = 15) survival to discharge 1 5 1 0 0 7 nonsurvival to discharge 3 3 0 1 1 8 p -value 0.57 0.62 0.47 1.00 1.00 table 2. fetal positioning and delivery method in sacs breech (n = 4) anterior presentation, dorsosacral position, with limb or neck mispositioned (n = 8) anterior presentation, ventro-sacral position, with flexed hind limbs (n = 1) posterior presentation, dorsosacral position, hindlimb flexed (n = 1) posterior presentation, dorsoilial position, with flexed hind limb (n = 1) total (n = 18) cesarian delivery 1 1 0 1 0 3 assisted vaginal delivery 3 7 1 0 1 15 p-value 1.00 1.00 1.00 0.17 1.00 table 3. cria survival status based on delivery type in sacs cesarian surgery (n = 9) assisted vaginal delivery (n = 16) total (n = 25) survival to discharge 6 10 16 nonsurvival to discharge 3 6 9 p-value 0.69 1.00 http://dx.doi.org/10.58292/ct.v17.12629 citation: clinical theriogenology 2025, 17, 12629, http://dx.doi.org/10.58292/ct.v17.12629 3 reported include difficulty nursing (n = 2), failure of passive transfer (n = 6), sepsis (n = 2), and death or euthanasia (n = 4). congenital abnormalities noted were: blindness (n = 2), bilateral luxating patella (n = 1), bilateral corneal ulcers (n = 1), and cervical scoliosis (n = 1). surgical correction was attempted in a cria with bilateral luxating patella, but it was euthanized due to lack of improvement and uncontrollable pain. one cria with cervical scoliosis received supportive care at home with passive range of motion exercises performed by the owner, and complete correction was achieved. overall 4/5 (80%) llama cria and 13/16 (81%) alpaca cria survived to discharge (tables 4 and 5, respectively). llamas were hospitalized for an average of 9.9 days (range 1-39 days). alpacas were hospitalized for an average of 4.9 days (range 1-30 days);  7/7 llamas and 22/23 alpacas were discharged from the hospital. follow up follow up information was collected for 24 patients (some was not collected due to inaccurate contact information or calls not returned). among crias discharged, 86% survived longer-term (> 1 year), including 88%, 14/16 alpaca and 100%, 4/4 llama. in total, 7 female cria (6 alpaca and 1 llama) and 11 male cria (8 alpaca and 3 llama) survived long-term. during follow up phone calls to owners, minimal breeding information was known due to new ownership; however, 2 female alpaca cria were bred with no history of dystocia. follow up regarding breeding soundness of male cria was limited due to new ownership. furthermore, 2 owners had castrated their male cria (no breeding soundness information was available). of the dams discharged from the hospital and those successfully contacted for follow up, 6 dams were successfully rebred whereas 4 dams were not bred due to age (16 years of age) (n = 1), owner preference (n = 2), or medical recommendation (n = 1). discussion  pregnancy length in sacs is 335-345 days2 and is 12.5 days longer in spring-bred compared to fall-bred dams.2 clinical management of sacs parturition is important. typical preparturient changes in other species (e.g. mammary gland development, sacropelvic ligament relaxation, and vulval relaxation) are not accurate predictors of parturition in camelids. additionally, parturition in camelids usually occurs during daytime hours, attributed to their mountainous geographic origins, where birthing in the morning/early afternoon allows drying before the cool evenings.8–10 in that regard, 49% of parturitions occurred between 08:00-12:00 hours in a large alpaca farm during an 11-year period.10 furthermore, it is theorized that parturition at night is secondary to either an extended birthing process or a pathologic stressor inappropriately triggering parturition.11 clinicians and owners should be aware of parturition characteristics in sacs. they have 3 stages of labor. stage 1 labor ranges 3-48 hours and involves fetal repositioning and cervical dilation.2 clinical signs observed include restlessness, isolation from the herd, and increased defecation. stage ii labor (fetal expulsion) should occur within 15 minutes after rupture of the amniotic sac.2 typically, the fetus is oriented in anterior presentation, dorsosacral position, with the head, forelimbs, and neck extended. stage iii labor (fetal membranes expulsion) should occur from 40 minutes to 4 hours after parturition; however, during dystocia, fetal membranes are often expelled with the fetus.2 in this study, a majority (8/15, 53%) of cria were oriented in anterior presentation, dorsosacral position, with limb or neck malposition. within this category of malposition, the most common defect was neck flexion (50%, n = 4), followed by head and bilateral front limb flexion (25%, n = 2), unilateral carpal flexion (12.5%, n = 1), and front limb crossing (12.5%, n = 1). clinical signs associated with dystocia in llamas and alpacas vary depending on the primary cause. equine dystocia principles apply to camelid dystocia; however, camelids have a narrower pelvic inlet; the cervix and vagina are at increased risk of laceration and secondary adhesions compared to equids; and crias are at increased risk of hypoxia due to forceful uterine and abdominal contractions.12 overall, special care is needed when attempting assisted vaginal delivery in sacs undergoing dystocia, to minimize iatrogenic trauma to the dam. understanding the inciting cause of dystocia is important in providing appropriate assistance. dystocia of fetal origin is most commonly associated with fetal malposition, typically carpal or shoulder flexure or head deviations.1,2 in this report, none of the cria were in anatomically correct positions. a full assessment of fetal position, viability (difficult to access in sacs), and the clinical picture of the dam should be taken into consideration before attempting vaginal delivery.1 it is important to ensure that the dam’s cervix, vulva, and vestibulum are fully dilated.2 if the fetal position does not allow manual manipulation (i.e. breech, transverse presentation, or severe head deviations), it is recommended to proceed straight to cesarian delivery.1 if the cria has mild limb or neck flexions, assisted vaginal delivery may be attempted only if the vulva, vestibular-sphincter, and cervix are all fully dilated.1 table 4. alpaca cria survival outcomes, based on referral status veterinarian referral (n = 8) owner referral (n = 14) total (n = 22) survival to discharge 4 9 13 nonsurvival to discharge 4 5 9 p-value 1.00 1.00 table 5. llama cria survival outcomes, based on referral status veterinarian referral (n = 3) owner referral (n = 4) total (n = 7) survival to discharge 1 3 4 nonsurvival to discharge 2 1 3 p-value 0.49 0.49 http://dx.doi.org/10.58292/ct.v17.12629 4 citation: clinical theriogenology 2025, 17, 12629, http://dx.doi.org/10.58292/ct.v17.12629 fetal manipulations be limited to 15-20 minutes, due to the high risk of vaginal tears.2 authors find it helpful to start a timer at the beginning of fetal manipulations, to ensure a timely transition to alternative methods if no progress is made. fetus should be manually manipulated to align the fetus to the largest part of the dam’s pelvis. if retropulsion is needed, elevating the hind limbs of the dam may be necessary, in conjunction with heavy sedation plus epidural analgesia and/or general anesthesia.1,2 additionally, depending on the patient’s analgesia level, there is a concern for secondary fetal hypoxia.2 manual manipulation predisposes to development of vaginal adhesions (a complication that should be prospectively communicated to owners).2 manual manipulation is performed under epidural anesthesia; either the first coccygeal intervertebral space or the sacrococcygeal intervertebral space depending on clinician preference.13,14 areas that are desensitized by caudal epidural include tail, anus, rectum, urethra, vagina, and vulva.13 before vaginal examination, rectal examination is recommended when possible.12 to prevent tears and iatrogenic trauma, use abundant lubricant (with lidocaine) in the rectum to promote relaxation and reduce straining.12 for sacs patients, lidocaine recommended dosage is 0.2 mg/kg with a maximum total dose of 4 mg/kg1,15,16 (maximum of ~ 2 or 2.5 ml of lidocaine per llama or alpaca, respectively).1,15 signs of lidocaine toxicity include ataxia, increased respiratory effort, tachypnea, weakness, hypotension, and altered mentation.16 rectal examination will assist in identifying the location and direction of uterine broad ligaments, for evaluating abnormalities (urinary bladder, pelvic region, and kidneys) and identifying uterine torsion.12 antimicrobial and antiinflammatory medications should be part of routine supportive care. most commonly used antimicrobial was ceftiofur and flunixin meglumine was the most commonly used antiinflammatory agent, similar to another retrospective report.17 if assisted vaginal delivery is not an option, cesarian delivery is recommended. increased duration of dystocia is associated with increased fetal and dam mortality in camels.4 the sooner a cesarian surgery is performed (recommended maximum 20-minutes of fetal vaginal manipulation), the fewer complications and better tissue viability.11 cria survival was not affected by delivery method, with 67% (6/9) of cria delivered via cesarian survived to discharge in this case review. surgical approaches in camelid species include left flank and low flank/ventrolateral and ventral midline, with left flank being common.14 for uterine torsion, a ventral midline incision under general anesthesia is recommended.14 complications of cesarian surgery include peritonitis, incisional seroma, hemorrhage, infection, dehiscence, and/or incisional herniation.1 complications are best minimized with good surgical techniques (careful tissue handling, appropriate homeostasis, and good apposition/reconstruction during closure).1 fetal membranes are removed during closure of the uterus; however, if they can be easily removed from the uterus, they may be removed at surgery.1 care should be taken during closure not to include fetal membranes in the uterine wall closure, due to the microcotyledonary placental structure.1 place an abdominal support band for 10-14 days to promote abdominal wall integrity and prevent complications.1 of the 9 patients that underwent cesarian surgery in this review, 1 llama did not have a belly band placed and the owner reported that a body wall hernia occurred after discharge. of the 9 cesarian deliveries, only 2 patients had complications; 1 had a body wall hernia and 1 died due to postoperative complications. hospitalization requirements vary, depending on dam and cria needs. retained fetal membranes after cesarian surgery are not uncommon (12.5%), can be managed medically (with oxytocin) and shedding usually occurs within 24 hours.14,15 however, it is important to ensure that the cervix is open.16 side effects of oxytocin include abdominal pain (i.e. colic) and patients should be monitored closely after treatment.1after cesarian delivery, 75.9% of dams were rebred and conceived again, implying breeding potential was not compromised in a majority of patients.14 in our population (where follow up was possible) 6 dams were successfully rebred, whereas rebreeding was not pursued with 4 dams. cria survivability after dystocia is important. at parturition, cria have increased fetal cortisol concentrations that promote surfactant production and increase catecholamines. however, this surge can subsequently mask clinical illness at birth or shortly after.16 half of cria deaths occurred within the first week, emphasizing routine monitoring and supportive care, especially after dystocia.2 following delivery, a physical examination is critical to assess cria stability and to detect congenital abnormalities. routine supportive care included camelid plasma transfusion if indicated, weight monitoring every 24 hours, dipping the umbilicus in disinfectant (chlorohexidine solution), nursing with milk replacer if necessary, and catheter care. it is recommended to test igg concentrations; due to high variability between assays, use 1 method consistently for any patient.17 our review of medical records was focused on the clinical practices used for the workup and care of dystocia patients presented to a tertiary care hospital. limitations included missing information (e.g. dam weight, height, breeding history, pelvic width, and sire dimensions). furthermore, as some patient care was lost to follow up, there may be some inaccuracy in long-term survivability and breeding of the dam and cria. due to high selling/adopting rates of cria within our demographic population, long-term survival was deemed at 1 year to facilitate follow up (extending this interval would provide more information). although we were only successful in gathering reproductive abilities of 1 female cria, future studies exploring their success is important, as many cria are sold as breeding stock in this region. the small sample size in comparison to the overall sacs caseload of this tertiary referral hospital was surprising, but may attest to the infrequent nature of dystocia in sacs. in 2024, our teaching hospital had an average caseload of 97 sacs/year compared to an average of only 1.7 sacs dystocia cases. further investigations into the long-term survivability of cria and their breeding soundness after dystocia would be beneficial to educate owners and to make proper treatment decisions.  additionally, as information regarding duration of dystocia was missing in many medical records evaluated, a future study investigating the relationship between dystocia duration and interval to patient assessment would better inform how intervention interval impacts patient outcome. furthermore, understanding individual predisposition for dystocia would benefit both owners and clinicians. http://dx.doi.org/10.58292/ct.v17.12629 citation: clinical theriogenology 2025, 17, 12629, http://dx.doi.org/10.58292/ct.v17.12629 5 conclusion effective case management of sacs dystocia requires swift action, appropriate knowledge of species anatomy and physiology, and species-specific approaches; when these practices are put into place, a favorable outcome is likely. it is evident from this report that favorable outcome and cria survival were, in general due to veterinarians’ and owners’ awareness of abnormalities in parturition, comprehensive case workup at the hospital, and effective case management (assisted vaginal delivery or cesarian delivery). cria survival did not differ between assisted vaginal delivery and cesarian delivery, was not affected by fetal malposition, with many still alive after at least 1 year later. finally, with appropriate management, dystocia in sacs has excellent prognosis for dam and cria. conflict of interest none to report. references 1. rodriquez j, pearson l, tibary a: parturition and obstetrics. in: cebra c, van saun rj, anderson de, et al: editors. llama and alpaca care: medicine, surgery, reproduction, nutrition, and herd health. ist edition, philadelphia; elsevier: 2013. p. 274-285. 2. tibary ad: camelid dystocia and neonatal care. large animal proceedings of the north american veterinary conference, orlando, florida, usa, 17-21 january, 2009; p. 349-352 3. tibary a, anouassi a, walker p: management of dystocia in camelids. proc am assoc bovine pract 2008; p. 166-176. 4. ali a, tharwat m, zeitoun mm, et al: dystocia in dromedary camels: prevalence, forms, risks and hematobiochemical changes. anim reprod sci 2016;170:149-156. doi: 10.1016/j. anireprosci.2016.05.004 5. mee jf: prevalence and risk factors for dystocia in dairy cattle: a review. vet j 2008;176:93-101. doi: 10.1016/j.tvjl.2007.12.032 6. ellerbrock m, wehrend a: definition, incidence and causes of dystocia in horses a review of the literature. tierarztl prax ausg g grosstiere nutztiere 2023;51:22-34. doi: 10.1055/a-2006-9248 7. misaka m, uematsu m, kitahara g, et al: association of herd size with stillbirth and dystocia rates in japanese black cattle. animals (basel) 2022;12:e1994. doi: 10.3390/ani12151994 8. davis gh, dodds kg, moore gh, et al: seasonal effects on gestation length and birth weight in alpacas. anim reprod sci 1997;46:297-303. doi: 10.1016/s0378-4320(96)01611-9 9. rossdale pd, short rv: the time of foaling of thoroughbred mares. j reprod fertil 1967;13:341-343. doi: 10.1530/jrf.0.0130341 10. tibary a, rodriguez j, sandoval s: reproductive emergencies in  camelids. theriogenology 2008;70:515-534. doi: 10.1016/j. theriogenology. 2008.04.024 11. ferrer ms, jones m, anderson de, et al: ultrasonographic parameters of fetal well-being and development in alpacas. theriogenology 2013;79:1236-1246. doi: 10.1016/j.theriogenology.2013.03.011 12. anderson de: uterine torsion and cesarean section in llamas and alpacas. vet clin north am food anim pract 2009;25:523-538. doi: 10.1016/j.cvfa.2009.02.002 13. ismail zb: epidural analgesia in cattle, buffalo, and camels. vet world 2016;9:1450-1455. doi: 10.58292/ct.v15.9639 14. waqas ms, campbell a, arroyo e, et al: cesarean section in camelids: indications, technique, survival, and postoperative fertility. clinical theriogenology 2023;15:82-86. doi: 10.58292/ct.v15.9639 15. anderson de, matt m, meredyth j: editors. veterinary techniques in llamas and alpacas. 2nd edition, ames; wiley blackwell: 2023. p. 243-246. 16. miller ba, brounts sh, anderson de, et al: cesarean section in alpacas and llamas: 34 cases (1997-2010). j am vet med assoc 2013;242:670-674. doi: 10.2460/javma.242.5.670 17. pinn tl, gagliardo lf, purdy sr, et al: comparison of three immunoglobulin g assays for the diagnosis of failure of passive transfer of immunity in neonatal alpacas. j vet diagn invest 2013;25:91-98. doi: 10.1177/1040638712472182 http://dx.doi.org/10.58292/ct.v17.12629 https://doi.org/10.1016/j.anireprosci.2016.05.004 https://doi.org/10.1016/j.anireprosci.2016.05.004 https://doi.org/10.1016/j.tvjl.2007.12.032 https://doi.org/10.1055/a-2006-9248 https://doi.org/10.3390/ani12151994 https://doi.org/10.1016/s0378-4320(96)01611-9 https://doi.org/10.1530/jrf.0.0130341 https://doi.org/10.1016/j.theriogenology.2008.04.024 https://doi.org/10.1016/j.theriogenology.2008.04.024 https://doi.org/10.1016/j.theriogenology.2013.03.011 https://doi.org/10.1016/j.cvfa.2009.02.002 https://doi.org/10.58292/ct.v15.9639 https://doi.org/10.58292/ct.v15.9639 https://doi.org/10.2460/javma.242.5.670 https://doi.org/10.1177/1040638712472182 2014: reproductive failure in female camelids reproductive failure in female camelids l.k. pearson, a. tibary comparative theriogenology, department of veterinary clinical sciences, college of veterinary medicine, washington state university, pullman, wa introduction diagnosis of the cause of reproductive failure in female camelids is a challenging task because of the numerous peculiarities of the reproductive anatomy and physiology in these species. the practitioner is often asked to investigate reproductive failure not only on the individual basis but also on the herd basis. this requires a thorough understanding of breeding management and a mastery of a wide range of clinical techniques for reproductive function investigation. in the present paper we discuss the main complaints, diagnostic and therapeutic approach in infertility in female south american camelids (sac) as well as camels with a primary focus on individual animal. keywords: infertility, ultrasonograhy, repeat breeding, early embryonic death reproductive management and common complaints in reproduction reproductive failure case definition relies on three main aspects: 1) the type of complaint, 2) signalment, and 3) a thorough breeding history. availability of accurate individual health and reproduction records is often a problem in large herds of sac and in camels. client education on record keeping is of paramount importance. although many sac breeders have computerized record systems, exact breeding dates and techniques used for diagnosis of pregnancy are often not precise. in addition, most “in-hand” mating is based on receptive behavior and not on specific ovarian follicular status. most breeders use behavioral signs for pregnancy diagnosis (“spiting off” in sac and “tail curling” in camels) at 14 days after mating. a small proportion of sac breeders uses progesterone assay for determination of pregnancy status in addition to the“spitting off” test. when used, progesterone levels are generally determined at 21 days after mating. an increase in diagnostic errors has been noted by the authors in recent years as more and more breeders have ventured into the use of sonograms and ultrasonography without veterinary supervision. herd reproductive efficiency may be assessed by annual pregnancy rate, abortion rate, birthing rate, distribution of births in the season, and weaning rate. these calculations are extremely valuable for the investigation of herd problems.1 client education should emphasize proper nutritional management, prophylactic treatments (especially vaccination and deworming programs), and male selection in order to maximize fertility. for individual animals, reproductive failure is defined based on number of unsuccessful mating cycles. females should be mated only once per week based on strong receptive behavior. if the female is not spitting off the male after a maximum of three matings to a proven male, she should be presented to the veterinarian for a breeding soundness examination. this will ensure an early diagnosis of the cause of infertility and reduce the risk of further complications or jeopardy to the animal welfare.2 in our referral practice, the most common complaints in 2435 sac females examined over the last 15 years were repeat breeding (76%), recurrent early pregnancy loss (18%), continuous spitting off the male (4%) and observed abnormalities of the vulva and perineum (2%).3 the repeat breeding complaints include different manifestations, from breeding continuously (every day or every other day), breeding every week or every two weeks. this is due to the variety of breeding management techniques used by different breeders. early pregnancy loss is generally defined as return to receptivity following one positive pregnancy diagnosis based either on behavior or progesterone level. repeat breeding syndrome can be due to many factors, some of which are pathological, whereas others are management errors. the pathological factors involved in repeat breeding include all conditions that may affect gamete or early embryo survival. management of reproduction in camelids is complicated because the signs of estrus are not very reliable and the timing of breeding in relationship to growth and 555 clinical theriogenology • volume 6, number 4 • december 2014 size of the follicle are very critical for induction of ovulation. diagnosis of the cause of repeat breeding can be challenging and a diagnosis can only be achieved with a complete and sound approach to the problem.4 female reluctance to breed may be due to pregnancy. behavioral problems are relatively common and may be associated to dominance or sexual differentiation problems. reluctance to breed may be caused by pain during mating, due to either congenital or acquired defects of the reproductive tract. visible abnormalities of the vulva or vagina can be very easy for an owner to detect. observed abnormalities include mainly vaginal discharge (bloody or mucopurulent) and abnormalities of development or perineal conformation. approach to diagnosis of reproductive failure the objectives of a breeding soundness examination are to attempt to answer the broad categories of causes of reproductive failure which are: is the failure due to breeding management errors? is the male a major factor? does the female have normal genitalia? does she have normal follicular dynamics and is she able to ovulate? is fertilization occurring and followed by pregnancy loss? judicious choice of and proper use of routine clinical examination techniques allows accurate diagnosis of the condition in the majority of cases(tables 1 and 2).4 timing of the initial examination is very important in order to maximize the information obtained. we recommend to our clients to present the female camelid for an evaluation 14 days following a mating.5 history and physical examination general and reproductive history should cover all parameters that may have an impact on reproductive performance including age, breed, parity, use, and breeding management system. a history of previous illnesses and treatments is important because of the possible effects on reproductive performance, and helps to identify females with high-risk pregnancies. complete clinical evaluation of the female, including body condition scoring, should be done before any manipulation of the genital tract. female behavior provides a relatively accurate and inexpensive means for pregnancy diagnosis. male rejection is very well correlated with high progestéronémie, while receptivity is not well correlated with ovarian status. external genitalia the vulva should be inspected for discharge or lesions and its size and conformation recorded. females presenting increased size and edema of the vulva should be examined for recent abortion. abnormal size and position of the vulva in maiden females may suggest presence of congenital disorders or intersexuality. intersex animals present ambiguous external genitalia, sometimes with a rudimentary penis. the distance from the anal sphincter to the vulvar opening is usually increased and the animal may have dysuria. copious vaginal discharge beyond seven days postpartum is almost always pathological.5 per rectum evaluation of the reproductive tract per rectum palpation of the genitalia is an essential part of the examination of the female llama and camel. the cervix is difficult to differentiate from the uterine body by palpation. however, its posterior orifice can be felt per rectum if the uterus is contracted. in the non-pregnant female, the uterus is “t” or “y” shaped with the horns curling slightly downward and backward at times. there is no intercornual ligament. uterine tone and edema increase during the follicular phase and become maximal in the presence of a mature follicle. the uterus is relaxed and thin-walled during the luteal phase and pregnancy. ovarian structures (corpus luteum, follicles) are palpable in the same manner as in cattle.5 ultrasonography transrectal ultrasonography of the genital tract is performed using a 7.5 mhz or 5 mhz linear transducer. for alpacas, the transducer is mounted on a handle to allow manipulation without inserting 556clinical theriogenology • volume 6, number 4 • december 2014 the hand in the rectum. ultrasonography allows a more precise evaluation of follicular and luteal activity within the ovary. in the early stage of the follicular wave, the ovary appears elliptical with several small (2 to 5 mm in diameter) follicles disposed along the periphery. dominance is established when follicular diameter reaches 6 mm. the dominant follicle continues to grow steadily (0.6 mm per day in sac and 1.5 to 2 mm/day in camels) until it reaches its maximal size (9 to 14 mm in llamas, 8 to 12 mm in alpacas, 12 to 25 mm in camels). the corpus luteum (cl) is easy to visualize by day 4 after mating of induction of ovulation. it is less echogenic than the ovarian stroma, protrudes from the ovarian surface, and measures 11 to 14 mm in sac and 15 to 25 mm in camels. a central anechoic cavity may be visualized in a significant proportion of cl.6-8 during the follicular phase, the uterus is toned, the uterine horns are straight, and ultrasonography there is increased edema of endometrial folds. during the luteal phase, the uterus is homogeneous with a medium degree of echogenicity. typical ultrasonographic features of the normal and abnormal reproductive tract are presented in figures 1-4.9 vaginal examination digital examination of the vestibulo-vaginal area should be performed on all maiden females to rule out persistent hymen and segmental aplasia, and all females suspected to have pyometra or hydrometra, due to vestibular or vaginal adhesions.5,10 vaginoscopy is best performed using a tube speculum (2 to 3 cm in diameter in llamas and camels) or a human sigmoidoscope in alpacas. the vaginal mucosa should be moist and pink. the cervix is clearly visible and should be open during the follicular phase. a small quantity of mucus may be observed in some females with peak follicular development. endometrial cytology and bacteriology endometrial cytology and culture should be part of the basic infertility workup. samples are taken from the uterine cavity using a mare double guarded swab. swabs are examined routinely for aerobic and anaerobic bacteria, ureaplasma spp., mycoplasma spp., and for fungus. the bacteria responsible for endometritis in camelids are essentially those found in the equine and bovine species. the best endometrial specimens for cytology are obtained with a double-guarded cytobrush.5 endometrial biopsy endometrial biopsy is essential in the diagnosis of inflammatory, degenerative, or neoplastic changes in the endometrium. endometrial biopsy should be considered on any female falling into the following categories: • barren female • infertility or unsuccessful breedings • embryo loss or abortion • pre-purchase evaluation of breeding females • screening of recipients for embryo transfer • abnormal echotexture of the uterus samples are preferably obtained during the peak follicular phase when the cervix is easy to bypass and uterus has maximum tone. an equine endometrial biopsy punch is appropriate for llamas and camels. for alpacas, we recommend the use of a turret® rectal biopsy punch (4 x 3 x 3 mm). the instrument is advanced into the uterine cavity with the aid of a vaginal speculum. endometrial samples may also be obtained through a flexible fiberoptic endoscope during hysteroscopy. this technique offers the advantage of samples specific areas of the endometrium but the samples obtained are often very small (2 mm) and difficult to interpret. a biopsy classification method has been proposed but has not been fully investigated (table 3).11,12 endocrinological evaluation 557 clinical theriogenology • volume 6, number 4 • december 2014 circulating progesterone concentration is the most widely used hormone assay in camelids. progesterone levels above 1.5 ng/ml indicate the presence of a functional corpus luteum or luteinized anovulatory follicle. progesterone assay may be used to confirm occurrence of ovulation seven to eight days after mating. pregnancy is suspected if progesterone level remains high in a second sample taken two weeks after breeding. this method of pregnancy diagnosis is relatively precise if breeding history is accurate. plasma estrogen levels above 10 pg/ml in plasma indicate presence of follicular activity. however, this assay provides little information compared to ultrasonography. follicular waves may be monitored by serial sampling (every 3 days for 10 days). testosterone and inhibin evaluation is critical for the diagnosis of granulosa-theca cell tumors. evaluation of amh has not been fully investigated in camelids. hysteroscopy hysteroscopy is performed under sedation using a 9 mm (outer diameter) flexible fiberoptic endoscope or videoendoscope (a human pediatric gastroscope is very appropriate for alpacas). the cervical canal and uterine cavity can be evaluated for the presence of adhesions, cystic dilation or abnormal content. targeted biopsy can be performed in combination with hysteroscopy. 13 laparoscopy and exploratory laparotomy laparoscopy is an invaluable technique for the confirmation of lesions suspected by ultrasonography or palpation (ovarian hypoplasia, hydrosalpinx, segmental aplasia, peri-uterine adhesions), particularly in alpacas. in our practice, this technique has been used to diagnose alpacas with suspected ovarian hypoplasia, ovarian masses, hydrosalpinx, pyosalpinx, ovarian and uterine adhesion, segmental aplasia of the uterus and uterine tubes. laparotomy is indicated for retrograde oviductal flushing to verify the patency of the uterine tube.4 cytogenetic evaluation the normal karyotype in camelids has 74 chromosomes. abnormal karyotype has been associated with different forms of reproductive problems in camelids. cytogenetic studies should be considered when external sexual characteristics are ambiguous or when there is extreme aplasia of the ovary or genitalia. cytogenetic abnormalities described in camelids include xo, xxx, xxy, xx/xy, and xx sex reversal. a minute chromosome has been described in camelids and may be implicated in infertility.14 evaluation of specific complaints repeat breeding repeat breeding complaints can be further subdivided into several categories based on initial evaluation: repeat breeding with no ovarian follicular activity, repeat breeding with no ovulation, or repeat breeding after a normal ovulation and without a positive pregnancy diagnosis (failure of fertilization or very early embryo loss). absence of ovarian follicular activity. absence of ovarian follicular activity can be confirmed by serial ultrasonographic evaluation or plasma estradiol levels (every two to three days for ten days) and may be congenital or acquired. ovarian hypoplasia was found in 16.8% of 155 infertile alpaca females on postmortem examination.15 the majority of affected females display continuous receptivity to the male. ovarian hypoplasia has been associated with various chromosomal abnormalities in about 30% of the cases (xxx, xo, xx/xy and presence of the minute chromosome).14 however, many females may have a normal karyotype. the uterus is often very small and flaccid. acquired ovarian inactivity is commonly observed in lactating camels and to a lesser degree in lactating llamas and alpacas. negative energy balance during lactation or due to poor nutrition is also involved.16,17 ovarian inactivity is also suspected to occur due to trace mineral deficiencies and 558clinical theriogenology • volume 6, number 4 • december 2014 hypothyroidism. follicular development may be seen in some females but the follicles fail to develop to an ovulatory size. poor ovarian activity is also observed with advanced age. ovulation failure. camelids are induced ovulators. ovulation is induced 30 to 36 hours following mating in response to the luteinizing hormone (lh) peak, which is elicited by a combination of uterine inflammation and the presence of ��nerve growth factor (��ngf) in seminal plasma.18,19 failure of ovulation may occur following mating to a less potent male, when the follicle is immature, or when the follicle is past its ovulatory peak (anovulatory follicles).20 anovulatory hemorrhagic follicles are not uncommon and some females seem to more prone to them than others. metabolic disorders (obese females) and endocrine disorders (hypothyroidism) seem to place a female at a higher risk of failure of ovulation and anovulatory hemorrhagic follicle development particularly in camels. ovarian disorders such as oophoritis and ovario-bursal adhesions have been diagnosed by the authors in females with failure of ovulation. ovulation failure and repeat breeding can be observed in females with abnormalities of the vagina and cervix that preclude normal mechanism of induction of ovulation (which is dependent on in utero semen deposition). these situations include imperforate hymen, vaginal aplasia, and vaginal or cervical adhesions. in all these cases accumulation of various quantities of fluid may be seen on ultrasonography of the uterus and/or vagina. some females may be extremely painful and may continue to strain after mating.21 fertilization failure. in the presence of adequate management and normal male fertility, the most common cause of fertilization failure in the female camelid is endometritis. uterine infection should be suspected in animals with a history of repeat breeding or early embryonic death. it is important to consider other primary reasons for reproductive failure as endometritis may be the result of repeat breeding in animals with ovarian or oviductal problems. clinical evaluation may reveal thick mucopurulent vaginal discharge. on ultrasonography, the uterine wall is often thickened and intrauterine fluid may be visualized. diagnosis is confirmed by uterine culture, uterine cytology, and eventually uterine biopsy.22 samples for bacteriology should be kept at room temperature and shipped overnight to the laboratory in special transport media depending on the type of culture desired (culturette®, aimes transport medium). samples should be examined routinely for aerobic and anaerobic bacteria, ureaplasma spp., mycoplasma spp., yeast and fungus. refrigeration and transport in special media should be considered for mycoplasma spp. and ureaplasma spp. and for campylobacter spp. (cary-blair transport medium). samples from camels should also be examined for tritrichomonas spp. and campylobacter spp. which are suspected to be responsible for fertility failure in the dromedary. bacteriological techniques should include sensitivity tests to the major antibiotics. the most common bacteria isolated form camelids with endometritis are escherichia coli, streptococcus equi zooepidemicus, β-hemolytic streptococci, enterococcus spp., coagulase negative staphyloccocus spp., proteus spp, enterobacter aerogenes, pseudomonas aeruginosa, klebsiella pneumoniae and trueperella (formerly arcanobacter) pyogenes. pseudomonas aeruginosa, campylobacter fetus fetus, and tritrichomonas foetus have been isolated from infertile camels and venereal transmission should be considered in cases of herd infertility or abortion outbreaks.23 aspergillus spp. and mucor spp. have been isolated from female dromedaries with endometritis. chlamydophila spp. and mycoplasma spp. have been found in a few cases. it is important to realize that a negative culture does not negate the presence of infection. 22 the authors have seen outbreaks of pseudomonas aeruginosa in some alpaca and camel herds due to venereal transmission by an infected male. treatment approaches are similar to those described for the equine and consist of uterine flushing followed by daily infusion of antibiotics for three to five days. uterine lavage with antiseptic solutions may compromise the local defense mechanisms. in recent years we have been satisfied with the use of equine uterine lavage solution® (bioniche animal health, pullman, wa) because it is buffered to ph 7.1 and contains a surfactant which helps clear mucus and cell debris. oxytocin (5 iu for alpacas, 10 iu for llamas and 20 iu for camels, im) is given to improve uterine clearance. if antibiotics are to be infused 559 clinical theriogenology • volume 6, number 4 • december 2014 in utero we prefer to infuse them a few hours after lavage. the antibiotic choice should be based on culture and sensitivity results. alternately, infusion with a broad spectrum antimicrobial may be used while laboratory cultures are pending. the most common antibiotics used are potassium penicillin (1.5x106 units for llamas and alpaca, 5x106 units for camels), gentamicin sulfate (200 to 300 mg for llamas and alpacas, 500 to 1000 mg for camels), ticarcillin and clavulonic acid (particularly for pseudomonas infections), amikacin sulfate (for pseudomanas spp. and klebsiella spp. infections) and ceftiofur sodium (250 to 500 mg for alpacas and llamas, 1 g for camels). the third generation cephalosporin ceftiofur has a broad spectrum and is effective against gram negative and gram positive bacteria. agents such as dmso or n-acetylcysteine may be used in chronic cases with thick mucopurulent discharge to help liquefy the mucus. infusion with 30% dmso solution prior to uterine lavage is used in our laboratory for chronic cases. biofilm formation is a characteristic of many organisms found in camelid endometritis including p. aeruginosa, s. epidermis, e. coli, enterobacter spp. and some fungi and yeast. studies in mares have shown that addition of buffered chelating agents (tris-edta and tricide®) help dissolve the biofilm and improve antimicrobial action. in camels, the authors have used specially formulated intrauterine infusion products such as metrijet® (1500 mg oxytetracycline hydrochloride solution) with good results. however, these products are not available in some countries. intrauterine foam (fatroximin®, fatro, bologna, italy) provides a release of rifaximin, a synthetic derivative of rifamycin, over 72 hours and has been used in camels for post-breeding control of re-infection. a 4% metacresol-sulfonic acid and formaldehyde solution (lotagen®) is commercially available in some countries and seems to provide good results for treatment of endometritis in camels.24 most antibiotics used in utero, as well as orally administered trimetrhoprim/sulfamethoxazole and iv enrofloxacin achieve very good concentration in uterine tissue. recently we have used long acting tetracyclines as well as ceftiofur (excede®) in the management of camels with high susceptibility to uterine infection. treated animals should be sexually rested for two weeks. the duration of mating should be minimized (12 minutes) during the peak of the follicular to reduce chance of recurrent endometritis. failure of fertilization may be due to uterine tube and bursal abnormalities. the most common disorders of the uterine tube in camelids are inflammation, pyosalpinx, or hydrosalpinx. the enlarged uterine tube can be visualized by ultrasonography . in severe cases, the ovary and ovarian bursa may be involved and adhere to each other. mucosal cysts and micro-abscesses are reported but relatively rare. diagnosis of these affections requires endoscopic evaluation of the uterine tube papillae and/or laparoscopy. prognosis is poor if bilateral and guarded if unilateral. in camels, ovario-bursal adhesions, ovariobursitis and hydrobursitis have been recently associated to chlamydophila infections but these reports have not been thoroughly investigated.25-27 recurrent pregnancy loss early embryonic death is commonly reported in camelids. incidence of early embryonic death in these species can approach 60%. most of the early embryonic loss occurs before day 45. some of the possible etiologies of embryonic death in camelids include early breeding of maiden females (less than 45 kg in body weight), genetic causes (fiber production and quality), environmental factors (heat stress), systemic diseases (acute respiratory syndrome, trypanosomiasis and pox in camels), corpus luteum dysfunction, and uterine pathology including infection or fibrosis. deficiencies in vitamins a, e, or selenium have been incriminated in increased early embryo loss. increased early embryonic death has been associated to early postpartum breeding (before three weeks). reduced ovulation rate and embryonic viability may be seen in females in a negative energy balance due to heavy milk production. twinning may contribute to early pregnancy loss. clinicians should always verify the location and number of cl during pregnancy diagnosis. most twin pregnancies will either reduce to a singleton or be completely lost by 60 days of pregnancy. 560clinical theriogenology • volume 6, number 4 • december 2014 management of recurrent pregnancy loss is best addressed by the use of embryo collection and transfer. females suspected of poor luteal function are often placed on progesterone supplementation in the form of hydroxyprogesterone caproate (250 mg every three weeks until 300 days). however this treatment should be used cautiously due to the risk of complications during pregnancy and at the time of parturition. poor luteal function is often observed in very obese animals. hypothyroidism is suspected to be a predisposing factor. other disorders of the reproductive tract that may be involved in failure of fertilization or recurrent pregnancy loss include segmental aplasia of the uterine tube or uterus, uterine cysts, uterine abscesses, peri-uterine adhesions, polyps, and uterine neoplasia. peri-uterine adhesions are suspected in llamas if difficulty is encountered when attempting to retract the uterus by palpation per rectum. these adhesions are usually a consequence of peritonitis and may be originate from post-surgical complications. uterine neoplasia (adenocarcinoma) has been described in the llama.11 endometrial hemangioma has been diagnosed by the authors in three females presented for reproductive failure and persistent bleeding following mating. abnormal behavior the main complaints regarding abnormal behavior in females are persistent rejection of the male or male-like behavior. persistent rejection of the male may be due to persistent luteal activity or to adverse reaction to mating (painful mating due to vaginal obstruction). persistence of cl may follow a sterile mating or a spontaneous ovulation. this problem is often encountered in the postpartum female or females with chronic degenerative uterine disorders. luteolysis is easily induced by administration of cloprostenol (250µg, im in sac, 500 µg in camels). persistent luteal function may also be due to luteinized hemorrhagic follicles. these structures do not readily respond to a single injection of cloprostenol. male-like behavior is normal to a certain degree and is observed in dominant females at the peak of follicular development. however, persistent aggressive male-like behavior may be due to testosteroneproducing ovarian tumors such as granulosa-theca cell tumors and interstitial cell tumors.21,28,29 unilateral ovariectomy is the best course of action. male-like behavior may also be observed in xx-sex reversed animals due to the presence of testicular tissue.30 abnormal external genitalia abnormalities of the external genitalia are typically congenital in origin. some of these abnormalities are obvious on inspection of the external genitalia but other may present as abnormal vaginal discharge (blood) or abnormal urination. abnormalities of the external genitalia can arise from cytogenetic abnormalities. ambiguous external genitalia with a long, prepuce-like structure is often encountered in xx sex reversal animals.30 intersex, freemartinism, and sry-negative xx sex reversal have been reported with anomalies of the vulva and clitoris.30-33 vulvar hypoplasia or aplasia is common. total vulvar aplasia with accumulation of large quantities of urine in the uterus is seen in neonates and can be lethal if not addressed quickly. vulvar hypoplasia is often observed in maiden females when mating is difficult due to the small vulvar opening.4,34-36 surgical correction is possible but the possible hereditary nature of the defect should be considered. animals with abnormal vaginal discharge may have congenital abnormalities or acquired vaginal or cervical adhesions that cause persistent inflammation during mating. double cervix with some degree of didelphia (double vagina) has been described in llamas and alpacas.37 the major complaints are infertility with a persistent vaginal discharge or repeat breeding. urinary incontinence as evidence by lack of good urine stream and/or urine scalding is often part of the clinical manifestation of congenital or acquired abnormalities of the caudal genitalia. these abnormalities may also include ectopic ureters. females presenting for persistent straining and vaginal and/or rectal prolapse following mating should be examined for vaginal adhesions or persistent imperforate hymen. 561 clinical theriogenology • volume 6, number 4 • december 2014 conclusion reproductive failure in the female camelid may be due to management as well as congenital or acquired disorders. the practitioner providing theriogenological services to camelid herds should be involved in client education. areas of focus should includebreeding management as well as the importance of breeding soundness examination of maiden females, for a timely diagnosis of congenital defects and to avoid compromise of animal welfare. timely and accurate diagnosis of acquired infertility is also important for prognosis for future fertility. practitioners providing reproductive services should master the basic clinical techniques used for evaluation of reproductive function such as ultrasonography. references 1. tibary a, abdelhaq a, abdelmalek s: factors affecting reproductive performance of camels at the herd and individual level. nato science series: life and behavioural sciences, vol.362. amsterdam: ios press; 2005. p. 97-114. 2. pearson lk, rodriguez js, tibary a: breeding management. in: cebra c, anderson d, tibary a, et al, editors. llama and alpaca care: medicine, surgery, reproduction, nutrition, and herd health. st louis: elsevier; 2014. 3. tibary a, rodriguez js, pearson lk. reproductive disorders in alpacas and llamas at a referral center. reprod domest anim 2010;45:51-51. 4. tibary a, anouassi a, memon ma: an approach to the diagnosis of infertility in camelids: retrospective study in alpaca, llamas and camels. j camel pract res 2001;8:167-179. 5. rodriguez js, pearson lk, tibary a: clinical examination of the female reproductive function. p. 168-187. 6. vaughan j: ovarian function in south american camelids (alpacas, llamas, vicunas, guanacos). anim reprod sci 2011;124:237-243. 7. cavilla mv, bianchi cp, maistruarena c, et al : ultrasonographic and endocrine characterization of follicular waves in llamas with a special reference to the overlapping phenomenon during successive waves. reprod domest anim 2013;48:923-930. 8. tibary a, anouassi a: ultrasonographic changes of the reproductive tract in the female camel (camelus dromedarius) during the follicular cycle and pregnancy. j camel pract res 1996;3:71-90. 9. tibary a, pearson lk: normal and abnormal reproductive ultrasonography in camelids. proc new york vet med assoc annu meet; 2012. 10. ferrer ms, anderson de, jones ml, et al: theriogenology question of the month. segmental aplasia of the uterine horn with mucometra. j am vet med assoc 2012;241:697-700. 11. powers be, johnson lw, linton lb, et al: endometrial biopsy technique and uterine pathologic findings in llamas. j am vet med assoc 1990;197:1157-1162. 12. tibary a: endometrial biopsy in camelids. proc north am vet conf; 2009. p. 355-356. 13. tibary a, anouassi a: videoendoscopic technique for genital tract examination in the female dromedary: findings in normal and barren animals. j camel pract res 2001;8:117-122. 14. pearson l, cargen k, sandoval s, et al: retrospective clinical and cytogenetic study on ovarian hypoplasia in alpacas. theriogenology 2007;68:509-510. 15. sumar j: studies on reproductive pathology in alpacas [dissertation]. uppsala: faculty of veterinary medicine, university of agricultural sciences; 1983. 16. sghiri a, driancourt ma: seasonal effects on fertility and ovarian follicular growth and maturation in camels (camelus dromedarius). anim reprod sci 1999;55:223-237. 17. norambuena mc, silva m, urra f, et al: effects of nutritional restriction on metabolic, endocrine, and ovarian function in llamas (lama glama). anim reprod sci 2013;138:252-260. 18. kershaw-young cm, druart x, vaughan j, et al: beta-nerve growth factor is a major component of alpaca seminal plasma and induces ovulation in female alpacas. reprod fertil dev 2012;24:1093-1097. 19. ratto mh, leduc ya, valderrama xp, et al: the nerve of ovulation-inducing factor in semen. proc natl acad sci usa 2012;109:15042-15047. 20. bravo pw, stabenfeldt gh, fowler me, et al: ovarian and endocrine patterns associated with reproductive abnormalities in llamas and alpacas. j am vet med assoc 1993;202:268-272. 21. rodriguez js, pearson lk, tibary a: infertility and subfertility in the female camelid. in: cebra c, anderson d, tibary a, et al, editors. llama and alpaca care: medicine, surgery, reproduction, nutrition, and herd health. st louis: elsevier; 2014. p. 216-242. 22. tibary a, pearson lk, rodriguez js, et al: uterine infection in camelidae: diagnostic and therapeutic approaches. proc intl camelid health conf vet; 2010. 23. mshelia gd, abba y, voltaire yac, et al: comparative uterine bacteriology and pathology of camels (camelus dromedarius) and cows in north-eastern nigeria. comp clin pathol 2013;22:1195-1200. 24. ali a, al-sobayil fa, al-hawas a: evaluating the effectiveness of different treatments of uterine infections in female camels (camelus dromedarius). theriogenology 2010;74:40-44. 562clinical theriogenology • volume 6, number 4 • december 2014 25. tibary a, anouassi a: retrospective study on an unusual form of ovario-bursal pathology in the camel (camelus dromedarius). theriogenology 2001;56:415-424. 26. ali a, al-sobayil fa, hassanein km, et al: ovarian hydrobursitis in female camels (camelus dromedarius): the role of chlamydophila abortus and a trial for medical treatment. theriogenology 2012;77:1754-1758. 27. ali a, al-sobayil fa, tharwat m, et al: ovarian hydrobursitis in female camels (camelus dromedarius): biochemical, bacterial and protozoal evaluation. theriogenology 2011;75:734-741. 28. anderson de, couto cg, oglesbee m: granulosa theca cell tumor with erythrocytosis in a llama. can vet j 2010;51:1157-1160. 29. gilbert r, kutzler m, valentine ba, et al: hyperandrogenism from an ovarian interstitial-cell tumor in an alpaca. j vet diag invest 2006;18:605-607. 30. drew ml, meyers-wallen vn, acland gm, et al: presumptive sry-negative xx sex reversal in a llama with multiple congenital anomalies. j am vet med assoc 1999;215:1134-1139. 31. wilker ce, meyers-wallen vn, schlafer dh, et al: xx sex reversal in a llama. j am vet medical assoc 1994;204:112-115. 32. hinrichs k, buoen lc, ruth gr: xx/xy chimerism and freemartinism in a female llama co-twin to a male. j am vet med assoc 1999;215:1140-1141. 33. hinrichs k, horin se, buoen lc, et al: x-chromosome monosomy in an infertile female llama. j am vet med assoc 1997;210:1503-1504. 34. schünemann m, böhm j, sendag s, et al: vulvahypoplasia in an alpaca cria first case report in germany. tierärztl prax ausg g grosstiere nutztiere 2012;40:177-181. 35. hallowell gd, potter tj, mills nj: labial fusion causing urinary tract obstruction in an alpaca cria. vet rec 2007;161:862. 36. wilkins pa, southwood ll, bedenice d: congenital vulvar deformity in 6 alpacas. j am vet med assoc 2006;229:263-265. 37. belknap eb, schmidt ar, carleton cl: double cervices in two llamas. j am vet med assoc 1990;197:1049-1053. 563 clinical theriogenology • volume 6, number 4 • december 2014 table 1: reproductive disorders, by organ, diagnosed in female camelids presented for infertility, and methods used to suspect (sm = suspicion method) and confirm (cm = confirmation method) the diagnosis diagnosis sac camels sm cm vulva and vagina ambiguous gender 5 1 external examination cytogenetics, post-mortem enlarged clitoris 8 2 external examination vulvar hypoplasia 8 2 external examination vaginal aplasia 7 1 vaginoscopy videoendoscopy vaginal adhesions 10 5 vaginoscopy videoendoscopy vaginitis 1 2 vaginoscopy double vagina 2 1 vaginoscopy videoendoscopy persistent hymen 9 3 digital palpation videoendoscopy recto-vaginal tears 8 6 external examination cervix cervicitis 5 3 vaginoscopy double cervix 2 1 vaginoscopy videoendoscopy cervical adhesions 3 0 manual examination videoendoscopy cervical rupture (incompetence) 11 3 vaginoscopy videoendoscopy uterus segmental aplasia uterus 5 1 ultrasonography laparoscopy endometrial abscess 1 1 ultrasonography videoendoscopy uterine cysts 8 4 ultrasonography videoendoscopy endometrial fibrosis 32 15 ultrasonography/palpation biopsy uterine luminal adhesions 12 7 ultrasonography/palpation videoendoscopy endometritis 83 102 history, vaginoscopy culture, cytology, biopsy intestinal-uterine adhesions 2 2 palpation laparoscopy mucometra pyometra 18 5 palpation ultrasonography mummification 1 2 palpation laparotomy uterine tube segmental aplasia 1 1 all usual techniques failed laparoscopy hydrosalpinx/ pyosalpinx 1 3 ultrasonography laparoscopy salpingitis 6 8 all usual techniques failed laparoscopy / laparotomy ovary and ovarian bursa ovarian hypoplasia 37 4 ultrasonography laparoscopy hemorrhagic follicles/cysts 41 86 ultrasonography ultrasonography ovulation failure 8 3 history endocrinology persistent luteal structures 15 4 ultrasonography endocrinology ovarian adhesions 3 11 ultrasonography laparotomy ovarian tumor 3 4 palpation, ultrasonography laparotomy ovario-bursal adhesions 3 117 palpation, ultrasonography laparotomy 564clinical theriogenology • volume 6, number 4 • december 2014 table 2: major complaints and value of specific examination procedures in the determination of the cause of infertility in camelids. condition to be considered major complaint* primary method of diagnosis confirmation method maiden females pregnancy 1 history, ultrasound ultrasound immature female 1, 3 history, physical examination ultrasound persistent hymen 1 vaginal examination videoendoscopy ovarian hypoplasia 1, 2 ultrasound endocrinology, laparoscopy segmental aplasia 1, 2 ultrasound laparoscopy/laparotomy ovulation failure 1, 2 ultrasound endocrinology behavioral problems 1 physical, ultrasound physical examination and endocrinology intersex 1, 4 physical laparoscopy, cytogenetics multiparous females anovulatory/hemorrhagic follicles 1 ultrasound endocrinology luteal activity/persistent corpus luteum 1 ultrasound endocrinology ovarian inactivity 1 ultrasound endocrinology, laparoscopy failure of ovulation 1 ultrasound endocrinology ovarian adhesions/ abscess 1, 2 ultrasound laparoscopy vaginal/cervical adhesion 1 vaginoscopy videoendoscopy endometritis 2, 3, 4 vaginoscopy, uterine culture and cytology uterine biopsy endometrial fibrosis 2, 3 history, ultrasonography uterine biopsy salpingitis 2 ultrasonography laparoscopy, laparotomy uterine adhesions 2,3 ultrasonography hysteroscopy systemic diseases 3 history, physical examination serology, hematology poor body condition 1, 2, 3, 4 physical examination vaginal conformation 4 physical examination *major complaints: inability to breed (1), repeat breeding (2), pregnancy loss (3), abnormal external genitalia (4) 565 clinical theriogenology • volume 6, number 4 • december 2014 table 3: classification of endometrial biopsy and potential effect on fertility (according to powers et al 1990) categories of biopsy histopathological characteristic effect on fertility grade 1a normal endometrium normal conception rates grade 1b few lymphocytes within the endometrium. siderophages present. low-grade infection or remnants of previous inflammation. mild surface irritations may indicate reaction to breeding. may be postpartum or post abortion (siderophages) grade 2a to 2b active and acute, chronic, or chronic active endometritis. chronic inflammation tended to be more deeply located in the endometrium, compared with active and chronic active inflammation interferes with conception and may cause early embryonic death. grade 3a chronic endometritis with glandular fibrosis interferes with implantation and placentation. may cause early embryonic death. grade 3b uterine neoplasia pregnancy loss or abortion figure 1: ultrasonographic imaging of follicular dynamics in camelids. a) quiescent ovary (arrows), ub= urinary bladder, b) and c) follicular recruitment, d) follicular dominance, e) mature follicle, f) regressing follicle. note the hyperechoic ovarian stroma and the more hypoechogenic cortex. 566clinical theriogenology • volume 6, number 4 • december 2014 figure 2: ultrasonogram of a mature corpus luteum. note the size and the small central cavity. 567 clinical theriogenology • volume 6, number 4 • december 2014 figure 3: ultrasonograms of the uterus during different stages of follicular development and after ovulation. a-b) increased edema and tone (follicular phase), c-d) relaxed and homogenous (luteal phase). 568clinical theriogenology • volume 6, number 4 • december 2014 figure 4: ultrasonographic appearance of some ovarian and tubal disorders in camelids. a) large anovulatory follicle, b) luteinized/hemorrhagic anovulatory follicle, c) cystic rete ovarii, d) ovarian teratoma, e) hydrobursitis, f) hydrosalpinx. 569 clinical theriogenology • volume 6, number 4 • december 2014 figure 5: ultrasonographic appearance of some disorders of the uterus. a) infantile uterus, b) endometritis, c) chronic endometritis,d) endometrial abscesses, e) cervical adhesions, f) pyometra; g) post-fetotomy pyometra; g) mucometra due vaginal adhesions, note the open cervical rings, i) mucometra due to vaginal aplasia, j-l) uterine cysts. 570clinical theriogenology • volume 6, number 4 • december 2014 1 contact margaret root kustritz peggy@drpeggyroot.com © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 13050, http://dx.doi.org/10.58292/ct.v17.13050 case report testicular microlithiasis in two dogs margaret root kustritz,a‡ michelle kissnerb adepartment of veterinary clinical sciences, college of veterinary medicine, university of minnesota, st. paul, mn, usa bveterinary ultrasound services, llc, eau claire, wi, usa abstract testicular microliths are small areas of calcification in testicular tissue, identified ultrasonographically as diffuse, punctate, nonshadowing, and hyperechoic foci. poor semen quality is associated with increasing numbers of testicular microliths in humans. there is much less description of this phenomenon in animals; we are describing 2 dogs that had microlithiasis. keywords: dog, testis, microliths, infertility, ultrasonogrpahy background testicular microlithiasis (tm) is well described in the human medical literature but is rarely described in the veterinary literature. these small calcified areas within the testicular parenchyma are described ultrasonographically as diffuse, punctate, and nonshadowing hyperechoic foci, generally of uniform size, throughout the testicular parenchyma.1 in humans, this is further differentiated into classic tm (> 5 microliths in at least 1 ultrasound image) and limited tm (< 5 microliths on all images).2 there is a great body of literature evaluating associations between testicular microlithiasis and reproductive disease in men, with greatest focus on possible associations with testicular neoplasia and with infertility. some studies evaluated tm in domestic and wild hoof stock in relation to semen quality.3–5 we are not aware of any reports of testicular microlithiasis in dogs. case presentation case 1: a 3½ year, intact male treeing walker coonhound, was presented for ultrasonographic evaluation of the abdomen and testes due to weight loss and polyphagia in july of 2021. semen had not been evaluated, but the dog had sired a litter in 2020. left testis appeared normal and had a coarse medium echogenicity throughout; right testis contained hyperechoic foci that were slightly larger than typical testicular microliths observed in human beings but were identical to tm in all other aspects (figure 1). other findings on abdominal ultrasonography included a complex cystic structure in the right kidney. ultrasonography of the testes was repeated in october of 2021 and july of 2022, with no changes noted from the original ultrasonography. in october of 2022, the dog was castrated, due to concern about testicular microlithiasis, a couple of episodes of prostatitis, and retirement from breeding. right testis was submitted for histopathology; 2 discrete and relatively small foci of intratubular mineralization accompanied by surrounding fibrosis were noted. in these foci, aggregates of fragmented basophilic material, interpreted to represent mineralization, were present within shrunken seminiferous tubules surrounded by a moderate amount of fibrosis (figures 2–4) without associated inflammation. case 2: a 3½ year, intact male rottweiler with a history of prostate disease was presented for ultrasonographic evaluation in february of 2022. testes were judged to be somewhat small and bilateral testicular microlithiasis was identified (figures 5 and 6). breeding soundness examination was performed in january of 2023. abnormalities noted on general physical examination were mild dental disease and a grade iii/vi cardiac murmur. both testes were small, with an estimate of them being half the size expected for a dog of this breed and size. semen was collected by manual ejaculation with no teaser female dog present but with exposure to estrous dog vaginal swabs. libido was high; 4.5 ml of semen was collected and that was azoospermic. semen alkaline phosphatase concentrations suggested incomplete ejaculation despite good libido and normal erection and reproductive behavior. dog was castrated and testes were submitted for histopathology in april 2023. both testes contained widely scattered foci (figures  7 and 8; blue arrows) of intratubular mineralization (500 µm in maximal diameter with < 200 µm in diameter) without associated inflammation. outside of these mineralized areas, there ‡ current affiliation: dr. peggy root llc, saint paul, mn 55101, usa mailto:peggy@drpeggyroot.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.13050 2 citation: clinical theriogenology 2025, 17, 13050, http://dx.doi.org/10.58292/ct.v17.13050 were rare small interstitial infiltrates of lymphocytes and plasma cells and rare individual atrophied seminiferous tubules. discussion there is debate in the human literature regarding the impact of tm. although there are reports of increased risk of testicular neoplasia in men with tm, it does not appear that tm is an independent risk factor for testicular neoplasia.6–10 however, because tm apparently can act as a marker for other risk factors for testicular neoplasia, it is recommended that men with an incidental finding of tm be reevaluated by ultrasonography every 6-12 months to screen for testicular neoplasia.11,12 testicular neoplasia in humans is more common in young men and is an aggressive, malignant tumor, whereas testicular neoplasia in dogs is more common in middle-aged and older dogs and is generally a more benign neoplasm that can be cured with castration. because of the difference in tumor biology and lack of evidence in the human literature of tm as an independent risk factor for tm, it is difficult to argue for the value of repeated ultrasonographic evaluation of the testes in dogs with tm. the other concern associated in men with tm that is described in the literature is poor semen quality, associated with subfertility or infertility. semen parameters altered with tm in men include decreased sperm concentration, decreased percentage total motility, and decreased percentage progressive motility.1,13,14 semen quality is negatively correlated with number of microliths in men.14 our 2 cases had very different histories and outcomes; 1 dog had unilateral and the other bilateral microlithiasis. future reports with associated semen evaluations and histopathology are required to permit correlation of extent of tm with fertility in male dogs. reports in cattle and elands support the above findings. in a study of 77 bulls (bos indicus, bos taurus, and crosses of these figure 1. testicular ultrasonographic image of case 1 figure 2. testicular histopathology (low magnification) of case 1 http://dx.doi.org/10.58292/ct.v17.13050 citation: clinical theriogenology 2025, 17, 13050, http://dx.doi.org/10.58292/ct.v17.13050 3 breeds), ~ 25% of the animals had some degree of tm and these animals had lower sperm concentration and percentage motility.3 in a report of 3 water buffalo bulls, 1 had tm whose semen quality was not reported.4 a survey of 24 eland in a wildlife park demonstrated histopathological evidence of tm and abnormal spermatogenesis in all animals, with evidence to suggest this was associated with environmental pollutants including phenols that are estrogenic.5 another species reported to have tm include is a cynomolgus monkey; this was an incidental finding at necropsy in a control animal in a toxicology study.15 evaluation by histopathology of testes from men with tm had changes similar to dogs in this case study. microliths are foci of calcification with a precipitate of a glycogen-protein matrix associated with the basement membrane of seminiferous tubules.1 cause of mineralized foci formation is not well described in humans or most other species. in men, no correlation has been demonstrated between development of tm and demographics, socioeconomic characteristics, or concurrent sexually transmitted disease.16 in mice with genetic alterations forming populations of cryptorchidic mice, hypogonadal mice, and mice lacking androgen receptors on sertoli cells or throughout their body, tm developed most commonly in hypogonadal mice with or without androgen receptors, suggesting that lack of endocrine support and subsequent testicular degeneration may have a role.17 tm has been demonstrated to be more common in men with a history of testicular neoplasia or torsion of the spermatic cord that also could be associated with testicular degeneration.16 in men with tm, semen culture yielded nanobacteria in almost 60% of samples.18 nanobacteria are the smallest known organisms with a cell wall and it is unknown whether they are living organisms as their minute size precludes them having all necessary figure 3. testicular histopathology (medium magnification) of case 1 figure 4. testicular histopathology (high magnification) of case 1 http://dx.doi.org/10.58292/ct.v17.13050 4 citation: clinical theriogenology 2025, 17, 13050, http://dx.doi.org/10.58292/ct.v17.13050 organelles for replication. rats injected with nanobacteria from men with tm demonstrated testicular calcification as observed in tm, possibly associated with particulate size of the nanobacteria providing a focus for mineralization.19 prevalence of tm in humans varied from 1.4-8.1%;7,13,14 62% of 226 men with tm were classified as having the classic variant.13 prevalence and extent of tm in veterinary species has only been reported in small populations and reported values likely are not reflective of true prevalence in a given species. it is possible that tm will be easier to diagnose going forward as the quality of ultrasound equipment available to veterinarians increases. to obtain the best images, the ultrasound transducer should be 8 mhz at a minimum. a linear transducer is best but one of the authors has identified these lesions with a curvilinear 8 mhz transducer. because the lesions are very small, a higher frequency transducer best permits identification and may even capture small shadows thrown by the microliths in the testicular tissue. appropriate transducers figure 5. left testicular ultrasonographic image of case 2 figure 6. right testicular ultrasonographic image of case 2 http://dx.doi.org/10.58292/ct.v17.13050 citation: clinical theriogenology 2025, 17, 13050, http://dx.doi.org/10.58292/ct.v17.13050 5 often are sold as the primary transducer used in small animal machines marketed at the time of this writing. experience may be required to permit the operator to see these lesions; review by an ultrasonographer may be warranted for accuracy of diagnosis. in dogs, identification of tm should spur a semen evaluation and a conversation with the client about possible association of tm with subfertility or infertility. regular recheck of the testes by ultrasonography probably is not warranted in dogs. learning points • testicular microlithiasis (tm) is accumulation of small foci of mineralization within the seminiferous tubules and is visible in ultrasonography • in humans and hoof stock, individuals with tm are more likely to have poor semen quality, with a greater decrease in semen quality in individuals with more microliths • cause of microliths formation is not defined but it may be a consequence of testicular degeneration • if tm is identified in dogs, owners should be made aware of a possible association with poor semen quality, and semen evaluation performed conflict of interest none to report. references 1. miller rl, wissman r, white s, et al: testicular microlithiasis: a benign condition with a malignant association. j clin ultrasound 1996;24:197-202. doi: 10.1002/(sici)1097-0096 (199605)24:4<197::aid-jcu6>3.0.co;2-a. figure 7. testicular histopathology (low magnification) of case 2 figure 8. testicular histopathology (high magnification) of case 2 http://dx.doi.org/10.58292/ct.v17.13050 https://doi.org/10.1002/(sici)1097-0096​(199605)24:4<197::aid-jcu6>3.0.co;2-a. https://doi.org/10.1002/(sici)1097-0096​(199605)24:4<197::aid-jcu6>3.0.co;2-a. 6 citation: clinical theriogenology 2025, 17, 13050, http://dx.doi.org/10.58292/ct.v17.13050 2. bennett hf, middleton wd, bullock ad, et al: testicular microlithiasis: us follow-up. radiol 2001;218:359-363. doi: 10.1148/ radiology.218.2.r01fe25359 3. saavedra-jimenez la, olvera-ramirez jf, nunez-martinez g, et al: exploratory study: testicular microlithiasis in cattle from the costa chica region. agro productividad 2022;15:89-94. doi: 10.32854/agrop.v15i4.2131 4. chandolia rk, singh g, kumar a, et al: testicular microlithiasis in a buffalo bull – a rare case. haryana vet 2018;57:122-123. available from: https://www.luvas.edu.in/haryana-veterinarian/ download/harvet2018-june/40.pdf 5. bornman ms, barnhoorn iej, dejager c, et al: testicular microlithiasis and neoplastic lesions in wild eland (tragelaphus oryx): possible effects of exposure to environmental pollutants? environ res 2010;110:327-333. doi: 10.1016/j.envres.2010.02.003  6. decastro bj, peterson ac, constable ra: a 5-year followup study of asymptomatic men with testicular microlithiasis. j urol 2008;179:1420-1423. doi: 10.1016/j.juro.2007.11.080  7. peterson ac, bauman jm, light de, et al: the prevalence of testicular microlithiasis in an asymptomatic population of men 18 to 35 years old. j urol 2001;166:2061-2064. doi: 10.1016/s0022-5347(05)65506-1 8. aoun f, slaoui a, naoum e, et al: testicular microlithiasis: systemic review and clinical guidelines. prog urol 2019;29:465-473. doi: 10.1016/j.purol.2019.07.001  9. pedersen mr, rafaelsen sr, moller h, et al: testicular microlithiasis and testicular cancer: review of the literature. intl j urol nephrol 2016;48:1079-1086. doi: 10.1007/s11255-016-1267-2 10. balawender k, orkisz s, wisz p: testicular microlithiasis: what urologists should know. a review of the current literature. cent european j urol 2018;71:310-314. doi: 10.5173/ceju.2018.1728  11. moran jm, moreno f, climent v, et al: idiopathic testicular microlithiasis: ultrastructural study. br j urol 1993;72:252-253. doi: 10.1111/j.1464-410x.1993.tb00702.x 12. leblanc l, lagrange f, lecoanet p, et al: testicular microlithiasis and testicular tumor: a review of the literature. basic clin androl 2018;28:8. doi: 10.1186/s12610-018-0073-3 13. xu c, liu m, zhang f-f, et al: the association between testicular microlithiasis and semen parameters in chinese adult men with fertility intention: experience of 226 cases. urol 2014;84:815-820. doi: 10.1016/j.urology.2014.03.021  14. hiramatsu i, tsujimura a, miyoshi m, et al: prevalence of testicular microlithiasis in healthy newlywed men trying for firsttime pregnancy. intl j urol 2020;27:990-995. doi: 10.1111/ iju.14342 15. shirai n, evans mg: testicular microlithiasis in a clinically healthy cynomolgus monkey (macaca fascicularis). j toxic pathol 2018;31:147-150. doi: 10.1293/tox.2017-0065 16. pedersen mr, moller h, rafaelsen sr, et al: association between risk factors and testicular microlithiasis. acta radiol open 2019;8:1-6. doi: 10.1177/2058460119870297 17. o’shaughnessy pj, monteiro a, verhoeven g, et al: occurrence of testicular microlithiasis in androgen insensitive hypogonadal mice. repro biol endocrinol 2009;7:88. doi: 10.1186/1477 7827-7-88 18. zhang qh, lu gs, shen xc, et al: nanobacteria may be linked to testicular microlithiasis in infertility. j androl 2010;31:121-125. doi: 10.2164/jandrol.109.007930 19. lin x-c, gao x, lu g-s, et al: role of calcifying nanoparticles in the development of testicular microlithiasis in vivo. bmc urol 2017;17:99. doi 10.1186/s12894-017-0289-0 http://dx.doi.org/10.58292/ct.v17.13050 https://doi.org/10.1148/radiology.218.2.r01fe25359 https://doi.org/10.1148/radiology.218.2.r01fe25359 https://doi.org/10.32854/agrop.v15i4.2131 https://www.luvas.edu.in/haryana-veterinarian/download/harvet2018-june/40.pdf https://www.luvas.edu.in/haryana-veterinarian/download/harvet2018-june/40.pdf https://doi.org/10.1016/j.envres.2010.02.003  https://doi.org/10.1016/j.juro.2007.11.080  https://doi.org/10.1016/s0022-5347(05)65506-1 https://doi.org/10.1016/j.purol.2019.07.001  https://doi.org/10.1007/s11255-016-1267-2 https://doi.org/10.5173/ceju.2018.1728  https://doi.org/10.1111/j.1464-410x.1993.tb00702.x http://doi: 10.1186/s12610-018-0073-3 https://doi.org/10.1016/j.urology.2014.03.021  https://doi.org/10.1111/iju.14342 https://doi.org/10.1111/iju.14342 https://doi.org/10.1293/tox.2017-0065 http://doi: 10.1177/2058460119870297 https://doi.org/10.1186/1477-7827-7-88 https://doi.org/10.1186/1477-7827-7-88 http://doi: 10.2164/jandrol.109.007930 http://doi 10.1186/s12894-017-0289-0 identifying risk factors for mastitis in postpartum bitches jennine lection, alyssa cornelius, mariana diel de amorim, soon hon cheong department of clinical sciences, college of veterinary medicine cornell university, ithaca ny mastitis in postpartum bitch is a disease that affects wellbeing of both dam and pups that rely on their mother's milk as the sole source of nutrition during a critical growth period. risk factors for canine mastitis have not been well-studied and this information could help veterinarians and breeders identify females that may need closer monitoring. goal of this study was to identify risk factors for postpartum mastitis in bitch. we hypothesized that age, breed, litter size, and whelping interventions are associated with risk of developing mastitis in postpartum bitch. a retrospective cohort study was performed using data from 2 guide dog colonies over a 13-year period. a total of 2,489 whelpings occurred during study period. all bitches were monitored closely and mastitis was diagnosed by colony veterinarian. risk factors examined were: colony, breed of dam, litter size, neonatal pup loss, age (in years), parity, dystocia, use of oxytocin during whelping, use of calcium during whelping, and if cesarean section was performed. risk factors were evaluated individually and offered to the model if p < 0.20. a final model was built using a backwards-stepwise method and colony was forced into the model to account for colony-specific differences. logistic regression anova was performed using jmp prov. 14 (sas institute). mastitis incidence was 8.9% (222 cases). 1bree significant risk factors were identified and offered to the model built; all were retained in the final model. first, mastitis risk increased as litter size increased and roc analysis identified 2: 9 pups as the optimal threshold. bitches that had 2: 9 pups (120/1,048 or 11.5% versus 7.1 % in litters < 9 pups) were more likely to develop mastitis (or = 1.67, 95% ci 1.26 2.21; p = 0.0003). second, there was an effect of dam breed; golden retrievers (12.9% or = 2.81, 95% c.i. 1.32 5.99; p = 0.0075) and labrador retrievers (8.6% or = 2.05, 95% c.i 1.03 4.11; p = 0.0418) were more likely to develop mastitis compared to german shepherd dogs (4.8%). finally, bitches that whelped naturally (9.4%) were more likely to develop mastitis compared to bitches that had cesarean section (5.7%) performed (or= 1.70, 95% ci 1.04 2.76; p = 0.0332); therefore, bitches that underwent cesarean section had lower rates of mastitis. surprisingly, bitches with singletons (3%) had lower mastitis risk than bitches with large litters, and neonatal (> 3 days) loss of pups did not affect mastitis risk. the dogma of older bitches having higher risk of mastitis was also not observed in our population. there is an effect of dam breed on mastitis risk and further studies examining more breeds are warranted. keywords: canine, mastitis, risk factors 354 clinical theriogenology • volume 12 number 3 • september 2020 fungal growth is more likely to be affected by hormones in equine uterine isolates compared to isolates from nonreproductive sites jennine lection, a theodore kapogiannis,8 erick bosire,b rebecca franklin-guild, b patrick crane, 8 mariana diel de amorim, 8 craig altier, b soon hon cheong8 8department of clinical sciences, ~epartment of population medicine and diagnostic sciences college of veterinary medicine, cornell university, ithaca, ny fungal endometritis is an important cause of infertility in mares. studies indicated that whereas estradiol may have a stimulatory effect on fungal growth, progesterone may have an inhibitory effect. however, both studies only examined 1 fungal isolate. hormone responsiveness of fungal isolates from mare reproductive tract has not been evaluated. objective was to determine if clinical fungal isolates from mare uteruses are hormone responsive; if so, are uterine isolates more likely to be hormone responsive compared to fungal isolates from nonreproductive sites. we hypothesized that estradiol has stimulatory effects whereas progesterone has inhibitory effects on fungal growth and that fungal isolates from mare uteruses are more likely hormone responsive compared to fungal isolates from nonreproductive sites. fungal isolates from mare uteruses (n = 7) and isolates from nonreproductive sites (n = 5) were evaluated for hormone responsiveness after being cultured in rpmi media for 48 hours at 30°c in 96-well plates. absorbance was read at 600 nm at seeding times, 24 and 48 hours. correlation between absorbance and fungal concentration was determined by hemocytometer. dependent variable was percent change in fungal concentration from initial seeding. estradiol treatment groups were: ethanol (vehicle) control and 10, 150, and 1,000 ng/ml whereas progesterone treatment groups were: ethanol control and 0.1 , 5, and 100 µg/ml . data were not normally distributed and nonparametric analyses were used. effects of hormone treatment and site on fungal growth were determined by kruskal-wallis test and treatment effects were determined using multiple comparison rank sum steel test with ethanol group as the control in jmp prov. 14 (sas institute, cary, nc). estradiol affected growth (p = 0.0004) at 48 hours with increased growth (p = 0.01 89) in 10 ng/ml compared to control. progesterone affected growth at 24 (p < 0.0001 ) and 48 hours (p < 0.0001) with decreased growth in 5 µg/ml (p < 0.0001) and 100 µg/ml (p < 0.0001) groups compared to controls. estradiol affected growth (p < 0.0001), with 3/7 (at 24 and 48 hours) isolates from uterus and 0/5 (at 24 hour) and 1/5 (at 48 hour) isolates from nonreproductive sites affected by estradiol. progesterone affected growth (p < 0.0001), with 6/7 (at 24 and 48 hour) isolates from mare uterus and 1/5 (24 hour) and 3/5 ( 48 hour) isolates from nonreproductive sites affected. majority of fungal isolates from mare uteruses were hormone responsive to estradiol (that increased growth rates) and progesterone (that decreased growth rates) whereas most nonreproductive isolates were not hormone responsive. this study highlighted potential interactions between hormone status and fungal infectivity in equine uterus. keywords: fungal, hormone responsiveness, horses acknowledgement part of this work was supported by theriogenology foundation clinical theriogenology • volume 12 number 3 • september 2020 355 a novel method to reduce egg laying in companion avian species using a hen model sandra ayres,8 jennifer graham,b yamin li,c qiaobing xue adepartment of biomedical sciences and b department of clinical sciences cummings school of veterinary medicine at tufts university, north grafton, ma coepartment of biomedical engineering, tufts university, medford, ma reproductive-related diseases in female avian species are a common problem in zoological companion animal medicine. of particular concern is a potentially serious condition called egg-binding where eggs are retained within female reproductive tract. in chronic cases, treatment involves preventing egg production with surgical removal of the ovary or administration of hormones such as leuprolide acetate, or deslorelin acetate. these approaches are problematic, as surgical gonadectomies in birds are difficult, if not impossible, and hormonal injections and implants are expensive, variably effective, and short-acting; therefore, an effective nonsurgical method of suppressing egg production is needed. we are developing a nonsurgical approach to reduce gonadal activity and germ cell production using an antimullerian hormone receptor 2 antibody (amhr2)-guided lipid nanocomplex carrying a cytotoxin (saporin) to induce apoptosis in gonadal support cells exhibiting amh2 receptor. this method disrupted gonadal architecture and germ cell development in rats and pigs. we hypothesized that this unique technology would reduce hens egg production. rhode island red laying hens (n = 12) formed control group (n = 6, 0.5 ml sterile intravenous saline) and treatment group ([n = 6], administered 0.5 ml of sterile intravenous saline containing 100 nmol of nanocomplex). hens were housed separately, weighed twice weekly and photographed once a week. general health and number of eggs laid were evaluated daily starting 13 days prior to treatment until 28 days after treatment. blood was collected prior to treatment and at end of study for concentrations of estradiol, progesterone and androstenedione. hens were euthanized via intravenous pentobarbital and left ovary was collected, measured, weighed, and formalin­ fixed for h&e histology. there were no differences between groups in general health, body weight, ovarian weight, or blood hormone concentrations. histologically, all ovaries had evidence of follicular activity. most obvious effect was cessation of egg laying in all treated hens starting 3 days postinjection and lasting at least 16 days in 4 of 6 treated hens. all injected hens eventually resumed laying eggs. eggs collected preinjection days 0-2 days 3-18 days 19-28 (13 days) (3 days) (16 days) (10 days) controls total eggs 59 12 52 31 --·-~·· "'•••tm•t-.eggs/bird/day 0.76 0.67 0.54 0.52 nano injected total eggs 57 12 10 28 eggs/bird/day 0.73 0.67 0.1 0.47 administration of nanocomplex resulted in short-term cessation of egg laying in all treated hens without impacting overall health of these birds supported the hypothesis. further studies are needed to determine if a different dose and/ or route of administration could increase the length of this cessation, or even lead to a permanent loss of egg production. keywords: gonadal suppression, fertility, avian egg laying, egg-binding, nanocomplex 356 clinical theriogenology • volume 12 number 3 • september 2020 sperm-bound antisperm antibodies are associated with poor cryosurvival of stallion sperm maria ferrer,8 igor canisso,b robyn ellerbrock,8 giorgia podicob 8department of large animal medicine, athens, ga 'department of veterinary clinical medicine, university of illinois, urbana, il semen freezing is a common practice in equine reproduction. however,~ 30 40% stallions' semen does not survive cryopreservation. reason for poor cryosurvival is not clearly understood. antisperm antibodies (asas) are associated with poor cooling ability of stallion sperm. we hypothesized that presence of sperm-bound asas is associated with poor cryosurvival. objective of this study was to evaluate asa binding in stallions with semen with good versus poor cryosurvival. ejaculates from stallions (n = 21) were extended in inra96 to 40 x 106 sperm/ml and shipped overnight to laboratory in a passive cooling device (equitainer~. after arrival, each ejaculate was divided into 3 aliquots, centrifuged, resuspended in botucrio~ (bc; botupharma, scottsdale, az), ez mixin cryomax modified french tm (mfr; ars, chino, ca) or ez mixin cryomax lactose-edt a tm (le; ars) and frozen following manufacturer's instructions. semen was stored in liquid nitrogen until evaluation. one straw from each aliquot was thawed at 38°c for 30 seconds and was assessed for total and progressive sperm motility (casa). in addition, acrosomal integrity (fitc-pna/pi), percentage of apoptotic and necrotic sperm (annexin v /pl), and percentage of sperm with lgg and iga binding ( antiequine igg and lga) were evaluated with flow cytometry. postthaw motility was considered acceptable if pm~ 30%. semen considered as good cryosurvival if they had acceptable postthaw motility with at least 2 extenders. there was no difference in asa binding among semen extenders and data were pooled. semen with good cryosurvival (n = 13) had a lower percentage oflgg (4.1 ± 0.5%) and iga bound sperm (2.9 ± 0.3%) compared to semen with poor cryosurvival (n = 8; igg 13.5 ± 2.3%, iga 11.2 ± 1.7%) (p < 0.0001; student's t-test). none of the semen with good cryosurvival were asa positive. however, 43.5% of semen with poor cryosurvival were asa positive (p < 0.0001, chi square). there was a negative correlation between percentage oflgg and iga bound sperm, and total motility (igg: p = 0.0045, r2 = -0.36; iga: p = 0.0003, r2 = -0.45) and progressive motility (igg: p = 0.0005, r2 = -0.44; iga: p < 0.0001, r2 = -0.49), and a positive correlation between percentage oflgg bound sperm and percentage of sperm with damaged acrosomes (igg: p = 0.049, r2 = -0.36). in summary, presence of asas was associated with poor cryosurvival of stallion sperm. keywords: stallion, semen, antisperm antibodies, cryopreservation, freezing clinical theriogenology • volume 12 number 3 • september 2020 357 effect of energy substrates on cool-stored stallion sperm camilo hemandez-aviles,8 charles love,8 luisa ramirez-agamez,8 macy friedrich,8 dale kelley,8 mariah pearson,8 alecia baker,8 sheila teague,8 anne beckham,8 katrina lacaze, a steven brinsko, a dickson v amer8 8department of large animal clinical sciences college of veterinary medicine & biomedical sciences texas a&m university, college station, tx commercially available equine semen extenders for cooled storage are typically formulated with glucose as an energy substrate. when incubated at 38.5°c, stallion sperm are reported to preferentially utilize oxidative phosphorylation (oxphos) substrates (lactate or pyruvate) for energy production. this study was conducted to determine the effect of 3 energy substrates (glucose, lactate, or pyruvate ), individually or in combination, on sperm quality after cooled storage for 24 hours. we hypothesized that inclusion of oxphos energy substrates in extender would increase sperm quality parameters compared to glucose alone. three ejaculates from each 1 of 6 stallions (n = 18) were processed by cushioned centrifugation using a milk-based extender containing various energy substrates, or their combinations, and 10% (v/v) homologous seminal plasma. treatment groups were: no substrate added (control); 40 mm added glucose (glu-40); 2 mm pyruvate (pyr-2); 2 mm lactate (lac-2); 19.8 mm pyruvate (pyr-19); 19.8 mm lactate (lac-19); 40 mm glucose+ 2 mm pyruvate + 2 mm lactate (glu-pyr-lac-2); or 40 mm glucose+ 19.8 mm lactate+ 19.8 mm pyruvate (glu-pyr-lac-19). extended semen was stored at 2 temperatures, 10 or 20°c, for 24 hours. after storage, semen was analyzed for % total motility (tmot) using casa; and % viable/acrosome intact (v ai), and % viable/lipid peroxidation positive (vlpp) using flow cytometry. data were rank-transformed prior to analysis using general linear model procedure. statistical significance was set at p < 0.05. at both storage temperatures, ¾tmot was similar (p > 0.05) among glu-40, glu-pyr-lac-2 and glu-pyr-lac-19 (57, 58, 54%), and higher (p < 0.05) in these than other treatment groups (31 39%; p < 0.05). percent v ai was lower (p < 0.05) in control (68%) and pyr-2 (69%), than in glu-40 (73%). percent vlpp was similar (p > 0.05) among glu-40, glu-pyr-lac-2 and pyr-19 (22, 24, 22%, respectively), but higher (p < 0.05) in these groups as compared to the control (17% ). these results suggest that at reduced storage temperatures, addition of oxphos substrates in extender do not enhance sperm motility over that of glucose alone. only slight differences on % v ai were observed, which might suggest that type of energy substrate does not have a substantial impact on stallion sperm membrane intactness. higher % vlpp was observed in treatment groups with higher motility values, which may be related to a higher metabolic rate, and resultant production of reactive oxygen species. keywords: stallion, sperm, low temperature, energy substrate, motility, plasma membrane 358 clinical theriogenology • volume 12 number 3 • september 2020 effects of recent feeding on canine serum progesterone william whitler, vanessa souz,8, charles estill department of clinical science, carlson college of veterinary medicine oregon state university, corvallis, or serum progesterone values are commonly used to estimate luteinizing hormone (lh) surge in the bitch to appropriately time insemination. known factors affecting serum progesterone values include type of sample tube used, size of dog, age of dog, anticoagulant, interval from blood collection to centrifugation ( especially if refrigerated before centrifugation), time of day and stage of estrus. in cattle and humans, serum progesterone values have been reported to decline following a meal. goal of this study was to determine the influence of a meal on serum progesterone in dogs. all procedures were approved by the osu animal care and use committee. client-owned female dogs (n = 7) of various breeds (body weight: 3.4 37 kg, mean: 17.65 kg) and ages (2 10 years, average 4.9 years) presented to osu lois bates acheson veterinary teaching hospital for breeding management were enrolled. dogs were fasted overnight prior to initial blood sampling between 0800 and 0900. blood was collected into plain red top tubes with no anticoagulant and submitted to laboratory within 20 minutes after sample collection. following initial sample, dog was fed a commercial dog food (aid® hills pet nutrition, topeka, ks), 10 15 ml/kg body weight (average 13.3 ml/kg). all dogs consumed food readily and no adverse effects were observed or reported by owners. a second blood sample was collected 60 7 5 minutes after feeding (average 68.5 minutes). serum progesterone was determined by a siemens lmmulite® 1000 (siemens usa, malvern, pa). serum progesterone concentrations were lower (p = 0.0005) in postprandial sample in all 7 patients. average reduction was 28.2% (11.3 68.4%), with an average decrease of 0.95 ng/ml (0.14 1.41 ng/ml). two samples of special interest had fasting concentrations of 1.96 and 2.26 ng/ml that dropped postprandial to 0.62 and 0.93 ng/ml, respectively. body weights of those 2 dogs were 5.8 and 14 kg, respectively. since lh surge occurs when serum progesterone is in 1 3 ng/ml range, interpretation of pre and postprandial samples might be different if the dog was not fasted at the initial sample. however, neither of these 2 dogs tested positive for lh test (witness®lh, zoetis, kalamazoo, mi) at first sampling. we recommend that samples for serum progesterone are collected from fasted patients to ensure most consistent results. cause for decreased serum progesterone concentrations following a meal in dogs has to be determined. keywords: progesterone, feeding, dog, ovulation clinical theriogenology • volume 12 number 3 • september 2020 359 effect of slide type to evaluate motility parameters of frozen-thawed equine sperm using computer aided sperm analysis paul loomis, karley milburn, kristin klohonatz select breeders services, chesapeake city, md in equine industry, motility assessment is the primary parameter currently used to evaluate sperm quality in an ejaculate. motility is determined either by visual subjective assessment or objectively via computer aided sperm analysis (casa) system. various types of slides and coverslips are used for sperm motility assessment. two main methods are a fixed coverslip slide ( e.g. leja® slide) or a drop coverslip slide ( e.g. cell-vu® slide or glass slide). both these slides can have a fixed chamber depth of 20 microns. previous studies in our laboratory demonstrated a negative effect on total and progressive motility with fixed coverslip slides compared to drop coverslip slides for analysis of fresh semen using casa. objective was to determine if there is a difference in casa derived motility parameters of frozen-thawed stallion sperm based on slide/chamber type. we evaluated the postthaw motility of 30 ejaculates of frozen semen. samples were thawed at 37°c for 30 seconds and placed into a prewarmed 5 ml tube. concentration was determined using a nucleocounter and samples were diluted to 30 x 106/ml in 2 ml standard skim milk semen extender. samples were incubated for 10 minutes and evaluated using the ceros ii casa system. then, 5 µ1 sample was placed in each chamber on either fixed coverslip slide or drop coverslip slide. to avoid any possible influence of an increased incubation time, the first slide type evaluated alternated with every sample. a minimum of 5 fields were selected for each sample and analysis continued until a minimum of 400 motile sperm were examined. each field was limited to have a maximum number of 150 objects per screen in order to minimize the impact of collisions. motility parameters evaluated were total motility (tm), progressive motility (pm), path velocity (v ap), curvilinear velocity (vcl), straight line velocity (vsl), amplitude oflateral head displacement (alh), beat cross frequency (bcf), linearity (lin), and straightness (str). a sperm was considered progressively motile if it had a str of::: 7 5% and a v ap ::: 50 µm/sec. a student's paired t-test was used to evaluate the difference in motility values between 2 slides and p < 0.05 indicated significance. there was a difference (p < 0.0001) for tm and pm between 2 slide types. both motility values were higher in drop coverslip slides. average tm for drop coverslip slides was 52% versus fixed coverslip slides was 35%. average pm for drop coverslip slides was 38% versus fixed coverslip slides was 24%. there was no difference between cell types for v ap, vsl, vcl, str, lin, alh, and bcf. these data agree with previous results in our laboratory obtained with fresh semen. although drop coverslip slides and fixed coverslip slides utilized 20 micron chamber for motility analysis, these results indicate that slide type has substantial impact on sperm motility and comparison of motility measures between samples using various chamber types is not valid. keywords: equine sperm, motility, casa, fixed coverslip, drop coverslip 360 clinical theriogenology • volume 12 number 3 • september 2020 effect of glycerol concentrations on a new extender for freezing dog semen juliana cunha,8 camila dell' aqua,8 sidnei de oliveira,8 felipe de sousa,8 laiza de camargo,8 rogerio souza,bmarco alvarenga,3 jose dell' aqua8 a sao paulo state university "julio de mesquita filho", botucatu, sao paulo, brazil ~otupharma us, phoenix, az glycerol is the most commonly used cryoprotectant for frozen semen in several species, because it alters colligative properties of water, decreasing freezing point and increasing sperm survival. many studies used various concentrations of glycerol for freezing dog semen, with no consensus regarding best glycerol concentration. aim was to evaluate glycerol concentrations ( 6, 7, and 8%) in dog freezing extender. two ejaculates from 12 adult dogs (2 5 years) of various breeds were used. ejaculates were collected by digital manipulation and divided into 3 aliquots, centrifuged at 600 x g for 7 minutes and pellet was resuspended (100 x 106 sperm per ml) in an egg yolk-based extender botudog® (botupharma®) with 3 glycerol concentrations: 6, 7, and 8% (g6, g7, and gs, respectively). samples were packaged in 0.5 ml straws (imv, france) and kept in a controlled temperature refrigerator (minitub®) for 60 minutes at 5°c. straws were placed in a styrofoam box and kept 3.5 cm above nitrogen level for 20 minutes, and finally were immersed. straws were thawed at 76°c for 8 seconds. sperm kinetics were evaluated using the casa system (hamilton thome researchivos 12, beverly, ma). integrity of plasma and acrosomal membranes, and stability of plasma membrane were determined by flow cytometry (bd lsr fortessa becton dickinson, mountain view, ca), at 10 and 30 minutes (tio and t30), after thawing with incubation at 37°c. data were analyzed using statistical program graph pad 6.0 and kolmogorv­ sminov test was used for normality. anova followed by tukey test were used and friedman test followed by dunns were used for nonparametric data. total motility was not different (p > 0.05) among groups in tio: g6 (52.5 ± 5.3), g7 (58.7 ± 3.7), and gs (57.3 ± 5.3) and in t30: g6 (35.1 ± 3.9), g7 (41.4 ± 4.1), and gs (38.2 ± 3.8), neither progressive motility in tio: g6 (39.5 ± 4.9), g7 (45.4 ± 3.8), and gs (44.1 ± 4.8) and in t30: g6 (26.3 ± 3.7), g7 (32.0 ± 3.8), and gs (29.7 ± 3.6). lntegrity of plasma and acrosomal membrane was higher in g7 (52.6 ± 3.4) compared to g6 (42.4 ± 3.7) at 10 minutes after thawing and after 30 minutes incubation in g7 (32.3 ± 2.8)compared to g6 (26.4 ± 2.7). we concluded that 7% glycerol in the botudog® freezing extender yielded better results for plasma and acrosomal membrane integrity than 6%, whereas 8% had intermediate results. keywords: cryopreservation, canine, casa, plasma and acrosomal membrane integrity clinical theriogenology • volume 12 number 3 • september 2020 361 sperm protein reactive with antisperm antibody is immunoexpressed in equine primordial, primary, secondary, and tertiary follicles brynley cozzi, hayder habeeb, michelle kutzler department of animal and rangeland sciences oregon state university, corvallis, or sperm protein reactive with antisperm antibody (sprasa) is present in all stages of ovarian follicles in humans, mice, cows, dogs, and cats, 1.2 and is localized to granulosa cells and ooplasm. 1• 2 objective was to determine if equine ovarian follicles expressed sprasa. we hypothesized that sprasa immune expression would be present in all stages of equine ovarian follicle, and localize to granulosa cells and ooplasm. routine immunohistochemistry was performed on formalin-fixed paraffin­ embedded ovarian sections ( 4 µm) from 3 adult grade quarter horse mares. briefly, sections were deparaffmized and rehydrated before antigen retrieval was accomplished by heat activation in a sodium citrate buffer. endogenous peroxidase activity was blocked using 3% hydrogen peroxide and nonspecific binding was blocked using serum-free protein block (dako #x0909, carpinteria, ca). polyclonal antisprasa primary antibody was applied to slides at a 1 :200 dilution. universal negative rabbit antibody (dako #s3022) was applied to adjacent sections to serve as negative control. horseradish peroxidase-conjugated antirabbit polymer secondary antibody (immuno bioscience, #ih-8064-osu-l 5, mukilteo, wa) was applied undiluted to all slides. peroxidase activity was detected using nova red kit (vector laboratories inc, #sk4800, burlingame, ca). slides were counterstained with hematoxylin, dehydrated, and cover slipped. digital images were captured at 10 and 40 x magnifications (qlmaging #qic-f-m-12-c). sprasa expressed in pregranulosa cells of primordial, and in granulosa cells of primary, secondary, and tertiary follicles in equine ovaries. sprasa was not expressed in equine ooplasm. there was no positive staining in negative controls. in us, wild horse and burro populations have drastically exceeded the carrying capacity of public lands where they are managed. with the exception of surgery (removal of ovaries), current methods for sterilizing wild female horses and burros ( e.g. porcine zona pellucida or gnrh immunization) are temporary reversible solutions. immunization against sprasa may prove to be a permanent nonsurgical sterilant method because it targets all follicles including primordial follicles. keywords: antisperm antibodies, contraceptive, horse, immunohistochemistry, oocyte, spaca3 references 1. chiu ww, erikson ek, sole ca, et al: sprasa, a novel sperm protein involved in immune-mediated infertility. hum reprod 2004;9:243-249. 2. wagner a, holland oj, tong m, et al: the role ofsprasa in female fertility. reprod sci 2015;22:452-461. 362 clinical theriogenology • volume 12 number 3 • september 2020 comparison of two methods to induce acrosome reaction in stallion sperm camilo hernandez-aviles, luisa ramirez-agamez, charles love, dale kelley, sheila teague, katrina lacaze, katrin hinrichs, dickson varner college of veterinary medicine & biomedical sciences texas a&m university, college station, tx acrosome reaction (ar) failure in stallion sperm has been associated with pronounced subfertility. acrosomal function in stallion sperm is typically assessed by sperm reaction to calcium ionophore, a23187 (cal). this method is considered "nonphysiological," because sperm viability is greatly reduced. we have recently demonstrated that incubation of stallion sperm under "capacitating-like conditions" (presence of bicarbonate, bovine serum albumin [bsa], and calcium) using a modified whitten's medium containing only lactate as an energy source (lac-mw) induces spontaneous ar in viable equine sperm (var). we compared rate of ar and var in stallion sperm induced by the cal and lac-mw methods. two ejaculates each from 4 fertile stallions (n = 8) were collected. sperm in lac-mw treatment were washed, resuspended in this medium, and incubated at 38.5°c in 5% co2 in air. sperm in cal treatment were diluted in inra-96 extender, treated with 10 µm cal, and incubated at 38.5°c in air. at 1, 2, 4, and 6 hours of incubation, sperm aliquots were stained using fitc-psa/propidium iodide (cal), or fitc-psa/fixable live-dead red stain (lac-mw), and analyzed by flow cytometry for percentages of viable (% v), total ar (%ar), and viable acrosome reacted (%var) sperm. fitc-psa/fixable live-dead red stain was used in lac-mw to reduce artifactual decrease in sperm populations due to agglutination after incubation. previous data from our laboratory indicated agreement between techniques is high (bias psa-pi versus fixable ld: -4.0%, sd: 5.3). data were rank­ transformed before analysis using a general linear model procedure. at all time periods, mean % v was higher in lac-mw than in cal ( e.g. 1 hour: 62 versus 28%; 6 hour: 59 versus 0%; p < 0.05). conversely, at all time periods, mean %ar was higher in cal than lac-mw (e.g. 1 hour: 40 versus18%; 6 hour: 95 versus 63%; p < 0.05). mean %v-ar was different in lac-mw than cal starting at 2 h (2 versus 0%), and was notably higher at 4 hour (24 versus 0%) and 6 hour (29 versus 0%; p < 0.05). use of lac-mw appeared to be preferable for induction of ar in stallion sperm if viability is desired, since higher var were obtained, and treatment did not reduce sperm viability. this suggested that lac-mw might create a more "physiological" condition for the ar. differences in %ar and % var observed suggested that these 2 methods induce acrosome reaction via different mechanisms. use of lac-mw could be useful to investigate the mechanism associated with ar failure in stallion. keywords: stallion sperm, acrosome reaction, viability, lactate, calcium ionophore clinical theriogenology • volume 12 number 3 • september 2020 363 sperm contact time in uterus and endometrial inflammation in mares bred by transrectally guided deep horn artificial insemination sofia kovacsy, a,b maria cadario, 8 marcelo miragaya, b juan sampere 0equine reproduction specialty practice, ocala, fl bpacultad de ciencias veterinarias, universidad de buenos aires, argentina cdepartment of large animal clinical sciences, university of florida, gainesville, fl sperm elicit an acute uterine inflammatory reaction that resolves within 24 48 hours in normal mares. delay in uterine clearance results in persistent inflammation, alters uterine environment, and decreases embryo survival, resulting in substantial economic losses. to improve embryo survival, uterus is lavaged within the first 4 12 hours after insemination. substantial reduction in pregnancy rate was observed if lavage is performed prior to 4 hours. deep (uterine) horn transrectally guided artificial insemination (dhai) is a technique that places sperm close to utero-tubal junction, ipsilateral to the ovulation side. advantages of dhai are reduction in volume and/or number of sperm inseminated, and reduction in sperm transport time, with faster oviductal sperm colonization. since severity of endometrial inflammation is directly related to number of sperm and duration of uterine-sperm interaction, we hypothesized that uterine lavage prior to conventional 4 hours would have an effect on uterine inflammation. objectives were to determine degree of inflammation at 1 and 4 hours after insemination in mares bred by dhai and determine pregnancy in mares lavaged earlier than 4 hours. four warmblood and 3 thoroughbred mares with normal reproductive histories were used over 14 estrous cycles in a cross-over experimental design. mares with obvious uterine edema and a dominant preovulatory follicle were induced to ovulate with 1 mg of deslorelin im. within o 6 hours after ovulation, mares were bred by dhai with 1 x 0.5 ml straw of frozen-thawed semen from 1 of 2 stallions of proven fertility and containing 120 160 x 106 total sperm. on the first estrous cycle, mares were randomly assigned for lavage at 1 or 4 hours following dhai. mares were rested for a cycle and assigned to opposite group. at lavage, mares were examined for presence and degree of endometrial edema. presence of uterine fluid and effluent samples were analyzed for presence or absence of pmn's, and pmn numbers. pregnancy diagnosis was conducted at 12 14 days following dhai. uterine semen contact time of 4 hours versus 1 hour had a significant effect on degree of uterine edema (p < 0.046), presence or absence of pmns (p < 0.0009), and pmn numbers (p < 0.0006), but not on amount of accumulated uterine fluid (p > 0.5) or pregnancy rate (p = 0.28). we concluded that presence of sperm in uterus of normal mare for 1 hour elicited lesser inflammatory reaction compared to 4 hours and pregnancy rate was not affected by early lavage in mares bred by dhai. keywords: endometritis, deep horn insemination, uterine lavage, pregnancy rate 364 clinical theriogenology • volume 12 number 3 • september 2020 inflammatory proteins as novel diagnostic biomarkers for endometritis in mare jennine lection,8 bettina wagner,b andrew miller,c tracey chenier,d soon hon cheong,8 mariana diel de amori.m8 8department of clinical sciences, ~epartment of population medicine and diagnostic sciences coepartment of biomedical sciences, college of veterinary medicine, cornell university, ithaca, ny ddepartment of population medicine, ontario veterinary college university of guelph, guelph, on canada equine endometritis is a costly, prevalent, and challenging disease for equine practitioners to diagnose, with clinical implications ranging from subfertility to infertility. currently, gold standard diagnostic test is endometrial biopsy subjected to kenney-doig grades; however, time to receive results can be variable. objective was to test for inflammatory proteins that are previously validated as equine diagnostic biomarkers in uterine low volume lavage (l vl) fluid of mares. we hypothesized that these inflammatory proteins would substantially increase in l vl fluid of mares with documented evidence of endometritis or poor biopsy grade. light breed mares (n = 29) 3 22 years old were used. each mare had an l vl (250 ml) followed by endometrial biopsy. endometrial cytology was performed from l vl and biopsy evaluation was graded by a board-certified veterinary pathologist. mares were either assigned to endometritis group (n = 12), or healthy group (n = 17) based on endometrial cytology(> 1 % pmns), and/or poor endometrial biopsy score (iib/iii). l vl was utilized in a multiplex bead assay (luminex corp. austin, tx) to quantify levels of following biomarkers: ifny, ill~, ill0, ill 7, scd14, tnfa, chemokine (c-c motif) ligand 2 (ccl2), ccl3, ccl5, and ccll 1. data were analyzed by stata (college station, tx). since data were not normally distributed, a mann-whitney u test was performed to compare levels of inflammatory markers between healthy and endometritis mares, with significance set at p value< 0.05. following inflammatory markers were higher in lvl from mares with endometritis compared to healthy mares: ifn-y (p = 0.0094), ill 7 (p = 0.0296), ccl2 (p = 0.0196), and ccl3 (p = 0.0118). these 4 biomarkers are all proinflammatory cytokines, which orchestrate response of various leukocytes to endometrial changes. further studies with a larger population are warranted; however, we suggest that proinflammatory markers identified may serve as potential diagnostic markers for endometritis in equine uterine fluid samples. use of lvl samples for detection of inflammatory biomarkers could provide a less invasive and efficient diagnostic test to practitioners during busy breeding season. keywords: biomarkers, endometritis, equine, low volume lavage clinical theriogenology • volume 12 number 3 • september 2020 365 oxytocin-induced secretion of 13,14-dihydro-15-keto-prostaglandin f2ain mares with prolonged corpus luteum function brendan samecky, dirk vanderwall, holly mason, caleb reichhardt, benson ambrose department of animal, dairy and veterinary sciences, school of veterinary medicine utah state university, logan, ut oxytocin treatment to prolong corpus luteum (cl) function for estrus suppression in mares is replacing use of intrauterine glass ball. most common oxytocin protocol involves 60 iu of oxytocin treatment given intramuscularly once daily from days 7 14 postovulation (day 0). about 70% of treated mares had prolonged cl function lasting for 60 90 days. however, a longer duration of cl function would be more favorable. serial treatment of human chorionic gonadotropin (hcg) during oxytocin-induced prolonged cl function would extend duration of cl function by having a luteotropic effect and/or by inducing ovulation of diestrous follicle(s) resulting in new cl(s) formation that would remain functional for additional 60 90 days (i.e. 120 180 days of cl function). 1 although diestrous ovulations were documented during prolonged cl function period, there was no difference between control and hcg-treated mares in duration of cl function. notably, in some mares, progesterone concentrations decreased abruptly after a diestrous ovulation, suggesting return of endogenous luteolytic mechanism responsible for terminating cl function. objective was to evaluate the ability of endometrium to secrete prostaglandin f2a (pgf2a) during oxytocin-induced prolonged cl function. oxytocin treatment was used to prolong cl function in mares that were subsequently assigned to 1 of 3 groups: 50 59 days (n = 4), 60 69 days (n = 4), or 70 79 days (n = 3); groups designated as 50s, 60s, and 70s, respectively). day14 mares served as control group (n = 3). endometrial secretion of pgf2a was evaluated by measuring systemic concentrations of 13,14-dihydro-15-keto-prostaglandin f2a(pgfm) following a single 10 iu intravenous oxytocin bolus (time 0) treatment to mares in all groups. blood samples were collected at -30, -15, 0, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, and 120 minutes. area under pgfm curve was higher in 70s group compared to 50s (p < 0.001) and 60s groups (p < 0.02), with no difference between 70s and control groups. cl function was maintained after oxytocin bolus in all 4 50s mares, 3 of 4 60s mares, and o of 3 70s mares. these results provided clear evidence for return of uterine luteolytic mechanism between days 50 70 in mares with prolonged cl function. keywords: equine, mare, estrus suppression, oxytocin, corpus luteum, pgfm reference 1. vanderwall dk, kinney dl, mason h, et al: effect of human chorionic gonadotropin (hcg) treatment on the duration of oxytocin-induced prolonged corpus luteum (cl) function in mares. clinical toeriogenology 2017;9:445. 366 clinical theriogenology • volume 12 number 3 • september 2020 pharmacodynamics of clomiphene citrate in cyclic mares cory anderson,3 candace lyman,3 carlos pinto,b adam bassett,3 reed holyoak3 3college of veterinary medicine, oklahoma state university, stillwater, ok bcollege of veterinary medicine, college of veterinary medicine, baton rouge, la clomiphene citrate (cc), a selective estrogen receptor modulator (serm), is an orally administered reproductive drug that improved follicular recruitment and development primarily in human but also in bovine, ovine, and lagomorph species. experimental data from mare after cc administration are slim; however, a 40 year-old, anecdotal case report describes some success after use of cc in 3 an.estrous mares. we hypothesized that administration of cc in mare would result in an increased number of ovarian follicles. objectives were to quantify amh, lh, and fsh serum concentrations, to characterize endometrial edema, and changes in follicular dynamics between treated and control mares. light-breed mares (n = 12) of 6 18 years were utilized from mid-july to early august. mares (n = 6) had oral treatment of cc (500 mg) once daily for 5 consecutive days and control mares (n = 6) had oral placebo. blood samples for amh concentrations were collected on days 1, 3, 6, and 9. blood samples for fsh and lh concentrations were collected prior to first dose of drug and then once daily through treatment period. transrectal ultrasonography and follicle mapping were performed in both treated and control mares. statistical analyses were done using two-way repeated measures anov a, with p :'.s 0.05. administration of cc did not induce changes in follicle numbers (p > 0.05). plasma amh concentrations did not differ between experimental groups; however, mean concentrations relative to time were different (p = 0.009). there were no differences for plasma fsh or lh concentrations, however, daily mean plasma lh varied (p < 0.001) with time within experimental groups. endometrial edema scores were higher (p = 0.041) in mares treated with cc than in control mares, after allowing for time effect. in conclusion, daily administration of cc for 5 consecutive days did not affect follicle numbers or elicit substantial endocrine response. in humans, efficacy of cc treatment in correcting ovulatory dysfunction derives from its blocking effect on hypothalamic estrogen receptors. that, in turn, causes a significant release of fsh and lh. in contrast, in the present study, cc treatment had substantial estrogenic effects in uterus by inducing endometrial edema. thus, it remains to be elucidated whether cc in mare has ability to modulate fsh and lh secretion. pharmacokinetics analyses of blood samples are pending, and could provide a basis for revised protocols for cc administration. keywords: equine, lh, fsh, amh, estrogen, follicle dynamics clinical theriogenology • volume 12 number 3 • september 2020 367 effect of mycobacterium cell wall fraction on histological, immunological, and clinical parameters of equine uterine involution carleigh fedorka,8 harutaka murase,8 shavahn loux,8 alan loynachan,8 olivia walker,b ed squires,8barry ball,8 mats troedsson3 3department of veterinary sciences, university of kentucky, lexington, ky bcollege of veterinary medicine, lincoln memorial university, harrogate, tn maintaining yearly foal production is important for economic success of broodmares and requires breeding to occur as quickly postpartum as possible. first estrus occurs within 5 20 days postpartum, during uterine involution uterus (repair from tissue alterations of pregnancy and parturition). attempts to hasten uterine involution have had minimal success. mycobacterium cell wall fraction (mcwf), an immunomodulator, reduced bacterial growth and altered aspects of immune response to breeding; however, it is unknown if mcwf hastened uterine involution. objectives were to: 1) investigate effect of mcwf on tissue remodeling; and 2) assess effect ofmcwf on cell-mediated immunity of uterus. we hypothesized that mcwf treatment in postpartum would hasten uterine involution. pregnant mixed breed mares (n = 10) were evaluated postpartum. control mares (n = 4) received 1.5 ml lrs intravenously on day 1 postpartum and again 7 days later and experimental mares (n = 6) received 1.5 ml settle® (mcwf) intravenously on day 1 and again 7 days later. all mares were assessed every 3 days for clinical, immunological, and histological parameters until day 15 postpartum. clinical parameters were assessed via transrectal ultrasonography and included assessment of ovarian activity, uterine fluid retention, and uterine wall diameter, in addition to obtaining endometrial culture. immunological parameters included endometrial biopsies for qpcr for expression of various cytokines (il1(3, illrn, il4, il6, il8, ill0, tnf, ifny, and gm-csf) and endometrial cytology. histological parameters were assessed on formalin-fixed endometrial biopsies and variables included retention of microcotyledons, endometrial glands dilation, and inflammation of mucosa, stratum compactum, and spongiosum. data were analyzed using sas 9.4, utilizing a mixed model for repeated measures, with treatment as a random effect. all posthoc analyses used tukey's hsd test. involution was considered complete by day 15 postpartum in all mares, and day postpartum had significant effect on all parameters investigated, indicating involvement of immunological process in uterine involution. treatment with mcwf decreased severity of bacterial growth, in addition to time to obtain clean culture. mcwf treatment increased expression of proinflammatory cytokines, namely, il1(3, ifny, and tnf. whereas treatment effect was minimal, there was histological evidence for decreased mucosal inflammation in mcwf-treated mares. in conclusion, uterine involution is heavily regulated by immune system. additionally, mcwf had bactericidal effect on postpartum mare; this may be due to increases in proinflammatory cytokine expression. further studies are needed to determine whether this immunomodulator will improve first estrous cycle fertility in postpartum mares. keywords: mare, uterine involution, mycobacterium cell wall fraction, postpartum, immunomodulator 368 clinical theriogenology • volume 12 number 3 • september 2020 transcriptomic analysis of equine chorioallantois reveals key regulators and pathways involved in ascending placentitis hossam el-sheikh ali, barry ball gluck equine research center, university of kentucky, lexington, ky although placentitis is the costliest disease in equine industry, its current standard treatment is not satisfactory and progress is slow in identifying efficient placentitis prevention, diagnostics, and treatment protocols. therefore, a faster, more holistic view of molecular mechanisms underlying equine placentitis holds potential for development of new diagnostic tools and therapies to forestall placentitis-induced preterm labor. our hypothesis was that characterizing transcriptome of equine chorioallantois (ca) during placentitis in comparison to pregnancy matched controls would elucidate key regulators and molecular mechanisms triggering equine placentitis. chorioallantois samples were collected after euthanasia at 290 days of pregnancy in mares with experimentally induced placentitis (placentitits group, n = 6) and uninoculated mares ( control group, n = 6). next generation rna sequencing was performed with illumina novaseq6000 and reads mapped to equcab3.0 (star-2.5.2b). differentially expressed genes (degs) were identified with cuffdiff-2.2.1 (fdr< 0.05). our study identified 2953 degs (2028 upregulated and 925 downregulated) in ca during placentitis compared to controls. pathway analysis of degs revealed that these genes were involved in relevant pathways, such as inflammatory signaling (inflammation mediated by chemokine and cytokine signaling pathway, interleukin signaling, toll-like receptor signaling, t cell activation, and b cell activation), angiogenesis­ related pathways (vascular endothelial growth factor signaling and angiogenesis pathway), cytostructural integrity (integrin signaling pathway), and apoptosis signaling pathway. upstream regulator analysis revealed central role of toll-like receptors (tlr2, tlr3, tlr5, and tlr7) in triggering inflammatory signaling after placental infection, and this consecutively resulted in placental inflammation and immune cells chemotaxis. increased leukocytic infiltration in ca was associated with upregulation of matrix metalloproteinase (mmp 1, mmp2, and mmp9) with subsequent extracellular matrix (ecm) degradation and apoptosis, as reflected by upregulation of several apoptosis-related genes such as caspases (casp3, casp4, casp7, and casplo) that are believed to be implicated in placental separation during disease course. additionally, activation of tlrs was associated with upregulation of a wide array of transcription factors and tlrs dependent downstream molecules in the inflammatory cascade. this in turn was associated with downregulation of transcripts coding for proteins essential for placental steroidogenesis (e.g. srd5al, akrlcl), angiogenesis (e.g. vegfa), nutrient transportation (e.g. glut12), and upregulation of hypoxia-related genes (e.g. hifia) that could explain placental insufficiency during placentitis. first time key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during equine placentitis were characterized. findings might lead to development of efficacious therapies by targeting key molecules and pathways. keywords: pregnancy, placentitis, chorioallantois, mare, rna-sequencing clinical theriogenology • volume 12 number 3 • september 2020 369 comparison of glucose and lactate concentrations between healthy equine and mule foals, and of an automated laboratory analyzer and accutrend® plus system yatta boakari,a maria alonso,b amanda riccio,b fernanda affonso,b joao losano,b marcilio nichi,h claudia fernandesb 3department of clinical sciences, college of veterinary medicine, auburn university, auburn, al hoepartment of animal reproduction, school of veterinary medicine and animal science university of sao paulo, sp, brazil mules are hybrids resulting from breeding between female horses (equus cabal/us) and male donkeys (equus asinus). information about physiological adaptation of newborn mules is scarce in literature. profiles of blood glucose (gluc) and lactate (lact) concentrations are important to evaluate neonates, as they indicate metabolic dysregulations, disease severity, and prognosis. thus, fast and affordable gluc and lact evaluations at farms using portable analyzers would be valuable. additionally, it is important to know the pattern of variation of these parameters to differentiate healthy and compromised equid neonates. we hypothesized that portable devices measure glucose and lactate concentrations accurately, that these parameters differ between equine and mule foals, and vary over time. aims of this study were to: 1) compare gluc and lact blood concentrations between equine and mule foals during their first 720 hours of life; 2) evaluate lact and gluc profiles from birth to 720 hours of life; and c) establish the correlation oflact and gluc concentrations using accutrend® plus system (acp) with whole blood and randox daytona automated analyzer (auto) with plasma in both species. healthy equine (n = 16) and mule foals (n = 15) were used and blood samples were collected immediately (to), 1 (tl), 6 (t6), 12 (t12), 24 (t24), 168 (t168), and 720 (t720) hours after birth. gluc and lact concentrations were evaluated with an auto and with an acp. data were analyzed by repeated measures using proc mixed and intraclass coefficient correlation (icc) between 2 analyzers was calculated. overall, gluc concentrations evaluated with auto were different between species; lact concentrations were higher (p < 0.05) in equine foals when compared to mule foals (2.14; 1.01 5.99 and 1.83; 0.61 9.04 mmol/1, respectively). there was no difference in gluc and lact results evaluated with acp for equine and mule foals. pattern of variation of gluc evaluated with both analyzers for equine and mule foals changed during first 720 hours of life, with an increase in gluc concentrations soon after nursing and then a decrease to baseline levels. lact pattern of variation was similar for all foals, with both analyzers detecting higher lact values at to (4.05; 1.91 9.04 mmol/1), with a progressive decrease until t720 (1.21; 0.61 5.88 mmol/1). the icc for equine and mule foals between auto and acp for gluc concentrations was low and was moderate for lact concentrations (icc < 0.90). we concluded that gluc and lact concentrations changed during first 720 hours after birth in mule and equine foals, with gluc values differing between species. additionally, we recommend standardization of the acp with specific samples before clinical use for gluc and lact analysis. keywords: neonate, hybrid, equine, hematological parameters, critical ill, prognosis acknowledgement supported by coordination for the improvement of higher education personnelbrazil (capes)-funding number 001 and the sao paulo research foundation (fapespproject 2017/05425-0) 370 clinical theriogenology • volume 12 number 3 • september 2020 effect of ejaculation frequency, prostaglandin f2a and cold storage on canine semen yield and postthaw quality kendra zelachowski, erin runcan, bryan blawut, marco coutinho da silva department of veterinary clinical sciences, college of veterinary medicine the ohio state university, columbus, oh number of sperm obtained from a single semen collection can vary widely among dogs, often requiring multiple ejaculates to produce a single breeding dose of cryopreserved sperm. alternative collection techniques and schedules have been reported to increase sperm yield, however, a direct comparison of these methods is warranted. objective of this study was to determine most effective collection regimen to increase total number of sperm available for cryopreservation without compromising postthaw motility parameters. we hypothesized that multiple collections and administration of prostaglandin f2a (pgf2a) could be used to increase sperm yield without detrimental effects to postthaw semen quality. sexually mature male dogs (n = 5) were all subjected to following 5 collection protocols in a random order: 1) 1 collection ( control); 2) 2 collections 1 hour apart; 3) 2 collections 24 hours apart; 4) 2 collections 48 hours apart; and 5) 1 collection 20 minutes after pgf2a (dinoprost, 0.05 mg/kg, im). total sperm count, total motility (tm) and progressive motility (pm) were assessed for each ejaculate using computer assisted sperm analysis (casa). semen was either frozen immediately after collection (protocols 1 and 5) or stored at 4°c, pooled with a second ejaculate (protocols 2, 3, and 4), and then frozen using a tris-egg yolk media. dogs were allowed 7 days of sexual rest between protocols. frozen samples were evaluated using casa and flow cytometry to determine tm, pm, viability (via) and acrosome integrity (acr). data were analyzed using a general linear mixed model and tukey's posthoc for pairwise mean comparisons. significance was set at p < 0.05. all collection protocols resulted in a higher sperm yield compared to control. postthaw semen parameters (presented in percent) were similar between control and protocols 2 and 5 (tm: 50.4 ± 5.1, 49.4 ± 5.1, 53.8 ± 5.1, pm: 36.8 ± 4.7, 35.4 ± 4.7, 40.2 ± 4.7, via: 61.9 ± 3.4, 65.3 ± 3.4, 61.7 ± 3.4, acr: 23.2 ± 2.7, 18.1± 2.7, 20.4 ± 2.7), respectively. protocol 4 resulted in poor postthaw semen parameters (tm: 30 ± 5.1, pm: 20.4 ± 4.7, via: 49.8 ± 3.4, acr: 29.8 ± 2.7). protocol 3 had intermediate results for all semen parameters (tm: 37.2 ± 5.1, pm: 24.4 ± 4.7, via: 55.2 ± 3.4, acr: 26.6 ± 2.7). in conclusion, 2 collections performed 1 hour apart with 1 dose of pgf2a dramatically increased sperm yield and also maintained postthaw quality. cooled storage before cryopreservation was increasingly detrimental with increasing time between collections. we provided 2 alternative semen collection protocols to practitioners to maximize number of breeding doses cryopreserved in 1 visit. further, this would enhance client satisfaction while also minimizing costs and veterinary visits. keywords: canine, spermatozoa, yield, cryopreservation, prostaglandin clinical theriogenology • volume 12 number 3 • september 2020 371 seroprevalence of canine brucellosis in the southeastern united states alyssa helms,3 clayton caswell,b kevin lahmers/ orsolya balogh,8 julie cecerea adepartment of small animal clinical sciences bdepartment of biomedical sciences and pathobiology virginia-maryland college ofveterinary medicine, blacksburg, va there are four brucella strains of zoonotic importance: smooth strains, i.e. brucella abortus (b. abortus), b. melitensis, b. suis, and a rough strain, i.e. b. canis . dogs can serve as hosts for all four zoonotic strains; however, standard serologic testing in dogs is limited to the identification of only b. canis antibodies. well known to be transmitted venereally, bruce/la are also readily shed in urine, feces, saliva, milk, and respiratory secretions, and can be easily transmitted through close contact. there is no approved treatment for the elimination of bruce/la from infected animals. human infection is a nationally notifiable condition, as treatment is not always effective in clearing the organism, resulting in recurrent infection. therefore, preventing spread of disease is paramount for public health. the goal of this pilot study was to estimate the number of dogs with circulating antibodies to any of the four zoonotic strains of bruce ila by sampling multiple subpopulations of hog hunting dogs, dogs presenting for routine spay and neuter, and imported animals throughout the southeastern united states. we hypothesized that the seroprevalence of smooth strains of brucellosis would be higher in imported and hog-hunting dog populations than dogs presenting for routine spay and neuter, and that the seroprevalence of the rough strain of brucellosis (b. canis) would be similar across all groups. to date, serum has been harvested from 65 client owned dogs (30 hog hunting and 35 companion dogs) and 19 shelter owned dogs of various ages, parities, and breeds. reproductive tissues, semen and/or vaginal swabs were collected when available. brucellosis serology was performed using the canine bruce/la slide agglutination test (sat), bruce/la canis agar gel immunodiffusion ii test (agid), bruce/la abortus card agglutination test (cag), and bruce/la abortus fluorescence polarization assay (fpa). to date, among 84 samples collected and assayed, one shelter dog tested positive for a rough brucella strain via agid. an imported dog tested positive for both a rough and a smooth strain of brucellosis via sat, agid and fp a, and two client owned dogs tested positive for a smooth brucella strain by a national veterinary services accredited laboratory via b. abortus plate and card agglutination. three attempts at culturing the organism from reproductive tissue samples of seropositive animals yielded contaminant growth only. all other samples (n = 80) were seronegative. although this project is still ongoing, at this time, 4 of 84 samples (5%) tested positive for at least one strain of brucella, with 3 of 4 positive samples (75%) testing positive for a smooth brucella strain. all hog-hunting dogs sampled were seronegative. our preliminary results support an overall low seroprevalence rate for brucellosis, with most samples testing positive for a smooth brucella strain, thus, supporting the need for a commercially available, validated test for the smooth strains in canines. to the authors' knowledge, this is the first study evaluating the seroprevalence of the smooth strains of brucellosis in dogs in the us. keywords: canine brucellosis, rough strains, smooth strains, seroprevalence 372 clinical theriogenology • volume 12 number 3 • september 2020 effect of polyacrylamide hydrogel injection into reproductive tract of mares stephanie walbornn,8 jamie kazcor,b christine bartley,b etta bradecamp,b charlie scoggin,b maria schnobrichb 8rood and riddle equine hospital in wellington, wellington, fl brood and riddle equine hospital, lexington, ky urogenital problems are common in older, multiparous broodmares. vesicovaginal reflux (vvr) or "urine pooling" is a condition where urine refluxes from urethra and accumulates within cranial vagina. currently, treatment for this condition involves medical management and surgery. caudal relocation of transverse fold or extension of urethra are surgical procedures utilized to treat vvr. currently, injectable therapies designed to bulk tissue surrounding transverse fold have not been utilized to treat vvr. use of a polyacrylamide hydrogel to alter vestibulovaginal fold anatomy may be an adjunctive treatment option for vvr in mares. polyacrylamide hydrogel is a nontoxic and nonimmunogenic biocompatible, nonabsorbable polymer gel that consists of 2.5% cross linked polyacrylamide and 97.5% sterile water.' 1bis compound has been utilized safely for soft tissue augmentation and urinary incontinence in women for several years.2 objective was to evaluate synamid® (contura international ns, sydmarken 23, 2860 soeborg, denmark) injection safety into vestibulovaginal mucosa of normal mares. ten mares ( aged 7 15 years) were utilized. prior to procedure, physical examinations and thorough reproductive evaluations were performed. subjects were administered 3 ml of saline and 3 ml of synamind® in separate sites, randomly assigned to 1 cm to either right or left of midline (urethra) in transverse fold. mares underwent daily physical examination, transrectal palpation and ultrasonography, vaginal speculum examination, and digital examination of vagina for first 7 days, then at days 15 and 30. daily physical examinations revealed no systemic abnormalities in all mares treated. hematoma formation at injection site occurred in 7 /10 mares, which was observed starting at 24 hours postinjection. hematomas persisted for an average of 3 days. no pain was elicited on palpation ofvestibulovaginal fold in any mares. a palpable bulge was present on synamid® injection site for 30 days in 9/10 mares. in conclusion, no severe complications were observed with injection of synamid® into transverse fold in mares. there is a potential for this long lasting product utilization in "urine pooling" mares to augment vestibulovaginal anatomy and in mares with issues ofvulvar incompetence. further research is needed to determine this product's efficacy in treating these conditions. keywords: mare, urine pooling, polyacrylamide hydrogel references i. broder k, cohen s: an overview of permanent and semipermanent fillers. plast reconstr surg 2006;118(3 suppl):7s-14s. 2. kasi ad, pergialiotis v, perrea dn, et al: polyacrylamide hydrogel (bulkamid®) for stress urinary incontinence in women: a systematic review of the literature. int urogynecol j 2016;27:367-375. clinical theriogenology • volume 12 number 3 • september 2020 373 uterine expression of leptin, rhoa and rho associated kinases in dogs with primary uterine inertia and with obstructive dystocia bianca frehner,3 iris reichler,8 mariusz kowalewski,b stefanie keller,3 *sandra goericke-pesch,c *orsolya balogha,d 3clinic of reproductive medicine, "institute of veterinary anatomy vetsuisse faculty university of zurich, zurich, switzerland 'reproductive unit of the clinics clinic for small animals university of veterinary medicine hannover, foundation, germany ddepartment of small animal clinical sciences virginia-maryland college of veterinary medicine, blacksburg, va *contributed equally primary uterine inertia (pui) is the most frequent dystocia in the bitch, but its etiology is still unclear. knowledge about underlying functional and molecular changes in the uterus is very scarce. obesity and high concentrations of leptin (major adipose derived hormone) are associated with complications of labor in women, and obese bitches are at increased risk of dystocia. in pregnant dogs, leptin is also produced by the uterus and placenta, so it may exert a negative paracrine/autocrine effect on uterine contractility during parturition. we hypothesized that high uterine leptin concentrations would adversely affect rhoa/rho kinase pathway, which is involved in calcium sensitization, leading to decreased uterine contractions in dogs with pui. bitches presenting with dystocia (n = 18) were assigned to pui (n = 11) or obstructive dystocia (od) group (n = 7). dogs in od group were still showing strong contractions and were designated as controls. dogs that received ecbolic or tocolytic medication were excluded. during cesarean section, a full thickness uterine biopsy was collected at an inter-placental site. relative gene expression (rge) oflep, lep receptor (lepr), rhoa and its effector kinases (rockl, rock2) was analyzed by real-time taqman qpcr. immunohistochemistry or in situ hybridization was used for protein or mrna localization, respectively. one-way anova was used to compare uterine rge between groups with body weight as covariate. statistical significance wasp < 0.05. dogs were not obese (body condition score recorded for 14 dogs 3-6/9). lep expression did not differ between groups. lepr mrna levels were below detection limit in 5 pui dogs and in all od dogs. rhoa expression did not differ, but uterine rockl and rock2 mrna levels were higher in pui dogs (p = 0.010 and p = 0.039, respectively). lep, rhoa, rockl, rock2 protein, and lepr mrna were all localized in myometrium, and staining intensity appeared similar between groups. protein and mrna signals were also present in endometrium and blood vessels. in conclusion, despite similar uterine leptin gene expression, undetectable lepr mrna levels in od dogs might indicate decreased responsiveness to potentially negative effects ofleptin. however, rockl and rock2 gene expression was lower in od than in pui dogs, which may be a physiological result oflong-lasting, strong uterine contractions, and as such, could be an indication of abnormal progression oflabor and insufficient uterine contractions in dogs with pui. keywords: uterine inertia, parturition, uterus, contractility, canine acknowledgement financial support by agria and skk research foundation (no. n2014-0002) 374 clinical theriogenology • volume 12 number 3 • september 2020 efficacy of a silicone y design intrauterine device as horse contraceptive in captive breeding cory anderson,8 reed holyoak,8 candace lyman,8 steve germaine,b adam bassett,8 julia baldrighi,8 grant rezabek,8 shuodao wang,c al kaned 3college of veterinary medicine, oklahoma state university, stillwater, ok ~s geological survey, fort collins, co cschool of mechanical & aerospace engineering, oklahoma state university, stillwater, ok dusda animal and plant health inspection service, fort collins, co due to continued population growth on western rangelands in us, there are now ~ 70,000 feral horses administered to by bureau for land management (blm) in an area where range ecologists have established 27,000 as desired population size. therefore, need for an effective contraceptive for feral horses on western range remains a blm priority. goal was to find a means of reducing unwanted pregnancies within us feral horse population, while maintaining reversibility so that genetic diversity can be maintained within population. multiple o ring designs were deemed unsuccessful due to their inability to resist uterine expulsatory forces in face of immediate and subsequent breeding behavior. we hypothesized that coadministration oflong acting progesterone at iud insertion would increase iud retention by preventing these immediate aftereffects. furthermore, discovering that a fundus seeking design in human iuds ameliorates normal uterine expulsion forces, we formulated an iud 'y' design to test for a desired minimum::'.: 75% retention rate. this current study compared 50 and 60 durometer (an indicator of material hardness) 'y' design iuds, using 2 breeding pods of 10 mares and 1 stallion in each group. in each pod, 5 mares received a 50 durometer iud and 5 mares received a 60 durometer iud. every other mare received an im injection of saline or long acting p4. after early data indication that 60 durometer 'y' shaped iuds had a higher retention rate, 50 durometer iuds were replaced with 60 durometer iuds. over next 2 years, all mares in both breeding pods were then monitored by transrectal ultrasonographic examination every 2 weeks to determine iud retention and pregnancy rates for those mares that lost their iuds. endometrial biopsies were obtained, and a kenney biopsy grade assigned, before and after research period in efforts to assess iud impact on endometrial health. five of 20 mares expelled their iud (75% retention rate) with each of these 5 mares becoming pregnant. seven of 20 had no change in kenney biopsy grade, 4 mares worsened by 1 grade, 5 mares worsened by more than one grade, and 4 mares improved by 1 grade. after 4 cycles following removal of iuds, 12 mares became pregnant. based on histopathologic and pregnancy results, mares that failed to become pregnant not due to a worsening endometrial grade but possibly due to involvement of other intrinsic factors. y design iud met intended parameters and achieved study goals. keywords: intrauterine device, feral horse, y design, endometrial grade, fertility clinical theriogenology • volume 12 number 3 • september 2020 375 canine brucellosis: serologically diagnosed positive cases soon hon cheong/ julie cecere,b isabelle schweitzer,c rebecca frankin-guild,c elisha frye,c linda mittel,c erin goodrich,c craig atier,c mariana diel de amorim8 8department of clinical sciences, college of veterinary medicine, cornell university, ithaca, ny bdepartment small animal clinical sciences virginia-maryland college of veterinary medicine, virginia tech, blacksburg, va cdepartment of population medicine, college of veterinary medicine, cornell university, ithaca, ny canine brucellosis is a devastating disease as it carries zoonotic potential and there are no effective treatments to clear infections in affected animals. thus, animals that test positive are commonly euthanized. it is thought to primarily affect intact animals and can be readily transmitted through oral or venereal route. in past few years, number of inquiries for advice on managing positive animals has increased sharply. goal was to evaluate serologically positive bruce/la canis submissions. we hypothesized that number and proportion of positive cases would vary by year, source of submission, and patient demographics. results from samples submitted for b. canis slide/agar gel immunodiffusion (agid ii) tests to cornell animal health diagnostic center from 2014 2019 from us were included in this retrospective study. submissions were classified as either from a referral laboratory or from a veterinarian. patient demographics (age, breed, and gender), source (referral laboratory or veterinarian, and state/region), and year were evaluated for association with positive serological diagnosis using logistic regression analysis sas (v9.4) proc logistic. all variables tested were associated with positive serology (p < 0.0001) and were retained in final model. number and proportion of positive cases were: 231 (16.8%) in 2014, 199 (10.3%) in 2015, 232 (11.4%) in 2016, 217(11.8%) in 2017, 414 (14.8%) in 2018, and 293 (10.0%) in 2019. referral laboratory submissions (n = 882; 17.4%) had more positive submissions compared to veterinarian submissions (n = 704; 9.0%). animals between 1 6 years of age and mix breeds were more likely to be serologically positive compared to pure breeds. most (83.0%) submissions were from intact animals, but proportion of positive animals was higher in spayed (33%) versus intact females (9%) and castrated (28%) versus intact males (8%). number and proportion of serologically positive submissions were significantly different among south (14%), west (1 2%), midwest (9%), and northeast (3%) regions. many submissions were likely prescreened by card agglutination test; thus, results should be interpreted with this in mind. years 2018 and 2019 had higher number of positive submissions compared to 2014 2017. submissions from young, mixed-breed animals were more likely to test positive, and intact animals had higher numbers but lower proportions of positive cases. regional difference was also observed. we recommend testing breeding animals and semen to reduce spread of disease. keywords: canine brucellosis, agid ii test 376 clinical theriogenology • volume 12 number 3 • september 2020 use of intravaginal progesterone releasing device and estradiol 17p is equivalent to estradiol l 7p and long acting progesterone in synchronizing acyclic embryo surrogate mares lorenzo segabinazzi,a.b luiz andrade jr.,8 igor canisso,h eunice oba,8 frederico papa,8 jose dell' aqua jr.,8 marco alvarenga8 8sao paulo state university, botucatu, sp, brazil ~niversity of illinois, urbana, il long-acting progesterone (la p4) is the most common hormone used to synchronize acyclic surrogate mares for embryo transfer (et). however, mares synchronized with la p4not used for et for various reasons ( e.g. donor yielding a negative flush or nonviable embryo) do not respond to a resynchronization in a short time, likely due to residual la p 4 in plasma. this results in economic losses to et programs due to days that the surrogate mare is not being utilized. intravaginal progesterone releasing devices (iprd) for cattle have been used in transitional mares to hasten cyclicity. however, iprd has not been tested to timely synchronize et surrogate mares. this study aimed to assess serum progesterone (p4) concentrations, pregnancy rates after et and pregnancy losses of embryo surrogate mares synchronized with iprd. in experiment 1, crossbred acyclic mares (n = 12) received estradiol l 7p ([17 peta, botupharma, brazil] 10, 20 and 10 mg, respectively, on 3 consecutive days. at 24 hours after the last estradiol injection, an iprd (sincrogest, ouro fino, brazil) containing 1 g of natural p4, was inserted vaginally and kept for 9 days. blood samples were collected daily for 11 consecutive days and p4 concentrations assessed using ria. experiment 2 was conducted for 2 breeding seasons (2016 2018), with crossbred embryo surrogate mares, randomly assigned to 3 groups: 1) cyclic mares (n = 43) having ovulation confirmed after induction (follicle 2'.: 35 mm) with histrelin acetate (250 µg, im, strelin, botupharma); 2) acyclic mares (n = 57) treated with estradiol l 7p for 3 days (as above) and then given a single dose of la p4 (p4-300, 1.5 g botupharma); or 3) acyclic mares (n = 57) treated with estradiol l 7p for 3 days and then given an iprd (sincrogest). day-8 embryos were transferred 4 8 days after ovulation or p 4 treatments, using a nonsurgical, transcervical technique. mares in group 3 had iprd removed immediately before et, and immediately after et, a new iprd was inserted. pregnancy diagnosis was performed at 5 days after et, and 30 and 60 days later. once pregnancy was first confirmed, mares in groups 2 and 3 received weekly injections of la p4 (1.5 g) until 120 days of pregnancy. mares in group 3 had the device removed 3 days after pregnancy diagnosis. data on p 4 concentrations were analyzed using anova-rm and tukey posthoc and data on pregnancy rates and losses were compared by multivariate logistic regression (p < 0.05). in experiment 1, p4 concentrations significantly increased from iprd insertion (0.5 ± 0.2 ng/ml) to next day (15.8 ± 3.1 ng/ml), and then maintained satisfactory p 4 concentrations deemed suitable for successful establishment of pregnancy until day 9 (7.7 ± 2.3 ng/ml). after iprd removal, p4 concentrations were reduced significantly to baseline (0.78 ± 0.4 and 0.67± 0.3 ng/ml on days 10 and 11, respectively). for groups 1-3, there was no difference in pregnancy rates (64, 66, and 69%, respectively) or pregnancy losses by 60 days of pregnancy (17, 13, and 10%). in conclusion, iprd used herein resulted in a rapid increase in p4 and sharp decline, upon its insertion and removal, respectively. in addition, this device can be used as a compatible alternative to la p4 to synchronize acyclic embryo surrogate mares. keywords: equine, recipient mare, embryo transfer, fertility, hormonal therapy clinical theriogenology • volume 12 number 3 • september 2020 377 effect of age on follicle stimulating hormone receptor expression in ovine endometrium hayder habeeb, a logan kleditz, b michelle kutzlerc adepartment of animal production, al-qasim green university, ba, iraq bcollege of engineering, oregon state university, corvallis, or cdepartment of animal and rangeland sciences, oregon state university, corvallis, or follicle stimulating hormone receptors (fshr) are present predominately in granulosa cells of secondary and preovulatory follicles. in addition, fshr are present within ovine endometrium. 1 in humans, ovarian fshr expression decreased with age.2 objective of this study was to compare endometrial fshr expression among ewes < 9 months of age (prepubertal/peripubertal; n = 6), 1.5 2 years of age (mature; n = 4), and > 3 years of age (aged; n = 4). we hypothesized that similar to human ovary, ovine endometrial fshr expression would decrease over time. intracaruncular endometrial samples were collected from crossbred ewes (n =14) at slaughter. age of ewe was determined by dental eruption and wear. ovine ovary served as positive control. samples were fixed in 10% formalin, embedded in paraffin, and sectioned (5 µm) onto charged slides. sections were deparaffinized in xylene and rehydrated in graded ethanol series. heat-induced antigen retrieval was performed with sodium citrate. endogenous peroxidases were blocked with 3% hydrogen peroxide and nonspecific binding was blocked with a serum-free protein block. rabbit antihuman fshr (f-3929, sigma aldrich) was applied to tissues at 1 :200 dilution and universal rabbit negative agent was applied to an adjacent tissue section to serve as control. horseradish peroxidase-conjugated polymer antirabbit igg was used as secondary antibody, followed by treatment with novared™ peroxidase substrate, and counterstained with hematoxylin. slides were dehydrated in graded ethanol series, passed through 3 xylene solutions, and then coverslipped. slides were evaluated by a blinded observer (hh) at 400 x magnification with a leica dm4000b microscope. intensity offshr expression was quantified from no staining (score of 0) to very strong staining (score of 4) for various endometrium compartments from 3 randomly selected fields per ewe.1 data on fshr expression between age groups were analyzed with one-way anov a. significance was defined asp < 0.05. although fshr was expressed in endometrial luminal epithelium, superficial and deep glandular epithelium, superficial and deep blood vessels, and superficial and deep stroma in both age groups, there were no differences (p > 0.05) in expression among age groups. it is not clear what role fshr plays in ovine endometrium, but it may regulate mucin-1 expression that is involved in early pregnancy establishment.3 keywords: immunohistochemistry, intracaruncular, sheep, uterus references i. grazul-bilska at, reyaz a, valkov v, et al: acta histochem 2018;120:420-428. 2. brodowska a, laszczynska m, brodowski j, et al: histol histopathol 2012;27:241-248. 3. constantinou pe, morgado m, & carson dd: adv anat embryo! cell biol 2015;216:51-68. 378 clinical theriogenology • volume 12 number 3 • september 2020 acupuncture reduces milk n-acetyl beta-d-glucosaminidase in dairy cows with mastitis elise ryan,8 michelle kutzlerb adepartment of integrative biology, ~epartment of animal and rangeland sciences oregon state university, corvallis, or acupuncture (ap) is used by dairy veterinarians as a complementary treatment for various conditions. 1 nagase is a lysosomal glycosidase released from mammary epithelial cells during mastitis.2 objective was to determine if ap reduces mammary inflammation. we hypothesized that dairy cattle with subclinical mastitis treated with an intramammary antibiotic and ap would have lower somatic cell count (scc) and milk n-acetyl beta-d-glucosaminidase (nagase) concentrations compared to cattle treated with only an intramammary antibiotic. lactating dairy cows at oregon state university dairy research center were selected ( cows with scc score of sec > 500,000 cells/ml) based on dairy herd improvement association reports. a california mastitis test (cmt) determined the quarter that had high scc (cmt score > 1 ). pretreatment milk samples (pre) were collected for scc scoring and na gase concentrations. scc was measured by delaval cell counter (tumba, sweden) and nagase concentrations were determined by an elisa kit (#mbs090625, mybiosource, san diego, ca). cows were treated with an intramammary infusion of ceftiofur hydrochloride (spectromast lc<i>, zoetis, parsippany, nj) and randomly assigned to ap group (n = 7) and nonap ( control) group (n = 7). both group cows were restrained for 30 minutes in a head catch 4 times, 12 hours apart. six ap points were used (bladder [bl] 30, bl 30-1, bl 49, kidney [ki] 10, conception vessel [cv] 2, cv 3). posttreatment milk samples (post) were collected 14 days after presample collection. pre cmt scores, nagase concentrations, and scc were compared to post scores within and between groups. significance was defined asp< 0.05. both control and ap groups had reduced post cmt scores (pre: 1.41 ± 0.49; post: 0.42 ± 0.66; p = 0.01 and pre: 1.21 ± 0.39; post: 0.57 ± 0.79; p = 0.039, respectively) but there was no significant change in scc in either group. in ap group but not in control group, nagase concentrations were reduced (pre: 19.70 ± 3.65 u/l; post: 16.70 ± 2.05 u/l; p = 0.04 and pre: 25.60 ± 12.90 u/l; post: 20.40 ± 6.04 u/l; p = 0.20, respectively). reduction in nagase provided evidence for ap as a complementary treatment. keywords: complementary treatment, california mastitis test, somatic cell count, subclinical acknowledgement authors thank oregon dairy farmers association and oregon state university's e. r. jackman friends and alumni and continuing researchers program for funding. references 1. riebel k, gentle l, menino a, et al . acupl.lllcture prevents the postpartum reduction in matrix metalloproteinase type2 immunoexpression, tissue concentration and enzyme activity in bovine caruncles. reprod domest anim 2019;54: 1010-1017. 2. kitchen bj, middleton g, salmon m. bovine milk n-acetyl b-d-glucosaminidase and its significance in the detection of abnormal udder secretions. j dairy res 1978;45: 15-20. clinical theriogenology • volume 12 number 3 • september 2020 379 dominant follicle removal prior to superovulation taylor mittleider,8 brianna price,8 john gibbons,8 jason antonb 8college of veterinary medicine, lincoln memorial university, harrogate, tn bovaflo genetics, tahlequah, ok superovulation enables cattle producers to reach reproductive, financial, and genetic goals. although knowledge of follicular development has improved, number of transferable embryos per collection has not been determined, leading to a high degree of unpredictability. follicle stimulating hormone (fsh) is a major cost of embryo transfer, and treatment must occur coincident with an endogenous fsh surge for effective superovulation and embryo recovery, which has not improved substantially in many years. following dominant follicle ablation, an fsh surge and associated follicular wave can be predicted and managed, which may lead to more consistent embryo collections and more transferable embryos. aim of this field trial was to evaluate dominant follicle ablation prior to superovulation with a minimal dose of fsh. cycling beef cattle, at random stages of estrous cycle, were subjected to transvaginal ultrasound-guided aspiration of all follicles(> 5 mm). following aspiration, pgf2a (25 mg) was administered and a cidr was placed. approximately 48 hours later, folltropin-v administration began and was given twice daily (am and pm). on third day of fsh administration, pgf2a was administered again and cidrs were removed that evening. cattle were inseminated at estrus. one week later, embryos were collected and corpora lutea (cl) were counted using transrectal ultrasonography. data were analyzed using anov a. neither number of follicles ablated, nor diameter of ablated follicles had any statistically detectable effect on embryo recovery; however, cattle (n = 24) with a cl > 20 mm at ablation, tended (p = 0.107) to produce more transferable quality embryos (mean ± sem; 7.8 ± 1.1) than those with a cl~ 20 mm (5.6 ± 0.6). cattle (n = 35) given~ 10 ml offsh had similar number of total ova (11.3 ± 1.3), transferable embryos (6.2 ± 0.9), and cl (14.2 ± 0.9) compared to cattle (n = 28) given< 10 ml offsh (12.3 ± 1.1, 6.3 ± 0.6, 15.1 ± 1.0, respectively). top quartile of embryo donors (n = 15) had a cl diameter= 22.2 ± 5.0 mm,:::; 8 follicles ablated per ovary, and largest ablated follicle was 11.5 ± 0.6 mm. donors from this group yielded following numbers: total ova (13.1 ± 1.3), transferable embryos (11.2 ± 1. 1), degenerate embryos (1.5 ± 0.4), and unfertilized (0.4 ± 0.2). total dose offsh for these donors was 9.9 ± 0.7 ml (i.e. 0.9 ml per transferable embryo). in conclusion, dominant follicle removal prior to superovulation required less exogenous fsh to achieve acceptable embryo recovery. this approach also facilitated acceptable results from consecutive embryo recoveries. these results indicated that ablation of follicles (> 5 mm) in cycling mid-diestrus beef cattle, prior to initiation of superovulation, may yield more consistent embryo production, perhaps due to a more tightly synchronized follicular wave emergence. further, characterization of dynamics of this follicular wave may facilitate more consistent superovulation results and reduce costs. keywords: bovine, superovulation, follicle, embryo 380 clinical theriogenology • volume 12 number 3 • september 2020 effects of zinc on maturation and fertilization of bovine oocytes brianna price, taylor mittleider, kayla grau, john gibbons, college of veterinary medicine, lincoln memorial university, harrogate, tn zinc is an essential trace mineral in many species and is present throughout the body (highest concentrations in eye and prostate gland) having key roles in reproduction. 'zink spark' (release of zinc) that occurs in oocyte following intracytoplasmic sperm injection or natural encounter with sperm influences embryo quality. in bovine oocyte, zinc is the most abundant transition element, with concentrations fluctuating occurring during maturation and fertilization events. zinc has an important role in dna stabilization during fertilization process. objective was to determine the role of zinc in in vitro maturation and fertilization of bovine oocytes. we hypothesized that dose-dependent zinc supplementation would enhance oocyte maturation, whereas chelation of zinc would inhibit fertilization and early embryonic development. bovine oocytes were obtained via follicular aspiration of postmortem ovaries harvested from an abattoir. selected oocytes contained::: 3 layers of cumulus cells and a homogenous cytoplasm. oocytes were separated into 4 maturation treatment groups, supplemented with 0, 5, 10, or 20 µm zinc. supplementation doses where determined from analysis of zinc concentrations in adult cow plasma and follicular fluid (10.55 and 11.47 µm, respectively) and commercial maturation and fertilization medias (1.07 µm for each). oocytes were considered mature if they had reached metaphase ii and had expelled their first polar body. data were analyzed using chi-square. rate of oocyte maturation was not different (78.1 ± 3.0, 59.5 ± 4.3, 69.8 ± 7.7 and 62.3 ± 3.2%, respectively). oocytes matured in 0 µm zinc, fertilized with frozen-thawed bull semen, were assigned to 2 groups. a zinc chelated group (2.7 mm of tpen (tetrakis [2-pyridinylmethyl]-1-2-ethanediamine) in fertilization media) and a control group with no tpen. following fertilization, presumptive zygotes were independently cultured for 7 days (no tpen). data analyzed using anova. tpen-treated group had lower (p < 0.05) cleavage rate compared to control group (46.1 ± 2.3 and 75.6 ± 3.4%, respectively). embryo development rate was also lower (p < 0.05) in tpen-treated group compared to control group (15.4 ± 0.03 and 37.8 ± 0.03%). average embryo developmental scores were lower (p < 0.001) in tpen-treated group compared to controls (2.2 ± 0.1 and 3 .4 ± 0.2). zinc supplementation had minimal effects on in vitro maturation of oocytes; however, removing zinc during in vitro fertilization significantly decreased cleavage rate and embryo development. keywords: bovine, zinc, fertilization, maturation, oocyte clinical theriogenology • volume 12 number 3 • september 2020 381 bull sperm morphology analysis varies greatly by reader ashley reeves, jessica klabnik, lew strickland, tulio prado, pablo jarrin-yepez, cheyenne dingemans, liesel schneider, brian whitlock department of large animal clinical sciences, college of veterinary medicine university of tennessee, knoxville, tn sperm morphology assessments are important for selection of a herd sire, with acceptable fertility influencing bovine industry economics. studies have evaluated effects of reader experience and evaluation method on sperm morphology in other species, but apparently no study evaluated bulls. semen was collected from 35 yearling bulls at a university bull test station and from each bull, 1 eosin-nigrosin morphology slide (with a monolayer of sperm) was created by a board-certified theriogenologist. seven individuals (blinded to bull ids) assessed slides: 4 board-certified theriogenologists (dact) and 3 fourth-year veterinary students (vs) who completed an advanced reproductive elective course. one-hundred cells from each slide were evaluated (2018 society for theriogenology classification) in oil immersion (100 x) objective utilizing a light microscope in 1 sitting. a second evaluation was completed by each individual 2'.: 1 week later. three dact's performed additional evaluations of 200 and 400 sperm from first 5 slides to determine if assessing a greater number of sperm would increase agreement of morphologic characteristics within and between reviewers. data were analyzed using separate mixed model analysis of variance (proc glimmix, sas v9 .4) to test fixed effects of reviewer type, reviewer, slide number or number of sperm read, and interactions. there was an interaction (p < 0.0001) ofreviewer type and slide (vs versus dact), indicating some slide ratings were very similar between reviewer types and some differed significantly. among slides that differed, vs identified larger number (p = 0.0001) of morphologically normal sperm compared to dact. in addition, there were differences (p < 0.0001) in coefficient of variation between reviewers, but no differences (p = 0.78) between reviewer type. within dacts, there were no effects (p = 0.96) of numbers read on percent normal sperm, indicating that evaluating additional sperm did not affect outcomes ofbse. there was an interaction (p < 0.0001) of number of sperm assessed and reviewer on time to complete and for some reviewers, there were significant increases in time to complete increased sperm assessment. standard cutoffs for adequate morphology assessment utilized for decades are questionable. bulls not classified as satisfactory potential breeders are possibly due to fewer number of sperm evaluated and variation between assessors. further investigation into slide preparation, microscope, and reader experience is important to validate this method of evaluating bovine sperm morphology. keywords: sperm, morphology, bull, assessment 382 clinical theriogenology • volume 12 number 3 • september 2020 effects of motility activation and cryopreservation on fish sperm glycocalyx bryan blawut,8barbara wolfe,b gustavo scheunemann,b chris premanandan,c stuart ludsin/ marco coutinho da silva8 a department of veterinary clinical sciences, ~epartment of veterinary preventive medicine c department of veterinary biosciences, d aquatic ecology laboratory the ohio state university, columbus, oh mammalian sperm are coated with a glycocalyx: that is formed during sperm development, maturation, and contact with seminal fluid. in contrast, little is known about sperm glycocalyx: of fish sperm. our objective was to describe fish sperm glycocalyx: characteristics in different sperm types (testicular, stripped, and cryopreserved), motility activation statuses (inactive versus activated), and determine its role in fertilization using sauger (sander canadensis) as a model. our hypothesis was that glycocalyx: characteristics would differ among sperm types, activation statuses, and be related to fertilization. stripped, testicular, and cryopreserved sperm were prepared using our laboratory's published methodology for sauger. 1 all 3 sperm types were assessed in both inactive state (in extender± dmso) and activated state (1 :20 dilution in hatchery water, 35 mosm/kg). three fluorescent lectins commonly used in mammalian sperm analysis (wheat germ agglutinin [wga], concanvalin a [cona], and peanut agglutinin [pna]) were used to evaluate presence and distribution of specific sugar moieties in sperm membrane (n acetyl glucosamine [glcnac], a-mannose, and p-galactose, respectively) using flow cytometry and fluorescent microscopy. fertilization was assessed in each sperm type and in stripped sperm pretreated with wga prior to insemination. linear mixed models were used to compare staining patterns(%) and fluorescent intensity (a.u.) among sperm types and activation statuses (a= 0.05, n = 10 12). glycocalyx: of sauger sperm contained glcnac and a-mannose but lacked p-galactose moieties. wga staining patterns differed among sperm types and with activation. apical staining of wga increased -10 fold in activated sperm, except that frozen sperm had fewest cells with apical staining post-activation (46% testicular, 50 % stripped, and 5% frozen). a majority(> 90%) of sperm stained homogenously with cona independent of sperm type or activation status but fluorescent intensity was elevated (2 3 fold) in frozen sperm compared to other sperm types. activation did not affect fluorescent intensity of either wga or cona. fertilization was similar for stripped (79.3 ± 4.2%) and testicular sperm (64.0 ± 5.7%); however, fertilization rates were significantly lower in both frozen sperm (17.4 ± 3.7%) and sperm pretreated with wga (14.0 ± 1.4%). in conclusion, motility activation caused changes in glycocalyx: of fish sperm. these changes, particularly in glcnac, were largely negated by cryopreservation, which could partially explain observed reduction in fertilization. moreover, blocking glcnac moieties in sperm prior to insemination further demonstrated glycocalyx: had a critical role in fertilization. keywords: milt, sperm, cryopreservation, activation, glycocalyx: references 1 blawut, b, wolfe b, moraes cr, et al: use ofhypertonic medium to cryopreserve sauger (sander canadensis) spermatozoa. n am j aquac 2020;82:84-91. clinical theriogenology • volume 12 number 3 • september 2020 383 platelet-rich plasma reduces endometrial macrophages in postpartum beef heifers ellie alves, michelle kutzler department of animal and rangeland sciences, oregon state university, corvallis, or postpartum endometritis in beef heifers can result in infertility and culling. methods used by beef producers to treat endometritis can result in meat residues. platelet-rich plasma (prp) is an effective therapeutic treatment for endometritis in horses. 1 therefore, we hypothesized that prp would decrease endometritis in beef heifers. objective was to compare endometrial responses of intrauterine prp treatment to responses of platelet-poor plasma (ppp) or saline (sal, 0.9% sodium chloride [ph= 5.0]) treatment in normal calving heifers. fifteen normal calving crossbred beef heifers were randomly assigned to 3 intrauterine treatment groups. heifers were examined at 14 and 28 days postpartum. each examination included transrectal ultrasonographic measurements of cervical diameter, assessment of cervical discharge score (cds) using a vaginal speculum, and quantitative aerobic bacterial culture and cytology of endometrial samples. after initial examination, heifers received 10 ml of either prp, ppp or sal via intrauterine infusion. prp was prepared using routine methods previously described. 2 no treatment was administered after second examination. cervical diameter, cds, bacterial count, percentage of macrophages (%mac), and percentage of neutrophils (%pmn) were compared between 2 time points using a student's t-test. groups were compared between time points using a repeated measures anova, with a tukey's post hoc test. significance was defined asp< 0.05. cervical diameter decreased in prp and ppp groups, but not in sal. cds decreased in prp and sal groups, but not in ppp group. there was no difference in number of total aerobic bacteria count or %pmn among groups. however, %mac decreased in prp group, but not in ppp or sal groups. prp reduces in vitro responsiveness of bovine endometrial cells to bacterial endotoxins by downregulating several proinflammatory cytokines (e.g. interleukin-8).3 research is needed to determine if prp downregulates endometrial pro-inflammatory cytokines in vivo. keywords: cervical discharge score, endometrial cytology, endometrial culture, endometritis, neutrophil, transrectal ultrasonography acknowledgement authors thank oregon dairy farmers association and oregon state university's e. r. jackman friends and alumni and continuing researchers program for funding. references: 1. segabinazzi lg, friso am, correa] sb, et al: uterine clinical findings, fertility rate, leucocyte migration, and cox-2 protein levels in the endometria1 tissue of susceptible mares treated with platelet-rich plasma before and after ai. theriogenology 2017;104:120-126. 2. lange-consiglio a, spelta c, garlappi r, et a1. intramammary administration of platelet concentrate as an unconventional therapy in bovine mastitis: first clinical application. j dairy sci 2014;97:6223-6230. 3. marini mg, perrini c, esposti p, et a1. effects of platelet-rich plasma in a model of bovine endometrial inflammation in vitro. reprod biol endocrin 2016; doi: 10.1l86/s12958-016-0195-4. 384 clinical theriogenology • volume 12 number 3 • september 2020 ultrafast cooling reduced oxidative stress in vitrified bovine oocytes patrick crane,8 jiajing teng,8 yifan shen,b johnna graham,8 david closs,c robert thorne,c soon hon cheong8 8department of clinical sciences, college of veterinary medicine, cornell university, ithaca, ny bdepartment of biochemistry and biophysics, school of medicine and dentistry university of rochester, rochester, ny ~tegen, llc., ithaca, ny cryopreservation of gametes has been a revolutionary tool in assisted reproductive technology field. traditionally, cryopreservation was performed by slowcooling (0.2 10°c/minute) method which induces ice formation in extracellular space and dehydration of intracellular space. recently, fastcooling or vitrification-based approaches replaced slow cooling in human and equine gamete cryopreservation, with higher cryosurvival rates and superior clinical outcomes. obstacles for cells to survive fastcooling include intracellular ice formation and exposure to high concentrations of cryoprotectants (cpas) required to achieve vitrification. increasing cooling rates should reduce ice formation, and ice formation during warming can be reduced by reducing volume of media around cell. cryotop system is industry leader, achieving cooling rates of -3,000°c per minute. nanuqtm hyperquenching cryocooler, designed to maximize cooling rate for protein crystallography, combines automated fast{< 2 minutes) plunging with removal of cold gas layer above liquid nitrogen, leading to ultra-fast cooling rates of~ 600,000°c per minute. we hypothesized that ultrafast cooling rates would reduce oxidative stress from cryodamage of vitrified/warmed bovine oocytes. bovine oocytes were collected from abattoir­ derived ovaries, matured in vitro, partially denuded of cumulus cells, then assigned to 1 of 4 groups: 1) negative control group which is not exposed to cpa and not cryocooled, 2) cpa control group which is exposed to cpa but not cooled, 3) cryotop group with cpa exposure and cryocooling on cryotops according to manufacturer protocols, and 4) nanuq group with cpa exposure and cryocooling on microloops using nanuq hyperquenching cooler. oocytes were warmed, completely denuded and cultured for 1 hour in holding medium. effect of treatment on oocyte oxidative stress was determined by fluorescent signal measured from cells after coincubation with green fluorescent general oxidative stress indicator cm-h2dcfda. images were transformed in imagej to derive corrected total cell fluorescence (ctcf) values, and these values were compared using anova with tukey multiple comparison on rstudio. a total of 281 oocytes were included. there were no differences (p > 0.05) in ctcf values between negative (n = 77) and vehicle control (n = 93) groups. ctcf values for cryocooled cryotop (n = 65) and nanuq (n = 46) groups were higher (p < 0.001) than control groups. for cryocooled groups, microloops cooled using nanuq gave a lower (p < 0.001) ctcf value compared to cryotop group. experiment was repeated and in vitro fertilization was performed, but vitrified groups had poor cleavage rates and no blastocysts were produced, whereas negative and vehicle control groups had similar (p > 0.05) acceptable embryo development rates. nanuqtm reduced postthaw oxidative stress of vitrified bovine oocytes compared to an industry standard vitrification device. exposure to cpa did not have a significant effect on development. keywords: automation, vitrification, in vitro fertilization, bovine acknowledgement supported by center for advanced technology, cornell university clinical theriogenology • volume 12 number 3 • september 2020 385 cooled-transported epididymides for donkey semen cryopreservation y amilka lago-alvarez, a giorgia podico, a lorenzo segabinazzi, a lais cunha, a leonardo barbosa,8 carolyn arnold,b fabio lima,8 luise king,c igor canissoa 8department of veterinary clinical medicine, university of illinois, urbana, il ~epartment of large animal clinical sciences, texas a&m university, college station, tx cdepartment of veterinary clinical medicine, university of arizona, oro valley, az in the event of death or unforeseen castration for medical reasons, epididymal semen harvesting represents the last opportunity to preserve valuable jacks' genetics. objective was to compare 2 semen cryopreservation methods ( centrifugation versus noncentrifugation) on donkey cooled-shipped epididymides. we hypothesized that both methods would result in equivalent postthaw semen parameters. experiment 1: donkeys (n = 7) housed at bureau of land management in tucson, az were surgically castrated. testes and epididymides were placed in a plastic bag containing skim milk-based extender (25 ml, botusemen, botupharma, scottsdale, az), and cooled-shipped overnight in an equitainer to university of illinois for semen cryopreservation. each pair of epididymides was submitted to retrograde flushing with 5 10 ml with a cooling extender botugold (botupharma), or a similar volume ofbotucrio (botupharma). botugold group samples were submitted to cushion-centrifugation (1000 g x 20 minutes), supernatant was discarded, and pellet was resuspended at 100x106sperm/ml in botucrio. botucrio group samples were resuspended to a similar final concentration. semen from both groups was loaded in 0.5 ml straws, cooled at 5°c for 20 minutes, placed 5 cm over ln2 for 15 minutes, and then plunged in ln2 for storage. experiment 2: donkeys (n = 20) housed at peaceful valley rescue in san angelo, tx were surgically castrated and epididymides dissected away from testes and each pair was shipped similar to experiment 1, except that a different shipping container was used (botuflex, botupharma). each pair was submitted to retrograde flushing with either botugold or botucrio and processed exactly as in experiment 1. for both experiments, 1 straw for each group was thawed at 38°c for 1 minute before sperm motility parameters assessments with casa. data were analyzed with r, using a mixed model tukey's as post hoc. motility parameters were affected by shipment method (p < 0.05) but not by cushion centrifugation (p > 0.05) processing (table). donkeys had poor postthaw sperm motility compared to what was expected for donkey ejaculates subjected to cryopreservation. this was apparently the first study involving epididymal semen in donkeys. table. sperm motility parameters 1m(%) pm(%) vcl (um/s) vsl (um/s) vap (um/s) experiment 1 centrifugation 32.7 ± 5.2 23.0 ± 4.4 115.9±3.3 45.1 ± 1.8 55.3 ± 0.9 noncentrifugation 26.0 ± 4.4 18.5 ± 4.9 109.5 ± 2.3 42.7 ± 2.6 52.7 ± 2.5 experiment 2 centrifugation 20.4 ± 2.0 11.0 ± 1.5 87.7 ± 3.3 33.9 ± 1.6 41.9 ± 0.9 noncentrifugation 26.0 ± 1.3 10.6 ± 1.3 88.9 ± 3.9 36.3 ± 1.7 44.9 ± 1.9 1m: total motility; pm: progressive motility; vcl curvilinear velocity; vsl straight-line velocity; v ap: average path velocity. keywords: epididymis, storage time, testis 386 clinical theriogenology • volume 12 number 3 • september 2020 intrauterine infusion of platelet-rich or platelet-poor plasma to modulate persistent breeding induced endometritis in embryo donor mares lorenzo segabinazzi,a,b igor canisso,a giorgia podico,8 lais leal,a guilherme novello,a,b leonardo barbosa,8 michael rosser,8 fabio lima,8 marco alvarengab 8department of veterinary clinical medicine, university of illinois, urbana, il bsao paulo state university (unesp), botucatu, brazil goal was to assess postbreeding endometrial inflammatory response and embryo recovery rates in mares susceptible to persistent breeding induced endometritis (pbie) treated with platelet-rich plasma (prp) and platelet-poor plasma (ppp). we hypothesized that administration of prp and ppp mitigate endometrial inflammation in mares susceptible to pbie and platelets have an additive ability to modulate this response. mares (n = 12) that had 3 cycles were classified as susceptible to pbie, based on a challenge with killed sperm and randomly assigned in a crossover design. mares received intrauterine infusions of 40 ml of lactate ringer's solution (lrs, control), or autologous prp or ppp at 48 and 24 hours before and 6 and 24 hours postbreeding. prp and ppp were prepared from blood, using a standard double centrifugation method. platelet count and viability were assessed with spectral flow cytometry (cd41/61 coupled with igg-pe conjugated). ovulation was induced (2: 35 mm follicle) with a gonadotropin releasing hormone agonist (histrelin) and mares were bred on subsequent day with fresh semen collected from a single stallion. at 6 hour postbreeding, uterine lavage (2 liters lrs) was performed immediately before infusion with treatment. mares had daily measurements (height and width) of intrauterine fluid accumulation (iuf) at uterine body for 96 hours. endometrial cytology was performed until 72 hours postovulation, and number of polymorphonuclear cells(# pmns) was counted. embryo flushing was performed 8 days postovulation with lrs. recovered uterine fluid was aerobically cultured. plasma was collected on the day of ovulation induction, 72 hours and 8 days postovulation to assess p4 concentrations. statistical analyses were performed with anova-rm and posthoc tukey's (iuf, # pmn s, p 4 concentrations), whereas fertility and number of positive bacterial cultures were assessed with multivariate regression. mean platelet concentrations were 608.7 ± 62 and 47.5 ± 12 x 103/µl in prp and ppp, respectively. in addition, there were no differences in platelet-viability between groups (97 ± 0.7 versus 97.2 ± 0.6%). both treatment groups significantly reduced the# pmns on endometrial cytology from 24 72 hours postbreeding. infusion of prp and ppp resulted in a significant reduction in iuf postbreeding when compared to control. estrous cycles assigned as control resulted in a higher percentage (p < 0.05) of positive cultures (42%), compared to prp cycles (0%), whereas cycles treated with ppp were not different (p > 0.05; 17%) from other groups. p4 concentrations substantially increased in both treatment groups on day 8 postovulation. mares treated with prp tended to have higher (p = 0.08) embryo recovery rates (83%) than mares in control group (42%), whereas ppp had intermediate embryo recovery ( 60% ). in conclusion, plasma infusion can be used as an alternate method to modulate inflammatory response in mares susceptible to pbie and prp apparently had additional antimicrobial properties compared to plasma ppp. keywords: embryo transfer, endometritis, reproduction, inflammation clinical theriogenology • volume 12 number 3 • september 2020 387 effect of pyruvate on lactate-induced spontaneous acrosome reaction in stallion sperm luisa ramirez-agamez, camilo hernandez-aviles, charles love, katrin hinrichs college of veterinary medicine & biomedical sciences texas a&m university, college station, tx stallion sperm metabolism is thought to be largely dependent on oxidative phosphorylation. our recent study suggested that under "capacitating-like conditions" (presence of calcium, bovine serum albumin (bsa), and bicarbonate) only samples with pyruvate (as an energy substrate) had higher motility of stallion sperm. presence of lactate alone was associated with substantial increases in spontaneous acrosome reaction rate in viable sperm, to almost half of all viable sperm at 4 hour incubation. however, motility was reduced in lactate treatment at 4 hours. we determined whether addition of pyruvate to a lactate-only-containing medium may increase stallion sperm motility while maintaining acrosome reaction rate in viable sperm. fresh stallion ejaculates (n = 9) were washed and diluted to 30 x 106 sperm/ml using a modified whitten's medium (mw) containing 7 mg/ml bsa and 10 mm lactate (control), or 10 mm lactate with 0.5, 1, 5, or 10 mm added pyruvate. to control the effect of added substrate treatments, additional lactate was added at similar concentrations. diluted sperm were incubated under a 5% co2 atmosphere for 4 hours. after incubation, samples were analyzed for total and progressive motility, %tmot, and pmot by casa. samples were stained with a fixable live/dead stain, followed by fitc-psa and assessed for viability and acrosome status by flow cytometry. data were rank-transformed prior to analysis by general linear model. the % tmot was higher (p < 0.05) in 5 and 10 mm pyruvate (47 50%) than in lactate-only media (30 37%). similarly,% pmot was higher (p < 0.05) in 5 and 10 mm pyruvate (16%) than in lactate-only media (10 11 %,).the% viable (55 57%) was not affected (p > 0.05) by any treatment tested. addition of pyruvate was associated with a dose-dependent decrease in the proportion of viable sperm that were acrosome-reacted (ar; 42, 3 6, 31, 18, and 11 % for 0, 0.5, 1, 5, or 10 mm added pyruvate ), as compared to lactate ( 42, 40, 42, 39, and 40%, respectively). results suggested that presence of pyruvate increased motility and decreased proportion of viable sperm undergoing spontaneous ar. increases in motility and decreases in ar occurred at the same pyruvate concentration (5 mm added pyruvate). increasing lactate concentration in the medium was not associated with an increase in ar. findings may help to formulate media for stallion sperm capacitation, and highlighted the delicate energy balance required to support key functions of stallion sperm. keywords: stallion sperm, acrosome reaction, energy substrate, pyruvate, lactate 388 clinical theriogenology • volume 12 number 3 • september 2020 streptococcus equi subspecies zooepidemicus endometritis in mares: culture, cytology, and antimicrobial susceptibility tests christina divine,a joshua daniels,b christian bisiau,8 julie storme,8 melissa prell, a patrick mccuea 8department of clinical sciences, ~epartment of microbiology, immunology, and pathology college of veterinary medicine and biomedical sciences colorado state university, fort collins, co infectious endometritis is a significant cause of subfertility in broodmares, leading to devastating economic and emotional effects on horse owners and breeders each year. aims of this observational, retrospective study were to report the percentage of positive uterine cultures and specific pathogens, evaluate the relationship between uterine culture and cytology results, and describe the results of antimicrobial susceptibility tests for streptococcus isolates at an equine reproduction center in colorado. we hypothesized that the most common bacterial pathogen would be streptococcus sp. and that all streptococcus isolates would be sensitive to beta-lactam antibiotics (i.e. penicillin g and ceftiofur), but not all isolates would be sensitive to other antibiotics. a double-guarded uterine swab and brush were used to collect 622 paired samples for culture and cytology, respectively, from mares at colorado state university between 2017 and 2019. swabs were plated onto spectrum™ 4-part (colorado) plates (vetlab supply, palmetto bay, fl), which consisted ofmacconkey agar, tryptic soy agar with 5% sheep blood, chromogenic gram-positive agar, and chromogenic gram-negative agar. antimicrobial susceptibility tests were performed using the kirby-bauer disk diffusion method, incorporating amikacin, ceftiofur, ciprofloxacin, enrofloxacin, gentamicin, and penicillin g. cytology slides were stained with a modified wright-giemsa stain (astral diagnostics inc., west deptford, nj) and evaluated under 400 and 1000 x microscopy. a total of 513 cultures (82.5%) exhibited no growth of microbial pathogens, whereas 109 cultures (17 .5%) had growth of 1 or more pathogens. most common bacterial pathogens were streptococcus spp. (73.4% of positive cultures), escherichia coli (34.9%), klebsiella spp. (5.5%), pseudomonas spp. (3.7%), staphylococcus spp. (0.9%), and enterococcusfaecalis (0.9%). a total of 20 positive cultures (18.3%) exhibited growth of more than 1 pathogen. a positive cytology was noted on 32.3% of samples collected from mares with a streptococcus spp. culture and 42.1 % of samples associated with an escherichia coli culture. antimicrobial susceptibility tests were performed on 38 isolates of streptococcus spp. percentages of susceptible isolates were as follows: amikacin (29. 7% ), ceftiofur (100%), ciprofloxacin (75%), enrofloxacin (61.1%), gentamicin (66.7%), and penicillin g (100%). in summary, streptococcus spp. and escherichia coli were the most common bacterial pathogens isolated. all streptococcus isolates were susceptible to beta-lactam antibiotics. use of antimicrobial agents to treat equine uterine infections should be based on antibiotic stewardship principles. keywords: mare, uterine, culture, cytology, endometritis, antibiotics clinical theriogenology • volume 12 number 3 • september 2020 389 pregnancy rates in mares with pre and postovulation versus only postovulation frozen semen inseminations christian bisiau, julie storme, melissa prell, patrick mccue department of clinical sciences, college of veterinary medicine and biomedical sciences colorado state university, fort collins, co mares may be bred by live cover or artificial insemination with fresh, cooled or frozen semen. aim of this retrospective study was to compare pregnancy rates in mares after insemination with 2 doses of frozen-thawed semen (pre and postovulation) versus 1 dose (postovulation), in a commercial equine breeding program. our hypothesis was that pregnancy rate will be similar. reproductive records from mares bred with frozen semen at colorado state university over a 3 year period (2017 2019) were evaluated retrospectively. only first breeding cycle of the year was evaluated for any given mare. mares were monitored by ultrasonography once daily when in estrus and administered 1 dose of 500 µg gonadotropin releasing hormone (gnrh) agonist (histrelin) at 8:00 pm when a dominant follicle of an appropriate size was present along with uterine edema. mares were subsequently monitored 2 4 times daily to predict and subsequently confirm ovulation. mares were bred by deep uterine horn insemination with either 2 doses of frozen-thawed semen, with 1 dose prior to anticipated ovulation plus a second dose after ovulation was detected, or only 1 dose of semen immediately after ovulation. selection of the stallion, number of breeding doses allocated per cycle and number of straws per insemination were made by the owner of the mare and/or stallion. a mare was considered pregnant if an embryo was collected during a uterine lavage 8 days after ovulation, or if an embryonic vesicle was visible on 14 days postovulation via transrectal ultrasonography. pregnancy rate was compared by chi square analysis. records from a total of 156 estrous cycles were evaluated. percentage of mares that ovulated within specific time frames after administration ofhistrelin were: :s 12 hours (8.3%), 13 24 hours (6.4%), 25 35 hours (2.6%), 36 40 hours (53.8%), 41 48 hours (19.2%), and> 48 hours (9.6%). pregnancy rate of 54 mares inseminated prior to and after ovulation ( 46%) was not different (p = 0.143) from 102 mares inseminated with 1 dose of frozen and thawed semen immediately after ovulation (34%). there was also no difference in pregnancy rates based on mare age (3 10, 11 15 or 16 20 years) or reproductive status (maiden, barren, open or foaling). however, there was a difference (p = 0.049) in pregnancy rate between mares bred with semen oflow progressive motility (n = 8; :s 15%) and high progressive motility (n = 25; > 60%). in summary, a numerical, but not statistical, difference in pregnancy rate was noted between mares bred with 2 doses of frozen semen (pre and postovulation) versus 1 dose of frozen semen, however, further studies are needed with more number in each group to confirm this finding. it is becoming increasingly common to breed mares with a single or partial dose of frozen semen, with veterinarians having little control over the quality and/or quantity of semen utilized. keywords: equine, frozen semen, pregnancy, dose, motility 390 clinical theriogenology • volume 12 number 3 • september 2020 use of serum amyloid a and other inflammatory markers to monitor inflammatory response in mares with periparturient complications mette christoffersen, sofie larsen, nina schmidt, hanne pedersen veterinary reproduction and obstetrics, department of veterinary clinical sciences faculty of health and medical science, university of copenhagen, copenhagen, denmark early recognition of excessive inflammation and infectious complications related to the peripartum period, leading to early institution of therapy, reduces postpartum discomfort and facilitates recovery. because serum amyloid a (saa) is a highly sensitive marker of inflammation, measurements of saa and other inflammatory markers in postpartum mares may be valuable in assisting clinical assessment of periparturient complications. we hypothesized that mares with peripartum complications substantially altered inflammatory responses compared to normal postpartum mares. aims were to: 1) determine if inflammatory markers ( serum amyloid a (saa), fibrinogen, white blood cell count (wbc), and iron are affected by normal parturition; and 2) investigate if parturition-related complications affect concentrations of wbc, saa, fibrinogen, and iron. a retrospective case-control study included 118 postpartum mares, 72 clinically healthy (ch) mares accompanying sick foals and 46 mares with periparturient complications (pc) admitted to the university of copenhagen large animal teaching hospital from 2008 2017. periparturient complications were divided into 3 groups: metritis (n = 9), dystocia (n = 13) and others (n =24). a multivariate linear regression analysis evaluated the effects of health status of mare (ch or pc), time after foaling (day pp) and individual mare on blood parameters. independent-samples student's t test analyzed differences in inflammatory parameters between ch and pc mares at different days after foaling. ch mares had saa, wbc, and iron concentrations within reference intervals for first week postpartum (pp). mean fibrinogen concentrations increased above upper reference limit in both ch and pc mares during the first week pp, but pc mares had higher (p < 0.05) concentrations compared to ch mares. health status of mares had substantial influence on concentrations of saa (p < 0.0001), fibrinogen (p < 0.0001), and iron (p = 0.009), and day pp had an effect (p = 0.02) on wbc in both ch and pc mares. fibrinogen concentrations increased (p < 0.05) on days 2, 3, and 7 and saa concentrations on days 1 7 (p < 0.05), and wbc and iron concentrations decreased (p < 0.05) on days 1 3 (p < 0.05) in pc mares compared to ch mares. iron and wbc concentrations were, however, within reference concentrations, for both groups of mares. mares diagnosed with metritis had lower (p = 0.008) iron concentrations compared to mares with other periparturient complications. inflammatory markers saa and wbc were not affected by normal parturition and can be used to monitor inflammation and infection in mares with peripartum complications. healthy postpartum mares had increased fibrinogen concentrations within first 7 days after parturition. periparturient complications elicited, however, substantial higher fibrinogen concentrations compared to concentrations in normal postpartum mare. a major fibrinogen response, therefore, still indicates periparturient complications in a postpartum mare, and can, together with saa and wbc, be used to monitor the inflammatory response related to periparturient complications. keywords: acute phase response, serum amyloid a, periparturient complications, postpartum clinical theriogenology • volume 12 number 3 • september 2020 391 clinical effects of prebreeding intrauterine platelet-rich plasma in mare lauren pasch, a andrew schmidt, a william kingb 8wisconsin equine clinic & hospital, oconomowoc, wi bowl manor medical, warsaw, in platelet-rich plasma (prp) is being used with increasing frequency in both human and veterinary medicine. by concentrating platelets, growth factors are recruited to aid in tissue healing and repair. in women, intrauterine prp treatment improved endometrial receptivity. in mares, a limited number of studies focused on treating subfertile mares with intrauterine autologous blood products. our aim was to examine the safety and efficacy of intrauterine administration of autologous prp prepared with a commercial platelet isolation device (restigen prp®, [platelet huffy coat concentration device], owl manor, warsaw, in). we hypothesized that the treatment would be both safe and effective in addressing intrauterine inflammation, based on the frequency of adverse events, intrauterine fluid grade and pregnancy rate following treatment. notably, we elected to focus on clinical end points rather than histologic or cytologic evaluations, and used frozen-thawed semen as opposed to fresh or cooled transported semen. in this clinical-based crossover study, inclusion criteria were failure to achieve pregnancy after artificial insemination with frozen semen, absence of clinical evidence of infectious endometritis, and normal physical exam parameters. eighteen mares of various breeds, ages and parities were used. each mare served as her own control and was bred to the same stallion as the previous cycle. breeding management was performed in a routine manner. autologous prp was prepared by an experienced operator using restigen prp kits and uterine body infusion was performed 12 48 hours prior to artificial insemination. postinsemination, intrauterine fluid was graded via transrectal ultrasonography using a predetermined scale based on volume and echogenicity. pregnancy status was determined via transrectal ultrasonography from 13 16 days postovulation or via embryo flush performed 7 8 days postovulation. no adverse events were recorded in association with intrauterine infusion of prp. data were analyzed using nonparametric tests. there was no difference (p > 0.05) in post-breeding intrauterine fluid score between treatment and control groups. postbreeding intrauterine fluid score improved in 7/1 8 mares (39%) and was unchanged in 6/18 (33%). there was substantial effect of treatment on pregnancy rate, as 11/18 mares ( 61 % ) became pregnant in the treated cycle as opposed to 0/18 (0%) in control cycle. results indicated that intrauterine infusion of autologous prp prepared with a commercial platelet isolation device in the periovulatory period was a safe procedure, and associated with improved breeding outcomes in this population of mares. to better determine efficacy, further studies are in progress to differentiate these results from those of second cycle pregnancy rates without prp use. keywords: mare, platelet-rich plasma, frozen semen, artificial insemination, safety, inflammation 392 clinical theriogenology • volume 12 number 3 • september 2020 expression of prostaglandin e2 and oxytocin receptors in stallion accessory sex glands robyn ellerbrock,a igor canisso,b mariano carossino,b udenyi balasuriyac acollege of veterinary medicine, university of georgia bcollege of veterinary medicine, university of illinois, urbana, il ccollege of veterinary medicine, louisiana state university it is unknown whether oxytocin and prostaglandin receptors are present in accessory sex glands. furthermore, if receptors are present, their importance and distribution is unclear. knowledge of normal receptor expression in intact and castrated males may help to improve semen collections in difficult stallions and may further our understanding of accessory sex gland physiology. objectives were to characterize expression of prostaglandin e2 (ep2, ep4) and oxytocin (oxtr) receptors' genes in equine accessory sex glands using immunohistochemistry (ihc) and to determine if localization varied based on age or reproductive status. we hypothesized that ep2, ep4, and oxtr are more strongly expressed in mature stallion glands than in gelding or fetal glands. at euthanasia, ampulla, prostate, vesicular, and bulbourethral gland tissue were collected from mature stallions (> 5 years old, n = 3), mature geldings (> 5 years old, n = 3), and male fetuses (280 days of pregnancy, n = 3). fresh tissues were fixed in 10% neutral buffered formalin, then embedded in paraffin until processing. tissues were sectioned in 5 µm sections and stained with rabbit antihuman polyclonal antibody for ep2 or ep4 (santa cruz biotechnology), or mouse antihuman monoclonal antibody for oxtr. slides were processed using an ihc select hrp/dab kit (millipore, burlington, ma). protein localization ofep2, ep4, and oxtr was evaluated by ihc, and staining was characterized as absent, mild, moderate, or strong. prostaglandin fa and oxytocin receptors' genes were expressed in all glands. ep2 expression was mild to moderate in luminal epithelium of all glands. ep4 was strongly expressed in luminal epithelium of all glands, moderately expressed in smooth muscle of ampulla and prostate, and mildly expressed in submucosa of vesicular gland. presence ofep2 in lumen should not come as a surprise, since pge:z/.ep2 play an important role in secretory epithelium in other organs (e.g. lungs and stomach). interestingly, upregulation ofep2/ep4 are associated with tumor development in accessory glands of humans and rodents and other tissues; however, neoplasia in accessory sex glands of stallions is extremely rare. ep2 and ep4 play an important role in contraction and relaxation in the esophagus and it is likely they play a similar role in the stallion accessory sex glands. moderate expression of oxtr in epithelium and mild expression in glandular stroma, and its receptor presence suggested that oxytocin may help regulate glandular secretions. presence in equine fetus suggested that these receptors may play a role in development of accessory sex glands. lack of apparent variation in these receptors in castrated horses suggested a steroid-independent role for receptor expression in glands. in conclusion, expression ofep2, ep4, and oxtr was confirmed in all male equine accessory sex glands, including equine fetus. relative roles of these receptors in stallion accessory sex glands physiology, during ejaculation, and disease remain to be studied. keywords: prostate, horse, ampulla, seminal vesicle clinical theriogenology • volume 12 number 3 • september 2020 393 fungal endometritis caused embryo loss in a maiden mare allan gunn, a.b brianna clark,8 cyril stephen, a,b elizabeth jonesa.b aschool of animal and veterinary sciences, bgraham centre for agricultural innovation charles strut university, wagga wagga, new south wales, australia a 6 year old, maiden, standardbred mare was inseminated with chilled, extended semen (from same stallion) during 2 consecutive estrous cycles. following first cycle insemination, traces (< 1 cm diameter) of fluid were occasionally detected in uterine body during routine transrectal ultrasonographic examination. day after second cycle insemination, transrectal ultrasonographic examination confirmed ovulation and absence of uterine fluid. thirteen days postovulation, she was reexamined for an early pregnancy diagnosis. embryonic vesicle (9 mm) was detected in uterine body with no free fluid in uterus. at scheduled midpregnancy diagnosis (28 days postovulation), 10 cm diameter, mainly hypoechogenic, fluid pocket with mixed echogenic contents was detected in uterine body. no uterine edema was detected and cervix was closed. uterine lumen was initially lavaged with sterile saline, followed by 1 liter of sterile saline with 20 ml of white vinegar. reflux from saline uterine lavage was turbid and included debris and round, "fluffy" white objects 1 3 mm in diameter. samples from lavage were sent for laboratory identification. a smear was stained with 'diffquick' and assessed in the examining area. debris, epithelial and inflammatory cells, stout rod­ shaped bacteria, and nonfruiting fungal hyphae were detected. multiple drug resistant enterobacter aerogenes was identified in culture. a fungus was cultured, but not definitively typed, although it was suspected to be acremonium spp. mare was treated with prostaglandin f2aanalogue (125 µg) and oxytocin (10 iu) im twice daily for 2 days, and reexamined 4 days later. minimal (< 0.5 cm diameter) intrauterine fluid was detected. a clitoral swab was collected for fungal isolation, but was unrewarding. a double-guarded uterine swab was collected prior to next insemination, but culture results were unrewarding. she was inseminated again 26 and 58 days after diagnosis of fungal endometritis. she was diagnosed pregnant 14 days following second insemination. mare had good perinea! conformation with no previous history of intrauterine antibiotics or immunomodulating pharmacological ( corticosteroids or nsaid' s) agents ( considered risk factors for fungal endometritis ). putative fungus, acremonium spp. is typically considered to be a plant associated symbiont or pathogen. fungal endometritis is considered most likely reason for embryonic loss in this mare. to the authors' knowledge, there are no reports of fungal endometritis as a likely cause of embryo loss (prior to 40 days) mmare. keywords: mare, fungal endometritis, embryo, embryonic loss 394 clinical theriogenology • volume 12 number 3 • september 2020 pyometra associated with hyperammonemia in a mare daniela orellana-guerrero, 8 emily berryhill,b emily schaefer,8 ghislaine dujovnec 1w'illiam r pritchard veterinary medical teaching hospital bdepartment of medicine and epidemiology tiepartment of population health and reproduction school of veterinary medicine university of california, davis, ca pyometra is an abnormal accumulation of purulent debris in uterine lumen. this accumulation may be due to cervical adhesions that interfere with drainage of uterine fluids. however, accumulation can also occur without cervical obstruction. in mares, clinical signs of systemic disease are rarely present. etiology of pyometra in mares remains to be fully determined. a 20 year old multiparous paint mare was presented for inappetence and lethargy. on physical examination, her mentation was obtunded with a low head carriage, head pressing, and facial grimace. she was underconditioned (bcs 3/9) with a distended abdomen. her vital parameters were unremarkable except decreased (97 .1 °f) temperature. on abdominal palpation per rectum, a mass filling entire abdominal space was palpated which was confirmed to be uterus on ultrasonographic examination. flocculent hyperechoic fluid (31 cm) was visualized. a complete blood cell count showed neutrophilia with toxic left shift, hyperfibrinogenemia and thrombocytosis. chemistry revealed a metabolic acidosis associated with a high anion gap, mild azotemia, hyperglobulinemia, hypoalbuminemia and moderately elevated alp, ggt, mildly elevated sdh, hypertriglyceridemia and hyperammonemia (300 µg/dl). uterine drainage and lavage were performed. thick, pale brown, and malodorous fluid (101 liters) was recovered from uterus. uterine fluid sample was submitted for culture, which yielded escherichia coli and anaerobes (fusobacterium necrophorum, prevotella sp, bacteroides sp, fusobacterium nuc/eatum). mare received antimicrobials for a total of 27 days, including initial gentamicin (6.6 mg/kg iv once daily) and pencillin g procaine (22,000 iu/kg im twice daily) for 3 days, followed by trimethoprim sulfamethoxazole (30 mg/kg po twice daily) on day 4 due to clinical improvement. metronidazole (20 mg/kg per rectum every 8 hours) was initiated on day 15. flunixin meglumine 0.3 mg/kg twice daily was also administered for 3 days, and 5 iu of oxytocin was administered im every 3 hours for 2 treatments. uterus was lavaged daily for first 3 days, followed by every other day for remainder of hospitalization. cervix developed adhesions, endometrium remained thickened, and fluid continued to accumulate between lavages ( 6 8 liters per lavage). a cervical wedge resection performed on day 27 of hospitalization resulted in full thickness vaginal tear. mare was humanely euthanized. clinical presentation of pyometra concurrent with signs of systemic disease observed in this mare provided new information regarding physiopathology of pyometra. keywords: uterine infection, hyperammonemia, mare pyometra clinical theriogenology • volume 12 number 3 • september 2020 395 diagnosis of omphalocele in a toggenburg goat fetus during pregnancy bret mcnabb,8 katherine watson,b joan rowea 8department of population health and reproduction banimal and health food safety lab school of veterinary medicine university of california, davis, ca multiple ovulation embryo transfer was performed on a 5 year old toggenburg donor doe. washed and trypsin-treated fresh embryos were transferred into 4 primiparous toggenburg does. pregnancy was confirmed via transabdominal ultrasonography at day 33 of pregnancy. does were regularly monitored throughout pregnancy. a doe that received 3 embryos, lost 1 in embryo and 1 fetal (male fetus) stages, respectively, and gave birth to 1 live kid. at day 61, transabdominal ultrasonography of this male fetus identified an umbilical abnormality that was monitored until its death at day 104 of pregnancy. this male fetus was delivered as a mummified fetus with its unaffected, full term twin at day 149 of pregnancy. omphalocele was diagnosed in this male fetus, based on ultrasonographic findings of a ventral abdominal wall defect with herniation of abdominal viscera into umbilical cord base. at parturition, an abdominal wall defect was apparent, but omphalocele was no longer observable, due to fetal autolysis. significant postmortem findings included a multivessel umbilical cord, concurrent mummification and maceration of affected fetus, long bone abnormalities, and cleft palate. infectious causes of caprine abortion were ruled out. this case demonstrates practitioner's ability to diagnose umbilical defects, including omphalocele, using transabdominal ultrasonography during routine pregnancy diagnosis. transabdominal ultrasonography can identify omphalocele's bilaminar sac presence to distinguish omphalocele from gastroschisis, which has similar appearance. this sac may tear prenatally or during delivery, making perinatal differentiation of these 2 conditions more challenging. keywords: omphalocele, mummification, maceration, pregnancy, umbilicus 396 clinical theriogenology • volume 12 number 3 • september 2020 presence of tritrichonwnasfoetus in a chronically infected bull's urethra jessica rush,8 julie gard,8 katelyn waters,8 richard hopper,8 thomas passler,8 misty edmondson,c soren rodning,b jenna stockler,8 lawerence cofield, a y atta boakari, 8 sue duran8 8department of clinical sciences, college of veterinary medicine, auburn university ~epartment of animal sciences, college of agriculture, auburn university, auburn, al calabama department of agriculture and industries, montgomery, al a 5 year old maine anjou angus cross bull was donated to auburn university college of veterinary medicine. bull's smegma was collected via preputial scraping with an artificial insemination pipette attached to a 20 ml syringe. smegma sample was placed into a vial with modified diamond's media and submitted to thompson bishop sparks alabama state diagnostic laboratory (tbsasdl) for testing for tritrichomonasfoetus (i'.foetus). bull was positive for t.foetus dna, based on real time polymerase chain reaction (rtpcr). bull was tested for t.foetus on numerous occasions annually over a period of 5 years and was deemed chronically infected with t. foetus. prior to euthanasia, smegma was collected and placed in modified diamond's media. bull and its smegma sample were submitted to tbsasdl for necropsy and rtpcr, respectively. reproductive tract was dissected free; 1 x 1 cm sections were cut from prostate, right and left ampullae, right and left vesicular glands, and sections of urethra. the urethra was sectioned at specified locations; 5 cm proximal from distal end of urethra; 23 cm proximal from distal end of urethra; 5 cm distal to last bend of sigmoid; and 13 cm proximal to sigmoid and urethra at ischium level. all samples were placed in modified diamond's media and submitted to tbsasdl for culture and testing for t.foetus dna via rtpcr. reproductive tract was sectioned from most proximal portion to most distal portion. gloves were changed between each collection of tissue sectioned. instruments were cleaned following collection of each tissue section to prevent accidental contamination. a new sterile blade was used when sectioning each portion of reproductive tract. prostate, vesicular glands, ampullae, and proximal urethra were all negative for t. foetus dna. smegma from preputial scraping and urethral sections 5 cm proximal from distal end of urethra, and 23 cm proximal from distal end of urethra were positive for t.foetus dna via rtpcr with a cycle threshold (ct) of 30, 34.3, and 36, respectively. ct values from 30 37 are considered a positive reaction, indicative of moderate amounts of target nucleic acid present in this case. this is first documentation of t. foetus in a more proximal location in urethra. a few studies identified t. foetus presence in distal urethra of some bulls, but not in every bull. however, those studies were performed prior to availability of pcr; therefore, there might have been some false positives from bulls tested. this case provided important information for researchers assessing clearance of t. foetus from infected bulls, as topical treatments alone will not be curative to all bulls. keywords: cattle, trichomonias, tritrichomonias foetus, urethra clinical theriogenology • volume 12 number 3 • september 2020 397 hypertestosteronism in an intact female alpaca secondary to an interstitial cell tumor katelyn waters, richard hopper, jenna stockier, thomas passler, lawerence cofield, yatta boakari, jessica rush, julie gard schnuelle department of clinical sciences, college of veterinary medicine auburn university, auburn, al an apparently healthy, 10 year old female alpaca was presented for infertility and male-like behavior. she had failed to conceive after exposure to intact males (multiple observed breeding) since her last parturition. all female alpacas housed with her were determined to be pregnant. she had been regularly mounting other female alpacas for over 1 year. a month prior to presentation, she had elevated serum testosterone concentrations. abdominal computed topography (ct) identified a soft tissue (attenuating mass like structure with multiple cystic areas) associated with right uterine horn and right ovary, consistent with a neoplastic process. bilateral ovariectomy was performed; left ovary was 6 cm in length and had 2 firm nodules (0.9 and 0.8 cm in diameter) and cystic areas were identified in right ovary. both ovaries were submitted for histopathologic evaluation. pre and postop blood samples were submitted to endocrine laboratory (university of california, davis, davis, ca) for inhibin b, progesterone, and testosterone. preop blood samples had following concentrations; inhibin pb: 40.1 pg/ml; progesterone: 1.2 ng/ml; and testosterone 1538.3 pg/ml; and postop blood samples had: inhibin pb: 4.9 pg/ml; progesterone: 0.11 ng/ml; and testosterone: 26.4 pg/ml. blood samples submitted from a healthy intact female alpaca (to provide a reference range for normal values) had following concentrations; inhibin pb: 41.7 pg/ml; progesterone: 0.01 ng/ml; and testosterone: 33.7 pg/ml. preop hormone concentrations confirmed hypertestosteronemia and its ovarian source was confirmed by postoperation hormone concentrations. lowering of inhibin pb and progesterone in postop sample was consistent with expected changes that occur following removal of ovaries. right ovary had multiple cysts lined with attenuated to ciliated cuboidal eosinophilic material and a diagnosis of cystic rete ovarii, whereas left ovary had 2 neoplastic structures composed of packed polygonal cells with indistinct borders and abundant eosinophilic cytoplasm. no mitotic figures observed (10 fields; 400 x magnification) indicating a benign nature of the tumor (interstitial cell tumor). testosterone produced by left ovary was most likely was responsible for cystic condition of right ovary and cystic rete ovarii. interstitial or leydig stromal cell tumors are rare ovarian tumors that belong to sex cord stromal tumors group. these tumors account for < 0.1 % of all ovarian tumors in women and are even more rare in camelids. interstitial cell tumors are characteristically benign and unilateral and produce testosterone. although interstitial tumors are considered benign neoplastic conditions, they have significant effects on fertility, often ending reproductive life, as in this case. it is important to determine hormone concentrations to diagnose hypertestosteronemiam, followed by histopathology of ovaries in females exhibiting male-like behavior and virilization, to assist in definitive identification of interstitial cell tumors. keywords: camelid, ovary, interstitial cell tumor, leydig cell, hypertestosteronemia 398 clinical theriogenology • volume 12 number 3 • september 2020 hemicastration for a suspected spermatic cord torsion in a dog audrey a. kelleman small animal reproduction service, department of large animal clinical sciences college of veterinary medicine, university of florida, gainesville, fl a 4 year old domestic black mouth cur dog, 32.5 kg, was evaluated for infertility. the dog had sired, with natural mating, 3 litters. approximately 9 months prior, dog was reportedly febrile ( 40 41 °q and had a stiff gait. leptospirosis was suspected, based upon a pet-side antibody detection test. veterinary care included subcutaneous fluid therapy and 3 week course of doxycycline. two months prior to presentation, a bitch to which the dog was mated did not achieve pregnancy and upon subsequent recent semen collection, oligospermia and asthenozoospermia were evident. on initial presentation, at physical examination, dog appeared clinically systemically healthy, but scrotal content was abnormal. the left testis was normal in size and texture, whereas the right testis was firm and enlarged. both cauda epididymides felt slightly firm and both testes apparently had normal orientation. scrotal skin was normal. prostate felt normal on transrectal examination and was nonpainful. on transcutaneous ultrasonographic examination, prostate had normal parenchyma, whereas right testis was abnormal, with mixed echogenicity. blood flow was evident in both spermatic cords. inhouse serum bruce/la canis rapid slide agglutination test (rsat) was positive and 2 mercaptoethanol-rsat was negative, whereas new yark state diagnostic laboratory agar gel immunodiffusion ii and sat results were negative. semen had 120 million sperm with asthenozoospermia (< 10% total motile) and teratozoospermia (24% normal). hemicastration of the right testis was recommended and performed 6 weeks later. absence of testicular parenchyma ( consistent with tissue necrosis) and structurally normal epididymis devoid of sperm were observed on histopathology. prior spermatic cord torsion as the etiology of initial illness with subsequent complete testicular degeneration was postulated. approximately 3 months postsurgery, an ejaculate contained 110 million sperm and showed significant improvement (50% progressively motile and 60% morphologically normal sperm). determination of precise ovulation timing of bitch was recommended for future breeding to maximize chance of pregnancy. hemicastration and reexamination at appropriate interval in management of unilateral testicular disease are necessary, as demonstrated by this case. keywords: canine, infertility, testicular degeneration, spermatic cord torsion, hemicastration clinical theriogenology • volume 12 number 3 • september 2020 399 repair of a full thickness uterine tear via iatrogenic uterine prolapse in an anesthetized thoroughbred broodmare justin mcnaughten,8 amanda watkins/ shannon roska,c gustavo abuja8 8rhinebeck equine l.l.p, rhinebeck, ny "new bolton center, university of pennsylvania, kennett square, pa cjohnson equine veterinary service, sullivan, wi a 9 year old multiparous thoroughbred mare at 34 7 days of pregnancy was presented to rhinebeck equine, llp for a delay in progression of stage ii parturition. farm foaling attendant reported that the foal was not present in vaginal vault and instead 2 feet and a nose were present within rectum. mare was transported to clinic immediately. on presentation, there was no evidence of a full thickness rectovaginal tear; however, the foal was displacing rectal mucosa dorsally. mare was induced under general anesthesia and a live foal was delivered via controlled vaginal delivery. following recovery of anesthesia, mare was assessed, based on farm history. there was no evidence of a rectovaginal fistula. transabdominal ultrasonography revealed presence of swirling echogenic free fluid. abdominocentesis was performed and hemorrhagic, nonclotting fluid obtained. fluid analysis revealed elevated total nucleated cell count (7.55 x 103/µl [reference range 1.5 5 x103 cells/µl]), elevated abdominal lactate (3.8 mmol/1 [reference range o 1.5 mmol/1]), and elevated total protein (2.6 g/dl [reference range < 2 g/dl]). perineum was aseptically prepared. digital manual transvaginal examination confirmed an 15 20 cm full-thickness uterine tear on dorsal aspect of uterine body, to right of midline. mare was induced under general anesthesia for uterine laceration correction. a ventral midline celiotomy was attempted, but due to caudodorsal location and size of the defect, surgical repair was not achieved. while in dorsal recumbency, uterus was manually prolapsed through vagina by placing gentle traction on attached fetal membranes. this approach allowed visualization and hand-sewn repair of laceration. the dutch method (umbilical vessel water infusion) was used to facilitate removal of fetal membranes prior to uterus replacement. recovery was uneventful. rebreeding was delayed until the following season. mare conceived on the second cycle and delivered a live normal colt at day 356 postovulation. to authors' knowledge, iatrogenic uterine prolapse and umbilical vessel water infusion have not been reported. this technique is used by authors with good outcomes in mares with large, miduterine body laceration. keywords: mare, dystocia, uterine tear 400 clinical theriogenology • volume 12 number 3 • september 2020 therapy and evaluation of early embryonic loss in a subfertile bitch hannah smith, audrey a. kelleman small animal reproduction service, department of large animal clinical sciences college of veterinary medicine, university of florida, gainesville, fl a five year old intact female cardigan corgi was presented with a history of infertility and pregnancy loss. interestrus interval reported was consistent ( every 6 7 months), except for the last interval, which was reduced to 4½ months. bitch was inseminated 5 times in 2 years. surgical insemination using frozen semen was successful, and 2 puppies were whelped. since then, bitch was inseminated with fresh or cooled semen 4 times (1 vaginal, 1 surgical, and 2 transcervical). it was suspected that pregnancy was either not established or lost in midpregnancy. last insemination was perfonned 2 months before presentation. local veterinarian diagnosed pregnancy via a relaxin assay and abdominal ultrasonography. due to history of infertility, another abdominal ultrasonography was performed the following week, and early embryonic death was noted. on presentation, bitch appeared healthy and bright with normal physical parameters, and there was no vulvar discharge. complete blood count and chemistry were within normal limits. vaginal cytology had scant cellularity; noncornified parabasal cells, scant red blood cells, with few neutrophils and bacteria noted. serum progesterone concentrations were 1.1 ng/ml. bruce/la canis serology was negative. vaginal samples were submitted for mycoplasma and aerobic cultures. scant growth was noted on mycoplasma culture. ultrasonographic evaluation revealed the presence of 8 mm echogenic fluid in right uterine horn and 2 mm fluid in left uterine horn. both uterine horns contained thickened foci with a hypoechoic central region and hyperechoic luminal margins, consistent with necrotic placental zones. uterus had multifocal cystic endometrial hyperplasia with several 4 mm cysts. ovaries were normal and had luteal tissue. renal cortical calcifications with normal abdominal content were noted. as bitch appeared clinically and systemically stable, 3 weeks of oral enrofloxacin (12 mg/kg) and clavamox® (14 mg/kg) were prescribed for possible risk of fulminant pyometra. two weeks of topical vaginal misoprostol, 1 µg daily, was prescribed for cervical dilation. future mibolerone administration was discussed to prolong anestrus, but was declined. electro-acupuncture was performed several times and bitch was given an oral "lotus formula". one month later, ultrasonographic examination revealed an involuted uterus with no free fluid present and 2 small cysts. two months later, no free fluid or cysts were present. bitch will be bred at next estrus to a proven male, with results pending. keywords: canine, infertility, cystic endometrial hyperplasia, early embryonic death clinical theriogenology • volume 12 number 3 • september 2020 401 generation of hormone-responsive organoids from fresh and cryopreserved equine endometrium: comparison between domestic and endangered przewalski's mares riley thompson,a.b aime johnson,c pouya dini,d margherita turco/ tulio prado/ christopher premanandan/ graham burton/ barry balv brian whitlock/ budhan pukazhenthib adepartment of large animal clinical sciences, university of tennessee, knoxville, tn bcenter for species survival, smithsonian conservation biology institute, front royal, va cauburn university, auburn, al; duniversity of kentucky, lexington, ky "university of cambridge, cambridge, uk; rthe ohio state university, columbus, oh endometrium is responsive to signals associated with reproductive cyclicity and pregnancy. organoids, 3-dimensional cultured cell structures, mimic in vivo tissue structure and function better than other cell culture methods and may improve ru1derstanding of endometrial physiology. however, there are no reports of endometrial organoids generated from equine tissues. our hypothesis was hormonally­ responsive organoids can be generated from fresh and cryopreserved endometrium from domestic (equusferus caballus) and przewalski's (e.f przewalskii) mares. our objective was to compare histology, immunohistochemistry (ihc), transmission electron microscopy (tem), and gene expression in organoids derived from fresh and frozen-thawed domestic horse and frozen-thawed przewalski's horse endometrial tissues. domestic (n = 11) and przewalski's (n = 3) mare endometrial biopsies were collected, bisected, and either cryopreserved in 10% dmso or dissociated enzymatically. endometrial gland fragments were embedded in matrigel and overlaid with dmem/f12 (cell culture) plus supplements. organoids (8 wells for each treatment) were cultured through 2 passages (6 days/passage) and then, for the third passage, incubated with no hormones (control; c), or supplemented with: 1) lµm progesterone (p4) for 6 days; 2) 10 nm estradiol-17r, (ez) for 6 days; 3) ez for 2 days then p4 for 4 days; 4) c for 5 days then 10-5m oxytocin (ot) for 24 hours; or 5) c for 5 days then 10-6m ot for 24 hours. at the end of culture, organoids were analyzed for histology, tem, ihc, and gene expression. gene expression was analyzed (r project) and kruskal-wallis and dunn were used as posthocs. organoids were derived from fresh and frozen-thawed domestic mare and frozen-thawed przewalski's mare endometrium with histology and tem revealing cystic structures of epithelial cells with microvilli and intact secretory apparatus. expression (ihc) of estrogen receptor-a (era) and progesterone receptor (pr) was restricted to the nuclei and prostaglandin endoperoxide synthase-2 (ptgs2) to the cytoplasm of endometrial cells. expression ofera and oxytocin receptor (oxtr) decreased (p < 0.05) while pr and prostaglandin e synthase (pges) increased (p < 0.05) in fresh-derived organoids exposed to certain treatments compared to c. era, e-cadherin, and ptgs2 expression decreased (p < 0.05) and pges increased (p < 0.05) in cryopreserved-derived organoids exposed to selected treatments compared to corresponding c. subspecies comparison revealed an increase (p < 0.05) in oxtr expression in the przewalski ' s horse. this is the first report of equine endometrial organoid generation and long-term culture. this novel method of in vitro equine endometrial culture may lead to development of improved in vitro evaluation of normal reproductive physiology, pathological conditions, and potential therapies for uterine diseases in both domestic and endangered equids. keywords: endometrium, organoid, mare, przewalski 's, in vitro 402 clinical theriogenology • volume 12 number 3 • september 2020 semen parameters and fertility of cooled stallion semen extended with sodium caseinate and phosphocaseinate based extenders guillherme novello, giorgia podico, lorenzo segabinazzi, igor canisso department of veterinary clinical medicine, university of illinois, urbana-champaign, il aim was to compare 2 commercially available equine semen extenders based on phosphocaseinate (inra 96 imv, us) and sodium caseinate associated with cholesterol-cyclodextrin (botugold, botupharma, us), on sperm parameters and fertility of stallion semen after cooling. we hypothesized that use of a sodium caseinate cholesterol-enriched extender and seminal plasma removal would result in superior parameters in cooled stallion semen. in experiment 1, 45 ejaculates collected from 9 mature stallions were extended to 50 x 106/ml (sp group) with inra96 or botugold and processed through cushion centrifugation (cc group) (1000 g x 20 minutes) before resuspension at 100 x 106/ml with respective extender. noncentrifuged and cc samples were placed in 3 (equitainer, botuflex, and equine-express ii) passive cooling devices for 24 or 48 hours. seminal parameters assessed at 0, 24, and 48 hours, included total motility (tm), progressive motility (pm) with casa (spermvision, mof a, verona, wi), and membrane integrity and mitochondrial membrane potential with spectral flow cytometry (zombie green and mitotracker deep red). in experiment 2, mares (n = 12 x 2 estrous cycles) were bred with 1 billion total sperm from 1 fertile stallion, 40 hours postinduction of ovulation with gonadotrophin releasing hormone agonist, histrelin. semen was collected, extended in inra96 and botugold, and stored for 48 hours in equitainer, and mares were randomly bred with 1 extender in an alternate order. mares had transcervical embryo flushing performed on day 8 postovulation. data were analyzed using mixed models (r project) and tukey's as posthoc. addition of cholesterol to sodium caseinate extender, resulted in superior (p < 0.05) semen parameters. there were no differences between container type. cushion centrifugation increased (p < 0.05) sperm kinetics parameters and mitochondrial membrane potential and not (p > 0.05) plasma membrane integrity (table). in experiment 2, embryo recovery rates were identical (p > 0.05) between extenders (50%). in conclusion, we inferred that novel extender (botugold) is a suitable commercially available product that can be used for stallion semen cooling with different containers, with superior sperm parameters than traditional inra96 and at least with comparable fertility. table. effect of cushion centrifugation on semen quality parameters time total motility(%) progressive motility(%) sperm with intact sperm with high in plasma membrane(%) mitochondrial hours membrane potential (%) inr96 botruwld inr96 botugold inra96 botruwld inra96 botugold 24 sp 70 ± 1•w 75 ± 1 bw 63 ± pw 67 ± 2bw 72 ± 3.78 76 ± 2.3b 23 ±3w 28 ±2w cc 74 ± 1"' 76 ± l.3b• 67 ± 1"' 69 ± lb• 68.4 ± 2.8 72 ±2.2 28 ± 2• 34 ±2" 48 sp 56 ± 2•w 66.2 ± 1.8bw 50 ± 2•w 58 ± 2bw 66 ± 2.2• 69 ± 1.7b 71 ± 3.3w 71 ±3w cc 70 ± 2ax 68.9 ± 1.6b• 63 ± 1.8 .. 65 ± 2bx 65 ± 2.3 66 ± 2.0 76±2.r 76 ± 3w sp: before centrifugation, cc: after centrifugation w.xwithin a column, means without a common superscript differed (p < 0.05) a,bwithin a parameter, means within a row without a common superscript differed (p < 0.05) keywords: stallion, semen, extender; cryopreservation, andrology clinical theriogenology • volume 12 number 3 • september 2020 403 sperm parameters after cushion centrifugation of stallion cooled-stored semen claire kaplan,8 giorgia podico,8 kevin kline,b fabio lima,8 igor canisso8 8department of veterinary clinical medicine ~epartment of animal sciences, university of illinois, urbana-champaign, il clinical practice suggests that some stallions may benefit from semen centrifugation and re­ extension after cooled storage and shipping. this study aimed to determine the effects of centrifugation and resuspension on stallion cooled semen. it was hypothesized that an egg-yolk based extender would improve sperm motilities parameters after cushion centrifugation (cc) of cooled-stored semen. ejaculates (n = 25) from 5 mature stallions were extended to 25 million/ml with a skim milk-based extender (cst, animal reproduction systems, chino, ca) and stored for 24 or 48 hours in a passive cooling device (equine-express ii; ee, exodus breeder, york, pa) or equitainer (eq; hamilton bio vet, ipswich, ma). after storage, semen was processed through cc ( 1000 x g, 20 minutes), supernatant was discarded, and pellet resuspended in 2 ml of inra 96 (imv, maple grove, mn) or botucrio (botupharma, phoenix, az,). noncentrifuged aliquots of cooled stored semen served as controls. motility parameters were assessed with a computer-assisted sperm analyzer (sperm.vision, mof a, verona, wi) before and after cc and in control samples that were not processed. assessment included total motility (tm) and progressive motility (pm). data were analyzed using a mixed model (r project) accounting for fixed (containers, time, and extenders) and random (stallion and ejaculate) effects. cc did not affect (p > 0.05), tm at 24 or 48 hours, whereas pm tended (p = 0.07) to decrease after centrifugation and 24 hours of storage (table). additionally, after 24 hours, none of the other sperm kinetics parameters were affected (p > 0.05) by the type of extender, processing with cc or the type of container. these results could either suggest that: 1) cc cannot be used to enhance sperm kinetics parameters of cooled-stored semen; 2) or it could be possible that apparent benefits of cc cannot be seen on normospermic stallions (as used herein). thus, it is reasonable to speculate that if stallions with poor semen cooling quality were used, results may be different. in addition, cc has another benefit that was not assessed here; upon arrival, semen needs to be concentrated in a small volume for deep uterine horn insemination. additional studies are warranted to assess effects of cc on sperm motility parameters on stallions with poor-quality semen cooled semen. table. motility parameters (mean ± sem) after cushion centrifugation in cooled stallion semen time (hours) 24 48 24 48 total motility(%) progressive motility(%) control inra96 botucrio control inra96 botucrio eq 64± 3"" 64 ±3"" 70±3"" 54 ± 4xb 52 ± 3xb 58 ± 3xb eq 54 ± 4yb 61 ± 3yb 64 ± 2yh 45 ± 4yb 50 ± 3yb 53 ± 3yb ee 60 ± 3xb 70 ± 2xb 70 ± 3xb 51 ± 4xb 60 ± 3xh 59 ± 2xb ee 55 ± 4yb 61 ± 3yb 64 ±3yh 45 ± 4yb 51 ± 4yh 50 ± 4yb eq: equitainer; ee: equine-express ii a,hwithin a row and parameter, means without a common superscript differed (p < 0.05) x,ywithin a column, means without a common superscript differed (p < 0.05) keywords: sperm motility, semen quality, horse, equine, andrology 404 clinical theriogenology • volume 12 number 3 • september 2020 evaluating the impact of a systemic treatment protocol on uterine biopsy grade and uterine microbiome in mares with pretreatment kenney grades ofiib-ill adam bassett,8 cory anderson,8 james bailey,b lulu guo,8 janna ruether,8 udaya desilva, c candace lyman,8 reed holyoaka acollege of veterinary medicine, oklahoma state university, stillwater, ok broyal vista southwest, purcell, ok coepartment of animal science, oklahoma state university, stillwater, ok various treatment strategies have been utilized to correct endometritis in broodmares. majority of described treatment protocols involve intrauterine administration of various pharmacologic compounds. one treatment protocol used by one of the co-authors, dr. james bailey, in embryo transfer donor mares and mares intended to carry their own pregnancies, involves a regimen ofdmso, antibiotics, and dexamethasone (all systemic treatments) administered at specific times during a course of 8 days. this treatment protocol has been anecdotally reported to produce positive results (successful embryo donors and also carrying their own pregnancies) in mares with uterine biopsy kenney grades of iib or iii. it was hypothesized that this treatment protocol would: 1) improve kenney biopsy grades iib or iii; and 2) alter uterine microbiome. objectives were to evaluate the impact of this treatment protocol on each mare's: 1) endometrial biopsy grade; and 2) uterine microbiome. mares (n = 13) with previous uterine biopsy grades ofiib-iii were included. eligibility was determined by first collecting an endometrial biopsy sample. once mares with biopsies that were graded as iib-iii were selected and a pretreatment uterine microbiome sample was obtained using triple-guarded uterine culture swabs. two swab samples were obtained from each mare. one swab was utilized for 16s-rdna microbiome analysis. the other swab was submitted for aerobic and anaerobic bacterial culture. once the pretreatment samples were obtained, the following treatment protocol began: intravenous administration of250 ml ofdmso mixed with 750 ml of saline on days 1 and 3 (day 1 being the first day of treatment), a daily, oral antibiotic (equisul-sdt, sulfadiazine-trimethoprime, 24 mg/kg, aurora pharmaceutical, northfield, mn) on day 4 and continuing through day 8, and finally 80 mg of intramuscular dexamethasone on days 6 and 8 ( day 8 was the last day of the treatment protocol). thirty days after the completion of the treatment protocol, posttreatment uterine biopsies and microbiome swab samples were once again obtained. results are currently pending (laboratory shutdown due to covid-19), but will be available for presentation at the society for theriogenology conference in july. this study will provide objective data to characterize the impact of this treatment protocol on the endometrial grade and the microbiome of treated mares. keywords: dmso, endometrial microbiome, 16s rdna, embryo recipient, sulfadiazine­ trimethoprim clinical theriogenology • volume 12 number 3 • september 2020 405 effect of platelet rich plasma lysate and fibroblast growth factor 2 on sperm motility in stallions fabio pinaffi, robyn wilborn, lindsey boone, aime johnson department of clinical sciences, auburn university, auburn, al semen extenders are continually tested to improve sperm motility, longevity, and, consequently, improve fertility. growth factors (gfs) modulate cell function, which could be advantageous to sperm by improving motility. in humans and mice, fibroblast growth factor 2 (fgf2) improved sperm motility. platelet rich plasma (prp), which is rich in growth factors (gfs) including fgf2, reduced postmating inflammatory response within the uterus when infused 24 hours before or after artificial insemination. effect of prp on sperm is not determined. objective was to evaluate the effect of adding either pooled prp lysate (prpl) or recombinant equine fgf2 (refgf2) at varying concentrations to semen extended in commercial equine semen extender (inra 96). prp lysate was used instead of prp, since it is more purified and highly concentrated in gfs. eight treatments were tested using concentrations of 1, 2.5, 5, and 10% of prpl containing 1 iu/ml of heparin, and 0.1, 1, 10, and 100 ng/ml ofrefgf2 and compared to control groups with and without 1 iu/ml of heparin. heparin use was based on a previous titration to prevent gel formation from prpl reaction with semen extender, precluding motility analysis. motility parameters were evaluated with samples standardized to 50 million sperm/ml using computer assisted semen analysis (casa) at hours 0, 0.5, 1, 1.5, 6, and 24 after treatment. for both prpl and refgf2 treatments, there were no differences (p = 0.99) in motility among groups at any time point, with ranges from 66 to 60% at hour o and 46 to 49% at hour 24. interestingly, results concentrations of prpl > 5% induced sperm agglutination via head-to-head attachment (hha), starting at hour 1, whereas prpl at concentrations below 2.5% did not induce hha nor affect sperm motility. in addition, hha was objectively detected by decreases in total number of cells counted per field (total cells) and estimated concentration measured by casa, assuming that only free sperm were counted, whereas sperm entrapped by hha not counted. decreases in total sperm and concentration were different (p < 0.0001) for 5 and 10% prp groups, being more pronounced for 10% prpl group, suggesting a dose-dependent characteristic of hha induced by prpl. one important finding was that prpl did not kill sperm, besides inducing hha. although no motility improvement was observed, present results suggests that direct addition of prpl in semen extender at doses below 5% could be implemented to reduce postmating uterine inflammatory response, without substantially affecting sperm motility. however, further research on uterine inflammatory response is needed to test this hypothesis. keywords: growth factors, sperm motility, endometritis, head-to-head attachment 406 clinical theriogenology • volume 12 number 3 • september 2020 prevalence of and potential impact on fertility of pars pituitary intermedia dysfunction in a thoroughbred broodmare population in england sarah moore rossdales llp, weston, hertfordshire, uk prevalence of pars pituitary intermedia dysfunction (ppid) has been reported in general equine population as 21.2% in horses and ponies aged ::: 15 years. 1 aim was to establish the prevalence of ppid in a thoroughbred broodmare population using seasonally adjusted cutoff values for basal plasma adrenocorticotrophic hormone (acth) concentrations and analyze the potential impact on fertility. venous blood samples were collected from 79 thoroughbred broodmares ~ 15 years old ( average 18 years) on first january 2019. samples were analyzed for plasma acth concentrations using immulite 1000 assay. mares with plasma acth concentrations above the seasonally adjusted reference range for nonautumn months (> 29. 7 pg/ml) were allocated into a followup group and underwent repeat sampling in 2019 autumn. prevalence of ppid in january 2019 was 16.4% (13/79), with mean plasma acth concentrations of 38.0 pg/ml (30.2 61.4) in affected mares. on repeat sampling, 23% (3/13) mares remained positive(> 47 pg/ml; autumn reference range), whereas 46% (6/13) had a reduction in basal plasma acth concentration. furthermore, 7% (1/13) had small increases in plasma acth that was still within normal limits for autumn, whereas 23% (3/13) were lost to followup. mean pregnancy rate per cycle for 13 mares in the followup group was 52.4%. five mares were not bred in the 2019 breeding season. two pregnancies were lost between 15 and 28 days. one mare was lost to followup. initial prevalence of ppid within this population was comparable to previous studies; however, followup data in autumn had a decrease. prevalence of ppid in this study population was lower than general equine population, with elevated acth concentrations having no impact on fertility. affected mares had consistent estrous cycles and were able to conceive and maintain pregnancy to 28 days at a pregnancy rate comparable to previous reports for aged mares. furthermore, these results also highlighted the requirement for repeat testing and need for further investigation into use of single basal plasma acth concentrations as a conclusive test for ppid. keywords: equine, ppid, acth, fertility, prevalence references 1. ireland jl, mcgowan cm: epidemiology of pituitary pars intermedia dysfunction: a systemic literature review of clinical presentation, disease prevalence and risk factors. vet j 2018;235:22-33. clinical theriogenology • volume 12 number 3 • september 2020 407 kisspeptin and rfamide-related peptide 3 neurons in bovine hypothalamus: estrogen receptor a expression and inputs to gonadotrophin releasing hormone neurons allan gunn,a,b jessica rose,b,c rebecca scott/ christopher scottb,c 8school of animal and veterinary sciences, charles sturt university bgraham centre for agricultural innovation, albert pugsley place cschool of biomedical sciences, charles sturt university driverina anglican college, farrer road, wagga wagga, new south wales, australia fertility in dairy cattle is considered low, most likely due to selection for high milk production resulting in extreme metabolic demands and a period of negative energy status. in most mammals, there appears to be an inverse relationship between metabolic status and reproduction. to address and manage this relationship in dairy cows, a better understanding of brain control of reproduction is required. final output signal of the brain is gonadotropin releasing hormone (gnrh), but it is evident that integration of internal and external signals, such as feed intake and metabolic status, occurs upstream of the gnrh neurons. the rf-amide neuropeptides, kisspeptin (kp), and rfamide-related peptide 3 (rfrp-3) may be involved in this integration and relaying this to the gnrh neurons. in cattle, gnrh secretion is stimulated by kp and inhibited by rfrp-3. we used immunohistochemistry (ihc) to map the distributions ofkp and rfrp-3 neurons in the hypothalamus of dairy cattle, determined their co­ expression with estrogen receptor alpha (era), and whether they made contact with gnrh neurons. the heads of male and female calves (n = 3 each), steers, heifers, and lactating cows (n = 2/group) were perfusion-fixed (4% paraformaldehyde) and the brains dissected out and frozen. dual-label ihc for kp or rfrp-3 with era or gnrh was performed on 40 micron thick cryostat sections, using fluorescently labelled secondary antibodies. gnrh neurons were identified in the preoptic area and anterior hypothalamic area. highest numbers of neurons expressing era were in the arcuate nucleus, ventromedial nucleus, preoptic area, and anterior hypothalamic area. kp neurons were located primarily in arcuate nucleus, with some cells in the preoptic area (although few were observed in calves). most kp neurons in adult animals of both sexes, but few in calves, expressed era. very few gnrh neurons, however, received close appositions from kp neuron fibers. rfrp-3 neurons were localised in the dorsomedial hypothalamus and paraventricular nucleus, with fewer cells observed in cows than heifers and steers, whereas era was not expressed in these regions. fewer than 20% ofgnrh neurons received close contact from rfrp-3 neurons. in conclusion, we inferred that feedback actions of estrogen on gnrh secretion may, in part, be relayed via kp neurons but unlike most other mammalian species studied, this is unlikely to be a direct action. rfrp-3 neurons likely have a small role in the regulation of gnrh neurons in cattle. further studies are required to clarify the physiological roles of kp and rfrp-3 in regulation of gnrh secretion in dairy cattle. keywords: kisspeptin, dairy cattle, gnrh, hypothalamus, neuroendocrine signalling 408 clinical theriogenology • volume 12 number 3 • september 2020 oviductal insemination by hysteroscopic hydrotubation in mares: a preliminary investigation yuji inoue inoue equine clinic, hokkaido, japan small numbers of sperm were recovered from the oviduct after natural mating or artificial insemination in mares. one advantage of oviductal insemination is that only small number of sperm may be required to achieve pregnancy in a mare compared to numbers required with standard intrauterine insemination. objective was to investigate the potential for oviductal insemination, using hysteroscopic hydrotubation1 with small number of sperm. we hypothesized that pregnancy can be established with a small number of sperm, obtained from frozen semen and infused into the oviduct directly through the uterotubal junction by hysteroscopic hydrotubation. ten mares were used; 5 mares each were assigned into 2 groups and inseminated when preovulatory follicle size reached 35 40 mm. ovulation was confirmed 48 hours after insemination. progressively motile sperm were selected using the swim-up technique with frozen semen of known fertile stallions (a and b). one straw of 0.5 ml frozen semen was thawed and mixed with 0.5 ml quinn's® sperm washing medium and 1 ml of the same medium was floated onto the mixed solution and incubated for 10 minutes at 3 7°c. number of sperm that swam up into the supernatant was counted with a thoma cytometer. progressively motile sperm concentrations were 1.3 and 1.2 x 105/ml in the supernatant of stallions a and b, respectively. in group 1 (5 mares) frozen semen of stallion a was used. from 0.1 to 1.0 ml of the supernatant including sperm was mixed with 1.0 or 2.0 ml of the same medium to adjust the number of sperm (1.3, 2.0, 2.0, 4.1, 4.1, and 9.8 x 104 ) and total volume of medium (1.1, 1.7, 1.7, 1.9, 1.9, and 1.5 ml). medium, including sperm, was infused into oviduct through uterotubal junction ipsilateral to ovulation site using hysteroscopic hydrotubation. one of 2 mares was pregnant in cases where 2.0 and 4.1 x 104 sperm were used, 1 mare was pregnant with 9.8 x 104 sperm. in group 2 (5 mares), 1.9 x 104 sperm from stallion bin 1.7 ml medium was infused into oviducts in 6 cycles of 5 mares by the same procedure used in group 1. three of 5 mares (3/6 cycles) in group 2 were pregnant. in conclusion, oviductal insemination of small number of sperm to achieve pregnancy in mares using hysteroscopic hydrotubation may be a clinically applicable technique. keywords: oviductal insemination, hysteroscopic hydrotubation, mare reference 1. inoue y: hysteroscopic hydrotubation of the equine oviduct. equine vet j 2013;45:761-765 clinical theriogenology • volume 12 number 3 • september 2020 409 effect of mid-diestrus oxytocin treatment on early pregnancy in mare justin mcnaughten,8 ross wallaceb 8rhinebeck equine l.l.p, rhinebeck, ny ~urray veterinary services, coolup, western australia, australia progesterone supplementation in early pregnancy is commonplace in equine reproduction. recently, there has been some controversy questioning the beneficial effects of routine progesterone administration and rising concerns as an occupational hazard for humans. although premature luteal regression and resulting low plasma progesterone concentrations is rare in the mare, select cases benefit from luteal support. luteal function can be extended following oxytocin administration during mid-di estrus. aim of this field study was to investigate the effects of oxytocin administration on early pregnancy in the mare. thoroughbred mares (n = 39) were enlisted and bred over 55 estrous cycles. prior to natural breeding, uterine cultures were obtained and mares were assigned to 1 of 2 groups: oxytocin treatment (ot); oxytocin 60 units, im, once daily, days 7 14 postovulation; or control (ct), given no treatment. mare reproductive cycles were monitored by transrectal palpation and ultrasonography. ovulation was documented and defined as day 0. pregnancy diagnosis was performed by transrectal ultrasonography 14 days postovulation. if pregnancy was confirmed, a blood sample was collected and assayed for plasma progesterone concentrations. mares that did not conceive were rebred and treatment was determined by the stage in the study; therefore, some mares underwent the same treatment more than once. subsequent pregnancy examinations were performed on days 28 and 45 of pregnancy. first and percycle pregnancy rates for ot and ct were 90.9 and 91.3% and 68.2 and 55.1 %, respectively. median progesterone concentrations for ot were 6.9 and 7.0 ng/ml for ct. there were no differences between ot and ct groups for first (p = 0.165) or percycle (p = 0.289) pregnancy rates, days 28 and 45 pregnancy rates (p = 1.0) or progesterone concentrations (p = 1.0). oxytocin administration during mid-diestrus in early pregnancy did not induce a negative effect. further investigation in this area may reveal potential beneficial effects of oxytocin administration in early pregnancy. keywords: mare, oxytocin, corpus luteum, progesterone, breeding management 410 clinical theriogenology • volume 12 number 3 • september 2020 combination of estradiol cypionate and altrenogest to control ovulation timing in mares william whitler,8 eleas wu,b vanessa souz.a,8 charles estil18 8carlson college of veterinary medicine, oregon state university, corvallis, or bcollege of veterinary medicine, iowa state university, ames, ia controlling ovulation timing in mares is used primarily for convenience when frozen or transported semen is used, or to provide recipients for an embryo transfer program. luteal or progestational phase termination with prostaglandin f2a or its analogs, results in too variable a time to ovulation to be of practical use (3 15 days). progestogens alone, although capable of inhibiting ovulation, have no control on follicular development. time from withdrawal of progestogen to ovulation is too variable to be useful in timed insemination with frozen semen or embryo reception. a combination of progesterone and estrogen (p & e) to inhibit both follicle stimulating hormone (fsh) and luteinizing hormone (lh) synthesis and release results in more precise control of follicular development and ovulation. this protocol requires daily intramuscular (im) injections and use of compounded products. estradiol-17 p is reported to be the best product, because estradiol cypionate (ecp) and estradiol benzoate are too slowly metabolized and cause delayed or erratic return to estrus. anecdotal reports dispute this claim. goal of this study was to determine the usefulness of a combination of commercially available products and minimize the number of im injections to control ovulation in the mare. procedures were approved by oregon state university animal care and use committee. ten mares of variable age ( 17 23 years, mean 19. 7 years) and size ( 515 626 kg, mean 570 kg) were given altrenogest liquid (regumate®, merck animal health, madison, nj) 0.044 mg/kg orally, once daily, for 10 days and estradiol cypionate (depo estradiol®, pfizer, new york, ny) 0.011 mg/kg im on day 1 and day 5. study was performed during normal breeding season for the latitude. none of the mares were examined prior to treatment to determine stage of estrous cycle. mares were evaluated by transrectal ultrasonography of reproductive tract, had serum progesterone concentrations measured, and received cloprostenol (estrumate®, merck animal health, madison, nj) 0.5 µg/kg im on day 10. reproductive examinations were performed daily or on alternate days, from days 15 or 16, to detection of ovulation. nine out of 10 mares had progesterone concentrations < 1 ng/ml at the end of 10 day study periods. largest follicle at cloprostenol treatment was 38 mm(< 20 38 mm) and second largest was 27 mm. eight mares ovulated 7 .25 7 .625 days after discontinuation of altrenogest. even a mare with 3 8 mm follicle did not ovulate until day 17, despite having < 0.2 ng/ml progesterone at cloprostenol treatment. two mares did not ovulate within 14 days of discontinuation of treatment. it is concluded that ovulation timing can be controlled in mares using commercial compounds readily available to the practicing veterinarian, although fertility of those ovulations remains to be determined. keywords: mare, ovulation, ecp, altrenogest clinical theriogenology • volume 12 number 3 • september 2020 411 prevalence of malignancy in canine mammary masses in a population of shelter dogs sharon pindar spca tampa bay, largo, fl a retrospective study was carried out on 11 bitches with mammary masses that were surrendered to a municipal shelter in florida from 2017 to 2019. ten bitches had reached sexual maturity, and 1 did not have an indicated age. objective was to determine the prevalence of malignancy of mammary masses in this population. mammary masses were removed at ovariohysterectomy and submitted to a commercial pathology service for analysis. ten bitches had solitary mammary masses and 1 had 2 mammary masses. a total of 12 mammary masses were submitted; 11 (92%) had no evidence of malignancy and 1 mass (8%) had low-grade neoplasia. most common masses identified were adenomas (n = 7). other masses identified included a parasitic granuloma, follicular cyst, lipoma, and mammary ductal ectasia. one neoplastic mass (grade 1 mammary carcinoma identified) was excised from a 7 year old chihuahua mix with multiple mammary masses. three (25%) masses in this patient were not mammary in origin. chihuahuas and chihuahua mixes (n = 6) were overrepresented in this study. in this small population, mammary masses were predominantly benign. further studies are needed with expanded sample size and involving a reproductive pathologist. keywords: mammary, neoplasia, canine, benign, malignant 412 clinical theriogenology • volume 12 number 3 • september 2020 intravaginal progesterone releasing device to hasten first ovulation in mares: side effects and pregnancy rates in a commercial thoroughbred farm david trundell dt veterinary services, c/o norman court stud, salisbury, united kingdom many protocols have been evaluated to hasten first ovulation of breeding season in mares, including use of progesterone releasing devices. however, use of these devices in clinical practice is not widespread. in the thoroughbred industry, there is immense pressure to have mares cycling in the nonphysiologic breeding season. thoroughbred mares (n = 20) over 2 consecutive breeding seasons in a commercial studfarm underwent treatment with a prid™ delta (1.55 g progesterone) inserted intravaginally for 10 days. mares ( 5 23 years) were not under artificial lights. mares at time of insertion were deemed to be in early transition via transrectal ultrasonography, having at least 1 follicle of2.0 cm, no uterine edema, and no previous ovulation noted for that breeding season. on removal of prid™ delta, mares had a preovulatory follicle of at least 3.5 cm. on day 11 (1 day after removal), mares had a uterine edema score of at least 1 (on a scale o 3) and were given deslorelin acetate to induce ovulation. all mares ( 100%) ovulated within 3 days after removal of prid™ delta ( day 13). eight mares ( 40%) cultured positive for endometrial streptococcus equi subspecies zooepidemicus. remaining 12 mares (60%) were inseminated via live cover, resulting in 10 established pregnancies (50%) diagnosed on day 14 by transrectal ultrasonography. no vaginal discharge was observed in any of the mares, although all mares were noted to have some increase in vascularization of the vaginal wall with some hyperemia (n = 20). no other complications were noted in the reproductive tract of any of the mares. discomfort upon removal of the device, including straining, mild colic-like signs, and weight shifting, were noted in some mares (6/20) all of which were transient in nature, and required no medical interventions. it is concluded that prid™ delta use to hasten first ovulation in mare is effective with minimal side effects and results in a fertile ovulation. use of intra vaginal progesterone releasing devices along with other methods to manipulate mare's physiologic breeding season, such as artificial lighting regimes, can hasten time to first ovulation and reduce associated costs. keywords: intravaginal progesterone, breeding management, mare, first ovulation clinical theriogenology • volume 12 number 3 • september 2020 413 individual variation of frozen-thawed sperm from stallions to survive dilution and cooling after thawing kristin klohonatz, karley milburn, paul loomis select breeders services, chesapeake city, md within the equine industry, there has been interest in the ability to thaw frozen semen in a semen production laboratory, dilute further in extender and ship it as a cooled dose to the farm for insemination. two previous studies concluded that acceptable retention of motility and fertility can be achieved using this technique, but was only done on a small set of samples. goal of this experiment was to measure semen quality after thawing and cooling over 48 hours on a large number of stallions and illustrate the variability in the success among individual stallions. we evaluated 22 doses of frozen semen of variable post-thaw quality, all of which previously resulted in pregnancies. motility analyses were performed using a hamilton thome ceros ii computer assisted sperm analysis system to evaluate at least 400 motile sperm from each sample at each time point. samples were analyzed for 40 video frames at 60 frames/second and progressive motility (pm) was defined as motile sperm moving with an average path velocity:::: 50 µm/second and a straightness value of:::: 75%. a full dose of eight (0.5 ml) straws per sample were thawed at 37°c for 30 seconds, then combined in a prewarmed 5 ml tube. concentration was determined with a nucleocounter sp-100 and an aliquot of the sample was added to pre-warmed cooling extender to a concentration of approximately 10 million/ml. after incubation for 3 0 minutes at 3 7°c, initial motility was determined. a second aliquot of thawed sample was incubated in modified whitens medium with sybr-14 and propidium iodide at a concentration of 5 million/ml. membrane integrity analysis was completed on a guava easycyte ht flow cytometer. frozen-thawed samples were diluted to 30 million/ml in extender previously determined to be optimum for cooling semen of that individual stallion (inra96® or a standard skim milk-glucose extender). motility and membrane integrity analyses were performed initially (time 0), 6, 10, 24, 32 and 48 hours postthaw. percent change in motility and membrane integrity was calculated from time o (initial) to 6, 24, and 48 hours after cooling. average percent decrease in pm across all 22 stallions was 23, 41, and 67% at 6, 24, and 48 hours. average percent decrease in sperm with intact membranes was 16, 16, and 23% at 6, 24, and 48 hours. individual variation in the success of this technique was substantial. decrease in semen quality after thawing and cooling for 24 hours, ranged among stallions from 2.5 to 77.6% for pm and oto 37% for membrane integrity. decrease in pm for top 5 stallions at 6, 24, and 48 hours were 5, 13, and 33% respectively whereas for bottom 5 stallions there was a 47%, 71 %, and 98% change in pm, respectively. it is concluded that although this technique is a viable option for some stallions, it is not appropriate for all stallions and therefore must be tested on every stallion. keywords: equine sperm, frozen-thawed, cooled, membrane integrity 414 clinical theriogenology • volume 12 number 3 • september 2020 comparison of pregnancy rates using new versus once used cidr in 7 day estrus synchronization protocol during breeding season in ewes robin stevens,° mohanathas gobikrushanth,b kamal gabadage,b fritz schu.mann,b colin palmer,b dinesh dadarwalb 8babine animal hospital, smithers, british columbia, canada 11,arge animal clinical sciences, western college ofveterinruy medicine university of saskatchewan, saskatoon, saskatchewan, canada small ruminant cidrs are labelled for use in estrus synchronization protocols for up to 14 days. protocols of shorter duration (5 9 days) include prostaglandin f2a (pgf2a) treatment at cidr removal to induce luteolysis. short duration protocols have led to anecdotal belief that a cidr may be used a second time. addition of gonadotropins to cidr based protocols are known to enhance follicular growth and facilitate multiple ovulations. our objective was to compare pregnancy status following natural breeding at a synchronized estrus using a 7 day exposure to either a new cidr or once used cidr, with pg600 treatment on day 6. canadian arcott ewes (mixture oflambs and mature) housed at 2 separate locations (flocks a and b) were used. in flock a, ewes (n = 61) were synchronized during 2018 2019 (n = 31) and 2019 2020 (n = 30) breeding seasons, whereas ewes (n = 71) in flock b were synchronized during 2018 2019 breeding season. on day 0, ewes were randomly allocated to have either a new cidr (ncidr) or used cidr (ucidr) placed intravaginally. on day 6, all ewes were treated with pg600 (400 iu ecg + 200 iu ofhcg/5 ml dose, im). on day 7, cidrs were removed and all ewes received a single dose of pgf2a (cloprostenol, 125 mg, 0.5 ml, im). on day 8, ewes were exposed to rams for 4 days. rams were identified as satisfactory breeders using breeding soundness evaluation and previous breeding history. ram to ewe ratio was maintained at 1 :5 (flock a) and 1 :5 to 1 :6 (flocks b). rams were provided 4 5 days of sexual rest before being used. pregnancy diagnosis was performed between days 45 60 postbreeding via transabdominal ultrasonography. fischer's exact test was used to compare pregnancy rates between treatment groups. all ewes in flock a were marked (ram marking harness) within 24 hours after ram introduction. overall, pregnancy rates (ncidr and ucidr combined) were different (p < 0.01) between flocks (85%, 52/61 versus 58%, 41/71; flocks a versus b, respectively); therefore, data were analyzed separately by flock. within flock, pregnancy rates were not different between treatment groups (flock a : ncidr versus ucidr, 25/30 versus 27/31, p = 0.75; flock b: ncidr21/34 versus 20/37, p = 0.63). lower pregnancy rates in flock b were believed to be due to nutritional status (as a general observation, body condition scores of ewes in flock b were lower than those in flock a). in conclusion, a short duration (s 7 days) once used cidr, can be successfully reused to obtain pregnancy rates comparable to new cidr. keywords: estrus synchronization, pg600, cidr, ewes, fertility clinical theriogenology • volume 12 number 3 • september 2020 415 retrospective review of uterine prolapse in mares jenny boye/ ghislaine dujovneb awilliam r. pritchard veterinary medical teaching hospital bdepartment of population health and reproduction school of veterinary medicine, university of california, davis, ca uterine prolapse ( eversion) is an infrequent but life-threatening emergency in mares. since uterine prolapse is so uncommon, there is very little information published in scientific journals. aim of this report is to describe uterine prolapse predispositions and case outcomes by reviewing clinical records from the uc davis veterinary medicine teaching hospital (vmth). mare history and outcome data were analyzed for correlation to mare survival with mann-whitney u calculation and fishers exact test. significance level of p < 0.05 was set for all analyses. during 30 year period (1989 2019), 24 mares were presented with uterine prolapse. age of mares ranged from 4 23 years (mean ± sd; 11.1 ± 4.6) and parity ranged from 1st to 13th foaling (mean ± sd; 3 ± 2. 7). there was a high representation of maiden mares (7 out of 24). investigation of interrelation between breed and uterine prolapse revealed that arabians were overrepresented among affected mares compared to general breed distribution at the vmth in the same time period. uterine prolapse was associated with parturition and occurred within 1 hour after foaling in 82% of the cases. fetal membranes were attached at the time of prolapse in 63% of the cases. contrary to common belief that dystocia is the greatest predisposing factor to prolapse, only 33.3% of prolapses occurred after dystocia and 16.6% prolapses followed an abortion. survival of the mares at discharge was 75%. regarding fatalities, 5 mares suffered acute hemorrhage associated with prolapse and 1 died from secondary peritonitis. fetal gender was overrepresented by males, 12 foals were colts and 5 were fillies (7 unknown). fifteen foals survived. we were able to obtain follow-up history for 14 mares; 5 were rebred, and 4 foaled successfully. there were no correlations between mare survival and parity, age, sex of the foal, retained fetal membranes, or occurrence of sepsis. based on the findings, we concluded that uterine prolapse occurred more after normal parturition (eutocia) than following abortions (16.6%) and dystocia (33.3%). uterine prolapse may also have some breed predisposition to arabians. colts are overrepresented among affected mares. no characteristics were correlated with survival of mare, but severe hemorrhage was correlated with nonsurvival. keywords: mare, uterine prolapse, normal parturition, abortion, dystocia 416 clinical theriogenology • volume 12 number 3 • september 2020 alfaxalone crossreactivity affecting progesterone concentrations in cats joshua_trumble, aime johnson, jacob johnson, robert kemppainen, stuart clark-price college of veterinary medicine, auburn university, al alfaxalone is a commonly used anesthetic agent in small animals. in cats, alfaxalone is used as an intramuscular agent to achieve clinically useful sedation or anesthesia, negating need for intravenous treatment, particularly in difficult patients. molecular structure of alfaxalone is similar to progesterone (p 4) hormone allowing for activation of gab aa complex through a progesterone receptor. on clinical observation, intact female cats demonstrating signs of estrus, following alfaxalone treatment had serum p 4 concentrations suggestive ofluteal activity. concern that alfaxalone may be interfering with the assay, we hypothesized that alfaxalone would crossreact with progesterone assay. eight domestic shorthair neutered male cats were administered 3 mg/kg of alfaxalone im. blood samples were collected at set time points (baseline, 30 and 60 minutes, and 3, 6 and 10 hours). serum concentrations of p4 immunoreactivity (ir) were determined using an automated immunoassay system (immulite 1000). data were analyzed using repeated measures anov a and a tukey-cramer multiple comparisons test, with p < 0.05 used for significance. serum p4 ir was elevated (p < 0.05) at 30 minutes, 1 hour, and 3 hours compared to baseline and returned to baseline at 6 hours. we concluded that intramuscular alfaxalone treatment in cats may interfere with immunoassay measurement of serum p 4 for up to 6 hours. caution should be exercised when interpreting serum p 4 ir results in cats treated with alfaxalone. keywords: alfaxalone, progesterone, immunoreactivity, cat clinical theriogenology • volume 12 number 3 • september 2020 417 influence of extender, temperature, and equilibration time on postthaw sperm motility in ram semen peri pelletier,8 rachael gately,b bruce bartone 8cummings school of veterinary medicine, tufts university, north grafton, ma ~ufts veterinary field service, woodstock, ct cpopulation and quantitative health sciences university of massachusetts medical school worcester, ma objective was to investigate effects of extender, loading temperature, and equilibration time on postthaw sperm motility. ejaculates were collected with an artificial vagina from 4 rams from may july. each ejaculate was split into 3 aliquots and extended in a 1 step protocol to a concentration of 200 x 106 sperm/ml with a liposome-based extender (optixcell™), an egg yolk-based extender (triladyl®), and a soy lecithin-based extender (andromed®). half of each extended aliquot was loaded into straws at ambient temperature (23°c) and left to equilibrate for either 2, 4, or 12 hours at 4°c before cryopreservation in liquid nitrogen. remaining half was loaded at 4°c after equilibration before freezing. concentration and motility were measured with isperm® semen analyzer. linear mixed models were used for statistical analysis. results are expressed as least square means ± sem. postthaw sperm motility differed (p < 0.0001) among optixcell™ (37.1 ± 1.5%), andromed® (26.3 ± 1.8%), and triladyi® (30 ± 0.8%) extenders. ambient loading temperature (32.6 ± 1.5%) also had appreciable positive effects (p < 0.014 7) on postthaw motility compared to 4 °c (29 ± 1.1 % ). samples with an equilibration time of 2 hours had lowest (p < 0.0001) postthaw sperm motility (23 ± 1.2%) compared to 4 hours (33.8 ± 2.3%) and 12 hours (35.6 ± 1.5%) equilibration. however, sperm postthaw sperm motility between 4 hours and 12 hours equilibration was not different (p < 0.4270). in conclusion, it is optimal to extend ram semen with optixcell™ to avoid risk of microbial contamination that comes with animal-based extenders, load at ambient temperature which is less labor intensive and may limit possibility for temperature fluctuations, and to equilibrate at 4°c for 4 hours which could lengthen the window of ai postthaw. this protocol is ideal for efficiency and attractive to those without experience or cuttingedge equipment to freeze semen. keywords: extender, cryopreservation, ram semen, motility, isperm, temperature 418 clinical theriogenology • volume 12 number 3 • september 2020 sonographic appearance of late pregnancy sheep fetal intestine and kidney elizabeth frieden,• victoria monroe," alyssa helms," sierra guynn," julie cecere," orsolya balogh,• sherrie clark,8 robyn ellerbrock,h jamie stewart8 "virginia-maryland college of veterinary medicine, blacksburg, va huniversity of georgia college of veterinary medicine, athens, ga objective was to identify changes in sonographic characteristics of late pregnancy ovine fetus associated with fetal maturity. ewes were synchronized, ram bred, and confirmed pregnant (n = 18) for potential cesarian section (c-section) at day 145 of pregnancy (d 0). transabdominal ultrasonography was performed at days (d) 22, 15, 10, 7, 4, 1, and o prepartum to assess fetal heart rate (hr), abdominal diameter, renal volume, corticomedullary distinction (cmd), and gastrointestinal peristalsis (gip). at day 1, 12 ewes were induced with 20 mg dexamethasone im and underwent elective c-section on day 0. at birth, lambs were assigned a vigor score (1 10) based on hr, respiratory rate, muscle tone, irritability reflex, and mucous membrane color. a total of 19 live and 3 dead lambs were delivered with an average vigor score of 7 ± 0.6. statistical analyses were performed using r. mean days prepartum were assessed using anov a. pearson's correlation analysis was used to assess associations. both cmd and gip were strongly correlated with days prepartum (r = 0.7, p < 0.001), whereas low correlation was observed with renal volume (r = 0.27, p = 0.006) and abdominal circumference (r = 0.25, p = 0.009). days prepartum differed for cmd categories (absent: 22 ± 0, indistinct: 9.1 ± 0.6, distinct: 0.5 ± 0.1; p < 0.001). days prepartum also differed for gip categories (no peristalsis: 12 ± 0.7, occasional peristalsis: 2.3 ± 0.4, frequent peristalsis: 0.6 ± 0.1; p < 0.001). days prepartum did not differ when fetal hr measured < 130bpm(7.1 ±0.7)or 130-149bpm(8.8± l.l;p = 0.43), butdifferedwhenfetalhrmeasured > 150 bpm (15 ± 1.5; p < 0.001). fetal hr prior to induction (day 1) was moderately correlated with vigor scores at birth (r = 0.55, p = 0.07). in summary, qualitative sonographic characteristics, such as onset of fetal gip and cmd were superior to quantitative measurements in determining fetal maturity. additionally, ultrasonographic assessment of fetal hr before induction may help to predict neonate survivability. keywords: ewe, pregnancy, gi peristalsis, renal development, ultrasonography clinical theriogenology • volume 12 number 3 • september 2020 419 efficacy of deslorelin acetate in advancing ovulation in timed artificial insemination in goats sarah legg, alyssa helms, kevin pelzer, sherrie clark, jamie stewart virginia-maryland college of veterinary medicine, blacksburg, va objective was to determine the efficacy of deslorelin acetate in advancing ovulation for use in timed artificial insemination {tai) protocols in goats. does received an intravaginal cidr containing 0.3 g progesterone and 10 mg dinoprost tromethamine im at day -5. after cidr removal (day 0), does received 0.2 mg deslorelin acetate (n = 9) or saline ( control, n = 10) im. transrectal ultrasonography was performed twice daily to measure follicular growth and determine time to ovulation. subsequently, does were synchronized similarly and left untreated ( control, n = 42), administered 0.2 mg deslorelin im (n = 42), or administered 5 ml p.g. 600® im (n = 42). tai was performed 48 56 hours after cidr removal using fresh semen from 1 of 3 bucks, randomly distributed among treatments. does were then housed with assigned bucks for natural breeding. transabdominal ultrasonography was performed at 50 and 90 days after tai to assess pregnancy status. statistical analyses were performed using r. time to ovulation was analyzed using a kruskal-wallis test and number of ovulations was analyzed by student's t-test. pregnancy rates were analyzed by anova using treatment and buck as variables. deslorelin-treated does had increased (p < 0.01) number of ovulations (2.9 ± 0.4) over control does (1.5 ± 0.2). hours to ovulation tended to be less in deslorelin-treated does (53 ± 11) than in control does (61 ± 11; p = 0.06). pregnancy per ai was affected by both treatment (p < 0.01) and buck (p = 0.04). control does bred by buck 1 had higher (p = 0.02) percent pregnancy rates (50) than those treated with deslorelin (7) or p.g. 600® (14). deslorelin-treated does also had decreased (p < 0.01) in overall pregnancy rates across all bucks. it is concluded that deslorelin may have superovulatory effects impacting does' cyclicity and ability to achieve pregnancy. further research is needed to investigate use of lower doses of deslorelin in goat estrus synchronization programs. keywords: artificial insemination, caprine, deslorelin, estrus synchronization, pregnancy 420 clinical theriogenology • volume 12 number 3 • september 2020 monozygotic twins in a thoroughbred mare bred by natural cover katelyn kimble, charles love college of veterinary medicine, texas a&m university, college station, tx twin pregnancies in mares are undesirable, with a majority resulting in abortion or neonatal death. 1 most twin pregnancies are dizygotic and originate from 2 oocytes rather than division of an embryo after fertilization (monozygotic ). the exact etiology of monozygotic twin occurrence is not well understood in humans2 • 3 or horses.4 it is known, however, that although the natural occurrence of monozygotic twins is rare in both horses and humans (only 0.4% of in utero conception pregnancies in humans), the rate more than doubles when using assisted reproductive technologies.2 • 5 • 6 a 13 year old thoroughbred mare was evaluated by transrectal ultrasonography while in estrus and was bred naturally (natural cover) the same day a 40 mm follicle was identified. a single ovulation was detected by ultrasonography on the following day. at 14, 15, 16, 20, 26, and 30 days postovulation, a single embryo presence was observed. on day 33, 2 heartbeats (apparently from 2 embryos) were noticed and the vesicle measured 36 mm. presence of2 embryos was confirmed on day 34. due to the inherent risks of twinning, her uterus was lavaged to remove and recover the embryos for analysis. two distinct embryos were present within a single chorionic membrane. tissue samples from both embryos were analyzed for genetic markers, specifically 13 microsatellites, and both embryos had identical genotypes. this case of monozygotic twins was unusual in that it was not associated with assisted reproductive technologies, but with natural cover. 1• 7 unlike the diagnosis of dizygotic twins, which can be determined around 14 days postovulation, monozygotic twins are often not diagnosed until around 30 days postovulation.8 it is therefore important that mares diagnosed with single vesicles early in pregnancy, be re-examined~ 30 days postovulation to ruleout the presence of monozygotic twins. 8• 9 even though monozygotic twins have been successfully reduced later in pregnancy, 10 most likely both embryos will be lost. keywords: mare, monozygotic twins, natural cover references 1. meadows sj, binns mm, newcombe jr, et al: identical triplets in a thoroughbred mare. equine vet j 1995;27:394-397. 2. vitthala s, gelbaya ta, brison dr, et al: the risk ofmonozygotic twins after assisted reproductive technology: a systematic review and meta-analysis. hum reprod update 2009;15:45-55. 3. toledo mg: is there increased monozygotic twinning after assisted reproductive technology? aust nz j obstet gyn 2005;45:360-364. 4. dijkstra a, cuervo-arango j, stout t ae, et al: monozygotic multiple pregnancies after transfer of single in vitro produced equine embryos. equine vet j 2019;0:l-4. 5. aston ki, peterson cm, & carrell dt: monozygotic twinning associated with assisted reproductive technologies: a review. reproduction 2008;136:377-386. 6. kanter jr, boulet sl, kawwass jf, et al: trends and correlates ofmonozygotic twinning after single embryo transfer. obstet gynecol 2015;125:111-117. 7. govaere j, hoogewijs m, de schauwer, et al: an abortion ofmonozygotic twins in a warmblood mare. reprod domest anim 2009;44:852-854. 8. mccue pm, thayer j, & squires el: twin pregnancies following transfer of single embryos in three mares: a case report. j equine vet sci 1998; 18:832-834. 9. roberts ma, london k, campos-chillon lf, et al: presumed monozygotic twins develop following transfer of an in vitro-produced equine embryo. j equine vet sci 2015;26:89-94. 10. sper rb, whitacre md, bailey cs, et al: successful reduction of a monozygotic equine twin pregnancy via transabdominal ultrasound-guided cardiac puncture. equine vet educ 2011;24:55-59. clinical theriogenology • volume 12 number 3 • september 2020 421 pregnancy toxemia in the bitch kalie beckers, viviane gomes, jennifer sones, carlos pinto veterinary clinical sciences, school of veterinary medicine louisiana state university, baton rouge, la canine pregnancy toxemia can be a life-threatening condition for the dam and fetuses. the condition is characterized by hypoglycemia, ketonemia, ketonuria, and hepatic lipidosis during late pregnancy and clinical signs include weakness, collapse, seizures, and coma. pregnant bitches bearing a large litter are more predisposed, especially if they develop anorexia during the last 2 weeks of pregnancy. the diagnosis is made by association of clinical signs, hypoglycemia and, importantly, high concentrations of ketone bodies in the blood and urine. 1 • 3 measurement of ketone bodies (p-hydroxybutyrate) is commonly performed in food animal medicine; however, not often in small animal practice, which may impair the diagnosis of pregnancy toxemia. if diagnosed early, the condition can be resolved with enteral and/or parenteral glucose supplementation. however, in severe cases, pregnancy termination may be required. a 2 year old female labrador retriever presented with 1-week history of vomiting and severe lethargy. the bitch had been accidentally bred 65 days prior to presentation. tachypnea, distended abdomen, and engorged mammary glands were noted initially on physical examination. hypoglycemia (38 mg/dl) and hypocalcemia (9.1 mg/dl) were observed. serum ketone measured 1.0 mmol/1 (normal range, 0.0 0.1 mmol/1). plasma progesterone concentration was 1.07 ng/ml. twelve fetal skeletons were seen radiographically. on ultrasonography, fetal viability and maturation were confirmed by fetal heart rates, presence of well-defined fetal renal corticomedullary distinction and intestinal peristalsis. based on those findings, cesarean section was elected. surgery was performed without complication and 12 healthy puppies were successfully delivered. dam received vetivex® (lactated ringer's with 2.5% dextrose) and 22 mg/kg calcium gluconate. this case highlighted the importance of maintaining proper nutrition and energy balance in late-term pregnancy and measuring ketone bodies in an anorexic pregnant bitch. keywords: canine, pregnancy, hypoglycemia, ketonemia references 1. johnson ca: glucose homeostasis during canine pregnancy: insulin resistance, ketosis, and hypoglycemia. theriogenology 2008;70: 1418-1423. 2. johnston sd, root kustritz mv, olson ps: in: canine and feline theriogenology, ist edition, philadelphia; saunders: 2001. p. 80. 3. kustritz mvr: pregnancy diagnosis and abnormalities of pregnancy in the dog. theriogenology 2005;64:755-765. 422 clinical theriogenology • volume 12 number 3 • september 2020 dystocia due to congenital hydrocephalus in a miniature dachshund collen kutzler,8 margaret root-kustritz,8 michelle kutzlerb °college of veterinary medicine, university of minnesota, st. paul, mn bcollege of agricultural sciences, oregon state university, corvallis, or congenital hydrocephalus (ch) is an active distension of the ventricular system that develops because of either an interruption of cerebrospinal fluid (csf) flow or absorption. chihuahua, pomeranian, pug, pekingese, english bulldog, lhasa apso, yorkshire, boston, and maltese terrier breeds are predisposed to ch. 1 although dachshunds have a relatively low frequency of congenital defects compared to other breeds,2 ch has been previously reported.3 however, there have been no published reports of ch resulting in dystocia in this breed. an 8 year old multiparous miniature dachshund presented with a dystocia persisting for > 8 hours. the bitch was clinically stable on physical examination and a digital vaginal examination revealed no fetal parts. transabdominal ultrasonography of uterus revealed 4 fetuses with heart rates between 150 250 bpm. a catheter was placed in the right cephalic vein and the bitch was anesthetized with propofol. anesthesia was maintained with sevoflurane. a routine hysterotomy was performed and all 4 fetuses were delivered alive, including a fetus with a large, dome­ shaped head that was lodged in the cranial vagina. no other congenital defects were present during gross postnatal examination of pups. puppies were followed to 9 weeks of age. toe pup with the ch developed normally and displayed no neurologic signs other than a consistent lack of menace reflex. transfontanelle ultrasonography at 4 weeks of age revealed the lateral ventricles were greatly distended with anechoic fluid and lined by a thin wall of cortical tissue. at 9 weeks of age, the pup (2 kg) began acutely and inconsolably crying and began circling with head pressing. euthanasia was performed and necropsy findings revealed 5 open fontanelles, occipital dysplasia, and symmetrical ventriculomegaly with periventricular cortical atrophy. prior to ossification of the cranial sutures, ch may cause abnormalities of skull development such as a thinning of the bone structure, a dome-shaped appearance to the head, and/or persistent fontanelles. 1 in the current case, each ventricle contained approximately 10 ml of cerebrospinal fluid. concurrent congenital abnormalities of the brain (e.g. intracranial arachnoid cyst, dandy-walker syndrome, chiari-like malformation) without ce were previously reported in dogs.4 no cerebellar or other abnormalities were present grossly and there was no histological evidence of neuronal degeneration or inflammation. toe most common reported malformation in ch is a stenotic mesencephalic aqueduct,4 which was the presumed cause in the current case, since no other lesions were present. in the current case, the formation of csf must have equilibrated with absorption, resulting in a compensated hydrocephalic state, until an acute blockage of the ventricular system occurred, resulting in rapid decompensation. keywords: canine, cranial deformity, dog, fetus, occipital dysplasia references 1. harrington mk, bagley rs, moore mp: hydrocephalus. vet clin north am small anim pract 1996;26:843-856. 2. nobre pacifico pereira kh, cruz dos santos correia le, ritir oliveira el, et al: incidence of congenital malformations and impact on the mortality ofneonatal canines. theriogenology 2019;140:52-57. 3. kobatake y, miyabayashi t, yada n, et al: magnetic resonance imaging diagnosis of dandy-walker-like syndrome in a wire-haired miniature dachshund. j vet med sci 2013;75:1379-81. 4. estey cm: congenital hydrocephalus. vet clin small anim 2016;46:217-229. clinical theriogenology • volume 12 number 3 • september 2020 423 fetal mummification and abortion secondary to umbilical cord torsion rachel doenges,8 heath king,8 richard hopper,b brittany baughman,8 darcie sidelinger8 8mississippi state college of veterinary medicine, mississippi state, ms b auburn university college of veterinary medicine, auburn, al a 14 year old, quarter horse, mare presented, having aborted the previous day. mare was bred by natural service and confirmed pregnant at 30 days of pregnancy. fetal crown rump length measured 24.8 cm and fetus weighed 318 grams. estimated fetal age was 4 5 months. fetus was partially mummified and visceral organs were autolyzed and appeared tan. there were > 20 counter-clockwise twists in the umbilical cord, extending from umbilicus to allantois. umbilical cord torsion is considered a major cause for noninfectious abortion in mares. reported frequency of abortion secondary to umbilical cord torsion varies, likely reflecting regional differences, as well as management differences leading to increased reporting. according to a retrospective study in uk, umbilical cord torsion accounted for 35.7% of all abortions and stillbirths. 1 some umbilical twisting were normal, averaging 4 5 twists.2 excessive torsion occludes umbilical vessels and the urachus, resulting in decreased perfusion and fetal death. umbilical edema, hemorrhage, thrombosis and fluid-filled sacculation were commonly observed along umbilical cord. 1• 3 long umbilical cords predisposed cords for excessive torsion, with cords > 84 cm having a higher risk. 1 • 4 etiology of abnormally long umbilical cords is unknown.2 mares that aborted due to umbilical cord torsion had successful subsequent pregnancies.2 fetuses were not expelled immediately after death and generally had autolytic changes. 3 abortions were most common at 6 8 months.2 infectious causes should be ruled out in all abortions to reduce transmission. in this case, fetus was tested for equine herpes virus 1 and leptospira interrogans using pcr and both were negative. a needle aspirate of lungs had only mucus. allantois was unremarkable. diagnosis of abortion secondary to umbilical cord torsion was confirmed. keywords: equine, mare, abortion, umbilical cord torsion, noninfectious references 1. smith kc, blunden as, whitwell ke et al: a survey of equine abortion, stillbirth and neonatal death in the u.k. between 1988 and 1997. equine vet j 2003:35:496-501. 2. frazer gs: umbilical cord compromise as a cause of abortion. equine vet educ 2007;19:535-537. 3. whitwell ke: morphology and pathology of the equine umbilical cord. j reprod fert suppl 1975:23;599-603 4. foote ak, ricketts sw, whitwell, ke: a racing start in life? toe hurdles of equine feto-placental pathology. equine vet j 2012;44:120-129. 424 clinical theriogenology • volume 12 number 3 • september 2020 left testicular rupture in a red angus bull sara dietz,8 heath king,8 richard hopper,b darcie sidelinger1 3mississippi state college of veterinary medicine, mississippi state, ms b auburn university college of veterinary medicine, auburn, al a 16 month old, red angus, bull presented with a history of bilateral scrotal swelling. on presentation, his scrotum was swollen and firm on palpation. there was a line of demarcation at the ventral aspect of scrotum, suggestive of trauma. pain was elicited on palpation of scrotum. ultrasonography of scrotum revealed a loss of normal homogenous echogenicity of testicular parenchyma and disruption of testicular architecture. no blood flow was detectable on the affected side with doppler examination. inguinal herniation was ruled out by ultrasonography of scrotum and transrectal palpation; however, diagnosis remained undetermined until surgery was performed. scrotal enlargement most commonly results from fluid accumulation in the vaginal cavity.1 typically this fluid can be readily observed by ultrasonography, but was not apparent in this case. traumatic rupture of testis has been reported to occur occasionally and can be diagnosed on palpation and confirmed by ultrasonography. unilateral castration was performed to conserve the function of the unaffected testis. it was readily apparent after incising the scrotum that the bull had suffered a traumatic rupture of left testis. there was a large blood clot contained within the scrotum and disruption of vaginal tunics and tunica albuginea. bull recovered uneventfully from surgery and was discharged with follow up appointments for suture removal and 90 days postsurgery for breeding soundness examination (bse). following unilateral castration, remaining testis hypertrophied and produced 75% of normal sperm capacity.2 although these bulls have been successfully used for breeding, they will not pass a bse with 1 testis. this bull returned to service and was maintained as a breeding bull for several years after his procedure. keywords: angus, bull, testis, hemicastration references 1. dwight f. wolfe: bull development: sexual development and puberty in bulls. in: hopper, rm: editor. bovine reproduction. 1st edition, ames; wiley-blackwell: 2015. p. 136-141. 2. wolfe d, unilateral castration for acquired conditions of the scrotum. in: wolfe df, moll hd: editors large animal urogenital surgery, 2nd edition, baltimore; williams and wilkins: 1998. p.313-320. clinical theriogenology • volume 12 number 3 • september 2020 425 successful treatment of ascending placentitis in a thoroughbred mare hannah neer, daniela orellana, ghislaine dujovne, stuart meyers w. pritchard veterinary teaching hospital, school of veterinary medicine university of california-davis, davis, ca ascending placentitis in mares is 1 of the leading causes of late-term abortions, premature delivery, and neonate morbidity and mortality. 1 a 5 year old nulliparous thoroughbred recipient mare presented on the 306'11 day of pregnancy for vulvar discharge without mammary development. fetal viability was confirmed by transrectal and transabdominal ultrasonography. an increase in the combined thickness of the uterus and placenta (ctup [17 mm]), was detected via transrectal ultrasonographic examination, although no discharge was evident on vaginal speculum examination. based on examination findings, treatment for ascending placentitis was initiated using an established treatment protocol. 2 antimicrobial (24 mg/kg oral sulfadiazine and trimethoprim twice daily), antiinflammatory (8.6 mg/kg oral pentoxifylline twice daily), and tocolytic (0.088 mg/kg of oral altrenogest daily) agents were given. in addition, 10 mg of estradiol cypionate (im, once every 3 days, 3 doses total) was given, as it had improved clinical outcome of ascending placentitis cases. 3 a decrease in ctup was observed on transrectal ultrasonographic examination 8 days after initial presentation. however, all measurements did not return to normal limits; therefore, treatment had to be continued. all measurements of ctup were within normal limits on transrectal ultrasonographic examination 16 days after initial presentation and the ascending placentitis was considered resolved. transrectal and transabdominal ultrasonographic examination continued throughout pregnancy to monitor fetal health and screen for recurrent ascending placentitis. on 350th day of pregnancy, the mare delivered a healthy colt and grossly normal fetal membranes. the colt stood and nursed without assistance < 2 hours after parturition. histopathological examination of fetal membranes demonstrated no evidence of placentitis. this case demonstrated that successful outcomes are possible in cases of ascending placentitis with early detection and aggressive treatment. keywords: mare, ascending placentitis, parturition references 1. leblanc mm: ascending placentitis in the mare: an update. reprod dom anim 2010;45:28-34. 2. bailey cs, macpherson ml, pozor ma, et al: treatment efficacy oftrimethoprim sulfamethoxazole, pentoxifylline, and altrenogest in experimentally induced equine placentitis. theriogenology 2010;74:402-412. 3. curcio br, canisso if, pazinato fm, et al: estradiol cypionate aided treatment for experimentally induced ascending placentitis in mares. theriogenology 2017; 102:98-i 07. 426 clinical theriogenology • volume 12 number 3 • september 2020 anasarca in a neonatal bulldog puppy, secondary to a ventricular septal defect claudia acevedo, jennine lection, lacey rosenberg, soon hon cheong, mariana diel de amorim department of clinical sciences, cornell university, ithaca, ny a 2 year old primiparous english bulldog presented for fetal monitoring prior to elective cesarean section. bitch was bred via transcervical insemination and her singleton pregnancy was confirmed via transabdominal ultrasonography and radiographs. due to risk of primary uterine inertia and fetal oversize with 1 fetus, and due to being a brachycephalic breed, a cesarean section was scheduled. first fetal monitoring was performed at 64 days post lh peak; at this visit, bitch had colostrum present within caudal mammary glands, and her progesterone concentration was 6.32 ng/ml. on ultrasonography, although the fetal heart rate was normal (226 232 bpm), fetus appeared to have pleural effusion and subcutaneous edema, consistent with an anasarca puppy. cesarean section was performed on the following day. a single pup with severe subcutaneous edema (confirmed ultrasonography findings) was removed. euthanasia was elected due to poor prognosis and necropsy followed. gross examination revealed a ventricular septal defect, a finding that has been associated with hydrops fetalis in human and veterinary species. 1 renal tubules and renal pelvis were mildly dilated, suggestive of urinary obstruction presumptively due to severe anasarca. on histologic examination, hepatocellular necrosis and congestion of various organs were noticed (lesions secondary to decreased left-sided cardiac output). in summary, ultrasonography was diagnostic (anasarca fetalis and pleural effusion) and findings enabled us to decide on a timely surgery. reference 1. huggon ic, cook ac, smeeton nc, et al: atrioventricular septal defects diagnosed in fetal life: associated cardiac and extra-cardiac abnormalities. j am coll cardiol 2000 36:593-601. clinical theriogenology • volume 12 number 3 • september 2020 427 ejaculatory failure in a halter stallion due to dorsal displacement of soft palate breanthony baker, charles love department of large animal clinical sciences college of veterinary medicine & biomedical sciences texas a&m university, college station, tx exercise-induced dorsal displacement of soft palate (ddsp) commonly occurs in racehorses, characterized by decreased performance; 1 however, it has not been identified as a cause for failure of ejaculation. in may 2019, a 5 year old maiden american quarter horse halter stallion was presented for failure of ejaculation. during breeding soundness examination, stallion demonstrated normal libido and mounting behavior during repeated mounts on phantom, but never ejaculated. while mounted, stallion expressed labored breathing that owner had noticed during previous breeding attempts. manual palpation of scrotum, accessory sex glands, and penis were normal. an echocardiogram and chemistry analysis for lactic acid, alkaline phosphatase, and creatine kinase were normal. static endoscopy of larynx was normal, but dynamic endoscopy during stallion's mount on phantom revealed 50 70% of airway became blocked by ddsp. to correct this condition, a laryngeal tie-forward procedure was performed. following 2-week postoperative recovery under owner's supervision, stallion was able to ejaculate normally and repeatedly. ejaculation disorders in stallions were attributed to physical, musculoskeletal, psychogenic, or neurologic causes;2 apparently, ddsp has not been reported before as a cause. halter-type stallions with ejaculatory dysfunction have been observed to exhibit labored breathing during and following breeding and was assumed to be a "common" breed characteristic. occurrence of ddsp is associated with exercise intolerance; however, exact etiology is unknown. 1 in this stallion, increased activity was associated with breeding-induced displacement of soft palate above epiglottis. as a result, stallion was unable to complete ejaculation, due to blockage of airway. following surgical correction, stallion was able to ejaculate. ejaculatory dysfunction in stallions can be difficult to assess; therefore, ddsp should be considered as an underlying cause, especially when abnormal respiratory sounds are heard during breeding. keywords: halter stallion, ejaculatory failure, ddsp references 1. cercone m, olsen e, perkins jd, et al: investigation into pathophysiology of naturally occurring palatal instability and intermittent dorsal displacement of the soft palate (ddsp) in racehorses: toyro-hyoid muscles fatigue during exercise. plos one 2019;14(10): e0224524. 2. mcdonnel sm: ejaculation. physiology and dysfunction. vet clin n am-equine 1992;8:57-70. 428 clinical theriogenology • volume 12 number 3 • september 2020 priapism in a quarter horse gelding hannah carter,8 heath king,8 richard hopper,b darcie sidelingei1 8mississippi state college of veterinary medicine, mississippi state, ms bauburn university college of veterinary medicine, auburn, al priapism is an uncommon finding in domestic animals; however, it is reported in horses. it has multiple etiologies, including use of phenothiazine tranquilizers for routine procedures such as sheath cleaning and dentistry. 1 priapism results in a selective engorgement of corpus cavemosum and corpus spongiosum that ultimately leads to venous occlusion. 2 exact mechanism for initial failure of detumescence is not known, but ultimately, red blood cells become sickle shaped in response to increased partial pressure of c02.3 this deformation in red blood cells can lead to permanent obstruction of venous drainage. a 25 year old, quarter horse gelding was presented with paraphimosis (noticed 2 days prior to presentation) secondary to priapism. his teeth had been floated 1 week earlier, under acepromazine sedation. initial efforts to address priapism included placement of 12 gauge trocar needles, 1 dorsally and 1 ventrally within corpus cavemosum for irrigation with heparinized saline. egress fluid from ventral trocar needles were thick, dark, and serosanguineous. as flush continued, fluid consistency changed to thin, bright red, serosanguineous to sanguineous, suggesting intact arterial supply. phenylephrine was also administered directly into corpus cavemosum to promote vasoconstriction and reduction of priapism. complete detumescence was not achieved by flush or phenylephrine treatment. a vascular shunt was established from corpus cavemosum to corpus spongiosum to relieve venous obstruction. two days after the procedure, there was minimal improvement. humane euthanasia was performed due to quality of life and financial constraints. at necropsy, vascular shunt (communication between corpus spongiosum and corpus cavemosum) appeared intact. chronicity of the disease might have prevented full resolution and recovery, despite creation of a functional vascular shunt. keywords: equine, gelding, priapism, acepromazine, corpus cavemosum, corpus spongiosum references 1. brinsko sp, blanchard tl, varner dd, et al: surgery of the stallion reproductive tract. in: brinsko sp, blanchard tl, varner dd, et al: editors. manual of equine reproduction. 3rd edition, mary land heights; elsevier: 2011. p. 263-272. 2. rochat, mc. priapism: a review. department of veterinary clinical sciences, college of veterinary medicine oklahoma state university. 2001. 3. hinman f. priapism: reasons for failure of therapy. j urol 1960;83:420. clinical theriogenology • volume 12 number 3 • september 2020 429 changes in milk ph, milk protein, and udder development in late-pregnant ewe and effects on lamb serum total protein concentrations mary clapham, victoria monroe, alyssa helms, sierra guynn, kevin pelzer, sherrie clark, jamie stewart virginia-maryland college of veterinary medicine, blacksburg, va objective was to describe changes in milk ph, milk protein concentrations, and udder development in late-pregnant ewes. ewes were synchronized, ram bred, and confirmed pregnant (n = 18) for potential cesarean section at 145 days of pregnancy (d 0). udder score was assessed on days 22, 15, 10, 7, 4, 1, and o prepartum. milk ph (digital ph meter) and protein concentrations (brix refractometer) were monitored once daily from days 6 to o prepartum. twelve ewes were selected for elective cesarean section and induced with 20 mg dexamethasone im on day 1. at birth, lambs were assigned a vigor score (1 10) based on heart rate, respiratory rate, muscle tone, irritability reflex, and mucous membrane color. a total of 19 live and 3 dead lambs were delivered. serum total protein (tp) was measured in lambs from 24 to 36 hours after birth. statistical analyses were performed using r. data on days prepartum and lamb tp were analyzed using anov a. pearson's correlation analysis was used to determine associations. there was moderate correlation in days prepartum with both udder development (r = 0.53, p < 0.001) and milk ph (r = 0.46; p < 0.001 ), but no correlation with milk protein concentrations (r = 0.11, p = 0.25). days prepartum did not differ between udder scores of o (no development: 14 ± 3 .8 days) and 1 (minor development: 12 ± 1.2 days; p = 0.96), but did change for scores of 2 (moderate development: 6.6 ± 0.5 days) and 3 (full development: 3.4±0.4 days; p :'.so .03). days prepartum differed when milk ph measured > 6.5 (4.4 ± 0.3 days), 6 6.5 (2.1 ± 0.2 days), and< 6 (0 ± 0 days; p < 0.001). lamb tp was not affected by milk protein concentrations at d o (p = 0.96), but tended to be affected by vigor score (p < 0.06). we concluded that udder score and milk ph may be useful for predicting parturition in ewes. additionally, assessing neonate vigor at birth can help influence colostrum management decisions. keywords: milk ph, milk protein, neonate vigor, parturition, udder development 430 clinical theriogenology • volume 12 number 3 • september 2020 canine herpes virus in a breeding kennel of wirehaired dachshund jordan farrell, jamie douglas, aime johnson, robyn wilborn college of veterinary medicine, auburn university, auburn, al a 3-year-old, wirehaired dachshund presented to auburn university veterinary hospital 53 days postovulation for dystocia and delivery of premature stillborn puppies. rapid slide agglutination testing (rsa t) was performed for suspicion of bruce/la can is infection, but was negative. seven days later, the patient's kennelmate was presented 57 days postovulation for abortion, and bruce/la rsat was again negative. canine herpesvirus ( cahv-1) was then investigated as a possible causative agent of serial abortions. a vaginal swab from the second patient was subjected to direct fluorescent antibody testing. results were positive for cahv-1 antigen. symptoms of cahv-1 infection include abortion, stillbirth, and infertility in bitches, whereas cahv-1 can also cause "fading puppy syndrome" in neonates. client returned 2 days later with 4 additional pregnant dams for testing. vaginal swabs from all females tested positive for cahv-1. all bitches also possessed a low or negative titer to cahv-1. based on these results, future litters were considered 'at risk' for contracting cahv-1 infection. recommendations included increasing the temperature in the neonatal area and implementation ofbiosecurity. pregnant dams were prescribed oral acyclovir (20 mg/kg), to be administered every 6 hours until puppies were 3 weeks old. once whelped, puppies received oral acyclovir solution at 15 mg/kg until 3 weeks old. with 3 successful litters, 13 puppies were born and alive at 6 months. owners revealed that a new stud dog had been imported from germany approximately 1 month prior to the initial abortion. this case highlighted the importance and magnitude of cahv-1 infection in breeding bitches and neonates. though most adult dogs are not clinically affected by cahv-1, the virus can rapidly result in widespread loss in a breeding kennel by compromising the health of serologically naive dams and neonates. in the case of this kennel's cahv-1 outbreak, a positive outcome was reached through proper biosecurity and veterinary intervention. keywords: canine herpes, abortion, stud dog references 1. dubovi ej, maclachlan nj: editors. fenner's veterinary virology. 5th edition. cambridge: academic press; 2017. p. 106-128 2. greene ce: canine herpes virus infection. in: greene ce, editor. infectious diseases of the dog and cat. 4th edition, st. louis: w.b. saunders; 2013. p. 48-54. clinical theriogenology • volume 12 number 3 • september 2020 431 a retrospective analysis on determinants of litter size in a colony of working dogs jordan farrell,8 jamie douglas,8pamela haney/ aime johnson,a robyn wilborn3 adepartment of clinical sciences, bcanine performance sciences college of veterinary medicine, auburn university, auburn, al successful breeding and increased litter size are paramount to increasing working dogs' numbers for national security. we determined key factors affecting pregnancy and litter size in canine performance sciences (cps) breeding colony. specific aims of this research were to elucidate factors that improved breeding outcomes in the colony. data from cps breeding population for 55 breeding cycles for 26 females over 5 years were analyzed. factors analyzed were: age of dam at breeding, semen type (fresh, cooled-shipped, frozen), insemination method (vaginal ai, transcervical insemination [tci], live cover), and total number of inseminations. multiple regression model was used. above factors influenced (p < 0.001) and explained 57% of the variance in litter size. addition of 1 tci increased (p = 0.005) litter size. addition of i live breeding, although not different (p = 0.07) had clinical relevance (increased litter size). average litter size of a 30 month old dam using fresh semen ai was 5.45 puppies and cooled­ shipped semen ai was 7 ± 1 puppies. age of dams at breeding ranged from 13 88 months and there was no effect of age or parity on litter size. results indicated that maximum litter size occurred when fresh semen was utilized, with cooled-shipped semen being equivalent. use of frozen semen was least successful, resulting in negative effects on pregnancy rate in this population. scrutiny of insemination methods revealed that addition of 1 live cover increased litter size by 2 puppies, whereas addition of 1 tci increased litters by 3 puppies. in summary, incorporation of i live breeding and 1 tci utilizing fresh semen increased average litter size in cps breeding colony. keywords: canine, semen, transcervical, litter, breeding 432 clinical theriogenology • volume 12 number 3 • september 2020 foal born using intracytoplasmic sperm injection to a friesian mare with unexplained subfertility alexandra grillos, daniela orellana, stuart meyers, ghislaine dujovne department of population health and reproduction university of california davis, davis, ca intracytoplasmic sperm injection (icsi) is an advanced reproductive technology wherein oocytes are retrieved from tertiary follicles and injected with a single sperm to develop into an embryo in vitro. unlike embryo transfer, icsi affords versatility for mares with a suboptimum uterine environment, blocked oviducts, or at postmortem where the opportunity for natural fertilization is not possible. icsi can also be used when limited numbers of poor-quality sperm are available. 1 this technique provides the possibility to obtain foals from mares that otherwise could not propagate their genetics. a 13 year old friesian mare presented to the uc davis theriogenology service in may 2013 for breeding with cooled semen after several years of unexplained subfertility. uterine cytology was taken at multiple intervals, with no clear evidence of endometritis. on ultrasonography, anechoic fluid was present in the uterus after all breeding attempts. treatments included uterine lavages and oxytocin to enhance uterine clearance. in 2016, after several unsuccessful breeding cycles, laparoscopic application of prostaglandin e2 in the oviducts was performed, with the suspicion that they could be blocked.2 after the procedure, there were 2 more years of unsuccessful breeding attempts. in 2018, it was decided that this mare was an ideal candidate for oocyte retrieval and icsi. in may 2018, transvaginal aspiration of oocytes was performed, where 15 follicles between 10 30 mm were aspirated from both ovaries. of the 15 follicles aspirated, 9 oocytes were recovered. four of these oocytes matured to the mii stage in vitro and underwent icsi. two embryos matured and were shipped as blastocysts to colorado state university where they were implanted into 2 recipient mares. both embryos survived until fetal heartbeats were detectable, with 1 pregnancy surviving to term. the 7 year old recipient mare was transported to uc davis where she foaled successfully and without complications. keywords: equine, intracytoplasmic sperm injection, mare subfertility references 1. colleoni s, barbacini s, necchi d, et al: application of ovum pick-up, intracytoplasmic sperm injection and embryo culture in equine practice. proc am assoc equine pract 2007;53:554-559. 2. allen wr. wilsher s, morris l, et al: laparoscopic application of pge2 to re-establish oviducal patency and fertility in infertile mares: a preliminary study. equine vet j 2006;38:454-459. clinical theriogenology • volume 12 number 3 • september 2020 433 granulosa cell tumor and prolonged estrus in a boxer bitch christina havrila,a alyssa helms,8 phillip sponenberg,b julie cecerea adepartment of small animal clinical sciences bdepartment of biomedical sciences and pathobiology virginia-maryland college of veterinary medicine, blacksburg, va an 11 year old, multiparous boxer bitch was presented to the virginia-maryland college of veterinary medicine theriogenology service for evaluation of persistent vulvar swelling and bloody vulvar discharge of 34 days duration. on physical examination, the vulva was markedly swollen, with a small amount of blood-tinged discharge. vaginal cytology revealed 100% superficial epithelial cells, confirming a diagnosis of estrogen influence. thorough history revealed no known exposure to exogenous estrogens. abdominal ultrasonography was performed to identify potential structural abnormalities of the reproductive tract and/or adrenals which could be responsible for estrogen influence. ultrasonography revealed a markedly thickened myometrium and endometrium, with unremarkable ovaries and adrenal glands. furthermore, serum progesterone concentrations via chemiluminescence werel.02 ng/ml. based on the clinical findings, our differential diagnoses were microscopic disease of the ovary or exogenous estrogen exposure. because the bitch had no history of exogenous estrogen exposure, microscopic ovarian disease, such as a granulosa cell tumor, was considered to be the most likely differential. despite ovarian neoplasia being rather uncommon, granulosa cell tumors account for approximately half of all ovarian tumors in bitches. 1 it is not unusual for granulosa cell tumors to produce estrogen and therefore cause clinical signs consistent with a prolonged estrus. definitive diagnosis of granulosa cell tumors relies on histopathology of ovaries. the owner elected not to pursue recommended ovariohysterectomy and instead elected humane euthanasia due to orthopedic comorbidities, with postmortem necropsy. histopathology of a small (2 mm in diameter) round mass within an ovary revealed a well demarcated, proliferative mass made of tubules and small cysts lined with foamy basophilic cells, consistent with a granulosa cell tumor. this case demonstrated the importance of considering microscopic disease when presented with a bitch exhibiting prolonged estrus. keywords: granulosa cell tumor, prolonged estrus, ovarian neoplasia, bitch reference 1. white rn, brearley m: bsa va manual of canine and feline oncology. dobson j, lascelles d. gloucester, england: british small animal veterinary association; 2016. p. 248-264. 434 clinical theriogenology • volume 12 number 3 • september 2020 detection of tritrichonwnas foetus in a beef herd cecilia hemandez,8 julie gard schnuelle,8 richard hopper,8 katelyn waters,a lawerence cofield,8 yatta boakari,8 jessica rush,8 thomas passler,8 soren rodning,b sue duran8 8department of clinical sciences, college of veterinary medicine, auburn university, auburn, al ~epartment of animal sciences, college of agriculture, auburn university, auburn, al a herd of 150 angus cows had transrectal palpation for pregnancy diagnosis at the end of april 2019. eighty two percent of cows were determined to be pregnant. eight nonpregnant cows had pyometra that was diagnosed by transrectal palpation and confirmed via transrectal ultrasonography. pregnancies ranged from 25 to 110 days of age. four angus bulls were with the cows during a 120 day breeding season. last year's weaning in the group was 77.57%. smegma was collected from these 4 bulls via preputial scraping utilizing pizzle stick (lane manufacturing, denver, co). samples were placed in modified diamonds media and submitted to bishop-thompson-sparks-alabama state diagnostic laboratory and tested for tritrichomonas foetus (t foetus) dna, via a reverse transcriptase polymerase chain reaction (rt-pcr) assay. two of the 4 bulls tested positive for tfoetus, with strong positive cycle threshold (ct) values of 33.3 and 34.2. two bulls that tested positive were sold to slaughter and 2 remaining bulls were retested via rt-pcr and were negative again. all cows with pyometra were sold for slaughter. remainder of nonpregnant cows were treated im 3 times with 5 ml of dinoprost tromethamine (lutalyse) at 45 day intervals beginning in october 2019 to aid in clearing infection. these cows were then bred via ai in january 2020. the 2 positive bulls had been previously tested for t.foetus via rt-pcr and were negative in 2017 2018. therefore, it was speculated that t. foetus entered the herd through replacements from another state. this case highlighted the importance of testing all bulls yearly for t foetus when the herd is not a closed herd. keywords: angus bulls, tritrichomonasfoetus, pyometra, smegma, preputial scarping, rt-pcr clinical theriogenology • volume 12 number 3 • september 2020 435 genetic causes of golden retriever congenital hypomyelinating polyneuropathy blair hooser,8 shawna cook,8 dayna dreger,8diane shelton,c jennifer koziol,b kari ekenstedt' 8department of basic medical sciences, ~epartment of veterinary clinical sciences college of veterinary medicine, purdue university, west lafayette, in cdepartment of pathology, school of medicine, university of california, san diego, ca myelin is an important component of both central nervous system (cns) and peripheral nervous system (pns) by surrounding nerve cells, protecting them and aiding in perpetuation of signal conduction. myelin deficits can occur either by demyelination (myelin that initially formed properly and was then degraded) or by hypomyelination (myelin never correctly/fully formed at all). two cases of littermate, full-sibling golden retriever (gr) puppies with very young onset (i.e. congenital) had neurological deficits. 1 peripheral nerve biopsies revealed myelin sheath changes consistent with a predominantly hypomyelinating neuropathy. uniquely, this syndrome affected only the pns. previously reported hypomyelination syndromes in veterinary patients had either exclusively involved the cns, or both the cns and the pns together. we acquired dna from 4 grs presenting with clinical signs as reported; 1 nerve biopsies confirmed these dogs to have a congenital pns hypomyelinating neuropathy. we performed whole genome sequencing on dna from each of these 4 dogs. three causative private variants were identified, each in an excellent functional candidate gene known to cause charcot-marie­ tooth disease in humans: a loss of donor splice site variant in 1 dog, a missense variant in 1 dog, and a premature stop codon shared by 2 dogs. these 3 variants were all absent from > 1,000 canine whole genome sequences of normal dogs, and from > 200 control grs. while this condition is rare in the gr breed, breeders could use genetic tests for these variants to avoid producing affected puppies. in conclusion, theriogenology contains within its purview inherited congenital diseases. four cases of congenital hereditary neurological disease in golden retrievers, wherein, using modem whole genome sequencing technology, we identified the likely causative mutation in each of them. therefore, this is important for theriogenology service, as we can now offer each of these 3 variants as genetic tests to golden retriever breeders and prevent any more puppies born affected with this condition. reference i . braund kg, mehta jr, toivio-kinnucan m, et al: congenital hypomyelinating polyneuropathy in two golden retriever littermates. vet pathol !989;26:202-208. acknowledgment supported by theriogenology foundation, morris animal foundation, and the office of the director, national institutes of health (n1h) under award number ko 10d027051. 436 clinical theriogenology • volume 12 number 3 • september 2020 delayed uterine prolapse in a thoroughbred mare with retained fetal membranes joanna kania, sherrie clark, sera moran, nadia saklou department of large animal clinical sciences, virginia-maryland college of veterinary medicine virginia polytechnic institute and state university, blacksburg, va a 6 year old thoroughbred mare was presented to virginia-maryland college of veterinary medicine' s (vmcvm) equine field service for evaluation ofretained fetal membranes (rfm) after aborting twins the night before. fetal membranes retention was observed by the owners, with no progression for 5 6 hours. initial treatment included gentle manual traction of fetal membranes, administration of oxytocin and flunixin meglumine, and lavage of uterus. despite expelling large amounts of fluid after lavage, there was no further progress in fetal membranes expulsion. mare was given another oxytocin injection which caused continued myometrial contractions; however, there was no progress. due to duration of retention, worsening of clinical signs (increased digital pulses and injected mucus membranes), and lack of improvement, mare was referred to the vmcvm veterinary teaching hospital. she was resedated, and her uterus was lavaged again without dislodging fetal membranes. mare was placed on iv fluids and broad-spectrum antimicrobials, and was under continued sedation and oxytocin treatment. at 8 hours after presentation, mare's uterus prolapsed ( everted). mare was given an epidural, uterus was rinsed with hypertonic saline and replaced. a caslick's procedure was performed to aid in uterine retention. there was still a small amount ofrfm observed after replacement. hospitalization for treatment included twice-daily uterine lavages, iv fluids, oxytocin, antimicrobials, flunixin meglumine, and omeprazole. mare was also treated with ice boots, vasodilators, and antiinflammatories to prevent laminitis. mare was discharged 4 days later with no medications and did not experience any further complications. uterine prolapse (eversion) is rare in mares, often fatal (from infection), and usually occurs immediately or within a few hours after parturition. aggressive and prompt treatment ofrfm in mares is critical. regardless, as observed in this case, rfm may be considered a predisposing factor for delayed uterine prolapse. keywords: equine, uterine prolapse, retained fetal membranes clinical theriogenology • volume 12 number 3 • september 2020 437 importance of breeding management in abnormal estrous cycles: a case study tokaj kozak, leanna mottus crestwood veterinary centre, edmonton, alberta, canada in the expanding small animal breeding industry, accurate estrus detection and ovulation timing are essential for breeding management. many clinics rely on inhouse progesterone tests for convenient and efficient results. despite widespread use of these tests, it is critical to monitor progesterone concentrations comprehensively due to potential abnormalities in the estrous cycles of individual patients. such an anomalous cycle was diagnosed in suzie, a 3.5 year old fawn chinese pug, who was presented to crestwood veterinary centre for artificial insemination. suzie's baseline progesterone concentration on december 18, 2019 was 0.8 nmol/1 (0.25 ng/ml) with concurrent clinical proestrus signs. regular progesterone monitoring at 2 3 day intervals continued until december 27, 2019, where an increase to 4.0 nmol/1 (1.26 ng/ml) indicated an impending luteinizing hormone (lh) surge. despite this, on december 30, 2019, progesterone concentration decreased abruptly and was undetectable. vaginal cytology had cornified cells confirming estrus. suzie was bred on january 1 and 3 via artificial insemination with fresh semen; however, pregnancy was not achieved. a theriogenologist reviewed this case and suggested that suzie's progesterone decrease after an apparent lh surge indicated she had not ovulated, and therefore should not have been bred. her progesterone readings suggested an atypical estrous cycle, possibly a split heat or an.ovulatory cycle. 1 it was recommended to monitor suzie's next cycle using serial examinations of serum progesterone and vaginal cytology, and to breed only after confirmation of ovulation via serum progesterone 2 3 days after the perceived lh surge. it was also suggested to induce ovulation using gonadotropin-releasing hormone or human chorionic gonadotropin in case of repeated an.ovulatory cycles. with advances in theriogenology, we are better able to understand dysfunctional or abnormal canine estrous cycles and to provide suitable advice to our clients in their management. keywords: estrus, progesterone, canine, luteinizing hormone, artificial insemination, ovulation reference 1. meyers-wallen vv: unusual and abnormal canine estrous cycles. toeriogenology 2007;68:1205-1210. 438 clinical theriogenology • volume 12 number 3 • september 2020 sertoli cell tumor in an alpaca samantha mccarter,8 jamie stewart,8 meghan brookhart,3 sherrie clark,3 tanya leroithb 8department of large animal clinical sciences, ~epartment of biomedical sciences and pathobiology virginia maryland college of veterinary medicine virginia polytechnic institute and state university, blacksburg, va a 9 year old intact male alpaca was presented with a unilaterally enlarged right testis that the owner noticed after annual shearing. he had no previous breeding history, nor history of health or behavioral problems. physical examination was normal except for visible unilateral scrotal enlargement. scrotal palpation findings were, a large turgid right testis and a soft left testis, with normal epididymides. palpation elicited no pain response and ruled out the presence of epididymitis, herniation, or testicular torsion. testicular ultrasonography revealed a heterogeneous appearance of right testis parenchyma with diffuse multifocal areas of hyperechoic foci and several anechoic pockets. left testis was comparatively homogenous in appearance with rare hyperechoic foci. at this point, differential diagnoses included neoplasia or orchitis. a bilateral elective castration was performed, since this alpaca was not intended for breeding. after castration, right testis measured 50 x 54 mm, with 2 cystic structures and a bulging cream.­ colored bulging structure grossly identified upon dissection. left testis measured 37 x 32 mm with no gross abnormalities. histopathology diagnosed a sertoli cell tumor that completely replaced the stroma in the right testis and diffuse degeneration with no tumor infiltration in left testis. spermatic cord was also free from tumor involvement. the alpaca recovered from castration uneventfully. testicular neoplasia in camelids is rare. while sertoli cell tumors have been reported in dogs, bulls, rams, cats, and horses, apparently, this is the first report in camelids. sertoli cell tumors are characteristically hormonally active. while no symptoms were present in this case, degeneration of contralateral testis suggested that the tumor might have been secreting estrogen. bilateral castration proved to be curative for this alpaca; however, since no metastasis was evident, unilateral castration might have preserved fertility, if there was an intention to use the animal for breeding. keywords: sertoli cell tumor, testicular enlargement, testicular degeneration, alpaca references 1. carreira vs, langohr im, ferrari hf, et al: pathology in practice. j am vet med assoc 2012;241:55-57. 2. jubb kvf, kennedy pc: palmer n: pathology of domestic animals. 5th edition, volume 3, cambridge; academic press: 2007. p. 506-507. 3. scott cj, christensen bw, dechant je, et al: theriogenology question of the month. j am vet med assoc 2015;24 7: 1105-1108. 4. tibary a, vaughan j: reproductive physiology and infertility in male south american camelids: a review and clinical observations. small rumin res 2006;61:283-298. clinical theriogenology • volume 12 number 3 • september 2020 439 scrotal hydrocele in an angus bull maryanna mcclure,8 heath king,8 richard hopper,b darcie sidelinger8 8mississippi state college of veterinary medicine, mississippi state, ms bauburn university college of veterinary medicine, auburn, al a 5 year old, angus, bull presented with a history of left testicular swelling. ultrasonography of scrotum revealed a normal appearing right testis and the presence of fluid containing strands of hyperechoic material surrounding left testis. no bowel was appreciated within the scrotum. semen evaluation revealed decreased numbers of sperm with adequate motility. white blood cells were present on the semen slide and proximal droplets were seen within individual sperm. the fluid surrounding left testis indicated unilateral scrotal hydrocele. hydrocele is an abnormal accumulation of serous or inflammatory fluid within the vaginal cavity of testis.1 in mature bulls, hydrocele results from local inflammation or trauma.2 most commonly, scrotal hydroceles present with swelling in scrotum.3 ultrasonographic examination reveals varying amounts of anechoic fluid surrounding the epididymis and testis with normal testicular echotexture.3 • 4 with fluid accumulation, testicular thermoregulation is disrupted, resulting in decreased semen quality.3 treatment of this condition is directed at removing the inciting cause of fluid accumulation.3 in this case, a hemicastration of left testis was performed with the possibility ofregaining reproductive potential. cystorelin (gnrh) was administered to increase spermatogenesis in the remaining testis. at re-examination, appropriate postoperative healing was noted. a semen sample obtained via electro-ejaculation had 16% normal cells, 79% cells having primary abnormalities and 5% cells having secondary abnormalities. other parameters of the breeding soundness examination were concluded to be within normal limits. the scrotal circumference was 29 cm. prognosis for reproductive performance in bulls with unilateral hydrocele is good with prompt removal of affected testis.3 since spermatogenesis can be impaired for 2 6 months following testicular insult, final recommendations regarding fertility should only be made after allowing adequate time for recovery.3 • 5 keywords: hydrocele, bull, castration, bse, ultrasonography references 1. bartholomew ab, caudle ab, peroni jf: unusual clinical presentation of scrotal hydrocele in a tennessee walking stallion. equine rounds compendium, 2003;632-634. 2. abbitt b, fiske r. craig t: scrotal hydrocele secondary to ascites in 28 bulls. j amer vet med educ 1995;207:533-756. 3. smith bp, van metre dc, pusterla n: large animal internal medicine (6th edition), 2020, st. louis, missouri, p. 1513. 4. abu-seida ama: ultrasonographic diagnosis of some scrotal swellings in bulls. pak vet j 2012;32:78-381. 5. shore md, bretzlaffkn, thomson ja, et al: outcome of scrotal hydrocele in 26 bulls. j amer vet med educ 1995; 207:6,757-760. 440 clinical theriogenology • volume 12 number 3 • september 2020 bilateral ovarian adenocarcinoma in a geriatric shih tzu miranda senft,8 lacey rosenberg,8 jen.nine lection,8 anna mitchell,8 kathleen kelly/ nicholas vetter, b soon hon cheong, a mariana diel de amorim a 8department of clinical sciences, ~epartment of biomedical sciences college of veterinary medicine, cornell university, ithaca, ny a 15 year old female intact shih tzu was presented due to vaginal discharge of 4 day duration that had been recurring for over 1 year. she had a history of an open pyometra twice in the past year. ovariohysterectomy had not been pursued, due to owner's concern for anesthetic risk. on presentation, the patient was moderately thin, had serosanguinous vaginal discharge, a swollen vulva, and a positive flagging response; physical examination was otherwise unremarkable. vaginal cytology revealed primarily intermediate cells with few parabasal cells. vaginal cultures revealed growth of enterococcus faecalis, escherichia coli, and staphylococcus pseudointermedius. progesterone concentration was 0.79 ng/ml. transrectal ultrasonography revealed cystic structures on both ovaries. followup examinations within next 2 weeks revealed similar findings on vaginal discharge and cytology, and progesterone concentrations rose slowly to 2.28 ng/ml. due to the abnormal ovarian findings and potential for hyperestrogenism, ovariohysterectomy was pursued. preoperative bloodwork revealed a mild leukocytosis characterized by a neutrophilia with a mild left shift. histopathology of reproductive tract revealed bilateral ovarian adenocarcinoma, severe, diffuse, chronic cystic endometrial hyperplasia, and mild pyometra. epithelial tumors, including ovarian adenomas and adenocarcinomas, were the most common primary ovarian tumor in dogs ( 46% ), with adenocarcinomas making up 20% of canine ovarian tumors. 1 the patient recovered without incident and was discharged to the care of her owner. followup examination 3 weeks postoperatively, including thoracic radiographs and abdominal ultrasonography, revealed no evidence of metastasis. this case presented a unique abnormal estrous cycle secondary to a neoplastic process in a geriatric bitch with an open pyometra. keywords: ovarian adenocarcinoma, geriatric dog, hyperestrogenism references 1. arlt s, haimerl p: cystic ovaries and ovarian neoplasia in the female dog a systematic review. reprod domest anim 2016;51 :3-11. clinical theriogenology • volume 12 number 3 • september 2020 441 bilateral cystadenoma with a granulosa cell tumor in a bernese mountain dog miranda senft,8 jennine lection,8 lacey rosenberg,8kathleen kelly,b ileana miranda, b soon hon cheong,8 mariana diel de amorim8 8department of clinical sciences, ~epartment of biomedical sciences college of veterinary medicine, cornell university, ithaca, ny a 7 year old bernese mountain dog presented for more than 5 weeks of persistent estrus and infertility. her history consisted of multiple attempts of unsuccessful breeding, with and without progesterone timing, and she had never produced a litter. on presentation, the bitch had normal physiological parameters with the presence of a swollen vulva and vaginal bleeding. vaginal cytology revealed 100% superficial cells, consistent with estrus. serum progesterone was 0.671 ng/ml, also consistent with estrus. transabdominal ultrasonography was elected to examine for the presence of follicular activity or any abnormalities. the right uterine horn presented with a small single cyst. the right ovary was larger and with a lobulated appearance and a questionable small mass. in addition, the right ovary had increased blood flow on doppler compared to the left ovary. due to her advanced age, history of infertility and ultrasonographic findings, an ovariohysterectomy was performed. morphological diagnosis on histopathology was bilateral papillary cystadenoma, a granulosa cell tumor (gct) on the right side and aggregates of small cells, indicative of an early dysplastic change over a gct on the left side. according to an analysis of studies of canine ovarian tumors from 1960 2004, granulosa cell tumors accounted for~ 25% of all ovarian tumors. 1 a case that had a history of infertility and presented with clinical signs of estrus was diagnosed to have bilateral papillary cystadenoma and a unilateral gct. keywords: bilateral cystadenoma, granulosa cell tumor, infertility reference 1. arlt s, haimerl p: cystic ovaries and ovarian neoplasia in the female dog a systematic review. reprod domest anim 2016;51:3-ll. 442 clinical theriogenology • volume 12 number 3 • september 2020 a third-degree rectovaginal tear in a primiparous mare joshua trumble, fred caldwell, aime johnson college of veterinary medicine, auburn university, auburn, al a 10 year old quarter horse mare was presented for recto vaginal tear and fistula. she had foaled unattended. the large foal was found dead but appeared grossly normal. a tear was present from the mare's vulva through her perinea! body and extended cranially 6 inches. the mare was medically managed with flunixin meglumine and femycin and hydrotherapy to reduce irritation from fecal contamination. the owner was advised to keep her on a soft stool diet and to continue hydrotherapy for 6 weeks prior to surgical reconstruction. two months after the initial visit, the mare was reevaluated for surgical correction of the rectovaginal tear. on rectal palpation, a 5 x 5 inch fistula was noted and the rectal sphincter was intact. fecal contamination of the vagina was present. surgical correction for this case was performed in 2 phases. in the first phase, the rectal sphincter was split and the fistula was converted to a grade 3 rectovaginal tear. in phase 2, the perinea! body and anal sphincter were repaired. due to the degree of fibrosis from the severity of her injury, additional foaling was not recommended. third-degree rectovaginal tears occur predominantly in primiparous animals, with the mare being over represented, due to the explosive nature of their parturition. 1 third-degree rectovaginal tears are a complete communication between the vestibule and the rectum. correction is important to either restore breeding function or to provide a better cosmetic appearance. reference 1. climent f, ribera t, arguelles d, et al: modified technique for the repair of third-degree rectovaginal lacerations in mares. vet rec 2009;164:393-396. clinical theriogenology • volume 12 number 3 • september 2020 443 cervical adenoma and excisional biopsy in a spayed labrador retriever bitch julia zuercher,8 alyssa helms/geoffrey saunders,b philip sponenberg,b julie cecerea 8department of small animal clinical sciences bdepartment of biomedical sciences & pathobiology virginia maryland college of veterinary medicine, blacksburg, va a 7 year old ( 42 kg), nulliparous, spayed female labrador retriever was presented following referral to the virginia maryland college of veterinary medicine for evaluation of chronic, intermittent, bloody vaginal discharge and acute tissue herniation from the vulva. physical examination revealed a hemorrhagic, multilobulated, irregularly shaped mass protruding from the vulva. a digital vaginal examination revealed the presence of a firm, prominent, pedunculated mass originating from a distinct stalk. an impression smear had a monomorphic population of large, undifferentiated round cells with anisocytosis, anisokaryosis, and coarse chromatin. following sedation with intravenous dexmedetomidine (7 µg/kg) and butorphanol (0.3 mg/kg) and administration of a lumbosacral epidural (0.25 mg/kg of 0.75% preservative free bupivacaine), digital vaginal examination revealed 2 pedunculated masses. the caudal mass originated from ventral vaginal mucosa located 5 mm cranial to the urethral orifice, and the cranial mass was located dorsolaterally, 1.5 cm cranial to the first. the owner elected excisional biopsy of both masses for histopathology performed with a vessel sealing device (ligasure) over an episiotomy with complete excision. although the caudal mass was easily exteriorized and excised, the cranial mass was more friable and tore during attempted exteriorization, leaving a remnant within the cranial vagina. minimal vaginal bleeding was observed postbiopsy. the dog was discharged with carprofen (2.2 mg/kg po, every12 hours) and trazodone (2.4 mg/kg, po, every 12 hours) awaiting histopathology. histopathology revealed a well-differentiated simple cuboidal and columnar epithelial population of cells, typical of the cervix, with secretory mucin-rich cystic glandular lesions consistent with cervical adenoma. vaginoscopy was repeated at 1 month postbiopsy. the remaining mass had not changed in size or appearance; however, the previous clinical signs had resolved. while benign epithelial tumors of the vaginal mucosa have been documented, this case presents the first reported benign epithelial tumor of the cervix, as well as a minimally invasive technique for treatment. keywords: cervical adenoma, ligasure, canine, vaginal bleeding 444 clinical theriogenology • volume 12 number 3 • september 2020 society for theriogenology "veterinarians dedicated to animal reproduction" p.o.box 168 mathews, al 36052 334-395-4666 (voice) 334-270-3399 (fax) http://www.therio.org 2009: what's known about selected sperm abnormalities in the bull? what’s known about selected sperm abnormalities in the bull? 1 2 j. d. smith 3 4 department of pathobiology and population medicine, college of veterinary medicine, 5 mississippi state university, mississippi state, ms, usa 6 7 abstract 8 the morphological evaluation of spermatozoa is an important and often 9 overlooked aspect of the bull breeding soundness examination and it is imperative that 10 practitioners not only be able to recognize defective spermatozoa but also to understand 11 the origins of observed defects and provide information related to their effects on the 12 bull’s fertility. this review describes the common causes of altered spermatogenesis. it 13 details many of the morphological abnormalities frequently encountered during routine 14 bull breeding soundness exams. insight is given into their causes as well as the potential 15 impact of each defect on the fertilization process. 16 keywords: bull, spermatozoa, acrosome, proximal droplet; breeding soundness 17 examination 18 introduction 19 the testicle of a bull is a very sensitive organ which is capable of responding to 20 an assortment of insults including heat, hypoxia, radiation, toxicity, and stress as well as 21 the influence of genetic disorders. the response of the testis to these insults often leads 22 to the formation of spermatozoa with observable defects that can be seen during a routine 23 spermiogram. disturbances in spermatogenesis result in the formation of a wide range of 24 defective spermatozoa which can be observed clinically. attempts to make both an 25 etiologic diagnosis as well as provide a prognosis for the bull’s potential recovery of 26 normal spermatogenesis is one of the goals of a complete breeding soundness 27 examination when excessive numbers of abnormal spermatozoa are observed. this paper 28 337 will review a select population of sperm defects which could be encountered in veterinary 29 practice and provide their common causes as well as the prognosis for fertility when 30 known. 31 causes of altered spermatogenesis 32 the two most common insults which affect bulls on pasture appear to be heat and 33 stress.1 the affects of trauma, elevated ambient temperature, fever, excessive deposition 34 of fat in the scrotal neck and scrotal frostbite can all lead to increased heat in the scrotum. 35 the impact of the excessive heat in the scrotum leads to altered spermatogenesis and the 36 eventual appearance of increased numbers of morphologically abnormal spermatozoa in 37 the ejaculate. the temperature of the scrotal contents in normal bulls generally varies 38 between 33.0 and 34.5 ºc with a temperature gradient of 6 ºc from that of core body 39 temperature.2 numerous studies have shown that even very small increases in testicular 40 temperature can have dramatic impact on spermatogenesis leading to the subsequent 41 appearance of abnormal spermatozoa in the ejaculate.1,3,4 the testicles of bulls appear to 42 function in a nearly hypoxic state which is in part due to the actions of the pampiniform 43 plexus which essentially eliminates the pulse pressure and slows the blood entering the 44 testicle.2 when scrotal temperatures increase, the metabolic demands of the testicles 45 increase as well, but there is no corresponding increase in testicular blood flow ultimately 46 leading to testicular hypoxia and subsequent alterations in spermatogenesis. 1 47 stress in the form of illness, pain, herd or social interactions, transportation, and 48 weather conditions can be experienced by bulls. there appear to be disturbances in the 49 endocrine pathways associated with normal spermatogenesis in bulls under stress.1 the 50 affect of stress has been measured by examining the relationship between cortisol, 51 338 luteinizing hormone (lh) and testosterone. high cortisol levels in bulls have been 52 associated with reduced levels of lh and testosterone when compared to bulls with 53 normal cortisol; the elevated cortisol levels may interfere with normal spermatogenesis.5 54 the toxic effects of gossypol on spermatogenesis are well-recognized and the 55 characterization of the of abnormal spermatozoa has been the subject of several studies.6,7 56 the spermatoxic effects of gossypol appear to be related to both the dose and duration of 57 consumption of the phenolic compound produced by the cotton plant.8 the incidence of 58 gossypol-induced altered spermatogenesis in bulls is likely low due to the rumen’s ability 59 to detoxify the gossypol. however, high intake of free gossypol, albeit rare, can 60 overwhelm this system and create toxicosis.7 61 genetic causes of sperm abnormalities are not as common as environmental 62 causes but are becoming more recognized because of improved diagnostic tools.9 these 63 defects have been shown to either consistently or occasionally have a genetic mode of 64 transmission. with the ability to scrutinize large numbers of ai sires and their progeny a 65 number of sperm defects have been classified as genetic in nature. 66 distal midpiece reflex 67 the distal midpiece reflex (dmr) is the most common tail abnormality 68 encountered when evaluating the morphology of bull sperm.10 the typical appearance of 69 a dmr is that of a distinct hairpin bend of the tail at the location of the distal midpiece, 70 however there can be varying bending patterns noted in the tail giving the affected 71 spermatozoa several different appearances. a consistent finding is the presence of a 72 cytoplasmic droplet noted within the bend of the tail.10 when this defect is observed in 73 339 live samples, the spermatozoa will appear to be swimming in a reverse motion often in a 74 circular pattern. 10 75 the dmr defect can be induced experimentally when spermatozoa are exposed 76 to hypotonic solutions or when cooled very rapidly.10 this is critically important when 77 preparing a slide for staining. many of the morphology stains used today (ie. eosin 78 nigrosin) are hypoosmotic and will create a similar defect if spermatozoa are exposed for 79 an extended time.10 it is critical that the slide be prepared properly and dried quickly so 80 as to reduce the chance of creating this defect iatrogenically. one striking difference in 81 the defects which occur naturally and those that are caused in vitro is the presence of a 82 cytoplasmic droplet within the reflex of the tail. when a large number of dmr defects 83 are noted without the concurrent presence of a cytoplasmic droplet the possibility of an 84 artificial cause should be investigated.10 85 the dmr defect is produced in the corpus and cauda epididymis where the 86 spermatozoa still have a distal cytoplamic droplet which is present in the bending of the 87 tail.10 the evidence which supports the epididymis as the origin of this defect is based on 88 appearance of this defect after known testicular insults. semen evaluation of 606 bulls 89 prior to a severe snowstorm showed the percentage of bulls with greater than 15% dmr 90 to be only 10.9% whereas of the 117 bulls examined 3-4 days after the snowstorm 45.3% 91 had greater than 15% dmr defects.10 a significant increase in the dmr defect was 92 observed 6-12 days after a brief period of scrotal insulation.10 93 other recognized causes of this defect include treatment with estrogens, induced 94 hypothyroidism and fever. all of these conditions have been shown to reduce 95 340 testosterone levels, which appears to adversely affect the epididymal environment leading 96 to the formation of the dmr defect.10 97 the fertility of ejaculates containing high numbers of spermatozoa with dmr 98 defects has not been critically evaluated however it has been observed that the defect 99 could be found in normal fertile bulls with a prevalence up to 25%.10 owing to the fact 100 that this defect is of epididymal origin, it would seem that any effect on fertility would be 101 short-lived provided the inciting insult was removed. since affected spermatozoa would 102 swim in a reverse fashion it is unlikely that they would be able to participate in 103 fertilization thereby could be compensated for by additional spermatozoa. there is no 104 evidence that spermatozoa with the dmr defect are capable of regaining normal 105 function.10 semen evaluation in affected bulls should be performed often to detect 106 changes in prevalence of the dmr defect that could help identify an inciting cause. 107 knobbed acrosome 108 the knobbed acrosome defect has been described as a refractile or dark-staining 109 area or eccentric thickening often giving a beaded appearance to the apex of affected 110 spermatozoa.10 more commonly however, it appears with the apex of the spermatozoa 111 having a flattened or indented acrosome.10 with the availability of electron microscopy, 112 studies have shown that with both the beaded and indented forms the abnormal acrosome 113 folds back on the sperm apex and a few affected spermatozoa may show a bead-like 114 protrusion from the apex of the spermatozoa. the folding back of the acrosome may also 115 result in a bending back of the apex of the nucleus which causes the apex to appear to 116 have an indentation.10,11 117 341 the knobbed acrosome defect results from altered spermatogenesis caused by 118 either environmental or genetic factors leading to abnormal development of the fine 119 structure of the plasma membrane making it more susceptible to structural and functional 120 changes.9,12 it has been shown that spermatozoa with the knobbed acrosome defect lack 121 membrane integrity which can lead to premature capacitation and subsequent acrosome 122 reaction however the exact mechanisms involved remain elusive.11,12 123 it is generally accepted that bulls whose semen contains a high percentage of 124 knobbed acrosome defects will have poor fertility.10 the exact mechanism by which this 125 defect causes reduced fertility has yet to be elucidated, however it has been theorized that 126 spermatozoa with abnormally shaped heads including the knobbed acrosome defect may 127 have altered motility characteristics which could impair the passage of spermatozoa 128 through the female reproductive tract.13 spermatozoa containing the knobbed acrosome 129 defect could also have altered sperm-oocyte binding and zona penetration.13 in vitro 130 fertilization (ivf) models have shown that knobbed acrosome affected spermatozoa have 131 a reduced ability to bind the zona pellucida and could not penetrate the zona pellucida.11 132 thus the effect of the knobbed acrosome defect on fertility appears to be related to both 133 altered passage as well as impaired plasma membrane function.12 in vitro studies also 134 indicate there could be compromised fertility associated with apparently normal 135 spermatozoa from males with many knobbed acrosome defects.14 these normal 136 appearing spermatozoa have been shown to undergo premature capacitation and have 137 spontaneous acrosome reaction as well as evidence of chromatin condensation.12 138 this sperm defect has been associated with infertility in a number of species 139 including bulls, boars, rams and stallions.11 in cattle this defect was first reported in the 140 342 friesian breed in the 1940’s however it has since been seen in charolais, simmental, 141 maine anjou, salers, horned hereford, angus and normande.9,15 this defect is 142 associated with an autosomal sex-linked mode of transmission in the friesian breed of 143 cattle and in boars.9 there appears to be evidence to support a genetic cause of this 144 defect in both the charolais breed and angus cattle in north america. 9,15 145 cytoplasmic droplet 146 the presence of a cytoplasmic droplet is common on a small number of 147 spermatozoa in the ejaculate of fertile bulls. the cytoplasmic droplet is a spherical mass 148 of cytoplasm that is typically found in one of two locations on the spermatozoa. it is 149 considered a proximal droplet when located in the proximal midpiece and a distal droplet 150 when surrounding the midpiece just proximal to the annulus.10 droplets are rarely 151 observed in an intermediate location due to the rapid migration of the proximal droplet to 152 the distal location prior to shedding. 153 cytoplasmic droplets are formed during spermiogenesis when the spermatid 154 changes from its round shape to an elongated shape and cytoplasm is pulled from the 155 head region towards the tail.10 during this transition the sertoli cell molds this cytoplasm 156 into a lobule called the residual body.10 at the time of spermiation the stalk connecting 157 this residual body to the spermatid is severed and leaving the droplet of cytoplasm in the 158 proximal neck region of the spermatozoa.10 159 as spermatozoa enter the caput epididymis almost 85 percent will have a 160 proximal droplet. as spermatozoa move through the epididymis and maturation occurs 161 the proximal droplet moves to a distal location and is eventually shed. by the time the 162 spermatozoa reach the caudal epididymis over 60 percent of the spermatozoa have a 163 343 distal droplet.10 the loss of the cytoplasmic droplet appears to be associated with the 164 gaining of motility in the epididymis. 165 it is very common to find a high incidence of proximal droplets in bulls that are 166 approaching puberty, however with repeated collections over the following months the 167 percentage of affected spermatozoa generally drops substantially. in yearling bulls a 168 major cause of failure to pass a breeding soundness examination is often the presence of a 169 large percentage of proximal droplets. in data collection from colorado, 12-26% of 170 yearling bulls failed to pass an initial breeding soundness examination with 6.3% of these 171 failures attributable to proximal droplets.16 as bulls mature however, the incidence of 172 cytoplasmic droplets tends to drop dramatically. when over 1500 bulls of various ages 173 and breeds were examined the percentage of bulls whose ejaculates contained proximal 174 droplets was 67 percent, with the number of affected spermatozoa averaging only 2.7 175 percent.10 in older bulls a high incidence of spermatozoa with proximal droplets points 176 towards abnormal spermiogenesis likely due to a degenerative process of the 177 seminiferous epithelium.10 178 the prognosis for bulls with a high percentage of spermatozoa with proximal 179 droplets varies depending on the underlying cause and the presence of other defects. 180 recovery has been seen in bulls with profound disturbances of spermatogenesis leading 181 to ejaculates which contain greater than 50 percent of spermatozoa with proximal 182 droplets, however recovery often requires months.10 in vitro fertilization was used to 183 evaluate the fertilizing potential of semen from young bulls with a high incidence of 184 proximal droplets. it was concluded that fertility was severely compromised but as the 185 bulls matured the incidence of proximal droplets decreased and fertility increased.17 this 186 344 study also showed the fertilizing potential of a bull whose semen contained ≥30% 187 spermatozoa with proximal droplets will be low until the incidence of proximal droplets 188 decreases. 17 189 distal droplets are not considered to be a major problem and often indicate 190 insufficient maturation in the epididymis. it has been noted that ejaculates containing a 191 high number of spermatozoa with distal droplets when allowed to incubate for several 192 minutes will almost be totally cleared of the defect.10 193 crater or diadem defect 194 the crater or diadem defect is seen in spermatozoa which have a nuclear vacuole 195 or invagination of the nuclear membrane into the nucleoplasm which occurs during 196 spermiogenesis.18,19 this defect can appear as a “string of pearls” at the acrosome-197 postacrosomal sheath or as round to elongated white spots which often appear to 198 sparkle.10 the defect often appears as a surface oriented crater and can range from 1 to 199 over 20 in number.10 this particular defect is often overlooked with light microscopy on 200 routine eosin-nigrosin stained spermiograms, whereas phase-contrast microscopy and 201 differential interference microscopy allow one to more easily detect this defect. the use 202 of a nucleus stain such as the feulgen stain should also allow excellent visualization of 203 the nucleus and associated vacuoles.10 204 the incidence of the crater or diadem defect in bulls is generally very high, often 205 approaching 100%; however the percentage of affected spermatozoa within an ejaculate 206 can vary greatly. in a czechoslovakian study of young and older bulls, nuclear vacuoles 207 were observed in all bulls with the incidence of affected spermatozoa ranging from 3-208 345 26%.10 in one study of bulls at an ai center, all bulls had some defects and 28% of the 209 bulls examined had an incidence of crater defects in excess of 20%.19 210 the precise pathogenesis of this defect has not been elucidated however there are 211 several theories put forth in the literature. coulter suggested a possible viral etiology 212 based on the presence of viral-like particles within the vacuoles with the thought that the 213 virus may attack the developing spermatozoa.10 others have shown that certain 214 insecticides when administered to bulls resulted in an increased incidence of nuclear 215 vacuoles.10 the most common and accepted theory involves the impact of stress on the 216 process of spermatogenesis. it has clearly been shown that administration of 217 dexamethasone to bulls to mimic a stressful event will significantly reduce their lh and 218 testosterone levels and is thought to impair spermatogenesis and lead to the formation of 219 defective sperm. studies of the sequential appearance of sperm defects after 220 administration of dexamethasone to bulls clearly show there is an increase in the presence 221 of nuclear vacuoles with the peak incidence occurring around 21 days after 222 dexamethasone treatment.1 whether the effects of dexamethasone on spermatogenesis 223 are direct or indirect have yet to be determined. when bulls were studied over long 224 periods of time, fluctuations were seen in the incidence of affected spermatozoa 225 indicating a nonheritable etiology. this is supported by the absence of the defect in 226 significant numbers in the ejaculates of bulls which are sons of a known affected bull.10 227 however there is reason to believe that there may be bulls which have a heritable 228 predisposition to produce spermatozoa with the crater defect in response to stress.10 229 it is clear that ejaculates containing high numbers of spermatozoa with nuclear 230 vacuoles are a cause of infertility in the bull. breeding trials have shown that 231 346 spermatozoa with multiple nuclear vacuoles have reduced fertilization characteristics 232 both in vivo and in vitro.18,20 it appears that affected spermatozoa have a reduced 233 capacity to penetrate the zona pellucida as well as reduced ability to form a male 234 pronucleus.18 with this knowledge, bulls whose semen contains high numbers of 235 spermatozoa with nuclear vacuoles should be monitored with successive semen 236 evaluations as it has been reported that occasionally bulls recover and regain normal 237 fertility.10 238 dag defect 239 the dag defect traces back to a jersey bull with this name in which this unique 240 defect was first discovered and reported. this heritable defect which occurs during late 241 spermiogenesis is characterized by severe coiling of the tail with fracture of the distal part 242 of the midpiece.10 a distal cytoplasmic droplet can also be seen associated with this 243 defect.9 other features which may be seen include a roughened appearance to the 244 mitochondrial sheath and fracturing of the axonal elements leading to disruption of the 245 mitochondrial arrangement.10 this defect has been reported as a cause of infertility in 246 several breeds including the jersey, polled and horned hereford, and the swedish red 247 and white. in these particular infertile bulls the incidence of the dag defect was 248 generally greater than 50%. this defect can be found in the ejaculates of bulls with 249 normal fertility however the incidence of affected spermatozoa rarely exceeds 5%.10 250 when the incidence of this defect approaches 50% there appears to be a profound impact 251 on fertility. 10 252 a genetic basis for this defect was proven in the danish jersey breed through 253 selected breeding of a suspected carrier to 120 of his daughters which produced 6 bulls 254 347 with the typical defective spermatoazoa.10 there may be breed specific differences in the 255 exact pathogenesis of this defect because investigators have noted distinct differences in 256 the microanatomy of affected spermatozoa from various breeds. 10 257 pyriform or tapered heads 258 the pyriform defect is the most common defect of head shape and it appears as a 259 pear-shaped head with a pronounced narrowing of the postacrosomal area.10 there are 260 many variations of the pyriform defect that range from almost imperceptible to those with 261 severe narrowing all of which can occur in the same ejaculate. the tapered head shape is 262 slightly different than the pyriform head shape in that the entire nucleus is narrow and the 263 head appears elongated.10 both of these head defects can be found in the same ejaculate 264 and affected spermatozoa always appear smaller than their normal counterparts. these 265 two defects appear closely related in origin and may be categorized together.10 266 the incidence of the pyriform or tapered head defect occurring in more than 15% 267 of spermatozoa in an evaluation of over 1300 range beef bulls was determined to be 268 8.7%. this was was lower than the 16.4% incidence found in another study of 216 dairy 269 and beef ai sires.10 the presence of this defect in low numbers however is a fairly 270 common finding in the ejaculate of many fertile bulls. 271 the pathogenesis of the pyriform or tapered head defect has not been proven but 272 appears to occur secondary to some disturbance of thermoregulation or an endocrine 273 aberration leading to impaired testicular function.10 over-conditioned bulls which have 274 excess fat in their inguinal and scrotal areas commonly have these defects. the 275 appearance of pyriform head defects in experimentally-induced scrotal insulation peaked 276 348 at 22 days post insulation and at 24 days in dexamethasone-treated bulls suggesting the 277 damaging effects occurred during nucleus condensation and shaping.1 278 the effects of spermatozoa with pyriform or tapered heads on fertility appear to 279 be related to their reduced ability to bind and penetrate the zona pellucida however there 280 does not appear to be an increased incidence of embryo or fetal loss provided fertilization 281 occurs.10 work by saacke investigating the accessory sperm population has also shown 282 that the severity of the head defect may dictate the accessibility of spermatozoa to the 283 ovum thereby limiting the possibility of zona binding.21,22 284 the prognosis for bulls exhibiting large numbers of pyriform or tapered heads in 285 their ejaculates varies depending on the underlying cause. when a cause such as altered 286 testicular thermoregulation can be identified and corrected, the prognosis is generally 287 good provided enough time is allowed to resume normal spermatogenesis. however in 288 bulls which have high numbers of pyriform or tapered heads in which there is no 289 apparent reason for the altered spermatogenesis the prognosis is generally poor for 290 recovery.10 the percentage of affected spermatozoa can generally be used as a 291 prognostic indicator. 292 detatched heads 293 the presence of detached heads on routine evaluation of a bull’s spermiogram is 294 relatively common. this defect is easy to identify during evaluation of a sperm 295 morphology slide. the incidence of this defect in bulls of normal fertility and various 296 ages appears to be around 5 percent.10 a number of conditions have been associated with 297 the presence of increased numbers of detached heads. in one study of eight bulls with 298 testicular hypoplasia the incidence of detached heads ranged between 39 and 93 percent. 299 349 however, of these eight bulls, seven were traced back to a common ancestor and the 300 possibility that the high incidence of detached heads was inherited could not be 301 overlooked.10 the relationship between detached heads and testicular hypoplasia is not 302 consistent among all bulls with this condition. in a study of 141 bulls with testicular 303 hypoplasia the percentage of detached heads was only 6.9 percent.10 304 there appears to be a condition in bulls that mimics the clinical appearance of 305 “plugged ampullae” in stallions. the incidence of this condition was found to be 1.1% 306 when over 1300 bulls were evaluated. the characteristics of the ejaculate consist of a 307 very large volume of highly concentrated semen which contains a high proportion of 308 dead spermatozoa as well as 15-45 percent detached heads.10 these bulls appeared to 309 improve with frequent repeated collections however the incidence of this defect would be 310 higher after periods of sexual rest. the most common theory is that these bulls have a 311 failure of normal sperm transport with accumulation of spermatozoa within the 312 epididymis and ampullae.10 313 other conditions that have been shown to cause a transient increase in the number 314 of detached heads include: seminal vesiculitis, epididymitis, or any condition which leads 315 to failure of normal testicular thermoregulation. lameness which causes the bull to lie 316 down for long periods of time with resultant failure of normal thermoregulation is known 317 to increase the number of detached heads.10 318 abaxial attachment of the tail 319 abaxial attachment of the tail is an infrequently encountered defect of bull 320 spermatozoa. in a retrospective analysis of semen analysis from 1049 range and ai bulls 321 the percentage of bulls found to have the defect was 10.5% and only 0.48% of bulls had 322 350 semen with greater than 50% abaxial attachment of the head.23 it is considered a normal 323 finding in the boar and stallion however its significance in the bull remains unclear. 324 there have been reports of sterile bulls that had an increased number of spermatozoa with 325 abaxial attachment of the head however there were no controlled breeding trials 326 performed.23 in three controlled experiments comparing the fertility of bulls with high 327 numbers of abaxial tail attachement to that of known fertile bulls there was no difference 328 in all fertility parameters measured between bulls.23 the combined results of these 329 experiments indicate that abaxial attachment of the tail does not impact fertility and 330 should be considered a normal morphological variation of bovine spermatozoa.23 331 conclusion 332 there has been a strong correlation between morphologically abnormal sperm and 333 some degree of infertility for many years. it is important to not only be able to recognize 334 morphological defects of spermatozoa but also to provide the client with possible 335 etiologic causes of the defects observed and make suggestions as to their impact on 336 fertility. with the use of routine staining procedures most defects can be observed when 337 examined at 1000x with light microscopy. electron microscopy may more accurately 338 identify the exact defect present in affected spermatozoa allowing a more in-depth 339 understanding of the sperm abnormalities. 340 references 341 342 1. barth ad, bowman pa: the sequential appearance of sperm abnormalities after 343 scrotal insulation or dexamethasone treatment in bulls. can vety j 1994;34:93-344 101. 345 351 2. senger pl: pathways to pregnancy and parturition. 2nd ed. pullman, wa: current 346 conceptions; 2003. 347 3. kastelic jp, cook rb, coulter gh, et al: insulating the scrotal neck affects semen 348 quality and scrotal/testicular temperatures in the bull. theriogenology 349 1996;45:935-942. 350 4. vogler cj, bame jh, dejarnette jm, et al: effects of elevated testicular 351 temperature on morphology characteristics of ejaculatated spermatozoa in the 352 bovine. theriogenology 1993;40:1207-1219. 353 5. welsh th, johnson bh: stress induced alterations in secretion of corticosteroids, 354 progesterone, lutenizing hormone and testosterone in bulls. endocrinology 355 1981;109:185-190. 356 6. chenoweth pj, chase cc, risco ca, et al: characterization of gossypol-induced 357 sperm abnormalities in bulls. theriogenology 2000;53:1193-1203. 358 7. chenoweth pj, risco ca, larsen re, et al: effects of dietary gossypol on aspects 359 of semen quality, sperm morphology and sperm production in young brahman 360 bulls. theriogenology 1994;42:1-13. 361 8. randel rd, chase cc, wyse sj: effects of gossypol and cottonseed products on 362 reproduction of mammals. j anim sci 1992;70:1628-1638. 363 9. chenoweth pj: genetic sperm defects. theriogenology 2005;64:457-468. 364 10. barth ad, oko rj: abnormal morphology of bovine spermatozoa. ames, ia: 365 iowa state univ press; 1989. 366 11. thundathil j, meyer r, palasz at, et al: effect of the knobbed acrosome defect in 367 bovine sperm on ivf and embryo production. theriogenology 2000;54:921-934. 368 352 12. thundathil j, palasz at, barth ad, et al: plasma membrane and acrosomal 369 integrity in bovine spermatozoa with knobbed acrosome defect. theriogenology 370 2002;58:87-102. 371 13. saacke rg, nadir s, nbel rl: relationship of semen quality to sperm transport, 372 fertility and embryo quality in ruminants. theriogenology 1994;41:45-50. 373 14. soderquist l: reduced fertility after artificial insemination in a ram with a high 374 incidence of knobbed acrosomes. vet rec 1998;143:227-228. 375 15. barth ad: the knobbed acrosome defect in beef bulls. can vet j 1986;27:379-376 384. 377 16. molnar e: evaluation of breeding soundness exam in performance-tested beef 378 bulls: colorado state university, 1989. 379 17. amann rp, seidel ge, mortimer rg: fertilizing potential in vitro of semen from 380 young beef bulls containing a high or low percentage of sperm with a proximal 381 droplet. theriogenology 2000;54:1499-1515. 382 18. pilip r, campo mrd, barth ad, et al: in vitro fertilization characteristics of 383 bovine spermatozoa with multiple nuclear vacuoles: a case study. theriogenology 384 1996;46:1-12. 385 19. coulter gh, oko rj, costerton jw: incidence and ultrastructure of "crater" defect 386 of bovine spermatozoa. theriogenology 1978;9:165-171. 387 20. miller bm, hrudka f, cates wf, et al: infertility in a bull with a nuclear sperm 388 defect: a case report. theriogenology 1982;17:611-619. 389 353 21. saacke rg, dejarnette jm, barne jh, et al: can spermatozoa with abnormal 390 heads gain access to the ovum in artificially inseminated superand single-391 ovulating cattle? theriogenology 1998;50:117-128. 392 22. evenson dp: loss of livestock breeding efficiency due to uncompensable sperm 393 nuclear defects. reprod fertil dev 1999;11:1-15. 394 23. barth ad: abaxial tail attachment of bovine spermatozoa and its effect on 395 fertility. can vet j 1989;30:656-662. 396 397 398 354 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments 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number of bulls suggested for successful pasture breeding of female cattle bull-to-cow ratios: practical formulae for estimating the number of bulls suggested for successful pasture breeding of female cattle james g.w. wenzel, robert l. carson, dwight f. wolfe food animal section, department of clinical sciences, college of veterinary medicine, auburn university, auburn, al abstract the literature contains inconsistent recommendations for bull-to-cow ratios used for pasture breeding in restricted breeding seasons. age of bulls and scrotal circumference are primary determinants when bulls are classified as satisfactory potential breeders according to the standards of the society for theriogenology. the auburn formulae are, for single-sire units, based on the bull’s age in months for young bulls, and on scrotal circumference in centimeters for adult bulls. the formula for multiple sire breeding groups also establishes a firm recommendation for larger breeding groups. keywords: breeding soundness, bull-to-cow ratio, natural service, pasture mating, scrotal circumference introduction the published literature is inconsistent regarding suggestions for the number of bulls required for natural service of varying numbers of cows and/or heifers, or conversely, the number of female cattle assigned to a bull or bulls for pasture breeding. the most common circumstance finds producers with an established number of females in a breeding group or groups; therefore we prefer the designation “bull-tocow ratio,” as this ideates the relatively fixed number of females as the denominator. this phrase is also occasionally found in the literature1,2 or electronic media.3 other potentially applicable phrases include “cow-to-bull” which is not widely used, or the colloquial “bull power,” but its meaning is inconsistent. “serving capacity” is widely referenced, especially in australian literature regarding rams and bulls, but typically includes an assessment of breeding behavior4,5 which is not a part of the bull breeding soundness examination commonly practiced in the united states. estimating an efficient bull-to-cow ratio seems to have been an inexact science, traditionally being 1:25-30 for mature bulls,6 but ranging from 1:10 to 1:60.7 several trials and observational studies should be considered. when bulls were selected for maturity and scrotal circumference, acceptable reproductive performance has been observed in single-sire, non-synchronized breeding groups of up to 60 females8,9 and synchronized breeding groups of up to 25 females.10 age of bull(s) should be considered when establishing breeding ratios. yearling bulls have traditionally been suggested to be limited to ratios of 1:10-20.6 this is not without support in the literature. for example, single-sire breeding groups averaging 20 females (range: 14-29) had higher pregnancy rates when bred by two-year-old bulls than by yearling bulls.1 however, 24 months is not necessarily the age of peak performance: two-year-old bulls exposed for 60 days to 50-cow, single-sire breeding groups achieved only 67-83% pregnancy rates, leading the authors to suggest that the typically recommended ratio of 1:25 could be expanded to 1:40 only for “high-serving-capacity” bulls.2 we read with interest that approximating breeding group size for young bulls by equating it to their age in months is “a time honored rule-of-thumb …”3 because it has been time-honored only since we developed the notion in the mid-1990’s. there is general agreement and reasonably good evidence that multiple-sire breeding groups are less efficient than single-sire units. social dominance, which is correlated with age,9 is alleged to play a part. this is supported by the finding that individual bulls in multiple-sire breeding systems sire disproportionate numbers of calves.11,12 breedings by more than one bull during each estrus may be inefficient and, interestingly, heifers in breeding groups of 1:20 were bred more times during each estrus by their single sire than were heifers in groups of 2:40 by the combined services of two bulls.13 we have noted some reluctance to accept minimum scrotal circumference standards established by the society for theriogenology,14 especially in the face of differences in average scrotal circumference among breeds. however, we suggest this undermines the notion of a unified standard for pasture breeding. interestingly, we have noted a decrease over time in the proportion of bulls deemed 477 clinical theriogenology • volume 4, number 4 • december 2012 unsatisfactory due to insufficient scrotal circumference during breeding soundness examinations.15 we believe that this has been the effect of selection for this moderately to highly heritable trait. we also suggest that breed associations should require scrotal circumferences which exceed the minimum standard if they wish to market bulls for natural service, and that we should discriminate against the minimum scrotal circumference in favor of bulls that well exceed that measurement. the auburn formulae for bull-to-cow ratios regardless of the pattern of use for breeding, each bull must be determined to be a satisfactory potential breeder by the standards of the society for theriogenology for breeding soundness examination of the bull. further consideration of scrotal circumference obtained during this examination is detailed, below. single sire breeding groups the size of a single-sire breeding group is n (1:n bull:cow ratio), meaning that the bull may be exposed to up to, approximately, n females: using a bull < 36 months of age, n = (is approximated by) the bull’s age in months; using a bull > 36 months of age, n = the bull’s scrotal circumference in centimeters. simply stated, the number of females per bull in single-sire units equals the bull’s age in months up to 36 months of age and, beginning at 36 months of age, the number of females equals the bull’s scrotal circumference.16 multiple sire breeding groups when it is not possible or reasonable to divide larger groups of females into single-sire breeding groups, some accommodation must be made for the inefficiency of multi-sire systems. our mentor, robert s. (“bob”) hudson, proffered this advice: each bull added to the first in a multiple sire breeding group may be expected to dependably serve only one-half of his compliment of cows or heifers16 (n, as calculated, above). thus, for example, a breeding group of 100 females would require four adult bulls, if each had a scrotal circumference of approximately 40 centimeters: 40 females for the first bull and 20 for each of the additional three bulls, or: b = 1 + [(t-n) / ½n] where b = number of bulls required, t= total female cattle in the breeding group, and n is the fair compliment of females calculated for single-sire groups, based on the characteristics of the bulls. this calculation depends on reasonable uniformity in size, age and scrotal circumference amongst the candidate bulls. discussion our formulae result in exposure of somewhat fewer females to each bull than the maximum numbers assigned to some bulls in the literature, but in some cases, more than allowed by the tentative, “traditional” estimates. we defend our suggested limits to the pressure placed on bulls by pointing to the value of each calf resulting from the goals of a satisfactory potential breeding: greater than a 90-95% calf crop from a restricted breeding season of 60-90 days. empirically, our recommendations have stood well against even greater pressure placed on bulls, for example, breeding seasons as short as 45 days. we are unaware of the source of the on-line information regarding exposure of young bulls to the number of females approximated by the bull’s age in months.3 this is not referenced, but is presented as common knowledge, and the author is not indicated. this was surprising to us, because we distinctly recall originating the idea in shared discussion. this means of estimating the reproductive pressure placed on young bulls is, to our knowledge, otherwise absent in the literature. it is possible, of course, 478clinical theriogenology • volume 4, number 4 • december 2012 that others divined but did not publish this notion and it became, indeed, common knowledge. we believe it perhaps more plausible that we, or our students who have been exposed to the idea for some 1520 years, have extolled it to the degree that it has entered the public sphere. our use of scrotal circumference to allow exposure of more females to bulls with larger scrotal circumferences is the result of our desire to increase the allowed minimum for a satisfactory potential breeder. continued pressure on this trait will increase the efficiency of cow/calf operations by allowing the use of fewer bulls. we foresee the possibility of reducing the age of conversion of the calculation from age-in-months to scrotal-circumference-in-centimeters based on improvements in this trait. the requirement of uniformity of age and scrotal circumference among bulls when calculating bull requirements for multi-sire breeding groups may be problematic. we insist that such uniformity remains a best-practice when multiple sires are required, but understand that it is not always possible. if bulls with such disparate characteristics must be utilized together, the combined wisdom of experienced veterinarians and herdsmen should be applied to minimize conflicts which might interfere with breeding efficiency. references 1. makarechian m, farid a, berg rt: evaluation of bull and cow fertility at pasture in single-sire mating. can j anim sci 1985;65:799-809. 2. sprott lr, carpenter bb, thrift ta: bull management for cow/calf producers. texas agrilife extension service pub#b-6064 2005;8 pp. 3. bull to cow ratio. purebred cattle pages. accessed june 6, 2012, at http://www.pbcattle.com/bull-to-cow-ratio.htm. 4. blockey madeb: development of a serving capacity test for beef bulls. app anim eth 1981;7:307-336. 5. bertram jd, fordyce g, mcgowan mr, et al: bull selection and use in northern australia 3. serving capacity tests. anim reprod sci 2002;71:51-66. 6. anderson l: section 4. managing reproduction, in the kentucky beef book 1997;p.68. accessed june 6, 2012 at http://www.uky.edu/ag/animalsciences/pubs/kybeefbook04.pdf. 7. perry g, walker j, daly r: reproductive fertility in herd bulls. south dakota state university cooperative extension service, 2008. accessed june 6, 2012, at http://www.thebeefsite.com/ articles/1491/reproductive-fertility-in-herd-bulls. 8. rupp gp, ball l, shoop mc, et al: reproductive efficiency of bulls in natural service: effects of male to female ratio and singlevs multiplesire breeding groups. j am vet med assoc 1977;171:639-642. 9. chenoweth pj: bull libido/serving capacity. vet clin north am food anim pract 1997;13:331-344. 10. healy vm, boyd gw, gutierrez ph, et al: investigating optimal bull:heifer ratios required for estrous-synchronized heifers. j anim sci 1993;71:291-297. 11. lehrer ar, brown mb, schindler h, et al: paternity tests in multisired beef herds by blood grouping. acta vet scand 1977;18:433-441. 12. holroyd rg, doogan vj, defaveri j, et al: bull selection and use in northern australia 4. calf output and predictors of fertility of bulls in multiple-sire herds. anim reprod sci 2002;71:67-79. 13. farin pw, chenoweth pj, mateos er, et al: beef bulls mated to estrus synchronized heifers: singlevs multi-sire breeding groups. theriogenology 1982;17:365-372. 14. chenoweth pj, spitzer js, hopkins fm: a new bull breeding soundness evaluation form. proc annu meet soc therio 1992; p.63-70. 15. carson rl: bull breeding soundness evaluation. proc ky vet med assoc annu meet and mid-amer vet conf 2011; compact disk. 16. carson rl: bull breeding problems: libidos, mating ability, and serving capacity. proc ky vet med assoc annu meet and mid-amer vet conf 2011; compact disk. 479 clinical theriogenology • volume 4, number 4 • december 2012 480clinical theriogenology • volume 4, number 4 • december 2012 omniblank: 2013: the effect of oxytocin or carbetocin administration during mid-diestrus on the interovulatory interval and estrous behavior of mares the effect of oxytocin or carbetocin administration during mid-diestrus on the interovulatory interval and estrous behavior of mares cassandra a. bare,a anne r. schramme,a* c. scott bailey,a jason m. heitzman,a scott whisnant,b renan d. sper,a kate archibald,a michael whitacrea acollege of veterinary medicine, north carolina state university, raleigh, nc; bcollege of agriculture and life sciences, department of animal science, north carolina state university, raleigh, nc abstract estrous behavior can negatively impact performance mares. prolongation of the interestrus interval (iei) or interovulatory interval (ioi) through oxytocin (oxy) injections represents a safe, reversible means of controlling estrous behavior. the purpose of this study was to determine whether an oxy analog, carbetocin (carb), would increase the ioi and iei similarly to oxy. we hypothesized that mares would have a longer ioi and iei after treatment with oxy or carb than they would during untreated cycles. twelve cycling mares were randomly assigned to one of two groups (carb and oxy). one normal cycle was documented in ten mares prior to the study and two mares after the study. during the treatment cycle, mares were administered either 1.19 mg of carb or 60 iu of oxy once daily by intramuscular (im) injection between day seven and 14 post-ovulation. blood was drawn from all mares once weekly for progesterone assay. mares were teased with a fertile stallion every other day to detect estrous behavior and subsequently examined by ultrasonography to detect ovulation between may and august. administration of carb decreased the ioi in all mares compared to the control cycle (18±1.1 vs. 20.8±1.8 days, respectively; p=0.00001). administration of oxy increased the ioi in four of six mares (67%) compared to the control cycle (37.3±17 vs. 21.8±1.5 days, respectively; p=0.0025) and substantially delayed the return of estrous behavior in two of six mares (33%). two mares failed to return to estrus before the end of the study; the diestral length at that time was used for calculations. luteal regression and subsequent ovulation were confirmed with serum progesterone concentration after treatment in both groups. this work suggests that carb, an analog of oxy, would not be useful for initiating long-term estrus suppression in the mare. keywords: mare, estrus suppression, oxytocin, carbetocin, interestrus interval, luteal function introduction estrous behavior in mares can be problematic especially during training and performance activities. the mare’s reproductive cycle is approximately 21 days in length and estrus expression can vary between five and seven days of the cycle. many methods for the suppression of estrous behavior have been explored, but no single, highly effective treatment for long term estrus suppression is available. methods of estrus suppression available today are administration of exogenous progesterone, suppressing ovarian follicular activity, extending the duration of corpra luteal function, and ovariectomy.1 administration of natural progesterone is very effective in suppression of estrus, but due to the inconvenience of daily dosing, tissue reactions and pain associated with daily injection, it is not routinely used.2 the only effective progestin for estrus suppression in the united states is altrenogest. altrenogest is administered orally once daily and has been successful in suppressing estrous behavior in 94% of treated mares.3 several other synthetic progestins, such as medroxyprogesterone acetate, hydroxyprogesterone caproate, norgestomet and megesterol acetate are available, but lack efficacy.2,4,5 presumably, this is due to failure to bind adequately to the progesterone receptors in the mare.4 finally, administration of exogenous progestins to competition horses is not permitted by many governing bodies. *current address: vet agro sup, campus vétérinaire de lyon, 1 avenue de bourgelat, 69280 marcy l’etoile, france 27 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 suppressing the ovarian follicular activity by vaccination against gonadotropin releasing hormone (gnrh) has also been demonstrated. mares vaccinated with an anti-gnrh vaccine four weeks apart had suppressed follicular development and lower progesterone levels than unvaccinated animals.6 mares in this study demonstrated reduced estrous behavior for a minimum of three months before returning to normal cyclicity by the following breeding season. however, gnrh vaccines are currently not approved for use or widely available in the united states. in addition, concern has arisen that gnrh vaccineinduced suppression of ovarian activity could be permanent, which obviously has dire implications for the subsequent fertility of mares treated in this manner.7 another method for long-term estrus suppression is a bilateral ovariectomy. research has shown a success rate of 82% in estrous behavior reduction in one study, and in another study client satisfaction was 78%.8-10 however, due to the permanent nature of the procedure, success rates, and surgical risks this is not an appealing solution for long-term estrous suppression. extension of luteal function through the use of intrauterine devices, such as glass or plastic balls, has met with variable success rates.11,12 another non-pharmacologic technique for prolonging luteal function is the manual disruption of conceptus development beyond day 16 of ovulation.13 in that study, the mares did not return to estrus for an average of 82 days. due to the relatively invasive nature of these techniques and a high degree of variability in effectiveness, they have not gained wide acceptance as long-term treatment methods for estrous suppression in mares. recently, an alternate means of extending luteal function pharmacologically has been sought by several research groups. hedberg and coworkers induced a prolonged luteal phase in a limited number of mares by inducing mid-diestrus ovulation after administration of human chorionic gonadotropin (hcg),14 while work in allen’s laboratory demonstrated prolongation of the luteal phase for at least 30 days after intrauterine administration of 1 ml of various plant oils.15 the use of oxy to disrupt normal luteolysis was first described by stout and allen.16,17 subsequent work demonstrated that twice daily injections of 60 iu oxy during the mid-luteal phase disrupted luteolysis by prolonging luteal function in 100% of mares.10 further work, comparing once daily and twice daily injections of oxy found no difference between groups with prolonged luteal function in 63% and 71% of animals, respectively.18 in each of these studies, progesterone levels were measured as a means of determining the length of luteal maintenance. although carb has not been used widely in equine medicine, its use has recently been studied in other species as a long-acting analogue of oxy. early work in rats demonstrated that carb and its metabolites bind to myometrial receptors similarly to oxy and have similar effects, although the maximal contractile effects were approximately 50% lower than those of oxy.19 in women, carb has been used in the treatment of life-threatening postpartum hemorrhage. in one study, carb had greater efficacy, increased half-life and a decreased administration frequency when compared with oxy.20 a study in cattle found no difference between groups in the incidence of postpartum endometritis after administration of saline, oxy or carb.21 recent work in our laboratory demonstrated that the half-life of this oxy analogue was 17.2 minutes,22 whereas previous work found a half-life of 6.8 minutes for oxy.23 thus, the objective of the current study was to compare the reproductive effects of oxy and carb administration in cycling mares. the first aim was to determine whether once daily administration of carb would effectively prevent luteolysis, similarly to oxy. the second aim was to determine whether either oxy or carb would effectively inhibit estrous behavior in mares, by assessing both endocrine patterns and behavioral patterns in treated and untreated cycles. we hypothesized that the ioi and iei would be significantly prolonged in mares administered 1.19 mg of carb or 60 iu of oxy once daily for eight days during diestrus, when compared to a control cycle. 28clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 materials and methods mares twelve cycling horse-mares between the ages of three and 20 years were enrolled in the study. eight of the mares were maintained on pastures at the north carolina state university veterinary school and four of the mares on pasture at the equine educational unit in raleigh. baseline inclusion data were gathered on each mare, including normal physical examination, normal ultrasonographic appearance of the reproductive tract, and a grade i or iia endometrial biopsy.24 each mare’s cyclicity was documented between february and july prior to enrollment into the experiment by transrectal ultrasonography. the ioi of ten mares was documented by repeated transrectal examination during a control cycle before treatment and for two mares it was documented during a control cycle after treatment. drug administration the mares were randomly assigned to one of two experimental groups, (carb or oxy). mares in group oxy received 60 iu of oxy (osborn®, bimeda inc., le sueur, mn) once daily between day seven and 14 post-ovulation as previously described.18 mares in group carb received 1.19 mg of carb (hypophysine la®, veyx-pharma gmbh, schwarzenborn, germany), administered once daily between day 7 and 14 post-ovulation. based on the pharmacokinetics, 0.175 mg of carb is equivalent to 8.75 iu of natural oxy; therefore, 1.19 mg carb is equivalent to 60 iu oxy.22 both medications were administered via intramuscular injection in the morning. the injection sites were alternated between neck and hip muscles on each day. body temperature, heart rate, and respiration rate were recorded immediately before each injection and again 20 minutes after injection. any signs of discomfort or reactions were noted. teasing normal estrous behavior of each mare was documented by teasing to a fertile stallion before enrollment into the study. during control and the treatment cycles, mares were teased to a fertile stallion on monday, wednesday and friday, beginning seven days after ovulation and ending when the mare exited the subsequent estrus or at the conclusion of the study on august 15th. behavioral response was given a score of 0 to 4 based on a model described by gorecka and coworkers.25 behavioral evaluation scores o score 0: non-receptive behavior o score 1: mare standing still indifferently (passive) o score 2: mare shows both estrous behavior and some non-receptive behavior o score 3: mare shows full estrous behavior and some non-receptive behavior o score 4: mare shows full estrus: no non-receptive behavior the iei was calculated from the day of ovulation until the first day that mares were given a behavior score of 2 or greater. ultrasonography when a behavioral score of 1 or greater was documented (standing passively, lifting tail, clitoral eversion, approaching stallion with pricked ears, frequent urination, and immobility in presence of stallion), mares were evaluated via transrectal ultrasonography once daily to confirm the physiological characteristics of estrus and to monitor the dominant follicle until ovulation (disappearance of the dominant follicle and presence of a corpus luteum). at each evaluation, the firmness of the cervix was noted and the amount of uterine edema was scored as previously described by samper and coworkers.26 the presence of any abnormality was also documented. ultrasonographic diagnosis of ovulation was used to determine the ioi for control and treatment cycles. 29 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 blood sample collection blood samples were collected from the mares every thursday after onset of treatment for the detection of progesterone (p4) until ovulation of the dominant follicle. seven mls of blood were collected from alternating jugular veins into evacuated glass tubes (bd vacutainers ®, bd, franklin lakes nj) with no additive. all blood samples were kept at room temperature (22°c) until clotted and were centrifuged at 3,000 revolutions per minute for three minutes. the serum was recovered and stored at -80°c until all samples were collected for analysis. radioimmunoassay for progesterone quantification of serum p4 levels was performed using a solid phase-radioimmunoassay kit (coat-a-count total progesterone kit, siemens medical diagnostics, los angeles, ca). the kit was validated for measuring p4 in equine serum in our laboratory.27 statistical analysis data were evaluated for normality using a shapiro-wilk normality test. homogeneity of variance was examined between groups using an f-test. differences in interestrus and ioi were compared between cycles using either a paired t-test or wilkoxon signed rank test, and between groups using a two-sample student’s t test or wilkoxon rank sum test, depending on the results of the shapiro wilk test. the software-program statistix 8.1 (statistix®, analytical software inc, tallahassee, fl) was used to analyze data. significance was assigned to all values p≤ 0.05. results interovulatory interval the ioi of both cycles was determined precisely in six of six mares in group carb and five of six mares in group oxy. one mare in group oxy experienced an ovulation without demonstrating behavioral estrus during the treatment cycle and the exact ovulation date was unknown. the last examination at which she did not have a corpus luteum was used for analysis (53 days). mares in group carb had an ioi of 20.8 ± 1.8 days in the control cycle and an ioi of 18.0 ± 1.1 days in the treatment cycle (p=0.00001). six of six mares in group carb had a shorter ioi after treatment than during the control cycle, with a reduction in ioi of one to five days. mares in group oxy had an ioi of 21.8 ± 1.5 days in the control cycle and 37.3 ± 17 days in the treatment cycle (p=0.0025). four of six mares (67%) had a longer ioi after treatment than during the control cycle, with an increase of five to 52 days. one mare had no numerical change in ioi between cycles and one mare had a slight numerical decrease in ioi after treatment (one day). mares in group carb had significantly shorter ioi during the treatment cycle than mares in group oxy (p=0.0043). no differences were detected in the ioi between control cycles of either group (p=0.3; figure 1). interestrus interval the iei of both cycles was determined precisely in six of six mares in group carb and four of six mares in group oxy. two mares in group oxy had not demonstrated any signs of estrus at the conclusion of the study and the interval from the onset of the cycle to the study conclusion (68 and 69 days) was used for statistical purposes. mares in group carb had an iei of 15.3 ± 6.2 days in the control cycle and 13.0 ± 1.1 days in the treatment cycle (p=0.0025). one mare in this group showed mild signs of estrus during diestrus in both the treatment and control cycle. she first had a behavioral score of 2 three days after ovulation during the control cycle and 12 days after ovulation during the treatment cycle. all other mares had a slight numerically shorter iei in the treatment cycle, compared to the control cycle (two to seven days). mares in group oxy had a mean iei of 14.8 ± 5.9 days in the control cycle and 33.2 ± 27.7 days in the treatment cycle (p=0.0025). one mare showed behavioral estrus intermittently during diestrus of both the treatment and control cycle. this mare first had a behavioral score of two or greater on day 3 of the 30clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 control cycle and day 8 of the treatment cycle. she continued to show intermittent estrus (scores 2-4) until the subsequent ovulation at 62 days. two mares (33%) in group oxy failed to show estrus before the conclusion of the study (68 and 69 days, respectively). each of these mares experienced a second ovulation during this period in the absence of estrous behavior. four mares (67%) had an iei of similar length after treatment as on the control cycle (figure 2). mares in group carb had numerically, but shorter iei during the treatment cycle than mares in group oxy, which was not statistically significant (p=0.07). progesterone luteolysis was confirmed by a serum progesterone concentration <1 ng/ml in six of six mares in group carb by week 2 after treatment onset. further, corpus luteum formation was confirmed by an increased serum progesterone concentration at subsequent measurement in five of five mares in group carb. the sample from the sixth mare was lost during processing. in group oxy, luteolysis was confirmed by measurement of serum progesterone concentrations <1 ng/ml by week 2 in two mares and by week 3 in an additional mare. a subsequent rise in serum progesterone concentrations confirmed ovulation and luteal development in all three of these mares. in one mare, progesterone concentrations remained above 1 ng/ml for eight weeks post-ovulation. this mare displayed intermittent estrus throughout the study despite serum progesterone concentrations consistent with diestrus (2.4-11.1 ng/ml). in two mares, serum progesterone concentrations remained above 1 ng/ml for the duration of the study (nine and 12 weeks, respectively). each of these mares had a secondary ovulation between week seven and week 11 after treatment onset in the presence of elevated progesterone concentrations (5.1-8.8 ng/ml) and without demonstrating any behavioral signs of estrus. drug-related complications in group carb, one mare showed slight bleeding at two of eight injection sites. in four of six mares there was moderate to severe sweating over the neck, flank, and axilla regions. in group oxy, one mare displayed mild sweating. one mare displayed urticaria around the site of injection after the first two injections. one mare had an onset of mild diarrhea after the second injection which continued a few days after the completion of the last injection; no elevation in body temperature was noted. physical examination body temperature, heart rate and respiratory rate were not different 20 minutes after administration of either carb or oxy than immediately before drug administration (figure 3). discussion the results of this study demonstrate that both oxy and carb can be administered safely during mid-diestrus at relatively high doses (60 iu and 1.19 mg respectively) without notable side-effects. this is an important consideration for owners and veterinarians seeking a medical option for estrus suppression. carb, an oxy analog, administered once daily between days seven and 14 post-ovulation did not increase either the ioi or iei. contrary to our hypothesis, carb administration resulted in a statistically significant decrease in the ioi and iei, compared to a control cycle. during the treatment cycle, 100% (six of six) of mares administered carb had a shorter ioi than during the control cycle, and 83% (five of six) of mares had a shorter iei. oxy administration increased the ioi and iei compared to the control cycle. during the treatment cycle, 67% (for of six) of mares had a longer ioi, with a delay in ovulation by five to 52 days compared to the control cycle. these findings are similar to those previously published by vanderwall and coworkers.18 interestingly, only 33% (two of six) of mares had a prolonged iei of greater than 30 days after oxy treatment, whereas 67% (four of six) had an iei of similar length during the control and 31 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 treatment cycles. this suggests that while the differences were statistically significant, oxy treatment may not effectively inhibit unwanted estrous behavior in a majority of mares. most previous work has focused on hormonal effects of oxy administration, and little information is available regarding behavioral effects. one recently published study utilized a protocol similar to the one described here and found “a strong agreement between teasing behavior, ultrasonographic assessment of ovarian activity, and concentration of progesterone in serum”.5 in that study, three groups of six mares were subjected to eight injections of either serum, 10 iu oxy im or 10 iu oxy iv. mares were subsequently monitored for behavioral estrus via teasing three times weekly, and for ovulation via transrectal ultrasound examination and serum progesterone assays. gee and coworkers reported that mares administered oxy iv had a longer duration of diestrus than control mares, but the proportion of mares in which diestrus was greater than 30 days was not statistically different between groups (5/6, 1/6 and 2/6 for the iv, im and control groups respectively).5 similarly, in the current study, im administration of oxy (60 iu once daily for eight days) delayed the return to estrus in only two of six mares. two mares in this study (one each from group carb and oxy), demonstrated estrus behavior during diestrus in both the control and treatment cycle, despite the fact that they had an ultrasonographically visible corpus luteum and adequate serum progesterone concentration to inhibit estrus. these mares may have been displaying submissive behavior in response to close contact with a stallion. it has been shown that submissive mares may show similar behaviors similar to estrus when placed in a stressful situation including urination and winking.28 although oxy administration did result in an increased ioi (from 23 to 62 days) in one of these mares, it did not eliminate estrus behavior. several questions remain at the conclusion of this study; it is unclear whether the estrous behavior demonstrated in response to direct contact with a stallion would also result in disruptive behavior in a performance setting. it is possible that oxy administration would result in adequate behavioral control in a performance setting without completely preventing them from “teasing” in response to direct contact with a stallion. furthermore, it has been suggested that some mares have impaired racing or show performance due to discomfort or pain from a large preovulatory follicle rather than directly from estrous behavior (bailey, unpublished data). in three of four mares with an increased ioi in this study, the presence of a large follicle or diestrus ovulation was documented. it is possible that these would result in decreased performance. a large randomized clinical trial of mares in training would be necessary to evaluate such effects. luteolytic mechanisms have been well-studied in mares and were reviewed in detail by shand.29 briefly, goff and coworkers demonstrated an increase in prostaglandin metabolites in the peripheral circulation of mares administered oxy after day 11 post-ovulation, with a peak response between 14 and 16 days.30 subsequently, work by stout and coworkers demonstrated that continuous intravenous administration of oxy, initiated prior to this period, could inhibit luteolysis, whereas initiation of treatment during this period hastened ovulation in two of five animals.16 a series of studies have strongly suggested that a major source of endogenous oxy for the induction of luteolysis is the endometrium itself.31-34 the biologic importance of uterine oxy for luteolysis was confirmed by stout and coworkers, who demonstrated that intrauterine infusion of oxy around the time of luteolysis resulted in a substantial rise in peripheral prostaglandin metabolites in the absence of elevated systemic oxy concentrations.16,32 the mechanism by which diestrus administration of oxy inhibits luteolysis is not fully understood. based on the information regarding luteolysis in the mare, it has been presumed by our group, as well as others, that mid-diestrus oxy administration to mares may prevent the increase in endometrial oxy receptor numbers or an increase in endometrial oxy receptor responsiveness, normally observed in diestrous mares.17,18 the prolonged luteal phase seen in many diestrus mares administered oxy in this study and several others has been presumed to result from a negative feedback loop, preventing the upregulation of oxy receptors in the uterus, as first proposed by stout.17,18 failure of carb to induce a prolonged luteal phase could also indirectly support this, as a previous study demonstrated that carb and its metabolites have both agonist and antagonist effects on oxy receptors.19 however, vanderwall and coworkers did not find a difference in endometrial oxy-binding capacity in mares administered oxy or saline twice 32clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 daily between seven and 14 days.18 thus the mechanism by which oxy, but not carb, disrupts normal luteolysis after mid-diestrus administration is not known at this time. the use of carb in addition to oxy may serve as a useful model for future research in this area. in conclusion, the current study demonstrated the safety of both oxy and carb in the mare, and found the effect of oxy to be similar to previous work. in contrast, carb administered between seven and 14 days post-ovulation effectively short-cycled the mares and decreased the ioi. neither carb, nor oxy reliably inhibited estrous behavior in mares exposed to a mature stallion. acknowledgements partial funding was provided by a grant from the north carolina horse council and by the north carolina state university college of veterinary medicine. the authors acknowledge the dedication of our clinical technician, jori vasgaard, in supporting the work described in this manuscript. the carbetocin used in this study was generously donated by veyx-pharma gmbh. figure 1. number of days (mean±sd) between successive ovulations of mares in group carb and group oxyduring control and treatment cycles. statistically significant differences between cycles within or between treatment groups are indicated by different letters. the ioi was shorter for treated mares in group carb, than during the control cycle (p=0.00001) and longer for treated mares in group oxy than during the control cycle (p=0.0025). treated mares in group carb had significantly shorter ioi than treated mares in group oxy (p=0.0043). 33 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 figure 2. number of days (mean±sd) with a behavior score <2 in group carb and group oxyduring control and treatment cycles. statistically significant differences between cycles within or between treatment groups are indicated by different letters. the iei was shorter for treated mares in group carb, than during the control cycle (p=0.0025) and longer for treated mares in group oxy than during the control cycle (p=0.0025). treated mares in group carb had a numerically shorter iei than treated mares in group oxy (p=0.07). figure 3. mean ± sd body temperature (panel a), heart-rate and respiratory rate (panel b) of mares in group carb and group oxy prior to drug administration and 20 minutes later. no differences were detected between groups or at different times in relation to treatment. references 1. mccue pm: estrus suppression in performance horses. j equine vet sci 2003;23:342-344. 2. loy rg, swan sm: effects of exogenous progestogens on reproductive phenomena in mares. j anim sci 1966;25:821826. 3. webel sk, squires el: control of the oestrous cycle in mares with altrenogest. j reprod fertil suppl 1982;32:193-198. 4. wiepz gj, squires el, chapman pl: effects of norgestomet, altrenogest, and/or estradiol on follicular and hormonal characteristics of late transitional mares. theriogenology 1988;30:181-193. 5. gee ek, gillespie l, bolwell cf: effect of oxytocin on suppression of oestrus in mares exhibiting normal oestrous cycles. n z vet j 2012;60:189-193. 6. elhay m, newbold a, britton a, et al: suppression of behavioural and physiological oestrus in the mare by vaccination against gnrh. aust vet j 2007;85:39-45. 7. robinson sj, mckinnon ao: prolonged ovarian inactivity in broodmares temporally associated with administration of equity. aust equine vet 2006;25:85-87. 8. hooper r, taylor t, varner d: et al. effects of bilateral ovariectomy via colpotomy in mares 23 cases (1984-1990). j am vet med assoc 1993;203:1043-1046. 34clinical theriogenology • volume 4 number 1 • march 2012clinical theriogenology • volume 5 number 1 • march 2013 9. kamm jl, hendrickson da: clients' perspectives on the effects of laparoscopic ovariectomy on equine behavior and medical problems. j equine vet sci 2007;27:435-438. 10. vanderwall dk, rasmussen dm, woods gl: effect of repeated administration of oxytocin during diestrus on duration of function of corpora lutea in mares. j am vet med assoc 2007;231:1864-1867. 11. rivera del alamo mm, reilas t, kindahl h, et al: mechanisms behind intrauterine device-induced luteal persistence in mares. anim reprod sci 2008;107:94-106. 12. nie gj, johnson ke, braden td, et al: use of an intra-uterine glass ball protocol to extend luteal function in mares. j equine vet sci 2003;23:266-273. 13. lefranc ac, allen wr: nonpharmacological suppression of oestrus in the mare. equine vet j 2004;36:183-185. 14. hedberg y, dalin a, santesson m, et al: a preliminary study on the induction of dioestrous ovulation in the mare a possible method for inducing prolonged luteal phase. acta vet scand 2006;48:12. 15. wilsher s, allen wr: intrauterine administration of plant oils inhibits luteolysis in the mare. equine vet j 2011;43:99105. 16. stout ta, lamming ge, allen wr: oxytocin administration prolongs luteal function in cyclic mares. j reprod fertil 1999;116:315-320. 17. stout t, allen w: the role of oxytocin in luteolysis in the cycling mare. reprod domest anim 1999;34:351-354. 18. vanderwall dk, rasmussen dm, carnahan kg, et al: effect of administration of oxytocin during diestrus on corpus luteum function and endometrial oxytocin receptor concentration in cycling mares. j equine vet sci 2012;32:536-541. 19. engstrøm t, barth t, melin p, et al: oxytocin receptor binding and uterotonic activity of carbetocin and its metabolites following enzymatic degradation. eur j pharmacol 1998;355:203-210. 20. dansereau j, joshi ak, helewa me, et al: double-blind comparison of carbetocin versus oxytocin in prevention of uterine atony after cesarean section. obstet gynecol 1999;180:670-676. 21. barrett aj, murray rd, christley rm, et al: effects of the administration of oxytocin or carbetocin to dairy cows at parturition on their subsequent fertility. vet rec 2009;165:623-626. 22. schramme ar, pinto crf, davis j, et al: pharmacokinetics of carbetocin, a long-acting oxytocin analogue, following intravenous administration in horses. equine vet j 2008;40:658-661. 23. paccamonti dl, pycock jf, taverne mam, et al: pgfm response to exogenous oxytocin and determination of the half-life of oxytocin in nonpregnant mares. equine vet j 1999;31:285-288. 24. kenney rm. prognostic value of endometrial biopsy of the mare. j reprod fertil suppl 1975;23:347-348. 25. gorecka a, jezierski ta, sloniewski k: relationships between sexual behaviour, dominant follicle area, uterus ultrasonic image and pregnancy rate in mares of two breeds differing in reproductive efficiency. anim reprod sci 2005;87:283-293. 26. samper jc: uterine edema in the mare. in: samper jc, editor. equine breeding management and artificial insemination. 2nd ed. st louis: saunders elsevier; 2009. p. 133-138. 27. pinto cr, kozink dm, whitacre md, et al: utilization of a semi-quantaitive elisa progesterone kit in broodmare management. anim reprod sci 2006;94:204-206. 28. mair t, divers t: equine internal medicine. london: manson publishing ltd; 1997. 29. shand n, irvine ch, turner je, et al: a detailed study of hormonal profiles in mares at luteolysis. j reprod fertil suppl 2000;56:271-279. 30. goff ak, pontbriand d, sirois j: oxytocin stimulation of plasma 15-keto-13,14-dihydro prostaglandin f-2 alpha during the oestrous cycle and early pregnancy in the mare. j reprod fertil suppl 1987;35:253-260. 31. behrendt-adam cy, adams mh, simpson ks, et al: effects of steroids on endometrial oxytocin mrna production. j reprod fertil suppl 2000;56:297-304. 32. stout tae, lamming ge, allen wr: the uterus as a source of oxytocin in cyclic mares. j reprod fertil suppl 2000;56:281-287. 33. watson ed, buckingham j, bjorksten t, et al: immunolocalization of oxytocin and neurophysin in the mare uterus. j reprod fertil suppl 2000;56:289-296. 34. bae se, watson ed: a light microscopic and ultrastructural study on the presence and location of oxytocin in the equine endometrium. theriogenology 2003;60:909-921. 35 clinical theriogenology • volume 5 number 1 • march 2013clinical theriogenology • volume 5 number 1 • march 2013 2010: dosing frozen bovine semen based on progressively motile spermatozoa per dose using an automated sperm quality analyzer dosing frozen bovine semen based on progressively motile spermatozoa per dose using an automated sperm quality analyzer u. shalit,a,c l. rabinovitch,c y. zeronb amedisoos veterinary practice, israel; bsion a.i. company, israel,cmedical electronic systems, caesarea, israel objective the objective of the study was to determine whether frozen bovine semen ai doses could be effectively and efficiently produced based on a constant number of progressively motile sperm cells per dose, using the sqa-vb analyzer as the measurement device and accompanying b-sperm software as the dosing tool. goal the goal was to produce frozen bovine semen doses with a constant number of post thaw progressively motile sperm cells per dose in a commercial farm environment. study overview in order to insure consistency in post thaw doses, a two phase trial was conducted using an automated bovine sperm quality analyzer and dosing software, sqa-vb and b-sperm software (mes, caesarea, israel). phase i: twenty five ejaculates from five different bulls (sion a.i. company, israel) were collected following standard procedures using an artificial vagina. the 25 ejaculates were tested for progressive motility using the sqa-vb in order to establish an average percent of progressively motile sperm for each bull. samples were then frozen and re-tested after thawing using the sqa-vb to determine the "freezing motility loss" factor unique to each bull. phase ii: ten thousand straws were frozen using the sqa-v for testing and b-sperm software to determine the dose of sperm. each ejaculate was split into four different target groups. three groups were dosed based on progressively motile sperm targets post thaw (pmst): 1.5, 3.5, and 7 million progressively motile sperm cells per dose. the “freezing motility loss factor” unique to each bull was taken into consideration according to the following formula: pmst x "freezing motility loss factor". one group was dosed based on the standard protocol of 15 million total sperm per dose and was used as the study control. a total of 380 straws were tested post thaw using the sqa-vb. pmst values were compared to the actual progressively motile sperm per dose results measured by the sqa-vb. the results are presented in the table below. phase 2 final results : measured mean values +/standard deviation vs. target values in post thaw straws 15.013.6 1.5 3.0 6.2 3.5 7.0 1.5 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 total # sperm # prog. mot. sperm # prog. mot. sperm # prog. mot. sperm control group group a group b group c experimental group # sp er m / st ra w , m il. measured mean value target value 41 summary it is possible to produce commercial frozen bovine semen doses with a consistent number of progressively motile sperm cells per post thaw dose, unique to each bull. the sqa-vb and b-sperm software can be effectively used as the technology to measure, dose and control the quality of bovine ai dose samples based on both progressive motility and total sperm concentration. 42 2012: stud dog infertility stud dog infertility margret l. casal section of medical genetics, school of veterinary medicine, university of pennsylvania, philadelphia, pa abstract the first step in determining the cause of infertility in a stud dog is a complete medical and breeding history. together with a thorough physical examination and semen evaluation, the problem can be narrowed down to a few specific causes. at this point, further diagnostic tests may be necessary, such as blood work, urinalysis, urine and semen cultures, ultrasound examination or even a testicular biopsy. once a diagnosis has been made, treatment, management, and/or a specific breeding plan will have to be implemented to improve fertility. key words: canine, testicle, male infertility introduction demonstration of normal fertility in the male dog requires proof of successful pregnancy and the birth of live puppies. infertility in the male dog is defined as complete failure to impregnate normal bitches that were bred appropriately. subfertility is defined as a whelping rate of less than 75% when bred appropriately to normal bitches. sterility is defined as the inability to produce offspring, i.e., through lack of an ejaculate or normal sperm cells. as with any presentation of infertility, confirmation, characterization, and localization of the problem needs to be ascertained before a diagnosis can be made. a complete workup is required including the overall health of the dog, his past medical history, and his breeding history, as well as that of his relatives. reasons for infertility can be localized to the prostate, testicles, epididymis, or scrotum. causes are trauma, tumors, autoimmune diseases and diseases outside of the genital system, such as bladder infections, immune disorders, other tumors, and endocrine diseases (e.g. diabetes mellitus, cushing’s disease, etc.). in some cases, the stud may just be reluctant to breed or has problems with ejaculation. depending on the cause of infertility, treatment options may be considered. most stud dogs presenting for infertility are not truly infertile but have perceived infertility due to poor breeding timing. the following gives a brief summary of 1) information that you should gather before going to see your reproductive specialist and 2) the diagnostics that your specialist may be performing to diagnose infertility in the stud dog. treatment is beyond the scope of this article but we will touch briefly on nutraceuticals at the end of these proceedings. signalment this information includes the age of the stud and the breed. in cases where there are different lines within the same breed (e.g. field trial versus show dog versus hunting dog), this information should be included. history a complete general medical history is vital. previous conditions such as car accidents, overheating, seizures, infections, and allergies are among some of the disorders that can have an effect on fertility. vaccination status, deworming protocols, nutrition, and current medications including supplements, heartworm, flea and tick preventatives may or may not have an effect on fertility but the specialist needs to know this information. current problems should be listed such as lameness; difficulty in producing feces; exercise intolerance; and excessively drinking water or urinating. information on kennel type and size, number of dogs present, and other animals on the premises can be helpful. if someone handling the dog is on hormone patches (estrogen, testosterone), these can have an effect on fertility in dogs. previous medical records should be copied and brought to the appointment. documentation of genetic testing appropriate for the breed should also be included in the medical records. 535 clinical theriogenology • volume 4, number 4 • december 2012 specific reproductive history includes the age, at which the stud was first bred, how many times he was used for breeding (successful and unsuccessful), if the bitch was timed properly (method?), how many puppies resulted, the overall health of the puppies, and the age at last breeding. it is also important to know if semen evaluations were done previously and if semen had been frozen. the history on the bitches used for breeding should include number of successful and unsuccessful breedings, number of litters produced with other dogs, health of the litters, and current status of the bitches. documentation of brucellosis testing of the stud is also very important. it is extremely helpful for the specialist if all of the events are written up and presented in chronological order. physical examination a general physical examination should take place first. this will alert the reproductive specialist to issues that could be contributing to infertility, such as problems with vision or hearing, back pain, arthritis, hip dysplasia, or signs of endocrine disorders, such as diabetes mellitus, addison’s or cushing’s disease, heart disease, lung disease or tumors. most veterinarians will perform a semen collection before performing a specific reproductive examination because it may be more difficult to collect semen after the prostate has been palpated, for example. the idea is to leave the most uncomfortable examinations for last. during semen collection, libido can be assessed as well as the ability to achieve an erection. some studs require a female in heat being present and some do fine with scents. some dogs will have trouble performing in an unfamiliar environment, especially if they have never been collected before. multiple visits may be required. after semen collection and evaluation, the scrotum is carefully inspected and the testicles and epididymis palpated for lumps and bumps. lesions on the scrotum may be an indication that the stud feels pain in his testicles, as he will lick the scrotum in an attempt to get at the testicles. the prostate is palpated for potential tumors or benign prostatic hyperplasia. semen evaluation a semen sample should be collected (see above) and evaluated. seminal fluid samples should be submitted for bacterial cultures. cytologic evaluation of seminal fluid should also be performed to determine if other cells (inflammatory cells or tumor cells) are present. alkaline phosphatase analysis of the seminal fluid can assist in determination of complete ejaculation, particularly in the diagnosis of azoospermia (absence of live sperm cells). in the event sperm cells are obtained, their motility, number, and morphology will be evaluated, along with the volume of the ejaculate to calculate total number of progressive forward motile normal sperm. other diagnostics in addition to history taking and physical examination, a minimum database (complete blood cell count, blood chemistry, and urinalysis), as well as thyroid and brucellosis testing should be included. a urine sample may also be collected by cystocentesis after semen collection to evaluate for the potential for retrograde ejaculation. ultrasound analysis of the prostate and the testicles should also be performed. chromosomal analysis (karyotype) and analysis for the presence of the sry (male specific) gene can be conducted, if intersex conditions are suspected. in some cases, advanced semen diagnostics are available that can directly evaluate specific parts of the sperm cells, such as the acrosome, which is crucial for fertilization. testicular biopsies may also be necessary to properly diagnose the cause and the degree of infertility and to be able to provide a prognosis. causes of infertility there are many causes for infertility, but benign prostatic hyperplasia/hypertrophy is without a doubt the most common disorder of the prostate in the intact male dog. the most common clinical signs include blood dripping from the penis and difficulty defecating (constipation or ribbon-like stools). other disorders include infections of the testicles, epididymides, or prostate. brucellosis is very important cause to rule out, as it can be transmitted to the entire kennel as well as to humans. overheating has been described in several papers as a cause. the most notable case occurred when dogs were left on an 536clinical theriogenology • volume 4, number 4 • december 2012 airplane in the blazing heat without air conditioning; the males that survived became sterile and never regained fertility. trauma and neoplasia (cancers) of any of the reproductive structures can cause infertility, as can immune conditions such as the production of antisperm antibodies. some dogs may present with testicular degeneration or atrophy. by that time, the changes may be secondary and the inciting cause may thus never be determined. failure to achieve an erection can be due to apprehension, psychological constraints, pain, or androgen insufficiency. the most common presentation for this is due to intact male dogs that have been disciplined their entire life for exhibiting mounting behavior. these as well as subordinate dogs may not exhibit normal breeding behavior when desired. failure to ejaculate in the presence of erection can be caused by lack of sexual maturity, pain, psychological factors, drug therapy (e.g., tricyclic antidepressants), or sympathetic neuropathy which can be idiopathic, secondary to systemic disease such as diabetes mellitus, or secondary to spinal cord injury. it is important to keep in mind that some experienced stud dogs will not ejaculate unless an estrous teaser bitch is present. causes for the failure to achieve normal copulation in intact male dogs include sexual immaturity, old age, sexual overuse, pain, psychological constraints, persistent frenulum, and idiopathic poor libido. female factors may include aggressive females, anatomical anomalies, and improper timing. dogs presented with infertility but normal copulation and normal semen quality are difficult to assess as there are currently no specific tests for function of apparently normal spermatozoa in dogs. when faced with this presentation, types of breeding as well as fertility of the bitch should also be assessed. in general when evaluating historical reports in the literature, conception rate is better with copulation than artificial insemination, with a decline in the conception rate with increasing manipulation of the semen. infertility with normal copulation and abnormal semen quality is a more common presentation of the infertile stud. the findings can be divided as follows: 1. azoospermia= ejaculation of seminal fluid that does not contain sperm. determination of high alkaline phosphatase in the seminal fluid confirms complete ejaculation. one possible cause is retrograde ejaculation; the semen will be present in the urine. 2. oligozoospermia= is a low total number of sperm in the ejaculate. the number of sperm a dog can produce daily is dependent upon the amount of testicular tissue present. normally, greater than 300 million sperm are present in a normal ejaculate. keep in mind that these dogs are not necessarily infertile. 3. hematospermia= blood in the ejaculate. it is seen most commonly with prostatic disease and penile trauma during collection. dogs with hematospermia are not necessarily infertile. 4. teratozoospermia= decreased number of morphologically normal sperm. normal dogs have 80% or greater morphologically normal sperm in the ejaculate. specific morphologic defects are poorly correlated with infertility in the dog. however, some defects when seen in large numbers have been associated with infertility such as proximal cytoplasmic droplets. 5. sperm agglutination occurs after induction of antisperm antibodies and has been reported in dogs with brucellosis. the blood-testis barrier normally protects the testicular tissue and the sperm cells from an immune response. any breakdown in this barrier can result in the formation of anti-sperm antibodies and resultant infertility. 6. asthenozoospermia= progressive motility less than 70% and is often associated with teratozoospermia. 7. infection/inflammation (see orchitis/ epididymitis/ prostatitis above) treatment treatment is based on the underlying cause of the infertility. it is important to consider that the prognosis of male dogs with confirmed infertility is very guarded and one report suggested that fewer than 10% of dogs that presented with infertility returned to fertility after a diagnostic work-up and 537 clinical theriogenology • volume 4, number 4 • december 2012 appropriate therapy. treatable conditions are transient insults, drug or hormone therapy, mild infection, retrograde ejaculation, and in some cases, neoplasia. untreatable or difficult to treat conditions include genetic, immune-mediated, idiopathic degeneration, and disorders that have destroyed primary spermatogonia (the precursors to sperm cells). nutraceuticals the word nutraceuticals is made up of nutrition and pharmaceutical, and are supplements that are reported to have medicinal properties. many of the nutraceuticals contain glucosamine, docosahexaenoic acid (dha), glycosaminoglycans (gag), vitamin c or e, or fatty acids. the idea is to provide antioxidants and the claims range from improvement in sperm motility and in the quality of cryopreserved or chilled semen after supplementation to a decrease in proximal droplets to improved numbers of sperm cells. however, the mechanism of action has not been documented in any of the nutraceuticals on the market and the effects of many supplements on canine semen quality are currently unsubstantiated. summary if faced with a stud in which infertility is suspected, all historical information should be collected before the appointment with the specialist. a general physical examination and a reproductive examination will help narrow down the possible causes. supplemental literature 1. freshman jl: clinical management of the subfertile stud dog. vet clin north am small anim pract. 2001;31:259-269. 2. hess m: documented and anecdotal effects of certain pharmaceutical agents used to enhance semen quality in the dog. theriogenology. 2006;66:613-617. 3. lopate c: the problem stud dog. vet clin north am small anim pract. 2012;42:469-488, vi. 538clinical theriogenology • volume 4, number 4 • december 2012 2009: antibiotic use in prostatic disease in dogs antibiotic use in prostatic disease in dogs 1 m. v. root kustritz 2 college of veterinary medicine, university of minnesota, st. paul, mn, usa 3 abstract 4 knowledge of pharmacokinetics of currently available antibiotics permits 5 veterinarians to choose antibiotic therapy based on microbial culture and sensitivity 6 results and ability of various classes of antibiotics to readily distribute through prostatic 7 tissue. this may replace other diagnostic tests used to support antibiotic choice, such as 8 measurement of ph of ejaculated prostatic fluid. 9 keywords: prostate, prostatitis, fluroquinolone 10 introduction 11 the prostate is the only accessory sex gland of dogs. it is a retroperitoneal organ, 12 encircling the neck of the urinary bladder. it is bilobed with a palpable median raphe. in 13 smaller breed and younger dogs, it is palpable per rectum. it may be difficult to palpate 14 per rectum in large or giant breed dogs of any age, and in dogs with age-related increase 15 in prostate size and subsequent cranial positioning of the prostate and urinary bladder. 16 the prostatic capsule is thick and contains smooth muscle fibers, some as an 17 extension of muscle fibers from the wall of the urinary bladder. some believe there is no 18 such thing as a true prostatic capsule; one study evaluating prostate histology after 19 prostatectomy in men with prostatic carcinoma instead defined a fibromuscular band of 20 varying thickness that was an inseparable continuation of the prostate stroma.1 the 21 prostatic capsule may be more of a functional than a histologic entity. 22 81 the canine prostate is made up of lobules of secretory tissue separated radially by 23 bands of connective tissue. these lobules are larger and contain more secretory tissue in 24 the dorsal and lateral areas of the prostate than in the ventral portion.2 there is little 25 smooth muscle and less stromal tissue in the canine prostate than in the human prostate. 26 the human prostate also can be separated into three distinct histologic zones (peripheral, 27 transitional, and central); these zones are not evident in dogs.3 28 the primary blood suppy arises via the internal pudental artery as the paired 29 prostatic arteries. the right and left lobes of the prostate are vascularized independently, 30 with the prostatic artery on each side separating into cranial, middle, and caudal arteries. 31 in general, vascularity is poor on the ventral prostate surface compared to the rest of the 32 gland.4 33 clinical prostatitis 34 in men, prostate infection or inflammation is classified as acute infectious 35 prostatitis, chronic infectious prostatitis, non-infectious chronic pelvic pain syndrome, or 36 asymptomatic prostate inflammation.5,6 in dogs, acute and chronic prostatitis are 37 recognized disease entities, with prostatic abscessation generally considered a companion 38 to either. chronic prostatitis without abscessation will be the focus of this discussion. 39 chronic prostatis occurs in the presence of some other prostate disease. the 40 normal canine prostate is protected from ascending infection by anatomic, functional, and 41 immunologic barriers, including secretion of prostatic fluid and urine flow, urethral 42 peristalsis, formation of a urethral high pressure zone at the area of the prostate, and 43 antibacterial properties of prostatic fluid.7,8 likelihood of infection is increased by any 44 process that increases the number of bacteria in the periprostatic urethra or urinary 45 82 bladder, or that compromises local or systemic immune response.9 specific examples 46 include urolithiasis, urethral neoplasia, urinary tract infection, squamous metaplasia of 47 the prostate due to hyperestrogenism with subsequent decrease in prostate secretion, and 48 concurrent prostate diseases such as benign prostatic hypertrophy (bph) or neoplasia.7 49 the class of organism most often associated with chronic prostatitis in dogs is the 50 gram negative enterobacteriaciae, including e. coli, klebsiella sp., and proteus sp. other 51 bacterial organisms reported include pseudomonas aeruginosa, staphylococcus aureus, 52 and brucella canis.10-12 mycoplasma has been reported as a cause of chronic prostatitis,11 53 as has blastomycosis.13 bacterial organisms implicated in human prostatitis are the same 54 as those in dogs. a complicating factor in human medicine, which may occur in 55 veterinary medicine, is formation of biofilms, a matrix of bacteria and polysaccharide that 56 permits creation of antibiotic-resistant microcolonies within tissue.14 57 clinical signs are mild, with disease often inapparent. reported clinical 58 presentations include infertility, poor semen quality, and signs of associated prostate 59 disease such as dripping of bloody fluid from the penis in dogs with concurrent bph.12 60 diagnosis is via rectal palpation, imaging, and collection of samples of prostatic 61 tissue or fluid for cytology or culture. rectal palpation is not an accurate diagnostic test; 62 in one study of 500 dogs, rectal palpation permitted correct identification of prostate 63 disease in only 53% of cases.15 rectal palpation can be used to judge prostate size and to 64 assess for a pain response, which usually will be absent in dogs with chronic disease. 65 there are no pathognomic changes visible by ultrasound that permit definitive diagnosis 66 by imaging alone.16 most often the infected prostate is described as locally to diffusely 67 hyperechoic.17 ultrasound may be best used to guide sample collection; ultrasound-68 83 guided fine-needle aspirate has been reported to be 75% accurate in identifying 69 prostatitis.18,19 70 culture of prostatic fluid or tissue is required for definitive diagnosis of 71 prostatitis. culture of ejaculated prostatic fluid is sensitive but not specific as it may be 72 contaminated with organisms from the urinary tract.20 results from culture of prostatic 73 fluid correlated well with culture of prostatic tissue in only 80% of instances in one 74 study.21 similarly, presence of inflammatory cells in ejaculated prostatic fluid is not well 75 correlated with culture results.21,22 for these reasons, cytology and culture of prostatic 76 tissue is preferred when possible. the only reported side-effect of fine-needle aspirate of 77 the prostate is transient hematuria.23 78 treatment involves management of concurrent prostatic or urinary tract disease 79 and antibiotic therapy, as described in detail below. it has been well demonstrated that 80 castration hastens resolution of chronic prostatitis in dogs without prostatic neoplasia as 81 an underlying cause of disease.11,24 medical treatment of underlying bph with 82 finasteride may hasten resolution of disease; the author is unaware of studies evaluating 83 effect of finasteride therapy as a component of treatment for prostatitis in dogs. 84 antibiotic pharmacokinetics relative to the prostate 85 antibiotics may be characterized by their absorption, distribution, metabolism, 86 and excretion. factors that affect these processes include lipid solubility, molecular 87 weight, and ionization of the drug molecule, and degree to which the drug is bound to 88 protein in circulation. for antibiotics to penetrate tissue, they must be able to pass 89 through the lipid-rich cell membrane, either by active transfer or diffusion. since most 90 antibiotics move into cells passively, lipid solubility and size of the molecule are crucial 91 84 determinants of ability to penetrate tissue. environmental ph alters ionization of the cell, 92 which also affect permeability as ionized drug cannot pass through the lipid bilayer. 93 finally, drug that is bound to protein in serum is unavailable compared to drug that is 94 dissolved and can freely cross cell membranes.25,26 95 ionization and trapping of drugs due to altered ph has long been upheld as a 96 component of the decision regarding which antibiotic to choose for treatment of chronic 97 prostatitis. the premise is that because charged (ionized) molecules cannot cross the cell 98 membrane and because there may be a ph gradient from serum into prostatic tissue as 99 disease develops within the prostate, uncharged molecules will equilibrate across that 100 membrane but charged molecules will get trapped on one side or the other. if you could 101 trap molecules within the tissue, you’d get a higher concentration of drug on that side 102 (uncharged plus charged portions of the total drug present).26 knowledge of the pka, or 103 ionization constant, would permit one to choose an appropriate antibiotic based on ph of 104 prostatic fluid. the value of this premise is decreased in small animal practice as it has 105 been demonstrated that ph of canine prostatic fluid does not significantly change in the 106 presence of prostate disease,21 and because of the availability of antibiotics that can 107 ionize at either acicid or alkaline ph. 108 fluoroquinolones 109 fluoroquinolones are the preferred class of antibiotics for treatment of chronic 110 infectious prostatitis in humans. the fluoroquinolones are bactericidal via inhibition of 111 dna gyrase, which is necessary for dna replication and repair.25 they are well 112 distributed throughout the body because they are lipid soluble and are amphoteric, with 113 both acidic and alkaline ionization constants.27-29 fluoroquinolones also move well into 114 85 biofilms, lessening chance of persistent or recurrent infection.27 products available for 115 oral use in dogs include enrofloxacin (baytril®; bayer animal health, shawnee mission, 116 ks, usa), ciprofloxacin (cipro®; schering-plough, kenilworth, nj, usa), 117 marbofloxacin (zeniquin®; pfizer animal health, new york, ny, usa), orbifloxacin 118 (orbax®; intervet/schering-plough animal health; summit, nj, usa), and difloxacin 119 (dicural®; fort dodge animal health, ft. dodge, ia, usa). of these compounds, 120 enrofloxacin shows superior movement into canine prostatic tissue, followed by 121 ciprofloxacin and marbofloxacin.28,30-32 122 macrolides 123 the macrolide antibiotics are bactericidal or bacteriostatic, depending on bacterial 124 species.25 erythromycin is lipid-soluble but is quickly broken down after oral 125 administration and is highly protein bound.27 azithromycin concentrates in phagocytes 126 and so is carried into tissue, from which it is slowly released, permitting less frequent 127 dosing.27,33-35 it has been demonstrated to penetrate the prostate well in humans.27 there 128 are no veterinary formulations of azithromycin available as of this writing. 129 chloramphenicol 130 chloramphenicol is bactericidal or bacteriostatic, depending on bacterial 131 species.25 it is very lipid-soluble and not highly protein bound.27 because tissue 132 concentrations generally are well below those in serum, high doses must be used, raising 133 concerns about toxic effects, including myelosuppression, with long-term use.7 134 trimethoprim-sulfas 135 trimethoprim-sulfa combinations are bacteriostatic as they inhibit dna 136 replication.25 these drugs are well absorbed orally and penetrate prostate tissue well.27 137 86 concerns of long-term use include keratoconjunctivitis sicca, anemia, and acute 138 neutrophilic hepatitis.27 the approved veterinary product for dogs is trimethoprim-139 sulfadiazine (tribrissen®; intervet/schering-plough). 140 other antibiotics 141 penicillins and cephalosporins are poorly lipid-soluble and do not penetrate the 142 chronically infected prostate.27 aminoglycosides (amikacin, gentamycin) show 143 variability tissue penetration and potential toxicity with long-term use.27,36 doxycycline 144 penetrates prostate tissue better than tetracycline but is not commonly used.27 145 metronidazole (flagyl®; pfizer, inc, new york, ny, usa) may be useful if anaerobic 146 infection is a component of disease; it is a lipid-soluble antibiotics that is well absorbed 147 orally and is not highly protein bound.27 neurologic signs and gastrointestinal signs may 148 be seen as side-effects.27 149 conclusion 150 the primary factor governing antibiotic choice must be the result of culture and 151 sensititivity testing, preferably of prostatic tissue. the chosen antibiotic should be 152 administered for 4 to 6 weeks, with rechecks of prostatic fluid or tissue culture at 7 days, 153 one month and 6 months after completion of therapy.29,37 antibiotic resistance may be a 154 growing concern with wide use of fluoroquinolone antibiotics;38 for this reason an effort 155 should be made to decrease risk of recurrence. concurrent disease of the urinary tract or 156 prostate must be addressed, including a recommendation for castration in dogs with bph 157 and recurrent or persistent prostatitis. 158 references 159 87 1. ayala ag, ro jy, babaian r, et al: the prostatic capsule: does it exist? its 160 importance in the staging and treatment of prostatic carcinoma. am j surg path 161 1989;13:21-27. 162 2. stefanov m, martin-alguacil n, martin-orti r: distinct vascular zones in the 163 canine prostate. micro res tech 2000;50:169-175. 164 3. lai c-l, van den ham r, van leenders g, et al: comparative characterization 165 of the canine normal prostate in intact and castrated animals. prostate 166 2008;68:498-507. 167 4. stefanov m: extraglandular and intraglandular vascularization of canine prostate. 168 micro res tech 2004;63:188-197. 169 5. taylor bc, noorbaloochi s, mcnaughton-collins m, et al: excessive antibiotic 170 use in men with prostatitis. am j med 2008;121:444-449. 171 6. wagenlehner fme, weidner w, sorgel f, et al: the role of antibiotics in 172 chronic bacterial prostatitis. intl j antimicrob agents 2005;26:1-7. 173 7. klausner js, osborne ca: management of canine bacterial prostatitis. j am vet 174 med assoc 1983;182:292-296. 175 8. fair wr, wehner n: the antibacterial action of canine prostatic fluid and human 176 seminal plasma in an agar diffusion assay system. invest urol 1973;10:262-265. 177 9. dorfman m, 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examination as a screening test. j so afr 192 vet assoc 2007;78:66-68. 193 16. ling gv, nyland tg, kennedy pc, et al: comparison of two sample collection 194 methods for quantitative bacteriologic culture of canine prostatic fluid. j am vet 195 med assoc 1990;196:1479-1482. 196 17. feeney da, johnston gr, klausner js, et al: canine prostatic disease – 197 comparison of ultrasonographic appearance with morphologic and microbiologic 198 findings: 30 cases (1981-1985). j am vet med assoc 1987;190:1027-1034. 199 18. powe jr, canfield pj, martin ra: evaluation of the cytologic diagnosis of 200 canine prostatic disorders. vet clin path 2004;33:150-154. 201 19. nickel rf, teske e: diagnosis of canine prostatic carcinoma. tidjschr voor 202 diergeen 1992;117 (suppl 1):32s. 203 20. barsanti ja, finco dr: evaluation of techniques for diagnosis of canine prostatic 204 diseases. j am vet med assoc 1984;185:198-200. 205 89 21. barsanti ja, prasse kw, crowell wa, et al: evaluation of various techniques for 206 diagnosis of chronic bacterial prostatitis in the dog. j am vet med assoc 207 1983;183:219-224. 208 22. root kustritz mv, johnston sd, olson pn, et al: relationship between 209 inflammatory cytology of canine seminal fluid and significant aerobic bacterial, 210 anaerobic bacterial or mycoplasma cultures of canine seminal fluid: 95 cases 211 (1987-2000). theriogenology 2005;64:1333-1339. 212 23. root kustritz mv: collection of tissue and culture samples from the canine 213 reproductive tract. theriogenology 2006;66:567-574. 214 24. cowan la, barsanti ja, crowell w, et al: effects of castration on chronic 215 bacterial prostatitis in dogs. j am vet med assoc 1991;199:346-350. 216 25. dawson js: pharmacology. new york: mosby; 2002. p. 10-15, 31-38. 217 26. naber kg, sorgel f: antibiotic therapy – rationale and evidence for optimal drug 218 concentrations in prostatic and seminal fluid and in prostatic tissue. andrologia 219 2003;35:331-335. 220 27. plumb dc: veterinary 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chronic bacterial prostatitis. am j vet res 235 1995;56:386-390. 236 33. bosnar m, kelneric z, munic v, et al: cellular uptake and efflux of 237 azithromycin, erythromycin, clarithromycin, telithromycin, and cethromycin. 238 antimicrob agents chemo 2005;49:2372-2377. 239 34. sheppard rm, falkner fc: pharmacokinetics of azimithromycin in rats and dogs. 240 j antimicrob chemther 1990;25(suppl a):49-60. 241 35. lode h: the pharmacokinetics of azithromycin and their clinical significance. 242 eur j clin micro infect dis 1991;10:807-812. 243 36. wang h, li zc, luo zh, et al: penetrability of amikacin into prostate tissues in 244 rat models of chronic bacterial prostatitis. zhonghua nan ke xue 2008;14:583-245 589. 246 37. naber kg: the role of quinolones in the treatment of chronic bacterial prostatitis. 247 infection 1991;19(suppl 3):5170-5177. 248 91 38. miura t, tanaka k, shigemura k, et al: levofloxacin resistant escherichia coli 249 sepsis following an ultrasound-guided transrectal prostate biopsy: report of four 250 cases and review of the literature. intl j urol 2008;15:457-459. 251 92 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true 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/flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /bleedoffset [ 0 0 0 0 ] /convertcolors /noconversion /destinationprofilename () /destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2014: twenty year trends of bull breeding soundness examimations at a teaching hospital twenty year trends of bull breeding soundness examimations at a teaching hospital robert carson, jennifer koziol, james wenzel, chance armstrong, jason edmondson, herris maxwell college of veterinary medicine, auburn university, auburn, al introduction: the origin of the society for theriogenology has its roots in breeding soundness examination (bse) of bulls as the western blizzard of 1954 lead to the evaluation of bulls by the founders of the society. today bull bses are still one of the more common procedures performed by food animal veterinarians. bull bses are one of the most economically strategic procedures required in natural service cattle herds. in well-managed herds with short breeding and calving seasons bull bses are vital to their productivity and economic success. since bull bses are such an important part of the food animal industry it remains extremely important to teach proper techniques to future food animal veterinarians as well as provide a service for local clientele. in the fall of 1992, the society for theriogenology adopted new individual and herd bse forms. under these guidelines, a satisfactory potential breeder must be acceptable on all four parts of the examination. the four parts consist of a physical examination, minimum scrotal circumference based on age, minimum progressive motility of 30%, and minimum normal morphology of 70%. after examination the final classifications are: satisfactory for bulls meeting minimum standards for the four parts of the examination and unsatisfactory or deferred for bulls not meeting one or more of the minimum standards. deferred classification allows for reevaluation at a specified time for conditions in the opinion of the examiner that might improve. the 1992 form replaced a form used in the 1970’s and 1980’s which evaluated the same criteria but was based on a point system and had final classifications of: satisfactory, questionable or unsatisfactory. prior to the 1970’s a point system was used which evaluated visual concentration, vigor (gross motility) and live dead ratio; obvious problems existed with this system. materials and methods breeding soundness examination records (1992 form) for client bulls from the auburn university teaching hospital were reviewed and data tabulated. for comparison three different time periods were evaluated: 1993-1995, 2006-2008 and 2009-2013. the 1993-1995 and 2006-2008 data were hand tabulated and recorded while the 2009-2013 utilized the society for theriogenology computer software for bull breeding soundness and excel spreadsheet for recording and tabulation. a complete bse was performed on each bull. bulls were restrained in a squeeze chute and examinations were performed by one or more clinicians. physical examination and scrotal circumference measurements were done prior to semen collection. semen was collected by electroejaculation. bulls with poor motility or high numbers of secondary abnormalities initially were immediately recollected. each time a bull was presented was considered one examination for tabulation purposes; therefore a small number of bulls may have been represented more than once due to reevaluations. the population consisted of bulls consigned to sales, recent purchases, bulls offered for sale at private treaty and herd bulls. herd bulls retained in the herd were often presented in multiple years. breed composition was primarily beef bulls with holsteins being the only dairy breed examined. examinations were conducted in the teaching hospital and in the field. for each time period the following data were tabulated and compared: 1. total number of bulls examined and number of bulls per year; 2. breeds of bulls represented 3. age of bulls when examined; 4. results of examinations all ages (satisfactory, unsatisfactory, deferred); 5. age and percent satisfactory; 6. age and percent unsatisfactory; 7. percent deferred by age; 8. combination of unsatisfactory and deferred by age; 9. reasons for unsatisfactory and deferred all ages; 10. combined reasons for unsatisfactory and deferred all bulls; 11. inadequate scrotal circumference all bulls; 12. inadequate scrotal circumference by breed; and 13. inadequate scrotal circumference by age for 2009-2013. since the three time periods were not the exact same in length the data in most cases are presented in percentages rather than exact numbers. data were statistically evaluated using a sas program. 495 clinical theriogenology • volume 6, number 4 • december 2014 results the total number of bulls examined for each of the time periods and the number of bulls per year is shown in table 1. the various breeds of bulls examined are shown in table 2. the age of the bulls when examined is illustrated in table 3. the results for all bulls for each time period appears in table 4. table 1 total number of bulls examined for each time period and number of bulls examined per year 1993-1995 2006-2008 2009-2013 1276 bulls 1076 bulls 1943 bulls 638 bulls/year 538 bulls/year 485 bulls/year table 2 the various breeds of bulls examined for each time period and percentage of total 1993-1995 (1276 total) 2006-2008 (1076 total) 2009-2013 (1943 total) an (323) 25% an (776) 72% * an (1322) 68% * ch (221) 17% ch (38) 3.5% ch (34) 1.75% sm (183) 14% sm (75) 7% sm (77) 3.96% ho (107) 8% ho (16) 1.5% ho (0) 0% lm (97) 8% lm (6) 0.5% lm (2) 0.1% he (89) 7% he (53) 5% he (86) 4.43% bm (51) 4% bm (3) 0.3% bm (2) 0.1% ba (47) 4% ba (61) 5.7% ba (204) 10.5% * gv (39) 3% gv (9) 0.8% gv (24) 1.25% ra (25) 2% ra (22) 2% ra (5) 0.26% other (102) 8% other (18) 1.7% other (187) 9.62% • difference p>.05 the ages of bulls examined both number and percent classified as satisfactory appears in table 5 while age of bull vs number and percent unsatisfactory is in table 6. age of bulls and number and percent deferred is represented in table 7 and the combined unsatisfactory and deferred classifications are in table 8. the reasons for unsatisfactory and deferred classifications combined are shown in table 9. combination of reasons for unsatisfactory and deferred are shown in table 10. numbers and percent of all bulls with insufficient scrotal circumference for each time period is shown in table 11. percent insufficient scrotal circumference for the most prevalent breeds for all time periods is represented in table 12. insufficient scrotal circumference by age for the 2009-2013 time period is illustrated in table 13. a breakdown of satisfactory and combined unsatisfactory and deferred by the more prevalent breeds for the geographic area and the reasons for unsatisfactory and deferred are shown by breed for the 20092013 time period in table 14. 496clinical theriogenology • volume 6, number 4 • december 2014 table 3 age of bulls and percent of total when presented for examination for each of three time periods age 1993-1995 (1276 total) 2006-2008 (1076 total) 2009-2013 (1943 total) </= 15 months (146) 11.4% (196) 18.2% (200) 10.3% >15 </= 18 months (179) 14% (164) 15.2% (144) 7.4% >18 </= 21 months (211) 16.5% (57) 5.3% (72) 3.7% >21 </= 24 months (129) 10.1% (38) 3.5% (557) 28.6% * >24 </= 36 months (273) 21.4% (198) 18.4% (339) 17.4% >36 </= 48 months (129) 10.1% (113) 10.5 (199) 10.2% 4-5 years (108) 8.5% (177) 16.5% (202) 10.4% 5-6 years (58) 4.5% (102) 9.5% (126) 6.5% 6-7 years (21) 1.6% (23) 2.1% (55) 2.8% >7 years (22) 1.7% (8) 0.74% (28) 1.4% *difference p>.05 table 4 examination results all ages combined for each time period 1993-1995 2006-2008 2009-2013 satisfactory (802/1276) 62.9% (806/1076) 74.9% (1372/1943) 70.6% unsatisfactory (369/1276) 28.9% (172/1076) 16% (482/1943) 24.8% deferred (105/1276) 8.23% (98/1076) 9.1% (89/1943) 4.6% unsatisfactory/deferred (474/1276) 37.2% (270/1076) 25.1% (571/1943) 29.4% table 5 age of bulls vs number and percent classified satisfactory for each time period age 1993-1995 2006-2008 2009-2013 </= 15 months (83/146) 56.8% (127/196) 64.8% (120/200) 60.0% >15 </=18 months (125/179) 69.8% (113/164) 69.0% (82/144) 57.2% >18 </= 21 months (125/211) 59.2% (38/57) 66.0% (48/72) 66.7% >21 </= 24 months (83/129) 64.3 % (31/38) 81.6% (375/557) 67.3% >24 </= 36 months (179/273) 65.5% (75/198) 76.8% (263/339) 77.6% >36 </= 48 months (86/129) 66.7% (87/113) 76.8% (159/199) 79.9% 4 -5 years (68/108) 63% (147/177) 83.1% (144/202) 71.3% 5 – 6 years (31/58) 53.5% (78/102) 76.5% (99/126) 78.6% 6 – 7 years (10/21) 47.6% (21/23) 90.1% (46/55) 83.6% >7years (12/22) 54.6% (4/8) 50.0% (20/28) 71.4% 497 clinical theriogenology • volume 6, number 4 • december 2014 table 6 age of bulls vs number and percent unsatisfactory for each time period age 1993-1995 2006-2008 2009-2013 </= 15 months (41/146) 28.1% (24/196) 12.2% (51/200) 25.5% >15 </= 18 months (40/179) 22.4% (31/164) 19.0% (52/144) 35.9% >18 </= 21 months (61/211) 28.9% (14/57) 23.7% (23/72) 31.9% >21 </= 24 months (37/129) 28.7% (4/38) 10.5% (166/557) 29.8% >24 </= 36 months (80/273) 29.3% (35/198) 17.7% (68/339) 20.1% >36 </= 48 months (35/129) 27.1% (15/113) 13.3% (32/199) 16.1% 4 – 5 years (35/108) 32.4% (25/177) 14.1% (48/202) 23.8% 5 – 6 years (31/58) 37.9% (18/102) 17.6% (23/126) 18.3% 6 – 7 years (10/21) 47.6% (2/23) 9.5% (8/55) 14.6% >7 years (8/22) 36.6% (4/8) 50.0% (6/28) 21.4% table 7 age of bulls vs number and percent deferred for each time period age 1993-1995 2006-2008 2009-2013 </=15 months (22/146) 15.1% (45/196) 23.0% (29/200) 14.5% >15 </= 18 months (14/179) 7.8% (18/164) 11.0% (10/144) 6.9% >18 </= 21 months (25/211) 11.9% (6/57) 10.5% (10/72) 1.4% >21 </= 24 months (9/129) 7.0% (3/38) 7.9% (16/557) 2.8% >24 </= 36 months (14/273) 5.2% (11/198) 5.6% (8/339) 2.3% >36</=48 months (8/129) 6.2% (6/113) 5.2% (10/199) 5.0% 4-5 years (5/108) 4.6% (5/177) 2.8% (8/202) 4.0% 5-6 years (5/58) 8.6% (6/102) 5.9% (4/126) 3.2% 6-7 years (1/21) 4.8% (23) 0% (1/55) 1.8% >7 years (2/22) 9.1% (8) 0% (2/28) 7.1% table 8 age of bulls and numbers and percent for combined unsatisfactory and deferred for each time period age 1993-1995 2006-2008 2009-2013 </=15 months (63/146) 43.1% (69/196) 35.2% (80/200) 40.0% >15 </=18 months (54/179) 30.2% (49/164) 30.0% (62/144) 42.8% >18 </= 21 months (86/211) 40.8% (19/57) 33.8% (24/72) 33.3% >21 </= 24 months 46/129) 35.7% (7/38) 18.4% (182/557) 32.7% >24 </= 36 months (94/273) 34.4% (46/198) 23.3% (76/339) 22.4% >36 </= 48 months (43/129) 33.3% (21/113) 18.3% (44/199) 21.9% 4-5 years (40/108) 37.0% (30/177) 16.9% (56/202) 27.8% 5-6 years (27/58) 46.5% (24/102) 23.5% (27/126) 21.4% 6-7 years (11/21) 52.4% (2/23) 9.5% (9/55) 16.4% >7 years (10/22) 45.4% (4/8) 50.0% (8/28) 28.6% 498clinical theriogenology • volume 6, number 4 • december 2014 table 9 reasons for combined unsatisfactory and deferred classification for all bulls for each time period 1993-1995 2006-2008 2009-2013 total unsat/deferred (474/1276) 37.2% (270/1076) 25.1% (571/1943) 29.4% physical 9.5% 10.4% 5.4% scrotal circumference 12.5% 3.7% 13.5% sc/morphology 11% 2.6% 0.35% morphology 52.1% 61.0% 72.7% physical/morphology 5.1% 7.4% 1.1% motility/morphology 4.2% 9.3% 5.4% table 10 percent combined reasons for unsatisfactory and deferred for all bulls for each time period 1993-1995 2006-2008 2009-2013 all physical 12.4% 20.8% 6.8% all scrotal circum. 27.0% 7.0% 14.8% all morphology 76.0% 84.4% 81.3% all motility 8.5% 14.8% 6.7% table 11 number and percent insufficient scrotal circumference all bulls for each time period 1993-1995 2006-2008 2009-2013 (59/1276) 4.6% (18/1076) 1.69% (77/1943) 3.96% table 12 percent insufficient scrotal circumference by most prevalent breeds for each time period breed 1993-1995 2006-2008 2009-2013 an 10% 1.2% 2.8% ch 15% 0% 2.9% sm 2% 0% 0% he 14% 5.7% 4.6% ba 17% 6.6% 13.9% * *difference p>.05 table 13 insufficient scrotal circumference by age 20009-2013 number and percent age number percent </= 15 months 6/200 3.0% >15</= 18 months 3/144 2.0% >18</= 21 months 4/72 5.0% >21</= 24 months 47/557 8% >24</= 36 months 14/339 4.1% >36</= 48 months 2/196 1.0% 4-5 years 0 0% 5-6 years 0 0% 6-7 years 0 0% >7 years 1/28 3.6% 499 clinical theriogenology • volume 6, number 4 • december 2014 table 14 most prevalent breeds results for the 4 criteria for bse and total 2009-2013 breed s u/d physical sc morph motility total an s u/d 1299/ 98.3% 23/ 1.7% 1284/97.1% 38/2.8% 1017/76.9% 305/23.1% 1299/98.3% 23/ 1.7% 958/72.5% 364/27.5% ch s u/d 34/ 82.9% 7/ 17.1% 33/ 97.1% 1/ 2.9% 27/ 79.4% 7 / 20.6% 34 / 100% 0 26 / 76.5% 8 / 23.5% sm s u/d 61 / 100% 0 61 / 100% 0 50 / 82% 11 / 18.0% 60 / 98.4% 1 / 1.6% 49 / 81.7% 12 / 20.0% he s u/d 83 / 100% 0 79 / 95.2% 4 / 4.8% 71 / 85.5% 12 / 14.5% 82 / 98.8% 1 / 1.2% 66 / 79.5% 17 / 20.5% ba s u/d 200 / 99% 2 / 1.0% 174 / 86.1% 28 / 13.9% 136 / 67.3% 66 / 32.7% 198 / 98.0% 4 / 2.0% 102 / 50.5% 100 / 49.5% * * * *difference p>.05 discussion the total number of bulls examined per year (table 1) declined steadily over the three periods. this decline in numbers is reflective of the decrease in the cattle population of the united states and similarly the southeast region. the breeds of bulls (table 2) changed dramatically over time with a variety of breeds being represented in period 1993-1995 but as time progressed angus bulls became by far the predominant breed in the two more recent time periods. in time period 2009-2013 the brangus breed made an increase in numbers as in the last few years the breed has become popular in the southeast. holsteins decreased from time period 1993-1995 as most natural service dairies in our area have dispersed. across all time periods (table 3) the most common age bulls were presented for examination was 24-36 months. in our area this appears to be the age that most producers elect for moderate service. a slight difference in this observation is in the period of 2009-2013 in which 21-24 month old bulls were numerous. two factors are represented in this increase as previously stated some producers want “2 year olds” and one farm presented a large number in this age category for presale examination. across all time periods lower numbers of bulls were presented after age 5 years. various factors contribute to this decline: avoidance of inbreeding, need for genetic change and musculoskeletal problems. however; in the two most recent time periods several 4-5 year olds remained in herds. the retention of these older bulls probably reflects owners desire to get one more season of genetics from these bulls. the percent of bulls classified as satisfactory (table 4) appears to have improved slightly over time with time period 2006-2008 having the best percent satisfactory, while the unsatisfactory percent is lowest for the same period of 2006-2008. percent deferred did not change between the first two time periods but is slightly lower in the period 2009-2013. since fewer bulls were deferred during period 2009-2013 they were likely placed in the unsatisfactory category therefore increasing the number unsatisfactory over time period 2006-2008. when unsatisfactory and deferred classifications are combined period 1993-1995 is higher than the other two periods which are only slightly different from each other. a reason speculated for the higher number of unsatisfactory and deferred in the first time period is the higher standards required by the new form which was introduced at that time. another positive speculation is that the quality of bulls presented improved over time. the percentages of satisfactory, unsatisfactory, deferred and combined unsatisfactory and deferred vs age are presented in tables 5,6,7 and 8. the percent satisfactory is lowest for all time periods in older bulls in period 1993-1995 that group of bulls also represents the highest unsatisfactory classification. a possible reason for this observation is in 1993-1995 there were a group of bulls in herds being used that were in the questionable category under the old system. interestingly in the more recent time periods bulls over two years of age had the highest satisfactory classification. most of these bulls 500clinical theriogenology • volume 6, number 4 • december 2014 represent bulls that had been in herds for an extended amount of time and had been examined annually since entering the herd. as expected the highest percent deferred across all time periods (table 7) are the younger bulls. most of the deferred classification in these bulls was due to immaturity. when unsatisfactory and deferred classifications are combined (table 8) in time period 1993-1995 the young bulls and older bulls performed the worst. however; in the more recent time periods the younger bulls were more likely to be in the unsatisfactory and deferred classification. the explanation for this change was stated earlier after the initial examination and bulls have entered herds they are evaluated on a once a year protocol with the satisfactory bulls remaining and frequently presented the following year. when reasons for unsatisfactory and deferred classifications (table 9) are examined it appears that physical problems were not very different in the first two time periods but improved slightly in period 2009-2013. insufficient scrotal circumference improved from the first time period to the second but became an issue again in the most recent period. bulls with insufficient scrotal circumference and unacceptable morphology improved in the two more recent time periods over time period 1993-1995. unacceptable morphology as an unsatisfactory or deferred reason exceeds all other reasons for every time period and appears to be higher for the time period 2009-2013. physical problems combined with morphology improved in the most recent time period. no real differences for motility and morphology as a reason was see between the three time periods. when reasons were combined (table 10) physical problems were greatest in time period 2006-2008. insufficient scrotal circumference was highest in the first period with great improvement in period 2006-2008, however; insufficient scrotal circumference became an issue again in period 2009-2013. unacceptable morphology was by far the greatest reason for unsatisfactory or deferred classification for all time periods. poor motility as a reason was slightly higher for the period 2006-2008. bulls of all ages presented with insufficient scrotal circumference (table 11) improved for period 2006-2008 but unfortunately was not different between 1993-1995 and 2009-2013.v when insufficient scrotal circumference is examined by the most prevalent breeds (table 12) it is obvious all breeds were making improvement until the angus and brangus breeds lost some progress in the last time period. most breeders have realized the importance of scrotal circumference and have made progress. however; with angus and brangus due to the demand for bulls in our area breeders have not been as selective as they were in 2006-2008. the age at which insufficient scrotal circumference was detected (table 13) for the time period 2009-2013 is in the younger bulls usually when they are present for their first examination. the age group >21</=24 months may be over represented as one farm presented a large group of bulls presale in that age group for their first evaluation. when the results for 2009-2013 were examined by the most prevalent breeds (table 14) there were no differences in physical problems among breeds. brangus were more likely to have insufficient scrotal circumference. brangus were also more likely to have unacceptable morphology while there were no differences among the other breeds. no differences were apparent among any of the breeds for motility. there was only one breed that was different for final classification and that was brangus. the brangus breed was classified satisfactory only 50.5% of examinations. a possible explanation for this poor performance has been stated earlier in that the demand for bulls of this breed is growing in our area and the breeders of these bulls have softened on selectivity. suggested reading 1. carson r, wenzel j: observations using the new bull-breeding soundness evaluation forms in young and adult bulls. vet clin north am food anim pract 1997; 13:305-311. 2. barth al, oko rj: abnormal morphology of bovine spermatozoa. ames: iowa state university press; 1989. 3. bierschwal cj: revised breeding soundness procedures. proc annu meet soc therio; 1996. p 128-130 4. carson r: bull breeding soundness examination. proc mid-am vet conf; 2012. 501 clinical theriogenology • volume 6, number 4 • december 2014 502clinical theriogenology • volume 6, number 4 • december 2014 omniblank: 2009: the heat is on: what's new for suppression of estrus in mares the heat is on: what’s new for suppression of estrus in mares 1 d. k. vanderwall 2 department of clinical studies, new bolton center, school of veterinary medicine, university 3 of pennsylvania, kennett square, pa, usa 4 5 abstract 6 therapeutic suppression of estrus in mares is routinely performed when exhibition of 7 estrous behavior is deemed undesirable or it interferes with intended uses such as recreational 8 and/or performance activities. various modalities are available for suppression of estrus in 9 mares, each with its own advantages, disadvantages and efficacy (or lack of). this paper will 10 review currently available methods of suppressing estrous behavior in mares, which includes 11 administration of exogenous progesterone/progestins, extending the duration of corpora luteal 12 function, suppressing ovarian follicular activity and ovariectomy. particular emphasis will be 13 placed on recent studies supporting or refuting the effectiveness of these methods of blocking 14 estrus, so the clinician can identify the most suitable and efficacious method of suppressing 15 estrus for horse owners and their animals. 16 17 keywords: equine, mare, estrus, estrous behavior, therapeutics 18 19 introduction 20 a relatively common complaint of horse owners and trainers is variable performance in 21 mares related to the estrous cycle.1 similarly, approximately 90% of over 750 veterinarians 22 507 responding to a survey had the clinical impression the estrous cycle impacted the performance of 23 mares, and the most frequently reported clinical sign associated with an effect of the 24 reproductive cycle on performance was attitude change, while other signs included tail swishing, 25 difficulty to train, squealing, “horsing”, excess urination, kicking, and a decrease in 26 performance.2 in some cases undesirable behavior has been associated with estrus and in other 27 cases with diestrus.3 it is important to note that some problematic behaviors displayed by mares 28 that are thought to be associated with estrus, are in fact not estrous behaviors; in particular, 29 submissive behavior may be most easily confused with estrous behavior. submissive behavior 30 includes leaning away from perceived threats, swishing/ringing the tail and actively squirting 31 urine, which collectively can give the impression of estrus.4 in contrast to submissive behavior, 32 true estrous behavior includes leaning towards the stallion (or other stimulus), a relaxed lifting 33 motion of the tail, stationary/squatting stance and passive urination (full stream or small amounts 34 in spurts).4 in some mares the signs of true estrous behavior are so strong as to directly impair 35 performance; for example, even under saddle some mares may “break down” and show estrus in 36 response to being around other horses and/or other stimuli.4 in other mares, the condition may 37 be much more subtle, simply causing owners and trainers to report the mare is less cooperative 38 or attentive during estrus.1 39 in an effort to evaluate the potential for an effect of stage of estrous cycle on the behavior 40 of mares, hedberg et al.5 conducted two behavior tests (novel object and isolation) on 12 mares 41 once when they were in estrus and once during diestrus in a cross-over design. five of the mares 42 served as controls, while the other seven mares were classified as “problem” mares based on 43 their owner’s perception of estrus-related behavioral problems. there were no significant 44 508 differences in the behavioral responses between estrus and diestrus within the control and 45 “problem” groups, nor were there differences in the behavioral responses between the two 46 groups of horses; however, as the authors note, the sample size was small and a cross-over study 47 design may not have been the most appropriate experimental design. therefore, further work in 48 this area is warranted. 49 ideally, a complaint of an estrous cycle-related behavior/performance problem in a mare 50 should be systematically evaluated in order to determine if the problematic behavior is or is not 51 related to a specific phase of the cycle (i.e., estrus or diestrus). a team approach to evaluating 52 and addressing the problem involving behavioral and reproductive expertise may be beneficial.6 53 in situations where there is evidence that the problematic behavior is associated with estrus, 54 suppression of estrus may be warranted. it is also common to suppress estrus in situations where 55 the signs of estrous behavior are simply perceived to be associated with performance problems or 56 to preemptively block the behavior to preclude the possibility of an adverse effect of estrus on 57 performance. this paper will review currently available methods of suppressing estrous 58 behavior in mares, with particular emphasis on recent data that support or refute various methods 59 of blocking estrus. the following methods of suppressing estrous behavior will be discussed: 1) 60 administration of exogenous progesterone/progestins, 2) extending the duration of corpora luteal 61 (cl) function, 3) suppressing ovarian follicular activity and 4) ovariectomy. 62 administration of exogenous progesterone/progestins 63 progesterone 64 it was first demonstrated in the 1960's that daily intramuscular administration of 100 mg 65 progesterone in oil (0.2 mg/kg) effectively suppressed signs of estrus in mares.7 intramuscular 66 509 administration of 100 mg progesterone in oil to ovariectomized mares produced peak systemic 67 blood levels of approximately 2 ng/ml, which then declined to 1 ng/ml or less 24 hours later.8 68 progesterone in oil is available from several compounding pharmacies; however, the need for 69 daily administration and the potential for soreness at the site of injection are limitations to its use. 70 it has also been demonstrated that intramuscular administration of a compounded long-acting 71 formulation of progesterone containing a total dose of 1.5 g progesterone will maintain blood 72 levels of progesterone above 1.0 ng/ml for approximately 10 days,9 which is a sufficient level of 73 progesterone to block estrous behavior;7,8 however, the potential for soreness at the injection site 74 is a limitation to its use, particularly in performance horses. 75 altrenogest 76 altrenogest (regu-mate®, intervet/schering-plough animal health, millsboro, de, 77 usa) is a synthetic progestin approved for use in horses for suppressing estrus, and is widely 78 considered to be the “gold-standard” method of inhibiting estrous behavior. daily oral 79 administration of altrenogest at a dose of 0.044 mg/kg (1 ml per 110 lbs of body weight) is very 80 efficacious for suppressing estrus in mares;10,11 however, the need for daily administration is a 81 drawback to its use. it was recently reported that intramuscular administration of a compounded 82 preparation containing 225 mg or 450 mg of altrenogest in a sustained-release vehicle blocked 83 estrous behavior for approximately 12 and 15 days, respectively, while administration of 500 mg 84 altrenogest in lactide-glycolide microparticles suppressed estrous behavior for approximately 30 85 days.12 86 medroxyprogesterone acetate 87 510 although there are anecdotal reports on the use of the synthetic progestin 88 medroxyprogesterone acetate (mpa) for estrus suppression in mares, it was recently reported 89 that intramuscular administration of an initial dose of 1,600 mg mpa followed by 400 mg once 90 weekly for five weeks did not suppress estrous behavior in mares.13 similarly, administration of 91 1,000 mg mpa in an aqueous suspension did not prolong the return to estrus compared to 92 control mares.12 on a related note, mpa was unable to maintain pregnancy in mares following 93 induced luteolysis when 1,000 mg was administered intramuscularly every seven days.14 94 therefore, the use of mpa for suppression of estrus cannot be advocated. 95 hydroxyprogesterone caproate 96 although it has not been tested for its efficacy for suppressing estrous behavior in mares, 97 mckinnon et al. demonstrated that intramuscular administration of 500 mg hydroxyprogesterone 98 caproate every other day was unable to maintain pregnancy in ovariectomized mares.15 99 similarly, the same synthetic progestin, repackaged as hydroxyprogesterone hexanoate, though 100 labeled for pregnancy maintenance was unable to maintain pregnancy in mares when 500 mg 101 was administered intramuscularly every four days following induced luteolysis.14 therefore, the 102 use of hydroxyprogesterone caproate for suppression of estrus cannot be advocated. 103 melengestrol acetate 104 melengestrol acetate (mga), a synthetic progestin, is labeled as a feed additive for estrus 105 suppression in cattle. however, when fed to mares at 10 or 20 mg/day for up to 15 days, it failed 106 to block estrus.7 it has been suggested higher doses (>100 mg/day) may be effective for 107 suppression of estrus in mares,16 which may be plausible, since it was recently reported that oral 108 administration of 100 mg or 150 mg mga to mares during the spring transitional phase 109 511 significantly hastened the onset of ovulatory activity compared to control mares indicating 110 apparent biological activity of higher doses of mga.17 therefore, further work to evaluate the 111 effect of higher doses of mga on estrous behavior is warranted. 112 implants 113 a variety of implants containing various progestins, labeled and marketed for use in other 114 species, have been used in mares with the intent of suppressing estrus. the most widely used 115 implant has been synovex s® (fort dodge animal health, ft. dodge, ia, usa), which contains 116 200 mg progesterone and 20 mg estradiol benzoate in each dose of eight pellets. mccue et al. 117 were unable to suppress estrus or cyclicity in mares that received 80 synovex s® pellets.18 118 similarly, although not critically tested for its ability to suppress estrous behavior, scheffrahn et 119 al. reported that following subcutaneous placement of a implant containing 6.0 mg of the 120 synthetic progestin norgestomet in six mares, two of the mares displayed estrous behavior two 121 days before the implants were removed 10 days after placement. 19 on a related note, placement 122 of five subcutaneous implants containing a total of 15 mg norgestomet was unable to maintain 123 pregnancy in mares following induced luteolysis.14 therefore, at this time, there is no evidence 124 that implants containing progesterone/progestins have efficacy for suppression of estrus in 125 mares, so their use cannot be advocated. 126 extending cl function 127 intra-uterine glass marble 128 one alternative to the use of exogenous progesterone/progestins for estrus suppression is 129 intrauterine insertion of a glass ball to extend cl function, which allows continued secretion of 130 endogenous progesterone to block estrus. nie et al. reported that placement of a 25 or 35 mm 131 512 sterile glass ball into the uterine body immediately following ovulation resulted in prolonged cl 132 function in seven of 18 (39%) mares that retained the glass ball after insertion (six of 12 mares 133 expelled the glass marble).20 in mares that developed prolonged cl function following 134 placement of the glass ball, cl function was maintained for approximately 90 days, during 135 which time progesterone levels remained above 1.0 ng/ml and estrous behavior was not 136 exhibited. in non-treated control mares, spontaneous prolongation of cl function occurred in 137 four of 32 (13%) mares. although placement of a glass ball appeared to be an efficacious means 138 of blocking estrous behavior for an extended period of time, it should be noted that in addition to 139 the 11 mares that retained the glass ball and never developed extended cl function (i.e., 140 continued to cycle normally), three of the seven glass ball treated mares with extended cl 141 function had one or two estrous cycles of normal duration after placement of the glass ball before 142 cl function was prolonged. therefore, on a “per-cycle” basis the incidence of prolonged cl 143 function was only 11% (7/62 cycles) in the glass ball treated mares compared to 8% (4/50 144 cycles) in the non-treated control mares, which was not significantly different between groups. 145 because of its variable efficacy among mares, and the need to physically remove the glass ball 146 when the resumption of cyclical reproductive activity is desired, placement of an intrauterine 147 glass ball does not appear to be an optimal method of suppressing estrous behavior in mares. 148 in a more recent study, rivera del alamo et al. examined the effect of intrauterine 149 placement of a 20 mm water-filled polyproplylene ball on the duration of cl function in mares 150 with the specific objective of investigating two potential mechanisms by which cl function is 151 extended: 1) the intrauterine device induces mild endometrial inflammation that completely 152 blocks or markedly attenuates prostaglandin (pg) f2α secretion (i.e., prevents high magnitude 153 513 luteolytic pulses) or 2) the physical presence of the device (movement and/or contact with the 154 endometrium) directly mimics the inhibitory effect of a conceptus on pgf2α secretion.21 155 corpora luteal function was extended in nine of 12 mares (75%) with an average duration of 57 156 days compared to zero of 12 control mares in which the average duration of cl function was 16 157 days. in six of the nine mares with extended cl function, small accumulations of intrauterine 158 fluid (≤10 mm x 20 mm) were identified during the luteal phase, but no neutrophils or bacteria 159 were recovered on uterine swabs when they were examined during the subsequent estrus. in 160 addition, changes in uterine biopsy scores for inflammation and glandular dilation preand post-161 treatment were similar for control and uterine device mares (with or without extended cl 162 function); therefore, there was no evidence the intrauterine device induced an inflammatory 163 response in the uterus. based on intensive blood sampling and measurement of pgf2α 164 metabolite (pgfm) levels in the systemic circulation on days 11 to 16 post-ovulation in four 165 control and eight uterine device mares, pgf2α secretion was attenuated in mares with prolonged 166 cl function, with the exception of two mares; one mare showed a single pgfm peak and 167 another showed two isolated pgfm peaks. because there was no evidence of inflammatory 168 changes caused by the intrauterine device, the authors concluded the physical presence of the 169 device in the uterine lumen somehow mimicked the effect of a conceptus by impairing 170 endometrial secretion of pgf2α; however, the exact mechanism remains unknown. 171 administration of exogenous oxytocin 172 in contrast to using an intrauterine device to extend cl function, administration of 173 exogenous oxytocin during diestrus is an alternative method of blocking luteolysis to prolong cl 174 function. endogenous oxytocin secretion is involved in regulating prostaglandin pgf2α 175 514 secretion from the endometrium during spontaneous luteolysis in the mare,22,23 and although 176 administration of exogenous oxytocin to mares around the time of luteolysis (i.e., days 11 to 15 177 post-ovulation) stimulates an acute onset of pgf2α secretion, 24-26 when oxytocin is administered 178 in the mid-luteal phase prior to the expected time of luteolysis (i.e., before day 10 post-ovulation) 179 it does not induce pgf2α secretion and often disrupts luteolysis causing prolonged cl 180 function.25 181 experimentally, continuous infusion of oxytocin using a subcutaneous osmotic minipump 182 from day eight to 20 post-ovulation blocked luteolysis in four of five mares, whereas luteolysis 183 occurred at the expected time in all four control mares that received saline infusion.27 although 184 it successfully induced prolonged cl function, continuous infusion of oxytocin to disrupt 185 luteolysis would not be a practical method of long-term suppression of estrous behavior. more 186 recently, vanderwall et al. showed that twice daily intramuscular administration of 60 units of 187 oxytocin on days seven to 14 post-ovulation was an efficacious method of disrupting luteolysis, 188 since it caused prolonged cl function through day 30 post-ovulation in six treated mares (figure 189 1), whereas six saline-treated control mares underwent luteolysis by day 16 post-ovulation 190 (figure 2).28 progesterone levels fell below 1.0 ng/ml between days 30 and 40 in two of the 191 mares with prolonged cl function, while the other four mares maintained progesterone levels 192 above 3.0 ng/ml through day 40 when blood sampling was discontinued. the cessation of cl 193 function before day 40 in two mares may have reflected a seasonal effect on cl function, since 194 the study was completed at the end of the physiological breeding season when gonadotropin 195 secretion wanes, and cl function (i.e., progesterone secretion) is dependent upon adequate 196 support from endogenous gonadotropin secretion.29-31 a follow-up study (vanderwall et al., 197 515 unpublished) was then performed to compare use of the same dose of oxytocin (60 units) given 198 twice daily compared to once daily on days seven to 14 post-ovulation; cl function was 199 maintained for 50 days post-ovulation in five of seven mares treated twice daily, five of eight 200 mares treated once daily and in one of seven untreated control mares. there was no difference 201 (p>0.05) in the proportion of mares with extended cl function between once daily and twice 202 daily administration of oxytocin, whereas collectively oxytocin treatment increased (p<0.05) the 203 proportion of mares with extended cl function. an advantage of using exogenous oxytocin 204 treatment to prolong cl function is that it can be readily reversed by the administration of a 205 luteolytic dose of pgf2α, in contrast to the need to physically remove an intrauterine device as 206 described above. 207 inducing late-diestrus ovulation 208 in 2006, hedberg et al. described the results of a preliminary study in which their 209 objective was to prolong the luteal phase in mares by using human chorionic gonadotropin 210 (hcg) to induce a late-diestrus ovulation to produce a new cl that would be too immature to 211 respond to the luteolytic effects of endogenous pgf2α secretion at the end of diestrus (i.e., day 212 14 to 15 after the initial ovulation).32 mares were randomly assigned to control (n=4) and 213 experimental groups (n=5), and beginning on approximately day eight after ovulation (or last 214 signs of estrous in three mares) their ovaries were examined with transrectal ultrasonography 215 every other day to determine the size(s) of their diestrus follicles. when a diestrus follicle ≥30 216 mm was detected, control mares were treated with saline and the experimental mares were 217 treated with 3,000 iu hcg im. after treatment the mares were followed with transrectal 218 ultrasonography for up to 72 hours or until ovulation was detected, and then once weekly for 219 516 three weeks. beginning on the day of treatment, blood samples were collected twice weekly for 220 at least one month and then once weekly for another two to four months for progesterone 221 determination. if a mare did not develop a diestrus follicle ≥30 mm during the first diestrus 222 period, they were monitored for a second, and if necessary a third diestrus period. 223 three of the nine mares developed a follicle ≥30 mm during the first diestrus period, 224 four mares during the second diestrus period and one mare in the third diestrus period. one 225 experimental mare never developed a diestrus follicle that was ≥30 mm during the three diestrus 226 periods that were monitored, and therefore, could not be treated with hcg. overall, three out of 227 the four (75%) experimental mares treated with hcg ovulated within 72 hours after treatment 228 with hcg, which resulted in luteal phases that lasted for 58 to 82 days after treatment. none of 229 the control mares ovulated during the luteal phase; however, one control mare had a 230 spontaneously prolonged luteal phase during both a non-treated cycle in which she never 231 developed a diestrus follicle ≥30 mm (cl function was terminated with exogenous pgf2α) and 232 during the subsequent cycle in which she was treated with saline when she had a large diestrus 233 follicle (that did not ovulate). 234 although, based on this study, the use of hcg to induce a late-diestrus ovulation looks 235 promising for prolonging cl function, it is important to note that as described above, for some 236 mares (five out of nine) it required multiple estrous cycles to develop a diestrus follicle ≥30 mm. 237 in addition, one mare never developed a large diestrus follicle during the three cycles that were 238 monitored, which precluded her from receiving the hcg treatment. therefore, in addition to the 239 effort (and expense) of monitoring mares in order to evaluate their suitability for treatment, the 240 fact that some mares may not develop a large enough diestrus follicle to warrant treatment, the 241 517 use of hcg to induce a late-diestrus ovulation does not appear to be a reliable, “on-demand” 242 method of blocking estrous behavior in mares. it is interesting to note that although the use of 243 hcg was apparently efficacious for inducing ovulation of diestrus follicles ≥30 mm in diameter 244 in this study by hedberg et al.,32 previous work by glazar et al.33 demonstrated that 245 administration of the gnrh agonist deslorelin acetate failed to induce ovulation and/or 246 luteinization of diestrus follicles > 30 mm in diameter. 247 pregnancy 248 pregnancy is another means of suspending cyclicity by taking advantage of the natural 249 ability of the conceptus to block luteolysis and maintain cl function/progesterone secretion. 250 although efficacious, this method has obvious disadvantages that may make it undesirable for 251 many horse owners. in addition to the time and expense necessary to establish pregnancy, is the 252 need to eventually terminate pregnancy (unless an offspring is ultimately desired). lefranc and 253 allen reported that manual transrectal rupture of the conceptus between days 16 and 22 of 254 gestation in 11 mares resulted in continued cl function for at least 60 days in all of the mares, 255 during which time they did not display estrous behavior.34 although efficacious, as noted above, 256 terminating a normal, healthy pregnancy may be untenable to many horse owners. 257 suppressing ovarian follicular activity 258 when considering the use of suppression of ovarian follicular activity as a method of 259 blocking estrous behavior in mares, it is important to recognize that mares are unique among 260 domestic animals, because in addition to the ovarian-derived estrogen-induced signs of estrous 261 behavior that occur when progesterone is at a basal level, many seasonally anovulatory (and 262 ovariectomized) mares exhibit paradoxical estrous behavior associated with hormone secretion 263 518 from the adrenal cortex.35,36 the intensity of this type of “unseasonable” estrous behavior was 264 judged to be equivalent to the behavior intact cycling mares display during the initial and 265 terminal days of estrus, but less intense than the behavior displayed near ovulation.35 such 266 behavioral receptivity to a stallion outside the breeding/ovulatory season that is independent of 267 ovarian estrogen secretion may have developed as a means of maintaining social bonds between 268 a harem stallion and his mares.35,37 this phenomenon has important implications for the clinical 269 management of estrous behavior in mares, since simply suppressing ovarian follicular activity 270 (or removing the ovaries) and its attendant estrogen production may not ensure the elimination of 271 estrous behavior. 272 down-regulation of the hypothalamic-pituitary-ovarian axis with gonadotropin releasing 273 hormone (gnrh) analogs 274 although a subcutaneous implant containing the potent gnrh analog deslorelin acetate 275 (ovuplant®, fort dodge animal health) was initially developed and found to be efficacious for 276 inducing timed ovulation for the breeding management of mares,38,39 it soon became evident the 277 implant caused prolonged anovulatory intervals in some mares.40-42 subsequent work 278 demonstrated the deslorelin implant caused reduced circulating fsh concentrations and absence 279 of the mid-cycle fsh peak that was associated with a prolonged inter-ovulatory interval.43 280 although problematic for the breeding management of mares, the down-regulating effect of the 281 deslorelin implant on pituitary function has been used clinically to suppress ovarian activity; for 282 example, placement of two deslorelin implants suppressed follicular development in mares used 283 as recipients for oocyte transfer.44 fitzgerald et al. reported suppressing ovulation for 30-90 days 284 in 15 of 20 mares that were treated with a subcutaneous implant containing another gnrh 285 519 analog (goserelin acetate).45 collectively, these data indicate that treating mares with a potent 286 gnrh analog may be efficacious for suspending cyclicity and suppressing estrus in mares for 287 extended periods of time. however, as noted previously, since anovulatory mares can show 288 estrous behavior, suppressing follicular activity may not ensure a complete absence of estrous 289 behavior. 290 immunologic 291 immunizing a mare against gnrh would eliminate the stimulus for gonadotropin release 292 from the pituitary resulting in suspension of cyclicity. tshewang et al. reported successfully 293 suspending ovarian activity and suppressing estrus for 25-30 weeks in mares after treating with a 294 gnrh vaccine.46 all of the vaccinated mares recovered from the effect of immunization with 295 normal cyclicity and estrous behavior. over the next two seasons, each mare also conceived and 296 produced a normal foal. subsequent studies have confirmed the efficacy of vaccination against 297 gnrh for suppressing ovarian follicular activity; however, not surprisingly, some of the 298 vaccinated mares continued to show estrous behavior in spite of their anovulatory state. it is 299 important to note, that in one study47 one vaccinated mare had not resumed normal ovarian 300 activity two years after initial vaccination; therefore, although most mares appear to regain 301 ovarian activity within a reasonable period of time post vaccination, some mares may not, which 302 could be particularly problematic. although a gnrh vaccine is not currently available in north 303 america, commercial preparations are available in europe and australia (equity®; pfizer 304 australia pty ltd, west ryde, nsw, australia). 305 ovariectomy 306 520 bilateral ovariectomy may be warranted in some circumstances for permanently 307 eliminating ovarian activity in mares. although the ovaries can be removed through a ventral 308 abdominal or flank laparotomy, laparoscopy or a colpotomy, at this time the latter two methods 309 are most commonly utilized.48,49 in an initial report, hooper et al. described the outcome of the 310 use of bilateral ovariectomy for treatment of objectionable behavior during estrus in 17 mares; 311 they found that after surgery owners reported that behavior was no longer a problem in 14 mares 312 (82%), while the remaining three mares continued to show estrous behavior.48 of the 23 mares 313 in that study that underwent bilateral ovariectomy for any reason, eight (35%) continued to show 314 estrous behavior after surgery. in a recent report, kamm and hendrickson evaluated clients’ 315 perspectives on the outcome following the use of laparoscopic ovariectomy for behavioral and 316 medical problems in mares.49 overall, client satisfaction (rated as very satisfied or satisfied) 317 with bilateral ovariectomy as a treatment for behavioral problems was 78% (18 of 23 of mares). 318 assessment of outcomes for specific behavioral problems showed that aggression problems 319 improved in 86% of cases; general disagreeable demeanor improved in 81%; excitability 320 improved in 75%; kicking and biting at other horses improved in 73%; problems in training 321 improved in 72%; frequent urination improved in 64%; and problems with other horses 322 improved in 64% of cases. the most common source of dissatisfaction for owners of patients 323 with behavioral problems was lack of behavioral change after surgery, including continued signs 324 of estrous behavior. although ovariectomy offers the advantage of being a potentially 325 permanent solution to a cycle-related behavior/performance problem, there are significant 326 disadvantages as well. the procedure is relatively expensive, though when compared to repeated 327 hormonal treatments, the cost may, in fact, be comparable. there are also risks associated with 328 521 the surgical procedure, though newer laparoscopic procedures have significantly reduced 329 them.48,49 the permanency of the procedure can also be a significant disadvantage because all 330 possibility for future reproduction is eliminated. and as noted above, because of the potential 331 for paradoxical estrous behavior, removing the ovaries may not ensure cessation of estrous 332 behavior. therefore, this option should be weighed carefully before proceeding. one way of 333 evaluating the potential effectiveness of ovariectomy for a behavioral problem is to evaluate the 334 mare’s behavior during either a natural (i.e., seasonal) or induced anovulatory state (e.g., down-335 regulation with a gnrh agonist); if the problem is not resolved or at least significantly improved 336 during the anovulatory state, it is unlikely ovariectomy will be any more efficacious. 337 summary 338 the primary indication for suppressing estrous behavior in mares is cycle-related 339 behavior/performance problems during estrus. when evaluating an owner/trainer complaint of 340 an estrous cycle-related problem in a mare, it should first be determined if the problematic 341 behavior is or is not related to a specific phase of the estrous cycle. in order to thoroughly 342 evaluate the mare, additional expertise may be needed in the form of consultation with or 343 referrals to behavior and/or reproduction experts. once a behavior/performance problem is 344 confidently defined as being related to estrus, the previously discussed methods (with proven 345 efficacy) of suppressing estrous behavior can be considered for use. each method has 346 advantages and potential disadvantages, that should be weighed for each individual 347 animal/owner/trainer. 348 references 349 522 1. mcdonnell s:. estrus cycle-related performance problems. j equine vet sci 350 1997;17:196. 351 2. jorgensen js, vivrette s, correa m,et al: significance of the estrous cycle on athletic 352 performance in mares. proc annu conv am assoc equine practnr 1996;42:98-100. 353 3. mcdonnell sm: evaluation and modification of mare behavior problems. proc annu mtg 354 soc therio 1993; p. 185-189. 355 4. mcdonnell s: performance problems in mares. the horse; april 2000:61-70. 356 5. hedberg y, dalin am, ohagen p, et al: effect of oestrous-cycle stage on the response of 357 mares in 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440 j 1993;25:321-323. 441 40. johnson ca, thompson dl, jr., kulinski km, et al: prolonged interovulatory interval 442 and hormonal changes in mares following the use of ovuplanttm to hasten ovulation. j 443 equine vet sci 2000;20:331-336. 444 41. morehead ja, blanchard tl. clinical experience with deslorelin (ovuplanttm) in a 445 kentucky thoroughbred broodmare practice (1999). j equine vet sci 2000;20:358-402. 446 42. vanderwall dk, juergens td, woods gl. reproductive performance of commercial 447 broodmares after induction of ovulation with hcg or ovuplanttm (deslorelin). j equine 448 vet sci 2001;21:539-542. 449 43. farquhar vj, mccue pm, nett tm, et al: effect of deslorelin acetate on gonadotropin 450 secretion and ovarian follicle development in cycling mares. j am vet med assoc 451 2001;218:749-752. 452 44. carnevale em, checura ch, coutinho da silva ma, et al: use of deslorelin acetate to 453 suppress follicular activity in mares used as recipients for oocyte transfer. 454 theriogenology 2001;55:358 (abstract). 455 45. fitzgerald bp, peterson kd, silvia pj. effect of constant administration of a 456 gonadotropin-releasing hormone agonist on reproductive activity in mares: preliminary 457 527 evidence on suppression of ovulation during the breeding season. am j vet res 458 1993;54:1746-1751. 459 46. tshewang u, dowsett kf, knott lm, et al: preliminary study of ovarian activity in fillies 460 treated with a gnrh vaccine. aust vet j 1997;75:663-667. 461 47. imboden i, janett f, burger d, et al: influence of immunization against gnrh on 462 reproductive cyclicity and estrous behavior in the mare. theriogenology 2006;66:1866-463 1875. 464 48. hooper rn, taylor ts, varner dd, et al: effects of bilateral ovariectomy via colpotomy 465 in mares: 23 cases (1984-1990). j am vet med assoc 1993;203:1043-1046. 466 49. kamm jl, hendrickson da. client's perspectives on the effects of laparoscopic 467 ovariectomy on equine behavioral and medical problems. j equine vet sci 2007;27:435-468 438. 469 470 471 528 figure 1. serum progesterone concentrations from the day of ovulation (day 0) through day 472 40 after ovulation in six mares treated with 60 units oxytocin intramuscularly 473 twice daily on days seven to 14 after ovulation (reprinted with permission from 474 javma 2007;231:1864-1867). 475 476 figure 2. serum progesterone concentrations from the day of ovulation (day 0) through day 477 40 after ovulation in six mares treated with 3 ml sterile saline intramuscularly 478 twice daily on days seven to 14 after ovulation (reprinted with permission from 479 javma 2007;231:1864-1867). 480 529 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages 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true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 2009: nutriceuticals and other drugs used to enhance fertility in stallions nutriceuticals and other drugs used to enhance fertility in stallions 1 s. p. brinsko 2 department of large animal clinical sciences, college of veterinary medicine, texas a&m 3 university, college station, tx, usa 4 abstract 5 until recently, there has been little information regarding the ability to improve semen 6 quality or fertility of stallions through alterations in diet. this paper presents evidence from work 7 in humans and other species, including the stallion, which indicates that dietary supplementation 8 with fatty acids, polyamines, vitamins or antioxidants may indeed have the potential of 9 improving semen quality, particularly in those individuals who have marginal semen quality 10 prior to supplementation. 11 keywords: stallion, semen quality, dietary supplementation, fatty acids 12 introduction 13 over the years, horsemen have been supplementing their animals’ diets with various 14 products in an attempt to enhance performance and overall well being. most of these products 15 have been geared toward improving stamina, hair coat, joint function and hoof growth. 16 historically, supplements touted to improve the breeding performance of stallions have not 17 proven to be efficacious. recently however, supplements have become available that show real 18 promise in this regard. 19 fatty acids 20 most fatty acids are straight-chain compounds, with an even number of carbon atoms. 21 chain-lengths can range from two to 80 but most commonly range from 12 up to 24. with a 22 chain length from two to six, they are called short-chain, from eight to 10 they are called 23 355 medium-chain and 12 up to 24 they are called long-chain fatty acids. among straight-chain fatty 24 acids, the simplest are referred to as saturated fatty acids. they have no double bonds and 25 cannot be altered by hydrogenation. when double bonds are present, fatty acids are said to be 26 unsaturated. they are called monounsaturated fatty acids (mufa) if only one double bond is 27 present and polyunsaturated fatty acids (pufa) when multiple double bonds are present. 28 the double bonds are counted from the methyl group determining the metabolic family, 29 noted by n-x (with n being the total number of carbon and x the position of the last double bond). 30 for example, linoleic acid is also named 18:2 n-6 in the shorthand nomenclature. therefore this 31 pufa has 18 carbon atoms, 2 double bonds and there are 6 carbon atoms from the last double 32 bond to the terminal methyl group. the number following "omega-" in omega-3 and omega-6 33 fatty acids indicates the position of the first double bond, counting from the terminal methyl 34 group on the molecule. hence, linoleic acid (18:2 n-6) is an omega-6 fatty acid. omega-3 fatty 35 acids cannot be converted to omega-6 fatty acids or vice-versa. 36 omega-3 fatty acids play an important role as structural membrane lipids in all cells. 37 they are also precursors to reactive substances such as prostaglandins and leukotrienes, and 38 possess anti-inflammatory, antiarrhythmic, antithrombotic and vasodilatory properties. there are 39 also data to indicate that dietary supplementation with omega-3 fatty acids to horses may modify 40 the response to endotoxin by reducing the synthesis of potentially harmful cellular mediators. a 41 plethora of equine dietary supplements containing omega-3 fatty acids and their precursors are 42 currently being marketed. 43 semen from virtually all species examined contains relatively large amounts of lipid. 44 semen lipids play a major role in motion characteristics, sensitivity to cold shock and fertilizing 45 356 capacity of sperm. phospholipids are the major lipid components found in semen and they are 46 largely composed of pufas.1 while spermatozoa from all mammals contain high 47 concentrations of pufas,1,2 the combination and distribution of pufa in semen varies among 48 species.3 for example, the distribution of long chain pufas in stallion spermatozoa is more 49 similar to boars than that of bulls or roosters.3 major differences in the lipid content of bull 50 spermatozoa compared to those of boars and stallions are the relative amounts of 22:5 and 22:6 51 fatty acids. spermatozoa of bulls have higher levels of 22:6 fatty acids whereas spermatozoa 52 from stallions and boars have higher levels of 22:5 fatty acids.3 bulls and roosters produce 53 spermatozoa that are very resistant to cold shock and freeze well, whereas spermatozoa from 54 boars and stallions have very low tolerance to cold shock and in general, freeze poorly. 55 in particular, docosahexaenoic acid (dha; 22:6 n-3, an omega-3 fatty acid) is the major 56 22:6 pufa in semen and docosapentaenoic acid (dpa; 22:5 n-6, an omega-6 fatty acid) is the 57 major 22:5 pufa. the majority of seminal pufas reside in spermatozoa. in men with poor 58 sperm motility, the level of dha in seminal plasma as well as the ratio of omega-3 to omega-6 59 fatty acids in their spermatozoa was found to be significantly lower than in men with normal 60 semen quality.4 studies in the boar and other species have shown that increasing the ratio of 61 dha to dpa in semen increases fertilizing capacity and semen quality.2,5 conversely, higher 62 levels of dpa relative to dha results in reduced fertility.2 the potential role of dha in 63 spermatozoal motility and membrane stability is supported by the fact that membranes high in 64 dha are noted for their flexibility, compressibility, elasticity and deformability.6 65 animals are unable to synthesize pufas from saturated or monounsaturated fatty acids. 66 therefore, they must acquire them from precursor pufas in their diet. transport of pufas 67 from the diet to semen has been shown to occur in a number of species including humans,7 fowl,5 68 357 boars,2 and rams.8 vegetable oils, such as corn and soybean oil, contain high levels of linoleic 69 acid, the parent compound of dpa. most proprietary equine rations are therefore, very high in 70 linoleic acid, as well as other omega-6 series fatty acids and their precursors while the 71 precursors for omega-3 fatty acids, such as dha, are very low. a diet of this nature would 72 favor the formation of dpa over dha since conversion of precursors to dpa and dha uses the 73 same competitive enzymatic pathway. since high dpa to dha ratios in semen have been 74 associated with reduced sperm quality and fertility, typical equine diets with an overwhelming 75 availability of n-6 precursors could have a negative impact on quality of stallion semen and its 76 tolerance to cooling and freezing. 77 while simply supplementing the stallion’s diet with precursors to omega-3 fatty acids 78 such as cod liver oil or flaxseed oil can increase the overall level of omega-3 fatty acids in 79 semen, this may not result in the desired effects of improved semen quality. for example, 80 supplementing boar diets with cod liver oil did not improve the freezability of semen.9 however, 81 when a supplement containing pre-formed dha and antioxidants was added to boar rations, 82 significant increases in semen quality and fertility were observed compared to boars fed a control 83 diet.2 feeding the supplement to boars resulted in a number of benefits including a higher dha 84 to dpa ratio in semen, as well as an increase in total spermatozoal number, spermatozoal 85 concentration, motility score, percentage of normal spermatozoa, and percentage of viable cells. 86 cooling and freezing of spermatozoa can induce cellular injury, which is associated with 87 a disruption of membrane lipids, resulting in damage to mitochondria and loss of integrity of 88 both the plasma and acrosomal membranes. these events are accompanied by a loss of motility, 89 viability and fertilizing capacity of sperm, a phenomenon commonly referred to as “cold shock”. 90 differences in the ability of spermatozoa from various animals to resist cold shock appear to be 91 358 related to their sperm membrane lipid composition.3 the lipid composition of spermatozoal 92 membranes not only influences the response of spermatozoa to cooling and freezing, but also 93 plays a major role in the physiologic changes leading to fertilization 94 most breed registries have allowed the use of cooled, transported semen, and/or frozen-95 thawed semen for a several years. the use of shipped semen in the cooled-liquid or frozen state 96 offers many advantages to breeders. unfortunately, there are many stallions that produce semen 97 that is unable to provide acceptable fertility after undergoing the rigors of cooling and storage, 98 and cryopreservation magnifies this reduction in fertility even further. this problem and the 99 promising results observed in pigs prompted equine researchers to investigate whether the 100 addition of a dha-enriched dietary supplement could result in improvements in equine semen 101 quality. 102 researchers at texas a&m used eight stallions in a 2 x 2 crossover study to determine if 103 feeding a supplement rich in dha would improve semen quality.10 the stallions were randomly 104 assigned to one of two treatment groups (n = 4/group). within these groups, the stallions were 105 fed either their normal diet (control) or their normal diet top-dressed with 250 grams of a dha-106 enriched supplement. the feeding trials lasted for 14 weeks after which a 14-week washout 107 period was imposed, during which only the normal diet was fed. following the wash out period, 108 the treatment groups were reversed for another 14 week feeding trial. feeding the supplement 109 resulted in a 3-fold increase in semen dha levels and a doubling of the ratio of dha to dpa in 110 the stallions’ semen. spermatozoal motion characteristics in fresh semen were unaffected by 111 feeding the supplement and after 24 h of cooling and semen storage, total and progressive 112 spermatozoal motility also did not differ between treatment groups. however, the spermatozoa 113 from stallions fed the supplement exhibited higher velocity and straighter trajectory than those of 114 359 stallions being fed the control diet. beneficial effects were more apparent after 48 hours of 115 cooling and storage, where increases in the percentages of spermatozoa exhibiting total motility, 116 progressive motility and rapid motility, were observed in the semen of stallions being fed the 117 supplement. total sperm numbers and percentage of spermatozoa with normal morphology were 118 unaffected by treatment. however, when stallions were being fed the supplement, the sperm 119 concentration in their ejaculates was almost double that of when they were fed the control diet. 120 therefore, it is possible that the observed improvements in semen quality after cooling and 121 storage could be attributed, at least in part, to the reduced exposure of sperm to seminal plasma 122 prior to and during processing. 123 in a subset of four stallions, whose progressive spermatozoal motility was <40% after 24 124 hours of cooling and storage when they were fed the control diet, feeding the supplement 125 resulted in improvements in mean progressive spermatozoal motility after both 24 hours and 48 126 hours of cooled storage. feeding the supplement resulted in similar improvements in motion 127 characteristics being observed in frozen-thawed semen. total spermatozoal motility, progressive 128 spermatozoal motility, and percentage of spermatozoa exhibiting rapid motility were 129 significantly higher in frozen-thawed semen of stallions being fed the supplement. 130 despite increasing the level of dha in semen by feeding the supplement, the level of 131 dpa did not decline. the level of dpa in semen remained higher than that of dha so that the 132 dha:dpa ratios were always less than one. because the stallion’s rations were typical equine 133 formulations containing corn and soybean oils, which are the precursors that favor the pathway 134 to dpa, the authors speculate that more dramatic improvements in semen quality may be 135 observed if modifications in the main fat content of the diet are incorporated with the dha 136 supplement. 137 360 the authors concluded that supplementing the diet of highly fertile stallions or those that 138 produce sperm that survive cooling does not appear warranted. however, stallions of marginal 139 fertility and those whose spermatozoa have poor tolerance to cooling and freezing would be 140 horses that might benefit most from being fed the supplement. optimizing levels of dha and its 141 precursors by altering the diet of marginally fertile stallions, may improve their semen quality 142 sufficiently enough to make them commercially viable for cooling or freezing. 143 similar studies were carried out at the university of arizona. in those studies, three 144 stallions were fed 550 grams of a dha-enriched supplement in addition to their normal diet for 145 90 days.11 dietary supplementation resulted in a 3.5-fold increase in semen dha 146 concentrations. unlike the study performed at texas a&m, motion characteristics of 147 spermatozoa in cooled and frozen-thawed semen did not differ between supplemented and non-148 supplemented stallions. however, in this study dietary n-3 pufa supplementation did result in a 149 46% increase in daily sperm output and a 15% increase in normal morphology. the most 150 dramatic improvements were observed for one stallion which had the lowest percentages of 151 morphologically normal and progressively motile spermatozoa prior to supplementation. 152 recently, workers in uruguay12 performed a 2 x 2 crossover study similar to the one performed 153 at texas a&m10 with 3 stallions/group and treatment consisting of 30 grams of dha being fed 154 as a supplement for 80 days. in that study, dietary dha supplementation improved total sperm 155 numbers, sperm morphology and percentages of live sperm. progressive motility in semen 156 cooled for 48 hours and frozen-thawed semen improved for some stallions. consistent with the 157 other studies, the improvements were most noticeable for stallions that initially had poorer semen 158 quality. improvements in semen quality have also been reported for stallions whose diets were 159 supplemented with rice oil.13 160 361 polyamines 161 both spermine and spermidine are polyamines found in all cells and thought to be 162 essential for replication, growth, and differentiation.14 it is believed that spermine and 163 spermidine are produced by the prostate and found in the semen of most mammals. early 164 investigations documented increased spermatozoal motility in vitro when spermine was added to 165 spermatozoa from mice, rats, guinea pigs and rabbits.12 however, the exact physiologic role of 166 spermine and spermidine in semen remains a matter of debate and their functions may be 167 concentration dependent. micromolar amounts of spermine in semen appear to enhance the 168 acrosome reaction15 while milimolar amounts appear to inhibit the acrosome reaction.15,16 169 in rams, ejaculates with spermatozoal motility greater than 85% had approximately two-170 fold higher spermine and total sperm polyamine content than ejaculates with lower motility. 171 compared to spermatozoa from lambs the spermatozoa of mature rams had approximately three-172 fold higher levels of spermidine, spermine, and total polyamines.17 lower levels of spermidine 173 are found in the seminal plasma of men with idiopathic asthenozoospermia (poor spermatozoal 174 motility) as well as those with asthenozoospermia associated with diabetes compared to 175 normozoospermic men.18 176 to date, no studies have been published which examined the effects of dietary 177 supplementation of polyamines on stallion semen. however, anecdotal information exists from 178 practitioners using an herbal supplement called “spermaid”, which was originally marketed to 179 increase fertility and libido in stallions, as well as to increase testicular hormone production. the 180 active ingredients in this product are the phytochemicals spermine and spermidine, which are 181 found in radish leaves, radish root, cucumber fruit, and oats. feeding of the supplement is 182 typically initiated three weeks prior to the breeding season. while significant improvements in 183 362 spermatozoal motility have not been reported with the use of this product, a number of slow 184 breeding stallions have anecdotally shown dramatic improvements in libido. 185 vitamins and antioxidants 186 dietary supplementation with antioxidants and vitamins has been shown to have 187 beneficial effect on semen quality. many of these vitamins exert their beneficial effects through 188 their antioxidant properties. however, as with most supplements examined, conflicting evidence 189 exists regarding their efficacy. the conflicting results can be related to the species of the male 190 subjects in the different studies as well as the doses of individual supplements or combination of 191 supplements investigated. 192 vitamins c and e are well known for their antioxidant properties and are those that have 193 been the most extensively examined. in rabbits, dietary supplementation with vitamin c, vitamin 194 e or a combination of vitamins c and e increased total spermatozoal output, spermatozoal 195 concentration and spermatozoal motility while decreasing dead and abnormal spermatozoa in the 196 ejaculate.19 analogous findings have been reported for humans and boars.19,20 in humans, 197 vitamin c was associated with higher spermatozoal numbers and concentrations in ejaculates, 198 whereas vitamin e appeared to exert its effects by improving spermatozoal motility.20 similarly, 199 there was tendency for semen production to be greater for boars supplemented with water soluble 200 vitamins, with the effect being less in boars supplemented with fat soluble vitamins.21 while the 201 intake of high levels of antioxidant vitamins was associated with better semen quality, moderate 202 intake did not appear to be effective.20 while other investigators were unable to demonstrate any 203 improvements in conventional semen quality parameters from infertile men,22,23 supplementation 204 with vitamins c and e did result in a significant reduction in dna fragmentation.23 205 363 work in the stallion is more limited. in 1978, a german investigator gave 10 stallions of 206 three different breeds an emulsion containing vitamins a and e for eight weeks.24 207 improvements in ejaculate volume, spermatozoal concentration, spermatozoal morphology and 208 spermatozoal motility, including post-thaw motility were reported. however, the improvements 209 were not universal and appeared to differ among breeds. more recently, russian workers 210 formulated a complex feed additive that included vitamins a, d and e.25 they reported 211 improved spermatozoal motility in fresh semen and that spermatozoa remained viable longer 212 after freezing and thawing. 213 another antioxidant, showing promise for improving semen quality is l-carnitine 214 (levocarnitine). along with its antioxidant properties, l-carnitine is essential for mitochondrial 215 energy metabolism. both l-carnitine and l-acetyl-carnintine are found in high concentrations in 216 the epididymis and both forms are accumulated by spermatozoa.26 in men with 217 asthenozoospemia, combined treatment with l-carnitine and l-acetyl-carnitine was effective in 218 increasing spermatozoal motility.24 the most significant improvements were seen in men with 219 the lowest numbers of motile spermatozoa prior to treatment. feeding l-carnitine to boars 220 resulted in higher semen volumes and spermatozoal concentrations thereby increasing the total 221 number of available spermatozoa in ejaculates for artificial insemination.27 222 223 conclusions 224 it is clear that dietary alterations can have an effect on semen quality and in some cases, 225 fertility. controlled studies in stallions are few, but those investigating fatty acids, in particular 226 omega-3 fatty acids such as dha, have shown real potential. based on work in other species, 227 364 further studies involving optimal levels of individual supplements and combinations of 228 supplements which could act synergistically to improve stallion semen quality are needed. 229 references 230 1. scott jw: lipid metabolism of spermatozoa. j reprod fertil 1973;18 (suppl):65-76. 231 2. penny pc, maldjian a, noble rc: an enhancement of boar fertility and reproductive 232 performance. proc14th intl cong anim reprod 2000; p. 109 (abstr). 233 3. parks je, lynch dv: lipid composition and thermotrophic phase behavior of boar, bull, 234 stallion, and rooster sperm membranes. cryobiology 1992;29:255-266. 235 4. conquer ja, martin jb, tummon i, et al: fatty acid analysis of blood, serum, seminal 236 plasma, and spermatozoa of normozoospermic vs. asthenozoospermic males. lipids 237 1999;34:793-799. 238 5. blesbois e, lessire m, grasseau i, et al: effect of dietary fat on the fatty acid composition 239 and fertilizing ability of fowl semen. biol reprod 1997;56:1216-1220. 240 6. salem n, kim h-y, yergey ja: docosohexaenoic acid: membrane function and metabolism, 241 in: simopolulos ap, kifer rr, martin re, editors. health effects of polyunsaturated fatty 242 acids in seafoods. new york: academic press; 1986, p 263-321. 243 7. conquer ja, martin jb, tummon i, et al: effect of dha supplementation on dha status and 244 sperm motility in asthenozoospermic males. lipids 2000;35:149-154. 245 8. drokin si, vaisberg tn, kopeika ef, et al : effect of cryopreservation on lipids and some 246 physiological features of spermatozoa from rams pastured in highlands and in valleys. 247 cytobios 1999;100:27-36. 248 9. paulenz h, taugbol o, kammisrud e, et al: effect of dietary supplementation with cod liver 249 oil on cold shock and freezability of boar semen. reprod domest anim 1999;34:431-435. 250 365 10. brinsko sp, varner dd, love cc, et al: effect of feeding a dha-enriched nutriceutical on 251 the quality of fresh, cooled and frozen stallion semen. theriogenology 2005;63:1519-1527. 252 11. harris ma, baumgard lh, arns, mj,et al: stallion spermatozoa membrane phospholipid 253 dynamics following dietary n-3 supplementation. anim reprod sci 2005;89:234-237 (abstr). 254 12. elhordoy dm, cazales n, costa g, et al: effect of dietary supplementation with dha on the 255 quality of fresh, cooled and frozen stallion semen. anim reprod sci 2008;107:18 (abstr). 256 13. arlas tr, pederzolli cd, terraciano pb, et al: sperm quality is improved feeding stallions 257 with a rice oil supplement. anim reprod sci 2008;107:5 (abstr). 258 14. tabor cw, rosenthal sm: pharmacology of spermine and spermidine: some effects of 259 animals and bacteria. j pharmacol exp ther 1956;i 16:139-155. 260 15. rubenstein s, lax y shalev y, et al: dual effect of spermine on acrosomal exocytosis in 261 capacitated bovine spermatozoa. biochimica et biophysica acta 1995;1266:196-200. 262 16. rubenstein s, breitbart h: role of spermine in mammalian sperm capacitation and acrosome 263 reaction. biochem. j 1991;278:25-28. 264 17. melendrez cs, ruttle jl, hallford dm, et al: polyamines in ejaculated ram spermatozoa and 265 their relationship with sperm motility. j androl 1992;13:293-296. 266 18. morales me: progressive motility increase caused by l-arginine and polyamines in sperm 267 from patients with idiopathic and diabetic asthenozoospermia. ginecol obstet mex 268 2003;71:297-303. 269 19. yousef, mi. effect of ascorbic acid and vitamin e supplementation on semen quality and 270 biochemical parameters of male rabbits. anim reprod sci 2003;76 :99. 271 20. eskenazi b, kidd s a, marks a r, et al: antioxidant intake is associated with semen quality 272 in healthy men. human reprod 2005;20:1006-1012. 273 366 21. audet i, laforest j p, martineau g p, et al: effect of vitamin supplements on some aspects of 274 performance, vitamin status, and semen quality in boars. j anim sci 2004;82:626-633. 275 22. rolf c, cooper t g, yeung c h, et al: antioxidant treatment of patients with 276 asthenozoospermia or moderate oligoasthenozoospermia with high-dose vitamin c and 277 vitamin e: a randomized, placebo-controlled, double-blind study. human reprod 278 1999;14:1028-1033. 279 23. greco e, iacobelli m, rienzi l, et al: reduction of the incidence of sperm dna 280 fragmentation by oral antioxidant treatment. j androl 2005;26:349-353. 281 24. prinz k: effect of a vitamin a –e emulsion on stallion semen. tierarztliche umschau 282 1978;33:27-30. 283 25. kalashnikov vv, ugadchikov st: the significance of biologically active substances in the 284 feeding of stud stallions. russian agricultural sciences 2001-2002;4:40-43. 285 26. lenzi a, sgro p, salacone p, et al: a placebo-controlled double-blind randomized trial of the 286 use of combined l-carnitine and l-acetyl-carnitine treatment in men with 287 asthenozoospermia. fertil steril 2004;81:1578-1584. 288 27. wahner m,. geyer m, hallfarth g, et al: the influence of vitamin emulsion with l-carnitine 289 on the sperm qualities of ai-boars. zuchtungskunde 2004;76:196-207. 290 291 367 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 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/pagesize [612.000 792.000] >> setpagedevice 2012: impact of body condition change post-breeding on reproductive performance of beef cows impact of body condition change post-breeding on reproductive performance of beef cows ramanathan kasimanickam,a phillip firth,a matthew asay,a ahmed tibary,a dale moore,a john hall,b william whittierc adepartment of veterinary clinical sciences, washington state university, pullman, wa; bnancy m cummins research extension and education center, university of idaho, carmen, id; cdepartment of large animal clinical sciences, virginia tech, blacksburg, va abstract body condition score (bcs) is a commonly used tool for evaluating the nutritional status of beef cows. good nutritional status of beef cows is one of the essential components for improved reproductive performance. the objective was to evaluate the impact of change in body condition during two months after breeding on the artificial insemination (ai) pregnancy rate in beef cows. angus cross beef cows (n = 2571) from 11 locations were included in this study. all cows were given a bcs (1-emaciated; 9obese), by the same clinician in each location at the initiation of synchronization (co-synch+cidr) protocol and again at pregnancy diagnosis, 55 to 70 days later. cows were fitted with kamar heat detection patches at cidr removal and observed three times daily for estrus expression until inseminated. pregnancy status was determined by per-rectal palpation or using ultrasonography. variables included in the model (proc mixed) were change in bcs (no change or gain vs. loss), cows expressed estrus at or prior to ai (activated, partially activated, or lost kamar) or not (intact kamar), age of the dam (2, 3 to 6 and >6 years), days post-calving at initiation of synchronization (30 to 60, 61 to 80 and > 81 days) and appropriate 2-way interactions. season (location) and ai sires were considered as random effects. the results showed ai pregnancy rate was influenced by change in the bcs [no change or gain: 55.3%; (1034/1870) vs loss: 50.1% (352/703); p<0.05] and cows that showed estrus at or prior to ai [estrus: 57.8% (825/1423) vs no estrus: 48.9% (560/1148); p<0.0001]. in conclusion, cows that lost body condition after breeding had lower ai pregnancy rates compared to cows that maintained or gained body condition. it is essential to feed cows following breeding to maintain their bcs and optimize their conception rates. keywords: body condition, pregnancy rate, timed artificial insemination, beef cows, fertility introduction variability in quantity and quality of pastures limits the energy intake in extensive rangeland based cow-calf systems. poor quality pasture negatively impacts reproductive performance of beef cows. nutritional program prior to calving reflects in body condition score (bcs) at calving and after calving. it is the most important factor determining the duration of the postpartum anestrus period beef cows.1,2 however, post-calving nutrition may partially overcome the negative effects of a restriction in pre-partum nutrition in thin to moderate bcs beef cows.1,2 the nutritional status of a pregnant beef cows throughout gestation is important for the development and health of fetus and nutritional status after calving is important for growth and health of calf. studies have focused bcs at calving, bcs change from calving to breeding and bcs at breeding on reproductive performance and not many studies focused on the effect of bcs change immediately after breeding on reproductive performance. increased energy intake for three to four weeks, before or during the mating period, in combination with temporary weaning (with or without separation of the calf) has been shown to increase pregnancy rates during the first half of the mating period in cows with low-to-moderate bcs.2 nutritional status is reflected in changes in body weight (bw) and bcs as well as in the levels of metabolites and hormones in blood. blood concentrations of glucose, insulin and insulin-like growth factor-i (igf-i) are indicative of the availability of energy, provide shortor long-term signals that mediate the effects of nutrition on reproduction.3 insulin-like growth factor-i plays a pivotal role in cattle fertility, acting as a monitoring signal that allows reproductive events to occur when nutritional conditions 469 clinical theriogenology • volume 4, number 4 • december 2012 for successful reproduction are reached.4 the objective of this study was to evaluate the impact of body condition change during two months after breeding on ai pregnancy rate in beef cows. materials and methods multiparous angus cross beef cows (n=1054) from 11 locations were included in this study. all cows were given a bcs (1-emaciated; 9-obese), by the same clinician in each location at the initiation of synchronization (co-synch+cidr; figure 1) protocol and again at pregnancy diagnosis, 55 to 70 days later. briefly, cows received 100 g gonadotropin releasing hormone (gnrh) im (cystorelin®, merial ltd. duluth, ga) and a controlled internal drug release insert (cidr; eazi-breed cidr cattle insert, pfizer animal health, new york, ny) on day 0. the cidr insert was removed and 25 mg prostaglandin f2α (pgf) im (lutalyse sterile solution; pfizer animal health) was given on day 7. on day 10, starting at 8:00 am, at 66 h (n=544) after cidr removal cows were inseminated and were given 100 µg of gnrh im concurrently. figure 1. diagrammatic representation of the co-synch+cidr synchronization protocol all cows received a kamar® heatmount detector (kamar, inc., steamboat springs, co) at the time of cidr insert removal on day 7. during the period from cidr insert removal to insemination, cows were observed three times daily for estrus and heat detector aid status (activated, partially activated and lost vs. intact). a cow was determined to be in estrus if the cow was observed to stand for mounting or if the cow had an activated, lost (with mount marks) or partially-activated heat detector aid. two weeks later, intact angus bulls were placed with cows (approximately1:40 bull: cow ratio), for the remainder of the 60 to70 d breeding season. cows were examined for pregnancy status at 55 to 70 days after fixed-time ai (ftai) by ultrasonography (aloka-500, sysmed lab inc., chicago, il) to identify time of conception. in all locations, cows were inseminated by ai technicians from stud companies and/or clinicians. the ai sires were selected and assigned to cows based on sire traits and to avoid inbreeding. the ai pregnancy rate was calculated as number of cows pregnant to ai divided by total number of cows inseminated. statistical analyses the data were analyzed using sas statistical software (sas version 9.12, sas institute inc., cary, nc). variables included in the model (proc mixed) were change in bcs (no change or gain vs. loss), cows expressed estrus at or prior to ai (activated, partially activated, or lost kamar) or not (intact kamar), age of the dam (2, 3 to 6 and >6 years), days post-calving at initiation of synchronization (30 to 60, 61 to 80 and > 81 days) and appropriate 2-way interactions. season (location) and ai sires were considered as random effects. general linear model procedure was sued to determine the differences in the ai pregnancy among season (spring and fall), locations (n=11), and ai sires (n=13). 470clinical theriogenology • volume 4, number 4 • december 2012 results there were 1227 spring cows and 1346fall cows. the mean body condition difference was different between seasons (spring: 0.45 ± 0.03 vs. fall -0.22 ± 0.03; p<0.05). the mean body condition scores difference among locations were significantly different (fig 2). the range for bcs difference was -3 to +3. the results showed ai pregnancy rate was influenced by change in the bcs [table 1; no change or gain: 55.3%; (1034/1870) vs. loss: 50.1% (352/703); p<0.05] and cows that showed estrus at or prior to ai [table; estrus: 57.8% (825/1423) vs. no estrus: 48.9% (560/1148); p<0.0001]. no interactions were observed (p>0.1). no differences in ai pregnancy (%) were observed for dam’s age (p>0.1) and days post-calving (p>0.1). mean ai pregnancy (%) for mean body condition score differences during first two months after breeding in beef cows were different (fig 3; p<0.05). there was no difference in ai pregnancy between seasons (fig 4; p>0.1). the ai pregnancy ranged from 50.4 to 66.7% for locations. among locations, the ai pregnancy differed significantly (fig 5; p<0.05). pregnancy for ai sires ranged from 49.0 to 71.3%. the ai pregnancy differed among ai sires (fig 6; p<0.05). pregnancy rates for bcs at initiation of synchronization were different (fig 7; p<0.05) discussion in this study, we observed that cows that lost body condition during the first two months after breeding had decreased odds of becoming pregnant to ai compared to cows that maintained or gained body condition. in spite of the facts that fertilization occurs in 90% of inseminated cows, but only 60% of those diagnosed to be pregnant at 60 days after breeding indicates that there is an estimated 30 % pregnancy wastage from fertilization to 60 days after breeding due to various reasons.5 several factors contribute to low ai pregnancy rates. the greatest of these risk factors is acute body condition loss. studies on body condition loss and reproductive performance determined the body condition loss from calving from breeding.6-8 these studies focused on the effect nutrition on resumption ovarian cyclicity, duration of the anestrus period after calving and/or proportion of anestrus cows at the beginning of the breeding season. duration of the anestrus period after calving is a key factor and is variable in beef cows. evidence suggests that cows that lose body condition have a longer postpartum interval to first ovulation. follicular growth generally resumes within a week or two in the majority of cows associated with a transient rise in follicle-stimulating hormone (fsh) that occurs within three to five days of parturition.5 beef cows with calf alongside in good body condition normally have 30 days to first ovulation whereas beef cows in poor body condition have 70 to 100 days to first ovulation.5 the lack of ovulation of dominant follicles during the postpartum period is associated with infrequent luteinizing hormone (lh) pulses, with both suckling and low level of nutrition being implicated in the prolonged suppression of lh pulses in the absence of progesterone. the key to optimizing resumption of ovulation in beef cows is appropriate pre-calving nutrition and management so that cows calve in an optimal body condition (bcs 5 to 6) with post-partum body condition loss restricted to <0.5 bcs units. in this study, we evaluated the effect of body condition loss during two months after breeding. there are several key factors regulating embryonic development during first 60 days of gestation. leptin, originally described as a regulator of food intake and energy balance, was found to play a critical role in reproductive function.9 leptin promotes preimplantation embryo development.10 this is supported by evidence that blastocysts cocultured with endometrial epithelial cells modulate leptin secretion.9 adiponectin is another cytokine that plays an important role in regulating energy homeostasis, specifically lipid and glucose metabolism.11 recent data suggest that adiponectin can also directly regulate reproductive and placental processes.12 adiponectin can play a complementary role in the regulation of several key female reproductive functions. it has been demonstrated that adiponectin is involved in the modulation of ovarian and endometrial functions, influencing periovulatory remodeling of the ovarian follicle, and steroid synthesis/secretion, as well as energy supply, and inflammatory response of endometrial cells.13-15 adiponectin has also been involved embryonic development and implantation supported by its expression in rat embryos and uterus and adiponectin receptors expression during the 471 clinical theriogenology • volume 4, number 4 • december 2012 mid-secretory phase that corresponds to the embryo implantation window in humans, mice and pigs.16-19 these findings strongly suggests that maintenance of body condition and energy status is critical during early gestation. artificial insemination pregnancy was higher for cows gaining bc 1 or 2 scores compared to cows gaining 3 scores. it is possible that cows gaining 3 scores may suffer from poor embryonic development due to metabolic changes in the uterine environment compared to cows gaining 1 or 2 scores. it should be noted that cows gaining 3 scores from 3 to 6 or 4 to 7 may benefit more than cows gaining 3 scores from 5 to 8. however, the ai pregnancy for cows gaining 3 scores was higher compared to cows losing bc. in this study, there was difference in ai pregnancy among locations. it should be noted that the number of cows that lost or gained body condition differed among locations. the increase in bcs might have been associated with the increase in forage availability and quality during winter and spring months is supported by the bcs difference was different between the fall and spring in this study. the bcs gains in cows could be explained by increased intake of energy and/or nutrients. in addition, increased forage availability and quality have been associated with reduced grazing energy cost by decreasing grazing time.20 it should be noted that there was no difference in ai pregnancy between seasons. it is plausible that the gain or loss in body condition was associated with the individual cow needs. blood concentrations of glucose, insulin and igf-i are indicative of the availability of energy, and provide shortor long-term signals that mediate the effects of nutrition on reproduction.3 in agreement with bcs maintenance, recovery or gain, the total protein and albumin concentrations increased and urea and non-esterified fatty acids (nefa) decreased which would indicate a better nutritional status21 and decreased mobilization of reserves from muscle22 and adipose tissue,23 respectively. similarly, serum nefa was reduced in beef cows as range forage quantity and quality improved.24 it should be noted that adipose depot depletion and associated increase in adiponectin alter cytokine production. this alteration in cytokines may initiate neuroendocrine signals to interfere with feed intake.21-24 so cows that loose body condition may not be interested in feed intake which may further prolong the decrease in essential metabolites involved and hence affect the reproductive performance. the economic benefit of a beef operation is directly related to pounds of beef produced per cow per year. in order to maximize the production not only the cow need to become pregnant but also it should become pregnant early in the breeding season. cows that loose body condition take more time to become pregnant and thus incurring economic loss to beef producers. it is also conceivable that cows with poor body condition may lose their pregnancy and need to become pregnant again after early embryonic death during the breeding season. several studies focus on alternative approach to address this by supplementing essential fatty acid and amino acids; however, further studies needed to focus on increasing key makers that are important for early preand peri-embryonic development, and placental development. in conclusion, cows that lost body condition after breeding had lower ai pregnancy rates compared to cows that maintained or gained body condition. it is essential to feed cows following breeding to maintain their bcs and optimize their reproductive performance. references 1. lalman dl, keisle dh, william je, et al: influence of postpartum weight and body condition change on duration of anestrus by undernourished suckled beef heifers. j anim sci 1997;75:2003-2008. 2. perry rc, corah lr, cochran rc, et al: influence of dietary energy on follicular development, serum gonadotropins, and first postpartum ovulation in suckled beef cows. j anim sci 1991;69:3762-3773. 3. bossis i, wettemann rp, welty sd, et al: nutritionally induced anovulation in beef heifers: ovarian and endocrine function during realimentation and resumption of ovulation. biol reprod 2000;62:1436-1444. 4. velazquez ma, spicer lj, wathes dc: the role of endocrine insulin-like growth factor-i (igf-i) in female bovine reproduction. domest anim endocrinol 2008:35:325-342. 5. crowe ma: resumption of ovarian cyclicity in post-partum beef and dairy cows. reprod domest anim 2008;43 suppl 5:20-28. 6. crowe ma, goulding d, baguisi, et al: induced ovulation of the first postpartum dominant follicle in beef suckler cows using a gnrh analogue. j reprod fertil 1993;99:551-555. 472clinical theriogenology • volume 4, number 4 • december 2012 7. crowe ma, padmanabhan v, mihm m, et al: resumption of follicular waves in beef cows is not associated with periparturient changes in follicle-stimulating hormone heterogeneity despite major changes in steroid and luteinizing hormone concentrations. biol reprod 1998;5:1445-1450. 8. duffy p, crowe ma, boland mp, et al: effect of exogenous lh pulses on the fate of the first dominant follicle in postpartum beef cows nursing calves. j reprod fertil 2000;118:9-17. 9. gonzalez rr, simon c, caballero-campo p, et al: leptin and reproduction. hum reprod update 2000;6:290-300. 10. kawamura k, sato n, fukuda j, et al: leptin promotes the development of mouse preimplantation embryos in vitro. endocrinology 2002;143:19221931. 11. yamauchi t, kamon j, waki h, et al: the fat derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. nat med 2001;7:941-946. 12. tilg h, moschen ar: adipocytokines: mediators linking adipose tissue, inflammation and immunity. nat rev immunol 2006;6:772-783. 13. takemura y, osuga y, yamauchi t, et al: expression of adiponectin receptors and its possible implication in the human endometrium. endocrinology 2006;147:3203-3210. 14. smolinska n, siawrys g, kaminski t, et al: leptin gene and protein expression in the trophoblast and uterine tissues during early pregnancy and the oestrous cycle of pigs. j physiol pharmacol 2007;58:563-581. 15. gosman gg, katcher hi, legro rs: obesity and the role of gut and adipose hormones in female reproduction. hum reprod update 2006;12:585-601. 16. dos santos e, serazin v, morvan c, et al: adiponectin and leptin systems in human endometrium during window of implantation. fertil steril 2012;97:771-778. 17. schmidt t, fischer s, tsikolia n, et al: expression of adipokines in preimplantation rabbit and mice embryos. histochem cell biol 2008;129:817-825. 18. chappaz e, albornoz ms, campos d, et al: adiponectin enhances in vitro development of swine embryos. domest anim endocrinol 2008;35:198-207. 19. maillard v, uzbekova s, guignot f, et al: effect of adiponectin on bovine granulosa cell steroidogenesis, oocyte maturation and embryo development. reprod biol endocrinol. 2010;8:23. 20. brosh a: heart rate measurements as an index of energy expenditure and energy balance in ruminants: a review. j anim sci 2007;85:1213-1227. 21. ndlovu t, chimonyo m, okoh ai, et al: assessing the nutritional status of beef cattle: current practices and future prospects. afr j biotechnol 2007;6:2727-2734. 22. chimonyo m, hamudikuwana h, kusina nt, et al: changes in stress-related plasma metabolite concentrations in working mashona cows on dietary supplementation. livest prod sci 2002;73:165-173. 23 meikle a, kulcsar m, chilliard y, et al: effects of parity and body condition at parturition on endocrine and reproductive parameters of the cow. reproduction 2004;127:727-737. 24. waterman rc, grings ee, geary t et al: influence of seasonal forage quality on glucose kinetics of young beef cows. j anim sci 2007;5:2582-2595. table. logistic regression for the effect of body condition change from breeding to pregnancy diagnosis at two months after ai and estrus status at or before the time of ai on the odds of pregnancy in beef cows predictor coefficient se coefficient z ‘p’ value odds ratio 95% ci lower upper constant -0.05555 0.059197 -0.94 0.348 estrus status 0.370487 0.079877 4.64 0.0001 1.45 1.24 1.69 body condition change 0.072708 0.037397 1.94 0.052 1.08 1 1.16 ci – confidence interval; se – standard error; accounted for season (location) and ai-sires; 473 clinical theriogenology • volume 4, number 4 • december 2012 figure 2. mean (± sem) body condition difference for cows in different locations. mean bcs difference differed among locations (p<0.05). figure 3. mean ai pregnancy (%) for mean body condition score differences during first 2 months after breeding in beef cows. abcdifferent superscripts are significantly different (p<0.05) -1.00 -0.80 -0.60 -0.40 -0.20 0.00 0.20 0.40 0.60 0.80 1.00 1 2 3 4 5 6 7 8 9 10 11 m ea n b c s d if fe re nc e locations 40.0 48.6 51.3 52.8 58.6 56.8 55.1 0.0 20.0 40.0 60.0 80.0 100.0 -3 -2 -1 0 1 2 3 a i pr eg nn ac y (% ) body condiiton score difference babab c bc bc a 474clinical theriogenology • volume 4, number 4 • december 2012 figure 4. mean ai pregnancy (%) differences in angus cross beef cows inseminated during fall and spring season. no differences in ai pregnancy (%) between seasons (p>0.1) figure 5: mean ai pregnancy (%) differences in angus cross beef cows inseminated at different locations differences in ai pregnancy (%) observed between different locations (p<0.05) 55.1 58.9 0 20 40 60 80 100 spring fall a i p re gn an cy (% ) 52.9 57.7 50.8 55.0 55.1 66.7 60.0 54.5 50.4 50.6 50.9 0 20 40 60 80 100 1 2 3 4 5 6 7 8 9 10 11 a i p re gn an cy ( % ) locations 475 clinical theriogenology • volume 4, number 4 • december 2012 figure 6: mean ai pregnancy (%) differences in angus cross beef cows inseminated using different ai sires differences in ai pregnancy (%) observed between different locations (p<0.05); locations 1, 2, 3, and 8 were both fall and spring locations. figure 7. mean ai pregnancy for body condition score at initiation of synchronization in angus cross beef cows. abcdifferent superscripts are significantly different (p<0.05) 61.4 51.4 51.6 55.5 53.2 48.0 56.1 71.3 48.7 51.3 60.4 52.1 49.0 0 20 40 60 80 100 1 2 3 4 5 6 7 8 9 10 11 12 13 a i p re gn an cy ( % ) ai sires 40.1 45.5 61.9 64.5 60.7 53.7 0 20 40 60 80 100 3 4 5 6 7 8 a i pr eg na nc y (% ) body conditon score at initaiton of synchronization a ab b c c bc 476clinical theriogenology • volume 4, number 4 • december 2012 36 contact ahmed tibary tibary@wsu.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/ct.v17.11652 review report manipulation of ovarian activity in camelids muhammad-salman waqas,a abdelhaq anouassi,b ahmed tibarya adepartment of veterinary clinical sciences, college of veterinary medicine, and center for reproductive biology, washington state university, pullman, wa, usa bveterinary research center, advanced scientific groups, abu dhabi, uae abstract camelid production has increased in importance in the world in recent years. the high demand for genetically superior males and the implementation of reproductive technologies requires the synchronization of ovarian follicular waves. this paper describes the state of knowledge regarding seasonality and follicular wave patterns in camelids. reproductive seasonality in camels can be manipulated using artificial photoperiod or melatonin treatment. as in other ruminants, protocols for follicular wave synchronization are based on the elimination of the dominant follicle (induction of ovulation) or progesterone treatment. these protocols are described for breeding management and ovarian superstimulation in embryo donors. induction of ovulation and initiation of superstimulation 2-4 days later (emergence of a new follicular wave) results in higher response in terms of number of ovulations and collected embryos. keywords: camels, alpacas, llamas, superovulation, follicular wave introduction camelids are a vital production animal in several areas of the world. the renewed interest in these species in the last 3 decades has increased our understanding of their reproductive biology. in camels, the importance of the racing industry and the intensification of camel dairy production have increased the demand for assisted reproductive technology. a similar situation is observed in south american camelids (sac), particularly alpacas, where the show and fiber market increased the genetic selection pressure. efficient reproductive management and the implementation of reproductive technologies such as artificial insemination (ai) and embryo transfer (et) in camelids require manipulation of ovarian activity. this paper reviews the mechanisms controlling ovarian follicular dynamics and methods to manipulate follicular dynamics and ovulation in camelids. follicular dynamics and ovulation in camelids endocrine and ultrasonographic studies in the mid to late 1990s helped to define follicular dynamics in several domestic camelid species.1–8 all camelid species are induced ovulators. in the absence of an ovulatory stimulus (mating or hormonal induction), follicular waves occur in an overlapping manner (figure 1).6,9 seasonal variation of ovarian activity of female camelids has been described in south american camelids (sac)10 but is more pronounced in camels.6,11,12 in camels, follicular activity (number and size) and oocyte quality are significantly lower during the nonbreeding season compared to the breeding season.12,13 however, a substantial proportion of females (20-60%) continue to have regular follicular activity outside the defined breeding season.6,14 recent research has revealed that photoperiod is involved in the control of ovarian follicular dynamics in the dromedary.15,16 ovarian follicular activity is also affected by nutritional status (i.e. body condition score) in sac17,18 and camels.19 restricted nutrition decreases ovarian follicular growth and corpus luteum development, and lowers plasma progesterone and leptin concentrations. however, the interaction among photoperiod, temperature, and nutrition in the control of seasonality in camels remains poorly studied.12,20 remarkable difference exists between camels and sac regarding the effect of lactation on follicular dynamics. resumption of ovarian activity occurs within 5-10 days after parturition in sac,21,22 whereas in camels, lactational anestrus can last from 45 days up to several months, and has substantial impact on the productivity of this species.6,23–26 mailto:tibary@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11652 citation: clinical theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/ct.v17.11652 37 ovarian follicular dynamics are regulated by follicle stimulating hormone (fsh) and luteinizing hormone (lh).1,27–29 duration of follicular development phases, ovulation, corpus luteum development, and luteolysis are presented (table). the presence of 2 or more codominant follicles is common in camelids and may occur in up to 40% of follicular waves.30–33 follicles ovulate in response to mating or hormonal treatment (gnrh or hcg) when they reach an appropriate size (camels: 12 mm; sac: 7-8 mm). receptivity to the male is not strongly correlated with the size of the follicle and readiness for ovulation (camels;33,34 sac35–38). therefore, the best method for monitoring follicular dynamics is transrectal ultrasonography. at the peak of follicular development, the uterus is toned and has a characteristic edema pattern on ultrasonography (figure 2). a sharp rise in serum lh concentration occurs minutes after mating (camels;2,3,39 sac28,29,40). ovulation occurs on average 30 hours (range: 26-72 hours) after mating. ovulation is induced by the β nerve growth factor (βngf) present in camelid seminal plasma (sac;41,42 camels43). the presence of βngf and endometrial inflammation are required to maximize the ovulatory response.41 the action of the ßngf involves kisspeptin neuron activation44,45 and a local mechanism on the ovary;46 ßngf treatment by various routes (intravenous [iv], intramuscular [im], or intrauterine induces lh surge. however, the dose is higher with intrauterine route.47 βngf appears to have a luteotrophic effect on the cl.48–50 however, this effect was not observed after intrauterine treatment.51 spontaneous ovulation has been described in up to 5% of the follicular waves in llamas and camels.52 this phenomenon appears to be more common in lactating animals.53 follicular recruitment starts 2-4 days after ovulation, and a mature follicle develops 4-6 days after completion of luteolysis, which occurs 9-10 days after a sterile mating. this short luteal phase results in a single follicular wave per cycle in most females. preliminary data in our laboratory revealed that 90% of females (camels and alpacas) have only 1 follicular wave per ovulatory cycle. thus, after a sterile mating or hormonal induction of ovulation, the average interval between 2 consecutive ovulatory follicles is 14 days (figure 3).6 in the absence of mating or other ovulatory stimuli (i.e. gnrh or hcg treatment), follicles continue to grow and reach a maximum diameter (25 mm in camels, 12 mm in alpacas, 13 mm in llamas) then undergo atresia. some follicles continue to grow and develop into large anovulatory follicles that may become hemorrhagic or luteinized (figure 2). the incidence of anovulatory haf is higher in camels,6,33 and llamas55 than other camelid species. there is an individual predisposition for haf in the absence of ovulation, but the exact pathophysiology of ahf is poorly understood.6 manipulation of ovarian follicular activity is an important aspect of reproductive management and implementation of synchronization, timed-ai, and multiple ovulation and embryo transfer technologies. several techniques have been used to control ovarian follicular dynamics, including inhibition of follicular activity, manipulation of seasonality, synchronization of follicular waves, and ovarian superstimulation. inhibition of follicular activity inhibition of follicular activity may be desired for management reasons (contraception) or to control the emergence of a new follicular wave. there are limited observations on hormonal inhibition of follicular activity in camelids. in alpacas, daily subcutaneous (sc) buserelin (50 µg/female) treatment for 10 days suppressed follicular activity, starting on day 6 after treatment.56 immunization against gnrh has not been studied thoroughly in camelids, but preliminary experiments in our laboratory in males and females produced variable results. manipulation of seasonality the seasonal reproductive pattern of camels is driven in large part by photoperiod. peak reproductive activity in this species is observed during the short photoperiod.57 the photoperiod effect on reproduction is confirmed by changes in the nocturnal melatonin secretion. melatonin secretion peak is shorter under an artificially long photoperiod.15,16,58 similar findings were reported in guanacos in the wild, indicating a circadian pattern of melatonin secretion.59 melatonin treatment advanced the breeding season in female dromedaries.60 dromedary females exposed to an artificial long photoperiod (16 light:8 dark) for 41 days and then treated with a subcutaneous melatonin implant (1 implant of 18 mg melatonin per 28 kg of body weight), displayed ovarian cyclicity 3.5 months earlier compared to females that did not receive melatonin.16 figure 1. schematic representation of follicular dynamics in camelids showing overlapping follicular waves in absence of ovulation http://dx.doi.org/10.58292/ct.v17.11652 38 citation: clinical theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/ct.v17.11652 induction of ovulation ovulation can be reliably induced by gnrh (sac buserelin 8 µg, camels 20 µg camels; sac gnrh 20-50 µg, camels 100 µg) or hcg (sac 500-750 iu iv; camels 1,500-3,000 iu iv) in the presence of a growing or mature follicle. the optimal ovulatory response after treatment is observed when the dominant follicle is 7-10 mm in alpaca, 8-12 mm in llamas, and 11-18 mm in camels.8,54,61–63 if given outside the optimal time, ovulation rate decreases. in llamas, there was no difference in ovulation rate, interval to ovulation, or luteal development when ovulation was induced by copulation or hormonal (lh or gnrh) treatment.64 synchronization of follicular waves synchronization of follicular waves is essential for timed-ai, superovulation, and embryo transfer.27,65 methods used in other ruminants have been adapted to camelids with varying degrees of success. several approaches have been used to control ovarian follicular dynamics and eliminate dominant follicles prior to gonadotropin treatment. these include manual ablation (not recommended), ultrasound-guided aspiration of the dominant follicles, and hormonal treatments.54,63,66,67 follicular ablation in the dromedary camel, a new follicular wave emerges 2.3 ± 0.5 days after ablation of the dominant follicle.68 in sac, a new follicular wave emerges 2-3 days after induction of ovulation of the dominant follicle. in camels, follicular wave emergence occurs 70.6 ± 1.4 h (range: 60-84 hours) after gnrh injection to induce ovulation, and follicular deviation occurs 58.6 ± 2.7 hours (range: 36-84 hours) after emergence.31 in dromedary camels, the percentages of females that ovulated within 14 days were: no treatment (47%), ultrasound-guided follicular ablation (40%), gnrh treatment (80%), or gnrh followed by a luteolytic dose of cloprostenol 1 week later (87%).68 in llamas, elimination of the dominant follicle by ultrasound-guided aspiration or lh treatment is effective in inducing follicular wave synchronization.69 eliminating dominant follicles by aspiration or induction of ovulation allows initiation of gonadotropin treatment for table. characteristics of the follicular wave, ovulation, and corpus luteum in camelids (adapted from54) dromedary camel (camelus dromedarius) bactrian camel (camelus bactrianus) llama (llama glama) alpaca (vicugna pacos) vicuna (vicugna vicuga) guanaco (llama guanaco) number of follicles at emergence 8-30 8-12 8-12 growth phase (days) 9-12 9-12 3-9 3-9 3-6 5-9 growth rate (mm/day) 1.6 1.6 0.7 1.4 1.8 1.0 plateau (days) 5-9 5-11 3-8 3-8 2-4 2-4 minimum ovulatory follicular size (mm) 8 9 8 7 7 8 average ovulatory follicular size (mm) 15 15 10 9 8 10 maximum ovulatory follicular diameter (mm) 25 22 14 12 10 13 regression phase (days) 10-12 7-15 3-8 3-8 3-5 3-7 hemorrhagic anovulatory follicle (haf) incidence (%) 40-55 4-22 haf size (mm) 30-90 30-50 14-35 13-35 haf regression (days) 8-45 4-22 codominance (%) 45 10-30 25 12 inter-wave interval (days) 18.2 ± 3.8 19.1 ± 0.6 15.8 ± 0.5 12-16 7.2 ± 0.5 12.6 ± 5.6 ovarian alternance (%) 77 93 interval mating to ovulation (hours) 32.-40 30-48 28-30 27-36 30 30 corpus luteum size (mm) 15-25 15-25 11-18 11-15 11-15 11-15 day at maximum size of corpus luteum 7.2±1.7 7.3 8 7-8 day of luteolysis 10 ± 1.2 10.5 10-12 10-12 return to receptivity (cycle-days) 12-16 12-16 12-15 12-15 http://dx.doi.org/10.58292/ct.v17.11652 citation: clinical theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/ct.v17.11652 39 superovulation that coincides with the emergence of a new follicular wave.70 embryo recovery rates are improved when gonadotropin treatment is started 2-4 days after induction of ovulation (sac;71,72 camels73–75) synchronization with a combination of gnrh and pgf2α ovulation synchronization using a combination of gnrh and pgf2α was investigated in camelids (figure 4). in camels, 2 gnrh injections given 14 days apart or 2 gnrh injections 14 days apart with pgf2α 7 days after the first gnrh was effective in synchronizing ovulation.68 timed-breeding on day 22 after a hormonal treatment protocol consisting of gnrh on day 0, pgf2α on day 7, gnrh on day 10, and pgf2α on day 17 resulted in 46-60% pregnancy rates.76–78 however, several studies lacked a proper control group. two injections of gnrh given 14 days apart followed by timed-mating (14 days after the second injection of gnrh) resulted in a 57.7% pregnancy rate.79 in bactrian camels, 2 injections of gnrh given 14 days apart resulted in better synchronization of follicular waves and response to ovarian superstimualtion with equine chorionic gonadotropin (ecg) or fsh.80 in llamas, gnrh treatment on day 0 and pgf2α after 7 days, then a second gnrh on day 10 resulted in a good synchronization of ovulation in females that ovulated after the first gnrh.81 there was no advantage in synchronization of the follicular wave, ovulation rate, or pregnancy rate using a treatment consisting of 2 injections of gnrh 1 week apart, followed by pgf2α on day 14.54,82 progesterone treatment several progestogens have been used to attempt to control follicular dynamics in camelids, including daily progesterone injection (50-100 mg in sac and 100-150 mg in camels), intravaginal devices (prids or cidrs with 1.38 g or 1.9 g progesterone in camels, cidrs 0.3 g progesterone and medroxyprogesterone acetate (map) sponges in llamas and alpacas) or sc implants of norgestomet (3 mg) in llamas and alpacas. in several studies, the length of treatment varied from 7 to 14 days.54,67,83,84 figure 2. ultrasonographic images of follicular dynamics in an alpaca: a. ovary quiescent stage; b. ovary with follicular recruitment and growth (follicles 3-4 mm in diameter); c. ovary with dominant follicle (9 mm in diameter); d. uterine horn during peak follicular growth (edema and tone); e. ovary with corpus luteum 7 days after ovulation; f. ovary with anovulatory follicle (19 mm) and g. ovary with hemorrhagic anovulatory follicle http://dx.doi.org/10.58292/ct.v17.11652 40 citation: clinical theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/ct.v17.11652 in camels, there are conflicting reports on the efficacy of prids85 and cidrs19,86 for synchronization of follicular waves. in addition, some studies have reported an increase in spontaneous ovulation when these devices are used.85 treatment with prids containing 1.55 g of progesterone for 7 days did not synchronize follicular waves.85,87 however, camels treated for 17 days with prids containing 1.9 g progesterone and receiving a large dose of ecg (3,000 iu) had a better synchrony of follicular growth than those not treated with prids.88 treatment with cidrs containing 1.38 g of progesterone for 10 days did not synchronize follicular waves in the nonbreeding season.19 in dromedary camels, 70 and 75% had a preovulatory follicle on days 16 and 18, respectively, after treatment with cidrs (1.9 g of progesterone) for 14 days.89 however, this study has no control (untreated) group. norgestomet implants were not efficacious in synchronizing follicular waves in bactrian camels.80 in dromedary camels, daily im progesterone injections (100 mg/day) for 10-16 days were used with relatively good results to synchronize recipients in an embryo transfer program.90 long-acting progesterone injection can be used in camels and may be more advantageous than daily injections or intravaginal devices, but this compound still needs to be thoroughly investigated for synchronization of follicular waves.54,91 daily im injection of progesterone (50 mg) for 12 days inhibited follicular growth by day 7.92 in llamas, 9 days of map treatment vaginal sponges (60 mg) synchronized follicular activity, resulting in the emergence of a preovulatory follicle 6 days after treatment.93 however, in another study, sponges containing 120, 240, or 480 mg map had no inhibitory effect on follicular development.94 treatment with cidrs (0.33 mg progesterone) for 16 days reduced follicular diameter starting on day 5 of the treatment.95 similar results were obtained in our laboratory in llamas and alpacas with a 14-day treatment.54 intravaginal devices containing 0.5 mg of progesterone appears to provide better control of follicular activity and better response to superovulation with ecg. the shape and area of contact of the vaginal device for progesterone delivery may affect the absorption of progesterone.54,96 vaginal devices containing 0.78 g progesterone (cue-mate®) inserted for 7 days reduced follicular development. a new dominant follicle was available 6 days after the removal of the device.97 daily im injections of progesterone (50 mg) for 12 days inhibited follicular growth by day 7.92 in vicuñas, treatment with cidrs for 5 days exerted a negative effect on follicular development and allowed a better superstimulation in response to ecg.98 in summary, progesterone therapy in camelids reduces the growth of large follicles and inhibits lh release,3 but does not figure 3. camelid cycle in presence of an ovulation figure 4. examples of camelid follicular wave synchronization using gnrh and pgf2α http://dx.doi.org/10.58292/ct.v17.11652 citation: clinical theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/ct.v17.11652 41 completely suppress follicular activity; therefore, its use for synchronization of follicular wave emergence and timed-breeding remains questionable.6,54,63,83,87 combination of progesterone and estrogens the combination of estradiol and progesterone is more effective in controlling follicular waves in some studies99 but not in others.84 in llamas, superstimulation at the end of a 5-day daily treatment with 100 or 150 mg progesterone after injection of estradiol benzoate (1 mg) resulted in a higher embryo recovery rate.100 in another study, 1 injection of estradiol-17ß (1 mg) and progesterone (25 mg) provided some synchronization of follicular waves, but not as good as that accomplished by the induction of ovulation or by follicle aspiration.69 in alpacas, daily estradiol benzoate (5 mg) and progesterone (50 mg) treatment for 7-10 days produced a more uniform follicular wave response; however, the ovulatory response was poor.101 in camels, 1 injection of estradiol benzoate (5 mg) and progesterone (100 mg) was ineffective for synchronizing follicular waves.68 ovarian superstimulation in vivo and in vitro embryo production are important technologies to multiply genetically superior females and have become common place in camels.27,102 additionally, these technologies are critical for the reproductive management and multiplication of endangered wild camelids using interspecies embryo transfer.73,103 ovarian superstimulation is a crucial step for in vivo production of embryos and oocyte collection. protocols for ovarian superstimulation used in camelids have been largely adapted from those used in ruminants with variable success (camels;27,54,85,87,90,104 sac65,84,105). the 2 main hormones used are fsh and ecg, either alone or in combination. as for other species, response to these hormones depends on the timing of treatment in relationship to follicular dynamics, dose, frequency of treatment, and individual animal variation. although fsh and ecg treatments have been initiated during the receptive or luteal phase of the cycle with some success, the best results in camels are obtained when the treatment is initiated in the absence of any follicles > 3 mm in diameter (camel).27 follicle stimulating hormone ovine (ofsh) and porcine (pfsh) fsh have been used for ovarian superstimulation with variable success.6,27 induction with camel fsh (cfsh), purified from camel pituitary extract, or equine pituitary extract, was not successful in the authors’ experience. the manner of fsh treatment (dose, frequency, and timing during the cycle) has been investigated to some degree; however, detailed descriptions of the treatment protocols are not always provided in publications. in the dromedary, a total dose of 20-30 mg of ofsh was given in 2 daily injections of decreasing doses over 6 days. the treatment starts 2 days before and continues up to 1 day after the completion of 7 days course of progesterone treatment by intravaginal device (prid).85 fsh was also given in a single dose (3.3 units) followed the next day by 1 injection of 3,500 iu of ecg, resulting in an average of 7 embryos recovered per treated female.87 in another study, ofsh was given twice daily (1-3 mg per injection) for 3-5 days after 10-15 days course of progesterone treatment (100 mg per day).90 single sc dose of ofsh has been tested with variable (5.7 ± 2.32 embryos recovered) results.106 in the dromedary, ovarian superstimulation has been obtained by twice daily efsh treatment in decreasing doses over 3, 5, or 7 days after 10-15 days progesterone treatment. (dromedary27,90,106–109; bactrian73,80). in practice, superovulation protocols are modified on an individual basis, based on ultrasonographic monitoring of the follicular response. the interval from pfsh treatment to the development of mature follicles (10-16 mm in diameter) varies between 6 and 8 days (figure 5).6,27,54,63 the number of fsh injections can be reduced using a slow-release fsh preparation (hyaluronan solution) resulting in the same superovulatory response and embryo collection rate.109 a single epidural dose of fsh resulted in multiple ovulations in camels; however, the response was lower than for traditional protocols using multiple injections.110 a recent study reported an adequate embryo recovery response in dromedaries superovulated with a single dose of recombinant bovine fsh given im (5.1 ± 3.6 embryos flushed per donor for 120 µg fsh and 5.0 ± 2.9 for 100 µg fsh) given 4 days after ovulation.111 in llamas and alpacas, ovarian superstimulation with pfsh alone or in combination with ecg has been used after 12 days of progesterone treatment.84,112,113 the best results were obtained with pfsh treatment (im) given twice daily for 5 days in decreasing doses (32, 27, 22, 17, and 12 mg).84,113 superovulation was obtained in 95% of the alpaca cycles after treatment with fsh (decreasing doses) starting 2 days after induction of ovulation.114 the number of embryos (4.2 ± 3.3) obtained after pfsh stimulation is generally lower compared to the number of ovulations (10 ± 4.4).114,115 generally, alpacas produce a more variable response to superstimulation protocols than llamas.84 equine chorionic gonadotropin ovarian superstimulation with ecg has been extensively used in camelids. in general, a single dose is given im a day before or on the day of completion of progesterone treatment for 5-15 days. the dose of ecg used varies from 1,000 to 6,000 iu in camels,27,75,80,85,90,104,106,107,110,116,117 from 500 to 2,000 iu in llamas,72,84,85,93,96,99,100,113,118,119 and from 500 to 750 iu in alpacas84,113 and vicuñas.93 in the dromedary, ecg is given as a single injection (2,000 iu, 2,500 iu, 3,000 iu, or 4,000 iu), 1 day before or 1 day after prid removal, resulted in superovulation in 40% of treated animals. however, only 42% of ovulating females yielded > 1 embryo. the interval from prid removal to mating was 5 and 4.5 days for females receiving 2,500 iu and 4,000 iu of ecg, respectively. this interval was 1 day shorter in females treated with ecg 1 day before the removal of prid.85,87 when ecg (2,000-3,000 iu) is given to females at the beginning of a follicular wave (no follicle > 3 mm in diameter), the interval from treatment to mating (follicular diameter of 12 mm) is relatively constant (8 days).27,120 follicular response is variable (0-19 follicles) and 20% of the females did not respond to ecg treatment.27 treatment of 2,500 iu ecg after a cidr http://dx.doi.org/10.58292/ct.v17.11652 42 citation: clinical theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/ct.v17.11652 (1.38 g progesterone for 3 days) produced superstimulation during the nonbreeding and transitional seasons, with follicles reaching ovulatory size 12-13 days after treatment.116,117 treatment with ecg is on 2-4 days after induction of ovulation. the ovulation and embryo recovery rates are improved with doses ranging 3,000-4,000 iu. higher doses (5,0006,000 iu) of ecg produced a larger number of follicles; however, the ovulation rate and the number of transferrable embryos collected were lower.109 a small number of animals had better response and fewer anovulatory follicles when 2,500 iu ecg was given at the end of a 13-day progesterone treatment in decreasing doses over 3 days compared to 1 dose.116 for the decreasing dose regimen, 2,500 iu ecg was splitted in 6 decreasing doses at 12-hour interval over 3 days as follows: 1,250, 600, 300, 200, 100, and 50 iu, respectively. in llamas, ecg (1,000 iu) is given after progesterone priming (luteal phase after ovulation induction with hcg or gnrh, cidr, or sc implants).72,96,119,121 follicular response is variable (0-13 follicles), and the number of ovulations ranged from 0-7 with a mean of 1.3, and an average of 2.3 embryos collected per donor (range: 0-6). follicles reached the mature size (9-13 mm) 5-11 days after ecg treatment. several treated females had premature luteinization 7-9 days after ecg treatment. increasing the dose of ecg to 2,000 iu increased the incidence of anovulatory follicles.54 supplementation with exogenous progesterone during the ecg treatment in the luteal phase appears to inhibit excessive follicular growth and improve ovulation rate and embryo quality.121 superstimulation with ecg and timed mating can be performed after synchronization with gnrh and pgf2α (figure 6).122 as ecg is not available in the usa, the use of pg600 can result in ovarian superstimulation in alpacas but the ovulation rate was low.123 in alpacas, the average number of recovered embryos per ovulating female was 3.7 after superovulation with 750 iu of ecg daily for 3 days.84,112 the main disadvantage of ecg is the high incidence of follicular luteinization and failure of ovulation, most likely due to its long half-life. additionally, females tend to become refractory to ecg after multiple use. this suggests that there is a risk of inducing anti-ecg antibodies.27,63 combination of fsh and ecg ovarian superstimulation protocols combining fsh and ecg have been published in alpacas, llamas,84,105,118 vicunas,32 and camels.27,80,110,124 these protocols consist of giving a figure 5. examples of fsh ovarian superstimulation protocols figure 6. example of ecg ovarian superstimulation protocol after induction of ovulation http://dx.doi.org/10.58292/ct.v17.11652 citation: clinical theriogenology 2025, 17, 11652, http://dx.doi.org/10.58292/ct.v17.11652 43 single dose of ecg followed by fsh injection twice daily in decreasing doses.124,125 generally, these protocols do not provide much superiority compared to protocols using fsh alone.123 human menopausal gonadotropin human menopausal gonadotropin (hmg) is a hormone with equal fsh and lh activity. its use for superovulation in camelids has been limited. although hmg produced superovulation in camels, ovulation rate (fsh: 22.4 ± 2.25; ecg + fsh: 11.6 ± 2.58; hmg: 7 ± 3.19) and embryo yield (fsh: 16.2 ± 2.72; ecg + fsh: 7.2 ± 3.1; hmg: 1.6 ± 1.17) were less predictable and lower than those obtained with superovulation protocols based on fsh or fsh + ecg.106,107 immunization against inhibin immunization against inhibin, inhibin subunits or recombinant dna vaccines result in high concentrations of circulating fsh, and consequently, an increase in the number of recruited and mature follicles. the vaccination was followed by a booster 28-231 days later, depending on the species and study. immunization against inhibin has been used successfully to improve ovarian superovulation in several species (cattle,126,127 horses,128 goats,129 sheep130). in the dromedary, a trial on immunization against inhibin gave encouraging results. an increase in ovulation number (4-10) was observed in 60% of the immunized females.63 further studies reported a high rate  of triple ovulations up to 5 months after the initial immunization.131,132 problems with superovulation in camelidae major problems encountered in superovulation of camelids are the high incidence of non-responsive females (20-30%), high incidence of follicular luteinization, overstimulation in some females, and loss of efficacy after multiple treatments. additionally, ovulation response and embryo yield remain highly variable.6,27,65,84 the recovery rate (number of embryos recovered/number of corpora lutea) is also variable, ranging from 30-90%. initiation of gonadotropin treatment 2 days after induction of ovulation or after a progesterone treatment results in the best superovulation and embryo recovery outcomes (figure 6). failure of ovulation may be due to premature regression of follicles and may be associated with inappropriate fsh dose or method of delivery.74 luteinization of follicles before ovulation may be due to increased concentrations of lh in response to high serum estradiol concentrations. preliminary trials in our laboratory using recombinant  highly purified fsh are encouraging. most females with overstimulated ovaries do not produce embryos, possibly because of alteration of gamete transport. finally, some females become refractory to superovulation with either fsh or ecg, perhaps because of the development of antibodies against these hormones. sources of variations in the response to superovulation in camelids that need to be investigated include species, breed, and individual animal. in alpacas, ovarian superstimulation response is positively correlated to serum antimüllerian hormone concentrations.133 a similar trend was reported recently in dromedary camels.134 conclusion research on camelid reproductive endocrinology and clinical monitoring of the reproductive function in female camelids led to the adaptation of several hormonal strategies to alter this function positively or negatively. knowledge gained on factors governing seasonality in camels, in particular photoperiod, allowed the use of artificial photoperiod or melatonin treatments to extend the breeding season in camels. multiple approaches to synchronize follicular waves have been adapted from other species, with variable results. progesterone therapy alone or in combination with estradiol has some efficacy for the control of follicular waves; however, it is still not optimal for timed-ai. synchronization based on induction of ovulation followed by natural or induced luteolysis provides better synchronization of follicular dynamics and allows optimization of ovarian superstimulation protocols. ovarian superstimulation treatments were adapted mainly from ruminant protocols. however, these treatments are still far 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http://dx.doi.org/10.58292/ct.v17.11652 128. roser jf, meyers-brown g: superovulation in the mare: a work in progress. j equine vet sci 2012;32:376-386. doi: 10.1016/j. jevs.2012.05.055 129. ge l, wu c, dai j, et al: effects of inhibin immunization on superovulation in chongming white goats. acta agric shanghai 2018;34:96-100. 130. khan su, jamal ma, su y, et al: towards improving the outcomes of multiple ovulation and embryo transfer in sheep, with particular focus on donor superovulation. vet sci 2022;9:117. doi: 10.3390/vetsci9030117 131. rateb sa, khalifa m, el-bahrawy ka: the influence of active immunization against inhibin on dromedary camel ovarian and hormonal dynamics. small rumi res 2015;132:32-36. doi: 10.1016/j.smallrumres.2015.10.003 132. rateb sa, el-bahrawy ka, khalifa ma: the prolonged reproductive response to immunization against inhibin and manipulating ovarian hyperactivity for timed ovulation in camels. small rumin res 2016;137:53-58. 133. patino c, arroyo e, ciccarelli m, et al: serum anti-müllerian hormone concentrations in female alpacas: variations during the reproductive cycle and correlation with ovarian superstimulation response. clinical theriogenology 2022;14:91-97. doi: 10.58292/ct.v14.9139 134. seyedasgari f, vidales lm, souza a, et al: anti-mullerian hormone in female dromedary camel and its association with super-ovulatory response in embryo donors. domest anim endocrinol 2024;86:106818. doi: 10.1016/j.domaniend. 2023.106818 http://dx.doi.org/10.58292/ct.v17.11652 https://doi.org/10.1016/j.jevs.2012.05.055 https://doi.org/10.1016/j.jevs.2012.05.055 https://doi.org/10.3390/vetsci9030117 https://doi.org/10.1016/j.smallrumres.2015.10.003 https://doi.org/10.58292/ct.v14.9139 https://doi.org/10.1016/j.domaniend.2023.106818 https://doi.org/10.1016/j.domaniend.2023.106818 2009: neonatology neonatology 1 2 a. p. davidson 3 school of veterinary medicine, university of california, davis, ca, usa 4 5 neonatal survival 6 average reported neonatal puppy and kitten mortality rates (greatest during the first week of life) 7 vary, ranging from 9-26%.1 prudent veterinary intervention in the prenatal, parturient and postpartum 8 periods can increase neonatal survival by controlling or eliminating factors contributing to puppy and 9 kitten morbidity and mortality. poor prepartum condition of the dam, dystocia, congenital malformations, 10 genetic defects, injury, environmental exposure, malnutrition, parasitism and infectious disease all 11 contribute to neonatal morbidity and mortality. 12 neonates that fail to survive to weaning are most commonly stillborn or die within the first three 13 days of life. factors implicated in perinatal deaths include prematurity, in utero infection with viruses such 14 as canine distemper, canine parvovirus, feline herpes, feline infectious peritonitis, panleukopenia, and 15 feline leukemia virus, as well as anatomic birth defects, birth trauma/dystocia, low birth weight, 16 inadequate nutrition, maternal neglect, and environmental stresses. optimal husbandry impacts neonatal 17 survival favorably by managing labor and delivery to reduce stillbirths, controlling parasitism and reducing 18 infectious disease, preventing injury and environmental exposure, and optimizing nutrition of the dam and 19 neonates. proper genetic screening for selection of sires and dams minimizes inherited defects.1-4 the 20 neonatal period here is defined as the first 4-6 weeks of life. 21 keywords: neonatal, pediatric, physiology, disease 22 neonatal physiology 23 cardiovascular system 24 1. the neonate has a low pressure, low volume, low peripheral resistance circulatory system. 25 2. higher heart rate, cardiac output, plasma volume and central venous pressure result. 26 3. sympathetic innervation of the heart is incompletely developed, response to anticholinergics 27 minimal. 28 127 4. baroreceptor reflexes are present after 4 days of age, prior to that hypotension results from 29 anoxia.5 30 clinical implications: one of the most important considerations of cardiovascular physiology in the 31 neonate is that in the fetus and during the first 4 days of life bradycardia is not vagally mediated and 32 is indicative of hypoxemia. although during this time the neonate appears able to resist circulatory 33 failure to a greater extent than the adult animal, it is far more appropriate to supplement oxygen than 34 to administer parasympatholytic agents such as atropine; administration of which will only exacerbate 35 cardiac hypoxemia via increasing oxygen demand in the face of hypoxemia. additionally, due to 36 incomplete maturity of the autonomic nervous system, the neonate is less able to respond to 37 physiological stresses. care should be given to maintain the neonate environment such that demands 38 on the cardiovascular system are minimal. 39 respiratory system 40 1. stimulation of the genital or umbilical region of the neonate induces reflex respiration in the first 41 three days after birth and may be clinically used to stimulate respiration in the immediate post 42 partum period. 43 2. normal respiratory rate in the neonate is low, ranging from 10 – 18 breaths per minute during the 44 first week, despite a high metabolic oxygen demand. 45 3. the mechanisms that control respiratory function (carotid body chemoreceptors) in the newborn 46 develop well before birth but require maturation in the post natal period. 47 4. the amount of work and pressure that is required by a neonate to maintain tidal breathing is 48 increased as compared to that of the adult due to the high compliance of the chest wall.5 49 clinical implications. the neonate is very susceptible to the development of hypoxemia and/or 50 jeopardized ventilation and gas exchange due to the immaturity of chemoreceptor responses to 51 hypoxia and chest wall construction. although there are adaptations present to help compensate for 52 this physiological state, such as an extremely low circulatory failure pressure until four days of age, it 53 is important to recognize that hypoxemia in the neonate may result in life threatening sequelae such 54 as septic shock due to bacterial translocation despite a lack of mucosal lesions. it is vital that the 55 environment be kept free of airway irritants and oxygenation is adequate. 56 128 hematopoietic system 57 1. at birth the neonate red blood cell exhibits macrocytosis with corpuscle volume decreasing to that 58 of the adult by four weeks of age as fetal red blood cells are replaced by adult red blood cells. 59 2. the hematocrit of the neonate may be as high as 60 per cent accounting for the red mucous 60 membrane color often noted at birth. by three days of age red blood cell counts have decreased 61 dramatically and continue to decrease for approximately three weeks. adult levels for red blood 62 cell count, hemoglobin, and hematocrit are generally not detected in most dogs until six months of 63 age. 64 3. neonatal isoerythrolysis is uncommon in the cat and rare in the dog. in the feline, the 65 phenomenon occurs in association with a type a kitten born to a type b queen that has anti-a 66 alloantibodies (agglutinating and hemolytic). white blood cell parameters in the canine and feline 67 neonate are typically consistent with those of their adult counterparts. lymphocytosis may also be 68 noted in the normal neonate.5 69 clinical implications. during the neonatal period, as fetal red blood cells are replaced 70 polychromasia and elevated reticulocyte counts may be noted. care must be taken to ensure 71 adequate ectoparasite control as iron demands are high; the presence of microcytosis is suggestive 72 of iron deficiency anemia. extramedullary hematopoeisis is commonly noted in the neonate liver. 73 urinary system 74 1. in the canine, the neonatal kidney is morphologically and functionally immature; nephrogenesis 75 continues for at least two weeks after birth. 76 2. the canine neonatal kidney is functionally characterized by a low glomerular filtration rate (gfr.), 77 low renal plasma flow (rpf), low filtration fraction (ff), depressed reabsorption of amino acids 78 and phosphate, exaggerated proximal tubule natriuresis and low concentrating ability. 79 3. serum creatinine levels and blood urea nitrogen (bun) concentrations are lower than in the adult 80 animal; typically 0.4 mg/dl and 8 – 10 mg/dl respectively. serum phosphorous concentrations are 81 elevated; typically 9 mg/dl, due to skeletal growth. 82 4. at birth arterial pressure is low (50 – 60 mmhg). during renal maturation increased blood 83 pressure and decreased vascular resistance result in an increase in gfr and rpf. in the 84 129 neonate, renal blood flow is directly correlated with arterial pressure and does not appear to be 85 altered by inhibition of angiotensin until approximately 6 weeks of age.5 86 clinical implications. a urine sample is easy to obtain from the neonate with gentle stimulation. 87 the immature nature of the kidney alters interpretation of urinalysis. low urine specific gravity (1.006 88 – 1.0017) is normal as is detection of protein, glucose and various amino acids due to the immaturity 89 of the proximal tubule. by three weeks of age urine protein and glucose concentrations approach that 90 of the adult dog and urine concentration is expected to compare to that of the adult dog by six to eight 91 weeks of age. 92 as the neonatal kidney is less able to concentrate or dilute urine, renal blood flow parallels blood 93 pressure, and there is altered sodium excretion by the proximal tubule. fluid therapy should be 94 administered with care to ensure adequate volume maintenance without over hydration or oncotic 95 loading. recommended daily fluid rates for the canine neonate range from 60–180 ml/kg/day. 96 caution must be exercised when administering renally excreted or metabolized antimicrobials 97 (penicillin, ampicillin, cephalosporins, fluroquinolones, and aminoglycosides) to neonates. generally, 98 ß-lactam antibiotics (penicillins, cephalosporins) are the antimicrobial drugs of choice, as although 99 the half-life may be prolonged there is a large therapeutic margin. ceftiofur, for example, 100 administered at 2.5mg/kg sq q 12hrs, maximum five days is an acceptable antimicrobial choice. due 101 to altered metabolism of nonsteroidal antiinflammatories the potential for renal toxicity from their use 102 in the neonate is far greater than in the adult animal.5 103 hepatobiliary system 104 1. during pregnancy, the maternal placenta supports many functions performed by the liver and 105 biliary system in the adult animal. prior to birth, the ductus venosis shunts blood through the 106 liver, effectively bypassing the neonatal sinusoid. the canine neonatal liver and biliary system is 107 functionally immature at birth. 108 2. there is a significant reduction in bile flow in the newborn puppy as compared to the adult dog, 109 and a complete failure of secretin and glucagon to stimulate bile flow at 3-28 days of age. despite 110 a relative functional cholestasis in the neonate, serum bile acids may be used to detect 111 hepatocirculatory abnormalities in puppies and kittens as young as four weeks. alkaline 112 130 phosphatase (alp) and gamma-glutamyltransferase (ggt) liver enzyme activities are markedly 113 elevated in neonates less than two weeks old and moderately elevated after two weeks of age. 114 elevations in alp and ggt enzyme activity have been attributed to placental, colostral, and 115 intestinal activity. aspartate aminotransferase (ast) and alanine aminotransferase (alt) are 116 typically comparable to that of the adult. alkaline phosphatase can be physiologically elevated 117 during skeletal growth. 118 3. postnatal hepatic microsomal enzyme activities at four weeks of age are 85% of that seen in an 119 adult dog. adult dog levels of microsomal enzyme activity are achieved by four and one-half 120 months of age.5 121 clinical implications. at birth, the neonate experiences functional cholestasis with altered liver 122 enzyme serum biochemical profiles. due to the absence of fully developed microsomal and p450 123 enzyme activity in the neonate until four to five months of age, caution must be exercised when 124 prescribing medication that requires hepatic metabolism or excretion. the detection of serum 125 increases in ggt and alp in the newborn may be indicative of colostrum intake and potentially 126 passive transfer. 127 gastrointestinal system 128 1. dentition eruption in the neonate first occurs at two to three weeks of age. all deciduous teeth 129 should be present by 12-16 weeks of age. 130 2. at birth, the gastrointestinal tract is sterile and has a neutral gastric ph. it is characterized by a 131 time-dependent increased permeability of the intestinal mucosa which decreases dramatically 132 after ten hours. normal nursing pup feces are semiformed and tan in color (acholic). gi motility 133 prior to 30-40 days of life is dependent upon pressure gradients rather than electrical intestinal 134 motility. 135 3. body temperature is known to have a dramatic effect on gastrointestinal movement in the 136 neonate. at rectal temperatures below 94 °f, ileus develops. as ileus progresses, the willingness 137 to nurse decreases and the necessity for tube feeding puppies increases. inherent to the tube 138 feeding process is the risk for aspiration and subsequent development of pneumonia.5 139 131 clinical implications. care should be taken to ensure adequate environmental conditions to 140 maintain normal body temperature in neonates to minimize gastrointestinal ileus. due to altered 141 absorption from increased gastrointestinal permeability and neutral gastric ph in the immediate post 142 natal period, care must be taken if administering oral drug therapy. diarrhea can result from 143 overeating, and is then complicated by subsequent bacterial overgrowth. 144 immune system 145 1. five to ten percent of canine neonatal serum antibodies are derived from trans-placental 146 transfer. at birth, the canine neonate is antibody deficient and immunologically incompetent. the 147 acquisition of passive immunity requires adequate ingestion and absorption of colostrum during 148 the first 24 hours of life. gastrointestinal absorption of colostral antibodies decreases markedly 149 after 12 hours. 150 2. providing adequate ingestion of quality colostrum, the puppy is protected by maternally derived 151 immunoglobulins during the neonatal period. puppies are capable of producing challenge 152 specific antibodies within two weeks and with repeated challenge can produce a secondary 153 immune response at 40 days. however, even by 40 days of age, t cell mitogenesis and 154 differentiation, and phagocytic cell function systems may not be fully mature.5 155 clinical implications. incompletely developed immune systems and inadequate thermoregulation 156 during the first days of life make neonates vulnerable to systemic infection (bacterial and viral). adequate 157 ingestion of colostrum must occur promptly post partum for puppies to acquire passive immunity. the 158 transmission of protective immunity (placental or colostral antibodies) between a bitch and her puppies 159 depends upon the prior existence of adequate serum maternal antibodies. when colostral intake is not 160 possible or is of questionable quality, pooled adult dog serum (20-150 ml/kg sc divided) may be 161 administered to elevate serum immunoglobulin concentrations in the puppy.5-7 162 neurologic system 163 1. the neonatal puppy’s main activities during the first two weeks of life are sleeping and nursing. 164 the rooting reflex orients the neonate to its source of food, the dam. vestibular function is present 165 at birth and is important for positioning during nursing. muscular coordination however is absent. 166 initial movements are characterized by swimming-like movements of the limbs, while sliding along 167 132 on the ventral abdomen and thorax. the ability to raise the head is present at birth in puppies and 168 the head may be used initially for righting reflex. an upright posture in puppies cannot be 169 maintained until ten to 14 days. 170 2. the eeg of the neonatal puppy initially is similar during periods of sleep and waking. 171 3. at birth the body posture is primarily one of flexion. if suspended by the head, flexor hypertonicity 172 is present. at four to five days in puppies, the flexor hypertonicity is replaced by extension until 173 three to four weeks of age when the puppies will begin to struggle to escape when held in 174 suspension. 175 4. the nociceptive threshold is much lower than in adults. this may be due to a lack of some of the 176 descending inhibitory mechanisms found in older animals. the coordination of motor responses 177 to noxious stimuli is not well-developed and the animal may have much wider receptive fields to 178 noxious events. neurotransmitters may not have reached full function.8 179 clinical implications. although the nervous system of the neonate is immature there is no doubt 180 that nociceptive pathways are present and that pain is perceived by the neonate subjected to noxious 181 stimuli. drugs which might be effective in adults may not be as effective in neonates. procedures 182 carried out on neonates with insufficient pain control produce greater stress responses than those 183 where analgesia has been provided. a local anesthetic (lidocaine, dose extrapolated from humans) 184 can be used and is very effective; the dose requirement is lower because of the immaturity of the 185 nerves but the neonate does not appear to be at any greater risk of toxic side effects with a single 186 dose of lidocaine. bupivicaine is not advised in the neonate due to the risk of cardiotoxicity with 187 overdosage. 188 the pharmacokinetics of the opioid analgesics are different in the neonate versus the adult. 189 lower doses of these drugs are required for analgesia at one day of age compared with 34 days 190 (three to four-fold differences). 191 metabolism 192 1. the normal birth weight of the puppy is breed dependent; generally, 500 gm ± 150 gm for a 193 medium breed dog. birth weights lower than 300 gm in the medium size dog are associated with 194 an increased risk of neonatal mortality. increased mortality in low birth weight puppies is most 195 133 likely associated with negative effects of chilling (higher body surface area: mass) and the ability 196 to nurse and maintain glucose concentrations. generally, there is a similar pattern of growth 197 amongst different breeds of dogs; the most rapid weight gain occurring during the first 12 weeks. 198 puppies should gain on average 10% of their body weight each day for the first few weeks of life. 199 2. unlike their homeothermic adult counterparts, neonates are poikilothermic. however, they have 200 well-developed behavioral heat-seeking responses which enable them to maintain a stable rectal 201 temperature providing sources of heat are available. shivering and vasoconstrictive reflexes are 202 not functional in the newborn. physiological responses noted during hypothermia include 203 bradycardia, cardiovascular failure, neuronal injury, and ileus. normal rectal temperatures in the 204 puppy are 95 – 99 °f (week one), 97 – 100 °f (weeks two and three) and by weaning rectal 205 temperatures approach that of the adult. 206 3. at birth the neonate must transition from placental support to endogenous food stores for glucose 207 production. during the first three to 24 hours after birth, hepatic glycogen stores decline by more 208 than 50% and there is a shift from glycogenolysis to a mixture of glycogenolysis and 209 gluconeogenesis. for maintenance of blood glucose concentrations, regular feeding is required. 210 in addition to regular nursing, the dam’s nutritional state must be adequate to provide for the 211 needs of her puppies.5 212 clinical implications. the neonate is susceptible to a wide variety of toxic, environmental, 213 infectious and congenital insults; however, the ability for them to respond is limited. one of the first 214 signs of illness in both the kitten and puppy is a failure to gain weight. this finding is often noted well 215 before any other clinical signs of disease are present. twice daily weighing of neonates during the 216 first week(s) of life dramatically facilitates early detection of illness, ensures adequate intervention in a 217 timely manner to prevent poor weight gain and positively impacts neonatal survival.1 218 neonatal/pediatric conundrums 219 fading puppies 220 a fading puppy commonly dies following the onset of rapidly progressive, vague signs of illness. 221 premortem diagnosis is challenging. immediate necropsy of a neonate dying without obvious cause is 222 134 warranted to provide proper veterinary care of the littermates. clients should be advised to refrigerate (not 223 freeze) deceased neonates and present them promptly for evaluation. 224 neonatal bacterial peritonitis with septicemia can cause rapid deterioration of the puppy resulting 225 in death if not recognized and treated promptly. factors shown to predispose a puppy to septicemia 226 include endometritis in the bitch, a prolonged (often not recognized or reported) delivery/dystocia, feeding 227 of replacement formulas, the use of ampicillin, stress, low birth weight (< 350 gms), and chilling with body 228 temperature <35.5 °c. the umbilicus of neonates should be treated with tincture of iodine immediately 229 after birth to reduce contamination and prevent ascent of environmental bacteria into the peritoneal cavity 230 (omphalitis-peritonitis). 231 the bacterial organisms most frequently associated with septicemia are e. coli, streptococci, 232 staphylococci, and klebsiella spp. commonly, a decrease in weight gain, failure to suckle, hematuria, 233 persistent diarrhea, unusual vocalization, abdominal distention and pain, and sloughing of the extremities 234 indicate septicemia may be present. 235 prompt therapy with broad spectrum, bactericidal antibiotics, optimal nutrition via 236 supported nursing, tube feeding or bottle-feeding, maintenance of body temperature, and 237 appropriate fluid replacement are indicated. the third generation antibiotic, ceftiofur 238 sodium, is an appropriate choice for neonatal septicemia as it alters normal intestinal flora 239 minimally and is usually effective against the causative organisms. the prognosis for septicemic 240 neonates is poor. failure to respond to antibiotic therapy should prompt consideration of canine herpes 241 virus infection. 242 canine herpesvirus (chv) is a widely recognized and commonly blamed cause of fading puppy 243 syndrome. premortem and postmortem diagnosis of chv infection in neonates can be challenging. 244 typical necropsy findings include multifocal petechial renal hemorrhages. confoundingly, these can be 245 also be present with bacterial septicemia. intranuclear inclusion bodies can be difficult to find. diagnosis 246 by virus isolation or chv-specific pcr is confirmatory. treatment has been reported to be unrewarding 247 and recovery is rare. recovery has been reported to result in residual cardiac and neurologic damage. 248 treatment with immune serum from affected dams is reported to be ineffective in infected puppies. one 249 case report of successful treatment with the antiviral drug, acyclovir exists. successful vaccine 250 135 development has been hampered by the poor immunogenicity of other herpesviral vaccines developed for 251 other species, as with feline and bovine rhinotracheitis. neonates of a naïve bitch exposed to chv during 252 the last two to three weeks of gestation or the first three weeks postpartum are at risk.9,10 253 acyclovir is an antiviral agent with activity against a variety of viruses including herpes 254 simplex. acylcovir is preferentially taken up by susceptible viruses and converted into 255 the active triphosphate form, which inhibits viral dna replication. acyclovir is poorly 256 absorbed after oral administration and is primarily metabolized by the liver. acyclovir 257 can increase the toxicity of nephrotoxic drugs. the half-life in humans is approximately 258 three hours. its use in veterinary medicine is not well established and it should be used 259 with caution and only in situations where indicated. the safety and effectiveness in 260 humans less than two weeks of age is not established. the dose is extrapolated from 261 that for humans.10 262 juvenile cellulitis 263 juvenile cellulitis (puppy strangles) is a progressive, granulomatous, pustular disorder of puppies, 264 most commonly occurring in dogs younger than four months of age, but it is occasionally reported in dogs 265 up to four years of age. the eyelids, pinnae, lips, chin, muzzle, paws, abdomen, thorax, vulva, prepuce 266 and anus can be affected with lesions that fistulate, drain and crust. lymphadenomegaly, most commonly 267 mandibular and superficial cervical, can be distant from the affected skin sites and is often painful. 268 pustules and lymph nodes are usually sterile when cultured. superficial cutaneous flora can be cultured 269 from open, draining lesions. pyrexia, anorexia, sterile suppurative painful arthritis and an inflammatory 270 hemogram can occur. the diagnosis is confirmed by histopathologic evaluation but is commonly made on 271 the basis of clinical appearance. the predominant inflammatory cell in juvenile cellulitis, characterized by 272 light and electron microscopy and immunohistochemical staining, is an epithelioid macrophage juvenile 273 cellulitis requires aggressive immunosuppressive therapy early in the course of the disease for resolution 274 and to avoid the sequellae of cicatricial lesions. traditionally, puppies have been placed on 275 immunosuppressive doses of prednisone (2.2 mg/kg/day), causing concerns with immunization efforts. 276 griseofulvin therapy offers an apparently effective treatment without the side effects associated with 277 corticosteroid administration, enabling discontinuation of corticosteroids sooner in the course of the 278 136 disease. it has been reported to be effective as sole immunomodulatory therapy (14.2 to 34 mg/kg po q 279 12 h). griseofulvin is postulated to induce down regulatory signals within the lesions. the use of 280 griseofulvin as sole therapy could be attempted in early cases. vaccination of puppies undergoing 281 immunosuppressive therapy is not advised and they must be strictly isolated from sources of infectious 282 disease.11,12 283 bacterial overgrowth syndrome-associated diarrhea 284 pediatric dogs and cats are often presented to the veterinarian for signs referable to the 285 abdominal cavity. dietary indiscretions, parasitism and infectious disease (primarily viral, less commonly 286 bacterial) account for most of these presentations. congenital and developmental disorders should also 287 be considered. 288 symbiotic colonic bacteria assist digestion. the upper gi tract was once believed to be sterile, 289 but normal colonization of the duodenum, jejunum, and ileum is now appreciated. bacterial overgrowth 290 syndrome (bos) occurs when the normally low bacterial colonization in the upper gi tract significantly 291 increases. neonates are particularly at risk for developing bos. mucosal injury resulting from a minor 292 viral or bacterial gastroenteritis can induce bos in these individuals if a proper post infectious dietary 293 regimen is not followed. 294 a particular bacterial pathogen has never been implicated; instead, abnormally large numbers of 295 normal or pathological flora appear to cause bos. under normal conditions, gram-positive bacteria and 296 fungi colonize the duodenum and jejunum in quantities less than 1 x 105 organisms per milliliter of fluid. 297 aerobic and anaerobic bacteria colonize the ileum in quantities less than 1 x 108 organisms per milliliter 298 of fluid. this is in sharp contrast to the 1 x 1011 organisms per milliliter of fluid that colonize the colon. 299 studies of duodenal aspirates have not identified any particular bacteria as a cause of bos; however, 1 x 300 105 organisms per milliliter of aspirate fluid is diagnostic for bos. usually, abnormally large numbers of 301 anaerobic bacteria and normal florae grow from cultured fluid of patients with bos. 302 137 the following are protective factors that stabilize the number and type of bacteria that colonize 303 the upper gi tract. abnormalities in these mechanisms put a patient at risk for bacterial overgrowth. 304 1. two coordinated motor phenomena produce the continuous propulsive peristaltic action of the 305 upper gi tract. both the migrating motor complex and the migrating action potential complex clear 306 the upper intestine of unwanted bacteria and undigested substances. desynchronization of these 307 complexes results in diarrhea and weight loss in animal models. neonates lack propulsive 308 peristaltic action. gut motility in neonates results from aboral pressure. 309 2. gastric acid normally reduces the proximal small intestine bacteria populations, particularly 310 anaerobic bacteria. the bowel mucosa integrity and mucin layer protect the gut from bacteria. 311 neonates have reduced gastric acidity. 312 3. malabsorption of bile acids, fats, carbohydrates, proteins, and vitamins causes many of the 313 symptoms of diarrhea and weight loss associated with bos. anaerobes and bacteroides fragilis 314 actively deconjugate bile acids, thereby preventing proper bile acid function and enterohepatic 315 circulation. fatty acid absorption is reduced because deconjugated bile acids cannot help micelle 316 formation. deconjugated bile acids directly inhibit carbohydrate transporters. these unabsorbed 317 sugars ferment into organic acids because of the intestinal flora, which reduces the intraluminal 318 ph and produces osmotic diarrhea. the unconjugated bile acids also damage intestinal 319 enterocytes and induce water secretion by the colonic mucosa. 320 4. fat, protein, carbohydrate, and vitamin malabsorption result from poor enterocyte function and 321 bacterial transformation of nutrients into nonabsorbable and toxic metabolites. toxic metabolites 322 damage the intestinal mucosa. malabsorption and enterocyte dysfunction further degrade the 323 health of the gut by reducing local and systemic nutrition delivery. 324 treatment of bos is aimed at reducing the damage caused by malabsorption and restoring 325 nutritional health and normal gut flora. prompt recognition and treatment can prevent the development of 326 138 severe malnutrition. the antimicrobials of choice for therapy of bos-associated diarrhea are ampicillin or 327 amoxicillin in the pediatric patient (due to the neurotoxicity associated with metronidazole overdosage).14 328 anasarca 329 anasarca, a lethal congenital edema, can occur with or without concurrent cardiovascular 330 abnormalities. generalized subcutaneous edema, with intrathoracic and intraperitoneal fluid accumulation is 331 present. congenital hereditary lymphedema causes edema of the extremities and sometimes head, and is 332 associated with morphologic lymphatic abnormalities. prepartum ultrasonographic evaluation of the fetuses 333 can be used to screen for this disorder. dystocia can result due to fetal oversize. anasarca is a problem 334 common in bulldogs, but recognized in other breeds as well (labrador retriever). it is suspected to have 335 a heritable component. its exact pathophysiology in the dog is not understood. the genetics are not 336 known; anasarca is thought to be inherited as an autosomal dominant trait. there are multiple anecdotal 337 remedies, none proven or reported in the scientific literature. it is debated and discussed on 338 theriogenology list serves and on the layman’s internet exhaustively. as well as causing dystocia, 339 anasarca usually results in stillborn puppies or puppies needing to be euthanized. some veterinarians 340 promote various therapies, usually doomed to failure. diuretic therapy of affected neonates can sometimes 341 cause slight normalization, but euthanasia is usually indicated if the neonate is not stillborn. environmental, 342 dietary, and pharmacologic contributory factors are not scientifically defined. anasarca has been 343 recognized for many years, yet its incidence remains unchanged. an attempt to recognize the presence 344 of anasarca prepartum with ultrasonography should be made in bitches with a history of affected puppies 345 or in breeds with high incidence, due to the higher incidence of dystocia associated with the syndrome. 346 “swimmer” puppies 347 swimmer puppies fail to develop normal ambulation at ten to 14 days of life, moving instead by paddling 348 their limbs laterally and caudally. compression and deformation of the sternum and thorax occur 349 concurrently. obese puppies from small litters, commonly raised on relatively slippery surfaces are 350 predisposed. treatment should be instituted immediately upon diagnosis, consisting of caloric restriction, 351 physical therapy, and improved traction in the nest box. if diagnosed early (three to five weeks of age) the 352 condition is reversible and does not require binding of puppies. 353 139 puppy vaginitis 354 puppy vaginitis is characterized by an apparently healthy female puppy presented with mucoid 355 vulvar discharge that is usually white to yellow, and sometimes copious. the discharge can be 356 accompanied by mild perivulvar dermatitis. the puppy is not typically attentive to the discharge, and 357 there is not any associated change in urinary behavior (dysuria or polakiura). clients often have a difficult 358 time deciding if a puppy has normal urinary behavior or not. the age of onset ranges from six weeks to 359 puberty, the duration is from days to months, and the disorder is often intermittent. 360 cytologic examination of the discharge finds suppurative inflammation. vaginal cultures (aerobic) 361 generally fail to grow anything but normal flora in small numbers, similar to unaffected littermates. a 362 urinalysis, acquired by cystocentesis, is characteristically normal (a decreased urine specific gravity is 363 typical for young dogs lacking adult concentrating abilities), and the urine culture negative. the clinician 364 needs to perform enough diagnostics to rule out more significant causes of vulvar discharge and feel 365 comfortable with the diagnosis of benign puppy vaginitis. 366 the specific etiology of puppy vaginitis is unknown. an imbalance of juvenile vaginal glandular 367 epithelium is postulated. the condition is reported in the literature to resolve both with puberty and with 368 ovariohysterectomy, two very different events endocrinologically, therefore neither likely to truly cause 369 resolution. puppy vaginitis diminishes with maturity. the term “puppy vaginitis” is a misnomer, as it is 370 asymptomatic and not indicative of inflammation. important rule-outs (some of which are associated with 371 inflammation) include urinary tract infection, urinary incontinence with associated mucosal scalding, the 372 onset of the initial estrous cycle, vaginal foreign bodies (i.e. foxtails) and urogenital anatomic anomalies 373 (ectopia, disorders of sexual differentiation, significant strictures). cleansing the perivulvar area with a 374 gentle solution (non-alcoholic otic preparations or “baby wipes”), benign neglect and tincture of time are 375 advised.14,15 376 references 377 1. johnston sd, root kustritz mv, olson pns: the neonate – from birth to weaning. canine and 378 feline theriogenology. philadelphia: wb saunders; 2001. p.146–167. 379 2. moon pf, erb hn, ludders jw, et al: perioperative risk factors for puppies delivered by 380 cesarean section in the united states and canada. j am anim hosp assoc 2000;36:359-368. 381 140 3. kelley rl: canine reproduction: factors influencing litter size. proc soc for therio annu conf, 382 2002. 383 4. davidson ap: nursing care of neonatal puppies and kittens. in: pratt p, editor. principles and 384 practice of veterinary technology. santa barbara: mosby-year book inc; 1997. 385 5. grundy sa: clinically relevant physiology of the neonate. vet clin north am small anim pract 386 2006;36:443-460. 387 6. center sa, randolph jf, manwarren t, et al : effect of colostrum ingestion of gamma-388 glutamylaminotransferase and alkaline phosphatase activity in neonatal pups. am j vet res 389 1991;52:499–504. 390 7. levy jk, crawford pc, collante wr, et al: use of adult cat serum to correct failure of passive 391 transfer in kittens. j am vet med assoc 2001;219:1401-5. 392 8. lavely ja: pediatric neurology of the dog and cat. vet clin north am small anim pract 393 2006;36:475-501. 394 9. carmichael l: neonatal viral infections of pups: canine herpesvirus and minute virus of canines 395 (canine parvovirus-1). in: carmichael l, editor. recent advances in canine infectious diseases. 396 ithaca: international veterinary information service (www.ivis.org);1999. 397 10. davidson ap, grundy sa, foley je: successful medical management of neonatal canine 398 herpesvirus: a case report. commun therio 2003;3:1. 399 11. shibata k, nagata m: efficacy of griseofulvin for juvenile cellulitis in dogs. vet dermatol 400 2004;15(suppl 1):26 (abstract). 401 12. white sd, rosychuck ra, stewart lj, et al: juvenile cellulitis in dogs: 15 cases. (1979-1988). j 402 am vet med assoc 1989;195:1609-1611. 403 13. davidson ap: juvenile cellulitis. north am vet conf clinician’s brief 2006;4:21-22. 404 14.davidson ap: topics in pediatrics. proc swedish soc small anim reprod annu meeting; 2005. 405 15. mosier je: canine pediatrics: the neonate. proc am anim hosp assoc 1981;48:339–347. 406 141 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true 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reproduction 1 j.j. dascanio 2 virginia-maryland regional college of veterinary medicine, virginia tech, phase ii, duck 3 pond drive, blacksburg, va, usa 4 abstract 5 antibiotics are used to treat a variety of reproductive tract infections in the mare. the 6 results of an on-line survey of veterinarians concerning the use of antibiotics in mare 7 reproduction are presented. listservs were used to acquire the data. most veterinarians follow 8 the recommendations in the literature for treating mares with reproductive tract infections. 9 however, some veterinarians may mix antibiotics inappropriately, treat mares too soon after 10 breeding, use inappropriate mixed lavage solutions, or use antibiotics in cases that do not warrant 11 their use. 12 keywords: mare, antibiotic, reproduction, uterine, systemic, fungal 13 introduction 14 antibiotics are used in the mare to treat potential or realized reproductive tract infections 15 including vaginitis, cervicitis, endometritis, metritis, pyometra, and placentitis. alternatively, 16 infections may be associated with or classified as sexually transmitted diseases, post-mating 17 induced endometritis, acute or chronic endometritis, abortion related, and/or bacterial and fungal 18 infections.1 antibiotics are administered either through intravenous/intramuscular routes or 19 directly into the reproductive tract lumen. antibiotics are naturally occurring or synthetic 20 substances that inhibit the growth of or kill microorganisms. the definition may be limited to 21 substances affecting bacteria or may also include fungi and protozoa. in this paper antifungals 22 will be considered part of the antibiotic class. 23 411 there are a few reviews of the use of antibiotics in mare reproduction.2-4 the choice of 24 antibiotic should be based on culture and sensitivity patterns when possible or based on the most 25 likely organism when not possible. the most common bacteria isolated from the mare’s 26 reproductive tract are streptococcus equi subspecies zooepidemicus (gram positive), escherichia 27 coli (gram negative), klebsiella pneumonia (gram negative), pseudomonas aeruginosa (gram 28 negative), staphylococcus aureus (gram positive), and bacteroides (gram negative, anaerobe).5-29 10 streptococcus equi subspecies zooepidemicus and escherichia coli are the number one and 30 two isolates in almost all reports. the most common fungi isolated from the mare’s reproductive 31 tract are candida spp and aspergillus spp.11 32 there are many factors that may affect antibiotic effectiveness/clearance such as 33 overwhelming microorganism numbers, presence of uterine fluid/debris, lack of uterine 34 contractility, use of ecbolics, normality of uterine mucocilliary clearance mechanisms, cervical 35 dilation, and dependency of the uterine horns.12-15 disruptions of natural barriers to infection, 36 such as previous cervical trauma/scarring, vestibulovaginal fold incompetence (windsucker), and 37 poor vulvar conformation, may also contribute to continued bacterial/fungal contamination.16 38 intrauterine antibiotic therapy appears to have decreased in use, most likely due to 39 concerns about inducing secondary fungal infections and/or antibiotic resistance and due to new 40 information on the effectiveness of uterine lavage and the use of ecbolics, such as oxytocin and 41 prostaglandin. antibiotic therapies are now more targeted at specific organisms, are used with 42 more specific disease processes or are used in conjunction with methods to disrupt biofilms or 43 after decreasing bacterial numbers with lavage techniques. this manuscript will describe the 44 results of an on-line survey of veterinarians concerning antibiotic use in mare reproduction and 45 correlate the results to the literature. 46 412 survey on antibiotic usage 47 two surveys were conducted with regards to the use of antibiotics in equine 48 reproduction. the surveys were initiated to see what is commonly used in practice vs. what is 49 recommended in the literature. both surveys were sent to the equine clinicians network listserv 50 (ecn@listserv.vetmed.wsu.edu), the american association of equine practitioner’s listserv 51 (aaep_discussion@list.aaep.org), the equine reproduction listserv (eqrepro-l@po.missouri.edu) 52 and the american college of theriogenologists listserv (actlist@lists.theriogenology.org). 53 the first was initiated in september 2008 (190 respondents) and the second was initiated in 54 march 2009 (109 respondents). the second survey was performed to augment the first survey 55 results. sixty-one percent of participants in the second survey partook in the first survey. 56 approximately 69% of survey participants stated that the primary way that mares are 57 bred in their practice is by the use of fresh cooled semen, 27% are bred primarily by natural 58 cover, 3% primarily with frozen semen and 2% did not provide an answer. the number of years 59 in practice was: <5 years – 7%, 5 to 10 years – 20%, 11 to 15 years – 13%, 16 to 20 years – 18%, 60 >20 years – 39%, no answer – 2%. the percentage of their practice that was devoted to equine 61 reproduction was: <10% 11%, 10 to 25% 21%, 25 to 50% 18%, 50 to 75% 15%, >75% 62 34%, no answer – 1%. the larger number of practitioners in the >75% category most likely 63 reflects the distribution of the survey to two predominately reproductively oriented listservs 64 (act and eqrepro). veterinarians from 14 countries participated in the survey, with 70% of 65 respondents practicing in the united states. 66 when asked which bacterial and fungal organisms they encountered most frequently, the 67 overwhelming answers for bacterial isolates were streptococcus zooepidemicus followed by 68 escherichia coli. one practice stated that they had 80% beta-hemolytic streptococcus isolated 69 413 from 1400 uterine cultures in their clinic. an antibiotic with both gram positive and gram 70 negative properties may be appropriate for the treatment of uterine infections in those cases 71 without culture. fungal cultures, according to survey results, yielded primarily candida spp 72 followed secondarily by aspergillus spp. 73 intrauterine usage of antibiotics. 74 antibiotics may be placed into the uterus prior to or after breeding or in association with 75 treatment of suspected or known uterine infections. dosages for antibiotics commonly used for 76 intrauterine infusion are presented in table 1. practitioners responding to the survey stated that 77 antibiotics administered prior to breeding were used for mares that were known to be problem 78 breeders, mares that were repeat breeders, mares with uterine fluid pre-breeding, mares with 79 excessive uterine edema, mares suspected of having an infection (awaiting culture/cytology 80 results), mares suspected of having an infection (owners decline culture/cytology) or strictly at 81 the owner’s request. 82 survey participants stated that they used post-breeding antibiotics in situations where 83 they knew the mare had previous problems, in mares with uterine fluid, in those mares 84 susceptible to post-mating induced endometritis, in mares bred late in the breeding season, in 85 mares with previous pregnancy loss or as a routine procedure with a single dose of antibiotics, 86 especially in natural cover situations. a study by pycock found that pregnancy rates were better 87 after a single dose of antibiotics (± oxytocin) post-breeding, especially in older mares (>12 88 years) and mares mated at the first estrus post-partum.17 some thoroughbred farms may 89 routinely use a single post-breeding antibiotic to limit bacterial contamination from natural 90 cover.18 91 414 when asked how many days mares were commonly treated with intrauterine antibiotics, 92 the responses were: one day (12%), two days (7%), three days (50%), four days (5%), five days 93 (7%), one week (1%), other (13%), and no answer (5%). those answering “other” may treat for 94 1 to 3 days, 3 to 5 days, number of days would depend on bacteria isolated, number of days 95 would depend on presence of fluid, or would never treat a mare with intrauterine antibiotics. it 96 has been recommended, based on endometrial biopsy, that treatment for mild intrauterine 97 infections be performed for 3 days, moderate infections for 5 days, and severe infections for 7 98 days.12 the determination of how mares fit into these categories may not be clear in practice and 99 would need to be subjectively based on clinical signs and possible cytologic examination as 100 biopsy results may not be returned for a number of days. it has also been suggested that mares 101 not be treated for more than 2 or 3 days post-ovulation so as to decrease possible negative effects 102 on corpus luteum progesterone secretion from prostaglandin released in response to endometrial 103 irritation caused by the antibiotic or vehicle.12 antibiotics should also not be used immediately 104 pre-breeding as high concentrations of antibiotics may negatively affect sperm function.12 105 forty-three percent of practitioners would increase the volume of antibiotic solution 106 infused to between 50 to 100 ml prior to infusion. eight percent used the antibiotic without 107 dilution; 19% added extra volume, but kept the total less than 50 ml; 11% added extra liquid so 108 that the final volume was >100 ml; 7% added the antibiotic to the lavage solution; 6% did not 109 answer the question; and 8% provided an alternative answer of “other” which included leaving 110 some lavage solution in the uterus and adding the antibiotic to that fluid, using a 250 ml bottle of 111 fluid for infusion with antibiotics added, or using a 10 ml or 20 ml total volume. the literature 112 has suggested intrauterine infusion volumes ranging from 30 to 200 ml to achieve distribution 113 throughout the uterine lumen.2 six grams of ticarcillin, for instance, has a higher intrauterine 114 415 concentration over time when a 250 ml volume is infused rather than a 60 ml volume.19 with 115 large volumes, however, reflux of fluid back through the cervix could occur, diminishing the 116 overall dose. a more appropriate recommendation may be to maximize the volume of an 117 antibiotic solution while considering the relative size and position of the uterus. multiparous 118 mares would naturally require a larger volume, while nulliparous mares should require less. 119 with a dependant uterus, infused fluids tend to pool in the base of the uterine horns making it 120 difficult to achieve uniform coverage of the endometrium; consequently, systemic antimicrobials 121 may be a good choice in these mares. 122 the most common antibiotic used for intrauterine treatment prior to receiving 123 culture/antibiotic sensitivity results by veterinarians who participated in the survey was ceftiofur 124 (21%), followed by gentamicin (19%), ticarcillin with clavulanic acid (13%), ampicillin (12%), 125 other (12%), procaine penicillin (5%), amikacin (5%), potassium penicillin (3%), and ticarcillin 126 (3%). nine percent of survey participants did not answer this question. the category “other” 127 included combination of penicillin and gentamicin (2%), penicillin and neomycin (2%), 128 ampicillin and gentamicin (1%), oxytetracycline, framomycin, framycetin, cefquinome, 129 cefazolin, or chloramphenicol. interestingly, procaine penicillin was used even though there are 130 no dosages reported in most published reviews. some practitioners had concern about residues 131 that may be left within the uterus with the use of the procaine penicillin suspension. 132 enrofloxacin has been administered by intrauterine infusion without causing more than a 133 moderate inflammatory response,20 but there are other reports that the basic ph of enrofloxacin is 134 very irritating to the endometrium.12 differences among reports may be due to the formulation 135 studied in various countries and dosage, thus caution should be exerted when considering 136 intrauterine enrofloxacin or, alternatively, enrofloxacin should be used systemically. 137 416 if we examine some of the more common antibiotics used in practice, one study found 138 that 19% of beta-hemolytic streptococcus isolates (includes streptococcus zooepidemicus) were 139 susceptible to gentamicin, whereas 96% of escherichia coli isolates were susceptible.10 in that 140 study, 100% of the beta-hemolytic streptococcal isolates were susceptible to ampicillin and 141 penicillin g, whereas 86% of escherichia coli isolates were susceptible to ampicillin. another 142 study evaluated intrauterine ceftiofur in mares and found that the drug had good antimicrobial 143 activity and caused no increase in uterine inflammation when compared to controls.21 ticarcillin 144 with clavulanic acid has been evaluated for intrauterine use and it was found that adequate 145 intrauterine concentrations of the clavulanic acid portion are not maintained. thus, this 146 formulation may be questionable for intrauterine use.22 these authors also reported that 147 concentrations of ticarcillin declined rapidly after intrauterine administration, and multiple daily 148 doses would be required. 149 aminoglycosides have an acid ph that will irritate the endometrium.3 it is suggested that 150 aminoglycosides be buffered to a neutral ph with an equal volume of 7.5% sodium bicarbonate. 151 forty-three percent of practitioners added sodium bicarbonate, while 38% increased the volume 152 of infusion as a means to moderate the acidic effects, and 10% did not add anything to the 153 aminoglycoside. if saline is used to increase the volume of infusion to reduce the effect of low 154 ph, it should be noted that saline has a ph of ≈5.5. a more suitable diluent may be lactated 155 ringer’s solution which has a neutral ph. 156 aminoglycosides should not be mixed with beta-lactam antibiotics. precipitates may 157 form when they are combined or, more importantly, aminoglycosides may cause a nucleophilic 158 opening of the beta-lactam ring which then combines with an amino group from the 159 aminoglycoside, resulting in a biologically inactive amide.23,24 while the two drugs are 160 417 synergistic in controlling gram positive (beta-lactams) and gram negative (aminoglycosides) 161 infections when given systemically, it is not completely understood how effective they are when 162 placed together into the uterine lumen. in addition, penicillin g (potassium or procaine) is 163 inactivated by acids, so if penicillin and an aminoglycoside are used together in an unbuffered 164 form, the penicillin may be less effective because of the low ph environment caused by the 165 aminoglycoside. from survey results it appears that quite a few practitioners (34%) mix the two 166 classes of drugs together either in the same syringe (20%) or the drugs are infused into the uterus 167 at the same time (14%). for maximum effectiveness, mixing these drugs within the uterus 168 should be discontinued and the drugs should be given either systemically or their administration 169 separated in time by an unknown number of hours if given by intrauterine infusion. it is also not 170 recommended to mix the two classes of drugs in lavage solutions. interestingly, there are many 171 semen extenders that combine potassium penicillin and amikacin. this practice may diminish 172 the effectiveness of the antibiotics. conversely, gentamicin has a high rate of inactivation when 173 mixed with certain beta-lactams, while amikacin is only slightly inactivated.25 174 when asked about which antifungal intrauterine drug they used prior to receiving culture 175 results, the majority of practitioners (32%) would not use an antifungal drug, but instead opted 176 for either a povidone-iodine solution lavage, lufenuron or, less commonly, a dilute vinegar 177 lavage. if an antifungal drug were used, then the most common responses included clotrimazole 178 (17%), nystatin (11%), miconazole (10%), fluconazole (8%) and amphotericin b (3%). no 179 answer was provided by 19% of the respondents. only 53% of practitioners submitted fungal 180 cultures for sensitivity assay. lack of antibiotic sensitivity patterns to determine the most 181 appropriate therapy may explain, in part, why fungal uterine infections are difficult to treat. the 182 reasons stated for not submitting fungal cultures for a sensitivity are: length of time to receive 183 418 results; had success with povidone-iodine lavage; all seem sensitive to amphotericin b; inability 184 to obtain fungal sensitivities from the laboratory; the relative infrequency with which fungal 185 infections were encountered precluded sensitivity testing; just treated candida infection with 186 nystatin; or treating seems to work just fine. there are a number of laboratories that offer fungal 187 sensitivity patterns including the laboratory at cornell university. there was also concern from 188 practitioners that in vitro sensitivity patterns may not correlate with in vivo effectiveness. it 189 would appear that within the group of polyene antifungal antibiotics, amphotericin b (96% 190 susceptibility of all fungal organisms) and nystatin (100% susceptibility) are good choices, 191 where as clotrimazole (80% susceptibility) or ketoconazole (81% susceptibility) are good 192 choices when using azole antifungal antibiotics. (personal communication, marco coutinho da 193 silva, cornell university) polyene antibiotics are generally considered fungicidal, whereas azole 194 antibiotics are fungistatic, except at higher doses. some practitioners try to avoid intrauterine 195 antifungal treatments with the concern that repeated intrauterine treatment may make the mare 196 more susceptible to re-infection or prolonged inflammation. an alternative would be oral 197 antifungal drugs which may be expensive. 198 lufenuron is a chitin inhibitor which has been used in an extra-label manner for treatment 199 of fungal uterine infections.26 it should be noted that lufenuron affects the wall of growing fungi 200 and may not be appropriate for treatment of mature infections. a better approach may be to treat 201 with an antifungal antibiotic and then at the end of treatment, place lufenuron in the uterus to 202 prevent new growth. the effectiveness of lufenuron still remains in question.27,28 203 uterine lavage with either iodine or vinegar is a component of therapy for many 204 veterinarians when treating fungal infections. forty percent of veterinarians used a dilute iodine 205 solution for lavage (24% added iodine to saline, 16% added iodine to lactated ringer’s solution). 206 419 the percent iodine in lavage solutions in the survey range from 0.02% (2 ml of 10% iodine per 207 liter) to 0.5% (50 ml of 10% iodine per liter). a 0.2% solution of iodine infused into the uterus 208 has been associated with endometrial inflammation and fibrosis.29 a 0.01% to 0.05% solution of 209 iodine maintains antimicrobial activity30 without causing inflammation and fibrosis.31 210 practitioners should be cautious of the higher concentrations of iodine in intrauterine infusions. 211 twenty-two percent of veterinarians used a dilute vinegar solution (15% of practitioners added 212 vinegar to saline, 7% added it to lactated ringer’s solution). when using vinegar, saline would 213 be a more appropriate lavage solution, if the desire is to lavage with a lower ph solution. 214 addition of 20 ml of white vinegar to 1000 ml of saline (2% v:v solution) will reduce the ph 215 from ≈5.5 to ≈3, whereas it has little effect on the ph of lactated ringer’s solution. 216 intrauterine antibiotics and lavage should be avoided within 4 hours of breeding12 so that 217 spermatozoa are not negatively affected by the drugs or the vehicles in which they are delivered. 218 after 4 hours post-insemination, spermatozoa are located in the oviduct and intrauterine 219 treatment at this time does not have a negative effect on fertility.32 most practitioners who 220 participated in the survey appeared to be aware of this, with only 16% of them infusing 221 antibiotics within 4 hours post-insemination. most practitioners (37%) withheld treatment for 222 more 4 hours post-breeding, because the next examination, and thus treatment of the mare, did 223 not occur until the day following insemination. 224 systemic antibiotics 225 the decision to use systemic antibiotics either in combination with intrauterine infusion, 226 after intrauterine infusion or instead of intrauterine infusion of antibiotics may be due to personal 227 preference, a desire to prolong the treatment period, because the organism is not susceptible to 228 non-irritating drugs, or to avoid manual manipulation of the reproductive tract. results of the 229 420 on-line survey indicated that systemic antibiotics are chosen when intrauterine treatments extend 230 beyond 3 to 5 days, when treating mares with metritis, when treating mares with contaminated 231 caudal reproductive tracts, when treating mares with anatomical defects of the caudal 232 reproductive tract or occasionally when treating mares with fungal infections. respondents felt 233 that systemic antibiotics negate the need to invade the uterus, possibly avoiding the chances of 234 iatrogenically placed bacteria or fungi. the disadvantages of using systemic antibiotics are 235 increased costs and inconvenience from having to dose at the animal’s full body weight and 236 possibly the need to treat multiple times per day. a very small number of practitioners felt that it 237 was not good veterinary practice to place antibiotics directly into the uterus, since systemic 238 antibiotics work well, do not cause endometrial irritation, and do not lead to further 239 contamination. 240 dosages for antibiotics commonly used systemically are presented in table 2. 241 trimethoprim sulfadiazine, ceftiofur, and a combination of penicillin and gentamicin were the 242 most common antibiotics administered by practitioners who participate in the survey. 243 trimethoprim sulfamethoxazole (30 mg/kg, per os, q12h) was found to provide adequate 244 antibiotic concentrations in fetal tissues in mares with placentitis.33 in a separate study, ceftiofur 245 dosed at 2 mg/kg q12h intramuscularly, did not result in adequate endometrial tissue levels;34 246 however, it has been suggested as a potential treatment for mares with placentitis.35 higher 247 dosage concentrations (recommended up to 4.4 mg/kg) and/or intravenous treatment could 248 perhaps result in adequate endometrial levels. in cattle, minimal inhibitory concentrations of 249 ceftiofur are achieved in endometrial tissue after subcutaneous administration.36 a study by 250 murchie, et al., found that intravenous administration of penicillin g potassium and gentamicin 251 sulfate resulted in adequate allantoic fluid concentrations in pregnant pony mares.37 252 421 enrofloxacin has also been used in mares with more resistant bacteria.20,38,39 enrofloxacin 253 should not be used in pregnant mares due to its effects on developing cartilage.40 doxycycline 254 has also been demonstrated to reach endometrial concentrations above the minimum inhibitory 255 concentration for streptococcus equi subspecies zooepidemicus.41 256 antifungal antibiotics may be administered systemically. amphotericin b is fairly 257 caustic due to a low ph and needs to be given via nasogastric intubation or diluted and given 258 slowly intravenously. oral fluconazole has been recommended for treatment of candida spp. 259 while oral itraconazole has been suggested for treatment of aspergillus spp. (personal 260 communication, marco coutinho da silva, cornell university). 261 uterine cytology 262 uterine cytology was performed in conjunction with 67% of uterine cultures. this is a 263 relatively easy procedure to perform and interpret.42 sixty-four percent of practitioners either 264 read their own (56%) or had someone in their practice (8%) read cytologies. by performing this 265 examination “in-house” results may be interpreted and therapy instituted without the delay of 266 sending the slides to an outside laboratory. only 9% of practitioners, however, used gram stain 267 to distinguish gram negative from gram positive bacteria in order to institute appropriate 268 antimicrobial therapy. 269 treatments to augment antimicrobial therapy 270 uterine lavage is recommended to remove uterine debris, bacteria and fungi and to 271 enhance uterine contractility. dmso lavages may be useful to augment tissue penetration and to 272 disrupt microbial biofilms.43 acetylcysteine and kerosene have also been suggested as possible 273 mucolytic agents. biofilms are aggregates of bacteria and/or fungi encased in an adherent 274 polymeric matrix which may inhibit antibiotic penetration.44-46 biofilms have been known to 275 422 form with pseudomonas aeruginosa, escherichia coli, staphylococcus aureus, klebsiella 276 pneumonia, and candida spp.47 277 tris-edta has been demonstrated to act synergistically with antimicrobials by 278 increasing the membrane permeability of bacteria to these drugs.48 uterine lavage with tris-279 edta, either alone or in combination with antibiotics, should be considered with resilient 280 infections or in cases with antibiotic-resistant organisms. 281 new intrauterine therapies 282 intrauterine foam (fatroximin®; fatro, bologna, italy) containing the antibiotic 283 rifaximin, a synthetic derivative of rifamycin, has been developed for use in cattle and horses.49 284 it has a spectrum of activity that includes gram negative, gram positive and anaerobic bacteria. 285 the drug, in the foam vehicle, has a 72+ hour residual effect and expands to cover the entire 286 uterine lumen. a single treatment is recommended for treatment of endometritis or the product 287 may be administered for two consecutive days for treatment of vulvovaginitis. this drug is 288 currently available in europe. 289 conclusions. 290 veterinarians should base antibiotic therapy on sensitivity tests. consideration should be 291 given to antibiotic therapy alternatives such as proper breeding management, use of uterine 292 lavage and oxytocin or prostaglandin treatment. biofilm formation should be appropriately 293 treated to enable antibiotics to access bacteria and fungi. with a plan, antibiotic usage can be 294 minimized and treatment success optimized. the main issues of concern identified from the on-295 line survey of veterinarians are: mixing beta-lactam and aminoglycoside antibiotics for 296 intrauterine infusion, intrauterine infusion of high concentrations of iodine solutions, use of 297 423 lactated ringer’s solution with vinegar for uterine lavage, and treatment of mares less than 4 298 hours post-breeding with intrauterine antibiotics. 299 300 table 1. intrauterine antibiotic dosages. 301 302 intrauterine antibiotics antibacterial antibiotics antibiotic dosage comments major bacterial susceptibility amikacin 1 to 2 grams buffer with sodium bicarbonate or 150 to 200 ml solution gram negative ampicillin 1 to 3 grams use soluble product, may be irritating when concentrated gram positive and e.coli ceftiofur sodium 1 gram gram positive and gram negative chloramphenicol 2 to 3 grams can be irritating gram positive and gram negative gentamicin 1 to 3 grams acidic – need to dilute and/or buffer gram negative neomycin 2 to 4 grams gram negative potassium penicillin 5 million gram positive 424 international units procaine penicillin 4.5 to 6 million international units concern about residue left in uterus gram positive ticarcillin 3 to 6 grams infuse with 150-200 ml solution gram positive, pseudomonas ticarcillin with clavulanic acid 3 to 6 grams beta-lactamase inhibitor, infuse with 150-200 ml solution same as ticarcillin plus more gram positive (staph, bacillus, enterobacter) antifungal antibiotics q24h for 7 days drug dosage comment amphotericin b 100-200 mg polyene, dilute in >100 ml solution, mix well clotrimazole 400 to 700 mg azole, tablets usually crushed and mixed with solution fluconazole 100 mg azole, may need to adjust ph to avoid acidic nature miconazole 500-700 mg azole nystatin 0.5 to 2.5 million international units polyene, dilute in sterile water, not saline to avoid precipitates, mix well 303 304 425 table 2. systemic antibiotic dosages. (iv-intravenous, im-intramuscular, po-per os, q-every, h-305 hour, iu-international units, kg-kilogram) 306 systemic antibiotics antibacterial antibiotics drug dosage route, comment amikacin 10 mg/kg q24h iv or im ampicillin 29 mg/kg q12-24h iv or im ceftiofur 2 to 4 mg/kg q12h iv or im doxycyline 10 mg/kg q12h po enrofloxacin 5.5 mg/kg q24h iv 7.5 mg/kg q24h per os 4.0 mg/kg q12h per os gentamicin 6.6 mg/kg q24h iv or im metronidazole 15 to 25 mg/kg po oxytetracycline 6.6 mg/kg q12h iv, dilute and give slowly potassium penicillin 22,000 iu/kg q6h iv procaine penicillin 22,000 iu/kg q12h im, only 10 ml per injection site trimethoprim sulfa 30 mg/kg q12h po antifungal antibiotics drug dosage route comments amphotericin b 0.3 to 0.9 mg/kg q24-48h iv polyene, dilute and give slowly fluconazole 14 mg/kg loading, iv or per os azole 426 then 5mg/kg q24h; alternatively, 2 grams q24h itraconazole 5 mg/kg q12-24h iv or per os azole, oral suspension more bioavailable than capsules ketoconazole 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/destinationprofileselector /na /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure true /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 6 /marksweight 0.250000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /na /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /leaveuntagged /usedocumentbleed false >> << /allowimagebreaks true /allowtablebreaks true /expandpage false /honorbaseurl true /honorrollovereffect false /ignorehtmlpagebreaks false /includeheaderfooter false /marginoffset [ 0 0 0 0 ] /metadataauthor () /metadatakeywords () /metadatasubject () /metadatatitle () /metricpagesize [ 0 0 ] /metricunit /inch /mobilecompatible 0 /namespace [ (adobe) (golive) (8.0) ] /openzoomtohtmlfontsize false /pageorientation /portrait /removebackground false /shrinkcontent true /treatcolorsas /mainmonitorcolors /useembeddedprofiles false /usehtmltitleasmetadata true >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 24 contact muhammad-salman waqas salman.waqas@wsu.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11653, http://dx.doi.org/10.58292/ct.v17.11653 review report manipulation of ovarian function in sheep and goats muhammad-salman waqas, ahmed tibary department of veterinary clinical sciences, college of veterinary medicine and center for reproductive biology, washington state university, pullman, wa, usa abstract sheep and goats are short-day breeders. melatonin, photoperiod, breed, nutrition, and geographical location influence seasonality. estrus synchronization is useful in maximizing the profitability of small ruminant production. this article discusses the endocrine control of seasonality and the reproductive cycle, and manipulation of ovarian function in sheep and goats. the ovarian follicular wave pattern in these species is described to help the reader understand the mechanisms involved in the protocols for estrus synchronization and superovulation in small ruminants. the estrus synchronization protocol in these species encompasses using the male effect, photoperiod manipulation, and hormonal treatments. the male effect is an efficient synchronization technique during the transition period. although not used routinely, photoperiod manipulation of seasonality is possible. hormonal control of the cycle can be grouped into 2 categories; protocols used during the breeding season and protocols used for out-of-season breeding. during the breeding season, prostaglandin f2α can be used for estrus synchronization but this protocol is not very efficient for timed artificial breeding. progestogen-based protocols are commonly used for estrus synchronization, for both inand out-of-season breeding. such protocols are often combined with the use of equine chorionic gonadotropin (ecg) to improve synchrony and ovulation rate. melatonin implants are used to induce cyclicity for out-of-season breeding and can be combined with progestogen synchronization protocols. synchronization protocols in combination with the use of follicle stimulating hormone or ecg allow superovulation for the purpose of embryo production. the most common synchronization and superovulation protocols are described. the availability of hormones varies among countries. keywords: small ruminants, estrus synchronization, superovulation, hormones, sheep, goat introduction the profitability of sheep and goat production systems relies on a timely establishment of pregnancy with a goal of > 90% pregnancy rate. estrus synchronization is a globally practiced technique in the breeding management of small ruminants.1 intensive production, particularly in sheep, relies on accelerated lambing systems. the consumer demand is higher during specific periods of the year. melatonin-driven, photoperiodic seasonality limits producers from meeting this market demand. estrus synchronization protocols for inand out-of-season breeding management maximize profitability,2 improve flock genetics and facilitate artificial insemination (ai) with the use of semen from superior sires. furthermore, it is useful to match the production of a lamb or kid crop with consumer demand and availability of feed and cheaper labor, and allows efficient application of ai and embryo transfer (et) programs.3 in small ruminants, various synchronization methods are used, ranging from manipulation of light to a combination of hormones coupled with photoperiodic manipulation and male effect. estrus synchronization protocols based on intravaginal delivery of progestogens followed by ecg treatment and luteolysis using prostaglandin f2α (pgf2α) are the most commonly used.4 current estrus synchronization protocols are not efficient enough to precisely synchronize time of ovulation. the protocols result in suboptimal fertility when applied to less prolific breeds and during out of season.5 compared to protocols involving natural mating, fertility following estrus synchronization is lesser for ai and et programs. genetic improvement requires the use of reproductive technologies such as ai, et using in vivo-derived embryos, and, more recently oocyte collection for in vitro production of embryos. all these technologies require control and manipulation of ovarian follicular dynamics. the protocols used to achieve this are based on our understanding of ovarian follicular wave dynamics and hormonal mechanisms controlling them. our aims are to: 1. describe the mechanisms controlling seasonality and estrous cycle; 2. discuss the hormonal and nonhormonal methods of induction and synchronization of cyclicity; and 3. describe common protocols for superovulation in sheep and goats. mailto:salman.waqas@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11653 citation: clinical theriogenology 2025, 17, 11653, http://dx.doi.org/10.58292/ct.v17.11653 25 seasonality of reproduction in sheep and goats the seasonality of reproduction in ewes and does is a wellknown biological phenomenon. in temperate zones, the breeding season corresponds to short days. at the end of this season, females undergo a transition period characterized by silent ovulations before entering a period of anestrus (i.e. absence of estrus and ovulation). seasonal anestrus is followed by a transition period characterized by an increasing percentage of ovulating females but often with a short luteal phase during the first cycle. the breeding season is characterized by regular estrous cycles and an increased ovulation rate.6–8 the mechanisms that control the seasonality of reproduction have been studied for several years but have yet to be fully understood. the transition from seasonal anestrus to reproductive cyclicity is determined by the decreased sensitivity of the hypothalamus to estrogen-negative feedback. this change is induced by the photoperiod perceived by the retina and then transmitted by the retinohypothalamic tract to the suprachiasmatic nuclei and other structures, including the paraventricular nuclei of the hypothalamus, the mediolateral horn of the thoracic spinal cord, the superior cervical ganglia, and then the pineal gland (epiphysis).1 the neural signal is then translated into an endocrine signal through melatonin secretion. melatonin is produced overnight from tryptophan that is converted into serotonin and then n-acetylserotonin. blood melatonin concentrations rise within 10 minutes after the onset of darkness and remain high throughout the night. the duration of melatonin secretion is proportional to dark hours of the night and is responsible for the seasonal variation in the photoperiod. plasma melatonin concentrations are low during the day (< 5 pg/ml) and high at night (100-500 pg/ml in sheep and 50-150 pg/ml in goats). melatonin is in much higher concentrations (100 times the plasma concentrations) in the cerebrospinal fluid of the 3rd ventricle.1 melatonin stimulates the increase in gnrh secretion that causes an increase in follicle stimulating hormone (fsh) and luteinizing hormone (lh) pulsatility, inducing follicular growth and ovulation. it is important to note that the effect of increasing melatonin on the hypothalamus, in response to decreasing daylight, takes 40-60 days.6,7 during seasonal anestrus, 2 mechanisms inhibit gnrh secretion. first, there is an increase in the sensitivity of the hypothalamus to the negative feedback of estradiol, involving the estradiol receptors within a population of glutamate neurons (preoptic and retrochiasmatic areas). these neurons stimulate a retrochiasmatic population of dopamine neurons, defined as the a15da population that inhibits gnrh neurons. this inhibition occurs through a direct action at the terminals of gnrh neurons in the median eminence and through an indirect action inhibiting kisspeptin neurons in the arcuate nucleus that project directly onto gnrh neurons in the medio-basal hypothalamus. these mechanisms lead to a decrease in gnrh pulses, reducing lh and fsh secretion and thus inhibiting ovarian activity. the second mechanism involves an estradiol-independent inhibition of gnrh neurons through a melatoninergic signal of long photoperiod (summer). this signal stimulates the synthesis of thyroid stimulating hormone (tsh) in the pars tuberalis of the pituitary gland, inducing the conversion of triiodothyronine to thyroxine. this action stimulates rfrp-3 neurons and inhibits kisspeptin neurons, inhibiting gnrh secretion.6,7 during the breeding season, the hypothalamus’s sensitivity to estradiol’s negative feedback is reduced, lifting the tsh inhibition pathway. this results in the activation of kisspeptin and gnrh neurons which culminate in the activation of the pituitary-gonadal axis. the seasonality of reproduction in small ruminants is governed by a circannual biological clock, enabling endogenous reproductive seasonality to be maintained over several years without the intervention of photoperiod (constant conditions). in fact, keeping females under a short photoperiod (short days) does not extend the breeding season. a refractory period to photostimulation is necessary for the initiation and cessation of sexual activity.6 seasonality is determined mainly by the photoperiod (high latitudes), but other factors can affect it, including ambient temperature, nutrition, altitude, age, and breed. in general, breeds of tropical (23°n-23°s) or subtropical (24°-34°n, 24°-34°s) origin have a longer breeding season than breeds from temperate zones (> 40°n or > 40°s). breeds from intermediate or mediterranean zones (35-40°n, 35-40°s) have a short anestrus period, during which a proportion of females continue to ovulate. meat breeds are very seasonal. prolific breeds, on the other hand, are known to have a more extended breeding season. boer does in south africa cycle practically all year round. this difference between breeds may be due to a difference in sensitivity to the negative feedback of estradiol.6,7 estrous cycle during the breeding season, females display regular cycles governed by the hypothalamic-pituitary-gonadal axis. short cycles (short luteal phase) characterize the first cycles of the breeding season. the length of the cycle, follicular dynamics, and luteal activity, differ markedly between sheep and goats. ovarian follicles develop in waves to preovulatory stage, even outside the breeding season.8,9 in ewes, estrous cycle length is 14-17 days. each estrous cycle has 3 or 4 follicular waves. the number of follicular waves per estrous cycle is not affected by the prolificacy of the breed. follicular waves emerge in response to increased fsh triggered by the pulsatile gnrh secretion from the hypothalamus. a cohort of follicles, 1-2 mm in diameter, appears every 4 days. ovulatory follicles generally emerge ~ on day 12 of the cycle. during the luteal phase, follicles reach a maximum diameter of 4-7 mm, then regress or become atretic after 5 and 12 days, respectively. follicular dynamics thus presents 3 phases: a growth phase of 2-4 days, a static phase of 1-4 days, and a regression phase of 1-5 days. at the end of each follicular wave, 1-4 follicles reach the preovulatory stage. follicles that ovulate during estrus may be from the last 2 waves. follicles from prolific breeds mature earlier and have a smaller diameter before ovulation. the antral follicles of prolific breeds have fewer granulosa cells, but their steroidogenic capacity is greater, resulting in higher serum estradiol concentrations.10 in the absence of progesterone, estradiol-17ß induces estrus behavior that lasts an average of 36 hours (24-72 hours). estrus is shorter in ewe-lambs and at the start of the breeding season and longer in prolific breeds. external signs of estrus are discreet in ewes, except for receptivity to the ram.9 the cervical mucus changes from clear and runny at the beginning of estrus to highly viscous and white as ovulation approaches. the final maturation of the follicle is obtained through the increasing lh sensitivity of the granulosa cells.9 the lh surge occurs in response to high estradiol-17ß concentrations, followed by ovulation 20-40 hours after the onset of estrus.9 a corpus hemorrhagicum forms after ovulation and undergoes http://dx.doi.org/10.58292/ct.v17.11653 26 citation: clinical theriogenology 2025, 17, 11653, http://dx.doi.org/10.58292/ct.v17.11653 histological and functional changes under the influence of lh. formation of the corpus luteum (cl) takes 3-4 days. the cl diameter increases, reaching a maximum of 11-14 mm on days 9-10 of the cycle (day 0 = ovulation). the cl size is smaller in prolific than in nonprolific breeds. the increase in cl size is due to an increase in the volume of large luteal cells from day 4 until day 10 of the cycle and to the mitotic multiplication of small luteal cells on days 4-8. progesterone concentrations rise progressively on days 3-7, reaching a plateau, then begin to decrease on days 12-14. serum progesterone concentrations are higher in nonprolific breeds. luteolysis is induced on days 14-16 of the cycle by pgf2α secreted by the endometrium and transported directly by the ovarian artery (from the uterine veins and lymphatic vessels) to the ipsilateral ovary. pgf2α causes vasoconstriction in the cl through the release of endothelin-1. also, pgf2α binds to the receptors of the large luteal cells, inducing a functional luteolysis by disrupting progesterone synthesis through the alteration of cholesterol transport across mitochondrial membranes. this mechanism of pgf2α release is exacerbated by the action of oxytocin from pituitary and luteal origin.9 in goats, the estrous cycle length is 17-25 days. short cycles, averaging 8 days, are common at the beginning of the breeding season. the goat estrous cycle has 2-6 follicular waves. in general, the follicles that ovulate during estrus are from the last wave. however, in some cases (prolific breeds), the ovulations are from follicles from the last 2 waves. a follicular wave begins with the recruitment of a cohort of 2-3 mm gonadotropin-sensitive antral follicles. from this cohort, 2-4 follicles grow to 4 mm and become dominant. during the luteal phase of the cycle, the dominant follicles continue to develop to reach a maximum size and then undergo atresia. in the absence of progesterone (i.e. after luteolysis), the dominant follicles continue to grow under the influence of lh and reach the preovulatory stage with a diameter of 6-9 mm. during this phase, serum estradiol concentrations rise, leading to onset of estrus. the length of estrus is 36 hours on average and ranges 22-48 hours depending on the breed, age of the female, season, and male presence. young nulliparous females have a shorter estrus than adult does. signs of estrus are more obvious in does than in ewes and occur in 2 phases: a proceptive phase followed by a receptive phase.8 during the proceptive phase, the doe actively seeks the buck but does not accept mounting. during the receptive phase, the doe exhibits rapid tail movements (wagging), vocalizations, frequent urination, and accepts mounting. estrous does may also mount each other. estrus is often accompanied by reduced appetite and milk production, and physical changes (vulvar edema) and a characteristic vaginal cytology (> 50% eosinophilic desquamated polyhedral epithelial cells). the interval from the onset of estrus to the lh peak varies among breeds (8 hours for the boer, 14.5 hours for the alpine). consequently, the interval from the onset of estrus to ovulation is also variable (9-37 hours). the ovulation rate varies from 1-5, depending on age, breed, and nutritional status. as described for the ewe, corpora hemorrhagica form after ovulation and progressively luteinize to become mature cls by day 5 after ovulation.8 methods of estrus synchronization in small ruminants male effect the use of hormones in livestock is prohibited in some countries and for organic animal production systems.11 the ‘male effect’ is an alternate tool for nonpharmaceutical reproductive management in small ruminants. combined use of the male effect and hormones is emerging as a more effective estrus synchronization tool as it seems to overcome some disadvantages of hormonal methods to stimulate and synchronize ovarian activity. the use of hormones can be followed by male effect alone or male effect coupled with photoperiodic manipulation, as an alternate choice. the male effect, being more cost-effective than hormonal methods, could be very useful for developing countries; however further studies are needed as geographical location and breed influences the outcome of efforts to induce estrus and ovulation.12 the male effect refers to a relatively synchronized physiological response of females (induction of estrus and ovulation) after the introduction of a male into the flock. because of the strong male effect in sheep and goats, these species are used as a model to study complex hypothalamo-hypophyseal endocrine changes in females induced by sociosexual stimuli from the male. by enhancing gnrh and thus lh, the male effect induces ovulation in cyclic and acyclic females. in acyclic animals that respond to the male effect, an lh surge is observed within 72 hours that may or may not induce ovulation. this ovulation may not be accompanied by behavioral estrus and is followed by normal or shortened luteal phases.13 in cyclic females, luteotropic effect of the male-effect-induced lh surges occurring in the early to mid-luteal phase may lead to a prolonged estrous cycle.14 the introduction of the male during the late luteal or follicular phase shortens the estrous cycle.1,15 generally, the male effect is assumed to be effective within the same species; however, increased lh pulse frequency has been observed in ewes exposed to buck hair and in does exposed to rams.16 recently, the buck-to-buck effect, where sexually active male goats stimulate the endocrine and sexual activities of other males, has been reported.17 the male effect is thought to be mediated primarily by chemical signaling of pheromones via olfaction, along with some supplementary effects of vision and memory. the main sites of pheromone production are the sebaceous glands of the head, neck, shoulder, and rump. the 5α-reductase in these sites converts testosterone to dihydroxy testosterone, which stimulates production of pheromones that impregnate the hair or wool in these areas. chemically, the derivatives of 4-ethyl octanoic acid act as primary pheromones in goats and are responsible for a range of social interactions within the species, including the male effect.1,18,19 however, the biomolecule causing the male effect is not a pheromone by definition.20 the main pathway for the ram effect is the stimulation of the gnrh nuclei in the brain through the major and minor pathways. the pheromone mediated stimulation through the olfactory and major olfactory bulb passes through the cortical nuclei in the amygdala before reaching the hypothalamus (major pathway). the stimulation through the vomeronasal organ and accessory olfactory bulb is processed through the medial nuclei of the hypothalamus (minor pathway). ultimately, these olfactory stimuli activate the ventromedial nucleus of the hypothalamus in the preoptic area to generate gnrh pulses. the male effect is mediated through kisspeptin and neurokinin b. the pheromone signals are conveyed from the amygdala to kisspeptin neurons in the arcuate nucleus. neurokinin b, involved in the cellular mechanism through gprotein-coupled receptors, causes an lh surge by activating the gnrh pulse generator.1,18,19,21 http://dx.doi.org/10.58292/ct.v17.11653 citation: clinical theriogenology 2025, 17, 11653, http://dx.doi.org/10.58292/ct.v17.11653 27 factors affecting response to the male effect include the number of cyclic females, season, geographical location, breed of the female, breed of male, male’s libido, depth of anestrus, body condition of female, parity, lactation, degree of anestrus of ewes or doe, male sexual activity, male novelty, and duration of contact with the male.7,22 male sexual behavior is also an important factor in stimulating the ovarian response and maintaining pulsatile lh secretion for ovulation.23,24 ewes with lower body condition scores have a lower level of lh surge in response to the male effect that delays subsequent estrus, ovulation, and progesterone concentrations.25,26 less seasonal breeds like the merino ovulate within 2-3 days, whereas more seasonal breeds gradually proceed to ovulation in response to the male effect.27 the male effect is more pronounced during the breeding season than during the nonbreeding season.28 response to the male effect does not seem to depend on follicular diameter at the time of male’s introduction.29 the male should be separated from the female for 15-34 days for optimum response.30 however, as sexual activity and novelty are more crucial for efficacy of the male effect, isolation is required only for the reintroduction of familiar males.1,31 visual and audio-visual stimuli, including male images or male vocalization, lead to some ovarian activity and/or weaker lh secretion compared to direct contact with the male.21,32 socially dominant does ovulate earlier than subordinate does in response to the male effect.33 a 4-hour contact per day with novel rams induced ovulation in anovulatory ewes without prior separation.34 however, ewes or does are unresponsive to the male effect if they are habitual/familiar to particular males.31 the familiarity occurred quickly within hours and is mediated via the hippocampus, the center for learning and memory.35 the separation of males from females diminish olfactory awareness of ewes to males, and the male can be reintroduced as a novel male. the reintroduction of familiar males after a separation period of 30 days led to a weaker lh surge than observed after the introduction of novel males.26 the use of sexually active novel males produces a more efficient male effect than sexually inactive novel males and does not require a period of separation of the females.26,36 interestingly, daily rotation of photo-stimulated bucks among groups of does increased sexual activity of the buck but did not increase ovulation or pregnancy rate in the does compared to does subjected to photo-stimulated but no buck rotation.37 for practical application in goat breeding, 2 hours per day of contact with a photo-stimulated, sexually active male for 15 consecutive days is sufficient to induce and synchronize ovulation. one photo-stimulated sexually active male is sufficient for 9-11 does.22 alternatively, photoperiod-melatonin induced, sexually activated males can be used for male effect. the sexual activation in rams in spring can be achieved by 2 months of long days (16 hours light/day) followed by 3 melatonin (18 mg each) subcutaneous implants at the end of the long days.38 combined use of male effect and hormones application of the male effect in combination with hormonal treatments and photoperiod management offers a more predictable way to synchronize estrus. for estrus synchronization, using 2 injections (13 days apart) of pgf2α with the introduction of the male on the day of the second pgf2α injection increased the ovulation rate in ewes. this protocol resulted in  better synchronization and ovulation than the sole introduction of males without the second injection.39 estrus synchronization of does in deep anestrus using artificial light (16 hours), 11-day progesterone treatment, and male effect resulted in an lh surge in 92% of the study goats within 4 days after the introduction of males, with a 98% ovulation rate.4 progesterone treatment coupled with the introduction of males eliminated the drawback of a short infertile estrus observed in response to using the male effect only to synchronize estrus.4,40 however, with the increasing demand for hormone and drug-free animal products, combined use of the male effect and photoperiodic manipulation would be a natural method to synchronize estrus and ovulation. the combined use of the male effect and hormones is emerging as the most effective tool for estrus synchronization in small ruminants. the male effect is more cost effective than hormonal methods and could be very beneficial for developing countries. however, studies are needed to evaluate the effects of breed and geographical location on the outcome.41 estrus synchronization using progestogens progestogens alter  the  secretion  of  lh by negative feedback, modifying the activity of hypothalamic gnrh. a surge in lh is observed after progesterone withdrawal.42 the progesterone source can be natural progesterone impregnated in silicon elastomers (e.g. cidr), sponges,  or  implants.  additionally,  synthetic  progesterone  analogs  such  as  norgestomet,  fluorogestone  acetate (fga), and medroxyprogesterone acetate (map) can  also  be used. natural progesterone is available as a controlled internal drug-releasing device (cidreasi-breed®) that is the only approved marketed progestogen treatment in the usa. progesterone is used in cidrs at the dose of 330 mg. fga is used at a dose of 45 mg in goats, 30 mg in ewe lambs, and 40 mg in ewes. map is used in sheep for 14 days as sponges containing 40, 50, or 60 mg. norgestomet (3 mg) is used as a subcutaneous implant. oral megestrol acetate (mga) is used in sheep at 0.125 mg twice daily for 8-14 days. estrus is observed on average 48 (range: 40-60) hours after removal of the progestogen.43 progesterone therapies are given for either short (5-7 days) or long (12-16 days for sheep; 14-21 days for goat) periods. progesterone treatment for 12-14 days is the most common application.44 the cidrs, or progestogen-impregnated sponges, are placed intravaginally. approximately 48 hours after the device/sponge removal, ewes exhibit estrus. artificial insemination can be performed 48 to 54 hours after progesterone removal. in general, progesterone treatment is recommended for 16-17 days during breeding season and 14 days during nonbreeding season for goats.45 the short-term progestogen treatment requires a luteolytic treatment because the duration of the treatment is shorter9 than the lifespan of cl. during the nonbreeding season, hormonal induction of follicular growth at progesterone removal is required to stimulate and increase endogenous pituitary gonadotropin production. such stimulation in cyclic animals enhances the ovulation rate. intravaginal progesterone delivery is associated with vaginitis with purulent vaginal discharge. intravaginal progesterone blocks the vaginal secretions, thus affecting vaginal microflora and stimulating the proliferation of microorganisms in the vagina.46 this vaginitis is not associated with infertility. the cidr devices result in a lower incidence of vaginitis, vaginal discharge, and drawstring breakage compared to sponges by allowing drainage of vaginal fluids.47–49 http://dx.doi.org/10.58292/ct.v17.11653 28 citation: clinical theriogenology 2025, 17, 11653, http://dx.doi.org/10.58292/ct.v17.11653 the most commonly used commercial progestogen sponges are fga (20 mg/sponge) and map (60 mg/sponge). the most commonly used dose of map is 60 mg, whereas the dose of fga is 40 mg. use of intravaginal sponges for either a short (5-7 days) or long (12-14 days for sheep; 14-16 for goat) period during the breeding season resulted in a similar estrous response.48 estrous response and pregnancy rate in merino ewes synchronized with various amounts of map (40, 50, and 60 mg) were similar.50 recommended doses of either map or fga did not affect fertility, pregnancy, or fecundity/lambing rates.51 fga treatment (11 versus 13 days) with 5 combinations of 10 mg of pgf2α, 330 iu of ecg, and 6 µg gnrh had no significant difference on estrus rate, sponge loss rate, vaginitis rate, total percentage of estrous ewes, conception rate, single lambing rate, twinning rate, and multiple lambing rates.52 vaginal sponges containing 30 or 60 mg of map were used for 6 or 12 days; estrus rate and pregnancy rate were higher for the 12 days treatment, independent of dose.53 the controlled internal drug release (cidr), an inert silicone elastomer molded over a nylon core, was designed in new zealand in the late 1980s.54,55 plasma progesterone concentrations peak 3 days after insertion and then gradually decrease.44 using either short (5 days) or long (14 days) estrus synchronization protocols is possible. short protocols are gaining popularity due to reduced progesterone concentrations observed when cidrs are placed for more than 7 days. to ensure cl absence, an initial dose of pgf2α is recommended at cidr insertion. in lengthy cidr protocols, pgf2α may be given 24-48 hours prior to the removal of cidr or completely omitted if the duration of treatment is equal or longer than the cl lifespan. estrous response was higher in ewes receiving cidr treatment for 9 and 12 days than those receiving treatment for 3 and 6 days.56 estrus synchronization using cidr and map had no significant difference in the average time to estrus expression (23 versus 21 hours, respectively), estrus and lambing rate.51 also, the estrus synchronization outcome in ewes was similar for fga and cidr.49 there was no significant difference in either estrus or conception rates among the ewes treated with cidr, fga, or map-based synchronization protocols.57 mga is an oral progesterone with a daily dose of 0.25 mg/ head/day, fed as 0.125 mg/head twice daily. the estrus induction was considerably lower (62 versus 89%) in ewes that received mga treatment for 9 days compared to those that received mga treatment for 12 days. however, the pregnancy rate was not different between groups (41 versus 44%).58 use of gonadotropins in progestogen-based protocols the initiation of pituitary activity after progestogen therapy can be expedited with chorionic gonadotropin (ecg), fsh or pg600® (merck animal health, summit, nj) treatment. gonadotropins have a crucial part in synchronization protocols by stimulating and achieving synchrony in the growth, maturity, and ovulation of follicles. the most commonly used hormone for induction of follicular growth and synchronization of ovulation in sheep and goats is ecg (figure 1). the dose of ecg used depends on the age, breed, and season, and varies (250-750 iu).44 higher doses (up to 1,000 iu) might be required for goats. several studies used 700-750 iu ecg, resulting in higher pregnancy and lambing rates, but estrus or pregnancy rates did not differ using either 300 or 600 iu ecg during the nonbreeding season.59 the unavailability of ecg in the usa has led to the off-label use of pg600 for estrus synchronization in small ruminants, especially ewes. pg600 is a gonadotropin preparation approved for estrus synchronization in sows. pg600 contains 80 iu/ml ecg and 40 iu/ml human chorionic gonadotropin that mimic fsh and lh, respectively. for anestrous ewes, pregnancy rates are higher for ewes that received 2.0 to 3.5 ml of intramuscular pg600 than receiving less < 2.00 ml or > 3.99 ml.60 in cyclic ewes, the pregnancy rate was significantly lower for ewes receiving 5 ml pg600 than ewes receiving 1.5 ml pg600. pregnancy rates were similar for ewes receiving 1.5 ml pg600 or normal saline.61 high pg600 doses lead to overstimulation of ovaries, increased follicular size, increased estradiol-17β concentrations at the time of ovulation and reduced fertilization rate.62 although not commonly used, porcine fsh (pfsh) can be injected as a single injection of 68 mg (folltropin®) 12 hours prior to progesterone removal.63 prostaglandin f2α and d-cloprostenol prostaglandin f2α (dinoprost tromethamine) or its anolog (cloprostenol) (pgs) act by inducing luteolysis and thereby figure 1. cidr/progestogen bases estrus synchronization protocol for small ruminants. artificial insemination with fresh semen can be performed 50-55 hours after cidr removal. laparoscopic artificial insemination should be performed 55-60 hours for frozen semen and 52-60 hours for fresh semen after cidr removal. in the usa, ecg can be substituted with pg600. http://dx.doi.org/10.58292/ct.v17.11653 citation: clinical theriogenology 2025, 17, 11653, http://dx.doi.org/10.58292/ct.v17.11653 29 increasing lh pulse frequency, causing a significant increase in estradiol from the dominant follicle and induction of estrus and ovulation. pgs are only effective during the breeding season with an active cl. the sheep cl is responsive to pgs on day 3, whereas the goat cl is responsive on day 5 after ovulation (day 0). estrus is observed 2-5 days after pgs treatment during the luteal phase.64 the interval from pgs treatment to onset of estrus depends on the stage of the follicular wave at the time of injection. pg treatment can result in impaired follicular function and asynchronous ovulation.15 to better synchronize estrus and remove the variability due to the ineffectiveness of pg treatment during the early and late luteal phases, a double pg protocol has been proposed. two pg injections are given 9-11 days apart (figure 2). most animals will be in the mid-luteal phase of the estrous cycle and will respond better to the second treatment. in goats, the second injection can be given 7-16 days apart.65 the fertility following single and double injection protocols is lower than that of progesterone-based protocols. this decrease in fertility is due to the negative influence of mid-cycle high progesterone on final follicular maturation, synchrony of ovulation after the second pg injection, and poor luteogenesis after estrus. injecting a second dose in the early or late luteal phase results in higher estrus intensity and ovulation rate. injecting the second dose at (hypothetical) day 5 of the induced diestrus (day 7 after first injection of pg) is recommended. this avoids animals being on the first or second day of the cycle and has a similar effect (luteolytic efficiency, percentage, and timing of estrus onset, preovulatory production of lh, ovulation, and functionality of subsequent corpora lutea) as the 3rd day of diestrus. most ewes will be in estrus within 30-48 hours, whereas does will be in estrus within 45-48 hours after the second injection. although timed ai is possible at 55 hours, fertility is variable.44 one study in tropical hair sheep had a higher response following 2 injections (9 days apart) of d-cloporstenol compared to dinoprost tromethamine (87 versus 57%); however, there was no difference in pregnancy rate.66 several other studies reported equal efficacy of dinoprost tromethamine and d-cloprostenol in inducing estrus in small ruminants. giving 2 pgf2α injections (d-cloprostenol; 0.15 mg; 10 days apart) along with gnrh (4.2 mg buserelin) before the first pgf2α treatment resulted in a higher lambing rate.67 the combination of pgf2α during the early luteal phase with the male effect may be an adequate alternative for synchronizing estrus prior to artificial insemination in the absence of previous estrus detection.44 melatonin melatonin has been used in several countries since the 1980s for ovarian stimulation in small ruminants.68 melatonin increased ovulation rate, lambing rate, progesterone synthesis and embryo survival.69 gnrh secretion started to increase on day 40, and gnrh and lh pulses increased from day 74 after melatonin treatment.44,70 melatonin enhances the release of gnrh and lh during anestrus by decreasing tyrosine hydroxylase activity and, consequently, the release of dopamine in the median eminence. the dopaminergic system has a crucial role in estradiol suppression of lh secretion during seasonal anestrus. therefore, decreased dopamine secretion during anestrus is linked to enhanced reproductive activity.71 melatonin implants (melawin®, regulin®) release melatonin continuously throughout the day (> 100 pg/ml of plasma) without affecting the night release and thus, create a short-day-like effect. the 18 mg melatonin implant releases effective melatonin concentration for 90-100 days after the implantation. each ram receives 3 melatonin (18 mg) implants subcutaneously at the base of the ear and is removed from the flock. ewes receive single implants 7 days later than rams, and 40 days after the sheep implants, rams are returned to the flock. the timing of melatonin use depends on geographical location (table 1).44 melatonin also increases litter size in sheep by 15-30%; however, the mechanism is not fully understood as melatonin receptors are ubiquitous. for sheep, melatonin treatment is recommended around the spring equinox. pretreatment with long daylight hours after the winter solstice enhances the efficacy of melatonin treatment in goats. the light treatment is recommended 06:00-09:00 and 22:00-24:00 per day.44 some of the commonly used protocols for estrus synchronization in sheep and goats are summarized in table 2. superovulation in small ruminants superovulation (also known as ovarian superstimulation) involves the targeted delivery of gonadotropins at a precise time point of the estrous cycle to increase the number of follicles reaching maturation and ovulation. the primary gonadotropins used are equine chorionic hormone (ecg), fsh, or a combination of both.79 the superovulation response is affected by the timing of the treatment, the type of hormone, breed, age, season, geographical location, and individual characteristics.79 multiple protocols for superovulation have been documented in sheep and goats. the most commonly used methods involve table 1. recommendations on the timing of melatonin use for estrus synchronization in small ruminants geographical location effective timing of insertion of melatonin implant more northern than n45° or more southern than s45° around summer solstice more southern than n40° or more northern than s40° spring equinox mediterranean region more southern than n35° or more northern than s35° after winter solstice latitudes between n20° and s20° limited efficacy (breeds are nonseasonal) figure 2. protocol for estrus synchronization in small ruminants using 2 doses of pgf2α http://dx.doi.org/10.58292/ct.v17.11653 30 citation: clinical theriogenology 2025, 17, 11653, http://dx.doi.org/10.58292/ct.v17.11653 the manipulation of the estrous cycle through progestogen therapy and gonadotropins treatment at a specified time.80 the progestogen treatments are comparable to those employed for estrus synchronization and can vary in duration. successful superovulation requires precise control of follicular waves. gonadotropin treatment has to be initiated before follicular deviation. various hormonal techniques have been investigated to decrease the variability in response to ovarian stimulation by gonadotropins. these methods encompass immunization against androstenedione or inhibin, synchronization of ovulation for superovulation during first wave. pretreatment with a gnrh antagonist from day 10 to fsh treatment to prevent follicular dominance and progesterone– estradiol cotreatment is used to synchronize follicular wave  emergence.81 nevertheless, these techniques are hardly employed in practical applications and will not be addressed. superovulation using equine chorionic gonadotropin (ecg) the hormone ecg exhibits fsh and lh activity. it is the oldest technique for inducing superovulation in ruminants. the hormone is given at a dosage ranging 750-1,500 iu in goats and 1,000-2,000 iu in sheep, commonly 1 or 2 days before progestogen removal; ecg-based superovulation protocols are no longer used due to significant fluctuations in response.82 the poor results are likely due to the elevated lh activity of ecg and its extended half-life. the strong lh activity results in the early initiation of oocyte meiosis. the prolonged halflife causes the recruitment of follicles that are not uniform in size. moreover, ecg-based superovulation disturbs the hormonal balance that affects the transfer of gametes and decreases the rates of ovulation and fertilization. the efficacy  of ecg treatment diminishes with repeated treatment, primarily because of the development of anti-ecg antibodies.83,84 superovulation using follicle stimulating hormone superovulation with fsh results in higher fertilization rates and embryo recovery. the fsh commonly used in practice for superovulation in ruminants is either of ovine (ofsh) or porcine (pfsh) origin. these preparations are made from pituitary gland extracts and remain costly. recombinant fsh formulations have been studied, although they are not yet readily available. fsh has a short half-life, necessitating repeated treatment to induce follicular stimulation. fsh is given in decreasing doses every 12 hours throughout the final 3 or 4 days of the progestogen therapy. the optimal dosage of fsh for sheep and goat varies among studies. comparison is often difficult because of the difference in measuring unit. the most reported doses are 10-14 μg of pure fsh (equivalent to 16-20 mg in armour units, or 200 mg in nih-fsh-p1 units). the dosage of fsh should be tailored according to the breed.82 prolific breeds typically exhibit a stronger response to smaller fsh doses. for instance, in goats, an adequate dosage of fsh (1 armour unit can contain 9.4-14.2 iu) ranges from 6-9 mg. the efficacy of porcine-derived fsh diminishes with repeated superovulation. the number of injections can be reduced by using a slow-release fsh preparation diluted in hyaluronic acid or an aluminum hydroxide gel.85,86 common superovulation protocols used in practice are illustrated (figures 3, 4, 5 and 6). in goats, to prevent the effect of luteal regression on embryo yield, a progestogen sponge/cidr must be reinserted after last ai or mating. this treatment has also been found beneficial in ewes87 in the short superovulation protocol, progestogen treatment lasts 6 days. the fsh treatment begins on day 4 and continues for 8 injections (36, 36/36, 34/34, 24/24, 16 mg) with pgf2α treatment in the evening of the day of progesterone withdrawal. the females are mated or artificially inseminated after estrus detection. a dose of gnrh is given on the morning of day 8. progestogen treatment is from day 10 until the embryos are harvested, especially in goats to counteract the regression of the cl. superovulation with equine chorionic gonadotropin/ follicle stimulating hormone combination several protocols combining fsh and ecg have been described. typically, ecg is injected in a low dose (200 iu) at the same time as the penultimate injection of the fsh.81 however, this is not a commonly practiced in the usa because of the unavailability of ecg. table 2. some of the commonly used protocols for estrus synchronization in sheep and goats protocol species synchrony and/or fertility results gnrh and pgf2α 72 sheep 20% lambing rate gnrh, p and pgf2α 72 sheep 47% lambing rate map and ecg41 sheep 32.5% lambing rate 90% estrus synchrony fga and ecg73 sheep 80% ovulation rate artificial light, p4 coupled with male effect4 goat 98.7% fga, ecg, pg and male introduction74 goat 66.7% estrus behavior male introduction, fga, ecg, and pg74 goat 71% estrus behavior long day, melatonin, and male effect75 goat 100% ovulation rate 81.3% pregnancy rate ovsynch and timed ai76 goat 58% kidding rate p4 sponges77 goat 100% estrus behavior 87.5% pregnancy rate fga, pg, ecg73 sheep 80% ovulation rate pg, fga, ecg73 sheep 100% ovulation rate pg, fga, gnrh73 sheep 60% ovulation rate gnrh, pgf2α, ecg73 sheep 80% ovulation rate map (7 days) + gnrh and pgf2α 78 goat 88% estrus rate 84% pregnancy rate map (12 days) + pmsg and gnrh78 goat 80% estrus rate 72% pregnancy rate fga + pgf2α + ecg + gnrh52 sheep 80% conception rate 3% lambing rate http://dx.doi.org/10.58292/ct.v17.11653 citation: clinical theriogenology 2025, 17, 11653, http://dx.doi.org/10.58292/ct.v17.11653 31 prediction of superovulation response with anti-müllerian hormone anti-müllerian hormone (amh) concentrations are indicative of ovarian follicular reserve.88 the amh can be a potential marker to predict the outcome of superovulatory treatment and to adjust the fsh dose before starting the fsh treatment. serum amh concentrations are positively correlated with the number of ovulations in the sheep and goat.89,90 fertilization and embryo recovery rates in ewes with figure 3. long protocol for superovulation in ewes. the progestogen treatment is for 14 days but the cidr is replaced at 7 days combined with pgf2α treatment. the fsh treatment lasts 4 days starting on day 12. the female is either bred naturally or artificially inseminated after estrus detection. the dose of fsh varies (200-250 mg) depending on the species and breed and given in a series of injections 20/20%, 15/15%, 10/10%, 5/5%. the cidr is removed in the evening on day 14 and an injection of the ecg (200 iu) can be given. figure 4. short protocol for superovulation. progestogen treatment lasts 6 days. fsh treatment begins on day 4 and 8 daily injections (36/36, 36/36, 34/34, 24/24, 16 mg) are given until pgf2α treatment in the evening of the day of progesterone withdrawal. the females are mated or artificially inseminated after estrus detection. a dose of gnrh is given on the morning of day 8. progestogen treatment is given from day 10 until the embryos are harvested, especially in goats to counteract the regression of the cl. figure 5. long protocol for superovulation in goats. progestogen treatment lasts 17 days. fsh treatment continues days 15-18 (50, 25/25, 25/25, 25/25 mg). ai is performed 36 and 48 hours after cidr removal. http://dx.doi.org/10.58292/ct.v17.11653 32 citation: clinical theriogenology 2025, 17, 11653, http://dx.doi.org/10.58292/ct.v17.11653 low serum amh can be enhanced by increasing to an 8-dose fsh treatment protocol instead of 6 doses. in ewes with high serum amh concentrations, the embryo recovery rate is not different regardless of the number of doses of fsh treatment.89 however, ewes with low amh have low follicular reserve and high circulating fsh concentrations. the refractoriness of the granulosa cells to fsh is responsible for a higher fsh dose required for higher superovulatory response in the ewes with low amh.91 conclusion estrus synchronization in ewes can be accomplished by manipulating the timing of estrus through progesterone and/ or prostaglandin treatment. these hormones are commonly coupled with gonadotropin, especially during the nonbreeding season. initial research on synchronization protocols relied on progestogen treatment alone, but that has been refined throughout the years. progestogens are still used as the primary method for estrus synchronization, particularly when timed ai is used. progestogens can be used during and outside the physiological breeding season, whereas pgs are effective only for in-season estrus synchronization. the various commercial preparations of progestogens are equally effective for estrus synchronization in small ruminants. not all hormonal products are available in all countries. if ecg is not available, pg600 may be used; however, there is still a need for studies to evaluate proper dose for different breeds. in general, pg600 is not as efficacious as ecg. the efficiency of estrus synchronization protocols may differ based on the specific breed of sheep. legal regulations and thus availability of certain hormonal products in various countries and consumer demand for organic products highlight the need for nonhormonal approaches (e.g. male effect). the male effect and photoperiod manipulation are a great option to avoid problems with residue and for organic farming. there is an increased demand for in vivo and in vitro production of embryos. the fsh-based superovulation protocols work well but need to be adjusted for each breed and individual. the discovery of the relationship between serum amh concentrations and potential response to superovulation may help in refining the fsh dose for superovulation. finally, none of these techniques for estrus synchronization or superovulation can be efficient unless other aspects of flock/herd health including nutritional flushing, 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https://iav.ac.ma/fr/reproduction-des-petites-ruminants-tome-2-la-femelle https://iav.ac.ma/fr/reproduction-des-petites-ruminants-tome-2-la-femelle https://doi.org/10.1095/biolreprod60.4.805 https://doi.org/10.1095/biolreprod61.1.209 https://doi.org/10.1016/j.ijpharm.2020.119904 https://www.researchgate.net/profile/jakub https://doi.org/10.1016/j.anireprosci.2023.107273 https://doi.org/10.1016/j.anireprosci.2023.107273 https://doi.org/10.58292/ct.v14.9139 https://doi.org/10.1016/j.theriogenology.2024.01.024 https://doi.org/10.1016/j.theriogenology.2024.01.024 https://doi.org/10.1530/rep-11-0211 https://doi.org/10.1071/rd11020 2009: theriogenology experiences and reflections the 2009 bartlett address 1 2 theriogenology experiences and reflections 3 b.e. seguin 4 professor emeritus, college of veterinary medicine, university of minnesota, st 5 paul, mn, usa 6 7 dr. bartlett’s influence 8 to start this story i go back to my first year in veterinary school at the 9 university of minnesota. i worked in the anatomy department, and one of my 10 duties was to make bone preparations for teaching and display purposes. one 11 day i was asked if i would like to take on some extra work in this area and if so i 12 should contact a dr. bartlett at american breeders service (abs) in deforest, 13 wi. i did not know dr. bartlett but i certainly knew of the abs company, and 14 was therefore very excited to find out what this might be about. 15 now i’d like to go back several years before to my teenage years in my 16 hometown of alma center in west-central wisconsin. i was a “city-kid” if that is 17 possible in a town of some 400 people. my dad introduced me to all kinds of 18 animals but he especially liked birds and we raised chickens, ducks, pheasants 19 and homing pigeons. i was more interested in dairy cattle. all six of the major 20 breeds were raised on farms in our area. but guernseys were my favorite as 21 they were raised by my good friend fred moseley and on the seguin family farm 22 near eau claire, wi. as artificial insemination (ai) increased in popularity, i 23 21 became intrigued with bull studs, semen technology and the work of ai 24 technicians. so i was well aware of the abs company. the local veterinarian 25 lived just a few houses away. i found his practice in general, but especially the 26 reproductive part, compelling. so my intention for going to veterinary school was 27 to work with cattle, and especially cattle reproduction in dairy practice or possibly 28 the ai industry. 29 so i was very excited to find out what dr. bartlett had in mind. (wouldn’t a 30 four-year scholarship be wonderful!)? as you can guess, what he was looking for 31 was someone to provide pelvis preparations for use in their training programs for 32 ai technicians. i was able to provide the specimens so my first paycheck for 33 theriogenology work was thanks to dr. bartlett! but the real reward for me was 34 the chance to get to know the veterinary staff at abs: drs. david bartlett, les 35 larson and willis parker. from that point on, the possibility of a career similar to 36 theirs was my motivation whenever needed to get through veterinary school. a 37 fact that is not well known is that dr. bartlett was the first head of the ob/gyn 38 department in the college of veterinary medicine at the university of minnesota. 39 in 1984 dr. bartlett gave the first of these addresses at this same meeting in 40 denver, colorado. i encourage you to read that paper as it is a classic reflection 41 of dr. bartlett’s professionalism, intellect and dedication to our profession and 42 specialty.1 so i am deeply honored to speak to you today as this is the 25th 43 anniversary of the first bartlett address given of course by dr. bartlett himself. 44 student at the university of minnesota veterinary college 45 22 as veterinary students at minnesota in the 1960’s most of us had heard 46 the legend of dr. ray zemjanis long before we were to have him as a teacher. 47 he was on sabbatical for our 3rd year when we started our theriogenology 48 courses. however we were not shortchanged, as a very capable group remained 49 to carry on: dick schultz, mel fahning, louis archibald, charles gibson, dennis 50 copeland, john ellery and bob wescott. they were all superb teachers, strong 51 mentors and excellent theriogenologists. during that year we took cow palpation 52 lab, which included several early morning trips to the bartusch slaughterhouse 53 about 3 miles from campus. on these trips cows to be slaughtered that morning 54 were palpated and findings recorded by each student. the reproductive tracts 55 were recovered and returned to the therio lab for review (and grading) that noon. 56 this nearly instant feedback helped us master the art and science of cow 57 palpation and reproductive physiology. this slaughterhouse resource was the 58 backbone of the minnesota program and where we really learned the techniques 59 described in the zemjanis textbook diagnostic and therapeutic techniques in 60 animal reproduction.2 ray zemjanis returned as we started our 4th year. those 61 of you who knew him you can imagine the surprise we experienced that first day 62 in lecture. even though his reputation had proceeded him and we knew we could 63 learn a great deal from this man, his hardcore, old school, no nonsense approach 64 was a shock to say the least. if we answered when doing cow palpation with “i 65 think i found ------“, he would respond, “your clients won’t care about what you 66 think you found, they expect you to know what you found.” of course he 67 23 was right and we gained great respect for the teaching system he established 68 and the principles he professed. 69 colorado state university 70 toward spring of my 4th year, a notice was posted that the veterinary 71 college at colorado state university (csu) was looking to fill an internship 72 position in bovine reproduction. anyone interested should contact dr. ed carroll 73 at the “bull farm”. that address sounded perfect! i went to the library to see 74 what i could learn about dr. carroll and found his paper on bse of 10,940 beef 75 bulls,3 which was the basis for the formation of the group that would become the 76 society for theriogenology. i applied and was fortunate to be offered the 77 position. the bull farm was an exciting place where i worked with drs. carroll, 78 les ball and lloyd faulkner. we did lots of bull work and more cow work (both 79 beef and dairy) than i had imagined. also present at csu were dr. bill pickett 80 with his equine research program and dr. jim wiltbank working on beef cow 81 reproductive management systems. their detailed approach to controlled 82 experimental design in animal reproductive studies made a strong impression. i 83 had many “interesting” mornings with dr. pickett and his graduate students 84 collecting semen in their seasonality study in stallions and in a boar project. 85 these experiences plus a growing concern at that time about seminal vesiculitis 86 in bulls started me thinking that research in bovine reproduction and possibly 87 even graduate school might be something to consider. dr. carroll and i did a 88 small trial on seminal vesiculitis in yearling bulls in the hopes of creating the 89 24 basis for a possible graduate degree project. [unfortunately ed’s life was far too 90 short; he was a valuable resource to our profession.] the days at csu flew by. 91 michigan state university 92 fortunately for me there was a bovine theriogenology residency/ms spot 93 at michigan state university with drs. dave morrow, wayne oxender and fayne 94 oberst. i got off to rocky start with dr. morrow when he wanted me to write an 95 application for an nih postdoctoral fellowship. my first reaction was to question if 96 a veterinarian was even eligible, but indeed we are (or were). about the last 97 thing i thought i wanted to be doing was to be confined to a desk when there 98 were field trips, clinic cases and experiments to be done. but i did the 99 application and have been grateful to dr. morrow ever since as it was funded 100 about 2 years later. my master’s thesis was on the cycle-altering effects of 101 endometrial irritation in cows,4 which started my career-long search for better 102 control of the timing of estrus and ovulation in cattle. two review papers by dr. 103 o.j. ginther were available on the utero-ovarian relationship in cattle.5,6 i still 104 feel these should be must read literature for graduate students and/or residents 105 in theriogenology. at that time we were still talking about the uterine luteolysin 106 and the luteolytic factor, but this would soon change. 107 the dairy science department at msu had a very active reproductive 108 physiology group that included drs. harold hafs, alan tucker, ed convey, jack 109 britt and wayne oxender plus a large number of graduate students. it was the 110 exciting place to be. i was accepted into a ph.d. program with the dairy science 111 group with wayne oxender as my advisor. graduate students were officed six 112 25 per room so there were many stimulating discussions. there were always lots of 113 trials being planned and conducted and many needed the assistance of a 114 veterinarian so my days were always busy. 115 if you know wayne oxender, you know that something interesting was 116 always going on when he was around. in those days, select sires and michigan 117 animal breeders had a bull stud on the edge of campus. they allowed us to use 118 25 two-year-old holstein bulls housed in tie stalls for our experiment. the 119 purpose was to study five anterior pituitary hormones possibly involved in the 120 process by which false mounting bulls increases the number of sperm per 121 ejaculate. each bull was treated and then bled via the tail vein eight times in a 122 24-hour period. that barn was all muscular energy, testosterone and flying 123 hooves. somehow we all survived and i was able to present the project at the 124 asas meeting in lincoln, ne. 125 prostaglandin! today the activity of prostaglandin f2α (pgf2α) in cattle 126 is taken for granted but in 1972 prostaglandin was pretty much unheard of. one 127 day at the weekly graduate seminar tom louis reviewed some recent work in lab 128 animals that showed some negative effects on corpus luteum (cl) function by a 129 compound, which was thought to be what we had been calling the uterine 130 luteolysin. as i remember many of us present thought “this is a dairy science 131 department, what in the world does this work have to do with cows?” little did 132 we appreciate where this was going to take us. shortly thereafter, dr. hafs went 133 to visit dr. jim lauderdale at the upjohn company about the possibility of some 134 trials with a new compound that was pgf2α. soon tom louis, john stellflug, 135 26 and i were sent to the university’s dairy to select heifers and cows for the initial 136 trials with this product. late one night we were in the research barn getting 137 everything ready for the start of the project when the manager of the dairy farm 138 walked through. it must have been obvious to him that we could hardly contain 139 our excitement. he asked what company was sponsoring the project. we were 140 only thinking about how the project would get us started on our graduate work 141 but he was looking for an investment opportunity! in the first trials cows were 142 treated by intrauterine infusion in the ipsilateral vs. contralateral horn to the cl 143 and the heifers by intravaginal deposit vs. im injection in diestrus vs. metestrus. 144 there were no adverse reactions and to our amazement, the cls that were 145 present at the time of treatment were soon disappearing. when the heifers went 146 out for heat detection on the third day after treatment they lined up in standing 147 heat like elephants in the circus. these two projects resulted in two of the first 148 publications on the action of pgf2α in cattle.7,8 many other experiments with 149 pgf2a followed to further define dose effects, routes of administration, intervals 150 to estrus and ovulation, etc. then it was time for fertility trials where we had very 151 good results.9 a review of potential uses of pgf2α in cattle was presented at the 152 first sft conference by wayne oxender.10 153 there were some other pgf2α experiments and experiences with 154 unexpected events. several surprises occurred at the chatham experiment 155 station in the upper peninsula of michigan where we used beef cattle for one of 156 the first pgf2α fertility trials. the first was that we would take a commercial 157 plane to chatham, which sounded great, but it was a very bumpy ride in a small 158 27 commuter plane and it made 3 or 4 stops! a second surprise was the weather. 159 we left lansing on a beautiful spring day but in the up it was still winter with 160 more than a foot of snow still on the ground. another surprise was the cattle. 161 some of you will remember the chianina beef breed that was imported from italy 162 for crossbreeding to add some size to our native beef cows. it turned out their 163 other highly heritable traits were how high they could jump and how slow they 164 were to reach puberty. the heifers used were ½ and ¾ chianina yearlings. 165 although well grown and in good condition, many were not yet cycling and they 166 all could jump. it was a long day. you have probably seen the warning on the 167 lutalyse® bottle that says exposure to this product may cause breathing 168 problems or an asthmatic attack in some people. i have never seen such a 169 problem with the commercial product but for these first trials pgf2α came to us 170 in powder form that we had to dissolve in pss. i had usually done this in a large 171 well-ventilated room but that day i did it in a closed up car. as i mixed the two, 172 some of the pgf2α powder became airborne and i experienced a weird 173 respiratory episode. this did not last long but i was convinced of two things: 174 pgf2α is a potent substance and it is true that it’s half is very short. on our last 175 trip to chatham an experiment station cowboy named blacky took us coho 176 fishing in a local river. when we could not get a bite by hook and line, he just 177 illegally used a dip net to catch a few under a state highway bridge. 178 there was also some mare reproductive research done in our group. pat 179 noden was another of the graduate students. her ph.d. thesis was on using 180 pgf2α for estrus control in mares.11 the day the first mare was treated i was at 181 28 the campus dairy barn. i received a phone call that there was an emergency at 182 the bennett farm where the research horses were kept. by the time i was 183 contacted and got over there, all was quiet and no one was around. the only 184 thing that seemed unusual was a large wet spot in the treatment area! while we 185 had never seen any side effects from pgf2α in cattle, pat had just seen the first 186 sweating reaction in a mare caused by pgf2α treatment. this reaction in mares 187 to pgf2α is well known now but was a shock that day. 188 another surprise occurred when dr. hafs wanted to try pgf2α in bulls. he 189 had a research interest in sperm output and collection efficiency of ai bulls. he 190 wanted to see if an injection of pgf2α might mimic the affect seen with false 191 mounting. the initial trials were done in rabbits where the most obvious affect 192 was a marked increase in fecal expulsion. 193 one disappointing aspect of the early pgf2α work for me personally was 194 that we did not have approval to do research trials in lactating dairy cows. this 195 was my major interest area and where most of us thought the greatest use would 196 occur. 197 gonadotropin releasing hormone: at the same time that the pgf2α 198 research was starting at msu, drs. jack britt and ed convey and several 199 graduate students were studying uses of gonadotropin-releasing hormone 200 (gnrh) in cattle reproduction. an early project by britt et al. described the basic 201 effect of gnrh on lh release and showed great promise for the use of gnrh to 202 induce early postpartum ovulation and cl formation in cows.12 another project 203 that became my ph.d. thesis compared the luteotropic effects of hcg and gnrh 204 29 in normal cattle and in dairy cows treated for ovarian follicular cysts.13,14 an 205 interesting part of this work happened when several bovine practitioners were 206 asked to find cows with follicular cysts for this trial. even though it was specified 207 that the cysts had to be follicular in type, 40% had enough progesterone to be 208 luteal cysts if not normal cls. when in 10 to 14 days i went to examine and treat 209 the cows, most of that 40% no longer had the ovarian structure originally thought 210 to be a follicular cyst. a review of potential uses of gnrh in cattle was presented 211 at the first sft conference.15 212 university of minnesota faculty 213 with completion of my graduate program at msu, it was time to find a real 214 job. back at the university of minnesota there was a temporary position to be 215 filled while ray zemjanis went to africa to teach for two years. the 216 theriogenology group had changed greatly since my graduation. now ed 217 mather, howard whitmore, borje gustafsson (newly hired from sweden) and 218 bob wescott were the faculty, and residents included shirley johnston, john 219 hurtgen and rolf larsen. also working closely with the theriogenology group 220 were the reproductive physiology group from the department of animal science: 221 ed graham, bo crabo, alan hunter and jon wheaton. members of both 222 departments participated in the theriogenology graduate program, in which 12 223 to 15 veterinarians were enrolled. later ed mather moved to michigan state 224 university and borje gustafsson and howard whitmore went to the university of 225 illinois. they were replaced by ray zemjanis returning from africa, norm 226 williamson from the university of melbourne and myself. after completing her 227 30 ph.d., shirley johnston joined the faculty as small animal theriogenologist. dr. 228 mel fahning returned to the program after several years in private practice and 229 bovine et business. these were rewarding times with an excellent teaching and 230 clinic program for veterinary students, and productive teaching and research with 231 veterinary residents and graduate students in theriogenology. 232 with a series of graduate students we continued studying applications for 233 pgf2α and gnrh in cattle. now we had two pgf2α products (estrumate® and 234 lutalyse®) to work with. despite ads to the contrary we could find no significant 235 difference in the luteolytic potency between these two products in dairy cows.16 236 we did however make some interesting observations on factors affecting results 237 with either product when treating unobserved estrus in dairy cows. we did 238 several fertility trials in cooperating private beef herds. cooperating farmers 239 would assure us that the entire herd was ready for the breeding season when we 240 planned for the first pgf2α injection. but a pretrial palpation would almost 241 always find at least a few pregnant cows! a good lesson for everyone involved 242 was that, at least in the first year of a pgf2α controlled breeding program for 243 beef herds, never treat cows prior to checking that some might still be pregnant. 244 we also looked at the efficacy of the pgf2α products to induce abortion in 245 feedlot heifers17 and causes of the variation in the timing of estrus after pgf2α 246 treatment. then the fun began when it became possible to do pgf2α 247 application trials in lactating dairy cows; first in individual cows with unobserved 248 estrus18 and later in herd breeding programs.19,20 today many dairy herds use 249 31 multi-product, multi-treatment programs for breeding that eliminate estrus 250 detection altogether. 251 one of the real highlights of my faculty career came in 1984-85 when i 252 was able to go on sabbatical leave at the swedish veterinary college. at that 253 time the ob/gyn department included drs. stig einarsson, kjell larsson, hans 254 kindahl as well as many excellent staff and graduate students. their courses, 255 facilities, clinical service and research were first class. i could not speak 256 swedish so i did very limited lecturing; rather i mostly helped the graduate 257 students with their research projects. my own research efforts that year were to 258 focus on measuring a pgf2α metabolite. this was thwarted by a small technical 259 detail that became a painful lesson. we changed to a new source of tubes for 260 blood collection without testing to see if the new tubes affected assay results. 261 after all samples had been collected, we learned that the new tubes somehow 262 interfered with the assay negating nearly a years’ work. my family and i will 263 never forget the hospitality and friendships we enjoyed that year. 264 we returned to minnesota to some changes. ray zemjanis retired and 265 was replaced by harry momont. norm williamson moved to the veterinary 266 school in new zealand and jerry olson joined us. shirley johnston moved into 267 administration. harry momont moved on to the veterinary school in wisconsin. 268 margaret root kustritz came on board to work in small animal theriogenology 269 and mats troedsson joined our group specializing in equine theriogenology. 270 one last trial 271 32 over the years we had done many projects looking at various means to 272 maximize control of the timing of estrus and ovulation in cattle. these included 273 endometrial irritation, estradiol cypionate (ecp), pgf2α, gnrh, hcg, removal of 274 the ovary containing the preovulatory follicle,21 etc. 275 the last experiments i undertook involved two protocols that were tested 276 for potential to improve ai submission and pregnancy rates achieved by using 277 pgf2α (lutalyse®, pfizer animal health, new york, ny, usa) in dairy cows with 278 unobserved estrus judged to have a pgf-responsive cl by ovarian palpation. in 279 expt 1, 151 cows were selected and treated with pgf2α. cows in the 280 experimental group were treated with 1.5 cc (3 mg) of ecp im at 30-34 hrs after 281 pgf2α while controls had no further treatment. significantly more cows treated 282 with pgf2α plus ecp were seen in estrus and ai’d within 6 days of pgf2α than 283 were cows treated only with pgf2α (91% vs. 53%). estrus behavior was more 284 tightly synchronized for cows treated with pgf2α plus ecp than for cows treated 285 only with pgf2α. resulting conception rates were similar (55% vs. 58%). 286 therefore pregnancy rate 6 days after pgf2α was higher for cows treated with 287 pgf2α plus ecp than for cows treated only with pgf2α (50% vs. 31%). in expt 288 2, 197 cows were similarly selected and treated but all cows were ai’d at 72-76 289 hrs after pgf2α without regard for estrus behavior. there was no significant 290 difference in pregnancy rates between groups, 40% for 98 pgf2α plus ecp 291 cows versus 35% for 99 cows treated only with pgf2α. results showed that a 3 292 mg dose of ecp 30-34 hrs after pgf2α treatment 1) can increase ai submission 293 and pregnancy rates in unobserved estrus dairy cows judged by palpation to 294 33 have a functional cl when ai’d based on detection of estrus, 2) can tighten the 295 estrus response pattern of responding cows, and 3) but did not improve 296 pregnancy rate results when cows were ai’d by appointment at 72-76 hrs after 297 pgf2α. 298 i did not publish this research although i believe it demonstrates significant 299 results for controlling estrus and ovulation with the combined use of pgf2α and 300 ecp. unfortunately, the work is now nearly irrelevant because of public health 301 concerns regarding estradiol in the human food supply and the fact that ecp is 302 no longer marketed for use in dairy cattle. nonetheless, these findings were a 303 rewarding culmination to 35 years of theriogenology research. i am pleased to 304 have had the opportunity to share them with you today. 305 graduate students 306 working with graduate students has probably been the most rewarding 307 part of my career. i totally enjoyed the chance to help them search for goals, 308 answers and their future. in fact most of these ladies and gentlemen gave more 309 to me than i gave to them. every one of them educated me in some way. i have 310 been able to visit many places and do many things. with ahmed tibary, i saw 311 great horsemanship and some of the world’s most beautiful arabian horses in 312 morocco. i saw beef production and the ai industry in argentina with miquel 313 fortin. i reviewed theriogenology curricula and graduate programs at 3 314 veterinary colleges in thailand with preeyphan udompresert. by the 315 recommendation of juan ramono, i visited dairy farms and gave ce lectures for 316 veterinary practitioners in uruguay. with translation help from stephano 317 34 romagnoli, i lectured at the veterinary school in pizza, italy and to practitioners 318 in sardinia. from ting q. zhang, i learned about china. when i started, my 319 world was quite small but by these relationships it is now much more complete. i 320 cannot list you all here but please know that i am grateful for the experiences we 321 shared. thank you! 322 retirement and beyond 323 bovine theriogenology and academia have been very good to me and my 324 family (wife trish, daughter ann, son tony, daughter-in-law sarah beth, and 325 granddaughter avery). i cherish the memories. after 35 years it was time to 326 walk away – while i could still hit a golf ball. since golf season is short in 327 minnesota, i soon needed another activity. it has been surprising what a 328 veterinary education and a theriogenology career prepared me for. for 6 years 329 now i have done corneal excisions on organ donors as an eye procurement 330 technician for the minnesota lions eye bank. this too has been an interesting 331 and rewarding experience. 332 if you have not already done so, i encourage you to register as an organ 333 donor, discuss your wishes with your next of kin and give the gift of life or sight to 334 someone in need. 335 references 336 1. bartlett de: theriogenology: from concept to actuality proc annu conf 337 soc therio 1984; p. 221-234. 338 2. zemjanis r: diagnostic and therapeutic techniques in animal 339 reproduction, 2nd ed. baltimore: the williams & wilkins co; 1970. 340 35 3. carroll ej, ball l, scott ja: breeding soundness in bulls—a summary of 341 10,940 examinations. j am vet med assoc 1963;142:1105-1111. 342 4. seguin be, morrow da, louis tm: luteolysis, luteostasis, and the effect 343 of prostaglandin f2α in cows after endometrial irritation. am j vet res 344 1974; 35:57-61. 345 5. ginther oj: utero-ovarian relationships in cattle: physiologic aspects. j 346 am vet med assoc 1968;153:1656-1664. 347 6. ginther oj: utero-ovarian relationships in cattle: applied veterinary 348 aspects. j am vet med assoc 1968;153:1665-1671. 349 7. louis tm, hafs hd, seguin be: progesterone, lh, estrus and ovulation 350 after prostaglandin f2α in heifers. proc soc exp biol med 1973; 143:152-351 155. 352 8. louis tm, hafs hd, morrowda: intrauterine administration of 353 prostaglandin f2α in cows: progesterone, estrogen, lh, estrus and 354 ovulation. j anim sci 1974;38:347-353. 355 9. lauderdale jw, seguin be, stellflug jn, et al: fertility of cattle following 356 pgf2α injection. j anim sci 1974;38:964-967. 357 10. oxender wd: prostaglandins in domestic animals. proc annu conf soc 358 for therio 1975. 359 11. noden pa, oxender wd, hafs hd: estrus, ovulation, progesterone and 360 luteinizing hormone after prostaglandin f2α in mares. proc soc expt biol 361 med 1974; p. 145. 362 36 12. britt jh, kittok r, harrison ds: ovulation, estrus and endocrine response 363 after gnrh in early postpartum cows. j anim sci 1974;39:915-919. 364 13 seguin be, oxender wd, brit jht: effect of human chorionic gonadotropin 365 and gonadotropin-releasing hormone on corpus luteum function and 366 estrous cycle duration in dairy heifers. am j vet res 1977;38:1153-367 1156,1977. 368 14. seguin be, convey em, oxender wd: effect of gonadotropin-releasing 369 hormone and human chorionic gonadotropin on cows with ovarian 370 follicular cysts. am j vet res 1976;37:153-157. 371 15. seguin be: applications of gonadotropin releasing hormone (gnrh) in 372 domestic animals. proc annu conf soc for therio 1975. 373 16. seguin b, momont h, baumann l: cloprostenol and dinoprost 374 tromethamine in experimental and field trials treating unobserved estrus in 375 dairy cows. bovine pract 1985;20:85-90. 376 17. refsal kr, seguin be: estradiol 17beta cyclopentylpropionate and 377 prostaglandin f2α for induction of abortion during the first trimester of 378 pregnancy in feedlot heifers. j am vet med assoc 1981;179:701-703. 379 18. seguin be, gustafsson bk, hurtgen jp, et al: use of the prostaglandin 380 f2α analog cloprostenol (ici 80,996) in dairy cattle with unobserved 381 estrus. theriogenology 1978;10:55-64. 382 19. seguin be, tate dj, otterby de: use of cloprostenol in a reproductive 383 management system for dairy cattle. j am vet med assoc 1985;183:533-384 537. 385 37 20. seguin be, momont hw, king vl, et al: breeding programs for dairy 386 herds using prostaglandin f2α products to save labor: field trial evaluation 387 of reproductive performance. bovine pract 1999;33:106-112. 388 21. fortin mr, seguin be, momont hw, et al: interestrous intervals in cows 389 after unilateral ovariectomy at estrus to prevent corpus luteum 390 development. theriogenology 1988;29:1357-1365. 391 38 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true 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setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 1 contact jenna dockweiler jenna.dockweiler@gmail.com © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12703, http://dx.doi.org/10.58292/ct.v17.12703 *abstract was presented at the society for theriogenology 2023 conference; (http://dx.doi.org/10.58292/ct.v15.10001) page 20. case report use of a direct-to-consumer genetic screening test for diagnosis of a chromosomal abnormality in a female dog* jenna dockweiler,a valeria rickard,b daniel leon-frank,b christopher premanandan,c adam boyko,d takeshi kawakamia aembark veterinary, inc., boston, ma, usa, bnorth oatlands animal hospital, leesburg, va, usa, cdepartment of veterinary biosciences, college of veterinary medicine, the ohio state university columbus, oh, usa, ddepartment of biomedical sciences, college of veterinary medicine, cornell university, ithaca, ny, usa abstract a 3-year phenotypically female siberian husky with primary anestrus, was screened for known genetic diseases using a direct to-consumer saliva test. dog had 2 distinct genetic profiles, suggesting a chimera or mosaic condition, ultimately confirmed with cytogenetic analysis. histopathology of the intraabdominal gonads revealed hypoplastic testicular and epididymal tissue. this case highlights the utility of a novel test that is both inexpensive and noninvasive to diagnose chromosomal anomalies in dogs. keywords: microarray, chromosomal anomaly, genetic, genetic test, dna test background this case highlights the clinical utility of genetic microarray -based testing to diagnose chromosomal abnormalities. currently, available tests for this condition are expensive and rely on blood or tissue samples to arrive at a diagnosis. given the increasing availability of noninvasive and inexpensive genetic testing options, this case offers valuable insights into applying such tests during the workup of suspected chromosomal anomalies in animals. case presentation a 3-year intact female siberian husky was genetically screened using a commercially available direct-to-consumer genetic test (embark® breed & health dog dna test, embark veterinary, inc., boston, ma, usa) to assess known disease-associated mutations before breeding. this testing was run on a dense dna genotyping microarray (illumina canine hd 270k genotyping array, illumina, inc., san diego, ca, usa) using a saliva sample to detect risk variants for 270 known genetic health conditions. genetic analysis failed to return results through the automated pipeline with 3 separate samples, suggesting structural abnormalities within dog’s genome. owner reported no signs of estrus had ever been noted in this dog, although a littermate had cycled thrice. at 6 months of age, the patient underwent a negative exploratory laparotomy for a suspected foreign body. no comment was made on the appearance of the internal reproductive tract at this time. at 14 months of age, the patient began to exhibit nocturnal enuresis. chemistry panel, cbc, and urinalysis were within normal limits. resting estradiol, progesterone, and testosterone concentrations were within normal limits for a spayed dog, an intact female dog, and a neutered male (table). testosterone concentrations were lower (< 15 ng/ml) compared to the reference range for intact males (19-2,630 ng/ml) (table). a right lateral radiograph revealed a suspected pelvic bladder (short urethra syndrome); phenylpropanolamine (1.2 mg/kg) once every 12 hours, was recommended. physical examination revealed juvenile mammary glands with indiscernible teats. external genitalia were consistent with that of a female, but close examination of the vulva revealed abnormal conformation with a possible rudimentary penile structure. genetic screening (embark® breed & health dog dna test) run on a customized caninehd 270k microarray genotyping platform (illumina, inc.) performed on a saliva sample could not identify disease-associated mutations for all health conditions tested due to difficulty detecting both paternal and mailto:jenna.dockweiler@gmail.com http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12703 http://dx.doi.org/10.58292/ct.v15.10001 2 citation: clinical theriogenology 2025, 17, 12703, http://dx.doi.org/10.58292/ct.v17.12703 maternal variants of all genetic markers on the microarray. a manual review of the fluorescent signal plots revealed abnormal cytogenetic patterns across all chromosomes. specifically, 4 copies of each autosome, 3 copies of each x chromosome, and 1 copy of the y chromosome were detected (figure 1), consistent with 2 distinct canine dna signatures, 1 male and 1 female, contributing to the sample. this pattern indicated xx/xy chimerism or mosaicism. treatment gonadectomy was recommended due to poor prognosis for fertility and possible neoplastic transformation of gonadal tissue. presurgical bloodwork (cbc, chemistry panel, pt, and aptt) was within normal limits. standard 3-port laparoscopy was used for abdominal exploration. the gonad-like structures caudal to each kidney did not have a discrete shape but appeared to have a blood supply like an ovarian pedicle and a suspensory ligament-like structure cranially (figure 2). the pedicle-like blood supply was cauterized, and each gonad was removed. broad ligament-like tissue extended toward the inguinal ring and uterus was not visualized. outcome surgically excised tissues were fixed in formalin and processed by routine methods for microscopic evaluation. small aggregates of interstitial cells and sporadic basement membranes (presumably of collapsed seminiferous tubules) were identified in the tissue excised from the left gonadal tissue. a separate region within the specimen contained small tubular profiles resembling epididymis. the tissue excised from the right contained similar tubules. in summary, testicular and epididymal tissue were hypoplastic in the left specimen, whereas only hypoplastic epididymal tissue was identified in the right (figure 3). a blood karyotype was performed on lymphocytes using giemsa staining and fluorescent in situ hybridization. blood was collected into sterile sodium heparin and ethylenediaminetetraacetic acid (edta) tubes and shipped refrigerated overnight to texas a&m university molecular cytogenetics laboratory. an almost equal number of normal female 78, xx (54%) and male 78, xy (46%) cells in blood lymphocytes were present, consistent with blood chimerism (figure 4). polymerase chain reaction testing for the y-linked sry gene was positive (consistent with xy cells presence). discussion a dna genotyping microarray is a genotyping platform consisting of thousands of known nucleic acid fragments (probes) bound to a solid surface (chip). the chip is bathed with a dna sample from a patient, and complementary base pairing between the patient sample and probes occurs. bound fragments are fluorescently labeled, and a machine reads the fluorescence and assigns a genotype at each locus. this technology can create a readout of genotype information at thousands of predetermined locations in the genome that can be used for health screening and to make animal breeding decisions. figure 1. allele frequency plots of the case dog (a) and a normal diploid xy male dog (b) generated by the microarray used for health screening in this case (embark® breed & health dog dna test). each dot denotes a genetic marker along a chromosome, and the plot’s alternating gray and black dots correspond to chromosomes 1-38 and the sex chromosomes. the vertical axis indicates the estimated frequencies of the non-reference allele. in panel a, four sets of chromosomes mean more than three possible combinations of genotypes (leading to multiple clusters in the middle of the plot instead of a single band). the absence of markers with allele frequencies around 0.5 on chromosome 40 (y) indicates the presence of a single y chromosome. in panel b, the cluster at the bottom represents homozygotes for a reference allele, the cluster at the top represents homozygotes for a non-reference allele, and the cluster in the middle represents the heterozygous state. this normal male dog is homozygous for some and heterozygous for other markers across all autosomes, and homozygous for all markers on chromosome 39 (x) and chromosome 40 (y) table. patient results and reference ranges for resting hormonal assays performed at 14 months of age. testing was performed at the university of tennessee college of veterinary medicine’s diagnostic endocrinology service. hormones patient spayed female intact female neutered male intact male estradiol (pg/ml) 32.1 30.8 – 69.9 31.5 – 65.4 23.1 – 65.1 30.5 – 66.6 progesterone (ng/ml) < 0.20 < 0.20 – 0.49 < 0.20 – 2.16 < 0.20 < 0.20 – 0.40 testosterone (ng/ml) < 15.0 < 15.0 – 32.0 < 15.0 < 15.0 – 24.0 19.0 – 2630.0 http://dx.doi.org/10.58292/ct.v17.12703 citation: clinical theriogenology 2025, 17, 12703, http://dx.doi.org/10.58292/ct.v17.12703 3 cytogenetic analysis in the dog is challenging due to a high diploid chromosome number (78 autosomes and a pair of sex chromosomes) and the one-arm morphology of the autosomes.1 microarrays can assess chromosomal imbalances and may detect small anomalies beyond the resolution of routine karyotyping.2 additionally, microarray technology may detect aneuploidy and the presence of multiple genotypes (such as chimerism or mosaicism).2 allele frequency plots are utilized to detect these anomalies.2 this plot visualizes the fluorescent signal ratio of reference and nonreference alleles, such that a value 1 or 0 in the plot indicates the complete absence of the alternate allele (a homozygous genotype of either reference or nonreference allele), and a value 0.5 indicates the presence of both alleles (the heterozygous state). in a diploid animal, 3 discrete clusters are observed in the allele frequency plot near 0, 0.5, and 1. a different number of bands will be noted in aneuploidy, mosaicism, and chimerism due to the possibility of greater than 3 genotype combinations (figure 1). a chimera is a single organism derived from the fusion of 2 or more zygotes, whereas a mosaic is a single organism with multiple cell lines derived from 1 zygote with subsequent mitotic errors during early embryogenesis.3 in humans, not all xx/xy individuals are chimeras; several case reports of a mosaic 46,xx/46,xy karyotype derived from an xxy zygote by either 2 nondisjunction or anaphase lag events exist.3 in our case, determining whether this individual is a chimera or a mosaic was not performed as it was not clinically relevant to patient outcome. disorders of sexual development (dsds) in dogs can be divided into 3 broad categories: sex chromosome dsds, xy dsds, and xx dsds.4 xx and xy dsds occur in animals with normal karyotypes but other anomalies, such as testicular or ovarian development disorders or cryptorchidism.4 furthermore, 78, xx/78, xy chimerism or mosaicism are examples of sex chromosome dsds.4 this type of dsd is reported less frequently in veterinary species than humans, potentially due to economic concerns of diagnostic testing.4 each year, a commercial genetic screening company (embark veterinary, inc.) generally identifies between 20 and 50 individuals with autosomal and/or sex chromosome anomalies, including monosomy, trisomy, and chimeras or mosaics. the overall frequency of chimeric/mosaic dogs and dogs with aneuploidy detected by this company is ~ 77 per 1 million dogs tested. other sex chromosome anomalies detected include xo, xxx, xxy, and xyy karyotypes. many more individuals with discrepancies between the genetic and phenotypic sex are identified (with a normal chromosome number) annually. these data imply abnormal karyotypes in dogs may be less rare than previously believed. the clinical relevance of most types of anomalies is unclear due to limited owner reporting of associated health outcomes. additionally, for sex chromosome anomalies, many affected individuals were altered figure 2. appearance of gonads intraoperatively (a) and after excision (b). g = gonad, s = suspensory ligament-like structure, p = pedicle-like structure, t = tissue extending toward and through the inguinal ring. marker = 1 cm figure 3. photomicrograph depicting the remnant interstitial cells from the gonadal tissue excised from the left side (a). marker = 50 µm. photomicrograph depicting the hypoplastic epididymal tubules excised from the right side (b). marker = 50 µm http://dx.doi.org/10.58292/ct.v17.12703 4 citation: clinical theriogenology 2025, 17, 12703, http://dx.doi.org/10.58292/ct.v17.12703 prior to adoption, so the manifestation of reproductive symptoms is unknown. direct-to-consumer genetic testing has continued to gain popularity, and veterinary professionals appreciate its clinical utility.5 however, knowledge gaps in interpreting results persist within the industry.5 genetic tests typically offer information on breed ancestry, health conditions, inbreeding, and physical traits. the present case describes the use of direct to-consumer genetic testing to diagnose a chromosomal abnormality (xx/xy chimerism or mosaicism) resulting in a dsd and primary anestrus (failure to cycle by 24 months of age). this case highlighted the utility of this cost-effective, noninvasive test to screen for potential chromosomal anomalies, a seldom-considered benefit of microarray-based testing. learning points • chromosomal anomalies may be more common in dogs than currently recognized • microarrays offer an inexpensive, noninvasive screening method for sex chromosome disorders including chimerism/mosaicism and phenotypic/genotypic sex mismatch • chromosomal anomalies may lead to primary anestrus acknowledgement authors thank dr. terje raudsepp and the molecular cytogenetics laboratory at texas a&m for excellent diagnostic results interpretation, reporting, and support. funding embark veterinary, inc. provided monetary support for continued diagnostics (histopathology and blood karyotyping) in this case. no further financial compensation was provided. conflict of interest one author (ab) cofounded embark veterinary, inc. two authors (jd and tk) are currently employed by embark veterinary, inc. author contributions and agreement all authors have made substantial contributions to the clinical (vr, dlf) or diagnostic (jd, cp, ab, tk) management of this case and have been involved in drafting and revising this manuscript. all authors have read and approved the final version of the manuscript and have agreed to its submission. references 1. szczerbal i, switonski m: clinical cytogenetics of the dog: a review. animals 2021;11:947. doi: 10.3390/ani11040947 2. levy b, burnside rd: are all chromosome microarrays the same? what clinicians need to know. prenat diagn 2019;39:157-164. doi: 10.1002/pd.5422 3. madan k: natural human chimeras: a review. eur j med genet 2020;63:103971. doi: 10.1016/j.ejmg.2020.103971 4. meyers-wallen vn: gonadal and sex differentiation abnormalities of dogs and cats. sex dev 2012;6:46-60. doi: 10.1159/000332740 5. bennett ne, johnson ea, gray pb: veterinary care providers recognize clinical utility of genetic testing but report limited confidence in interpreting direct-to-consumer results. am j vet res 2024. 2024;86:e0265 doi: 10.2460/ajvr.24.09.0265 figure 4. giemsa-stained karyotype image to show chromosome number and morphology of 2 separate lymphocytes from the case dog. the x and y chromosomes are indicated by magenta and blue arrows, respectively. one lymphocyte exhibits a karyotype of xx (a), while another exhibits a karyotype of xy (b). karyotyping was performed at the texas a&m university molecular cytogenetics laboratory http://dx.doi.org/10.58292/ct.v17.12703 https://doi.org/10.3390/ani11040947 https://doi.org/10.1002/pd.5422 https://doi.org/10.1016/j.ejmg.2020.103971 https://doi.org/10.1159/000332740 https://doi.org/10.2460/ajvr.24.09.0265 1 contact maria ferrer msferrer@uga.edu supplemental data for this article can be accessed online at http://dx.doi.org/10.58292/ct.v17.12705 © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12705, http://dx.doi.org/10.58292/ct.v17.12705 research report constitutive expression of toll-like receptor genes and signaling pathways in stallion testis and epididymis maria ferrer,a karen moran,b maria bilbao,b michele barletta,a jared williams,a julie gordon,a julian bartoloméb adepartment of large animal medicine, college of veterinary medicine, university of georgia, athens, ga, usa bfacultad de ciencias veterinarias, universidad nacional de la pampa, la pampa, argentina abstract toll-like receptors (tlrs) expression pattern and their associated molecules have not been described in stallions. understanding the tlr expression and their responses to activation is key in interpreting the interactions between immune system and reproductive system in healthy and ill animals. objective of this study was to describe the constitutive expression pattern of tlrs and their signaling pathways in stallion’s testis and epididymis. it was hypothesized that all tlrs and downstream signaling molecules are constitutively expressed in these tissues in reproductively normal stallions. transcriptome analysis was performed in testicular and epididymal samples from reproductively normal stallions (n = 8). a detectable constitutive expression of all tlrs, costimulatory molecules, and most downstream adaptors and effectors was identified in stallion reproductive tissues. most significant pathways were associated with tlr3, tlr2/tlr1, tlr2/tlr6, and the effector molecules c-c motif ligand 5 (ccl5) and cd40. most genes were overexpressed in the epididymis compared to testis. widespread and abundant expression of these genes supports an important function of the innate immunity in testicular and epididymal defense. these tlrs may also have a physiological role in maintaining the tolerogenic environment, supporting posttesticular maturation, or removing defective sperm from the tubular system. keywords: horse, testis, epididymis, toll-like receptor, innate immunity, transcriptome introduction equine toll-like receptor (tlr) family includes 12 reported transmembrane proteins localized on the plasma membrane (tlr1, tlr2, tlr4, tlr5, tlr6, tlr10, tlr11) or cytosolic endosomal membranes (tlr3, tlr7, tlr8, tlr9, tlr12).1,2 these receptors are essential for the initiation of the innate immune response through recognition of conserved pathogen-associated molecular patterns (pamps). although surface tlrs identify bacterial, fungal and protozoal peptides and lipopolysaccharides, cytosolic tlrs identify viral and bacterial nucleic acids. in mammals, most tlrs bind several ligands by forming homodimers; however, tlr2 forms heterodimers with tlr1 or tlr6. dimer type influences the affinity and magnitude of the tlr response to its ligand. the heterodimer tlr2/1 has higher affinity and a more potent response to bacterial lipopeptides than tlr2/6 in the horse.3 upon engagement with the ligand, tlr dimers initiate the myeloid differentiation protein 88 (myd88)-dependent pathway. recruitment of the adaptor proteins myd88 and tir domain containing adaptor protein (tirap) results in activation of the cytosolic nuclear factor kappa b (nf-kb), its translocation to the nucleus, and transcription of proinflammatory cytokine genes, such as tnfα, il-6, il-1, and il-12.4 the exception to this pathway is tlr3, whose activation triggers the toll/il-1r domain-containing adaptor inducing interferon β (trif)-dependent pathway. recruitment of trif and trif-related adaptor molecule culminates with activation and nuclear translocation of interferon regulatory factor 3 and expression of type 1 interferons (ifn-α and ifn-β).2,4 the tlr4 has the ability to activate both pathways and requires the presence of the co-stimulatory molecules cd14 and myeloid differentiation. although the innate immunity has a critical role in the initial control of infectious agents, tlrs can also be activated by endogenous damage-associated molecular patterns (damps), resulting in sterile inflammation.5 equid peripheral leucocytes express the 12 tlrs, whereas equine thymus, lung, liver, jejunum, colon, kidney, and lymph nodes express tlr1 to tlr10.2,6 there is also constitutive expression of tlr4 in the equine gingiva, hoof lamella, eye, mailto:msferrer@uga.edu http://dx.doi.org/10.58292/ct.v17.12705 http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12705 2 citation: clinical theriogenology 2025, 17, 12705, http://dx.doi.org/10.58292/ct.v17.12705 skeletal muscle, cerebrum, cerebellum and adipose tissue, with increased expression induced by exercise, cortisol and heat in skeletal muscle and peripheral leucocytes, and by insulin resistance in adipose tissue.7–11 in the mare reproductive tract, the chorioallantois, endometrial glandular and luminal epithelium, stromal cells, leucocytes, endothelium, and vascular smooth muscle express tlr2, tlr4, tlr6, and tlr7 constitutively.12–16 inducible expression increased in mares with endometritis and placentitis.14–16 expression of tlrs in the stallion reproductive tract has not been reported. most tlrs and signaling components are expressed in immune cells and somatic and germ cells of the testis, epididymis, vas deferens, and accessory sex glands of rodents.17 rodent sertoli cells, leydig cells, and testicular macrophages have the widest tlr expression patterns and express proinflammatory cytokines upon activation.18 microbial pamps and endogenous damps released by germ cells induce inflammatory cytokine production by sertoli cells through tlr activation.17,19,20 activation of inflammatory pathways in sertoli cells inhibits their response to follicle stimulating hormone and androgens and disassembles junctional complexes of the blood-testis barrier.17 the activation of tlrs in leydig cells suppresses steroidogenesis, altering testicular function through decreased intratesticular testosterone concentrations, whereas the activation of tlr2 and tlr4 in germ cells directly induces germ cell apoptosis.17,21 activation of tlrs is also critical for the development of autoimmune orchitis in mice.20,22 although the expression and activation pattern of tlrs in the rodent reproductive tract has been characterized, their expression in the genital tract of stallions has not been described. understanding the expression patterns, ligands, and responses to activation is key to interpreting the interactions between the immune system and the reproductive system in healthy and ill animals. objective of this study was to describe the constitutive expression of tlrs and their signaling pathways in stallion’s testis and epididymis. it was hypothesized that the testis and epididymis of reproductively normal stallions constitutively express all tlrs and downstream signaling molecules. materials and methods animals and tissue processing testicular and epididymal samples were collected from 8 client-owned stallions presenting to the veterinary medical center of the university of georgia for routine castration as part of the clinical services provided by the hospital. our institutional animal care and use committee does not require an approved animal use protocol for the use of discarded clinical samples. stallions were 2-13 years american quarter horse (n = 5), arabian (n = 2) and american paint horse breed (n = 1); 4 stallions were castrated in april and 4 in february. testes and epididymides were placed on ice immediately after castration and processed within 10 minutes. sperm were collected from the vas deferens. sperm morphology was evaluated using a hancock stain and light microscopy;23 stallions’ fertility was not known. given that sperm morphology is associated with pregnancy rate, this parameter was chosen as an inclusion criterion.23–25 various threshold values have been suggested for fertile or highly fertile stallions, ranging from 30-60%.26–30 the highest value was used here to include stallions assumed to have normal testicular and epididymal function; thus, tissues from stallions with ≥ 60% morphologically normal sperm were included. vaginal tunics and connective tissue were removed, and the organs were rinsed with sterile saline solution. a 1 x 1 cm tissue sample was collected with sterile instruments from the testicular parenchyma and the epididymal head, body, and tail; 1 testis and epididymis were included per stallion. the side (left versus right) was randomly selected. each tissue was placed in individual tubes, snap-frozen in liquid nitrogen, and stored in liquid nitrogen until processing. total rna was extracted using a commercial kit (rneasy mini kit, qiagen, redwood city, ca, usa) following the manufacturer’s protocol. approximately 1 cm3 of tissue from each sample was cryopulverized and homogenized with an omni th homogenizer (vwr) in rlt lysis buffer containing 1% 2 mercaptoethanol. the tissue suspension was centrifuged at 11,000 × g for 1 minute. supernatant containing the total rna was loaded on a rneasy spin column to remove any dna contamination and to elute the total rna. total rna was frozen at –80°c and shipped on dry ice to a commercial laboratory (novogene, sacramento, ca, usa) for quality control and rna sequencing. library preparation for transcriptome sequencing degradation and contamination of the rna were monitored on 1% agarose gels. purity of the rna was evaluated using the nanophotometer® spectrophotometer (implen, westlake village, ca, usa). the rna integrity and quantification was assessed using the rna nano 6000 assay kit of the bioanalyzer 2100 system (agilent technologies, santa clara, ca, usa). a total amount of 1 μg of rna per sample was used as input material for the rna sample preparations. sequencing libraries were generated using nebnext® ultra™ rna library prep kit for illumina® (illumina, san diego, ca, usa) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. briefly, mrna was purified from total rna using poly-t oligo-attached magnetic beads. fragmentation was carried out using divalent cations under elevated temperature in neb and next in first strand synthesis reaction buffer (5 x). first strand cdna was synthesized using random hexamer primer and m-mulv reverse transcriptase (rnase h). second strand cdna synthesis was subsequently performed using dna polymerase i and rnase h. remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. after adenylation of 3’ ends of dna fragments, neb and next adaptor with hairpin loop structure were ligated to prepare for hybridization. to select cdna fragments of preferentially 150-200 base pair (bp) in length, the library fragments were purified with ampure xp system (beckman coulter, brea, ca usa); 3 µl of user enzyme (neb, usa) were used with size-selected, adaptor-ligated cdna at 37°c for 15 minutes, followed by 5 minutes at 95°c before pcr. then pcr was performed with phusion high-fidelity dna polymerase, universal pcr primers, and index (x) primer. the pcr products were purified (ampure xp system), and the library quality was assessed on the agilent bioanalyzer 2100 system. the clustering of the index-coded samples was performed on a cbot cluster generation system using pe cluster kit cbot-hs (illumina), according to the manufacturer’s instructions. after cluster generation, the library preparations were sequenced on an illumina platform and 125 bp/150 bp paired-end reads were generated. http://dx.doi.org/10.58292/ct.v17.12705 citation: clinical theriogenology 2025, 17, 12705, http://dx.doi.org/10.58292/ct.v17.12705 3 sequencing data analysis raw data (raw reads) of fastq format were first processed and clean data (clean reads) were obtained by removing reads containing adapters, reads containing ploy-n, and low-quality reads from the raw data. all the downstream analyses were based on the clean data with high quality. the reference genome (equus caballus, equcab3) and gene model annotation files were directly downloaded from the genome website. the index of the reference genome was built using hisat2 2.1.0 and paired-end clean reads were aligned to the reference genome using hisat2 (johns hopkins university, baltimore, md, usa). featurecounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. the expected number of fragments per kilobase of transcript sequence per millions base pairs sequenced (fpkm) of each gene was calculated based on the length of the gene and reads count mapped to this gene.31 differential expression analysis was performed comparing the testicular tissue to each segment of the epididymis using the deseq2 r package (1.14.1, r core team, vienna, austria). deseq2 provided statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. the p values were adjusted using the benjamini and hochberg’s approach for controlling the false discovery rate. genes with an adjusted p value < 0.05 by deseq2 were considered differentially expressed.32 the differential expression of tlrs was evaluated within each tissue sample by comparing mean fpkm among tlrs. from the entire data set of transcripts, genes involved in the toll-like receptor pathway (https://www.genome.jp/pathway/hsa04620) were selected and reported. reverse transcription and real time polymerase chain reaction real time polymerase chain reaction (rt-pcr) was performed in a subset of 3 stallion testicular samples to confirm expression of tlrs 1-8 for quality control of the sequencing analysis. tissue collected from equine lymph nodes was used as positive control. total rna yield and purity were estimated spectrophotometrically at 260 nm using a nanodrop (thermofisher scientific, waltham, ma, usa). complementary dna (cdna) was synthesized from 1 µg total rna using qscript™ cdna supermix (quanta biosciences, gaithersburg, md, usa) and nuclease-free water, following the manufacturer’s instructions. reverse transcription was performed at 25°c for 5 minutes, 42°c for 30 minutes, followed by 90°c for 5 minutes. the cdnas obtained were stored at –20°c until further investigation. reactions without reverse transcriptase were run in parallel to rt to confirm the absence of any genomic dna or contamination. the tlr gene-specific primers (table) were designed using sequences published in the national center for biotechnology information database (ncbi, bethesda, md, usa) with the aid of primer3 software and purchased from sigma. amplification of cdna was performed using the following conditions: an initial denaturation at 94°c for 5 minutes; followed by 39 cycles of 94°c for 30 seconds, and annealing at the temperature specified for each gene for 60 seconds; with a final extension at 72°c for 4 minutes. a dissociation curve was added for 15 seconds at 95°c to ensure the presence of a single amplicon. gene expression was analyzed using sybr green quantitative rt-pcr, and expression data for the different genes were obtained in the form of threshold cycle (ct) values. figure 1. toll-like receptors and their associated pathways, gray boxes represent genes that were not constitutively expressed (fpkm = 0) within the stallion’s reproductive tract; fpkm = fragments per kilobase of transcript sequence per millions base pairs sequenced http://dx.doi.org/10.58292/ct.v17.12705 https://www.genome.jp/pathway/hsa04620 4 citation: clinical theriogenology 2025, 17, 12705, http://dx.doi.org/10.58292/ct.v17.12705 results gene expression was not affected by stallion age or season (p > 0.05); therefore, data from all stallions were grouped. detectable constitutive expression of all tlrs, costimulatory molecules, and most downstream adaptors and effectors was identified in stallion reproductive tissues, including the ortholog gene tlr12 (supplemental table; figure 1). within the testes, transcription of all tlrs and most of their associated adaptors and effectors was identified (supplemental table and figure 2). however, not all genes were equally expressed. the testicular expression of tlr3 (fpkm 1.57 ± 0.4) was higher (p = 0.002) than all other tlrs, followed by tlr2 (0.57 ± 0.1), tlr6 (0.28 ± 0.05), and tlr1 (0.24 ± 0.05). the transcription of tlr4 (0.01 ± 0.01) and 10 (0.03 ± 0.01) was minimal (figure 2). furthermore, there was an active expression of most adaptor protein genes and downstream mediators from all signaling pathways, except for phosphoinositide 3-kinase regulatory subunit 5 (pik3r5). genes for most of the resulting cytokines and effector proteins were also expressed in the testis, except for interleukin 8 (il8), interferon alpha 2  (ifna2), interferon beta 1 (ifnb1), and c-x-c motif ligand  9  (cxcl9) (figure 1 and supplemental table). the most significantly expressed effector chemokine was ccl5 (supplemental table). level of expression of most genes differed (p < 0.05; supplemental table). expression of tlr1, tlr2, tlr3, and tlr5-9 was downregulated in the testes compared to epididymis (figure 2). expression of tlr4 and tlr10-12 did not differ among tissues. expression of most genes was upregulated in several segments of the epididymis compared to testis. the exception was some proteins of the pik3-akt and myd88dependent signaling pathways that were downregulated in the epididymis (supplemental table). similar to testes, epididymal expression of tlr3 was higher than all other tlrs. the tlr1, tlr2, tlr5, tlr6, tlr7 and tlr10 were abundantly expressed (fpkm > 1) in at least 1 segment of the epididymis, whereas tlr4 and tlr11 had a minimal level of expression (fpkm < 0.1). there was also active expression of most adaptor proteins and downstream mediators from all signaling figure 2. differential expression of equine toll-like receptors in the testis, epididymal head (ephead), body (epbody), and tail (eptail). fpkm = fragments per kilobase of transcript sequence per millions base pairs sequenced; a-cwithin a tissue, means without a common superscript differed (p < 0.05) table. forward and reverse primer sequences for tlr 1-8 genes for rt-pcr gene name gene symbol accession no. forward reverse product size (bp) toll-like receptor 1 tlr1 nm_001256899.1 gcccatatgcaaagagtttggc tctgtgttaaggtgtcgaaggc 183 toll-like receptor 2 tlr2 nm_001081796.1 catgctttgtggacggtgtg aagacttttcacagctgccg 168 toll-like receptor 3 tlr3 nm_001081798.1 gagactgttgcccttttggg ccgttatgtttgctgggagg 125 toll-like receptor 4 tlr4 nm_001099769.2 acatccccacatcaaccaagg atggttgaggccctgatatgc 158 toll-like receptor 5 tlr5 kwon, et al. 2011 tccatggagggttgtgatga ccccggaactttgtgacaat – toll-like receptor 6 tlr6 nm_001257142.1 gacttgccaccagaaaccaag aggtaccggatgctattacgg 128 toll-like receptor 7 tlr7 nm_001081771.2 acttctgtagcaggtcacgg acttaggtccaaggtctgcc 159 toll-like receptor 8 tlr8 nm_001111301.1 gccgttttggaacttggtgg ggcatctgaaacacacgtcg 186 http://dx.doi.org/10.58292/ct.v17.12705 citation: clinical theriogenology 2025, 17, 12705, http://dx.doi.org/10.58292/ct.v17.12705 5 figure 3. rt-pcr amplification curves of toll-like receptor (tlr) genes tlr1 (a), tlr2 (b), tlr3 (c), tlr4 (d), tlr5 (e), tlr6 (f), tlr7 (g), and tlr8 (h) and cq values of individual samples (i). a = nontemplate negative control, b = positive control (lymph node tissue), c = testicular tissue samples http://dx.doi.org/10.58292/ct.v17.12705 6 citation: clinical theriogenology 2025, 17, 12705, http://dx.doi.org/10.58292/ct.v17.12705 pathways, except for pik3r5. the resulting cytokines involved in inflammation, chemotaxis, and stimulation of the immune system were upregulated in the epididymis, except for il8, ifna1, ifna2, ifnb1, and cxcl9 that were not detected. the most significantly expressed effector chemokine was ccl5 (supplement table). constitutive testicular expression of tlrs 1-8 was confirmed using rt-pcr (figure 3). discussion to our knowledge, this is the first description of constitutive expression of tlr genes and their downstream adaptors and effector cytokines in the stallion’s reproductive tract, confirming that the equine testis and epididymis are fully equipped with the machinery needed for tlr stimulation and function. limitations of our study are: first, unavailability of stallions’ fertility history and second, histologic evaluation of the tissue. however, a strict criterion was used to select stallions with a high percentage of morphologically normal sperm that were assumed to have normal testicular and epididymal function.26–30 in this group of stallions, tlr3 was the most abundant transcript within all segments of the stallion’s genital tract evaluated. a third limitation is that we did not attempt to identify the cell types expressing each gene. in rodents, tlr3 was abundantly expressed in germ cells, sertoli cells, and peritubular cells.17,33 toll-like receptor 3 also had the most widespread and abundant expression in the rodent and human testis, suggesting an important role in testicular function.17,33,34 this cytosolic receptor recognizes viral and mammalian double-stranded dna. it was speculated that physiological activation of tlr3 during phagocytosis of germ cells by sertoli cells induced an inflammation-like activation of sertoli cells that triggered the release of critical spermatogenic regulators.35 during inflammation, tlr3 activates the trif-dependent pathway, resulting in release of interferons. despite the abundance of tlr3 and its mediators, interferons were not abundantly transcribed in the reproductive tract of reproductively normal stallions, suggesting that tlr3 may indeed serve a physiological function in the absence of inflammation. several snps were identified in the coding regions of the horse tlr3 gene, as well as tlr7 and tlr8, but their effect on pattern recognition or function is not known.36 the next most abundantly expressed receptors were tlr1, tlr2, and tlr6, together with their associated mediators of the pik3-akt signaling pathway. toll-like receptor 2 was ubiquitously expressed in rodent germ cells, leydig cells, and sertoli cells, whereas tlr6 was only expressed in mouse sertoli cells.17,33 cellular localization of tlr1 in the testis has not been described. these tlrs are located in cell membranes.1 heterodimers tlr1/tlr2 and tlr2/tlr6 recognize lipoproteins in gram positive bacteria. although their activation induces the release of several cytokines, those involved in innate chemotaxis, particularly ccl5, were the most abundantly expressed in stallion’s testis and epididymis. macrophages in the basal region of the epididymal epithelium constitutively express ccl5. these macrophages present antigens to lymphocytes and help eliminate abnormal sperm.37 concentration of ccl5 in seminal plasma was lower in men with immune-mediated infertility.38 it was suspected that reduced ccl5 concentration decreased the chemoattraction of scavenger leukocytes involved in the physiological elimination of abnormal sperm during storage in the genital tract, leading to increased exposure to autoantigens and autoimmune reactions.38 furthermore, ccl5positive macrophages are involved in the acidification of the epididymal luminal fluid. this low ph is necessary for posttesticular sperm maturation and inhibition of sperm motility during epididymal storage.37 role of ccl5 in the testicular medium is less well characterized; however, since the testicular luminal ph is also acidic, ccl5 may serve a similar function in the testis.39 effect of tlr2 stimulation depends on the relative expression of tlr1 and tlr6 that determines heterodimer predominates. heterodimer tlr2/tlr1 was more predominant in stallion’s epididymis than tlr2/tlr6; heterodimer is thought to have an antiinflammatory role by inducing the production of il-10 and favoring the differentiation of regulatory t cells.40 this immunosuppressive effect can be synergized by low level stimulation of cd40 in tolerogenic dendritic cells that leads to maintenance of the homeostasis of the immune system and a prevalence of a tolerogenic environment.40–43 of the costimulatory molecules evaluated here, cd40 was the most abundantly expressed. altogether, it is possible that the prevalent tlrs and the effectors identified in our study have a physiological role maintaining the homeostasis of the immune system and removing abnormal sperm. conversely, the expression of tlr4 and its costimulatory molecule cd14 were minimal in the stallion’s reproductive tract, contradicting findings in other species. expression of tlr4 was high in rodent and human germ cells, and sertoli cells.17,33,34,44 conversely, as in other species, tlr10 and tlr11 were also less abundant.17,33,44 it is possible that this low level of expression indicates a less important role of these tlrs in testicular and epididymal immunity. alternatively, these tlrs may be inducible, and may acquire a more active role in the face of infection. furthermore, the low transcriptional levels may be due to the cellular distribution of these tlrs since the expression of tlr8, tlr9 and tlr11 was limited to myeloid cells within the rodent testis.17,33 the majority of the genes were overexpressed in the epididymis compared to testis. despite the higher gene expression of the tlrs and adaptors, cytokine expression was also relatively low (fpkm < 1) within the epididymis. the overexpression emphasizes the importance of the innate immunity in controlling and preventing ascending infections of the reproductive tract. conversely, the innate immunity may have a role in the removal of abnormal sperm from the epididymis, preventing their transport through the tubular system into the ejaculate. in the rat epididymis, all tlrs, except for tlr10, were localized in the epithelial cells.44 although seasonal variations were reported in the donkey epididymal epithelium morphology and presence of inflammatory cells, no differences in gene expression were identified in our study between samples collected in the spring versus winter.45 our study is the first to identify the expression of tlr12 in equine tissues. expression of tlr12 was only recently described in equine peripheral leucocytes.2 in mice, tlr12 was described as an intracellular receptor acting in cooperation with tlr11 in the recognition of toxoplasma gondii profilin.46 equine tlr12 is poorly characterized, and its role in equine immunity is not known. future studies should consider evaluating inducible expression of tlrs and associated genes in the testes and epididymis of stallions with testicular or epididymal dysfunction or suspected immune-mediated infertility to evaluate the role of these components of the innate immune system on the pathophysiology of these conditions. in summary, constitutive gene expression of most tlrs and associated proteins was identified in stallion’s testis and http://dx.doi.org/10.58292/ct.v17.12705 citation: clinical theriogenology 2025, 17, 12705, http://dx.doi.org/10.58292/ct.v17.12705 7 epididymis. their widespread expression supports an important function of the innate immunity in testicular and epididymal defense. the most significant pathways were associated with tlr3, tlr2/tlr1, and tlr2/tlr6, and the effector molecules ccl5 and cd40. these tlr pathways may have a physiological role maintaining the tolerogenic environment, supporting posttesticular maturation or removing defective sperm from the tubular system. conflict of interest authors have no conflict of interest that could be perceived as prejudicing against the impartiality of the research reported. funding for the love of the horse competitive research grants, university of georgia. references 1. werling d, coffey tj: pattern recognition receptors in companion and farm animals the key to unlocking the door to animal disease? vet j 2007;174:240-251. doi: 10.1016/j.tvjl.2006.10.010 2. stejskalova k, janova e, splichalova p, et al: twelve toll-like receptor (tlr) 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the irf8-dependent il-12 response to toxoplasma gondii profilin. j immunol 2013;191:4818-4827. doi: 10.4049/ jimmunol.1301301 http://dx.doi.org/10.58292/ct.v17.12705 https://doi.org/10.1038/nbt.1621 https://doi.org/10.1186/gb-2010-11-10-r106 https://doi.org/10.1210/en.2007-1776 https://doi.org/10.1248/bpb.28.886 https://doi.org/10.1038/sj.icb.7100086 https://doi.org/10.1038/sj.icb.7100086 https://doi.org/10.1007/s00251-009-0381-z https://doi.org/10.1007/s00251-009-0381-z https://doi.org/10.3389/fimmu.2020.583274 https://doi.org/10.1111/j.8755-8920.2000.440402.x https://doi.org/10.1080/15287399109531464 https://doi.org/10.3389/fimmu.2014.00220 https://doi.org/10.1111/j.1600-0625.2009.00975.x https://doi.org/10.2337/db13-1657 https://doi.org/10.1111/j.1600-0897.2008.00654.x https://doi.org/10.1002/jemt.24436 https://doi.org/10.4049/jimmunol.1301301 https://doi.org/10.4049/jimmunol.1301301 1 contact ky pohler ky.pohler@ag.tamu.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11037, http://dx.doi.org/10.58292/ct.v17.11037 review report highlighting factors contributing to pregnancy loss in beef cattle* brette poliakiwski, damon smith, zachary seekford, ky pohler department of animal science, texas a&m university, college station, tx, usa abstract pregnancy loss in beef cattle remains a costly problem for producers, leading to diminished calf crop uniformity and reduced percentages of cows with a calf at the end of calving season. although several tools exist to ascertain pregnancy status, the first 30 days of pregnancy encompasses the period with the greatest proportion of pregnancy losses and these losses often occur before traditional methods permit pregnancy determination. the ability to accurately predict pregnancy failure remains a major limitation. blood-based assays detecting chemical changes in maternal circulation have provided insight into embryonic and fetal monitoring and are used to make predictions for pregnancy loss. although there are certain unknown aspects to the etiology of pregnancy loss, there is growing body of work to identify physiological biomarkers within the maternal, paternal, and embryonic systems to clarify risk factors for pregnancy failure. this review highlights a few of the factors contributing to pregnancy loss and the rapidly evolving methods utilized to predict pregnancy failure. further, this review highlights a few of the changes to parental physiology after exposure to various environmental factors, the consequences on the physiology of pregnancy and the likelihood of pregnancy success. keywords: cattle, pregnancy, placentation, maternal, sire, environmental introduction1 embryonic and fetal mortality remain as major issues in domestic livestock production. although embryonic mortality and pregnancy loss research have garnered industry-wide attention, a major limitation within is the inability to accurately predict pregnancy failure and determine early embryonic and fetal viability. additionally, predicting pregnancy loss in beef cattle is difficult in enterprises that are less intensely managed than dairy operations that collect data to compile reproductive outcomes. early pregnancy contains several critical developmental milestones and pregnancy losses can occur throughout this period. there are continued discussions within reproductive physiology to determine the period of most substantial embryonic loss. fertilization rates in beef cattle are estimated quite high (~ 90%) but attrition occurring throughout pregnancy indicated that the underlying issue of pregnancy loss extends beyond the period of successful conception.1 indeed, embryonic mortality (0-45 days after insemination) is estimated to affect 54% of beef cattle after a single insemination.1 within this period, roughly 16% of this embryonic loss *presented at the 2023 society for theriogenology conference, published after peer review. occurs between days 16-32 and ~ 6% of these losses occurs between days 29-45.1 some suggest that embryonic mortality is most substantial between embryo hatching and elongation although others suggest that it is most substantial during and after maternal recognition of pregnancy and early placentation.2,3 these periods of embryonic development encompass the processes of maternal recognition of pregnancy and early placentation, possibly indicating that pregnancy loss may be attributed to either failed embryonic signaling or improper placentation events. therefore, the issue of pregnancy loss is likely multifactorial. although the timing of embryonic mortality and pregnancy loss in cattle can vary substantially based on production status, parity, genetic composition, breeding method, and management conditions there are apparent embryonic, maternal, paternal, and environmental factors known to contribute to pregnancy failure (figure 1). aim of this review is to highlight some of the current known and hypothesized contributions to pregnancy loss experienced in beef cattle and the evolving methods on how pregnancy loss is predicted. measuring reproductive efficiency before discussing various components of pregnancy loss, it is important to first understand how to measure and manage mailto:ky.pohler@ag.tamu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11037 2 citation: clinical theriogenology 2025, 17, 11037, http://dx.doi.org/10.58292/ct.v17.11037 these losses. pregnancy determination is a vital tool to measure reproductive efficiency; the generated data are used to directly monitor farm reproductive success or to indirectly study trends in national fertility. it has been estimated that pregnancy loss in beef cattle in the usa results in a gross loss of $3.7 billion in profit per year.4 despite this, only 20% of usa beef operations utilize pregnancy diagnosis. thus, there remains a clear disconnect between the financial benefits and the utilization of pregnancy data. the lack of adoption of pregnancy diagnosis likely stems from a lack of trained technicians, increased input costs associated with labor and service fees, the need for adequate handling facilities, and/or the effects of handling stress on cattle. fortunately for producers, there are 3 primary methods to consider for pregnancy diagnosis; each method has advantages and disadvantages over the others. the most traditional method is transrectal palpation of uterine contents. transrectal palpation is often completed by certified personnel at least 40 days after breeding and offers an affordable means to detect conceptus and associated membranes without the use of costly equipment. despite the cost effectiveness of transrectal palpation, the accuracy of pregnancy determination remains dependent on the skill of the technician. the precise determination of aging via transrectal palpation before day 40 is dependent solely on the detection of the cardinal signs of pregnancy (displacement of the chorio-allantoic membrane, palpation of amniotic vesicle, palpation of fetus or palpation of placentomes).5 although transrectal palpation of uterine contents are used to estimate age of pregnancy, this method is limited in its ability to accurately estimate embryonic viability as assessed by the heartbeat.6 ultrasonography is becoming quickly adopted amongst beef producers. unlike transrectal palpation of uterine contents, ultrasonography can determine pregnancy as early as 28 days after breeding and is a more accurate method to determine fetal age through the measurement of crown rump length.7,8 ultrasonography also provides producers an earlier opportunity to make a management decision compared to transrectal palpation.6,7 another benefit of ultrasonography is the ability to determine fetal sex from days 60-90 of pregnancy. knowing fetal sex is an advantage for seedstock producers who can plan what sex of breeding animals they will market in upcoming sales. although ultrasonography is becoming more economical and more accurate as technology advances, it remains a costly tool for practitioners that imparts costs to producers. likewise, although ultrasonography can detect pregnancy earlier than transrectal palpation of uterine contents, it does not prevent the possibility of loss after pregnancy diagnosis. lastly, one of the newest technologies utilized for pregnancy determination are blood-based pregnancy tests. similar to figure 1. proposed factors contributing to pregnancy loss in beef cattle; pregnancy loss is a multifactorial issue and is likely a reflection of inadequacy at the embryonic, maternal, paternal and environmental levels (ega: embryonic genome activation, mrp: maternal recognition of pregnancy, pag: pregnancy associated glycoproteins, ifnt: interferon-tau, p4: progesterone pg: prostaglandins). http://dx.doi.org/10.58292/ct.v17.11037 citation: clinical theriogenology 2025, 17, 11037, http://dx.doi.org/10.58292/ct.v17.11037 3 ultrasonography, blood or milk-based tests are performed as early as 28 days after breeding and rely on the detection of a family of proteins called pregnancy associated glycoprotein (pag) in maternal circulation. detectable concentrations of pag are only present in maternal circulation when the animal is pregnant or just recently calved or aborted. one advantage of blood or milk tests is that producers do not require substantial training or, in the case of rapid tests, expensive equipment to perform. additionally, blood or milk-based tests also offer pregnancy status determination for operations that are located far away from a technician capable of performing transrectal palpation or ultrasonography; furthermore, travel expenses for technicians to get to an operation are eliminated. blood or milk tests are performed either chute side or shipped to laboratories across the country for pag quantification. chute-side results are generated within 20 minutes, whereas blood shipped to the laboratory generally have results within a week. therefore, the ability of producers to quickly make management decisions pertaining to pregnancy status relies on the type of test utilized. commercially available blood-based pregnancy tests have true-positive rates of 93-98% and false-positive rates of 1-7%.9,10 thus, one clear limitation of blood or milk-based pregnancy determination is the risk of false-positives for animals experiencing embryonic mortality occurring after collection of blood or milk used for initial pregnancy status determination and the period of secondary confirmation via blood or milk test. cows that recently experienced embryonic mortality or immediately after parturition maintained elevated concentrations of pags.11,12 collectively, each of these methods of pregnancy determination are suited for various operations but each of them ultimately results in the same outcome, increased reproductive efficiency by determining nonpregnant status earlier. ability to accurately determine pregnancy status allows producers to make management decisions, ultimately improving farm profitability and sustainability of beef production. embryonic contributions to pregnancy loss first week of pregnancy encompasses the greatest proportion of pregnancy loss in beef and dairy cattle.1,2 during this period there are major developmental milestones that are critical for pregnancy success such as fertilization, embryonic genome activation, blastocyst formation, and hatching from the zona pellucida. a meta-analysis compiling data from 12 studies investigating pregnancy loss in beef cattle through day 7 of pregnancy was 28.4% and within these studies, before day 4, pregnancy loss was 23%.1 in dairy cattle, it has been estimated that only ~ 50% of ovulated oocytes will generate viable embryos between days 6 and 8 after insemination.2 given the large proportion of attrition occurring during the first week of pregnancy, discussing specific embryonic factors associated with pregnancy failure is warranted. estimated rates of fertilization in beef and dairy cattle are predicted to be ~ 90%, indicating that substantial losses occur after gametic syngamy.13-15 although the consensus is that ovulated oocytes do not differ in the ability to undergo fertilization, given compliance with accurate artificial insemination (ai) techniques, there are factors prior to ovulation that can impact oocyte developmental competence. indeed, manipulating the periovulatory hormonal environment can influence fertility outcomes. one such example is in cows that ovulated smaller follicles at estrus had fewer pregnancies per ai compared to cows that ovulated larger follicles.16 evidence from dairy cattle indicated that follicular growth is altered when progesterone concentrations are lower, hindering oocyte maturation and ability to establish pregnancy.17 although it should be acknowledged that many determinants of embryonic competence are influenced by preovulatory factors, the activation of the embryonic genome also presents a critical period of embryonic mortality. embryonic genome activation, also called the maternal-to-embryonic transition, is a transitory period occurring around the 8-16 cell stage of embryonic development whereby the early embryo degrades maternal rnas and proteins and begins transcription and translation of the newly formed genomic products.18,19 the molecular mechanisms regulating embryonic genome activation have yet to be fully elucidated; however, failure of the embryo to complete this transition results in the inability of the embryo to continue. collectively, identifying optimal periovulatory physiology and regulators of the maternal-embryonic transition present opportunities to improve embryonic survival and mitigate pregnancy losses. the high incidence of pregnancy loss during the first weeks of pregnancy has driven concerted efforts to try and predict embryonic competence to sustain pregnancy to term.20 these efforts have been focused on identifying biomarkers indicative of pregnancy success during the preimplantation and postattachment periods of development. early statistical models utilizing calving data from more than 4,500 embryo transfers attempted to predict embryo survivability to term and revealed that only 50-70% of embryos and recipients are competent to result in a calf.21 these authors concluded that factors external to the embryo (e.g. recipient) are critical regulators to embryonic survival or loss.21 as technology has advanced, the tools utilized to predict embryonic competence have also expanded. indeed, the use of machine learning to incorporate highly detailed models with developmental outcomes has been explored to identify embryonic genes predictive of competence. one study that combined transcriptomic data from blastocysts of known developmental competence with transcriptomic data of long and short conceptuses identified differentially expressed genes amongst the populations and integrated these genes into pathways predictive of embryonic competence and conceptus elongation.20 the 341 differentially expressed genes associated with embryonic competence were annotated to pathways relating to metabolic processes, glycolysis/gluconeogenesis, and glycerolipid metabolism.20 similarly, 669 genes associated with embryonic incompetence were annotated to the spliceosome, rna processing, and cell cycle regulation.20 together, these authors suggest that specific transcriptional patterns within the first weeks of pregnancy are identified as predictors of embryonic success, but also, the pathways identified may reveal novel targets for reducing pregnancy failure. the authors further utilized machine learning to discriminate differentially expressed genes into genes predictive of pregnancy success or failure and identified 8 genes (chsy1, gsto1, tpi1, ccna2, cdk7, eif4a3, lsm4, and ywhag); the first 3 are predicted to be expressed within competent blastocysts and last 5 expressed in incompetent blastocysts.20 collectively, this work has provided insights into the molecular signature of embryos predicted to lead to pregnancy success. further works remain to test the validity of these candidate biomarker genes and to interrogate the pathways regulating embryonic competency; however, the increased utilization of next generation sequencing and machine learning will permit further understanding of the processes leading to pregnancy success. failure to elicit maternal recognition of pregnancy around day 16 of the normal estrous cycle, follicular estradiol concentrations begin to increase that stimulate the actions of estrogen receptor alpha leading to increased http://dx.doi.org/10.58292/ct.v17.11037 4 citation: clinical theriogenology 2025, 17, 11037, http://dx.doi.org/10.58292/ct.v17.11037 expression of oxytocin receptors in the endometrium, ultimately perpetuating pulsatile secretion of prostaglandin f2α (pgf2α).22 these pgf2α pulses act as the primary luteolytic signaling molecule causing regression of the corpus luteum, thus beginning a new estrous cycle. when a conceptus is present within the uterine lumen, however, interferon tau (ifnt) secreted by the trophoblast cells in the placenta work to block the luteolytic cascade and maintain the corpus luteum. this process, deemed maternal recognition of pregnancy, is also heavily reliant on embryonic secreted factors. although ifnt is primarily attributed to its role in cycle extension, ifnt also elicits robust changes in the local endometrial landscape and to peripheral physiology. for example, ifnt secreted by the conceptus will exit from the uterine vein and increase the expression of interferon stimulated genes (isg) such as isg15, mx1, mx2, and oas1 in extrauterine tissues such as circulating blood cells and the corpus luteum compared to nonpregnant controls.23-25 the dynamic changes to extrauterine expression in isgs have been utilized as a predictor of pregnancy loss. indeed, recent works have demonstrated an association between elevated concentrations of isgs and pregnancy maintenance in dairy cattle.26 currently, the physiological implication of ifnt-induced changes to maternal immune cells in the periphery are not clear. transcriptomic analyses of peripheral leukocytes collected 21 days after embryo transfer have been conducted to compare between cows that maintained or lost pregnancy.27 sequencing revealed that the top upregulated pathways were related to inflammatory chemokine activity and immune defense response, suggesting that ifnt has a role in modulating immune tolerance.27 the roles of conceptus-derived ifnt promoting immune tolerance and recognition of pregnancy in extrauterine tissues require further study, and understanding the mechanisms of immune tolerance in cattle presents a unique opportunity to mitigate pregnancy failure by identifying key regulatory pathways. another consideration when discussing ifnt is the abundance or dose of ifnt secreted by the trophoblast cells. it is known that endometrium responds differently to shorter and longer conceptuses.28 this finding is interesting given recent in vitro data indicating that endometrial explants had doseand period-dependent changes to gene expression in response to ifnt.29 taken together, these data implied that inadequate or inappropriately timed secretion of ifnt by the conceptus can fail to elicit a response within the endometrium and ultimately fail to rescue corpus luteum. thus, failure of the conceptus to secrete sufficient amounts of ifnt could contribute to pregnancy loss. given that secretion of ifnt is dependent on the embryo, future studies should investigate whether the conceptus must secrete baseline concentrations of ifnt to elicit a maternal response. lastly, although ifnt is known to induce changes to the endometrial transcriptome, endometrium also responds to other secreted factors independent of ifnt.30 a novel experiment cultured endometrial explants with either a day 15 conceptus derived from in vitro fertilization, day 15 conceptus derived from ai, 100 ng/ml of recombinant ifnt, or medium alone revealed 240 differentially expressed genes in endometrial explants cultured in the presence of conceptus, regardless of origin.30 indeed, infusion of conceptus-derived prostaglandins into the uterine lumen of cyclic ewes induced changes in the expression of classical type i interferon-stimulated genes isg15 and rsad2.31 collectively, roles of prostaglandins during the periattachment period warrant further study. in particular, roles of prostaglandins secreted by the conceptus and the endometrium as they pertain to attachment need further exploration. attachment failure at the embryonic-maternal interface although evidence strongly indicates that the proportion of pregnancy loss decreases as pregnancy progresses, the incidence of pregnancy loss during the late embryonic and early fetal period is limited in the characterization.1 this period of embryonic development occurs after maternal recognition of pregnancy ~ days 21-28 of pregnancy, encompassing the earliest phases of placental apposition, adhesion, and attachment.32,33 during this period of pregnancy, the placental trophoblast cells begin to differentiate into 2 morphologically recognizable populations delineated by the number of nuclei (mononucleated or binucleated).32 binucleated trophoblast cells comprise 15-20% of trophoblast cells and will migrate through microvillar junctions of the uterine luminal epithelium, ultimately forming a fetal-maternal syncytium.34 trophoblast binucleate cells upon final maturation begin to rapidly express and secrete pag.35 the pag family of proteins represent a group of aspartic proteinases that are highly expressed products of the cetartiodactyla placenta.35 at the placental-epithelial interface, there is an abundance of pagpositive cells beginning around day 21.33 functional roles of pag are still explored; a study treated endometrial explants isolated from pregnant and nonpregnant animals 18 days after estrus with an equal mixture of pag 4, 6, and 9 resulted in transcriptional changes for proteins associated with matrix remodeling, chemokine production and prostaglandin release.36 thus, changes elicited in response to pag locally in the endometrium and peripherally need to be investigated. in particular, the immunomodulatory roles of pag during the periattachment period warrant further study. as aforementioned, pag concentrations within maternal circulation are an accurate marker to predict embryonic mortality.37 pregnant cows with higher peripheral concentrations of pag experienced increased embryonic survival compared to those with lower circulating pag concentrations (figure 2).11,38 additionally, it has been documented that cows undergoing late embryonic mortality had differing circulating pag patterns compared to those that maintained pregnancy (figure 2).11 given that pag concentrations in maternal circulation are associated with embryonic competence or mortality, roles of pag in the physiology of pregnancy competence are explored. recent evidence has indicated that cows with higher placentome blood perfusion have elevated concentrations of circulating pag compared to cows with lower placentome blood perfusion.39 in lactating dairy cattle, delayed increases in pag was indicative of inappropriately timed or insufficient embryonic attachment.26 collectively, the pagmediated physiological changes at the placental-maternal interface are still early in the characterization. the literature has established that pags, an embryonic product, are a strong measure of fetal monitoring and are incorporated in parametric analyses to predict pregnancy failure. however, the specific molecular and cellular functions of pag have not been completely elucidated and therefore researchers are interested in understanding the roles of pag in quintessential aspects of placentation, such as tissue remodeling. in summary, the embryo represents a major contributing factor to the success or failure of pregnancy. therefore, there are many pivotal risk periods for determining embryonic success for pregnancy. current and future works should investigate and test more precise predictors of embryonic competence. furthermore, understanding the molecular and cellular physiology regulating positive pregnancy outcomes could be used http://dx.doi.org/10.58292/ct.v17.11037 citation: clinical theriogenology 2025, 17, 11037, http://dx.doi.org/10.58292/ct.v17.11037 5 to study the supplementation of factors that may be lacking in embryos progressing towards failure. contributions of the maternal uterine environment to pregnancy loss maternal-embryonic communication is critical in achieving reproductive success, and the uterus responds dynamically to the embryo throughout pregnancy. secretion of specific proteins, hormones, and growth factors by the embryo and endometrium within the lumen are essential for the establishment and maintenance of pregnancy. therefore, understanding this dialogue is necessary for elucidating the underlying reasons for pregnancy loss. the fundamental role of the endometrium prior to the development of a functioning cotyledonary placenta is to support rapid embryonic development and conceptus growth.40 thus, the uterine environment is a critical regulator in pregnancy success or failure. prior to conceptus attachment to the luminal epithelium, the conceptus relies on endometrial secretions, also called histotroph, to support cell proliferation, migration and the morphological changes that occur during elongation.41 perturbing the native uterine histotroph composition by flushing the uterine lumen with saline on days 4 or 7 after estrus reduced pregnancy per embryo transfer by 33.1 and 30.6%, respectively.42 this evidence is supported by ovine models of uterine gland ablation, whereby histotroph composition is dramatically altered, and hatched blastocysts are unable to undergo elongation.43 furthermore, machine learning has been utilized to integrate the endometrial transcriptome 7 days after estrus with pregnancy outcomes to predict genes linked to uterine receptivity.44 in this discriminate analysis, 50 genes were identified and could predict uterine receptivity with an overall accuracy of 96.1%, regardless of the breed of the animal.44 therefore, inadequate transcription or secretion of necessary factors within the uterus can contribute to pregnancy loss. coincidentally, identifying secreted factors within the uterine lumen that are critical for embryonic development presents an opportunity to improve pregnancy outcomes and mitigate pregnancy losses. failure to respond to conceptus-derived signals and maintain the corpus luteum as previously mentioned, the bovine conceptus begins to secrete ifnt around day 16, and this type 1 interferon elicits robust responses in the endometrium, ultimately extending the life of the corpus luteum. the signaling actions of ifnt are perpetuated in a paracrine manner are reviewed.45 evidence from sheep has demonstrated that ifnt signals via the interferon alpha and beta receptors on luminal endometrial epithelial cells to inhibit expression of esr1 and oxtr, ultimately diminishing the pulsatile release of endometrial pgf2α. 45,46 thus, the ability of the endometrium to respond to ifnt is critical to successful maternal recognition of pregnancy and maintenance of the corpus luteum. recent work in dairy heifers has revealed that the endometrial responsiveness to ifnt is variable among individuals, and this variability is associated with subsequent fertility.47 after intrauterine infusion of recombinant ifnt, the endometrial transcriptome had differences in expression between heifers classified as highly fertile and subfertile and these genes were associated with cell signaling, metabolism, attachment, migration, and extracellular matrix proteins.47 further, subfertile heifers had lower concentrations of glycerol and oxylipins derived from arachidonic acid within uterine luminal fluid after ifnt infusion.47 together, this work indicated that individual animals may have differential responsiveness to ifnt signaling. in the context of pregnancy loss, identifying regulators of ifnt sensitivity present an opportunity to improve fertility outcomes, and although the secretion of ifnt is dependent on the embryo, it is worthy of considering if the endometrium has a threshold of sensitivity to various doses of ifnt that is sufficient to elicit a signaling response. roles of aberrant maternal prostaglandins in pregnancy loss prostaglandins are lipid-based signaling molecules that have essential roles in regulating bodily processes. prostaglandins are synthesized from arachidonic acid, and the synthesis of various prostaglandins is mediated by the cyclooxygenase (cox) family of proteins. in cattle, prostaglandins (f2α and e2) begin a definitive increase between days 31-35 of pregnancy and increases in the concentrations of these hormones have been positively correlated with pregnancy.48,49 the expression of ptgs2 (cox2) is upregulated during ovine implantation and is directly related to the degree of invasion of the trophoblast cells.50 furthermore, ptgs2 in the mouse model had upregulated during placental development and is therefore involved in decidualization and angiogenesis, promoting placental development.51 as mentioned above, estrus cyclicity in figure 2. relationship between peripheral pag and embryonic survival: a. cows with lower concentrations of plasma pag on day 28 of pregnancy experience higher pregnancy loss compared to cows that maintained pregnancy (adapted11); b. cows experiencing embryonic mortality have lower concentrations of plasma pag on day 30 of pregnancy compared to cows with embryonic survival (adapted38). http://dx.doi.org/10.58292/ct.v17.11037 6 citation: clinical theriogenology 2025, 17, 11037, http://dx.doi.org/10.58292/ct.v17.11037 cattle is highly dependent on appropriate secretion and regulation of prostaglandins from the endometrium. indeed, during the maternal recognition period, the ablation of pgf2α pulses is necessary for the maintenance and prolonged lifespan of the corpus luteum; however, during normal cyclic scenarios in the absence of a conceptus, prostaglandin will reach the corpus luteum and initiate luteal regression. although pgf2α is of critical importance to maintain normal cyclicity, the exact roles of pgf2α during placentation are unknown. the conceptus begins to synthesize and secrete prostaglandins around day 13 of pregnancy, eliciting changes to endometrial gene expression.31 further, circulating pgf2α concentrations increased during days 31-35 in beef cows that maintained pregnancy compared to cows that underwent embryonic mortality.48 this period of pregnancy corresponds to the period of active placentation, suggesting that pgf2α may be necessary in facilitating proper attachment of the bovine embryo. indeed, placental cells are under strict hormonal control; therefore, these hormones tightly regulate the abundance and activity of proteins involved in extracellular matrix remodeling at the placental-endometrial interface.52 treating caruncular epithelial cells isolated from pregnant cows with pgf2α increased cell viability and adhesion.52 therefore it is likely that appropriate secretion of prostaglandins from either the conceptus and/or endometrium are required to facilitate early placentation events. further, bovine endometrial cells treated in vitro with indomethacin and aspirin (prostaglandin inhibitors), experienced decreases in cell proliferation and cell viability.53 collectively, it is becoming more apparent that prostaglandins are having a critical role in facilitating dialogue between the conceptus and endometrium, having a role in placentation events such as extracellular matrix remodeling and adhesion, and therefore may be targets for improving pregnancy loss. there are still many unknowns regarding the maternal contribution to pregnancy loss in cattle. likewise, there are consistently novel findings published relating to the structure and establishment of the bovine placenta, the dynamic endocrine milieu of pregnancy, and the delicate cell-cell interactions at the placental-endometrial interface that will aid in the understanding of the mechanisms leading to late embryonic/fetal loss in bovine. a proposed model of key maternal factors contributing to pregnancy failure is depicted in figure 3. paternal contributions to pregnancy loss although the sire is traditionally speculated to only contribute half of the genetic material to the oocyte during fertilization, this line of thinking is rapidly challenged as more information pertaining to paternal contributions to pregnancy are revealed. further, although research regarding pregnancy loss has mainly focused on maternal and embryonic contributions, there is considerable variation in pregnancy loss among sires.54–57 consequently, variation in genetic indices to measure sire fertility have been developed and rapidly utilized in dairy bulls; however, genomic-based tools have been less utilized in beef herds because of the lack of data and validation of quantitative trait loci.58,59 consequently, sire phenotypic data have been utilized to make associations with fertility. indeed, associations among bull libido, scrotal circumference, backfat thickness, testis weight, semen quality parameters and sire residual feed intake have all been linked with variation in reproductive function.60,61 given that genetic selection parameters for beef bull fertility are limiting, other avenues have been explored to evaluate sire fertility and predict sires that will support embryonic development. although poor sperm morphometric parameters are useful for chute-side breeding soundness exams to quickly rule out unacceptable bulls, other more quantitative measurements are utilized to improve sire fertility. computer assisted sperm analysis (casa) monitors sperm movement and applies algorithms to the behavior of sperm cells to estimate sperm cell morphokinetic defects in real time62; casa is used to predict beef bull fertility in timed ai systems.63 compared to other figure 3. conceptual model of maternal factors contributing to pregnancy failure. perturbations in luminal histotroph composition, variance in the endometrial response to interferon-tau, and altered prostaglandin metabolism may all contribute to pregnancy failure at various stages of pregnancy. identifying key regulators of embryonic survival during milestones of pregnancy will permit a deeper understanding of pregnancy loss. http://dx.doi.org/10.58292/ct.v17.11037 citation: clinical theriogenology 2025, 17, 11037, http://dx.doi.org/10.58292/ct.v17.11037 7 various qualitative measurements assessed in the study, casa had the greatest coefficient of determination for fertility.63 this study also generated a composite model by incorporating casa data with image-based flow cytometric analyses, and together these 2 metrics were successful in predicting bull fertility.63 one recent study classified 2 groups of bulls as high or low fertility sires and attempted to objectively measure sperm quality.64 one promising method was the detection of aggresomes located in the head of the sperm. aggresomes are a buildup of unwanted proteins that occur after a failure in protein modification and are associated with cell death.64 interestingly, low fertility sires had a significantly higher amount of aggresomes located in the head of the sperm cell (via fluorescent microscopy) prior to a gradient purification commonly used for in vitro fertilization, but this difference between fertility groups disappeared after purification.64 further study of aggresome defects identified that the low fertility sires had a much higher percentage of aggresome defects via image-based flow cytometry.64 collectively, new molecular insights are developed to quantitatively evaluate sperm cell dynamics and integrate these data into predictive indices independent of paternal genomics. advancing sperm cell diagnostic and predictive abilities will work in part to reduce pregnancy failure by minimizing errors attributed to inherent sperm defects. as mentioned above, indices such as sire conception rate (scr) (defined as the probability of a single straw of semen to yield a pregnancy as compared to the means of other bulls in the population) have been developed and are heavily utilized as proxies to estimate sire fertility.65 despite these indices, the relationship between estimated sire fertility and the paternal contributions to pregnancy failure has remained elusive. one study utilized 10 sires classified as either high or low scr for in vitro and in vivo embryo production; low scr sires produced fewer blastocysts compared to high scr.66 after superovulation, low scr sires produced a higher percentage of unfertilized oocytes and degenerated embryos than high scr sires.66 together, these data indicated that low scr sires may have altered ability to fertilize oocytes and support early embryonic development, ultimately reducing pregnancy establishment. although the relationship between scr and the ability of the sire to support embryonic development during early pregnancy is interesting, a further point should be highlighted. in these studies, there are presumed no differences in sperm cell morphology and motility such that the sperm utilized between high and low fertility bulls would pass routine screening examinations, indicating that differences at the molecular level may be facilitating the reduced ability to support embryonic development. research into the molecular contributions and alterations of sperm to embryonic development will permit more robust fertility screening types and minimize the proportion of early embryonic failure as a result of improper paternal factors. sire contributions to placentation one of the proposed reasons why individual sires have variable incidence of pregnancy loss is the possibility of variation in the paternal contributions to placentation during the late embryonic period of development. in this hypothesis, it is proposed that differences in pag expression or transmission of pag to maternal circulation (via placental vasculature) may be a major component behind the variation in pregnancy losses amongst sires. indeed, pregnancies sired by some sires consistently yielded higher peripheral concentrations of pag and experienced lower percentages of pregnancy loss compared to pregnancies generated by sires yielding lower concentrations of pag (figure 4).11,37,56,67 given that complete and successful placentation is required for the continuation of pregnancy to term, one of the tools utilized to further understand the contributions of the sire in successful pregnancy and placentation is the use of uniparental embryos. parthenogenic (pa) embryos are embryos that contain only the maternal genome and are therefore lacking the entire paternal genome.68 seminal experiments conducted in mice demonstrated that zygotes generated using 2 male pronuclei yielded poorly developed embryos with normally developing trophoblast, whereas zygotes produced with 2 female pronuclei generate relatively normal embryos, but poor extraembryonic tissue.69,70 previous studies in cattle have suggested that the sire may be one of the primary contributing factors to a fully developed and functioning placenta.56,71 some evidence indicated that although pa embryos are capable of survival past the period of maternal recognition of pregnancy, the transfer of a single pa embryo on day 8 after estrus failed to increase isg15 expression in peripheral granulocytes compared to an in vivo-produced embryo.72 further, the transfer of multiple pa was sufficient to increase uterine ifnt protein concentrations, indicating that pa embryos are capable of transcribing and translating the signal required to extend the estrous cycle.72 despite the ability of pa embryos to extend the estrous cycle, the role of the sire in specific aspects of placentation requires further research. collectively these data strongly suggest that sires have critical roles in pregnancy that expand beyond fertilization but also that unknown sire factors are required for complete and successful attachment, implantation, and overall placentation. environmental contributions to pregnancy loss there is a close relationship between environmental exposures and reproductive success in cattle. some of the factors that have garnered the most attention in beef cattle reproductive physiology are the interactions of pregnancy and nutritional status, exposure to elevated environmental temperatures, figure 4. relationship between sire and pag concentrations in maternal circulation; sires classified as low embryonic loss have higher pag concentrations compared to sires classified as high embryonic loss (adapted37). http://dx.doi.org/10.58292/ct.v17.11037 8 citation: clinical theriogenology 2025, 17, 11037, http://dx.doi.org/10.58292/ct.v17.11037 and disease. these factors have drawn attention because of the negative relationship with fertility. although many of the studies investigating the relationship between nutritional status and fertility have been conducted in dairy cattle, beef cattle also face drastic challenges relating to nutritional availability. adequate nutrition is critical not only during the periconceptual and postpartum period, but proper management of nutrition for prepubertal heifers can impact lifetime reproductive performance.73,74 indeed, nutritional requirements are influenced by cow’s breed, season, and parity, but are also dynamic depending on the physiological status of the cow. for example, nutrient requirements increase as pregnancy progresses.75 the importance of available nutrients is evident through experiments in cows with a lower body condition score (bcs) had lower pregnancy rates after ai.76 this relationship is due to the imbalance between the hypothalamic-gonadal axis, whereby cows with lower bcs did not return to appropriate cyclicity.73 cows supplemented with 100 or 150% of their energy and protein requirement during the third trimester of pregnancy had faster ovarian follicular growth and more ovulatory follicles 21 days postpartum compared to cows maintained on pasture.77 this change in ovulatory capacity directly related to increased pregnancy rates in cows fed 150% of their energy and protein requirement, ultimately increasing offspring sale value.77 although fertility increases in supplemented cows, it must be noted that over supplementation of nutrients can also yield negative effects on fertility. for example, cows with elevated bcs (obese) had lower pregnancy rates after embryo transfer, and lower blastocyst rates after superstimulation compared to cows with a moderate bcs.24,25 these data provide strong evidence that metabolic status and nutrient supply are 2 factors that require close management for optimal reproductive efficiency. approximately 70% of the world’s cattle population are in locations considered tropical or subtropical. reproductive processes are in cattle are sensitive to hyperthermic condition.78 indeed, heat stress is attributed to reduced estrous behavior, impaired follicular development, reduced oocyte competence, and inhibited embryonic development.78 therefore, cattle that are well adapted to these environments are necessary to remain efficient and sustainable. for example, oocytes collected from nelore (bos indicus) cows were more tolerant of artificial heat stress compared to jersey and angus (bos taurus) cows.79 this was evident by nelore-derived embryos having higher blastocyst production, higher expression of cell division markers, and lower expression of apoptotic markers when compared to bos taurus-derived embryos79; furthermore, there was a tendency for angus-derived embryos to yield fewer pregnancies compared to nelore embryos.79 another source of environmental influence of pregnancy loss is the role of infectious diseases in populations of cattle. although infectious agents can hinder embryonic development, the most pronounced and costly period of abortion occurs between 42 and 260 days. recent reviews identified the most common infectious agents associated with abortion in cattle and revealed that neospora caninum, trueperella pyogenes, bovine viral diarrhea virus, infectious bovine rhinotracheitis, leptospira species and fungal infections are the most diagnosed abortive agents figure 5. schematic of critical factors contributing to pregnancy success and failure. pregnancy loss is multifactorial, and the incidence of pregnancy failure changes depending on the timepoint of pregnancy. there are specific temporal failures that contribute collectively to pregnancy loss, and reducing these losses at each point will contribute to higher reproductive efficiency and sustainability of beef production. http://dx.doi.org/10.58292/ct.v17.11037 citation: clinical theriogenology 2025, 17, 11037, http://dx.doi.org/10.58292/ct.v17.11037 9 identified in beef cattle.80,81 the best management practices to mitigate the impacts of disease-induced abortion are to prevent transmission via rapid diagnostics, maintaining strong biosecurity practices, and appropriate management of positive cattle. monitoring and reducing the incidence of infectious diseases in cattle populations will aid in minimizing pregnancy loss. conclusion pregnancy success is highly dependent on numerous conditions, and therefore, the etiology of pregnancy loss is multifactorial. a schematic of critical factors contributing to pregnancy success and failure is depicted in figure 5. the maternal contribution to pregnancy begins well before the period of fertilization, whereby complete maturation and final development of the oocyte must be completed. from here on, the maternal endocrine milieu must be conducive for ovulation and rapid reprogramming of ovarian function to prepare for pregnancy. indeed, the changes to the uterine luminal environment are likely driven by changes to circulating ovarian steroids, and these changes are paramount for supporting the earliest phases of embryonic development. shortly afterwards, a symbiosis must occur where the embryonic and maternal systems must work together in concert through cell signaling and proliferation to allow for complete attachment and placentation. although the maternal and 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https://doi.org/10.1093/jas/skaa358 https://doi.org/10.17226/19014 https://doi.org/10.1016/j.theriogenology.2019.06.024 https://doi.org/10.1016/j.animal.2020.100089 https://doi.org/10.1016/j.animal.2020.100089 https://doi.org/10.1016/s0093-691x(00)​00448-9 https://doi.org/10.1016/s0093-691x(00)​00448-9 https://doi.org/10.1016/j.theriogenology.2012.10.003 https://doi.org/10.1016/j.prevetmed.2021.105366 https://doi.org/10.1016/j.prevetmed.2021.105366 https://doi.org/10.1111/rda.14366 https://doi.org/10.1111/rda.14366 2012: factors affecting the reproductive capacity of water buffalo (bubalus bubalis) factors affecting the reproductive capacity of water buffalo (bubalus bubalis) mona radheshwar, mushtaq a. memon department of veterinary clinical sciences, washington state university, pullman, wa abstract the water buffalo has historically been one of the most important agriculture animals of asia and today herds are now raised across the mediterranean regions as well as the americas. of great economic value for the milk’s high fat content and lean meat, buffalo production lags behind other domestic cattle species in terms of reproductive efficiency. water buffalo have a reputation as being poor breeders due to a propensity for long calving intervals, high incidence of postpartum anestrous and late age of maturity. the objectives of this investigation were to examine the environmental and genetic factors that affect male and female reproductive potential. when implemented, simple management practices such as proper nutrition year round, prevention of heat stress and the housing of male and females together have the potential to overcome the water buffalo’s apparent reproductive shortcomings. the maintenance of production and progeny testing records are essential for genetic improvement programs, which are slowing being more prominent at institutional farms across the world. keywords: water buffalo, reproductive capacity, reproduction introduction while water buffalo (bubalus bubalis) are not commonly raised in the united states, it is fast becoming the most important agricultural animal of asia, serving as both a dairy and meat animal as well as a beast of burden. measures of production capacity and reproductive potential of the buffalo are often compared to the domestic cow bos taurus and bos indicus, to which buffalo exhibit a comparatively excellent feed conversion rate and an innate ability to adapt and thrive in hot humid conditions of the tropics. the main issue affecting the farmer is the buffalo’s relatively poor reproductive potential which is most commonly attributed to a late age at first calving, high incidence of postpartum anestrous and seasonal depression of reproduction. by discussing the genetic and environmental factors effecting male and female reproductive potential with the global buffalo community, management solutions may be shared and adapted to promote water buffalo production in the various diverse region in which they thrive. water buffalo reproduction: the bull breeding soundness examinations for buffalo bulls have yet to be standardized and long term progeny testing records are scarce to non-existent in most parts of the world. italy has the most advanced genetic improvement program with milking records kept on 27.8% of the population.1 natural mating continues to be the main mode of breeding in buffalo herds, largely due to weak estrus expression and subsequent difficulties associated with the timing of artificial insemination (ai). in italy and pakistan, only 5-10% of buffalo farmers utilize ai.1 small herd size, long generational intervals and scarcity of production records are major factors limiting the use of progeny testing of buffalo herds for genetic improvement.2 factors affecting male reproductive capacity genetic factors. the male buffalo typically reaches sexual maturity between 16 and 30 months of age, depending on breed and management system.3 peak reproductive potential is reached at five years of age after which a decline in sperm output per gram of testis per ejaculate may be observed.2,5 as in cattle, scrotal circumference (sc) is strongly correlated with semen quality and quantity in murrah buffalo bulls.24 scrotal circumference in murrah bulls aged 37 to 48 months is on average 29.2 cm; nearly 40% lower than corresponding mean value of 40.0 cm observed in holstein bulls aged 34-42 months. the maximum sc (32.1cm +/1.5) is typically reached by five years of age in buffalo. ejaculate volume ranges from 2.5 to 3.6 ml and sperm concentration ranges from 1475 to 1666 x 106/ml. in the 2009 study 487 clinical theriogenology • volume 4, number 4 • december 2012 by ahmad et al, bulls were collected with an artificial vagina twice a day for five weeks. average daily output was 2.18 x 109 to 3.37 x 109 sperm/day, significantly lower than that observed in holstein bulls. there was no significant difference between holstein and murrah buffalo bulls when sperm output per gram of testis per ejaculate (15.3 x 106 vs. 14.8 x 106) was compared. this information highlights the potential for buffalo breeders to increase bull fertility by selecting individual bulls with greater sc and maintaining long term progeny testing records. table 1. characteristics of murrah buffalo bull testis size and semen output collected as compared to age.5 attributes 25-36 months 37-48 months 49-60 months >60 months scrotal circumference (cm) 26.9 29.2 30.4 32.4 estimated testis weight (g) 261 427 498 652 sperm volume (ml) 2.5 3.1 3.5 3.6 sperm concentration (x106/ml) 1,677 1,493 1,475 1,666 breed variability. the murrah bull has been shown to reach sexual maturity by two to three years of age while murrah x mediterranean crossbred buffalos raised in intensive management systems in brazil may reach sexual maturity between 16 to 24 months.5,6 the average age at sexual maturity of niliravi bulls found in one study was 22.8 months.2 several studies document onset of puberty earlier in crossbred buffalos (mediterranean x jaffarabadi) at 10-14 months and (murrah x jaffarabadi) 13 months.6 in most swamp buffalo bulls, puberty is attained by 24 months of age, when sc is approximately 16 cm while sexual maturity, defined as the attainment of adult levels of daily sperm production per gram of testis parenchyma (14 x 106) occurs at 30 to 33 months of age, when sc is in the 17-to 20-cm range.7 the two subspecies of agricultural interest; the river buffalo (bubalus bubalis bubalis) and the swamp buffalo (bubalus bubalis carbanesis) have different karyotypes with 2n= 50 and 48 chromosomes for the river and swamp buffalo, respectively. cross breeding between the two subspecies produced subfertile f1 hydids.8,9 histological examination of hybrid f1 (murrah x swamp; 2n=49) testis revealed a large proportion of degenerating spermatocytes and abnormal spermatids in the process of spermatogenesis, presumably a result of abnormal pairing configurations f1, f2 and backcrosses with 2n=48, 49 or 50 had a significantly higher frequency of morphologically abnormal spermatid (48-72%) when compared to purebred river (29%) and swamp (16%) buffalo.8,10 prevalence of interbreeding between subspecies in rural areas could be a contributing factor to decreased male fertility and further studies may be warranted. environmental factors. buffalo bulls are capable of breeding all year round but seasonal fluctuations in reproductive output are consistently observed in most countries. not only is the male libido decreased during the warmer months, examination of semen showed significant differences in the post-thaw sperm motility of semen frozen according to season, with motility being significantly lower during summer months compared to winter and/or monsoon season.11,12,13 in a 2007 study examining post-thaw plasma membrane stability in thai swamp water buffalo spermatozoa, koonjaenak et al14 found the percentage of live sperm to be significantly higher (54.6%) when collected during the winter months than the rainy season (43.5%) or summer season (46.7%). the authors recommended that semen only be collected and processed during spring and winter.14 a bull’s access to females has been correlated with a positive effect on blood testosterone levels. in the study by malfatti et al,15 bulls were divided into two groups and blood testosterone levels were measured monthly for one year. bulls in group a were housed with females, while bulls in group b were held separate from the cows during the winter months, october to february. blood testosterone levels of group a showed no significant difference between winter and summer months while mean serum 488clinical theriogenology • volume 4, number 4 • december 2012 testosterone concentrations were significantly lower in the bulls during the separation period (octoberfebruary) than during the rest of the year. it may be concluded that the contact with females has a stimulating effect on testicular androgen secretion in buffalo bulls even when considering seasonal factors affecting reproduction. 15 in tropical countries such as pakistan, male libido has been shown to significantly decrease during the hot dry season.16 bulls housed in intensive management systems of italy, do not show a significant difference in blood testosterone levels or libido during winter vs. summer months. the seasonal depression in libido and reproduction in general can most likely be attributed to environmental factors such as heat stress, lack of green fodder and decreased food availability as apposed to being influenced by an increased photoperiod.15 it may be concluded that access to adequate feed, wallowing ponds, and cooling misters as well as presence of females during the hot dry season may help to overcome problems associated with buffalo reproductive seasonality.15,16 water buffalo reproduction: the buffalo cow buffalos are commonly known as “poor breeders” when compared to other bovine species.17 this is often attributed to late onset of puberty and sexual maturity, weak or silent estrus expression, prolonged calving interval, postpartum anestrus, seasonality of reproductive cycles and obstetrical problems such as uterine torsions.18 on the other hand, current research has shown that fertility parameters such as conception rate, pregnancy rate and calving interval under intensive management systems are comparable to the rates achieved in cattle.19 while water buffalo, as compared to bos taurus, may be relatively late in reaching sexual maturity, they are renowned for their longevity. in italy, it is common to find highly productive buffalo cows with three, four, or more daughters with registered lactations.20 factors affecting female reproductive capacity genetic factors. the productive life of a buffalo, while often lasting seven to ten lactations, is often considered reduced due to the late age at onset of puberty.21 buffalos typically reach sexual maturity at 55-60% of their final body weight.18 age at onset of puberty in the buffalo shows a wide range — between 13 to 40 months depending on breed and management system (table 3). under favorable conditions puberty typically occurs at 15 to 18 months in river buffalo and 21 to 24 months in swamp buffalo.22 table 2. effect of farming system on reproductive parameters of buffalo cows. adapted from pasha et al23, borghese et al28, marai ifm et al24, zicarelli l et al20 reproductive parameters pakistan: rural subsistence pakistan: rural market oriented pakistan: periurban pakistan: comercial urban commercial: italy intensive: egypt age at puberty 34.2 mo 35.6mo 33.7mo 27.6mo 17-20mo n/a calving interval 18.4mo 17.2mo 16.9mo 15.2mo 13.3mo (400) days 13.5mo use of ai 15% 14.70% 33.40% 23.10% 5% n/a in female buffalos, estrus behaviors such as homosexual activity are rare compared to bos taurus species making heat detection significantly more challenging.25,26 while subtle signs of heat may include clear mucus, restlessness, frequent voiding of small amounts of urine, bellowing and swelling of the vulvar lips, 100% of females in estrus will exhibit standing heat. when attempting to use natural estrus for artificial insemination (ai), the use of a teaser animal (buffalo male with penile deviation and vasectomy or androgenized female) with a chin-ball marker device is necessary to identify females in estrus. the ideal time to inseminate is six to 18 hours after the start of estrus as ovulation takes place ten to 16 hours after the end of heat.40 489 clinical theriogenology • volume 4, number 4 • december 2012 estrus duration can range between 10 and 20 hours in normally cycling buffalos during the breeding season, although the period of courtship or interest showed by the male has been reported to range anywhere from nine to 68 hours.20 the luteinizing hormone (lh) surge typically occurs between one and 12 hours after the start of estrus and ovulation occurs between 26 and 33 hours after the lh surge.29 due to the absence of consistent signs of behavioral estrus and variability in time of ovulation in relation to estrus, ai at observed natural heats is of debatable value in buffalo. post partum anestrous and prolonged calving interval. gestation length in river buffalo averages 310 days while swamp buffalo may gestate up to 330 days; relatively long when compared to the holstein cows.18 buffalo embryos enter the uterus at four to five days after ovulation and may hatch as early as day 5; much earlier than in bos taurus.28 compact morulae are observed five days after estrus with blastocysts typically developing by day 6.29 circulating progesterone levels ≥ 1.5 ng/ml, an indication of a functional corpus luteum (cl), occur at day 6 of the estrous cycle. embryonic development and cl formation in the buffalo are thus more tightly coupled than in cattle. research suggests a positive correlation between pregnancy rates and circulating progesterone levels on day 5 after ovulation. the use of exogenous hormonal protocols to support early embryonic development is currently being investigated.29 long calving intervals and postpartum anestrus are major causes of economic loss to buffalo breeders. based on anatomical features, the uterus of the buffalo is completely involuted by 45 days postpartum.30 studies in egypt, india, and pakistan demonstrated that only 34-49% of buffaloes showed estrus during the first 90 days after calving and 31-42% remained anestrus for more than 150 days.30 several factors inherent to traditional farming practices negatively affect return to cyclicity. there is sufficient evidence to conclude that the presence of a suckling calf significantly prolongs the period of postpartum anestrus.30 on the intensively managed dairy farms of italy, it is common practice to remove calves from their dam at one day of age. this early weaning results in a calving interval of 400-430 days as compared to an average range of 480-600 days found in many areas of rural india. 12,20 studies carried out in postpartum anestrous cows have demonstrated that progesterone treatment stimulates an increase in lh pulse frequency resulting in greater follicular fluid and circulating concentrations of estradiol and increased numbers of receptors for lh in granulosa and theca cells as compared to untreated animals.17 baruselli et al17 found the addition of a progesterone-releasing implant (cidr) combined with a fixed time ai protocol was successful in combating the depressive effects of seasonality and could also be used in the postpartum anestrous period to hasten a return to cyclicity.17 the following protocol was used in postpartum females with a 50% pregnancy rate at first insemination:  day 0: cidr plus 2.0 mg estradiol benzoate im  day 9: cidr is removed and prostaglandin f2α and 500 iu equine chorionic gonadotropin (ecg) im  day 11: 1500 iu ecg with timed ai 16 hours later complications of pregnancy. prepartum cervico-vaginal prolapse is hereditary and eradication may be obtained by removing predisposed females from breeding stock. uterine torsion is the most common cause of dystocia (70%) in the buffalo. use of sharma's detorsion method (rolling of the dam with external fixation of the uterus) and anti-stress measures increase survival rates in cases presented within 36 hours.31 in a recent study, most cases of uterine torsion were postcervical torsion (85.7%; the fold palpated caudal to the cervix) and only a few cases were precervical torsion (14.3%; the fold palpated cranial to the cervix).32 traditionally, many farmers associate uterine torsion with wallowing behavior during late gestation. environmental factors seasonality. buffalos are polyestrous animals capable of breeding throughout the year but a distinct seasonal reproductive pattern has been noted in several countries.18 when buffalo are bred in non-equatorial regions, they demonstrate reproductive behavior that is positively influenced by a decreasing photoperiod and is mediated by melatonin secretions.17,18 the endocrine profile of a buffalo 490clinical theriogenology • volume 4, number 4 • december 2012 during summer anestrous is characterized by low plasma concentrations of gonadotropic and gonadal hormones and high levels of prolactin leading to reduced lh pulse frequency, poor follicle maturation and decreased estradiol production.12,33 heat, humidity and availability of forage are the main contributing factors to lack of cyclicity and poor fertility in equatorial regions.13 heat stress. buffalos are adapted to hot, humid, muddy terrains but exhibit fewer physiological adaptations to extreme heat from direct solar radiation than other cattle breeds.24 the black color of buffalo skin combined with only one-sixth the density of sweat glands in cattle skin, contribute to the buffalo’s poor ability to dissipate heat through sweating.12 under natural conditions, buffalos prefer to wallow in water rather than seeking shade during periods of intense heat. management systems to prevent the deleterious effects of heat stress such as access to wallowing ponds or sprinkler systems have been shown to have a positive effect on dry matter intake, milk production and fertility.24 bull effect. the presence of bulls in a herd has been shown to have a biostimulatory effect on ovarian activity of buffalo cows.20 in order to combat the seasonal depression on cyclicity, italian farmers have developed a management practice technique termed out-of-breeding-season-mating (obsm), which takes advantage of the bull’s biostimulatory effect. this is especially important in italy where demand for milk is highest during a period of the year when calving is less frequent.34 in this technique (figure), bulls are removed from the herd in october (fall) and returned between march and the end of september (spring-summer) so that most calvings occur between the end of january and the beginning of august (spring-summer). figure: out of breeding season mating technique implemented in italy to combat the seasonal depression on reproduction. 491 clinical theriogenology • volume 4, number 4 • december 2012 in a 2010 study by gokuldas et al.35 the authors were able to show that bull exposure during the postpartum period accelerates resumption of ovarian cyclicity, reduces incidence of silent ovulation and enhances first service ai conception rates (table 3). table 3. comparison of reproductive parameters of female buffalos when exposed to vasectomized bull vs. when isolated from the bull between 40 and 90 days postpartum.35 reproductive parameter exposure to vasectomised bull from 40th to 90th day postpartum isolation from the bull resumption of ovarian cyclicity 47 ± 2.58 days 56 ± 2.37 days observation of behavioral estrus 57 ± 3.61 days 71 ± 5.13 days conception by 60 days post-partum (ai) 54% 15% first service ai conception rate 100% 37.50% nutrition. when heifers are exposed to intensive management and feeding systems, the age at puberty can be drastically reduced from those recorded at the rural farm level. when 30 heifers in italy received 4.5-5.5 milk forage units (mfu)/day, all achieved cyclicity before 20 months of age at a body weight of 421 kg or less. on the other hand, when heifers received 3.7-4.4 mfu/day, only 24% reached puberty by 20 months.36 these trials highlight the importance of a proper feeding system and early weight gain to hasten puberty and thus increase the productive life span of a buffalo. body condition score (bcs) and overall plane of nutrition are extremely important factors affecting reproductive efficiency. baruselli et al17 reported earlier first postpartum estrus in buffaloes (49±1 day) with bcs of 3.5 or higher than in those with a bcs of 3.5 or lower (77±1 day). adequate nutrition is essential for not only the onset of sexual maturity and cyclicity but also a timely return to normal cyclicity after parturition.30 in a recent study, buffalo heifers undergoing regular estrous cycles were randomly assigned to high energy diet group (he; 5.8 mfu/day) or a low energy diet group (le; 3.6 mfu/day). metabolic substrates and reproductive hormones were monitored weekly over a period of 19 weeks. results showed no significant difference in levels of circulating reproductive hormones between the two groups. heifers fed the low energy diet had a negative calculated daily energy provision yet were able to maintain body weight and reproductive activity. it may be concluded that buffalos have the capacity to undergo metabolic adjustments and reduce their energy requirements when dietary energy is scare which may explain why buffalos have the ability to stay productive in environments unsuitable for other ruminants.37 infertility and repeat breeding. clinical features of repeat breeding vary across the world and production systems. kishore et al established several infertility camps to examine features of repeat breeding in the chitwan district of nepal. of 85 buffalo presented to the camp, 51 (60%) were heifers with 29% being in poor body condition; 46% of the animals presented had a functioning cl; and 51% had a dominant follicle, indicating that about 50% of the animals had normal ovarian function. twenty percent of the animals were diagnosed with cervicitis, while only a few cases of endometritis were observed. animals were given three weeks of vitamin/mineral supplement and those with cervicitis were treated by washing the vagina with lugol’s solution. animals with a cl were injected with prostaglandin f2α (pgf) and those with a dominant follicle were given a dose of gonadotropin releasing hormone (gnrh). females responded well to hormonal treatment and vitamin supplementation; at the end of 6 492clinical theriogenology • volume 4, number 4 • december 2012 months, 100% of the females initially presented to the infertility camp were pregnant.38 one might infer that rural farmers could benefit greatly from education on proper nutrition of heifers, maintenance of body condition score and proper heat detection methods in order to decrease incidence of cervicitis. conclusions the reputation of water buffalos as having a low reproductive potential appears to be highly dependent on inefficient management practices and environmental factors rather than on their natural physiology. puberty can be hastened with highly managed feeding programs to encourage rapid weight gain. systems used for estrus detection in cows cannot be directly applied to buffalos but the use of a teaser bull can accurately identify females in standing heat as well as impart a biostimulatory effect on cyclicity. by ensuring animals are kept in good body condition and by removing sucking calves at one day of age, a return to cyclicty by 60 to 90 days postpartum can be reliably achieved. ovsynch and cidrsynch protocols developed for use in cattle can be applied to the buffalo with pregnancy rates comparable to or exceeding those in cattle despite the depressive effects of seasonality on buffalo fertility. references 1. borghese a, mazzi m: buffalo population and strategies in the world. in: borghese a, editor. buffalo production and research. rome: food and animal organization of the united nations; 2005. p 1-39. 2. sethi rk: progeny testing for genetic improvement in buffalos. proc int buffalo conf; 2010. p. 85-91. 3. das gk, khan fa: summer anestrus in buffalo-a review. reprod domest anim 2010;45:483-494. 4. ahmad ns, umair b, shahabm, et al: testicular development and establishment of spermatogenesis in nili-ravi buffalo bulls. theriogenology 2010;73:20-25. 5. pant hc, sharma rk, patel sh, et al: testicular development and its relationship to semen production in murrah buffalo bulls. theriogenology 2003;60:27-34. 6. da luz pac, andrighetto c, santos prs, et al: daily sperm production and evaluation of morphological reproductive parameters of murrah buffaloes in an extensive breeding system. spermatogenesis 2012;2:88-93. 7. mccool cj, entwistle kw: the development of puberty and sexual maturity in the australian swamp buffalo bull. theriogenology 1989;32:171-184. 8. dai k, gillies cb, dollin ae, et al: synaptonemal complex analysis of hybrid and purebred water buffaloes (bubalus bubalis). hereditas 1994;121:171-84. 9. yadav ps, singh b, singh i, et al: reproductive biotechnology in buffalo. dehli: satish serial publishing house; 2010. 10. bongso ta, hilmi m, basrur pk: testicular cells in hybrid water buffaloes (bubalus bubalis). res vet sci 1983;35:253-258. 11. andrabi smh: factors affecting the quality of cryopreserved buffalo (bubalus bubalis) bull spermatozoa. reprod domest anim 2009;44:552-569. 12. das d: buffalo breeding in north eastern region in india. proc int buffalo conf; 2010. p. 109-112. 13. sansone g, nastri mjf, fabbrocini a: storage of buffalo (bubalus bubalis) semen. anim reprod sci 2000;62:55-76. 14. koonjaenak s, pongpeng p, wirojwuthikul s, et al: seasonality affects post-thaw plasma membrane intactness and sperm velocities in spermatozoa from thai ai swamp buffalos (bubalus bubalis) theriogenology 2007; 67:1424-1435. 15. malfatti a, barbato o, todini l, et al: blood testosterone levels in italian mediterranean buffalo bulls managed in two different breeding conditions. theriogenology 2006;65:1137-1144. 16. younis m, samas h.a. ahmad n, et al: effects of age and season on the body weight, scrotal circumference and libido in nili-ravi buffalo bulls maintained at the semen production unit, qadirabad. pak vet j 2003;23:59-65. 17. baruselli ps, gimenes lu, carvalho nat, et al: folliculogenesis applied to reproductive biotechnologies in buffalos. proc int buffalo conf; 2010. p. 167-176. 18. perera bmao: reproduction in domestic buffalo. reprod domest anim 2008;43:200-206. 19. taylor u, rath d: flow cytometrical sex-sorting of buffalo semen: status and opportunities. proc int buffalo conf; 2010. p. 190-195. 20. zicarelli l, campanile g: constraints in application of ett in buffalos. proc int buffalo conf; 2010. p. 177-189. 21. madan ml: concerns and conflicts in buffalo production. proc int buffalo conf; 2010. p. 1-6. 22. perera bmao: reproductive cycles in buffalo. anim reprod sci 2011;124:194-199. 23. pasha tn: buffalo production systems in pakistan. proc int buffalo conf; 2010. p. 26-30. 24. marai ifm, haeeb aam: optimizing buffalo productivity through conventional and novel technologies. proc int buffalo conf; 2010. p. 15-25. 25. drost m: bubaline versus bovine reproduction. theriogenology 2007;68:447-449. 26. drost m: advanced reproductive technology in the water buffalo. theriogenology 2007;68:450-453. 27. vale wg: artificial insemination in buffaloes in latin america: state of the art. proc int buffalo conf; 2010.p. 61-69. 28. borghese a: buffalo production systems in europe and the near east. proc int buffalo conf; 2010. p. 31-39. 493 clinical theriogenology • volume 4, number 4 • december 2012 29. campanile g, pietro sb, negliaa g, et al: ovarian function in the buffalo and implications for embryo development and assisted reproduction. anim reprod sci 2010;121:1-11. 30. el-wishy ab: the postpartum buffalo ii. acyclicty and anestrus. anim reprod sci 2007;97:216-236. 31. nanda as, brar ps, prabhakar s: enhancing reproductive performance in dairy buffalo: major constraints and achievements. reprod suppl 2003;61:27-36. 32. swelum aa, amin se, eidaroos as, et al: prognosis prediction of uterine torsion mechanical treatment (rolling) after estimation of calcium and creatinine level in the serum of buffaloes (bubalus bubalis). theriogenology 2012;78:10481055. 33. rensis fd, lopez-gatius f: protocols for synchoronizing estrus and ovulation in buffalo (bubalus bubalis): a review. theriogenology 2007;67:209-216. 34. presicce ga: reproduction in water buffalo. reprod domest anim 2007;42(suppl 2):24-32. 35. gokuldasa pp, yadava mc, kumara h: resumption of ovarian cyclicity and fertility response in bull-exposed postpartum buffaloes. anim reprod sci 2010;121:236-241. 36. terramoccia s, bartocci a, borghese a: nutritional requirements in buffalo cows and heifers. in: borghese a, editor. buffalo production and research. rome: food and animal organization of the united nations; 2005. p 145-160. 37. campanile g, baruselli ps, vecchio d, et al: growth, metabolic status and ovarian function in buffalo (bubalus bubalis) heifers fed a low energy or high energy diet. anim reprod sci 2010;122:74-81. 38. shyam kishore sah, toshihiko n: characteristics of repeat breeding buffaloes in nepal. j reprod dev 2006;52:335341. 494clinical theriogenology • volume 4, number 4 • december 2012 2009: legal and ethical veterinary compounding legal and ethical veterinary compounding s. d. stanley school of veterinary medicine, k.l. maddy equine analytical chemistry laboratory, university of california, davis, ca usa food and drug administration the food and drug administration (fda) first made a distinction between human and veterinary-labeled drugs in 1968. as with most regulations since that time, the distinction reflected a concern for human food safety. at that point in time, an animal drug was adulterated if used in an extralabel manner. consequently, veterinarians were not permitted to use any drug except according to the label claim, without being in violation of both criminal and civil law. in an ideal world, pharmaceutical dosage formulations would exist to treat all disease conditions in humans as well as all species of animals. the economic reality does not permit the establishment of numerous pharmaceutical solutions for most disease states and relatively few pharmaceuticals have been developed for animal species. the development of a new animal drug typically averages ten years and cost $40 million. this combined with the narrow profit margins for most products makes for a challenging market place. by definition, a new animal drug is “any drug intended for use in animals other than man…”.1,2 veterinarians must by necessity on occasion utilize products that are compounded to meet a specific medical need. however, the avma guidelines for pharmaceutical compounding state that compounded products “may be used only when a need has been established and fda-approved products are not available or clinically effective.”2,3 while fda-approved products are extensively tested for efficacy, quality, 51 purity, strength, bioavailability, and stability, the testing that compounded formulations are subjected to is extremely variable. the fda’s center for veterinary medicine (cvm) allows for the compounding of animal drugs under the 1994 animal medicinal drug use clarification act (amduca).2 this act became a law in 1996 and extended the veterinarian’s authority to use human drugs in an off-label manner, including the right to compound with the use of fda-approved dosage forms. amduca does not allow for the compounding of drugs from bulk active substances, and any products resulting from such substances are considered new animal drugs and are subject to the fda drug-approval process. the fda modernization act of 1997 allows for the compounding of human drugs from bulk chemicals as long as the bulk substance is an ingredient of a currently approved product that appears on an fda list of drugs that can be compounded.3 it is important to note, however, that ingredients that are on the list of bulk substances withdrawn from the market for safety reasons may not be compounded. human compounding regulations are sharp contrast to current animal compounding regulations. these regulations become an important consideration for veterinarians who out-source compounding to pharmacies that may compound exclusively from bulk active chemicals. bulk chemicals are defined as active ingredients used in the manufacture of finished dosage forms of the drug. bulk chemicals are also referred to as the active pharmaceutical ingredients (api). compounding from bulk is allowed in instances where the health of the animal is at risk and there are no other remedies. drug: the fda defines a drug as any substance, food or nonfood; intended for diagnosis, cure, mitigation, or prevention of disease in humans or other animals; intended 52 to affect body structure or function; or any substance administered by injection.3 this broad definition effectively means that any substance used to treat an animal can be considered a drug. veterinary compounding drug compounding can be defined as the art and science of mixing ingredients, which may be active, inactive or both, to create a specific dosage form to meet a particular patient’s needs. for example, mixing two injectable drugs is compounding. compounding can be performed by a veterinarian, or by a pharmacist upon receipt of a veterinarian's prescription for a particular patient. a veterinarian must have a valid veterinarian-client-patient relationship in order to legally prescribe or prepare a compounded product. federal regulations require that legally compound drugs meet the following criteria:3 • a valid veterinarian-client-patient relationship (vcpr) must exist. • the health of an animal must be threatened or suffering or death may result from failure to treat. • there must be no fda-approved, commercially available animal or human drug that, when used as labeled or in an extra-label fashion in its available dosage form and concentration, will appropriately treat the patient. • the product must be made from an fda-approved commercially available animal or human drug. 53 • the product must be compounded by a licensed veterinarian or a licensed pharmacist on the order of a veterinarian within the practice of veterinary medicine. • the compounded product must be safe and effective. • the amount of product compounded must be commensurate with the need of the animal identified in the vcpr-based prescription. • for animals produced for human consumption, the veterinarian must establish an extended withdrawal interval for the compounded product and ensure food safety. compounding is not permitted if it results in violative food residue, or any residue that may present a risk to public health. • no drug may be compounded for food animals from drugs listed on the prohibited list. • veterinarians must comply with all aspects of the federal extra-label drug use regulations including record-keeping and labeling requirements. pharmacies specializing in veterinary compounding have been growing exponentially aided by the ability to reach a larger number of consumers via the internet. many commercial websites market directly to the owner offering subjective treatments based on testimonials and compounded therapies that are not permitted by the criteria established for compounded drugs. currently, the fda does not have the resources to enforce these regulations; however, veterinarians should be aware that abuse of these regulations can result in legal action (e.g., fda warning letters, confiscation of inventory and other enforcement action). the fda has determined that it will seriously consider taking action 54 when the scope and nature of activities of veterinarians and pharmacists raise concerns normally associated with a drug manufacturer resulting in significant violations of the new animal drug, including adulteration, or misbranding. compounded drugs are not the same as generic drugs. generic drugs are fdaapproved. to receive fda approval, generic drugs must demonstrate bioequivalence to the "pioneer brand name" drug. generic drugs can be identified by the anada number on their label and by cross-checking with a drug reference found in the fda green book of approved animal drug products. in contrast, compounded drugs are extemporaneously prepared products that lack fda approval. the idea is that compounded drugs with their possible inadequacies are better than no drug at all and suitable for a small patient population. equine practitioners using compounded products are put in a position of evaluating the integrity of the compounding pharmacy as well as the quality and consistency of the pharmaceuticals they produce. lack of regulatory approval means that not all veterinary compounding pharmacies follow good manufacturing practices (gmps) guidelines simply because they are not required to. in some instances, loose oversight has allowed negligent compounders to prepare products from unregulated raw materials with no quality standards. other compounding pharmacies distribute medication without a valid prescription. veterinarians are schooled on quality patient care but few pharmacists are. veterinarians who frequently use compounded products would be well advised to learn more about pharmacy issues related to veterinary medical therapy. for example: 55 • it is illegal to compound a specific product when there is an approved drug form of that specific product, except to make a different dosing form. however, the approved product must be used to make the compounded new dose form. • it is illegal to mark up prices on compounded drugs • as a veterinarian, if you use a compounded product, you assume liability for any adverse effects or efficacy failure. • drug manufacturers are required to carry product liability insurance, pharmacies are not • it is illegal to place expiration dates on compounded products • it is illegal to have a drug compounded in order to obtain the drug at a lower price problems and concerns regarding the use of compounded drugs omeprazole . this study was undertaken to determine the efficacy of commercially available omeprazole paste and a compounded omeprazole suspension to heal gastric ulcers.4 results from this study suggested that while administration of the commercially available omeprazole formulation was effective in promoting healing of gastric ulcers in horses, administration of the compounded omeprazole suspension was ineffective. differences in the source of omeprazole and partial inactivation of omeprazole by the vehicle or by gastric contents following administration of the suspension were cited as possible reasons for the poor results seen with the compounded product. pharmaceutical equivalence. the pharmaceutical potency of compounded preparations of ketoprofen, amikacin and boldenone were compared with commercially 56 available fda approved products.5 the fda requires any manufactured pharmaceutical to have a concentration (potency) of not less than ±10% of the expected concentration as stated on the product label. the results of this study found that 11 of the 22 compounded products failed to meet the fda requirement for potency. of the eleven products that failed to meet the fda standard, the range in percentage potency was as low as 50% and as high as 150% of what was stated on the prescription label. compounded clenbuterol. in 2006, concentrated counterfeit clenbuterol was determined as the cause of death for several thoroughbred horses in louisiana. the compounded clenbuterol solution was analyzed and found to be extremely potent, ~70 times greater than the fda approved commercial product ventipulmin® (boehringer ingelheim vetmedica, inc, st. joseph, mo usa). the source of the compounded clenbuterol solution was not determined but the trainer stated the product was “…just like ventipulim® but cheaper…”. compounded chloramphenicol palmitate. six owners sued a new jersey veterinary pharmacy alleging that a defective antibiotic led to the deaths of three horses, including saratoga county and egg head, both stakes winners. they alleged negligence, breach of warranty, and strict products liability against the pharmacy involving the antibiotic product chloramphenicol palmitate, that they claimed necessitated the euthanasia of three horses. the case cites improper design, manufacture, compounding, formulation, mixing and/or labeling, (which) "led to the slow, painful demise of these horses." following a two year legal battle the case was dismissed by the united states district court in new york. 57 conclusion products are required to treat hundreds of conditions and diseases in dozens of species. compounding of drugs for use in animals is a necessary and beneficial component of veterinary practice. licensed veterinarians may legally use or dispense prescription drug products only within the course of their professional practice where a valid vcpr exists. fda compliance policy guides permit licensed practitioners to manufacture, prepare, propagate, compound or process drugs during the regular course of business, as long as the compounded product is not a new animal drug. references: 1. avma symposium on compounding. j am vet med assoc 1994;205:189-301. 2. fda-cvm. fda-cvm’s compliance policy guide on compounding of drugs. j am vet med assoc 1996; 209:2025-2029. 3. fda-cvm. fda seeks to clear up confusion about compounding. j am vet med. assoc 2003;223:1103-1106. 4. nieto je, spier s, piper f, et al: comparison of paste and suspension formulations of omeprazole in the healing of gastric ulcers in racehorses in active training. j am vet med assoc 2002;221:1139-1143. 5. stanley s, thomasy s, skinner w: comparison of pharmaceutical equivalence of fda-approved products and compounded preparations of ketoprofen, amikacin and boldenone, proc am assoc equine. pract 2003; p. 238-242. 58 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /all /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /tags /compresspages false /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false 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jsmit604@utk.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 11729, http://dx.doi.org/10.58292/ct.v17.11729 case report surgical management of pyometra in a miniature sow joe smith,a cassandra klostermann,a jessica garcia,a christopher smith,b matthew aviles,a eduardo pradoa alarge animal clinical sciences, university of tennessee, knoxville, tn, usa bsmall animal clinical sciences, college of veterinary medicine, university of tennessee, knoxville, tn, usa abstract an american miniature pig sow (~ 5-year) was presented for abdominal distention and lethargy of 2 weeks duration. initial diagnostics revealed distended fluid-filled structures in the caudal abdomen on ultrasonography, and minor alterations in biochemistry and complete blood count profiles. exploratory laparotomy under general anesthesia revealed a large uterus distended with purulent material; ovariohysterectomy was performed. the uterine horns were friable and inadvertent rupture of the left horn occurred that was managed intraoperatively. postoperative management included intravenous fluids, antiinflammatory and antimicrobial therapies. complications encountered during hospitalization were mild hypotension under anesthesia and constipation/ileus during recovery. culture of the uterine contents revealed mixed growth including escherichia coli, schaalia hyovaginalis, and trueperella abortisuis. the sow was discharged 6 days after surgery and no complications were reported. comparative management as described for other species was useful in the treatment. keywords: sow, pyometra, surgery, transabdominal ultrasonography background miniature pigs, such as american miniature pigs (‘mini pigs’), vietnamese potbellied pigs, kunekune pigs, and other breeds are rapidly increasing in popularity as pets.1 with this increasing popularity there is an increased demand for veterinary services and consultation. nevertheless, there are few published resources for management of reproductive-related disorders of miniature companion pigs. this case presents an american miniature pig diagnosed with pyometra that was managed utilizing a comparative medicine approach that may provide clinicians with a case management example for similar clinical cases, as miniature pigs are increasing in popularity in the usa and canada. case presentation an american miniature pig sow (~ 5-year) was presented for concerns of abdominal distention, anorexia, and decreased activity of 2 weeks duration. owners obtained the sow 5  months ago after farrowing. sow’s previous medical and vaccination history was unknown. no medical issues were reported from other pigs on the farm. initial diagnostics included physical examination, serum biochemistry, complete blood count (cbc), and transabdominal ultrasonography. sow weighed 66.4 kg, temperature was 38.7ºc (reference: 37.6-39.1ºc), pulse was 56 per minute (reference: 60-80) and respiratory rate was 20 breadths per minute (reference:12-18), and sow had marked abdominal distention (figure 1). sow was alert and had normal urination and defecation, but was inappetent. initial cbc was unremarkable with exception of increased total protein of 9.6 g/dl (reference: 6.5-8.9) and decreased absolute lymphocytes of 0.32 x 103/µl (reference: 1.5-7.6). chemistry findings included decreased sodium (126 mmol/l; reference: 135-145), chloride (90 mmol/l; reference: 94-107) and phosphorous (3.0 mmol/l; reference: 3.9-7.5); as well as mildly increased blood urea nitrogen (22 mg/dl; reference: 2-19). abdominal ultrasonography revealed distended uterine horns filled with mildly echogenic fluid (figure 2). due to the combination of clinical and diagnostic findings, the top differential diagnosis was pyometra. treatment initial treatment included intramuscular tulathromycin (2.5  mg/kg; draxxin, zoetis, parsippany, nj) and flunixin mailto:jsmit604@utk.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.11729 2 citation: clinical theriogenology 2025, 17, 11729, http://dx.doi.org/10.58292/ct.v17.11729 meglumine (2.2 mg/kg; banamine s, merck animal health, rahway, nj) and intravenous fluid therapy with a balanced polyionic fluid at maintenance rate (2-3 ml/kg/hour). due to sow’s stable condition and presumptive diagnosis of pyometra, anesthesia and ovariohysterectomy were planned for the following day. after several hours of fluid therapy, the sow had mild appetite for grapes and was observed drinking. feed and water were withheld for 8 hours prior to surgery. the morning of surgery, several hours prior to premedication,2,3 the sow was given oral clonazepam (0.4 mg/kg) and maropitant (2 mg/kg) as an oral sedative prior to preanesthesia induction manipulation and as a preanesthetic antiemetic, respectively. anesthesia based on sow’s presentation and clinical status, american society of anesthesiologists score of iii was assigned prior to anesthesia. intramuscular alfaxalone (1 mg/kg; alfaxan multidose, zoetis) and ketamine (8 mg/kg; ketaset, zoetis) were given as premedications that resulted in moderate sedation. sow was subsequently preoxygenated via mask and anesthesia machine and a 20 g x 1.5” catheter was placed in an auricular vein. intravenous lidocaine (2 mg/kg: hospira inc, lake forest, il) and propofol (1 mg/kg; propoflo, zoetis) were given to induce anesthesia. sow was orotracheally intubated using an 8 mm internal diameter, cuffed figure 1. sow’s standing caudal view at presentation (note distended left abdomen) http://dx.doi.org/10.58292/ct.v17.11729 citation: clinical theriogenology 2025, 17, 11729, http://dx.doi.org/10.58292/ct.v17.11729 3 endotracheal tube. anesthesia was maintained with isoflurane (etiso 0.75-1.25%; piramal critical care, inc, bethlehem, pa) in oxygen (via a rebreathing circuit and small animal anesthesia machine) and lidocaine as constant rate (3 mg/kg/hour) infusion. in addition, intravenous morphine (0.3 mg/kg, hikma, berkeley heights, nj) was given along with lactated ringers (5 ml/kg/hour; lrs, icu medical, inc). transversus abdominis plane (tap) block was performed, using bupivacaine 0.25% (1 ml/kg; hospira inc) and dexmedetomidine (1 µg/kg; dexdomitor, zoetis), delivered in 1 subcostal and 1 preiliac injection site, per hemiabdomen. as alpha 2 agonists such as dexmedetomidine prolong the duration of local anesthetics, this protocol was chosen to maximize the duration of analgesia provided by the local anesthetic to the abdominal wall to reduce the need for systemic analgesics.4 sow exhibited hypotension (mean arterial pressure < 60 mm hg) ~ 40 minutes after induction. a 10 ml/kg fluid bolus or lrs (over 10 minutes) was given, and a constant infusion rate (5 µg/kg/minute) of dobutamine (cri; hospira inc) was started that transiently resolved the hypotension. the bolus was repeated ~ 30 minutes later when hypotension occurred again. hypotension occurred once more ~ 30 minutes after the second fluid bolus, and a dose of intravenous ephedrine (0.1 mg/kg; fresenius kabi, lake zurich, il) was given that resolved hypotension for ~ 20 minutes. finally, intravenous hypertonic saline bolus (2  ml/kg) was given over 15 minutes that maintained normotension till the procedure was completed and anesthesia was terminated. sow recovered from anesthesia uneventfully. the premedication protocol consisted of alfaxalone (a neurosteroid with gabaa agonistic properties) and ketamine (n-methyl-d-aspartate receptor antagonist) were chosen to facilitate sedation, muscle relaxation, and for intravenous catheter placement; these 3 goals were accomplished. the induction protocol consisted of a loading dose of lidocaine, a sodium channel blocker, for the subsequent cri and, together with the premedications, allowed for a small dose of propofol (a gabaa agonist) to induce further muscle relaxation, hypnosis, and general anesthesia. this multimodal protocol allowed several classes of drugs use, lowering the required dose of each in attempts to mitigate any undesired effects (e.g. dose-dependent vasodilation) associated with propofol and/ or isoflurane. the lidocaine cri, morphine dose and tap block were further efforts to reduce the mac of isoflurane required for general anesthesia and surgery in efforts to mitigate isoflurane’s dose-dependent cardiovascular depression. accomplishing this was evidenced by the percentage of isoflurane required to maintain general anesthesia (well below reported mac).5 however, there were several episodes of hypotension that were successfully treated with boluses of isotonic and hypertonic crystalloids, as well as sympathomimetics; dobutamine (positive inotrope via β agonism) and ephedrine (direct and indirect action on α and β receptors). sow recovered uneventfully and had normal hemoglobin oxygen saturation, blood pressure, and temperature in the recovery stall. sow also visually appeared comfortable and exhibited no clinical signs of pain associated with abdominal palpation, therefore no additional analgesics were given immediately after surgery. surgery sow received an additional dose of intravenous flunixin meglumine (2.2 mg/kg, ~ 24 hours after first), and respiratory (respisure-one/er bac, zoetis) and tetanus (tetanus toxoid, colorado serum company, denver, co) vaccinations figure 2. transabdominal ultrasonographic image of sow’s uterus at presentation (note distended uterine horns [blue arrows] filled with mildly echogenic fluid) http://dx.doi.org/10.58292/ct.v17.11729 4 citation: clinical theriogenology 2025, 17, 11729, http://dx.doi.org/10.58292/ct.v17.11729 prior to surgery. sow was subsequently placed in dorsal recumbency, clipped, and aseptically prepared and draped from xiphoid to pubis. approximately 15 cm ventral midline skin incision was made with size 10 scalpel blade on the caudal abdominal midline. the subcutaneous adipose tissue was incised with a scalpel blade and bluntly dissected digitally. once the linea alba was visualized, it was incised by tenting the tissue with thumb forceps and making a stab incision with an inverted size 10 scalpel blade. this incision was extended with metzenbaum scissors cranially and caudally until peritoneum was visualized. the peritoneum was incised by tenting and making a stab incision as described for the linea alba. metzenbaum scissors were used to extend linea alba and peritoneum the length of the skin incision. the distended uterine horns were visualized immediately upon entry into the abdomen (figure 3) and were used to locate ovaries (had several cor pus lutea). the left ovarian pedicle was ligated with 3 encircling ligatures placed proximally to the ovary by introducing the swagged portion of the needle first to minimize the risk of inadvertently puncturing a vessel within the broad ligament using 0 polydioxanone (pds, ethicon, bridgewater, nj). these were placed ~ 1 cm apart from each other, and a bipolar electrosurgical device (ligasure, stryker, portage, mi) was used to ligate and seal the pedicle between the remaining ligatures as previously described for bipolar pedicle ligation.6,7 the ligasure was used to transect along the broad ligament toward the uterine body to allow for exteriorization of the left uterine horn. one transfixing ligature was placed around each uterine artery adjacent to the uterine body using 2-0 pds suture. attempts to locate the right ovary resulted in ~ 2 cm tear in the abaxial uterine wall of exteriorized left uterine horn, clear to white cloudy malodourous liquid leaked. saline-soaked laparotomy sponges were packed below the left uterine horn to prevent figure 3. uterus during surgery prior to completion of ovariohysterectomy as partially exteriorized on a mayo stand (note the large distended horns and darkened vessels) http://dx.doi.org/10.58292/ct.v17.11729 citation: clinical theriogenology 2025, 17, 11729, http://dx.doi.org/10.58292/ct.v17.11729 5 leakage into the abdomen. intrauterine suction was performed with a poole suction tip to remove remaining fluid from the uterus. doyen forceps were placed adjacent to the defect and was closed with 0 pds in a simple continuous pattern to prevent further spillage. abdomen was lavaged with warm saline and suctioned. the abdominal incision was repacked with  sterile saline-soaked laparotomy sponges. new gloves were used by the surgery team to minimize abdominal contamination. the right ovary was identified, and the process was repeated for the right ovarian pedicle and right broad ligament without further contamination of the surgical site. three miller’s knots were placed using 1 polyglactin 910 (vicryl, ethicon) suture around the body of the uterus ~ 1 cm apart from each other distal to the cervix. the abdomen adjacent to uterus body was packed with 2 saline-soaked laparotomy sponges. between the 2 most proximal ligatures, an 18 g needle was inserted and suction applied to remove purulent material from the uterine body. the ligasure was used to transect between these 2 ligatures. the uterine body stump was closed with 0 pds in a simple continuous pattern. after removal of uterus, the laparotomy sponges were removed, the abdominal cavity was lavaged with 5 liters of warm saline, which was then removed with suction. no hemorrhage was observed prior to closing. a new surgery pack was opened prior to closure. peritoneum and linea alba were closed with a cruciate pattern using 0 pds; subcutaneous layer was closed with 0 pds using a simple continuous pattern and the skin was closed with 2-0 pds with an intradermal pattern. an aluminum aerosol wound dressing (aluspray, neogen inc, lansing, mi) was sprayed over the incision and postanesthetic recovery was prolonged. postoperative management several hours after recovery, due to concerns of pain, an additional intravenous dose (0.2 mg/kg) of morphine was given. postoperatively intramuscular ceftiofur crystalline-free acid (5 mg/kg; excede for swine, zoetis) was given due to possible contamination from the uterine contents. for the next 3 days after surgery sow was maintained on daily intravenous balanced polyionic fluids and flunixin meglumine (decreased to 1.1 mg/kg). appetite returned to normal 24 hours after surgery and sow was maintained on twice-daily oral amoxicillin-clavulanic acid (20.5 mg/kg; (clavamox, zoetis). during recovery, scant manure production was noted; sow was given once 100 mg of ducosate sodium (generic) with continuation of intravenous fluids and feed was provided in mash form. culture of the uterine fluid collected at surgery identified escherichia coli (e. coli), schaalia hyovaginalis, trueperella abortisuis (table 1). two days after surgery, the sow had complete resolution to normal behavior and attitude. repeat cbc was unremarkable, with the exception of elevated fibrinogen (700 mg/dl; reference: 0-600) expected after surgery. intravenous fluids were tapered and discontinued 4 days after surgery. sow was discharged 6 days after surgery with instructions to complete the 10-day therapy of amoxicillin-clavulanate. follow up on days 14, 29, and 91 after discharge suggested sow’s return to normal activity with no additional health concerns. discussion there is limited information on pyometra cases in pigs, although it was reported in a wild boar sow8 and a production sow.9 a previous retrospective study of complications from ovariohysterectomy in sows described pyometra cases as least likely to survive after discharge from the hospital among groups in that study, as only 3/5 survived.6 an additional retrospective investigation identified 3 pigs with lesions consistent with pyometra in 24 pigs with uterine lesions observed during ovariohysterectomy.10 with this lack of clinical reporting, it is important for clinicians to consider comparative measures for pyometra management information available in small and large animal literature. we utilized a comparative approach derived from reports on the management of pyometra in other species. pyometra is defined as the accumulation of purulent content within the lumen of the uterus in an intact or ovariectomized female.11,12 the causes of this pathology are sometimes unknown; however, there are several factors (e.g. age, parity, complicated parturition) involved. there are 2 main clinical presentations of this pathology; these are described as open or closed pyometra, depending on cervical patency.11 the type of presentation depends on the presence of an active corpus luteum. physiopathology varies depending on the species, age, reproductive status, or history. although the accumulation of purulent material is due to a bacterial interaction, perhaps there are other causes. for example, in the mare, some common predisposing factors are failure to relax/open the cervix due to internal or external adhesions, incompetent uterine clearance, and reproductive tract position.13 in cattle, pyometra is most common in the postpartum stage due to an ovulation that occurred before uterine clearance is completed.14 additionally, dystocia, fetal membrane retention, and metritis could lead to the accumulation of purulent material in the uterus. multiple bacterial species are associated with pyometra in veterinary medicine. in cattle the pathogen most isolated is trueperella pyogenes (t. pyogenes),14 whereas the main organism associated with pyometra in the mare is streptococcus equi ssp. zooepidemicus.13 pyometra in dogs is strongly associated with hormone interactions.2 progesterone causes endometrial changes and in addition to the decreased immune system and closed cervix lead to an ideal environment for bacteria to proliferate and cause pyometra. dogs tend to become systemically ill because the majority of the infections are caused by e. coli that release endotoxins, causing a systemic inflammatory response.15 pigs with substantial endometrial changes had cystic endometrial hyperplasia16 leading to pyometra due to the rupture of these fluid-filled structures providing an ideal medium for bacterial growth. additionally, accumulation of purulent material is caused by previous endometritis or metritis. chronic endometritis led to pyometra in a sow;9 some of the pathogens isolated were e. coli and t. pyogenes, similar to our case (e. coli and a trueperella species). table 1. uterine fluid (collected intraoperatively) culture results culture component identified organisms aerobic escherichia coli aerobic schaalia hyovaginalis aerobic trueperella abortisuis anaerobic unidentifiable gram-negative rod anaerobic unidentifiable gram-positive coccus http://dx.doi.org/10.58292/ct.v17.11729 6 citation: clinical theriogenology 2025, 17, 11729, http://dx.doi.org/10.58292/ct.v17.11729 sow received multiple medications, all were extralabel, as none is labelled for pigs pyometra treatment. although some drugs used were labelled for pigs, they were not for the indications of management of pyometra (e.g. flunixin labeled for pyrexia). other drugs used were not labelled for pigs; however, were used before (e.g. clonazepam and maropitant for perioperative management).2,3 extralabel drug use is allowed in the usa as long there is a valid veterinary-client-patient relationship and possibility of making decision as outlined in the animal medicinal drug use clarification act. this is common in companion miniature pig cases.17 in the usa, food animal status is determined by species, not role, and this sow was given withdrawal instructions after consultation with the food animal residue avoidance databank (farad). it should be noted that when utilizing farad for advice, it is meant for the specific case it was requested for, and may not be accurate for other cases. although uncommon, there are documented cases of miniature companion pigs entering the food chain in the usa.18 ceftiofur (excede for swine, zoetis) was used extralabelly in this case in a manner permitted for 3rd generation cephalosporin order of prohibition (label dose, route, and duration), and the indication was for infection determined by examination and initial diagnostics. in small animal medicine, medical management of pyometra is possible. the ideal patients for medical management of pyometra are young and otherwise healthy cats and dogs with open cervix.19 close monitoring is critical for these cases as severe complications, such as sepsis, is possible. it is important to note that in these small animal cases, although antimicrobials may reduce disease progression, they alone will not result in uterine healing.19 medical management without surgical intervention was not considered as a preferential option for the sow, due to the perceived disease progression, cervix in an apparently closed state and the depressed nature at presentation indicating suspected systemic illness. comparative antimicrobial selection for companion miniature pigs can be challenging. for the isolates identified in this case, there are no validated clinical and laboratory standards institute (clsi) breakpoints. for example, there are no e. coli breakpoints for swine, so laboratory interpretation of susceptibility data is likely extrapolated from other species, and may not be entirely accurate. amoxicillin was used in this case based on susceptibility information for ampicillin and clsi recommendations on the interchangeability of ampicillin and amoxicillin for antimicrobial susceptibility testing results (clsi, vet01s, 7th edition).20 furthermore, we are unaware of any disposition data for amoxicillin or ampicillin in the uterus or uterine fluid of the pig after oral administration of the drug. this case illustrates the approach for practitioners to integrate diagnostic findings, patient information, as well as available references to extrapolate a treatment of an infection in a species where there is not enough evidence to interpret antimicrobial susceptibility testing results based solely on the susceptibility report. limitations of this report are its singular and retrospective nature. although this case had a successful outcome, variation in similar cases in the future may dictate the need for alternate approaches. future studies could evaluate less-invasive surgical procedures in pigs, such as the assisted laparoscopic surgical glove port technique for ovariohysterectomy in the female dog21 or the hand-assisted laparoscopic ovariohysterectomy described for resolution of chronic pyometra in a mare.22 however, these endeavors should be considered keeping in mind the possibility of encountering a friable uterus, as in this case. in conclusion, a comparative approach described for pyometra in other veterinary species was useful for the management of this sow. specifically, a comparative approach was utilized in this case for diagnostic measures, intravenous fluid therapy, ovariohysterectomy, and medical management with antimicrobials and pain management. clinicians should keep in mind the friable nature of uterus, if surgery is planned for pyometra in pigs. learning points • pyometra can account for ~ 25% of body weight • pyometra can be managed with surgical removal of the uterus and medical management • comparative species approach can be applied, with awareness to extralabel drug regulations • careful surgical handling of diseased uterus is necessary to prevent rupture • hypotension during anesthesia and ileus or constipation during recovery are possible complications references 1. curnutte m: the big problem with mini-pigs. washington, dc; national geographic: 2014. 2. hampton ce, kleine sa, smith js, et al: pharmacokinetics of oral clonazepam in growing commercial pigs (sus scrofa domestica). j vet pharmacol ther 2024;47:365-371. doi: 10.1111/jvp.13451 3. smith js, gebert je, ebner ls, et al: pharmacokinetics of intramuscular maropitant in pigs (sus scrofa domesticus). j vet pharmacol ther 2023;46:158-164. doi: 10.1111/jvp.13120 4. brummett cm, williams ba: additives to local anesthetics for peripheral nerve blockade. int anesthesiol clin 2011;49:104-116. doi: 10.1097/aia.0b013e31820e4a49 5. eger ei, ii, johnson bh, weiskopf rb, et al: minimum alveolar concentration of i-653 and isoflurane in pigs: definition of a supramaximal stimulus. anesth analg 1988;67:1174-1176. doi: 10.1213/00000539-198812000-00012 6. cypher e, videla r, pierce r, et al: clinical prevalence and associated intraoperative surgical complications of reproductive tract lesions in pot-bellied pigs undergoing ovariohysterectomy: 298 cases (2006-2016). vet rec 2017;181:685. doi: 10.1136/vr.104294 7. smith j, salazar g, peace c, et al: electrosurgical bipolar vessel sealing for a standing flank ovariectomy in beef heifers. clinical theriogenology 13;202:60-67. doi: 10.58292/ct.v13.9364 8. garcês a, soeiro v, lóio s, et al: description of a closed pyometra on a wild boar (sus scrofa, linnaeus 1758). vet stn 2022;53: 343-346. doi: 10.46419/vs.53.3.3 9. morris c, watson k, schwartz d, et al: nonpuerperal chronic endometritis with pyometra causing systemic illness in a production sow. clinical theriogenology 2023;15:9806. doi: 10.58292/ct.v15.9806 10. ilha mrs, newman sj, van amstel s, et al: uterine lesions in 32 female miniature pet pigs. vet pathol 2010;47:1071-1075. doi: 10.1177/0300985810382522 http://dx.doi.org/10.58292/ct.v17.11729 https://doi.org/10.1111/jvp.13451 https://doi.org/10.1111/jvp.13120 https://doi.org/10.1097/aia.0b013e31820e4a49 https://doi.org/10.1213/00000539-198812000-00012 https://doi.org/10.1136/vr.104294 https://doi.org/10.58292/ct.v13.9364 https://doi.org/10.46419/vs.53.3.3 https://doi.org/10.58292/ct.v15.9806 https://doi.org/10.1177/0300985810382522 citation: clinical theriogenology 2025, 17, 11729, http://dx.doi.org/10.58292/ct.v17.11729 7 11. youngquist rs, threlfall wr: current therapy in large animal theriogenology. amsterdam; elsevier: 2006. p. 339-343. 12. van goethem b, schaefers-okkens a, kirpensteijn j: making a rational choice between ovariectomy and ovariohysterectomy in the dog: a discussion of the benefits of either technique. vet surg 2006;35:136-143. doi: 10.1111/j.1532950x. 2006.00124.x 13. mckinnon ao, mccue pm: induction of ovulation. in: mckinnon ao, squires el, vaala we, et al: editors. equine reproduction. 2nd edition, ames; wiley-blackwell: 2011. p. 1858-1869. 14. palmer c: postpartum uterine infection. in: hopper rm: editor. bovine reproduction. ames; john wiley & sons, inc.: 2014. p. 440-448. 15. xavier rgc, santana ch, de castro yg, et al: canine pyometra: a short review of current advances. animals 2023;13:3310. doi: 10.3390/ani13213310 16. dürlinger s, knecht c, gumpenberger m, et al: cystic endometrial hyperplasia and uterine adenocarcinoma in two miniature pigs. tierarztl prax ausg g grosstiere nutztiere 2022;50:333-339. doi: 10.1055/a-1877-4487 17. smith j, merkatoris p, breuer rm, et al: letter regarding “gastrointestinal foreign bodies in pet pigs: 17 cases”. j vet intern med 2022;36:1199-1200. doi: 10.1111/jvim.16479 18. lord lk, wittum te: survey of humane organizations and slaughter plants regarding experiences with vietnamese potbellied pigs. j am vet med assoc 1997;211:562-565. doi: 10.2460/javma. 1997.211.05.562 19. hagman r: pyometra in small animals 2.0. vet clin north am small anim pract 2022;52:631-657. doi: 10.1016/j.cvsm.2022.01.004 20. clinical and laboratory standards institute: performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. in: diaz-campos d, burbick c: editors. clsi vet01s. 7th edition, clinical and laboratory standards institute: 2024. p. 1-288. 21. becher-deichsel a, aurich je, schrammel n, et al: a surgical glove port technique for laparoscopic-assisted ovariohysterectomy for pyometra in the bitch. theriogenology 2016;86:619-625. doi: 10.1016/j.theriogenology.2016.02.010 22. thurston cc, sertich pl, mcdonnell sm, et al: standing hand-assisted laparoscopic ovariohysterectomy to treat chronic pyometra in a mare. j am vet med assoc 2024;262:1-3. doi: 10.2460/javma. 23.11.0646 http://dx.doi.org/10.58292/ct.v17.11729 https://doi.org/10.1111/j.1532-950x.2006.00124.x https://doi.org/10.1111/j.1532-950x.2006.00124.x https://doi.org/10.3390/ani13213310 https://doi.org/10.1055/a-1877-4487 https://doi.org/10.1111/jvim.16479 https://doi.org/10.2460/javma.1997.211.05.562 https://doi.org/10.1016/j.cvsm.2022.01.004 https://doi.org/10.1016/j.theriogenology.2016.02.010 https://doi.org/10.2460/javma.23.11.0646 2012: surgical aspiration of a 19 centimeter ovarian follicular cyst in an embryo donor cow surgical aspiration of a 19 centimeter ovarian follicular cyst in an embryo donor cow elizabeth a. coffman, marco a. coutinho da silva, andrew j. niehaus, rebecca l. pentecost, christopher premanandan, carlos r.f. pinto the ohio state veterinary medical center, college of veterinary medicine, the ohio state university, columbus, oh summary a two and a half year-old holstein cow presented for assessment of a markedly enlarged ovary, with a fluid-filled structure measuring approximately 19 centimeters in diameter following superstimulation with follicle stimulating hormone (fsh) for embryo collection and transfer. clinical evaluation, including transrectal palpation and ultrasonography, measurement of steroid hormone concentrations in serum and follicular fluid, and histopathologic examination of a sample obtained from the wall of the follicle-like structure during surgery were consistent with an ovarian follicular cyst. surgical drainage of the fluid combined with medical treatment was effective in inducing cyclicity and did not result in complications (adhesion formation). new follicular cysts occurring within two weeks following the surgical aspiration were responsive to treatment with transvaginal ultrasound-guided aspiration followed by a 7-day cidr-synch protocol. the cow returned to cyclicity and subsequently became pregnant, producing twin bull calves within a year of initial presentation. background while cystic ovarian degeneration (cod) is a relatively common disorder, particularly in cows subjected to superovulation protocols, this case is notable in the unusual size of the ovarian cyst. it demonstrates that surgical drainage may be an effective treatment in cows with markedly large ovarian cysts that preclude drainage via transvaginal ultrasound-guided aspiration or do not respond to hormonal therapies. case presentation a two and a half year-old holstein cow admitted to the ohio state university veterinary medical center theriogenology service for evaluation of an enlarged left ovary approximately four months after calving. the cow was previously enrolled in an embryo transfer program that included conventional treatments with fsh (folltropin®-v; 16ml total equivalent to 320 mg of nih-fsh-p1 in decreasing doses over four days) to induce superovulation for embryo collection and transfer. no reproductive examination (transrectal palpation or ultrasound) was performed before beginning superstimulation with fsh beginning 102 days post-calving. at the time of embryo collection attempt, both ovaries were classified as having corpora lutea too numerous to count based on transrectal palpation. in addition, the left ovary was greatly enlarged (cantaloupe-sized) and the right ovary was classified as cystic; nonetheless, the uterus was lavaged and sixteen embryos were recovered; eleven of transferrable quality (six grade 1, five grade 2) and five degenerate embryos. nine embryos were transferred to appropriate recipients and two were frozen. having failed to respond to conventional treatment utilizing gonadotropin releasing hormone (gnrh) and prostaglandin f2α (pgf; details of regimen employed not provided by owners or referring veterinarian), the cow was referred to the ohio state university veterinary medical center approximately one month after embryo collection. the initial examination revealed an apparently healthy animal in good body condition, and blood chemistry and complete blood count were within reference ranges. on palpation per rectum, both ovaries were appreciably enlarged and soft. palpation per rectum revealed the left ovary was markedly increased in size and could not be grasped in its entirety; the right ovary, albeit smaller than the left ovary, also contained cystic structures. on transrectal ultrasonography (aloka ssd 500 v ultrasound scanner equipped with a 5.0 mhz linear array transrectal transducer; aloka co. ltd., wellingford, ct), the left ovary consisted mainly of a large fluid-filled structure, consisting of thin walls containing hypoechoic fluid (figure 1). the left ovary could not be observed completely on the ultrasound monitor owing to the size of the cyst, but its diameter was estimated at approximately > 13 centimeters using the ultrasound 495 clinical theriogenology • volume 4, number 4 • december 2012 calipers (13 cm maximum depth allowed in the ultrasound unit used). the right ovary also contained two follicular cysts, measuring 3.1 and 3.0 cm in diameter. differential diagnosis the most likely cause of ovarian enlargement on the left and right ovaries based on history and findings of the clinical reproductive examination was cod or ovarian follicular cysts. other potential diagnoses considered included luteal cysts, ovarian neoplasia, and ovarian abscess. treatment a presumptive diagnosis of bilateral follicular cysts was made and 5000 iu of human chorionic gonadotropin (hcg) was administered intravenously to induce luteinization of the cystic structures. although gnrh or its analogues can also be administered for the same pharmacologic effect, hcg was elected due to its ability to bypass the adenohypophysis and bind directly to luteinizing hormone (lh) receptors in the ovary; the authors often elect this agent for chronic cases of cod that fail to respond to treatment with gnrh. transvaginal ultrasound-guided aspiration of the right ovarian follicular cyst was attempted. one cyst was successfully drained and the second cyst spontaneously ruptured during the manipulation per rectum. owing to the large size of the left ovarian cyst, which precluded adequate manual manipulation per rectum, a decision was made to not attempt to aspirate it using transvaginal ultrasound-guided aspiration and a left flank laparotomy was elected to better access the left ovary and to ensure complete drainage of the accumulated fluid. following left flank laparotomy, the left ovary was fully exteriorized to obtain diagnostic samples and facilitate drainage (figure 2). samples of the cystic fluid and cyst wall were collected for hormone analysis and histopathology, respectively. biopsy samples were collected by insertion of an endoscopic biopsy instrument through a puncture created with a 14-gauge needle to drain the cystic fluid. after complete drainage of the fluid contained within the cyst, the ovary was repositioned within the abdominal cavity and no further resection of the cyst walls was performed; the abdominal wall and skin were closed in routine fashion. post-operatively, multiple doses of ceftiofur (excede™, pfizer animal health, new york, ny; 6.6 mg/kg sc at the ear base weekly for 4 doses total) were prescribed to reduce the risk of oophoritis.* the wall of the cyst was composed of loosely arranged fibrous tissue of varying degrees of maturity (figure 3). small arterioles and capillaries were interspersed within this tissue. the luminal aspect of the cyst was lined by multiple layers of round to cuboidal cells with slightly basophilic cytoplasm and round nuclei containing dispersed chromatin. there was no evidence of luteinized tissue, inflammation or neoplasia. additional sections were submitted for vimentin and cytokeratin immunohistochemical staining. rare cytokeratin staining was observed in the lining cells. strong staining for vimentin was observed in the spindle shaped stromal cells as well as the cells lining the cyst described above (figure 3). the morphology and immunohistochemical staining pattern suggested that the lining of the cyst was composed of granulosa cells. the expression of the intermediate filament proteins, vimentin and cytokeratin in ovarian structures can vary between species and stage of follicular development. in cattle, ovarian surface epithelium demonstrate positive cytokeratin and negative vimentin staining while rete ovarii demonstrate dual staining for cytokeratin and vimentin.1 however, while positive vimentin staining has been shown to be a consistent finding in granulosa cells, keratin staining appears to be restricted to preantral follicles.2 thus, the staining pattern demonstrated supports the diagnosis of a cystic ovarian follicle. serum and follicular fluid samples collected at the time of surgery were submitted to the university of california-davis endocrinology laboratory and were analyzed by enzymeimmunoassay (progesterone) and radioimmunoassay (estradiol, inhibin, testosterone). the results obtained for both serum and follicular fluid are listed in the table.                                                              *at the time of case presentation, the described use of excede was within amduca guidelines. as of april 5, 2012, extra-label use of ceftiofur in disease prevention in food animals is prohibited by the food and drug administration.  496clinical theriogenology • volume 4, number 4 • december 2012 thirty three days following surgery, the cow was again presented for a reproductive examination. on transrectal ultrasonography, the left ovary contained a 4.0 cm follicular cyst. the right ovary had a 2.5 cm cavitated corpus luteum and a 2.5 cm cystic follicle. no adhesions were detected. at that time, a single injection of a gnrh analog (1.8 mg of deslorelin acetate) was administered intramuscularly. one week later, the cysts were still present without ultrasonographic signs of ongoing luteinization and measured approximately 4.1 cm and 2.3 cm on the left and right ovaries, respectively; the corpus luteum was still present on the right ovary and concentrations of serum progesterone were 3.5 ng/ml. transvaginal ultrasound-guided follicular aspiration was performed, and 25 ml of follicular fluid was aspirated from the left ovary and 6 ml from the right. the owner was instructed to place a controlled internal drug-releasing insert (cidr) for seven days, administer pgf at removal, and breed when the cow was seen in estrus. outcome the owners reported that following the final treatment, the cow returned to cyclicity and became pregnant approximately two months following surgery. the following year, she produced twin bull calves but succumbed to severe pneumonia within a week of parturition. discussion the case described in this report is notable in the marked size of the follicular cyst and the successful restoration of reproductive function following surgical aspiration. cystic ovarian disease occurs relatively frequently, affecting approximately 10% of dairy cows annually;3 true incidence may actually be higher as a proportion of cows recover spontaneously, and some herds reportedly experience higher prevalence of this disorder. the economic impact of this condition is significant, as, on average, it prolongs inter-calving interval by 50 or more days3 and may necessitate culling animals unresponsive to treatment. due to its prevalence and impact on production, this disorder is very important to the dairy industry. traditionally, ovarian follicular cysts have been defined as anovulatory fluid-filled structures greater than 24 mm in diameter that persist for more than 7-10 days in the absence of a normal corpus luteum (cl);4 however, cysts may also occur in conjunction with a cl.5 large cysts, up to 5 cm in diameter, have been described.5 ovarian cysts are relatively common in the postpartum period, with the highest incidence 30-60 days after parturition.7 application of ultrasonography for reproductive evaluation and enhanced understanding of bovine follicular dynamics have contributed to knowledge regarding cystic ovarian disease by allowing serial monitoring and more accurate documentation of ultrasonographic characteristics. the pathological condition actually entails a dynamic process with three potential circumstances: a single cyst may persist, the cyst may spontaneously regress (20-50%), or cysts may be cyclically replaced by others.8-10 follicular wave turnover takes longer (13.0 + 1.1 days) in cystic cows as compared to normal, cycling animals (8.5 + 0.5 days).10 it is noteworthy that superstimulation with exogenous of fsh in the cow described in this case was implemented in the postpartum period without reproductive examination prior to initiation of treatment; therefore, it is possible that there was some pre-existing cystic condition that was exacerbated by hormonal treatment. despite the failure to characterize the etiological basis of cod, neuroendocrine dysfunction and alterations of the hypothalamic-hypophyseal-ovarian (hhpo) axis appear to play a significant role in the pathogenesis. a fundamental disruption in normal endocrine function and feedback mechanisms is apparently involved in the pathophysiology,3,11,12 specifically, failure of positive feedback of estradiol-17β to the surge center of the hypothalamus. cows with cystic ovaries show no or delayed lh response to the administration of exogenous estradiol compared to normal animals.12,13 at the hypothalamic level, assessment of gnrh concentrations in homogenates of hypothalamic-suprachiasmatic nuclei from cystic cows revealed lower content in cystic versus control animals and there tended to be higher content in the median eminence of cystic cows.14 increased release of gnrh from the median eminence could account for higher serum concentration of lh and more frequent peaks in cystic as compared to normal cows; despite these changes, a preovulatory lh surge is not achieved.10,14 while failure of positive feedback of 497 clinical theriogenology • volume 4, number 4 • december 2012 estradiol-17β and subsequent absence of an lh surge is widely accepted to be the underlying endocrine event leading to cyst formation, there is some evidence that animals chronically affected with cod may display similar lh pulsatility profiles compared to normal animals.15 by contrast, fsh levels do not differ in cystic versus control cows, and cystic cows show a normal increase in fsh prior to initiation of a follicular wave.10,14 administration of fsh to cows with chronic cod results in increased expression of lh and fsh receptors in ovarian tissue and increased intrafollicular estradiol-17β compared to untreated cows.15 the administration of supra-physiological levels of fsh as commonly done in superovulation protocols may contribute to the development of cod. in one study, incidence of cod in beef embryo donor cows in japan was 20% compared to a reported rate of 4.2% in normal dairy cows in the same area.16 ovarian steroid expression in follicular cysts may be altered as compared to normal follicles. expression of gonadotropin receptors and steroidogenic enzymes is altered in the development of cysts as compared to normal follicles.17 cows with cystic ovaries generally have elevated serum concentrations of estradiol-17β as compared to normal cows.10,14 the cow described in present report did not show elevated steroid hormone levels in serum, and estrogen concentrations were low in follicular fluid as compared to normal developing follicles. it is important to note that different cysts may exhibit different steroid hormone profiles, and various classification systems have been derived according to these characteristics. actual concentrations of serum and follicular steroid hormones in normal and cystic animals have been reported previously;15,17,18 however, the reported values show marked variation among individuals and even within the same animal in addition to different approaches and hormonal assays.19 therefore, consideration of the ratio of hormone expression rather than the actual values20 or total steroid hormone concentration (calculated based on follicle diameter)17 provide alternate approaches in interpretation of results. for the cyst described here, the concentration of estradiol-17β in the follicular fluid was relatively low (86.4 pg/ml), but the total content (310 ng) was significant when accounting for total follicular fluid volume [based on the volume of a sphere according to the formula 4/3*(πr3)]. this value is within the range reported for total estrogen in follicular cysts.21 while it is interesting to consider these physiological parameters and characterize the cyst according to its hormonal profile, it might be prudent to consider that cod is a dynamic process and that such information on steroid composition of the follicular fluid may only provide a snapshot of the cyst endocrine function at the time of sampling. due to the apparently defective gnrh secretion and lh surge in cows affected by cystic ovarian disease, medical therapy has aimed at use of these hormones or their analogs to induce luteinization. administration of hcg was originally employed with reported success varying from 65-80%,9 and this was administered on initial assessment in the case presented here because of the previous history of unsuccessful gnrh use. the use of hcg has increasingly been replaced by gnrh and gnrh analogs with reported success rates of about 80%.9 because these approaches aim to luteinize the cystic structure, administration of pgf seven to nine days following gnrh or gnrh analog administration may shorten the interval between initiation of treatment and estrus. an alternate approach employs the use of intervaginal progesterone-releasing devices and ovulation or estrus synchronization protocols as treatment to increase the chances to return to cyclicity. pretreatment with gnrh increases the number of cows with a cl at the time of prostaglandin administration, and insertion of a progesterone releasing device results in acceptable pregnancy rates in treated cows.22 furthermore, management of cows with a synchronization protocol employing a progesterone releasing device results in decreased incidence of cod compared to previous occurrence using pgf only.16 the use of transvaginal needle aspiration of cystic fluid has been more recently employed as a treatment for follicular cysts,23 and it can be effective when employed even in cows who are unresponsive to traditional treatment with synchronization protocols.24,25 transvaginal ultrasound guided aspiration of follicular cysts may be considered as a modification and improvement on the older approach of manual rupture of ovarian cysts in that it carries a decreased risk of adhesions and the added benefit of acute removal of hormone-rich cystic fluid and, therefore, potentially fast recovery and resumption of normal cyclicity. cairoli et al. reported a 75.6% recovery rate and 64.7% conception rate when aspiration was combined with administration of the gnrh analogue buserelin.23 in another study, 100% of cystic cows 498clinical theriogenology • volume 4, number 4 • december 2012 treated with transvaginal ultrasound guided aspiration in combination with gnrh and pgf recovered, including animals which did not respond to aspiration alone (5.5%) or gnrh and pgf alone (16%) initially; the average interval from treatment to estrus was 12.4 + 1.1 days.24 thus, transvaginal aspiration of follicular cysts may be considered in animals that seem refractory to gnrh and pgf. in the case reported here, surgical aspiration was elected initially to allow drainage of the very large structure, and to obtain a biopsy sample for histopathologic examination. thirty-three days following the initial surgical aspiration, bilateral ovarian cysts and a cavitated cl were diagnosed and the cow was treated with an intramuscular injection of 1.8 ml deslorelin acetate. on re-evaluation a week later, the cysts were still present and drained via transvaginal ultrasound-guided aspiration; no other follicular structures were detected at the time of the transvaginal aspiration. because the removal of follicles via ultrasonographic ablation should elicit the emergence of a new follicular wave, the cow was then subjected to a 7-day cidr-synch protocol. although ovarian follicular cysts are not an infrequent occurrence in dairy cattle, this case is remarkable due to the sheer size of the lesion. in the present case, it is probable that the serial administration of exogenous fsh to induce superovulation contributed to the marked growth of the cystic follicle beyond the typical size reported for follicular cysts. this experience may therefore emphasize the need to closely examine cows undergoing superovulation protocols, especially in the post-partum period, and to provide prompt intervention when cows develop cod and remain unresponsive to conventional hormonal treatments. learning points  cystic ovarian disease is a frequent occurrence with dramatic economic impact on the dairy industry. the administration of exogenous fsh in superovulation protocols for embryo transfer can contribute to increasing incidence and severity of this process. embryo donor cows subjected to fsh superovulation protocols should be monitored for developments of cysts during fsh treatment or shortly after. in our clinic, a subsequent examination of the ovaries is performed with ultrasonography at the time of embryo collection.  while most animals with cod respond to medical treatment (administration of a gnrh analogue or hcg, in conjunction or not with an intervaginal progesterone releasing device and pgf), transvaginal aspiration and removal of cystic contents has been shown to be successful in refractory cows that do not respond to conventional hormonal treatments; transvaginal aspiration in association with hormonal treatments is currently the method of choice for treatment of cod in our clinic. the present case illustrates that surgical drainage and removal of cystic contents may be warranted in cows afflicted with unusually large ovarian cysts that preclude adequate ovarian manipulation per rectum and transvaginal ultrasound-guided aspiration, and that have not responded to conventional hormonal treatments. references 1. perez-martinez c, garcia-fernandez ra, escudero a, et al: expression of cytokeratins and vimentin in normal and neoplastic tissue from the bovine female reproductive tract. j comp pathol 2001;124:70-78. 2. van den hurk r, dijkstra g, van mil fn, et al: distribution of the intermediate filament proteins vimentin, keratin, and desmin in the bovine ovary. mol reprod dev 1995;41:459-467. 3. garverick ha: ovarian follicular cysts in dairy cows. j dairy sci 1997;80:995-1004. 4. peter at, levine h, drost m, et al: compilation of classical and contemporary terminology used to describe morphological aspects of ovarian dynamics in cattle. theriogenology 2009;71:1343-1357. 5. aldahash sya, david jse: anatomical features of cystic ovaries in cattle found during an abattoir survey. vet rec 1977;101:320-324. 6. farin pw, estill ct: infertility due to abnormalities of the ovaries in cattle. vet clin north am-food anim pract 1993;9:291-308. 7. erb hn, white me: incidence rates of cystic follicles in holstein cows according to 15-day and 30-day intervals. cornell vet 1981;71: 326-331. 8. cook dl, smith ca, parfet jr, et al: fate and turnover rate of ovarian follicular cysts in dairy-cattle. j reprod fertil 1990;90: 37-46. 499 clinical theriogenology • volume 4, number 4 • december 2012 9. garverick ha: ovarian follicular cysts in dairy cows. j dairy sci 1997;80:995-1004. 10. hamilton sa, garverick ha, keisler dh, et al: characterization of ovarian follicular cysts and associated endocrine profiles in dairy-cows. biol reprod 1995;53:890-898. 11. kesler dj, garverick ha: ovarian cysts in dairy cattle: a review. j anim sci 1982;55:1147-1159. 12. zaied aa, garverick ha, kesler dj, et al: luteinizing hormone response to estradiol benzoate in cows postpartum and cows with ovarian cysts. theriogenology 1981;16:349-358. 13. refsal kr, jarrin-maldonado jh, nachreiner rf: basal and estradiol-induced release of gonadotropins in dairy cows with naturally occurring ovarian cysts. theriogenology 1988;30:679-693. 14. cook dl, parfet jr, smith ca, et al: secretory patterns of lh and fsh during development and hypothalamic and hypophyseal characteristics following development of steroid-induced ovarian follicular cysts in dairy-cattle. j reprod fertil 1991;91:19-28. 15. brown jl, schoenemann hm, reeves jj: effect of fsh treatment on lh and fsh receptors in chronic cystic-ovariandiseased dairy cows. j anim sci 1986;62:1063-1071. 16. todoroki j, noguchi j, kikuchi k, et al: retrospective analysis of the efficacy of controlled internal drug release in follicular cysts in an embryo donor beef herd. j reprod dev 2004;50:369-373. 17. calder md, manikkam m, salfen be, et al: dominant bovine ovarian follicular cysts express increased levels of messenger rnas for luteinizing hormone receptor and 3 beta-hydroxysteroid dehydrogenase delta(4), delta(5) isomerase compared to normal dominant follicles. biol reprod 2001;65:471-476. 18. boryczko z, bostedt h, hoffmann b: comparison of the hormonal and chemical-composition of the fluid from bovine ovarian follicles and cysts. reprod domest anim 1995;30:36-38. 19. lopezdiaz mc, bosu wtk: a review and and an update of cystic ovarian degeneration in ruminants. theriogenology 1992;37:1163-1183. 20. kruip tam, dieleman sj: steroid-hormone concentrations in the fluid of bovine follicles relative to size, quality and stage of the estrous cycle. theriogenology 1985;24:395-408. 21. hernandez-ledezma jj, garverick ha, elmore rg, et al: gonadotropin-releasing hormone-treatment of dairycows with ovarian cysts. 3. steroids in ovarian follicular-fluid and ovarian-cyst fluid. theriogenology 1982;17:697-707. 22. bartolome ja, thatcher ww, melendez p, et al: strategies for the diagnosis and treatment of ovarian cysts in dairy cattle. j am vet med assoc 2005;227:1409-1414. 23. cairoli f, vigo d, battocchio m, et al: 17beta-estradiol, progesterone and testosterone concentrations in cystic fluids and response to gnrh treatment after emptying of ovarian cysts in dairy cows. reprod domest anim 2002;37:294298. 24. amiridis gs: comparison of aspiration and hormonal therapy for the treatment of ovarian cysts in cows. acta vet hung 2009;57:521-529. 25. cruz ce, corbellini lg, driemeier d: simple procedure for emptying long-term ovarian cysts in cattle. vet rec 2004;155:599-601.   figure 1. ultrasonograph of the left ovary. the ovary was markedly enlarged (too large to be effectively imaged with a 5.0mhz linear transducer probe) with a single cystic structure, filled with hypoechoic fluid. (aloka, 5.0mhz linear transducer, hashmarks represent 1cm) 500clinical theriogenology • volume 4, number 4 • december 2012 figure 2. left flank laparotomy. exteriorization of the ovary allowed observation of a large cystic structure (19 cm in diameter) with distended, well-vascularized wall. fluid contents were straw-colored. figure 3. immunohistochemical staining profile of tissue submitted from the ovarian cyst. a. hematoxylin and eosin stain. b. cytokeratin stain. c. vimentin stain. the brown color indicates positive cytoplasmic staining. bar represents 100 μm. a.  b.  c.  501 clinical theriogenology • volume 4, number 4 • december 2012 table: concentration of hormones in serum and follicular fluid collected at the time of flank laparotomy. hormone serum follicular fluid progesterone (ng/ml) 0.2 76.0 estradiol (pg/ml) 24.2 86.4 testosterone (pg/ml) 22.7 45.4 inhiin (ng/ml) 1.1 >10.0 (editor’s note: the photographs in this paper appear in color in the online edition of clinical theriogenology.) 502clinical theriogenology • volume 4, number 4 • december 2012 2012: just because i can, doesn't mean i should: the application of evidence-based medicine to small animal theriogenology just because i can, doesn’t mean i should: the application of evidence-based medicine to small animal theriogenology melissa goodman veterinary reproductive services, west chester, pa “you ask of my companions. hills, sir, and the sundown, and a dog…… they are better than beings, because they know, but do not tell.” emily dickinson introduction the stresses and demands of modern american society have triggered an increase in dog ownership; as stated by the american veterinary medical association, our canine counterparts provide companionship, joy, unconditional love, a sense of safety, and often a service. this same trend has caused pet owners to become careful consumers, desiring a healthy, stable dog with traits compatible with an individual’s way of life. a pedigreed dog that is carefully bred and raised is the best guarantee that a rewarding and long lasting relationship will develop, and likewise the best guarantee that fewer animals end up unwanted, in shelters or on the street. as the importance of purebred dog ownership is increasingly appreciated, dog breeders are challenged to produce quality animals. the veterinary profession, however, had largely neglected the field of canine reproduction; despite progress in the study of reproduction in human medicine and livestock since the 1960’s, little technical progress was made on behalf of dog breeders. recent years, however, have seen a dramatic change. proper reproductive management is recognized as the foundation of a successful breeding program; genetic screening is seen to be an important factor in planned breedings; technologically advanced breeding systems have been developed to give dog breeders wider options; the pathology and treatment of canine infertility is better understood. most importantly, the veterinarian has become an active participant in the dog breeding process. keywords: evidence-based medicine, artificial insemination, vaginal insemination, transcervical insemination, surgical insemination the importance of evidence while the field has grown, and the demand has increased exponentially, training and information have remained limited. the time sensitive and multidisciplinary nature of small animal theriogenology is difficult to fit into the practical limitations of an academic system; as a result veterinary students, interns and residents have limited exposure to the type of cases that are routinely managed in a practice setting. logistical, financial and ethical constraints severely restrict the amount and quality of research performed, and with it the evidence available to aid in clinical decision making. nonetheless, it remains our obligation to recognize these inherent limitations, and to use the best available resources in a disciplined, science-based approach, to determine both the benefits and potential adverse effects of the procedures and treatments we recommend. by combining our collective knowledge and reasoning and maintaining the “science based” approach available to us, we can do justice to our clients and patients. by ignoring them, we risk succumbing to quackery. veterinary medicine offers some unique ethical challenges not faced by other health professionals. we must serve the needs of both the animals and the humans who own them, in areas as diverse as the beloved pet being given medical care as sophisticated as that provided to their human counterparts, as well as cattle in the feedlot, and laboratory animals used in biochemical research. the leaders of the profession are just beginning to learn how to address these issues. today we come together as veterinarians who work specifically with purebred dog breeders. our society wants and demands healthy pets who fit a specific lifestyle and are both mentally and physically sound, but at the same time unwanted pets fill our shelters. purpose-bred dogs are needed for military service, guide dogs and service dogs. wealthy pet owners want to clone a beloved pet. anti-dog and 521 clinical theriogenology • volume 4, number 4 • december 2012 anti-dog breeding legislation is on the rise at the same time that “designer breeds” are flooding the consumer market. veterinarians must use the advancing technology in reproduction and genetics to help their clients make good breeding decisions to achieve their goals but not neglect the health and well-being of the individual animals or their offspring. evidence based medicine has been described as "the conscientious, explicit and judicious use of the best scientific evidence to inform clinical judgments with a view to improving clinical outcome at the level of the individual case."1 in small animal reproduction, “best scientific evidence” is often absent or lacking. a recent review of the quality of current literature in theriogenology found that only 7 % of publications in canine reproduction were graded adequate to draw sound conclusions.2 an earlier review found that only 31 % could be classified as clinical trials, with the remaining 69 % being case reports or personal opinion. meta-analysis (the integration of data from a number of independent studies to increase the statistical relevance) could not be found in the literature of canine reproduction, and in half, generally accepted and science-based conclusions could not be legitimately drawn from the collected data.3 nonetheless, although they are few and far between, quality research studies that are both critically designed and statistically evaluated are the best predictors of the results likely to occur in our patients. as clinicians, we are forced to make decisions regarding treatment options without significant randomized and controlled scientific studies to guide us. to deal with these limitations, it is helpful to rank the information available into categories that reflect the relative strength of the evidence, and to use this type of methodical and systematic approach to formulate clinical decisions. several reasonable scoring systems have been described, with an example shown below:4 grade i: at least one properly designed randomized controlled clinical study performed in patients of the target species grade ii: evidence from properly designed randomized controlled studies in animals of the target species with spontaneous disease in a laboratory or research colony setting grade iii: appropriately controlled studies without randomization appropriately designed case-controlled epidemiologic studies studies using models of disease or simulations in the target species dramatic results from uncontrolled studies case series grade iv: studies conducted in other species reports of expert committees descriptive studies; case reports pathophysiologic justification/rationale opinions of respected experts many treatment modalities and techniques have become commonplace in small animal reproduction based on anecdotal information, and without proof of efficacy or a critical evaluation of potential risks and benefits. while dog breeders and veterinarians clamor for “newer, better, cheaper”, we must resist the temptation to embrace unproven treatments “just because we can”. applying evidence based reproduction: choosing an insemination method three insemination techniques are widely used in canine artificial insemination, namely (1) vaginal, (2) intrauterine deposition via surgical insemination, and (3) intrauterine deposition via a transcervical approach, either through the use of a rigid catheter or with endoscopic guidance. the techniques involved are well described elsewhere; the specifics will not be discussed here. the choice of insemination technique should be made based on the clinical history of the animals, the quality of the semen being used, the goals of the client, the safety of the procedures, and the skills of the operator. a 522clinical theriogenology • volume 4, number 4 • december 2012 technique should be chosen that is likely to give the desired results using the method that is the least invasive, the least expensive, and safest. how to decide? first evaluate the evidence and grade it according to scientific standards. then apply the evidence to your patient. are the outcomes of the study applicable to your circumstance? are there differences between your patient and the animals in the study that may alter the expected response? are there differences in the semen quality or available insemination dose from that used in the study that may alter the expected response? will the patient’s behavior, breed, or history influence the decision? how will the owner’s values or financial situation affect your decision? what are the patient’s likely benefits and risks from the various options? does the patient have other health conditions that alter the potential benefits and risks of each option? vaginal insemination involves deposition of semen into the cranial portion of the vagina, essentially approximating the site of semen deposition that occurs in a natural mating. this method is indicated for most instances of artificial insemination. this simple technique requires minimal equipment, experience and training, and is relatively noninvasive, with little risk to the bitch regardless of her degree of cooperation (or lack thereof). since it is as close as possible to natural breeding, it will appeal to owners who desire a more natural approach. studies have shown efficacy with fresh, chilledextended and frozen semen of high quality, with conception rates of between 60 and 95%. relatively large numbers of healthy sperm are required, when available this should be the method of choice. surgical intrauterine insemination involves deposition of semen into the cranial uterine horns, close to the site of fertilization in the oviducts, which should maximize conception. this method is indicated when using semen of low quality (either in motility, count, longevity and/or vigor), or in females in which fertility is expected to be compromised. the procedure is quick and simple, and easily performed safely by a veterinarian with basic surgical skills. it can be assumed that only bitches in good general health are voluntarily bred, and with the safe and short acting anesthetics available today, the risks of general anesthesia are low. the procedure is performed using sterile technique, so the risk of bacterial introduction into the uterus is eliminated. a surgical approach allows reliable access to the uterus, so that deposition of semen into the cranial uterine horns is done with confidence, regardless of volume, and without tissue trauma to the vagina, cervix or uterus. visualization and gentle palpation of reproductive organs can be performed to assess possible pathology in subfertile bitches. general anesthesia will minimize stress in nervous, excitable or pain sensitive bitches. however, a surgical approach for an elective procedure may be morally objectionable for some clients. studies and anecdotal reports give the highest success rates with this method. transcervical insemination (tci) involves deposition of semen into the uterine body, avoiding general anesthesia and surgery, although sedation is sometimes necessary. this method is indicated when it is desirable to get past the cervix but not necessary to get close to the site of conception in the oviducts. the procedure requires specialized equipment and can be difficult and time-consuming to learn, as well as frustrating and time-consuming to perform. the size and anatomy of some bitches create challenges that may be difficult to overcome. since the uterine body is small and the myometrium of the uterus has significant resistance, only a small volume of semen can be used without backflow through the cervix into the vagina. this small volume may limit the insemination dose unless semen is concentrated by centrifugation, which will frequently lower sperm quality, especially in samples that are already compromised, or by lowering extender to semen dilution ratios which may also decrease sperm quality. since the cervix is not directly accessible to the operator, tci has an increased risk of trauma of the reproductive tract, especially with the rigid catheter technique. in addition, since the catheter is taken from the nonsterile vagina through the cervix into the uterus, the procedure should be used with caution in bitches who are at increased of pyometra. studies have shown acceptable conception rates with fresh, chilled and frozen semen, although results have been somewhat contradictory from study to study. what is the evidence? a literature search showed no properly designed randomized controlled clinical studies performed in dogs comparing insemination techniques. 523 clinical theriogenology • volume 4, number 4 • december 2012 there were three properly designed studies performed in laboratory or research animal colony settings. in one study, both fresh semen and frozen/thawed semen were used, with both vaginal and surgical intrauterine insemination methods. the semen parameters for all groups were in range of what would be considered adequate numbers and quality. conception rates were similar to other studies of fresh semen (100% ) and frozen/thawed semen (60%). no significant difference was found between vaginal and surgical insemination.5 in another study, two different semen freezing extenders were compared with both intravaginal and transcervical inseminations. again, generally accepted adequate semen parameters for both sperm numbers and quality were used. no significant differences in pregnancy rate or number of fetuses were found between the two insemination methods.6 the third study used a cross-over design to evaluate vaginal vs. surgical intrauterine insemination technique in bitches with decreased fertility. surgical insemination showed a significant increase in conception over vaginal insemination when female fertility was compromised.7 several other experimental studies looking at conception with varying insemination methods were found in the literature, but other variables affecting fertility were not controlled. as a result, these studies could not be used to give evidence concerning the choice of insemination technique. numerous cohort studies, both prospective and retrospective, evaluating canine conception rates were found in the literature. some of these compared insemination techniques, and some evaluated conception using fresh, chilled or frozen/thawed semen using one insemination method. the usefulness of these studies was limited by the number of variables that were poorly controlled or uncontrolled. for example, factors such as ovulation timing methods, number of inseminations performed per cycle, numbers of viable sperm per insemination dose can and do have a profound effect on conception. even still, when evaluating the data of these studies, insemination method appeared to have little effect on conception providing adequate numbers of viable sperm were used. this held true for fresh, chilled and frozen/thawed semen inseminations. studies in other species (equine, porcine and human)8-10 comparing site of deposition of compromised semen largely show an increase in conception with “deep uterine insemination” into the anterior portion of the uterine horns or close to the oviducts. similar results may be expected in the dog, suggesting that surgical insemination might be expected to yield superior results over transcervical and intravaginal insemination when compromised sperm numbers or semen quality is used. safety and side effects with different insemination techniques have not been studied but are discussed as points to consider in several reports. these include the risk of anesthesia and/or surgical complications with surgical insemination, as well as the risk of bacterial introduction, trauma, pain and stress with transcervical insemination. interestingly, in spite of voicing these concerns, several authors suggested doing transcervical inseminations for all breedings, so that the veterinarian could become competent in performing the procedure.11,12 pathophysiologic knowledge may also be used as evidence and rationale to justify the choice of insemination method. since it is known that conception occurs in the oviducts, it follows that the goal of any insemination would be to place semen in a location such that adequate numbers of sperm are present in the oviducts at a time that conception can occur. the anatomy of the bitch presents limitations that must be dealt with when approaching this challenge. in a properly performed vaginal insemination, semen is deposited in the cranial vagina, close to the site nature intended with natural mating. sperm will then travel through the cervix, the uterine body, the uterine horns, and into the oviducts. the canine uterine horns are long, and the lumen is often tortuous, greatly increasing the length the sperm must traverse to reach the oviducts. nonetheless, normal dogs have an extremely high efficiency of reproduction with natural mating, and it should be assumed that vaginal insemination would be successful as well. however, when the fertility of the dogs is compromised, it would also follow that the goal of the insemination technique chosen would be to place the semen as close to the site of conception in the oviducts as is necessary for the individual case. at the same time, insemination method should not further compromise any pathology that exists. for example, transcervical insemination and to a lesser extent surgical insemination, limit insemination volume. to achieve this small volume, centrifugation of the semen may be required, which has a high chance of causing further damage and/or loss of sperm 524clinical theriogenology • volume 4, number 4 • december 2012 numbers in an already compromised semen sample. bitches with decreased fertility may be more susceptible to trauma to the reproductive tract, which is difficult to control with transcervical insemination. likewise these bitches may be less able to clear bacteria that are introduced via transcervical insemination. another consideration might be a negative effect of mild trauma, that may occur with transcervical insemination, to cervical competence. recommendations vaginal insemination should be used for all cases involving normal fertile males and females to minimize risks but maintain high efficacy. this should hold true for chilled and frozen/thawed semen breedings, providing adequate numbers of normal sperm are available. surgical insemination should be used in cases involving dogs and/or bitches with compromised fertility, or when sperm numbers and/or quality is limited. transcervical insemination should be used in cases involving normal fertile bitches but semen quality is not good enough to expect success with vaginal insemination, but not poor enough to require surgical insemination. for ethical considerations, the safest and least invasive method with the lowest risk of side effects, that is likely to achieve the desired results, should be chosen. conclusion as veterinarians, we strive to achieve the best results for our clients. when making therapeutic decisions, we must consider the quality of the evidence available to support our options. improvement in the quality of the studies performed in small animal theriogenology is necessary for our field to legitimately progress. references 1. cochrane al: effectiveness and efficiency : random reflections on health services. london: nuffield provincial hospitals trust: 1972. 2. simoneit c, heuwieser w, arlt s: evidence-based medicine in bovine, equine and canine reproduction: quality of current literature. theriogenology. 2010;76:1042-1050. 3. arlt s, dicty v, heuwieser w: evidence based medicine in small animal reproduction: current available literature. proc 6th int symp canine feline reprod; 2008. 4. roudebush p, allen ta, dodd ce, et al: application of evidence-based medicine to veterinary clinical nutrition. j am vet med assoc 2004;224:1766-1771. 5. silva ld, onclin k, lejeune b, et al: comparisons of intravaginal and intrauterine insemination of bitches with fresh or frozen semen. vet rec 1996;138:154-157. 6. rota a, iguer-ouada m, verstegen j, et al: fertility after vaginal or uterine deposition of dog semen frozen in a tris extender with or without equex stm paste. theriogenolgy 1999;51:1045-1058. 7. brittain d, concannon pw, flanders ja, et al: use of surgical insemination to manage infertility in a colony of research german shepherd dogs. lab anim sci 1995;45:404-407. 8. roca j, rodŕiguez-martínez h, vázquez jm, et al: strategies to improve the fertility of frozen-thawed boar semen for artificial insemination. soc reprod fertil suppl 2006;62:261-275. 9. samper jc: management and fertility of mares bred with frozen semen. anim reprod sci 2001;68:219-228. 10. merviel p, heraud mh, grenier n, et al: predictive factors for pregnancy after intrauterine insemination (iui): an analysis of 1038 cycles and a review of the literature. fertil steril 2010;93:79-88. 11. wilson ms: endoscopic transcervical insemination in the bitch. in: concannon pw, england g, verstegen j, et al, editors. recent advances in small animal reproduction. ithaca (ny): international veterinary information service; 2003. a1232.1203. available at: www/ivis.org. 12. linde-forsberg c: intra-uterine insemination in the dog using the scandanavian trans-cervical catheter and a comparison with other methods. in: concannon pw, england g, verstegen j, et al, editors. recent advances in small animal reproduction. ithaca (ny): international veterinary information service; 2001. a1207.0201. available at: www/ivis.org. 525 clinical theriogenology • volume 4, number 4 • december 2012 526clinical theriogenology • volume 4, number 4 • december 2012 omniblank: 1 contact ramanathan kasimanickam ramkasi@wsu.edu © 2025 the author(s). this is an open access article distributed under the terms of the creative commons attribution-noncommercial 4.0 international license (http:// creativecommons.org/licenses/by-nc/4.0/), permitting all noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 review report selenium in cattle diseases and reproductive health carol majors,‡ andrew myers,† ramanathan kasimanickam college of veterinary medicine, washington state university, pullman, wa, usa ‡current address: soda springs animal clinic, soda springs, id, usa †current address: verona veterinary medical service, verona, wi, usa abstract selenium (se) is a trace mineral critical for antioxidant activity, immune function, thyroid hormone conversion, fetal development, and reproduction. the goal is to emphasize the importance of preventing se deficiency in cattle through year-round supplementation, soil analysis, and appropriate treatments to avoid conditions like white muscle disease, nutritional muscular dystrophy, and reproductive issues. certain us regions have se-deficient soils that may produce se-deficient forages. in these areas, se deficiency-related syndromes are common. cattle require 0.1 mg/kg of se for maintenance and more during pregnancy or lactation. across age groups, deficiency results in various clinical manifestations, including white muscle disease in neonates, nutritional muscular dystrophy in calves and yearlings, and reproductive dysfunction in adults. diagnosis involves soil and pasture analyses, se concentrations in blood and serum, erythrocyte glutathione peroxidase activity, liver biopsies, or tissue recovery at slaughter or necropsy. treatment is generally ineffective for neonates, but can be successful in older cattle, with injectable se and ongoing supplementation being key. prevention through year-round supplementation is crucial in se-deficient regions. keywords: nutrition, selenium, food animal, health, reproduction introduction selenium (se) is a trace element with important roles in animal health and performance. in cattle, se deficiency can have economically important impacts such as reduced fertility, retained fetal membranes (rfm) and increased mastitis and metritis.1 this review includes the importance of meeting se requirements for cattle, how to recognize clinical manifestations across age groups, and using yearround se supplementation to prevent deficiency-related conditions, such as enzootic (nutritional) muscular dystrophy (nmd) and reproductive dysfunction in adult cattle.2,3 selenium is a naturally occurring trace mineral with a critical role in bovine health; however, its availability in soil and forage varies widely across the us. areas with se-deficient soils (e.g. the pacific northwest) are at particularly high risk for se deficiency in cattle, leading to a range of issues, from neonatal diseases to reproductive and metabolic problems in adults.4 understanding the role of se in cattle, clinical signs of deficiency, and methods for diagnosis, prevention, and treatment, are essential for effective herd management. there is considerable variation in soil se concentrations across the continental us (figure 1),5 with markedly lower soil se concentrations in the northwest, northeast, southeast, and great lakes states. soils with concentrations < 0.05 ppm are considered critically low, whereas concentrations above this threshold are generally considered adequate. however, se deficiency does not depend solely on soil se concentrations, as deficiency in cattle occurs because of se absorbed by plants that is influenced by soil ph and availability of se compounds in soil. critically low soil se concentrations lead to forages with < 0.1 ppm se in > 80% of cases, often resulting in deficiencies in unsupplemented cattle. in areas with variable soil se concentrations (figure 2),6 forages are roughly split between deficient and adequate; therefore, some cattle may still develop se deficiencies despite adequate soil concentrations. as forages obtain their se from soil, the distribution of se-deficient areas in cattle generally mirrors the distribution of soil se concentrations, albeit not perfectly. seleniumdeficient soils substantially increase the risk, but do not guarantee se-deficient cattle. mailto:ramkasi@wsu.edu http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ http://dx.doi.org/10.58292/ct.v17.12015 2 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 selenium deficiency in south atlantic seaboard’s soil is largely attributed to costal deposits washed from highly weathered land masses. the northeastern states’ soil predates cretaceous period selenization; seleniferous cretaceous shales did not weather in these areas, resulting in low soil se concentrations. much of these cretaceous sedimentary shale rocks weathered into the central and northcentral states from montana and north dakota, through wyoming, colorado, and south dakota, resulting in soils that produce forages with variable or adequate se.7 washington state is riddled with volcanoes; 5 are active and have erupted relatively recently, with volcanic deposits creating very low soil se concentrations.7 current agricultural practices (e.g. intensive cropping) can further decrease soil se concentrations. figure 1. color-coded map of relative se concentrations in soil samples for counties in the continental us. soil se < 0.05 ppm is critically low; higher values are considered adequate, depending on soil ph and se availability to plants4-9 figure 2. distribution of forages generally deficient, relatively low, and adequate in se6 http://dx.doi.org/10.58292/ct.v17.12015 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 3 selenium requirements daily se requirements for cattle are 100 μg/kg dry matter (dm) for beef cattle, 300 μg/kg dm for dairy cows and 100 μg/ kg dm for calves.1 there is a close link between se and vitamin e status, as well as antioxidant status.8 vitamin e has a key role in se absorption and utilization.9 selenium and vitamin e have interdependent antioxidant functions; therefore, low vitamin e intake may increase the se required to prevent some conditions.9 the national academies of sciences, engineering, and medicine (nasem; formerly national research council, the operating arm of the nasem) recommends a daily intake of 15-60 iu of vitamin e for adult cattle, whereas nursing calves require 40-60 iu.10,11 deficiencies in either se or vitamin e can impair thyroid metabolism, decrease growth rate, reduce fertility, alter phagocytic response, and lower disease resistance.12 certain dietary components can negatively impact se absorption in cattle. diets rich in carbohydrates, nitrates, sulfates, calcium, or hydrogen cyanide (e.g. from clover forage or flax seeds) can interfere with se utilization. sulfur competes with se for absorption at concentrations > 2.4 g/kg dm. similarly, ferric iron can reduce se absorption by forming insoluble complexes that are not assimilated by enterocytes.13 calcium also influences se absorption; concentration of 0.8% dm in the feed supports optimal se absorption in dairy cows during late pregnancy.14 however, calcium supplementation in beef cattle, especially at concentrations typically used in intensive production, significantly reduces muscle se concentrations.15 additionally, high concentrations of lead in a calf diet can decrease both serum and tissue se concentrations.16 it should also be noted that any lead exposure sufficient to affect se concentrations is likely to present a food safety risk. the primary concern for the practitioner should be the adulterated meat from these animals, rather than se deficiency. iodine deficiency can exacerbate se deficiency.17 in cattle, iodine deficiency leads to marked induction of selenoprotein-d1, accompanied by elevated glutathione peroxidase (gpx) activity. finally, diets containing adequate levels of crude protein and cellulose can enhance animal’s ability to absorb and utilize se more efficiently. selenium intake without supplementation most se for beef cattle is obtained through forage, with soluble se compounds in water a much poorer source.15 in areas with moderately low or variable soil se concentrations, certain plants, (e.g. alfalfa) incorporate more se than other forages (e.g. red clover, timothy, or brome grasses).18 therefore, feeding alfalfa in these regions can reduce the need for supplementation. additionally, neutral or acidic soils can impair a plant’s ability to uptake se. forage testing is an important tool in evaluating herd se status.19 organic se compounds in plant matter are easily absorbed by the gastrointestinal tract and have more prolonged retention. in contrast, inorganic se (e.g. selenate) in water is readily absorbed but also rapidly excreted. excess se is primarily excreted in urine, with lesser amounts excreted in feces and breath. selenium is mainly stored in the liver and, to a lesser extent, in the kidney.19 selenium utilization selenium is present in nearly all tissues, but it is most concentrated in tissues that are rapidly breaking down, growing, or dividing, including heart, skeletal muscle, ovaries, and testes. it is also present in immune system tissues (including bone marrow, thymus, liver, intestines, spleen, and lymph nodes), thyroid, and other organs.20 there are many selenoproteins, each with a specific location and function. selenoproteins involved in reproduction are listed (table 1).21,22 selenium has a crucial role as a functional component of a group of antioxidants known as gpx,23,24 a water-soluble cytosolic enzyme that protects cellular membranes and membranes of lipid-containing organelles, from irreversible damage caused by peroxides. vitamin e has a similar function, but as a lipid-soluble antioxidant in cell and organelle membranes. peroxides can denature cellular proteins, causing cell degeneration and death. glutathione peroxidase catalyzes breakdown of hydrogen peroxide (h2o2) and related hydroperoxides.23,24 based on a cell membrane as the central structure, the role of oxidative stress (os), generation of reactive oxygen species (ros), antioxidant defenses, and lipid peroxidation are summarized (figure 3). cell membrane and phospholipids the cell membrane’s phospholipid bilayer, with hydrophilic heads and hydrophobic tails, maintains membrane integrity and selective permeability.25 unsaturated fatty acids in the tails create bends that promote fluidity, crucial for membrane flexibility, protein movement, and communication. lipid peroxidation from ros damages the membrane, triggering inflammation and cell death if uncontrolled.26 reactive oxidative stress generation mitochondria are the main source of ros during atp production, leaking electrons that form superoxide anions (o2• –), which convert to h2o2. 28,29 nadph oxidases and enzymes like cytochrome p450 and lipoxygenases also contribute to ros production, particularly in immune responses and inflammation.30-32 enzyme actions in reactive oxidative stress detoxification enzymes like gpx, superoxide dismutase (sod), and catalase protect cells by neutralizing ros. glutathione peroxidase reduces lipid peroxides, sod converts superoxides to h2o2, and catalase converts h2o2 to water and oxygen, preventing oxidative damage.27,33,34 micronutrient protection against reactive oxidative stress vitamin e, a fat-soluble antioxidant, protects membrane lipids from oxidative damage.36,37,38 vitamin c scavenges ros and regenerates antioxidants,35,36 whereas coq10 neutralizes lipid-soluble ros and supports mitochondrial function and atp production.38 oxidative stress and membrane damage oxidative stress occurs when ros production exceeds antioxidant defenses, causing membrane damage, destabilizing fluidity, and impairing function.39 this can lead to protein http://dx.doi.org/10.58292/ct.v17.12015 4 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 dysfunction, increased permeability, and cell death.40 antioxidants and membrane repair mechanisms help prevent and restore membrane integrity. role of selenium in immunity selenium has a crucial role in maintaining immune function and supporting defenses against infections. as a key antioxidant, it is integral to several cellular processes that regulate immune responses, particularly through its incorporation into enzymes like gpx that manages os and inflammation.41 given the close relationship between se and vitamin e, it is not surprising that many se-responsive diseases may also respond to vitamin e supplementation. selenium’s role in the immune system is summarized below. table 1. selenoproteins, principal location, and functions nomenclature selenoprotein location function gpx1 glutathione peroxidase 1 follicle antioxidant protection gpx2 glutathione peroxidase 2 embryo antioxidant protection gpx3 glutathione peroxidase 3 follicle, endometrium antioxidant protection gpx4 glutathione peroxidase 4 embryo, testis, sperm mitochondrial sheath antioxidant protection gpx5 glutathione peroxidase 5 epididymis, sperm antioxidant protection txnrd1 thioredoxin reductase 1 embryo antioxidant role, redox regulation, cell signaling txnrd2 thioredoxin reductase 2 embryo antioxidant role, redox regulation, cell signaling txnrd3 thioredoxin-glutathione reductase sperm antioxidant role, redox regulation, cell signaling dio1,2 &3 iodothyronine deiodinase 1, 2, and 3 utero-placental unit t3 production selenoh selenoprotein h trophoblast, placenta glutathione synthesis selenop selenoprotein p seminal plasma, utero placental unit se transport, antioxidant mcsep mitochondrial selenoprotein sperm mitochondrial capsule store for gpx4 tgr thioredoxin-glutathione reductase spermatid antioxidant figure 3. schematic presentation of cell membrane oxidative metabolism/antioxidant interactions, indicating how gpx 1, 2, and 4 and other micronutrients protect membranes and organelles from peroxidation23,27 http://dx.doi.org/10.58292/ct.v17.12015 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 5 cell-mediated and humoral immunity selenium, in conjunction with vitamin e, is essential for both cell-mediated and humoral immunity. deficiencies in se compromise resistance to infections, reduce neutrophil function, and decrease antibody production. these deficiencies affect proliferation of t and b lymphocytes in response to mitogens and impair the function of natural killer cells.42 neutrophil function neutrophils have a key role in the innate immune response and rely heavily on se to maintain high gpx enzyme activity.43 this enzyme is vital for managing os during phagocytosis (process by which neutrophils engulf and kill pathogens). without adequate se, neutrophils have a reduced ability to eliminate pathogens and manage peroxides generated during this process.43,44 selenium supplementation in colostrum significantly increased igg concentrations in calves45; a single feeding of colostrum supplemented with 3.0 ppm se caused a 40% increase in peripheral igg that persisted at least 2 weeks, implying a prolonged benefit to immunity. increases in igg were attributed to se’s ability to promote pinocytosis (the process by which cells absorb antibodies from colostrum) within intestinal epithelial cells.46 this mechanism ensures more efficient absorption of immunoglobulins, providing calves with better passive immunity. improved humoral response selenium supplementation enhanced the humoral immune response in cattle experimentally infected with the infectious bovine rhinotracheitis virus.47 selenium-supplemented cattle produced more antibodies and had a stronger immune response compared to control groups. although se deficiency may increase susceptibility to infections, naturally se-deficient herds have not had a consistent increase in infection rates or severity.45,48 in addition to se, other factors also influence disease incidence. selenium is transferred from dam to fetus during pregnancy, ensuring a calf is born with adequate se. prenatal se supplementation in dams helps produce se-sufficient calves and improves immune status at birth.49 after birth, se concentrations in calves deplete rapidly, typically within 18 days. however, the initial boost in se from the dam may provide early-life protection against neonatal morbidity and disease. milk se concentrations are highly correlated with maternal blood se concentrations. selenium-adequate dams can provide se-sufficient milk to their calves until weaning, supporting calf’s immune system until they are able to consume solid feed.50,51 in cattle, se supplementation can enhance passive immunity (igg transfer), improve immune responses during infections, and provide essential nutrients to offspring through placenta and milk. ensuring dams and calves are sufficient to promote a strong immune system, reduces susceptibility to infections and improves herd health. role of selenium in thyroid function in cattle, se has a critical role in thyroid function by supporting synthesis, metabolism, and regulation of thyroid hormones.52 thyroid gland produces hormones, primarily thyroxine (t4) and triiodothyronine (t3) that regulate metabolism, growth, and development. selenium is essential for enzymes involved in the activation and deactivation of these hormones. selenium is incorporated into several selenoproteins that are crucial for thyroid function, most notably the deiodinase enzymes, in cattle, mice, and humans.52,53 these enzymes are responsible for the activation and inactivation of thyroid hormones: type 1 deiodinase: this enzyme converts t4 into the more active t3 that is essential for regulating metabolism and energy balance; it is highly active in liver and kidneys. type 2 deiodinase: this enzyme converts t4 to t3, but it is mainly active in the brain, pituitary, and brown adipose tissue. in the brain, it has an important role in regulating local concentrations of t3 to support neurodevelopment and cognition. type 3 deiodinase: this enzyme converts t4 and t3 to inactive forms; it has an important role in regulating thyroid hormone action, especially in placenta and brain. in cattle, inadequate se reduces the efficiency of these enzymes and the conversion of t4 into active t3.52 antioxidant defense in the thyroid selenium is a key component of gpx, an enzyme that helps protect the thyroid from os54 when it generates h2o2 during thyroid hormone synthesis. excess h2o2 can cause oxidative damage to thyroid cells.55 glutathione peroxidase, with the help of se, neutralizes this peroxide, prevents damage and ensures thyroid function.54 the antioxidant role of se protects the thyroid from damage during high hormone production or stress. thyroid hormone synthesis selenium is involved in synthesis of thyroid hormones; adequate se concentrations ensure that the thyroid can produce sufficient t4 and t3.52,56 although iodine is a key component for thyroid hormone synthesis, se promotes thyroid function, contributing to activation of thyroid hormones. in cattle, selenium affects pituitary release of thyroid-stimulating hormone (tsh) that stimulates thyroid to produce t4 and t3.58 further, se deficiency can increase tsh concentrations to compensate for low thyroid hormone concentrations, potentially causing subclinical hypothyroidism (marginal thyroid hormone concentrations).57 selenium deficiency and thyroid hormone imbalance when se concentrations are inadequate, the conversion of t4 to t3 is impaired due to reduced activity of the deiodinase enzymes; this can reduce the t3/t4 ratio that can affect various metabolic processes. a se-deficient diet reduces t3 concentrations, although t4 concentrations may increase. this imbalance can lead to suboptimal metabolic function and growth issues, as t3 is the active hormone responsible for regulating energy expenditure, cell growth, and development.58 selenium deficiency in calves is often associated with poor growth and performance,12,59 due in part due to reduced thyroid function and hormonal imbalance. since t3 is involved http://dx.doi.org/10.58292/ct.v17.12015 6 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 in regulation of growth, se deficiency could impair growth rates and developmental processes in young animals.1,12,59 selenium-deficient calves often have slower growth rates, likely due to impaired thyroid function and reduced t3.12,59 in these calves, se supplements can restore normal thyroid function, improve t3 production, and promote growth. however, se supplementation typically does not affect growth in well-nourished calves. selenium supplementation and improved thyroid function milk selenium and neonatal growth the correlation between milk se concentrations and neonatal t3 concentrations highlights the importance of se-rich milk for supporting newborn development. since t3 has a critical role in growth, adequate se intake during the neonatal period is essential for promoting growth and development in offspring. positive effects in ewes and lambs selenium supplementation can increase t3 in both ewes and their lambs. for example, se-supplemented ewes had higher t3 in their serum and milk that positively affected thyroid hormone concentrations in their lambs. in these lambs, there was a strong positive correlation between milk se concentration and serum t3 (r = 0.84, p < 0.01).60,61 in contrast, a negative relationship was observed in the control (unsupplemented) group; lower se intake reduced t3 and increased the t4/t3 ratio. role in fetal development selenium transfer from dam to fetus selenium is primarily incorporated into selenoproteins (e.g. gpx and thioredoxin reductase) that have critical roles in reducing os and protecting cells. the fetus relies on maternal se to ensure proper development and to establish its own antioxidant defenses. selenium is actively transported across the placenta from maternal blood to the fetus.62 the placenta has specialized transport systems to facilitate transfer of various nutrients and trace elements, including se, to support fetal development. selenium is transported in the blood as selenomethionine or selenate and transferred from dam to fetus via the placenta, facilitated by placental transporters, including sodium-dependent active transport mechanisms.63,64 fetal se concentrations largely depend on maternal se intake.62,64 if a dam has adequate se, the fetus is typically well supplied. however, maternal se deficiency can reduce se transfer to the fetus, with detrimental effects on fetal development.65 selenium, through its role in selenoproteins (gpx), protects fetal tissues from oxidative damage, crucial during rapid cell division and organ development.66 selenium’s role in embryonic development and maternal health is critical, especially during pregnancy, where it influences antioxidant defense, hormonal regulation, and fetal growth.66-68 although much of se’s function is understood in terms of postnatal development, its importance during pregnancy is equally important, particularly in protecting the fetus from os and supporting placental function. the role of se in embryonic development, fetal health, and maternal nutrition during pregnancy is as follows: selenium’s role in embryonic development and antioxidant protection • dna methylation and gene expression: selenium affects dna methylation and mrna expression during embryonic development. these processes are essential for cell differentiation, proliferation, and apoptosis, critical in early development.69 disruptions in these processes, especially those involving os, can negatively affect embryo development and survival. • oxidative stress in embryonic development: during embryonic development, os is an important concern. the embryo, despite developing in a relatively low-oxygen environment, has low antioxidant capacity and is particularly vulnerable to oxidative damage. selenium added to the culture media can improve embryonic development.70 cocultured 2-cell stage embryos and the oviductal epithelial cells had a significantly increased cleavage rate and enhanced blastocyst development,71-73 suggesting se may promote embryogenesis (figure 4). • selenium’s antioxidant role: selenium has a crucial role in mitigating os by being a key component of selenoproteins (e.g. gpx) that neutralize ros. selenium supplementation during pregnancy ensures the proper balance of antioxidants in both the dam and fetus, reducing cellular damage that can impair development. selenium and pregnancy complications • low se and pregnancy complications: in small ruminants and humans, low se concentrations during pregnancy are linked to low birth weight, impaired fetal growth, and increased likelihood of a small for gestational age (sga) fetus (figure 4).74,75 in humans, se’s antioxidant properties help protect against oxidative damage in the developing fetus, and insufficient se during pregnancy can lead to os that harms both maternal and fetal health.76 • progesterone production: selenium also has a role in progesterone production that is essential for maintaining pregnancy (critical for maintaining uterine conditions conducive to fetal development and for preventing premature labor). in a study with heifers, se supplementation increased plasma progesterone concentrations during the late stages of pregnancy (29-39 weeks), indicating that se supports the function of the corpus luteum (cl) and the placenta during pregnancy (figure 4).77 placental development and selenium’s influence on oxidative balance • oxidative stress and placental function: during pregnancy, the placenta is the interface between the dam and fetus, facilitating nutrient and oxygen exchange. during placental development, os can affect placental function (figure 4).78 placenta changes over time, including increased nadph oxidase-mediated ros generation, that affects trophoblast proliferation, spiral artery remodeling, and placental growth.79 excessive ros can compromise placental function, especially if antioxidant function is inadequate. • complications from oxidative imbalance: imbalances in ros and antioxidant capacity can lead to insufficient remodeling of spiral arteries by extra-villous trophoblasts that is essential for proper blood flow and nutrient exchange.80 in humans, disruptions in this process may result in pregnancy complications such as preeclampsia and intrauterine growth restriction (iugr), both are associated with fetal hypoxia.65 http://dx.doi.org/10.58292/ct.v17.12015 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 7 effects of selenium supplementation on maternal health and fetal development • selenium and maternal health: selenium supplementation during pregnancy can promote antioxidant balance and support placental function, reducing pregnancy complications.65,81 it is particularly beneficial in preventing conditions related to os, protecting maternal immune system and tissues. • impact on fetal development: in humans and animals, adequate se intake during pregnancy supports fetal growth and development, reduces the risk of iugr and promotes development of critical tissues, e.g. brain and lungs.8,82 selenium’s antioxidant properties protect fetal tissues from oxidative injury, promoting organ development.8,65,82 effects on lactation and offspring growth • selenium and colostrum: selenium supplementation during pregnancy and lactation increases gpx activity and reduces lipid oxidation in colostrum, improving antioxidant capacity.60 this provides the newborn with essential antioxidants, prevents oxidative damage and promotes immune function (figure 4). • selenium and growth of calves and piglets: supplementing se in cattle and pigs improved milk composition and colostrum quality and enhanced transfer of immunoglobulins (iga, igm) to calves and pigelets.1,81,83 this enhanced preweaning survival, immune function, and piglet growth and development. • selenium and heat stress: selenium supplementation of heat-stressed cows and sows, improved antioxidant capacity and immunoglobulin transfer.81,83 this promoted maternal health and fetal development, despite exposure to high temperatures. selenium is vital for supporting healthy fetal development, placental function, and maternal health during pregnancy. its role in reducing os and supporting hormone production is indispensable to support pregnancy and promote growth and development of the offspring. role in female reproduction selenium offers a broad range of health benefits for livestock, especially cattle, having a crucial role in reproductive health. deficiencies in se can cause large economic losses, due to impaired fertility, increased incidence of mastitis, metritis, rfm, and others.13,84 selenium has a vital role in oocyte and ovary function. in an in vitro study using a buffalo oocyte cell line, se significantly improved the rate of nuclear maturation and reduced the number of cells in the germinal vesicle stage. although se did not affect cell development at other meiotic stages, it altered expression of several genes involved in oocyte development, including downregulating casp3 and amh, and upregulating gpx4 and sod.85 selenium also reduced transcript levels of pla2g3, a phospholipase involved in lipid metabolism.85,86 similarly, in a study on primary luteinized granulosa cells from goat ovarian follicles, se stimulated cell proliferation and increased markers associated with estradiol production, such as 3β-hsd, p-akt, camp, and steroidogenic acute figure 4. roles of selenium in female reproduction 1 2 3 4 5 6 7 8 9 10 11 12 13 5 66 7 88 999 1100 12 1133 http://dx.doi.org/10.58292/ct.v17.12015 8 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 regulatory protein.87 therefore, se may regulate the hypothalamic-pituitary-gonadal (hpg) axis by influencing sex hormone production through its effects on redox balance and lipid metabolism. in terms of reproductive health, se is critical for fertility and early embryonic development. adequate se reduces os during ovarian follicular and cl development, thereby protecting reproductive organs from ros (figure 4).77 selenium also has a key role in maintaining progesterone production in heifers that is essential for pregnancy maintenance (figure 4).77 without sufficient se, the cl cannot function properly that can lead to reproductive failure. selenium supplementation reduces the incidence of rfm, metritis, endometritis, and ovarian cysts, all negatively affect cow health and herd productivity. ensuring se adequacy before and after calving is crucial to optimize reproduction.88-91 cows with adequate se also have fewer services per conception, a higher pregnancy rate at first service, and a shorter interval to conception,90,91 all improve reproductive efficiency. selenium also impacts mastitis and metritis. selenium deficiency can impair milk quality, alter colostrum composition, and hinder immune transfer to the offspring.81,92-97 inadequate se increases the risk of mastitis.98 selenium’s antioxidant properties protect the mammary gland from oxidative damage, reducing mastitis incidence (figure 4).99 selenium supplementation also reduces the risk of metritis, a uterine infection that can follow calving13,91 and lead to prolonged postpartum anestrus, delaying conception. by improving immune function and reducing inflammation, se helps prevent these conditions, enhancing reproductive health.100,101 regarding se supplementation sources, the form of se (sodium selenite or selenized yeast) has little impact on conception rates or calving intervals.102 however, sources may vary in bioavailability and effectiveness in various farming systems. regardless of the source, ensuring adequate se in the diet is essential for maintaining overall reproductive health in cattle. from an economic perspective, se supplementation improves reproductive efficiency by reducing the number of services per conception, improving first-service success rates, and shortening calving intervals.103 these benefits increase herd productivity and profitability. selenium also lowers the incidence of metritis, mastitis, and rfm, reducing veterinary costs and leading to healthier cows1,56,88,103 with greater milk yields, longevity, and long-term profitability. further, the bovine ovary contains high levels of se that are localized to healthy preovulatory follicles, but not atretic follicles.72 the presence of se in healthy follicles implies involvement in preparing the oocyte for fertilization, embryo development, and postnatal life (figure 4). adding se to in vitro fertilization (ivf) cultures for cattle, dogs, pigs, and yaks improved embryo development, reduced ros, and decreased dna damage.104-108 cattle fed organically bound se have better conceptus length and differential expression of genes related to pregnancy recognition (figure 4).109,110 furthermore, ewes supplemented with se throughout pregnancy have increased cell density and cell proliferation in the placenta during late pregnancy that correlates with improved lamb birth weights.111 these effects on placental development may partially explain effects of se on fetal growth and development.111-114 in humans, low se in plasma, follicular fluid, or amniotic fluid, as well as low tissue gpx, are associated with infertility, miscarriage, preterm birth, gestational diabetes, and sga fetuses,75,115-118 emphasizing the importance of se for reproductive success across species. 1. regulates preantral and preovulatory follicle function, enhancing fertility competence of oocytes; 2. enhances cl function, ensuring sufficient progesterone to support pregnancy; 3. promotes successful fertilization; 4 and 5. improves early embryonic development in the oviduct by increasing the cleavage rate and 8-cell embryo development; 6. enhances blastocyst rate; 7 and 8. increases conceptus length by enhancing the expression of genes related to maternal recognition of pregnancy, including interferon-stimulated genes and progesterone-stimulated genes; 9. promotes early embryo and fetal development; 10. increases cell density and cell proliferation in the placenta; 11. as the placenta is the primary site for the exchange of nutrients, respiratory gases, and metabolic wastes, se promotes fetal growth and development; 12. protects mammary gland from oxidative damage and reduces incidence of mastitis; 13. enhances immune function in neonate. selenium is an essential micronutrient that has a crucial role in immune function and reproductive health in cattle. it helps protect against os and inflammation, supports fertility, and improves milk quality. supplementing cattle with se, especially in areas where the soil is deficient, can improve reproductive efficiency, health outcomes, and overall economic performance. ensuring adequate se before and during pregnancy, as well as in the postpartum period, can significantly reduce the incidence of reproductive disorders such as rfm, metritis, and ovarian cysts, while also enhancing immune function and disease resistance. role in male reproduction selenium has a crucial role in sperm health, sperm parameters and fertility,119 particularly in bulls (figure 5). selenium, through its incorporation into selenoproteins like gpx4,120 has several protective and structural functions in sperm, promoting viability, motility, and integrity that are critical for fertility. selenium is an essential component of gpx4, a key antioxidant enzyme protecting sperm from oxidative stress-induced damage.119 selenium (via gpx4) contributes to the structural integrity of the sperm’s midpiece, critical for mitochondrial function and motility.121 oxidative stress caused by ros can damage sperm membranes and impair motility.122 adequate se protects sperm mitochondria producing energy that is essential for sperm motility.123,124 poor motility reduces sperm fertilizing ability and fertility. although gpx4 activity is not significant in sperm, it is crucial for protecting sperm at various stages of development. in the early stages of spermatogenesis, gpx4 protects sperm dna from oxidative damage. gpx4 is strongly expressed in seminiferous tubules of healthy bulls, particularly in spermatogonia, round spermatids, and elongated spermatids, suggesting a critical role in sperm maturation. immuno-signals for gpx4 are also detected in the acrosomal region of epididymal and ejaculated sperm, further emphasizing its importance in sperm function.119 http://dx.doi.org/10.58292/ct.v17.12015 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 9 impact on sperm quality selenium supplementation of holstein bulls improved various sperm quality parameters, including viability, motility, membrane integrity, and postthaw quality. these improvements are particularly notable after semen cryopreservation.125 supplementation also reduced lipid peroxidation and apoptosis/necrosis rates in sperm that are commonly associated with oxidative damage. selenium supplementation elevates antioxidant biomarkers in seminal plasma, including total antioxidant capacity (taoc) and malondialdehyde (mda), markers of os. selenium reduces oxidative damage to the sperm, particularly in winter and autumn, when gpx concentrations in the testes naturally increase.126 selenium deficiency can increase abnormal sperm morphology (e.g. deformed heads or tails) that reduces fertilizing ability.127 selenium, through its antioxidant properties, helps prevent oxidative damage to sperm dna that is crucial for fertilization and development.128 selenium has a multifaceted role in protecting sperm from oxidative damage, improving sperm quality, and contributing to overall fertility and reproductive health in bulls. by incorporating se into selenoproteins (e.g. gpx4), sperm maintain structural integrity, motility, and viability, making se supplementation a useful tool for improving sperm health and fertility. selenium nanoparticles (senps) protect against aflatoxin b1 induced toxicity, reducing morphologically abnormal sperm and dna fragmentation129 and preserving testicular architecture, mitigating atrophy of the seminiferous tubules.130 furthermore, senps protected against deltamethrin-induced reproductive toxicity and negative effects such as sperm characteristics, tst, and antioxidant biomarkers, as well as behavioral and histopathological alterations. bulls given senps had improved semen parameters, antioxidant status, and sexual performance.131 in addition, senps improved the quality of semen in rats and goats.131-133 selenium’s role in testosterone production hormonal regulation: selenium has an indirect but essential role in testosterone (tst) production by supporting testicular function. testosterone is a key hormone for spermatogenesis and overall bull fertility. selenium and the hypothalamic-pituitary-gonadal axis: selenium is involved in the proper functioning of the hpg axis that regulates the release of hormones, including lh and follicle stimulating hormone (fsh).134 these hormones stimulate the testes to produce tst and sperm. selenium also helps regulate the enzymes involved in testicular function. selenium is also important for maintaining production of sex steroid hormones, including tst, estradiol, and progesterone. selenium depletion lowered tst concentrations while decreasing testicular mass and adversely affecting testicular morphology.135 although testicular mass of first-generation selenium-deficient rats was not significantly changed, each successive generation had increasingly worsening symptoms. exogenous gnrh or lh induced a reduced tst secretion in se-deficient rats; perhaps tst production was impaired or receptors or signaling pathways for gnrh and lh were affected. selenium supported tst synthesis by activating the extracellular signal-regulated kinase (erk) signaling pathway in ovine leydig cells.136 selenium affects proliferation and apoptosis of leydig cells by regulating os and expression of cell cycle and apoptosis-related genes.131,132 in addition, se can enhance tst production by activating the erk signaling pathway and expression of downstream genes (star and 3β-hsd) that could be related to regulatory roles of se in male fertility and spermatogenesis.136,137 selenium has an important role in immune system function, enhancing the ability of white blood cells (e.g. neutrophils figure 5. effects of selenium supplementation on sperm and fertility http://dx.doi.org/10.58292/ct.v17.12015 10 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 and lymphocytes) to respond to infections.133 a healthy immune system is crucial for maintaining reproductive health in bulls, as it reduces risk of reproductive infections (seminal vesiculitis, epididymitis, or orchitis) that can impair sperm production and quality.138 selenium deficiency can suppress immune responses, increasing susceptibility to reproductive tract infections that reduce sperm quality. seminal plasma selenium is present in the seminal plasma (457.4 ± 108.7 μg/ liter) and its antioxidant activity protects sperm from oxidative damage during ejaculation and transport through the female reproductive tract.136,137 selenium is also involved in production of seminal plasma proteins that support sperm viability and function. selenium-dependent enzymes promote the health of sperm and improve their ability to survive in the female reproductive tract. fertility and in vitro findings selenium supplementation enhances sperm function in ivf. it significantly improves mitochondrial activity in sperm and sperm acrosome integrity and promotes sperm binding to the zona pellucida (zp), critical for fertilization.21,139 beef cattle selenium requirements concentrations of blood se > 100 mg/liter are required to maintain optimum immunocompetence in growing beef cattle.140 dietary requirements to avoid nmd are 0.1 mg/kg dm. regular se analysis of forages and feeds is necessary to detect deficiencies, and supplementation may be needed, in the form of salt blocks, loose minerals, or selenium-enriched yeasts.3 the fda permits selenium to be included in the total diet of beef cattle at a maximum level of 0.3 ppm of dry matter. in areas where deficiencies are common, the maximum allowable level should be used (https://www.ecfr. gov/current/title-21/chapter-i/subchapter-e/part-573/subpart-b/section-573.920 [cited april 8 2025]). additionally, the fda allows up to 120 ppm of se to be incorporated into a salt-mineral mixture for free-choice feeding. selenium deficiency is unlikely if adequate amounts are consumed through the mineral supplement. however, the se concentration in the supplement and the labeled intake must ensure that the total daily intake does not exceed 3 mg. for example, a mineral supplement with a recommended intake of 4 ounces per head per day cannot contain more than 26 ppm of se. clinical presentation of selenium deficiency in various age groups selenium deficiency, often paired with a lack of vitamin e, can lead to various nmd in cattle, with clinical signs that can range from acute to subacute.141 these disorders affect a wide array of conditions, including poor growth in pastured calves, lowered milk production and subclinical mastitis in dairy cows, decreased fertility, embryonic death, rfm, metritis, poor uterine involution, cystic ovaries, impaired immune function, and even premature death, especially in cases of marginal se deficiency.3 in neonates, se is crucial for fetal development, and its deficiency in calves can lead to severe health issues, as se crosses the placenta from dam to fetus. selenium-deficient calves may suffer from myocardial necrosis, heart failure, and other systemic issues due to insufficient antioxidant activity from gpx, an enzyme dependent on se. clinical signs include premature birth, perinatal death, heart or respiratory failure, and spontaneous abortion. postmortem findings will typically include myocardial and skeletal muscle damage. timely se supplementation to both dams and calves is essential for preventing further damage.8,20 in beef calves aged 2-8 months, se deficiency often manifests as acute or subacute nmd after unaccustomed exercise. calves raised indoors and then exposed to pastures or those moved long distances may develop nmd due to se-deficient diets. the os from exercise accelerates muscle degeneration, leading to muscle calcification and widespread tissue damage. acute nmd can lead to death from heart failure or respiratory distress, with affected calves being weak, recumbent, and unable to stand.8,141 subacute nmd, more common in rapidly growing calves, typically affects skeletal muscles, causing weakness and tremors but often resolves with treatment.3,142 muscle degeneration releases several enzymes, including creatinine kinase (ck); this can be useful in supporting a diagnosis of nmd.1,136 subclinical se deficiency in older calves may manifest as ill thrift, with stunted growth, poor coat condition, and poor nutrient absorption, potentially leading to death.1,20 yearling cattle, particularly those raised on se-deficient diets or grain-based feeds that cause vitamin e degradation, are also susceptible to nmd.3 subclinical cases often present as weakened immune systems, with an increased susceptibility to diseases like respiratory infections. subacute nmd in yearlings can cause muscle degeneration, similar to clinical signs in older calves. severe cases may lead to ‘flying scapula’143 or a spreading of toes, and muscle weakness may impair feeding or swallowing.1 acute cases can cause myoglobinuria, stiffness, and even sudden death.3 selenium supplementation alleviates some clinical signs, especially in beef steers, where it can improve prolactin and mitigate fescue toxicosis.144-149 in adult cattle, se deficiency often manifests as reproductive issues, due to its crucial role in follicular development, progesterone production, and antioxidant defense during high metabolic stress (e.g. pregnancy and lactation). a se-deficient herd may have higher rates of rfm, infertility, metritis, cystic ovaries, and early embryonic death, even with marginal deficiencies.3,20,150 bulls are also affected, with reduced sperm motility and viability due to impaired selenoproteins in sperm tails and acrosomes, leading to decreased fertility.124,125 selenium toxicosis selenium deficiency is more common than toxicosis, but the latter does occur. mining operations can expose se-bearing elements to air and water, mobilizing se into aquatic systems where it bioaccumulates in the ground water, soil, grasses and forages, amplifying hazardous effects.151 selenium toxicosis occurs when an animal’s diet exceeds 3-8 ppm of se (threshold varies by species).152 some animals can tolerate higher concentrations. forage plants that lead to toxicosis typically contain 30-160 times the amount of se required for nutrition. poisoning can also result from consuming grains with elevated se, affecting livestock such as poultry and swine.152 http://dx.doi.org/10.58292/ct.v17.12015 https://www.ecfr.gov/current/title-21/chapter-i/subchapter-e/part-573/subpart-b/section-573.920 https://www.ecfr.gov/current/title-21/chapter-i/subchapter-e/part-573/subpart-b/section-573.920 https://www.ecfr.gov/current/title-21/chapter-i/subchapter-e/part-573/subpart-b/section-573.920 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 11 selenium poisoning is classified into acute and chronic forms.153,154 acute poisoning typically results from shortterm ingestion of highly seleniferous plants, particularly when animals are hungry and consume low-palatable plants with high se content. symptoms of acute poisoning include abnormal posture, unsteady gait, abdominal pain, and, in severe cases, death.153,154 chronic selenosis is associated with hair and hoof abnormalities. bilateral alopecia or hair fragility and breakage along the mane, tail, and the nape are described in production animals. chronic se poisoning is further categorized into alkali disease and blind staggers.153,154 alkali disease occurs when animals consume seleniferous forages (typically 5-40 ppm of se) over extended intervals. symptoms of alkali disease include dullness, emaciation, rough coats, hair loss, and lameness. blind staggers result from grazing se indicator plants containing water-soluble se, with impaired vision, weakness, paralysis, and death from respiratory failure. there is debate whether blind staggers is caused solely by se, with other factors or toxins in plants suspected as having a role.155 similar symptoms have been observed in conditions such as forage poisoning in colorado, tansy mustard poisoning in the southwest, and kochia poisoning.156 high sulfate concentrations in water or feed may also contribute to these symptoms, implying blind staggers may have multiple causes. ultimately, alkali disease appears to be the primary chronic manifestation of se poisoning, whereas blind staggers remain a more ambiguous condition that could involve additional toxins or unknown factors. reproductive effects of se toxicity selenium toxicity can negatively affect reproduction in cattle in several ways. at higher concentrations, se can become prooxidant, leading to os and damage to cellular components. elevated se can impair sperm quality157 in bulls. selenium controls development of granulosa cells and biosynthesis of estradiol-17β in adult ovaries in vitro.158 selenosis can disrupt oocyte development leading to reduced fertility. high se concentrations can cause spontaneous abortions, particularly in later stages of pregnancy.159 calves born to cows with high se exposure may have birth defects, particularly musculoskeletal issues.160 selenium toxicity can decrease milk yield, possibly due to damage to mammary tissue or an overall decline in health. diagnosis of selenium deficiency testing soil, feed, and live cattle if herd problems are primarily reproductive, general unthriftiness or subtle and deficient se status is suspected, a diagnosis is typically made based on clinical signs and response to se therapy. testing is an important adjunct for diagnosis and is vital for future supplementation planning. deficient, marginal, and normal se concentrations standards for the analysis of different samples are provided (table 2). representative analysis of pasture/forage/herd samples are critical. diagnosis of selenosis diagnosis generally involves observation of symptoms such as hoof abnormalities, hair loss, and reproductive problems.161,162 blood, serum samples or liver tissues are tested for se.163 normal se concentrations in cattle are generally 0.050.10 ppm, with > 0.25 ppm considered toxic. testing for se content in the forage and feed is also critical.164 liver and kidney are the most common postmortem samples used for se analysis.165 table 2. selenium concentration standards: deficient, marginal, and normal levels for various sample analyses sample types for testing selenium level deficient marginal normal soil 0.5 mg/kg pasture < 0.02 mg/kg dm of pasture forages and grains with 0.1 mg/kg dm are considered adequate3 serum† 0.002-0.025 mg/ml 0.03-0.07 mg/ml 0.08-0.3 mg/ml3 whole blood‡ < 0.05 ppm 0.05-0.1 ppm > 0.11 ppm* serum erythrocyte gpx activity** 0.2-10 10-19 19-36 serum ck levels*** 21-31 iu/l§ liver biopsy 0.07-0.6 mg/ gram dm 0.45-0.9 mg/ gram dm 0.9-1.75 mg/ gram dm3 †it responds more rapidly to se treatment than whole blood se; ‡there is a delayed response to se treatment compared to serum se; *(> 5 ppm high; se toxicity)20 **this test is faster and more economical than testing for se directly; however, the enzyme is unstable, and improper handling can significantly skew the results. the units are expressed as micromoles per minute at 37°c per gram of hemoglobin; ***it is the most common diagnostic aid for nmd, though it is not specific to se deficiency syndromes. its half-life is 2-4 hours, rapidly declining after myodegeneration, but it can remain elevated for up to 3 days following unaccustomed exercise. levels typically return to normal within a few days of treatment; §after a myodegeneration event, levels are commonly > 5,000-10,000 iu/liter3. http://dx.doi.org/10.58292/ct.v17.12015 12 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 postmortem testing necropsy should be done on aborted fetuses or acute deaths, especially if nmd is suspected. the earlier the diagnosis and intervention, the more subclinical and subacute cases can be remedied. because nmd is not an inflammatory process but rather degeneration of hyaline followed by coagulation necrosis +/mineralization, there will be no muscle inflammation.1 no specific skeletal muscle is always affected, but groups of muscles are always affected bilaterally and symmetrically. they contain localized degenerative and necrotic areas that are white to gray. the affected area is often a streak through the center of an otherwise normal muscle or may be flanking either side of a normal piece of muscle.3 diaphragm also has a streaked appearance but in a radiating pattern with a central focus of muscle degeneration/ necrosis. the diaphragm will be very friable and edematous, with or without mineralization.3 if the muscles of the throat or chest are affected, secondary pneumonia is common.3 in calves with myocardium degeneration, the endocardium of the left ventricle is commonly affected. white to gray lesions may extend to involve interventricular septum and papillary muscles. pulmonary congestion and edema are common with heart involvement.3 treatment of selenium deficiency due to the close relationship vitamin e and se in protecting cells and having difficulties in determining, which is the primary deficiency, se and α-tocopherol combinations are recommended.3 treating nmd is calves is best done with an injectable se (3 mg) and dl alpha-tocopherol acetate (150 iu/ml) injection at 2 ml/45 kg bw im. once is usually sufficient unless it is the acute myocardial form where mortality can reach 90%. if there is at least 1 case of se deficiency, treatment of the entire herd is recommended. as unaccustomed exercise is a predisposing factor for clinical nmd, cattle should be carefully handled during treatment. subacute cases of nmd should improve by 3 days and regain mobility within 1 week.3 treatment of selenosis if se toxicity is suspected, stop the use of any se-containing feed supplements. in recovering steers, half-lives for se elimination from serum, whole blood, liver, and muscle were 40.5 ± 8.2, 115.6 ± 25.1, 38.2 ± 5.0, and 98.5 ± 19.1 days, respectively (relatively long).166 treatment may involve fluids, antioxidants, and liver-protective substances, but recovery can be slow, and severe cases may have permanent damage.167 medications or supplements like vitamin e and antioxidants may reduce os in the liver. preventing selenium deficiency with routine mineral supplementation selenium supplementation needs to be tailored to each specific operation. current se status must be known to determine how and when to supplement the herd.168 first, determine if the area in question is considered se deficient; this can be gained from local extension offices or the us geological survey website.5 consider testing pasture or feed and check labels of any packaged products.152 are there any signs of se deficiency, as described above? if so, consider blood testing for serum se concentrations, whole-blood se concentrations, or erythrocyte gpx concentrations, on a representative sample or all cattle.168 when developing a supplementation program, consider economics and logistics. there are inorganic and organic forms of se. the inorganic form is usually sodium selenite that gives a short-duration increase in se but does not persist. however, if applied to soil, it is readily taken up by plants and incorporated as organic se. organic se is usually selenomethionine and typically provides a long-standing boost in se status.168 selenium injections, typically paired with vitamin e or copper, utilize inorganic sodium selenite, and are therefore best used as prevention in young livestock. in heifers and cows, injecting a se and vitamin e supplement 3 weeks before calving can greatly decrease fertility problems, rfm, poor uterine involution, and cystic ovaries.3 oral se supplementation during pregnancy provides protection for the fetus due to placental transfer and may decrease neonatal morbidity and mortality. selenium concentrations in colostrum and milk reflect the dam’s se status. however, milk se concentrations decrease 7 days after calving, so treatment of calves is often needed.3 mineral supplementation is commonly used, typically in loose or block form. loose is more common as it is more readily consumed, but se can be leached by rain. due to individual differences in intake and potential losses due to rain, results are often inconsistent.169 for calf nmd prevention, loose salt and mineral mix can be fed ad libitum. the recommended 0.1 mg/kg se and 1 gram/ day/head alpha-tocopherol can be achieved with 14.8 mg/kg of mixture and 2,700 iu/kg mixture of vitamin e. cow and calf health and productivity are optimized by using this for the last 2 trimesters of pregnancy and first month of lactation.3,169 fortified feed supplements have inorganic and/or organic sources of se; the latter will produce a more prolonged increase in blood and liver se concentrations than the former. furthermore, organic sources result in more consistent intake on an individual basis.167 seleno-yeast is an excellent option for supplementation in beef cattle. in pregnant cattle, seleno-yeast increased gpx activity better than sodium selenite when offered as a free-choice mineral.3 selenium pellets are available as 10% se and 90% iron grit. they can increase gpx activity for > 2 years and are effective when fed to pregnant cows in the last 3 months of pregnancy, increasing se milk content to prevent calf nmd.3 these pellets were also used for growing cattle and did not cause toxicosis at 2-4 times the recommended dose. alternatively, to ensure no problems upon turn out to pasture in the spring after being fed hay, straw, or high-moisture grain through the winter, supplementing with 0.1 mg/kg dm se and 150 mg/day/head alpha-tocopherol can be used.3 selenium boluses are only approved in california. they provide a consistent amount of se that is slowly released from the bolus into the rumen, making them a good option for grazing animals where supplementation is not feasible.69 these supplementation options should be used year-round in se-deficient areas to improve reproductive and immune health. http://dx.doi.org/10.58292/ct.v17.12015 citation: clinical theriogenology 2025, 17, 12015, http://dx.doi.org/10.58292/ct.v17.12015 13 agricultural practices can also increase soil se concentrations, though they very rarely make a large impact. applying manure of farm animals fed imported se-adequate feed can slowly increase soil concentrations, although this can be a lengthy and expensive process.3 finally, there are licensed products to fertilize se-deficient soils. it is typically recommended to fertilize 5-10 grams of actual se per acre; an application rate of 12-24 grams of sodium selenate per acre will provide the recommended 5-10 grams of actual se per acre.167 prevention of selenosis ensure that se supplementation is done within the recommended levels; total se intake for cattle should not exceed 0.3 mg/kg body weight per day. regularly test forage and pasture soil for se content,170 especially in areas known to have se-rich soils. forage or feed that contains 2-5 ppm se poses a marginal threat to livestock. forage with > 5 ppm se will cause acute toxic conditions in livestock and should be avoided.170 high se forage can be mixed with low se forages to balance total se content.171 in seleniferous areas with high se concentrations, cattle should not be allowed to drink water with a se concentration > 0.5 ppm.170 conclusion selenium is a critical micronutrient with an indispensable role in cellular function; therefore, its importance cannot be overstated. selenium, a powerful antioxidant, supports a variety of physiological processes, including immune function, thyroid hormone metabolism, and reproductive health. in the northwestern us, where soil and forage se concentrations can be low or highly variable, this nutrient is particularly important. deficiencies in se are common in these regions, making it essential to ensure that animal health and productivity are not compromised. inadequate se intake can reduce fertility and the immune response, increase susceptibility to infectious diseases, and compromise growth rates in calves. furthermore, se deficiency can lead to more severe conditions, including nmd and cause death. therefore, it is crucial to thoroughly assess se status of animals and the local environment to determine potential deficiencies. understanding how se concentrations in the soil, forage, and livestock affect herd health will 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https://danr.sd.gov/agriculture/inspection/feedremedy/docs/selenium-brochure.pdf https://doi.org/10.1146/annurev.arplant.51.1.401 2012: maximizing success using fresh chilled semen maximizing success using fresh chilled semen steven escobar springfield veterinary center, glen allen, va the practice of using fresh chilled semen has been done for many years to allow breeding of dogs from different geographical areas. this helps avoid the inconvenience and often stress to the animal of shipping a bitch or stud, or allows a breeding when availability of a stud is difficult. as the popularity of this modality has increased over the years, understanding how to maximize success with this technique is an important consideration. several factors should be discussed. as with any breeding, both the bitch and the stud need to be considered. criteria for the bitch include appropriate health examination, brucellosis testing, and ovulation timing. these will help ensure pregnancy in any insemination attempt. ovulation timing is paramount, as longevity of viability of fresh chilled semen is potentially lower than that of naturally inseminated semen. one may have easier success without specific ovulation timing with natural breedings or “side by side” breedings when multiple inseminations are convenient over the fertile period. even in these breedings, however, appropriate timing increases not only the level of success of pregnancy but also helps to predict gestational length and determine whelping dates. in fresh chilled breedings, quantitative progesterone testing, and when convenient, luteinizing hormone testing are critical to optimize insemination at an appropriate time postovulation. interpretation of these tests as to when ovulation has occurred is a matter of understanding when luteinizing hormone has peaked, stimulating the ovulatory event, and based on the luteinizing hormone surge, and or a concomitant rise in progesterone levels. it has often been simplified by many that ovulation occurs at 5 nanograms of progesterone, or insemination should occur at 15-18 nanograms for example. these statements are not accurate and actual values can vary from bitch to bitch and cycle to cycle of the same bitch. it is important to note that ovulation does not occur at a specific value, nor should insemination be done at a particular value of progesterone. rather, it is the surge in luteinizing hormone, and rise in progesterone, and “the slope of the curve” of that rise which helps determine when ovulation has likely occurred. this requires a bit of experience, and should be done by a veterinarian familiar with ovulation timing. luteinizing hormone testing requires daily serum samples to be drawn and held, whereas progesterone levels can be drawn less frequently such as every other day. for this reason, many veterinarians, based on the convenience of the breeder to present the bitch for daily blood drawings, may opt to do only progesterone timing unless convenient to draw daily luteinizing hormone levels. once it is determined when the ovulatory event has occurred, coordinated communication with the collecting individual is important. for determining when to ship semen, one needs to know that the canine egg is unique in that it requires maturation, and meiotic division causing a polar body shed before it is viable for conception. this takes twenty four to forty eight hours post-ovulation, and for the purpose of fresh chilled insemination, shipment should be arranged for the first sample to be sent to arrive at least twenty four and preferably forty eight hours post-ovulation. therefore, appropriate progesterone testing, and timely results help the individual collecting semen to be informed in time to collect and ship the sample. constant communication between parties during ovulation timing is paramount to achieve this. in terms of ensuring viable semen is available when the bitch has been appropriately timed for ovulation, similar criteria exist for the male. health examination, brucellosis testing, semen evaluation, and his current semen’s viability as a fresh chilled sample are critical. importantly, not all males that can produce a litter with a natural breeding, or “side by side” breeding will necessarily do well with a fresh chilled shipment. semen performance in a fresh chilled extender can be variable, and affected by several factors. ideally, either knowledge of viability in a chilled extender based on recent shipment experience, or a fresh chill test done when the decision has been made to use this male and the bitch is soon expected to be in estrus, or has just started her cycle, ensure viable semen at the time of insemination. therefore a current semen evaluation and or chill test are important. semen production is a dynamic changing event, and simply because a male has produced a litter in the last several months or year, does not guarantee 539 clinical theriogenology • volume 4, number 4 • december 2012 current viability of his semen. many factors can contribute to semen viability. ambient heat during summer months, if the male is kept in outdoor kennels can affect sperm viability. age of the male, disease, and genetic factors can also affect his viability. the sperm cycle from spermatogenesis to sperm maturity is about two months in the male dog. if traumatic events such as heat damage or infection occur, it can take two to three months to see a full cycle of sperm development and improvement of the ejaculate. a current sperm evaluation which includes progressive motility, morphology, and viability in the fresh chilled extender to be used, are important to maximize success. this will require the use of a microscope, and experience in evaluating morphological abnormalities, grading progressive motility, and evaluating concentration of a semen sample. a semen count requiring specific equipment such as micropipetters, and hemocytometers, or machines is desirable when inseminating with fresh chilled semen, but not required for this particular procedure if the analyzing individual is experienced in estimating concentration levels. chill testing to determine viability for shipment is the next step after determining initial viability. many veterinarians practicing reproductive medicine will have a variety of extenders available, and can do a chill test in each of them to determine in which extender the male’s semen best performs. both initial evaluation and chill testing should be done as close to the potential shipment as possible to ensure no changes or insult to the semen may have occurred affecting its structure or viability in an extender prior to shipment. with inexperienced males, the initial semen evaluation and chill test also gives the collector a chance to make sure the male is amenable to collection of a semen sample. it is not uncommon if the male has not been previously collected, for the bitch owner to call for a collection, and receive a call back that the male was not able to be collected due to inexperience, stress with a particular facility or collector, or other factors. this can be devastating to the bitch owner unless a standby male is available. there are several techniques to allow a male dog to be collected, from use of teaser bitches, to the use of and injection of the prostaglandin f2-alpha if a teaser bitch is not available. some male dogs are not amenable to collection though, and this should be determined ahead of time. once the individual collecting the semen has been notified of the need for shipment, several factors are to be considered. ideally, the inseminating individual has notified the collector of what type of insemination is expected, allowing for proper extension in the appropriate volume of extender. for surgical insemination, or transcervical insemination a smaller amount of total volume of sample may be desired than for vaginal insemination. this volume will depend on the preference of the individual inseminating, and should be discussed prior to collection. preparation of the semen rich fraction of the ejaculate may include centrifugation to remove undesired volumes of seminal or prostatic fluid, and allow for proper final volume as discussed earlier. please note that most canine reproductive tracts (uterine body and horns) will hold no more than up to two to three milliliters, and any larger volume will simply flush back out into the vaginal tract taking sperm cells with it. too large a volume shipped to the inseminating individual may require centrifugation, and re-extension to a smaller volume because of this fact. proper labeling of the sample tube, and inclusion in the shipment of a record of semen morphology, and progressive motility will help the individual inseminating to determine how well the sample survived the shipping process, and allow for determination of viability, and which type of insemination to perform. while determination of which type of insemination will be done depends on personal preference and is often subjective, samples with lower overall or progressive motility, low percentage of morphologically normal sperm, or low total concentration often will be inseminated by transcervical or surgical insemination to avoid losing viable numbers of sperm more distally in the reproductive tract such as the vaginal vault. it is advisable on shipment that the collecting individual note the tracking number of the shipment and inform the inseminating individual so they may track the shipment on the receiving end. several entities exist for shipment of semen, but in the domestic us, federal express is the most commonly used company. alternatively, direct “counter to counter” shipment can be used to ship semen “same day”. this requires the collector to be identified as a “known shipper” however, which will require a facility inspection and registry with the shipping agent. the author has had success with delta dash in the continental us for this purpose. the process of becoming 540clinical theriogenology • volume 4, number 4 • december 2012 a “known shipper” must be done well previously to an expected collection and shipment and can be a cumbersome process. the success of becoming a “known shipper” can also depend on the airline being used. it can be difficult to achieve this status. if possible, “counter to counter” semen shipment provides for sending semen for example on weekends when federal express cannot deliver. it also allows the semen to be inseminated in a timely fashion on the same day it was collected, when ovulation timing does not ensure enough time for a twenty four hour shipment to be effective. pick up at the local airport, however, is required and should be planned as a part of the process. what method of shipment to be used will also be affected by the environmental conditions currently present geographically. if extreme heat or cold are expected, “counter to counter” shipment in a well-insulated shipping container may be preferable over a delivery with an overnight shipper where samples may not be kept in a controlled environment during shipment. if the collecting individual does not have “known shipper” status, then packaging, and type of container are critical. semen should be in a well-insulated container, and never be placed directly in contact with a frozen “cooling block” as it may freeze the sample and kill the sperm. the sample should always be kept separated from the cooling block by newspaper, bubble wrap, or some other insulating material. cold ambient temperature has also been known to freeze a chilled sample. an equitainer®, a shipping vessel commonly used by equine veterinarians, can be used to protect the sample. there are several shipping containers available from various manufacturers, and personal preference, based on experience can help determine which to utilize. how many shipments should be sent with the use of fresh chilled semen often arises as a common query. of course, the more samples shipped, the more expensive the venture, but generally multiple samples covering the fertile period offer the best opportunity for success. for the purpose of vaginal insemination, which is the most common method used, two samples are often sent to inseminate the bitch on day one and three, or more commonly day two and four after suspected ovulation. at diestrus, which is usually expected at day five to six after ovulation, the cervix closes, and vaginal delivery of sperm to the uterus and horns can be compromised. diestrus may be determined to some extent by reading a vaginal smear. the infiltration of segmented neutrophils into the vaginal lumen is a hallmark of diestrus and can be visualized from a vaginal swab evaluation under a microscope. for this reason, it is usually not recommended to inseminate after day four post-ovulation. this can be circumvented by surgical insemination, effectively bypassing the closed cervix, and allowing for conception sometimes up to eight or nine days post-ovulation. while this has been documented, it is not advisable to make such a late attempt at insemination if possible. if it is not possible to collect two samples for insemination at day one and three, or two and four post-ovulation, and only one shipment is to be made, it is best to do this single insemination at day two or three post-ovulation. which type of insemination to perform is again personal preference of the inseminating individual, and may be decided based on the overall viability of the sample as determined by overall count, progressive motility percentage, and morphology. a common practice in the related field of equine medicine is sometimes to “split” a single collection into two samples to be sent separately a couple of days apart. this is not advisable with canine semen. the differences lie in the fact that equine samples contain tremendously higher numbers of sperm than canine samples. by splitting a canine sample, you are simply decreasing the number of sperm introduced by half for each insemination, and with lower overall numbers than equine sperm you are decreasing your success rate for conception. it is better to inseminate a full collection and collect a second sample for insemination a couple of days later, effectively doubling total sperm inseminated, and providing a more nurturing environment in the bitch’s reproductive tract, than in a refrigerated test tube. the chilled sample in a test tube sample will still be two days older and in a less hospitable environment than in the bitch’s reproductive tract which is designed to maintain the sperm cells appropriately. if only one shipment is expected, then all of the sample should be inseminated at the same time as mentioned above. extenders do exist currently that allow for chilled samples to be held for longer periods. these are advised to only be used when availability of the male for collection when the bitch is ready is simply not achievable. they are not to be used routinely with fresh chilled insemination when the male will be available to be collected when needed. the best environment for semen is still either the male’s 541 clinical theriogenology • volume 4, number 4 • december 2012 epididymis, or the bitch’s reproductive tract. long term extension in a test tube should be avoided unless necessary as described above. once received, the semen sample should be kept at a constant cool temperature until it is inseminated. refrigeration is advisable and if the container is still at appropriate temperature and the cooling blocks are still cold, it may be kept in the shipment container. alternatively the sample may be kept in the refrigerator (not the freezer) until the bitch is available for insemination. insemination should, however, be done as soon as possible upon shipment arrival, unless there is specific reason as mentioned earlier such as use of a longer effective extender in case the male will not be available when the bitch is ready, and semen has been shipped a bit early. on insemination, directions if provided with the shipment, for warming or activation should be followed as they usually have been developed and tested for that particular extender. insemination can be achieved as mentioned by vaginal, transcervical, or surgical insemination as determined by the individual inseminating. this again will be decided based on several factors such as viability and concentration of the semen, how many inseminations are to be performed, and personal experience and preference. it therefore is very important for the sample to again at insemination be evaluated for viability. this is no less critical at this point, than it was on initial collection. semen progressive motility, morphology, and concentration should be analyzed. if less than desirable, several interventions can be achieved, such as different insemination technique, different extender for the next sample, or an increased number of shipments during the fertile period. sometimes re-suspending the sample in fresh extender has helped to boost the sample’s motility. while fresh chilled semen has been collected and shipped in a variety of ways by a variety of individuals, with or without thorough evaluation, ovulation timing, or careful packaging, maximizing the effectiveness of this technique can be achieved by consideration of the techniques and factors listed in this text. due to the costs of collecting, packaging, and shipping fresh chilled semen, and often the desire to achieve a pregnancy on a particular cycle, it is advisable to be as thorough and careful in the process as possible. when performed in this manner, fresh chilled insemination is a very effective means of achieving pregnancy in the canine. 542clinical theriogenology • volume 4, number 4 • december 2012